{"article_id": "PMC5075302", "article_text": ["anxiety affects quality of life in those living with parkinson 's disease ( pd ) more so than overall cognitive status , motor deficits , apathy , and depression [ 13 ] .", "although anxiety and depression are often related and coexist in pd patients , recent research suggests that anxiety rather than depression is the most prominent and prevalent mood disorder in pd [ 5 , 6 ] . yet ,", "our current understanding of anxiety and its impact on cognition in pd , as well as its neural basis and best treatment practices , remains meager and lags far behind that of depression .", "overall , neuropsychiatric symptoms in pd have been shown to be negatively associated with cognitive performance .", "for example , higher depression scores have been correlated with lower scores on the mini - mental state exam ( mmse ) [ 8 , 9 ] as well as tests of memory and executive functions ( e.g. , attention ) [ 1014 ] .", "likewise , apathy and anhedonia in pd patients have been associated with executive dysfunction [ 10 , 1523 ] .", "however , few studies have specifically investigated the relationship between anxiety and cognition in pd .", "one study showed a strong negative relationship between anxiety ( both state and trait ) and overall cognitive performance ( measured by the total of the repeatable battery for the assessment of neuropsychological status index ) within a sample of 27 pd patients .", "furthermore , trait anxiety was negatively associated with each of the cognitive domains assessed by the rbans ( i.e. , immediate memory , visuospatial construction , language , attention , and delayed memory ) .", "two further studies have examined whether anxiety differentially affects cognition in patients with left - sided dominant pd ( lpd ) versus right - sided dominant pd ( rpd ) ; however , their findings were inconsistent .", "the first study found that working memory performance was worse in lpd patients with anxiety compared to rpd patients with anxiety , whereas the second study reported that , in lpd , apathy but not anxiety was associated with performance on nonverbally mediated executive functions and visuospatial tasks ( e.g. , tmt - b , wms - iii spatial span ) , while in rpd , anxiety but not apathy significantly correlated with performance on verbally mediated tasks ( e.g. , clock reading test and boston naming test ) .", "furthermore , anxiety was significantly correlated with neuropsychological measures of attention and executive and visuospatial functions . taken together ,", "it is evident that there are limited and inconsistent findings describing the relationship between anxiety and cognition in pd and more specifically how anxiety might influence particular domains of cognition such as attention and memory and executive functioning .", "it is also striking that , to date , no study has examined the influence of anxiety on cognition in pd by directly comparing groups of pd patients with and without anxiety while excluding depression .", "given that research on healthy young adults suggests that anxiety reduces processing capacity and impairs processing efficiency , especially in the central executive and attentional systems of working memory [ 26 , 27 ] , we hypothesized that pd patients with anxiety would show impairments in attentional set - shifting and working memory compared to pd patients without anxiety .", "furthermore , since previous work , albeit limited , has focused on the influence of symptom laterality on anxiety and cognition , we also explored this relationship .", "seventeen pd patients with anxiety and thirty - three pd patients without anxiety were included in this study ( see table 1 ) .", "the cross - sectional data from these participants was taken from a patient database that has been compiled over the past 8 years ( since 2008 ) at the parkinson 's disease research clinic at the brain and mind centre , university of sydney .", "inclusion criteria involved a diagnosis of idiopathic pd according to the united kingdom parkinson 's disease society brain bank criteria   and were confirmed by a neurologist ( sjgl ) .", "patients also had to have an adequate proficiency in english and have completed a full neuropsychological assessment .", "ten patients in this study ( 5 pd with anxiety ; 5 pd without anxiety ) were taking psychotropic drugs ( i.e. , benzodiazepine or selective serotonin reuptake inhibitor ) .", "patients were also excluded if they had other neurological disorders , psychiatric disorders other than affective disorders ( such as anxiety ) , or if they reported a score greater than six on the depression subscale of the hospital anxiety and depression scale ( hads ) .", "thus , all participants who scored within a  depressed  ( hads - d > 6 ) range were excluded from this study , in attempt to examine a refined sample of pd patients with and without anxiety in order to determine the independent effect of anxiety on cognition .", "this research was approved by the human research ethics committee of the university of sydney , and written informed consent was obtained from all participants .", "self - reported hads was used to assess anxiety in pd and has been previously shown to be a useful measure of clinical anxiety in pd .", "a cut - off score of > 8 on the anxiety subscale of the hads ( hads - a ) was used to identify pd cases with anxiety ( pda+ ) , while a cut - off score of < 6 on the hads - a was used to identify pd cases without anxiety ( pda ) .", "this criterion was more stringent than usual ( > 7 cut - off score ) , in effort to create distinct patient groups .", "the neurological evaluation rated participants according to hoehn and yahr ( h&y ) stages   and assessed their motor symptoms using part iii of the revised mds task force unified parkinson 's disease rating scale ( updrs ) . in a similar way", "this was determined by calculating a total left and right score from rigidity items 3035 , voluntary movement items 3643 , and tremor items 5057 from the mds - updrs part iii ( see table 1 ) .", "processing speed was assessed using the trail making test , part a ( tmt - a , z - score ) .", "attentional set - shifting was measured using the trail making test , part b ( tmt - b , z - score ) .", "working memory was assessed using the digit span forward and backward subtest of the wechsler memory scale - iii ( raw scores ) .", "language was assessed with semantic and phonemic verbal fluency via the controlled oral word associated test ( cowat animals and letters , z - score ) .", "the ability to retain learned verbal memory was assessed using the logical memory subtest from the wechsler memory scale - iii ( lm - i z - score , lm - ii z - score , % lm retention z - score ) . the mini - mental state examination ( mmse )", "demographic , clinical , and neuropsychological variables were compared between the two groups with the independent t - test or mann  whitney u test , depending on whether the variable met parametric assumptions .", "chi - square tests were used to examine gender and symptom laterality differences between groups .", "all analyses employed an alpha level of p < 0.05 and were two - tailed .", "spearman correlations were performed separately in each group to examine associations between anxiety and/or depression ratings and cognitive functions .", "as expected , the pda+ group reported significant greater levels of anxiety on the hads - a ( u = 0 , p < 0.001 ) and higher total score on the hads ( u = 1 , p < 0.001 ) compared to the pda group ( table 1 ) .", "groups were matched in age ( t(48 ) = 1.31 , p = 0.20 ) , disease duration ( u = 259 , p = 0.66 ) , updrs - iii score ( u = 250.5 , p = 0.65 ) , h&y ( u = 245 , p = 0.43 ) , ledd ( u = 159.5 , p = 0.80 ) , and depression ( hads - d ) ( u = 190.5 , p = 0.06 ) .", "additionally , all groups were matched in the distribution of gender (  = 0.098 , p = 0.75 ) and side - affected (  = 0.765 , p = 0.38 ) .", "there were no group differences for tmt - a performance ( u = 256 , p = 0.62 ) ( table 2 ) ; however , the pda+ group had worse performance on the trail making test part b ( t(46 ) = 2.03 , p = 0.048 ) compared to the pda group ( figure 1 ) .", "the pda+ group also demonstrated significantly worse performance on the digit span forward subtest ( t(48 ) = 2.22 , p = 0.031 ) and backward subtest ( u = 190.5 , p = 0.016 ) compared to the pda group ( figures 2(a ) and 2(b ) ) .", "neither semantic verbal fluency ( t(47 ) = 0.70 , p = 0.49 ) nor phonemic verbal fluency ( t(47 ) = 0.39 , p = 0.70 ) differed between groups .", "logical memory i immediate recall test ( u = 176 , p = 0.059 ) showed a trend that the pda+ group had worse new verbal learning and immediate recall abilities than the pda group . however , logical memory ii test performance ( u = 219 , p = 0.204 ) and logical memory % retention ( u = 242.5 , p = 0.434 ) did not differ between groups .", "there were also no differences between groups in global cognition ( mmse ) ( u = 222.5 , p = 0.23 ) .", "participants were split into lpd and rpd , and then further group differences were examined between pda+ and pda. importantly , the groups remained matched in age , disease duration , updrs - iii , dde , h&y stage , and depression but remained significantly different on self - reported anxiety .", "lpda+ demonstrated worse performance on the digit span forward test ( t(19 ) = 2.29 , p = 0.033 ) compared to lpda , whereas rpda+ demonstrated worse performance on the digit span backward test ( u = 36.5 , p = 0.006 ) , lm - i immediate recall ( u = 37.5 , p = 0.008 ) , and lm - ii ( u = 45.0 , p = 0.021 ) but not lm % retention ( u = 75.5 , p = 0.39 ) compared to rpda.", "this study is the first to directly compare cognition between pd patients with and without anxiety .", "the findings confirmed our hypothesis that anxiety negatively influences attentional set - shifting and working memory in pd .", "more specifically , we found that pd patients with anxiety were more impaired on the trail making test part b which assessed attentional set - shifting , on both digit span tests which assessed working memory and attention , and to a lesser extent on the logical memory test which assessed memory and new verbal learning compared to pd patients without anxiety . taken together ,", "these findings suggest that anxiety in pd may reduce processing capacity and impair processing efficiency , especially in the central executive and attentional systems of working memory in a similar way as seen in young healthy adults [ 26 , 27 ] .", "although the neurobiology of anxiety in pd remains unknown , many researchers have postulated that anxiety disorders are related to neurochemical changes that occur during the early , premotor stages of pd - related degeneration [ 37 , 38 ] such as nigrostriatal dopamine depletion , as well as cell loss within serotonergic and noradrenergic brainstem nuclei ( i.e. , raphe nuclei and locus coeruleus , resp . , which provide massive inputs to corticolimbic regions ) . over time", ", chronic dysregulation of adrenocortical and catecholamine functions can lead to hippocampal damage as well as dysfunctional prefrontal neural circuitries [ 39 , 40 ] , which play a key role in memory and attention .", "recent functional neuroimaging work has suggested that enhanced hippocampal activation during executive functioning and working memory tasks may represent compensatory processes for impaired frontostriatal functions in pd patients compared to controls . therefore , chronic stress from anxiety ,", "for example , may disrupt compensatory processes in pd patients and explain the cognitive impairments specifically in working memory and attention seen in pd patients with anxiety .", "it has also been suggested that hyperactivation within the putamen may reflect a compensatory striatal mechanism to maintain normal working memory performance in pd patients ; however , losing this compensatory activation has been shown to contribute to poor working memory performance .", "anxiety in mild pd has been linked to reduced putamen dopamine uptake which becomes more extensive as the disease progresses .", "this further supports the notion that anxiety may disrupt compensatory striatal mechanisms as well , providing another possible explanation for the cognitive impairments observed in pd patients with anxiety in this study .", "noradrenergic and serotonergic systems should also be considered when trying to explain the mechanisms by which anxiety may influence cognition in pd . although these neurotransmitter systems are relatively understudied in pd cognition , treating the noradrenergic and serotonergic systems has shown beneficial effects on cognition in pd .", "selective serotonin reuptake inhibitor , citalopram , was shown to improve response inhibition deficits in pd , while noradrenaline reuptake blocker , atomoxetine , has been recently reported to have promising effects on cognition in pd [ 45 , 46 ] .", "overall , very few neuroimaging studies have been conducted in pd in order to understand the neural correlates of pd anxiety and its underlying neural pathology .", "future research should focus on relating anatomical changes and neurochemical changes to neural activation in order to gain a clearer understanding on how these pathologies affect anxiety in pd . to further understand how anxiety and cognitive dysfunction are related ,", "future research should focus on using advanced structural and function imaging techniques to explain both cognitive and neural breakdowns that are associated with anxiety in pd patients .", "research has indicated that those with amnestic mild cognitive impairment who have more neuropsychiatric symptoms have a greater risk of developing dementia compared to those with fewer neuropsychiatric symptoms .", "future studies should also examine whether treating neuropsychiatric symptoms might impact the progression of cognitive decline and improve cognitive impairments in pd patients .", "previous studies have used pd symptom laterality as a window to infer asymmetrical dysfunction of neural circuits .", "for example , lpd patients have greater inferred right hemisphere pathology , whereas rpd patients have greater inferred left hemisphere pathology .", "thus , cognitive domains predominantly subserved by the left hemisphere ( e.g. , verbally mediated tasks of executive function and verbal memory ) might be hypothesized to be more affected in rpd than lpd ; however , this remains controversial .", "it has also been suggested that since anxiety is a common feature of left hemisphere involvement [ 48 , 49 ] , cognitive domains subserved by the left hemisphere may also be more strongly related to anxiety .", "results from this study showed selective verbal memory deficits in rpd patients with anxiety compared to rpd without anxiety , whereas lpd patients with anxiety had greater attentional / working memory deficits compared to lpd without anxiety .", "although these results align with previous research , interpretations of these findings should be made with caution due to the small sample size in the lpd comparison specifically .", "recent work has suggested that the hads questionnaire may underestimate the burden of anxiety related symptomology and therefore be a less sensitive measure of anxiety in pd [ 30 , 50 ] . in addition , our small sample size also limited the statistical power for detecting significant findings .", "based on these limitations , our findings are likely conservative and underrepresent the true impact anxiety has on cognition in pd . additionally , the current study employed a very brief neuropsychological assessment including one or two tests for each cognitive domain .", "future studies are encouraged to collect a more complex and comprehensive battery from a larger sample of pd participants in order to better understand the role anxiety plays on cognition in pd .", "another limitation of this study was the absence of diagnostic interviews to characterize participants ' psychiatric symptoms and specify the type of anxiety disorders included in this study .", "future studies should perform diagnostic interviews with participants ( e.g. , using dsm - v criteria ) rather than relying on self - reported measures to group participants , in order to better understand whether the type of anxiety disorder ( e.g. , social anxiety , phobias , panic disorders , and generalized anxiety ) influences cognitive performance differently in pd .", "one advantage the hads questionnaire provided over other anxiety scales was that it assessed both anxiety and depression simultaneously and allowed us to control for coexisting depression .", "although there was a trend that the pda+ group self - reported higher levels of depression than the pda group , all participants included in the study scored < 6 on the depression subscale of the hads .", "controlling for depression while assessing anxiety has been identified as a key shortcoming in the majority of recent work .", "considering many previous studies have investigated the influence of depression on cognition in pd without accounting for the presence of anxiety and the inconsistent findings reported to date , we recommend that future research should try to disentangle the influence of anxiety versus depression on cognitive impairments in pd . considering the growing number of clinical trials for treating depression , there are few if any for the treatment of anxiety in pd .", "anxiety is a key contributor to decreased quality of life in pd and greatly requires better treatment options .", "moreover , anxiety has been suggested to play a key role in freezing of gait ( fog ) , which is also related to attentional set - shifting [ 52 , 53 ] .", "future research should examine the link between anxiety , set - shifting , and fog , in order to determine whether treating anxiety might be a potential therapy for improving fog ."], "abstract_text": ["<S> research on the implications of anxiety in parkinson 's disease ( pd ) has been neglected despite its prevalence in nearly 50% of patients and its negative impact on quality of life . </S>", "<S> previous reports have noted that neuropsychiatric symptoms impair cognitive performance in pd patients ; however , to date , no study has directly compared pd patients with and without anxiety to examine the impact of anxiety on cognitive impairments in pd . </S>", "<S> this study compared cognitive performance across 50 pd participants with and without anxiety ( 17 pda+ ; 33 pda ) , who underwent neurological and neuropsychological assessment . </S>", "<S> group performance was compared across the following cognitive domains : simple attention / visuomotor processing speed , executive function ( e.g. , set - shifting ) , working memory , language , and memory / new verbal learning . </S>", "<S> results showed that pda+ performed significantly worse on the digit span forward and backward test and part b of the trail making task ( tmt - b ) compared to the pda group . </S>", "<S> there were no group differences in verbal fluency , logical memory , or tmt - a performance . in conclusion , </S>", "<S> anxiety in pd has a measurable impact on working memory and attentional set - shifting . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["anxiety affects quality of life in those living with parkinson 's disease ( pd ) more so than overall cognitive status , motor deficits , apathy , and depression [ 13 ] .", "although anxiety and depression are often related and coexist in pd patients , recent research suggests that anxiety rather than depression is the most prominent and prevalent mood disorder in pd [ 5 , 6 ] . yet ,", "our current understanding of anxiety and its impact on cognition in pd , as well as its neural basis and best treatment practices , remains meager and lags far behind that of depression .", "overall , neuropsychiatric symptoms in pd have been shown to be negatively associated with cognitive performance .", "for example , higher depression scores have been correlated with lower scores on the mini - mental state exam ( mmse ) [ 8 , 9 ] as well as tests of memory and executive functions ( e.g. , attention ) [ 1014 ] .", "likewise , apathy and anhedonia in pd patients have been associated with executive dysfunction [ 10 , 1523 ] .", "however , few studies have specifically investigated the relationship between anxiety and cognition in pd .", "one study showed a strong negative relationship between anxiety ( both state and trait ) and overall cognitive performance ( measured by the total of the repeatable battery for the assessment of neuropsychological status index ) within a sample of 27 pd patients .", "furthermore , trait anxiety was negatively associated with each of the cognitive domains assessed by the rbans ( i.e. , immediate memory , visuospatial construction , language , attention , and delayed memory ) .", "two further studies have examined whether anxiety differentially affects cognition in patients with left - sided dominant pd ( lpd ) versus right - sided dominant pd ( rpd ) ; however , their findings were inconsistent .", "the first study found that working memory performance was worse in lpd patients with anxiety compared to rpd patients with anxiety , whereas the second study reported that , in lpd , apathy but not anxiety was associated with performance on nonverbally mediated executive functions and visuospatial tasks ( e.g. , tmt - b , wms - iii spatial span ) , while in rpd , anxiety but not apathy significantly correlated with performance on verbally mediated tasks ( e.g. , clock reading test and boston naming test ) .", "furthermore , anxiety was significantly correlated with neuropsychological measures of attention and executive and visuospatial functions . taken together ,", "it is evident that there are limited and inconsistent findings describing the relationship between anxiety and cognition in pd and more specifically how anxiety might influence particular domains of cognition such as attention and memory and executive functioning .", "it is also striking that , to date , no study has examined the influence of anxiety on cognition in pd by directly comparing groups of pd patients with and without anxiety while excluding depression .", "given that research on healthy young adults suggests that anxiety reduces processing capacity and impairs processing efficiency , especially in the central executive and attentional systems of working memory [ 26 , 27 ] , we hypothesized that pd patients with anxiety would show impairments in attentional set - shifting and working memory compared to pd patients without anxiety .", "furthermore , since previous work , albeit limited , has focused on the influence of symptom laterality on anxiety and cognition , we also explored this relationship ."], ["seventeen pd patients with anxiety and thirty - three pd patients without anxiety were included in this study ( see table 1 ) .", "the cross - sectional data from these participants was taken from a patient database that has been compiled over the past 8 years ( since 2008 ) at the parkinson 's disease research clinic at the brain and mind centre , university of sydney .", "inclusion criteria involved a diagnosis of idiopathic pd according to the united kingdom parkinson 's disease society brain bank criteria   and were confirmed by a neurologist ( sjgl ) .", "patients also had to have an adequate proficiency in english and have completed a full neuropsychological assessment .", "ten patients in this study ( 5 pd with anxiety ; 5 pd without anxiety ) were taking psychotropic drugs ( i.e. , benzodiazepine or selective serotonin reuptake inhibitor ) .", "patients were also excluded if they had other neurological disorders , psychiatric disorders other than affective disorders ( such as anxiety ) , or if they reported a score greater than six on the depression subscale of the hospital anxiety and depression scale ( hads ) .", "thus , all participants who scored within a  depressed  ( hads - d > 6 ) range were excluded from this study , in attempt to examine a refined sample of pd patients with and without anxiety in order to determine the independent effect of anxiety on cognition .", "this research was approved by the human research ethics committee of the university of sydney , and written informed consent was obtained from all participants .", "self - reported hads was used to assess anxiety in pd and has been previously shown to be a useful measure of clinical anxiety in pd .", "a cut - off score of > 8 on the anxiety subscale of the hads ( hads - a ) was used to identify pd cases with anxiety ( pda+ ) , while a cut - off score of < 6 on the hads - a was used to identify pd cases without anxiety ( pda ) .", "this criterion was more stringent than usual ( > 7 cut - off score ) , in effort to create distinct patient groups .", "the neurological evaluation rated participants according to hoehn and yahr ( h&y ) stages   and assessed their motor symptoms using part iii of the revised mds task force unified parkinson 's disease rating scale ( updrs ) . in a similar way", "this was determined by calculating a total left and right score from rigidity items 3035 , voluntary movement items 3643 , and tremor items 5057 from the mds - updrs part iii ( see table 1 ) .", "processing speed was assessed using the trail making test , part a ( tmt - a , z - score ) .", "attentional set - shifting was measured using the trail making test , part b ( tmt - b , z - score ) .", "working memory was assessed using the digit span forward and backward subtest of the wechsler memory scale - iii ( raw scores ) .", "language was assessed with semantic and phonemic verbal fluency via the controlled oral word associated test ( cowat animals and letters , z - score ) .", "the ability to retain learned verbal memory was assessed using the logical memory subtest from the wechsler memory scale - iii ( lm - i z - score , lm - ii z - score , % lm retention z - score ) . the mini - mental state examination ( mmse )", "demographic , clinical , and neuropsychological variables were compared between the two groups with the independent t - test or mann  whitney u test , depending on whether the variable met parametric assumptions .", "chi - square tests were used to examine gender and symptom laterality differences between groups .", "all analyses employed an alpha level of p < 0.05 and were two - tailed .", "spearman correlations were performed separately in each group to examine associations between anxiety and/or depression ratings and cognitive functions ."], ["as expected , the pda+ group reported significant greater levels of anxiety on the hads - a ( u = 0 , p < 0.001 ) and higher total score on the hads ( u = 1 , p < 0.001 ) compared to the pda group ( table 1 ) .", "groups were matched in age ( t(48 ) = 1.31 , p = 0.20 ) , disease duration ( u = 259 , p = 0.66 ) , updrs - iii score ( u = 250.5 , p = 0.65 ) , h&y ( u = 245 , p = 0.43 ) , ledd ( u = 159.5 , p = 0.80 ) , and depression ( hads - d ) ( u = 190.5 , p = 0.06 ) .", "additionally , all groups were matched in the distribution of gender (  = 0.098 , p = 0.75 ) and side - affected (  = 0.765 , p = 0.38 ) .", "there were no group differences for tmt - a performance ( u = 256 , p = 0.62 ) ( table 2 ) ; however , the pda+ group had worse performance on the trail making test part b ( t(46 ) = 2.03 , p = 0.048 ) compared to the pda group ( figure 1 ) .", "the pda+ group also demonstrated significantly worse performance on the digit span forward subtest ( t(48 ) = 2.22 , p = 0.031 ) and backward subtest ( u = 190.5 , p = 0.016 ) compared to the pda group ( figures 2(a ) and 2(b ) ) .", "neither semantic verbal fluency ( t(47 ) = 0.70 , p = 0.49 ) nor phonemic verbal fluency ( t(47 ) = 0.39 , p = 0.70 ) differed between groups .", "logical memory i immediate recall test ( u = 176 , p = 0.059 ) showed a trend that the pda+ group had worse new verbal learning and immediate recall abilities than the pda group . however , logical memory ii test performance ( u = 219 , p = 0.204 ) and logical memory % retention ( u = 242.5 , p = 0.434 ) did not differ between groups .", "there were also no differences between groups in global cognition ( mmse ) ( u = 222.5 , p = 0.23 ) .", "participants were split into lpd and rpd , and then further group differences were examined between pda+ and pda. importantly , the groups remained matched in age , disease duration , updrs - iii , dde , h&y stage , and depression but remained significantly different on self - reported anxiety .", "lpda+ demonstrated worse performance on the digit span forward test ( t(19 ) = 2.29 , p = 0.033 ) compared to lpda , whereas rpda+ demonstrated worse performance on the digit span backward test ( u = 36.5 , p = 0.006 ) , lm - i immediate recall ( u = 37.5 , p = 0.008 ) , and lm - ii ( u = 45.0 , p = 0.021 ) but not lm % retention ( u = 75.5 , p = 0.39 ) compared to rpda."], ["this study is the first to directly compare cognition between pd patients with and without anxiety .", "the findings confirmed our hypothesis that anxiety negatively influences attentional set - shifting and working memory in pd .", "more specifically , we found that pd patients with anxiety were more impaired on the trail making test part b which assessed attentional set - shifting , on both digit span tests which assessed working memory and attention , and to a lesser extent on the logical memory test which assessed memory and new verbal learning compared to pd patients without anxiety . taken together ,", "these findings suggest that anxiety in pd may reduce processing capacity and impair processing efficiency , especially in the central executive and attentional systems of working memory in a similar way as seen in young healthy adults [ 26 , 27 ] .", "although the neurobiology of anxiety in pd remains unknown , many researchers have postulated that anxiety disorders are related to neurochemical changes that occur during the early , premotor stages of pd - related degeneration [ 37 , 38 ] such as nigrostriatal dopamine depletion , as well as cell loss within serotonergic and noradrenergic brainstem nuclei ( i.e. , raphe nuclei and locus coeruleus , resp . , which provide massive inputs to corticolimbic regions ) . over time", ", chronic dysregulation of adrenocortical and catecholamine functions can lead to hippocampal damage as well as dysfunctional prefrontal neural circuitries [ 39 , 40 ] , which play a key role in memory and attention .", "recent functional neuroimaging work has suggested that enhanced hippocampal activation during executive functioning and working memory tasks may represent compensatory processes for impaired frontostriatal functions in pd patients compared to controls . therefore , chronic stress from anxiety ,", "for example , may disrupt compensatory processes in pd patients and explain the cognitive impairments specifically in working memory and attention seen in pd patients with anxiety .", "it has also been suggested that hyperactivation within the putamen may reflect a compensatory striatal mechanism to maintain normal working memory performance in pd patients ; however , losing this compensatory activation has been shown to contribute to poor working memory performance .", "anxiety in mild pd has been linked to reduced putamen dopamine uptake which becomes more extensive as the disease progresses .", "this further supports the notion that anxiety may disrupt compensatory striatal mechanisms as well , providing another possible explanation for the cognitive impairments observed in pd patients with anxiety in this study .", "noradrenergic and serotonergic systems should also be considered when trying to explain the mechanisms by which anxiety may influence cognition in pd . although these neurotransmitter systems are relatively understudied in pd cognition , treating the noradrenergic and serotonergic systems has shown beneficial effects on cognition in pd .", "selective serotonin reuptake inhibitor , citalopram , was shown to improve response inhibition deficits in pd , while noradrenaline reuptake blocker , atomoxetine , has been recently reported to have promising effects on cognition in pd [ 45 , 46 ] .", "overall , very few neuroimaging studies have been conducted in pd in order to understand the neural correlates of pd anxiety and its underlying neural pathology .", "future research should focus on relating anatomical changes and neurochemical changes to neural activation in order to gain a clearer understanding on how these pathologies affect anxiety in pd . to further understand how anxiety and cognitive dysfunction are related ,", "future research should focus on using advanced structural and function imaging techniques to explain both cognitive and neural breakdowns that are associated with anxiety in pd patients .", "research has indicated that those with amnestic mild cognitive impairment who have more neuropsychiatric symptoms have a greater risk of developing dementia compared to those with fewer neuropsychiatric symptoms .", "future studies should also examine whether treating neuropsychiatric symptoms might impact the progression of cognitive decline and improve cognitive impairments in pd patients .", "previous studies have used pd symptom laterality as a window to infer asymmetrical dysfunction of neural circuits .", "for example , lpd patients have greater inferred right hemisphere pathology , whereas rpd patients have greater inferred left hemisphere pathology .", "thus , cognitive domains predominantly subserved by the left hemisphere ( e.g. , verbally mediated tasks of executive function and verbal memory ) might be hypothesized to be more affected in rpd than lpd ; however , this remains controversial .", "it has also been suggested that since anxiety is a common feature of left hemisphere involvement [ 48 , 49 ] , cognitive domains subserved by the left hemisphere may also be more strongly related to anxiety .", "results from this study showed selective verbal memory deficits in rpd patients with anxiety compared to rpd without anxiety , whereas lpd patients with anxiety had greater attentional / working memory deficits compared to lpd without anxiety .", "although these results align with previous research , interpretations of these findings should be made with caution due to the small sample size in the lpd comparison specifically .", "recent work has suggested that the hads questionnaire may underestimate the burden of anxiety related symptomology and therefore be a less sensitive measure of anxiety in pd [ 30 , 50 ] . in addition , our small sample size also limited the statistical power for detecting significant findings .", "based on these limitations , our findings are likely conservative and underrepresent the true impact anxiety has on cognition in pd . additionally , the current study employed a very brief neuropsychological assessment including one or two tests for each cognitive domain .", "future studies are encouraged to collect a more complex and comprehensive battery from a larger sample of pd participants in order to better understand the role anxiety plays on cognition in pd .", "another limitation of this study was the absence of diagnostic interviews to characterize participants ' psychiatric symptoms and specify the type of anxiety disorders included in this study .", "future studies should perform diagnostic interviews with participants ( e.g. , using dsm - v criteria ) rather than relying on self - reported measures to group participants , in order to better understand whether the type of anxiety disorder ( e.g. , social anxiety , phobias , panic disorders , and generalized anxiety ) influences cognitive performance differently in pd .", "one advantage the hads questionnaire provided over other anxiety scales was that it assessed both anxiety and depression simultaneously and allowed us to control for coexisting depression .", "although there was a trend that the pda+ group self - reported higher levels of depression than the pda group , all participants included in the study scored < 6 on the depression subscale of the hads .", "controlling for depression while assessing anxiety has been identified as a key shortcoming in the majority of recent work .", "considering many previous studies have investigated the influence of depression on cognition in pd without accounting for the presence of anxiety and the inconsistent findings reported to date , we recommend that future research should try to disentangle the influence of anxiety versus depression on cognitive impairments in pd . considering the growing number of clinical trials for treating depression , there are few if any for the treatment of anxiety in pd .", "anxiety is a key contributor to decreased quality of life in pd and greatly requires better treatment options .", "moreover , anxiety has been suggested to play a key role in freezing of gait ( fog ) , which is also related to attentional set - shifting [ 52 , 53 ] .", "future research should examine the link between anxiety , set - shifting , and fog , in order to determine whether treating anxiety might be a potential therapy for improving fog ."]]}
{"article_id": "PMC3309138", "article_text": ["small non - coding rnas are transcribed into mrna but remain untranslated in eukaryotic cells .", "they include sirna ( small interfering rna ) , mirna ( microrna ) , pirna ( piwi - interacting rna ) and snorna ( small nucleolar rna ) .", "mirnas are a class of multifunctional singled - stranded small rna which are ~20  nt in length and regulate the stability or translational efficiency of targeted messenger rna depending on the base - pairing complementarity between the mirna and its target mrna [ 1 , 2 ] . although over 1,000 mirna sequences have been identified from the tissues or cells of human origin and other species , as many as 1,000 to 10,000 mirnas per genome have been predicted [ 3 , 4 ] .", "mirnas regulate a broad range of biological processes including timing of development , cell cycle progression , stem cell self - renewal , differentiation , cancer initiation , cancer cell proliferation , metastasis and apoptosis [ 511 ] .", "cancer is caused by multiple processes including uncontrolled cellular proliferation and inappropriate survival of apoptotic cells .", "many regulatory factors switch on or off genes that govern cell division and direct cellular proliferation .", "mirnas regulate gene expression and play important roles in the onset and progression of tumorigenesis .", "emerging evidence demonstrates the involvement of mirna in mammary gland tumorigenesis , functioning either as tumor suppressors or oncogenes .", "although the current treatment of radiation therapy , chemotherapy and hormone therapy slow mammary gland tumor growth , prolong survival and improve the quality of patients life , metastatic breast cancer still remains incurable due to our limited understanding of the molecular mechanisms through which tumorigenesis and metastasis occur .", "as small non - coding rnas regulate gene expression and tumorigenesis , they may represent a novel cancer therapy .", "unlike mrna , mirnas are transcribed but never translated . some mirnas are transcribed from non - coding regions between genes , deriving from independent transcription unit .", "other mirnas are transcribed together with coding mrnas from the coding region of the genome , deriving from the introns of gene transcripts [ 13 , 14 ] .", "mirna gene copy number gain / loss and mirna gene mutation have been observed in breast cancer resulting in the aberrant expression of mirna .", "the first study about the altered expression of mirnas in human breast cancer patients and human breast cancer cell lines was reported in 2005 by lorio et al . ,", "in which 29 mirnas were identified with aberrant expression based on microarray and northern blot analysis of 76 breast tumor samples and 14 human breast cell lines .", "zhang and colleagues analyzed 283 human mirna genes on 55 human breast primary tumors and 18 human breast cancer cell lines using array - based comparative genomic hybridization .", "the results demonstrated a high frequency ( ~72.8% ) of gene copy number abnormality in mirna - containing regions in human breast cancer .", "wang et al . collected 68 patients with newly diagnosed breast cancer and examined the expression of selected mirnas in tumor and adjacent non - tumor tissues .", "mir-21 , mir-106a and mir-155 were significantly over - expressed in the tumor specimens compared with normal controls , whereas mir-126 , mir-199a and mir-335 were significantly decreased in expression in the tumor samples .", "our studies of the mir-17 - 92 cluster demonstrated decreased expression of mir-17/20 in human breast cancer specimens compared with matching normal breast tissue from the same patient .", "subsequent analysis identified reduced mir-17/20 expression in node - positive compared with node - negative breast cancers and demonstrated that mir-17/20 inhibited breast cancer cell migration and invasion via a heterotypic signaling .", "although the tendency for a global decrease of mirna expression in human cancers originally suggested a general tumor suppressor function of mirnas , subsequent studies showing the aberrant expression of specific mirnas in breast cancer suggest mirna - specific roles in breast cancer onset and progression .", "many distinct mirnas have been shown to regulate breast cancer cell proliferation , apoptosis , cancer stem cell expansion , and tumorigenesis .", "mirna may function as either tumor suppressors or oncogenes depending on the cell type , culture conditions , target genes and pathway .", "the involvement of mirna in mammary gland tumorigenesis has been reviewed recently [ 21 , 22 ] .", "le et al . described the expression pattern and regulatory network of key mirnas in breast cancer , including let-7 , mir-34 , mir-125 , mir-200 family , mir-205 , mir-21 , mir-10 and the mir-17 - 92 cluster .", "adams et al . reviewed the mirna regulation of estrogen signaling pathway and erbb2/her signaling pathway in breast cancer .", "the understanding of how mirnas are involved in breast cancer through regulating the cell cycle remains rudimentary .", "herein we summarize the recent literature and research progress on the mechanism by which mirnas regulate the breast cancer cell cycle and cellular proliferation ( fig . ", "1mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and", "let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and", "let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint cyclin d1 is either overexpressed or amplified in ~50% of breast cancer .", "the abundance of cyclin d1 is rate - limiting in breast cancer cellular proliferation and g1-s phase transition [ 23 , 24 ] .", "in addition , cyclin d1 is a critical downstream target of erbb2- , ras- and -catenin- induced breast cancers , and is sufficient for the induction of mammary tumors when targeted to the mammary gland of mice .", "antisense inhibition of cyclin d1 expression in vivo suppressed the growth of neut - transformed mammary adenocarcinoma cells in nude mice .", "conserved sequences of the cyclin d1 3utr contain potential binding sites for multiple mirnas including mir-17/20/106 , mir-15/16 , mir-23 and let-7 .", "mir-17/20 binds the cyclin d1 3utr , inhibiting the expression of cyclin d1 , resulting in cell cycle arrest at the g1 phase and suppression of mcf-7 cell proliferation [ 18 , 26 ] .", "the regulation of cyclin d1 expression by mir-17 - 92 , as well as mir-15/16 , was confirmed by deshpande et al . .", "the let-7 family functions as a tumor suppressor in a variety of cancers including lung , colon , ovarian   and breast cancer .", "schultz et al . demonstrated the downregulation of cyclin d1 by mirna let-7 in control of cancer cell growth .", "the regulation of cyclin d1 by mirna is likely of broad importance as cyclin d1 encodes the regulatory subunit of a kinase that phosphorylates and inactivates the prb family proteins to inhibit dna synthesis , and phosphorylates nuclear respiratory factor 1 ( nrf-1 ) to inhibit mitochondria biogenesis [ 32 , 33 ] .", "furthermore , cyclin d1 promotes breast epithelial cell angiogenesis and migration , and promotes chromosomal instability which in turn contributes to tumorigenesis .", "the mir-221/222 cluster regulates the cell cycle , cell growth and epithelial - to - mesenchymal transition ( emt ) in breast cancer .", "mir-221/222 inhibited p27 and p57 abundance , facilitating g1-s phase transition , thereby promoting cancer cell proliferation [ 36 , 37 ] .", "moreover , mir-221/222 may contribute to the aggressive clinical behavior of basal - like breast cancers .", "the breast cancer basal - like subtype - specific mirnas , mir-221 and mir-222 , promote emt in breast cancer by targeting trps1 ( trichorhinophalangeal syndrome type 1 ) which inhibits emt by repressing zeb2 expression .", "mir-221 and/or mir-222 expression in mcf-7 and t47d breast cancer cells decreased er expression associated with tamoxifen resistance .", "the onco - mirna mir-21 is overexpressed in a wide variety of cancers including breast cancer [ 40 , 41 ] .", "mir-21 induced cellular proliferation , migration , invasion , emt , cancer stem cell characteristics and chemotherapy resistance in human breast cancer [ 42 , 43 ] .", "high mir-21 level is associated with poor prognosis , advanced stage , positive lymph node status and reduced survival time in breast cancer .", "mir-21 promotes mcf-7 cellular proliferation in part through inhibiting the expression of a tumor suppressor gene programmed cell death 4 ( pdcd4 ) . in colon cancer ,", "mir-21 participates in a dna damage - induced g2-m checkpoint through suppressing the cell cycle regulator cdc25a .", "a recent report demonstrated the mir-21 regulates the cell cycle through targeting cdc25a in mcf-7 breast cancer cells . with a potential anti - cancer chemical 3,3-diindolylmethane treatment ,", "mir-27a expression is upregulated in human breast cancer cell lines . in mda - mb-231 cells ,", "mir-27a negatively regulated the zinc finger zbtb10 gene and myt-1 , thereby promoting breast cancer cell proliferation .", "mir-27a suppressed myt-1 , increased cdc2/cyclin b activity and promoted the g2-m checkpoint in mda - mb-231 cells . thus , distinct mirnas affect key genetic targets that govern distinct cell - cycle checkpoints including cyclins , cdks , cdk inhibitors and the g2-m regulation apparatus .", "in addition to cell - cycle control , breast tumor onset and progression and breast tumor stem cells are also regulated by distinct mirnas .", "cancer stem cells ( cscs ) are characterized by their self - renewal capacity , an ability to differentiate into non - tumorigenic cell progeny , and their ability to seed tumors when transplanted into animal hosts .", "cell surface markers such as cd44 , cd24 , cd133 , epithelial - specific antigen and aldehyde dehydrogenase-1 are frequently used to isolate and enrich cscs .", "the involvement of mirnas in regulating tumor formation by cscs or tumor - initiating cells ( t - ic ) has been widely investigated .", "let-7 expression is very low to undetectable level in embryonic stem cells ( es cells ) and increases with differentiation .", "a comparison of mirna expression between breast t - ic and non - t - ic demonstrated reduced let-7 expression in t - ic and increased abundance with differentiation .", "transduction of breast cscs with let-7 reduced the proportion of undifferentiated cells , inhibited cell proliferation , mammosphere formation , and tumor formation in vivo .", "clarke and colleagues identified 37 mirnas which were differentially expressed between human breast cscs ( cd44cd24lineage ) and lineage nontumorigenic breast cancer cells .", "the mir-200 family members were downregulated in human breast cscs and normal mammary stem / progenitor cells .", "expression of mir-200 inhibited breast cancer stem cell expansion in vitro , and suppressed the tumor formation ability of human breast cancer stem cell in vivo .", "ectopic mir-34c expression reduced breast t - ics self - renewal , inhibited emt and suppressed tumor cell invasiveness via silencing notch4 .", "zhu et al . found the reduced mir-128 expression in human breast t - ic was accompanied by bmi-1 and abcc5 overexpression , and associated with chemotherapeutic resistance and poor survival .", "enforced mir-128 expression increased the sensitivity of breast cancer cells to doxorubicin - induced apoptosis and dna damage .", "emerging evidence has demonstrated the importance of cscs in cancer initiation , cancer metastasis and drug resistance .", "cscs are believed to be one of the most promising targets for cure of cancer .", "the discovery that non - coding rnas regulate cscs widens our understanding of cscs , and may provide potential novel strategies for breast cancer therapy .", "deletion of chromosome 6q , including region 6q14-q16 , is frequently observed in breast cancer .", "the small non - coding snorna u50 is a candidate tumor suppressor gene in the 6q14 - 16 region , playing a role in the development and/or progression of breast cancer .", "genomic deletion and transcriptional downregulation of snorna u50 was detected in breast cancer cell lines .", "re - expression of snorna u50 inhibited colony formation of the human breast cancer cells hs578 t and mda - mb-231 .", "pirnas are small non - coding rna that form rna - protein complexes through interactions with piwi proteins .", "pirna was initially discovered in germ line cells , and considered as germ line - specific small rnas .", "emerging evidence indicates that pirna expression occurs in somatic cells [ 57 , 58 ] and piwil2 expression has been identified in human breast cancer cells .", "high - throughput deep sequencing identified a group of small rnas matching pirna sequences in human breast cancer tissues and breast cancer cell lines .", "the study of these non - coding small rnas in human cancer is just starting .", "the identification of the expression signature of these non - coding small rnas in breast cancer subtypes , and an understanding of their functional significance to oncogene expression , tumor initiation and tumor cell metastasis may shed important new perspectives on the role of these non - coding small rnas in breast cancer .", "small non - coding rna - based diagnostic and therapeutic applications for human cancer are expected in the near future . although tumor - targeted delivery and local administration are still major challenges to the practical application of gene therapy for cancer , mirna - based cancer therapeutic approaches are being established and tested in animal models .", "synthetic mirna mimics or mirna expression vectors have been successfully applied to restore or overexpress mirna in vitro . chemically modified lna anti - sense mirna inhibitor and other approaches have been used to block mirna function in cells .", "kim et al . recently reported significant anti - tumor effect of virus - mediated delivery of mir-145 combined with 5-fu to treat breast cancer .", "intranasal delivery of let-7   and intravenous delivery of mir-34a mimics   for non - small - cell lung cancer treatment and a virus - mediated delivery of mir-26a for liver cancer treatment in mouse model   demonstrate the promise of mirna for treatment of cancer .", "dysregulated expression of mirnas has implicated components of the non - coding genome as either oncogenes or tumor suppressors of breast cancer .", "experimental evidence has shown specific mirnas regulating the initiation , progression , metastasis and drug resistance of breast cancer via control of the cell cycle , altering cellular proliferation , altering cellular apoptosis and/or controlling the population of tumor stem cells . dysregulated", "mirna expression has also been observed in cancer associated fibroblasts ( caf ) and in the systemic circulation [ 65 , 66 ] .", "the circulating mirnas have the potential to serve as novel diagnostic and prognostic biomarkers for breast cancer .", "a specific subset of dysregulated mirnas in breast cancer cells may serve as targets for gene therapy either alone or as an adjuvant treatment to current clinical protocols for breast cancer patients ."], "abstract_text": ["<S> small non - coding rnas include sirna , mirna , pirna and snorna . </S>", "<S> the involvement of mirnas in the regulation of mammary gland tumorigenesis has been widely studied while the role for other small non - coding rnas remains unclear . here </S>", "<S> we summarize the involvement of mirna in breast cancer onset and progression through regulating the cell cycle and cellular proliferation . </S>", "<S> the regulation of breast cancer stem cells and tumor regeneration by mirna is reviewed . </S>", "<S> in addition , the emerging evidence demonstrating the involvement of pirna and snorna in breast cancer is briefly described . </S>"], "labels": null, "section_names": ["Introduction", "Aberrant Expression of miRNA in Breast Cancer", "miRNA Regulation of Cell Cycle and Mammary Gland Tumorigenesis", "miRNA Regulation of Breast Cancer Stem Cells", "Other Non-coding Small RNAs in Breast Cancer", "Therapeutic Application of miRNA", "Concluding Remarks"], "sections": [["small non - coding rnas are transcribed into mrna but remain untranslated in eukaryotic cells .", "they include sirna ( small interfering rna ) , mirna ( microrna ) , pirna ( piwi - interacting rna ) and snorna ( small nucleolar rna ) .", "mirnas are a class of multifunctional singled - stranded small rna which are ~20  nt in length and regulate the stability or translational efficiency of targeted messenger rna depending on the base - pairing complementarity between the mirna and its target mrna [ 1 , 2 ] . although over 1,000 mirna sequences have been identified from the tissues or cells of human origin and other species , as many as 1,000 to 10,000 mirnas per genome have been predicted [ 3 , 4 ] .", "mirnas regulate a broad range of biological processes including timing of development , cell cycle progression , stem cell self - renewal , differentiation , cancer initiation , cancer cell proliferation , metastasis and apoptosis [ 511 ] .", "cancer is caused by multiple processes including uncontrolled cellular proliferation and inappropriate survival of apoptotic cells .", "many regulatory factors switch on or off genes that govern cell division and direct cellular proliferation .", "mirnas regulate gene expression and play important roles in the onset and progression of tumorigenesis .", "emerging evidence demonstrates the involvement of mirna in mammary gland tumorigenesis , functioning either as tumor suppressors or oncogenes .", "although the current treatment of radiation therapy , chemotherapy and hormone therapy slow mammary gland tumor growth , prolong survival and improve the quality of patients life , metastatic breast cancer still remains incurable due to our limited understanding of the molecular mechanisms through which tumorigenesis and metastasis occur .", "as small non - coding rnas regulate gene expression and tumorigenesis , they may represent a novel cancer therapy ."], ["unlike mrna , mirnas are transcribed but never translated . some mirnas are transcribed from non - coding regions between genes , deriving from independent transcription unit .", "other mirnas are transcribed together with coding mrnas from the coding region of the genome , deriving from the introns of gene transcripts [ 13 , 14 ] .", "mirna gene copy number gain / loss and mirna gene mutation have been observed in breast cancer resulting in the aberrant expression of mirna .", "the first study about the altered expression of mirnas in human breast cancer patients and human breast cancer cell lines was reported in 2005 by lorio et al . ,", "in which 29 mirnas were identified with aberrant expression based on microarray and northern blot analysis of 76 breast tumor samples and 14 human breast cell lines .", "zhang and colleagues analyzed 283 human mirna genes on 55 human breast primary tumors and 18 human breast cancer cell lines using array - based comparative genomic hybridization .", "the results demonstrated a high frequency ( ~72.8% ) of gene copy number abnormality in mirna - containing regions in human breast cancer .", "wang et al . collected 68 patients with newly diagnosed breast cancer and examined the expression of selected mirnas in tumor and adjacent non - tumor tissues .", "mir-21 , mir-106a and mir-155 were significantly over - expressed in the tumor specimens compared with normal controls , whereas mir-126 , mir-199a and mir-335 were significantly decreased in expression in the tumor samples .", "our studies of the mir-17 - 92 cluster demonstrated decreased expression of mir-17/20 in human breast cancer specimens compared with matching normal breast tissue from the same patient .", "subsequent analysis identified reduced mir-17/20 expression in node - positive compared with node - negative breast cancers and demonstrated that mir-17/20 inhibited breast cancer cell migration and invasion via a heterotypic signaling .", "although the tendency for a global decrease of mirna expression in human cancers originally suggested a general tumor suppressor function of mirnas , subsequent studies showing the aberrant expression of specific mirnas in breast cancer suggest mirna - specific roles in breast cancer onset and progression ."], ["many distinct mirnas have been shown to regulate breast cancer cell proliferation , apoptosis , cancer stem cell expansion , and tumorigenesis .", "mirna may function as either tumor suppressors or oncogenes depending on the cell type , culture conditions , target genes and pathway .", "the involvement of mirna in mammary gland tumorigenesis has been reviewed recently [ 21 , 22 ] .", "le et al . described the expression pattern and regulatory network of key mirnas in breast cancer , including let-7 , mir-34 , mir-125 , mir-200 family , mir-205 , mir-21 , mir-10 and the mir-17 - 92 cluster .", "adams et al . reviewed the mirna regulation of estrogen signaling pathway and erbb2/her signaling pathway in breast cancer .", "the understanding of how mirnas are involved in breast cancer through regulating the cell cycle remains rudimentary .", "herein we summarize the recent literature and research progress on the mechanism by which mirnas regulate the breast cancer cell cycle and cellular proliferation ( fig . ", "1mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and", "let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint mirna regulation of mammary gland tumorigenesis in control of the cell cycle . through targeting different genes and different cyclin / cdk complexes , mir-17/20 and", "let-7 regulate the g1-s transition ; mir-21 and mir-27a regulate the g2-m checkpoint cyclin d1 is either overexpressed or amplified in ~50% of breast cancer .", "the abundance of cyclin d1 is rate - limiting in breast cancer cellular proliferation and g1-s phase transition [ 23 , 24 ] .", "in addition , cyclin d1 is a critical downstream target of erbb2- , ras- and -catenin- induced breast cancers , and is sufficient for the induction of mammary tumors when targeted to the mammary gland of mice .", "antisense inhibition of cyclin d1 expression in vivo suppressed the growth of neut - transformed mammary adenocarcinoma cells in nude mice .", "conserved sequences of the cyclin d1 3utr contain potential binding sites for multiple mirnas including mir-17/20/106 , mir-15/16 , mir-23 and let-7 .", "mir-17/20 binds the cyclin d1 3utr , inhibiting the expression of cyclin d1 , resulting in cell cycle arrest at the g1 phase and suppression of mcf-7 cell proliferation [ 18 , 26 ] .", "the regulation of cyclin d1 expression by mir-17 - 92 , as well as mir-15/16 , was confirmed by deshpande et al . .", "the let-7 family functions as a tumor suppressor in a variety of cancers including lung , colon , ovarian   and breast cancer .", "schultz et al . demonstrated the downregulation of cyclin d1 by mirna let-7 in control of cancer cell growth .", "the regulation of cyclin d1 by mirna is likely of broad importance as cyclin d1 encodes the regulatory subunit of a kinase that phosphorylates and inactivates the prb family proteins to inhibit dna synthesis , and phosphorylates nuclear respiratory factor 1 ( nrf-1 ) to inhibit mitochondria biogenesis [ 32 , 33 ] .", "furthermore , cyclin d1 promotes breast epithelial cell angiogenesis and migration , and promotes chromosomal instability which in turn contributes to tumorigenesis .", "the mir-221/222 cluster regulates the cell cycle , cell growth and epithelial - to - mesenchymal transition ( emt ) in breast cancer .", "mir-221/222 inhibited p27 and p57 abundance , facilitating g1-s phase transition , thereby promoting cancer cell proliferation [ 36 , 37 ] .", "moreover , mir-221/222 may contribute to the aggressive clinical behavior of basal - like breast cancers .", "the breast cancer basal - like subtype - specific mirnas , mir-221 and mir-222 , promote emt in breast cancer by targeting trps1 ( trichorhinophalangeal syndrome type 1 ) which inhibits emt by repressing zeb2 expression .", "mir-221 and/or mir-222 expression in mcf-7 and t47d breast cancer cells decreased er expression associated with tamoxifen resistance .", "the onco - mirna mir-21 is overexpressed in a wide variety of cancers including breast cancer [ 40 , 41 ] .", "mir-21 induced cellular proliferation , migration , invasion , emt , cancer stem cell characteristics and chemotherapy resistance in human breast cancer [ 42 , 43 ] .", "high mir-21 level is associated with poor prognosis , advanced stage , positive lymph node status and reduced survival time in breast cancer .", "mir-21 promotes mcf-7 cellular proliferation in part through inhibiting the expression of a tumor suppressor gene programmed cell death 4 ( pdcd4 ) . in colon cancer ,", "mir-21 participates in a dna damage - induced g2-m checkpoint through suppressing the cell cycle regulator cdc25a .", "a recent report demonstrated the mir-21 regulates the cell cycle through targeting cdc25a in mcf-7 breast cancer cells . with a potential anti - cancer chemical 3,3-diindolylmethane treatment ,", "mir-27a expression is upregulated in human breast cancer cell lines . in mda - mb-231 cells ,", "mir-27a negatively regulated the zinc finger zbtb10 gene and myt-1 , thereby promoting breast cancer cell proliferation .", "mir-27a suppressed myt-1 , increased cdc2/cyclin b activity and promoted the g2-m checkpoint in mda - mb-231 cells . thus , distinct mirnas affect key genetic targets that govern distinct cell - cycle checkpoints including cyclins , cdks , cdk inhibitors and the g2-m regulation apparatus .", "in addition to cell - cycle control , breast tumor onset and progression and breast tumor stem cells are also regulated by distinct mirnas ."], ["cancer stem cells ( cscs ) are characterized by their self - renewal capacity , an ability to differentiate into non - tumorigenic cell progeny , and their ability to seed tumors when transplanted into animal hosts .", "cell surface markers such as cd44 , cd24 , cd133 , epithelial - specific antigen and aldehyde dehydrogenase-1 are frequently used to isolate and enrich cscs .", "the involvement of mirnas in regulating tumor formation by cscs or tumor - initiating cells ( t - ic ) has been widely investigated .", "let-7 expression is very low to undetectable level in embryonic stem cells ( es cells ) and increases with differentiation .", "a comparison of mirna expression between breast t - ic and non - t - ic demonstrated reduced let-7 expression in t - ic and increased abundance with differentiation .", "transduction of breast cscs with let-7 reduced the proportion of undifferentiated cells , inhibited cell proliferation , mammosphere formation , and tumor formation in vivo .", "clarke and colleagues identified 37 mirnas which were differentially expressed between human breast cscs ( cd44cd24lineage ) and lineage nontumorigenic breast cancer cells .", "the mir-200 family members were downregulated in human breast cscs and normal mammary stem / progenitor cells .", "expression of mir-200 inhibited breast cancer stem cell expansion in vitro , and suppressed the tumor formation ability of human breast cancer stem cell in vivo .", "ectopic mir-34c expression reduced breast t - ics self - renewal , inhibited emt and suppressed tumor cell invasiveness via silencing notch4 .", "zhu et al . found the reduced mir-128 expression in human breast t - ic was accompanied by bmi-1 and abcc5 overexpression , and associated with chemotherapeutic resistance and poor survival .", "enforced mir-128 expression increased the sensitivity of breast cancer cells to doxorubicin - induced apoptosis and dna damage .", "emerging evidence has demonstrated the importance of cscs in cancer initiation , cancer metastasis and drug resistance .", "cscs are believed to be one of the most promising targets for cure of cancer .", "the discovery that non - coding rnas regulate cscs widens our understanding of cscs , and may provide potential novel strategies for breast cancer therapy ."], ["deletion of chromosome 6q , including region 6q14-q16 , is frequently observed in breast cancer .", "the small non - coding snorna u50 is a candidate tumor suppressor gene in the 6q14 - 16 region , playing a role in the development and/or progression of breast cancer .", "genomic deletion and transcriptional downregulation of snorna u50 was detected in breast cancer cell lines .", "re - expression of snorna u50 inhibited colony formation of the human breast cancer cells hs578 t and mda - mb-231 .", "pirnas are small non - coding rna that form rna - protein complexes through interactions with piwi proteins .", "pirna was initially discovered in germ line cells , and considered as germ line - specific small rnas .", "emerging evidence indicates that pirna expression occurs in somatic cells [ 57 , 58 ] and piwil2 expression has been identified in human breast cancer cells .", "high - throughput deep sequencing identified a group of small rnas matching pirna sequences in human breast cancer tissues and breast cancer cell lines .", "the study of these non - coding small rnas in human cancer is just starting .", "the identification of the expression signature of these non - coding small rnas in breast cancer subtypes , and an understanding of their functional significance to oncogene expression , tumor initiation and tumor cell metastasis may shed important new perspectives on the role of these non - coding small rnas in breast cancer ."], ["small non - coding rna - based diagnostic and therapeutic applications for human cancer are expected in the near future . although tumor - targeted delivery and local administration are still major challenges to the practical application of gene therapy for cancer , mirna - based cancer therapeutic approaches are being established and tested in animal models .", "synthetic mirna mimics or mirna expression vectors have been successfully applied to restore or overexpress mirna in vitro . chemically modified lna anti - sense mirna inhibitor and other approaches have been used to block mirna function in cells .", "kim et al . recently reported significant anti - tumor effect of virus - mediated delivery of mir-145 combined with 5-fu to treat breast cancer .", "intranasal delivery of let-7   and intravenous delivery of mir-34a mimics   for non - small - cell lung cancer treatment and a virus - mediated delivery of mir-26a for liver cancer treatment in mouse model   demonstrate the promise of mirna for treatment of cancer ."], ["dysregulated expression of mirnas has implicated components of the non - coding genome as either oncogenes or tumor suppressors of breast cancer .", "experimental evidence has shown specific mirnas regulating the initiation , progression , metastasis and drug resistance of breast cancer via control of the cell cycle , altering cellular proliferation , altering cellular apoptosis and/or controlling the population of tumor stem cells . dysregulated", "mirna expression has also been observed in cancer associated fibroblasts ( caf ) and in the systemic circulation [ 65 , 66 ] .", "the circulating mirnas have the potential to serve as novel diagnostic and prognostic biomarkers for breast cancer .", "a specific subset of dysregulated mirnas in breast cancer cells may serve as targets for gene therapy either alone or as an adjuvant treatment to current clinical protocols for breast cancer patients ."]]}
{"article_id": "PMC4086000", "article_text": ["ohss is a serious complication of ovulation induction , occurring in 1 - 10% of in vitro fertilization patients  ( rizk and aboulghar , 1991 ; brinsden et al . , 1995 ) .", "this iatrogenic condition has a spectrum of clinical and laboratory manifestations ranging from mild to severe , even life - threatening conditions  ( rizk et al . , 1990 ) . among the serious manifestations of ohss are ascites and pleural effusion  ( rizk and smitz , 1992 ) .", "vascular endothelial growth factor has emerged as one of the factors most likely involved in the pathophysiology of ohss  ( geva and jaffe , 2000 ; rizk and aboulghar , 2010 ) .", "vascular endothelial growth factor is an angiogenic cytokine that is a potent stimulator of the vascular endothelium and appears to play an integral role in follicular growth , corpus luteum function and ovarian angiogenesis  ( rizk et al . , 1997 ) .", "vascular endothelial growth factor causes increased capillary permeability with its sequelae of ascites and possible pleural effusion  ( rizk et al . , 1997 ; gomez et al . , 2002 ) .", "human chorionic gonadotrophin is thought to play a crucial role in the development of ohss  ( rizk et al .", "two distinct forms of severe ohss have been described : early and late forms  ( papanikolaou et al . , 2005 ; mathur et al . , 2000 ) .", "early ohss is caused by the administration of exogenous human chorionic gonadotrophin  ( hcg ) and it starts before the 10th day after oocyte retrieval , while the late form is the result of endogenous human chorionic gonadotrophin release in the event of pregnancy , and it starts after the 10th day following oocyte aspiration  ( papanikolaou et al . , 2005 ;", "such strategies included the use of low dose gonadotropins and frequent monitoring during controlled ovarian stimulation , cycle cancellation , coasting , freeze all embryos and the use of gonadotropin releasing hormone  ( gnrh ) agonist as an oocyte trigger in gnrh antagonist cycles  ( rizk et al .", "the use of antagonist in the luteal phase with total freeze of embryos to prevent severe ohss has also been suggested  ( lainas et al . , 2012 ) .", "such strategies should be used especially in patients at high risk for developing severe ohss , including those who are young , have low body weight , have polycystic ovary syndrome , those who experience high estradiol levels , rapidly increasing estradiol levels , high number of follicles during controlled ovarian stimulation and those with high number of oocytes retrieved  ( rizk and aboulghar , 1999 ; delvigne , 2009 ) .", "dopamine agonists have been proposed for the prevention of ohss in women at high risk . however , the study by lvarez et al . ", "( 2007 ) suggested that it was only effective in reducing the incidence of moderate ohss , but not in its severe form  ( alvarez et al . , 2007 ) .", "low - dose aspirin therapy and doxycycline have also been suggested to inhibit angiogenesis  ( fainaru et al . , 2009 ) .", "( 2011 ) believes that the concept of an ohss - free clinic has become a reality .", "the authors suggest that this approach should include pituitary down - regulation using a gnrh antagonist , ovulation triggering with a gnrh agonist and vitrification of oocytes or embryos  ( devroey et al . , 2011 ) .", "severe and critical forms of ohss are associated with significant ascites  ( golan et al . , 1989 ; rizk et al . ,", "ascites results in an increased abdominal pressure compromising venous return , cardiac output and renal perfusion  ( navot et al .", "paracentesis is frequently needed , as prompt drainage of fluid produces a significant clinical and biochemical improvement , including spontaneous diuresis and hastening the resolution process  ( rizk et al .", ", multiple paracenteses may be required to relieve symptoms and avoid serious sequelae of hemoconcentration , hypotension , decreased renal perfusion and severe respiratory compromise . instead of multiple interventions ,", "the placement of a catheter temporarily for a few days will permit complete drainage through one procedure  ( rizk and aboulghar , 1999 ) .", "pigtail catheters have been used for drainage in various clinical situations . in a previous study we described the use of percutaneous placement of a pigtail catheter for drainage of ascites caused by severe / critical ohss  ( abuzeid et al . , 2003 ) .", "in this study we established the efficacy and safety of pigtail catheter drainage in the management of severe / critical ohss in patients who underwent in vitro fertilization and embryo transfer at our centre between 1999 and 2001 on both inpatient and outpatient basis .", "the aim of the current study was to evaluate the role of the pigtail catheter drainage in outpatient management of patients with early onset severe ohss .", "this is a retrospective study that included all patients who underwent in vitro fertilization and developed severe or critical ohss by the golan classification  ( golan et al . , 1989 ) in the period between 2004 and 2009 .", "all patients underwent an ultrasound evaluation which documented the presence of severe ascites and bilateral ovarian enlargement .", "all patients presented with shortness of breath , marked abdominal distension , nausea , weight gain and lower abdominal pain .", "thirteen patients complained of vomiting , and 3 patients reported decreased urine output  ( less than 720 cc per 24  hours ) and had haemoglobin   15 g / dl .", "all 33 patients were offered treatment using pigtail catheter insertion as an alternative to trans - vaginal paracentesis .", "blood studies including a complete blood count , creatinine , electrolytes , serum albumin and total protein , and coagulation panel were ordered prior to the procedure .", "all 33  patients were rehydrated with intravenous crystalloid solutions  ( 0.9% normal saline ) .", "care was taken to give the patient the same amount of fluid as the amount that was drained from the peritoneal cavity .  ", "all pigtail catheter placements were performed in the operating room at our surgical centre . the catheter  ( 6 - 0 french , 2.0 mm , boston scientific , quincy , ma , usa ) was introduced utilizing a non - disposable metal gamete intra - fallopian treatment  ( gift ) trocar and cannula  ( embryon gift catheter , ivf online , gilford , ct , usa ) which was introduced into the largest accessible ascitic fluid pocket in the upper outer quadrant of the abdominal wall under local anaesthesia and trans - abdominal ultrasound guidance . the trocar was then removed and a glide wire  ( boston scientific corp . , watertown , ma , usa ) was threaded through the gift canula . the canula was removed with care so as to keep the glide wire in the peritoneal cavity .", "the pigtail catheter was guided into the peritoneal cavity by the wire after removal of the stylet of the pigtail catheter .", "the catheter was anchored to the skin using 2 - 0 silk suture and it was supported by gauze , which was covered by a tegaderm .", "the catheter was connected to a three - way stop cock which was connected to a drainage bag .", "initial amount of drained fluid was calculated and subsequently the patients were trained to drain 1000 cc per day  ( 500 cc in the morning and 500 cc in the evening ) .", "one liter normal saline and 500 cc of 6% hydroxyethyle starch solution  ( hespan ) was administered to the patient on the same day of pigtail catheter placement .", "any patient with critical ohss according to the golan classification , or those in whom the presenting symptoms and signs did not completely subside after drainage of ascitic fluid and iv fluid replacement , were admitted to the hospital for observation and further management .", "all other patients were sent home . once there was minimal or no drainage from the catheter , the patient returned to the office for removal of the catheter .", "the patients were encouraged to drink plenty of fluids and to increase their protein intake .", "all patients kept a diary for body weight , abdominal girth , and urine output before and after the procedure .", "patients were seen in the office as needed for evaluation and hydration with normal saline .", "demographic data including mean age  ( years ) , duration of infertility  ( years ) , bmi  ( kg / m2 ) and aetiology of infertility are illustrated in table i. ovarian stimulation data including number of treatment days , total dose of recombinant follicle stimulating hormone , hormone serum levels , endometrial thickness , number of follicles and number of patients receiving only 5000 iu of human chorionic gonadotrophin is illustrated in table ii .", "our data include the number of patients requiring early versus late placement of pigtail catheter , mean amount of ascitic fluid drained immediately after pigtail placement , number of days of pigtail catheter drainage and the number of days between oocyte retrieval and the onset of severe ohss symptoms requiring pigtail placement .", "table iii also illustrates the number of patients who underwent coasting , number of patients who received prophylactic hespan on day of egg retrieval .", "values are expressed as mean + standard deviation values are expressed as mean  +  standard deviation : rfsh  =  recombinant fsh ; hmg  = human menopausal gonadotropins ; hcg  =  human chorionic gonadotropin ; e2  =  estradiol values are expressed as mean + standard deviation three patients  ( 9% ) required an early placement of a pigtail catheter while 30 patients  ( 91% ) had late placement of the catheter  ( table iii ) .", "twenty - nine patients  ( 88% ) were managed on an outpatient basis without any complications .", "four patients  ( 12% ) required hospital admission for 1 - 7 days  ( 3 + 2.7 ) , three for associated severe pleural effusion requiring thoracentesis .", "these three patients were admitted to the hospital immediately after insertion of the pigtail catheter for management of severe pleural effusion and careful observation .", "one patient was admitted 48 hours after insertion of the pigtail catheter for work up for chest pain , all tests were negative .", "navot et al . , 1992 ; rizk and aboulghar , 1999 ) , but perhaps the most accepted classification of ohss is the one described by golan et al as mild , moderate and severe  ( golan et al . , 1989 ) .", "however , clinicians should be aware that ohss is a dynamic condition and moderate ohss may progress to severe ohss within hours  ( rizk and aboulghar , 1999 ; rizk , 2009 ) .", "therefore , the practice committee of the american society for reproductive medicine  ( asrm ) proposed a classification for ohss of mild , worsening and serious  ( the practice committee of the american society for reproductive medicine , 2008 ) . recognizing the patients at high - risk , prevention and early recognition are the most important steps in the management of ohss  ( rizk , 2006 ) .", "however , severe forms of ohss , especially late onset type that occurs when patients are pregnant , most likely will continue to happen , albeit at much lower rate , unless one reverts to a total freeze of all embryos after the use of gnrh agonist as a trigger shot in a gnrh antagonist cycle  ( donoso and devroey , 2013 ; devroey et al . , 2011 ) .", "traditional treatment consists mainly of supportive management until spontaneous resolution occurs  ( rizk , 1994 ) .", "although moderate ohss does not usually require intervention , outpatient monitoring is essential to identify progression to a severe form  ( rizk , 2006 ) .", "patients with moderate ohss can be managed on an outpatient basis by adequate hydration and close daily monitoring of weight gain and abdominal girth to detect progression to severe ohss , and to provide intervention , if required  ( rizk , 2010 ) .", "patients presenting with severe forms of ohss are usually managed on inpatient basis and most patients with critical ohss are admitted to intensive care units for aggressive supportive measures to avoid maternal morbidity and mortality  ( the practice committee of the american society for reproductive medicine , 2008 ) .", "correction of hypovolemia , electrolyte imbalance and hypoalbuminemia are the basic principles during the treatment of severe ohss  ( rizk , 1994 , 2006 ; myrianthefs et al . , 2000 ) .", "( 1992 ) suggested that paracentesis is the single most important treatment modality in life - threatening ohss not controlled by medical therapy .", "paracentesis and removal of ascites result in relieving the pressure on the inferior vena cava and improvement in venous return , cardiac output and renal perfusion  ( padilla et al . , 1990 ) .", "therefore , prompt drainage of fluid produces significant clinical and biochemical improvement , including spontaneous diuresis and hastening the resolution process  ( abuzeid et al .", ", 2003 ; the practice committee of the american society for reproductive medicine , 2008 ) .", "it is also possible that removal of ascites decreases the amount of vegf and its effects on vascular permeability .", "therefore ultrasound guided drainage of the ascitic fluid should be performed in severe cases of ohss with respiratory distress , oliguria or severe abdominal pain  ( padilla et al . , 1990 ) .", "this can be done either trans - vaginally  ( aboulghar et al . , 1990 ) or trans - abdominally  ( padilla et al . ,", "1990 ) . however , recurrence of ascites is common in severe cases of ohss .", "placement of a pigtail catheter has been proposed by some investigators to allow drainage of ascitic fluid whenever it accumulates without the need for further surgical procedures  ( abuzeid et al . , 2003 ) .", "in a previous study we reported the use of percutaneous pigtail catheter for drainage of ascites in severe ohss as an alternative to repeated paracentesis  ( abuzeid et al . , 2003 ) .", "this procedure is relatively simple and requires a short period of time as compared to the time needed to gradually aspirate any fluid accumulation transvaginally .", "immediate relief of symptoms and signs of severe ohss after pigtail catheter insertion was an important finding .", "in addition , the ease by which any accumulated ascitic fluid was drained resulted in a faster resolution of ohss  ( abuzeid et al . ,", "no infection was reported  ( abuzeid et al . , 2003 ) . in that study the first 13 patients", "they were discharged home with the pigtail catheter in place and managed on an outpatient basis until ohss subsided ; thereafter the catheter was removed .", "the last 13 patients were managed on an outpatient basis without any problem  ( abuzeid mi , et al . , 2003 ) .", "our previous study confirmed the safety and effectiveness of the pigtail catheter for the management of severe ohss .", "therefore , we embarked on the current study to evaluate the role of pigtail catheter drainage in outpatient management of patients with severe ohss syndrome . in the current study we demonstrated that patients who were diagnosed very early with severe ohss can be managed safely and effectively on outpatient basis using pigtail catheter drainage , iv fluid replacement and careful monitoring .", "although all 33 patients presented with shortness of breath , marked abdominal distension , nausea , weight gain and lower abdominal pain , and 13 of them complained of vomiting ,  ( all are criteria for hospital admission according to the practice committee of the american society for reproductive medicine on ohss ) , 30 patients were sent home as their presenting symptoms were relieved .", "early diagnosis and intervention resulted in rapid elimination of the symptoms of severe ohss and normalization of vital signs , which allow us to manage the patients on outpatient basis .", "the pigtail catheter placements were performed without complications and patients were discharged home after a few hours of observation .", "the patients who needed hospital admission were those with associated severe pleural effusion requiring thoracentesis .", "another patient was admitted to the hospital for 1 day for work up for chest pain and possible diagnosis of pulmonary embolism .", "once pulmonary embolism was ruled out , she was discharged home with a pigtail catheter in place and she was managed on outpatient basis .", "outpatient management of ohss has no place in those who fail to have complete resolutions of their symptoms , or those with severe hyperproteinuria or electrolyte imbalance .", "certainly it has no place in patients with critical ohss such as those with severe pleural effusion , diminished urine output , severe hemoconcentration and those at increased risk of thromboembolic risks  ( rizk , 2010 ) .", "it is imperative to stress that such a protocol can only be used in patients who are motivated , reliable and have the necessary support at home to allow them to return to the clinic or go to the emergency room if the need arises .", "it also requires dedicated in vitro fertilization nurses to educate and follow up the patient on a daily basis .", "contrary to other reports that state that access to intravenous albumin is a critical part of outpatient management for severe ohss , we did not use any albumin replacement  ( lincoln et al . , 2002 ) .", "the reason behind the use of intravenous albumin is to increase intravascular oncotic pressure , thereby curtailing the exodus of free water from the intravascular space . in our study", "we used hespan instead of albumin , as some investigators have shown that it is beneficial in reducing the incidence of severe ohss  ( youssef et al . , 2011 ) . by increasing the intravascular oncotic pressure", "in addition , the majority of our patients had late onset ohss as a result of pregnancy .", "hespan should be used with caution in patients with late onset ohss only when benefits outweigh risks.it is a fact that outpatient management of early severe ohss is a common practice among infertility specialists after trans - vaginal aspiration of ascitic fluid and iv fluid hydration .", "outpatient management by repeated abdominal or trans - vaginal paracentesis has been reported to be safe , effective and cost effective  ( lincoln et al .", ", 2002 ; shrivastav et al . , 1994 ; fluker et al . , 2000", "( 1994 ) were the first to suggest that day care management in patients with severe ohss with abdominal paracentesis and iv hydration was simple , safe and effective compared with traditional treatment with in - patient hospitalization and supportive measures .", "( 2002 ) concluded that the need for hospitalization for hyperstimulated patients can be minimized by outpatient management of severe ohss with trans - vaginal culdocentesis and rehydration with crystalloids and albumin .", "( 2000 ) advocated in a pilot study an active management strategy for moderate ohss , which involved early paracentesis designed to minimize the progression from moderate to severe ohss , instead of late paracentesis aimed at speeding up recovery in severely ill patients .", "( 2010 ) concluded that early outpatient paracentesis for moderate to severe ohss is the most cost effective management plan when compared with traditional conservative inpatient therapy .", "( 2009 ) reported a large series of patients with severe ohss in whom the vast majority were successfully managed as outpatients with use of aggressive trans - vaginal paracentesis  ( smith et al . , 2009 ) . despite the findings in our study", "it is important to emphasize that ohss remains a serious disorder with the potential for rapid deterioration , requiring hospitalization and intensive treatment of a critically ill patient .", "the life - threatening complications of ohss are secondary to thromboembolism or compromised pulmonary or cardiovascular function and multi - organ failure", ". there have been isolated reports of women dying from the complications associated with ohss .", "the mortality rate related to ohss is very low and was estimated by brinsden et al . ", "more recently , reports regarding maternal mortality rates due to ohss in the netherlands and united kingdom demonstrate an incidence of approximately 3 deaths per 100,000 in ivf cycles  ( braat et al .", ", 2006 ; lewis , 2007 ) . eleven lethal complications of assisted reproductive technology have been documented and summarized by braat et al . ", "several investigators reported sporadic fatality as a result of ohss due to adult respiratory distress syndrome , massive pulmonary oedema , myocardial infarction , and carotid artery occlusion resulting in cerebral infarction . in this study", "we describe a modified technique of what interventional radiologists usually use for pigtail catheter placement .", "we observed that in the obese patients the metal introducer provided in the pigtail catheter kit is difficult to use ; instead we used the non disposable gamete intra - fallopian treatment trocar and cannula to facilitate the introduction of the catheter through the abdominal wall of such patients .", "this technique helps to manage the patients in the office without the need for sending her to the hospital for an interventional radiologist to place the catheter .", "however , the patients in this study were identified from our in vitro fertilization database ; the study included every patient who developed severe / critical ohss .", "in addition , the lack of a control group that was managed without pigtail catheter placement limits the conclusions that can be drawn from our study . in summary ,", "drainage of ascitic fluid in patients with severe / critical ohss is an important step in the management of this condition  ( rizk , 2010 ) .", "the placement of a pigtail catheter resulted in a safe and effective outpatient management of the majority of patients with severe ohss .", "however , outpatient management of this potentially fatal condition requires clear understanding that rapid deterioration may occur , which may necessitate hospitalization and intensive treatment of a critically ill patient  ( rizk , 2006 , 2010 ) .", "patients with critical ohss and those with persistence of symptoms of severe ohss despite insertion of a pigtail catheter and drainage of ascitic fluid should be admitted to the hospital for inpatient management ."], "abstract_text": ["<S> objective : to evaluate the efficacy and safety of outpatient management of severe ovarian hyperstimulation syndrome  ( ohss ) requiring placement of a pigtail catheter.methods : retrospective analysis of thirty - three consecutive patients who underwent in - vitro fertilization  ( 2003 - 2009 ) and developed severe / critical ohss requiring placement of a pigtail catheter . </S>", "<S> patients who were managed on outpatient basis were monitored by frequent office visits , daily phone calls , and received iv normal saline for hydration when required.results : in 3 patients  ( 9.1% ) ohss started early , requiring placement of a pigtail catheter 4.3 + 0.6 days after retrieval . in 30 patients  ( 90.9% ) </S>", "<S> ohss started late  ( 14  4 days after retrieval ) . </S>", "<S> the mean amount of ascitic fluid drained immediately after placement of the catheter was 2085  1018 cc . </S>", "<S> the pigtail catheter was removed after 7.8  5.3 days . </S>", "<S> of the 31 patients who had embryo transfer  ( two had total freeze ) , 84% conceived . </S>", "<S> twenty - nine patients  ( 88% ) were managed on outpatient basis without any complications . </S>", "<S> four patients required hospital admission for 1 - 7 days  ( 3.0  2.7 ) . </S>", "<S> one patient with severe ohss was admitted for work up for chest pain . </S>", "<S> three patients with critical ohss with severe pleural effusion requiring thoracentesis were admitted for supportive measures.conclusion : the placement of a pigtail catheter resulted in safe and effective outpatient management for the majority of patients with severe ohss . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion"], "sections": [["ohss is a serious complication of ovulation induction , occurring in 1 - 10% of in vitro fertilization patients  ( rizk and aboulghar , 1991 ; brinsden et al . , 1995 ) .", "this iatrogenic condition has a spectrum of clinical and laboratory manifestations ranging from mild to severe , even life - threatening conditions  ( rizk et al . , 1990 ) . among the serious manifestations of ohss are ascites and pleural effusion  ( rizk and smitz , 1992 ) .", "vascular endothelial growth factor has emerged as one of the factors most likely involved in the pathophysiology of ohss  ( geva and jaffe , 2000 ; rizk and aboulghar , 2010 ) .", "vascular endothelial growth factor is an angiogenic cytokine that is a potent stimulator of the vascular endothelium and appears to play an integral role in follicular growth , corpus luteum function and ovarian angiogenesis  ( rizk et al . , 1997 ) .", "vascular endothelial growth factor causes increased capillary permeability with its sequelae of ascites and possible pleural effusion  ( rizk et al . , 1997 ; gomez et al . , 2002 ) .", "human chorionic gonadotrophin is thought to play a crucial role in the development of ohss  ( rizk et al .", "two distinct forms of severe ohss have been described : early and late forms  ( papanikolaou et al . , 2005 ; mathur et al . , 2000 ) .", "early ohss is caused by the administration of exogenous human chorionic gonadotrophin  ( hcg ) and it starts before the 10th day after oocyte retrieval , while the late form is the result of endogenous human chorionic gonadotrophin release in the event of pregnancy , and it starts after the 10th day following oocyte aspiration  ( papanikolaou et al . , 2005 ;", "such strategies included the use of low dose gonadotropins and frequent monitoring during controlled ovarian stimulation , cycle cancellation , coasting , freeze all embryos and the use of gonadotropin releasing hormone  ( gnrh ) agonist as an oocyte trigger in gnrh antagonist cycles  ( rizk et al .", "the use of antagonist in the luteal phase with total freeze of embryos to prevent severe ohss has also been suggested  ( lainas et al . , 2012 ) .", "such strategies should be used especially in patients at high risk for developing severe ohss , including those who are young , have low body weight , have polycystic ovary syndrome , those who experience high estradiol levels , rapidly increasing estradiol levels , high number of follicles during controlled ovarian stimulation and those with high number of oocytes retrieved  ( rizk and aboulghar , 1999 ; delvigne , 2009 ) .", "dopamine agonists have been proposed for the prevention of ohss in women at high risk . however , the study by lvarez et al . ", "( 2007 ) suggested that it was only effective in reducing the incidence of moderate ohss , but not in its severe form  ( alvarez et al . , 2007 ) .", "low - dose aspirin therapy and doxycycline have also been suggested to inhibit angiogenesis  ( fainaru et al . , 2009 ) .", "( 2011 ) believes that the concept of an ohss - free clinic has become a reality .", "the authors suggest that this approach should include pituitary down - regulation using a gnrh antagonist , ovulation triggering with a gnrh agonist and vitrification of oocytes or embryos  ( devroey et al . , 2011 ) .", "severe and critical forms of ohss are associated with significant ascites  ( golan et al . , 1989 ; rizk et al . ,", "ascites results in an increased abdominal pressure compromising venous return , cardiac output and renal perfusion  ( navot et al .", "paracentesis is frequently needed , as prompt drainage of fluid produces a significant clinical and biochemical improvement , including spontaneous diuresis and hastening the resolution process  ( rizk et al .", ", multiple paracenteses may be required to relieve symptoms and avoid serious sequelae of hemoconcentration , hypotension , decreased renal perfusion and severe respiratory compromise . instead of multiple interventions ,", "the placement of a catheter temporarily for a few days will permit complete drainage through one procedure  ( rizk and aboulghar , 1999 ) .", "pigtail catheters have been used for drainage in various clinical situations . in a previous study we described the use of percutaneous placement of a pigtail catheter for drainage of ascites caused by severe / critical ohss  ( abuzeid et al . , 2003 ) .", "in this study we established the efficacy and safety of pigtail catheter drainage in the management of severe / critical ohss in patients who underwent in vitro fertilization and embryo transfer at our centre between 1999 and 2001 on both inpatient and outpatient basis .", "the aim of the current study was to evaluate the role of the pigtail catheter drainage in outpatient management of patients with early onset severe ohss ."], ["this is a retrospective study that included all patients who underwent in vitro fertilization and developed severe or critical ohss by the golan classification  ( golan et al . , 1989 ) in the period between 2004 and 2009 .", "all patients underwent an ultrasound evaluation which documented the presence of severe ascites and bilateral ovarian enlargement .", "all patients presented with shortness of breath , marked abdominal distension , nausea , weight gain and lower abdominal pain .", "thirteen patients complained of vomiting , and 3 patients reported decreased urine output  ( less than 720 cc per 24  hours ) and had haemoglobin   15 g / dl .", "all 33 patients were offered treatment using pigtail catheter insertion as an alternative to trans - vaginal paracentesis .", "blood studies including a complete blood count , creatinine , electrolytes , serum albumin and total protein , and coagulation panel were ordered prior to the procedure .", "all 33  patients were rehydrated with intravenous crystalloid solutions  ( 0.9% normal saline ) .", "care was taken to give the patient the same amount of fluid as the amount that was drained from the peritoneal cavity .  ", "all pigtail catheter placements were performed in the operating room at our surgical centre . the catheter  ( 6 - 0 french , 2.0 mm , boston scientific , quincy , ma , usa ) was introduced utilizing a non - disposable metal gamete intra - fallopian treatment  ( gift ) trocar and cannula  ( embryon gift catheter , ivf online , gilford , ct , usa ) which was introduced into the largest accessible ascitic fluid pocket in the upper outer quadrant of the abdominal wall under local anaesthesia and trans - abdominal ultrasound guidance . the trocar was then removed and a glide wire  ( boston scientific corp . , watertown , ma , usa ) was threaded through the gift canula . the canula was removed with care so as to keep the glide wire in the peritoneal cavity .", "the pigtail catheter was guided into the peritoneal cavity by the wire after removal of the stylet of the pigtail catheter .", "the catheter was anchored to the skin using 2 - 0 silk suture and it was supported by gauze , which was covered by a tegaderm .", "the catheter was connected to a three - way stop cock which was connected to a drainage bag .", "initial amount of drained fluid was calculated and subsequently the patients were trained to drain 1000 cc per day  ( 500 cc in the morning and 500 cc in the evening ) .", "one liter normal saline and 500 cc of 6% hydroxyethyle starch solution  ( hespan ) was administered to the patient on the same day of pigtail catheter placement .", "any patient with critical ohss according to the golan classification , or those in whom the presenting symptoms and signs did not completely subside after drainage of ascitic fluid and iv fluid replacement , were admitted to the hospital for observation and further management .", "all other patients were sent home . once there was minimal or no drainage from the catheter , the patient returned to the office for removal of the catheter .", "the patients were encouraged to drink plenty of fluids and to increase their protein intake .", "all patients kept a diary for body weight , abdominal girth , and urine output before and after the procedure .", "patients were seen in the office as needed for evaluation and hydration with normal saline ."], ["demographic data including mean age  ( years ) , duration of infertility  ( years ) , bmi  ( kg / m2 ) and aetiology of infertility are illustrated in table i. ovarian stimulation data including number of treatment days , total dose of recombinant follicle stimulating hormone , hormone serum levels , endometrial thickness , number of follicles and number of patients receiving only 5000 iu of human chorionic gonadotrophin is illustrated in table ii .", "our data include the number of patients requiring early versus late placement of pigtail catheter , mean amount of ascitic fluid drained immediately after pigtail placement , number of days of pigtail catheter drainage and the number of days between oocyte retrieval and the onset of severe ohss symptoms requiring pigtail placement .", "table iii also illustrates the number of patients who underwent coasting , number of patients who received prophylactic hespan on day of egg retrieval .", "values are expressed as mean + standard deviation values are expressed as mean  +  standard deviation : rfsh  =  recombinant fsh ; hmg  = human menopausal gonadotropins ; hcg  =  human chorionic gonadotropin ; e2  =  estradiol values are expressed as mean + standard deviation three patients  ( 9% ) required an early placement of a pigtail catheter while 30 patients  ( 91% ) had late placement of the catheter  ( table iii ) .", "twenty - nine patients  ( 88% ) were managed on an outpatient basis without any complications .", "four patients  ( 12% ) required hospital admission for 1 - 7 days  ( 3 + 2.7 ) , three for associated severe pleural effusion requiring thoracentesis .", "these three patients were admitted to the hospital immediately after insertion of the pigtail catheter for management of severe pleural effusion and careful observation .", "one patient was admitted 48 hours after insertion of the pigtail catheter for work up for chest pain , all tests were negative ."], ["navot et al . , 1992 ; rizk and aboulghar , 1999 ) , but perhaps the most accepted classification of ohss is the one described by golan et al as mild , moderate and severe  ( golan et al . , 1989 ) .", "however , clinicians should be aware that ohss is a dynamic condition and moderate ohss may progress to severe ohss within hours  ( rizk and aboulghar , 1999 ; rizk , 2009 ) .", "therefore , the practice committee of the american society for reproductive medicine  ( asrm ) proposed a classification for ohss of mild , worsening and serious  ( the practice committee of the american society for reproductive medicine , 2008 ) . recognizing the patients at high - risk , prevention and early recognition are the most important steps in the management of ohss  ( rizk , 2006 ) .", "however , severe forms of ohss , especially late onset type that occurs when patients are pregnant , most likely will continue to happen , albeit at much lower rate , unless one reverts to a total freeze of all embryos after the use of gnrh agonist as a trigger shot in a gnrh antagonist cycle  ( donoso and devroey , 2013 ; devroey et al . , 2011 ) .", "traditional treatment consists mainly of supportive management until spontaneous resolution occurs  ( rizk , 1994 ) .", "although moderate ohss does not usually require intervention , outpatient monitoring is essential to identify progression to a severe form  ( rizk , 2006 ) .", "patients with moderate ohss can be managed on an outpatient basis by adequate hydration and close daily monitoring of weight gain and abdominal girth to detect progression to severe ohss , and to provide intervention , if required  ( rizk , 2010 ) .", "patients presenting with severe forms of ohss are usually managed on inpatient basis and most patients with critical ohss are admitted to intensive care units for aggressive supportive measures to avoid maternal morbidity and mortality  ( the practice committee of the american society for reproductive medicine , 2008 ) .", "correction of hypovolemia , electrolyte imbalance and hypoalbuminemia are the basic principles during the treatment of severe ohss  ( rizk , 1994 , 2006 ; myrianthefs et al . , 2000 ) .", "( 1992 ) suggested that paracentesis is the single most important treatment modality in life - threatening ohss not controlled by medical therapy .", "paracentesis and removal of ascites result in relieving the pressure on the inferior vena cava and improvement in venous return , cardiac output and renal perfusion  ( padilla et al . , 1990 ) .", "therefore , prompt drainage of fluid produces significant clinical and biochemical improvement , including spontaneous diuresis and hastening the resolution process  ( abuzeid et al .", ", 2003 ; the practice committee of the american society for reproductive medicine , 2008 ) .", "it is also possible that removal of ascites decreases the amount of vegf and its effects on vascular permeability .", "therefore ultrasound guided drainage of the ascitic fluid should be performed in severe cases of ohss with respiratory distress , oliguria or severe abdominal pain  ( padilla et al . , 1990 ) .", "this can be done either trans - vaginally  ( aboulghar et al . , 1990 ) or trans - abdominally  ( padilla et al . ,", "1990 ) . however , recurrence of ascites is common in severe cases of ohss .", "placement of a pigtail catheter has been proposed by some investigators to allow drainage of ascitic fluid whenever it accumulates without the need for further surgical procedures  ( abuzeid et al . , 2003 ) .", "in a previous study we reported the use of percutaneous pigtail catheter for drainage of ascites in severe ohss as an alternative to repeated paracentesis  ( abuzeid et al . , 2003 ) .", "this procedure is relatively simple and requires a short period of time as compared to the time needed to gradually aspirate any fluid accumulation transvaginally .", "immediate relief of symptoms and signs of severe ohss after pigtail catheter insertion was an important finding .", "in addition , the ease by which any accumulated ascitic fluid was drained resulted in a faster resolution of ohss  ( abuzeid et al . ,", "no infection was reported  ( abuzeid et al . , 2003 ) . in that study the first 13 patients", "they were discharged home with the pigtail catheter in place and managed on an outpatient basis until ohss subsided ; thereafter the catheter was removed .", "the last 13 patients were managed on an outpatient basis without any problem  ( abuzeid mi , et al . , 2003 ) .", "our previous study confirmed the safety and effectiveness of the pigtail catheter for the management of severe ohss .", "therefore , we embarked on the current study to evaluate the role of pigtail catheter drainage in outpatient management of patients with severe ohss syndrome . in the current study we demonstrated that patients who were diagnosed very early with severe ohss can be managed safely and effectively on outpatient basis using pigtail catheter drainage , iv fluid replacement and careful monitoring .", "although all 33 patients presented with shortness of breath , marked abdominal distension , nausea , weight gain and lower abdominal pain , and 13 of them complained of vomiting ,  ( all are criteria for hospital admission according to the practice committee of the american society for reproductive medicine on ohss ) , 30 patients were sent home as their presenting symptoms were relieved .", "early diagnosis and intervention resulted in rapid elimination of the symptoms of severe ohss and normalization of vital signs , which allow us to manage the patients on outpatient basis .", "the pigtail catheter placements were performed without complications and patients were discharged home after a few hours of observation .", "the patients who needed hospital admission were those with associated severe pleural effusion requiring thoracentesis .", "another patient was admitted to the hospital for 1 day for work up for chest pain and possible diagnosis of pulmonary embolism .", "once pulmonary embolism was ruled out , she was discharged home with a pigtail catheter in place and she was managed on outpatient basis .", "outpatient management of ohss has no place in those who fail to have complete resolutions of their symptoms , or those with severe hyperproteinuria or electrolyte imbalance .", "certainly it has no place in patients with critical ohss such as those with severe pleural effusion , diminished urine output , severe hemoconcentration and those at increased risk of thromboembolic risks  ( rizk , 2010 ) .", "it is imperative to stress that such a protocol can only be used in patients who are motivated , reliable and have the necessary support at home to allow them to return to the clinic or go to the emergency room if the need arises .", "it also requires dedicated in vitro fertilization nurses to educate and follow up the patient on a daily basis .", "contrary to other reports that state that access to intravenous albumin is a critical part of outpatient management for severe ohss , we did not use any albumin replacement  ( lincoln et al . , 2002 ) .", "the reason behind the use of intravenous albumin is to increase intravascular oncotic pressure , thereby curtailing the exodus of free water from the intravascular space . in our study", "we used hespan instead of albumin , as some investigators have shown that it is beneficial in reducing the incidence of severe ohss  ( youssef et al . , 2011 ) . by increasing the intravascular oncotic pressure", "in addition , the majority of our patients had late onset ohss as a result of pregnancy .", "hespan should be used with caution in patients with late onset ohss only when benefits outweigh risks.it is a fact that outpatient management of early severe ohss is a common practice among infertility specialists after trans - vaginal aspiration of ascitic fluid and iv fluid hydration .", "outpatient management by repeated abdominal or trans - vaginal paracentesis has been reported to be safe , effective and cost effective  ( lincoln et al .", ", 2002 ; shrivastav et al . , 1994 ; fluker et al . , 2000", "( 1994 ) were the first to suggest that day care management in patients with severe ohss with abdominal paracentesis and iv hydration was simple , safe and effective compared with traditional treatment with in - patient hospitalization and supportive measures .", "( 2002 ) concluded that the need for hospitalization for hyperstimulated patients can be minimized by outpatient management of severe ohss with trans - vaginal culdocentesis and rehydration with crystalloids and albumin .", "( 2000 ) advocated in a pilot study an active management strategy for moderate ohss , which involved early paracentesis designed to minimize the progression from moderate to severe ohss , instead of late paracentesis aimed at speeding up recovery in severely ill patients .", "( 2010 ) concluded that early outpatient paracentesis for moderate to severe ohss is the most cost effective management plan when compared with traditional conservative inpatient therapy .", "( 2009 ) reported a large series of patients with severe ohss in whom the vast majority were successfully managed as outpatients with use of aggressive trans - vaginal paracentesis  ( smith et al . , 2009 ) . despite the findings in our study", "it is important to emphasize that ohss remains a serious disorder with the potential for rapid deterioration , requiring hospitalization and intensive treatment of a critically ill patient .", "the life - threatening complications of ohss are secondary to thromboembolism or compromised pulmonary or cardiovascular function and multi - organ failure", ". there have been isolated reports of women dying from the complications associated with ohss .", "the mortality rate related to ohss is very low and was estimated by brinsden et al . ", "more recently , reports regarding maternal mortality rates due to ohss in the netherlands and united kingdom demonstrate an incidence of approximately 3 deaths per 100,000 in ivf cycles  ( braat et al .", ", 2006 ; lewis , 2007 ) . eleven lethal complications of assisted reproductive technology have been documented and summarized by braat et al . ", "several investigators reported sporadic fatality as a result of ohss due to adult respiratory distress syndrome , massive pulmonary oedema , myocardial infarction , and carotid artery occlusion resulting in cerebral infarction . in this study", "we describe a modified technique of what interventional radiologists usually use for pigtail catheter placement .", "we observed that in the obese patients the metal introducer provided in the pigtail catheter kit is difficult to use ; instead we used the non disposable gamete intra - fallopian treatment trocar and cannula to facilitate the introduction of the catheter through the abdominal wall of such patients .", "this technique helps to manage the patients in the office without the need for sending her to the hospital for an interventional radiologist to place the catheter .", "however , the patients in this study were identified from our in vitro fertilization database ; the study included every patient who developed severe / critical ohss .", "in addition , the lack of a control group that was managed without pigtail catheter placement limits the conclusions that can be drawn from our study . in summary ,", "drainage of ascitic fluid in patients with severe / critical ohss is an important step in the management of this condition  ( rizk , 2010 ) .", "the placement of a pigtail catheter resulted in a safe and effective outpatient management of the majority of patients with severe ohss .", "however , outpatient management of this potentially fatal condition requires clear understanding that rapid deterioration may occur , which may necessitate hospitalization and intensive treatment of a critically ill patient  ( rizk , 2006 , 2010 ) .", "patients with critical ohss and those with persistence of symptoms of severe ohss despite insertion of a pigtail catheter and drainage of ascitic fluid should be admitted to the hospital for inpatient management ."]]}
{"article_id": "PMC3603086", "article_text": ["congenital adrenal hyperplasia ( cah ) refers to a group of autosomal recessive disorders caused by an enzyme deficiency which leads to defects in biosynthesis of steroid precursors .", "depending on the severity and degree of 21 hydroxylase deficiency , the clinical spectrum may vary from mild form of non classical cah to classic cah .", "however , the non classical cah variant is more common with a prevalence rate of 1 in 1000 .", "it also helps in maintaining normal levels of precursors by suppressing adreno cortico trophic hormone ( acth ) . during childhood", ", the management is largely focused on achieving normal growth and attaining appropriate final adult height .", "johns medical college hospital , bangalore by the department of endocrinology on patients diagnosed to have cah and seen in the outpatient clinic between january 2012 and october 2012 . during this period", "data regarding demography , clinical presentation at time of diagnosis , treatment details , height sds and bmi were collected .", "all patients underwent biochemical testing for 17 hydroxy progesterone ( 17 ohp ) levels for assessment of adequacy of therapy . bone age assessment with left hand and wrist", "bmi was calculated for all patients and obesity was defined using who charts as values above 95th percentile .", "17 ohp levels between 1 ng / ml and 12 ng / ml were considered appropriate ; values below 1 ng / ml suggested suppression and over treatment and values above 12 ng / ml suggested under treatment .", "29 patients were included in the study of which 22 were females ( 76% ) and 7 were males ( 24% ) . based on the cross sectional data collected , 11 patients were adults ( age > 18 ) and 18 patients were children ( 62% ) . among the males ,", "one child was identified at birth via a neonatal screening program , one child presented with early pubarche , the other 5 infants presented between their 3rd and 6th week of life with features suggestive of adrenal crisis - poor feeding , vomiting and failure to gain weight .", "one of the male patients incidentally also had a penoscrotal hypospadias which was surgically corrected . among the females ,", "9 infants were identified at birth due to presence of genital ambiguity ( 40% ) , 1 presented with symptoms of adrenal crisis at 4 weeks of life , 4 patients presented in the pre pubertal period due to early onset adrenarche ( 18% ) , 5 patients presented in the late adolescent period with marked virilization ( 23% ) and 3 patients presented with features of poly cystic ovarian disease ( pcos ) .", "all the five patients who presented in the late adolescent period had obvious genital ambiguity from birth , however , they sought medical attention only much later due to marked virilization and failure to attain menarche .", "one among them had actually been evaluated and even underwent a clitoroplasty at a young age , but unfortunately , the diagnosis of cah was missed as she was mistakenly categorized as probable ovo testicular dsd due to presence of mullerian structures with a phallic length of around 6 cm .", "all these 5 women had a masculine built at presentation with poor breast development , severe hirsutism , muscular body habitus , temporal balding and varying degrees of deepening of voice .", "the non classical cah ( nccah ) patients presented in adulthood with complaints of irregular cycles and hirsutism ; they had no features of virilisation .", "simple virilizing was the most common sub class seen among the women ( 81% ) whereas the salt wasting type was predominant among boys ( 85% ) .", "around 35% of patients had short stature as defined by height sds < -2.0 ; 2 patients who presented with adrenarche had increase in height sds , however , their bone age was also correspondingly advanced .", "all the children were treated with hydrocortisone ; almost all adult patients were on dexamethasone .", "none of the nccah patients were treated with glucocorticoids ; they were on anti - androgen therapy along with oral contraceptive pills .", "most adult classic cah patients with complaints of hirsutism were treated with either spironolactone or finasteride .", "pubertal induction with ethinyl estradiol was initiated for three of the adult patients who sought medical care late .", "analysis of 17 ohp levels revealed that 32% were suppressed with levels less than 1ng / ml ; appropriate in 47% and inadequate in 21% of patients .", "we report the phenotypic features of a cohort of patients with cah being evaluated and managed in a tertiary centre in southern india . in this series ,", "the typical presentation of male babies were with adrenal crisis between the 3 and 6 week of life . around 20% of female patients with classic cah", "were identified and appropriately treated only after late adolescence even when genital ambiguity was present since birth .", "short stature was seen in one third of patients on therapy and the average final adult height among patients with classic cah was 142.37 , which was significantly lower than the mean adult height for females in our population .", "the mean final adult height of 157.5 cm for those with nccah was better than their classic cah counterpart .", "obesity and hypertension were not found to be significantly higher in this cohort of cah patients .", "one third of patients had suppressed 17 ohp levels suggestive of over replacement therapy which may also contribute to the reduction in the final adult stature ."], "abstract_text": ["<S> congenital adrenal hyperplasia is a group of autosomal recessive disorders caused by enzyme deficiency which leads to defects in biosynthesis of steroid precursors . </S>", "<S> most common is 21 hydroxylase deficiency . </S>", "<S> clinical spectrum varies from non - classical cah to classic cah , and it may be simple virilising form or salt - wastinfg type . </S>", "<S> 29 patients were included in our study from january 2012 to october 2012 . </S>", "<S> 76% were females . </S>", "<S> male babies typically presented with adrenal crisis between 3rd to 6th week of life . around 20% of females </S>", "<S> were identified and appropriately treated only after late adolescence . </S>", "<S> short stature was seen in 1/3rd of patients . </S>", "<S> 1/3rd of patients had suppressed 17 ohp levels suggestive of over - replacement therapy which may contribute to final reduction in adult height . </S>"], "labels": null, "section_names": ["I", "M", "R", "D"], "sections": [["congenital adrenal hyperplasia ( cah ) refers to a group of autosomal recessive disorders caused by an enzyme deficiency which leads to defects in biosynthesis of steroid precursors .", "depending on the severity and degree of 21 hydroxylase deficiency , the clinical spectrum may vary from mild form of non classical cah to classic cah .", "however , the non classical cah variant is more common with a prevalence rate of 1 in 1000 .", "it also helps in maintaining normal levels of precursors by suppressing adreno cortico trophic hormone ( acth ) . during childhood", ", the management is largely focused on achieving normal growth and attaining appropriate final adult height ."], ["johns medical college hospital , bangalore by the department of endocrinology on patients diagnosed to have cah and seen in the outpatient clinic between january 2012 and october 2012 . during this period", "data regarding demography , clinical presentation at time of diagnosis , treatment details , height sds and bmi were collected .", "all patients underwent biochemical testing for 17 hydroxy progesterone ( 17 ohp ) levels for assessment of adequacy of therapy . bone age assessment with left hand and wrist", "bmi was calculated for all patients and obesity was defined using who charts as values above 95th percentile .", "17 ohp levels between 1 ng / ml and 12 ng / ml were considered appropriate ; values below 1 ng / ml suggested suppression and over treatment and values above 12 ng / ml suggested under treatment ."], ["29 patients were included in the study of which 22 were females ( 76% ) and 7 were males ( 24% ) . based on the cross sectional data collected , 11 patients were adults ( age > 18 ) and 18 patients were children ( 62% ) . among the males ,", "one child was identified at birth via a neonatal screening program , one child presented with early pubarche , the other 5 infants presented between their 3rd and 6th week of life with features suggestive of adrenal crisis - poor feeding , vomiting and failure to gain weight .", "one of the male patients incidentally also had a penoscrotal hypospadias which was surgically corrected . among the females ,", "9 infants were identified at birth due to presence of genital ambiguity ( 40% ) , 1 presented with symptoms of adrenal crisis at 4 weeks of life , 4 patients presented in the pre pubertal period due to early onset adrenarche ( 18% ) , 5 patients presented in the late adolescent period with marked virilization ( 23% ) and 3 patients presented with features of poly cystic ovarian disease ( pcos ) .", "all the five patients who presented in the late adolescent period had obvious genital ambiguity from birth , however , they sought medical attention only much later due to marked virilization and failure to attain menarche .", "one among them had actually been evaluated and even underwent a clitoroplasty at a young age , but unfortunately , the diagnosis of cah was missed as she was mistakenly categorized as probable ovo testicular dsd due to presence of mullerian structures with a phallic length of around 6 cm .", "all these 5 women had a masculine built at presentation with poor breast development , severe hirsutism , muscular body habitus , temporal balding and varying degrees of deepening of voice .", "the non classical cah ( nccah ) patients presented in adulthood with complaints of irregular cycles and hirsutism ; they had no features of virilisation .", "simple virilizing was the most common sub class seen among the women ( 81% ) whereas the salt wasting type was predominant among boys ( 85% ) .", "around 35% of patients had short stature as defined by height sds < -2.0 ; 2 patients who presented with adrenarche had increase in height sds , however , their bone age was also correspondingly advanced .", "all the children were treated with hydrocortisone ; almost all adult patients were on dexamethasone .", "none of the nccah patients were treated with glucocorticoids ; they were on anti - androgen therapy along with oral contraceptive pills .", "most adult classic cah patients with complaints of hirsutism were treated with either spironolactone or finasteride .", "pubertal induction with ethinyl estradiol was initiated for three of the adult patients who sought medical care late .", "analysis of 17 ohp levels revealed that 32% were suppressed with levels less than 1ng / ml ; appropriate in 47% and inadequate in 21% of patients ."], ["we report the phenotypic features of a cohort of patients with cah being evaluated and managed in a tertiary centre in southern india . in this series ,", "the typical presentation of male babies were with adrenal crisis between the 3 and 6 week of life . around 20% of female patients with classic cah", "were identified and appropriately treated only after late adolescence even when genital ambiguity was present since birth .", "short stature was seen in one third of patients on therapy and the average final adult height among patients with classic cah was 142.37 , which was significantly lower than the mean adult height for females in our population .", "the mean final adult height of 157.5 cm for those with nccah was better than their classic cah counterpart .", "obesity and hypertension were not found to be significantly higher in this cohort of cah patients .", "one third of patients had suppressed 17 ohp levels suggestive of over replacement therapy which may also contribute to the reduction in the final adult stature ."]]}
{"article_id": "PMC4414990", "article_text": ["type 1 diabetes ( t1d ) results from the destruction of insulin producing pancreatic  cells by a  cell specific autoimmune process ( 1 ) . chronic pancreatic inflammation ( insulitis ) and destruction of islet -cells in type 1 diabetes", "is mediated by the immune cells , particularly autoreactive cd4 and cd8 t lymphocytes , b cells , macrophages and dendritic cells ( 2 ) . in order to obtain insight into type 1 diabetes pathogenic mechanisms in humans and to test novel therapeutic approaches for its treatment , different preclinical models of the disease such as spontaneous and accelerated diabetes in the non - obese diabetic ( nod ) mice ,", "biobreeding rats , or diabetes induced in susceptible rodent strains by multiple low doses of streptozotocin ( mlds ) are now available ( 3 ) . in the pathogenesis of t1d , several proinflamma - tory cytokines including ifn -  , tnf- , il-1 , as well as il-17 ( 4 - 6 ) , have been implicated .", "it is also thought that the production of anti - inflammatory cytokines such as il-4 , il-10 and tgf- correlates with protection from t1d ( 6 ) .", "it has been shown that some drugs such as pentoxifylline ( ptx ) have immunomodulatory and anti - inflammatory activity , which might represent a potential preventive therapy for autoimmune diseases .", "ptx is a methyl xanthine - derived general phosphodiesterase ( pde ) inhibitor that has been available for many years to treat vascular disorder ( 7 ) .", "phosphodiesterases ( pdes ) are a family of enzymes that regulate intracellular levels of the cyclic nucleotides cyclic adenosine monophosphate ( camp ) and cyclic guanosine monophosphate ( cgmp ) by catalyzing their breakdown to inactive metabolites .", "drugs that increase the levels of camp , tend to reduce the production of proinflammatory mediators and increase the production of anti - inflammatory mediators by immune cells ( 8) . in recent years , the potential of ptx as an immunomodulatory and anti - inflammatory agent gained interest as it has been shown to effectively suppress the synthesis of tnf- and other pro inflammatory cytokines such as ifn -  and il-12 in vitro and in vivo ( 9 - 10 ) .", "ptx has been successfully used for the treatment of experimental autoimmune diseases including experimental autoimmune myocarditis ( eam ) ( 11 ) , experimental autoimmune encephalomyelitis ( 12 ) , and adjuvant arthritis ( 13 ) . in the present study", ", we hypothesized that pentoxifylline , due to its anti - inflammatory and immunosuppressive activity , may affect autoimmune diabetes .", "consequently , we decided to investigate whether pentoxifylline treatment could prevent the development of mlds - induced diabetes in mice .", "streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) .", "male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle .", "diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice .", "stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days .", "the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) .", "mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) .", "subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip )", "plasma , separated from blood by centrifugation , was stored at -80c until insulin assay .", "mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test .", "non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) . before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes .", "mice were euthanized and their spleens were removed on day 21 for cytokine production assay .", "after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle .", "a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris  hcl and 1% nh4cl ( ph 7.2 ) .", "subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes .", "remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2", "the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn -  were measured by elisa kits according to the manufacturers instructions .", "mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr .", "after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a. one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups .", "streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) .", "male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle .", "diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice .", "stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days .", "the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) .", "mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) .", "subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip ) was initiated in treatment group when they were considered diabetic . at the same time , the control groups received saline vehicle alone with the same schedule .", "plasma , separated from blood by centrifugation , was stored at -80c until insulin assay .", "mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test .", "non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) .", "before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes .", "mice were euthanized and their spleens were removed on day 21 for cytokine production assay .", "after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle .", "a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris  hcl and 1% nh4cl ( ph 7.2 ) .", "subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes .", "remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2 mmol / l l - glutamine .", "the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn -  were measured by elisa kits according to the manufacturers instructions .", "mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr . the plate was then centrifuged and followed by removal of medium .", "after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a.", "one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups .", "all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group .", "however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) .", "effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) .", "ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage .", "effect of pentoxifylline treatment on plasma levels of insulin ( * p<0.05 ) treatment of mice with ptx significantly decreased mlds - induced production of ifn-  and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) .", "effect of pentoxifylline on productions of ifn  il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group ) the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) .", "effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 )", "all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group .", "however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) .", "effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) .", "ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage .", "treatment of mice with ptx significantly decreased mlds - induced production of ifn-  and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) .", "effect of pentoxifylline on productions of ifn  il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group )", "the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) .", "effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 )", "in rodents , experimental insulin - dependent diabetes can be induced by multiple low doses of streptozotocin ( mlds ) ( 15 ) .", "this model is a commonly used animal model that has many histological and clinical features similar to those of human type 1 diabetes and involves the participation of macrophages and t cells .", "streptozotocin ( stz ) is a pancreatic  cell toxin that induces inflammation of the islets by immune cells when it is given in multiple low doses ( 16 ) .", "this model of diabetes offers some advantages , such as simultaneous appearance of diabetes mellitus in all animals and not having immune abnormalities that may complicate studies in spontaneous diabetes in non - obese diabetic ( nod ) mice or bio breeding ( bb ) rats .", "therefore , this model as a good model has been applied extensively to investigate the type 1 diabetes mellitus , especially study the immune pathways and modulations of autoimmune insulitis and  cell death ( 17 - 18 ) .", "c57bl/6 mice , also called  c57 black 6  or simply  black 6  has the advantage of strain stability and easy breeding .", "the most common application of c57bl/6 mice is to serve as physiological or pathological models for in vivo experiments ( 19 ) . as such", ", mlds induced c57bl/6j mice were chosen to evaluate the anti - hyperglycemic activity of ptx and investigate the immunotherapeutic molecular mechanisms of how ptx attenuated the development of t1d .", "ptx is commonly used for the treatment of microcirculatory disorders and yields only minimal side effects in patients .", "in addition , due to its varied immunomodulatory effects , ptx has been used in several autoimmune diseases including rheumatoid arthritis ( 20 ) , multiple sclerosis ( 21 ) , and systemic lupus erythematosus ( 22 ) .", "drugs that increase the level of camp , including pde inhibitors , are known to prevent or attenuate inflammatory cell responses ( 20 ) .", "previous studies have also shown that ptx lowers blood glucose levels in diabetic animals ( 23 ) , and the results from the present studies also indicated that ptx possessed an anti - hyperglycemic property .", "more importantly , the present data demonstrated that ptx could increase serum insulin concentrations in type 1 diabetic mice .", "it has been shown that ptx could reduce ( but not abrogate ) the insulin requirements or lengthen the  honeymoon  ( non - insulin - requiring ) period in a study of 21 children with new - onset type 1diabetes ( 24 ) .", "t helper 1 cells produce proinflammatory cytokines ( tnf- , ifn -  , il-1 , il-6 , and il-12 ) , which activate macrophages and cytotoxic t cells to destroy -cells , whereas anti - inflammatory cytokines ( il-4 and il-10 ) that are produced by activated t helper 2 cells , prevent -cell destructive insulitis ( 25 ) .", "il-10 has been shown to prevent the onset of diabetes in mice ( 26 ) . in response to cytokine stimulation , -cells generate reactive oxygen species ( ross ) and reactive nitrogen species such as nitric oxide ( no ) , which facilitate their destruction ( 27 ) .", "no is also synthesized within cytokine - activated macrophages by inducible nitric oxide synthase ( inos ) ( 28 ) .", "more recently , the role of il-17-producing t cells have been demonstrated in the development of several autoimmune diseases , including multiple sclerosis , rheumatoid arthritis and type 1 diabetes .", "il-17 is a proinflammatory cytokine that is detrimental to pancreatic islet cells ( 29 ) .", "il-17 promotes infiltration of neutrophils and macrophages , stimulates the production of other proinflammatory cytokines , including tnf- , il-1 , il-6 and il-12 , by activated macrophages and induces nitric oxide release from  cells that interferes with their function and cause -cell destruction ( 30 , 5 ) .", "it has been assumed that ptx similar to camp elevating agents , selectively suppresses the production of t helper type 1(il-2 , ifn -  ) , but not t helper type 2(il-4 , il-6 , il-10 ) cytokines ( 31 ) .", "however , it has also been demonstrated that the effect of ptx on cytokine production is cell - specific ( 32 ) .", "the present study shows that ptx is not only able to inhibit the release of proinflammatory cytokines ( ifn -  and il-17 ) , but can also increase the production of anti - inflammatory cytokines ( il-10 ) . since ptx has been found to be able to inhibit the release of proinflammatory cytokines ( 33 ) , and favor th2 response ( 34 ) , it may indirectly interfere with the induction of inos .", "it has been shown that pde inhibitors elevate camp , prevent or attenuate inflammatory cell responses ( 35 ) and suppress proliferation of lymphocytes ( 36 ) .", "our findings are consistent with previous studies , showing that ptx treatment prevented lymphocyte proliferation .", "in summary , results of the present work demonstrated that the suppressive effect of the ptx treatment on t1d was accompanied by decreased blood glucose level , increased plasma insulin level , suppression of t cell proliferation , down - regulation of th1 and th17 cytokines ( ifn -  and il-17 ) and increase in the production of il-10 in supernatant of splenic culture of the treated mice .", "it seems that pentoxifylline treatment has a therapeutic effect against mlds - induced diabetes in mice ."], "abstract_text": ["<S> objective(s):pentoxifylline is an immunomodulatory and anti - inflammatory agent and is used in vascular disorders . </S>", "<S> it has been shown that pentoxifylline inhibits proinflammatory cytokines production . </S>", "<S> the purpose of this study was to investigate the therapeutic effects of pentoxifylline on the treatment of autoimmune diabetes in mice.materials and methods : diabetes was induced by multiple low dose of streptozotocin ( mlds ) injection ( 40 mg / kg / day for 5 consecutive days ) in male c57bl/6 mice . </S>", "<S> after induction of diabetes , mice were treated with pentoxifylline ( 100 mg / kg / day ip ) for 21 days . </S>", "<S> blood glucose levels and plasma levels of insulin were measured . </S>", "<S> splenocytes were tested for proliferation by mtt test and cytokine production by elisa.results:pentoxifylline treatment prevented hyperglycemia and increased plasma insulin levels in the diabetic mice . aside from reducing lymphocyte proliferation , pentoxifylline significantly inhibited the production of proinflammatory interleukin 17 ( il-17 ) as well as interferon gamma ( ifn- ) , while increased anti - inflammatory cytokine il-10 as compared with those in mlds group ( diabetic control group).conclusion : these findings indicate that pentoxifylline may have therapeutic effect against the autoimmune destruction of the pancreatic beta - cells during the development of mlds - induced type 1 diabetes in mice . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Drug and Reagent", "Animals and materials", "Animal treatments", "Quantification of glucose in the blood", "Plasma insulin determination", "Splenocyte culture and cytokine determination", "MTT proliferation assay", "Statistical analysis", "Results", "Effects of pentoxifylline on MLDS-induced hyperglycemia and plasma insulin level", "Effects of pentoxifylline on cytokine production in type 1 diabetic murine splenocytes", "Effects of pentoxifylline on the proliferation of splenic lymphocytes", "Discussion", "Conclusion"], "sections": [["type 1 diabetes ( t1d ) results from the destruction of insulin producing pancreatic  cells by a  cell specific autoimmune process ( 1 ) . chronic pancreatic inflammation ( insulitis ) and destruction of islet -cells in type 1 diabetes", "is mediated by the immune cells , particularly autoreactive cd4 and cd8 t lymphocytes , b cells , macrophages and dendritic cells ( 2 ) . in order to obtain insight into type 1 diabetes pathogenic mechanisms in humans and to test novel therapeutic approaches for its treatment , different preclinical models of the disease such as spontaneous and accelerated diabetes in the non - obese diabetic ( nod ) mice ,", "biobreeding rats , or diabetes induced in susceptible rodent strains by multiple low doses of streptozotocin ( mlds ) are now available ( 3 ) . in the pathogenesis of t1d , several proinflamma - tory cytokines including ifn -  , tnf- , il-1 , as well as il-17 ( 4 - 6 ) , have been implicated .", "it is also thought that the production of anti - inflammatory cytokines such as il-4 , il-10 and tgf- correlates with protection from t1d ( 6 ) .", "it has been shown that some drugs such as pentoxifylline ( ptx ) have immunomodulatory and anti - inflammatory activity , which might represent a potential preventive therapy for autoimmune diseases .", "ptx is a methyl xanthine - derived general phosphodiesterase ( pde ) inhibitor that has been available for many years to treat vascular disorder ( 7 ) .", "phosphodiesterases ( pdes ) are a family of enzymes that regulate intracellular levels of the cyclic nucleotides cyclic adenosine monophosphate ( camp ) and cyclic guanosine monophosphate ( cgmp ) by catalyzing their breakdown to inactive metabolites .", "drugs that increase the levels of camp , tend to reduce the production of proinflammatory mediators and increase the production of anti - inflammatory mediators by immune cells ( 8) . in recent years , the potential of ptx as an immunomodulatory and anti - inflammatory agent gained interest as it has been shown to effectively suppress the synthesis of tnf- and other pro inflammatory cytokines such as ifn -  and il-12 in vitro and in vivo ( 9 - 10 ) .", "ptx has been successfully used for the treatment of experimental autoimmune diseases including experimental autoimmune myocarditis ( eam ) ( 11 ) , experimental autoimmune encephalomyelitis ( 12 ) , and adjuvant arthritis ( 13 ) . in the present study", ", we hypothesized that pentoxifylline , due to its anti - inflammatory and immunosuppressive activity , may affect autoimmune diabetes .", "consequently , we decided to investigate whether pentoxifylline treatment could prevent the development of mlds - induced diabetes in mice ."], ["streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) .", "male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle .", "diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice .", "stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days .", "the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) .", "mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) .", "subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip )", "plasma , separated from blood by centrifugation , was stored at -80c until insulin assay .", "mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test .", "non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) . before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes .", "mice were euthanized and their spleens were removed on day 21 for cytokine production assay .", "after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle .", "a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris  hcl and 1% nh4cl ( ph 7.2 ) .", "subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes .", "remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2", "the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn -  were measured by elisa kits according to the manufacturers instructions .", "mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr .", "after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a. one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups ."], ["streptozotocin ( stz ) , citrate buffer , pentoxifylline ( ptx ) , rpmi 1640 , l - glutamine , dimethylsul- foxide ( dmso ) , concanavalin a , mtt ( ( 3-(4,5-dimethyl - thiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) and fetal calf serum fetal were purchased from sigma - aldrich ( st.louis , mo ) ."], ["male c57bl/6 mice , weighing 20 to 25 g , were housed in a room with a 12-hr light / dark cycle ."], ["diabetes was induced by multiple low - dose of streptozotocin ( mlds ) injection in male c57bl/6 mice .", "stz was dissolved in 0.1 m citrate buffer ( ph 4.5 ) and injected intraperitoneally ( ip ) , within 10 min of preparation , at a dose of 40 mg / kg / day for 5 consecutive days .", "the blood samples were obtained from the tail vein of non - fasted mice , and glucose was measured using a glucometer ( accu - chech active ) .", "mice were considered diabetic when their non - fasting blood glucose level was > 250 mg / dl ( 14 ) .", "subsequently , the mice were allocated to three therapeutic groups ( n=7 per group ) ( normal control group , mlds group ( diabetic control group ) and treatment group ) . treatment with ptx ( 100 mg / kg / day for 21 days , ip ) was initiated in treatment group when they were considered diabetic . at the same time , the control groups received saline vehicle alone with the same schedule .", "plasma , separated from blood by centrifugation , was stored at -80c until insulin assay .", "mice were euthanized and their splenic tissues were removed on day 21 for cytokine assay and mtt test ."], ["non - fasting blood sugar level was measured in 0 , 7 , 14 and 21 days after streptozotocin induced diabetes by using a glucometer ( accu - chech active).the blood samples were obtained from the tail vein of non - fasted mice ( 14 ) ."], ["before mice were euthanized , blood was collected from each mouse on day 21 in heparinized tubes ."], ["mice were euthanized and their spleens were removed on day 21 for cytokine production assay .", "after aseptic removal , spleens were placed in cold hanks solution and teased apart with a pair of forceps and a needle .", "a single cell suspension was obtained by passing it through a 200-mesh net and hemolyzed by the buffer solution containing 1 mmol / l tris  hcl and 1% nh4cl ( ph 7.2 ) .", "subsequently , the macrophage cell content was depleted by incubation of the cell suspension in tissue culture dishes at 37c ( air+5% co2 ) to allow these cells to adhere to the bottom of the culture dishes .", "remaining free floating cells were seeded on culture dishes at a density of 510cells / ml in rpmi 1640 with 10% fetal calf serum , and 2 mmol / l l - glutamine .", "the splenocytes ( 510cells / ml ) were treated with 2 g / ml concanavalin a for 72 hr , and cell supernatants were collected , then the levels of il-10 , il17 and ifn -  were measured by elisa kits according to the manufacturers instructions ."], ["mouse splenic lymphocytes ( 110cells / ml ) were incubated in the absence or presence of concanavalin a ( 1.25 g / ml ) in a 96-well flat - bottom microculture plat in triplicate for 72 hr . thirty microliters of 5 mg / ml mtt ( ( 3-(4,5-dimethylthiazolyl)-2,5-diphenyl - tetrazolium bromide ) ) in pbs was added to each well , and the plate was incubated at 37c for 4 hr . the plate was then centrifuged and followed by removal of medium .", "after incubation at 37c for 5 min , absorbance was measured spectro - photometrically at 490 nm . the stimulation index si ) was calculated according to the following formula : si= od concanavalin a ( con a ) stimulated lymphocyte proliferation / od spontaneous lymphocyte proliferation without con a."], ["one - way analysis of variance ( anova ) followed by tukey s test were used for multiple comparisons between groups ."], ["all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group .", "however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) .", "effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) .", "ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage .", "effect of pentoxifylline treatment on plasma levels of insulin ( * p<0.05 ) treatment of mice with ptx significantly decreased mlds - induced production of ifn-  and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) .", "effect of pentoxifylline on productions of ifn  il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group ) the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) .", "effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 )"], ["all stz - induced diabetic mice with ptx treatment remained hyperglycemic on day 14 , and no significant difference in blood glucose levels was demonstrated between ptx and mlds(diabetic control group ) group .", "however , the levels of glucose in diabetic mice were significantly reduced after treatment with ptx ( p<0.05 ) , for 21day , when compared to the values of diabetic control mice ( figure 1 ) .", "effect of pentoxifylline treatment on blood glucose levels ( * p<0.05 ) to evaluate -cell function in terms of insulin release , we measured the plasma insulin levels on day 21 ( figure 2 ) .", "ptx prevented the mlds - induced reduction in plasma insulin , indicating a possible protective effect of ptx against -cell damage ."], ["treatment of mice with ptx significantly decreased mlds - induced production of ifn-  and il-17 , while increased il-10 as compared with those in mlds group ( diabetic control group ) ( p<0.05)(figure 3 ) .", "effect of pentoxifylline on productions of ifn  il-10 and il-17 of diabetic murine splenocytes induced by con a ex vivo ( * p<0.05 versus diabetic control group )"], ["the stimulation index ( si ) in treatment group with ptx showed a significant decrease as compared with that in mlds group ( diabetic control group ) ( p<0.05)(figure 4 ) .", "effects of pentoxifylline on the proliferation of splenic lymphocytes induced by con a after 72 hr ( * p<0.05 )"], ["in rodents , experimental insulin - dependent diabetes can be induced by multiple low doses of streptozotocin ( mlds ) ( 15 ) .", "this model is a commonly used animal model that has many histological and clinical features similar to those of human type 1 diabetes and involves the participation of macrophages and t cells .", "streptozotocin ( stz ) is a pancreatic  cell toxin that induces inflammation of the islets by immune cells when it is given in multiple low doses ( 16 ) .", "this model of diabetes offers some advantages , such as simultaneous appearance of diabetes mellitus in all animals and not having immune abnormalities that may complicate studies in spontaneous diabetes in non - obese diabetic ( nod ) mice or bio breeding ( bb ) rats .", "therefore , this model as a good model has been applied extensively to investigate the type 1 diabetes mellitus , especially study the immune pathways and modulations of autoimmune insulitis and  cell death ( 17 - 18 ) .", "c57bl/6 mice , also called  c57 black 6  or simply  black 6  has the advantage of strain stability and easy breeding .", "the most common application of c57bl/6 mice is to serve as physiological or pathological models for in vivo experiments ( 19 ) . as such", ", mlds induced c57bl/6j mice were chosen to evaluate the anti - hyperglycemic activity of ptx and investigate the immunotherapeutic molecular mechanisms of how ptx attenuated the development of t1d .", "ptx is commonly used for the treatment of microcirculatory disorders and yields only minimal side effects in patients .", "in addition , due to its varied immunomodulatory effects , ptx has been used in several autoimmune diseases including rheumatoid arthritis ( 20 ) , multiple sclerosis ( 21 ) , and systemic lupus erythematosus ( 22 ) .", "drugs that increase the level of camp , including pde inhibitors , are known to prevent or attenuate inflammatory cell responses ( 20 ) .", "previous studies have also shown that ptx lowers blood glucose levels in diabetic animals ( 23 ) , and the results from the present studies also indicated that ptx possessed an anti - hyperglycemic property .", "more importantly , the present data demonstrated that ptx could increase serum insulin concentrations in type 1 diabetic mice .", "it has been shown that ptx could reduce ( but not abrogate ) the insulin requirements or lengthen the  honeymoon  ( non - insulin - requiring ) period in a study of 21 children with new - onset type 1diabetes ( 24 ) .", "t helper 1 cells produce proinflammatory cytokines ( tnf- , ifn -  , il-1 , il-6 , and il-12 ) , which activate macrophages and cytotoxic t cells to destroy -cells , whereas anti - inflammatory cytokines ( il-4 and il-10 ) that are produced by activated t helper 2 cells , prevent -cell destructive insulitis ( 25 ) .", "il-10 has been shown to prevent the onset of diabetes in mice ( 26 ) . in response to cytokine stimulation , -cells generate reactive oxygen species ( ross ) and reactive nitrogen species such as nitric oxide ( no ) , which facilitate their destruction ( 27 ) .", "no is also synthesized within cytokine - activated macrophages by inducible nitric oxide synthase ( inos ) ( 28 ) .", "more recently , the role of il-17-producing t cells have been demonstrated in the development of several autoimmune diseases , including multiple sclerosis , rheumatoid arthritis and type 1 diabetes .", "il-17 is a proinflammatory cytokine that is detrimental to pancreatic islet cells ( 29 ) .", "il-17 promotes infiltration of neutrophils and macrophages , stimulates the production of other proinflammatory cytokines , including tnf- , il-1 , il-6 and il-12 , by activated macrophages and induces nitric oxide release from  cells that interferes with their function and cause -cell destruction ( 30 , 5 ) .", "it has been assumed that ptx similar to camp elevating agents , selectively suppresses the production of t helper type 1(il-2 , ifn -  ) , but not t helper type 2(il-4 , il-6 , il-10 ) cytokines ( 31 ) .", "however , it has also been demonstrated that the effect of ptx on cytokine production is cell - specific ( 32 ) .", "the present study shows that ptx is not only able to inhibit the release of proinflammatory cytokines ( ifn -  and il-17 ) , but can also increase the production of anti - inflammatory cytokines ( il-10 ) . since ptx has been found to be able to inhibit the release of proinflammatory cytokines ( 33 ) , and favor th2 response ( 34 ) , it may indirectly interfere with the induction of inos .", "it has been shown that pde inhibitors elevate camp , prevent or attenuate inflammatory cell responses ( 35 ) and suppress proliferation of lymphocytes ( 36 ) .", "our findings are consistent with previous studies , showing that ptx treatment prevented lymphocyte proliferation ."], ["in summary , results of the present work demonstrated that the suppressive effect of the ptx treatment on t1d was accompanied by decreased blood glucose level , increased plasma insulin level , suppression of t cell proliferation , down - regulation of th1 and th17 cytokines ( ifn -  and il-17 ) and increase in the production of il-10 in supernatant of splenic culture of the treated mice .", "it seems that pentoxifylline treatment has a therapeutic effect against mlds - induced diabetes in mice ."]]}
{"article_id": "PMC5094872", "article_text": ["determinar a presena de anticorpos ige especficos para superantgenos estafiloccicos e o grau de sensibilizao mediada por esses , assim como se esses esto associados  gravidade da asma em pacientes adultos .", "estudo transversal incluindo asmticos adultos em acompanhamento ambulatorial em um hospital universitrio tercirio no rio de janeiro ( rj ) .", "os pacientes foram alocados consecutivamente em dois grupos de gravidade da asma segundo critrios da global initiative for asthma : asma leve ( al ) , com asmticos leves intermitentes ou persistentes , e asma moderada ou grave ( amg ) .", "foram determinados os nveis sricos de anticorpos ige antitoxinas estafiloccicas , e os resultados foram comparados por anlise estatstica .", "foram includos 142 pacientes no estudo : 72 no grupo al ( mediana de idade = 46 anos ; 59 do sexo feminino ) e 70 do grupo amg ( mediana de idade = 56 anos ; 60 do sexo feminino ) .", "na amostra geral , 62 pacientes ( 43,7% ) apresentaram resultados positivos para dosagens de anticorpos ige antitoxinas estafiloccicas : enterotoxina ( tx ) a , em 29 ( 20,4% ) ; txb , em 35 ( 24,6% ) ; txc , em 33 ( 23,2% ) ; e toxic shock syndrome toxin ( tsst ) , em 45 ( 31,7% ) . as mdias das dosagens sricas de anticorpos ige especficos anti - txa , txb , txc e tsst foram , respectivamente , de 0,96 u / l , 1,09 u / l , 1,21 u / l , e 1,18 u / l .", "a presena de anticorpos ige sricos anti - txa , txb , txc e tsst , foi detectada em 43,7% nessa amostra de pacientes , mas no houve associao estatisticamente significativa entre seus resultados qualitativos ou quantitativos e gravidade clnica da asma .", "\n staphylococcus aureus is a gram - positive bacterium that can colonize the human skin and respiratory tract .", "the most important are toxic shock syndrome toxin ( tsst ) , staphylococcal enterotoxin a ( sea ) , seb , sec , sed , see , seg , seh , and sei , the activities of which include superantigen activity , pyrogenicity , and potentiation of lethality of other toxins . \n\n\n\n ", "the superantigen activity of staphylococcal toxins consists of direct stimulation of class ii mhc receptors and t cells , independently of antigen presentation by antigen - presenting cells , stimulating the proliferation and activity of cd4 and cd8 t lymphocytes .", "this mechanism is related to the worsening of allergic diseases by the production of staphylococcal toxin - specific ige antibodies , as well as by a direct effect on tissue mast cells , leading to their degranulation . \n\n\n\n  in asthma patients", ", staphylococcal toxins also act as superantigens , stimulating cd4 t lymphocyte proliferation and activity and leading to an increased production of staphylococcal toxin - specific ige antibodies , causing an allergic - type reaction by biding to mast cells in the respiratory tract .", "this reaction results in the release of mediators such as histamine , kinins , platelet - activating factor , and arachidonic acid metabolites ( prostaglandins and leukotrienes ) , as well as of chemokines , eliciting immediate and late inflammatory responses ( by the recruitment and activation of neutrophils and eosinophils ) and culminating in asthma worsening . \n\n\n\n ", "staphylococcal superantigens have been shown to play roles in atopic dermatitis , rhinosinusitis , and asthma , being correlated with their severity . \n\n\n  with regard to asthma , kowalski et al . found ige antibodies to sea , sec , and tsst in 89.7% of 237 asthma patients ( mean levels of 1.096  3.25 ku / l ) ; although there was no significant difference between those with severe asthma and those with non - severe asthma in terms of the prevalence of staphylococcal toxin - specific ige antibodies ( 81.4% vs. 69.2% ) , mean levels were higher in the former than in the latter ( 1.65  3.25 ku / l vs. 0.54  0.72 ku / l ) . \n  in another study ( n = 210 ) , the same authors obtained similar results , the prevalence of staphylococcal toxin - specific ige antibodies being 76.1% in patients with severe asthma and 71.1% in those with non - severe asthma , mean levels being three times higher in the former than in the latter . \n ", "bachert et al . found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe asthma when compared with those with mild asthma and controls ( n = 70 ) . \n  in a more recent study ( n = 387 ) , the same group of authors found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe uncontrolled asthma ( 59.6% ) when compared with those with controlled asthma ( 40.8% ) and controls ( 13.0% ) .", "high levels of staphylococcal toxin - specific ige antibodies have been found to be a risk factor for asthma ( or = 7.6 ) and severe asthma ( or = 11.09 ) . \n\n  in latin", "america , there have been no studies correlating staphylococcal superantigens with the severity of asthma .", "therefore , we investigated a population of asthma patients treated at a university hospital in the city of rio de janeiro , brazil , and having no risk factors for increased staphylococcal colonization or infection in order to correlate the clinical severity of asthma with the presence of staphylococcal toxin - specific ige antibodies and degree of ige - mediated sensitization .", "this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 ,", "consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria \n\n\n : the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group .   according to the global initiative for asthma", ", \n\n\n  asthma severity can be evaluated on the basis of the treatment required in order to control the disease .", "patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two .", "patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses .", "patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control .  ", "the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma , \n\n\n  regardless of the presence of rhinitis and positive skin test results to aeroallergens .  ", "the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent .  ", "the sample size calculation was based on a study by kowalski et al . \n  and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% ,", "the required sample size was calculated to be 140 ( 70 per group ) .  ", "procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) .", "spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines \n\n\n  and the reference values proposed by knudson et al . \n ", "a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma . \n\n\n  for pef measurement , a peak flow meter ( mini - wright afs ;", "clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg . \n ", "skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens . \n  for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used . \n ", ", we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables", ", we used the chi - square test or fisher 's exact test , as appropriate .", "the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant .  ", "the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital .", "all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision .", "this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 ,", "consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria \n\n\n : the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group .   according to the global initiative for asthma", ", \n\n\n  asthma severity can be evaluated on the basis of the treatment required in order to control the disease .", "patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two .", "patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses .", "patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control .  ", "the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma , \n\n\n  regardless of the presence of rhinitis and positive skin test results to aeroallergens .  ", "the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent .  ", "the sample size calculation was based on a study by kowalski et al . \n  and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% ,", "procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) . spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines \n\n\n  and the reference values proposed by knudson et al . \n ", "a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma . \n\n\n  for pef measurement , a peak flow meter ( mini - wright afs ;", "clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg . \n ", "skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens . \n  for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used . \n ", "in order to compare numerical variables , we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables", ", we used the chi - square test or fisher 's exact test , as appropriate .", "the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant .", "the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital .", "all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision .", "a total of 142 patients were studied . of those , 72 ( 17 with mild intermittent asthma and 55 with mild persistent asthma )", "were allocated to the ma group and 70 ( 53 with moderate asthma and 17 with severe asthma ) were allocated to the msa group .", "the median age was 52.5 years ( 46 years in the ma group and 56 years in the msa group ) , females and white individuals having predominated . in the sample as a whole , the mean body mass index ( bmi ) was 27.09 kg / m ,", "128 patients had rhinitis , 131 had positive skin test results to aeroallergens , and 99 had a family history of atopy . only 37 ( 26.1% ) had a history of smoking .", "mean percent predicted pef was 72.59% , mean percent predicted pre - bronchodilator fev1 was 71.55% , and mean percent predicted post - bronchodilator fev1 was 81.48% .", "mean eosinophil count was 4.4% , and mean total ige levels were 574.92 iu / ml .", "table 1 shows the distribution of sociodemographic and clinical variables , and table 2 shows lung function parameters and laboratory findings in the ma and msa groups .  \n", "student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) . \n ", "* student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) . \n", "table 2lung function parameters and laboratory findings in the study population , by asthma severity . \n", "student 's t - test or mann - whitney test . of the sample as a whole ,", "62 patients ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies : sea , in 29 ( 20.4% ) ; seb , in 35 ( 24.6% ) ; sec , in 33 ( 23.2% ) ; and tsst , in 45 ( 31.7% ) .", "the mean serum levels of ige antibodies to sea , seb , sec , and tsst were 0.96 u / l , 1.09 u / l , 1.21 u / l , and 1.18 u / l , respectively .  ", "as can be seen in tables 3 and \n , there were no statistically significant differences between the two groups regarding the frequency of ige - mediated sensitization and serum levels of staphylococcal toxin - specific ige antibodies .  \n", "table 3frequency of ige - mediated sensitization to staphylococcal toxins in the study population , by asthma severity .", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin .", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin .", "table 4serum levels of staphylococcal toxin - specific ige antibodies in the study population , by asthma severity .", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test . \n ", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test .", "there were statistically significant differences between the two groups of patients in the present study regarding their clinical and sociodemographic characteristics ( including age , bmi , and prevalence of rhinitis ) .", "the fact that the patients in the msa were significantly older than were those in the ma group might be due to the fact that asthma tends to be more severe in older individuals , especially those in whom the onset of asthma occurred at an older age . \n\n\n ", "our finding of a significantly higher bmi in the msa group is consistent with the literature , obesity having been reported to be a risk factor for and an aggravator of asthma .", "recent studies have established a relationship between obesity - induced changes in the gastrointestinal and respiratory microbiome and the etiopathogenesis of obesity - related asthma . \n\n\n\n\n ", "our finding of a higher prevalence of rhinitis in the ma group suggests that atopy was more common in the ma group than in the msa group , supporting the concept that atopic manifestations tend to be less common in asthma patients with disease that is more severe . \n\n\n\n  with regard to the lung function parameters assessed in the present study , absolute and percent predicted pef", "were significantly lower in the msa group , as were percent predicted pre - bronchodilator fev1 and percent predicted post - bronchodilator fev1 .", "these findings were expected and are consistent with the literature , showing the usual correlation between clinical severity and lung function parameters . \n\n\n\n  of the 142 patients studied , 62 ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies , ige antibodies to tsst being the most prevalent .", "our findings are different from those of two studies in the literature and similar to those of two other studies . in one study , kowalski et al . found an 89.7% prevalence of positivity for staphylococcal toxin - specific ige antibodies in severe and non - severe asthma patients ; in another study , they found a 76.1% prevalence in patients with severe refractory asthma and a 71.1% prevalence in patients with non - severe asthma . \n\n\n ", "in one study , bachert et al . found a 38.1% prevalence of positivity for staphylococcal toxin - specific ige antibodies in patients with asthma ( independently of disease severity ) and a 62% prevalence in patients with severe asthma ; in another study , they found a 59.6% prevalence in patients with severe uncontrolled asthma and a 40.8% prevalence in patients with controlled asthma , \n\n\n  the latter prevalence being closer to that found in the present study .  ", "aureus , such as sinonasal polyposis , bronchiectasis , chronic bronchitis , and atopic dermatitis , were not included in the present study . by facilitating colonization or infection with s .", "aureus , the aforementioned conditions can lead to increased quantities of staphylococcal toxins in the body , resulting in increased ige - mediated sensitization and , consequently , a heterogeneous population of asthma patients .", "the studies conducted by kowalski et al . \n\n\n  and bachert et al . \n\n\n  had no such exclusion criteria and included patients with chronic sinus disease and sinonasal polyposis , as was the case with the most recent study by kowalski et al . , \n  who nevertheless found no statistically significant differences between asthma patients with polyposis and those without regarding their levels of staphylococcal toxin - specific ige antibodies .", "this might explain the differences between our results and those obtained by the two aforementioned groups of authors regarding the degree of ige - mediated sensitization .", "aureus in the brazilian population ; therefore , it is currently impossible to determine whether or not it is lower than that in the european population .", "it is also impossible to determine whether the allergic immune response to staphylococcal toxins is lower in asthma patients in brazil than in those in other countries .   in the present study", ", there were no statistically significant differences between the two groups regarding the frequency of staphylococcal toxin - specific ige antibodies .", "our results are qualitatively similar to but quantitatively different from those obtained by kowalski et al . , \n\n\n  who found significantly higher levels of staphylococcal toxin - specific ige antibodies in patients with severe asthma .", "in addition , our results are qualitatively and quantitatively different from those obtained by bachert et al . \n\n\n ", "these differences are also due to the exclusion criteria used in the present study , which were not used in any of the aforementioned studies , and to specific characteristics of our study population , as previously mentioned .   in the present study , ige antibodies to sea , seb , sec , and tsst", "were found in 62 ( 43.7% ) of the 142 asthma patients analyzed , and neither the frequency of staphylococcal toxin - specific ige antibodies nor the serum levels of those antibodies were associated with the clinical severity of asthma .", "these results are extremely relevant because this is the first study on this topic in latin america , the results of which differed from those of previous studies conducted in europe , indicating a \" negative \" association", ".   the limitations of the present study lie in the fact that it was a single - center study conducted at a tertiary care university hospital , in a single demographic area of brazil .", "our primary objective was to evaluate the influence of serum levels of staphylococcal toxin - specific ige antibodies and their association with asthma severity , meaning that this was not a population prevalence study of ige sensitization in asthma patients and healthy individuals in our region . if that had been the case , a much larger sample size would have been required , and it would have been impossible to obtain that in a single - center study .", "therefore , in the present study , each group served as the control group for the other , without the use of a third group , comprising healthy individuals .  ", "multicenter studies in brazil and other latin american countries are needed in order to determine more accurately the role of ige - mediated sensitization to staphylococcal toxins as an aggravator of asthma , as well as to determine its prevalence in asthma patients and healthy individuals .", "such studies should include asthma patients with and without diseases that can lead to increased colonization or infection with s ."], "abstract_text": ["<S> abstractobjective : to determine the presence of staphylococcal superantigen - specific ige antibodies and degree of ige - mediated sensitization , as well as whether or not those are associated with the severity of asthma in adult patients . </S>", "<S> methods : this was a cross - sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of rio de janeiro , brazil . </S>", "<S> consecutive patients were divided into two groups according to the severity of asthma based on the global initiative for asthma criteria : mild asthma ( ma ) , comprising patients with mild intermittent or persistent asthma ; and moderate or severe asthma ( msa ) . </S>", "<S> we determined the serum levels of staphylococcal toxin - specific ige antibodies , comparing the results and performing a statistical analysis . </S>", "<S> results : the study included 142 patients : 72 in the ma group ( median age = 46 years ; 59 females ) and 70 in the msa group ( median age = 56 years ; 60 females ) . in the sample as a whole , 62 patients ( 43.7% ) presented positive results for staphylococcal toxin - specific ige antibodies : staphylococcal enterotoxin a ( sea ) , in 29 ( 20.4% ) ; seb , in 35 ( 24.6% ) ; sec , in 33 ( 23.2% ) ; and toxic shock syndrome toxin ( tsst ) , in 45 ( 31.7% ) . </S>", "<S> the mean serum levels of ige antibodies to sea , seb , sec , and tsst were 0.96 u / l , 1.09 u / l , 1.21 u / l , and 1.18 u / l , respectively . </S>", "<S> there were no statistically significant differences between the two groups in terms of the qualitative or quantitative results . </S>", "<S> conclusions : serum ige antibodies to sea , seb , sec , and tsst were detected in 43.7% of the patients in our sample . </S>", "<S> however , neither the qualitative nor quantitative results showed a statistically significant association with the clinical severity of asthma . </S>"], "labels": null, "section_names": ["Objetivo:", "Mtodos:", "Resultados:", "Concluses:", "INTRODUCTION", "METHODS", " Patients ", " Procedures ", " Statistical analysis ", " Ethical aspects ", "RESULTS", "DISCUSSION"], "sections": [["determinar a presena de anticorpos ige especficos para superantgenos estafiloccicos e o grau de sensibilizao mediada por esses , assim como se esses esto associados  gravidade da asma em pacientes adultos ."], ["estudo transversal incluindo asmticos adultos em acompanhamento ambulatorial em um hospital universitrio tercirio no rio de janeiro ( rj ) .", "os pacientes foram alocados consecutivamente em dois grupos de gravidade da asma segundo critrios da global initiative for asthma : asma leve ( al ) , com asmticos leves intermitentes ou persistentes , e asma moderada ou grave ( amg ) .", "foram determinados os nveis sricos de anticorpos ige antitoxinas estafiloccicas , e os resultados foram comparados por anlise estatstica ."], ["foram includos 142 pacientes no estudo : 72 no grupo al ( mediana de idade = 46 anos ; 59 do sexo feminino ) e 70 do grupo amg ( mediana de idade = 56 anos ; 60 do sexo feminino ) .", "na amostra geral , 62 pacientes ( 43,7% ) apresentaram resultados positivos para dosagens de anticorpos ige antitoxinas estafiloccicas : enterotoxina ( tx ) a , em 29 ( 20,4% ) ; txb , em 35 ( 24,6% ) ; txc , em 33 ( 23,2% ) ; e toxic shock syndrome toxin ( tsst ) , em 45 ( 31,7% ) . as mdias das dosagens sricas de anticorpos ige especficos anti - txa , txb , txc e tsst foram , respectivamente , de 0,96 u / l , 1,09 u / l , 1,21 u / l , e 1,18 u / l ."], ["a presena de anticorpos ige sricos anti - txa , txb , txc e tsst , foi detectada em 43,7% nessa amostra de pacientes , mas no houve associao estatisticamente significativa entre seus resultados qualitativos ou quantitativos e gravidade clnica da asma ."], ["\n staphylococcus aureus is a gram - positive bacterium that can colonize the human skin and respiratory tract .", "the most important are toxic shock syndrome toxin ( tsst ) , staphylococcal enterotoxin a ( sea ) , seb , sec , sed , see , seg , seh , and sei , the activities of which include superantigen activity , pyrogenicity , and potentiation of lethality of other toxins . \n\n\n\n ", "the superantigen activity of staphylococcal toxins consists of direct stimulation of class ii mhc receptors and t cells , independently of antigen presentation by antigen - presenting cells , stimulating the proliferation and activity of cd4 and cd8 t lymphocytes .", "this mechanism is related to the worsening of allergic diseases by the production of staphylococcal toxin - specific ige antibodies , as well as by a direct effect on tissue mast cells , leading to their degranulation . \n\n\n\n  in asthma patients", ", staphylococcal toxins also act as superantigens , stimulating cd4 t lymphocyte proliferation and activity and leading to an increased production of staphylococcal toxin - specific ige antibodies , causing an allergic - type reaction by biding to mast cells in the respiratory tract .", "this reaction results in the release of mediators such as histamine , kinins , platelet - activating factor , and arachidonic acid metabolites ( prostaglandins and leukotrienes ) , as well as of chemokines , eliciting immediate and late inflammatory responses ( by the recruitment and activation of neutrophils and eosinophils ) and culminating in asthma worsening . \n\n\n\n ", "staphylococcal superantigens have been shown to play roles in atopic dermatitis , rhinosinusitis , and asthma , being correlated with their severity . \n\n\n  with regard to asthma , kowalski et al . found ige antibodies to sea , sec , and tsst in 89.7% of 237 asthma patients ( mean levels of 1.096  3.25 ku / l ) ; although there was no significant difference between those with severe asthma and those with non - severe asthma in terms of the prevalence of staphylococcal toxin - specific ige antibodies ( 81.4% vs. 69.2% ) , mean levels were higher in the former than in the latter ( 1.65  3.25 ku / l vs. 0.54  0.72 ku / l ) . \n  in another study ( n = 210 ) , the same authors obtained similar results , the prevalence of staphylococcal toxin - specific ige antibodies being 76.1% in patients with severe asthma and 71.1% in those with non - severe asthma , mean levels being three times higher in the former than in the latter . \n ", "bachert et al . found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe asthma when compared with those with mild asthma and controls ( n = 70 ) . \n  in a more recent study ( n = 387 ) , the same group of authors found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe uncontrolled asthma ( 59.6% ) when compared with those with controlled asthma ( 40.8% ) and controls ( 13.0% ) .", "high levels of staphylococcal toxin - specific ige antibodies have been found to be a risk factor for asthma ( or = 7.6 ) and severe asthma ( or = 11.09 ) . \n\n  in latin", "america , there have been no studies correlating staphylococcal superantigens with the severity of asthma .", "therefore , we investigated a population of asthma patients treated at a university hospital in the city of rio de janeiro , brazil , and having no risk factors for increased staphylococcal colonization or infection in order to correlate the clinical severity of asthma with the presence of staphylococcal toxin - specific ige antibodies and degree of ige - mediated sensitization ."], ["this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 ,", "consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria \n\n\n : the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group .   according to the global initiative for asthma", ", \n\n\n  asthma severity can be evaluated on the basis of the treatment required in order to control the disease .", "patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two .", "patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses .", "patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control .  ", "the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma , \n\n\n  regardless of the presence of rhinitis and positive skin test results to aeroallergens .  ", "the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent .  ", "the sample size calculation was based on a study by kowalski et al . \n  and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% ,", "the required sample size was calculated to be 140 ( 70 per group ) .  ", "procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) .", "spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines \n\n\n  and the reference values proposed by knudson et al . \n ", "a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma . \n\n\n  for pef measurement , a peak flow meter ( mini - wright afs ;", "clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg . \n ", "skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens . \n  for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used . \n ", ", we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables", ", we used the chi - square test or fisher 's exact test , as appropriate .", "the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant .  ", "the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital .", "all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision ."], ["this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 ,", "consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria \n\n\n : the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group .   according to the global initiative for asthma", ", \n\n\n  asthma severity can be evaluated on the basis of the treatment required in order to control the disease .", "patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two .", "patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses .", "patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control .  ", "the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma , \n\n\n  regardless of the presence of rhinitis and positive skin test results to aeroallergens .  ", "the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent .  ", "the sample size calculation was based on a study by kowalski et al . \n  and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% ,"], ["procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) . spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines \n\n\n  and the reference values proposed by knudson et al . \n ", "a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma . \n\n\n  for pef measurement , a peak flow meter ( mini - wright afs ;", "clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg . \n ", "skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens . \n  for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used . \n "], ["in order to compare numerical variables , we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables", ", we used the chi - square test or fisher 's exact test , as appropriate .", "the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant ."], ["the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital .", "all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision ."], ["a total of 142 patients were studied . of those , 72 ( 17 with mild intermittent asthma and 55 with mild persistent asthma )", "were allocated to the ma group and 70 ( 53 with moderate asthma and 17 with severe asthma ) were allocated to the msa group .", "the median age was 52.5 years ( 46 years in the ma group and 56 years in the msa group ) , females and white individuals having predominated . in the sample as a whole , the mean body mass index ( bmi ) was 27.09 kg / m ,", "128 patients had rhinitis , 131 had positive skin test results to aeroallergens , and 99 had a family history of atopy . only 37 ( 26.1% ) had a history of smoking .", "mean percent predicted pef was 72.59% , mean percent predicted pre - bronchodilator fev1 was 71.55% , and mean percent predicted post - bronchodilator fev1 was 81.48% .", "mean eosinophil count was 4.4% , and mean total ige levels were 574.92 iu / ml .", "table 1 shows the distribution of sociodemographic and clinical variables , and table 2 shows lung function parameters and laboratory findings in the ma and msa groups .  \n", "student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) . \n ", "* student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) . \n", "table 2lung function parameters and laboratory findings in the study population , by asthma severity . \n", "student 's t - test or mann - whitney test . of the sample as a whole ,", "62 patients ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies : sea , in 29 ( 20.4% ) ; seb , in 35 ( 24.6% ) ; sec , in 33 ( 23.2% ) ; and tsst , in 45 ( 31.7% ) .", "the mean serum levels of ige antibodies to sea , seb , sec , and tsst were 0.96 u / l , 1.09 u / l , 1.21 u / l , and 1.18 u / l , respectively .  ", "as can be seen in tables 3 and \n , there were no statistically significant differences between the two groups regarding the frequency of ige - mediated sensitization and serum levels of staphylococcal toxin - specific ige antibodies .  \n", "table 3frequency of ige - mediated sensitization to staphylococcal toxins in the study population , by asthma severity .", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin .", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin .", "table 4serum levels of staphylococcal toxin - specific ige antibodies in the study population , by asthma severity .", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test . \n ", "sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test ."], ["there were statistically significant differences between the two groups of patients in the present study regarding their clinical and sociodemographic characteristics ( including age , bmi , and prevalence of rhinitis ) .", "the fact that the patients in the msa were significantly older than were those in the ma group might be due to the fact that asthma tends to be more severe in older individuals , especially those in whom the onset of asthma occurred at an older age . \n\n\n ", "our finding of a significantly higher bmi in the msa group is consistent with the literature , obesity having been reported to be a risk factor for and an aggravator of asthma .", "recent studies have established a relationship between obesity - induced changes in the gastrointestinal and respiratory microbiome and the etiopathogenesis of obesity - related asthma . \n\n\n\n\n ", "our finding of a higher prevalence of rhinitis in the ma group suggests that atopy was more common in the ma group than in the msa group , supporting the concept that atopic manifestations tend to be less common in asthma patients with disease that is more severe . \n\n\n\n  with regard to the lung function parameters assessed in the present study , absolute and percent predicted pef", "were significantly lower in the msa group , as were percent predicted pre - bronchodilator fev1 and percent predicted post - bronchodilator fev1 .", "these findings were expected and are consistent with the literature , showing the usual correlation between clinical severity and lung function parameters . \n\n\n\n  of the 142 patients studied , 62 ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies , ige antibodies to tsst being the most prevalent .", "our findings are different from those of two studies in the literature and similar to those of two other studies . in one study , kowalski et al . found an 89.7% prevalence of positivity for staphylococcal toxin - specific ige antibodies in severe and non - severe asthma patients ; in another study , they found a 76.1% prevalence in patients with severe refractory asthma and a 71.1% prevalence in patients with non - severe asthma . \n\n\n ", "in one study , bachert et al . found a 38.1% prevalence of positivity for staphylococcal toxin - specific ige antibodies in patients with asthma ( independently of disease severity ) and a 62% prevalence in patients with severe asthma ; in another study , they found a 59.6% prevalence in patients with severe uncontrolled asthma and a 40.8% prevalence in patients with controlled asthma , \n\n\n  the latter prevalence being closer to that found in the present study .  ", "aureus , such as sinonasal polyposis , bronchiectasis , chronic bronchitis , and atopic dermatitis , were not included in the present study . by facilitating colonization or infection with s .", "aureus , the aforementioned conditions can lead to increased quantities of staphylococcal toxins in the body , resulting in increased ige - mediated sensitization and , consequently , a heterogeneous population of asthma patients .", "the studies conducted by kowalski et al . \n\n\n  and bachert et al . \n\n\n  had no such exclusion criteria and included patients with chronic sinus disease and sinonasal polyposis , as was the case with the most recent study by kowalski et al . , \n  who nevertheless found no statistically significant differences between asthma patients with polyposis and those without regarding their levels of staphylococcal toxin - specific ige antibodies .", "this might explain the differences between our results and those obtained by the two aforementioned groups of authors regarding the degree of ige - mediated sensitization .", "aureus in the brazilian population ; therefore , it is currently impossible to determine whether or not it is lower than that in the european population .", "it is also impossible to determine whether the allergic immune response to staphylococcal toxins is lower in asthma patients in brazil than in those in other countries .   in the present study", ", there were no statistically significant differences between the two groups regarding the frequency of staphylococcal toxin - specific ige antibodies .", "our results are qualitatively similar to but quantitatively different from those obtained by kowalski et al . , \n\n\n  who found significantly higher levels of staphylococcal toxin - specific ige antibodies in patients with severe asthma .", "in addition , our results are qualitatively and quantitatively different from those obtained by bachert et al . \n\n\n ", "these differences are also due to the exclusion criteria used in the present study , which were not used in any of the aforementioned studies , and to specific characteristics of our study population , as previously mentioned .   in the present study , ige antibodies to sea , seb , sec , and tsst", "were found in 62 ( 43.7% ) of the 142 asthma patients analyzed , and neither the frequency of staphylococcal toxin - specific ige antibodies nor the serum levels of those antibodies were associated with the clinical severity of asthma .", "these results are extremely relevant because this is the first study on this topic in latin america , the results of which differed from those of previous studies conducted in europe , indicating a \" negative \" association", ".   the limitations of the present study lie in the fact that it was a single - center study conducted at a tertiary care university hospital , in a single demographic area of brazil .", "our primary objective was to evaluate the influence of serum levels of staphylococcal toxin - specific ige antibodies and their association with asthma severity , meaning that this was not a population prevalence study of ige sensitization in asthma patients and healthy individuals in our region . if that had been the case , a much larger sample size would have been required , and it would have been impossible to obtain that in a single - center study .", "therefore , in the present study , each group served as the control group for the other , without the use of a third group , comprising healthy individuals .  ", "multicenter studies in brazil and other latin american countries are needed in order to determine more accurately the role of ige - mediated sensitization to staphylococcal toxins as an aggravator of asthma , as well as to determine its prevalence in asthma patients and healthy individuals .", "such studies should include asthma patients with and without diseases that can lead to increased colonization or infection with s ."]]}
{"article_id": "PMC3702150", "article_text": ["the family is the cornerstone of human social support network and its presence is essential in everyone s life . changes inevitably occur in families with illness and hospitalization of a family member . in other words , among the sources of stress for families are accidents leading to hospitalization particularly intensive care unit ( icu ) .", "statistics show that 8% of hospital beds in the united states are occupied by the intensive care units .", "stress in the family while the patient is in the icu can disrupt the harmony power of the family members and finally , it may causes disturbances in the support of the patient .", "in addition to the various sources of stress in intensive care units such as the patient s fear of death , financial problems , lack of awareness about the environment and etc . , their satisfaction level is another important source of stress for the patient s family .", "today , the family needs of hospitalized patients in the icu are summarized in five sections .", "these factors may include receiving assurance , staying close to the patient , receiving information , feeling comfortable and receiving support .", "however , in many cases , the patient s family needs and their expectations in the icu will not be fulfilled which will cause dissatisfaction . since , how families deal with mental stress is the most important components of comprehensive care , it should be known that attention to the family needs of the patients hospitalized in intensive care units could increase their satisfaction and thus a reduction in stress disorders .", "studies showed that the nurses in particular sections focus primarily on the patient and the disease .", "therefore , attention to the families should be considered as an important part in patient management .", "effective interventions targeted at the needs and expectations of family members should help to reduce the stress and improve their satisfaction .", "fox et al . stated that the needs of the family members were not often overlooked by the nurses .", "however , the nurses spend a lot of time with the patients and their families and are in a good position to assess their needs and plan for meeting these needs with appropriate interventions .", "the reason of investigating the satisfaction of the families of the patients in the icu is that the patients are facing life - threatening illness experiences and complex treatments , different technologies and different equipment are used for them .", "these cases could potentially lead to the dissatisfaction of their families . in the meantime ,", "the families of trauma and neurosurgical patients are more vulnerable than the other groups of patients .", "they need supports that are more emotional and should be accepted by the icu staff . today , there are many theories expressed that in intensive care units , the patient and his family should be considered as a single unit . by providing care to this unit ,", "the aim of this study was to analyze the satisfaction of the families of icu patients .", "the tool of society of critical care medicine s family needs assessment ( sccmfna ) ( which has been validated in 1998 by johnson ) was used in that study .", "the results showed that most of the family satisfaction was related to the communication and patient care and the lowest level of satisfaction was about the ability of staff to patients family comfort .", "bailey et al . have conducted a study in 2009 with the purpose of investigating the relationship between informational support to families of special care patients and their anxiety and satisfaction .", "they showed that there is a direct link between the informational support and their satisfaction .", "the study of fumis et al . in 2008 also showed that the increase in the availability of physicians for giving the information to the patient s family and as well as nurses efforts to provide understandable explanations about the patient s condition to them will increase the patient s family satisfaction .", "this clinical trial was performed with respect to the existing gap for a study carried out for investigating the interventions for the satisfaction of the patients in the special care units and their families in iran .", "the purpose of the study was to determine the effectiveness of nursing interventions based on family needs on family satisfaction level of hospitalized patients in the neurosurgery intensive care unit of al - zahra hospital in 2010 .", "the statistical research community was the families of hospitalized patients in neurosurgery intensive care unit of al - zahra ( sa ) hospital , isfahan , iran from may to september 2010 .", "with respect to inclusion criteria , the families of 64 patients were selected with simple sampling . after obtaining the informed consents from the hospitalized patients , by using the envelopes , which were prepaid by using a table of random numbers , they were divided into two groups ( intervention and control ) .", "the inclusion criteria was included as the following cases : to be a first - degree member of the family , to be older than 18 years , willing to participate in the study , having the power to speak and write in farsi , to be hospitalized one day before the study , lack of physical or mental problem in the family member , no responsibility for the care of another patient , introduced as the first person responsible for the patient care .", "obtaining the written informed consent , completion of the questionnaires about demographic information and the families satisfaction were performed in the second day of hospitalization of the patients in icu in both groups .", "johnson questionnaire for the satisfaction of the family of icu patients was used to measure the family satisfaction .", "the rating of the questionnaire was performed by using the likert scale with four options of 0 to 4 as follows : almost all the time ( 3 scores ) , often ( 2 scores ) , only occasionally ( 1 score ) and never ( zero score ) .", "johnson s questionnaire was the modified type of multer patient s family needs , which was evaluated in various scientific researches and there are extensive evidences for its validity .", "the interventions were performed in the second and the third day of hospitalization of the patient in the neurosurgery icu by the researcher in the morning shift for the intervention group .", "thus , the researcher provided understandable explanations about the disease to the patient s family and answered honestly to the questions about the patient and the disease .", "the researcher ensured them for providing the best care in the section and spoke about the prognosis of the disease .", "the researcher took the family member to the patient s room and gave the necessary information about the room space , equipment , personnel departments and the actions that were done for the patient .", "the patient family member was contacted if there was a change in the patient condition .", "they let the family help the patient in some cases of the public health ( such as hand and foot massage and giving food ) .", "locations of rest , nutrition , prayer room , bathroom and buffet of the hospital were introduced to them .", "the telephone numbers of hospitals and wards ( and if necessary , the social worker and supervisor ) were given .", "various departments and personnel of the hospital were taught to accept the families and communicate with them . the hospital social worker was introduced and if necessary , the patient s family was allowed to express their feelings .", "these interventions were performed in the evening shift of the second and the third day of the patient reception .", "the actions were carried out by the fellow researcher . while , for the control group , only the routine work was done . on the fourth day , the satisfaction survey was completed by the selected family member in both of the intervention and control groups .", "statistical methods used in this study were 2 test , man - whitney , independent student s t - test and paired t - tests .", "the findings did not show statistically significant difference in the demographic characteristics of the intervention and control groups , such as : age ( p < 0.99 ) , gender ( p < 0.79 ) , marital status ( p < 0.41 ) , educational level ( p < 0.12 ) and relation with patient ( p < 0.54 ) . as shown in table 1", ", there was no significant difference in the mean satisfaction score before the intervention in the intervention and control groups ( p > 0.05 ) .", "the mean satisfaction score after the intervention in the intervention group was significantly more than the control group ( p < 0.001 ) .", "the mean satisfaction score in the intervention group after the intervention was significantly higher than before the intervention ( p < 0.001 ) .", "there was no significant difference in the mean satisfaction score in the control group before and after the intervention ( p > 0.05 ) ( table 2 ) .", "comparison of mean satisfaction score ( 100 * ) of participants in the intervention and control groups the mean of satisfaction score changes of the studied subjects in the intervention and control groups after intervention", "the results of the present study showed that the nursing interventions based on the family needs increased the patient s family satisfaction in the neurosurgery intensive care unit of al - zahra hospital .", "due to the differences in the study design , study population , the number of samples and methods of intervention , it would be difficult to compare the results . in chien et al .", "study , the results showed that performing the training based on the patients family needs in the icu , decreased the anxiety and increased their satisfaction . in the study of myhren et al . indicated that effective communication of the staff with the families of icu patients would increase their satisfaction .", "in the study by rosher and robinson , it showed that involving the families in patient care led to increase their satisfaction than before the intervention .", "the mentioned results were consistent with the results of this study , because all of these interventions were some parts of the interventions performed in this study .", "however , the study of steele et al . indicated that clinical training had no impact on the family satisfaction of the patient hospitalized in the icu", ". therefore , there was no statistically significant difference in the satisfaction level in the control and intervention group .", "in this study , it was shown that the use of nursing interventions based on family needs ( confidence , support , information , proximity and convenience ) had significant impact on the family satisfaction of the patient hospitalized in intensive care unit .", "they should be based on the family investigation and recognition and their priority needs should be determined in order to cause the greatest satisfaction .", "the icu nurses must also accept their role in the care of patient s family members .", "the findings of this study could be a basis for performing further studies about family needs of the patients in the icu especially with the different forms of culture and economy .", "consequently , the increase in family satisfaction of patients can reduce the stress disorders and improve their mental state and ultimately better support of the patient by the family .", "this study only examined the effect of nursing interventions in the first days of hospitalization on the satisfaction of the families but the effects on the patient s entire hospitalization period has not been assessed . by performing other researches in this field", ", it would be possible to compare the differences in the effects of nursing interventions based on the needs of families in the early days and the whole period of the patient s hospitalization ."], "abstract_text": ["<S> background : since the family is a social system , the impairment in each of its component members may disrupt the entire family system . </S>", "<S> one of the stress sources for families is accidents leading to hospitalization particularly in the intensive care unit ( icu ) . in many cases , </S>", "<S> the families needs in patient care are not met that cause dissatisfaction . since the nurses spend a lot of time with patients and their families , they are in a good position to assess their needs and perform appropriate interventions . </S>", "<S> therefore , this study was conducted to determine the effectiveness of nursing interventions based on family needs on family satisfaction level of hospitalized patients in the neurosurgery icu.materials and methods : this clinical trial was conducted in the neurosurgery icu of al - zahra hospital , isfahan , iran in 2010 . </S>", "<S> sixty four families were selected by simple sampling method and were randomly placed in two groups ( test and control ) using envelopes . in the test group , </S>", "<S> some interventions were performed to meet their needs . in the control group , </S>", "<S> the routine actions were only carried out . </S>", "<S> the satisfaction questionnaire was completed by both groups two days after admission and again on the fourth day.findings:both of the intervention and control groups were compared in terms of the mean satisfaction scores before and after intervention . </S>", "<S> there was no significant difference in mean satisfaction scores between test and control groups before the intervention . </S>", "<S> the mean satisfaction score significantly increased after the intervention compared to the control group.conclusions:nursing interventions based on family needs of hospitalized patients in the icu increase their satisfaction . </S>", "<S> attention to family nursing should be planned especially in the icus . </S>"], "labels": null, "section_names": ["I", "M", "F", "D", "C"], "sections": [["the family is the cornerstone of human social support network and its presence is essential in everyone s life . changes inevitably occur in families with illness and hospitalization of a family member . in other words , among the sources of stress for families are accidents leading to hospitalization particularly intensive care unit ( icu ) .", "statistics show that 8% of hospital beds in the united states are occupied by the intensive care units .", "stress in the family while the patient is in the icu can disrupt the harmony power of the family members and finally , it may causes disturbances in the support of the patient .", "in addition to the various sources of stress in intensive care units such as the patient s fear of death , financial problems , lack of awareness about the environment and etc . , their satisfaction level is another important source of stress for the patient s family .", "today , the family needs of hospitalized patients in the icu are summarized in five sections .", "these factors may include receiving assurance , staying close to the patient , receiving information , feeling comfortable and receiving support .", "however , in many cases , the patient s family needs and their expectations in the icu will not be fulfilled which will cause dissatisfaction . since , how families deal with mental stress is the most important components of comprehensive care , it should be known that attention to the family needs of the patients hospitalized in intensive care units could increase their satisfaction and thus a reduction in stress disorders .", "studies showed that the nurses in particular sections focus primarily on the patient and the disease .", "therefore , attention to the families should be considered as an important part in patient management .", "effective interventions targeted at the needs and expectations of family members should help to reduce the stress and improve their satisfaction .", "fox et al . stated that the needs of the family members were not often overlooked by the nurses .", "however , the nurses spend a lot of time with the patients and their families and are in a good position to assess their needs and plan for meeting these needs with appropriate interventions .", "the reason of investigating the satisfaction of the families of the patients in the icu is that the patients are facing life - threatening illness experiences and complex treatments , different technologies and different equipment are used for them .", "these cases could potentially lead to the dissatisfaction of their families . in the meantime ,", "the families of trauma and neurosurgical patients are more vulnerable than the other groups of patients .", "they need supports that are more emotional and should be accepted by the icu staff . today , there are many theories expressed that in intensive care units , the patient and his family should be considered as a single unit . by providing care to this unit ,", "the aim of this study was to analyze the satisfaction of the families of icu patients .", "the tool of society of critical care medicine s family needs assessment ( sccmfna ) ( which has been validated in 1998 by johnson ) was used in that study .", "the results showed that most of the family satisfaction was related to the communication and patient care and the lowest level of satisfaction was about the ability of staff to patients family comfort .", "bailey et al . have conducted a study in 2009 with the purpose of investigating the relationship between informational support to families of special care patients and their anxiety and satisfaction .", "they showed that there is a direct link between the informational support and their satisfaction .", "the study of fumis et al . in 2008 also showed that the increase in the availability of physicians for giving the information to the patient s family and as well as nurses efforts to provide understandable explanations about the patient s condition to them will increase the patient s family satisfaction .", "this clinical trial was performed with respect to the existing gap for a study carried out for investigating the interventions for the satisfaction of the patients in the special care units and their families in iran .", "the purpose of the study was to determine the effectiveness of nursing interventions based on family needs on family satisfaction level of hospitalized patients in the neurosurgery intensive care unit of al - zahra hospital in 2010 ."], ["the statistical research community was the families of hospitalized patients in neurosurgery intensive care unit of al - zahra ( sa ) hospital , isfahan , iran from may to september 2010 .", "with respect to inclusion criteria , the families of 64 patients were selected with simple sampling . after obtaining the informed consents from the hospitalized patients , by using the envelopes , which were prepaid by using a table of random numbers , they were divided into two groups ( intervention and control ) .", "the inclusion criteria was included as the following cases : to be a first - degree member of the family , to be older than 18 years , willing to participate in the study , having the power to speak and write in farsi , to be hospitalized one day before the study , lack of physical or mental problem in the family member , no responsibility for the care of another patient , introduced as the first person responsible for the patient care .", "obtaining the written informed consent , completion of the questionnaires about demographic information and the families satisfaction were performed in the second day of hospitalization of the patients in icu in both groups .", "johnson questionnaire for the satisfaction of the family of icu patients was used to measure the family satisfaction .", "the rating of the questionnaire was performed by using the likert scale with four options of 0 to 4 as follows : almost all the time ( 3 scores ) , often ( 2 scores ) , only occasionally ( 1 score ) and never ( zero score ) .", "johnson s questionnaire was the modified type of multer patient s family needs , which was evaluated in various scientific researches and there are extensive evidences for its validity .", "the interventions were performed in the second and the third day of hospitalization of the patient in the neurosurgery icu by the researcher in the morning shift for the intervention group .", "thus , the researcher provided understandable explanations about the disease to the patient s family and answered honestly to the questions about the patient and the disease .", "the researcher ensured them for providing the best care in the section and spoke about the prognosis of the disease .", "the researcher took the family member to the patient s room and gave the necessary information about the room space , equipment , personnel departments and the actions that were done for the patient .", "the patient family member was contacted if there was a change in the patient condition .", "they let the family help the patient in some cases of the public health ( such as hand and foot massage and giving food ) .", "locations of rest , nutrition , prayer room , bathroom and buffet of the hospital were introduced to them .", "the telephone numbers of hospitals and wards ( and if necessary , the social worker and supervisor ) were given .", "various departments and personnel of the hospital were taught to accept the families and communicate with them . the hospital social worker was introduced and if necessary , the patient s family was allowed to express their feelings .", "these interventions were performed in the evening shift of the second and the third day of the patient reception .", "the actions were carried out by the fellow researcher . while , for the control group , only the routine work was done . on the fourth day , the satisfaction survey was completed by the selected family member in both of the intervention and control groups .", "statistical methods used in this study were 2 test , man - whitney , independent student s t - test and paired t - tests ."], ["the findings did not show statistically significant difference in the demographic characteristics of the intervention and control groups , such as : age ( p < 0.99 ) , gender ( p < 0.79 ) , marital status ( p < 0.41 ) , educational level ( p < 0.12 ) and relation with patient ( p < 0.54 ) . as shown in table 1", ", there was no significant difference in the mean satisfaction score before the intervention in the intervention and control groups ( p > 0.05 ) .", "the mean satisfaction score after the intervention in the intervention group was significantly more than the control group ( p < 0.001 ) .", "the mean satisfaction score in the intervention group after the intervention was significantly higher than before the intervention ( p < 0.001 ) .", "there was no significant difference in the mean satisfaction score in the control group before and after the intervention ( p > 0.05 ) ( table 2 ) .", "comparison of mean satisfaction score ( 100 * ) of participants in the intervention and control groups the mean of satisfaction score changes of the studied subjects in the intervention and control groups after intervention"], ["the results of the present study showed that the nursing interventions based on the family needs increased the patient s family satisfaction in the neurosurgery intensive care unit of al - zahra hospital .", "due to the differences in the study design , study population , the number of samples and methods of intervention , it would be difficult to compare the results . in chien et al .", "study , the results showed that performing the training based on the patients family needs in the icu , decreased the anxiety and increased their satisfaction . in the study of myhren et al . indicated that effective communication of the staff with the families of icu patients would increase their satisfaction .", "in the study by rosher and robinson , it showed that involving the families in patient care led to increase their satisfaction than before the intervention .", "the mentioned results were consistent with the results of this study , because all of these interventions were some parts of the interventions performed in this study .", "however , the study of steele et al . indicated that clinical training had no impact on the family satisfaction of the patient hospitalized in the icu", ". therefore , there was no statistically significant difference in the satisfaction level in the control and intervention group ."], ["in this study , it was shown that the use of nursing interventions based on family needs ( confidence , support , information , proximity and convenience ) had significant impact on the family satisfaction of the patient hospitalized in intensive care unit .", "they should be based on the family investigation and recognition and their priority needs should be determined in order to cause the greatest satisfaction .", "the icu nurses must also accept their role in the care of patient s family members .", "the findings of this study could be a basis for performing further studies about family needs of the patients in the icu especially with the different forms of culture and economy .", "consequently , the increase in family satisfaction of patients can reduce the stress disorders and improve their mental state and ultimately better support of the patient by the family .", "this study only examined the effect of nursing interventions in the first days of hospitalization on the satisfaction of the families but the effects on the patient s entire hospitalization period has not been assessed . by performing other researches in this field", ", it would be possible to compare the differences in the effects of nursing interventions based on the needs of families in the early days and the whole period of the patient s hospitalization ."]]}
{"article_id": "PMC4262794", "article_text": ["development of human societies and industrialization as well as changes in stress sources has changed disease pattern in civilized societies . as a result , the disease pattern has changed from traditional diseases such as infectious diseases and malnutrition to diseases such as heart disease , diabetes , accidents and so forth . among them", "coronary artery disease is the most cardiovascular disorder as a health problem in developing and developed countries .", "in fact , this is not only a chronic disease associated with high mortality , but it causes limitations in life and disability in a large part of the productive forces of the country .", "it is also associated with reduced production and increased medical costs [ 3 - 5 ] . nowadays ,", "cardiovascular diseases are among the most widespread chronic diseases in most countries . according to forecasts ,", "the mortality rate in eastern mediterranean countries including iran will be 30 - 35% [ 6 , 7 ] . despite the emphasis on prevention and development of newtreatments , surgery is the only choice for many patients with cardiovascular disease .", "one of the surgical procedures for the treatment of cardiovascular patients is coronary artery bypass surgery .", "in fact , heart surgery significantly influences on the quality of life of patients with cardiovascular diseases .", "hence , heart surgery is an important event in the lives of patients causing collapse of economic , professional and personal life . on the other hand , each surgical procedure", "is associated with several psychological complications for patients . of the most important complications are anxiety and depression so that approximately 65% of cardiovascular patients experience them after surgical and medical interventions .", "in fact , anxiety and depression complicate the treatment process [ 7 , 8 ] .", "some scientists believe that anxiety is one of the essential elements of human life . however , acute and long - term anxiety is integral part of all psychological diseases . according to nemati and colleagues ,", "in fact , anxiety is a common psychological response of cardiovascular patients which is associated with reduced quality of life and psychological morbidity [ 11 , 12 ] . on the other hand ,", "anxiety causes increased heart rate and breathing as well as high blood pressure and even mortality .", "it is associated with increased risk of mortality , disability , increased medical care and functional impairment in daily activities [ 12 , 13 ] . clinical depression has been reported in 54% of patients after bypass surgery . according to world health organization ( who ) , depression is the fourth chronic disease where its disability is comparable with 8 major chronic disorders .", "the outcome of depression in patients with cardiovascular disease is deterioration of physical and emotional state . given the incidence of such complications in patients with cardiovascular disease", "the best practice in this area is appropriate communication with patient [ 13 , 15 ] .", "good and effective communication is very valuable from the perspective of patients . in general , communication is a set of learning skills .", "in fact , poor communication is the cause of most problems in treatment of patients .", "in fact , communication at the bedside is a therapeutic and professional communication . in health professionals ,", "communication and communication skills play a very important role in satisfaction of patients and solving their problems .", "this is more important , especially in patients with chronic diseases or those requiring long - term care .", "the therapeutic communication is formed from the first encounter of the patient with the medical team [ 21 , 22 ] .", "however , studies have shown that the communication between patient and medical team is not efficient .", "the health personnel don not have adequate communication skills . in fact , nursing and medical team spend very little time to communicate with patients .", "accordingly , patients are not often satisfied with received information as well as the level and method of communication .", "hence , to achieve best therapeutic results , special attention should be paid to communication [ 16 , 19 , 22 ] .", "since nursing is a practical discipline based on professional knowledge , it is necessary to use knowledge infrastructure to develop new approaches in clinical practice .", "theories proposed by nursing scientists can be useful in this area [ 16 , 19 , 23 ] . for this purpose", "the nurse - patient communication is an essential element of this theory [ 21 , 24 ] . according to peplau theory ,", "the purpose of nursing care is to achieve a common good nurse - patient communication . according to peplau ,", "peplau refers to the importance of therapeutic communication with patients and its important role in reducing anxiety [ 21 , 23 , 24 ] .", "in fact , this theory provides a framework for nurse - patient communication . following this framework", ", the nurse will be able to respond communication needs of patients through establishing a good therapeutic communication with the patient [ 16 , 19 , 23 ] .", "hospitalization and a complex surgery such as heart surgery is a stressful process leading to depression in patients .", "furthermore , the need for security and psychological comfort is one of the basic needs of patients achieved through a proper structured therapeutic communication with low cost .", "accordingly , the objective of the present study is to examine the impact of peplau therapeutic communication model on anxiety and depression in patients who were candidate for coronary bypass surgery .", "this is a clinical trial with pre / post testing scheme examined the impact of independent variable ( peplau therapeutic communication ) on the dependent variables ( anxiety and depression ) .", "the population consisted of all patients who were candidates for coronary artery bypass referred to the research environment on non - emergency basis .", "the research environment was al - zahra heart hospital in shiraz due to the easy access to subjects and sufficient number of patients .", "data were collected using the hospital anxiety and depression scale ( kanter et al . ) .", "the reliability and validity of the scale have been studied by montazeri et al in 2003 .", "an internal consistency of 78% and 86% was respectively calculated for anxiety and depression using cronbach 's alpha .", "there are 14 four - option questions , each with a score of zero to 3 .", "odd and even questions are used to measure anxiety and depression , respectively ( seven questions to assess anxiety and seven questions to assess depression ) .", "a score of zero to 21 is given to each patient based on responses in each area ( a score of zero - seven : normal , eight-10 : moderate and > 10 : disease ) .", "due to the lack of access to error rate to calculate the sample size , 10 patients were selected as pilot ( with a depression and anxiety score of 10 or more based on the hospital anxiety and depression scale ) . after performing tests ,", "the subjects were randomly divided into test and control groups ( using a coin ( toss ) ) .", "z12+z12s12+s22x1x22 the inclusion criteria were : being in a bypass list , having moderate to severe depression and anxiety scores , no history of mental illness , interesting in participating in the study ( i.e. the tendency of the patient and his family to participate in the intervention ) , lack of previous bypass surgery , aged between 35 and 70 years , ability to communicate verbally and ability to speak persian .", "the exclusion criteria included : lack of cooperation of patients and families during the intervention , failure to perform coronary artery bypass surgery for various reasons , mortality during the study , failure to attend therapeutic communication sessions ( at least the absence in two sessions ) .", "therapeutic communication sessions were held for the intervention group based on peplau 's model at four stages including : orientation , identification , exploitation and resolution . in total , seven sessions were held individually with the consent of the patient and his family at the hospital and patient 's home", ". it should be notedthat during therapeutic communication , duration of each session was variable given the location and patient 's needs .", "the place of each meeting was determined with the consent of the patient . at all meetings , the researcher used verbal and nonverbal communication skills to communicate with patients . to ensure the correct application of therapeutic communication skills , a self - control researcher - made tool approved by experts was used .", "the depression and anxiety of patients in both groups were assessed using the hospital anxiety and depression scale ( hads ) at baseline and then two and four months after surgery .", "the obtained data were analyzed using descriptive statistics as well as covariance analysis with the help of spss 16 .", "this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code .", "the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details .", "furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) .", "this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code .", "the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details .", "furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) .", "the percentage of male and female patients in the intervention group was 70.3% and 29.7% , respectively .", "the percentage of males and females in the control group was 51.4% and 48.6% , respectively .", "the status of the underlying disease was depicted in both intervention and control groups . according to results", ", the majority of patients were suffering from high blood pressure in both groups with a high frequency of 35.1% ( table 1 ) .", "as shown in table 2 , anxiety was reduced in the intervention group , while postoperative anxiety in the control group was increased . the hospital anxiety in the intervention group", "this indicates the role of peplau 's therapeutic communication in reducing hospital anxiety of patients .", "analysis of covariance was used to determine the effect of peplau 's therapeutic communication on anxiety level of the subjects . since", "hospital anxiety scale was used to measure anxiety , analysis of variance was performed using hospital anxiety scores .", "the results showed significant differences between experimental and control groups in terms of anxiety level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test .", "adjusted mean anxiety scores suggest that anxiety in the intervention group was lower than the control group .", "in fact , the mean anxiety scores of patients in the intervention group in pre - test and post - test stages represent the independent role of peplau 's therapeutic communication model in reducing anxiety in the intervention group ( f=174.02 , p=0.000 ) .", "moreover , the anxiety level in the intervention group two and four months after the intervention was decreased compared to the control group ( f=38.37 , p=0.000 ; f=11.58 , p=0.000 ) ( table 3 ) . as shown in table 4 , the mean depression in the intervention group was reduced after the surgery . the mean postoperative depression in the control group was increased .", "in fact , the hospital depression in the intervention group was reduced after the therapeutic communication .", "this indicates that the role of peplau 's therapeutic communication model in reducing hospital depression .", "analysis of covariance was used to determine the effect of peplau 's therapeutic communication on depression level of the subjects . since", "hospital depression scale was used to measure depression , analysis of variance was performed using hospital depression scores .", "the results showed significant differences between experimental and control groups in terms of depression level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test .", "adjusted mean depression scores suggest that anxiety in the intervention group was lower than the control group .", "in fact , the mean depression scores of patients in the intervention group in pre - test and post - test indicate the independent role of peplau 's therapeutic communication model in reducing depression in the intervention group ( f=163.27 , p=0.000 ) . such a difference is not observed in the control group .", "moreover , the depression level in the intervention group four months after the intervention was decreased compared to the control group ( f=20.58 , p=0.000 ) . however , no significant difference was found between two groups in terms of hospital depression two months after intervention ( p=0.61 ) ( table 5 ) .", "according to the results , peplau 's therapeutic communication is effective in reducing anxiety and depression in patients who were candidate for coronary artery bypass .", "it resulted in a dramatic decrease in anxiety and depression scores in the intervention group .", "depression and anxiety are the most important preventive factors in cardiovascular diseases   causing disease rejection by the patient and reduce the incentive to treat and increase the likelihood of disease recurrence [ 25 - 27 ] .", "most patients with coronary heart disease who survive after surgery resume their normal life after several weeks or several months .", "previous studies show that patients suffer from anxiety and depression several months and sometimes several years after the surgery [ 26 , 27 , 29 ] .", "therefore , follow - up and strategies to deal with postoperative psychological complications are of utmost importance .", "it is not possible except through establishing a coherent and targeted therapeutic communication . indeed , effective communication enables the medical team to identify patients needs and take steps to solve problems and meet their needs . according to dadashi , hosseini and moghaddam ( 2009 ) , staff had close encounters with patients in 85.5% of cases .", "according to mastaneh and mouseli ( 2013 ) , the rate of respecting patient rights in terms of communication was 60% indicating a moderate level of communication skills . on the other hand , duration and how to communicate with patients , particularly in dealing with patients with chronic diseases are essential in the hope and positive attitude to treatment and follow - up after discharge [ 29 - 31 ] .", "in fact , the patient gains motivation to cope with illness and trust in nurses through proper therapeutic communication , because the care of patients with either physical or mental illness requires correct and consistent communication [ 30 , 31 ] .", "therefore , implementing a communication pattern will help medical team to establish a proper communication .", "peplau 's model is one of these communication patterns . to communicate with patients , especially patients with chronic diseases ,", "peplau 's described four stages and defined roles for the nurse at every step as someone who is in close relationship with the patient [ 16 , 23 ] .", "peplau 's communication model helps nurses and patient to identify the disease , concerns and questions of the patient . through defining different roles for the nurse", "this model refers to nursing skills and abilities to establish a simple convenient therapeutic communication .", "the model enables nurses help the patient after discharge and to spend more time with patient [ 16 , 23 , 24 ] .", "for example , it has been used in communicating with cancer patients and solving systematic problems of families [ 32 , 33 ] . in this study ,", "peplau 's communication models were employed as communication sessions for patients who were candidates for coronary artery bypass through applying nursing roles .", "the results showed that holding communication meetings with patients immediately after surgery dramatically reduced hospital anxiety and depression .", "accordingly , to improve the nurse - patient communication and establish more effective therapeutic communication processes which improves clinical basis and can have a positive impact on the treatment and discharge processes and rehabilitation of patients , especially in patients with chronic diseases , training courses should be included in in - service courses to train and introduce medical team with simple and inexpensive communication skills .", "furthermore , there is evidence that workshops on communication skills for health team can improve health outcomes and satisfaction of patients .", "another limitation of this study is the lower percentage of females ( 29.7% ) in the intervention group with respect to the control group ( 51.4% ) .", "the lower anxiety score in the intervention group could be due to therapeutic communication but also to gender differences .", "to write this article not contributed any financial resources and costs are the responsibility of the authors .", "all rights of this article reserved is owned by the ahvaz jundishapur university of medical sciences ."], "abstract_text": ["<S> background and objective :   anxiety and depression are among the psychological disorders in heart \n surgeries . establishing a simple communication is essential to reduce anxiety and depression . </S>", "<S> hence , the objective of the present studywas to examine the impact of peplau therapeutic communication model on anxiety and depression in patients , who were candidate for coronary artery bypass in al - zahra heart hospital , shiraz during 2012 - 2013 . \n </S>", "<S> methods :   this is a clinical trial in which 74 patients were randomly divided into intervention and control groups , each consisted of 37 patients . anxiety and depression levels were assessed before , and two and four months after intervention using the hospital anxiety and depression scale ( hads ) . </S>", "<S> seven therapeutic communication sessions were held in four stages . </S>", "<S> data were analyzed with the spss ( version 16 ) using analysis of covariance . </S>", "<S> results :   the mean anxiety and depression levels decreased in the intervention group after the therapeutic communication ( p<0.01 ) . </S>", "<S> anxiety scores in the intervention group before and after intervention were 10.23 and 9.38 , respectively . while the corresponding scores in the control group were 10.26 and 11.62 , respectively . </S>", "<S> depression scores in the intervention group before and after intervention were 11 and 9.13 , respectively . </S>", "<S> the corresponding scores in the control group were 11.30 and 12.08 , respectively . </S>", "<S> conclusion :   the results demonstrated the positive role of therapeutic communication in reducing anxiety and depression of the patients . </S>", "<S> therefore , the therapeutic communication is recommended as a simple , cost effective and efficient method in this area . \n </S>"], "labels": null, "section_names": ["INTRODUCTION ", "METHODS ", "ETHICAL CONSIDERATIONS", "RESULTS ", "DISCUSSION ", "CONFLICT OF INTEREST", "FUNDING", "AUTHORSHIP", "RESEARCH ETHICS STATEMENT"], "sections": [["development of human societies and industrialization as well as changes in stress sources has changed disease pattern in civilized societies . as a result , the disease pattern has changed from traditional diseases such as infectious diseases and malnutrition to diseases such as heart disease , diabetes , accidents and so forth . among them", "coronary artery disease is the most cardiovascular disorder as a health problem in developing and developed countries .", "in fact , this is not only a chronic disease associated with high mortality , but it causes limitations in life and disability in a large part of the productive forces of the country .", "it is also associated with reduced production and increased medical costs [ 3 - 5 ] . nowadays ,", "cardiovascular diseases are among the most widespread chronic diseases in most countries . according to forecasts ,", "the mortality rate in eastern mediterranean countries including iran will be 30 - 35% [ 6 , 7 ] . despite the emphasis on prevention and development of newtreatments , surgery is the only choice for many patients with cardiovascular disease .", "one of the surgical procedures for the treatment of cardiovascular patients is coronary artery bypass surgery .", "in fact , heart surgery significantly influences on the quality of life of patients with cardiovascular diseases .", "hence , heart surgery is an important event in the lives of patients causing collapse of economic , professional and personal life . on the other hand , each surgical procedure", "is associated with several psychological complications for patients . of the most important complications are anxiety and depression so that approximately 65% of cardiovascular patients experience them after surgical and medical interventions .", "in fact , anxiety and depression complicate the treatment process [ 7 , 8 ] .", "some scientists believe that anxiety is one of the essential elements of human life . however , acute and long - term anxiety is integral part of all psychological diseases . according to nemati and colleagues ,", "in fact , anxiety is a common psychological response of cardiovascular patients which is associated with reduced quality of life and psychological morbidity [ 11 , 12 ] . on the other hand ,", "anxiety causes increased heart rate and breathing as well as high blood pressure and even mortality .", "it is associated with increased risk of mortality , disability , increased medical care and functional impairment in daily activities [ 12 , 13 ] . clinical depression has been reported in 54% of patients after bypass surgery . according to world health organization ( who ) , depression is the fourth chronic disease where its disability is comparable with 8 major chronic disorders .", "the outcome of depression in patients with cardiovascular disease is deterioration of physical and emotional state . given the incidence of such complications in patients with cardiovascular disease", "the best practice in this area is appropriate communication with patient [ 13 , 15 ] .", "good and effective communication is very valuable from the perspective of patients . in general , communication is a set of learning skills .", "in fact , poor communication is the cause of most problems in treatment of patients .", "in fact , communication at the bedside is a therapeutic and professional communication . in health professionals ,", "communication and communication skills play a very important role in satisfaction of patients and solving their problems .", "this is more important , especially in patients with chronic diseases or those requiring long - term care .", "the therapeutic communication is formed from the first encounter of the patient with the medical team [ 21 , 22 ] .", "however , studies have shown that the communication between patient and medical team is not efficient .", "the health personnel don not have adequate communication skills . in fact , nursing and medical team spend very little time to communicate with patients .", "accordingly , patients are not often satisfied with received information as well as the level and method of communication .", "hence , to achieve best therapeutic results , special attention should be paid to communication [ 16 , 19 , 22 ] .", "since nursing is a practical discipline based on professional knowledge , it is necessary to use knowledge infrastructure to develop new approaches in clinical practice .", "theories proposed by nursing scientists can be useful in this area [ 16 , 19 , 23 ] . for this purpose", "the nurse - patient communication is an essential element of this theory [ 21 , 24 ] . according to peplau theory ,", "the purpose of nursing care is to achieve a common good nurse - patient communication . according to peplau ,", "peplau refers to the importance of therapeutic communication with patients and its important role in reducing anxiety [ 21 , 23 , 24 ] .", "in fact , this theory provides a framework for nurse - patient communication . following this framework", ", the nurse will be able to respond communication needs of patients through establishing a good therapeutic communication with the patient [ 16 , 19 , 23 ] .", "hospitalization and a complex surgery such as heart surgery is a stressful process leading to depression in patients .", "furthermore , the need for security and psychological comfort is one of the basic needs of patients achieved through a proper structured therapeutic communication with low cost .", "accordingly , the objective of the present study is to examine the impact of peplau therapeutic communication model on anxiety and depression in patients who were candidate for coronary bypass surgery ."], ["this is a clinical trial with pre / post testing scheme examined the impact of independent variable ( peplau therapeutic communication ) on the dependent variables ( anxiety and depression ) .", "the population consisted of all patients who were candidates for coronary artery bypass referred to the research environment on non - emergency basis .", "the research environment was al - zahra heart hospital in shiraz due to the easy access to subjects and sufficient number of patients .", "data were collected using the hospital anxiety and depression scale ( kanter et al . ) .", "the reliability and validity of the scale have been studied by montazeri et al in 2003 .", "an internal consistency of 78% and 86% was respectively calculated for anxiety and depression using cronbach 's alpha .", "there are 14 four - option questions , each with a score of zero to 3 .", "odd and even questions are used to measure anxiety and depression , respectively ( seven questions to assess anxiety and seven questions to assess depression ) .", "a score of zero to 21 is given to each patient based on responses in each area ( a score of zero - seven : normal , eight-10 : moderate and > 10 : disease ) .", "due to the lack of access to error rate to calculate the sample size , 10 patients were selected as pilot ( with a depression and anxiety score of 10 or more based on the hospital anxiety and depression scale ) . after performing tests ,", "the subjects were randomly divided into test and control groups ( using a coin ( toss ) ) .", "z12+z12s12+s22x1x22 the inclusion criteria were : being in a bypass list , having moderate to severe depression and anxiety scores , no history of mental illness , interesting in participating in the study ( i.e. the tendency of the patient and his family to participate in the intervention ) , lack of previous bypass surgery , aged between 35 and 70 years , ability to communicate verbally and ability to speak persian .", "the exclusion criteria included : lack of cooperation of patients and families during the intervention , failure to perform coronary artery bypass surgery for various reasons , mortality during the study , failure to attend therapeutic communication sessions ( at least the absence in two sessions ) .", "therapeutic communication sessions were held for the intervention group based on peplau 's model at four stages including : orientation , identification , exploitation and resolution . in total , seven sessions were held individually with the consent of the patient and his family at the hospital and patient 's home", ". it should be notedthat during therapeutic communication , duration of each session was variable given the location and patient 's needs .", "the place of each meeting was determined with the consent of the patient . at all meetings , the researcher used verbal and nonverbal communication skills to communicate with patients . to ensure the correct application of therapeutic communication skills , a self - control researcher - made tool approved by experts was used .", "the depression and anxiety of patients in both groups were assessed using the hospital anxiety and depression scale ( hads ) at baseline and then two and four months after surgery .", "the obtained data were analyzed using descriptive statistics as well as covariance analysis with the help of spss 16 .", "this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code .", "the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details .", "furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) ."], ["this study was approved by the ethics committee of the jondi shapoor medical university of ahvaz with rec.1392.58 code .", "the ethical issues considered in this study include an adequate explanation for the patients and their permission to participate in the study , the lack of any compulsion for patients to participate in the study , refusing to disclose secrets or private matters of patients and confidentiality of information , announcing the results without names and personal details .", "furthermore , the present study was registered with the irct2013072214110n1 code in clinical trial center ( irct.ir ) ."], ["the percentage of male and female patients in the intervention group was 70.3% and 29.7% , respectively .", "the percentage of males and females in the control group was 51.4% and 48.6% , respectively .", "the status of the underlying disease was depicted in both intervention and control groups . according to results", ", the majority of patients were suffering from high blood pressure in both groups with a high frequency of 35.1% ( table 1 ) .", "as shown in table 2 , anxiety was reduced in the intervention group , while postoperative anxiety in the control group was increased . the hospital anxiety in the intervention group", "this indicates the role of peplau 's therapeutic communication in reducing hospital anxiety of patients .", "analysis of covariance was used to determine the effect of peplau 's therapeutic communication on anxiety level of the subjects . since", "hospital anxiety scale was used to measure anxiety , analysis of variance was performed using hospital anxiety scores .", "the results showed significant differences between experimental and control groups in terms of anxiety level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test .", "adjusted mean anxiety scores suggest that anxiety in the intervention group was lower than the control group .", "in fact , the mean anxiety scores of patients in the intervention group in pre - test and post - test stages represent the independent role of peplau 's therapeutic communication model in reducing anxiety in the intervention group ( f=174.02 , p=0.000 ) .", "moreover , the anxiety level in the intervention group two and four months after the intervention was decreased compared to the control group ( f=38.37 , p=0.000 ; f=11.58 , p=0.000 ) ( table 3 ) . as shown in table 4 , the mean depression in the intervention group was reduced after the surgery . the mean postoperative depression in the control group was increased .", "in fact , the hospital depression in the intervention group was reduced after the therapeutic communication .", "this indicates that the role of peplau 's therapeutic communication model in reducing hospital depression .", "analysis of covariance was used to determine the effect of peplau 's therapeutic communication on depression level of the subjects . since", "hospital depression scale was used to measure depression , analysis of variance was performed using hospital depression scores .", "the results showed significant differences between experimental and control groups in terms of depression level immediately after surgery , two and four months after surgery after adjustment for post - test scores by eliminating the effect of pre - test .", "adjusted mean depression scores suggest that anxiety in the intervention group was lower than the control group .", "in fact , the mean depression scores of patients in the intervention group in pre - test and post - test indicate the independent role of peplau 's therapeutic communication model in reducing depression in the intervention group ( f=163.27 , p=0.000 ) . such a difference is not observed in the control group .", "moreover , the depression level in the intervention group four months after the intervention was decreased compared to the control group ( f=20.58 , p=0.000 ) . however , no significant difference was found between two groups in terms of hospital depression two months after intervention ( p=0.61 ) ( table 5 ) ."], ["according to the results , peplau 's therapeutic communication is effective in reducing anxiety and depression in patients who were candidate for coronary artery bypass .", "it resulted in a dramatic decrease in anxiety and depression scores in the intervention group .", "depression and anxiety are the most important preventive factors in cardiovascular diseases   causing disease rejection by the patient and reduce the incentive to treat and increase the likelihood of disease recurrence [ 25 - 27 ] .", "most patients with coronary heart disease who survive after surgery resume their normal life after several weeks or several months .", "previous studies show that patients suffer from anxiety and depression several months and sometimes several years after the surgery [ 26 , 27 , 29 ] .", "therefore , follow - up and strategies to deal with postoperative psychological complications are of utmost importance .", "it is not possible except through establishing a coherent and targeted therapeutic communication . indeed , effective communication enables the medical team to identify patients needs and take steps to solve problems and meet their needs . according to dadashi , hosseini and moghaddam ( 2009 ) , staff had close encounters with patients in 85.5% of cases .", "according to mastaneh and mouseli ( 2013 ) , the rate of respecting patient rights in terms of communication was 60% indicating a moderate level of communication skills . on the other hand , duration and how to communicate with patients , particularly in dealing with patients with chronic diseases are essential in the hope and positive attitude to treatment and follow - up after discharge [ 29 - 31 ] .", "in fact , the patient gains motivation to cope with illness and trust in nurses through proper therapeutic communication , because the care of patients with either physical or mental illness requires correct and consistent communication [ 30 , 31 ] .", "therefore , implementing a communication pattern will help medical team to establish a proper communication .", "peplau 's model is one of these communication patterns . to communicate with patients , especially patients with chronic diseases ,", "peplau 's described four stages and defined roles for the nurse at every step as someone who is in close relationship with the patient [ 16 , 23 ] .", "peplau 's communication model helps nurses and patient to identify the disease , concerns and questions of the patient . through defining different roles for the nurse", "this model refers to nursing skills and abilities to establish a simple convenient therapeutic communication .", "the model enables nurses help the patient after discharge and to spend more time with patient [ 16 , 23 , 24 ] .", "for example , it has been used in communicating with cancer patients and solving systematic problems of families [ 32 , 33 ] . in this study ,", "peplau 's communication models were employed as communication sessions for patients who were candidates for coronary artery bypass through applying nursing roles .", "the results showed that holding communication meetings with patients immediately after surgery dramatically reduced hospital anxiety and depression .", "accordingly , to improve the nurse - patient communication and establish more effective therapeutic communication processes which improves clinical basis and can have a positive impact on the treatment and discharge processes and rehabilitation of patients , especially in patients with chronic diseases , training courses should be included in in - service courses to train and introduce medical team with simple and inexpensive communication skills .", "furthermore , there is evidence that workshops on communication skills for health team can improve health outcomes and satisfaction of patients .", "another limitation of this study is the lower percentage of females ( 29.7% ) in the intervention group with respect to the control group ( 51.4% ) .", "the lower anxiety score in the intervention group could be due to therapeutic communication but also to gender differences ."], [""], ["to write this article not contributed any financial resources and costs are the responsibility of the authors ."], [""], ["all rights of this article reserved is owned by the ahvaz jundishapur university of medical sciences ."]]}
{"article_id": "PMC3320503", "article_text": ["sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp .", "throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) .", "antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) .", "rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification ,", "5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3  and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3  ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) .", "pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min .", "all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination .", "the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3  ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3  ) .", "unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations .", "nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications .", "multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . ,", "three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) .", "ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos .", "sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp .", "throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) .", "antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) .", "rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification ,", "5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3  and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3  ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) .", "pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min .", "all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination .", "the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3  ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3  ) .", "unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations .", "nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications .", "multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . , madison , wi , usa ) software package .", "three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) .", "ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos .", "clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) .", "of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals .", "most of the patients were < 10 years of age ; 36 were male and 28 were female .", "none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture .", "the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 .", "( map courtesy of http://www.maps.com ) hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera .", "six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) .", "* test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference .  a", "rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype .", "twenty - six of the 42 influenza a  positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene .", "the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) .", "alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) .", "all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . *", "n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine . ", "consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal .", "of the 26 nepal strains examined , 24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) .", "this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position .", "prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 .", "additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season .", "two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively .", "both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) .", "the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) .", "the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions .", "unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains .", "the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow )", "was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season .", "nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no .", "the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 .", "three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively .", "the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution .", "this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) . located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features .", "this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) .", "interestingly , this proline residue is located within a  barrel ( a protein motif consisting of an antiparallel  sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 .", "three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 .", "most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) .", "magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 .", "hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) .", "clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) .", "of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals .", "most of the patients were < 10 years of age ; 36 were male and 28 were female .", "none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture .", "the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 .", "hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera .", "six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) .", "* test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference .  a", "rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype .", "twenty - six of the 42 influenza a  positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene .", "the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) .", "alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) .", "all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . *", "n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine .  consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal . of the 26 nepal strains examined ,", "24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) .", "this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position .", "prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 .", "additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season .", "two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively .", "both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) .", "the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) .", "the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions .", "unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains .", "the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow )", "was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season .", "nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no .", "the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 .", "three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively .", "the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution .", "this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) .", "located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features .", "this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) .", "interestingly , this proline residue is located within a  barrel ( a protein motif consisting of an antiparallel  sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 .", "three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 .", "most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) .", "magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 .", "hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) .", "the 4 substitutions described represent a growing lineage of influenza a ( h3n2 ) viruses characterized since july 2004 .", "three amino acid changes are confined within known antibody - binding sites , i.e. , the s189n change within antibody - binding site b ( 4,5 ) and the v226i and s227p changes residing in antibody - binding site d ( 4,5 ) . because of rotational restrictions , a proline substitution at position 227 ( s227p ) would typically give rise to considerable conformation change ; however , this particular substitution is located within a  barrel motif and therefore has little effect on regional protein conformation . cumulatively , field isolates characterized subsequent to this outbreak continue to exhibit these 4 changes , and they appear to constitute a distinct branch in the phylogeny of ha sequences when compared with h3n2 isolates from the 20032004 season .", ". this change may affect protein - protein interactions since it is immediately adjacent to antibody - binding site a , where neutralizing antibodies have been shown to bind ( 4,5 ) .", "furthermore , since the k145n substitution is located within a glycosylation site , the charge alteration may affect glycosyl transferase activity , which results in altered glycosylation .", "differences in glycosylation have been shown to contribute to antigenic variation by preventing antibody binding to antigenic sites ( 15 ) .", "additionally , 3-dimensional analysis suggests this amino acid substitution may also promote enhanced receptor binding since the asparagine r group is shorter , which may make binding requirements less stringent and the receptor cleft more accessible .", "the 3-dimensional depiction provides a unique regional residue perspective , demonstrating how the rapidly evolving ha surface antigens in the vaccine strain differ at the molecular level .", "collectively , the clinical isolates obtained from this outbreak in nepal can not be considered antigenically distinct from the a / wyoming/3/03 vaccine strain because only 4 of 9 isolates evaluated exhibited 4-fold lower titers by hi ( table 1 ) .", "furthermore , the varying reactivity noted in several isolates from this outbreak having identical ha1 sequences is suggestive that other viral antigens aside from the ha1 protein may have contributed to the antigenic variability observed in the hi panel . with the exception of a / nepal/1670/2004 and a / nepal/1672/2004 , all isolates evaluated by hi ( table 1 ) exhibited identical ha1 amino acid sequences and varying antigenicity profiles to a / wyoming/03/2003 reference antisera .", "one explanation for this observation is that genetic differences in other influenza surface proteins contribute to the observed immunoreactivity .", "alternative viral surface protein candidates include the neuraminidase , ha2 , and m2 glycoproteins , which have been shown to exhibit antigenic properties ( 1619 ) . in this report", ", we describe the genetic analysis of the ha proteins from viruses obtained from an early season outbreak and compare them to current vaccine strains .", "three amino acids changes ( s189n , i226v , and s227p ) were noted in known ( 4,5 ) antibody - binding sites ( table 2 ) . the fourth change ( k145n ) , which was located within a glycosylation site , may enhance viral binding since the smaller asparagine r group is located close to the ha receptor - binding cleft ( figure 3 ) .", "phylogenetic analyses show that the nepal isolates make up a distinct branch in the evolution of h3n2 viruses when they are compared with vaccine and reference strains ( figure 2 ) . however , antigenic data appear more ambiguous , suggesting a multigenic effect that can not solely be attributed to properties of the influenza ha ( table 1 ) .", "studies are in progress to characterize the neuraminidase , m2 , and ha2 proteins to determine the molecular basis responsible for antigenicity differences observed within isolates from this outbreak .", "the k145n substitution change has become a marker for an increasingly large subset of the fujian - like viruses .", "cdc and the us department of defense have recently characterized viruses with the k145n change in singapore , taiwan , china , australia , canada , and the united states . in february 2005 , who reported the emergence of a new influenza h3n2 strain in the united states .", "the a / california/7/2004 strain , which was first identified in the united states in september 2004 , contains all 4 changes observed in isolates from this nepalese outbreak .", "the a / california/7/2004 strain differs by only 1 amino acid in ha1 ( which is of no immunologic importance ) from most isolates from the outbreak in nepal .", "all viruses characterized ( 150 globally isolated strains ) subsequent to the preparation of this report ( march 2005 ) by the us department of defense are genetically similar in amino acid sequence to these nepalese strains ( and the a / california strain ) .", "most of the isolates ( 80% ) analyzed by cdc since october 2004 are antigenically related to a / california ( 20 ) , which indicates that this strain has emerged as the dominant influenza a h3n2 strain .", "these data indicate that these viruses may persist as the dominant strain at the onset of the 20052006 influenza season . in february 2005 , who recommended inclusion of an a / california/7/2004-like strain in the 20052006 trivalent influenza vaccine to afford immunologic protection from this variant h3n2 virus .", "our findings emphasize the importance of continued molecular surveillance for characterizing emerging influenza drift variants ."], "abstract_text": ["<S> worldwide emergence of variant viruses has prompted a change in the 20052006 h3n2 influenza a vaccine strain . </S>"], "labels": null, "section_names": ["Materials and Methods", "Sample Collection and Antigenic Analysis", "Molecular Analysis", "Results", "Epidemiologic and Laboratory Assessment", "Antigenic Analysis", "Molecular Analysis", "Discussion"], "sections": [["sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp .", "throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) .", "antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) .", "rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification ,", "5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3  and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3  ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) .", "pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min .", "all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination .", "the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3  ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3  ) .", "unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations .", "nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications .", "multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . ,", "three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) .", "ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos ."], ["sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp .", "throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) .", "antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) ."], ["rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification ,", "5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3  and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3  ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) .", "pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min .", "all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination .", "the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3  ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3  ) .", "unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations .", "nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications .", "multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . , madison , wi , usa ) software package .", "three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) .", "ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos ."], ["clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) .", "of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals .", "most of the patients were < 10 years of age ; 36 were male and 28 were female .", "none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture .", "the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 .", "( map courtesy of http://www.maps.com ) hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera .", "six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) .", "* test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference .  a", "rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype .", "twenty - six of the 42 influenza a  positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene .", "the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) .", "alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) .", "all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . *", "n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine . ", "consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal .", "of the 26 nepal strains examined , 24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) .", "this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position .", "prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 .", "additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season .", "two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively .", "both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) .", "the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) .", "the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions .", "unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains .", "the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow )", "was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season .", "nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no .", "the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 .", "three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively .", "the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution .", "this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) . located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features .", "this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) .", "interestingly , this proline residue is located within a  barrel ( a protein motif consisting of an antiparallel  sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 .", "three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 .", "most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) .", "magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 .", "hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) ."], ["clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) .", "of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals .", "most of the patients were < 10 years of age ; 36 were male and 28 were female .", "none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture .", "the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 ."], ["hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera .", "six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) .", "* test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference .  a"], ["rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype .", "twenty - six of the 42 influenza a  positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene .", "the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) .", "alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) .", "all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . *", "n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine .  consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal . of the 26 nepal strains examined ,", "24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) .", "this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position .", "prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 .", "additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season .", "two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively .", "both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) .", "the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) .", "the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions .", "unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains .", "the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow )", "was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season .", "nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no .", "the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 .", "three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively .", "the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution .", "this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) .", "located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features .", "this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) .", "interestingly , this proline residue is located within a  barrel ( a protein motif consisting of an antiparallel  sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 .", "three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 .", "most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) .", "magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 .", "hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) ."], ["the 4 substitutions described represent a growing lineage of influenza a ( h3n2 ) viruses characterized since july 2004 .", "three amino acid changes are confined within known antibody - binding sites , i.e. , the s189n change within antibody - binding site b ( 4,5 ) and the v226i and s227p changes residing in antibody - binding site d ( 4,5 ) . because of rotational restrictions , a proline substitution at position 227 ( s227p ) would typically give rise to considerable conformation change ; however , this particular substitution is located within a  barrel motif and therefore has little effect on regional protein conformation . cumulatively , field isolates characterized subsequent to this outbreak continue to exhibit these 4 changes , and they appear to constitute a distinct branch in the phylogeny of ha sequences when compared with h3n2 isolates from the 20032004 season .", ". this change may affect protein - protein interactions since it is immediately adjacent to antibody - binding site a , where neutralizing antibodies have been shown to bind ( 4,5 ) .", "furthermore , since the k145n substitution is located within a glycosylation site , the charge alteration may affect glycosyl transferase activity , which results in altered glycosylation .", "differences in glycosylation have been shown to contribute to antigenic variation by preventing antibody binding to antigenic sites ( 15 ) .", "additionally , 3-dimensional analysis suggests this amino acid substitution may also promote enhanced receptor binding since the asparagine r group is shorter , which may make binding requirements less stringent and the receptor cleft more accessible .", "the 3-dimensional depiction provides a unique regional residue perspective , demonstrating how the rapidly evolving ha surface antigens in the vaccine strain differ at the molecular level .", "collectively , the clinical isolates obtained from this outbreak in nepal can not be considered antigenically distinct from the a / wyoming/3/03 vaccine strain because only 4 of 9 isolates evaluated exhibited 4-fold lower titers by hi ( table 1 ) .", "furthermore , the varying reactivity noted in several isolates from this outbreak having identical ha1 sequences is suggestive that other viral antigens aside from the ha1 protein may have contributed to the antigenic variability observed in the hi panel . with the exception of a / nepal/1670/2004 and a / nepal/1672/2004 , all isolates evaluated by hi ( table 1 ) exhibited identical ha1 amino acid sequences and varying antigenicity profiles to a / wyoming/03/2003 reference antisera .", "one explanation for this observation is that genetic differences in other influenza surface proteins contribute to the observed immunoreactivity .", "alternative viral surface protein candidates include the neuraminidase , ha2 , and m2 glycoproteins , which have been shown to exhibit antigenic properties ( 1619 ) . in this report", ", we describe the genetic analysis of the ha proteins from viruses obtained from an early season outbreak and compare them to current vaccine strains .", "three amino acids changes ( s189n , i226v , and s227p ) were noted in known ( 4,5 ) antibody - binding sites ( table 2 ) . the fourth change ( k145n ) , which was located within a glycosylation site , may enhance viral binding since the smaller asparagine r group is located close to the ha receptor - binding cleft ( figure 3 ) .", "phylogenetic analyses show that the nepal isolates make up a distinct branch in the evolution of h3n2 viruses when they are compared with vaccine and reference strains ( figure 2 ) . however , antigenic data appear more ambiguous , suggesting a multigenic effect that can not solely be attributed to properties of the influenza ha ( table 1 ) .", "studies are in progress to characterize the neuraminidase , m2 , and ha2 proteins to determine the molecular basis responsible for antigenicity differences observed within isolates from this outbreak .", "the k145n substitution change has become a marker for an increasingly large subset of the fujian - like viruses .", "cdc and the us department of defense have recently characterized viruses with the k145n change in singapore , taiwan , china , australia , canada , and the united states . in february 2005 , who reported the emergence of a new influenza h3n2 strain in the united states .", "the a / california/7/2004 strain , which was first identified in the united states in september 2004 , contains all 4 changes observed in isolates from this nepalese outbreak .", "the a / california/7/2004 strain differs by only 1 amino acid in ha1 ( which is of no immunologic importance ) from most isolates from the outbreak in nepal .", "all viruses characterized ( 150 globally isolated strains ) subsequent to the preparation of this report ( march 2005 ) by the us department of defense are genetically similar in amino acid sequence to these nepalese strains ( and the a / california strain ) .", "most of the isolates ( 80% ) analyzed by cdc since october 2004 are antigenically related to a / california ( 20 ) , which indicates that this strain has emerged as the dominant influenza a h3n2 strain .", "these data indicate that these viruses may persist as the dominant strain at the onset of the 20052006 influenza season . in february 2005 , who recommended inclusion of an a / california/7/2004-like strain in the 20052006 trivalent influenza vaccine to afford immunologic protection from this variant h3n2 virus .", "our findings emphasize the importance of continued molecular surveillance for characterizing emerging influenza drift variants ."]]}
{"article_id": "PMC3679767", "article_text": ["rising levels of obesity are becoming a worldwide phenomenon and are increasingly identified as a health problem across the globe [ 14 ] .", "higher weight has been associated with adverse health indicators and outcomes , including cardiovascular disease [ 512 ] , stroke [ 5 , 13 ] , cognitive and functional decline [ 1418 ] , metabolic syndrome [ 19 , 20 ] , inflammation [ 21 , 22 ] , and mortality [ 20 , 2325 ] .", "obesity among aging populations is relatively recent and aging among people who have been obese for much of their lives is also a new phenomenon . from 1980 to 2004 , the prevalence of obesity in the us has continued to rise from about 17% to 25% for men aged 5059 . while obesity in england has also increased during this period , from approximately 9% in 1980 to 15% in 2004 for men aged 5564 , the level of obesity remains much lower in england .", "additionally , the difference in obesity between the us and england is more pronounced for women .", "the level of obesity in us women was about 24% in 1980 and rose to 37% in 2004 ( age 5059 ) ; in england , levels for women were 9% in 1980 and 14% in 2004 ( age 5564 ) .", "the aim of this paper is to investigate differences in how obesity relates to indicators of physiological dysregulation in men and women of diverse populations .", "this comparison will lead to an improved understanding of how obesity might be differentially related to health and mortality across cultures and lifestyles .", "obesity was relatively rare in most populations until the second part of the last century , but it has now become common in many countries", ". the us population is recognized as among the most obese in the world , although many other countries are now approaching the us level and most countries are experiencing increasing obesity .", "this worldwide obesity epidemic began with the epidemiological revolution and the virtual elimination of infectious disease ; the decline in manual labor needed to provide sustenance with the industrial revolution ; and the increasing availability and decreasing cost of food [ 2729 ] .", "obesity reflects some combination of calorie intake , diet content , and amount of physical activity . in some cultures ,", "lack of physical activity can be a more important determinant of obesity ; in other cultures , overeating or food composition may be the more important determinant of obesity .", "it is also true that within countries , individuals could differ in the causes of obesity .", "for instance , changes in activity might be more characteristic of women or men resulting in different reasons for obesity by gender .", "these differences may affect how obesity is related to other risk factors for poor health , and it may determine the overall health risk associated with obesity . if physical activity is maintained , the overall effect of obesity may be less than if the activity is not .", "one indication of the cause of obesity may be the relationship between waist size and weight [ 30 , 31 ] . in societies where obese people are more physically active , waist size of the obese", "waist circumference has also been linked to late life mortality , where high waist circumference has been associated with increased mortality among men and women in the netherlands , while high bmi was not associated with mortality .", "this paper builds upon current obesity research by using both bmi and waist circumference to quantify obesity in order to determine how a combined indicator of weight and adiposity is related to physiological dysregulation in populations with different cultures , diets , behaviors , and epidemiological histories .", "obesity has been related to many indicators of physiological dysregulation including cardiovascular risk factors such as hypertension   and metabolic dysregulation in lipid levels or insulin resistance .", "most studies that investigate the differences in biological risk associated with excess weight have examined western populations [ 33 , 36 ] .", "comparative studies on the health risks associated with obesity that examined the us and england reported that obese americans had an increased risk of diabetes and a higher waist circumference [ 37 , 38 ] .", "these studies suggested that differences in physical activity , diet , and social environments may explain these national differences .", "while these differences have been observed between the us and england , these two western countries have roughly similar life expectancy , levels of living , history , and culture even while the us has poorer health by a number of indicators of disease prevalence and biological risk [ 36 , 39 ] .", "comparative studies of the links between obesity and health outcomes and risk factors for obesity comparing western and non - western countries indicate important differences in the causes and consequences of obesity .", "a comparison of the association of disease with overweight and obesity in japan and the us indicated that the associations were stronger in the us than in japanese women and that there was no association in japanese men .", "links between social , demographic , and behavioral risk factors for obesity also differ markedly in japan , korea , and the us .", "the availability of biomarker data from taiwan  a middle - income country undergoing rapid economic growth , increasing obesity , and with life expectancy recently increasing to levels similar to that of the us and england  allows for investigation of the biological risk associated with obesity in a population characterized by very different cultural , behavioral , socioeconomic , and dietary parameters .", "we examine how elevated weight and obesity ( using an indicator that considers both bmi and waist circumference ) relate to having levels defined as clinical risk for cardiovascular , metabolic , and inflammatory markers in three aging societies that are now relatively similar in life expectancy but that differ in the timing of the epidemiological transition and obesity epidemic , history of economic development , socioeconomic levels , general lifestyle habits , health behaviors , and health care systems : the us , england , and taiwan .", "finding differences in the relationship between obesity and indicators of physiological dysregulation in these three aging societies will clarify whether increases in weight gain are equally problematic across all countries .", "we use data from three nationally representative samples : the us national health and nutrition examination survey ( nhanes , 20032006 ; n = 3855 ) , the english longitudinal study of ageing ( elsa , 2004 - 2005 ; n = 9139 ) , and the social environment and biomarkers of aging study ( sebas ) in taiwan ( 2000 ; n = 1023 ) .", "these surveys collect information on demographics , as well as anthropometric , physical , and biological measures .", "every year , approximately 5,000 individuals undergo detailed interviews and medical examinations , which include collection of several physiological measures .", "nhanes utilizes a complex sampling design , and when weights are applied , the sample is representative of the noninstitutionalized american population .", "we use the 20032006 data since nhanes data is released in two - year intervals , and this sample is centered on 2004 - 2005 , which matches the period in which elsa was collected .", "for nhanes , we use individual - level data based on a sample of 1,513 fasting individuals aged 54 and older .", "elsa includes participants drawn from households responding to the health survey for england ( hse ) in 1998 , 1999 , and 2001   and is representative of the english population aged 50 and older .", "the core elsa sample ( wave 1 : 2002 - 2003 ) includes people living in an hse responding household who were born prior to march 1 , 1952 , and their partners who could be under age 50 . wave 4 of elsa ( 2008 - 2009 ) , which includes a nurse visit , includes wave 1 core members , if they are still alive and do not refuse further contact after the first interview at wave 1 .", "it also includes a refresher sample to maintain the age structure of the sample ( in waves 3 and 4 ) , and their partners . for elsa , we use individual - level data based on a sample of 7,384 individuals aged 54 and older .", "sebas is drawn from a follow - up survey of the survey of health and living status of the near elderly and elderly in taiwan ( also known as the taiwan longitudinal study of aging ( tlsa ) ) , a nationally representative survey of taiwanese adults ( including institutionalized individuals ) collected in 1989 , 1993 , 1996 , 1999 , and 2000 . in 2000 , a subsample of individuals was randomly selected for inclusion in sebas .", "sebas consists of adults aged 54 and older in 2000 , with in - home interviews and medical exams taken in a hospital . for sebas ,", "the sample averages 66.8 years of age in england , and the us and taiwan mean age is about the same ( table 1 ) .", "there are more men in taiwan ( 56% ) and england ( 53% ) and fewer in the us ( 44% ) .  ", "we examine the following indicators of physiological dysregulation often associated with obesity and also associated with increased risk for multiple adverse health outcomes and obesity [ 43 , 44 ] : ( 1 ) cardiovascular markers : high systolic ( sbp ) and diastolic blood pressure ( dbp ) ; ( 2 ) metabolic markers : high levels of blood lipids ( total and low - density lipoprotein ( ldl ) cholesterol , and fasting triglycerides ) , low levels of high - density lipoprotein ( hdl ) cholesterol , and high fasting glucose and glycated hemoglobin ; ( 3 ) high levels of inflammatory markers c - reactive protein ( crp ; available in nhanes and elsa ) and interleukin-6 ( il-6 ; available in sebas ) , as crp and il-6 have been positively associated with bmi . for each indicator", "we use clinical cutpoints or widely used research - based cutpoints to indicate high levels of risk which are shown in table 1 [ 39 , 43 , 45 ] .", "there has been debate as to the best indicator of obesity : body mass index ( bmi ) or waist circumference .", "waist circumference is thought to be a better measure of abdominal adiposity than bmi and a better indicator of risk of poor health outcomes , including all - cause and cardiovascular mortality [ 46 , 47 ] . for this reason", "we investigate the association between bmi and biomarkers across categories of bmi ( underweight < 18.5  kg / m , normal and overweight 18.529.9  kg / m , obese 3034.9  kg / m , and very obese 35  kg / m ) and waist circumference categorized as normal or high waist ( high waist : men 120  cm , women 88  cm ) . we create a composite measure of obesity and adiposity by categorizing individuals into five groups : ( 1 ) underweight and normal waist ( all underweight individuals had a normal waist circumference ) , ( 2 ) normal / overweight bmi ( termed normal bmi ) and normal waist circumference ( reference group ) , ( 3 ) normal / overweight bmi ( termed normal bmi ) and high waist circumference ( termed high waist ) , ( 4 ) obese and high waist ( all obese individuals had a high waist circumference ) , and ( 5 ) very obese .", "we also evaluate an alternate definition for obesity in taiwan based on bmi 27 , as it has been suggested by some that obesity levels should be differentially defined for asians [ 48 , 49 ] .", "a similar composite measure of obesity and adiposity was calculated using this alternate definition of bmi in taiwan . because these risk factors are all assumed to be associated with obesity and because dysregulation in multiple physiological systems has been shown to predict many of the poor health risk outcomes associated with aging , we also create two summary measures of risk based on the total number of at - risk levels of biomarkers , either 9 or 8 . because crp values for sebas are not available , this measure", "is not included in the 9-item summary measure for taiwan , but a summary measure ( range 09 ) was calculated for taiwan using il-6 , another indicator of inflammation , instead of the crp values included for the us and england . a second alternate summary measure of biological risk , excluding the inflammatory marker ( range 08 ) ,", "biological risk summary scores were computed for individuals who had missing values on no more than 3 biological markers .", "we examine multiple covariates in our investigation of the relationship between obesity and biological risk .", "self - reported use of antihypertensives was determined in all three countries , and use of lipid - lowering statins was only asked in the us sample .", "dichotomous variables were created to indicate whether the respondent reported being a current smoker and participating in at least moderate physical activity for exercise ( e.g. , brisk walking , running , or swimming ) in the past 30 days ( for the us and england ) or generally exercising once a week ( for taiwan ) .  ", "we use logistic regression models to determine the odds of having at - risk levels of a specific biomarker for obese men and women among the three populations . for all countries ,", "the comparison group for bmi and waist circumference is the normal bmi and normal waist group .", "the regressions included indicators of age , use of antihypertensives , current smoking status , and having exercised in the past 30 days .", "ordinary least squares ( ols ) regression models were used to determine the relationship between the summary measures of biological risk and the composite measure of obesity and adiposity .", "we begin by examining national differences in the high risk levels of individual biomarkers ( table 1 ) .", "low levels  or high risk levels  of hdl cholesterol are also more common in taiwan .", "high total and ldl cholesterol is more common among the english ; lower levels of plasma glucose , crp , and glycated hemoglobin are also characteristic of the english .", "few adults in england have elevated levels of fasting glucose ( 2.2% ) , while this is observed in 17.3% and 13.2% of american and taiwanese adults . \n", "most people in this age range are in the normal to overweight category ( 64.7% , 67.2% , and 89.4% in the us , england , and taiwan , respectively ) ( table 2(a ) ) .", "americans are more likely to be obese ( 33.7% ) compared to the english ( 32.1% ) and taiwanese ( 7.2% ) . among the obese , americans are much more likely to be very obese : 13.4% of the total us sample , 10.1% in england , and about 1% in taiwan .", "both among the obese and very obese , the average bmi is higher in the us and england compared to taiwan .", "few are underweight in any country ( 1.7% , 0.8% , and 3.4% in the us , england , and taiwan , resp . ) .  ", "when we examine waist circumference , the us has the highest average waist circumference , with 65.5% of americans , 55.9% of english , and 15.8% of taiwanese having a high waist size ( table 2(a ) ) .", "this means that high waist characterizes a substantial number of those who would be categorized as normal weight in the us and england . among those in the normal and overweight group about half ( 49.5% ) of americans and a third ( 36.4% ) of the english", "almost all obese individuals have a high waist in the us and england ( 98.3% in the us and 96.5% in england ) , but only 78.4% of the obese in taiwan also have a high waist . when we use the alternate obese cutpoint of 27  kg / m in taiwan , less than half of the obese individuals have a high waist ( not shown here ) . \n", "americans exhibit the highest proportion of the older population taking antihypertensive medication ( 47.1% ) ( table 1 ) .", "the percentage who reports taking antihypertensives is lower in england ( 32.0% ) and taiwan ( 28.6% ) .", "americans are more likely to be current smokers ( 24.5% ) than persons in taiwan ( 22.5% ) and england ( 13.9% ) .", "more than half of the population in all countries report having exercised in the past 30 days , with more english exercising ( 82.2% ) compared to taiwan ( 61.4% ) and the us ( 58.5% ) . \n", "men with normal bmi and high waist have a greater likelihood than men with normal bmi and normal waist size of having high - risk levels of triglycerides in all three countries . in the us and england ,", "men with high waist are more likely to have high levels of glycosylated hemoglobin and higher crp ; fasting glucose is also elevated among this group in the us ( table 3(a ) ) .", "men who are obese in the us have fewer elevated risk factors than those with high waist who are not obese ; in the us , obese men are only more likely than normal weight men without high waist to have elevated glycated hemoglobin , fasting glucose and crp .", "english men who are obese have more elevated risk : both blood pressure indicators , hdl cholesterol , triglycerides , glycated hemoglobin , and crp .", "very obese men in england have the same elevated risk factors with the exception of dbp .", "very obese men in the us are more likely to have elevated fasting glucose in addition to crp and glycated hemoglobin .", "english and taiwanese women with normal weight and high waist are more likely to have elevated sbp , dbp , and glycosylated hemoglobin ; only british women with higher waist have significantly elevated triglycerides and only the taiwanese women had more hdl risk .", "high risk crp is more common among both american and english women with normal bmi and high waist , and this risk of elevated crp is also higher in the obese and very obese ( table 3(b ) ) .", "obese women in britain had elevations in the same markers as normal bmi and high waist english women , while obese women in the us only have elevated fasting glucose , glycated hemoglobin , and crp ; obese women in taiwan only had high dbp . with the exception of taiwan , levels of the inflammatory markers ( crp in the us and england ; il-6 in taiwan ) are more likely to be elevated among persons with a high waist and normal bmi , obese or very obese , compared to their normal bmi and normal waist counterparts . among men ,", "an increase in the biological risk summary score ( range 09 ) is associated with having a high waist relative to being of normal bmi and normal waist in all three countries ( table 4(a ) ) .", "being obese or very obese is also related to a higher biological risk summary score ( 09 ) for men in the us and england .", "these equations explain 6 to 11 percent of the variance in the summary indicator of biological risk .", "these relationships are similar for women ( table 4(b ) ) , with one exception : obese taiwanese women do not have a significantly increased biological risk compared to their normal weight and normal waist counterparts .", "when we consider the alternate summary score that excludes our indicators of inflammation ( range 08 ) , being obese or very obese is no longer associated with a higher biological risk summary score in us men compared to men with a normal bmi and normal waist when controls for health behaviors and medication use are included ( table 5(a ) ) .", "the size of the effects of the obesity categories is reduced on the 8-indicator summary measure in both england and the us , indicating the strong link between crp and obesity .", "the r is also reduced in these equations for england and the us . for taiwan ,", "women in england and taiwan , but not women in the us , with a higher waist but who are not obese have significantly higher physiological dysregulation compared to their normal bmi and normal waist counterparts .", "obese women in all three countries have elevated risk and the very obese have even higher risk ( table 5(b ) ) . the alternate biological risk summary measure ( 08 )", "yields different relationships between weight and physiological dysregulation in the us and taiwan . in the us ,", "only very obese women have a higher alternate biological risk summary score ( 08 ) . in taiwan , obese and normal bmi and high waist women exhibit higher alternate biological risk summary scores compared to their normal bmi and normal waist counterparts , except when smoking status , physical activity , and use of hypertensives are included .   figures 1(a ) and 1(b ) illustrate the predicted alternate biological risk score ( 08 ) for each weight category for men and women ( respectively ) aged 65 who are nonsmokers , do not engage in physical activity , and are currently taking antihypertensive medication .", "this figure allows for country comparisons of individuals with these characteristics within each weight category and across weight categories .", "the predicted values indicate that the us has the highest biological risk score within each respective weight category among men and women of the same age and lifestyle behaviors . with the exception of women in the normal bmi and high waist group ,", "england has the second highest biological risk score within each weight category , followed by taiwan . among 65-year - old women with the noted lifestyle behaviors and with a normal bmi and high waist", "when we consider the alternate bmi cutpoint for obesity in taiwan ( bmi  27  kg / m ) , our findings for the individual biomarkers and summary measures of biological risk are similar to using the bmi  30  kg / m cutoff for taiwan except that the category normal weight with high waist no longer differs from the omitted category ( results not shown ) .", "this study observes three general findings about how biological risk is associated with obesity in three countries that differ in lifestyle and culture .", "first , obesity is associated with physiological dysregulation in all countries with differences in the links between specific indicators of biological risk and obesity .", "generally , obesity in england is associated with hypertension , dyslipidemia , and elevated glycated hemoglobin ; americans who are obese are not more likely to have hypertension . in taiwan ,", "obese women are more likely to have elevated dbp and obese men have an increased risk of elevated triglycerides and glycated hemoglobin compared to their nonobese , normal waist counterparts .", "our biological risk summary scores indicate that at all levels of weight physiological dysregulation was highest in the us , followed by england ( with one exception ) , with taiwanese exhibiting the lowest biological risk in all groups among the three countries .", "second , these relationships remain after controlling for demographic factors , participation in physical activity , and other behavioral factors .", "third , similar to obese older adults , high waist individuals with normal bmi also exhibit greater physiological dysregulation in all countries compared to their normal bmi and normal waist counterparts .", "our finding of a higher physiological dysregulation , as shown by the alternate biological risk summary score , in taiwan compared to the us and england could be due to a couple of potential explanations .", "the prevalence of obesity in the us and england is much higher than in taiwan , indicating an earlier initial rise in obesity relative to taiwan . from 1978 to 2002 , the proportion of obese americans and britons exhibited stark increases ( 1332% and 623% for men and women , resp . ) .", "the estimates for obesity prevalence in taiwan indicate a recent increase for men but not women . from 19931996 to 2000 - 2001 , the age - adjusted prevalence of obesity rose from 10.5% to 15.9% for men and declined from 13.2% to 10.7% in women .", "it may be that the lower levels of risk among older adults who have lived longer years with obesity could be a reflection of better pharmacologic control of physiological dysregulation ( e.g. , through statin use ) , which may in turn confer less biological risk in these populations compared to populations of currently obese taiwanese adults who may have more recently begun living with obesity .", "a second reason for the observed country differences in obesity may be due to differences in dietary habits and lifestyle .", "the us and england are two modern , western populations whose diets have been influenced by increased industrialization and have over time come to be characterized by high glycemic loads and high fatty acid composition .", "taiwan , on the other hand , represents a country that has experienced the effects of the industrial and scientific revolutions later than that of the us and england but is currently rapidly undergoing economic development and demographic change .", "the recent economic changes in taiwan may indicate that obese older adults in taiwan have more recently begun to consume high - fat diets , which could result in greater initial physiological dysregulation associated with access to western - influenced dietary habits . despite controlling for lifestyle behaviors thought to be linked with health , the country differences in obesity and physiological regulation remain .", "moreover , the consideration of antihypertensives does not alter our substantive conclusions on these associations .", "this suggests that despite the greater use of medications to treat hypertension in the us , obesity among americans is associated with greater overall biological risk than the other two countries .", "this is supported by findings from the general population of americans relative to england , which report that the us is faced with greater health disadvantages than england in adulthood   and across the life span .", "we also note differences in biological profiles of obese individuals between the two westernized countries : the us and england . the excess risk of hypertension associated with obesity in england was not found in the us .", "these differences may be due to the higher use of medications among americans compared to the english , with about 16% more men and 18% more women in the us aged 65 + taking antihypertensive medication compared to their british counterparts .", "the greater use of hypertensive medications in the us is also noted when compared to japan and countries across europe .  ", "two notable differences in country patterns of the relationship between obesity and physiological dysregulation by sex are found . among men in england and taiwan ,", "the order of magnitude of physiological dysregulation increases with higher weight categories ; however , this is not observed for us men .", "this difference may be due to our inability to consider statin use in england and taiwan , which may be particularly important in the relationship between obesity and physiological dysregulation for men .", "conversely , the importance of considering statin use may be less vital to understanding the country differences in the association between obesity and physiological dysregulation among women , given that the relationship for women is more consistent across countries , namely in the us and england .", "women , underweight corresponds with higher biological risk ( though nonsignificant ) compared to women with normal bmi and normal waist .", "underweight among men in taiwan is significantly associated with much lower biological risk than their normal bmi and normal weight counterparts .", "further studies will be required to explore possible explanations for these differences in physiological dysregulation .", "the higher biological risk observed among normal bmi and high waist individuals relative to normal bmi and normal waist older adults builds upon previous studies that report on alternate indicators of body shape , which vary across countries .", "the importance of waist circumference is underscored by our current study , as well as a growing body of literature on the predictive value of waist circumference on indicators of health .", "higher rates of diabetes among older americans compared to britons have been accounted for by high waist circumference as opposed to bmi differences . additionally , increasing waist circumference is more predictive of greater risk of incident diabetes than bmi in middle - aged british men ( and the european prospective investigation into cancer and nutrition ( epic)-potsdam study ) .", "waist circumference , as an indicator of central fat mass , is thought to be more strongly associated with disease risk , and in our case with physiological dysregulation , compared to bmi , which is considered a cruder index of adiposity .", "banks and colleagues   cite differences in physical activity , diet and greater psychosocial environmental challenges in america compared to england as potential mechanisms linking central adiposity and type 2 diabetes .", "our study considers some of these possible mechanisms ( e.g. , physical activity and antihypertensive use ) but finds that they explain little of the relationship between biological risk and adiposity among the three countries .", "together , these results highlight the importance of considering waist circumference in investigating the links between indicators of health and adiposity .", "our finding of the biological risks associated with obesity among older taiwanese adults underscores the growing concern for risks associated with obesity in countries rapidly undergoing modernization . in comparing the biological risk of obese individuals among the three countries , we are able to use these international comparisons to our advantage to examine how differences in modernization influence the health of older adults in different populations", "this may have potential health policy implications that underscore the importance of addressing and controlling the rising obesity epidemic that has become most widespread in countries , like the us and england , that have long experienced high economic growth and in countries currently undergoing rapid economic development .", "the increasing use of biological information to inform our understanding of health represents an innovative method in biodemography that will further contribute to the testing of current comparative theory and the potential creation of new paradigms surrounding the influence of modernization on health .", "first is the use of a broad range of biological markers across three large - scale population surveys . the inclusion of biological information as objective precursors of health allows , to some extent , a fairly comparable comparison of indicators of health across the different populations .", "an exception to this uniform comparison of biomarkers across the three surveys is our use of inflammatory marker crp in the us and england and our inclusion of a different marker of inflammation ( il-6 ) in taiwan . of note , crp seems to be more strongly associated with obesity than il-6 .", "a growing body of literature has made distinctions between bmi and waist circumference , namely , suggesting that waist circumference is a better indicator of abdominal obesity , which in turn has been associated with obesity - related health risks .", "our findings generally report a similar association between increased biological risk and ( 1 ) normal bmi and high waist and ( 2 ) obese and high waist .", "using the us nhanes ,   reported that when both waist circumference and bmi were included in their analyses , only waist circumference was a significant predictor of comorbidity . although this and other studies have suggested that waist circumference may be a better indicator of obesity and risk for adverse health outcomes , our study finds the two indicators to be similarly associated with biological risk across the three countries .", "first , we examine population - based data from three countries at a single time point .", "future studies of longitudinal data will allow for further investigations of the potential role of obesity on biological risk observed in the current associations .", "second , we do not have measures of some lifestyle and medical behaviors for some of the datasets ( e.g. , statin use ) , which likely influence the relationship between obesity and biological risk .", "as such , we are unable to include such factors in our analyses of all three countries .", "it is possible that these lifestyle behaviors are key explanatory factors to the noted cross - country differences in obesity - related biological risk .", "the cross - country differences in the relationship between increased biological risk for individuals who are obese and have a high waist underscore potential differences in health and lifestyle behaviors .", "these behaviors may be a result of country differences in economic development that we are not able to observe in this study .", "the country differences in the links between obesity and physiological dysregulation are particularly marked when comparing obesity among taiwanese older adults relative to westernized populations , such as the us and england .", "further examination of these relationships over time and across other countries will contribute to our understanding of the potential factors responsible for these country - specific variations in biological risk , as obesity becomes increasingly more prevalent and older adults in various countries live more years with obesity and increased adiposity ."], "abstract_text": ["<S> excess weight has generally been associated with adverse health outcomes ; however , the link between overweight and health outcomes may vary with socioeconomic , cultural , and epidemiological conditions . </S>", "<S> we examine associations of weight with indicators of biological risk in three nationally representative populations : the us national health and nutrition examination survey , the english longitudinal study of ageing , and the social environment and biomarkers of aging study in taiwan . </S>", "<S> indicators of biological risk were compared for obese ( defined using body mass index ( bmi ) and waist circumference ) and normal weight individuals aged 54 + . generally , obesity in england </S>", "<S> was associated with elevated risk for more markers examined ; obese americans also had elevated risks except that they did not have elevated blood pressure ( bp ) . including waist circumference in our consideration of bmi indicated different links between obesity and waist size across countries ; we found higher physiological dysregulation among those with high waist but normal bmi compared to those with normal waist and normal bmi . </S>", "<S> americans had the highest levels of biological risk in all weight / waist groups . </S>", "<S> cross - country variation in biological risk associated with obesity may reflect differences in health behaviors , lifestyle , medication use , and culture . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["rising levels of obesity are becoming a worldwide phenomenon and are increasingly identified as a health problem across the globe [ 14 ] .", "higher weight has been associated with adverse health indicators and outcomes , including cardiovascular disease [ 512 ] , stroke [ 5 , 13 ] , cognitive and functional decline [ 1418 ] , metabolic syndrome [ 19 , 20 ] , inflammation [ 21 , 22 ] , and mortality [ 20 , 2325 ] .", "obesity among aging populations is relatively recent and aging among people who have been obese for much of their lives is also a new phenomenon . from 1980 to 2004 , the prevalence of obesity in the us has continued to rise from about 17% to 25% for men aged 5059 . while obesity in england has also increased during this period , from approximately 9% in 1980 to 15% in 2004 for men aged 5564 , the level of obesity remains much lower in england .", "additionally , the difference in obesity between the us and england is more pronounced for women .", "the level of obesity in us women was about 24% in 1980 and rose to 37% in 2004 ( age 5059 ) ; in england , levels for women were 9% in 1980 and 14% in 2004 ( age 5564 ) .", "the aim of this paper is to investigate differences in how obesity relates to indicators of physiological dysregulation in men and women of diverse populations .", "this comparison will lead to an improved understanding of how obesity might be differentially related to health and mortality across cultures and lifestyles .", "obesity was relatively rare in most populations until the second part of the last century , but it has now become common in many countries", ". the us population is recognized as among the most obese in the world , although many other countries are now approaching the us level and most countries are experiencing increasing obesity .", "this worldwide obesity epidemic began with the epidemiological revolution and the virtual elimination of infectious disease ; the decline in manual labor needed to provide sustenance with the industrial revolution ; and the increasing availability and decreasing cost of food [ 2729 ] .", "obesity reflects some combination of calorie intake , diet content , and amount of physical activity . in some cultures ,", "lack of physical activity can be a more important determinant of obesity ; in other cultures , overeating or food composition may be the more important determinant of obesity .", "it is also true that within countries , individuals could differ in the causes of obesity .", "for instance , changes in activity might be more characteristic of women or men resulting in different reasons for obesity by gender .", "these differences may affect how obesity is related to other risk factors for poor health , and it may determine the overall health risk associated with obesity . if physical activity is maintained , the overall effect of obesity may be less than if the activity is not .", "one indication of the cause of obesity may be the relationship between waist size and weight [ 30 , 31 ] . in societies where obese people are more physically active , waist size of the obese", "waist circumference has also been linked to late life mortality , where high waist circumference has been associated with increased mortality among men and women in the netherlands , while high bmi was not associated with mortality .", "this paper builds upon current obesity research by using both bmi and waist circumference to quantify obesity in order to determine how a combined indicator of weight and adiposity is related to physiological dysregulation in populations with different cultures , diets , behaviors , and epidemiological histories .", "obesity has been related to many indicators of physiological dysregulation including cardiovascular risk factors such as hypertension   and metabolic dysregulation in lipid levels or insulin resistance .", "most studies that investigate the differences in biological risk associated with excess weight have examined western populations [ 33 , 36 ] .", "comparative studies on the health risks associated with obesity that examined the us and england reported that obese americans had an increased risk of diabetes and a higher waist circumference [ 37 , 38 ] .", "these studies suggested that differences in physical activity , diet , and social environments may explain these national differences .", "while these differences have been observed between the us and england , these two western countries have roughly similar life expectancy , levels of living , history , and culture even while the us has poorer health by a number of indicators of disease prevalence and biological risk [ 36 , 39 ] .", "comparative studies of the links between obesity and health outcomes and risk factors for obesity comparing western and non - western countries indicate important differences in the causes and consequences of obesity .", "a comparison of the association of disease with overweight and obesity in japan and the us indicated that the associations were stronger in the us than in japanese women and that there was no association in japanese men .", "links between social , demographic , and behavioral risk factors for obesity also differ markedly in japan , korea , and the us .", "the availability of biomarker data from taiwan  a middle - income country undergoing rapid economic growth , increasing obesity , and with life expectancy recently increasing to levels similar to that of the us and england  allows for investigation of the biological risk associated with obesity in a population characterized by very different cultural , behavioral , socioeconomic , and dietary parameters .", "we examine how elevated weight and obesity ( using an indicator that considers both bmi and waist circumference ) relate to having levels defined as clinical risk for cardiovascular , metabolic , and inflammatory markers in three aging societies that are now relatively similar in life expectancy but that differ in the timing of the epidemiological transition and obesity epidemic , history of economic development , socioeconomic levels , general lifestyle habits , health behaviors , and health care systems : the us , england , and taiwan .", "finding differences in the relationship between obesity and indicators of physiological dysregulation in these three aging societies will clarify whether increases in weight gain are equally problematic across all countries ."], ["we use data from three nationally representative samples : the us national health and nutrition examination survey ( nhanes , 20032006 ; n = 3855 ) , the english longitudinal study of ageing ( elsa , 2004 - 2005 ; n = 9139 ) , and the social environment and biomarkers of aging study ( sebas ) in taiwan ( 2000 ; n = 1023 ) .", "these surveys collect information on demographics , as well as anthropometric , physical , and biological measures .", "every year , approximately 5,000 individuals undergo detailed interviews and medical examinations , which include collection of several physiological measures .", "nhanes utilizes a complex sampling design , and when weights are applied , the sample is representative of the noninstitutionalized american population .", "we use the 20032006 data since nhanes data is released in two - year intervals , and this sample is centered on 2004 - 2005 , which matches the period in which elsa was collected .", "for nhanes , we use individual - level data based on a sample of 1,513 fasting individuals aged 54 and older .", "elsa includes participants drawn from households responding to the health survey for england ( hse ) in 1998 , 1999 , and 2001   and is representative of the english population aged 50 and older .", "the core elsa sample ( wave 1 : 2002 - 2003 ) includes people living in an hse responding household who were born prior to march 1 , 1952 , and their partners who could be under age 50 . wave 4 of elsa ( 2008 - 2009 ) , which includes a nurse visit , includes wave 1 core members , if they are still alive and do not refuse further contact after the first interview at wave 1 .", "it also includes a refresher sample to maintain the age structure of the sample ( in waves 3 and 4 ) , and their partners . for elsa , we use individual - level data based on a sample of 7,384 individuals aged 54 and older .", "sebas is drawn from a follow - up survey of the survey of health and living status of the near elderly and elderly in taiwan ( also known as the taiwan longitudinal study of aging ( tlsa ) ) , a nationally representative survey of taiwanese adults ( including institutionalized individuals ) collected in 1989 , 1993 , 1996 , 1999 , and 2000 . in 2000 , a subsample of individuals was randomly selected for inclusion in sebas .", "sebas consists of adults aged 54 and older in 2000 , with in - home interviews and medical exams taken in a hospital . for sebas ,", "the sample averages 66.8 years of age in england , and the us and taiwan mean age is about the same ( table 1 ) .", "there are more men in taiwan ( 56% ) and england ( 53% ) and fewer in the us ( 44% ) .  ", "we examine the following indicators of physiological dysregulation often associated with obesity and also associated with increased risk for multiple adverse health outcomes and obesity [ 43 , 44 ] : ( 1 ) cardiovascular markers : high systolic ( sbp ) and diastolic blood pressure ( dbp ) ; ( 2 ) metabolic markers : high levels of blood lipids ( total and low - density lipoprotein ( ldl ) cholesterol , and fasting triglycerides ) , low levels of high - density lipoprotein ( hdl ) cholesterol , and high fasting glucose and glycated hemoglobin ; ( 3 ) high levels of inflammatory markers c - reactive protein ( crp ; available in nhanes and elsa ) and interleukin-6 ( il-6 ; available in sebas ) , as crp and il-6 have been positively associated with bmi . for each indicator", "we use clinical cutpoints or widely used research - based cutpoints to indicate high levels of risk which are shown in table 1 [ 39 , 43 , 45 ] .", "there has been debate as to the best indicator of obesity : body mass index ( bmi ) or waist circumference .", "waist circumference is thought to be a better measure of abdominal adiposity than bmi and a better indicator of risk of poor health outcomes , including all - cause and cardiovascular mortality [ 46 , 47 ] . for this reason", "we investigate the association between bmi and biomarkers across categories of bmi ( underweight < 18.5  kg / m , normal and overweight 18.529.9  kg / m , obese 3034.9  kg / m , and very obese 35  kg / m ) and waist circumference categorized as normal or high waist ( high waist : men 120  cm , women 88  cm ) . we create a composite measure of obesity and adiposity by categorizing individuals into five groups : ( 1 ) underweight and normal waist ( all underweight individuals had a normal waist circumference ) , ( 2 ) normal / overweight bmi ( termed normal bmi ) and normal waist circumference ( reference group ) , ( 3 ) normal / overweight bmi ( termed normal bmi ) and high waist circumference ( termed high waist ) , ( 4 ) obese and high waist ( all obese individuals had a high waist circumference ) , and ( 5 ) very obese .", "we also evaluate an alternate definition for obesity in taiwan based on bmi 27 , as it has been suggested by some that obesity levels should be differentially defined for asians [ 48 , 49 ] .", "a similar composite measure of obesity and adiposity was calculated using this alternate definition of bmi in taiwan . because these risk factors are all assumed to be associated with obesity and because dysregulation in multiple physiological systems has been shown to predict many of the poor health risk outcomes associated with aging , we also create two summary measures of risk based on the total number of at - risk levels of biomarkers , either 9 or 8 . because crp values for sebas are not available , this measure", "is not included in the 9-item summary measure for taiwan , but a summary measure ( range 09 ) was calculated for taiwan using il-6 , another indicator of inflammation , instead of the crp values included for the us and england . a second alternate summary measure of biological risk , excluding the inflammatory marker ( range 08 ) ,", "biological risk summary scores were computed for individuals who had missing values on no more than 3 biological markers .", "we examine multiple covariates in our investigation of the relationship between obesity and biological risk .", "self - reported use of antihypertensives was determined in all three countries , and use of lipid - lowering statins was only asked in the us sample .", "dichotomous variables were created to indicate whether the respondent reported being a current smoker and participating in at least moderate physical activity for exercise ( e.g. , brisk walking , running , or swimming ) in the past 30 days ( for the us and england ) or generally exercising once a week ( for taiwan ) .  ", "we use logistic regression models to determine the odds of having at - risk levels of a specific biomarker for obese men and women among the three populations . for all countries ,", "the comparison group for bmi and waist circumference is the normal bmi and normal waist group .", "the regressions included indicators of age , use of antihypertensives , current smoking status , and having exercised in the past 30 days .", "ordinary least squares ( ols ) regression models were used to determine the relationship between the summary measures of biological risk and the composite measure of obesity and adiposity ."], ["we begin by examining national differences in the high risk levels of individual biomarkers ( table 1 ) .", "low levels  or high risk levels  of hdl cholesterol are also more common in taiwan .", "high total and ldl cholesterol is more common among the english ; lower levels of plasma glucose , crp , and glycated hemoglobin are also characteristic of the english .", "few adults in england have elevated levels of fasting glucose ( 2.2% ) , while this is observed in 17.3% and 13.2% of american and taiwanese adults . \n", "most people in this age range are in the normal to overweight category ( 64.7% , 67.2% , and 89.4% in the us , england , and taiwan , respectively ) ( table 2(a ) ) .", "americans are more likely to be obese ( 33.7% ) compared to the english ( 32.1% ) and taiwanese ( 7.2% ) . among the obese , americans are much more likely to be very obese : 13.4% of the total us sample , 10.1% in england , and about 1% in taiwan .", "both among the obese and very obese , the average bmi is higher in the us and england compared to taiwan .", "few are underweight in any country ( 1.7% , 0.8% , and 3.4% in the us , england , and taiwan , resp . ) .  ", "when we examine waist circumference , the us has the highest average waist circumference , with 65.5% of americans , 55.9% of english , and 15.8% of taiwanese having a high waist size ( table 2(a ) ) .", "this means that high waist characterizes a substantial number of those who would be categorized as normal weight in the us and england . among those in the normal and overweight group about half ( 49.5% ) of americans and a third ( 36.4% ) of the english", "almost all obese individuals have a high waist in the us and england ( 98.3% in the us and 96.5% in england ) , but only 78.4% of the obese in taiwan also have a high waist . when we use the alternate obese cutpoint of 27  kg / m in taiwan , less than half of the obese individuals have a high waist ( not shown here ) . \n", "americans exhibit the highest proportion of the older population taking antihypertensive medication ( 47.1% ) ( table 1 ) .", "the percentage who reports taking antihypertensives is lower in england ( 32.0% ) and taiwan ( 28.6% ) .", "americans are more likely to be current smokers ( 24.5% ) than persons in taiwan ( 22.5% ) and england ( 13.9% ) .", "more than half of the population in all countries report having exercised in the past 30 days , with more english exercising ( 82.2% ) compared to taiwan ( 61.4% ) and the us ( 58.5% ) . \n", "men with normal bmi and high waist have a greater likelihood than men with normal bmi and normal waist size of having high - risk levels of triglycerides in all three countries . in the us and england ,", "men with high waist are more likely to have high levels of glycosylated hemoglobin and higher crp ; fasting glucose is also elevated among this group in the us ( table 3(a ) ) .", "men who are obese in the us have fewer elevated risk factors than those with high waist who are not obese ; in the us , obese men are only more likely than normal weight men without high waist to have elevated glycated hemoglobin , fasting glucose and crp .", "english men who are obese have more elevated risk : both blood pressure indicators , hdl cholesterol , triglycerides , glycated hemoglobin , and crp .", "very obese men in england have the same elevated risk factors with the exception of dbp .", "very obese men in the us are more likely to have elevated fasting glucose in addition to crp and glycated hemoglobin .", "english and taiwanese women with normal weight and high waist are more likely to have elevated sbp , dbp , and glycosylated hemoglobin ; only british women with higher waist have significantly elevated triglycerides and only the taiwanese women had more hdl risk .", "high risk crp is more common among both american and english women with normal bmi and high waist , and this risk of elevated crp is also higher in the obese and very obese ( table 3(b ) ) .", "obese women in britain had elevations in the same markers as normal bmi and high waist english women , while obese women in the us only have elevated fasting glucose , glycated hemoglobin , and crp ; obese women in taiwan only had high dbp . with the exception of taiwan , levels of the inflammatory markers ( crp in the us and england ; il-6 in taiwan ) are more likely to be elevated among persons with a high waist and normal bmi , obese or very obese , compared to their normal bmi and normal waist counterparts . among men ,", "an increase in the biological risk summary score ( range 09 ) is associated with having a high waist relative to being of normal bmi and normal waist in all three countries ( table 4(a ) ) .", "being obese or very obese is also related to a higher biological risk summary score ( 09 ) for men in the us and england .", "these equations explain 6 to 11 percent of the variance in the summary indicator of biological risk .", "these relationships are similar for women ( table 4(b ) ) , with one exception : obese taiwanese women do not have a significantly increased biological risk compared to their normal weight and normal waist counterparts .", "when we consider the alternate summary score that excludes our indicators of inflammation ( range 08 ) , being obese or very obese is no longer associated with a higher biological risk summary score in us men compared to men with a normal bmi and normal waist when controls for health behaviors and medication use are included ( table 5(a ) ) .", "the size of the effects of the obesity categories is reduced on the 8-indicator summary measure in both england and the us , indicating the strong link between crp and obesity .", "the r is also reduced in these equations for england and the us . for taiwan ,", "women in england and taiwan , but not women in the us , with a higher waist but who are not obese have significantly higher physiological dysregulation compared to their normal bmi and normal waist counterparts .", "obese women in all three countries have elevated risk and the very obese have even higher risk ( table 5(b ) ) . the alternate biological risk summary measure ( 08 )", "yields different relationships between weight and physiological dysregulation in the us and taiwan . in the us ,", "only very obese women have a higher alternate biological risk summary score ( 08 ) . in taiwan , obese and normal bmi and high waist women exhibit higher alternate biological risk summary scores compared to their normal bmi and normal waist counterparts , except when smoking status , physical activity , and use of hypertensives are included .   figures 1(a ) and 1(b ) illustrate the predicted alternate biological risk score ( 08 ) for each weight category for men and women ( respectively ) aged 65 who are nonsmokers , do not engage in physical activity , and are currently taking antihypertensive medication .", "this figure allows for country comparisons of individuals with these characteristics within each weight category and across weight categories .", "the predicted values indicate that the us has the highest biological risk score within each respective weight category among men and women of the same age and lifestyle behaviors . with the exception of women in the normal bmi and high waist group ,", "england has the second highest biological risk score within each weight category , followed by taiwan . among 65-year - old women with the noted lifestyle behaviors and with a normal bmi and high waist", "when we consider the alternate bmi cutpoint for obesity in taiwan ( bmi  27  kg / m ) , our findings for the individual biomarkers and summary measures of biological risk are similar to using the bmi  30  kg / m cutoff for taiwan except that the category normal weight with high waist no longer differs from the omitted category ( results not shown ) ."], ["this study observes three general findings about how biological risk is associated with obesity in three countries that differ in lifestyle and culture .", "first , obesity is associated with physiological dysregulation in all countries with differences in the links between specific indicators of biological risk and obesity .", "generally , obesity in england is associated with hypertension , dyslipidemia , and elevated glycated hemoglobin ; americans who are obese are not more likely to have hypertension . in taiwan ,", "obese women are more likely to have elevated dbp and obese men have an increased risk of elevated triglycerides and glycated hemoglobin compared to their nonobese , normal waist counterparts .", "our biological risk summary scores indicate that at all levels of weight physiological dysregulation was highest in the us , followed by england ( with one exception ) , with taiwanese exhibiting the lowest biological risk in all groups among the three countries .", "second , these relationships remain after controlling for demographic factors , participation in physical activity , and other behavioral factors .", "third , similar to obese older adults , high waist individuals with normal bmi also exhibit greater physiological dysregulation in all countries compared to their normal bmi and normal waist counterparts .", "our finding of a higher physiological dysregulation , as shown by the alternate biological risk summary score , in taiwan compared to the us and england could be due to a couple of potential explanations .", "the prevalence of obesity in the us and england is much higher than in taiwan , indicating an earlier initial rise in obesity relative to taiwan . from 1978 to 2002 , the proportion of obese americans and britons exhibited stark increases ( 1332% and 623% for men and women , resp . ) .", "the estimates for obesity prevalence in taiwan indicate a recent increase for men but not women . from 19931996 to 2000 - 2001 , the age - adjusted prevalence of obesity rose from 10.5% to 15.9% for men and declined from 13.2% to 10.7% in women .", "it may be that the lower levels of risk among older adults who have lived longer years with obesity could be a reflection of better pharmacologic control of physiological dysregulation ( e.g. , through statin use ) , which may in turn confer less biological risk in these populations compared to populations of currently obese taiwanese adults who may have more recently begun living with obesity .", "a second reason for the observed country differences in obesity may be due to differences in dietary habits and lifestyle .", "the us and england are two modern , western populations whose diets have been influenced by increased industrialization and have over time come to be characterized by high glycemic loads and high fatty acid composition .", "taiwan , on the other hand , represents a country that has experienced the effects of the industrial and scientific revolutions later than that of the us and england but is currently rapidly undergoing economic development and demographic change .", "the recent economic changes in taiwan may indicate that obese older adults in taiwan have more recently begun to consume high - fat diets , which could result in greater initial physiological dysregulation associated with access to western - influenced dietary habits . despite controlling for lifestyle behaviors thought to be linked with health , the country differences in obesity and physiological regulation remain .", "moreover , the consideration of antihypertensives does not alter our substantive conclusions on these associations .", "this suggests that despite the greater use of medications to treat hypertension in the us , obesity among americans is associated with greater overall biological risk than the other two countries .", "this is supported by findings from the general population of americans relative to england , which report that the us is faced with greater health disadvantages than england in adulthood   and across the life span .", "we also note differences in biological profiles of obese individuals between the two westernized countries : the us and england . the excess risk of hypertension associated with obesity in england was not found in the us .", "these differences may be due to the higher use of medications among americans compared to the english , with about 16% more men and 18% more women in the us aged 65 + taking antihypertensive medication compared to their british counterparts .", "the greater use of hypertensive medications in the us is also noted when compared to japan and countries across europe .  ", "two notable differences in country patterns of the relationship between obesity and physiological dysregulation by sex are found . among men in england and taiwan ,", "the order of magnitude of physiological dysregulation increases with higher weight categories ; however , this is not observed for us men .", "this difference may be due to our inability to consider statin use in england and taiwan , which may be particularly important in the relationship between obesity and physiological dysregulation for men .", "conversely , the importance of considering statin use may be less vital to understanding the country differences in the association between obesity and physiological dysregulation among women , given that the relationship for women is more consistent across countries , namely in the us and england .", "women , underweight corresponds with higher biological risk ( though nonsignificant ) compared to women with normal bmi and normal waist .", "underweight among men in taiwan is significantly associated with much lower biological risk than their normal bmi and normal weight counterparts .", "further studies will be required to explore possible explanations for these differences in physiological dysregulation .", "the higher biological risk observed among normal bmi and high waist individuals relative to normal bmi and normal waist older adults builds upon previous studies that report on alternate indicators of body shape , which vary across countries .", "the importance of waist circumference is underscored by our current study , as well as a growing body of literature on the predictive value of waist circumference on indicators of health .", "higher rates of diabetes among older americans compared to britons have been accounted for by high waist circumference as opposed to bmi differences . additionally , increasing waist circumference is more predictive of greater risk of incident diabetes than bmi in middle - aged british men ( and the european prospective investigation into cancer and nutrition ( epic)-potsdam study ) .", "waist circumference , as an indicator of central fat mass , is thought to be more strongly associated with disease risk , and in our case with physiological dysregulation , compared to bmi , which is considered a cruder index of adiposity .", "banks and colleagues   cite differences in physical activity , diet and greater psychosocial environmental challenges in america compared to england as potential mechanisms linking central adiposity and type 2 diabetes .", "our study considers some of these possible mechanisms ( e.g. , physical activity and antihypertensive use ) but finds that they explain little of the relationship between biological risk and adiposity among the three countries .", "together , these results highlight the importance of considering waist circumference in investigating the links between indicators of health and adiposity .", "our finding of the biological risks associated with obesity among older taiwanese adults underscores the growing concern for risks associated with obesity in countries rapidly undergoing modernization . in comparing the biological risk of obese individuals among the three countries , we are able to use these international comparisons to our advantage to examine how differences in modernization influence the health of older adults in different populations", "this may have potential health policy implications that underscore the importance of addressing and controlling the rising obesity epidemic that has become most widespread in countries , like the us and england , that have long experienced high economic growth and in countries currently undergoing rapid economic development .", "the increasing use of biological information to inform our understanding of health represents an innovative method in biodemography that will further contribute to the testing of current comparative theory and the potential creation of new paradigms surrounding the influence of modernization on health .", "first is the use of a broad range of biological markers across three large - scale population surveys . the inclusion of biological information as objective precursors of health allows , to some extent , a fairly comparable comparison of indicators of health across the different populations .", "an exception to this uniform comparison of biomarkers across the three surveys is our use of inflammatory marker crp in the us and england and our inclusion of a different marker of inflammation ( il-6 ) in taiwan . of note , crp seems to be more strongly associated with obesity than il-6 .", "a growing body of literature has made distinctions between bmi and waist circumference , namely , suggesting that waist circumference is a better indicator of abdominal obesity , which in turn has been associated with obesity - related health risks .", "our findings generally report a similar association between increased biological risk and ( 1 ) normal bmi and high waist and ( 2 ) obese and high waist .", "using the us nhanes ,   reported that when both waist circumference and bmi were included in their analyses , only waist circumference was a significant predictor of comorbidity . although this and other studies have suggested that waist circumference may be a better indicator of obesity and risk for adverse health outcomes , our study finds the two indicators to be similarly associated with biological risk across the three countries .", "first , we examine population - based data from three countries at a single time point .", "future studies of longitudinal data will allow for further investigations of the potential role of obesity on biological risk observed in the current associations .", "second , we do not have measures of some lifestyle and medical behaviors for some of the datasets ( e.g. , statin use ) , which likely influence the relationship between obesity and biological risk .", "as such , we are unable to include such factors in our analyses of all three countries .", "it is possible that these lifestyle behaviors are key explanatory factors to the noted cross - country differences in obesity - related biological risk .", "the cross - country differences in the relationship between increased biological risk for individuals who are obese and have a high waist underscore potential differences in health and lifestyle behaviors .", "these behaviors may be a result of country differences in economic development that we are not able to observe in this study .", "the country differences in the links between obesity and physiological dysregulation are particularly marked when comparing obesity among taiwanese older adults relative to westernized populations , such as the us and england .", "further examination of these relationships over time and across other countries will contribute to our understanding of the potential factors responsible for these country - specific variations in biological risk , as obesity becomes increasingly more prevalent and older adults in various countries live more years with obesity and increased adiposity ."]]}
{"article_id": "PMC3830274", "article_text": ["male macroprolactinomas ( mprl ) are usually revealed by headaches , visual troubles and gonadal insufficiency .", "suppurative meningitis ( sm ) , a life - threatening condition , is scarcely observed in subjects with macro tumors secreting prolactin ( prl ) and in other pituitary tumors ( pt ) .", "however , in some very rare cases it can be a primary presentation or appear after radiotherapy or medical treatment used for tumors destroying the sellar floor and/or the skull base .", "this destruction leads to cerebral spinal fluid ( csf ) leak , which can act as an entry portal for organisms predisposing to meningitis .", "our aim was to analyze sm frequency among male mprl deemed to be very invasive tumors , to report our cases and analyze the circumstances under which the dangerous neurological complication appeared .", "in this retrospective study , we analyzed 82 subjects with mprl to look for symptoms , clinical signs and biological proof of sm .", ", we took into account medical history , clinical examination , routine exploration , csf analysis and hormonal assessment .", "that one was based on prl , growth hormone ( gh ) , insulin growth hormone ( igf1 ) , cortisol , adrenocorticotropic hormone , testosterone , follicle stimulating hormone , luteinizing hormone , thyroid stimulating hormone and free thyroxin .", "radiological assessment was based on cerebral computed tomography scan and/or magnetic resonance imaging ( mri ) .", "three were first hospitalized in the department of infectious diseases for typical symptoms and signs of sm ; that one was proved by csf biochemical analyses .", "the fourth one was hospitalized in our department for the fourth episode of sm which was proved by csf analysis after lumbar puncture .", "cerebral mri showed a pt invading cavernous sinuses , the sphenoid sinus and the brain [ figure 1 ] . hormonal assessment [ table 1 ] demonstrated high prl with gonadotroph deficit .", "then , the pt was treated with dopamine agonists that were successful on prl ( 25 ng / ml ) and on tumor size [ figure 1 ] . after a follow - up of 7 years ,", "invasive and aggressive pituitary tumor destroying the sella floor , responsible for suppurative meningitis ( sm ) in a 22-year - old man .", "( a ) before bromocriptine ; ( b ) after treatment , bromocriptine intake and sm disappearance tumor size , hormonal assessment of our 4 patients , and bromocriptine dose used to reduce tumor volume and normalize prolactin .", "cerebral mri showed an invasive and multidirectional pt measuring 47 mm  40 mm  30 mm destroying the sella floor and filling the sphenoid sinus [ figure 2 ] .", "the pt was secreting prl [ table 1 ] . that one was normalized by bromocriptine and the tumor size was significantly reduced .", "large invasive tumor destroying the sella floor , responsible for suppurative meningitis in a 49-year - old man .", "( a ) before medical treatment ; ( b ) liquification of the tumor after bromocriptine a man aged 25 , with a history of chronic otitis and arrested puberty , was diagnosed as a multidirectional prolactinoma destroying the sella floor and measuring 30 mm  30 mm  30 mm [ figure 3 ] .", "sm was sensitive to antibiotics . under dopamine agonists prl was normalized and the tumor volume decreased .", "the pituitary tumor filling the sphenoid sinus before bromocriptine ( a ) and after bromocriptine ( b ) a 29-year - old man was sent to our unit for a prolactinoma revealed by epilepsy crises .", "the pt was very large ( 68 mm  50 mm  50 mm ) and multidirectional with an extension to the chiasm , cavernous sinuses , posterior and nasal areas [ figure 4 ] .", "after 3 months he had rhinorrhea , then one episode of sm sterilized by antibiotics . sm never relapsed after a follow - up of over 2 years .", "mri showing a large tumor invading cavernous sinuses , the brain and sphenoid sinus ( a ) .", "after dopamine agonists ( b ) the tumor size was reduced the table 1 shows tumor sizes , hormonal parameters and bromocriptine dose which was used to treat the 4 prolactinomas .", "cerebral mri showed a pt invading cavernous sinuses , the sphenoid sinus and the brain [ figure 1 ] . hormonal assessment [ table 1 ] demonstrated high prl with gonadotroph deficit .", "then , the pt was treated with dopamine agonists that were successful on prl ( 25 ng / ml ) and on tumor size [ figure 1 ] . after a follow - up of 7 years ,", "invasive and aggressive pituitary tumor destroying the sella floor , responsible for suppurative meningitis ( sm ) in a 22-year - old man .", "( a ) before bromocriptine ; ( b ) after treatment , bromocriptine intake and sm disappearance tumor size , hormonal assessment of our 4 patients , and bromocriptine dose used to reduce tumor volume and normalize prolactin .", "the previous ones were proved in 1984 , 1988 and 1990 . clinical examination confirmed the meningeal syndrome and noticed rhinorrhea .", "cerebral mri showed an invasive and multidirectional pt measuring 47 mm  40 mm  30 mm destroying the sella floor and filling the sphenoid sinus [ figure 2 ] .", "the pt was secreting prl [ table 1 ] . that one was normalized by bromocriptine and the tumor size was significantly reduced .", "large invasive tumor destroying the sella floor , responsible for suppurative meningitis in a 49-year - old man .", "( a ) before medical treatment ; ( b ) liquification of the tumor after bromocriptine", "a man aged 25 , with a history of chronic otitis and arrested puberty , was diagnosed as a multidirectional prolactinoma destroying the sella floor and measuring 30 mm  30 mm  30 mm [ figure 3 ] .", "sm was sensitive to antibiotics . under dopamine agonists prl was normalized and the tumor volume decreased .", "the pituitary tumor filling the sphenoid sinus before bromocriptine ( a ) and after bromocriptine ( b )", "a 29-year - old man was sent to our unit for a prolactinoma revealed by epilepsy crises .", "the pt was very large ( 68 mm  50 mm  50 mm ) and multidirectional with an extension to the chiasm , cavernous sinuses , posterior and nasal areas [ figure 4 ] .", "after 3 months he had rhinorrhea , then one episode of sm sterilized by antibiotics .", "mri showing a large tumor invading cavernous sinuses , the brain and sphenoid sinus ( a ) .", "after dopamine agonists ( b ) the tumor size was reduced the table 1 shows tumor sizes , hormonal parameters and bromocriptine dose which was used to treat the 4 prolactinomas .", "when it happens , it is usually observed in post - operative period or after tumor reduction by radiotherapy , apoplexy or medicine intake .", "the situation is observed in people in whom meninges and sella floor are destroyed by an aggressive pt or an ectopic one arising from the skull base .", "our 4 cases can be explained by the fact that prolactinomas are the largest and the most invasive pt , especially in males .", "male prolactinomas often destroy the sellar floor and invade the sphenoid sinus , then the nasopharynx .", "the meningeal and bony breaches lead to intra and extradural spaces communication , which is responsible for bacterial infection of meninges and encephalic structures .", "the most difficult differential diagnosis with meningitis occurring in people with pt is pituitary apoplexy , which mimics perfectly bacterial meningitis symptoms .", "however , a real microbial meningitis with or without rhinorrhea can be observed in apoplexy .", "the true microbial meningitis secondary to pt is an exceptional phenomenon compared to pituitary apoplexy , which is relatively more frequent . to our best knowledge", "only 15 sm , revealing pituitary adenomas or appearing under medical treatment have been reported so far .", "csf leak may proceed or not sm , and can be totally unnoticed . according to lam et al . who analyzed 29 articles published between 1980 and 2011 there were only 52 pt with csf leak .", "the seven cases were divided in two groups : the primitive meningitis or meningitis occurring prior to any treatment of the pt , and secondary meningitis occurring after surgery and/or dopamine agonists intake as in our 4 observation .", "our personal research found 15 cases meningitis secondary to pt . if we add our 4 cases , one can totalize 19 cases . in this group", "most pt were prolactinomas , especially males ones . among this group 14 were primitive .", "secondary ones ( n = 5 ) appeared under medical treatment , preceded or not by surgery or radiotherapy .", "sellar floor destruction was obvious on radiographies in all except one case [ table 2 ] .", "their age ( varying between 22 and 69 years ) did not seem to be a determining factor , but the nature of the adenoma and sphenoid sinus invasion seem fundamental . according to ciatto and to lascelles et al .", "we find only one case concerning gh adenoma treated by somatostatin 's analogs . a case of recurrent meningitis which appeared many years after neurosurgery was observed in nelson syndrome too .", "curiously sm was not reported in mixed pt and gonadotroph adenomas which are usually very large and very invasive .", "contrary to many authors and in agreement with ciatto , we think the association of pituitary adenoma and meningitis is more common than it is generally supposed as we observed it in nearly 5% of our male mprl .", "so , one should think about it more often even if a cause of sm seems obvious from the first sight as in our 4 case who suffered from chronic otitis .", "radiological research for pt should be systematic , especially if headaches , visual troubles , and gonadal abnormalities with or without anterior or posterior pituitary deficiencies are present .", "physicians who deal with large pt should be aware of high risk spontaneous meningitis , but also of secondary sm which can appear 1 month to 4 years or even more after medical treatment initiation by dopamine agonists or somatostatin 's analogs or radiotherapy . on another hand", ", medical treatment initiation should be gradual , especially for huge tumors as rapid shrinkage may lead to csf leak apparition .", "if this situation appears , a medical solution may be tried using temporary lumbar puncture or a lumboperitoneal shunt until the bony defect is clogged by fibrosis .", "if this attitude fails , surgical repair is recommended although the reparation may represent a great challenge because of the many potential sites of the csf around the tumor . if surgical repair in not feasible some authors recommend a vaccination against pathogens of the upper respiratory tract such as streptococcus pneumonia , haemophilus influenza and neisseria meningitidis to prevent sm .", "three revealed the pt , and one was observed after medical treatment . in 2 cases", "the described cases emphasize the necessity of an early diagnosis and treatment of large and invasive pt , especially male mprl . medical treatment , which is now the gold standard for prolactinomas ,", "should be gradual and well monitored to avoid rhinorrhea or otorrhea which can lead to potentially fatal bacterial meningitis ."], "abstract_text": ["<S> background : suppurative meningitis ( sm ) or bacterial meningitis is a life - threatening condition , which is exceptionally due to pituitary tumors ( pt ) . our aim was to analyze its frequency among male macroprolactinomas ( mprl ) deemed to be aggressive , to report the cases we observed in our practice and describe the circumstances under which sm appeared.materials and methods : we retrospectively analyzed 82 male mprl in order to look for a history of well proved sm and the circumstances under which sm appeared . </S>", "<S> we also took into account the possibility of sm relapsing.results:four out of 82 male mprl had sm = 4.87% . </S>", "<S> three consulted for sm symptoms . </S>", "<S> sm was confirmed in infectious diseases department , but only one had rhinorrhea . hormonal assessment and </S>", "<S> cerebral magnetic resonance imaging pleaded for aggressive prolactinomas . </S>", "<S> after antibiotics , sm was sterilized . </S>", "<S> then , mprl were treated with bromocriptine , which normalized prolactin and reduced pt . </S>", "<S> sm never relapsed . </S>", "<S> the 4th case was hospitalized for a large multidirectional prolactinoma invading and/or arising from the skull base . </S>", "<S> he was operated on 3 times and then he was given bromocriptine . </S>", "<S> after 3 months , he had rhinorrhea and then sm which was successfully treated by antibiotics . </S>", "<S> sm never relapsed after tumor reduction.conclusion:sm was demonstrated in 4.87% . </S>", "<S> sm has revealed mprl in 3 cases and appeared after bromocriptine intake in the 4th one . </S>", "<S> endocrinologists should be aware of this severe condition , which can be avoided by repairing as soon as possible the bony defect secondary to aggressive tumors , unless it is clogged by fibrosis : what probably happened in our cases . </S>"], "labels": null, "section_names": ["I", "M", "R", "C", "Case 1", "Case 2", "Case 3", "Case 4", "D", "C"], "sections": [["male macroprolactinomas ( mprl ) are usually revealed by headaches , visual troubles and gonadal insufficiency .", "suppurative meningitis ( sm ) , a life - threatening condition , is scarcely observed in subjects with macro tumors secreting prolactin ( prl ) and in other pituitary tumors ( pt ) .", "however , in some very rare cases it can be a primary presentation or appear after radiotherapy or medical treatment used for tumors destroying the sellar floor and/or the skull base .", "this destruction leads to cerebral spinal fluid ( csf ) leak , which can act as an entry portal for organisms predisposing to meningitis .", "our aim was to analyze sm frequency among male mprl deemed to be very invasive tumors , to report our cases and analyze the circumstances under which the dangerous neurological complication appeared ."], ["in this retrospective study , we analyzed 82 subjects with mprl to look for symptoms , clinical signs and biological proof of sm .", ", we took into account medical history , clinical examination , routine exploration , csf analysis and hormonal assessment .", "that one was based on prl , growth hormone ( gh ) , insulin growth hormone ( igf1 ) , cortisol , adrenocorticotropic hormone , testosterone , follicle stimulating hormone , luteinizing hormone , thyroid stimulating hormone and free thyroxin .", "radiological assessment was based on cerebral computed tomography scan and/or magnetic resonance imaging ( mri ) ."], ["three were first hospitalized in the department of infectious diseases for typical symptoms and signs of sm ; that one was proved by csf biochemical analyses .", "the fourth one was hospitalized in our department for the fourth episode of sm which was proved by csf analysis after lumbar puncture ."], ["cerebral mri showed a pt invading cavernous sinuses , the sphenoid sinus and the brain [ figure 1 ] . hormonal assessment [ table 1 ] demonstrated high prl with gonadotroph deficit .", "then , the pt was treated with dopamine agonists that were successful on prl ( 25 ng / ml ) and on tumor size [ figure 1 ] . after a follow - up of 7 years ,", "invasive and aggressive pituitary tumor destroying the sella floor , responsible for suppurative meningitis ( sm ) in a 22-year - old man .", "( a ) before bromocriptine ; ( b ) after treatment , bromocriptine intake and sm disappearance tumor size , hormonal assessment of our 4 patients , and bromocriptine dose used to reduce tumor volume and normalize prolactin .", "cerebral mri showed an invasive and multidirectional pt measuring 47 mm  40 mm  30 mm destroying the sella floor and filling the sphenoid sinus [ figure 2 ] .", "the pt was secreting prl [ table 1 ] . that one was normalized by bromocriptine and the tumor size was significantly reduced .", "large invasive tumor destroying the sella floor , responsible for suppurative meningitis in a 49-year - old man .", "( a ) before medical treatment ; ( b ) liquification of the tumor after bromocriptine a man aged 25 , with a history of chronic otitis and arrested puberty , was diagnosed as a multidirectional prolactinoma destroying the sella floor and measuring 30 mm  30 mm  30 mm [ figure 3 ] .", "sm was sensitive to antibiotics . under dopamine agonists prl was normalized and the tumor volume decreased .", "the pituitary tumor filling the sphenoid sinus before bromocriptine ( a ) and after bromocriptine ( b ) a 29-year - old man was sent to our unit for a prolactinoma revealed by epilepsy crises .", "the pt was very large ( 68 mm  50 mm  50 mm ) and multidirectional with an extension to the chiasm , cavernous sinuses , posterior and nasal areas [ figure 4 ] .", "after 3 months he had rhinorrhea , then one episode of sm sterilized by antibiotics . sm never relapsed after a follow - up of over 2 years .", "mri showing a large tumor invading cavernous sinuses , the brain and sphenoid sinus ( a ) .", "after dopamine agonists ( b ) the tumor size was reduced the table 1 shows tumor sizes , hormonal parameters and bromocriptine dose which was used to treat the 4 prolactinomas ."], ["cerebral mri showed a pt invading cavernous sinuses , the sphenoid sinus and the brain [ figure 1 ] . hormonal assessment [ table 1 ] demonstrated high prl with gonadotroph deficit .", "then , the pt was treated with dopamine agonists that were successful on prl ( 25 ng / ml ) and on tumor size [ figure 1 ] . after a follow - up of 7 years ,", "invasive and aggressive pituitary tumor destroying the sella floor , responsible for suppurative meningitis ( sm ) in a 22-year - old man .", "( a ) before bromocriptine ; ( b ) after treatment , bromocriptine intake and sm disappearance tumor size , hormonal assessment of our 4 patients , and bromocriptine dose used to reduce tumor volume and normalize prolactin ."], ["the previous ones were proved in 1984 , 1988 and 1990 . clinical examination confirmed the meningeal syndrome and noticed rhinorrhea .", "cerebral mri showed an invasive and multidirectional pt measuring 47 mm  40 mm  30 mm destroying the sella floor and filling the sphenoid sinus [ figure 2 ] .", "the pt was secreting prl [ table 1 ] . that one was normalized by bromocriptine and the tumor size was significantly reduced .", "large invasive tumor destroying the sella floor , responsible for suppurative meningitis in a 49-year - old man .", "( a ) before medical treatment ; ( b ) liquification of the tumor after bromocriptine"], ["a man aged 25 , with a history of chronic otitis and arrested puberty , was diagnosed as a multidirectional prolactinoma destroying the sella floor and measuring 30 mm  30 mm  30 mm [ figure 3 ] .", "sm was sensitive to antibiotics . under dopamine agonists prl was normalized and the tumor volume decreased .", "the pituitary tumor filling the sphenoid sinus before bromocriptine ( a ) and after bromocriptine ( b )"], ["a 29-year - old man was sent to our unit for a prolactinoma revealed by epilepsy crises .", "the pt was very large ( 68 mm  50 mm  50 mm ) and multidirectional with an extension to the chiasm , cavernous sinuses , posterior and nasal areas [ figure 4 ] .", "after 3 months he had rhinorrhea , then one episode of sm sterilized by antibiotics .", "mri showing a large tumor invading cavernous sinuses , the brain and sphenoid sinus ( a ) .", "after dopamine agonists ( b ) the tumor size was reduced the table 1 shows tumor sizes , hormonal parameters and bromocriptine dose which was used to treat the 4 prolactinomas ."], ["when it happens , it is usually observed in post - operative period or after tumor reduction by radiotherapy , apoplexy or medicine intake .", "the situation is observed in people in whom meninges and sella floor are destroyed by an aggressive pt or an ectopic one arising from the skull base .", "our 4 cases can be explained by the fact that prolactinomas are the largest and the most invasive pt , especially in males .", "male prolactinomas often destroy the sellar floor and invade the sphenoid sinus , then the nasopharynx .", "the meningeal and bony breaches lead to intra and extradural spaces communication , which is responsible for bacterial infection of meninges and encephalic structures .", "the most difficult differential diagnosis with meningitis occurring in people with pt is pituitary apoplexy , which mimics perfectly bacterial meningitis symptoms .", "however , a real microbial meningitis with or without rhinorrhea can be observed in apoplexy .", "the true microbial meningitis secondary to pt is an exceptional phenomenon compared to pituitary apoplexy , which is relatively more frequent . to our best knowledge", "only 15 sm , revealing pituitary adenomas or appearing under medical treatment have been reported so far .", "csf leak may proceed or not sm , and can be totally unnoticed . according to lam et al . who analyzed 29 articles published between 1980 and 2011 there were only 52 pt with csf leak .", "the seven cases were divided in two groups : the primitive meningitis or meningitis occurring prior to any treatment of the pt , and secondary meningitis occurring after surgery and/or dopamine agonists intake as in our 4 observation .", "our personal research found 15 cases meningitis secondary to pt . if we add our 4 cases , one can totalize 19 cases . in this group", "most pt were prolactinomas , especially males ones . among this group 14 were primitive .", "secondary ones ( n = 5 ) appeared under medical treatment , preceded or not by surgery or radiotherapy .", "sellar floor destruction was obvious on radiographies in all except one case [ table 2 ] .", "their age ( varying between 22 and 69 years ) did not seem to be a determining factor , but the nature of the adenoma and sphenoid sinus invasion seem fundamental . according to ciatto and to lascelles et al .", "we find only one case concerning gh adenoma treated by somatostatin 's analogs . a case of recurrent meningitis which appeared many years after neurosurgery was observed in nelson syndrome too .", "curiously sm was not reported in mixed pt and gonadotroph adenomas which are usually very large and very invasive .", "contrary to many authors and in agreement with ciatto , we think the association of pituitary adenoma and meningitis is more common than it is generally supposed as we observed it in nearly 5% of our male mprl .", "so , one should think about it more often even if a cause of sm seems obvious from the first sight as in our 4 case who suffered from chronic otitis .", "radiological research for pt should be systematic , especially if headaches , visual troubles , and gonadal abnormalities with or without anterior or posterior pituitary deficiencies are present .", "physicians who deal with large pt should be aware of high risk spontaneous meningitis , but also of secondary sm which can appear 1 month to 4 years or even more after medical treatment initiation by dopamine agonists or somatostatin 's analogs or radiotherapy . on another hand", ", medical treatment initiation should be gradual , especially for huge tumors as rapid shrinkage may lead to csf leak apparition .", "if this situation appears , a medical solution may be tried using temporary lumbar puncture or a lumboperitoneal shunt until the bony defect is clogged by fibrosis .", "if this attitude fails , surgical repair is recommended although the reparation may represent a great challenge because of the many potential sites of the csf around the tumor . if surgical repair in not feasible some authors recommend a vaccination against pathogens of the upper respiratory tract such as streptococcus pneumonia , haemophilus influenza and neisseria meningitidis to prevent sm ."], ["three revealed the pt , and one was observed after medical treatment . in 2 cases", "the described cases emphasize the necessity of an early diagnosis and treatment of large and invasive pt , especially male mprl . medical treatment , which is now the gold standard for prolactinomas ,", "should be gradual and well monitored to avoid rhinorrhea or otorrhea which can lead to potentially fatal bacterial meningitis ."]]}
{"article_id": "PMC3270611", "article_text": ["many studies have not been reported in literature for lumbar discectomy by destandau endospine system .", "we report a series of 300 patients operated for lumbar dissectomy by destandau endospine system .", "a total of 300 patients suffering from lumbar disc herniations were operated between january 2002 and december 2008 .", "technique comprised localization of symptomatic level followed by insertion of an endospine system devise through a 15 mm skin and fascial incision .", "endoscopic discectomy is then carried out by conventional micro disc surgery instruments by minimal invasive route .", "the results were evaluated by macnab 's criteria after a minimum followup of 12 months and maximum up to 24 months .", "based on modified macnab 's criteria , 90% patients had excellent to good , 8% had fair , and 2% had poor results .", "the complications observed were discitis and dural tear in five patients each and nerve root injury in two patients .", "90% patients were able to return to light and sedentary work with an average delay of 3 weeks and normal physical activities after 2 months .", "the advantages of use of minimal invasive spinal surgical techniques in treatment of lumbar disc herniation is small incision , limited tissue disruption , enhanced visualization due to better magnification and illumination , shorter hospital stay , and faster recovery time.13 among many posterior spinal endoscopic systems used for disc surgery , destandau endospine system and foley and smith 's metrx system are seen as viable alternatives to open disc surgery.46 the aim of this study was to present results in 300 patients operated by edoscopic discectomy and to discuss technical points to shorten the learning curve .", "a total of 475 patients suffering from different type and level of lumbar disc herniation with radiculopathy and degenerative lumbar canal stenosis were operated between january 2002 and december 2008 .", "the inclusion criteria were patients having lumbar disc prolapse with unilateral radiculopathy , on clinical evaluation , positive straight leg raise or femoral stretch test , and identification of a single nerve root lesion on mri .", "patients with bilateral symptoms , double root involvement , cauda equina syndrome and whose clinical symptoms did not match mri picture were excluded from present study .", "all these patients had fair trial of conservative treatment in the form of rest , medication ( nsaid ) , activity modification , and physiotherapy ( minimum 6 weeks ) before they were advised to undergo surgery .", "however , in present study , none of the patients opted for surgery at 6 weeks after completion of conservative treatment .", "there were 206 males and 94 females aged between 18 to 72 years ( mean , 38.4 years ) .", "levels operated upon included l1-l2 ( n=3 ) , l2-l3 ( n=2 ) , l3-l4 ( n=6 ) , l4-l5 ( n=205 ) , and l5-s1 ( n=84 ) .", "there were 235 extruded , 20 contained , 15 foraminal , and 30 sequestrated herniations .", "results were evaluated as poor , fair , and good or excellent using modified macnab 's criteria .", "modified macnab 's grading was as follows : excellent - no pain / restriction of activity and being able to do all activities ; good - occasional pain with relief of presenting symptoms and returning to work with some modification ; fair - some improved functional capacity but still handicapped or unemployed ; and poor results - having objective symptoms of root involvement or repeat surgery at the index level .", "the clinical material included preoperative history , physical examination , plain x - rays and mri studies of lumbosacral spine , laboratory tests , and intraoperative video documentation .", "postoperative follow - up was carried out on third day , 2 weeks , 6 weeks , 3 , 6 , 12 , and 24 months .", "it consists of endospine tube , trocar , and working insert [ figure 1a and b ] .", "one port for 0 degree endoscope , second for suction cannula , third port ( biggest ) for working instrument , and fourth port for dural and nerve root retractor .", "the procedure of discectomy can be carried out under general , spinal , epidural , or local anesthesia .", "the operative technique consists of knee chest positioning after administration of anesthesia followed by level localization by localization devise [ figure 1c ] . at marked point ,", "15 mm skin incision is made aponeurosis is incised using mayo 's scissors ; 1.5 cm wide periosteal elevator is used to elevate paravertebral muscles subperiostealy , thus exposing the interlaminar window and part of the affected side facet . (", "a ) clinical photograph showing patient positioning and level marking ( b ) destandau endospine system ( c ) iitv picture of marked level ( d ) position of endospine tube ( e ) endoscopic view of decompressed nerve root the endospine tube with trocar is pushed through the incision in the direction of posterior arch over interlaminar window followed by withdrawal of trocar . the working insert", "any soft tissue bulging in the mouth of tube is removed till boundaries of interlaminar window such as superior and inferior lamina , facet joint are clearly visualized [ figure 1e ] .", "this follows part resection of inferior margin of the superior lamina followed by excision of ligamentum flavum leading to exposure of the dural sac and nerve root under endoscopic vision .", "once the nerve root has been accurately identified , it is retracted using a nerve root retractor .", ". it also helps to keep the field dry . depending on local findings , discectomy involving the extraction of the nucleus pulposus", "once satisfactory nerve root decompression is achieved , endospine tube along with working insert is withdrawn .", "aponeurosis is sutured using vicryl fine suture followed by closure of the skin in a subcuticular fashion . a water - impermeable dressing", "( a ) preoperative sagittal t2wi mri of prolapsed l5-s1 disc ( b ) postoperative sagittal t2wi mri of l5-s1 disc after endoscopic discectomy at 2 years follow up ( c ) axial t2wi mri section at l5-s1 disc after endoscopic discectomy at 2 years follow up shows no compression these patients were followed up on third day , 2 weeks , 6 weeks , 3 , 6 , 12 , and 24 months .", "patients were followed up for minimum of one year and maximum of 2-year duration . on second visit on third day ,", "wound was inspected for any drainage or evidence of infection . complains about fever , backache , and leg discomfort", "at final follow - up , 90% patients were relieved of sciatica and were satisfied with procedure .", "285 patients were operated as day care cases and were mobilized and discharged same evening from day care facility .", "based on modified macnab 's criteria 90% patients had excellent to good , 8% had fair , and 2% had poor results .", "five patients who had interaoperative minor dural tears were hospitalized and were observed for any dural leak .", "causative factor for dural tears in present study were as follows : three patients had dural rent due to forceful retraction of dura and nerve root by dural and nerve root retractor .", "this was observed in patients in whom there was significant posterolateral herniation resulting in tenting of dura and nerve root at recess .", "in this situation , authors have found gentle mobilization of nerve root and dura by nerve root hook or approaching and debulking the offending disc through axilla before proceeding with retraction of nerve root and discectomy .", "this happened when kerrison rongeur was used to open the tight recess resulting in dural tear and nerve root injury .", "these dural tears were managed by water tight closure of muscle , fascia , and skin and bed rest for duration of one week .", "superficial delayed wound healing was observed in 20 patients , which healed in 21 days by regular dressings rest and administration of antibiotics .", "the diagnosis of postsurgical discitis was based on mainly clinical grounds and laboratory evidence of raised counts , esr , and c - reactive proteins .", "clinical criteria included recurrence of severe and unrelenting back pain within first week of surgery , keeping patient awake at night after initial recovery .", "no biopsy of disc was resorted to ; however , mri of lumbosacral spine was ordered in all these patients which did not contribute much to the diagnosis .", "lizolid 600 mg / bd ) for first week followed by oral antibiotics for 5 weeks .", "all these patients responded well to antibiotics and no further intervention of any kind was carried out .", "after initial back pain for 6 weeks , these patients had occasional residual backache which was treated by analgesics , activity modification , lumbar support , and rest during subsequent follow - up visits .", "nerve root injuries ( n=2 ) were encountered while trying to do a medial facetectomy to open the recess by a kerrison rongeur causing severe laceration of nerve root . however , nerve root was in continuity .", ", patties were used for gentle retraction and procedure of discectomy was successfully completed endoscopically without resorting to open discectomy .", "276 patients were able to return to sedentary work with an average delay of 2 weeks , except 24 patients who had complains of backache , occasional leg discomfort , discitis , and nerve root injuries .", "medial facetectomy was resorted to in 59 patients to open the recess to decompress the nerve root in addition to discectomy .", "mixter and barr7 discovered the pathophysiology of discogenic sciatica and suggested laminectomy and discectomy as operative treatment .", "the overall results of standard discectomy range from 68 to 95% in different series.8 the operative microscope and microsurgical techniques were developed in mid-1960 's by yasargil and krayenbuhl910 and these techniques revolutionized spine surgery leading to smaller incisions , less blood loss , increased visualization of site of pathology , decreased hospitalization , shorter postoperative recovery , and earlier return to activities compared with previous operative interventional techniques .", "the results of microdiscectomy also range from 85 to 98%.1113 katayama et al.14 compared the results of open vs gold standard microdiscectomy and observed no difference between the surgical outcomes in both the groups but microdiscectomy gave better lighting , magnification , and therefore decreased the length of incision and tissue invasion .", "microdiscectomy also allowed the patients to return to early work with lesser use of narcotic medication .", "microendoscopic dissectomy ( med ) combines standard microsurgical technique with an endoscope , enabling the surgeons to address all types of disc herniations including decompression of nerve root and lateral recess .", "chemonucleolysis was reported by smith.15 nonetheless , based upon various randomized clinical trials , the efficacy of chemonucleolysis compared with more traditional and open procedures for the operative treatment of lumbar disc herniations remained speculative.16 the use of percutaneous nucleotomy , laser discectomy , and intradiscal electrothermal annuloplasty ( idet ) compared with microdiscectomy remains unclear , and it is attributed to the lack of high - quality studies.17 conclusions about the efficacy of some of the aforementioned minimally invasive procedures ( e.g. , chemonucleolysis , apd , idet ) were questionable with regard to disc - related pathology.18 therefore , lumbar microdiscectomy remained the gold standard for addressing a herniated or sequestrated intervertebral disc ; however , a movement toward more minimally invasive approaches that would yield superior outcomes , while minimizing excessive soft and bony tissue removal and minimizing soft tissue trauma , were sought . as such ,", "an evolution in procedures toward smaller incisions , less tissue trauma , and quicker return to daily activities took center stage in spine surgery .", "the use of muscular retractor system was reported initially by faubert and caspar.1920 perez - curet and fessler,21 described for the first time a myriad of spine pathologies that could be addressed using tubeology .", "though from our initial experience , endospine technique is minimal invasive , but limitation of study has been lack of comparison with gold standard microscopic discectomy technique .", "however , in a study by shin et al.,22 15 cases each were compared of med and microscopic group ( md ) .", "the mean cpk - mm levels were lower for the med group than for the md group at both 3 ( 576.1286.3 iu / l compared with 968.1377.8", "iu / l ) and 5 days ( 348.1231.0 iu / l compared with 721.7463.2 ) postoperatively ( p<0.05 ) . the mean vas scores for postoperative back pain were lower in the med group than in the md group , both at 1 ( 3.32.3 compared with 5.81.5 ) and 5 days ( 1.91.1 compared with 3.61.1 ) postoperatively ( p<0.01 ) .", "aforementioned authors concluded that the med procedure is less invasive than md , and causes less muscle damage and backache .", "the 90% excellent results in present study is comparable with other surgical procedures for herniated lumbar discs such as those of destandau , perez - cruet et al . , and", "their average surgical time was 66 minutes , average blood loss was 22 ml , average hospital stay was 7.7 hours , complication rate was 5% , reoperation rate was 4% , and average return to work was 17 days with excellent result in 94% patients .", "average operative time was 50 minutes average blood loss was 45 ml ( range , 30 - 70 ml ) .", "return to work ( 21 days ) and overall results ( 90% ) which are comparable . in another prospective and randomized evaluation of surgical treatment for lumbar disc herniation by hermantin et al.,24 satisfactory results of 97% in endoscopic group ( n=30 ) and 93% in open laminectomy group ( n=30 ) were reported .", "however , in endoscopic group , these authors had excluded large central herniations and extra ligamentous herniations between l5 and first sacral vertebra .", "however , present endoscopic technique could be used for all levels and all type of herniations . in our current series , there was 5% discitis and 5% incidence of dural injury .", "caspar and ebling,2526 authors have reported reoperation rate of 5.5 , 5.7 , and 3% , respectively .", "another measure of success is reflected by the patient 's ability to return to previous employment .", "our patients returned to previous employment on an average at 15 days with restriction to avoid heavy manual work for 2 months .", "discectomy ( med ) by endospine system has claimed even lesser tissue invasion than microdiscectomy with even smaller skin incision , lesser use of analgesics , and early return to work .", "least tissue invasion is established by many reports comparing the postoperative mri signal of paraspinal muscles,27 intraoperative electromyographic findings establishing less invasion to nerve roots,28 and by measuring serum levels of biochemical parameters reflective of a postoperative inflammatory reaction and damage to the paravertebral muscles.29 our personal opinion is similar , though this was not the parameter studied in our series .", "minimal invasive microendoscopic decompression technique has been used not only for paracentral disc herniations , but also for all types including far lateral , cephalad , caudal migrated , and central and recurrent disc herniations.3032 one of the driving forces behind the minimal invasive spine surgery is economics , shorter hospital stay , reduced postoperative morbidity , and quicker recovery times . in our series , 90% patients were operated as day care cases .", "posterior paraspinous process endoscopic access to lumbar disc herniation requires creation of working space where no or little space existed before .", "internal view of operating site is magnified and well illuminated . with advent of this system ,", "discectomy can be done as day care procedure ensuring reduced postoperative morbidity , minimal or no hospitalization , less pain , and faster recovery .", "with proper patient selection , discectomy and adequate nerve root decompression by doing foraminotomy or opening a lateral recess stenosis by minimally invasive technique can be achieved with this system .", "however , the endospine system has been excellent modality to address discogenic radiculopathy and to decompress lumbar canal stenosis .", "many surgeons are convinced of advantages of the system and have included this system as part of their inventry . however , due to difficulty in orientation with scope and two - dimensional vision , availability of less space , frustrating and steep learning curve , and inability to master hand eye coordination , majority of surgeons are not able to continue with the technique .", "the patience and persvrance to work through narrow confines and work closely with a surgeon who has mastered the technique is the key to learn .", "second step would be to become comfortable with 2 dimensional vision of endoscopic camera and to master orientation , triangulation .", "depth perception in these techniques comes from experience rather than observation ; hence , surgeon keen to learn these techniques must combine these procedures during early phase of learning with standard procedures he is doing in his clinical practice . gradually , as surgeons master the learning curve , he will be able to use this as treatment method for his patients .", "there is also a need to establish cadaveric labs and dummy models on line of arthroscopic learning centers where surgeons can practice hands - on cadavers and models to improve triangulation , depth perception , and hand eye coordination ."], "abstract_text": ["<S> background : posterior endoscopic discectomy is an established method for treatment of lumbar disc herniation . </S>", "<S> many studies have not been reported in literature for lumbar discectomy by destandau endospine system . </S>", "<S> we report a series of 300 patients operated for lumbar dissectomy by destandau endospine system.materials and methods : a total of 300 patients suffering from lumbar disc herniations were operated between january 2002 and december 2008 . </S>", "<S> all patients were operated as day care procedure . </S>", "<S> technique comprised localization of symptomatic level followed by insertion of an endospine system devise through a 15 mm skin and fascial incision . </S>", "<S> endoscopic discectomy is then carried out by conventional micro disc surgery instruments by minimal invasive route . </S>", "<S> the results were evaluated by macnab 's criteria after a minimum followup of 12 months and maximum up to 24 months.results:based on modified macnab 's criteria , 90% patients had excellent to good , 8% had fair , and 2% had poor results . </S>", "<S> the complications observed were discitis and dural tear in five patients each and nerve root injury in two patients . </S>", "<S> 90% patients were able to return to light and sedentary work with an average delay of 3 weeks and normal physical activities after 2 months.conclusion:edoscopic discectomy provides a safe and minimal access corridor for lumbar discectomy . </S>", "<S> the technique also allows early postoperative mobilization and faster return to work . </S>"], "labels": null, "section_names": ["Background:", "Materials and Methods:", "Results:", "Conclusion:", "I", "M", "R", "D"], "sections": [["many studies have not been reported in literature for lumbar discectomy by destandau endospine system .", "we report a series of 300 patients operated for lumbar dissectomy by destandau endospine system ."], ["a total of 300 patients suffering from lumbar disc herniations were operated between january 2002 and december 2008 .", "technique comprised localization of symptomatic level followed by insertion of an endospine system devise through a 15 mm skin and fascial incision .", "endoscopic discectomy is then carried out by conventional micro disc surgery instruments by minimal invasive route .", "the results were evaluated by macnab 's criteria after a minimum followup of 12 months and maximum up to 24 months ."], ["based on modified macnab 's criteria , 90% patients had excellent to good , 8% had fair , and 2% had poor results .", "the complications observed were discitis and dural tear in five patients each and nerve root injury in two patients .", "90% patients were able to return to light and sedentary work with an average delay of 3 weeks and normal physical activities after 2 months ."], [""], ["the advantages of use of minimal invasive spinal surgical techniques in treatment of lumbar disc herniation is small incision , limited tissue disruption , enhanced visualization due to better magnification and illumination , shorter hospital stay , and faster recovery time.13 among many posterior spinal endoscopic systems used for disc surgery , destandau endospine system and foley and smith 's metrx system are seen as viable alternatives to open disc surgery.46 the aim of this study was to present results in 300 patients operated by edoscopic discectomy and to discuss technical points to shorten the learning curve ."], ["a total of 475 patients suffering from different type and level of lumbar disc herniation with radiculopathy and degenerative lumbar canal stenosis were operated between january 2002 and december 2008 .", "the inclusion criteria were patients having lumbar disc prolapse with unilateral radiculopathy , on clinical evaluation , positive straight leg raise or femoral stretch test , and identification of a single nerve root lesion on mri .", "patients with bilateral symptoms , double root involvement , cauda equina syndrome and whose clinical symptoms did not match mri picture were excluded from present study .", "all these patients had fair trial of conservative treatment in the form of rest , medication ( nsaid ) , activity modification , and physiotherapy ( minimum 6 weeks ) before they were advised to undergo surgery .", "however , in present study , none of the patients opted for surgery at 6 weeks after completion of conservative treatment .", "there were 206 males and 94 females aged between 18 to 72 years ( mean , 38.4 years ) .", "levels operated upon included l1-l2 ( n=3 ) , l2-l3 ( n=2 ) , l3-l4 ( n=6 ) , l4-l5 ( n=205 ) , and l5-s1 ( n=84 ) .", "there were 235 extruded , 20 contained , 15 foraminal , and 30 sequestrated herniations .", "results were evaluated as poor , fair , and good or excellent using modified macnab 's criteria .", "modified macnab 's grading was as follows : excellent - no pain / restriction of activity and being able to do all activities ; good - occasional pain with relief of presenting symptoms and returning to work with some modification ; fair - some improved functional capacity but still handicapped or unemployed ; and poor results - having objective symptoms of root involvement or repeat surgery at the index level .", "the clinical material included preoperative history , physical examination , plain x - rays and mri studies of lumbosacral spine , laboratory tests , and intraoperative video documentation .", "postoperative follow - up was carried out on third day , 2 weeks , 6 weeks , 3 , 6 , 12 , and 24 months .", "it consists of endospine tube , trocar , and working insert [ figure 1a and b ] .", "one port for 0 degree endoscope , second for suction cannula , third port ( biggest ) for working instrument , and fourth port for dural and nerve root retractor .", "the procedure of discectomy can be carried out under general , spinal , epidural , or local anesthesia .", "the operative technique consists of knee chest positioning after administration of anesthesia followed by level localization by localization devise [ figure 1c ] . at marked point ,", "15 mm skin incision is made aponeurosis is incised using mayo 's scissors ; 1.5 cm wide periosteal elevator is used to elevate paravertebral muscles subperiostealy , thus exposing the interlaminar window and part of the affected side facet . (", "a ) clinical photograph showing patient positioning and level marking ( b ) destandau endospine system ( c ) iitv picture of marked level ( d ) position of endospine tube ( e ) endoscopic view of decompressed nerve root the endospine tube with trocar is pushed through the incision in the direction of posterior arch over interlaminar window followed by withdrawal of trocar . the working insert", "any soft tissue bulging in the mouth of tube is removed till boundaries of interlaminar window such as superior and inferior lamina , facet joint are clearly visualized [ figure 1e ] .", "this follows part resection of inferior margin of the superior lamina followed by excision of ligamentum flavum leading to exposure of the dural sac and nerve root under endoscopic vision .", "once the nerve root has been accurately identified , it is retracted using a nerve root retractor .", ". it also helps to keep the field dry . depending on local findings , discectomy involving the extraction of the nucleus pulposus", "once satisfactory nerve root decompression is achieved , endospine tube along with working insert is withdrawn .", "aponeurosis is sutured using vicryl fine suture followed by closure of the skin in a subcuticular fashion . a water - impermeable dressing", "( a ) preoperative sagittal t2wi mri of prolapsed l5-s1 disc ( b ) postoperative sagittal t2wi mri of l5-s1 disc after endoscopic discectomy at 2 years follow up ( c ) axial t2wi mri section at l5-s1 disc after endoscopic discectomy at 2 years follow up shows no compression these patients were followed up on third day , 2 weeks , 6 weeks , 3 , 6 , 12 , and 24 months .", "patients were followed up for minimum of one year and maximum of 2-year duration . on second visit on third day ,", "wound was inspected for any drainage or evidence of infection . complains about fever , backache , and leg discomfort"], ["at final follow - up , 90% patients were relieved of sciatica and were satisfied with procedure .", "285 patients were operated as day care cases and were mobilized and discharged same evening from day care facility .", "based on modified macnab 's criteria 90% patients had excellent to good , 8% had fair , and 2% had poor results .", "five patients who had interaoperative minor dural tears were hospitalized and were observed for any dural leak .", "causative factor for dural tears in present study were as follows : three patients had dural rent due to forceful retraction of dura and nerve root by dural and nerve root retractor .", "this was observed in patients in whom there was significant posterolateral herniation resulting in tenting of dura and nerve root at recess .", "in this situation , authors have found gentle mobilization of nerve root and dura by nerve root hook or approaching and debulking the offending disc through axilla before proceeding with retraction of nerve root and discectomy .", "this happened when kerrison rongeur was used to open the tight recess resulting in dural tear and nerve root injury .", "these dural tears were managed by water tight closure of muscle , fascia , and skin and bed rest for duration of one week .", "superficial delayed wound healing was observed in 20 patients , which healed in 21 days by regular dressings rest and administration of antibiotics .", "the diagnosis of postsurgical discitis was based on mainly clinical grounds and laboratory evidence of raised counts , esr , and c - reactive proteins .", "clinical criteria included recurrence of severe and unrelenting back pain within first week of surgery , keeping patient awake at night after initial recovery .", "no biopsy of disc was resorted to ; however , mri of lumbosacral spine was ordered in all these patients which did not contribute much to the diagnosis .", "lizolid 600 mg / bd ) for first week followed by oral antibiotics for 5 weeks .", "all these patients responded well to antibiotics and no further intervention of any kind was carried out .", "after initial back pain for 6 weeks , these patients had occasional residual backache which was treated by analgesics , activity modification , lumbar support , and rest during subsequent follow - up visits .", "nerve root injuries ( n=2 ) were encountered while trying to do a medial facetectomy to open the recess by a kerrison rongeur causing severe laceration of nerve root . however , nerve root was in continuity .", ", patties were used for gentle retraction and procedure of discectomy was successfully completed endoscopically without resorting to open discectomy .", "276 patients were able to return to sedentary work with an average delay of 2 weeks , except 24 patients who had complains of backache , occasional leg discomfort , discitis , and nerve root injuries .", "medial facetectomy was resorted to in 59 patients to open the recess to decompress the nerve root in addition to discectomy ."], ["mixter and barr7 discovered the pathophysiology of discogenic sciatica and suggested laminectomy and discectomy as operative treatment .", "the overall results of standard discectomy range from 68 to 95% in different series.8 the operative microscope and microsurgical techniques were developed in mid-1960 's by yasargil and krayenbuhl910 and these techniques revolutionized spine surgery leading to smaller incisions , less blood loss , increased visualization of site of pathology , decreased hospitalization , shorter postoperative recovery , and earlier return to activities compared with previous operative interventional techniques .", "the results of microdiscectomy also range from 85 to 98%.1113 katayama et al.14 compared the results of open vs gold standard microdiscectomy and observed no difference between the surgical outcomes in both the groups but microdiscectomy gave better lighting , magnification , and therefore decreased the length of incision and tissue invasion .", "microdiscectomy also allowed the patients to return to early work with lesser use of narcotic medication .", "microendoscopic dissectomy ( med ) combines standard microsurgical technique with an endoscope , enabling the surgeons to address all types of disc herniations including decompression of nerve root and lateral recess .", "chemonucleolysis was reported by smith.15 nonetheless , based upon various randomized clinical trials , the efficacy of chemonucleolysis compared with more traditional and open procedures for the operative treatment of lumbar disc herniations remained speculative.16 the use of percutaneous nucleotomy , laser discectomy , and intradiscal electrothermal annuloplasty ( idet ) compared with microdiscectomy remains unclear , and it is attributed to the lack of high - quality studies.17 conclusions about the efficacy of some of the aforementioned minimally invasive procedures ( e.g. , chemonucleolysis , apd , idet ) were questionable with regard to disc - related pathology.18 therefore , lumbar microdiscectomy remained the gold standard for addressing a herniated or sequestrated intervertebral disc ; however , a movement toward more minimally invasive approaches that would yield superior outcomes , while minimizing excessive soft and bony tissue removal and minimizing soft tissue trauma , were sought . as such ,", "an evolution in procedures toward smaller incisions , less tissue trauma , and quicker return to daily activities took center stage in spine surgery .", "the use of muscular retractor system was reported initially by faubert and caspar.1920 perez - curet and fessler,21 described for the first time a myriad of spine pathologies that could be addressed using tubeology .", "though from our initial experience , endospine technique is minimal invasive , but limitation of study has been lack of comparison with gold standard microscopic discectomy technique .", "however , in a study by shin et al.,22 15 cases each were compared of med and microscopic group ( md ) .", "the mean cpk - mm levels were lower for the med group than for the md group at both 3 ( 576.1286.3 iu / l compared with 968.1377.8", "iu / l ) and 5 days ( 348.1231.0 iu / l compared with 721.7463.2 ) postoperatively ( p<0.05 ) . the mean vas scores for postoperative back pain were lower in the med group than in the md group , both at 1 ( 3.32.3 compared with 5.81.5 ) and 5 days ( 1.91.1 compared with 3.61.1 ) postoperatively ( p<0.01 ) .", "aforementioned authors concluded that the med procedure is less invasive than md , and causes less muscle damage and backache .", "the 90% excellent results in present study is comparable with other surgical procedures for herniated lumbar discs such as those of destandau , perez - cruet et al . , and", "their average surgical time was 66 minutes , average blood loss was 22 ml , average hospital stay was 7.7 hours , complication rate was 5% , reoperation rate was 4% , and average return to work was 17 days with excellent result in 94% patients .", "average operative time was 50 minutes average blood loss was 45 ml ( range , 30 - 70 ml ) .", "return to work ( 21 days ) and overall results ( 90% ) which are comparable . in another prospective and randomized evaluation of surgical treatment for lumbar disc herniation by hermantin et al.,24 satisfactory results of 97% in endoscopic group ( n=30 ) and 93% in open laminectomy group ( n=30 ) were reported .", "however , in endoscopic group , these authors had excluded large central herniations and extra ligamentous herniations between l5 and first sacral vertebra .", "however , present endoscopic technique could be used for all levels and all type of herniations . in our current series , there was 5% discitis and 5% incidence of dural injury .", "caspar and ebling,2526 authors have reported reoperation rate of 5.5 , 5.7 , and 3% , respectively .", "another measure of success is reflected by the patient 's ability to return to previous employment .", "our patients returned to previous employment on an average at 15 days with restriction to avoid heavy manual work for 2 months .", "discectomy ( med ) by endospine system has claimed even lesser tissue invasion than microdiscectomy with even smaller skin incision , lesser use of analgesics , and early return to work .", "least tissue invasion is established by many reports comparing the postoperative mri signal of paraspinal muscles,27 intraoperative electromyographic findings establishing less invasion to nerve roots,28 and by measuring serum levels of biochemical parameters reflective of a postoperative inflammatory reaction and damage to the paravertebral muscles.29 our personal opinion is similar , though this was not the parameter studied in our series .", "minimal invasive microendoscopic decompression technique has been used not only for paracentral disc herniations , but also for all types including far lateral , cephalad , caudal migrated , and central and recurrent disc herniations.3032 one of the driving forces behind the minimal invasive spine surgery is economics , shorter hospital stay , reduced postoperative morbidity , and quicker recovery times . in our series , 90% patients were operated as day care cases .", "posterior paraspinous process endoscopic access to lumbar disc herniation requires creation of working space where no or little space existed before .", "internal view of operating site is magnified and well illuminated . with advent of this system ,", "discectomy can be done as day care procedure ensuring reduced postoperative morbidity , minimal or no hospitalization , less pain , and faster recovery .", "with proper patient selection , discectomy and adequate nerve root decompression by doing foraminotomy or opening a lateral recess stenosis by minimally invasive technique can be achieved with this system .", "however , the endospine system has been excellent modality to address discogenic radiculopathy and to decompress lumbar canal stenosis .", "many surgeons are convinced of advantages of the system and have included this system as part of their inventry . however , due to difficulty in orientation with scope and two - dimensional vision , availability of less space , frustrating and steep learning curve , and inability to master hand eye coordination , majority of surgeons are not able to continue with the technique .", "the patience and persvrance to work through narrow confines and work closely with a surgeon who has mastered the technique is the key to learn .", "second step would be to become comfortable with 2 dimensional vision of endoscopic camera and to master orientation , triangulation .", "depth perception in these techniques comes from experience rather than observation ; hence , surgeon keen to learn these techniques must combine these procedures during early phase of learning with standard procedures he is doing in his clinical practice . gradually , as surgeons master the learning curve , he will be able to use this as treatment method for his patients .", "there is also a need to establish cadaveric labs and dummy models on line of arthroscopic learning centers where surgeons can practice hands - on cadavers and models to improve triangulation , depth perception , and hand eye coordination ."]]}
{"article_id": "PMC4841868", "article_text": ["medical tourism is illustrated as occurrence in which individuals travel abroad to receive healthcare services ( 1 ) .", "it is a multi - billion dollar industry and countries like india , thailand , singapore , malaysia , belgium , costa rica , cuba , dubai , hungary , israel , jordan , south africa and many others are being benefited in their economy by this recent phenomenon ( 2 , 3 ) .", "the prime driving factors in medical tourism are increased medical costs , increased insurance premiums , increasing number of uninsured or partially insured individuals in developed countries , long waiting lists for procedures in countries having public healthcare system , availability of high quality services at affordable price , and cheaper airfare . increased communication and internet access in developing countries", "are other supporting factors , which help patients to develop awareness about international travel for medical care ( 47 ) .", "estimations can vary but still some of reliable sources claim ; gross medical tourism revenue worldwide was more than us$ 40 billion in year 2004 and reached up to us$ 100 billion by year 2012 ( 8) . in year 2007", ", it was estimated that around 750,000 us residents traveled abroad . accordingly , the base case estimated form 2007 to 2010 for the annual growth rate for outbound patients was 100 per cent in us ( 9 ) .", "forbes business estimated around 1.25 million americans were expected to travel outside for medical treatment in year 2014 ( 10 ) .", "after thorough review it was found that there is huge gap in literature available in medical tourism .", "accounts detailing size and market of the industry are large in number yet there is scarcity of literature detailing about role and significance of different variables and linking them to form conceptual and theoretical framework which reflects decision making of international patients for taking part in medical tourism .", "available literature explore various factors related to patients travel such as source of information , perceived risks , benefits of medical tourism and attributes of medical tourism destinations in sufficient amount but almost all of these related accounts are exploratory in nature and do not provide conceptual frameworks for testing .", "many researchers ( 2 , 6 , 11 , 12 ) made calls to explore more about the decision making process based on conceptual models and test them empirically , so managers in medical tourism industry will be more acquainted about the needs and requirements of patients and design their future strategies accordingly and at the same time patients will also be benefited by receiving more quality services from the providers .", "this article will draw a conceptual model based on patients source of information , perceived benefits , perceived risks , and medical tourism destination image with available literature that will be helpful to mangers to draw their future course of action for competitive advantage and at the same time fulfill the gap in available literature .", "image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place .", "various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) .", "consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) .", "amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) .", "source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) .", "there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation .", "medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) .", "judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) .", "these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own .", "many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) .", "literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) .", "word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors .", "studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) .", "websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) .", "printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) .", "major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations .", "some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) .", "print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel .", "los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients .", "fox , cbs s  60 minutes  and cnn aired their segments on patients traveling to medical services .", "magazines like forbes and wall street journal analyzed the business aspect of medical tourism as  brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) .", "literature revealed that not every physical attribute of a destination has influence on image formation process .", "there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) .", "it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one .", "this is due to dimensions of liking may not be agree with the dimensions of similarity .", "literature revealed that many researchers made distinction between physical and beneficial aspect of a product .", "few researchers explained it as  characteristic and  benefits to the physical and beneficial aspect of product respectively ( 41 ) .", "the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery .", "there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) .", "the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds .", "benefits offered by a product or service are considered as consumption value of the same ( 44 ) .", "sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) .", "conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values .", "consumption value model define the characteristics of  functional value as price , credibility , and durability .", "medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) .", "quality of medical services is one of the aspects which drive medical tourism high towards success .", "most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) .", "high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) .", "although availability of literature explain  social value in medical tourism is less , little available literature clearly define the ", "social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) .", "senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) .", "most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) .", "however , the concept of social value and epistemic value may not be associated with patients from developed regions .", "many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) .", "such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) .", "conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value .", "perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision ,", "making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) .", "researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) .", "however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) .", "in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) .", "researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) .", "hence , perceived risks and cognitive image should consider individual variables for image related studies in travel .", "credence goods as the quality of these good can not be assessed accurately even after consumption .", "patients may vulnerable to many risks if consider to take medical services at medical tourism destinations .", "researchers described six dimension of risks associated with health travelers visited israel as  human - induced risks , ", "financial risks ,  service quality risks ,  socio - psychological risk ,  natural disasters and car accident risk , and ", "after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as  physical - health related risks ,  service related risks , and  destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) .", "in medical tourism , international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism .", "health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) .", "literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) .", "researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel .", "apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) . according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) .", "destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel", ", researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) .", "attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination .", "later on , a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) .", "the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) .", "researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing ", "furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) .", "thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) .", "it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination .", "the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) .", "after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ;  medical amenities ,  general infra - structure ,  tourism attractions , and ", "social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency .", "majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff .", "command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) .", "based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services .", "physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians .", "prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk .", "the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) .", "collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules .", "most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) .", "most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients .", "many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world .", "language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) .", "facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients .", "essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination .", "thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) .", "potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability", "image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place .", "various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) .", "consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) .", "amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) .", "source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) .", "there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation .", "medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) .", "judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) .", "these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own .", "many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) .", "literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) .", "word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors .", "studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) .", "websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) .", "printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) .", "major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations .", "some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) .", "print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel .", "los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients .", "fox , cbs s  60 minutes  and cnn aired their segments on patients traveling to medical services .", "magazines like forbes and wall street journal analyzed the business aspect of medical tourism as  brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) .", "literature revealed that not every physical attribute of a destination has influence on image formation process .", "there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) .", "it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one .", "this is due to dimensions of liking may not be agree with the dimensions of similarity .", "literature revealed that many researchers made distinction between physical and beneficial aspect of a product .", "few researchers explained it as  characteristic and  benefits to the physical and beneficial aspect of product respectively ( 41 ) .", "the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery .", "there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) .", "the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds .", "benefits offered by a product or service are considered as consumption value of the same ( 44 ) .", "sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) .", "conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values .", "consumption value model define the characteristics of  functional value as price , credibility , and durability .", "medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) .", "quality of medical services is one of the aspects which drive medical tourism high towards success .", "most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) .", "high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) .", "although availability of literature explain  social value in medical tourism is less , little available literature clearly define the  social value factor with - in the patient groups especially from less developed regions .", "social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) .", "senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) .", "most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) .", "however , the concept of social value and epistemic value may not be associated with patients from developed regions .", "many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) . such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) .", "conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value .", "perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision ,", "making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) .", "researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) .", "however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) .", "in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) .", "researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) .", "hence , perceived risks and cognitive image should consider individual variables for image related studies in travel .", "credence goods as the quality of these good can not be assessed accurately even after consumption .", "patients may vulnerable to many risks if consider to take medical services at medical tourism destinations .", "researchers described six dimension of risks associated with health travelers visited israel as  human - induced risks , ", "financial risks ,  service quality risks ,  socio - psychological risk ,  natural disasters and car accident risk , and ", "after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as  physical - health related risks ,  service related risks , and ", "destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) . in medical tourism ,", "international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism .", "health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) .", "literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) .", "researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel .", "apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) .", "according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) .", "destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel", ", researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) .", "attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination . later on ,", "a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) .", "the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) .", "researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing ", "sleepy , pleasant  unpleasant , exciting gloomy , and relaxing  distressing ( 70 ) .", "furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) .", "thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) .", "it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination .", "the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) .", "after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ;  medical amenities ,  general infra - structure , ", "social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency .", "majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff .", "command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) . based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services .", "physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians .", "prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk .", "the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) .", "collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules .", "most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) .", "most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients .", "many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world .", "language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) .", "facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients .", "essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination .", "thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) .", "potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability", "prominent databases ( 2013 , 2014 , 2015 ) have been searched to obtain desire literature related to medical tourism including science direct , utmj.org , nih.gov , nchu.edu.tw , palgrave - journals , medretreat , biomedcentral and so on and the keywords used are medical tourism , information sources , medical travel , health travel and so on .", "google search was performed to get latest data related to medical tourism . to know in detail about the variables used in the framework scientific research databases", "the key words used to find out research variables as well as relationship between researches variables were destination image , destination information sources , beneficial image , perceived travel risks , cognitive image , affective image , consumption values and so on . to obtain data about medical tourism various types of available literature were reviewed such as industry reports , market research reports , media reports and internet sources , however for identification and explanation of variables scientific literature published in reputed journals only were included .", "the theory of planned ( tpb ) is an established and comprehensively tested model details the relationship between beliefs , attitude , intention , and actual behavior of consumers ( 85 ) . the model is perfectly applied in a variety of studies including leisure and tourism travel , and hospitality ( 36 , 8688 ) .", "these research studies emphases on the factors such as motivations , information sources , attitudes , and visit intentions , thus authors argue that attitudinal theory provides a sound foundation to understand travel intentions of international patients and underpin the conceptual model discussed in this article .", "studies explained that consumers access more information in case of high association of risks or benefits or both in a particular act ( 89 , 90 ) and perform rigorous information search in case of planning first time trip ( 91 ) because first time trip is associated with unknown risks as well as unknown leisure activities .", "in general consumer behaviour practices risk - handling activity increases in case of high level of perceived risks .", "importance of risk handling activity also determines by associated benefits gained after engaging with risk taking activity ( 92 ) .", "the benefits of information search includes possibility of finding superior alternative to those already considered and the reduction in risk achieved from eliminating inferior , but a priori uncertain , alternatives ( 93 ) . with the discussion of above literature related to tourism", "it could be assumed that medical tourism information sources will have positive influence on perceived travel benefits of international patients . whereas , information sources of medical tourism will negatively influence perceived travel risks of international patients .", "destination image is considered as perceptions or impressions of destination held by tourists with respect to the expected benefit or consumption values .", "the benefits sought by the travelers are highly associated with the image of destination and the affective image of destination is largely highly influenced by the motivations / benefits sought by individuals ( 13 , 44 ) .", "the affective image of a destination is more positive in individual s mind when emotions evoked by the place coincide with the benefits sought ( 95 ) .", "risks and constraints associated with travel have significant impact on destination image formation at the time of early decision - making process ( 96 ) . according to researchers ,", "two dimensions significantly influence cognitive and affective image of destination ( 58 ) . on the basis of previous studies in tourism literature", "it can be assumed that perceived travel benefits of international patients will positively influence the destination image , whereas perceived travel risks of international patients will negatively influence the destination image .", "intention of visit related to travel is considered as tourists perceived likelihood to visit particular destination within a specific time period ( 97 ) .", "thus , to be closely correlated to the travel behaviour intention of visit believes as an important outcome variable in tourism research ( 16 , 97 ) .", "intention to visit a destination is highly influenced by cognitive / perceptual and affective evaluation ( 98 ) .", "destination image is a direct antecedent of perceived quality , satisfaction and revisit intention and recommendation ( 99 ) . according to researchers", ", there is significant theoretical and empirical link between cognitive destination image and behavioral intention ( 100 ) .", "based on above discussion it could be assumed that image of medical tourism destination will positively influence visit intention of international patients . conceptual framework  international patients travel decision making", "the article is a sincere effort by authors to establish conceptual framework which explains decision - making process of international patients .", "though , there has been advance in explaining different factors play important roles in international patients travel , however , there is scarcity of literature conceptualizing these factors into a framework and test it empirically .", "literature reveals about associated benefits and risks related to travel abroad for medical purposes but do not conceptualize it in the process of decision - making .", "sources of information in medical tourism play significant role in development of attitude as well as intention , has already been established in consumer behavior .", "however , need is to test its significance in medical tourism decision - making process .", "the proposed framework will encourage future researchers to test different variables empirically and establish the model of international patients travel behavior .", "ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors ."], "abstract_text": ["<S> background : role of information source , perceived benefits and risks , and destination image has significantly been examined in travel and tourism literature ; however , in medical tourism it is yet to be examined thoroughly . </S>", "<S> the concept discussed in this article is drawn form well established models in tourism literature.methods:the purpose of this research was to identify the source of information , travel benefits and perceived risks related to movement of international patients and develop a conceptual model based on well - established theory . </S>", "<S> thorough database search ( science direct , utmj.org , nih.gov , nchu.edu.tw , palgrave - journals , medretreat , biomedcentral ) was performed to fulfill the objectives of the study.results:international patients always concern about benefits and risks related to travel . </S>", "<S> these benefits and risks form images of destination in the minds of international patients . </S>", "<S> different sources of information make international patients acquaint about the associated benefits and risks , which later leads to development of intention to visit . </S>", "<S> this conceptual paper helps in establishing model for decision - making process of international patients in developing visit intention.conclusion:ample amount of literature is available detailing different factors involved in travel decision making of international patients ; however literature explaining relationship between these factors is scarce . </S>"], "labels": null, "section_names": ["Introduction", "Literature Review", "International Patients Source of Information", "International Patients Perceived Travel Benefits", "Consumption Value of Medical Tourism", "International Patients Perceived Travel Risks", "Medical Tourism Destination Image", "Methods", "Results", "Conclusion", "\nEthical considerations\n"], "sections": [["medical tourism is illustrated as occurrence in which individuals travel abroad to receive healthcare services ( 1 ) .", "it is a multi - billion dollar industry and countries like india , thailand , singapore , malaysia , belgium , costa rica , cuba , dubai , hungary , israel , jordan , south africa and many others are being benefited in their economy by this recent phenomenon ( 2 , 3 ) .", "the prime driving factors in medical tourism are increased medical costs , increased insurance premiums , increasing number of uninsured or partially insured individuals in developed countries , long waiting lists for procedures in countries having public healthcare system , availability of high quality services at affordable price , and cheaper airfare . increased communication and internet access in developing countries", "are other supporting factors , which help patients to develop awareness about international travel for medical care ( 47 ) .", "estimations can vary but still some of reliable sources claim ; gross medical tourism revenue worldwide was more than us$ 40 billion in year 2004 and reached up to us$ 100 billion by year 2012 ( 8) . in year 2007", ", it was estimated that around 750,000 us residents traveled abroad . accordingly , the base case estimated form 2007 to 2010 for the annual growth rate for outbound patients was 100 per cent in us ( 9 ) .", "forbes business estimated around 1.25 million americans were expected to travel outside for medical treatment in year 2014 ( 10 ) .", "after thorough review it was found that there is huge gap in literature available in medical tourism .", "accounts detailing size and market of the industry are large in number yet there is scarcity of literature detailing about role and significance of different variables and linking them to form conceptual and theoretical framework which reflects decision making of international patients for taking part in medical tourism .", "available literature explore various factors related to patients travel such as source of information , perceived risks , benefits of medical tourism and attributes of medical tourism destinations in sufficient amount but almost all of these related accounts are exploratory in nature and do not provide conceptual frameworks for testing .", "many researchers ( 2 , 6 , 11 , 12 ) made calls to explore more about the decision making process based on conceptual models and test them empirically , so managers in medical tourism industry will be more acquainted about the needs and requirements of patients and design their future strategies accordingly and at the same time patients will also be benefited by receiving more quality services from the providers .", "this article will draw a conceptual model based on patients source of information , perceived benefits , perceived risks , and medical tourism destination image with available literature that will be helpful to mangers to draw their future course of action for competitive advantage and at the same time fulfill the gap in available literature ."], ["image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place .", "various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) .", "consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) .", "amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) .", "source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) .", "there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation .", "medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) .", "judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) .", "these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own .", "many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) .", "literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) .", "word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors .", "studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) .", "websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) .", "printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) .", "major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations .", "some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) .", "print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel .", "los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients .", "fox , cbs s  60 minutes  and cnn aired their segments on patients traveling to medical services .", "magazines like forbes and wall street journal analyzed the business aspect of medical tourism as  brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) .", "literature revealed that not every physical attribute of a destination has influence on image formation process .", "there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) .", "it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one .", "this is due to dimensions of liking may not be agree with the dimensions of similarity .", "literature revealed that many researchers made distinction between physical and beneficial aspect of a product .", "few researchers explained it as  characteristic and  benefits to the physical and beneficial aspect of product respectively ( 41 ) .", "the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery .", "there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) .", "the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds .", "benefits offered by a product or service are considered as consumption value of the same ( 44 ) .", "sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) .", "conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values .", "consumption value model define the characteristics of  functional value as price , credibility , and durability .", "medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) .", "quality of medical services is one of the aspects which drive medical tourism high towards success .", "most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) .", "high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) .", "although availability of literature explain  social value in medical tourism is less , little available literature clearly define the ", "social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) .", "senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) .", "most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) .", "however , the concept of social value and epistemic value may not be associated with patients from developed regions .", "many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) .", "such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) .", "conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value .", "perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision ,", "making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) .", "researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) .", "however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) .", "in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) .", "researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) .", "hence , perceived risks and cognitive image should consider individual variables for image related studies in travel .", "credence goods as the quality of these good can not be assessed accurately even after consumption .", "patients may vulnerable to many risks if consider to take medical services at medical tourism destinations .", "researchers described six dimension of risks associated with health travelers visited israel as  human - induced risks , ", "financial risks ,  service quality risks ,  socio - psychological risk ,  natural disasters and car accident risk , and ", "after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as  physical - health related risks ,  service related risks , and  destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) .", "in medical tourism , international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism .", "health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) .", "literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) .", "researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel .", "apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) . according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) .", "destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel", ", researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) .", "attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination .", "later on , a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) .", "the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) .", "researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing ", "furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) .", "thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) .", "it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination .", "the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) .", "after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ;  medical amenities ,  general infra - structure ,  tourism attractions , and ", "social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency .", "majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff .", "command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) .", "based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services .", "physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians .", "prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk .", "the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) .", "collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules .", "most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) .", "most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients .", "many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world .", "language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) .", "facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients .", "essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination .", "thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) .", "potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability"], ["image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place .", "various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) .", "consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) .", "amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) .", "source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) .", "there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation .", "medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) .", "judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) .", "these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own .", "many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) .", "literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) .", "word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors .", "studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) .", "websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) .", "printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) .", "major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations .", "some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) .", "print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel .", "los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients .", "fox , cbs s  60 minutes  and cnn aired their segments on patients traveling to medical services .", "magazines like forbes and wall street journal analyzed the business aspect of medical tourism as  brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) ."], ["literature revealed that not every physical attribute of a destination has influence on image formation process .", "there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) .", "it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one .", "this is due to dimensions of liking may not be agree with the dimensions of similarity .", "literature revealed that many researchers made distinction between physical and beneficial aspect of a product .", "few researchers explained it as  characteristic and  benefits to the physical and beneficial aspect of product respectively ( 41 ) .", "the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery .", "there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) .", "the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds .", "benefits offered by a product or service are considered as consumption value of the same ( 44 ) .", "sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) .", "conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values ."], ["consumption value model define the characteristics of  functional value as price , credibility , and durability .", "medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) .", "quality of medical services is one of the aspects which drive medical tourism high towards success .", "most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) .", "high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) .", "although availability of literature explain  social value in medical tourism is less , little available literature clearly define the  social value factor with - in the patient groups especially from less developed regions .", "social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) .", "senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) .", "most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) .", "however , the concept of social value and epistemic value may not be associated with patients from developed regions .", "many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) . such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) .", "conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value ."], ["perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision ,", "making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) .", "researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) .", "however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) .", "in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) .", "researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) .", "hence , perceived risks and cognitive image should consider individual variables for image related studies in travel .", "credence goods as the quality of these good can not be assessed accurately even after consumption .", "patients may vulnerable to many risks if consider to take medical services at medical tourism destinations .", "researchers described six dimension of risks associated with health travelers visited israel as  human - induced risks , ", "financial risks ,  service quality risks ,  socio - psychological risk ,  natural disasters and car accident risk , and ", "after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as  physical - health related risks ,  service related risks , and ", "destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) . in medical tourism ,", "international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism .", "health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) .", "literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) .", "researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel .", "apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) ."], ["according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) .", "destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel", ", researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) .", "attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination . later on ,", "a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) .", "the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) .", "researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing ", "sleepy , pleasant  unpleasant , exciting gloomy , and relaxing  distressing ( 70 ) .", "furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) .", "thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) .", "it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination .", "the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) .", "after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ;  medical amenities ,  general infra - structure , ", "social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency .", "majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff .", "command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) . based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services .", "physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians .", "prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk .", "the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) .", "collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules .", "most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) .", "most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients .", "many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world .", "language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) .", "facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients .", "essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination .", "thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) .", "potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability"], ["prominent databases ( 2013 , 2014 , 2015 ) have been searched to obtain desire literature related to medical tourism including science direct , utmj.org , nih.gov , nchu.edu.tw , palgrave - journals , medretreat , biomedcentral and so on and the keywords used are medical tourism , information sources , medical travel , health travel and so on .", "google search was performed to get latest data related to medical tourism . to know in detail about the variables used in the framework scientific research databases", "the key words used to find out research variables as well as relationship between researches variables were destination image , destination information sources , beneficial image , perceived travel risks , cognitive image , affective image , consumption values and so on . to obtain data about medical tourism various types of available literature were reviewed such as industry reports , market research reports , media reports and internet sources , however for identification and explanation of variables scientific literature published in reputed journals only were included ."], ["the theory of planned ( tpb ) is an established and comprehensively tested model details the relationship between beliefs , attitude , intention , and actual behavior of consumers ( 85 ) . the model is perfectly applied in a variety of studies including leisure and tourism travel , and hospitality ( 36 , 8688 ) .", "these research studies emphases on the factors such as motivations , information sources , attitudes , and visit intentions , thus authors argue that attitudinal theory provides a sound foundation to understand travel intentions of international patients and underpin the conceptual model discussed in this article .", "studies explained that consumers access more information in case of high association of risks or benefits or both in a particular act ( 89 , 90 ) and perform rigorous information search in case of planning first time trip ( 91 ) because first time trip is associated with unknown risks as well as unknown leisure activities .", "in general consumer behaviour practices risk - handling activity increases in case of high level of perceived risks .", "importance of risk handling activity also determines by associated benefits gained after engaging with risk taking activity ( 92 ) .", "the benefits of information search includes possibility of finding superior alternative to those already considered and the reduction in risk achieved from eliminating inferior , but a priori uncertain , alternatives ( 93 ) . with the discussion of above literature related to tourism", "it could be assumed that medical tourism information sources will have positive influence on perceived travel benefits of international patients . whereas , information sources of medical tourism will negatively influence perceived travel risks of international patients .", "destination image is considered as perceptions or impressions of destination held by tourists with respect to the expected benefit or consumption values .", "the benefits sought by the travelers are highly associated with the image of destination and the affective image of destination is largely highly influenced by the motivations / benefits sought by individuals ( 13 , 44 ) .", "the affective image of a destination is more positive in individual s mind when emotions evoked by the place coincide with the benefits sought ( 95 ) .", "risks and constraints associated with travel have significant impact on destination image formation at the time of early decision - making process ( 96 ) . according to researchers ,", "two dimensions significantly influence cognitive and affective image of destination ( 58 ) . on the basis of previous studies in tourism literature", "it can be assumed that perceived travel benefits of international patients will positively influence the destination image , whereas perceived travel risks of international patients will negatively influence the destination image .", "intention of visit related to travel is considered as tourists perceived likelihood to visit particular destination within a specific time period ( 97 ) .", "thus , to be closely correlated to the travel behaviour intention of visit believes as an important outcome variable in tourism research ( 16 , 97 ) .", "intention to visit a destination is highly influenced by cognitive / perceptual and affective evaluation ( 98 ) .", "destination image is a direct antecedent of perceived quality , satisfaction and revisit intention and recommendation ( 99 ) . according to researchers", ", there is significant theoretical and empirical link between cognitive destination image and behavioral intention ( 100 ) .", "based on above discussion it could be assumed that image of medical tourism destination will positively influence visit intention of international patients . conceptual framework  international patients travel decision making"], ["the article is a sincere effort by authors to establish conceptual framework which explains decision - making process of international patients .", "though , there has been advance in explaining different factors play important roles in international patients travel , however , there is scarcity of literature conceptualizing these factors into a framework and test it empirically .", "literature reveals about associated benefits and risks related to travel abroad for medical purposes but do not conceptualize it in the process of decision - making .", "sources of information in medical tourism play significant role in development of attitude as well as intention , has already been established in consumer behavior .", "however , need is to test its significance in medical tourism decision - making process .", "the proposed framework will encourage future researchers to test different variables empirically and establish the model of international patients travel behavior ."], ["ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors ."]]}
{"article_id": "PMC3135278", "article_text": ["squamous cell carcinoma of the head and neck ( hnscc ) is a heterogeneous disease that includes tumors arising from the mucosal epithelial surface of the oral cavity , oropharynx , hypopharynx , and larynx . although these tumors originate within different anatomic sites within the upper aerodigestive tract , they are histologically identical ( 95% of hnscc are squamous cell carcinomas ) , share common etiologic risk factors and overlapping metastatic target site profiles ( reviewed in [ 13 ] ) .", "recent genetic analysis of human head and neck tumors has revealed common molecular alterations including p53 mutation , p14arf , and p16 methylation , as well as cyclin d and egfr amplification [ 36 ] . despite these similarities ,", "the distinct anatomic subsites are associated with differing rates of regional metastasis  for example , vocal cord lesions tend to metastasize less frequently than oropharyngeal or hypopharyngeal lesions . this variation may be attributed to differing densities of lymph draining vessels within each of the relevant subsites .", "patients who exhibit metastases into the regional nodal basin exhibit a 50% decrease in survival irrespective of treatment [ 715 ] .", "currently , it is the 5th leading cause of cancer by incidence and the 6th leading cause of cancer mortality in the world [ 16 , 17 ] .", "recurrent and/or metastatic hnscc patients have a poor prognosis , with a median survival of less than 1 - 2 years [ 18 , 19 ] .", "several lines of evidence indicate that cancer is a disease resulting from dynamic changes in the genome that promote the progressive transformation of normal human cells into highly malignant derivatives [ 20 , 21 ] . during this process ,", "cancer cells acquire several unique capabilities including self - sufficiency in response to growth signals , insensitivity to antigrowth signals , evasion of programmed death ( apoptosis ) , limitless replicative potential , sustained angiogenesis as well as invasion and metastasis , reprogramming of energy metabolism , and avoiding immune destruction [ 21 , 22 ] .", "detailed global genomic analyses of several human tumors has revealed that certain classes of signaling proteins appear to be targeted more frequently by oncogenic mutations .", "receptor tyrosine kinases ( rtks ) are a good example . of the 59 transmembrane rtks identified to date ,", "dysregulation of ~30 rtks are associated with neoplastic transformation and cancer progression [ 2325 ] .", "interestingly , ninety percent of primary head and neck squamous cell cancers , irrespective of subsite , have alterations in members of the epidermal growth factor ( egf ) family of receptor tyrosine kinases ( erbbs ) , in particular erbb1/egfr .", "ten to fifteen percent of tumors will also have an alteration in another egfr family member , the erbb2/her2/neu receptor [ 27 , 28 ] .", "these findings suggest a strong etiologic role for rtk dysregulation in this type of tumors . given this association , patients with head and neck squamous cell cancers are well positioned to benefit from existing and future molecular targeted agents directed against oncogenic rtks such as egfr ( reviewed in ) .     ", "rtks are a family of transmembrane proteins that mediate many important physiological processes in both normal and cancerous cells .", "ligand binding to the extracellular domain of rtks induces receptor dimerization and activation of rtk activity .", "subsequent autophosphorylation of the receptor at specific tyrosine residues within the cytoplasmic domain generates binding sites for proteins that relay downstream biological signals to regulate protein function , protein - protein interactions , and gene expression . under physiological conditions ,", "rtk dysregulation can occur through several mechanisms including gene amplification or rtk overexpression , chromosomal translocation to produce constitutively active rtks , gain of function mutations or deletions that promote ligand - independent rtk activity , escape from negative regulatory mechanisms or local environmental changes , all of which lead to potent oncogenic signaling and hence neoplastic growth .", "these complex signaling networks use multiple factors to drive the outcome of rtk signaling . although often depicted as linear pathways , they actually represent an integrated network with various modes of cross - talk , overlapping and distinct functions .", "known signaling pathways involved in head and neck tumorigenesis include the phosphatidylinositol-3-kinase ( pi3k)-akt - mammalian target of rapamycin ( mtor ) , signal transducer and activator of transcription ( stats ) and raf kinase - mitogen - activated protein kinase kinase ( mek)-p42/p44 mitogen activated protein kinase ( mapk ) signaling pathways [ 1 , 30 ] .", "this review highlights three rtk signaling pathways involved in head and neck squamous cell carcinoma ; egfr , the type 1 insulin - like growth factor receptor ( igf-1r ) and the hepatocyte growth factor ( hgf ) receptor ( met ) .", "this short review will explore the relative contribution of each signaling axis to disease progression , potential modes of cross - talk , and targeted clinical approaches under investigation for disease management .", "the egfr family of rtks is comprised of four different receptors known as erbb1 ( also referred to as egfr ) , erbb2 ( her2/neu in rodents ) , erbb3 ( her3 ) , and erbb4 ( her4 ) ( reviewed in [ 3133 ] ) . each receptor , with the exception of erbb3 , contain an intracellular tyrosine kinase domain that is activated by binding to extracellular egf - like ligands , which result in receptor dimerization and hence activation of downstream signaling cascades including mapk , pi3k / akt and stat signaling .", "eleven egf - like ligands have been identified to date that can be categorized into four groups  those that bind egfr only ( egf , transforming growth factor alpha ( tgf ) , and amphiregulin ) , those that bind to egfr and her4 ( heparin binding - egf , betacellulin and epiregulin ) , those binding directly to either her3 and her4 ( neuregulin 1 and neuregulin 2 ) and her4 binding only ( neuregulin 3 and neuregulin 4 ) ( reviewed in ) .", "epigen , the most recently discovered member of the egf - like ligand family appears to be a low affinity and broad specificity ligand that effectively activates egfr .", "erbb2 is considered a ligand - less coreceptor as it does not have any known ligands that bind directly with high affinity , despite its established role as a potent oncogene in several cancer types including breast , colorectal , nonsmall cell lung carcinoma ( nsclc ) and hnscc [ 36 , 37 ] .  ", "aberrant egfr activity has been strongly linked to the etiology of 5890% of hnscc [ 26 , 38 ] .", "these rates can vary due to the inclusion of cancers from different subsites within the head and neck , methods used to assess gene amplification and tumor scoring methods . in contrast to lung adenocarcinomas in which activating egfr mutations result in ligand - independent signaling [ 3943 ] , such activating egfr mutations are infrequent in hnscc [ 44 , 45 ] .", "egfr gene amplification resulting in upwards of 12 copies per cell has been reported in hnscc patients compared to copy numbers detected in normal mucosa from noncancer patients .", "this and other pathways of ligand - independent receptor activation that do not require egfr overexpression have been characterized as the likely drivers of egfr activity in hnscc .  ", "egfr gene amplification remains a strong indicator for poor patient survival , radioresistance , and locoregional failure [ 4749 ] .", "egfr overexpression is detected in healthy mucosa in cancer patients ( field cancerization ) that will increase in proportion to observed histological abnormalities such as hyperplasia , carcinoma in situ and invasive carcinoma , indicating that it is an early event in hnscc .", "accordingly , significant effort has focused on egfr signaling as a therapeutic target for treating hnscc patients .", "cetuximab , matuzumab and nimotuzumab represent humanized antiegfr antibodies , whereas gefitinib and erlotinib are small tyrosine kinase inhibitors ( tkis ) ( figure 1 ) .", "cetuximab ( erbitux ) competitively inhibits endogenous ligand - binding to egfr and thereby inhibits subsequent receptor activation [ 5053 ] .", "cetuximab is a valuable treatment option in head and neck patients as it synergizes with current treatment modalities .", "cetuximab enhances the effects of many standard cytotoxic agents , including cisplatin ( the conventional platinum - fluorouracil chemotherapeutic ) , and in combination with chemotherapy it can elicit antitumor responses in tumors that previously failed to respond to that chemotherapy .", "notably , cetuximab did not dramatically exacerbate the common toxic effects associated with radiotherapy of the head and neck , including mucositis , xerostomia , dysphagia , pain , weight loss , and performance status deterioration .", "cetuximab has been approved for use in combination with radiation for treating patients with locally advanced hnscc   and as monotherapy for patients with recurrent hnscc .", "matuzumab ( formerly emd 72000 ) binds to egfr with high specificity and affinity to block receptor signaling , and also modulates antibody - dependent cellular cytotoxicity ( adcc ) when combined with cetuximab [ 5860 ] .", "phase i clinical trials report excellent antitumor activity of matuzumab against several human tumor types including head and neck cancers .", "a randomized phase iib , four - arm , open - label study recently assessed the safety and efficacy of nimotuzumab in combination with radiation therapy ( rt ) or chemoradiation therapy ( crt ) in patients with advanced ( stage iii or iva ) hnscc .", "the addition of nimotuzumab to both the radiation and chemoradiation regimens was reported to improve the overall response rate , survival rate at 30 months , median progression - free survival and median overall survival .", "a combined group analysis of the nimotuzumab arms versus the non - nimotuzumab arms demonstrated a significant difference in overall survival favoring nimotuzumab .", "this study is compelling as patient response rates compare favorably with studies combining cetuximab with radiotherapy , but with fewer side effects .", "gefitinib ( iressa ) is a small molecule tki - targeted to the intracellular active site for phosphorylation that has been tested in clinical trials involving hnscc patients , as a single agent or in combination with radiation treatment .", "unfortunately , gefitinib has shown limited clinical efficacy with response rates of 1015% [ 63 , 64 ] .", "erlotinib is a selective inhibitor of the egfr that also shows antitumor activity in hnscc comparable to standard combination chemotherapy .", "another promising rtk under preclinical and clinical evaluation for head and neck cancers includes the igf-1r ( reviewed in [ 66 , 67 ] ) .", "two ligands , insulin - like growth factor 1 ( igf1 ) and igf2 bind to igf-1r .", "ligand binding to the igf-1r stimulates its intrinsic tyrosine kinase activity , activating downstream signaling networks including ras - raf , mapk and erk , and pi3k ( figure 1 ) to drive cellular functions such as cell growth , survival and differentiation .", "it is widely accepted that the igf - axis activates antiapoptotic signaling , which in turn upregulates the pi3k - akt and mapk pathways in cancer cells .", "additionally , igf - ir also regulates vascular endothelial growth factor ( vegf ) production , suggesting a role in tumor angiogenesis .", "several studies indicate that igf-1r is overexpressed and functional in 94% of hnscc patient samples [ 70 , 71 ] .", "consistent with this , igf - ir signaling significantly enhances the proliferation , motility and tumorigenicity of human head and neck cancer cell lines . igf-1r", "down regulation in a hnscc cell line using antisense oligonucleotides resulted in a dose - dependent decrease in cellular proliferation , induction of apoptosis , caspase activation and reduced expression of proangiogenic cytokines such as vegf .", "interest in targeting the igf-1r in hnscc was bolstered by the observation that treatment of head and neck cancer cells with either igf or egf resulted in igf - ir and egfr heterodimerization [ 71 , 72 ] .", "however , only igf resulted in the phosphorylation of both receptors . using a mouse xenograft model for hnscc , treatment with antibodies against igf-1r , egfr or", "it remains to be determined whether cellular cross - talk between igf-1r and egfr has an important role in determining the biological aggressiveness of hnscc or resistance to egfr - targeted therapies .  ", "several monoclonal antibodies and tkis for igf-1r have been tested in preclinical studies and early phase clinical studies .", "however , the efficacy of igf-1r - targeted therapy for treating patients with hnscc , particularly cross - talk with egfr , warrants further investigation . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer cell lines [ 7375 ] .", "treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] . targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) . similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) .", "these studies will be critical for evaluating whether the use of anti - igf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc .", "the met receptor is a single pass transmembrane protein that upon binding its ligand hgf  also known as scatter factor - promotes increased cell proliferation , survival and motility ( reviewed in [ 78 , 79 ] ) .", "hgf is the only physiological ligand for met and is secreted as an inactive precursor polypeptide chain by mesenchymal cells .", "hgf is proteolytically cleaved to form an active / heterodimer by a number of serine proteases including urokinase plasminogen activator ( upa ) , tissue - type plasminogen activator ( tpa ) , coagulation factors x. xi and xii .", "met is a disulphide - linked / heterodimer derived from the proteolytic cleavage of a 170  kda precursor .", "the  chain and n - terminal region of the -chain form  sema domain , a seven -propeller structure in which blades 2 and 3 bind to hgf .", "the sema domain is flanked by a cysteine - rich region followed by four immunoglobulin repeats .", "it is proposed that the cysteine - rich region and immunoglobulin repeat domains undergo a conformational change following hgf binding allowing for met dimerization [ 80 , 81 ] .", "binding of hgf to met results in receptor autophosphorylation at key catalytic residues and subsequent recruitment of several cytosolic signaling molecules that are shared with the egfr and igf-1r signaling pathways , including the grb2/sos complex , the p85 regulatory subunit of pi3k , gab1 and jak / stat3 ( figure 1 ) .", "subsequent activation of the mapk and jun - n - terminal kinase ( jnk ) pathways is responsible for the mitogenic and motogenic properties of met / hgf signaling resulting in  invasive growth  , depending on the physiological setting .", "increased met signaling in human cancers can be the result of enhanced ligand - binding ( autocrine and paracrine ) , met overexpression or missense mutations that often induce constitutive kinase activity , failure of met down regulation and interactions with other cell surface receptors such as egfr ( reviewed in [ 8284 ] ) . met is overexpressed in 84% of hnscc patient samples .", "interestingly , amplification of the met gene ( > 10 copies per cell ) is present only in 3 of 23 ( 13% ) tumor tissues .", "hgf overexpression is detected in 45% of hnsccs , suggesting that hgf functions predominantly in a paracrine manner to drive met signaling in these cancers .", "moreover , high levels of hgf are detected in hnscc patient plasma samples   supporting the idea that ligand availability is not a limiting factor for met activation .", "mutations in the met ligand - binding domain ( t230m / e168d ) , transmembrane or jm domain ( r988c , t1010i ) and the tyrosine kinase domain ( t1275i , v14333i ) have also been identified in hnscc tumor samples , although their relative contribution to hnscc progression remains to be determined .", "two somatic met mutations have been detected in hnscc that result in constitutively active receptor signaling that confers an invasive phenotype when ectopically expressed in cell lines .", "the y1230c mutation confers anchorage - independent growth and an invasive phenotype in transfected cells , whereas the y1235d met mutation stimulates epithelial cells to invade reconstituted basement membrane in the absence of hgf . in the case of the mety1235d mutation", ", genomic analyses of hnscc patient samples detected the presence of this mutant allele in 50% of metastatic tumors versus 26% in primary tumors , raising the possibility that this could be a critical genetic lesion for the acquisition of a metastatic phenotype .", "alternatively , increased met signaling could afford hnscc a selective advantage for growth and/or survival in metastatic sites , such as the lymph node and lung .", "indeed several studies indicate that met overexpression correlates highly with lymph node metastasis , pathologic stage , and disease reoccurrence [ 8891 ] .", "moreover , patient survival was significantly reduced in biopsy samples with positive met expression relative to negative met expression , suggesting the association of met with hnscc disease progression .", "consistent with these findings , treatment with the tki pf-2341066 caused a significant reduction in tumor growth , a high level of apoptosis and cellular debris within the tumor using a xenograft animal model for hnscc .", "selective inhibitors of met / hgf signaling include humanized monoclonal antibodies for hgf and met , and small - molecule tyrosine kinase inhibitors directed against met ( figure 1 ) .", "although their efficacy for treating a variety of solid tumors is increasingly recognized , we await results of preclinical and clinical trials for head and neck cancer that are ongoing .", "the humanized antibody amg 102 shows high potency towards the mature and processed form of hgf with no detected effects on proteolytic activation of prohgf .", "amg 102 interferes with met signaling , by competing with hgf for binding to the  chain of the met receptor . in phase", "i clinical studies in patients with advanced solid tumors , 70% of patients had a best response in terms of achieving stable disease [ 93 , 94 ] .", "importantly , no antiamg 102 antibodies were detected and circulating hgf levels were dose dependent .", "another promising clinical therapeutic is the one - armed 5d5 humanized antibody ( oa5d5/metmab ) directed against met .", "metmab binds met with high affinity , preventing hgf binding , met phosphorylation , receptor internalization and downstream signaling events and has been shown to inhibit tumor growth in animal models by more than 95% [ 95 , 96 ] .", "metmab is currently in phase i / ii human clinical trials in comparison with erlotinib in patients with nsclc ( http://www.clinicaltrials.gov/ , identifier nct00854308 ) .", "future clinical trials will be required to determine the suitability of amg102 and metmab as either single agents or combinatorial therapeutics for treating hnscc patients .", "foretinib ( formerly xl880 ) is a tki whose primary targets include met and vegf , and to a lesser extent the platelet - derived growth factor ( pdgf ) receptor , ron , kit and tie2 rtks .", "foretinib recently completed phase ii clinical trials in head and neck patients ( http://www.clinicaltrials.gov/ , identifier nct00725764 ) .", "interim results suggest that after 12 months , 12 of 18 patients had stable disease .", "a phase i dose - escalation study of the safety and pharmacokinetics of xl184 administered orally to patients with advanced malignancies ( showed that , on average , patients survived for more than 3 months with several up to 6 months while on treatment ) ( reviewed in ) . due to", "encouraging data from this study , a randomized phase iii trial of xl184 in hnscc patients was initiated to investigate xl184 as a first - line treatment ( compared with placebo ) for survival benefit to patients with hnscc ( http://www.clinicaltrials.gov/ , identifier nct00704730 ) .", "arq197 ( arqule ) is a nonatp - site competitive , selective small molecule inhibitor of the met intracellular region .", "although the mechanism of arq197 is presently unknown , the results of phase i trials suggest potential antiinvasive activity for this compound .", "overall , met , and hgf - targeted therapies have been well tolerated in clinical trials with negligible toxicities .", "however , it remains to be determined whether met is a better therapeutic target than hgf .", "clearly , in patients where met is activated by autocrine hgf secretion , both hgf and met targeted therapies may prove to be more efficacious treatment options .", "acquired resistance is likely the result of several mechanisms including ( 1 ) egfr mutations initially present as well as those acquired during therapy , ( 2 ) receptor independent activation of downstream signaling cascades , ( 3 ) cross - talk with other rtks and converging signaling pathways and ( 4 ) environmental factors including inflammatory agents and viral infection .", "resistance to cetuximab has been associated with the coexpression of the truncated egfr mutant , egfrviii with wild - type egfr .", "egfrviii is the result of an in frame deletion of exons 27 spanning the extracellular ligand - binding domain .", "the deletion results in a truncated egfr receptor that signals in a ligand - independent manner .", "egfrviii expression has been detected in 42% of hnscc patient samples , and closely correlates with increased hnscc cell proliferation in vitro and increased tumor growth using in vivo xenograft models .", "egfrviii preferentially activates the pi3k pathway instead of the ras / raf / mek pathway , which is activated by wild - type egfr . of particular interest to the therapeutic treatment of hnscc ,", "egfrviii expression decreases the proliferative response of egfr expressing tumor cells to cetuximab treatment relative to vector control cells . in a recent study ,", "egfrviii cells were shown to be resistant to the antiinvasive effects of cetuximab due to an increase in phosphorylation of stat3 rather than increased pi3k signaling .", "egf - induced expression of the stat3 target gene hif1 was abolished by cetuximab in hnscc cells expressing wild - type egfr under hypoxic conditions , but not in egfrviii - expressing hnscc cells [ 102 , 103 ] .", "these data suggest a role for egfrviii in mediating hnscc resistance to cetuximab .   despite egfrs critical role in the development of hnscc", ", clinical data indicate modest clinical benefits for locoregional control and survival of head and neck cancer patients treated with egfr - targeted therapies .", "hnscc patients resistant to cetuximab , often succumb to local tumor recurrence as well as regional and distant metastasis .", "the addition of cetuximab to radiation therapy was reported to show improved locoregional disease control , progression - free survival , and overall survival in patients with locally advanced hnscc .", "however the data revealed a disproportionate benefit of cetuximab with radiotherapy to oropharyngeal cancer patients when compared to patients treated with hyperfractionated radiotherapy .", "accumulating evidence suggests that human papilloma virus ( hpv ) 16 status ( hpv+ ) is an important prognostic factor associated with a favorable outcome in a subset of head and neck cancers , including oropharyngeal and tonsilar cancers .", "hpv+ tumors tend to have unique genetic aberrations including decreased egfr expression , whereas increased igf-1r levels characteristic of hnscc appear to be independent of hpv status .", "clinically , hpv+ tumors are characterized by more favorable patient prognosis regarding disease - free survival as well as overall survival [ 104 , 105 ] , possibly as a result of increased genomic stability associated with global gene hypermethylation in hpv+ tumors .", "thus it will be interesting to determine whether hpv+ status explains some of the benefits derived from the addition of cetuximab to radiotherapy in this subset of hnscc patients . at present", ", there are few clinical indicators of which hnscc patients will most likely respond to egfr - targeted therapies .", "accordingly , strategies to optimize egfr - targeted therapy remain an active area of research .", "additional mechanisms that result in egfr activation include activating mutations in downstream signaling components or cross - talk between different rtk pathways . activating mutations in the pi3ka oncogene", "occurs in 10% of hnscc tumors   whereas elevated levels of phosphorylated stat3 correlates with lymph node metastasis and poor patient prognosis [ 108110 ] .", "conversely , h - ras mutations are infrequent in hnscc cases ( less than 5% ) , although a higher incidence has been detected in asian populations and correlates with areca nut chewing [ 111 , 112 ] .", "met signaling has been shown to contribute to resistance in cell lines derived from multiple tumor types including breast , gastric and lung .", "in one key study , nsclc with activating mutations in the egfr acquire resistance to the tki gefitinib and erlotinib , by amplification of the met gene to maintain akt and her3 signaling .", "these studies underscore the role of cross - talk between rtks to preferentially signal through the pi3k - akt survival pathway as a mechanism for acquired drug resistance .", "the relevance of met as a mechanism for escape from egfr - targeted therapy in head and neck cancers remains to be determined .", "hypoxia results in the transcriptional upregulation of met gene expression via hif1 in a number of tumors including head and neck , often downstream of egfr signaling . in normoxia", ", hydroxylation of 2 prolines in hif1 enables its binding to the von hippel - lindau tumor suppressor protein ( pvhl ) linking hif1 to a ubiquitin ligase complex . during hypoxia , minimal or no hydroxylation occurs enabling hif1 to avoid proteasomal degradation and", "dimerize to other hif family members such as hif1   and coactivators , to form an active transcriptional hif complex on the hypoxia response element ( hre ) of target genes such as met .", "the ubiquitin ligase catalyzes polyubiquitination of hif1 targeting it for proteasomal degradation . under hypoxic conditions ,", "increased met signaling directs the invasive growth program , enabling cells to invade more oxygenated tissues .", "since met has been reported to promote invasive and angiogenic effects in the tumor microenvironment , the use of hgf / met inhibitors may afford a means of impairing tissue colonization as well as tumor vascularization in head and neck cancer patients .     ", "studies on other solid tumor types , most notably glioblastoma , indicate a role for igf-1r upregulation in resistance to egfr - targeted therapies .", "igf-1r mediates resistance to anti - egfr therapy in primary glioblastoma through the continued activation of the pi3k / akt survival pathway .", "the apparent cooperation between igf-1r and egfr in promoting hnscc pathogenesis as well as resistance to egfr - targeted therapy , suggests an advantage to cotargeting these signaling axes for the treatment of head and neck cancers . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer lines .", "treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] .", "targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) .", "similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) .", "these studies will be critical for evaluating whether the use of antiigf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc .", "targeted therapies that block egfr , met , and igf-1r signaling in head and neck cancers continue to show promising results in preclinical studies and clinical trials .", "however , it is difficult to predict which patients are most likely to benefit from these therapeutics and potential side effects during long - term in vivo use . given the interplay between these rtk signaling pathways and the mediocre results obtained with monotherapy regimens thus far , clinical trials will be required to determine how egfr- , met- , and igf-1r - targeted therapies can be used in combination in order to definitively abrogate their common downstream oncogenic signaling networks .", "although gaps in our knowledge concerning the role of met and igf-1r in head and neck tumorigenesis , as well as acquired resistance to antiegfr therapies remain to be addressed , efforts to translate current information towards clinical applications continue to be impressive ."], "abstract_text": ["<S> molecular therapeutics for treating epidermal growth factor receptor-(egfr- ) expressing cancers are a specific method for treating cancers compared to general cell loss with standard cytotoxic therapeutics .   </S>", "<S> however , the finding that resistance to such therapy is common in clinical trials now dampens the initial enthusiasm over this targeted treatment .   </S>", "<S> yet an improved molecular understanding of other receptor tyrosine kinases known to be active in cancer has revealed a rich network of cross - talk between receptor pathways with a key finding of common downstream signaling pathways . </S>", "<S> such cross talk may represent a key mechanism for resistance to egfr - directed therapy .   </S>", "<S> here we review the interplay between egfr and met and the type 1 insulin - like growth factor receptor ( igf-1r ) tyrosine kinases , as well as their contribution to anti - egfr therapeutic resistance in the context of squamous cell cancer of the head and neck , a tumor known to be primarily driven by egfr - related oncogenic signals . </S>"], "labels": null, "section_names": ["1. Introduction", "2. EGFR Amplification in Head and Neck Cancers", "3. Targeting IGF-1R Signaling in Head and Neck Cancers", "4. A Role for Met/HGF Signaling in Head and Neck Cancers", "5. Understanding Resistance to EGFR-Targeted Therapies in HNSCC", "6. Conclusions"], "sections": [["squamous cell carcinoma of the head and neck ( hnscc ) is a heterogeneous disease that includes tumors arising from the mucosal epithelial surface of the oral cavity , oropharynx , hypopharynx , and larynx . although these tumors originate within different anatomic sites within the upper aerodigestive tract , they are histologically identical ( 95% of hnscc are squamous cell carcinomas ) , share common etiologic risk factors and overlapping metastatic target site profiles ( reviewed in [ 13 ] ) .", "recent genetic analysis of human head and neck tumors has revealed common molecular alterations including p53 mutation , p14arf , and p16 methylation , as well as cyclin d and egfr amplification [ 36 ] . despite these similarities ,", "the distinct anatomic subsites are associated with differing rates of regional metastasis  for example , vocal cord lesions tend to metastasize less frequently than oropharyngeal or hypopharyngeal lesions . this variation may be attributed to differing densities of lymph draining vessels within each of the relevant subsites .", "patients who exhibit metastases into the regional nodal basin exhibit a 50% decrease in survival irrespective of treatment [ 715 ] .", "currently , it is the 5th leading cause of cancer by incidence and the 6th leading cause of cancer mortality in the world [ 16 , 17 ] .", "recurrent and/or metastatic hnscc patients have a poor prognosis , with a median survival of less than 1 - 2 years [ 18 , 19 ] .", "several lines of evidence indicate that cancer is a disease resulting from dynamic changes in the genome that promote the progressive transformation of normal human cells into highly malignant derivatives [ 20 , 21 ] . during this process ,", "cancer cells acquire several unique capabilities including self - sufficiency in response to growth signals , insensitivity to antigrowth signals , evasion of programmed death ( apoptosis ) , limitless replicative potential , sustained angiogenesis as well as invasion and metastasis , reprogramming of energy metabolism , and avoiding immune destruction [ 21 , 22 ] .", "detailed global genomic analyses of several human tumors has revealed that certain classes of signaling proteins appear to be targeted more frequently by oncogenic mutations .", "receptor tyrosine kinases ( rtks ) are a good example . of the 59 transmembrane rtks identified to date ,", "dysregulation of ~30 rtks are associated with neoplastic transformation and cancer progression [ 2325 ] .", "interestingly , ninety percent of primary head and neck squamous cell cancers , irrespective of subsite , have alterations in members of the epidermal growth factor ( egf ) family of receptor tyrosine kinases ( erbbs ) , in particular erbb1/egfr .", "ten to fifteen percent of tumors will also have an alteration in another egfr family member , the erbb2/her2/neu receptor [ 27 , 28 ] .", "these findings suggest a strong etiologic role for rtk dysregulation in this type of tumors . given this association , patients with head and neck squamous cell cancers are well positioned to benefit from existing and future molecular targeted agents directed against oncogenic rtks such as egfr ( reviewed in ) .     ", "rtks are a family of transmembrane proteins that mediate many important physiological processes in both normal and cancerous cells .", "ligand binding to the extracellular domain of rtks induces receptor dimerization and activation of rtk activity .", "subsequent autophosphorylation of the receptor at specific tyrosine residues within the cytoplasmic domain generates binding sites for proteins that relay downstream biological signals to regulate protein function , protein - protein interactions , and gene expression . under physiological conditions ,", "rtk dysregulation can occur through several mechanisms including gene amplification or rtk overexpression , chromosomal translocation to produce constitutively active rtks , gain of function mutations or deletions that promote ligand - independent rtk activity , escape from negative regulatory mechanisms or local environmental changes , all of which lead to potent oncogenic signaling and hence neoplastic growth .", "these complex signaling networks use multiple factors to drive the outcome of rtk signaling . although often depicted as linear pathways , they actually represent an integrated network with various modes of cross - talk , overlapping and distinct functions .", "known signaling pathways involved in head and neck tumorigenesis include the phosphatidylinositol-3-kinase ( pi3k)-akt - mammalian target of rapamycin ( mtor ) , signal transducer and activator of transcription ( stats ) and raf kinase - mitogen - activated protein kinase kinase ( mek)-p42/p44 mitogen activated protein kinase ( mapk ) signaling pathways [ 1 , 30 ] .", "this review highlights three rtk signaling pathways involved in head and neck squamous cell carcinoma ; egfr , the type 1 insulin - like growth factor receptor ( igf-1r ) and the hepatocyte growth factor ( hgf ) receptor ( met ) .", "this short review will explore the relative contribution of each signaling axis to disease progression , potential modes of cross - talk , and targeted clinical approaches under investigation for disease management ."], ["the egfr family of rtks is comprised of four different receptors known as erbb1 ( also referred to as egfr ) , erbb2 ( her2/neu in rodents ) , erbb3 ( her3 ) , and erbb4 ( her4 ) ( reviewed in [ 3133 ] ) . each receptor , with the exception of erbb3 , contain an intracellular tyrosine kinase domain that is activated by binding to extracellular egf - like ligands , which result in receptor dimerization and hence activation of downstream signaling cascades including mapk , pi3k / akt and stat signaling .", "eleven egf - like ligands have been identified to date that can be categorized into four groups  those that bind egfr only ( egf , transforming growth factor alpha ( tgf ) , and amphiregulin ) , those that bind to egfr and her4 ( heparin binding - egf , betacellulin and epiregulin ) , those binding directly to either her3 and her4 ( neuregulin 1 and neuregulin 2 ) and her4 binding only ( neuregulin 3 and neuregulin 4 ) ( reviewed in ) .", "epigen , the most recently discovered member of the egf - like ligand family appears to be a low affinity and broad specificity ligand that effectively activates egfr .", "erbb2 is considered a ligand - less coreceptor as it does not have any known ligands that bind directly with high affinity , despite its established role as a potent oncogene in several cancer types including breast , colorectal , nonsmall cell lung carcinoma ( nsclc ) and hnscc [ 36 , 37 ] .  ", "aberrant egfr activity has been strongly linked to the etiology of 5890% of hnscc [ 26 , 38 ] .", "these rates can vary due to the inclusion of cancers from different subsites within the head and neck , methods used to assess gene amplification and tumor scoring methods . in contrast to lung adenocarcinomas in which activating egfr mutations result in ligand - independent signaling [ 3943 ] , such activating egfr mutations are infrequent in hnscc [ 44 , 45 ] .", "egfr gene amplification resulting in upwards of 12 copies per cell has been reported in hnscc patients compared to copy numbers detected in normal mucosa from noncancer patients .", "this and other pathways of ligand - independent receptor activation that do not require egfr overexpression have been characterized as the likely drivers of egfr activity in hnscc .  ", "egfr gene amplification remains a strong indicator for poor patient survival , radioresistance , and locoregional failure [ 4749 ] .", "egfr overexpression is detected in healthy mucosa in cancer patients ( field cancerization ) that will increase in proportion to observed histological abnormalities such as hyperplasia , carcinoma in situ and invasive carcinoma , indicating that it is an early event in hnscc .", "accordingly , significant effort has focused on egfr signaling as a therapeutic target for treating hnscc patients .", "cetuximab , matuzumab and nimotuzumab represent humanized antiegfr antibodies , whereas gefitinib and erlotinib are small tyrosine kinase inhibitors ( tkis ) ( figure 1 ) .", "cetuximab ( erbitux ) competitively inhibits endogenous ligand - binding to egfr and thereby inhibits subsequent receptor activation [ 5053 ] .", "cetuximab is a valuable treatment option in head and neck patients as it synergizes with current treatment modalities .", "cetuximab enhances the effects of many standard cytotoxic agents , including cisplatin ( the conventional platinum - fluorouracil chemotherapeutic ) , and in combination with chemotherapy it can elicit antitumor responses in tumors that previously failed to respond to that chemotherapy .", "notably , cetuximab did not dramatically exacerbate the common toxic effects associated with radiotherapy of the head and neck , including mucositis , xerostomia , dysphagia , pain , weight loss , and performance status deterioration .", "cetuximab has been approved for use in combination with radiation for treating patients with locally advanced hnscc   and as monotherapy for patients with recurrent hnscc .", "matuzumab ( formerly emd 72000 ) binds to egfr with high specificity and affinity to block receptor signaling , and also modulates antibody - dependent cellular cytotoxicity ( adcc ) when combined with cetuximab [ 5860 ] .", "phase i clinical trials report excellent antitumor activity of matuzumab against several human tumor types including head and neck cancers .", "a randomized phase iib , four - arm , open - label study recently assessed the safety and efficacy of nimotuzumab in combination with radiation therapy ( rt ) or chemoradiation therapy ( crt ) in patients with advanced ( stage iii or iva ) hnscc .", "the addition of nimotuzumab to both the radiation and chemoradiation regimens was reported to improve the overall response rate , survival rate at 30 months , median progression - free survival and median overall survival .", "a combined group analysis of the nimotuzumab arms versus the non - nimotuzumab arms demonstrated a significant difference in overall survival favoring nimotuzumab .", "this study is compelling as patient response rates compare favorably with studies combining cetuximab with radiotherapy , but with fewer side effects .", "gefitinib ( iressa ) is a small molecule tki - targeted to the intracellular active site for phosphorylation that has been tested in clinical trials involving hnscc patients , as a single agent or in combination with radiation treatment .", "unfortunately , gefitinib has shown limited clinical efficacy with response rates of 1015% [ 63 , 64 ] .", "erlotinib is a selective inhibitor of the egfr that also shows antitumor activity in hnscc comparable to standard combination chemotherapy ."], ["another promising rtk under preclinical and clinical evaluation for head and neck cancers includes the igf-1r ( reviewed in [ 66 , 67 ] ) .", "two ligands , insulin - like growth factor 1 ( igf1 ) and igf2 bind to igf-1r .", "ligand binding to the igf-1r stimulates its intrinsic tyrosine kinase activity , activating downstream signaling networks including ras - raf , mapk and erk , and pi3k ( figure 1 ) to drive cellular functions such as cell growth , survival and differentiation .", "it is widely accepted that the igf - axis activates antiapoptotic signaling , which in turn upregulates the pi3k - akt and mapk pathways in cancer cells .", "additionally , igf - ir also regulates vascular endothelial growth factor ( vegf ) production , suggesting a role in tumor angiogenesis .", "several studies indicate that igf-1r is overexpressed and functional in 94% of hnscc patient samples [ 70 , 71 ] .", "consistent with this , igf - ir signaling significantly enhances the proliferation , motility and tumorigenicity of human head and neck cancer cell lines . igf-1r", "down regulation in a hnscc cell line using antisense oligonucleotides resulted in a dose - dependent decrease in cellular proliferation , induction of apoptosis , caspase activation and reduced expression of proangiogenic cytokines such as vegf .", "interest in targeting the igf-1r in hnscc was bolstered by the observation that treatment of head and neck cancer cells with either igf or egf resulted in igf - ir and egfr heterodimerization [ 71 , 72 ] .", "however , only igf resulted in the phosphorylation of both receptors . using a mouse xenograft model for hnscc , treatment with antibodies against igf-1r , egfr or", "it remains to be determined whether cellular cross - talk between igf-1r and egfr has an important role in determining the biological aggressiveness of hnscc or resistance to egfr - targeted therapies .  ", "several monoclonal antibodies and tkis for igf-1r have been tested in preclinical studies and early phase clinical studies .", "however , the efficacy of igf-1r - targeted therapy for treating patients with hnscc , particularly cross - talk with egfr , warrants further investigation . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer cell lines [ 7375 ] .", "treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] . targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) . similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) .", "these studies will be critical for evaluating whether the use of anti - igf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc ."], ["the met receptor is a single pass transmembrane protein that upon binding its ligand hgf  also known as scatter factor - promotes increased cell proliferation , survival and motility ( reviewed in [ 78 , 79 ] ) .", "hgf is the only physiological ligand for met and is secreted as an inactive precursor polypeptide chain by mesenchymal cells .", "hgf is proteolytically cleaved to form an active / heterodimer by a number of serine proteases including urokinase plasminogen activator ( upa ) , tissue - type plasminogen activator ( tpa ) , coagulation factors x. xi and xii .", "met is a disulphide - linked / heterodimer derived from the proteolytic cleavage of a 170  kda precursor .", "the  chain and n - terminal region of the -chain form  sema domain , a seven -propeller structure in which blades 2 and 3 bind to hgf .", "the sema domain is flanked by a cysteine - rich region followed by four immunoglobulin repeats .", "it is proposed that the cysteine - rich region and immunoglobulin repeat domains undergo a conformational change following hgf binding allowing for met dimerization [ 80 , 81 ] .", "binding of hgf to met results in receptor autophosphorylation at key catalytic residues and subsequent recruitment of several cytosolic signaling molecules that are shared with the egfr and igf-1r signaling pathways , including the grb2/sos complex , the p85 regulatory subunit of pi3k , gab1 and jak / stat3 ( figure 1 ) .", "subsequent activation of the mapk and jun - n - terminal kinase ( jnk ) pathways is responsible for the mitogenic and motogenic properties of met / hgf signaling resulting in  invasive growth  , depending on the physiological setting .", "increased met signaling in human cancers can be the result of enhanced ligand - binding ( autocrine and paracrine ) , met overexpression or missense mutations that often induce constitutive kinase activity , failure of met down regulation and interactions with other cell surface receptors such as egfr ( reviewed in [ 8284 ] ) . met is overexpressed in 84% of hnscc patient samples .", "interestingly , amplification of the met gene ( > 10 copies per cell ) is present only in 3 of 23 ( 13% ) tumor tissues .", "hgf overexpression is detected in 45% of hnsccs , suggesting that hgf functions predominantly in a paracrine manner to drive met signaling in these cancers .", "moreover , high levels of hgf are detected in hnscc patient plasma samples   supporting the idea that ligand availability is not a limiting factor for met activation .", "mutations in the met ligand - binding domain ( t230m / e168d ) , transmembrane or jm domain ( r988c , t1010i ) and the tyrosine kinase domain ( t1275i , v14333i ) have also been identified in hnscc tumor samples , although their relative contribution to hnscc progression remains to be determined .", "two somatic met mutations have been detected in hnscc that result in constitutively active receptor signaling that confers an invasive phenotype when ectopically expressed in cell lines .", "the y1230c mutation confers anchorage - independent growth and an invasive phenotype in transfected cells , whereas the y1235d met mutation stimulates epithelial cells to invade reconstituted basement membrane in the absence of hgf . in the case of the mety1235d mutation", ", genomic analyses of hnscc patient samples detected the presence of this mutant allele in 50% of metastatic tumors versus 26% in primary tumors , raising the possibility that this could be a critical genetic lesion for the acquisition of a metastatic phenotype .", "alternatively , increased met signaling could afford hnscc a selective advantage for growth and/or survival in metastatic sites , such as the lymph node and lung .", "indeed several studies indicate that met overexpression correlates highly with lymph node metastasis , pathologic stage , and disease reoccurrence [ 8891 ] .", "moreover , patient survival was significantly reduced in biopsy samples with positive met expression relative to negative met expression , suggesting the association of met with hnscc disease progression .", "consistent with these findings , treatment with the tki pf-2341066 caused a significant reduction in tumor growth , a high level of apoptosis and cellular debris within the tumor using a xenograft animal model for hnscc .", "selective inhibitors of met / hgf signaling include humanized monoclonal antibodies for hgf and met , and small - molecule tyrosine kinase inhibitors directed against met ( figure 1 ) .", "although their efficacy for treating a variety of solid tumors is increasingly recognized , we await results of preclinical and clinical trials for head and neck cancer that are ongoing .", "the humanized antibody amg 102 shows high potency towards the mature and processed form of hgf with no detected effects on proteolytic activation of prohgf .", "amg 102 interferes with met signaling , by competing with hgf for binding to the  chain of the met receptor . in phase", "i clinical studies in patients with advanced solid tumors , 70% of patients had a best response in terms of achieving stable disease [ 93 , 94 ] .", "importantly , no antiamg 102 antibodies were detected and circulating hgf levels were dose dependent .", "another promising clinical therapeutic is the one - armed 5d5 humanized antibody ( oa5d5/metmab ) directed against met .", "metmab binds met with high affinity , preventing hgf binding , met phosphorylation , receptor internalization and downstream signaling events and has been shown to inhibit tumor growth in animal models by more than 95% [ 95 , 96 ] .", "metmab is currently in phase i / ii human clinical trials in comparison with erlotinib in patients with nsclc ( http://www.clinicaltrials.gov/ , identifier nct00854308 ) .", "future clinical trials will be required to determine the suitability of amg102 and metmab as either single agents or combinatorial therapeutics for treating hnscc patients .", "foretinib ( formerly xl880 ) is a tki whose primary targets include met and vegf , and to a lesser extent the platelet - derived growth factor ( pdgf ) receptor , ron , kit and tie2 rtks .", "foretinib recently completed phase ii clinical trials in head and neck patients ( http://www.clinicaltrials.gov/ , identifier nct00725764 ) .", "interim results suggest that after 12 months , 12 of 18 patients had stable disease .", "a phase i dose - escalation study of the safety and pharmacokinetics of xl184 administered orally to patients with advanced malignancies ( showed that , on average , patients survived for more than 3 months with several up to 6 months while on treatment ) ( reviewed in ) . due to", "encouraging data from this study , a randomized phase iii trial of xl184 in hnscc patients was initiated to investigate xl184 as a first - line treatment ( compared with placebo ) for survival benefit to patients with hnscc ( http://www.clinicaltrials.gov/ , identifier nct00704730 ) .", "arq197 ( arqule ) is a nonatp - site competitive , selective small molecule inhibitor of the met intracellular region .", "although the mechanism of arq197 is presently unknown , the results of phase i trials suggest potential antiinvasive activity for this compound .", "overall , met , and hgf - targeted therapies have been well tolerated in clinical trials with negligible toxicities .", "however , it remains to be determined whether met is a better therapeutic target than hgf .", "clearly , in patients where met is activated by autocrine hgf secretion , both hgf and met targeted therapies may prove to be more efficacious treatment options ."], ["acquired resistance is likely the result of several mechanisms including ( 1 ) egfr mutations initially present as well as those acquired during therapy , ( 2 ) receptor independent activation of downstream signaling cascades , ( 3 ) cross - talk with other rtks and converging signaling pathways and ( 4 ) environmental factors including inflammatory agents and viral infection .", "resistance to cetuximab has been associated with the coexpression of the truncated egfr mutant , egfrviii with wild - type egfr .", "egfrviii is the result of an in frame deletion of exons 27 spanning the extracellular ligand - binding domain .", "the deletion results in a truncated egfr receptor that signals in a ligand - independent manner .", "egfrviii expression has been detected in 42% of hnscc patient samples , and closely correlates with increased hnscc cell proliferation in vitro and increased tumor growth using in vivo xenograft models .", "egfrviii preferentially activates the pi3k pathway instead of the ras / raf / mek pathway , which is activated by wild - type egfr . of particular interest to the therapeutic treatment of hnscc ,", "egfrviii expression decreases the proliferative response of egfr expressing tumor cells to cetuximab treatment relative to vector control cells . in a recent study ,", "egfrviii cells were shown to be resistant to the antiinvasive effects of cetuximab due to an increase in phosphorylation of stat3 rather than increased pi3k signaling .", "egf - induced expression of the stat3 target gene hif1 was abolished by cetuximab in hnscc cells expressing wild - type egfr under hypoxic conditions , but not in egfrviii - expressing hnscc cells [ 102 , 103 ] .", "these data suggest a role for egfrviii in mediating hnscc resistance to cetuximab .   despite egfrs critical role in the development of hnscc", ", clinical data indicate modest clinical benefits for locoregional control and survival of head and neck cancer patients treated with egfr - targeted therapies .", "hnscc patients resistant to cetuximab , often succumb to local tumor recurrence as well as regional and distant metastasis .", "the addition of cetuximab to radiation therapy was reported to show improved locoregional disease control , progression - free survival , and overall survival in patients with locally advanced hnscc .", "however the data revealed a disproportionate benefit of cetuximab with radiotherapy to oropharyngeal cancer patients when compared to patients treated with hyperfractionated radiotherapy .", "accumulating evidence suggests that human papilloma virus ( hpv ) 16 status ( hpv+ ) is an important prognostic factor associated with a favorable outcome in a subset of head and neck cancers , including oropharyngeal and tonsilar cancers .", "hpv+ tumors tend to have unique genetic aberrations including decreased egfr expression , whereas increased igf-1r levels characteristic of hnscc appear to be independent of hpv status .", "clinically , hpv+ tumors are characterized by more favorable patient prognosis regarding disease - free survival as well as overall survival [ 104 , 105 ] , possibly as a result of increased genomic stability associated with global gene hypermethylation in hpv+ tumors .", "thus it will be interesting to determine whether hpv+ status explains some of the benefits derived from the addition of cetuximab to radiotherapy in this subset of hnscc patients . at present", ", there are few clinical indicators of which hnscc patients will most likely respond to egfr - targeted therapies .", "accordingly , strategies to optimize egfr - targeted therapy remain an active area of research .", "additional mechanisms that result in egfr activation include activating mutations in downstream signaling components or cross - talk between different rtk pathways . activating mutations in the pi3ka oncogene", "occurs in 10% of hnscc tumors   whereas elevated levels of phosphorylated stat3 correlates with lymph node metastasis and poor patient prognosis [ 108110 ] .", "conversely , h - ras mutations are infrequent in hnscc cases ( less than 5% ) , although a higher incidence has been detected in asian populations and correlates with areca nut chewing [ 111 , 112 ] .", "met signaling has been shown to contribute to resistance in cell lines derived from multiple tumor types including breast , gastric and lung .", "in one key study , nsclc with activating mutations in the egfr acquire resistance to the tki gefitinib and erlotinib , by amplification of the met gene to maintain akt and her3 signaling .", "these studies underscore the role of cross - talk between rtks to preferentially signal through the pi3k - akt survival pathway as a mechanism for acquired drug resistance .", "the relevance of met as a mechanism for escape from egfr - targeted therapy in head and neck cancers remains to be determined .", "hypoxia results in the transcriptional upregulation of met gene expression via hif1 in a number of tumors including head and neck , often downstream of egfr signaling . in normoxia", ", hydroxylation of 2 prolines in hif1 enables its binding to the von hippel - lindau tumor suppressor protein ( pvhl ) linking hif1 to a ubiquitin ligase complex . during hypoxia , minimal or no hydroxylation occurs enabling hif1 to avoid proteasomal degradation and", "dimerize to other hif family members such as hif1   and coactivators , to form an active transcriptional hif complex on the hypoxia response element ( hre ) of target genes such as met .", "the ubiquitin ligase catalyzes polyubiquitination of hif1 targeting it for proteasomal degradation . under hypoxic conditions ,", "increased met signaling directs the invasive growth program , enabling cells to invade more oxygenated tissues .", "since met has been reported to promote invasive and angiogenic effects in the tumor microenvironment , the use of hgf / met inhibitors may afford a means of impairing tissue colonization as well as tumor vascularization in head and neck cancer patients .     ", "studies on other solid tumor types , most notably glioblastoma , indicate a role for igf-1r upregulation in resistance to egfr - targeted therapies .", "igf-1r mediates resistance to anti - egfr therapy in primary glioblastoma through the continued activation of the pi3k / akt survival pathway .", "the apparent cooperation between igf-1r and egfr in promoting hnscc pathogenesis as well as resistance to egfr - targeted therapy , suggests an advantage to cotargeting these signaling axes for the treatment of head and neck cancers . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer lines .", "treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] .", "targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) .", "similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) .", "these studies will be critical for evaluating whether the use of antiigf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc ."], ["targeted therapies that block egfr , met , and igf-1r signaling in head and neck cancers continue to show promising results in preclinical studies and clinical trials .", "however , it is difficult to predict which patients are most likely to benefit from these therapeutics and potential side effects during long - term in vivo use . given the interplay between these rtk signaling pathways and the mediocre results obtained with monotherapy regimens thus far , clinical trials will be required to determine how egfr- , met- , and igf-1r - targeted therapies can be used in combination in order to definitively abrogate their common downstream oncogenic signaling networks .", "although gaps in our knowledge concerning the role of met and igf-1r in head and neck tumorigenesis , as well as acquired resistance to antiegfr therapies remain to be addressed , efforts to translate current information towards clinical applications continue to be impressive ."]]}
{"article_id": "PMC4727599", "article_text": ["this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals .", "the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation .", "primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) .", "then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated .", "the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium .", "dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) .", "the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div ,", "half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem .", "serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day . a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm )", "four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls .", "the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons .", "the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) .", "the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10  ) , and 10 m of mpp on the tenth div for 48 h. dopaminergic neurons were identified immunocytochemically by staining tyrosine hydroxylase .", "cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature .", "cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures", ", cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages .", "the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification .", "cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium .", "the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing  - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes .", "the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored .", "the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period .", "the ldh release is proportional to the number of damaged or destroyed cells.11,12 lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes .", "cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div ,", "culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs", ", cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m )", "the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells .", "the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) .", "after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software .", "cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) ,", "the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark .", "after washing with dpbs , 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) .", "nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software .", "data was obtained from 12 wells ( from 2 repeats ) for each treatment condition .", "comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . ,", "this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals .", "the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation .", "primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) .", "then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated .", "the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium .", "dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) .", "the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div ,", "half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem .", "serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day .", "a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm ) was prepared in dimethyl sulfoxide ( dmso ) .", "four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls .", "the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons .", "the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) .", "the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10  ) , and 10 m of mpp on the tenth div for 48 h.", "cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature .", "cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures", ", cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages .", "the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification .", "cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium .", "the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing  - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes .", "the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored .", "the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period .", "lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes .", "cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div ,", "culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs ,", "cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m )", "the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells .", "the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) .", "after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software .", "cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) ,", "the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark . after washing with dpbs", ", 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) .", "nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software .", "data was obtained from 12 wells ( from 2 repeats ) for each treatment condition .", "comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . ,", "treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) .", "treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) .", "surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) .", "co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) .", "anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures .", "100% corresponds to the total number of thir neurons after 12 div in untreated controls .", "values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) .", "the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) .", "treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures .", "th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture .", "the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) .", "the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) .", "div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture .", "lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) .", "lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures .", "100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div .", "fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures .", "div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m .", "cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) .", "as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures .", "100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div .", "red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) .", "against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) .", "4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures .", "100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture .", "treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) .", "treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) .", "surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) .", "co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) .", "anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures .", "100% corresponds to the total number of thir neurons after 12 div in untreated controls .", "values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) .", "the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) .", "treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures .", "th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture .", "the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) .", "the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) .", "div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture .", "lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) .", "lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures .", "100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div .", "fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures .", "div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m .", "cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) .", "as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures .", "100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div .", "red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) .", "against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) .", "4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures .", "100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture .", "in the present study , tq was investigated to ascertain whether it protected mesencephalic dopaminergic neurons against mpp - induced cell death through activation of enzymatic degradation , preservation of mitochondrial function , and inhibition of apoptotic cell death .", "clearly , mpp was found to significantly decrease the survival of dopaminergic neurons and increase the release of ldh into the culture medium .", "the mpp toxicity involves its selective uptake by dopaminergic neurons through the dopamine transporter and inhibition of mitochondrial complex i activity with subsequent mitochondrial depolarization.14 in parallel , the use of jc-1 fluorescence dye in our current study showed that mpp significantly decreased the m of cultured cells as indicated by the decreasing red : green fluorescence ratio of jc-1 .", "similar mpp - induced reduction of m was reported in other in vitro disease models.15,16 mitochondrial damage has long been implicated in the death of nigrostriatal dopaminergic neurons in both pd patients and experimental models.17,18 staining of primary dopaminergic cultures with blue - fluorescent dapi nucleic acid stain showed that a significant number of the cells displayed features of apoptosis , most notably chromatin condensation , and fragmentation .", "previously , tang et al19 and xu et al16 demonstrated that mpp caused apoptotic cell death in pc12 and sh - sy5y cells .", "the mpp - induced apoptosis was reported to occur as the result of disruption of mitochondrial transmembrane potential and opening of the permeability transition pore.20 similar to our previous report,9 co - treatment of primary mesencephalic cell cultures with tq and mpp was found to protect dopaminergic neurons and decreased the release of ldh into the culture medium . since that time , no evidence in the literature has shown how tq protected dopaminergic neurons in the primary mesencephalic cell culture .", "staining of cultures with lysotracker deep red showed that tq significantly increased the red fluorescence of the dye compared with mpp - treated cultures , indicating enhancement of the formation of many autophagolysosomes , the sites of lysosomal degradation , by tq .", "this is supported by the findings of he and klionsky21 who correlated the fluorescent signals of lysotracker deep red to the upregulation of autophagy in zebrafish . increased red fluorescence of lysotracker deep red", "is attributed to the formation of many autophagosomes and autophagolysosomes that retain much dye as the result of increasing their acidification . using jc-1 fluorescent dye showed that tq significantly enhanced m as it increased the red : green fluorescence ratio of jc-1 compared with mpp - treated cultures .", "the tq was similarly found to protect rat cortical neurons against ethanol- and a1 - 42-induced neurotoxicity through inhibition of mitochondrial membrane depolarization.22,23 counting of apoptotic nuclei using blue - fluorescent dapi nucleic acid stain indicated that tq decreased mpp - induced apoptotic cell death in primary mesencephalic cell cultures . in accordance ,", "ullah et al22 reported that tq inhibited apoptotic cell death in ethanol - treated rat cortical neurons and attributed this effect of tq to the preservation of mitochondrial integrity .", "zhang et al24 reported that mitochondrial clearance protected cultured cortical neurons against ischemia - reperfusion - induced cell damage . in conclusion , correlating such results would therefore suggest that tq might activate a lysosomal degradative process in dopaminergic neurons , where clearance of damaged mitochondria results in reduced mitochondria - mediated apoptotic cell death .", "this might raise the possibility of using tq as a potentially therapeutic intervention in pd patients .", "whenever a manuscript contains material ( tables , figures , etc . ) which is protected by copyright ( previously published ) , it is the obligation of the author to obtain written permission from the holder of the copyright ( usually the publisher ) to reproduce the material in neurosciences .", "please submit copies of the material from the source in which it was first published ."], "abstract_text": ["<S> objectives : to investigate potential mechanisms mediating the neuroprotective effect of thymoquinone ( tq ) on dopaminergic neurons.methods:this study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 . </S>", "<S> primary cultures were prepared from embryonic mouse mesencephala ( ofi / spf ) at gestation day 14 . </S>", "<S> four sets of cultures were kept untreated , treated with tq on the eighth day in vitro ( div ) for 4 days , treated with 1-methyl-4-phenylpyridinium ( mpp+ ) on the tenth div for 48 hours and co - treated with thymoquinone and mpp+ . on the twelfth div </S>", "<S> , cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using lysotracker deep red , 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazolylcarbocyanine ( jc-1 ) and 4,6-diamidino-2-phenylindole stains.results:the mpp+ decreased the number of dopaminergic neurons by 40% , and increased the release of lactate dehydrogenase ( ldh ) into the culture medium . </S>", "<S> the tq significantly rescued dopaminergic neurons and decreased the release of ldh at the concentrations of 0.1 and 1 m . the tq significantly shifted the red fluorescent intensity of the lysotracker deep red , increased the mitochondrial membrane potential as it increased the red : green florescent ratio of jc-1 , and decreased mpp+-induced apoptotic cell death.conclusion:the tq protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria - mediated apoptotic cell death . </S>"], "labels": null, "section_names": ["Methods", "Preparation of primary mesencephalic cell culture", "Study design and treatment of primary mesencephalic cell cultures", "Identification of tyrosine hydroxylase immunoreactive (THir) neurons", "Measurement of lactate dehydrogenase activity", "Fluorescence staining", "Results", "Effect of TQ on the survival of dopaminergic neurons", "Discussion", "None"], "sections": [["this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals .", "the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation .", "primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) .", "then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated .", "the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium .", "dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) .", "the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div ,", "half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem .", "serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day . a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm )", "four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls .", "the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons .", "the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) .", "the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10  ) , and 10 m of mpp on the tenth div for 48 h. dopaminergic neurons were identified immunocytochemically by staining tyrosine hydroxylase .", "cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature .", "cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures", ", cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages .", "the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification .", "cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium .", "the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing  - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes .", "the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored .", "the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period .", "the ldh release is proportional to the number of damaged or destroyed cells.11,12 lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes .", "cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div ,", "culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs", ", cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m )", "the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells .", "the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) .", "after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software .", "cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) ,", "the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark .", "after washing with dpbs , 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) .", "nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software .", "data was obtained from 12 wells ( from 2 repeats ) for each treatment condition .", "comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . ,"], ["this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals .", "the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation .", "primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) .", "then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated .", "the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium .", "dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) .", "the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div ,", "half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem .", "serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day ."], ["a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm ) was prepared in dimethyl sulfoxide ( dmso ) .", "four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls .", "the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons .", "the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) .", "the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10  ) , and 10 m of mpp on the tenth div for 48 h."], ["cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature .", "cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures", ", cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages .", "the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification ."], ["cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium .", "the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing  - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes .", "the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored .", "the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period ."], ["lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes .", "cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div ,", "culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs ,", "cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m )", "the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells .", "the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) .", "after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .", "fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software .", "cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) ,", "the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark . after washing with dpbs", ", 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) .", "nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software .", "data was obtained from 12 wells ( from 2 repeats ) for each treatment condition .", "comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . ,"], ["treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) .", "treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) .", "surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) .", "co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) .", "anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures .", "100% corresponds to the total number of thir neurons after 12 div in untreated controls .", "values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) .", "the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) .", "treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures .", "th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture .", "the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) .", "the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) .", "div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture .", "lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) .", "lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures .", "100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div .", "fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures .", "div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m .", "cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) .", "as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures .", "100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div .", "red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) .", "against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) .", "4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures .", "100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture ."], ["treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) .", "treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) .", "surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) .", "co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) .", "anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures .", "100% corresponds to the total number of thir neurons after 12 div in untreated controls .", "values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) .", "the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) .", "treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures .", "th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture .", "the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) .", "the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) .", "div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture .", "lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) .", "lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures .", "100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div .", "fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures .", "div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m .", "cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) .", "as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone .", "5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures .", "100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div .", "red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) .", "against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) .", "4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures .", "100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div .", "( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures .", "tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture ."], ["in the present study , tq was investigated to ascertain whether it protected mesencephalic dopaminergic neurons against mpp - induced cell death through activation of enzymatic degradation , preservation of mitochondrial function , and inhibition of apoptotic cell death .", "clearly , mpp was found to significantly decrease the survival of dopaminergic neurons and increase the release of ldh into the culture medium .", "the mpp toxicity involves its selective uptake by dopaminergic neurons through the dopamine transporter and inhibition of mitochondrial complex i activity with subsequent mitochondrial depolarization.14 in parallel , the use of jc-1 fluorescence dye in our current study showed that mpp significantly decreased the m of cultured cells as indicated by the decreasing red : green fluorescence ratio of jc-1 .", "similar mpp - induced reduction of m was reported in other in vitro disease models.15,16 mitochondrial damage has long been implicated in the death of nigrostriatal dopaminergic neurons in both pd patients and experimental models.17,18 staining of primary dopaminergic cultures with blue - fluorescent dapi nucleic acid stain showed that a significant number of the cells displayed features of apoptosis , most notably chromatin condensation , and fragmentation .", "previously , tang et al19 and xu et al16 demonstrated that mpp caused apoptotic cell death in pc12 and sh - sy5y cells .", "the mpp - induced apoptosis was reported to occur as the result of disruption of mitochondrial transmembrane potential and opening of the permeability transition pore.20 similar to our previous report,9 co - treatment of primary mesencephalic cell cultures with tq and mpp was found to protect dopaminergic neurons and decreased the release of ldh into the culture medium . since that time , no evidence in the literature has shown how tq protected dopaminergic neurons in the primary mesencephalic cell culture .", "staining of cultures with lysotracker deep red showed that tq significantly increased the red fluorescence of the dye compared with mpp - treated cultures , indicating enhancement of the formation of many autophagolysosomes , the sites of lysosomal degradation , by tq .", "this is supported by the findings of he and klionsky21 who correlated the fluorescent signals of lysotracker deep red to the upregulation of autophagy in zebrafish . increased red fluorescence of lysotracker deep red", "is attributed to the formation of many autophagosomes and autophagolysosomes that retain much dye as the result of increasing their acidification . using jc-1 fluorescent dye showed that tq significantly enhanced m as it increased the red : green fluorescence ratio of jc-1 compared with mpp - treated cultures .", "the tq was similarly found to protect rat cortical neurons against ethanol- and a1 - 42-induced neurotoxicity through inhibition of mitochondrial membrane depolarization.22,23 counting of apoptotic nuclei using blue - fluorescent dapi nucleic acid stain indicated that tq decreased mpp - induced apoptotic cell death in primary mesencephalic cell cultures . in accordance ,", "ullah et al22 reported that tq inhibited apoptotic cell death in ethanol - treated rat cortical neurons and attributed this effect of tq to the preservation of mitochondrial integrity .", "zhang et al24 reported that mitochondrial clearance protected cultured cortical neurons against ischemia - reperfusion - induced cell damage . in conclusion , correlating such results would therefore suggest that tq might activate a lysosomal degradative process in dopaminergic neurons , where clearance of damaged mitochondria results in reduced mitochondria - mediated apoptotic cell death .", "this might raise the possibility of using tq as a potentially therapeutic intervention in pd patients ."], ["whenever a manuscript contains material ( tables , figures , etc . ) which is protected by copyright ( previously published ) , it is the obligation of the author to obtain written permission from the holder of the copyright ( usually the publisher ) to reproduce the material in neurosciences .", "please submit copies of the material from the source in which it was first published ."]]}
{"article_id": "PMC2656958", "article_text": ["the femoral head often leads to healing complications , while the more predictable prosthetic replacements are associated with poorer function and significant complications .", "the treatment of these fractures depends on the age of the patient , fracture displacement , bone quality , timing of surgery and activity level of the patient .", "displaced fractures in healthy , active patients are best treated by reduction and internal fixation .", "there are however , complications unique to femoral neck fractures which are almost impossible to predict . non - union and avascular necrosis", "non - union usually can definitely diagnosed within a year of fracture fixation with the same being achieved within 3  months at times . after non - union has been established , intervention is inevitable .", "the decision to proceed in the management of failed fixation is based on the careful consideration of various factors . in young patients", "revision internal fixation with cancellous or muscle pedicle bone grafting ( vascularised bone graft ) or an osteotomy results in useful outcome .", "we report a case of non - union of the fracture of the neck of the femur , who refused additional procedures after his non - union had been established .", "the study conforms to the declaration of helsinki and was approved by the institutional ethical board .", "a 38-year old male businessman reported to the out door department of our hospital with a history of a fall from height .", "clinical and radiological examination revealed a displaced fracture of the neck of the femur which was graded as garden type 4 ( fig .", "the patient was operated within 24  h. intraoperatively the garden alignment index was used as a yardstick of acceptable reduction .", "the fracture was fixed with 3 ao 6.5  mm cannulated screws in an inverted triangle .", "onward the patient complained of persistent but mild groin pain on ambulation which was assisted .", "the patients conservative line was continued till 1  year when his symptoms continued and radiologically there was no progress . at this point", "twenty - six  months into the post operative followup the patients pain subsided and radiographs started showing signs of union . a radiograph taken 32  months after the fixation showed full union .", "non - union after femoral neck fracture can be defined as a lack of radiographic evidence of union 6  months after the fracture .", "delayed or non - union often manifests as continued pain with weight bearing beyond 3  months post fixation .", "incidence of non - union of femoral neck fractures has been reported to be between 2 and 22% and generally becomes apparent within 1  year [ 69 ] .", "3radiograph at 32  months depicting union garden 4 type fracture of the neck of the femur showing persistent non - union at 2  years radiograph at 32  months depicting union anatomical reduction and rigid internal fixation are the two important surgeon controlled factors that may contribute to outcome .", "the preferred treatment for symptomatic non - unions in the elderly is prosthetic replacement . in the young active patients ,", "if failure is due to technical errors , revision open reduction and internal fixation may be adequate .", "our case demonstrates that as long as the implant is holding and the patient is regularly followed up good results might be expected in cases as far as 32  months into the post fixation period ."], "abstract_text": ["<S> fracture of the femoral neck continues to be a vexing clinical and therapeutic challenge for the orthopedic surgeon . </S>", "<S> the fracture has a propensity for non - union and avascular necrosis . </S>", "<S> it is a challenge for the orthopedic surgeon to decide when to intervene in a case with non - union where the implant continues to be in place . </S>", "<S> we present a case with persistent clinical and radiological non - union signs where the fracture eventually united after 32  months . </S>", "<S> the case bolsters the view that a continued conservative regime might entail good results in such situations . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["the femoral head often leads to healing complications , while the more predictable prosthetic replacements are associated with poorer function and significant complications .", "the treatment of these fractures depends on the age of the patient , fracture displacement , bone quality , timing of surgery and activity level of the patient .", "displaced fractures in healthy , active patients are best treated by reduction and internal fixation .", "there are however , complications unique to femoral neck fractures which are almost impossible to predict . non - union and avascular necrosis", "non - union usually can definitely diagnosed within a year of fracture fixation with the same being achieved within 3  months at times . after non - union has been established , intervention is inevitable .", "the decision to proceed in the management of failed fixation is based on the careful consideration of various factors . in young patients", "revision internal fixation with cancellous or muscle pedicle bone grafting ( vascularised bone graft ) or an osteotomy results in useful outcome .", "we report a case of non - union of the fracture of the neck of the femur , who refused additional procedures after his non - union had been established .", "the study conforms to the declaration of helsinki and was approved by the institutional ethical board ."], ["a 38-year old male businessman reported to the out door department of our hospital with a history of a fall from height .", "clinical and radiological examination revealed a displaced fracture of the neck of the femur which was graded as garden type 4 ( fig .", "the patient was operated within 24  h. intraoperatively the garden alignment index was used as a yardstick of acceptable reduction .", "the fracture was fixed with 3 ao 6.5  mm cannulated screws in an inverted triangle .", "onward the patient complained of persistent but mild groin pain on ambulation which was assisted .", "the patients conservative line was continued till 1  year when his symptoms continued and radiologically there was no progress . at this point", "twenty - six  months into the post operative followup the patients pain subsided and radiographs started showing signs of union . a radiograph taken 32  months after the fixation showed full union ."], ["non - union after femoral neck fracture can be defined as a lack of radiographic evidence of union 6  months after the fracture .", "delayed or non - union often manifests as continued pain with weight bearing beyond 3  months post fixation .", "incidence of non - union of femoral neck fractures has been reported to be between 2 and 22% and generally becomes apparent within 1  year [ 69 ] .", "3radiograph at 32  months depicting union garden 4 type fracture of the neck of the femur showing persistent non - union at 2  years radiograph at 32  months depicting union anatomical reduction and rigid internal fixation are the two important surgeon controlled factors that may contribute to outcome .", "the preferred treatment for symptomatic non - unions in the elderly is prosthetic replacement . in the young active patients ,", "if failure is due to technical errors , revision open reduction and internal fixation may be adequate .", "our case demonstrates that as long as the implant is holding and the patient is regularly followed up good results might be expected in cases as far as 32  months into the post fixation period ."]]}
{"article_id": "PMC3461795", "article_text": ["granuloma faciale ( gf ) is an uncommon , benign , inflammatory skin disorder of unknown etiology .", "it is characterized by single or multiple , grey - brown or violaceous nodules or plaques primarily occurring on the face and occasionally at extra - facial sites .", "the disease is notoriously resistant to therapies and often tends to relapse when treatment is discontinued .", "we present a patient with multiple lesions of gf and its response to topical tacrolimus .", "a 35-year - old female presented to our department with a 5 year history of single , asymptomatic , grey - brown pigmented , nodule over the left cheek [ figure 1 ] .", "it started as a pin head sized papule which gradually increased to 2.5 cm  1.5 cm in size .", "two years later similar lesions appeared on the forehead , both arms and upper back .", "past and personal history was unremarkable . before treatment  single , grey - brown nodule with prominent follicular orifices over left cheek .", "after treatment  residual lesion after three months of tacrolimus application general physical and systemic examination was normal .", "cutaneous examination revealed multiple , well - defined , grey - brown , indurated , non - tender plaques , varying in size from 0.5 cm  0.5 cm to 1.5 cm  2.5 cm , present on the left cheek , left forehead , both arms and upper back .", "multiple grey - brown plaques over upper back routine hematological and biochemical investigations were normal .", "skin biopsy ( 4 mm ) from plaque revealed normal epidermis with clear sub epidermal ", "grenz zone and pan dermal dense infiltrate comprising of neutrophils , lymphocytes , histiocytes and plasma cells .", "small dermal vessels showed infiltration of neutrophils in the vessel wall along with peri - appendageal and peri - neural infiltrate in subcutaneous fat [ figure 3 ] .", "( h and e , 100  ) skin biopsy with normal epidermis and dense , mixed inflammatoey infiltrate beneath a narrow grenz zone in the dermis .", "infiltrate is composed of mononuclear cells with neutrophils and eosinophils she was started on intralesional triamcilone acetonide 10 mg / ml injection monthly with tab .", "six sessions of cryotherapy were performed once monthly after which she developed erythema and itching over the plaques and discontinued treatment .", "the lesions showed 40 - 50% improvement after 3 months of therapy [ figure 1 ] .", "classically , red - brown or violaceous nodules or plaques with associated telangiectasia and follicular accentuation are seen on the face over sun - exposed sites .", "differential diagnosis includes lupus pernio , lupus vulgaris , lymphoma , discoid lupus erythematosus and deep mycotic infection .", "skin biopsy is characterized by a mixed inflammatory infiltrate with a predominance of neutrophils and eosinophils in the dermis , in conjunction with small vessel vasculitis .", "there is a grenz zone that separates the infiltrate from the epidermis and pilosebaceous units .", "the disease is notoriously resistant to therapies and often tends to relapse once the treatment is discontinued .", "several medical and surgical modalities like topical and intralesional corticosteroids , cryotherapy , pulsed dye laser , puva , systemic corticosteroids , dapsone and antimalarials have been tried with variable success rates .", "carbon dioxide laser has also been used in a case of recurrent gf . surgical excision has been performed with often unsatisfactory results .", "ablative procedures may leave residual pigmentation and scarring , whereas long - term application of corticosteroids is associated with skin atrophy , telangiectasia and other possible adverse effects . in recent years successes with topical calcineurin inhibitors has been reported .", "several authors have reported complete or near - complete resolution of lesions after application of topical tacrolimus 0.1% ointment.[48 ] treatment regimens , duration and time to resolution of lesions have varied in these case reports [ table 1 ] .", "others have found time to resolution to be between 4 and 6 months . in our patient ,", "treatment with tacrolimus 0.1% ointment twice daily for 3 months has resulted in improvement . reported cases of successful treatment of granuloma faciale ( gf ) with topical tacrolimus tacrolimus", "inhibits t - cell proliferation , production and release of several pro - inflammatory cytokines like interleukin-2 ( il-2 ) , il-4 , tumor necrosis factor - alpha , and interferon - gamma ( ifn - gamma ) .", "although the pathogenesis is still unknown , it has been suggested that gf may be an ifn - gamma mediated disease .", "in addition , an increased production of il-5 , probably induced by the clonal expansion of a locally recruited t - cell population may enhance the attraction of eosinophils into the lesions of gf .", "therefore , a possible mechanism of action of topical tacrolimus in this condition may be the inhibition of ifn - gamma and il-5 production and release , induced by the down - regulation of the t - cell activity , primarily involving the calcineurin binding and inactivation .", "however , we did observe eosinophils in skin biopsy in our case , probably since the biopsy was taken after one year of oral dapsone .", "our patient experienced a relevant improvement within 3 months of treatment with tacrolimus ointment after no response with intra lesional steroids , dapsone and cryo therapy . in conclusion", ", the previous reports and our observation suggest that topical tacrolimus may be a well - tolerated , efficacious therapy for gf ."], "abstract_text": ["<S> granuloma faciale ( gf ) is a chronic condition characterized by red - brown plaques with follicular accentuation present usually on the face . </S>", "<S> we present a case of 35-year - old female with 5 year history of plaques over cheek and extra facial sites consistent with gf and its response to topical tacrolimus . </S>", "<S> this case supports previous reports of successful treatment of gf with topical tacrolimus . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["granuloma faciale ( gf ) is an uncommon , benign , inflammatory skin disorder of unknown etiology .", "it is characterized by single or multiple , grey - brown or violaceous nodules or plaques primarily occurring on the face and occasionally at extra - facial sites .", "the disease is notoriously resistant to therapies and often tends to relapse when treatment is discontinued .", "we present a patient with multiple lesions of gf and its response to topical tacrolimus ."], ["a 35-year - old female presented to our department with a 5 year history of single , asymptomatic , grey - brown pigmented , nodule over the left cheek [ figure 1 ] .", "it started as a pin head sized papule which gradually increased to 2.5 cm  1.5 cm in size .", "two years later similar lesions appeared on the forehead , both arms and upper back .", "past and personal history was unremarkable . before treatment  single , grey - brown nodule with prominent follicular orifices over left cheek .", "after treatment  residual lesion after three months of tacrolimus application general physical and systemic examination was normal .", "cutaneous examination revealed multiple , well - defined , grey - brown , indurated , non - tender plaques , varying in size from 0.5 cm  0.5 cm to 1.5 cm  2.5 cm , present on the left cheek , left forehead , both arms and upper back .", "multiple grey - brown plaques over upper back routine hematological and biochemical investigations were normal .", "skin biopsy ( 4 mm ) from plaque revealed normal epidermis with clear sub epidermal ", "grenz zone and pan dermal dense infiltrate comprising of neutrophils , lymphocytes , histiocytes and plasma cells .", "small dermal vessels showed infiltration of neutrophils in the vessel wall along with peri - appendageal and peri - neural infiltrate in subcutaneous fat [ figure 3 ] .", "( h and e , 100  ) skin biopsy with normal epidermis and dense , mixed inflammatoey infiltrate beneath a narrow grenz zone in the dermis .", "infiltrate is composed of mononuclear cells with neutrophils and eosinophils she was started on intralesional triamcilone acetonide 10 mg / ml injection monthly with tab .", "six sessions of cryotherapy were performed once monthly after which she developed erythema and itching over the plaques and discontinued treatment .", "the lesions showed 40 - 50% improvement after 3 months of therapy [ figure 1 ] ."], ["classically , red - brown or violaceous nodules or plaques with associated telangiectasia and follicular accentuation are seen on the face over sun - exposed sites .", "differential diagnosis includes lupus pernio , lupus vulgaris , lymphoma , discoid lupus erythematosus and deep mycotic infection .", "skin biopsy is characterized by a mixed inflammatory infiltrate with a predominance of neutrophils and eosinophils in the dermis , in conjunction with small vessel vasculitis .", "there is a grenz zone that separates the infiltrate from the epidermis and pilosebaceous units .", "the disease is notoriously resistant to therapies and often tends to relapse once the treatment is discontinued .", "several medical and surgical modalities like topical and intralesional corticosteroids , cryotherapy , pulsed dye laser , puva , systemic corticosteroids , dapsone and antimalarials have been tried with variable success rates .", "carbon dioxide laser has also been used in a case of recurrent gf . surgical excision has been performed with often unsatisfactory results .", "ablative procedures may leave residual pigmentation and scarring , whereas long - term application of corticosteroids is associated with skin atrophy , telangiectasia and other possible adverse effects . in recent years successes with topical calcineurin inhibitors has been reported .", "several authors have reported complete or near - complete resolution of lesions after application of topical tacrolimus 0.1% ointment.[48 ] treatment regimens , duration and time to resolution of lesions have varied in these case reports [ table 1 ] .", "others have found time to resolution to be between 4 and 6 months . in our patient ,", "treatment with tacrolimus 0.1% ointment twice daily for 3 months has resulted in improvement . reported cases of successful treatment of granuloma faciale ( gf ) with topical tacrolimus tacrolimus", "inhibits t - cell proliferation , production and release of several pro - inflammatory cytokines like interleukin-2 ( il-2 ) , il-4 , tumor necrosis factor - alpha , and interferon - gamma ( ifn - gamma ) .", "although the pathogenesis is still unknown , it has been suggested that gf may be an ifn - gamma mediated disease .", "in addition , an increased production of il-5 , probably induced by the clonal expansion of a locally recruited t - cell population may enhance the attraction of eosinophils into the lesions of gf .", "therefore , a possible mechanism of action of topical tacrolimus in this condition may be the inhibition of ifn - gamma and il-5 production and release , induced by the down - regulation of the t - cell activity , primarily involving the calcineurin binding and inactivation .", "however , we did observe eosinophils in skin biopsy in our case , probably since the biopsy was taken after one year of oral dapsone .", "our patient experienced a relevant improvement within 3 months of treatment with tacrolimus ointment after no response with intra lesional steroids , dapsone and cryo therapy . in conclusion", ", the previous reports and our observation suggest that topical tacrolimus may be a well - tolerated , efficacious therapy for gf ."]]}
{"article_id": "PMC5002941", "article_text": ["chronic myelomonocytic leukaemia ( cmmol ) is a rare disease with an incidence of 0.370.72 per 100 000 population . the median age at diagnosis varies between 65 years and 75 years [ 14 ] .", "the radical treatment is allogeneic stem cell transplantation , although this treatment is unsuitable for most patients because of their advanced age [ 6 , 7 ] .", "the median survival of patients with cmmol is 1520 months [ 5 , 6 , 8 ] .", "several case reports have indicated that excess surgical stress causes an acute blastic crisis , which contributes to a poor outcome [ 9 , 10 ] .", "a 68-year - old man complained of having a reduced appetite for approximately 5 months and weight loss during the previous 4 months . on 12 may 2012", ", he visited a nearby general internal medicine clinic for the evaluation of left lower quadrant abdominal pain .", "he was referred to the department of internal medicine at asahi general hospital ( asahi , japan ) for investigation and treatment .", "he began taking oral hydroxyurea ( hydrea ; bristol - myers squibb , princeton , nj , usa ) .", "he was unable to stop the gingival bleeding that had been ongoing for 5 hours .", "he then visited the emergency department of asahi general hospital and was referred to the department of dentistry and oral surgery . at the initial visit", "the right molar teeth ( 47 and 48 ) showed grade iii mobility , and their gingivae were painful with continuous bleeding .", "1a ) . the right molar teeth ( 46 and 47 ) showed class iii alveolar ridge deficiency on panoramic radiographs and were mobile teeth ( fig .", "we packed an absorbable haemostat ( surgicel ; ethicon , somerville , nj , usa ) into the gingivae and sutured 30 silk threads above it . finally , we used surgical dressing packs ( coe - pak ; g.c .", "figure 1:(a ) the oral photograph at the initial visit , immediately after the gauze with the astringent has been removed .", "( b ) the oral photograph after the extraction of teeth 47 and 48 . \n", "( a ) the oral photograph at the initial visit , immediately after the gauze with the astringent has been removed .", "the internal medicine department assigned a clinical diagnosis of cmmol , based on the blood and bone marrow examinations conducted on 13 june 2012 .", "computed tomography on june 25 indicated that bone absorption had nearly reached the right inferior alveolar canal , and a ring of sequestrum was present under tooth 47 ( fig .", "we diagnosed right mandibular osteomyelitis , based on these clinical features . on 11 july 2012 ,", "the patient was hospitalized in the department of haematology ward because of significant anaemia and decreased platelet numbers . on the same day ,", "the internal medicine specialist placed him on intravenous  piperacillin  tazobactam ( zosyn ; pfizer , new york , ny , usa ) .", "he afterwards complained of severe pain in the socket ( 47 and 48 ) , despite receiving the potent opioid analgesic dihydrohydroxycodeinone ( fentanyl ; daiichi - sankyo , chiyoda - ku , tokyo , japan ) .", "hence , we decided to apply a surgical anti - inflammatory treatment in consideration of an acute blastic crisis .", "a bone marrow examination on july 13 showed no signs of acute blastic crisis of his cmmol .", "after we consulted an internal medicine specialist , we planned a surgical procedure to clean the inflamed region in his mouth . on the day before the surgery", ", he was administered a transfusion of platelet concentrate ( 20 units ) and piperacillin  tazobactam to prevent bleeding and further infection . on july 20 , with the patient under local anaesthesia , we surgically resected the right lower sequestrum and extracted molar 37 , which displayed severe caries .", "the blood examination indicated improvement in the platelet count after the transfusion . after performing an inferior alveolar block", ", we removed the necrotic bone with ultrasonic bone surgery ( variosurg ; nsk , ashikaga , tochigi , japan ) until there was bleeding from the bone surface ( fig .", "we placed gauze with tetracycline ointment in the socket and sutured 30 silk thread to compress it ( fig .", "( b ) we removed necrotic bone with ultrasonic bone surgery until there was bleeding from the bone surface .", "( b ) we removed necrotic bone with ultrasonic bone surgery until there was bleeding from the bone surface .", "( d ) we used 30 silk thread sutures to compress the gauze . on august 11", "the formation of granulation tissue had nearly covered the surface of the bone , and all oral discomfort had disappeared ( fig .", "we later placed a bridge in the upper anterior teeth and added a partial denture ( fig .", "we placed a bridge for upper anterior teeth 12 , 11 , 22 and 23 , and a partial denture .", "we placed a bridge for upper anterior teeth 12 , 11 , 22 and 23 , and a partial denture . on 3 january 2013", ", he admitted himself to the hospital in the haematology ward because of exertional dyspnoea .", "the internal medicine specialists diagnosed cmmol that was undergoing acute leukaemic transformation by bone marrow examination .", "he died on 10 february 2013 because of cardiac arrest , following haemorrhagic shock and cardiovascular failure due to cmmol .", "in all patients with cmmol , every effort must be made to use the least invasive procedure possible . in the present patient ,", "our deepest concerns were to have a proper balance between invasiveness and healing , and to avoid evoking an acute blastic crisis [ 9 , 10 ] .", "our most important goal was to alleviate the patient 's discomfort by using a minimally invasive procedure to eliminate infection . based on haematology results ,", "the oral surgery did not trigger the acute blastic crisis of cmmol in this patient .", "the surgery was necessary to improve his oral environment and to prevent further infection and more pain . because cmmol is associated with a risk of acute blastic crisis and a poor prognosis , any surgical procedure in these patients must be considered on a case - by - case basis ."], "abstract_text": ["<S> we report a case of chronic myelomonocytic leukaemia ( cmmol ) in a 68-year - old man who developed osteomyelitis of the mandible . at the initial visit </S>", "<S> , he reported uncontrolled gingival bleeding , despite self - administered haemostasis . </S>", "<S> he complained of severe pain in the socket , despite potent opioid analgesia . </S>", "<S> after consultation with the internal medicine specialists , we undertook a surgical anti - inflammatory approach that included sequestrectomy with massive blood transfusion . </S>", "<S> his physical condition was ameliorated after the surgical procedure , and he was discharged from the hospital . </S>", "<S> however , 3 months later , he died because of cardiac arrest after haemorrhagic shock and cardiovascular failure because his cmmol had developed to an acute blastic crisis . </S>", "<S> this experience demonstrates that the most important goal in such cases is to alleviate a patient 's discomfort by applying minimally invasive actions to eliminate infection and improve the quality of life without causing deterioration in the cmmol status . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conflict of Interest Statement"], "sections": [["chronic myelomonocytic leukaemia ( cmmol ) is a rare disease with an incidence of 0.370.72 per 100 000 population . the median age at diagnosis varies between 65 years and 75 years [ 14 ] .", "the radical treatment is allogeneic stem cell transplantation , although this treatment is unsuitable for most patients because of their advanced age [ 6 , 7 ] .", "the median survival of patients with cmmol is 1520 months [ 5 , 6 , 8 ] .", "several case reports have indicated that excess surgical stress causes an acute blastic crisis , which contributes to a poor outcome [ 9 , 10 ] ."], ["a 68-year - old man complained of having a reduced appetite for approximately 5 months and weight loss during the previous 4 months . on 12 may 2012", ", he visited a nearby general internal medicine clinic for the evaluation of left lower quadrant abdominal pain .", "he was referred to the department of internal medicine at asahi general hospital ( asahi , japan ) for investigation and treatment .", "he began taking oral hydroxyurea ( hydrea ; bristol - myers squibb , princeton , nj , usa ) .", "he was unable to stop the gingival bleeding that had been ongoing for 5 hours .", "he then visited the emergency department of asahi general hospital and was referred to the department of dentistry and oral surgery . at the initial visit", "the right molar teeth ( 47 and 48 ) showed grade iii mobility , and their gingivae were painful with continuous bleeding .", "1a ) . the right molar teeth ( 46 and 47 ) showed class iii alveolar ridge deficiency on panoramic radiographs and were mobile teeth ( fig .", "we packed an absorbable haemostat ( surgicel ; ethicon , somerville , nj , usa ) into the gingivae and sutured 30 silk threads above it . finally , we used surgical dressing packs ( coe - pak ; g.c .", "figure 1:(a ) the oral photograph at the initial visit , immediately after the gauze with the astringent has been removed .", "( b ) the oral photograph after the extraction of teeth 47 and 48 . \n", "( a ) the oral photograph at the initial visit , immediately after the gauze with the astringent has been removed .", "the internal medicine department assigned a clinical diagnosis of cmmol , based on the blood and bone marrow examinations conducted on 13 june 2012 .", "computed tomography on june 25 indicated that bone absorption had nearly reached the right inferior alveolar canal , and a ring of sequestrum was present under tooth 47 ( fig .", "we diagnosed right mandibular osteomyelitis , based on these clinical features . on 11 july 2012 ,", "the patient was hospitalized in the department of haematology ward because of significant anaemia and decreased platelet numbers . on the same day ,", "the internal medicine specialist placed him on intravenous  piperacillin  tazobactam ( zosyn ; pfizer , new york , ny , usa ) .", "he afterwards complained of severe pain in the socket ( 47 and 48 ) , despite receiving the potent opioid analgesic dihydrohydroxycodeinone ( fentanyl ; daiichi - sankyo , chiyoda - ku , tokyo , japan ) .", "hence , we decided to apply a surgical anti - inflammatory treatment in consideration of an acute blastic crisis .", "a bone marrow examination on july 13 showed no signs of acute blastic crisis of his cmmol .", "after we consulted an internal medicine specialist , we planned a surgical procedure to clean the inflamed region in his mouth . on the day before the surgery", ", he was administered a transfusion of platelet concentrate ( 20 units ) and piperacillin  tazobactam to prevent bleeding and further infection . on july 20 , with the patient under local anaesthesia , we surgically resected the right lower sequestrum and extracted molar 37 , which displayed severe caries .", "the blood examination indicated improvement in the platelet count after the transfusion . after performing an inferior alveolar block", ", we removed the necrotic bone with ultrasonic bone surgery ( variosurg ; nsk , ashikaga , tochigi , japan ) until there was bleeding from the bone surface ( fig .", "we placed gauze with tetracycline ointment in the socket and sutured 30 silk thread to compress it ( fig .", "( b ) we removed necrotic bone with ultrasonic bone surgery until there was bleeding from the bone surface .", "( b ) we removed necrotic bone with ultrasonic bone surgery until there was bleeding from the bone surface .", "( d ) we used 30 silk thread sutures to compress the gauze . on august 11", "the formation of granulation tissue had nearly covered the surface of the bone , and all oral discomfort had disappeared ( fig .", "we later placed a bridge in the upper anterior teeth and added a partial denture ( fig .", "we placed a bridge for upper anterior teeth 12 , 11 , 22 and 23 , and a partial denture .", "we placed a bridge for upper anterior teeth 12 , 11 , 22 and 23 , and a partial denture . on 3 january 2013", ", he admitted himself to the hospital in the haematology ward because of exertional dyspnoea .", "the internal medicine specialists diagnosed cmmol that was undergoing acute leukaemic transformation by bone marrow examination .", "he died on 10 february 2013 because of cardiac arrest , following haemorrhagic shock and cardiovascular failure due to cmmol ."], ["in all patients with cmmol , every effort must be made to use the least invasive procedure possible . in the present patient ,", "our deepest concerns were to have a proper balance between invasiveness and healing , and to avoid evoking an acute blastic crisis [ 9 , 10 ] .", "our most important goal was to alleviate the patient 's discomfort by using a minimally invasive procedure to eliminate infection . based on haematology results ,", "the oral surgery did not trigger the acute blastic crisis of cmmol in this patient .", "the surgery was necessary to improve his oral environment and to prevent further infection and more pain . because cmmol is associated with a risk of acute blastic crisis and a poor prognosis , any surgical procedure in these patients must be considered on a case - by - case basis ."], [""]]}
{"article_id": "PMC3580606", "article_text": ["in a recent issue of critical care , we presented a series of 13 patients undergoing post - cardiotomy extracorporeal membrane oxygenation ( ecmo ) with bivalirudin - based anticoagulation .", "after this series , we satisfactorily continued this experience , reaching about 20 patients treated .", "however , we think that a word of caution should be added to our recently published experience . owing to its pharmacological profile", ", bivalirudin is rapidly cleaved by proteolytic enzymes , and its half - life is about 25 minutes when the renal function is normal .", "the use of bivalirudin for cardiopulmonary bypass ( cpb )   and ecmo   is a feasible option .", "however , it is recommended that , during cpb , blood stagnation in the circuit be avoided because the rapid cleavage of bivalirudin may result in thrombosis .", "the ecmo circuit is devoid of a reservoir , and therefore blood stagnation is usually not a circuit- related problem .", "conversely , under some circumstances , the cardiac chambers may act as a ' natural reservoir ' , which entails blood stagnation and the risk for spontaneous intracardiac thrombosis .", "this is particularly true in the case of a very large right or left atrium with insufficient venous drainage or the case of very poor ventricular systolic function with intraventricular blood stagnation . to avoid this condition", ", we think that maintaining a partial ecmo support , leaving a minimal degree of intracardiac blood flow , may be a reasonable choice .", "this may be checked by echocardiographic view of heart valve movement or simply by the observation of some degree of pulsatile arterial blood pressure .", "we think that , in the event of echocardiographic evidence of a ' smoke effect ' within one or more cardiac chambers , bivalirudin should be replaced by standard heparin anticoagulation .", "intracardiac thrombus formation during ecmo with heparin anticoagulation has been described as well , but the pharmacokinetic properties of heparin may limit the risk of thrombus formation due to blood stagnation ."], "abstract_text": ["<S> bivalirudin has been proposed as the sole anticoagulant in patients under extracorporeal membrane oxygenation ( ecmo ) or cardiopulmonary bypass . </S>", "<S> owing to the pharmacodynamic properties of bivalirudin , areas of blood stagnation should be carefully avoided in order to limit the risk of thrombosis . </S>", "<S> the ecmo circuit has no reservoir and is usually devoid of blood stagnation areas . </S>", "<S> conversely , under some circumstances , intracardiac blood stagnation areas may exist . in this case </S>", "<S> , there is a potential risk for the spontaneous formation of an intracardiac thrombus . </S>", "<S> we suggest that , under bivalirudin anticoagulation , a minimal degree of intracardiac blood flow with left heart valve movement is allowed . </S>"], "labels": null, "section_names": ["None", "Abbreviations", "Competing interests"], "sections": [["in a recent issue of critical care , we presented a series of 13 patients undergoing post - cardiotomy extracorporeal membrane oxygenation ( ecmo ) with bivalirudin - based anticoagulation .", "after this series , we satisfactorily continued this experience , reaching about 20 patients treated .", "however , we think that a word of caution should be added to our recently published experience . owing to its pharmacological profile", ", bivalirudin is rapidly cleaved by proteolytic enzymes , and its half - life is about 25 minutes when the renal function is normal .", "the use of bivalirudin for cardiopulmonary bypass ( cpb )   and ecmo   is a feasible option .", "however , it is recommended that , during cpb , blood stagnation in the circuit be avoided because the rapid cleavage of bivalirudin may result in thrombosis .", "the ecmo circuit is devoid of a reservoir , and therefore blood stagnation is usually not a circuit- related problem .", "conversely , under some circumstances , the cardiac chambers may act as a ' natural reservoir ' , which entails blood stagnation and the risk for spontaneous intracardiac thrombosis .", "this is particularly true in the case of a very large right or left atrium with insufficient venous drainage or the case of very poor ventricular systolic function with intraventricular blood stagnation . to avoid this condition", ", we think that maintaining a partial ecmo support , leaving a minimal degree of intracardiac blood flow , may be a reasonable choice .", "this may be checked by echocardiographic view of heart valve movement or simply by the observation of some degree of pulsatile arterial blood pressure .", "we think that , in the event of echocardiographic evidence of a ' smoke effect ' within one or more cardiac chambers , bivalirudin should be replaced by standard heparin anticoagulation .", "intracardiac thrombus formation during ecmo with heparin anticoagulation has been described as well , but the pharmacokinetic properties of heparin may limit the risk of thrombus formation due to blood stagnation ."], [""], [""]]}
{"article_id": "PMC5353405", "article_text": ["global and regional left ventricular ( lv ) functions are well - known indicators of cardiac disease . quantitative values of ventricular volumes and of myocardial mass are independent predictors of morbidity and mortality in patients with coronary artery disease .", "classically , echo has been used to evaluate lv volume and function because it is relatively inexpensive and noninvasive . however ,", "a component of operator dependence and poor contrast between blood and myocardium are considerable limitations of this technique . cardiac magnetic resonance imaging ( mri )", "is considered the clinical  gold standard for lv function assessment , but it is expensive , of limited availability , and can not be performed in patients with implanted pacemakers or defibrillators . in recent years , multidetector ct ( mdct ) has gained acceptance as a promising imaging method for coronary arteries .", "mdct acquired in a single breath - hold with retrospective electrocardiogram ( ecg ) gating can cover the entire heart with 1-mm slice thickness with a temporal resolution of 125 - 250 ms .", "when performed for coronary imaging , this method provides excellent opportunity to create , image reformation in any desired plane , including anatomically optimized long axis , short axis , or four - chamber views .", "diastolic and systolic images can easily be produced from the same data set with a retrospective ecg - gating technique , thus obtaining lv end - diastolic and end - systolic volumes ( edvs and esvs ) .", "mdct has a potential of being utilized as tool for the combined assessment of the coronary anatomy and lv function .", "in addition , ventricular wall motion can be assessed visually by the use of cine loop displays of multiple cardiac phases .", "recently we observed increasing tendency for utilizing low radiation dose , prospective gating for coronary angiography , thus limiting possibility of volumetric assessment of ventricular function .", "however , a small number of patients may need a retrospective gating , thus providing possibility of reconstructions in various phases of cardiac activity . according to published reports ,", "measurements for various lv functional parameters with mdct were well - correlated and agree with measurements obtained with mri , two - dimensional transthoracic echocardiography ( 2d - tte ) , and ecg - gated single photon emission ct ( spect ) .", "experience with 64-slice mdct for cardiac function assessment remains limited by small patient numbers and the inclusion of homogeneous patient populations .", "the purpose of this study was to assess lv ejection fraction ( lvef ) using 64-slice as a byproduct of mdct coronary examination and to compare efficacy of technique with 2d - tte in a heterogeneous patient population . also , review the role of mdct lv function with a relation to evolution in the technology of coronary mdct imaging .", "study included 113 patients referred for 64-slice mdct coronary angiography for evaluation of coronary artery disease .", "all patients were scanned on 64 slice ge - helical ct ( ge high speed advantage ) scanner and had an echo done within 1 week of the ct scan .", "this prospective study was approved by the institutional review board and written informed consent was obtained from all patients .", "patients with absolute contraindication to contrast or radiation were excluded from the study . in patients with relative contraindications such as atopy , asthma , and renal failure scan was performed if the benefit of examination outweighed the risk in such patients .", "patients with arrhythmias and ectopic heart beats were excluded as stable heart rate is required for ct coronary angiogram .", "apart from the routine contraindications , patients with pacemaker and ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool is not possible in these patients due to artifacts and incorrect segmentation by software .", "all patients undergoing ct coronary angiogram , who had heart rate of more than 60 bpm , were premedicated with 50 - 200 mg oral b adrenergic blocking agent : metoprolol , 1-h prior to the study . a 60 - 120 mg calcium channel blocker : diltiazem ,", "cta was performed with contrast volume of 1.2 ml / kg body weight of iohexol 350 .", "the intravenous contrast agent was followed by 30 ml of saline chaser bolus at the same injection rate .", "scan parameters were 0.35 s rotation time , 120 kv tube voltage , 600 - 800 effective ma , 0.6 mm collimation , and a helical pitch of 0.22:1 .", "the image acquisition was caudocranial for post - coronary artery bypass grafting ( cabg ) patients and craniocaudal for the rest of the patients .", "no complications encountered in any of the patients . retrospectively , ecg - correlated image reconstruction was performed .", "the reconstruction was performed with the reconstruction window starting at 10% of r - r interval and up to 90% r - r interval with increment of 10% .", "this included data sets reconstructed in systole , if diastolic data sets showed motion artifact .", "diastolic and systolic axial image sets were then transferred to the scanner 's workstation - ge advanced workstation advantage windows 4.4 p. image data were evaluated with a prototype version of a commercially available program ( auto ejection fraction , circulation ; ge medical solutions ) that performs a fully automatic segmentation of the blood volume in the lv by defining the mitral valve plane and the lv .", "the software uses this mitral valve plane as an upper boundary for the segmentation of the lv .", "the software identifies the hinges of the mitral and aortic valve leaflets closest to the ventricle wall and selects these as defining points for the plane [ figure 1a ] .", "all ct scans were analyzed according to this method , which allowed for optimal segmentation of the lv .", "papillary muscles were automatically excluded from the blood pool , which allows for precise determination of blood volume in the lv .", "multiplanar reformats are then performed by the software in long and short axes of left ventricle .", "the long axis image is obtained parallel to the interventricular septum connecting the lv apex and the middle level of mitral valve .", "the short axis images are obtained parallel to the plane of mitral valve [ figure 1b ] .", "lv = left ventricular ( b ) line perpendicular to long axis of left ventricle ( parallel to mitral valve ) ", "plane for short axis images once the region of interest is finalized , the edv and esv are measured by this software using simpson 's method by summing the endocardial area of all lv ed and es short - axis slices multiplied by the slice thickness [ figure 2a and b ] .", "the stroke volume ( sv ) and ef were automatically calculated from these values and displayed by the software [ figure 3a  d ] .", "( a ) lv short axis image in diastole ( b ) short axis image in systole 3d display of volumetric data in different patients . ( a ) patient 1 : left ventricular ejection fraction by ct  58.4% ( echo  58% ) . ( b )", "patient 2 : left ventricular ejection fraction by ct  55.7% ( echo  53% ) .", "( c ) patient 3 : left ventricular ejection fraction by ct  60.0% ( echo  60% ) .", "( d ) patient 4 : left ventricular ejection fraction by ct  38.3% ( echo  35% ) .", "3d = three - dimensional , echo = echocardiography , ct = computed tomography two - dimensional echo examination was performed either 1 week before or after coronary cta in either of two ultrasound units , an acuson sequoia ( siemens medical systems usa , mountain view , ca ) or a ge vivid 3 ( ge healthcare , milwaukee , wi ) .", "images were obtained using a 3.5 mhz transducer and images were acquired in standard apical and parasternal two- and four - chamber views .", "the chamber and wall dimensions were measured using standard recommendations for chamber quantification in consensus .", "results on continuous measurements are presented on mean  sd ( min  max ) and results on categorical measurements are presented in number ( % ) . the mean  standard deviation ( sd ) lvef calculated by clinical echo", "similarly mean  sd of lvef calculated by fully automated software based on cta data was obtained [ table 2 ] .", "agreement for lvef was determined by the use of pearson 's regression analysis [ figure 4 ] and calculating correlation coefficient ( r ) .", "bland - altman analysis [ figure 5 ] was used to compare the lvef measured with mdct and that with 2d - tte .", "mountain plot [ figure 6 ] was used to see the relationship between two groups .", "echo - ejection fraction ( ef ) mean  standard deviation ( sd ) : 58.67  4.53 , echo = echocardiography ct - ejection fraction ( ef ) mean  standard deviation ( sd ) : 58.93  5.43 , ct = computed tomography pearson regression analysis between echo - ef and ct - ef .", "r = pearson correlation coefficient , p = p - value , ef = ejection fraction bland and altman showing the correlation of echo - ef and ct - ef", "sd = standard deviation mountain plot showing the correlation of echo - ef and ct - ef .", "in our study , 86 ( 76.1% ) were males and 27 ( 23.9% ) were females .", "the mean age of the patients was 51.19  10.10 years and majority of subjects belonged to age group between 51 and 60 years .", "28.3% were between 41 and 50 years , 15.9% subjects were between 61 and 70 years , and 12.4% subjects between 31 and 40 years .", "most of the patients had at least one symptom , the commonest being chest pain in 88 ( 77.9% ) cases .", "the most common coronary risk factor association was hypertension , accounting for 77.9% of the cases .", "the mean heart rate of the patients at the time of scan was 61.5  8.6 bpm with maximum patients having a heart rate range of 61 - 70 bpm .", "the mean lvef calculated by clinical echo was 58.67  4.53% with maximum number of patients having an ef range of 56 - 60% .", "the mean lvef calculated by fully automated software based on cta data was 58.93  5.43% with maximum number of patients having an ef range of 61 - 65% . in our study using the fully automated software , the pearson 's regression analysis showed a good interstudy correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) .", "bland - altman analysis showed a trend towards mdct resulting in slightly higher values for lvef when compared with echo ; however , this observation was not statistically significant .", "the mountain plot analysis reinforced that ef measured by ct correlated well with that measured by echo .", "mdct coronary angiography has emerged as a valuable technique for the evaluation of coronary artery disease in patients with low to intermediate pretest probability of ischemic heart disease . utilizing analytic software ,", "gated volumetric ct data can be processed to provide quantitative functional analysis of the lv in patients with coronary artery disease .", "we were able to obtain satisfactory artifact free datasets from 113 consecutive subjects who underwent coronary cta .", "all of our patients were either known cases of coronary artery disease or had suggestive clinical symptoms .", "patients with pacemaker , ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool was not possible . achieving a stable heart rate for", "the examination was variable component of the examination . however , no examination had to be postponed or cancelled due to this limitation .", "our study using automated software showed a good interstudy correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) .", "the bland - altman plot revealed a slight mean difference between ef measurements on ct and echo with most differences falling within two sds of the mean .", "hence , we found that software is user - friendly and capable of providing good reproducibility for ef measurements in comparison with echo . in a previous study by krishnam et al .", ", similar results were recorded , though the number of subjects was small . in a study by cury et al . , a trend of mdct slightly underestimating lvef compared with tte was observed .", "we observed a trend towards mdct resulting in slightly higher values for lvef when compared with echo , contrary to expected mild reduction in beta blocked patients .", "trend however was not statistically significant , could be due to recognized limitation of evaluation technique leading to over or underestimation .", "mean difference in ef measurements between cta and echo is small ; although standard deviation of the mean difference is quite high , leading to wide limits of agreement .", "possibly observation results from the fact that the ef measurements from echo were obtained in a clinical setting , on visual estimation and calculation of ef using simpson 's method based on geometrical assumptions .", "our observations are in accordance with the previous studies of 64-slice coronary ct , confirming that lvef estimation is feasible with the mdct data and may be regarded as a useful clinical index , correlating with results of echo .", "there are studies with semiautomated software for quantitative functional analysis of lv with user defined mitral valve plane and an arbitrary point within the lv , with the option to expand or reduce the area of segmentation .", "many earlier studies have compared the use of 4- , 8- , and 16-slice ct scanners for evaluation of lv volumes .", "larger detector configuration in 64-slice ct scanner , has the advantage of being faster , capable of smaller slice thickness and higher temporal resolution .", "relatively higher radiation dose results from the protocol optimized for thin slice high - resolution imaging of the coronary arteries .", "the ecg - dependent tube current modulation is currently the most effective tool for dose reduction and may reduce patient dose by up to 50% .", "it is important to note that two points of the cardiac cycle ( end - systole and end - diastole ) with modulation of tube current were not used in our study because ecg - gated dose modulation was only applicable to 50 - 90% of the rr interval on ecg .", "if the aim is to evaluate coronary arteries only , it is recommended to use an ecg - dependent dose modulation technique or newer prospective gated techniques .", "new developments in mdct technology is allowing examination of patients with higher heart rates and reducing the dose of beta - blockers . presently , mdct examination is possible which contains all the phases with a considerably lower dose of the order of 2 - 3.3 msv .", "the software identifies the lv blood pool based on hounsfield unit values and continuity of adjacent voxels . in patients with a ventricular septal defect", ", there is contrast opacification in both ventricles and a bridge of contrast through the septal defect .", "therefore , the software identifies the right and lv blood pools as a single chamber , resulting in incorrect segmentation and thus inaccurate assessment of lv functional parameters . in patient with pacemakers", ", software identifies a pacemaker wire as high - density contrast and segments the pacing wire as part of the ventricle , resulting in failed segmentation . the version of the software used for this study it was not able to segment the myocardium in order to quantify the lv myocardial mass , which may be important in certain cardiac diseases such as hypertrophic cardiomyopathy .", "it is important to note that cardiac mri is considered as a  gold standard for lv function assessment .", "cardiac mri ( cmri ) provides excellent temporal and spatial resolution , image acquisition in any desired plane , and a high degree of accuracy and reproducibility . concerning quantitative measurements", "cine mri technique is potentially the most comprehensive cardiac imaging modality available because of its excellent contrast between blood - filled ventricles and the surrounding myocardium .", "yamamuro et al . , have shown high linear correlation between ef measurements on ct and on mri .", "the temporal resolution of many available mdct is still considerably lower than that of echo and cine mri .", "this lower temporal resolution can make evaluation of isovolumetric ed and es phases of the cardiac cycle , and thus ef , less precise .", "there is considerable improvement in temporal resolution of mdct with improvement in gantry rotation , multiple and partial segment processing techniques .", "tr of 80 - 250 ms has been achieved in state of the art units .", "though it is short of fluoroscopic resolution , currently available options are more than sufficient for motion free systolic and diastolic cardiac imaging with heart rate below 100/min . additionally , the use of a b - blocker to reduce the heart rate to less than 65 bpm can influence the functional parameters that are to be measured . beta blocker do influence the lvef , lead to underestimation .", "overestimation or underestimation of the lv volume has been reported because of the different criteria for selecting the endocardial boundary or the inclusion / exclusion of papillary muscle .", "ideally estimation of the real - ef from all 20 phases is more precise ; however , this significantly increases effort and processing time .", "effect of beta - blocker has to be factored in interpretation of ef by mdct .", "introduction of new ct imaging methods , including dual source ct in clinical practice will overcome these two problems significantly owing to its two - fold increase in temporal resolution . also , newer options in mdct technology may partially obviate need for use of beta blockers .", "functional parameters derived from 2d - tte are compared with ct derived 3d volumetric data , which are not strictly comparable .", "our study design did not allow realistic comparison of mdct and echo lv volume data .", "it would be interesting to compare the same for assessment of accuracy of respective data .", "with evolution of speckle - tracking 3d echography and new low radiation , high tr scanning such studies are possible .", "the delay time between ct and echo and premedication with -blockers could have changed myocardial contraction and lv volumes as measured with the two methods . in the present", "set up , the number of patients referred to coronary angiography who will have volumetric data will be significantly smaller , limiting the application of this utility to a smaller offset of patients .", "the radiation issue will certainly be an important consideration to use this technique in the larger group of patients .", "emerging new applications of echography in the form of 2d and 3d speckle - tracking echo certainly will have a greater role to play in the future noninvasive assessment of the myocardial function . in conclusion", "our study confirms useful complementary functional information in coronary cta datasets , using fully automated analysis software for rapid assessment of lvef .", "it is irrational to utilize mdct alone to assess lv function in clinical patients , given the radiation exposure involved .", "however , additional clinically useful information from a clinically indicated coronary ct examination with a lowest possible radiation dose is invaluable in patients known or suspected of ischemic heart disease .", "validating consistency of results with mri will further lend support to the use of mdct derived results .", "going forward with changing trends in ccta imaging , it is conceivable that number of patients undergoing cta with retrospective gating will substantially be reduced , thus limiting the functionality to a small group of patients ."], "abstract_text": ["<S> background : coronary computed tomography angiography ( ccta ) is a frequently performed examination for coronary artery disease . when performed with retrospective gating , there is an opportunity to derive functional parameters of left ventricle utilizing automated software . </S>", "<S> complementary information , if validated with established standards , will enhance the total value of study.objective:study evaluates the usefulness of fully automated software for the assessment of left ventricular ejection fraction ( lvef ) using 64-slice ccta data and to correlate ct results with echocardiography ( echo ) . </S>", "<S> role of ct derived lv function is reviewed in the light of emerging technologies and recent developments in multidetector ct ( mdct).materials and methods : a total of 113 patients referred for mdct ccta for evaluation of coronary artery disease . </S>", "<S> all patients were scanned on 64 slice ge - helical ct scanner and had an echo done within 1 week of the ct scan . retrospectively electrocardiogram ( </S>", "<S> ecg)-correlated image reconstruction was performed with the reconstruction at 10% r - r interval increment . </S>", "<S> axial image sets were analyzed with advanced workstation using a program - auto ejection fraction , circulation : ge medical solutions.results:the mean lvef calculated by clinical echo was 58.6  4.5% and by fully automated software based on cta data was 58.9  5.4% . </S>", "<S> the pearson 's regression analysis showed a large correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) . </S>", "<S> bland - altman analysis showed a trend towards mdct resulting in slightly higher values for lvef when compared with echo.conclusion:the fully automated software is simple , reliable , and user - friendly , and can provide rapid assessment of lv functional parameters with good reproducibility . </S>", "<S> despite of good correlation , fewer patients are likely to benefit , in future , from this function due to smaller number of patients undergoing ccta with retrospective gating . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["global and regional left ventricular ( lv ) functions are well - known indicators of cardiac disease . quantitative values of ventricular volumes and of myocardial mass are independent predictors of morbidity and mortality in patients with coronary artery disease .", "classically , echo has been used to evaluate lv volume and function because it is relatively inexpensive and noninvasive . however ,", "a component of operator dependence and poor contrast between blood and myocardium are considerable limitations of this technique . cardiac magnetic resonance imaging ( mri )", "is considered the clinical  gold standard for lv function assessment , but it is expensive , of limited availability , and can not be performed in patients with implanted pacemakers or defibrillators . in recent years , multidetector ct ( mdct ) has gained acceptance as a promising imaging method for coronary arteries .", "mdct acquired in a single breath - hold with retrospective electrocardiogram ( ecg ) gating can cover the entire heart with 1-mm slice thickness with a temporal resolution of 125 - 250 ms .", "when performed for coronary imaging , this method provides excellent opportunity to create , image reformation in any desired plane , including anatomically optimized long axis , short axis , or four - chamber views .", "diastolic and systolic images can easily be produced from the same data set with a retrospective ecg - gating technique , thus obtaining lv end - diastolic and end - systolic volumes ( edvs and esvs ) .", "mdct has a potential of being utilized as tool for the combined assessment of the coronary anatomy and lv function .", "in addition , ventricular wall motion can be assessed visually by the use of cine loop displays of multiple cardiac phases .", "recently we observed increasing tendency for utilizing low radiation dose , prospective gating for coronary angiography , thus limiting possibility of volumetric assessment of ventricular function .", "however , a small number of patients may need a retrospective gating , thus providing possibility of reconstructions in various phases of cardiac activity . according to published reports ,", "measurements for various lv functional parameters with mdct were well - correlated and agree with measurements obtained with mri , two - dimensional transthoracic echocardiography ( 2d - tte ) , and ecg - gated single photon emission ct ( spect ) .", "experience with 64-slice mdct for cardiac function assessment remains limited by small patient numbers and the inclusion of homogeneous patient populations .", "the purpose of this study was to assess lv ejection fraction ( lvef ) using 64-slice as a byproduct of mdct coronary examination and to compare efficacy of technique with 2d - tte in a heterogeneous patient population . also , review the role of mdct lv function with a relation to evolution in the technology of coronary mdct imaging ."], ["study included 113 patients referred for 64-slice mdct coronary angiography for evaluation of coronary artery disease .", "all patients were scanned on 64 slice ge - helical ct ( ge high speed advantage ) scanner and had an echo done within 1 week of the ct scan .", "this prospective study was approved by the institutional review board and written informed consent was obtained from all patients .", "patients with absolute contraindication to contrast or radiation were excluded from the study . in patients with relative contraindications such as atopy , asthma , and renal failure scan was performed if the benefit of examination outweighed the risk in such patients .", "patients with arrhythmias and ectopic heart beats were excluded as stable heart rate is required for ct coronary angiogram .", "apart from the routine contraindications , patients with pacemaker and ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool is not possible in these patients due to artifacts and incorrect segmentation by software .", "all patients undergoing ct coronary angiogram , who had heart rate of more than 60 bpm , were premedicated with 50 - 200 mg oral b adrenergic blocking agent : metoprolol , 1-h prior to the study . a 60 - 120 mg calcium channel blocker : diltiazem ,", "cta was performed with contrast volume of 1.2 ml / kg body weight of iohexol 350 .", "the intravenous contrast agent was followed by 30 ml of saline chaser bolus at the same injection rate .", "scan parameters were 0.35 s rotation time , 120 kv tube voltage , 600 - 800 effective ma , 0.6 mm collimation , and a helical pitch of 0.22:1 .", "the image acquisition was caudocranial for post - coronary artery bypass grafting ( cabg ) patients and craniocaudal for the rest of the patients .", "no complications encountered in any of the patients . retrospectively , ecg - correlated image reconstruction was performed .", "the reconstruction was performed with the reconstruction window starting at 10% of r - r interval and up to 90% r - r interval with increment of 10% .", "this included data sets reconstructed in systole , if diastolic data sets showed motion artifact .", "diastolic and systolic axial image sets were then transferred to the scanner 's workstation - ge advanced workstation advantage windows 4.4 p. image data were evaluated with a prototype version of a commercially available program ( auto ejection fraction , circulation ; ge medical solutions ) that performs a fully automatic segmentation of the blood volume in the lv by defining the mitral valve plane and the lv .", "the software uses this mitral valve plane as an upper boundary for the segmentation of the lv .", "the software identifies the hinges of the mitral and aortic valve leaflets closest to the ventricle wall and selects these as defining points for the plane [ figure 1a ] .", "all ct scans were analyzed according to this method , which allowed for optimal segmentation of the lv .", "papillary muscles were automatically excluded from the blood pool , which allows for precise determination of blood volume in the lv .", "multiplanar reformats are then performed by the software in long and short axes of left ventricle .", "the long axis image is obtained parallel to the interventricular septum connecting the lv apex and the middle level of mitral valve .", "the short axis images are obtained parallel to the plane of mitral valve [ figure 1b ] .", "lv = left ventricular ( b ) line perpendicular to long axis of left ventricle ( parallel to mitral valve ) ", "plane for short axis images once the region of interest is finalized , the edv and esv are measured by this software using simpson 's method by summing the endocardial area of all lv ed and es short - axis slices multiplied by the slice thickness [ figure 2a and b ] .", "the stroke volume ( sv ) and ef were automatically calculated from these values and displayed by the software [ figure 3a  d ] .", "( a ) lv short axis image in diastole ( b ) short axis image in systole 3d display of volumetric data in different patients . ( a ) patient 1 : left ventricular ejection fraction by ct  58.4% ( echo  58% ) . ( b )", "patient 2 : left ventricular ejection fraction by ct  55.7% ( echo  53% ) .", "( c ) patient 3 : left ventricular ejection fraction by ct  60.0% ( echo  60% ) .", "( d ) patient 4 : left ventricular ejection fraction by ct  38.3% ( echo  35% ) .", "3d = three - dimensional , echo = echocardiography , ct = computed tomography two - dimensional echo examination was performed either 1 week before or after coronary cta in either of two ultrasound units , an acuson sequoia ( siemens medical systems usa , mountain view , ca ) or a ge vivid 3 ( ge healthcare , milwaukee , wi ) .", "images were obtained using a 3.5 mhz transducer and images were acquired in standard apical and parasternal two- and four - chamber views .", "the chamber and wall dimensions were measured using standard recommendations for chamber quantification in consensus .", "results on continuous measurements are presented on mean  sd ( min  max ) and results on categorical measurements are presented in number ( % ) . the mean  standard deviation ( sd ) lvef calculated by clinical echo", "similarly mean  sd of lvef calculated by fully automated software based on cta data was obtained [ table 2 ] .", "agreement for lvef was determined by the use of pearson 's regression analysis [ figure 4 ] and calculating correlation coefficient ( r ) .", "bland - altman analysis [ figure 5 ] was used to compare the lvef measured with mdct and that with 2d - tte .", "mountain plot [ figure 6 ] was used to see the relationship between two groups .", "echo - ejection fraction ( ef ) mean  standard deviation ( sd ) : 58.67  4.53 , echo = echocardiography ct - ejection fraction ( ef ) mean  standard deviation ( sd ) : 58.93  5.43 , ct = computed tomography pearson regression analysis between echo - ef and ct - ef .", "r = pearson correlation coefficient , p = p - value , ef = ejection fraction bland and altman showing the correlation of echo - ef and ct - ef", "sd = standard deviation mountain plot showing the correlation of echo - ef and ct - ef ."], ["in our study , 86 ( 76.1% ) were males and 27 ( 23.9% ) were females .", "the mean age of the patients was 51.19  10.10 years and majority of subjects belonged to age group between 51 and 60 years .", "28.3% were between 41 and 50 years , 15.9% subjects were between 61 and 70 years , and 12.4% subjects between 31 and 40 years .", "most of the patients had at least one symptom , the commonest being chest pain in 88 ( 77.9% ) cases .", "the most common coronary risk factor association was hypertension , accounting for 77.9% of the cases .", "the mean heart rate of the patients at the time of scan was 61.5  8.6 bpm with maximum patients having a heart rate range of 61 - 70 bpm .", "the mean lvef calculated by clinical echo was 58.67  4.53% with maximum number of patients having an ef range of 56 - 60% .", "the mean lvef calculated by fully automated software based on cta data was 58.93  5.43% with maximum number of patients having an ef range of 61 - 65% . in our study using the fully automated software , the pearson 's regression analysis showed a good interstudy correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) .", "bland - altman analysis showed a trend towards mdct resulting in slightly higher values for lvef when compared with echo ; however , this observation was not statistically significant .", "the mountain plot analysis reinforced that ef measured by ct correlated well with that measured by echo ."], ["mdct coronary angiography has emerged as a valuable technique for the evaluation of coronary artery disease in patients with low to intermediate pretest probability of ischemic heart disease . utilizing analytic software ,", "gated volumetric ct data can be processed to provide quantitative functional analysis of the lv in patients with coronary artery disease .", "we were able to obtain satisfactory artifact free datasets from 113 consecutive subjects who underwent coronary cta .", "all of our patients were either known cases of coronary artery disease or had suggestive clinical symptoms .", "patients with pacemaker , ventricular septal defect were excluded from the study as successful segmentation of the lv blood pool was not possible . achieving a stable heart rate for", "the examination was variable component of the examination . however , no examination had to be postponed or cancelled due to this limitation .", "our study using automated software showed a good interstudy correlation , with a correlation coefficient of 0.503 ( p < 0.001 ) .", "the bland - altman plot revealed a slight mean difference between ef measurements on ct and echo with most differences falling within two sds of the mean .", "hence , we found that software is user - friendly and capable of providing good reproducibility for ef measurements in comparison with echo . in a previous study by krishnam et al .", ", similar results were recorded , though the number of subjects was small . in a study by cury et al . , a trend of mdct slightly underestimating lvef compared with tte was observed .", "we observed a trend towards mdct resulting in slightly higher values for lvef when compared with echo , contrary to expected mild reduction in beta blocked patients .", "trend however was not statistically significant , could be due to recognized limitation of evaluation technique leading to over or underestimation .", "mean difference in ef measurements between cta and echo is small ; although standard deviation of the mean difference is quite high , leading to wide limits of agreement .", "possibly observation results from the fact that the ef measurements from echo were obtained in a clinical setting , on visual estimation and calculation of ef using simpson 's method based on geometrical assumptions .", "our observations are in accordance with the previous studies of 64-slice coronary ct , confirming that lvef estimation is feasible with the mdct data and may be regarded as a useful clinical index , correlating with results of echo .", "there are studies with semiautomated software for quantitative functional analysis of lv with user defined mitral valve plane and an arbitrary point within the lv , with the option to expand or reduce the area of segmentation .", "many earlier studies have compared the use of 4- , 8- , and 16-slice ct scanners for evaluation of lv volumes .", "larger detector configuration in 64-slice ct scanner , has the advantage of being faster , capable of smaller slice thickness and higher temporal resolution .", "relatively higher radiation dose results from the protocol optimized for thin slice high - resolution imaging of the coronary arteries .", "the ecg - dependent tube current modulation is currently the most effective tool for dose reduction and may reduce patient dose by up to 50% .", "it is important to note that two points of the cardiac cycle ( end - systole and end - diastole ) with modulation of tube current were not used in our study because ecg - gated dose modulation was only applicable to 50 - 90% of the rr interval on ecg .", "if the aim is to evaluate coronary arteries only , it is recommended to use an ecg - dependent dose modulation technique or newer prospective gated techniques .", "new developments in mdct technology is allowing examination of patients with higher heart rates and reducing the dose of beta - blockers . presently , mdct examination is possible which contains all the phases with a considerably lower dose of the order of 2 - 3.3 msv .", "the software identifies the lv blood pool based on hounsfield unit values and continuity of adjacent voxels . in patients with a ventricular septal defect", ", there is contrast opacification in both ventricles and a bridge of contrast through the septal defect .", "therefore , the software identifies the right and lv blood pools as a single chamber , resulting in incorrect segmentation and thus inaccurate assessment of lv functional parameters . in patient with pacemakers", ", software identifies a pacemaker wire as high - density contrast and segments the pacing wire as part of the ventricle , resulting in failed segmentation . the version of the software used for this study it was not able to segment the myocardium in order to quantify the lv myocardial mass , which may be important in certain cardiac diseases such as hypertrophic cardiomyopathy .", "it is important to note that cardiac mri is considered as a  gold standard for lv function assessment .", "cardiac mri ( cmri ) provides excellent temporal and spatial resolution , image acquisition in any desired plane , and a high degree of accuracy and reproducibility . concerning quantitative measurements", "cine mri technique is potentially the most comprehensive cardiac imaging modality available because of its excellent contrast between blood - filled ventricles and the surrounding myocardium .", "yamamuro et al . , have shown high linear correlation between ef measurements on ct and on mri .", "the temporal resolution of many available mdct is still considerably lower than that of echo and cine mri .", "this lower temporal resolution can make evaluation of isovolumetric ed and es phases of the cardiac cycle , and thus ef , less precise .", "there is considerable improvement in temporal resolution of mdct with improvement in gantry rotation , multiple and partial segment processing techniques .", "tr of 80 - 250 ms has been achieved in state of the art units .", "though it is short of fluoroscopic resolution , currently available options are more than sufficient for motion free systolic and diastolic cardiac imaging with heart rate below 100/min . additionally , the use of a b - blocker to reduce the heart rate to less than 65 bpm can influence the functional parameters that are to be measured . beta blocker do influence the lvef , lead to underestimation .", "overestimation or underestimation of the lv volume has been reported because of the different criteria for selecting the endocardial boundary or the inclusion / exclusion of papillary muscle .", "ideally estimation of the real - ef from all 20 phases is more precise ; however , this significantly increases effort and processing time .", "effect of beta - blocker has to be factored in interpretation of ef by mdct .", "introduction of new ct imaging methods , including dual source ct in clinical practice will overcome these two problems significantly owing to its two - fold increase in temporal resolution . also , newer options in mdct technology may partially obviate need for use of beta blockers .", "functional parameters derived from 2d - tte are compared with ct derived 3d volumetric data , which are not strictly comparable .", "our study design did not allow realistic comparison of mdct and echo lv volume data .", "it would be interesting to compare the same for assessment of accuracy of respective data .", "with evolution of speckle - tracking 3d echography and new low radiation , high tr scanning such studies are possible .", "the delay time between ct and echo and premedication with -blockers could have changed myocardial contraction and lv volumes as measured with the two methods . in the present", "set up , the number of patients referred to coronary angiography who will have volumetric data will be significantly smaller , limiting the application of this utility to a smaller offset of patients .", "the radiation issue will certainly be an important consideration to use this technique in the larger group of patients .", "emerging new applications of echography in the form of 2d and 3d speckle - tracking echo certainly will have a greater role to play in the future noninvasive assessment of the myocardial function . in conclusion", "our study confirms useful complementary functional information in coronary cta datasets , using fully automated analysis software for rapid assessment of lvef .", "it is irrational to utilize mdct alone to assess lv function in clinical patients , given the radiation exposure involved .", "however , additional clinically useful information from a clinically indicated coronary ct examination with a lowest possible radiation dose is invaluable in patients known or suspected of ischemic heart disease .", "validating consistency of results with mri will further lend support to the use of mdct derived results .", "going forward with changing trends in ccta imaging , it is conceivable that number of patients undergoing cta with retrospective gating will substantially be reduced , thus limiting the functionality to a small group of patients ."]]}
{"article_id": "PMC4745564", "article_text": ["\n moraxella catarrhalis is a gram - negative , aerobic diplococcus human mucosal pathogen which causes middle ear infections in infants and children [ 13 ] , and it is one of the three major causes of otitis media along with streptococcus pneumonia and haemophilus influenzae . although moraxella catarrhalis is frequently found as a commensal of the upper respiratory tract , recently it has emerged as a genuine pathogen and is now considered an important cause of upper respiratory tract infections in healthy children and elderly people , lower respiratory tract infections in adults with chronic obstructive pulmonary disease [ 1 , 5 ] , and hospital - acquired pneumonia .", "amikacin , cefixime , fosfomycin , cefuroxime , cotrimoxazole , doxycycline , and erythromycin resistant strains of moraxella catarrhalis were isolated and the widespread production of a -1actamase enzyme renders the bacterium resistant to the penicillin [ 79 ] .", "this has led to the search for new and effective therapeutic alternatives among natural compounds .", "plants remain an important source of diverse chemical entities which have been used as drugs or provide scaffolds from which new drugs have been derived .", "the selection of a suitable candidate species for investigations can be done on the basis of long - term use by humans .", "this approach is based on an assumption that the active compounds isolated from such plants are likely to be safer than those derived from plant species with no history of human use .", "aristolochia is an important genus in the family of aristolochiaceae and is widespread across tropical asia , africa , and south america .", "it is used in traditional medicine as a gastric stimulant and in the treatment of cancer , lung inflammation , dysentery , and snakebites .", "it is also used in the treatment of tumors and malaria and for fevers , but its usage as an antimalarial is not recommended in its crude form .", "aristolochia bracteolata showed a definite positive effect on wound healing , with significant increase in the level of powerful antioxidant enzymes .", "the whole plant was used as a purgative , antipyretic , and anti - inflammatory .", "organic solvent extracts of the plant showed antibacterial activities while the water extract showed antifungal activity .", "although aristolochia has been used for thousands of years in many cultures for many indications due to its various pharmacological activities , it was later discovered that consuming these plants can certainly be dangerous .", "the genus of aristolochia contains a naturally carcinogenic compound aa which has been shown to be the cause of so - called chinese herb nephropathy or aa nephropathy [ 19 , 20 ] , and mutations in the cells of people who consume it , causes more mutations than two of the best - known environmental carcinogens : tobacco smoke and uv light [ 21 , 22 ] .", "there are many cases of nephropathy reported in the literature caused by the systemic and long term application of chinese snakeroot ( aristolochia fangchi ) ; this highlighted the risk of using preparations which contain aristolochic acids .", "although aristolochia is being known in many countries that is containing a toxic compound aa , but this has not stopped it from being a popular herbal remedy for thousands of years .", "it is still extensively used in india and in traditional chinese medicine for slimming , menstrual symptoms , and rheumatism .", "it is also widespread used in sudan and other african countries as one of the most effective herbal remedies for infectious diseases .", "therefore , it was our objective to assess the potential antimicrobial activity of aristolochia bracteolata using a bioassay - guided fractionation , in order to produce pure compound that can act as the lead compound in developing new , safe , and effective drug to replace the use of the harmful crude plant material .", "sephadex lh-20 ( pharmacia fine chemical co. ltd ) was used for column chromatography . precoated silica gel plates ( merck , kieselgel 60 f254 , 0.25  mm ) and precoated rp-18 f254s plates ( merck ) were used for thin - layer chromatography ( tlc ) analysis .", "high resolution fab - ms and esi - ms were recorded on jeol jms700n and jms-100td , respectively .", "h- and c - nmr , h - h cosy , noesy , hsqc , and hmbc spectra were recorded with a unity plus 500 spectrometer ( varian inc . , u.s.a . ) operating at 500  mhz for h and 125  mhz for c , respectively .", "h - nmr chemicals shifts are expressed in  values referring to the solvent peak h 2.49 for dmso and coupling constants are expressed in hz .", "c - nmr chemical shifts are expressed in  values referring to the solvent peak c 39.5 for dmso .", "piperonylic acid was purchased from commercial sources ( tci ) and used without further purification . the plant material ( whole )", "was collected in the period from ( october to december 2012 ) from khartoum state in sudan .", "standard strains : moraxella catarrhalis ( gtc 01544 ) , klebsiella pneumoniae ( atcc 13883 ) , escherichia coli ( k12 ) , salmonella typhimurium ( atcc 14028 ) , streptococcus pyogenes ( atcc 19615 ) , streptococcus agalactiae ( atcc 13813 ) , staphylococcus epidermidis ( atcc 12228 ) , neisseria lactamicus ( atcc 23970 ) , enterobacter cloacae , ( atcc 23355 ) , bacillus subtilis ( atcc 6633 ) , staphylococcus aureus ( 209p ) , and pseudomonas aeruginosa ( ifo 3445).clinical strains : moraxella catarrhalis , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica . \n  standard strains : moraxella catarrhalis ( gtc 01544 ) , klebsiella pneumoniae ( atcc 13883 ) , escherichia coli ( k12 ) , salmonella typhimurium ( atcc 14028 ) , streptococcus pyogenes ( atcc 19615 ) , streptococcus agalactiae ( atcc 13813 ) , staphylococcus epidermidis ( atcc 12228 ) , neisseria lactamicus ( atcc 23970 ) , enterobacter cloacae , ( atcc 23355 ) , bacillus subtilis ( atcc 6633 ) , staphylococcus aureus ( 209p ) , and pseudomonas aeruginosa ( ifo 3445 ) . clinical strains : moraxella catarrhalis , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica .", "in addition to a sea urchin ( anthocidaris crassispina ) derived bacillus sp . which obtained from the laboratory in medical plants garden , nagasaki university.(c)fungal strains : the fungal strains used were aspergillus niger ( nbrc 33023 ) , penicillium crustosum ( nbrc 33015 ) , schizophyllum commune ( nbrc 30749 ) , trichophyton concentricum ( nbrc 31068 ) , and candida albicans ( nbrc 10108 ) .", "fungal strains : the fungal strains used were aspergillus niger ( nbrc 33023 ) , penicillium crustosum ( nbrc 33015 ) , schizophyllum commune ( nbrc 30749 ) , trichophyton concentricum ( nbrc 31068 ) , and candida albicans ( nbrc 10108 ) .", "the air - dried powdered whole plant ( 200  g ) was exhaustively extracted with cold maceration method with sufficient quantity of 70% methanol for 7 days at room temperature .", "the methanolic extract was passed through whatman number 1 filter paper ( whatman england ) and the concentrated extract ( 40  g ) was digested with 100  ml distilled water and successively partitioned with n - hexane ( 4  400  ml ) , chloroform ( 3  400  ml ) , ethyl acetate ( 5  400  ml ) , and n - butanol ( 2  400  ml ) .", "each fraction was concentrated under reduced pressure to a constant weight to give the corresponding n - hexane fraction ( 0.4  g ) , chloroform fraction ( 2  g ) , ethyl acetate fraction ( 0.7  g ) , n - butanol fraction ( 6  g ) , and aqueous fraction ( 30  g ) . the most active fraction against bacillus sp . and", "m. catarrhalis ( chloroform fraction ) was subjected to sephadex lh20 column chromatography to give three subfractions ( a - c ) .", "fraction ( b ) was resubjected again to sephadex lh20 to afford very active and pure compound aa-1 ( 150  mg ) . to the solution of aa-1 ( 50  mg ; 0.23  mmol ) in dimethylformamide ( dmf )", ", 1  ml potassium carbonate was added ( 95  mg ; 0.69  mmol ) . to the resulting suspension iodomethane", "was added ( 72  l ; 1.15  mmol ) and stirred for 8 hours .", "the reaction mixture was poured onto water ( 10  ml ) and ethyl acetate ( 20  ml ) .", "the organic layer was washed with 1  m hcl ( 10  ml ) three times and then with brine ( 10  ml ) once .", "after removal of solvent under reduced pressure , the residue was purified by silica - gel chromatography ( chloroform - methanol ) to afford the titled ester ( 88% ) .", "h - nmr ( 400  mhz , cdcl3 , tms , r.t . )  ( ppm ) : 3.88 ( 3h , s ) , 4.06 ( 3hs ) , 6.38 ( 2h , s ) , 7.11 ( 1h , d , j = 7.8  hz ) , 7.72 ( 1h , dd , j = 7.8  hz , 8.0  hz ) , 7.77 ( 1h , s ) , 8.70 ( 1h , d , j = 8.0  hz ) , and 8.83 ( 1h , s ) .", "suspension of the tested bacteria ( 100  l of 10  cfu / ml ) was spread onto solid media plates .", "the sterile paper discs ( 6  mm in diameter ) which were impregnated with the plant extract ( 14  mg ) and pure compound ( 10100  g ) were placed aseptically over the bacterial culture on nutrient agar plates .", "after incubation at 37c for 24 hours , the zone of inhibition around the discs was measured by millimeter scale .", "sterile , blank paper discs impregnated with only sterile solvents served as negative control each time .", "the sterile paper discs ( 6  mm in diameter ) which were impregnated with individual extract were placed on the inoculated plates .", "these plates were incubated for 2472  h at 2528c and the growth was evaluated visually by comparing a particular plate with the negative control plates ( having only test fungi ) .", "the antifungal activity was evaluated by measuring the inhibition zone diameter ( in millimeter ) observed .", "the mic and mbc values were determined by broth dilution method in accordance with clsi methodology .", "bacterial strains were cultured for 24  h at 37c on nutrient broth and then suspended in sterile distilled water to give a final inoculum concentration of 1.5  1.0  10  cfu / ml .", "dilutions ranging from 1.56 to 400  g / ml of the compound were prepared in tubes including broth and dmso 10% ( v / v ) , in addition to one negative control ( broth + dmso 10% v / v + test microorganism ) to ensure that the final concentration of dmso in the assays did not interfere with the bacterial growth and one sterility control ( broth + dmso 10% v / v + test compound ) .", "a 100  l suspension of test microorganism was added to individual tubes and incubated at 37c for 24  h. the mic of the compound was defined as the lowest concentration that inhibited the visible bacterial growth and the mbc was defined as the lowest concentration that prevented the growth of the organism after subculture onto antibiotic - free plates .", "initial steps in new drug discovery involve identification of new chemical entities , which can be either sourced through chemical synthesis or can be isolated from natural products through biological activity guided fractionation .", "bioassay - guided fractionation of the identified plant may lead to standardized extract or isolated bioactive lead compounds as the new drug .", "the whole plant of aristolochia bracteolata was extracted successively with meoh and subjected to liquid - liquid fractionation with n - hexane , chloroform , ethyl acetate , and n - butanol .", "the resulting fractions were tested for antibacterial and antifungal activities . the crude extract and chloroform fraction were significantly active against sea urchin - derived bacillus sp . and both standard strain and clinical isolates of moraxella catarrhalis and were moderately active against s. aureus , b. subtilis , and ps .", "the n - hexane fraction had moderate activity against s. aureus and b. subtilis while ethyl acetate fraction showed moderate activity against ps .", "the crude extract failed to inhibit the growth of all test fungi in addition to the following bacterial strains : klebsiella pneumoniae , escherichia coli , salmonella typhimurium , streptococcus pyogenes , streptococcus agalactiae , staphylococcus epidermidis , neisseria lactamicus , enterobacter cloacae , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica .  ", "the chloroform soluble fraction was therefore selected for further chromatographic separations and resulted in the isolation of known compound aa-1 ( figure 1 ) .", "aa-1 showed strong activity against moraxella catarrhalis ( standard strain and clinical isolates ) and sea urchin - derived bacillus sp .", "( table 2 ) , with equal mic and mbc values of 25 and 50  g / ml . both the piperonylic acid moiety of aa-1 ( figure 1 ) and aa-1-methyl ester showed no activity against bacteria ( table 2 ) , which suggests that the coexistence of phenanthrene ring and free carboxylic acid is essential for aa-1 antibacterial activity .", "bioguided fractionation of methanolic extract of aristolochia bracteolata led to isolation of aa-1 and its structure was elucidated by interpretation of its nmr and ms data and by comparison with those reported in the literature .", "electrospray ionization mass spectrometry ( esims ) showed pseudomolecular ion signal at m / z 364.03 [ m + na ] and high resolution fast atom bombardment mass spectrometry ( hr - fabms ) afforded m+1 ion signal at m / z 342.0620 which was corresponding to the molecular formula c17h12no7 ( calculated for 342.06138 ) .", "h nmr and c - nmr spectra were matched with those of aa-1 which were previously reported .", "two weak signals which did not show any correlation with proton in hsqc and hmbc spectroscopy were considered as quaternary carbons at positions 5 (  124.3  ppm ) and 6 (  143.5  ppm ) , respectively .", "laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all types of microorganisms in vitro .", "more of these compounds should be subjected to animal and human studies to determine their effectiveness in whole - organism systems , including in particular toxicity studies and an examination of their effects on beneficial normal microbiota . in spite of", "the fact that herbal remedy is a mixture of many chemicals in unknown doses and might result in unpleasant side effects , many people believe that treatments that are natural are somehow magically safe and effective . \n aristolochia is used in traditional medicine for the treatment of various diseases [ 13 , 15 ] , including those associated with bacteria .", "this study showed clearly that the excellent effect of aristolochia in treating such diseases is due to the toxic compound aa-1 .", "although aa-1 is highly effective in killing m. catarrhalis , it is ineffective against the other microorganisms tested .", "this highlights the importance of m. catarrhalis in discovering the cellular target of aa-1 and the mechanism of aa-1 toxicity .", "the widespread use of aristolochia is not sufficient to ensure that it is effective or even that it is safe .", "therefore , hit - to - lead exploration is necessary to identify related compounds with low toxicity , low cost , and improved potency that can replace the use of the harmful crude plant material .", "it is impossible to ban the use of these remedies , especially in the rural areas in sudan and other african countries ; therefore , we strongly recommend educating the public of the risks versus the benefits of aristolochia and gradually replacing them with either economical new drugs or standardized extracts and homogenous batches of other plant material with known levels of safe active constituents .", "using bioassay - guided fractionation technique , the present study directly linked the antibacterial activity of aristolochia bracteolata to the aa-1 .", "although aa-1 had strong activity against m. catarrhalis , it had a narrow spectrum of activity than expected based on the activity of the crude extract from which it was isolated or from its traditional usage .", "this may be the result of synergism between different compounds in the complex extracts or may be due to placebo effect ."], "abstract_text": ["<S> a bioassay - guided fractionation of methanol extract of aristolochia bracteolata whole plant was carried out in order to evaluate its antimicrobial activity and to identify the active compounds in this extract . </S>", "<S> antibacterial and antifungal activities of methanol extract against gram - positive , gram - negative , and fungal strains were investigated by the agar disk diffusion method . among the strains tested , moraxella catarrhalis and sea urchin - derived bacillus sp . </S>", "<S> showed the highest sensitivity towards the methanol extract and hence they are used as test organisms for the bioassay - guided fractionation . from this </S>", "<S> extract , aristolochic acid 1 ( aa-1 ) has been isolated and has showed the greatest antibacterial activity against both standard strain and clinical isolates of moraxella catarrhalis with equal minimum inhibitory concentration ( mic ) and minimum bactericidal concentration ( mbc ) values of 25 and 50  g / ml . modification of the aa-1 to aa-1 methyl ester completely abolished the antibacterial activity of the compound and the piperonylic acid moiety of aa-1 which suggested that the coexistence of phenanthrene ring and free carboxylic acid is essential for aa-1 antibacterial activity . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusion"], "sections": [["\n moraxella catarrhalis is a gram - negative , aerobic diplococcus human mucosal pathogen which causes middle ear infections in infants and children [ 13 ] , and it is one of the three major causes of otitis media along with streptococcus pneumonia and haemophilus influenzae . although moraxella catarrhalis is frequently found as a commensal of the upper respiratory tract , recently it has emerged as a genuine pathogen and is now considered an important cause of upper respiratory tract infections in healthy children and elderly people , lower respiratory tract infections in adults with chronic obstructive pulmonary disease [ 1 , 5 ] , and hospital - acquired pneumonia .", "amikacin , cefixime , fosfomycin , cefuroxime , cotrimoxazole , doxycycline , and erythromycin resistant strains of moraxella catarrhalis were isolated and the widespread production of a -1actamase enzyme renders the bacterium resistant to the penicillin [ 79 ] .", "this has led to the search for new and effective therapeutic alternatives among natural compounds .", "plants remain an important source of diverse chemical entities which have been used as drugs or provide scaffolds from which new drugs have been derived .", "the selection of a suitable candidate species for investigations can be done on the basis of long - term use by humans .", "this approach is based on an assumption that the active compounds isolated from such plants are likely to be safer than those derived from plant species with no history of human use .", "aristolochia is an important genus in the family of aristolochiaceae and is widespread across tropical asia , africa , and south america .", "it is used in traditional medicine as a gastric stimulant and in the treatment of cancer , lung inflammation , dysentery , and snakebites .", "it is also used in the treatment of tumors and malaria and for fevers , but its usage as an antimalarial is not recommended in its crude form .", "aristolochia bracteolata showed a definite positive effect on wound healing , with significant increase in the level of powerful antioxidant enzymes .", "the whole plant was used as a purgative , antipyretic , and anti - inflammatory .", "organic solvent extracts of the plant showed antibacterial activities while the water extract showed antifungal activity .", "although aristolochia has been used for thousands of years in many cultures for many indications due to its various pharmacological activities , it was later discovered that consuming these plants can certainly be dangerous .", "the genus of aristolochia contains a naturally carcinogenic compound aa which has been shown to be the cause of so - called chinese herb nephropathy or aa nephropathy [ 19 , 20 ] , and mutations in the cells of people who consume it , causes more mutations than two of the best - known environmental carcinogens : tobacco smoke and uv light [ 21 , 22 ] .", "there are many cases of nephropathy reported in the literature caused by the systemic and long term application of chinese snakeroot ( aristolochia fangchi ) ; this highlighted the risk of using preparations which contain aristolochic acids .", "although aristolochia is being known in many countries that is containing a toxic compound aa , but this has not stopped it from being a popular herbal remedy for thousands of years .", "it is still extensively used in india and in traditional chinese medicine for slimming , menstrual symptoms , and rheumatism .", "it is also widespread used in sudan and other african countries as one of the most effective herbal remedies for infectious diseases .", "therefore , it was our objective to assess the potential antimicrobial activity of aristolochia bracteolata using a bioassay - guided fractionation , in order to produce pure compound that can act as the lead compound in developing new , safe , and effective drug to replace the use of the harmful crude plant material ."], ["sephadex lh-20 ( pharmacia fine chemical co. ltd ) was used for column chromatography . precoated silica gel plates ( merck , kieselgel 60 f254 , 0.25  mm ) and precoated rp-18 f254s plates ( merck ) were used for thin - layer chromatography ( tlc ) analysis .", "high resolution fab - ms and esi - ms were recorded on jeol jms700n and jms-100td , respectively .", "h- and c - nmr , h - h cosy , noesy , hsqc , and hmbc spectra were recorded with a unity plus 500 spectrometer ( varian inc . , u.s.a . ) operating at 500  mhz for h and 125  mhz for c , respectively .", "h - nmr chemicals shifts are expressed in  values referring to the solvent peak h 2.49 for dmso and coupling constants are expressed in hz .", "c - nmr chemical shifts are expressed in  values referring to the solvent peak c 39.5 for dmso .", "piperonylic acid was purchased from commercial sources ( tci ) and used without further purification . the plant material ( whole )", "was collected in the period from ( october to december 2012 ) from khartoum state in sudan .", "standard strains : moraxella catarrhalis ( gtc 01544 ) , klebsiella pneumoniae ( atcc 13883 ) , escherichia coli ( k12 ) , salmonella typhimurium ( atcc 14028 ) , streptococcus pyogenes ( atcc 19615 ) , streptococcus agalactiae ( atcc 13813 ) , staphylococcus epidermidis ( atcc 12228 ) , neisseria lactamicus ( atcc 23970 ) , enterobacter cloacae , ( atcc 23355 ) , bacillus subtilis ( atcc 6633 ) , staphylococcus aureus ( 209p ) , and pseudomonas aeruginosa ( ifo 3445).clinical strains : moraxella catarrhalis , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica . \n  standard strains : moraxella catarrhalis ( gtc 01544 ) , klebsiella pneumoniae ( atcc 13883 ) , escherichia coli ( k12 ) , salmonella typhimurium ( atcc 14028 ) , streptococcus pyogenes ( atcc 19615 ) , streptococcus agalactiae ( atcc 13813 ) , staphylococcus epidermidis ( atcc 12228 ) , neisseria lactamicus ( atcc 23970 ) , enterobacter cloacae , ( atcc 23355 ) , bacillus subtilis ( atcc 6633 ) , staphylococcus aureus ( 209p ) , and pseudomonas aeruginosa ( ifo 3445 ) . clinical strains : moraxella catarrhalis , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica .", "in addition to a sea urchin ( anthocidaris crassispina ) derived bacillus sp . which obtained from the laboratory in medical plants garden , nagasaki university.(c)fungal strains : the fungal strains used were aspergillus niger ( nbrc 33023 ) , penicillium crustosum ( nbrc 33015 ) , schizophyllum commune ( nbrc 30749 ) , trichophyton concentricum ( nbrc 31068 ) , and candida albicans ( nbrc 10108 ) .", "fungal strains : the fungal strains used were aspergillus niger ( nbrc 33023 ) , penicillium crustosum ( nbrc 33015 ) , schizophyllum commune ( nbrc 30749 ) , trichophyton concentricum ( nbrc 31068 ) , and candida albicans ( nbrc 10108 ) .", "the air - dried powdered whole plant ( 200  g ) was exhaustively extracted with cold maceration method with sufficient quantity of 70% methanol for 7 days at room temperature .", "the methanolic extract was passed through whatman number 1 filter paper ( whatman england ) and the concentrated extract ( 40  g ) was digested with 100  ml distilled water and successively partitioned with n - hexane ( 4  400  ml ) , chloroform ( 3  400  ml ) , ethyl acetate ( 5  400  ml ) , and n - butanol ( 2  400  ml ) .", "each fraction was concentrated under reduced pressure to a constant weight to give the corresponding n - hexane fraction ( 0.4  g ) , chloroform fraction ( 2  g ) , ethyl acetate fraction ( 0.7  g ) , n - butanol fraction ( 6  g ) , and aqueous fraction ( 30  g ) . the most active fraction against bacillus sp . and", "m. catarrhalis ( chloroform fraction ) was subjected to sephadex lh20 column chromatography to give three subfractions ( a - c ) .", "fraction ( b ) was resubjected again to sephadex lh20 to afford very active and pure compound aa-1 ( 150  mg ) . to the solution of aa-1 ( 50  mg ; 0.23  mmol ) in dimethylformamide ( dmf )", ", 1  ml potassium carbonate was added ( 95  mg ; 0.69  mmol ) . to the resulting suspension iodomethane", "was added ( 72  l ; 1.15  mmol ) and stirred for 8 hours .", "the reaction mixture was poured onto water ( 10  ml ) and ethyl acetate ( 20  ml ) .", "the organic layer was washed with 1  m hcl ( 10  ml ) three times and then with brine ( 10  ml ) once .", "after removal of solvent under reduced pressure , the residue was purified by silica - gel chromatography ( chloroform - methanol ) to afford the titled ester ( 88% ) .", "h - nmr ( 400  mhz , cdcl3 , tms , r.t . )  ( ppm ) : 3.88 ( 3h , s ) , 4.06 ( 3hs ) , 6.38 ( 2h , s ) , 7.11 ( 1h , d , j = 7.8  hz ) , 7.72 ( 1h , dd , j = 7.8  hz , 8.0  hz ) , 7.77 ( 1h , s ) , 8.70 ( 1h , d , j = 8.0  hz ) , and 8.83 ( 1h , s ) .", "suspension of the tested bacteria ( 100  l of 10  cfu / ml ) was spread onto solid media plates .", "the sterile paper discs ( 6  mm in diameter ) which were impregnated with the plant extract ( 14  mg ) and pure compound ( 10100  g ) were placed aseptically over the bacterial culture on nutrient agar plates .", "after incubation at 37c for 24 hours , the zone of inhibition around the discs was measured by millimeter scale .", "sterile , blank paper discs impregnated with only sterile solvents served as negative control each time .", "the sterile paper discs ( 6  mm in diameter ) which were impregnated with individual extract were placed on the inoculated plates .", "these plates were incubated for 2472  h at 2528c and the growth was evaluated visually by comparing a particular plate with the negative control plates ( having only test fungi ) .", "the antifungal activity was evaluated by measuring the inhibition zone diameter ( in millimeter ) observed .", "the mic and mbc values were determined by broth dilution method in accordance with clsi methodology .", "bacterial strains were cultured for 24  h at 37c on nutrient broth and then suspended in sterile distilled water to give a final inoculum concentration of 1.5  1.0  10  cfu / ml .", "dilutions ranging from 1.56 to 400  g / ml of the compound were prepared in tubes including broth and dmso 10% ( v / v ) , in addition to one negative control ( broth + dmso 10% v / v + test microorganism ) to ensure that the final concentration of dmso in the assays did not interfere with the bacterial growth and one sterility control ( broth + dmso 10% v / v + test compound ) .", "a 100  l suspension of test microorganism was added to individual tubes and incubated at 37c for 24  h. the mic of the compound was defined as the lowest concentration that inhibited the visible bacterial growth and the mbc was defined as the lowest concentration that prevented the growth of the organism after subculture onto antibiotic - free plates ."], ["initial steps in new drug discovery involve identification of new chemical entities , which can be either sourced through chemical synthesis or can be isolated from natural products through biological activity guided fractionation .", "bioassay - guided fractionation of the identified plant may lead to standardized extract or isolated bioactive lead compounds as the new drug .", "the whole plant of aristolochia bracteolata was extracted successively with meoh and subjected to liquid - liquid fractionation with n - hexane , chloroform , ethyl acetate , and n - butanol .", "the resulting fractions were tested for antibacterial and antifungal activities . the crude extract and chloroform fraction were significantly active against sea urchin - derived bacillus sp . and both standard strain and clinical isolates of moraxella catarrhalis and were moderately active against s. aureus , b. subtilis , and ps .", "the n - hexane fraction had moderate activity against s. aureus and b. subtilis while ethyl acetate fraction showed moderate activity against ps .", "the crude extract failed to inhibit the growth of all test fungi in addition to the following bacterial strains : klebsiella pneumoniae , escherichia coli , salmonella typhimurium , streptococcus pyogenes , streptococcus agalactiae , staphylococcus epidermidis , neisseria lactamicus , enterobacter cloacae , bacillus cereus , aeromonas hydrophila , salmonella typhi , vibrio cholerae , and yersinia enterocolitica .  ", "the chloroform soluble fraction was therefore selected for further chromatographic separations and resulted in the isolation of known compound aa-1 ( figure 1 ) .", "aa-1 showed strong activity against moraxella catarrhalis ( standard strain and clinical isolates ) and sea urchin - derived bacillus sp .", "( table 2 ) , with equal mic and mbc values of 25 and 50  g / ml . both the piperonylic acid moiety of aa-1 ( figure 1 ) and aa-1-methyl ester showed no activity against bacteria ( table 2 ) , which suggests that the coexistence of phenanthrene ring and free carboxylic acid is essential for aa-1 antibacterial activity .", "bioguided fractionation of methanolic extract of aristolochia bracteolata led to isolation of aa-1 and its structure was elucidated by interpretation of its nmr and ms data and by comparison with those reported in the literature .", "electrospray ionization mass spectrometry ( esims ) showed pseudomolecular ion signal at m / z 364.03 [ m + na ] and high resolution fast atom bombardment mass spectrometry ( hr - fabms ) afforded m+1 ion signal at m / z 342.0620 which was corresponding to the molecular formula c17h12no7 ( calculated for 342.06138 ) .", "h nmr and c - nmr spectra were matched with those of aa-1 which were previously reported .", "two weak signals which did not show any correlation with proton in hsqc and hmbc spectroscopy were considered as quaternary carbons at positions 5 (  124.3  ppm ) and 6 (  143.5  ppm ) , respectively .", "laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all types of microorganisms in vitro .", "more of these compounds should be subjected to animal and human studies to determine their effectiveness in whole - organism systems , including in particular toxicity studies and an examination of their effects on beneficial normal microbiota . in spite of", "the fact that herbal remedy is a mixture of many chemicals in unknown doses and might result in unpleasant side effects , many people believe that treatments that are natural are somehow magically safe and effective . \n aristolochia is used in traditional medicine for the treatment of various diseases [ 13 , 15 ] , including those associated with bacteria .", "this study showed clearly that the excellent effect of aristolochia in treating such diseases is due to the toxic compound aa-1 .", "although aa-1 is highly effective in killing m. catarrhalis , it is ineffective against the other microorganisms tested .", "this highlights the importance of m. catarrhalis in discovering the cellular target of aa-1 and the mechanism of aa-1 toxicity .", "the widespread use of aristolochia is not sufficient to ensure that it is effective or even that it is safe .", "therefore , hit - to - lead exploration is necessary to identify related compounds with low toxicity , low cost , and improved potency that can replace the use of the harmful crude plant material .", "it is impossible to ban the use of these remedies , especially in the rural areas in sudan and other african countries ; therefore , we strongly recommend educating the public of the risks versus the benefits of aristolochia and gradually replacing them with either economical new drugs or standardized extracts and homogenous batches of other plant material with known levels of safe active constituents ."], ["using bioassay - guided fractionation technique , the present study directly linked the antibacterial activity of aristolochia bracteolata to the aa-1 .", "although aa-1 had strong activity against m. catarrhalis , it had a narrow spectrum of activity than expected based on the activity of the crude extract from which it was isolated or from its traditional usage .", "this may be the result of synergism between different compounds in the complex extracts or may be due to placebo effect ."]]}
{"article_id": "PMC5216144", "article_text": ["anti - neutrophil cytoplasmic antibodies ( ancas ) correlate well with a wide spectrum of vasculitis manifestations , including wegener 's granulomatosis , microscopic polyangiitis , and churg - strauss syndrome , all of which are commonly referred to as anca - associated vasculitis ( 1 ) .", "several infections , particularly infective endocarditis , have been reported to show positive findings on anca tests ; furthermore , their clinical features have been acknowledged to mimic anca - associated vasculitis , which may lead to a misdiagnosis and inappropriate treatment ( 2,3 ) .", "however , to our knowledge , infected aortic aneurysms have not been reported to show positive findings on anca tests .", "we herein report a patient with an infected thoracic aortic aneurysm mimicking anti - proteinase 3-antineutrophil cytoplasmic antibody ( pr3-anca)-associated vasculitis .", "vegetations in the descending aorta , which were detected using transesophageal echocardiography , contributed to the diagnosis in this case .", "a woman in her 60s was admitted to the nagoya city university hospital complaining of a fever , which had persisted for three weeks , along with a loss of appetite , proteinuria , hematuria , and renal dysfunction .", "her renal function test values had been in the normal range three months prior to the admission .", "she also had a history of orbital mucosa - associated lymphoid tissue lymphoma , which was in remission after treatment with only radiation therapy .", "a physical examination at the time of admission revealed a body temperature of 38.1 , pulse rate 109 beats / min , and blood pressure 104/53 mmhg .", "the laboratory tests revealed a white blood cell count of 18,900/mm , hemoglobin 6.6 g / dl , blood urea nitrogen 30.8 mg / dl , serum creatinine 2.24 mg / dl , and c - reactive protein ( crp ) 9.44 mg / dl ( table ) .", "the pr3-anca titer was 47.4 iu / ml ( normal range < 10 iu / ml ) , although her myeloperoxidase - antineutrophil cytoplasmic antibody ( mpo - anca ) titer was not elevated .", "a urinalysis gave results of 2 + for protein and 3 + for occult blood in a dipstick examination , and a microscopic examination detected more than 100 red blood cells per high - power field .", "alt : alanine aminotransferase , ana : antinuclear antibodies , ast : aspartate transaminase , c3 : compliment 3 , c4 : compliment 4 , ch50 : 50% hemolytic unit of complement , crp : c - reactive protein , dna : deoxyribonucleic acid , ldh : lactate dehydrogenase , mpo - anca : myeloperoxidase - antineutrophil cytoplasmic antibody , rf : rheumatic factor , pr3-anca : anti - proteinase 3-antineutrophil cytoplasmic antibody she was initially diagnosed with pr3-anca - associated vasculitis and was thereafter treated with methylprednisolone pulse therapy ( 500 mg / day ) for three days ( fig .", "however , streptococcus sanguis was serially detected in blood cultures ; therefore , the administration of steroids was stopped .", "transesophageal echocardiography also revealed no vegetation in the heart ; however , it showed many oscillating masses attached to the intima in the descending aorta ( fig .", "2 ) . contrast - enhanced computed tomography ( ct ) revealed a new descending aortic aneurysm ( fig .", "effects of antibiotics on fever and c - reactive protein during hospitalization . the patient finally became afebrile and has c - reactive protein levels within the normal range after receiving antibiotics .", "crp : c - reactive protein , dap : daptomycin , pcg : penicillin g , taz / pipc : tazobactam / piperacillin , tx : therapy transesophageal echocardiographic images of the descending aorta , with sectioning planes at 90 ( a ) and 0 ( b ) .", "many oscillating masses attached to the intima were seen in the lumen of the descending aorta .", "contrast - enhanced computed tomographic images in the axial ( a ) and frontal ( b ) sections .", "these images show a descending aortic pseudoaneurysm and a contrast defect ( arrow ) beside the aneurysm that is compatible with echo - documented vegetation .", "based on the results of microbiological testing , antibiotic treatment with intravenous penicillin g ( 24,000,000 unit / day ) was started , after which she became afebrile .", "we added intravenous daptomycin ( 300 mg every 24 hours ) and tazobactam / piperacillin ( 2.25 g every 6 hours ) to the penicillin g because recurrent fever was observed two weeks later .", "this additional antibiotic treatment made her afebrile again , and her renal function was recovered ( fig .", ", she developed a janeway lesion , which was confirmed by the pathological finding of a skin biopsy specimen ( fig .", "4 ) . transesophageal echocardiography finally demonstrated that the vegetation had disappeared after the antibiotic treatment ( fig .", "5 ) , but contrast - enhanced ct revealed that the aneurysm remained unchanged in form . a nontender hemorrhagic macular on the sole of the foot ( a ) .", "photomicrograph of the macular lesion shows microembolization with fibrin and infiltration of neutrophilic cells hematoxylin and eosin staining ( b , c ) .", "a transesophageal echocardiographic image of the descending aorta after the antibiotic treatment , with the sectioning plane at 90. many oscillating masses have disappeared . on the 54th hospital day , she underwent endovascular aortic repair ( evar ) of the aneurysm ( fig .", "6 ) after confirmation that her crp level was in the normal range and once blood cultures were consistently negative .", "one year after the procedure , she continues to take oral antibiotics ; no complications related to stent - graft deployment or recurrent infections have been encountered .", "her pr3-anca titer has normalized . a contrast - enhanced computed tomographic image after endovascular stent graft repair .", "in this case , an infected aortic aneurysm exhibited elevated the patient 's pr3-anca level , and its clinical course mimicked pr3-anca - associated vasculitis .", "the detection of ancas is highly specific for a diagnosis of anca - associated vasculitis ( 4 ) ; however , a number of infections can result in a positive anca test .", "there are several reports of infective endocarditis with the presence of ancas that mimic the clinical manifestations of an anca - associated vasculitis such as glomerulonephritis ( 2,3 ) . in the present case , based on the finding of positive blood cultures", ", we suspected that the patient was experiencing not anca - associated vasculitis but infective endocarditis .", "however , no vegetations were detected in the cardiac chambers using transthoracic echocardiography or transesophageal echocardiography .", "instead , contrast - enhanced ct revealed a pseudoaneurysm in the descending aorta , which suggested that the patient had an infected aneurysm .", "her renal dysfunction and elevated pr3-anca levels normalized with clinical resolution of the infected aneurysm after the administration of appropriate antibiotic therapy and evar .", "the patient has remained free of any evidence of systemic vasculitis during follow - up .", "the presence of pr3-anca may have been falsely positive in the present patient . to our knowledge , this is the first report of an infected aortic aneurysm with a positive pr3-anca test and clinical features mimicking anca - associated vasculitis .", "the presence of vegetations in the heart is one of the most characteristic findings of infective endocarditis .", "these vegetations are composed of bacteria , platelets , and inflammatory cells interspersed with fibrin mesh , either on damaged endocardium , including native cardiac valves , or prosthetic valves ( 5 ) . in the present case ,", "because these masses disappeared after antibiotic treatment , they were considered to be vegetations . a pathological examination of the macular hemorrhages on the patient 's sole indicated not a cholesterol embolization but rather a janeway lesion , which was the consequence of septic embolic events .", "we therefore suspected that these masses were not mobile plaques but bacterial vegetations . to our knowledge ,", "few cases of infected native aortic aneurysm manifesting vegetations in the aneurysm have been reported ( 6 - 8 ) .", "several mechanisms have been proposed regarding how ancas are formed during the course of infection , such as autoantigen complementarity , molecular mimicry , epigenetics , neutrophil extracellular traps , and microbial - sensing proteins called toll - like receptors ( 9 ) . there may be a great degree of overlap in the clinical and laboratory manifestations between anca - associated vasculitis and anca - positive infected aortic aneurysm , potentially leading to a misdiagnosis .", "it is important to correctly diagnose these cases in order to avoid administering inappropriate therapy , such as immunosuppressive treatment , which might have life - threatening consequences in a patient with an infected aortic aneurysm .", "positive blood cultures and imaging findings , such as a pseudoaneurysm and/or vegetations in the aorta , suggest the presence of an infected aortic aneurysm . a combination of conventional surgical treatment with resection of the aneurysm , extensive local debridement , and revascularization by in situ reconstruction or extra - anatomic bypass may be the gold standard , but such a treatment strategy is accompanied by a high mortality and morbidity . in the present case ,", "it was recently reported that evar of an infected aortic aneurysm is feasible and , for most patients , a durable treatment option ( 10 ) .", "given that the continuous administration of antibiotics for the infected aneurysm should prevent a recurrence of local infection , we continued using antibiotics until the infection was controlled and carefully monitored the shape of the pseudoaneurysm on contrast - enhanced ct .", "we were ready to perform an emergency surgical correction at any point during this patient 's treatment course . in conclusion", ", we herein reported an unusual case of infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation and with serological findings and clinical features mimicking pr3-anca - associated vasculitis ."], "abstract_text": ["<S> we herein report an unusual case of an infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation with serological findings and clinical features mimicking anti - proteinase 3-antineutrophil cytoplasmic antibody - associated vasculitis . the positive blood cultures and imaging findings , including a pseudoaneurysm and vegetations in the aorta , suggested the presence of an infected aortic aneurysm . </S>", "<S> the patient was successfully treated with antibiotics and endovascular aortic repair . </S>", "<S> a precise diagnosis is crucial in order to avoid inappropriate therapy such as immunosuppressive treatment , which could result in life - threatening consequences in a patient with an infected aortic aneurysm . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "The authors state that they have no Conflict of Interest (COI)."], "sections": [["anti - neutrophil cytoplasmic antibodies ( ancas ) correlate well with a wide spectrum of vasculitis manifestations , including wegener 's granulomatosis , microscopic polyangiitis , and churg - strauss syndrome , all of which are commonly referred to as anca - associated vasculitis ( 1 ) .", "several infections , particularly infective endocarditis , have been reported to show positive findings on anca tests ; furthermore , their clinical features have been acknowledged to mimic anca - associated vasculitis , which may lead to a misdiagnosis and inappropriate treatment ( 2,3 ) .", "however , to our knowledge , infected aortic aneurysms have not been reported to show positive findings on anca tests .", "we herein report a patient with an infected thoracic aortic aneurysm mimicking anti - proteinase 3-antineutrophil cytoplasmic antibody ( pr3-anca)-associated vasculitis .", "vegetations in the descending aorta , which were detected using transesophageal echocardiography , contributed to the diagnosis in this case ."], ["a woman in her 60s was admitted to the nagoya city university hospital complaining of a fever , which had persisted for three weeks , along with a loss of appetite , proteinuria , hematuria , and renal dysfunction .", "her renal function test values had been in the normal range three months prior to the admission .", "she also had a history of orbital mucosa - associated lymphoid tissue lymphoma , which was in remission after treatment with only radiation therapy .", "a physical examination at the time of admission revealed a body temperature of 38.1 , pulse rate 109 beats / min , and blood pressure 104/53 mmhg .", "the laboratory tests revealed a white blood cell count of 18,900/mm , hemoglobin 6.6 g / dl , blood urea nitrogen 30.8 mg / dl , serum creatinine 2.24 mg / dl , and c - reactive protein ( crp ) 9.44 mg / dl ( table ) .", "the pr3-anca titer was 47.4 iu / ml ( normal range < 10 iu / ml ) , although her myeloperoxidase - antineutrophil cytoplasmic antibody ( mpo - anca ) titer was not elevated .", "a urinalysis gave results of 2 + for protein and 3 + for occult blood in a dipstick examination , and a microscopic examination detected more than 100 red blood cells per high - power field .", "alt : alanine aminotransferase , ana : antinuclear antibodies , ast : aspartate transaminase , c3 : compliment 3 , c4 : compliment 4 , ch50 : 50% hemolytic unit of complement , crp : c - reactive protein , dna : deoxyribonucleic acid , ldh : lactate dehydrogenase , mpo - anca : myeloperoxidase - antineutrophil cytoplasmic antibody , rf : rheumatic factor , pr3-anca : anti - proteinase 3-antineutrophil cytoplasmic antibody she was initially diagnosed with pr3-anca - associated vasculitis and was thereafter treated with methylprednisolone pulse therapy ( 500 mg / day ) for three days ( fig .", "however , streptococcus sanguis was serially detected in blood cultures ; therefore , the administration of steroids was stopped .", "transesophageal echocardiography also revealed no vegetation in the heart ; however , it showed many oscillating masses attached to the intima in the descending aorta ( fig .", "2 ) . contrast - enhanced computed tomography ( ct ) revealed a new descending aortic aneurysm ( fig .", "effects of antibiotics on fever and c - reactive protein during hospitalization . the patient finally became afebrile and has c - reactive protein levels within the normal range after receiving antibiotics .", "crp : c - reactive protein , dap : daptomycin , pcg : penicillin g , taz / pipc : tazobactam / piperacillin , tx : therapy transesophageal echocardiographic images of the descending aorta , with sectioning planes at 90 ( a ) and 0 ( b ) .", "many oscillating masses attached to the intima were seen in the lumen of the descending aorta .", "contrast - enhanced computed tomographic images in the axial ( a ) and frontal ( b ) sections .", "these images show a descending aortic pseudoaneurysm and a contrast defect ( arrow ) beside the aneurysm that is compatible with echo - documented vegetation .", "based on the results of microbiological testing , antibiotic treatment with intravenous penicillin g ( 24,000,000 unit / day ) was started , after which she became afebrile .", "we added intravenous daptomycin ( 300 mg every 24 hours ) and tazobactam / piperacillin ( 2.25 g every 6 hours ) to the penicillin g because recurrent fever was observed two weeks later .", "this additional antibiotic treatment made her afebrile again , and her renal function was recovered ( fig .", ", she developed a janeway lesion , which was confirmed by the pathological finding of a skin biopsy specimen ( fig .", "4 ) . transesophageal echocardiography finally demonstrated that the vegetation had disappeared after the antibiotic treatment ( fig .", "5 ) , but contrast - enhanced ct revealed that the aneurysm remained unchanged in form . a nontender hemorrhagic macular on the sole of the foot ( a ) .", "photomicrograph of the macular lesion shows microembolization with fibrin and infiltration of neutrophilic cells hematoxylin and eosin staining ( b , c ) .", "a transesophageal echocardiographic image of the descending aorta after the antibiotic treatment , with the sectioning plane at 90. many oscillating masses have disappeared . on the 54th hospital day , she underwent endovascular aortic repair ( evar ) of the aneurysm ( fig .", "6 ) after confirmation that her crp level was in the normal range and once blood cultures were consistently negative .", "one year after the procedure , she continues to take oral antibiotics ; no complications related to stent - graft deployment or recurrent infections have been encountered .", "her pr3-anca titer has normalized . a contrast - enhanced computed tomographic image after endovascular stent graft repair ."], ["in this case , an infected aortic aneurysm exhibited elevated the patient 's pr3-anca level , and its clinical course mimicked pr3-anca - associated vasculitis .", "the detection of ancas is highly specific for a diagnosis of anca - associated vasculitis ( 4 ) ; however , a number of infections can result in a positive anca test .", "there are several reports of infective endocarditis with the presence of ancas that mimic the clinical manifestations of an anca - associated vasculitis such as glomerulonephritis ( 2,3 ) . in the present case , based on the finding of positive blood cultures", ", we suspected that the patient was experiencing not anca - associated vasculitis but infective endocarditis .", "however , no vegetations were detected in the cardiac chambers using transthoracic echocardiography or transesophageal echocardiography .", "instead , contrast - enhanced ct revealed a pseudoaneurysm in the descending aorta , which suggested that the patient had an infected aneurysm .", "her renal dysfunction and elevated pr3-anca levels normalized with clinical resolution of the infected aneurysm after the administration of appropriate antibiotic therapy and evar .", "the patient has remained free of any evidence of systemic vasculitis during follow - up .", "the presence of pr3-anca may have been falsely positive in the present patient . to our knowledge , this is the first report of an infected aortic aneurysm with a positive pr3-anca test and clinical features mimicking anca - associated vasculitis .", "the presence of vegetations in the heart is one of the most characteristic findings of infective endocarditis .", "these vegetations are composed of bacteria , platelets , and inflammatory cells interspersed with fibrin mesh , either on damaged endocardium , including native cardiac valves , or prosthetic valves ( 5 ) . in the present case ,", "because these masses disappeared after antibiotic treatment , they were considered to be vegetations . a pathological examination of the macular hemorrhages on the patient 's sole indicated not a cholesterol embolization but rather a janeway lesion , which was the consequence of septic embolic events .", "we therefore suspected that these masses were not mobile plaques but bacterial vegetations . to our knowledge ,", "few cases of infected native aortic aneurysm manifesting vegetations in the aneurysm have been reported ( 6 - 8 ) .", "several mechanisms have been proposed regarding how ancas are formed during the course of infection , such as autoantigen complementarity , molecular mimicry , epigenetics , neutrophil extracellular traps , and microbial - sensing proteins called toll - like receptors ( 9 ) . there may be a great degree of overlap in the clinical and laboratory manifestations between anca - associated vasculitis and anca - positive infected aortic aneurysm , potentially leading to a misdiagnosis .", "it is important to correctly diagnose these cases in order to avoid administering inappropriate therapy , such as immunosuppressive treatment , which might have life - threatening consequences in a patient with an infected aortic aneurysm .", "positive blood cultures and imaging findings , such as a pseudoaneurysm and/or vegetations in the aorta , suggest the presence of an infected aortic aneurysm . a combination of conventional surgical treatment with resection of the aneurysm , extensive local debridement , and revascularization by in situ reconstruction or extra - anatomic bypass may be the gold standard , but such a treatment strategy is accompanied by a high mortality and morbidity . in the present case ,", "it was recently reported that evar of an infected aortic aneurysm is feasible and , for most patients , a durable treatment option ( 10 ) .", "given that the continuous administration of antibiotics for the infected aneurysm should prevent a recurrence of local infection , we continued using antibiotics until the infection was controlled and carefully monitored the shape of the pseudoaneurysm on contrast - enhanced ct .", "we were ready to perform an emergency surgical correction at any point during this patient 's treatment course . in conclusion", ", we herein reported an unusual case of infected descending aortic pseudoaneurysm with luminal pathognomonic oscillating vegetation and with serological findings and clinical features mimicking pr3-anca - associated vasculitis ."], [""]]}
{"article_id": "PMC5189720", "article_text": ["endometriosis is a prevalent cause of infertility , pelvic pain , dysmenorrhea , and dyspareunia in reproductive - age women .", "its diagnosis is by inspection of the pelvis during laparoscopy . in women with pelvic pain and infertility ,", "endometriosis mimics some autoimmune and malignant diseases , including familial occurrence and immunological abnormalities in b and t cells , increased angiogenesis , invasion of endometrial cells to adjacent organs ( e.g. , bladder and bowels ) , and need for repeat surgeries due to recurrence [ 1,57 ] .", "multiple mechanisms for etiology and improvement of endometriosis are suggested and the treatments are based on these unclear mechanisms . therefore , progestin , gnrh agonists and antagonists , and drugs related to lipid metabolism ( e.g. , simvastatin ) are used for treatment .", "it has been shown that inflammation is important in the pathogenesis of endometriosis , so endometriosis treatment should not be different from that of other inflammatory disorders", ". there may be a correlation between vitamin d levels and the risk of polycystic ovarian disease , endometriosis , breast and ovarian cancer , increased arterial stiffness in older patients , and myasthenia gravis [ 1218 ] .", "it has been found that vitamin d has a role in normal cellular growth regulation .", "vitamin d increases anti - inflammatory cytokines production and decreases pro - inflammatory cytokines [ 1922 ] .", "vitamin d induces apoptosis and suppression of angiogenesis in vitro and in vivo [ 2528 ] .", "although an indirect relationship between vitamin d and endometriosis has been reported in multiple studies , here has been no published randomized clinical trial on endometriosis and vitamin d treatment in women .", "we explored the relationship between vitamin d and endometriosis in a double - blind , randomized clinical trial looking at the effect of vitamin d supplementation on cessation of pain in proven endometriosis after laparoscopic diagnosis and treatment .", "this randomized , double - blind clinical trial was performed in a single tertiary university hospital from nov 2014 to feb 2016 . to find patients with endometriosis , we did laparoscopy for various indications , including ovarian cyst , infertility , pelvic pain , and dysmenorrhea .", "using laparoscopy , the surgeons diagnosed patients with endometriosis and tried to excise or ablate all diseased tissue . the day before laparoscopy", ", a data collection form was completed by a physician , including the reason for laparoscopy , and the severity of pelvic pain and dysmenorrhea were estimated using a visual analogue scale test ( vas test ) , with a score of 0 being no pain and a score 10 being the worst pain ever experienced .", "the laparoscopies were done by 2 gynecologic laparoscopic surgeons ; both were involved in each operation and they recorded the severity of endometriosis according to the revised american society for reproductive medicine ( asrm ) classification . in the patients with endometriosis in the second menses after laparoscopic diagnosis and treatment , the vas test was repeated .", "patients with vas scores of at least 3 for dysmenorrhea and/or pelvic pain were invited to participate in this clinical trial , and after consultation we asked them to sign the informed consent .", "the dyspareunia score was not a criterion for entering the study because some of the patients were not married and had not had intercourse .", "women aged 1540 years with proven endometriosis by laparoscopy and a vas test score of 3 or more for dysmenorrhea and/or pelvic pain at second menses after operative laparoscopy .", "patients with vitamin d treatment in the last 6 months prior to surgery ; patients with known systemic diseases ( e.g. , hypertension , diabetes , coronary , renal , and hepatic diseases ) ; patients with known malignancy ; patients with hormonal treatment , including oral contraceptive pills , in the last 6 months . after authorization by the university ethics committee ,", "eligible patients were assigned by simple randomization to receive either vitamin d or placebo . in the vitamin d group ( d group ) , we prescribed oral vitamin d 50 000 iu / weekly for 12 weeks ( capsule d - vigel , vitamin d3 50 000 iu , daana pharma co. tabriz - iran ) and in the placebo group ( p group ) we prescribed 1 capsule of placebo ( daana pharma co. tabriz - iran ) weekly for 12 weeks .", "four weeks after the end of the intervention ( 24 weeks after surgical treatment ) , the vas test was repeated for the 2 groups .", "we used the ks test ( one - sample kolmogorov - smirnov test ) for normality of data distribution , levine s test for equality of variances and independent samples , and the t test for equality of means for comparing quantitative normal data between the 2 groups .", "we used the paired - samples t test for comparing quantitative normal data between before and after treatment in each group and the pearson chi - square test for matching and comparing categorical variables between the 2 groups . due to the small sample size", ", we conducted mann - whitney non - parametric analysis between the 2 groups and wilcoxon signed ranks test for comparing before and after treatment data in each group .", "this study was funded and supported by iran university of medical sciences ( iums ) , grant no .", "women aged 1540 years with proven endometriosis by laparoscopy and a vas test score of 3 or more for dysmenorrhea and/or pelvic pain at second menses after operative laparoscopy .", "patients with vitamin d treatment in the last 6 months prior to surgery ; patients with known systemic diseases ( e.g. , hypertension , diabetes , coronary , renal , and hepatic diseases ) ; patients with known malignancy ; patients with hormonal treatment , including oral contraceptive pills , in the last 6 months . after authorization by the university ethics committee ,", "eligible patients were assigned by simple randomization to receive either vitamin d or placebo . in the vitamin d group ( d group ) , we prescribed oral vitamin d 50 000 iu / weekly for 12 weeks ( capsule d - vigel , vitamin d3 50 000 iu , daana pharma co. tabriz - iran ) and in the placebo group ( p group ) we prescribed 1 capsule of placebo ( daana pharma co. tabriz - iran ) weekly for 12 weeks .", "four weeks after the end of the intervention ( 24 weeks after surgical treatment ) , the vas test was repeated for the 2 groups .", "we used the ks test ( one - sample kolmogorov - smirnov test ) for normality of data distribution , levine s test for equality of variances and independent samples , and the t test for equality of means for comparing quantitative normal data between the 2 groups .", "we used the paired - samples t test for comparing quantitative normal data between before and after treatment in each group and the pearson chi - square test for matching and comparing categorical variables between the 2 groups . due to the small sample size", ", we conducted mann - whitney non - parametric analysis between the 2 groups and wilcoxon signed ranks test for comparing before and after treatment data in each group .", "this study was funded and supported by iran university of medical sciences ( iums ) , grant no .", "we did 146 laparoscopies for different indications in gynecologic patients , and endometriosis was diagnosed in 75 . at the second menses after diagnostic and therapeutic laparoscopy , 40 cases met the inclusion criteria for our study .", "the remaining 39 cases were randomly assigned in vitamin d ( n=19 ) or placebo treatment ( n=20 ) groups .", "one patient in the placebo group became pregnant at the third month after the operation and discontinued placebo treatment . at the end of the study , we had 19 patients in each group ( figure 1 ) .", "table 1 shows a baseline comparison between the 2 study groups for general characteristics , reason for laparoscopy , severity of endometriosis , and severity of dysmenorrhea and pelvic pain .", "causes of laparoscopy in women with endometriosis diagnosed by laparoscopy were dysmenorrhea ( n=28 ) , ovarian cyst ( n=23 ) , chronic pelvic pain ( n=19 ) , and infertility ( n=9 ) . in some patients , there was more than 1 reason for laparoscopy .", "severity of endometriosis in 45% was moderate ( n=17 ) and 47% had severe endometriosis ( n=18 ) . before laparoscopy ,", "the mean pelvic pain score in the vitamin d group was 4.053.45 and 4.824.1(p=0.513 ) in the placebo group . before laparoscopy , the mean dysmenorrhea pain score in the vitamin d group was 7.372.61 and in placebo group it was 6.423.04 ( p=0.325 ) . table 2 shows a comparison between the 2 groups for severity of pelvic pain and/or dysmenorrhea at different time points ( before laparoscopy , in second menses after laparoscopy , and at 24 weeks after laparoscopy ) . at the second menses after laparoscopy , there was no significant difference between the 2 groups for pelvic pain ( p=0.583 ) and dysmenorrhea ( p=0.365 ) , and at 24 weeks after laparoscopy there was no significant difference between mean pain scores in the 2 groups . mean pelvic pain at 24 weeks after laparoscopy in the vitamin d group was 0.841.74 and in placebo group it was 0.681.70 ( p=0.513 ) .", "mean dysmenorrhea was 2.102.33 in the vitamin d group and 2.732.84 in the placebo group ( p=0.45 ) .", "all of the patients had good cooperation for follow - up and continued their treatment until the end of the study .", "in this double - blind , randomized clinical trial , at 24 weeks after laparoscopic treatment of endometriosis there was no significant difference between effect of vitamin d3 ( cholecalciferol ) and placebo on severity of dysmenorrhea and/or pelvic pain .", "this is the first clinical trial on women with endometriosis to explore the possible relationship between vitamin d treatment and relief of endometriosis - related pain .", "our literature search found only studies that were indirectly related to vitamin d and endometriosis [ 2743 ] .", "the etiology of endometriosis is poorly understood , and many etiologic factors have been suggested , including the serum level of 1 , 25-dihydroxy vitamin d3 .", "1 , 25-dihydroxy vitamin d3 is a fat - soluble vitamin with an unclear role in endometriosis .", "it is suggested that nuclear vitamin d receptors can have a regulatory role in inflammation and can be used as an index of cellular metabolic health . a study on vitamin d receptor gene polymorphism in endometriosis compared 132 infertile women with endometriosis with 132 fertile women , reporting no significant difference and suggesting that vitamin d receptor gene polymorphism does not play an important role in the pathogenesis of endometriosis . in some studies ,", "higher plasma levels of 1 , 25-dihydroxy vitamin d3 and higher intake of dairy foods was associated with lower risk of endometriosis .", "conversely , another study compared serum vitamin d levels of 87 women with endometriosis with 53 women without endometriosis ; the mean serum levels of 1 , 25-dihydroxy vitamin d3 in women with and without endometriosis were 24.914.8 ng / ml and 20.411.8 , respectively ( p=0.05 ) and the study concluded that endometriosis is associated with higher serum levels of vitamin d .", "a systematic review of 10 case - control studies and 1 cohort study on women s diet found that women with endometriosis had lower consumption of vegetables and omega-3 , and reported a significant association between diet and endometriosis .", "vitamin d binding protein ( dbp ) is a plasma glycoprotein that modulates immune and inflammatory responses and also controls transport of vitamin d metabolites and bone development . in a study", "comparing 13 ectopic endometrial tissues and 6 normal endometrial tissues , vitamin d binding protein was significantly higher in the ectopic endometrial tissues ( p<0.05 ) .", "a systematic review of research from 1946 to 2013 on vitamin d and endometriosis reported that women with endometriosis had higher serum levels of vitamin d binding protein .", "another study compared serum and peritoneal levels of dbp in 26 women with endometriosis and 17 women with other benign gynecological conditions and reported that women with endometriosis had higher serum levels of dbp than in the control group .", "a study comparing urinary levels of dbp in 57 women with endometriosis with levels in 38 controls found that the urinary level of dbp was significantly higher in patients with endometriosis .", "a study using a rat model of endometriosis reported that treatment with vitamin d3 produced fibrosis and apoptosis in the stroma of tissues with endometriosis . a study on induced endometriosis in adult balb female mice reported that administration of 100 g / kg / day elocalcitol ( a vitamin d receptor agonist ) for 3 weeks reduced total lesion weight . because a relationship between vitamin d and endometriosis has been suggested by multiple studies , and since there has been no randomized clinical trial on endometriosis and vitamin d treatment in women , we decided to explore this relationship . in the present study on endometriosis - related pain", ", we found no significant difference in results of vitamin d treatment vs. placebo at 24 weeks after surgical diagnosis and treatment .", "vitamin d is a fat - soluble vitamin mainly produced in the body from food and supplements and cutaneous sun exposure .", "vitamin d deficiency is defined as serum 1.25-dihydroxy vitamin d3 levels under 20 ng / ml .", "it is reported in 52% of black and hispanic adolescents in boston and in 48% of girls in maine , and it is seen in 40100% of elderly men and women in the usa and europe . a study in germany found that 57% of people", "1879 years old were vitamin d deficient . the prevalence of vitamin d deficiency was reported to be 90% in healthy subjects in delhi , india . in a systematic review of 195 studies in 44 countries found that 37.3% of studies found that the mean serum vitamin d levels were less than 20 ng / ml .", "the prevalence in pregnant turkish women was 81.4% . in a study of high school students in iran ,", "the serum mean vitamin d level was 14.79.4 ng / ml . in another study in university students in shiraz , iran , 51.2% of female students had low serum levels of vitamin d . because the incidence of vitamin d deficiency in iran is high and we did not check the serum vitamin d levels in samples before intervention , it is possible that this dose and duration of vitamin d prescription was beneficial only for treatment of vitamin d deficiency and not endometriosis . in 1072 women attending an infertility center ,", "an in vitro study compared the effect of vitamin d3 on 25 human endometriosis stromal cell cultures ( ovarian endometrioma ) with the effect of vitamin d3 on culture of 20 endometrial samples of non - endometriosis women ; vitamin d3 inhibited proliferation , invasion , and pro - inflammatory cytokine production in endometriosis and reduced production of interleukin 6 and other inflammatory cytokines that stimulate adhesion of endometrial cells to the peritoneal cavity . a study on in vitro effects of vitamin d3 on human endometriosis stromal cells found that vitamin d3 significantly reduced interleukin 1 and tumor necrotizing factor- inflammatory responses , and also reported fewer endometrial stromal cells and reduced dna synthesis .", "the study found significantly lower serum vitamin d3 levels in severe endometriosis compared to normal controls and patients with mild endometriosis . a study in italy investigated the effect of vitamin d on primary dysmenorrhea .", "the samples were 40 women aged 1840 years old with 4 consecutive painful periods in the past 6 months .", "they measured the serum levels of 25 hydroxy vitamin d with high - performance liquid chromatography .", "then the women were randomized into a group of 20 women who received a single oral dose of 300 000 iu vitamin d ( cholecalciferol ) at 5 days before their next menstrual cycle and another group of 20 women received placebo .", "there was a negative correlation between the baseline dysmenorrhea pain score and the level of 25 hydroxy vitamin d3 ( r=0.36 , p=0.2 ) .", "the researchers found a significant reduction of dysmenorrhea pain in the vitamin d group in comparison with the placebo group in the next 2 menstruations ( p<0.01 ) .", "they suggested this significant reduction of dysmenorrhea pain in vitamin d prescription group was due to decreased levels of pro - inflammatory cytokines and decreased the biological activity of prostaglandins .", "the samples were not assessed for existing endometriosis . because low levels of serum vitamin d are common in healthy italian pre - menopausal women , these results only show that vitamin d was effective in relieving dysmenorrhea at least for 2 months after treatment in women with or without endometriosis and also in women with or without vitamin d deficiency .", "there may be a relationship between vitamin d and pathogenesis of endometriosis , but in our study vitamin d was not effective in treatment of endometriosis - related pain .", "larger clinical trials are needed to determine the possible effects of vitamin d supplementation in endometriosis treatment .", "the first limitation of this study is the high prevalence of vitamin d deficiency in our country and worldwide , meaning that a high percentage of our samples may have had vitamin d deficiency , which may have affected the results of our research .", "further clinical trials are needed on the role of vitamin d treatment for endometriosis - related pain .", "future studies should assess the serum levels of vitamin d before enrolling study subjects , and those with vitamin d deficiency should be excluded .", "the first limitation of this study is the high prevalence of vitamin d deficiency in our country and worldwide , meaning that a high percentage of our samples may have had vitamin d deficiency , which may have affected the results of our research .", "further clinical trials are needed on the role of vitamin d treatment for endometriosis - related pain .", "future studies should assess the serum levels of vitamin d before enrolling study subjects , and those with vitamin d deficiency should be excluded ."], "abstract_text": ["<S> backgroundendometriosis is a disabling disease of reproductive - age women . </S>", "<S> dysmenorrhea , dyspareunia , and pelvic pain are the main symptoms of endometriosis . </S>", "<S> its etiology is not clear . </S>", "<S> endometriosis may have various causes , including vitamin d deficiency , but its effect is controversial.material/methodsin this double - blind clinical trial , we enrolled patients with endometriosis diagnosed and treated by laparoscopy , with scores of at least 3 for of dysmenorrhea and/or pelvic pain at 8 weeks after surgical treatment . </S>", "<S> they were randomly prescribed vitamin d ( 50 000 iu weekly for 12 weeks ) or placebo . </S>", "<S> severity of pain in the 2 groups ( placebo and treatment ) was compared by vas test at 24 weeks after surgical treatment.resultsthere were 19 patients in the vitamin d group and 20 in the placebo group . </S>", "<S> baseline characteristics in the 2 groups were similar . </S>", "<S> following the treatment with vitamin d or placebo , we did not find significant differences in severity of pelvic pain ( p=0.24 ) and dysmenorrhea ( p=0.45 ) between the 2 groups . </S>", "<S> mean pelvic pain at 24 weeks after laparoscopy in the vitamin d group was 0.841.74 and in placebo group it was 0.681.70 ( p=0.513 ) . </S>", "<S> mean dysmenorrhea was 2.102.33 in the vitamin d group and 2.732.84 in the placebo group ( p=0.45).conclusionsafter ablative surgery for endometriosis , vitamin d treatment did not have a significant effect in reducing dysmenorrhea and/or pelvic pain . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Inclusion criteria", "Exclusion criteria", "Statistical analysis", "Results", "Discussion", "Conclusions", "Study limitations"], "sections": [["endometriosis is a prevalent cause of infertility , pelvic pain , dysmenorrhea , and dyspareunia in reproductive - age women .", "its diagnosis is by inspection of the pelvis during laparoscopy . in women with pelvic pain and infertility ,", "endometriosis mimics some autoimmune and malignant diseases , including familial occurrence and immunological abnormalities in b and t cells , increased angiogenesis , invasion of endometrial cells to adjacent organs ( e.g. , bladder and bowels ) , and need for repeat surgeries due to recurrence [ 1,57 ] .", "multiple mechanisms for etiology and improvement of endometriosis are suggested and the treatments are based on these unclear mechanisms . therefore , progestin , gnrh agonists and antagonists , and drugs related to lipid metabolism ( e.g. , simvastatin ) are used for treatment .", "it has been shown that inflammation is important in the pathogenesis of endometriosis , so endometriosis treatment should not be different from that of other inflammatory disorders", ". there may be a correlation between vitamin d levels and the risk of polycystic ovarian disease , endometriosis , breast and ovarian cancer , increased arterial stiffness in older patients , and myasthenia gravis [ 1218 ] .", "it has been found that vitamin d has a role in normal cellular growth regulation .", "vitamin d increases anti - inflammatory cytokines production and decreases pro - inflammatory cytokines [ 1922 ] .", "vitamin d induces apoptosis and suppression of angiogenesis in vitro and in vivo [ 2528 ] .", "although an indirect relationship between vitamin d and endometriosis has been reported in multiple studies , here has been no published randomized clinical trial on endometriosis and vitamin d treatment in women .", "we explored the relationship between vitamin d and endometriosis in a double - blind , randomized clinical trial looking at the effect of vitamin d supplementation on cessation of pain in proven endometriosis after laparoscopic diagnosis and treatment ."], ["this randomized , double - blind clinical trial was performed in a single tertiary university hospital from nov 2014 to feb 2016 . to find patients with endometriosis , we did laparoscopy for various indications , including ovarian cyst , infertility , pelvic pain , and dysmenorrhea .", "using laparoscopy , the surgeons diagnosed patients with endometriosis and tried to excise or ablate all diseased tissue . the day before laparoscopy", ", a data collection form was completed by a physician , including the reason for laparoscopy , and the severity of pelvic pain and dysmenorrhea were estimated using a visual analogue scale test ( vas test ) , with a score of 0 being no pain and a score 10 being the worst pain ever experienced .", "the laparoscopies were done by 2 gynecologic laparoscopic surgeons ; both were involved in each operation and they recorded the severity of endometriosis according to the revised american society for reproductive medicine ( asrm ) classification . in the patients with endometriosis in the second menses after laparoscopic diagnosis and treatment , the vas test was repeated .", "patients with vas scores of at least 3 for dysmenorrhea and/or pelvic pain were invited to participate in this clinical trial , and after consultation we asked them to sign the informed consent .", "the dyspareunia score was not a criterion for entering the study because some of the patients were not married and had not had intercourse .", "women aged 1540 years with proven endometriosis by laparoscopy and a vas test score of 3 or more for dysmenorrhea and/or pelvic pain at second menses after operative laparoscopy .", "patients with vitamin d treatment in the last 6 months prior to surgery ; patients with known systemic diseases ( e.g. , hypertension , diabetes , coronary , renal , and hepatic diseases ) ; patients with known malignancy ; patients with hormonal treatment , including oral contraceptive pills , in the last 6 months . after authorization by the university ethics committee ,", "eligible patients were assigned by simple randomization to receive either vitamin d or placebo . in the vitamin d group ( d group ) , we prescribed oral vitamin d 50 000 iu / weekly for 12 weeks ( capsule d - vigel , vitamin d3 50 000 iu , daana pharma co. tabriz - iran ) and in the placebo group ( p group ) we prescribed 1 capsule of placebo ( daana pharma co. tabriz - iran ) weekly for 12 weeks .", "four weeks after the end of the intervention ( 24 weeks after surgical treatment ) , the vas test was repeated for the 2 groups .", "we used the ks test ( one - sample kolmogorov - smirnov test ) for normality of data distribution , levine s test for equality of variances and independent samples , and the t test for equality of means for comparing quantitative normal data between the 2 groups .", "we used the paired - samples t test for comparing quantitative normal data between before and after treatment in each group and the pearson chi - square test for matching and comparing categorical variables between the 2 groups . due to the small sample size", ", we conducted mann - whitney non - parametric analysis between the 2 groups and wilcoxon signed ranks test for comparing before and after treatment data in each group .", "this study was funded and supported by iran university of medical sciences ( iums ) , grant no ."], ["women aged 1540 years with proven endometriosis by laparoscopy and a vas test score of 3 or more for dysmenorrhea and/or pelvic pain at second menses after operative laparoscopy ."], ["patients with vitamin d treatment in the last 6 months prior to surgery ; patients with known systemic diseases ( e.g. , hypertension , diabetes , coronary , renal , and hepatic diseases ) ; patients with known malignancy ; patients with hormonal treatment , including oral contraceptive pills , in the last 6 months . after authorization by the university ethics committee ,", "eligible patients were assigned by simple randomization to receive either vitamin d or placebo . in the vitamin d group ( d group ) , we prescribed oral vitamin d 50 000 iu / weekly for 12 weeks ( capsule d - vigel , vitamin d3 50 000 iu , daana pharma co. tabriz - iran ) and in the placebo group ( p group ) we prescribed 1 capsule of placebo ( daana pharma co. tabriz - iran ) weekly for 12 weeks .", "four weeks after the end of the intervention ( 24 weeks after surgical treatment ) , the vas test was repeated for the 2 groups ."], ["we used the ks test ( one - sample kolmogorov - smirnov test ) for normality of data distribution , levine s test for equality of variances and independent samples , and the t test for equality of means for comparing quantitative normal data between the 2 groups .", "we used the paired - samples t test for comparing quantitative normal data between before and after treatment in each group and the pearson chi - square test for matching and comparing categorical variables between the 2 groups . due to the small sample size", ", we conducted mann - whitney non - parametric analysis between the 2 groups and wilcoxon signed ranks test for comparing before and after treatment data in each group .", "this study was funded and supported by iran university of medical sciences ( iums ) , grant no ."], ["we did 146 laparoscopies for different indications in gynecologic patients , and endometriosis was diagnosed in 75 . at the second menses after diagnostic and therapeutic laparoscopy , 40 cases met the inclusion criteria for our study .", "the remaining 39 cases were randomly assigned in vitamin d ( n=19 ) or placebo treatment ( n=20 ) groups .", "one patient in the placebo group became pregnant at the third month after the operation and discontinued placebo treatment . at the end of the study , we had 19 patients in each group ( figure 1 ) .", "table 1 shows a baseline comparison between the 2 study groups for general characteristics , reason for laparoscopy , severity of endometriosis , and severity of dysmenorrhea and pelvic pain .", "causes of laparoscopy in women with endometriosis diagnosed by laparoscopy were dysmenorrhea ( n=28 ) , ovarian cyst ( n=23 ) , chronic pelvic pain ( n=19 ) , and infertility ( n=9 ) . in some patients , there was more than 1 reason for laparoscopy .", "severity of endometriosis in 45% was moderate ( n=17 ) and 47% had severe endometriosis ( n=18 ) . before laparoscopy ,", "the mean pelvic pain score in the vitamin d group was 4.053.45 and 4.824.1(p=0.513 ) in the placebo group . before laparoscopy , the mean dysmenorrhea pain score in the vitamin d group was 7.372.61 and in placebo group it was 6.423.04 ( p=0.325 ) . table 2 shows a comparison between the 2 groups for severity of pelvic pain and/or dysmenorrhea at different time points ( before laparoscopy , in second menses after laparoscopy , and at 24 weeks after laparoscopy ) . at the second menses after laparoscopy , there was no significant difference between the 2 groups for pelvic pain ( p=0.583 ) and dysmenorrhea ( p=0.365 ) , and at 24 weeks after laparoscopy there was no significant difference between mean pain scores in the 2 groups . mean pelvic pain at 24 weeks after laparoscopy in the vitamin d group was 0.841.74 and in placebo group it was 0.681.70 ( p=0.513 ) .", "mean dysmenorrhea was 2.102.33 in the vitamin d group and 2.732.84 in the placebo group ( p=0.45 ) .", "all of the patients had good cooperation for follow - up and continued their treatment until the end of the study ."], ["in this double - blind , randomized clinical trial , at 24 weeks after laparoscopic treatment of endometriosis there was no significant difference between effect of vitamin d3 ( cholecalciferol ) and placebo on severity of dysmenorrhea and/or pelvic pain .", "this is the first clinical trial on women with endometriosis to explore the possible relationship between vitamin d treatment and relief of endometriosis - related pain .", "our literature search found only studies that were indirectly related to vitamin d and endometriosis [ 2743 ] .", "the etiology of endometriosis is poorly understood , and many etiologic factors have been suggested , including the serum level of 1 , 25-dihydroxy vitamin d3 .", "1 , 25-dihydroxy vitamin d3 is a fat - soluble vitamin with an unclear role in endometriosis .", "it is suggested that nuclear vitamin d receptors can have a regulatory role in inflammation and can be used as an index of cellular metabolic health . a study on vitamin d receptor gene polymorphism in endometriosis compared 132 infertile women with endometriosis with 132 fertile women , reporting no significant difference and suggesting that vitamin d receptor gene polymorphism does not play an important role in the pathogenesis of endometriosis . in some studies ,", "higher plasma levels of 1 , 25-dihydroxy vitamin d3 and higher intake of dairy foods was associated with lower risk of endometriosis .", "conversely , another study compared serum vitamin d levels of 87 women with endometriosis with 53 women without endometriosis ; the mean serum levels of 1 , 25-dihydroxy vitamin d3 in women with and without endometriosis were 24.914.8 ng / ml and 20.411.8 , respectively ( p=0.05 ) and the study concluded that endometriosis is associated with higher serum levels of vitamin d .", "a systematic review of 10 case - control studies and 1 cohort study on women s diet found that women with endometriosis had lower consumption of vegetables and omega-3 , and reported a significant association between diet and endometriosis .", "vitamin d binding protein ( dbp ) is a plasma glycoprotein that modulates immune and inflammatory responses and also controls transport of vitamin d metabolites and bone development . in a study", "comparing 13 ectopic endometrial tissues and 6 normal endometrial tissues , vitamin d binding protein was significantly higher in the ectopic endometrial tissues ( p<0.05 ) .", "a systematic review of research from 1946 to 2013 on vitamin d and endometriosis reported that women with endometriosis had higher serum levels of vitamin d binding protein .", "another study compared serum and peritoneal levels of dbp in 26 women with endometriosis and 17 women with other benign gynecological conditions and reported that women with endometriosis had higher serum levels of dbp than in the control group .", "a study comparing urinary levels of dbp in 57 women with endometriosis with levels in 38 controls found that the urinary level of dbp was significantly higher in patients with endometriosis .", "a study using a rat model of endometriosis reported that treatment with vitamin d3 produced fibrosis and apoptosis in the stroma of tissues with endometriosis . a study on induced endometriosis in adult balb female mice reported that administration of 100 g / kg / day elocalcitol ( a vitamin d receptor agonist ) for 3 weeks reduced total lesion weight . because a relationship between vitamin d and endometriosis has been suggested by multiple studies , and since there has been no randomized clinical trial on endometriosis and vitamin d treatment in women , we decided to explore this relationship . in the present study on endometriosis - related pain", ", we found no significant difference in results of vitamin d treatment vs. placebo at 24 weeks after surgical diagnosis and treatment .", "vitamin d is a fat - soluble vitamin mainly produced in the body from food and supplements and cutaneous sun exposure .", "vitamin d deficiency is defined as serum 1.25-dihydroxy vitamin d3 levels under 20 ng / ml .", "it is reported in 52% of black and hispanic adolescents in boston and in 48% of girls in maine , and it is seen in 40100% of elderly men and women in the usa and europe . a study in germany found that 57% of people", "1879 years old were vitamin d deficient . the prevalence of vitamin d deficiency was reported to be 90% in healthy subjects in delhi , india . in a systematic review of 195 studies in 44 countries found that 37.3% of studies found that the mean serum vitamin d levels were less than 20 ng / ml .", "the prevalence in pregnant turkish women was 81.4% . in a study of high school students in iran ,", "the serum mean vitamin d level was 14.79.4 ng / ml . in another study in university students in shiraz , iran , 51.2% of female students had low serum levels of vitamin d . because the incidence of vitamin d deficiency in iran is high and we did not check the serum vitamin d levels in samples before intervention , it is possible that this dose and duration of vitamin d prescription was beneficial only for treatment of vitamin d deficiency and not endometriosis . in 1072 women attending an infertility center ,", "an in vitro study compared the effect of vitamin d3 on 25 human endometriosis stromal cell cultures ( ovarian endometrioma ) with the effect of vitamin d3 on culture of 20 endometrial samples of non - endometriosis women ; vitamin d3 inhibited proliferation , invasion , and pro - inflammatory cytokine production in endometriosis and reduced production of interleukin 6 and other inflammatory cytokines that stimulate adhesion of endometrial cells to the peritoneal cavity . a study on in vitro effects of vitamin d3 on human endometriosis stromal cells found that vitamin d3 significantly reduced interleukin 1 and tumor necrotizing factor- inflammatory responses , and also reported fewer endometrial stromal cells and reduced dna synthesis .", "the study found significantly lower serum vitamin d3 levels in severe endometriosis compared to normal controls and patients with mild endometriosis . a study in italy investigated the effect of vitamin d on primary dysmenorrhea .", "the samples were 40 women aged 1840 years old with 4 consecutive painful periods in the past 6 months .", "they measured the serum levels of 25 hydroxy vitamin d with high - performance liquid chromatography .", "then the women were randomized into a group of 20 women who received a single oral dose of 300 000 iu vitamin d ( cholecalciferol ) at 5 days before their next menstrual cycle and another group of 20 women received placebo .", "there was a negative correlation between the baseline dysmenorrhea pain score and the level of 25 hydroxy vitamin d3 ( r=0.36 , p=0.2 ) .", "the researchers found a significant reduction of dysmenorrhea pain in the vitamin d group in comparison with the placebo group in the next 2 menstruations ( p<0.01 ) .", "they suggested this significant reduction of dysmenorrhea pain in vitamin d prescription group was due to decreased levels of pro - inflammatory cytokines and decreased the biological activity of prostaglandins .", "the samples were not assessed for existing endometriosis . because low levels of serum vitamin d are common in healthy italian pre - menopausal women , these results only show that vitamin d was effective in relieving dysmenorrhea at least for 2 months after treatment in women with or without endometriosis and also in women with or without vitamin d deficiency ."], ["there may be a relationship between vitamin d and pathogenesis of endometriosis , but in our study vitamin d was not effective in treatment of endometriosis - related pain .", "larger clinical trials are needed to determine the possible effects of vitamin d supplementation in endometriosis treatment .", "the first limitation of this study is the high prevalence of vitamin d deficiency in our country and worldwide , meaning that a high percentage of our samples may have had vitamin d deficiency , which may have affected the results of our research .", "further clinical trials are needed on the role of vitamin d treatment for endometriosis - related pain .", "future studies should assess the serum levels of vitamin d before enrolling study subjects , and those with vitamin d deficiency should be excluded ."], ["the first limitation of this study is the high prevalence of vitamin d deficiency in our country and worldwide , meaning that a high percentage of our samples may have had vitamin d deficiency , which may have affected the results of our research .", "further clinical trials are needed on the role of vitamin d treatment for endometriosis - related pain .", "future studies should assess the serum levels of vitamin d before enrolling study subjects , and those with vitamin d deficiency should be excluded ."]]}
{"article_id": "PMC3881782", "article_text": ["it is generally accepted that microleakage between the filling materials and root canal \n walls might adversely affect the outcome of root canal treatment .", "therefore , it is critical the complete \n sealing of the root canal system after cleaning and shaping in order to avoid the \n bacterial penetration and re - infection of the root and periapical tissues .", "the association of gutta - percha cones \n and root canal sealer has been traditionally used for this purpose . however , in the last \n decade , the dentin adhesive technology has been incorporated into the root canal filling \n techniques to reduce apical and coronal leakage by bonding to root canal walls .", "etch - and - rinse adhesives have been \n tested with resin cements and the \n combination of a dentin - bonding agent and an epoxy resin - based root canal sealer \n significantly reduced apical leakage . in restorative dentistry , the self - etch adhesive systems have shown less technique \n sensitivity , with reliable long - term performance of a two - step mild self - etch \n adhesive . following this trend ,", "pentron clinical technologies \n ( wallingford , ct , usa ) has developed the epiphany system , which contains a self - etch \n primer , a dual - cured composite resin sealer and a polyester - based thermoplastic \n root - filling material ( resilon ; resilon research llc , madison , ct , usa ) . \n according to shipper , et al .", "( 2004 ) \n this material has been shown to be more resistant to bacterial leakage than epoxy \n resin - based sealers for filling root canals . in endodontics ,", "the controversy about the performance of adhesive systems inside the \n root canal remains . despite that , \n other possibility is combining epiphany primer with ah plus in an attempt to add the \n hybridization capacity to the gold standard endodontic sealer .", "the reason for this is \n that the removal of the smear layer with ethylenediamine tetraacetic acid ( edta ) does \n not provide the same etching pattern usually associated with the hybrid layer , which is \n considered an important factor for dentin bonding .", "the majority of studies have evaluated apical or coronal microleakage , and few have \n focused gap formation at the dentin / sealer interface .", "so far , no correlation between microleakage and gap \n formation has been established in the literature .", "thus , it is reasonable to believe that \n the association of both methods would provide a more precise evaluation of the adhesive \n interface .", "the aim of this study was to compare the apical sealing and gap formation of ah \n plus / gutta - percha with epiphany system .", "in addition , the opportunity was taken to assess \n the effect of the association of epiphany primer and ah plus / gutta - percha on apical \n sealing and gap formation .", "the null hypotheses tested were as follows : ( 1 ) there is no \n difference in apical microleakage or ( 2 ) apical gap formation among the experimental \n groups and ( 3 ) there is no correspondence between apical microleakage and the presence \n of gaps .", "thirty - nine lower single - rooted human premolars with straight root canals and fully \n developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted \n to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo , \n sp , brazil ) , and stored in saline solution at 4c .", "after endodontic access , \n the real working length ( rwl ) was established 1 mm short of the apical foramen .", "a \n crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5% \n naocl .", "the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5 \n ml ) followed by saline \n solution ( 15 ml ) .", "three coats of nail polish were applied to \n external root surfaces except for the apical 2 mm .", "the teeth were randomly ( http://www.random.org ) divided into \n 3 experimental groups ( n=11 ) .", "the endodontic sealers were \n prepared according to the manufacturer 's instructions and the cold lateral \n condensation filling technique ( lc ) was used according with the following \n description : an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah \n plus ; dentsply detrey , konstanz , germany ) and placed into the \n canal to rwl .", "a size b finger spreader ( dentsply maillefer , ballaigues , switzerland ) \n was then inserted into the canal to a level approximately 1 mm short of rwl .", "lc with \n accessory gutta - percha cones was performed until the entirely filled root canal .", "the \n excess gutta - percha was removed with a heated plugger and then compacted \n vertically .", "adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a \n paper point soaked with epiphany primer", "was used to etch dentin ( 30 s ) and the excess \n was removed with paper points .", "the coronal surface of the root filling was \n light - cured for 40 s ( 600 mw / cm ) .  ", "the positive controls ( n=3 ) were left unfilled and coated as described earlier . the \n negative controls ( n=3 ) were filled and totally coated , including the apical \n foramina .", "the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and \n stored in a chamber held at 100% humidity and 37c for 7 days .", "next , all teeth \n were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 ) \n in the darkness that was buffered using naoh 0.1 n for 24 h at 37c .", "the silver - impregnated teeth \n were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the \n silver ions into metallic silver . \n", "lake bluff , \n il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low \n speed ( 200 rpm ) with a water - cooled diamond blade .", "the interfaces were etched with a \n 35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently \n air - dried .", "then , specimen preparation followed the protocol 1 for apical microleakage \n analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ; \n or the protocol 2 for gaps analysis using sem according to the following \n description : specimens were fixed , dehydrated in ascending grades of ethanol and final chemical \n drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . ,", "louis , mo , usa ) for 10 \n min , and covered with \n carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) .", "the apical 5 mm \n of the root canal filling were divided into 5 regions of 1 mm to evaluate \n microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . ,", "cambridge , \n england ) using back scattered electrons ( bse ) mode ( figure 1a ) .", "the eds ( inca software , oxford , uk , england ) was performed in \n lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the \n identification of silver was made punctually to determine its exact location ( figure 1c ) .", "each 1 mm region was classified \n according to the following scores : 0 ( absence of leakage in both interfaces ) and 1 \n ( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e", "scanning electron microscopy ( sem ) micrographs using back scattered electrons \n ( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of \n agno3 leakage : ( a ) - the apical 5 mm of the root canal filling \n divided into 1 mm - regions .", "pointer at the detected metallic silver ; ( b )  area \n ( 300 m ) scanned for the existence of silver and \n respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation \n of its exact location .", "gp = gutta - percha ; d = dentin impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were \n made of the interfaces and the surfaces replicated with epoxy resin \n ( epon - thin , buhler ) .", "the replicas were covered with carbon to \n investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as \n described previously , using secondary electrons ( se ) mode .", "as the gaps were not \n continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were \n gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present .", "therefore , \n for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and \n accordingly classified .", "scanning electron microscopy ( sem ) micrographs using secondary electrons ( se ) \n and back scattered electrons ( bse ) mode of the replica and the section of the \n same specimen , respectively .", "( a ) - the apical 5 mm of the root canal filling \n divided into 1 mmregions .", "classification of the types of gaps : ( b )  type 1 : \n gap between dentin / sealer ( pointer ) ; air voids were present within the sealer \n ( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer \n interface , into dentinal tubules in a reticular form and granular aspect on the \n dentin surface ( arrowhead ) ; ( c )  type 2 : gap between sealer / cone ( pointer ) - \n cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure \n c at higher magnification : silver deposition into the dentinal tubules in a \n reticular form and granular aspect within the sealer layer ( arrowhead ) could be \n seen ; ( the sealer thickness was indicated between black / white arrowheads . \n", "gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically \n analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software \n program ( minitab inc .", "the counting of regions analyzed for gaps was also computed and adjusted in a \n generalized linear model with poisson distribution and logarithmic link function .", "the \n sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered \n as explanatory variables and ah plus and the type 0 as references .", "thirty - nine lower single - rooted human premolars with straight root canals and fully \n developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted \n to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo , \n sp , brazil ) , and stored in saline solution at 4c .", "after endodontic access , \n the real working length ( rwl ) was established 1 mm short of the apical foramen .", "a \n crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5% \n naocl .", "the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5 \n ml ) followed by saline \n solution ( 15 ml ) .", "three coats of nail polish were applied to \n external root surfaces except for the apical 2 mm .", "the teeth were randomly ( http://www.random.org ) divided into \n 3 experimental groups ( n=11 ) .", "the endodontic sealers were \n prepared according to the manufacturer 's instructions and the cold lateral \n condensation filling technique ( lc ) was used according with the following \n description :", "an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah \n plus ; dentsply detrey , konstanz , germany ) and placed into the \n canal to rwl .", "a size b finger spreader ( dentsply maillefer , ballaigues , switzerland ) \n was then inserted into the canal to a level approximately 1 mm short of rwl .", "lc with \n accessory gutta - percha cones was performed until the entirely filled root canal .", "the \n excess gutta - percha was removed with a heated plugger and then compacted \n vertically .", "adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a \n paper point soaked with epiphany primer", "was used to etch dentin ( 30 s ) and the excess \n was removed with paper points .", "the coronal surface of the root filling was \n light - cured for 40 s ( 600 mw / cm ) .", "the positive controls ( n=3 ) were left unfilled and coated as described earlier . the \n negative controls ( n=3 ) were filled and totally coated , including the apical \n foramina .", "the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and \n stored in a chamber held at 100% humidity and 37c for 7 days .", "next , all teeth \n were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 ) \n in the darkness that was buffered using naoh 0.1 n for 24 h at 37c .", "the silver - impregnated teeth \n were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the \n silver ions into metallic silver . \n", "lake bluff , \n il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low \n speed ( 200 rpm ) with a water - cooled diamond blade .", "the interfaces were etched with a \n 35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently \n air - dried .", "then , specimen preparation followed the protocol 1 for apical microleakage \n analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ; \n or the protocol 2 for gaps analysis using sem according to the following \n description :", "specimens were fixed , dehydrated in ascending grades of ethanol and final chemical \n drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . , st .", "louis , mo , usa ) for 10 \n min , and covered with \n carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) .", "the apical 5 mm \n of the root canal filling were divided into 5 regions of 1 mm to evaluate \n microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . ,", "cambridge , \n england ) using back scattered electrons ( bse ) mode ( figure 1a ) .", "the eds ( inca software , oxford , uk , england ) was performed in \n lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the \n identification of silver was made punctually to determine its exact location ( figure 1c ) .", "each 1 mm region was classified \n according to the following scores : 0 ( absence of leakage in both interfaces ) and 1 \n ( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e", "scanning electron microscopy ( sem ) micrographs using back scattered electrons \n ( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of \n agno3 leakage : ( a ) - the apical 5 mm of the root canal filling \n divided into 1 mm - regions .", "pointer at the detected metallic silver ; ( b )  area \n ( 300 m ) scanned for the existence of silver and \n respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation \n of its exact location .", "impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were \n made of the interfaces and the surfaces replicated with epoxy resin \n ( epon - thin , buhler ) .", "the replicas were covered with carbon to \n investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as \n described previously , using secondary electrons ( se ) mode .", "as the gaps were not \n continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were \n gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present .", "therefore , \n for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and \n accordingly classified .", "scanning electron microscopy ( sem ) micrographs using secondary electrons ( se ) \n and back scattered electrons ( bse ) mode of the replica and the section of the \n same specimen , respectively .", "( a ) - the apical 5 mm of the root canal filling \n divided into 1 mmregions .", "classification of the types of gaps : ( b )  type 1 : \n gap between dentin / sealer ( pointer ) ; air voids were present within the sealer \n ( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer \n interface , into dentinal tubules in a reticular form and granular aspect on the \n dentin surface ( arrowhead ) ; ( c )  type 2 : gap between sealer / cone ( pointer ) - \n cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure \n c at higher magnification : silver deposition into the dentinal tubules in a \n reticular form and granular aspect within the sealer layer ( arrowhead ) could be \n seen ; ( the sealer thickness was indicated between black / white arrowheads . \n", "gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically \n analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software \n program ( minitab inc .", "the counting of regions analyzed for gaps was also computed and adjusted in a \n generalized linear model with poisson distribution and logarithmic link function .", "the \n sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered \n as explanatory variables and ah plus and the type 0 as references .", "the ordinal logistic regression analysis demonstrated that there was no statistically \n significant difference ( p>0.05 ) among the groups tested and types of gaps over \n the logit of microleakage .", "the model was adjusted to identify the \n effect of the type of gap on microleakage .", "the type 3 data were not considered isolated \n in the analysis ( very few regions ) but joined with type 2 data ( figure 2c ; table 1 ) .", "the \n adjustment tests of the model presented high p - values for pearson 's test ( p=0.900 ) and \n deviance ( p=0.852 ) .", "the adjusted model demonstrated that the effect of the type of gap \n on the logit of microleakage is significant when compared to the \n presence of types 1 and 2 ( figure 2b and 2c , respectively ) of gap with the absence of gap \n ( p=0.047 ) .", "the correspondence analysis also showed the association between microleakage \n and both types of apical gaps ( figure 3 ) .", "correspondence analysis of microleakage vs. type of gap : ( inf.0 ) - absence of \n leakage ; ( inf.1 ) - leakage up to the 1 millimeter ; ( inf.2 ) ", "leakage \n up to the 2 mm ; ( inf.3 )  leakage up to the 3 \n millimeter ; ( inf.5 )  leakage up to the 5 millimeter ; ( f.0 )  absence \n of gaps ; ( f.1 )  gaps type 1 ; ( f.2 ) - gaps types 1 and 2 .", "the absence of leakage \n is associated with the absence of gap ; leakage up to the 1 millimeter \n is associated with type 1 ; and more extensive leakage is associated with types 1 \n and 2 frequency and type of gaps in the apical 5 mm of root canal fillings percentage of regions with gaps .", "type 0 : absence of gaps ; type 1 : gaps between \n dentin and sealer ; type 2 gaps between sealer and cone the generalized linear model with poisson distribution ( table 1 ) found no difference between ah plus and ah primer ( p=0.497 ) in \n distribution of each type of gap , but they presented more type 0 regions \n ( p<0.001 ) .", "there was a significant interaction between epiphany and the type of \n gap ( p<0.003 ) and for this group , there were more type 1 regions .", "based on the findings of this study , the first null hypothesis could not be rejected . \n", "recently some studies have shown similar results between ah plus and epiphany on apical \n sealing ability using the fluid filtration method .", "consistent with these \n studies , our results indicate that the epiphany system is as effective as ah \n plus / gutta - percha in preventing microleakage based on the chemical tracer penetration \n analysis by sem and the use of epiphany primer does not reduce the microleakage of ah \n plus . nevertheless , other researches pointed out that the epiphany system provided the \n greatest resistance to the movement of fluids or dye leakage \n test , when compared to epoxy \n resin - based root canal sealers , but others have just indicated the opposite .", "these apparent discrepancies can perhaps be explained by the \n methodology used and their variables .  ", "these results agree with those of previous studies in which , microleakage markers were \n used to evaluate apical sealing of the same endodontic sealers .", "the tracer \n selected was agno3 , which may penetrate into dentinal tubules due to their \n physical and chemical characteristics including : concentration , smaller molecular size , \n its neutral ph , diffusion coefficient and service life up to 168 h post - preparation time of \n solution .", "another reason for \n this choice is that metallic silver deposits may be observed by sem using back scattered \n electrons ( bse ) mode . on the other hand ,", "the results of the current study disagree with the findings of \n shipper , et al .  ", "an \n explanation for this would be that their studies have evaluated coronal and not apical \n bacterial leakage . indeed , coronal seal might be favorably influenced by photoactivation \n of the material which is unlikely to occur in the apical third .", "furthermore , it is speculated that in in \n vivo studies ( dogs ) , the high release of calcium hydroxide ( 41.46 mg / l ) that \n would occur during the process of epiphany sealer solubilization , would make the medium \n alkaline , resulting in acceleration of the periapical tissue repair process .", "this study has shown that the presence of apical gaps at the sealer / dentin or \n sealer / cone interfaces had an effect on apical microleakage .", "the epiphany group \n presented more type 1 gaps than the other groups ( table \n 1 ) , rejecting the second null hypothesis .", "these results corroborate with \n previous observation of gaps in the apical 4 mm of epiphany or ah plus - filled root \n canals .", "stress generated \n during the polymerization shrinkage of epiphany sealer has probably influenced the \n integrity loss at the sealer / dentin interface .", "in addition , the cavity configuration factor ( c - factor ) is \n highly unfavorable for adhesion inside root canals .", "moreover , it is known that analysis of gap formation of \n vertically sectioned root filled teeth hides the risk of artifacts during sectioning . \n", "therefore , in this study , the sections were made at low speed under water cooling .", "the \n resin - epoxy replicas at the dentin / sealer and sealer / cone interfaces were made before \n the specimens had been prepared for sem examination in order to differentiate genuine \n gaps from artifactual gaps created after vacuum desiccation in conventional scanning \n electron microscopes .", "few studies have focused on apical sealing from two separate perspectives , such as \n microleakage and apical gap formation .", "sem was used to evaluate these perspectives , but it was restricted to a descriptive \n approach .", "a systematization of the observations , complemented by a statistical analysis , such as \n the one performed in this study , could provide greater methodological accuracy and \n impartiality for comparing the experimental groups .", "the correspondence of analysis \n between the types of gaps and linear extent of agno3 leakage ( fig .", "3 ) confirms the effect of gaps on apical \n microleakage , expressed by the silver deposition into gaps that extended deeply inside \n dentinal tubules ( fig .", "regarding to statistical analysis , the ordinal logistic regression analysis model was \n used due to microleakage was considered categorized ordinal response variable ( ranging \n from 0 - no leakage to 5 - leakage up to the fifth millimeter ) .", "the correspondence \n analysis is a method that leads to visualize the association between two categorical \n variables , in this case microleakage and the type of gap .", "the generalized linear model \n with poisson which is an extension of usual regression model was performed to quantify \n the distribution of types of gaps .", "this result could be explained by two hypotheses : ( 1 ) \n the low degree of conversion of the epiphany sealer and ( 2 ) the formation of hydrogel at the bond interface resulting \n from the incomplete evaporation of the epiphany primer solvent .", "the second hypothesis could also explain the inefficacy \n of epiphany primer in improving the sealing capacity of ah plus .", "moreover , there is a \n chemical incompatibility between these two materials , since the epoxy resin sealers do \n not copolymerize with methacrylate resin - based adhesives .", "other studies should be carried out to clarify the \n sealing ability of the root - filled materials studied .", "it may be concluded that none of the tested materials completely sealed the apical 5 mm . \n", "the presence \n of gaps had an effect on apical microleakage for all materials . comparing the materials , \n epiphany system presented more regions containing gaps between the dentin and the sealer \n ( type 1 ) . in view of these findings ,", "clinically , it can be suggested that ah plus would \n provide a better apical seal .", "this study was partially supported by capes ( coordenao de \n aperfeioamento de pessoal de nvel superior)/institutional \n qualification program ( pqi n : 0090/03 - 4 ) ."], "abstract_text": ["<S> objectivethe aim of this study was to compare the correspondence between gap formation and \n apical microleakage in root canals filled with epoxy resin - based ( ah plus ) \n combined or not with resinous primer or with a dimethacrylate - based root canal \n sealer ( epiphany ) . </S>", "<S> material and methodsthirty - nine lower single - rooted human premolars were filled by the lateral \n condensation technique ( lc ) and immersed in a 50-wt% aqueous silver nitrate \n solution at 37c ( 24 h ) . </S>", "<S> after longitudinal sectioning , epoxy resin \n replicas were made from the tooth specimens . </S>", "<S> both the replicas and the specimens \n were prepared for scanning electron microscopy ( sem ) . </S>", "<S> the gaps were observed in \n the replicas . </S>", "<S> apical microleakage was detected in the specimens by sem / energy \n dispersive spectroscopy ( sem / eds ) . </S>", "<S> the data were analyzed statistically using an \n ordinal logistic regression model and analysis of correspondence ( =0.05 ) . \n </S>", "<S> resultsepiphany presented more regions containing gaps between dentin and sealer \n ( p<0.05 ) . </S>", "<S> there was correspondence between the presence of gaps and \n microleakage ( p<0.05 ) . </S>", "<S> microleakage was similar among the root - filling \n materials ( p>0.05 ) . </S>", "<S> conclusionsthe resinous primer did not improve the sealing ability of ah plus sealer and the \n presence of gaps had an effect on apical microleakage for all materials . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "Specimen preparation", "Group AH Plus", "Group AH Primer", "Group epiphany", "Positive and negative controls", "Protocol 1", "Protocol 2", "RESULTS", "DISCUSSION", "CONCLUSIONS", "ACKNOWLEDGEMENTS"], "sections": [["it is generally accepted that microleakage between the filling materials and root canal \n walls might adversely affect the outcome of root canal treatment .", "therefore , it is critical the complete \n sealing of the root canal system after cleaning and shaping in order to avoid the \n bacterial penetration and re - infection of the root and periapical tissues .", "the association of gutta - percha cones \n and root canal sealer has been traditionally used for this purpose . however , in the last \n decade , the dentin adhesive technology has been incorporated into the root canal filling \n techniques to reduce apical and coronal leakage by bonding to root canal walls .", "etch - and - rinse adhesives have been \n tested with resin cements and the \n combination of a dentin - bonding agent and an epoxy resin - based root canal sealer \n significantly reduced apical leakage . in restorative dentistry , the self - etch adhesive systems have shown less technique \n sensitivity , with reliable long - term performance of a two - step mild self - etch \n adhesive . following this trend ,", "pentron clinical technologies \n ( wallingford , ct , usa ) has developed the epiphany system , which contains a self - etch \n primer , a dual - cured composite resin sealer and a polyester - based thermoplastic \n root - filling material ( resilon ; resilon research llc , madison , ct , usa ) . \n according to shipper , et al .", "( 2004 ) \n this material has been shown to be more resistant to bacterial leakage than epoxy \n resin - based sealers for filling root canals . in endodontics ,", "the controversy about the performance of adhesive systems inside the \n root canal remains . despite that , \n other possibility is combining epiphany primer with ah plus in an attempt to add the \n hybridization capacity to the gold standard endodontic sealer .", "the reason for this is \n that the removal of the smear layer with ethylenediamine tetraacetic acid ( edta ) does \n not provide the same etching pattern usually associated with the hybrid layer , which is \n considered an important factor for dentin bonding .", "the majority of studies have evaluated apical or coronal microleakage , and few have \n focused gap formation at the dentin / sealer interface .", "so far , no correlation between microleakage and gap \n formation has been established in the literature .", "thus , it is reasonable to believe that \n the association of both methods would provide a more precise evaluation of the adhesive \n interface .", "the aim of this study was to compare the apical sealing and gap formation of ah \n plus / gutta - percha with epiphany system .", "in addition , the opportunity was taken to assess \n the effect of the association of epiphany primer and ah plus / gutta - percha on apical \n sealing and gap formation .", "the null hypotheses tested were as follows : ( 1 ) there is no \n difference in apical microleakage or ( 2 ) apical gap formation among the experimental \n groups and ( 3 ) there is no correspondence between apical microleakage and the presence \n of gaps ."], ["thirty - nine lower single - rooted human premolars with straight root canals and fully \n developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted \n to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo , \n sp , brazil ) , and stored in saline solution at 4c .", "after endodontic access , \n the real working length ( rwl ) was established 1 mm short of the apical foramen .", "a \n crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5% \n naocl .", "the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5 \n ml ) followed by saline \n solution ( 15 ml ) .", "three coats of nail polish were applied to \n external root surfaces except for the apical 2 mm .", "the teeth were randomly ( http://www.random.org ) divided into \n 3 experimental groups ( n=11 ) .", "the endodontic sealers were \n prepared according to the manufacturer 's instructions and the cold lateral \n condensation filling technique ( lc ) was used according with the following \n description : an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah \n plus ; dentsply detrey , konstanz , germany ) and placed into the \n canal to rwl .", "a size b finger spreader ( dentsply maillefer , ballaigues , switzerland ) \n was then inserted into the canal to a level approximately 1 mm short of rwl .", "lc with \n accessory gutta - percha cones was performed until the entirely filled root canal .", "the \n excess gutta - percha was removed with a heated plugger and then compacted \n vertically .", "adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a \n paper point soaked with epiphany primer", "was used to etch dentin ( 30 s ) and the excess \n was removed with paper points .", "the coronal surface of the root filling was \n light - cured for 40 s ( 600 mw / cm ) .  ", "the positive controls ( n=3 ) were left unfilled and coated as described earlier . the \n negative controls ( n=3 ) were filled and totally coated , including the apical \n foramina .", "the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and \n stored in a chamber held at 100% humidity and 37c for 7 days .", "next , all teeth \n were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 ) \n in the darkness that was buffered using naoh 0.1 n for 24 h at 37c .", "the silver - impregnated teeth \n were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the \n silver ions into metallic silver . \n", "lake bluff , \n il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low \n speed ( 200 rpm ) with a water - cooled diamond blade .", "the interfaces were etched with a \n 35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently \n air - dried .", "then , specimen preparation followed the protocol 1 for apical microleakage \n analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ; \n or the protocol 2 for gaps analysis using sem according to the following \n description : specimens were fixed , dehydrated in ascending grades of ethanol and final chemical \n drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . ,", "louis , mo , usa ) for 10 \n min , and covered with \n carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) .", "the apical 5 mm \n of the root canal filling were divided into 5 regions of 1 mm to evaluate \n microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . ,", "cambridge , \n england ) using back scattered electrons ( bse ) mode ( figure 1a ) .", "the eds ( inca software , oxford , uk , england ) was performed in \n lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the \n identification of silver was made punctually to determine its exact location ( figure 1c ) .", "each 1 mm region was classified \n according to the following scores : 0 ( absence of leakage in both interfaces ) and 1 \n ( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e", "scanning electron microscopy ( sem ) micrographs using back scattered electrons \n ( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of \n agno3 leakage : ( a ) - the apical 5 mm of the root canal filling \n divided into 1 mm - regions .", "pointer at the detected metallic silver ; ( b )  area \n ( 300 m ) scanned for the existence of silver and \n respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation \n of its exact location .", "gp = gutta - percha ; d = dentin impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were \n made of the interfaces and the surfaces replicated with epoxy resin \n ( epon - thin , buhler ) .", "the replicas were covered with carbon to \n investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as \n described previously , using secondary electrons ( se ) mode .", "as the gaps were not \n continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were \n gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present .", "therefore , \n for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and \n accordingly classified .", "scanning electron microscopy ( sem ) micrographs using secondary electrons ( se ) \n and back scattered electrons ( bse ) mode of the replica and the section of the \n same specimen , respectively .", "( a ) - the apical 5 mm of the root canal filling \n divided into 1 mmregions .", "classification of the types of gaps : ( b )  type 1 : \n gap between dentin / sealer ( pointer ) ; air voids were present within the sealer \n ( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer \n interface , into dentinal tubules in a reticular form and granular aspect on the \n dentin surface ( arrowhead ) ; ( c )  type 2 : gap between sealer / cone ( pointer ) - \n cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure \n c at higher magnification : silver deposition into the dentinal tubules in a \n reticular form and granular aspect within the sealer layer ( arrowhead ) could be \n seen ; ( the sealer thickness was indicated between black / white arrowheads . \n", "gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically \n analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software \n program ( minitab inc .", "the counting of regions analyzed for gaps was also computed and adjusted in a \n generalized linear model with poisson distribution and logarithmic link function .", "the \n sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered \n as explanatory variables and ah plus and the type 0 as references ."], ["thirty - nine lower single - rooted human premolars with straight root canals and fully \n developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted \n to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo , \n sp , brazil ) , and stored in saline solution at 4c .", "after endodontic access , \n the real working length ( rwl ) was established 1 mm short of the apical foramen .", "a \n crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5% \n naocl .", "the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5 \n ml ) followed by saline \n solution ( 15 ml ) .", "three coats of nail polish were applied to \n external root surfaces except for the apical 2 mm .", "the teeth were randomly ( http://www.random.org ) divided into \n 3 experimental groups ( n=11 ) .", "the endodontic sealers were \n prepared according to the manufacturer 's instructions and the cold lateral \n condensation filling technique ( lc ) was used according with the following \n description :"], ["an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah \n plus ; dentsply detrey , konstanz , germany ) and placed into the \n canal to rwl .", "a size b finger spreader ( dentsply maillefer , ballaigues , switzerland ) \n was then inserted into the canal to a level approximately 1 mm short of rwl .", "lc with \n accessory gutta - percha cones was performed until the entirely filled root canal .", "the \n excess gutta - percha was removed with a heated plugger and then compacted \n vertically ."], ["adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a \n paper point soaked with epiphany primer", "was used to etch dentin ( 30 s ) and the excess \n was removed with paper points ."], ["the coronal surface of the root filling was \n light - cured for 40 s ( 600 mw / cm ) ."], ["the positive controls ( n=3 ) were left unfilled and coated as described earlier . the \n negative controls ( n=3 ) were filled and totally coated , including the apical \n foramina .", "the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and \n stored in a chamber held at 100% humidity and 37c for 7 days .", "next , all teeth \n were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 ) \n in the darkness that was buffered using naoh 0.1 n for 24 h at 37c .", "the silver - impregnated teeth \n were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the \n silver ions into metallic silver . \n", "lake bluff , \n il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low \n speed ( 200 rpm ) with a water - cooled diamond blade .", "the interfaces were etched with a \n 35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently \n air - dried .", "then , specimen preparation followed the protocol 1 for apical microleakage \n analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ; \n or the protocol 2 for gaps analysis using sem according to the following \n description :"], ["specimens were fixed , dehydrated in ascending grades of ethanol and final chemical \n drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . , st .", "louis , mo , usa ) for 10 \n min , and covered with \n carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) .", "the apical 5 mm \n of the root canal filling were divided into 5 regions of 1 mm to evaluate \n microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . ,", "cambridge , \n england ) using back scattered electrons ( bse ) mode ( figure 1a ) .", "the eds ( inca software , oxford , uk , england ) was performed in \n lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the \n identification of silver was made punctually to determine its exact location ( figure 1c ) .", "each 1 mm region was classified \n according to the following scores : 0 ( absence of leakage in both interfaces ) and 1 \n ( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e", "scanning electron microscopy ( sem ) micrographs using back scattered electrons \n ( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of \n agno3 leakage : ( a ) - the apical 5 mm of the root canal filling \n divided into 1 mm - regions .", "pointer at the detected metallic silver ; ( b )  area \n ( 300 m ) scanned for the existence of silver and \n respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation \n of its exact location ."], ["impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were \n made of the interfaces and the surfaces replicated with epoxy resin \n ( epon - thin , buhler ) .", "the replicas were covered with carbon to \n investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as \n described previously , using secondary electrons ( se ) mode .", "as the gaps were not \n continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were \n gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present .", "therefore , \n for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and \n accordingly classified .", "scanning electron microscopy ( sem ) micrographs using secondary electrons ( se ) \n and back scattered electrons ( bse ) mode of the replica and the section of the \n same specimen , respectively .", "( a ) - the apical 5 mm of the root canal filling \n divided into 1 mmregions .", "classification of the types of gaps : ( b )  type 1 : \n gap between dentin / sealer ( pointer ) ; air voids were present within the sealer \n ( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer \n interface , into dentinal tubules in a reticular form and granular aspect on the \n dentin surface ( arrowhead ) ; ( c )  type 2 : gap between sealer / cone ( pointer ) - \n cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure \n c at higher magnification : silver deposition into the dentinal tubules in a \n reticular form and granular aspect within the sealer layer ( arrowhead ) could be \n seen ; ( the sealer thickness was indicated between black / white arrowheads . \n", "gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically \n analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software \n program ( minitab inc .", "the counting of regions analyzed for gaps was also computed and adjusted in a \n generalized linear model with poisson distribution and logarithmic link function .", "the \n sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered \n as explanatory variables and ah plus and the type 0 as references ."], ["the ordinal logistic regression analysis demonstrated that there was no statistically \n significant difference ( p>0.05 ) among the groups tested and types of gaps over \n the logit of microleakage .", "the model was adjusted to identify the \n effect of the type of gap on microleakage .", "the type 3 data were not considered isolated \n in the analysis ( very few regions ) but joined with type 2 data ( figure 2c ; table 1 ) .", "the \n adjustment tests of the model presented high p - values for pearson 's test ( p=0.900 ) and \n deviance ( p=0.852 ) .", "the adjusted model demonstrated that the effect of the type of gap \n on the logit of microleakage is significant when compared to the \n presence of types 1 and 2 ( figure 2b and 2c , respectively ) of gap with the absence of gap \n ( p=0.047 ) .", "the correspondence analysis also showed the association between microleakage \n and both types of apical gaps ( figure 3 ) .", "correspondence analysis of microleakage vs. type of gap : ( inf.0 ) - absence of \n leakage ; ( inf.1 ) - leakage up to the 1 millimeter ; ( inf.2 ) ", "leakage \n up to the 2 mm ; ( inf.3 )  leakage up to the 3 \n millimeter ; ( inf.5 )  leakage up to the 5 millimeter ; ( f.0 )  absence \n of gaps ; ( f.1 )  gaps type 1 ; ( f.2 ) - gaps types 1 and 2 .", "the absence of leakage \n is associated with the absence of gap ; leakage up to the 1 millimeter \n is associated with type 1 ; and more extensive leakage is associated with types 1 \n and 2 frequency and type of gaps in the apical 5 mm of root canal fillings percentage of regions with gaps .", "type 0 : absence of gaps ; type 1 : gaps between \n dentin and sealer ; type 2 gaps between sealer and cone the generalized linear model with poisson distribution ( table 1 ) found no difference between ah plus and ah primer ( p=0.497 ) in \n distribution of each type of gap , but they presented more type 0 regions \n ( p<0.001 ) .", "there was a significant interaction between epiphany and the type of \n gap ( p<0.003 ) and for this group , there were more type 1 regions ."], ["based on the findings of this study , the first null hypothesis could not be rejected . \n", "recently some studies have shown similar results between ah plus and epiphany on apical \n sealing ability using the fluid filtration method .", "consistent with these \n studies , our results indicate that the epiphany system is as effective as ah \n plus / gutta - percha in preventing microleakage based on the chemical tracer penetration \n analysis by sem and the use of epiphany primer does not reduce the microleakage of ah \n plus . nevertheless , other researches pointed out that the epiphany system provided the \n greatest resistance to the movement of fluids or dye leakage \n test , when compared to epoxy \n resin - based root canal sealers , but others have just indicated the opposite .", "these apparent discrepancies can perhaps be explained by the \n methodology used and their variables .  ", "these results agree with those of previous studies in which , microleakage markers were \n used to evaluate apical sealing of the same endodontic sealers .", "the tracer \n selected was agno3 , which may penetrate into dentinal tubules due to their \n physical and chemical characteristics including : concentration , smaller molecular size , \n its neutral ph , diffusion coefficient and service life up to 168 h post - preparation time of \n solution .", "another reason for \n this choice is that metallic silver deposits may be observed by sem using back scattered \n electrons ( bse ) mode . on the other hand ,", "the results of the current study disagree with the findings of \n shipper , et al .  ", "an \n explanation for this would be that their studies have evaluated coronal and not apical \n bacterial leakage . indeed , coronal seal might be favorably influenced by photoactivation \n of the material which is unlikely to occur in the apical third .", "furthermore , it is speculated that in in \n vivo studies ( dogs ) , the high release of calcium hydroxide ( 41.46 mg / l ) that \n would occur during the process of epiphany sealer solubilization , would make the medium \n alkaline , resulting in acceleration of the periapical tissue repair process .", "this study has shown that the presence of apical gaps at the sealer / dentin or \n sealer / cone interfaces had an effect on apical microleakage .", "the epiphany group \n presented more type 1 gaps than the other groups ( table \n 1 ) , rejecting the second null hypothesis .", "these results corroborate with \n previous observation of gaps in the apical 4 mm of epiphany or ah plus - filled root \n canals .", "stress generated \n during the polymerization shrinkage of epiphany sealer has probably influenced the \n integrity loss at the sealer / dentin interface .", "in addition , the cavity configuration factor ( c - factor ) is \n highly unfavorable for adhesion inside root canals .", "moreover , it is known that analysis of gap formation of \n vertically sectioned root filled teeth hides the risk of artifacts during sectioning . \n", "therefore , in this study , the sections were made at low speed under water cooling .", "the \n resin - epoxy replicas at the dentin / sealer and sealer / cone interfaces were made before \n the specimens had been prepared for sem examination in order to differentiate genuine \n gaps from artifactual gaps created after vacuum desiccation in conventional scanning \n electron microscopes .", "few studies have focused on apical sealing from two separate perspectives , such as \n microleakage and apical gap formation .", "sem was used to evaluate these perspectives , but it was restricted to a descriptive \n approach .", "a systematization of the observations , complemented by a statistical analysis , such as \n the one performed in this study , could provide greater methodological accuracy and \n impartiality for comparing the experimental groups .", "the correspondence of analysis \n between the types of gaps and linear extent of agno3 leakage ( fig .", "3 ) confirms the effect of gaps on apical \n microleakage , expressed by the silver deposition into gaps that extended deeply inside \n dentinal tubules ( fig .", "regarding to statistical analysis , the ordinal logistic regression analysis model was \n used due to microleakage was considered categorized ordinal response variable ( ranging \n from 0 - no leakage to 5 - leakage up to the fifth millimeter ) .", "the correspondence \n analysis is a method that leads to visualize the association between two categorical \n variables , in this case microleakage and the type of gap .", "the generalized linear model \n with poisson which is an extension of usual regression model was performed to quantify \n the distribution of types of gaps .", "this result could be explained by two hypotheses : ( 1 ) \n the low degree of conversion of the epiphany sealer and ( 2 ) the formation of hydrogel at the bond interface resulting \n from the incomplete evaporation of the epiphany primer solvent .", "the second hypothesis could also explain the inefficacy \n of epiphany primer in improving the sealing capacity of ah plus .", "moreover , there is a \n chemical incompatibility between these two materials , since the epoxy resin sealers do \n not copolymerize with methacrylate resin - based adhesives .", "other studies should be carried out to clarify the \n sealing ability of the root - filled materials studied ."], ["it may be concluded that none of the tested materials completely sealed the apical 5 mm . \n", "the presence \n of gaps had an effect on apical microleakage for all materials . comparing the materials , \n epiphany system presented more regions containing gaps between the dentin and the sealer \n ( type 1 ) . in view of these findings ,", "clinically , it can be suggested that ah plus would \n provide a better apical seal ."], ["this study was partially supported by capes ( coordenao de \n aperfeioamento de pessoal de nvel superior)/institutional \n qualification program ( pqi n : 0090/03 - 4 ) ."]]}
{"article_id": "PMC4573445", "article_text": ["neonatal diabetes mellitus ( ndm ) presenting within the first 6 months of life includes permanent neonatal diabetes mellitus ( pndm ) , which require lifelong therapy , and transient neonatal diabetes mellitus ( tndm ) where the condition shows remission during infancy but relapses in adolescence .", "almost all cases of neonatal diabetes have monogenic etiology in contrast to the autoimmune diabetes presenting in children beyond 6 months of age .", "pndm is associated with defects in pancreatic beta cell development and function . activating mutations in the kcnj11 gene , encoding the subunit kir6.2 , and abcc8 gene ,", "encoding the sulfonylurea receptor 1 ( sur1 ) of atp - sensitive potassium ( katp ) channel , which has a key role in insulin secretion in glucose metabolism , are the most common causes and account for approximately 40% of all cases of pndm12 ) .", "mutations in the glucokinase ( gck ) and insulin ( ins ) genes have also been reported in patients with pndm .", "sulfonylureas close the katp channel by an atp - independent route , leading to increase insulin secretion4 ) .", "therefore , in many patients with pndm with kir6.2 and sur1 mutations , insulin therapy can be replaced by oral sulfonylureas which offer more improvement in glycemic control and better quality of life35 ) .", "we report a case of pndm caused by a novel p.h186d heterozygous mutation in the kcnj11 gene whose treatment was successfully transitioned from insulin to oral sulfonylurea .", "her parents were unrelated , and her family was healthy and had no history of diabetes .", "laboratory results at diagnosis were : blood glucose , 1,041 mg / dl ; ph , 7.025 ; hco3 , 5.1", "mmol / l ; pco2 , 19.8 mmhg ; sodium , 147 mmol / l ; potassium , 5.6 mmol / l ; chloride , 113 mmol / l ; blood urea nitrogen , 31 mg / dl ; creatinine , 1.2 mg / dl ; urine ketone , + + + ; glycosylated hemoglobin ( hba1c ) , 7.7% ; and c - peptide , 0.2 ng / ml . after recovery from diabetic ketoacidosis , she had injections of neutral protamine hagedorn ( nph ) insulin two times a day and regular insulin ( ri ) four times a day .", "her maximal daily insulin dose was at 1.5 u / kg . her insulin requirement was gradually decreased to 0.44 u / kg / day by 4 months of age .", "although there were several hypoglycemic events , insulin injection for glucose control after the infancy period could not be stopped .", "genetic study for neonatal diabetes using genomic dna extracted from peripheral lymphocyte was done at 4.5 years of age , and a novel heterozygous mutation of the kcnj11 gene located on chromosome 11p15.1 and encoding kir6.2 , c.556c > g ( p.his186asp ) , was found ( fig .", "transition of treatment from insulin to sulfonylurea was attempted at the age of 4.8 . before the trial ,", "laboratory findings were as follows : hba1c , 6.9% ; fasting glucose , 205 mg / dl ; c - peptide , 0.84 ng / ml ; 24-hour urine c - peptide , 2.2 g ; negative for islet cell antibody ; anti - insulin antibody ; and anti - gad antibody .", "she was 103.2 cm in height ( 25th-50th percentile ) , 17 kg in weight ( 50th-75th percentile ) and did not have any specific neurological deficit .", "oral sulfonylurea ( glibenclamide ) was initiated at a dose of 0.1 mg / kg / day divided into two equal doses .", "the glibenclamide dose was gradually increased to 0.4 mg / kg / day over 1 week and the insulin was stopped .", "oral glucose tolerance test ( glucose , 1.75 g / kg ) was done at 6 months and 1 year after completion of the sulfonylurea transition .", "insulinogenic index [ insulin ( 30 min-0 min)/glucose ( 30 min-0 min ) ] , a marker of beta cell function , became increased from 0.10 to 0.18 , and acute c - peptide response [ c - peptide ( 30 min-0 min)1,000/glucose ( 30 min-0 min ) ] became increased from 6.48 to 8.29 ( table 1 ) .", "the patient is now 7.5 years of age with a height of 121.4 cm ( 50th percentile ) and weight of 23.4 kg ( 50th percentile ) .", "blood glucose was well controlled without episodes of hypoglycemia and the hba1c has been lower than during insulin injection ( fig .", "ndm is a monogenic form of diabetes that presents within the first 6 months of life .", "heterozygous activating mutations in the kcnj11 and abcc8 gene encoding the subunits kir6.2 and sur1 of katp channel in pancreatic beta cells are the most common and account for nearly half of all cases of pndm12 ) .", "mutations in the glucokinase ( gck ) , insulin ( ins ) genes have also been reported in patients with pndm .", "the causes of rare syndromic pndm includes recessive mutations in several genes , such as pdx1 , eif2ak3 , gck , ptf1a , foxp3 , neurog3 , neurod1 , rfx6 , ier3ip1 , hnf1b , glis3 , pax6 , slc19a2 , slc2a2 , and wfs167 ) . in tndm", ", imprinted locus at chromosome 6q24 seen in more than half of cases8 ) , and kcnj11 and abcc8 gene mutations are also found in some cases .", "glucose increases intracellular atp level and it induces the closure of katp channels that lead to insulin secretion by pancreatic beta cells . activating mutations in the kcnj11 or abcc8 gene of katp channel lead to katp channels remaining open despite the presence of glucose , thus , insulin secretion can not be increased1 ) . on the contrary , loss of function mutations cause congenital hyperinsulinemia due to the closure of the katp channel and lead to increased insulin secretion", ". sulfonylureas ( su ) can close the katp channel and increase insulin secretion4 ) .", "treatment response is not expected in patients with mutations in other genes , such as glucokinase gene , foxp3 , and ipf1 .", "therefore , molecular diagnosis in neonatal diabetes may help identify patients that are likely to respond to oral sulfonylureas .", "pearson et al.3 ) reported the responses to sulfonylurea according to mutation types in diabetic patients caused by kcnj11 mutation .", "there were no differences in blood insulin , c - peptide level , and insulin injection dose between the successful group and unsuccessful group .", "v59 m , r201c , f35v , h46y , r50q , g53r , r201l , e322k , y330s , f333i mutations also belong to the successful group .", "switching to su was unsuccessful in patients with q52r , i296l , and l164p mutations .", "in addition , failure of switching to su was more related with the presence of neurologic features ( 14% in successful group and 80% in unsuccessful group ) and older age at initiation of su .", "median age was 6 years old ( intraquartile range , 3 - 12 years ) in the successful group and 18 years old ( intraquartile range , 6 - 35 years ) in the unsuccessful group3 ) .", "the report about a mother and daughter carrying the same kcnj11 mutation showed that the daughter could be switched from insulin to su at age 8.5 years , but her mother had an imcomplete response9 ) .", "the trial of su switching in a poorly controlled diabetic 19 years old patient with r201h mutation , a mutation type expected to respond successfully , failed10 ) .", "these observations suggest that factors that can affect the success of switching to su include ; mutation type , severity of mutation , and duration of the diabetes .", "a long - standing diabetes may lead to islet beta cell exhaustion resulting in nonresponse to su9 ) .", "ndm caused by kcnj11 gene mutations is inherited in autosomal dominant pattern , but most of these cases result from new mutations without family history . the patient in this case has a novel mutation ( p.h186d ) in the kcnj11 gene and presented with a mild form of pndm .", "switching to su with an initial dose 0.4 mg / kg / day was successful at 4.8 years of age .", "oral glucose tolerance test in index patient showed improvement in insulin secretion during the treatment at 6 months and 12 months after the use of su .", "although she had hypoglycemia every 2 or 3 weeks and experienced a few episodes of hypoglycemia with mental change during insulin treatment , there was no more hypoglycemia after changing to su treatment . oral sulfonylurea therapy is both safe and better than insulin for metabolic control .", "reported side effects of oral su are transitory diarrhea and tooth discoloration in a few patients311 ) .", "mean hba1c was 8.1% in patients with insulin injection and 6.4% in patients with su therapy312 ) .", "chronic su treatment seems to develop no beta cell desensitisation in pndm patients with kcnj11 mutation13 ) .", "early su therapy at disease onset can permit insulin hypersensitivity and maintained basal insulin secretion , then provide long - term remission in animal subset14 ) . allowing for the potential beneficial effect on neurodevelopmental outcome and glycemic control , empiric tiral of su before genetic testing in neonatal diabetes patients can be considered15 ) . in conclusion , as the mutation type , severity of mutation , and duration of the diabetes are the major factors affecting the success of switching to su , early genetic analysis and trial of sulfonylurea is important in the management of neonatal diabetes .", "further studies looking at the result of su treatment and long term follow in pndm patients are needed ."], "abstract_text": ["<S> permanent neonatal diabetes mellitus refers to diabetes that occurs before the age of 6 months and persists through life . </S>", "<S> it is a rare disorder affecting one in 0.2 - 0.5 million live births . </S>", "<S> mutations in the gene kcnj11 , encoding the subunit kir6.2 , and abcc8 , encoding sur1 of the atp - sensitive potassium ( katp ) channel , are the most common causes of permanent neonatal diabetes mellitus . </S>", "<S> sulfonylureas close the katp channel and increase insulin secretion . kcnj11 and abcc8 mutations have important therapeutic implications because sulfonylurea therapy can be effective in treating patients with mutations in the potassium channel subunits . </S>", "<S> the mutation type , the presence of neurological features , and the duration of diabetes are known to be the major factors affecting the treatment outcome after switching to sulfonylurea therapy . </S>", "<S> more than 30 mutations in the kcnj11 gene have been identified . here , </S>", "<S> we present our experience with a patient carrying a novel p.h186d heterozygous mutation in the kcnj11 gene who was successfully treated with oral sulfonylurea . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["neonatal diabetes mellitus ( ndm ) presenting within the first 6 months of life includes permanent neonatal diabetes mellitus ( pndm ) , which require lifelong therapy , and transient neonatal diabetes mellitus ( tndm ) where the condition shows remission during infancy but relapses in adolescence .", "almost all cases of neonatal diabetes have monogenic etiology in contrast to the autoimmune diabetes presenting in children beyond 6 months of age .", "pndm is associated with defects in pancreatic beta cell development and function . activating mutations in the kcnj11 gene , encoding the subunit kir6.2 , and abcc8 gene ,", "encoding the sulfonylurea receptor 1 ( sur1 ) of atp - sensitive potassium ( katp ) channel , which has a key role in insulin secretion in glucose metabolism , are the most common causes and account for approximately 40% of all cases of pndm12 ) .", "mutations in the glucokinase ( gck ) and insulin ( ins ) genes have also been reported in patients with pndm .", "sulfonylureas close the katp channel by an atp - independent route , leading to increase insulin secretion4 ) .", "therefore , in many patients with pndm with kir6.2 and sur1 mutations , insulin therapy can be replaced by oral sulfonylureas which offer more improvement in glycemic control and better quality of life35 ) .", "we report a case of pndm caused by a novel p.h186d heterozygous mutation in the kcnj11 gene whose treatment was successfully transitioned from insulin to oral sulfonylurea ."], ["her parents were unrelated , and her family was healthy and had no history of diabetes .", "laboratory results at diagnosis were : blood glucose , 1,041 mg / dl ; ph , 7.025 ; hco3 , 5.1", "mmol / l ; pco2 , 19.8 mmhg ; sodium , 147 mmol / l ; potassium , 5.6 mmol / l ; chloride , 113 mmol / l ; blood urea nitrogen , 31 mg / dl ; creatinine , 1.2 mg / dl ; urine ketone , + + + ; glycosylated hemoglobin ( hba1c ) , 7.7% ; and c - peptide , 0.2 ng / ml . after recovery from diabetic ketoacidosis , she had injections of neutral protamine hagedorn ( nph ) insulin two times a day and regular insulin ( ri ) four times a day .", "her maximal daily insulin dose was at 1.5 u / kg . her insulin requirement was gradually decreased to 0.44 u / kg / day by 4 months of age .", "although there were several hypoglycemic events , insulin injection for glucose control after the infancy period could not be stopped .", "genetic study for neonatal diabetes using genomic dna extracted from peripheral lymphocyte was done at 4.5 years of age , and a novel heterozygous mutation of the kcnj11 gene located on chromosome 11p15.1 and encoding kir6.2 , c.556c > g ( p.his186asp ) , was found ( fig .", "transition of treatment from insulin to sulfonylurea was attempted at the age of 4.8 . before the trial ,", "laboratory findings were as follows : hba1c , 6.9% ; fasting glucose , 205 mg / dl ; c - peptide , 0.84 ng / ml ; 24-hour urine c - peptide , 2.2 g ; negative for islet cell antibody ; anti - insulin antibody ; and anti - gad antibody .", "she was 103.2 cm in height ( 25th-50th percentile ) , 17 kg in weight ( 50th-75th percentile ) and did not have any specific neurological deficit .", "oral sulfonylurea ( glibenclamide ) was initiated at a dose of 0.1 mg / kg / day divided into two equal doses .", "the glibenclamide dose was gradually increased to 0.4 mg / kg / day over 1 week and the insulin was stopped .", "oral glucose tolerance test ( glucose , 1.75 g / kg ) was done at 6 months and 1 year after completion of the sulfonylurea transition .", "insulinogenic index [ insulin ( 30 min-0 min)/glucose ( 30 min-0 min ) ] , a marker of beta cell function , became increased from 0.10 to 0.18 , and acute c - peptide response [ c - peptide ( 30 min-0 min)1,000/glucose ( 30 min-0 min ) ] became increased from 6.48 to 8.29 ( table 1 ) .", "the patient is now 7.5 years of age with a height of 121.4 cm ( 50th percentile ) and weight of 23.4 kg ( 50th percentile ) .", "blood glucose was well controlled without episodes of hypoglycemia and the hba1c has been lower than during insulin injection ( fig ."], ["ndm is a monogenic form of diabetes that presents within the first 6 months of life .", "heterozygous activating mutations in the kcnj11 and abcc8 gene encoding the subunits kir6.2 and sur1 of katp channel in pancreatic beta cells are the most common and account for nearly half of all cases of pndm12 ) .", "mutations in the glucokinase ( gck ) , insulin ( ins ) genes have also been reported in patients with pndm .", "the causes of rare syndromic pndm includes recessive mutations in several genes , such as pdx1 , eif2ak3 , gck , ptf1a , foxp3 , neurog3 , neurod1 , rfx6 , ier3ip1 , hnf1b , glis3 , pax6 , slc19a2 , slc2a2 , and wfs167 ) . in tndm", ", imprinted locus at chromosome 6q24 seen in more than half of cases8 ) , and kcnj11 and abcc8 gene mutations are also found in some cases .", "glucose increases intracellular atp level and it induces the closure of katp channels that lead to insulin secretion by pancreatic beta cells . activating mutations in the kcnj11 or abcc8 gene of katp channel lead to katp channels remaining open despite the presence of glucose , thus , insulin secretion can not be increased1 ) . on the contrary , loss of function mutations cause congenital hyperinsulinemia due to the closure of the katp channel and lead to increased insulin secretion", ". sulfonylureas ( su ) can close the katp channel and increase insulin secretion4 ) .", "treatment response is not expected in patients with mutations in other genes , such as glucokinase gene , foxp3 , and ipf1 .", "therefore , molecular diagnosis in neonatal diabetes may help identify patients that are likely to respond to oral sulfonylureas .", "pearson et al.3 ) reported the responses to sulfonylurea according to mutation types in diabetic patients caused by kcnj11 mutation .", "there were no differences in blood insulin , c - peptide level , and insulin injection dose between the successful group and unsuccessful group .", "v59 m , r201c , f35v , h46y , r50q , g53r , r201l , e322k , y330s , f333i mutations also belong to the successful group .", "switching to su was unsuccessful in patients with q52r , i296l , and l164p mutations .", "in addition , failure of switching to su was more related with the presence of neurologic features ( 14% in successful group and 80% in unsuccessful group ) and older age at initiation of su .", "median age was 6 years old ( intraquartile range , 3 - 12 years ) in the successful group and 18 years old ( intraquartile range , 6 - 35 years ) in the unsuccessful group3 ) .", "the report about a mother and daughter carrying the same kcnj11 mutation showed that the daughter could be switched from insulin to su at age 8.5 years , but her mother had an imcomplete response9 ) .", "the trial of su switching in a poorly controlled diabetic 19 years old patient with r201h mutation , a mutation type expected to respond successfully , failed10 ) .", "these observations suggest that factors that can affect the success of switching to su include ; mutation type , severity of mutation , and duration of the diabetes .", "a long - standing diabetes may lead to islet beta cell exhaustion resulting in nonresponse to su9 ) .", "ndm caused by kcnj11 gene mutations is inherited in autosomal dominant pattern , but most of these cases result from new mutations without family history . the patient in this case has a novel mutation ( p.h186d ) in the kcnj11 gene and presented with a mild form of pndm .", "switching to su with an initial dose 0.4 mg / kg / day was successful at 4.8 years of age .", "oral glucose tolerance test in index patient showed improvement in insulin secretion during the treatment at 6 months and 12 months after the use of su .", "although she had hypoglycemia every 2 or 3 weeks and experienced a few episodes of hypoglycemia with mental change during insulin treatment , there was no more hypoglycemia after changing to su treatment . oral sulfonylurea therapy is both safe and better than insulin for metabolic control .", "reported side effects of oral su are transitory diarrhea and tooth discoloration in a few patients311 ) .", "mean hba1c was 8.1% in patients with insulin injection and 6.4% in patients with su therapy312 ) .", "chronic su treatment seems to develop no beta cell desensitisation in pndm patients with kcnj11 mutation13 ) .", "early su therapy at disease onset can permit insulin hypersensitivity and maintained basal insulin secretion , then provide long - term remission in animal subset14 ) . allowing for the potential beneficial effect on neurodevelopmental outcome and glycemic control , empiric tiral of su before genetic testing in neonatal diabetes patients can be considered15 ) . in conclusion , as the mutation type , severity of mutation , and duration of the diabetes are the major factors affecting the success of switching to su , early genetic analysis and trial of sulfonylurea is important in the management of neonatal diabetes .", "further studies looking at the result of su treatment and long term follow in pndm patients are needed ."]]}
{"article_id": "PMC3459524", "article_text": ["radiocontrast - induced nephropathy ( rin ) can lead to acute renal failure ( arf ) , which may require dialysis therapy .", "arf increases treatment cost due to sepsis , hemorrhage , respiratory failure , and a long hospitalization.[13 ] rin is an important cause of hospital - acquired arf and is responsible for 12% of cases .", "renal medullary hypoxia and the direct toxic effects of iodinated contrast agents on renal tubules are possible mechanisms responsible for the pathophysiology of rin", ". identified specific risk factors for rin are current renal insufficiency , diabetes mellitus , and high contrast volume , dehydration , advanced age ( > 70 years ) , congestive heart failure ( chf ) , and nephrotoxic drug use ( angiotensin converting enzyme inhibitor and nonsteroidal antiinflammatory drugs ) .", "patients at risk for radiocontrast nephropathy are recommended to use nonionic iso - osmolar or nonionic low osmolar contrast agents .", "increases in serum creatinine levels are useful for detecting rin . in the majority of patients ,", "plasma creatinine levels rise within the first 2448 h after administering the radiocontrast agent , reach a peak within 35 days , and return to normal after 13 weeks .", "recent studies have reported that urine levels of il-18 ( a pro - inflammatory cytokine ) , kidney injury molecule-1 , and neutrophil gelatinase - associated lipocalin ( ngal ) levels are important for early detection of rin .", "some reports have shown that il-18 levels start to increase within 46 h and peak at 12 h in patients with acute renal injury . in this study", ", we aimed to compare the plasma creatinine levels with spot urine il-18 levels following radiocontrast administration .", "twenty patients ( 11 males and 9 females ) underwent diagnostic and therapeutic contrast - enhanced examinations at the department of internal medicine from january 2009 to march 2009 .", "the study was approved by the institute ethics committee and written consent was obtained from the selected patients based on a low mehran risk score ( 5 ) .", "patient demographic characteristics a precontrast - enhanced examination of serum blood urea nitrogen ( bun ) , creatinine , na , k , cl , ca , p , creatinine clearance was analyzed and they were repeated at 24 , 48 , and 72 h following contrast administration .", "spot urine il-18 levels were measured before and 6 and 24 h after radiocontrast administration with a human il-18 elisa kit ( biosource invitrogen human il-18 , california ) .", "intravenous iopromid ( 623 mg / ml , 1.5 ml / kg ; ultravist 300 ) , a three - way oral and rectal contrast material for abdominal ct scans , and 650 mg / ml meglumine diatrizoate ( urovist , 100 ml ) were used for every patient . glomerular filtration rate was calculated using the cockcroft ", "1 h before the procedure , 8.4% nahco3 plus 5% dextrose ( 3 ml / kg / h ) with 1200 mg / day n - acetylcysteine was given to all patients prophylactically .", "after radiocontrast agent administration , the same prophylactic treatment was continued ( 1 ml / kg / h ) for 6 h. during this time , hydration and urine output were followed by monitoring the intake and release of fluids .", "patients with no history of kidney disease , plasma creatinine values < 1.2 mg / dl , gfr60 ml / min , nondiabetic , no urinary infection , and no decompensated heart failure were included .", "urinary il-18 levels were measured with a human il-18 elisa kit ( biosource invitrogen human il-18 , carlsbad , ca , usa ) .", "the statistical analysis was performed with the ncss pass 2007 and 2008 statistical software ( kaysville , ut , usa ) .", "serum creatinine levels increased after radiocontrast administration compared with precontrast levels , although the result was not statistically significant [ table 2 ] .", "a slight increase in creatinine levels occurred at 48 h after radiocontrast administration but they fell to precontrast values at 72 h. a slight increase in plasma creatinine levels at 24 and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant ( p=0.052 and p=0.285 , respectively ) [ table 2 ] .", "plasma creatinine levels in patients before and after radiocontrast agent administration compared with precontrast urine spot il-18 levels , postcontrast 6 and 24 h urinary levels of il-18 increased significantly ( p=0.048 and p=0.028 , respectively ; table 3 ) . a tendency for postcontrast 24-h urinary il-18 levels to increase", "was observed compared with 6 h , but the increase was not statistically significant ( p=0.808 ; table 3 ) .", "spot urine il-18 levels in patients before and after radiocontrast agent administration plasma creatinine levels and spot urine il-18 were weakly but positively correlated with those during the precontrast period , although this finding was not statistically significant ( r=0.246 , p=0.126 ) .", "similarly , postcontrast 24-h plasma creatinine levels and spot urine il-18 levels were weakly but positively correlated , although this result was also not statistically significant ( r=0.254 , p=0.276 ) .", "there was no difference between pre- and postcontrast values of serum blood urea nitrogen ( bun ) , creatinine , na , k , cl , ca , p , and creatinine clearance .", "the most common definition of rin is plasma creatinine levels of 0.5 mg / dl or higher 72 h after contrast administration or 25% higher than the basal plasma creatinine level .", "plasma creatinine levels began to rise within 24 h in 80% of the patients with rin , peaking at 4872 h , and returning to baseline after 2 weeks .", "the first 24 h remains unclear in patients with acute renal injury , but il-18 levels start to increase within 46 h , peaking at 12 h. additionally , plasma creatinine is affected by age , body weight , total body volume , gender , race , drug use , muscle mass , and protein intake so researchers are looking for a diagnostic marker for rin that can be measured easily and is not affected by nonrenal factors .", "most studies related to these parameters include serum and urine cystatin c , serum and urine ngal , and urine il-18 in the analysis .", "as proinflammatory cytokine il-18 levels increase in urine , tubular inflammation , such as ischemia , reperfusion injury , allograft rejection , cisplatin toxicity , and endotoxemia occur .", "parikh et al . , found that 72 patients with acute tubular necrosis and delayed graft reaction have significantly higher il-18 levels than other kidney diseases ( urinary tract infection , chronic renal failure , nephrotic syndrome , or prerenal azotemia ) .", "reported that il-18 generally showed a low sensitivity but high specificity , respectively , for assessing an acute kidney injury diagnosis and risk classification . in our study , we examined spot urinary il-18 levels in comparison with plasma creatinine levels .", "a weak positive correlation was found between precontrast creatinine and urine il-18 levels , although it was not statistically significant . furthermore , we also found a weak positive correlation between postcontrast 24 h creatinine and urine il-18 levels , although this was not statistically significant either .", "a slight increase in plasma creatinine levels at 24 h and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant which was regressed to precontrast values at 72 h. a statistically significant increase in the level of spot urinary il-18 levels at 6 and 24 h postcontrast was observed , compared with precontrast spot urine il-18 levels and difference between 6 and 24 hour levels were not statistically significant .", "one of the limitations of this study is that there are other early biomarkers of acute kidney injury such as ngal and kim-1 .", "another limitation is that urine il-18 measurement was indexed to serum creatinine instead of urine creatinine since serum creatinine is considered to be a better and commonly used marker in the diagnosis of acute kidney injury . in conclusion ,", "spot urine il-18 levels at sixth hour following radiocontrast administration suggesting that it may be an earlier parameter for identifying kidney injury ."], "abstract_text": ["<S> radiocontrast administration is an important cause of acute renal failure . in this study , </S>", "<S> compared the plasma creatinine levels with spot urine il-18 levels following radiocontrast administration . </S>", "<S> twenty patients ( 11 males , 9 females ) underwent radiocontrast diagnostic and therapeutic - enhanced examinations . </S>", "<S> the rin mehran risk score was low ( 5 ) . </S>", "<S> the radiocontrast agents used were 623 mg / ml iopromid ( 1.5 ml / kg ) , and 100 ml of 650 mg / ml meglumine diatrizoate as three - way oral and rectal contrast material for abdominal computed tomography ( ct ) scans . </S>", "<S> serum blood urea nitrogen , creatinine , na , k , cl , ca , p , creatinine clearance , and spot urine il-18 levels were analyzed before and repeated at 24 , 48 , and 72 h after radiocontrast administration . </S>", "<S> six and 24-h urinary il-18 levels were measured with a human il-18 elisa kit following radiocontrast administration . </S>", "<S> an increase in plasma creatinine 24 and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant ( p=0.052 and p=0.285 , respectively ) . a statistically significant increase in il-18 levels was observed at 6 and 24 h , compared with precontrast values ( p=0.048 and p=0.028 , respectively ) . </S>", "<S> a tendency for postcontrast 24-h urinary il-18 levels to increase was observed compared with 6 h , but the increase was not statistically significant ( p=0.808 ) . </S>", "<S> our results show that plasma creatinine starts to increase at 24th hour ; however , spot urine il-18 levels go up at 6th hour following radiocontrast administration implying urine il-18 to be an earlier parameter for kidney injury . </S>"], "labels": null, "section_names": ["Introduction", "Patients and Methods", "Results", "Discussion"], "sections": [["radiocontrast - induced nephropathy ( rin ) can lead to acute renal failure ( arf ) , which may require dialysis therapy .", "arf increases treatment cost due to sepsis , hemorrhage , respiratory failure , and a long hospitalization.[13 ] rin is an important cause of hospital - acquired arf and is responsible for 12% of cases .", "renal medullary hypoxia and the direct toxic effects of iodinated contrast agents on renal tubules are possible mechanisms responsible for the pathophysiology of rin", ". identified specific risk factors for rin are current renal insufficiency , diabetes mellitus , and high contrast volume , dehydration , advanced age ( > 70 years ) , congestive heart failure ( chf ) , and nephrotoxic drug use ( angiotensin converting enzyme inhibitor and nonsteroidal antiinflammatory drugs ) .", "patients at risk for radiocontrast nephropathy are recommended to use nonionic iso - osmolar or nonionic low osmolar contrast agents .", "increases in serum creatinine levels are useful for detecting rin . in the majority of patients ,", "plasma creatinine levels rise within the first 2448 h after administering the radiocontrast agent , reach a peak within 35 days , and return to normal after 13 weeks .", "recent studies have reported that urine levels of il-18 ( a pro - inflammatory cytokine ) , kidney injury molecule-1 , and neutrophil gelatinase - associated lipocalin ( ngal ) levels are important for early detection of rin .", "some reports have shown that il-18 levels start to increase within 46 h and peak at 12 h in patients with acute renal injury . in this study", ", we aimed to compare the plasma creatinine levels with spot urine il-18 levels following radiocontrast administration ."], ["twenty patients ( 11 males and 9 females ) underwent diagnostic and therapeutic contrast - enhanced examinations at the department of internal medicine from january 2009 to march 2009 .", "the study was approved by the institute ethics committee and written consent was obtained from the selected patients based on a low mehran risk score ( 5 ) .", "patient demographic characteristics a precontrast - enhanced examination of serum blood urea nitrogen ( bun ) , creatinine , na , k , cl , ca , p , creatinine clearance was analyzed and they were repeated at 24 , 48 , and 72 h following contrast administration .", "spot urine il-18 levels were measured before and 6 and 24 h after radiocontrast administration with a human il-18 elisa kit ( biosource invitrogen human il-18 , california ) .", "intravenous iopromid ( 623 mg / ml , 1.5 ml / kg ; ultravist 300 ) , a three - way oral and rectal contrast material for abdominal ct scans , and 650 mg / ml meglumine diatrizoate ( urovist , 100 ml ) were used for every patient . glomerular filtration rate was calculated using the cockcroft ", "1 h before the procedure , 8.4% nahco3 plus 5% dextrose ( 3 ml / kg / h ) with 1200 mg / day n - acetylcysteine was given to all patients prophylactically .", "after radiocontrast agent administration , the same prophylactic treatment was continued ( 1 ml / kg / h ) for 6 h. during this time , hydration and urine output were followed by monitoring the intake and release of fluids .", "patients with no history of kidney disease , plasma creatinine values < 1.2 mg / dl , gfr60 ml / min , nondiabetic , no urinary infection , and no decompensated heart failure were included .", "urinary il-18 levels were measured with a human il-18 elisa kit ( biosource invitrogen human il-18 , carlsbad , ca , usa ) .", "the statistical analysis was performed with the ncss pass 2007 and 2008 statistical software ( kaysville , ut , usa ) ."], ["serum creatinine levels increased after radiocontrast administration compared with precontrast levels , although the result was not statistically significant [ table 2 ] .", "a slight increase in creatinine levels occurred at 48 h after radiocontrast administration but they fell to precontrast values at 72 h. a slight increase in plasma creatinine levels at 24 and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant ( p=0.052 and p=0.285 , respectively ) [ table 2 ] .", "plasma creatinine levels in patients before and after radiocontrast agent administration compared with precontrast urine spot il-18 levels , postcontrast 6 and 24 h urinary levels of il-18 increased significantly ( p=0.048 and p=0.028 , respectively ; table 3 ) . a tendency for postcontrast 24-h urinary il-18 levels to increase", "was observed compared with 6 h , but the increase was not statistically significant ( p=0.808 ; table 3 ) .", "spot urine il-18 levels in patients before and after radiocontrast agent administration plasma creatinine levels and spot urine il-18 were weakly but positively correlated with those during the precontrast period , although this finding was not statistically significant ( r=0.246 , p=0.126 ) .", "similarly , postcontrast 24-h plasma creatinine levels and spot urine il-18 levels were weakly but positively correlated , although this result was also not statistically significant ( r=0.254 , p=0.276 ) .", "there was no difference between pre- and postcontrast values of serum blood urea nitrogen ( bun ) , creatinine , na , k , cl , ca , p , and creatinine clearance ."], ["the most common definition of rin is plasma creatinine levels of 0.5 mg / dl or higher 72 h after contrast administration or 25% higher than the basal plasma creatinine level .", "plasma creatinine levels began to rise within 24 h in 80% of the patients with rin , peaking at 4872 h , and returning to baseline after 2 weeks .", "the first 24 h remains unclear in patients with acute renal injury , but il-18 levels start to increase within 46 h , peaking at 12 h. additionally , plasma creatinine is affected by age , body weight , total body volume , gender , race , drug use , muscle mass , and protein intake so researchers are looking for a diagnostic marker for rin that can be measured easily and is not affected by nonrenal factors .", "most studies related to these parameters include serum and urine cystatin c , serum and urine ngal , and urine il-18 in the analysis .", "as proinflammatory cytokine il-18 levels increase in urine , tubular inflammation , such as ischemia , reperfusion injury , allograft rejection , cisplatin toxicity , and endotoxemia occur .", "parikh et al . , found that 72 patients with acute tubular necrosis and delayed graft reaction have significantly higher il-18 levels than other kidney diseases ( urinary tract infection , chronic renal failure , nephrotic syndrome , or prerenal azotemia ) .", "reported that il-18 generally showed a low sensitivity but high specificity , respectively , for assessing an acute kidney injury diagnosis and risk classification . in our study , we examined spot urinary il-18 levels in comparison with plasma creatinine levels .", "a weak positive correlation was found between precontrast creatinine and urine il-18 levels , although it was not statistically significant . furthermore , we also found a weak positive correlation between postcontrast 24 h creatinine and urine il-18 levels , although this was not statistically significant either .", "a slight increase in plasma creatinine levels at 24 h and 48 h following radiocontrast administration was observed compared with precontrast values , but it was not statistically significant which was regressed to precontrast values at 72 h. a statistically significant increase in the level of spot urinary il-18 levels at 6 and 24 h postcontrast was observed , compared with precontrast spot urine il-18 levels and difference between 6 and 24 hour levels were not statistically significant .", "one of the limitations of this study is that there are other early biomarkers of acute kidney injury such as ngal and kim-1 .", "another limitation is that urine il-18 measurement was indexed to serum creatinine instead of urine creatinine since serum creatinine is considered to be a better and commonly used marker in the diagnosis of acute kidney injury . in conclusion ,", "spot urine il-18 levels at sixth hour following radiocontrast administration suggesting that it may be an earlier parameter for identifying kidney injury ."]]}
{"article_id": "PMC5423572", "article_text": ["according to the u.s . national institutes of health , up to 80% of human bacterial infections involve biofilm - associated microorganisms 1 . among these , implant - related infections", "do still have a tremendous impact in orthopaedics and trauma 2 , with high social and economic costs 3 , 4 , posing challenging diagnostic and therapeutic dilemmas 5 .", "in fact , peri - prosthetic joint infection ( pji ) remains one of the most feared complications in orthopaedic surgery and among the first reasons for implant failure 6 .", "moreover , given the increasing number of hip and knee arthroplasties performed , the prevalence of this complication is rising , with increasing costs for national health systems and increasing biological costs for the patients , such as loss or reduced joint function and deterioration in their physical and psychological health 7 . according to the widely accepted model of the ' race for the surface ' for pji development ,", "host and bacterial cells compete for surface colonization , with a low probability of bacterial attachment if host cells adhere to implant first , and vice versa . in the event of bacterial adhesion to an implant ,", "in addition , the matrix protects the biofilm cells from various microbicidal agents and stresses , including dehydration , toxins , ultraviolet light , chemical disinfectants , temperature and osmotic shock , and lead them to increased resistance against antimicrobials 9 , 10 . to address the limited efficacy of existing antibiotics in the treatment of established bacterial biofilms , novel approaches are required to prevent bacterial adhesion and biofilm formation 11 .", "adhesion is a necessary first step in microbial colonization and pathogenesis and provides a good theoretical target for new preventive and treatment strategies 12 .", "bacterial adhesion to surfaces can be divided into a first , reversible phase and a second , irreversible phase .", "once an implant is inserted into the body , it is covered by a conditional protein layer composed of host proteins , such as albumin and complement , that act as a reservoir of several receptors for bacterial adhesive ligands , mediating adhesion of free - floating bacteria to the surface of the biomaterials 14 , 15 ; these first adhesions are , however mechanically and biologically unstable .", "few minutes after this first , reversible phase , bacterial clusters attached to the surface starts to express biofilm related genes , produce glycocalyx and form mature biofilm , thus transforming the adhesion from reversible to irreversible 16 .", "full - formed biofilm can be found few hours after the first bacterial adhesion 17 .", "antimicrobial surface coatings can be based on an anti - adhesive principle that prevents bacteria to adhere and form biofilms 18 .", "in fact , some polymer coatings , like the hydrophilic polymethacrylic acid , polyethylene oxide or protein - resistant polyethylene glycol can be applied to the surface of titanium implants and result in significant inhibition of bacterial adhesion 19 - 22 .", "hydrophobic and superhydrophobic surface treatment technologies have also shown a great repellent antibacterial effect in preclinical studies 23 - 25 .", "however , clinical application of completely novel coating technologies and compounds , not otherwise previously tested in humans , appears particularly challenging 26 .", "bacterial colonization can also be blocked by an inhibitor interfering with ligand - receptor interaction for bacterial attachment .", "one of these inhibitors could be hyaluronic acid ( ha ) , a glycosaminoglycan made up of glucuronic acid and n - acetylglucosamine disaccharide units .", "it is a uniform , linear and unbranched molecule , with highly variable length and molecular weight ( up to 106 da ) .", "it is abundant in skin ( up to 56% ) and in connective tissues , with a turnover ranging from several hours to a few days depending on tissues .", "hyaluronic acid constitutes one of the main components of extracellular matrices . because of its biological properties , ha has several clinical applications ( aesthetic surgery , dermatology , dentistry , orthopedics and opthalmology ) 27 .", "extensive studies on the chemical and physicochemical properties of ha and its physiological role in humans , together with its versatile properties , such as its biocompatibility , non - immunogenicity , biodegradability , and viscoelasticity , have proved that it is an ideal biomaterial for medical and pharmaceutical applications 28 , 29 . among its various properties ,", "several studies have recently shown the ability of ha to protect against various infectious agents 30 , depending on ha concentration and molecular weight 31 , 32 , while more recently ha interference on bacterial adhesion and biofilm formation has been extensively investigated 33 .", "given its high biocompatibility and well known safety profile and the anti - adhesive capabilities , ha and its composites represent an attractive , non - antibiotic , option to mitigate the impact of biofilm - related infections in various clinical settings including implant - related infections . aim of this review is to provide an update of the current evidence concerning ha ability to reduce / prevent bacterial adhesion and biofilm formation .", "nearly two decades ago , pavesio et al . 34 were probably the first to describe the ability of ha to resist bacterial adhesion , with particular reference to staphylococcus epidermidis , and its non - fouling properties 35 , proposing coated polymeric medical devices ( e.g. , intraocular lenses , stents and catheters ) to reduce implant - related infections .", "in particular , a hydrophilic ha overlayer , linked to the surface of polymethylmethacrylate intraocular lenses ( iols ) , was shown to be able to prevent fibroblasts adhesion and to greatly reduce staphyloccous epidermidis adhesion to the implant surface 36 .", "the impact of slime dispersants and anti - adhesives on in vitro biofilm formation on iols was further investigated by kadry and co - workers 37 , using a staphyloccous epidermidis wild strain , isolated from a patient with endophtalmitis ; the authors reported the ability of hyaluronan to reduce bacterial adhesion to iols to 30% , compared with untreated control cells .", "the authors suggested the use of adjuvant therapy such as dispersants or anti - adhesives , in addition to the antibiotics in irrigating solutions for bacterial ocular infections .", "more recently , the in vitro antiadhesive and antibiofilm activity of hyaluronic acid towards bacterial species commonly isolated from respiratory infections was investigated by drago et al .", "33 . in this study , the interference exerted on bacterial adhesion was evaluated by using hep-2 cells , while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet .", "the experimental findings clearly demonstrated how hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration - dependent manner .", "moreover , staphylococcus aureus biofilm was found to be more sensitive to the action of ha , compared to that produced by haemophilus influenzae and moraxella catarrhalis . concerning more specifically the antimicrobial activity , ha", "has also been shown to exert varied bacteriostatic , but not bactericidal , dose - dependent effects on different microorganisms in the planktonic phase 31 , 38 . in this", "regard , radaeva et al . reported the inhibiting activity of ha with respect to some pseudomonas species 39 , while ardizzoni and co - workers 30 investigated the effects of ha on 15 atcc bacterial strains , representative of clinically relevant bacterial and fungal species .", "their results showed that different microbial species and , sometimes , different strains belonging to the same species , are differently affected by ha . in particular , staphylococci , enterococci , streptococcus mutans , two escherichia coli strains , pseudomonas aeruginosa , candida glabrata and c. parapsilosis displayed a ha dose - dependent growth inhibition , while no ha effects were detected in e. coli atcc 13768 and c. albicans and s. sanguinis was favoured by the highest ha dose . comparing the potential bacteriostatic effect of some of the most commonly used biomatrix materials ( collagen type i , hyaluronic acid , hydroxyapatite , polylactic acid and polyglycolic acid ) on the growth over the first 12h of exposure of some of the most common orthopaedic bacterial pathogens ( staphylococcus aureus , staphylococcus epidermidis , -hemolytic streptococcus , pseudomonas aeruginosa ) , carlson and co - workers 38 found that ha had the most significant bacteriostatic properties on the studied organisms .", "31 investigated the potential bacteriostatic effect of hyaluronic acid in different concentrations and molecular weight on oral and non - oral microorganisms ( staphylococcus aureus , propionibacterium acnes , actinobacillus actinomycetemcomitans , pavotella oris and porphyromonas gingivalis ) with potential application in dentistry surgery ; the results showed that different hyaluronan solutions exerted varied bacteriostatic effects on all the bacterial strains .", "the authors concluded that the clinical application of hyaluronan in form of membranes , gels , or sponges during surgical therapy may reduce bacterial contamination of the surgical wound , thereby lessening the risk of postsurgical infection and promoting more predictable regeneration .", "concerning possible orthopaedic applications , in 2004 harris and richards 40 showed the visualization and quantification of s. aureus adhering to a variety of different treated / coated titanium surfaces . in their study ,", "coating titanium with sodium hyaluronate significantly decreased the density of s. aureus adhering to the surfaces and its potential use in osteosynthesis , orthopaedics or dental applications was suggested out . in a very recent review on polysaccharide - based coatings , that have been proposed over the last ten years to impede biofilm formation on material surfaces exposed to bacterial contamination ,", "hyaluronic acid was discussed as one of the most studied , with demonstrated non - fouling properties on glass surfaces 41 ; displaying hydrophilic characteristics ( contact angle of 22 ) , this coating was in fact reported to reduce adhesion of s. epidermidis and e. coli by several orders of magnitude compared to the unmodified glass slide .", "similarly , adhesion of s. aureus on ti foils functionalized with hyaluronic acid - catechol was lower than on pristine substrates .", "based on ha antiadhesive properties , a novel ha - based hydrogel has been recently proposed , in order to protect implanted biomaterials in orthopaedics , trauma and dental surgery from bacterial colonization 42 ; this fast - resorbable hydrogel coating , composed of covalently linked hyaluronan and poly - d , l - lactide (  defensive antibacterial coating  , dac , novagenit srl , mezzolombardo , italy ) , has been found to have a synergistic antibiofilm activity with various antibacterials and able to be effectively manually spread onto the surface of various biomaterials commonly used in orthopaedics , trauma and dental surgery 43 ( fig .", "1 ) . the ability to completely cover even sand - blasted titanium surface and resist scraping has in fact been confirmed by scanning electron microscopy ( sem ) analysis ( cf .", "this is an important requirement in order to reduce the exposed surface of a biomaterial , thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria . in unpublished experiments ( novagenit srl , data on file ) , in order to evaluate dac ability to prevent bacterial adhesion , 200 mg of hydrogel were homogenously spread on the surface of sterile titanium discs .", "hydrogel - coated substrates and uncoated substrates ( controls ) were then placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells for 15 , 30 , 60 and 120 minutes .", "the remaining adhered cells were detached by adding a solution of 0.1% w / v dithiothreitol ( dtt ) ( sigma - aldrich , milan , italy ) to each well and stirring for 15 minutes at room temperature .", "then , 100 l of each sample were plated onto tryptic soy agar ( tsa ; merck , darmstadt , germany ) and incubated at 37c for 24 hours for cfu counts .", "the results showed that the adhesion density of s. aureus on titanium discs pre - treated with dac , was significantly lower than adhesion on untreated controls at each time point ( fig .", "2 ) . in particular , reductions of adhered bacteria equal to 86.8% , 80.4% ,", "74.6% and 66.7% vs untreated discs were observed after 15 , 30 , 60 and 120 minutes of incubation , respectively , while an increase of adhesion density during time was observed for both control and pre - treated discs ( fig .", "further analyses were conducted to show the ability to dislodge previously adhered bacteria ; to this aim , titanium discs were placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells in order to allow the adhesion of bacterial cells .", "afterwards , 200 mg of hydrogel were spread on the surface of contaminated titanium discs in order to remove previously adhered bacteria .", "non - adherent bacteria were removed by rinsing with sterile saline , while the remaining adhered cells were detached by adding 0.1% dtt as previously described .", "then , 100 l of each sample were plated onto tsa and incubated at 37c for 24 hours for cfu counts .", "the results showed that dac hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15 , 30 , 60 and 120 minutes of 84.0% , 72.8% , 72.3% and 64.3% , respectively ( fig.4 ) .", "once again , an increase of adhesion density during time was observed for both control and treated discs ( fig .", "dac hydrogel showed similar or superior in vitro activity , compared to various antibacterials and a synergistic activity when used in combination ( fig .", "were grown on chrome - cobalt devices in 6-wells polystyrene plates containing tsb for 24 hours at 37c .", "then , growth medium was removed together with non - adherent bacteria and new broth added .", "the plates were incubated at 37c in ambient air , until a visible biofilm was obtained .", "gentamycin and vancomycin were tested at a final concentration of 20 mg / ml . similarly , when mixed with the hydrogel , 60 mg of gel powder were reconstituted with 1 ml of water for injections containing gentamicin or vancomycin at 20 mg / ml concentration .", "amount of biofilm at each time was determined before hydrogel and antibiotic agents addition and after 0.5 , 1 , 2 , 4 , 6 , 24 and 48 hours of incubation by a spectrophotometric assay .", "in particular , at each time , broth was removed and biofilm stained with crystal violet .", "after elution of the stain from implants with absolute ethanol , the amount of biofilm was quantified by reading optical density ( o.d . ) at a wavelength of 595 nm against blank ( consisting of ethanol ) .", "amount of biofilm at each time was compared with that formed on the same type of implant before treatment .", "each assay was performed in duplicate and repeated for three times . at each time point , both for gentamycin and vancomycin showed only a partial inhibition of biofilm formation ( ca .", "40 - 50% for vancomycin ) , with minor difference between the two studied microorganisms . on the other side ,", "50% in comparison to the untreated controls , while a combination of the hydrogel with either antibacterial resulted in a larger reduction of biofilm formation ( approximately 75 to 80% in comparison with untreated controls ) .", "both these experimental studies show the ability of the dac hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens , thus potentially providing an effective protection of the implant ; however , these data also point out how , in the clinical setting , in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics , a passive antiadhesive coating 18 like ha can be overcome by the remaining bacteria in a time - dependent manner .", "for this reason , any passive antiadhesive coating of implants 44 should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree , also depending on the local environment , the contaminating bacterial species and initial bacterial load ; this may still provide an additional advantage to the host 's cells to first colonize the implanted biomaterial and win the competition with the microorganisms that may eventually be present , thus contributing to reduce the occurrence of implant - related infections .", "several clinical local applications of ha to reduce the impact of biofilm - related infections have been reported , in different clinical settings , with favourable results and no adverse events .", "45 recently described topical administration of hyaluronic acid in children with recurrent or chronic middle ear inflammations and chronic adenoiditis . in this prospective , single - blind , randomised controlled study , otoscopy , tympanometry and pure - tone audiometry in children which received the daily topical administration of normal 0.9% sodium chloride saline solution ( control group ) or 9 mg of sodium hyaluronate in 3 ml of a 0.9% sodium saline solution was performed .", "the final analysis was based on 116 children ( 49.1% boys ; mean age , 62.9  17.9 months ) : 58 in the control group and 58 in the study group . at the end of follow - up ,", "the prevalence of patients with impaired otoscopy was significantly lower in the study group ( p value = 0.024 ) compared to baseline but not in the control group . in comparison with baseline ,", "the prevalence of patients with impaired tympanometry at the end of the follow - up period was significantly lower in the study group ( p value = 0.047 ) but not in the control group .", "the reduction in the prevalence of patients with conductive hearing loss ( chl ) ( p value = 0.008 ) and those with moderate chl ( p value = 0.048 ) was significant in the study group , but not in the control group .", "the mean auditory threshold had also significantly improved by the end of treatment in the study group ( p value = 0.004 ) but not in the control group .", "several studies have also reported the beneficial effect of topical ha in chronic urinary tract infections ( uti ) .", "in contrast to traditional antibiotic therapy , which aims at eradicating pathogens , treatment with ha targets bacterial adherence to the bladder mucosa with the presumption that a damaged glycosaminoglycan mucous layer facilitates bacterial adherence and therefore recurrent uti 46 . among others 47 , 48 ,", "lipovac and colleagues evaluated the efficacy of nine ha bladder instillations over 6 months in 20 women with a history of recurrent uti .", "their status was assessed prospectively but compared with a retrospective review of patients ' charts .", "the number of infections per year per patient was significantly reduced ( from 4.990.92 to 0.560.82 , p>0.001 ) and the mean time to recurrence ( from 76.724.6 to 178.325.5 days , p>0.001 ) was prolonged significantly .", "nevertheless 65% of treated patients were free of recurrences until the end of study ( 47.6 weeks ) 49 .", "were able to provide a higher level of evidence by reporting a prospective , randomized , double - blind , placebo - controlled study , in which a significant reduction of 77% ( p<0.0002 ) in the uti rate per patient per year versus placebo was observed at the end of the study .", "moreover , mean time to uti recurrence was significantly prolonged ( 185.278.7 versus 52.733.4 days , p<0.001 ) after treatment compared with placebo .", "overall urinary symptoms and quality of life measured by questionnaires significantly improved compared with placebo 50 .", "very recently a multicentre european study confirmed the efficacy of intravesical administration of combined hyaluronic acid and chondroitin sulphate ( cs ) for the treatment of female recurrent urinary tract infections 51 .", "a total of 276 adult women received intravesical administration of ha+cs or standard of care ( antimicrobial/ immunoactive prophylaxis/ probiotics / cranberry ) . at follow - up ,", "181 patients treated with ha+cs and 95 patients treated with standard of care from 7 centres were available .", "the crude and adjusted ors ( 95% ci ) for bacteriologically confirmed recurrence within 12 months were 0.77 ( 0.46 to 1.28 ) and 0.51 ( 0.27 to 0.96 ) , respectively .", "studies were also undertaken to determine the effect on clinical variables , sub - gingival bacteria and local immune response brought about by application of hyaluronan - containing gels in early wound healing after scaling and root planing ( srp ) in dentistry 52 , 53 . in the study reported from eick et al .", "the exclusion criteria were : antibiotics intake in the 6 months before the study , periodontal treatment received during the previous year , pregnancy , nursing , smoking , chronic diseases such as diabetes mellitus or rheumatoid arthritis , and allergy to ingredients in the drug . in the test group ( n=17 ) , a 0.8% hyaluronan - containing gels ( ha ) was introduced into all periodontal pockets during srp and a 0.2% ha gel was applied by the patients onto the gingival margin twice daily during the following 2 weeks while the control group ( n=17 ) was treated with srp only ; no placebo was used . probing depth ( pd ) and clinical attachment level ( cal )", "were recorded at baseline and after 3 and 6 months , and subgingival plaque and sulcus fluid samples were taken for microbiologic and biochemical analysis . in both groups , pd and cal", "the changes in pd and the reduction of the number of pockets with pd5 mm were significantly higher in the test group after 3 ( p=0.014 and 0.021 ) and 6 ( p=0.046 and 0.045 ) months .", "six months after srp , the counts of treponema denticola were significantly reduced in both groups ( both p=0.043 ) , as were those of campylobacter rectus in the test group only ( p=0.028 ) .", "although to date no surface modification has been reported to be able to fully prevent bacterial adhesion and biofilm formation 55 , available data show that hyaluronic acid has a proven in vitro antiadhesive / antibiofilm effect against some of the most common pathogens and it has been used safely , alone or in combination with other polymers , with satisfactory results in different conditions associated with biofilm - related chronic infections .", "clinical data in various applications , including dentistry , urology , wound management , dermatology and orthopedics , allow to consider the potential use of ha as a protective coating barrier of implants particularly safe and feasible on a large scale basis .", "while antibacterial coatings to mitigate the occurrence of implant- and biofilm - related infections are regarded as one of the most needed technology , currently only few and insufficient options are available for clinical use in orthopedics and trauma surgery 18 .", "considering the pathogenesis of implant - related infections , any protection offered by a fully biocompatible antiadhesive barrier , like ha and some of its derivatives , could be extremely useful to reduce the tremendous burden of implant - related infections .", "on the other hand , it should be noted that hyaluronic acid as a passive protective barrier has some limits . among others ,", "the antiadhesive / antibiofilm effect is limited and may vary , depending on the type of the microorganism , the bacterial load , the local environment , etc .", "; moreover , ha protection may be neutralized by the possible ability of some bacteria to produce hyaluronidase , an enzyme that catalyzes the degradation of hyaluronic acid 56 , while collagen and hyaluronan may even become possible ligands for microbial attachment in particular situations 57 , 58 .", "to overcome at least some of these limits , possible loading of hyaluronic - based hydrogels with antibiotics is technically feasible and has been proposed by different authors 59 - 62 , being a possible option for future developments and large scale clinical applications , provided that regulatory requirements can be met ."], "abstract_text": ["<S> living in biofilms is probably the most common condition for bacteria and fungi and biofilm - related infections account for the majority of bacterial infectious diseases worldwide.among others biofilm - related infections , those associated with implanted biomaterials have an enormous and still largely underestimated impact in orthopaedics and trauma , cardio - surgery and several other surgical disciplines.given the limited efficacy of existing antibiotics in the prevention and treatment of bacterial biofilms , new strategies are needed to protect implants and host tissues , overcoming the striking ability of the microorganisms to adhere on different surfaces and to immediately protect themselves by forming the biofilm matrix.adhesion is a necessary first step in microbial colonization and pathogenesis and provides a potential target for new preventive and treatment approach.among various polymers , tested as antibacterial coatings , hyaluronic acid and some of its composites do offer a well - established long - term safety profile and a proven ability to reduce bacterial adhesion and biofilm formation.aim of the present review is to summarize the available evidence concerning the antiadhesion / antibiofilm activity of hyaluronic acid and some of its derivatives to reduce / prevent bacterial adhesion and biofilm formation in various experimental and clinical settings . </S>"], "labels": null, "section_names": ["Introduction", "Anti-adhesive and anti-biofilm properties of hyaluronic acid and its composites", "Clinical applications of hyaluronic acid to prevent bacterial adhesion", "Conclusion"], "sections": [["according to the u.s . national institutes of health , up to 80% of human bacterial infections involve biofilm - associated microorganisms 1 . among these , implant - related infections", "do still have a tremendous impact in orthopaedics and trauma 2 , with high social and economic costs 3 , 4 , posing challenging diagnostic and therapeutic dilemmas 5 .", "in fact , peri - prosthetic joint infection ( pji ) remains one of the most feared complications in orthopaedic surgery and among the first reasons for implant failure 6 .", "moreover , given the increasing number of hip and knee arthroplasties performed , the prevalence of this complication is rising , with increasing costs for national health systems and increasing biological costs for the patients , such as loss or reduced joint function and deterioration in their physical and psychological health 7 . according to the widely accepted model of the ' race for the surface ' for pji development ,", "host and bacterial cells compete for surface colonization , with a low probability of bacterial attachment if host cells adhere to implant first , and vice versa . in the event of bacterial adhesion to an implant ,", "in addition , the matrix protects the biofilm cells from various microbicidal agents and stresses , including dehydration , toxins , ultraviolet light , chemical disinfectants , temperature and osmotic shock , and lead them to increased resistance against antimicrobials 9 , 10 . to address the limited efficacy of existing antibiotics in the treatment of established bacterial biofilms , novel approaches are required to prevent bacterial adhesion and biofilm formation 11 .", "adhesion is a necessary first step in microbial colonization and pathogenesis and provides a good theoretical target for new preventive and treatment strategies 12 .", "bacterial adhesion to surfaces can be divided into a first , reversible phase and a second , irreversible phase .", "once an implant is inserted into the body , it is covered by a conditional protein layer composed of host proteins , such as albumin and complement , that act as a reservoir of several receptors for bacterial adhesive ligands , mediating adhesion of free - floating bacteria to the surface of the biomaterials 14 , 15 ; these first adhesions are , however mechanically and biologically unstable .", "few minutes after this first , reversible phase , bacterial clusters attached to the surface starts to express biofilm related genes , produce glycocalyx and form mature biofilm , thus transforming the adhesion from reversible to irreversible 16 .", "full - formed biofilm can be found few hours after the first bacterial adhesion 17 .", "antimicrobial surface coatings can be based on an anti - adhesive principle that prevents bacteria to adhere and form biofilms 18 .", "in fact , some polymer coatings , like the hydrophilic polymethacrylic acid , polyethylene oxide or protein - resistant polyethylene glycol can be applied to the surface of titanium implants and result in significant inhibition of bacterial adhesion 19 - 22 .", "hydrophobic and superhydrophobic surface treatment technologies have also shown a great repellent antibacterial effect in preclinical studies 23 - 25 .", "however , clinical application of completely novel coating technologies and compounds , not otherwise previously tested in humans , appears particularly challenging 26 .", "bacterial colonization can also be blocked by an inhibitor interfering with ligand - receptor interaction for bacterial attachment .", "one of these inhibitors could be hyaluronic acid ( ha ) , a glycosaminoglycan made up of glucuronic acid and n - acetylglucosamine disaccharide units .", "it is a uniform , linear and unbranched molecule , with highly variable length and molecular weight ( up to 106 da ) .", "it is abundant in skin ( up to 56% ) and in connective tissues , with a turnover ranging from several hours to a few days depending on tissues .", "hyaluronic acid constitutes one of the main components of extracellular matrices . because of its biological properties , ha has several clinical applications ( aesthetic surgery , dermatology , dentistry , orthopedics and opthalmology ) 27 .", "extensive studies on the chemical and physicochemical properties of ha and its physiological role in humans , together with its versatile properties , such as its biocompatibility , non - immunogenicity , biodegradability , and viscoelasticity , have proved that it is an ideal biomaterial for medical and pharmaceutical applications 28 , 29 . among its various properties ,", "several studies have recently shown the ability of ha to protect against various infectious agents 30 , depending on ha concentration and molecular weight 31 , 32 , while more recently ha interference on bacterial adhesion and biofilm formation has been extensively investigated 33 .", "given its high biocompatibility and well known safety profile and the anti - adhesive capabilities , ha and its composites represent an attractive , non - antibiotic , option to mitigate the impact of biofilm - related infections in various clinical settings including implant - related infections . aim of this review is to provide an update of the current evidence concerning ha ability to reduce / prevent bacterial adhesion and biofilm formation ."], ["nearly two decades ago , pavesio et al . 34 were probably the first to describe the ability of ha to resist bacterial adhesion , with particular reference to staphylococcus epidermidis , and its non - fouling properties 35 , proposing coated polymeric medical devices ( e.g. , intraocular lenses , stents and catheters ) to reduce implant - related infections .", "in particular , a hydrophilic ha overlayer , linked to the surface of polymethylmethacrylate intraocular lenses ( iols ) , was shown to be able to prevent fibroblasts adhesion and to greatly reduce staphyloccous epidermidis adhesion to the implant surface 36 .", "the impact of slime dispersants and anti - adhesives on in vitro biofilm formation on iols was further investigated by kadry and co - workers 37 , using a staphyloccous epidermidis wild strain , isolated from a patient with endophtalmitis ; the authors reported the ability of hyaluronan to reduce bacterial adhesion to iols to 30% , compared with untreated control cells .", "the authors suggested the use of adjuvant therapy such as dispersants or anti - adhesives , in addition to the antibiotics in irrigating solutions for bacterial ocular infections .", "more recently , the in vitro antiadhesive and antibiofilm activity of hyaluronic acid towards bacterial species commonly isolated from respiratory infections was investigated by drago et al .", "33 . in this study , the interference exerted on bacterial adhesion was evaluated by using hep-2 cells , while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet .", "the experimental findings clearly demonstrated how hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration - dependent manner .", "moreover , staphylococcus aureus biofilm was found to be more sensitive to the action of ha , compared to that produced by haemophilus influenzae and moraxella catarrhalis . concerning more specifically the antimicrobial activity , ha", "has also been shown to exert varied bacteriostatic , but not bactericidal , dose - dependent effects on different microorganisms in the planktonic phase 31 , 38 . in this", "regard , radaeva et al . reported the inhibiting activity of ha with respect to some pseudomonas species 39 , while ardizzoni and co - workers 30 investigated the effects of ha on 15 atcc bacterial strains , representative of clinically relevant bacterial and fungal species .", "their results showed that different microbial species and , sometimes , different strains belonging to the same species , are differently affected by ha . in particular , staphylococci , enterococci , streptococcus mutans , two escherichia coli strains , pseudomonas aeruginosa , candida glabrata and c. parapsilosis displayed a ha dose - dependent growth inhibition , while no ha effects were detected in e. coli atcc 13768 and c. albicans and s. sanguinis was favoured by the highest ha dose . comparing the potential bacteriostatic effect of some of the most commonly used biomatrix materials ( collagen type i , hyaluronic acid , hydroxyapatite , polylactic acid and polyglycolic acid ) on the growth over the first 12h of exposure of some of the most common orthopaedic bacterial pathogens ( staphylococcus aureus , staphylococcus epidermidis , -hemolytic streptococcus , pseudomonas aeruginosa ) , carlson and co - workers 38 found that ha had the most significant bacteriostatic properties on the studied organisms .", "31 investigated the potential bacteriostatic effect of hyaluronic acid in different concentrations and molecular weight on oral and non - oral microorganisms ( staphylococcus aureus , propionibacterium acnes , actinobacillus actinomycetemcomitans , pavotella oris and porphyromonas gingivalis ) with potential application in dentistry surgery ; the results showed that different hyaluronan solutions exerted varied bacteriostatic effects on all the bacterial strains .", "the authors concluded that the clinical application of hyaluronan in form of membranes , gels , or sponges during surgical therapy may reduce bacterial contamination of the surgical wound , thereby lessening the risk of postsurgical infection and promoting more predictable regeneration .", "concerning possible orthopaedic applications , in 2004 harris and richards 40 showed the visualization and quantification of s. aureus adhering to a variety of different treated / coated titanium surfaces . in their study ,", "coating titanium with sodium hyaluronate significantly decreased the density of s. aureus adhering to the surfaces and its potential use in osteosynthesis , orthopaedics or dental applications was suggested out . in a very recent review on polysaccharide - based coatings , that have been proposed over the last ten years to impede biofilm formation on material surfaces exposed to bacterial contamination ,", "hyaluronic acid was discussed as one of the most studied , with demonstrated non - fouling properties on glass surfaces 41 ; displaying hydrophilic characteristics ( contact angle of 22 ) , this coating was in fact reported to reduce adhesion of s. epidermidis and e. coli by several orders of magnitude compared to the unmodified glass slide .", "similarly , adhesion of s. aureus on ti foils functionalized with hyaluronic acid - catechol was lower than on pristine substrates .", "based on ha antiadhesive properties , a novel ha - based hydrogel has been recently proposed , in order to protect implanted biomaterials in orthopaedics , trauma and dental surgery from bacterial colonization 42 ; this fast - resorbable hydrogel coating , composed of covalently linked hyaluronan and poly - d , l - lactide (  defensive antibacterial coating  , dac , novagenit srl , mezzolombardo , italy ) , has been found to have a synergistic antibiofilm activity with various antibacterials and able to be effectively manually spread onto the surface of various biomaterials commonly used in orthopaedics , trauma and dental surgery 43 ( fig .", "1 ) . the ability to completely cover even sand - blasted titanium surface and resist scraping has in fact been confirmed by scanning electron microscopy ( sem ) analysis ( cf .", "this is an important requirement in order to reduce the exposed surface of a biomaterial , thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria . in unpublished experiments ( novagenit srl , data on file ) , in order to evaluate dac ability to prevent bacterial adhesion , 200 mg of hydrogel were homogenously spread on the surface of sterile titanium discs .", "hydrogel - coated substrates and uncoated substrates ( controls ) were then placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells for 15 , 30 , 60 and 120 minutes .", "the remaining adhered cells were detached by adding a solution of 0.1% w / v dithiothreitol ( dtt ) ( sigma - aldrich , milan , italy ) to each well and stirring for 15 minutes at room temperature .", "then , 100 l of each sample were plated onto tryptic soy agar ( tsa ; merck , darmstadt , germany ) and incubated at 37c for 24 hours for cfu counts .", "the results showed that the adhesion density of s. aureus on titanium discs pre - treated with dac , was significantly lower than adhesion on untreated controls at each time point ( fig .", "2 ) . in particular , reductions of adhered bacteria equal to 86.8% , 80.4% ,", "74.6% and 66.7% vs untreated discs were observed after 15 , 30 , 60 and 120 minutes of incubation , respectively , while an increase of adhesion density during time was observed for both control and pre - treated discs ( fig .", "further analyses were conducted to show the ability to dislodge previously adhered bacteria ; to this aim , titanium discs were placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells in order to allow the adhesion of bacterial cells .", "afterwards , 200 mg of hydrogel were spread on the surface of contaminated titanium discs in order to remove previously adhered bacteria .", "non - adherent bacteria were removed by rinsing with sterile saline , while the remaining adhered cells were detached by adding 0.1% dtt as previously described .", "then , 100 l of each sample were plated onto tsa and incubated at 37c for 24 hours for cfu counts .", "the results showed that dac hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15 , 30 , 60 and 120 minutes of 84.0% , 72.8% , 72.3% and 64.3% , respectively ( fig.4 ) .", "once again , an increase of adhesion density during time was observed for both control and treated discs ( fig .", "dac hydrogel showed similar or superior in vitro activity , compared to various antibacterials and a synergistic activity when used in combination ( fig .", "were grown on chrome - cobalt devices in 6-wells polystyrene plates containing tsb for 24 hours at 37c .", "then , growth medium was removed together with non - adherent bacteria and new broth added .", "the plates were incubated at 37c in ambient air , until a visible biofilm was obtained .", "gentamycin and vancomycin were tested at a final concentration of 20 mg / ml . similarly , when mixed with the hydrogel , 60 mg of gel powder were reconstituted with 1 ml of water for injections containing gentamicin or vancomycin at 20 mg / ml concentration .", "amount of biofilm at each time was determined before hydrogel and antibiotic agents addition and after 0.5 , 1 , 2 , 4 , 6 , 24 and 48 hours of incubation by a spectrophotometric assay .", "in particular , at each time , broth was removed and biofilm stained with crystal violet .", "after elution of the stain from implants with absolute ethanol , the amount of biofilm was quantified by reading optical density ( o.d . ) at a wavelength of 595 nm against blank ( consisting of ethanol ) .", "amount of biofilm at each time was compared with that formed on the same type of implant before treatment .", "each assay was performed in duplicate and repeated for three times . at each time point , both for gentamycin and vancomycin showed only a partial inhibition of biofilm formation ( ca .", "40 - 50% for vancomycin ) , with minor difference between the two studied microorganisms . on the other side ,", "50% in comparison to the untreated controls , while a combination of the hydrogel with either antibacterial resulted in a larger reduction of biofilm formation ( approximately 75 to 80% in comparison with untreated controls ) .", "both these experimental studies show the ability of the dac hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens , thus potentially providing an effective protection of the implant ; however , these data also point out how , in the clinical setting , in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics , a passive antiadhesive coating 18 like ha can be overcome by the remaining bacteria in a time - dependent manner .", "for this reason , any passive antiadhesive coating of implants 44 should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree , also depending on the local environment , the contaminating bacterial species and initial bacterial load ; this may still provide an additional advantage to the host 's cells to first colonize the implanted biomaterial and win the competition with the microorganisms that may eventually be present , thus contributing to reduce the occurrence of implant - related infections ."], ["several clinical local applications of ha to reduce the impact of biofilm - related infections have been reported , in different clinical settings , with favourable results and no adverse events .", "45 recently described topical administration of hyaluronic acid in children with recurrent or chronic middle ear inflammations and chronic adenoiditis . in this prospective , single - blind , randomised controlled study , otoscopy , tympanometry and pure - tone audiometry in children which received the daily topical administration of normal 0.9% sodium chloride saline solution ( control group ) or 9 mg of sodium hyaluronate in 3 ml of a 0.9% sodium saline solution was performed .", "the final analysis was based on 116 children ( 49.1% boys ; mean age , 62.9  17.9 months ) : 58 in the control group and 58 in the study group . at the end of follow - up ,", "the prevalence of patients with impaired otoscopy was significantly lower in the study group ( p value = 0.024 ) compared to baseline but not in the control group . in comparison with baseline ,", "the prevalence of patients with impaired tympanometry at the end of the follow - up period was significantly lower in the study group ( p value = 0.047 ) but not in the control group .", "the reduction in the prevalence of patients with conductive hearing loss ( chl ) ( p value = 0.008 ) and those with moderate chl ( p value = 0.048 ) was significant in the study group , but not in the control group .", "the mean auditory threshold had also significantly improved by the end of treatment in the study group ( p value = 0.004 ) but not in the control group .", "several studies have also reported the beneficial effect of topical ha in chronic urinary tract infections ( uti ) .", "in contrast to traditional antibiotic therapy , which aims at eradicating pathogens , treatment with ha targets bacterial adherence to the bladder mucosa with the presumption that a damaged glycosaminoglycan mucous layer facilitates bacterial adherence and therefore recurrent uti 46 . among others 47 , 48 ,", "lipovac and colleagues evaluated the efficacy of nine ha bladder instillations over 6 months in 20 women with a history of recurrent uti .", "their status was assessed prospectively but compared with a retrospective review of patients ' charts .", "the number of infections per year per patient was significantly reduced ( from 4.990.92 to 0.560.82 , p>0.001 ) and the mean time to recurrence ( from 76.724.6 to 178.325.5 days , p>0.001 ) was prolonged significantly .", "nevertheless 65% of treated patients were free of recurrences until the end of study ( 47.6 weeks ) 49 .", "were able to provide a higher level of evidence by reporting a prospective , randomized , double - blind , placebo - controlled study , in which a significant reduction of 77% ( p<0.0002 ) in the uti rate per patient per year versus placebo was observed at the end of the study .", "moreover , mean time to uti recurrence was significantly prolonged ( 185.278.7 versus 52.733.4 days , p<0.001 ) after treatment compared with placebo .", "overall urinary symptoms and quality of life measured by questionnaires significantly improved compared with placebo 50 .", "very recently a multicentre european study confirmed the efficacy of intravesical administration of combined hyaluronic acid and chondroitin sulphate ( cs ) for the treatment of female recurrent urinary tract infections 51 .", "a total of 276 adult women received intravesical administration of ha+cs or standard of care ( antimicrobial/ immunoactive prophylaxis/ probiotics / cranberry ) . at follow - up ,", "181 patients treated with ha+cs and 95 patients treated with standard of care from 7 centres were available .", "the crude and adjusted ors ( 95% ci ) for bacteriologically confirmed recurrence within 12 months were 0.77 ( 0.46 to 1.28 ) and 0.51 ( 0.27 to 0.96 ) , respectively .", "studies were also undertaken to determine the effect on clinical variables , sub - gingival bacteria and local immune response brought about by application of hyaluronan - containing gels in early wound healing after scaling and root planing ( srp ) in dentistry 52 , 53 . in the study reported from eick et al .", "the exclusion criteria were : antibiotics intake in the 6 months before the study , periodontal treatment received during the previous year , pregnancy , nursing , smoking , chronic diseases such as diabetes mellitus or rheumatoid arthritis , and allergy to ingredients in the drug . in the test group ( n=17 ) , a 0.8% hyaluronan - containing gels ( ha ) was introduced into all periodontal pockets during srp and a 0.2% ha gel was applied by the patients onto the gingival margin twice daily during the following 2 weeks while the control group ( n=17 ) was treated with srp only ; no placebo was used . probing depth ( pd ) and clinical attachment level ( cal )", "were recorded at baseline and after 3 and 6 months , and subgingival plaque and sulcus fluid samples were taken for microbiologic and biochemical analysis . in both groups , pd and cal", "the changes in pd and the reduction of the number of pockets with pd5 mm were significantly higher in the test group after 3 ( p=0.014 and 0.021 ) and 6 ( p=0.046 and 0.045 ) months .", "six months after srp , the counts of treponema denticola were significantly reduced in both groups ( both p=0.043 ) , as were those of campylobacter rectus in the test group only ( p=0.028 ) ."], ["although to date no surface modification has been reported to be able to fully prevent bacterial adhesion and biofilm formation 55 , available data show that hyaluronic acid has a proven in vitro antiadhesive / antibiofilm effect against some of the most common pathogens and it has been used safely , alone or in combination with other polymers , with satisfactory results in different conditions associated with biofilm - related chronic infections .", "clinical data in various applications , including dentistry , urology , wound management , dermatology and orthopedics , allow to consider the potential use of ha as a protective coating barrier of implants particularly safe and feasible on a large scale basis .", "while antibacterial coatings to mitigate the occurrence of implant- and biofilm - related infections are regarded as one of the most needed technology , currently only few and insufficient options are available for clinical use in orthopedics and trauma surgery 18 .", "considering the pathogenesis of implant - related infections , any protection offered by a fully biocompatible antiadhesive barrier , like ha and some of its derivatives , could be extremely useful to reduce the tremendous burden of implant - related infections .", "on the other hand , it should be noted that hyaluronic acid as a passive protective barrier has some limits . among others ,", "the antiadhesive / antibiofilm effect is limited and may vary , depending on the type of the microorganism , the bacterial load , the local environment , etc .", "; moreover , ha protection may be neutralized by the possible ability of some bacteria to produce hyaluronidase , an enzyme that catalyzes the degradation of hyaluronic acid 56 , while collagen and hyaluronan may even become possible ligands for microbial attachment in particular situations 57 , 58 .", "to overcome at least some of these limits , possible loading of hyaluronic - based hydrogels with antibiotics is technically feasible and has been proposed by different authors 59 - 62 , being a possible option for future developments and large scale clinical applications , provided that regulatory requirements can be met ."]]}
{"article_id": "PMC4749793", "article_text": ["in 2011 , who declared  combat drug resistance : no action today , no cure tomorrow .  . in recent years", "presently , antimicrobial resistance ( amr ) poses a major threat to patient 's treatment as it leads to increased morbidity and mortality , increased hospital stay , and severe economic loss to the patient and nation [ 3 , 4 ] .", "the clinical isolates such as pseudomonas aeruginosa , methicillin resistant staphylococcus aureus ( mrsa ) , enterococci especially vancomycin resistant enterococci ( vre ) , and members of family enterobacteriaceae , for example , klebsiella pneumoniae , e. coli , and proteus sp .", " in the last two decades , there were so much increase of infectious diseases that the standard of public health in many parts of the world is equivalent to preantibiotic era .", "as per standardized international terminology created by european centre for disease control ( ecdc ) and centre for disease control & prevention ( cdc ) , atlanta , the multidrug - resistant ( mdr ) , extensively drug - resistant ( xdr ) , and pandrug - resistant ( pdr ) bacteria have been well defined .", "multidrug resistant ( mdr ) was defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories . extensively drug", "resistant ( xdr ) was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories ( i.e. , bacterial isolates remain susceptible to only one or two antimicrobial categories ) .", "pandrug resistant ( pdr ) was defined as nonsusceptibility to all agents in all antimicrobial categories .", "hence , this short term study was undertaken to detect the incidence of mdr , xdr , and pdr bacterial isolates in a tertiary care hospital of central india .", "this short term cross - sectional study was conducted in the department of microbiology from 15th of april to 15th of july , 2014 .", "the bacterial strains were isolated from different clinical samples and were identified by conventional methods .", "the clinical specimens from indoor patient departments ( ipd ) only were included in the study .", "antibiotic susceptibility test of bacterial strains was done by kirby bauer disc diffusion method   as per clinical laboratory standard institute ( clsi ) guidelines .", "antibiotics used for gram positive cocci ( gpc ) were penicillin , erythromycin , ciprofloxacin , tetracycline , amikacin , vancomycin , and linezolid and for gram negative bacilli ( gnb ) were amikacin , ceftazidime , ceftazidime - clavulanic acid , ciprofloxacin , imipenem , and colistin , respectively .", "linezolid and colistin were used as supplemental drugs . for urine sample , instead of ciprofloxacin and tetracycline ,", "for routine quality control of antibiotic susceptibility test , s. aureus atcc 25923 , e. coli atcc 25922 , and pseudomonas aeruginosa atcc 27853 were used .", "mdr , xdr , and pdr strains were detected as per criteria described by ecdc and cdc .", "methicillin resistant staphylococcus aureus ( mrsa ) strains were detected by meca - mediated oxacillin resistance using cefoxitin disk ( 30  g ) on mueller hinton ( mh ) agar plate inoculated with test strains as per standard disk diffusion recommendations and incubated at 3335c for 1618 hours .", "inhibition zone 21  mm with cefoxitin disk was interpreted as meca positive according to clsi guidelines .", "extended spectrum -lactamases ( esbl ) producing strains were detected by combined disk method using ceftazidime ( 30  g ) and ceftazidime plus clavulanic acid ( 30  g plus 10  g ) .", "an increase in diameter of 5  mm with ceftazidime plus clavulanic acid as compared to ceftazidime disk alone was considered positive for esbl detection .", "138 clinical samples were received from intensive care unit ( icu ) and 742 clinical samples were received from wards of different clinical specialities .", "698 clinical samples had single bacterial growth and 182 had mixed bacterial growth . out of these 182 clinical samples , 172 samples had 2 bacterial isolates , 4 samples had 3 bacterial isolates , and 6 samples had 1 bacterial isolate along with candida albicans .", "figure 1 shows that 314 ( 29.6% ) bacterial strains were gram positive cocci ( gpc ) and 746 ( 70.4% ) were gram negative bacilli ( gnb ) .", "out of 314 gpc , 252 ( 80.3% ) were coagulase positive staphylococci . amongst 746 gnb , 261 ( 35% ) were e. coli , followed by pseudomonas aeruginosa 212 ( 28.4% ) . during the study period ,", "figure 2 shows the incidence of mdr and xdr strains isolated . out of total 1060 bacterial strains", "studied , 393 ( 37.1% ) bacterial strains were mdr and 146 ( 13.8% ) strains were xdr .", "amongst 314 gpc strains isolated , 143 ( 45.5% ) and 56 ( 17.8% ) were mdr and xdr , respectively .", "out of 746 gnb isolates , 250 ( 33.5% ) strains were mdr and 90 ( 12.1% ) were xdr .", "out of total 9304 patients admitted , 393 ( 4.2% ) and 146 ( 1.6% ) were positive for mdr and xdr strains , respectively . \n", "figure 3 shows the incidence of mdr and xdr gram positive cocci isolated . out of total 252 coagulase positive staphylococci", "isolated , 125 ( 49.6% ) were mdr and 38 ( 15.1% ) were xdr .", "10 coagulase negative staphylococci were isolated and 5 ( 50% ) were mdr , whereas 2 ( 20% ) were xdr .", "79 ( 31.3% ) coagulase positive staphylococci strains were mrsa and 2 ( 20% ) coagulase negative staphylococci were mrcons . out of total 45 enterococci isolated , 13 ( 28.9% ) were mdr and 16 ( 35.6% ) were xdr .", "no vancomycin intermediate staphylococcus aureus ( visa ) , vancomycin resistant staphylococcus aureus ( vrsa ) , or vancomycin resistant enterococci ( vre ) were isolated .", "all streptococcus species including group a , nongroup a , and pneumococcus were sensitive to penicillin .", "no mdr or xdr strain was isolated from streptococcus sp . all ( 100% ) gram positive cocci were sensitive to vancomycin and linezolid . \n", "figure 4 shows incidence of mdr and xdr strains isolated from each species of gram negative bacilli . in the present study ,", "e. coli was the commonest isolate 261 ( 35% ) , followed by pseudomonas aeruginosa 212 ( 28.4% ) .", "79 ( 30.3% ) and 22 ( 8.4% ) e. coli strains were mdr and xdr , respectively .", "out of 200 klebsiella pneumoniae strains isolated , 75 ( 37.5% ) and 25 ( 12.5% ) were detected as mdr and xdr , respectively . out of 42 acinetobacter and other nonfermenter species isolated , 19 ( 45.2% ) and 8 ( 19% ) were mdr and xdr strains , respectively . amongst 250 gnb - mdr strains isolated ,", "the commonest mdr strains were detected from e. coli 79/250 ( 31.6% ) , followed by klebsiella pneumoniae 75/250 ( 30% ) .", "similarly , out of 90 gnb - xdr strains isolated , the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 ( 32.2% ) , followed by klebsiella pneumoniae 25/90 ( 27.8% ) . in the present study , 137 ( 18.4% )", "others include wards like dermatology , pulmonary medicine , orthopedics , and cardiovascular and thoracic surgery ( cvts ) .", "the different icus include neonatal icu ( nicu ) , medicine icu ( micu ) , operation theatre icu ( ot icu ) , and paediatric icu ( picu ) .", "out of total 393 mdr strains detected , 127 ( 32.3% ) ( the highest number ) mdr strains were isolated from surgery wards followed by 72 ( 18.3% ) mdr strains from different icus .", "amongst total 146 xdr strains isolated , 41 ( 28.1% ) ( the highest number ) were isolated from surgery wards also .", "out of 72 mdr strains detected from different icus , 29 ( 40.3% ) ( the highest number ) mdr strains were isolated from nicu , followed by 20 ( 27.8% ) and 18 ( 25% ) from ot icu , and only 5 ( 6.9% ) from picu . even in the total 26 xdr strains isolated from different icus , 10 ( 38.5% ) ( the highest number )", "the percentage of mdr and xdr strains isolated from different icus was 72/138 ( 52.2% ) and 26/138 ( 18.8% ) , respectively , which were again much more than mdr and xdr strains isolated from wards 321/742 ( 43.3% ) and 120/742 ( 16.2% ) , respectively .", "275 patients were admitted to nicu , of whom 29 ( 10.5% ) were positive for mdr strains and 10 ( 3.6% ) were positive for xdr strains . out of total 1907", "patients admitted to surgery ward , 127 ( 6.7% ) were positive for mdr strains whereas 41 ( 2.1% ) were positive for xdr strains . in micu ,", "545 patients were admitted and 20 ( 3.7% ) were positive for mdr strains and 8 ( 1.5% ) were positive for xdr strains .", "the clinical and financial burden to patients and health care providers for mdros is really challenging .", "barbara soule , joint commission resources practice leader , infection prevention and control services , has told , ", "patients who are infected with mdros often have an increased risk of prolonged illness and mortality .", "the cost of care for these patients can be more than double as compared to those without mdro infection  . since the year 2000 , only 4 new classes of antibiotics have been approved by food and drug administration ( fda ) , us , for example , linezolid , streptogramins , daptomycin , and tigecycline .", "the problem is that the bacteria are developing resistance at a much faster pace than the new drug development . regarding public health attention , mdros are described as superbugs having very limited treatment options . for some mdros", ", only 1 or 2 antibiotics can be effective with toxic side effects . in 2009 , boucher et al .", "have reported eskape organisms as  bad bugs ,  where e stands for enterococcus faecium , s for staphylococcus aureus , k for klebsiella pneumoniae , a for acinetobacter baumannii , p for pseudomonas aeruginosa , and e for enterobacter species . in the year 2009 only", ", peterson has reported the escape group of organism , which was the same as the above list but k was replaced by c , that is , clostridium difficile , and the last e stands for enterobacteriaceae . in the present study , amongst 250 gnb - mdr strains isolated , the commonest mdr strains were detected from e. coli 79/250 ( 31.6% ) , followed by klebsiella pneumoniae 75/250 ( 30% ) .", "similarly , out of 90 gnb - xdr strains isolated , the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 ( 32.2% ) , followed by klebsiella pneumoniae 25/90 ( 27.8% ) .", "aly and balkhy reported that most prevalent mdro in their study was e. coli followed by klebsiella pneumoniae . in another study , carried out in a tertiary care hospital in riyadh", ", it has been reported that most frequent mdr pathogens were pseudomonas aeruginosa followed by e. coli .", "the percentage of mdr e. coli strains was more than klebsiella pneumoniae and even pseudomonas aeruginosa in our study probably because a total number of e. coli strains isolated ( 261 ) were also higher .", "the slightly increased incidence of drug resistant strains observed in our study may be because our hospital is a tertiary care center in a rural setup and patients from adjoining districts and even villages are admitted for treatment . before attending the hospital , most of the patients get different antibiotics from general practitioners or due to over - the - counter sell of antibiotics often in improper dose .", "the limitation of this study is that this is a single center study for only three - month period in a tertiary care hospital in central india . to reflect the trend of infections caused by mdr and xdr strains of bacteria in the region , a multicenter study involving all types of healthcare setups for a minimum period of one year", "there is paucity of data regarding mdros in health care setup not only in india but also worldwide .", "unless and until multidrug resistant organisms are detected and their incidence is known , the strategies for their control can not be adopted properly in healthcare setup . hence , detection , prevention of transmission of mdros by following infection control practices , antimicrobial surveillance , and stewardship are need of the hour .", "misuse and overuse of antibiotics , over - the - counter selling of antibiotics without prescription to common people , must be stopped by strict implementations of rules and regulations .", "we hereby conclude that early detection and close monitoring of mdr , xdr , or even pdr bacterial strains must be started by all clinical microbiology laboratories to reduce the menace of antimicrobial resistance which is now a global problem ."], "abstract_text": ["<S> \n background and objective . </S>", "<S> antimicrobial resistance is now a major challenge to clinicians for treating patients . </S>", "<S> hence , this short term study was undertaken to detect the incidence of multidrug - resistant ( mdr ) , extensively drug - resistant ( xdr ) , and pandrug - resistant ( pdr ) bacterial isolates in a tertiary care hospital . </S>", "<S> material and methods . </S>", "<S> the clinical samples were cultured and bacterial strains were identified in the department of microbiology . </S>", "<S> the antibiotic susceptibility profile of different bacterial isolates was studied to detect mdr , xdr , and pdr bacteria . </S>", "<S> results . the antibiotic susceptibility profile of 1060 bacterial strains was studied . </S>", "<S> 393 ( 37.1% ) bacterial strains were mdr , 146 ( 13.8% ) strains were xdr , and no pdr was isolated . </S>", "<S> all ( 100% ) gram negative bacterial strains were sensitive to colistin whereas all ( 100% ) gram positive bacterial strains were sensitive to vancomycin . </S>", "<S> conclusion . </S>", "<S> close monitoring of mdr , xdr , or even pdr must be done by all clinical microbiology laboratories to implement effective measures to reduce the menace of antimicrobial resistance . </S>"], "labels": null, "section_names": ["1. Introduction ", "2. Material and Methods", "3. Observations and Results", "4. Discussion", "5. Conclusion"], "sections": [["in 2011 , who declared  combat drug resistance : no action today , no cure tomorrow .  . in recent years", "presently , antimicrobial resistance ( amr ) poses a major threat to patient 's treatment as it leads to increased morbidity and mortality , increased hospital stay , and severe economic loss to the patient and nation [ 3 , 4 ] .", "the clinical isolates such as pseudomonas aeruginosa , methicillin resistant staphylococcus aureus ( mrsa ) , enterococci especially vancomycin resistant enterococci ( vre ) , and members of family enterobacteriaceae , for example , klebsiella pneumoniae , e. coli , and proteus sp .", " in the last two decades , there were so much increase of infectious diseases that the standard of public health in many parts of the world is equivalent to preantibiotic era .", "as per standardized international terminology created by european centre for disease control ( ecdc ) and centre for disease control & prevention ( cdc ) , atlanta , the multidrug - resistant ( mdr ) , extensively drug - resistant ( xdr ) , and pandrug - resistant ( pdr ) bacteria have been well defined .", "multidrug resistant ( mdr ) was defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories . extensively drug", "resistant ( xdr ) was defined as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories ( i.e. , bacterial isolates remain susceptible to only one or two antimicrobial categories ) .", "pandrug resistant ( pdr ) was defined as nonsusceptibility to all agents in all antimicrobial categories .", "hence , this short term study was undertaken to detect the incidence of mdr , xdr , and pdr bacterial isolates in a tertiary care hospital of central india ."], ["this short term cross - sectional study was conducted in the department of microbiology from 15th of april to 15th of july , 2014 .", "the bacterial strains were isolated from different clinical samples and were identified by conventional methods .", "the clinical specimens from indoor patient departments ( ipd ) only were included in the study .", "antibiotic susceptibility test of bacterial strains was done by kirby bauer disc diffusion method   as per clinical laboratory standard institute ( clsi ) guidelines .", "antibiotics used for gram positive cocci ( gpc ) were penicillin , erythromycin , ciprofloxacin , tetracycline , amikacin , vancomycin , and linezolid and for gram negative bacilli ( gnb ) were amikacin , ceftazidime , ceftazidime - clavulanic acid , ciprofloxacin , imipenem , and colistin , respectively .", "linezolid and colistin were used as supplemental drugs . for urine sample , instead of ciprofloxacin and tetracycline ,", "for routine quality control of antibiotic susceptibility test , s. aureus atcc 25923 , e. coli atcc 25922 , and pseudomonas aeruginosa atcc 27853 were used .", "mdr , xdr , and pdr strains were detected as per criteria described by ecdc and cdc .", "methicillin resistant staphylococcus aureus ( mrsa ) strains were detected by meca - mediated oxacillin resistance using cefoxitin disk ( 30  g ) on mueller hinton ( mh ) agar plate inoculated with test strains as per standard disk diffusion recommendations and incubated at 3335c for 1618 hours .", "inhibition zone 21  mm with cefoxitin disk was interpreted as meca positive according to clsi guidelines .", "extended spectrum -lactamases ( esbl ) producing strains were detected by combined disk method using ceftazidime ( 30  g ) and ceftazidime plus clavulanic acid ( 30  g plus 10  g ) .", "an increase in diameter of 5  mm with ceftazidime plus clavulanic acid as compared to ceftazidime disk alone was considered positive for esbl detection ."], ["138 clinical samples were received from intensive care unit ( icu ) and 742 clinical samples were received from wards of different clinical specialities .", "698 clinical samples had single bacterial growth and 182 had mixed bacterial growth . out of these 182 clinical samples , 172 samples had 2 bacterial isolates , 4 samples had 3 bacterial isolates , and 6 samples had 1 bacterial isolate along with candida albicans .", "figure 1 shows that 314 ( 29.6% ) bacterial strains were gram positive cocci ( gpc ) and 746 ( 70.4% ) were gram negative bacilli ( gnb ) .", "out of 314 gpc , 252 ( 80.3% ) were coagulase positive staphylococci . amongst 746 gnb , 261 ( 35% ) were e. coli , followed by pseudomonas aeruginosa 212 ( 28.4% ) . during the study period ,", "figure 2 shows the incidence of mdr and xdr strains isolated . out of total 1060 bacterial strains", "studied , 393 ( 37.1% ) bacterial strains were mdr and 146 ( 13.8% ) strains were xdr .", "amongst 314 gpc strains isolated , 143 ( 45.5% ) and 56 ( 17.8% ) were mdr and xdr , respectively .", "out of 746 gnb isolates , 250 ( 33.5% ) strains were mdr and 90 ( 12.1% ) were xdr .", "out of total 9304 patients admitted , 393 ( 4.2% ) and 146 ( 1.6% ) were positive for mdr and xdr strains , respectively . \n", "figure 3 shows the incidence of mdr and xdr gram positive cocci isolated . out of total 252 coagulase positive staphylococci", "isolated , 125 ( 49.6% ) were mdr and 38 ( 15.1% ) were xdr .", "10 coagulase negative staphylococci were isolated and 5 ( 50% ) were mdr , whereas 2 ( 20% ) were xdr .", "79 ( 31.3% ) coagulase positive staphylococci strains were mrsa and 2 ( 20% ) coagulase negative staphylococci were mrcons . out of total 45 enterococci isolated , 13 ( 28.9% ) were mdr and 16 ( 35.6% ) were xdr .", "no vancomycin intermediate staphylococcus aureus ( visa ) , vancomycin resistant staphylococcus aureus ( vrsa ) , or vancomycin resistant enterococci ( vre ) were isolated .", "all streptococcus species including group a , nongroup a , and pneumococcus were sensitive to penicillin .", "no mdr or xdr strain was isolated from streptococcus sp . all ( 100% ) gram positive cocci were sensitive to vancomycin and linezolid . \n", "figure 4 shows incidence of mdr and xdr strains isolated from each species of gram negative bacilli . in the present study ,", "e. coli was the commonest isolate 261 ( 35% ) , followed by pseudomonas aeruginosa 212 ( 28.4% ) .", "79 ( 30.3% ) and 22 ( 8.4% ) e. coli strains were mdr and xdr , respectively .", "out of 200 klebsiella pneumoniae strains isolated , 75 ( 37.5% ) and 25 ( 12.5% ) were detected as mdr and xdr , respectively . out of 42 acinetobacter and other nonfermenter species isolated , 19 ( 45.2% ) and 8 ( 19% ) were mdr and xdr strains , respectively . amongst 250 gnb - mdr strains isolated ,", "the commonest mdr strains were detected from e. coli 79/250 ( 31.6% ) , followed by klebsiella pneumoniae 75/250 ( 30% ) .", "similarly , out of 90 gnb - xdr strains isolated , the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 ( 32.2% ) , followed by klebsiella pneumoniae 25/90 ( 27.8% ) . in the present study , 137 ( 18.4% )", "others include wards like dermatology , pulmonary medicine , orthopedics , and cardiovascular and thoracic surgery ( cvts ) .", "the different icus include neonatal icu ( nicu ) , medicine icu ( micu ) , operation theatre icu ( ot icu ) , and paediatric icu ( picu ) .", "out of total 393 mdr strains detected , 127 ( 32.3% ) ( the highest number ) mdr strains were isolated from surgery wards followed by 72 ( 18.3% ) mdr strains from different icus .", "amongst total 146 xdr strains isolated , 41 ( 28.1% ) ( the highest number ) were isolated from surgery wards also .", "out of 72 mdr strains detected from different icus , 29 ( 40.3% ) ( the highest number ) mdr strains were isolated from nicu , followed by 20 ( 27.8% ) and 18 ( 25% ) from ot icu , and only 5 ( 6.9% ) from picu . even in the total 26 xdr strains isolated from different icus , 10 ( 38.5% ) ( the highest number )", "the percentage of mdr and xdr strains isolated from different icus was 72/138 ( 52.2% ) and 26/138 ( 18.8% ) , respectively , which were again much more than mdr and xdr strains isolated from wards 321/742 ( 43.3% ) and 120/742 ( 16.2% ) , respectively .", "275 patients were admitted to nicu , of whom 29 ( 10.5% ) were positive for mdr strains and 10 ( 3.6% ) were positive for xdr strains . out of total 1907", "patients admitted to surgery ward , 127 ( 6.7% ) were positive for mdr strains whereas 41 ( 2.1% ) were positive for xdr strains . in micu ,", "545 patients were admitted and 20 ( 3.7% ) were positive for mdr strains and 8 ( 1.5% ) were positive for xdr strains ."], ["the clinical and financial burden to patients and health care providers for mdros is really challenging .", "barbara soule , joint commission resources practice leader , infection prevention and control services , has told , ", "patients who are infected with mdros often have an increased risk of prolonged illness and mortality .", "the cost of care for these patients can be more than double as compared to those without mdro infection  . since the year 2000 , only 4 new classes of antibiotics have been approved by food and drug administration ( fda ) , us , for example , linezolid , streptogramins , daptomycin , and tigecycline .", "the problem is that the bacteria are developing resistance at a much faster pace than the new drug development . regarding public health attention , mdros are described as superbugs having very limited treatment options . for some mdros", ", only 1 or 2 antibiotics can be effective with toxic side effects . in 2009 , boucher et al .", "have reported eskape organisms as  bad bugs ,  where e stands for enterococcus faecium , s for staphylococcus aureus , k for klebsiella pneumoniae , a for acinetobacter baumannii , p for pseudomonas aeruginosa , and e for enterobacter species . in the year 2009 only", ", peterson has reported the escape group of organism , which was the same as the above list but k was replaced by c , that is , clostridium difficile , and the last e stands for enterobacteriaceae . in the present study , amongst 250 gnb - mdr strains isolated , the commonest mdr strains were detected from e. coli 79/250 ( 31.6% ) , followed by klebsiella pneumoniae 75/250 ( 30% ) .", "similarly , out of 90 gnb - xdr strains isolated , the commonest xdr strains were detected from pseudomonas aeruginosa 29/90 ( 32.2% ) , followed by klebsiella pneumoniae 25/90 ( 27.8% ) .", "aly and balkhy reported that most prevalent mdro in their study was e. coli followed by klebsiella pneumoniae . in another study , carried out in a tertiary care hospital in riyadh", ", it has been reported that most frequent mdr pathogens were pseudomonas aeruginosa followed by e. coli .", "the percentage of mdr e. coli strains was more than klebsiella pneumoniae and even pseudomonas aeruginosa in our study probably because a total number of e. coli strains isolated ( 261 ) were also higher .", "the slightly increased incidence of drug resistant strains observed in our study may be because our hospital is a tertiary care center in a rural setup and patients from adjoining districts and even villages are admitted for treatment . before attending the hospital , most of the patients get different antibiotics from general practitioners or due to over - the - counter sell of antibiotics often in improper dose .", "the limitation of this study is that this is a single center study for only three - month period in a tertiary care hospital in central india . to reflect the trend of infections caused by mdr and xdr strains of bacteria in the region , a multicenter study involving all types of healthcare setups for a minimum period of one year", "there is paucity of data regarding mdros in health care setup not only in india but also worldwide .", "unless and until multidrug resistant organisms are detected and their incidence is known , the strategies for their control can not be adopted properly in healthcare setup . hence , detection , prevention of transmission of mdros by following infection control practices , antimicrobial surveillance , and stewardship are need of the hour .", "misuse and overuse of antibiotics , over - the - counter selling of antibiotics without prescription to common people , must be stopped by strict implementations of rules and regulations ."], ["we hereby conclude that early detection and close monitoring of mdr , xdr , or even pdr bacterial strains must be started by all clinical microbiology laboratories to reduce the menace of antimicrobial resistance which is now a global problem ."]]}
{"article_id": "PMC4815391", "article_text": ["ureterocele is a congenital urinary abnormality characterized by the presence of an intrabladder hernia or cystic ballooning of the lower end of a ureter lying between the mucosa and muscle of the bladder .", "the abnormality leads to urinary retention and recurrent urinary tract infection ( uti ) , which can cause irreversible damage to the kidney .", "this abnormality can be suspected in the fetus by antenatal ultrasonography ( usg ) and confirmed by other x - ray investigations after birth .", "the incidence of ureterocele is variable with the highest rate of 1:500 and it is generally found in females with duplex system association ( 95% ) .", "ureteroceles can have different clinical presentations , such as antenatal hydronephrosis , uti , vesicoureteral reflux ( vur ) , bladder outlet obstruction , prolapsed urethral mass , etc .", "usg and voiding cystourethrography ( vcug ) are essential initial procedures for a child suspected of having a ureteral anomaly .", "a 5-year - old female child was referred to our department for tc - diethylene triamine pentaacetic acid ( dtpa ) scan .", "she was a follow - up case of right - sided hydroureteronephrosis with pyonephrosis with percutaneous tube ( pcn ) insertion done on the right side .", "patient 's blood urea and serum creatinine were 29 and 1.3 mg / dl , respectively .", "usg of the abdomen revealed left - sided mild hydroureteronephrosis and gross enlargement of the right kidney , reaching up to lower abdomen with dilated pelvis and ureter .", "the child was referred for evaluation of glomerular filtration rate and excretory function of kidneys .", "tc - dtpa scan was done in our department with right pcn tube in situ .", "pcn tube was clamped during the acquisition of dynamic and prevoid static image and clamp released thereafter . on tc - dtpa scan , left kidney showed good perfusion and adequate cortical radiotracer concentration followed by good drainage into dilated ureter .", "right ureter and right pelvicalyceal system ( pcs ) were visualized in the postvoid image ( indirect evidence of vur ) [ figure 2b , thin arrow ] .", "retention of radiotracer was noted in dilated pcs and dilated ureter in delayed static images acquired till 4 h [ figure 2c and d ] .", "faint visualization of radiotracer was noted through the pcn tube after removal of clamp [ figure 2b , bold arrow ] .", "a large photopenia was noted in the suprapubic region in the urinary bladder ( ub ) during dynamic [ figure 1 ] as well as delayed static images acquired till 4 h [ figure 2a  d ] .", "an usg of the abdomen was done in the radiology department of our hospital to find out the cause of this persistent photopenia in the ub .", "usg of the abdomen showed evidence of left - sided mild hydroureteronephrosis and grossly enlarged hydronephrotic right kidney with dilated right ureter and an intracystic ureterocele arising from the right side [ figure 3a and b ] .", "perfusion and dynamic images of 99mtc - diethylene triamine pentaacetic acid scan scintigraphy , posterior view with percutaneous clamp in situ , showing good perfusion and adequate cortical function and good drainage of left kidney .", "photopenia noted in the urinary bladder ( bold arrow ) ( a ) prevoid static image with right pcn clamp in situ .", "right ureter and right pelvicalyceal system visualization is noted suggesting indirect evidence of vesico - ureteric reflux ( thin arrow ) .", "( c and d ) 3 and 4 h delayed static images showing persistent photopenia in the urinary bladder ( arrow marked ) ( a and b ) ultrasonography images depicting right ureterocele within the urinary bladder ( ub ) and dilated distal right ureter", "according to literature , 90% of the patients with ureterocele are diagnosed before the age of 3 years .", "most of the patients with ureterocele are classically diagnosed during the investigation for uti , asymptomatic hydronephrosis , and abdominal mass .", "although the age of diagnosis is decreasing , uti is still the most common clinical presentation of ureterocele in 50% of the patients promoting physician to make the thorough evaluation of the urinary system .", "the whole nephronourinary system could have already been negatively affected at the time of diagnosis . in our case ,", "usg is an easy method to perform , noninvasive , and probably the best imaging modality for making the diagnosis .", "reflux can occur in the ipsilateral lower pole in almost half of the patients , but the contralateral system is also affected at a rate of 25% . the tc - dimercapto succinic acid ( dmsa ) scan should be undertaken routinely to assess the distribution of function in the duplex kidney and for detecting and follow - up of scarred tissue and nonfunctioning upper poles in cases of ureterocele .", "did a study on pediatric ureteroceles in 19 patients , on the diagnosis , management and treatment options .", "vcug was able to detect vur in 13 out of 17 patients ( 33% ) .", "dmsa scintigraphy showed ipsilateral renal scarring and nonfunctioning upper pole images in 7 out of 13 patients . computed tomography and mag3 scintigraphy", "did a study on the management of 36 patients with varied presentation of complicated ureteroceles .", "usg , micturating cystourethrogram , isotope renogram were done preoperatively in all the babies . in one of the cases ,", "initial usg was unable to detect ureterocele in most of the cases in this study .", "they said that unless the usg is done in well - hydrated , cooperative patient , ureterocele is likely to be missed on usg .", "hence , an intravenous pyelogram , radioisotope renogram , and micturating cystourethrogram are invaluable in the complete understanding of ureterocele . in our patient also , the diagnosis of ureterocele was missed on initial usg .", "photopenia was noted in the ub on our scan , and this prompted us to investigate the patient further .", "possible causes for photopenic defects in the ub on nuclear scintigraphic studies include bladder papilloma , bladder polyp , carcinoma bladder , bladder calculus , foreign body in ub , intravesical ureterocele , etc .", "repeat usg revealed the cause of photopenia in ub in our case to be an intravesical ureterocele . on reviewing the literature we found that tc - dtpa scan is useful in the evaluation of renal function in patient with known ureteroceles .", "however , in our case on finding the photopenia in the ub on tc - dtpa scan , intravesical ureterocele was detected on usg ."], "abstract_text": ["<S> ureterocele is a common ureteric anomaly detected in pediatric population . </S>", "<S> ureterocele diagnosis and evaluation need a variety of radiological methods . </S>", "<S> we report a case of 5-year - old female child sent for 99mtc - diethylene triamine pentaacetic acid scan for evaluation of glomerular filtration rate and excretory function of kidneys in view of right - sided hydroureteronephrosis and pyonephrosis with percutaneous tube in situ . </S>", "<S> incidental photopenia was noted in the urinary bladder . </S>", "<S> on ultrasonography of abdomen cause of this photopenia was found to be an intravesical ureterocele . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["ureterocele is a congenital urinary abnormality characterized by the presence of an intrabladder hernia or cystic ballooning of the lower end of a ureter lying between the mucosa and muscle of the bladder .", "the abnormality leads to urinary retention and recurrent urinary tract infection ( uti ) , which can cause irreversible damage to the kidney .", "this abnormality can be suspected in the fetus by antenatal ultrasonography ( usg ) and confirmed by other x - ray investigations after birth .", "the incidence of ureterocele is variable with the highest rate of 1:500 and it is generally found in females with duplex system association ( 95% ) .", "ureteroceles can have different clinical presentations , such as antenatal hydronephrosis , uti , vesicoureteral reflux ( vur ) , bladder outlet obstruction , prolapsed urethral mass , etc .", "usg and voiding cystourethrography ( vcug ) are essential initial procedures for a child suspected of having a ureteral anomaly ."], ["a 5-year - old female child was referred to our department for tc - diethylene triamine pentaacetic acid ( dtpa ) scan .", "she was a follow - up case of right - sided hydroureteronephrosis with pyonephrosis with percutaneous tube ( pcn ) insertion done on the right side .", "patient 's blood urea and serum creatinine were 29 and 1.3 mg / dl , respectively .", "usg of the abdomen revealed left - sided mild hydroureteronephrosis and gross enlargement of the right kidney , reaching up to lower abdomen with dilated pelvis and ureter .", "the child was referred for evaluation of glomerular filtration rate and excretory function of kidneys .", "tc - dtpa scan was done in our department with right pcn tube in situ .", "pcn tube was clamped during the acquisition of dynamic and prevoid static image and clamp released thereafter . on tc - dtpa scan , left kidney showed good perfusion and adequate cortical radiotracer concentration followed by good drainage into dilated ureter .", "right ureter and right pelvicalyceal system ( pcs ) were visualized in the postvoid image ( indirect evidence of vur ) [ figure 2b , thin arrow ] .", "retention of radiotracer was noted in dilated pcs and dilated ureter in delayed static images acquired till 4 h [ figure 2c and d ] .", "faint visualization of radiotracer was noted through the pcn tube after removal of clamp [ figure 2b , bold arrow ] .", "a large photopenia was noted in the suprapubic region in the urinary bladder ( ub ) during dynamic [ figure 1 ] as well as delayed static images acquired till 4 h [ figure 2a  d ] .", "an usg of the abdomen was done in the radiology department of our hospital to find out the cause of this persistent photopenia in the ub .", "usg of the abdomen showed evidence of left - sided mild hydroureteronephrosis and grossly enlarged hydronephrotic right kidney with dilated right ureter and an intracystic ureterocele arising from the right side [ figure 3a and b ] .", "perfusion and dynamic images of 99mtc - diethylene triamine pentaacetic acid scan scintigraphy , posterior view with percutaneous clamp in situ , showing good perfusion and adequate cortical function and good drainage of left kidney .", "photopenia noted in the urinary bladder ( bold arrow ) ( a ) prevoid static image with right pcn clamp in situ .", "right ureter and right pelvicalyceal system visualization is noted suggesting indirect evidence of vesico - ureteric reflux ( thin arrow ) .", "( c and d ) 3 and 4 h delayed static images showing persistent photopenia in the urinary bladder ( arrow marked ) ( a and b ) ultrasonography images depicting right ureterocele within the urinary bladder ( ub ) and dilated distal right ureter"], ["according to literature , 90% of the patients with ureterocele are diagnosed before the age of 3 years .", "most of the patients with ureterocele are classically diagnosed during the investigation for uti , asymptomatic hydronephrosis , and abdominal mass .", "although the age of diagnosis is decreasing , uti is still the most common clinical presentation of ureterocele in 50% of the patients promoting physician to make the thorough evaluation of the urinary system .", "the whole nephronourinary system could have already been negatively affected at the time of diagnosis . in our case ,", "usg is an easy method to perform , noninvasive , and probably the best imaging modality for making the diagnosis .", "reflux can occur in the ipsilateral lower pole in almost half of the patients , but the contralateral system is also affected at a rate of 25% . the tc - dimercapto succinic acid ( dmsa ) scan should be undertaken routinely to assess the distribution of function in the duplex kidney and for detecting and follow - up of scarred tissue and nonfunctioning upper poles in cases of ureterocele .", "did a study on pediatric ureteroceles in 19 patients , on the diagnosis , management and treatment options .", "vcug was able to detect vur in 13 out of 17 patients ( 33% ) .", "dmsa scintigraphy showed ipsilateral renal scarring and nonfunctioning upper pole images in 7 out of 13 patients . computed tomography and mag3 scintigraphy", "did a study on the management of 36 patients with varied presentation of complicated ureteroceles .", "usg , micturating cystourethrogram , isotope renogram were done preoperatively in all the babies . in one of the cases ,", "initial usg was unable to detect ureterocele in most of the cases in this study .", "they said that unless the usg is done in well - hydrated , cooperative patient , ureterocele is likely to be missed on usg .", "hence , an intravenous pyelogram , radioisotope renogram , and micturating cystourethrogram are invaluable in the complete understanding of ureterocele . in our patient also , the diagnosis of ureterocele was missed on initial usg .", "photopenia was noted in the ub on our scan , and this prompted us to investigate the patient further .", "possible causes for photopenic defects in the ub on nuclear scintigraphic studies include bladder papilloma , bladder polyp , carcinoma bladder , bladder calculus , foreign body in ub , intravesical ureterocele , etc .", "repeat usg revealed the cause of photopenia in ub in our case to be an intravesical ureterocele . on reviewing the literature we found that tc - dtpa scan is useful in the evaluation of renal function in patient with known ureteroceles .", "however , in our case on finding the photopenia in the ub on tc - dtpa scan , intravesical ureterocele was detected on usg ."], [""], [""]]}
{"article_id": "PMC3521802", "article_text": ["health - related quality of life ( hrqol ) is a multidimensional concept that defines a person 's health based on specific aspects , such as physical , emotional , and social functioning and general welfare ( 1 ) .", "the assessment of hrqol consists of an evaluation of the degree to which these aspects are decreased by symptoms , incapacities , and limitations caused by disease ( 2 ) .", "the assessment of hrqol has been used as a measure complementary to bone mineral density to evaluate and monitor the burden of osteoporosis on a patient 's daily life ( 3 ) .", "there are several instruments that can be used to assess the quality of life of individuals with osteoporosis , including the osteoporosis assessment questionnaire , the quality of life questionnaire for osteoporosis ( optqol ) , the osteoporosis quality of life questionnaire , and the questionnaire of the european foundation for osteoporosis ( qualeffo ) ( 4 - 7 ) .", "the qualeffo , a specific tool used to evaluate subjects with vertebral fractures and that includes questions on pain , physical functioning , social functioning , general health perception and mental functioning , has been shown to be repeatable and consistent ( 7 - 8 ) .", "vertebral fractures are the most frequent osteoporotic fractures , occurring in at least 30% of the elderly population ( 9 ) , and have important clinical implications ( 10 - 14 ) .", "these fractures are associated with increased risks of new osteoporotic fractures and mortality , especially in older women ( 10 - 11 ) .", "only one - third of vertebral fractures are symptomatic ; therefore , patients may be unaware of their presence .", "indeed , in studies based on the radiographic screening of populations , the incidence of all vertebral fractures has been estimated to be three times higher than the incidence of hip fractures , and only 30% of people with vertebral fractures were found to have received medical attention ( 12 ) . women with vertebral fractures can also experience decreased hrqol due to physical limitations and psychosocial disabilities ( 13 - 14 ) .", "some studies have assessed the impact of vertebral fractures on hrqol in older women in many countries ( 15,16 ) , but few such epidemiological studies have been conducted in brazil .", "moreover , most of the studies conducted in this country have been performed in ambulatory or institutionalized individuals ( 17,18 ) .", "therefore , this study evaluated the impact of vertebral fractures on the quality of life of healthy , community - dwelling women aged 65 years or older using the qualeffo .", "this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) .", "the inclusion and exclusion criteria were the same as those of the core study ( 9 ) .", "all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer .", "current or previous bisphosphonate use was also an exclusion criterion ( 9 ) . quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) .", "the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol .", "radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 \" between the tube and the film .", "the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures .", "they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached .", "the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 .", "each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height .", "the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer .", "the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale .", "the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight  =  bmi<25 ; overweight  =  25bmi<30 ; and obese  =  bmi30 ( 20 ) .", "information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews .", "women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) .", "physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) .", "regarding concomitant diseases , those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination . systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) .", "participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) .", "the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc .", "bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur .", "anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) .", "the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) .", "we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density .", "thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) .", "the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level .", "the study had 95% power to detect a difference of 10 points in the total qualeffo score .", "the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies .", "demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables .", "the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test .", "correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) .", "generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score .", "variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model .", "this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations .", "this study was approved by the local ethics and research committee ( research protocol 1110/07 ) .", "this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) .", "the inclusion and exclusion criteria were the same as those of the core study ( 9 ) .", "all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer .", "quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) .", "the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol .", "radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 \" between the tube and the film .", "the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures .", "they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached .", "the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 .", "each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height .", "the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer .", "the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale .", "the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight  =  bmi<25 ; overweight  =  25bmi<30 ; and obese  =  bmi30 ( 20 ) .", "information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews .", "women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) .", "physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) . regarding concomitant diseases ,", "those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination .", "systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) .", "participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) .", "the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc .", "bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur .", "anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) .", "the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) .", "we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density .", "thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) .", "the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level .", "the study had 95% power to detect a difference of 10 points in the total qualeffo score .", "the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies .", "demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables .", "the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test .", "correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) .", "generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score .", "variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model .", "this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations .", "this study was approved by the local ethics and research committee ( research protocol 1110/07 ) .", "the demographic , anthropometric and clinical data for all participants in the study , grouped based on the presence ( vertebral fracture ) or absence of vertebral fractures ( no vertebral fracture ) , are shown in table 1 .", "there were no significant differences with respect to the mean bmi or the percentage of caucasian individuals between the groups ( p>0.05 ) ( table 1 ) .", "regarding the bmi classification , 22.8% and approximately 38% of the subjects were classified as normal and obese , respectively , with no significant difference between the two groups ( p>0.05 ) .", "a tendency of older age in the vertebral fracture group was observed ( p  =  0.057 ) .", "the frequencies of hypertension ( p  =  0.224 ) , diabetes ( p  =  0.672 ) , hypothyroidism ( p  =  0.723 ) , and two or more concomitant diseases ( p  =  0.216 ) and the average number of medications used ( p  =  0.497 ) were comparable between the groups .", "interestingly , the vertebral fracture group contained a higher frequency of women defined as chronic fallers than the no vertebral fracture group ( 64.7 vs. 32.1% , p  =  0.017 ) .", "as expected , the vertebral fracture group had a higher frequency of osteoporosis ( 73.2 vs. 51.1% , p  =  0.012 ) and a lower frequency of osteopenia ( 19.5 vs. 38.1% , p  =  0.027 ) than the no vertebral fracture group .", "the results for each domain of the qualeffo and the total qualeffo score in both groups are shown in table 2 .", "the total qualeffo score was lower in the vertebral fracture group than in the no vertebral fracture group [ 61.4 ( 15.4 ) vs. 67.1 ( 14.2 ) , p  =  0.031 ] .", "likewise , the physical function domain score of the qualeffo was worse in the vertebral fracture group compared with the no vertebral fracture group [ 69.5 ( 20.1 ) vs. 77.3 ( 17.1 ) , p  =  0.018 ] .", "no difference was observed regarding the other qualeffo domains ( pain , social functioning , health perception , and mental functioning ) ( table 2 ) .", "the total qualeffo score was inversely related to bmi ( rs  =  -0.21 ,", "p  =  0.005 ) and weight ( rs  =  -0.22 , p  =  0.009 ) .", "the total qualeffo score was worse in women classified as obese than in those classified as overweight or normal [ 61.7 ( 15.4 ) vs. 66.4 ( 13.8 ) vs. 70.8 ( 13.5 ) , respectively , p  =  0.008 ] .", "a lower total qualeffo score was also observed in women with low physical activity than in those with moderate or high activity [ 51.8 ( 19.1 ) vs. 64.8 ( 14.3 ) vs. 70.3 ( 13.2 ) , respectively , p  =  0.010 ] ( table 3 ) .", "similarly , the physical function domain of the qualeffo was inversely related to the bmi ( rs  =  -0.24 , p  =  0.001 ) . in this domain , women classified as obese were found to have lower scores than women classified as overweight or normal [ 70.7 ( 19.3 ) vs. 76.1 ( 16.1 ) vs. 81.4 ( 17.4 ) , respectively , p  =  0.002 ] . finally , lower scores for the physical function domain were found among women with low physical activity compared with those with moderate or high activity [ 49.6(24.9 ) vs. 75.1(18.1 ) vs. 80.7(12.6 ) , respectively , p  =  0.002 ] ( table 3 ) . a generalized linear model with gamma distributions and logarithmic link functions was developed to identify patient characteristics that were related to the total and physical function domain scores of the qualeffo .", "variables with a p<0.10 in the univariate analysis ( age , bmi classification , physical activity , diabetes , presence of at least one fracture ) were included as independent variables .", "the presence of obesity was negatively associated with the total qualeffo score ( p  =  0.001 ) .", "a high physical activity level was positively associated with the total qualeffo score ( p  =  0.001 ) .", "the presence of at least one fracture was associated with a worse total qualeffo score , independent of age , bmi classification and physical activity level ( p  =  0.030 ) .", "likewise , the presence of at least one fracture was negatively associated with the physical function domain , independent of these same variables ( p  =  0.041 ) ( table 4 ) .", "this study was the first study conducted in brazil that specifically assessed the impact of vertebral fractures on the quality of life in older , community - dwelling women using a specific questionnaire . in this study", ", we demonstrated that the presence of vertebral fractures in this population is related to worse hrqol , particularly with respect to physical functioning .", "the major advantage of the present study is the homogenous selection of community - dwelling women , unlike previous studies in which individuals were recruited from clinics or from populations included in clinical trials .", "studies showing worse hrqol in patients with vertebral fractures have been published in several countries ( 15,16 ) .", "there are only two studies evaluating the hrqol in patients with vertebral fractures in brazil , but neither was specific to older community - dwelling individuals ( 17,18 ) .", "the first of these two other studies was performed in 55 outpatient women divided into three groups : 1- women without osteoporosis , 2- women without osteoporosis and no vertebral fractures , 3- women with osteoporosis and vertebral fractures . in that study ,", "the quality of life was assessed with the sf-36 , and no difference was found among the three groups .", "one of the reasons for this finding was the inclusion of only women who were able to perform the spirometric tests , resulting in the exclusion of women in worse conditions who would most likely belong to the fracture group ( 17 ) .", "later , de oliveira et al . evaluated the quality of life in ambulatory women with osteoporosis and found similar results for those who had vertebral fractures and those who did not .", "however , that study was not designed to assess the impact of vertebral fractures on quality of life , and the number of women with fractures was too small to enable an accurate assessment ( 18 ) .", "the assessment of quality of life in relation to the qualeffo pain domain was similar in women with and without vertebral fractures in our study .", "some studies have found that the pain domain is worse in women with fractures ; however , the patients included in those studies were recruited based on clinical symptoms related to fractures and were compared with those without back pain ( 28 ) .", "vertebral fractures do not always manifest with symptoms and are often diagnosed based on radiographs taken for other reasons .", "evaluated the chest radiographs of older women who had been hospitalized for several causes , and they found that only a few of the vertebral fractures present had been previously identified by clinicians ( 29 ) .", "indeed , in our previous study performed in brazil , 29.4% subjects had vertebral fractures , and none of these patients had prior knowledge of their vertebral fractures ( 9 ) .", "as observed in other studies , the mental function , social function , and health perception domains were not significantly different between women with and without fractures ( 30 ) .", "the relatively small differences between the groups with and without fractures may be the result of the acceptance of poor health conditions due to the natural expectation of physical decline in older women ( 13 ) . in our study", ", we found that obesity and low physical activity were associated with lower quality of life .", "other authors have reported that higher bmi and a sedentary lifestyle are factors that influence the quality of life in patients with osteoporosis ( 18,31 - 32 ) .", "it is important to highlight the fact that both obesity and a sedentary lifestyle are preventable factors and can be controlled by a change in lifestyle .", "some authors observed that physical exercise is associated with a better quality of life ( 33 ) and have demonstrated that a home exercise program for women with vertebral fractures ( 60 min / d , 3x / week for 12 months ) significantly improves the hrqol ( 34 ) . a study performed at our institution in osteoporosis patients showed that an exercise program improved the quality of life in these women ( 35 ) .", "therefore , our data reinforce the need for all older women to be advised about the benefits of exercise .", "these women should be encouraged to exercise regularly to reduce their bmi and improve their general welfare .", "our study is the first in brazil to conduct a thorough assessment of the relationship between quality of life and vertebral fractures .", "an important characteristic of this study was the use of standardized and reliable instruments for both the vertebral fracture assessment ( genant semi - quantitative method ) and the hrqol assessment ( qualeffo ) .", "although the qualeffo has not been validated in brazil , which is a limitation of the present study , this tool is the most frequently used for assessing quality of life in osteoporosis and was recommended for the investigation of vertebral fracture subjects in multicentric studies that included centers in brazil ( 36 ) .", "this questionnaire is more sensitive in detecting differences between groups , and it provides a better discrimination between individuals with and without vertebral fractures compared with generic hrqol instruments , such as the sf-36 , particularly with respect to physical functioning , which is significantly affected in these patients ( 8) . in conclusion , this study demonstrated the negative impact of vertebral fractures on the quality of life in older women , independent of other factors such as bmi and physical activity .", "although the clinical relevance of vertebral fractures is well established , these results are important for assessing the burden of this disease and reinforce the need to reduce the underdiagnosis and undertreatment of these fractures . the results of this study also highlight the need for awareness of the importance of maintaining proper weight and promoting changes in lifestyle through physical activity and dietary control .", "this study was supported by fundao de amparo e pesquisa do estado de so paulo ( fapesp ) # 03/09313 - 0 ; conselho nacional de cincia e tecnologia ( cnpq ) # 305691/2006 - 6 ( rmrp ) and # 119601/2009 - 5 ( lf ) ; federico foundation grants ( rmrp ) ; and coordenao de aperfeioamento de pessoal de nvel superior ( capes ) ( jbl , cpf ) ."], "abstract_text": ["<S> objectives : the aim of this study was to investigate the impact of asymptomatic vertebral fractures on the quality of life in older women as part of the sao paulo ageing & health study.methods:this study was a cross - sectional study with a random sample of 180 women 65 years of age or older with or without vertebral fractures . </S>", "<S> the quality of life questionnaire of the european foundation for osteoporosis was administered to all subjects . </S>", "<S> anthropometric data were obtained by physical examination , and the body mass index was calculated . </S>", "<S> lateral thoracic and lumbar spine x - ray scans were obtained to identify asymptomatic vertebral fractures using a semi - quantitative method.results:women with asymptomatic vertebral fractures had lower total scores [ 61.4(15.3 ) vs. 67.1(14.2 ) , p  =  0.03 ] and worse physical function domain scores [ 69.5(20.1 ) vs. 77.3(17.1 ) , p  =  0.02 ] for the quality of life questionnaire of the european foundation for osteoporosis compared with women without fractures . </S>", "<S> the total score of this questionnaire was also worse in women classified as obese than in women classified as overweight or normal . </S>", "<S> high physical activity was related to a better total score for this questionnaire ( p  =  0.01 ) . likewise , lower physical function scores were observed in women with higher body mass index values ( p<0.05 ) and lower physical activity levels ( p<0.05 ) . </S>", "<S> generalized linear models with gamma distributions and logarithmic link functions , adjusted for age , showed that lower total scores and physical function domain scores for the quality of life questionnaire of the european foundation for osteoporosis were related to a high body mass index , lower physical activity , and the presence of vertebral fractures ( p<0.05).conclusion : vertebral fractures are associated with decreased quality of life mainly physical functioning in older community - dwelling women regardless of age , body mass index , and physical activity . </S>", "<S> therefore , the results highlight the importance of preventing and controlling asymptomatic vertebral fractures to reduce their impact on quality of life among older women . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "Population", "Quality of life assessment", "Assessment of vertebral fractures", "Anthropometry", "Other variables and definitions", "Bone mineral density (BMD) assessment", "Statistical analysis", "Ethics", "RESULTS", "DISCUSSION", "ACKNOWLEDGMENTS"], "sections": [["health - related quality of life ( hrqol ) is a multidimensional concept that defines a person 's health based on specific aspects , such as physical , emotional , and social functioning and general welfare ( 1 ) .", "the assessment of hrqol consists of an evaluation of the degree to which these aspects are decreased by symptoms , incapacities , and limitations caused by disease ( 2 ) .", "the assessment of hrqol has been used as a measure complementary to bone mineral density to evaluate and monitor the burden of osteoporosis on a patient 's daily life ( 3 ) .", "there are several instruments that can be used to assess the quality of life of individuals with osteoporosis , including the osteoporosis assessment questionnaire , the quality of life questionnaire for osteoporosis ( optqol ) , the osteoporosis quality of life questionnaire , and the questionnaire of the european foundation for osteoporosis ( qualeffo ) ( 4 - 7 ) .", "the qualeffo , a specific tool used to evaluate subjects with vertebral fractures and that includes questions on pain , physical functioning , social functioning , general health perception and mental functioning , has been shown to be repeatable and consistent ( 7 - 8 ) .", "vertebral fractures are the most frequent osteoporotic fractures , occurring in at least 30% of the elderly population ( 9 ) , and have important clinical implications ( 10 - 14 ) .", "these fractures are associated with increased risks of new osteoporotic fractures and mortality , especially in older women ( 10 - 11 ) .", "only one - third of vertebral fractures are symptomatic ; therefore , patients may be unaware of their presence .", "indeed , in studies based on the radiographic screening of populations , the incidence of all vertebral fractures has been estimated to be three times higher than the incidence of hip fractures , and only 30% of people with vertebral fractures were found to have received medical attention ( 12 ) . women with vertebral fractures can also experience decreased hrqol due to physical limitations and psychosocial disabilities ( 13 - 14 ) .", "some studies have assessed the impact of vertebral fractures on hrqol in older women in many countries ( 15,16 ) , but few such epidemiological studies have been conducted in brazil .", "moreover , most of the studies conducted in this country have been performed in ambulatory or institutionalized individuals ( 17,18 ) .", "therefore , this study evaluated the impact of vertebral fractures on the quality of life of healthy , community - dwelling women aged 65 years or older using the qualeffo ."], ["this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) .", "the inclusion and exclusion criteria were the same as those of the core study ( 9 ) .", "all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer .", "current or previous bisphosphonate use was also an exclusion criterion ( 9 ) . quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) .", "the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol .", "radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 \" between the tube and the film .", "the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures .", "they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached .", "the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 .", "each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height .", "the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer .", "the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale .", "the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight  =  bmi<25 ; overweight  =  25bmi<30 ; and obese  =  bmi30 ( 20 ) .", "information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews .", "women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) .", "physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) .", "regarding concomitant diseases , those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination . systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) .", "participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) .", "the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc .", "bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur .", "anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) .", "the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) .", "we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density .", "thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) .", "the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level .", "the study had 95% power to detect a difference of 10 points in the total qualeffo score .", "the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies .", "demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables .", "the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test .", "correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) .", "generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score .", "variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model .", "this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations .", "this study was approved by the local ethics and research committee ( research protocol 1110/07 ) ."], ["this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) .", "the inclusion and exclusion criteria were the same as those of the core study ( 9 ) .", "all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer ."], ["quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) .", "the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol ."], ["radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 \" between the tube and the film .", "the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures .", "they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached .", "the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 .", "each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height ."], ["the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer .", "the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale .", "the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight  =  bmi<25 ; overweight  =  25bmi<30 ; and obese  =  bmi30 ( 20 ) ."], ["information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews .", "women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) .", "physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) . regarding concomitant diseases ,", "those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination .", "systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) .", "participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) ."], ["the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc .", "bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur .", "anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) .", "the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) .", "we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density .", "thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) ."], ["the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level .", "the study had 95% power to detect a difference of 10 points in the total qualeffo score .", "the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies .", "demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables .", "the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test .", "correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) .", "generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score .", "variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model ."], ["this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations .", "this study was approved by the local ethics and research committee ( research protocol 1110/07 ) ."], ["the demographic , anthropometric and clinical data for all participants in the study , grouped based on the presence ( vertebral fracture ) or absence of vertebral fractures ( no vertebral fracture ) , are shown in table 1 .", "there were no significant differences with respect to the mean bmi or the percentage of caucasian individuals between the groups ( p>0.05 ) ( table 1 ) .", "regarding the bmi classification , 22.8% and approximately 38% of the subjects were classified as normal and obese , respectively , with no significant difference between the two groups ( p>0.05 ) .", "a tendency of older age in the vertebral fracture group was observed ( p  =  0.057 ) .", "the frequencies of hypertension ( p  =  0.224 ) , diabetes ( p  =  0.672 ) , hypothyroidism ( p  =  0.723 ) , and two or more concomitant diseases ( p  =  0.216 ) and the average number of medications used ( p  =  0.497 ) were comparable between the groups .", "interestingly , the vertebral fracture group contained a higher frequency of women defined as chronic fallers than the no vertebral fracture group ( 64.7 vs. 32.1% , p  =  0.017 ) .", "as expected , the vertebral fracture group had a higher frequency of osteoporosis ( 73.2 vs. 51.1% , p  =  0.012 ) and a lower frequency of osteopenia ( 19.5 vs. 38.1% , p  =  0.027 ) than the no vertebral fracture group .", "the results for each domain of the qualeffo and the total qualeffo score in both groups are shown in table 2 .", "the total qualeffo score was lower in the vertebral fracture group than in the no vertebral fracture group [ 61.4 ( 15.4 ) vs. 67.1 ( 14.2 ) , p  =  0.031 ] .", "likewise , the physical function domain score of the qualeffo was worse in the vertebral fracture group compared with the no vertebral fracture group [ 69.5 ( 20.1 ) vs. 77.3 ( 17.1 ) , p  =  0.018 ] .", "no difference was observed regarding the other qualeffo domains ( pain , social functioning , health perception , and mental functioning ) ( table 2 ) .", "the total qualeffo score was inversely related to bmi ( rs  =  -0.21 ,", "p  =  0.005 ) and weight ( rs  =  -0.22 , p  =  0.009 ) .", "the total qualeffo score was worse in women classified as obese than in those classified as overweight or normal [ 61.7 ( 15.4 ) vs. 66.4 ( 13.8 ) vs. 70.8 ( 13.5 ) , respectively , p  =  0.008 ] .", "a lower total qualeffo score was also observed in women with low physical activity than in those with moderate or high activity [ 51.8 ( 19.1 ) vs. 64.8 ( 14.3 ) vs. 70.3 ( 13.2 ) , respectively , p  =  0.010 ] ( table 3 ) .", "similarly , the physical function domain of the qualeffo was inversely related to the bmi ( rs  =  -0.24 , p  =  0.001 ) . in this domain , women classified as obese were found to have lower scores than women classified as overweight or normal [ 70.7 ( 19.3 ) vs. 76.1 ( 16.1 ) vs. 81.4 ( 17.4 ) , respectively , p  =  0.002 ] . finally , lower scores for the physical function domain were found among women with low physical activity compared with those with moderate or high activity [ 49.6(24.9 ) vs. 75.1(18.1 ) vs. 80.7(12.6 ) , respectively , p  =  0.002 ] ( table 3 ) . a generalized linear model with gamma distributions and logarithmic link functions was developed to identify patient characteristics that were related to the total and physical function domain scores of the qualeffo .", "variables with a p<0.10 in the univariate analysis ( age , bmi classification , physical activity , diabetes , presence of at least one fracture ) were included as independent variables .", "the presence of obesity was negatively associated with the total qualeffo score ( p  =  0.001 ) .", "a high physical activity level was positively associated with the total qualeffo score ( p  =  0.001 ) .", "the presence of at least one fracture was associated with a worse total qualeffo score , independent of age , bmi classification and physical activity level ( p  =  0.030 ) .", "likewise , the presence of at least one fracture was negatively associated with the physical function domain , independent of these same variables ( p  =  0.041 ) ( table 4 ) ."], ["this study was the first study conducted in brazil that specifically assessed the impact of vertebral fractures on the quality of life in older , community - dwelling women using a specific questionnaire . in this study", ", we demonstrated that the presence of vertebral fractures in this population is related to worse hrqol , particularly with respect to physical functioning .", "the major advantage of the present study is the homogenous selection of community - dwelling women , unlike previous studies in which individuals were recruited from clinics or from populations included in clinical trials .", "studies showing worse hrqol in patients with vertebral fractures have been published in several countries ( 15,16 ) .", "there are only two studies evaluating the hrqol in patients with vertebral fractures in brazil , but neither was specific to older community - dwelling individuals ( 17,18 ) .", "the first of these two other studies was performed in 55 outpatient women divided into three groups : 1- women without osteoporosis , 2- women without osteoporosis and no vertebral fractures , 3- women with osteoporosis and vertebral fractures . in that study ,", "the quality of life was assessed with the sf-36 , and no difference was found among the three groups .", "one of the reasons for this finding was the inclusion of only women who were able to perform the spirometric tests , resulting in the exclusion of women in worse conditions who would most likely belong to the fracture group ( 17 ) .", "later , de oliveira et al . evaluated the quality of life in ambulatory women with osteoporosis and found similar results for those who had vertebral fractures and those who did not .", "however , that study was not designed to assess the impact of vertebral fractures on quality of life , and the number of women with fractures was too small to enable an accurate assessment ( 18 ) .", "the assessment of quality of life in relation to the qualeffo pain domain was similar in women with and without vertebral fractures in our study .", "some studies have found that the pain domain is worse in women with fractures ; however , the patients included in those studies were recruited based on clinical symptoms related to fractures and were compared with those without back pain ( 28 ) .", "vertebral fractures do not always manifest with symptoms and are often diagnosed based on radiographs taken for other reasons .", "evaluated the chest radiographs of older women who had been hospitalized for several causes , and they found that only a few of the vertebral fractures present had been previously identified by clinicians ( 29 ) .", "indeed , in our previous study performed in brazil , 29.4% subjects had vertebral fractures , and none of these patients had prior knowledge of their vertebral fractures ( 9 ) .", "as observed in other studies , the mental function , social function , and health perception domains were not significantly different between women with and without fractures ( 30 ) .", "the relatively small differences between the groups with and without fractures may be the result of the acceptance of poor health conditions due to the natural expectation of physical decline in older women ( 13 ) . in our study", ", we found that obesity and low physical activity were associated with lower quality of life .", "other authors have reported that higher bmi and a sedentary lifestyle are factors that influence the quality of life in patients with osteoporosis ( 18,31 - 32 ) .", "it is important to highlight the fact that both obesity and a sedentary lifestyle are preventable factors and can be controlled by a change in lifestyle .", "some authors observed that physical exercise is associated with a better quality of life ( 33 ) and have demonstrated that a home exercise program for women with vertebral fractures ( 60 min / d , 3x / week for 12 months ) significantly improves the hrqol ( 34 ) . a study performed at our institution in osteoporosis patients showed that an exercise program improved the quality of life in these women ( 35 ) .", "therefore , our data reinforce the need for all older women to be advised about the benefits of exercise .", "these women should be encouraged to exercise regularly to reduce their bmi and improve their general welfare .", "our study is the first in brazil to conduct a thorough assessment of the relationship between quality of life and vertebral fractures .", "an important characteristic of this study was the use of standardized and reliable instruments for both the vertebral fracture assessment ( genant semi - quantitative method ) and the hrqol assessment ( qualeffo ) .", "although the qualeffo has not been validated in brazil , which is a limitation of the present study , this tool is the most frequently used for assessing quality of life in osteoporosis and was recommended for the investigation of vertebral fracture subjects in multicentric studies that included centers in brazil ( 36 ) .", "this questionnaire is more sensitive in detecting differences between groups , and it provides a better discrimination between individuals with and without vertebral fractures compared with generic hrqol instruments , such as the sf-36 , particularly with respect to physical functioning , which is significantly affected in these patients ( 8) . in conclusion , this study demonstrated the negative impact of vertebral fractures on the quality of life in older women , independent of other factors such as bmi and physical activity .", "although the clinical relevance of vertebral fractures is well established , these results are important for assessing the burden of this disease and reinforce the need to reduce the underdiagnosis and undertreatment of these fractures . the results of this study also highlight the need for awareness of the importance of maintaining proper weight and promoting changes in lifestyle through physical activity and dietary control ."], ["this study was supported by fundao de amparo e pesquisa do estado de so paulo ( fapesp ) # 03/09313 - 0 ; conselho nacional de cincia e tecnologia ( cnpq ) # 305691/2006 - 6 ( rmrp ) and # 119601/2009 - 5 ( lf ) ; federico foundation grants ( rmrp ) ; and coordenao de aperfeioamento de pessoal de nvel superior ( capes ) ( jbl , cpf ) ."]]}
{"article_id": "PMC4334055", "article_text": ["the majority of women with menorrhagia , postcoital bleeding , intermenstrual bleeding , or postmenopausal bleeding ultimately undergo diagnostic hysteroscopy with endometrial sampling as part of their assessment , particularly if symptoms persist or pelvic imaging suggests a uterine abnormality .", "dilatation and curettage ( d&c ) has been widely considered to be the method of choice for obtaining endometrial samples for histopathological evaluation", ". however , the needs for admission and general anesthesia and their associated costs have made this option less favorable . in the outpatient setting , endometrial sampling is an effective and acceptable method for obtaining endometrial samples for histopathological assessment [ 3 , 4 ] .", "inadequate sampling is more problematic in postmenopausal women , for whom up to 68% of endometrial samples are reported to be inadequate . in our institution , the only sampling tool available to perform the outpatient sampling procedure is the uterine explora model i - mx120 ( http://www.coopersurgical.com/ ) ( figure 1 ) .", "in addition , the device is sterile and disposable ( one - time use ) .", "the advantages of using explora rather than d&c as a sampling device include a reduction in hospitalization costs , extra convenience for the patient and physician , and the minimal complications of the procedure .", "the purpose of this study is to compare the effectiveness of the explora model i tool with the conventional d&c technique for obtaining adequate endometrial samples that are capable of providing specific and informative histopathologic diagnoses .", "after obtaining the approval of our institutional review board , all endometrial samples received at the histopathology department in king khalid university hospital ( kkuh , riyadh , ksa ) between january 2007 and december 2010 were included in this study . a total of 1270 endometrial samples were included ( table 1 ) .", "two hundred seventy - four samples ( 21.6% ) were obtained by conventional d&c in the surgical theater , while the remaining 996 samples ( 78.4% ) were obtained by senior obstetrics and gynecology residents who used a standardized biopsy technique in the outpatient procedure rooms . during the usage of the explora model", "i , the syringe provided with the instrument was used to create a negative pressure , and the explora was rotated as it was withdrawn . after withdrawal , the tip was cut off , and the tissue was placed in 10% buffered formalin saline fixative and was sent for pathological examination .", "the pathologists who interpreted the endometrial samples were blinded to the instrument or method used to obtain the samples .", "an inadequate sample was defined as consisting of only blood , cervical mucus , endocervical epithelium , or blood with fragments of endometrial glands or stroma insufficient for histopathological assessment and diagnosis .", "the age , gravidity , parity , menstrual history , uterine size , hysteroscopy findings ( when available ) , and the presence or absence of any cervical abnormality were recorded on the request forms , which were reviewed by the investigators . for each of the two methods used ( explora model i and d&c ) , the numbers and percentages of inadequate samples and age group clustering were calculated and statistically analyzed .", "of the 1270 endometrial samples obtained , 253 samples ( 19.9% ) were scored as inadequate . of these samples ,", "the explora sampler was used to collect 224 samples ( 88.5% ) , whereas 29 samples ( 11.5% ) were obtained by d&c ( figure 2 ) .", "thus , the insufficient tissue percentage was higher with the explora ( 17.6% ) than with d&c ( 2.2% ) , which was a statistically significant difference ( p < 0.0001 )", ". age group clustering ( i.e. , numbers of premenopausal and postmenopausal women ) of inadequate sample results was also calculated ( figure 3 ) . of the 253 inadequate samples ,", "82.6% were from women 45 years of age and older ( i.e. , postmenopausal ) compared to 17.4% in premenopausal women ; the age difference was significant ( p < 0.0001 ) .", "the detection rates of endometrial hyperplasia and carcinoma using both methods were assessed and calculated .", "of the 73 samples with a diagnosis of endometrial hyperplasia , 50 ( 68.5% ) were diagnosed by d&c , and 23 ( 31.5% ) were diagnosed using the explora sampler .", "however , of the 18 samples with a diagnosis of endometrial cancer , the rates of detection were similar between the two methods .", "endometrial sampling for the evaluation of dysfunctional uterine bleeding and the diagnosis of endometrial hyperplasia and carcinoma and other indications remains one of the most commonly performed gynecological procedures [ 14 ] . in recent years", ", less hazardous and more inexpensive and convenient outpatient sampling methods have replaced the traditional , in - hospital , endometrial curettage .", "the advantages of outpatient endometrial biopsy include reduced cost and less risk for the patient , as no anesthesia is required .", "furthermore , the discomfort and pain produced by sampling have been reported to be minimal .", "however , it is essential to ensure that outpatient endometrial sampling is quantitatively adequate and comparably accurate to conventional dilatation and curettage .", "a sample is judged as adequate if a specific diagnosis can be given from the histological examination of the endometrial fragments obtained .", "adequacy can be measured by comparison of either outpatient biopsy with curettage histological evaluation or outpatient biopsy with the results of pathological examination of hysterectomy specimens [ 3 , 4 ] .", "many techniques for obtaining an endometrial sample without the need for curettage have been described in the literature .", "chicago , il , usa ) and the novak biopsy curette with a 10  ml syringe functioning as an aspiratory device , which have been shown to be equally effective compared to d&c in detecting an endometrial pathology [ 69 ] .", "however , the vabra aspirator and novak biopsy curette , although widely available and relatively inexpensive , have several disadvantages , including the need for an electric vacuum pump to perform the aspiration in the former technique and the pain caused by both methods . as a result of these drawbacks , smaller inexpensive and", "self - contained instruments have been developed and the prototype of this class of endometrial samplers is the pipelle . the pipelle has been shown to have a diagnostic accuracy comparable to that of vabra aspiration and the novak curettage while causing less pain [ 911 ] .", "all of these instruments ( i.e. , the vabra aspirator , the novak biopsy curette , and the pipelle ) have low rates of false - negative and insufficient tissue results for the detection of endometrial abnormalities , as determined by comparison to hysterectomy specimens [ 1113 ] .", "it was found that pipelle biopsy had a sensitivity of 99.2% in pinpointing high grade cancer and a sensitivity of 93% in detecting low grade malignancies ; the sensitivities defined for d&c were 100% and 97% , respectively . while ", "excellent agreement  was generally noted between preoperative histology and grade and the final pathology , pre - operative endometrial sampling more commonly provided underestimates of final grade ( low grade versus high grade ) than overestimates .", "the explora is somewhat similar in its design to the pipelle , but clinical studies on its effectiveness are scarce , with the effectiveness ranging between 14.6 and 15% according to various studies [ 6 , 15 ] .", "our own findings revealed that the rate of obtaining inadequate samples using the explora was much higher ( 17.6% ) than the rates reported in the literature .", "however , most of these cases ( 82.6% ) were obtained from postmenopausal women with atrophic endometrial status .", "this finding is in keeping with the rates reported by other investigators [ 58 , 16 ] .", "this retrospective study suggests that traditional d&c produces better endometrial sample adequacy than the explora technique .", "this finding indicates that clinicians performing endometrial sampling would benefit from more experience and training using the explora technique .", "additional studies comparing the adequacy of samples obtained with different endometrial sampling techniques and devices are warranted .", "furthermore , we recommend using the d&c procedure when the explora - obtained samples are inconclusive or when the use of the explora sampler is accompanied by ultrasound findings that are suspicious of endometrial hyperplasia or carcinoma ."], "abstract_text": ["<S> \n aims . our aim is to compare the adequacy and diagnostic yield of samples obtained by the endometrial explora sampler i - mx120 with endometrial specimens obtained by conventional dilatation and curettage ( d&c ) </S>", "<S> . methods . a total of 1270 endometrial samples </S>", "<S> were received in the histopathology laboratories at the king khalid university hospital , riyadh , saudi arabia , between 2007 and 2010 . in the outpatient clinic , </S>", "<S> the uterine explora model i was used to obtain 996 samples . </S>", "<S> the remaining 274 samples were obtained by conventional d&c . </S>", "<S> sample adequacy and the clustering of inadequate specimens according to age groups by the two different techniques were compared and statistically analyzed . results . out of 1270 endometrial samples , 253 ( 19.9% ) were inadequate . </S>", "<S> the uterine explora was used in 88.5% of these inadequate samples ( 253 samples ) , and the remaining 11.5% were obtained by d&c . </S>", "<S> the insufficient tissue incidence was higher with the explora ( 17.6% ) than with the d&c ( 2.2% ) and the difference was statistically significant ( p < 0.0001 ) . </S>", "<S> the ages of the patients , as well as the clinical indications for the procedures , were recorded . conclusion . </S>", "<S> this retrospective study demonstrated better specimen adequacy when d&c was used compared to the higher rate of sample insufficiency obtained with the explora . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["the majority of women with menorrhagia , postcoital bleeding , intermenstrual bleeding , or postmenopausal bleeding ultimately undergo diagnostic hysteroscopy with endometrial sampling as part of their assessment , particularly if symptoms persist or pelvic imaging suggests a uterine abnormality .", "dilatation and curettage ( d&c ) has been widely considered to be the method of choice for obtaining endometrial samples for histopathological evaluation", ". however , the needs for admission and general anesthesia and their associated costs have made this option less favorable . in the outpatient setting , endometrial sampling is an effective and acceptable method for obtaining endometrial samples for histopathological assessment [ 3 , 4 ] .", "inadequate sampling is more problematic in postmenopausal women , for whom up to 68% of endometrial samples are reported to be inadequate . in our institution , the only sampling tool available to perform the outpatient sampling procedure is the uterine explora model i - mx120 ( http://www.coopersurgical.com/ ) ( figure 1 ) .", "in addition , the device is sterile and disposable ( one - time use ) .", "the advantages of using explora rather than d&c as a sampling device include a reduction in hospitalization costs , extra convenience for the patient and physician , and the minimal complications of the procedure .", "the purpose of this study is to compare the effectiveness of the explora model i tool with the conventional d&c technique for obtaining adequate endometrial samples that are capable of providing specific and informative histopathologic diagnoses ."], ["after obtaining the approval of our institutional review board , all endometrial samples received at the histopathology department in king khalid university hospital ( kkuh , riyadh , ksa ) between january 2007 and december 2010 were included in this study . a total of 1270 endometrial samples were included ( table 1 ) .", "two hundred seventy - four samples ( 21.6% ) were obtained by conventional d&c in the surgical theater , while the remaining 996 samples ( 78.4% ) were obtained by senior obstetrics and gynecology residents who used a standardized biopsy technique in the outpatient procedure rooms . during the usage of the explora model", "i , the syringe provided with the instrument was used to create a negative pressure , and the explora was rotated as it was withdrawn . after withdrawal , the tip was cut off , and the tissue was placed in 10% buffered formalin saline fixative and was sent for pathological examination .", "the pathologists who interpreted the endometrial samples were blinded to the instrument or method used to obtain the samples .", "an inadequate sample was defined as consisting of only blood , cervical mucus , endocervical epithelium , or blood with fragments of endometrial glands or stroma insufficient for histopathological assessment and diagnosis .", "the age , gravidity , parity , menstrual history , uterine size , hysteroscopy findings ( when available ) , and the presence or absence of any cervical abnormality were recorded on the request forms , which were reviewed by the investigators . for each of the two methods used ( explora model i and d&c ) , the numbers and percentages of inadequate samples and age group clustering were calculated and statistically analyzed ."], ["of the 1270 endometrial samples obtained , 253 samples ( 19.9% ) were scored as inadequate . of these samples ,", "the explora sampler was used to collect 224 samples ( 88.5% ) , whereas 29 samples ( 11.5% ) were obtained by d&c ( figure 2 ) .", "thus , the insufficient tissue percentage was higher with the explora ( 17.6% ) than with d&c ( 2.2% ) , which was a statistically significant difference ( p < 0.0001 )", ". age group clustering ( i.e. , numbers of premenopausal and postmenopausal women ) of inadequate sample results was also calculated ( figure 3 ) . of the 253 inadequate samples ,", "82.6% were from women 45 years of age and older ( i.e. , postmenopausal ) compared to 17.4% in premenopausal women ; the age difference was significant ( p < 0.0001 ) .", "the detection rates of endometrial hyperplasia and carcinoma using both methods were assessed and calculated .", "of the 73 samples with a diagnosis of endometrial hyperplasia , 50 ( 68.5% ) were diagnosed by d&c , and 23 ( 31.5% ) were diagnosed using the explora sampler .", "however , of the 18 samples with a diagnosis of endometrial cancer , the rates of detection were similar between the two methods ."], ["endometrial sampling for the evaluation of dysfunctional uterine bleeding and the diagnosis of endometrial hyperplasia and carcinoma and other indications remains one of the most commonly performed gynecological procedures [ 14 ] . in recent years", ", less hazardous and more inexpensive and convenient outpatient sampling methods have replaced the traditional , in - hospital , endometrial curettage .", "the advantages of outpatient endometrial biopsy include reduced cost and less risk for the patient , as no anesthesia is required .", "furthermore , the discomfort and pain produced by sampling have been reported to be minimal .", "however , it is essential to ensure that outpatient endometrial sampling is quantitatively adequate and comparably accurate to conventional dilatation and curettage .", "a sample is judged as adequate if a specific diagnosis can be given from the histological examination of the endometrial fragments obtained .", "adequacy can be measured by comparison of either outpatient biopsy with curettage histological evaluation or outpatient biopsy with the results of pathological examination of hysterectomy specimens [ 3 , 4 ] .", "many techniques for obtaining an endometrial sample without the need for curettage have been described in the literature .", "chicago , il , usa ) and the novak biopsy curette with a 10  ml syringe functioning as an aspiratory device , which have been shown to be equally effective compared to d&c in detecting an endometrial pathology [ 69 ] .", "however , the vabra aspirator and novak biopsy curette , although widely available and relatively inexpensive , have several disadvantages , including the need for an electric vacuum pump to perform the aspiration in the former technique and the pain caused by both methods . as a result of these drawbacks , smaller inexpensive and", "self - contained instruments have been developed and the prototype of this class of endometrial samplers is the pipelle . the pipelle has been shown to have a diagnostic accuracy comparable to that of vabra aspiration and the novak curettage while causing less pain [ 911 ] .", "all of these instruments ( i.e. , the vabra aspirator , the novak biopsy curette , and the pipelle ) have low rates of false - negative and insufficient tissue results for the detection of endometrial abnormalities , as determined by comparison to hysterectomy specimens [ 1113 ] .", "it was found that pipelle biopsy had a sensitivity of 99.2% in pinpointing high grade cancer and a sensitivity of 93% in detecting low grade malignancies ; the sensitivities defined for d&c were 100% and 97% , respectively . while ", "excellent agreement  was generally noted between preoperative histology and grade and the final pathology , pre - operative endometrial sampling more commonly provided underestimates of final grade ( low grade versus high grade ) than overestimates .", "the explora is somewhat similar in its design to the pipelle , but clinical studies on its effectiveness are scarce , with the effectiveness ranging between 14.6 and 15% according to various studies [ 6 , 15 ] .", "our own findings revealed that the rate of obtaining inadequate samples using the explora was much higher ( 17.6% ) than the rates reported in the literature .", "however , most of these cases ( 82.6% ) were obtained from postmenopausal women with atrophic endometrial status .", "this finding is in keeping with the rates reported by other investigators [ 58 , 16 ] ."], ["this retrospective study suggests that traditional d&c produces better endometrial sample adequacy than the explora technique .", "this finding indicates that clinicians performing endometrial sampling would benefit from more experience and training using the explora technique .", "additional studies comparing the adequacy of samples obtained with different endometrial sampling techniques and devices are warranted .", "furthermore , we recommend using the d&c procedure when the explora - obtained samples are inconclusive or when the use of the explora sampler is accompanied by ultrasound findings that are suspicious of endometrial hyperplasia or carcinoma ."]]}
{"article_id": "PMC4599590", "article_text": ["the geological and ( palaeo-)biological evolution of lowland amazonia during neogene and quaternary times remains fascinating since the early days of natural sciences ( for summaries of research history see e.g. , loczy , 1963 and wesselingh , 2008 ) .", "many , but partly highly controversial models have been introduced to explain its historical development ( for recent compilations see e.g. , lundberg et  al . , 1998 ;", ", 2006 ; wesselingh and salo , 2006 ; rossetti and mann de toledo , 2007 ; haffer , 2008 ; hoorn and wesselingh , 2010 ; hoorn et  al . , 2010 ;", "generally , it is widely accepted that around the onset of the miocene ( 23  ma ) a mega - wetland (  pebas system  , also called  lake pebas  ) developed in western amazonia due to the subsiding subandean foreland ( e.g. , hoorn , 1994 , 2006 ; wesselingh et  al . , 2002 ; wesselingh and salo , 2006 ; shephard et  al . , 2010 ) .", "short - lived marine incursions or even a transcontinental seaway from the caribbean sea through the venezuelan / columbian llanos basin southwards to the argentinean paran basin are proposed ( e.g. , rsnen et  al . , 1995 ;", "2009 ) but heavily disputed ( e.g. , campbell et  al . , 2006 ; cozzuol , 2006 ; westaway , 2006 ; latrubesse et  al . , 2007 , 2010 ;", ", 2003 ; hoorn and vonhof , 2006 ; wesselingh , 2006 ) . in the late miocene this mega - wetland disintegrated due to enhanced uplift of the northern / central andes .", "the drainage pattern of northern south america started to reverse completely to today s easterly course and the modern  amazon system  became established during the early pliocene ( hoorn , 2006 ; figueiredo et  al .", ", 2009 ; hoorn et  al . , 2010 ; latrubesse et  al . ,", "2010 ) . beside the vast size of amazonia and the still fragmentary regional coverage with field surveys ,", "there are considerable inconsistencies in palaeoenvironmental reconstructions and , in particular , in the chronology and correlation of scattered outcrops .", "wesselingh ( 2008 , p. 5 ) stated :  the lack of geological data has led to the emergence of many grand theories about the origin of present - day amazon system and its highly diversity , often based on dubious interpretations of the little data available  .", "the present paper aims to contribute basic sedimentological data from a barely studied region ( eirunep , 2.000  km sw manaus ; fig . ", "1a ) , which is supposed to be placed at the south - eastern margin of the  pebas system  ( wesselingh and ramos , 2010 ) .", "we demonstrate that there is no evidence for a long - lived lake ( sensu gorthner , 1994 ) or any marine influx .", "conversely , we document a well - structured , aggrading fluvial system of late miocene age , which is in agreement with the sedimentation model and chronology proposed by latrubesse et  al .", "the studied sections are located along the juru and tarauac river , ne respectively se of the city eirunep ( state of amazonia , western brazil ; 245  km  s of benjamin constant ; fig . ", "delineations and subdivisions of basins in western amazonia diverge notably and several authors attribute the region of eirunep to the solimes basin ( eirunep subbasin ; e.g. , caputo , 1991 ; roddaz et  al . , 2005 ; ramos , 2006 ; wesselingh and salo , 2006 ; wesselingh et  al . ,", "however , based on subsurface information , obtained by extensive hydrocarbon and coal exploration campaigns during the 1970 s , this region is situated west of a basement high ( miura , 1972 ;  iquitos arch  ; corresponds to the  carauari arch  of caputo , 1991 ) on the eastern margin of the acre basin ( e.g. , del arco et  al . , 1977 ; maia et  al . , 1977 ; latrubesse et  al . , 2010 ; fig . ", "beside quaternary deposits ( terraces , alluvium ) , the scattered outcrops along river banks expose sediments of the upper parts of the solimes formation ( del arco et  al . , 1977 ;", "maia et  al . , 1977 ; paz et  al . , in press ) . the solimes fm .", "comprises more than 1000  m thick , largely pelitic  sandy alternations with lignitic intercalations and covers a huge part of western amazonia ( fig . ", ". uncertainties in its definition , its stratigraphical and geographical extent as well as its depositional environments basically reflect the ongoing debate about amazonia s history through neogene times .", "( 1977 ) , hoorn ( 1993 , 1994 ) , latrubesse et  al .", "the sections were vertically logged by visual inspection of the lithofacies ( including colour , grain size , bedding planes , sedimentary structures , macrofossil content ) . for lithofacies coding the scheme of miall ( 1996 )", "was used : capital letters  =  dominant grain size ( g , gravel ; s , sand ; f , fine sand  clay ) followed by a lowercase letter  =  sedimentary structures and/or biogenic features ( c , clast - supported ; m , massive / faint lamination ; h , horizontal bedding / lamination ; t and p , trough and planar cross bedding ; r , ripple bedding ; s , scour fill ; l , lamination ; r , rooted ; c , coal ; p , pedogenic overprint ) .", "additionally the outcrops were mapped laterally as far as possible . due to poor outcrop conditions", "an application of the  architectural element concept ( miall , 1996 ) was only loosely possible .", "for micropalaeontological investigations bulk samples ( 12  kg ) were taken from all outcrops .", "500  g of dried sediment ( 40  c , 24  h ) were washed by using diluted hydrogen superoxide for disintegration through standard sieves ( h2o2 : h2o  =  1 : 5 ; 63/125/250/500  m ) .", "wet sieve residuals were washed with ethanol prior to drying ( 40  c , 24  h ) .", "an in - depth taxonomic examination of the obtained microfossils ( including the sieve fractions < 250  m ) will be subject of further studies .", "for preliminary stable isotope analyses ( o , c ; 18 samples ) two or three ostracod valves ( 4060  g ; cyprideis spp . ) from earlier sampling campaigns were used ( collected by m.i.r ) . for analyses", "a thermo - finnigan kiel ii automated reaction system and a thermo - finnigan delta plus isotope - ratio mass spectrometer were used ( university of graz ; standard deviation  =  0.1 relative to nbs-19 ; results in per mille relative to vpdb ) .", "more detailed investigations are in preparation ( m. fonseca / belm , m. caporaletti / graz ) .", "location : 12.6  km ne eirunep ( s 063355.5/w 0694611.7  , altitude : 108  m ) , left cutbank of the juru river ( fig .  2 ) .", "description : the basal 1.8  m ( layers 19 ) of the section consists of partly ripple bedded sands and laminated silts ; some cm - thick clay intercalations were observed . above", "follows a 0.65  m thick alternation of clay with incorporated calcareous nodules and laminated silty sand or ripple bedded sand ( 1017 ; fig . ", "the top is rich in vertebrate remains ( crocodiles , turtles ; 17 ; fig . ", "massive to finely laminated , occasionally bivalves - bearing ( silty ) clay with coaly and sandy layers forms the next 1.14  m ( 1823 ; fig . ", "beds 2425 ( 1.85  m thick ) comprise massive , horizontally or trough cross bedded sand and finely laminated or massive ( sandy  silty ) clay / fine sand ( fig . ", "grey mottled , rooted and massive clay ( paleosol ) with a calcareous horizon on its top ( 26 ) .", "up - section , a 4.2  m ( 2739 ) thick alternation of massive or laminated clay , silt and partly ripple bedded fine sand is developed ( a slight coarsening - upward trend is anticipated ) .", "bed 32 displays a slightly erosive base with intraclasts in the lower part ( mud pebbles ) .", "layers 3139 are cut by a channel ( 40 ) with a multiphase fill . large scale cross bedded sands ( -cross stratification ) with sporadic intraclasts ( mud pebbles ) on reactivation surfaces and trough cross bedded and ripple bedded fine ", "microfossil contents : samples from the basal part of the section ( pd2 ) yielded only scarce valves of cyprideis spp . and rare fish elements .", "interpretation : the lithofacies of the basal part of the outcrop ( 117 ) refers to a deposition in a fluvial overbank environment .", "the diffuse layers of calcareous nodules ( 17 ) are supposed to be largely a diagenetic feature ( groundwater caliche ) .", "however , it may also hint to soil - forming processes at seasonally dry floodplain areas ( retallack , 2001 ) .", "up - section the sediments become finer and contain several ( sandy  silty ) coal - rich layers ( 1823 ) .", "these beds are assigned to the formation of a floodplain pond , which was inhabited by opportunistic ostracods , freshwater bivalves and fishes .", "however , crevasse splays influenced episodically that shallow lacustrine environment ( sandy and organic matter - rich layers ) .", "later ( layers 2425 ) , the influx of crevasse splays into the pond , which is still populated by ostracod faunas and fishes increased ( more prominent sandy beds , partly scour - fills ) .", "pedogenic processes are indicated by incipient soil formation in layer 26 ( mottled , roots ) .", "floodplain pond conditions ended above ( beds 2739 ) due to enhanced clastic input from an approaching channel", ". finally ( bed 40 ) , the overbank fines were intersected by a channel of at least 23  m in depth , which was filled up by large scale cross bedded sand .", "more or less perpendicular to the main accretion surfaces oriented ripple bedding indicates deposition by lateral accretion of a point bar .", "location : 17.9  km ne eirunep ( s 063251.1/w 0694239.4  , altitude : 107  m ) , left cutbank of the juru river ( fig .  4 ) .", "description : the lower 5  m of the outcrop are formed by ( section 1 ) : ( 1 ) greenish  grey , mottled clay with ferran cutans and cm - sized carbonate nodules ( fig . ", "5a ) ; ( 2 ) dark grey , massive or indistinctly laminated clay with cm - sized carbonate concretions ( fig . ", "5b ) , thin , pyrite - rich coaly layers , plant fragments , root traces and vertebrate remains ( crocodiles , turtles ) ; ( 3 ) greenish  yellowish , mottled clay with ferran cutans ( passes gradually into bed 4 ) ; ( 4 ) violet - red  green - grey , mottled clay / paleosol ( fig . ", "5c ) ; and ( 5 ) green  grey , mottled clay with vertebrate remains ( reptiles ) on top .", "a trough cross bedded sandy layer with coaly interlayers in the se part of the site mentioned by paz et  al .", "( in press ) was not exposed during our observation ( section 4 ) . in the nw part ( section 1 ) , above layer 5 , ripple bedded fine sand with intraclasts ( mud pebbles ) at its base is recorded , which contains bivalves and coalified wood remains ( 6 ) .", "laminated sand alternations ( 7 ) and massive sandy clay , which is rich in molluscs in its lower and upper part ( 8) . up - section ( 9 ) , an alternation of clay and coal layers , rich in molluscs ( i.e. , mycetopoda sp . ) , as well as a thinly bedded succession of laminated sand and thin clay interlayers ( 10 ) follow . towards the se ( section 24 )", "an up to 2  m thick , large scale cross bedded sandy unit is developed ( fig . ", "occasionally , intraclasts and vertebrate fragments are found at the base of this element or on accretion surfaces , which dip around ne .", "the dip of ripple foresets is approximately perpendicular oriented to these surfaces ( sw ) .", "this unit is overlain by a layer of calcareous nodules ( 11 ; section 3 ) and laminated clay ( silt)coal alternations with bivalve and vertebrate remains ( 13 and 14  =  equivalent to 9 ) .", "bed 12 ( plastic , strongly weathered clay ) is an equivalent of layer 10 .", "microfossil contents : samples mn1 and mn2 lack microfossils , in mn8a and mn8b only scattered valves of cypria sp . and heterocypris ?", "mn9 and mn 14 yielded many fish , gastropod and bivalve ( sphaeriids ) remains as well as abundant ostracod valves ( cyprideis spp . ) .", "earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) documented the following ostracod taxa : cyprideis graciosa , cyprideis lacrimata , cyprideis longispina , cyprideis machadoi , cyprideis pebasae , cyprideis olivencai , cyprideis sp . 1 and 2 .", "further findings ( plants , fishes , reptiles ) are mentioned in del arco et  al .", "the later authors also reported the occurrence of cyathecidites annulatus , echitricolporites spinosus and grimsdalea magnaclavata pollen .", "greenish to yellowish and dark grey colouration might indicate more or less water - logged conditions , whereas reddish colours may hint to rather well - drained , oxidizing environments .", "however , diagenetic alteration can hamper such an interpretation significantly ( retallack , 2001 ) .", "layer 2 is less affected by pedogenic processes ( faint lamination , coaly layers , plant and reptile fragments ) and perhaps represents floodplain lake / backswamp deposits .", "the paleosols ( overbank fines ; 15 ) are overlain by a sandy channel fill ( point bar ) with lag deposits at its base and repeatedly occurring intraclasts at its lateral accretion surfaces ( channel depth > 2  m based on the preserved height of this unit ) . in the nw part of morada nova layers", "68 represent an abandoned channel fill , which could be the fill of a subordinate channel or the fill of a chute channel .", "up - section , the influx of the active channel decreased and a floodplain lake developed ( 9 , 13 , 14 ) , which enabled the establishment of plentiful ostracod and aquatic molluscs faunas .", "the pond itself or its surroundings were richly vegetated ( coaly layers ) and settled by semi - aquatic reptiles . finally , enhanced input of sandy matter points to an increasing influence of the channel , probably due to crevasse splays .", "( s 063140.8/w 0693952.0  ; altitude : 106  m ) , left cutbank of the juru river ( fig . ", "description : section 1 ( w part ) starts with a 1.6  m thick succession of laminated , ripple and cross bedded silty sand with coaly intercalations ( layers 16/1 ) .", "up - section ( 714/1 ) , a 2.5  m thick sequence of ripple bedded silty fine sand layers with commonly erosive basal boundaries ( incl .", "convolute bedding and m - thick concretions are frequent in its upper part ( 1114/1 ) .", "silty clay ; 1520/1 ) and comprise coal and mollusc - rich pelitic ( 16/1 , 19/1 ) and coaly layers ( 18/1 ) as well as carbonate - cemented clay ( 20/1 ) .", "layer 21/1 ( 0.75  m thick ) is composed of greenish  violet , mottled or poorly laminated clay with scattered bivalve shells .", "intercalated are several layers ( 10  cm - thick ) , which consist of 12  mm large , rounded clay clasts ( fig . ", "it is overlain by 1  m of massive clay with abundant mollusc ( e.g. , sheppardiconcha septencincta ; fig . ", "leaves ; 22/1 ) and a > 0.5  m thick ( weathered ) coaly layer ( 23/1 ) with mollusc fragments ( e.g. , anodontites ?", "section 2 ( e part ; 120  m downstream of section 2 ) starts with > 0.35  m thick , laminated fine sand with coaly interlayers ( layer 1/2 ) .", "it is cut by a 4  m wide and 0.8  m deep channel fill ( 25/2 ; fining - upward , from base to top : laminated , coal - rich fine sand with rare mollusc and plant remains ; ripple bedded fine sand , rich in plant fragments ; massive clay , rich in bivalves ( sphaeriids ) , several wood remains ; fig . ", "this channel fill is topped by a 0.5  m thick , massive  poorly laminated clay with thin fine sand interlayers ( 6/2 ) and 0.2  m of massive , partly indurated ( cemented ) clay ( 7/2 ) .", "coalified wood remains are rare ; bivalves ( sphaeriids ) are abundant ( especially upper part of 6/2 ) .", "layer 6/2 and 7/2 taper off towards the w. up - section ( 1  m thickness ) , follow massive , silty  sandy clays ( 89/2 ) and ripple bedded silty fine sand with silt interlayers ( 1014/2 ) .", "especially layer 8/2 is rich in molluscs ( sphaeriids ) , 1314/2 contain coaly fragments ; clay intraclasts are observed at the base of 13/2 .", "these beds are topped by 2.7  m of mottled silty fine sand with coaly fragments in the lower 0.3  m ( 15/2 ) .", ". layer 16/2 ( > 1.2  m thick ) comprises coal  coaly fine sand  finely laminated clay alternations ( fig . ", "7f ) and is rich in bivalves ( sphaeriids ) in the lowermost 57  cm .", "whereas samples aq5/1 and aq6/1 contain only rare ostracod remains , aq16/1 yields , beside fish fragments , some valves of cytheridella sp . and darwinulids , accompanied by rare specimens of cypria sp . , cyprideis spp . and", ", darwinulids , cypria sp . , ilyocypridids ) and fish bones . several ( aq22a/1 ) respectively rare ( aq22b/1 ) cyprideis spp .", "valves are recorded in the samples of section 1 above , together with fish remains and gastropods ( aq22b/1 ) .", "sample aq3/2 of section 2 delivered only rarely cyprideis spp . and some fish remains . in the samples aq 5 - 6 - 8 - 13/2 plentiful ostracod faunas ( cytheridella sp . , ilyocyprinids , darwinulids , cyprideis spp .", "are documented along with fish remains and sphaeriid bivalve shells ( aq8/2 and aq13/2 ) . aq15/2 and aq16/2 are almost barren of ostracods but contain fish and bivalve ( sphaeriids ) fossils . earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) recorded the following ostracod taxa : alicenula ( darwinula ) fragilis , cypria aqualica , c.  longispina , c.  pebasae , cyprideis sp . 3 , cytheridella purperae , heterocypris ?", "( 2006b ) re - examined mollusc material studied by roxo ( 1937 ) from aquidab and described : ampullariidae sp .", "sheppardiconcha septemcincta . additional findings ( including vertebrates and plants ) are mentioned by del arco et  al .", "the basal , sandy  silty layers ( 16/1  =  1/2 ) possibly represent crevasse splay deposits , which are followed by a series of crevasse splay and/or crevasse channel sediments ( 714/1 respectively 25/2 ) .", "alternatively , these layers could represent channel deposits of an avulsive river arm . in section 2 a subordinate abandoned channel fill ( floodplain pond ; 67/2 ) is developed .", "afterwards ( 1523/1 and 816/2 ) the influx of crevassing respectively the active channel successively decreased and led to the formation of a floodplain lake , which was only influenced by flash floods in more proximal settings ( section 2 ; e.g. , 12/2 ) . aquatic faunas ( bivalves , ostracods ,", "fishes ) and semi - aquatic crocodilians inhabited that lake , which was surrounded by a densely vegetated backswamp .", "these beds display a mottled appearance and contain ( 21/1 ) layers of crumb peds ( clast - supported layers of rounded but not interlocked tiny mud balls ) , which indicate bioturbation due to invertebrate activity ( retallack , 2001 ) .", "finally , the lake became replaced by a swampy environment ( 23/1 and 16/2 ) .", "location : 27.4  km ne eirunep ( s 063122.0/w 0693542.8  ; altitude : 105  m ) , left cutbank of the juru river ( fig . ", "description : at the base > 2  m of slightly southwards inclined ( < 10 ) rippled bedded silty fine sand ( layer 1 ) are followed by 2.7  m thick violet - brown ( 2 ) and yellowish ( 3 ) mottled paleosols with calcareous nodules ( between 2 and 3 a pedogenically overprinted clay plug is intercalated ) .", "layers 13 are cut by a channel , whose fill starts ( 4 ) with > 1.5  m thick , massive  poorly laminated silty clay with gastropods , rare bivalves and plant ( leaves ) remains .", "bed 4 grades into a 0.8  m thick , brownish - grey , blocky structured silty clay ( 5 ) with a calcareous crust on its top .", "it is overlain by 2.4  m of violet - brown , mottled clay ( 6 ) with intercalated layers of calcareous nodules .", "strata 13 as well as the channel fill ( 46 ) are topped by a 0.4  m thick alternation of laminated silty clay and laminated or rippled bedded fine ", "medium sand with a high amount of plant detritus ( also wood remains ) and abundant vertebrates ( turtles ; 7 ) .", "the badly exposed , 7.2  m thick beds up - section ( 8) consist of cross and ripple bedded fine sand with intercalations of clayey lenses ( 2  m lateral extension , 20  cm thickness ) , which are formed by clay intraclasts ( mud pebbles , 1  cm diameter ) .", "repeatedly coaly layers are intercalated and rarely vertebrate ( crocodiles ) remains were found in the intraclast conglomerate .", "layer 9 represents a 1.2  m thick alternation of horizontally or ripple bedded fine ", "the topmost 1.2  m thick layer 10 consists of ripple bedded silty fine sand and silty clay alternations , which form cm - thick , fining - upward couplets .", "microfossil contents : sample re4 yielded only a few valves of cyprideis spp . as well as some fish and gastropod remains .", "interpretation : layer 1 could represent a crevasse splay or levee deposit , which is overlain by pedogenically altered floodplain fines ( 23 ) with intercalated minor channel fills .", "these overbank deposits are cut by a channel ( ? 20  m wide and 5  m deep ) , which became filled by pelitic , afterwards pedogenically overprinted sediments .", "the rare ( a matter of preservation ? ) mollusc and ostracod record indicates at least at the beginning ( 4 ) aquatic life in the abandoned channel . above , a crevasse splay ( 7 ) points to rising proximity of the active channel .", "large wood remains and vertebrate fragments were accumulated by this splay . the sandy succession up - section (", "8) can only be attributed to a sandy bedform sensu miall ( 1996 ) due to insufficient outcrop conditions .", "intercalated shallow scours , filled up with mud balls indicate deposition of reworked material and variations of the flow regime .", "the more or less rhythmically stratified , sandy - pelitic alternations at the top of remanso ( 910 ) mark a shift towards overbank environment and might represent a bar - top assemblage . location : 17.5  km se eirunep ( s 064923/w 0694704  , altitude : 117  m ) , left cutbank of the tarauac river ( fig . ", "description : the basal 1.7  m ( layers 13 ) consist of massive or laminated pelite- and fine sand with rare bivalve remains .", "they are topped by 0.2  m of reddish - brown , massive clay ( 4 ) .", "the sandy  silty layers 59 display a general coarsening - upward trend and contain only rarely bivalve fragments .", "up - section ( 10 ) , a 0.25  m thick intraclast conglomerate ( mudstone pebbles ) with abundant mollusc ( e.g. , ampullariidae ?", "this stratum is overlain by thin sand and clay layers ( 1117 ) with abundant mollusc remains ( 12 , 14 , 16 , lower part of 17 ) .", "layer 14 is rich in coal fragments ( wood , leaves ) at its top .", "the 2.57  m thick succession above ( 1823 ) starts with thin , ripple bedded fine sand beds ( 1819 ) , followed by massive silty / sandy clay with coaly fragments ( 20 ) and cm - thick alternations of fine sandy silt and silty fine sand ( 21 ; forming couplets of small - scale fining - upward cycles ) .", "beds 22 and 23 comprise alternations of massive , fine sandy silt and ripple bedded , silty fine sand .", "24 consists of 10  cm - thick alternations of indistinctly ripple bedded silty fine sand and massive or poorly laminated pelite . the upper part ( 1.5  m ) is mottled and primary sedimentary structures are obscured due to pedogenic processes .", "these samples contain several fish remains and in to12 rare characean gyrogonites were found additionally . the samples to20 and to 23 delivered only rare ostracod ( cypria sp .", "earlier reports of fossil material and facies : ramos ( 2006 ) mentioned the following ostracod taxa : c.  aqualica , c.  graciosa , c.  lacrimata , c.  longispina , c.  pebasae , c.  purperae , cytheridella sp . , darwinula fragilis as well as additional vertebrate and crab findings .", "interpretation : the lowermost sandy - pelitic layers ( 14 ) might be deposited in a floodplain pond / lake , which was influenced by crevasse splays .", "the coarsening - upward trend of layers 59 hints to a progressive influx of a crevasse channel ( layer 10 ) .", "it remains unclear if the ostracods of this layer are allochthonous or if they are incorporated in that layer due to subaquatic inflow of the crevasse splay ( crevasse delta ) into a floodplain pond / lake .", "up - section ( 1124 ) , plentiful ostracod and mollusc faunas ( especially in the lower part : 1117 ; compare ramos , 2006 ) indicate the return to an aquatic environment and the abandonment of the crevasse channel .", "small - scale fining - upward alternations of ripple bedded sand and pelite document individual flooding events or surges .", "location : 22.1  km sse eirunep ( s 065218.3/w 0694705.1  ; altitude : 120  m ) , left cutbank of the tarauac river ( fig . ", "description : layers 16 represent a 3.2  m thick succession of greenish ( 1 ) , reddish ( 2 ) , yellowish ( 3 , 5 , 6 ) or violet grey ( 4 ) coloured , mottled , frequently calcareous nodules - bearing paleosols . in layer 6 vertebrate remains ( crocodile teeth ) and gastropods were found .", "above follows a 1.1  m thick , indistinctly bedded clay to fine sand ( 7 ) , which contains in its lower half coaly plant fragments and becomes towards the top and laterally progressively mottled ( paleosol ) .", "the 0.65  m thick sediment column up - section ( 811 ) is composed of massive clay ( 8) , ripple bedded fine sand ( 9 ) , laminated silty fine sand to silt ( 10 ) and fine sand ( 11 ) .", "each of these layers displays a slightly erosive base and an internal fining - upward trend .", "the beds 8 , 9 and 11 each start with a basal layer of concretionary mud clasts in which ( layer 8 and 11 ) abundant vertebrate remains were found .", "layer 12 is formed by 1.2  m thick , ripple bedded fine sand with cm - thick , laminated silty clay interlayers .", "up - section a 0.7  m thick , orange - grey , mottled paleosol with mud clasts and rare vertebrate remains at its base ( 13 ) as well as a > 1.5  m thick , violet - purple coloured , mottled paleosol are developed ( 14 ) .", "microfossil contents : the samples ba7a and ba7b contained several ostracod valves ( mainly cyprideis spp . , some specimens of cypria sp .", "interpretation : the basal layers ( 16 ) represent a succession of paleosols , which could be seriously altered floodplain deposits . due to the occurrence of ostracods , probably bed 7 was deposited in a floodplain lake environment .", "afterwards , a series of crevasse splay ( maybe partly also crevasse channel ) deposits follows ( 813 ) , which is topped by pedogenically overprinted floodplain fines ( 14 ) .", "the sedimentary record of the investigated outcrops documents vertically as well as laterally highly variable fine - grained clastic successions ( predominantly clay  fine sand , along with fine  medium sand ) . based on the lithofacies and , as far as possible , lateral observations , these sediments represent fluvial deposition within active channels as well as in overbank environments .", "macroforms , generated within the active channel include lateral accretion deposits ( sandy point bars ; figs .  2 and 4 ) and undifferentiated sandy bedforms ( stacked dune fields ? ; fig . ", "8) , which are probably partly intersected by chute channels or are overlain by overbank fines .", "these comprise pelite  sand - alternations of levee , crevasse splay and crevasse channel deposits .", "frequently , a lag of mud pebbles and vertebrate fragments is found at the base of crevasse channels / splays . in part", ", some splays may have entered floodplain ponds / lakes via crevasse deltas ( e.g. , figs .  6 and 9 ) .", "abandoned , fine - grained channel fills ( mud plugs ) were observed ( fig . ", "decimetre - thick , clay  silt beds with rich ostracod and mollusc faunas indicate the presence of short - lived , shallow ponds or lakes within the floodplain ( floodbasin ; e.g. , fig . ", "6 ) point to partly swampy , poorly - drained conditions and a densely vegetated floodplain , which is , however , influenced by high clastic input .", "intensively mottled paleosols with root casts and calcareous nodules are ubiquitous ( e.g. , fig . ", "differences in colour and occasional calcic horizons may reflect diverging water - logging due to e.g. , local topography and/or seasonal fluctuations in discharge or climate ( kaandorp et  al .", "nevertheless , diagenesis may have altered significantly primary features and more  specific investigations are required ( retallack , 2001 ) .", "we are aware that restricted outcrop conditions and the limited areal and stratigraphical extent ( 30  m stratigraphical thickness ) of the current investigation hamper conclusions about the nature of fluvial style .", "reconstructions of fluvial geomorphology , based solely on lithofacies assemblages and fragmentary known fluvial elements , remain to some degree tentative ( miall , 1996 ; bridge , 2003 )", ". however , it seems obvious that we are dealing with a suspended load river system due to the high amount of overbank fines , probably within a tropical to subtropical wet  dry climate ( kaandorp et  al . , 2005 ; latrubesse et  al .", "we observed : ( a ) a large proportion of overbank deposits , ( b ) laterally extensive , heterogeneous , fine - grained avulsion deposits ( including crevasse splays ) , ( c ) predominantly sandy channel deposits , which change laterally and vertically rapidly into overbank deposits as well as ( d ) lacustrine and coaly deposits . some sandy point bars ( of secondary channels ? ) indicate subordinate lateral sediment accretion .", "these features are not exclusive to anastomosing river deposits , however , they largely coincide with the  standard  model of an anastomosing river sensu miall ( 1996 ) as well as with characteristics of such a system as reviewed by makaske ( 2001 ; compare also smith , 1983 ) .", "the general setting ( subsiding foreland basin , extensive sediment input from the andes , possibly incompletely framed by tectonically active basement highs towards the east ) as well as a wet  dry seasonal climate ( seasonal floods , channel banks stabilized by vegetation ) would provide the conditions for anastomosing river systems ( smith , 1983 ; nanson and knighton , 1996 ; makaske , 2001 ; latrubesse et  al .", "( late miocene ) in the state of acre ( sw brazil ) led to similar assumptions ( latrubesse et  al .", "these authors proposed a megafan system , which originates in the andes and encompass a complex mosaic of avulsive rivers and associated wetlands ( including abandoned channels , floodplain lakes , floodbasin deltas , backswamps and well - drained floodplains ; see also wilkinson et  al .", "this system is suggested to reach to the east at least as far as 67 w ( iquitos arch  sensu del arco et  al . , 1977 ) and probably also beyond (  purus arch  ; latrubesse et  al . , 1997 , 2010 ; fig", "according to that , the sections around eirunep were located in a more distal position , which could be the reason for a more pronounced anastomosing - anabranching pattern due to a lower gradient ( compare also interpretations of the late miocene tariquia formation , southern bolivia ; uba et  al .", "( 2007 , 2010 ) compared the depositional environment of the late miocene solimes fm . in acre with i.e. , the quaternary megafans of the chaco plain and the pantanal wetland ( iriondo , 1993 ; horton and decelles , 2001", "the mollusc fauna , recorded from floodplain pond / lake deposits , is dominated by sphaeriids and the pachychilid sheppardiconcha ( fig . ", "7b ) and indicates freshwater conditions within a fluvio - lacustrine environment ( wesselingh et  al . , 2006b ; wesselingh and ramos , 2010 ) .", "typical freshwater ostracods ( darwinulids , ilyocyprinids , cypria , cytheridella ) are associated with diverse species of cyprideis .", "this genus is a holoeuryhaline taxon , able to cope with fluctuating salinities , aberrant water chemistries , variable temperatures and oxygenation ( e.g. , meisch , 2000 ; keyser , 2005 ) . as it could adapt to freshwater conditions ( e.g. , lake tanganyika ; wouters and martens , 2001 ) and co - occurs here with exclusively freshwater taxa", ", there is no constraint to conclude a brackish water environment ( ramos , 2006 ) .", "preliminary stable isotope data ( o , c ) measured on cyprideis valves from aquidab , morada nova and torre da lua , yielded very negative values ( o :  5.7 to  9.7 , c :  10.3 to  12.5 ) , which exclude any marine influx .", "there is still intensive debate on definition and timing of palynological biozonation concepts ( e.g. , hoorn , 1993 , 1994 ; latrubesse et  al . , 2007 , 2010 ; jaramillo et  al . , 2010 ; silva - caminha et  al . ,", "however , palynostratigraphy remains a cornerstone for correlation across amazonia up to now and provides at least a rough stratigraphical framework .", "( 2006a ) are highly linked to the concept established by hoorn ( 1993 ) .", "based on the presence of some index taxa ( c.  annulatus , e.  spinosus , g.  magnaclavata ) a late miocene age ( probably asteraceae zone sensu lorente , 1986 ) is proposed by paz et  al .", "( in press ) for the outcrops morada nova and torre da lua recently . the vertebrate fauna from torre da lua ( ramos , 2006 ) is related to the so - called  acre fauna  , which is one of the best documented faunas of northern south america ( cozzuol , 2006 ; latrubesse et  al .", "the acre vertebrate assemblage displays significant affinities with the  mesopotamian  faunas of argentina and uruguay as well as with the urumaco assemblages ( venezuela ) and is dated to the late miocene ( huayquerian south american land mammal age ; cozzuol , 2006 ; latrubesse et  al .", "is still poor and has to be extended by later works ( wesselingh and ramos , 2010 ; see roxo , 1937 ; wesselingh et  al .", ", 2006b ; wesselingh , 2008 , and outcrop descriptions herein ) . from a stratigraphical point of view the mollusc faunas of aquidab and torre da lua", "( 2006b ) to be of late miocene origin and post - date the  pebas fauna ", "( compare celestino and ramos , 2007 ; wesselingh , 2008 ; wesselingh and ramos , 2010 , which suggested a late middle miocene age based on the ostracod record ) .", "a late miocene age seems to be in agreement with the lack of typical endemic pebasian molluscs ( wesselingh and ramos , 2010 ) . the recorded cyprideis - species from aquidab , morada nova and torre da lua ( ramos , 2006 ; celestino and ramos , 2007 ; wesselingh and ramos , 2010 ) have generally long ranges according to muoz - torres et  al .", "( 1998 , 2006 ; see also whatley et  al . , 1998 ) .", "the occurrence of c.  lacrimata and c.  pebasae might be indicative for an age not younger than the ( early ) late miocene .", "nonetheless , due to some contradictions ( e.g. , c.  lacrimata is supposed to appear first after the last appearance of c.  longispina but both are co - occurring at morada nova and torre da lua ) and the lack of key taxa , the current ostracod zonation needs some adjustment and is not further stressed here .", "however , to date , a late miocene age seems most plausible for all outcrops described herein by considering the palaeontological record .", "moreover , this fits well to the palaeogeographic context of this region as proposed by latrubesse et  al .", "sedimentological observations derived from outcrops around eirunep ( south - western part of the state of amazonia ) document various subenvironments of a fluvial system ( upper part of the solimes formation , late miocene ) . beside sandy channel deposits ,", "the main part comprises overbank sediments of levees , crevasse splays / channels / deltas , abandoned channels , backswamps and floodplain paleosols .", "based on the lithofacies and the general geological setting , this system can be possibly related to an anastomosing river style .", "there is not any indication for a long - lived lake (  lake pebas  ) or any marine influx in this region during the late miocene .", "( 1997 , 2007 , 2010 ; compare also cozzuol , 2006 ; westaway , 2006 ) ."], "abstract_text": ["<S> in miocene times a vast wetland existed in western amazonia . </S>", "<S> whereas the general development of this amazing ecosystem is well established , many questions remain open on sedimentary environments , stratigraphical correlations as well as its palaeogeographical configuration . </S>", "<S> several outcrops located in a barely studied region around eirunep ( sw amazonas state , brazil ) were investigated to obtain basic sedimentological data . </S>", "<S> the observed deposits belong to the upper part of the solimes formation and are biostratigraphically dated to the late miocene . </S>", "<S> vertically as well as laterally highly variable fine - grained clastic successions were recorded . </S>", "<S> based on the lithofacies assemblages , these sediments represent fluvial deposits , possibly of an anastomosing river system . </S>", "<S> sand bodies formed within active channels and dominant overbank fines are described ( levees , crevasse splays / channels / deltas , abandoned channels , backswamps , floodplain paleosols ) . </S>", "<S> lacustrine environments are restricted to local floodplain ponds / lakes . </S>", "<S> the mollusc and ostracod content as well as very light 18o and 13c values , measured on ostracod valves , refer to exclusively freshwater conditions . based on palaeontological and geological results the existence of a long - lived lake (  lake pebas  ) or </S>", "<S> any influx of marine waters can be excluded for that region during the late miocene . </S>"], "labels": null, "section_names": ["Introduction", "Working area and geological background", "Field and laboratory work", "Results  sedimentology of the outcrops", "Discussion", "Conclusions"], "sections": [["the geological and ( palaeo-)biological evolution of lowland amazonia during neogene and quaternary times remains fascinating since the early days of natural sciences ( for summaries of research history see e.g. , loczy , 1963 and wesselingh , 2008 ) .", "many , but partly highly controversial models have been introduced to explain its historical development ( for recent compilations see e.g. , lundberg et  al . , 1998 ;", ", 2006 ; wesselingh and salo , 2006 ; rossetti and mann de toledo , 2007 ; haffer , 2008 ; hoorn and wesselingh , 2010 ; hoorn et  al . , 2010 ;", "generally , it is widely accepted that around the onset of the miocene ( 23  ma ) a mega - wetland (  pebas system  , also called  lake pebas  ) developed in western amazonia due to the subsiding subandean foreland ( e.g. , hoorn , 1994 , 2006 ; wesselingh et  al . , 2002 ; wesselingh and salo , 2006 ; shephard et  al . , 2010 ) .", "short - lived marine incursions or even a transcontinental seaway from the caribbean sea through the venezuelan / columbian llanos basin southwards to the argentinean paran basin are proposed ( e.g. , rsnen et  al . , 1995 ;", "2009 ) but heavily disputed ( e.g. , campbell et  al . , 2006 ; cozzuol , 2006 ; westaway , 2006 ; latrubesse et  al . , 2007 , 2010 ;", ", 2003 ; hoorn and vonhof , 2006 ; wesselingh , 2006 ) . in the late miocene this mega - wetland disintegrated due to enhanced uplift of the northern / central andes .", "the drainage pattern of northern south america started to reverse completely to today s easterly course and the modern  amazon system  became established during the early pliocene ( hoorn , 2006 ; figueiredo et  al .", ", 2009 ; hoorn et  al . , 2010 ; latrubesse et  al . ,", "2010 ) . beside the vast size of amazonia and the still fragmentary regional coverage with field surveys ,", "there are considerable inconsistencies in palaeoenvironmental reconstructions and , in particular , in the chronology and correlation of scattered outcrops .", "wesselingh ( 2008 , p. 5 ) stated :  the lack of geological data has led to the emergence of many grand theories about the origin of present - day amazon system and its highly diversity , often based on dubious interpretations of the little data available  .", "the present paper aims to contribute basic sedimentological data from a barely studied region ( eirunep , 2.000  km sw manaus ; fig . ", "1a ) , which is supposed to be placed at the south - eastern margin of the  pebas system  ( wesselingh and ramos , 2010 ) .", "we demonstrate that there is no evidence for a long - lived lake ( sensu gorthner , 1994 ) or any marine influx .", "conversely , we document a well - structured , aggrading fluvial system of late miocene age , which is in agreement with the sedimentation model and chronology proposed by latrubesse et  al ."], ["the studied sections are located along the juru and tarauac river , ne respectively se of the city eirunep ( state of amazonia , western brazil ; 245  km  s of benjamin constant ; fig . ", "delineations and subdivisions of basins in western amazonia diverge notably and several authors attribute the region of eirunep to the solimes basin ( eirunep subbasin ; e.g. , caputo , 1991 ; roddaz et  al . , 2005 ; ramos , 2006 ; wesselingh and salo , 2006 ; wesselingh et  al . ,", "however , based on subsurface information , obtained by extensive hydrocarbon and coal exploration campaigns during the 1970 s , this region is situated west of a basement high ( miura , 1972 ;  iquitos arch  ; corresponds to the  carauari arch  of caputo , 1991 ) on the eastern margin of the acre basin ( e.g. , del arco et  al . , 1977 ; maia et  al . , 1977 ; latrubesse et  al . , 2010 ; fig . ", "beside quaternary deposits ( terraces , alluvium ) , the scattered outcrops along river banks expose sediments of the upper parts of the solimes formation ( del arco et  al . , 1977 ;", "maia et  al . , 1977 ; paz et  al . , in press ) . the solimes fm .", "comprises more than 1000  m thick , largely pelitic  sandy alternations with lignitic intercalations and covers a huge part of western amazonia ( fig . ", ". uncertainties in its definition , its stratigraphical and geographical extent as well as its depositional environments basically reflect the ongoing debate about amazonia s history through neogene times .", "( 1977 ) , hoorn ( 1993 , 1994 ) , latrubesse et  al ."], ["the sections were vertically logged by visual inspection of the lithofacies ( including colour , grain size , bedding planes , sedimentary structures , macrofossil content ) . for lithofacies coding the scheme of miall ( 1996 )", "was used : capital letters  =  dominant grain size ( g , gravel ; s , sand ; f , fine sand  clay ) followed by a lowercase letter  =  sedimentary structures and/or biogenic features ( c , clast - supported ; m , massive / faint lamination ; h , horizontal bedding / lamination ; t and p , trough and planar cross bedding ; r , ripple bedding ; s , scour fill ; l , lamination ; r , rooted ; c , coal ; p , pedogenic overprint ) .", "additionally the outcrops were mapped laterally as far as possible . due to poor outcrop conditions", "an application of the  architectural element concept ( miall , 1996 ) was only loosely possible .", "for micropalaeontological investigations bulk samples ( 12  kg ) were taken from all outcrops .", "500  g of dried sediment ( 40  c , 24  h ) were washed by using diluted hydrogen superoxide for disintegration through standard sieves ( h2o2 : h2o  =  1 : 5 ; 63/125/250/500  m ) .", "wet sieve residuals were washed with ethanol prior to drying ( 40  c , 24  h ) .", "an in - depth taxonomic examination of the obtained microfossils ( including the sieve fractions < 250  m ) will be subject of further studies .", "for preliminary stable isotope analyses ( o , c ; 18 samples ) two or three ostracod valves ( 4060  g ; cyprideis spp . ) from earlier sampling campaigns were used ( collected by m.i.r ) . for analyses", "a thermo - finnigan kiel ii automated reaction system and a thermo - finnigan delta plus isotope - ratio mass spectrometer were used ( university of graz ; standard deviation  =  0.1 relative to nbs-19 ; results in per mille relative to vpdb ) .", "more detailed investigations are in preparation ( m. fonseca / belm , m. caporaletti / graz ) ."], ["location : 12.6  km ne eirunep ( s 063355.5/w 0694611.7  , altitude : 108  m ) , left cutbank of the juru river ( fig .  2 ) .", "description : the basal 1.8  m ( layers 19 ) of the section consists of partly ripple bedded sands and laminated silts ; some cm - thick clay intercalations were observed . above", "follows a 0.65  m thick alternation of clay with incorporated calcareous nodules and laminated silty sand or ripple bedded sand ( 1017 ; fig . ", "the top is rich in vertebrate remains ( crocodiles , turtles ; 17 ; fig . ", "massive to finely laminated , occasionally bivalves - bearing ( silty ) clay with coaly and sandy layers forms the next 1.14  m ( 1823 ; fig . ", "beds 2425 ( 1.85  m thick ) comprise massive , horizontally or trough cross bedded sand and finely laminated or massive ( sandy  silty ) clay / fine sand ( fig . ", "grey mottled , rooted and massive clay ( paleosol ) with a calcareous horizon on its top ( 26 ) .", "up - section , a 4.2  m ( 2739 ) thick alternation of massive or laminated clay , silt and partly ripple bedded fine sand is developed ( a slight coarsening - upward trend is anticipated ) .", "bed 32 displays a slightly erosive base with intraclasts in the lower part ( mud pebbles ) .", "layers 3139 are cut by a channel ( 40 ) with a multiphase fill . large scale cross bedded sands ( -cross stratification ) with sporadic intraclasts ( mud pebbles ) on reactivation surfaces and trough cross bedded and ripple bedded fine ", "microfossil contents : samples from the basal part of the section ( pd2 ) yielded only scarce valves of cyprideis spp . and rare fish elements .", "interpretation : the lithofacies of the basal part of the outcrop ( 117 ) refers to a deposition in a fluvial overbank environment .", "the diffuse layers of calcareous nodules ( 17 ) are supposed to be largely a diagenetic feature ( groundwater caliche ) .", "however , it may also hint to soil - forming processes at seasonally dry floodplain areas ( retallack , 2001 ) .", "up - section the sediments become finer and contain several ( sandy  silty ) coal - rich layers ( 1823 ) .", "these beds are assigned to the formation of a floodplain pond , which was inhabited by opportunistic ostracods , freshwater bivalves and fishes .", "however , crevasse splays influenced episodically that shallow lacustrine environment ( sandy and organic matter - rich layers ) .", "later ( layers 2425 ) , the influx of crevasse splays into the pond , which is still populated by ostracod faunas and fishes increased ( more prominent sandy beds , partly scour - fills ) .", "pedogenic processes are indicated by incipient soil formation in layer 26 ( mottled , roots ) .", "floodplain pond conditions ended above ( beds 2739 ) due to enhanced clastic input from an approaching channel", ". finally ( bed 40 ) , the overbank fines were intersected by a channel of at least 23  m in depth , which was filled up by large scale cross bedded sand .", "more or less perpendicular to the main accretion surfaces oriented ripple bedding indicates deposition by lateral accretion of a point bar .", "location : 17.9  km ne eirunep ( s 063251.1/w 0694239.4  , altitude : 107  m ) , left cutbank of the juru river ( fig .  4 ) .", "description : the lower 5  m of the outcrop are formed by ( section 1 ) : ( 1 ) greenish  grey , mottled clay with ferran cutans and cm - sized carbonate nodules ( fig . ", "5a ) ; ( 2 ) dark grey , massive or indistinctly laminated clay with cm - sized carbonate concretions ( fig . ", "5b ) , thin , pyrite - rich coaly layers , plant fragments , root traces and vertebrate remains ( crocodiles , turtles ) ; ( 3 ) greenish  yellowish , mottled clay with ferran cutans ( passes gradually into bed 4 ) ; ( 4 ) violet - red  green - grey , mottled clay / paleosol ( fig . ", "5c ) ; and ( 5 ) green  grey , mottled clay with vertebrate remains ( reptiles ) on top .", "a trough cross bedded sandy layer with coaly interlayers in the se part of the site mentioned by paz et  al .", "( in press ) was not exposed during our observation ( section 4 ) . in the nw part ( section 1 ) , above layer 5 , ripple bedded fine sand with intraclasts ( mud pebbles ) at its base is recorded , which contains bivalves and coalified wood remains ( 6 ) .", "laminated sand alternations ( 7 ) and massive sandy clay , which is rich in molluscs in its lower and upper part ( 8) . up - section ( 9 ) , an alternation of clay and coal layers , rich in molluscs ( i.e. , mycetopoda sp . ) , as well as a thinly bedded succession of laminated sand and thin clay interlayers ( 10 ) follow . towards the se ( section 24 )", "an up to 2  m thick , large scale cross bedded sandy unit is developed ( fig . ", "occasionally , intraclasts and vertebrate fragments are found at the base of this element or on accretion surfaces , which dip around ne .", "the dip of ripple foresets is approximately perpendicular oriented to these surfaces ( sw ) .", "this unit is overlain by a layer of calcareous nodules ( 11 ; section 3 ) and laminated clay ( silt)coal alternations with bivalve and vertebrate remains ( 13 and 14  =  equivalent to 9 ) .", "bed 12 ( plastic , strongly weathered clay ) is an equivalent of layer 10 .", "microfossil contents : samples mn1 and mn2 lack microfossils , in mn8a and mn8b only scattered valves of cypria sp . and heterocypris ?", "mn9 and mn 14 yielded many fish , gastropod and bivalve ( sphaeriids ) remains as well as abundant ostracod valves ( cyprideis spp . ) .", "earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) documented the following ostracod taxa : cyprideis graciosa , cyprideis lacrimata , cyprideis longispina , cyprideis machadoi , cyprideis pebasae , cyprideis olivencai , cyprideis sp . 1 and 2 .", "further findings ( plants , fishes , reptiles ) are mentioned in del arco et  al .", "the later authors also reported the occurrence of cyathecidites annulatus , echitricolporites spinosus and grimsdalea magnaclavata pollen .", "greenish to yellowish and dark grey colouration might indicate more or less water - logged conditions , whereas reddish colours may hint to rather well - drained , oxidizing environments .", "however , diagenetic alteration can hamper such an interpretation significantly ( retallack , 2001 ) .", "layer 2 is less affected by pedogenic processes ( faint lamination , coaly layers , plant and reptile fragments ) and perhaps represents floodplain lake / backswamp deposits .", "the paleosols ( overbank fines ; 15 ) are overlain by a sandy channel fill ( point bar ) with lag deposits at its base and repeatedly occurring intraclasts at its lateral accretion surfaces ( channel depth > 2  m based on the preserved height of this unit ) . in the nw part of morada nova layers", "68 represent an abandoned channel fill , which could be the fill of a subordinate channel or the fill of a chute channel .", "up - section , the influx of the active channel decreased and a floodplain lake developed ( 9 , 13 , 14 ) , which enabled the establishment of plentiful ostracod and aquatic molluscs faunas .", "the pond itself or its surroundings were richly vegetated ( coaly layers ) and settled by semi - aquatic reptiles . finally , enhanced input of sandy matter points to an increasing influence of the channel , probably due to crevasse splays .", "( s 063140.8/w 0693952.0  ; altitude : 106  m ) , left cutbank of the juru river ( fig . ", "description : section 1 ( w part ) starts with a 1.6  m thick succession of laminated , ripple and cross bedded silty sand with coaly intercalations ( layers 16/1 ) .", "up - section ( 714/1 ) , a 2.5  m thick sequence of ripple bedded silty fine sand layers with commonly erosive basal boundaries ( incl .", "convolute bedding and m - thick concretions are frequent in its upper part ( 1114/1 ) .", "silty clay ; 1520/1 ) and comprise coal and mollusc - rich pelitic ( 16/1 , 19/1 ) and coaly layers ( 18/1 ) as well as carbonate - cemented clay ( 20/1 ) .", "layer 21/1 ( 0.75  m thick ) is composed of greenish  violet , mottled or poorly laminated clay with scattered bivalve shells .", "intercalated are several layers ( 10  cm - thick ) , which consist of 12  mm large , rounded clay clasts ( fig . ", "it is overlain by 1  m of massive clay with abundant mollusc ( e.g. , sheppardiconcha septencincta ; fig . ", "leaves ; 22/1 ) and a > 0.5  m thick ( weathered ) coaly layer ( 23/1 ) with mollusc fragments ( e.g. , anodontites ?", "section 2 ( e part ; 120  m downstream of section 2 ) starts with > 0.35  m thick , laminated fine sand with coaly interlayers ( layer 1/2 ) .", "it is cut by a 4  m wide and 0.8  m deep channel fill ( 25/2 ; fining - upward , from base to top : laminated , coal - rich fine sand with rare mollusc and plant remains ; ripple bedded fine sand , rich in plant fragments ; massive clay , rich in bivalves ( sphaeriids ) , several wood remains ; fig . ", "this channel fill is topped by a 0.5  m thick , massive  poorly laminated clay with thin fine sand interlayers ( 6/2 ) and 0.2  m of massive , partly indurated ( cemented ) clay ( 7/2 ) .", "coalified wood remains are rare ; bivalves ( sphaeriids ) are abundant ( especially upper part of 6/2 ) .", "layer 6/2 and 7/2 taper off towards the w. up - section ( 1  m thickness ) , follow massive , silty  sandy clays ( 89/2 ) and ripple bedded silty fine sand with silt interlayers ( 1014/2 ) .", "especially layer 8/2 is rich in molluscs ( sphaeriids ) , 1314/2 contain coaly fragments ; clay intraclasts are observed at the base of 13/2 .", "these beds are topped by 2.7  m of mottled silty fine sand with coaly fragments in the lower 0.3  m ( 15/2 ) .", ". layer 16/2 ( > 1.2  m thick ) comprises coal  coaly fine sand  finely laminated clay alternations ( fig . ", "7f ) and is rich in bivalves ( sphaeriids ) in the lowermost 57  cm .", "whereas samples aq5/1 and aq6/1 contain only rare ostracod remains , aq16/1 yields , beside fish fragments , some valves of cytheridella sp . and darwinulids , accompanied by rare specimens of cypria sp . , cyprideis spp . and", ", darwinulids , cypria sp . , ilyocypridids ) and fish bones . several ( aq22a/1 ) respectively rare ( aq22b/1 ) cyprideis spp .", "valves are recorded in the samples of section 1 above , together with fish remains and gastropods ( aq22b/1 ) .", "sample aq3/2 of section 2 delivered only rarely cyprideis spp . and some fish remains . in the samples aq 5 - 6 - 8 - 13/2 plentiful ostracod faunas ( cytheridella sp . , ilyocyprinids , darwinulids , cyprideis spp .", "are documented along with fish remains and sphaeriid bivalve shells ( aq8/2 and aq13/2 ) . aq15/2 and aq16/2 are almost barren of ostracods but contain fish and bivalve ( sphaeriids ) fossils . earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) recorded the following ostracod taxa : alicenula ( darwinula ) fragilis , cypria aqualica , c.  longispina , c.  pebasae , cyprideis sp . 3 , cytheridella purperae , heterocypris ?", "( 2006b ) re - examined mollusc material studied by roxo ( 1937 ) from aquidab and described : ampullariidae sp .", "sheppardiconcha septemcincta . additional findings ( including vertebrates and plants ) are mentioned by del arco et  al .", "the basal , sandy  silty layers ( 16/1  =  1/2 ) possibly represent crevasse splay deposits , which are followed by a series of crevasse splay and/or crevasse channel sediments ( 714/1 respectively 25/2 ) .", "alternatively , these layers could represent channel deposits of an avulsive river arm . in section 2 a subordinate abandoned channel fill ( floodplain pond ; 67/2 ) is developed .", "afterwards ( 1523/1 and 816/2 ) the influx of crevassing respectively the active channel successively decreased and led to the formation of a floodplain lake , which was only influenced by flash floods in more proximal settings ( section 2 ; e.g. , 12/2 ) . aquatic faunas ( bivalves , ostracods ,", "fishes ) and semi - aquatic crocodilians inhabited that lake , which was surrounded by a densely vegetated backswamp .", "these beds display a mottled appearance and contain ( 21/1 ) layers of crumb peds ( clast - supported layers of rounded but not interlocked tiny mud balls ) , which indicate bioturbation due to invertebrate activity ( retallack , 2001 ) .", "finally , the lake became replaced by a swampy environment ( 23/1 and 16/2 ) .", "location : 27.4  km ne eirunep ( s 063122.0/w 0693542.8  ; altitude : 105  m ) , left cutbank of the juru river ( fig . ", "description : at the base > 2  m of slightly southwards inclined ( < 10 ) rippled bedded silty fine sand ( layer 1 ) are followed by 2.7  m thick violet - brown ( 2 ) and yellowish ( 3 ) mottled paleosols with calcareous nodules ( between 2 and 3 a pedogenically overprinted clay plug is intercalated ) .", "layers 13 are cut by a channel , whose fill starts ( 4 ) with > 1.5  m thick , massive  poorly laminated silty clay with gastropods , rare bivalves and plant ( leaves ) remains .", "bed 4 grades into a 0.8  m thick , brownish - grey , blocky structured silty clay ( 5 ) with a calcareous crust on its top .", "it is overlain by 2.4  m of violet - brown , mottled clay ( 6 ) with intercalated layers of calcareous nodules .", "strata 13 as well as the channel fill ( 46 ) are topped by a 0.4  m thick alternation of laminated silty clay and laminated or rippled bedded fine ", "medium sand with a high amount of plant detritus ( also wood remains ) and abundant vertebrates ( turtles ; 7 ) .", "the badly exposed , 7.2  m thick beds up - section ( 8) consist of cross and ripple bedded fine sand with intercalations of clayey lenses ( 2  m lateral extension , 20  cm thickness ) , which are formed by clay intraclasts ( mud pebbles , 1  cm diameter ) .", "repeatedly coaly layers are intercalated and rarely vertebrate ( crocodiles ) remains were found in the intraclast conglomerate .", "layer 9 represents a 1.2  m thick alternation of horizontally or ripple bedded fine ", "the topmost 1.2  m thick layer 10 consists of ripple bedded silty fine sand and silty clay alternations , which form cm - thick , fining - upward couplets .", "microfossil contents : sample re4 yielded only a few valves of cyprideis spp . as well as some fish and gastropod remains .", "interpretation : layer 1 could represent a crevasse splay or levee deposit , which is overlain by pedogenically altered floodplain fines ( 23 ) with intercalated minor channel fills .", "these overbank deposits are cut by a channel ( ? 20  m wide and 5  m deep ) , which became filled by pelitic , afterwards pedogenically overprinted sediments .", "the rare ( a matter of preservation ? ) mollusc and ostracod record indicates at least at the beginning ( 4 ) aquatic life in the abandoned channel . above , a crevasse splay ( 7 ) points to rising proximity of the active channel .", "large wood remains and vertebrate fragments were accumulated by this splay . the sandy succession up - section (", "8) can only be attributed to a sandy bedform sensu miall ( 1996 ) due to insufficient outcrop conditions .", "intercalated shallow scours , filled up with mud balls indicate deposition of reworked material and variations of the flow regime .", "the more or less rhythmically stratified , sandy - pelitic alternations at the top of remanso ( 910 ) mark a shift towards overbank environment and might represent a bar - top assemblage . location : 17.5  km se eirunep ( s 064923/w 0694704  , altitude : 117  m ) , left cutbank of the tarauac river ( fig . ", "description : the basal 1.7  m ( layers 13 ) consist of massive or laminated pelite- and fine sand with rare bivalve remains .", "they are topped by 0.2  m of reddish - brown , massive clay ( 4 ) .", "the sandy  silty layers 59 display a general coarsening - upward trend and contain only rarely bivalve fragments .", "up - section ( 10 ) , a 0.25  m thick intraclast conglomerate ( mudstone pebbles ) with abundant mollusc ( e.g. , ampullariidae ?", "this stratum is overlain by thin sand and clay layers ( 1117 ) with abundant mollusc remains ( 12 , 14 , 16 , lower part of 17 ) .", "layer 14 is rich in coal fragments ( wood , leaves ) at its top .", "the 2.57  m thick succession above ( 1823 ) starts with thin , ripple bedded fine sand beds ( 1819 ) , followed by massive silty / sandy clay with coaly fragments ( 20 ) and cm - thick alternations of fine sandy silt and silty fine sand ( 21 ; forming couplets of small - scale fining - upward cycles ) .", "beds 22 and 23 comprise alternations of massive , fine sandy silt and ripple bedded , silty fine sand .", "24 consists of 10  cm - thick alternations of indistinctly ripple bedded silty fine sand and massive or poorly laminated pelite . the upper part ( 1.5  m ) is mottled and primary sedimentary structures are obscured due to pedogenic processes .", "these samples contain several fish remains and in to12 rare characean gyrogonites were found additionally . the samples to20 and to 23 delivered only rare ostracod ( cypria sp .", "earlier reports of fossil material and facies : ramos ( 2006 ) mentioned the following ostracod taxa : c.  aqualica , c.  graciosa , c.  lacrimata , c.  longispina , c.  pebasae , c.  purperae , cytheridella sp . , darwinula fragilis as well as additional vertebrate and crab findings .", "interpretation : the lowermost sandy - pelitic layers ( 14 ) might be deposited in a floodplain pond / lake , which was influenced by crevasse splays .", "the coarsening - upward trend of layers 59 hints to a progressive influx of a crevasse channel ( layer 10 ) .", "it remains unclear if the ostracods of this layer are allochthonous or if they are incorporated in that layer due to subaquatic inflow of the crevasse splay ( crevasse delta ) into a floodplain pond / lake .", "up - section ( 1124 ) , plentiful ostracod and mollusc faunas ( especially in the lower part : 1117 ; compare ramos , 2006 ) indicate the return to an aquatic environment and the abandonment of the crevasse channel .", "small - scale fining - upward alternations of ripple bedded sand and pelite document individual flooding events or surges .", "location : 22.1  km sse eirunep ( s 065218.3/w 0694705.1  ; altitude : 120  m ) , left cutbank of the tarauac river ( fig . ", "description : layers 16 represent a 3.2  m thick succession of greenish ( 1 ) , reddish ( 2 ) , yellowish ( 3 , 5 , 6 ) or violet grey ( 4 ) coloured , mottled , frequently calcareous nodules - bearing paleosols . in layer 6 vertebrate remains ( crocodile teeth ) and gastropods were found .", "above follows a 1.1  m thick , indistinctly bedded clay to fine sand ( 7 ) , which contains in its lower half coaly plant fragments and becomes towards the top and laterally progressively mottled ( paleosol ) .", "the 0.65  m thick sediment column up - section ( 811 ) is composed of massive clay ( 8) , ripple bedded fine sand ( 9 ) , laminated silty fine sand to silt ( 10 ) and fine sand ( 11 ) .", "each of these layers displays a slightly erosive base and an internal fining - upward trend .", "the beds 8 , 9 and 11 each start with a basal layer of concretionary mud clasts in which ( layer 8 and 11 ) abundant vertebrate remains were found .", "layer 12 is formed by 1.2  m thick , ripple bedded fine sand with cm - thick , laminated silty clay interlayers .", "up - section a 0.7  m thick , orange - grey , mottled paleosol with mud clasts and rare vertebrate remains at its base ( 13 ) as well as a > 1.5  m thick , violet - purple coloured , mottled paleosol are developed ( 14 ) .", "microfossil contents : the samples ba7a and ba7b contained several ostracod valves ( mainly cyprideis spp . , some specimens of cypria sp .", "interpretation : the basal layers ( 16 ) represent a succession of paleosols , which could be seriously altered floodplain deposits . due to the occurrence of ostracods , probably bed 7 was deposited in a floodplain lake environment .", "afterwards , a series of crevasse splay ( maybe partly also crevasse channel ) deposits follows ( 813 ) , which is topped by pedogenically overprinted floodplain fines ( 14 ) ."], ["the sedimentary record of the investigated outcrops documents vertically as well as laterally highly variable fine - grained clastic successions ( predominantly clay  fine sand , along with fine  medium sand ) . based on the lithofacies and , as far as possible , lateral observations , these sediments represent fluvial deposition within active channels as well as in overbank environments .", "macroforms , generated within the active channel include lateral accretion deposits ( sandy point bars ; figs .  2 and 4 ) and undifferentiated sandy bedforms ( stacked dune fields ? ; fig . ", "8) , which are probably partly intersected by chute channels or are overlain by overbank fines .", "these comprise pelite  sand - alternations of levee , crevasse splay and crevasse channel deposits .", "frequently , a lag of mud pebbles and vertebrate fragments is found at the base of crevasse channels / splays . in part", ", some splays may have entered floodplain ponds / lakes via crevasse deltas ( e.g. , figs .  6 and 9 ) .", "abandoned , fine - grained channel fills ( mud plugs ) were observed ( fig . ", "decimetre - thick , clay  silt beds with rich ostracod and mollusc faunas indicate the presence of short - lived , shallow ponds or lakes within the floodplain ( floodbasin ; e.g. , fig . ", "6 ) point to partly swampy , poorly - drained conditions and a densely vegetated floodplain , which is , however , influenced by high clastic input .", "intensively mottled paleosols with root casts and calcareous nodules are ubiquitous ( e.g. , fig . ", "differences in colour and occasional calcic horizons may reflect diverging water - logging due to e.g. , local topography and/or seasonal fluctuations in discharge or climate ( kaandorp et  al .", "nevertheless , diagenesis may have altered significantly primary features and more  specific investigations are required ( retallack , 2001 ) .", "we are aware that restricted outcrop conditions and the limited areal and stratigraphical extent ( 30  m stratigraphical thickness ) of the current investigation hamper conclusions about the nature of fluvial style .", "reconstructions of fluvial geomorphology , based solely on lithofacies assemblages and fragmentary known fluvial elements , remain to some degree tentative ( miall , 1996 ; bridge , 2003 )", ". however , it seems obvious that we are dealing with a suspended load river system due to the high amount of overbank fines , probably within a tropical to subtropical wet  dry climate ( kaandorp et  al . , 2005 ; latrubesse et  al .", "we observed : ( a ) a large proportion of overbank deposits , ( b ) laterally extensive , heterogeneous , fine - grained avulsion deposits ( including crevasse splays ) , ( c ) predominantly sandy channel deposits , which change laterally and vertically rapidly into overbank deposits as well as ( d ) lacustrine and coaly deposits . some sandy point bars ( of secondary channels ? ) indicate subordinate lateral sediment accretion .", "these features are not exclusive to anastomosing river deposits , however , they largely coincide with the  standard  model of an anastomosing river sensu miall ( 1996 ) as well as with characteristics of such a system as reviewed by makaske ( 2001 ; compare also smith , 1983 ) .", "the general setting ( subsiding foreland basin , extensive sediment input from the andes , possibly incompletely framed by tectonically active basement highs towards the east ) as well as a wet  dry seasonal climate ( seasonal floods , channel banks stabilized by vegetation ) would provide the conditions for anastomosing river systems ( smith , 1983 ; nanson and knighton , 1996 ; makaske , 2001 ; latrubesse et  al .", "( late miocene ) in the state of acre ( sw brazil ) led to similar assumptions ( latrubesse et  al .", "these authors proposed a megafan system , which originates in the andes and encompass a complex mosaic of avulsive rivers and associated wetlands ( including abandoned channels , floodplain lakes , floodbasin deltas , backswamps and well - drained floodplains ; see also wilkinson et  al .", "this system is suggested to reach to the east at least as far as 67 w ( iquitos arch  sensu del arco et  al . , 1977 ) and probably also beyond (  purus arch  ; latrubesse et  al . , 1997 , 2010 ; fig", "according to that , the sections around eirunep were located in a more distal position , which could be the reason for a more pronounced anastomosing - anabranching pattern due to a lower gradient ( compare also interpretations of the late miocene tariquia formation , southern bolivia ; uba et  al .", "( 2007 , 2010 ) compared the depositional environment of the late miocene solimes fm . in acre with i.e. , the quaternary megafans of the chaco plain and the pantanal wetland ( iriondo , 1993 ; horton and decelles , 2001", "the mollusc fauna , recorded from floodplain pond / lake deposits , is dominated by sphaeriids and the pachychilid sheppardiconcha ( fig . ", "7b ) and indicates freshwater conditions within a fluvio - lacustrine environment ( wesselingh et  al . , 2006b ; wesselingh and ramos , 2010 ) .", "typical freshwater ostracods ( darwinulids , ilyocyprinids , cypria , cytheridella ) are associated with diverse species of cyprideis .", "this genus is a holoeuryhaline taxon , able to cope with fluctuating salinities , aberrant water chemistries , variable temperatures and oxygenation ( e.g. , meisch , 2000 ; keyser , 2005 ) . as it could adapt to freshwater conditions ( e.g. , lake tanganyika ; wouters and martens , 2001 ) and co - occurs here with exclusively freshwater taxa", ", there is no constraint to conclude a brackish water environment ( ramos , 2006 ) .", "preliminary stable isotope data ( o , c ) measured on cyprideis valves from aquidab , morada nova and torre da lua , yielded very negative values ( o :  5.7 to  9.7 , c :  10.3 to  12.5 ) , which exclude any marine influx .", "there is still intensive debate on definition and timing of palynological biozonation concepts ( e.g. , hoorn , 1993 , 1994 ; latrubesse et  al . , 2007 , 2010 ; jaramillo et  al . , 2010 ; silva - caminha et  al . ,", "however , palynostratigraphy remains a cornerstone for correlation across amazonia up to now and provides at least a rough stratigraphical framework .", "( 2006a ) are highly linked to the concept established by hoorn ( 1993 ) .", "based on the presence of some index taxa ( c.  annulatus , e.  spinosus , g.  magnaclavata ) a late miocene age ( probably asteraceae zone sensu lorente , 1986 ) is proposed by paz et  al .", "( in press ) for the outcrops morada nova and torre da lua recently . the vertebrate fauna from torre da lua ( ramos , 2006 ) is related to the so - called  acre fauna  , which is one of the best documented faunas of northern south america ( cozzuol , 2006 ; latrubesse et  al .", "the acre vertebrate assemblage displays significant affinities with the  mesopotamian  faunas of argentina and uruguay as well as with the urumaco assemblages ( venezuela ) and is dated to the late miocene ( huayquerian south american land mammal age ; cozzuol , 2006 ; latrubesse et  al .", "is still poor and has to be extended by later works ( wesselingh and ramos , 2010 ; see roxo , 1937 ; wesselingh et  al .", ", 2006b ; wesselingh , 2008 , and outcrop descriptions herein ) . from a stratigraphical point of view the mollusc faunas of aquidab and torre da lua", "( 2006b ) to be of late miocene origin and post - date the  pebas fauna ", "( compare celestino and ramos , 2007 ; wesselingh , 2008 ; wesselingh and ramos , 2010 , which suggested a late middle miocene age based on the ostracod record ) .", "a late miocene age seems to be in agreement with the lack of typical endemic pebasian molluscs ( wesselingh and ramos , 2010 ) . the recorded cyprideis - species from aquidab , morada nova and torre da lua ( ramos , 2006 ; celestino and ramos , 2007 ; wesselingh and ramos , 2010 ) have generally long ranges according to muoz - torres et  al .", "( 1998 , 2006 ; see also whatley et  al . , 1998 ) .", "the occurrence of c.  lacrimata and c.  pebasae might be indicative for an age not younger than the ( early ) late miocene .", "nonetheless , due to some contradictions ( e.g. , c.  lacrimata is supposed to appear first after the last appearance of c.  longispina but both are co - occurring at morada nova and torre da lua ) and the lack of key taxa , the current ostracod zonation needs some adjustment and is not further stressed here .", "however , to date , a late miocene age seems most plausible for all outcrops described herein by considering the palaeontological record .", "moreover , this fits well to the palaeogeographic context of this region as proposed by latrubesse et  al ."], ["sedimentological observations derived from outcrops around eirunep ( south - western part of the state of amazonia ) document various subenvironments of a fluvial system ( upper part of the solimes formation , late miocene ) . beside sandy channel deposits ,", "the main part comprises overbank sediments of levees , crevasse splays / channels / deltas , abandoned channels , backswamps and floodplain paleosols .", "based on the lithofacies and the general geological setting , this system can be possibly related to an anastomosing river style .", "there is not any indication for a long - lived lake (  lake pebas  ) or any marine influx in this region during the late miocene .", "( 1997 , 2007 , 2010 ; compare also cozzuol , 2006 ; westaway , 2006 ) ."]]}
{"article_id": "PMC4369954", "article_text": ["with increasing availability , widened indications , and technical refinements of computed tomography ( ct ) and magnetic resonance imaging ( mri ) , the number of incidentally discovered adrenal lesions , such as adrenal incidentalomas and incidental adrenal enlargement , is increasing . in a recent study by tang et al .", ", among a total of 564 eligible ct studies ( patients undergoing ct without prior known malignancy , trauma , or endocrine disease ) , adrenal hyperplasia was detected in 64 cases , giving a prevalence of 11.3% .", "this indicated that incidental adrenal enlargement had a significant prevalence and has become a common clinical problem .", "it should be emphasized that the term incidental adrenal enlargement represents the way the lesion was detected ( incidentally ) , rather than the etiology or diagnosis .", "it is a common term for a variety of adrenal disorders , but its cause must be properly assessed so that patients needing treatment , such as those with hormone hypersecretion or malignant disease , can receive appropriate care . however , there is a lack of literature on functional status and its follow - up to provide comprehensive insight to these findings .", "patients with incidental adrenal enlargement were evaluated in a tertiary referral hospital with endocrinological departments in china .", "this study aimed to determine the primary clinical presentation that most frequently leads to the discovery of adrenal enlargement , to evaluate clinical characteristics and functional status of these patients , and to analyze risk factors for functional lesions .", "this retrospective study included 578 patients with adrenal imaging features showing adrenal enlargement who were hospitalized at the department of endocrinology in pla general hospital ( beijing , china ) between january 1993 and july 2013 .", "data retrieved included patient demographics , final functional diagnosis , adrenal imaging features , and concomitant diseases .", "patients were classified as having incidental adrenal enlargement when abdominal imaging was performed for indications unrelated to adrenal disease .", "patients with diseases known to cause adrenal enlargement , such as known endocrine disorders which could affect adrenal size , trauma , and underlying malignancy , were excluded . among all enrolled patients , 78 presented with incidental adrenal enlargement .", "the ct imaging technique used was not standardized due to the various clinical indications . however , to be included in their entirety on a maximum of 5  mm section thickness , the upper limit of normal was set as 10  mm for the body of the gland and 5  mm for each limb as documented by vincent et al . .", "the type of enlargement , based on subjective evaluation of the adrenal glands , was recorded as either smooth or nodular .", "nodular enlargement was diagnosed if the adrenal gland had an irregular contour , contained nodules , and had normal adrenal tissue interspersed between the nodules .", "smooth enlargement was defined as enlargement of the gland with a smooth contour and no measureable or diffuse nodules . after obtaining patient history and physical examination , all patients underwent biochemical evaluation to assess their functional status .", "patients with elevated 24-hour urine - free cortisol level ( 2 times ) or those in whom plasma cortisol levels did not decrease after an overnight low - dose dexamethasone test ( 1  mg dst ; cutoff 50 ", "patients with an aldosterone - rennin ratio ( arr ) > 20 underwent any 1 of 3 confirmatory tests ( saline infusion , captopril challenge , or postural stimulation ) to confirm or exclude definitively primary hyperaldosteronism ( pa ) .", "categorical data such as gender and clinical / radiologic features were compared using -test or fisher 's exact test .", "variables that resulted in a p < 0.05 in the univariate analyses were entered into logistic regression analysis to assess the risk factors of functional lesions .", "the hospital ethics committee approved this study , and written informed consent was obtained from all patients or their parents .", "of 578 patients with adrenal enlargement , 78 cases ( 13.49% ) were detected incidentally .", "every 2 years , the numbers of total cases were 17 , 11 , 14 , 24 , 33 , 31 , 54 , 55 , 114 , and 225 , respectively .", "the numbers of incidental adrenal enlargement cases were 0 , 0 , 0 , 1 , 1 , 2 , 5 , 4 , 16 , and 49 , respectively .", "in addition , the proportion of incidental adrenal enlargement gradually increased ( 0 , 0 , 0 , 4.17% , 3.03% , 7.32% , 9.26% , 7.27% , 14.04% , and 21.33% ) .", "39 cases had unilateral enlargement on the left side and 3 on the right side , and the remaining 36 were bilateral enlargement .", "as shown in table 1 , routine medical checkup was found to have the greatest chance ( 43.59% ) of revealing clinical onsets leading to the discovery of adrenal enlargement .", "predominant complaints included low back pain ( 10.26% ) and abdominal pain ( 3.85% ) . in addition , there were 30 ( 38.46% ) cases in which the lesions were incidentally detected during hospitalization for underlying diseases , such as diabetes mellitus , hypertension , and coronary heart disease , among others . biochemical and functional evaluation revealed that 54 ( 69.23% ) cases were nonfunctional and 12 ( 15.38% ) were subclinical cushing syndrome ( scs ) ; among these patients , 10 cases were diagnosed as aimah , the other 2 were diagnosed as adenomas , and they were all confirmed by pathology results .", "primary hyperaldosteronism 6 ( 7.69% ) , metastatic 1 ( 1.28% ) , the primary cancer was gastric cancer .", "there were 5 patients ( 6.41% ) whose functional status remained unclear because of failure to finish the functional evaluation ( figure 2 ) .", "nodular adrenal enlargement ( or 7.306 ; 95% ci , 1.72728.667 ; p = 0.006 ) was the risk factor for functional lesions .", "as outlined above , incidental adrenal enlargement is detected with increasing frequency , most likely due to widespread increase in cross - sectional imaging , and is gradually emerging as a common clinical problem .", "our study shows that the proportion of incidental adrenal enlargement has gradually increased by year .", "mean age at diagnosis was 50.32 years , which is in line with other incidentally detected adrenal findings , namely , adrenal incidentaloma .", "the increasing age of the general population and a research trend towards more advanced investigations in the elderly population may be contributing to the high detection rate in this age group .", "our results indicate that , for the elderly patients , it is essential to place emphasis on these incidental findings .", "'s study   indicated that , of the total 64 patients , of which 40 ( 63% ) were men and 24 ( 37% ) were women , 43 ( 67% ) cases were bilateral enlargement and 21 ( 33% ) cases were unilateral .", "in addition , smooth enlargement was more common , in 53 ( 83% ) cases , and together these statistics reflect the likelihood that adrenal enlargement will be bilateral , smooth , and found in men .", "however , our study did not show this tendency , likely because the research goals and thus , study populations , differed between the 2 studies .", "'s study aimed to explore prevalence , while the present study aimed to evaluate functional status .", "in addition , patients enrolled in our study were hospitalized at the department of endocrinology . it should be noted that admitting was more or less selective , especially in tertiary referral hospitals , and that economic considerations in parts of china were still a problem .", "clinically , upon discovery of incidental adrenal enlargement , 2 issues arise : functionality and malignancy . in the relevant literature [ 48 ] , adrenal enlargement can result from endocrine disorders , such as adrenocorticotropic hormone- ( acth- ) dependent or independent cushing syndrome , pa , multiple endocrine neoplasia type 1 ( men-1 ) , and congenital adrenal hyperplasia .", "other potential causes include nonfunctional lesions , defined as a radiographic adrenal enlargement without clinical or biochemical manifestations , inflammation , neoplastic processes , obesity , or depression .", "12 patients were found to have subclinical cushing syndrome ( originated from the adrenal gland ) and 6 patients were diagnosed with pa . however , the variety of disease spectrum in the study was only moderate , perhaps due , in part , to the limited number of included cases .", "it is important to note that even though reported prevalence was up to 11.3% , patient referrals to endocrinologists are relatively rare .", "this is likely related to the poor radiological awareness of this issue and its potential clinical significance .", "the clinical significance of lesion location and patient gender is smaller . in the present study ,", "acth - independent macronodular hyperplasia ( aimah ) and primary pigmented nodular adrenal hyperplasia often manifest as adrenal hyperplasia . the clinical features of aimah tended to be atypical .", "nodules usually distorted and completely obscured the normal adrenal glands and were characteristically  ginger - like  .", "as for pa , adrenal glands affected by idiopathic hyperaldosteronism ( iha ) may be normal on the ct scan or show nodular changes , and small aldosterone producing adenoma ( apas ) may be interpreted incorrectly by the radiologists as ", "iha  on the basis of ct findings of bilateral nodularity or normal - appearing adrenal glands [ 1013 ] .", "thus , it is a simple matter to explain why nodular enlargement could be a predictive factor of functional lesions .", "meanwhile , this also suggests that if incidentally detected lesions were nodular enlargements , evaluating its functional status should be a priority .", "in addition , the present study suggests that lesions on the left side were likely to be nonfunctional .", "in addition , there was only one patient with malignant lesion in the present study , and thus we were unable to analyze incidence of malignancy .", "incidental adrenal enlargement is a frequent radiographic finding and it is accompanied by diverse clinical factors that require proper diagnostic evaluation and management . in functional evaluation ,"], "abstract_text": ["<S> \n aim . to investigate incidental adrenal enlargement clinical characteristics and functional status and analyze functional lesion risk factors . materials and methods . </S>", "<S> this retrospective study included 578 patients with adrenal imaging features showing enlargement . </S>", "<S> incidental adrenal enlargement cases ( 78 ) were considered eligible . </S>", "<S> demographics , functional diagnosis , adrenal imaging features , and concomitant diseases were analyzed . </S>", "<S> results . </S>", "<S> the number of adrenal enlargements and proportion of incidental adrenal enlargement increased each year . </S>", "<S> mean patient age was 50.32 years . </S>", "<S> thirty - nine cases had unilateral enlargement on the left side and 3 on the right side ; 36 had bilateral enlargement . </S>", "<S> routine medical checkup was found to have the greatest chance ( 43.59% ) of revealing clinical onsets leading to discovery . </S>", "<S> biochemical and functional evaluation revealed 54 ( 69.23% ) cases of nonfunctional lesions , 12 ( 15.38% ) of subclinical cushing syndrome , 6 ( 7.69% ) of primary hyperaldosteronism , 1 ( 1.28% ) of metastasis , and 5 ( 6.41% ) of unknown functional status . </S>", "<S> nodular adrenal enlargement ( or , 7.306 ; 95% ci , 1.72728.667 ; </S>", "<S> p = 0.006 ) was a risk factor for functional lesions . </S>", "<S> age and lesion location were not significant factors . </S>", "<S> conclusion . </S>", "<S> incidental adrenal enlargement is a frequent radiographic finding and is accompanied by diverse clinical factors that require proper evaluation and management . </S>", "<S> nodular adrenal enlargement was a risk factor . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["with increasing availability , widened indications , and technical refinements of computed tomography ( ct ) and magnetic resonance imaging ( mri ) , the number of incidentally discovered adrenal lesions , such as adrenal incidentalomas and incidental adrenal enlargement , is increasing . in a recent study by tang et al .", ", among a total of 564 eligible ct studies ( patients undergoing ct without prior known malignancy , trauma , or endocrine disease ) , adrenal hyperplasia was detected in 64 cases , giving a prevalence of 11.3% .", "this indicated that incidental adrenal enlargement had a significant prevalence and has become a common clinical problem .", "it should be emphasized that the term incidental adrenal enlargement represents the way the lesion was detected ( incidentally ) , rather than the etiology or diagnosis .", "it is a common term for a variety of adrenal disorders , but its cause must be properly assessed so that patients needing treatment , such as those with hormone hypersecretion or malignant disease , can receive appropriate care . however , there is a lack of literature on functional status and its follow - up to provide comprehensive insight to these findings .", "patients with incidental adrenal enlargement were evaluated in a tertiary referral hospital with endocrinological departments in china .", "this study aimed to determine the primary clinical presentation that most frequently leads to the discovery of adrenal enlargement , to evaluate clinical characteristics and functional status of these patients , and to analyze risk factors for functional lesions ."], ["this retrospective study included 578 patients with adrenal imaging features showing adrenal enlargement who were hospitalized at the department of endocrinology in pla general hospital ( beijing , china ) between january 1993 and july 2013 .", "data retrieved included patient demographics , final functional diagnosis , adrenal imaging features , and concomitant diseases .", "patients were classified as having incidental adrenal enlargement when abdominal imaging was performed for indications unrelated to adrenal disease .", "patients with diseases known to cause adrenal enlargement , such as known endocrine disorders which could affect adrenal size , trauma , and underlying malignancy , were excluded . among all enrolled patients , 78 presented with incidental adrenal enlargement .", "the ct imaging technique used was not standardized due to the various clinical indications . however , to be included in their entirety on a maximum of 5  mm section thickness , the upper limit of normal was set as 10  mm for the body of the gland and 5  mm for each limb as documented by vincent et al . .", "the type of enlargement , based on subjective evaluation of the adrenal glands , was recorded as either smooth or nodular .", "nodular enlargement was diagnosed if the adrenal gland had an irregular contour , contained nodules , and had normal adrenal tissue interspersed between the nodules .", "smooth enlargement was defined as enlargement of the gland with a smooth contour and no measureable or diffuse nodules . after obtaining patient history and physical examination , all patients underwent biochemical evaluation to assess their functional status .", "patients with elevated 24-hour urine - free cortisol level ( 2 times ) or those in whom plasma cortisol levels did not decrease after an overnight low - dose dexamethasone test ( 1  mg dst ; cutoff 50 ", "patients with an aldosterone - rennin ratio ( arr ) > 20 underwent any 1 of 3 confirmatory tests ( saline infusion , captopril challenge , or postural stimulation ) to confirm or exclude definitively primary hyperaldosteronism ( pa ) .", "categorical data such as gender and clinical / radiologic features were compared using -test or fisher 's exact test .", "variables that resulted in a p < 0.05 in the univariate analyses were entered into logistic regression analysis to assess the risk factors of functional lesions .", "the hospital ethics committee approved this study , and written informed consent was obtained from all patients or their parents ."], ["of 578 patients with adrenal enlargement , 78 cases ( 13.49% ) were detected incidentally .", "every 2 years , the numbers of total cases were 17 , 11 , 14 , 24 , 33 , 31 , 54 , 55 , 114 , and 225 , respectively .", "the numbers of incidental adrenal enlargement cases were 0 , 0 , 0 , 1 , 1 , 2 , 5 , 4 , 16 , and 49 , respectively .", "in addition , the proportion of incidental adrenal enlargement gradually increased ( 0 , 0 , 0 , 4.17% , 3.03% , 7.32% , 9.26% , 7.27% , 14.04% , and 21.33% ) .", "39 cases had unilateral enlargement on the left side and 3 on the right side , and the remaining 36 were bilateral enlargement .", "as shown in table 1 , routine medical checkup was found to have the greatest chance ( 43.59% ) of revealing clinical onsets leading to the discovery of adrenal enlargement .", "predominant complaints included low back pain ( 10.26% ) and abdominal pain ( 3.85% ) . in addition , there were 30 ( 38.46% ) cases in which the lesions were incidentally detected during hospitalization for underlying diseases , such as diabetes mellitus , hypertension , and coronary heart disease , among others . biochemical and functional evaluation revealed that 54 ( 69.23% ) cases were nonfunctional and 12 ( 15.38% ) were subclinical cushing syndrome ( scs ) ; among these patients , 10 cases were diagnosed as aimah , the other 2 were diagnosed as adenomas , and they were all confirmed by pathology results .", "primary hyperaldosteronism 6 ( 7.69% ) , metastatic 1 ( 1.28% ) , the primary cancer was gastric cancer .", "there were 5 patients ( 6.41% ) whose functional status remained unclear because of failure to finish the functional evaluation ( figure 2 ) .", "nodular adrenal enlargement ( or 7.306 ; 95% ci , 1.72728.667 ; p = 0.006 ) was the risk factor for functional lesions ."], ["as outlined above , incidental adrenal enlargement is detected with increasing frequency , most likely due to widespread increase in cross - sectional imaging , and is gradually emerging as a common clinical problem .", "our study shows that the proportion of incidental adrenal enlargement has gradually increased by year .", "mean age at diagnosis was 50.32 years , which is in line with other incidentally detected adrenal findings , namely , adrenal incidentaloma .", "the increasing age of the general population and a research trend towards more advanced investigations in the elderly population may be contributing to the high detection rate in this age group .", "our results indicate that , for the elderly patients , it is essential to place emphasis on these incidental findings .", "'s study   indicated that , of the total 64 patients , of which 40 ( 63% ) were men and 24 ( 37% ) were women , 43 ( 67% ) cases were bilateral enlargement and 21 ( 33% ) cases were unilateral .", "in addition , smooth enlargement was more common , in 53 ( 83% ) cases , and together these statistics reflect the likelihood that adrenal enlargement will be bilateral , smooth , and found in men .", "however , our study did not show this tendency , likely because the research goals and thus , study populations , differed between the 2 studies .", "'s study aimed to explore prevalence , while the present study aimed to evaluate functional status .", "in addition , patients enrolled in our study were hospitalized at the department of endocrinology . it should be noted that admitting was more or less selective , especially in tertiary referral hospitals , and that economic considerations in parts of china were still a problem .", "clinically , upon discovery of incidental adrenal enlargement , 2 issues arise : functionality and malignancy . in the relevant literature [ 48 ] , adrenal enlargement can result from endocrine disorders , such as adrenocorticotropic hormone- ( acth- ) dependent or independent cushing syndrome , pa , multiple endocrine neoplasia type 1 ( men-1 ) , and congenital adrenal hyperplasia .", "other potential causes include nonfunctional lesions , defined as a radiographic adrenal enlargement without clinical or biochemical manifestations , inflammation , neoplastic processes , obesity , or depression .", "12 patients were found to have subclinical cushing syndrome ( originated from the adrenal gland ) and 6 patients were diagnosed with pa . however , the variety of disease spectrum in the study was only moderate , perhaps due , in part , to the limited number of included cases .", "it is important to note that even though reported prevalence was up to 11.3% , patient referrals to endocrinologists are relatively rare .", "this is likely related to the poor radiological awareness of this issue and its potential clinical significance .", "the clinical significance of lesion location and patient gender is smaller . in the present study ,", "acth - independent macronodular hyperplasia ( aimah ) and primary pigmented nodular adrenal hyperplasia often manifest as adrenal hyperplasia . the clinical features of aimah tended to be atypical .", "nodules usually distorted and completely obscured the normal adrenal glands and were characteristically  ginger - like  .", "as for pa , adrenal glands affected by idiopathic hyperaldosteronism ( iha ) may be normal on the ct scan or show nodular changes , and small aldosterone producing adenoma ( apas ) may be interpreted incorrectly by the radiologists as ", "iha  on the basis of ct findings of bilateral nodularity or normal - appearing adrenal glands [ 1013 ] .", "thus , it is a simple matter to explain why nodular enlargement could be a predictive factor of functional lesions .", "meanwhile , this also suggests that if incidentally detected lesions were nodular enlargements , evaluating its functional status should be a priority .", "in addition , the present study suggests that lesions on the left side were likely to be nonfunctional .", "in addition , there was only one patient with malignant lesion in the present study , and thus we were unable to analyze incidence of malignancy ."], ["incidental adrenal enlargement is a frequent radiographic finding and it is accompanied by diverse clinical factors that require proper diagnostic evaluation and management . in functional evaluation ,"]]}
{"article_id": "PMC3533150", "article_text": ["pursuing the goal of improved health literacy requires more alliances between health and education sectors to improve literacy levels in the population .", "it is important for health educators to know about knowledge transfer to meet their needs in transferring their knowledge to public", ". knowledge transfer , which means the synthesis , exchange and ethically application of knowledge within a complex system of relationship among researchers and users , has become one of the recent priorities in research centers . the word ", "the method of knowledge transfer , apart from its characteristics , requires active interactions between researchers and users .", "caplan proposes the two- communities theory indicating a gap between researchers and policymakers , whereas these days another gap has been formed between researchers and other users .", "in addition to illiteracy in developing countries that hampers effective health education , there are multitudes of non - medical specialists who do not have enough information about daily health affairs , and this lack is more remarkable in countries that welcome immigrants .", "immigrants often have significant language and health literacy difficulties , which are further exacerbated by cultural barriers .", "alongside with sophisticated methods like computer and internet , using simple methods help us bring to achieve our health goals[68 ] . also , the lack of health literacy needs more attention when it is manifested in caregivers .", "it is believed that some of the undesirable health outcomes in children are because of inadequate health knowledge among caregivers . in the usa ,", "therefore researchers have considered different methods to come up with this deficiency by having children to cooperate in their health programs .", "a research group in india endeavored to transfer knowledge on leprosy in cooperation with children and informed their parents through them .", "jacob et al ( 1994 ) started conducting a similar research that yields promising results .", "rimal and flora ( 1998 ) express that parental dietary behavior is partially affected by children .", "the findings from studies in this field have encouraged other experts to apply similar methods to conduct their research projects , allahverdipour and bashirian , onyango - ouma and mwangagain from the influence of children in order to teach different parts of their society[1316 ] . despite the researcher 's trends in using novel methods in this field ,", "none of them have used childish poem as a medium for knowledge transferring . in this study", ", we try to raise families health knowledge about infectious diseases by a method that uses children as health agents .", "infectious diseases are still the leading cause of mortality in children less than 60 months in developing countries .", "juvenile age is accompanied by learning childish poems , which can be sung contnuously at home and potentially become as part of both children 's and parents memories .", "we decided to transfer health knowledge to families through altering this information to childish poems and teaching children at kindergartens . in this method", "the outcome of transferring health knowledge through childish poems has been assessed in this study .", "in this study , we use the method of interventional pre and post series to conduct our research .", "the proposal of this non - invasive project was approved by the research committee of hamadan medical university in january 2009 .", "it was also accepted by hamadan welfare organization , which is the responsible organization of kindergartens in hamadan .", "our methodology is briefly explained as follows . at first , a sub specialist in pediatric infectious diseases provides seven short texts about health and common pediatric infectious diseases prevention .", "then , a poet transfers these conceptions to childish poems for the first time ( appendix 1&2 ) . in the process of preparing an accurate questionnaire , at first 30 questions with three choices of ", ",  false  and  do not know  were made , but 24 items of those are confirmed as valid questions by two experts of health education and infectious diseases . to assess", "the reliability of the questionnaire a pilot study on a 40 participant sample was performed and cronbach 's alpha of the questionnaire was determined as 83 percent .", "in addition , we defined knowledge mark ( km ) as a parent mark in both the pre - test and/or post - tests . among 35 kindergartens in hamadan , we selected 7 kindergartens by simple random method .", "the parents ( either father or mother , the one who is responsible to take her / his child ) of all five to six year old children of the selected kindergartens are asked to participate in the survey and a verbal consent is obtained from those who accepted .", "we excluded the following children from our sample : those whose parents are physicians , nurses , health - care workers , and those children whose parents work at the kindergarten ( exclusion criteria ) .", "we asked the parents of the sample survey to participate in a pretest by answering to a questionnaire when they are in the kindergartens .", "also , they were asked to come to the kindergartens personally one more time when it is required ( to answer to the post test ) . then , we asked our kindergarten tutors to teach children seven musical poems about hydatid cyst , antibiotic misuse , botulinum toxin in home - canned foods , dysentery , the importance of sixth tooth , brucellosis and tetanus . in this step , children were not allowed to take home these texts , but they were asked to sing the poems at home and want their parents to re - write the poems on a paper and give them the papers to take to the kindergarten .", "the teaching stage took between three to four months . during the next stage , parents took a post - test survey with the same 24 questions .", "parents of 115 kids participated in the pre - test and 103 of them completed the post - test .", "statistical analysis is done on 103 who completed both tests by paired t - test .", "the sample data consists of 103 parents who provided solutions to both the pre- and post - test question survey . of the participants 77 were female and 26 male . from the level of education point of view", ", 19 were below high school diploma , 45 had high school diploma , 8 had associate degree , 27 had ba , 3 had ma , and 1 had phd .", "the results of the survey show a significant difference between the correct answers in both pre - test and post - test stages ( fig .", "1 ) . the frequency of correct answers to the tests \n table 1 shows the mean of the correct answers in the pre - test stage is 59.22 while the same figure for the post - test step is 81 ( p < 0.001 ) .", "in addition , we define knowledge mark ( km ) as a parent mark in both the pre - test and/or post - test stages . the mean km shows a significant increase ( 5.01 ) from 13.62 in the pre - test to 18.63 in the post - test ( table 2 ) . \n ", "mean percent of correct answers to pre - test and post - test categories \n  analyzed by paired t - test sd : standard deviation mean of knowledge mark in pre - test and post test analysed by paired t - test the difference of km promotion among females and males was not significant ( 21.6120.17 in males and 20.331.61 in females , p=0.8 ) . the km was increased to 19.9325.06 among parents who at most had high school diploma and 23.4720.84 among parents with upper educational levels ( p=0.5 ) .", "health has been the concern of poets such as fiona sampson for years , however , these poems never had educational purposes . the results of this study", "confirm our hypothesis about the role of children in raising family 's knowledge on health .", "the results show that neither gender nor the grade of knowledge degree ( below and upper high school diploma or below and upper ba ) affect answers .", "therefore , people with different educational status have received the information similarly implying that this method can be extended to general cases in a straightforward manner . \n", "1 shows that the percentage of correct answers has increased in 23 out of 24 questions .", "the percent - age of correct answer to post - test has decreased only in one item ( item 11 ) which compares the risk of brucellosis transmission via unpasteurized milk and yogurt . although the term yogurt has not been mentioned directly in the related poem , but there is a hint about boiling mechanism and we expect that the audience discover the answer by logical thinking about the necessitation of boiling milk for preparing yogurt .", "the decrease in answering is 2 percent which is not significant ; however , it shows that it would be better to mention the conceptions directly in these types of poems in order to avoid any misconceptions .", "moreover , the percentage of correct answers to the questions of selected topics in pre - test and post - test has been compared ( table 1 ) and shows that parents have done better in post - test about all topics except one .", "the difference of correct answers to the questions about brucellosis does not show a significant increase in post - test .", "this finding is probably due to higher basic knowledge about brucellosis among parents . in other words , parents have had acceptable information about brucellosis even without our educational program , so we observe lower contrast between pre - test and post - test about brucellosis", ". it should be taken into consideration that brucellosis is an endemic disease in hamadan and people have been educated in different ways about this disease in recent years . although the lowest contrast between pre - test and post - test correct answers belongs to a question about botulism , the overall assessment shows that parent 's knowledge about this topic has been promoted significantly ( p<0.001 , table 1 ) . the significant difference between the pre - test and post - test km , based on the results of table 2 , is the sign of successful knowledge transfer through childish poems .", "the researcher 's method is impressively successful ; however , the fact that he mentions that some mothers can not distinguish the semantic difference between information and advice , needs serious attention .", "in addition , since daughters may misunderstand the information , it would be probable to transfer the misconceived information to their mothers and this fact threats the successfulness of study in applying . while , in our study ; firstly , childish poems do not imply sententious , secondly , if the poem is sung wrong , the disturbance in rhythm will be appeared , so the information which is transferred through poems is not at risk of alteration . in evans research ,", "parents get familiar with educated topics with the help of their children in homework assignments designed for asthma management , and are taught about asthma indirectly .", "the advantage of our study in comparison with evans is that in evans method knowledge transfer brings to bear via homework assignment , while in our study there is no obligation for doing home works , and parents are educated while enjoying their child 's singing . on account of the fact that making communication with younger children is not as difficult as with adolescents for parents , we are sure that knowledge transfers within a family through a continuous and friendly communication will work .", "christensen expressed the importance of children 's role in promoting the family health status . in his article , he emphasized on the activities that children can perform to enhance their health and promote their family health situation via the health efforts for themselves .", "we showed that children , additionally to what christensen mentioned , are able to affect their family 's health status directly . they can improve their family 's health as little teachers in health .", "it should be taken into consideration that the limitations of this study was the impact of other media on participant 's knowledge , which was not preventable .", "one of the acceptable results of this survey was publishing the poems as a book for children which was republished and welcomed by public .", "this study 's results suggest that health knowledge transfer to families through childish poems is an applicable method that has many advantages . by applying this joyful , cost - effective and easy - to - use method", "however , it should be taken into consideration that this method is applicable for families with trainable children , for teaching in larger scales more general ways should be added to this method .", "moreover , the simplicity of poems and expressing the topics in a less elaborative form are the key factors that affect the successfulness of this way ."], "abstract_text": ["<S> objectivethe purpose of this study is to propose an innovative method of knowledge transfer that aims to improve health literacy about pediatric infectious diseases prevention in families . </S>", "<S> children have an appreciable role in this scheme.methodsthis study is a before and after trial that has been conducted in hamedan in 2009 . after changing seven infectious disease topics into childish poems , we selected five kindergartens randomly and taught these poetries to the children . teaching process held after a pretest containing 24 questions that examined 103 of parents about mentioned topics . </S>", "<S> the same post - test was given after 4 months of teaching process.findingsthe mean of correct answers to the pretest was 59.22% comparable with 81.00% for post - test ( p<0.00 ) . </S>", "<S> gender and knowledge degree could not change the results significantly . </S>", "<S> assuming one 's correct answers to the questions as his / her knowledge mark , the mean of this variable increased to 5.32 by this method.conclusionthis cost - effective and joyful method had successful results in promoting health knowledge . </S>", "<S> children are able to play an active role in family 's health situation . </S>", "<S> learning within family atmosphere without any obligations makes our scheme a solution for paving the knowledge transferring way . </S>"], "labels": null, "section_names": ["Introduction", "Subjects and Methods", "Findings", "Discussion", "Conclusion", "Conflict of Interest"], "sections": [["pursuing the goal of improved health literacy requires more alliances between health and education sectors to improve literacy levels in the population .", "it is important for health educators to know about knowledge transfer to meet their needs in transferring their knowledge to public", ". knowledge transfer , which means the synthesis , exchange and ethically application of knowledge within a complex system of relationship among researchers and users , has become one of the recent priorities in research centers . the word ", "the method of knowledge transfer , apart from its characteristics , requires active interactions between researchers and users .", "caplan proposes the two- communities theory indicating a gap between researchers and policymakers , whereas these days another gap has been formed between researchers and other users .", "in addition to illiteracy in developing countries that hampers effective health education , there are multitudes of non - medical specialists who do not have enough information about daily health affairs , and this lack is more remarkable in countries that welcome immigrants .", "immigrants often have significant language and health literacy difficulties , which are further exacerbated by cultural barriers .", "alongside with sophisticated methods like computer and internet , using simple methods help us bring to achieve our health goals[68 ] . also , the lack of health literacy needs more attention when it is manifested in caregivers .", "it is believed that some of the undesirable health outcomes in children are because of inadequate health knowledge among caregivers . in the usa ,", "therefore researchers have considered different methods to come up with this deficiency by having children to cooperate in their health programs .", "a research group in india endeavored to transfer knowledge on leprosy in cooperation with children and informed their parents through them .", "jacob et al ( 1994 ) started conducting a similar research that yields promising results .", "rimal and flora ( 1998 ) express that parental dietary behavior is partially affected by children .", "the findings from studies in this field have encouraged other experts to apply similar methods to conduct their research projects , allahverdipour and bashirian , onyango - ouma and mwangagain from the influence of children in order to teach different parts of their society[1316 ] . despite the researcher 's trends in using novel methods in this field ,", "none of them have used childish poem as a medium for knowledge transferring . in this study", ", we try to raise families health knowledge about infectious diseases by a method that uses children as health agents .", "infectious diseases are still the leading cause of mortality in children less than 60 months in developing countries .", "juvenile age is accompanied by learning childish poems , which can be sung contnuously at home and potentially become as part of both children 's and parents memories .", "we decided to transfer health knowledge to families through altering this information to childish poems and teaching children at kindergartens . in this method", "the outcome of transferring health knowledge through childish poems has been assessed in this study ."], ["in this study , we use the method of interventional pre and post series to conduct our research .", "the proposal of this non - invasive project was approved by the research committee of hamadan medical university in january 2009 .", "it was also accepted by hamadan welfare organization , which is the responsible organization of kindergartens in hamadan .", "our methodology is briefly explained as follows . at first , a sub specialist in pediatric infectious diseases provides seven short texts about health and common pediatric infectious diseases prevention .", "then , a poet transfers these conceptions to childish poems for the first time ( appendix 1&2 ) . in the process of preparing an accurate questionnaire , at first 30 questions with three choices of ", ",  false  and  do not know  were made , but 24 items of those are confirmed as valid questions by two experts of health education and infectious diseases . to assess", "the reliability of the questionnaire a pilot study on a 40 participant sample was performed and cronbach 's alpha of the questionnaire was determined as 83 percent .", "in addition , we defined knowledge mark ( km ) as a parent mark in both the pre - test and/or post - tests . among 35 kindergartens in hamadan , we selected 7 kindergartens by simple random method .", "the parents ( either father or mother , the one who is responsible to take her / his child ) of all five to six year old children of the selected kindergartens are asked to participate in the survey and a verbal consent is obtained from those who accepted .", "we excluded the following children from our sample : those whose parents are physicians , nurses , health - care workers , and those children whose parents work at the kindergarten ( exclusion criteria ) .", "we asked the parents of the sample survey to participate in a pretest by answering to a questionnaire when they are in the kindergartens .", "also , they were asked to come to the kindergartens personally one more time when it is required ( to answer to the post test ) . then , we asked our kindergarten tutors to teach children seven musical poems about hydatid cyst , antibiotic misuse , botulinum toxin in home - canned foods , dysentery , the importance of sixth tooth , brucellosis and tetanus . in this step , children were not allowed to take home these texts , but they were asked to sing the poems at home and want their parents to re - write the poems on a paper and give them the papers to take to the kindergarten .", "the teaching stage took between three to four months . during the next stage , parents took a post - test survey with the same 24 questions .", "parents of 115 kids participated in the pre - test and 103 of them completed the post - test .", "statistical analysis is done on 103 who completed both tests by paired t - test ."], ["the sample data consists of 103 parents who provided solutions to both the pre- and post - test question survey . of the participants 77 were female and 26 male . from the level of education point of view", ", 19 were below high school diploma , 45 had high school diploma , 8 had associate degree , 27 had ba , 3 had ma , and 1 had phd .", "the results of the survey show a significant difference between the correct answers in both pre - test and post - test stages ( fig .", "1 ) . the frequency of correct answers to the tests \n table 1 shows the mean of the correct answers in the pre - test stage is 59.22 while the same figure for the post - test step is 81 ( p < 0.001 ) .", "in addition , we define knowledge mark ( km ) as a parent mark in both the pre - test and/or post - test stages . the mean km shows a significant increase ( 5.01 ) from 13.62 in the pre - test to 18.63 in the post - test ( table 2 ) . \n ", "mean percent of correct answers to pre - test and post - test categories \n  analyzed by paired t - test sd : standard deviation mean of knowledge mark in pre - test and post test analysed by paired t - test the difference of km promotion among females and males was not significant ( 21.6120.17 in males and 20.331.61 in females , p=0.8 ) . the km was increased to 19.9325.06 among parents who at most had high school diploma and 23.4720.84 among parents with upper educational levels ( p=0.5 ) ."], ["health has been the concern of poets such as fiona sampson for years , however , these poems never had educational purposes . the results of this study", "confirm our hypothesis about the role of children in raising family 's knowledge on health .", "the results show that neither gender nor the grade of knowledge degree ( below and upper high school diploma or below and upper ba ) affect answers .", "therefore , people with different educational status have received the information similarly implying that this method can be extended to general cases in a straightforward manner . \n", "1 shows that the percentage of correct answers has increased in 23 out of 24 questions .", "the percent - age of correct answer to post - test has decreased only in one item ( item 11 ) which compares the risk of brucellosis transmission via unpasteurized milk and yogurt . although the term yogurt has not been mentioned directly in the related poem , but there is a hint about boiling mechanism and we expect that the audience discover the answer by logical thinking about the necessitation of boiling milk for preparing yogurt .", "the decrease in answering is 2 percent which is not significant ; however , it shows that it would be better to mention the conceptions directly in these types of poems in order to avoid any misconceptions .", "moreover , the percentage of correct answers to the questions of selected topics in pre - test and post - test has been compared ( table 1 ) and shows that parents have done better in post - test about all topics except one .", "the difference of correct answers to the questions about brucellosis does not show a significant increase in post - test .", "this finding is probably due to higher basic knowledge about brucellosis among parents . in other words , parents have had acceptable information about brucellosis even without our educational program , so we observe lower contrast between pre - test and post - test about brucellosis", ". it should be taken into consideration that brucellosis is an endemic disease in hamadan and people have been educated in different ways about this disease in recent years . although the lowest contrast between pre - test and post - test correct answers belongs to a question about botulism , the overall assessment shows that parent 's knowledge about this topic has been promoted significantly ( p<0.001 , table 1 ) . the significant difference between the pre - test and post - test km , based on the results of table 2 , is the sign of successful knowledge transfer through childish poems .", "the researcher 's method is impressively successful ; however , the fact that he mentions that some mothers can not distinguish the semantic difference between information and advice , needs serious attention .", "in addition , since daughters may misunderstand the information , it would be probable to transfer the misconceived information to their mothers and this fact threats the successfulness of study in applying . while , in our study ; firstly , childish poems do not imply sententious , secondly , if the poem is sung wrong , the disturbance in rhythm will be appeared , so the information which is transferred through poems is not at risk of alteration . in evans research ,", "parents get familiar with educated topics with the help of their children in homework assignments designed for asthma management , and are taught about asthma indirectly .", "the advantage of our study in comparison with evans is that in evans method knowledge transfer brings to bear via homework assignment , while in our study there is no obligation for doing home works , and parents are educated while enjoying their child 's singing . on account of the fact that making communication with younger children is not as difficult as with adolescents for parents , we are sure that knowledge transfers within a family through a continuous and friendly communication will work .", "christensen expressed the importance of children 's role in promoting the family health status . in his article , he emphasized on the activities that children can perform to enhance their health and promote their family health situation via the health efforts for themselves .", "we showed that children , additionally to what christensen mentioned , are able to affect their family 's health status directly . they can improve their family 's health as little teachers in health .", "it should be taken into consideration that the limitations of this study was the impact of other media on participant 's knowledge , which was not preventable .", "one of the acceptable results of this survey was publishing the poems as a book for children which was republished and welcomed by public ."], ["this study 's results suggest that health knowledge transfer to families through childish poems is an applicable method that has many advantages . by applying this joyful , cost - effective and easy - to - use method", "however , it should be taken into consideration that this method is applicable for families with trainable children , for teaching in larger scales more general ways should be added to this method .", "moreover , the simplicity of poems and expressing the topics in a less elaborative form are the key factors that affect the successfulness of this way ."], [""]]}
{"article_id": "PMC5040566", "article_text": ["septic arthritis of the shoulder is uncommon in adults . it is a surgical emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality .", "a 43 years old male came to our outpatient department with complaints of pain and stiffness of his left shoulder . on examination , his shoulder movements were severely restricted .", "further evaluation with mri revealed septic arthritis of left gleno - humeral joint for which emergency arthroscopic debridement was done .", "septic arthritis of shoulder may not present with classical clinical features . hence , a through clinical and radiological evaluation will help us prognosticate and treat accordingly thereby preventing complications like septic shock , osteomyelitis .", "patients with septic arthritis usually develop moderate to severe joint pain , warmth , tenderness , effusion , restricted active and passive motion , and sometimes redness .", "we report an unusual presentation of shoulder septic arthritis in a 43 years old man with no other clinical signs and symptoms of classical septic arthritis .", "a 43-year - old man presented to the orthopaedic outpatient clinic with 2 months history of pain and limited range of motion in his left shoulder .", "his pain was insidious in onset , mild to moderate in intensity , aggravated by activities and associated with moderate rest and night pain .", "patient was diagnosed as frozen shoulder at an outside facility and had been given intraarticular depomedrol 40 mg injection for the same 40 days ago with no improvement in his symptoms .", "he is diabetic and has liver cirrhosis and is on treatment . on physical examination ,", "the skin colour and temperature of the left shoulder were normal , but the shoulder was tender to touch over the anterior joint line .", "mri was obtained [ fig 2 ] which showed extraosseous soft tissue enhancements around left shoulder with soft tissue abscess in inter muscular planes of supraspinatous , infraspinatous , subscapularis .", "blood test revealed normal white cell count with normal differential count , crp-13.4 mg / l ( normal<5.0 mg", "the erythrocyte sedimentation rate was raised , at 88 mm / hour ( normal range , 0 - 20 mm / hour ) aspiration of shoulder joint was performed and fluid was sent for aerobic and anaerobic culture , afb staining , mycobacterium culture , and mycobacterium genetic testing .", "arthroscopic lavage of the joint was done and articular fluid sent for repeat aerobic and anaerobic culture .", "joint visualization was markedly limited because of severe inflammation and fibrinous changes of the joint .", "the articular cartilages of glenoid and humeral head were completely eroded down to the bone .", "susceptibility of the isolate was determined with the disk diffusion method and it was susceptible only to colistin .", "3 ) fibrocollagenous and fibrovascular inflammed connective tissue containing proliferated blood vessels and perivascular mixed inflammatory cells and lymphoplasmacytoid cells .", "based on preoperative and intraoperative culture report , a definitive diagnosis of polymicrobial septic arthritis of the shoulder was established and patient was treated with combination antimicrobial treatment .", "our patient had muted inflammatory response probably due to immunocompromised state ( cirrhosis and diabetes ) which is not uncommon .", "most often it is hematogenous seeding of shoulder joint , however it can also happen after intraarticular steroid injections .", "septic arthritis of the shoulder is more common in immunocompromised patients and intravenous drug abusers . in this case report , the patient had atypical clinical symptoms and is immunocompromised and his primary source of infection seems to be hematogenous .", "our experience with this patient highlights the importance of maintaining high index of suspicion for septic arthritis in immunocompromised patients presenting with atypical shoulder pain associated with stiffness . in the setting of suspected septic arthritis ,", "our initial pre - investigation diagnosis was atypical shoulder pain and stiffness of non specific etiology in an immunocompromised patient and infection was part of the differential diagnosis not the only diagnosis .", "esenwein et al in his report highlighted the importance of early intervention to prevent chondral damage , osteomyelitis and also to prevent systemic spread .", "various authors have highlighted the importance of early diagnosis and management of septic arthritis failing which could lead to osteomyelitis and septic shock . in our patient", ", the diagnosis was delayed due to atypical presentation and over reliance on clinical findings at an outside center .", "we strongly recommend early imaging studies in immunocompromised patients presenting with shoulder pain and practitioners should avoid loosely diagnosing as shoulder pain with associated stiffness as frozen shoulders .", "klinger et al . did a retrospective study on 21 patients who underwent surgical treatment for septic arthritis of the shoulder joint between 2000 and 2007 , and he concluded that patients with symptoms for less than 2 weeks did well with arthroscopic approach and early infection can be managed arthroscopically .", "our reports show that arthroscopic washout can give good result even after 2 weeks of clinical symptoms if there is no evidence of osteomyelitis .", "septic arthritis of the shoulder is very often due to hematogenous spread and diagnosis is often clinical .", "laboratory investigations and imaging studies like mri and usg may be useful in establishing the diagnosis but confirmation is usually by joint aspiration .", "patients who are immunocompromised and have insidious onset of moderate to severe pain and which fails to respond to trial of conservative treatment should be subjected to either ultrasound or mri instead of x - ray because very often in these group of patients it is primarily a soft tissue pathology like impingement , rc tear , calcific tendinitis or rarely infections and malignancies .", "we recommend that moderate pain of more than 4 weeks duration with severe stiffness in immunocompromised patient ( liver cirrhosis , renal failure , steroid treatment ) should be further evaluated with mri or ultrasound in the setting of normal x - ray and should not be loosely diagnosed as frozen shoulder .", "goldenberg in his report emphasized the role of local as well as systemic factors that predispose patients with cirrhosis to gram - negative bacterial joint infections .", "malnick also reported a case of spontaneous septic arthritis in a cirhottic patient that was due to e.coli .", "our study as well as other studies by goldenberg and malnick highlight the importance of including broad spectrum antibiotics with gram negative cover whilst waiting for final culture sensitivity .", "septic arthritis of shoulder may not present with classic clinical features . a through clinical and radiological evaluation should be executed and treatment initiated promptly to prevent complications like septic shock and osteomyelitis .", "the case highlights the importance of establishing anatomical and pathological diagnosis using mri in patients with shoulder pain instead of loosely diagnosing them as impingement or frozen shoulder .", "the case also challenges the practice of routine shoulder depomedrol steroid injection , in the setting of secondary frozen shoulder , atleast in immunocompromised individuals .", "primary idiopathic frozen shoulder is a rare condition and secondary frozen shoulder cases are often due to underlying shoulder pathology ."], "abstract_text": ["<S> introduction : septic arthritis of the shoulder is uncommon in adults . it is a surgical emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality . </S>", "<S> accurate diagnosis can be particularly challenging in patients with underlying liver disease . </S>", "<S> mri is a useful adjunct in early detection of atypical causes of shoulder pain.case report : a 43 years old male came to our outpatient department with complaints of pain and stiffness of his left shoulder . on examination , </S>", "<S> his shoulder movements were severely restricted . further evaluation with mri revealed septic arthritis of left gleno - humeral joint for which emergency arthroscopic debridement was done.conclusion:septic arthritis of shoulder may not present with classical clinical features . </S>", "<S> hence , a through clinical and radiological evaluation will help us prognosticate and treat accordingly thereby preventing complications like septic shock , osteomyelitis . </S>"], "labels": null, "section_names": ["Introduction:", "Case report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["septic arthritis of the shoulder is uncommon in adults . it is a surgical emergency as joint destruction occurs rapidly and can lead to significant morbidity and mortality ."], ["a 43 years old male came to our outpatient department with complaints of pain and stiffness of his left shoulder . on examination , his shoulder movements were severely restricted .", "further evaluation with mri revealed septic arthritis of left gleno - humeral joint for which emergency arthroscopic debridement was done ."], ["septic arthritis of shoulder may not present with classical clinical features . hence , a through clinical and radiological evaluation will help us prognosticate and treat accordingly thereby preventing complications like septic shock , osteomyelitis ."], ["patients with septic arthritis usually develop moderate to severe joint pain , warmth , tenderness , effusion , restricted active and passive motion , and sometimes redness .", "we report an unusual presentation of shoulder septic arthritis in a 43 years old man with no other clinical signs and symptoms of classical septic arthritis ."], ["a 43-year - old man presented to the orthopaedic outpatient clinic with 2 months history of pain and limited range of motion in his left shoulder .", "his pain was insidious in onset , mild to moderate in intensity , aggravated by activities and associated with moderate rest and night pain .", "patient was diagnosed as frozen shoulder at an outside facility and had been given intraarticular depomedrol 40 mg injection for the same 40 days ago with no improvement in his symptoms .", "he is diabetic and has liver cirrhosis and is on treatment . on physical examination ,", "the skin colour and temperature of the left shoulder were normal , but the shoulder was tender to touch over the anterior joint line .", "mri was obtained [ fig 2 ] which showed extraosseous soft tissue enhancements around left shoulder with soft tissue abscess in inter muscular planes of supraspinatous , infraspinatous , subscapularis .", "blood test revealed normal white cell count with normal differential count , crp-13.4 mg / l ( normal<5.0 mg", "the erythrocyte sedimentation rate was raised , at 88 mm / hour ( normal range , 0 - 20 mm / hour ) aspiration of shoulder joint was performed and fluid was sent for aerobic and anaerobic culture , afb staining , mycobacterium culture , and mycobacterium genetic testing .", "arthroscopic lavage of the joint was done and articular fluid sent for repeat aerobic and anaerobic culture .", "joint visualization was markedly limited because of severe inflammation and fibrinous changes of the joint .", "the articular cartilages of glenoid and humeral head were completely eroded down to the bone .", "susceptibility of the isolate was determined with the disk diffusion method and it was susceptible only to colistin .", "3 ) fibrocollagenous and fibrovascular inflammed connective tissue containing proliferated blood vessels and perivascular mixed inflammatory cells and lymphoplasmacytoid cells .", "based on preoperative and intraoperative culture report , a definitive diagnosis of polymicrobial septic arthritis of the shoulder was established and patient was treated with combination antimicrobial treatment ."], ["our patient had muted inflammatory response probably due to immunocompromised state ( cirrhosis and diabetes ) which is not uncommon .", "most often it is hematogenous seeding of shoulder joint , however it can also happen after intraarticular steroid injections .", "septic arthritis of the shoulder is more common in immunocompromised patients and intravenous drug abusers . in this case report , the patient had atypical clinical symptoms and is immunocompromised and his primary source of infection seems to be hematogenous .", "our experience with this patient highlights the importance of maintaining high index of suspicion for septic arthritis in immunocompromised patients presenting with atypical shoulder pain associated with stiffness . in the setting of suspected septic arthritis ,", "our initial pre - investigation diagnosis was atypical shoulder pain and stiffness of non specific etiology in an immunocompromised patient and infection was part of the differential diagnosis not the only diagnosis .", "esenwein et al in his report highlighted the importance of early intervention to prevent chondral damage , osteomyelitis and also to prevent systemic spread .", "various authors have highlighted the importance of early diagnosis and management of septic arthritis failing which could lead to osteomyelitis and septic shock . in our patient", ", the diagnosis was delayed due to atypical presentation and over reliance on clinical findings at an outside center .", "we strongly recommend early imaging studies in immunocompromised patients presenting with shoulder pain and practitioners should avoid loosely diagnosing as shoulder pain with associated stiffness as frozen shoulders .", "klinger et al . did a retrospective study on 21 patients who underwent surgical treatment for septic arthritis of the shoulder joint between 2000 and 2007 , and he concluded that patients with symptoms for less than 2 weeks did well with arthroscopic approach and early infection can be managed arthroscopically .", "our reports show that arthroscopic washout can give good result even after 2 weeks of clinical symptoms if there is no evidence of osteomyelitis .", "septic arthritis of the shoulder is very often due to hematogenous spread and diagnosis is often clinical .", "laboratory investigations and imaging studies like mri and usg may be useful in establishing the diagnosis but confirmation is usually by joint aspiration .", "patients who are immunocompromised and have insidious onset of moderate to severe pain and which fails to respond to trial of conservative treatment should be subjected to either ultrasound or mri instead of x - ray because very often in these group of patients it is primarily a soft tissue pathology like impingement , rc tear , calcific tendinitis or rarely infections and malignancies .", "we recommend that moderate pain of more than 4 weeks duration with severe stiffness in immunocompromised patient ( liver cirrhosis , renal failure , steroid treatment ) should be further evaluated with mri or ultrasound in the setting of normal x - ray and should not be loosely diagnosed as frozen shoulder .", "goldenberg in his report emphasized the role of local as well as systemic factors that predispose patients with cirrhosis to gram - negative bacterial joint infections .", "malnick also reported a case of spontaneous septic arthritis in a cirhottic patient that was due to e.coli .", "our study as well as other studies by goldenberg and malnick highlight the importance of including broad spectrum antibiotics with gram negative cover whilst waiting for final culture sensitivity ."], ["septic arthritis of shoulder may not present with classic clinical features . a through clinical and radiological evaluation should be executed and treatment initiated promptly to prevent complications like septic shock and osteomyelitis .", "the case highlights the importance of establishing anatomical and pathological diagnosis using mri in patients with shoulder pain instead of loosely diagnosing them as impingement or frozen shoulder .", "the case also challenges the practice of routine shoulder depomedrol steroid injection , in the setting of secondary frozen shoulder , atleast in immunocompromised individuals .", "primary idiopathic frozen shoulder is a rare condition and secondary frozen shoulder cases are often due to underlying shoulder pathology ."]]}
{"article_id": "PMC2809321", "article_text": ["split - liver   transplantation ( slt ) is an   attractive alternative procedure to expand the donor pool in patients waiting for liver transplantation .", "the standard split - liver procedure for a child and an adult ( adult / pediatric split liver ttransplantation , a / p slt ) , by splitting segment ii - iii for pediatric recipient and segment i - iv - v - vi - vii - viii for an adult , is an accepted surgical option with good results both for the adult and for pediatric recipient .", "biliary complications continue to be the major cause of morbidity after slt with a reported high incidence ranging between 10% and 32% [ 1 , 2 ] . as a consequence of bile duct anatomic variations", ", slt requires a precise knowledge of the liver anatomy [ 3 , 4 ] .", "the challenge of this procedure is represented by a preoperative radiological assessment of the biliary anatomy often unavailable at the donor 's hospital .", "thus the risk of biliary duct injury during the splitting procedure is usually considered higher than during living donor procedure .   in this report", "we describe an uncommon late biliary complication that occurred after slt and was successfully treated by a multidisciplinary approach .  ", "a 63-year - old male with hepatitis c - related cirrhosis was referred for liver transplantation to our institution .", "we performed a conventional a / p slt with in situ technique providing the left lateral segment for a child ( segments ii - iii ) and leaving the right lobe graft ( segment i - iv - v - vi - vii - viii ) for an adult recipient .", "the celiac trunk was left on the left graft while the right hepatic artery remained on the right graft .", "the patient was transplanted using the piggy - back technique without a veno - venous bypass .", "the biliary tract was reconstructed performing a duct - to - duct anastomosis using a t - tube by our standard technique previously described .", "a cholangiography through the t - tube was performed on postoperative day 14 , and the t - tube was clamped before patient discharge .", "three months after a / pslt the t - tube was removed after a cholangiography with normal findings .", "one year after transplant the patient showed abnormal liver function tests , hyperbilirubinemia , leukocytosis , and elevated g - glutamyl transpeptidase ( ggt ) , and mild elevation in alanine transaminase ( alt ) .  ", "the patient underwent a doppler ultrasound that showed ( a patent hepatic artery )   an intrahepatic bile duct dilatation and an anastomotic biliary stricture .", "the anastomotic stricture was treated by stenting the main biliary duct during an endoscopic retrograde cholangiopancreatoghaphy ( ercp ) without any evidence of intrahepatic biliary dilatation .", "after this procedure the patient was submitted to a percutaneous transhepatic cholangiography ( ptc ) showing a complete obstruction of segments vi and vii biliary branches near the duct - to - duct biliary anastomosis ( figure 1 ) .  ", "the patient was discharged leaving the external biliary drain open allowing bile drainage and an easy access for repeated radiologic treatment and an internal stent in the common bile duct .", "three months later the patient underwent a surgical revision because of repeated episodes of cholangitis . during surgery", "an intraoperative cholangiography was performed through the biliary drain   confirming a bile duct dilatation at the level of segments vi and vii .  ", "it was impossible to cross the biliary stricture by a torque catheter and by hydrophilic guide wire ( figure 2 ) .", "we supposed that the segmentary bile duct branch of   posterior segments vi and vii draining in the left bile duct was unidentified and tied at the time of the in situ split - liver procedure during the parenchymal transection .", "a primary biliary reconstruction by biliary repair or biliodigestive anastomosis was considered at high risk because of the presence of postsurgical   adhesions   sand due to fibrotic tissue replacing   the parenchymal transected area . a liver resection of the dilated segments vi and vii was considered at high risk of leaving an inadequate liver mass .  ", "we decided to perform a permanent intraoperative obliteration of the dilated intrahepatic ducts by a percutaneous embolization using a nonresorbable agent . with a fluoroscopic guidance through the transhepatic access we positioned a 5-french polyethylene catheter inside the ducts , preliminary flushed by a nonionic dextrose solution .", "we then injected the tissue adhesive agent n - butyl cyanoacrylate ( nbca , glubran 2 , gem , viareggio , italy ) mixed with ionized oil ( lipiodol , guebert , aulnay - sous - bois , france )   for opacization in a ratio of 1:5 .", "this solution completely filled the biliary duct , and the occlusion was totally accomplished in a few seconds ( figure 3 ) . during the first 3 days after the chemical bile duct embolization , the patient had a low fever with a slightly abnormal liver function test .", "a computed tomography ( ct ) scan performed 6 months later showed no sign of hepatic abscesses , and the bile duct dilatation was completely occupied by the nbca - lipiodol mixture .", "one year after the procedure patient showed normal liver function tests without no episodes of cholangitis .", "chemical bile duct embolization treatment could represent a valuable   solution to treat uncommon biliary complications .", "these tissue adhesive glues are low - viscosity liquid monomers that undergo rapid polymerization and solidification when they come into contact with organic fluids such as bile .", "nbca is a permanent liquid embolic material that produces long - term occlusion in vessels of various size through an inflammatory tissue response resulting in vessel thrombosis or tissue   atrophy .", "little is known about the use of cyanoacrylate compounds , and unlike european countries   the use of glubran has not been approved by the food and drug administration yet .  ", "other authors have described the efficacy of biliary duct ablation by nbca in patients with persistent postsurgical bile leaks after lobectomy or cholecystectomy .", "vu et al . treated six patients with persistent postsurgical bile leaks as a complication after hepatic lobectomy or cholecistectomy using nbca glue for the obliteration of isolated segmental bile ducts in four cases .", "endoscopic treatment of biliary leakage by nbca has been described by seewald in nine patients in whom primary stent placement or nasobiliary drain was unsuccessful .", "described the use of a cyanoacrlylate in the treatment of a pancreatic fistula after distal pancreatectomy .", "the percutaneous interventional technique represents an effective valuable approach to reduce mortality and morbidity in the treatment of biliary complications after liver transplantation .", "the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection above all in high - risk patients with posttransplant bile duct injuries , decreasing the morbidity associated with chronic external biliary drainage .", "futher studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and costs ."], "abstract_text": ["<S> biliary complications continue to be a major cause of morbidity after split - liver transplantation ( slt ) . in this report </S>", "<S> we describe an uncommon late biliary complication . </S>", "<S> one year after slt the patient showed an intrahepatic bile dicy dilatation with severe cholangitis episodes . </S>", "<S> the segmentary bile duct of hepatic segment vi - vii draining in the left duct was unidentified and tied at the time of the in situ split - liver procedure . </S>", "<S> we perform a permanent obliteration of the dilated intrahepatic ducts by a percutaneous embolization using an n - butyl cyanoacrylate ( nabc ) . the management of biliary complications after slt requires a multidisciplinary approach . </S>", "<S> the use of   nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection and decreasing the morbidity associated with chronic external biliary drainage . </S>", "<S> further studies are needed to determine   whether this approach is effective and safe and whether it could reduce hospital stay and cost . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Case Report", "3. Discussion"], "sections": [["split - liver   transplantation ( slt ) is an   attractive alternative procedure to expand the donor pool in patients waiting for liver transplantation .", "the standard split - liver procedure for a child and an adult ( adult / pediatric split liver ttransplantation , a / p slt ) , by splitting segment ii - iii for pediatric recipient and segment i - iv - v - vi - vii - viii for an adult , is an accepted surgical option with good results both for the adult and for pediatric recipient .", "biliary complications continue to be the major cause of morbidity after slt with a reported high incidence ranging between 10% and 32% [ 1 , 2 ] . as a consequence of bile duct anatomic variations", ", slt requires a precise knowledge of the liver anatomy [ 3 , 4 ] .", "the challenge of this procedure is represented by a preoperative radiological assessment of the biliary anatomy often unavailable at the donor 's hospital .", "thus the risk of biliary duct injury during the splitting procedure is usually considered higher than during living donor procedure .   in this report", "we describe an uncommon late biliary complication that occurred after slt and was successfully treated by a multidisciplinary approach .  "], ["a 63-year - old male with hepatitis c - related cirrhosis was referred for liver transplantation to our institution .", "we performed a conventional a / p slt with in situ technique providing the left lateral segment for a child ( segments ii - iii ) and leaving the right lobe graft ( segment i - iv - v - vi - vii - viii ) for an adult recipient .", "the celiac trunk was left on the left graft while the right hepatic artery remained on the right graft .", "the patient was transplanted using the piggy - back technique without a veno - venous bypass .", "the biliary tract was reconstructed performing a duct - to - duct anastomosis using a t - tube by our standard technique previously described .", "a cholangiography through the t - tube was performed on postoperative day 14 , and the t - tube was clamped before patient discharge .", "three months after a / pslt the t - tube was removed after a cholangiography with normal findings .", "one year after transplant the patient showed abnormal liver function tests , hyperbilirubinemia , leukocytosis , and elevated g - glutamyl transpeptidase ( ggt ) , and mild elevation in alanine transaminase ( alt ) .  ", "the patient underwent a doppler ultrasound that showed ( a patent hepatic artery )   an intrahepatic bile duct dilatation and an anastomotic biliary stricture .", "the anastomotic stricture was treated by stenting the main biliary duct during an endoscopic retrograde cholangiopancreatoghaphy ( ercp ) without any evidence of intrahepatic biliary dilatation .", "after this procedure the patient was submitted to a percutaneous transhepatic cholangiography ( ptc ) showing a complete obstruction of segments vi and vii biliary branches near the duct - to - duct biliary anastomosis ( figure 1 ) .  ", "the patient was discharged leaving the external biliary drain open allowing bile drainage and an easy access for repeated radiologic treatment and an internal stent in the common bile duct .", "three months later the patient underwent a surgical revision because of repeated episodes of cholangitis . during surgery", "an intraoperative cholangiography was performed through the biliary drain   confirming a bile duct dilatation at the level of segments vi and vii .  ", "it was impossible to cross the biliary stricture by a torque catheter and by hydrophilic guide wire ( figure 2 ) .", "we supposed that the segmentary bile duct branch of   posterior segments vi and vii draining in the left bile duct was unidentified and tied at the time of the in situ split - liver procedure during the parenchymal transection .", "a primary biliary reconstruction by biliary repair or biliodigestive anastomosis was considered at high risk because of the presence of postsurgical   adhesions   sand due to fibrotic tissue replacing   the parenchymal transected area . a liver resection of the dilated segments vi and vii was considered at high risk of leaving an inadequate liver mass .  ", "we decided to perform a permanent intraoperative obliteration of the dilated intrahepatic ducts by a percutaneous embolization using a nonresorbable agent . with a fluoroscopic guidance through the transhepatic access we positioned a 5-french polyethylene catheter inside the ducts , preliminary flushed by a nonionic dextrose solution .", "we then injected the tissue adhesive agent n - butyl cyanoacrylate ( nbca , glubran 2 , gem , viareggio , italy ) mixed with ionized oil ( lipiodol , guebert , aulnay - sous - bois , france )   for opacization in a ratio of 1:5 .", "this solution completely filled the biliary duct , and the occlusion was totally accomplished in a few seconds ( figure 3 ) . during the first 3 days after the chemical bile duct embolization , the patient had a low fever with a slightly abnormal liver function test .", "a computed tomography ( ct ) scan performed 6 months later showed no sign of hepatic abscesses , and the bile duct dilatation was completely occupied by the nbca - lipiodol mixture .", "one year after the procedure patient showed normal liver function tests without no episodes of cholangitis ."], ["chemical bile duct embolization treatment could represent a valuable   solution to treat uncommon biliary complications .", "these tissue adhesive glues are low - viscosity liquid monomers that undergo rapid polymerization and solidification when they come into contact with organic fluids such as bile .", "nbca is a permanent liquid embolic material that produces long - term occlusion in vessels of various size through an inflammatory tissue response resulting in vessel thrombosis or tissue   atrophy .", "little is known about the use of cyanoacrylate compounds , and unlike european countries   the use of glubran has not been approved by the food and drug administration yet .  ", "other authors have described the efficacy of biliary duct ablation by nbca in patients with persistent postsurgical bile leaks after lobectomy or cholecystectomy .", "vu et al . treated six patients with persistent postsurgical bile leaks as a complication after hepatic lobectomy or cholecistectomy using nbca glue for the obliteration of isolated segmental bile ducts in four cases .", "endoscopic treatment of biliary leakage by nbca has been described by seewald in nine patients in whom primary stent placement or nasobiliary drain was unsuccessful .", "described the use of a cyanoacrlylate in the treatment of a pancreatic fistula after distal pancreatectomy .", "the percutaneous interventional technique represents an effective valuable approach to reduce mortality and morbidity in the treatment of biliary complications after liver transplantation .", "the use of nbca in obliteration of a dilated bile duct seems to be a safe procedure with good results providing a less invasive option than hepatic resection above all in high - risk patients with posttransplant bile duct injuries , decreasing the morbidity associated with chronic external biliary drainage .", "futher studies are needed to determine whether this approach is effective and safe and whether it could reduce hospital stay and costs ."]]}
{"article_id": "PMC3356024", "article_text": ["ribonucleotide reductase ( rr ) inhibitors have been studied as radiation sensitizers for over 30  years in both the lab and the clinic .", "the first of these , hydroxyurea , has been studied in both cervical and head and neck cancers , among others .", "although initially promising , many of the clinical trials produced negative results , or those that were difficult to interpret . there has recently been a significant advance in our understanding of this pathway from a number of well - done pre - clinical studies .", "in addition , the discovery of new rr inhibitors with increased potency and improved binding characteristics has produced a significant increase in interest in this area .", "this review will synthesize the data detailing the response of rr to ionizing radiation ( ir ) and will provide a perspective on the use of rr inhibitors as radiosensitizers in the treatment of human cancers . over the years , many groups have explored the use of rr inhibitors as chemotherapeutics in their own right , or as adjuncts to dna damaging molecules , particularly in hematologic malignancies . while this area looks promising , this subject will not be reviewed here .", "ribonucleotide reductase is the rate limiting enzyme in the synthesis and repair of dna and is the only enzyme responsible for the conversion of ribonucleoside diphosphates to deoxyribonucleotide diphosphates , the fundamental building blocks of dna synthesis and repair .", "r1 ( also called rrm1 or m1 ) is the larger , regulatory subunit that is constitutively expressed throughout the cell cycle .", "it binds allosteric modulators , ribonucleoside diphosphates , and the nucleoside analogs gemcitabine and fludarabine .", "there are currently two known smaller subunits that bind r1 to form the active enzyme ; r2 ( also called rrm2 or m2 ) and a more recently discovered p53 inducible homolog of the r2 subunit , known as p53r2 .", "both contain a tyrosine free radical stabilized by a non - heme iron complex that is critical in the reduction of ribonucleotides .", "r2 is known to be cell cycle regulated , with the highest levels during s phase , however the precise roles of the r2 and p53r2 subunit in the response to ir are an area of active debate , as outlined below ( figure 1 ) .", "given the pivotal role of rr in dna synthesis and repair , many studies have investigated the effect of dna damaging agents , including ir , on rr and its subunits .", "even so , there is currently still a great deal of controversy surrounding the exact mechanism of rr response to ir .", "the first is that the small subunit r2 is up - regulated and provides dntps for dna repair in addition to its usual role in dna synthesis during s phase .", "this is supported by a number of studies , including one by kuo et al . , who characterized the response of rr in the human cervical cancer cell line , caski .", "they demonstrated an increase in r2 protein levels after treating cells with ir , which was correlated with an increase in rr activity .", "however , there was no increase in the transcription of r2 , implying that the protein increase was due to post - transcriptional regulation ( kuo and kinsella , 1998 ) .", "this finding was reinforced by studies that demonstrated dna damage dependent stabilization of the r2 protein without any change in mrna after ir exposure in mouse balb/3t3 cells ( chabes and thelander , 2000 ) ; however it was in contrast to earlier work showing transcriptional activation of the r1 and r2 promoters after exposure to ir in the same cell line ( filatov et al . , 1996 ) . even though the precise mechanism of r2 response to ir damage is unclear , work has shown that human nasopharyngeal cancer cells overexpressing r2 demonstrate a significant increase in radioresistance and fewer dna double strand breaks ( dsb ) after exposure to ir ( kuo et al . , 2003 ) , confirming the concept that r2 is important in the cellular response to ir", "the second theory involves the p53 inducible subunit , p53r2 , first reported by tanaka et al .", "they showed that p53r2 was not cell cycle regulated ( unlike r2 ) , but was significantly induced after exposure of normal fibroblasts to ir ( tanaka et al . , 2000 ) and was found to translocate to the nucleus , the proposed site of most important dntp production ( r2 did not ) . in cells that lacked wild - type ( wt ) p53 ,", "there was no induction , indicating that functional p53 was necessary for p53r2 up - regulation .", "cells transfected with p53r2 were resistant to dna damage induced cell death . in agreement with other studies , they found no increase in r2 mrna , but did not measure protein levels .", "additional studies have subsequently shown that p53r2 forms an active complex with r1 ( guitett et al . , 2001 ) and", "rr activity increased in correlation with the increase in p53r2 , however the response of r2 was variable , with decrease in some lines and a moderate increase in the others ( yamaguchi et al . , 2001 ) .", "finally , other models are possible , including one described by xue and colleagues . in their study , both subunits were found to bind p53 in human oropharyngeal carcinoma cells , and in response to ir , were released to bind r1 in the nucleus ( xue et al . , 2003 ) , highlighting a third possibility in contrast to those previously presented .", "clearly , there is still work to be done in elucidating the response of rr to ir .", "many of the differences seen in the studies discussed can likely be attributed to the use of different techniques , materials , and especially cell lines .", "what should be clear is that rr is involved in the cellular response to ir and targeting it is both rational and likely desirable in enhancing the treatment of cancers with ir .", "it is likely that both r2 and p53r2 are involved to a different degree in different cancers , with cellular phenotype likely playing a key role in determining their relative significance .", "hydroxyurea ( hu ) is a hydroxylated analog of urea and was the first rr inhibitor to be extensively studied .", "it has directed activity at the tyrosine radical moiety of r2 and was first found to be active against cancer cells in 1963 ( stearns et al . , 1963 ) .", "subsequent experiments in vitro showed that in addition to its direct inhibition of dna synthesis , it was also a sensitizer of cell killing by x - rays , particularly if given before or after ir ( sinclair , 1968 ) .", "later experiments showed that this was also the case in in vivo animal tumor models using isotransplants of spontaneous c3h / he mouse mammary carcinomas ( piver et al . , 1972 ) .", "the total dose of ir to cure 50% of tumors was reduced when hu was combined with fractionated ir , although this effect was nt seen with single fraction ir treatments . given these encouraging pre - clinical results during the 19601970s ,", "hu was subsequently examined in a number of clinical trials in a variety of human cancers .", "the majority of these trials have occurred in cervical cancer , most commonly in locally advanced disease . in particular , there were a number of prospective randomized controlled trials in the 1970s and 1980s that examined the effect of hu plus radiotherapy vs. radiotherapy alone .", "and the gynecologic oncology group ( gog ) enrolled 190 women with figo stage iiib or iva cervical carcinoma .", "hu was administered orally at 80  mg / kg starting on the first day of irradiation and every 3  days thereafter for 12  weeks .", "patients received at least 50  gy minimum tumor dose to the whole pelvis followed by a single brachytherapy treatment of 20  gy to point a. in spite of the large number of patients enrolled , only 90 were eligible for assessment of response .", "this was due to ineligibility ( wrong stage , wrong cell type , etc . ) and those that were inevaluable ( refused treatment , periaortic node irradiation , improper field , etc . ) .", "the data were impressive , with a complete response ( cr ) of 68.1% in the hu group vs. 48.8% in the placebo ( p  =  <  0.05 ) , and a median progression free survival ( pfs ) of 13.6 vs. 7.6  months ( hreshchyshyn et al . , 1979 ) . however , myelosuppression was more common in the hu group , with seven grade iii or iv myelotoxicities .", "they recruited 148 women with figo iib or iiib cervical carcinoma and again compared hu to placebo in the setting of conventional radiotherapy , with hu given every 3  days for 12  weeks .", "of the stage iib patients , 74% receiving hu had no evidence of disease at the completion of therapy compared with 43.5% of the placebo ( p  =  <  0.01 ) .", "of the stage iiib patients , the cr rates were 52.5 vs. 33% ( p  =  0.22 ; piver et al . , 1977 ) .", "again , 78% of patients in the hu group developed leucopenia vs. 11% in the placebo group indicating a significant toxicity in addition to the improved clinical effect . at the time these studies were published", ", it was felt that hu added significant benefit to the treatment of locally advanced cervical carcinoma , and hu plus radiotherapy became the standard of care for the gog .", "however , over time , much of the data in these studies has been challenged , particularly in a systematic review by symonds et al .", "they found a number of methodological problems with the studies such as small sample size , large numbers of exclusions post randomization , subgroup analyses of already small groups and questionable censoring .", "they concluded that hu  appears to add to acute toxicity and probably increases late complications  and that  there is no convincing evidence of sufficient quality to suggest a therapeutic effect of this drug ", "although the gog initially used hu as its standard of care in the treatment of locally advanced cervical cancer , it was nt long before this combination was supplanted by a new adjunct to radiation therapy . one of the most important studies prompting this paradigm shift was the gog 120 trial , first reported in 1999 ( rose et al . , 1999 ) and recently updated ( rose et al . , 2007 ) .", "gog 120 was a randomized phase iii study comparing cisplatin alone vs. cisplatin , fluorouracil , and hu vs. hu alone , in conjunction with pelvic irradiation for patients with locally advanced cervical cancer and pathologically negative para - aortic nodes .", "they reported a significant improvement in pfs and overall survival ( os ) in both cisplatin containing arms ( p  <  0.001 ) , with relative risks for progression of disease or death of 0.57 and 0.51 for cisplatin alone and cisplatin , fluorouracil and hu , respectively , when compared with hu alone . in addition , toxicities were similar in all groups .", "further , a similar study by whitney et al . compared the efficacy of standard radiotherapy ( rt ) plus hu with standard rt plus fluorouracil ( 5-fu ) and cisplatin in a randomized controlled trial . in 368 women with figo stage iib , iii , or iva cancer of the uterine cervix , there were significant improvements in pfs and os with the 5-fu and cisplatin combination ( whitney et al . , 1999 ) .", "adverse effects included leucopenia : 4% of 5-fu / cisplatin patients and 24% of hu patients experienced grade iii or iv toxicity .", "given the findings of these studies , concurrent cisplatin and radiotherapy became the standard of care in locally advanced cervical cancer , spelling the end of hu use in the treatment of cervical cancer .", "in addition to its study in cervical carcinoma , hu has also been extensively investigated in other cancers , particularly head and neck .", "while early studies had significant flaws in methodology , a phase i study showed a 90% response rate in patients treated with hu , 5-fu , and palliative dose fractionated ir ( vokes et al .", ", 1989 ) , prompting a series of trials by the same group and others .", "this included work by mantz et al . , who examined the benefit of hu when added to a more extensive chemoradiation protocol including cisplatin , 5-fu , leucovorin , and interferon-2b induction chemotherapy followed by 5-fu and hu with fractionated radiotherapy in 32 laryngeal cancer patients .", "median follow up was 44.5  months , and , after completion of all therapy , the cr rate was 94% .", "median os was 44.5  months , and median pfs was 86 , 78 , and 78% at 1 , 3 , and 5  years , respectively , which compared favorably with other published data and for the first time saw patients with stage iv head and neck cancer failing distantly more often than locally ( mantz et al .", "the increased local control , unfortunately , was associated with increased toxicity , which has also been seen in other studies .", "one phase i study with prolonged infusion of hu with hyperfractionated , accelerated , external radiation in patients with advanced squamous cell cancer of the head and neck showed an increase in the severity of swallowing toxicity compared with previous trials , with severe edema , and reductions in motility and mobility of pharyngeal and laryngeal structures ( beitler et al . , 1998 ) .", "the same group later published a study examining the long term impact on swallowing that showed persistent , severe swallowing dysfunction ( smith et al . , 2000 ) .", "interestingly , these studies examined continuous hu infusions based on pre - clinical data that suggested that hu should always be given concurrently with ir to maximize effect . even though local control was excellent , with just 4 of 26 patients experiencing recurrence ,", "quality of life is of great importance in these patients , and the late follow up reported esophageal strictures for the first time after chemoradiotherapy , suggesting that this particular regimen may be too aggressive .", "the state of hu in head and neck cancer is well reviewed by argiris et al .", "( 2003 ) and while the authors are optimistic about the future of chemoradiation in head and neck cancer , it remains to be seen where hu and other rr inhibitors will fit in the future treatment of this disease site . even as hu has slowly progressed in the clinic", ", work has continued on examining the mechanism by which it sensitizes cells to the effects of ir . in particular , work by kuo et al .", "has shown that the sequence with which cells are treated with hu plays an important role .", "they demonstrated that in the caski cervical cancer cell line , clinically relevant concentrations of hu had a significant interaction with ir , with post - ir exposure  >  pre - ir ( kuo et al . , 1997 ) .", "this was associated with increased g2 delay , suggesting a decrease in the repair of damaged dna .", "in addition , in cells overexpressing the r2 subunit , hu is able to return ir sensitivity to baseline ( kuo et al . , 2003 ) , demonstrating that r2 inhibition is the likely mechanism for hu radiosensitization in these cells .", "these findings are potentially informative about the failure of hu to become established as a radiosensitizer in cervical cancer . in the majority of the early trials ,", "given that hu works best in vitro when dosed immediately after ir exposure , one could conclude that these trials were not optimized for best effect .", "in addition , hu has recently been shown to have a significant effect on the mechanism of dna dsb repair employed by cells after exposure to ir .", "burkhalter et al . showed that cells pre - incubated with hu were unable to use homologous recombination ( hr ) to repair dsb , and instead relied on non - homologous end joining ( nhej ) .", "in addition , cells that were nhej deficient had significantly more dsb after hu treatment ( burkhalter et al . , 2009 ) .", "given that nhej is thought to be the dominant dsb repair mechanism in cells treated with hu , rr inhibitors are likely to have enhanced activity in tumors that are nhej deficient .", "even with new studies on its mechanism of action , hu will likely remain consigned to history due to the many inadequacies it has as a drug molecule .", "while its oral absorption is almost complete and it is completely distributed in the water compartments of the body , hu has a short half - life ( between 1.6 and 4.45  h ; gwilt and tracewell , 1998 ) and its effectiveness is limited by relatively low affinity for rr and by the development of resistance .", "one area where it could potentially find use in the future is in cns neoplasms , as it does cross the blood ", "recent studies have examined its use in progressive meningioma in combination with 3d - conformal radiotherapy and adjuvant chemotherapy . in one trial ,", "pfs at 1 and 2  years was 84 and 77% , which is similar to other adjuvant studies ( hahn et al .", "in spite of the mixed clinical data for hu , there is sufficient proof of concept to suggest that a rr inhibitor can be efficacious as a radiosensitizer in human cancers .", "thus , there has been a concerted effort to discover more potent molecules with more favorable pharmacokinetics and pharmacodynamics for this purpose .", "one of the more promising of these is triapine , a thiosemicarbazone that destroys the tyrosyl radical in r2/p53r2 by forming a redox active complex with iron , producing reactive oxygen species . in studies comparing it with hu in vitro", ", triapine was shown to have significantly higher potency in both enzyme and cell assays .", "in addition , it was fully active against hu and gemcitabine resistant cells and was equally potent against r2 and p53r2 , whereas hu was approximately threefold less potent at binding p53r2 ( zhu et al . , 2009 ) .", "in addition , in in vivo models , triapine was active against hu resistant l1210 and kb cell lines and caused significant inhibition of solid tumor growth in mouse xenograft models ( finch et al .", "further studies have examined the radiosensitizing properties of triapine in a number of human cell lines .", "used a panel of three human tumor cell lines , including glioma , pancreatic , and prostate cancer cells , with triapine enhancing radiosensitivity when delivered 16  h before or immediately after ir by 1.5- to 2-fold .", "ir interaction was associated with a reduction in the repair of dna dsb as evidenced by a persistence of h2ax foci at 24  h ( barker et al . , 2006 ) .", "a similar effect was seen in mouse tumor xenografts , again , with greater effect if triapine was dosed just after ir .", "the most effective temporal relationship between triapine dosing and ir is similar to that seen with hu in earlier pre - clinical studies .", "interestingly , normal human fibroblasts were only sensitized when triapine was given before , not after ir , suggesting a potential for an improved therapeutic index for ir  triapine sequencing that may be incorporated into future clinical studies .", "are ic50 ( concentration of compounds producing 50% inhibition of recombinant rr activity ) values for both compounds in an in vitro rr activity assay with r2 or p53r2 bound to r1 ( zhu et al . , 2009 ) .", "also shown are elimination half - lives ( t1/2 ) for both compounds ( gwilt and tracewell , 1998 ; kunos et al . ,", "2010b ) . of note , many cancers have virally or mutationally silenced p53 that allows rr activity to continue unchecked . in these cancers , it is potentially of increased importance to inhibit the r2 and p53r2 subunits that have lost p53 regulatory control .", "this is the case in cervical cancer , where the vast majority have dysfunctional p53 due to hpv infection . in one study , three cervical cancer cell lines with mutated or dysfunctional p53", "were irradiated 6  h after triapine exposure . in all cases , the cell lines were sensitized to ir and sustained dna damage as measured by persistence of h2ax foci . in addition , by measuring dctp levels , the investigators were able to show reduced rr activity in cells with and without functional p53 , demonstrating that the inhibition of rr is p53 independent ( kunos et al . , 2009 ) .", "these findings were reinforced by further work of the same group that also showed a synergistic effect when cisplatin was added in addition to triapine and ir ( kunos et al . , 2010a ) .", "these promising pre - clinical data have prompted the initiation of a number of clinical trials .", "indeed , triapine has so far been studied in 27 clinical trials in the usa at various stages of recruitment ( www.clinicaltrials.gov ) , including those in both solid and hematologic malignancies .", "in particular there are three trials investigating the radiosensitizing potential of triapine , with data from one phase i study being published by kunos et al .", "the purpose of their study was to assess the safety / tolerability , pharmacokinetics , and clinical activity of triapine three times weekly in concert with once weekly cisplatin and pelvic radiation in 11 patients with gynecologic malignancies .", "triapine was dosed in 2  h infusions and the half - life was found to be 2  h. all 10 patients with advanced stage ib to ivb cervical cancer achieved complete clinical response , with a median 18  month follow up showing no disease progression in any of the patients .", "five of the 10 patients had pet / ct evident pelvic or para - aortic lymphadenopathy before treatment , with complete resolution on follow up imaging after treatment ( kunos et al .", "in addition to the clinical data , the authors also examined objective markers of disease response .", "late responders had a significantly higher rr activity on day 10 vs. day 1 and there was no temporal change in rr activity in the early responders , indicating that the expected spike in rr activity after ir was suppressed by triapine .", "the fact that the late responders experienced durable responses in spite of elevated rr activity at day 10 suggests that there may be other mechanisms of triapine activity that require further study .", "the promising results from this phase i trial prompted a phase ii trial which is now underway .", "over the last 10  years , there have been a number of major leaps forward in the field of radiation oncology that allow us to deliver higher doses of radiation in ever more specific ways to our patients .", "still , in the vast majority of cases , we are constrained by dose limiting toxicities that must be accounted for when designing treatment plans and balanced against the therapeutic benefit realized .", "therefore , the field of radio modulation and specifically radiosensitization will continue to grow in importance in the coming years as we search for ways to maximize the therapeutic index of our treatments .", "while there are many different radiosensitizers currently being studied , the rr inhibitors are among the oldest of targets , and are getting a new lease of life in recent times with the development of more modern drug molecules .", "as outlined in the review above , the story began with the study of hu in combination with ir in a number of pre - clinical trials in the 1960s , leading to a large amount of interest in the clinic , culminating with the gog adopting hu and ir as its standard of care in locally advanced cervical cancer . while its success in this arena was short", "lived , many parallel studies investigating the mechanism of action of hu were carried out , resulting in a far clearer understanding of the biological interactions between ir and rr inhibitors .", "even as hu was fading from view , investigators were working on successors including the intriguing molecule , triapine .", "as shown above , this drug works in a similar fashion to hu , but has significantly improved potency , pharmacokinetics , and pharmacodynamics .", "the phase i study in cervical cancers that was recently published includes some very encouraging data , and the field waits in anticipation for the publication of further trials .", "it remains to be seen what the best dosing regimen for triapine will be , however the weight of pre - clinical data , including that with hu , suggests that daily dosing shortly after radiation therapy will be most effective and provide the greatest therapeutic window .", "these authors would encourage studies incorporating this type of dosing schedule in the clinic , although this must obviously be weighed carefully against the potential for increased toxicity .", "in addition to triapine , there are other new rr inhibitors being tested at various stages , including trimidox and motexafin gadolinium .", "( 1994 ) , in a paper that reported 100-fold more potency at rr than hu in a cell based assay .", "subsequent in vitro studies have demonstrated that while it acts as a radiosensitizer in panc-1 human pancreatic cancer cells ( ahmed and hassan , 2000 ) , it is less potent than hu and further work is ongoing to fully assess its potential .", "motexafin gadolinium is a texaphyrin molecule that targets thioredoxin reductase in addition to rr . in in vitro experiments , it was shown to inhibit rr with an ic50 of 26  m ( hashemy et al . , 2006 ) , although given that it has a number of other cellular effects , it is unclear how much of its potency as a radiosensitizer is due to rr inhibition alone .", "it is likely that the coming years will continue to see the emergence of new rr inhibitors with unique properties .", "the recent advances in the understanding of rr biology and the development of new inhibitors places us at an important crossroads in the story of rr inhibitors .", "there are sufficient data to provide proof of concept for the target from a biological standpoint , however clinical trials to this point have not been wholly convincing .", "it will be interesting to follow the development of the field in the next 510  years , particularly with the clinical trials of triapine in cervical cancer , and hopefully at least one of the many rr inhibitors being studied will eventually bring additional therapeutic benefit to patients in the near future .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S> ribonucleotide reductase ( rr ) , the rate limiting enzyme in the synthesis and repair of dna , has been studied as a target for inhibition in the treatment of cancer for many years . </S>", "<S> while some researchers have focused on rr inhibitors as chemotherapeutic agents , particularly in hematologic malignancies , some of the most promising data has been generated in the field of radiosensitization . </S>", "<S> early pre - clinical studies demonstrated that the addition of the first of these drugs , hydroxyurea , to ionizing radiation ( ir ) produced a synergistic effect in vitro , leading to a large number of clinical studies in the 19701980s . </S>", "<S> these studies , mainly in cervical cancer , initially produced a great deal of interest , leading to the incorporation of hydroxyurea in the treatment protocols of many institutions . </S>", "<S> however , over time , the conclusions from these studies have been called into question and hydroxyurea has been replaced in the standard of care of cervical cancer . over the last 10  years </S>", "<S> , a number of well - done pre - clinical studies have greatly advanced our understanding of rr as a target . </S>", "<S> those advances include the elucidation of the role of p53r2 and our understanding of the temporal relationship between the delivery of ir and the response of rr . at the same time </S>", "<S> , new inhibitors with increased potency and improved binding characteristics have been discovered , and pre - clinical and early clinical data look promising . here </S>", "<S> we present a comprehensive review of the pre - clinical and clinical data in the field to date and provide some discussion of future areas of research . </S>"], "labels": null, "section_names": ["Introduction", "Hydroxyurea", "Triapine", "Conclusion", "Conflict of Interest Statement"], "sections": [["ribonucleotide reductase ( rr ) inhibitors have been studied as radiation sensitizers for over 30  years in both the lab and the clinic .", "the first of these , hydroxyurea , has been studied in both cervical and head and neck cancers , among others .", "although initially promising , many of the clinical trials produced negative results , or those that were difficult to interpret . there has recently been a significant advance in our understanding of this pathway from a number of well - done pre - clinical studies .", "in addition , the discovery of new rr inhibitors with increased potency and improved binding characteristics has produced a significant increase in interest in this area .", "this review will synthesize the data detailing the response of rr to ionizing radiation ( ir ) and will provide a perspective on the use of rr inhibitors as radiosensitizers in the treatment of human cancers . over the years , many groups have explored the use of rr inhibitors as chemotherapeutics in their own right , or as adjuncts to dna damaging molecules , particularly in hematologic malignancies . while this area looks promising , this subject will not be reviewed here .", "ribonucleotide reductase is the rate limiting enzyme in the synthesis and repair of dna and is the only enzyme responsible for the conversion of ribonucleoside diphosphates to deoxyribonucleotide diphosphates , the fundamental building blocks of dna synthesis and repair .", "r1 ( also called rrm1 or m1 ) is the larger , regulatory subunit that is constitutively expressed throughout the cell cycle .", "it binds allosteric modulators , ribonucleoside diphosphates , and the nucleoside analogs gemcitabine and fludarabine .", "there are currently two known smaller subunits that bind r1 to form the active enzyme ; r2 ( also called rrm2 or m2 ) and a more recently discovered p53 inducible homolog of the r2 subunit , known as p53r2 .", "both contain a tyrosine free radical stabilized by a non - heme iron complex that is critical in the reduction of ribonucleotides .", "r2 is known to be cell cycle regulated , with the highest levels during s phase , however the precise roles of the r2 and p53r2 subunit in the response to ir are an area of active debate , as outlined below ( figure 1 ) .", "given the pivotal role of rr in dna synthesis and repair , many studies have investigated the effect of dna damaging agents , including ir , on rr and its subunits .", "even so , there is currently still a great deal of controversy surrounding the exact mechanism of rr response to ir .", "the first is that the small subunit r2 is up - regulated and provides dntps for dna repair in addition to its usual role in dna synthesis during s phase .", "this is supported by a number of studies , including one by kuo et al . , who characterized the response of rr in the human cervical cancer cell line , caski .", "they demonstrated an increase in r2 protein levels after treating cells with ir , which was correlated with an increase in rr activity .", "however , there was no increase in the transcription of r2 , implying that the protein increase was due to post - transcriptional regulation ( kuo and kinsella , 1998 ) .", "this finding was reinforced by studies that demonstrated dna damage dependent stabilization of the r2 protein without any change in mrna after ir exposure in mouse balb/3t3 cells ( chabes and thelander , 2000 ) ; however it was in contrast to earlier work showing transcriptional activation of the r1 and r2 promoters after exposure to ir in the same cell line ( filatov et al . , 1996 ) . even though the precise mechanism of r2 response to ir damage is unclear , work has shown that human nasopharyngeal cancer cells overexpressing r2 demonstrate a significant increase in radioresistance and fewer dna double strand breaks ( dsb ) after exposure to ir ( kuo et al . , 2003 ) , confirming the concept that r2 is important in the cellular response to ir", "the second theory involves the p53 inducible subunit , p53r2 , first reported by tanaka et al .", "they showed that p53r2 was not cell cycle regulated ( unlike r2 ) , but was significantly induced after exposure of normal fibroblasts to ir ( tanaka et al . , 2000 ) and was found to translocate to the nucleus , the proposed site of most important dntp production ( r2 did not ) . in cells that lacked wild - type ( wt ) p53 ,", "there was no induction , indicating that functional p53 was necessary for p53r2 up - regulation .", "cells transfected with p53r2 were resistant to dna damage induced cell death . in agreement with other studies , they found no increase in r2 mrna , but did not measure protein levels .", "additional studies have subsequently shown that p53r2 forms an active complex with r1 ( guitett et al . , 2001 ) and", "rr activity increased in correlation with the increase in p53r2 , however the response of r2 was variable , with decrease in some lines and a moderate increase in the others ( yamaguchi et al . , 2001 ) .", "finally , other models are possible , including one described by xue and colleagues . in their study , both subunits were found to bind p53 in human oropharyngeal carcinoma cells , and in response to ir , were released to bind r1 in the nucleus ( xue et al . , 2003 ) , highlighting a third possibility in contrast to those previously presented .", "clearly , there is still work to be done in elucidating the response of rr to ir .", "many of the differences seen in the studies discussed can likely be attributed to the use of different techniques , materials , and especially cell lines .", "what should be clear is that rr is involved in the cellular response to ir and targeting it is both rational and likely desirable in enhancing the treatment of cancers with ir .", "it is likely that both r2 and p53r2 are involved to a different degree in different cancers , with cellular phenotype likely playing a key role in determining their relative significance ."], ["hydroxyurea ( hu ) is a hydroxylated analog of urea and was the first rr inhibitor to be extensively studied .", "it has directed activity at the tyrosine radical moiety of r2 and was first found to be active against cancer cells in 1963 ( stearns et al . , 1963 ) .", "subsequent experiments in vitro showed that in addition to its direct inhibition of dna synthesis , it was also a sensitizer of cell killing by x - rays , particularly if given before or after ir ( sinclair , 1968 ) .", "later experiments showed that this was also the case in in vivo animal tumor models using isotransplants of spontaneous c3h / he mouse mammary carcinomas ( piver et al . , 1972 ) .", "the total dose of ir to cure 50% of tumors was reduced when hu was combined with fractionated ir , although this effect was nt seen with single fraction ir treatments . given these encouraging pre - clinical results during the 19601970s ,", "hu was subsequently examined in a number of clinical trials in a variety of human cancers .", "the majority of these trials have occurred in cervical cancer , most commonly in locally advanced disease . in particular , there were a number of prospective randomized controlled trials in the 1970s and 1980s that examined the effect of hu plus radiotherapy vs. radiotherapy alone .", "and the gynecologic oncology group ( gog ) enrolled 190 women with figo stage iiib or iva cervical carcinoma .", "hu was administered orally at 80  mg / kg starting on the first day of irradiation and every 3  days thereafter for 12  weeks .", "patients received at least 50  gy minimum tumor dose to the whole pelvis followed by a single brachytherapy treatment of 20  gy to point a. in spite of the large number of patients enrolled , only 90 were eligible for assessment of response .", "this was due to ineligibility ( wrong stage , wrong cell type , etc . ) and those that were inevaluable ( refused treatment , periaortic node irradiation , improper field , etc . ) .", "the data were impressive , with a complete response ( cr ) of 68.1% in the hu group vs. 48.8% in the placebo ( p  =  <  0.05 ) , and a median progression free survival ( pfs ) of 13.6 vs. 7.6  months ( hreshchyshyn et al . , 1979 ) . however , myelosuppression was more common in the hu group , with seven grade iii or iv myelotoxicities .", "they recruited 148 women with figo iib or iiib cervical carcinoma and again compared hu to placebo in the setting of conventional radiotherapy , with hu given every 3  days for 12  weeks .", "of the stage iib patients , 74% receiving hu had no evidence of disease at the completion of therapy compared with 43.5% of the placebo ( p  =  <  0.01 ) .", "of the stage iiib patients , the cr rates were 52.5 vs. 33% ( p  =  0.22 ; piver et al . , 1977 ) .", "again , 78% of patients in the hu group developed leucopenia vs. 11% in the placebo group indicating a significant toxicity in addition to the improved clinical effect . at the time these studies were published", ", it was felt that hu added significant benefit to the treatment of locally advanced cervical carcinoma , and hu plus radiotherapy became the standard of care for the gog .", "however , over time , much of the data in these studies has been challenged , particularly in a systematic review by symonds et al .", "they found a number of methodological problems with the studies such as small sample size , large numbers of exclusions post randomization , subgroup analyses of already small groups and questionable censoring .", "they concluded that hu  appears to add to acute toxicity and probably increases late complications  and that  there is no convincing evidence of sufficient quality to suggest a therapeutic effect of this drug ", "although the gog initially used hu as its standard of care in the treatment of locally advanced cervical cancer , it was nt long before this combination was supplanted by a new adjunct to radiation therapy . one of the most important studies prompting this paradigm shift was the gog 120 trial , first reported in 1999 ( rose et al . , 1999 ) and recently updated ( rose et al . , 2007 ) .", "gog 120 was a randomized phase iii study comparing cisplatin alone vs. cisplatin , fluorouracil , and hu vs. hu alone , in conjunction with pelvic irradiation for patients with locally advanced cervical cancer and pathologically negative para - aortic nodes .", "they reported a significant improvement in pfs and overall survival ( os ) in both cisplatin containing arms ( p  <  0.001 ) , with relative risks for progression of disease or death of 0.57 and 0.51 for cisplatin alone and cisplatin , fluorouracil and hu , respectively , when compared with hu alone . in addition , toxicities were similar in all groups .", "further , a similar study by whitney et al . compared the efficacy of standard radiotherapy ( rt ) plus hu with standard rt plus fluorouracil ( 5-fu ) and cisplatin in a randomized controlled trial . in 368 women with figo stage iib , iii , or iva cancer of the uterine cervix , there were significant improvements in pfs and os with the 5-fu and cisplatin combination ( whitney et al . , 1999 ) .", "adverse effects included leucopenia : 4% of 5-fu / cisplatin patients and 24% of hu patients experienced grade iii or iv toxicity .", "given the findings of these studies , concurrent cisplatin and radiotherapy became the standard of care in locally advanced cervical cancer , spelling the end of hu use in the treatment of cervical cancer .", "in addition to its study in cervical carcinoma , hu has also been extensively investigated in other cancers , particularly head and neck .", "while early studies had significant flaws in methodology , a phase i study showed a 90% response rate in patients treated with hu , 5-fu , and palliative dose fractionated ir ( vokes et al .", ", 1989 ) , prompting a series of trials by the same group and others .", "this included work by mantz et al . , who examined the benefit of hu when added to a more extensive chemoradiation protocol including cisplatin , 5-fu , leucovorin , and interferon-2b induction chemotherapy followed by 5-fu and hu with fractionated radiotherapy in 32 laryngeal cancer patients .", "median follow up was 44.5  months , and , after completion of all therapy , the cr rate was 94% .", "median os was 44.5  months , and median pfs was 86 , 78 , and 78% at 1 , 3 , and 5  years , respectively , which compared favorably with other published data and for the first time saw patients with stage iv head and neck cancer failing distantly more often than locally ( mantz et al .", "the increased local control , unfortunately , was associated with increased toxicity , which has also been seen in other studies .", "one phase i study with prolonged infusion of hu with hyperfractionated , accelerated , external radiation in patients with advanced squamous cell cancer of the head and neck showed an increase in the severity of swallowing toxicity compared with previous trials , with severe edema , and reductions in motility and mobility of pharyngeal and laryngeal structures ( beitler et al . , 1998 ) .", "the same group later published a study examining the long term impact on swallowing that showed persistent , severe swallowing dysfunction ( smith et al . , 2000 ) .", "interestingly , these studies examined continuous hu infusions based on pre - clinical data that suggested that hu should always be given concurrently with ir to maximize effect . even though local control was excellent , with just 4 of 26 patients experiencing recurrence ,", "quality of life is of great importance in these patients , and the late follow up reported esophageal strictures for the first time after chemoradiotherapy , suggesting that this particular regimen may be too aggressive .", "the state of hu in head and neck cancer is well reviewed by argiris et al .", "( 2003 ) and while the authors are optimistic about the future of chemoradiation in head and neck cancer , it remains to be seen where hu and other rr inhibitors will fit in the future treatment of this disease site . even as hu has slowly progressed in the clinic", ", work has continued on examining the mechanism by which it sensitizes cells to the effects of ir . in particular , work by kuo et al .", "has shown that the sequence with which cells are treated with hu plays an important role .", "they demonstrated that in the caski cervical cancer cell line , clinically relevant concentrations of hu had a significant interaction with ir , with post - ir exposure  >  pre - ir ( kuo et al . , 1997 ) .", "this was associated with increased g2 delay , suggesting a decrease in the repair of damaged dna .", "in addition , in cells overexpressing the r2 subunit , hu is able to return ir sensitivity to baseline ( kuo et al . , 2003 ) , demonstrating that r2 inhibition is the likely mechanism for hu radiosensitization in these cells .", "these findings are potentially informative about the failure of hu to become established as a radiosensitizer in cervical cancer . in the majority of the early trials ,", "given that hu works best in vitro when dosed immediately after ir exposure , one could conclude that these trials were not optimized for best effect .", "in addition , hu has recently been shown to have a significant effect on the mechanism of dna dsb repair employed by cells after exposure to ir .", "burkhalter et al . showed that cells pre - incubated with hu were unable to use homologous recombination ( hr ) to repair dsb , and instead relied on non - homologous end joining ( nhej ) .", "in addition , cells that were nhej deficient had significantly more dsb after hu treatment ( burkhalter et al . , 2009 ) .", "given that nhej is thought to be the dominant dsb repair mechanism in cells treated with hu , rr inhibitors are likely to have enhanced activity in tumors that are nhej deficient .", "even with new studies on its mechanism of action , hu will likely remain consigned to history due to the many inadequacies it has as a drug molecule .", "while its oral absorption is almost complete and it is completely distributed in the water compartments of the body , hu has a short half - life ( between 1.6 and 4.45  h ; gwilt and tracewell , 1998 ) and its effectiveness is limited by relatively low affinity for rr and by the development of resistance .", "one area where it could potentially find use in the future is in cns neoplasms , as it does cross the blood ", "recent studies have examined its use in progressive meningioma in combination with 3d - conformal radiotherapy and adjuvant chemotherapy . in one trial ,", "pfs at 1 and 2  years was 84 and 77% , which is similar to other adjuvant studies ( hahn et al ."], ["in spite of the mixed clinical data for hu , there is sufficient proof of concept to suggest that a rr inhibitor can be efficacious as a radiosensitizer in human cancers .", "thus , there has been a concerted effort to discover more potent molecules with more favorable pharmacokinetics and pharmacodynamics for this purpose .", "one of the more promising of these is triapine , a thiosemicarbazone that destroys the tyrosyl radical in r2/p53r2 by forming a redox active complex with iron , producing reactive oxygen species . in studies comparing it with hu in vitro", ", triapine was shown to have significantly higher potency in both enzyme and cell assays .", "in addition , it was fully active against hu and gemcitabine resistant cells and was equally potent against r2 and p53r2 , whereas hu was approximately threefold less potent at binding p53r2 ( zhu et al . , 2009 ) .", "in addition , in in vivo models , triapine was active against hu resistant l1210 and kb cell lines and caused significant inhibition of solid tumor growth in mouse xenograft models ( finch et al .", "further studies have examined the radiosensitizing properties of triapine in a number of human cell lines .", "used a panel of three human tumor cell lines , including glioma , pancreatic , and prostate cancer cells , with triapine enhancing radiosensitivity when delivered 16  h before or immediately after ir by 1.5- to 2-fold .", "ir interaction was associated with a reduction in the repair of dna dsb as evidenced by a persistence of h2ax foci at 24  h ( barker et al . , 2006 ) .", "a similar effect was seen in mouse tumor xenografts , again , with greater effect if triapine was dosed just after ir .", "the most effective temporal relationship between triapine dosing and ir is similar to that seen with hu in earlier pre - clinical studies .", "interestingly , normal human fibroblasts were only sensitized when triapine was given before , not after ir , suggesting a potential for an improved therapeutic index for ir  triapine sequencing that may be incorporated into future clinical studies .", "are ic50 ( concentration of compounds producing 50% inhibition of recombinant rr activity ) values for both compounds in an in vitro rr activity assay with r2 or p53r2 bound to r1 ( zhu et al . , 2009 ) .", "also shown are elimination half - lives ( t1/2 ) for both compounds ( gwilt and tracewell , 1998 ; kunos et al . ,", "2010b ) . of note , many cancers have virally or mutationally silenced p53 that allows rr activity to continue unchecked . in these cancers , it is potentially of increased importance to inhibit the r2 and p53r2 subunits that have lost p53 regulatory control .", "this is the case in cervical cancer , where the vast majority have dysfunctional p53 due to hpv infection . in one study , three cervical cancer cell lines with mutated or dysfunctional p53", "were irradiated 6  h after triapine exposure . in all cases , the cell lines were sensitized to ir and sustained dna damage as measured by persistence of h2ax foci . in addition , by measuring dctp levels , the investigators were able to show reduced rr activity in cells with and without functional p53 , demonstrating that the inhibition of rr is p53 independent ( kunos et al . , 2009 ) .", "these findings were reinforced by further work of the same group that also showed a synergistic effect when cisplatin was added in addition to triapine and ir ( kunos et al . , 2010a ) .", "these promising pre - clinical data have prompted the initiation of a number of clinical trials .", "indeed , triapine has so far been studied in 27 clinical trials in the usa at various stages of recruitment ( www.clinicaltrials.gov ) , including those in both solid and hematologic malignancies .", "in particular there are three trials investigating the radiosensitizing potential of triapine , with data from one phase i study being published by kunos et al .", "the purpose of their study was to assess the safety / tolerability , pharmacokinetics , and clinical activity of triapine three times weekly in concert with once weekly cisplatin and pelvic radiation in 11 patients with gynecologic malignancies .", "triapine was dosed in 2  h infusions and the half - life was found to be 2  h. all 10 patients with advanced stage ib to ivb cervical cancer achieved complete clinical response , with a median 18  month follow up showing no disease progression in any of the patients .", "five of the 10 patients had pet / ct evident pelvic or para - aortic lymphadenopathy before treatment , with complete resolution on follow up imaging after treatment ( kunos et al .", "in addition to the clinical data , the authors also examined objective markers of disease response .", "late responders had a significantly higher rr activity on day 10 vs. day 1 and there was no temporal change in rr activity in the early responders , indicating that the expected spike in rr activity after ir was suppressed by triapine .", "the fact that the late responders experienced durable responses in spite of elevated rr activity at day 10 suggests that there may be other mechanisms of triapine activity that require further study .", "the promising results from this phase i trial prompted a phase ii trial which is now underway ."], ["over the last 10  years , there have been a number of major leaps forward in the field of radiation oncology that allow us to deliver higher doses of radiation in ever more specific ways to our patients .", "still , in the vast majority of cases , we are constrained by dose limiting toxicities that must be accounted for when designing treatment plans and balanced against the therapeutic benefit realized .", "therefore , the field of radio modulation and specifically radiosensitization will continue to grow in importance in the coming years as we search for ways to maximize the therapeutic index of our treatments .", "while there are many different radiosensitizers currently being studied , the rr inhibitors are among the oldest of targets , and are getting a new lease of life in recent times with the development of more modern drug molecules .", "as outlined in the review above , the story began with the study of hu in combination with ir in a number of pre - clinical trials in the 1960s , leading to a large amount of interest in the clinic , culminating with the gog adopting hu and ir as its standard of care in locally advanced cervical cancer . while its success in this arena was short", "lived , many parallel studies investigating the mechanism of action of hu were carried out , resulting in a far clearer understanding of the biological interactions between ir and rr inhibitors .", "even as hu was fading from view , investigators were working on successors including the intriguing molecule , triapine .", "as shown above , this drug works in a similar fashion to hu , but has significantly improved potency , pharmacokinetics , and pharmacodynamics .", "the phase i study in cervical cancers that was recently published includes some very encouraging data , and the field waits in anticipation for the publication of further trials .", "it remains to be seen what the best dosing regimen for triapine will be , however the weight of pre - clinical data , including that with hu , suggests that daily dosing shortly after radiation therapy will be most effective and provide the greatest therapeutic window .", "these authors would encourage studies incorporating this type of dosing schedule in the clinic , although this must obviously be weighed carefully against the potential for increased toxicity .", "in addition to triapine , there are other new rr inhibitors being tested at various stages , including trimidox and motexafin gadolinium .", "( 1994 ) , in a paper that reported 100-fold more potency at rr than hu in a cell based assay .", "subsequent in vitro studies have demonstrated that while it acts as a radiosensitizer in panc-1 human pancreatic cancer cells ( ahmed and hassan , 2000 ) , it is less potent than hu and further work is ongoing to fully assess its potential .", "motexafin gadolinium is a texaphyrin molecule that targets thioredoxin reductase in addition to rr . in in vitro experiments , it was shown to inhibit rr with an ic50 of 26  m ( hashemy et al . , 2006 ) , although given that it has a number of other cellular effects , it is unclear how much of its potency as a radiosensitizer is due to rr inhibition alone .", "it is likely that the coming years will continue to see the emergence of new rr inhibitors with unique properties .", "the recent advances in the understanding of rr biology and the development of new inhibitors places us at an important crossroads in the story of rr inhibitors .", "there are sufficient data to provide proof of concept for the target from a biological standpoint , however clinical trials to this point have not been wholly convincing .", "it will be interesting to follow the development of the field in the next 510  years , particularly with the clinical trials of triapine in cervical cancer , and hopefully at least one of the many rr inhibitors being studied will eventually bring additional therapeutic benefit to patients in the near future ."], ["the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC4941920", "article_text": ["the study of long bones and the cells of the bone marrow is central to a myriad of research disciplines , including , but not limited to , bone biology , cancer biology , immunology , hematology , and biomechanics .", "the bone is a highly dynamic organ that together with the cartilage forms the skeleton to provide mechanical support against loading and protection of the internal organs .", "in addition , the mineral components of bone are a storage sink for the critical signaling molecules calcium and phosphorus , as well as other factors .", "finally , bones house the bone marrow and , together with metabolically active bone forming osteoblasts and bone resorbing osteoclasts , provide the stem cell niche necessary for the maintenance of hematopoietic and lymphoid cell populations .", "bone and bone marrow are affected in many disorders , often leading to bone marrow dysfunction , severe bone pain , and pathologic fracture .", "bone is a common site of metastasis in many solid tumors , most notably breast cancer and prostate cancer , where tumor cells directly engage the bone marrow niche to initiate the vicious cycle of bone metastasis and displace hematopoietic stem cells .", "hematopoietic malignancies including myeloma and leukemia are characterized by bone marrow dysfunction as well as deregulation of healthy bone remodeling .", "other non - malignant skeletal disorders are also active areas of research , such as osteoarthritis , osteoporosis , scoliosis , and rickets . even in an otherwise healthy individual , biomechanical failure in a bone", "all of these disorders represent active areas of research with the goal of identifying new preventative measures and treatment regimens to reduce morbidity and mortality . to research the plethora of roles of the bone and the bone marrow , both under physiologic and pathologic conditions", ", it is critical for researchers to have a simple and efficient standardized method for dissection of the mouse long bones for rapid processing of large in vivo experiments .", "the dissection protocol outlined here is suitable for all long bone analyses including ex vivo imaging , histology , histomorphometry , and strength testing , among others .", "similarly , a standardized bone marrow isolation method with high bone marrow cell recovery and low inter - user variability is important for experimental analysis such as fluorescence - activated cell sorting ( facs ) or quantitative pcr ( qpcr ) as well as downstream applications such as primary cell culture of bone marrow cells .", "all animal work was approved by the institutional animal care and use committee in accordance with the recommendations outlined in the guide for the care and use of laboratory animals of the national institutes of health .", "position the mouse in a supine position and affix by pinning all four legs through the mouse paw pads below the ankle joint . spray the mouse with 70% ethanol , thoroughly dousing the legs . make a small incision to the right of midline in the lower abdomen , just above the hip .", "pull back the skin and cut the quadriceps muscle anchored to proximal end of the femur to expose the anterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the blade of the scissors against the posterior side of the femur , cut the hamstrings away from the knee joint . pull back the skin and the hamstring muscles anchored to proximal end of the femur to expose the posterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the forceps , hold the distal end of the femur , just above the knee joint . guide the blades of the scissors on either side of the femoral shaft towards the hip joint , being careful not to cut into the femur itself . after reaching the femoral head , indicated by the scissors opening slightly ,", "twist the scissors with the top blade of the scissors moving directly over the femoral head to dislocate the femur , being careful not to snap the bone below the femoral head .", "grasp the top of the femoral shaft with the forceps , cut the soft tissue away from the femoral head to release it from the acetabulum .", "pull the entire leg bone , including femur , knee , and tibia , up and away from the body , carefully cutting away the connective tissue and muscle connecting the leg to the skin .", "overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia . grasping the distal end of the tibia , taking care not to sever the tendons , pull the tibia up and away from the body and the pin board .", "cut any remaining connective tissue attaching the long bone to the mouse at the knee . remove any additional muscle or connective tissue attached to the femur and the tibia .", "for any applications that require the bone to remain intact ( histology , histomorphometry , biomechanical testing , etc . ) , proceed with standard in - house protocols ( as in ) . to isolate bone marrow , proceed to section 2 . using the forceps ,", "grasp the femur with the patella facing away and the proximal end ( femoral head ) down .", "overextend the knee joint and use the scissors in a twisting motion to dislocate the tibia and femur .", "grasp the femur with the anterior side facing away and the proximal end ( femoral head end ) down .", "rotate the scissors back and forth to remove the condyles , the patella , and the epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the femur using forceps , scissors , and kimwipes . using the forceps ,", "grasp the tibia with the anterior side facing away and the distal end ( ankle end ) down .", "if the tibial epiphysis is intact , guide the scissors up the tibia shaft to the condyles . gently rotate the scissors back and forth to remove the condyles and epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the tibia using forceps , scissors , and kimwipes .", "push an 18 g needle through the bottom of a 0.5 ml  microcentrifuge tube . place the long bones ( maximum of 2 femurs and 2 tibiae ) into the tube , knee - end", "verify that the bone marrow has been spun out of the bones by visual inspection .", "the bones should appear white and there should be a large visual pellet in the larger tube .", "suspend the bone marrow in appropriate solution ( e.g. , pbs , culture media , facs buffer ) and proceed with experimental protocol ( dna , rna , or protein isolation , facs analysis , or primary cell culture ) .", "euthanize the mouse in accordance with institutional guidelines . position the mouse in a supine position and affix by pinning all four legs through the mouse paw pads below the ankle joint . spray the mouse with 70% ethanol , thoroughly dousing the legs . make a small incision to the right of midline in the lower abdomen , just above the hip .", "pull back the skin and cut the quadriceps muscle anchored to proximal end of the femur to expose the anterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the blade of the scissors against the posterior side of the femur , cut the hamstrings away from the knee joint .", "pull back the skin and the hamstring muscles anchored to proximal end of the femur to expose the posterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the forceps , hold the distal end of the femur , just above the knee joint .", "guide the blades of the scissors on either side of the femoral shaft towards the hip joint , being careful not to cut into the femur itself . after reaching the femoral head , indicated by the scissors opening slightly ,", "twist the scissors with the top blade of the scissors moving directly over the femoral head to dislocate the femur , being careful not to snap the bone below the femoral head . grasp the top of the femoral shaft with the forceps , cut the soft tissue away from the femoral head to release it from the acetabulum . pull the entire leg bone , including femur , knee , and tibia , up and away from the body , carefully cutting away the connective tissue and muscle connecting the leg to the skin .", "overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia . grasping the distal end of the tibia , taking care not to sever the tendons , pull the tibia up and away from the body and the pin board .", "cut any remaining connective tissue attaching the long bone to the mouse at the knee . remove any additional muscle or connective tissue attached to the femur and the tibia .", "for any applications that require the bone to remain intact ( histology , histomorphometry , biomechanical testing , etc . ) , proceed with standard in - house protocols ( as in ) . to isolate bone marrow , proceed to section 2 .", "using the forceps , grasp the femur with the patella facing away and the proximal end ( femoral head ) down .", "overextend the knee joint and use the scissors in a twisting motion to dislocate the tibia and femur .", "grasp the femur with the anterior side facing away and the proximal end ( femoral head end ) down .", "rotate the scissors back and forth to remove the condyles , the patella , and the epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the femur using forceps , scissors , and kimwipes . using the forceps ,", "grasp the tibia with the anterior side facing away and the distal end ( ankle end ) down .", "if the tibial epiphysis is intact , guide the scissors up the tibia shaft to the condyles . gently", "rotate the scissors back and forth to remove the condyles and epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the tibia using forceps , scissors , and kimwipes .", "push an 18 g needle through the bottom of a 0.5 ml  microcentrifuge tube . place the long bones ( maximum of 2 femurs and 2 tibiae ) into the tube , knee - end", "verify that the bone marrow has been spun out of the bones by visual inspection .", "the bones should appear white and there should be a large visual pellet in the larger tube .", "suspend the bone marrow in appropriate solution ( e.g. , pbs , culture media , facs buffer ) and proceed with experimental protocol ( dna , rna , or protein isolation , facs analysis , or primary cell culture ) .", "the protocol described here is optimized for rapid dissection of the mouse femur and tibia with a minimum of damage to the bone tissue .", "this technique is suitable for a number of downstream analyses , including biomechanics studies , histomorphometry ( figure 1a - b ) , and histology ( figure 1c ) .", "the representative histomophometric micoct 3d reconstruction ( figure 1a - b ) demonstrates that both the cancellous bone and cortical shell are maintained that allows for accurate quantitation of the standardized structural parameters for bone histomorphometry , including trabecular number , thickness , and spacing ;  bone volume ;  and cortical thickness , among other measures .", "the representative histologic section shows an h&e stained formalin - fixed and decalcified tibia ( figure 1c ) .", "the image demonstrates the integrity of both the calcified bone and cellular bone marrow for histologic analysis .", "the bone marrow isolation procedure preserves the sterility of the bone marrow space , has low handling to reduce contamination , and does not require cutting of the long bone , thus reducing loss of bone marrow yield .", "this bone marrow is suitable for many downstream applications , including flow cytometry and pcr analyses . in addition", ", this procedure can be used to isolate bone marrow for primary cell culture of bone marrow cells , including osteoclasts and osteoblasts ( figure 2a - b ) .", "three - dimensional microct reconstruction of a mouse tibia showing ( a ) the outer cortical shell and ( b ) trabecular bone ( scale bar = 0.5 mm ) .", "( c ) histological h&e stain of a decalcified and sectioned tibia ( 4x ) .", "( a ) trap staining for multinucleated osteoclasts after 7 days in osteoclastogenic media ( 4x ) .", "( b ) alkaline phosphatase ( purple color ) for osteoblasts and alizarin red ( red color ) stain for mineralization after 21 days in osteogenic media .", "we present a simple and efficient method for removal of mouse hind long bones and subsequent bone marrow isolation .", "this method maintains the high structural and cellular integrity of the bones and bone marrow and has low handling time , minimizing the likelihood of user - induced fracture or bone scoring that may influence downstream analyses .", "in addition , the centrifugation method for isolating bone marrow does not require cutting the bone to expose the bone marrow space or fluid to flush the bone marrow , reducing potential points of contamination .", "moreover , the centrifuge technique is relatively high - throughput with lower hands - on time than other methods , thus reducing processing time .", "high variation is inherent to in vivo mouse studies due to high mouse - to - mouse phenotypic variation . in order to maximize the research impact of expensive and labor - intensive mouse studies , it is critical to minimize technical experimental error .", "time from animal sacrifice to downstream analysis or tissue fixation introduces experimental variation that may overcome subtle changes and reduce large differences between groups .", "the long bone dissection and bone marrow isolation techniques described here are optimized for rapid processing of animals and samples to reduce technical variation .", "this protocol can be widely applied to many research fields , including investigation of the bone tissue itself or interrogation of the cells of the bone marrow .", "in addition , this straightforward approach to long bone dissection will enable researchers in related fields to directly interrogate bone contributions in order to expand our knowledge of bone marrow dysfunction in otherwise understudied pathologies ."], "abstract_text": ["<S> investigation of the bone and the bone marrow is critical in many research fields including basic bone biology , immunology , hematology , cancer metastasis , biomechanics , and stem cell biology . despite the importance of the bone in healthy and pathologic states , however , it is a largely under - researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation . </S>", "<S> mice are a common model organism to study effects on bone and bone marrow , necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts . </S>", "<S> we describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications , including but not limited to histomorphologic analysis and strength testing . </S>", "<S> in addition , we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time , ideal for cell sorting , primary cell culture , or dna , rna , and protein extraction . </S>", "<S> the protocol is streamlined for rapid processing of samples to limit experimental error , and is standardized to minimize user - to - user variability . </S>"], "labels": null, "section_names": ["Introduction", "Protocol", "1. Hind Limb Long Bone Dissection", "2. Long Bone Preparation for Bone Marrow Isolation", "3. Bone Marrow Isolation", "Representative Results", "Discussion", "Disclosures"], "sections": [["the study of long bones and the cells of the bone marrow is central to a myriad of research disciplines , including , but not limited to , bone biology , cancer biology , immunology , hematology , and biomechanics .", "the bone is a highly dynamic organ that together with the cartilage forms the skeleton to provide mechanical support against loading and protection of the internal organs .", "in addition , the mineral components of bone are a storage sink for the critical signaling molecules calcium and phosphorus , as well as other factors .", "finally , bones house the bone marrow and , together with metabolically active bone forming osteoblasts and bone resorbing osteoclasts , provide the stem cell niche necessary for the maintenance of hematopoietic and lymphoid cell populations .", "bone and bone marrow are affected in many disorders , often leading to bone marrow dysfunction , severe bone pain , and pathologic fracture .", "bone is a common site of metastasis in many solid tumors , most notably breast cancer and prostate cancer , where tumor cells directly engage the bone marrow niche to initiate the vicious cycle of bone metastasis and displace hematopoietic stem cells .", "hematopoietic malignancies including myeloma and leukemia are characterized by bone marrow dysfunction as well as deregulation of healthy bone remodeling .", "other non - malignant skeletal disorders are also active areas of research , such as osteoarthritis , osteoporosis , scoliosis , and rickets . even in an otherwise healthy individual , biomechanical failure in a bone", "all of these disorders represent active areas of research with the goal of identifying new preventative measures and treatment regimens to reduce morbidity and mortality . to research the plethora of roles of the bone and the bone marrow , both under physiologic and pathologic conditions", ", it is critical for researchers to have a simple and efficient standardized method for dissection of the mouse long bones for rapid processing of large in vivo experiments .", "the dissection protocol outlined here is suitable for all long bone analyses including ex vivo imaging , histology , histomorphometry , and strength testing , among others .", "similarly , a standardized bone marrow isolation method with high bone marrow cell recovery and low inter - user variability is important for experimental analysis such as fluorescence - activated cell sorting ( facs ) or quantitative pcr ( qpcr ) as well as downstream applications such as primary cell culture of bone marrow cells ."], ["all animal work was approved by the institutional animal care and use committee in accordance with the recommendations outlined in the guide for the care and use of laboratory animals of the national institutes of health .", "position the mouse in a supine position and affix by pinning all four legs through the mouse paw pads below the ankle joint . spray the mouse with 70% ethanol , thoroughly dousing the legs . make a small incision to the right of midline in the lower abdomen , just above the hip .", "pull back the skin and cut the quadriceps muscle anchored to proximal end of the femur to expose the anterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the blade of the scissors against the posterior side of the femur , cut the hamstrings away from the knee joint . pull back the skin and the hamstring muscles anchored to proximal end of the femur to expose the posterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the forceps , hold the distal end of the femur , just above the knee joint . guide the blades of the scissors on either side of the femoral shaft towards the hip joint , being careful not to cut into the femur itself . after reaching the femoral head , indicated by the scissors opening slightly ,", "twist the scissors with the top blade of the scissors moving directly over the femoral head to dislocate the femur , being careful not to snap the bone below the femoral head .", "grasp the top of the femoral shaft with the forceps , cut the soft tissue away from the femoral head to release it from the acetabulum .", "pull the entire leg bone , including femur , knee , and tibia , up and away from the body , carefully cutting away the connective tissue and muscle connecting the leg to the skin .", "overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia . grasping the distal end of the tibia , taking care not to sever the tendons , pull the tibia up and away from the body and the pin board .", "cut any remaining connective tissue attaching the long bone to the mouse at the knee . remove any additional muscle or connective tissue attached to the femur and the tibia .", "for any applications that require the bone to remain intact ( histology , histomorphometry , biomechanical testing , etc . ) , proceed with standard in - house protocols ( as in ) . to isolate bone marrow , proceed to section 2 . using the forceps ,", "grasp the femur with the patella facing away and the proximal end ( femoral head ) down .", "overextend the knee joint and use the scissors in a twisting motion to dislocate the tibia and femur .", "grasp the femur with the anterior side facing away and the proximal end ( femoral head end ) down .", "rotate the scissors back and forth to remove the condyles , the patella , and the epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the femur using forceps , scissors , and kimwipes . using the forceps ,", "grasp the tibia with the anterior side facing away and the distal end ( ankle end ) down .", "if the tibial epiphysis is intact , guide the scissors up the tibia shaft to the condyles . gently rotate the scissors back and forth to remove the condyles and epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the tibia using forceps , scissors , and kimwipes .", "push an 18 g needle through the bottom of a 0.5 ml  microcentrifuge tube . place the long bones ( maximum of 2 femurs and 2 tibiae ) into the tube , knee - end", "verify that the bone marrow has been spun out of the bones by visual inspection .", "the bones should appear white and there should be a large visual pellet in the larger tube .", "suspend the bone marrow in appropriate solution ( e.g. , pbs , culture media , facs buffer ) and proceed with experimental protocol ( dna , rna , or protein isolation , facs analysis , or primary cell culture ) ."], ["euthanize the mouse in accordance with institutional guidelines . position the mouse in a supine position and affix by pinning all four legs through the mouse paw pads below the ankle joint . spray the mouse with 70% ethanol , thoroughly dousing the legs . make a small incision to the right of midline in the lower abdomen , just above the hip .", "pull back the skin and cut the quadriceps muscle anchored to proximal end of the femur to expose the anterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the blade of the scissors against the posterior side of the femur , cut the hamstrings away from the knee joint .", "pull back the skin and the hamstring muscles anchored to proximal end of the femur to expose the posterior side of the femur and pin out from the leg , placing the pin at a 45-degree angle from the board . with the forceps , hold the distal end of the femur , just above the knee joint .", "guide the blades of the scissors on either side of the femoral shaft towards the hip joint , being careful not to cut into the femur itself . after reaching the femoral head , indicated by the scissors opening slightly ,", "twist the scissors with the top blade of the scissors moving directly over the femoral head to dislocate the femur , being careful not to snap the bone below the femoral head . grasp the top of the femoral shaft with the forceps , cut the soft tissue away from the femoral head to release it from the acetabulum . pull the entire leg bone , including femur , knee , and tibia , up and away from the body , carefully cutting away the connective tissue and muscle connecting the leg to the skin .", "overextend the ankle joint and again use the scissors in a twisting motion to dislocate the tibia . grasping the distal end of the tibia , taking care not to sever the tendons , pull the tibia up and away from the body and the pin board .", "cut any remaining connective tissue attaching the long bone to the mouse at the knee . remove any additional muscle or connective tissue attached to the femur and the tibia .", "for any applications that require the bone to remain intact ( histology , histomorphometry , biomechanical testing , etc . ) , proceed with standard in - house protocols ( as in ) . to isolate bone marrow , proceed to section 2 ."], ["using the forceps , grasp the femur with the patella facing away and the proximal end ( femoral head ) down .", "overextend the knee joint and use the scissors in a twisting motion to dislocate the tibia and femur .", "grasp the femur with the anterior side facing away and the proximal end ( femoral head end ) down .", "rotate the scissors back and forth to remove the condyles , the patella , and the epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the femur using forceps , scissors , and kimwipes . using the forceps ,", "grasp the tibia with the anterior side facing away and the distal end ( ankle end ) down .", "if the tibial epiphysis is intact , guide the scissors up the tibia shaft to the condyles . gently", "rotate the scissors back and forth to remove the condyles and epiphysis to expose the metaphysis .", "remove any additional muscle or connective tissue attached to the tibia using forceps , scissors , and kimwipes ."], ["push an 18 g needle through the bottom of a 0.5 ml  microcentrifuge tube . place the long bones ( maximum of 2 femurs and 2 tibiae ) into the tube , knee - end", "verify that the bone marrow has been spun out of the bones by visual inspection .", "the bones should appear white and there should be a large visual pellet in the larger tube .", "suspend the bone marrow in appropriate solution ( e.g. , pbs , culture media , facs buffer ) and proceed with experimental protocol ( dna , rna , or protein isolation , facs analysis , or primary cell culture ) ."], ["the protocol described here is optimized for rapid dissection of the mouse femur and tibia with a minimum of damage to the bone tissue .", "this technique is suitable for a number of downstream analyses , including biomechanics studies , histomorphometry ( figure 1a - b ) , and histology ( figure 1c ) .", "the representative histomophometric micoct 3d reconstruction ( figure 1a - b ) demonstrates that both the cancellous bone and cortical shell are maintained that allows for accurate quantitation of the standardized structural parameters for bone histomorphometry , including trabecular number , thickness , and spacing ;  bone volume ;  and cortical thickness , among other measures .", "the representative histologic section shows an h&e stained formalin - fixed and decalcified tibia ( figure 1c ) .", "the image demonstrates the integrity of both the calcified bone and cellular bone marrow for histologic analysis .", "the bone marrow isolation procedure preserves the sterility of the bone marrow space , has low handling to reduce contamination , and does not require cutting of the long bone , thus reducing loss of bone marrow yield .", "this bone marrow is suitable for many downstream applications , including flow cytometry and pcr analyses . in addition", ", this procedure can be used to isolate bone marrow for primary cell culture of bone marrow cells , including osteoclasts and osteoblasts ( figure 2a - b ) .", "three - dimensional microct reconstruction of a mouse tibia showing ( a ) the outer cortical shell and ( b ) trabecular bone ( scale bar = 0.5 mm ) .", "( c ) histological h&e stain of a decalcified and sectioned tibia ( 4x ) .", "( a ) trap staining for multinucleated osteoclasts after 7 days in osteoclastogenic media ( 4x ) .", "( b ) alkaline phosphatase ( purple color ) for osteoblasts and alizarin red ( red color ) stain for mineralization after 21 days in osteogenic media ."], ["we present a simple and efficient method for removal of mouse hind long bones and subsequent bone marrow isolation .", "this method maintains the high structural and cellular integrity of the bones and bone marrow and has low handling time , minimizing the likelihood of user - induced fracture or bone scoring that may influence downstream analyses .", "in addition , the centrifugation method for isolating bone marrow does not require cutting the bone to expose the bone marrow space or fluid to flush the bone marrow , reducing potential points of contamination .", "moreover , the centrifuge technique is relatively high - throughput with lower hands - on time than other methods , thus reducing processing time .", "high variation is inherent to in vivo mouse studies due to high mouse - to - mouse phenotypic variation . in order to maximize the research impact of expensive and labor - intensive mouse studies , it is critical to minimize technical experimental error .", "time from animal sacrifice to downstream analysis or tissue fixation introduces experimental variation that may overcome subtle changes and reduce large differences between groups .", "the long bone dissection and bone marrow isolation techniques described here are optimized for rapid processing of animals and samples to reduce technical variation .", "this protocol can be widely applied to many research fields , including investigation of the bone tissue itself or interrogation of the cells of the bone marrow .", "in addition , this straightforward approach to long bone dissection will enable researchers in related fields to directly interrogate bone contributions in order to expand our knowledge of bone marrow dysfunction in otherwise understudied pathologies ."], [""]]}
{"article_id": "PMC3649198", "article_text": ["tuberculosis ( tb ) is a major health issue in developing countries with over two billion people being infected with tuberculosis bacilli world wide ( 1 ) . although tb was recently regarded primarily as a disease of the developing countries it is now also on the increase in developed countries such as the united kingdom , mainly as a result of globalisation as well as an increase in immunosuppressed patients .", "figures by the health protection agency ( hpa ) show that the cases of tb in the uk increased by 5.5% in 2009 when compared to those in 2008 ( 2 ) .", "this caucasian patient presented to the emergency department with sudden pain localised to the ulnar aspect of her wrist .", "x - ray of the forearm was normal and she was treated conservatively but was lost to follow up .", "she presented again at the orthopaedic clinic 26 months later with continuing swelling and pain .", "ultrasound showed a synovial tumour on the dorso - medial aspect of the wrist ( figure 1 ) .", "ultrasound showing a synovial tumour on the dorsal medial aspect of the wrist however , special stains for fungi and acid fast bacilli were negative .", "subsequently the patient developed a sinus and continued to have a chronic discharge from her left wrist .", "x ray this time showed destruction of the ulnar styloid consistent with a bony infection ( figure 2 ) .", "mri showed destruction localised around the ulnar head with oedematous changes extending up the shaft of the ulna ( figure 3 ) .", "ap xray of the wrist showing destruction of the ulnar styloid consistent with a bony infection .", "soft tissue swelling can also be noted over the dorsum of the wrist mri showing localised destruction around the ulnar head .", "the mri also shows thickening around the extensor carpi ulnaris tendon in keeping with marked tenosynovitis at this site .", "the patient agreed to a repeat biopsy and excision of the necrotic bone ( figure 4 ) .", "microscopy of the specimen again showed a florid granulomatous inflammatory process with areas of fibrinoid necrosis .", "post - operative xray of the wrist showing excision of the ulnar styloid further enquiry revealed that our patient had been experiencing lethargy but no other features to suggest tuberculosis .", "following the diagnosis of tb , the patient recalled that in her former post as a nursing assistant , when she was much younger , she was exposed to patients with tb .", "although improvements in its management and treatment are continuously occurring an estimated 1.8 million people died of tb in 2008 ( 1 ) .", "extra - spinal tuberculous osteomyelitis is rare and comprises about 2 - 3% of all cases of osteoarticular tuberculosis , with the hip and knee joints being the most common following spinal involvement ( 3 ) .", "tuberculous involvement of the ulna is uncommon and tends to be more common in the diaphysis .", "a high index of suspicion of tb is important to obtain the correct diagnosis . in chronic soft tissue and bony infection", "the diagnosis of tb may not be evident from the history and examination and indeed no obvious risk factors may be present as in our case . in our patient", "the infection could have been contracted at a much younger age , when she was caring for patients infected with tuberculosis , and remained latent .", "however no other risk factors were evident that could explain why the disease was activated after such a long period of latency .", "it is also interesting to note that the actual distal radio - ulnar joint was not involved , most of the destruction being situated around the ulnar process .", "although localisation of the tuberculous infection in the bone without joint involvement can occur , this is rare ( 3 ) ."], "abstract_text": ["<S> tuberculosis ( tb ) is a major health issue in both developing and developed countries such as the uk . </S>", "<S> healthy individuals who contract the infection have only 5 - 10% chance of converting to the active disease over their lifetime . </S>", "<S> we present the clinical history of a 76 year old female who attended the emergency department complaining of wrist pain , and was only diagnosed with tb after three years . </S>", "<S> this case report emphasises the importance of including tb in the differential diagnosis when treating patients with an infection resistant to conventional antibiotics , even though risk factors for tb may not be evident or special stains for tb are negative . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["tuberculosis ( tb ) is a major health issue in developing countries with over two billion people being infected with tuberculosis bacilli world wide ( 1 ) . although tb was recently regarded primarily as a disease of the developing countries it is now also on the increase in developed countries such as the united kingdom , mainly as a result of globalisation as well as an increase in immunosuppressed patients .", "figures by the health protection agency ( hpa ) show that the cases of tb in the uk increased by 5.5% in 2009 when compared to those in 2008 ( 2 ) ."], ["this caucasian patient presented to the emergency department with sudden pain localised to the ulnar aspect of her wrist .", "x - ray of the forearm was normal and she was treated conservatively but was lost to follow up .", "she presented again at the orthopaedic clinic 26 months later with continuing swelling and pain .", "ultrasound showed a synovial tumour on the dorso - medial aspect of the wrist ( figure 1 ) .", "ultrasound showing a synovial tumour on the dorsal medial aspect of the wrist however , special stains for fungi and acid fast bacilli were negative .", "subsequently the patient developed a sinus and continued to have a chronic discharge from her left wrist .", "x ray this time showed destruction of the ulnar styloid consistent with a bony infection ( figure 2 ) .", "mri showed destruction localised around the ulnar head with oedematous changes extending up the shaft of the ulna ( figure 3 ) .", "ap xray of the wrist showing destruction of the ulnar styloid consistent with a bony infection .", "soft tissue swelling can also be noted over the dorsum of the wrist mri showing localised destruction around the ulnar head .", "the mri also shows thickening around the extensor carpi ulnaris tendon in keeping with marked tenosynovitis at this site .", "the patient agreed to a repeat biopsy and excision of the necrotic bone ( figure 4 ) .", "microscopy of the specimen again showed a florid granulomatous inflammatory process with areas of fibrinoid necrosis .", "post - operative xray of the wrist showing excision of the ulnar styloid further enquiry revealed that our patient had been experiencing lethargy but no other features to suggest tuberculosis .", "following the diagnosis of tb , the patient recalled that in her former post as a nursing assistant , when she was much younger , she was exposed to patients with tb ."], ["although improvements in its management and treatment are continuously occurring an estimated 1.8 million people died of tb in 2008 ( 1 ) .", "extra - spinal tuberculous osteomyelitis is rare and comprises about 2 - 3% of all cases of osteoarticular tuberculosis , with the hip and knee joints being the most common following spinal involvement ( 3 ) .", "tuberculous involvement of the ulna is uncommon and tends to be more common in the diaphysis .", "a high index of suspicion of tb is important to obtain the correct diagnosis . in chronic soft tissue and bony infection", "the diagnosis of tb may not be evident from the history and examination and indeed no obvious risk factors may be present as in our case . in our patient", "the infection could have been contracted at a much younger age , when she was caring for patients infected with tuberculosis , and remained latent .", "however no other risk factors were evident that could explain why the disease was activated after such a long period of latency .", "it is also interesting to note that the actual distal radio - ulnar joint was not involved , most of the destruction being situated around the ulnar process .", "although localisation of the tuberculous infection in the bone without joint involvement can occur , this is rare ( 3 ) ."]]}
{"article_id": "PMC3420518", "article_text": ["the value of the ketogenic diet ( kd ) has been recognized in the treatment of epilepsy , although the exact mechanisms by which it exerts its effect remain an enigma .", "they seem to be different from those of regular antiepileptic medications ( aeds ) , and discovering what they are may lead to its use in clinical situations other than epilepsy as well .", "the kd is comprised of four elements , the changes of any of them can potentially lead to losing its anticonvulsant effect : ( 1 ) increased amount of fat , usually in a ratio of 3 to 4 grams of fat for each gram of protein and carbohydrates , ( 2 ) as low a consumption of glucose as possible , ( 3 ) caloric restriction , and ( 4 ) fluid restriction .", "although there is some debate about the last component , clinical practice has shown that stopping fluid restriction can lead to seizure recurrence much in the same way as when stopping glucose restriction .  ", "the most important result of observing the diet is the increased blood level of free fatty acids ( ffas ) .", "the ffas are transferred into the mitochondria , a process which requires the presence of an appropriate amount of carnitine , where they are degraded into ketone bodies through  oxidation .", "these ketone bodies include  hydroxybutyrate , acetoacetate , and acetone   ( figure 1 ) .  ", "the effect of the increased amount of ketone bodies seems to be the most prominent of all other suggested antiepileptic mechanisms of the kd .", "the degradation of the ketone bodies delivers acetyl - coa directly to the tricarboxylic acid cycle , thus increasing its turnover while bypassing the need to depend on acetyl - coa coming from glycolysis to produce atp . unlike glucose , which requires a transporter to cross the blood brain barrier ,", "the ketone bodies penetrate it easily . when this transporter is deficient , as in glut 1 deficiency , the kd is the preferred way of antiepileptic treatment , since it allows for bypassing the need for glucose .", "children in whom the conversion of pyruvate to acetyl - coa is blocked , for instance , in pyruvate dehydrogenase ( pdh ) deficiency , will also benefit from bypassing this route .", " hydroxybutyrate is the predominant ketone body measured in the blood , and it is used to monitor the degree of ketosis during therapy .", "the breath of a patient who is ketotic while on the diet will often even smell of acetone .", "acetone is one of the ketone bodies that have an anticonvulsant effect in several types of mouse seizure models .", "the mechanism of this effect is unknown , although an effect on k2p channels has been proposed .   by means of the tca cycle ,", "acetyl - coa increases the level of the neurotransmitters glutamate and -aminobutyric acid ( gaba ) and the major excitatory and inhibitory neurotransmitters in brain , respectively .", "another product of an elevated level of free fatty acids is polyunsaturated fatty acids ( pufas ) .", "the potential ability of pufas to block seizure activity in the brain is speculated to be associated with some rather more complicated mechanisms , including ( 1 ) directly inhibiting voltage - gated sodium and calcium channels , ( 2 ) activating a lipid - sensitive potassium channel , ( 3 ) enhancing the activity of the sodium pump to limit neuronal excitability , ( 4 ) activating peroxisome proliferator - activated receptor- ( ppar ) , and ( 5 ) inducing the expression and activity of brain - specific uncoupling proteins in the mitochondria , thereby inducing a neuroprotective effect .", "neuroprotection can contribute to the anticonvulsant effect , but it may have other effects as well , which can lead to other clinical uses of the kd .", "appleton and de vivo   reported that the kd increased the total quantity of bioenergetic substrates ( adenosine triphosphate ( atp ) ) and elevated the energy charge in rat brain .", "acetoacetate , a product of  hydroxybutyrate dehydrogenation , is transferred into acetyl - coa which enters the tricarboxylic acid ( tca ) cycle .", "the increased turnover of the tca cycle generates protons and electrons that are channeled to the electron transport chain .", "this , in turn , drives the formation of atp from adenosine phosphate ( adp ) by atp synthase .", "enhanced atp can either be converted to phosphocreatine for energy storage or broken into adenosine .", "enhanced atp levels provide energy reserves for a neuron to continue functioning under stress . increased extracellular adenosine offers a neuroprotective buffer against insults , reduces excitation , and averts excessive atp demands , thus providing local seizure control and neuroprotection .  ", "it was also suggested that the kd influences toward an upregulation of transcripts encoding energy metabolism enzymes and increase in the density of mitochondria in neuronal process , leading to heightened energy reserves .", "an improved energetic status can support seizure prevention , for instance , by supporting gabaergic inhibition .", "it is suggested that adaptive processes to the metabolic changes induced by the diet lead to changes in gene expression which in turn result in some of the above - noted changes .", "other path of neuroprotection is modulated through decreased generation of ros which is considered to be related to pufas effect on uncoupling proteins .", "the fact that the kd is considered a proven therapy with relatively few adverse affects and wide clinical experience , particularly in children , led to recent studies investigating new potential uses for other neurological disorders .", "one of the most intriguing and active fields of research is the effect of a high - fat caloric - restricted diet on the survival of brain tumors cells .", "it is hypothesized that mitochondrial abnormalities impair the ability of brain tumors to generate energy from ketone bodies . unlike normal cells ,", "malignant tumor cells have impaired genetic adaptability due to their genetic abnormalities and , therefore , increased susceptibility to environmental stress , such as fasting or caloric restriction .", "the same genomic defects that are involved in the creation of brain tumors can be exploited for their destruction [ 3 , 10 , 11 ] .   in 1995 , nebeling et al", ".   reported two young girls with unresectable advanced stage brain tumors who had poor response to radiation and chemotherapy .", "they were treated with a kd and their response was remarkable , both clinically and according to positron emission tomography follow - up scans .", "described a patient with glioblastoma multiforme whose tumor , which is very malignant , improved on the kd . surprisingly , despite the appealing efficacy of this treatment , no further human studies or clinical trials on the kd as a therapy for brain tumors have been conducted .", "several laboratory studies in mouse and rat models have recently confirmed that inhibition of brain tumor growth is directly related with reduced levels of glucose and elevated levels of ketone bodies .", "moreover , the kd was shown to reduce reactive oxygen species ( ros ) in the brain .", "cancer cells need high levels of ros for the induction of angiogenesis and the production of tumor growth factors , thus , through this mechanism the kd can be protective .", "the clinical manifestations were mainly intractable seizures that necessitated repeated admissions to the intensive care unit , as well as severe cognitive and alertness decline .", "after the oncologists decided that antitumor treatment would be ineffective , she was started on the kd .", "after four weeks of trial , it did not have any effect on the tumor progression .", "however , it did have a notable improving effect on cognition , alertness , and mood of the girl , in spite of her devastating condition .", "the beneficial effect of the kd on cognition , alertness , and mood is well recognized , and clinical experience shows that in many times it is not less important than the anticonvulsant effect .", "the potential neuroprotective effect of the kd was what motivated investigations into its potential as a treatment option in other neurologic disorders .", "there are increasing numbers of reports that ketosis achieved by starvation or administration of a kd has a consistent neuroprotective effect after various brain injuries in animal models .", "one human pilot study and several animal model studies have shown improvement in autistic behavior parameters with kd treatment .", "it remains to be further clarified whether this improvement is related to reduced epileptic activity found in up to 30% of these patients or to a primary effect of the kd .  ", "a factor that can be crucial for the application of the kd in medical conditions other than intractable epilepsy is the inherent difficulties in its use .", "dietary restriction can pose a significant problem in a child with a progressive tumor that undergoes massive chemotherapy , who may already be cachectic .", "the kd may not be an option in a grown - up hyperactive autistic child .", "thus , the neurologist must evaluate the appropriate clinical , familial , and environmental situations very cautiously prior to the recommendation of the kd . in conclusion ,", "the major metabolic effect of the kd is in supplying the brain with an increased amount of free fatty acids .", "their degradation into ketone bodies , together with the load of pufas , leads to major changes in the metabolic , bioenergetic , mitochondrial , and even genetic constellation .", "whether it may also be effective in other pathologies , especially in treating malignancies , awaits future research ."], "abstract_text": ["<S> although the ketogenic diet ( kd ) has been widely accepted as a legitimate and successful therapy for epilepsy and other neurological disorders , its mechanism of action remains an enigma . </S>", "<S> the use of the kd causes major metabolic changes . </S>", "<S> the most significant of them seems to be the situation of chronic ketosis , but there are others as well , for instance , high level of polyunsaturated fatty acids ( pufas ) . </S>", "<S> these  primary  influences lead to  secondary  , in part adaptive , effects , for instance changes in mitochondrial density and gene expression . </S>", "<S> clinically , the influences of the diet are considered as anticonvulsive and neuroprotective , although neuroprotection can also lead to prevention of seizures . </S>", "<S> potential clinical implications of these mechanisms are discussed . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Illustrative Case"], "sections": [["the value of the ketogenic diet ( kd ) has been recognized in the treatment of epilepsy , although the exact mechanisms by which it exerts its effect remain an enigma .", "they seem to be different from those of regular antiepileptic medications ( aeds ) , and discovering what they are may lead to its use in clinical situations other than epilepsy as well .", "the kd is comprised of four elements , the changes of any of them can potentially lead to losing its anticonvulsant effect : ( 1 ) increased amount of fat , usually in a ratio of 3 to 4 grams of fat for each gram of protein and carbohydrates , ( 2 ) as low a consumption of glucose as possible , ( 3 ) caloric restriction , and ( 4 ) fluid restriction .", "although there is some debate about the last component , clinical practice has shown that stopping fluid restriction can lead to seizure recurrence much in the same way as when stopping glucose restriction .  ", "the most important result of observing the diet is the increased blood level of free fatty acids ( ffas ) .", "the ffas are transferred into the mitochondria , a process which requires the presence of an appropriate amount of carnitine , where they are degraded into ketone bodies through  oxidation .", "these ketone bodies include  hydroxybutyrate , acetoacetate , and acetone   ( figure 1 ) .  ", "the effect of the increased amount of ketone bodies seems to be the most prominent of all other suggested antiepileptic mechanisms of the kd .", "the degradation of the ketone bodies delivers acetyl - coa directly to the tricarboxylic acid cycle , thus increasing its turnover while bypassing the need to depend on acetyl - coa coming from glycolysis to produce atp . unlike glucose , which requires a transporter to cross the blood brain barrier ,", "the ketone bodies penetrate it easily . when this transporter is deficient , as in glut 1 deficiency , the kd is the preferred way of antiepileptic treatment , since it allows for bypassing the need for glucose .", "children in whom the conversion of pyruvate to acetyl - coa is blocked , for instance , in pyruvate dehydrogenase ( pdh ) deficiency , will also benefit from bypassing this route .", " hydroxybutyrate is the predominant ketone body measured in the blood , and it is used to monitor the degree of ketosis during therapy .", "the breath of a patient who is ketotic while on the diet will often even smell of acetone .", "acetone is one of the ketone bodies that have an anticonvulsant effect in several types of mouse seizure models .", "the mechanism of this effect is unknown , although an effect on k2p channels has been proposed .   by means of the tca cycle ,", "acetyl - coa increases the level of the neurotransmitters glutamate and -aminobutyric acid ( gaba ) and the major excitatory and inhibitory neurotransmitters in brain , respectively .", "another product of an elevated level of free fatty acids is polyunsaturated fatty acids ( pufas ) .", "the potential ability of pufas to block seizure activity in the brain is speculated to be associated with some rather more complicated mechanisms , including ( 1 ) directly inhibiting voltage - gated sodium and calcium channels , ( 2 ) activating a lipid - sensitive potassium channel , ( 3 ) enhancing the activity of the sodium pump to limit neuronal excitability , ( 4 ) activating peroxisome proliferator - activated receptor- ( ppar ) , and ( 5 ) inducing the expression and activity of brain - specific uncoupling proteins in the mitochondria , thereby inducing a neuroprotective effect .", "neuroprotection can contribute to the anticonvulsant effect , but it may have other effects as well , which can lead to other clinical uses of the kd .", "appleton and de vivo   reported that the kd increased the total quantity of bioenergetic substrates ( adenosine triphosphate ( atp ) ) and elevated the energy charge in rat brain .", "acetoacetate , a product of  hydroxybutyrate dehydrogenation , is transferred into acetyl - coa which enters the tricarboxylic acid ( tca ) cycle .", "the increased turnover of the tca cycle generates protons and electrons that are channeled to the electron transport chain .", "this , in turn , drives the formation of atp from adenosine phosphate ( adp ) by atp synthase .", "enhanced atp can either be converted to phosphocreatine for energy storage or broken into adenosine .", "enhanced atp levels provide energy reserves for a neuron to continue functioning under stress . increased extracellular adenosine offers a neuroprotective buffer against insults , reduces excitation , and averts excessive atp demands , thus providing local seizure control and neuroprotection .  ", "it was also suggested that the kd influences toward an upregulation of transcripts encoding energy metabolism enzymes and increase in the density of mitochondria in neuronal process , leading to heightened energy reserves .", "an improved energetic status can support seizure prevention , for instance , by supporting gabaergic inhibition .", "it is suggested that adaptive processes to the metabolic changes induced by the diet lead to changes in gene expression which in turn result in some of the above - noted changes .", "other path of neuroprotection is modulated through decreased generation of ros which is considered to be related to pufas effect on uncoupling proteins .", "the fact that the kd is considered a proven therapy with relatively few adverse affects and wide clinical experience , particularly in children , led to recent studies investigating new potential uses for other neurological disorders .", "one of the most intriguing and active fields of research is the effect of a high - fat caloric - restricted diet on the survival of brain tumors cells .", "it is hypothesized that mitochondrial abnormalities impair the ability of brain tumors to generate energy from ketone bodies . unlike normal cells ,", "malignant tumor cells have impaired genetic adaptability due to their genetic abnormalities and , therefore , increased susceptibility to environmental stress , such as fasting or caloric restriction .", "the same genomic defects that are involved in the creation of brain tumors can be exploited for their destruction [ 3 , 10 , 11 ] .   in 1995 , nebeling et al", ".   reported two young girls with unresectable advanced stage brain tumors who had poor response to radiation and chemotherapy .", "they were treated with a kd and their response was remarkable , both clinically and according to positron emission tomography follow - up scans .", "described a patient with glioblastoma multiforme whose tumor , which is very malignant , improved on the kd . surprisingly , despite the appealing efficacy of this treatment , no further human studies or clinical trials on the kd as a therapy for brain tumors have been conducted .", "several laboratory studies in mouse and rat models have recently confirmed that inhibition of brain tumor growth is directly related with reduced levels of glucose and elevated levels of ketone bodies .", "moreover , the kd was shown to reduce reactive oxygen species ( ros ) in the brain .", "cancer cells need high levels of ros for the induction of angiogenesis and the production of tumor growth factors , thus , through this mechanism the kd can be protective ."], ["the clinical manifestations were mainly intractable seizures that necessitated repeated admissions to the intensive care unit , as well as severe cognitive and alertness decline .", "after the oncologists decided that antitumor treatment would be ineffective , she was started on the kd .", "after four weeks of trial , it did not have any effect on the tumor progression .", "however , it did have a notable improving effect on cognition , alertness , and mood of the girl , in spite of her devastating condition .", "the beneficial effect of the kd on cognition , alertness , and mood is well recognized , and clinical experience shows that in many times it is not less important than the anticonvulsant effect .", "the potential neuroprotective effect of the kd was what motivated investigations into its potential as a treatment option in other neurologic disorders .", "there are increasing numbers of reports that ketosis achieved by starvation or administration of a kd has a consistent neuroprotective effect after various brain injuries in animal models .", "one human pilot study and several animal model studies have shown improvement in autistic behavior parameters with kd treatment .", "it remains to be further clarified whether this improvement is related to reduced epileptic activity found in up to 30% of these patients or to a primary effect of the kd .  ", "a factor that can be crucial for the application of the kd in medical conditions other than intractable epilepsy is the inherent difficulties in its use .", "dietary restriction can pose a significant problem in a child with a progressive tumor that undergoes massive chemotherapy , who may already be cachectic .", "the kd may not be an option in a grown - up hyperactive autistic child .", "thus , the neurologist must evaluate the appropriate clinical , familial , and environmental situations very cautiously prior to the recommendation of the kd . in conclusion ,", "the major metabolic effect of the kd is in supplying the brain with an increased amount of free fatty acids .", "their degradation into ketone bodies , together with the load of pufas , leads to major changes in the metabolic , bioenergetic , mitochondrial , and even genetic constellation .", "whether it may also be effective in other pathologies , especially in treating malignancies , awaits future research ."]]}
{"article_id": "PMC5371447", "article_text": ["the endotracheal tube ( ett ) should be placed at the optimal level to avoid inadvertent complication .", "if the ett is too deep , it increases the risk of unintended single lung ventilation .", "on the other hand , if the ett is too shallow , it may cause vocal cord injury by the ett balloon or accidental extubation .", "there are many methods for determining the appropriate depth of ett in adults ; fixed insertion depth according to sex ( 23 and 21  cm from the upper incisors in adult males and females , respectively ) , the use of depth marks on the ett , suprasternal palpation of the ett tip or cuff , and bilateral auscultation .", "although chest radiography and bronchoscopy are considered an accurate method , they are not always feasible and the costs are considerable . considering the individual variation in the length of the trachea , using fixed depths or marks on the ett may result in inadequate placement of the ett . to reduce the risk of single lung ventilation or vocal cord injury , the segment between the proximal edge of the cuff and the ett tip should be placed at the mid - trachea level . if the length of the trachea can be predicted at bedside before intubation , the ett can be placed at a safe depth for each patient .", "the purpose of this study was to determine whether surface anatomical landmarks can be used to predict the mid - tracheal level in adult patients .", "neck computed tomography ( ct ) images of adults obtained between 2009 and 2014 were reviewed by a single reviewer after obtaining approval form seoul national university hospital institutional review board ( july 17 , 2015/no .", "patients with laryngeal , tracheal , or thoracic abnormalities , tracheostomy or significant tracheal deviation caused by mass lesions were excluded .", "in addition , poor ct image quality , absent sagittal ct images , ct images that did not cover the entire trachea , or ct images taken in neck hyperextension ( boidin angle > 130 on scout view ) or hyperflexion ( boidin angle < 110 on scout view ) position were also excluded .", "levels of subglottic and tracheal airway segments that include the vocal cords ( vc ) , the cricoid cartilage ( cc ) , and the carina were identified using the method described by sirisopana et al .", "the vocal cords were identified as the most cranial level of the upper airway , with a teardrop shape below the laryngeal ventricle . anatomic level of suprasternal notch ( ssn )", "the level of manubriosternal junction ( msj ) was defined as the middle level between the manubrium and the body of the sternum where the second rib attaches to the sternum .", "the level of carina was defined as the bifurcation of right and left main bronchus and identified by the figure 8 shaped lumen .", "the external locations of the cc , ssn , and msj were defined on the skin surface .", "the distances between the external points of the cc and the ssn ( extcc - ssn ) , and those of the ssn and the msj ( extssn - msj ) were measured .", "the distances between the subglottic structures at the levels of the vc , the cc , ssn , and carina were measured .", "tracheal length was calculated as the summation of the distances measured from the vc to the carina .", "the mid - trachea level was calculated from tracheal length and the ideal position of ett is shown in fig .", "2 . identification of subglottic segments on axial view ( right ) , and defining the distances between subglottic segments on sagittal view ( left ) in a 45-year - old male patient .", "cc  =  cricoid cartilage , extcc - ssn  =  the surface distances from the cricoid cartilage to the suprasternal notch , extssn - msj  =  the surface distances from the suprasternal notch to the manubriosternal junction , msj  =  manubriosternal juction , ssn  =  suprasternal notch , vc  =  vocal cord , tracheal length  =  a  +  b  +  c  +  d. the ideal position of endotracheal tube ( ett ) to minimize the risk of endobronchial intubation and vocal cord injury .", "the mid - point between the proximal edge of the cuff and the ett tip was matched to the level of the middle of the trachea ( mtl ) , which was calculated from the tracheal length .", "cc  =  cricoid cartilage , ett  =  endotracheal tube , msj  =  manubriosternal juction , mtl  =  the level of the middle of the trachea , ssn  =  suprasternal notch , vc  =  vocal cord .", "the correlations between age , height , weight , and tracheal length were analyzed using linear regression .", "the relationships between surface measurements and the mid - tracheal level were analyzed with bland ", "the distance between the cc and the ssn was longer in females ( p  <  0.001 ) , whereas distances between the vc and the cc , the ssn and the carina , and the tracheal length were longer in males ( p  <  0.001 ) .", "the extcc - ssn was longer in females ( p  <  0.001 ) , whereas extssn - msj was longer in males ( p  <  0.001 ) .", "demographic data and measured distances between subglottic structures of trachea , external landmarks , and calculated tracheal length .", "the calculated tracheal length was 131.9 ( 10.3 ) mm and ranged from 106.0 to 169.8  mm .", "tracheal length showed weak correlation with height ( r  =  0.2188 ) and weaker correlation with age ( r  =  0.0004 ) and weight ( r  =  0.0466 ) ( fig .", "the calculated mid - tracheal level was 66.0    5.1  mm from the vc .", "altman plots showing the difference between the mid - tracheal level and the surface measurements are shown in fig .", "the bias was calculated by subtracting the mid - tracheal level from the surface measurements .", "the difference between the extcc - ssn and the mid - tracheal level was 6.6 ( 12.5 ) mm , and the difference between the extssn - msj and the mid - tracheal level was 19.2 ( 6.1 ) mm .", "altman plots showing the agreement between the mid - tracheal level ( mtl ) and the surface measurements of surface anatomical landmarks .", "( left ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 6.6 [ 31.1 to 17.9 ] ( mm ) .", "( right ) the bias ( subtraction of the mid - tracheal level from the surface distance from the suprasternal notch to the manubriosternal junction ) and 95% limit of agreement as the mean difference were 19.2 [ 31.1 to 7.2 ] ( mm ) .", "extcc - ssn  =  the surface distances from the cricoid cartilage to the suprasternal notch , extssn - msj  =  the surface distances from the suprasternal notch to the manubriosternal junction , mtl  =  the level of the middle of the trachea .", "altman plots of the difference between the extcc - ssn and the mid - tracheal level in female and male patients .", "the calculated mid - tracheal level was 64.5 ( 4.6 ) mm in females and 67.9 ( 5.0 ) mm in males .", "the difference between the extcc - ssn and the mid - tracheal level was 1.7 ( 11.7 ) mm in females and 12.8 ( 10.7 ) mm in males ( p  <  0.001 ) .", "altman plots of female ( left ) and male ( right ) patients showing the agreement between the mid - tracheal level ( mtl ) and the surface the surface distance from the cricoid cartilage from the suprasternal notch .", "( left ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 1.7 [ 24.6 to 21.2 ] ( mm ) in females .", "( right ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 12.8 [ 33.8 to 8.2 ] ( mm ) in males .", "extcc - ssn  =  the surface distances from the cricoid cartilage to the suprasternal notch , mtl  =  the level of the middle of the trachea .", "this study showed that mid - tracheal level could be estimated by the surface distance between the cc and the ssn ( extcc - ssn ) . compared with males , extcc - ssn more", "accurately predicts the mid - tracheal level in females . in clinical practice , physicians can use extcc - ssn to determine the depth of ett so that the segment between the proximal edge of the cuff and the ett tip lies at the mid - tracheal level .", "there are several studies that used the distance between the surface anatomical landmarks to estimate the airway length and the optimal insertion depth of endotracheal tube .", "lee et al showed that the distance between the upper incisor and the manubriosternal angle in the neck extension position correlates with the distance between the upper incisor and the carina in the neutral neck position .", "evron et al used the sum of the 2 distances from the mouth angle to the jaw angle and from the jaw angle to the sternal manubrium to determine the depth of ett", ". however , these methods are complicated than single measurement of extcc - ssn due to the difficulty in locating the sternal manubrium , more than 1 measurement , and the need for full neck extension .", "the physicians can easily apply our method with a simple measurement of the extcc - ssn to estimate the mid - tracheal level , and apply this depth during endotracheal intubation .", "however , according to previous studies in adults , flexion and extension of the neck from neutral position are associated with the inward and outward movements of the ett .", "conrardy et al showed that the orally intubated ett can move 1.5 ( range 0.52.0 ) cm toward the carina during flexion and 2.4 ( range 1.34.3 ) cm away from the carina during extension .", "kim et al showed that the ett can migrate 1.3 ( sd 0.6 ; range 0.52.5 ) cm toward the carina during flexion and 1.7 ( sd 0.8 ; range 0.43.1 ) cm during extension .", "therefore , the position of the neck should be considered when determining the optimal depth of the ett .", "first , this study was a retrospective analysis of ct images and the distances were measured in the neutral position .", "second , since the vc is used as a baseline of the depth of intubation in our method , it can be difficult to apply our method to patients with high cormack grades .", "lastly , the efficacy of this method is yet to be demonstrated in clinical practice . in conclusion ,", "the mid - tracheal level , which is considered the optimal depth of ett , can be predicted by the surface distance between the cc and ssn in adults , especially in females ."], "abstract_text": ["<S> abstractendotracheal tube ( ett ) should be placed at the optimal level to avoid single lung ventilation or accidental extubation . </S>", "<S> this study was performed to estimate the mid - tracheal level by using surface anatomical landmarks in adult patients.neck computed tomography images of 329 adult patients between the ages of 16 and 79 years were reviewed . in the midline sagittal plane , the levels corresponding to the vocal cords , cricoid cartilage , suprasternal notch , manubriosternal junction , and carina were identified . </S>", "<S> the surface distances from the cricoid cartilage to the suprasternal notch ( extcc - ssn ) and that from the suprasternal notch to the manubriosternal junction ( extssn - msj ) were measured . </S>", "<S> the relationship between mid - tracheal level and the surface distances was analyzed using bland  </S>", "<S> altman plot.the difference between the extcc - ssn and the mid - tracheal level was 6.6 ( 12.5 ) mm , and the difference between the extssn - msj and the mid - tracheal level was 19.2 ( 6.1 ) mm . </S>", "<S> the difference between the extcc - ssn and the mid - tracheal level was smaller in females compared with males [ 1.7 ( 11.7 ) mm vs 12.8 ( 10.7 ) mm ; p  <  0.001].the mid - tracheal level , which is helpful in planning the insertion depth of an ett , can be predicted by the surface distance between the cricoid cartilage and suprasternal notch in adults , especially in females . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Analysis of CT images", "Statistical analysis", "Results", "Discussion"], "sections": [["the endotracheal tube ( ett ) should be placed at the optimal level to avoid inadvertent complication .", "if the ett is too deep , it increases the risk of unintended single lung ventilation .", "on the other hand , if the ett is too shallow , it may cause vocal cord injury by the ett balloon or accidental extubation .", "there are many methods for determining the appropriate depth of ett in adults ; fixed insertion depth according to sex ( 23 and 21  cm from the upper incisors in adult males and females , respectively ) , the use of depth marks on the ett , suprasternal palpation of the ett tip or cuff , and bilateral auscultation .", "although chest radiography and bronchoscopy are considered an accurate method , they are not always feasible and the costs are considerable . considering the individual variation in the length of the trachea , using fixed depths or marks on the ett may result in inadequate placement of the ett . to reduce the risk of single lung ventilation or vocal cord injury , the segment between the proximal edge of the cuff and the ett tip should be placed at the mid - trachea level . if the length of the trachea can be predicted at bedside before intubation , the ett can be placed at a safe depth for each patient .", "the purpose of this study was to determine whether surface anatomical landmarks can be used to predict the mid - tracheal level in adult patients ."], ["neck computed tomography ( ct ) images of adults obtained between 2009 and 2014 were reviewed by a single reviewer after obtaining approval form seoul national university hospital institutional review board ( july 17 , 2015/no .", "patients with laryngeal , tracheal , or thoracic abnormalities , tracheostomy or significant tracheal deviation caused by mass lesions were excluded .", "in addition , poor ct image quality , absent sagittal ct images , ct images that did not cover the entire trachea , or ct images taken in neck hyperextension ( boidin angle > 130 on scout view ) or hyperflexion ( boidin angle < 110 on scout view ) position were also excluded ."], ["levels of subglottic and tracheal airway segments that include the vocal cords ( vc ) , the cricoid cartilage ( cc ) , and the carina were identified using the method described by sirisopana et al .", "the vocal cords were identified as the most cranial level of the upper airway , with a teardrop shape below the laryngeal ventricle . anatomic level of suprasternal notch ( ssn )", "the level of manubriosternal junction ( msj ) was defined as the middle level between the manubrium and the body of the sternum where the second rib attaches to the sternum .", "the level of carina was defined as the bifurcation of right and left main bronchus and identified by the figure 8 shaped lumen .", "the external locations of the cc , ssn , and msj were defined on the skin surface .", "the distances between the external points of the cc and the ssn ( extcc - ssn ) , and those of the ssn and the msj ( extssn - msj ) were measured .", "the distances between the subglottic structures at the levels of the vc , the cc , ssn , and carina were measured .", "tracheal length was calculated as the summation of the distances measured from the vc to the carina .", "the mid - trachea level was calculated from tracheal length and the ideal position of ett is shown in fig .", "2 . identification of subglottic segments on axial view ( right ) , and defining the distances between subglottic segments on sagittal view ( left ) in a 45-year - old male patient .", "cc  =  cricoid cartilage , extcc - ssn  =  the surface distances from the cricoid cartilage to the suprasternal notch , extssn - msj  =  the surface distances from the suprasternal notch to the manubriosternal junction , msj  =  manubriosternal juction , ssn  =  suprasternal notch , vc  =  vocal cord , tracheal length  =  a  +  b  +  c  +  d. the ideal position of endotracheal tube ( ett ) to minimize the risk of endobronchial intubation and vocal cord injury .", "the mid - point between the proximal edge of the cuff and the ett tip was matched to the level of the middle of the trachea ( mtl ) , which was calculated from the tracheal length .", "cc  =  cricoid cartilage , ett  =  endotracheal tube , msj  =  manubriosternal juction , mtl  =  the level of the middle of the trachea , ssn  =  suprasternal notch , vc  =  vocal cord ."], ["the correlations between age , height , weight , and tracheal length were analyzed using linear regression .", "the relationships between surface measurements and the mid - tracheal level were analyzed with bland "], ["the distance between the cc and the ssn was longer in females ( p  <  0.001 ) , whereas distances between the vc and the cc , the ssn and the carina , and the tracheal length were longer in males ( p  <  0.001 ) .", "the extcc - ssn was longer in females ( p  <  0.001 ) , whereas extssn - msj was longer in males ( p  <  0.001 ) .", "demographic data and measured distances between subglottic structures of trachea , external landmarks , and calculated tracheal length .", "the calculated tracheal length was 131.9 ( 10.3 ) mm and ranged from 106.0 to 169.8  mm .", "tracheal length showed weak correlation with height ( r  =  0.2188 ) and weaker correlation with age ( r  =  0.0004 ) and weight ( r  =  0.0466 ) ( fig .", "the calculated mid - tracheal level was 66.0    5.1  mm from the vc .", "altman plots showing the difference between the mid - tracheal level and the surface measurements are shown in fig .", "the bias was calculated by subtracting the mid - tracheal level from the surface measurements .", "the difference between the extcc - ssn and the mid - tracheal level was 6.6 ( 12.5 ) mm , and the difference between the extssn - msj and the mid - tracheal level was 19.2 ( 6.1 ) mm .", "altman plots showing the agreement between the mid - tracheal level ( mtl ) and the surface measurements of surface anatomical landmarks .", "( left ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 6.6 [ 31.1 to 17.9 ] ( mm ) .", "( right ) the bias ( subtraction of the mid - tracheal level from the surface distance from the suprasternal notch to the manubriosternal junction ) and 95% limit of agreement as the mean difference were 19.2 [ 31.1 to 7.2 ] ( mm ) .", "extcc - ssn  =  the surface distances from the cricoid cartilage to the suprasternal notch , extssn - msj  =  the surface distances from the suprasternal notch to the manubriosternal junction , mtl  =  the level of the middle of the trachea .", "altman plots of the difference between the extcc - ssn and the mid - tracheal level in female and male patients .", "the calculated mid - tracheal level was 64.5 ( 4.6 ) mm in females and 67.9 ( 5.0 ) mm in males .", "the difference between the extcc - ssn and the mid - tracheal level was 1.7 ( 11.7 ) mm in females and 12.8 ( 10.7 ) mm in males ( p  <  0.001 ) .", "altman plots of female ( left ) and male ( right ) patients showing the agreement between the mid - tracheal level ( mtl ) and the surface the surface distance from the cricoid cartilage from the suprasternal notch .", "( left ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 1.7 [ 24.6 to 21.2 ] ( mm ) in females .", "( right ) the bias ( subtraction of the mid - tracheal level from the surface distance from the cricoid cartilage to the suprasternal notch ) and 95% limit of agreement as the mean difference were 12.8 [ 33.8 to 8.2 ] ( mm ) in males .", "extcc - ssn  =  the surface distances from the cricoid cartilage to the suprasternal notch , mtl  =  the level of the middle of the trachea ."], ["this study showed that mid - tracheal level could be estimated by the surface distance between the cc and the ssn ( extcc - ssn ) . compared with males , extcc - ssn more", "accurately predicts the mid - tracheal level in females . in clinical practice , physicians can use extcc - ssn to determine the depth of ett so that the segment between the proximal edge of the cuff and the ett tip lies at the mid - tracheal level .", "there are several studies that used the distance between the surface anatomical landmarks to estimate the airway length and the optimal insertion depth of endotracheal tube .", "lee et al showed that the distance between the upper incisor and the manubriosternal angle in the neck extension position correlates with the distance between the upper incisor and the carina in the neutral neck position .", "evron et al used the sum of the 2 distances from the mouth angle to the jaw angle and from the jaw angle to the sternal manubrium to determine the depth of ett", ". however , these methods are complicated than single measurement of extcc - ssn due to the difficulty in locating the sternal manubrium , more than 1 measurement , and the need for full neck extension .", "the physicians can easily apply our method with a simple measurement of the extcc - ssn to estimate the mid - tracheal level , and apply this depth during endotracheal intubation .", "however , according to previous studies in adults , flexion and extension of the neck from neutral position are associated with the inward and outward movements of the ett .", "conrardy et al showed that the orally intubated ett can move 1.5 ( range 0.52.0 ) cm toward the carina during flexion and 2.4 ( range 1.34.3 ) cm away from the carina during extension .", "kim et al showed that the ett can migrate 1.3 ( sd 0.6 ; range 0.52.5 ) cm toward the carina during flexion and 1.7 ( sd 0.8 ; range 0.43.1 ) cm during extension .", "therefore , the position of the neck should be considered when determining the optimal depth of the ett .", "first , this study was a retrospective analysis of ct images and the distances were measured in the neutral position .", "second , since the vc is used as a baseline of the depth of intubation in our method , it can be difficult to apply our method to patients with high cormack grades .", "lastly , the efficacy of this method is yet to be demonstrated in clinical practice . in conclusion ,", "the mid - tracheal level , which is considered the optimal depth of ett , can be predicted by the surface distance between the cc and ssn in adults , especially in females ."]]}
{"article_id": "PMC3714553", "article_text": ["embryos generated through assisted reproductive technologies ( art ) are deemed surplus when , for some reason , they are not used for reproductive purposes ( svendsen and koch 2008 ; haimes and taylor 2011 ) ; instead , they are then disposed of or , where this is allowed by a country s art regulation , may be donated for a variety of research purposes .", "thus , the generation and subsequent fate of embryos that are designated surplus are determined as much by legislation as by embryo biology or technological constraints .", "for example , legislatures differ in whether cryopreservation of embryos is permitted , meaning that in some countries any ivf embryos not immediately transferred for pregnancy are surplus , while in others they may be frozen for later transfer . in some countries , the law considers surplus embryos as the primary source of material for human embryonic stem cell ( hesc ) research , while in others this use of embryos is expressly forbidden .", "although hesc research has been subjected to exhaustive ethical scrutiny , the lack of consensus on the ontological or ethical status of surplus embryos means that the uses to which they may then be put remain contested ( deckers 2007 ; guenin 2008 ; moller 2009 ) .", "even if using human embryos for stem cell research is deemed permissible , ethical issues continue to emerge as new social practices and roles develop around the act of embryo donation .", "the relative novelty of these practices and roles , complicated by the pace of change in technical possibility and regulation , mean that there is still uncertainty about how to conceptualise embryo donation , not just in terms of systematic ethics but as a sociomoral practice in everyday social life and morality ( banks , scully , and shakespeare 2006 ) . from a sociological perspective , and also to understand more generally how everyday morality deals with unprecedented ethical dilemmas , it is important to examine which conceptualisations emerge as salient and how they are stabilised and used .", "for example , one way of thinking about the donation of embryos to research is as a bodily gift relationship , one of the many established by modern biomedicine such as the donation of eggs and sperm for reproductive purposes , organs or tissues for transplantation , blood for transfusion , and other biological tissues for research .", "drawing analogies to existing practices in this way has proved helpful in other instances of bioethical novelty ( hofmann , solbakk , and holm 2006 ) .", "however , analogies can equally well be misleading , if there are morally relevant but unacknowledged differences between situations .", "the contexts in which different organs and tissues are donated vary significantly , and these differences influence the sociomoral understanding of donation in each case and make them noncomparable in ethical terms", ". reproductive tissue , for example , is generally distinguished from other types of donated tissue because eggs , sperm , and embryos have the potential to give rise to new individuals , not just to prolong the lives of existing individuals , or to be used for research . because embryos are generally considered to have a different moral status from other tissues , the use of surplus embryos in research raises moral unease about the instrumentalization of human life that is not raised in quite the same way by the donation of either ova or sperm .", "it is therefore unclear whether an embryo can meaningfully be treated as a  gift  (  donation  ) without blurring the morally relevant differences between embryos and other tissues .", "similarly ,  hesc research ,  to which the embryo may be given , is a domain of biomedical practices and not an entity . as such", ", hesc research is not a subject with which a gift relationship can be established ( unlike , for instance , an organ recipient ) .", "understanding the social and ethical meanings that are emerging for the practices associated with embryo donation calls for a detailed empirical examination of people s reasoning behind donation decisions .", "however , most empirical studies of embryo donation have not focused on people s donation rationales in depth ( with some exceptions ; for example , haimes et al . 2008 ; de lacey 2003 ) .", "the scarcity of data on donor rationales means there is a corresponding lack of information on contextual and other factors that influence donor decisions and how these can be related to the evaluations of theoretical ethics .", "the authors of this paper have been involved in a series of linked qualitative studies of practices of embryo donation , first in the united kingdom ( researcher haimes and colleagues ) , then switzerland ( scully , rehmann - sutter , and porz ) , and in a smaller pilot project in china ( mitzkat , rehmann - sutter , and haimes ) .", "all three studies shared an interest in understanding reasons for the donation or non - donation of embryos . while the studies in switzerland and china drew upon the original u.k .", "design , each study was conducted independently , and the details of each project , including interview design , differed in light of the varying regulatory , clinical , and cultural contexts . however , by looking across the three data sets , we hope to gain cross - cultural insights into donation and non - donation rationales and the moral understandings on which they are based .", "the three studies all used open - ended , semi - structured , one - off interviews between the researcher and people who had been in the position of deciding whether to donate their surplus embryos to research .", "interviews were designed to explore in depth not just the interviewees decision about donation but also the background to that decision , such as their ivf story , their family and other relationships , their relationship with the clinic and its staff , and so on .", "the interviews were transcribed , coded , and initially analysed to identify the reasons for donation decisions given by participants in each study , using an interpretative approach that all of us have previously found useful for identifying key themes around decision - making and implicit or explicit ethical judgements ( charmaz 2006 ; smith , flowers , and larkin 2009 ) .", "study ran for more than three years ; after some familiarisation and observation in the collaborating clinic , 44 in - depth interviews were conducted with couples who had been asked to donate their surplus fresh embryos , generated through ongoing ivf treatment , to hesc research ( haimes and taylor 2009 ) . in the united kingdom , it has been possible for some years to donate unused embryos to stem cell and other kinds of research and also to other couples .", "the swiss study was carried out shortly after a change in the law that permitted fresh or cryopreserved surplus ivf embryos , under strictly defined conditions , to be donated to stem cell research only . in this study , 17 individuals who had variously chosen to donate or not to donate were interviewed ; thus , the decision concerned the fate of cryopreserved embryos some time after the ivf treatment that had produced them ( scully , rehmann - sutter , and porz 2010 ) . the chinese work involved a much smaller three - month pilot study carried out in a large art hospital .", "it was designed as a pilot to provide a comparison with the u.k . and swiss material and is , therefore , included here despite the low number of participants , but with no attempt to draw general conclusions from it .", "legislation in china does not allow the donation of embryos to other couples , but consent can be given for a surplus embryo to be used in stem cell research or for it to be discarded .", "the study included participant observation of the information and consent procedures and qualitative interviews with five ivf patients , three choosing to donate to stem cell research and two refusing ( mitzkat , haimes , and rehmann - sutter 2010 ) .", "for the purposes of this paper , the authors involved in the studies jointly compared the rationales for donation decisions given by their participants .", "we also independently reexamined interview transcripts for material relevant to the discussion of gratitude . in the rest of this paper , we first identify and compare the main reasons given by participants in these three studies for their decisions to donate or to refuse to donate their surplus embryos to research .", "we then look in more detail at the implications of our findings for one area of potential ethical concern : the possible role of gratitude in making embryo disposition decisions . in this way", ", we not only collect empirical data to help understand the emerging moral meaning of a new practice , but also give an example to show how empirical data can be used to question and then refine the conceptual basis of bioethical theory .", "participants who chose to donate their surplus embryos to research had a background premise that donation is fundamentally permissible because embryos do not have the sort of ontological or moral status that would forbid it .", "the swiss study discovered people stating this explicitly :  [ embryos ] are not , they are not yet people ; they are nt beings with souls as far as i m concerned ,  as one swiss participant said.1 but this did not mean that the surplus embryos were considered as morally equivalent to any other tissue , and participants in both the swiss and u.k .", "interviewees stopped themselves midway through sentences referring to the  left over embryos ,  which they clearly felt , on reflection , was an inappropriate phrase .", "so the embryos did not have the sort of moral status that would have made it wrong to donate them , but neither were they completely disposable .", "to some of our participants , embryos had a value ; they were a precious resource ( haimes et al .", "2008 ) that made simply throwing them out an unjustifiable waste . as another u.k .", "[ i]t just really seemed a waste , really , not to have them used .", " it was not always clear , however , quite how interviewees were using this notion of  precious  : whether with reference to economic  bio - value  ( waldby 2000 ; waldby and mitchell 2006 ) or because they had been obtained at intense personal and emotional cost , or because they had high moral value , or possibly a combination of all these aspects", ". particularly in the swiss study , which involved cryopreserved embryos stored for some time , participants referred to embryos moral status in a nuanced way that was highly sensitive to the developmental stage of the embryo , its physical location ( whether inside or outside the body ) , and its state ( whether cryopreserved or not ) ( see scully , rehmann - sutter , and porz 2010 ) .", "studies seemed less interested in explicitly defining  the  moral status of an abstract embryo than working out what their own particular embryos meant for them at defined points in their own story , and what this meaning then indicated it was morally permissible for them to do with that embryo .", "across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research .", "such research was seen as a valuable endeavour by those like the swiss participant who said ,  i feel that , fundamentally , research has to go on , and i support that .", " donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k .", "interviewee said ,  it was quite simply that  we ve been helped by the system , we should help the system back.  not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress .  in the u.k .", "study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science : ", "i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it .", " this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here", "in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility .", "a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children .", " [ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research .", " note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as ", " one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland ,", "the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies", "were participants asked outright if they thought their donated embryo was being used in infertility research .", "however , the fact that they used phrases such as  helping others as we have been helped  ( italics added ) indicates a potential misunderstanding .", "it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation . the u.k .", "participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues .", "these participants , as in the chinese study , were current patients donating fresh embryos .", "the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k .", "study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing .", "it is possible that this level of engagement with the detail of the research underlies the difference .", "it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial . as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it .", "for example , one interviewee said :  if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it .", " i feel a little bit guilty sometimes , not very much , but a little bit like  if we can at least give these embryos for a good reason , then it was nt completely in vain .  using similar wording , another said ,  if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it .", " our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in  creating life  but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong .", "( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . )", "they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal .", "for these swiss participants , then , their donation decision appears to have been driven in part by what we could call a reparative urge .", "no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below .", "across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research .", "such research was seen as a valuable endeavour by those like the swiss participant who said ,  i feel that , fundamentally , research has to go on , and i support that .", " donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k .", "we ve been helped by the system , we should help the system back.  not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress .  in the u.k .", "study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science : ", "i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it .", " this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here", "in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility . for instance , one participant said , ", "a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children .", " [ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research .", " note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as  scientific research  for the ivf treatment . ", "one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland ,", "the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies", "were participants asked outright if they thought their donated embryo was being used in infertility research .", "however , the fact that they used phrases such as  helping others as we have been helped  ( italics added ) indicates a potential misunderstanding .", "it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation .", "participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues .", "these participants , as in the chinese study , were current patients donating fresh embryos .", "the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k .", "study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing .", "it is possible that this level of engagement with the detail of the research underlies the difference", ". it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial .", "as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it .", "for example , one interviewee said :  if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it .", " i feel a little bit guilty sometimes , not very much , but a little bit like  if we can at least give these embryos for a good reason , then it was nt completely in vain .  using similar wording , another said , ", "if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it .", " our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in  creating life  but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong .", "( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . )", "they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal . for these swiss participants ,", "then , their donation decision appears to have been driven in part by what we could call a reparative urge .", "no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below .", "approximately half of the swiss participants who were interviewed turned out to have decided against donation and instead have their surplus cryopreserved embryo(s ) destroyed .", "none of these explicitly used the moral status of the embryo as the fundamental basis for rejecting donation to research .", "the scientists  would do with their embryo , which was articulated either as disapproval of specific types of research ( e.g. ,  not if it s for cloning  ) or as a more general unwillingness to relinquish responsibility of the embryo to unknown others . in both sets of reasoning , therefore , participants were expressing unease about the loss of control over what would happen to their embryo .", "the two chinese interviewees who had opted against donation also did so because of reservations about lack of knowledge of exactly what the embryos would be used for , explicitly mentioning the fear that they would be donated to ( i.e. , used to generate pregnancy in ) other women .", "in addition , two of the swiss participants gave financial reasons , that is , they expressed anger that they would not be compensated for giving up their embryo and that through donation researchers were  getting something for nothing .", "study , speculation about why some people might refuse to donate raised concerns about what might be done with the embryos and whether a baby would be developed through experimentation ; others wondered if  refusers  were worried that another infertile couple would receive their embryos and have a baby when they , the embryo providers , failed to do so .", "sample contained only two actual refusers , who objected on the grounds of possible use in animal research ( haimes and taylor 2009 ; hug 2008 ) .", "these observations provide material for an empirically grounded bioethics of the ivf  stem cell interface ( franklin 2006 ) . in the rest of this paper , we consider a single aspect in detail : the potential  problem  of gratitude influencing informed consent .", "empirically , the main reasons given by our participants for donation were : ( 1 ) to avoid the waste of a precious resource , ( 2 ) to give something back to research , and ( 3 ) to compensate for a perceived moral wrong . in none of the three countries", "did our participants indicate that their decision arose out of any sense of gratitude to the physician who had given them ivf treatment .", "this is an important observation , because the possible impact of gratitude on informed consent in embryo donation has previously been raised by bioethical commentators .", "it has been suggested that if there is any institutional or procedural link between ivf treatment and hesc research , the potential donors gratitude toward the clinicians who have helped them to conceive may steer them toward opting for donation ( haimes and luce 2006 ; parry 2006 ; mcleod and baylis 2007 ; also discussed in roberts and throsby 2008 ) .", "the bioethical concern is that parents who receive the  gift of a baby  will feel a sense of gratitude ; feeling grateful might then cause parents to feel that they should respond with a gift in return ; and if a request for donation of surplus embryos is made in that context , potential donors might inadvertently be encouraged to see donation as an appropriate form of return gift . in this way", "the moral emotion of gratitude could compromise the potential donor s freedom to weigh the pros and cons of the request , which in turn means compromising the capacity to give fully voluntary consent .", "even where the clinicians and researchers themselves are scrupulous about not using persuasion , gratitude might be persuading potential donors to do something they would not otherwise choose to do . to avoid this possibility , countries that allow embryo donation for research may attempt a strict separation of ivf treatment and the stem cell research domain .", "( not all legislatures do this , however : china is one country that does not . ) in the united kingdom , for example , it is accepted good practice that requests for donation of spare embryos should not be made by the physician(s ) who delivered the original ivf treatment . in switzerland ,", "a predominant interpretation of the current law on embryo donation is that a couple undergoing ivf treatment should not be told even of the theoretical possibility that a surplus embryo might be generated unless it actually is ( porz et al .", "regulators hope to rule out any conflict of interest on the part of the treating physician as well as the possibility of gratitude on the part of the patient .", "there are several theoretical questions that can be asked about a claim that the  risk  of gratitude affects potential donors decisions .", "for example , moral psychologists might want to examine the emotional exceptionalism in which gratitude is seen as potentially compromising , while other emotions , or even the absence of emotion , is not .", "there is also an obvious empirical question which does not seem to have been closely examined : whether potential embryo donors actually do experience a sense of personal gratitude to the physician  or one strong enough to sway their donation decision .", "however , they do indicate that participants rationales for donation indicate a desire to  give back  in a more complex way .", "first , our empirical results show the importance of distinguishing between the three moral emotions of gratitude , indebtedness , and solidarity .", "what we normally mean by gratitude is an emotion primarily associated with gifts or with help that is undeserved .", "gratitude has been defined as  a feeling of thankful appreciation for favours received  ( guralnik 1971 , 327 ) and is experienced as a positive emotion .", "( 2006 ) provide a basis from experimental psychology for distinguishing between indebtedness and gratitude .", "one important distinction is that  indebtedness is an emotion of exchange , whereas gratitude is not  ( watkins et al .", "even if a debt of gratitude is felt ,  it does not appear to be analogous to an economic debt ", "indebtedness , however , is , literally , a debt : a  state of obligation to repay another  ( mauss 2001 , 2 ) .", "a further feature associated with indebtedness but not with gratitude is the inherent imbalance of power , so that a hierarchy is created in which those who are indebted are rendered more vulnerable . the empirical social psychological work of watkins et al .", "indicates that , in experimental settings at least , a person s feelings of gratitude diminish as the expectation of return ( indebtedness ) increases . the more reciprocity is expected or demanded ,", "importantly for considering the effects of these social emotions on donation decisions , watkins et al . found that the greater the expectation that something will be given back in return , the less motivated the participants were actually to comply with the norm of reciprocity ( watkins et al .", "these results suggest that if patients were to sense any expectation of return by an individual physician or researcher , it would if anything lessen any gratitude they might have felt . in the ivf context , what people have received ( the thing for which they might feel grateful ) is their baby or pregnancy , or at the very least , treatment . donors who achieve this have not received a gift , but a medical service , which will have been paid for in one way or another . in switzerland , where ivf is not covered by the mandatory health insurance", ", this will often be direct payment from the patients to the clinic , as it will also often be in china . in the united kingdom ,", "patients who do not already have children commonly receive two  free  cycles of nhs treatment ( paid for through taxes on the population ) , and then they pay for future cycles directly through fees to the clinics . whatever the system of payment , however , the point is that the ivf physician or clinic has already been paid , directly or indirectly , for the treatment which produced the outcome .", "so in the case of embryo donation , it could be argued that neither gratitude nor indebtedness should be anticipated .", "gratitude would not be expected , because what patients have received was not a favour or unexpected gift ; and indebtedness would not be expected , because there is no debt if payment has already been made . in our three studies , although we found appreciation and high esteem were expressed for the work of the treating hospital or team , we found no evidence of gratitude being expressed toward an individual physician .", "this is despite the fact that in both the united kingdom and china mention was made of the well - known head physicians who led each clinic ; the mentions were in appreciation of their reputations , not as a debt of gratitude .", "participants wanted to do something with the surplus embryo that would be of benefit to research and medical treatment for infertility , but this was not directly associated with the physician who provided it originally . indeed , some of our interviewees were critical of aspects of their own ivf treatment , while still wanting to support the infertility research enterprise overall . in this context , it should be remembered that about half the swiss participants interviewed in fact chose not to donate , primarily out of anxiety about losing control over the fate of their embryos , but also in some cases because it meant researchers were  getting something for nothing .", "study , a few patients were suspicious that research was being prioritised over treatment and that embryos might have been kept back for the research rather than frozen .", "although only a small number of participants expressed this view , it has particular relevance here as it indicates clearly the absence of either gratitude or indebtedness : indeed , it suggests that these participants felt that , if anything , donation would mean the researchers were receiving something beyond their entitlement or were indebted to the patients rather than the reverse .", "social exchanges may entail giving back not to an individual , because the individual may be unknowable or because the benefit may not have come from a single identifiable person , but to  the pattern of social life ", "( becker 1986)for example , giving back to the institutional biomedical research that had helped the participants . in serial reciprocity , person a receives something from person b , but pays back not to person b but to some other third party ( moody 2008 ) . serial reciprocity accounts for the way that altruistic blood donors often do describe their action in terms of reciprocity , even though they have not received anything from the person to whom their blood eventually goes .", "what drives serial reciprocity is not gratitude or indebtedness to an individual or an organisation but a sense of solidarity with unknown others in the community . in the case of embryo donation , what appears to be a directly reciprocal act of gratitude  a surplus embryo in return for treatment  may thus be understood as something quite different .", "participants in our studies used a rationale for donation based not on reciprocal exchange between individuals , but on  giving back to  a research enterprise from which they had benefited and which they hoped would benefit unknown others in the future . for the swiss and chinese donors who choose to donate because they think they are supporting infertility research , these unknown others are not completely anonymous : they are  known  because of imaginatively shared experience .  giving something back  to the research that had helped them ( as they think they are doing )", "was not considered an obligation but , rather , a form of return that was meaningful and seemed especially fitting to them because of the experiences they had gone through . in the u.k .", "study , although there was greater clarity about the purpose of the research being contributed to , there was still a sense among embryo providers that they were benefiting from the fact that others had participated in research in the past which had improved ivf ; they were clear in expressing solidarity with this imagined community of previous ivf research contributors by making their own contributions to research , albeit research in another domain .", "at least some of our participants , then , also see their donation to research as an indirect way of passing on the benefit they have received from research to someone else , whose situation in some way resembles their own .", "the social meaning of this indirect reciprocity is neither gratitude nor indebtedness , but solidarity with other present and future patients .", "to summarise : our interpretation of the material suggests that donors reasons for donating were not connected in any straightforward way to either gratitude or indebtedness . for some people", "the desire not to waste a valuable ( in more than one sense ) resource is foremost .", "a generally positive stance toward biomedical science means that donation is a good use of a valuable resource . with some of the swiss participants", ", we also identified a reparative urge coming from a sense of moral unease , and here donation seems to offer a route through which the urge to make reparation can be satisfied .", "all of this suggests that neither gratitude nor indebtedness per se are present in decision - making at the ivf  stem cell interface , at least not in terms of undue inducement or of compromising the capacity for informed consent .", "the reparative urge foregrounds a different set of ethical questions about the sociomoral meaning of the generation of spare embryos and the act of donation .", "for example , the perceived need for reparation that appears to drive some of the swiss participants donation choices arises because people felt some sense of wrongdoing at creating embryos that are not used for pregnancy .", "the spirit of the swiss legal framework strongly reinforces the sense that the occurrence of embryos not used for pregnancy is to be considered as wrong . from this point of view", ", one possible conclusion could be that the felt desire for reparation is in fact an ethically appropriate response : if the act of deliberately creating an embryo is a morally significant one , which needs to be justified by the good of its intended outcome ( pregnancy ) , then not using it for the purpose that justifies it does indeed present a genuine ethical difficulty .", "on the other hand , the novelty of surplus embryos in the swiss legal and social context ( scully and rehmann - sutter 2006 ) meant that many patients would have been unaware at the time of their ivf of the possibility of generating a surplus embryo .", "moreover , in most cases there were perfectly valid clinical or legal reasons why the embryos could not be transferred , which fully justified the failure to use them as originally intended . that these factors were not in the participants control", "the swiss participants were quite aware of the valid reasons why their embryos could not be transferred , and yet some of them still said that they had fallen short of some moral standard .", "we suggest that this persistent unease ( which does not appear so prevalent in the u.k .", "these are captured in swiss legislation s highly defensive attitude toward embryos in general and the generation of surplus embryos in particular .", "swiss law operates from the presumption that a surplus embryo is an exceptional event that will happen only through the failure of regulation and practice specifically set up to prevent it ( porz et al .", "in addition , as we discussed earlier , the generation of surplus embryos is still an unfamiliar social practice and the role of the embryo donor is one that lacks widespread cultural recognition . in the swiss context of legal exceptionalism , then , producing one of these culturally unfamiliar entities may more readily be understood as something ", "wrong  for which reparation is in order . in the more permissive cultural and legislative context of the united kingdom , although some interviewees expressed guilt about other aspects of the process ( for example , inappropriate styles of speech about embryos ) , there was no sense that not using the embryos as intended in itself constituted a moral failing .", "if our interpretation is correct , as the creation of surplus embryos becomes normalised in swiss society and the role of embryo donor becomes normalised in both the u.k . and", "swiss ( and other ) societies , there will be a shift in ideas about the moral meaning of surplus embryos , and this should be empirically testable . for the majority of the participants in switzerland and the united kingdom who chose to donate their surplus embryos , donation decisions were not driven by a reparative urge but by the feeling that donation expressed solidarity with research and/or other patients .", "what our empirical work shows is that gratitude to an individual must be distinguished from a solidarity - based desire to  give something back  to medical research .", "however , this does not necessarily mean that solidarity - based reciprocity has no relevant impact on the capacity for voluntary consent .", "one possible consequence , for example , is that if potential donors feel solidarity with the research enterprise , or with present and future patients , then they might also experience an obligation to support it .", "however , in all three studies there were people who chose not to donate ( about half of the swiss interviewees did not donate , while in the united kingdom approximately 46 percent of those asked have been shown to opt against donation [ choudhary et al .", "2004 ] ) , which suggests either that there is no sense of obligation , or that it is neither universal nor irresistible .", "there is a second problem if solidarity - based reciprocity is taken as something to be prevented .", "if there is a risk that gratitude felt by an individual could influence donation decisions , then the risk can be minimised by separating ivf and stem cell research physically and procedurally , as bioethicists have suggested and some clinics and guidelines have implemented .", "but if instead there is a desire to benefit research or to show solidarity with collective  others  rather than an individual , then what sort of physical or procedural barriers could be set up to prevent it having an influence", "? there are further theoretical questions to be explored here about the assumption that contextual emotions such as gratitude or solidarity compromise the decision to donate .", "the situation in which donation of a surplus embryo is a possibility comes about as a result of a variety of social , cultural , political , legal , and emotional features that combine to make the situation what it is .", "it will be important to identify carefully the features that are constitutive of the situation of having a surplus embryo ( including a sense of solidarity to similar others , emotional bonds with the embryo that change over time , regret over the failure to use all embryos for pregnancy , and so on ) and to consider whether these constitutive features can or should also be treated as factors that compromise the responsible , autonomous decision - making capacity of the people involved .", "this introduces the question of the extent to which any decision to donate can be detached from its context and the contextual factors that shape how people respond to donation requests .", "finally , we should not forget that the possibility that some donors misunderstand the kind of research they are donating to raises some ethical difficulty .", "it means that donors may be deciding for donation out of a misplaced sense of solidarity  a different ethical problem .", "the deliberative processes of the donor participants in our three studies incorporated their appreciation of the particular moral value of their own embryos and of the value of biomedical research .", "these deliberations are complex , and the participants own dependency on pre - existing medical research and their affiliation with others in similar situations must be recognised , we suggest , in order to understand how they might configure donation as a means to cope with the moral ambivalence of the situation in which they find themselves .", "their decisions are not driven by some unacknowledged sense of debt , but rather are responsive to the emerging social and moral reality of embryo donation in the first decade of the 21st century ."], "abstract_text": ["<S> this paper is based on linked qualitative studies of the donation of human embryos to stem cell research carried out in the united kingdom , switzerland , and china . </S>", "<S> all three studies used semi - structured interview protocols to allow an in - depth examination of donors and non - donors rationales for their donation decisions , with the aim of gaining information on contextual and other factors that play a role in donor decisions and identifying how these relate to factors that are more usually included in evaluations made by theoretical ethics . </S>", "<S> our findings have implications for one factor that has previously been suggested as being of ethical concern : the role of gratitude . </S>", "<S> our empirical work shows no evidence that interpersonal gratitude is an important factor , but it does support the existence of a solidarity - based desire to  give something back  to medical research . </S>", "<S> thus , we use empirical data to expand and refine the conceptual basis of bioethically theorizing the ivf  stem cell interface . </S>"], "labels": null, "section_names": ["Embryo Donation as a Sociomoral Practice", "Empirical Exploration of Donation Decisions", "Reasons for Donating", "Contribution to Research", "Reparation", "Reasons Not to Donate", "The Problem of Gratitude", "Concluding Comments on Reparation, Solidarity, and Consent"], "sections": [["embryos generated through assisted reproductive technologies ( art ) are deemed surplus when , for some reason , they are not used for reproductive purposes ( svendsen and koch 2008 ; haimes and taylor 2011 ) ; instead , they are then disposed of or , where this is allowed by a country s art regulation , may be donated for a variety of research purposes .", "thus , the generation and subsequent fate of embryos that are designated surplus are determined as much by legislation as by embryo biology or technological constraints .", "for example , legislatures differ in whether cryopreservation of embryos is permitted , meaning that in some countries any ivf embryos not immediately transferred for pregnancy are surplus , while in others they may be frozen for later transfer . in some countries , the law considers surplus embryos as the primary source of material for human embryonic stem cell ( hesc ) research , while in others this use of embryos is expressly forbidden .", "although hesc research has been subjected to exhaustive ethical scrutiny , the lack of consensus on the ontological or ethical status of surplus embryos means that the uses to which they may then be put remain contested ( deckers 2007 ; guenin 2008 ; moller 2009 ) .", "even if using human embryos for stem cell research is deemed permissible , ethical issues continue to emerge as new social practices and roles develop around the act of embryo donation .", "the relative novelty of these practices and roles , complicated by the pace of change in technical possibility and regulation , mean that there is still uncertainty about how to conceptualise embryo donation , not just in terms of systematic ethics but as a sociomoral practice in everyday social life and morality ( banks , scully , and shakespeare 2006 ) . from a sociological perspective , and also to understand more generally how everyday morality deals with unprecedented ethical dilemmas , it is important to examine which conceptualisations emerge as salient and how they are stabilised and used .", "for example , one way of thinking about the donation of embryos to research is as a bodily gift relationship , one of the many established by modern biomedicine such as the donation of eggs and sperm for reproductive purposes , organs or tissues for transplantation , blood for transfusion , and other biological tissues for research .", "drawing analogies to existing practices in this way has proved helpful in other instances of bioethical novelty ( hofmann , solbakk , and holm 2006 ) .", "however , analogies can equally well be misleading , if there are morally relevant but unacknowledged differences between situations .", "the contexts in which different organs and tissues are donated vary significantly , and these differences influence the sociomoral understanding of donation in each case and make them noncomparable in ethical terms", ". reproductive tissue , for example , is generally distinguished from other types of donated tissue because eggs , sperm , and embryos have the potential to give rise to new individuals , not just to prolong the lives of existing individuals , or to be used for research . because embryos are generally considered to have a different moral status from other tissues , the use of surplus embryos in research raises moral unease about the instrumentalization of human life that is not raised in quite the same way by the donation of either ova or sperm .", "it is therefore unclear whether an embryo can meaningfully be treated as a  gift  (  donation  ) without blurring the morally relevant differences between embryos and other tissues .", "similarly ,  hesc research ,  to which the embryo may be given , is a domain of biomedical practices and not an entity . as such", ", hesc research is not a subject with which a gift relationship can be established ( unlike , for instance , an organ recipient ) ."], ["understanding the social and ethical meanings that are emerging for the practices associated with embryo donation calls for a detailed empirical examination of people s reasoning behind donation decisions .", "however , most empirical studies of embryo donation have not focused on people s donation rationales in depth ( with some exceptions ; for example , haimes et al . 2008 ; de lacey 2003 ) .", "the scarcity of data on donor rationales means there is a corresponding lack of information on contextual and other factors that influence donor decisions and how these can be related to the evaluations of theoretical ethics .", "the authors of this paper have been involved in a series of linked qualitative studies of practices of embryo donation , first in the united kingdom ( researcher haimes and colleagues ) , then switzerland ( scully , rehmann - sutter , and porz ) , and in a smaller pilot project in china ( mitzkat , rehmann - sutter , and haimes ) .", "all three studies shared an interest in understanding reasons for the donation or non - donation of embryos . while the studies in switzerland and china drew upon the original u.k .", "design , each study was conducted independently , and the details of each project , including interview design , differed in light of the varying regulatory , clinical , and cultural contexts . however , by looking across the three data sets , we hope to gain cross - cultural insights into donation and non - donation rationales and the moral understandings on which they are based .", "the three studies all used open - ended , semi - structured , one - off interviews between the researcher and people who had been in the position of deciding whether to donate their surplus embryos to research .", "interviews were designed to explore in depth not just the interviewees decision about donation but also the background to that decision , such as their ivf story , their family and other relationships , their relationship with the clinic and its staff , and so on .", "the interviews were transcribed , coded , and initially analysed to identify the reasons for donation decisions given by participants in each study , using an interpretative approach that all of us have previously found useful for identifying key themes around decision - making and implicit or explicit ethical judgements ( charmaz 2006 ; smith , flowers , and larkin 2009 ) .", "study ran for more than three years ; after some familiarisation and observation in the collaborating clinic , 44 in - depth interviews were conducted with couples who had been asked to donate their surplus fresh embryos , generated through ongoing ivf treatment , to hesc research ( haimes and taylor 2009 ) . in the united kingdom , it has been possible for some years to donate unused embryos to stem cell and other kinds of research and also to other couples .", "the swiss study was carried out shortly after a change in the law that permitted fresh or cryopreserved surplus ivf embryos , under strictly defined conditions , to be donated to stem cell research only . in this study , 17 individuals who had variously chosen to donate or not to donate were interviewed ; thus , the decision concerned the fate of cryopreserved embryos some time after the ivf treatment that had produced them ( scully , rehmann - sutter , and porz 2010 ) . the chinese work involved a much smaller three - month pilot study carried out in a large art hospital .", "it was designed as a pilot to provide a comparison with the u.k . and swiss material and is , therefore , included here despite the low number of participants , but with no attempt to draw general conclusions from it .", "legislation in china does not allow the donation of embryos to other couples , but consent can be given for a surplus embryo to be used in stem cell research or for it to be discarded .", "the study included participant observation of the information and consent procedures and qualitative interviews with five ivf patients , three choosing to donate to stem cell research and two refusing ( mitzkat , haimes , and rehmann - sutter 2010 ) .", "for the purposes of this paper , the authors involved in the studies jointly compared the rationales for donation decisions given by their participants .", "we also independently reexamined interview transcripts for material relevant to the discussion of gratitude . in the rest of this paper , we first identify and compare the main reasons given by participants in these three studies for their decisions to donate or to refuse to donate their surplus embryos to research .", "we then look in more detail at the implications of our findings for one area of potential ethical concern : the possible role of gratitude in making embryo disposition decisions . in this way", ", we not only collect empirical data to help understand the emerging moral meaning of a new practice , but also give an example to show how empirical data can be used to question and then refine the conceptual basis of bioethical theory ."], ["participants who chose to donate their surplus embryos to research had a background premise that donation is fundamentally permissible because embryos do not have the sort of ontological or moral status that would forbid it .", "the swiss study discovered people stating this explicitly :  [ embryos ] are not , they are not yet people ; they are nt beings with souls as far as i m concerned ,  as one swiss participant said.1 but this did not mean that the surplus embryos were considered as morally equivalent to any other tissue , and participants in both the swiss and u.k .", "interviewees stopped themselves midway through sentences referring to the  left over embryos ,  which they clearly felt , on reflection , was an inappropriate phrase .", "so the embryos did not have the sort of moral status that would have made it wrong to donate them , but neither were they completely disposable .", "to some of our participants , embryos had a value ; they were a precious resource ( haimes et al .", "2008 ) that made simply throwing them out an unjustifiable waste . as another u.k .", "[ i]t just really seemed a waste , really , not to have them used .", " it was not always clear , however , quite how interviewees were using this notion of  precious  : whether with reference to economic  bio - value  ( waldby 2000 ; waldby and mitchell 2006 ) or because they had been obtained at intense personal and emotional cost , or because they had high moral value , or possibly a combination of all these aspects", ". particularly in the swiss study , which involved cryopreserved embryos stored for some time , participants referred to embryos moral status in a nuanced way that was highly sensitive to the developmental stage of the embryo , its physical location ( whether inside or outside the body ) , and its state ( whether cryopreserved or not ) ( see scully , rehmann - sutter , and porz 2010 ) .", "studies seemed less interested in explicitly defining  the  moral status of an abstract embryo than working out what their own particular embryos meant for them at defined points in their own story , and what this meaning then indicated it was morally permissible for them to do with that embryo .", "across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research .", "such research was seen as a valuable endeavour by those like the swiss participant who said ,  i feel that , fundamentally , research has to go on , and i support that .", " donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k .", "interviewee said ,  it was quite simply that  we ve been helped by the system , we should help the system back.  not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress .  in the u.k .", "study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science : ", "i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it .", " this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here", "in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility .", "a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children .", " [ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research .", " note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as ", " one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland ,", "the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies", "were participants asked outright if they thought their donated embryo was being used in infertility research .", "however , the fact that they used phrases such as  helping others as we have been helped  ( italics added ) indicates a potential misunderstanding .", "it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation . the u.k .", "participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues .", "these participants , as in the chinese study , were current patients donating fresh embryos .", "the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k .", "study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing .", "it is possible that this level of engagement with the detail of the research underlies the difference .", "it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial . as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it .", "for example , one interviewee said :  if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it .", " i feel a little bit guilty sometimes , not very much , but a little bit like  if we can at least give these embryos for a good reason , then it was nt completely in vain .  using similar wording , another said ,  if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it .", " our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in  creating life  but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong .", "( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . )", "they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal .", "for these swiss participants , then , their donation decision appears to have been driven in part by what we could call a reparative urge .", "no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below ."], ["across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research .", "such research was seen as a valuable endeavour by those like the swiss participant who said ,  i feel that , fundamentally , research has to go on , and i support that .", " donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k .", "we ve been helped by the system , we should help the system back.  not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress .  in the u.k .", "study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science : ", "i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it .", " this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here", "in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility . for instance , one participant said , ", "a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children .", " [ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research .", " note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as  scientific research  for the ivf treatment . ", "one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland ,", "the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies", "were participants asked outright if they thought their donated embryo was being used in infertility research .", "however , the fact that they used phrases such as  helping others as we have been helped  ( italics added ) indicates a potential misunderstanding .", "it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation .", "participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues .", "these participants , as in the chinese study , were current patients donating fresh embryos .", "the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k .", "study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing .", "it is possible that this level of engagement with the detail of the research underlies the difference", ". it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial ."], ["as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it .", "for example , one interviewee said :  if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it .", " i feel a little bit guilty sometimes , not very much , but a little bit like  if we can at least give these embryos for a good reason , then it was nt completely in vain .  using similar wording , another said , ", "if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it .", " our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in  creating life  but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong .", "( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . )", "they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal . for these swiss participants ,", "then , their donation decision appears to have been driven in part by what we could call a reparative urge .", "no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below ."], ["approximately half of the swiss participants who were interviewed turned out to have decided against donation and instead have their surplus cryopreserved embryo(s ) destroyed .", "none of these explicitly used the moral status of the embryo as the fundamental basis for rejecting donation to research .", "the scientists  would do with their embryo , which was articulated either as disapproval of specific types of research ( e.g. ,  not if it s for cloning  ) or as a more general unwillingness to relinquish responsibility of the embryo to unknown others . in both sets of reasoning , therefore , participants were expressing unease about the loss of control over what would happen to their embryo .", "the two chinese interviewees who had opted against donation also did so because of reservations about lack of knowledge of exactly what the embryos would be used for , explicitly mentioning the fear that they would be donated to ( i.e. , used to generate pregnancy in ) other women .", "in addition , two of the swiss participants gave financial reasons , that is , they expressed anger that they would not be compensated for giving up their embryo and that through donation researchers were  getting something for nothing .", "study , speculation about why some people might refuse to donate raised concerns about what might be done with the embryos and whether a baby would be developed through experimentation ; others wondered if  refusers  were worried that another infertile couple would receive their embryos and have a baby when they , the embryo providers , failed to do so .", "sample contained only two actual refusers , who objected on the grounds of possible use in animal research ( haimes and taylor 2009 ; hug 2008 ) ."], ["these observations provide material for an empirically grounded bioethics of the ivf  stem cell interface ( franklin 2006 ) . in the rest of this paper , we consider a single aspect in detail : the potential  problem  of gratitude influencing informed consent .", "empirically , the main reasons given by our participants for donation were : ( 1 ) to avoid the waste of a precious resource , ( 2 ) to give something back to research , and ( 3 ) to compensate for a perceived moral wrong . in none of the three countries", "did our participants indicate that their decision arose out of any sense of gratitude to the physician who had given them ivf treatment .", "this is an important observation , because the possible impact of gratitude on informed consent in embryo donation has previously been raised by bioethical commentators .", "it has been suggested that if there is any institutional or procedural link between ivf treatment and hesc research , the potential donors gratitude toward the clinicians who have helped them to conceive may steer them toward opting for donation ( haimes and luce 2006 ; parry 2006 ; mcleod and baylis 2007 ; also discussed in roberts and throsby 2008 ) .", "the bioethical concern is that parents who receive the  gift of a baby  will feel a sense of gratitude ; feeling grateful might then cause parents to feel that they should respond with a gift in return ; and if a request for donation of surplus embryos is made in that context , potential donors might inadvertently be encouraged to see donation as an appropriate form of return gift . in this way", "the moral emotion of gratitude could compromise the potential donor s freedom to weigh the pros and cons of the request , which in turn means compromising the capacity to give fully voluntary consent .", "even where the clinicians and researchers themselves are scrupulous about not using persuasion , gratitude might be persuading potential donors to do something they would not otherwise choose to do . to avoid this possibility , countries that allow embryo donation for research may attempt a strict separation of ivf treatment and the stem cell research domain .", "( not all legislatures do this , however : china is one country that does not . ) in the united kingdom , for example , it is accepted good practice that requests for donation of spare embryos should not be made by the physician(s ) who delivered the original ivf treatment . in switzerland ,", "a predominant interpretation of the current law on embryo donation is that a couple undergoing ivf treatment should not be told even of the theoretical possibility that a surplus embryo might be generated unless it actually is ( porz et al .", "regulators hope to rule out any conflict of interest on the part of the treating physician as well as the possibility of gratitude on the part of the patient .", "there are several theoretical questions that can be asked about a claim that the  risk  of gratitude affects potential donors decisions .", "for example , moral psychologists might want to examine the emotional exceptionalism in which gratitude is seen as potentially compromising , while other emotions , or even the absence of emotion , is not .", "there is also an obvious empirical question which does not seem to have been closely examined : whether potential embryo donors actually do experience a sense of personal gratitude to the physician  or one strong enough to sway their donation decision .", "however , they do indicate that participants rationales for donation indicate a desire to  give back  in a more complex way .", "first , our empirical results show the importance of distinguishing between the three moral emotions of gratitude , indebtedness , and solidarity .", "what we normally mean by gratitude is an emotion primarily associated with gifts or with help that is undeserved .", "gratitude has been defined as  a feeling of thankful appreciation for favours received  ( guralnik 1971 , 327 ) and is experienced as a positive emotion .", "( 2006 ) provide a basis from experimental psychology for distinguishing between indebtedness and gratitude .", "one important distinction is that  indebtedness is an emotion of exchange , whereas gratitude is not  ( watkins et al .", "even if a debt of gratitude is felt ,  it does not appear to be analogous to an economic debt ", "indebtedness , however , is , literally , a debt : a  state of obligation to repay another  ( mauss 2001 , 2 ) .", "a further feature associated with indebtedness but not with gratitude is the inherent imbalance of power , so that a hierarchy is created in which those who are indebted are rendered more vulnerable . the empirical social psychological work of watkins et al .", "indicates that , in experimental settings at least , a person s feelings of gratitude diminish as the expectation of return ( indebtedness ) increases . the more reciprocity is expected or demanded ,", "importantly for considering the effects of these social emotions on donation decisions , watkins et al . found that the greater the expectation that something will be given back in return , the less motivated the participants were actually to comply with the norm of reciprocity ( watkins et al .", "these results suggest that if patients were to sense any expectation of return by an individual physician or researcher , it would if anything lessen any gratitude they might have felt . in the ivf context , what people have received ( the thing for which they might feel grateful ) is their baby or pregnancy , or at the very least , treatment . donors who achieve this have not received a gift , but a medical service , which will have been paid for in one way or another . in switzerland , where ivf is not covered by the mandatory health insurance", ", this will often be direct payment from the patients to the clinic , as it will also often be in china . in the united kingdom ,", "patients who do not already have children commonly receive two  free  cycles of nhs treatment ( paid for through taxes on the population ) , and then they pay for future cycles directly through fees to the clinics . whatever the system of payment , however , the point is that the ivf physician or clinic has already been paid , directly or indirectly , for the treatment which produced the outcome .", "so in the case of embryo donation , it could be argued that neither gratitude nor indebtedness should be anticipated .", "gratitude would not be expected , because what patients have received was not a favour or unexpected gift ; and indebtedness would not be expected , because there is no debt if payment has already been made . in our three studies , although we found appreciation and high esteem were expressed for the work of the treating hospital or team , we found no evidence of gratitude being expressed toward an individual physician .", "this is despite the fact that in both the united kingdom and china mention was made of the well - known head physicians who led each clinic ; the mentions were in appreciation of their reputations , not as a debt of gratitude .", "participants wanted to do something with the surplus embryo that would be of benefit to research and medical treatment for infertility , but this was not directly associated with the physician who provided it originally . indeed , some of our interviewees were critical of aspects of their own ivf treatment , while still wanting to support the infertility research enterprise overall . in this context , it should be remembered that about half the swiss participants interviewed in fact chose not to donate , primarily out of anxiety about losing control over the fate of their embryos , but also in some cases because it meant researchers were  getting something for nothing .", "study , a few patients were suspicious that research was being prioritised over treatment and that embryos might have been kept back for the research rather than frozen .", "although only a small number of participants expressed this view , it has particular relevance here as it indicates clearly the absence of either gratitude or indebtedness : indeed , it suggests that these participants felt that , if anything , donation would mean the researchers were receiving something beyond their entitlement or were indebted to the patients rather than the reverse .", "social exchanges may entail giving back not to an individual , because the individual may be unknowable or because the benefit may not have come from a single identifiable person , but to  the pattern of social life ", "( becker 1986)for example , giving back to the institutional biomedical research that had helped the participants . in serial reciprocity , person a receives something from person b , but pays back not to person b but to some other third party ( moody 2008 ) . serial reciprocity accounts for the way that altruistic blood donors often do describe their action in terms of reciprocity , even though they have not received anything from the person to whom their blood eventually goes .", "what drives serial reciprocity is not gratitude or indebtedness to an individual or an organisation but a sense of solidarity with unknown others in the community . in the case of embryo donation , what appears to be a directly reciprocal act of gratitude  a surplus embryo in return for treatment  may thus be understood as something quite different .", "participants in our studies used a rationale for donation based not on reciprocal exchange between individuals , but on  giving back to  a research enterprise from which they had benefited and which they hoped would benefit unknown others in the future . for the swiss and chinese donors who choose to donate because they think they are supporting infertility research , these unknown others are not completely anonymous : they are  known  because of imaginatively shared experience .  giving something back  to the research that had helped them ( as they think they are doing )", "was not considered an obligation but , rather , a form of return that was meaningful and seemed especially fitting to them because of the experiences they had gone through . in the u.k .", "study , although there was greater clarity about the purpose of the research being contributed to , there was still a sense among embryo providers that they were benefiting from the fact that others had participated in research in the past which had improved ivf ; they were clear in expressing solidarity with this imagined community of previous ivf research contributors by making their own contributions to research , albeit research in another domain .", "at least some of our participants , then , also see their donation to research as an indirect way of passing on the benefit they have received from research to someone else , whose situation in some way resembles their own .", "the social meaning of this indirect reciprocity is neither gratitude nor indebtedness , but solidarity with other present and future patients ."], ["to summarise : our interpretation of the material suggests that donors reasons for donating were not connected in any straightforward way to either gratitude or indebtedness . for some people", "the desire not to waste a valuable ( in more than one sense ) resource is foremost .", "a generally positive stance toward biomedical science means that donation is a good use of a valuable resource . with some of the swiss participants", ", we also identified a reparative urge coming from a sense of moral unease , and here donation seems to offer a route through which the urge to make reparation can be satisfied .", "all of this suggests that neither gratitude nor indebtedness per se are present in decision - making at the ivf  stem cell interface , at least not in terms of undue inducement or of compromising the capacity for informed consent .", "the reparative urge foregrounds a different set of ethical questions about the sociomoral meaning of the generation of spare embryos and the act of donation .", "for example , the perceived need for reparation that appears to drive some of the swiss participants donation choices arises because people felt some sense of wrongdoing at creating embryos that are not used for pregnancy .", "the spirit of the swiss legal framework strongly reinforces the sense that the occurrence of embryos not used for pregnancy is to be considered as wrong . from this point of view", ", one possible conclusion could be that the felt desire for reparation is in fact an ethically appropriate response : if the act of deliberately creating an embryo is a morally significant one , which needs to be justified by the good of its intended outcome ( pregnancy ) , then not using it for the purpose that justifies it does indeed present a genuine ethical difficulty .", "on the other hand , the novelty of surplus embryos in the swiss legal and social context ( scully and rehmann - sutter 2006 ) meant that many patients would have been unaware at the time of their ivf of the possibility of generating a surplus embryo .", "moreover , in most cases there were perfectly valid clinical or legal reasons why the embryos could not be transferred , which fully justified the failure to use them as originally intended . that these factors were not in the participants control", "the swiss participants were quite aware of the valid reasons why their embryos could not be transferred , and yet some of them still said that they had fallen short of some moral standard .", "we suggest that this persistent unease ( which does not appear so prevalent in the u.k .", "these are captured in swiss legislation s highly defensive attitude toward embryos in general and the generation of surplus embryos in particular .", "swiss law operates from the presumption that a surplus embryo is an exceptional event that will happen only through the failure of regulation and practice specifically set up to prevent it ( porz et al .", "in addition , as we discussed earlier , the generation of surplus embryos is still an unfamiliar social practice and the role of the embryo donor is one that lacks widespread cultural recognition . in the swiss context of legal exceptionalism , then , producing one of these culturally unfamiliar entities may more readily be understood as something ", "wrong  for which reparation is in order . in the more permissive cultural and legislative context of the united kingdom , although some interviewees expressed guilt about other aspects of the process ( for example , inappropriate styles of speech about embryos ) , there was no sense that not using the embryos as intended in itself constituted a moral failing .", "if our interpretation is correct , as the creation of surplus embryos becomes normalised in swiss society and the role of embryo donor becomes normalised in both the u.k . and", "swiss ( and other ) societies , there will be a shift in ideas about the moral meaning of surplus embryos , and this should be empirically testable . for the majority of the participants in switzerland and the united kingdom who chose to donate their surplus embryos , donation decisions were not driven by a reparative urge but by the feeling that donation expressed solidarity with research and/or other patients .", "what our empirical work shows is that gratitude to an individual must be distinguished from a solidarity - based desire to  give something back  to medical research .", "however , this does not necessarily mean that solidarity - based reciprocity has no relevant impact on the capacity for voluntary consent .", "one possible consequence , for example , is that if potential donors feel solidarity with the research enterprise , or with present and future patients , then they might also experience an obligation to support it .", "however , in all three studies there were people who chose not to donate ( about half of the swiss interviewees did not donate , while in the united kingdom approximately 46 percent of those asked have been shown to opt against donation [ choudhary et al .", "2004 ] ) , which suggests either that there is no sense of obligation , or that it is neither universal nor irresistible .", "there is a second problem if solidarity - based reciprocity is taken as something to be prevented .", "if there is a risk that gratitude felt by an individual could influence donation decisions , then the risk can be minimised by separating ivf and stem cell research physically and procedurally , as bioethicists have suggested and some clinics and guidelines have implemented .", "but if instead there is a desire to benefit research or to show solidarity with collective  others  rather than an individual , then what sort of physical or procedural barriers could be set up to prevent it having an influence", "? there are further theoretical questions to be explored here about the assumption that contextual emotions such as gratitude or solidarity compromise the decision to donate .", "the situation in which donation of a surplus embryo is a possibility comes about as a result of a variety of social , cultural , political , legal , and emotional features that combine to make the situation what it is .", "it will be important to identify carefully the features that are constitutive of the situation of having a surplus embryo ( including a sense of solidarity to similar others , emotional bonds with the embryo that change over time , regret over the failure to use all embryos for pregnancy , and so on ) and to consider whether these constitutive features can or should also be treated as factors that compromise the responsible , autonomous decision - making capacity of the people involved .", "this introduces the question of the extent to which any decision to donate can be detached from its context and the contextual factors that shape how people respond to donation requests .", "finally , we should not forget that the possibility that some donors misunderstand the kind of research they are donating to raises some ethical difficulty .", "it means that donors may be deciding for donation out of a misplaced sense of solidarity  a different ethical problem .", "the deliberative processes of the donor participants in our three studies incorporated their appreciation of the particular moral value of their own embryos and of the value of biomedical research .", "these deliberations are complex , and the participants own dependency on pre - existing medical research and their affiliation with others in similar situations must be recognised , we suggest , in order to understand how they might configure donation as a means to cope with the moral ambivalence of the situation in which they find themselves .", "their decisions are not driven by some unacknowledged sense of debt , but rather are responsive to the emerging social and moral reality of embryo donation in the first decade of the 21st century ."]]}
{"article_id": "PMC4803041", "article_text": ["thyroid cancer is the most common endocrine neoplasia which accounts for about 1% of human cancers .", "mtc originates from calcitonin - producing cells ( c - cells ) of the thyroid gland and accounts for 35% of thyroid cancers .", "mtc is relatively slow - growing tumor but , if metastasized or relapsed , it becomes very aggressive causing more than 13% of all thyroid cancer - related mortality . in the united states ,", "mtc has an incidence in children of 0.03 per 100 000 population per year with a fairly equal female to male ratio [ 4 , 5 ] .", "mtc occurs either sporadically or in an inherited autosomal dominant manner . in adults , sporadic mtc accounts for 6575% of mtc , but in children ,", "sporadic mtc is very rare ; the vast majority of mtc diagnosed in the childhood is hereditary .", "hereditary mtc occurs as a familial mtc ( fmtc ) or as a part of multiple endocrine neoplasia ( men ) type 2a and b syndromes , wherein other endocrine glands are also affected .", "mtc diagnosed during childhood almost always results from a dominantly inherited or de novo activating mutations in the ret proto - oncogene , which encodes the ret receptor tyrosine kinase [ 69 ] .", "advances in predictive genetic testing for ret mutations have enabled early diagnosis of hereditary men syndromes and prophylactic thyroidectomy in presymptomatic patients to prevent mtc .", "the early onset of mtc in hereditary syndromes makes it an important endocrine disease that is increasingly managed by pediatric providers [ 1012 ] . in this review", ", we discuss the etiology of pediatric mtc and currently available therapeutic modality for the cancer .", "ret encodes a receptor tyrosine - kinase which is expressed in the neural crest - derived cell types , including thyroid parafollicular cells , neuronal cells , and adrenal medullary chromaffin cells . in these cell types", ", ret plays a central role in regulating cell proliferation , growth , differentiation , migration and survival . in humans", "after alternative splicing at the 3 end , ret transcripts encode three protein isoforms with distinct c - terminal ends that contain either 9 ( ret9 ) , 51 ( ret51 ) , or 43 ( ret43 ) amino acids .", "ret exon 19 is present in all transcripts and its differential splicing at the 3 end produces distinct transcripts wherein exon 19 is either unspliced , spliced to exon 20 , or spliced to exon 21 .", "all three resulting ret isoforms commonly contain a tyrosine ( tyr1062 ) whose phosphorylation is critical for their activation .", "the major ret isoforms in vivo are ret9 and ret51 , which consist of 1072 and 1114 amino acids , respectively , and are usually co - expressed .", "ret consists of an extracellular ligand binding domain , a trans - membrane domain , and an intracellular kinase domain ( figure 1 ) .", "the extracellular domain includes four cadherin - like repeats and a highly conserved cysteine - rich region , which is located near the cell membrane .", "the intracellular domain consists of two tyrosine - kinase subdomains , tk1 and tk2 , which contain multiple tyrosine residues that are phosphorylated during receptor activation and are required for the activation of different downstream signaling pathways of ret [ 19 , 20 ] .", "the ligands for ret are the glial cell line - derived neurotrophic factor ( gdnf ) family proteins , including gdnf , neurturin , artemin , and perseptin .", "activation of ret also requires the formation of a heterodimeric complex recruiting a gdnf - family receptor alpha ( gfr ) . when unbound by a ligand , ret is monomeric , unphosphorylated , and inactive .", "when a ligand and the gfr co - receptor bind to the extracellular domain of ret , ret undergoes dimerization and autophosphorylation of the tyrosine residues in their kinase domains .", "this generates the docking sites for their downstream effectors that contain the src homology 2 domain .", "for example , gdnf - mediated stimulation of ret results in activation of the pathways regulated by phosphatidylinositol 3-kinase ( pi3k ) and different mitogen - activated protein kinases ( mapks ) , including the extracellular regulated kinases ( erks ) , c - jun amino - terminal protein kinases ( jnks ) , the p38 mapk and the big map kinase ( bmk1 ) erk5 [ 22 , 23 ] .", "ret is one of the first receptor tyrosine - kinases ( rtks ) that have been found to play a role in neoplasia , being most well - known as a key etiological factor for thyroid cancer [ 6 , 24 ] . activating mutations of ret", "abnormally enhance ret activity and can trigger tumorigenesis in certain organs although the exact underlying mechanisms are as of yet unclear .", "first , mutations of the six cysteine residues ( cys609 , 611 , 618 , 620 , 630 , and 634 ) in the extracellular domains can promote ret dimerization via disulfide bonds and result in constitutive ligand - independent activation of ret .", "second , mutations affecting the tyrosine kinase domains can also confer ligand - independent catalytic activity to monomeric ret .", "these ret mutants exhibit different patterns of autophosphorylation and altered substrate specificity [ 2628 ] . indeed ,", "activation of different downstream signaling pathways is associated with different clinical features of ret mutant thyroid cancers , as observed in men2 syndromes discussed below .", "for example , the hirschprung disease , a congenital disorder of neural crest development is caused by a loss - of - function ret mutation .", "of note , the hirschprung disease is closely associated with men2a , demanding a genetic screening for men2a for children with familial hirschsprung 's disease .", "a strict correlation exists between specific ret mutations and the onset of hereditary mtc ( table 1 ) [ 31 , 32 ] .", "the detailed and up - to - date information of ret sequence variations can be obtained from the men2 ret database ( www.arup.utah.edu/database/men2/men2_welcome.php ) , which also contains links to selected men2 literature reviews , gene and protein information , and ret reference sequences .", "the men2a subtype , accounting for 9095% of the men type 2 cases , is a highly penetrant , autosomal dominant endocrine tumor syndrome characterized by the development of mtc in > 90% of ret mutation carriers . in association with mtc", "in addition , rare variants of men2a are also associated with cutaneous lichen amyloidosis and hirschsprung disease [ 35 , 36 ] .", "patients with men2a usually have mutations in the extracellular cysteine - rich region of the ret tyrosine kinase receptor , usually in exon 10 ( codons 609 , 611 , 618 or 620 ) or exon 11 ( codon 634 ) ( table 1 , figure 1 ) [ 31 , 37 ] .", "more than 80% of men2a patients exhibit a specific substitution , i.e. , cys634arg , on exon 11 .", "mtc is generally the first manifestation of men2a syndrome and develops during early childhood , usually before age six and sometimes before age two . the men2b subtype accounts for approximately 510% of the men type 2 cases .", "men2b patients typically feature early coincident onsets of mtc , pheochromocytoma , and gastrointestinal mucosal ganglioneuromas .", "visible physical symptoms include mucosal neuromas of lips ( bumpy lips ) and tongue , and asthenic marfanoid body habitus [ 36 , 40 ] .", "men2b patients usually have mutations in the tyrosine kinase domain 2 ( tk2 ) in the intracellular region of ret , which almost always ( > 95% ) lead to a single substitution , i.e. , met918thr , in exon 16 ( figure 1 ) [ 31 , 37 ] . de novo mutations , which usually occur on the paternal allele , are also common in men2b .", "men2b is characterized by the early development of an aggressive form of mtc in all affected individuals , typically during the first year of life .", "thus , apart from the genetic testing of ret mutations in children born to a parent with men2b , early diagnosis of men2b remains challenging .", "individuals with men2b are likely to develop metastatic mtc at an early age if they do not undergo prophylactic thyroidectomy before age one . without this intervention ,", "fmtc is considered as the least aggressive clinical variant of men2a with decreased penetrance and/or delayed onset of the other endocrine pathologic manifestations [ 39 , 43 ] . similarly to sporadic cases , familial mtcs are isolated and are not associated with other endocrine tumors .", "patients with fmtc harbor mutations similar to men2a in either the extracellular or intracellular region of the ret tyrosine kinase receptor [ 6 , 44 ] .", "the onset of fmtc is relatively late , not appearing until the second or the third decade of life , and its penetrance is lower than the mtc caused by men2a and men2b [ 31 , 39 , 45 , 46 ] .", "therefore , it is often difficult to determine fmtc based upon a family history and only careful genetic screening can distinguish between inherited and sporadic forms of mtc .", "mtc cells secrete the polypeptide hormone , calcitonin , and the glycoprotein carcinoembryonic antigen ( cea ) , and these are used as diagnostic biomarkers for mtc .", "mtc is most commonly diagnosed by immunohistochemical staining of fine - needle aspiration of a new thyroid nodule for calcitonin , chromogranin a , or cea .", "serum calcitonin is the primary biochemical marker used for detection , staging , postoperative management , and prognosis for mtc patients .", "however , in very rare cases , certain mtc cells do not secrete calcitonin , which makes diagnosis and patient follow - up difficult .", "symptoms of mtc include neck pain , a palpable neck mass , and/or diarrhea resulting from hypercalcitoninemia .", "the clinical course of mtc in men2 patients is variable and is determined by the codon specific mutations . in hereditary form , an age - related progression of malignant disease is observed , with lymph - node and distant metastases being typically detected years after the onset of tumorigenesis .", "metastatic spread to cervical and regional lymph nodes ( i.e. , parathyroid , paratracheal , jugular chain , and upper mediastinum ) or to distant sites including the liver , lungs , or bone is common and is frequently present in individuals with a palpable thyroid mass or diarrhea .", "positive lymph - node status and higher stage at diagnosis predict lower disease - free survival and higher mortality [ 5 , 5456 ] .", "men2 is one of few hereditary cancer syndromes for which predictive genetic testing is recommended at childhood .", "genetic testing for hereditary mtc syndromes has had an enormous impact on reducing the incidence of mtc in the families affected by these hereditary syndromes [ 57 , 58 ] .", "genetic counseling is indicated for all children diagnosed with mtc and others who either carry or are at risk of inheriting a ret mutation .", "children of patients with men2b should undergo ret analysis at birth , and children of patients with men2a or fmtc should undergo ret analysis before age six [ 59 , 60 ] .", "even 610% of apparently sporadic cases of mtc demonstrate de novo germ - line ret mutations , thus making genetic testing worthwhile in all patients with mtc .", "thus , all children with an affected parent in this setting retain a 50% risk of mtc , and surgical decisions must rely solely on clinical testing .", "the standard treatment for mtc is surgical removal of all thyroid tissue including the posterior capsule [ 39 , 62 ] .", "early thyroidectomy in all men2 patients can change the course of disease , either in a preventive or a curative fashion .", "the american thyroid association guidelines task force has classified mutations based upon a model that uses the genotypephenotype correlations to rank the mutations into risk levels for the development of aggressive mtc from the lowest  a  to the highest  d  ( table 1 ) .", "this classification may be used to predict phenotype , to recommend the timing of prophylactic thyroidectomy and the extent of surgical intervention , and to begin biochemical screening for pheochromocytoma and hyperparathyroidism .", "the ages at which the prophylactic thyroidectomy is recommended for the children tested positive for the ret gene mutation are as follows : ages 01 for ret mutations that carry the highest risk for aggressive metastatic mtc at young ages , i.e. , classified as  ata - d  ; before age 5 for ret mutations that carry a lower , yet still high risk of aggressive mtc at any age , i.e. , classified as  ata - c  ; after age 5 for ret mutations that carry a lower risk of aggressive mtc , i.e. , classified as  ata - b  or  ata - a ,  so long as the affected children have no other clinical signs of mtc development .", "there is ongoing debate on what age the thyroidectomy should be recommended for fmtc patients .", "some clinical institutes suggest the prophylactic surgery at age 1015 , depending upon the exact mutation and family history , while recommending yearly test of calcitonin levels prior to deciding the surgery .", "indeed , the ata management guideline for mtc has been very recently revised . in the setting of a prophylactic thyroidectomy ,", "the lymph - nodes are not routinely removed since metastases are not expected to occur at this stage . in the case of clinically apparent mtc , whether sporadic or hereditary , thyroidectomy and concomitant central and compartment - oriented lateral neck dissection should be performed to increase clinical outcomes .", "primary hyperthyroidism is rare during childhood ; therefore , parathyroidectomy is usually avoided , particularly during a prophylactic procedure .", "dissection and autotransplantation of parathyroid tissue is not typically performed at the time of thyroidectomy unless there is enough biochemical evidence for hyperparathyroidism .", "thyroidectomy in children is usually associated with a higher rate of complications , such as recurrent laryngeal nerve injury and hypoparathyroidism , as compared to the surgery in adults .", "therefore , pediatric thyroidectomy must be performed by highly experienced thyroid surgeons . for individuals with a ret mutation who have not had a thyroidectomy , annual biochemical screening of calcitonin levels is recommended and , if the results are abnormal , immediate thyroidectomy is required .", "annual serum calcitonin screening should begin at age six months for children with men2b and at age 35 for children with men2a or fmtc .", "biochemical evidence of disease recurrence includes elevation of calcitonin and cea levels . all individuals who have undergone thyroidectomy", "need thyroid hormone replacement therapy along with annual screening for pheochromocytoma and hyperparathyroidism depending upon the ret mutation present in the patients .", "mtc does not respond well to radiation therapy or the standard cytotoxic chemotherapeutic agents , including doxorubicin , dacarbazine , capacitabine , and 5-fluorouracil [ 43 , 71 ] . of note ,", "the mechanism - based targeted therapies that inhibit ret and other receptor tyrosine kinases have become available for the treatment of surgically inoperable progressive mtc .", "these include the multi - kinase inhibitors , vandetanib ( zd6474 , caprelsa ) and cabozantinib ( xl-184 , cometriq ) , which have been recently approved by the us food and drug administration [ 72 , 73 ] . a recent phase i / ii trial of vandetanib in children with mtc reported partial responses in 47% patients .", "in general , the drug efficiency and the primary side effects , i.e. , diarrhea , rash , headache , hypertension , and nausea , were similar between children and adults .", "phase iii trial of cabozantinib demonstrated a 28% response rate in adults with significant adverse effects .", "therefore , there is a critical need for more effective therapies for patients with advanced mtc .", "characterization of additional molecular pathways responsible for mtc development may allow the discovery of therapeutic targets that can be exploited to induce reduction of tumor size , disease stabilization , and symptomatic improvement [ 7782 ] .", "mtc and the men type 2 syndromes are rare but significant endocrine diseases that are increasingly encountered by pediatricians .", "our understanding of mtc has been greatly increased by the discovery of ret and the genotype ", "genetic tests according to the established guidelines should be performed whenever diagnosis of mtc is made .", "due to limited adjuvant treatment options , adequate surgical treatment is critical for initial control of the disease and prophylactic thyroidectomy is recommended for children with men2a and men2b at an early age , sometimes during infancy .", "emerging newer treatments are expected to better treat this rare but life - threatening malignancy ."], "abstract_text": ["<S> medullary thyroid carcinoma ( mtc ) , which originates from thyroid parafollicular c cells , accounts for 3 to 5% of thyroid malignancies . </S>", "<S> mtc occurs either sporadically or in an inherited autosomal dominant manner . </S>", "<S> hereditary mtc occurs as a familial mtc or as a part of multiple endocrine neoplasia ( men ) type 2a and b syndromes . </S>", "<S> a strong genotype - phenotype correlation has been observed between hereditary mtc and germ - line  gain of function  mutations of the ret proto - oncogene . </S>", "<S> most cases of pediatric mtc are hereditary whereas sporadic mtc is rare in children and is usually diagnosed in adults . </S>", "<S> therefore , mtc in children is most often diagnosed in the course of a familial genetic investigation . </S>", "<S> the standard treatment of mtc mainly requires surgery involving total thyroidectomy and central neck node dissection before extrathyroidal extension occurs . to prevent mtc development in hereditary syndromes , </S>", "<S> prophylactic thyroidectomy is performed in presymptomatic patients . </S>", "<S> an appropriate age at which the surgery should take place is determined based upon the data from genotyping , serum calcitonin measurements , and ultrasonography . for the treatment of advanced mtc cases , the broad spectrum receptor tyrosine kinase inhibitors vandetanib and cabozantinib , which also inhibit ret , </S>", "<S> are used although they are not always effective . </S>"], "labels": null, "section_names": ["1. INTRODUCTION", "2. STRUCTURE AND FUNCTION OF RET", "3. MEN TYPE 2 SYNDROMES", "4. DIAGNOSIS AND TREATMENT", "5. CONCLUSION"], "sections": [["thyroid cancer is the most common endocrine neoplasia which accounts for about 1% of human cancers .", "mtc originates from calcitonin - producing cells ( c - cells ) of the thyroid gland and accounts for 35% of thyroid cancers .", "mtc is relatively slow - growing tumor but , if metastasized or relapsed , it becomes very aggressive causing more than 13% of all thyroid cancer - related mortality . in the united states ,", "mtc has an incidence in children of 0.03 per 100 000 population per year with a fairly equal female to male ratio [ 4 , 5 ] .", "mtc occurs either sporadically or in an inherited autosomal dominant manner . in adults , sporadic mtc accounts for 6575% of mtc , but in children ,", "sporadic mtc is very rare ; the vast majority of mtc diagnosed in the childhood is hereditary .", "hereditary mtc occurs as a familial mtc ( fmtc ) or as a part of multiple endocrine neoplasia ( men ) type 2a and b syndromes , wherein other endocrine glands are also affected .", "mtc diagnosed during childhood almost always results from a dominantly inherited or de novo activating mutations in the ret proto - oncogene , which encodes the ret receptor tyrosine kinase [ 69 ] .", "advances in predictive genetic testing for ret mutations have enabled early diagnosis of hereditary men syndromes and prophylactic thyroidectomy in presymptomatic patients to prevent mtc .", "the early onset of mtc in hereditary syndromes makes it an important endocrine disease that is increasingly managed by pediatric providers [ 1012 ] . in this review", ", we discuss the etiology of pediatric mtc and currently available therapeutic modality for the cancer ."], ["ret encodes a receptor tyrosine - kinase which is expressed in the neural crest - derived cell types , including thyroid parafollicular cells , neuronal cells , and adrenal medullary chromaffin cells . in these cell types", ", ret plays a central role in regulating cell proliferation , growth , differentiation , migration and survival . in humans", "after alternative splicing at the 3 end , ret transcripts encode three protein isoforms with distinct c - terminal ends that contain either 9 ( ret9 ) , 51 ( ret51 ) , or 43 ( ret43 ) amino acids .", "ret exon 19 is present in all transcripts and its differential splicing at the 3 end produces distinct transcripts wherein exon 19 is either unspliced , spliced to exon 20 , or spliced to exon 21 .", "all three resulting ret isoforms commonly contain a tyrosine ( tyr1062 ) whose phosphorylation is critical for their activation .", "the major ret isoforms in vivo are ret9 and ret51 , which consist of 1072 and 1114 amino acids , respectively , and are usually co - expressed .", "ret consists of an extracellular ligand binding domain , a trans - membrane domain , and an intracellular kinase domain ( figure 1 ) .", "the extracellular domain includes four cadherin - like repeats and a highly conserved cysteine - rich region , which is located near the cell membrane .", "the intracellular domain consists of two tyrosine - kinase subdomains , tk1 and tk2 , which contain multiple tyrosine residues that are phosphorylated during receptor activation and are required for the activation of different downstream signaling pathways of ret [ 19 , 20 ] .", "the ligands for ret are the glial cell line - derived neurotrophic factor ( gdnf ) family proteins , including gdnf , neurturin , artemin , and perseptin .", "activation of ret also requires the formation of a heterodimeric complex recruiting a gdnf - family receptor alpha ( gfr ) . when unbound by a ligand , ret is monomeric , unphosphorylated , and inactive .", "when a ligand and the gfr co - receptor bind to the extracellular domain of ret , ret undergoes dimerization and autophosphorylation of the tyrosine residues in their kinase domains .", "this generates the docking sites for their downstream effectors that contain the src homology 2 domain .", "for example , gdnf - mediated stimulation of ret results in activation of the pathways regulated by phosphatidylinositol 3-kinase ( pi3k ) and different mitogen - activated protein kinases ( mapks ) , including the extracellular regulated kinases ( erks ) , c - jun amino - terminal protein kinases ( jnks ) , the p38 mapk and the big map kinase ( bmk1 ) erk5 [ 22 , 23 ] .", "ret is one of the first receptor tyrosine - kinases ( rtks ) that have been found to play a role in neoplasia , being most well - known as a key etiological factor for thyroid cancer [ 6 , 24 ] . activating mutations of ret", "abnormally enhance ret activity and can trigger tumorigenesis in certain organs although the exact underlying mechanisms are as of yet unclear .", "first , mutations of the six cysteine residues ( cys609 , 611 , 618 , 620 , 630 , and 634 ) in the extracellular domains can promote ret dimerization via disulfide bonds and result in constitutive ligand - independent activation of ret .", "second , mutations affecting the tyrosine kinase domains can also confer ligand - independent catalytic activity to monomeric ret .", "these ret mutants exhibit different patterns of autophosphorylation and altered substrate specificity [ 2628 ] . indeed ,", "activation of different downstream signaling pathways is associated with different clinical features of ret mutant thyroid cancers , as observed in men2 syndromes discussed below .", "for example , the hirschprung disease , a congenital disorder of neural crest development is caused by a loss - of - function ret mutation .", "of note , the hirschprung disease is closely associated with men2a , demanding a genetic screening for men2a for children with familial hirschsprung 's disease .", "a strict correlation exists between specific ret mutations and the onset of hereditary mtc ( table 1 ) [ 31 , 32 ] .", "the detailed and up - to - date information of ret sequence variations can be obtained from the men2 ret database ( www.arup.utah.edu/database/men2/men2_welcome.php ) , which also contains links to selected men2 literature reviews , gene and protein information , and ret reference sequences ."], ["the men2a subtype , accounting for 9095% of the men type 2 cases , is a highly penetrant , autosomal dominant endocrine tumor syndrome characterized by the development of mtc in > 90% of ret mutation carriers . in association with mtc", "in addition , rare variants of men2a are also associated with cutaneous lichen amyloidosis and hirschsprung disease [ 35 , 36 ] .", "patients with men2a usually have mutations in the extracellular cysteine - rich region of the ret tyrosine kinase receptor , usually in exon 10 ( codons 609 , 611 , 618 or 620 ) or exon 11 ( codon 634 ) ( table 1 , figure 1 ) [ 31 , 37 ] .", "more than 80% of men2a patients exhibit a specific substitution , i.e. , cys634arg , on exon 11 .", "mtc is generally the first manifestation of men2a syndrome and develops during early childhood , usually before age six and sometimes before age two . the men2b subtype accounts for approximately 510% of the men type 2 cases .", "men2b patients typically feature early coincident onsets of mtc , pheochromocytoma , and gastrointestinal mucosal ganglioneuromas .", "visible physical symptoms include mucosal neuromas of lips ( bumpy lips ) and tongue , and asthenic marfanoid body habitus [ 36 , 40 ] .", "men2b patients usually have mutations in the tyrosine kinase domain 2 ( tk2 ) in the intracellular region of ret , which almost always ( > 95% ) lead to a single substitution , i.e. , met918thr , in exon 16 ( figure 1 ) [ 31 , 37 ] . de novo mutations , which usually occur on the paternal allele , are also common in men2b .", "men2b is characterized by the early development of an aggressive form of mtc in all affected individuals , typically during the first year of life .", "thus , apart from the genetic testing of ret mutations in children born to a parent with men2b , early diagnosis of men2b remains challenging .", "individuals with men2b are likely to develop metastatic mtc at an early age if they do not undergo prophylactic thyroidectomy before age one . without this intervention ,", "fmtc is considered as the least aggressive clinical variant of men2a with decreased penetrance and/or delayed onset of the other endocrine pathologic manifestations [ 39 , 43 ] . similarly to sporadic cases , familial mtcs are isolated and are not associated with other endocrine tumors .", "patients with fmtc harbor mutations similar to men2a in either the extracellular or intracellular region of the ret tyrosine kinase receptor [ 6 , 44 ] .", "the onset of fmtc is relatively late , not appearing until the second or the third decade of life , and its penetrance is lower than the mtc caused by men2a and men2b [ 31 , 39 , 45 , 46 ] .", "therefore , it is often difficult to determine fmtc based upon a family history and only careful genetic screening can distinguish between inherited and sporadic forms of mtc ."], ["mtc cells secrete the polypeptide hormone , calcitonin , and the glycoprotein carcinoembryonic antigen ( cea ) , and these are used as diagnostic biomarkers for mtc .", "mtc is most commonly diagnosed by immunohistochemical staining of fine - needle aspiration of a new thyroid nodule for calcitonin , chromogranin a , or cea .", "serum calcitonin is the primary biochemical marker used for detection , staging , postoperative management , and prognosis for mtc patients .", "however , in very rare cases , certain mtc cells do not secrete calcitonin , which makes diagnosis and patient follow - up difficult .", "symptoms of mtc include neck pain , a palpable neck mass , and/or diarrhea resulting from hypercalcitoninemia .", "the clinical course of mtc in men2 patients is variable and is determined by the codon specific mutations . in hereditary form , an age - related progression of malignant disease is observed , with lymph - node and distant metastases being typically detected years after the onset of tumorigenesis .", "metastatic spread to cervical and regional lymph nodes ( i.e. , parathyroid , paratracheal , jugular chain , and upper mediastinum ) or to distant sites including the liver , lungs , or bone is common and is frequently present in individuals with a palpable thyroid mass or diarrhea .", "positive lymph - node status and higher stage at diagnosis predict lower disease - free survival and higher mortality [ 5 , 5456 ] .", "men2 is one of few hereditary cancer syndromes for which predictive genetic testing is recommended at childhood .", "genetic testing for hereditary mtc syndromes has had an enormous impact on reducing the incidence of mtc in the families affected by these hereditary syndromes [ 57 , 58 ] .", "genetic counseling is indicated for all children diagnosed with mtc and others who either carry or are at risk of inheriting a ret mutation .", "children of patients with men2b should undergo ret analysis at birth , and children of patients with men2a or fmtc should undergo ret analysis before age six [ 59 , 60 ] .", "even 610% of apparently sporadic cases of mtc demonstrate de novo germ - line ret mutations , thus making genetic testing worthwhile in all patients with mtc .", "thus , all children with an affected parent in this setting retain a 50% risk of mtc , and surgical decisions must rely solely on clinical testing .", "the standard treatment for mtc is surgical removal of all thyroid tissue including the posterior capsule [ 39 , 62 ] .", "early thyroidectomy in all men2 patients can change the course of disease , either in a preventive or a curative fashion .", "the american thyroid association guidelines task force has classified mutations based upon a model that uses the genotypephenotype correlations to rank the mutations into risk levels for the development of aggressive mtc from the lowest  a  to the highest  d  ( table 1 ) .", "this classification may be used to predict phenotype , to recommend the timing of prophylactic thyroidectomy and the extent of surgical intervention , and to begin biochemical screening for pheochromocytoma and hyperparathyroidism .", "the ages at which the prophylactic thyroidectomy is recommended for the children tested positive for the ret gene mutation are as follows : ages 01 for ret mutations that carry the highest risk for aggressive metastatic mtc at young ages , i.e. , classified as  ata - d  ; before age 5 for ret mutations that carry a lower , yet still high risk of aggressive mtc at any age , i.e. , classified as  ata - c  ; after age 5 for ret mutations that carry a lower risk of aggressive mtc , i.e. , classified as  ata - b  or  ata - a ,  so long as the affected children have no other clinical signs of mtc development .", "there is ongoing debate on what age the thyroidectomy should be recommended for fmtc patients .", "some clinical institutes suggest the prophylactic surgery at age 1015 , depending upon the exact mutation and family history , while recommending yearly test of calcitonin levels prior to deciding the surgery .", "indeed , the ata management guideline for mtc has been very recently revised . in the setting of a prophylactic thyroidectomy ,", "the lymph - nodes are not routinely removed since metastases are not expected to occur at this stage . in the case of clinically apparent mtc , whether sporadic or hereditary , thyroidectomy and concomitant central and compartment - oriented lateral neck dissection should be performed to increase clinical outcomes .", "primary hyperthyroidism is rare during childhood ; therefore , parathyroidectomy is usually avoided , particularly during a prophylactic procedure .", "dissection and autotransplantation of parathyroid tissue is not typically performed at the time of thyroidectomy unless there is enough biochemical evidence for hyperparathyroidism .", "thyroidectomy in children is usually associated with a higher rate of complications , such as recurrent laryngeal nerve injury and hypoparathyroidism , as compared to the surgery in adults .", "therefore , pediatric thyroidectomy must be performed by highly experienced thyroid surgeons . for individuals with a ret mutation who have not had a thyroidectomy , annual biochemical screening of calcitonin levels is recommended and , if the results are abnormal , immediate thyroidectomy is required .", "annual serum calcitonin screening should begin at age six months for children with men2b and at age 35 for children with men2a or fmtc .", "biochemical evidence of disease recurrence includes elevation of calcitonin and cea levels . all individuals who have undergone thyroidectomy", "need thyroid hormone replacement therapy along with annual screening for pheochromocytoma and hyperparathyroidism depending upon the ret mutation present in the patients .", "mtc does not respond well to radiation therapy or the standard cytotoxic chemotherapeutic agents , including doxorubicin , dacarbazine , capacitabine , and 5-fluorouracil [ 43 , 71 ] . of note ,", "the mechanism - based targeted therapies that inhibit ret and other receptor tyrosine kinases have become available for the treatment of surgically inoperable progressive mtc .", "these include the multi - kinase inhibitors , vandetanib ( zd6474 , caprelsa ) and cabozantinib ( xl-184 , cometriq ) , which have been recently approved by the us food and drug administration [ 72 , 73 ] . a recent phase i / ii trial of vandetanib in children with mtc reported partial responses in 47% patients .", "in general , the drug efficiency and the primary side effects , i.e. , diarrhea , rash , headache , hypertension , and nausea , were similar between children and adults .", "phase iii trial of cabozantinib demonstrated a 28% response rate in adults with significant adverse effects .", "therefore , there is a critical need for more effective therapies for patients with advanced mtc .", "characterization of additional molecular pathways responsible for mtc development may allow the discovery of therapeutic targets that can be exploited to induce reduction of tumor size , disease stabilization , and symptomatic improvement [ 7782 ] ."], ["mtc and the men type 2 syndromes are rare but significant endocrine diseases that are increasingly encountered by pediatricians .", "our understanding of mtc has been greatly increased by the discovery of ret and the genotype ", "genetic tests according to the established guidelines should be performed whenever diagnosis of mtc is made .", "due to limited adjuvant treatment options , adequate surgical treatment is critical for initial control of the disease and prophylactic thyroidectomy is recommended for children with men2a and men2b at an early age , sometimes during infancy .", "emerging newer treatments are expected to better treat this rare but life - threatening malignancy ."]]}
{"article_id": "PMC3263624", "article_text": ["neurosurgery is characterized by the delicate balance between surgical success and potential for devastating side effects . thanks to multiple technological improvements", ", the morbidity of neurosurgical interventions has substantially decreased over the last decades , allowing for the resection of previously inoperable lesions . in particular , image - guided neurosurgery ( igns ) devices allow the use of coregistered and fused multimodality 3d images to guide the surgeon 's hand and help define preoperatively the boundaries of pathological and predefined functional structures . meanwhile", ", new modes of medical imaging have also improved the localization of pathological lesions and their characterization .", "medical imaging nowadays includes a wealth of different techniques , such as computed tomography ( ct ) , structural and functional magnetic resonance imaging ( smri and fmri ) , diffusion tensor imaging ( dti ) , and positron emission tomography ( pet ) .", "although the overall accuracy of igns is estimated to be 12  mm , current neuronavigation systems can not , however , adapt to changing conditions over time .", "skull - opening brain shift , brain retraction , cerebrospinal fluid suction , lesion resection , perfusion , and pharmacological manipulation during surgery indeed all alter the 3d morphology of the structures [ 25 ] .", "these changes can lead to localization errors that are one order of magnitude larger that igns accuracy [ 1 , 2 , 6 ] and may result in incomplete resections or unexpected damage to normal brain .", "such inaccuracies could be reduced if one could acquire , throughout surgery , fresh images of the same modalities and quality as the preoperative ones", "intraoperative images such as intraoperative mr ( imr ) images are  with the exception of a handful surgical facilities  usually acquired using low - field mri scanners that provide lower resolution and contrast than their preoperative counterparts , and , to this date , several useful imaging modalities , such as pet and possibly meg , can not be acquired intraoperatively .", "one solution is to  bring over  the high - quality preoperative multimodality images into the intraoperative configuration of the brain using a nonrigid registration technique [ 710 ] .", "one category of nonrigid registration techniques uses physics - based models , where landmarks are tracked in successive reduced - quality intraoperative images , and their displacement fields drive the deformation of a biomechanical model .", "so far , most of the mechanical conditions of the brain can not be estimated in the operating room , such as the volume of cerebrospinal fluid flowing out of the skull cavity , intercellular fluid volume changes that result from mannitol injection , or changes in blood volume and vessel permeability .", "the fact that an intraoperative image can provide the knowledge of the current state of the brain after some deformation partly eliminates the need for a complete evaluation of these mechanical conditions .", "the nonrigid registration technique replaces them with the landmark displacements evaluated from successive intraoperative images . using a nonrigid registration technique based on a biomechanical model , three types of brain deformations", "have been identified that require specific modeling , although they depend on common parameters , such as csf suction , perfusion , or pharmacological manipulation .", "the first deformation is the brain shift , which appears at the beginning of surgery with the opening of the skull and dura .", "the suction or leakage of csf , as well as the release of intracranial pressure caused by tumor growth , generally cause such shift of the brain ( note that in this work , we name  brain shift  the specific shift of the brain that occurs after the opening of the skull and dura , before any other surgical act has happened ) .", "however , for these deformations , we will consider that the shift is a part of these two deformations .", "the second deformation is the retraction ; when target tissues are located deep inside the brain , the surgeon incises brain tissues and inserts a retractor to spread out the tissues , and to create a path towards the target .", "the third deformation is the resection , that is , the removal , of lesion tissues .", "three deformations can thus be defined in terms of the two elemental actions that change the topology of the brain : the introduction of a discontinuity and the removal of some tissues .", "most studies of brain deformation based on biomechanical models have focused on shifts ( the topology of the brain is not modified ) , that occurs just after the opening of the skull and dura [ 1125 ] .", "a good review of these different studies can be found in [ 24 , 2628 ] .", "resection and retraction are more complex to model than ( brain ) shift . until recently ,", "their modeling for the specific application of preoperative image update has received much less attention .", "one of the difficulty for modeling resection and retraction is that both induce a topological change of the brain because some tissue are cut .", "a method of mesh adaptation [ 2931 ] or remeshing [ 3235 ] must be used in conjunction with fem if an accurate representation of the location of the cut , for example , the resection cavity or retraction path , is needed to deform the model . indeed , fem can not directly handle discontinuities that go through the fes , and requires to realign the discontinuity with fe boundaries . in the field of fracture mechanics , which studies the growth and propagation of cracks in mechanical parts", ", some methods were developed to avoid using fem in conjunction with mesh adaptation or remeshing .", "the extended finite element method ( xfem or x - fem ) appeared in 1999   and has been the object of considerable research since then .", "xfem works by allowing the displacement field to be discontinuous within some fes of the mesh .", "the mesh does not have to conform to the discontinuities , so that these can be arbitrarily located with respect to the underlying fe mesh . because xfem allows an accurate representation of the discontinuities", "while avoiding mesh adaption or remeshing , and because of the similarity between cracks in mechanical parts and cuts in tissue , we proposed the use of xfem for handling cut , resection , and retraction in the updating of preoperative images .", "this paper presents a complete 3d framework for updating multimodal preoperative images with respect to surgical brain deformations , due to brain shift and successive resections , followed and quantified using imr images .", "our approach is modular , and is applied iteratively each time a new intraoperative image is acquired .", "we take into account successive deformations based on a linear elastic biomechanical model which is deformed using fem or xfem , depending on the type of deformation occurring between the pair of imr images under consideration , namely , brain shift or resection .", "while some 3d results have already been presented for brain shift , and initial 3d results for resection   modelings , this paper is the first complete and detailed account of the generalization to 3d of our 2d previous work .", "we present the state - of - the - art of resection modeling for preoperative image update . in section 3", ", we describe our basic strategy for updating preoperative images based on successive intraoperative images . in section 4 , we give detail about our methods and algorithms . in section 5 ,", "we consider two patient cases that illustrate our approach for handling brain shift followed by successive resections .", "among studies that take into account resection for preoperative image update , one should distinguish two categories .", "the first category of studies models brain deformation using two time - point images , the first image being acquired before surgery has started , the second image being acquired after resection . in this category ,", "the methods that existed for a second image showing some brain shift are adapted for a second image showing some resection .", "the second category of studies models brain deformation using more than two time - point images , and models at least two successive resections . among the first category of studies ,", "hagemann et al .   developed a 2d method for modeling brain deformation between a preoperative mr image and a postoperative mr image , the postoperative image showing a complete resection .", "the 2d mesh of the biomechanical model corresponded to the underlying pixel grid of the 2d image .", "the biomechanical model included four different linear elastic laws for the skull / skin region , the whole - brain region , the csf region , and the image background .", "they computed the correspondence of the skull boundary , the whole - brain region boundary in the neighborhood of the tumor , and the posterior midline between the two images .", "they also computed the correspondence between the internal tumor region boundary visible in the preoperative image , and the resection cavity boundary visible in the postoperative image , both boundaries corresponding under the assumption that the resection is complete .", "the displacements fields of these landmarks drove the deformation of the biomechanical model . as a result , the biomechanical model presented high deformation in the tumor region , which is not physically plausible .", "however , the resection was complete , and , thus , they were not interested by the displacement field of the biomechanical model in the tumor region itself .", "clatz et al .   developed a 3d method for modeling the brain deformation between a preoperative mr image and an imr image , the latter showing partial or complete resection .", "the biomechanical model was deformed based on a sparse volume displacement field evaluated from the two images , using a block matching algorithm . in their algorithm , blocks of voxels that presented discriminant structures were selected in the preoperative image .", "the blocks were then matched to blocks in the imr image using a similarity criterion , for example , a coefficient of correlation .", "the value of the similarity criterion was used as a value of confidence in the displacement measured by the block matching algorithm .", "the biomechanical model was then deformed iteratively , driven by the sparse displacement field of the matched blocks , where a block rejection step was included for measured block displacements initially selected but considered as outliers . in the imr image , a part , or the totality , of the tumor tissues", "the blocks were thus selected and matched in only the healthy - brain region of the two images .", "they tested their algorithm on six patient cases , and used for validation nine landmarks picked up manually in each image .", "they found a mean and maximum error on displacements of 0.75  mm and 2.5  mm , respectively .", "they explained this phenomenon by the fact that a substantial number of block matchings were rejected in the tumor neighborhood .", "the deformation of the biomechanical model in the tumor neighborhood was thus essentially governed by the linear elastic law , and the result might show the limitation of this model .", "archip et al .   also tested the nonrigid registration method presented in   on eleven patient cases , and used the 95% hausdorff distance   for evaluating the alignment of the nonrigidly registered images . as a result", ", they obtained a mean error of 1.82  mm . among the second category of studies , miga et al .  ", "they built a linear poroelastic biomechanical model and preoperatively tagged the tetrahedron fes that were going to be removed to simulate the brain deformation due to successive resections .", "second , a boundary condition was applied to the new boundary of the resection cavity , in order to model the relaxation of strain energy , induced by preoperative tumor growth or surgery acts , stored in the resected tissues , and released after their removal . in this approach ,", "the tissue discontinuity was represented as best as possible with a jagged topology defined by the fe facets defining the boundary of the resection cavity .", "[ 45 , 46 ] also modeled the removal of tetrahedra in order to model the action of an ultrasonic aspirator in the context of real - time surgery simulation .", "[ 13 , 47 , 48 ] modeled successive resections based on several time - point imr images . between two successive images", ", they deformed the biomechanical model , in its current state of update , to take into account the ( partial ) resections(s ) that took place between these two images .", "first , the biomechanical model , in its current state of update , was deformed in accordance with the displacement field of the healthy - brain boundary between the pair of images under consideration .", "second , the fes that fell into the resection cavity in the second image of the pair were removed , while the fes that laid across the resection - cavity boundary were cut . to ensure the link between the successive deformed configuration of the biomechanical model , their algorithm kept track of the topology modification between fes and nodes of the mesh before and after the removal of fes .", "they tested their algorithm on one patient case including five imr images ( the first two imr images being used for brain shift modeling ) , and used for validation thirty - two landmarks picked up manually in each image .", "they found a mean and maximum error on the displacements of 0.9  0.7  mm ( mean  standard deviation ) and 3.7  mm , respectively .", "they explained this phenomenon by the limited accuracy in the process of picking landmarks in that region , and because the retraction occurring between the second and third images was modeled as a resection , that is , a removal of tissues , even though the tissues were not removed but simply spread out .", "the methods described above have been all developed using an fem - based biomechanical model for intraoperative image registration .", "the objective of a surgical simulator is to provide an interactive manipulation with force feedback of the anatomical part to be operated using various surgical instruments . in order to model a large range surgical procedures ,", "jebkov and kuhlen   have applied nonlinear xfem for simulating cut , and have shown that xfem is successfully efficient for such purpose .", "the block diagram of figure 1 shows our global approach for updating preoperative images using successive imr images acquired at different critical points during surgery .", "although the principles of the approach are quite general , they are tailored for use based on images acquired with a 0.5  tesla intraoperative ge signa scanner , which guarantees that the full volume of brain tissues is included in the image field of view . in our present strategy ,", "the preoperative images are updated incrementally . at the end of each update , the preoperative images should be in the best possible alignment with the last imr image acquired .", "prior to surgery , a patient - specific biomechanical model is built from the set of preoperative images . because the patient does not necessarily lie in the same position during the acquisition of each of the preoperative images", ", one may need to perform a rigid registration ( involving translations , rotations , and scales ) to bring these images into correspondence , assuming , in first approximation , there is no local , that is , nonuniform , brain deformation between preoperative images .", "once the 1st imr image has been acquired prior to the opening of the skull , the set of registered preoperative images and the biomechanical model are registered to the 1st imr image via a rigid transformation . in the present situation , it is assumed that the patient 's brain imaged in the 1st imr image has the same physical shape as the brain imaged in the preoperative images ( note that in the following , when an imr image is defined by a number , this number is the index of the imr image in the series for a specific patient case .", "the 1st imr image thus corresponds to the very first imr image of the series ) . as each imr image", "is acquired , this new image and the preceding imr image are used to estimate the deformation of the brain .", "the update of the preoperative images is done incrementally with each new pair of successive imr images . for each pair , we proceed as follows . a set of common anatomical landmarks", "the use of surface structures rather than volume structures   seems more appropriate given the reduced - quality of typical intraoperative images , and would be more easily adapted to intraoperative modalities other than imr , such as ius .", "the landmark surface displacement fields resulting from the matching are then applied to the biomechanical model , which is deformed using fem or xfem , depending on the type of deformation occurring between the acquisition times of the imr images in the pair under consideration , namely , brain shift , or resection .", "the resulting displacement field of the biomechanical model is finally used to warp the set of preoperative images in their current state of updating .", "note that , for each deformation modeling , the biomechanical model is deformed in accordance with the landmark displacements tracked between the pair of successive imr images under consideration . because intraoperative deformation can follow a reverse direction , it is important to track the landmarks between the next - to - last and the last acquired imr images , rather than track the landmarks between the first and the last acquired imr images . for the patient cases treated in section 5 , we assume that the brain undergoes relatively small deformations ( small strains and small displacements ) , and we use a linear finite - element formulation in the biomechanical model .", "a consequence of using this linear formulation ( linear elasticity ) is that the equations of solid mechanics can be solved based on the initial configuration of the solid .", "actually , knowing the displacement field increment un = u  u at the anatomical landmarks between configuration n and increment n + 1 , one can apply this constrained displacement field increment un to the initial configuration , and the finite element analysis will lead to the deformation tensor increment n between the configuration n and n + 1 .", "the final deformation tensor or the body is thus simply obtained from  = k=0k .", "remark that rigorously , the increment of constrained displacement field at the landmark should be applied to the balanced solution of the solid reached after increment n , but as we are using a linear elasticity model , this step can be skipped owing to the superposition principle : if  = c , then  = k=0k = ck=0k = c , where c is the hooke tensor . as a summary , with this approach ,", "the process of deformations is modeled as a succession of deformations k , for example , brain deformation composed of shift followed by successive resections and the current configuration of the brain biomechanical model , after a specific deformation can then be recovered by adding the computed volume displacements for all successive incremental deformations . remark", "that this is not a limitation of the method as we could easily extend it to nonlinear model by simply keeping in memory the previous deformed configuration n and adding the constrained displacement field increment un to compute the new deformed configuration at increment n + 1 , simply this would be less computationally efficient . because we use a linear formulation ( and , thus , the incremental volume displacement fields can be added to recover the current configuration of the biomechanical model ) , we could theoretically obtain an identical deformed configuration of the biomechanical model using the two following approaches .", "the first one would consist of computing and adding the successive incremental deformations of the biomechanical model based on the landmarks tracked between the next - to - last and the last acquired imr images .", "the second approach would consist in computing directly the deformed configuration of the biomechanical model based on the landmarks tracked between the first and the last acquired imr images .", "however , the landmarks selected to drive the deformation of the biomechanical model vary depending on the type of deformation , namely , brain shift or resection .", "in addition , part of the biomechanical model is  cut ,  using xfem , to model resection .", "consequently , we would not get an identical deformed configuration of the biomechanical model by these two approaches .", "in order to use a maximum of information from the imr images , we track , as explained for the first approach , the landmarks between the next - to - last and the last acquired imr images .", "the problem of updating preoperative images between more than two critical points during surgery , that is , based on more than two imr images , is addressed in only a small number of studies . in our previous work [ 39 , 41 ] , and in", ", the biomechanical model was successively deformed , and this was done using a linear formulation .", "the framework proposed here , where the initial biomechanical model is always used , instead of using it in its successive states of deformation , has the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively deteriorates with each successive deformation modeling , and which would require remeshing or mesh adaptation for getting back good fe quality . to summarize ,", "for each deformation , the landmarks are tracked between the two successive imr images under consideration . because we use a linear formulation ,", "the displacement fields of these landmarks are applied to the initial , rather than current , configuration of the biomechanical model .", "the resulting volume displacement field corresponds to the deformation that the brain undergoes between the two imr images .", "this volume displacement field is used to deform the preoperative images in their current state of update , that is , registered ( at the previous step , if any ) to the first imr image of the pair .", "after the deformation , the preoperative images are thus in as good as possible registration to the second imr image of the pair . in all the rest of this work ,", "we make a simplification of the approach just presented , by using the 1st imr image as a substitute for the preoperative images .", "the biomechanical model is thus built based on structures visible in the 1st imr image , instead of in the preoperative images , and the structures used in the model are limited to the ones visible in the intraoperative image . except for the rigid registration between the preoperative images , the biomechanical model , and the 1st imr image , this simplified approach allows us to discuss , illustrate , and test all key aspects of the system .", "the above strategy allows us to focus on the main issue of this paper , that is , the estimation and handling of 3d deformations .", "even though the issues involved in the update of preoperative images will need to be addressed in a operational image update system , the present strategy of deforming the imr images remains useful for calibration purpose , even in the operating room .", "this section details the different methods that are commonly used for updating preoperative images in presence of brain shift and resection .", "more specifically , the block diagram of figure 2(a ) shows the building of the biomechanical model from the preoperative images .", "specific regions from the preoperative images are segmented , meshed , and assigned appropriate constitutive laws .", "the block diagram of figure 2(b ) shows , for any pair of successive imr images , a detailed view of the calculation of the volume displacement field of the initial biomechanical model that corresponds to the deformation that has occurred between the acquisition time of these images . all along surgery , the patient is lying inside the 0.5 tesla intraoperative ge signa scanner .", "although the patient 's head is fixed , one can not totally rule out the possibility of slight head motion .", "imr images thus have to be rigidly coregistered to take into account this potential rigid motion .", "the rigid registration that we use is the point - based landmark transform available in vtk ( http://www.vtk.org/ ) .", "the segmentation of imr images into specific regions , for example , healthy - brain and tumor regions , is first performed manually using 3d slicer ( http://www.slicer.org/ ) and then smoothed to minimize the dependance of the results on segmentation roughness .", "it is clear that performing a manual segmentation in the operating room is not acceptable , and that this process needs to be automated as completely as possible to test the feasibility of our framework online .", "however , while there exist sophisticated segmentation algorithms that could be used [ 5052 ] , in particular for extracting the whole - brain region ( skull and external cerebrospinal fluid masked out ) , the segmentation of the tumor region is still challenging . as mentioned above , the biomechanical model is built , in the present context , from the 1st imr image rather than from the preoperative images . thanks to the use of xfem instead of fem for modeling discontinuities , this biomechanical model can be built offline before the operation starts and does not need to be repeated ( through remeshing ) during the surgery . with respect to fem - based approaches ,", "the execution time thus ceases to be a limiting parameter , which is a remarkable advantage of our approach .", "it thus requires specific techniques , and we use the meshing software tool isosurf ( http://svr-www.eng.cam.ac.uk/~gmt11/software/isosurf/isosurf.html ) . our goal is to model the boundaries of healthy - brain and tumor regions as two connected surfaces meshes .", "however , isosurf can only mesh the boundaries of one or several separate regions , and , thus , does not allow one to mesh connected region boundaries with common nodes at their intersections .", "we thus start by building two separate surfaces meshes that we connect using our own routines based on vtk .", "the two connected triangle surfaces are then jointly meshed into a single volume mesh of tetrahedra that conform to the two surface meshes using gmsh ( http://www.geuz.org/gmsh/ ) .", "further details on the building of the biomechanical model , in particular the building of the connected surface meshes , can be found in .", "a linear elastic law is assigned to the biomechanical model , with young modulus e = 3000  pa and poisson ratio  = 0.45 . because displacements , rather than forces , are applied to the model using a linear formulation , the fem or xfem solution is independent of young modulus e .", "we choose as surface landmarks the whole - brain and internal tumor region boundaries . to evaluate the surface deformations of these region boundaries between two imr images , we use an active surface algorithm [ 55 , 56 ] .", "because these region boundaries to match must be closed surfaces , we thus use as surface landmarks the whole - brain and healthy - brain region boundaries .", "the surface deformation of the internal tumor region boundary will be derived from the active surface algorithm of the healthy - brain region boundary . in our active surface algorithm coming from [ 13 , 47 , 48 ] , the external forces f(x ) are computed using a gradient descent on a distance map of the region boundary . with such external forces , the active surface algorithm is not able to take correctly into account local rigid motion due , as an example , to lateral or tangential movement depending on the head orientation . for the whole - brain region , any rigid transformation that could have occurred", "has already been taken into account by the rigid registration of the imr images ( section 4.1 ) . however , for the healthy - brain region , it can happen that the internal tumor region boundary moves partly in a rigid way .", "therefore , the active surface , initialized from the healthy - brain region boundary in the first imr image , is first locally transformed in a rigid way along the internal tumor region boundary using the iterative closest point transform available in vtk .", "then , this resulting surface is deformed using the active surface algorithm as explained above .", "further details on the local rigid registration of the healthy - brain region boundary can be found in . before applying the displacements whole - brain and internal tumor region boundaries to the biomechanical model nodes ,", "the two surface displacement fields are smoothed based on a weighted - distance average , that is , the displacement of each node is averaged with the displacements of its n closest neighbor nodes .", "this smoothing will make them consistent with each other , and compatible with the volume mesh in order to avoid element flipping , in particular at the intersections between whole - brain and internal tumor region boundaries .", "the displacement fields of the surface landmarks are applied to the biomechanical model , which deforms according to the laws of solid mechanics .", "the equations of solid mechanics are solved using fem or xfem , depending upon the type of circumstances , namely , brain shift or resection .", "we use the fem - software tool metafor ( http://metafor.ltas.ulg.ac.be/ )   developed in our mechanical - engineering department , to which we have added an xfem module .", "the initial stress state of the brain is unknown and is thus set to zero for each fem or xfem computation , as in [ 10 , 13 ] .", "fem discretizes the solid of interest into a mesh , that is , into a set of fes interconnected by nodes , and approximates the displacement field u(x ) by the fem displacement field u(x ) defined as \n ( 1)ufem(x)=iii(x)ui , \n\t\t\t\t\t\t\t where i is the set of nodes , the i(x ) 's are the nodal shape functions ( nsfs ) , and the ui 's are the nodal degrees of freedom ( dofs ) .", "each i(x ) is defined as being continuous on its compact support i , which corresponds to the union of the domains of the fes connected to node i . in our approach , we use linear nsfs .", "in contrast , xfem handles a discontinuity by allowing the displacement field to be discontinuous within fes [ 37 , 5860 ] .", "the xfem displacement field generalises the fem displacement field ( 1 ) with \n ( 2)uxfem(x)=iii(x)ui+iji(x)j=1neigj(x)aji . \n\t\t\t\t\t\t\t", "the first term corresponds to the fem displacement field ( 1 ) , where i is the set of nodes , the i(x ) 's are the fem nsfs , and the ui 's are the nodal fem dofs . the heart of xfem is the  enrichment  that adds a number , n , of dofs aji to each node i of the set j , which is the subset of nodes of i whose support is intersected by the discontinuity of interest .", "these dofs are multiplied by the nsfs i(x ) and the discontinuous functions gj(x ) .", "the use of specific xfem enrichment functions gj(x ) for a node i  j depends on the type of discontinuity , for example , crack , hole , material interface , and so forth , to be modeled .", "suppose that our goal is to model a crack , characterized by a discontinuity in the displacement field ( as opposed to a material interface for instance , characterized by a discontinuity in the derivative of the displacement field ) .", "when the crack fully intersects the support of the node , a simple choice is a piecewise - constant unit function that changes sign at the boundary across the crack , that is , the heaviside function \n ( 3)h(x)={1for  ( xx)en>0,1for  ( xx)en<0 , \n\t\t\t\t\t\t\t where x is again the position of a point of the solid , x * is the position of the point on the crack that is the closest to x , and en is the outward normal to the crack at x * . in case of resection deformation , the goal is to model a discontinuity such that the part of tissues corresponding to tissue removed by the resection has no influence on the deformation of the remaining part of the tissues .", "one is actually interested in the deformation of the remaining part of the tissues only . in that sense ,", "the hole function    as the following equation :  \n ( 4)v(x)={1for  ( xx)en>0,0for  ( xx)en<0 , \n\t\t\t\t\t\t\t could be used as xfem enrichment function , instead of the heaviside function , and would be totally sufficient .", "the results that we would obtain on the remaining part of the tissues would be identical .", "however , because the heaviside function is necessary for retraction modeling , we have used the same function for the resection modeling even if it was not strictly necessary . when minimizing the total deformation energy , the resulting xfem equations remain sparse and symmetric as for fem .", "whereas fem requires a remeshing and the duplication of the nodes along the crack to take into account any discontinuity , xfem requires the identification of the nodes whose support is intersected by the crack and the addition of dofs : ( 1 ) any node whose support is not intersected by the discontinuity remains unaffected and thus possesses three dofs ; ( 2 ) any node whose support is fully intersected by the discontinuity is enriched with three heaviside dofs and thus possesses six dofs . to qualitatively estimate the similarity between two images , we compare the edges extracted from these images using the canny edge detector available in itk ( http://www.itk.org/ ) .  ", "indeed , although potentially useful for the sake of comparing methods on a mathematical basis and defining unique correspondences , landmark - based target analysis presents several relevant limitations in the present setting . having experts picking landmarks introduces significant intra- and interobserver variability . picking landmark points , as ferrant et al .  ", "did , is rather difficult when it comes to define enough visible landmarks  especially in the tumor region  on the 5 different images ( and not 2 images only , as majority of studies focusing on brain shift are using ) . rather than point targets , linear tumor contours , and limits between structures and potential eloquent structures matter most in the practical case of tumor ablation neurosurgery . \n\t\t\t\t\t", "these are the reason why we chose to use the canny edges in order to evaluate the registration . besides", ", while it is true that these edges do not necessarily physically correspond between the successive imr images , these images have been acquired with the same image protocol ( mr sequence , voxel size , grayscale value range ) , which should limit this problem .", "to quantitatively estimate the similarity of the two edge maps , we compute the modified hausdorff distance between the sets of edge points , that is , voxels representing the edges , in these two images .", "the modified hausdorff distance (a , b )   between two sets of points a and b is defined as \n ( 5)(a , b)=max(h(a , b),h(b , a ) )  with  h(a , b)=1naaad(a , b ) , \n\t\t\t\t\t\t\t where the directed hausdorff distance h(a , b ) is a measure of the distance of the point set a to the point set b , na is the number of points in set a , and d(a , b ) is the distance of point a  a to the closest point in b , that is , d(a , b ) = minbb||a  b|| , where ||a  b|| is the euclidean distance .", "the directed hausdorff distance h(a , b ) thus computes the average distance of points of a to points of b. the averaging minimizes the effects of outlier points , for example , due to image noise .", "the value of the modified hausdorff distance (a , b ) increases with the amount of difference between the two sets of edges points . in the following ,", "we denote by (ia , ib ) the modified hausdorff distance of the edges extracted from the whole - brain region of the images ia and ib , that is , with the skull and external cerebrospinal fluid masked out from them .", "in this section , we apply our methods , respectively , of brain shift and resection ( imr images are acquired with the 0.5  tesla intraoperative ge signa scanner of the brigham and women 's hospital , boston , usa .", "imr image size is 256  256  60 voxels , and voxel size is 0.9375  0.9375  2.5  mm ) .", "two patient cases , each including five imr images , are treated to illustrate our modeling and brain shift followed by successive resections . in both cases , the 1st imr image was acquired prior to the opening of the skull ; the 2nd imr image was acquired after the opening of the skull and dura , and shows some brain shift ; the 3rd , 4th , and 5th imr images were acquired after successive resections .", "the modelings of brain shift , 1st , 2nd , and 3rd resection are performed using different techniques , as detailed below . except where otherwise noted", ", the following discussion applies to both patient cases ( the result of each deformation modeling is shown for the two patient cases at the end of section 5.2.3 ) . to model brain shift based on the 1st and 2nd imr images", ", we estimate the surface displacement fields of the whole - brain region boundary and the internal tumor region boundary from the two imr images .", "no tissue discontinuity is involved in the brain shift deformation , so the biomechanical model is deformed using fem .", "this results in the volume displacement field of the biomechanical model , which is illustrated in figure 3 for the first patient case .", "this volume displacement field is used to warp the part of the 1st imr image corresponding to the whole - brain region . in the following sections ,", "the three successive resections are modeled separately , because they require different types of processing .", "matching two region boundaries to get a displacement field makes sense only if they correspond to the same physical entity .", "once the resection has started , we can no longer rely on the entirety of the whole - brain region boundary , since a part of it is now missing . for modeling the successive resections , we thus evaluate the displacement field for the boundary of the healthy - brain region only .", "the 1st resection occurs between the times the 2nd and 3rd imr images are acquired .", "however , since the corresponding removal of tissues is most likely accompanied by deformation , one can not exactly determine what tissue is removed based just on the two imr images .", "we thus decided to model the 1st resection by still relying on the displacement fields of key surfaces , here the healthy - brain region boundary , to deform the biomechanical model .", "this indeed appears to be the only reliable information concerning the deformation due to resection that we can extract from the 2nd and 3rd imr images .", "consequently , we do not model explicitly the removal of tissue , but we model directly the deformation resulting from it , without introducing any tissue discontinuity . using the surface displacement field of the healthy - brain region boundary , we compute the deformation of the biomechanical model via fem . then , using the resulting volume displacement field , we warp the part of the 2nd imr image corresponding to the whole - brain region , in the same way as we did in the case of for brain shift .", "the image resulting from the 1st resection modeling is now registered to the 3rd imr image , except outside of the healthy - brain region boundary , that is , for the tumor region . finally , we alter the resulting image to reflect the effect of resection . for this", ", we assign the background color to the voxels corresponding to the resected tissue volume ", "the significant feature of the 2nd resection is that some tissue has already been removed by the 1st resection , which means that this tissue can not have any physical influence on subsequent brain deformations because it does not ", "recall that the biomechanical model has been deformed to model the brain shift and the 1st resection and is thus registered to the 3rd imr image .", "so , using the 3rd imr image , we can define the boundary of the 1st resection , that is , the tissue discontinuity to include in the deformed biomechanical model ( figures 4(a ) and 4(b ) ) .", "we then enrich the nodes whose supports are intersected by the discontinuity with heaviside dofs .", "consequently , when the xfem - based biomechanical model deforms , the part corresponding to tissue removed by the 1st resection has no influence on the deformation of the remaining part of the brain .", "for the first patient case illustrated in figure 4 , the tetrahedron mesh consists of 3,317 nodes , which corresponds to 9,951 fem dofs .", "the biomechanical model is deformed in accordance with the displacement field of the healthy - brain region boundary evaluated from the 3rd and 4th imr images .  ", "the bottom part of the mesh , representing the tissue remaining after the 1st resection , has been deformed according to the displacement field of the healthy - brain region boundary , while the top part , representing the tissue removed by the 1st resection , has been subjected to a translation , but only for visualization purposes . even though the mesh is displayed as two separate parts , it is , in fact , a single entity .", "indeed , a main feature of xfem is its ability to handle the effect of a discontinuity without modifying the underlying mesh , that is , without remeshing . for modeling the 2nd resection , the edges of fes straddling the discontinuity", "have been made discontinuous and their nodes moved apart . using the xfem volume displacement field , we warp the part of the 3rd imr image corresponding to the whole - brain region .", "the resulting image is then masked out with the whole - brain region segmented out from the 4th imr image .", "one significant feature of the procedure described for modeling the 2nd resection is that it can be applied repetitively for each subsequent resection visible on successive imr images , no matter how many there are .", "the modeling of the 3rd resection is thus identical to the modeling of the 2nd resection .", "the tissue discontinuity due to the 2nd resection is defined from the 4th imr image , and used to appropriately enrich the nodes of the biomechanical model .", "then , this biomechanical model is deformed using xfem , in accordance with the displacement field of the healthy - brain region boundary evaluated from the 4th and 5th imr images . for the first patient case ,", "a simplification for the modeling of the 3rd resection can be made because , by the time the 5th imr image is acquired , the resection is complete .", "this means that we only need to compute the volume displacement field of the healthy - brain region .", "since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem will be identical .", "using the fem ( for the first patient case ) or xfem ( for the second patient case ) volume displacement field , we warp the part of the 4th imr image corresponding to the whole - brain region . the resulting image is then masked out with the whole - brain region segmented out from the 5th imr image .", "figures 5 and 6 show the results of warping the imr images , as well as the edges extracted from them , after brain shift and each successive resection modeling for the two patient cases . as explained in section 5.2.3 , since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem are identical in the healthy - brain region .", "one can deduce that using xfem for modeling resection is interesting when the neurosurgeon needs to have an accurate displacement field of the remaining tumor tissues . in this case", ", it is interesting to evaluate the impact of using fem , instead of xfem , to model the resection as if no resection was performed before .", "using fem for modeling resection is equivalent to ignoring the presence of resection on intraoperative images . to illustrate the comparison between fem and xfem results , we choose the 3rd resection modeling of the second patient case", "indeed , it is the deformation with remaining tumor tissues that shows the largest magnitude , and , thus , that is likely to give a maximum difference between the two computations .  ", "the healthy - brain and tumor regions segmented out from the 4th and 5th imr images are respectively shown in figures 7(a ) and 7(b ) .", "the volume displacement fields of the biomechanical model using xfem and fem are respectively shown in figures 7(c ) and 7(d ) .", "the part of the 4th imr image corresponding to the whole - brain region is warped , first with the volume displacement field obtained via fem , and then with that obtained via xfem .", "the difference between the two warped images is shown in figure 7(e ) . as expected", "however , the difference between the two volume displacement fields is smaller than the image resolution ( although the difference between the two volume displacement fields is smaller than the image resolution , the difference between the images resulting of the warping using these two volume displacement fields is nonzero .", "this is explained by the fact that the ( gray ) value of each voxel of the warped image is defined as a weighted - value of voxels of the original image .", "the weights are defined based on the overlapping ratio of the voxel of the warped image , with voxels ( determined using the volume displacement field ) of the original image ) .", "in addition , the deformed 4th imr images , using the xfem- and the fem - based deformations of the biomechanical model , show the same similarity , computed based on the modified hausdorff distance , with the 5th imr .", "two reasons explain that the differences between the fem and xfem results are so small .", "first , the brain deformation itself due to the 3rd resection is small , and , thus , it is expected to obtain small differences between the two resulting brain deformations .", "second , in the case the remaining tumor tissues are close to the healthy - brain region boundary , it implies that they are close to the boundary where surface displacement fields are applied to drive the deformation of the biomechanical model .", "although this comparison between fem and xfem should be done on more patient cases , we suggest that , in first approximation , fem could be used for modeling resection cases with small brain deformations .", "nevertheless , the presentation of the successive resections using xfem shows the generality of our framework , and details how xfem is implemented .", "note that in section 6 devoted to validation , the warped images are the ones deformed with xfem .", "for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images that are already rigidly registered , we compare the similarity between these ik and ik+1 images , as well as the similarity between the ik and ik+1 images , where ik is the result of warping ik .", "this gives us an estimate of how well we are able to capture , and compensate for , the local deformations between ik and ik+1 .", "the goal of the nonrigid registration is , however , to deform the preoperative images . by warping ik for each deformation modeling", ", we do not take into account the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . to evaluate the effect of this error amplification on the results", ", we also perform the required succession of warpings on i1 , and we denote the resulting image by i1,k .", "we then compare , for each deformation modeling , the similarity between i1 and ik+1 , together with the similarity between i1,k and ik+1 .", "this allows one to evaluate the propagation , that is , the amplification , of alignment error on the results .", "the modified hausdorff distance computed for each pair of imr images are given in tables 1 and 2 . \n", "table 1 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (ik , ik+1 ) and (ik , ik+1 ) .", "these values are computed using the canny edges extracted from the pair of images ( ik , ik+1 ) ( figures 5 ( d ) and 6 ( d ) ) and ( ik , ik+1 ) ( figures 5 ( e ) and 6 ( e ) ) .", "we observe that the values for the images nonrigidly registered are relatively constant , that is , 1  mm , for each deformation modeling .", "six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are ( rigidly and subsequently ) nonrigidly registered .", "however , the modified hausdorff distance increases for the 3rd resection modeling of the first patient case , as well as for the brain shift modeling of the second patient case . to understand", "if the nonrigid registration is responsible for the increase of the misalignment of the two imr images everywhere in the whole - brain region , or if this effect is localized , we compute the modified hausdorff distance in the region and neighborhood of the tumor only ( volume region that extents by 25  mm the tumor region segmented in i1 for both patient cases ) .", "the modified hausdorff distance decreases from (i4 , i5 ) = 1.70  mm to (i4 , i5 ) = 1.37  mm for the first patient case , while it decreases from (i1 , i2 ) = 1.36  mm to (i1 , i2 ) = 1.28  mm for the second patient case .", "this indicates that the nonrigid registration enhances the alignment of the two imr images within the tumor region and its neighborhood , which is in fact the location requiring the best modeling accuracy .", "this behavior could be explained by the fact that a maximum of information from the imr images is used in this region , that is , one or two ( in case of brain shift modeling ) surface displacement fields are applied around it .", "the increase of misalignment elsewhere in the brain volume could be explained by two reasons .", "first , the landmarks tracked from the imr images are surfaces . as a consequence , the nonrigid registration is expected to give better results near the tracked surfaces than far from them in the volume .", "the volume misalignment could point out the need for better parameters values and/or other constitutive laws . \n", "table 2 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (i1 , ik+1 ) and (i1,k , ik+1 ) .", "so far , igns systems allow one to rigidly register preoperative and successive imr images .", "(i1 , ik+1 ) thus represents the navigation accuracy that we can obtain with an igns system at the present time .", "the comparison of (i1 , ik+1 ) with (i1,k , ik+1 ) gives the improvement that could be practically achieved in the alignment with our approach .", "as expected , table 2 shows that the igns accuracy decreases through the successive deformations .", "indeed , the modified hausdorff distance increases from (i1 , i2 ) = 1.24  mm to (i1 , i5 ) = 1.78  mm for the first patient case , and from (i1 , i2 ) = 1.01  mm to (i1 , i5 ) = 1.68  mm for the second patient case .", "six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are nonrigidly registered .", "to understand if the modified hausdorff distance increases everywhere in the whole - brain region for the brain shift and 1st resection modeling of the second patient case , we compute the modified hausdorff distance in the neighborhood of the tumor region ( in the same way as explained for table 1 ) , and observe the improvement of the alignment within the tumor region and its neighborhood . as opposed to the values of the modified hausdorff distances in table 1 , the values for the images nonrigidly registered in table 2 increase through the successive resection modeling .", "this amplification error is due to the fact that , after having modeled brain deformation between a pair of imr images , the deformed biomechanical model is not in perfect alignment with the second image of the pair .", "since , for the subsequent deformation modeling , the surface landmarks are initialized based on the deformed biomechanical model , this can thus ampliy a misregistration error .", "we developed a complete 3d framework for serial preoperative image update in the presence of brain shift followed by successive resections .", "the nonrigid registration technique used an homogeneous linear elastic biomechanical model , driven by the deformations of whole - brain and internal tumor region boundaries for brain shift modeling , and healthy - brain region boundary for resection modelings , tracked between successive imr images .", "the biomechanical model was deformed using fem for brain shift modeling , and fem or xfem for resection modeling , depending upon whether some brain tissues were previously resected or not .", "we showed that our approach was modular , and could be applied each time a new imr image is acquired .", "we used a linear formulation to characterize the deformation of the brains of both patients because the brains underwent relatively small deformations and displacements .", "while nonlinear biomechanical models have proven effective to decrease  yet do not abolish  the inaccuracies of fem - based modeling methods of large brain deformations , the deformations observed in our patients during surgery remained moderate ( 47  mm ) , thus reducing the theoretical benefit of using nonlinear models .", "this allowed us to use simpler linear models and focus on the added value of xfem to simultaneously account for surgical deformations , namely , shift and resection . using a linear formulation", "implied that , for each new deformation modeling , one could use the initial configuration rather than the last - deformed configuration of the biomechanical model .", "this had the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively degraded with each successive deformation modeling .", "this also had the advantage that we did no longer need to reconnect the deformed mesh for each new xfem calculation , which was one drawback of our previous method , presented in [ 39 , 41 ] , where the biomechanical model was successively deformed .", "we also showed how xfem could handle a discontinuity for modeling resection without any remeshing or mesh adaptation while the representation of the discontinuity remained accurate , that is , the representation of the discontinuity was not based on a jagged topology using fe facets .", "xfem thus also avoided making the mesh resolution richer in the neighborhood of the resection - cavity boundary for improving the accuracy of the representation of the discontinuity for that purpose only .", "we showed that our nonrigid registration technique improved the alignment of the successive imr images for most of the deformation modeling of both patient cases . when our nonrigid registration failed , it still improved the alignment locally , that is , within the tumor region and its neighborhood .", "we tested the explicit modeling of the lateral ventricles ' region with a soft , compressible law in addition to the whole - brain region law used in the homogeneous biomechanical model .", "in addition to the validation that is usually performed for successive deformation modelings , that is , validation between pairs of successive intraoperative images , shown in table 1 of section 6 or in the work of ferrant et al .", "[ 13 , 47 , 48 ] , we also evaluated the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . as a result , shown in table 2 of section 6", ", we showed that our approach suffered from the propagation of misregistration through the successive deformation modelings .", "we expected that this was due , at least partly , to the algorithms used to evaluate intraoperative surface displacements fields from the whole - brain and healthy - brain region boundaries .", "the surface displacement fields were computed using active surface algorithms , and smoothed to make them compatible with the biomechanical model .", "because of these two smoothings , the deformed biomechanical model was likely to not be in a perfect alignment with the imr image to which it was registered .", "because the surface displacement fields evaluated for the next deformation modeling were initialized based on the deformed biomechanical model , we expected to observe an amplification of the misregistration , which was confirmed by our quantitative evaluation . at the present time", "though , commercial igns systems allow one to register preoperative images and successive imr images , but in a rigid way only .", "consequently , although the effect of error amplification exists , our technique still enhances the current capabilities of commercial igns systems .", "future work on modeling of brain shift followed by successive resection is required in five main areas .", "first , the effect of error amplification through the successive brain deformation modelings calls for further research .", "consequently , the segmentation , and the subsequent smoothing , as well as the evaluation of surface displacement fields , should be improved to minimize the effect of error amplification .", "second , further research is required to include additional structures in the biomechanical model in general , and to study the best way to include the lateral ventricles in particular .", "the use of a poroelastic model in order to model the cerebrospinal fluid filling the ventricles could be considered [ 17 , 18 ] .", "indeed , these images provide volume information ( rather than surface information only ) , are of good quality in comparison to other intraoperative modalities , and possess a field of view that includes the full volume of brain tissues ( for the 0.5 tesla ge signa scanner ) .", "these images thus allow one to evaluate what , and how , new structures of the brain could be used , to enhance the modeling of brain shift .", "some regions , for example , the lateral ventricles ' region , could be extracted from the two imr images , and used as surface landmarks to drive the deformation of the biomechanical model [ 13 , 62 ] . indeed , the workflow presented in this paper has the advantage of being easily adaptable . in case", "the tumor region would not be visible ( enough ) on the imr images , these new structures , easier to segment , could also adequately replace the tumor for driving the deformation .", "fourth , our global approach should no longer be based on the 1st imr image used as a substitute for preoperative images , but on the preoperative images themselves .", "fifth , we should implement , for the surgery cases involving large deformations of the brain , a nonlinear formulation of fem [ 63 , 64 ] , and , particularly , a nonlinear formulation of xfem , which is the subject of recent research [ 65 , 66 ] ."], "abstract_text": ["<S> current neuronavigation systems can not adapt to changing intraoperative conditions over time . to overcome this limitation </S>", "<S> , we present an experimental end - to - end system capable of updating 3d preoperative images in the presence of brain shift and successive resections . the heart of our system is a nonrigid registration technique using a biomechanical model , driven by the deformations of key surfaces tracked in successive intraoperative images . </S>", "<S> the biomechanical model is deformed using fem or xfem , depending on the type of deformation under consideration , namely , brain shift or resection . </S>", "<S> we describe the operation of our system on two patient cases , each comprising five intraoperative mr images , and we demonstrate that our approach significantly improves the alignment of nonrigidly registered images . </S>"], "labels": null, "section_names": ["1. Introduction", "2. State-of-the-Art", "3. Basic Strategy for Serial Preoperative Image  Update", "4. Methods", "5. Results", "6. Validation", "7. Conclusions and Future Work"], "sections": [["neurosurgery is characterized by the delicate balance between surgical success and potential for devastating side effects . thanks to multiple technological improvements", ", the morbidity of neurosurgical interventions has substantially decreased over the last decades , allowing for the resection of previously inoperable lesions . in particular , image - guided neurosurgery ( igns ) devices allow the use of coregistered and fused multimodality 3d images to guide the surgeon 's hand and help define preoperatively the boundaries of pathological and predefined functional structures . meanwhile", ", new modes of medical imaging have also improved the localization of pathological lesions and their characterization .", "medical imaging nowadays includes a wealth of different techniques , such as computed tomography ( ct ) , structural and functional magnetic resonance imaging ( smri and fmri ) , diffusion tensor imaging ( dti ) , and positron emission tomography ( pet ) .", "although the overall accuracy of igns is estimated to be 12  mm , current neuronavigation systems can not , however , adapt to changing conditions over time .", "skull - opening brain shift , brain retraction , cerebrospinal fluid suction , lesion resection , perfusion , and pharmacological manipulation during surgery indeed all alter the 3d morphology of the structures [ 25 ] .", "these changes can lead to localization errors that are one order of magnitude larger that igns accuracy [ 1 , 2 , 6 ] and may result in incomplete resections or unexpected damage to normal brain .", "such inaccuracies could be reduced if one could acquire , throughout surgery , fresh images of the same modalities and quality as the preoperative ones", "intraoperative images such as intraoperative mr ( imr ) images are  with the exception of a handful surgical facilities  usually acquired using low - field mri scanners that provide lower resolution and contrast than their preoperative counterparts , and , to this date , several useful imaging modalities , such as pet and possibly meg , can not be acquired intraoperatively .", "one solution is to  bring over  the high - quality preoperative multimodality images into the intraoperative configuration of the brain using a nonrigid registration technique [ 710 ] .", "one category of nonrigid registration techniques uses physics - based models , where landmarks are tracked in successive reduced - quality intraoperative images , and their displacement fields drive the deformation of a biomechanical model .", "so far , most of the mechanical conditions of the brain can not be estimated in the operating room , such as the volume of cerebrospinal fluid flowing out of the skull cavity , intercellular fluid volume changes that result from mannitol injection , or changes in blood volume and vessel permeability .", "the fact that an intraoperative image can provide the knowledge of the current state of the brain after some deformation partly eliminates the need for a complete evaluation of these mechanical conditions .", "the nonrigid registration technique replaces them with the landmark displacements evaluated from successive intraoperative images . using a nonrigid registration technique based on a biomechanical model , three types of brain deformations", "have been identified that require specific modeling , although they depend on common parameters , such as csf suction , perfusion , or pharmacological manipulation .", "the first deformation is the brain shift , which appears at the beginning of surgery with the opening of the skull and dura .", "the suction or leakage of csf , as well as the release of intracranial pressure caused by tumor growth , generally cause such shift of the brain ( note that in this work , we name  brain shift  the specific shift of the brain that occurs after the opening of the skull and dura , before any other surgical act has happened ) .", "however , for these deformations , we will consider that the shift is a part of these two deformations .", "the second deformation is the retraction ; when target tissues are located deep inside the brain , the surgeon incises brain tissues and inserts a retractor to spread out the tissues , and to create a path towards the target .", "the third deformation is the resection , that is , the removal , of lesion tissues .", "three deformations can thus be defined in terms of the two elemental actions that change the topology of the brain : the introduction of a discontinuity and the removal of some tissues .", "most studies of brain deformation based on biomechanical models have focused on shifts ( the topology of the brain is not modified ) , that occurs just after the opening of the skull and dura [ 1125 ] .", "a good review of these different studies can be found in [ 24 , 2628 ] .", "resection and retraction are more complex to model than ( brain ) shift . until recently ,", "their modeling for the specific application of preoperative image update has received much less attention .", "one of the difficulty for modeling resection and retraction is that both induce a topological change of the brain because some tissue are cut .", "a method of mesh adaptation [ 2931 ] or remeshing [ 3235 ] must be used in conjunction with fem if an accurate representation of the location of the cut , for example , the resection cavity or retraction path , is needed to deform the model . indeed , fem can not directly handle discontinuities that go through the fes , and requires to realign the discontinuity with fe boundaries . in the field of fracture mechanics , which studies the growth and propagation of cracks in mechanical parts", ", some methods were developed to avoid using fem in conjunction with mesh adaptation or remeshing .", "the extended finite element method ( xfem or x - fem ) appeared in 1999   and has been the object of considerable research since then .", "xfem works by allowing the displacement field to be discontinuous within some fes of the mesh .", "the mesh does not have to conform to the discontinuities , so that these can be arbitrarily located with respect to the underlying fe mesh . because xfem allows an accurate representation of the discontinuities", "while avoiding mesh adaption or remeshing , and because of the similarity between cracks in mechanical parts and cuts in tissue , we proposed the use of xfem for handling cut , resection , and retraction in the updating of preoperative images .", "this paper presents a complete 3d framework for updating multimodal preoperative images with respect to surgical brain deformations , due to brain shift and successive resections , followed and quantified using imr images .", "our approach is modular , and is applied iteratively each time a new intraoperative image is acquired .", "we take into account successive deformations based on a linear elastic biomechanical model which is deformed using fem or xfem , depending on the type of deformation occurring between the pair of imr images under consideration , namely , brain shift or resection .", "while some 3d results have already been presented for brain shift , and initial 3d results for resection   modelings , this paper is the first complete and detailed account of the generalization to 3d of our 2d previous work .", "we present the state - of - the - art of resection modeling for preoperative image update . in section 3", ", we describe our basic strategy for updating preoperative images based on successive intraoperative images . in section 4 , we give detail about our methods and algorithms . in section 5 ,", "we consider two patient cases that illustrate our approach for handling brain shift followed by successive resections ."], ["among studies that take into account resection for preoperative image update , one should distinguish two categories .", "the first category of studies models brain deformation using two time - point images , the first image being acquired before surgery has started , the second image being acquired after resection . in this category ,", "the methods that existed for a second image showing some brain shift are adapted for a second image showing some resection .", "the second category of studies models brain deformation using more than two time - point images , and models at least two successive resections . among the first category of studies ,", "hagemann et al .   developed a 2d method for modeling brain deformation between a preoperative mr image and a postoperative mr image , the postoperative image showing a complete resection .", "the 2d mesh of the biomechanical model corresponded to the underlying pixel grid of the 2d image .", "the biomechanical model included four different linear elastic laws for the skull / skin region , the whole - brain region , the csf region , and the image background .", "they computed the correspondence of the skull boundary , the whole - brain region boundary in the neighborhood of the tumor , and the posterior midline between the two images .", "they also computed the correspondence between the internal tumor region boundary visible in the preoperative image , and the resection cavity boundary visible in the postoperative image , both boundaries corresponding under the assumption that the resection is complete .", "the displacements fields of these landmarks drove the deformation of the biomechanical model . as a result , the biomechanical model presented high deformation in the tumor region , which is not physically plausible .", "however , the resection was complete , and , thus , they were not interested by the displacement field of the biomechanical model in the tumor region itself .", "clatz et al .   developed a 3d method for modeling the brain deformation between a preoperative mr image and an imr image , the latter showing partial or complete resection .", "the biomechanical model was deformed based on a sparse volume displacement field evaluated from the two images , using a block matching algorithm . in their algorithm , blocks of voxels that presented discriminant structures were selected in the preoperative image .", "the blocks were then matched to blocks in the imr image using a similarity criterion , for example , a coefficient of correlation .", "the value of the similarity criterion was used as a value of confidence in the displacement measured by the block matching algorithm .", "the biomechanical model was then deformed iteratively , driven by the sparse displacement field of the matched blocks , where a block rejection step was included for measured block displacements initially selected but considered as outliers . in the imr image , a part , or the totality , of the tumor tissues", "the blocks were thus selected and matched in only the healthy - brain region of the two images .", "they tested their algorithm on six patient cases , and used for validation nine landmarks picked up manually in each image .", "they found a mean and maximum error on displacements of 0.75  mm and 2.5  mm , respectively .", "they explained this phenomenon by the fact that a substantial number of block matchings were rejected in the tumor neighborhood .", "the deformation of the biomechanical model in the tumor neighborhood was thus essentially governed by the linear elastic law , and the result might show the limitation of this model .", "archip et al .   also tested the nonrigid registration method presented in   on eleven patient cases , and used the 95% hausdorff distance   for evaluating the alignment of the nonrigidly registered images . as a result", ", they obtained a mean error of 1.82  mm . among the second category of studies , miga et al .  ", "they built a linear poroelastic biomechanical model and preoperatively tagged the tetrahedron fes that were going to be removed to simulate the brain deformation due to successive resections .", "second , a boundary condition was applied to the new boundary of the resection cavity , in order to model the relaxation of strain energy , induced by preoperative tumor growth or surgery acts , stored in the resected tissues , and released after their removal . in this approach ,", "the tissue discontinuity was represented as best as possible with a jagged topology defined by the fe facets defining the boundary of the resection cavity .", "[ 45 , 46 ] also modeled the removal of tetrahedra in order to model the action of an ultrasonic aspirator in the context of real - time surgery simulation .", "[ 13 , 47 , 48 ] modeled successive resections based on several time - point imr images . between two successive images", ", they deformed the biomechanical model , in its current state of update , to take into account the ( partial ) resections(s ) that took place between these two images .", "first , the biomechanical model , in its current state of update , was deformed in accordance with the displacement field of the healthy - brain boundary between the pair of images under consideration .", "second , the fes that fell into the resection cavity in the second image of the pair were removed , while the fes that laid across the resection - cavity boundary were cut . to ensure the link between the successive deformed configuration of the biomechanical model , their algorithm kept track of the topology modification between fes and nodes of the mesh before and after the removal of fes .", "they tested their algorithm on one patient case including five imr images ( the first two imr images being used for brain shift modeling ) , and used for validation thirty - two landmarks picked up manually in each image .", "they found a mean and maximum error on the displacements of 0.9  0.7  mm ( mean  standard deviation ) and 3.7  mm , respectively .", "they explained this phenomenon by the limited accuracy in the process of picking landmarks in that region , and because the retraction occurring between the second and third images was modeled as a resection , that is , a removal of tissues , even though the tissues were not removed but simply spread out .", "the methods described above have been all developed using an fem - based biomechanical model for intraoperative image registration .", "the objective of a surgical simulator is to provide an interactive manipulation with force feedback of the anatomical part to be operated using various surgical instruments . in order to model a large range surgical procedures ,", "jebkov and kuhlen   have applied nonlinear xfem for simulating cut , and have shown that xfem is successfully efficient for such purpose ."], ["the block diagram of figure 1 shows our global approach for updating preoperative images using successive imr images acquired at different critical points during surgery .", "although the principles of the approach are quite general , they are tailored for use based on images acquired with a 0.5  tesla intraoperative ge signa scanner , which guarantees that the full volume of brain tissues is included in the image field of view . in our present strategy ,", "the preoperative images are updated incrementally . at the end of each update , the preoperative images should be in the best possible alignment with the last imr image acquired .", "prior to surgery , a patient - specific biomechanical model is built from the set of preoperative images . because the patient does not necessarily lie in the same position during the acquisition of each of the preoperative images", ", one may need to perform a rigid registration ( involving translations , rotations , and scales ) to bring these images into correspondence , assuming , in first approximation , there is no local , that is , nonuniform , brain deformation between preoperative images .", "once the 1st imr image has been acquired prior to the opening of the skull , the set of registered preoperative images and the biomechanical model are registered to the 1st imr image via a rigid transformation . in the present situation , it is assumed that the patient 's brain imaged in the 1st imr image has the same physical shape as the brain imaged in the preoperative images ( note that in the following , when an imr image is defined by a number , this number is the index of the imr image in the series for a specific patient case .", "the 1st imr image thus corresponds to the very first imr image of the series ) . as each imr image", "is acquired , this new image and the preceding imr image are used to estimate the deformation of the brain .", "the update of the preoperative images is done incrementally with each new pair of successive imr images . for each pair , we proceed as follows . a set of common anatomical landmarks", "the use of surface structures rather than volume structures   seems more appropriate given the reduced - quality of typical intraoperative images , and would be more easily adapted to intraoperative modalities other than imr , such as ius .", "the landmark surface displacement fields resulting from the matching are then applied to the biomechanical model , which is deformed using fem or xfem , depending on the type of deformation occurring between the acquisition times of the imr images in the pair under consideration , namely , brain shift , or resection .", "the resulting displacement field of the biomechanical model is finally used to warp the set of preoperative images in their current state of updating .", "note that , for each deformation modeling , the biomechanical model is deformed in accordance with the landmark displacements tracked between the pair of successive imr images under consideration . because intraoperative deformation can follow a reverse direction , it is important to track the landmarks between the next - to - last and the last acquired imr images , rather than track the landmarks between the first and the last acquired imr images . for the patient cases treated in section 5 , we assume that the brain undergoes relatively small deformations ( small strains and small displacements ) , and we use a linear finite - element formulation in the biomechanical model .", "a consequence of using this linear formulation ( linear elasticity ) is that the equations of solid mechanics can be solved based on the initial configuration of the solid .", "actually , knowing the displacement field increment un = u  u at the anatomical landmarks between configuration n and increment n + 1 , one can apply this constrained displacement field increment un to the initial configuration , and the finite element analysis will lead to the deformation tensor increment n between the configuration n and n + 1 .", "the final deformation tensor or the body is thus simply obtained from  = k=0k .", "remark that rigorously , the increment of constrained displacement field at the landmark should be applied to the balanced solution of the solid reached after increment n , but as we are using a linear elasticity model , this step can be skipped owing to the superposition principle : if  = c , then  = k=0k = ck=0k = c , where c is the hooke tensor . as a summary , with this approach ,", "the process of deformations is modeled as a succession of deformations k , for example , brain deformation composed of shift followed by successive resections and the current configuration of the brain biomechanical model , after a specific deformation can then be recovered by adding the computed volume displacements for all successive incremental deformations . remark", "that this is not a limitation of the method as we could easily extend it to nonlinear model by simply keeping in memory the previous deformed configuration n and adding the constrained displacement field increment un to compute the new deformed configuration at increment n + 1 , simply this would be less computationally efficient . because we use a linear formulation ( and , thus , the incremental volume displacement fields can be added to recover the current configuration of the biomechanical model ) , we could theoretically obtain an identical deformed configuration of the biomechanical model using the two following approaches .", "the first one would consist of computing and adding the successive incremental deformations of the biomechanical model based on the landmarks tracked between the next - to - last and the last acquired imr images .", "the second approach would consist in computing directly the deformed configuration of the biomechanical model based on the landmarks tracked between the first and the last acquired imr images .", "however , the landmarks selected to drive the deformation of the biomechanical model vary depending on the type of deformation , namely , brain shift or resection .", "in addition , part of the biomechanical model is  cut ,  using xfem , to model resection .", "consequently , we would not get an identical deformed configuration of the biomechanical model by these two approaches .", "in order to use a maximum of information from the imr images , we track , as explained for the first approach , the landmarks between the next - to - last and the last acquired imr images .", "the problem of updating preoperative images between more than two critical points during surgery , that is , based on more than two imr images , is addressed in only a small number of studies . in our previous work [ 39 , 41 ] , and in", ", the biomechanical model was successively deformed , and this was done using a linear formulation .", "the framework proposed here , where the initial biomechanical model is always used , instead of using it in its successive states of deformation , has the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively deteriorates with each successive deformation modeling , and which would require remeshing or mesh adaptation for getting back good fe quality . to summarize ,", "for each deformation , the landmarks are tracked between the two successive imr images under consideration . because we use a linear formulation ,", "the displacement fields of these landmarks are applied to the initial , rather than current , configuration of the biomechanical model .", "the resulting volume displacement field corresponds to the deformation that the brain undergoes between the two imr images .", "this volume displacement field is used to deform the preoperative images in their current state of update , that is , registered ( at the previous step , if any ) to the first imr image of the pair .", "after the deformation , the preoperative images are thus in as good as possible registration to the second imr image of the pair . in all the rest of this work ,", "we make a simplification of the approach just presented , by using the 1st imr image as a substitute for the preoperative images .", "the biomechanical model is thus built based on structures visible in the 1st imr image , instead of in the preoperative images , and the structures used in the model are limited to the ones visible in the intraoperative image . except for the rigid registration between the preoperative images , the biomechanical model , and the 1st imr image , this simplified approach allows us to discuss , illustrate , and test all key aspects of the system .", "the above strategy allows us to focus on the main issue of this paper , that is , the estimation and handling of 3d deformations .", "even though the issues involved in the update of preoperative images will need to be addressed in a operational image update system , the present strategy of deforming the imr images remains useful for calibration purpose , even in the operating room ."], ["this section details the different methods that are commonly used for updating preoperative images in presence of brain shift and resection .", "more specifically , the block diagram of figure 2(a ) shows the building of the biomechanical model from the preoperative images .", "specific regions from the preoperative images are segmented , meshed , and assigned appropriate constitutive laws .", "the block diagram of figure 2(b ) shows , for any pair of successive imr images , a detailed view of the calculation of the volume displacement field of the initial biomechanical model that corresponds to the deformation that has occurred between the acquisition time of these images . all along surgery , the patient is lying inside the 0.5 tesla intraoperative ge signa scanner .", "although the patient 's head is fixed , one can not totally rule out the possibility of slight head motion .", "imr images thus have to be rigidly coregistered to take into account this potential rigid motion .", "the rigid registration that we use is the point - based landmark transform available in vtk ( http://www.vtk.org/ ) .", "the segmentation of imr images into specific regions , for example , healthy - brain and tumor regions , is first performed manually using 3d slicer ( http://www.slicer.org/ ) and then smoothed to minimize the dependance of the results on segmentation roughness .", "it is clear that performing a manual segmentation in the operating room is not acceptable , and that this process needs to be automated as completely as possible to test the feasibility of our framework online .", "however , while there exist sophisticated segmentation algorithms that could be used [ 5052 ] , in particular for extracting the whole - brain region ( skull and external cerebrospinal fluid masked out ) , the segmentation of the tumor region is still challenging . as mentioned above , the biomechanical model is built , in the present context , from the 1st imr image rather than from the preoperative images . thanks to the use of xfem instead of fem for modeling discontinuities , this biomechanical model can be built offline before the operation starts and does not need to be repeated ( through remeshing ) during the surgery . with respect to fem - based approaches ,", "the execution time thus ceases to be a limiting parameter , which is a remarkable advantage of our approach .", "it thus requires specific techniques , and we use the meshing software tool isosurf ( http://svr-www.eng.cam.ac.uk/~gmt11/software/isosurf/isosurf.html ) . our goal is to model the boundaries of healthy - brain and tumor regions as two connected surfaces meshes .", "however , isosurf can only mesh the boundaries of one or several separate regions , and , thus , does not allow one to mesh connected region boundaries with common nodes at their intersections .", "we thus start by building two separate surfaces meshes that we connect using our own routines based on vtk .", "the two connected triangle surfaces are then jointly meshed into a single volume mesh of tetrahedra that conform to the two surface meshes using gmsh ( http://www.geuz.org/gmsh/ ) .", "further details on the building of the biomechanical model , in particular the building of the connected surface meshes , can be found in .", "a linear elastic law is assigned to the biomechanical model , with young modulus e = 3000  pa and poisson ratio  = 0.45 . because displacements , rather than forces , are applied to the model using a linear formulation , the fem or xfem solution is independent of young modulus e .", "we choose as surface landmarks the whole - brain and internal tumor region boundaries . to evaluate the surface deformations of these region boundaries between two imr images , we use an active surface algorithm [ 55 , 56 ] .", "because these region boundaries to match must be closed surfaces , we thus use as surface landmarks the whole - brain and healthy - brain region boundaries .", "the surface deformation of the internal tumor region boundary will be derived from the active surface algorithm of the healthy - brain region boundary . in our active surface algorithm coming from [ 13 , 47 , 48 ] , the external forces f(x ) are computed using a gradient descent on a distance map of the region boundary . with such external forces , the active surface algorithm is not able to take correctly into account local rigid motion due , as an example , to lateral or tangential movement depending on the head orientation . for the whole - brain region , any rigid transformation that could have occurred", "has already been taken into account by the rigid registration of the imr images ( section 4.1 ) . however , for the healthy - brain region , it can happen that the internal tumor region boundary moves partly in a rigid way .", "therefore , the active surface , initialized from the healthy - brain region boundary in the first imr image , is first locally transformed in a rigid way along the internal tumor region boundary using the iterative closest point transform available in vtk .", "then , this resulting surface is deformed using the active surface algorithm as explained above .", "further details on the local rigid registration of the healthy - brain region boundary can be found in . before applying the displacements whole - brain and internal tumor region boundaries to the biomechanical model nodes ,", "the two surface displacement fields are smoothed based on a weighted - distance average , that is , the displacement of each node is averaged with the displacements of its n closest neighbor nodes .", "this smoothing will make them consistent with each other , and compatible with the volume mesh in order to avoid element flipping , in particular at the intersections between whole - brain and internal tumor region boundaries .", "the displacement fields of the surface landmarks are applied to the biomechanical model , which deforms according to the laws of solid mechanics .", "the equations of solid mechanics are solved using fem or xfem , depending upon the type of circumstances , namely , brain shift or resection .", "we use the fem - software tool metafor ( http://metafor.ltas.ulg.ac.be/ )   developed in our mechanical - engineering department , to which we have added an xfem module .", "the initial stress state of the brain is unknown and is thus set to zero for each fem or xfem computation , as in [ 10 , 13 ] .", "fem discretizes the solid of interest into a mesh , that is , into a set of fes interconnected by nodes , and approximates the displacement field u(x ) by the fem displacement field u(x ) defined as \n ( 1)ufem(x)=iii(x)ui , \n\t\t\t\t\t\t\t where i is the set of nodes , the i(x ) 's are the nodal shape functions ( nsfs ) , and the ui 's are the nodal degrees of freedom ( dofs ) .", "each i(x ) is defined as being continuous on its compact support i , which corresponds to the union of the domains of the fes connected to node i . in our approach , we use linear nsfs .", "in contrast , xfem handles a discontinuity by allowing the displacement field to be discontinuous within fes [ 37 , 5860 ] .", "the xfem displacement field generalises the fem displacement field ( 1 ) with \n ( 2)uxfem(x)=iii(x)ui+iji(x)j=1neigj(x)aji . \n\t\t\t\t\t\t\t", "the first term corresponds to the fem displacement field ( 1 ) , where i is the set of nodes , the i(x ) 's are the fem nsfs , and the ui 's are the nodal fem dofs . the heart of xfem is the  enrichment  that adds a number , n , of dofs aji to each node i of the set j , which is the subset of nodes of i whose support is intersected by the discontinuity of interest .", "these dofs are multiplied by the nsfs i(x ) and the discontinuous functions gj(x ) .", "the use of specific xfem enrichment functions gj(x ) for a node i  j depends on the type of discontinuity , for example , crack , hole , material interface , and so forth , to be modeled .", "suppose that our goal is to model a crack , characterized by a discontinuity in the displacement field ( as opposed to a material interface for instance , characterized by a discontinuity in the derivative of the displacement field ) .", "when the crack fully intersects the support of the node , a simple choice is a piecewise - constant unit function that changes sign at the boundary across the crack , that is , the heaviside function \n ( 3)h(x)={1for  ( xx)en>0,1for  ( xx)en<0 , \n\t\t\t\t\t\t\t where x is again the position of a point of the solid , x * is the position of the point on the crack that is the closest to x , and en is the outward normal to the crack at x * . in case of resection deformation , the goal is to model a discontinuity such that the part of tissues corresponding to tissue removed by the resection has no influence on the deformation of the remaining part of the tissues .", "one is actually interested in the deformation of the remaining part of the tissues only . in that sense ,", "the hole function    as the following equation :  \n ( 4)v(x)={1for  ( xx)en>0,0for  ( xx)en<0 , \n\t\t\t\t\t\t\t could be used as xfem enrichment function , instead of the heaviside function , and would be totally sufficient .", "the results that we would obtain on the remaining part of the tissues would be identical .", "however , because the heaviside function is necessary for retraction modeling , we have used the same function for the resection modeling even if it was not strictly necessary . when minimizing the total deformation energy , the resulting xfem equations remain sparse and symmetric as for fem .", "whereas fem requires a remeshing and the duplication of the nodes along the crack to take into account any discontinuity , xfem requires the identification of the nodes whose support is intersected by the crack and the addition of dofs : ( 1 ) any node whose support is not intersected by the discontinuity remains unaffected and thus possesses three dofs ; ( 2 ) any node whose support is fully intersected by the discontinuity is enriched with three heaviside dofs and thus possesses six dofs . to qualitatively estimate the similarity between two images , we compare the edges extracted from these images using the canny edge detector available in itk ( http://www.itk.org/ ) .  ", "indeed , although potentially useful for the sake of comparing methods on a mathematical basis and defining unique correspondences , landmark - based target analysis presents several relevant limitations in the present setting . having experts picking landmarks introduces significant intra- and interobserver variability . picking landmark points , as ferrant et al .  ", "did , is rather difficult when it comes to define enough visible landmarks  especially in the tumor region  on the 5 different images ( and not 2 images only , as majority of studies focusing on brain shift are using ) . rather than point targets , linear tumor contours , and limits between structures and potential eloquent structures matter most in the practical case of tumor ablation neurosurgery . \n\t\t\t\t\t", "these are the reason why we chose to use the canny edges in order to evaluate the registration . besides", ", while it is true that these edges do not necessarily physically correspond between the successive imr images , these images have been acquired with the same image protocol ( mr sequence , voxel size , grayscale value range ) , which should limit this problem .", "to quantitatively estimate the similarity of the two edge maps , we compute the modified hausdorff distance between the sets of edge points , that is , voxels representing the edges , in these two images .", "the modified hausdorff distance (a , b )   between two sets of points a and b is defined as \n ( 5)(a , b)=max(h(a , b),h(b , a ) )  with  h(a , b)=1naaad(a , b ) , \n\t\t\t\t\t\t\t where the directed hausdorff distance h(a , b ) is a measure of the distance of the point set a to the point set b , na is the number of points in set a , and d(a , b ) is the distance of point a  a to the closest point in b , that is , d(a , b ) = minbb||a  b|| , where ||a  b|| is the euclidean distance .", "the directed hausdorff distance h(a , b ) thus computes the average distance of points of a to points of b. the averaging minimizes the effects of outlier points , for example , due to image noise .", "the value of the modified hausdorff distance (a , b ) increases with the amount of difference between the two sets of edges points . in the following ,", "we denote by (ia , ib ) the modified hausdorff distance of the edges extracted from the whole - brain region of the images ia and ib , that is , with the skull and external cerebrospinal fluid masked out from them ."], ["in this section , we apply our methods , respectively , of brain shift and resection ( imr images are acquired with the 0.5  tesla intraoperative ge signa scanner of the brigham and women 's hospital , boston , usa .", "imr image size is 256  256  60 voxels , and voxel size is 0.9375  0.9375  2.5  mm ) .", "two patient cases , each including five imr images , are treated to illustrate our modeling and brain shift followed by successive resections . in both cases , the 1st imr image was acquired prior to the opening of the skull ; the 2nd imr image was acquired after the opening of the skull and dura , and shows some brain shift ; the 3rd , 4th , and 5th imr images were acquired after successive resections .", "the modelings of brain shift , 1st , 2nd , and 3rd resection are performed using different techniques , as detailed below . except where otherwise noted", ", the following discussion applies to both patient cases ( the result of each deformation modeling is shown for the two patient cases at the end of section 5.2.3 ) . to model brain shift based on the 1st and 2nd imr images", ", we estimate the surface displacement fields of the whole - brain region boundary and the internal tumor region boundary from the two imr images .", "no tissue discontinuity is involved in the brain shift deformation , so the biomechanical model is deformed using fem .", "this results in the volume displacement field of the biomechanical model , which is illustrated in figure 3 for the first patient case .", "this volume displacement field is used to warp the part of the 1st imr image corresponding to the whole - brain region . in the following sections ,", "the three successive resections are modeled separately , because they require different types of processing .", "matching two region boundaries to get a displacement field makes sense only if they correspond to the same physical entity .", "once the resection has started , we can no longer rely on the entirety of the whole - brain region boundary , since a part of it is now missing . for modeling the successive resections , we thus evaluate the displacement field for the boundary of the healthy - brain region only .", "the 1st resection occurs between the times the 2nd and 3rd imr images are acquired .", "however , since the corresponding removal of tissues is most likely accompanied by deformation , one can not exactly determine what tissue is removed based just on the two imr images .", "we thus decided to model the 1st resection by still relying on the displacement fields of key surfaces , here the healthy - brain region boundary , to deform the biomechanical model .", "this indeed appears to be the only reliable information concerning the deformation due to resection that we can extract from the 2nd and 3rd imr images .", "consequently , we do not model explicitly the removal of tissue , but we model directly the deformation resulting from it , without introducing any tissue discontinuity . using the surface displacement field of the healthy - brain region boundary , we compute the deformation of the biomechanical model via fem . then , using the resulting volume displacement field , we warp the part of the 2nd imr image corresponding to the whole - brain region , in the same way as we did in the case of for brain shift .", "the image resulting from the 1st resection modeling is now registered to the 3rd imr image , except outside of the healthy - brain region boundary , that is , for the tumor region . finally , we alter the resulting image to reflect the effect of resection . for this", ", we assign the background color to the voxels corresponding to the resected tissue volume ", "the significant feature of the 2nd resection is that some tissue has already been removed by the 1st resection , which means that this tissue can not have any physical influence on subsequent brain deformations because it does not ", "recall that the biomechanical model has been deformed to model the brain shift and the 1st resection and is thus registered to the 3rd imr image .", "so , using the 3rd imr image , we can define the boundary of the 1st resection , that is , the tissue discontinuity to include in the deformed biomechanical model ( figures 4(a ) and 4(b ) ) .", "we then enrich the nodes whose supports are intersected by the discontinuity with heaviside dofs .", "consequently , when the xfem - based biomechanical model deforms , the part corresponding to tissue removed by the 1st resection has no influence on the deformation of the remaining part of the brain .", "for the first patient case illustrated in figure 4 , the tetrahedron mesh consists of 3,317 nodes , which corresponds to 9,951 fem dofs .", "the biomechanical model is deformed in accordance with the displacement field of the healthy - brain region boundary evaluated from the 3rd and 4th imr images .  ", "the bottom part of the mesh , representing the tissue remaining after the 1st resection , has been deformed according to the displacement field of the healthy - brain region boundary , while the top part , representing the tissue removed by the 1st resection , has been subjected to a translation , but only for visualization purposes . even though the mesh is displayed as two separate parts , it is , in fact , a single entity .", "indeed , a main feature of xfem is its ability to handle the effect of a discontinuity without modifying the underlying mesh , that is , without remeshing . for modeling the 2nd resection , the edges of fes straddling the discontinuity", "have been made discontinuous and their nodes moved apart . using the xfem volume displacement field , we warp the part of the 3rd imr image corresponding to the whole - brain region .", "the resulting image is then masked out with the whole - brain region segmented out from the 4th imr image .", "one significant feature of the procedure described for modeling the 2nd resection is that it can be applied repetitively for each subsequent resection visible on successive imr images , no matter how many there are .", "the modeling of the 3rd resection is thus identical to the modeling of the 2nd resection .", "the tissue discontinuity due to the 2nd resection is defined from the 4th imr image , and used to appropriately enrich the nodes of the biomechanical model .", "then , this biomechanical model is deformed using xfem , in accordance with the displacement field of the healthy - brain region boundary evaluated from the 4th and 5th imr images . for the first patient case ,", "a simplification for the modeling of the 3rd resection can be made because , by the time the 5th imr image is acquired , the resection is complete .", "this means that we only need to compute the volume displacement field of the healthy - brain region .", "since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem will be identical .", "using the fem ( for the first patient case ) or xfem ( for the second patient case ) volume displacement field , we warp the part of the 4th imr image corresponding to the whole - brain region . the resulting image is then masked out with the whole - brain region segmented out from the 5th imr image .", "figures 5 and 6 show the results of warping the imr images , as well as the edges extracted from them , after brain shift and each successive resection modeling for the two patient cases . as explained in section 5.2.3 , since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem are identical in the healthy - brain region .", "one can deduce that using xfem for modeling resection is interesting when the neurosurgeon needs to have an accurate displacement field of the remaining tumor tissues . in this case", ", it is interesting to evaluate the impact of using fem , instead of xfem , to model the resection as if no resection was performed before .", "using fem for modeling resection is equivalent to ignoring the presence of resection on intraoperative images . to illustrate the comparison between fem and xfem results , we choose the 3rd resection modeling of the second patient case", "indeed , it is the deformation with remaining tumor tissues that shows the largest magnitude , and , thus , that is likely to give a maximum difference between the two computations .  ", "the healthy - brain and tumor regions segmented out from the 4th and 5th imr images are respectively shown in figures 7(a ) and 7(b ) .", "the volume displacement fields of the biomechanical model using xfem and fem are respectively shown in figures 7(c ) and 7(d ) .", "the part of the 4th imr image corresponding to the whole - brain region is warped , first with the volume displacement field obtained via fem , and then with that obtained via xfem .", "the difference between the two warped images is shown in figure 7(e ) . as expected", "however , the difference between the two volume displacement fields is smaller than the image resolution ( although the difference between the two volume displacement fields is smaller than the image resolution , the difference between the images resulting of the warping using these two volume displacement fields is nonzero .", "this is explained by the fact that the ( gray ) value of each voxel of the warped image is defined as a weighted - value of voxels of the original image .", "the weights are defined based on the overlapping ratio of the voxel of the warped image , with voxels ( determined using the volume displacement field ) of the original image ) .", "in addition , the deformed 4th imr images , using the xfem- and the fem - based deformations of the biomechanical model , show the same similarity , computed based on the modified hausdorff distance , with the 5th imr .", "two reasons explain that the differences between the fem and xfem results are so small .", "first , the brain deformation itself due to the 3rd resection is small , and , thus , it is expected to obtain small differences between the two resulting brain deformations .", "second , in the case the remaining tumor tissues are close to the healthy - brain region boundary , it implies that they are close to the boundary where surface displacement fields are applied to drive the deformation of the biomechanical model .", "although this comparison between fem and xfem should be done on more patient cases , we suggest that , in first approximation , fem could be used for modeling resection cases with small brain deformations .", "nevertheless , the presentation of the successive resections using xfem shows the generality of our framework , and details how xfem is implemented .", "note that in section 6 devoted to validation , the warped images are the ones deformed with xfem ."], ["for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images that are already rigidly registered , we compare the similarity between these ik and ik+1 images , as well as the similarity between the ik and ik+1 images , where ik is the result of warping ik .", "this gives us an estimate of how well we are able to capture , and compensate for , the local deformations between ik and ik+1 .", "the goal of the nonrigid registration is , however , to deform the preoperative images . by warping ik for each deformation modeling", ", we do not take into account the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . to evaluate the effect of this error amplification on the results", ", we also perform the required succession of warpings on i1 , and we denote the resulting image by i1,k .", "we then compare , for each deformation modeling , the similarity between i1 and ik+1 , together with the similarity between i1,k and ik+1 .", "this allows one to evaluate the propagation , that is , the amplification , of alignment error on the results .", "the modified hausdorff distance computed for each pair of imr images are given in tables 1 and 2 . \n", "table 1 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (ik , ik+1 ) and (ik , ik+1 ) .", "these values are computed using the canny edges extracted from the pair of images ( ik , ik+1 ) ( figures 5 ( d ) and 6 ( d ) ) and ( ik , ik+1 ) ( figures 5 ( e ) and 6 ( e ) ) .", "we observe that the values for the images nonrigidly registered are relatively constant , that is , 1  mm , for each deformation modeling .", "six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are ( rigidly and subsequently ) nonrigidly registered .", "however , the modified hausdorff distance increases for the 3rd resection modeling of the first patient case , as well as for the brain shift modeling of the second patient case . to understand", "if the nonrigid registration is responsible for the increase of the misalignment of the two imr images everywhere in the whole - brain region , or if this effect is localized , we compute the modified hausdorff distance in the region and neighborhood of the tumor only ( volume region that extents by 25  mm the tumor region segmented in i1 for both patient cases ) .", "the modified hausdorff distance decreases from (i4 , i5 ) = 1.70  mm to (i4 , i5 ) = 1.37  mm for the first patient case , while it decreases from (i1 , i2 ) = 1.36  mm to (i1 , i2 ) = 1.28  mm for the second patient case .", "this indicates that the nonrigid registration enhances the alignment of the two imr images within the tumor region and its neighborhood , which is in fact the location requiring the best modeling accuracy .", "this behavior could be explained by the fact that a maximum of information from the imr images is used in this region , that is , one or two ( in case of brain shift modeling ) surface displacement fields are applied around it .", "the increase of misalignment elsewhere in the brain volume could be explained by two reasons .", "first , the landmarks tracked from the imr images are surfaces . as a consequence , the nonrigid registration is expected to give better results near the tracked surfaces than far from them in the volume .", "the volume misalignment could point out the need for better parameters values and/or other constitutive laws . \n", "table 2 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (i1 , ik+1 ) and (i1,k , ik+1 ) .", "so far , igns systems allow one to rigidly register preoperative and successive imr images .", "(i1 , ik+1 ) thus represents the navigation accuracy that we can obtain with an igns system at the present time .", "the comparison of (i1 , ik+1 ) with (i1,k , ik+1 ) gives the improvement that could be practically achieved in the alignment with our approach .", "as expected , table 2 shows that the igns accuracy decreases through the successive deformations .", "indeed , the modified hausdorff distance increases from (i1 , i2 ) = 1.24  mm to (i1 , i5 ) = 1.78  mm for the first patient case , and from (i1 , i2 ) = 1.01  mm to (i1 , i5 ) = 1.68  mm for the second patient case .", "six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are nonrigidly registered .", "to understand if the modified hausdorff distance increases everywhere in the whole - brain region for the brain shift and 1st resection modeling of the second patient case , we compute the modified hausdorff distance in the neighborhood of the tumor region ( in the same way as explained for table 1 ) , and observe the improvement of the alignment within the tumor region and its neighborhood . as opposed to the values of the modified hausdorff distances in table 1 , the values for the images nonrigidly registered in table 2 increase through the successive resection modeling .", "this amplification error is due to the fact that , after having modeled brain deformation between a pair of imr images , the deformed biomechanical model is not in perfect alignment with the second image of the pair .", "since , for the subsequent deformation modeling , the surface landmarks are initialized based on the deformed biomechanical model , this can thus ampliy a misregistration error ."], ["we developed a complete 3d framework for serial preoperative image update in the presence of brain shift followed by successive resections .", "the nonrigid registration technique used an homogeneous linear elastic biomechanical model , driven by the deformations of whole - brain and internal tumor region boundaries for brain shift modeling , and healthy - brain region boundary for resection modelings , tracked between successive imr images .", "the biomechanical model was deformed using fem for brain shift modeling , and fem or xfem for resection modeling , depending upon whether some brain tissues were previously resected or not .", "we showed that our approach was modular , and could be applied each time a new imr image is acquired .", "we used a linear formulation to characterize the deformation of the brains of both patients because the brains underwent relatively small deformations and displacements .", "while nonlinear biomechanical models have proven effective to decrease  yet do not abolish  the inaccuracies of fem - based modeling methods of large brain deformations , the deformations observed in our patients during surgery remained moderate ( 47  mm ) , thus reducing the theoretical benefit of using nonlinear models .", "this allowed us to use simpler linear models and focus on the added value of xfem to simultaneously account for surgical deformations , namely , shift and resection . using a linear formulation", "implied that , for each new deformation modeling , one could use the initial configuration rather than the last - deformed configuration of the biomechanical model .", "this had the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively degraded with each successive deformation modeling .", "this also had the advantage that we did no longer need to reconnect the deformed mesh for each new xfem calculation , which was one drawback of our previous method , presented in [ 39 , 41 ] , where the biomechanical model was successively deformed .", "we also showed how xfem could handle a discontinuity for modeling resection without any remeshing or mesh adaptation while the representation of the discontinuity remained accurate , that is , the representation of the discontinuity was not based on a jagged topology using fe facets .", "xfem thus also avoided making the mesh resolution richer in the neighborhood of the resection - cavity boundary for improving the accuracy of the representation of the discontinuity for that purpose only .", "we showed that our nonrigid registration technique improved the alignment of the successive imr images for most of the deformation modeling of both patient cases . when our nonrigid registration failed , it still improved the alignment locally , that is , within the tumor region and its neighborhood .", "we tested the explicit modeling of the lateral ventricles ' region with a soft , compressible law in addition to the whole - brain region law used in the homogeneous biomechanical model .", "in addition to the validation that is usually performed for successive deformation modelings , that is , validation between pairs of successive intraoperative images , shown in table 1 of section 6 or in the work of ferrant et al .", "[ 13 , 47 , 48 ] , we also evaluated the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . as a result , shown in table 2 of section 6", ", we showed that our approach suffered from the propagation of misregistration through the successive deformation modelings .", "we expected that this was due , at least partly , to the algorithms used to evaluate intraoperative surface displacements fields from the whole - brain and healthy - brain region boundaries .", "the surface displacement fields were computed using active surface algorithms , and smoothed to make them compatible with the biomechanical model .", "because of these two smoothings , the deformed biomechanical model was likely to not be in a perfect alignment with the imr image to which it was registered .", "because the surface displacement fields evaluated for the next deformation modeling were initialized based on the deformed biomechanical model , we expected to observe an amplification of the misregistration , which was confirmed by our quantitative evaluation . at the present time", "though , commercial igns systems allow one to register preoperative images and successive imr images , but in a rigid way only .", "consequently , although the effect of error amplification exists , our technique still enhances the current capabilities of commercial igns systems .", "future work on modeling of brain shift followed by successive resection is required in five main areas .", "first , the effect of error amplification through the successive brain deformation modelings calls for further research .", "consequently , the segmentation , and the subsequent smoothing , as well as the evaluation of surface displacement fields , should be improved to minimize the effect of error amplification .", "second , further research is required to include additional structures in the biomechanical model in general , and to study the best way to include the lateral ventricles in particular .", "the use of a poroelastic model in order to model the cerebrospinal fluid filling the ventricles could be considered [ 17 , 18 ] .", "indeed , these images provide volume information ( rather than surface information only ) , are of good quality in comparison to other intraoperative modalities , and possess a field of view that includes the full volume of brain tissues ( for the 0.5 tesla ge signa scanner ) .", "these images thus allow one to evaluate what , and how , new structures of the brain could be used , to enhance the modeling of brain shift .", "some regions , for example , the lateral ventricles ' region , could be extracted from the two imr images , and used as surface landmarks to drive the deformation of the biomechanical model [ 13 , 62 ] . indeed , the workflow presented in this paper has the advantage of being easily adaptable . in case", "the tumor region would not be visible ( enough ) on the imr images , these new structures , easier to segment , could also adequately replace the tumor for driving the deformation .", "fourth , our global approach should no longer be based on the 1st imr image used as a substitute for preoperative images , but on the preoperative images themselves .", "fifth , we should implement , for the surgery cases involving large deformations of the brain , a nonlinear formulation of fem [ 63 , 64 ] , and , particularly , a nonlinear formulation of xfem , which is the subject of recent research [ 65 , 66 ] ."]]}
{"article_id": "PMC4189468", "article_text": ["the contribution to human health of the specific fatty acid ( fa ) composition of the diet has received considerable attention in the literature .", "fatty acids are key nutrients that affect early growth and development as well as the prevention of chronic disease in later life.1 among the fas , omega-3 polyunsaturated fatty acids ( n-3 pufa ) and omega-6 polyunsaturated fatty acids ( n-6 pufa ) have been suggested to decrease and increase several human diseases , respectively .", "pufa that contains more than one carbon double bond consists of two major classes such as n-6 and n-3 ( fig .", "linoleic acid ( la ) is a representative n-6 pufa , serving as a substrate to be converted into an arachidonic acid ( aa ) affecting the prevalence and severity of inflammation .", "n-6 pufa can induce cardiovascular disease , diabetes , cancer , and age - related disease.1 \n -linolenic acid ( ala ) , eicosapentaenoic acid ( epa 20:5 ) and docosahexaenoic acid ( dha 22:6 ) are important n-3 pufa involved in human .", "they are essential fatty acids that can not be synthesized by mammals , by which must be obtained from dietary sources such as cold - water fish , certain seeds ( flax ) and nuts ( walnuts ) .", "a lot of studies suggest that n-3 pufa , as diet - dependent factors , may be critical to preventing disease backed up with authentic antioxidative and anti - inflammatory actions . especially , n-3", "pufa has been shown to exert beneficial effects on some chronic degenerative diseases such as cardiovascular disease,2,3 rheumatoid arthritis,4 diabetes,5 other autoimmune diseases,6,7 and cancer.8,9 increased fat consumption by western diet has been associated with the development of cancer such as breast , colon , pancreatic , and prostate cancers with the notable exception of n-3 pufa , which show to have multiple beneficial anti - tumor actions that affect the essential alterations that dictate malignant growth in a number of studies.10 a diet rich in n-3 pufa may protect from cancer , at least at certain sites .", "studies on the fatty acid status of patients with several cancer types including bladder , pancreatic , lung and esophageal cancer show low concentrations of plasma phospholipid n-3 pufa , ranging from 55 to 88% of amounts in healthy individuals.1113 recent studies have found a positive association between n-6 pufa and cancer risk , whereas in the same model , n-3 pufa were shown to reduce the development of cancer .", "epidemiological studies suggest that a high n-3 pufa to n-6 pufa ratio may be the optimal strategy to decrease breast cancer risk.14 solid epidemiological study shows that consumption of n-3 pufa appears to protect against the development of hepatocellular carcinoma , even among patients with hepatitis b virus ( hbv ) and/or hepatitis c virus ( hcv ) infection.15 recently , zhennan et al .", "has reviewed prospective studies investigating the possible protective effects of the dietary intake of n-3 pufa on prostate cancer development.16 fasano et al also has reviewed a lot of in vivo and in vitro experimental studies providing strong indications of the anti - tumor action of n-3 pufa against lung cancer.17 the purpose of this review is to discuss the potential role of n-3 pufa in gastrointestinal ( gi ) cancer development .", "we believe that increased consumption of n-3 pufa may lower the risk of gi cancer development via various chemopreventive activities .", "future studies should include combination treatment of n-3 pufa and nutrients with different and complementary mechanisms of chemopreventive action .", "among various cancers , most of the gi cancers including esophageal cancer , stomach cancer , and colon cancer , have a natural history of multi - step transition from precursor lesions to malignant lesions , inflammation , adenoma formation , dysplastic changes.18 therefore , gi cancers usually have premalignant lesions before developing invasive cancers , for instances , barrett s esophagus for esophageal cancer , chronic atrophic gastritis accompanied with intestinal metaplasia for gastric cancer , and adenoma or dysplasia originating from chronic ulcerative colitis for colon cancer .", "because the western diet contains disproportionally high amounts of n-6 pufa and low amounts of n-3 pufa , denoted as a high n-6 to n-3 pufa ratio , n-3 pufa may feasibly play a role in several stages of gi cancers management .", "esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) ,", "esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades .", "suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus .", "in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) .", "moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa . gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease .", "some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer .", "recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23 \n h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer .", "it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 \n in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach .", "colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer", ", crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer . recently", "there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer .", "although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk .", "dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer .", "kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) .", "there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer .", "the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours .", "studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies .", "similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc .", "the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9", "esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) ,", "esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades .", "suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus .", "in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) .", "moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa .", "gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease . some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer .", "recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23 \n h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer .", "it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 \n in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach .", "colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer ,", "crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer .", "there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer .", "although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk .", "dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer .", "kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) .", "there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer .", "the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours .", "studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies .", "similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc .", "the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9", "n-3 pufa , especially , epa and dha , have been shown to have multiple anti - tumor actions .", "current knowledge of the anti - tumor activity of n-3 pufa has been comprehensively reviewed elsewhere.9,16,39 recently , stephenson et al.10 has reviewed more recent underlying mechanisms providing strong indications of the anti - tumor actions of n-3 pufa on the hallmarks of cancer .", "n-3 pufa appear to down - regulate epidermal growth factor receptor ( egfr ) , protein kinase c ( pkc ) , ras , and nf-b , insulin like growth factor ( igf ) , which are important cell signaling mediators often found to be elevated in carcinogenesis .", "secondly , n-3 pufa induces cancer cell apoptosis via modulation of peroxisome proliferator - activated receptors ( ppars ) , the bcl-2 family , and nf-b cell signaling .", "thirdly , n-3 pufa decreases sprouting angiogenesis by suppressing vascular endothelial growth factor ( vegf)- and platelet derived growth factor ( pdgf)-stimulated endothelial cell proliferation , migration , and tube formation and by inhibition of mmps via no production and nf-b and -catenin cell signaling .", "fourthly , n-3 pufa also decreases cell - cell adhesion via down regulation of rho - gtpase , which inhibits cytoskeleton reorganisation , and reduction in intercellular adhesion molecule ( icam)-1 and vascular cell adhesion molecule ( vcam)-1 expression .", "cockbain et al.9 has also proposed the four main anti - tumor actions of n-3 pufa ( i ) modulation of cox activity ; ( ii ) alteration of membrane dynamics and cell surface receptor function ; ( iii ) increased cellular oxidative stress and ( iiii ) derived anti - inflammatory lipid mediators .", "firstly , n-3 pufa can act as an alternative substrate for cox-2 , instead of aa , leading to a reduction in formation of pro - tumorigenic ", "secondly , incorporation of n-3 pufa into cell membranes alters the fluidity , structure and/or function of lipid rafts or calveolae .", "especially , the localization of cell surface receptors , such as g protein - coupled receptors ( gpcrs ) , toll - like receptors ( tlrs ) , and epidermal growth factor receptor ( egfr ) , in lipid rafts is believed to be crucial for downstream receptor signaling , controlling proliferation and apoptosis .", "thirdly , n-3 pufa may have an anti - tumor effect through alteration in the cellular redox state .", "n-3 pufa can increase reactive oxygen species ( ros ) because it is highly peroxidisable .", "therefore , n-3 pufa can induce cancer cell apoptosis via elevation of intracellular ros levels .", "fourthly , n-3 pufa can be metabolized novel anti - inflammatory lipid mediators including resolvins , protectins and maresins .", "it is known that resolvins exhibit antineoplastic activity via anti - inflammatory and inflammation resolution activity in animal models of acute inflammation . besides these multiple anti - tumor actions", ", it is also reported recently that n-3 pufa can activate nrf2 and induced nrf2-directed gene expression40,41 and can suppress lipopolysaccharide - induced inflammation through induction of nrf2 expression.42 nuclear factor erythroid 2-related factor 2 ( nrf2 ) is a redox - sensitive master regulatory transcriptional factor that plays an important protective role in cells by regulating cellular redox balance.43 moreover , n-3 pufa significantly reduces oxidative stress - induced endothelial cell ca influx .", "this effect might be associated , at least in part , with altered lipid composition in membrane lipid rafts.44 recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate .", "this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations .", "hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis .", "since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice .", "nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties .", "song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice .", "recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice .", "they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers ,", "the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) .", "fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice .", "a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown . moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion , several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development .", "the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55", "recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate .", "this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations .", "hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis .", "since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice .", "nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties .", "song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice .", "recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice .", "they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers , the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) .", "fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice .", "a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown .", "moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion ,", "several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development .", "the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55", "gi cancer incidence and mortality are increasing in the eastern world and a high n-6 to n-3 pufa ratio in the western style diet may be a contributing factor .", "there is much evidence to suggest that higher consumption of dietary n-3 pufa is associated with a lower risk of gi cancer in animal models and humans .", "especially , recent studies suggest that endogenous n-3 pufa delay the progression of colon and stomach cancer and elevating n-3 pufa may be an important strategy to delay / prevent gastrointestinal cancer in high - risk patients via various mechanisms mediating cancer prevention by n-3 pufa ( fig .", "2 ) . in addition , using n-3 pufa in combination with other agents with complementary antitumor action may improve their efficacy in gi cancer prevention . recently ,", "manson et al . has started the vitamin d and omega-3 trial ( vital ) , a large randomized , double - blind , placebo - controlled , 22 factorial trial of vitamin d and n-3 pufa supplements in the primary prevention of cancer among a multi - ethnic population of 20,000 u.s .", "men aged 50 and women aged 55.56 we expect that new findings in combination consumption of n-3 pufa with other nutrients will provide new approaches to public health implications with regard to prevention of gi cancer through dietary and lifestyle interventions ."], "abstract_text": ["<S> omega-3 polyunsaturated fatty acids ( n-3 pufas ) , particularly eicosapentanoic acid ( epa ) and docosahexanoic acid ( dha ) , has been acknowledged as essential very long - chain fatty acids contributing to either achieving optimal health or protection against diseases , and even longevity . </S>", "<S> recent high impact studies dealing with epa and dha have sparked a renewed interest in using n-3 pufas for cancer prevention and cancer treatment , for which n-3 pufas may exert their anticancer actions by influencing multiple targets implicated in various stages of cancer development , including cell proliferation , cell survival , angiogenesis , inflammation , and metastasis against various cancers . </S>", "<S> however , gastrointestinal cancers develop implicated with the close connection between inflammation and cancer and n-3 pufas especially imposed excellent actions of antiinflammation and antioxidation as well as their restorative actions . in detail , these beneficial lipids can restore or modify inflammation - associated lipid distorsion and alteration of lipid rafts . although the chemopreventive effect of n-3 pufas has been studied in various experimental models , our understanding regarding the underlying mechanisms of n-3 pufas against gi cancer is still limited . in this review article </S>", "<S> , we described the in - detailed perspective and underlying mechanism of n-3 pufas application for gi cancers and added in vivo efficacy of n-3 pufas with fat-1 transgenic mice experience . </S>", "<S> we suggest that future work should consider the n-6/n-3 fa ratio , combination treatment of other nutritions and alteration of lipid rafts to be a key element in experimental design and analysis . </S>"], "labels": null, "section_names": ["INTRODUCTION", "THE EFFICACIES OF N-3 PUFA IN PREVENTION OF GI CANCERS", "Esophageal cancer", "Gastric cancer", "Colorectal cancer", "MECHANISMS OF N-3 PUFAS ON CHEMOPREVENTION", "Useful animal model to study chemopreventive mechanism of n-3 PUFA: ", "CONCLUSIONS"], "sections": [["the contribution to human health of the specific fatty acid ( fa ) composition of the diet has received considerable attention in the literature .", "fatty acids are key nutrients that affect early growth and development as well as the prevention of chronic disease in later life.1 among the fas , omega-3 polyunsaturated fatty acids ( n-3 pufa ) and omega-6 polyunsaturated fatty acids ( n-6 pufa ) have been suggested to decrease and increase several human diseases , respectively .", "pufa that contains more than one carbon double bond consists of two major classes such as n-6 and n-3 ( fig .", "linoleic acid ( la ) is a representative n-6 pufa , serving as a substrate to be converted into an arachidonic acid ( aa ) affecting the prevalence and severity of inflammation .", "n-6 pufa can induce cardiovascular disease , diabetes , cancer , and age - related disease.1 \n -linolenic acid ( ala ) , eicosapentaenoic acid ( epa 20:5 ) and docosahexaenoic acid ( dha 22:6 ) are important n-3 pufa involved in human .", "they are essential fatty acids that can not be synthesized by mammals , by which must be obtained from dietary sources such as cold - water fish , certain seeds ( flax ) and nuts ( walnuts ) .", "a lot of studies suggest that n-3 pufa , as diet - dependent factors , may be critical to preventing disease backed up with authentic antioxidative and anti - inflammatory actions . especially , n-3", "pufa has been shown to exert beneficial effects on some chronic degenerative diseases such as cardiovascular disease,2,3 rheumatoid arthritis,4 diabetes,5 other autoimmune diseases,6,7 and cancer.8,9 increased fat consumption by western diet has been associated with the development of cancer such as breast , colon , pancreatic , and prostate cancers with the notable exception of n-3 pufa , which show to have multiple beneficial anti - tumor actions that affect the essential alterations that dictate malignant growth in a number of studies.10 a diet rich in n-3 pufa may protect from cancer , at least at certain sites .", "studies on the fatty acid status of patients with several cancer types including bladder , pancreatic , lung and esophageal cancer show low concentrations of plasma phospholipid n-3 pufa , ranging from 55 to 88% of amounts in healthy individuals.1113 recent studies have found a positive association between n-6 pufa and cancer risk , whereas in the same model , n-3 pufa were shown to reduce the development of cancer .", "epidemiological studies suggest that a high n-3 pufa to n-6 pufa ratio may be the optimal strategy to decrease breast cancer risk.14 solid epidemiological study shows that consumption of n-3 pufa appears to protect against the development of hepatocellular carcinoma , even among patients with hepatitis b virus ( hbv ) and/or hepatitis c virus ( hcv ) infection.15 recently , zhennan et al .", "has reviewed prospective studies investigating the possible protective effects of the dietary intake of n-3 pufa on prostate cancer development.16 fasano et al also has reviewed a lot of in vivo and in vitro experimental studies providing strong indications of the anti - tumor action of n-3 pufa against lung cancer.17 the purpose of this review is to discuss the potential role of n-3 pufa in gastrointestinal ( gi ) cancer development .", "we believe that increased consumption of n-3 pufa may lower the risk of gi cancer development via various chemopreventive activities .", "future studies should include combination treatment of n-3 pufa and nutrients with different and complementary mechanisms of chemopreventive action ."], ["among various cancers , most of the gi cancers including esophageal cancer , stomach cancer , and colon cancer , have a natural history of multi - step transition from precursor lesions to malignant lesions , inflammation , adenoma formation , dysplastic changes.18 therefore , gi cancers usually have premalignant lesions before developing invasive cancers , for instances , barrett s esophagus for esophageal cancer , chronic atrophic gastritis accompanied with intestinal metaplasia for gastric cancer , and adenoma or dysplasia originating from chronic ulcerative colitis for colon cancer .", "because the western diet contains disproportionally high amounts of n-6 pufa and low amounts of n-3 pufa , denoted as a high n-6 to n-3 pufa ratio , n-3 pufa may feasibly play a role in several stages of gi cancers management .", "esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) ,", "esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades .", "suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus .", "in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) .", "moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa . gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease .", "some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer .", "recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23 \n h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer .", "it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 \n in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach .", "colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer", ", crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer . recently", "there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer .", "although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk .", "dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer .", "kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) .", "there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer .", "the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours .", "studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies .", "similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc .", "the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9"], ["esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) ,", "esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades .", "suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus .", "in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) .", "moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa ."], ["gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease . some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer .", "recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23 \n h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer .", "it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 \n in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach ."], ["colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer ,", "crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer .", "there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer .", "although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk .", "dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer .", "kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) .", "there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer .", "the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours .", "studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies .", "similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc .", "the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9"], ["n-3 pufa , especially , epa and dha , have been shown to have multiple anti - tumor actions .", "current knowledge of the anti - tumor activity of n-3 pufa has been comprehensively reviewed elsewhere.9,16,39 recently , stephenson et al.10 has reviewed more recent underlying mechanisms providing strong indications of the anti - tumor actions of n-3 pufa on the hallmarks of cancer .", "n-3 pufa appear to down - regulate epidermal growth factor receptor ( egfr ) , protein kinase c ( pkc ) , ras , and nf-b , insulin like growth factor ( igf ) , which are important cell signaling mediators often found to be elevated in carcinogenesis .", "secondly , n-3 pufa induces cancer cell apoptosis via modulation of peroxisome proliferator - activated receptors ( ppars ) , the bcl-2 family , and nf-b cell signaling .", "thirdly , n-3 pufa decreases sprouting angiogenesis by suppressing vascular endothelial growth factor ( vegf)- and platelet derived growth factor ( pdgf)-stimulated endothelial cell proliferation , migration , and tube formation and by inhibition of mmps via no production and nf-b and -catenin cell signaling .", "fourthly , n-3 pufa also decreases cell - cell adhesion via down regulation of rho - gtpase , which inhibits cytoskeleton reorganisation , and reduction in intercellular adhesion molecule ( icam)-1 and vascular cell adhesion molecule ( vcam)-1 expression .", "cockbain et al.9 has also proposed the four main anti - tumor actions of n-3 pufa ( i ) modulation of cox activity ; ( ii ) alteration of membrane dynamics and cell surface receptor function ; ( iii ) increased cellular oxidative stress and ( iiii ) derived anti - inflammatory lipid mediators .", "firstly , n-3 pufa can act as an alternative substrate for cox-2 , instead of aa , leading to a reduction in formation of pro - tumorigenic ", "secondly , incorporation of n-3 pufa into cell membranes alters the fluidity , structure and/or function of lipid rafts or calveolae .", "especially , the localization of cell surface receptors , such as g protein - coupled receptors ( gpcrs ) , toll - like receptors ( tlrs ) , and epidermal growth factor receptor ( egfr ) , in lipid rafts is believed to be crucial for downstream receptor signaling , controlling proliferation and apoptosis .", "thirdly , n-3 pufa may have an anti - tumor effect through alteration in the cellular redox state .", "n-3 pufa can increase reactive oxygen species ( ros ) because it is highly peroxidisable .", "therefore , n-3 pufa can induce cancer cell apoptosis via elevation of intracellular ros levels .", "fourthly , n-3 pufa can be metabolized novel anti - inflammatory lipid mediators including resolvins , protectins and maresins .", "it is known that resolvins exhibit antineoplastic activity via anti - inflammatory and inflammation resolution activity in animal models of acute inflammation . besides these multiple anti - tumor actions", ", it is also reported recently that n-3 pufa can activate nrf2 and induced nrf2-directed gene expression40,41 and can suppress lipopolysaccharide - induced inflammation through induction of nrf2 expression.42 nuclear factor erythroid 2-related factor 2 ( nrf2 ) is a redox - sensitive master regulatory transcriptional factor that plays an important protective role in cells by regulating cellular redox balance.43 moreover , n-3 pufa significantly reduces oxidative stress - induced endothelial cell ca influx .", "this effect might be associated , at least in part , with altered lipid composition in membrane lipid rafts.44 recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate .", "this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations .", "hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis .", "since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice .", "nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties .", "song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice .", "recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice .", "they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers ,", "the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) .", "fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice .", "a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown . moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion , several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development .", "the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55"], ["recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate .", "this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations .", "hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis .", "since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice .", "nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties .", "song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice .", "recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice .", "they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers , the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) .", "fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice .", "a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown .", "moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion ,", "several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development .", "the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55"], ["gi cancer incidence and mortality are increasing in the eastern world and a high n-6 to n-3 pufa ratio in the western style diet may be a contributing factor .", "there is much evidence to suggest that higher consumption of dietary n-3 pufa is associated with a lower risk of gi cancer in animal models and humans .", "especially , recent studies suggest that endogenous n-3 pufa delay the progression of colon and stomach cancer and elevating n-3 pufa may be an important strategy to delay / prevent gastrointestinal cancer in high - risk patients via various mechanisms mediating cancer prevention by n-3 pufa ( fig .", "2 ) . in addition , using n-3 pufa in combination with other agents with complementary antitumor action may improve their efficacy in gi cancer prevention . recently ,", "manson et al . has started the vitamin d and omega-3 trial ( vital ) , a large randomized , double - blind , placebo - controlled , 22 factorial trial of vitamin d and n-3 pufa supplements in the primary prevention of cancer among a multi - ethnic population of 20,000 u.s .", "men aged 50 and women aged 55.56 we expect that new findings in combination consumption of n-3 pufa with other nutrients will provide new approaches to public health implications with regard to prevention of gi cancer through dietary and lifestyle interventions ."]]}
{"article_id": "PMC4855955", "article_text": ["sexual development in males is a multistep process , implying a delicate network of molecular events that directs the bipotential gonad to differentiate into testis ( sex determination ) and consequent differentiation of internal and external genitalia in the presence of testicular hormones ( sex differentiation ) .", "alteration of any genetic or endocrine factors involved in testicular development may lead to 46 xy disorders of sexual development ( dsd ) .", "genital ambiguity in patients with 46 xy dsd may either occur due to the disorders in gonadal development or due to the disruption in androgen synthesis or action .", "patients with disorders of gonadal development may present with either complete or partial forms of gonadal dysgenesis .", "swyer 's syndrome or complete gonadal dysgenesis ( cgd ) is characterized by unambiguous female genitalia , bilateral streak gonads , and elevated gonadotropins .", "partial gonadal dysgenesis ( pgd ) may have a wide spectrum of phenotypes associated with ambiguous genitalia , varying degree of hypospadias and testis of variable size and echotexture present along the path of decent .", "ovotesticular dsd ( ot - dsd ) is a rare condition marked by the presence of both ovarian and testicular tissue in an individual while congenital bilateral anorchia ( cba ) is characterized by the complete absence of testicular tissue in genotypic males .", "disorders of androgen synthesis may occur due to deficiency of various enzymes involved in the steroidogenesis .", "congenital adrenal hyperplasia ( cah ) due to 17-hydroxysteroid dehydrogenase 3 ( 17-hsd3 ) is caused by the mutations in hsd17b3 gene which is required for the conversion of androstenedione to testosterone .", "deficiency of this enzyme results in feminized genitalia in newborns due to the low testosterone levels .", "mutations in srd5a2 gene impairs the activity of enzyme 5-reductase and hence the conversion of testosterone to its more potent form dihydrotestosterone ( dht ) .", "individuals with 5-reductase deficiency ( 5-rd ) are often raised as females due to undermasculinized genitalia but the gender role changes soon after attaining puberty .", "mutations in androgen receptor gene may lead to the androgen insensitivity syndrome ( ais ) which can be partial androgen insensitivity syndrome ( pais ) or complete androgen insensitivity syndrome ( cais ) and is the most common cause of 46xy dsd .", "the spectrum of phenotypes associated with ais may range from completely female through mixed male / female to completely male type . in 1995 ,", "charmian quigley and frank french proposed new grading system for the phenotypic features in ais based on prader classification for cah .", "individuals diagnosed with 46xy dsd show an overlap in clinical and biochemical parameters which emphasizes the need for comprehensive evaluation with a multidisciplinary approach .", "this is an ambispective study where all new and old follow - up patients diagnosed with 46xy dsd , being managed at endocrine opd in a period of 16 months ( june 2014october 2015 ) were included .", "external masculinization score ( ems ) were calculated based on the external genitalia of patients .", "abdominopelvic ultrasound was done for the localization of gonads ( magnetic resonance imaging ( mri ) also , when required ) . for patients on follow - up , previous records were reviewed to establish a diagnosis as they were on medication while in new patients , samples were collected and hormonal measurements included serum luteinizing hormone , follicle - stimulating hormone , testosterone , androstenedione ( a ) , cortisol , dehydroepiandrostenedione , and plasma adrenocorticotropic hormone levels by electrochemiluminescence immunoassay using commercial kits ( roche , germany ) .", "radioimmunoassay kit - based method was used for evaluating 17-hydroxyprogesterone ( 17-ohp ) levels ( diagnostic systems laboratories , inc . , webster , tx , usa ) .", "5dht was measured using radioimmunoassay ( immunotech ; prague , czech republic ) after extraction from other hormones with diethyl ether and celite chromatography .", "the short - term human chorionic gonadotropin ( hcg ) stimulation test was carried out to check the functioning of testicular tissue .", "testosterone levels were measured before and 2448 h after the last injection of a series of three intramuscular injections of hcg on alternate days ( < 1-year - old , 500 units ; 110 years , 1000 units ; > 10 years , 2000 units ) .", "testosterone : androstenedione ( t : a ) and testosterone : 5 dht ( t:5dht ) ratios were calculated as and when required to rule out 17 beta hsd deficiency and 5 alpha reductase deficiency respectively .", "on first evaluation , 11 patients had presented with the primary complaint of ambiguous genitalia , three ( who were being reared as female ) with primary amenorrhea , three with undescended gonads and one with fever and vomiting .", "the presenting complaints , clinical , hormonal , and radiological findings are summarized in table 1 .", "five ( 26% ) of them were reared females and 14 ( 74% ) as males .", "ten patients ( s.5 , s.7 , s.8 , s.9 , s.10 , s.14 , s.15 , s.16 , s.17 , and s.18 ) had undergone corrective surgeries for ambiguous genitalia .", "clinical profile of patients patient s.1 reared as female presented with primary amenorrhea , delayed development of secondary sexual characteristics and unambiguous female genitalia .", "patients s.2 , s.3 , and s.5 had ambiguous genitalia , undescended gonads , raised fsh and low testosterone levels while patient s.4 had unilateral undescended testis with raised fsh and low testosterone levels , all three were suspected to be cases of pgd .", "abdominopelvic ultrasonography ( usg ) and mri could not locate gonads in s.7 while in patient s.6 , a nubbin was seen in the scrotal area .", "inguinal exploration was done to locate testis but it revealed the absence of any testicular tissue .", "fsh was raised in both [ table 2 ] and there was no testosterone response to hcg stimulation .", "hormonal profile of patients patient s.8 being reared as female had presented with ambiguous genitalia and raised gonadotropins .", "she had undergone bilateral gonadectomy and on histological evaluation ot tissue was found , she was diagnosed with ot - dsd .", "patient s.9 being reared as female presented with primary amenorrhea , genital ambiguity and delayed development of secondary sexual characteristics .", "she underwent bilateral gonadectomy , histopathological examination revealed testicular tissue and was diagnosed with cais .", "hormonal profile showed normal testosterone levels with t : dht ratio of 9.7 and t : a ratio of 5.8 .", "patient s.11 being reared as female came with a complaint of hirsutism and delayed development of secondary sexual characteristics .", "bilateral gonadectomy was done and histological studies showed the presence of testicular tissue which confirmed the diagnosis of pais as t : dht and t : a ratios were also normal .", "testosterone levels were undetectable , but showed a good response to hcg stimulation with t : dht ratio of 7.25 , t : a ratio of 5.8 and was diagnosed with pais .", "patients s.13 , s.14 , s.15 , and s.16 came with ambiguous genitalia and low baseline testosterone levels .", "testosterone to dht ratio in s.13 , s.14 , and s.15 were 31.4 , 18 , and 13.9 , respectively .", "due to high t : dht ratio post - hcg stimulation , all three were diagnosed with 5rd while in s.16 , t : dht ratio was not calculated as b / l gonadectomy was done at 2 years of age .", "molecular analysis of srd5a2 gene in this patient revealed r246q mutation commonly seen in 5-rd .", "records show that his post hcg serum t : a ratio was 0.06 , he was thought to be a case of 17hsd deficiency . at 2 years of age he had fever , vomiting and loose stools with hyponatremia , hyperkalemia and hypoglycemia .", "patient s.18 was admitted in pediatrics with a complaint of poor feeding and poor activity since day one of life . on evaluation", ", he was found to have hypernatremia , hypokalemia , and genital ambiguity with bifid scrotum and hypospadias . in view of electrolyte imbalance", ", incomplete masculinization , low cortisol , and elevated 17-ohp , he was suspected to be a case of cah and treatment was started .", "an accurate diagnosis could not be established when he came to us as he was already on steroids and had a history of salt wasting , so steroids could not be stopped for evaluation of the cause of cah .", "he had undergone three genital surgeries for hypospadias correction but still had difficulty in urination .", "the patient is kept on antihypertensives , but no definitive diagnosis could be made as the baseline hormonal reports are missing . with the help of clinical , biochemical and radiological evaluation ,", "accurate diagnosis was made in 16 patients while rest 2 are on follow - up and managed in our clinic .", "discordance between genetic ( xx or xy ) , gonadal ( testis or ovaries ) , external genital ( vulva or penis ) , and internal sex ( wolffian or mullerian ducts ) results in dsd . in humans ,", "the bipotential gonad differentiates into testis in the presence of sry gene present on y - chromosome .", "testicular differentiation is followed by the development of internal sex ducts and external genitalia in the presence of fetal hormones produced by the testis .", "sertoli cells secrete anti - mullerian hormone , which causes irreversible regression of paramesonephric or mullerian ducts around the sixth week of gestational age .", "testosterone produced by leydig cells of fetal testis promotes the development of mesonephric or wolffian ducts and hence male internal genitalia ; epididymis , vas deferens , and vesicular seminalis .", "the development of external male genitalia depends on the conversion of testosterone to a more potent steroid dht in the presence of 5-reductase enzyme . in 2006 , the term  intersex  or  hermaphrodite  was replaced by  dsd  and", "46xy dsd was subcategorized as disorders of testicular development and disorders of androgen synthesis or action .", "first - line testing in newborns includes karyotyping , imaging ( usg / mri pelvis ) , serum electrolytes , and serum gonadotropins .", "molecular diagnosis has been made in approximately 20% of cases with 46xy dsd , although confirmatory , but is limited by cost and accessibility .", "the inability of bipotential gonad to differentiate into testis results in testicular dysgenesis or agenesis which occurs due to the disruption of various genetic factors involved in the process of sex determination . in our study , four patients were diagnosed with pgd , one with cgd , one with ot - dsd , and two with testicular agenesis or cba . in the absence of gonadal development ,", "patients with cgd usually present with unambiguous female genitalia due to the absence of gonadal steroid production while in pgd , the external phenotype depends upon the degree of testicular tissue present . in our study , out of four patients with pgd , three ( s.2 , s.3 , and s.5 ) presented with ambiguous genitalia and one with undescended gonad ( s.4 ) . in such cases , corrective surgeries are done if required , and testosterone supplementations are given at pubertal age .", "patient s.5 being reared as female had presented with ambiguous genitalia at 4 years of age . with parents consent clitorovaginoplasty and bilateral orchidectomy", "orchidopexy and biopsy are usually recommended for undescended gonads as the risk of malignancy ranges from 8.3% to 54% in patients with xy pgd . in 46xy", "cba is a rare condition and is marked by the absence of testis since birth .", "previous studies have shown the occurrence of pure or partial forms of 46xy gonadal dysgenesis in families of patients with cba , but the genetic cause is still not known . in our study ,", "two patients ( s.6 and s.7 ) with cba were born out of nonconsanguineous marriage and had no family history of genital ambiguity but the father of patient s.7 gave history of oligospermia and assisted reproduction .", "46xy ot - dsd is a very rare condition marked by the presence of ot tissue and is confirmed by histopathological examination .", "parents of patient s.8 had sought medical attention for ambiguous genitalia at the age of 13 years .", "bilateral gonadectomy was done , and the corrective surgeries for hypospadias and phallus are awaited .", "the complete or partial resistance to the action of androgens may result in cais or pais in xy individuals .", "patients with cais may present with primary amenorrhea in adolescence or inguinal swellings in infants .", "patient s.9 was being reared as female , parents sought medical attention at the age of 18 years for primary amenorrhea and delayed development of secondary sexual characteristics .", "bilateral gonadectomy was recommended after confirming the gender identity of the patient to be female .", "a recent study done on 102 phenotypic women with y - chromosome showed an incidence of 17.6% malignancy .", "of these 9 out of 30 patients ( 30% ) of cais with a mean age of 20.7 years were found to have gonadoblastoma on histological analysis .", "therefore , adult cais patients who receive late intervention are recommended to undergo immediate gonadectomy in order to avoid the risk of malignancy .", "the phenotype of patients presenting with pais depends on the responsiveness of genital tissues to androgens .", "infants with pais present with the genital ambiguity of varying degrees while adults may present with gynecomastia .", "however , the risk of malignancy in individuals with pais is 15% if the testis is not scrotal in position .", "hormonal profile showed normal testosterone levels with t : dht ratio of 9.7 and t : a ratio of 5.8 .", "patient s.11 being reared as female sought medical attention for hirsutism at the age of 19 years .", "biochemical examination showed normal t : dht and t : a ratios with testosterone levels in male range .", "gonadectomy was done and histopathological examination showed the presence of testicular tissue , she was then diagnosed with pais .", "she had female gender orientation and is now on estrogen replacement therapy for breast development .", "his t : dht ratio was 7.25 and t : a ratio was 5.8 post - hcg stimulation , which excluded the diagnosis of 5rd and 17hsd .", "though the presumptive diagnosis of patients s.10 , s.11 , and s.12 was pais , as t : dht ratio and t : a ratio were normal , [ figure 1 ] diagnosis with needs further confirmation by molecular studies . flow chart for", "diagnosis of 46xy disorders of sexual development the interrupted conversion of testosterone to dht due to the mutations in gene encoding 5-reductase enzyme may lead to 5rd .", "the differentiation of internal and external male genitalia occurs in the presence of testosterone ( t ) and dht .", "the t : dht ratio is used to diagnose this condition but interpreting these results is not always straightforward .", "the diagnosis of 5rd is suspected in newborns with undermasculinized genitalia ( depending upon the activity of enzyme 5-reductase ) , low serum testosterone levels , good response to hcg stimulation and high testosterone to dht ratio ( > 1012 ) . a good response to hcg stimulation ( rise by more than twice the baseline value ) is observed in these patients .", "the patients may present with microphallus , inguinal gonads or gonads in labioscrotal folds and varying degree of hypospadias .", "treatment with percutaneous dht increases the size of the phallus in infants and children with 5-reductase 2 deficiency .", "most of them are reared as males and gender reassignment is required in those who are reared as females due to the spontaneous virilization at puberty .", "the decisions regarding management of masculinizing puberty need to be taken before puberty with a multidisciplinary approach .", "female gender assignment in these patients by surgery should be undertaken postpuberty with full caution and counseling with parents . in our study ,", "patient s.16 had severely undervirilized genitalia , bilateral gonadectomy had been done at 2 years of age and female gender had been assigned . at the age of 17 years , he sought medical attention for gender dysphoria . at this time , the presence of common r246q mutation on molecular analysis of srd5a2 gene confirmed the diagnosis of 5rd .", "46xy patients with deficiency of 17-hsd enzyme usually present with female - like genitalia at birth with clitoromegaly and blind - ending vagina or male type genitalia with micropenis and hypospadias . at puberty , these patients experience significant virilization due to the abnormally elevated levels of androstenedione or extragonadal synthesis of testosterone , the exact cause of which is unknown .", "the diagnosis of 17-hsd3 can be suspected if testosterone to androstenedione ratio is < 0.8 .", "the overlapping phenotype of individuals with 17-hsd and pais can be differentiated by molecular analysis or on the basis of hcg stimulated t : a ratio , which is < 0.8 in cases of 17-hsd .", "patient s.17 initially predicted to have 17 hsd deficiency , but later presented with features of mineralocorticoid and glucocorticoid deficiency at 2 years of age .", "patient s.19 is presently 49 years of age and gave a history of ambiguous genitalia .", "he was on dexamethasone , sustanon and had undergone three corrective surgeries for hypospadias but still has a problem in urination . routine examination revealed raised blood pressure for which he is on hypertensives .", "limitations of this study are that , being an ambispective study , all tests were not done in all patients and baseline records / tests to make a diagnosis could not be obtained in two patients .", "nonetheless , this gives an insight into how to diagnose and manage 46xy dsd , who may present at different ages , with different etiologies and different gender of rearing in our set up .", "there is overlap in the clinical , hormonal and anatomic profile of different subsets of 46xy dsd .", "the diagnosis of 46xy dsd needs to be confirmed by cytogenetic , hormonal , radiological , and genetic tests .", "phenotype varies depending on the degree of genetic , hormonal and anatomic defect . at times , in spite of all the tests a definitive diagnosis may not be possible .", "the pediatrician , endocrinologist , surgeon , psychologist , and clinical geneticist need to work as a team for multifaceted management of these patients .", "this study was supported by department of biotechnology , ministry of science and technology ( bt / pr7681/med/12/597/2013 ) and indian council of medical research ( dhr / gia/1/2014 ) , new delhi , india .", "this study was supported by department of biotechnology , ministry of science and technology ( bt / pr7681/med/12/597/2013 ) and indian council of medical research ( dhr / gia/1/2014 ) , new delhi , india ."], "abstract_text": ["<S> background and objectives:46 xy disorders of sexual development ( dsd ) cover a wide spectrum of phenotypes ranging from unambiguous female genitalia to ambiguous male genitalia with hypospadias or dysgenetic gonads . </S>", "<S> management of these patients depends on the cause of dsd , degree of feminization , age at presentation , and gender orientation . </S>", "<S> the aim of this study was to evaluate the presentation and management of patients with 46xy dsd at our center.patients and methods : all new and old patients of 46xy dsd attending the endocrine opd in a period of 16 months were included in this study . clinical , cytogenetic , hormonal , and radiological evaluation </S>", "<S> were done to identify the cause of dsd.results:among 19 patients , eight were diagnosed with disorders of gonadal development ( one with complete gonadal dysgenesis , four with partial gonadal dysgenesis , two with congenital bilateral anorchia , and one with ovotesticular dsd ) and eight with disorders of androgen synthesis and action ( one with complete androgen insensitivity syndrome [ ais ] , three with partial ais and four with 5 reductase deficiency ) . in three patients , a definitive diagnosis could not be made.conclusions:management of patients with dsd depends on etiology , gender assignment , gender orientation , hormonal treatment , genital surgery , and consequent psychosocial implications . </S>", "<S> due to the overlapping clinical and biochemical parameters in different subsets of dsd , only a preliminary etiological diagnosis can be made in some cases . </S>", "<S> genetic studies with long - term follow - up are required for an accurate diagnosis . </S>"], "labels": null, "section_names": ["I", "P", "R", "D", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["sexual development in males is a multistep process , implying a delicate network of molecular events that directs the bipotential gonad to differentiate into testis ( sex determination ) and consequent differentiation of internal and external genitalia in the presence of testicular hormones ( sex differentiation ) .", "alteration of any genetic or endocrine factors involved in testicular development may lead to 46 xy disorders of sexual development ( dsd ) .", "genital ambiguity in patients with 46 xy dsd may either occur due to the disorders in gonadal development or due to the disruption in androgen synthesis or action .", "patients with disorders of gonadal development may present with either complete or partial forms of gonadal dysgenesis .", "swyer 's syndrome or complete gonadal dysgenesis ( cgd ) is characterized by unambiguous female genitalia , bilateral streak gonads , and elevated gonadotropins .", "partial gonadal dysgenesis ( pgd ) may have a wide spectrum of phenotypes associated with ambiguous genitalia , varying degree of hypospadias and testis of variable size and echotexture present along the path of decent .", "ovotesticular dsd ( ot - dsd ) is a rare condition marked by the presence of both ovarian and testicular tissue in an individual while congenital bilateral anorchia ( cba ) is characterized by the complete absence of testicular tissue in genotypic males .", "disorders of androgen synthesis may occur due to deficiency of various enzymes involved in the steroidogenesis .", "congenital adrenal hyperplasia ( cah ) due to 17-hydroxysteroid dehydrogenase 3 ( 17-hsd3 ) is caused by the mutations in hsd17b3 gene which is required for the conversion of androstenedione to testosterone .", "deficiency of this enzyme results in feminized genitalia in newborns due to the low testosterone levels .", "mutations in srd5a2 gene impairs the activity of enzyme 5-reductase and hence the conversion of testosterone to its more potent form dihydrotestosterone ( dht ) .", "individuals with 5-reductase deficiency ( 5-rd ) are often raised as females due to undermasculinized genitalia but the gender role changes soon after attaining puberty .", "mutations in androgen receptor gene may lead to the androgen insensitivity syndrome ( ais ) which can be partial androgen insensitivity syndrome ( pais ) or complete androgen insensitivity syndrome ( cais ) and is the most common cause of 46xy dsd .", "the spectrum of phenotypes associated with ais may range from completely female through mixed male / female to completely male type . in 1995 ,", "charmian quigley and frank french proposed new grading system for the phenotypic features in ais based on prader classification for cah .", "individuals diagnosed with 46xy dsd show an overlap in clinical and biochemical parameters which emphasizes the need for comprehensive evaluation with a multidisciplinary approach ."], ["this is an ambispective study where all new and old follow - up patients diagnosed with 46xy dsd , being managed at endocrine opd in a period of 16 months ( june 2014october 2015 ) were included .", "external masculinization score ( ems ) were calculated based on the external genitalia of patients .", "abdominopelvic ultrasound was done for the localization of gonads ( magnetic resonance imaging ( mri ) also , when required ) . for patients on follow - up , previous records were reviewed to establish a diagnosis as they were on medication while in new patients , samples were collected and hormonal measurements included serum luteinizing hormone , follicle - stimulating hormone , testosterone , androstenedione ( a ) , cortisol , dehydroepiandrostenedione , and plasma adrenocorticotropic hormone levels by electrochemiluminescence immunoassay using commercial kits ( roche , germany ) .", "radioimmunoassay kit - based method was used for evaluating 17-hydroxyprogesterone ( 17-ohp ) levels ( diagnostic systems laboratories , inc . , webster , tx , usa ) .", "5dht was measured using radioimmunoassay ( immunotech ; prague , czech republic ) after extraction from other hormones with diethyl ether and celite chromatography .", "the short - term human chorionic gonadotropin ( hcg ) stimulation test was carried out to check the functioning of testicular tissue .", "testosterone levels were measured before and 2448 h after the last injection of a series of three intramuscular injections of hcg on alternate days ( < 1-year - old , 500 units ; 110 years , 1000 units ; > 10 years , 2000 units ) .", "testosterone : androstenedione ( t : a ) and testosterone : 5 dht ( t:5dht ) ratios were calculated as and when required to rule out 17 beta hsd deficiency and 5 alpha reductase deficiency respectively ."], ["on first evaluation , 11 patients had presented with the primary complaint of ambiguous genitalia , three ( who were being reared as female ) with primary amenorrhea , three with undescended gonads and one with fever and vomiting .", "the presenting complaints , clinical , hormonal , and radiological findings are summarized in table 1 .", "five ( 26% ) of them were reared females and 14 ( 74% ) as males .", "ten patients ( s.5 , s.7 , s.8 , s.9 , s.10 , s.14 , s.15 , s.16 , s.17 , and s.18 ) had undergone corrective surgeries for ambiguous genitalia .", "clinical profile of patients patient s.1 reared as female presented with primary amenorrhea , delayed development of secondary sexual characteristics and unambiguous female genitalia .", "patients s.2 , s.3 , and s.5 had ambiguous genitalia , undescended gonads , raised fsh and low testosterone levels while patient s.4 had unilateral undescended testis with raised fsh and low testosterone levels , all three were suspected to be cases of pgd .", "abdominopelvic ultrasonography ( usg ) and mri could not locate gonads in s.7 while in patient s.6 , a nubbin was seen in the scrotal area .", "inguinal exploration was done to locate testis but it revealed the absence of any testicular tissue .", "fsh was raised in both [ table 2 ] and there was no testosterone response to hcg stimulation .", "hormonal profile of patients patient s.8 being reared as female had presented with ambiguous genitalia and raised gonadotropins .", "she had undergone bilateral gonadectomy and on histological evaluation ot tissue was found , she was diagnosed with ot - dsd .", "patient s.9 being reared as female presented with primary amenorrhea , genital ambiguity and delayed development of secondary sexual characteristics .", "she underwent bilateral gonadectomy , histopathological examination revealed testicular tissue and was diagnosed with cais .", "hormonal profile showed normal testosterone levels with t : dht ratio of 9.7 and t : a ratio of 5.8 .", "patient s.11 being reared as female came with a complaint of hirsutism and delayed development of secondary sexual characteristics .", "bilateral gonadectomy was done and histological studies showed the presence of testicular tissue which confirmed the diagnosis of pais as t : dht and t : a ratios were also normal .", "testosterone levels were undetectable , but showed a good response to hcg stimulation with t : dht ratio of 7.25 , t : a ratio of 5.8 and was diagnosed with pais .", "patients s.13 , s.14 , s.15 , and s.16 came with ambiguous genitalia and low baseline testosterone levels .", "testosterone to dht ratio in s.13 , s.14 , and s.15 were 31.4 , 18 , and 13.9 , respectively .", "due to high t : dht ratio post - hcg stimulation , all three were diagnosed with 5rd while in s.16 , t : dht ratio was not calculated as b / l gonadectomy was done at 2 years of age .", "molecular analysis of srd5a2 gene in this patient revealed r246q mutation commonly seen in 5-rd .", "records show that his post hcg serum t : a ratio was 0.06 , he was thought to be a case of 17hsd deficiency . at 2 years of age he had fever , vomiting and loose stools with hyponatremia , hyperkalemia and hypoglycemia .", "patient s.18 was admitted in pediatrics with a complaint of poor feeding and poor activity since day one of life . on evaluation", ", he was found to have hypernatremia , hypokalemia , and genital ambiguity with bifid scrotum and hypospadias . in view of electrolyte imbalance", ", incomplete masculinization , low cortisol , and elevated 17-ohp , he was suspected to be a case of cah and treatment was started .", "an accurate diagnosis could not be established when he came to us as he was already on steroids and had a history of salt wasting , so steroids could not be stopped for evaluation of the cause of cah .", "he had undergone three genital surgeries for hypospadias correction but still had difficulty in urination .", "the patient is kept on antihypertensives , but no definitive diagnosis could be made as the baseline hormonal reports are missing . with the help of clinical , biochemical and radiological evaluation ,", "accurate diagnosis was made in 16 patients while rest 2 are on follow - up and managed in our clinic ."], ["discordance between genetic ( xx or xy ) , gonadal ( testis or ovaries ) , external genital ( vulva or penis ) , and internal sex ( wolffian or mullerian ducts ) results in dsd . in humans ,", "the bipotential gonad differentiates into testis in the presence of sry gene present on y - chromosome .", "testicular differentiation is followed by the development of internal sex ducts and external genitalia in the presence of fetal hormones produced by the testis .", "sertoli cells secrete anti - mullerian hormone , which causes irreversible regression of paramesonephric or mullerian ducts around the sixth week of gestational age .", "testosterone produced by leydig cells of fetal testis promotes the development of mesonephric or wolffian ducts and hence male internal genitalia ; epididymis , vas deferens , and vesicular seminalis .", "the development of external male genitalia depends on the conversion of testosterone to a more potent steroid dht in the presence of 5-reductase enzyme . in 2006 , the term  intersex  or  hermaphrodite  was replaced by  dsd  and", "46xy dsd was subcategorized as disorders of testicular development and disorders of androgen synthesis or action .", "first - line testing in newborns includes karyotyping , imaging ( usg / mri pelvis ) , serum electrolytes , and serum gonadotropins .", "molecular diagnosis has been made in approximately 20% of cases with 46xy dsd , although confirmatory , but is limited by cost and accessibility .", "the inability of bipotential gonad to differentiate into testis results in testicular dysgenesis or agenesis which occurs due to the disruption of various genetic factors involved in the process of sex determination . in our study , four patients were diagnosed with pgd , one with cgd , one with ot - dsd , and two with testicular agenesis or cba . in the absence of gonadal development ,", "patients with cgd usually present with unambiguous female genitalia due to the absence of gonadal steroid production while in pgd , the external phenotype depends upon the degree of testicular tissue present . in our study , out of four patients with pgd , three ( s.2 , s.3 , and s.5 ) presented with ambiguous genitalia and one with undescended gonad ( s.4 ) . in such cases , corrective surgeries are done if required , and testosterone supplementations are given at pubertal age .", "patient s.5 being reared as female had presented with ambiguous genitalia at 4 years of age . with parents consent clitorovaginoplasty and bilateral orchidectomy", "orchidopexy and biopsy are usually recommended for undescended gonads as the risk of malignancy ranges from 8.3% to 54% in patients with xy pgd . in 46xy", "cba is a rare condition and is marked by the absence of testis since birth .", "previous studies have shown the occurrence of pure or partial forms of 46xy gonadal dysgenesis in families of patients with cba , but the genetic cause is still not known . in our study ,", "two patients ( s.6 and s.7 ) with cba were born out of nonconsanguineous marriage and had no family history of genital ambiguity but the father of patient s.7 gave history of oligospermia and assisted reproduction .", "46xy ot - dsd is a very rare condition marked by the presence of ot tissue and is confirmed by histopathological examination .", "parents of patient s.8 had sought medical attention for ambiguous genitalia at the age of 13 years .", "bilateral gonadectomy was done , and the corrective surgeries for hypospadias and phallus are awaited .", "the complete or partial resistance to the action of androgens may result in cais or pais in xy individuals .", "patients with cais may present with primary amenorrhea in adolescence or inguinal swellings in infants .", "patient s.9 was being reared as female , parents sought medical attention at the age of 18 years for primary amenorrhea and delayed development of secondary sexual characteristics .", "bilateral gonadectomy was recommended after confirming the gender identity of the patient to be female .", "a recent study done on 102 phenotypic women with y - chromosome showed an incidence of 17.6% malignancy .", "of these 9 out of 30 patients ( 30% ) of cais with a mean age of 20.7 years were found to have gonadoblastoma on histological analysis .", "therefore , adult cais patients who receive late intervention are recommended to undergo immediate gonadectomy in order to avoid the risk of malignancy .", "the phenotype of patients presenting with pais depends on the responsiveness of genital tissues to androgens .", "infants with pais present with the genital ambiguity of varying degrees while adults may present with gynecomastia .", "however , the risk of malignancy in individuals with pais is 15% if the testis is not scrotal in position .", "hormonal profile showed normal testosterone levels with t : dht ratio of 9.7 and t : a ratio of 5.8 .", "patient s.11 being reared as female sought medical attention for hirsutism at the age of 19 years .", "biochemical examination showed normal t : dht and t : a ratios with testosterone levels in male range .", "gonadectomy was done and histopathological examination showed the presence of testicular tissue , she was then diagnosed with pais .", "she had female gender orientation and is now on estrogen replacement therapy for breast development .", "his t : dht ratio was 7.25 and t : a ratio was 5.8 post - hcg stimulation , which excluded the diagnosis of 5rd and 17hsd .", "though the presumptive diagnosis of patients s.10 , s.11 , and s.12 was pais , as t : dht ratio and t : a ratio were normal , [ figure 1 ] diagnosis with needs further confirmation by molecular studies . flow chart for", "diagnosis of 46xy disorders of sexual development the interrupted conversion of testosterone to dht due to the mutations in gene encoding 5-reductase enzyme may lead to 5rd .", "the differentiation of internal and external male genitalia occurs in the presence of testosterone ( t ) and dht .", "the t : dht ratio is used to diagnose this condition but interpreting these results is not always straightforward .", "the diagnosis of 5rd is suspected in newborns with undermasculinized genitalia ( depending upon the activity of enzyme 5-reductase ) , low serum testosterone levels , good response to hcg stimulation and high testosterone to dht ratio ( > 1012 ) . a good response to hcg stimulation ( rise by more than twice the baseline value ) is observed in these patients .", "the patients may present with microphallus , inguinal gonads or gonads in labioscrotal folds and varying degree of hypospadias .", "treatment with percutaneous dht increases the size of the phallus in infants and children with 5-reductase 2 deficiency .", "most of them are reared as males and gender reassignment is required in those who are reared as females due to the spontaneous virilization at puberty .", "the decisions regarding management of masculinizing puberty need to be taken before puberty with a multidisciplinary approach .", "female gender assignment in these patients by surgery should be undertaken postpuberty with full caution and counseling with parents . in our study ,", "patient s.16 had severely undervirilized genitalia , bilateral gonadectomy had been done at 2 years of age and female gender had been assigned . at the age of 17 years , he sought medical attention for gender dysphoria . at this time , the presence of common r246q mutation on molecular analysis of srd5a2 gene confirmed the diagnosis of 5rd .", "46xy patients with deficiency of 17-hsd enzyme usually present with female - like genitalia at birth with clitoromegaly and blind - ending vagina or male type genitalia with micropenis and hypospadias . at puberty , these patients experience significant virilization due to the abnormally elevated levels of androstenedione or extragonadal synthesis of testosterone , the exact cause of which is unknown .", "the diagnosis of 17-hsd3 can be suspected if testosterone to androstenedione ratio is < 0.8 .", "the overlapping phenotype of individuals with 17-hsd and pais can be differentiated by molecular analysis or on the basis of hcg stimulated t : a ratio , which is < 0.8 in cases of 17-hsd .", "patient s.17 initially predicted to have 17 hsd deficiency , but later presented with features of mineralocorticoid and glucocorticoid deficiency at 2 years of age .", "patient s.19 is presently 49 years of age and gave a history of ambiguous genitalia .", "he was on dexamethasone , sustanon and had undergone three corrective surgeries for hypospadias but still has a problem in urination . routine examination revealed raised blood pressure for which he is on hypertensives .", "limitations of this study are that , being an ambispective study , all tests were not done in all patients and baseline records / tests to make a diagnosis could not be obtained in two patients .", "nonetheless , this gives an insight into how to diagnose and manage 46xy dsd , who may present at different ages , with different etiologies and different gender of rearing in our set up .", "there is overlap in the clinical , hormonal and anatomic profile of different subsets of 46xy dsd .", "the diagnosis of 46xy dsd needs to be confirmed by cytogenetic , hormonal , radiological , and genetic tests .", "phenotype varies depending on the degree of genetic , hormonal and anatomic defect . at times , in spite of all the tests a definitive diagnosis may not be possible .", "the pediatrician , endocrinologist , surgeon , psychologist , and clinical geneticist need to work as a team for multifaceted management of these patients .", "this study was supported by department of biotechnology , ministry of science and technology ( bt / pr7681/med/12/597/2013 ) and indian council of medical research ( dhr / gia/1/2014 ) , new delhi , india ."], ["this study was supported by department of biotechnology , ministry of science and technology ( bt / pr7681/med/12/597/2013 ) and indian council of medical research ( dhr / gia/1/2014 ) , new delhi , india ."], [""]]}
{"article_id": "PMC5411178", "article_text": ["urinary tract infection ( uti ) is a frequent cause of morbidity both in the community and in the hospital setting .", "the causative pathogen can vary greatly geographically , and so it is prudent to identify those with resistant strains and have current data on the appropriate empirical therapy within a region .", "the most frequently encountered organisms associated with utis include enteric gram - negative bacteria ( with escherichia coli being the most predominant ) , coagulase negative staphylococcus saprophyticus along with proteus mirabilis , klebsiella , and enterococcus , which account for less than 5% . however , recent studies have reported the increasing prevalence of staphylococcus aureus ( sa ) in utis .", "sa is an opportunistic pathogen affecting both immune competent and immunocompromised individuals , frequently resulting in significant morbidity", ". many strains of sa carry a wide variety of multidrug - resistant genes on plasmids , which aid the spread of resistance among species .", "methicillin - resistant sa ( mrsa ) is widespread in many irish hospitals and is increasingly seen in community health care units such as nursing homes .", "globally , it is considered that there has been an epidemic of mrsa within health care institutions . bacteriuria with sa is hypothesized to occur through a number of mechanisms that includes catheterization , urologic procedures , or seeding of the genitourinary tract  including nephrologically excreted bacteria in overt bacteremia .", "bacteremia itself is associated with bacteriuria in patients infected with sa , which suggests that bacteremia is an important precursor for bacteriuria in some patient groups .", "herein , we chronologically assessed the source , patient demographics , and antimicrobial susceptibilities of mrsa - positive isolates from the hospital and community setting over a 5-year period in university hospital waterford  a tertiary referral hospital with the primary microbiology laboratory for 4 acute hospitals , serving a total catchment area of almost 500,000 people .", "the microbiology laboratory in university hospital waterford processes all inpatient urines in the catchment area of almost 500,000 , excluding 2 small private elective inpatient institutions .", "it also processes all urine samples sent from the community ( i.e. , all samples from general practice and nursing homes ) and all specimens from the catchments emergency departments .", "laboratory diagnosis of mrsa bacteriuria was performed using accredited microbiological microscopy analysis with culture identification criteria and standardized susceptibility testing protocols .", "our laboratory utilized clinical and laboratory standards institute ( clsi ; 20102014 ) and european committee on antimicrobial susceptibility testing ( eucast ; 2014 ) methodologies .", "a retrospective data extraction was performed , from the hospital 's electronic microbiological system  cognos  and de - duplication was executed .", "we considered a  separate episode  as a urine specimen culturing mrsa at least 6 months following a previous positive culture .", "further urine specimens sent within a 6-month period following an initial laboratory diagnosis mrsa bacteriuria were excluded .", "resistance rates were then calculated for the pathogen 's susceptibility to 5 commonly used antimicrobial agents  ciprofloxacin , coamoxiclav , flucloxacillin , nitrofurantoin , and trimethoprim . for unwell patients ,", "that is , those in whom there was clinical suspicion of sepsis or invasive infection , teicoplanin and vancomycin were also tested for sensitivity profiling . in order to obtain further clinical information from our patient group , we also assessed those patients who had an mrsa - positive swab ( usually from groin , nose , or perineum ) , those who had documented mrsa bacteremia , and we analyzed their demographic details .", "ethical approval was waived for this retrospective observational study in light of its non - interventional nature .", "over a consecutive 5-year period , the laboratory cultured 425,013 urine samples from all sources , with a mean number of 85,003 specimens per year received ( table 1 ) .", "a total of 542 unique urine isolates cultured sa and 151 of species ( 27.9% ) were methicillin resistant , meaning that 0.04% of all urine samples tested cultured mrsa .", "number of urine cultures by year . of these 151 samples that tested positive for mrsa , 92 ( 61% ) were from mid - stream urine ( msu ) samples , 50 ( 33% ) from catheter specimen urine ( csu ) samples , and 9 ( 6% ) were from an unspecified source ( fig .", "csu  =  catheter specimen urine , mrsa  =  methicillin resistant staphylococcus aureus , msu  =  mid - stream urine .", "mrsa was slightly more common in msu samples ; however , the findings were not statistically significant ( 18.2% vs 13.9% , p  =  0.388 ) .", "forty - nine ( 32.5% ) specimens were from an inpatient cohort , 9 ( 6% ) were from the emergency department , 79 ( 52.3% ) from general practitioners , 12 ( 7.9% ) were from nursing homes , with 2 ( 1.3% ) from an unrecorded source ( fig .", "mrsa was seen in a similar proportion of inpatient and outpatient samples indicating that this is not solely a hospital - acquired phenomenon ( 29.1% vs 26.9% , p  =  0.587 ) .", "mrsa  =  methicillin resistant staphylococcus aureus . the mean age of our patients who cultured mrsa within their urine was 72.7 years ( range 10.9790.2 ) and most patients were aged between 70 and 90 years ( table 2 ) .", "patients who cultured an mrsa bacterium were older than patients with mssa , and this was statistically significant ( 53 vs 73 years , p    0.001 ) .", "the number of sa isolates grown over the 5-year study period doubled from 77 in 2010 to 161 in 2014 .", "the number of mrsa isolates had also increased ; however , the percentage of sa isolates that were methicillin resistant has decreased ( p  =  0.145 ) .", "this is true for both inpatient and outpatient samples ( p  =  0.313 , p  =  0.254 , figs .", "mrsa  =  methicillin resistant staphylococcus aureus , mssa  =  methicillin sensitive staphylococcus aureus , sa  =  staphylococcus aureus . inpatient trends in sa", "mrsa  =  methicillin resistant staphylococcus aureus , mssa  =  methicillin sensitive staphylococcus aureus , sa  =  staphylococcus aureus .", "mrsa  =  methicillin resistant staphylococcus aureus , mssa  =  methicillin sensitive staphylococcus aureus , sa  =  staphylococcus aureus .", "regarding the clinical stance of patients within the cohort , 9 patients ( 7% ) with mrsa - positive urine cultures had a documented history of mrsa bacteremia at that time .", "each of these patients cultured mrsa isolates that were sensitive to nitrofurantoin , and 8 of the 9 patients ( 88.9% ) were sensitive to trimethoprim .", "it was noted that all 9 patients in this cohort were aged 70 years or above ( p  =  0.079 ) , although this did not reach statistical significance .", "further antimicrobials agents were introduced to the testing panel in 59 ( 39% ) samples , where there was a clinical concern regarding invasive infection or sepsis  including the 9 patients with mrsa bacteremia . all 59 samples ( 100% ) were sensitive to both vancomycin and teicoplanin .", "eighty - eight ( 66.8% ) of our 128 patients cultured mrsa - positive isolates on routine swabs from either mucosal or skin sites .", "all 3 patients with resistance to nitrofurantoin in their urine had a positive swab from either groin or nose .", "eight of the 10 patients ( 80% ) who had trimethoprim - resistant mrsa bacteriuria and 87 of the 125 ( 69.6% ) of the ciprofloxacin - resistant mrsa bacteriuria also had mrsa - positive mucosal or skin swabs .", "there was no association between age and having an mrsa - positive swab in our cohort . throughout the study period", ", there was 100% resistance of all mrsa isolates to flucloxacillin and coamoxiclav , as expected .", "ninety - eight percent of isolates were resistant to ciprofloxacin , but of these samples , there was 97.2% sensitivity for nitrofurantoin and 92.4% sensitivity for trimethoprim ( fig .", "overall , there was only 2.7% resistance for nitrofurantoin , but all of these samples were sensitive to trimethoprim .", "trimethoprim itself had a 7.4% resistance level , but all of these samples were sensitive to nitrofurantoin , meaning that no sample was resistant to both oral agents , that is , nitrofurantoin and trimethoprim .", "there was a statistically significant association between nitrofurantoin resistance and younger patients , in that 75% of patients with nitrofurantoin resistance were younger than 70 years ( p  =  0.025 ) .", "there was no statistically significant association between age and resistance profile in any other of the antimicrobial panel .", "the impact of mrsa is considerable ; in ireland , approximately 40% to 50% of isolates sa recovered from bloodstream infections are methicillin resistant . despite available publications and guidelines on the management of mrsa skin - colonized patients ,", "little has been published on the colonization of urine from patients with indwelling urinary catheters .", "the identification of an mrsa isolate in a urine culture has important ramifications for patients , both in the community and in the hospital setting .", "a recent study demonstrated that 22% of patients with mrsa bacteriuria went on to develop invasive mrsa infection within 12 months .", "mrsa in urine clearly warrants treatment in symptomatic patients , but even in asymptomatic patients , it may require eradication before certain elective procedures  such as endourological surgery .", "it also has consequences for patients and health care providers when it comes to providing isolation and other barrier precautions that should be administered in a nosocomial setting .", "the european association of urology guidelines outlines that colonization with microorganisms is a special risk factor for urological procedures ( http://uroweb.org/guideline/urological-infections/ ) , and furthermore , that an indwelling catheter is one of the most important risk factors for complications .", "patients with asymptomatic bacteriuria who undergo traumatic genitourinary procedures associated with mucosal bleeding have a high rate of post - procedure bacteremia and sepsis .", "bacteremia occurs in up to 60% of bacteriuric patients who undergo transurethral prostatic resection , and sepsis is clinically confirmed in 6% to 10% of these patients .", "retrospective analysis and prospective , randomized clinical trials support the effectiveness of antimicrobial treatment in preventing these complications in bacteriuric men undergoing transurethral resection of the prostate .", "there is little information relevant to other genitourinary procedures , but any intervention with a high probability of mucosal bleeding should be considered as a risk for post - procedure sepsis  and treatment of mrsa bacteriuria should be considered .", "some studies have documented that eradication of asymptomatic bacteriuria is not required before nonurologic procedures , such as arthroplasty  but s. aureus is the most pathogenic of all staphylococci and this study did not include patients with mrsa bacteriuria .", "our study would suggest that when it comes to urine - colonizing mrsa eradication , or for treating a patient who is not critically unwell , oral therapy with nitrofurantoin or trimethoprim would be a suitable first - line agent , as none of the patients in our cohort were resistant to both trimethoprim and nitrofurantoin . in the unwell or septic patient , vancomycin or teicoplanin", "interestingly , less than one - third ( 32.5% ) of our mrsa urine samples came from hospital inpatient sources , implying that mrsa bacteriuria diagnosis is more frequently a community - based phenomenon .", "national recommendations dictate that hospital patients colonized with mrsa should be isolated in single rooms  and expert opinion would surmise that there is an increased risk of spread from mrsa - colonized catheterized patients  due to increased interventions and manipulations required from staff ( e.g. changing catheter drainage devices ) .", "however , within the community , these patients are not recommended to be isolated as would happen within the nosocomial environment and this could contribute to further bacterial spread", ". the limitations of our study include its retrospective nature  which meant that causation was not addressed , and not all key information was available to our research group .", "also , acting as a potential confounder is the fact that mrsa bacteriuria is not a common pathology ", "meaning we had only small number of urine isolates ( 167 from 106 patients ) despite the large sample size ( 425,013 ) . to this author 's knowledge", ", there are no prospective mrsa bacteriuria studies underway  and a supra - regional or national study of this type could provide further relevant data in this field ."], "abstract_text": ["<S> abstracturinary tract infections are one of the most common infectious diseases diagnosed in the community and in the hospital setting . </S>", "<S> their treatment is complicated by drug - resistant pathogens and the colonization by microbes of indwelling urinary catheters . </S>", "<S> this study assessed the occurrence and antimicrobial susceptibility of methicillin - resistant staphylococcus aureus ( mrsa ) uropathogens isolated for 5 consecutive years at university hospital waterford between 2010 and 2014 . </S>", "<S> we created 4 clinically relevant subdivisions , based on urine source : hospital inpatients , patients from the emergency department , patients referred from their general practitioner , and nursing home patients . </S>", "<S> we performed a retrospective review from the hospital 's electronic microbiological system and calculated resistance rates for each of the standard antimicrobial agents . during the 5-year study period </S>", "<S> , we studied 151 urine isolates obtained from 128 patients who had an mrsa cultured in their urine sample . </S>", "<S> there was 100% resistance of all mrsa isolates to flucloxacillin and coamoxiclav . </S>", "<S> ninety - eight percent of isolates were resistant to ciprofloxacin . </S>", "<S> the resistance rate for trimethoprim was 7.4% and there was only 2.7% resistance for nitrofurantoin . for a clinical subset of patients </S>", "<S> , we also demonstrated 100% sensitivity for samples tested against teicoplanin and vancomycin . </S>", "<S> urinary mrsa is an infrequently studied phenomenon , but with the rising trend of hospital superbugs nationally , its management is of critical importance . </S>", "<S> suitable agents to address this within our population include nitrofurantoin in the well patient requiring urinary mrsa eradication or vancomycin / teicoplanin in the unwell patient requiring intravenous therapy . in all groups , fluoroquinolones should be avoided due to significant resistance rates . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion"], "sections": [["urinary tract infection ( uti ) is a frequent cause of morbidity both in the community and in the hospital setting .", "the causative pathogen can vary greatly geographically , and so it is prudent to identify those with resistant strains and have current data on the appropriate empirical therapy within a region .", "the most frequently encountered organisms associated with utis include enteric gram - negative bacteria ( with escherichia coli being the most predominant ) , coagulase negative staphylococcus saprophyticus along with proteus mirabilis , klebsiella , and enterococcus , which account for less than 5% . however , recent studies have reported the increasing prevalence of staphylococcus aureus ( sa ) in utis .", "sa is an opportunistic pathogen affecting both immune competent and immunocompromised individuals , frequently resulting in significant morbidity", ". many strains of sa carry a wide variety of multidrug - resistant genes on plasmids , which aid the spread of resistance among species .", "methicillin - resistant sa ( mrsa ) is widespread in many irish hospitals and is increasingly seen in community health care units such as nursing homes .", "globally , it is considered that there has been an epidemic of mrsa within health care institutions . bacteriuria with sa is hypothesized to occur through a number of mechanisms that includes catheterization , urologic procedures , or seeding of the genitourinary tract  including nephrologically excreted bacteria in overt bacteremia .", "bacteremia itself is associated with bacteriuria in patients infected with sa , which suggests that bacteremia is an important precursor for bacteriuria in some patient groups .", "herein , we chronologically assessed the source , patient demographics , and antimicrobial susceptibilities of mrsa - positive isolates from the hospital and community setting over a 5-year period in university hospital waterford  a tertiary referral hospital with the primary microbiology laboratory for 4 acute hospitals , serving a total catchment area of almost 500,000 people ."], ["the microbiology laboratory in university hospital waterford processes all inpatient urines in the catchment area of almost 500,000 , excluding 2 small private elective inpatient institutions .", "it also processes all urine samples sent from the community ( i.e. , all samples from general practice and nursing homes ) and all specimens from the catchments emergency departments .", "laboratory diagnosis of mrsa bacteriuria was performed using accredited microbiological microscopy analysis with culture identification criteria and standardized susceptibility testing protocols .", "our laboratory utilized clinical and laboratory standards institute ( clsi ; 20102014 ) and european committee on antimicrobial susceptibility testing ( eucast ; 2014 ) methodologies .", "a retrospective data extraction was performed , from the hospital 's electronic microbiological system  cognos  and de - duplication was executed .", "we considered a  separate episode  as a urine specimen culturing mrsa at least 6 months following a previous positive culture .", "further urine specimens sent within a 6-month period following an initial laboratory diagnosis mrsa bacteriuria were excluded .", "resistance rates were then calculated for the pathogen 's susceptibility to 5 commonly used antimicrobial agents  ciprofloxacin , coamoxiclav , flucloxacillin , nitrofurantoin , and trimethoprim . for unwell patients ,", "that is , those in whom there was clinical suspicion of sepsis or invasive infection , teicoplanin and vancomycin were also tested for sensitivity profiling . in order to obtain further clinical information from our patient group , we also assessed those patients who had an mrsa - positive swab ( usually from groin , nose , or perineum ) , those who had documented mrsa bacteremia , and we analyzed their demographic details .", "ethical approval was waived for this retrospective observational study in light of its non - interventional nature ."], ["over a consecutive 5-year period , the laboratory cultured 425,013 urine samples from all sources , with a mean number of 85,003 specimens per year received ( table 1 ) .", "a total of 542 unique urine isolates cultured sa and 151 of species ( 27.9% ) were methicillin resistant , meaning that 0.04% of all urine samples tested cultured mrsa .", "number of urine cultures by year . of these 151 samples that tested positive for mrsa , 92 ( 61% ) were from mid - stream urine ( msu ) samples , 50 ( 33% ) from catheter specimen urine ( csu ) samples , and 9 ( 6% ) were from an unspecified source ( fig .", "csu  =  catheter specimen urine , mrsa  =  methicillin resistant staphylococcus aureus , msu  =  mid - stream urine .", "mrsa was slightly more common in msu samples ; however , the findings were not statistically significant ( 18.2% vs 13.9% , p  =  0.388 ) .", "forty - nine ( 32.5% ) specimens were from an inpatient cohort , 9 ( 6% ) were from the emergency department , 79 ( 52.3% ) from general practitioners , 12 ( 7.9% ) were from nursing homes , with 2 ( 1.3% ) from an unrecorded source ( fig .", "mrsa was seen in a similar proportion of inpatient and outpatient samples indicating that this is not solely a hospital - acquired phenomenon ( 29.1% vs 26.9% , p  =  0.587 ) .", "mrsa  =  methicillin resistant staphylococcus aureus . the mean age of our patients who cultured mrsa within their urine was 72.7 years ( range 10.9790.2 ) and most patients were aged between 70 and 90 years ( table 2 ) .", "patients who cultured an mrsa bacterium were older than patients with mssa , and this was statistically significant ( 53 vs 73 years , p    0.001 ) .", "the number of sa isolates grown over the 5-year study period doubled from 77 in 2010 to 161 in 2014 .", "the number of mrsa isolates had also increased ; however , the percentage of sa isolates that were methicillin resistant has decreased ( p  =  0.145 ) .", "this is true for both inpatient and outpatient samples ( p  =  0.313 , p  =  0.254 , figs .", "mrsa  =  methicillin resistant staphylococcus aureus , mssa  =  methicillin sensitive staphylococcus aureus , sa  =  staphylococcus aureus . inpatient trends in sa", "mrsa  =  methicillin resistant staphylococcus aureus , mssa  =  methicillin sensitive staphylococcus aureus , sa  =  staphylococcus aureus .", "mrsa  =  methicillin resistant staphylococcus aureus , mssa  =  methicillin sensitive staphylococcus aureus , sa  =  staphylococcus aureus .", "regarding the clinical stance of patients within the cohort , 9 patients ( 7% ) with mrsa - positive urine cultures had a documented history of mrsa bacteremia at that time .", "each of these patients cultured mrsa isolates that were sensitive to nitrofurantoin , and 8 of the 9 patients ( 88.9% ) were sensitive to trimethoprim .", "it was noted that all 9 patients in this cohort were aged 70 years or above ( p  =  0.079 ) , although this did not reach statistical significance .", "further antimicrobials agents were introduced to the testing panel in 59 ( 39% ) samples , where there was a clinical concern regarding invasive infection or sepsis  including the 9 patients with mrsa bacteremia . all 59 samples ( 100% ) were sensitive to both vancomycin and teicoplanin .", "eighty - eight ( 66.8% ) of our 128 patients cultured mrsa - positive isolates on routine swabs from either mucosal or skin sites .", "all 3 patients with resistance to nitrofurantoin in their urine had a positive swab from either groin or nose .", "eight of the 10 patients ( 80% ) who had trimethoprim - resistant mrsa bacteriuria and 87 of the 125 ( 69.6% ) of the ciprofloxacin - resistant mrsa bacteriuria also had mrsa - positive mucosal or skin swabs .", "there was no association between age and having an mrsa - positive swab in our cohort . throughout the study period", ", there was 100% resistance of all mrsa isolates to flucloxacillin and coamoxiclav , as expected .", "ninety - eight percent of isolates were resistant to ciprofloxacin , but of these samples , there was 97.2% sensitivity for nitrofurantoin and 92.4% sensitivity for trimethoprim ( fig .", "overall , there was only 2.7% resistance for nitrofurantoin , but all of these samples were sensitive to trimethoprim .", "trimethoprim itself had a 7.4% resistance level , but all of these samples were sensitive to nitrofurantoin , meaning that no sample was resistant to both oral agents , that is , nitrofurantoin and trimethoprim .", "there was a statistically significant association between nitrofurantoin resistance and younger patients , in that 75% of patients with nitrofurantoin resistance were younger than 70 years ( p  =  0.025 ) .", "there was no statistically significant association between age and resistance profile in any other of the antimicrobial panel ."], ["the impact of mrsa is considerable ; in ireland , approximately 40% to 50% of isolates sa recovered from bloodstream infections are methicillin resistant . despite available publications and guidelines on the management of mrsa skin - colonized patients ,", "little has been published on the colonization of urine from patients with indwelling urinary catheters .", "the identification of an mrsa isolate in a urine culture has important ramifications for patients , both in the community and in the hospital setting .", "a recent study demonstrated that 22% of patients with mrsa bacteriuria went on to develop invasive mrsa infection within 12 months .", "mrsa in urine clearly warrants treatment in symptomatic patients , but even in asymptomatic patients , it may require eradication before certain elective procedures  such as endourological surgery .", "it also has consequences for patients and health care providers when it comes to providing isolation and other barrier precautions that should be administered in a nosocomial setting .", "the european association of urology guidelines outlines that colonization with microorganisms is a special risk factor for urological procedures ( http://uroweb.org/guideline/urological-infections/ ) , and furthermore , that an indwelling catheter is one of the most important risk factors for complications .", "patients with asymptomatic bacteriuria who undergo traumatic genitourinary procedures associated with mucosal bleeding have a high rate of post - procedure bacteremia and sepsis .", "bacteremia occurs in up to 60% of bacteriuric patients who undergo transurethral prostatic resection , and sepsis is clinically confirmed in 6% to 10% of these patients .", "retrospective analysis and prospective , randomized clinical trials support the effectiveness of antimicrobial treatment in preventing these complications in bacteriuric men undergoing transurethral resection of the prostate .", "there is little information relevant to other genitourinary procedures , but any intervention with a high probability of mucosal bleeding should be considered as a risk for post - procedure sepsis  and treatment of mrsa bacteriuria should be considered .", "some studies have documented that eradication of asymptomatic bacteriuria is not required before nonurologic procedures , such as arthroplasty  but s. aureus is the most pathogenic of all staphylococci and this study did not include patients with mrsa bacteriuria .", "our study would suggest that when it comes to urine - colonizing mrsa eradication , or for treating a patient who is not critically unwell , oral therapy with nitrofurantoin or trimethoprim would be a suitable first - line agent , as none of the patients in our cohort were resistant to both trimethoprim and nitrofurantoin . in the unwell or septic patient , vancomycin or teicoplanin", "interestingly , less than one - third ( 32.5% ) of our mrsa urine samples came from hospital inpatient sources , implying that mrsa bacteriuria diagnosis is more frequently a community - based phenomenon .", "national recommendations dictate that hospital patients colonized with mrsa should be isolated in single rooms  and expert opinion would surmise that there is an increased risk of spread from mrsa - colonized catheterized patients  due to increased interventions and manipulations required from staff ( e.g. changing catheter drainage devices ) .", "however , within the community , these patients are not recommended to be isolated as would happen within the nosocomial environment and this could contribute to further bacterial spread", ". the limitations of our study include its retrospective nature  which meant that causation was not addressed , and not all key information was available to our research group .", "also , acting as a potential confounder is the fact that mrsa bacteriuria is not a common pathology ", "meaning we had only small number of urine isolates ( 167 from 106 patients ) despite the large sample size ( 425,013 ) . to this author 's knowledge", ", there are no prospective mrsa bacteriuria studies underway  and a supra - regional or national study of this type could provide further relevant data in this field ."]]}
{"article_id": "PMC3220812", "article_text": ["acquired angioedema ( aae ) , an acquired deficiency of c1esterase inhibitor , is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen .", "this disorder is strongly associated with chronic lymphocytic leukemia ( cll ) and other indolent lymphoplasmacytic disorders .", "we describe a patient with known cll who developed incapacitating , recurrent severe abdominal pains , culminating in partial bowel resection .", "wider appreciation of the possibility of aae , particularly in patients with lymphoproliferative disorders , could lead to preventive therapy and spare unnecessary surgery .", "acquired angioedema ( aae ) is due to acquired deficiency of c1 inhibitor ( c1inh ) , resulting in excessive complement and bradykinin activities .", "blood vessel permeability is increased ; thus , angioedema occurs . just as with hereditary angioedema ( hereditary c1inh deficiency ; hae ) , common clinical manifestations are skin swelling , laryngeal edema , and/or abdominal pain.1,2 aae often occurs in the context of lymphoplasmacytic disorders , such as monoclonal gammopathy of unknown significance ( mgus ) , non - hodgkin s lymphoma , or chronic lymphocytic leukemia ( cll).1,3 among 32 patients with aae , castelli found that 13 ( 40% ) had mgus and 9 ( 28% ) had lymphoproliferative disease.3 therefore , all cases of aae should be evaluated for the possibility of underlying lymphoplasmacytic disorder .", "conversely , when patients with known lymphoproliferative disease manifest compatible symptoms , aae should be expeditiously considered .", "this is important because aae can be effectively treated medically , but delayed diagnosis can lead to unnecessary diagnostic procedures , therapeutic interventions , or life - threatening complications , well - illustrated by our case .", "a 78-year - old woman with atherosclerotic vascular disease was transferred to our hospital with abdominal pain and underwent emergent laparotomy .", "one year earlier , she had been diagnosed with rai stage i cll which had been observed without treatment .", "two months earlier , she presented with severe abdominal pain , nausea , and vomiting . over the next 8  weeks , she had six emergency room and/or hospital admissions for identical symptoms .", "pains would begin at rest in the lower abdomen , spread to the upper abdomen , described as  gas - like  , non - radiating , constant .", "extensive evaluation included colonoscopy , endoscopic retrograde cholangiopancreatography , magnetic resonance angiography , and abdominal aortography .", "benign colon polyps and small gallstones were removed , mesenteric stenoses ruled out , yet pains recurred unabated .", "there was no dysphagia , change in bowel habits , gi bleeding , fever , or sweats .", "physical examination on transfer revealed blood pressure 130/88  mmhg , pulse 88 beats per minute , respiratory rate 20  cycles per minute , pulse oximetry of 95% on ambient air , and temperature 97.0f .", "hemoglobin was 18.1  g / dl , hematocrit 55% , platelets 146    10/l , and leukocytes elevated to 34,500 cells/l with 47% neutrophils and 48% lymphocytes .", "ct scan of abdomen and pelvis with iv contrast showed multiple abnormal loops of small bowel with contrast - enhanced bowel wall edema ( fig . ", ". 1a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema .", "b a section of small bowel shows massive submucosal edema . there is no infiltration of the wall by lymphocytes a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema .", "b a section of small bowel shows massive submucosal edema . there is no infiltration of the wall by lymphocytes at laparotomy , massively swollen small bowel was encountered and resected .", "c4 was 3  mg / dl ( normal 1746 ) , c3 66  mg / dl ( 85200 ) , and c1inh activity reportedly 83% ( 68200% ) .", "serum protein electrophoresis revealed two faint bands immunofixing as monoclonal igm kappa and igg kappa .", "lymphocytosis and lymphadenopathy improved and c1inh activity increased to 110% . over 3  years of follow - up , abdominal symptoms never recurred .", "approximately 145 cases have been reported of aae,3 and this is one of four cases that we have diagnosed in the last decade with abdominal pains from aae with associated cll .", "table  1 shows characteristics of patients with cll and aae we have seen ( some briefly mentioned in a prior report).4 this is our only case to undergo surgery , allowing unique and dramatic demonstration of massive bowel edema visible radiographically , on surgical inspection and on histopathology .", "an initial c1inh activity was reported low normal , but there can be no doubt about the diagnosis based on radiologic / surgical / histologic findings , further laboratory results , and the clinical course .", "characteristic are very low c4 level and low c3 ; diagnostic recommendations currently add c1q and anti - c1inh antibody levels .", "autoantibody is demonstrable in up to 70% with aae.1 our patient had monoclonal gammopathy , which frequently corresponds to the c1inh autoantibody .", "patients with the hereditary form ( hae ) usually manifest before age 20 and give a family history of symptoms . in all our patients with cll and aae , chemotherapy and androgens increased c1inh and produced durable remission.table 1characteristics of patients with aaepatientunderlying diagnosismanifestations of acquired angioedemamonths before diagnosislaboratory valuesinterventions prior to diagnosistreatmentangioeedema episodes post - treatment ( years of follow - up)pretreatmentpost - treatment78  years old / fcllrecurrent abdominal pain2c1 inh activity : 83% ( 68200)c1 inh activity : 110% ( 68200)exploratory laparotomy with small bowel resectionchemotherapydanazolnone ( 3  years)c4 : 3  mg / dl ( 1746)ct scanc3 : 66  mg / dl ( 85200)colonoscopysmall monoclonal gammopathyercpmraabdominal aortography74  years old / fsll / cllrecurrent abdominal pain24c1 inh activity : 1% ( 68200%)c1 inh activity : 117% ( 68200%)ct scanchemotherapynone ( 6  years)episodic oropharyngeal swellingc1 inh quantitative : 6.7  mg / dl ( > 11  mg / dl)c1 inh quantitative : 21  mg / dl ( 1025)colonoscopydanazolc4 : 13  mg / dl ( 1647)c3 : 70  mg / dl ( 75161)small monoclonal gammopathy61  years old / mcllrecurrent abdominal pain5c1 inh activity : 4% ( 68200%)not availablect scanchemotherapynone ( 10  years)small monoclonal gammopathycolonoscopydanazol67  years", "old / mcllrecurrent abdominal pain3c1 inh activity : 6%(68200)c1 inh quantitative : 38  mg / dl ( 2139)colonoscopychemotherapynone ( 3  months)oropharyngeal swellingc1 inh quantitative : 3  mg / dl ( 2139)laryngoscopydanazolc1q : 3.6  mg / dl ( 58.6)c4 : < 2  mg / dl ( 1746)small monoclonal gammopathy characteristics of patients with aae angioedema should be borne in mind among  medical  illnesses that can mimic acute surgical abdomen , along with such other disorders as porphyria , familial mediterranean fever and sickle cell disease ( fig .  2 ) .", "one may need to discern whether a true surgical emergency might supervene even when one of these disorders is present .", "our patient had typical symptoms of acute bowel edema , including diffuse abdominal pain , occasionally rebound tenderness and vomiting , with spontaneous resolution within 15  days.1,2 some patients with aae have cutaneous or upper respiratory edema in addition , or instead of , bowel symptoms .", "because of cardiovascular comorbidities , there was a high suspicion for ischemic bowel in our patient , but radiography and endoscopy did not support this . in the differential diagnosis ,", "angiotensin - converting enzyme inhibitors rarely precipitate angioedema , but our patient had taken benazepril many years and continued it without incident after aae therapy.fig .", "2algorithm for diagnosis and treatment for suspected aae algorithm for diagnosis and treatment for suspected aae in 2009 , the us fda approved the c1inh concentrate berinert p and the kallikrein inhibitor ecallantide ( kalbitor ) for treatment of acute attacks , and the c1inh concentrate cinryze for prophylaxis in severely affected patients.5 fresh frozen plasma can be given when these are unavailable .", "c1inh concentrates are highly safe and effective ; the standard of care for decades in other countries , but us approval was delayed by concerns of virus transmissibility.1,59 doses used in the clinical hae trials may need to be higher for aae because of increased enzyme clearance .", "a bradykinin b2 receptor antagonist icatibant ( firazyr ) is under investigation . for long - term control ,", "commonly used for prophylaxis are anti - fibrinolytic drugs or the attenuated androgen danazol , which increases c1inh synthesis at low cost.10 in conclusion , earlier suspicion for aae in our known cll patient could have spared her the morbidities of recurrent abdominal pains , hospitalizations , morbid interventions , and bowel resection .", "wider appreciation of this disorder takes on added importance as our ability to effectively treat the problem has grown ."], "abstract_text": ["<S> introductionacquired angioedema ( aae ) , an acquired deficiency of c1esterase inhibitor , is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen . </S>", "<S> this disorder is strongly associated with chronic lymphocytic leukemia ( cll ) and other indolent lymphoplasmacytic disorders.discussionwe describe a patient with known cll who developed incapacitating , recurrent severe abdominal pains , culminating in partial bowel resection . </S>", "<S> signs , symptoms , laboratory and pathologic findings demonstrated aae.conclusionwider appreciation of the possibility of aae , particularly in patients with lymphoproliferative disorders , could lead to preventive therapy and spare unnecessary surgery . </S>", "<S> this is more important now that more effective medical therapies are available . </S>"], "labels": null, "section_names": ["Introduction", "Discussion", "Conclusion", "Introduction", "Case Presentation", "Discussion"], "sections": [["acquired angioedema ( aae ) , an acquired deficiency of c1esterase inhibitor , is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen .", "this disorder is strongly associated with chronic lymphocytic leukemia ( cll ) and other indolent lymphoplasmacytic disorders ."], ["we describe a patient with known cll who developed incapacitating , recurrent severe abdominal pains , culminating in partial bowel resection ."], ["wider appreciation of the possibility of aae , particularly in patients with lymphoproliferative disorders , could lead to preventive therapy and spare unnecessary surgery ."], ["acquired angioedema ( aae ) is due to acquired deficiency of c1 inhibitor ( c1inh ) , resulting in excessive complement and bradykinin activities .", "blood vessel permeability is increased ; thus , angioedema occurs . just as with hereditary angioedema ( hereditary c1inh deficiency ; hae ) , common clinical manifestations are skin swelling , laryngeal edema , and/or abdominal pain.1,2 aae often occurs in the context of lymphoplasmacytic disorders , such as monoclonal gammopathy of unknown significance ( mgus ) , non - hodgkin s lymphoma , or chronic lymphocytic leukemia ( cll).1,3 among 32 patients with aae , castelli found that 13 ( 40% ) had mgus and 9 ( 28% ) had lymphoproliferative disease.3 therefore , all cases of aae should be evaluated for the possibility of underlying lymphoplasmacytic disorder .", "conversely , when patients with known lymphoproliferative disease manifest compatible symptoms , aae should be expeditiously considered .", "this is important because aae can be effectively treated medically , but delayed diagnosis can lead to unnecessary diagnostic procedures , therapeutic interventions , or life - threatening complications , well - illustrated by our case ."], ["a 78-year - old woman with atherosclerotic vascular disease was transferred to our hospital with abdominal pain and underwent emergent laparotomy .", "one year earlier , she had been diagnosed with rai stage i cll which had been observed without treatment .", "two months earlier , she presented with severe abdominal pain , nausea , and vomiting . over the next 8  weeks , she had six emergency room and/or hospital admissions for identical symptoms .", "pains would begin at rest in the lower abdomen , spread to the upper abdomen , described as  gas - like  , non - radiating , constant .", "extensive evaluation included colonoscopy , endoscopic retrograde cholangiopancreatography , magnetic resonance angiography , and abdominal aortography .", "benign colon polyps and small gallstones were removed , mesenteric stenoses ruled out , yet pains recurred unabated .", "there was no dysphagia , change in bowel habits , gi bleeding , fever , or sweats .", "physical examination on transfer revealed blood pressure 130/88  mmhg , pulse 88 beats per minute , respiratory rate 20  cycles per minute , pulse oximetry of 95% on ambient air , and temperature 97.0f .", "hemoglobin was 18.1  g / dl , hematocrit 55% , platelets 146    10/l , and leukocytes elevated to 34,500 cells/l with 47% neutrophils and 48% lymphocytes .", "ct scan of abdomen and pelvis with iv contrast showed multiple abnormal loops of small bowel with contrast - enhanced bowel wall edema ( fig . ", ". 1a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema .", "b a section of small bowel shows massive submucosal edema . there is no infiltration of the wall by lymphocytes a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema .", "b a section of small bowel shows massive submucosal edema . there is no infiltration of the wall by lymphocytes at laparotomy , massively swollen small bowel was encountered and resected .", "c4 was 3  mg / dl ( normal 1746 ) , c3 66  mg / dl ( 85200 ) , and c1inh activity reportedly 83% ( 68200% ) .", "serum protein electrophoresis revealed two faint bands immunofixing as monoclonal igm kappa and igg kappa .", "lymphocytosis and lymphadenopathy improved and c1inh activity increased to 110% . over 3  years of follow - up , abdominal symptoms never recurred ."], ["approximately 145 cases have been reported of aae,3 and this is one of four cases that we have diagnosed in the last decade with abdominal pains from aae with associated cll .", "table  1 shows characteristics of patients with cll and aae we have seen ( some briefly mentioned in a prior report).4 this is our only case to undergo surgery , allowing unique and dramatic demonstration of massive bowel edema visible radiographically , on surgical inspection and on histopathology .", "an initial c1inh activity was reported low normal , but there can be no doubt about the diagnosis based on radiologic / surgical / histologic findings , further laboratory results , and the clinical course .", "characteristic are very low c4 level and low c3 ; diagnostic recommendations currently add c1q and anti - c1inh antibody levels .", "autoantibody is demonstrable in up to 70% with aae.1 our patient had monoclonal gammopathy , which frequently corresponds to the c1inh autoantibody .", "patients with the hereditary form ( hae ) usually manifest before age 20 and give a family history of symptoms . in all our patients with cll and aae , chemotherapy and androgens increased c1inh and produced durable remission.table 1characteristics of patients with aaepatientunderlying diagnosismanifestations of acquired angioedemamonths before diagnosislaboratory valuesinterventions prior to diagnosistreatmentangioeedema episodes post - treatment ( years of follow - up)pretreatmentpost - treatment78  years old / fcllrecurrent abdominal pain2c1 inh activity : 83% ( 68200)c1 inh activity : 110% ( 68200)exploratory laparotomy with small bowel resectionchemotherapydanazolnone ( 3  years)c4 : 3  mg / dl ( 1746)ct scanc3 : 66  mg / dl ( 85200)colonoscopysmall monoclonal gammopathyercpmraabdominal aortography74  years old / fsll / cllrecurrent abdominal pain24c1 inh activity : 1% ( 68200%)c1 inh activity : 117% ( 68200%)ct scanchemotherapynone ( 6  years)episodic oropharyngeal swellingc1 inh quantitative : 6.7  mg / dl ( > 11  mg / dl)c1 inh quantitative : 21  mg / dl ( 1025)colonoscopydanazolc4 : 13  mg / dl ( 1647)c3 : 70  mg / dl ( 75161)small monoclonal gammopathy61  years old / mcllrecurrent abdominal pain5c1 inh activity : 4% ( 68200%)not availablect scanchemotherapynone ( 10  years)small monoclonal gammopathycolonoscopydanazol67  years", "old / mcllrecurrent abdominal pain3c1 inh activity : 6%(68200)c1 inh quantitative : 38  mg / dl ( 2139)colonoscopychemotherapynone ( 3  months)oropharyngeal swellingc1 inh quantitative : 3  mg / dl ( 2139)laryngoscopydanazolc1q : 3.6  mg / dl ( 58.6)c4 : < 2  mg / dl ( 1746)small monoclonal gammopathy characteristics of patients with aae angioedema should be borne in mind among  medical  illnesses that can mimic acute surgical abdomen , along with such other disorders as porphyria , familial mediterranean fever and sickle cell disease ( fig .  2 ) .", "one may need to discern whether a true surgical emergency might supervene even when one of these disorders is present .", "our patient had typical symptoms of acute bowel edema , including diffuse abdominal pain , occasionally rebound tenderness and vomiting , with spontaneous resolution within 15  days.1,2 some patients with aae have cutaneous or upper respiratory edema in addition , or instead of , bowel symptoms .", "because of cardiovascular comorbidities , there was a high suspicion for ischemic bowel in our patient , but radiography and endoscopy did not support this . in the differential diagnosis ,", "angiotensin - converting enzyme inhibitors rarely precipitate angioedema , but our patient had taken benazepril many years and continued it without incident after aae therapy.fig .", "2algorithm for diagnosis and treatment for suspected aae algorithm for diagnosis and treatment for suspected aae in 2009 , the us fda approved the c1inh concentrate berinert p and the kallikrein inhibitor ecallantide ( kalbitor ) for treatment of acute attacks , and the c1inh concentrate cinryze for prophylaxis in severely affected patients.5 fresh frozen plasma can be given when these are unavailable .", "c1inh concentrates are highly safe and effective ; the standard of care for decades in other countries , but us approval was delayed by concerns of virus transmissibility.1,59 doses used in the clinical hae trials may need to be higher for aae because of increased enzyme clearance .", "a bradykinin b2 receptor antagonist icatibant ( firazyr ) is under investigation . for long - term control ,", "commonly used for prophylaxis are anti - fibrinolytic drugs or the attenuated androgen danazol , which increases c1inh synthesis at low cost.10 in conclusion , earlier suspicion for aae in our known cll patient could have spared her the morbidities of recurrent abdominal pains , hospitalizations , morbid interventions , and bowel resection .", "wider appreciation of this disorder takes on added importance as our ability to effectively treat the problem has grown ."]]}
{"article_id": "PMC3590702", "article_text": ["distal clavicle fractures associated with partial and complete coracoclavicular ligament disruption are potentially unstable , and non - operative treatment of these fractures is associated with a high non - union rate.[13 ] partial ligament disruptions ( conoid or trapezoid ) retain some inherent stability , and should be distinguished from complete dual ligament disruptions that result in an uncommon and highly unstable subgroup", ". internal fixation of these fractures necessitates the use of acromioclavicular joint spanning implants ( clavicle hook plate , synthes , usa ) that overcome distraction forces by subacromial leverage ; however , subacromial / acromioclavicular joint encroachment with these implants is associated with subacromial pathology , and this may negatively influence the final outcomes . to overcome the shortcomings of the joint - spanning implants , acromioclavicular joint - sparing implants ( 2.4 mm lcp distal radius plates , synthes , usa ) have been suggested .", "additionally , a combination of this implant with some form of coracoclavicular fixation using sutures / anchors / endobutton devices ( tightrope , arthrex , fl , usa ) has been recently described for use in the highly unstable fracture subgroup .", "however , comparative clinico - radiological outcomes of the two techniques are lacking in literature .", "the purpose of this study was ( 1 ) to analyze the overall and comparative clinico - radiological outcomes ( radiographic and ultrasonographic ) of surgical treatment of distal clavicle fractures associated with complete coracoclavicular ligament disruption , and ( 2 ) to identify and analyze radiographic patterns of fracture and comminution that are associated with this injury .", "the hospital records of all patients who were surgically treated for lateral clavicle fractures between 2005 and 2008 at three hospitals were retrospectively evaluated to identify the study group .", "inclusion criteria for this study was defined as : ( i ) acute , isolated , non - comminuted and comminuted fractures of the lateral clavicle , ( ii ) complete disruption of both coracoclavicular ligaments , confirmed on radiographs and at surgery , ( iii ) absence of concomitant or pre - existing subacromial pathology ( rotator cuff tears , acromial undersurface degeneration ) , ( iv ) surgical treatment with either a joint - spanning implant alone ( group 1 ) , or combination of a joint - sparing implant and coracoclavicular fixation with either endobutton device , suture anchor , or coracoid cerclage ( group 2 ) [ figures 1a and b ] , and ( v ) follow - up period of at least 6 months for radiographic evidence of union .", "exclusion criteria was defined as : ( i ) partial disruption of the coracoclavicular ligaments at surgery , ( ii ) subacromial pathology ( iii ) concomitant injuries to the ipsilateral shoulder girdle , ( iv ) surgical treatment with any technique other than those described in the inclusion criteria , and ( v ) follow - up period inadequate for complete union .", "surgical treatment of an unstable distal clavicle fracture with a joint - spanning implant ( group 1 ) .", "( hk : hook plate , cl : clavicle , m : medial fragment , l : lateral fragment , arrow : fracture line , ac : acromion , h : humeral head , co : coracoid process ) surgical treatment of an unstable distal clavicle fracture with a joint - sparing implant and coracoclavicular ligament reconstruction using endobutton device ( group 2 ) .", "( rp : locking distal radius plate , arrow : acromioclavicular joint , ac : acromion , cl : clavicle , en : endobutton , h : humeral head ) the clinical records and operative notes were analyzed to identify patients that satisfied the criteria described above , and all other lateral clavicle fracture patients were excluded .", "pre - operative radiographs of the study group were obtained and analyzed with a graphic analysis software ; fracture lines were traced to identify involvement of the acromioclavicular joint , lateral fragment integrity , and presence / absence of comminution .", "the follow - up clinical protocol included a subjective evaluation of pain and functional status by interview , evaluation of active and passive range of motion by clinical examination , and evaluation of muscle strength in elevation and external rotation using a portable dynamometer .", "clinical acromioclavicular joint tests ( tenderness and paxinos sign ) and rotator cuff tests ( lag signs , bear - hug test , impingement signs ) were used to evaluate these structures.[811 ] clinical outcome measures included ( i ) simple shoulder test , ( ii ) constant and murley shoulder score , and ( iii ) walch acromioclavicular joint score . the follow - up radiographic protocol consisted of standardized radiographs that included a true glenohumeral anteroposterior view ( neutral rotation , elbow by the side ) , and a weight bearing comparative cephalic - tilt view ; these were analyzed for implant migration , acromioclavicular joint pathology ( degeneration , instability ) , subacromial changes ( degeneration , osteolysis ) , acromiohumeral interval measurement ( normal interval = 7 mm or more ) , peri - coracoid changes ( abnormal bone formation ) , and glenohumeral changes .", "in addition , in those cases where implant had been previously removed , past radiographs were obtained from the records and individually analyzed as described above .", "the follow - up ultrasonographic protocol included evaluation of the acromioclavicular joint , subacromial space , glenohumeral joint , bicipital groove and biceps long tendon , and suprascapular and spinoglenoid notches ; ultrasound assessment was performed by a senior radiologist experienced in shoulder ultrasound .", "statistical analysis was performed using a statistical software ( spss inc , chicago , usa ) to determine mean values and range of measured parameters of the overall and implant specific groups .", "significant differences between clinical and radiological parameters were determined using the non - parametric mann - whitney / wilcoxon rank - sum test for numerical data , and the fisher 's exact test for categorical data .", "the average age of the patients in the study group was 34.5 years ( range , 20 to 57 years ) .", "the mechanism of injury was a direct impact to the ipsilateral shoulder sustained during a fall ; in 8 patients , the fracture was sustained during a cycling - related sporting activity ( competitive or recreational ) .", "ten patients were operated using a joint - spanning implant ( group 1 ) and 5 were operated using a joint - sparing implant technique ( group 2 ) .", "the mean follow - up period was 26.1 months ( range , 12 to 40 months ) .", "the overall and group - specific clinico - radiological outcomes and the types and distribution of fracture patterns are summarized in table 1 .", "overall and group.specific clinico.radiological outcomes and fracture patterns clinical outcomes : the mean cs was marginally higher in group 2 , while the mean sst score was marginally higher in group 1 ; none of these differences were statistically significant .", "acromioclavicular joint signs were positive in half of the group 1 patients , and in none in group 2 ; however , the walch scores for the acromioclavicular joint were not significantly different for the two groups .", "return to pre - operative level of recreational and competitive sports was seen in approximately two - thirds of the patients of group 1 , and all patients of group 2 ; this difference was not found to be statistically significant .", "reoperation rates related to implant removal were significantly higher ( p < 0.05 ) in group 1 patients ( 90 % ) as compared to group 2 ( 0% ) .", "radiographic outcomes [ figure 2a - d ] : radiographic union was observed in 93.3% of fractures ( 9 in group 1 , 5 in group 2 ) with only one non - union with subsequent fragment resorption ( group 1 ) .", "radiographic acromioclavicular joint degeneration was present in 3 patients ( group 1 ) ; all 3 patients had a clinically symptomatic ac joint .", "radiographic acromioclavicular joint superior subluxation was present in 4 patients ( group 1 = 1 patient , 10% ; group 2 = 3 patients , 60% ) ; none of these were clinically symptomatic .", "subacromial osteolysis and hook migration were seen in 5 patients ( group 1 ) ; implant removal was necessary in 4 of these 5 patients , and at final follow - up , resolution of the radiographic subacromial osteolytic lesions was observed .", "signs of progressive implant loosening ( screws and/or plate disengagement ) were seen in 3 patients ( group 1 ) .", "abnormal bone formation was present in 7 patients ( group 1 = 6 , group 2 = 1 ) .", "( b ) acromioclavicular joint subluxation ( arrows ) , ( c ) hook migration and osteolysis of acromial undersurface ( arrow ) , ( d ) peri - coracoid ossification ( arrows ) .", "( ac : acromion , cl : clavicle , co : coracoid , p : plate , an : suture anchor , g : glenoid , h : humeral head , hk : hook plate , ahi : acromiohumeral interval ) ultrasonographic outcomes [ figure 3a and b ] : supraspinatus lesions were seen in 3 patients ; these included partial articular - side tears ( group 1 = 1 patient , 10% ; group 2 = 1 patient , 20% ) , and supraspinatus bursal - side degenerative changes ( group 1 = 1 patient , 10% ) .", "acromioclavicular joint screw penetration was observed in 1 patient ( group 2 ) ; this penetration was not apparent on plain radiographs and the patient was asymptomatic at the final follow - up .", "no abnormalities were detected in the subacromial bursa on static ultrasound evaluation ; dynamic testing revealed abnormal subacromial bursal \" bunching \" with arm abduction in 2 patients ( group 1 = 1 , group 2 = 1 ) .", "( a ) partial articular - surface supraspinatus tendon avulsion ( black arrows ) ( b ) screw penetration into the acromioclavicular joint ( arrows ) .", "( gt : greater tuberosity , h : humeral head , ss : supraspinatus , ac : acromion , cl : clavicle , asterix : acromioclavicular joint ) radiographic evaluation of fracture comminution revealed 5 non - comminuted fractures ( group 1 = 2 , group 2 = 3 ) , and 10 comminuted fractures ( group 1 = 8 , group 2 = 2 ) .", "analysis of the fracture lines and fragment cortices revealed repetitive patterns in the fracture geometry , and four types could be identified : ( i ) type 1 pattern ( n = 5 , 33% ) involved a clean vertical or a short oblique type bicortical fracture line without any comminution [ figure 4a ] , ( ii ) type 2 pattern ( n = 4 , 27% ) involved a zone of segmental comminution , between intact bicortical lateral and medial fragments [ figure 4b ] , ( iii ) type 3 pattern ( n = 5 , 33% ) involved propogation of at least one fracture line into the juxta - articular cortex of the ac joint [ figure 4c ] and ( iv ) type 4 pattern ( n = 1 , 7% ) consisted of fracture line propogation into the acromioclavicular joint [ figure 4d ] .", "radiographic fracture patterns : ( a ) type 1 , ( b ) type 2 , ( c ) type 3 , and ( d ) type 4 .", "( ac : acromion , cl : clavicle , co : coracoid , g : glenoid , h : humeral head , arrows : fracture lines )", "the present study is the first study that describes overall and comparative outcomes ( clinical , radiographic , and ultrasonographic ) of surgical treatment of an uncommon lateral clavicular bony - ligamentous injury with joint - spanning and joint - sparing implants .", "in addition , four radiographic patterns of bone injury were identified to develop guidelines for choice of implants in these injuries . clinical outcomes and union rates after operative treatment of this injury", "are satisfactory , and this has been shown in other studies in literature . in the present study , both types of implants resulted in satisfactory and comparable clinical outcome scores , irrespective of the radiographic outcomes .", "however , a significant difference in the reoperation rate for implant removal ( p < 0.05 ) in patients treated with a joint - spanning implant probably implies that a joint - sparing implant could be preferentially used whenever fracture geometry permits .", "radiological outcomes , especially those associated with joint sparing techniques , have not been well documented in other studies .", "subluxation of the acromioclavicular joint , ( present in 60% of group 2 patients ) may be related to either concomitant injury to the acromioclavicular ligaments , or to a partial failure of the coracoclavicular ligament reconstruction ; transfer of distraction forces to ac ligaments after fracture union may then induce gradual subluxation of this joint .", "this complication was rare in the other group , probably due to the joint spanning implant design ; however , other radiological abnormalities like hook migration , subacromial osteolysis , implant loosening , and new bone formation were frequent .", "the acromioclavicular joint spanning hook plate is a commonly used implant , and most studies have utilized this as the sole implant of choice .", "although clinical outcomes in these studies have been satisfactory , the effect of the subacromial hook on bursal tissues has been debated and use of the distal clavicle plate suggested . by using ultrasonographic evaluation , this study demonstrated the safety of the joint - spanning hook plate in relation to rotator cuff and subacromial bursa .", "persistent pain in the post - operative period probably resulted from acromial undersurface irritation ; early plate removal after fracture union is recommended , and long - term clinical outcomes seem to be satisfactory after implant removal .", "an important finding of this study is the grouping of the radiographic fracture patterns into four surgically relevant types .", "types 1 and 2 are ideal indications for use of a joint - sparing implant ; adequate bone stock in the distal fragment in these types will theoretically permit secure fixation across the fracture site . in types 3 and 4", ", inadequate distal fragment size may not permit secure bicortical fixation , thereby necessitating use of joint - spanning implants .", ". the retrospective nature and small sample size does not permit identification of all possible fracture configurations , and statistical analysis may differ with larger numbers .", "however , the clinical results are similar to other literature studies that have analyzed joint - spanning implants . also , the current radiographic patterns described provide guidelines for implant choice , and are not intended to be used as a prognostic classification .", "we attribute the small size of the study group to the stringent inclusion and exclusion criteria that eliminated several other patients with lateral clavicle fractures , and this was necessary to obtain meaningful conclusions", ". lateral clavicle fractures with complete disruption of the coracoclavicular ligaments should be regarded as a distinct entity that is perhaps biomechanically and prognostically different from similar fractures with partial ligament disruption .", "a combination of a locking distal radius plate with coracoclavicular ligament reconstruction resulted in stable fixation and significantly lower reoperation rates , and should be used preferentially in lesser comminuted fractures ( types 1 and 2 ) ."], "abstract_text": ["<S> purpose : distal clavicle fracture associated with complete coracoclavicular ligament disruption represents an unstable injury , and osteosynthesis is recommended . </S>", "<S> this study was performed ( 1 ) to retrospectively analyse the clinico - radiological outcomes of two internal fixation techniques , and ( 2 ) to identify and analyse radiographic fracture patterns of fracture that are associated with this injury.materials and methods : a total of 15 patients underwent osteosynthesis with either ( 1 ) acromioclavicular joint - spanning implants ( group 1 , hook plate device , n = 10 ) or ( 2 ) joint - sparing implants ( group 2 , distal radius plate , n = 5 ) ; these were reviewed at a mean period of 26.1 months ( 12 to 40 months ) . </S>", "<S> clinical outcomes were measured using constant score ( cs ) , simple shoulder test ( sst ) , and walch acj score ( ws ) . </S>", "<S> radiographs and ultrasonography were used to assess the glenohumeral and acromioclavicular joints , and the subacromial space . </S>", "<S> preoperative radiographs were analyzed for assessment of fracture lines to identify radiographic patterns . </S>", "<S> statistical analysis of the data was performed to determine any significant differences between the two groups.results:the overall clinical outcome was satisfactory ( cs 80.8 , sst 11.3 , ws 17.6 ) and a high union rate ( 93.3% ) was observed . </S>", "<S> radiographic complications ( acromioclavicular degeneration and subluxation , hook migration , abnormal ossification ) did not negatively influence the final clinical outcomes . </S>", "<S> four distinct radiographic fracture patterns were observed . </S>", "<S> a statistically significant difference ( p < 0.05 ) was observed in the reoperation rates between the two groups.conclusions:internal fixation of this fracture pattern is associated with a high union rate and favorable clinical outcomes with both techniques . </S>", "<S> a combination of distal radius plate and ligament reconstruction device resulted in stable fixation and significantly lower reoperation rates , and should be used when fracture geometry permits ( types 1 and 2).design : retrospective review of a consecutive clinical case series.setting:level 1 academic trauma service , public hospital . </S>"], "labels": null, "section_names": ["Design:", "Setting:", "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [[""], [""], ["distal clavicle fractures associated with partial and complete coracoclavicular ligament disruption are potentially unstable , and non - operative treatment of these fractures is associated with a high non - union rate.[13 ] partial ligament disruptions ( conoid or trapezoid ) retain some inherent stability , and should be distinguished from complete dual ligament disruptions that result in an uncommon and highly unstable subgroup", ". internal fixation of these fractures necessitates the use of acromioclavicular joint spanning implants ( clavicle hook plate , synthes , usa ) that overcome distraction forces by subacromial leverage ; however , subacromial / acromioclavicular joint encroachment with these implants is associated with subacromial pathology , and this may negatively influence the final outcomes . to overcome the shortcomings of the joint - spanning implants , acromioclavicular joint - sparing implants ( 2.4 mm lcp distal radius plates , synthes , usa ) have been suggested .", "additionally , a combination of this implant with some form of coracoclavicular fixation using sutures / anchors / endobutton devices ( tightrope , arthrex , fl , usa ) has been recently described for use in the highly unstable fracture subgroup .", "however , comparative clinico - radiological outcomes of the two techniques are lacking in literature .", "the purpose of this study was ( 1 ) to analyze the overall and comparative clinico - radiological outcomes ( radiographic and ultrasonographic ) of surgical treatment of distal clavicle fractures associated with complete coracoclavicular ligament disruption , and ( 2 ) to identify and analyze radiographic patterns of fracture and comminution that are associated with this injury ."], ["the hospital records of all patients who were surgically treated for lateral clavicle fractures between 2005 and 2008 at three hospitals were retrospectively evaluated to identify the study group .", "inclusion criteria for this study was defined as : ( i ) acute , isolated , non - comminuted and comminuted fractures of the lateral clavicle , ( ii ) complete disruption of both coracoclavicular ligaments , confirmed on radiographs and at surgery , ( iii ) absence of concomitant or pre - existing subacromial pathology ( rotator cuff tears , acromial undersurface degeneration ) , ( iv ) surgical treatment with either a joint - spanning implant alone ( group 1 ) , or combination of a joint - sparing implant and coracoclavicular fixation with either endobutton device , suture anchor , or coracoid cerclage ( group 2 ) [ figures 1a and b ] , and ( v ) follow - up period of at least 6 months for radiographic evidence of union .", "exclusion criteria was defined as : ( i ) partial disruption of the coracoclavicular ligaments at surgery , ( ii ) subacromial pathology ( iii ) concomitant injuries to the ipsilateral shoulder girdle , ( iv ) surgical treatment with any technique other than those described in the inclusion criteria , and ( v ) follow - up period inadequate for complete union .", "surgical treatment of an unstable distal clavicle fracture with a joint - spanning implant ( group 1 ) .", "( hk : hook plate , cl : clavicle , m : medial fragment , l : lateral fragment , arrow : fracture line , ac : acromion , h : humeral head , co : coracoid process ) surgical treatment of an unstable distal clavicle fracture with a joint - sparing implant and coracoclavicular ligament reconstruction using endobutton device ( group 2 ) .", "( rp : locking distal radius plate , arrow : acromioclavicular joint , ac : acromion , cl : clavicle , en : endobutton , h : humeral head ) the clinical records and operative notes were analyzed to identify patients that satisfied the criteria described above , and all other lateral clavicle fracture patients were excluded .", "pre - operative radiographs of the study group were obtained and analyzed with a graphic analysis software ; fracture lines were traced to identify involvement of the acromioclavicular joint , lateral fragment integrity , and presence / absence of comminution .", "the follow - up clinical protocol included a subjective evaluation of pain and functional status by interview , evaluation of active and passive range of motion by clinical examination , and evaluation of muscle strength in elevation and external rotation using a portable dynamometer .", "clinical acromioclavicular joint tests ( tenderness and paxinos sign ) and rotator cuff tests ( lag signs , bear - hug test , impingement signs ) were used to evaluate these structures.[811 ] clinical outcome measures included ( i ) simple shoulder test , ( ii ) constant and murley shoulder score , and ( iii ) walch acromioclavicular joint score . the follow - up radiographic protocol consisted of standardized radiographs that included a true glenohumeral anteroposterior view ( neutral rotation , elbow by the side ) , and a weight bearing comparative cephalic - tilt view ; these were analyzed for implant migration , acromioclavicular joint pathology ( degeneration , instability ) , subacromial changes ( degeneration , osteolysis ) , acromiohumeral interval measurement ( normal interval = 7 mm or more ) , peri - coracoid changes ( abnormal bone formation ) , and glenohumeral changes .", "in addition , in those cases where implant had been previously removed , past radiographs were obtained from the records and individually analyzed as described above .", "the follow - up ultrasonographic protocol included evaluation of the acromioclavicular joint , subacromial space , glenohumeral joint , bicipital groove and biceps long tendon , and suprascapular and spinoglenoid notches ; ultrasound assessment was performed by a senior radiologist experienced in shoulder ultrasound .", "statistical analysis was performed using a statistical software ( spss inc , chicago , usa ) to determine mean values and range of measured parameters of the overall and implant specific groups .", "significant differences between clinical and radiological parameters were determined using the non - parametric mann - whitney / wilcoxon rank - sum test for numerical data , and the fisher 's exact test for categorical data ."], ["the average age of the patients in the study group was 34.5 years ( range , 20 to 57 years ) .", "the mechanism of injury was a direct impact to the ipsilateral shoulder sustained during a fall ; in 8 patients , the fracture was sustained during a cycling - related sporting activity ( competitive or recreational ) .", "ten patients were operated using a joint - spanning implant ( group 1 ) and 5 were operated using a joint - sparing implant technique ( group 2 ) .", "the mean follow - up period was 26.1 months ( range , 12 to 40 months ) .", "the overall and group - specific clinico - radiological outcomes and the types and distribution of fracture patterns are summarized in table 1 .", "overall and group.specific clinico.radiological outcomes and fracture patterns clinical outcomes : the mean cs was marginally higher in group 2 , while the mean sst score was marginally higher in group 1 ; none of these differences were statistically significant .", "acromioclavicular joint signs were positive in half of the group 1 patients , and in none in group 2 ; however , the walch scores for the acromioclavicular joint were not significantly different for the two groups .", "return to pre - operative level of recreational and competitive sports was seen in approximately two - thirds of the patients of group 1 , and all patients of group 2 ; this difference was not found to be statistically significant .", "reoperation rates related to implant removal were significantly higher ( p < 0.05 ) in group 1 patients ( 90 % ) as compared to group 2 ( 0% ) .", "radiographic outcomes [ figure 2a - d ] : radiographic union was observed in 93.3% of fractures ( 9 in group 1 , 5 in group 2 ) with only one non - union with subsequent fragment resorption ( group 1 ) .", "radiographic acromioclavicular joint degeneration was present in 3 patients ( group 1 ) ; all 3 patients had a clinically symptomatic ac joint .", "radiographic acromioclavicular joint superior subluxation was present in 4 patients ( group 1 = 1 patient , 10% ; group 2 = 3 patients , 60% ) ; none of these were clinically symptomatic .", "subacromial osteolysis and hook migration were seen in 5 patients ( group 1 ) ; implant removal was necessary in 4 of these 5 patients , and at final follow - up , resolution of the radiographic subacromial osteolytic lesions was observed .", "signs of progressive implant loosening ( screws and/or plate disengagement ) were seen in 3 patients ( group 1 ) .", "abnormal bone formation was present in 7 patients ( group 1 = 6 , group 2 = 1 ) .", "( b ) acromioclavicular joint subluxation ( arrows ) , ( c ) hook migration and osteolysis of acromial undersurface ( arrow ) , ( d ) peri - coracoid ossification ( arrows ) .", "( ac : acromion , cl : clavicle , co : coracoid , p : plate , an : suture anchor , g : glenoid , h : humeral head , hk : hook plate , ahi : acromiohumeral interval ) ultrasonographic outcomes [ figure 3a and b ] : supraspinatus lesions were seen in 3 patients ; these included partial articular - side tears ( group 1 = 1 patient , 10% ; group 2 = 1 patient , 20% ) , and supraspinatus bursal - side degenerative changes ( group 1 = 1 patient , 10% ) .", "acromioclavicular joint screw penetration was observed in 1 patient ( group 2 ) ; this penetration was not apparent on plain radiographs and the patient was asymptomatic at the final follow - up .", "no abnormalities were detected in the subacromial bursa on static ultrasound evaluation ; dynamic testing revealed abnormal subacromial bursal \" bunching \" with arm abduction in 2 patients ( group 1 = 1 , group 2 = 1 ) .", "( a ) partial articular - surface supraspinatus tendon avulsion ( black arrows ) ( b ) screw penetration into the acromioclavicular joint ( arrows ) .", "( gt : greater tuberosity , h : humeral head , ss : supraspinatus , ac : acromion , cl : clavicle , asterix : acromioclavicular joint ) radiographic evaluation of fracture comminution revealed 5 non - comminuted fractures ( group 1 = 2 , group 2 = 3 ) , and 10 comminuted fractures ( group 1 = 8 , group 2 = 2 ) .", "analysis of the fracture lines and fragment cortices revealed repetitive patterns in the fracture geometry , and four types could be identified : ( i ) type 1 pattern ( n = 5 , 33% ) involved a clean vertical or a short oblique type bicortical fracture line without any comminution [ figure 4a ] , ( ii ) type 2 pattern ( n = 4 , 27% ) involved a zone of segmental comminution , between intact bicortical lateral and medial fragments [ figure 4b ] , ( iii ) type 3 pattern ( n = 5 , 33% ) involved propogation of at least one fracture line into the juxta - articular cortex of the ac joint [ figure 4c ] and ( iv ) type 4 pattern ( n = 1 , 7% ) consisted of fracture line propogation into the acromioclavicular joint [ figure 4d ] .", "radiographic fracture patterns : ( a ) type 1 , ( b ) type 2 , ( c ) type 3 , and ( d ) type 4 .", "( ac : acromion , cl : clavicle , co : coracoid , g : glenoid , h : humeral head , arrows : fracture lines )"], ["the present study is the first study that describes overall and comparative outcomes ( clinical , radiographic , and ultrasonographic ) of surgical treatment of an uncommon lateral clavicular bony - ligamentous injury with joint - spanning and joint - sparing implants .", "in addition , four radiographic patterns of bone injury were identified to develop guidelines for choice of implants in these injuries . clinical outcomes and union rates after operative treatment of this injury", "are satisfactory , and this has been shown in other studies in literature . in the present study , both types of implants resulted in satisfactory and comparable clinical outcome scores , irrespective of the radiographic outcomes .", "however , a significant difference in the reoperation rate for implant removal ( p < 0.05 ) in patients treated with a joint - spanning implant probably implies that a joint - sparing implant could be preferentially used whenever fracture geometry permits .", "radiological outcomes , especially those associated with joint sparing techniques , have not been well documented in other studies .", "subluxation of the acromioclavicular joint , ( present in 60% of group 2 patients ) may be related to either concomitant injury to the acromioclavicular ligaments , or to a partial failure of the coracoclavicular ligament reconstruction ; transfer of distraction forces to ac ligaments after fracture union may then induce gradual subluxation of this joint .", "this complication was rare in the other group , probably due to the joint spanning implant design ; however , other radiological abnormalities like hook migration , subacromial osteolysis , implant loosening , and new bone formation were frequent .", "the acromioclavicular joint spanning hook plate is a commonly used implant , and most studies have utilized this as the sole implant of choice .", "although clinical outcomes in these studies have been satisfactory , the effect of the subacromial hook on bursal tissues has been debated and use of the distal clavicle plate suggested . by using ultrasonographic evaluation , this study demonstrated the safety of the joint - spanning hook plate in relation to rotator cuff and subacromial bursa .", "persistent pain in the post - operative period probably resulted from acromial undersurface irritation ; early plate removal after fracture union is recommended , and long - term clinical outcomes seem to be satisfactory after implant removal .", "an important finding of this study is the grouping of the radiographic fracture patterns into four surgically relevant types .", "types 1 and 2 are ideal indications for use of a joint - sparing implant ; adequate bone stock in the distal fragment in these types will theoretically permit secure fixation across the fracture site . in types 3 and 4", ", inadequate distal fragment size may not permit secure bicortical fixation , thereby necessitating use of joint - spanning implants .", ". the retrospective nature and small sample size does not permit identification of all possible fracture configurations , and statistical analysis may differ with larger numbers .", "however , the clinical results are similar to other literature studies that have analyzed joint - spanning implants . also , the current radiographic patterns described provide guidelines for implant choice , and are not intended to be used as a prognostic classification .", "we attribute the small size of the study group to the stringent inclusion and exclusion criteria that eliminated several other patients with lateral clavicle fractures , and this was necessary to obtain meaningful conclusions", ". lateral clavicle fractures with complete disruption of the coracoclavicular ligaments should be regarded as a distinct entity that is perhaps biomechanically and prognostically different from similar fractures with partial ligament disruption ."], ["a combination of a locking distal radius plate with coracoclavicular ligament reconstruction resulted in stable fixation and significantly lower reoperation rates , and should be used preferentially in lesser comminuted fractures ( types 1 and 2 ) ."]]}
{"article_id": "PMC4971116", "article_text": ["a 68-year - old woman visited the clinic with the chief complaints of pain , swelling , and a warm sensation in her left thigh for over 2 days prior to her visit .", "the patient also complained about a warm sensation during the nighttime but body temperature was normal when measured in the clinic .", "she had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to the current visit and was wearing compression stockings to prevent the occurrence of a deep vein thrombosis .", "she had been taking aspirin 100 mg daily , fexonadine 180 mg daily , and hydrea 500 mg twice a day for a diagnosis of polycythemia vera for 1 year and was phlebotomized in a hemato - oncology setting on an irregular basis . in advance of the operation , her erythrocyte sedimentation rate ( esr ) , and c - reactive protein ( crp ) levels were 30 mm / hr and 4.99 mg / dl , respectively .", "three days after her hip surgery , her esr and crp levels were 23 mm / hr and 6.35 mg / dl , respectively .", "medical care was administered in close cooperation with the hemato - oncology department and the level of hemoglobin was maintained at < 12 g / dl to prevent a thrombosis prior to her operation .", "the patient did not have a fever and her skin color seemed normal despite swelling and a slight warm sensation in the left hip and proximal femur .", "results of a blood test showed that her complete blood count , electrolyte levels , and liver function were all within normal ranges ( white blood cell count [ wbc ] : 4,700/l , hemoglobin : 10.6 g / dl , hematocrit : 32.3% , platelet : 213,000/l , neutrophil count 67.2% , lymphocyte 24.1% , monocyte 6.8% ) whereas the esr and crp level were 38 mm / hr and 8.25 mg / dl , respectively . when compared to tests performed previously , no further observations of additional fractures , signs of infection , or loosening of inserts after the surgery", "acute or sub - acute inflammation was diagnosed given the perfusion image of soft tissue in the left hip area as well as the blood pool image ( fig .", "the patient was injected intravenously with antibiotics for 1 week while staying in the hospital , however , symptoms were not alleviated . in order to prevent a deep vein thrombosis , the patient started to move with the aid of a wheelchair and exercised her knee joints 3 days after the operation .", "g / dl , a color doppler ultrasonography was performed due to her history of polycythemia vera .", "results revealed a deep vein thrombosis in the common femoral vein as well as in the superficial femoral vein ( fig .", "symptoms were alleviated following the administration of low - molecular - weight heparin ( enoxaparin , 40 mg ) and warfarin ( 5 mg ) for 5 days .", "additional warfarin was prescribed ( 2.5 mg daily ) for maintenance at discharge . during her first follow - up visit 2 weeks later , no symptoms were noted and all blood test parameters were within normal ranges .", "it has been reported that patients with polycythemia vera present with various complications related to thrombosis due to excessive blood viscosity1 ) .", "several putative reasons have been postulated to explain such complications including hypervolemia , an increase in the volume of red blood cells , telangiectasia due to decreased blood flow velocity as well as vascular elasticity , thrombocytosis , and complex hemostatic disorders attributable to inefficient blood clotting processes2 ) .", "thrombosis is the leading cause of death in those with polycythemia vera and has been associated with high morbidity as well .", "approximately 12 - 49% of patients with polycythemia vera experience thrombosis and 20 - 40% will die as a result2 ) .", "thromboembolism is one of the major complications of hip surgery and contributes to its poor prognosis .", "warwick et al.3 ) reported that the incidence rate for a deep vein thrombosis was about 1.89% if proper medication was not provided after total hip arthroplasty .", "therefore , in the case of the patient described in the current case report , it was reasonable to expect that she would be susceptible to the high incidence rate of thrombosis as she underwent the hip surgery based on her history of polycythemia vera . for prophylactic purposes ,", "aspirin was administrated ( 100 mg daily ) and early exercise and active movements were recommended for the patient in order to lower the risk of venous thromboembolism . in the current case report , a patient with polycythemia vera exhibited clinical symptoms and signs of a postoperative infection following bipolar hemiarthroplasty due to femoral neck fractures .", "there was , however , a deep vein thrombosis which rendered the intravenous administration of antibiotics ineffective in the alleviation of symptoms .", "early symptoms of a deep vein thrombosis , including swelling , pain , oppressive pain , and a warm sensation , can mimic those of a postoperative infection .", "furthermore , features of systematic inflammatory responses such as increased levels of interleukin-6 , interleukin-8 , and crp may be observed in patients with a deep vein thrombosis .", "collectively , these findings suggest that appropriate clinical examinations are warranted at an early stage in order to avoid making an inaccurate diagnosis4,5 ) . on the first visit after the operation ,", "the patient was suspected to have had a postoperative infection due to the simultaneous increase in esr and crp levels", ". however , the specificity of esr and cpr levels pertaining to the diagnosis of infections is questionable , as most patients display elevated levels of esr and crp following a hip surgery6 ) .", "these inflammatory responses stimulate the production of cytokines due to the fractures and operation per se , thus biochemical parameters for inflammation are known to be elevated in a non - specific manner . in cases without postoperative infections , elevated crp levels recover to the normal range within 3 weeks of the operation6,7 ) . in the current case report , the elevated crp level that was maintained for up to 24 days after the operation led us to suspect a postoperative infection .", "however , the abnormally increased level of acute phase reactants ( e.g. , crp ) may be observed in patients with chronic myeloproliferative diseases ( e.g. , polycythemia vera ) and therefore further specific clinical examinations are required for an infectious diagnosis8 ) . regarding the diagnosis of a postoperative infection ,", "elevated level of serum procalcitonin may be more valuable than wbcs and crp levels7 ) .", "regrettably , the level of serum procalcitonin was not measured in the current case but could have been informative in distinguishing between postoperative infection and a deep vein thrombosis . despite the presentation of similar clinical symptoms between a postoperative infection and a deep vein thrombosis , their etiological mechanisms are completely distinct . therefore ,", "different treatments are warranted , as patients receiving inappropriate medical treatments may experience exacerbated symptoms and conditions .", "the potential for a deep vein thrombosis requires continuous thought and attention regarding relevant examinations for patients at high risk for thrombosis as well as for embolism ( e.g. , polycythemia vera ) ."], "abstract_text": ["<S> a 68-year - old woman who suffered from polycythemia vera presented at our clinic with the chief complaints of pain , swelling , and a warm sensation in her left thigh . </S>", "<S> she had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to this presentation . </S>", "<S> her erythrocyte sedimentation rate and c - reactive protein ( crp ) levels were elevated . </S>", "<S> in addition , a postoperative infection was suspected in the 3-phase bone scan ; therefore , she received intravenous antibiotic therapy . </S>", "<S> this approach proved to be ineffective and she was subsequently diagnosed with a deep vein thrombosis via color doppler ultrasonography . </S>", "<S> it is interesting to note that a deep vein thrombosis can present with symptoms similar to those of a postoperative infection . </S>", "<S> furthermore , an elevated crp level is frequently observed in patients suffering from polycythemia vera . </S>", "<S> therefore , the two conditions , which require completely different treatments , can be confused . </S>", "<S> we report on this case with a review of the relevant literature . </S>"], "labels": null, "section_names": ["CASE REPORT", "DISCUSSION"], "sections": [["a 68-year - old woman visited the clinic with the chief complaints of pain , swelling , and a warm sensation in her left thigh for over 2 days prior to her visit .", "the patient also complained about a warm sensation during the nighttime but body temperature was normal when measured in the clinic .", "she had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to the current visit and was wearing compression stockings to prevent the occurrence of a deep vein thrombosis .", "she had been taking aspirin 100 mg daily , fexonadine 180 mg daily , and hydrea 500 mg twice a day for a diagnosis of polycythemia vera for 1 year and was phlebotomized in a hemato - oncology setting on an irregular basis . in advance of the operation , her erythrocyte sedimentation rate ( esr ) , and c - reactive protein ( crp ) levels were 30 mm / hr and 4.99 mg / dl , respectively .", "three days after her hip surgery , her esr and crp levels were 23 mm / hr and 6.35 mg / dl , respectively .", "medical care was administered in close cooperation with the hemato - oncology department and the level of hemoglobin was maintained at < 12 g / dl to prevent a thrombosis prior to her operation .", "the patient did not have a fever and her skin color seemed normal despite swelling and a slight warm sensation in the left hip and proximal femur .", "results of a blood test showed that her complete blood count , electrolyte levels , and liver function were all within normal ranges ( white blood cell count [ wbc ] : 4,700/l , hemoglobin : 10.6 g / dl , hematocrit : 32.3% , platelet : 213,000/l , neutrophil count 67.2% , lymphocyte 24.1% , monocyte 6.8% ) whereas the esr and crp level were 38 mm / hr and 8.25 mg / dl , respectively . when compared to tests performed previously , no further observations of additional fractures , signs of infection , or loosening of inserts after the surgery", "acute or sub - acute inflammation was diagnosed given the perfusion image of soft tissue in the left hip area as well as the blood pool image ( fig .", "the patient was injected intravenously with antibiotics for 1 week while staying in the hospital , however , symptoms were not alleviated . in order to prevent a deep vein thrombosis , the patient started to move with the aid of a wheelchair and exercised her knee joints 3 days after the operation .", "g / dl , a color doppler ultrasonography was performed due to her history of polycythemia vera .", "results revealed a deep vein thrombosis in the common femoral vein as well as in the superficial femoral vein ( fig .", "symptoms were alleviated following the administration of low - molecular - weight heparin ( enoxaparin , 40 mg ) and warfarin ( 5 mg ) for 5 days .", "additional warfarin was prescribed ( 2.5 mg daily ) for maintenance at discharge . during her first follow - up visit 2 weeks later , no symptoms were noted and all blood test parameters were within normal ranges ."], ["it has been reported that patients with polycythemia vera present with various complications related to thrombosis due to excessive blood viscosity1 ) .", "several putative reasons have been postulated to explain such complications including hypervolemia , an increase in the volume of red blood cells , telangiectasia due to decreased blood flow velocity as well as vascular elasticity , thrombocytosis , and complex hemostatic disorders attributable to inefficient blood clotting processes2 ) .", "thrombosis is the leading cause of death in those with polycythemia vera and has been associated with high morbidity as well .", "approximately 12 - 49% of patients with polycythemia vera experience thrombosis and 20 - 40% will die as a result2 ) .", "thromboembolism is one of the major complications of hip surgery and contributes to its poor prognosis .", "warwick et al.3 ) reported that the incidence rate for a deep vein thrombosis was about 1.89% if proper medication was not provided after total hip arthroplasty .", "therefore , in the case of the patient described in the current case report , it was reasonable to expect that she would be susceptible to the high incidence rate of thrombosis as she underwent the hip surgery based on her history of polycythemia vera . for prophylactic purposes ,", "aspirin was administrated ( 100 mg daily ) and early exercise and active movements were recommended for the patient in order to lower the risk of venous thromboembolism . in the current case report , a patient with polycythemia vera exhibited clinical symptoms and signs of a postoperative infection following bipolar hemiarthroplasty due to femoral neck fractures .", "there was , however , a deep vein thrombosis which rendered the intravenous administration of antibiotics ineffective in the alleviation of symptoms .", "early symptoms of a deep vein thrombosis , including swelling , pain , oppressive pain , and a warm sensation , can mimic those of a postoperative infection .", "furthermore , features of systematic inflammatory responses such as increased levels of interleukin-6 , interleukin-8 , and crp may be observed in patients with a deep vein thrombosis .", "collectively , these findings suggest that appropriate clinical examinations are warranted at an early stage in order to avoid making an inaccurate diagnosis4,5 ) . on the first visit after the operation ,", "the patient was suspected to have had a postoperative infection due to the simultaneous increase in esr and crp levels", ". however , the specificity of esr and cpr levels pertaining to the diagnosis of infections is questionable , as most patients display elevated levels of esr and crp following a hip surgery6 ) .", "these inflammatory responses stimulate the production of cytokines due to the fractures and operation per se , thus biochemical parameters for inflammation are known to be elevated in a non - specific manner . in cases without postoperative infections , elevated crp levels recover to the normal range within 3 weeks of the operation6,7 ) . in the current case report , the elevated crp level that was maintained for up to 24 days after the operation led us to suspect a postoperative infection .", "however , the abnormally increased level of acute phase reactants ( e.g. , crp ) may be observed in patients with chronic myeloproliferative diseases ( e.g. , polycythemia vera ) and therefore further specific clinical examinations are required for an infectious diagnosis8 ) . regarding the diagnosis of a postoperative infection ,", "elevated level of serum procalcitonin may be more valuable than wbcs and crp levels7 ) .", "regrettably , the level of serum procalcitonin was not measured in the current case but could have been informative in distinguishing between postoperative infection and a deep vein thrombosis . despite the presentation of similar clinical symptoms between a postoperative infection and a deep vein thrombosis , their etiological mechanisms are completely distinct . therefore ,", "different treatments are warranted , as patients receiving inappropriate medical treatments may experience exacerbated symptoms and conditions .", "the potential for a deep vein thrombosis requires continuous thought and attention regarding relevant examinations for patients at high risk for thrombosis as well as for embolism ( e.g. , polycythemia vera ) ."]]}
{"article_id": "PMC4130329", "article_text": ["brachial plexus surgery using the da vinci surgical robot is a new procedure . to evaluate the advantages and the restrictions of the technique , a cadaveric study of supraclavicular and axillary approaches was conducted .", "we found that the axillary approach was useful and advantageous for lower roots , particularly for thoracic outlet syndrome ( tos ) .", "this report will focus on the evaluation of axillary robotic approach as the advantages and disadvantages of supraclavicular robotic intervention have been widely discussed in the literature .", "a human cadaver was subjected to this experiment in paris university ecole europenne de chirurgie anatomy laboratory and da vinci robot system was used .", "the left arm was tucked along the side and the right arm was placed in a semiflexed position extending toward the anesthesia location near the head , supported by foam and blankets ( figure 1 ) .", "a 6  cm long incision was made at the right axillar line , lateral to the edge of the pectoralis major muscle ( figure 2 ) .", "a self - retaining chung retractor was placed into the incision to elevate the pectoralis major muscle flap .", "the robot was docked as a camera ; right and left robotic arm were adapted in the incision area ( figure 3 ) . a 10  mm 0 downlooking scope , maryland forceps , and a curved scissors", "the working space was maintained with the self - retaining retractor , without co2 insufflation ( figure 4 ) .", "the subclavian artery was seen in front of the truncus and was positioned to the posterior of the working space .", "subclavian artery was dissected from the plexus and truncus of the lower plexus was exposed with blunt dissection .", "the plexus was exposed thoroughly from t1 to c7 levels . in this surgical setting , the operating surgeon , who has", "a wide experience in open brachial surgery of the brachial plexus , reported that lower brachial plexus exposure was easier from the axillary working area and a more wide range of motion was achieved to manipulate the robotic tools compared to the supraclavicular exposure for lower part of the brachial plexus .", "the development of robotic - assisted minimally invasive techniques began in urology , general surgery , and gynecology because of the generally large working spaces available in the abdomen for these types of surgeries [ 14 ] .", "since then , other surgeons have sought to use robotic devices in other areas , such as the brachial plexus [ 5 , 6 ] .", "brachial plexus dysfunction can be the result of shoulder trauma [ 7 , 8 ] .", "it can also occur with tos , which encompasses three separate disorders involving compression of the subclavian artery , subclavian vein , or brachial plexus in the triangular space bordered by the first rib , clavicle , and scalene muscles [ 9 , 10 ] .", "compression of the vessel - nerve package at the thoracic inlet has been treated with soft - tissue ( scalene muscle ) release and/or bone ( first rib ) resection .", "surgical approaches to first rib resection may be transthoracic , transaxillary , supraclavicular , infraclavicular , or thoracoscopic [ 9 , 10 ] . however , these approaches are typically associated with incomplete resection of the most medial portion of the first rib and neurovascular complications .", "theoretically , a minimally invasive transthoracic approach can obviate these problems , enabling complete resection of the offending portion of the first rib without neurovascular complication .", ", respectively , reported successful results of robotic en bloc first rib resection for tos treatment via transthoracic and transaxillary approaches [ 9 , 11 ] .", "'s techniques were only bony interventions and as being intrathoracic these need to be lung collapsed and lung complication can be waiting risk .", "although liverneaux et al . reported techniques and results of upper brachial plexus injury intervention via robotic surgery with a supraclavicular approach , they described the disadvantages as a narrow working space and difficulty to expose the c7 vertebra [ 1 , 5 , 12 ] . to our knowledge , this report is the first to objectively describe robotic axillar brachial plexus exposure .", "thus , we discuss the theoretical and clinical advantages and disadvantages of the axillary approach in the present report .", "the development of robot - assisted surgery has revealed new perspectives in peripheral nerve microsurgery . minimally invasive", "robot - assisted surgery could lead to modification of the classic algorithm for the treatment of traumatic brachial plexus lesions [ 6 , 8 ] . to date , exploration of these lesions has not been attempted less than 3 months after the traumatic event because clinical examination can not provide an accurate diagnosis or reliable prognosis in these first weeks .", "early intervention may enable initial assessment of the lesion and repair of potentially graftable nerve roots .", "several robotics properties are particularly adapted to microsurgery , such as high - resolution three - dimensional ( 3d ) visualization with up to 40 magnification , up to 10-fold magnification of surgical movements , elimination of physiological tremors , and the provision of ergonomic work conditions for otherwise uncomfortable surgery .", "robotic surgical systems allow high - definition magnified 3d visualization of the operative field , provide significant instrument maneuverability , even within a confined space , and may overcome the shortcomings of conventional approaches [ 2 , 5 ] .", "axillary ( infraclavicular ) brachial plexus intervention via robotic surgery has not been described previously .", "axillary intervention was previously performed as an open procedure to expose the plexus or resect the first rib for the treatment of tos .", "martinez et al . described first rib resection via robotic surgery but not to address plexus injury without transthoracic exposure , a novel minimally invasive approach to the first rib from inside of the chest .", "in addition , gharagozloo et al . reported first rib resection via transthoracic robotic surgery for paget - schroetter disease .", "'s techniques were considered more useful for lower brachial plexus viewing and assessing according to gharagozloo et al .", "the experience of the whole surgical team with robotic technology is important for the procedure . during learning curve period ,", "two staff surgeons are required to participate in all procedures to ensure the safety of the program [ 5 , 9 ] .", "martinez et al . reported importance of the learning curve , not only for the surgeon but also for the entire surgical team and 180 minutes for the initial 10 cases .", "reported exclusion criteria include a history of previous incision in the same area and obesity , which present difficulties in robotic surgery initiation .", "other drawbacks of this new surgical approach are the increased cost of surgical equipment and longer operating time , especially during the learning curve period .", "however , we believe that the avoidance of a classic incision leads to significant patient satisfaction for cosmetic reasons and we believe that demand for this procedure from a select group of patients justifies the exploration of alternative ways to avoid classic brachial plexus exposure .", "this report presents our initial experience with robot - assisted axillary exposure of the brachial plexus region . in our opinion", "robotic surgery will be used routinely in the future for brachial plexus surgery and particularly for tos that is caused by bone and/or soft tissue . however , newer dedicated surgical instruments need to be developed and further studies should be conducted to evaluate in vivo application and results of this novel approach ."], "abstract_text": ["<S> brachial plexus surgery using the da vinci surgical robot is a new procedure . </S>", "<S> although the supraclavicular approach is a well known described and used procedure for robotic surgery , axillary approach was unknown for brachial plexus surgery . </S>", "<S> a cadaveric study was planned to evaluate the robotic axillary approach for brachial plexus surgery . </S>", "<S> our results showed that robotic surgery is a very useful method and should be used routinely for brachial plexus surgery and particularly for thoracic outlet syndrome . </S>", "<S> however , we emphasize that new instruments should be designed and further studies are needed to evaluate in vivo results . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Discussion", "3. Conclusion"], "sections": [["brachial plexus surgery using the da vinci surgical robot is a new procedure . to evaluate the advantages and the restrictions of the technique , a cadaveric study of supraclavicular and axillary approaches was conducted .", "we found that the axillary approach was useful and advantageous for lower roots , particularly for thoracic outlet syndrome ( tos ) .", "this report will focus on the evaluation of axillary robotic approach as the advantages and disadvantages of supraclavicular robotic intervention have been widely discussed in the literature .", "a human cadaver was subjected to this experiment in paris university ecole europenne de chirurgie anatomy laboratory and da vinci robot system was used .", "the left arm was tucked along the side and the right arm was placed in a semiflexed position extending toward the anesthesia location near the head , supported by foam and blankets ( figure 1 ) .", "a 6  cm long incision was made at the right axillar line , lateral to the edge of the pectoralis major muscle ( figure 2 ) .", "a self - retaining chung retractor was placed into the incision to elevate the pectoralis major muscle flap .", "the robot was docked as a camera ; right and left robotic arm were adapted in the incision area ( figure 3 ) . a 10  mm 0 downlooking scope , maryland forceps , and a curved scissors", "the working space was maintained with the self - retaining retractor , without co2 insufflation ( figure 4 ) .", "the subclavian artery was seen in front of the truncus and was positioned to the posterior of the working space .", "subclavian artery was dissected from the plexus and truncus of the lower plexus was exposed with blunt dissection .", "the plexus was exposed thoroughly from t1 to c7 levels . in this surgical setting , the operating surgeon , who has", "a wide experience in open brachial surgery of the brachial plexus , reported that lower brachial plexus exposure was easier from the axillary working area and a more wide range of motion was achieved to manipulate the robotic tools compared to the supraclavicular exposure for lower part of the brachial plexus ."], ["the development of robotic - assisted minimally invasive techniques began in urology , general surgery , and gynecology because of the generally large working spaces available in the abdomen for these types of surgeries [ 14 ] .", "since then , other surgeons have sought to use robotic devices in other areas , such as the brachial plexus [ 5 , 6 ] .", "brachial plexus dysfunction can be the result of shoulder trauma [ 7 , 8 ] .", "it can also occur with tos , which encompasses three separate disorders involving compression of the subclavian artery , subclavian vein , or brachial plexus in the triangular space bordered by the first rib , clavicle , and scalene muscles [ 9 , 10 ] .", "compression of the vessel - nerve package at the thoracic inlet has been treated with soft - tissue ( scalene muscle ) release and/or bone ( first rib ) resection .", "surgical approaches to first rib resection may be transthoracic , transaxillary , supraclavicular , infraclavicular , or thoracoscopic [ 9 , 10 ] . however , these approaches are typically associated with incomplete resection of the most medial portion of the first rib and neurovascular complications .", "theoretically , a minimally invasive transthoracic approach can obviate these problems , enabling complete resection of the offending portion of the first rib without neurovascular complication .", ", respectively , reported successful results of robotic en bloc first rib resection for tos treatment via transthoracic and transaxillary approaches [ 9 , 11 ] .", "'s techniques were only bony interventions and as being intrathoracic these need to be lung collapsed and lung complication can be waiting risk .", "although liverneaux et al . reported techniques and results of upper brachial plexus injury intervention via robotic surgery with a supraclavicular approach , they described the disadvantages as a narrow working space and difficulty to expose the c7 vertebra [ 1 , 5 , 12 ] . to our knowledge , this report is the first to objectively describe robotic axillar brachial plexus exposure .", "thus , we discuss the theoretical and clinical advantages and disadvantages of the axillary approach in the present report .", "the development of robot - assisted surgery has revealed new perspectives in peripheral nerve microsurgery . minimally invasive", "robot - assisted surgery could lead to modification of the classic algorithm for the treatment of traumatic brachial plexus lesions [ 6 , 8 ] . to date , exploration of these lesions has not been attempted less than 3 months after the traumatic event because clinical examination can not provide an accurate diagnosis or reliable prognosis in these first weeks .", "early intervention may enable initial assessment of the lesion and repair of potentially graftable nerve roots .", "several robotics properties are particularly adapted to microsurgery , such as high - resolution three - dimensional ( 3d ) visualization with up to 40 magnification , up to 10-fold magnification of surgical movements , elimination of physiological tremors , and the provision of ergonomic work conditions for otherwise uncomfortable surgery .", "robotic surgical systems allow high - definition magnified 3d visualization of the operative field , provide significant instrument maneuverability , even within a confined space , and may overcome the shortcomings of conventional approaches [ 2 , 5 ] .", "axillary ( infraclavicular ) brachial plexus intervention via robotic surgery has not been described previously .", "axillary intervention was previously performed as an open procedure to expose the plexus or resect the first rib for the treatment of tos .", "martinez et al . described first rib resection via robotic surgery but not to address plexus injury without transthoracic exposure , a novel minimally invasive approach to the first rib from inside of the chest .", "in addition , gharagozloo et al . reported first rib resection via transthoracic robotic surgery for paget - schroetter disease .", "'s techniques were considered more useful for lower brachial plexus viewing and assessing according to gharagozloo et al .", "the experience of the whole surgical team with robotic technology is important for the procedure . during learning curve period ,", "two staff surgeons are required to participate in all procedures to ensure the safety of the program [ 5 , 9 ] .", "martinez et al . reported importance of the learning curve , not only for the surgeon but also for the entire surgical team and 180 minutes for the initial 10 cases .", "reported exclusion criteria include a history of previous incision in the same area and obesity , which present difficulties in robotic surgery initiation .", "other drawbacks of this new surgical approach are the increased cost of surgical equipment and longer operating time , especially during the learning curve period .", "however , we believe that the avoidance of a classic incision leads to significant patient satisfaction for cosmetic reasons and we believe that demand for this procedure from a select group of patients justifies the exploration of alternative ways to avoid classic brachial plexus exposure ."], ["this report presents our initial experience with robot - assisted axillary exposure of the brachial plexus region . in our opinion", "robotic surgery will be used routinely in the future for brachial plexus surgery and particularly for tos that is caused by bone and/or soft tissue . however , newer dedicated surgical instruments need to be developed and further studies should be conducted to evaluate in vivo application and results of this novel approach ."]]}
{"article_id": "PMC3748574", "article_text": ["older people have particular spiritual needs that are distinct from those of others , particularly in times of ill health and death and dying .", "it has been reported that spirituality has a positive effect on health outcomes and welfare of the elderly .", "nowadays , aging phenomenon and relevant problems with its complexities are important issues that have attracted the attention of social and medical scientists to investigate the spiritual needs of the elderly .  ", "spiritual health is a dimension of health that is portrayed as the validation of life in relation to god , fellow human beings , and the environment .", "spirituality is the representative of the basic values that guide a person in searching to find answers to the crucial questions of life , such as the purpose and meaning of life , reality , love , good and bad , disease , and death .   over the past three decades , spirituality , religion , and prayer", "have turned into a subject of large interest for researchers in medicine , nursing , gerontology , physical health and mental health disciplines , often in conjunction with complementary and alternative medicine .", "this tendency has been stimulated by research demonstrating physical and psychological benefits of spiritual and religious involvements .  ", "in fact , approximately 96% of adults in the united states expressed their belief in god , and 72% of identified religion and spirituality as having the most important influence in their lives . saying prayers is a regular daily task for muslims which begins at the age of puberty and continues throughout the life .", "it is a time getting rid of the material world and approaching the world beyond , as well as , to the inner self . in iran , elderly people are more religious than younger ones , and this is a norm of this culture .", "a recent national survey of the iranians values and attitudes found that over 80% of the iranians practice prayer regularly as a part of their religion .", "one might argue that this is a true reflection of individual culture where prayer and religious beliefs are a part of people 's everyday life .", "however , prayer can go beyond just performing a religious duty , and based on one 's wishes , take different forms of formal and informal practices .", "it has been shown that disease prevalence , incidence and mortality rates differ from one population to another .", "this is partly ascribed to their disparities in lifestyle , dietary habits and other characteristics that are somehow affected by culture , especially religious traditions and experiences .   during the past two decades", "some studies have been carried out and the associations between religious beliefs or experiences and various types of physical illnesses , mental health , and mortality have been examined .", "spirituality is viewed as personally important matter to the older people and it has been proved that it is highly related to older people 's health outcomes .  ", "however , there has not been adequate experiential research on the relationship between religious or spiritual factors and healthy lifestyle behaviors , and their impact on the adult 's well - being .", "scientists believe that probing the effects of religious and spiritual factors as well as healthy lifestyle behaviors may be decisive .", "additionally , social science and medical research have concurrently reported that older adults are more inclined to take spiritual chase than younger people are .  ", "kotrotsiou - barboutal et al.(2006 ) indicated that older adults are remaining members of religious associations without necessarily participating regularly in services , as there are numerous issues that can diminish the active participation in religious events , such as transportation intricacies , bad sound , or the illegibility of small font text in books of prayers available at mosques or religious centers . in spite of those complexities ,", "many elderly manage to find out a way to come up to god through prayer .", "it is stated that prayer increases the feelings of personal value of an elderly by reducing the feelings of loneliness and desertation , while television and radio provide support for spiritual life .   in this regard , researchers have shown that some groups of elderly people with inadequate and difficult livelihood situations communicate their problems by putting their trust in god . moreover , more than half of the elder patients practiced spiritual needs throughout their hospitalization period , with the most prominent need of being in the search of the meaning of life .  ", "extensive epidemiological studies have shown that higher levels of religiousness are related to lower mortality rates .", "studies identified the multidimensional nature of religiousness and spirituality and investigated various religious dimensions , for instance private religious behaviors , devotion , spiritual transcendence , and religious adaptation .", "for example , it was indicated that some aspects of religiousness predicted lower rates of disability and illness , alcoholism , cardiovascular disease , hypertension , and myocardial infarction .", "furthermore , clinical research has suggested that spirituality can play a vital role in the recovery of patients suffering from physical and psychological diseases .  ", "it seems that religious obligation has a crucial role in averting physical and mental diseases , recovery promotion , and disease adaptation .", "numerous studies have revealed the positive involvement of spirituality and religious indexes on different aspects of health such as cardiac surgery , mortality , immunity system function and self esteem .  ", "above all , religious involvement can be useful for psychological well - being and physical health .", "additionally , supporting the effect of spirituality may become more and more prominent in later life as a result of declining health , social and financial sources .", "researchers have proposed numerous psychological , social , and physiological interveners that may explain the spirituality - health association .  ", "quantitative measurements and the evaluation of spiritual beliefs are difficult because quantitative studies neither reveal the content , components of the beliefs , and its factors , nor the contexts that might be included . therefore , implementing qualitative research that explores the personal and experiential aspects of beliefs seems crucial , and may shed light on a range of important issues which can be investigated in detail on larger samples by further quantitative studies in future .  ", "spiritual or religious beliefs may affect the decisions of individuals that make about their health and illness .", "thus , recognizing and understanding the spiritual and religious behaviors within the context of cultural influence by a qualitative research is critical for nurse and other health - care providers .", "the main strengths of the present study are that it focuses on the spiritual life of healthy elderly people .", "therefore , the present study aimed to explore the concept of spirituality from the perspectives of iranian healthy elderly people . the central question of the paper was  what characterizes the spirituality in the iranian healthy older people ?", "in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 .", "it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects .  ", "initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data .", "the study consisted of 17 healthy elders within the age range of 65 - 86 .", "criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study .", "all participants were shia muslims . in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions .", "based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection .", "interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences .", "the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about  spirituality in elderly adults  .", "what is your experience concerning the spirituality in later age? and  what is the meaning of spirituality in your experience ?", " each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next .", "coding was carried out line by line , and comparative analysis of the quotations was carried out .", "all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions .", "each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes .", "data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories .", "the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories .", "credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking .", "the findings and explanations of this study were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data .", "this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability .", "permission to conduct this study was obtained from the ethics committee of tarbiat modares university .", "other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses .", "all participants were aware of the purpose of the study , and their participation in the study was optional .", "informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki .", "in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 .", "it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects .", "initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data .", "the study consisted of 17 healthy elders within the age range of 65 - 86 .", "criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study .", "in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions . the interview was scheduled according to the participant 's agreement .", "based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection .", "interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences .", "the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about  spirituality in elderly adults  .", "what is your experience concerning the spirituality in later age? and  what is the meaning of spirituality in your experience ? ", "each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next .", "coding was carried out line by line , and comparative analysis of the quotations was carried out .", "all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions .", "each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes .", "data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories .", "the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories .", "credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking . the findings and explanations of this study", "were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data .", "this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability .", "permission to conduct this study was obtained from the ethics committee of tarbiat modares university .", "other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses .", "all participants were aware of the purpose of the study , and their participation in the study was optional .", "informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki .", "the participants of the study were 11 males and 10 females , within the age range of 65 - 86 .", "also , 15 out of 21 were married , four were widowed , and one was single .", "five participants were illiterate , four were primary degree holders , eight had a diploma , two had bachelor degrees , and two had msc degree in human sciences . in terms of their occupation status , two were employees , 13 were pensioners , two was manual worker , and three were housewives .", "three main categories emerged from the data and 3 - 4 distinctive subcategories within each category were identified .", "these categories and their subcategories represent the main factors influencing the spirituality in iranian elderly people .", "main categories and subcategories one of the main categories that emerged from data analysis was  spiritual health  . four subcategories ;", " saying prayer as a calming factor  ,  beneficence as a way to god  ,  loss of psychological and spiritual support  , and  faith , a way to happiness  , emerged from the participants experiences .", "also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them .", " i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day  .", "( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers .", "they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives .", "they said that man would receive god 's blessings as a consequence of his / her deeds in the world .", "additionally , they believed that the ultimate consequence of good deeds was good in return .", "if she asks me to fix her air conditioner you can be sure that i ll do it .", "this is because i think that by doing this i make her happy and she prays for me .", ", the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support .", " when i think about my life , i feel that i do n't have any support from my children .", "also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . ", "( female ) the elderly participants lived experiences disclosed the subcategory of  faith , a way to happiness  in life .", "one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs .", "also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness . ", "the one who has strong faith and beliefs is always happy and confident . in my opinion", "worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free  .", "( male )  spiritual beliefs  was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including  seeking help from god in difficulties ", " doing good deeds is the god 's will  . regarding the subcategory of  seeking help from god in difficulties ", ", the participants believed that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses .", "this desire results in god 's attention to elderly and also creates sense of trust in older adults .", "some statements said by participants are :  i really believe in having trust in god . when i m trapped in a complicated situation", ", i believe that this is god who helps me and i only trust in god .  ( male ) one participant believed that spirituality was a vital component of older people lives .", "also , he believed that god was the ultimate source to affect my life or death and he controls everything .", " we re all waiting to see when the angel of death arrives and takes us away .", "( male ) concerning the subcategory of  doing good deeds is the god 's will  , the participants believed that trust in god guided them to many positive activities in their lives .", "they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity .", "one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems .", " when i want to get out of my house for work , e first thing that comes to mind is that ", " my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody .  ( male ) another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage .", "the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities .", "for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness .", "we go to quran classes on friday mornings also.(male )  i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced .", "i go to the religious rites with the my friends and peers , and i want to get out of loneliness .", "( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque .", "one of the main categories that emerged from data analysis was  spiritual health  .", "four subcategories ;  saying prayer as a calming factor  ,  beneficence as a way to god  ,  loss of psychological and spiritual support  , and  faith , a way to happiness  , emerged from the participants experiences .", "also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them .", " i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day  .", "( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers .", "they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives .", "they said that man would receive god 's blessings as a consequence of his / her deeds in the world .", "additionally , they believed that the ultimate consequence of good deeds was good in return .", "if she asks me to fix her air conditioner you can be sure that i ll do it .", "this is because i think that by doing this i make her happy and she prays for me .", "( female ) moreover regarding  loss of psychological and spiritual support  , the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support .", " when i think about my life , i feel that i do n't have any support from my children .", "also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . ", "( female ) the elderly participants lived experiences disclosed the subcategory of  faith , a way to happiness  in life .", "one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs .", "also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness . ", "the one who has strong faith and beliefs is always happy and confident . in my opinion", "worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free  .", " spiritual beliefs  was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including  seeking help from god in difficulties  ,  only god 's power over life and death  , and ", "doing good deeds is the god 's will  . regarding the subcategory of  seeking help from god in difficulties  , the participants believed", "that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses .", "this desire results in god 's attention to elderly and also creates sense of trust in older adults .", "some statements said by participants are :  i really believe in having trust in god . when i m trapped in a complicated situation", ", i believe that this is god who helps me and i only trust in god .  ( male ) one participant believed that spirituality was a vital component of older people lives .", "also , he believed that god was the ultimate source to affect my life or death and he controls everything .", " we re all waiting to see when the angel of death arrives and takes us away .", "( male ) concerning the subcategory of  doing good deeds is the god 's will  , the participants believed that trust in god guided them to many positive activities in their lives .", "they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity .", "one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems .", " when i want to get out of my house for work , e first thing that comes to mind is that ", " my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody .  ( male )", "another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage .", "the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities .", "for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness .", "we go to quran classes on friday mornings also.(male )  i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced .", "i go to the religious rites with the my friends and peers , and i want to get out of loneliness .", "( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque .", "this qualitative study describes the concept of spirituality and its impression on iranian elderly people daily living behaviors .", "the findings are significant as they reveal that the living experiences of saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , are the most important domain and subcategory of spiritual health of elderly people , which can positively affect their health . on the other hand spiritual health", "is an important dimension of human health and it can determine the individual integrity . also spiritual health is a power that coordinates physical , mental and social dimensions and is necessary in coping with diseases . when spiritual health will is at serious risk , individuals may be stricken by mental disorders such as loneliness , depression , and loss of meaning .", "there are a number of studies that support the beneficial effects of spirituality on health .", "behaviors such as reliance on god , pilgrimage and praying create hope and positive attitudes that lead to internal serenity , peacefulness and meaningful life .", "most faithful people describe their relationship with god as a relationship with an intimate friend and believe that they can control the effect of irrepressible situations by having recourse to god .", "all in all , religious adaptation relies on beliefs and religious activities and help people to control their stress and physical disorders . having meaning and goal in life , sense of belonging to a sublime source , hope for gods support in time of trouble and social and spiritual support are among the sources that help religious people to suffer less from hardships of life . in the iranian society context , where the majority of the population are muslim , and", "a religious culture is dominant in the society , it is expected that tendency to spirituality could be affect their health .", "praying is the expression of soul and a deep human instinct that arises from human soul and is uttered with profound words .", "it is neither dependent upon any particular spirituality nor entailed in any time and place .", "the content of praying includes confession of sins and weakness , pleading for forgiveness , bliss and closeness to god .", "praying reduces mental and psychological pains , releases personal emotions , leading individual to attain eternal source of power .", "weeping that often comes with praying greatly helps a person to release his or her distress and emotions .", "also prayer helped participants to cope with disease and gave them calm , hope and inner strength .", "in this regard , quran has put it magnificently :  those who have faith in god and inundate their hearts with name of god , surely god remembrance ascertains their hearts  ( ( quran , chapter 13 : sureh 13 , al - rad ) verse 28 ) .", "many americans , regardless of their health status , rely on their religious and spiritual beliefs to cope with stressful life events .", "the present study has demonstrated that one of the most important factors that help elderly in confronting with difficulties was seeking help from god .", "several studies noted that spirituals beliefs had positive influence on welfare and personal and social life of elderly people .", "effective spiritual coping strategies help individuals find meaning and purpose in their life , problems and difficulties . for instance", ", one study showed that healthy older adults believed that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being .", "thus , spirituality plays an important role in the lives of healthy individuals , too .", "believing in support from spiritual / religious resource and connectedness with a higher power is beneficial and it can affect issues of control , quality of life , spiritual well - being , coping , depression , decision - making , and possibly health outcomes .", "for instance , it has been reported that healthy older adults believe that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being .", "thus , spirituality plays an important role in the lives of healthy individuals . in the result of our study", "a sense of god 's power over life and death and doing good deeds is the god 's will were derived from participants experience around spiritual beliefs .", "all the participants talked about the importance of god and belief to god 's power in their life .", "moreover , the findings reveal that god 's power was very important in personal and private experiences . in this regard , roff et al.(2009 ) in a qualitative study through a thematic content analysis identified god 's power in the lived experiences of women who were suffering from breast cancer . also , in another qualitative study by taleghani et al .", "( 2006 ) belief to god 's will was a important key in acceptance of disease .", "2002 ) suggested that personal variables such as the concept of god and perception of others beliefs played important mediating roles in religious coping with stress situations .", "religious practices were a subcategory that emerged from the lived experiences of elderly participants in this research . furthermore , the results of study by keefe et al .", "( 2001 ) showed that individuals who reported frequent daily spiritual experiences had higher levels of positive mood , lower levels of daily negative mood , and higher levels of all of the social support domains .", "another subcategory that emerged from participants experience was going to mosque and religious ceremonies . in this regard , palinkas ( 1982 ) found that religious ceremonies are supportive for individuals because liberating and curative friendships relevant to spiritual states brings about psychological health for them . attending religious ceremonies and shrines also reduces individuals anxieties and sense of loneliness .", "these results are in accordance with our study findings which show that elderly women who participate in religious ceremonies , are relieved from loneliness . also koenig ( 1998 )", "suggested that religious practices , attitudes and coping behaviors are prevalent among hospitalized medically ill older adults and are related to social , psychological and physical health outcomes .", "morris ( 1983 ) examined the relationship between anxiety and pilgrimage and found that people with religious devotion experienced significantly lower rates of anxiety disorder compared to the non - religious group .", "every muslim learns their prayers from a young age and prays five times a day .", "in addition , those who seek guidance from the holy quran and believe that it has power over every aspect of their lives , expect and welcome any eventualities in their lives ( quran , chapter balad , verse 4 ) .", "it seems that elderly people have more tendencies to religious and spiritual subjects than young people .", "koenig et al . ( 2004 ) in his study showed spirituality and religion as an important factor in their lives more often than younger ones .", "several experimental studies have been conducted that showed positive effects of prayer on the management of physical symptoms of illness .", "kwilecki ( 1986 ) assessed the effect of prayer on stress and anxiety and found that 42% of participants report that saying prayer can diminish the stress and anxiety . also salehi ( 2001 ) found that individuals who pray regularly , experienced significantly lower rates of anxiety disorder and depression , psychological balance and hopefulness , compared to the non - prayer group .", "azizi ( 1996 ) found that more than 90% of prayers attained psychological tranquility and relaxation after saying prayer .", "several studies noted moderately positive correlations between indicators of physical health and prayer activities . also several studies have shown that prayer can play an important role in the recovery of patients who suffer from cardiac disease , hiv , arthritis rheumatoid , and cva attack , cancer cases , treatment of addiction , and physical , mental and social well - being .", "furthermore , evidence suggests that patients with strong religious beliefs and high levels of religious activity experience lower levels of pain , have better immune function , lower death rates from cancer , fewer incidences of heart disease , lower blood pressure and levels of cholesterol , better health behaviors , and greater compliance with medical treatment .", "the practical relevance of this study may conduct future work to further explicate and clarify the linkages between spirituality and physical and mental health and life style .", "future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area ,", "future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects .", "it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) .", "future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area ,", "future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects .", "it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) .", "in summary , we found that elderly people describe several elements in their illustration of spirituality in healthcare settings .", "spiritual health , spiritual beliefs , and religious practice intentionality were the main components of concept of spirituality in this group experiences .", "also , the findings of this study made it clear that spirituality has a considerable effect on the health and life of iranian elderly people and is a major supportive resource for their physical and psychosocial health .", "it can reduce mental distress and induce inner peace and hopefulness . because of the significant influence of spirituality on all domains of health of elderly people , it is critically important that health care providers understand how spirituality can considerably influence elderly people throughout their life .", "these findings will assist health professionals such as nurses , physicians , and social workers to recognize the spiritual needs and value the role of spirituality in promoting health and well - being among elderly people from different religions and cultures worldwide ."], "abstract_text": ["<S> background : spirituality is recognized as a personally important matter to the elderly , and there are evidences of its impact on their health . </S>", "<S> the aim of this study was to explore the concept of spirituality from the perspectives of iranian healthy elderly individuals.materials and methods : a conventional qualitative content analysis of carried out with 21 healthy elderly people from both male and female genders were chosen using a purposive sampling method in tehran in 2010 - 2011 . </S>", "<S> data collection was done through semi structured interviews . </S>", "<S> a qualitative content analysis was used to analyze the participants experiences and perceptions on spirituality , using a central question  what characterizes the spirituality in the iranian healthy elderly people?results : the following categories emerged from the data analysis : ( 1 ) spiritual health , with four sub categories including saying prayer as a calming factor ; beneficence as a way to god ; loss of psychological and spiritual support ; faith as a way to happiness ; ( 2 ) spiritual beliefs , with three sub categories including seeking help from god in difficulties ; god 's power over life and death ; doing good deeds is the god 's will ; and ( 3 ) religious practice with three sub categories including saying prayer ; reading quran ; and going to mosque , religious ceremonies and pilgrimage.conclusions:in this study was found that spirituality was a fundamental element in elderly individuals lives that help them to adapt with daily living conditions . </S>"], "labels": null, "section_names": ["I", "M", "Design", "Selection", "Data collection", "Analysis", "Data trustworthiness", "Ethical considerations", "R", "Spiritual health", "Spiritual beliefs", "Religious practice", "D", "Suggestions", "C"], "sections": [["older people have particular spiritual needs that are distinct from those of others , particularly in times of ill health and death and dying .", "it has been reported that spirituality has a positive effect on health outcomes and welfare of the elderly .", "nowadays , aging phenomenon and relevant problems with its complexities are important issues that have attracted the attention of social and medical scientists to investigate the spiritual needs of the elderly .  ", "spiritual health is a dimension of health that is portrayed as the validation of life in relation to god , fellow human beings , and the environment .", "spirituality is the representative of the basic values that guide a person in searching to find answers to the crucial questions of life , such as the purpose and meaning of life , reality , love , good and bad , disease , and death .   over the past three decades , spirituality , religion , and prayer", "have turned into a subject of large interest for researchers in medicine , nursing , gerontology , physical health and mental health disciplines , often in conjunction with complementary and alternative medicine .", "this tendency has been stimulated by research demonstrating physical and psychological benefits of spiritual and religious involvements .  ", "in fact , approximately 96% of adults in the united states expressed their belief in god , and 72% of identified religion and spirituality as having the most important influence in their lives . saying prayers is a regular daily task for muslims which begins at the age of puberty and continues throughout the life .", "it is a time getting rid of the material world and approaching the world beyond , as well as , to the inner self . in iran , elderly people are more religious than younger ones , and this is a norm of this culture .", "a recent national survey of the iranians values and attitudes found that over 80% of the iranians practice prayer regularly as a part of their religion .", "one might argue that this is a true reflection of individual culture where prayer and religious beliefs are a part of people 's everyday life .", "however , prayer can go beyond just performing a religious duty , and based on one 's wishes , take different forms of formal and informal practices .", "it has been shown that disease prevalence , incidence and mortality rates differ from one population to another .", "this is partly ascribed to their disparities in lifestyle , dietary habits and other characteristics that are somehow affected by culture , especially religious traditions and experiences .   during the past two decades", "some studies have been carried out and the associations between religious beliefs or experiences and various types of physical illnesses , mental health , and mortality have been examined .", "spirituality is viewed as personally important matter to the older people and it has been proved that it is highly related to older people 's health outcomes .  ", "however , there has not been adequate experiential research on the relationship between religious or spiritual factors and healthy lifestyle behaviors , and their impact on the adult 's well - being .", "scientists believe that probing the effects of religious and spiritual factors as well as healthy lifestyle behaviors may be decisive .", "additionally , social science and medical research have concurrently reported that older adults are more inclined to take spiritual chase than younger people are .  ", "kotrotsiou - barboutal et al.(2006 ) indicated that older adults are remaining members of religious associations without necessarily participating regularly in services , as there are numerous issues that can diminish the active participation in religious events , such as transportation intricacies , bad sound , or the illegibility of small font text in books of prayers available at mosques or religious centers . in spite of those complexities ,", "many elderly manage to find out a way to come up to god through prayer .", "it is stated that prayer increases the feelings of personal value of an elderly by reducing the feelings of loneliness and desertation , while television and radio provide support for spiritual life .   in this regard , researchers have shown that some groups of elderly people with inadequate and difficult livelihood situations communicate their problems by putting their trust in god . moreover , more than half of the elder patients practiced spiritual needs throughout their hospitalization period , with the most prominent need of being in the search of the meaning of life .  ", "extensive epidemiological studies have shown that higher levels of religiousness are related to lower mortality rates .", "studies identified the multidimensional nature of religiousness and spirituality and investigated various religious dimensions , for instance private religious behaviors , devotion , spiritual transcendence , and religious adaptation .", "for example , it was indicated that some aspects of religiousness predicted lower rates of disability and illness , alcoholism , cardiovascular disease , hypertension , and myocardial infarction .", "furthermore , clinical research has suggested that spirituality can play a vital role in the recovery of patients suffering from physical and psychological diseases .  ", "it seems that religious obligation has a crucial role in averting physical and mental diseases , recovery promotion , and disease adaptation .", "numerous studies have revealed the positive involvement of spirituality and religious indexes on different aspects of health such as cardiac surgery , mortality , immunity system function and self esteem .  ", "above all , religious involvement can be useful for psychological well - being and physical health .", "additionally , supporting the effect of spirituality may become more and more prominent in later life as a result of declining health , social and financial sources .", "researchers have proposed numerous psychological , social , and physiological interveners that may explain the spirituality - health association .  ", "quantitative measurements and the evaluation of spiritual beliefs are difficult because quantitative studies neither reveal the content , components of the beliefs , and its factors , nor the contexts that might be included . therefore , implementing qualitative research that explores the personal and experiential aspects of beliefs seems crucial , and may shed light on a range of important issues which can be investigated in detail on larger samples by further quantitative studies in future .  ", "spiritual or religious beliefs may affect the decisions of individuals that make about their health and illness .", "thus , recognizing and understanding the spiritual and religious behaviors within the context of cultural influence by a qualitative research is critical for nurse and other health - care providers .", "the main strengths of the present study are that it focuses on the spiritual life of healthy elderly people .", "therefore , the present study aimed to explore the concept of spirituality from the perspectives of iranian healthy elderly people . the central question of the paper was  what characterizes the spirituality in the iranian healthy older people ?"], ["in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 .", "it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects .  ", "initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data .", "the study consisted of 17 healthy elders within the age range of 65 - 86 .", "criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study .", "all participants were shia muslims . in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions .", "based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection .", "interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences .", "the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about  spirituality in elderly adults  .", "what is your experience concerning the spirituality in later age? and  what is the meaning of spirituality in your experience ?", " each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next .", "coding was carried out line by line , and comparative analysis of the quotations was carried out .", "all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions .", "each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes .", "data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories .", "the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories .", "credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking .", "the findings and explanations of this study were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data .", "this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability .", "permission to conduct this study was obtained from the ethics committee of tarbiat modares university .", "other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses .", "all participants were aware of the purpose of the study , and their participation in the study was optional .", "informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki ."], ["in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 .", "it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects ."], ["initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data .", "the study consisted of 17 healthy elders within the age range of 65 - 86 .", "criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study ."], ["in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions . the interview was scheduled according to the participant 's agreement .", "based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection .", "interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences .", "the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about  spirituality in elderly adults  .", "what is your experience concerning the spirituality in later age? and  what is the meaning of spirituality in your experience ? "], ["each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next .", "coding was carried out line by line , and comparative analysis of the quotations was carried out .", "all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions .", "each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes .", "data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories .", "the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories ."], ["credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking . the findings and explanations of this study", "were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data .", "this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability ."], ["permission to conduct this study was obtained from the ethics committee of tarbiat modares university .", "other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses .", "all participants were aware of the purpose of the study , and their participation in the study was optional .", "informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki ."], ["the participants of the study were 11 males and 10 females , within the age range of 65 - 86 .", "also , 15 out of 21 were married , four were widowed , and one was single .", "five participants were illiterate , four were primary degree holders , eight had a diploma , two had bachelor degrees , and two had msc degree in human sciences . in terms of their occupation status , two were employees , 13 were pensioners , two was manual worker , and three were housewives .", "three main categories emerged from the data and 3 - 4 distinctive subcategories within each category were identified .", "these categories and their subcategories represent the main factors influencing the spirituality in iranian elderly people .", "main categories and subcategories one of the main categories that emerged from data analysis was  spiritual health  . four subcategories ;", " saying prayer as a calming factor  ,  beneficence as a way to god  ,  loss of psychological and spiritual support  , and  faith , a way to happiness  , emerged from the participants experiences .", "also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them .", " i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day  .", "( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers .", "they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives .", "they said that man would receive god 's blessings as a consequence of his / her deeds in the world .", "additionally , they believed that the ultimate consequence of good deeds was good in return .", "if she asks me to fix her air conditioner you can be sure that i ll do it .", "this is because i think that by doing this i make her happy and she prays for me .", ", the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support .", " when i think about my life , i feel that i do n't have any support from my children .", "also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . ", "( female ) the elderly participants lived experiences disclosed the subcategory of  faith , a way to happiness  in life .", "one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs .", "also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness . ", "the one who has strong faith and beliefs is always happy and confident . in my opinion", "worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free  .", "( male )  spiritual beliefs  was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including  seeking help from god in difficulties ", " doing good deeds is the god 's will  . regarding the subcategory of  seeking help from god in difficulties ", ", the participants believed that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses .", "this desire results in god 's attention to elderly and also creates sense of trust in older adults .", "some statements said by participants are :  i really believe in having trust in god . when i m trapped in a complicated situation", ", i believe that this is god who helps me and i only trust in god .  ( male ) one participant believed that spirituality was a vital component of older people lives .", "also , he believed that god was the ultimate source to affect my life or death and he controls everything .", " we re all waiting to see when the angel of death arrives and takes us away .", "( male ) concerning the subcategory of  doing good deeds is the god 's will  , the participants believed that trust in god guided them to many positive activities in their lives .", "they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity .", "one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems .", " when i want to get out of my house for work , e first thing that comes to mind is that ", " my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody .  ( male ) another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage .", "the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities .", "for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness .", "we go to quran classes on friday mornings also.(male )  i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced .", "i go to the religious rites with the my friends and peers , and i want to get out of loneliness .", "( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque ."], ["one of the main categories that emerged from data analysis was  spiritual health  .", "four subcategories ;  saying prayer as a calming factor  ,  beneficence as a way to god  ,  loss of psychological and spiritual support  , and  faith , a way to happiness  , emerged from the participants experiences .", "also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them .", " i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day  .", "( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers .", "they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives .", "they said that man would receive god 's blessings as a consequence of his / her deeds in the world .", "additionally , they believed that the ultimate consequence of good deeds was good in return .", "if she asks me to fix her air conditioner you can be sure that i ll do it .", "this is because i think that by doing this i make her happy and she prays for me .", "( female ) moreover regarding  loss of psychological and spiritual support  , the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support .", " when i think about my life , i feel that i do n't have any support from my children .", "also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . ", "( female ) the elderly participants lived experiences disclosed the subcategory of  faith , a way to happiness  in life .", "one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs .", "also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness . ", "the one who has strong faith and beliefs is always happy and confident . in my opinion", "worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free  ."], [" spiritual beliefs  was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including  seeking help from god in difficulties  ,  only god 's power over life and death  , and ", "doing good deeds is the god 's will  . regarding the subcategory of  seeking help from god in difficulties  , the participants believed", "that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses .", "this desire results in god 's attention to elderly and also creates sense of trust in older adults .", "some statements said by participants are :  i really believe in having trust in god . when i m trapped in a complicated situation", ", i believe that this is god who helps me and i only trust in god .  ( male ) one participant believed that spirituality was a vital component of older people lives .", "also , he believed that god was the ultimate source to affect my life or death and he controls everything .", " we re all waiting to see when the angel of death arrives and takes us away .", "( male ) concerning the subcategory of  doing good deeds is the god 's will  , the participants believed that trust in god guided them to many positive activities in their lives .", "they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity .", "one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems .", " when i want to get out of my house for work , e first thing that comes to mind is that ", " my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody .  ( male )"], ["another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage .", "the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities .", "for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness .", "we go to quran classes on friday mornings also.(male )  i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced .", "i go to the religious rites with the my friends and peers , and i want to get out of loneliness .", "( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque ."], ["this qualitative study describes the concept of spirituality and its impression on iranian elderly people daily living behaviors .", "the findings are significant as they reveal that the living experiences of saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , are the most important domain and subcategory of spiritual health of elderly people , which can positively affect their health . on the other hand spiritual health", "is an important dimension of human health and it can determine the individual integrity . also spiritual health is a power that coordinates physical , mental and social dimensions and is necessary in coping with diseases . when spiritual health will is at serious risk , individuals may be stricken by mental disorders such as loneliness , depression , and loss of meaning .", "there are a number of studies that support the beneficial effects of spirituality on health .", "behaviors such as reliance on god , pilgrimage and praying create hope and positive attitudes that lead to internal serenity , peacefulness and meaningful life .", "most faithful people describe their relationship with god as a relationship with an intimate friend and believe that they can control the effect of irrepressible situations by having recourse to god .", "all in all , religious adaptation relies on beliefs and religious activities and help people to control their stress and physical disorders . having meaning and goal in life , sense of belonging to a sublime source , hope for gods support in time of trouble and social and spiritual support are among the sources that help religious people to suffer less from hardships of life . in the iranian society context , where the majority of the population are muslim , and", "a religious culture is dominant in the society , it is expected that tendency to spirituality could be affect their health .", "praying is the expression of soul and a deep human instinct that arises from human soul and is uttered with profound words .", "it is neither dependent upon any particular spirituality nor entailed in any time and place .", "the content of praying includes confession of sins and weakness , pleading for forgiveness , bliss and closeness to god .", "praying reduces mental and psychological pains , releases personal emotions , leading individual to attain eternal source of power .", "weeping that often comes with praying greatly helps a person to release his or her distress and emotions .", "also prayer helped participants to cope with disease and gave them calm , hope and inner strength .", "in this regard , quran has put it magnificently :  those who have faith in god and inundate their hearts with name of god , surely god remembrance ascertains their hearts  ( ( quran , chapter 13 : sureh 13 , al - rad ) verse 28 ) .", "many americans , regardless of their health status , rely on their religious and spiritual beliefs to cope with stressful life events .", "the present study has demonstrated that one of the most important factors that help elderly in confronting with difficulties was seeking help from god .", "several studies noted that spirituals beliefs had positive influence on welfare and personal and social life of elderly people .", "effective spiritual coping strategies help individuals find meaning and purpose in their life , problems and difficulties . for instance", ", one study showed that healthy older adults believed that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being .", "thus , spirituality plays an important role in the lives of healthy individuals , too .", "believing in support from spiritual / religious resource and connectedness with a higher power is beneficial and it can affect issues of control , quality of life , spiritual well - being , coping , depression , decision - making , and possibly health outcomes .", "for instance , it has been reported that healthy older adults believe that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being .", "thus , spirituality plays an important role in the lives of healthy individuals . in the result of our study", "a sense of god 's power over life and death and doing good deeds is the god 's will were derived from participants experience around spiritual beliefs .", "all the participants talked about the importance of god and belief to god 's power in their life .", "moreover , the findings reveal that god 's power was very important in personal and private experiences . in this regard , roff et al.(2009 ) in a qualitative study through a thematic content analysis identified god 's power in the lived experiences of women who were suffering from breast cancer . also , in another qualitative study by taleghani et al .", "( 2006 ) belief to god 's will was a important key in acceptance of disease .", "2002 ) suggested that personal variables such as the concept of god and perception of others beliefs played important mediating roles in religious coping with stress situations .", "religious practices were a subcategory that emerged from the lived experiences of elderly participants in this research . furthermore , the results of study by keefe et al .", "( 2001 ) showed that individuals who reported frequent daily spiritual experiences had higher levels of positive mood , lower levels of daily negative mood , and higher levels of all of the social support domains .", "another subcategory that emerged from participants experience was going to mosque and religious ceremonies . in this regard , palinkas ( 1982 ) found that religious ceremonies are supportive for individuals because liberating and curative friendships relevant to spiritual states brings about psychological health for them . attending religious ceremonies and shrines also reduces individuals anxieties and sense of loneliness .", "these results are in accordance with our study findings which show that elderly women who participate in religious ceremonies , are relieved from loneliness . also koenig ( 1998 )", "suggested that religious practices , attitudes and coping behaviors are prevalent among hospitalized medically ill older adults and are related to social , psychological and physical health outcomes .", "morris ( 1983 ) examined the relationship between anxiety and pilgrimage and found that people with religious devotion experienced significantly lower rates of anxiety disorder compared to the non - religious group .", "every muslim learns their prayers from a young age and prays five times a day .", "in addition , those who seek guidance from the holy quran and believe that it has power over every aspect of their lives , expect and welcome any eventualities in their lives ( quran , chapter balad , verse 4 ) .", "it seems that elderly people have more tendencies to religious and spiritual subjects than young people .", "koenig et al . ( 2004 ) in his study showed spirituality and religion as an important factor in their lives more often than younger ones .", "several experimental studies have been conducted that showed positive effects of prayer on the management of physical symptoms of illness .", "kwilecki ( 1986 ) assessed the effect of prayer on stress and anxiety and found that 42% of participants report that saying prayer can diminish the stress and anxiety . also salehi ( 2001 ) found that individuals who pray regularly , experienced significantly lower rates of anxiety disorder and depression , psychological balance and hopefulness , compared to the non - prayer group .", "azizi ( 1996 ) found that more than 90% of prayers attained psychological tranquility and relaxation after saying prayer .", "several studies noted moderately positive correlations between indicators of physical health and prayer activities . also several studies have shown that prayer can play an important role in the recovery of patients who suffer from cardiac disease , hiv , arthritis rheumatoid , and cva attack , cancer cases , treatment of addiction , and physical , mental and social well - being .", "furthermore , evidence suggests that patients with strong religious beliefs and high levels of religious activity experience lower levels of pain , have better immune function , lower death rates from cancer , fewer incidences of heart disease , lower blood pressure and levels of cholesterol , better health behaviors , and greater compliance with medical treatment .", "the practical relevance of this study may conduct future work to further explicate and clarify the linkages between spirituality and physical and mental health and life style .", "future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area ,", "future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects .", "it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) ."], ["future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area ,", "future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects .", "it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) ."], ["in summary , we found that elderly people describe several elements in their illustration of spirituality in healthcare settings .", "spiritual health , spiritual beliefs , and religious practice intentionality were the main components of concept of spirituality in this group experiences .", "also , the findings of this study made it clear that spirituality has a considerable effect on the health and life of iranian elderly people and is a major supportive resource for their physical and psychosocial health .", "it can reduce mental distress and induce inner peace and hopefulness . because of the significant influence of spirituality on all domains of health of elderly people , it is critically important that health care providers understand how spirituality can considerably influence elderly people throughout their life .", "these findings will assist health professionals such as nurses , physicians , and social workers to recognize the spiritual needs and value the role of spirituality in promoting health and well - being among elderly people from different religions and cultures worldwide ."]]}
{"article_id": "PMC3761089", "article_text": ["according to the national regulations regarding diagnostic criteria of brain death ( bd ) , instrumental confirmatory tests should be used in certain clinical situations , such as intoxications , infratentorial processes , extensive facial damage , children younger than 2  years of age , or any case in which clinical examinations are inadequate .", "electrophysiological tests are often unavailable due to limited access to proper equipment and competent specialists . therefore ,", "two of them , catheter angiography and computed tomographic angiography ( cta ) , are based on the detection of contrast enhancement of cerebral vessels . in these examinations ,", "first is a non - filling phenomenon , which means a complete absence of contrast above the carotid siphons and foramen magnum .", "second is a delayed , weak , and persistent opacification of proximal segments of the cerebral arteries not reaching cortical branches .", "stasis filling causes a significant problem in interpretation of cta results in the diagnosis of bd . since 1998 ,", "when cta was proposed as the new imaging technique in the diagnostics of bd , a consensus on its interpretation criteria has not been reached .", "an analysis of stasis filling in a dynamic series of computed tomographic perfusion ( ctp ) can provide valuable information on the interpretation of cta results and relation of this phenomenon to brain perfusion .", "the aim of this prospective study was to characterize stasis filling phenomenon in the diagnosis of bd . to achieve this", ", we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and control subjects with the use of the 40-s series of ctp scans .", "we also confronted stasis filling pattern with ctp findings to assess the relevance of this phenomenon to brain perfusion .", "we examined 67 patients who fulfilled clinical bd criteria according to the national regulations ( coma , apnea , and no brainstem reflexes ) . the examination protocol consisted of cta , followed by ctp and catheter angiography .", "thirty - seven subjects were excluded due to lack of contrast enhancement in the intracranial arteries in ctp series .", "finally , 30 ( 44.8  % ) patients , in whom analysis of ctp series enabled us to identify contrast - enhanced intracranial arteries , were enrolled in the study .", "the population consisted of 18 men and 12 women with a median age of 54.5  years ( range , 2284  years ) .", "initial causes of coma were intracerebral hemorrhage ( n  =  16 ) , subarachnoid hemorrhage ( n  =  11 ) , cerebral edema ( n  =  2 ) , and ischemic stroke ( n  =  1 ) .", "decompressive craniectomy was performed in 19 ( 63  % ) included patients . among 37 excluded patients ,", "all patients were managed in the intensive care units of two university hospitals and one multi - profile provincial hospital . during all the examinations ,", "the patients were normoventilated and mean arterial blood pressure ( mabp ) maintained at greater than 80  mmhg .", "the elapsed time between the onset of bd symptoms and the radiological examination ranged between 6 and 48  h. delay was sometimes caused by a prolonged initial observation due to prior use of sedatives and sometimes the limited availability of an angiographic team . the majority of patients became actual organ donors .", "in some cases , organ donation was not possible for medical reasons and occasionally because of a failure to obtain permission from relatives .", "in such situations , families were informed about the termination of futile therapy , and ventilators were legally switched off .", "the demographic and epidemiological characteristics of bd patients are presented in table  1.table 1demographic and clinical characteristics of bd patientsnumbersexage ( years)diagnosiscraniectomyexamined arterystasis filling in angiography1m56ichmca2m71sahmca+3m73ich+mca+4f49sah+aca5f29sahaca6m40isaca7f66ichmca8m84sah+mca9m53ichmca10f67ich+mca+11m63cemca12f58sah+mca13m34sah+aca14m34sah+mca+15m78ichmca+16m39ich+mca+17f38sah+mca18f74sahaca+19f75ichmca+20f40sah+mca+21f22ich+mca+22f34ich+mca23m50ich+aca+24m44sah+mca+25m51ich+pca+26m71ich+mca27f41ce+mca28m59ichmca29m62ich+mca30m56ich+mca+ \n is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery demographic and clinical characteristics of bd patients \n is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery the control group consisted of 30 patients who underwent surgical clipping of an intracranial aneurysm .", "the population consisted of 14 men and 16 women with a median age of 53  years ( range , 1865  years ) .", "ctp was performed 810  days after neurosurgical clipping of the middle cerebral or anterior communicating artery aneurysms . during all the examinations , mabp stayed above 80  mmhg .", "none of the control subjects presented any signs of bd , with normal pco2 and po2 .", "all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] .", "we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta .", "injection of 80  ml of iodinated contrast medium at a flow rate of 4  ml / s .", "then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60  s after contrast injection .", "siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15  min .", "administration of 50  ml of the contrast medium at a flow rate of 5  ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns .", "detailed protocol is presented in table  2.table 2technical parameters of ctp examinationacquisition  tube voltage ( kvp)80  tube current ( mas)270  rotation time ( s)1.0  collimation ( mm)24    1.2  scan range ( mm)28.8  cycle time ( s)1.0  scan time", "( s)40  automatic dose modulationoffreconstruction  slice width ( mm)3    9.6  fov ( mm)220  matrix512    512  recon algorithmh30scontrast injection  volume ( ml)50  flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing , two small circular 0.250.3-cm regions of interest ( rois )", "the second roi covered the cerebral artery in the most distal visible segment ( see fig .  1 ) .", "this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table  1 ) .", "rois were automatically propagated over the entire series of scans . for each roi , time", "calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig .", "1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) .", "this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation .", "therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases .", "we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs .", "cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels .", "angiography was performed with a delay of 15  min to 3  h after ctp .", "we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) .", "after positioning a pigtail 45-f catheter in the ascending aorta , 30  ml of contrast was injected at a flow rate of 15  ml / s .", "we registered 3050  s series with a frequency of 2  f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines ,", "cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow   non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow   demographic data recorded for bd patients and control subjects were age and sex .", "in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series", ", the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling .", "whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p  <  0.05 , shapiro ", "all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] .", "we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta .", "injection of 80  ml of iodinated contrast medium at a flow rate of 4  ml / s .", "then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60  s after contrast injection .", "we used the same scanner  siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15  min .", "administration of 50  ml of the contrast medium at a flow rate of 5  ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns .", "detailed protocol is presented in table  2.table 2technical parameters of ctp examinationacquisition  tube voltage ( kvp)80  tube current ( mas)270  rotation time ( s)1.0  collimation ( mm)24    1.2  scan range ( mm)28.8  cycle time ( s)1.0  scan time ( s)40  automatic dose modulationoffreconstruction  slice width ( mm)3    9.6  fov ( mm)220  matrix512    512  recon algorithmh30scontrast injection  volume ( ml)50  flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing ,", "two small circular 0.250.3-cm regions of interest ( rois ) were positioned in each ctp series of scans .", "the second roi covered the cerebral artery in the most distal visible segment ( see fig .  1 ) .", "this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table  1 ) .", "rois were automatically propagated over the entire series of scans . for each roi , time  density curve ( tdc ) was plotted .", "calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig .", "1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) .", "this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation .", "therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases .", "we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs .", "cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels .", "angiography was performed with a delay of 15  min to 3  h after ctp .", "we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) .", "a typical femoral approach was used in all cases . after positioning a pigtail 45-f catheter in the ascending aorta ,", "30  ml of contrast was injected at a flow rate of 15  ml / s .", "we registered 3050  s series with a frequency of 2  f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines ,", "cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow   non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow", "in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series", ", the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling .", "whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p  <  0.05 , shapiro ", "for cerebral arteries , tdcs in bd patients represented flat curves in contrast to tdcs in the control group , which formed steep and narrow gaussian curves ( see fig .  2 ) .", "baseline density for cerebral and extracranial arteries was significantly higher in bd patients in comparison to the control group ( 60.5 vs. 41.5  hu ( hounsfield unit ) ;", "we found significantly longer time to peak enhancement in cerebral arteries in bd patients than in controls ( median , 32 vs. 21  s ; p  < ", "in bd patients , peak enhancement in cerebral arteries occurred with a median delay of 14.5  s to peak in extracranial arteries while practically no delay was noted in controls ( it occurred 1  s earlier , which is represented as median 1  s value ; p  < ", "cerebral arteries in bd patients showed significantly lower peak enhancement in comparison to the control group ( 34.5 vs. 81.5  hu ; p  <  0.0001 ) ( see fig . ", "density curves in a bd patient ( a ) and in a control subject ( b ) .", "3distribution of baseline density values in bd patients and controls in cerebral and extracranial arteriesfig .", "4distribution of time to peak in cerebral and extracranial arteries in bd patients and controlsfig .", "6distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls time ", "density curves in a bd patient ( a ) and in a control subject ( b ) .", "time to peak ( ttp ) enhancement distribution of baseline density values in bd patients and controls in cerebral and extracranial arteries distribution of time to peak in cerebral and extracranial arteries in bd patients and controls distribution of delay of cerebral peak values in bd patients and controls distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls for extracranial arteries , tdcs represented a similar shape in both groups .", "however , we observed higher enhancement in bd patients at 20 , 30 , and 40  s ( see table  3 ) .", "peak enhancement was significantly higher and earlier in bd patients compared to controls ( p  =  0.0087 and p  <  0.0001 , respectively ) ( see figs .  4 and 6 ) .", "the intensity of peak enhancement in cerebral arteries in relation to extracranial arteries expressed as c / e peak ratio appeared to be significantly lower in bd patients in comparison to the control group ( 0.31 vs. 0.87 ; p  <  0.0001 ) ( see fig . ", "7).table 3variables calculated from dynamic ctp series in bd patients vs. controlsparameterbd ( n  =  30)controls ( n  =  30 ) \n p valueenhancement at 20  s ( hu )  cerebral artery7.5 ( 24)41 ( 25)<0.0001  extracranial artery53.5 ( 40)41.5 ( 30)nsenhancement at 30  s ( hu )  cerebral artery18.5 ( 14)25 ( 19)ns  extracranial artery36 ( 23)26 ( 20)0.0225enhancement at 40  s ( hu )  cerebral artery20 ( 15)16.5 ( 8)ns  extracranial artery36.5 ( 18)17 ( 10)<0.0001values expressed as median ( interquartile range ) \n ns not significantfig", ". 7distribution of cerebral / extracranial peak ratio values in bd patients and controls variables calculated from dynamic ctp series in bd patients vs. controls values expressed as median ( interquartile range ) distribution of cerebral / extracranial peak ratio values in bd patients and controls analyzing the influence of demographic and clinical features on dynamic ct parameters , we revealed significant differences in time to peak and delay of peak enhancement in cerebral arteries between subgroups of bd patients with and without craniectomy . in the subgroup with craniectomy , time to peak and delay of cerebral peak were shorter in comparison to the subgroup without craniectomy ( see table  4 ) .", "a statistically insignificant trend was found in the analysis of tdcs in bd patients with subarachnoid hemorrhage ( sah ) and intracerebral hemorrhage ( ich ) .", "we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich ( see table  5 ) .", "we did not observe any significant differences between ct parameters in relation to sex or examined artery.table 4variables calculated from dynamic ctp series in bd patients without vs. with craniectomyparameterbd patients without craniectomy ( n  =  11)bd patients with craniectomy ( n  =  19 ) \n p valuetime to peak in cerebral artery ( s)34 ( 6)26 ( 9)0.007delay of cerebral peak ( s)18 ( 8)13 ( 9)0.04values expressed as median ( interquartile range)table 5variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhageparameterbd patients with ich ( n  =  16)bd patients with sah ( n  =  11 ) \n p valuetime to peak in cerebral artery ( s)27.5 ( 10)33 ( 9)0.34 ( ns)delay of cerebral peak ( s)13.5 ( 8)18 ( 13)0.16 ( ns)values expressed as median ( interquartile range ) \n ns not significant variables calculated from dynamic ctp series in bd patients without vs. with craniectomy values expressed as median ( interquartile range ) variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhage values expressed as median ( interquartile range ) in all 30 bd patients , ctp revealed zero values of cbf and cbv in all rois .", "stasis filling pattern was observed in 14 ( 46.7  % ) and non - filling in 16 ( 53.3  % ) cases . in 37", "excluded bd patients , angiography revealed a non - filling pattern in all cases ; no stasis filling was noted .", "the term stasis filling for describing a delayed , weak , and persistent intracranial opacification in patients with bd was used for the first time by kricheff et al . in 1978 .", "munari et al . reported stasis filling in 5  % ( one out of 20 ) , braun et al . in 11  % ( 15/140 ) , bradac et al . in 12.5  % ( two out of 16 ) , kricheff et al . in 15  % ( three out of 20 ) , and savard et al . in 28  % ( nine out of 32 ) of cases [ 2 , 69 ] .", "noted stasis filling in 43  % ( six out of 14 ) and welschehold et al . in 59  % ( 37/63 ) of cases [ 3 , 10 ] .", "stasis filling , as specific angiographic pattern of cerebral circulatory arrest , is a consequence of two major factors : raised intracranial pressure ( icp ) and high cerebrovascular resistance ( cvr ) .", "they both cause reduction of cbf , according to poiseuille s law : cbf  =  cpp / cvr  =  ( mabp    icp)/cvr , where cpp stands for cerebral perfusion pressure . increased cvr in extreme cerebral hemodynamic disturbances", "was revealed in studies using a transcranial doppler ( tcd ) [ 11 , 12 ] .", "this is caused mainly by altered cerebral autoregulation mechanisms , which are sufficient to preserve almost constant cbf when cpp is above 50  mmhg .", "cessation of capillary circulation is consistent with cerebral circulatory arrest while proximal arterial segments are still patent . at this stage", ", tcd frequently shows  to and fro  or narrow systolic spike patterns , which reflect blood movement [ 11 , 12 ] . in such circumstances ,", "heartbeat driven slow propagation of contrast column in proximal cerebral arteries is possible . in the present study ,", "recently revealed a potential efficacy of ctp for assessing brain death . in this material ,", "ctp detected complete absence of brain perfusion reflected by zero values of cbf and cbv in all cases .", "these findings clearly show that stasis filling is not an indicator of any residual brain perfusion but only ineffective propagation of contrast in cerebral vessels ; thus , it does not preclude diagnosis of brain death .", "ctp results are complementary to the recent report of selcuk et al . , which revealed global reduction of adc consistent with necrosis of neurons using diffusion - weighted mri technique .", "the findings showed two features of stasis filling phenomenon : delay and weakness of intracranial opacification .", "tdcs for cerebral arteries in bd patients were characterized by significantly longer times to peak ( median , 32  s ) compared to controls ( 21  s ) . moreover", ", intracranial peak enhancement occurred with a median delay of 14.5  s to extracranial peak .", "this is consistent with observations in cta studies , in which intracranial filling in bd patients is usually detected in the late phase ( performed 60  s after contrast injection ) and very rarely in the early phase of scanning [ 3 , 10 , 17 ] .", "correlation with ctp results shows that such delayed vascular opacification does not provide any brain perfusion , thus can not preclude diagnosis of brain death .", "typical cerebral tdc in bd patients was a flat curve with a low peak ( median value of 34.5  hu ) ; about three times lower compared to the peak in the extracranial artery .", "in contrast , extra- and intracranial tdcs in the control group represented steep and narrow gaussian curves , and both peaks were of similar height .", "such low peak values could be the reason of lower detectability of stasis filling by catheter angiography compared to ct ( 14/67  =  21  % vs. 30/67  =  45  % cases ) observed in the present study .", "these findings are consistent with results of others as was presented in the first paragraph of discussion .", "also observed underestimation of intracranial filling in catheter angiography compared to cta in four out of seven cases .", "although , a larger amount of contrast was used in dynamic ct than in catheter angiography ( 50 vs. 30  ml ) , but in angiography , it was injected intra - arterially , which minimizes the effect of dilution .", "this discrepancy could be a result of advanced reconstruction algorithms , systems of noise reduction , and signal amplification used in ct scanners , which reduce noise , improve spatial resolution , and low contrast detectability", ". the other explanation could be the time sequence in performing ctp and catheter angiography .", "angiography was carried out with a delay from 15  min to 3  h after ctp . during this period", ", slow rising icp could stop the propagation of contrast at the level of skull base . analyzing the influence of demographic and clinical features on tdc s parameters", ", we found a statistically significant trend towards shorter time to peak and shorter delay of peak in the cerebral arteries of bd patients with craniectomy compared to those without it .", "moreover , the incidence of craniectomy was much lower in bd patients excluded from the study due to lack of intracranial opacification ( four out of 37  =  11  % ) compared to included patients who presented intracranial filling ( 19/30  =  63  % ) .", "several authors previously reported a relationship between skull defect and preserved intracranial filling in bd patients [ 7 , 18 , 19 ] .", "this can be explained by a decreased icp enabling propagation of contrast in the cerebral vessels .", "a statistically insignificant trend was found in the analysis of tdcs in bd patients with sah and intracerebral hemorrhage .", "we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich .", "this difference was probably caused by vasospasm after sah , which independently increases cvr and additionally contributes to slowing down the propagation of contrast .", "the major drawback of this study is a consequence of performing ctp shortly after cta in bd patients .", "the influence of residual contrast injected for cta was reflected by higher baseline density in the cerebral and extracranial arteries , higher and earlier peak enhancement , and shorter time to peak in the extracranial vessels in bd patients compared to controls .", "however , this contrast contamination should not significantly change values of delay of cerebral peak or c / e peak ratio as they were calculated on the basis of both cerebral and extracranial tcds .", "in this study , we assessed the characteristic features of intracranial filling in bd patients  delay and weakness of cerebrovascular opacification .", "it led to the conclusion that delayed and weak opacification of cerebral arteries do not necessarily mean the presence of cerebral perfusion , thus does not preclude diagnosis of bd ."], "abstract_text": ["<S> introductionstasis filling , defined as delayed , weak , and persistent opacification of proximal segments of the cerebral arteries , is frequently found in brain dead patients . </S>", "<S> this phenomenon causes a major problem in the development of reliable computed tomographic angiography ( cta ) protocol in the diagnosis of brain death ( bd ) . </S>", "<S> the aim of our study was to characterize stasis filling in the diagnosis of bd . to achieve this </S>", "<S> , we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and controls.methodsstudy population included 30 bd patients , who showed stasis filling in computed tomographic perfusion ( ctp ) series . </S>", "<S> thirty patients , after clipping of an intracranial aneurysm , constituted the control group . </S>", "<S> the study protocol consisted of cta , ctp , and angiography . </S>", "<S> time  </S>", "<S> density curves ( tdcs ) of cerebral and extracranial arteries were generated using 40-s series of ctp.resultscerebral tdcs in bd patients represented flat curves in contrast to tdcs in controls , which formed steep and narrow gaussian curves . </S>", "<S> we found longer time to peak enhancement in bd patients than in controls ( 32 vs. 21  s ; p  <  0.0001 ) . in bd patients , peak enhancement in the cerebral arteries occurred with a median delay of 14.5  s to peak in extracranial arteries , while no delay was noted in controls ( p  <  0.0001 ) . </S>", "<S> cerebral arteries in bd patients showed lower peak enhancement than controls ( 34.5 vs. 81.5  hu ; p  <  0.0001 ) . in all bd patients </S>", "<S> , ctp revealed zero values of cerebral blood flow and volume . </S>", "<S> angiography showed stasis filling in 14 ( 46.7  % ) and non - filling in 16 ( 53.3  % ) cases.conclusiona confrontation of stasis filling with ctp results showed that stasis filling is not consistent with preserved cerebral perfusion , thus does not preclude diagnosis of bd . </S>"], "labels": null, "section_names": ["Background", "Material and methods", "CTA", "CTP", "Catheter angiography", "Data collection", "Statistical analysis", "Results", "Discussion", "Limitations", "Conclusions", "Conflict of interest"], "sections": [["according to the national regulations regarding diagnostic criteria of brain death ( bd ) , instrumental confirmatory tests should be used in certain clinical situations , such as intoxications , infratentorial processes , extensive facial damage , children younger than 2  years of age , or any case in which clinical examinations are inadequate .", "electrophysiological tests are often unavailable due to limited access to proper equipment and competent specialists . therefore ,", "two of them , catheter angiography and computed tomographic angiography ( cta ) , are based on the detection of contrast enhancement of cerebral vessels . in these examinations ,", "first is a non - filling phenomenon , which means a complete absence of contrast above the carotid siphons and foramen magnum .", "second is a delayed , weak , and persistent opacification of proximal segments of the cerebral arteries not reaching cortical branches .", "stasis filling causes a significant problem in interpretation of cta results in the diagnosis of bd . since 1998 ,", "when cta was proposed as the new imaging technique in the diagnostics of bd , a consensus on its interpretation criteria has not been reached .", "an analysis of stasis filling in a dynamic series of computed tomographic perfusion ( ctp ) can provide valuable information on the interpretation of cta results and relation of this phenomenon to brain perfusion .", "the aim of this prospective study was to characterize stasis filling phenomenon in the diagnosis of bd . to achieve this", ", we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and control subjects with the use of the 40-s series of ctp scans .", "we also confronted stasis filling pattern with ctp findings to assess the relevance of this phenomenon to brain perfusion ."], ["we examined 67 patients who fulfilled clinical bd criteria according to the national regulations ( coma , apnea , and no brainstem reflexes ) . the examination protocol consisted of cta , followed by ctp and catheter angiography .", "thirty - seven subjects were excluded due to lack of contrast enhancement in the intracranial arteries in ctp series .", "finally , 30 ( 44.8  % ) patients , in whom analysis of ctp series enabled us to identify contrast - enhanced intracranial arteries , were enrolled in the study .", "the population consisted of 18 men and 12 women with a median age of 54.5  years ( range , 2284  years ) .", "initial causes of coma were intracerebral hemorrhage ( n  =  16 ) , subarachnoid hemorrhage ( n  =  11 ) , cerebral edema ( n  =  2 ) , and ischemic stroke ( n  =  1 ) .", "decompressive craniectomy was performed in 19 ( 63  % ) included patients . among 37 excluded patients ,", "all patients were managed in the intensive care units of two university hospitals and one multi - profile provincial hospital . during all the examinations ,", "the patients were normoventilated and mean arterial blood pressure ( mabp ) maintained at greater than 80  mmhg .", "the elapsed time between the onset of bd symptoms and the radiological examination ranged between 6 and 48  h. delay was sometimes caused by a prolonged initial observation due to prior use of sedatives and sometimes the limited availability of an angiographic team . the majority of patients became actual organ donors .", "in some cases , organ donation was not possible for medical reasons and occasionally because of a failure to obtain permission from relatives .", "in such situations , families were informed about the termination of futile therapy , and ventilators were legally switched off .", "the demographic and epidemiological characteristics of bd patients are presented in table  1.table 1demographic and clinical characteristics of bd patientsnumbersexage ( years)diagnosiscraniectomyexamined arterystasis filling in angiography1m56ichmca2m71sahmca+3m73ich+mca+4f49sah+aca5f29sahaca6m40isaca7f66ichmca8m84sah+mca9m53ichmca10f67ich+mca+11m63cemca12f58sah+mca13m34sah+aca14m34sah+mca+15m78ichmca+16m39ich+mca+17f38sah+mca18f74sahaca+19f75ichmca+20f40sah+mca+21f22ich+mca+22f34ich+mca23m50ich+aca+24m44sah+mca+25m51ich+pca+26m71ich+mca27f41ce+mca28m59ichmca29m62ich+mca30m56ich+mca+ \n is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery demographic and clinical characteristics of bd patients \n is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery the control group consisted of 30 patients who underwent surgical clipping of an intracranial aneurysm .", "the population consisted of 14 men and 16 women with a median age of 53  years ( range , 1865  years ) .", "ctp was performed 810  days after neurosurgical clipping of the middle cerebral or anterior communicating artery aneurysms . during all the examinations , mabp stayed above 80  mmhg .", "none of the control subjects presented any signs of bd , with normal pco2 and po2 .", "all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] .", "we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta .", "injection of 80  ml of iodinated contrast medium at a flow rate of 4  ml / s .", "then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60  s after contrast injection .", "siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15  min .", "administration of 50  ml of the contrast medium at a flow rate of 5  ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns .", "detailed protocol is presented in table  2.table 2technical parameters of ctp examinationacquisition  tube voltage ( kvp)80  tube current ( mas)270  rotation time ( s)1.0  collimation ( mm)24    1.2  scan range ( mm)28.8  cycle time ( s)1.0  scan time", "( s)40  automatic dose modulationoffreconstruction  slice width ( mm)3    9.6  fov ( mm)220  matrix512    512  recon algorithmh30scontrast injection  volume ( ml)50  flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing , two small circular 0.250.3-cm regions of interest ( rois )", "the second roi covered the cerebral artery in the most distal visible segment ( see fig .  1 ) .", "this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table  1 ) .", "rois were automatically propagated over the entire series of scans . for each roi , time", "calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig .", "1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) .", "this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation .", "therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases .", "we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs .", "cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels .", "angiography was performed with a delay of 15  min to 3  h after ctp .", "we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) .", "after positioning a pigtail 45-f catheter in the ascending aorta , 30  ml of contrast was injected at a flow rate of 15  ml / s .", "we registered 3050  s series with a frequency of 2  f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines ,", "cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow   non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow   demographic data recorded for bd patients and control subjects were age and sex .", "in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series", ", the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling .", "whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p  <  0.05 , shapiro "], ["all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] .", "we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta .", "injection of 80  ml of iodinated contrast medium at a flow rate of 4  ml / s .", "then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60  s after contrast injection ."], ["we used the same scanner  siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15  min .", "administration of 50  ml of the contrast medium at a flow rate of 5  ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns .", "detailed protocol is presented in table  2.table 2technical parameters of ctp examinationacquisition  tube voltage ( kvp)80  tube current ( mas)270  rotation time ( s)1.0  collimation ( mm)24    1.2  scan range ( mm)28.8  cycle time ( s)1.0  scan time ( s)40  automatic dose modulationoffreconstruction  slice width ( mm)3    9.6  fov ( mm)220  matrix512    512  recon algorithmh30scontrast injection  volume ( ml)50  flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing ,", "two small circular 0.250.3-cm regions of interest ( rois ) were positioned in each ctp series of scans .", "the second roi covered the cerebral artery in the most distal visible segment ( see fig .  1 ) .", "this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table  1 ) .", "rois were automatically propagated over the entire series of scans . for each roi , time  density curve ( tdc ) was plotted .", "calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig .", "1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .", "rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) .", "this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation .", "therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases .", "we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs .", "cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels ."], ["angiography was performed with a delay of 15  min to 3  h after ctp .", "we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) .", "a typical femoral approach was used in all cases . after positioning a pigtail 45-f catheter in the ascending aorta ,", "30  ml of contrast was injected at a flow rate of 15  ml / s .", "we registered 3050  s series with a frequency of 2  f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines ,", "cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow   non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling  delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow"], ["in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series", ", the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40  s : difference between density at the time point of 20 , 30 , and 40  s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling ."], ["whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p  <  0.05 , shapiro "], ["for cerebral arteries , tdcs in bd patients represented flat curves in contrast to tdcs in the control group , which formed steep and narrow gaussian curves ( see fig .  2 ) .", "baseline density for cerebral and extracranial arteries was significantly higher in bd patients in comparison to the control group ( 60.5 vs. 41.5  hu ( hounsfield unit ) ;", "we found significantly longer time to peak enhancement in cerebral arteries in bd patients than in controls ( median , 32 vs. 21  s ; p  < ", "in bd patients , peak enhancement in cerebral arteries occurred with a median delay of 14.5  s to peak in extracranial arteries while practically no delay was noted in controls ( it occurred 1  s earlier , which is represented as median 1  s value ; p  < ", "cerebral arteries in bd patients showed significantly lower peak enhancement in comparison to the control group ( 34.5 vs. 81.5  hu ; p  <  0.0001 ) ( see fig . ", "density curves in a bd patient ( a ) and in a control subject ( b ) .", "3distribution of baseline density values in bd patients and controls in cerebral and extracranial arteriesfig .", "4distribution of time to peak in cerebral and extracranial arteries in bd patients and controlsfig .", "6distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls time ", "density curves in a bd patient ( a ) and in a control subject ( b ) .", "time to peak ( ttp ) enhancement distribution of baseline density values in bd patients and controls in cerebral and extracranial arteries distribution of time to peak in cerebral and extracranial arteries in bd patients and controls distribution of delay of cerebral peak values in bd patients and controls distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls for extracranial arteries , tdcs represented a similar shape in both groups .", "however , we observed higher enhancement in bd patients at 20 , 30 , and 40  s ( see table  3 ) .", "peak enhancement was significantly higher and earlier in bd patients compared to controls ( p  =  0.0087 and p  <  0.0001 , respectively ) ( see figs .  4 and 6 ) .", "the intensity of peak enhancement in cerebral arteries in relation to extracranial arteries expressed as c / e peak ratio appeared to be significantly lower in bd patients in comparison to the control group ( 0.31 vs. 0.87 ; p  <  0.0001 ) ( see fig . ", "7).table 3variables calculated from dynamic ctp series in bd patients vs. controlsparameterbd ( n  =  30)controls ( n  =  30 ) \n p valueenhancement at 20  s ( hu )  cerebral artery7.5 ( 24)41 ( 25)<0.0001  extracranial artery53.5 ( 40)41.5 ( 30)nsenhancement at 30  s ( hu )  cerebral artery18.5 ( 14)25 ( 19)ns  extracranial artery36 ( 23)26 ( 20)0.0225enhancement at 40  s ( hu )  cerebral artery20 ( 15)16.5 ( 8)ns  extracranial artery36.5 ( 18)17 ( 10)<0.0001values expressed as median ( interquartile range ) \n ns not significantfig", ". 7distribution of cerebral / extracranial peak ratio values in bd patients and controls variables calculated from dynamic ctp series in bd patients vs. controls values expressed as median ( interquartile range ) distribution of cerebral / extracranial peak ratio values in bd patients and controls analyzing the influence of demographic and clinical features on dynamic ct parameters , we revealed significant differences in time to peak and delay of peak enhancement in cerebral arteries between subgroups of bd patients with and without craniectomy . in the subgroup with craniectomy , time to peak and delay of cerebral peak were shorter in comparison to the subgroup without craniectomy ( see table  4 ) .", "a statistically insignificant trend was found in the analysis of tdcs in bd patients with subarachnoid hemorrhage ( sah ) and intracerebral hemorrhage ( ich ) .", "we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich ( see table  5 ) .", "we did not observe any significant differences between ct parameters in relation to sex or examined artery.table 4variables calculated from dynamic ctp series in bd patients without vs. with craniectomyparameterbd patients without craniectomy ( n  =  11)bd patients with craniectomy ( n  =  19 ) \n p valuetime to peak in cerebral artery ( s)34 ( 6)26 ( 9)0.007delay of cerebral peak ( s)18 ( 8)13 ( 9)0.04values expressed as median ( interquartile range)table 5variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhageparameterbd patients with ich ( n  =  16)bd patients with sah ( n  =  11 ) \n p valuetime to peak in cerebral artery ( s)27.5 ( 10)33 ( 9)0.34 ( ns)delay of cerebral peak ( s)13.5 ( 8)18 ( 13)0.16 ( ns)values expressed as median ( interquartile range ) \n ns not significant variables calculated from dynamic ctp series in bd patients without vs. with craniectomy values expressed as median ( interquartile range ) variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhage values expressed as median ( interquartile range ) in all 30 bd patients , ctp revealed zero values of cbf and cbv in all rois .", "stasis filling pattern was observed in 14 ( 46.7  % ) and non - filling in 16 ( 53.3  % ) cases . in 37", "excluded bd patients , angiography revealed a non - filling pattern in all cases ; no stasis filling was noted ."], ["the term stasis filling for describing a delayed , weak , and persistent intracranial opacification in patients with bd was used for the first time by kricheff et al . in 1978 .", "munari et al . reported stasis filling in 5  % ( one out of 20 ) , braun et al . in 11  % ( 15/140 ) , bradac et al . in 12.5  % ( two out of 16 ) , kricheff et al . in 15  % ( three out of 20 ) , and savard et al . in 28  % ( nine out of 32 ) of cases [ 2 , 69 ] .", "noted stasis filling in 43  % ( six out of 14 ) and welschehold et al . in 59  % ( 37/63 ) of cases [ 3 , 10 ] .", "stasis filling , as specific angiographic pattern of cerebral circulatory arrest , is a consequence of two major factors : raised intracranial pressure ( icp ) and high cerebrovascular resistance ( cvr ) .", "they both cause reduction of cbf , according to poiseuille s law : cbf  =  cpp / cvr  =  ( mabp    icp)/cvr , where cpp stands for cerebral perfusion pressure . increased cvr in extreme cerebral hemodynamic disturbances", "was revealed in studies using a transcranial doppler ( tcd ) [ 11 , 12 ] .", "this is caused mainly by altered cerebral autoregulation mechanisms , which are sufficient to preserve almost constant cbf when cpp is above 50  mmhg .", "cessation of capillary circulation is consistent with cerebral circulatory arrest while proximal arterial segments are still patent . at this stage", ", tcd frequently shows  to and fro  or narrow systolic spike patterns , which reflect blood movement [ 11 , 12 ] . in such circumstances ,", "heartbeat driven slow propagation of contrast column in proximal cerebral arteries is possible . in the present study ,", "recently revealed a potential efficacy of ctp for assessing brain death . in this material ,", "ctp detected complete absence of brain perfusion reflected by zero values of cbf and cbv in all cases .", "these findings clearly show that stasis filling is not an indicator of any residual brain perfusion but only ineffective propagation of contrast in cerebral vessels ; thus , it does not preclude diagnosis of brain death .", "ctp results are complementary to the recent report of selcuk et al . , which revealed global reduction of adc consistent with necrosis of neurons using diffusion - weighted mri technique .", "the findings showed two features of stasis filling phenomenon : delay and weakness of intracranial opacification .", "tdcs for cerebral arteries in bd patients were characterized by significantly longer times to peak ( median , 32  s ) compared to controls ( 21  s ) . moreover", ", intracranial peak enhancement occurred with a median delay of 14.5  s to extracranial peak .", "this is consistent with observations in cta studies , in which intracranial filling in bd patients is usually detected in the late phase ( performed 60  s after contrast injection ) and very rarely in the early phase of scanning [ 3 , 10 , 17 ] .", "correlation with ctp results shows that such delayed vascular opacification does not provide any brain perfusion , thus can not preclude diagnosis of brain death .", "typical cerebral tdc in bd patients was a flat curve with a low peak ( median value of 34.5  hu ) ; about three times lower compared to the peak in the extracranial artery .", "in contrast , extra- and intracranial tdcs in the control group represented steep and narrow gaussian curves , and both peaks were of similar height .", "such low peak values could be the reason of lower detectability of stasis filling by catheter angiography compared to ct ( 14/67  =  21  % vs. 30/67  =  45  % cases ) observed in the present study .", "these findings are consistent with results of others as was presented in the first paragraph of discussion .", "also observed underestimation of intracranial filling in catheter angiography compared to cta in four out of seven cases .", "although , a larger amount of contrast was used in dynamic ct than in catheter angiography ( 50 vs. 30  ml ) , but in angiography , it was injected intra - arterially , which minimizes the effect of dilution .", "this discrepancy could be a result of advanced reconstruction algorithms , systems of noise reduction , and signal amplification used in ct scanners , which reduce noise , improve spatial resolution , and low contrast detectability", ". the other explanation could be the time sequence in performing ctp and catheter angiography .", "angiography was carried out with a delay from 15  min to 3  h after ctp . during this period", ", slow rising icp could stop the propagation of contrast at the level of skull base . analyzing the influence of demographic and clinical features on tdc s parameters", ", we found a statistically significant trend towards shorter time to peak and shorter delay of peak in the cerebral arteries of bd patients with craniectomy compared to those without it .", "moreover , the incidence of craniectomy was much lower in bd patients excluded from the study due to lack of intracranial opacification ( four out of 37  =  11  % ) compared to included patients who presented intracranial filling ( 19/30  =  63  % ) .", "several authors previously reported a relationship between skull defect and preserved intracranial filling in bd patients [ 7 , 18 , 19 ] .", "this can be explained by a decreased icp enabling propagation of contrast in the cerebral vessels .", "a statistically insignificant trend was found in the analysis of tdcs in bd patients with sah and intracerebral hemorrhage .", "we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich .", "this difference was probably caused by vasospasm after sah , which independently increases cvr and additionally contributes to slowing down the propagation of contrast ."], ["the major drawback of this study is a consequence of performing ctp shortly after cta in bd patients .", "the influence of residual contrast injected for cta was reflected by higher baseline density in the cerebral and extracranial arteries , higher and earlier peak enhancement , and shorter time to peak in the extracranial vessels in bd patients compared to controls .", "however , this contrast contamination should not significantly change values of delay of cerebral peak or c / e peak ratio as they were calculated on the basis of both cerebral and extracranial tcds ."], ["in this study , we assessed the characteristic features of intracranial filling in bd patients  delay and weakness of cerebrovascular opacification .", "it led to the conclusion that delayed and weak opacification of cerebral arteries do not necessarily mean the presence of cerebral perfusion , thus does not preclude diagnosis of bd ."], [""]]}
{"article_id": "PMC3687400", "article_text": ["gastric cancer is the second most frequent neoplasm of the alimentary tract after the large intestine .", "5,103 people in poland were affected by it in 2008 . the case - to - death ratio of around 1 indicates unfavourable prognosis as to recovery from this disease [ 1 , 2 ] .", "this poor result is determined by the fact that it is rarely ( only in around 8% ) detected in the form of early gastric cancer , in the mildly symptomatic or asymptomatic phase . in most patients", "it is diagnosed at a higher degree than the 1st degree of disease progression and its classic symptoms are weight loss , continuous and dull pain in the epigastrium , loss of appetite , nausea , vomiting and chronic bleeding [ 3 , 4 ] .", "the existence of gastric cancer metastasis to the ureter has been described twice in the literature to date .", "a female patient , age 67 , was diagnosed at the district hospital ( 22.04 - 30.04.2010 ) because of intensified symptoms of left - sided renal colic . based on the conducted usg and single - phase computed tomography tests of the abdominal cavity and the pelvis , dilation of the ureter", "was found because of its infiltration by a pathological focus with the dimensions of 28 mm  15 mm .", "another lesion was located nearby at the level of the left iliac muscle  30 mm  27 mm  20 mm , adhering to the sigmoid colon .", "tissue infiltration of the pelvis minor wall was found descending in the direction of the left appendages .", "colonoscopy was conducted and in this test the large intestine was described without pathology , while the gynaecological usg test confirmed the presence of fluid in the pelvis . during her stay the patient was treated with analgesic and diastolic medication and then referred for further treatment at the regional oncology centre . because of reported pain complaints , she was immediately admitted to the department of oncological surgery on the day of her visit to the outpatient clinic ( 04.05.2010 ) .", "the urologist consulting the patient indicated the possibility of kidney damage due to ureteral obstruction with recommendation for an accelerated operation .", "therefore , pre - operative diagnostics were not extended beyond the tests received from the district hospital .", "the patient underwent surgery on 10.05.2010 and intraoperatively , besides the expected neoplastic tumour of the left ovary with infiltration of the ureter , numerous neoplastic foci were also found : a sigmoid colon tumour , a caecum tumour , a tumour of the body of the stomach and two single tumours in the omentum .", "because of the resectability of the neoplastic foci described above , the operation plan adopted earlier was changed and the following were performed in succession with palliative intention : partial gastrectomy by the rydygier method , right - sided hemicolectomy , left ovariectomy and sigmoid resection . after restoring the continuity of the alimentary tract ,", "the tumour occluded the lumen , which is why segmental ureterectomy was performed with end - to - end anastomosis over a pigtail catheter .", "the patient passed the post - operative period without complications , except a two - day fever .", "because of the expected alimentary tract failure , parenteral nutrition was included on the 1st day after the procedure and blood deficits were supplemented with 2 units of erythrocyte mass and 7 units of plasma .", "on the 9th day after the operation , the patient was discharged home in a good general condition with recommendations for further treatment .", "the received postoperative histopathological protocol indicated that the stomach was the origin of the neoplastic process .", "to lauren ) , type i ( acc . to goseki ) , of a stomach adenocarcinoma with a g2 malignancy degree , with occupation of the whole thickness of the stomach wall , with the following immunohistochemical characteristics : ck7(+++ ) , ck20(++ ) , ca125(+ ) , mucicarmine(+ ) .", "neoplastic infiltrations along nerves and neoplastic embolisms of blood vessels , as well as metastatic foci in the greater omentum , were observed in the specimen . because of the palliative partial gastrectomy type , only 3 lymph nodes were described in this specimen and all of them contained neoplastic cells . besides the above", ", histopathological confirmations were obtained of the metastatic character of the foci in the ovary , caecum and sigmoid colon .", "the cancer occupied large and small intestine walls without infiltration of the mucous membrane and the immunohistochemical characteristics were identical as for the primary lesion . according to the above protocol , excision within tissues with a healthy margin of these organs was achieved .", "the widening of the left ureter is visible ( marked by arrow ) focus of metastatic carcinoma in the ureter wall", ". microscopic section , he staining , magnification 40 neoplastic invasion in the ureter wall .", "microscopic section , he staining , magnification 100 it is interesting biologically and constitutes the basis for this paper that the ureteral tumour was described by the pathologists not as a neoplastic infiltration encroaching on the ureter , but as a metastatic focus to the ureteral wall .", "after the operative treatment , the patient was qualified for palliative chemotherapy and received it from 28.07.2010 .", "the first treatment course was according to the eox regimen ( oxaliplatin and capecitabine ) , but because of the occurrence of neutropenic fever the regimen was changed to pf ( cisplatin and 5-fluorouracil ) .", "she again received only one course and the treatment was changed once more because newly formed metastatic foci in the liver were located in imaging tests .", "next , three chemotherapy courses were administered according to the folfiri regimen ( irinotecan , leucovorin and 5-fluorouracil ) , but only until 11.11.2010 because due to progression of changes in the liver described in examinations the chemotherapy was discontinued and the patient was qualified for symptomatic treatment . according to our knowledge", "treatment of gastric cancer in the disseminated phase of the disease aims at extension of life and achieving a good palliative effect .", "the recommended methods , allowing the above to be achieved , are chemotherapy or combination radiochemotherapy . in an advanced stage ,", "palliative operations on this organ are performed for life reasons and serve to eliminate complications such as bleeding , perforation or obstruction of the organ .", "it is indicated that the above complications forced surgical intervention in 1/4 of the patients previously disqualified from operative treatment because of the presence of metastatic foci . according to literature data , patients operated on by palliative resection in the disseminated phase of the disease achieve a survival time of between 9 and 15 months . the survival time is limited by the number of metastatic foci ; when their number is higher than two foci , no statistically significant differences are observed in this scope", ". the value of these procedures is increasing because of the reported low perioperative mortality and the observed significant improvement in the further quality of life for these patients [ 513 ] .", "it seems that the operative procedure conducted in the presented patient allowed a good palliative effect to be achieved along with loss of severe colic and maintenance of kidney function .", "the literature often presents , as characteristic of gastric cancer , blood - borne metastasis to the ovary , termed a krukenberg tumour .", "this is a term generally defining metastasis to the ovary , mainly gastric cancer and next colon cancer .", "metastases from other organs , such as the lungs , the mammary gland and the uterus , are also possible , though much rarer .", "krukenberg tumours are encountered in the course of 2 - 4% of disseminated neoplastic processes .", "this is a negative prognostic factor , with varying median survival time after its diagnosis : 12 - 13 months in the course of gastric cancer compared to 17 - 29 months in the course of large intestine cancer .", "this undoubtedly results from biological differences in the course of these neoplasms [ 1423 ] . in summary ,", "the available literature sources have described this only twice to date ( in 1976 and 2000 ) .", "however , nephroureterectomy was conducted in these quoted cases [ 24 , 25 ] . in the presented patient", "the kidney was spared and healing of the ureter after end - to - end suture was achieved .", "the dissemination of a neoplasm with extremely rarely encountered symptomatology described above is an interesting experience which we wanted to share .", "the beginning of the symptomatic disease as a left - sided renal colic is , in itself , a previously unreported case ."], "abstract_text": ["<S> the typical symptoms of advanced cancer of the stomach are well known in clinical practice . </S>", "<S> the presented case concerns a patient with symptoms of left - sided renal colic , caused by a malignant tumour involving the ureter , which was diagnosed with a ct scan . </S>", "<S> the multifocal process , involving the stomach , two parts of the colon , the left ovary and the side of the pelvis , was confirmed only during surgery.the resection or partial resection of the above - mentioned organs involved by the malignant process and reconstruction of the alimentary tract as well as the ureter were performed at time of this operation . </S>", "<S> the patient 's recovery was without any complications . </S>", "<S> the histopathological findings support the diagnosis of this malignant process as disseminated stomach cancer.in the available literature only two cases of stomach cancer metastasis to the ureter have been described . in both cited examples resection of the ureter with nephrectomy was performed . </S>", "<S> the review of the literature supports the value of stomach palliative resection in prolonging life and improving quality of life . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["gastric cancer is the second most frequent neoplasm of the alimentary tract after the large intestine .", "5,103 people in poland were affected by it in 2008 . the case - to - death ratio of around 1 indicates unfavourable prognosis as to recovery from this disease [ 1 , 2 ] .", "this poor result is determined by the fact that it is rarely ( only in around 8% ) detected in the form of early gastric cancer , in the mildly symptomatic or asymptomatic phase . in most patients", "it is diagnosed at a higher degree than the 1st degree of disease progression and its classic symptoms are weight loss , continuous and dull pain in the epigastrium , loss of appetite , nausea , vomiting and chronic bleeding [ 3 , 4 ] .", "the existence of gastric cancer metastasis to the ureter has been described twice in the literature to date ."], ["a female patient , age 67 , was diagnosed at the district hospital ( 22.04 - 30.04.2010 ) because of intensified symptoms of left - sided renal colic . based on the conducted usg and single - phase computed tomography tests of the abdominal cavity and the pelvis , dilation of the ureter", "was found because of its infiltration by a pathological focus with the dimensions of 28 mm  15 mm .", "another lesion was located nearby at the level of the left iliac muscle  30 mm  27 mm  20 mm , adhering to the sigmoid colon .", "tissue infiltration of the pelvis minor wall was found descending in the direction of the left appendages .", "colonoscopy was conducted and in this test the large intestine was described without pathology , while the gynaecological usg test confirmed the presence of fluid in the pelvis . during her stay the patient was treated with analgesic and diastolic medication and then referred for further treatment at the regional oncology centre . because of reported pain complaints , she was immediately admitted to the department of oncological surgery on the day of her visit to the outpatient clinic ( 04.05.2010 ) .", "the urologist consulting the patient indicated the possibility of kidney damage due to ureteral obstruction with recommendation for an accelerated operation .", "therefore , pre - operative diagnostics were not extended beyond the tests received from the district hospital .", "the patient underwent surgery on 10.05.2010 and intraoperatively , besides the expected neoplastic tumour of the left ovary with infiltration of the ureter , numerous neoplastic foci were also found : a sigmoid colon tumour , a caecum tumour , a tumour of the body of the stomach and two single tumours in the omentum .", "because of the resectability of the neoplastic foci described above , the operation plan adopted earlier was changed and the following were performed in succession with palliative intention : partial gastrectomy by the rydygier method , right - sided hemicolectomy , left ovariectomy and sigmoid resection . after restoring the continuity of the alimentary tract ,", "the tumour occluded the lumen , which is why segmental ureterectomy was performed with end - to - end anastomosis over a pigtail catheter .", "the patient passed the post - operative period without complications , except a two - day fever .", "because of the expected alimentary tract failure , parenteral nutrition was included on the 1st day after the procedure and blood deficits were supplemented with 2 units of erythrocyte mass and 7 units of plasma .", "on the 9th day after the operation , the patient was discharged home in a good general condition with recommendations for further treatment .", "the received postoperative histopathological protocol indicated that the stomach was the origin of the neoplastic process .", "to lauren ) , type i ( acc . to goseki ) , of a stomach adenocarcinoma with a g2 malignancy degree , with occupation of the whole thickness of the stomach wall , with the following immunohistochemical characteristics : ck7(+++ ) , ck20(++ ) , ca125(+ ) , mucicarmine(+ ) .", "neoplastic infiltrations along nerves and neoplastic embolisms of blood vessels , as well as metastatic foci in the greater omentum , were observed in the specimen . because of the palliative partial gastrectomy type , only 3 lymph nodes were described in this specimen and all of them contained neoplastic cells . besides the above", ", histopathological confirmations were obtained of the metastatic character of the foci in the ovary , caecum and sigmoid colon .", "the cancer occupied large and small intestine walls without infiltration of the mucous membrane and the immunohistochemical characteristics were identical as for the primary lesion . according to the above protocol , excision within tissues with a healthy margin of these organs was achieved .", "the widening of the left ureter is visible ( marked by arrow ) focus of metastatic carcinoma in the ureter wall", ". microscopic section , he staining , magnification 40 neoplastic invasion in the ureter wall .", "microscopic section , he staining , magnification 100 it is interesting biologically and constitutes the basis for this paper that the ureteral tumour was described by the pathologists not as a neoplastic infiltration encroaching on the ureter , but as a metastatic focus to the ureteral wall .", "after the operative treatment , the patient was qualified for palliative chemotherapy and received it from 28.07.2010 .", "the first treatment course was according to the eox regimen ( oxaliplatin and capecitabine ) , but because of the occurrence of neutropenic fever the regimen was changed to pf ( cisplatin and 5-fluorouracil ) .", "she again received only one course and the treatment was changed once more because newly formed metastatic foci in the liver were located in imaging tests .", "next , three chemotherapy courses were administered according to the folfiri regimen ( irinotecan , leucovorin and 5-fluorouracil ) , but only until 11.11.2010 because due to progression of changes in the liver described in examinations the chemotherapy was discontinued and the patient was qualified for symptomatic treatment . according to our knowledge"], ["treatment of gastric cancer in the disseminated phase of the disease aims at extension of life and achieving a good palliative effect .", "the recommended methods , allowing the above to be achieved , are chemotherapy or combination radiochemotherapy . in an advanced stage ,", "palliative operations on this organ are performed for life reasons and serve to eliminate complications such as bleeding , perforation or obstruction of the organ .", "it is indicated that the above complications forced surgical intervention in 1/4 of the patients previously disqualified from operative treatment because of the presence of metastatic foci . according to literature data , patients operated on by palliative resection in the disseminated phase of the disease achieve a survival time of between 9 and 15 months . the survival time is limited by the number of metastatic foci ; when their number is higher than two foci , no statistically significant differences are observed in this scope", ". the value of these procedures is increasing because of the reported low perioperative mortality and the observed significant improvement in the further quality of life for these patients [ 513 ] .", "it seems that the operative procedure conducted in the presented patient allowed a good palliative effect to be achieved along with loss of severe colic and maintenance of kidney function .", "the literature often presents , as characteristic of gastric cancer , blood - borne metastasis to the ovary , termed a krukenberg tumour .", "this is a term generally defining metastasis to the ovary , mainly gastric cancer and next colon cancer .", "metastases from other organs , such as the lungs , the mammary gland and the uterus , are also possible , though much rarer .", "krukenberg tumours are encountered in the course of 2 - 4% of disseminated neoplastic processes .", "this is a negative prognostic factor , with varying median survival time after its diagnosis : 12 - 13 months in the course of gastric cancer compared to 17 - 29 months in the course of large intestine cancer .", "this undoubtedly results from biological differences in the course of these neoplasms [ 1423 ] . in summary ,", "the available literature sources have described this only twice to date ( in 1976 and 2000 ) .", "however , nephroureterectomy was conducted in these quoted cases [ 24 , 25 ] . in the presented patient", "the kidney was spared and healing of the ureter after end - to - end suture was achieved .", "the dissemination of a neoplasm with extremely rarely encountered symptomatology described above is an interesting experience which we wanted to share .", "the beginning of the symptomatic disease as a left - sided renal colic is , in itself , a previously unreported case ."]]}
{"article_id": "PMC4676670", "article_text": ["eosinophilic gastritis is an extremely rare disease that is characterized by eosinophilic infiltration of the various layers of the gastrointestinal tract in the absence of any definite causes of eosinophilia .", "patients with eosinophilic gastritis have diverse symptoms , including abdominal pain , emesis , abdominal distension , and weight loss .", "these symptoms are associated with eosinophilic infiltration of the various layers of the gastrointestinal tract .", "the disease has to be distinguished from generalized eosinophilic disorder presenting with involvement of other organs .", "a 52-year - old female patient visited another hospital because of epigastric pain and tenderness .", "she had no history of allergic diseases such as eczema or atopy , or food or drug allergies .", ", she had undergone esophagogastroduodenoscopy ( egd ) , which revealed multiple focal ulcerative lesions with diffuse discoloration and edematous change of the rugae in the gastric fundus , cardia , and upper body ( fig .", "the symptoms had been recalcitrant to treatment with proton pump inhibitors , fasting , and fluid .", "she had reported no nausea , vomiting , hematemesis , or melena . on admission ,", "her blood pressure was 110/60 mm hg , pulse rate 76 beats per minute , respiratory rate 22 breaths per minute , and body temperature 36.2. physical examination revealed tenderness of the epigastric area .", "her white blood cell count was 22,770/mm with markedly increased eosinophils ( 5,009/mm , 22% ) .", "the c - reactive protein concentration was 13.95 mg / dl . her other blood chemistry test results were normal .", "the tests for viral markers included hepatitis a , b , and c virus and human immunodeficiency virus ; those for autoimmune antibodies included anti - nuclear , anti - double stranded dna , and anti - neutrophilic cytoplasmic antibodies ; and those for tumor markers included -fetoprotein , carbohydrate antigen 19 - 9 , and carcinoembryonic antigen .", "the serological test for toxocara antibodies ( igg ) was positive , whereas those for echinococcus , paragonimus westermani , sparganumi , and trichinella antibodies were negative .", "the chest and abdomen radiographic examinations were normal . to investigate the cause of the epigastric pain and tenderness ,", "abdominal computed tomography was performed , which revealed severe edematous wall thickening with focal localized low attenuation of the fundus and cardia of the stomach ( fig .", "she underwent a repeated egd , which showed diffuse necrotic change in the fundus , cardia , and upper body ( fig .", "a biopsy specimen was obtained during egd ; a rapid urease test ( clotest ) revealed no evidence of helicobacter pylori . on histopathologic evaluation ,", "the gastric surface was found to be eroded and the underlying lamina propria showed dense eosinophilic infiltration .", "she was treated with empirical intravenous antibiotics ( cefoperazone and metronidazole ) immediately after her transfer because infectious gastritis was not ruled out .", "although she was treated with a broad - spectrum anthelmintic ( albendazole ) , antibiotics , proton pump inhibitor , and prokinetic agent during the next 5 days , her symptoms became worse .", "eosinophilic gastritis was diagnosed according to the clinical pictures , laboratory findings , and endoscopic findings .", "she was immediately started on methylprednisolone ( 62.5 mg / day ; this dose was maintained for 7 days ) .", "therefore , she underwent a repeated short - term follow - up egd that showed regenerative epithelial tissue with peeling off , of the necrotic tissue ( fig .", "she underwent a repeated egd that showed the replacement of white color scar tissue ( fig .", "currently , she is being treated with 30 mg / day prednisone and is showing considerable clinical improvement .", "eosinophilic gastroenteritis is an uncommon and rarely reported disorder characterized by eosinophilic infiltration of the various tissue layers of the digestive tract in the absence of any definite causes of eosinophilia , without eosinophilic infiltration in other organs .", "the diagnosis of eosinophilic gastroenteritis is based on the following criteria : the presence of gastrointestinal symptoms , histological presentation of eosinophilic infiltration in tissue layers of the digestive tract , presence of high eosinophil count in ascites , and no evidence of parasitic infection or eosinophilic involvement of extraintestinal organs .", "eosinophilic gastroenteritis can involve any portion of the gastrointestinal tract from the esophagus to the rectum , and the stomach is the most commonly involved organ , especially the antrum . in eosinophilic gastritis ,", "the endoscopic features are rather extensive : rugal fold thickening , erythema , friability , nodularity , gastric outlet obstruction , gastric ulcer , and even a normal mucosa .", "although not clearly defined , the common pathophysiological mechanism of this disease is associated with eosinophilic infiltration and degranulation in specific tissue layers of the digestive tract .", "this eosinophilic recruitment and activation regulated by diverse cytokines is a part of the host immune mechanism in the gastrointestinal mucosa ; however , it can be a type of serious allergic or inflammatory reaction in the deeper tissue layers of the gastrointestinal tract . although there is no treatment consensus on eosinophilic gastroenteritis ,", "several studies report good results with steroids in dosages from 20 to 40 mg / day , for 6 to 8 weeks [ 7 - 10 ] . in some case studies ,", "leukotriene modifiers such as montelukast or mast cell stabilizers such as sodium cromoglycate have been proposed to be helpful for symptomatic improvement .", "antihistamines such as ketotifen or immunosuppressants such as mycophenolate mofetil are also used in the treatment of eosinophilic gastroenteritis ; however , their therapeutic effects are not clear and would require more studies .", "although rare , eosinophilic gastritis should be considered in the differential diagnosis of patients with gastrointestinal symptoms and peripheral blood eosinophilia .", "lymphoma of the stomach , gastric cancer , and crohn disease involving the stomach may demonstrate endoscopic features similar to those of eosinophilic gastritis .", "gastrointestinal parasitic infection should be considered in patients with abdominal discomfort , weight loss , and peripheral eosinophilia . in particular , infestations by hookworms ,", "ascaris , strongyloides , toxocara , trichuris , and intestinal capillaria should be considered in patients from endemic areas . in this case , the concentration of the antigen - specific ige to the a. simplex was 0.57", "moreover , the serology for toxocara antibodies ( igg ) was positive . however , this is not clinically significant in korea because koreans often consume raw fish , which causes repeated exposures to anisakis .", "in addition , she had not had contact with any animals , including dogs and cats , at least for several years .", "moreover , she was treated with albendazole for 5 days , and her symptoms had become worse . after steroid treatment , the symptoms disappeared and the eosinophil count decreased to the reference range .", "moreover , the follow - up egd showed regenerative epithelial tissue with peeling off , of the necrotic tissue", ". she had undergone several egds that showed diffuse necrotic change in the fundus , cardia , and upper body .", "the etiology includes thromboembolism and occlusion of major arterial supply , ingestion of corrosive agents , volvulus of the stomach , endoscopic hemostatic injections , and infectious gastritis . in this case", "thus , the possible cause of gangrene could be infection , and she was treated with empirical intravenous antibiotics ( cefoperazone and metronidazole ) .", "however , she was treated with broad - spectrum antibiotics during the next 5 days , and her symptoms became worse .", "the necrotic portion of the gastric high body is very vulnerable site of retching injury .", "this retching injury is called prolapse gastropathy syndrome , a clinical syndrome involving the invagination of part of the gastric mucosa into the lower esophagus .", "direct trauma to the mucosa occurs when the gastric mucosa becomes incarcerated through the lower esophageal sphincter .", "in addition , the endoscopic findings and histopathologic results were not compatible to prolapse gastropathy syndrome . on the basis of the clinical picture , laboratory findings , and therapeutic results , we concluded the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis in our patient .", "this case highlights the reality of eosinophilic gastritis presenting as necrotizing gastritis , and that endoscopy and histopathological examination of the biopsies are the most useful tools for the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis .", "eosinophilic gastritis should be considered in the differential diagnosis in patients with necrotic gastritis who do not respond to empirical treatment ."], "abstract_text": ["<S> eosinophilic gastroenteritis is very rare disorder that is characterized by eosinophilic infiltration of the gastrointestinal tract in the absence of any definite causes of eosinophilia . </S>", "<S> it is associated with various clinical gastrointestinal manifestations , and depends on the involved layer and site . </S>", "<S> we report a case of eosinophilic gastritis presenting with severe necrosis . </S>", "<S> the symptoms disappeared immediately after beginning steroid treatment , and the eosinophil count decreased to the reference range . </S>", "<S> the patient showed eosinophilic gastritis characterized by necrotic change such as necrotizing gastritis . </S>", "<S> it is a unique presentation of eosinophilic gastritis . to the best of our knowledge , </S>", "<S> no case of eosinophilic gastritis characterized by necrotic change such as necrotizing gastritis has been previously reported in korea . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["eosinophilic gastritis is an extremely rare disease that is characterized by eosinophilic infiltration of the various layers of the gastrointestinal tract in the absence of any definite causes of eosinophilia .", "patients with eosinophilic gastritis have diverse symptoms , including abdominal pain , emesis , abdominal distension , and weight loss .", "these symptoms are associated with eosinophilic infiltration of the various layers of the gastrointestinal tract .", "the disease has to be distinguished from generalized eosinophilic disorder presenting with involvement of other organs ."], ["a 52-year - old female patient visited another hospital because of epigastric pain and tenderness .", "she had no history of allergic diseases such as eczema or atopy , or food or drug allergies .", ", she had undergone esophagogastroduodenoscopy ( egd ) , which revealed multiple focal ulcerative lesions with diffuse discoloration and edematous change of the rugae in the gastric fundus , cardia , and upper body ( fig .", "the symptoms had been recalcitrant to treatment with proton pump inhibitors , fasting , and fluid .", "she had reported no nausea , vomiting , hematemesis , or melena . on admission ,", "her blood pressure was 110/60 mm hg , pulse rate 76 beats per minute , respiratory rate 22 breaths per minute , and body temperature 36.2. physical examination revealed tenderness of the epigastric area .", "her white blood cell count was 22,770/mm with markedly increased eosinophils ( 5,009/mm , 22% ) .", "the c - reactive protein concentration was 13.95 mg / dl . her other blood chemistry test results were normal .", "the tests for viral markers included hepatitis a , b , and c virus and human immunodeficiency virus ; those for autoimmune antibodies included anti - nuclear , anti - double stranded dna , and anti - neutrophilic cytoplasmic antibodies ; and those for tumor markers included -fetoprotein , carbohydrate antigen 19 - 9 , and carcinoembryonic antigen .", "the serological test for toxocara antibodies ( igg ) was positive , whereas those for echinococcus , paragonimus westermani , sparganumi , and trichinella antibodies were negative .", "the chest and abdomen radiographic examinations were normal . to investigate the cause of the epigastric pain and tenderness ,", "abdominal computed tomography was performed , which revealed severe edematous wall thickening with focal localized low attenuation of the fundus and cardia of the stomach ( fig .", "she underwent a repeated egd , which showed diffuse necrotic change in the fundus , cardia , and upper body ( fig .", "a biopsy specimen was obtained during egd ; a rapid urease test ( clotest ) revealed no evidence of helicobacter pylori . on histopathologic evaluation ,", "the gastric surface was found to be eroded and the underlying lamina propria showed dense eosinophilic infiltration .", "she was treated with empirical intravenous antibiotics ( cefoperazone and metronidazole ) immediately after her transfer because infectious gastritis was not ruled out .", "although she was treated with a broad - spectrum anthelmintic ( albendazole ) , antibiotics , proton pump inhibitor , and prokinetic agent during the next 5 days , her symptoms became worse .", "eosinophilic gastritis was diagnosed according to the clinical pictures , laboratory findings , and endoscopic findings .", "she was immediately started on methylprednisolone ( 62.5 mg / day ; this dose was maintained for 7 days ) .", "therefore , she underwent a repeated short - term follow - up egd that showed regenerative epithelial tissue with peeling off , of the necrotic tissue ( fig .", "she underwent a repeated egd that showed the replacement of white color scar tissue ( fig .", "currently , she is being treated with 30 mg / day prednisone and is showing considerable clinical improvement ."], ["eosinophilic gastroenteritis is an uncommon and rarely reported disorder characterized by eosinophilic infiltration of the various tissue layers of the digestive tract in the absence of any definite causes of eosinophilia , without eosinophilic infiltration in other organs .", "the diagnosis of eosinophilic gastroenteritis is based on the following criteria : the presence of gastrointestinal symptoms , histological presentation of eosinophilic infiltration in tissue layers of the digestive tract , presence of high eosinophil count in ascites , and no evidence of parasitic infection or eosinophilic involvement of extraintestinal organs .", "eosinophilic gastroenteritis can involve any portion of the gastrointestinal tract from the esophagus to the rectum , and the stomach is the most commonly involved organ , especially the antrum . in eosinophilic gastritis ,", "the endoscopic features are rather extensive : rugal fold thickening , erythema , friability , nodularity , gastric outlet obstruction , gastric ulcer , and even a normal mucosa .", "although not clearly defined , the common pathophysiological mechanism of this disease is associated with eosinophilic infiltration and degranulation in specific tissue layers of the digestive tract .", "this eosinophilic recruitment and activation regulated by diverse cytokines is a part of the host immune mechanism in the gastrointestinal mucosa ; however , it can be a type of serious allergic or inflammatory reaction in the deeper tissue layers of the gastrointestinal tract . although there is no treatment consensus on eosinophilic gastroenteritis ,", "several studies report good results with steroids in dosages from 20 to 40 mg / day , for 6 to 8 weeks [ 7 - 10 ] . in some case studies ,", "leukotriene modifiers such as montelukast or mast cell stabilizers such as sodium cromoglycate have been proposed to be helpful for symptomatic improvement .", "antihistamines such as ketotifen or immunosuppressants such as mycophenolate mofetil are also used in the treatment of eosinophilic gastroenteritis ; however , their therapeutic effects are not clear and would require more studies .", "although rare , eosinophilic gastritis should be considered in the differential diagnosis of patients with gastrointestinal symptoms and peripheral blood eosinophilia .", "lymphoma of the stomach , gastric cancer , and crohn disease involving the stomach may demonstrate endoscopic features similar to those of eosinophilic gastritis .", "gastrointestinal parasitic infection should be considered in patients with abdominal discomfort , weight loss , and peripheral eosinophilia . in particular , infestations by hookworms ,", "ascaris , strongyloides , toxocara , trichuris , and intestinal capillaria should be considered in patients from endemic areas . in this case , the concentration of the antigen - specific ige to the a. simplex was 0.57", "moreover , the serology for toxocara antibodies ( igg ) was positive . however , this is not clinically significant in korea because koreans often consume raw fish , which causes repeated exposures to anisakis .", "in addition , she had not had contact with any animals , including dogs and cats , at least for several years .", "moreover , she was treated with albendazole for 5 days , and her symptoms had become worse . after steroid treatment , the symptoms disappeared and the eosinophil count decreased to the reference range .", "moreover , the follow - up egd showed regenerative epithelial tissue with peeling off , of the necrotic tissue", ". she had undergone several egds that showed diffuse necrotic change in the fundus , cardia , and upper body .", "the etiology includes thromboembolism and occlusion of major arterial supply , ingestion of corrosive agents , volvulus of the stomach , endoscopic hemostatic injections , and infectious gastritis . in this case", "thus , the possible cause of gangrene could be infection , and she was treated with empirical intravenous antibiotics ( cefoperazone and metronidazole ) .", "however , she was treated with broad - spectrum antibiotics during the next 5 days , and her symptoms became worse .", "the necrotic portion of the gastric high body is very vulnerable site of retching injury .", "this retching injury is called prolapse gastropathy syndrome , a clinical syndrome involving the invagination of part of the gastric mucosa into the lower esophagus .", "direct trauma to the mucosa occurs when the gastric mucosa becomes incarcerated through the lower esophageal sphincter .", "in addition , the endoscopic findings and histopathologic results were not compatible to prolapse gastropathy syndrome . on the basis of the clinical picture , laboratory findings , and therapeutic results , we concluded the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis in our patient .", "this case highlights the reality of eosinophilic gastritis presenting as necrotizing gastritis , and that endoscopy and histopathological examination of the biopsies are the most useful tools for the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis .", "eosinophilic gastritis should be considered in the differential diagnosis in patients with necrotic gastritis who do not respond to empirical treatment ."]]}
{"article_id": "PMC2234247", "article_text": ["the skin is the largest organ of the body and protects the organism against external physical , chemical , and biological insults such as wounding , uvb radiation , and microorganisms .", "this major barrier resides in the upper layers of the epidermis ( for review see segre , 2006 ) . the epidermis is the upper part of the skin that is continuously renewed .", "the basal layer , or stratum basale , of the epidermis contains proliferating keratinocytes ( fig .", "1 ) . upon withdrawal from the cell cycle , these basal keratinocytes detach from the basement membrane and undergo a terminal differentiation program to become corneocytes in the outer layers of the epidermis .", "the cells reinforce their cytoskeletal keratin filament network , and adjacent cells interact via many desmosomes , a specialized type of cell junction , to resist physical trauma . in the stratum granulosum ,", "the keratinocytes become more flattened and express certain proteins such as profilaggrin and loricrin , which aggregate to form the typical keratohyalin granules of the stratum granulosum . in addition , lipids are produced and stored in lamellar bodies . at the final stage of differentiation , the keratinocytes lose their organelles , including the nucleus , and become the dead , flattened corneocytes of the stratum corneum . during cornification , proteins are cross - linked at the inner side of the cytoplasmic membrane to form a cornified envelope ( for review see candi et al . , 2005 ) . in the transitional layer between the stratum granulosum and the stratum corneum , lipids are extruded to form a water - repelling envelope around the cornified envelope , thereby assuring an adequate permeability barrier function of the mammalian epidermis .", "improper formation of these envelopes results in an impaired epidermal barrier that can not protect against dehydration , uvb , and infection .", "the signaling cascades involved in epidermal barrier formation are largely unknown , but the many proteases that seem to be involved are currently being intensively studied .", "see introduction for details . since the cloning of caspase-14 in the late nineties , it has become clear that this protease is a unique member of the caspase family .", "unlike apoptotic caspases , which evolved in common ancestors such as hydra , echinodermata , insects , nematodes , and chordates , caspase-14 has so far been found only in terrestrial mammals ( lamkanfi et al . , 2002 ) .", "in contrast to the ubiquitously expressed other members of the caspase family , caspase-14 is expressed and activated mainly in the epidermis and is absent from most other adult tissues ( eckhart et al .", "recently , caspase-14 was found to be involved in epidermal barrier formation ( denecker et al . , 2007 ) .", "in this review , we discuss current knowledge of the expression , regulation , and function of caspase-14 .", "the expression pattern of caspase-14 is unique among the caspases , as it is present mainly in cornifying epithelia , such as the epidermis , the hassall 's bodies of the thymus , and the forestomach of rodents ( lippens et al . , 2000 , 2005 ;", "in skin , caspase-14 is expressed only in the differentiating and cornifying layers of the epidermis and the hair follicle ( lippens et al . , 2000 ;", "this is consistent with the observation that , in vitro , caspase-14 is only expressed in differentiating but not in proliferating keratinocytes ( lippens et al .", "remarkably , nail matrix keratinocytes that differentiate into specialized nail corneocytes , the building blocks of the nail plate , do not express caspase-14 ( jager et al .", "in addition , caspase-14 is not expressed in the noncornifying keratinocytes of the sweat gland or the mouth epithelium ( lippens et al . , 2000 ;", "ultrastructural analysis demonstrated that spatial distribution of caspase-14 in the epidermis and hair follicles is strongly conserved among several mammalian species ( alibardi et al .", "caspase-14 was found to be associated with the nucleus , the keratohyalin granules , and the desmosomes , whereas in corneocytes , caspase-14 was found in the cytoplasm and was associated with corneodesmosomes ( a modified version of desmosomes ) and nuclear remnants .", "these observations suggested a role for caspase-14 in nuclear degradation during cornification , but nuclear degradation was not affected in caspase-14deficient mice ( denecker et al . , 2007 ) .", "the expression of caspase-14 in the hassall 's bodies of the thymus and in the forestomach of rodents is somewhat expected , as they are cornifying structures and express the typical late differentiation markers , such as profilaggrin and loricrin , which are also found in the epidermis ( laster and haynes , 1986 ; favre , 1989 ; jarnik et al . , 1996 ) .", "protein expression of caspase-14 has also been reported in several noncornifying tissues ( lippens et al . , 2003 ; krajewska et al . , 2004 , 2005 ; kam et al . , 2005", "however , these observations should be interpreted carefully , as we have recently shown that the reported expression of caspase-14 in such tissues can be the result of aspecific staining ( denecker et al . , 2007 ) .", "remarkably , so far caspase-14 has been found only in terrestrial mammals but not in birds or reptiles .", "whereas birds and reptiles have a stiff , dry , scaly epidermis , mammals have a soft stratum corneum because of the larger amounts of histidine - rich late differentiation markers ( e.g. , profilaggrin ; alibardi , 2003 ) .", "interestingly , profilaggrin is a direct substrate of caspase-14 ( denecker et al . , 2007 ) .", "this could indicate that the occurrence of a soft stratum corneum and the caspase-14 gene are associated during evolution .", "although the expression of caspase-14 is very restricted , little is known about the transcriptional regulation of its gene . in vitro", ", caspase-14 is only expressed when keratinocytes are forced to differentiate by growing them postconfluently or in suspension or by adding vitamin d3 ( eckhart et al .", "in contrast , adding ca at high concentrations to the medium , a method frequently used to induce differentiation , did not induce caspase-14 expression ( eckhart et al .", "retinoids , which suppress keratinocyte differentiation , down - regulate caspase-14 expression ( rendl et al . ,", "these results indicate that transcription factors that are specifically active during terminal differentiation are required to regulate caspase-14 expression . whether caspase-14 expression levels can be regulated at the posttranscriptional level is not known .", "down - regulation of several differentiation - associated genes by retinoids has been shown to be mediated by the transrepression of activator protein 1 ( ap-1)mediated gene activation ( fisher and voorhees , 1996 ) .", "indeed , the caspase-14 promoter contains at least two potential ap-1binding sites ( unpublished data ) .", "the green tea phenol ( )-epigallocatechin-3-gallate ( egcg ) is a potent activator of ap-1 and has been shown to up - regulate caspase-14 in a p38- and jnk - dependent way ( hsu et al . , 2005 , 2007 ) .", "ap-1 alone is probably not sufficient to drive caspase-14 expression because tnf and 12-o - tetradecanoyl - phorbol 13-acetate , two potent activators of ap-1 in keratinocytes ( arnott et al . , 2002 )", ", did not induce caspase-14 expression in keratinocytes ( lippens et al . , 2004 ) .", "differentiation - dependent expression of caspase-14 in keratinocytes could also result from a strong transcriptional repression in proliferating keratinocytes .", "this possibility is supported by the observation that mice deficient in nuclear receptor corepressor hairless ( hr ) had 510-fold higher levels of caspase-14 and profilaggrin mrna , starting from postnatal day 6 and progressing during development ( zarach et al . , 2004 ) .", "these alterations in gene expression were detected mainly in the keratinocytes of the utricle , an abnormal pouch - like structure at the upper part of the hair follicle . increased", "gene expression occurred before the morphologically distinct utricle could be identified , indicating that the up - regulation was probably a cause rather than a consequence of utricle formation", ". it would be interesting to analyze both caspase-14 activation and filaggrin processing in these mice .", "multiple mutant hr alleles in mice and in humans show phenotypic variations that include congenital hair loss , skin wrinkling , and papular rash ( cichon et al . , 1998 ;", "1998 ) . whether caspase-14 overexpression is important for the observed phenotypes could be addressed by generating epidermis - specific caspase-14 transgenic mice or by crossing the hr mice with caspase-14deficient mice .", "procaspases consist of a prodomain , a large subunit ( p20 ) , and a small subunit ( p10 ) .", "activation of caspases is induced by dimerization , ( auto-)proteolytic cleavage at asp residues , and/or conformational changes ( lamkanfi et al . , 2003 ) .", "so far , maturation of caspase-14 by proteolytic cleavage into p20 and p10 subunits has been consistently observed only in cornifying epithelia such as the epidermis and the rodent forestomach ( lippens et al . , 2000 ;", "although some investigators suggested that caspase-8 and -10 can activate caspase-14 in vitro ( ahmad et al .", ", 1998 ; van de craen et al . , 1998 ) , this could not be confirmed by others ( lippens et al .", "furthermore , caspase-14 is probably not proteolytically activated by a caspase in vivo , as other caspases are not activated during epidermal differentiation ( eckhart et al .", "2000 ; raymond et al . , 2007 ) , and caspase-14 is not processed at an aspartate residue like other caspases but is processed at ile in man and presumably at leu in the mouse ( chien et al . , 2002 ) .", "alignment of the protease - sensitive loop between the p20 and p10 subunits of the known mammalian procaspase-14 amino acid sequences ( fig .", "2 ) reveals a conserved hydrophobic patch that is n terminal of the caspase-14 cleavage site .", "this patch contains p1-preferred amino acids of elastase - like serine proteases , such as val , ala , leu , and ile ( mallory and travis , 1975 ; vered et al . , 1985 ; takahashi et al . ,", "1989 ) , suggesting that a serine protease with elastase - like properties could be involved in caspase-14 activation ( unpublished data ) . all together ,", "these data indicate that during skin homeostasis , the caspase-14activating protease is not a caspase , separating caspase-14 activation from the apoptotic and inflammatory caspase cascades that could be detrimental to epidermal integrity .", "the precise epidermal layer in which caspase-14 is processed and activated is unknown because antiserum specifically recognizing activated caspase-14 is not yet available .", "however , the following findings indicate that activation of caspase-14 occurs at the interface between the granular and cornified layers of the epidermis or early during cornification .", "first , both the proform and activated form of caspase-14 can be found in total epidermal extracts , whereas in the cornified layer only activated caspase-14 is found ( fischer et al . , 2004 ) .", "second , caspase-14 activation coincides with stratum corneum formation both during embryonic development and in organotypic skin cultures ( eckhart et al .", "this implies that the main biological function of caspase-14 is exercised in the stratum corneum , as proven by the phenotype of the caspase-14deficient mice ( see the next section ) .", "sequence analysis indicates that a hydrophobic patch in the protease - sensitive loop is conserved .", "only part of the alignment is shown here , including the c - terminal part of the p20 subunit , the protease - sensitive loop , and the n - terminal part of the p10 subunit .", "the darker the yellow , the more the amino acids are conserved between species . the catalytic qacrg box is delineated with a green box .", "the conserved hydrophobic patch is delineated with a red box , and the cleavage site in human caspase-14 is indicated with a red arrow .", "the alignment was performed using clustalw ( mega version 3.1 ; kumar et al . , 2004 ) and", "identification of caspase-14 substrates has been hampered for a long time by the unavailability of enzymatically active caspase-14 .", "however , it was recently shown that proteolytically processed caspase-14 requires high concentrations of kosmotropic salt to be active in vitro , such as sodium citrate , in addition to proteolytic cleavage between the p20 and p10 subunit ( mikolajczyk et al . , 2004 ) .", "these salts induce both dimerization and ordering of active site loops by partial desolvation of the protein to a more compact , catalytically active protease .", "the cellular environment of the stratum corneum of the epidermis probably favors caspase-14 activity in a similar way .", "indeed , the water content decreases from 45% at the transitional layer to 1525% at the skin surface ( warner et al . , 1988 ; caspers et al . , 2001 ) .", "human caspase-14 preferentially accommodates tryptophan or tyrosine in the s4 subsite , whereas mouse caspase-14 is more tolerant , with almost equal preferences for -branched and aromatic amino acids ( mikolajczyk et al . , 2004 ) .", "for example , both human and mouse caspase-14 efficiently cleave the fluorescent peptide substrate wehd - amc , but only mouse caspase-14 cleaves ietd - amc as efficiently ( fischer et al . , 2004 ; mikolajczyk et al . ,", "these substrate preferences would classify human caspase-14 as an inflammatory caspase and mouse caspase-14 as an inflammatory and apoptotic initiator caspase ( thornberry et al .", ", 1997 ; thornberry , 1998 ) . however , human caspase-14 can not proteolytically activate the inflammatory cytokines pro ", "2004 ) , and there are no data supporting a direct role for caspase-14 in apoptosis ( lippens et al . , 2000 ; denecker et al . , 2007 ) .", "whether the substrate preferences of human and murine caspase-14 observed in vitro on peptide substrates also occur in vivo is not clear .", "importantly , profilaggrin , a major structural protein in the differentiating epidermis , has been shown to be a physiological substrate of caspase-14 ( denecker et al . , 2007 ) .", "identification of additional substrates and determination of the cleavage sites will provide more insight into the preferred recognition sequence of caspase-14 in the context of a protein .", "keratinocytes can die by two different processes : apoptotic cell death induced by damaging agents such as uvb , chemicals , and cytotoxic cytokines or by a continuous process of differentiation leading to the formation of corneocytes .", "these processes are clearly distinct pathways executed by different players ( for review see lippens et al . , 2005 ) , but the role of caspases in these two cell death programs has long been controversial .", "although almost all procaspases are constitutively expressed in the epidermis , only caspase-14 has consistently been shown to be activated during epidermal cornification ( eckhart et al .", ", 2000b ; lippens et al . , 2000 ; raymond et al . , 2007 ) .", "in addition , knockouts for apoptotic caspases were not reported to have a phenotypic skin anomaly except for caspase-3deficient mice , in which keratinocyte differentiation is delayed in the embryo but normalized at birth ( okuyama et al . , 2004 ) .", "however , others could not confirm the activation of caspase-3 during embryonic epidermal development ( fischer et al .", "although they are not activated during cornification , apoptotic caspases , in contrast to caspase-14 , become activated during uvb- , staurosporine- , tnf- , and tnf - related apoptosis - inducing ligand  induced apoptosis of keratinocytes and , thereby , play a role in the apoptotic cell death of keratinocytes ( for review see lippens et al . , 2005 ) .", "thus , we conclude that apoptotic caspases are not involved in the physiological keratinocyte cell death program leading to cornification .", "furthermore , caspase-14deficient epidermal cells can undergo classical apoptosis , which genetically demonstrates that caspase-14 is dispensable for the apoptosis of keratinocytes . during development , caspase-14 protein expression is detectable from embryonic day ( e ) 15.5 on , and its processing is observed from e17.5 ( hu et al . , 1998 ; van de craen et al . , 1998 ; fischer et al . , 2005 ) , which coincides with stratum corneum formation and establishment of the epidermal barrier .", "however , no differences in outside - in barrier formation of the skin of caspase-14deficient mice during embryogenesis were observed ( denecker et al . , 2007 ) .", "furthermore , caspase-14deficient mice were born at the expected mendelian ratios , were fertile , and had long survival rates .", "detailed analysis of caspase-14deficient mice indicated that caspase-14 has an important role in cornification , hydration , and uvb protection .", "the skin of caspase-14deficient mice was shinier , characterized by deeper skin lines , and had larger scales ( denecker et al . , 2007 ) even though the shape and size of the cornified envelopes themselves were not altered .", "biochemical analysis indicated that caspase-14 was responsible for the correct processing and degradation of ( pro)filaggrin , as epidermis lacking caspase-14 was characterized by an altered profilaggrin processing and staining pattern ( fig .", "3 ) and by the presence of aberrant keratohyalin granules , the profilaggrin storage granules . profilaggrin is a large , insoluble protein consisting of a calcium - binding a domain , a b domain , and several tandem repeats of filaggrin units . in the transitional layer", "4 ) , which aid in the bundling of keratin intermediate filaments and formation of the cornified envelope ( for review see candi et al . , 2005 ) .", "subsequently , filaggrin is deiminated ( conversion of arginine to citrulline by elimination of the imino group of arginine by peptidylarginine deiminases ) , causing its release from keratin and allowing its degradation into free hygroscopic amino acids that act as natural moisturizing factors of the stratum corneum ( scott and harding , 1986 ; rawlings and matts , 2005 ) .", "therefore , filaggrin plays an important role in skin hydration . although the filaggrin unit was detected in caspase-14deficient epidermis by western blot analysis , lower molecular weight filaggrin fragments were also present ( denecker et al . , 2007 ) .", "immunofluorescence analysis showed that in these mice , filaggrin immunoreactive fragments accumulated in the upper layers of the stratum corneum ( fig .", "this indicates that the correct degradation of filaggrin into free amino acids was affected in caspase-14deficient skin .", "interestingly , caspase-14 was found to be associated with keratohyalin granules in the stratum granulosum and to remain cytoplasmic in the stratum corneum ( alibardi et al . , 2004 ) , which could correlate with its possible involvement in the generation of free amino acids . expression of caspase-14 and ( pro)filaggrin in wild - type and caspase-14deficient skin .", "immunofluorescence staining for caspase-14 ( red ) and ( pro)filaggrin ( green ) on paraffin sections of 5.5-d - old skin of both wild - type ( + /+ ) and caspase-14deficient ( / ) mice ( denecker et al . , 2007 ) .", "fluorescence microscopy was performed on a cellm system ( olympus ) with an upright microscope ( bx61 ; olympus ) .", "a specific dapi emission band - pass filter ( 450470 nm ) and a gfp emission band - pass filter ( 510550 nm ) were used .", "image acquisition and processing were performed with the cellm software using a cooled ccd camera with a 1,344  1,024 pixel resolution .", "image intensity scaling and color conversion were completed in imagej ( national institutes of health ) .", "caspase-14 is expressed mainly in the spinous , granular , and cornified layers of wild - type mice and is absent in caspase-14deficient mice .", "( pro)filaggrin is expressed in the granular layer and in the lower cornified layer in wild - type skin . in caspase-14deficient skin , additional filaggrin", "caspase-14 protects the skin against uvb photo damage and water loss and is involved in the processing of ( pro)filaggrin .", "caspase-14 expression starts in the spinous layer ( indicated in shades of red ) , and cleavage into its p20 and p10 subunits occurs at the transition of the granular to the cornified layer .", "caspase-14 is active in the dehydrating environment of the cornified layer , where it has an important function in formation of the epidermal barrier leading to protection against uvb and water loss ( denecker et al . , 2007 ) .", "profilaggrin is a large structural molecule consisting of an n - terminal a domain and a b domain followed by multiple filaggrin repeats and a unique c - terminal sequence ( for review see candi et al . , 2005 ) .", "profilaggrin undergoes many posttranslational modifications , eventually leading to release from the keratin intermediate filaments ( see the section on the function of caspase-14 for details ) . in the lower stratum corneum", "these amino acids compose 40% of the natural moisturizing factors present in the stratum corneum and are important for maintaining epidermal hydration ( rawlings and matts , 2005 ) . in caspase-14deficient skin ,", "accumulating filaggrin fragments are present ( denecker et al . , 2007 ) , indicating that an unidentified protease ( asterisk ) cleaves the filaggrin monomer into these fragments and that caspase-14 is responsible for the further processing and degradation of these fragments into free amino acids .", "as it is very unlikely that caspase-14 is directly responsible for degradation of the filaggrin fragments into free amino acids , we propose two possible mechanisms : ( 1 ) caspase-14 could first cleave these filaggrin fragments , leading to further degradation into free amino acids by another endo- and/or exopeptidase ; or ( 2 ) caspase-14 could directly or indirectly ( by inactivating an inhibitor ) activate an endo- and/or exopeptidase that further processes the smaller filaggrin fragments .", "kg , keratohyalin granule ; kif , keratin intermediate filament ; nmf , natural moisturizing factors ; sb , stratum basale ; sc , stratum corneum ; sg , stratum granulosum ; ss , stratum spinosum ; tg , transglutaminase .", "these results , together with the finding that caspase-14 can directly cleave ( pro)filaggrin in vitro , demonstrate that caspase-14 has a critical role in the correct processing of ( pro)filaggrin during cornification . whether the degradation of other differentiation - associated proteins is also affected in caspase-14deficient mice", "first , caspase-14 may cleave the filaggrin fragments and , thereby , expose cleavage sites that can be recognized by other endo- and/or exopeptidases for further degradation .", "second , caspase-14 may be the activator of an endo- and/or exopeptidase that cleaves and degrades filaggrin .", "direct degradation of filaggrin fragments into free amino acids by caspase-14 can be ruled out , as caspases only cleave after aspartate residues .", "the lack of filaggrin processing into free hygroscopic amino acids in caspase-14deficient mice may lead to the reduced epidermal hydration and increased trans - epidermal water loss observed in these mice ( denecker et al . , 2007 ) .", "these results point to an important function of caspase-14 in the maintenance of epidermal hydration .", "although a profilaggrin - deficient mouse has not been generated , it has been demonstrated that flaky tail ( ft / ft ) mice , which have an autosomal recessive mutation in the flaky tail gene ( probably the profilaggrin gene ) , lack a functional filaggrin monomer ( presland et al . , 2000 ) .", "these mice have been proposed as a model for the filaggrin - deficient skin disease ichthyosis vulgaris because they have dry , flaky skin and irregular scales of variable size .", "the importance of filaggrin has been underscored recently by human genetic studies demonstrating that loss - of - function mutations in the profilaggrin gene are the primary cause of the skin disease ichthyosis vulgaris ( smith et al . , 2006 ) , which is characterized by silvery scales on the abdomen and palmar hyperlinearity .", "2006 ) , possibly as a result of a defect in epidermal barrier function that allows the increased entry of allergens and infectious agents .", "two of the important functions of the skin are prevention of water loss and protection against environmental stress , such as protection against uvb radiation , which are essential for terrestrial life .", "the development of caspase-14deficient mice revealed that the absence of caspase-14 enhances sensitivity toward uvb - induced photo damage and apoptosis of the skin ( denecker et al . , 2007 ) .", "importantly , this is not caused by cell - autonomous differences in dna damage sensitivity and apoptosis between wild - type and caspase-14deficient keratinocytes .", "instead , the uvb - filtering capacity of the stratum corneum is severely reduced in caspase-14deficient skin , as higher levels of cyclobutane pyrimidine dimers are detected immediately after uvb irradiation .", "this indicates that caspase-14 has an indispensable role in the photoprotective function of the stratum corneum .", "interestingly , topical application of egcg , an inducer of caspase-14 expression , has been shown to be photoprotective ( elmets et al . , 2001 ) . how caspase-14 alters the structural and biochemical properties of the stratum corneum is currently under investigation .", "caspase-14 was shown to be expressed at the protein level in several cancer cell lines ( pistritto et al . , 2002 ; koenig et al . , 2005 ; krajewska et al . , 2005 ) .", "in addition , caspase-14 mrna , together with keratin 1 and profilaggrin mrna , was decreased in murine uvb - induced squamous cell carcinoma , possibly reflecting reduced differentiation in the tumor ( rundhaug et al . , 2005 ) .", "furthermore , in some cases , caspase-14 protein expression was associated with highly differentiated cornified areas of lung squamous cell carcinoma and cervix carcinoma ( koenig et al . , 2005 ) . however", ", caspase-14 activation in tumors has not been shown , and so it might not be responsible for the tumor phenotype .", "presumably , the ectopic caspase-14 expression is caused by the changed transcriptional activity in these epithelial tumors .", "mutations in the caspase-14 gene have not been found in human carcinomas except very rarely in colorectal tumors , which most probably were not the cause of altered caspase-14 expression ( koenig et al . , 2005 ;", "we as well as other investigators demonstrated that caspase-14 expression is substantially down - regulated in psoriatic lesions but is unaffected in the nonlesional epidermis ( lippens et al . , 2000 , 2004 ;", "psoriasis is an autoimmune disease characterized by the uncontrolled proliferation of keratinocytes and impaired cornification , which results in the aberrant presence of nuclei in the cornified layer , also called parakeratosis .", "although caspase-14 is absent in these parakeratotic regions , this is probably not the cause of the development of parakeratotic plaques , as caspase-14deficient mice did not show spontaneous parakeratosis ( denecker et al . , 2007 ) .", "more likely , caspase-14 down - regulation results from the impairment of terminal differentiation or up - regulation of transcriptional repressors .", "the absence of caspase-14 in psoriatic plaques may lead to the formation of a defective barrier and , therefore , to the aggravation of psoriatic lesions . treating the parakeratotic plaques of patients with a vitamin d3 analogue results in the up - regulation of caspase-14 and", "likewise , in the flaky skin ( fsn / fsn ) mouse model of psoriasis , topical egcg treatment causes the up - regulation of caspase-14 and the amelioration of psoriasis ( hsu et al . , 2007 ) .", "interestingly , the expression of junb is strongly down - regulated in psoriatic lesions , and inducible epidermal deletion of both junb and c - jun in mice results in a psoriatic phenotype ( zenz et al . , 2005 ) . because caspase-14 might be regulated by these transcription factors", ", it would be interesting to elucidate whether caspase-14 is down - regulated in junb / c - jun  deficient mice .", "recent evidence sheds light on the crucial role of caspase-14 in the skin , but several major questions remain .", "activation of caspase-14 occurs most probably at the interface between the granular and cornified layer , implicating a role for caspase-14 in the stratum corneum .", "it is now clear that the caspase-14activating protease is not a caspase but probably an epidermis - specific serine protease with elastase - like properties .", "this is not surprising , as it has been known for a long time that serine proteases are of major importance in epidermal homeostasis .", "determination of the in vitro conditions for caspase-14 activity that mimic stratum corneum conditions , together with the generation of caspase-14deficient mice , led to the identification of ( pro)filaggrin as the first known physiological caspase-14 substrate .", "importantly , caspase-14 seems to be involved in the correct processing of filaggrin preceding its degradation into free hygroscopic amino acids , which might explain its role in the prevention of water loss from the epidermis .", "proteomic approaches could lead to the identification of additional caspase-14 substrates , which would contribute to understanding the role of caspase-14 in the skin .", "caspase-14 also protects against uvb - induced damage , which means that it is involved in the establishment of the biochemical or structural properties of the stratum corneum as a uvb filter . how caspase-14 establishes the uvb - filtering capacity of the corneum is not completely understood .", "an extensive biochemical analysis of caspase-14deficient epidermis could reveal these mechanisms . whether caspase-14 , its activating protease , or its substrates could be used as therapeutic agents or as targets to improve formation of the epidermal barrier is a challenging research goal ."], "abstract_text": ["<S> caspase-14 is a unique member of the evolutionarily conserved family of cysteinyl aspartate </S>", "<S>  specific proteinases , which are mainly involved in inflammation and apoptosis . </S>", "<S> however , recent evidence also implicates these proteases in proliferation and differentiation . </S>", "<S> although most caspases are ubiquitously expressed , caspase-14 expression is confined mainly to cornifying epithelia , such as the skin . moreover , </S>", "<S> caspase-14 activation correlates with cornification , indicating that it plays a role in terminal keratinocyte differentiation . </S>", "<S> the determination of in vitro conditions for caspase-14 activity paved the way to identifying its substrates . </S>", "<S> the recent development of caspase-14deficient mice underscored its importance in the correct degradation of ( pro)filaggrin and in the formation of the epidermal barrier that protects against dehydration and uvb radiation . </S>", "<S> here , we review the current knowledge on caspase-14 in skin homeostasis and disease . </S>"], "labels": null, "section_names": ["Introduction", "Caspase-14 expression and regulation", "Activation of caspase-14", "Function: caspase-14 is involved in cornification, hydration, and protection against UVB", "Caspase-14 and disease", "Conclusions"], "sections": [["the skin is the largest organ of the body and protects the organism against external physical , chemical , and biological insults such as wounding , uvb radiation , and microorganisms .", "this major barrier resides in the upper layers of the epidermis ( for review see segre , 2006 ) . the epidermis is the upper part of the skin that is continuously renewed .", "the basal layer , or stratum basale , of the epidermis contains proliferating keratinocytes ( fig .", "1 ) . upon withdrawal from the cell cycle , these basal keratinocytes detach from the basement membrane and undergo a terminal differentiation program to become corneocytes in the outer layers of the epidermis .", "the cells reinforce their cytoskeletal keratin filament network , and adjacent cells interact via many desmosomes , a specialized type of cell junction , to resist physical trauma . in the stratum granulosum ,", "the keratinocytes become more flattened and express certain proteins such as profilaggrin and loricrin , which aggregate to form the typical keratohyalin granules of the stratum granulosum . in addition , lipids are produced and stored in lamellar bodies . at the final stage of differentiation , the keratinocytes lose their organelles , including the nucleus , and become the dead , flattened corneocytes of the stratum corneum . during cornification , proteins are cross - linked at the inner side of the cytoplasmic membrane to form a cornified envelope ( for review see candi et al . , 2005 ) . in the transitional layer between the stratum granulosum and the stratum corneum , lipids are extruded to form a water - repelling envelope around the cornified envelope , thereby assuring an adequate permeability barrier function of the mammalian epidermis .", "improper formation of these envelopes results in an impaired epidermal barrier that can not protect against dehydration , uvb , and infection .", "the signaling cascades involved in epidermal barrier formation are largely unknown , but the many proteases that seem to be involved are currently being intensively studied .", "see introduction for details . since the cloning of caspase-14 in the late nineties , it has become clear that this protease is a unique member of the caspase family .", "unlike apoptotic caspases , which evolved in common ancestors such as hydra , echinodermata , insects , nematodes , and chordates , caspase-14 has so far been found only in terrestrial mammals ( lamkanfi et al . , 2002 ) .", "in contrast to the ubiquitously expressed other members of the caspase family , caspase-14 is expressed and activated mainly in the epidermis and is absent from most other adult tissues ( eckhart et al .", "recently , caspase-14 was found to be involved in epidermal barrier formation ( denecker et al . , 2007 ) .", "in this review , we discuss current knowledge of the expression , regulation , and function of caspase-14 ."], ["the expression pattern of caspase-14 is unique among the caspases , as it is present mainly in cornifying epithelia , such as the epidermis , the hassall 's bodies of the thymus , and the forestomach of rodents ( lippens et al . , 2000 , 2005 ;", "in skin , caspase-14 is expressed only in the differentiating and cornifying layers of the epidermis and the hair follicle ( lippens et al . , 2000 ;", "this is consistent with the observation that , in vitro , caspase-14 is only expressed in differentiating but not in proliferating keratinocytes ( lippens et al .", "remarkably , nail matrix keratinocytes that differentiate into specialized nail corneocytes , the building blocks of the nail plate , do not express caspase-14 ( jager et al .", "in addition , caspase-14 is not expressed in the noncornifying keratinocytes of the sweat gland or the mouth epithelium ( lippens et al . , 2000 ;", "ultrastructural analysis demonstrated that spatial distribution of caspase-14 in the epidermis and hair follicles is strongly conserved among several mammalian species ( alibardi et al .", "caspase-14 was found to be associated with the nucleus , the keratohyalin granules , and the desmosomes , whereas in corneocytes , caspase-14 was found in the cytoplasm and was associated with corneodesmosomes ( a modified version of desmosomes ) and nuclear remnants .", "these observations suggested a role for caspase-14 in nuclear degradation during cornification , but nuclear degradation was not affected in caspase-14deficient mice ( denecker et al . , 2007 ) .", "the expression of caspase-14 in the hassall 's bodies of the thymus and in the forestomach of rodents is somewhat expected , as they are cornifying structures and express the typical late differentiation markers , such as profilaggrin and loricrin , which are also found in the epidermis ( laster and haynes , 1986 ; favre , 1989 ; jarnik et al . , 1996 ) .", "protein expression of caspase-14 has also been reported in several noncornifying tissues ( lippens et al . , 2003 ; krajewska et al . , 2004 , 2005 ; kam et al . , 2005", "however , these observations should be interpreted carefully , as we have recently shown that the reported expression of caspase-14 in such tissues can be the result of aspecific staining ( denecker et al . , 2007 ) .", "remarkably , so far caspase-14 has been found only in terrestrial mammals but not in birds or reptiles .", "whereas birds and reptiles have a stiff , dry , scaly epidermis , mammals have a soft stratum corneum because of the larger amounts of histidine - rich late differentiation markers ( e.g. , profilaggrin ; alibardi , 2003 ) .", "interestingly , profilaggrin is a direct substrate of caspase-14 ( denecker et al . , 2007 ) .", "this could indicate that the occurrence of a soft stratum corneum and the caspase-14 gene are associated during evolution .", "although the expression of caspase-14 is very restricted , little is known about the transcriptional regulation of its gene . in vitro", ", caspase-14 is only expressed when keratinocytes are forced to differentiate by growing them postconfluently or in suspension or by adding vitamin d3 ( eckhart et al .", "in contrast , adding ca at high concentrations to the medium , a method frequently used to induce differentiation , did not induce caspase-14 expression ( eckhart et al .", "retinoids , which suppress keratinocyte differentiation , down - regulate caspase-14 expression ( rendl et al . ,", "these results indicate that transcription factors that are specifically active during terminal differentiation are required to regulate caspase-14 expression . whether caspase-14 expression levels can be regulated at the posttranscriptional level is not known .", "down - regulation of several differentiation - associated genes by retinoids has been shown to be mediated by the transrepression of activator protein 1 ( ap-1)mediated gene activation ( fisher and voorhees , 1996 ) .", "indeed , the caspase-14 promoter contains at least two potential ap-1binding sites ( unpublished data ) .", "the green tea phenol ( )-epigallocatechin-3-gallate ( egcg ) is a potent activator of ap-1 and has been shown to up - regulate caspase-14 in a p38- and jnk - dependent way ( hsu et al . , 2005 , 2007 ) .", "ap-1 alone is probably not sufficient to drive caspase-14 expression because tnf and 12-o - tetradecanoyl - phorbol 13-acetate , two potent activators of ap-1 in keratinocytes ( arnott et al . , 2002 )", ", did not induce caspase-14 expression in keratinocytes ( lippens et al . , 2004 ) .", "differentiation - dependent expression of caspase-14 in keratinocytes could also result from a strong transcriptional repression in proliferating keratinocytes .", "this possibility is supported by the observation that mice deficient in nuclear receptor corepressor hairless ( hr ) had 510-fold higher levels of caspase-14 and profilaggrin mrna , starting from postnatal day 6 and progressing during development ( zarach et al . , 2004 ) .", "these alterations in gene expression were detected mainly in the keratinocytes of the utricle , an abnormal pouch - like structure at the upper part of the hair follicle . increased", "gene expression occurred before the morphologically distinct utricle could be identified , indicating that the up - regulation was probably a cause rather than a consequence of utricle formation", ". it would be interesting to analyze both caspase-14 activation and filaggrin processing in these mice .", "multiple mutant hr alleles in mice and in humans show phenotypic variations that include congenital hair loss , skin wrinkling , and papular rash ( cichon et al . , 1998 ;", "1998 ) . whether caspase-14 overexpression is important for the observed phenotypes could be addressed by generating epidermis - specific caspase-14 transgenic mice or by crossing the hr mice with caspase-14deficient mice ."], ["procaspases consist of a prodomain , a large subunit ( p20 ) , and a small subunit ( p10 ) .", "activation of caspases is induced by dimerization , ( auto-)proteolytic cleavage at asp residues , and/or conformational changes ( lamkanfi et al . , 2003 ) .", "so far , maturation of caspase-14 by proteolytic cleavage into p20 and p10 subunits has been consistently observed only in cornifying epithelia such as the epidermis and the rodent forestomach ( lippens et al . , 2000 ;", "although some investigators suggested that caspase-8 and -10 can activate caspase-14 in vitro ( ahmad et al .", ", 1998 ; van de craen et al . , 1998 ) , this could not be confirmed by others ( lippens et al .", "furthermore , caspase-14 is probably not proteolytically activated by a caspase in vivo , as other caspases are not activated during epidermal differentiation ( eckhart et al .", "2000 ; raymond et al . , 2007 ) , and caspase-14 is not processed at an aspartate residue like other caspases but is processed at ile in man and presumably at leu in the mouse ( chien et al . , 2002 ) .", "alignment of the protease - sensitive loop between the p20 and p10 subunits of the known mammalian procaspase-14 amino acid sequences ( fig .", "2 ) reveals a conserved hydrophobic patch that is n terminal of the caspase-14 cleavage site .", "this patch contains p1-preferred amino acids of elastase - like serine proteases , such as val , ala , leu , and ile ( mallory and travis , 1975 ; vered et al . , 1985 ; takahashi et al . ,", "1989 ) , suggesting that a serine protease with elastase - like properties could be involved in caspase-14 activation ( unpublished data ) . all together ,", "these data indicate that during skin homeostasis , the caspase-14activating protease is not a caspase , separating caspase-14 activation from the apoptotic and inflammatory caspase cascades that could be detrimental to epidermal integrity .", "the precise epidermal layer in which caspase-14 is processed and activated is unknown because antiserum specifically recognizing activated caspase-14 is not yet available .", "however , the following findings indicate that activation of caspase-14 occurs at the interface between the granular and cornified layers of the epidermis or early during cornification .", "first , both the proform and activated form of caspase-14 can be found in total epidermal extracts , whereas in the cornified layer only activated caspase-14 is found ( fischer et al . , 2004 ) .", "second , caspase-14 activation coincides with stratum corneum formation both during embryonic development and in organotypic skin cultures ( eckhart et al .", "this implies that the main biological function of caspase-14 is exercised in the stratum corneum , as proven by the phenotype of the caspase-14deficient mice ( see the next section ) .", "sequence analysis indicates that a hydrophobic patch in the protease - sensitive loop is conserved .", "only part of the alignment is shown here , including the c - terminal part of the p20 subunit , the protease - sensitive loop , and the n - terminal part of the p10 subunit .", "the darker the yellow , the more the amino acids are conserved between species . the catalytic qacrg box is delineated with a green box .", "the conserved hydrophobic patch is delineated with a red box , and the cleavage site in human caspase-14 is indicated with a red arrow .", "the alignment was performed using clustalw ( mega version 3.1 ; kumar et al . , 2004 ) and", "identification of caspase-14 substrates has been hampered for a long time by the unavailability of enzymatically active caspase-14 .", "however , it was recently shown that proteolytically processed caspase-14 requires high concentrations of kosmotropic salt to be active in vitro , such as sodium citrate , in addition to proteolytic cleavage between the p20 and p10 subunit ( mikolajczyk et al . , 2004 ) .", "these salts induce both dimerization and ordering of active site loops by partial desolvation of the protein to a more compact , catalytically active protease .", "the cellular environment of the stratum corneum of the epidermis probably favors caspase-14 activity in a similar way .", "indeed , the water content decreases from 45% at the transitional layer to 1525% at the skin surface ( warner et al . , 1988 ; caspers et al . , 2001 ) .", "human caspase-14 preferentially accommodates tryptophan or tyrosine in the s4 subsite , whereas mouse caspase-14 is more tolerant , with almost equal preferences for -branched and aromatic amino acids ( mikolajczyk et al . , 2004 ) .", "for example , both human and mouse caspase-14 efficiently cleave the fluorescent peptide substrate wehd - amc , but only mouse caspase-14 cleaves ietd - amc as efficiently ( fischer et al . , 2004 ; mikolajczyk et al . ,", "these substrate preferences would classify human caspase-14 as an inflammatory caspase and mouse caspase-14 as an inflammatory and apoptotic initiator caspase ( thornberry et al .", ", 1997 ; thornberry , 1998 ) . however , human caspase-14 can not proteolytically activate the inflammatory cytokines pro ", "2004 ) , and there are no data supporting a direct role for caspase-14 in apoptosis ( lippens et al . , 2000 ; denecker et al . , 2007 ) .", "whether the substrate preferences of human and murine caspase-14 observed in vitro on peptide substrates also occur in vivo is not clear .", "importantly , profilaggrin , a major structural protein in the differentiating epidermis , has been shown to be a physiological substrate of caspase-14 ( denecker et al . , 2007 ) .", "identification of additional substrates and determination of the cleavage sites will provide more insight into the preferred recognition sequence of caspase-14 in the context of a protein ."], ["keratinocytes can die by two different processes : apoptotic cell death induced by damaging agents such as uvb , chemicals , and cytotoxic cytokines or by a continuous process of differentiation leading to the formation of corneocytes .", "these processes are clearly distinct pathways executed by different players ( for review see lippens et al . , 2005 ) , but the role of caspases in these two cell death programs has long been controversial .", "although almost all procaspases are constitutively expressed in the epidermis , only caspase-14 has consistently been shown to be activated during epidermal cornification ( eckhart et al .", ", 2000b ; lippens et al . , 2000 ; raymond et al . , 2007 ) .", "in addition , knockouts for apoptotic caspases were not reported to have a phenotypic skin anomaly except for caspase-3deficient mice , in which keratinocyte differentiation is delayed in the embryo but normalized at birth ( okuyama et al . , 2004 ) .", "however , others could not confirm the activation of caspase-3 during embryonic epidermal development ( fischer et al .", "although they are not activated during cornification , apoptotic caspases , in contrast to caspase-14 , become activated during uvb- , staurosporine- , tnf- , and tnf - related apoptosis - inducing ligand  induced apoptosis of keratinocytes and , thereby , play a role in the apoptotic cell death of keratinocytes ( for review see lippens et al . , 2005 ) .", "thus , we conclude that apoptotic caspases are not involved in the physiological keratinocyte cell death program leading to cornification .", "furthermore , caspase-14deficient epidermal cells can undergo classical apoptosis , which genetically demonstrates that caspase-14 is dispensable for the apoptosis of keratinocytes . during development , caspase-14 protein expression is detectable from embryonic day ( e ) 15.5 on , and its processing is observed from e17.5 ( hu et al . , 1998 ; van de craen et al . , 1998 ; fischer et al . , 2005 ) , which coincides with stratum corneum formation and establishment of the epidermal barrier .", "however , no differences in outside - in barrier formation of the skin of caspase-14deficient mice during embryogenesis were observed ( denecker et al . , 2007 ) .", "furthermore , caspase-14deficient mice were born at the expected mendelian ratios , were fertile , and had long survival rates .", "detailed analysis of caspase-14deficient mice indicated that caspase-14 has an important role in cornification , hydration , and uvb protection .", "the skin of caspase-14deficient mice was shinier , characterized by deeper skin lines , and had larger scales ( denecker et al . , 2007 ) even though the shape and size of the cornified envelopes themselves were not altered .", "biochemical analysis indicated that caspase-14 was responsible for the correct processing and degradation of ( pro)filaggrin , as epidermis lacking caspase-14 was characterized by an altered profilaggrin processing and staining pattern ( fig .", "3 ) and by the presence of aberrant keratohyalin granules , the profilaggrin storage granules . profilaggrin is a large , insoluble protein consisting of a calcium - binding a domain , a b domain , and several tandem repeats of filaggrin units . in the transitional layer", "4 ) , which aid in the bundling of keratin intermediate filaments and formation of the cornified envelope ( for review see candi et al . , 2005 ) .", "subsequently , filaggrin is deiminated ( conversion of arginine to citrulline by elimination of the imino group of arginine by peptidylarginine deiminases ) , causing its release from keratin and allowing its degradation into free hygroscopic amino acids that act as natural moisturizing factors of the stratum corneum ( scott and harding , 1986 ; rawlings and matts , 2005 ) .", "therefore , filaggrin plays an important role in skin hydration . although the filaggrin unit was detected in caspase-14deficient epidermis by western blot analysis , lower molecular weight filaggrin fragments were also present ( denecker et al . , 2007 ) .", "immunofluorescence analysis showed that in these mice , filaggrin immunoreactive fragments accumulated in the upper layers of the stratum corneum ( fig .", "this indicates that the correct degradation of filaggrin into free amino acids was affected in caspase-14deficient skin .", "interestingly , caspase-14 was found to be associated with keratohyalin granules in the stratum granulosum and to remain cytoplasmic in the stratum corneum ( alibardi et al . , 2004 ) , which could correlate with its possible involvement in the generation of free amino acids . expression of caspase-14 and ( pro)filaggrin in wild - type and caspase-14deficient skin .", "immunofluorescence staining for caspase-14 ( red ) and ( pro)filaggrin ( green ) on paraffin sections of 5.5-d - old skin of both wild - type ( + /+ ) and caspase-14deficient ( / ) mice ( denecker et al . , 2007 ) .", "fluorescence microscopy was performed on a cellm system ( olympus ) with an upright microscope ( bx61 ; olympus ) .", "a specific dapi emission band - pass filter ( 450470 nm ) and a gfp emission band - pass filter ( 510550 nm ) were used .", "image acquisition and processing were performed with the cellm software using a cooled ccd camera with a 1,344  1,024 pixel resolution .", "image intensity scaling and color conversion were completed in imagej ( national institutes of health ) .", "caspase-14 is expressed mainly in the spinous , granular , and cornified layers of wild - type mice and is absent in caspase-14deficient mice .", "( pro)filaggrin is expressed in the granular layer and in the lower cornified layer in wild - type skin . in caspase-14deficient skin , additional filaggrin", "caspase-14 protects the skin against uvb photo damage and water loss and is involved in the processing of ( pro)filaggrin .", "caspase-14 expression starts in the spinous layer ( indicated in shades of red ) , and cleavage into its p20 and p10 subunits occurs at the transition of the granular to the cornified layer .", "caspase-14 is active in the dehydrating environment of the cornified layer , where it has an important function in formation of the epidermal barrier leading to protection against uvb and water loss ( denecker et al . , 2007 ) .", "profilaggrin is a large structural molecule consisting of an n - terminal a domain and a b domain followed by multiple filaggrin repeats and a unique c - terminal sequence ( for review see candi et al . , 2005 ) .", "profilaggrin undergoes many posttranslational modifications , eventually leading to release from the keratin intermediate filaments ( see the section on the function of caspase-14 for details ) . in the lower stratum corneum", "these amino acids compose 40% of the natural moisturizing factors present in the stratum corneum and are important for maintaining epidermal hydration ( rawlings and matts , 2005 ) . in caspase-14deficient skin ,", "accumulating filaggrin fragments are present ( denecker et al . , 2007 ) , indicating that an unidentified protease ( asterisk ) cleaves the filaggrin monomer into these fragments and that caspase-14 is responsible for the further processing and degradation of these fragments into free amino acids .", "as it is very unlikely that caspase-14 is directly responsible for degradation of the filaggrin fragments into free amino acids , we propose two possible mechanisms : ( 1 ) caspase-14 could first cleave these filaggrin fragments , leading to further degradation into free amino acids by another endo- and/or exopeptidase ; or ( 2 ) caspase-14 could directly or indirectly ( by inactivating an inhibitor ) activate an endo- and/or exopeptidase that further processes the smaller filaggrin fragments .", "kg , keratohyalin granule ; kif , keratin intermediate filament ; nmf , natural moisturizing factors ; sb , stratum basale ; sc , stratum corneum ; sg , stratum granulosum ; ss , stratum spinosum ; tg , transglutaminase .", "these results , together with the finding that caspase-14 can directly cleave ( pro)filaggrin in vitro , demonstrate that caspase-14 has a critical role in the correct processing of ( pro)filaggrin during cornification . whether the degradation of other differentiation - associated proteins is also affected in caspase-14deficient mice", "first , caspase-14 may cleave the filaggrin fragments and , thereby , expose cleavage sites that can be recognized by other endo- and/or exopeptidases for further degradation .", "second , caspase-14 may be the activator of an endo- and/or exopeptidase that cleaves and degrades filaggrin .", "direct degradation of filaggrin fragments into free amino acids by caspase-14 can be ruled out , as caspases only cleave after aspartate residues .", "the lack of filaggrin processing into free hygroscopic amino acids in caspase-14deficient mice may lead to the reduced epidermal hydration and increased trans - epidermal water loss observed in these mice ( denecker et al . , 2007 ) .", "these results point to an important function of caspase-14 in the maintenance of epidermal hydration .", "although a profilaggrin - deficient mouse has not been generated , it has been demonstrated that flaky tail ( ft / ft ) mice , which have an autosomal recessive mutation in the flaky tail gene ( probably the profilaggrin gene ) , lack a functional filaggrin monomer ( presland et al . , 2000 ) .", "these mice have been proposed as a model for the filaggrin - deficient skin disease ichthyosis vulgaris because they have dry , flaky skin and irregular scales of variable size .", "the importance of filaggrin has been underscored recently by human genetic studies demonstrating that loss - of - function mutations in the profilaggrin gene are the primary cause of the skin disease ichthyosis vulgaris ( smith et al . , 2006 ) , which is characterized by silvery scales on the abdomen and palmar hyperlinearity .", "2006 ) , possibly as a result of a defect in epidermal barrier function that allows the increased entry of allergens and infectious agents .", "two of the important functions of the skin are prevention of water loss and protection against environmental stress , such as protection against uvb radiation , which are essential for terrestrial life .", "the development of caspase-14deficient mice revealed that the absence of caspase-14 enhances sensitivity toward uvb - induced photo damage and apoptosis of the skin ( denecker et al . , 2007 ) .", "importantly , this is not caused by cell - autonomous differences in dna damage sensitivity and apoptosis between wild - type and caspase-14deficient keratinocytes .", "instead , the uvb - filtering capacity of the stratum corneum is severely reduced in caspase-14deficient skin , as higher levels of cyclobutane pyrimidine dimers are detected immediately after uvb irradiation .", "this indicates that caspase-14 has an indispensable role in the photoprotective function of the stratum corneum .", "interestingly , topical application of egcg , an inducer of caspase-14 expression , has been shown to be photoprotective ( elmets et al . , 2001 ) . how caspase-14 alters the structural and biochemical properties of the stratum corneum is currently under investigation ."], ["caspase-14 was shown to be expressed at the protein level in several cancer cell lines ( pistritto et al . , 2002 ; koenig et al . , 2005 ; krajewska et al . , 2005 ) .", "in addition , caspase-14 mrna , together with keratin 1 and profilaggrin mrna , was decreased in murine uvb - induced squamous cell carcinoma , possibly reflecting reduced differentiation in the tumor ( rundhaug et al . , 2005 ) .", "furthermore , in some cases , caspase-14 protein expression was associated with highly differentiated cornified areas of lung squamous cell carcinoma and cervix carcinoma ( koenig et al . , 2005 ) . however", ", caspase-14 activation in tumors has not been shown , and so it might not be responsible for the tumor phenotype .", "presumably , the ectopic caspase-14 expression is caused by the changed transcriptional activity in these epithelial tumors .", "mutations in the caspase-14 gene have not been found in human carcinomas except very rarely in colorectal tumors , which most probably were not the cause of altered caspase-14 expression ( koenig et al . , 2005 ;", "we as well as other investigators demonstrated that caspase-14 expression is substantially down - regulated in psoriatic lesions but is unaffected in the nonlesional epidermis ( lippens et al . , 2000 , 2004 ;", "psoriasis is an autoimmune disease characterized by the uncontrolled proliferation of keratinocytes and impaired cornification , which results in the aberrant presence of nuclei in the cornified layer , also called parakeratosis .", "although caspase-14 is absent in these parakeratotic regions , this is probably not the cause of the development of parakeratotic plaques , as caspase-14deficient mice did not show spontaneous parakeratosis ( denecker et al . , 2007 ) .", "more likely , caspase-14 down - regulation results from the impairment of terminal differentiation or up - regulation of transcriptional repressors .", "the absence of caspase-14 in psoriatic plaques may lead to the formation of a defective barrier and , therefore , to the aggravation of psoriatic lesions . treating the parakeratotic plaques of patients with a vitamin d3 analogue results in the up - regulation of caspase-14 and", "likewise , in the flaky skin ( fsn / fsn ) mouse model of psoriasis , topical egcg treatment causes the up - regulation of caspase-14 and the amelioration of psoriasis ( hsu et al . , 2007 ) .", "interestingly , the expression of junb is strongly down - regulated in psoriatic lesions , and inducible epidermal deletion of both junb and c - jun in mice results in a psoriatic phenotype ( zenz et al . , 2005 ) . because caspase-14 might be regulated by these transcription factors", ", it would be interesting to elucidate whether caspase-14 is down - regulated in junb / c - jun  deficient mice ."], ["recent evidence sheds light on the crucial role of caspase-14 in the skin , but several major questions remain .", "activation of caspase-14 occurs most probably at the interface between the granular and cornified layer , implicating a role for caspase-14 in the stratum corneum .", "it is now clear that the caspase-14activating protease is not a caspase but probably an epidermis - specific serine protease with elastase - like properties .", "this is not surprising , as it has been known for a long time that serine proteases are of major importance in epidermal homeostasis .", "determination of the in vitro conditions for caspase-14 activity that mimic stratum corneum conditions , together with the generation of caspase-14deficient mice , led to the identification of ( pro)filaggrin as the first known physiological caspase-14 substrate .", "importantly , caspase-14 seems to be involved in the correct processing of filaggrin preceding its degradation into free hygroscopic amino acids , which might explain its role in the prevention of water loss from the epidermis .", "proteomic approaches could lead to the identification of additional caspase-14 substrates , which would contribute to understanding the role of caspase-14 in the skin .", "caspase-14 also protects against uvb - induced damage , which means that it is involved in the establishment of the biochemical or structural properties of the stratum corneum as a uvb filter . how caspase-14 establishes the uvb - filtering capacity of the corneum is not completely understood .", "an extensive biochemical analysis of caspase-14deficient epidermis could reveal these mechanisms . whether caspase-14 , its activating protease , or its substrates could be used as therapeutic agents or as targets to improve formation of the epidermal barrier is a challenging research goal ."]]}
{"article_id": "PMC4445174", "article_text": ["gossypiboma ( retained surgical sponge ) describes a mass of cotton or sponge that is left behind in the body cavity during a surgical operation .", "these foreign bodies can often mimic tumors or abscesses when detected clinically or with radiation .", "in fact , surgical sponges without opaque markers are the leading cause for the difficulty of a correct diagnosis . here , we present a case in which a foreign body , gauze without radiopaque markers , was left behind during a procedure that involved the removal of an intrapelvic tumor . after a computed tomography ( ct )", "scan had revealed information leading to an inaccurate diagnosis , an accurate diagnosis of gossypiboma was successfully made using endoscopic ultrasound - fine - needle aspiration ( eus - fna ) .", "a 60-year - old female patient was referred with an unexpected detection of an intrapelvic tumor during a routine ct scan .", "the patient 's medical history included a hysterectomy and a left side ovariectomy at the age of 27 .", "the ct scan revealed a 45 mm tumor in the patient 's pelvis [ figure 1a and b ] , which was diagnosed as a gastrointestinal stromal tumor ( gist ) , a chronic expanding hematoma or leiomyoma . a forward - view eus ( tgf - uc260j , olympus optical co. , tokyo , japan ) identified a low echoic lesion when scanned from the sigmoid colon .", "fna was performed with a 22-gauge needle ( ezshot2 , olympus optical co. , tokyo , japan ) by using the forward - view eus [ figure 2 ] .", "examination of the fna specimen revealed a foreign body ( string of gauze ) displaying macrophage cells and necrotic tissue [ figure 3 ; hematoxylin and eosin , original magnification 40 ] . during the surgery ,", "histological examination of the tissue adjacent to the foreign body revealed granuloma formations with fragmented silken threads [ figure 4 ; hematoxylin and eosin , original magnification , 100 and 200 ] .", "a : sagittal section ( black arrow ) ; b : horizontal section ( black arrow ) .", "endoscopic ultrasound ( eus)-fine - needle aspiration using forward - view eus a foreign body ( string of gauze ) obtained using the 22-gauge needle , with macrophage cells and necrotic tissue granuloma formations with fragmented silken threads", "gossypiboma describes a mass of cotton or a sponge that is retained in the body after a surgery .", "gossypiboma causes foreign body reactions of the surrounding tissue since they are inert and show no specific decomposition . in this case ,", "radiopaque sponges were not used , and there were no adverse symptoms in the 30 years postsurgery .", "the typical appearance of gossypiboma shows a spongiform pattern with gas bubbles . in typical cases ,", "however , it can be difficult to diagnose the disorder with ct scans in cases without gas bubbles . in the case described in this paper ,", "radiologists using findings from a ct scan , initially and incorrectly diagnosed the lesion as gist with fibrosis .", "however , the disorder was correctly diagnosed by a team of gastroenterologists using eus - fna .", "the eus - fna procedure revealed a segment of string in a fixed foreign body in the pelvis .", "the application of eus using curved linear array ( cla ) endoscopes in the lower gi tract has been limited to the rectum and distal sigmoid colon because of the oblique - viewing optics .", "there were case reports using oblique - viewing cla echoendoscopes to evaluate lesions proximal to the sigmoid colon , but advancement of the cla echoendoscope beyond the sigmoid required the use of an overtube or a guide wire previously placed with a colonoscope .", "binmoeller et al . have reported the feasibility and safety of the front - view forward - array echoendoscope for evaluating right colon lesions .", "thus , we used a front - view forward - array echoendoscope to safely evaluate the intrapelvic tumor and sample the tissue .", "a correct diagnosis was obtained using this method . in summary , this is the first known case report diagnosing gossypiboma by eus - fna ."], "abstract_text": ["<S> gossypiboma is a term used to describe a foreign mass within the body that is composed of a cotton matrix . </S>", "<S> gossypiboma can cause infection or abscess formation from an early stage , or it can remain clinically silent for many years . </S>", "<S> gossypiboma can be diagnosed using several methods including abdominal x - ray films , us of the abdomen , contrast - enhanced computed tomography of the abdomen , and during surgery . </S>", "<S> however , the variable appearance of gossypiboma can lead to diagnostic misinterpretations . here , we report a successful diagnosis of gossypiboma using a novel method , that is , endoscopic ultrasound - fine - needle aspiration . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["gossypiboma ( retained surgical sponge ) describes a mass of cotton or sponge that is left behind in the body cavity during a surgical operation .", "these foreign bodies can often mimic tumors or abscesses when detected clinically or with radiation .", "in fact , surgical sponges without opaque markers are the leading cause for the difficulty of a correct diagnosis . here , we present a case in which a foreign body , gauze without radiopaque markers , was left behind during a procedure that involved the removal of an intrapelvic tumor . after a computed tomography ( ct )", "scan had revealed information leading to an inaccurate diagnosis , an accurate diagnosis of gossypiboma was successfully made using endoscopic ultrasound - fine - needle aspiration ( eus - fna ) ."], ["a 60-year - old female patient was referred with an unexpected detection of an intrapelvic tumor during a routine ct scan .", "the patient 's medical history included a hysterectomy and a left side ovariectomy at the age of 27 .", "the ct scan revealed a 45 mm tumor in the patient 's pelvis [ figure 1a and b ] , which was diagnosed as a gastrointestinal stromal tumor ( gist ) , a chronic expanding hematoma or leiomyoma . a forward - view eus ( tgf - uc260j , olympus optical co. , tokyo , japan ) identified a low echoic lesion when scanned from the sigmoid colon .", "fna was performed with a 22-gauge needle ( ezshot2 , olympus optical co. , tokyo , japan ) by using the forward - view eus [ figure 2 ] .", "examination of the fna specimen revealed a foreign body ( string of gauze ) displaying macrophage cells and necrotic tissue [ figure 3 ; hematoxylin and eosin , original magnification 40 ] . during the surgery ,", "histological examination of the tissue adjacent to the foreign body revealed granuloma formations with fragmented silken threads [ figure 4 ; hematoxylin and eosin , original magnification , 100 and 200 ] .", "a : sagittal section ( black arrow ) ; b : horizontal section ( black arrow ) .", "endoscopic ultrasound ( eus)-fine - needle aspiration using forward - view eus a foreign body ( string of gauze ) obtained using the 22-gauge needle , with macrophage cells and necrotic tissue granuloma formations with fragmented silken threads"], ["gossypiboma describes a mass of cotton or a sponge that is retained in the body after a surgery .", "gossypiboma causes foreign body reactions of the surrounding tissue since they are inert and show no specific decomposition . in this case ,", "radiopaque sponges were not used , and there were no adverse symptoms in the 30 years postsurgery .", "the typical appearance of gossypiboma shows a spongiform pattern with gas bubbles . in typical cases ,", "however , it can be difficult to diagnose the disorder with ct scans in cases without gas bubbles . in the case described in this paper ,", "radiologists using findings from a ct scan , initially and incorrectly diagnosed the lesion as gist with fibrosis .", "however , the disorder was correctly diagnosed by a team of gastroenterologists using eus - fna .", "the eus - fna procedure revealed a segment of string in a fixed foreign body in the pelvis .", "the application of eus using curved linear array ( cla ) endoscopes in the lower gi tract has been limited to the rectum and distal sigmoid colon because of the oblique - viewing optics .", "there were case reports using oblique - viewing cla echoendoscopes to evaluate lesions proximal to the sigmoid colon , but advancement of the cla echoendoscope beyond the sigmoid required the use of an overtube or a guide wire previously placed with a colonoscope .", "binmoeller et al . have reported the feasibility and safety of the front - view forward - array echoendoscope for evaluating right colon lesions .", "thus , we used a front - view forward - array echoendoscope to safely evaluate the intrapelvic tumor and sample the tissue .", "a correct diagnosis was obtained using this method . in summary , this is the first known case report diagnosing gossypiboma by eus - fna ."]]}
{"article_id": "PMC3124455", "article_text": ["to demonstrate the usefulness of enhanced depth imaging optical coherence tomography ( edi - oct ) in investigating choroidal lesions inaccessible to ultrasound sonography .", "in a 60-year - old woman with an asymptomatic choroidal nevus , normal oct was used to observe the macula and edi - oct to image the choroidal nevus that was inaccessible to ultrasound .", "the exact location of the lesion in the choroid and the dimensions of the nevus were measured .", "the lesion was located in the superior macula , and the nevus was homogeneous in its reflectivity .", "we observed a thickened choroid delineated by the shadow cone behind it , measuring 1,376  325 m in the larger vertical cut and 1,220  325 m in the larger horizontal cut in an image with a 1:1 pixel mapping and automatic zoom .", "edi - oct appears to be an excellent technique for measuring choroidal nevi and all choroidal lesions accessible to oct imaging by depicting their exact location in the choroid , their dimensions , and their demarcation from the surrounding healthy tissue , thus allowing for a more efficient and accurate follow - up .", "choroidal nevus is the most common tumor of the ocular fundus , present in 7% of the white population [ 1 , 2 ] . despite its benign nature", ", it presents a risk of visual loss and visual field loss , and can rarely transform into malignant melanoma .", "optical coherence tomography ( oct ) and ultrasonography have been used for imaging choroidal nevi . recently", ", enhanced depth imaging oct ( edi - oct ) has been used in order to measure choroidal thickness in normal and pathologic eyes [ 3 , 4 ] . here", ", we report a case of a flat choroidal nevus inaccessible to ultrasound sonography that was evaluated by edi - oct .", "a 60-year - old woman was referred to our department with an asymptomatic choroidal nevus in her left eye . her best - corrected visual acuity was 20/20 in both eyes . slit lamp and fundus examinations of the right eye were normal . in the left eye , the anterior segment was normal ; however , on fundoscopy a choroidal nevus located in the superior macula was observed ( fig .", "spectral - domain ( sd ) oct ( heidelberg engineering , heidelberg , germany ) revealed normal foveal thickness ( fig .", "2 ) . the flat nevus was inaccessible to ultrasound sonography , and edi - oct was used to image its posterior surface ( fig .", "edi - oct is a new technique that consists of positioning a heidelberg sd - oct close enough to the eye in order to obtain an inverted image , averaged for 100 scans .", "the advantage of this technique is that the sensitivity of the imaging in deeper tissue layers is increased , and thus the obtained measurements are more accurate .", "we employed this technique to image the location of the nevus in the choroid and obtain its dimensions as a reference measure for the follow - up examination ( fig .", "3 ) . heidelberg sd - oct provides an exact correlation between the oct image and the infrared photograph . as infrared light", "is absorbed by melanin , the nevus appears brighter . in the edi - oct image", ", the lesion appears homogeneous and hyperreflective at the level of the large choroidal vessels , masking the underlying choroidal vasculature .", "the choroidal thickness at the level of the nevus appears greater than the neighbor choroid ( fig .", "the diameter of the nevus was calculated based on the presence of a shadow cone created by the lesion 's lateral borders .", "the choroidal thickness was measured based on the hyperreflectivity between the bruch 's membrane and the beginning of the shadow cone ( fig .", "the choroid measured 1,220  325 m in the larger horizontal cut and 1,376  325 m in the larger vertical cut in an image with a 1:1 pixel mapping and automatic zoom ( fig .", "edi - oct appears to be an excellent technique for evaluating flat choroidal nevi that are inaccessible to ultrasound sonography and accessible to oct imaging , displaying simultaneously the lesion and the retinal changes .", "it is a reproducible examination that allows depiction of the exact location of the nevi in the choroid , their dimensions and their demarcation from the surrounding healthy tissue . to date , the software available for sd - oct does not allow for objective measures .", "the subjective measures obtained are useful upon comparison with the follow - up images . in order to limit the possibility of bias", ", these follow - up images must be obtained and compared with the same sd - oct machine .", "we believe that this technique is clinically useful and , together with color fundus photography , can provide a more efficient and accurate follow - up of choroidal nevi ."], "abstract_text": ["<S> purposeto demonstrate the usefulness of enhanced depth imaging optical coherence tomography ( edi - oct ) in investigating choroidal lesions inaccessible to ultrasound sonography.methodsin a 60-year - old woman with an asymptomatic choroidal nevus , normal oct was used to observe the macula and edi - oct to image the choroidal nevus that was inaccessible to ultrasound . </S>", "<S> the exact location of the lesion in the choroid and the dimensions of the nevus were measured.resultsthe lesion was located in the superior macula , and the nevus was homogeneous in its reflectivity . </S>", "<S> we observed a thickened choroid delineated by the shadow cone behind it , measuring 1,376  325 m in the larger vertical cut and 1,220  325 m in the larger horizontal cut in an image with a 1:1 pixel mapping and automatic zoom . </S>", "<S> the macular profile and thickness were both normal.conclusionsedi-oct appears to be an excellent technique for measuring choroidal nevi and all choroidal lesions accessible to oct imaging by depicting their exact location in the choroid , their dimensions , and their demarcation from the surrounding healthy tissue , thus allowing for a more efficient and accurate follow - up . </S>"], "labels": null, "section_names": ["Purpose", "Methods", "Results", "Conclusions", "Introduction", "Case Report", "Conclusion", "Disclosure Statement"], "sections": [["to demonstrate the usefulness of enhanced depth imaging optical coherence tomography ( edi - oct ) in investigating choroidal lesions inaccessible to ultrasound sonography ."], ["in a 60-year - old woman with an asymptomatic choroidal nevus , normal oct was used to observe the macula and edi - oct to image the choroidal nevus that was inaccessible to ultrasound .", "the exact location of the lesion in the choroid and the dimensions of the nevus were measured ."], ["the lesion was located in the superior macula , and the nevus was homogeneous in its reflectivity .", "we observed a thickened choroid delineated by the shadow cone behind it , measuring 1,376  325 m in the larger vertical cut and 1,220  325 m in the larger horizontal cut in an image with a 1:1 pixel mapping and automatic zoom ."], ["edi - oct appears to be an excellent technique for measuring choroidal nevi and all choroidal lesions accessible to oct imaging by depicting their exact location in the choroid , their dimensions , and their demarcation from the surrounding healthy tissue , thus allowing for a more efficient and accurate follow - up ."], ["choroidal nevus is the most common tumor of the ocular fundus , present in 7% of the white population [ 1 , 2 ] . despite its benign nature", ", it presents a risk of visual loss and visual field loss , and can rarely transform into malignant melanoma .", "optical coherence tomography ( oct ) and ultrasonography have been used for imaging choroidal nevi . recently", ", enhanced depth imaging oct ( edi - oct ) has been used in order to measure choroidal thickness in normal and pathologic eyes [ 3 , 4 ] . here", ", we report a case of a flat choroidal nevus inaccessible to ultrasound sonography that was evaluated by edi - oct ."], ["a 60-year - old woman was referred to our department with an asymptomatic choroidal nevus in her left eye . her best - corrected visual acuity was 20/20 in both eyes . slit lamp and fundus examinations of the right eye were normal . in the left eye , the anterior segment was normal ; however , on fundoscopy a choroidal nevus located in the superior macula was observed ( fig .", "spectral - domain ( sd ) oct ( heidelberg engineering , heidelberg , germany ) revealed normal foveal thickness ( fig .", "2 ) . the flat nevus was inaccessible to ultrasound sonography , and edi - oct was used to image its posterior surface ( fig .", "edi - oct is a new technique that consists of positioning a heidelberg sd - oct close enough to the eye in order to obtain an inverted image , averaged for 100 scans .", "the advantage of this technique is that the sensitivity of the imaging in deeper tissue layers is increased , and thus the obtained measurements are more accurate .", "we employed this technique to image the location of the nevus in the choroid and obtain its dimensions as a reference measure for the follow - up examination ( fig .", "3 ) . heidelberg sd - oct provides an exact correlation between the oct image and the infrared photograph . as infrared light", "is absorbed by melanin , the nevus appears brighter . in the edi - oct image", ", the lesion appears homogeneous and hyperreflective at the level of the large choroidal vessels , masking the underlying choroidal vasculature .", "the choroidal thickness at the level of the nevus appears greater than the neighbor choroid ( fig .", "the diameter of the nevus was calculated based on the presence of a shadow cone created by the lesion 's lateral borders .", "the choroidal thickness was measured based on the hyperreflectivity between the bruch 's membrane and the beginning of the shadow cone ( fig .", "the choroid measured 1,220  325 m in the larger horizontal cut and 1,376  325 m in the larger vertical cut in an image with a 1:1 pixel mapping and automatic zoom ( fig ."], ["edi - oct appears to be an excellent technique for evaluating flat choroidal nevi that are inaccessible to ultrasound sonography and accessible to oct imaging , displaying simultaneously the lesion and the retinal changes .", "it is a reproducible examination that allows depiction of the exact location of the nevi in the choroid , their dimensions and their demarcation from the surrounding healthy tissue . to date , the software available for sd - oct does not allow for objective measures .", "the subjective measures obtained are useful upon comparison with the follow - up images . in order to limit the possibility of bias", ", these follow - up images must be obtained and compared with the same sd - oct machine .", "we believe that this technique is clinically useful and , together with color fundus photography , can provide a more efficient and accurate follow - up of choroidal nevi ."], [""]]}
{"article_id": "PMC4204167", "article_text": ["kelch - like ech - associated protein 1 ( keap1)-nuclear factor e2-related factor 2 ( nrf2 ) is a major cellular pathway that protect normal cells against oxidative and xenobiotic damage.1 nrf2 is an essential transcription factor for antioxidant and detoxification genes and is crucial for the chemopreventive effect of various phytochemicals against carcinogenesis .", "representative chemopreventive agents that induce nrf2 include carotenoids , curcumins , cyclic lactones , diterpenes , dithiolethiones , epithionitriles , flavonoids , indoles , isothiocyanates , organosulfides , and phenols.2,3 accumulating evidences have demonstrated that phytochemicals can protect cells from oxidative stress related diseases including inflammatory , cardiovascular , and neurodegenerative diseases and cancer , through nrf2-dependent responses .", "paradoxically , recent researches demonstrated the dark side of nrf2.4 cancer cells acquire a growth advantage by eliminating keap1-mediated negative control of nrf2 , which leads to activation of the nrf2-dependent defense response.4 constitutive upregulation of nrf2 has been found in many types of cancers , including skin , breast , prostate , lung , head and neck , and endometrial cancer.5 overexpressed nrf2 provides a growth advantage for cancer cells by protecting those cells from oxidative stress and anticancer agents , thus contributing to chemoresistance.5 in this review , we summarize the therapeutic development of nrf2 inhibitors that enhance the efficacy of the current cancer treatments .", "nrf2 is a basic leucine zipper transcription factor with 7 domains from neh1 to neh7 .", "neh1 has dna binding motifs.6 neh3 , neh4 , and neh5 have domains involved in the transactivation of nrf2 target genes by binding coactivators .", "neh2 has a major regulatory domain with 2 binding sites for keap1 named etge and dlg ( fig .", "keap1 has 5 functional domains including kelch , intervening region ( ivr ) , broad complex / tramtrack , bric - a - brac domain ( btb ) , an n - terminal region ( ntr ) , and a c - terminal region ( ctr).3 cysteine residues of ivr play as a sensor for reactive oxygen species ( ros ) .", "the btb domain has a cys151 residue and is responsible for nrf2 ubiquitination through binding to cullin e3 ubiquitin ligase ( cul3)-based ubiquitin e3 ligase ( fig .", "1a ) . under unstressed conditions , nrf2 forms a complex with keap1 , leading to polyubiquitination and subsequent degradation by the 26s proteasome ( fig", ". 2a).7 upon exposure to a variety of stressors , including ros , toxicants , and carcinogens , nrf2 is released due to modification of the cysteine residues of keap1 ( fig .", "2b).8 two mechanistic models have been proposed for the regulation of nrf2 : the hinge and latch model and the keap - cul3 dissociation model.810 in the hinge and latch model , 2 binding motifs of the neh2 domain in nrf2 have different affinities.9 for the ubiquitination , both the dlg and etgf motifs should be occupied by keap1 proteins .", "ros modify the cysteine residues of keap1 , leading to the release of the dlg motif ( latch ) without changing the etgf motif ( hinge ) on neh2 .", "another model is the keap - cul3 dissociation model in which oxidative stress disrupts the keap1-cul3 e3 ligase interaction without changing the conformation of keap1.8 nrf2 stabilization with de novo synthesis increases the cytoplasmic level of nrf2 .", "nrf2 translocates into the nucleus and binds to an antioxidant response element ( are ) as a heterodimer with musculo - aponeurotic fibrosarcoma ( maf ) to recruit a transcriptional coactivator and promote thetranscription of various cytoprotective genes that are associated with antioxidant and detoxification enzymes . upon recovery of cellular redox homeostasis , keap1 translocates to the nucleus to dissociate nrf2 from the are resulting in degradation of nrf2.11", "nrf2 binds to ares in promoter regions of various genes that regulate the cellular response to oxidative and xenobiotic stress .", "nrf2 target genes include antioxidant genes and phase ii enzymes such as heme oxygenase-1 ( ho-1 ) , nad(p)h : quinone oxidoreductase 1 ( nqo1 ) , glutathione s - transferase ( gst ) , and glutathione peroxidases .", "the are - bearing genes that are regulated by nrf2 in humans have been studied using microarray analysis of genes induced by chemopreventive agents or knockout of keap1 in human cell lines.2 these genes can be categorized into 5 groups : antioxidant genes ( gclc , gclm , gpx2 , gsr , slc7a11 , srxn1 , and txnd1 ) , nadph - generating enzymes ( g6pd , me1 , and pgd ) , metal - binding proteins ( fth1 , ftl , mt1 , and mt2 ) , drug - metabolizing enzymes and drug transporters ( akr1b1 , akr1b10 , akr1c1 , akr1c2 , akr1c3 , akr1c4 , cbr1 , gstm3 , mrp2 , nq1 , and ptgr1 ) , and stress response proteins ( gadd45 , hmox1 , hsp40 , and hsp70).2 knockdown of keap1 in human keratinocytes resulted in upregulation of 23 mrnas while akr1b1 , akr1b10 , and akr1c1 were induced to the greatest extent , showing increases of between 12- and 16-fold indicating that aldo - keto reductases could be useful biomarkers for nrf2 activation.12 as keap1 is responsible for inhibitor kappa b ( ib ) kinase beta ( ikk- ) ubiquitination , upregulation of some of these genes could be attributable to the increase of nuclear factor - kappa b ( nf-b ) activity.13 expression profiling in mice has revealed that nrf2 regulates approximately 100 genes and that two - thirds of nrf2-target genes are not involved in detoxication or antioxidation . instead , many of the target genes are associated with inflammation and immunity proteins.2", "the aryl hydrocarbon receptor ( ahr ) is a transcription factors that mediates the biological effects of its xenobiotic ligands including dioxin.14 the ahr activates the transcription of nqo1 and other genes through the xenobiotic - responsive element ( xre).15 ahr induces the direct binding of nrf2 to promoters or indirectly produces reactive intermediates that trigger the nrf2 signaling pathway.16 the cross - talk between nf-b and nrf2 is more complex .", "several chemopreventive agents , such as sulforaphane , epigallocatechin-3-gallate , and curcumin , induce nrf2 signaling with concomitant repression of nf-b.14 the nf-b p65 subunit re - presses the nrf2-are pathway by preventing creb binding protein ( cbp ) from binding nrf2 through competitive interaction or by promoting the recruitment of histone deacetylase 3 ( hdac3 ) , a corepressor , to the are.17 in chondrocyte apoptosis , shear - induced cyclooxygenase-2 , an nk-b target gene , can suppress phosphatidyl inositol 3-kinase ( pi3k ) activity , causing a reduction in thenrf2-mediated transcriptional response.18 in contrast , nrf2 inhibits the activity of nf-b by attenuating phosphorylated ib .", "nrf-2 target genes , including ho-1 have been reported to negatively affect nf-b activation.19,20 p53 has been reported to suppress the nrf2-dependent transcription of antioxidant response genes by directly interacting with are - containing promoters .", "previous studies have suggested that the function of this negative regulation is to prevent the formation of antioxidant environment , which could hinder the induction of apoptosis by p53.21 in contrast , direct targets of p53 could upregulate nrf2 activity .", "studies have shown that p21 inhibits the degradation of nrf2 by interacting with the dlg motif , leading to upregulation of antioxidant genes.22 these dual functions of p53 toward nrf2 indicate that p53 may decide the fate of damaged cells whether to repair the damaged cells or apoptosis .", "nrf2 is also involved in autophagy by inducing p62 which is a receptor for autophagic degradation .", "as p62 could block binding between nrf2 and keap1 , p62 contributes to the activation of nrf2 target genes in response to oxidative stress creating a positive feedback loop.23", "the association of nrf2 overexpression and chemoresistance has been reported in many cancers including non - small cell lung cancer , stomach cancer , endometrial cancer , and osteosarcoma.2427 overexpression of nrf2 target genes was also found in a variety of solid tumors.28 ho-1 overexpression was found in brain cancer , prostate cancer , and renal cancer.29,30 nqo1 is known to be overexpressed in hepatoblastoma , colon cancer , breast cancer , and non - small cell lung cancer.31 several mechanisms have been proposed for the activation of keap1-nrf2 signaling in cancer .", "mutation of keap1 can result in accumulation of nrf2 in nucleus through decreased degradation or inhibition of nuclear export.28 in non - small cell lung cancer , heterozygous mutation of keap1 ( 8% of patients ) was reported to be sufficient for nrf2 overexpression.32 in addition to lung cancer , mutations in the keap1 gene were found in various cancers including breast ( 2% ) , colon ( 8% ) , gastric ( 11% ) , liver ( 9% ) , ovary ( 19% ) , and prostate ( 8%).3336 mutations in the neh2 domain of nrf2 were found in lung ( 11% ) , cancer ( 6% ) , and head and neck cancers ( 25%).37,38 all of these mutations were somatic mutations .", "mutation of egfr , kras , braf , myc , and the bcr - abl fusion can activate nrf2 , resulting in enhancement of ros detoxification and other oncogenic roles of nrf2 , including chemoresistance.3941 furthermore , posttranslational modifications can activate the keap1-nrf2 signaling pathway .", "hypermethylation of the keap1 promoter was found in 47% of lung cancer patients and this feature was associated with poor outcome.42 epigenetic regulation of keap1 was also found in malignant glioma , colon cancer , and breast cancer.4345", "in addition to the upregulation of cytoprotective genes , constitutive expression of nrf2 may confer a survival advantage to cancer cells by promotion of cell proliferation , chemoresistance and inhibition of apoptosis .", "an active pi3k - akt pathway augments the nuclear accumulation of nrf2 which then re - directs glucose into the anabolic pathway to increase metabolism , indicating the reinforcement of metabolic reprogramming by nrf2.46 nrf2 could mediate cell proliferation with dual regulation through epidermal growth factor receptor ( egfr ) signaling and keap1 interactions.39 in cells with the keap1 gene mutation ( a549 cells ) , activated nrf2 promotes cell proliferation independent of egfr signaling .", "therefore , egfr tyrosine - kinase inhibitors are intrinsically ineffective in these types of non - small cell lung cancer.39 cancer metastasis and tumor progression requires the epithelial - mesenchymal transition ( emt ) and the loss of e - cadherin is considered to be a main event in emt . in hek293 cells , complex of e - cadherin and beta - catenin could bind to c - terminus of nrf2 preventing nuclear translocation of nrf2.47 as e - cadherin inhibits nrf2-mediated transcription , loss of e - cadherin could promote nrf2 translocation and confer an additional survival advantage to cancer cells.47 the keap1-nrf2 pathway is involved in the inhibition of apoptosis by interacting with p53 and b cell lymphoma-2 ( bcl-2 ) .", "p53 inhibits the activation of nrf2 target genes by direct interacting with are - containing promoters or activating p21.21 considering that p53-induced apoptosis requires the accumulation of ros , increased activity of antioxidant genes by nrf2 in cancer cells can inhibit p53 dependent apoptosis .", "bcl-2 can repress cell death by dimerization with bcl-2-associated x protein ( bax ) and the bcl-2 homology 2 ( bh2 ) domain of bcl-2 is required for this heterodimerization.48 it was found that keap1 binds to the bh2 domain and facilitates the ubiquitination of bcl-2 leading , to bax accumulation and enhanced apoptosis .", "thus , mutations in the keap1 binding site for bcl2 are responsible for the anti - apoptotic effect as well as overexpression of nrf2.49 moreover , it has been reported that nrf2 can directly activate the transcription of bcl-2 and bcl - xl.50,51 many studies have reported the association between nrf2 upregulation and chemoresistance in various cancers , including gastric cancer , osteosarcoma , non - small cell lung cancer , endometrial cancer , bladder cancer , and neuroblastoma.2427,52 platinum drugs generate electrophilic molecules that damage cancer cells .", "chemoresistance to these drugs can be explained by high expression of antioxidant nrf2 target genes.4 another mechanism of chemoresistance is the induction of the drug efflux pump family , which includes the mdr , by nrf2.53 some drugs , including hdac inhibitors , oxaliplatin , and proteasome inhibitors can induce nrf2 and thus decrease the effectiveness of chemotherapy.5456", "in light of the data presented in this review , nrf2 is an attractive molecular target for the inhibition of cancer .", "in contrast to nrf2 activators , including numerous phytochemicals , only a small number of nrf2 inhibitors have been identified ( table ) .", "trigonelline , a coffee alkaloid , efficiently decreased basal and tertiary butylhydroquinone ( tbhq)-induced nrf2 activity in chemoresistant pancreatic carcinoma cell lines ( panc1 , colo357 , and miapaca2 ) which have high nrf2 activity . along with inhibiting nrf2", "the sensitivity of all cell lines to anticancer drugs and tumor necrosis factor - related apoptosis inducing ligand ( trail)-induced apoptosis was enhanced by trigonelline.57 chrysin ( 5,7-dihydroxyflavone ) is a natural flavonoid that is found in many plant extracts . in doxorubicin", "resistant hepatocellular carcinoma cell line ( bel-7402/adm ) , chrysin significantly reduced nrf2 expression by downregulating the pi3k - akt and erk pathways.58 in that study , chrysin restored chemosensitivity by downregulating the nrf2-downstream genes such as ho-1 , akr1b10 , and mrp5 .", "apigenin ( 4,5,7-trihydroxyflavone ) is a natural flavone that is present in many fruits and vegetables and has various biological activities , such as anti - inflammatory and antioxidant properties . in a study", "using bel-7402/adm cell lines , apigenin reduced nrf2 expression as well as the expression of nrf2-downstream genes .", "when cells were treated with doxorubicin , apigenin showed a synergistic anti - tumor effect on bel-7402/adm cell lines , resulting in reduced cell proliferation and more substantially induced apoptosis.59 brusatol is a quassinoid that is extracted from brucea javanica .", "brusatol reduced nrf-2 protein level without changing the keap1 level and sensitized various cells including hela , mda - mb-231 , and a549 to chemotherapeutic agents such as carboplatin , 5-fluorouracil , etoposide , and paclitaxel.60 the anticancer effect of brustalol mediated by nrf2 was confirmed in a xenograft model indicating that brusatol could be used to combat intrinsic resistance .", "ascorbic acid is an antioxidant that can suppress the level of nrf2 . in the imatinib - resistant chronic myelogenous leukemia cell line kcl22/sr ,", "binding of nrf2 to the are of the gamma - glutamylcysteine synthetase gene promoter was much stronger than in the parental imatinib - sensitive cell line kcl22.61 furthermore , addition of ascorbic acid to kcl22/sr cells resulted in a decrease in nrf2-dna binding and partial restoration of imatinib sensitivity to kcl22/sr.61 luteolin ( 3,4,5,7-tetrahydroxyflavone ) is a polyphenolic flavonoid found in high concentrations in celery , green pepper , and parsley . in a study using a cell - based are - reporter assay ,", "luteolin was found to be a potent nrf2 inhibitor.62 in that study , luteolin significantly sensitized a549 cells to the anticancer drugs oxaliplatin , bleomycin , and doxorubicin indicating the potential application of luteolin as a natural sensitizer in chemotherapy .", "constitutively expressed nrf2 can promote cancer cell proliferation and protect cells against oxidative stress and therapeutic agents .", "it must be kept in mind that chemopreventive agents including various phytochemicals can induce chemoresistance and tumor progression by activating the keap1-nrf2 pathway .", "however , it will be necessary to understand the molecular regulation of nrf2 and identifying the individualized status of nrf2 expression ."], "abstract_text": ["<S> nuclear factor e2-related factor 2 ( nrf2 ) is a transcription factor that upregulates expression of a battery of genes to combat oxidative and electrophilic stress . </S>", "<S> modification of kelch - like ech - associated protein 1 ( keap1 ) by reactive oxygen species stabilizes nrf2 by escaping from degradation . </S>", "<S> nrf2 then binds to antioxidant response elements ( ares ) on the promoter region of various genes . </S>", "<S> activation of the keap1-nrf2-are pathway plays critical roles in the chemopreventive effect of various phytochemicals . </S>", "<S> however , nrf2 can protect cancer cells from oxidative stress and promote cell proliferation . </S>", "<S> moreover , recent studies reveal that activation of the nrf2 pathway is critical for resistance to chemotherapeutic agents . </S>", "<S> the aim of this review is to provide a molecular basis for the use of nrf2 inhibitors in overcoming chemoresistance . </S>"], "labels": null, "section_names": ["INTRODUCTION", "REGULATION OF Nrf2 BY Keap1", "Nrf2 TARGET GENES", "CROSS-TALK BETWEEN Nrf2 AND OTHER SIGNALING PATHWAYS", "DEREGULATION OF Nrf2 IN CANCER", "ONCOGENIC ROLE OF Nrf2 IN CANCER", "INHIBITORS OF THE Nrf2", "CONCLUSION"], "sections": [["kelch - like ech - associated protein 1 ( keap1)-nuclear factor e2-related factor 2 ( nrf2 ) is a major cellular pathway that protect normal cells against oxidative and xenobiotic damage.1 nrf2 is an essential transcription factor for antioxidant and detoxification genes and is crucial for the chemopreventive effect of various phytochemicals against carcinogenesis .", "representative chemopreventive agents that induce nrf2 include carotenoids , curcumins , cyclic lactones , diterpenes , dithiolethiones , epithionitriles , flavonoids , indoles , isothiocyanates , organosulfides , and phenols.2,3 accumulating evidences have demonstrated that phytochemicals can protect cells from oxidative stress related diseases including inflammatory , cardiovascular , and neurodegenerative diseases and cancer , through nrf2-dependent responses .", "paradoxically , recent researches demonstrated the dark side of nrf2.4 cancer cells acquire a growth advantage by eliminating keap1-mediated negative control of nrf2 , which leads to activation of the nrf2-dependent defense response.4 constitutive upregulation of nrf2 has been found in many types of cancers , including skin , breast , prostate , lung , head and neck , and endometrial cancer.5 overexpressed nrf2 provides a growth advantage for cancer cells by protecting those cells from oxidative stress and anticancer agents , thus contributing to chemoresistance.5 in this review , we summarize the therapeutic development of nrf2 inhibitors that enhance the efficacy of the current cancer treatments ."], ["nrf2 is a basic leucine zipper transcription factor with 7 domains from neh1 to neh7 .", "neh1 has dna binding motifs.6 neh3 , neh4 , and neh5 have domains involved in the transactivation of nrf2 target genes by binding coactivators .", "neh2 has a major regulatory domain with 2 binding sites for keap1 named etge and dlg ( fig .", "keap1 has 5 functional domains including kelch , intervening region ( ivr ) , broad complex / tramtrack , bric - a - brac domain ( btb ) , an n - terminal region ( ntr ) , and a c - terminal region ( ctr).3 cysteine residues of ivr play as a sensor for reactive oxygen species ( ros ) .", "the btb domain has a cys151 residue and is responsible for nrf2 ubiquitination through binding to cullin e3 ubiquitin ligase ( cul3)-based ubiquitin e3 ligase ( fig .", "1a ) . under unstressed conditions , nrf2 forms a complex with keap1 , leading to polyubiquitination and subsequent degradation by the 26s proteasome ( fig", ". 2a).7 upon exposure to a variety of stressors , including ros , toxicants , and carcinogens , nrf2 is released due to modification of the cysteine residues of keap1 ( fig .", "2b).8 two mechanistic models have been proposed for the regulation of nrf2 : the hinge and latch model and the keap - cul3 dissociation model.810 in the hinge and latch model , 2 binding motifs of the neh2 domain in nrf2 have different affinities.9 for the ubiquitination , both the dlg and etgf motifs should be occupied by keap1 proteins .", "ros modify the cysteine residues of keap1 , leading to the release of the dlg motif ( latch ) without changing the etgf motif ( hinge ) on neh2 .", "another model is the keap - cul3 dissociation model in which oxidative stress disrupts the keap1-cul3 e3 ligase interaction without changing the conformation of keap1.8 nrf2 stabilization with de novo synthesis increases the cytoplasmic level of nrf2 .", "nrf2 translocates into the nucleus and binds to an antioxidant response element ( are ) as a heterodimer with musculo - aponeurotic fibrosarcoma ( maf ) to recruit a transcriptional coactivator and promote thetranscription of various cytoprotective genes that are associated with antioxidant and detoxification enzymes . upon recovery of cellular redox homeostasis , keap1 translocates to the nucleus to dissociate nrf2 from the are resulting in degradation of nrf2.11"], ["nrf2 binds to ares in promoter regions of various genes that regulate the cellular response to oxidative and xenobiotic stress .", "nrf2 target genes include antioxidant genes and phase ii enzymes such as heme oxygenase-1 ( ho-1 ) , nad(p)h : quinone oxidoreductase 1 ( nqo1 ) , glutathione s - transferase ( gst ) , and glutathione peroxidases .", "the are - bearing genes that are regulated by nrf2 in humans have been studied using microarray analysis of genes induced by chemopreventive agents or knockout of keap1 in human cell lines.2 these genes can be categorized into 5 groups : antioxidant genes ( gclc , gclm , gpx2 , gsr , slc7a11 , srxn1 , and txnd1 ) , nadph - generating enzymes ( g6pd , me1 , and pgd ) , metal - binding proteins ( fth1 , ftl , mt1 , and mt2 ) , drug - metabolizing enzymes and drug transporters ( akr1b1 , akr1b10 , akr1c1 , akr1c2 , akr1c3 , akr1c4 , cbr1 , gstm3 , mrp2 , nq1 , and ptgr1 ) , and stress response proteins ( gadd45 , hmox1 , hsp40 , and hsp70).2 knockdown of keap1 in human keratinocytes resulted in upregulation of 23 mrnas while akr1b1 , akr1b10 , and akr1c1 were induced to the greatest extent , showing increases of between 12- and 16-fold indicating that aldo - keto reductases could be useful biomarkers for nrf2 activation.12 as keap1 is responsible for inhibitor kappa b ( ib ) kinase beta ( ikk- ) ubiquitination , upregulation of some of these genes could be attributable to the increase of nuclear factor - kappa b ( nf-b ) activity.13 expression profiling in mice has revealed that nrf2 regulates approximately 100 genes and that two - thirds of nrf2-target genes are not involved in detoxication or antioxidation . instead , many of the target genes are associated with inflammation and immunity proteins.2"], ["the aryl hydrocarbon receptor ( ahr ) is a transcription factors that mediates the biological effects of its xenobiotic ligands including dioxin.14 the ahr activates the transcription of nqo1 and other genes through the xenobiotic - responsive element ( xre).15 ahr induces the direct binding of nrf2 to promoters or indirectly produces reactive intermediates that trigger the nrf2 signaling pathway.16 the cross - talk between nf-b and nrf2 is more complex .", "several chemopreventive agents , such as sulforaphane , epigallocatechin-3-gallate , and curcumin , induce nrf2 signaling with concomitant repression of nf-b.14 the nf-b p65 subunit re - presses the nrf2-are pathway by preventing creb binding protein ( cbp ) from binding nrf2 through competitive interaction or by promoting the recruitment of histone deacetylase 3 ( hdac3 ) , a corepressor , to the are.17 in chondrocyte apoptosis , shear - induced cyclooxygenase-2 , an nk-b target gene , can suppress phosphatidyl inositol 3-kinase ( pi3k ) activity , causing a reduction in thenrf2-mediated transcriptional response.18 in contrast , nrf2 inhibits the activity of nf-b by attenuating phosphorylated ib .", "nrf-2 target genes , including ho-1 have been reported to negatively affect nf-b activation.19,20 p53 has been reported to suppress the nrf2-dependent transcription of antioxidant response genes by directly interacting with are - containing promoters .", "previous studies have suggested that the function of this negative regulation is to prevent the formation of antioxidant environment , which could hinder the induction of apoptosis by p53.21 in contrast , direct targets of p53 could upregulate nrf2 activity .", "studies have shown that p21 inhibits the degradation of nrf2 by interacting with the dlg motif , leading to upregulation of antioxidant genes.22 these dual functions of p53 toward nrf2 indicate that p53 may decide the fate of damaged cells whether to repair the damaged cells or apoptosis .", "nrf2 is also involved in autophagy by inducing p62 which is a receptor for autophagic degradation .", "as p62 could block binding between nrf2 and keap1 , p62 contributes to the activation of nrf2 target genes in response to oxidative stress creating a positive feedback loop.23"], ["the association of nrf2 overexpression and chemoresistance has been reported in many cancers including non - small cell lung cancer , stomach cancer , endometrial cancer , and osteosarcoma.2427 overexpression of nrf2 target genes was also found in a variety of solid tumors.28 ho-1 overexpression was found in brain cancer , prostate cancer , and renal cancer.29,30 nqo1 is known to be overexpressed in hepatoblastoma , colon cancer , breast cancer , and non - small cell lung cancer.31 several mechanisms have been proposed for the activation of keap1-nrf2 signaling in cancer .", "mutation of keap1 can result in accumulation of nrf2 in nucleus through decreased degradation or inhibition of nuclear export.28 in non - small cell lung cancer , heterozygous mutation of keap1 ( 8% of patients ) was reported to be sufficient for nrf2 overexpression.32 in addition to lung cancer , mutations in the keap1 gene were found in various cancers including breast ( 2% ) , colon ( 8% ) , gastric ( 11% ) , liver ( 9% ) , ovary ( 19% ) , and prostate ( 8%).3336 mutations in the neh2 domain of nrf2 were found in lung ( 11% ) , cancer ( 6% ) , and head and neck cancers ( 25%).37,38 all of these mutations were somatic mutations .", "mutation of egfr , kras , braf , myc , and the bcr - abl fusion can activate nrf2 , resulting in enhancement of ros detoxification and other oncogenic roles of nrf2 , including chemoresistance.3941 furthermore , posttranslational modifications can activate the keap1-nrf2 signaling pathway .", "hypermethylation of the keap1 promoter was found in 47% of lung cancer patients and this feature was associated with poor outcome.42 epigenetic regulation of keap1 was also found in malignant glioma , colon cancer , and breast cancer.4345"], ["in addition to the upregulation of cytoprotective genes , constitutive expression of nrf2 may confer a survival advantage to cancer cells by promotion of cell proliferation , chemoresistance and inhibition of apoptosis .", "an active pi3k - akt pathway augments the nuclear accumulation of nrf2 which then re - directs glucose into the anabolic pathway to increase metabolism , indicating the reinforcement of metabolic reprogramming by nrf2.46 nrf2 could mediate cell proliferation with dual regulation through epidermal growth factor receptor ( egfr ) signaling and keap1 interactions.39 in cells with the keap1 gene mutation ( a549 cells ) , activated nrf2 promotes cell proliferation independent of egfr signaling .", "therefore , egfr tyrosine - kinase inhibitors are intrinsically ineffective in these types of non - small cell lung cancer.39 cancer metastasis and tumor progression requires the epithelial - mesenchymal transition ( emt ) and the loss of e - cadherin is considered to be a main event in emt . in hek293 cells , complex of e - cadherin and beta - catenin could bind to c - terminus of nrf2 preventing nuclear translocation of nrf2.47 as e - cadherin inhibits nrf2-mediated transcription , loss of e - cadherin could promote nrf2 translocation and confer an additional survival advantage to cancer cells.47 the keap1-nrf2 pathway is involved in the inhibition of apoptosis by interacting with p53 and b cell lymphoma-2 ( bcl-2 ) .", "p53 inhibits the activation of nrf2 target genes by direct interacting with are - containing promoters or activating p21.21 considering that p53-induced apoptosis requires the accumulation of ros , increased activity of antioxidant genes by nrf2 in cancer cells can inhibit p53 dependent apoptosis .", "bcl-2 can repress cell death by dimerization with bcl-2-associated x protein ( bax ) and the bcl-2 homology 2 ( bh2 ) domain of bcl-2 is required for this heterodimerization.48 it was found that keap1 binds to the bh2 domain and facilitates the ubiquitination of bcl-2 leading , to bax accumulation and enhanced apoptosis .", "thus , mutations in the keap1 binding site for bcl2 are responsible for the anti - apoptotic effect as well as overexpression of nrf2.49 moreover , it has been reported that nrf2 can directly activate the transcription of bcl-2 and bcl - xl.50,51 many studies have reported the association between nrf2 upregulation and chemoresistance in various cancers , including gastric cancer , osteosarcoma , non - small cell lung cancer , endometrial cancer , bladder cancer , and neuroblastoma.2427,52 platinum drugs generate electrophilic molecules that damage cancer cells .", "chemoresistance to these drugs can be explained by high expression of antioxidant nrf2 target genes.4 another mechanism of chemoresistance is the induction of the drug efflux pump family , which includes the mdr , by nrf2.53 some drugs , including hdac inhibitors , oxaliplatin , and proteasome inhibitors can induce nrf2 and thus decrease the effectiveness of chemotherapy.5456"], ["in light of the data presented in this review , nrf2 is an attractive molecular target for the inhibition of cancer .", "in contrast to nrf2 activators , including numerous phytochemicals , only a small number of nrf2 inhibitors have been identified ( table ) .", "trigonelline , a coffee alkaloid , efficiently decreased basal and tertiary butylhydroquinone ( tbhq)-induced nrf2 activity in chemoresistant pancreatic carcinoma cell lines ( panc1 , colo357 , and miapaca2 ) which have high nrf2 activity . along with inhibiting nrf2", "the sensitivity of all cell lines to anticancer drugs and tumor necrosis factor - related apoptosis inducing ligand ( trail)-induced apoptosis was enhanced by trigonelline.57 chrysin ( 5,7-dihydroxyflavone ) is a natural flavonoid that is found in many plant extracts . in doxorubicin", "resistant hepatocellular carcinoma cell line ( bel-7402/adm ) , chrysin significantly reduced nrf2 expression by downregulating the pi3k - akt and erk pathways.58 in that study , chrysin restored chemosensitivity by downregulating the nrf2-downstream genes such as ho-1 , akr1b10 , and mrp5 .", "apigenin ( 4,5,7-trihydroxyflavone ) is a natural flavone that is present in many fruits and vegetables and has various biological activities , such as anti - inflammatory and antioxidant properties . in a study", "using bel-7402/adm cell lines , apigenin reduced nrf2 expression as well as the expression of nrf2-downstream genes .", "when cells were treated with doxorubicin , apigenin showed a synergistic anti - tumor effect on bel-7402/adm cell lines , resulting in reduced cell proliferation and more substantially induced apoptosis.59 brusatol is a quassinoid that is extracted from brucea javanica .", "brusatol reduced nrf-2 protein level without changing the keap1 level and sensitized various cells including hela , mda - mb-231 , and a549 to chemotherapeutic agents such as carboplatin , 5-fluorouracil , etoposide , and paclitaxel.60 the anticancer effect of brustalol mediated by nrf2 was confirmed in a xenograft model indicating that brusatol could be used to combat intrinsic resistance .", "ascorbic acid is an antioxidant that can suppress the level of nrf2 . in the imatinib - resistant chronic myelogenous leukemia cell line kcl22/sr ,", "binding of nrf2 to the are of the gamma - glutamylcysteine synthetase gene promoter was much stronger than in the parental imatinib - sensitive cell line kcl22.61 furthermore , addition of ascorbic acid to kcl22/sr cells resulted in a decrease in nrf2-dna binding and partial restoration of imatinib sensitivity to kcl22/sr.61 luteolin ( 3,4,5,7-tetrahydroxyflavone ) is a polyphenolic flavonoid found in high concentrations in celery , green pepper , and parsley . in a study using a cell - based are - reporter assay ,", "luteolin was found to be a potent nrf2 inhibitor.62 in that study , luteolin significantly sensitized a549 cells to the anticancer drugs oxaliplatin , bleomycin , and doxorubicin indicating the potential application of luteolin as a natural sensitizer in chemotherapy ."], ["constitutively expressed nrf2 can promote cancer cell proliferation and protect cells against oxidative stress and therapeutic agents .", "it must be kept in mind that chemopreventive agents including various phytochemicals can induce chemoresistance and tumor progression by activating the keap1-nrf2 pathway .", "however , it will be necessary to understand the molecular regulation of nrf2 and identifying the individualized status of nrf2 expression ."]]}
{"article_id": "PMC5417286", "article_text": ["a 3-year - old male , known to have isolated speech delay , presented to the emergency department with severe body aches and inability to walk .", "few days prior to his presentation , the child started to have flu - like symptoms in the form of rhinorrhea and low - grade fever .", "this was followed by irritableness , hypoactivity , and inability to move his legs and arms .", "he was seen in a private hospital where computed tomography of the brain and a lumber puncture were done that showed no abnormalities . in the next day , his condition continued to worsen .", "he developed difficulty in swallowing and dark - colored urine , so he was transferred to our institution . upon physical examination ,", "the laboratory tests showed elevated creatine kinase level ( 1 778 856 u / l ; normal range 20 - 200", "u / l ) , elevated serum transaminases ( serum glutamic - pyruvic transaminase [ sgpt ] = 1857", "u / l [ normal range 0 - 50 u / l ] , serum glutamic - oxaloacetic transaminase [ sgot ] = 8626 u / l [ normal range 0 - 50 u / l ] ) , and creatinine 0.72 mg / dl ( 0.3 - 0.7 mg / dl ) .", "the child started on intravenous hydration and received 1 dose of intravenous immunoglobulin . on the second day ,", "the creatine kinase and serum transaminase levels improved , with creatinine reaching 0.94 mg / dl , the highest level reached .", "the creatine kinase and serum transaminase levels continued to improve gradually over the following days , with slow improvement in the clinical condition .", "he received another 2 doses of intravenous immunoglobulin on days 13 and 14 of admission .", "muscle biopsy showed endomysial inflammatory infiltrates , mostly composed of cd8 and cd4 lymphocytes ; myofibers necrosis mostly ventral portion of myofibers ; residual granular microcalcifications ; and terminal complement complex deposits that outline necrotic myofibers , all features compatible with necrotic myopathy and early rhabdomyolysis ( figure 2 ) .", "the arrows showed the degenerated myofibers . upon discharge , the child had mild muscle aches , was able to sit , and move his arms and feet .", "he had persistent weakness in the lower limbs , but was unable to walk , with a follow - up examination 1 month after discharge , revealing that the child was able to walk normally with positive deep tendon reflex .", "two months after discharge , the creatine kinase level dropped to 193 iu / l .", "rhabdomyolysis results from skeletal muscle breakdown due to various causes . viral myositis , trauma , and inherited disorders are the most common causes in the pediatric age group . \n", "the breakdown releases the normal cell contents into the bloodstream , including creatine kinase , myoglobin , phosphorus , and potassium .", "rhabdomyolysis is suspected clinically when muscle aches , muscle weakness , and tea - colored urine are present .", "the diagnosis is based on the high creatine kinase level , which is the most sensitive marker . although there is no established cutoff levels , a concentration 5 to 10 times higher than the upper limit of normal reference range ( ie , 500 - 1000", "other laboratory indicators include serum and urine myoglobin concentrations that may be useful but not essential for the diagnosis . \n ", "the most common complication of rhabdomyolysis is kidney damage . in recent large pediatric studies reported by mannix", "et al and wu et al , it was shown that the rate of acute renal failure in the pediatric patients with rhabdomyolysis ranged from 5% to 8.7% .", "the risk factors that predispose a patient with rhabdomyolysis for developing acute renal injury are creatine kinase level concentration more than 5000 u / l , creatine kinase value upon admission , and slower decline in the serum creatine kinase level .", "other factors include high myoglobin level , persistent or abrupt increase in the potassium or calcium level , as well as persistent metabolic acidosis .", "on the other hand , a recent study by fernandez et al showed that the most reliable predictor of acute renal failure and the need for dialysis is the creatinine level above 1.7 , despite the peak creatine kinase .", "the mainstay for the prevention and treatment of acute kidney injury is early and aggressive volume resuscitation .", "other management options include alkalization of the urine , forced diuresis with mannitol , and loop diuretics . in severe cases or", "in our case , the child developed extremely high creatine kinase levels that were not reported in the literature .", "a thorough literature review was done searching for high creatine kinase levels in patients with acute rhabdomyolysis . in 2014 , a case report described a pediatric patient with mcardle disease who developed rhabdomyolysis with a high 6-digit creatine kinase level reaching 500 000", "2 pediatric cases were reported . in both , the creatine kinase levels reached were 60 000", "a case report of an adolescent with rhabdomyolysis due to undiagnosed hypothyroidism was reported with a creatine kinase level that reached around 34 000", "a research article in 2013 examined the clinical spectrum of patients with rhabdomyolysis presenting to the pediatric emergency department , in which the peak serum level of creatine kinase was 9825.1  23 079.1", "the only risk factor for developing acute renal injury that our patient had was a high peak creatine kinase level upon admission . on the other hand ,", "he did not develop metabolic acidosis and had a normal albumin and electrolytes levels throughout his hospitalization .", "he responded very well to volume resuscitation with rapid decline in the creatine kinase level .", "although he received intravenous immunoglobulin for the fear of guillain - barre syndrome or other underlying immune - mediated diseases , neither the creatine kinase level decline nor the clinical status improvement was affected by the intravenous immunoglobulin administration . the sharp decline in the creatine kinase level", "there are no clues in the family history or the patient s personal history that may suggest an underlying inherited metabolic , muscular , or genetic disease .", ", this could be the first presentation of lipin-1 mutation that causes recurrent rhabdomyolysis in children .", "lipin-1 gene encodes the muscle - specific phosphatidic acid phosphatase , a key enzyme in triglyceride and membrane phospholipid biosynthesis .", "the episodes of rhabdomyolysis are mostly triggered by intercurrent infections and fever and to a lesser extent by fasting or exercise .", "the prognosis of lipin-1 deficiency is poor , with up to one - third of patients dying during an episode of rhabdomyolysis . in about 60% of patients with recurrent rhabdomyolysis ,"], "abstract_text": ["<S> rhabdomyolysis is an acute life - threatening condition that can occur in childhood secondary to many causes . </S>", "<S> the authors report the case of a 3-year - old male child who presented with acute rhabdomyolysis . </S>", "<S> the peak plasma creatine kinase level was extremely high . </S>", "<S> the 2 main causes of rhabdomyolysis in childhood are viral myositis and trauma , which can sometimes lead to acute renal failure . the highest creatine kinase levels reported in the literature so far was a 6-digit level in 2014 case report . in this study , the authors report the case of a 7-digit creatine kinase level in a child secondary to viral myositis who did not require renal dialysis . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 3-year - old male , known to have isolated speech delay , presented to the emergency department with severe body aches and inability to walk .", "few days prior to his presentation , the child started to have flu - like symptoms in the form of rhinorrhea and low - grade fever .", "this was followed by irritableness , hypoactivity , and inability to move his legs and arms .", "he was seen in a private hospital where computed tomography of the brain and a lumber puncture were done that showed no abnormalities . in the next day , his condition continued to worsen .", "he developed difficulty in swallowing and dark - colored urine , so he was transferred to our institution . upon physical examination ,", "the laboratory tests showed elevated creatine kinase level ( 1 778 856 u / l ; normal range 20 - 200", "u / l ) , elevated serum transaminases ( serum glutamic - pyruvic transaminase [ sgpt ] = 1857", "u / l [ normal range 0 - 50 u / l ] , serum glutamic - oxaloacetic transaminase [ sgot ] = 8626 u / l [ normal range 0 - 50 u / l ] ) , and creatinine 0.72 mg / dl ( 0.3 - 0.7 mg / dl ) .", "the child started on intravenous hydration and received 1 dose of intravenous immunoglobulin . on the second day ,", "the creatine kinase and serum transaminase levels improved , with creatinine reaching 0.94 mg / dl , the highest level reached .", "the creatine kinase and serum transaminase levels continued to improve gradually over the following days , with slow improvement in the clinical condition .", "he received another 2 doses of intravenous immunoglobulin on days 13 and 14 of admission .", "muscle biopsy showed endomysial inflammatory infiltrates , mostly composed of cd8 and cd4 lymphocytes ; myofibers necrosis mostly ventral portion of myofibers ; residual granular microcalcifications ; and terminal complement complex deposits that outline necrotic myofibers , all features compatible with necrotic myopathy and early rhabdomyolysis ( figure 2 ) .", "the arrows showed the degenerated myofibers . upon discharge , the child had mild muscle aches , was able to sit , and move his arms and feet .", "he had persistent weakness in the lower limbs , but was unable to walk , with a follow - up examination 1 month after discharge , revealing that the child was able to walk normally with positive deep tendon reflex .", "two months after discharge , the creatine kinase level dropped to 193 iu / l ."], ["rhabdomyolysis results from skeletal muscle breakdown due to various causes . viral myositis , trauma , and inherited disorders are the most common causes in the pediatric age group . \n", "the breakdown releases the normal cell contents into the bloodstream , including creatine kinase , myoglobin , phosphorus , and potassium .", "rhabdomyolysis is suspected clinically when muscle aches , muscle weakness , and tea - colored urine are present .", "the diagnosis is based on the high creatine kinase level , which is the most sensitive marker . although there is no established cutoff levels , a concentration 5 to 10 times higher than the upper limit of normal reference range ( ie , 500 - 1000", "other laboratory indicators include serum and urine myoglobin concentrations that may be useful but not essential for the diagnosis . \n ", "the most common complication of rhabdomyolysis is kidney damage . in recent large pediatric studies reported by mannix", "et al and wu et al , it was shown that the rate of acute renal failure in the pediatric patients with rhabdomyolysis ranged from 5% to 8.7% .", "the risk factors that predispose a patient with rhabdomyolysis for developing acute renal injury are creatine kinase level concentration more than 5000 u / l , creatine kinase value upon admission , and slower decline in the serum creatine kinase level .", "other factors include high myoglobin level , persistent or abrupt increase in the potassium or calcium level , as well as persistent metabolic acidosis .", "on the other hand , a recent study by fernandez et al showed that the most reliable predictor of acute renal failure and the need for dialysis is the creatinine level above 1.7 , despite the peak creatine kinase .", "the mainstay for the prevention and treatment of acute kidney injury is early and aggressive volume resuscitation .", "other management options include alkalization of the urine , forced diuresis with mannitol , and loop diuretics . in severe cases or", "in our case , the child developed extremely high creatine kinase levels that were not reported in the literature .", "a thorough literature review was done searching for high creatine kinase levels in patients with acute rhabdomyolysis . in 2014 , a case report described a pediatric patient with mcardle disease who developed rhabdomyolysis with a high 6-digit creatine kinase level reaching 500 000", "2 pediatric cases were reported . in both , the creatine kinase levels reached were 60 000", "a case report of an adolescent with rhabdomyolysis due to undiagnosed hypothyroidism was reported with a creatine kinase level that reached around 34 000", "a research article in 2013 examined the clinical spectrum of patients with rhabdomyolysis presenting to the pediatric emergency department , in which the peak serum level of creatine kinase was 9825.1  23 079.1", "the only risk factor for developing acute renal injury that our patient had was a high peak creatine kinase level upon admission . on the other hand ,", "he did not develop metabolic acidosis and had a normal albumin and electrolytes levels throughout his hospitalization .", "he responded very well to volume resuscitation with rapid decline in the creatine kinase level .", "although he received intravenous immunoglobulin for the fear of guillain - barre syndrome or other underlying immune - mediated diseases , neither the creatine kinase level decline nor the clinical status improvement was affected by the intravenous immunoglobulin administration . the sharp decline in the creatine kinase level", "there are no clues in the family history or the patient s personal history that may suggest an underlying inherited metabolic , muscular , or genetic disease .", ", this could be the first presentation of lipin-1 mutation that causes recurrent rhabdomyolysis in children .", "lipin-1 gene encodes the muscle - specific phosphatidic acid phosphatase , a key enzyme in triglyceride and membrane phospholipid biosynthesis .", "the episodes of rhabdomyolysis are mostly triggered by intercurrent infections and fever and to a lesser extent by fasting or exercise .", "the prognosis of lipin-1 deficiency is poor , with up to one - third of patients dying during an episode of rhabdomyolysis . in about 60% of patients with recurrent rhabdomyolysis ,"]]}
{"article_id": "PMC4218759", "article_text": ["the study was approved by the institutional ethics committee , and informed consent was obtained from all patients in accordance with the tenets of the declaration of helsinki .", "the donors were selected based on these inclusion criteria : myopic spherical refractive error ranging from 0.5 d to 10.00 d with no astigmatism or astigmatism less than 0.5 d ; healthy eyes free from significant systemic and ocular diseases ; and ability to understand the content of the consent form and willingness to undergo serological testing .", "eligible candidates underwent the relex smile procedure with the standard technique11 by the same surgeon ( s.g . ) using the visumax fs laser .", "the cap thickness was 100 m , and the optical zone varied from 6 to 6.5 mm .", "after dissection of both anterior and posterior planes , the lenticule was extracted through a superior 2-mm incision .", "bites were taken from one end to the diagonally opposite end , with the knot tied on the superior aspect of the lenticule to determine the side at the time of subsequent implantation .", "the lenticule was immediately transferred to a sterile plastic vial containing phosphate - buffered solution , labeled with the patient 's identification number , eye , and date , and sent to the cryobank for processing and cryopreservation .", "two types of solutions ( washing solution and cryopreservation solution ) were prepared . the washing solution comprised 10 ml of phosphate - buffered saline ( 1 ) , 200 l of penicillin  streptomycin ( 100 ) , and 200 l of amphotericin b ( 100 ) . the cryopreservation solution comprised 9 ml of tissue culture media ( cryobank wash media ) and 1 ml of 20% dimethyl sulfoxide ( dmso ) .", "the tissue culture medium is a commercially available medium used for semen processing in reproductive biology ( cryocell india pvt ltd , new delhi , india ) .", "the medium contains sodium chloride , potassium chloride , magnesium sulfate , anhydrous potassium phosphate monobasic , calcium chloride , anhydrous sodium bicarbonate , hepes , glucose , sodium pyruvate , sodium lactate , phenol red , and human albumin in company - specified compositions .", "extracted lenticules were washed twice for 10 minutes each in the washing solution ( fig .", "the lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . using a liquid dropper , a stock freezing solution containing 10% human tissue culture medium and 20% dmso ( sigma - aldrich , st louis , mo )", "was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso .", "the cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers ( ibp ; indian oil corporation limited , nasik , india ) .", ", the tissue was transported from the cryobank to the hospital in a liquid nitrogen container .", "the vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied .", "the lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents .", "a visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision .", "the incision was opened with a seibel spatula and the plane of the pocket was dissected .", "the cryopreserved lenticule ( which was matched for the refractive error after correcting for back vertex distance ) was placed on the patient 's cornea with its center marked with gentian violet dye .", "after insertion , the lenticule was spread out , and the center was aligned with the pupillary center .", "a depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes , the novelty of the nomograms , and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required .", "postoperatively , a topical steroid ( 1% prednisolone acetate ; allergan , irvine , ca ) was prescribed for 3 months in a tapering dosage .", "in addition , 5% hypertonic saline drops ( hypersol-5 ; jawa pharmaceuticals , india ) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing .", "patients were examined postoperatively on days 1 and 15 and at 1 , 3 , and 6 months .", "a slit - lamp examination was performed to check for lenticule clarity , position , and wound healing .", "postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward , the following assessments were performed : uncorrected visual acuity , best - corrected visual acuity ( bcva ) , retinoscopy for residual refractive error , topography using orbscan(bausch & lomb - technolas , munchen , germany ) and sirius ( schwind eye - tech solutions , kleinostheim , germany ) , anterior segment optical coherence tomography ( optovue , fremont , ca ) , clinical photography , specular microscopy ( tomey , japan ) , dry eye assessment ( schirmer 's i ) , and aberrometry ( itrace ; tracey technologies , houston , tx ) .", "two types of solutions ( washing solution and cryopreservation solution ) were prepared . the washing solution comprised 10 ml of phosphate - buffered saline ( 1 ) , 200 l of penicillin  streptomycin ( 100 ) , and 200 l of amphotericin b ( 100 ) .", "the cryopreservation solution comprised 9 ml of tissue culture media ( cryobank wash media ) and 1 ml of 20% dimethyl sulfoxide ( dmso ) .", "the tissue culture medium is a commercially available medium used for semen processing in reproductive biology ( cryocell india pvt ltd , new delhi , india ) .", "the medium contains sodium chloride , potassium chloride , magnesium sulfate , anhydrous potassium phosphate monobasic , calcium chloride , anhydrous sodium bicarbonate , hepes , glucose , sodium pyruvate , sodium lactate , phenol red , and human albumin in company - specified compositions .", "extracted lenticules were washed twice for 10 minutes each in the washing solution ( fig .", "the lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . using a liquid dropper , a stock freezing solution containing 10% human tissue culture medium and 20% dmso ( sigma - aldrich , st louis , mo )", "was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso .", "the cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers ( ibp ; indian oil corporation limited , nasik , india ) .", "on the day of surgery , the tissue was transported from the cryobank to the hospital in a liquid nitrogen container .", "the vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied .", "the lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents .", "a visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision .", "the incision was opened with a seibel spatula and the plane of the pocket was dissected .", "the cryopreserved lenticule ( which was matched for the refractive error after correcting for back vertex distance ) was placed on the patient 's cornea with its center marked with gentian violet dye .", "the lenticule was held with 2 forceps and inserted into the pocket . after insertion ,", "the lenticule was spread out , and the center was aligned with the pupillary center .", "a depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes , the novelty of the nomograms , and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required .", "postoperatively , a topical steroid ( 1% prednisolone acetate ; allergan , irvine , ca ) was prescribed for 3 months in a tapering dosage .", "in addition , 5% hypertonic saline drops ( hypersol-5 ; jawa pharmaceuticals , india ) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing .", "patients were examined postoperatively on days 1 and 15 and at 1 , 3 , and 6 months .", "a slit - lamp examination was performed to check for lenticule clarity , position , and wound healing .", "postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward , the following assessments were performed : uncorrected visual acuity , best - corrected visual acuity ( bcva ) , retinoscopy for residual refractive error , topography using orbscan(bausch & lomb - technolas , munchen , germany ) and sirius ( schwind eye - tech solutions , kleinostheim , germany ) , anterior segment optical coherence tomography ( optovue , fremont , ca ) , clinical photography , specular microscopy ( tomey , japan ) , dry eye assessment ( schirmer 's i ) , and aberrometry ( itrace ; tracey technologies , houston , tx ) .", "table 2 provides the details of donors and lenticules used for fili in the 9 eyes treated .", "demographic details of patients who underwent fili ( 7 patients , 9 eyes ) details of donors and lenticules used for fili ( n = 9 ) on day 1 , in the first 2 consecutive eyes , the implanted lenticules had mild to moderate descemet membrane folds that resolved spontaneously within 1 week .", "subsequently , the treated eyes were prescribed hypertonic saline drops , which showed clear lenticules within 48 hours and until the last follow - up time point .", "figure 2 shows serial clinical photographs and anterior segment optical coherence tomography of the eye treated with fili at day 15 and at 6 months post - fili showing clear lenticules with good centration .", "a , serial digital clinical photographs ( 16 ) of 32-year - old woman operated for + 6.5 d hyperopia in the right eye with fili .", "distance from edge of the lenticule to limbus was measured at 3 points and verified at every visit to check for centering and any shift in position .", "b , six - month postoperative anterior segment optical coherence tomography of an eye treated for + 6.5 d hyperopia showing a clear and well - centered lenticule .", "all eyes had an uncorrected visual acuity equal to or better than the preoperative bcva .", "the refractive predictability for hyperopia was fairly accurate , and all eyes had a residual spherical equivalent within 1.0 diopter ( d ) , although in the aphakic eye , the residual spherical equivalent was + 4.1 d. there was an average of 0.75 to 1.00 d against - the - rule astigmatism in all eyes due to the superior 4 mm incision , which reduced and stabilized over time .", "postoperative results of eyes treated with fili ( n = 9 ) table 4 provides changes in anterior and posterior curvature , pachymetry and asphericity ( q value ) following fili .", "all eyes had central corneal steepening with a mean change in anterior keratometry of 3.5 d in the central 3-mm zone .", "there was an average 0.33 d flattening of posterior corneal curvature in all eyes , which was seen more in thicker lenticules compared with the thinner ones .", "figure 3 illustrates changes in anterior and posterior curvature post - fili in an eye , over time .", "changes in anterior and posterior topography , pachymetry , and asphericity post - fili ( n = 9 ) orbscan anterior corneal surface topography ( top ) and sirius posterior elevation ( bottom ) over the 6-month postoperative course after fili for eye with + 6.5 d hyperopia .", "the postoperative q values in all eyes became more negative , suggesting a hyperprolate shift .", "table 5 provides analysis of the epithelial thickness profile , which suggested mild epithelial thinning in the 5-mm zone in all hyperopic eyes , except for the aphakic eye , which showed mild epithelial thickening in the corresponding zone .", "figure 4 illustrates the epithelial thickness map of an eye treated with fili at 6 months .", "epithelial thickness profile on anterior segment optical coherence tomography in the central 5-mm zone epithelial thickness profile with anterior segment optical coherence tomography of an eye 6 months post - fili for + 6.5 d hyperopia .", "higher order aberrations ( hoa ) did not show a significant increase in root mean square values postoperatively in the 8 hyperopic eyes ( p > 0.05 ) .", "endothelial cell count and schirmer i test scores were normal and the changes were not significant at the last follow - up ( p > 0.05 ) ( table 6 ) .", "total hoas at 5-mm pupil size ( n = 8) , ecd , and schirmer 's i scores , pre- and post - fili ( n = 9 )", "the potential advantages of cryopreservation of refractive lenticules have been largely described for use in the same patient , in the event of future ectasia or presbyopia by restoring corneal volume.3 with the increasing number of eyes undergoing relex smile for myopia , and thus , the extracted lenticules as a by - product , it may be a novel idea to preserve these lenticules on a long - term basis using cryopreservation for potential future use and research .", "following establishment of one such tissue bank , we wanted to explore the possibility of use of such cryopreserved refractive lenticules for potential treatment of hyperopia and study the feasibility , safety , efficacy , and reproducibility of this new treatment .", "initially , the amblyopic eyes were studied mainly for changes in refraction and topography following tissue addition .", "there was also the goal of evaluating any loss of bcva and complications , such as haze and tissue rejection .", "even these patients benefited from the surgery , as their refractive error was corrected to their best spectacle - corrected visual acuity and reduced their dependence on eyeglasses . after reviewing the results for 6 months , the indications were extended to normal eyes and bilateral surgery .", "unlike myopia , the correction of hyperopia involves creation of a furrow - like ring zone in the paracentral zone of the cornea .", "both photorefractive keratectomy and lasik are subtractive procedures that steepen the cornea by ablating the midperipheral tissue.14,15 this creates an abnormal hyperprolate shape of the cornea that is associated with significant regression , especially in higher degrees of refractive errors.16 it potentially leads to the induction of hoas like coma and spherical aberrations and loss of bcva.17 fili is an innovative technique based on barraquer 's law of thickness,18 whereby the cornea is made steeper by addition of a lenticule of known thickness and power into a pocket created in a patient 's cornea using the fs laser .", "the concept is similar to epikeratophakia , used to treat aphakia in the past , which had its own disadvantages.19 a fs laser overcomes these problems by creating precise lenticules and incisions at an accurate depth . the lenticular tissue processing technique described by mohamed - noriega et al10", "was modified in this study by replacing bovine serum albumin with human tissue culture media to eliminate the risk of transmission of zoonotic diseases or unexpected adverse reactions due to bovine serum .", "the protocols of gradual staged thawing , which are essential for the whole cornea to prevent endothelial cell injury due to sudden thawing , were not strictly followed in this study .", "microbiological and cellular cultures were not deemed necessary , as the tissue processing and preservation were conducted under strict sterile conditions , and the tissue did not contain vital endothelial cells . also , previous studies have suggested that stromal keratocytes within the extracted lenticules remain viable and can be stimulated to proliferate in appropriate cell culture conditions , even after a prolonged period of cryopreservation.9,10 the change in asphericity after fili suggests an increase in the prolateness of the cornea , which is expected after tissue addition , although the observed change was not seen as much as it is after hyperopic lasik.20 the shape of the cornea is more natural after tissue addition compared with tissue subtraction , which does not lead to significant induction of hoas.21 the total hoas after fili remain within normal acceptable values and may be better than hyperopic lasik / photorefractive keratectomy.22,23 also , tissue addition may be more forgiving for mild decentration , which is not the case with hyperopic lasik", ". the rate of enhancement following hyperopic lasik can be up to 10% or more,24 especially in younger patients .", "after tissue addition , significant changes in topography or regression are not expected ; thus , retreatment rates may be lower .", "epithelial thickness profile is more favorable , which , unlike hyperopic lasik , does not cause midperipheral epithelial hyperplasia to compensate for loss of stromal tissue , hence reducing chances of regression.25 problems of haze and flap - related issues like delayed healing , infection , and dislocation are eliminated , thereby leading to greater patient comfort and faster recovery .", "a fairly good refractive predictability was observed with this technique in the treatment of moderate hyperopia , although results were not highly accurate for correcting very high hyperopia ( eg , aphakia ) . in a case study reported by pradhan", "et al,4 a 10 d lenticule was implanted to achieve correction of aphakia but had a high residual refraction of + 5 d. there are possible explanations for this result .", "first , the technique of pradhan et al of pocket creation was different from the technique used in this study . in the said study ,", "a smile lenticule was first created , followed by dissection of the anterior plane to create a pocket , thus making 2 planes that could possibly cause weakening of the cornea .", "second , the optical zone of the lenticule in the study of pradhan et al was 5.7 mm compared with 6 mm in the current aphakia case , causing comparatively less volume of tissue addition .", "the correction is expected to be relatively higher in larger lenticules because there is more volume of the tissue added . in this hyperopic series ,", "the lenticule size ranged from 6.3 to 6.8 mm . also , the posterior curvature changes as described in the case study of pradhan et al reduced the effect on anterior curvature .", "this was also probably the cause of the high + 4.1 d residual refraction in the current study .", "after compensation , implantation of lenticule of approximately 12.5 d is required , and this is currently not possible with visumax .", "the biomechanics of corneas in these patients were not studied due to lack of equipment such as the corvis ( oculus optikgerte gmbh , wetzlar , germany ) . however , because most of the bowman layer is preserved and tissue is being added , it is assumed that the biomechanics are more stable than lasik , where a flap is cut and tissue is subtracted from the periphery.26 the procedure certainly amounts to corneal transplantation and thus , theoretically carries the risk of future rejection .", "however , unlike traditional full thickness grafts , which have a graft  host junction proximal to host limbus , in this technique , the lenticule was well protected in the host corneal pocket and did not come in direct contact with the host 's limbal vasculature and immune system , making the risk of allogeneic graft rejection remotely possible.27 also , the lenticule was mainly stromal collagen tissue ; the incidence of rejection is significantly low in such a scenario compared with endothelial rejection .", "because no sutures are applied , there are no suture - related complications such as loosening , vascularization , or infection that would potentially trigger graft rejection.28 nevertheless , there is a need to monitor for signs of rejection and treat it like allogeneic graft rejection should rejection occur .", "because it is a reversible procedure , the lenticule can always be explanted in the event of any adverse effects or rejection in future .", "the preliminary results of fili in hyperopia indicate that this may be a better technique than hyperopic lasik for moderate hyperopia in terms of stability , regression , aberrations , and postoperative dry eye .", "although it is a bit early to exactly predict the refractive outcomes , initial data indicate that the cryopreservation technique used is safe in heterologous individuals .", "further studies with a larger cohort and modification of the surgical technique with a goal of reducing postoperative astigmatism and refining nomograms are suggested to determine the long - term safety and refractive effects on the cornea and establish the technique as a valid option to treat hyperopia ."], "abstract_text": ["<S> purpose : to describe the technique of cryopreservation of corneal lenticules extracted after small incision refractive lenticule extraction ( relex smile ) and initial results of femtosecond laser intrastromal lenticular implantation for hyperopia.methods:lenticules were collected from patients undergoing relex smile for the correction of myopia and subjected to a tissue processing technique and cryopreservation . </S>", "<S> these lenticules were subsequently used to treat 8 hyperopic eyes and 1 aphakic eye . </S>", "<S> a femtosecond laser was used to create a pocket into each patient 's cornea , followed by implantation of a cryopreserved lenticule . </S>", "<S> the patients were monitored through follow - up examinations for a mean 155.4 days ( 38310 days).results : the mean interval from storage of lenticules to removal from liquid nitrogen was 96 days ( range , 19178 days ) . </S>", "<S> mean spherical equivalent of hyperopic eyes treated was + 4.50  1.1 diopter ( d ) . </S>", "<S> mean keratometry and pachymetry changed from preoperative 43.9 d and 531.6 m to 47.4 d and 605.2 m , respectively , postoperatively . </S>", "<S> mean residual spherical equivalent for hyperopic eyes was + 0.6 d and + 4.1 d for the aphakic eye . </S>", "<S> none of the eyes showed evidence of rejection or loss of best - corrected visual acuity at the end of the follow - up period.conclusions:the cryopreservation technique seems to be a safe method of long - term storage of refractive lenticules extracted after relex smile for use in allogeneic human subjects . </S>", "<S> it may potentially be a safe and effective alternative to excimer laser ablation for hyperopia because of the low risks of regression , haze , flap - related complications , postoperative dry eye , and higher - order aberrations.clinical trial registration  </S>", "<S> url : http://www.clinicaltrials.gov . </S>", "<S> unique identifier : ctri/2014/01/004331 . </S>"], "labels": null, "section_names": ["MATERIALS AND METHODS", "Tissue Processing and Cryopreservation", "Tissue Processing Protocol", "Preparation of Lenticules for Implantation", "Lenticular Implantation Technique", "RESULTS", "DISCUSSION"], "sections": [["the study was approved by the institutional ethics committee , and informed consent was obtained from all patients in accordance with the tenets of the declaration of helsinki .", "the donors were selected based on these inclusion criteria : myopic spherical refractive error ranging from 0.5 d to 10.00 d with no astigmatism or astigmatism less than 0.5 d ; healthy eyes free from significant systemic and ocular diseases ; and ability to understand the content of the consent form and willingness to undergo serological testing .", "eligible candidates underwent the relex smile procedure with the standard technique11 by the same surgeon ( s.g . ) using the visumax fs laser .", "the cap thickness was 100 m , and the optical zone varied from 6 to 6.5 mm .", "after dissection of both anterior and posterior planes , the lenticule was extracted through a superior 2-mm incision .", "bites were taken from one end to the diagonally opposite end , with the knot tied on the superior aspect of the lenticule to determine the side at the time of subsequent implantation .", "the lenticule was immediately transferred to a sterile plastic vial containing phosphate - buffered solution , labeled with the patient 's identification number , eye , and date , and sent to the cryobank for processing and cryopreservation .", "two types of solutions ( washing solution and cryopreservation solution ) were prepared . the washing solution comprised 10 ml of phosphate - buffered saline ( 1 ) , 200 l of penicillin  streptomycin ( 100 ) , and 200 l of amphotericin b ( 100 ) . the cryopreservation solution comprised 9 ml of tissue culture media ( cryobank wash media ) and 1 ml of 20% dimethyl sulfoxide ( dmso ) .", "the tissue culture medium is a commercially available medium used for semen processing in reproductive biology ( cryocell india pvt ltd , new delhi , india ) .", "the medium contains sodium chloride , potassium chloride , magnesium sulfate , anhydrous potassium phosphate monobasic , calcium chloride , anhydrous sodium bicarbonate , hepes , glucose , sodium pyruvate , sodium lactate , phenol red , and human albumin in company - specified compositions .", "extracted lenticules were washed twice for 10 minutes each in the washing solution ( fig .", "the lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . using a liquid dropper , a stock freezing solution containing 10% human tissue culture medium and 20% dmso ( sigma - aldrich , st louis , mo )", "was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso .", "the cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers ( ibp ; indian oil corporation limited , nasik , india ) .", ", the tissue was transported from the cryobank to the hospital in a liquid nitrogen container .", "the vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied .", "the lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents .", "a visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision .", "the incision was opened with a seibel spatula and the plane of the pocket was dissected .", "the cryopreserved lenticule ( which was matched for the refractive error after correcting for back vertex distance ) was placed on the patient 's cornea with its center marked with gentian violet dye .", "after insertion , the lenticule was spread out , and the center was aligned with the pupillary center .", "a depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes , the novelty of the nomograms , and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required .", "postoperatively , a topical steroid ( 1% prednisolone acetate ; allergan , irvine , ca ) was prescribed for 3 months in a tapering dosage .", "in addition , 5% hypertonic saline drops ( hypersol-5 ; jawa pharmaceuticals , india ) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing .", "patients were examined postoperatively on days 1 and 15 and at 1 , 3 , and 6 months .", "a slit - lamp examination was performed to check for lenticule clarity , position , and wound healing .", "postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward , the following assessments were performed : uncorrected visual acuity , best - corrected visual acuity ( bcva ) , retinoscopy for residual refractive error , topography using orbscan(bausch & lomb - technolas , munchen , germany ) and sirius ( schwind eye - tech solutions , kleinostheim , germany ) , anterior segment optical coherence tomography ( optovue , fremont , ca ) , clinical photography , specular microscopy ( tomey , japan ) , dry eye assessment ( schirmer 's i ) , and aberrometry ( itrace ; tracey technologies , houston , tx ) ."], ["two types of solutions ( washing solution and cryopreservation solution ) were prepared . the washing solution comprised 10 ml of phosphate - buffered saline ( 1 ) , 200 l of penicillin  streptomycin ( 100 ) , and 200 l of amphotericin b ( 100 ) .", "the cryopreservation solution comprised 9 ml of tissue culture media ( cryobank wash media ) and 1 ml of 20% dimethyl sulfoxide ( dmso ) .", "the tissue culture medium is a commercially available medium used for semen processing in reproductive biology ( cryocell india pvt ltd , new delhi , india ) .", "the medium contains sodium chloride , potassium chloride , magnesium sulfate , anhydrous potassium phosphate monobasic , calcium chloride , anhydrous sodium bicarbonate , hepes , glucose , sodium pyruvate , sodium lactate , phenol red , and human albumin in company - specified compositions ."], ["extracted lenticules were washed twice for 10 minutes each in the washing solution ( fig .", "the lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . using a liquid dropper , a stock freezing solution containing 10% human tissue culture medium and 20% dmso ( sigma - aldrich , st louis , mo )", "was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso .", "the cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers ( ibp ; indian oil corporation limited , nasik , india ) ."], ["on the day of surgery , the tissue was transported from the cryobank to the hospital in a liquid nitrogen container .", "the vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied .", "the lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents ."], ["a visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision .", "the incision was opened with a seibel spatula and the plane of the pocket was dissected .", "the cryopreserved lenticule ( which was matched for the refractive error after correcting for back vertex distance ) was placed on the patient 's cornea with its center marked with gentian violet dye .", "the lenticule was held with 2 forceps and inserted into the pocket . after insertion ,", "the lenticule was spread out , and the center was aligned with the pupillary center .", "a depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes , the novelty of the nomograms , and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required .", "postoperatively , a topical steroid ( 1% prednisolone acetate ; allergan , irvine , ca ) was prescribed for 3 months in a tapering dosage .", "in addition , 5% hypertonic saline drops ( hypersol-5 ; jawa pharmaceuticals , india ) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing .", "patients were examined postoperatively on days 1 and 15 and at 1 , 3 , and 6 months .", "a slit - lamp examination was performed to check for lenticule clarity , position , and wound healing .", "postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward , the following assessments were performed : uncorrected visual acuity , best - corrected visual acuity ( bcva ) , retinoscopy for residual refractive error , topography using orbscan(bausch & lomb - technolas , munchen , germany ) and sirius ( schwind eye - tech solutions , kleinostheim , germany ) , anterior segment optical coherence tomography ( optovue , fremont , ca ) , clinical photography , specular microscopy ( tomey , japan ) , dry eye assessment ( schirmer 's i ) , and aberrometry ( itrace ; tracey technologies , houston , tx ) ."], ["table 2 provides the details of donors and lenticules used for fili in the 9 eyes treated .", "demographic details of patients who underwent fili ( 7 patients , 9 eyes ) details of donors and lenticules used for fili ( n = 9 ) on day 1 , in the first 2 consecutive eyes , the implanted lenticules had mild to moderate descemet membrane folds that resolved spontaneously within 1 week .", "subsequently , the treated eyes were prescribed hypertonic saline drops , which showed clear lenticules within 48 hours and until the last follow - up time point .", "figure 2 shows serial clinical photographs and anterior segment optical coherence tomography of the eye treated with fili at day 15 and at 6 months post - fili showing clear lenticules with good centration .", "a , serial digital clinical photographs ( 16 ) of 32-year - old woman operated for + 6.5 d hyperopia in the right eye with fili .", "distance from edge of the lenticule to limbus was measured at 3 points and verified at every visit to check for centering and any shift in position .", "b , six - month postoperative anterior segment optical coherence tomography of an eye treated for + 6.5 d hyperopia showing a clear and well - centered lenticule .", "all eyes had an uncorrected visual acuity equal to or better than the preoperative bcva .", "the refractive predictability for hyperopia was fairly accurate , and all eyes had a residual spherical equivalent within 1.0 diopter ( d ) , although in the aphakic eye , the residual spherical equivalent was + 4.1 d. there was an average of 0.75 to 1.00 d against - the - rule astigmatism in all eyes due to the superior 4 mm incision , which reduced and stabilized over time .", "postoperative results of eyes treated with fili ( n = 9 ) table 4 provides changes in anterior and posterior curvature , pachymetry and asphericity ( q value ) following fili .", "all eyes had central corneal steepening with a mean change in anterior keratometry of 3.5 d in the central 3-mm zone .", "there was an average 0.33 d flattening of posterior corneal curvature in all eyes , which was seen more in thicker lenticules compared with the thinner ones .", "figure 3 illustrates changes in anterior and posterior curvature post - fili in an eye , over time .", "changes in anterior and posterior topography , pachymetry , and asphericity post - fili ( n = 9 ) orbscan anterior corneal surface topography ( top ) and sirius posterior elevation ( bottom ) over the 6-month postoperative course after fili for eye with + 6.5 d hyperopia .", "the postoperative q values in all eyes became more negative , suggesting a hyperprolate shift .", "table 5 provides analysis of the epithelial thickness profile , which suggested mild epithelial thinning in the 5-mm zone in all hyperopic eyes , except for the aphakic eye , which showed mild epithelial thickening in the corresponding zone .", "figure 4 illustrates the epithelial thickness map of an eye treated with fili at 6 months .", "epithelial thickness profile on anterior segment optical coherence tomography in the central 5-mm zone epithelial thickness profile with anterior segment optical coherence tomography of an eye 6 months post - fili for + 6.5 d hyperopia .", "higher order aberrations ( hoa ) did not show a significant increase in root mean square values postoperatively in the 8 hyperopic eyes ( p > 0.05 ) .", "endothelial cell count and schirmer i test scores were normal and the changes were not significant at the last follow - up ( p > 0.05 ) ( table 6 ) .", "total hoas at 5-mm pupil size ( n = 8) , ecd , and schirmer 's i scores , pre- and post - fili ( n = 9 )"], ["the potential advantages of cryopreservation of refractive lenticules have been largely described for use in the same patient , in the event of future ectasia or presbyopia by restoring corneal volume.3 with the increasing number of eyes undergoing relex smile for myopia , and thus , the extracted lenticules as a by - product , it may be a novel idea to preserve these lenticules on a long - term basis using cryopreservation for potential future use and research .", "following establishment of one such tissue bank , we wanted to explore the possibility of use of such cryopreserved refractive lenticules for potential treatment of hyperopia and study the feasibility , safety , efficacy , and reproducibility of this new treatment .", "initially , the amblyopic eyes were studied mainly for changes in refraction and topography following tissue addition .", "there was also the goal of evaluating any loss of bcva and complications , such as haze and tissue rejection .", "even these patients benefited from the surgery , as their refractive error was corrected to their best spectacle - corrected visual acuity and reduced their dependence on eyeglasses . after reviewing the results for 6 months , the indications were extended to normal eyes and bilateral surgery .", "unlike myopia , the correction of hyperopia involves creation of a furrow - like ring zone in the paracentral zone of the cornea .", "both photorefractive keratectomy and lasik are subtractive procedures that steepen the cornea by ablating the midperipheral tissue.14,15 this creates an abnormal hyperprolate shape of the cornea that is associated with significant regression , especially in higher degrees of refractive errors.16 it potentially leads to the induction of hoas like coma and spherical aberrations and loss of bcva.17 fili is an innovative technique based on barraquer 's law of thickness,18 whereby the cornea is made steeper by addition of a lenticule of known thickness and power into a pocket created in a patient 's cornea using the fs laser .", "the concept is similar to epikeratophakia , used to treat aphakia in the past , which had its own disadvantages.19 a fs laser overcomes these problems by creating precise lenticules and incisions at an accurate depth . the lenticular tissue processing technique described by mohamed - noriega et al10", "was modified in this study by replacing bovine serum albumin with human tissue culture media to eliminate the risk of transmission of zoonotic diseases or unexpected adverse reactions due to bovine serum .", "the protocols of gradual staged thawing , which are essential for the whole cornea to prevent endothelial cell injury due to sudden thawing , were not strictly followed in this study .", "microbiological and cellular cultures were not deemed necessary , as the tissue processing and preservation were conducted under strict sterile conditions , and the tissue did not contain vital endothelial cells . also , previous studies have suggested that stromal keratocytes within the extracted lenticules remain viable and can be stimulated to proliferate in appropriate cell culture conditions , even after a prolonged period of cryopreservation.9,10 the change in asphericity after fili suggests an increase in the prolateness of the cornea , which is expected after tissue addition , although the observed change was not seen as much as it is after hyperopic lasik.20 the shape of the cornea is more natural after tissue addition compared with tissue subtraction , which does not lead to significant induction of hoas.21 the total hoas after fili remain within normal acceptable values and may be better than hyperopic lasik / photorefractive keratectomy.22,23 also , tissue addition may be more forgiving for mild decentration , which is not the case with hyperopic lasik", ". the rate of enhancement following hyperopic lasik can be up to 10% or more,24 especially in younger patients .", "after tissue addition , significant changes in topography or regression are not expected ; thus , retreatment rates may be lower .", "epithelial thickness profile is more favorable , which , unlike hyperopic lasik , does not cause midperipheral epithelial hyperplasia to compensate for loss of stromal tissue , hence reducing chances of regression.25 problems of haze and flap - related issues like delayed healing , infection , and dislocation are eliminated , thereby leading to greater patient comfort and faster recovery .", "a fairly good refractive predictability was observed with this technique in the treatment of moderate hyperopia , although results were not highly accurate for correcting very high hyperopia ( eg , aphakia ) . in a case study reported by pradhan", "et al,4 a 10 d lenticule was implanted to achieve correction of aphakia but had a high residual refraction of + 5 d. there are possible explanations for this result .", "first , the technique of pradhan et al of pocket creation was different from the technique used in this study . in the said study ,", "a smile lenticule was first created , followed by dissection of the anterior plane to create a pocket , thus making 2 planes that could possibly cause weakening of the cornea .", "second , the optical zone of the lenticule in the study of pradhan et al was 5.7 mm compared with 6 mm in the current aphakia case , causing comparatively less volume of tissue addition .", "the correction is expected to be relatively higher in larger lenticules because there is more volume of the tissue added . in this hyperopic series ,", "the lenticule size ranged from 6.3 to 6.8 mm . also , the posterior curvature changes as described in the case study of pradhan et al reduced the effect on anterior curvature .", "this was also probably the cause of the high + 4.1 d residual refraction in the current study .", "after compensation , implantation of lenticule of approximately 12.5 d is required , and this is currently not possible with visumax .", "the biomechanics of corneas in these patients were not studied due to lack of equipment such as the corvis ( oculus optikgerte gmbh , wetzlar , germany ) . however , because most of the bowman layer is preserved and tissue is being added , it is assumed that the biomechanics are more stable than lasik , where a flap is cut and tissue is subtracted from the periphery.26 the procedure certainly amounts to corneal transplantation and thus , theoretically carries the risk of future rejection .", "however , unlike traditional full thickness grafts , which have a graft  host junction proximal to host limbus , in this technique , the lenticule was well protected in the host corneal pocket and did not come in direct contact with the host 's limbal vasculature and immune system , making the risk of allogeneic graft rejection remotely possible.27 also , the lenticule was mainly stromal collagen tissue ; the incidence of rejection is significantly low in such a scenario compared with endothelial rejection .", "because no sutures are applied , there are no suture - related complications such as loosening , vascularization , or infection that would potentially trigger graft rejection.28 nevertheless , there is a need to monitor for signs of rejection and treat it like allogeneic graft rejection should rejection occur .", "because it is a reversible procedure , the lenticule can always be explanted in the event of any adverse effects or rejection in future .", "the preliminary results of fili in hyperopia indicate that this may be a better technique than hyperopic lasik for moderate hyperopia in terms of stability , regression , aberrations , and postoperative dry eye .", "although it is a bit early to exactly predict the refractive outcomes , initial data indicate that the cryopreservation technique used is safe in heterologous individuals .", "further studies with a larger cohort and modification of the surgical technique with a goal of reducing postoperative astigmatism and refining nomograms are suggested to determine the long - term safety and refractive effects on the cornea and establish the technique as a valid option to treat hyperopia ."]]}
{"article_id": "PMC3941582", "article_text": ["sepsis is the major cause of death in the intensive care unit . despite improvement of antibiotics treatment and supportive techniques , the mortality of septic shock increases to approximately 60% .", ", it not only helps doctors to make an early diagnosis of sepsis , but also predicts outcomes .", "there have been some biomarkers and cytokines used in both the clinical practice and laboratory including soluble triggering receptor expressed on myeloid cells-1 ( strem-1 ) , procalcitonin ( pct ) , n - terminal probrain natriuretic peptide ( nt - pro - bnp ) , c - reactive protein ( crp ) , interleukin-6 ( il-6 ) , and interleukin-10 ( il-10 ) .", "trem-1 is a recently discovered member of the immunoglobulin superfamily of receptors that is specifically expressed on the surfaces of neutrophils and monocytes .", "strem-1 is a soluble form of trem-1 and is upregulated when exposed to infectious diseases .", "pct is a polypeptide consisting of 116 amino acids and is the precursor of calcitonin ; it was proven useful to identify nonsystemic inflammatory response syndrome and was firstly used in sepsis .", "nt - pro - bnp is a biologically inactive form that is cleaved from the prohormone probrain natriuretic peptide ( pro - bnp ) by proteolytic enzymes before secretion .", "crp is a widely used biomarker to discriminate the inflammatory response to sepsis . il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines during sepsis course .", ".   indicated that strem-1 was more accurate than pct and crp in the diagnosis of sepsis , but others showed that the prognostic utility of serum strem-1 in septic shock was inferior to that of pct .", "the purpose of the study was to compare the prognostic value of biomarkers and cytokines versus clinical severity scores and improved death risk prediction .", "a total of 102 patients with sepsis from single centre hospital intensive care unit were enrolled from december 2010 to august 2012 according to the 2001 international sepsis definition conference .", "exclusion criteria included : age younger than 18 years , preexisting thyroid disease and lung cancer that influence procalcitonin levels , patients with acute coronary syndromes and renal dysfunction , and patients staying in icu less than 24 hours .", "the study was approved by the hospital 's ethics committee and either the patients or their relatives provided informed consent .", "demographic and disease data of patients included age , gender , chief complaints for admission , vital signs , length of stay in icu , infection sites , microorganisms , routine blood test results , liver and kidney functions , coagulation indicators , blood gas analysis , acute physiologic assessment and chronic health evaluation ( apache ) ii scores , and sequential organ failure assessment ( sofa ) scores .", "these were recorded on 3 days ( days 1 , 3 , and 5 ) .", "serum was collected at these same time points and pct , strem-1 , nt - pro - bnp , crp , il-6 , and il-10 levels were determined in the end .", "pct was measured using an enzyme - linked fluorescence analysis kit ( elfa , vidas brahms pct kit , biomerieux sa , france ) .", "strem-1 was determined using a double antibody sandwich elisa ( quantikine human trem-1 immunoassay elisa kit , r & d systems , minneapolis , mn , usa ) .", "nt - pro - bnp was measured with an available immunoassay analyzer ( elecsys 2010 ; roche diagnostics , mannheim , germany ) .", "crp was determined using scattering using a nephelometric assay ( dade - behring , sa paris , france ) . il-6 and", "il-10 were determined using elisa ( immulite ; diagnostics products corporation , los angeles , ca ) .", "quantitative data with normal distributions are given as means  standard deviations ( sd ) .", "quantitative data that were not normally distributed were summarized as medians ( interquartile ranges ) and compared by nonparametric tests ( mann - whitney u test ) .", "we made a logarithmic conversion for the nondistribution data when we did dynamic comparison in figure 2 .", "proportions were used to express qualitative data and the differences in proportions between groups were compared using a chi - square test .", "we compared the characteristics of survivors versus nonsurvivors using univariate analysis and used receiver operating characteristics ( roc ) curves to evaluate prognostic value of the biomarkers and cytokines predicted 28-day mortality . those variables with p values less than 0.05 on univariate analysis were then entered into a multivariate logistic regression analysis to further identify the independent predictors of 28-day mortality .", "there were no significant difference in age and sex of these two groups ( p > 0.05 ) .", "the apache ii and sofa scores of patients in the nonsurvival group were higher than those of patients in the survival group ( p = 0.000 , p = 0.000 , resp . ) , ( table 1 ) .", "serum pct , strem-1 , il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ( p < 0.001 ) .", "there were no differences in nt - pro - bnp and crp , il-10 levels between the two groups ( p > 0.05 ) ( table 2 ) .", "the roc analysis showed that the accuracy of the pct , strem-1 , il-6 , apacheii , and sofa scores on day 1 for the prediction of 28-day mortality was moderate ( auc > 0.7 , p < 0.01 ) , whereas the accuracy of nt - pro - bnp , crp , and il-10 was low ( auc < 0.7 , p > 0.05 ) ( figure 1 ) . comparing auc of pct , strem-1 , and il-6", ", we found that there was no significant difference of auc between strem-1 and pct ( p = 0.2910 ) , and the auc of the two markers were higher than that of il-6 ( p > 0.05 ) .", "meanwhile , there was no significant difference of auc between apacheii and sofa scores ( p = 0.3753 ) .", "the auc of apacheii and sofa scores were higher than those of strem-1 and pct ( p < 0.05 ) ( table 3 ) .", "the baseline day 1 variables that were found to be significantly different between survivors and nonsurvivors on univariate analysis ( pct , strem-1 , il-6 , apacheii , and sofa scores ) were entered into a logistic regression model . among these variables ,", "three variables remained independently associated with 28-day mortality : strem-1 , pct , and sofa score ( table 4 ) .", "median serum biomarkers and cytokines levels were determined on days 1 , 3 , and 5 and were compared between the survival and nonsurvival groups .", "serum pct , strem-1 , and il-6 levels in the nonsurvival group were higher than those in the survival group on days 1 , 3 , and 5 ( p < 0.01 ) .", "there was no difference in nt - pro - bnp levels on day 1 ( p > 0.05 ) , but later the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 ( p < 0.05 ) .", "there were no differences in crp and il-10 levels on days 1 , 3 , and 5 .", "serum pct , strem-1 , il-6 , and nt - pro - bnp levels showed a decrease trend in the survival group ( p < 0.05 ) , but there was no decrease tendency in the nonsurviving group for these four biomarkers ; strem-1 even had a increase trend ( p < 0.05 ) .", "serum crp levels in both surviving and nonsurviving groups had decrease tendency ( p < 0.05 ) ( figure 2 ) .", "recently , pct , strem-1 , crp , and nt - pro - bnp cytokines were widely used to diagnose sepsis and reflect the severity , but the results were not the same .", "meanwhile , there were few studies to put so many biomarkers in one study , particularly how to combine the biomarkers , and clinical severity scores remained unclear .", "the present study showed that the serum levels of strem-1 and pct in nonsurvival group were higher than those in the survival group ; meanwhile , they decreased in survival group , but stayed in high levels even increased in the nonsurvival group during sepsis time course .", "many previous studies have shown that dynamic changes in strem-1 levels could predict survival and mortality of patients at the early stage of sepsis [ 10 , 11 ] .", "strem-1 is widely used to diagnose sepsis [ 7 , 12 ] . in the present study ,", "serum strem-1 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ; it decreased in survival group , but it even increased in the nonsurvival group .", "some studies failed to find the association between strem-1 and poor outcome [ 8 , 13 ] . at a cutoff of 252.05 ", "pg / ml , strem-1 measurements yielded a sensitivity of 85.7% , specificity of 75.7% , positive predictive value of 70.6% , negative predictive value of 88.2% , and an accuracy of 79.4% for differentiating nonsurvivors from survivors .", "pct is normally produced in the c cells of the thyroid gland ; plasma pct levels in healthy humans are approximately 550  pg / ml in normal state ; its half - time is about 2233 hours in serum .", "many tissues and cells except thyroid gland produce and release that pct on systemic inflammation . several previous studies reported pct could serve as a useful tool to distinguish sepsis from systemic inflammatory response syndrome [ 15 , 16 ] . on the other hand , pct could reflect the severity of sepsis and outcome .", "a study by christophe clec'h and coworkers found that serum pct on day 1 was significantly higher in patients with than without septic shock .", "meanwhile , among patients with sepsis , pct concentrations were significantly higher in those who died than in the survivors , at all four measurement time points .", "similar results were drawn from other investigations [ 16 , 18 ] . very few studies failed to find the prognostic value . at a cutoff of 10.64  ng", "/ ml , procalcitonin measurements yielded a sensitivity of 76.2% , specificity of 81.7% , positive predictive value of 53.5% , negative predictive value of 67.8% , and an accuracy of 61.8% for differentiating nonsurvivors from survivors .", "nt - pro - bnp has been found to be a useful markers in the diagnosis , management , and prognosis of patients with congestive heart failure and was secreted into blood in response to atrial or ventricular wall stretch .", "a recent meta - analysis suggested that an elevated nt - pro - bnp level may prove to be a powerful predictor of mortality in septic patients . in our study", ", there was no difference in nt - pro - bnp level between groups on day 1 , but the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 .", "meanwhile , serum nt - pro - bnp level showed a decreased trend in the survival group , but there was no decrease tendency in the nonsurvival group .", "we concluded that nt - pro - bnp may predict sepsis 28-day mortality in different stages .", "one research demonstrated that elevated serum nt - pro - bnp value represented an independent predictor for poor icu outcome in the presence of clinical severity scores ; the cut - off in admission nt - pro - bnp that best predicted outcome was 941  pg / ml .", "crp is an acute phase protein and a sensitive systemic marker of inflammation and tissue damage .", "the secretion of crp begins within 46  h after stimulus , doubles every 8  h thereafter , and peaks at 3650  h .", "the role of crp in sepsis prognostic value seemed different . in our study , there was no significant difference between survivors and nonsurvivors during the three measures , similar to previous study , indicating that crp was just an inflammatory biomarker and failed in reflecting sepsis severity .", "have reported that crp levels in severe sepsis were lower than those in sepsis , suggesting that crp levels did not reflect the severity of sepsis . il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines in sepsis . in our study , serum il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on days 1 , 3 , and 5 .", "the above results showed that il-6 had the prognostic utility for sepsis , whereas il-10 did not show the power .", "surez - santamar and coworkers   enrolled 253 hospitalized septic patients ; they found that il-10 and il-6 were the best predictors , whereas pct showed only moderate predictive value for mortality .", "another study investigated the prognostic value of il-6 , pct , and crp in critically ill patients during the first increase of fever ; only il-6 levels were significantly higher in nonsurvivors compared with survivors , in which prognostic value was superior to pct and crp . in contrast , kawczyski and polakowska   indicated that the predictive value of il-10 plasma concentration was better than that of il-6 . to sum up ,", "strem-1 , pct , and il-6 serum values attribute to the prognosis of sepsis during the time course .", "the dynamic changes of biomarkers and cytokines were more meaningful for predicting the sepsis procession .", "schneider and coworkers   retrospectively analyzed the relationships between serum pct , il-6 , and apacheii score and prognosis of 220 patients on the first day after operation .", "they found that pct was the sole independent predictor of 28-day mortality , in which prognostic ability was superior to those of il-6 and apacheii score .", "zhang et al .   suggested that serum strem-1 levels reflected the severity of sepsis more accurately than those of pct and crp and were more sensitive for dynamic evaluations of sepsis prognosis .", "facing the results , we wonder which was the best predictor and how to combine them together and which was more valuable compared to clinical severity scores .", "apache ii and sofa scores have been widely used to validate mortality risk stratification . in our study , we used roc and logistic regression model to search for the best predictor .", "based on roc analysis , strem-1 and pct showed the equal prognostic ability ( 0.792 for pct , 0.862 for strem-1 , p = 0.291 ) , whereas their prognostic utility was inferior to that of apacheii and sofa scores which had equal power to predict outcome ( 0.923 for apacheii score , 0.953 for sofa score , p = 0.375 ) .", "logistic regression model showed that serum strem-1 , pct , and sofa score were the independent predictors of 28-day mortality , which was supported by other result .", "as far as we know , the interrelationship between strem-1 , pct , nt - pro - bnp , cytokines , and clinical severity scores for mortality prediction in general icu patients has not been previously evaluated .", "our research firstly discovered that strem-1 and pct had the equal prognostic ability for sepsis mortality and were superior to other parameters .", "previous study has indicated that the iteraction of trem-1 and interact adaptor protein dap12 can stimulate neutrophil and monocyte - mediated inflammatory response via the triggering and release of pro - inflammatory cytokines and chemokines .", "strem-1 increases quickly when exposued to infection , and its half - time is short . in bacterial infections ,", "serum pct levels start to rise at 4  h after the onset of systemic infection and peak at between 8 and 24  h ; it decreased 50% every 24 hours along with therapy .", "in contrast , crp rises slowly and peaks 36  h after an endotoxin challenge .", "the mechanism of nt - pro - bnp release in sepsis is complex , and kinetics characteristic is unknown . il-6 and il-10 rise quickly and peak at 24 hours and maintain a short time .", "the patients admitted to icu often delayed more than 24 hours , either crp or cytokines serum concentration was unable to reach the peak at the period of sepsis .", "of course , the exact roles of biomarkers and cytokines in sepsis process are not clear , and need to be further studied .", "firstly , our study chosen a part of sepsis biomarkers and did not put all biomarkers in the research .", "of course , it was a costly and unnecessary task to do so . secondly , every biomarker has its own dynamic characteristics ; meanwhile the patients were not in the same sepsis stages in the study ; thus the explanation for the results would be influenced .", "thirdly , we excluded patients with previous heart diseases history , but we did not perform the ucg to evaluate cardiac function .", "finally , the sample size of the study was small and larger studies are needed .", "in summary , elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers . combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic for sepsis mortality . in the future , in order to improve the accuracy of the prognosis of sepsis , the combination of novel biomarkers and traditional markers of sepsis , reflecting different aspects of sepsis , is an attractive advice and is worthy of further investigation ."], "abstract_text": ["<S> \n objective . to investigate the prognostic significance of serum soluble triggering receptor expressed on myeloid cells-1 ( strem-1 ) , procalcitonin ( pct ) , n - terminal probrain natriuretic peptide ( nt - pro - bnp ) , c - reactive protein ( crp ) , cytokines , and clinical severity scores in patients with sepsis . </S>", "<S> methods . </S>", "<S> a total of 102 patients with sepsis were divided into survival group ( n = 60 ) and nonsurvival group ( n = 42 ) based on 28-day mortality . </S>", "<S> serum levels of biomarkers and cytokines were measured on days 1 , 3 , and 5 after admission to an icu , meanwhile the acute physiology and chronic health evaluation ii ( apache ii ) and sequential organ failure assessment ( sofa ) scores were calculated . results . </S>", "<S> serum strem-1 , pct , and il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ( p < 0.01 ) . </S>", "<S> the area under a roc curve for the prediction of 28 day mortality was 0.792 for pct , 0.856 for strem-1 , 0.953 for sofa score , and 0.923 for apache ii score . </S>", "<S> multivariate logistic analysis showed that serum baseline strem-1 pct levels and sofa score were the independent predictors of 28-day mortality . </S>", "<S> serum pct , strem-1 , and il-6 levels showed a decrease trend over time in the survival group ( p < 0.05 ) . </S>", "<S> serum nt - pro - bnp levels showed the predictive utility from days 3 and 5 ( p < 0.05 ) . conclusion . in summary , elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers . </S>", "<S> combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic utility for sepsis mortality . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Result", "4. Discussion", "5. Conclusions"], "sections": [["sepsis is the major cause of death in the intensive care unit . despite improvement of antibiotics treatment and supportive techniques , the mortality of septic shock increases to approximately 60% .", ", it not only helps doctors to make an early diagnosis of sepsis , but also predicts outcomes .", "there have been some biomarkers and cytokines used in both the clinical practice and laboratory including soluble triggering receptor expressed on myeloid cells-1 ( strem-1 ) , procalcitonin ( pct ) , n - terminal probrain natriuretic peptide ( nt - pro - bnp ) , c - reactive protein ( crp ) , interleukin-6 ( il-6 ) , and interleukin-10 ( il-10 ) .", "trem-1 is a recently discovered member of the immunoglobulin superfamily of receptors that is specifically expressed on the surfaces of neutrophils and monocytes .", "strem-1 is a soluble form of trem-1 and is upregulated when exposed to infectious diseases .", "pct is a polypeptide consisting of 116 amino acids and is the precursor of calcitonin ; it was proven useful to identify nonsystemic inflammatory response syndrome and was firstly used in sepsis .", "nt - pro - bnp is a biologically inactive form that is cleaved from the prohormone probrain natriuretic peptide ( pro - bnp ) by proteolytic enzymes before secretion .", "crp is a widely used biomarker to discriminate the inflammatory response to sepsis . il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines during sepsis course .", ".   indicated that strem-1 was more accurate than pct and crp in the diagnosis of sepsis , but others showed that the prognostic utility of serum strem-1 in septic shock was inferior to that of pct .", "the purpose of the study was to compare the prognostic value of biomarkers and cytokines versus clinical severity scores and improved death risk prediction ."], ["a total of 102 patients with sepsis from single centre hospital intensive care unit were enrolled from december 2010 to august 2012 according to the 2001 international sepsis definition conference .", "exclusion criteria included : age younger than 18 years , preexisting thyroid disease and lung cancer that influence procalcitonin levels , patients with acute coronary syndromes and renal dysfunction , and patients staying in icu less than 24 hours .", "the study was approved by the hospital 's ethics committee and either the patients or their relatives provided informed consent .", "demographic and disease data of patients included age , gender , chief complaints for admission , vital signs , length of stay in icu , infection sites , microorganisms , routine blood test results , liver and kidney functions , coagulation indicators , blood gas analysis , acute physiologic assessment and chronic health evaluation ( apache ) ii scores , and sequential organ failure assessment ( sofa ) scores .", "these were recorded on 3 days ( days 1 , 3 , and 5 ) .", "serum was collected at these same time points and pct , strem-1 , nt - pro - bnp , crp , il-6 , and il-10 levels were determined in the end .", "pct was measured using an enzyme - linked fluorescence analysis kit ( elfa , vidas brahms pct kit , biomerieux sa , france ) .", "strem-1 was determined using a double antibody sandwich elisa ( quantikine human trem-1 immunoassay elisa kit , r & d systems , minneapolis , mn , usa ) .", "nt - pro - bnp was measured with an available immunoassay analyzer ( elecsys 2010 ; roche diagnostics , mannheim , germany ) .", "crp was determined using scattering using a nephelometric assay ( dade - behring , sa paris , france ) . il-6 and", "il-10 were determined using elisa ( immulite ; diagnostics products corporation , los angeles , ca ) .", "quantitative data with normal distributions are given as means  standard deviations ( sd ) .", "quantitative data that were not normally distributed were summarized as medians ( interquartile ranges ) and compared by nonparametric tests ( mann - whitney u test ) .", "we made a logarithmic conversion for the nondistribution data when we did dynamic comparison in figure 2 .", "proportions were used to express qualitative data and the differences in proportions between groups were compared using a chi - square test .", "we compared the characteristics of survivors versus nonsurvivors using univariate analysis and used receiver operating characteristics ( roc ) curves to evaluate prognostic value of the biomarkers and cytokines predicted 28-day mortality . those variables with p values less than 0.05 on univariate analysis were then entered into a multivariate logistic regression analysis to further identify the independent predictors of 28-day mortality ."], ["there were no significant difference in age and sex of these two groups ( p > 0.05 ) .", "the apache ii and sofa scores of patients in the nonsurvival group were higher than those of patients in the survival group ( p = 0.000 , p = 0.000 , resp . ) , ( table 1 ) .", "serum pct , strem-1 , il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ( p < 0.001 ) .", "there were no differences in nt - pro - bnp and crp , il-10 levels between the two groups ( p > 0.05 ) ( table 2 ) .", "the roc analysis showed that the accuracy of the pct , strem-1 , il-6 , apacheii , and sofa scores on day 1 for the prediction of 28-day mortality was moderate ( auc > 0.7 , p < 0.01 ) , whereas the accuracy of nt - pro - bnp , crp , and il-10 was low ( auc < 0.7 , p > 0.05 ) ( figure 1 ) . comparing auc of pct , strem-1 , and il-6", ", we found that there was no significant difference of auc between strem-1 and pct ( p = 0.2910 ) , and the auc of the two markers were higher than that of il-6 ( p > 0.05 ) .", "meanwhile , there was no significant difference of auc between apacheii and sofa scores ( p = 0.3753 ) .", "the auc of apacheii and sofa scores were higher than those of strem-1 and pct ( p < 0.05 ) ( table 3 ) .", "the baseline day 1 variables that were found to be significantly different between survivors and nonsurvivors on univariate analysis ( pct , strem-1 , il-6 , apacheii , and sofa scores ) were entered into a logistic regression model . among these variables ,", "three variables remained independently associated with 28-day mortality : strem-1 , pct , and sofa score ( table 4 ) .", "median serum biomarkers and cytokines levels were determined on days 1 , 3 , and 5 and were compared between the survival and nonsurvival groups .", "serum pct , strem-1 , and il-6 levels in the nonsurvival group were higher than those in the survival group on days 1 , 3 , and 5 ( p < 0.01 ) .", "there was no difference in nt - pro - bnp levels on day 1 ( p > 0.05 ) , but later the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 ( p < 0.05 ) .", "there were no differences in crp and il-10 levels on days 1 , 3 , and 5 .", "serum pct , strem-1 , il-6 , and nt - pro - bnp levels showed a decrease trend in the survival group ( p < 0.05 ) , but there was no decrease tendency in the nonsurviving group for these four biomarkers ; strem-1 even had a increase trend ( p < 0.05 ) .", "serum crp levels in both surviving and nonsurviving groups had decrease tendency ( p < 0.05 ) ( figure 2 ) ."], ["recently , pct , strem-1 , crp , and nt - pro - bnp cytokines were widely used to diagnose sepsis and reflect the severity , but the results were not the same .", "meanwhile , there were few studies to put so many biomarkers in one study , particularly how to combine the biomarkers , and clinical severity scores remained unclear .", "the present study showed that the serum levels of strem-1 and pct in nonsurvival group were higher than those in the survival group ; meanwhile , they decreased in survival group , but stayed in high levels even increased in the nonsurvival group during sepsis time course .", "many previous studies have shown that dynamic changes in strem-1 levels could predict survival and mortality of patients at the early stage of sepsis [ 10 , 11 ] .", "strem-1 is widely used to diagnose sepsis [ 7 , 12 ] . in the present study ,", "serum strem-1 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 ; it decreased in survival group , but it even increased in the nonsurvival group .", "some studies failed to find the association between strem-1 and poor outcome [ 8 , 13 ] . at a cutoff of 252.05 ", "pg / ml , strem-1 measurements yielded a sensitivity of 85.7% , specificity of 75.7% , positive predictive value of 70.6% , negative predictive value of 88.2% , and an accuracy of 79.4% for differentiating nonsurvivors from survivors .", "pct is normally produced in the c cells of the thyroid gland ; plasma pct levels in healthy humans are approximately 550  pg / ml in normal state ; its half - time is about 2233 hours in serum .", "many tissues and cells except thyroid gland produce and release that pct on systemic inflammation . several previous studies reported pct could serve as a useful tool to distinguish sepsis from systemic inflammatory response syndrome [ 15 , 16 ] . on the other hand , pct could reflect the severity of sepsis and outcome .", "a study by christophe clec'h and coworkers found that serum pct on day 1 was significantly higher in patients with than without septic shock .", "meanwhile , among patients with sepsis , pct concentrations were significantly higher in those who died than in the survivors , at all four measurement time points .", "similar results were drawn from other investigations [ 16 , 18 ] . very few studies failed to find the prognostic value . at a cutoff of 10.64  ng", "/ ml , procalcitonin measurements yielded a sensitivity of 76.2% , specificity of 81.7% , positive predictive value of 53.5% , negative predictive value of 67.8% , and an accuracy of 61.8% for differentiating nonsurvivors from survivors .", "nt - pro - bnp has been found to be a useful markers in the diagnosis , management , and prognosis of patients with congestive heart failure and was secreted into blood in response to atrial or ventricular wall stretch .", "a recent meta - analysis suggested that an elevated nt - pro - bnp level may prove to be a powerful predictor of mortality in septic patients . in our study", ", there was no difference in nt - pro - bnp level between groups on day 1 , but the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 .", "meanwhile , serum nt - pro - bnp level showed a decreased trend in the survival group , but there was no decrease tendency in the nonsurvival group .", "we concluded that nt - pro - bnp may predict sepsis 28-day mortality in different stages .", "one research demonstrated that elevated serum nt - pro - bnp value represented an independent predictor for poor icu outcome in the presence of clinical severity scores ; the cut - off in admission nt - pro - bnp that best predicted outcome was 941  pg / ml .", "crp is an acute phase protein and a sensitive systemic marker of inflammation and tissue damage .", "the secretion of crp begins within 46  h after stimulus , doubles every 8  h thereafter , and peaks at 3650  h .", "the role of crp in sepsis prognostic value seemed different . in our study , there was no significant difference between survivors and nonsurvivors during the three measures , similar to previous study , indicating that crp was just an inflammatory biomarker and failed in reflecting sepsis severity .", "have reported that crp levels in severe sepsis were lower than those in sepsis , suggesting that crp levels did not reflect the severity of sepsis . il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines in sepsis . in our study , serum il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on days 1 , 3 , and 5 .", "the above results showed that il-6 had the prognostic utility for sepsis , whereas il-10 did not show the power .", "surez - santamar and coworkers   enrolled 253 hospitalized septic patients ; they found that il-10 and il-6 were the best predictors , whereas pct showed only moderate predictive value for mortality .", "another study investigated the prognostic value of il-6 , pct , and crp in critically ill patients during the first increase of fever ; only il-6 levels were significantly higher in nonsurvivors compared with survivors , in which prognostic value was superior to pct and crp . in contrast , kawczyski and polakowska   indicated that the predictive value of il-10 plasma concentration was better than that of il-6 . to sum up ,", "strem-1 , pct , and il-6 serum values attribute to the prognosis of sepsis during the time course .", "the dynamic changes of biomarkers and cytokines were more meaningful for predicting the sepsis procession .", "schneider and coworkers   retrospectively analyzed the relationships between serum pct , il-6 , and apacheii score and prognosis of 220 patients on the first day after operation .", "they found that pct was the sole independent predictor of 28-day mortality , in which prognostic ability was superior to those of il-6 and apacheii score .", "zhang et al .   suggested that serum strem-1 levels reflected the severity of sepsis more accurately than those of pct and crp and were more sensitive for dynamic evaluations of sepsis prognosis .", "facing the results , we wonder which was the best predictor and how to combine them together and which was more valuable compared to clinical severity scores .", "apache ii and sofa scores have been widely used to validate mortality risk stratification . in our study , we used roc and logistic regression model to search for the best predictor .", "based on roc analysis , strem-1 and pct showed the equal prognostic ability ( 0.792 for pct , 0.862 for strem-1 , p = 0.291 ) , whereas their prognostic utility was inferior to that of apacheii and sofa scores which had equal power to predict outcome ( 0.923 for apacheii score , 0.953 for sofa score , p = 0.375 ) .", "logistic regression model showed that serum strem-1 , pct , and sofa score were the independent predictors of 28-day mortality , which was supported by other result .", "as far as we know , the interrelationship between strem-1 , pct , nt - pro - bnp , cytokines , and clinical severity scores for mortality prediction in general icu patients has not been previously evaluated .", "our research firstly discovered that strem-1 and pct had the equal prognostic ability for sepsis mortality and were superior to other parameters .", "previous study has indicated that the iteraction of trem-1 and interact adaptor protein dap12 can stimulate neutrophil and monocyte - mediated inflammatory response via the triggering and release of pro - inflammatory cytokines and chemokines .", "strem-1 increases quickly when exposued to infection , and its half - time is short . in bacterial infections ,", "serum pct levels start to rise at 4  h after the onset of systemic infection and peak at between 8 and 24  h ; it decreased 50% every 24 hours along with therapy .", "in contrast , crp rises slowly and peaks 36  h after an endotoxin challenge .", "the mechanism of nt - pro - bnp release in sepsis is complex , and kinetics characteristic is unknown . il-6 and il-10 rise quickly and peak at 24 hours and maintain a short time .", "the patients admitted to icu often delayed more than 24 hours , either crp or cytokines serum concentration was unable to reach the peak at the period of sepsis .", "of course , the exact roles of biomarkers and cytokines in sepsis process are not clear , and need to be further studied .", "firstly , our study chosen a part of sepsis biomarkers and did not put all biomarkers in the research .", "of course , it was a costly and unnecessary task to do so . secondly , every biomarker has its own dynamic characteristics ; meanwhile the patients were not in the same sepsis stages in the study ; thus the explanation for the results would be influenced .", "thirdly , we excluded patients with previous heart diseases history , but we did not perform the ucg to evaluate cardiac function .", "finally , the sample size of the study was small and larger studies are needed ."], ["in summary , elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers . combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic for sepsis mortality . in the future , in order to improve the accuracy of the prognosis of sepsis , the combination of novel biomarkers and traditional markers of sepsis , reflecting different aspects of sepsis , is an attractive advice and is worthy of further investigation ."]]}
{"article_id": "PMC4342312", "article_text": ["nine male competitive cyclists participated in this study . their mean  sd age , height , and body mass were 33.3  7.5 yr , 184  4 cm , and 77.3  7.0 kg , respectively .", "they had a maximal oxygen consumption ( vo2max ) of 4.8  0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418  16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .", "all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .", "the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .", "the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .", "they trained 14 h 40 min  4 h 40 min per week before the acclimatization and 13 h 1 min  1 h 6 min during heat acclimatization .", "they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .", "the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .", "the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .", "the participants spent a minimum of 4 hd outside ( average temperature , 34c  3c ; relative humidity , 18%  5% ) but slept , rested , and ate indoors in an air - conditioned facility .", "two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .", "the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .", "cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .", "the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt", ", the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .", "the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,", "the riders had access to hr , speed , distance , and power output data .", "wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .", "environmental temperature was 8.2c  3.5c during ttc and 36.0c  0.4c , 37.4c  0.8c , and 36.2c  1.6c during tth-1 , -2 , and -3 , respectively .", "relative humidity was 30%  8% during ttc and 13%  1% , 16%  2% , and 12%  3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )", ", the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .", "power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .", "all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .", "hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .", "rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .", "in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .", "body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .", "continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions  10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .", "anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse  geisser corrections were applied where appropriate . in case of post hoc comparisons ,", "reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .", "effect sizes are described in terms of partial eta - squared (  ; with   0.06 representing moderate difference and   0.14 , large difference ) .", "data are presented as mean  sd along with the mean differences ( 95% confidence interval ) .", "nine male competitive cyclists participated in this study . their mean  sd age , height , and body mass were 33.3  7.5 yr , 184  4 cm , and 77.3  7.0 kg , respectively .", "they had a maximal oxygen consumption ( vo2max ) of 4.8  0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418  16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .", "all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .", "the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .", "the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .", "they trained 14 h 40 min  4 h 40 min per week before the acclimatization and 13 h 1 min  1 h 6 min during heat acclimatization .", "they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .", "the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .", "the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .", "the participants spent a minimum of 4 hd outside ( average temperature , 34c  3c ; relative humidity , 18%  5% ) but slept , rested , and ate indoors in an air - conditioned facility .", "two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .", "the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .", "cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .", "the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt", ", the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .", "the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,", "the riders had access to hr , speed , distance , and power output data .", "wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .", "environmental temperature was 8.2c  3.5c during ttc and 36.0c  0.4c , 37.4c  0.8c , and 36.2c  1.6c during tth-1 , -2 , and -3 , respectively .", "relative humidity was 30%  8% during ttc and 13%  1% , 16%  2% , and 12%  3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )", ", the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .", "power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .", "all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .", "hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .", "rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .", "in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .", "body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .", "continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions  10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .", "anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse  geisser corrections were applied where appropriate . in case of post hoc comparisons ,", "reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .", "effect sizes are described in terms of partial eta - squared (  ; with   0.06 representing moderate difference and   0.14 , large difference ) .", "data are presented as mean  sd along with the mean differences ( 95% confidence interval ) .", "nine male competitive cyclists participated in this study . their mean  sd age , height , and body mass were 33.3  7.5 yr , 184  4 cm , and 77.3  7.0 kg , respectively .", "they had a maximal oxygen consumption ( vo2max ) of 4.8  0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418  16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .", "all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .", "the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .", "the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .", "they trained 14 h 40 min  4 h 40 min per week before the acclimatization and 13 h 1 min  1 h 6 min during heat acclimatization .", "they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .", "the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .", "the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .", "the participants spent a minimum of 4 hd outside ( average temperature , 34c  3c ; relative humidity , 18%  5% ) but slept , rested , and ate indoors in an air - conditioned facility .", "two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .", "the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .", "cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .", "the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt", ", the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .", "the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,", "the riders had access to hr , speed , distance , and power output data .", "wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .", "environmental temperature was 8.2c  3.5c during ttc and 36.0c  0.4c , 37.4c  0.8c , and 36.2c  1.6c during tth-1 , -2 , and -3 , respectively .", "relative humidity was 30%  8% during ttc and 13%  1% , 16%  2% , and 12%  3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )", ", the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .", "power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .", "all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .", "hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .", "rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .", "in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .", "body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .", "continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions  10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .", "anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse  geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .", "effect sizes are described in terms of partial eta - squared (  ; with   0.06 representing moderate difference and   0.14 , large difference ) .", "data are presented as mean  sd along with the mean differences ( 95% confidence interval ) .", "there was a large (  = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s  3 min 26 s ) and tth-3 ( 65 min 37 s  3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s  6 min 26 s ) and tth-2 ( 69 min 25 s  4 min 37 s ) ( all p < 0.01 ) .", "speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .", "speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .", "average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .", "this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .", "1 ) (  = 0.90 , p < 0.001 ) and showed a large (  = 0.51 ) and significant ( p < 0.001 ) time  condition interaction .", "the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .", "1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .", "power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .", "power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .", "power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .", "ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .", "2 , hr significantly increased during the tt (  = 0.67 , p < 0.001 ) relative to testing conditions (  = 0.24 , p < 0.001 ) .", "the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .", "consequently , hr was different between conditions (  = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .", "hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *", "rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (  = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .", "2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .", "2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,", "the differences in body mass loss ( p = 0.071 ,  = 0.28 ) and fluid consumption ( p = 0.095 ,  = 0.30 ) between the tth did not reach significance .", "there was a large (  = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s  3 min 26 s ) and tth-3 ( 65 min 37 s  3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s  6 min 26 s ) and tth-2 ( 69 min 25 s  4 min 37 s ) ( all p < 0.01 ) .", "speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .", "speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .", "average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .", "this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .", "1 ) (  = 0.90 , p < 0.001 ) and showed a large (  = 0.51 ) and significant ( p < 0.001 ) time  condition interaction .", "the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .", "1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .", "power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .", "power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .", "power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .", "ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .", "2 , hr significantly increased during the tt (  = 0.67 , p < 0.001 ) relative to testing conditions (  = 0.24 , p < 0.001 ) .", "the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .", "consequently , hr was different between conditions (  = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .", "hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *", "rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (  = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .", "2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .", "2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,", "the differences in body mass loss ( p = 0.071 ,  = 0.28 ) and fluid consumption ( p = 0.095 ,  = 0.30 ) between the tth did not reach significance .", "there was a large (  = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s  3 min 26 s ) and tth-3 ( 65 min 37 s  3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s  6 min 26 s ) and tth-2 ( 69 min 25 s  4 min 37 s ) ( all p < 0.01 ) .", "speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .", "speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .", "average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .", "this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .", "1 ) (  = 0.90 , p < 0.001 ) and showed a large (  = 0.51 ) and significant ( p < 0.001 ) time  condition interaction .", "the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .", "1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .", "power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .", "power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .", "power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .", "ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .", "2 , hr significantly increased during the tt (  = 0.67 , p < 0.001 ) relative to testing conditions (  = 0.24 , p < 0.001 ) .", "the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .", "consequently , hr was different between conditions (  = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .", "hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *", "rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (  = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .", "2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .", "2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,", "the differences in body mass loss ( p = 0.071 ,  = 0.28 ) and fluid consumption ( p = 0.095 ,  = 0.30 ) between the tth did not reach significance .", "the current study is the first to determine the effects of acute heat exposure and heat acclimatization on performance and pacing during outdoor cycling tt in experienced cyclists .", "the cyclists initiated all tt in the heat with a similar power output as maintained during the first 20% of ttc .", "however , while maintaining similar hr , they subsequently experienced a marked decrease in power output and speed in the heat .", "these decrements were progressively restored with heat acclimatization , despite core temperature in all tt in the heat increasing significantly more than that in cool conditions .", "our data showed that mean power output during tth-1 decreased by 16%  5% in unacclimatized cyclists .", "this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .", "in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .", "these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .", "however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .", "our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16%  5% on the first day of heat exposure ( tth-1 )", "relative to ttc , the decrement was reduced to 8%  4% after 1 wk of training in the heat ( tth-2 ) and to 3%  4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement", ", our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .", "one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,", "this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .", "1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .", "our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .", "indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .", "although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .", "it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .", "moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .", "rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .", "given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .", "notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .", "this similar work rate adopted seems to correspond to a critical power ( 16 ) .", "given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .", "however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .", "initial power output was similar between trials and decreased thereafter in relation to acclimatization state .", "indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .", "consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .", "the current study is the first to investigate cycling performance during outdoor tt in a hot environment .", ". however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,", "the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) .", "therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .", "interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .", "the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level .", "in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .", "consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,", "air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .", "thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different .", "our data showed that mean power output during tth-1 decreased by 16%  5% in unacclimatized cyclists .", "this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .", "in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages .", "to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .", "these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .", "however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .", "our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16%  5% on the first day of heat exposure ( tth-1 )", "relative to ttc , the decrement was reduced to 8%  4% after 1 wk of training in the heat ( tth-2 ) and to 3%  4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement", ", our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .", "one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,", "this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .", "1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .", "our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .", "indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .", "although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .", "it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .", "moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .", "rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .", "given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .", "notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .", "this similar work rate adopted seems to correspond to a critical power ( 16 ) .", "given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .", "however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .", "initial power output was similar between trials and decreased thereafter in relation to acclimatization state .", "indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .", "consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .", "the current study is the first to investigate cycling performance during outdoor tt in a hot environment .", "however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,", "the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore ,", "it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .", "interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .", "the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast", ", air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .", "consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,", "air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .", "thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different .", "our data showed that mean power output during tth-1 decreased by 16%  5% in unacclimatized cyclists .", "this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .", "in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages .", "to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .", "these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .", "however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .", "our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16%  5% on the first day of heat exposure ( tth-1 )", "relative to ttc , the decrement was reduced to 8%  4% after 1 wk of training in the heat ( tth-2 ) and to 3%  4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement", ", our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .", "one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,", "this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .", "1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .", "our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .", "indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .", "although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .", "it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .", "moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .", "rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .", "given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .", "notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .", "this similar work rate adopted seems to correspond to a critical power ( 16 ) .", "given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .", "however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .", "initial power output was similar between trials and decreased thereafter in relation to acclimatization state .", "indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .", "consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .", "the current study is the first to investigate cycling performance during outdoor tt in a hot environment .", "however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,", "the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) .", "therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .", "interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .", "the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level .", "in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .", "consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,", "air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .", "thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different .", "the novel findings of this investigation are that competitive cyclists performing an outdoor tt undertake their effort at the same power output , irrespective of the environmental conditions or previous experience .", "consequently , non  heat - acclimatized cyclists are incapable of sustaining this absolute effort .", "however , the decrement in power output is partly recovered after 1 wk of heat acclimatization and almost fully restored after 2 wk .", "furthermore , our data seem to confirm that sustainable power output is related to a given relative intensity ( 24 ) , which is partly reflected in the maintenance of hr within a certain range . finally , because of the reduction in air density associated with cycling in hot ambient conditions , speed was not different between tth-3 and ttc ."], "abstract_text": ["<S> abstractpurposethis study aimed to determine the effects of heat acclimatization on performance and pacing during outdoor cycling time trials ( tt , 43.4 km ) in the heat.methodsnine cyclists performed three tt in hot ambient conditions ( tth , approximately 37c ) on the first ( tth-1 ) , sixth ( tth-2 ) , and 14th ( tth-3 ) days of training in the heat . data were compared with the average of two tt in cool condition ( approximately 8c ) performed before and after heat acclimatization ( ttc).resultstth-1 ( 77  6 min ) was slower ( p = 0.001 ) than tth-2 ( 69  5 min ) , and both were slower ( p < 0.01 ) than ttc and tth-3 ( 66  3 and 66  4 min , respectively ) , without differences between ttc and tth-3 ( p > 0.05 ) . </S>", "<S> the cyclists initiated the first 20% of all tt at a similar power output , irrespective of climate and acclimatization status ; however , during tth-1 , they subsequently had a marked decrease in power output , which was partly attenuated after 6 d of acclimatization and was further reduced after 14 d. hr was higher during the first 20% of tth-1 than that in the other tt ( p < 0.05 ) , but there were no differences between conditions from 30% onward . </S>", "<S> final rectal temperature was similar in all tth ( 40.2c  0.4c , p = 1.000 ) and higher than that in ttc ( 38.5c  0.6c , p < 0.001).conclusionsafter 2 wk of acclimatization , trained cyclists are capable of completing a prolonged tt in a similar time in the heat compared with cool conditions , whereas in the unacclimatized state , they experienced a marked decrease in power output during the tth . </S>"], "labels": null, "section_names": ["METHODS", "None", "Participants", "General procedure", "Measures", "Statistical analyses", "RESULTS", "None", "Performance", "Pacing of power output", "HR and temperature responses", "DISCUSSION", "None", "Power output and pacing", "Effect of air density and air movement while cycling in the heat", "CONCLUSIONS"], "sections": [["nine male competitive cyclists participated in this study . their mean  sd age , height , and body mass were 33.3  7.5 yr , 184  4 cm , and 77.3  7.0 kg , respectively .", "they had a maximal oxygen consumption ( vo2max ) of 4.8  0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418  16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .", "all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .", "the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .", "the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .", "they trained 14 h 40 min  4 h 40 min per week before the acclimatization and 13 h 1 min  1 h 6 min during heat acclimatization .", "they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .", "the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .", "the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .", "the participants spent a minimum of 4 hd outside ( average temperature , 34c  3c ; relative humidity , 18%  5% ) but slept , rested , and ate indoors in an air - conditioned facility .", "two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .", "the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .", "cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .", "the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt", ", the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .", "the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,", "the riders had access to hr , speed , distance , and power output data .", "wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .", "environmental temperature was 8.2c  3.5c during ttc and 36.0c  0.4c , 37.4c  0.8c , and 36.2c  1.6c during tth-1 , -2 , and -3 , respectively .", "relative humidity was 30%  8% during ttc and 13%  1% , 16%  2% , and 12%  3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )", ", the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .", "power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .", "all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .", "hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .", "rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .", "in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .", "body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .", "continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions  10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .", "anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse  geisser corrections were applied where appropriate . in case of post hoc comparisons ,", "reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .", "effect sizes are described in terms of partial eta - squared (  ; with   0.06 representing moderate difference and   0.14 , large difference ) .", "data are presented as mean  sd along with the mean differences ( 95% confidence interval ) ."], ["nine male competitive cyclists participated in this study . their mean  sd age , height , and body mass were 33.3  7.5 yr , 184  4 cm , and 77.3  7.0 kg , respectively .", "they had a maximal oxygen consumption ( vo2max ) of 4.8  0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418  16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .", "all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .", "the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .", "the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .", "they trained 14 h 40 min  4 h 40 min per week before the acclimatization and 13 h 1 min  1 h 6 min during heat acclimatization .", "they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .", "the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .", "the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .", "the participants spent a minimum of 4 hd outside ( average temperature , 34c  3c ; relative humidity , 18%  5% ) but slept , rested , and ate indoors in an air - conditioned facility .", "two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .", "the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .", "cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .", "the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt", ", the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .", "the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,", "the riders had access to hr , speed , distance , and power output data .", "wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .", "environmental temperature was 8.2c  3.5c during ttc and 36.0c  0.4c , 37.4c  0.8c , and 36.2c  1.6c during tth-1 , -2 , and -3 , respectively .", "relative humidity was 30%  8% during ttc and 13%  1% , 16%  2% , and 12%  3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )", ", the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .", "power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .", "all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .", "hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .", "rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .", "in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .", "body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .", "continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions  10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .", "anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse  geisser corrections were applied where appropriate . in case of post hoc comparisons ,", "reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .", "effect sizes are described in terms of partial eta - squared (  ; with   0.06 representing moderate difference and   0.14 , large difference ) .", "data are presented as mean  sd along with the mean differences ( 95% confidence interval ) ."], ["nine male competitive cyclists participated in this study . their mean  sd age , height , and body mass were 33.3  7.5 yr , 184  4 cm , and 77.3  7.0 kg , respectively .", "they had a maximal oxygen consumption ( vo2max ) of 4.8  0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418  16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .", "all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .", "the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .", "the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .", "they trained 14 h 40 min  4 h 40 min per week before the acclimatization and 13 h 1 min  1 h 6 min during heat acclimatization .", "they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) ."], ["the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .", "the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .", "the participants spent a minimum of 4 hd outside ( average temperature , 34c  3c ; relative humidity , 18%  5% ) but slept , rested , and ate indoors in an air - conditioned facility .", "two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .", "the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .", "cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .", "the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt", ", the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .", "the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,", "the riders had access to hr , speed , distance , and power output data .", "wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .", "environmental temperature was 8.2c  3.5c during ttc and 36.0c  0.4c , 37.4c  0.8c , and 36.2c  1.6c during tth-1 , -2 , and -3 , respectively .", "relative humidity was 30%  8% during ttc and 13%  1% , 16%  2% , and 12%  3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )", ", the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively ."], ["power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .", "all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .", "hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .", "rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .", "in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .", "body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) ."], ["continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions  10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .", "anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse  geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .", "effect sizes are described in terms of partial eta - squared (  ; with   0.06 representing moderate difference and   0.14 , large difference ) .", "data are presented as mean  sd along with the mean differences ( 95% confidence interval ) ."], ["there was a large (  = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s  3 min 26 s ) and tth-3 ( 65 min 37 s  3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s  6 min 26 s ) and tth-2 ( 69 min 25 s  4 min 37 s ) ( all p < 0.01 ) .", "speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .", "speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .", "average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .", "this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .", "1 ) (  = 0.90 , p < 0.001 ) and showed a large (  = 0.51 ) and significant ( p < 0.001 ) time  condition interaction .", "the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .", "1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .", "power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .", "power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .", "power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .", "ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .", "2 , hr significantly increased during the tt (  = 0.67 , p < 0.001 ) relative to testing conditions (  = 0.24 , p < 0.001 ) .", "the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .", "consequently , hr was different between conditions (  = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .", "hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *", "rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (  = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .", "2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .", "2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,", "the differences in body mass loss ( p = 0.071 ,  = 0.28 ) and fluid consumption ( p = 0.095 ,  = 0.30 ) between the tth did not reach significance ."], ["there was a large (  = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s  3 min 26 s ) and tth-3 ( 65 min 37 s  3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s  6 min 26 s ) and tth-2 ( 69 min 25 s  4 min 37 s ) ( all p < 0.01 ) .", "speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .", "speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .", "average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .", "this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .", "1 ) (  = 0.90 , p < 0.001 ) and showed a large (  = 0.51 ) and significant ( p < 0.001 ) time  condition interaction .", "the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .", "1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .", "power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .", "power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .", "power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .", "ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .", "2 , hr significantly increased during the tt (  = 0.67 , p < 0.001 ) relative to testing conditions (  = 0.24 , p < 0.001 ) .", "the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .", "consequently , hr was different between conditions (  = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .", "hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *", "rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (  = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .", "2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .", "2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,", "the differences in body mass loss ( p = 0.071 ,  = 0.28 ) and fluid consumption ( p = 0.095 ,  = 0.30 ) between the tth did not reach significance ."], ["there was a large (  = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s  3 min 26 s ) and tth-3 ( 65 min 37 s  3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s  6 min 26 s ) and tth-2 ( 69 min 25 s  4 min 37 s ) ( all p < 0.01 ) .", "speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .", "speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) ."], ["average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .", "this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .", "1 ) (  = 0.90 , p < 0.001 ) and showed a large (  = 0.51 ) and significant ( p < 0.001 ) time  condition interaction .", "the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .", "1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .", "power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .", "power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .", "power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .", "ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively ."], ["2 , hr significantly increased during the tt (  = 0.67 , p < 0.001 ) relative to testing conditions (  = 0.24 , p < 0.001 ) .", "the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .", "consequently , hr was different between conditions (  = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .", "hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *", "rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (  = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .", "2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .", "2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,", "the differences in body mass loss ( p = 0.071 ,  = 0.28 ) and fluid consumption ( p = 0.095 ,  = 0.30 ) between the tth did not reach significance ."], ["the current study is the first to determine the effects of acute heat exposure and heat acclimatization on performance and pacing during outdoor cycling tt in experienced cyclists .", "the cyclists initiated all tt in the heat with a similar power output as maintained during the first 20% of ttc .", "however , while maintaining similar hr , they subsequently experienced a marked decrease in power output and speed in the heat .", "these decrements were progressively restored with heat acclimatization , despite core temperature in all tt in the heat increasing significantly more than that in cool conditions .", "our data showed that mean power output during tth-1 decreased by 16%  5% in unacclimatized cyclists .", "this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .", "in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .", "these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .", "however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .", "our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16%  5% on the first day of heat exposure ( tth-1 )", "relative to ttc , the decrement was reduced to 8%  4% after 1 wk of training in the heat ( tth-2 ) and to 3%  4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement", ", our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .", "one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,", "this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .", "1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .", "our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .", "indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .", "although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .", "it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .", "moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .", "rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .", "given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .", "notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .", "this similar work rate adopted seems to correspond to a critical power ( 16 ) .", "given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .", "however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .", "initial power output was similar between trials and decreased thereafter in relation to acclimatization state .", "indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .", "consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .", "the current study is the first to investigate cycling performance during outdoor tt in a hot environment .", ". however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,", "the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) .", "therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .", "interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .", "the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level .", "in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .", "consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,", "air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .", "thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different ."], ["our data showed that mean power output during tth-1 decreased by 16%  5% in unacclimatized cyclists .", "this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .", "in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages .", "to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .", "these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .", "however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .", "our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16%  5% on the first day of heat exposure ( tth-1 )", "relative to ttc , the decrement was reduced to 8%  4% after 1 wk of training in the heat ( tth-2 ) and to 3%  4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement", ", our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .", "one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,", "this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .", "1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .", "our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .", "indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .", "although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .", "it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .", "moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .", "rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .", "given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .", "notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .", "this similar work rate adopted seems to correspond to a critical power ( 16 ) .", "given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .", "however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .", "initial power output was similar between trials and decreased thereafter in relation to acclimatization state .", "indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .", "consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .", "the current study is the first to investigate cycling performance during outdoor tt in a hot environment .", "however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,", "the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore ,", "it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .", "interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .", "the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast", ", air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .", "consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,", "air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .", "thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different ."], ["our data showed that mean power output during tth-1 decreased by 16%  5% in unacclimatized cyclists .", "this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .", "in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages .", "to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .", "these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .", "however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .", "our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16%  5% on the first day of heat exposure ( tth-1 )", "relative to ttc , the decrement was reduced to 8%  4% after 1 wk of training in the heat ( tth-2 ) and to 3%  4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement", ", our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .", "one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,", "this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .", "1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .", "our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .", "indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .", "although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .", "it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .", "moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .", "rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .", "given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .", "notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .", "this similar work rate adopted seems to correspond to a critical power ( 16 ) .", "given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .", "however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .", "initial power output was similar between trials and decreased thereafter in relation to acclimatization state .", "indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .", "consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow ."], ["the current study is the first to investigate cycling performance during outdoor tt in a hot environment .", "however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,", "the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) .", "therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .", "interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .", "the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level .", "in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .", "consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,", "air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .", "thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different ."], ["the novel findings of this investigation are that competitive cyclists performing an outdoor tt undertake their effort at the same power output , irrespective of the environmental conditions or previous experience .", "consequently , non  heat - acclimatized cyclists are incapable of sustaining this absolute effort .", "however , the decrement in power output is partly recovered after 1 wk of heat acclimatization and almost fully restored after 2 wk .", "furthermore , our data seem to confirm that sustainable power output is related to a given relative intensity ( 24 ) , which is partly reflected in the maintenance of hr within a certain range . finally , because of the reduction in air density associated with cycling in hot ambient conditions , speed was not different between tth-3 and ttc ."]]}
{"article_id": "PMC4960498", "article_text": ["worksites in glass and metal works , foundries , rolling mills , in the vicinity of hardening furnaces , rotary kilns in the cement industry , high - temperature laboratory furnaces , as well as firefighting , are associated , in addition to common mechanical hazards ( e.g. , due to splashes of molten metals and slag ) , sparks and direct contact with open fires , with hazards caused by emission of harmful optic radiation within the visible ( vis ) and infrared ( ir ) spectrum range .", "excessive exposure to ir radiation leads to dryness of the mucous membranes , burns of the eyeball and development of cataracts .", "protection against thermal hazards due to excessive amounts of ir radiation reaching the eyes is provided by protective optic filters installed in spectacles , goggles or face protections . besides protecting the user against hazardous ir radiation", ", such filters should ensure that the object / area of work can be seen clearly . if the level of ir radiation is very high ,", "the use of filters reflecting radiation is recommended , as it allows the temperature increase in the filter itself to be reduced .", "the filters currently used for the protection of the face and eyes against hazardous ir radiation are of metallic reflective type , made of glass or organic material ( mainly polycarbonate ) bases coated with a single metallic layer for reflectance of ir radiation or absorption filters .", "absorption filters are manufactured from dyed glass obtained in a mass staining process by introduction of a factor modifying the radiation within the vis and near infrared ( nir ) spectrum range passing through the filter .", "the effect of a protective optical filter obtained as a result of this process is based on absorption of a large proportion of optic radiation , whereas only a small proportion of radiation the filter is exposed to is reflected .", "this is an undesirable phenomenon , contributing to deterioration of the filter user 's comfort .", "reflective metallic filters are obtained by deposition of a metallic layer of copper ( cu ) or gold ( au ) on mineral or organic bases .", "such filters take advantage of the characteristic properties of the metals used in their production , i.e. , significant reflection coefficient for ir radiation and significant transmittance for the vis spectrum . despite the fact that the technique of deposition of interference layers has been known for many years   the application of thin film ir filters in eye protections to block", "the previous papers published by the authors of this study   contain descriptions of the structure of interference filters designed for application in eye protections ", "highly effective in reduction of ir radiation with high level of transmission of the vis spectrum .", "the authors investigated also the optical and mechanical parameters of the newly developed protective optic filters manufactured with the use of interference technologies .", "the analysis of the results of the conducted tests demonstrated numerous advantages of interference technology application in the construction of protective filters , including : improved resistance to damage of the external protective filter surface ( cracking , scratching etc . ) as well as high reflection coefficients for the nir spectrum .", "the aim of the present study was to compare the optic properties of protective filters manufactured by taking advantage of the interference technology and those coated with a single metallic layer reflecting ir radiation .", "the coefficients characterizing their optic parameters , allowing to assess the protective properties within the vis and ir spectrum range were compared .", "the following coefficients were analysed : \n luminous transmittance (  \n v),ir transmittance within the 7801400  nm range (  \n a),ir transmittance within the 7802000  nm range (  \n n),ir reflectance within the 7802000  nm range ( r \n n),ir absorptance within the 7802000  nm range ( a \n n),shade number ( n ) . \n ", "luminous transmittance (  \n v ) , ir transmittance within the 7801400  nm range (  \n a ) , ir transmittance within the 7802000  nm range (  \n n ) , ir reflectance within the 7802000  nm range ( r \n n ) , ir absorptance within the 7802000  nm range ( a \n n ) , the level of protection ( shade number ) is determined on the basis of luminous transmittance ( the higher protection level correlates with the higher level of ir radiation blocked and appropriately lower vis light transmittance ) .", "protection level consists of a code number ( e.g. , 4 ) for ir filters and a shade number ( e.g. , 3 , 5 , 7 ) calculated according to the following equation : \n ( 1 ) \n where  \n v \n =  luminous transmittance . for the particular level of protection , the mean ir transmittance within the 7801400 and 7802000  nm ranges as well as the ir reflectance and absorptance within the 7802000  nm range are determined .", "for comparative analysis of the metallic reflective filters available in the market versus the interference filters developed by the authors , the following samples were prepared for the tests : \n off the shelf metallic reflective filters on a polycarbonate base ( flat parallel plate of 50  mm diameter and 2  mm thickness with a metallic layer of copper ( cu )  protection levels : 4 - 3 , 4 - 5 , 4 - 7);interference filters on a polycarbonate base ( flat parallel plate of 50  mm diameter with interference coating made up of the following materials : aluminium , h4  latio3 substance and silicon dioxide ) . \n  off the shelf metallic reflective filters on a polycarbonate base ( flat parallel plate of 50  mm diameter and 2  mm thickness with a metallic layer of copper ( cu )  protection levels : 4 - 3 , 4 - 5 , 4 - 7 ) ; interference filters on a polycarbonate base ( flat parallel plate of 50  mm diameter with interference coating made up of the following materials : aluminium , h4  latio3 substance and silicon dioxide ) .", "the protection levels , luminous transmittance and symbols used for marking the tested filters are presented in table 1 . \n", "characteristics of the filters.symbolluminous transmittance v ( % ) protection levelfilter typecu 4 - 314.1334 - 3metallic reflectiveitf 4 - 39.0824 - 3interferencecu 4 - 52.0214 - 5metallic reflectiveitf 4 - 51.4704 - 5interferencecu 4 - 70.3534 - 7metallic reflectiveitf", "4 - 70.1604 - 7interferencenote : protection level consists of code number ( 4 ) for infrared filters and shade number ( 3 , 5 , 7 ) calculated according to equation ( 1 ) . \n ", "note : protection level consists of code number ( 4 ) for infrared filters and shade number ( 3 , 5 , 7 ) calculated according to equation ( 1 ) .", "optical properties of the filters were measured with a cary 5000 spectrophotometer ( varian , australia ) . the spectrophotometer enabled transmittance or reflectance of optical radiation within the vis and nir spectrum to be recorded with 1  nm steps and 0.001% resolution level .", "spectral transmittance and reflectance measurements were carried out within the wavelength range of 7802000  nm .", "the quartz halogen lamp was used as a source of radiation . to detect transmitted or reflected radiation", "the photomultiplier tube and lead sulphide detector were used for vis and nir spectrum , respectively .", "spectral transmittance was measured with incident radiation falling normally on the filter surface in the visual centre ( defined as in ) . for spectral reflectance", "the measurements were taken every second owing to the high absorption of investigated filters within the nir region .", "since the minimum averaging time of the cary 5000 spectrophotometer was 0.0125  s , the values of transmittance or reflectance were measured 125 times for a given wavelength .", "the aforementioned coefficients were calculated from the following equations [ 1012 ] : \n luminous transmittance \n ( 2 ) \n\n where ( )  =  spectral transmittance , v( )  =  spectral visibility function of the average human eye for daylight or night vision , s \n d65( )  =  spectral energy distribution of standard illuminant d65 ; \n ir transmittance within the 7801400  nm range \n ( 3 ) \n\n where ( )  =  spectral transmittance ; \n ir transmittance within the 7802000  nm range \n ( 4 ) \n\n where ( )  =  spectral transmittance ; \n ir reflectance within the 7802000  nm range \n ( 5 ) \n\n where ( )  =  spectral reflectance ; \n ir absorptance within the 7802000  nm range \n ( 6 ) \n\n where r \n n  =  ir reflectance within the 7802000  nm range ,  \n n  =  ir transmittance within the 7802000  nm range .", "luminous transmittance \n ( 2 ) \n  ir transmittance within the 7801400  nm range \n ( 3 ) \n  ir transmittance within the 7802000  nm range \n ( 4 ) \n  ir reflectance within the 7802000  nm range \n ( 5 ) \n  ir absorptance within the 7802000  nm range \n ( 6 ) \n  the factor before integrals corresponding to the value of the measurement step at the measurement of spectral characteristics .", "transmittance and reflectance characteristics of the studied filters.note : cu 4 - 3  =  metallic reflective filter ; itf 4 - 3  =  interference filter ; cu 4 - 5  =  metallic reflective filter ; itf 4 - 5  =  interference filter ; cu 4 - 7  =  metallic reflective filter ; itf 4 - 7  =  interference filter . \n ", "table 2 presents the values of coefficients determined on the basis of equations ( 3)(6 ) . \n", "infrared transmittance within the 7801400 and 7802000  nm range , infrared reflectance within the 7802000  nm range and infrared absorptance within the 7802000  nm range . ", "infrared transmittance a and n ( % ) infrared reflectance rn ( % ) infrared absorptance an ( % ) symbola 7801400 ( nm)n 7802000 ( nm)rn 7802000 ( nm)an 7802000 ( nm)cu 4 - 31.4860.94283.51615.542itf 4 - 31.0030.56382.08417.353cu 4 - 50.2160.13487.31912.547itf 4 - 50.0380.00691.0378.957cu 4 - 70.0040.02388.30811.669itf 4 - 70.0040.03994.3125.649 \n", "optical radiation within the 7801400  nm spectrum range passes through the anterior compartment of the eye , the lens and reaches the retina . exposure to an excessive amount of radiation from this range", "can result in burns of the retina and , in extreme cases , its permanent damage .", ", chronic long - term exposure may lead to the development of cataracts , referred to as ir or glass worker 's cataracts . in view of the above", "the results presented on the graphs in figure 1 and the determined transmittance values ( see table 2 ) indicate unequivocally more effective blocking of ir radiation provided by interference filters .", "the values of  \n a and  \n n for interference filters are lower even by an order of magnitude than those obtained for the metallic reflective filters .", "for instance , the mean value of ir transmittance within the 7802000  nm range for interference filters with protection level 4 - 5 ( one of the most common protection levels used in the metallurgic industry ) equals 0.006% and is significantly lower than the maximum acceptable value specified in , which is 10.6% .", "for all the investigated filters , the values of r \n n exceed 60% , which classifies them in the group of filters with increased ir reflectance .", "the levels of the ir absorptance for metallic reflective filters and interference filters of 4 - 3 and 4 - 5 protection class are similar . for the highest of the investigated protection levels  4 - 7 ", "the above findings are important for selection of a filter appropriate for the radiation source .", "the selection of filter protection level is dependent on the temperature of the radiation source .", "the 4 - 7 level is used for radiation sources whose temperature exceeds 1600  k ( e.g. , occurs in the metallurgical industry ) . with such high temperatures ,", "reduction of filter heating as a result of exposure to ir radiation is important . as it follows from comparative analysis of the optic properties of filters taking advantage of the interference technology and filters coated with a single metallic layer reflecting ir radiation", ", the interference filters provide a more effective blocking of ir radiation in comparison with the currently used protective filters ."], "abstract_text": ["<S> the paper analyses the selected optical parameters of protective optic filters used for protection of the eyes against hazardous radiation within the visible ( vis ) and near infrared ( nir ) spectrum range . </S>", "<S> the indexes characterizing transmission and reflection of optic radiation incident on the filter are compared . </S>", "<S> as it follows from the completed analysis , the newly developed interference filters provide more effective blocking of infrared radiation in comparison with the currently used protective filters . </S>"], "labels": null, "section_names": ["Introduction", "Test samples  filters protecting against hazardous IR radiation", "Testing methodology", "Results", "Discussion", "Disclosure statement"], "sections": [["worksites in glass and metal works , foundries , rolling mills , in the vicinity of hardening furnaces , rotary kilns in the cement industry , high - temperature laboratory furnaces , as well as firefighting , are associated , in addition to common mechanical hazards ( e.g. , due to splashes of molten metals and slag ) , sparks and direct contact with open fires , with hazards caused by emission of harmful optic radiation within the visible ( vis ) and infrared ( ir ) spectrum range .", "excessive exposure to ir radiation leads to dryness of the mucous membranes , burns of the eyeball and development of cataracts .", "protection against thermal hazards due to excessive amounts of ir radiation reaching the eyes is provided by protective optic filters installed in spectacles , goggles or face protections . besides protecting the user against hazardous ir radiation", ", such filters should ensure that the object / area of work can be seen clearly . if the level of ir radiation is very high ,", "the use of filters reflecting radiation is recommended , as it allows the temperature increase in the filter itself to be reduced .", "the filters currently used for the protection of the face and eyes against hazardous ir radiation are of metallic reflective type , made of glass or organic material ( mainly polycarbonate ) bases coated with a single metallic layer for reflectance of ir radiation or absorption filters .", "absorption filters are manufactured from dyed glass obtained in a mass staining process by introduction of a factor modifying the radiation within the vis and near infrared ( nir ) spectrum range passing through the filter .", "the effect of a protective optical filter obtained as a result of this process is based on absorption of a large proportion of optic radiation , whereas only a small proportion of radiation the filter is exposed to is reflected .", "this is an undesirable phenomenon , contributing to deterioration of the filter user 's comfort .", "reflective metallic filters are obtained by deposition of a metallic layer of copper ( cu ) or gold ( au ) on mineral or organic bases .", "such filters take advantage of the characteristic properties of the metals used in their production , i.e. , significant reflection coefficient for ir radiation and significant transmittance for the vis spectrum . despite the fact that the technique of deposition of interference layers has been known for many years   the application of thin film ir filters in eye protections to block", "the previous papers published by the authors of this study   contain descriptions of the structure of interference filters designed for application in eye protections ", "highly effective in reduction of ir radiation with high level of transmission of the vis spectrum .", "the authors investigated also the optical and mechanical parameters of the newly developed protective optic filters manufactured with the use of interference technologies .", "the analysis of the results of the conducted tests demonstrated numerous advantages of interference technology application in the construction of protective filters , including : improved resistance to damage of the external protective filter surface ( cracking , scratching etc . ) as well as high reflection coefficients for the nir spectrum .", "the aim of the present study was to compare the optic properties of protective filters manufactured by taking advantage of the interference technology and those coated with a single metallic layer reflecting ir radiation .", "the coefficients characterizing their optic parameters , allowing to assess the protective properties within the vis and ir spectrum range were compared .", "the following coefficients were analysed : \n luminous transmittance (  \n v),ir transmittance within the 7801400  nm range (  \n a),ir transmittance within the 7802000  nm range (  \n n),ir reflectance within the 7802000  nm range ( r \n n),ir absorptance within the 7802000  nm range ( a \n n),shade number ( n ) . \n ", "luminous transmittance (  \n v ) , ir transmittance within the 7801400  nm range (  \n a ) , ir transmittance within the 7802000  nm range (  \n n ) , ir reflectance within the 7802000  nm range ( r \n n ) , ir absorptance within the 7802000  nm range ( a \n n ) , the level of protection ( shade number ) is determined on the basis of luminous transmittance ( the higher protection level correlates with the higher level of ir radiation blocked and appropriately lower vis light transmittance ) .", "protection level consists of a code number ( e.g. , 4 ) for ir filters and a shade number ( e.g. , 3 , 5 , 7 ) calculated according to the following equation : \n ( 1 ) \n where  \n v \n =  luminous transmittance . for the particular level of protection , the mean ir transmittance within the 7801400 and 7802000  nm ranges as well as the ir reflectance and absorptance within the 7802000  nm range are determined ."], ["for comparative analysis of the metallic reflective filters available in the market versus the interference filters developed by the authors , the following samples were prepared for the tests : \n off the shelf metallic reflective filters on a polycarbonate base ( flat parallel plate of 50  mm diameter and 2  mm thickness with a metallic layer of copper ( cu )  protection levels : 4 - 3 , 4 - 5 , 4 - 7);interference filters on a polycarbonate base ( flat parallel plate of 50  mm diameter with interference coating made up of the following materials : aluminium , h4  latio3 substance and silicon dioxide ) . \n  off the shelf metallic reflective filters on a polycarbonate base ( flat parallel plate of 50  mm diameter and 2  mm thickness with a metallic layer of copper ( cu )  protection levels : 4 - 3 , 4 - 5 , 4 - 7 ) ; interference filters on a polycarbonate base ( flat parallel plate of 50  mm diameter with interference coating made up of the following materials : aluminium , h4  latio3 substance and silicon dioxide ) .", "the protection levels , luminous transmittance and symbols used for marking the tested filters are presented in table 1 . \n", "characteristics of the filters.symbolluminous transmittance v ( % ) protection levelfilter typecu 4 - 314.1334 - 3metallic reflectiveitf 4 - 39.0824 - 3interferencecu 4 - 52.0214 - 5metallic reflectiveitf 4 - 51.4704 - 5interferencecu 4 - 70.3534 - 7metallic reflectiveitf", "4 - 70.1604 - 7interferencenote : protection level consists of code number ( 4 ) for infrared filters and shade number ( 3 , 5 , 7 ) calculated according to equation ( 1 ) . \n ", "note : protection level consists of code number ( 4 ) for infrared filters and shade number ( 3 , 5 , 7 ) calculated according to equation ( 1 ) ."], ["optical properties of the filters were measured with a cary 5000 spectrophotometer ( varian , australia ) . the spectrophotometer enabled transmittance or reflectance of optical radiation within the vis and nir spectrum to be recorded with 1  nm steps and 0.001% resolution level .", "spectral transmittance and reflectance measurements were carried out within the wavelength range of 7802000  nm .", "the quartz halogen lamp was used as a source of radiation . to detect transmitted or reflected radiation", "the photomultiplier tube and lead sulphide detector were used for vis and nir spectrum , respectively .", "spectral transmittance was measured with incident radiation falling normally on the filter surface in the visual centre ( defined as in ) . for spectral reflectance", "the measurements were taken every second owing to the high absorption of investigated filters within the nir region .", "since the minimum averaging time of the cary 5000 spectrophotometer was 0.0125  s , the values of transmittance or reflectance were measured 125 times for a given wavelength .", "the aforementioned coefficients were calculated from the following equations [ 1012 ] : \n luminous transmittance \n ( 2 ) \n\n where ( )  =  spectral transmittance , v( )  =  spectral visibility function of the average human eye for daylight or night vision , s \n d65( )  =  spectral energy distribution of standard illuminant d65 ; \n ir transmittance within the 7801400  nm range \n ( 3 ) \n\n where ( )  =  spectral transmittance ; \n ir transmittance within the 7802000  nm range \n ( 4 ) \n\n where ( )  =  spectral transmittance ; \n ir reflectance within the 7802000  nm range \n ( 5 ) \n\n where ( )  =  spectral reflectance ; \n ir absorptance within the 7802000  nm range \n ( 6 ) \n\n where r \n n  =  ir reflectance within the 7802000  nm range ,  \n n  =  ir transmittance within the 7802000  nm range .", "luminous transmittance \n ( 2 ) \n  ir transmittance within the 7801400  nm range \n ( 3 ) \n  ir transmittance within the 7802000  nm range \n ( 4 ) \n  ir reflectance within the 7802000  nm range \n ( 5 ) \n  ir absorptance within the 7802000  nm range \n ( 6 ) \n  the factor before integrals corresponding to the value of the measurement step at the measurement of spectral characteristics ."], ["transmittance and reflectance characteristics of the studied filters.note : cu 4 - 3  =  metallic reflective filter ; itf 4 - 3  =  interference filter ; cu 4 - 5  =  metallic reflective filter ; itf 4 - 5  =  interference filter ; cu 4 - 7  =  metallic reflective filter ; itf 4 - 7  =  interference filter . \n ", "table 2 presents the values of coefficients determined on the basis of equations ( 3)(6 ) . \n", "infrared transmittance within the 7801400 and 7802000  nm range , infrared reflectance within the 7802000  nm range and infrared absorptance within the 7802000  nm range . ", "infrared transmittance a and n ( % ) infrared reflectance rn ( % ) infrared absorptance an ( % ) symbola 7801400 ( nm)n 7802000 ( nm)rn 7802000 ( nm)an 7802000 ( nm)cu 4 - 31.4860.94283.51615.542itf 4 - 31.0030.56382.08417.353cu 4 - 50.2160.13487.31912.547itf 4 - 50.0380.00691.0378.957cu 4 - 70.0040.02388.30811.669itf 4 - 70.0040.03994.3125.649 \n"], ["optical radiation within the 7801400  nm spectrum range passes through the anterior compartment of the eye , the lens and reaches the retina . exposure to an excessive amount of radiation from this range", "can result in burns of the retina and , in extreme cases , its permanent damage .", ", chronic long - term exposure may lead to the development of cataracts , referred to as ir or glass worker 's cataracts . in view of the above", "the results presented on the graphs in figure 1 and the determined transmittance values ( see table 2 ) indicate unequivocally more effective blocking of ir radiation provided by interference filters .", "the values of  \n a and  \n n for interference filters are lower even by an order of magnitude than those obtained for the metallic reflective filters .", "for instance , the mean value of ir transmittance within the 7802000  nm range for interference filters with protection level 4 - 5 ( one of the most common protection levels used in the metallurgic industry ) equals 0.006% and is significantly lower than the maximum acceptable value specified in , which is 10.6% .", "for all the investigated filters , the values of r \n n exceed 60% , which classifies them in the group of filters with increased ir reflectance .", "the levels of the ir absorptance for metallic reflective filters and interference filters of 4 - 3 and 4 - 5 protection class are similar . for the highest of the investigated protection levels  4 - 7 ", "the above findings are important for selection of a filter appropriate for the radiation source .", "the selection of filter protection level is dependent on the temperature of the radiation source .", "the 4 - 7 level is used for radiation sources whose temperature exceeds 1600  k ( e.g. , occurs in the metallurgical industry ) . with such high temperatures ,", "reduction of filter heating as a result of exposure to ir radiation is important . as it follows from comparative analysis of the optic properties of filters taking advantage of the interference technology and filters coated with a single metallic layer reflecting ir radiation", ", the interference filters provide a more effective blocking of ir radiation in comparison with the currently used protective filters ."], [""]]}
{"article_id": "PMC3781519", "article_text": ["this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) .", "the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010", "the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s .", "veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas .", "it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history .", "as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) .", "the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems .", "adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 .", "patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date .", "all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements .", "the final sample included patients who were enrolled in the database for at least 12 months after the index date .", "average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) .", "patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status .", "demographic information included age on index date , sex , race , bmi , and year of index date .", "the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes .", "medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits .", "characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and  tests for categorical variables .", "clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) .", "for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle .", "the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use .", "all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle .", "the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle .", "diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) .", "dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications .", "the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis .", "the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a  distribution , and adjusted results are reported as annualized incremental cost differences .", "all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use .", "sas software version 9.2 was used to conduct statistical analyses , and a two - tailed  level of 0.05 was used to determine statistical significance .", "this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) .", "the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010", "the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s .", "veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas .", "it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history .", "as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) .", "the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems .", "adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 .", "patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date .", "all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements .", "the final sample included patients who were enrolled in the database for at least 12 months after the index date .", "average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) .", "patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status .", "demographic information included age on index date , sex , race , bmi , and year of index date .", "the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes .", "medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits .", "characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and  tests for categorical variables .", "clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) . for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle .", "the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use .", "all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle .", "the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle .", "diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) .", "dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications .", "the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis .", "the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a  distribution , and adjusted results are reported as annualized incremental cost differences .", "all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use .", "sas software version 9.2 was used to conduct statistical analyses , and a two - tailed  level of 0.05 was used to determine statistical significance .", "of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis .", "as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m .", "almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) .", "rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) .", "patient baseline characteristics , demographics , comorbidities , complications , medications , and resource use the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone ,", "was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig .", "dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg .", "in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers . after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization ,", "dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig .", "2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) . similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . *", "p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig .", "diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal .", "of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis .", "as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m .", "almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) .", "rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) .", "the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone ,", "was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig .", "dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg .", "in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers .", "after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig .", "2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) .", "similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal .", "* p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 .", "after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig .", "diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . *", "the study results showed that , compared with the achievement of only the ldl - c goal , achievement of both hba1c and ldl - c goals is associated with a lower risk of microvascular complications , acs , and cardiovascular surgeries ( pci or cabg ) , lower utilization of health care resources , and lower costs of care , but no additional effect was observed for the composite cardiovascular - related end point .", "in addition , dual - goal achievement relative to hba1c goal achievement is associated with a lower risk of the composite cardiovascular - related end point , cabg , and outpatient visits . to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label ,", "randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) .", "a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study ,", "lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) .", "these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement .", "in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c .", "the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) .", "we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements .", "current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration .", "our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals .", "the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) .", "this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) .", "our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers .", "our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study ,", "achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) .", "this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes .", "our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone .", "our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings . for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s .", "patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) .", "a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm , compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) .", "first , the data used for this study include laboratory measurements for patients over time .", "second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements .", "this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models .", "first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model .", "second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions .", "third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records .", "fourth , because we used vha data , the patients in our study were predominantly male .", "although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed .", "finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c , ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion ,", "veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone", ". these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes .", "to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label ,", "randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) .", "a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study ,", "lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) .", "these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement .", "in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c .", "the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) .", "we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements .", "current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration .", "our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals .", "the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) .", "this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) .", "our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers .", "our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study ,", "achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) .", "this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes .", "our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone .", "our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings .", "for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s .", "patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) . a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm ,", "compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) .", "first , the data used for this study include laboratory measurements for patients over time .", "second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements .", "this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models .", "first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model .", "second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions .", "third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records .", "fourth , because we used vha data , the patients in our study were predominantly male .", "although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed . finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c", ", ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion , this retrospective study among u.s .", "veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone .", "these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes ."], "abstract_text": ["<S> objectivethis study compared the clinical and economic benefits associated with dual - goal achievement , glycated hemoglobin ( hba1c ) < 7% ( 53 mmol / mol ) and ldl cholesterol ( ldl - c ) < 100 mg / dl , with achievement of only the ldl - c goal or only the hba1c goal in veterans with type 2 diabetes mellitus ( t2dm).research design and methodsthis retrospective cohort analysis evaluated electronic medical records ( veterans integrated service network 16 ) in adult t2 dm patients with two or more measurements of ldl - c and hba1c between 1 january 2004 and 30 june 2010 ( n = 75,646 ) . </S>", "<S> cox proportional hazards models were used to compare microvascular and cardiovascular outcomes by goal achievement status ; generalized linear regression models were used to assess diabetes - related resource utilization ( hospitalization days and number of outpatient visits ) and medical service costs.resultsrelative to achievement of only the ldl - c goal , dual - goal achievement was associated with lower risk of microvascular complications ( adjusted hazard ratio [ ahr ] 0.79 ) , acute coronary syndrome ( 0.88 ) , percutaneous coronary intervention ( 0.78 ) , and coronary artery bypass graft ( cabg ) ( 0.74 ) ; it was also associated with fewer hospitalization days ( adjusted incidence rate ratio [ airr ] 0.93 ) and outpatient visits ( 0.88 ) , as well as lower diabetes - related annual medical costs ( $130.89 ) . </S>", "<S> compared with achievement of only the hba1c goal , dual - goal achievement was associated with lower risk of the composite cardiovascular - related end point ( ahr 0.87 ) and cabg ( ahr 0.62 ) , as well as fewer outpatient visits ( airr 0.98).conclusionsachieving both hba1c and ldl - c goals in diabetes care is associated with additional clinical and economic benefits , as compared with the achievement of either goal alone . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Data source", "Sample selection", "Data preparation", "Patient characteristics", "Clinical outcomes", "Economic outcomes", "RESULTS", "Patient characteristics", "Clinical outcomes", "Resource utilization", "Medical service costs", "CONCLUSIONS", "Clinical outcomes: dual- vs. single-goal achievement", "Clinical outcomes: single- vs. no-goal achievement", "Economic outcomes"], "sections": [["this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) .", "the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010", "the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s .", "veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas .", "it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history .", "as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) .", "the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems .", "adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 .", "patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date .", "all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements .", "the final sample included patients who were enrolled in the database for at least 12 months after the index date .", "average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) .", "patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status .", "demographic information included age on index date , sex , race , bmi , and year of index date .", "the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes .", "medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits .", "characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and  tests for categorical variables .", "clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) .", "for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle .", "the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use .", "all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle .", "the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle .", "diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) .", "dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications .", "the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis .", "the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a  distribution , and adjusted results are reported as annualized incremental cost differences .", "all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use .", "sas software version 9.2 was used to conduct statistical analyses , and a two - tailed  level of 0.05 was used to determine statistical significance ."], ["this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) .", "the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010", "the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s .", "veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas .", "it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history .", "as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) .", "the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems ."], ["adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 .", "patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date .", "all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements .", "the final sample included patients who were enrolled in the database for at least 12 months after the index date ."], ["average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) ."], ["patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status .", "demographic information included age on index date , sex , race , bmi , and year of index date .", "the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes .", "medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits .", "characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and  tests for categorical variables ."], ["clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) . for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle .", "the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use .", "all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle ."], ["the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle .", "diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) .", "dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications .", "the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis .", "the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a  distribution , and adjusted results are reported as annualized incremental cost differences .", "all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use .", "sas software version 9.2 was used to conduct statistical analyses , and a two - tailed  level of 0.05 was used to determine statistical significance ."], ["of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis .", "as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m .", "almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) .", "rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) .", "patient baseline characteristics , demographics , comorbidities , complications , medications , and resource use the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone ,", "was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig .", "dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg .", "in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers . after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization ,", "dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig .", "2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) . similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . *", "p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig .", "diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ."], ["of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis .", "as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m .", "almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) .", "rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) ."], ["the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone ,", "was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig .", "dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg .", "in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers ."], ["after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig .", "2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) .", "similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal .", "* p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 ."], ["after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig .", "diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal .", "dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . *"], ["the study results showed that , compared with the achievement of only the ldl - c goal , achievement of both hba1c and ldl - c goals is associated with a lower risk of microvascular complications , acs , and cardiovascular surgeries ( pci or cabg ) , lower utilization of health care resources , and lower costs of care , but no additional effect was observed for the composite cardiovascular - related end point .", "in addition , dual - goal achievement relative to hba1c goal achievement is associated with a lower risk of the composite cardiovascular - related end point , cabg , and outpatient visits . to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label ,", "randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) .", "a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study ,", "lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) .", "these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement .", "in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c .", "the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) .", "we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements .", "current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration .", "our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals .", "the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) .", "this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) .", "our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers .", "our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study ,", "achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) .", "this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes .", "our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone .", "our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings . for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s .", "patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) .", "a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm , compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) .", "first , the data used for this study include laboratory measurements for patients over time .", "second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements .", "this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models .", "first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model .", "second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions .", "third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records .", "fourth , because we used vha data , the patients in our study were predominantly male .", "although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed .", "finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c , ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion ,", "veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone", ". these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes ."], ["to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label ,", "randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) .", "a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study ,", "lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) .", "these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement .", "in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c .", "the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) .", "we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements .", "current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration ."], ["our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals .", "the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) .", "this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) .", "our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers .", "our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study ,", "achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) ."], ["this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes .", "our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone .", "our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings .", "for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s .", "patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) . a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm ,", "compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) .", "first , the data used for this study include laboratory measurements for patients over time .", "second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements .", "this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models .", "first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model .", "second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions .", "third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records .", "fourth , because we used vha data , the patients in our study were predominantly male .", "although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed . finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c", ", ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion , this retrospective study among u.s .", "veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone .", "these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes ."]]}
{"article_id": "PMC4247231", "article_text": ["endotracheal intubation is one of the most important skills for anesthesiologists in securing the airway during general anesthesia and resuscitation .", "failure to secure the airway can cause anesthesia - related life - threatening morbidity and mortality .", "however , the difficult laryngoscopy and tracheal intubation rate still remains at 1.513% due to poor reliability of traditional protocols , algorithms , and combinations of screening tools in identifying a potentially difficult airway .", "due to the portable , noninvasive characteristics , point - of - care ultrasound ( us ) technique has been widely used in the operating room for ultrasound - guided nerve block , central venous access , and pneumothorax diagnosis . with improved visualization of airway structures ,", "prasad et al . first found that us can reliably image all of the structures visualized by ct , and the infrahyoid airway structure parameters measured by ultrasound agree well with the parameters measured by ct .", "adhikari et al . further demonstrated that the anterior neck soft tissue thickness measured by ultrasound at hyoid bone and thyrohyoid membrane levels can be used as an index to predict difficult laryngoscopy , but only the anterior neck soft tissue thickness at thyrohyoid membrane levels can be used as an independent predictor of difficult laryngoscopy .", "interestingly , they did not find a correlation between us measurements and clinical screening tests .", ".   also found that an abundance of fat tissue at the anterior neck region as detected by ultrasound in israeli obesity patients was a very good independent predictor of difficult laryngoscopy , being a much better predictor than body mass index ( bmi ) per se .", "however , a similar study in the unites states showed that us quantification of anterior soft tissue and general bedside screening tests failed to predict difficult laryngoscopy in american obese patients , suggesting that racial or body shape differences might exist . to define the role of airway us in predicting difficult laryngoscopy", ", we evaluated the feasibility of ultrasound in predicting difficult laryngoscopy in a chinese han population .", "after approval of the research protocol by the local hospital ethics committee for human studies and obtaining personal informed consent , 203 american society of anesthesiologists ( asa ) grade i and ii adult patients undergoing elective surgeries and receiving general anesthesia were included in this study .", "exclusion criteria included patients who had facial , cervical , pharyngeal and epiglottis surgical or trauma history , patients with most teeth lost , and patients with arthritis .", "the modified mallampati score ( mms ) was specified according to the visibility of pharyngeal structures with the patient in an upright sitting position , head in neutral position , mouth wide open , and tongue protruding to its maximum without phonation ( figure 1a ) .", "class i is visualization of the hard palate , soft palate , fauces , uvula , and pillars .", "class ii is visualization of the hard palate , soft palate , fauces , and base of uvula .", "thyromental distance ( tmd ) was measured from the mental prominence to the thyroid cartilage with the patient s neck extended fully .", "interincisor gap ( iig ) was measured from the upper central incisors to the lower central incisors while the patient s mouth was fully opened .", "ultrasound measurements were performed by the primary investigator with the patient supine and the head and neck in neutral position .", "the thicknesses of anterior neck soft tissue at hyoid bone , thyrohyoid membrane , and anterior commissure levels were obtained transversely across the anterior surface of the neck with a 136 mhz hfl38 linear array ultrasound probe attached to a sonosite s - nerve machine ( sonosite inc . ,", "the minimal distance from the hyoid bone to skin surface ( dshb ) was measured ( figure 2a , 2b ) . at thyrohyoid membrane level ,", "the distance from skin to epiglottis midway ( dsem ) between the hyoid bone and thyroid cartilage was measured ( figure 2c , 2d ) . at anterior commissure level ,", "the minimal distance from skin to anterior commissure ( dsac ) was obtained ( figure 2e , 2f ) .", "after anesthesia induction with midazolam 0.04 mg / kg , propofol 22.5 mg / kg , fentanyl 24 g / kg , and succinylcholine 2 mg / kg , endotracheal intubation was carried out by anesthesia providers with a minimum of 2 years of endotracheal intubation experience .", "the macintosh blades were used to expose the target larynx , and no external laryngeal pressure was used to facilitate this process .", "classification of laryngoscopic views was based on the method described by cormack and lehane ( figure 1b ) .", "continuous data are expressed as means ( standard deviation [ sd ] ) ; categorical data are expressed as numbers of occurrences ( percents ) .", "comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests , as appropriate , for non - continuous variables .", "the best fit lines of dshb , dsem , dsac , mms , iig , and tmd were determined using a least - squares regression technique .", "receiver operating characteristic ( roc ) analyses were used to calculate the comparable threshold values of dshb , dsem , dsac , tmd , iig , and mms .", "optimal cutoff values were calculated using the youden index ( calculated as : sensitivity + specificity  100 ) .", "statistical analyses were performed using medcalc for windows , version 12.6.1.0 ( medcalc software , ostend , belgium ) .", "the level of statistical significance was p<0.05 , and p<0.0001 was considered to be very statistically significant .", "continuous data are expressed as means ( standard deviation [ sd ] ) ; categorical data are expressed as numbers of occurrences ( percents ) .", "comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests , as appropriate , for non - continuous variables .", "the best fit lines of dshb , dsem , dsac , mms , iig , and tmd were determined using a least - squares regression technique .", "receiver operating characteristic ( roc ) analyses were used to calculate the comparable threshold values of dshb , dsem , dsac , tmd , iig , and mms .", "optimal cutoff values were calculated using the youden index ( calculated as : sensitivity + specificity  100 ) .", "statistical analyses were performed using medcalc for windows , version 12.6.1.0 ( medcalc software , ostend , belgium ) .", "the level of statistical significance was p<0.05 , and p<0.0001 was considered to be very statistically significant .", "a total of 203 eligible patients ( 120 females , 83 males ) were included in this study .", "there were 28 of 203 patients ( 13.8% ) categorized as difficult laryngoscopy , and 50% of patients with difficult laryngoscopy in our study were males .", "no differences were noted in sex , age , and height , but bw and bmi values in the difficult laryngoscopy group were higher . the bmi value was 25.632.80 kg / m for the difficult laryngoscopy group , and 23.613.43 kg / m for the easy laryngoscopy group ( p<0.05 ) .", "airway evaluation parameters , including mms , tmd , iig , dshb , dsem , and dsac , are shown in table 2 .", "the thicknesses of anterior neck soft tissue measured by us were greater in the difficult laryngoscopy group compared to the easy laryngoscopy group at the level of the hyoid bone , thyrohyoid membrane , and anterior commissure .", "however , no statistically significant difference was found between easy and difficult laryngoscopy groups for tmd and iig .", "a strong positive correlation existed between dsem and dshb ; moderate positive correlations between dsem and dsac , dshb and dsac ; small positive correlations between mms and dshb , mms and dsem , mms and dsac ; and very small positive correlations between tmd and iig .", "the correlation coefficients along with 95% confidence interval ( ci ) between dsem and dshb , dsem and dsac , dshb and dsac , mms and dshb , mms and dsem , mms and dsac , and between tmd and iig were 0.74 ( 0.670.80 , p<0.0001 ) , 0.60 ( 0.500.68 , p<0.0001 ) , 0.69 ( 0.610.75 , p<0.0001 ) , 0.32 ( 0.190.44 , p<0.0001 ) , 0.27 ( 0.140.40 , p=0.0001 ) , 0.32 ( 0.190.44 , p<0.0001 ) , 0.18 ( 0.05 to 0.31 , p=0.0089 ) , respectively ( figure 3a3 g ) .", "small negative correlation were found between iig and mms ( r=0.27 , p=0.0001 ) ; and very small negative correlations were found between mms and tmd ( r=0.20 , p=0.0042 ) , iig and dsac ( r=0.18 , p=0.0113 ) , and iig and dshb ( r=0.15 , p=0.0344 ) ( figure 3h3k ) .", "no correlations were found between tmd and dsem , tmd and dshb , and tmd and dsac ( data not shown ) . to further assess the roles of mms , iig , tmd , dshb , dsem , and dsac in predicting difficult laryngoscopy ,", "the roc curves ( figure 4 ) were drawn using medcalc software with laryngoscopy grade over ii as the threshold of difficult laryngoscopy . as determined by the youden index , the optimal cutoff values ( with sensitivity and specificity in parentheses ) for mms , iig , tmd , dshb , dsem , and dsac to predict difficult laryngoscopy were over 2 ( 50.0% , 82.3% ) , 3.8 cm ( 75.0% , 48.0% ) , 5.9 cm ( 21.4% , 93.7% ) , 1.28 cm ( 85.7% , 85.1% ) , 1.78 cm ( 100.0% , 66.3% ) , and 1.1 cm ( 75.0% , 80.6% ) , respectively .", "the areas under the roc curve ( aucs ) were significantly different from the area under the reference line ( area=0.5 ) for mms , dshb , dsem , and dsac ; and the p values of mms , dshb , dsem , and dsac were all less than 0.0001 .", "no difference between the aucs of tmd , iig , and the area under the reference line were identified , suggesting that mms , dshb , dsem , and dsac might be better than tmd and iig in predicting difficult laryngoscopy ( table 3 ) .", "with the rapid expansion of portable us in anesthesia for guiding the placement of central venous catheters and the performance of regional block [ 1113 ] , some preliminary results exploring the feasibility of portable us in airway evaluation and management have been reported , even though the air attenuates us transmission and thereby creates artifacts [ 35,14,15 ] . in the present study , we compared the role of the us measurements and traditional screening tests , including iig , tmd , and mms , in predicting difficult laryngoscopy in a chinese han population .", "we found that strong positive linear correlations existed among the thicknesses of anterior neck soft tissue measured by us at hyoid bone , thyrohyoid membrane , and anterior commissure levels .", "the aucs of mms , dshb , dsem , and dsac are all over 0.7 , indicating they are all good parameters in predicting difficult laryngoscopy .", "the aucs of tmd and iig were less than 0.7 , suggesting that tmd and iig were poor parameters in predicating difficult laryngoscopy . to clearly visualize the glottis and smoothly finish the intubation procedure , anesthesiologists need to place the laryngoscope blade to the base of the epiglottis , and then lift it up", "therefore , in addition to the performer s skills and experience , many factors , including dental status , mouth opening , oropharyngeal space , mandibular space , and neck motility , are all involved this complicated procedure .", "mms mainly reflects the size of the tongue relative to the oral cavity , or the oropharyngeal space [ 1820 ] , iig means the mouth opening , and tmd can reflect the length of the neck .", "the mallapati score was first introduced by mallampati et al . in 1985 , and then modified by samsoon and young in 1987 . due to the simple and clear evaluation standards , mms has become one of the most commonly used tools to predict a difficult airway .", "however , mms mainly depends on patient cooperation and body position , which can greatly affect the accuracy of airway evaluation .", "therefore , mms has been reported to be a good predictor by many , but was found to be of limited value by others . a recent meta - analysis involving 177 088 patients", "demonstrate that mms is inadequate as a stand - alone test to predict difficult laryngoscopy or tracheal intubation , but it may well be a part of a multifactorial model for the prediction of a difficult tracheal intubation .", "iig has been repeatedly reported to be associated with difficult intubation , but our results showed that there was no significant difference in mean iig between difficult and easy laryngoscopy groups ( 3.740.43 vs. 3.860.47 cm ) .", "furthermore , the auc of iig was 0.580.06 , which was far less than 0.7 , suggesting that iig might be not a good screening parameter for predicting difficult laryngoscopy in our study .", "our iig conclusion was similar to the previous report by savva , but different from results of other studies .", "whether this difference was really caused by racial factors requires further investigation because the measurements of iig need patients to fully cooperate .", "tmd is also used as a screening test to predict difficult airway , but our results suggest that tmd is not a useful screening test .", "however , we could not determine whether this was because of limited patient numbers or because tmd was not useful in predicting difficult airway due to the small sample size .", "ct , mri , and other imaging techniques can accurately measure the thickness of anterior neck soft tissue , but they are expensive and unavailable in many operating rooms .", "portable us is inexpensive , rapid , and convenient to perform in the operating room , and most importantly , it can quantify the neck fat thickness as accurately as mi .", "however , the results of 2 prior studies focusing on soft tissue thickness at and below the level of the vocal cords in a specific group of obese patients are inconsistent .", "further measured the anterior neck soft tissue at the hyoid bone and thyrohyoid membrane levels , which is important to displace the glottis by the laryngoscopic blade , and found that us measurements of anterior neck soft tissue thickness at the level of hyoid bone and thyrohyoid membrane can be used to predict difficult laryngoscopies , and a 2.8-cm us measurement at the thyrohyoid membrane was a good independent predictor of difficult laryngoscopy .", "our results show that the thicknesses of anterior neck soft tissue at the level of the hyoid bone ( 1.51 [ 95% ci=1.401.61 ] cm vs. 0.98 [ 95% ci=0.941.02 ] cm ) , the thyrohyoid membrane ( 2.39 [ 95% ci=2.172.62 ] cm vs. 1.49 [ 95% ci=1.431.55 ] cm ) , and the anterior commissure ( 1.30 [ 95% ci=1.181.42 ] cm vs. 0.92 [ 95% ci=0.870.94 ] cm ) were greater in the difficult laryngoscopy group and were significantly correlated .", "furthermore , the ranges of anterior neck soft tissue for those with difficult laryngoscopy were mutually exclusive from those patients with an easy laryngoscopy , indicating that they are independent predictors of difficult laryngoscopy , even though the ranges of these 3 parameters were smaller than in a previous report .", "glottis exposure by place laryngoscope is a very complicated procedure , and many subjective and objective factors such as the provider s skills and experience , airway secretions , and abnormalities of anatomical structures are involved in this procedure .", "the investigators were not totally blinded to the study purpose , and some clinical signs might indicate the possibility of difficult laryngoscopy , which can cause some bias during us measurements . due to the low incidence of difficult laryngoscopy , it is impossible for us to randomly assign an equal number of patients to both groups , thus there were 28 patients in the difficult group and 175 patients in the easy group .", "anterior neck soft tissue thicknesses measured by us at the hyoid bone , thyrohyoid membrane , and anterior commissure levels are independent predictors of difficult laryngoscopy . the commonly used screening tests for difficult intubation have only poor - to - moderate predictive power when used alone .", "combinations of these screening tests or risk factors with us measurements might increase the ability to predict difficult laryngoscopy ."], "abstract_text": ["<S> backgroundthe aim of this study was to determine if ultrasound ( us ) measurements of anterior neck soft tissue thickness at hyoid bone ( dshb ) , thyrohyoid membrane ( dsem ) , and anterior commissure ( dsac ) levels can be used to predict difficult laryngoscopy.material/methodswe included 203 patients age 2065 years scheduled to undergo general anesthesia in this prospective observational study . </S>", "<S> correlation analysis and receiver operating characteristic curve ( roc ) analysis were used to determine the roles of screening tests [ interincisor gap ( iig ) , thyromental distance ( tmd ) , modified mallampati score ( mms ) ] and us measurements ( dshb , dsem , dsac ) in predicting difficult laryngoscopy.resultsthere were 28 out of 203 patients categorized as difficult laryngoscopy . </S>", "<S> dshb , dsem , dsac , and mms were greater in the difficult laryngoscopy group ( p<0.0001 ) . </S>", "<S> there was a strong positive correlation between dsem and dshb ( r=0.74 ) ; moderate positive correlations between dsem and dsac ( r=0.60 ) , dshb and dsac ( r=0.69 ) ; small positive correlations between mms and dshb ( r=0.32 ) , mms and dsem ( r=0.27 ) , mms and dsac ( r=0.32 ) , all p values 0.0001 ; very small positive correlation between tmd and iig ( r=0.18 , p=0.0089 ) ; small negative correlation between iig and mms ( r=0.27 , p=0.0001 ) ; and very small negative correlations between mms and tmd ( r=0.20 , p=0.004 ) , iig and dsac ( r=0.18 , p=0.011 ) , iig and dshb ( r=0.15 , p=0.034 ) . </S>", "<S> the areas under the roc curve ( aucs ) of mms , dshb , dsem , and dsac were significantly larger compared with the reference line ( p<0.0001).conclusionsanterior neck soft tissue thicknesses measured by us at hyoid bone , thyrohyoid membrane , and anterior commissure levels are independent predictors of difficult laryngoscopy . </S>", "<S> combinations of those screening tests or risk factors with us measurements might increase the ability to predict difficult laryngoscopy . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Statistical analysis", "Results", "Discussion", "Conclusions"], "sections": [["endotracheal intubation is one of the most important skills for anesthesiologists in securing the airway during general anesthesia and resuscitation .", "failure to secure the airway can cause anesthesia - related life - threatening morbidity and mortality .", "however , the difficult laryngoscopy and tracheal intubation rate still remains at 1.513% due to poor reliability of traditional protocols , algorithms , and combinations of screening tools in identifying a potentially difficult airway .", "due to the portable , noninvasive characteristics , point - of - care ultrasound ( us ) technique has been widely used in the operating room for ultrasound - guided nerve block , central venous access , and pneumothorax diagnosis . with improved visualization of airway structures ,", "prasad et al . first found that us can reliably image all of the structures visualized by ct , and the infrahyoid airway structure parameters measured by ultrasound agree well with the parameters measured by ct .", "adhikari et al . further demonstrated that the anterior neck soft tissue thickness measured by ultrasound at hyoid bone and thyrohyoid membrane levels can be used as an index to predict difficult laryngoscopy , but only the anterior neck soft tissue thickness at thyrohyoid membrane levels can be used as an independent predictor of difficult laryngoscopy .", "interestingly , they did not find a correlation between us measurements and clinical screening tests .", ".   also found that an abundance of fat tissue at the anterior neck region as detected by ultrasound in israeli obesity patients was a very good independent predictor of difficult laryngoscopy , being a much better predictor than body mass index ( bmi ) per se .", "however , a similar study in the unites states showed that us quantification of anterior soft tissue and general bedside screening tests failed to predict difficult laryngoscopy in american obese patients , suggesting that racial or body shape differences might exist . to define the role of airway us in predicting difficult laryngoscopy", ", we evaluated the feasibility of ultrasound in predicting difficult laryngoscopy in a chinese han population ."], ["after approval of the research protocol by the local hospital ethics committee for human studies and obtaining personal informed consent , 203 american society of anesthesiologists ( asa ) grade i and ii adult patients undergoing elective surgeries and receiving general anesthesia were included in this study .", "exclusion criteria included patients who had facial , cervical , pharyngeal and epiglottis surgical or trauma history , patients with most teeth lost , and patients with arthritis .", "the modified mallampati score ( mms ) was specified according to the visibility of pharyngeal structures with the patient in an upright sitting position , head in neutral position , mouth wide open , and tongue protruding to its maximum without phonation ( figure 1a ) .", "class i is visualization of the hard palate , soft palate , fauces , uvula , and pillars .", "class ii is visualization of the hard palate , soft palate , fauces , and base of uvula .", "thyromental distance ( tmd ) was measured from the mental prominence to the thyroid cartilage with the patient s neck extended fully .", "interincisor gap ( iig ) was measured from the upper central incisors to the lower central incisors while the patient s mouth was fully opened .", "ultrasound measurements were performed by the primary investigator with the patient supine and the head and neck in neutral position .", "the thicknesses of anterior neck soft tissue at hyoid bone , thyrohyoid membrane , and anterior commissure levels were obtained transversely across the anterior surface of the neck with a 136 mhz hfl38 linear array ultrasound probe attached to a sonosite s - nerve machine ( sonosite inc . ,", "the minimal distance from the hyoid bone to skin surface ( dshb ) was measured ( figure 2a , 2b ) . at thyrohyoid membrane level ,", "the distance from skin to epiglottis midway ( dsem ) between the hyoid bone and thyroid cartilage was measured ( figure 2c , 2d ) . at anterior commissure level ,", "the minimal distance from skin to anterior commissure ( dsac ) was obtained ( figure 2e , 2f ) .", "after anesthesia induction with midazolam 0.04 mg / kg , propofol 22.5 mg / kg , fentanyl 24 g / kg , and succinylcholine 2 mg / kg , endotracheal intubation was carried out by anesthesia providers with a minimum of 2 years of endotracheal intubation experience .", "the macintosh blades were used to expose the target larynx , and no external laryngeal pressure was used to facilitate this process .", "classification of laryngoscopic views was based on the method described by cormack and lehane ( figure 1b ) .", "continuous data are expressed as means ( standard deviation [ sd ] ) ; categorical data are expressed as numbers of occurrences ( percents ) .", "comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests , as appropriate , for non - continuous variables .", "the best fit lines of dshb , dsem , dsac , mms , iig , and tmd were determined using a least - squares regression technique .", "receiver operating characteristic ( roc ) analyses were used to calculate the comparable threshold values of dshb , dsem , dsac , tmd , iig , and mms .", "optimal cutoff values were calculated using the youden index ( calculated as : sensitivity + specificity  100 ) .", "statistical analyses were performed using medcalc for windows , version 12.6.1.0 ( medcalc software , ostend , belgium ) .", "the level of statistical significance was p<0.05 , and p<0.0001 was considered to be very statistically significant ."], ["continuous data are expressed as means ( standard deviation [ sd ] ) ; categorical data are expressed as numbers of occurrences ( percents ) .", "comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests , as appropriate , for non - continuous variables .", "the best fit lines of dshb , dsem , dsac , mms , iig , and tmd were determined using a least - squares regression technique .", "receiver operating characteristic ( roc ) analyses were used to calculate the comparable threshold values of dshb , dsem , dsac , tmd , iig , and mms .", "optimal cutoff values were calculated using the youden index ( calculated as : sensitivity + specificity  100 ) .", "statistical analyses were performed using medcalc for windows , version 12.6.1.0 ( medcalc software , ostend , belgium ) .", "the level of statistical significance was p<0.05 , and p<0.0001 was considered to be very statistically significant ."], ["a total of 203 eligible patients ( 120 females , 83 males ) were included in this study .", "there were 28 of 203 patients ( 13.8% ) categorized as difficult laryngoscopy , and 50% of patients with difficult laryngoscopy in our study were males .", "no differences were noted in sex , age , and height , but bw and bmi values in the difficult laryngoscopy group were higher . the bmi value was 25.632.80 kg / m for the difficult laryngoscopy group , and 23.613.43 kg / m for the easy laryngoscopy group ( p<0.05 ) .", "airway evaluation parameters , including mms , tmd , iig , dshb , dsem , and dsac , are shown in table 2 .", "the thicknesses of anterior neck soft tissue measured by us were greater in the difficult laryngoscopy group compared to the easy laryngoscopy group at the level of the hyoid bone , thyrohyoid membrane , and anterior commissure .", "however , no statistically significant difference was found between easy and difficult laryngoscopy groups for tmd and iig .", "a strong positive correlation existed between dsem and dshb ; moderate positive correlations between dsem and dsac , dshb and dsac ; small positive correlations between mms and dshb , mms and dsem , mms and dsac ; and very small positive correlations between tmd and iig .", "the correlation coefficients along with 95% confidence interval ( ci ) between dsem and dshb , dsem and dsac , dshb and dsac , mms and dshb , mms and dsem , mms and dsac , and between tmd and iig were 0.74 ( 0.670.80 , p<0.0001 ) , 0.60 ( 0.500.68 , p<0.0001 ) , 0.69 ( 0.610.75 , p<0.0001 ) , 0.32 ( 0.190.44 , p<0.0001 ) , 0.27 ( 0.140.40 , p=0.0001 ) , 0.32 ( 0.190.44 , p<0.0001 ) , 0.18 ( 0.05 to 0.31 , p=0.0089 ) , respectively ( figure 3a3 g ) .", "small negative correlation were found between iig and mms ( r=0.27 , p=0.0001 ) ; and very small negative correlations were found between mms and tmd ( r=0.20 , p=0.0042 ) , iig and dsac ( r=0.18 , p=0.0113 ) , and iig and dshb ( r=0.15 , p=0.0344 ) ( figure 3h3k ) .", "no correlations were found between tmd and dsem , tmd and dshb , and tmd and dsac ( data not shown ) . to further assess the roles of mms , iig , tmd , dshb , dsem , and dsac in predicting difficult laryngoscopy ,", "the roc curves ( figure 4 ) were drawn using medcalc software with laryngoscopy grade over ii as the threshold of difficult laryngoscopy . as determined by the youden index , the optimal cutoff values ( with sensitivity and specificity in parentheses ) for mms , iig , tmd , dshb , dsem , and dsac to predict difficult laryngoscopy were over 2 ( 50.0% , 82.3% ) , 3.8 cm ( 75.0% , 48.0% ) , 5.9 cm ( 21.4% , 93.7% ) , 1.28 cm ( 85.7% , 85.1% ) , 1.78 cm ( 100.0% , 66.3% ) , and 1.1 cm ( 75.0% , 80.6% ) , respectively .", "the areas under the roc curve ( aucs ) were significantly different from the area under the reference line ( area=0.5 ) for mms , dshb , dsem , and dsac ; and the p values of mms , dshb , dsem , and dsac were all less than 0.0001 .", "no difference between the aucs of tmd , iig , and the area under the reference line were identified , suggesting that mms , dshb , dsem , and dsac might be better than tmd and iig in predicting difficult laryngoscopy ( table 3 ) ."], ["with the rapid expansion of portable us in anesthesia for guiding the placement of central venous catheters and the performance of regional block [ 1113 ] , some preliminary results exploring the feasibility of portable us in airway evaluation and management have been reported , even though the air attenuates us transmission and thereby creates artifacts [ 35,14,15 ] . in the present study , we compared the role of the us measurements and traditional screening tests , including iig , tmd , and mms , in predicting difficult laryngoscopy in a chinese han population .", "we found that strong positive linear correlations existed among the thicknesses of anterior neck soft tissue measured by us at hyoid bone , thyrohyoid membrane , and anterior commissure levels .", "the aucs of mms , dshb , dsem , and dsac are all over 0.7 , indicating they are all good parameters in predicting difficult laryngoscopy .", "the aucs of tmd and iig were less than 0.7 , suggesting that tmd and iig were poor parameters in predicating difficult laryngoscopy . to clearly visualize the glottis and smoothly finish the intubation procedure , anesthesiologists need to place the laryngoscope blade to the base of the epiglottis , and then lift it up", "therefore , in addition to the performer s skills and experience , many factors , including dental status , mouth opening , oropharyngeal space , mandibular space , and neck motility , are all involved this complicated procedure .", "mms mainly reflects the size of the tongue relative to the oral cavity , or the oropharyngeal space [ 1820 ] , iig means the mouth opening , and tmd can reflect the length of the neck .", "the mallapati score was first introduced by mallampati et al . in 1985 , and then modified by samsoon and young in 1987 . due to the simple and clear evaluation standards , mms has become one of the most commonly used tools to predict a difficult airway .", "however , mms mainly depends on patient cooperation and body position , which can greatly affect the accuracy of airway evaluation .", "therefore , mms has been reported to be a good predictor by many , but was found to be of limited value by others . a recent meta - analysis involving 177 088 patients", "demonstrate that mms is inadequate as a stand - alone test to predict difficult laryngoscopy or tracheal intubation , but it may well be a part of a multifactorial model for the prediction of a difficult tracheal intubation .", "iig has been repeatedly reported to be associated with difficult intubation , but our results showed that there was no significant difference in mean iig between difficult and easy laryngoscopy groups ( 3.740.43 vs. 3.860.47 cm ) .", "furthermore , the auc of iig was 0.580.06 , which was far less than 0.7 , suggesting that iig might be not a good screening parameter for predicting difficult laryngoscopy in our study .", "our iig conclusion was similar to the previous report by savva , but different from results of other studies .", "whether this difference was really caused by racial factors requires further investigation because the measurements of iig need patients to fully cooperate .", "tmd is also used as a screening test to predict difficult airway , but our results suggest that tmd is not a useful screening test .", "however , we could not determine whether this was because of limited patient numbers or because tmd was not useful in predicting difficult airway due to the small sample size .", "ct , mri , and other imaging techniques can accurately measure the thickness of anterior neck soft tissue , but they are expensive and unavailable in many operating rooms .", "portable us is inexpensive , rapid , and convenient to perform in the operating room , and most importantly , it can quantify the neck fat thickness as accurately as mi .", "however , the results of 2 prior studies focusing on soft tissue thickness at and below the level of the vocal cords in a specific group of obese patients are inconsistent .", "further measured the anterior neck soft tissue at the hyoid bone and thyrohyoid membrane levels , which is important to displace the glottis by the laryngoscopic blade , and found that us measurements of anterior neck soft tissue thickness at the level of hyoid bone and thyrohyoid membrane can be used to predict difficult laryngoscopies , and a 2.8-cm us measurement at the thyrohyoid membrane was a good independent predictor of difficult laryngoscopy .", "our results show that the thicknesses of anterior neck soft tissue at the level of the hyoid bone ( 1.51 [ 95% ci=1.401.61 ] cm vs. 0.98 [ 95% ci=0.941.02 ] cm ) , the thyrohyoid membrane ( 2.39 [ 95% ci=2.172.62 ] cm vs. 1.49 [ 95% ci=1.431.55 ] cm ) , and the anterior commissure ( 1.30 [ 95% ci=1.181.42 ] cm vs. 0.92 [ 95% ci=0.870.94 ] cm ) were greater in the difficult laryngoscopy group and were significantly correlated .", "furthermore , the ranges of anterior neck soft tissue for those with difficult laryngoscopy were mutually exclusive from those patients with an easy laryngoscopy , indicating that they are independent predictors of difficult laryngoscopy , even though the ranges of these 3 parameters were smaller than in a previous report .", "glottis exposure by place laryngoscope is a very complicated procedure , and many subjective and objective factors such as the provider s skills and experience , airway secretions , and abnormalities of anatomical structures are involved in this procedure .", "the investigators were not totally blinded to the study purpose , and some clinical signs might indicate the possibility of difficult laryngoscopy , which can cause some bias during us measurements . due to the low incidence of difficult laryngoscopy , it is impossible for us to randomly assign an equal number of patients to both groups , thus there were 28 patients in the difficult group and 175 patients in the easy group ."], ["anterior neck soft tissue thicknesses measured by us at the hyoid bone , thyrohyoid membrane , and anterior commissure levels are independent predictors of difficult laryngoscopy . the commonly used screening tests for difficult intubation have only poor - to - moderate predictive power when used alone .", "combinations of these screening tests or risk factors with us measurements might increase the ability to predict difficult laryngoscopy ."]]}
{"article_id": "PMC4283862", "article_text": ["badana grupa skadaa si z 96 pacjentw ( 77 mczyzn i 19 kobiet ) w wieku rednio 58,1  9,8 roku .", "pacjenci zostali losowo przydzieleni do jednej z trzech grup : grupy pacjentw poddanych perikardiotomii tylnej ( grupa", "i , n = 30 ) , grupy kontrolnej ( grupa ii , n = 33 ) oraz pacjentw , u ktrych zastosowano tylny drena osierdziowy , ktrzy jednak nie zostali poddani zabiegowi perikardiotomii tylnej ( grupa iii , n = 33 ) .", "czas trwania hospitalizacji pooperacyjnej ( p = 0,03 ; 11,56  10,64 ) oraz czsto reoperacji z powodu tamponady ( p = 0,019 ; 12,1% ) byy znaczco wysze w grupie ii .", "znaczny wysik osierdziowy wykryto u jednego pacjenta ( grupa ii , n = 1 ) w pierwszym dniu po operacji , u jednego pacjenta w pitym dniu po operacji ( grupa iii , n = 1 ) oraz u jednego pacjenta w 30", "dniu po operacji ( p = 0,028 ) w grupie i odnotowano regresj wysiku osierdziowego .", "i stwierdzono rwnie wyszy wspczynnik pooperacyjnego migotania przedsionkw , jednake w tym wzgldzie midzy grupami nie byo znaczcych rnic .", "pacjenci , ktrzy nie zostali poddani perikardiotomii tylnej lub u ktrych nie zastosowano tylnego drenau klatki piersiowej , wykazywali szcztkowy wysik osierdziowy , wymagali duszej hospitalizacji i musieli by ponownie operowani z powodu tamponady .", "zarwno perikardiotomia tylna , jak i tylny drena klatki piersiowej okazay si skuteczne we wczesnym okresie pooperacyjnym .", "pericardial effusion , which is a common complication after coronary artery surgery ( cas ) , may be the cause of significant morbidity .", "it is often benign and small in volume , but can lead to life - threatening events , such as cardiac tamponade .", "delayed postoperative cardiac tamponade following an open - heart operation is an infrequent , but potentially serious complication .", "although previous reports have dealt with the clinical features and management of this problem , an optimal approach to prevent postoperative pericardial effusion and related problems has not been clearly defined [ 3 , 5 ] .", "this study aims to investigate the effects of posterior pericardiotomy ( pp ) on the development of pericardial effusion , tamponade , and atrial fibrillation following cas .", "this prospective randomized case - controlled study was carried out on 96 patients ( 77 men and 19 women ; age : 35 - 78 years ; mean : 58.1 years ) .", "the patients , undergoing cas performed by the same surgical team between march 2012 and january 2013 in istanbul mehmet akif ersoy thoracic and cardiovascular surgery research and training hospital , were assigned to 3 groups .", "after the study had received the approval of the institutional review board , informed consent was obtained from all patients .", "the treating physician did not know the groups to which individual patients had been assigned . patients with renal failure ,", "hyperthyroidism , emergency cas , history of cardiac operations associated with valvular heart disease , low ejection fraction ( < 35% ) , and preoperative atrial fibrillation or other rhythm disorders , as well as patients who did not provide research authorization , were excluded from the study .", "the study groups included patients after pp ( group i , n = 30 ) , a control group ( group ii , n = 33 ) , and patients without pp who received posterior pericardial tubes ( the tube was placed behind the heart and sutured to the adjacent pericardial tissue to avoid tube - induced ventricular arrhythmias ) ( group iii , n = 33 ) .", "i , a 4-cm longitudinal incision was made parallel and posterior to the left phrenic nerve , extending from the left inferior pulmonary vein to the diaphragm , as described by mulay et al . .", "in group i , the relationship between the location of the distal part of circumflex anastomosis and pp was also carefully considered .", "moreover , all patients underwent pleurotomy and lateral pericardiotomy ( approximately 3.5 - 4.5 cm in size ) in which the internal thoracic artery ( ita ) was used for anastomosis and positioned medially to the lung , which allowed the ita to run without kinking or twisting .", "the inferior part of the pericardium was left open ( 2 cm ) in our study .", "a straight tube was placed in the anterior mediastinum and an angled tube was placed in the left hemithorax in all patients .", "the same postoperative pain management protocol was provided for all patients . after a routine closure of the chest", ", drains were frequently milked and stripped to ensure tube patency during the intensive care unit stay .", "the chest tubes were removed on the 2 postoperative day when the drainage became serous ; otherwise , they remained in use for one or more days .", "pericardial effusion was defined as the accumulation of fluid in the pericardial space determined by transthoracic echocardiography ( tte ) .", "the presence of pericardial effusion on 2-dimensional tte was assessed using the criteria previously described by bakhshandeh et al . .", "two - dimensional echocardiography was performed on postoperative days 1 and 5 , as well as 1 month after surgery .", "the maximum diastolic separation between the pericardium and epicardium was measured at the tip of the mitral valve leaflet .", "when the diastolic echo - free space between the left ventricular posterior wall and the pericardium was < 10 mm , the pericardial effusion was classified as small .", "when the space was 10 - 20 mm , the effusion was classified as moderate , while for spaces > 20 mm , the effusion was classified as severe .", "if the diagnosis of evident pericardial effusion ( moderate or severe ) was established , tte was performed daily , and the volume of the effusion was observed closely .", "we compared the groups with regard to the volume of effusion on postoperative days 1 , 5 , and 30 .", "patients with no or small pericardial effusion and an uneventful postoperative course were discharged from the hospital on postoperative day 5 .", "patients with moderate and severe pericardial effusion were treated with non - steroidal anti - inflammatory drugs . during hospitalization ,", "all patients were continuously monitored with the use of a portable electrocardiogram ( ecg , telemetry ) to detect atrial fibrillation .", "atrial fibrillation was considered to be persistent and clinically significant when it lasted for more than 5 min .", "statistical analysis was performed with the ncss ( number cruncher statistical system ) 2007 statistical software ( utah , usa ) .", "one - way anova and tukey hsd tests were used for the statistical analysis of the groups parameters .", "the differences were also analyzed using  tests and independent t - tests where applicable .", "the hospital mortality in the study was 2% ( n = 2 ) ; both patients were in group ii .", "one of these patients experienced low cardiac output during and following reoperation due to cardiac tamponade and died on postoperative day 12 .", "the other patient had an uneventful postoperative course , but was readmitted to the hospital on postoperative day 11 due to cardiac tamponade and acute kidney failure .", "pericardial drainage was performed , but the patient died of sepsis on postoperative day 45 .", "the groups did not differ in terms of demographic parameters , which are summarized in table i. the average total volume of drainage ( mediastinal and thoracic ) during the first 24 hours was similar in all groups ( 536.67  237.873 cc , 656.97  407.634 cc , and 577.27  374.349 cc , respectively ; p = 0.383 ) ( table ii ) .", "in group i , the volume of the fluid evacuated by mediastinal drainage was lower than in groups ii and iii , but the difference was not statistically significant ( table ii ) .", "two patients in group ii required reoperation due to excessive mediastinal hemorrhage and cardiac tamponade .", "the incidence of cardiac tamponade was higher in group ii ( n = 4 , 12% ) , and the differences between the groups were statistically significant ( p = 0.019 ) .", "two of the patients with pericardial tamponade were managed by subxiphoid pericardial drainage alone ; 400 to 1000 ml of fluid was removed .", "two patients in group iii were reoperated due to excessive hemorrhage in the early postoperative period . \n ", "demographic variables in groups differential drainage volume from each chest tube the volume of pericardial drainage was added to the volume of mediastinal drainage for group iii .", "the average volume of pericardial effusion significantly decreased with time in groups i and ii ( fig .", "approximately 50% of the patients had small pericardial effusion on the first postoperative day ; no difference between the groups was found .", "moderate pericardial effusion was detected in 8 patients ( group i , n = 1 ; group ii , n = 3 ; group iii , n = 4 ) on the first postoperative day , in 11 patients ( group ii , n = 7 ; group iii , n = 4 ) on the fifth postoperative day , and in 7 patients on the 30 postoperative day ( group ii , n = 6 ; group iii , n = 1 ) during the study period .", "severe pericardial effusion was detected in one patient ( group ii , n = 1 ) on the first postoperative day , in one patient ( group iii , n = 1 ) on the fifth postoperative day , and in one patient ( group iii , n = 1 ) on the 30 postoperative day .", "the incidence of moderate to severe pericardial effusion in group i was significantly lower than in the other groups on the 30 postoperative day ( n = 0 , p = 0.028 ) .", "figure 1 shows a comparison of the groups with regard to the volume of pericardial effusion .", "four of these patients ( group ii , n = 4 ) underwent drainage of severe pericardial effusion on postoperative days 0 , 1 , 11 , and 17 ; one patient ( group ii ) underwent sternal dehiscence repair .", "comparison of the groups with regard to the volume of pericardial effusion atrial fibrillation was detected in 24 of the 96 patients ( 25% : 6 patients in group i , 11 patients in group ii , 7 patients in group iii ) .", "no statistically significant difference between the groups ( p = 0.392 ) was present in this respect .", "the length of hospital stay was significantly shorter in group i ( 6.63  2.71 , p = 0.03 ) .", "there are a number of articles favoring the use of posterior pericardiotomy , but no comprehensive comparison has been conducted between the use of a pericardial tube and pp in patients undergoing cas .", "pericardial effusion and the associated complications are common after all kinds of cardiac surgery due to postoperative bleeding or post - cardiotomy syndrome .", "however , clinically significant pericardial effusion occurs rarely and can be a crucial risk factor for cardiac tamponade ( 1% ) .", "posterior pericardiotomy has been reported to reduce the prevalence of pericardial effusion from 40% to 8% with a simultaneous reduction in the prevalence of svt ( supraventricular tachycardia ) .", "the semi - sitting position helps thoracic drainage in the early postoperative period by supporting the patient 's back . despite all the above - mentioned measures , the posterior wall of the pericardial sac forms an unprotected space that may cause pericardial effusion to develop septae .", "this leads to the appearance of adhesions preventing blood drainage , leading to cardiac compression .", "moreover , the collection of fluid may persist due to bleeding resulting from the use of anticoagulants .", "in fact , anti - coagulants ( low - molecular - weight heparin ) were used postoperatively in our patients with atrial fibrillation .", "posterior pericardial effusion can be evacuated by placing a tube in the posterior pericardial space .", "the placement of the pericardial tube is simple to perform , but it may have certain drawbacks . the tube may come into contact with coronary grafts during cardiac activity or movement .", "however , the patients included in the study ( in group iii as well as the other groups ) did not exhibit any arrhythmias ( apart from af ) or ischemic changes", ". this may be due to the fact that we fixed the pericardial tube to the surface of the diaphragmatic pericardium with polypropylene .", "pp or the use of posterior tubes may lessen the volume of pericardial effusion after cas .", "moreover , patients in group i exhibited regression of pericardial effusion during 30 postoperative days .", "this proved that pp had a lasting effect on pericardial effusion , as presented by figure 1 .", "what stands out in our results is that pericardial effusion may occur if no intervention is performed on the posterior part of the pericardium .", "this technique is suggested in both cas and valve surgery , but it is not routinely used [ 13 , 14 ] .", "what is more , pp is recommended by the american college of chest physicians guidelines and by some authors as a preventive technique for postoperative atrial fibrillation [ 15 , 16 ] .", "supraventricular tachyarrhythmias , mainly in the form of atrial fibrillation , occur in up to 40% of patients undergoing cas .", "although af is usually benign , it can cause hemodynamic instability , prolonged hospital stay , and increased cost and , in rare cases , predispose to cerebrovascular accidents .", "the antiarrhythmic effect of pp may , however , be a theoretical issue , as we observed similar results regarding atrial fibrillation in all the groups .", "in fact , the results of our study were different than expected with regard to atrial fibrillation compared to other researchers [ 4 , 6 , 16 ] .", "additionally , asimakopoulos et al . also found that the prevalence of atrial fibrillation was not significantly reduced ( 20% ) in comparison to the conventional technique ( 26% ) .", "another issue is that humans have increasingly longer life spans , which may result in higher numbers of reoperations .", "ellman et al . found that patent coronary grafts were the most commonly injured structures during reoperation ( 46% ) .", "we routinely cover the aorta and proximal anastomoses of grafts with thymic fat tissue and the anterior wall of the pericardial cavity .", "we also place an anterior mediastinal tube over the thymic fat tissue before closing the sternum in all patients . behind the heart ,", "adhesions between the inferior surface of the heart and the diaphragm can cause difficulties when dissecting the tissue layers during reoperation .", "mulay et al . demonstrated that pp provides an effective drainage pathway from the left pleural cavity , thereby reducing the prevalence of pericardial effusion .", "for this reason , pp may be advantageous for reoperated patients in terms of reducing adhesions related to pericardial effusion .", "echocardiography can be useful in confirming and identifying the presence of pericardial effusion , hematoma , and tamponade . according to the results of our study , it is important to diagnose pericardial effusion at an early stage .", "hemodynamic deterioration due to pericardial fluid can be divided into four stages : pericardial fluid , hemodynamic tamponade , echocardiographic tamponade , clinical tamponade .", "echocardiographic follow - up of patients with pericardial effusion might be a factor extending the duration of hospital stay .", "we recommend the pp technique and routine echocardiographic monitoring during the postoperative period in patients undergoing cas .", "there is not sufficient research regarding the effects of pp on long - term results .", "the present study also demonstrated that patients undergoing pp required a significantly shorter hospital stay and experienced lower rates of pericardial effusion and cardiac tamponade than the other groups .", "this research was carried out on a small set of patients ( n = 96 ) ; therefore , there was an imbalance between men and women enrolled in this trial .", "group ii had a 2-fold elevation in the number of patients going into af postoperatively .", "in conclusion , pp may be a useful technique in the prevention of pericardial effusion and cardiac tamponade after cas because it is simple , safe , and effective .", ". \n oral presentation at the 22 \n annual meeting of the asian society for cardiovascular and thoracic surgery that will be held in istanbul on april 3 - 6 , 2014 ."], "abstract_text": ["<S> introductionpericardial effusion and atrial fibrillation occur commonly after coronary artery surgery.aim of the studya prospective randomized clinical trial was conducted to evaluate the effects of posterior pericardiotomy on the occurrence of pericardial effusion , tamponade , and atrial fibrillation.material and methodsthe study group consisted of 96 patients ( 77 male and 19 female ) at a mean age of 58.1  9.8 years . </S>", "<S> the patients were randomly assigned to one of three study groups : patients undergoing posterior pericardiotomy ( group i , n = 30 ) , controls ( group ii , n = 33 ) , and patients with additional posterior pericardial drainage tubes ( 28 mm ) who did not undergo posterior pericardiotomy ( group iii , n = 33).resultspostoperative hospitalization ( p = 0.03 ; 11.56  10.64 ) and reoperation due to tamponade ( p = 0.019 ; 12.1% ) were significantly higher in group ii . </S>", "<S> extensive pericardial effusions were detected in one patient on the first postoperative day ( group ii , n = 1 ) , in one patient on the fifth postoperative day ( group iii , n = 1 ) , and in one patient on the 30th day after the operation ( group iii , n = 1 ) . </S>", "<S> pericardial effusion exhibited regression in group i on postoperative day 30 ( p = 0.028 ) . a higher rate of postoperative atrial fibrillation was noted in group i , but no significant differences were found between the groups with regard to postoperative atrial fibrillation.conclusionspatients who did not undergo posterior pericardiotomy or did not receive posterior chest tubes exhibited residual pericardial effusion , required longer hospitalization , and had to be reoperated due to tamponade . </S>", "<S> both posterior pericardiotomy and the use of posterior tubes are effective in the early postoperative period . </S>"], "labels": null, "section_names": ["Wstp", "Cel pracy", "Materia i metody", "Wyniki", "Wnioski", "Introduction", "Material and methods", "Results", "Discussion", "Limitations", "Conclusions", "Disclosure"], "sections": [[""], ["w celu oceny wpywu perikardiotomii tylnej na wystpowanie wysiku osierdziowego , tamponady oraz migotania przedsionkw przeprowadzono prospektywne badanie kliniczne z randomizacj ."], ["badana grupa skadaa si z 96 pacjentw ( 77 mczyzn i 19 kobiet ) w wieku rednio 58,1  9,8 roku .", "pacjenci zostali losowo przydzieleni do jednej z trzech grup : grupy pacjentw poddanych perikardiotomii tylnej ( grupa", "i , n = 30 ) , grupy kontrolnej ( grupa ii , n = 33 ) oraz pacjentw , u ktrych zastosowano tylny drena osierdziowy , ktrzy jednak nie zostali poddani zabiegowi perikardiotomii tylnej ( grupa iii , n = 33 ) ."], ["czas trwania hospitalizacji pooperacyjnej ( p = 0,03 ; 11,56  10,64 ) oraz czsto reoperacji z powodu tamponady ( p = 0,019 ; 12,1% ) byy znaczco wysze w grupie ii .", "znaczny wysik osierdziowy wykryto u jednego pacjenta ( grupa ii , n = 1 ) w pierwszym dniu po operacji , u jednego pacjenta w pitym dniu po operacji ( grupa iii , n = 1 ) oraz u jednego pacjenta w 30", "dniu po operacji ( p = 0,028 ) w grupie i odnotowano regresj wysiku osierdziowego .", "i stwierdzono rwnie wyszy wspczynnik pooperacyjnego migotania przedsionkw , jednake w tym wzgldzie midzy grupami nie byo znaczcych rnic ."], ["pacjenci , ktrzy nie zostali poddani perikardiotomii tylnej lub u ktrych nie zastosowano tylnego drenau klatki piersiowej , wykazywali szcztkowy wysik osierdziowy , wymagali duszej hospitalizacji i musieli by ponownie operowani z powodu tamponady .", "zarwno perikardiotomia tylna , jak i tylny drena klatki piersiowej okazay si skuteczne we wczesnym okresie pooperacyjnym ."], ["pericardial effusion , which is a common complication after coronary artery surgery ( cas ) , may be the cause of significant morbidity .", "it is often benign and small in volume , but can lead to life - threatening events , such as cardiac tamponade .", "delayed postoperative cardiac tamponade following an open - heart operation is an infrequent , but potentially serious complication .", "although previous reports have dealt with the clinical features and management of this problem , an optimal approach to prevent postoperative pericardial effusion and related problems has not been clearly defined [ 3 , 5 ] .", "this study aims to investigate the effects of posterior pericardiotomy ( pp ) on the development of pericardial effusion , tamponade , and atrial fibrillation following cas ."], ["this prospective randomized case - controlled study was carried out on 96 patients ( 77 men and 19 women ; age : 35 - 78 years ; mean : 58.1 years ) .", "the patients , undergoing cas performed by the same surgical team between march 2012 and january 2013 in istanbul mehmet akif ersoy thoracic and cardiovascular surgery research and training hospital , were assigned to 3 groups .", "after the study had received the approval of the institutional review board , informed consent was obtained from all patients .", "the treating physician did not know the groups to which individual patients had been assigned . patients with renal failure ,", "hyperthyroidism , emergency cas , history of cardiac operations associated with valvular heart disease , low ejection fraction ( < 35% ) , and preoperative atrial fibrillation or other rhythm disorders , as well as patients who did not provide research authorization , were excluded from the study .", "the study groups included patients after pp ( group i , n = 30 ) , a control group ( group ii , n = 33 ) , and patients without pp who received posterior pericardial tubes ( the tube was placed behind the heart and sutured to the adjacent pericardial tissue to avoid tube - induced ventricular arrhythmias ) ( group iii , n = 33 ) .", "i , a 4-cm longitudinal incision was made parallel and posterior to the left phrenic nerve , extending from the left inferior pulmonary vein to the diaphragm , as described by mulay et al . .", "in group i , the relationship between the location of the distal part of circumflex anastomosis and pp was also carefully considered .", "moreover , all patients underwent pleurotomy and lateral pericardiotomy ( approximately 3.5 - 4.5 cm in size ) in which the internal thoracic artery ( ita ) was used for anastomosis and positioned medially to the lung , which allowed the ita to run without kinking or twisting .", "the inferior part of the pericardium was left open ( 2 cm ) in our study .", "a straight tube was placed in the anterior mediastinum and an angled tube was placed in the left hemithorax in all patients .", "the same postoperative pain management protocol was provided for all patients . after a routine closure of the chest", ", drains were frequently milked and stripped to ensure tube patency during the intensive care unit stay .", "the chest tubes were removed on the 2 postoperative day when the drainage became serous ; otherwise , they remained in use for one or more days .", "pericardial effusion was defined as the accumulation of fluid in the pericardial space determined by transthoracic echocardiography ( tte ) .", "the presence of pericardial effusion on 2-dimensional tte was assessed using the criteria previously described by bakhshandeh et al . .", "two - dimensional echocardiography was performed on postoperative days 1 and 5 , as well as 1 month after surgery .", "the maximum diastolic separation between the pericardium and epicardium was measured at the tip of the mitral valve leaflet .", "when the diastolic echo - free space between the left ventricular posterior wall and the pericardium was < 10 mm , the pericardial effusion was classified as small .", "when the space was 10 - 20 mm , the effusion was classified as moderate , while for spaces > 20 mm , the effusion was classified as severe .", "if the diagnosis of evident pericardial effusion ( moderate or severe ) was established , tte was performed daily , and the volume of the effusion was observed closely .", "we compared the groups with regard to the volume of effusion on postoperative days 1 , 5 , and 30 .", "patients with no or small pericardial effusion and an uneventful postoperative course were discharged from the hospital on postoperative day 5 .", "patients with moderate and severe pericardial effusion were treated with non - steroidal anti - inflammatory drugs . during hospitalization ,", "all patients were continuously monitored with the use of a portable electrocardiogram ( ecg , telemetry ) to detect atrial fibrillation .", "atrial fibrillation was considered to be persistent and clinically significant when it lasted for more than 5 min .", "statistical analysis was performed with the ncss ( number cruncher statistical system ) 2007 statistical software ( utah , usa ) .", "one - way anova and tukey hsd tests were used for the statistical analysis of the groups parameters .", "the differences were also analyzed using  tests and independent t - tests where applicable ."], ["the hospital mortality in the study was 2% ( n = 2 ) ; both patients were in group ii .", "one of these patients experienced low cardiac output during and following reoperation due to cardiac tamponade and died on postoperative day 12 .", "the other patient had an uneventful postoperative course , but was readmitted to the hospital on postoperative day 11 due to cardiac tamponade and acute kidney failure .", "pericardial drainage was performed , but the patient died of sepsis on postoperative day 45 .", "the groups did not differ in terms of demographic parameters , which are summarized in table i. the average total volume of drainage ( mediastinal and thoracic ) during the first 24 hours was similar in all groups ( 536.67  237.873 cc , 656.97  407.634 cc , and 577.27  374.349 cc , respectively ; p = 0.383 ) ( table ii ) .", "in group i , the volume of the fluid evacuated by mediastinal drainage was lower than in groups ii and iii , but the difference was not statistically significant ( table ii ) .", "two patients in group ii required reoperation due to excessive mediastinal hemorrhage and cardiac tamponade .", "the incidence of cardiac tamponade was higher in group ii ( n = 4 , 12% ) , and the differences between the groups were statistically significant ( p = 0.019 ) .", "two of the patients with pericardial tamponade were managed by subxiphoid pericardial drainage alone ; 400 to 1000 ml of fluid was removed .", "two patients in group iii were reoperated due to excessive hemorrhage in the early postoperative period . \n ", "demographic variables in groups differential drainage volume from each chest tube the volume of pericardial drainage was added to the volume of mediastinal drainage for group iii .", "the average volume of pericardial effusion significantly decreased with time in groups i and ii ( fig .", "approximately 50% of the patients had small pericardial effusion on the first postoperative day ; no difference between the groups was found .", "moderate pericardial effusion was detected in 8 patients ( group i , n = 1 ; group ii , n = 3 ; group iii , n = 4 ) on the first postoperative day , in 11 patients ( group ii , n = 7 ; group iii , n = 4 ) on the fifth postoperative day , and in 7 patients on the 30 postoperative day ( group ii , n = 6 ; group iii , n = 1 ) during the study period .", "severe pericardial effusion was detected in one patient ( group ii , n = 1 ) on the first postoperative day , in one patient ( group iii , n = 1 ) on the fifth postoperative day , and in one patient ( group iii , n = 1 ) on the 30 postoperative day .", "the incidence of moderate to severe pericardial effusion in group i was significantly lower than in the other groups on the 30 postoperative day ( n = 0 , p = 0.028 ) .", "figure 1 shows a comparison of the groups with regard to the volume of pericardial effusion .", "four of these patients ( group ii , n = 4 ) underwent drainage of severe pericardial effusion on postoperative days 0 , 1 , 11 , and 17 ; one patient ( group ii ) underwent sternal dehiscence repair .", "comparison of the groups with regard to the volume of pericardial effusion atrial fibrillation was detected in 24 of the 96 patients ( 25% : 6 patients in group i , 11 patients in group ii , 7 patients in group iii ) .", "no statistically significant difference between the groups ( p = 0.392 ) was present in this respect .", "the length of hospital stay was significantly shorter in group i ( 6.63  2.71 , p = 0.03 ) ."], ["there are a number of articles favoring the use of posterior pericardiotomy , but no comprehensive comparison has been conducted between the use of a pericardial tube and pp in patients undergoing cas .", "pericardial effusion and the associated complications are common after all kinds of cardiac surgery due to postoperative bleeding or post - cardiotomy syndrome .", "however , clinically significant pericardial effusion occurs rarely and can be a crucial risk factor for cardiac tamponade ( 1% ) .", "posterior pericardiotomy has been reported to reduce the prevalence of pericardial effusion from 40% to 8% with a simultaneous reduction in the prevalence of svt ( supraventricular tachycardia ) .", "the semi - sitting position helps thoracic drainage in the early postoperative period by supporting the patient 's back . despite all the above - mentioned measures , the posterior wall of the pericardial sac forms an unprotected space that may cause pericardial effusion to develop septae .", "this leads to the appearance of adhesions preventing blood drainage , leading to cardiac compression .", "moreover , the collection of fluid may persist due to bleeding resulting from the use of anticoagulants .", "in fact , anti - coagulants ( low - molecular - weight heparin ) were used postoperatively in our patients with atrial fibrillation .", "posterior pericardial effusion can be evacuated by placing a tube in the posterior pericardial space .", "the placement of the pericardial tube is simple to perform , but it may have certain drawbacks . the tube may come into contact with coronary grafts during cardiac activity or movement .", "however , the patients included in the study ( in group iii as well as the other groups ) did not exhibit any arrhythmias ( apart from af ) or ischemic changes", ". this may be due to the fact that we fixed the pericardial tube to the surface of the diaphragmatic pericardium with polypropylene .", "pp or the use of posterior tubes may lessen the volume of pericardial effusion after cas .", "moreover , patients in group i exhibited regression of pericardial effusion during 30 postoperative days .", "this proved that pp had a lasting effect on pericardial effusion , as presented by figure 1 .", "what stands out in our results is that pericardial effusion may occur if no intervention is performed on the posterior part of the pericardium .", "this technique is suggested in both cas and valve surgery , but it is not routinely used [ 13 , 14 ] .", "what is more , pp is recommended by the american college of chest physicians guidelines and by some authors as a preventive technique for postoperative atrial fibrillation [ 15 , 16 ] .", "supraventricular tachyarrhythmias , mainly in the form of atrial fibrillation , occur in up to 40% of patients undergoing cas .", "although af is usually benign , it can cause hemodynamic instability , prolonged hospital stay , and increased cost and , in rare cases , predispose to cerebrovascular accidents .", "the antiarrhythmic effect of pp may , however , be a theoretical issue , as we observed similar results regarding atrial fibrillation in all the groups .", "in fact , the results of our study were different than expected with regard to atrial fibrillation compared to other researchers [ 4 , 6 , 16 ] .", "additionally , asimakopoulos et al . also found that the prevalence of atrial fibrillation was not significantly reduced ( 20% ) in comparison to the conventional technique ( 26% ) .", "another issue is that humans have increasingly longer life spans , which may result in higher numbers of reoperations .", "ellman et al . found that patent coronary grafts were the most commonly injured structures during reoperation ( 46% ) .", "we routinely cover the aorta and proximal anastomoses of grafts with thymic fat tissue and the anterior wall of the pericardial cavity .", "we also place an anterior mediastinal tube over the thymic fat tissue before closing the sternum in all patients . behind the heart ,", "adhesions between the inferior surface of the heart and the diaphragm can cause difficulties when dissecting the tissue layers during reoperation .", "mulay et al . demonstrated that pp provides an effective drainage pathway from the left pleural cavity , thereby reducing the prevalence of pericardial effusion .", "for this reason , pp may be advantageous for reoperated patients in terms of reducing adhesions related to pericardial effusion .", "echocardiography can be useful in confirming and identifying the presence of pericardial effusion , hematoma , and tamponade . according to the results of our study , it is important to diagnose pericardial effusion at an early stage .", "hemodynamic deterioration due to pericardial fluid can be divided into four stages : pericardial fluid , hemodynamic tamponade , echocardiographic tamponade , clinical tamponade .", "echocardiographic follow - up of patients with pericardial effusion might be a factor extending the duration of hospital stay .", "we recommend the pp technique and routine echocardiographic monitoring during the postoperative period in patients undergoing cas .", "there is not sufficient research regarding the effects of pp on long - term results .", "the present study also demonstrated that patients undergoing pp required a significantly shorter hospital stay and experienced lower rates of pericardial effusion and cardiac tamponade than the other groups ."], ["this research was carried out on a small set of patients ( n = 96 ) ; therefore , there was an imbalance between men and women enrolled in this trial .", "group ii had a 2-fold elevation in the number of patients going into af postoperatively ."], ["in conclusion , pp may be a useful technique in the prevention of pericardial effusion and cardiac tamponade after cas because it is simple , safe , and effective .", ". \n oral presentation at the 22 \n annual meeting of the asian society for cardiovascular and thoracic surgery that will be held in istanbul on april 3 - 6 , 2014 ."], [""]]}
{"article_id": "PMC5223030", "article_text": ["cancer places a huge burden on society and has been identified as the leading cause of death in both more and less economically developed countries .", "projections based on the globocan 2012 estimates predict a substantive increase to 19.3 million new cancer cases per year by 2025 , due to growth and ageing of the global population .", "south africa , like other developing countries , is also experiencing an increase in the overall burden of disease attributable to cancer , with the number of new cancer cases predicted to increase by 46% by 2030 .", "this is likely to result in an increase in the use of cancer chemotherapy agents , which assist in preventing the proliferation , invasion , and metastases of the cancer cells .", "the basis for chemotherapy is anticancer drugs containing platinum , that is , cisplatin ( cis - diamminedichloroplatinum ii ) and carboplatin ( cis - diammine 1,1-cyclobutane dicarboxylatoplatinum ii ) .", "other chemotherapy drugs include nitrogen mustard , amino - nicotinamide , dichloromethotrexate , bleomycin , and 5-fluorouracil [ 5 , 6 ] .", "the first of these drugs , that is , cisplatin , consists of a divalent pt ( ii ) central atom and four ligands of cis - positioned pairs of chlorine atoms or amine groups .", "since its discovery in the 1970s , cisplatin continues to be hailed as one of the most potent cancer chemotherapeutics in children and adults , as it is unique and unmatched in its effectiveness against many cancers , namely , osteogenic sarcoma , medulloblastoma , testicular , cervical , and ovarian cancers .", "similarly , its toxicity profile is expansive , involving the gastrointestinal , hematologic , renal , and auditory systems .", "while the use of saline hydration and mannitol diuresis may prevent nephrotoxicity , neurotoxicity is still not curable or preventable .", "ototoxicity refers to the hearing disorder that results from the temporary or permanent inner ear dysfunction after treatment with an ototoxic drug .", "other drug classes known to have ototoxic properties include aminoglycosides , loop diuretics , quinine , nonsteroidal anti - inflammatory drugs , and antiretroviral therapy ( art ) .", "this is of concern in south africa , as it is estimated that 12.2% of the population ( 6.4 million persons ) were hiv positive in 2012 , which is 1.2 million more people living with hiv than in 2008 ( 10.6% , or 5.2 million ) .", "resultantly , art exposure had almost doubled from 16.6% in 2008 to 31.2% in 2012 .", "not only will many infected people be at risk for ototoxicity due to arts , but a large number will also be susceptible to hiv - related cancers , such as kaposi 's sarcoma , non - hodgkin 's lymphoma , and cervical cancer , as well as infectious diseases such as tuberculosis , conditions that often require pharmacological therapy with the adverse side effect of ototoxicity .", "it is possible that their treatments could consist of simultaneous use of more than one ototoxic drug , increasing the likelihood of ototoxicity .", "all health care professionals managing patients with cancer should therefore be knowledgeable about the ototoxic properties of cisplatin .", "however , malhotra   indicated that most oncologists in india do not make referrals for audiological evaluations of patients receiving cisplatin , while a study in south africa revealed that the effects of ototoxicity , the role of audiologists , and need for their expertise were not fully realized by the oncologists sampled .", "this is further supported by evidence from the south african study of khoza - shangase and jina   which indicated that most general practitioners sampled also do not appear to carry out ototoxicity monitoring strategies , despite being aware of their own role within an ototoxicity monitoring programme .", "this review therefore aims to serve as resource for health professionals to enhance their understanding of ototoxicity as well as their roles within an ototoxicity monitoring programme by providing an overview and description of this condition in patients diagnosed with cancer and receiving cisplatin chemotherapy .", "the review identified peer - reviewed articles available from january 1975 to july 2015 on the topic of cisplatin - associated ototoxicity and ototoxicity monitoring and included english articles only .", "the same researcher conducted the literature search and reviewed the abstracts and articles for inclusion in the study .", "studies were identified using keyword and mesh term searches of electronic databases depicted in table 1 . a manual search of relevant authors and journals", "the references cited by each publication , review , or book chapter were reviewed in order to locate additional potential publications . in order to be selected , the article had to present data on either cisplatin ototoxicity and/or ototoxicity monitoring in human participants , and no research designs were excluded . running these searches yielded a total of 2106 records , of which 1581 were excluded based on the title and/or abstract as well as duplication .", "eighty - five relevant articles , comprising six national and 79 international articles , were selected .", "a perusal of narrative reviews of other auditory pathologies was conducted in an attempt to determine areas of significance for an overview of cisplatin ototoxicity .", "this resulted in the following eight areas being included : the mechanisms of cisplatin ototoxicity , clinical presentation , risk factors , incidence rates in adults and children , the effect on quality of life , ototoxicity monitoring , otoprotective strategies , and management of an ototoxic hearing loss .", "one such mechanism , the antioxidant model , involves the formation of reactive oxygen species ( ros ) within the cochlea and consequent reduction in antioxidant enzymes following exposure to cisplatin chemotherapy [ 1620 ] .", "another mechanism of cisplatin ototoxicity involves the significant contribution of nicotinamide adenine dinucleotide phosphate oxidase 3 isoform ( nox3 ) to the generation of reactive oxygen species within the cochlea , when activated by cisplatin [ 17 , 21 ] , while a third mechanism relates to the activation of transient receptor potential vanilloid 1 channel ( trpv1 ) [ 2224 ] .", "the molecular mechanisms of cisplatin ototoxicity therefore include the following:creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation ands - nitrosylation of cochlear proteins  .with the cellular mechanisms of cisplatin - associated ototoxicity including damage to the outer hair cells , supporting cells , marginal cells of the stria vascularis , spiral ligament , and the spiral ganglion cells , it is evident that the structures of the inner ear are most susceptible to damage by cisplatin chemotherapy , with apoptotic degeneration of the hair cell in the organ of corti being most prominent .", "the outer hair cells in the basal turn of the cochlea are most affected [ 27 , 28 ] .", "this leads to an initial elevation of high frequency audiometric thresholds , followed by a progressive loss into the lower frequencies with continued therapy [ 27 , 28 ] .", "knowledge of the different mechanisms of cisplatin ototoxicity is important for health care professionals as it will create an awareness of its complexity and the resulting clinical presentation . ", "creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation and s - nitrosylation of cochlear proteins  .", "cisplatin - associated ototoxicity usually manifests as irreversible , progressive , bilateral , high frequency sensorineural hearing loss   with tinnitus .", "the latter may occur with or without a hearing loss   and may be permanent or transient , sometimes disappearing a few hours after treatment   or alternatively persisting a week after treatment . while most of the hearing loss is permanent , there is sometimes sporadic and partial recovery . in addition ,", "rare cases of unilateral hearing loss have been reported , which are usually explained by tumour location and surgical or therapeutic intervention on the affected side . moreover", ", the hearing loss is not always symmetrical [ 33 , 34 ] , with jenkins et al .   finding that 75% of women on cisplatin chemotherapy displayed an asymmetry of hearing thresholds of at least 10  db between ears posttreatment .", "schmidt et al . , in their investigation of 55 children on cisplatin chemotherapy , found that the high frequency hearing thresholds were slightly elevated in the left ear and that males had a greater degree of hearing loss than the females .", "the degree of hearing loss is often variable and is related to the dose ; that is , the higher the cumulative dose , the greater the ototoxic effect [ 35 , 36 ] .", "the duration , number of cycles administered , and method of administration   also influence cisplatin - associated ototoxicity .", "additional factors that may increase ototoxicity include exposure to concomitant noise , chemicals , and other ototoxic medications .", "furthermore , evidence also shows that melanin content is related to an increased risk of cisplatin - associated ototoxicity .", "individuals with dark eyes and therefore a higher melanin content in the cochlear are at greater risk of ototoxic damage , as the melanin causes retention of the platinum within the cochlear and subsequently increases the risk of damage [ 41 , 42 ] .", "individuals presenting with renal insufficiency , that is , high levels of serum creatinine , are at a greater risk for cisplatin - associated ototoxicity . genetic risk factors , such as megalin and glutathione s - transferases gene polymorphism , have also been reported to influence cisplatin ototoxicity , as do physiological factors such as age , with younger children   and older adults ( older than 46 years )   presenting with a greater severity of hearing damage .", "awareness of these risk factors may assist health care professionals with informational counselling of the patient receiving cisplatin chemotherapy .", "the incidence of cisplatin ototoxicity is variable in adults ( table 2 ) and children ( table 3 ) .", "the variations may be due to a number of factors , such as differences in the dose , both within a cycle and the total amount administered over multiple cycles , time interval between courses , method of administration , and treatment duration , as well as differences in patient population .", "ototoxicity poses a major problem to the cancer patient , as the quality of life after receiving cisplatin chemotherapy may be negatively affected due to hearing loss , resulting in social , emotional , and vocational difficulties , as effective communication is often hindered .", "tasks that normal hearing persons take for granted may become challenging and frustrating . in addition , an individual 's safety may be compromised due to the hearing loss , as appropriate response to alarms and warning signals may be delayed .", "furthermore , a hearing loss may also result in psychosocial and physical health problems , as well as depression and social isolation .", "hence , hearing loss , often referred to as the  invisible condition ,  has serious visible ramifications on the quality of life of a hearing impaired individual .", "this is particularly relevant if the individual has already experienced the hearing world , as the hearing function is never restored to normal , even though patients may benefit from the use of assistive listening devices , such as hearing aids and cochlear implants .", "the impact of an ototoxic hearing loss may be more profound for infants and young children who are at a critical stage of their speech and language development .", "furthermore , the high frequency nature of an ototoxic hearing loss may result in speech recognition and comprehension being compromised , resulting in possible neurocognitive and psychosocial delays .", "there is also an elevated risk for academic learning problems and psychosocial difficulties in school - aged children and adolescents .", "literature indicated that childhood survivors of neuroblastoma had twice the rate of difficulties , as indicated by parent reports , with reading and math skills , and/or attention and a higher risk of a general learning disability than those without a hearing loss .", "there was also poorer self - reported quality of life scores in these children with regard to school functioning .", "hence , cisplatin - associated ototoxicity further complicates the morbidity of cancer patients , as it would also isolate them from family members and significant others at a time when they require the greatest support .", "advancements in medical knowledge and technology , such as screening and early detection of several cancers , have resulted in notable improvements in relative five - year survival rates for cancer [ 64 , 65 ] .", "therefore , improving the quality of life after cisplatin - based chemotherapy becomes increasingly important , and resulting comorbidities such as ototoxicity can be managed appropriately and immediately   if adequate monitoring is in place .", "the nature of ototoxicity is such that it often goes undetected until speech intelligibility is affected   and is usually detected when a communication problem becomes evident .", "communication problems , such as constantly asking for repetition or not responding when spoken to , signify that the hearing loss has progressed to the frequencies important for understanding speech . in this case ,", "an audiological monitoring programme can avert , to a large extent , the reduced quality of life as a result of hearing loss , as patients on cisplatin chemotherapy can be identified early , counselled , monitored , and managed appropriately through interventions in a logical , systematic , and coherent manner .", "audiological monitoring should aim to identify the hearing loss early and reduce its impact on the individual 's life by means of proper medical and hearing intervention .", "prospective audiological evaluations remain the only reliable method for detecting ototoxicity before it becomes symptomatic .", "an ototoxicity monitoring programme should involve a health care team comprising of an oncology nurse , oncologists , audiologist , and pharmacist to ensure effective sustainability of such a programme , if implemented , with the patient being the central focus .", "the audiologist is involved in identifying an ototoxic hearing loss , informing the oncologist of such a development , counselling the patient and their family , and prescribing amplification devices , such as hearing aids and cochlear implants .", "early identification of an ototoxic hearing loss provides oncologists with an opportunity to adjust the chemotherapy regimen in order to reduce or prevent further deterioration of hearing . the oncologist and nurses should also counsel patients on the side effects of cisplatin , including ototoxicity , in an attempt to prepare them for treatment outcomes and help them set realistic expectations .", "pharmacists who have access to a patient 's medication list may also alert the oncologists and audiologists to those who are on other ototoxic medication and therefore at a greater risk for cisplatin - induced ototoxicity .", "effective management of such patients using evidence - based practices may improve management of those with cancer , ensuring that they and their families are counselled and appropriate interventions are timeously implemented .", "the principles of early identification and early intervention are a part of ototoxicity monitoring , and the audiologist can manage such a programme . in countries without ototoxicity management guidelines , the  guidelines for the audiological management of individuals receiving cochleotoxic drug therapy  developed by the american association of speech - language - hearing association   may , consequently , guide the audiologist in the implementation of an ototoxicity monitoring programme within a local , regional , or national setting . for widespread acceptance and use ,", "ototoxicity monitoring programmes need to incorporate efficient and cost - effective ototoxicity identification techniques , while considering the health care system and demographics of the patient population being managed .", "for any population receiving ototoxic medication , the following should be considered :  ( 1 ) the patient 's level of alertness or ability to respond reliably ; ( 2 ) the most appropriate times during the treatment protocol for test administration , and ; ( 3 ) the test should comprise the baseline , monitoring and post - treatment evaluations  .", "appropriate time intervals for audiological assessments may differ depending on the type of cancer as well as the frequency and dose of cisplatin ( figure 2 ) .", "the audiological assessments should incorporate a detailed case history , otoscopic examination , immittance audiometry , speech audiometry , dpoaes , and conventional and extended high frequency audiometry ( i.e. , up to 20  000  hz ) ( hfa ) [ 69 , 73 ] . these procedures are all conducted for the baseline assessment and the six - month follow - up evaluation [ 69 , 73 ] . while auditory brainstem response may be used , it is not considered a standard procedure for monitoring ototoxicity . monitoring audiological evaluations during treatment and the one- and three - month follow - up evaluations include case interview , otoscopy , and immittance audiometry as well as air conduction pure tone and objective testing . however , full - frequency threshold testing is impractical for many patients on cisplatin chemotherapy , as these individuals are often extremely ill and easily fatigued . the use of abbreviated threshold monitoring procedures that are clinically practical for these patients is therefore recommended .", "one such method involves the use of the sensitive range for ototoxicity ( sro ) .", "this is  the highest frequency with a threshold at or below 100  db spl followed by the next six lower adjacent frequencies in 1/6-octave steps or the one octave range near the highest audible frequency  .", "sro is usually determined during the baseline evaluation and is dependent on each patient 's hearing threshold configuration . during monitoring evaluations", "however , full - frequency testing should be conducted within the same session if an asha significant hearing change is noted within the sro .", "if a patient on cisplatin chemotherapy is still responsive and alert , the protocol presented above would be suitable .", "however , a patient who has limited responsiveness may be required to undergo the same audiological evaluations , except speech audiometry .", "patients who are responsive as well as those who have limited responses can undergo both behavioural and objective testing . however , those patients who are too ill or too young to respond should undergo only objective testing , such as otoscopy , tympanometry , acoustic reflexes , and dpoaes or abrs . while pure tone audiometry in the conventional frequency range is suitable for evaluating hearing in the range responsible for speech understanding , as well as for differential diagnosis , it is less sensitive to detecting early ototoxic change [ 11 , 70 ] .", "the two tests identified as being the most important for the early detection of cisplatin ototoxicity are hfas and oaes , each also having limitations ( see table 4 )", ". therefore , using each test in isolation may not be as effective as utilizing a test battery approach , as it increases the chances of obtaining reliable audiologic monitoring data over time .", "in addition , these two tests could be used to complement one another in every cycle of chemotherapy to ensure the earliest detection of ototoxicity . the ototoxicity monitoring protocol proposed by", "asha   represents an aggressive and ideal approach for monitoring ototoxicity and is dependent on a country 's infrastructure and resource constraints .", "the asha   guidelines may therefore not be generalized to a country without considering the contextual factors that may influence its applicability to that country .", "however , it does provide guidance towards creating a roadmap that countries , such as south africa , may aspire towards in implementing an ototoxicity monitoring programme .", "similar to india , no programmes have been formally implemented to identify and monitor ototoxicity in patients on cancer chemotherapy in south africa . as a result , there is no contextually relevant research to steer the implementation of an accountable and effective ototoxicity monitoring program in the country .", "this is probably one of the main reasons for ototoxicity monitoring programmes not being commonplace in local hospitals and clinics .", "in addition , the health of south africans is characterized by a quadruple burden of disease , encompassing the occurrence of infectious diseases , the rise of noncommunicable diseases , and perinatal and maternal disorders , as well as injuries and violence , which may result in cancer receiving low priority for health care services . however", ", the creation of an audiological monitoring programme allows for better control of cancer related comorbidities . over the years", ", a number of studies have investigated the use of otoprotectants with cisplatin , their purpose being to protect the inner ear from any injury while not interfering with the antitumor effects of cisplatin .", "otoprotective strategies include reducing the formation of free radicals by maintaining glutathione levels and antioxidant activity .", "three mechanisms may provide protection against cisplatin , these being endogenous molecules , exogenous agents , or a combination of exogenous agents that trigger endogenous protective mechanisms .", "however , endogenous agents are not effective against cisplatin when the dose exceeds a certain threshold [ 17 , 81 ] .", "nearly all of the otoprotective agents are sulfur- or sulfhydryl - containing compounds ( thio compounds ) , known as antioxidants , and potent heavy metal chelators .", "the numerous otoprotective agents utilized in clinical and animal studies include amifostine , d - or l - methionine , methylthiobenzoic acid , lipoic acid , tiopronin , glutathione ester , sodium thiosulfate , melatonin , vitamin e , n - acetylcysteine , dexamethasone , and resveratrol . however", ", none of these agents have been found to be unequivocally beneficial in preventing cisplatin ototoxicity and no agent is currently recommended for routine use . further research is therefore needed to find new methods and optimize old ones to prevent and/or treat hearing loss during cisplatin therapy .", "in addition , intratympanic administration of medication together with gene therapy needs to be further explored .", "intratympanic administration involves the diffusion of the otoprotective agent across the round window into the inner ear , where its therapeutic effect is exerted .", "an advantage of this method of administration is that there are higher concentrations of the otoprotective agent in the inner ear , this being in comparison to the use of oral or parenteral administration , without potentially reducing the efficacy of the cisplatin treatment [ 89 , 90 ] .", "the disadvantage of this procedure , however , is that each ear would have to be treated with a moderately invasive procedure . alternatively", ", gene therapy may prove to be beneficial in protecting an individual against cisplatin - induced hearing loss as several genes , namely , megalin , glutathione - s - transferases , thiopurine s - methyltransferase , and catechol - o - methyl transferase , may be responsible for susceptibility to hearing loss .", "if a cisplatin - associated hearing loss results in communication difficulties , it is the audiologist 's ethical responsibility to begin or recommend aural rehabilitation .", "however , this intervention should not only occur once hearing loss has been detected but before the patient begins the cisplatin chemotherapy .", "aural rehabilitation techniques such as speech reading and counselling on compensatory communication strategies should be conducted .", "the counselling should include spouses and significant other , as hearing loss may not only impact the person with cancer but also frequent communication partners .", "patients with sensorineural hearing loss due to the use of cisplatin may also benefit from the use of assistive listening devices such as hearing aids or cochlear implants .", "children with ototoxic hearing loss may also require the use of personal frequency modulated systems in the classroom .", "furthermore , with the recent developments in hearing aid technology , a patient with an ototoxic hearing loss is more likely to receive the desired amplification benefit .", "these hearing aids are able to amplify sounds at and above 8000  hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology .hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping .", "frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound .", "while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds .", "commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) .", "these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively . ", "these hearing aids are able to amplify sounds at and above 8000  hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology .", "hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping .", "frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound .", "while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds .", "commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) .", "these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively .", "this review has highlighted that cisplatin ototoxicity is a frequent adverse event of cisplatin chemotherapy that may negatively affect the quality of life of patients with cancer .", "the different molecular and cellular mechanisms involved in cisplatin - associated ototoxicity highlight the complexity of this condition and the consequent difficulty in identifying an effective otoprotective agent .", "the varying incidence rates reported in both adults and paediatrics may be due to the different audiological tests employed in the monitoring of the cancer patient 's hearing status and therefore highlight the importance of the use of extended high frequency audiometry and dpoaes in ototoxicity monitoring .", "an audiological monitoring programme comprising a team of health care professionals , knowledgeable about cisplatin ototoxicity , may therefore serve to improve evidence - based service delivery to these patients ."], "abstract_text": ["<S> cisplatin is an effective drug used in the treatment of many cancers , yet its ototoxic potential places cancer patients , exposed to this drug , at risk of hearing loss , thus negatively impacting further on a patient 's quality of life . </S>", "<S> it is paramount for health care practitioners managing such patients to be aware of cisplatin 's ototoxic properties and the clinical signs to identify patients at risk of developing hearing loss . </S>", "<S> english peer - reviewed articles from january 1975 to july 2015 were assessed from pubmed , science direct , and ebscohost . </S>", "<S> seventy - nine articles and two books were identified for this review , using mesh terms and keywords such as  ototoxicity  ,  cisplatin  ,  hearing loss  , and  ototoxicity monitoring  . </S>", "<S> this review provides an up - to - date overview of cisplatin - associated ototoxicity , namely , its clinical features , incidence rates , and molecular and cellular mechanisms and risk factors , to health care practitioners managing the patient with cancer , and highlights the need for a team - based approach to complement an audiological monitoring programme to mitigate any further loss in the quality of life of affected patients , as there is currently no otoprotective agent recommended routinely for the prevention of cisplatin - associated ototoxicity . </S>", "<S> it also sets the platform for effective dialogue towards policy formulation and strengthening of health systems in developing countries . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Method", "3. Conclusion"], "sections": [["cancer places a huge burden on society and has been identified as the leading cause of death in both more and less economically developed countries .", "projections based on the globocan 2012 estimates predict a substantive increase to 19.3 million new cancer cases per year by 2025 , due to growth and ageing of the global population .", "south africa , like other developing countries , is also experiencing an increase in the overall burden of disease attributable to cancer , with the number of new cancer cases predicted to increase by 46% by 2030 .", "this is likely to result in an increase in the use of cancer chemotherapy agents , which assist in preventing the proliferation , invasion , and metastases of the cancer cells .", "the basis for chemotherapy is anticancer drugs containing platinum , that is , cisplatin ( cis - diamminedichloroplatinum ii ) and carboplatin ( cis - diammine 1,1-cyclobutane dicarboxylatoplatinum ii ) .", "other chemotherapy drugs include nitrogen mustard , amino - nicotinamide , dichloromethotrexate , bleomycin , and 5-fluorouracil [ 5 , 6 ] .", "the first of these drugs , that is , cisplatin , consists of a divalent pt ( ii ) central atom and four ligands of cis - positioned pairs of chlorine atoms or amine groups .", "since its discovery in the 1970s , cisplatin continues to be hailed as one of the most potent cancer chemotherapeutics in children and adults , as it is unique and unmatched in its effectiveness against many cancers , namely , osteogenic sarcoma , medulloblastoma , testicular , cervical , and ovarian cancers .", "similarly , its toxicity profile is expansive , involving the gastrointestinal , hematologic , renal , and auditory systems .", "while the use of saline hydration and mannitol diuresis may prevent nephrotoxicity , neurotoxicity is still not curable or preventable .", "ototoxicity refers to the hearing disorder that results from the temporary or permanent inner ear dysfunction after treatment with an ototoxic drug .", "other drug classes known to have ototoxic properties include aminoglycosides , loop diuretics , quinine , nonsteroidal anti - inflammatory drugs , and antiretroviral therapy ( art ) .", "this is of concern in south africa , as it is estimated that 12.2% of the population ( 6.4 million persons ) were hiv positive in 2012 , which is 1.2 million more people living with hiv than in 2008 ( 10.6% , or 5.2 million ) .", "resultantly , art exposure had almost doubled from 16.6% in 2008 to 31.2% in 2012 .", "not only will many infected people be at risk for ototoxicity due to arts , but a large number will also be susceptible to hiv - related cancers , such as kaposi 's sarcoma , non - hodgkin 's lymphoma , and cervical cancer , as well as infectious diseases such as tuberculosis , conditions that often require pharmacological therapy with the adverse side effect of ototoxicity .", "it is possible that their treatments could consist of simultaneous use of more than one ototoxic drug , increasing the likelihood of ototoxicity .", "all health care professionals managing patients with cancer should therefore be knowledgeable about the ototoxic properties of cisplatin .", "however , malhotra   indicated that most oncologists in india do not make referrals for audiological evaluations of patients receiving cisplatin , while a study in south africa revealed that the effects of ototoxicity , the role of audiologists , and need for their expertise were not fully realized by the oncologists sampled .", "this is further supported by evidence from the south african study of khoza - shangase and jina   which indicated that most general practitioners sampled also do not appear to carry out ototoxicity monitoring strategies , despite being aware of their own role within an ototoxicity monitoring programme .", "this review therefore aims to serve as resource for health professionals to enhance their understanding of ototoxicity as well as their roles within an ototoxicity monitoring programme by providing an overview and description of this condition in patients diagnosed with cancer and receiving cisplatin chemotherapy ."], ["the review identified peer - reviewed articles available from january 1975 to july 2015 on the topic of cisplatin - associated ototoxicity and ototoxicity monitoring and included english articles only .", "the same researcher conducted the literature search and reviewed the abstracts and articles for inclusion in the study .", "studies were identified using keyword and mesh term searches of electronic databases depicted in table 1 . a manual search of relevant authors and journals", "the references cited by each publication , review , or book chapter were reviewed in order to locate additional potential publications . in order to be selected , the article had to present data on either cisplatin ototoxicity and/or ototoxicity monitoring in human participants , and no research designs were excluded . running these searches yielded a total of 2106 records , of which 1581 were excluded based on the title and/or abstract as well as duplication .", "eighty - five relevant articles , comprising six national and 79 international articles , were selected .", "a perusal of narrative reviews of other auditory pathologies was conducted in an attempt to determine areas of significance for an overview of cisplatin ototoxicity .", "this resulted in the following eight areas being included : the mechanisms of cisplatin ototoxicity , clinical presentation , risk factors , incidence rates in adults and children , the effect on quality of life , ototoxicity monitoring , otoprotective strategies , and management of an ototoxic hearing loss .", "one such mechanism , the antioxidant model , involves the formation of reactive oxygen species ( ros ) within the cochlea and consequent reduction in antioxidant enzymes following exposure to cisplatin chemotherapy [ 1620 ] .", "another mechanism of cisplatin ototoxicity involves the significant contribution of nicotinamide adenine dinucleotide phosphate oxidase 3 isoform ( nox3 ) to the generation of reactive oxygen species within the cochlea , when activated by cisplatin [ 17 , 21 ] , while a third mechanism relates to the activation of transient receptor potential vanilloid 1 channel ( trpv1 ) [ 2224 ] .", "the molecular mechanisms of cisplatin ototoxicity therefore include the following:creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation ands - nitrosylation of cochlear proteins  .with the cellular mechanisms of cisplatin - associated ototoxicity including damage to the outer hair cells , supporting cells , marginal cells of the stria vascularis , spiral ligament , and the spiral ganglion cells , it is evident that the structures of the inner ear are most susceptible to damage by cisplatin chemotherapy , with apoptotic degeneration of the hair cell in the organ of corti being most prominent .", "the outer hair cells in the basal turn of the cochlea are most affected [ 27 , 28 ] .", "this leads to an initial elevation of high frequency audiometric thresholds , followed by a progressive loss into the lower frequencies with continued therapy [ 27 , 28 ] .", "knowledge of the different mechanisms of cisplatin ototoxicity is important for health care professionals as it will create an awareness of its complexity and the resulting clinical presentation . ", "creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation and s - nitrosylation of cochlear proteins  .", "cisplatin - associated ototoxicity usually manifests as irreversible , progressive , bilateral , high frequency sensorineural hearing loss   with tinnitus .", "the latter may occur with or without a hearing loss   and may be permanent or transient , sometimes disappearing a few hours after treatment   or alternatively persisting a week after treatment . while most of the hearing loss is permanent , there is sometimes sporadic and partial recovery . in addition ,", "rare cases of unilateral hearing loss have been reported , which are usually explained by tumour location and surgical or therapeutic intervention on the affected side . moreover", ", the hearing loss is not always symmetrical [ 33 , 34 ] , with jenkins et al .   finding that 75% of women on cisplatin chemotherapy displayed an asymmetry of hearing thresholds of at least 10  db between ears posttreatment .", "schmidt et al . , in their investigation of 55 children on cisplatin chemotherapy , found that the high frequency hearing thresholds were slightly elevated in the left ear and that males had a greater degree of hearing loss than the females .", "the degree of hearing loss is often variable and is related to the dose ; that is , the higher the cumulative dose , the greater the ototoxic effect [ 35 , 36 ] .", "the duration , number of cycles administered , and method of administration   also influence cisplatin - associated ototoxicity .", "additional factors that may increase ototoxicity include exposure to concomitant noise , chemicals , and other ototoxic medications .", "furthermore , evidence also shows that melanin content is related to an increased risk of cisplatin - associated ototoxicity .", "individuals with dark eyes and therefore a higher melanin content in the cochlear are at greater risk of ototoxic damage , as the melanin causes retention of the platinum within the cochlear and subsequently increases the risk of damage [ 41 , 42 ] .", "individuals presenting with renal insufficiency , that is , high levels of serum creatinine , are at a greater risk for cisplatin - associated ototoxicity . genetic risk factors , such as megalin and glutathione s - transferases gene polymorphism , have also been reported to influence cisplatin ototoxicity , as do physiological factors such as age , with younger children   and older adults ( older than 46 years )   presenting with a greater severity of hearing damage .", "awareness of these risk factors may assist health care professionals with informational counselling of the patient receiving cisplatin chemotherapy .", "the incidence of cisplatin ototoxicity is variable in adults ( table 2 ) and children ( table 3 ) .", "the variations may be due to a number of factors , such as differences in the dose , both within a cycle and the total amount administered over multiple cycles , time interval between courses , method of administration , and treatment duration , as well as differences in patient population .", "ototoxicity poses a major problem to the cancer patient , as the quality of life after receiving cisplatin chemotherapy may be negatively affected due to hearing loss , resulting in social , emotional , and vocational difficulties , as effective communication is often hindered .", "tasks that normal hearing persons take for granted may become challenging and frustrating . in addition , an individual 's safety may be compromised due to the hearing loss , as appropriate response to alarms and warning signals may be delayed .", "furthermore , a hearing loss may also result in psychosocial and physical health problems , as well as depression and social isolation .", "hence , hearing loss , often referred to as the  invisible condition ,  has serious visible ramifications on the quality of life of a hearing impaired individual .", "this is particularly relevant if the individual has already experienced the hearing world , as the hearing function is never restored to normal , even though patients may benefit from the use of assistive listening devices , such as hearing aids and cochlear implants .", "the impact of an ototoxic hearing loss may be more profound for infants and young children who are at a critical stage of their speech and language development .", "furthermore , the high frequency nature of an ototoxic hearing loss may result in speech recognition and comprehension being compromised , resulting in possible neurocognitive and psychosocial delays .", "there is also an elevated risk for academic learning problems and psychosocial difficulties in school - aged children and adolescents .", "literature indicated that childhood survivors of neuroblastoma had twice the rate of difficulties , as indicated by parent reports , with reading and math skills , and/or attention and a higher risk of a general learning disability than those without a hearing loss .", "there was also poorer self - reported quality of life scores in these children with regard to school functioning .", "hence , cisplatin - associated ototoxicity further complicates the morbidity of cancer patients , as it would also isolate them from family members and significant others at a time when they require the greatest support .", "advancements in medical knowledge and technology , such as screening and early detection of several cancers , have resulted in notable improvements in relative five - year survival rates for cancer [ 64 , 65 ] .", "therefore , improving the quality of life after cisplatin - based chemotherapy becomes increasingly important , and resulting comorbidities such as ototoxicity can be managed appropriately and immediately   if adequate monitoring is in place .", "the nature of ototoxicity is such that it often goes undetected until speech intelligibility is affected   and is usually detected when a communication problem becomes evident .", "communication problems , such as constantly asking for repetition or not responding when spoken to , signify that the hearing loss has progressed to the frequencies important for understanding speech . in this case ,", "an audiological monitoring programme can avert , to a large extent , the reduced quality of life as a result of hearing loss , as patients on cisplatin chemotherapy can be identified early , counselled , monitored , and managed appropriately through interventions in a logical , systematic , and coherent manner .", "audiological monitoring should aim to identify the hearing loss early and reduce its impact on the individual 's life by means of proper medical and hearing intervention .", "prospective audiological evaluations remain the only reliable method for detecting ototoxicity before it becomes symptomatic .", "an ototoxicity monitoring programme should involve a health care team comprising of an oncology nurse , oncologists , audiologist , and pharmacist to ensure effective sustainability of such a programme , if implemented , with the patient being the central focus .", "the audiologist is involved in identifying an ototoxic hearing loss , informing the oncologist of such a development , counselling the patient and their family , and prescribing amplification devices , such as hearing aids and cochlear implants .", "early identification of an ototoxic hearing loss provides oncologists with an opportunity to adjust the chemotherapy regimen in order to reduce or prevent further deterioration of hearing . the oncologist and nurses should also counsel patients on the side effects of cisplatin , including ototoxicity , in an attempt to prepare them for treatment outcomes and help them set realistic expectations .", "pharmacists who have access to a patient 's medication list may also alert the oncologists and audiologists to those who are on other ototoxic medication and therefore at a greater risk for cisplatin - induced ototoxicity .", "effective management of such patients using evidence - based practices may improve management of those with cancer , ensuring that they and their families are counselled and appropriate interventions are timeously implemented .", "the principles of early identification and early intervention are a part of ototoxicity monitoring , and the audiologist can manage such a programme . in countries without ototoxicity management guidelines , the  guidelines for the audiological management of individuals receiving cochleotoxic drug therapy  developed by the american association of speech - language - hearing association   may , consequently , guide the audiologist in the implementation of an ototoxicity monitoring programme within a local , regional , or national setting . for widespread acceptance and use ,", "ototoxicity monitoring programmes need to incorporate efficient and cost - effective ototoxicity identification techniques , while considering the health care system and demographics of the patient population being managed .", "for any population receiving ototoxic medication , the following should be considered :  ( 1 ) the patient 's level of alertness or ability to respond reliably ; ( 2 ) the most appropriate times during the treatment protocol for test administration , and ; ( 3 ) the test should comprise the baseline , monitoring and post - treatment evaluations  .", "appropriate time intervals for audiological assessments may differ depending on the type of cancer as well as the frequency and dose of cisplatin ( figure 2 ) .", "the audiological assessments should incorporate a detailed case history , otoscopic examination , immittance audiometry , speech audiometry , dpoaes , and conventional and extended high frequency audiometry ( i.e. , up to 20  000  hz ) ( hfa ) [ 69 , 73 ] . these procedures are all conducted for the baseline assessment and the six - month follow - up evaluation [ 69 , 73 ] . while auditory brainstem response may be used , it is not considered a standard procedure for monitoring ototoxicity . monitoring audiological evaluations during treatment and the one- and three - month follow - up evaluations include case interview , otoscopy , and immittance audiometry as well as air conduction pure tone and objective testing . however , full - frequency threshold testing is impractical for many patients on cisplatin chemotherapy , as these individuals are often extremely ill and easily fatigued . the use of abbreviated threshold monitoring procedures that are clinically practical for these patients is therefore recommended .", "one such method involves the use of the sensitive range for ototoxicity ( sro ) .", "this is  the highest frequency with a threshold at or below 100  db spl followed by the next six lower adjacent frequencies in 1/6-octave steps or the one octave range near the highest audible frequency  .", "sro is usually determined during the baseline evaluation and is dependent on each patient 's hearing threshold configuration . during monitoring evaluations", "however , full - frequency testing should be conducted within the same session if an asha significant hearing change is noted within the sro .", "if a patient on cisplatin chemotherapy is still responsive and alert , the protocol presented above would be suitable .", "however , a patient who has limited responsiveness may be required to undergo the same audiological evaluations , except speech audiometry .", "patients who are responsive as well as those who have limited responses can undergo both behavioural and objective testing . however , those patients who are too ill or too young to respond should undergo only objective testing , such as otoscopy , tympanometry , acoustic reflexes , and dpoaes or abrs . while pure tone audiometry in the conventional frequency range is suitable for evaluating hearing in the range responsible for speech understanding , as well as for differential diagnosis , it is less sensitive to detecting early ototoxic change [ 11 , 70 ] .", "the two tests identified as being the most important for the early detection of cisplatin ototoxicity are hfas and oaes , each also having limitations ( see table 4 )", ". therefore , using each test in isolation may not be as effective as utilizing a test battery approach , as it increases the chances of obtaining reliable audiologic monitoring data over time .", "in addition , these two tests could be used to complement one another in every cycle of chemotherapy to ensure the earliest detection of ototoxicity . the ototoxicity monitoring protocol proposed by", "asha   represents an aggressive and ideal approach for monitoring ototoxicity and is dependent on a country 's infrastructure and resource constraints .", "the asha   guidelines may therefore not be generalized to a country without considering the contextual factors that may influence its applicability to that country .", "however , it does provide guidance towards creating a roadmap that countries , such as south africa , may aspire towards in implementing an ototoxicity monitoring programme .", "similar to india , no programmes have been formally implemented to identify and monitor ototoxicity in patients on cancer chemotherapy in south africa . as a result , there is no contextually relevant research to steer the implementation of an accountable and effective ototoxicity monitoring program in the country .", "this is probably one of the main reasons for ototoxicity monitoring programmes not being commonplace in local hospitals and clinics .", "in addition , the health of south africans is characterized by a quadruple burden of disease , encompassing the occurrence of infectious diseases , the rise of noncommunicable diseases , and perinatal and maternal disorders , as well as injuries and violence , which may result in cancer receiving low priority for health care services . however", ", the creation of an audiological monitoring programme allows for better control of cancer related comorbidities . over the years", ", a number of studies have investigated the use of otoprotectants with cisplatin , their purpose being to protect the inner ear from any injury while not interfering with the antitumor effects of cisplatin .", "otoprotective strategies include reducing the formation of free radicals by maintaining glutathione levels and antioxidant activity .", "three mechanisms may provide protection against cisplatin , these being endogenous molecules , exogenous agents , or a combination of exogenous agents that trigger endogenous protective mechanisms .", "however , endogenous agents are not effective against cisplatin when the dose exceeds a certain threshold [ 17 , 81 ] .", "nearly all of the otoprotective agents are sulfur- or sulfhydryl - containing compounds ( thio compounds ) , known as antioxidants , and potent heavy metal chelators .", "the numerous otoprotective agents utilized in clinical and animal studies include amifostine , d - or l - methionine , methylthiobenzoic acid , lipoic acid , tiopronin , glutathione ester , sodium thiosulfate , melatonin , vitamin e , n - acetylcysteine , dexamethasone , and resveratrol . however", ", none of these agents have been found to be unequivocally beneficial in preventing cisplatin ototoxicity and no agent is currently recommended for routine use . further research is therefore needed to find new methods and optimize old ones to prevent and/or treat hearing loss during cisplatin therapy .", "in addition , intratympanic administration of medication together with gene therapy needs to be further explored .", "intratympanic administration involves the diffusion of the otoprotective agent across the round window into the inner ear , where its therapeutic effect is exerted .", "an advantage of this method of administration is that there are higher concentrations of the otoprotective agent in the inner ear , this being in comparison to the use of oral or parenteral administration , without potentially reducing the efficacy of the cisplatin treatment [ 89 , 90 ] .", "the disadvantage of this procedure , however , is that each ear would have to be treated with a moderately invasive procedure . alternatively", ", gene therapy may prove to be beneficial in protecting an individual against cisplatin - induced hearing loss as several genes , namely , megalin , glutathione - s - transferases , thiopurine s - methyltransferase , and catechol - o - methyl transferase , may be responsible for susceptibility to hearing loss .", "if a cisplatin - associated hearing loss results in communication difficulties , it is the audiologist 's ethical responsibility to begin or recommend aural rehabilitation .", "however , this intervention should not only occur once hearing loss has been detected but before the patient begins the cisplatin chemotherapy .", "aural rehabilitation techniques such as speech reading and counselling on compensatory communication strategies should be conducted .", "the counselling should include spouses and significant other , as hearing loss may not only impact the person with cancer but also frequent communication partners .", "patients with sensorineural hearing loss due to the use of cisplatin may also benefit from the use of assistive listening devices such as hearing aids or cochlear implants .", "children with ototoxic hearing loss may also require the use of personal frequency modulated systems in the classroom .", "furthermore , with the recent developments in hearing aid technology , a patient with an ototoxic hearing loss is more likely to receive the desired amplification benefit .", "these hearing aids are able to amplify sounds at and above 8000  hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology .hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping .", "frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound .", "while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds .", "commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) .", "these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively . ", "these hearing aids are able to amplify sounds at and above 8000  hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology .", "hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping .", "frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound .", "while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds .", "commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) .", "these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively ."], ["this review has highlighted that cisplatin ototoxicity is a frequent adverse event of cisplatin chemotherapy that may negatively affect the quality of life of patients with cancer .", "the different molecular and cellular mechanisms involved in cisplatin - associated ototoxicity highlight the complexity of this condition and the consequent difficulty in identifying an effective otoprotective agent .", "the varying incidence rates reported in both adults and paediatrics may be due to the different audiological tests employed in the monitoring of the cancer patient 's hearing status and therefore highlight the importance of the use of extended high frequency audiometry and dpoaes in ototoxicity monitoring .", "an audiological monitoring programme comprising a team of health care professionals , knowledgeable about cisplatin ototoxicity , may therefore serve to improve evidence - based service delivery to these patients ."]]}
{"article_id": "PMC4353012", "article_text": ["the synthetic modification \n of proteins enables the construction \n of biomolecular hybrids that can be used to study protein function , deliver potent therapeutics to cellular targets , and build new materials .", "the synthesis of these constructs requires a suite of chemoselective \n bioconjugation reactions that proceed under mild , aqueous conditions \n in the presence of the native functional groups that are present on \n protein surfaces .", "the most common methods for protein modification \n target the nucleophilic side - chains of lysine and cysteine . however , these strategies can \n result in complex product mixtures , as lysine is typically found in \n high abundance on the protein surface and \n uniquely reactive cysteine labeling sites can be difficult to install \n in many instances ( such as thiol proteases and proteins produced via \n the eukaryotic secretory pathway , for example ) . many newer approaches \n for the site - selective modification of proteins \n involve the introduction of artificial amino acids with reactivities \n that are orthogonal to those of the native \n amino acids . along these lines ,", "a number of powerful methods have \n been developed for the selective modification of azide , alkyne , alkene , carbonyl , and aniline moieties .", "however , the difficulty \n of introducing a non - canonical amino acid can limit the application \n of these methods .", "complementary approaches rely on the site - selective \n modification of native amino acids by enzymes .", "in addition , a reliable method for the modification of c - terminal \n thioesters with n - terminal cysteines , termed  native chemical \n ligation  , has been developed by kent and co - workers .", "this method has been used for the semi- and total synthesis of complex \n protein substrates , including the chemical synthesis of a single glycoform \n of human erythropoietin . as an alternative \n strategy ,", "we and others have developed methods \n for the selective modification of the n - terminal amino group .", "methods that target the n - terminus can offer significant advantages \n for bioconjugate preparation , as they can be used for a wide range \n of protein targets produced by virtually any expression system . conceptually \n powerful as they are , however , these methods can be hampered by long \n reaction times , often require large excesses of reagent , and/or involve \n at least two - steps for the attachment of synthetic molecules .", "we have \n therefore sought to develop new techniques that can achieve n - terminal \n modification with similarly high positional selectivity , but with \n significantly improved efficiency .", "herein , we report an oxidative \n coupling pathway that can preferentially \n modify the n - terminus of proteins with fast kinetics .", "peptide substrates \n were first used to screen reaction conditions and identify the site \n of modification . a peptide panel with varying n - terminal residues \n", "was then evaluated to determine the sequence specificity of the reaction , \n leading to the identification of proline as the optimal n - terminal \n amino acid .", "the reaction was next applied to protein substrates , showing \n similarly high levels of conversion when an n - terminal proline residue \n was present .", "this mild bioconjugation reaction enables the facile , \n rapid modification of proteins to create a well - defined and stable \n linkage in a single position , and thus should be useful for many different \n applications in chemical biology and the construction of biomolecular \n materials .", "we have previously reported \n the chemoselective coupling of aniline \n moieties on proteins to electron - rich aromatic rings , such as o - aminophenols , at slightly acidic ph ( 6.06.5 ) .", "these reactions require the addition of naio4 or k3fe(cn)6 as a terminal oxidant , with the latter \n reagent exhibiting improved compatibility with glycoproteins and substrates \n with free sulfhydryl groups . the use of ferricyanide as the oxidant \n also yields a single reaction product ( 1 ) , whereas periodate \n leads to the formation of a ring contracted species as a competing \n pathway .", "the ferricyanide - based reactions \n are presumed to involve an o - iminoquinone as the \n reactive intermediate , as suggested in scheme 1 , or could involve the corresponding o - quinone after \n imine hydrolysis . taken together , the oxidative coupling strategies \n have demonstrated excellent functional group compatibility and the \n ability to join large unprotected biomolecules at low concentrations , \n as demonstrated for the coupling of peptides , polymers , and nucleic \n acids to specific locations on viral capsids and antibody fc domains . while these coupling reactions \n were found to be highly aniline - selective \n under the conditions used , several studies have reported the reaction \n of o - aminophenols and o - catechols \n with native amino acids , dating back to 1949 .", "in addition , recent work by messersmith has shown the ability of \n proteins to be coupled to o - quinone moieties present \n on polydopamine - coated surfaces .", "these reports \n suggested that secondary coupling pathways could be developed to achieve \n the modification of native amino acids with o - aminophenols \n ( scheme 1 ) , and thus initial experiments were \n designed to identify the optimal reaction conditions for achieving \n this with complex molecules . in our previous \n work", ", we noted that low amounts of background reactivity could be \n observed in aniline - based oxidative coupling reactions when higher \n ph conditions ( > ph 6.5 ) were used . in \n an initial effort to characterize this alternative reaction pathway , \n conditions and reaction times", "were first screened to increase the \n reaction yields for peptides that did not contain aniline groups . \n", "angiotensin i and melittin were used as substrates , as they contain \n many reactive amino acids , including lys , arg , his , trp , and tyr . \n", "the peptides were exposed to 2-amino - p - cresol using \n k3fe(cn)6 as the oxidant .", "the reaction ph was \n varied from 5.5 to 8.5 , and the reaction mixtures were analyzed using \n maldi - tof ms ( supporting information figure \n s1 ) .", "the level of modification increased with the basicity of the \n reaction , with near quantitative modification of angiotensin i after \n 20 min at ph 7.5 and higher . throughout these initial investigations , \n it was noted that angiotensin and melittin showed significant differences \n in reactivity , with angiotensin consistently demonstrating better \n conversion .", "ms / ms analysis of the angiotensin product was used to \n identify the participating residue , and revealed that the n - terminal \n amino group was responsible for the observed reactivity ( supporting information figure s2 ) . as further \n confirmation of the site - selectivity ,", "several peptide substrates were \n screened for reactivity ( figure 1a and supporting information figure s3 ) .", "consistent \n with the n - terminal reaction selectivity , the only peptide that did \n not react had a pyroglutamate in this position , and therefore no free \n amino group .", "( a ) \n modification of commercially available peptides was monitored by maldi - tof \n ms .", "( b ) a positional scan of the n - terminal amino acid was \n evaluated .", "peptides with the sequence xadswag were tested for reactivity with 2-amino - p - cresol .", "the reactions were run with 100 m peptide , 200 m \n aminophenol , and 5 mm ferricyanide at ph 7.5 and analyzed by lc  ms . \n", "shown is the average percent modification with error bars representing \n the standard deviation of three reactions .", "the same peptides were \n used for a screen of coupling partner equivalents ( c ) and a time screen \n ( d ) .", "most buffers did not alter the reactivity , but \n imidazole and buffers containing a morpholine or piperazine ring ( pipes , \n hepes , hepps , and mops ) significantly impeded the reaction ( supporting information figure s4 ) .", "this is possibly \n due to small amounts of buffer impurities that react competitively \n with the oxidized intermediate .", "the time course of the reaction was \n also investigated ( supporting information figure s5 ) .", "in addition , it was found that the peptides could be modified \n using naio4 as the oxidant , or 4-methylcatechol as the \n coupling partner ( supporting information figures s67 ) . both of these reactions showed the same dependence \n on ph ; however , moderate levels of modification were still observed \n at acidic ph ( 5.56.5 ) when using these alternative coupling \n conditions .", "these observations suggested that the peptides were reacting \n with the o - quinone intermediate formed in \n situ from either the catechol or the iminoquinone precursor \n ( after imine hydrolysis ) .", "given the differential \n reactivity observed on peptide substrates , we synthesized peptides \n with varied n - terminal residues ( xadswag ) to determine the specificity of the reaction .", "the base sequence \n was selected to increase the mass of the peptide , impart water solubility , \n and include a tryptophan residue for quantitation using uv monitoring . \n", "the peptides were synthesized on the solid phase using standard fmoc \n synthesis , cleaved from the resin , and purified by hplc .", "after purification , \n the peptides were resuspended in phosphate buffer , ph 7.5 , adjusted \n to a concentration of 1 mm , and stored at 20 c until \n use .", "to assess the effect of the n - terminal residue on reactivity , \n the peptides ( 100 m ) were reacted with 2 equiv of 2-amino - p - cresol ( 200 m ) in the presence of k3fe(cn)6 ( 5 mm ) in phosphate buffer , ph 7.5 ( figure 1b ) .", "ms ( see supporting information figure s8 for representative ms data \n for the modified peptides ) .", "most n - terminal amino acids showed good - to - high \n levels of conversion ( 6090% ) , but proline stood out as the \n only residue that showed nearly complete modification ( 90100% ) . \n", "a second observation of this screen was the fact that tryptophan , \n tyrosine , and methionine residues were not oxidized by the ferricyanide \n reagent , consistent with our previous report of oxidative coupling \n with this oxidant ( see supporting information figure s8 ) .", "however , free cysteine residues can be oxidized to various \n species , potentially including disulfides and sulfenic acids , and \n thus it is recommended that they be protected as disulfides before \n oxidative coupling is attempted ( vide infra ) .", "to optimize the reagent ratios ( specifically the equivalents of o - aminophenol ) , the peptides ( 100 m ) were reacted \n with 110 equiv of the o - aminophenol ( 1001000 \n m ) in the presence of ferricyanide ( 10 mm ) . after 30 min , the \n reactions were quenched with excess tris(2-carboxyethyl)phosphine \n ( tcep ) .", "it was demonstrated that conversion was highest using 25 \n equiv of the coupling partner ( figure 1c ) . \n using more than 5 equiv of", "this was most likely due to the ability of \n the aminophenol to react with itself at higher concentrations ( 1 \n mm ) . consistent with this , when using 10 equiv of the o - aminophenol , a byproduct was observed with a mass that corresponded \n to the condensation of 3 aminophenols ( 344 da ) .", "we also investigated \n the differences in coupling rates for representative \n n - termini .", "the reaction of 2-amino - p - cresol with \n three different peptides was monitored over the course of 1 h ( figure 1d ) .", "the peptides ( 100 m ) were reacted with \n 2 equiv of the o - aminophenol ( 200 m ) in the \n presence of ferricyanide ( 5 mm ) , and aliquots were quenched with excess \n tcep at the indicated time points .", "the proline terminal peptide not \n only reached the highest level of conversion , but also did so in a \n significantly shorter time than the other termini . despite efforts \n to optimize conditions for all n - termini", "the reaction of n - terminal \n amines with o - aminophenols \n was characterized using small molecule mimics .", "the methyl esters of \n phenylalanine ( h - phe - ome ) and proline ( h - pro - ome ) were coupled to \n 2-amino - p - cresol using ferricyanide at ph 7.5 .", "the \n crude products were characterized using two - dimensional nmr and high - resolution \n mass spectrometry .", "the primary amine of h - phe - ome formed p - iminoquinone product 2 , which was analogous to the \n one formed with aniline coupling partners ( scheme 1 , supporting information figure \n s9 ) . however , the secondary amine of proline prevented the formation \n of the p - iminoquinone tautomer , and thus favored o - quinone product 3 ( scheme 1 , supporting information figure \n s10 ) .", "given the different linkage obtained with proline , we verified \n the stability of the product to a variety of conditions .", "the proline \n terminal peptide , padswag , was first modified with 2-amino - p - cresol .", "after purification , the modified peptide ( 100 \n m ) was exposed to reductants , nucleophiles and acidic and basic \n ph ( 10 mm additives or buffer ) .", "no loss of product was \n observed under any of the conditions tested , demonstrating the hydrolytic \n stability of the product ( supporting information figure s11 ) .", "the ability of the linkage to withstand these conditions \n renders this method quite useful for the construction of biomolecular \n materials for a variety of applications . with a view toward in vivo applications ,", "current efforts are examining the \n stability of the linkage in blood plasma , as well as evaluating the \n intrinsic immunogenicity of the o - quinone group . in the process of characterizing the reaction products ,", "it was observed \n that the colored products absorbed light at wavelengths greater than \n 500 nm ( with max between 505 and 525 nm depending \n on the amine coupling partner ) . as the starting coupling partners \n and ferricyanide did not absorb at these wavelengths , this unique \n absorbance provided a means to monitor the reaction progress . the \n different amine coupling partners ( p - toluidine , h - pro - ome , \n and h - phe - ome )", "were reacted with 4-methylcatechol in the presence \n of 10 mm ferricyanide , and the absorbance of the resulting solution \n was monitored at 520 nm to determine the relative rates of reactivity \n ( figure 2a ; for unnormalized data see supporting information figure s12 ) .", "the catechol \n substrate was used for these studies to simplify the reaction pathway \n by eliminating the imine hydrolysis step .", "the reactions were run under \n pseudo - first order conditions with 0.1 mm catechol and 1 mm amine \n coupling partner . when the reaction was carried out at ph 6.0 ,", "the \n reaction with the proline analogue reached completion nearly as rapidly \n ( 2 min ) , but the reaction with the primary aliphatic amine \n of phenylalanine required longer reaction times ( 10 min ) . \n", "( a ) amine coupling partners were reacted with 4-methylcatechol \n as a model substrate .", "reactions were run under pseudo - first \n order conditions with 100 m catechol , 1 mm amine , 10 mm ferricyanide \n in 50 mm phosphate buffer .", "( b ) a peptide containing both an n - terminal \n proline and a p - aminophenylalanine residue ( pad(paf)swag ) was tested for reactivity with 2 equiv of 2-amino - p - cresol at ph 6 .", "the remainder of the reaction was purified \n and then reacted with 2 equiv of the aminophenol at ph 7.5 and analyzed \n by lc  ms . by quantifying product \n formation by absorbance", ", we were also able \n to measure the second - order rate constant for the proline - based coupling \n ( supporting information figure s13 ) .", "the \n reaction of 1 equiv of h - pro - ome ( 100 m ) with 1 equiv of 4-methylcatechol \n ( 100 m ) and 100 equiv of k3fe(cn)6 ( 10 \n mm ) was performed in triplicate at 25 c .", "the second - order rate \n constant for the coupling was determined to be 44  4 m s. while proline reacted rapidly with the electron - rich \n coupling partner , the small molecule studies indicated that aniline \n should react faster .", "the rate for the aniline reaction was too fast \n under these conditions to determine the second - order rate constant \n accurately . given the differences in reactivity observed \n at ph 6.0 and 7.5 , \n we hypothesized that it would be possible to modify the aniline side \n chain of p - aminophenylalanine ( paf ) and the n - terminal proline amine sequentially . to test this hypothesis , \n we synthesized a peptide containing both reactive moieties ( pad(paf)swag ) .", "only one modification was observed when the peptide \n was reacted with 2-amino - p - cresol at ph 6.0 ( figure 2b ) .", "ms / ms analysis of the modified peptide confirmed \n that the single modification occurred on the aniline side chain ( supporting information figure s14 ) . after this \n step , the peptide was purified and subsequently reacted with 2-amino - p - cresol at ph 7.5 .", "reaction at the higher ph enabled a \n second modification of the peptide substrate , at the n - terminal proline \n residue .", "the differential reactivity \n was investigated by comparing the reactivity of a protein containing \n a paf residue to proteins without the artificial \n amino acid .", "the paf residue was introduced into the \n coat protein of bacteriophage ms2 , which self - assembles into a spherical , \n hollow protein shell .", "myoglobin and a mutant of the tobacco mosaic \n virus ( tmv ) coat protein were used as native protein substrates .", "reactions \n with 2-amino - p - cresol were either performed on the \n isolated , individual proteins or with the aniline containing protein \n mixed with the native protein substrate ( supporting \n information figure s15 ) .", "addition of the aniline containing \n protein to the native protein decreased the n - terminal reactivity , \n indicating that the aniline residues react more rapidly than n - terminal \n residues with the o - aminophenols .", "in addition , ms2 \n showed significantly higher reactivity at all of the phs tested , confirming \n preference for aniline residues .", "the oxidative coupling reaction \n with n - terminal amino groups was \n first tested on proteins with native n - termini .", "several proteins were \n reacted with o - aminophenol - functionalized 5 kda peg \n under the optimized reaction conditions ( figure 3 ) .", "the native proteins showed moderate levels of reactivity , which \n could be attributed to inaccessible n - termini or simply to the less \n reactive n - terminal residues . to test", "if proline terminal proteins \n were more reactive , a proline residue was introduced to the n - terminus \n of gfp and the tobacco mosaic virus ( tmv ) coat protein .", "the n - terminus \n of tmv was also slightly extended from the native sequence ( addition \n of pag ) .", "the proline - gfp was treated with a variety of conditions \n to determine the specificity of the reaction ( figure 4a ) . only at basic ph in the presence of both the o - aminophenol substrate and", "the oxidant was modification observed . \n additionally , the proline - terminal variant showed significantly improved \n reactivity compared to that of the wild - type n - terminus .", "these high \n levels of modification were maintained even when only 12 equiv \n of the o - aminophenol peg was used .", "the site of modification \n was confirmed to be the n - terminal proline by lc ", "ms / ms analysis \n of a tryptic digest of proline - gfp modified with 2-amino - p - cresol ( supporting information figure \n s16 ) .", "( a ) \n the n - terminus of several proteins was pegylated using o - aminophenol - functionalized 5 kda peg and ferricyanide .", "( b ) modification \n of wild type proteins with 5 kda o - aminophenol - peg \n was monitored by sds - page .", "( a ) a proline \n was introduced to the n - terminus of gfp .", "the proline terminal \n variant showed much higher levels of modification than the wild - type \n protein .", "the band doubling is due to a gel artifact , and appears in all \n lanes .", "( b ) mutants of tmv were reacted with 5 equiv of 2-amino - p - cresol and 1 mm k3fe(cn)6 for 30 min and analyzed by lc  ms .", "reaction conditions for both native and proline terminal \n proteins \n were optimized by evaluating reactivity with myoglobin and proline - gfp . \n", "the reaction time , buffer , and ph were screened ( supporting information figures s17 and s18 ) .", "similar to the \n results obtained with peptide substrates , the reaction reached its \n highest level of conversion after about 1530 min .", "in addition , \n most buffer salts tested were compatible with the reaction with the \n exception of buffers containing morpholine ( mops ) or piperazine moieties \n ( hepes ) , as was observed with peptide substrates .", "these buffers decreased \n the level of modification slightly , but did not completely inhibit \n reactivity .", "the effect of reaction ph was tested using both k3fe(cn)6 and naio4 as the oxidants .", "little \n reactivity was observed at acidic ph , and the level of conversion \n increased between ph 7.0 and 8.0 . at higher reaction ph ( 8.0 ) \n", "a second modification was observed , indicating that lysines may also \n participate in the reaction .", "however , it is also possible that under \n more forcing conditions , such as higher reaction ph or increased concentration \n of aminophenol substrate , the aminophenol reacts with both itself \n and the n - terminal amino group resulting in double modification of \n the n - terminus .", "n - terminal mutants of tmv were also evaluated \n for their reactivity \n with o - aminophenols .", "the tmv monomers assemble into \n well - known double disk structures , displaying 34 copies of the n - terminal \n groups on their peripheries .", "two n - terminal \n mutants ( pag and ag ) were reacted with 5 equiv of 2-amino - p - cresol ( 100 m ) and 1 mm k3fe(cn)6 for 30 min .", "ms demonstrated that the \n proline terminal mutant reached nearly complete conversion , while \n the alanine terminal mutant showed low levels of modification under \n these coupling conditions ( figure 4b , see supporting information figure s19 for wider mass \n range and ion series ) .", "the compatibility of the reaction with \n cysteine residues was also \n tested using tmv . a single cysteine residue ( s123c )", "was introduced \n into the tmv coat protein with a proline n - terminus ( pag s123c tmv ) . \n", "this mutant was reacted with 2-amino - p - cresol and \n analyzed by lc ", "the cysteine residue also reacted with the o - aminophenol , resulting in two modifications .", "however , it was found that the n - terminal proline could be modified \n selectively if the cysteine was first capped ( figure 5a , b , see supporting information figures s2122 for wider mass range and ion series ) . to do \n this ,", "the cysteine residue was protected as a disulfide bond by reaction \n with 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb , ellman s \n reagent ) .", "after the oxidative coupling step the disulfide bond was \n readily reduced by tcep , leaving the free cysteine and the modified \n n - terminus .", "alternatively , the cysteine residue was modified with \n a maleimide , followed by modification at the n - terminus with an o - aminophenol reagent .", "a ) pag s123c tmv was reacted with small molecule substrates and analyzed \n by lc ", "cysteine residues were protected as a disulfide using \n ellman s reagent ( dtnb ) before oxidative coupling .", "subsequent \n reduction of the disulfide resulted in selective modification of the \n n - terminus .", "the n - terminal \n proline was then modified with a rhodamine - functionalized o - aminophenol .", "this strategy allowed for the direct , dual modification of \n the \n protein at both the cysteine residue and the n - terminus .", "two fluorophores \n paired for frster resonance energy transfer ( fret , alexa fluor \n 488 c5-maleimide and o - aminophenol functionalized \n rhodamine b ) were thus conjugated to tmv using this strategy to create \n a templated array of chromophores for light harvesting applications .", "the free cysteine was first quantitatively labeled with an alexa \n fluor maleimide ( supporting information figure s22 ) .", "the n - terminal proline was then coupled to a fluorescent o - aminophenol resulting in 50% modification of the \n tmv monomers with both fluorphores ( figure 5b ) .", "complete modification of the n - terminus was not observed as tmv \n precipitated from solution with increasing levels of modification \n with the rhodamine dye . in current experiments", ", we are using this \n dual - labeling strategy to introduce more soluble chromophores .", "the oxidative coupling \n reaction was also compared to the reaction \n of protein amines with activated esters .", "this acylation methodology \n is commonly employed , and can be targeted to the n - terminus by controlling \n the reaction ph in some cases .", "the reactions \n were compared on creatine kinase , a protein with a native proline \n n - terminus .", "reaction with 15 equiv of o - aminophenol \n peg resulted in good levels of modification of creatine kinase ( 5060% ) , \n while reaction with 15 equiv of n - hydroxysuccinimide \n ( nhs ) peg resulted in low levels of modification ( 525% , figure 6 ) .", "only when a vast excess of the nhs peg was used \n were moderate levels of modification achieved . as was the case with \n proline - gfp ,", "some over modification was observed under the oxidative \n coupling conditions when using five or more equivalents of aminophenol . \n", "this could result from dimerization of the oxidized species before \n protein coupling , but has yet to be characterized due to the low abundance \n of this product . in any case , lowering the reaction ph slightly or \n using fewer equivalents of the o - aminophenol substrate \n prevented the over modification from occurring .", "the modification of the \n n - terminus of creatine kinase with aminophenol \n peg was compared to the reaction of creatine kinase with nhs peg . \n", "in this study , we have identified conditions \n for the oxidative \n coupling of o - aminophenols to n - terminal amino acids . \n", "proline residues work particularly well with this strategy , and are \n therefore strongly recommended when using it .", "these groups can be \n introduced readily in n - terminal positions using site - directed mutagenesis \n and escherichia coli expression , especially \n since the methionine residue resulting from the start codon is cleaved \n when proline is in the second position .", "the fast kinetics of the reaction allow it to be successful even \n at low reagent and substrate concentrations , and suggest that it can \n be used for sterically demanding bioconjugations .", "the oxidative \n coupling strategy reported here offers two distinct \n advantages over other n - terminal labeling methods .", "first , the modification \n occurs in a single step and does not require initial oxidation of \n the n - terminus .", "second , the fast second - order kinetics allow for low \n concentrations of the coupling partners to be used .", "however , to achieve \n high levels of modification on protein substrates , proline was required \n as the n - terminal residue .", "this new protein modification strategy is currently being explored \n in our lab for the generation of protein - based materials . in the larger \n context ,", "new techniques for the introduction of a single functional \n group in a specific position on a protein surface are always in demand . \n", "the ability of the n - terminal oxidative coupling method to achieve \n this in a single , brief reaction step is highly advantageous , and \n the fact that it can be combined with cysteine modification chemistry \n provides new opportunities for complex bioconjugate synthesis ."], "abstract_text": ["<S> the \n synthetic modification of proteins plays an important role \n in chemical biology and biomaterials science . </S>", "<S> these fields provide \n a constant need for chemical tools that can introduce new functionality \n in specific locations on protein surfaces . in this work </S>", "<S> , an oxidative \n strategy is demonstrated for the efficient modification of n - terminal \n residues on peptides and n - terminal proline residues on proteins . \n </S>", "<S> the strategy uses o - aminophenols or o - catechols that are oxidized to active coupling species in \n situ using potassium ferricyanide . </S>", "<S> peptide screening results \n have \n revealed that many n - terminal amino acids can participate in this \n reaction , and that proline residues are particularly reactive . </S>", "<S> when \n applied to protein substrates , the reaction shows a stronger requirement \n for the proline group . </S>", "<S> key advantages of the reaction include its \n fast second - order kinetics and ability to achieve site - selective modification \n in a single step using low concentrations of reagent . </S>", "<S> although free \n cysteines are also modified by the coupling reaction , they can be \n protected through disulfide formation and then liberated after n - terminal \n coupling is complete . </S>", "<S> this allows access to doubly functionalized \n bioconjugates that can be difficult to access using other methods . </S>"], "labels": null, "section_names": ["Introduction", "Results and Discussion", "Conclusion"], "sections": [["the synthetic modification \n of proteins enables the construction \n of biomolecular hybrids that can be used to study protein function , deliver potent therapeutics to cellular targets , and build new materials .", "the synthesis of these constructs requires a suite of chemoselective \n bioconjugation reactions that proceed under mild , aqueous conditions \n in the presence of the native functional groups that are present on \n protein surfaces .", "the most common methods for protein modification \n target the nucleophilic side - chains of lysine and cysteine . however , these strategies can \n result in complex product mixtures , as lysine is typically found in \n high abundance on the protein surface and \n uniquely reactive cysteine labeling sites can be difficult to install \n in many instances ( such as thiol proteases and proteins produced via \n the eukaryotic secretory pathway , for example ) . many newer approaches \n for the site - selective modification of proteins \n involve the introduction of artificial amino acids with reactivities \n that are orthogonal to those of the native \n amino acids . along these lines ,", "a number of powerful methods have \n been developed for the selective modification of azide , alkyne , alkene , carbonyl , and aniline moieties .", "however , the difficulty \n of introducing a non - canonical amino acid can limit the application \n of these methods .", "complementary approaches rely on the site - selective \n modification of native amino acids by enzymes .", "in addition , a reliable method for the modification of c - terminal \n thioesters with n - terminal cysteines , termed  native chemical \n ligation  , has been developed by kent and co - workers .", "this method has been used for the semi- and total synthesis of complex \n protein substrates , including the chemical synthesis of a single glycoform \n of human erythropoietin . as an alternative \n strategy ,", "we and others have developed methods \n for the selective modification of the n - terminal amino group .", "methods that target the n - terminus can offer significant advantages \n for bioconjugate preparation , as they can be used for a wide range \n of protein targets produced by virtually any expression system . conceptually \n powerful as they are , however , these methods can be hampered by long \n reaction times , often require large excesses of reagent , and/or involve \n at least two - steps for the attachment of synthetic molecules .", "we have \n therefore sought to develop new techniques that can achieve n - terminal \n modification with similarly high positional selectivity , but with \n significantly improved efficiency .", "herein , we report an oxidative \n coupling pathway that can preferentially \n modify the n - terminus of proteins with fast kinetics .", "peptide substrates \n were first used to screen reaction conditions and identify the site \n of modification . a peptide panel with varying n - terminal residues \n", "was then evaluated to determine the sequence specificity of the reaction , \n leading to the identification of proline as the optimal n - terminal \n amino acid .", "the reaction was next applied to protein substrates , showing \n similarly high levels of conversion when an n - terminal proline residue \n was present .", "this mild bioconjugation reaction enables the facile , \n rapid modification of proteins to create a well - defined and stable \n linkage in a single position , and thus should be useful for many different \n applications in chemical biology and the construction of biomolecular \n materials ."], ["we have previously reported \n the chemoselective coupling of aniline \n moieties on proteins to electron - rich aromatic rings , such as o - aminophenols , at slightly acidic ph ( 6.06.5 ) .", "these reactions require the addition of naio4 or k3fe(cn)6 as a terminal oxidant , with the latter \n reagent exhibiting improved compatibility with glycoproteins and substrates \n with free sulfhydryl groups . the use of ferricyanide as the oxidant \n also yields a single reaction product ( 1 ) , whereas periodate \n leads to the formation of a ring contracted species as a competing \n pathway .", "the ferricyanide - based reactions \n are presumed to involve an o - iminoquinone as the \n reactive intermediate , as suggested in scheme 1 , or could involve the corresponding o - quinone after \n imine hydrolysis . taken together , the oxidative coupling strategies \n have demonstrated excellent functional group compatibility and the \n ability to join large unprotected biomolecules at low concentrations , \n as demonstrated for the coupling of peptides , polymers , and nucleic \n acids to specific locations on viral capsids and antibody fc domains . while these coupling reactions \n were found to be highly aniline - selective \n under the conditions used , several studies have reported the reaction \n of o - aminophenols and o - catechols \n with native amino acids , dating back to 1949 .", "in addition , recent work by messersmith has shown the ability of \n proteins to be coupled to o - quinone moieties present \n on polydopamine - coated surfaces .", "these reports \n suggested that secondary coupling pathways could be developed to achieve \n the modification of native amino acids with o - aminophenols \n ( scheme 1 ) , and thus initial experiments were \n designed to identify the optimal reaction conditions for achieving \n this with complex molecules . in our previous \n work", ", we noted that low amounts of background reactivity could be \n observed in aniline - based oxidative coupling reactions when higher \n ph conditions ( > ph 6.5 ) were used . in \n an initial effort to characterize this alternative reaction pathway , \n conditions and reaction times", "were first screened to increase the \n reaction yields for peptides that did not contain aniline groups . \n", "angiotensin i and melittin were used as substrates , as they contain \n many reactive amino acids , including lys , arg , his , trp , and tyr . \n", "the peptides were exposed to 2-amino - p - cresol using \n k3fe(cn)6 as the oxidant .", "the reaction ph was \n varied from 5.5 to 8.5 , and the reaction mixtures were analyzed using \n maldi - tof ms ( supporting information figure \n s1 ) .", "the level of modification increased with the basicity of the \n reaction , with near quantitative modification of angiotensin i after \n 20 min at ph 7.5 and higher . throughout these initial investigations , \n it was noted that angiotensin and melittin showed significant differences \n in reactivity , with angiotensin consistently demonstrating better \n conversion .", "ms / ms analysis of the angiotensin product was used to \n identify the participating residue , and revealed that the n - terminal \n amino group was responsible for the observed reactivity ( supporting information figure s2 ) . as further \n confirmation of the site - selectivity ,", "several peptide substrates were \n screened for reactivity ( figure 1a and supporting information figure s3 ) .", "consistent \n with the n - terminal reaction selectivity , the only peptide that did \n not react had a pyroglutamate in this position , and therefore no free \n amino group .", "( a ) \n modification of commercially available peptides was monitored by maldi - tof \n ms .", "( b ) a positional scan of the n - terminal amino acid was \n evaluated .", "peptides with the sequence xadswag were tested for reactivity with 2-amino - p - cresol .", "the reactions were run with 100 m peptide , 200 m \n aminophenol , and 5 mm ferricyanide at ph 7.5 and analyzed by lc  ms . \n", "shown is the average percent modification with error bars representing \n the standard deviation of three reactions .", "the same peptides were \n used for a screen of coupling partner equivalents ( c ) and a time screen \n ( d ) .", "most buffers did not alter the reactivity , but \n imidazole and buffers containing a morpholine or piperazine ring ( pipes , \n hepes , hepps , and mops ) significantly impeded the reaction ( supporting information figure s4 ) .", "this is possibly \n due to small amounts of buffer impurities that react competitively \n with the oxidized intermediate .", "the time course of the reaction was \n also investigated ( supporting information figure s5 ) .", "in addition , it was found that the peptides could be modified \n using naio4 as the oxidant , or 4-methylcatechol as the \n coupling partner ( supporting information figures s67 ) . both of these reactions showed the same dependence \n on ph ; however , moderate levels of modification were still observed \n at acidic ph ( 5.56.5 ) when using these alternative coupling \n conditions .", "these observations suggested that the peptides were reacting \n with the o - quinone intermediate formed in \n situ from either the catechol or the iminoquinone precursor \n ( after imine hydrolysis ) .", "given the differential \n reactivity observed on peptide substrates , we synthesized peptides \n with varied n - terminal residues ( xadswag ) to determine the specificity of the reaction .", "the base sequence \n was selected to increase the mass of the peptide , impart water solubility , \n and include a tryptophan residue for quantitation using uv monitoring . \n", "the peptides were synthesized on the solid phase using standard fmoc \n synthesis , cleaved from the resin , and purified by hplc .", "after purification , \n the peptides were resuspended in phosphate buffer , ph 7.5 , adjusted \n to a concentration of 1 mm , and stored at 20 c until \n use .", "to assess the effect of the n - terminal residue on reactivity , \n the peptides ( 100 m ) were reacted with 2 equiv of 2-amino - p - cresol ( 200 m ) in the presence of k3fe(cn)6 ( 5 mm ) in phosphate buffer , ph 7.5 ( figure 1b ) .", "ms ( see supporting information figure s8 for representative ms data \n for the modified peptides ) .", "most n - terminal amino acids showed good - to - high \n levels of conversion ( 6090% ) , but proline stood out as the \n only residue that showed nearly complete modification ( 90100% ) . \n", "a second observation of this screen was the fact that tryptophan , \n tyrosine , and methionine residues were not oxidized by the ferricyanide \n reagent , consistent with our previous report of oxidative coupling \n with this oxidant ( see supporting information figure s8 ) .", "however , free cysteine residues can be oxidized to various \n species , potentially including disulfides and sulfenic acids , and \n thus it is recommended that they be protected as disulfides before \n oxidative coupling is attempted ( vide infra ) .", "to optimize the reagent ratios ( specifically the equivalents of o - aminophenol ) , the peptides ( 100 m ) were reacted \n with 110 equiv of the o - aminophenol ( 1001000 \n m ) in the presence of ferricyanide ( 10 mm ) . after 30 min , the \n reactions were quenched with excess tris(2-carboxyethyl)phosphine \n ( tcep ) .", "it was demonstrated that conversion was highest using 25 \n equiv of the coupling partner ( figure 1c ) . \n using more than 5 equiv of", "this was most likely due to the ability of \n the aminophenol to react with itself at higher concentrations ( 1 \n mm ) . consistent with this , when using 10 equiv of the o - aminophenol , a byproduct was observed with a mass that corresponded \n to the condensation of 3 aminophenols ( 344 da ) .", "we also investigated \n the differences in coupling rates for representative \n n - termini .", "the reaction of 2-amino - p - cresol with \n three different peptides was monitored over the course of 1 h ( figure 1d ) .", "the peptides ( 100 m ) were reacted with \n 2 equiv of the o - aminophenol ( 200 m ) in the \n presence of ferricyanide ( 5 mm ) , and aliquots were quenched with excess \n tcep at the indicated time points .", "the proline terminal peptide not \n only reached the highest level of conversion , but also did so in a \n significantly shorter time than the other termini . despite efforts \n to optimize conditions for all n - termini", "the reaction of n - terminal \n amines with o - aminophenols \n was characterized using small molecule mimics .", "the methyl esters of \n phenylalanine ( h - phe - ome ) and proline ( h - pro - ome ) were coupled to \n 2-amino - p - cresol using ferricyanide at ph 7.5 .", "the \n crude products were characterized using two - dimensional nmr and high - resolution \n mass spectrometry .", "the primary amine of h - phe - ome formed p - iminoquinone product 2 , which was analogous to the \n one formed with aniline coupling partners ( scheme 1 , supporting information figure \n s9 ) . however , the secondary amine of proline prevented the formation \n of the p - iminoquinone tautomer , and thus favored o - quinone product 3 ( scheme 1 , supporting information figure \n s10 ) .", "given the different linkage obtained with proline , we verified \n the stability of the product to a variety of conditions .", "the proline \n terminal peptide , padswag , was first modified with 2-amino - p - cresol .", "after purification , the modified peptide ( 100 \n m ) was exposed to reductants , nucleophiles and acidic and basic \n ph ( 10 mm additives or buffer ) .", "no loss of product was \n observed under any of the conditions tested , demonstrating the hydrolytic \n stability of the product ( supporting information figure s11 ) .", "the ability of the linkage to withstand these conditions \n renders this method quite useful for the construction of biomolecular \n materials for a variety of applications . with a view toward in vivo applications ,", "current efforts are examining the \n stability of the linkage in blood plasma , as well as evaluating the \n intrinsic immunogenicity of the o - quinone group . in the process of characterizing the reaction products ,", "it was observed \n that the colored products absorbed light at wavelengths greater than \n 500 nm ( with max between 505 and 525 nm depending \n on the amine coupling partner ) . as the starting coupling partners \n and ferricyanide did not absorb at these wavelengths , this unique \n absorbance provided a means to monitor the reaction progress . the \n different amine coupling partners ( p - toluidine , h - pro - ome , \n and h - phe - ome )", "were reacted with 4-methylcatechol in the presence \n of 10 mm ferricyanide , and the absorbance of the resulting solution \n was monitored at 520 nm to determine the relative rates of reactivity \n ( figure 2a ; for unnormalized data see supporting information figure s12 ) .", "the catechol \n substrate was used for these studies to simplify the reaction pathway \n by eliminating the imine hydrolysis step .", "the reactions were run under \n pseudo - first order conditions with 0.1 mm catechol and 1 mm amine \n coupling partner . when the reaction was carried out at ph 6.0 ,", "the \n reaction with the proline analogue reached completion nearly as rapidly \n ( 2 min ) , but the reaction with the primary aliphatic amine \n of phenylalanine required longer reaction times ( 10 min ) . \n", "( a ) amine coupling partners were reacted with 4-methylcatechol \n as a model substrate .", "reactions were run under pseudo - first \n order conditions with 100 m catechol , 1 mm amine , 10 mm ferricyanide \n in 50 mm phosphate buffer .", "( b ) a peptide containing both an n - terminal \n proline and a p - aminophenylalanine residue ( pad(paf)swag ) was tested for reactivity with 2 equiv of 2-amino - p - cresol at ph 6 .", "the remainder of the reaction was purified \n and then reacted with 2 equiv of the aminophenol at ph 7.5 and analyzed \n by lc  ms . by quantifying product \n formation by absorbance", ", we were also able \n to measure the second - order rate constant for the proline - based coupling \n ( supporting information figure s13 ) .", "the \n reaction of 1 equiv of h - pro - ome ( 100 m ) with 1 equiv of 4-methylcatechol \n ( 100 m ) and 100 equiv of k3fe(cn)6 ( 10 \n mm ) was performed in triplicate at 25 c .", "the second - order rate \n constant for the coupling was determined to be 44  4 m s. while proline reacted rapidly with the electron - rich \n coupling partner , the small molecule studies indicated that aniline \n should react faster .", "the rate for the aniline reaction was too fast \n under these conditions to determine the second - order rate constant \n accurately . given the differences in reactivity observed \n at ph 6.0 and 7.5 , \n we hypothesized that it would be possible to modify the aniline side \n chain of p - aminophenylalanine ( paf ) and the n - terminal proline amine sequentially . to test this hypothesis , \n we synthesized a peptide containing both reactive moieties ( pad(paf)swag ) .", "only one modification was observed when the peptide \n was reacted with 2-amino - p - cresol at ph 6.0 ( figure 2b ) .", "ms / ms analysis of the modified peptide confirmed \n that the single modification occurred on the aniline side chain ( supporting information figure s14 ) . after this \n step , the peptide was purified and subsequently reacted with 2-amino - p - cresol at ph 7.5 .", "reaction at the higher ph enabled a \n second modification of the peptide substrate , at the n - terminal proline \n residue .", "the differential reactivity \n was investigated by comparing the reactivity of a protein containing \n a paf residue to proteins without the artificial \n amino acid .", "the paf residue was introduced into the \n coat protein of bacteriophage ms2 , which self - assembles into a spherical , \n hollow protein shell .", "myoglobin and a mutant of the tobacco mosaic \n virus ( tmv ) coat protein were used as native protein substrates .", "reactions \n with 2-amino - p - cresol were either performed on the \n isolated , individual proteins or with the aniline containing protein \n mixed with the native protein substrate ( supporting \n information figure s15 ) .", "addition of the aniline containing \n protein to the native protein decreased the n - terminal reactivity , \n indicating that the aniline residues react more rapidly than n - terminal \n residues with the o - aminophenols .", "in addition , ms2 \n showed significantly higher reactivity at all of the phs tested , confirming \n preference for aniline residues .", "the oxidative coupling reaction \n with n - terminal amino groups was \n first tested on proteins with native n - termini .", "several proteins were \n reacted with o - aminophenol - functionalized 5 kda peg \n under the optimized reaction conditions ( figure 3 ) .", "the native proteins showed moderate levels of reactivity , which \n could be attributed to inaccessible n - termini or simply to the less \n reactive n - terminal residues . to test", "if proline terminal proteins \n were more reactive , a proline residue was introduced to the n - terminus \n of gfp and the tobacco mosaic virus ( tmv ) coat protein .", "the n - terminus \n of tmv was also slightly extended from the native sequence ( addition \n of pag ) .", "the proline - gfp was treated with a variety of conditions \n to determine the specificity of the reaction ( figure 4a ) . only at basic ph in the presence of both the o - aminophenol substrate and", "the oxidant was modification observed . \n additionally , the proline - terminal variant showed significantly improved \n reactivity compared to that of the wild - type n - terminus .", "these high \n levels of modification were maintained even when only 12 equiv \n of the o - aminophenol peg was used .", "the site of modification \n was confirmed to be the n - terminal proline by lc ", "ms / ms analysis \n of a tryptic digest of proline - gfp modified with 2-amino - p - cresol ( supporting information figure \n s16 ) .", "( a ) \n the n - terminus of several proteins was pegylated using o - aminophenol - functionalized 5 kda peg and ferricyanide .", "( b ) modification \n of wild type proteins with 5 kda o - aminophenol - peg \n was monitored by sds - page .", "( a ) a proline \n was introduced to the n - terminus of gfp .", "the proline terminal \n variant showed much higher levels of modification than the wild - type \n protein .", "the band doubling is due to a gel artifact , and appears in all \n lanes .", "( b ) mutants of tmv were reacted with 5 equiv of 2-amino - p - cresol and 1 mm k3fe(cn)6 for 30 min and analyzed by lc  ms .", "reaction conditions for both native and proline terminal \n proteins \n were optimized by evaluating reactivity with myoglobin and proline - gfp . \n", "the reaction time , buffer , and ph were screened ( supporting information figures s17 and s18 ) .", "similar to the \n results obtained with peptide substrates , the reaction reached its \n highest level of conversion after about 1530 min .", "in addition , \n most buffer salts tested were compatible with the reaction with the \n exception of buffers containing morpholine ( mops ) or piperazine moieties \n ( hepes ) , as was observed with peptide substrates .", "these buffers decreased \n the level of modification slightly , but did not completely inhibit \n reactivity .", "the effect of reaction ph was tested using both k3fe(cn)6 and naio4 as the oxidants .", "little \n reactivity was observed at acidic ph , and the level of conversion \n increased between ph 7.0 and 8.0 . at higher reaction ph ( 8.0 ) \n", "a second modification was observed , indicating that lysines may also \n participate in the reaction .", "however , it is also possible that under \n more forcing conditions , such as higher reaction ph or increased concentration \n of aminophenol substrate , the aminophenol reacts with both itself \n and the n - terminal amino group resulting in double modification of \n the n - terminus .", "n - terminal mutants of tmv were also evaluated \n for their reactivity \n with o - aminophenols .", "the tmv monomers assemble into \n well - known double disk structures , displaying 34 copies of the n - terminal \n groups on their peripheries .", "two n - terminal \n mutants ( pag and ag ) were reacted with 5 equiv of 2-amino - p - cresol ( 100 m ) and 1 mm k3fe(cn)6 for 30 min .", "ms demonstrated that the \n proline terminal mutant reached nearly complete conversion , while \n the alanine terminal mutant showed low levels of modification under \n these coupling conditions ( figure 4b , see supporting information figure s19 for wider mass \n range and ion series ) .", "the compatibility of the reaction with \n cysteine residues was also \n tested using tmv . a single cysteine residue ( s123c )", "was introduced \n into the tmv coat protein with a proline n - terminus ( pag s123c tmv ) . \n", "this mutant was reacted with 2-amino - p - cresol and \n analyzed by lc ", "the cysteine residue also reacted with the o - aminophenol , resulting in two modifications .", "however , it was found that the n - terminal proline could be modified \n selectively if the cysteine was first capped ( figure 5a , b , see supporting information figures s2122 for wider mass range and ion series ) . to do \n this ,", "the cysteine residue was protected as a disulfide bond by reaction \n with 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb , ellman s \n reagent ) .", "after the oxidative coupling step the disulfide bond was \n readily reduced by tcep , leaving the free cysteine and the modified \n n - terminus .", "alternatively , the cysteine residue was modified with \n a maleimide , followed by modification at the n - terminus with an o - aminophenol reagent .", "a ) pag s123c tmv was reacted with small molecule substrates and analyzed \n by lc ", "cysteine residues were protected as a disulfide using \n ellman s reagent ( dtnb ) before oxidative coupling .", "subsequent \n reduction of the disulfide resulted in selective modification of the \n n - terminus .", "the n - terminal \n proline was then modified with a rhodamine - functionalized o - aminophenol .", "this strategy allowed for the direct , dual modification of \n the \n protein at both the cysteine residue and the n - terminus .", "two fluorophores \n paired for frster resonance energy transfer ( fret , alexa fluor \n 488 c5-maleimide and o - aminophenol functionalized \n rhodamine b ) were thus conjugated to tmv using this strategy to create \n a templated array of chromophores for light harvesting applications .", "the free cysteine was first quantitatively labeled with an alexa \n fluor maleimide ( supporting information figure s22 ) .", "the n - terminal proline was then coupled to a fluorescent o - aminophenol resulting in 50% modification of the \n tmv monomers with both fluorphores ( figure 5b ) .", "complete modification of the n - terminus was not observed as tmv \n precipitated from solution with increasing levels of modification \n with the rhodamine dye . in current experiments", ", we are using this \n dual - labeling strategy to introduce more soluble chromophores .", "the oxidative coupling \n reaction was also compared to the reaction \n of protein amines with activated esters .", "this acylation methodology \n is commonly employed , and can be targeted to the n - terminus by controlling \n the reaction ph in some cases .", "the reactions \n were compared on creatine kinase , a protein with a native proline \n n - terminus .", "reaction with 15 equiv of o - aminophenol \n peg resulted in good levels of modification of creatine kinase ( 5060% ) , \n while reaction with 15 equiv of n - hydroxysuccinimide \n ( nhs ) peg resulted in low levels of modification ( 525% , figure 6 ) .", "only when a vast excess of the nhs peg was used \n were moderate levels of modification achieved . as was the case with \n proline - gfp ,", "some over modification was observed under the oxidative \n coupling conditions when using five or more equivalents of aminophenol . \n", "this could result from dimerization of the oxidized species before \n protein coupling , but has yet to be characterized due to the low abundance \n of this product . in any case , lowering the reaction ph slightly or \n using fewer equivalents of the o - aminophenol substrate \n prevented the over modification from occurring .", "the modification of the \n n - terminus of creatine kinase with aminophenol \n peg was compared to the reaction of creatine kinase with nhs peg . \n"], ["in this study , we have identified conditions \n for the oxidative \n coupling of o - aminophenols to n - terminal amino acids . \n", "proline residues work particularly well with this strategy , and are \n therefore strongly recommended when using it .", "these groups can be \n introduced readily in n - terminal positions using site - directed mutagenesis \n and escherichia coli expression , especially \n since the methionine residue resulting from the start codon is cleaved \n when proline is in the second position .", "the fast kinetics of the reaction allow it to be successful even \n at low reagent and substrate concentrations , and suggest that it can \n be used for sterically demanding bioconjugations .", "the oxidative \n coupling strategy reported here offers two distinct \n advantages over other n - terminal labeling methods .", "first , the modification \n occurs in a single step and does not require initial oxidation of \n the n - terminus .", "second , the fast second - order kinetics allow for low \n concentrations of the coupling partners to be used .", "however , to achieve \n high levels of modification on protein substrates , proline was required \n as the n - terminal residue .", "this new protein modification strategy is currently being explored \n in our lab for the generation of protein - based materials . in the larger \n context ,", "new techniques for the introduction of a single functional \n group in a specific position on a protein surface are always in demand . \n", "the ability of the n - terminal oxidative coupling method to achieve \n this in a single , brief reaction step is highly advantageous , and \n the fact that it can be combined with cysteine modification chemistry \n provides new opportunities for complex bioconjugate synthesis ."]]}
{"article_id": "PMC4006624", "article_text": ["\n alcohol withdrawal syndrome ( aws ) is characterized by varied symptoms that range from mild to severe intensity depending on several factors including the quantity , frequency and duration of alcohol intake , and the number of prior withdrawal episodes , as well as individual differences in the vulnerability [ 14 ] .", "symptoms usually present themselves within 6 to 24 hours after cessation of alcohol intake [ 5 , 6 ] .", "subtyping of the aws has been attempted in the past , as gross   conceptualized and proposed 3 constellations of alcohol withdrawal symptoms : factor 1 hallucinogenic that consisted of nausea , tinnitus , visual disturbance , pruritus , parasthesia , muscle pain , agitation , sleep disturbance , tactile hallucinations , and hallucinations which are auditory or visual or both ; factor 2 affective and physiological that consisted of anxiety , depression , tremor , and sweats ; and factor 3 delirium that consisted of clouding of the sensorium , impairment of consciousness , and impairment of contact with the observer .", "a cluster analytic study   identified three different symptoms clusters of alcohol withdrawal , namely , cns excitation , adrenergic hyperactivity , and delirium .", "several rating instruments have been used to measure severity of alcohol withdrawal . among them ,", "the most commonly used observer - rated scale is the 10-item clinical institute withdrawal assessment - alcohol , revised ( ciwa - ar ) .", "it has been proposed that alcohol withdrawal symptoms in ciwa - ar appear multidimensional . a pubmed search supplemented with", "the study by pittman et al .   was to explore the relationship between awsc and ciwa - ar , for which they carried out study on 127 male inpatients of alcohol dependence with principle components factor extraction with varimax rotation of ciwa - ar , a self - rated  alcohol withdrawal symptoms checklist ( awsc ) and on combined items of ciwa - ar and awsc .", "they found three , five , and seven factor solution , respectively , for ciwa - ar , awsc , and combination of ciwa - ar and awsc .", "the analysis of ciwa - ar was done on 7 items as 3 items had zero variance . the first factor ( variance 23.9% ) was  tension / anxiety  which consisted of anxiety , agitation , and tactile disturbances . the second factor ( variance 22.9% ) was  autonomic arousal  which consisted of paroxysmal sweats , tremor , and headache or fullness in head , whereas the third factor ( variance 17.4% , eigenvalue less than 1 ) was  nausea and vomiting  which consisted of a single item , nausea , and vomiting . in our setup", ", we use ciwa - ar as part of the measure for the management of alcohol withdrawal symptoms .", "it is generally observed that alcohol withdrawal symptoms fluctuate in presentation and severity across time .", "the present study was carried out to explore the dimensionality of this scale in an attempt to identify a set of underlying factors that exist and can explain the interrelationships among various manifestations of acute alcohol withdrawal symptoms .", "the knowledge of these underlying factors may enhance our understanding of aws and better prediction of complications thus management plans .", "this was a cross - sectional hospital - based study , conducted at centre for addiction psychiatry , central institute of psychiatry , ranchi , india , a tertiary care referral centre during may 2005 to june 2006 .", "study sample included 201 , only male fulfilling icd-10 dcr ( world health organization )   for alcohol dependence with currently withdrawal state , aged between 18 and 60 years , admitted within 24 hours of abstinence and patient himself or his guardian consenting for the study .", "information on patient 's demographics , treatment history , past history , and family history was obtained from interviews with patients and accompanying person .", "detailed physical and neurological examinations were done to exclude any comorbid general medical condition , comorbid other psychiatric disorder , and any other comorbid substance use disorders except caffeine and tobacco . \n", "the sociodemographic data sheet included age , marital status , religion , community , education , and economic status . whereas clinical variables recorded were age of onset of drinking alcohol , duration of dependence , past history of detoxification , number of previous detoxification , past history of withdrawal seizure , past history of delirium tremens , family history of alcohol or substance dependence , degree of relationship if family history of alcohol or substance dependence is present , and family history of mental illness .  \n", "it is the most widely used and studied 10-item alcohol withdrawal monitoring scale , which excludes vital sign abnormalities .", "it was developed from the 18-item clinical institute withdrawal assessment for alcohol and it has been studied across various geographic locations .", "it has a good reliability , validity , and it is considered as one of the most widely used alcohol withdrawal assessment scale for symptom - triggered therapy .", "each sign and symptom item of ciwa ar is evaluated on a 07 point likert scale except for one item  orientation and clouding of sensorium  , which is scored on a 04 point likert scale .", "a score of 8 points or less indicates mild withdrawal and patients scoring less than 10 do not usually need additional medication for withdrawal .", "patients admitted with alcohol dependence syndrome with acute withdrawal were evaluated with ciwa - ar immediately after admission then every six hours , as a routine protocol of the ward .", "detailed physical examination , mental status examination , and planned screening laboratory investigation were done to ensure conformity of study criteria .", "the patients get admitted with varying lengths of abstinence , ranging from hours to days , so we initially took all the ratings of all the patients and arranged them as rating at first 6 hours of abstinence and at every six hours like at 6 , 12 , 18 , 24 , 30 , 36 , 42 , 48 , 52 , 58 , and 64 hours till ciwa - ar scoring reaches below 10 .", "the averages of each rating of ciwa - ar scores were computed to see the severity of withdrawal symptoms across time span of abstinence .", "many patients were admitted after overnight , 12 to 18 hours of abstinence , so we included the patients who were admitted with at least 24 hours of abstinence .", "meanwhile management and medications were continued as per ward protocol and no adjustment for study purpose was done .", "the standard detoxification protocol included thiamine supplementation , benzodiazepines either lorazepam or diazepam , and correction of fluids and electrolytes if any and other symptomatic treatment of associated conditions like dyspepsia or concurrent injury , wound , and infections .", "the collected data on 201 patients was statistically analyzed , using statistical package for social sciences ( spss , inc . ,", "exploratory factor analysis ( maximum likelihood method ) was carried out to identify factor structure on all items of ciwa - ar for day three .", "kaiser - meyer - olkin measure of sample adequacy and bartlett 's test of sphericity were also done to assess appropriateness of conducting factor analysis .", "two criteria for retaining the number of components were considered : kaiser 's criterion   to retain eigenvalues greater than unity and cattell 's   scree plot inspection for the point of inflexion .", "the mean age of the group was 37.18 ( sd 9.35 , range 1869 ) years .", "most of them were married ( 83.3% ) , educated ( 90% ) , residing in urban background ( 64.7% ) , belonging to middle socioeconomic status ( 60.3% ) , and earning a livelihood ( 80.4% ) .", "the mean age of starting alcohol was 21.63 ( sd 4.99 ) years , whereas mean duration of dependence on alcohol was 6.49 ( sd 5.06 ) years .", "22.5% had history of withdrawal seizures , in their course of alcoholism , and only 2% reported history of delirium tremens in the past .", "family history of substance dependence was present in 33.3% of the total sample , out of which alcohol dependence was present in 56.9% of subjects and cannabis dependence in 8.3% .", "the item frequency and mean of all six hourly ciwa - ar ratings were calculated ; the mean scores of ciwa - ar at 24 hours and at 36 hours are shown in table 2 .", "the mean ciwa - ar score at 24 hours was 13.32 ( sd 9.27 ) and 20.4 ( sd 9.09 ) at 36 hours .", "based on the frequency variance and total ciwa - ar score we decided to carry out factor analysis with the 10 items of ciwa - ar as on scoring at 36 hours of abstinence .", "bartlett 's test of sphericity was significant ( 2 = 1.044 , df = 45 , p < .001 ) , indicating that a factor analysis is appropriate .", "factor analysis ( extraction method - maximum likelihood ) with the 10 items of ciwa - ar for day three , resulted in initial three factors with eigenvalues greater than unity .", "the scree plot was also showing clear inflexion , supporting three factors ( figure 1 ) .", "therefore , three factors were retained , which captured 68.74% of variance . following varimax rotation with kaiser 's standardization ,", "the factors and their item loadings , with absolute values greater than 0.1 , are shown in table 3 .", "none of the items loaded on more than one factor . the first factor named as ", "delirious factor ,  which had highest loading from tactile disturbances ( .999 ) , followed by auditory disturbances , orientation and clouding of sensorium , and agitation .", "it explained 34.34% of variance and showed good internal consistency ( cronbach 's alpha = .91 ) . the second factor named as ", "autonomic factor  reflected four - item loading , highest from anxiety , followed by paroxysmal sweats , tremor , and headache or fullness in head .", "it explained 24.25% of variance and showed moderate internal consistency ( cronbach 's alpha = .66 ) . the third factor named as ", "nonspecific factor  reflected two - item loadings , nausea , and visual disturbances that explained 10.04% of variance and a cronbach 's alpha of .26 .", "we examined the factor structure of the ciwa - ar in a population of adult men hospitalized to a tertiary psychiatric institute for treatment of alcohol dependence .", "the ideal study of aws could have been the assessment before starting medication , but this was practically not possible for many reasons .", "firstly , many of the patients come for admission after 12 to 18 hours of abstinence and severe withdrawal , so keeping them drug free was ethically not possible .", "thus natural aws presentation and its severity were masked by routine benzodiazepam administration and thiamine supplement .", "secondly , many other patients were referred from primary care centers with initial management , including long acting benzodiazepines like diazepam that masks the aws .", "thirdly , many other patients came even before onset of withdrawal and in a state of intoxication ; the aws was not fully evolved in terms of range of symptoms and severity .", "for all these reasons , the mean ciwa - ar score for the initial 24 hours of abstinence ( first day ) of admission was only 13.32 ( sd 9.27 ) .", "later the sequential rating found more prominent withdrawal symptoms reaching highest mean score of 20.4 at thirty - six hours then gradually decreased .", "as drug free aws was not possible , we consider that the higher mean score of ciwa - ar represents the aws better than low score . also as both aws medications and alcohol itself are cns depressant and act in a similar way , either medication or alcohol intake should not make much difference in clinical picture .", "so we decided to proceed for factor analysis with highest mean ciwa - ar scoring at the 36th hour .", "the severity of withdrawal symptoms and appearance of complete sets of withdrawal symptoms at the 36th hour may have been influenced by plasma half - life of benzodiazepams being used for detoxification . in this study ,", "choice of medication was with treating team of the hospital ; however , only either intermediate acting lorazepam or long acting diazepam was used for this purpose .", "there was no use of short acting benzodiazepines , which causes varying and rebound withdrawal symptoms across different time frame with its dosing and changing plasma concentration .", "another more important influencing reason for varying withdrawal symptoms across different time frame could have been the dose of benzodiazepines , but we did not interfered with any medications or dosing and it was continued as per ward protocol to ensure naturalistic conditions . however , the equivalent benzodiazepines mean dose at 36 hour was 30  mg of diazepam per day .", "but this equivalent dose may not be accurate as many patients were on oral medications and others were on parenteral benzodiazepines .", "we excluded any other comorbid substance use disorders or polysubstance dependence but a few patients may also have undisclosed benzodiazepine or organic inhalant abuse or addiction .", "we also excluded any comorbid general medical condition especially epilepsy for that reason , patients on antiepileptic either taking it regularly or skipping will modify the alcohol withdrawal symptoms . for", "that matter any psychotropic drugs causing cns depression or any effecting stimulants will alter the withdrawal symptoms .", "most of the patients needed proton pump inhibitor drugs like pantoprazole or omeprazole for the alcohol induced dyspepsia , peptic ulcer disease , or gastroesophageal reflux disease , but these medications do not impose any effect on alcohol withdrawal symptoms . in a previous study by pittman et al . , the mean ciwa - ar score for day one was 13.2 ( sd 3.7 ) which was similar to our study , 13.32 ( sd 9.27 ) at 24 hours ; they continued with analysis on data collected on the first study day to exclude medication effects .", "but we analyzed the data as of 36 hours of abstinence , showing higher mean ciwa - ar score and all ten items had variance above 10% , thus representing fully developed withdrawal syndrome .", "however we could not control the medication effect used to control the withdrawal symptoms for ethical reasons , the used medications were benzodiazepine and thiamine supplementation for all the patients .", "two items of ciwa - ar , namely , auditory disturbances and tactile disturbances were very infrequent in our sample ( 9.5 and 10% ) on day one which increased to 55.1 and 65.7% at 36 hours , other items scoring raised on day two like agitation , orientation and clouding of sensorium , visual disturbances , and paroxysmal sweats ; few other items remained unchanged on day one and two like anxiety and tremor ; however scoring of only this item  headache or fullness in head  improved on day two from 28.9 to 12.9% ( table 2 ) .", "this indicates the importance of time duration of abstinence to study the alcohol withdrawal symptoms .", "we found a three - factor solution based on rotated eigenvalues and scree plot analysis .", "the first factor explained 34.34% variance and consisted of four items , namely , tactile disturbances , auditory disturbances , orientation and clouding of sensorium , and agitation .", "this factor appears to represent perceptual abnormality and delirium and may be considered as subclinical spectrum of delirium .", "however , the only other factor analytic study of ciwa - ar by pittman et al .   found these items ( except agitation ) were infrequent in their sample and were not included in analysis .", "the difference may be due to the time span of withdrawal on which data was collected .", "study analyzed ciwa - ar for day one and also our data on day one showed very low variance for these items .", "this suggests that ratings on alcohol withdrawal symptoms on the very first day may miss certain set of symptoms , which appears on and around day two and are characterized by perceptual abnormality and delirium like picture .", "further progression of alcohol withdrawal , we could not found beyond 36 hours , may be due to effects of continued medications for withdrawal suppression .", "surprisingly , visual disturbances item did not have high loading on this factor , whereas tactile and auditory disturbances had maximum factor loading ( 0.999 and 0.873 , resp . ) .", "the etiological basis for these two disturbances includes cns rebound excitation which alters the perceptual quality .", "furthermore , some contribution of nutritional and specific vitamin deficiencies , such as thiamine and folate , and associated peripheral neuropathy probably add to these perceptual disturbances .", "the other two items , orientation and clouding of sensorium and agitation with factor loadings of 0.851 and 0.777 , respectively , represent delirium .", "the perceptual alteration and delirium being loaded in a single factor may have some predictive association and hence need to be studied for management plan .", "the second  autonomic  factor explained 24.25% of variance and consisted of four items : anxiety , paroxysmal sweats , tremor , and headache and fullness in head with factor scores of .998 , .660 , .528 , and .245 respectively .", "cronbach 's alpha for this factor was 0.66 showing adequate internal consistency . within this factor", "this factor may be a result of the practice of not using adrenergic medication on routine basis at our institute .", "all items ( paroxysmal sweats , tremor , and headache or fullness in head ) were loaded similarly .", "we had additional anxiety to this factor ; hence , we share the name of this factor with pittman et al .   as  autonomic  .", "the third  nonspecific  factor explained 10.04% of variance and consisted of two items : nausea and vomiting and visual disturbances , with low factor scores of .51 and .27 , respectively .", "there are three proposed physiologic bases for the symptom manifestation of alcohol withdrawal symptoms : cns excitation , adrenergic hyperactivity , and delirium , which may be attributed to different neurotransmitters and they respond selectively to different pharmacotherapy", ". the cns excitation may be secondary to deficiency in gaba activity , whereas increase in cns epinephrine level causes adrenergic hyperactivity .", "the nmda receptor hypersensitivity and overactivity of certain subtypes of nmda receptors are associated with delirium .", "though we did not find a factor structure of aws in accordance with very strict pathophysiological manifestation of either autonomic , or cns excitation or psychological / affective , or perceptual / hallucinogenic in our sample , the obtained factors suggest existence of subgroups of patients with different set of symptoms .", "this was expected as alcohol withdrawal manifests by simultaneous involvement of all mechanisms rather than any mutually exclusive mechanism .", "this simultaneous involvement of several other neurotransmitters besides gaba and nmda , like noradrenaline , acetylcholine , and dopamine as well as hormones and electrolytes [ 17 , 18 ] will affect the symptom presentation .", "additionally our data analysis had ciwa - ar ratings of day two ( 36 hours ) of admission with suppression of withdrawal symptoms from detoxification medications , that is , benzodiazepines .", "this would have differential effect on withdrawal symptoms manifestation in terms of gaba suppression only and selective unopposed action on other neurotransmitters or lacking adrenergic activity . though avoiding pharmacological suppression or modification was not possible on ethical grounds to understand completely natural unmodified withdrawal manifestation .", "there may also be possible influence of different types of alcoholic beverage and percent content of alcohol .", "strength of our study includes large sample size and not interfering with any medications or management strategies thus providing setting of naturalistic conditions .", "the use ciwa - ar is the most widely accepted alcohol withdrawal assessment scale and selection of abstinent hours was important to allow time for full appearance of symptoms , even though under cover of detoxification medication .", "there was for better coverage and inclusion of withdrawal symptoms at 36 hours as indicated by total ciwa - ar score and item frequency .", "these results have wider implications for the recognition and management of aws , particularly , for better understanding and identification of the symptom profiles for and differential management plans across subtypes of aws .", "the use of adrenergic antagonists may have a valuable role in addition to benzodiazepines , in a set of patients with autonomic features .", "one of the limitations in our study is that it includes male only patients ; however , gender can be an important issue in aws presentation and its severity .", "even studies found that sex hormone affects the aws by modulating the function of the gaba - a receptor .", "also low levels of testosterone are associated with symptoms like indecision , excessive worrying , fatigability , and lassitude .", "another limitation of this study is patients sample with severe aws requiring inpatient management , thus limiting generalizability of our findings across gender and to mild to moderate severity cases .", "another limitation was that diagnosis was made clinically with icd-10 dcr criteria for  alcohol dependence with currently withdrawal state  , but not by a validated clinical interview .", "it is known that autonomic arousal is an important mechanism in aws ; thus , other physiological measures and biological markers for objective assessment may be included in future studies .", "further studies may also be carried out including cases of mild to moderate severity and in both sex to uncover the differences .", "also , factor analysis depends heavily on the population studied ; therefore , studies on different population may be required to generalize our findings . for the clinical practice ,", "it is advisable not to overdepend on rating scales and it must not replace a thorough clinical evaluation of the patient 's medical status in prediction of those at risk of severe alcohol withdrawal .", "the acute alcohol withdrawal symptoms was most severe at 36 hours of abstinence in our sample .", "this study finds multidimensionality of alcohol withdrawal symptoms as measured with ciwa - ar ; we found three factors explaining 68.74 percentage of variance and named as delirious , autonomic and nonspecific ."], "abstract_text": ["<S> \n objective . to identify the underlying factor structure of alcohol withdrawal syndrome , as measured with ciwa - ar </S>", "<S> . methods . </S>", "<S> exploratory factor analysis was conducted on the items of ciwa - ar . on 201 alcohol - dependent male patients seeking treatment for alcohol withdrawal at 36 hours of abstinence . </S>", "<S> results . </S>", "<S> a three - factor solution was obtained that accounted for 68.74% of total variance . </S>", "<S> first factor had loading from four items ( 34.34% variance ) , second factor also had four items ( 24.25% variance ) , and the third had two items ( 10.04% variance ) . </S>", "<S> conclusions . factor analysis reveals the existence of multidimensionality of alcohol withdrawal as measured with ciwa - ar and we found three factors that can be named as delirious , autonomic and nonspecific factors . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods ", "3. Results ", "4. Discussion ", "5. Conclusion"], "sections": [["\n alcohol withdrawal syndrome ( aws ) is characterized by varied symptoms that range from mild to severe intensity depending on several factors including the quantity , frequency and duration of alcohol intake , and the number of prior withdrawal episodes , as well as individual differences in the vulnerability [ 14 ] .", "symptoms usually present themselves within 6 to 24 hours after cessation of alcohol intake [ 5 , 6 ] .", "subtyping of the aws has been attempted in the past , as gross   conceptualized and proposed 3 constellations of alcohol withdrawal symptoms : factor 1 hallucinogenic that consisted of nausea , tinnitus , visual disturbance , pruritus , parasthesia , muscle pain , agitation , sleep disturbance , tactile hallucinations , and hallucinations which are auditory or visual or both ; factor 2 affective and physiological that consisted of anxiety , depression , tremor , and sweats ; and factor 3 delirium that consisted of clouding of the sensorium , impairment of consciousness , and impairment of contact with the observer .", "a cluster analytic study   identified three different symptoms clusters of alcohol withdrawal , namely , cns excitation , adrenergic hyperactivity , and delirium .", "several rating instruments have been used to measure severity of alcohol withdrawal . among them ,", "the most commonly used observer - rated scale is the 10-item clinical institute withdrawal assessment - alcohol , revised ( ciwa - ar ) .", "it has been proposed that alcohol withdrawal symptoms in ciwa - ar appear multidimensional . a pubmed search supplemented with", "the study by pittman et al .   was to explore the relationship between awsc and ciwa - ar , for which they carried out study on 127 male inpatients of alcohol dependence with principle components factor extraction with varimax rotation of ciwa - ar , a self - rated  alcohol withdrawal symptoms checklist ( awsc ) and on combined items of ciwa - ar and awsc .", "they found three , five , and seven factor solution , respectively , for ciwa - ar , awsc , and combination of ciwa - ar and awsc .", "the analysis of ciwa - ar was done on 7 items as 3 items had zero variance . the first factor ( variance 23.9% ) was  tension / anxiety  which consisted of anxiety , agitation , and tactile disturbances . the second factor ( variance 22.9% ) was  autonomic arousal  which consisted of paroxysmal sweats , tremor , and headache or fullness in head , whereas the third factor ( variance 17.4% , eigenvalue less than 1 ) was  nausea and vomiting  which consisted of a single item , nausea , and vomiting . in our setup", ", we use ciwa - ar as part of the measure for the management of alcohol withdrawal symptoms .", "it is generally observed that alcohol withdrawal symptoms fluctuate in presentation and severity across time .", "the present study was carried out to explore the dimensionality of this scale in an attempt to identify a set of underlying factors that exist and can explain the interrelationships among various manifestations of acute alcohol withdrawal symptoms .", "the knowledge of these underlying factors may enhance our understanding of aws and better prediction of complications thus management plans ."], ["this was a cross - sectional hospital - based study , conducted at centre for addiction psychiatry , central institute of psychiatry , ranchi , india , a tertiary care referral centre during may 2005 to june 2006 .", "study sample included 201 , only male fulfilling icd-10 dcr ( world health organization )   for alcohol dependence with currently withdrawal state , aged between 18 and 60 years , admitted within 24 hours of abstinence and patient himself or his guardian consenting for the study .", "information on patient 's demographics , treatment history , past history , and family history was obtained from interviews with patients and accompanying person .", "detailed physical and neurological examinations were done to exclude any comorbid general medical condition , comorbid other psychiatric disorder , and any other comorbid substance use disorders except caffeine and tobacco . \n", "the sociodemographic data sheet included age , marital status , religion , community , education , and economic status . whereas clinical variables recorded were age of onset of drinking alcohol , duration of dependence , past history of detoxification , number of previous detoxification , past history of withdrawal seizure , past history of delirium tremens , family history of alcohol or substance dependence , degree of relationship if family history of alcohol or substance dependence is present , and family history of mental illness .  \n", "it is the most widely used and studied 10-item alcohol withdrawal monitoring scale , which excludes vital sign abnormalities .", "it was developed from the 18-item clinical institute withdrawal assessment for alcohol and it has been studied across various geographic locations .", "it has a good reliability , validity , and it is considered as one of the most widely used alcohol withdrawal assessment scale for symptom - triggered therapy .", "each sign and symptom item of ciwa ar is evaluated on a 07 point likert scale except for one item  orientation and clouding of sensorium  , which is scored on a 04 point likert scale .", "a score of 8 points or less indicates mild withdrawal and patients scoring less than 10 do not usually need additional medication for withdrawal .", "patients admitted with alcohol dependence syndrome with acute withdrawal were evaluated with ciwa - ar immediately after admission then every six hours , as a routine protocol of the ward .", "detailed physical examination , mental status examination , and planned screening laboratory investigation were done to ensure conformity of study criteria .", "the patients get admitted with varying lengths of abstinence , ranging from hours to days , so we initially took all the ratings of all the patients and arranged them as rating at first 6 hours of abstinence and at every six hours like at 6 , 12 , 18 , 24 , 30 , 36 , 42 , 48 , 52 , 58 , and 64 hours till ciwa - ar scoring reaches below 10 .", "the averages of each rating of ciwa - ar scores were computed to see the severity of withdrawal symptoms across time span of abstinence .", "many patients were admitted after overnight , 12 to 18 hours of abstinence , so we included the patients who were admitted with at least 24 hours of abstinence .", "meanwhile management and medications were continued as per ward protocol and no adjustment for study purpose was done .", "the standard detoxification protocol included thiamine supplementation , benzodiazepines either lorazepam or diazepam , and correction of fluids and electrolytes if any and other symptomatic treatment of associated conditions like dyspepsia or concurrent injury , wound , and infections .", "the collected data on 201 patients was statistically analyzed , using statistical package for social sciences ( spss , inc . ,", "exploratory factor analysis ( maximum likelihood method ) was carried out to identify factor structure on all items of ciwa - ar for day three .", "kaiser - meyer - olkin measure of sample adequacy and bartlett 's test of sphericity were also done to assess appropriateness of conducting factor analysis .", "two criteria for retaining the number of components were considered : kaiser 's criterion   to retain eigenvalues greater than unity and cattell 's   scree plot inspection for the point of inflexion ."], ["the mean age of the group was 37.18 ( sd 9.35 , range 1869 ) years .", "most of them were married ( 83.3% ) , educated ( 90% ) , residing in urban background ( 64.7% ) , belonging to middle socioeconomic status ( 60.3% ) , and earning a livelihood ( 80.4% ) .", "the mean age of starting alcohol was 21.63 ( sd 4.99 ) years , whereas mean duration of dependence on alcohol was 6.49 ( sd 5.06 ) years .", "22.5% had history of withdrawal seizures , in their course of alcoholism , and only 2% reported history of delirium tremens in the past .", "family history of substance dependence was present in 33.3% of the total sample , out of which alcohol dependence was present in 56.9% of subjects and cannabis dependence in 8.3% .", "the item frequency and mean of all six hourly ciwa - ar ratings were calculated ; the mean scores of ciwa - ar at 24 hours and at 36 hours are shown in table 2 .", "the mean ciwa - ar score at 24 hours was 13.32 ( sd 9.27 ) and 20.4 ( sd 9.09 ) at 36 hours .", "based on the frequency variance and total ciwa - ar score we decided to carry out factor analysis with the 10 items of ciwa - ar as on scoring at 36 hours of abstinence .", "bartlett 's test of sphericity was significant ( 2 = 1.044 , df = 45 , p < .001 ) , indicating that a factor analysis is appropriate .", "factor analysis ( extraction method - maximum likelihood ) with the 10 items of ciwa - ar for day three , resulted in initial three factors with eigenvalues greater than unity .", "the scree plot was also showing clear inflexion , supporting three factors ( figure 1 ) .", "therefore , three factors were retained , which captured 68.74% of variance . following varimax rotation with kaiser 's standardization ,", "the factors and their item loadings , with absolute values greater than 0.1 , are shown in table 3 .", "none of the items loaded on more than one factor . the first factor named as ", "delirious factor ,  which had highest loading from tactile disturbances ( .999 ) , followed by auditory disturbances , orientation and clouding of sensorium , and agitation .", "it explained 34.34% of variance and showed good internal consistency ( cronbach 's alpha = .91 ) . the second factor named as ", "autonomic factor  reflected four - item loading , highest from anxiety , followed by paroxysmal sweats , tremor , and headache or fullness in head .", "it explained 24.25% of variance and showed moderate internal consistency ( cronbach 's alpha = .66 ) . the third factor named as ", "nonspecific factor  reflected two - item loadings , nausea , and visual disturbances that explained 10.04% of variance and a cronbach 's alpha of .26 ."], ["we examined the factor structure of the ciwa - ar in a population of adult men hospitalized to a tertiary psychiatric institute for treatment of alcohol dependence .", "the ideal study of aws could have been the assessment before starting medication , but this was practically not possible for many reasons .", "firstly , many of the patients come for admission after 12 to 18 hours of abstinence and severe withdrawal , so keeping them drug free was ethically not possible .", "thus natural aws presentation and its severity were masked by routine benzodiazepam administration and thiamine supplement .", "secondly , many other patients were referred from primary care centers with initial management , including long acting benzodiazepines like diazepam that masks the aws .", "thirdly , many other patients came even before onset of withdrawal and in a state of intoxication ; the aws was not fully evolved in terms of range of symptoms and severity .", "for all these reasons , the mean ciwa - ar score for the initial 24 hours of abstinence ( first day ) of admission was only 13.32 ( sd 9.27 ) .", "later the sequential rating found more prominent withdrawal symptoms reaching highest mean score of 20.4 at thirty - six hours then gradually decreased .", "as drug free aws was not possible , we consider that the higher mean score of ciwa - ar represents the aws better than low score . also as both aws medications and alcohol itself are cns depressant and act in a similar way , either medication or alcohol intake should not make much difference in clinical picture .", "so we decided to proceed for factor analysis with highest mean ciwa - ar scoring at the 36th hour .", "the severity of withdrawal symptoms and appearance of complete sets of withdrawal symptoms at the 36th hour may have been influenced by plasma half - life of benzodiazepams being used for detoxification . in this study ,", "choice of medication was with treating team of the hospital ; however , only either intermediate acting lorazepam or long acting diazepam was used for this purpose .", "there was no use of short acting benzodiazepines , which causes varying and rebound withdrawal symptoms across different time frame with its dosing and changing plasma concentration .", "another more important influencing reason for varying withdrawal symptoms across different time frame could have been the dose of benzodiazepines , but we did not interfered with any medications or dosing and it was continued as per ward protocol to ensure naturalistic conditions . however , the equivalent benzodiazepines mean dose at 36 hour was 30  mg of diazepam per day .", "but this equivalent dose may not be accurate as many patients were on oral medications and others were on parenteral benzodiazepines .", "we excluded any other comorbid substance use disorders or polysubstance dependence but a few patients may also have undisclosed benzodiazepine or organic inhalant abuse or addiction .", "we also excluded any comorbid general medical condition especially epilepsy for that reason , patients on antiepileptic either taking it regularly or skipping will modify the alcohol withdrawal symptoms . for", "that matter any psychotropic drugs causing cns depression or any effecting stimulants will alter the withdrawal symptoms .", "most of the patients needed proton pump inhibitor drugs like pantoprazole or omeprazole for the alcohol induced dyspepsia , peptic ulcer disease , or gastroesophageal reflux disease , but these medications do not impose any effect on alcohol withdrawal symptoms . in a previous study by pittman et al . , the mean ciwa - ar score for day one was 13.2 ( sd 3.7 ) which was similar to our study , 13.32 ( sd 9.27 ) at 24 hours ; they continued with analysis on data collected on the first study day to exclude medication effects .", "but we analyzed the data as of 36 hours of abstinence , showing higher mean ciwa - ar score and all ten items had variance above 10% , thus representing fully developed withdrawal syndrome .", "however we could not control the medication effect used to control the withdrawal symptoms for ethical reasons , the used medications were benzodiazepine and thiamine supplementation for all the patients .", "two items of ciwa - ar , namely , auditory disturbances and tactile disturbances were very infrequent in our sample ( 9.5 and 10% ) on day one which increased to 55.1 and 65.7% at 36 hours , other items scoring raised on day two like agitation , orientation and clouding of sensorium , visual disturbances , and paroxysmal sweats ; few other items remained unchanged on day one and two like anxiety and tremor ; however scoring of only this item  headache or fullness in head  improved on day two from 28.9 to 12.9% ( table 2 ) .", "this indicates the importance of time duration of abstinence to study the alcohol withdrawal symptoms .", "we found a three - factor solution based on rotated eigenvalues and scree plot analysis .", "the first factor explained 34.34% variance and consisted of four items , namely , tactile disturbances , auditory disturbances , orientation and clouding of sensorium , and agitation .", "this factor appears to represent perceptual abnormality and delirium and may be considered as subclinical spectrum of delirium .", "however , the only other factor analytic study of ciwa - ar by pittman et al .   found these items ( except agitation ) were infrequent in their sample and were not included in analysis .", "the difference may be due to the time span of withdrawal on which data was collected .", "study analyzed ciwa - ar for day one and also our data on day one showed very low variance for these items .", "this suggests that ratings on alcohol withdrawal symptoms on the very first day may miss certain set of symptoms , which appears on and around day two and are characterized by perceptual abnormality and delirium like picture .", "further progression of alcohol withdrawal , we could not found beyond 36 hours , may be due to effects of continued medications for withdrawal suppression .", "surprisingly , visual disturbances item did not have high loading on this factor , whereas tactile and auditory disturbances had maximum factor loading ( 0.999 and 0.873 , resp . ) .", "the etiological basis for these two disturbances includes cns rebound excitation which alters the perceptual quality .", "furthermore , some contribution of nutritional and specific vitamin deficiencies , such as thiamine and folate , and associated peripheral neuropathy probably add to these perceptual disturbances .", "the other two items , orientation and clouding of sensorium and agitation with factor loadings of 0.851 and 0.777 , respectively , represent delirium .", "the perceptual alteration and delirium being loaded in a single factor may have some predictive association and hence need to be studied for management plan .", "the second  autonomic  factor explained 24.25% of variance and consisted of four items : anxiety , paroxysmal sweats , tremor , and headache and fullness in head with factor scores of .998 , .660 , .528 , and .245 respectively .", "cronbach 's alpha for this factor was 0.66 showing adequate internal consistency . within this factor", "this factor may be a result of the practice of not using adrenergic medication on routine basis at our institute .", "all items ( paroxysmal sweats , tremor , and headache or fullness in head ) were loaded similarly .", "we had additional anxiety to this factor ; hence , we share the name of this factor with pittman et al .   as  autonomic  .", "the third  nonspecific  factor explained 10.04% of variance and consisted of two items : nausea and vomiting and visual disturbances , with low factor scores of .51 and .27 , respectively .", "there are three proposed physiologic bases for the symptom manifestation of alcohol withdrawal symptoms : cns excitation , adrenergic hyperactivity , and delirium , which may be attributed to different neurotransmitters and they respond selectively to different pharmacotherapy", ". the cns excitation may be secondary to deficiency in gaba activity , whereas increase in cns epinephrine level causes adrenergic hyperactivity .", "the nmda receptor hypersensitivity and overactivity of certain subtypes of nmda receptors are associated with delirium .", "though we did not find a factor structure of aws in accordance with very strict pathophysiological manifestation of either autonomic , or cns excitation or psychological / affective , or perceptual / hallucinogenic in our sample , the obtained factors suggest existence of subgroups of patients with different set of symptoms .", "this was expected as alcohol withdrawal manifests by simultaneous involvement of all mechanisms rather than any mutually exclusive mechanism .", "this simultaneous involvement of several other neurotransmitters besides gaba and nmda , like noradrenaline , acetylcholine , and dopamine as well as hormones and electrolytes [ 17 , 18 ] will affect the symptom presentation .", "additionally our data analysis had ciwa - ar ratings of day two ( 36 hours ) of admission with suppression of withdrawal symptoms from detoxification medications , that is , benzodiazepines .", "this would have differential effect on withdrawal symptoms manifestation in terms of gaba suppression only and selective unopposed action on other neurotransmitters or lacking adrenergic activity . though avoiding pharmacological suppression or modification was not possible on ethical grounds to understand completely natural unmodified withdrawal manifestation .", "there may also be possible influence of different types of alcoholic beverage and percent content of alcohol .", "strength of our study includes large sample size and not interfering with any medications or management strategies thus providing setting of naturalistic conditions .", "the use ciwa - ar is the most widely accepted alcohol withdrawal assessment scale and selection of abstinent hours was important to allow time for full appearance of symptoms , even though under cover of detoxification medication .", "there was for better coverage and inclusion of withdrawal symptoms at 36 hours as indicated by total ciwa - ar score and item frequency .", "these results have wider implications for the recognition and management of aws , particularly , for better understanding and identification of the symptom profiles for and differential management plans across subtypes of aws .", "the use of adrenergic antagonists may have a valuable role in addition to benzodiazepines , in a set of patients with autonomic features .", "one of the limitations in our study is that it includes male only patients ; however , gender can be an important issue in aws presentation and its severity .", "even studies found that sex hormone affects the aws by modulating the function of the gaba - a receptor .", "also low levels of testosterone are associated with symptoms like indecision , excessive worrying , fatigability , and lassitude .", "another limitation of this study is patients sample with severe aws requiring inpatient management , thus limiting generalizability of our findings across gender and to mild to moderate severity cases .", "another limitation was that diagnosis was made clinically with icd-10 dcr criteria for  alcohol dependence with currently withdrawal state  , but not by a validated clinical interview .", "it is known that autonomic arousal is an important mechanism in aws ; thus , other physiological measures and biological markers for objective assessment may be included in future studies .", "further studies may also be carried out including cases of mild to moderate severity and in both sex to uncover the differences .", "also , factor analysis depends heavily on the population studied ; therefore , studies on different population may be required to generalize our findings . for the clinical practice ,", "it is advisable not to overdepend on rating scales and it must not replace a thorough clinical evaluation of the patient 's medical status in prediction of those at risk of severe alcohol withdrawal ."], ["the acute alcohol withdrawal symptoms was most severe at 36 hours of abstinence in our sample .", "this study finds multidimensionality of alcohol withdrawal symptoms as measured with ciwa - ar ; we found three factors explaining 68.74 percentage of variance and named as delirious , autonomic and nonspecific ."]]}
{"article_id": "PMC2799323", "article_text": ["this included only workers who had worked more than 50% of each day 's working period and had worked for at least 10 days of the follow - up period .", "information regarding the amount of liquid intake expressed ( in litres , l ) and daily productivity output ( in tons ) were recorded .", "the rehydration strategies were re - structured by decision makers participating in the project and the author of this paper .", "for an eight working hour schedule , all workers were instructed to drink 1 l of the rehydration solutions or tap water at least 30 minutes before they began to work .", "they were also encouraged to drink water and rehydration solutions ( 250 ml ) every 3045 minutes . in total , they were each asked to drink 10 l of liquid ( tap water and rehydration solutions ) per day . in order to facilitate the monitoring of basic heat stress indexes during the 2007/2008 harvesting season , temperature ,", "humidity and dew point were measured by using a portable temperature monitoring device (  easylog , model el - usb-2 ) .", "variables such as heart rate and weight were evaluated at the beginning and at the end of the working day . neither the amount of liquid intake nor productivity was validated .", "wet bulb globe temperature ( wbgt ) values during the 2007 harvest season at montelimar farm descriptive statistics were used to analyse climate data ( temperature ( c ) ; relative humidity ( % ) ; dew point ( c ) ) ; liquid intake and output production .", "chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 .", "descriptive statistics were used to analyse climate data ( temperature ( c ) ; relative humidity ( % ) ; dew point ( c ) ) ; liquid intake and output production .", "chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 .", "1 shows in situ hourly climate variation direct from the sugarcane fields while workers harvested the crop .", "as can be seen , temperature and relative humidity values oscillated from 23.5 to 34.5c and from 40% to 64% , respectively , reaching maximum values as early as 810 am .", "this meant that water distribution had to be started at around that time on an hourly basis .", "climate measurements during 160 working hours of follow - up for 22 sugarcane workers at the montelimar farm .", "table 2 shows the amount of liquid intake ( including both regular tap water and specially formulated rehydration drinks ) .", "what is of great concern is that , although temperature increased to maximum values early in the morning , many workers did not follow the rehydration measures and drank less than 6 l , a potentially dangerously low volume .", "comparison between temperature measurements and daily water intake for 22 sugarcane workers at the montelimar farm .", "this showed that 13 workers who had the highest production output ( range 68 tons , average 7.45 tons ) were those who drank more than 6 l of liquid ( regular tap water and/or specially formulated rehydration solutions ) in comparison to those who drank less liquid and had a smaller production output .", "relationship between production output and daily water intake for 22 sugarcane workers at the montelimar farm .", "measurements of heart rate and body weight at the start and end of the working day showed that workers experienced increases in heart rate and loss of body weight as they worked in these hot conditions .", "historically , monitoring of toxins in the work environment has been the primary focus for identifying risks .", "some potential biomarkers linked to cell injury are immunological factors , lymphokines , growth factors , prostaglandins , endothelins , collagen , adhesion molecules , thromboxanes , leukotrienes , platelet activating factors and heat shock proteins ( 10 ) . as mentioned earlier , heat illness is a major cause of preventable morbidity worldwide ( 1 ) and although human beings possess considerable ability to compensate for naturally occurring heat stress , many occupational environments and/or physical activities expose workers to heat loads which are so excessive as to threaten their health and productivity ( 11 ) .", "it is important to remember that the normal human body contains approximately 60% of water ,", "the present project evaluated climatic conditions at sugarcane plantations located at sea level , on the southwest coast of nicaragua .", "monitored climate indices ( temperature and relative humidity ) values varied from 23.5 to 34.5c and from 40% to 64% , respectively , reaching maximum values as early as 8 am . according to the nicaraguan institute of territorial studies ( ineter ) ( 13 ) , relative humidity values for the whole month of april varied between 69 and 79% all along nicaragua 's pacific coastline from san juan del sur ( southern region ) to chinandega ( western region ) .", "managua lies between the two regions ; the montelimar sugar farm is located 62 km from managua .", "however , the ineter humidity data are quite different from the relative humidity registered on the farm located in western managua .", "temperature data were not available at ineter 's website . although only 22 subjects were followed - up for a short period of time in this study , important results were obtained .", "other authors have shown the relationship between heat stress health effects and the ability to perform different tasks , as well as the increased risk of suffering work - related injuries ( 14 ) . in this study , the workers drank more liquid as temperature values increased to maximum peaks .", "this was part of a rehydration process which was well planned in advance by the company 's decision makers . unlike during past harvesting seasons , when water and rehydration solutions were distributed quite randomly , during this harvesting season cool water and specially formulated drinks were distributed or intended to be distributed to workers , who received 1 l just before they began their working day and then 500 ml every 30 minutes .", "the basis of this principle is that drinking to satisfy thirst is not enough to keep a person well hydrated .", "most of the people become aware of thirst once they have lost 12 l of body water and persons highly motivated to perform hard work may incur losses of 34 l before serious thirst forces them to stop and drink .", "since dehydration reduces the capacity for absorption from the gut , workers must be educated regarding the importance of drinking enough water during work and continuing generous rehydration during off - duty hours ( 14 ) .", "there was a significant increase of production , with up to 8 tons per worker during the follow - up period compared to the normal 5.5 tons per worker prior to the change in rehydration measures .", "this important change in rehydration policies and increase in production output is the result of various efforts of training workers , foremen and managers on heat stress prevention , proper hydration measures and quality of ( working ) life carried out by occupational health and safety professionals ( physicians and engineers ) , human resources departments and top management at sugarcane farms .", ". often workers rejected the new rehydration measures , most of the time because it was difficult for them to understand thoroughly the dehydration and physiological compensatory mechanisms .", "some of the reasons for this can be attributed to their low educational level , and feeling that  nothing bad has ever happened to me before , etc .", "certainly more effort in terms of intervention strategies and scientific investigation needs to be carried out among workers in nicaragua who perform jobs in which they are exposed to high ambient temperatures .", "these include farm workers , construction workers , miners and fishermen , especially those employed in the informal sector , which occupies about half of nicaragua 's economically active population .", "more funds should also be designated by companies decision makers for improving basic working conditions , in order to increase overall productivity ( and workers satisfaction in terms of better wages ) .", "this would also translate into safer and healthier workers , less absenteeism from sick leave , fewer accidents and other incidents .", "the author has not received any funding or benefits from industry to conduct this study ."], "abstract_text": ["<S> backgroundheat illness is a major cause of preventable morbidity worldwide . </S>", "<S> workers exposed to intense heat can become unable to activate compensation mechanisms , putting their health at risk . </S>", "<S> heat stress also has a direct impact on production by causing poor task performance and it increases the possibility of work - related morbidity and injuries . during the sugarcane harvest period , workers are exposed to excessive sunlight and heat from approximately 6 am to 3 pm . </S>", "<S> a first assessment of heat stress during the 2006/2007 harvesting season served to redesign the existing rehydration measures . in this project , </S>", "<S> sugarcane workers were provided with more rehydration solutions and water during their work schedule.objectiveto assess heat stress preventive measures in order to improve existing rehydration strategies as a means of increasing productivity.methodsa small group of 22 workers were followed up for 15 days during working hours , from 6 am to 3 pm . </S>", "<S> selection criteria were defined : to have worked more than 50% of the day 's working schedule and to have worked for at least 10 days of the follow - up period . </S>", "<S> a simple data recollection sheet was used . </S>", "<S> information regarding the amount of liquid intake was registered . </S>", "<S> production output data was also registered . </S>", "<S> temperature measurements were recorded by using a portable temperature monitoring device (  easylog , model el - usb-2).resultsthe average temperature measurements were above the nicaraguan ministry of labour thresholds . </S>", "<S> seven workers drank 78 l of liquid , improving their production . </S>", "<S> output production increased significantly ( p=0.005 ) among those best hydrated , from 5.5 to 8 tons of cut sugarcane per worker per day.conclusionsproductivity improved with the new rehydration measures . </S>", "<S> awareness among workers concerning heat stress prevention was increased . </S>"], "labels": null, "section_names": ["Methods", "Statistical analysis", "Results", "Discussion", "Conflict of interest and funding"], "sections": [["twenty - two workers were followed up from 15th april to 30th april 2008 during working hours , from approximately 6 am to 3 pm , for a total of 160 working hours .", "this included only workers who had worked more than 50% of each day 's working period and had worked for at least 10 days of the follow - up period .", "information regarding the amount of liquid intake expressed ( in litres , l ) and daily productivity output ( in tons ) were recorded .", "the rehydration strategies were re - structured by decision makers participating in the project and the author of this paper .", "for an eight working hour schedule , all workers were instructed to drink 1 l of the rehydration solutions or tap water at least 30 minutes before they began to work .", "they were also encouraged to drink water and rehydration solutions ( 250 ml ) every 3045 minutes . in total , they were each asked to drink 10 l of liquid ( tap water and rehydration solutions ) per day . in order to facilitate the monitoring of basic heat stress indexes during the 2007/2008 harvesting season , temperature ,", "humidity and dew point were measured by using a portable temperature monitoring device (  easylog , model el - usb-2 ) .", "variables such as heart rate and weight were evaluated at the beginning and at the end of the working day . neither the amount of liquid intake nor productivity was validated .", "wet bulb globe temperature ( wbgt ) values during the 2007 harvest season at montelimar farm descriptive statistics were used to analyse climate data ( temperature ( c ) ; relative humidity ( % ) ; dew point ( c ) ) ; liquid intake and output production .", "chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 ."], ["descriptive statistics were used to analyse climate data ( temperature ( c ) ; relative humidity ( % ) ; dew point ( c ) ) ; liquid intake and output production .", "chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 ."], ["1 shows in situ hourly climate variation direct from the sugarcane fields while workers harvested the crop .", "as can be seen , temperature and relative humidity values oscillated from 23.5 to 34.5c and from 40% to 64% , respectively , reaching maximum values as early as 810 am .", "this meant that water distribution had to be started at around that time on an hourly basis .", "climate measurements during 160 working hours of follow - up for 22 sugarcane workers at the montelimar farm .", "table 2 shows the amount of liquid intake ( including both regular tap water and specially formulated rehydration drinks ) .", "what is of great concern is that , although temperature increased to maximum values early in the morning , many workers did not follow the rehydration measures and drank less than 6 l , a potentially dangerously low volume .", "comparison between temperature measurements and daily water intake for 22 sugarcane workers at the montelimar farm .", "this showed that 13 workers who had the highest production output ( range 68 tons , average 7.45 tons ) were those who drank more than 6 l of liquid ( regular tap water and/or specially formulated rehydration solutions ) in comparison to those who drank less liquid and had a smaller production output .", "relationship between production output and daily water intake for 22 sugarcane workers at the montelimar farm .", "measurements of heart rate and body weight at the start and end of the working day showed that workers experienced increases in heart rate and loss of body weight as they worked in these hot conditions ."], ["historically , monitoring of toxins in the work environment has been the primary focus for identifying risks .", "some potential biomarkers linked to cell injury are immunological factors , lymphokines , growth factors , prostaglandins , endothelins , collagen , adhesion molecules , thromboxanes , leukotrienes , platelet activating factors and heat shock proteins ( 10 ) . as mentioned earlier , heat illness is a major cause of preventable morbidity worldwide ( 1 ) and although human beings possess considerable ability to compensate for naturally occurring heat stress , many occupational environments and/or physical activities expose workers to heat loads which are so excessive as to threaten their health and productivity ( 11 ) .", "it is important to remember that the normal human body contains approximately 60% of water ,", "the present project evaluated climatic conditions at sugarcane plantations located at sea level , on the southwest coast of nicaragua .", "monitored climate indices ( temperature and relative humidity ) values varied from 23.5 to 34.5c and from 40% to 64% , respectively , reaching maximum values as early as 8 am . according to the nicaraguan institute of territorial studies ( ineter ) ( 13 ) , relative humidity values for the whole month of april varied between 69 and 79% all along nicaragua 's pacific coastline from san juan del sur ( southern region ) to chinandega ( western region ) .", "managua lies between the two regions ; the montelimar sugar farm is located 62 km from managua .", "however , the ineter humidity data are quite different from the relative humidity registered on the farm located in western managua .", "temperature data were not available at ineter 's website . although only 22 subjects were followed - up for a short period of time in this study , important results were obtained .", "other authors have shown the relationship between heat stress health effects and the ability to perform different tasks , as well as the increased risk of suffering work - related injuries ( 14 ) . in this study , the workers drank more liquid as temperature values increased to maximum peaks .", "this was part of a rehydration process which was well planned in advance by the company 's decision makers . unlike during past harvesting seasons , when water and rehydration solutions were distributed quite randomly , during this harvesting season cool water and specially formulated drinks were distributed or intended to be distributed to workers , who received 1 l just before they began their working day and then 500 ml every 30 minutes .", "the basis of this principle is that drinking to satisfy thirst is not enough to keep a person well hydrated .", "most of the people become aware of thirst once they have lost 12 l of body water and persons highly motivated to perform hard work may incur losses of 34 l before serious thirst forces them to stop and drink .", "since dehydration reduces the capacity for absorption from the gut , workers must be educated regarding the importance of drinking enough water during work and continuing generous rehydration during off - duty hours ( 14 ) .", "there was a significant increase of production , with up to 8 tons per worker during the follow - up period compared to the normal 5.5 tons per worker prior to the change in rehydration measures .", "this important change in rehydration policies and increase in production output is the result of various efforts of training workers , foremen and managers on heat stress prevention , proper hydration measures and quality of ( working ) life carried out by occupational health and safety professionals ( physicians and engineers ) , human resources departments and top management at sugarcane farms .", ". often workers rejected the new rehydration measures , most of the time because it was difficult for them to understand thoroughly the dehydration and physiological compensatory mechanisms .", "some of the reasons for this can be attributed to their low educational level , and feeling that  nothing bad has ever happened to me before , etc .", "certainly more effort in terms of intervention strategies and scientific investigation needs to be carried out among workers in nicaragua who perform jobs in which they are exposed to high ambient temperatures .", "these include farm workers , construction workers , miners and fishermen , especially those employed in the informal sector , which occupies about half of nicaragua 's economically active population .", "more funds should also be designated by companies decision makers for improving basic working conditions , in order to increase overall productivity ( and workers satisfaction in terms of better wages ) .", "this would also translate into safer and healthier workers , less absenteeism from sick leave , fewer accidents and other incidents ."], ["the author has not received any funding or benefits from industry to conduct this study ."]]}
{"article_id": "PMC3836973", "article_text": ["choroid plexus papilloma ( cpp ) is a rare benign tumor accounting for 0.5% of all intracranial tumors that is more common in childhood , consisting of 1.5 to 6.4% of pediatric intracranial tumors.1 \n", "2 the majority of cpps occur during the first 2 years of life , with 12.5 to 20% ( 10 to 12% ) reported in infants under 1  year of age.1 \n 3 there have been some reports of  calcified cpp ,   cpp in an adult , ", " cpp in the cerebellopontine angle ( cpa ) ,  and  atypical form of cpp.4 \n 5 \n 6 \n 7 however , a case combining with these four features  adult , dense calcification , atypical form , and cpa  is considered to be extremely rare ; such a case has never been encountered among the 8,540 skull base tumors on which the senior author ( t.f . ) has operated in the past 20 years .", "we report an adult case of densely calcified , atypical cpp originated from the cpa without connection to the forth ventricle .", "a 41-year - old man presented with a chief complaint of headache , dizziness , and difficulty with gait that had been gradually worsening for 8 months .", "neurological examination revealed blurred vision , nystagmus with lateral gaze , and mild right facial weakness .", "intermittent difficulty with balance and gait were also present ; however , no motor weakness was observed .", "computed tomography ( ct ) of the head without contrast revealed a high - density multilobular mass in the right cpa causing mass effect and slight ventricular enlargement ( fig .", "magnetic resonance ( mr ) images demonstrated a trilobed solid mass that measured 4.1 cm in maximum diameter ( fig .", "the mass was extra - axial in location and revealed isointensity on both t1-weighted images ( t1-wi ) and t2-weighted images ( t2-wi ) .", "ct and mr images did not reveal any connection between the tumor and the fourth ventricle .", "the surface of the tumor was roughly irregular and looked grayish pink . since the tumor was friable , gelatinous , vascular , and adherent to surrounding structures , it was hard to separate from cranial nerves .", "cranial nerves vi , vii , viii , ix , and x were enveloped in the tumor mass , which included calcification .", "the patient was discharged after recovery from the dysphagia by appropriate rehabilitation . \n computed tomography without contrast revealed a high - density multilobular mass in the right cerebellopontine angle .", "magnetic resonance images demonstrated a trilobed solid mass that was in an extra - axial location .", "the tumor revealed isointensity on both t1-weighted images ( upper left ) and t2-weighted images ( upper right ) .", "homogeneous intense enhancement was identified ( axial in lower left , sagittal in lower right ) .", "cpp reported in this article primarily occurred in the cpa with close proximity to the foramen of luschka lacking continuity of the fourth ventricle .", "the majority of choroid plexus tumors ( cpts ) originate in the ventricular system , 43 to 67% in the lateral ventricle , 24 to 39% in the fourth ventricle , and 9.5 to 11% in the third ventricle , respectively.8 \n 9 the common site of origin differs in each pediatric and adult group . in the pediatric age group ,", "60 to 80% of such tumors occur in the lateral ventricle , especially in the trigone , because the vascular stalk of the choroid plexus in the lateral ventricle is usually attached to the inferior portion of the trigone . on the other hand ,", "most cpp in adults occurs within the fourth ventricle.9 \n 10 \n 11 a small number of the tumors have been reported to be located in extraventricular regions , the cpa , or the cerebellar hemisphere or the suprasellar cistern.8 \n 10 \n 12 cpp within the cpa was first described by cushing in 1917.13 to date , cpp within the cpa has been reported in 7 to 9% of all cpts and is most exclusively found in adults.9 \n 11 \n 14 \n 15 it is divided into two categories according to the origin of the tumor ; one is primary extraventricular cpts and the other is a result of a direct extension of fourth ventricular tumors through the foramen of luschka.15 because the tumor of our case revealed no connection from the fourth ventricle , it is considered to originate from small choroid tufts that normally project from each recess at the foramen luschka into the cpa or from ectopic choroidal islets  unconnected  with the choroid plexus.16 \n  the cpp of our case indicated dense calcification with ventricular enlargement .", "the bulbous tufts extended from the fourth ventricular choroid plexus through the foramen of luschka have been known as the bochdalek flower basket , which could be identified in 75% on ct and 96% on mr imaging.17 calcification of the region was found in 38% on normal imaging anatomy.17 in pathological states , calcification of cpp was reported in 4.1% on plain skull radiographs and 23.8% on ct examinations in patients of all", "9 the feature of the calcification was commonly described as a stippled or patchy configuration that may include some degrees of hemorrhage.8 \n 9 \n 10 dense calcification was exceptionally reported by sarkar et al.2 the rate of hydrocephalus in association with cpp is 90% and the rate increases up to 100% in malignant cpps.8 the cause of hydrocephalus was considered a combination of cerebrospinal fluid ( csf ) overproduction ( four to five times that in healthy persons ) , obstruction of csf pathways by the tumor mass , impaired csf absorption , increased protein content of csf around the tumor , subarachnoid scarring or granulations related to recurrent bleeding from the tumor , elevated intraventricular pulse pressure , and adhesions around the exit foramina of the fourth ventricle caused by highly proteinaceous or hemorrhagic csf.2 \n 10 \n 18 the finding of tumor calcification in the cpa generally required consideration of differential diagnosis with papillary or psammomatous meningioma , melanotic schwannoma , and myxopapillary ependymoma . among these tumors ,", "association of ventricular enlargement with the finding of calcification might suggest the possible presence of cpts .", "gross total removal was performed in our case of atypical cpp , although the tumor adhered to adjacent structures . in the 2007 world health organization classification of tumors of the central nervous system", ", cpts are classified into cpp as grade i , atypical cpp as grade ii , and choroid plexus carcinoma ( cpc ) as grade iii . among cpts ,", "total removal of cpts is recommended and seems to be feasible since a significant prognostic factor was relevant to the extent of tumor removal , and no definitive evidence of the beneficial effect of radiotherapy has been reported6 \n 18 \n 19 \n 20 . however , it is not always possible because of tumor bleeding and firm adhesion to adjacent structures , especially cranial nerves .", "the rate of complete resection of benign cpp was 80% and the 5-year survival rate indicated is nearly 100% .", "the majority of survivors are neurologically intact.19 because of the good prognosis , the preservation of cranial nerve functions should be primarily considered in surgery for benign cpp . as for the atypical cpp", ", the rate of total removal was decreased to 50 from 63%.11 \n 21 atypical cpp showed a recurrence rate of 29% , which was much higher than the 6% seen in cpp .", "in addition , seeding within the central nervous system , spontaneous pulmonary metastasis , or rapid regrowth of atypical cpp were also reported.7 \n 22 \n 23 total or gross total removal should be advocated , especially in malignant cpts even though the tumor encases the cranial nerves .", "the finding of calcification in the cpa should suggest the possible presence of cpp , especially if ventricular enlargement is present ."], "abstract_text": ["<S> choroid plexus papilloma is a rare benign tumor accounting for 0.5% of all intracranial tumors . the majority of choroid plexus papillomas occur during the first 2 years of life in the ventricular system . </S>", "<S> moreover , dense calcification of the tumor is uncommon . </S>", "<S> we report an adult case of densely calcified , atypical choroid plexus papilloma that originated from the cerebellopontine angle without connection to the forth ventricle . because the case involves a rare combination of four features  adult , dense calcification , atypical form , and cerebellopontine angle  each characteristic associated with the choroid plexus papilloma was discussed . </S>", "<S> the finding of calcification at the cerebellopontine angle in an adult should suggest the possible presence of choroid plexus papilloma . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["choroid plexus papilloma ( cpp ) is a rare benign tumor accounting for 0.5% of all intracranial tumors that is more common in childhood , consisting of 1.5 to 6.4% of pediatric intracranial tumors.1 \n", "2 the majority of cpps occur during the first 2 years of life , with 12.5 to 20% ( 10 to 12% ) reported in infants under 1  year of age.1 \n 3 there have been some reports of  calcified cpp ,   cpp in an adult , ", " cpp in the cerebellopontine angle ( cpa ) ,  and  atypical form of cpp.4 \n 5 \n 6 \n 7 however , a case combining with these four features  adult , dense calcification , atypical form , and cpa  is considered to be extremely rare ; such a case has never been encountered among the 8,540 skull base tumors on which the senior author ( t.f . ) has operated in the past 20 years .", "we report an adult case of densely calcified , atypical cpp originated from the cpa without connection to the forth ventricle ."], ["a 41-year - old man presented with a chief complaint of headache , dizziness , and difficulty with gait that had been gradually worsening for 8 months .", "neurological examination revealed blurred vision , nystagmus with lateral gaze , and mild right facial weakness .", "intermittent difficulty with balance and gait were also present ; however , no motor weakness was observed .", "computed tomography ( ct ) of the head without contrast revealed a high - density multilobular mass in the right cpa causing mass effect and slight ventricular enlargement ( fig .", "magnetic resonance ( mr ) images demonstrated a trilobed solid mass that measured 4.1 cm in maximum diameter ( fig .", "the mass was extra - axial in location and revealed isointensity on both t1-weighted images ( t1-wi ) and t2-weighted images ( t2-wi ) .", "ct and mr images did not reveal any connection between the tumor and the fourth ventricle .", "the surface of the tumor was roughly irregular and looked grayish pink . since the tumor was friable , gelatinous , vascular , and adherent to surrounding structures , it was hard to separate from cranial nerves .", "cranial nerves vi , vii , viii , ix , and x were enveloped in the tumor mass , which included calcification .", "the patient was discharged after recovery from the dysphagia by appropriate rehabilitation . \n computed tomography without contrast revealed a high - density multilobular mass in the right cerebellopontine angle .", "magnetic resonance images demonstrated a trilobed solid mass that was in an extra - axial location .", "the tumor revealed isointensity on both t1-weighted images ( upper left ) and t2-weighted images ( upper right ) .", "homogeneous intense enhancement was identified ( axial in lower left , sagittal in lower right ) ."], ["cpp reported in this article primarily occurred in the cpa with close proximity to the foramen of luschka lacking continuity of the fourth ventricle .", "the majority of choroid plexus tumors ( cpts ) originate in the ventricular system , 43 to 67% in the lateral ventricle , 24 to 39% in the fourth ventricle , and 9.5 to 11% in the third ventricle , respectively.8 \n 9 the common site of origin differs in each pediatric and adult group . in the pediatric age group ,", "60 to 80% of such tumors occur in the lateral ventricle , especially in the trigone , because the vascular stalk of the choroid plexus in the lateral ventricle is usually attached to the inferior portion of the trigone . on the other hand ,", "most cpp in adults occurs within the fourth ventricle.9 \n 10 \n 11 a small number of the tumors have been reported to be located in extraventricular regions , the cpa , or the cerebellar hemisphere or the suprasellar cistern.8 \n 10 \n 12 cpp within the cpa was first described by cushing in 1917.13 to date , cpp within the cpa has been reported in 7 to 9% of all cpts and is most exclusively found in adults.9 \n 11 \n 14 \n 15 it is divided into two categories according to the origin of the tumor ; one is primary extraventricular cpts and the other is a result of a direct extension of fourth ventricular tumors through the foramen of luschka.15 because the tumor of our case revealed no connection from the fourth ventricle , it is considered to originate from small choroid tufts that normally project from each recess at the foramen luschka into the cpa or from ectopic choroidal islets  unconnected  with the choroid plexus.16 \n  the cpp of our case indicated dense calcification with ventricular enlargement .", "the bulbous tufts extended from the fourth ventricular choroid plexus through the foramen of luschka have been known as the bochdalek flower basket , which could be identified in 75% on ct and 96% on mr imaging.17 calcification of the region was found in 38% on normal imaging anatomy.17 in pathological states , calcification of cpp was reported in 4.1% on plain skull radiographs and 23.8% on ct examinations in patients of all", "9 the feature of the calcification was commonly described as a stippled or patchy configuration that may include some degrees of hemorrhage.8 \n 9 \n 10 dense calcification was exceptionally reported by sarkar et al.2 the rate of hydrocephalus in association with cpp is 90% and the rate increases up to 100% in malignant cpps.8 the cause of hydrocephalus was considered a combination of cerebrospinal fluid ( csf ) overproduction ( four to five times that in healthy persons ) , obstruction of csf pathways by the tumor mass , impaired csf absorption , increased protein content of csf around the tumor , subarachnoid scarring or granulations related to recurrent bleeding from the tumor , elevated intraventricular pulse pressure , and adhesions around the exit foramina of the fourth ventricle caused by highly proteinaceous or hemorrhagic csf.2 \n 10 \n 18 the finding of tumor calcification in the cpa generally required consideration of differential diagnosis with papillary or psammomatous meningioma , melanotic schwannoma , and myxopapillary ependymoma . among these tumors ,", "association of ventricular enlargement with the finding of calcification might suggest the possible presence of cpts .", "gross total removal was performed in our case of atypical cpp , although the tumor adhered to adjacent structures . in the 2007 world health organization classification of tumors of the central nervous system", ", cpts are classified into cpp as grade i , atypical cpp as grade ii , and choroid plexus carcinoma ( cpc ) as grade iii . among cpts ,", "total removal of cpts is recommended and seems to be feasible since a significant prognostic factor was relevant to the extent of tumor removal , and no definitive evidence of the beneficial effect of radiotherapy has been reported6 \n 18 \n 19 \n 20 . however , it is not always possible because of tumor bleeding and firm adhesion to adjacent structures , especially cranial nerves .", "the rate of complete resection of benign cpp was 80% and the 5-year survival rate indicated is nearly 100% .", "the majority of survivors are neurologically intact.19 because of the good prognosis , the preservation of cranial nerve functions should be primarily considered in surgery for benign cpp . as for the atypical cpp", ", the rate of total removal was decreased to 50 from 63%.11 \n 21 atypical cpp showed a recurrence rate of 29% , which was much higher than the 6% seen in cpp .", "in addition , seeding within the central nervous system , spontaneous pulmonary metastasis , or rapid regrowth of atypical cpp were also reported.7 \n 22 \n 23 total or gross total removal should be advocated , especially in malignant cpts even though the tumor encases the cranial nerves ."], ["the finding of calcification in the cpa should suggest the possible presence of cpp , especially if ventricular enlargement is present ."]]}
{"article_id": "PMC3332071", "article_text": ["multipath signals occur in numerous microwave and rf applications when an unwanted portion of the original transmission propagates along any alternate path and ultimately couples to the receiver distorting the amplitude and phase of the desired signal [ 14 ] .", "if the amplitude of the multipath signal is sufficiently large , its impact can be considerable . in multistatic radar and communication systems ,", "these types of interference are most often caused by reflections of either the main beam or side lobes with objects near or actually in the beam path .", "classic examples include main beam propagation close to the earth 's surface with associated reflections off of the ground or water ( figure 1(a ) ) .", "various approaches can be used to filter or compensate for these reflections through time - gating   and signal time synchronization .", "the potential for interference from multipath signals increases substantially in near - field applications ( figure 1(b ) ) , especially in situations where the receiving and transmitting hardware are integrated .", "commercially available multichannel network analyzers ( e.g. , zvt8 by rohde & schwarz ; munich , germany ) utilize robust strategies to minimize these signal coupling problems .", "we are developing multichannel transceiving arrays for medical microwave imaging which exploit near - field concepts to produce electrical property maps ( permittivity and conductivity ) of tissues of interest [ 8 , 9 ] and have addressed the issue by incorporating ( a ) dedicated mixers for each channel , ( b ) additional solid state switches for isolation , ( c ) double- and triple - braided coaxial cables , and ( d ) compartmentalized rf circuitry .", "the implementation has proven effective for our application achieving channel / signal isolation greater than 130  db .", "an alternative data acquisition strategy integrates a commercial , 2-port network analyzer with an electronic switching network to feed an array of antennas [ 1012 ] which is effective but also has limitations because ( i ) dynamic range is constrained by the provisions of the network analyzer , ( ii ) two - way signal loss is incurred through the network , and ( iii ) the switching matrix has relatively poor cross - channel isolation .", "equally important in near - field imaging is the multiple paths a signal can take within the imaging zone .", "figures 2(a ) and 2(b ) show a photograph of our clinical breast imaging tank and a schematic diagram of the antenna configuration , respectively .", "in this situation , the array of 16 monopole antennas surrounds the breast and can be moved to multiple vertical positions .", "the antennas and target are submerged in a solution of glycerin and water which is lossy over the operating frequency range ( 700  mhz3  ghz ) .", "early empirical tests have indicated that reflections off the tank side walls do not impact the desired signals for an array with the antennas mounted on a 15.2  cm diameter circle .", "likewise , analysis of the monopole beam patterns as a function of frequency has shown that artifacts are minimal when the array approaches the liquid interface at the top of the tank .", "with respect to reflections off the bottom surface , the base of the tank was at least 1.8 wavelengths ( at the lowest frequency ) below the active sections of the antennas in our initial clinical installation when the array was located at its lowest position during an exam .", "since minimal multipath signals resulted from reflections off the top liquid surface , symmetry would suggest that the same should be true for the base of the tank ( figure 1(b ) ) .", "however , surface waves can cause multipath signals that can be especially difficult to eliminate in near - field systems", ". their excitation can be complex , but their propagation characteristics along two material interfaces , whether planar or along cylindrically shaped structures , have previously been studied in depth [ 1520 ] .", "surface waves can readily propagate at the interface of two dielectric materials or one conductor juxtaposed directly with a dielectric .", "their propagation and attenuation characteristics are nominally determined by the electrical properties of the two materials .", "in addition , their amplitude decays exponentially away from the interface in the perpendicular direction as a function of the lossiness of the complementary materials .", "it should be noted that these investigations have stemmed partially from our efforts to perform microwave tomographic images on patients in an actual mr scanner for the purpose of exploiting the refined spatial resolution of the mr along with the more specific nature of the tissue dielectric properties .", "the mr bore is quite small and places a significant constraint of space for the microwave system .", "this was where we first encountered multipath signal corruption which subsequently led to this study . in the following sections , we discuss the theoretical underpinnings of these modes for the geometries present in our system .", "we demonstrate cases from our current imaging system , where the measurements indicate corruption of the desired signals from multipath signals associated with the base of the tank .", "we then show experiments that allow us to partially isolate the effects to surface waves propagating along other pathways .", "we realize that there are a number of propagation modes around the antennas , their feedlines and the tank surfaces , of which the surface waves are only one possible contributor , but understanding these contributions is important .", "we present an initial strategy for minimizing the effects of these signals which may be instructive for designing other near - field imaging systems , including simulations confirming the earlier theoretical discussion and validating our feedline design strategy .", "a challenging situation occurs when a portion of the transmitted signal propagates along an unwanted path and recombines with the original signal at the receiver .", "time - gating strategies can sometimes be effective when the nature of the multipath signal is well understood [ 1 , 2 ] .", "the potential influence of a multipath signal when the original transmission is a continuous wave can be written as \n ( 1)resultant  signal = acombcos(t+comb ) , \n where \n ( 2)comb = acombtan1(b1a1),acomb = a12+b12,a1=[adecosde+ampcosmp],b1=[adesinde+ampsinmp ] . \n here ,", "ade , amp , de , and mp are the desired and multipath signal amplitudes and phases , respectively ,  is the operating frequency , and t is time .", "for example , if the magnitude of the multipath signal is 25  db below that of the desired signal , the maximum possible amplitude and phase errors in the resultant signal would be 0.48  db and 3.22 , respectively . for a 15  db multipath signal", ", these values increase to 1.42  db and 10.24. clearly , the resultant phase and amplitude errors can become very significant for multipath signals that are on the same order of magnitude as the desired signals .", "it should be noted that there can be many multipath contributions with a range of amplitude and phase contributions .", "this single contributor analysis serves to give a flavor that the unwanted effects can be significant and is generalizable to multiple sources .", "the first surface mode to be considered involves propagation along the interface between the tank base and coupling liquid . following the analysis by stratton , figure 3 shows a plane wave in region 1 impinging on an interface ( x = 0 ) with region 2 . in this case", ", the magnetic field ( hy ) is only oriented in the y - direction ( out of the page ) .", "the complex relative dielectric properties in the two regions are 1 = 1  j1 and 2 = 2  j2 , respectively .", "the classic surface wave solution occurs when the reflection coefficient is zero ( at the brewster angle ) , which is complex - valued in this instance .", "if a is the amplitude of the incident plane wave , then the magnetic component of the incident and transmitted waves can be represented as \n ( 3)hy = ae[jh1xjz ] ,  x>0,hy = ae[jh1xjz ] ,  x<0 , \n where h1 +  = k1 and h2 +  = k2 are needed to satisfy the wave equation , k1 and k2 are the wave numbers for the two regions , and  is the propagation constant .  ", "the wave impedances for the two regions are given by \n ( 4)z1=h1k1z1=h1k1z011j1,z2=h2k2z2=h2k2z012j2 , \n where z1 and z2 are the free space impedances in the corresponding regions , and z1 and z2 are the associated wave impedances [ 21 , 22 ] .", "this yields \n ( 5)h1=h2 , \n where \n ( 6)=21=2j21j1. \n from these relationships , we can solve for \n ( 7)h1=k01(1)12,h2=k012(1)12,=k01(1)12 . \n ", "in this situation , we are primarily interested in surface waves propagating along the outside of a coaxial cable , and their associated attenuation as a function of distance after the mode has been sufficiently established . for this analysis , we will consider the case of a coaxial line which is abruptly terminated by an open end ( figure 4 ) .", "a coaxial cable supports a tem - mode electromagnetic field which is incident on the cable opening .", "part of the signal is partially reflected into the cable , while a second portion is transmitted as a surface wave propagating along the outside of the surrounding cable .", "the fields transmitted into the surrounding space can be determined from the distribution at the coaxial opening which can be found by solving the integral equation for the radial component of the electric field over the opening : \n ( 8)14+jcabe(,0)kc(,)d  = j1abe(,0)kc(,)d0cosejkrrd , \n where c and 1 are the complex - valued permittivity of the coaxial cable insulator and the surrounding dielectric materials , respectively , a and b are inner and outer coaxial radii , respectively ,  is the operating frequency in radians , and k is the wavenumber in the coupling liquid , where 0 is the free space magnetic permeability .  and  are the radial cylindrical coordinates within and outside of the coaxial cable , respectively ,  is the angular coordinate within the coaxial cable , and r is defined as r=2+2 - 2cos. the variable kc( ,  ) is represented as \n ( 9)kc(,)=jn=0n()n()an2n , \n where \n ( 10)n()=y0(na)j1(n)j0(na)y1(n),n={kc2n2,kc>njn2kc2,kc>n , an2=22n2[j02(na)j02(nb)1 ] ,  n>0,a02=lnba . \n", "the eigenvalue n are solutions of the characteristic equation : \n ( 11)y0(na)j1(nb)=j0(na)y1(nb ) , \n where jn and yn are bessel functions of the first and second kind of order n , respectively , and kc is the wavenumber inside the coaxial line .", "once e( , z = 0 ) is determined , then electric and magnetic fields can be found at any point (  , z ) in the surrounding medium from \n ( 12)e(,z)=1abe(,0)0(jk+1r )  zcosejkrrdd , \n or \n ( 13)e(,z)=1abe(,0)  0[1(jk+1r)]cosrejkrrddh(,z)=j1abe(,0)  0cosejkrrdd , \n where \n ( 14)r=z2+2+22cos. \n from these equations it follows that the electromagnetic fields inside the surrounding medium decay approximately as e. \n figure 2(a ) shows the illumination tank used in our current clinical breast imaging system .", "each monopole antenna consists of an exposed length ( 3.8  cm ) of 2.2  mm diameter semirigid coaxial cable with only the center conductor and insulating teflon layer intact . for mechanical robustness", ", the coaxial feed line is enclosed in a 6.4  mm diameter rigid stainless steel tube , and the active section of the antenna is covered with an accompanying length of a delrin cylinder acting as a protective radome .", "the space between the copper coaxial outer conductor and the stainless steel sleeve is sealed at either end with silver epoxy to eliminate wave propagation along the gap .", "the antennas have a nominal return loss of 10  db over the bandwidth of 7003000  mhz .", "the black delrin fittings at the antenna / tank base contain hydraulic seals through which the antenna feeds pass to allow vertical motion of the array while eliminating any coupling fluid leakage .", "the 16 antennas are positioned on a 15.2  cm diameter circle , and both sets of 8 interleaved antennas are supported by individual mounting plates under the tank which provide independent motion of the array in groups of 8 . the tank is fabricated out of plexiglas with an inner wall diameter of 27.3  cm and thickness of 1.3  cm , and the base has a thickness of 2.5  cm .", "all connecting cables are double - braided to minimize stray radiation . in these experiments ,", "the antennas were positioned at heights close to the tank base ( that were not used in any clinical exams ) to study the multipath phenomenon in detail .", "figures 5(a ) , 5(b ) , and 5(c ) show three illumination tanks with different heights , arrays of monopole antennas , and coaxial feed lines that were fabricated from the same plexiglas and had identical diameters and wall / base thicknesses as figure 2(a ) tank .", "the feed lines passed through holes in the base of each tank and were fastened to sma flange connectors which were bolted to the tank floor .", "the tanks were filled with an 80  :  20 glycerin / water mixture with the liquid level 1.5  cm above the antenna tips . in figures 5(a ) and 5(c ) , the feed lines were both 10  cm long ; however , the latter was bent in a serpentine shape such that the top height of the feed line was only 5  cm above the tank floor . the feed line in figure 5(b ) was straight and was only 5  cm long . in these experiments", ", we used plexiglas for the tank materials with a dielectric constant of r = 2.7 that was effectively lossless in this frequency range .", "the dielectric properties of the 80  :  20 glycerin / water bath are plotted in figure 6 as a function of frequency .", "utilizing the clinical system described in section 2.2 , a + 5  dbm signal was transmitted at multiple frequencies over the 7002500  mhz range from a single antenna and received at the remaining 15 antennas .", "this sequence was repeated for the array positioned at multiple heights above the tank base .", "receive antenna amplitudes are plotted for representative frequencies in figure 7 . at 900  mhz , the measured levels are high for the receivers closest to the transmitter ( relative receiver numbers 1 , 2 , 14 , and 15 ) compared to the rest of the array and do not change dramatically with changes in antenna height .", "however , the amplitudes are considerably lower for the more distant receive antennas . for antenna heights 7  cm or greater ( above the tank floor )", ", the attenuation follows a smooth curve hitting a maximum at antenna 8 ( which is furthest away from the transmitter being located on the opposite side of the array ) . at antenna heights 5  cm and lower ,", "the behavior is consistent with the three frequencies shown in figure 7 . given that the distance from the antennas to the tank side walls did not change during the experiments , and that the antennas were sufficiently far from the liquid / air interface at all times for any array heights ( 10  cm in the worst case ) , the corruption of these most distantly received signals appears to be caused by multipath propagation associated with antenna tip proximity to the base of the tank most probably due to reflections off of the tank base or surface wave propagation along the dielectric interfaces . in this set of experiments", "figures 8(a ) and 8(b ) show the received signals for a single transmitter over a range of frequencies for straight feed line lengths of 10  cm and 5  cm ( tanks in figures 5(a ) and 5(b ) ) , respectively . for the longer ( 10  cm ) lines in figure 8(a ) ,", "the field patterns appear well - behaved like those in the previous section , when the array was positioned at the largest heights above the tank floor . however , the patterns for the shorter ( 5  cm ) line lengths in figure 8(b ) exhibit corruption of the signals resulting from the longer propagation distances similarly to when the array heights were closest to the base of the tank for the clinical system ( in figure 7 ) .", "these corrupted signals are nominally between 50 to 70  dbm and occur in uneven patterns relative to the same signals from the longer feed lines which reach 80 to 90  dbm at the furthest antenna . for the shorter propagation distances ( i.e. , the signals received at antennas 14 and 1215 ) , the attenuation patterns from the shorter and longer feed lines are similar ( figure 8(b ) versus 8(a ) ) .", "these results suggest that the unwanted multipath signal effect is the same in both the experimental tanks and the clinical system tests in the previous section and depends on the antenna height from the base of the tank .", "however , in figure 8(c ) , the antenna feed length is exactly the same as in figure 8(a ) , but is curved such that the antenna tip is the same height above the tank floor as the antennas in figure 8(b ) ( which led to signal corruption ) ; yet , the measurement results emulate those in figure 8(a ) ( which are not corrupted ) . here", ", the active part of the antennas is still positioned on the same 15.2  cm diameter circle as in the other two tanks .", "these findings show that the principle signal corruption observed in figure 8(b ) is not due to reflections off the tank floor ; otherwise it would have appeared in figure 8(c ) since the antennas are at the same position above the base for both figures 8(b ) and 8(c ) .", "it should be noted that the signal disruptions for this situation appear more substantial than that for the antennas used in the clinical system as discussed in section 3.1 .", "this is very likely due to differences in the designs of the hydraulic seals , feedline shielding , and antenna radome for the other system .", "more likely , the multipath signals in figure 8(b ) ( with short feed lines ) result from surface waves traveling along the outside of the coaxial lines , across the plexiglas / liquid and/or plexiglas / air interface and back up the outside of the receiver coaxial feed .", "the theoretical considerations in section 2.2 indicate that the attenuation along the coaxial lines is far more substantial than from the planar tank - base surface wave modes .  ", "figure 9(a ) shows a plot of attenuation as a function of frequency for 15.2  cm of plexiglas / liquid surface waves and indicates that very little attenuation of a surface wave propagating along this interface occurs at the frequencies we used .", "thus , in our experiments , the real source of surface wave attenuation comes from propagation along the outside of the coaxial lines .  ", "figure 9(b ) shows plots of the attenuation that results from single 5 and 10  cm lengths of feed line in the coupling fluid .", "the theoretical predictions of attenuation along the two 5  cm antenna feed lengths ( transmit and receive ) and the path along the plexiglas base / liquid interface are approximately 70  db ( { 2  34  db } + 2  db ) for the 1  ghz case ( values interpolated from figures 9(a ) and 9(b ) ) . given a transmit power level of + 5  dbm , the resultant 65  dbm multipath signal for the shorter line would easily corrupt the desired 81  dbm signal ( figure 8(a ) ) .", "only when the feed lines are nearly doubled in length , and the associated surface wave attenuation increased accordingly is the corruption of the desired signals reduced to an acceptable level .", "along with the analytical discussion in sections 2.1 and 2.2 , we have also performed simulations of the configurations described in section 2.4 .", "figures 10(a ) , 10(b ) , and 10(c ) show the 900  mhz electric field magnitude distributions for the long and short , straight antenna feed and the longer , serpentine structure , respectively .", "these simulations were computed using ansys ( burlington , ma , usa ) hfss version 13.0 . for all cases , there is a reasonably broad antenna pattern emanating outwards from the active part of the antennas , and this feature is reasonably similar for all feed line types . for the straight feeds  especially the longer one ,", "it is clear that there are considerable surface currents generated along the coaxial lines . for the shorter", "straight one , there is a high degree of field strength along the coaxial line within the plexiglas volume below the horizontal interface .", "the fields along the interface generally agree with our previous notion that the surface waves preferentially propagate within the lower - loss medium ( in this case the plexiglas ) as can be seen by the substantially greater amplitudes directly under the line .", "the results for the serpentine feedlines are consistent with previous results in that the field strength in the plexiglas is lower than that for the short , straight line case .  ", "figure 11 shows a plot of the field strength just below the liquid interface from a point directly underneath the antenna extending 140  cm to the right for all three corresponding plots .", "the field values are considerably less for the longer straight line but are also less for the serpentine cases compared to the short , straight cases . for the serpentine case", ", there seems to be some signal coupling between the lower feedline bend and the plexiglas .", "as discussed in section 1 , surface waves do decay exponentially from the surfaces , and given the proximity of the feedlines and the liquid / plexiglas interface , some coupling is expected .", "the potentially debilitating effects of unwanted multipath signals is a critical consideration in translating near - field microwave imaging approaches into clinical and commercial systems . for our noncontacting antenna approach ,", "surface waves ( relative to signal reflections from the imaging tank walls ) appear to cause the biggest effects as they propagate along the outside of the transmitting coaxial line across the illumination tank floor and back up the coaxial feed lines of the receivers .", "when the imaging tank is deep and the transmitting / receiving antenna tips are sufficiently far above the tank base , the surface wave signals are adequately suppressed relative to the transmissions through tissue .", "the results presented here indicate that 9 - 10  cm of distance along the feed line is adequate .", "however , reducing the tank depth is of interest for practical reasons and is essential in some settings and appears possible because reflections from the floor of the tank are still too small to degrade the measured signals propagating through tissue .", "indeed , we found that antenna tip distances as little as 5  cm from the tank floor maintain receiver signal fidelity across the array provided the surface wave contributions are attenuated through an equivalent feedline length approaching 10  cm .", "these findings are significant because they indicate that the antenna array and imaging tank geometry can be altered substantially by manipulating the shape of the antenna feed line , which can be exploited to ensure sufficient surface wave attenuation .", "there are certainly other mechanisms for multipath propagation including coupling of fields from the feedlines directly to portions of the breast tissue outside the immediate plane of propagation and are certainly good topics for further investigation ."], "abstract_text": ["<S> microwave imaging techniques are prone to signal corruption from unwanted multipath signals . </S>", "<S> near - field systems are especially vulnerable because signals can scatter and reflect from structural objects within or on the boundary of the imaging zone . </S>", "<S> these issues are further exacerbated when surface waves are generated with the potential of propagating along the transmitting and receiving antenna feed lines and other low - loss paths . in this paper </S>", "<S> , we analyze the contributions of multi - path signals arising from surface wave effects . </S>", "<S> specifically , experiments were conducted with a near - field microwave imaging array positioned at variable heights from the floor of a coupling fluid tank . </S>", "<S> antenna arrays with different feed line lengths in the fluid were also evaluated . </S>", "<S> the results show that surface waves corrupt the received signals over the longest transmission distances across the measurement array . however , the surface wave effects can be eliminated provided the feed line lengths are sufficiently long independently of the distance of the transmitting / receiving antenna tips from the imaging tank floor . </S>", "<S> theoretical predictions confirm the experimental observations . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results and Discussion", "4. Conclusion"], "sections": [["multipath signals occur in numerous microwave and rf applications when an unwanted portion of the original transmission propagates along any alternate path and ultimately couples to the receiver distorting the amplitude and phase of the desired signal [ 14 ] .", "if the amplitude of the multipath signal is sufficiently large , its impact can be considerable . in multistatic radar and communication systems ,", "these types of interference are most often caused by reflections of either the main beam or side lobes with objects near or actually in the beam path .", "classic examples include main beam propagation close to the earth 's surface with associated reflections off of the ground or water ( figure 1(a ) ) .", "various approaches can be used to filter or compensate for these reflections through time - gating   and signal time synchronization .", "the potential for interference from multipath signals increases substantially in near - field applications ( figure 1(b ) ) , especially in situations where the receiving and transmitting hardware are integrated .", "commercially available multichannel network analyzers ( e.g. , zvt8 by rohde & schwarz ; munich , germany ) utilize robust strategies to minimize these signal coupling problems .", "we are developing multichannel transceiving arrays for medical microwave imaging which exploit near - field concepts to produce electrical property maps ( permittivity and conductivity ) of tissues of interest [ 8 , 9 ] and have addressed the issue by incorporating ( a ) dedicated mixers for each channel , ( b ) additional solid state switches for isolation , ( c ) double- and triple - braided coaxial cables , and ( d ) compartmentalized rf circuitry .", "the implementation has proven effective for our application achieving channel / signal isolation greater than 130  db .", "an alternative data acquisition strategy integrates a commercial , 2-port network analyzer with an electronic switching network to feed an array of antennas [ 1012 ] which is effective but also has limitations because ( i ) dynamic range is constrained by the provisions of the network analyzer , ( ii ) two - way signal loss is incurred through the network , and ( iii ) the switching matrix has relatively poor cross - channel isolation .", "equally important in near - field imaging is the multiple paths a signal can take within the imaging zone .", "figures 2(a ) and 2(b ) show a photograph of our clinical breast imaging tank and a schematic diagram of the antenna configuration , respectively .", "in this situation , the array of 16 monopole antennas surrounds the breast and can be moved to multiple vertical positions .", "the antennas and target are submerged in a solution of glycerin and water which is lossy over the operating frequency range ( 700  mhz3  ghz ) .", "early empirical tests have indicated that reflections off the tank side walls do not impact the desired signals for an array with the antennas mounted on a 15.2  cm diameter circle .", "likewise , analysis of the monopole beam patterns as a function of frequency has shown that artifacts are minimal when the array approaches the liquid interface at the top of the tank .", "with respect to reflections off the bottom surface , the base of the tank was at least 1.8 wavelengths ( at the lowest frequency ) below the active sections of the antennas in our initial clinical installation when the array was located at its lowest position during an exam .", "since minimal multipath signals resulted from reflections off the top liquid surface , symmetry would suggest that the same should be true for the base of the tank ( figure 1(b ) ) .", "however , surface waves can cause multipath signals that can be especially difficult to eliminate in near - field systems", ". their excitation can be complex , but their propagation characteristics along two material interfaces , whether planar or along cylindrically shaped structures , have previously been studied in depth [ 1520 ] .", "surface waves can readily propagate at the interface of two dielectric materials or one conductor juxtaposed directly with a dielectric .", "their propagation and attenuation characteristics are nominally determined by the electrical properties of the two materials .", "in addition , their amplitude decays exponentially away from the interface in the perpendicular direction as a function of the lossiness of the complementary materials .", "it should be noted that these investigations have stemmed partially from our efforts to perform microwave tomographic images on patients in an actual mr scanner for the purpose of exploiting the refined spatial resolution of the mr along with the more specific nature of the tissue dielectric properties .", "the mr bore is quite small and places a significant constraint of space for the microwave system .", "this was where we first encountered multipath signal corruption which subsequently led to this study . in the following sections , we discuss the theoretical underpinnings of these modes for the geometries present in our system .", "we demonstrate cases from our current imaging system , where the measurements indicate corruption of the desired signals from multipath signals associated with the base of the tank .", "we then show experiments that allow us to partially isolate the effects to surface waves propagating along other pathways .", "we realize that there are a number of propagation modes around the antennas , their feedlines and the tank surfaces , of which the surface waves are only one possible contributor , but understanding these contributions is important .", "we present an initial strategy for minimizing the effects of these signals which may be instructive for designing other near - field imaging systems , including simulations confirming the earlier theoretical discussion and validating our feedline design strategy ."], ["a challenging situation occurs when a portion of the transmitted signal propagates along an unwanted path and recombines with the original signal at the receiver .", "time - gating strategies can sometimes be effective when the nature of the multipath signal is well understood [ 1 , 2 ] .", "the potential influence of a multipath signal when the original transmission is a continuous wave can be written as \n ( 1)resultant  signal = acombcos(t+comb ) , \n where \n ( 2)comb = acombtan1(b1a1),acomb = a12+b12,a1=[adecosde+ampcosmp],b1=[adesinde+ampsinmp ] . \n here ,", "ade , amp , de , and mp are the desired and multipath signal amplitudes and phases , respectively ,  is the operating frequency , and t is time .", "for example , if the magnitude of the multipath signal is 25  db below that of the desired signal , the maximum possible amplitude and phase errors in the resultant signal would be 0.48  db and 3.22 , respectively . for a 15  db multipath signal", ", these values increase to 1.42  db and 10.24. clearly , the resultant phase and amplitude errors can become very significant for multipath signals that are on the same order of magnitude as the desired signals .", "it should be noted that there can be many multipath contributions with a range of amplitude and phase contributions .", "this single contributor analysis serves to give a flavor that the unwanted effects can be significant and is generalizable to multiple sources .", "the first surface mode to be considered involves propagation along the interface between the tank base and coupling liquid . following the analysis by stratton , figure 3 shows a plane wave in region 1 impinging on an interface ( x = 0 ) with region 2 . in this case", ", the magnetic field ( hy ) is only oriented in the y - direction ( out of the page ) .", "the complex relative dielectric properties in the two regions are 1 = 1  j1 and 2 = 2  j2 , respectively .", "the classic surface wave solution occurs when the reflection coefficient is zero ( at the brewster angle ) , which is complex - valued in this instance .", "if a is the amplitude of the incident plane wave , then the magnetic component of the incident and transmitted waves can be represented as \n ( 3)hy = ae[jh1xjz ] ,  x>0,hy = ae[jh1xjz ] ,  x<0 , \n where h1 +  = k1 and h2 +  = k2 are needed to satisfy the wave equation , k1 and k2 are the wave numbers for the two regions , and  is the propagation constant .  ", "the wave impedances for the two regions are given by \n ( 4)z1=h1k1z1=h1k1z011j1,z2=h2k2z2=h2k2z012j2 , \n where z1 and z2 are the free space impedances in the corresponding regions , and z1 and z2 are the associated wave impedances [ 21 , 22 ] .", "this yields \n ( 5)h1=h2 , \n where \n ( 6)=21=2j21j1. \n from these relationships , we can solve for \n ( 7)h1=k01(1)12,h2=k012(1)12,=k01(1)12 . \n ", "in this situation , we are primarily interested in surface waves propagating along the outside of a coaxial cable , and their associated attenuation as a function of distance after the mode has been sufficiently established . for this analysis , we will consider the case of a coaxial line which is abruptly terminated by an open end ( figure 4 ) .", "a coaxial cable supports a tem - mode electromagnetic field which is incident on the cable opening .", "part of the signal is partially reflected into the cable , while a second portion is transmitted as a surface wave propagating along the outside of the surrounding cable .", "the fields transmitted into the surrounding space can be determined from the distribution at the coaxial opening which can be found by solving the integral equation for the radial component of the electric field over the opening : \n ( 8)14+jcabe(,0)kc(,)d  = j1abe(,0)kc(,)d0cosejkrrd , \n where c and 1 are the complex - valued permittivity of the coaxial cable insulator and the surrounding dielectric materials , respectively , a and b are inner and outer coaxial radii , respectively ,  is the operating frequency in radians , and k is the wavenumber in the coupling liquid , where 0 is the free space magnetic permeability .  and  are the radial cylindrical coordinates within and outside of the coaxial cable , respectively ,  is the angular coordinate within the coaxial cable , and r is defined as r=2+2 - 2cos. the variable kc( ,  ) is represented as \n ( 9)kc(,)=jn=0n()n()an2n , \n where \n ( 10)n()=y0(na)j1(n)j0(na)y1(n),n={kc2n2,kc>njn2kc2,kc>n , an2=22n2[j02(na)j02(nb)1 ] ,  n>0,a02=lnba . \n", "the eigenvalue n are solutions of the characteristic equation : \n ( 11)y0(na)j1(nb)=j0(na)y1(nb ) , \n where jn and yn are bessel functions of the first and second kind of order n , respectively , and kc is the wavenumber inside the coaxial line .", "once e( , z = 0 ) is determined , then electric and magnetic fields can be found at any point (  , z ) in the surrounding medium from \n ( 12)e(,z)=1abe(,0)0(jk+1r )  zcosejkrrdd , \n or \n ( 13)e(,z)=1abe(,0)  0[1(jk+1r)]cosrejkrrddh(,z)=j1abe(,0)  0cosejkrrdd , \n where \n ( 14)r=z2+2+22cos. \n from these equations it follows that the electromagnetic fields inside the surrounding medium decay approximately as e. \n figure 2(a ) shows the illumination tank used in our current clinical breast imaging system .", "each monopole antenna consists of an exposed length ( 3.8  cm ) of 2.2  mm diameter semirigid coaxial cable with only the center conductor and insulating teflon layer intact . for mechanical robustness", ", the coaxial feed line is enclosed in a 6.4  mm diameter rigid stainless steel tube , and the active section of the antenna is covered with an accompanying length of a delrin cylinder acting as a protective radome .", "the space between the copper coaxial outer conductor and the stainless steel sleeve is sealed at either end with silver epoxy to eliminate wave propagation along the gap .", "the antennas have a nominal return loss of 10  db over the bandwidth of 7003000  mhz .", "the black delrin fittings at the antenna / tank base contain hydraulic seals through which the antenna feeds pass to allow vertical motion of the array while eliminating any coupling fluid leakage .", "the 16 antennas are positioned on a 15.2  cm diameter circle , and both sets of 8 interleaved antennas are supported by individual mounting plates under the tank which provide independent motion of the array in groups of 8 . the tank is fabricated out of plexiglas with an inner wall diameter of 27.3  cm and thickness of 1.3  cm , and the base has a thickness of 2.5  cm .", "all connecting cables are double - braided to minimize stray radiation . in these experiments ,", "the antennas were positioned at heights close to the tank base ( that were not used in any clinical exams ) to study the multipath phenomenon in detail .", "figures 5(a ) , 5(b ) , and 5(c ) show three illumination tanks with different heights , arrays of monopole antennas , and coaxial feed lines that were fabricated from the same plexiglas and had identical diameters and wall / base thicknesses as figure 2(a ) tank .", "the feed lines passed through holes in the base of each tank and were fastened to sma flange connectors which were bolted to the tank floor .", "the tanks were filled with an 80  :  20 glycerin / water mixture with the liquid level 1.5  cm above the antenna tips . in figures 5(a ) and 5(c ) , the feed lines were both 10  cm long ; however , the latter was bent in a serpentine shape such that the top height of the feed line was only 5  cm above the tank floor . the feed line in figure 5(b ) was straight and was only 5  cm long . in these experiments", ", we used plexiglas for the tank materials with a dielectric constant of r = 2.7 that was effectively lossless in this frequency range .", "the dielectric properties of the 80  :  20 glycerin / water bath are plotted in figure 6 as a function of frequency ."], ["utilizing the clinical system described in section 2.2 , a + 5  dbm signal was transmitted at multiple frequencies over the 7002500  mhz range from a single antenna and received at the remaining 15 antennas .", "this sequence was repeated for the array positioned at multiple heights above the tank base .", "receive antenna amplitudes are plotted for representative frequencies in figure 7 . at 900  mhz , the measured levels are high for the receivers closest to the transmitter ( relative receiver numbers 1 , 2 , 14 , and 15 ) compared to the rest of the array and do not change dramatically with changes in antenna height .", "however , the amplitudes are considerably lower for the more distant receive antennas . for antenna heights 7  cm or greater ( above the tank floor )", ", the attenuation follows a smooth curve hitting a maximum at antenna 8 ( which is furthest away from the transmitter being located on the opposite side of the array ) . at antenna heights 5  cm and lower ,", "the behavior is consistent with the three frequencies shown in figure 7 . given that the distance from the antennas to the tank side walls did not change during the experiments , and that the antennas were sufficiently far from the liquid / air interface at all times for any array heights ( 10  cm in the worst case ) , the corruption of these most distantly received signals appears to be caused by multipath propagation associated with antenna tip proximity to the base of the tank most probably due to reflections off of the tank base or surface wave propagation along the dielectric interfaces . in this set of experiments", "figures 8(a ) and 8(b ) show the received signals for a single transmitter over a range of frequencies for straight feed line lengths of 10  cm and 5  cm ( tanks in figures 5(a ) and 5(b ) ) , respectively . for the longer ( 10  cm ) lines in figure 8(a ) ,", "the field patterns appear well - behaved like those in the previous section , when the array was positioned at the largest heights above the tank floor . however , the patterns for the shorter ( 5  cm ) line lengths in figure 8(b ) exhibit corruption of the signals resulting from the longer propagation distances similarly to when the array heights were closest to the base of the tank for the clinical system ( in figure 7 ) .", "these corrupted signals are nominally between 50 to 70  dbm and occur in uneven patterns relative to the same signals from the longer feed lines which reach 80 to 90  dbm at the furthest antenna . for the shorter propagation distances ( i.e. , the signals received at antennas 14 and 1215 ) , the attenuation patterns from the shorter and longer feed lines are similar ( figure 8(b ) versus 8(a ) ) .", "these results suggest that the unwanted multipath signal effect is the same in both the experimental tanks and the clinical system tests in the previous section and depends on the antenna height from the base of the tank .", "however , in figure 8(c ) , the antenna feed length is exactly the same as in figure 8(a ) , but is curved such that the antenna tip is the same height above the tank floor as the antennas in figure 8(b ) ( which led to signal corruption ) ; yet , the measurement results emulate those in figure 8(a ) ( which are not corrupted ) . here", ", the active part of the antennas is still positioned on the same 15.2  cm diameter circle as in the other two tanks .", "these findings show that the principle signal corruption observed in figure 8(b ) is not due to reflections off the tank floor ; otherwise it would have appeared in figure 8(c ) since the antennas are at the same position above the base for both figures 8(b ) and 8(c ) .", "it should be noted that the signal disruptions for this situation appear more substantial than that for the antennas used in the clinical system as discussed in section 3.1 .", "this is very likely due to differences in the designs of the hydraulic seals , feedline shielding , and antenna radome for the other system .", "more likely , the multipath signals in figure 8(b ) ( with short feed lines ) result from surface waves traveling along the outside of the coaxial lines , across the plexiglas / liquid and/or plexiglas / air interface and back up the outside of the receiver coaxial feed .", "the theoretical considerations in section 2.2 indicate that the attenuation along the coaxial lines is far more substantial than from the planar tank - base surface wave modes .  ", "figure 9(a ) shows a plot of attenuation as a function of frequency for 15.2  cm of plexiglas / liquid surface waves and indicates that very little attenuation of a surface wave propagating along this interface occurs at the frequencies we used .", "thus , in our experiments , the real source of surface wave attenuation comes from propagation along the outside of the coaxial lines .  ", "figure 9(b ) shows plots of the attenuation that results from single 5 and 10  cm lengths of feed line in the coupling fluid .", "the theoretical predictions of attenuation along the two 5  cm antenna feed lengths ( transmit and receive ) and the path along the plexiglas base / liquid interface are approximately 70  db ( { 2  34  db } + 2  db ) for the 1  ghz case ( values interpolated from figures 9(a ) and 9(b ) ) . given a transmit power level of + 5  dbm , the resultant 65  dbm multipath signal for the shorter line would easily corrupt the desired 81  dbm signal ( figure 8(a ) ) .", "only when the feed lines are nearly doubled in length , and the associated surface wave attenuation increased accordingly is the corruption of the desired signals reduced to an acceptable level .", "along with the analytical discussion in sections 2.1 and 2.2 , we have also performed simulations of the configurations described in section 2.4 .", "figures 10(a ) , 10(b ) , and 10(c ) show the 900  mhz electric field magnitude distributions for the long and short , straight antenna feed and the longer , serpentine structure , respectively .", "these simulations were computed using ansys ( burlington , ma , usa ) hfss version 13.0 . for all cases , there is a reasonably broad antenna pattern emanating outwards from the active part of the antennas , and this feature is reasonably similar for all feed line types . for the straight feeds  especially the longer one ,", "it is clear that there are considerable surface currents generated along the coaxial lines . for the shorter", "straight one , there is a high degree of field strength along the coaxial line within the plexiglas volume below the horizontal interface .", "the fields along the interface generally agree with our previous notion that the surface waves preferentially propagate within the lower - loss medium ( in this case the plexiglas ) as can be seen by the substantially greater amplitudes directly under the line .", "the results for the serpentine feedlines are consistent with previous results in that the field strength in the plexiglas is lower than that for the short , straight line case .  ", "figure 11 shows a plot of the field strength just below the liquid interface from a point directly underneath the antenna extending 140  cm to the right for all three corresponding plots .", "the field values are considerably less for the longer straight line but are also less for the serpentine cases compared to the short , straight cases . for the serpentine case", ", there seems to be some signal coupling between the lower feedline bend and the plexiglas .", "as discussed in section 1 , surface waves do decay exponentially from the surfaces , and given the proximity of the feedlines and the liquid / plexiglas interface , some coupling is expected ."], ["the potentially debilitating effects of unwanted multipath signals is a critical consideration in translating near - field microwave imaging approaches into clinical and commercial systems . for our noncontacting antenna approach ,", "surface waves ( relative to signal reflections from the imaging tank walls ) appear to cause the biggest effects as they propagate along the outside of the transmitting coaxial line across the illumination tank floor and back up the coaxial feed lines of the receivers .", "when the imaging tank is deep and the transmitting / receiving antenna tips are sufficiently far above the tank base , the surface wave signals are adequately suppressed relative to the transmissions through tissue .", "the results presented here indicate that 9 - 10  cm of distance along the feed line is adequate .", "however , reducing the tank depth is of interest for practical reasons and is essential in some settings and appears possible because reflections from the floor of the tank are still too small to degrade the measured signals propagating through tissue .", "indeed , we found that antenna tip distances as little as 5  cm from the tank floor maintain receiver signal fidelity across the array provided the surface wave contributions are attenuated through an equivalent feedline length approaching 10  cm .", "these findings are significant because they indicate that the antenna array and imaging tank geometry can be altered substantially by manipulating the shape of the antenna feed line , which can be exploited to ensure sufficient surface wave attenuation .", "there are certainly other mechanisms for multipath propagation including coupling of fields from the feedlines directly to portions of the breast tissue outside the immediate plane of propagation and are certainly good topics for further investigation ."]]}
{"article_id": "PMC5410432", "article_text": ["enucleation generally induces bone regeneration within a cystic lesion less than 3 cm in diameter and eliminates the lesion ; however , it is difficult to postoperatively predict lesion recurrence and bone regeneration in cases of larger cysts . for this reason", ", a surgeon can choose between conservative and aggressive treatments for a large , aggressive , cystic tumor12 .", "it has been reported that marsupialization and decompression , both of which are conservative treatments that create an opening to reduce pressure within a cystic cavity and induce bone formation , generate few postoperative complications and produce great therapeutic effects3456 .", "these procedures not only provide good surgical access to smaller lesions , but they also preserve intraoral tissues , maintain pulp vitality , and reduce recurrence rates . for this reason , many surgeons prefer these methods for treating large cysts . this study aimed to analyze and assess the therapeutic effects of decompression for patients who underwent decompression followed by enucleation .", "we reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital ( gwangju , korea ) and who had undergone enucleation after decompression .", "we collected data on their age and gender , location and size of the lesions , preoperative and postoperative histopathological findings , gap in time between decompression and enucleation , and variation in the size of the lesions , complications , and recurrence according to the time gap .", "patients were excluded from group review if they had no radiological data , if they had cured soft tissues due to the failure to maintain the window , or if they had been observed for less than six months after enucleation .", "we measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient , measured width and length with a caliper , and multiplied them together to determine the lesion index ( li ) with the aim of simplifying measurement of irregularly sized lesions . to determine the efficacy of decompression , lis were divided into initial and final lis , which were then graded on the basis of nakamura et al .", "'s formula7 . to assess the extent of reaction to decompression for each lesion , the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period . before conducting a biopsy ,", "clinical and radiographic data were used to determine if patients should undergo decompression . in decompression ,", "the window was formed in the buccal alveolar bone region of the jaw , and a biopsy was conducted after excising some portion of the cyst wall .", "1 ) after the treatment , patients were educated on how to wear and remove the device , and they were instructed to clean the device twice a day ( morning and night ) using saline solution .", "panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph , and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing .", "we reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital ( gwangju , korea ) and who had undergone enucleation after decompression .", "we collected data on their age and gender , location and size of the lesions , preoperative and postoperative histopathological findings , gap in time between decompression and enucleation , and variation in the size of the lesions , complications , and recurrence according to the time gap .", "patients were excluded from group review if they had no radiological data , if they had cured soft tissues due to the failure to maintain the window , or if they had been observed for less than six months after enucleation .", "we measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient , measured width and length with a caliper , and multiplied them together to determine the lesion index ( li ) with the aim of simplifying measurement of irregularly sized lesions . to determine the efficacy of decompression , lis were divided into initial and final lis , which were then graded on the basis of nakamura et al .", "'s formula7 . to assess the extent of reaction to decompression for each lesion , the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period .", "before conducting a biopsy , clinical and radiographic data were used to determine if patients should undergo decompression . in decompression ,", "the window was formed in the buccal alveolar bone region of the jaw , and a biopsy was conducted after excising some portion of the cyst wall .", "1 ) after the treatment , patients were educated on how to wear and remove the device , and they were instructed to clean the device twice a day ( morning and night ) using saline solution .", "panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph , and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing .", "in total , 17 patients ( 7 males and 10 females ) were reviewed , and their average age was 33.1 years ( range , 13 - 76 years ) .", "one patient was diagnosed with nevoid basal cell carcinoma syndrome ( nbccs ) and showed multiple keratocystic odontogenic tumors ( kcot ) in the maxilla and mandible .", "two of these cysts were treated with marsupialization.(table 1 ) jaw cysts were observed in 10 patients ( 58.8% ) in their teens or 20s , which was a higher rate compared to other age groups , and the sex ratio was 1:1.4 with seven males ( 41.2% ) and 10 females ( 58.8%).(fig .", "2 ) radiographic results showed that all lesions were unilocular and were either pushing or intruding into the surrounding structures ( maxillary sinus or mandibular canal ) in 15 patients ( 88.2% ) . based on the results of the histopathologic examination , 10 patients were diagnosed with kcot ( 58.8% ) , five patients with dentigerous cysts ( dc ) ( 29.4% ) , one patient with radicular cyst ( rc ) ( 5.9% ) , and one patient with nasopalatine duct cyst ( 5.9% ) .", "only one patient ( 5.9% ) was treated with marsupialization only , and 16 patients ( 94.1% ) were treated with marsupialization followed by enucleation . there was no case where the preoperative histopathologic results changed after the operation .", "the average time of decompression was 8.13.4 months , with 5.54.8 months for a cyst in the mandible and 8.33.1 months for the maxilla .", "the average time of decompression based on lesion type was 8.83.2 months for kcot and 6.02.7 months for dc , suggesting that kcots require approximately three more months of treatment .", "the li was measured at an average of 1,349 mm before decompression and 463 mm after decompression , showing an average size decrease of 64% after decompression .", "according to nakamura 's formula , five patients ( 29.4% ) showed a greater than 80% decrease in size , nine patients ( 52.9% ) showed a 50%-80% decrease in size , and three patients ( 17.6% ) showed a less than 50% decrease in size.(fig .", "3 ) the mandible decreased by 10% more in size than the maxilla , and it had a 1.7-fold higher reduction rate in the same period . also , when comparing size reduction based on patient age , the decrease was greater in patients in their teens or 20s compared to the other age groups , but the reduction rates were not significantly different .", "males were observed to have a smaller final lesion size , while the reduction ratio was higher in females ; however , no significant differences were observed .", "additionally , larger lesions reduced faster that smaller ones , and kcots showed more positive results for decompression than other lesion types.(table 2 ) one patient 's recovery was complicated by hypoesthesia after marsupialization , but this naturally recovered over time .", "the total follow - up period was 1 to 8 years , and 3 out of 17 patients were suspected recurrence .", "however , one patient was diagnosed with scar tissue after re - examination , and one patient was diagnosed with periapical cyst due to re - infection of an incomplete root canal treatment .", "a true recurrence appeared on one nbccs patient 's left maxilla and is currently being followed - up after re - operation .", "since decompression was introduced as a conservative treatment for odontogenic cyst , many cases have been treated with decompression , and various studies have reported high success rates89 . however , the reaction varies depending on the cyst .", "anavi et al.10 conducted decompression on 57 patients who were diagnosed with kcot , dc , or rc , and no statistically significant differences in reduction rate were reported based on the histologic diagnosis . in present study ,", "the reduction rate in kcot was higher than the other cysts , with an average of 131.84 mm / month , with the final reduction being the highest .", "this is because kcots are aggressive , so the size of the lesion tends to be greater than that of other cystic lesions . according to this study , cases of li larger than 1,000 mm showed twice the sensitivity of li smaller than 1,000 mm .", "therefore , in this study , kcot patients seemed to have a higher sensitivity to decompression since the li was larger than 1,000 mm .", "there are no formal criteria for the decompression period or change in size ; however , according to previous studies , a 65% reduction was reported when maintained for an average of 8.4 months .", "additionally , an 81% reduction was observed when decompression was maintained for an average of 17.5 months911 .", "another study reported that it is preferable to perform enucleation if the size of the lesion after decompression decreases more than 50%-60%8 . in this study , the average decompression period was 8.1 months , and the lesions showed an average reduction of 64% .", "the effects of decompression based on age are controversial , as it was reported that younger patients had higher reduction rates10 , but it also was reported that decompression is not correlated with age12 .", "therefore , even at older ages , if a large cystic lesion exists within the jaw , postoperative complications can be reduced by decreasing size via decompression .", "brndum and jensen13 performed enucleation after decompression in 12 patients with kcot ; during 7 to 17 years of follow - up , no recurrence was reported .", "such low recurrence rates were reported in many studies , and some of them reported biopsy results indicating that the histological characteristics of the residual cyst epithelium changed from parakeratinization to orthokeratinization or were no longer observed14 .", "based on these results , august et al.11 reported that histological changes are likely to appear when decompression is maintained for longer than nine months , and he proposed a decompression period of at least nine months .", "this study used a panoramic radiograph to two - dimensionally evaluate the lesion by measuring the maximum vertical and horizontal widths of the lesion .", "this method is widely used because of ease , but it has a disadvantage in accuracy , as it two - dimensionally evaluates a three - dimensional lesion .", "therefore , a recent method of measuring volume via three - dimensional analysis using a computer program was introduced15 .", "however , this method requires a special program and skilled technique , so it can be difficult to apply clinically . although plane analysis was performed in the present study due to lack of data for three - dimensional analysis , a more accurate analysis is expected once sufficient data is obtained .", "this study was limited in that statistical analysis was not performed due to lack of samples .", "thus , when a lesion is large , reducing its size via decompression not only allows for conservative treatment , but it can also minimize complications .", "according to this study , all patients treated with decompression were reported to have shown a reduction in lesion size and higher sensitivity with a larger lesion size .", "there was no difference in the effect of decompression based on age , and only one patient experienced recurrence of the cyst ."], "abstract_text": ["<S> objectivesthe purpose of this study is to evaluate the treatment efficacy of enucleation after decompression.materials and methodsa total of 17 patients with cystic lesion of the jaw were treated with decompression followed by enucleation . </S>", "<S> pre- and postdecompression panoramic radiographs were analyzed.resultsthe mean percentage of reduction after decompression was 64% . </S>", "<S> the reaction was graded as good ( > 80% ) in five patients ( 29.4% ) , moderate ( 50%-80% ) in nine patients ( 52.9% ) , and poor ( < 50% ) in three patients ( 17.6% ) . </S>", "<S> the reduction rate of larger cystic lesions was faster than that of smaller lesions . </S>", "<S> however , the reduction rate was not affected by age . </S>", "<S> the duration of follow - up ranged from one to eight years . </S>", "<S> there were no complications , and one case recurred.conclusiondecompression is an effective method for the initial treatment of jaw cysts . </S>"], "labels": null, "section_names": ["I. Introduction", "II. Materials and Methods", "1. Subject screening", "2. Treatment protocol", "III. Results", "IV. Discussion", "V. Conclusion"], "sections": [["jaw cysts are usually painless , but they can increase in size as they absorb neighboring structures .", "enucleation generally induces bone regeneration within a cystic lesion less than 3 cm in diameter and eliminates the lesion ; however , it is difficult to postoperatively predict lesion recurrence and bone regeneration in cases of larger cysts . for this reason", ", a surgeon can choose between conservative and aggressive treatments for a large , aggressive , cystic tumor12 .", "it has been reported that marsupialization and decompression , both of which are conservative treatments that create an opening to reduce pressure within a cystic cavity and induce bone formation , generate few postoperative complications and produce great therapeutic effects3456 .", "these procedures not only provide good surgical access to smaller lesions , but they also preserve intraoral tissues , maintain pulp vitality , and reduce recurrence rates . for this reason , many surgeons prefer these methods for treating large cysts . this study aimed to analyze and assess the therapeutic effects of decompression for patients who underwent decompression followed by enucleation ."], ["we reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital ( gwangju , korea ) and who had undergone enucleation after decompression .", "we collected data on their age and gender , location and size of the lesions , preoperative and postoperative histopathological findings , gap in time between decompression and enucleation , and variation in the size of the lesions , complications , and recurrence according to the time gap .", "patients were excluded from group review if they had no radiological data , if they had cured soft tissues due to the failure to maintain the window , or if they had been observed for less than six months after enucleation .", "we measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient , measured width and length with a caliper , and multiplied them together to determine the lesion index ( li ) with the aim of simplifying measurement of irregularly sized lesions . to determine the efficacy of decompression , lis were divided into initial and final lis , which were then graded on the basis of nakamura et al .", "'s formula7 . to assess the extent of reaction to decompression for each lesion , the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period . before conducting a biopsy ,", "clinical and radiographic data were used to determine if patients should undergo decompression . in decompression ,", "the window was formed in the buccal alveolar bone region of the jaw , and a biopsy was conducted after excising some portion of the cyst wall .", "1 ) after the treatment , patients were educated on how to wear and remove the device , and they were instructed to clean the device twice a day ( morning and night ) using saline solution .", "panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph , and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing ."], ["we reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital ( gwangju , korea ) and who had undergone enucleation after decompression .", "we collected data on their age and gender , location and size of the lesions , preoperative and postoperative histopathological findings , gap in time between decompression and enucleation , and variation in the size of the lesions , complications , and recurrence according to the time gap .", "patients were excluded from group review if they had no radiological data , if they had cured soft tissues due to the failure to maintain the window , or if they had been observed for less than six months after enucleation .", "we measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient , measured width and length with a caliper , and multiplied them together to determine the lesion index ( li ) with the aim of simplifying measurement of irregularly sized lesions . to determine the efficacy of decompression , lis were divided into initial and final lis , which were then graded on the basis of nakamura et al .", "'s formula7 . to assess the extent of reaction to decompression for each lesion , the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period ."], ["before conducting a biopsy , clinical and radiographic data were used to determine if patients should undergo decompression . in decompression ,", "the window was formed in the buccal alveolar bone region of the jaw , and a biopsy was conducted after excising some portion of the cyst wall .", "1 ) after the treatment , patients were educated on how to wear and remove the device , and they were instructed to clean the device twice a day ( morning and night ) using saline solution .", "panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph , and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing ."], ["in total , 17 patients ( 7 males and 10 females ) were reviewed , and their average age was 33.1 years ( range , 13 - 76 years ) .", "one patient was diagnosed with nevoid basal cell carcinoma syndrome ( nbccs ) and showed multiple keratocystic odontogenic tumors ( kcot ) in the maxilla and mandible .", "two of these cysts were treated with marsupialization.(table 1 ) jaw cysts were observed in 10 patients ( 58.8% ) in their teens or 20s , which was a higher rate compared to other age groups , and the sex ratio was 1:1.4 with seven males ( 41.2% ) and 10 females ( 58.8%).(fig .", "2 ) radiographic results showed that all lesions were unilocular and were either pushing or intruding into the surrounding structures ( maxillary sinus or mandibular canal ) in 15 patients ( 88.2% ) . based on the results of the histopathologic examination , 10 patients were diagnosed with kcot ( 58.8% ) , five patients with dentigerous cysts ( dc ) ( 29.4% ) , one patient with radicular cyst ( rc ) ( 5.9% ) , and one patient with nasopalatine duct cyst ( 5.9% ) .", "only one patient ( 5.9% ) was treated with marsupialization only , and 16 patients ( 94.1% ) were treated with marsupialization followed by enucleation . there was no case where the preoperative histopathologic results changed after the operation .", "the average time of decompression was 8.13.4 months , with 5.54.8 months for a cyst in the mandible and 8.33.1 months for the maxilla .", "the average time of decompression based on lesion type was 8.83.2 months for kcot and 6.02.7 months for dc , suggesting that kcots require approximately three more months of treatment .", "the li was measured at an average of 1,349 mm before decompression and 463 mm after decompression , showing an average size decrease of 64% after decompression .", "according to nakamura 's formula , five patients ( 29.4% ) showed a greater than 80% decrease in size , nine patients ( 52.9% ) showed a 50%-80% decrease in size , and three patients ( 17.6% ) showed a less than 50% decrease in size.(fig .", "3 ) the mandible decreased by 10% more in size than the maxilla , and it had a 1.7-fold higher reduction rate in the same period . also , when comparing size reduction based on patient age , the decrease was greater in patients in their teens or 20s compared to the other age groups , but the reduction rates were not significantly different .", "males were observed to have a smaller final lesion size , while the reduction ratio was higher in females ; however , no significant differences were observed .", "additionally , larger lesions reduced faster that smaller ones , and kcots showed more positive results for decompression than other lesion types.(table 2 ) one patient 's recovery was complicated by hypoesthesia after marsupialization , but this naturally recovered over time .", "the total follow - up period was 1 to 8 years , and 3 out of 17 patients were suspected recurrence .", "however , one patient was diagnosed with scar tissue after re - examination , and one patient was diagnosed with periapical cyst due to re - infection of an incomplete root canal treatment .", "a true recurrence appeared on one nbccs patient 's left maxilla and is currently being followed - up after re - operation ."], ["since decompression was introduced as a conservative treatment for odontogenic cyst , many cases have been treated with decompression , and various studies have reported high success rates89 . however , the reaction varies depending on the cyst .", "anavi et al.10 conducted decompression on 57 patients who were diagnosed with kcot , dc , or rc , and no statistically significant differences in reduction rate were reported based on the histologic diagnosis . in present study ,", "the reduction rate in kcot was higher than the other cysts , with an average of 131.84 mm / month , with the final reduction being the highest .", "this is because kcots are aggressive , so the size of the lesion tends to be greater than that of other cystic lesions . according to this study , cases of li larger than 1,000 mm showed twice the sensitivity of li smaller than 1,000 mm .", "therefore , in this study , kcot patients seemed to have a higher sensitivity to decompression since the li was larger than 1,000 mm .", "there are no formal criteria for the decompression period or change in size ; however , according to previous studies , a 65% reduction was reported when maintained for an average of 8.4 months .", "additionally , an 81% reduction was observed when decompression was maintained for an average of 17.5 months911 .", "another study reported that it is preferable to perform enucleation if the size of the lesion after decompression decreases more than 50%-60%8 . in this study , the average decompression period was 8.1 months , and the lesions showed an average reduction of 64% .", "the effects of decompression based on age are controversial , as it was reported that younger patients had higher reduction rates10 , but it also was reported that decompression is not correlated with age12 .", "therefore , even at older ages , if a large cystic lesion exists within the jaw , postoperative complications can be reduced by decreasing size via decompression .", "brndum and jensen13 performed enucleation after decompression in 12 patients with kcot ; during 7 to 17 years of follow - up , no recurrence was reported .", "such low recurrence rates were reported in many studies , and some of them reported biopsy results indicating that the histological characteristics of the residual cyst epithelium changed from parakeratinization to orthokeratinization or were no longer observed14 .", "based on these results , august et al.11 reported that histological changes are likely to appear when decompression is maintained for longer than nine months , and he proposed a decompression period of at least nine months .", "this study used a panoramic radiograph to two - dimensionally evaluate the lesion by measuring the maximum vertical and horizontal widths of the lesion .", "this method is widely used because of ease , but it has a disadvantage in accuracy , as it two - dimensionally evaluates a three - dimensional lesion .", "therefore , a recent method of measuring volume via three - dimensional analysis using a computer program was introduced15 .", "however , this method requires a special program and skilled technique , so it can be difficult to apply clinically . although plane analysis was performed in the present study due to lack of data for three - dimensional analysis , a more accurate analysis is expected once sufficient data is obtained .", "this study was limited in that statistical analysis was not performed due to lack of samples .", "thus , when a lesion is large , reducing its size via decompression not only allows for conservative treatment , but it can also minimize complications ."], ["according to this study , all patients treated with decompression were reported to have shown a reduction in lesion size and higher sensitivity with a larger lesion size .", "there was no difference in the effect of decompression based on age , and only one patient experienced recurrence of the cyst ."]]}
{"article_id": "PMC4475663", "article_text": ["for the first time , the presented data suggest that a variant in the retn gene plays a significant role in acne pathogenesis .", "we also demonstrate an excess transmission of mutant allele g at retn-420 from 130 trio families using tdt analysis .", "therefore , we suggest that inherited variation in retn at -420 appears to be associated with heritable acne vulgaris ."], "abstract_text": ["<S> resistin ( retn ) , recently found to be relevant to inflammation and inflammatory disorders . </S>", "<S> we , therefore , aimed to investigate the potential role of retn gene polymorphism in pathogenesis of acne vulgaris with familial history . </S>", "<S> we investigated the retn-420c / g polymorphism in 180 patients with acne vulgaris and 180 healthy individuals in a case - control association analysis . in this study </S>", "<S> , we also investigated the heritability of the retn susceptible allele from 140 trio families with acne affected offspring . </S>", "<S> the genotyping was performed by polymerase chain reaction and direct dna sequencing . </S>", "<S> the retn-420c / g polymorphism was significantly associated with acne in patients compared with healthy controls ( p=0.014 ) . </S>", "<S> the minor allele g at -420 was more prevalent in cases vs. controls ( p=0.002 ) . </S>", "<S> the retn-420c / g polymorphism was significantly associated with severity of acne vulgaris in patients ( p=0.0097 ) . </S>", "<S> the results of a transmission disequilibrium test revealed a significant association between the retn-420c / g polymorphism and acne vulgaris ( p<0.001 ) . </S>", "<S> for the first time in the literature , to our knowledge , we demonstrate a significant association of the retn-420c / g functional polymorphism with familial acne vulgaris . </S>"], "labels": null, "section_names": ["Conclusion"], "sections": [["for the first time , the presented data suggest that a variant in the retn gene plays a significant role in acne pathogenesis .", "we also demonstrate an excess transmission of mutant allele g at retn-420 from 130 trio families using tdt analysis .", "therefore , we suggest that inherited variation in retn at -420 appears to be associated with heritable acne vulgaris ."]]}
{"article_id": "PMC2976581", "article_text": ["many studies over the past 15 years have focused on discovering solvents that help promote efficient cc bond formation under benign reaction conditions .", "new solvents in dielsalder(2 ) and barbier reactions have been sought to help streamline the one - pot transformations further , reducing synthetic steps and organic waste .", "polar media often accelerate reactions of this type which has led to the use of water as an acceptable solvent for such organic conversions .", "recent contributions from our laboratory to this field have centered on coupling reactions between propargyl aldehydes and allyl or propargyl halides under barbier conditions to form homoallylic and homopropargylic propargyl alcohols .", "these compounds find wide use as synthetic templates and show a propensity for oxy - cope rearrangements ( scheme 1 ) .", "the use of -chloropropargylphenyl sulfide ( 4 ) , coupled with a propargyl aldehyde should offer an easy route into formation of enediyne and epoxydiyne skeletal structures ( scheme 2 ) .", "previous studies revealed that formation of 2 or 3 under indium - promoted barbier conditions is solvent dependent .", "use of an organic solvent for the coupling reaction results in a mixture of 2 and 3 while the use of water as a solvent yields 2 as the sole product .", "it is possible that water protonates the coupled product to form 2 quickly , quenching the alkoxide intermediate , thereby suppressing the possible oxy - cope pathway shown in scheme 1 .", "it may also be possible that the increased polarity of the solvent itself stabilizes the anion formed upon coupling , thus raising the relative rate of cope rearrangement in this system . with a less polar , aprotic solvent ,", "the anion will not be quenched , nor will it be stabilized to the same extent as witnessed in water .", "a combination of these phenomena may be why an oxy - cope rearrangement occurs under less polar conditions , forming product 3 ( scheme 1 ) .", "a coupling reaction between 1 and 4 ( scheme 2 ) was conducted under aqueous conditions to form 2 ( y = sph ) , with good regioselectivity ; however , the rate of this reaction is surprisingly slow .", "although reactions of the type shown in scheme 1 are reported in the literature to be accelerated by polar solvents such as water , coupling reactions between 1 and 4 , as shown in scheme 2 , are quite slow . indeed ,", "when water is used as the solvent , reactions often require 1036 h for completion .", "this oil has a density greater than that of water so it is presumed to be an organometallic species formed by interaction of the indium metal with the halide species .", "isolation of the oil followed by workup in an acidic thf / h2o mixture leads to isolation of propargylphenyl sulfide and , to a smaller extent , the aldehyde .", "we surmise that low solubility of this organometallic complex leads to poor reagent mixing , slowing the barbier coupling under aqueous conditions .", "second , indium - promoted coupling reactions conducted in water become more acidic as the reaction proceeds .", "we believe it is possible that the aldehyde is in equilibrium with its corresponding hydrate species , reducing the electrophilicity of the carbon center .", "a combination of these two reasons would lead to a decrease in the rate of reaction between 1 and 4 when conducted in an aqueous solvent .", "the reaction proceeds more quickly ( 812 h ) in dmf , a less polar solvent , due to better solubility of all reagents , however , a mixture of 2 and 3 was isolated .", "it was originally expected that a mixture of water and dmf would lead to faster reaction times while still maintaining regiocontrol of product formation . the use of a water / dmf solvent system , however , resulted in a mixture of products with water constituting up to 50% v / v of the solvent system .", "when water exceeds 50% v / v of the solvent system , only product 2 is isolated , but the rate of reaction slows perceptively once again . to help expedite this reaction sequence ,", "we began a search for solvent with polarity similar to , or greater than that of water .", "it was expected that an extremely polar organic solvent would promote the same type of acceleration normally witnessed in barbier couplings carried out under aqueous conditions .", "an organic solvent could also help solubilize the intermediate and reduce possible formation of the hydrate species , thus allowing for a faster coupling reaction to occur .", "if increased polarity stabilizes the anion formed by coupling of 1 and 4 , oxy - cope rearrangement should also be suppressed . with a dielectric constant of 186.9 at 25 c , significantly greater than that of water ( 78.37(7 ) ) , and dmf ( 38.3(7b ) ) ,", "n - methylformamide ( nmf ) has found occasional use in previous reactions requiring polar organic solvents ; however , it has historically found more widespread use as an organic reagent . with a pka of approximately 24,(7d )", "nmf is reactive under a range of organometallic reagent conditions , limiting its use as a solvent .", "indium - promoted coupling reactions , however , have been shown to be accelerated under aqueous conditions,(1 ) and the relative acidity of nmf , being higher than that of water , should not hinder the proposed barbier couplings proposed within this paper .", "we were especially intrigued by the fact that the dielectric constant of nmf increases to 220 as the temperature is lowered from 25 to 0 c .", "we were interested in examining what effect the increase in dielectric constant at lower temperature could have on a barbier - style reaction of the type shown in scheme 2 among others .", "this paper reports on the use of nmf in barbier - style reactions with a focus on rates of reaction , stereochemistry , and its use to form enediyne and epoxydiyne skeletal structures.(9 )", "although nmf has characteristics similar to those of dmf , it also has characteristics similar to water , such as a polar xh bond.(7 ) thus , having found a very polar solvent with properties similar to both water and organic systems , we hypothesized that the 1,2-coupling product could be formed in high yield under short reaction times .", "a series of reactions was set up to test this hypothesis . as shown in table 1 , use of either water or", "nmf resulted in formation of the 1,2-product ( 2 ) solely with no trace of 3 detected in the product mixture .", "although the yield of product was moderate to high in either solvent , product formation in reactions conducted in nmf proceeded at a much greater rate .", "we believe that there are two possible reasons behind the rate increase of coupling reactions carried out in nmf .", "the ratio of reagent was as follows : 1.0:1.5:1.1 ( aldehyde / halide / indium ) .", "the reaction was run at 0.1 m with respect to indium . in the lewis acid catalyzed reaction , 10 molar % of the lewis acid was used .", "all species completely dissolved in nmf , unlike what was observed for reactions in water . as such , we believe mixing is much more efficient , allowing the reaction to proceed at a faster rate .", "has also been noted in many literature examples to lead to rate enhancement in indium - promoted barbier reactions .", "we believe a mixture of better solubility coupled with greater solvent polarity results in an additive effect toward increasing the rate of reactions conducted in nmf when compared to water .", "h - bonding capabilities of nmf may explain the greater regioselectivity for reactions conducted in nmf when compared to those observed in dmf and thf ; however , the h - bonding capacity of nmf is much lower than that of water.(7d ) increased solvent polarity , thus increasing the stability of the intermediate anion and raising the relative energy of the oxy - cope rearrangement , may seem more likely at this time . in an effort to exclude the solvent as a direct participant in intermediate formation when nmf is used , nmr spectroscopy studies were performed to determine if a possible hemiaminal or imminium species was forming in situ ( figure 1 ) .", "the imminium functionality carries a positive charge and would increase the electrophilicity of the reactive carbon .", "mixing of 1 and nmf under sonication for periods of up to 24 h , however , revealed the presence of only 1 and nmf by nmr spectroscopy . possible formation of hemiaminal or imminium species . the presence or absence of a lewis acid has shown to help control stereoselectivity in indium - promoted cc bond - forming reactions .", "previous studies in our laboratory have shown that the addition of indium(iii ) chloride as a lewis acid favors formation of the syn - product in indium - promoted cc bond - forming reactions between 4 and aldehyde functional groups . in the absence of the lewis acid ,", "the anti - product is favored ( figure 2).(5b ) it was our hope that similar effects would be witnessed in the present system , especially while using nmf as the reaction solvent , and we were pleased to see this trend continue .", "absence of a lewis acid in the reagent mixture favored a nonchelated pathway , leading to formation of anti-2 .", "use of indium chloride to control stereochemistry.(5b ) the presence of a lewis acid in the reaction mixture favored a chelated pathway , leading to formation of syn-2 ( figure 3 ) .", "diastereoselectivities were determined either by direct measurement of vicinal coupling constants in the hydroxyl sulfide or by corresponding conversion to the epoxide compounds as shown in figure 4.(11 ) general j values for hydroxyl sulfides and epoxide structures .", "entries 1 , 5 , and 13 ( table 1 ) describe reactions run in nmf in the absence of a lewis acid .", "good anti - selectivity is witnessed with ratios as high as 15:85 ( syn / anti ) . in all cases , with", "the exception of r = phenyl , systems , with and without incl3 , run in nmf gave better selectivity than systems run in water .", "both systems showed poor selectivity when r = tms for reasons of which we are not quite sure . when incl3 was added as a lewis acid , the chelated route was favored in ratios as high as 90:10 ( syn : anti , entry 15 ) .", "n - methylformamide was shown to increase the rate of reaction compared with water and offered better stereocontrol in the systems studied .", "enediyne and epoxydiyne functionalities contain either a ( z)-3-ene-1,5-diyne or ( z)-3-epoxy-1,5-diyne unit which leads to their noted activity.(9 ) these unique functional groups cyclize to form a p - benzyne- or p - benzyne - like intermediate that interacts with dna by abstracting hydrogen.(9 ) the dna diradical then either couples with itself , or cleaves , stopping replication .", "natural product enediyne and epoxydiynes have found use as anticarcinogenic compounds.(9 ) this area of study is slowed , however , by complicated access into the skeletal systems required.(9 ) literature procedures that find use in epoxy alkyne formation such as halohydrin cylization,(12)m - cpba,(13 ) or oxone(14 ) have been shown to be successful , but limited examples are known with these systems at present . under the barbier coupling conditions described in this paper , formation of compound 2 offers an efficient and controlled method for the formation of either an enediyne or epoxydiyne skeletal structure under benign conditions by allowing easy conversion to either the cis - epoxydiyne ( from syn-2 , scheme 3 ) or the cis - enediyne ( from anti-2 , scheme 4 ) .", "facile synthesis of epoxydiyne and enediyne functional groups under the reported conditions would allow for their use as template structures in organic synthesis , opening access to more sophisticated units for further exploration . in the following pages , we describe the transformation of 1 into either an epoxydiyne ( 5 ) or enediyne ( 6 ) skeletal structure in two steps with high regio- and stereocontrol using nmf as a new solvent to form 2 under barbier conditions , followed by a one - step transformation to yield either 5 or 6 . as shown in scheme 3 , syn-2 can adopt the correct conformation to form a cis - oxirane upon reaction with either me3obf4 or tloet .", "conversion of the hydroxy sulfide starting material ( 2 ) to the epoxyalkyne product ( 5 ) was quite successful as shown in table 2 . in all cases ,", "this fact is worth mentioning as barbier reactions with propargyl halides can lead to rearrangement of the organometallic intermediate into an allenyl product .", "reagent 4 has been previously shown to offer one of the few examples of a barbier reaction with a propargyl halide where rearrangement does not occur.(5b ) use of tloet(17 ) gave yields similar to that of me3obf4(18 ) in all cases of oxirane formation , with stereoselectivity preserved under both sets of conditions .", "use of the anti-2 allows formation of the ( e)-alkene ( the alkyne functional groups are cis to each other ) functional group by the pathway shown in scheme 4 .", "formation of the enediyne product was possible under slightly acidic conditions . a catalytic amount of p - tsoh is added to anti-2 in a mixture of water and thf ( 10% water ) with stirring for 37 h. the reaction was also conducted under basic conditions after transformation of the oh group to a tosylate .", "yields were quite low under basic conditions , presumably due to the competing acidity of the alkynyl proton , leading to decomposition and side product formation . due to low yields ,", "results of the acid - catalyzed conversion of anti-2 to ( e)-6 are shown in table 3 .", "a slight amount of isomerization was witnessed under acidic conditions ( about 3% of the product mixture ) , but overall , good stereocontrol was realized in these conversions , allowing for easy entry into the cis - enediyne ( ( e)-alkene ) skeletal structure in a two - step sequence .", "the assignment of e- and z - isomers was made by comparison of the -vinyl proton shift .", "alignment of the -vinyl proton and the heteroatom cis to each other gives rise to a lower field proton resonance than trans alignment of these two groups ( figure 5).(19 ) the assignments for the enediyne products are compared correctly with the stereochemistry of compounds 2 and 3 within this study .", "coupled with the original indium - promoted barbier reaction , the transformation of starting materials 1 and 4 into enediyne or epoxydiyne compounds proceeded in two steps in an overall 6080% overall yield , offering an efficient , benign , and good - yielding formation of these important skeletal structures .", "the use of n - methylformamide is shown to be a nice alternative to aqueous conditions in indium promoted coupling reactions between propargyl aldehydes ( 1 ) and -chloropropargylphenyl sulfide ( 4 ) .", "this paper introduces the use of nmf as s viable solvent in a barbier coupling reaction , revealing increased rates and better selectivity when compared to reactions conducted in water and other organic solvents .", "the increased efficiency of the barbier coupling reaction as described in this paper allows for facile formation of epoxy - and enediyne skeletal structures in an overall two - step procedure .", "the introduction of nmf as an alternative solvent in barbier reactions will open new pathways to organic synthetic chemists . further studies to test other halides and aldehyde functional groups under barbier coupling reaction conditions in nmf are currently underway and will be reported in due time .", "tetrahydrofuran was purified by distillation under an argon atmosphere over sodium and benzophenone prior to use .", "radial chromatography was performed using a chromatatron . to a solution of 30 mmol of alkyne in 80 ml of thf that had been cooled to  60 c under nitrogen", "was added 15 ml of n - buli ( 2.0 m in thf ) with stirring . to this solution", "was added n , n - dimethylformamide ( 4.66 ml , 60 mmol ) dropwise over 10 min .", "after 20 min of further stirring , the solution was added to a mixture of 75 ml of tert - butyl methyl ether and 160 ml of 10% kh2po4 solution which had been previously cooled to 0 c .", "the entire solution was stirred at 0 c for 30 min and then allowed to warm to room temperature .", "the organic layer was dried over mgso4 , and the solvent removed under vacuum to give a clear oil .", "all propargyl aldehydes were used without further purification and could be stored at 0 c for 23 weeks .", "yields for this reaction ranged from 85 to 95% . a magnetically stirred solution of aldehyde ( 1 ) ( 2.0 mmol ) in deionized water ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) .", "the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) .", "the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2  10 ml ) .", "the combined organic layers were dried over mgso4 and evaporated to leave a dark yellow to brown oil .", "purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil .", "purification was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 471 mg ( 1.76 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 )  0.91.5 ( m , 7h ) , 1.82 ( d , j = 1.7 hz , 0.2h ) , 1.87 ( d , j = 1.6 hz , 0.8h ) , 2.13 ( t , j = 6.5 hz , 2h ) , 4.09 ( m , 1h ) , 4.86 ( m , 1h ) , 7.17.8 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  15.5 , 18.2 , 21.3 , 34.2 , ( 41.5 , 42.9 ) , ( 65.0 , 66.1 ) , 71.3 , 78.5 , 80.2 , 84.7 , 125.4 , 126.1 , 126.3 , 127.6 , 128.0 , 136.4 ; ms m / z ( m ) calcd 258.1078 , obsd 258.0998 .", "calcd for c16h18os : c , 74.38 ; h , 7.02 . found : c , 74.61 ; h , 6.99 .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : total yield = 489 mg ( 1.78 mmol ) = 80% .", "anti isomer : yield = 391 mg ( 1.41 mmol ) ; h nmr ( 300 mhz , cdcl3 )  1.94 ( d , j = 1.8 hz , 1h ) , 4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  43.8 , 66.0 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 .", "h nmr ( 300 mhz , cdcl3 )  1.83 ( d , j = 1.8 hz , 1h ) , 3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  44.1 , 65.8 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 50:50 syn / anti ratio : total yield = 418 mg ( 1.54 mmol ) = 70% ; anti isomer : yield = 209 mg ( 7.7 mmol ) ; h nmr ( 300 mhz , cdcl3 )  0.24 ( s , 9h ) , 1.84 ( d , j = 1.6 hz , 1h ) , 4.23 ( dd , j = 1.6 , 5.3 hz , 1h ) , 4.80 ( d , j = 5.3 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  0.28(3 ) , 44.4 , 66.5 , 67.5 , 74.1 , 84.3 , 86.4 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 ; ms m / z ( m ) calcd 274.0848 , obsd 274.0819 .", "calcd for c15h18ossi : c , 65.64 ; h , 6.61 . found : c , 65.17 ; h , 6.55 .", "syn isomer : yield = 209 mg ( 7.7 mmol ) = 70% ; h nmr ( 300 mhz , cdcl3 )  0.24 ( s , 9h ) , 1.79 ( d , j = 1.7 hz , 1h ) , 4.15 ( dd , j = 1.7 , 7.8 hz , 1h ) , 4.69 ( d , j = 7.8 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  0.28(3 ) , 44.1 , 67.9 , 68.1 , 75.0 , 83.2 , 86.9 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides : yield = 633 mg ( 1.69 mmol ) = 77% ; h nmr ( 300 mhz , cdcl3 )  0.25 ( s , 6h ) , 0.97 ( s , 9h ) , 1.25 ( m , 2h ) ,", "1.802.00 ( m , 3h ) , 3.83 ( t , j = 6.5 , 2h ) , 4.11 ( m , 1h ) , 4.99 ( m , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  0.28 ( 2 ) , 14.5 , 15.1 , 21.8 ( 2 ) , 21.9 33.4 , 47.0 , 64.6 , 65.1 , 67.9 , 75.2 , 81.9 , 87.3 , 125.4 , 126.6 ( 2 ) , 127.1 , 128.4 , 135.9 ; ms m / z ( m+ ) calcd 374.1736 , obsd 365.1633 ( loss of h2o ) .", "the general procedure was followed as described in section i.a with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used .", "separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 437 mg ( 1.63 mmol ) = 77% .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 75:25 syn / anti ratio : yield = total = 458 mg ( 1.65 mmol ) = 75% .", "anti isomer : yield = 115 mg ( 0.41 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  1.94 ( d , j = 1.8 hz , 1h ) , 4.12 ( dd , j = 1,8 , 4.5 hz ,", "syn isomer : yield = 343 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  1.83 ( d , j = 1.8 hz , 1h ) , 3.92 ( dd , j = 1.8 , 8.0 hz , 1h ) , 4.94 ( d , j = 8.1 hz , 1h ) processing and analysis of this reaction proceeded as described in section i.b . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 60:40 syn / anti ratio : yield = total = 434 mg ( 1.58 mmol ) = 72% .", "anti isomer : yield = 174 mg ( 0.63 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) .", "syn isomer : yield = 260 mg ( 0.95 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield 583 mg ( 1.55 mmol ) = 71% .", "analysis as shown in section i.a.4 . a magnetically stirred solution of aldehyde 1 ( 2.0 mmol ) in nmf ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) .", "the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) .", "the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2  10 ml ) .", "the combined organic layers were washed with 1 n hcl ( 3  20 ml ) , dried over mgso4 , and evaporated to leave a dark yellow to brown oil .", "purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil .", "separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 522 mg ( 2.02 mmol ) = 92% .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : yield = total = 556 mg ( 2.00 mmol ) = 91% .", "anti isomer : yield = 445 mg ( 1.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) .", "syn isomer : yield = 111 mg ( 0.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 40:60 syn / anti ratio : yield = total = 566 mg ( 2.06 mmol ) = 94% .", "anti isomer : yield = 340 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) .", "syn isomer : yield = 226 mg ( 0.82 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 717 mg ( 1.91 mmol ) = 87% .", "the general procedure was followed as described in section i.c with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used .", "separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 527 mg ( 2.05 mmol ) = 93% .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 85:15 syn / anti ratio : yield = total = 568 mg ( 2.05 mmol ) = 93% .", "anti isomer : yield = 85 mg ( 0.31 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.13 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.08 ( d , j = 4.7 hz , 1h ) .", "syn isomer : yield = 483 mg ( 1.74 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  3.92 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.97 ( d , j = 8.1 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 70:30 syn / anti ratio : yield = total = 548 mg ( 2.00 mmol ) = 91% .", "anti isomer : : yield = 164 mg ( 0.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.23 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.80 ( d , j = 5.5 hz , 1h ) .", "syn isomer : yield = 384 mg ( 1.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.68 ( d , j = 7.8 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 732 mg ( 1.96 mmol ) = 89% .", "a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 100 mg , 0.39 mmol ) in dichloromethane ( 10 ml ) was treated with trimethyloxonium tetrafluoroborate ( 90 mg ( 0.60 mmol ) .", "the solution was stirred at room temperature for 8 h and diluted with 7% sodium hydroxide solution ( aqueous , 8 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 80:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 85:15 syn / anti ratio : yield = 52 mg ( 0.35 mmol ) = 90% ; h nmr ( 300 mhz , cdcl3 )  0.9  1.5 ( m , 7h ) , 1.82 ( d , j = 1.8 hz , 0.15h ) , 1.86 ( d , j = 1.9 hz , 0.85h ) , 2.17 ( m , 2h ) , 3.25 ( d , j = 4.6 hz , 0.85h ) , 3.40 ( d , j = 2.3 hz , 0.15h ) , 3.50 ( dd , j = 1.8 , 2.3 hz , 0.15h ) , 3.61 ( dd , j = 1.9 , 4.6 hz , 0.85h ) ; c nmr ( 75 mhz , cdcl3 )  ( 14.0 , 14.1 ) , ( 17.2 , 17.4 ) , 21.6 , ( 31.7 , 31.8 ) , 44.5 , ( 48.2 , 48.3 ) , 66.7 , ( 77.5 , 77.9 ) , ( 79.5 , 79.7 ) , ( 85.2 , 85.5 )", "calcd for c10h12o : c , 81.04 ; h , 8.16 . found : c , 80.99 ; h , 8.19 .", "a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) .", "the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 85:15 syn / anti ratio : yield = total = 110 mg ( 0.64 mmol ) = 89% .", "anti isomer : yield = 12 mg ( 0.07 mmol ) ; h nmr ( 300 mhz , cdcl3 )  2.44 ( d , j = 1.8 hz , 1h ) , 3.51 ( dd , j = 1.8 , 2.4 hz , 1h ) , 3.97 ( d , j = 2.4 hz , 1h ) , 7.157.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  48.2 , 53.1 , 66.2 , 80.3 , 87.3 , 91.7 , 123.5 , 128.4 ( 2 ) , 129.0 , 133.1 ( 2 ) .", "calcd for c12h8o = c , 85.69 h , 4.79 . found : c , 86.00 h , 4.71 .", "syn isomer : yield = 98 mg ( 0.57 mmol ) ; h nmr ( 300 mhz , cdcl3 )  2.43 ( d , j = 1.8 hz , 1h ) , 3.46 ( dd , j = 1.8 , 4.7 hz , 1h ) , 4.15 ( d , j = 4.7 hz , 1h ) , 7.157.23 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  48.7 , 53.1 , 66.5 , 80.1 , 87.3 , 92.0 , 123.0 , 128.3 ( 2 ) , 128.7 , 132.3(2 ) . a solution of the hydroxy sulfide mixture ( 70:30 syn / anti ) ( 200 mg 0.73 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) .", "the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 70:30 syn / anti ratio : yield = total = 108 mg ( 0.66 mmol ) = 90% ; anti isomer : yield = 32 mg ( 0.30 mmol ) ; h nmr ( 300 mhz , cdcl3 )  0.13 ( s , 9h ) ; 2.21 ( d , j = 2.0 hz , 1h ) , 3.25 ( dd , j = 2.0 , 2.3 hz , 1h ) , 3.55 ( d , j = 2.3 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 )  0.10 ( 3 ) , 44.9 , 49.9 , 69.9 , 81.7 , 86.0 , 102.0 ; ms m / z ( m ) calcd = 164.0657 , obsd = 164.0656 . syn isomer : yield = 76 mg ( 0.46 mmol ) ; h nmr ( 300 mhz , cdcl3 )  0.13 ( s , 9h ) , 2.19 ( d , j = 2.0 hz , 1h ) , 3.32 ( dd , j = 2.0 , 4.9 hz , 1h ) , 3.63 ( d , j = 4.9 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 )  0.10 ( 3 ) , 45.1 , 49.3 , 70.1 , 81.5 , 85.6 , 101.2 . a solution of the hydroxy sulfide mixture ( 90:10 syn / anti ) ( 250 mg 0.67 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 150 mg ( 1.00 mmol ) .", "the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 12 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) to give a mixture of two diastereomers as a colorless oil in a 90:10 syn / anti ratio : yield = 148 mg ( 0.56 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 )  0.22 ( s , 6h ) ; 0.97 ( s , 9h ) , 1.27 ( m , 2h ) , 1.82.0 ( m , 3h ) , 3.23 ( d , j = 4.4 hz , 0.9h ) , 3.31 ( d , j = 2.5 hz , 0.1h ) , 3.53 ( dd , j = 1.9 , 2.5 hz , 0.1 hz ) , 3.69 ( dd , j = 1.9 , 4.4 hz , 0.9h ) , 3.83 ( m , 2h ) ; c nmr ( 75 mhz , cdcl3 )  0.28 ( 2 ) , 14.5 , 14.7 , 21.8 ( 2 ) , ( 22.0 , 22.3 ) , ( 33.9 , 34.2 ) , ( 43.8 , 44.0 ) , ( 47.1 , 47.9 ) , ( 64.7 , 65.0 ) , 67.3 , ( 78.7 , 79.1 ) , 81.8 , ( 85.4 , 85.8 ) ; ms m / z ( m ) calcd = 264.1546 , obsd = 264.1545", "calcd for c15h24osi : c , 68.13 ; h , 9.15 . found : c , 67.79 ; h , 9.4 .", "a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in chloroform ( 20 ml ) was treated with tloet ( 234 mg , 0.93 mmol ) .", "after 20 min of stirring , the insolubles were removed by filtration through a short celite pad . the resulting organic solution was washed with a saturated nahco3 solution ( aq ) and a saturated nacl solution ( aq ) . the organic layer was dried and concentrated .", "purification and analysis of products proceeded in the manner previously described ( section ii.a ) .", "subsequent hydroxy sulfides were treated in a fashion similar to that in ii.b.1 and analyzed in the manner previously described in section ii.a .", "a solution of the hydroxy sulfide mixture ( 25:75 syn / anti ) ( 100 mg , 0.39 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) .", "after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 73:27 cis / trans ratio : yield = 82 mg ( 0.34 mmol ) = 87% ; h nmr ( 300 mhz , cdcl3 )  0.901.51 ( m , 7h ) , 2.07 ( m , 2h ) , 2.63 ( s , 0.27h ) , 2.71 ( s , 0.73h ) , 5.90 ( s , 0.73h ) , 6.21 ( s , 0.27h ) 7.107.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  ( 13.9 , 14.1 ) , 17.1 , 22.9 , 31.6 , 77.2 , ( 79.5 , 80.3 ) , ( 83.1 , 83.7 ) , ( 90.5 , 91.7 ) , ( 113.3 , 113.9 ) , 125.1 , 128.4(2 ) , 129.3(2 ) , 134.0 , ( 145.9 , 146.2 ) ; ms m / z ( m ) calcd 240.0973 , obsd 240.0973 .", "calcd for c16h16s : c , 79.95 ; h , 6.71 . found : c , 80.09 ; h , 6.60 .", "a solution of the hydroxy sulfide ( 200 mg 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : ield = 156 mg ( 0.60 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 )  0.2.95 ( s , 1h ) , 6.01 ( s , 1h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 )  81.7 , 82.9 , 89.3 , 92.6 , 114.0 , 122.0 , 125.7 , 128.1 ( 2 ) , 128.3 , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , 134.0 , 144.6 .", "a solution of the hydroxy sulfide mixture ( 20:80 syn / anti ) ( 200 mg , 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 77:23 e / z ratio : yield = 160 mg ( 0.61 mmol ) = 85% ; h nmr ( 300 mhz , cdcl3 )  0.2.95 ( s , 0.77h ) , 3.01 ( s , 0.23h ) , 6.04 ( s , 0.77h ) , 6.35 ( s , 0.27h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 )  ( 81.5 , 81.7 ) , 83.0 , 89.3 , ( 92.6 , 93.0 ) , ( 114.0 , 119.2 ) , 122.0 , 125.7 , 128.1 ( 2 ) , ( 128.3 , 128.5 ) , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , ( 134.0 , 134.5 ) , ( 144.6 , 145.1 ) . a solution of the hydroxy sulfide ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : yield = 123 mg ( 0.0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 )  0.0.11 ( s , 9h ) , 2.87 ( s , 1h ) , 6.20 ( s , 1h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  0.09 ( 3 ) , 78.7 , 81.2 , 95.3 , 100.9 , 112.5 , 124.8 , 127.2(2 ) , 128.7(2 ) , 131.8 , 142.1 .", "calcd for c15h16ssi : c , 70.25 ; h , 6.29 . found : c , 69.99 ; h , 6.43 .", "a solution of the hydroxy sulfide mixture ( 40:60 syn / anti ) ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 50:50 e / z ratio : yield = 120 mg ( 0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 )  0.09 ( s , 4.5h ) 0.10 ( s , 4.5h ) 2.81 ( s , 0.5h ) , 2.87 ( s , 0.5h ) , 5.77 ( s , 0.5h ) , 6.20 ( s , 0.5h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  0.09 ( 3 ) , ( 78.7 , 79.1 ) , ( 81.2 , 81.8 ) , ( 95.3 , 95.9 ) , 100.9 , ( 112.5 , 116.1 ) , 124.8 , 127.2(2 ) , 128.7(2 ) , ( 131.8 , 132.9 ) , ( 142.1 , 143.5 ) .", "a solution of the hydroxy sulfide mixture ( 15:85 syn / anti ) ( 250 mg 0.67 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) .", "after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 10:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 82:18 cis / trans ratio : yield = 192 mg ( 0.54 mmol ) = 80% ; h nmr ( 300 mhz , cdcl3 )  0.15 ( s , 6h ) , 0.93 ( s , 9h ) , 1.27 ( m , 2h ) , 2.11(m , 2h ) , 2.71 ( s , 0.82h ) , 2.80 ( s , 0.18h ) , 3.75 ( m , 2h ) , 5.93 ( s , 0.82h ) , 6.16 ( s , 0.18h ) , 7.077.26 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  0.13(2 ) , ( 13.9 , 14.1 ) , ( 15.0 , 15.1 ) , 21.3(3 ) , 33.9 , ( 64.9 , 65.3 ) , ( 77.3 , 78.1 ) , ( 79.7 , 80.1 ) , ( 83.2 , 84.1 ) , ( 93.1 , 93.7 ) , ( 114.1 , 115.0 ) , 125.4 , 127.7(2 ) , 129.1 , 129.3 , ( 133.7 , 134.0 ) , ( 144.9 , 145.4 ) ; ms m / z ( m ) calcd 356.1630 , obsd 355.9891", "calcd for c21h28ossi : c , 70.73 ; h , 7.91 . found : c , 70.81 ; h , 8.01 ."], "abstract_text": ["<S> indium - promoted coupling reactions between propargyl aldehydes ( 1 ) and -chloropropargylphenyl sulfide are reported . </S>", "<S> although water has been shown to accelerate indium metal promoted reactions , the reverse pattern was observed in this series . </S>", "<S> use of n - methylformamide ( nmf ) , which has not previously been a solvent known for use in indium - promoted reactions , afforded an acceleration of these barbier - style reactions compared to water . </S>", "<S> indium - promoted reactions in this study also showed excellent regiocontrol and good stereocontrol , allowing for easy entry into the formation of epoxydiyne and enediyne skeletal structures . </S>", "<S> this paper also describes use of the barbier coupled product ( 2 ) as a new , and easy , entry into the formation of enediyne and epoxydiyne skeletal structures . </S>"], "labels": null, "section_names": ["Indroduction", "Results and Discussion", "Conclusion", "Experimental Section"], "sections": [["many studies over the past 15 years have focused on discovering solvents that help promote efficient cc bond formation under benign reaction conditions .", "new solvents in dielsalder(2 ) and barbier reactions have been sought to help streamline the one - pot transformations further , reducing synthetic steps and organic waste .", "polar media often accelerate reactions of this type which has led to the use of water as an acceptable solvent for such organic conversions .", "recent contributions from our laboratory to this field have centered on coupling reactions between propargyl aldehydes and allyl or propargyl halides under barbier conditions to form homoallylic and homopropargylic propargyl alcohols .", "these compounds find wide use as synthetic templates and show a propensity for oxy - cope rearrangements ( scheme 1 ) .", "the use of -chloropropargylphenyl sulfide ( 4 ) , coupled with a propargyl aldehyde should offer an easy route into formation of enediyne and epoxydiyne skeletal structures ( scheme 2 ) .", "previous studies revealed that formation of 2 or 3 under indium - promoted barbier conditions is solvent dependent .", "use of an organic solvent for the coupling reaction results in a mixture of 2 and 3 while the use of water as a solvent yields 2 as the sole product .", "it is possible that water protonates the coupled product to form 2 quickly , quenching the alkoxide intermediate , thereby suppressing the possible oxy - cope pathway shown in scheme 1 .", "it may also be possible that the increased polarity of the solvent itself stabilizes the anion formed upon coupling , thus raising the relative rate of cope rearrangement in this system . with a less polar , aprotic solvent ,", "the anion will not be quenched , nor will it be stabilized to the same extent as witnessed in water .", "a combination of these phenomena may be why an oxy - cope rearrangement occurs under less polar conditions , forming product 3 ( scheme 1 ) .", "a coupling reaction between 1 and 4 ( scheme 2 ) was conducted under aqueous conditions to form 2 ( y = sph ) , with good regioselectivity ; however , the rate of this reaction is surprisingly slow .", "although reactions of the type shown in scheme 1 are reported in the literature to be accelerated by polar solvents such as water , coupling reactions between 1 and 4 , as shown in scheme 2 , are quite slow . indeed ,", "when water is used as the solvent , reactions often require 1036 h for completion .", "this oil has a density greater than that of water so it is presumed to be an organometallic species formed by interaction of the indium metal with the halide species .", "isolation of the oil followed by workup in an acidic thf / h2o mixture leads to isolation of propargylphenyl sulfide and , to a smaller extent , the aldehyde .", "we surmise that low solubility of this organometallic complex leads to poor reagent mixing , slowing the barbier coupling under aqueous conditions .", "second , indium - promoted coupling reactions conducted in water become more acidic as the reaction proceeds .", "we believe it is possible that the aldehyde is in equilibrium with its corresponding hydrate species , reducing the electrophilicity of the carbon center .", "a combination of these two reasons would lead to a decrease in the rate of reaction between 1 and 4 when conducted in an aqueous solvent .", "the reaction proceeds more quickly ( 812 h ) in dmf , a less polar solvent , due to better solubility of all reagents , however , a mixture of 2 and 3 was isolated .", "it was originally expected that a mixture of water and dmf would lead to faster reaction times while still maintaining regiocontrol of product formation . the use of a water / dmf solvent system , however , resulted in a mixture of products with water constituting up to 50% v / v of the solvent system .", "when water exceeds 50% v / v of the solvent system , only product 2 is isolated , but the rate of reaction slows perceptively once again . to help expedite this reaction sequence ,", "we began a search for solvent with polarity similar to , or greater than that of water .", "it was expected that an extremely polar organic solvent would promote the same type of acceleration normally witnessed in barbier couplings carried out under aqueous conditions .", "an organic solvent could also help solubilize the intermediate and reduce possible formation of the hydrate species , thus allowing for a faster coupling reaction to occur .", "if increased polarity stabilizes the anion formed by coupling of 1 and 4 , oxy - cope rearrangement should also be suppressed . with a dielectric constant of 186.9 at 25 c , significantly greater than that of water ( 78.37(7 ) ) , and dmf ( 38.3(7b ) ) ,", "n - methylformamide ( nmf ) has found occasional use in previous reactions requiring polar organic solvents ; however , it has historically found more widespread use as an organic reagent . with a pka of approximately 24,(7d )", "nmf is reactive under a range of organometallic reagent conditions , limiting its use as a solvent .", "indium - promoted coupling reactions , however , have been shown to be accelerated under aqueous conditions,(1 ) and the relative acidity of nmf , being higher than that of water , should not hinder the proposed barbier couplings proposed within this paper .", "we were especially intrigued by the fact that the dielectric constant of nmf increases to 220 as the temperature is lowered from 25 to 0 c .", "we were interested in examining what effect the increase in dielectric constant at lower temperature could have on a barbier - style reaction of the type shown in scheme 2 among others .", "this paper reports on the use of nmf in barbier - style reactions with a focus on rates of reaction , stereochemistry , and its use to form enediyne and epoxydiyne skeletal structures.(9 )"], ["although nmf has characteristics similar to those of dmf , it also has characteristics similar to water , such as a polar xh bond.(7 ) thus , having found a very polar solvent with properties similar to both water and organic systems , we hypothesized that the 1,2-coupling product could be formed in high yield under short reaction times .", "a series of reactions was set up to test this hypothesis . as shown in table 1 , use of either water or", "nmf resulted in formation of the 1,2-product ( 2 ) solely with no trace of 3 detected in the product mixture .", "although the yield of product was moderate to high in either solvent , product formation in reactions conducted in nmf proceeded at a much greater rate .", "we believe that there are two possible reasons behind the rate increase of coupling reactions carried out in nmf .", "the ratio of reagent was as follows : 1.0:1.5:1.1 ( aldehyde / halide / indium ) .", "the reaction was run at 0.1 m with respect to indium . in the lewis acid catalyzed reaction , 10 molar % of the lewis acid was used .", "all species completely dissolved in nmf , unlike what was observed for reactions in water . as such , we believe mixing is much more efficient , allowing the reaction to proceed at a faster rate .", "has also been noted in many literature examples to lead to rate enhancement in indium - promoted barbier reactions .", "we believe a mixture of better solubility coupled with greater solvent polarity results in an additive effect toward increasing the rate of reactions conducted in nmf when compared to water .", "h - bonding capabilities of nmf may explain the greater regioselectivity for reactions conducted in nmf when compared to those observed in dmf and thf ; however , the h - bonding capacity of nmf is much lower than that of water.(7d ) increased solvent polarity , thus increasing the stability of the intermediate anion and raising the relative energy of the oxy - cope rearrangement , may seem more likely at this time . in an effort to exclude the solvent as a direct participant in intermediate formation when nmf is used , nmr spectroscopy studies were performed to determine if a possible hemiaminal or imminium species was forming in situ ( figure 1 ) .", "the imminium functionality carries a positive charge and would increase the electrophilicity of the reactive carbon .", "mixing of 1 and nmf under sonication for periods of up to 24 h , however , revealed the presence of only 1 and nmf by nmr spectroscopy . possible formation of hemiaminal or imminium species . the presence or absence of a lewis acid has shown to help control stereoselectivity in indium - promoted cc bond - forming reactions .", "previous studies in our laboratory have shown that the addition of indium(iii ) chloride as a lewis acid favors formation of the syn - product in indium - promoted cc bond - forming reactions between 4 and aldehyde functional groups . in the absence of the lewis acid ,", "the anti - product is favored ( figure 2).(5b ) it was our hope that similar effects would be witnessed in the present system , especially while using nmf as the reaction solvent , and we were pleased to see this trend continue .", "absence of a lewis acid in the reagent mixture favored a nonchelated pathway , leading to formation of anti-2 .", "use of indium chloride to control stereochemistry.(5b ) the presence of a lewis acid in the reaction mixture favored a chelated pathway , leading to formation of syn-2 ( figure 3 ) .", "diastereoselectivities were determined either by direct measurement of vicinal coupling constants in the hydroxyl sulfide or by corresponding conversion to the epoxide compounds as shown in figure 4.(11 ) general j values for hydroxyl sulfides and epoxide structures .", "entries 1 , 5 , and 13 ( table 1 ) describe reactions run in nmf in the absence of a lewis acid .", "good anti - selectivity is witnessed with ratios as high as 15:85 ( syn / anti ) . in all cases , with", "the exception of r = phenyl , systems , with and without incl3 , run in nmf gave better selectivity than systems run in water .", "both systems showed poor selectivity when r = tms for reasons of which we are not quite sure . when incl3 was added as a lewis acid , the chelated route was favored in ratios as high as 90:10 ( syn : anti , entry 15 ) .", "n - methylformamide was shown to increase the rate of reaction compared with water and offered better stereocontrol in the systems studied .", "enediyne and epoxydiyne functionalities contain either a ( z)-3-ene-1,5-diyne or ( z)-3-epoxy-1,5-diyne unit which leads to their noted activity.(9 ) these unique functional groups cyclize to form a p - benzyne- or p - benzyne - like intermediate that interacts with dna by abstracting hydrogen.(9 ) the dna diradical then either couples with itself , or cleaves , stopping replication .", "natural product enediyne and epoxydiynes have found use as anticarcinogenic compounds.(9 ) this area of study is slowed , however , by complicated access into the skeletal systems required.(9 ) literature procedures that find use in epoxy alkyne formation such as halohydrin cylization,(12)m - cpba,(13 ) or oxone(14 ) have been shown to be successful , but limited examples are known with these systems at present . under the barbier coupling conditions described in this paper , formation of compound 2 offers an efficient and controlled method for the formation of either an enediyne or epoxydiyne skeletal structure under benign conditions by allowing easy conversion to either the cis - epoxydiyne ( from syn-2 , scheme 3 ) or the cis - enediyne ( from anti-2 , scheme 4 ) .", "facile synthesis of epoxydiyne and enediyne functional groups under the reported conditions would allow for their use as template structures in organic synthesis , opening access to more sophisticated units for further exploration . in the following pages , we describe the transformation of 1 into either an epoxydiyne ( 5 ) or enediyne ( 6 ) skeletal structure in two steps with high regio- and stereocontrol using nmf as a new solvent to form 2 under barbier conditions , followed by a one - step transformation to yield either 5 or 6 . as shown in scheme 3 , syn-2 can adopt the correct conformation to form a cis - oxirane upon reaction with either me3obf4 or tloet .", "conversion of the hydroxy sulfide starting material ( 2 ) to the epoxyalkyne product ( 5 ) was quite successful as shown in table 2 . in all cases ,", "this fact is worth mentioning as barbier reactions with propargyl halides can lead to rearrangement of the organometallic intermediate into an allenyl product .", "reagent 4 has been previously shown to offer one of the few examples of a barbier reaction with a propargyl halide where rearrangement does not occur.(5b ) use of tloet(17 ) gave yields similar to that of me3obf4(18 ) in all cases of oxirane formation , with stereoselectivity preserved under both sets of conditions .", "use of the anti-2 allows formation of the ( e)-alkene ( the alkyne functional groups are cis to each other ) functional group by the pathway shown in scheme 4 .", "formation of the enediyne product was possible under slightly acidic conditions . a catalytic amount of p - tsoh is added to anti-2 in a mixture of water and thf ( 10% water ) with stirring for 37 h. the reaction was also conducted under basic conditions after transformation of the oh group to a tosylate .", "yields were quite low under basic conditions , presumably due to the competing acidity of the alkynyl proton , leading to decomposition and side product formation . due to low yields ,", "results of the acid - catalyzed conversion of anti-2 to ( e)-6 are shown in table 3 .", "a slight amount of isomerization was witnessed under acidic conditions ( about 3% of the product mixture ) , but overall , good stereocontrol was realized in these conversions , allowing for easy entry into the cis - enediyne ( ( e)-alkene ) skeletal structure in a two - step sequence .", "the assignment of e- and z - isomers was made by comparison of the -vinyl proton shift .", "alignment of the -vinyl proton and the heteroatom cis to each other gives rise to a lower field proton resonance than trans alignment of these two groups ( figure 5).(19 ) the assignments for the enediyne products are compared correctly with the stereochemistry of compounds 2 and 3 within this study .", "coupled with the original indium - promoted barbier reaction , the transformation of starting materials 1 and 4 into enediyne or epoxydiyne compounds proceeded in two steps in an overall 6080% overall yield , offering an efficient , benign , and good - yielding formation of these important skeletal structures ."], ["the use of n - methylformamide is shown to be a nice alternative to aqueous conditions in indium promoted coupling reactions between propargyl aldehydes ( 1 ) and -chloropropargylphenyl sulfide ( 4 ) .", "this paper introduces the use of nmf as s viable solvent in a barbier coupling reaction , revealing increased rates and better selectivity when compared to reactions conducted in water and other organic solvents .", "the increased efficiency of the barbier coupling reaction as described in this paper allows for facile formation of epoxy - and enediyne skeletal structures in an overall two - step procedure .", "the introduction of nmf as an alternative solvent in barbier reactions will open new pathways to organic synthetic chemists . further studies to test other halides and aldehyde functional groups under barbier coupling reaction conditions in nmf are currently underway and will be reported in due time ."], ["tetrahydrofuran was purified by distillation under an argon atmosphere over sodium and benzophenone prior to use .", "radial chromatography was performed using a chromatatron . to a solution of 30 mmol of alkyne in 80 ml of thf that had been cooled to  60 c under nitrogen", "was added 15 ml of n - buli ( 2.0 m in thf ) with stirring . to this solution", "was added n , n - dimethylformamide ( 4.66 ml , 60 mmol ) dropwise over 10 min .", "after 20 min of further stirring , the solution was added to a mixture of 75 ml of tert - butyl methyl ether and 160 ml of 10% kh2po4 solution which had been previously cooled to 0 c .", "the entire solution was stirred at 0 c for 30 min and then allowed to warm to room temperature .", "the organic layer was dried over mgso4 , and the solvent removed under vacuum to give a clear oil .", "all propargyl aldehydes were used without further purification and could be stored at 0 c for 23 weeks .", "yields for this reaction ranged from 85 to 95% . a magnetically stirred solution of aldehyde ( 1 ) ( 2.0 mmol ) in deionized water ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) .", "the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) .", "the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2  10 ml ) .", "the combined organic layers were dried over mgso4 and evaporated to leave a dark yellow to brown oil .", "purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil .", "purification was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 471 mg ( 1.76 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 )  0.91.5 ( m , 7h ) , 1.82 ( d , j = 1.7 hz , 0.2h ) , 1.87 ( d , j = 1.6 hz , 0.8h ) , 2.13 ( t , j = 6.5 hz , 2h ) , 4.09 ( m , 1h ) , 4.86 ( m , 1h ) , 7.17.8 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  15.5 , 18.2 , 21.3 , 34.2 , ( 41.5 , 42.9 ) , ( 65.0 , 66.1 ) , 71.3 , 78.5 , 80.2 , 84.7 , 125.4 , 126.1 , 126.3 , 127.6 , 128.0 , 136.4 ; ms m / z ( m ) calcd 258.1078 , obsd 258.0998 .", "calcd for c16h18os : c , 74.38 ; h , 7.02 . found : c , 74.61 ; h , 6.99 .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : total yield = 489 mg ( 1.78 mmol ) = 80% .", "anti isomer : yield = 391 mg ( 1.41 mmol ) ; h nmr ( 300 mhz , cdcl3 )  1.94 ( d , j = 1.8 hz , 1h ) , 4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  43.8 , 66.0 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 .", "h nmr ( 300 mhz , cdcl3 )  1.83 ( d , j = 1.8 hz , 1h ) , 3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  44.1 , 65.8 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 50:50 syn / anti ratio : total yield = 418 mg ( 1.54 mmol ) = 70% ; anti isomer : yield = 209 mg ( 7.7 mmol ) ; h nmr ( 300 mhz , cdcl3 )  0.24 ( s , 9h ) , 1.84 ( d , j = 1.6 hz , 1h ) , 4.23 ( dd , j = 1.6 , 5.3 hz , 1h ) , 4.80 ( d , j = 5.3 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  0.28(3 ) , 44.4 , 66.5 , 67.5 , 74.1 , 84.3 , 86.4 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 ; ms m / z ( m ) calcd 274.0848 , obsd 274.0819 .", "calcd for c15h18ossi : c , 65.64 ; h , 6.61 . found : c , 65.17 ; h , 6.55 .", "syn isomer : yield = 209 mg ( 7.7 mmol ) = 70% ; h nmr ( 300 mhz , cdcl3 )  0.24 ( s , 9h ) , 1.79 ( d , j = 1.7 hz , 1h ) , 4.15 ( dd , j = 1.7 , 7.8 hz , 1h ) , 4.69 ( d , j = 7.8 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  0.28(3 ) , 44.1 , 67.9 , 68.1 , 75.0 , 83.2 , 86.9 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides : yield = 633 mg ( 1.69 mmol ) = 77% ; h nmr ( 300 mhz , cdcl3 )  0.25 ( s , 6h ) , 0.97 ( s , 9h ) , 1.25 ( m , 2h ) ,", "1.802.00 ( m , 3h ) , 3.83 ( t , j = 6.5 , 2h ) , 4.11 ( m , 1h ) , 4.99 ( m , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 )  0.28 ( 2 ) , 14.5 , 15.1 , 21.8 ( 2 ) , 21.9 33.4 , 47.0 , 64.6 , 65.1 , 67.9 , 75.2 , 81.9 , 87.3 , 125.4 , 126.6 ( 2 ) , 127.1 , 128.4 , 135.9 ; ms m / z ( m+ ) calcd 374.1736 , obsd 365.1633 ( loss of h2o ) .", "the general procedure was followed as described in section i.a with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used .", "separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 437 mg ( 1.63 mmol ) = 77% .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 75:25 syn / anti ratio : yield = total = 458 mg ( 1.65 mmol ) = 75% .", "anti isomer : yield = 115 mg ( 0.41 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  1.94 ( d , j = 1.8 hz , 1h ) , 4.12 ( dd , j = 1,8 , 4.5 hz ,", "syn isomer : yield = 343 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  1.83 ( d , j = 1.8 hz , 1h ) , 3.92 ( dd , j = 1.8 , 8.0 hz , 1h ) , 4.94 ( d , j = 8.1 hz , 1h ) processing and analysis of this reaction proceeded as described in section i.b . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 60:40 syn / anti ratio : yield = total = 434 mg ( 1.58 mmol ) = 72% .", "anti isomer : yield = 174 mg ( 0.63 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) .", "syn isomer : yield = 260 mg ( 0.95 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield 583 mg ( 1.55 mmol ) = 71% .", "analysis as shown in section i.a.4 . a magnetically stirred solution of aldehyde 1 ( 2.0 mmol ) in nmf ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) .", "the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) .", "the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2  10 ml ) .", "the combined organic layers were washed with 1 n hcl ( 3  20 ml ) , dried over mgso4 , and evaporated to leave a dark yellow to brown oil .", "purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil .", "separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 522 mg ( 2.02 mmol ) = 92% .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : yield = total = 556 mg ( 2.00 mmol ) = 91% .", "anti isomer : yield = 445 mg ( 1.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) .", "syn isomer : yield = 111 mg ( 0.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 40:60 syn / anti ratio : yield = total = 566 mg ( 2.06 mmol ) = 94% .", "anti isomer : yield = 340 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) .", "syn isomer : yield = 226 mg ( 0.82 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 717 mg ( 1.91 mmol ) = 87% .", "the general procedure was followed as described in section i.c with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used .", "separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 527 mg ( 2.05 mmol ) = 93% .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 85:15 syn / anti ratio : yield = total = 568 mg ( 2.05 mmol ) = 93% .", "anti isomer : yield = 85 mg ( 0.31 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.13 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.08 ( d , j = 4.7 hz , 1h ) .", "syn isomer : yield = 483 mg ( 1.74 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  3.92 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.97 ( d , j = 8.1 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 70:30 syn / anti ratio : yield = total = 548 mg ( 2.00 mmol ) = 91% .", "anti isomer : : yield = 164 mg ( 0.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.23 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.80 ( d , j = 5.5 hz , 1h ) .", "syn isomer : yield = 384 mg ( 1.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 )  4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.68 ( d , j = 7.8 hz , 1h ) .", "separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .", "stereoselectivity was determined upon transformation to the epoxide product : yield = 732 mg ( 1.96 mmol ) = 89% .", "a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 100 mg , 0.39 mmol ) in dichloromethane ( 10 ml ) was treated with trimethyloxonium tetrafluoroborate ( 90 mg ( 0.60 mmol ) .", "the solution was stirred at room temperature for 8 h and diluted with 7% sodium hydroxide solution ( aqueous , 8 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 80:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 85:15 syn / anti ratio : yield = 52 mg ( 0.35 mmol ) = 90% ; h nmr ( 300 mhz , cdcl3 )  0.9  1.5 ( m , 7h ) , 1.82 ( d , j = 1.8 hz , 0.15h ) , 1.86 ( d , j = 1.9 hz , 0.85h ) , 2.17 ( m , 2h ) , 3.25 ( d , j = 4.6 hz , 0.85h ) , 3.40 ( d , j = 2.3 hz , 0.15h ) , 3.50 ( dd , j = 1.8 , 2.3 hz , 0.15h ) , 3.61 ( dd , j = 1.9 , 4.6 hz , 0.85h ) ; c nmr ( 75 mhz , cdcl3 )  ( 14.0 , 14.1 ) , ( 17.2 , 17.4 ) , 21.6 , ( 31.7 , 31.8 ) , 44.5 , ( 48.2 , 48.3 ) , 66.7 , ( 77.5 , 77.9 ) , ( 79.5 , 79.7 ) , ( 85.2 , 85.5 )", "calcd for c10h12o : c , 81.04 ; h , 8.16 . found : c , 80.99 ; h , 8.19 .", "a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) .", "the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 85:15 syn / anti ratio : yield = total = 110 mg ( 0.64 mmol ) = 89% .", "anti isomer : yield = 12 mg ( 0.07 mmol ) ; h nmr ( 300 mhz , cdcl3 )  2.44 ( d , j = 1.8 hz , 1h ) , 3.51 ( dd , j = 1.8 , 2.4 hz , 1h ) , 3.97 ( d , j = 2.4 hz , 1h ) , 7.157.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  48.2 , 53.1 , 66.2 , 80.3 , 87.3 , 91.7 , 123.5 , 128.4 ( 2 ) , 129.0 , 133.1 ( 2 ) .", "calcd for c12h8o = c , 85.69 h , 4.79 . found : c , 86.00 h , 4.71 .", "syn isomer : yield = 98 mg ( 0.57 mmol ) ; h nmr ( 300 mhz , cdcl3 )  2.43 ( d , j = 1.8 hz , 1h ) , 3.46 ( dd , j = 1.8 , 4.7 hz , 1h ) , 4.15 ( d , j = 4.7 hz , 1h ) , 7.157.23 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  48.7 , 53.1 , 66.5 , 80.1 , 87.3 , 92.0 , 123.0 , 128.3 ( 2 ) , 128.7 , 132.3(2 ) . a solution of the hydroxy sulfide mixture ( 70:30 syn / anti ) ( 200 mg 0.73 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) .", "the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 70:30 syn / anti ratio : yield = total = 108 mg ( 0.66 mmol ) = 90% ; anti isomer : yield = 32 mg ( 0.30 mmol ) ; h nmr ( 300 mhz , cdcl3 )  0.13 ( s , 9h ) ; 2.21 ( d , j = 2.0 hz , 1h ) , 3.25 ( dd , j = 2.0 , 2.3 hz , 1h ) , 3.55 ( d , j = 2.3 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 )  0.10 ( 3 ) , 44.9 , 49.9 , 69.9 , 81.7 , 86.0 , 102.0 ; ms m / z ( m ) calcd = 164.0657 , obsd = 164.0656 . syn isomer : yield = 76 mg ( 0.46 mmol ) ; h nmr ( 300 mhz , cdcl3 )  0.13 ( s , 9h ) , 2.19 ( d , j = 2.0 hz , 1h ) , 3.32 ( dd , j = 2.0 , 4.9 hz , 1h ) , 3.63 ( d , j = 4.9 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 )  0.10 ( 3 ) , 45.1 , 49.3 , 70.1 , 81.5 , 85.6 , 101.2 . a solution of the hydroxy sulfide mixture ( 90:10 syn / anti ) ( 250 mg 0.67 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 150 mg ( 1.00 mmol ) .", "the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 12 ml ) .", "purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) to give a mixture of two diastereomers as a colorless oil in a 90:10 syn / anti ratio : yield = 148 mg ( 0.56 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 )  0.22 ( s , 6h ) ; 0.97 ( s , 9h ) , 1.27 ( m , 2h ) , 1.82.0 ( m , 3h ) , 3.23 ( d , j = 4.4 hz , 0.9h ) , 3.31 ( d , j = 2.5 hz , 0.1h ) , 3.53 ( dd , j = 1.9 , 2.5 hz , 0.1 hz ) , 3.69 ( dd , j = 1.9 , 4.4 hz , 0.9h ) , 3.83 ( m , 2h ) ; c nmr ( 75 mhz , cdcl3 )  0.28 ( 2 ) , 14.5 , 14.7 , 21.8 ( 2 ) , ( 22.0 , 22.3 ) , ( 33.9 , 34.2 ) , ( 43.8 , 44.0 ) , ( 47.1 , 47.9 ) , ( 64.7 , 65.0 ) , 67.3 , ( 78.7 , 79.1 ) , 81.8 , ( 85.4 , 85.8 ) ; ms m / z ( m ) calcd = 264.1546 , obsd = 264.1545", "calcd for c15h24osi : c , 68.13 ; h , 9.15 . found : c , 67.79 ; h , 9.4 .", "a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in chloroform ( 20 ml ) was treated with tloet ( 234 mg , 0.93 mmol ) .", "after 20 min of stirring , the insolubles were removed by filtration through a short celite pad . the resulting organic solution was washed with a saturated nahco3 solution ( aq ) and a saturated nacl solution ( aq ) . the organic layer was dried and concentrated .", "purification and analysis of products proceeded in the manner previously described ( section ii.a ) .", "subsequent hydroxy sulfides were treated in a fashion similar to that in ii.b.1 and analyzed in the manner previously described in section ii.a .", "a solution of the hydroxy sulfide mixture ( 25:75 syn / anti ) ( 100 mg , 0.39 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) .", "after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 73:27 cis / trans ratio : yield = 82 mg ( 0.34 mmol ) = 87% ; h nmr ( 300 mhz , cdcl3 )  0.901.51 ( m , 7h ) , 2.07 ( m , 2h ) , 2.63 ( s , 0.27h ) , 2.71 ( s , 0.73h ) , 5.90 ( s , 0.73h ) , 6.21 ( s , 0.27h ) 7.107.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  ( 13.9 , 14.1 ) , 17.1 , 22.9 , 31.6 , 77.2 , ( 79.5 , 80.3 ) , ( 83.1 , 83.7 ) , ( 90.5 , 91.7 ) , ( 113.3 , 113.9 ) , 125.1 , 128.4(2 ) , 129.3(2 ) , 134.0 , ( 145.9 , 146.2 ) ; ms m / z ( m ) calcd 240.0973 , obsd 240.0973 .", "calcd for c16h16s : c , 79.95 ; h , 6.71 . found : c , 80.09 ; h , 6.60 .", "a solution of the hydroxy sulfide ( 200 mg 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : ield = 156 mg ( 0.60 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 )  0.2.95 ( s , 1h ) , 6.01 ( s , 1h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 )  81.7 , 82.9 , 89.3 , 92.6 , 114.0 , 122.0 , 125.7 , 128.1 ( 2 ) , 128.3 , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , 134.0 , 144.6 .", "a solution of the hydroxy sulfide mixture ( 20:80 syn / anti ) ( 200 mg , 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 77:23 e / z ratio : yield = 160 mg ( 0.61 mmol ) = 85% ; h nmr ( 300 mhz , cdcl3 )  0.2.95 ( s , 0.77h ) , 3.01 ( s , 0.23h ) , 6.04 ( s , 0.77h ) , 6.35 ( s , 0.27h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 )  ( 81.5 , 81.7 ) , 83.0 , 89.3 , ( 92.6 , 93.0 ) , ( 114.0 , 119.2 ) , 122.0 , 125.7 , 128.1 ( 2 ) , ( 128.3 , 128.5 ) , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , ( 134.0 , 134.5 ) , ( 144.6 , 145.1 ) . a solution of the hydroxy sulfide ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : yield = 123 mg ( 0.0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 )  0.0.11 ( s , 9h ) , 2.87 ( s , 1h ) , 6.20 ( s , 1h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  0.09 ( 3 ) , 78.7 , 81.2 , 95.3 , 100.9 , 112.5 , 124.8 , 127.2(2 ) , 128.7(2 ) , 131.8 , 142.1 .", "calcd for c15h16ssi : c , 70.25 ; h , 6.29 . found : c , 69.99 ; h , 6.43 .", "a solution of the hydroxy sulfide mixture ( 40:60 syn / anti ) ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .", "after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 50:50 e / z ratio : yield = 120 mg ( 0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 )  0.09 ( s , 4.5h ) 0.10 ( s , 4.5h ) 2.81 ( s , 0.5h ) , 2.87 ( s , 0.5h ) , 5.77 ( s , 0.5h ) , 6.20 ( s , 0.5h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  0.09 ( 3 ) , ( 78.7 , 79.1 ) , ( 81.2 , 81.8 ) , ( 95.3 , 95.9 ) , 100.9 , ( 112.5 , 116.1 ) , 124.8 , 127.2(2 ) , 128.7(2 ) , ( 131.8 , 132.9 ) , ( 142.1 , 143.5 ) .", "a solution of the hydroxy sulfide mixture ( 15:85 syn / anti ) ( 250 mg 0.67 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .", "the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) .", "after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min .", "the organic layers were combined , washed with 3  10 ml of water , dried over mgso4 , and concentrated .", "purification was accomplished by radial chromatography on silica gel ( elution with 10:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 82:18 cis / trans ratio : yield = 192 mg ( 0.54 mmol ) = 80% ; h nmr ( 300 mhz , cdcl3 )  0.15 ( s , 6h ) , 0.93 ( s , 9h ) , 1.27 ( m , 2h ) , 2.11(m , 2h ) , 2.71 ( s , 0.82h ) , 2.80 ( s , 0.18h ) , 3.75 ( m , 2h ) , 5.93 ( s , 0.82h ) , 6.16 ( s , 0.18h ) , 7.077.26 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 )  0.13(2 ) , ( 13.9 , 14.1 ) , ( 15.0 , 15.1 ) , 21.3(3 ) , 33.9 , ( 64.9 , 65.3 ) , ( 77.3 , 78.1 ) , ( 79.7 , 80.1 ) , ( 83.2 , 84.1 ) , ( 93.1 , 93.7 ) , ( 114.1 , 115.0 ) , 125.4 , 127.7(2 ) , 129.1 , 129.3 , ( 133.7 , 134.0 ) , ( 144.9 , 145.4 ) ; ms m / z ( m ) calcd 356.1630 , obsd 355.9891", "calcd for c21h28ossi : c , 70.73 ; h , 7.91 . found : c , 70.81 ; h , 8.01 ."]]}
{"article_id": "PMC5207012", "article_text": ["sepsis is the systemic inflammation caused by infection with bacteria , viruses , fungi , parasites , or toxic products , and has become one of the leading causes of mortality in children . in developed countries ,", "the mortality rate of pediatric sepsis is about 210% and is as high as 50% in developing countries .", "pediatric sepsis usually leads to tachycardia , tachypnea , peripheral vasodilation , fever or hypothermia , and even multiple organ dysfunction syndrome ( mods ) .", "frequently - used applications in pediatric sepsis treatment concentrate on maintaining metabolic , circulatory , and respiratory stabilities , and antimicrobial agents , as well as surgical and adjunctive therapies such as nitric oxide and corticosteroids , which show great benefits in the outcome of pediatric sepsis treatment .", "acute kidney injury ( aki ) is a severe consequence of sepsis and mods , and about 1527% of aki in children is generated from pediatric sepsis .", "the pathophysiology of septic aki is related to the altered global renal blood flow , as well as the comprehensive effects of inflammation , microvasculature blood flow , and bioenergetics factors .", "for example , glomerular endothelial injury during aki progression in a lipopolysaccharide - induced mouse sepsis model is closely related to tumor necrosis factor and receptor 1 . due to the increased risk of mortality caused by septic aki , it is imperative to explore effective therapies for controlling aki in pediatric sepsis .", "baicalin is one of the flavonoid constituents in scutellaria baicalensis georgi , a kind of perennial herb with protective effects against diseases like hydrogen peroxide - induced oxidative stress and cerebral ischemia injury [ 911 ] .", "specifically , baicalin has been revealed to protect against oxidants , anxiety , acute hepatic injury , experimental periodontitis , and sepsis .", "it also alleviates reperfusion - induced damage to the myocardium , the mechanism of which may be associated with its anti - apoptotic roles . moreover", ", a recent study found that baicalin can inhibit inflammation and cell apoptosis , which allows it to protect against ischemia - reperfusion injuries in the kidney .", "however , few studies have investigated the role of baicalin in septic aki or pediatric sepsis .", "this study aimed to uncover the effects of baicalin in treating aki in pediatric sepsis patients .", "the baicalin adjunctive therapy was tested in pediatric sepsis patients , after which the effect was compared based on the renal function assessment from bun and serum creatinine ( cr ) .", "we also performed experiments in the cecal ligation and puncture ( clp)-induced mouse sepsis model to investigate the effects and potential mechanisms of baicalin in septic aki .", "this study lays the foundation for future application of baicalin in adjunctive therapies for aki in pediatric sepsis .", "a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 .", "all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) .", "the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure .", "the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients .", "no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) .", "patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions .", "the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital .", "these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital .", "clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report .", "thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin .", "the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity .", "then the cecum was relocated to the abdominal cavity , and the abdomen was closed .", "the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) .", "mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling .", "cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions .", "briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices .", "fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark .", "then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min .", "the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min .", "slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) .", "total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions .", "dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse ,", "1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) . then the synthesized complementary dna ( 20 ng )", "was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) .", "data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control .", "protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions .", "the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes .", "the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c .", "after washing in pbs 5 times , the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature .", "ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals .", "comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) .", "a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 .", "all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) .", "the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure .", "the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients .", "no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) .", "patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions .", "the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital .", "these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital .", "clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report .", "thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin .", "the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity .", "then the cecum was relocated to the abdominal cavity , and the abdomen was closed .", "the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) .", "mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling .", "cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions .", "briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices .", "fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark .", "then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min .", "the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min .", "slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) .", "total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions .", "dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse ,", "1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) .", "then the synthesized complementary dna ( 20 ng ) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) .", "data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control .", "protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions .", "the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes .", "the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c .", "gapdh ( ab181602 ) was used as an internal control . after washing in pbs 5 times ,", "the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature .", "ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals .", "comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) .", "in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies .", "results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) .", "it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy .", "we next performed experiments in the mouse model to analyze the function of baicalin in septic aki .", "mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp . both bun and cr levels", "were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model .", "the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis .", "tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) .", "thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model .", "bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis .", "qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) .", "bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) .", "protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2", "are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model .", "in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies .", "results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) .", "it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy .", "we next performed experiments in the mouse model to analyze the function of baicalin in septic aki .", "mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp .", "both bun and cr levels were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model .", "the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis .", "tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) .", "thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model .", "bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis .", "qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) .", "bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) .", "protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2", "are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model .", "this study performed investigations in pediatric sepsis patients and in a clp - induced mouse sepsis model to reveal the role of baicalin in aki of pediatric sepsis .", "baicalin adjunctive therapy in the patients and treatment in the mouse model decreased bun and cr levels .", "further histological and expression analyses suggested the suppressive function of baicalin in renal cell apoptosis .", "the 50 pediatric patients participating in this study were chosen from pediatric sepsis patients diagnosed with aki , and the mouse model induced by clp was detected with elevated bun and cr levels , which suggested potential kidney injury .", "previous studies using mouse sepsis models used various baicalin treatment methods , including topical application on the mouse skin to analyze the effect of baicalin on epidermis , intraperitoneal injection to investigate the role of baicalin in mammary glands , and baicalin - containing diets to study liver injury , inflammation in the kidney , and lung carcinoma [ 22,2729 ] . in these studies ,", "the dose of baicalin was usually 100200 mg/(kgd ) , and the duration ranged from 3 to 50 days .", "thus , in the present study we chose to perform baicalin treatment in the model mouse via intragastric administration of 200 mg/(kgd ) for 6 days , and the significantly elevated bun and cr levels in the clp group compared to the sham group indicated that this treatment method effectively induced kidney injury in the mouse sepsis model .", "clinical trials with baicalin adjunctive therapy have focused on its protective roles in ulcerative colitis , hepatic fibrosis , and diabetes mellitus [ 3032 ] , and several studies have investigated the potential of baicalin in treating patients with early diabetic nephropathy , in which baicalin inhibits aldose reductase activity and reduces urinary albumin excretion rate and the blood 2-microglobulin .", "the present study further discovered the protective role of baicalin against aki in pediatric sepsis patients , which was reflected in the reduced bun and cr levels .", "moreover , the experiments in the mouse model confirmed that baicalin treatment in a sepsis animal model could improve renal functions .", "these findings show the potential of using baicalin to attenuate aki in pediatric sepsis patients , but the proper dosage and possible complications need to be assessed in future research .", "tunel results showed that baicalin treatment in the mouse model significantly suppressed renal cell apoptosis , and western blot and qrt - pcr analyses indicated consistent changes in the expression of bax and bcl2 .", "a similar mechanism has been reported in mouse mammary glands , where baicalin inhibits cell apoptosis via regulating bax and bcl2 .", "in addition , baicalin inhibits hepatocyte apoptosis in mouse kidney injury induced by concanavalin a ; therefore , suppressing renal cell apoptosis is likely to be one of the mechanisms by which baicalin attenuates septic kidney injury .", "however , the anti - proliferative role of baicalin has also been reported in studies on lung cancer and mouse embryonic stem cells , and it is converted to baicalin by intestinal gut flora to act as a pro - apoptotic and anti - proliferative substance .", "baicalin is capable of promoting neuronal differentiation , and thus may benefit therapies for parkinson and alzheimer diseases .", "we speculate that there are multiple potential mechanisms behind the effects of baicalin in various diseases ; therefore , it is necessary to uncover the detailed mechanisms in order to optimize use of baicalin to control aki in pediatric sepsis patients .", "the investigations of baicalin in pediatric sepsis patients and the clp - induced mouse sepsis model reveal the protective role of baicalin against aki in pediatric sepsis .", "baicalin can inhibit renal cell apoptosis , which may be one of its functional mechanisms .", "this study provides a potential adjunctive therapy for treating aki in pediatric sepsis , and future research will concentrate on more detailed mechanisms of baicalin ."], "abstract_text": ["<S> backgroundpediatric sepsis has high morbidity in children , may lead to acute kidney injury ( aki ) , and further aggravate the disease . </S>", "<S> baicalin is a kind of flavonoid in scutellaria baicalensis georgi and has been reported to protect against several diseases , but its roles in septic aki remain unclear . </S>", "<S> this study aimed to uncover the effects of baicalin in aki during pediatric sepsis.material/methodsblood urea nitrogen ( bun ) and serum creatinine ( cr ) levels were detected in 50 pediatric patients , who underwent basic therapy with or without baicalin adjunctive therapy . </S>", "<S> mouse sepsis models were constructed by cecal ligation and puncture ( clp ) and treated with baicalin intragastrically , after which bun and cr examination , tunel apoptosis assay , and expression analyses of bax and bcl2 were performed.resultsbaicalin adjunctive therapy significantly decreased bun and cr levels in pediatric sepsis patients ( p<0.05 ) . </S>", "<S> clp led to elevated bun and cr levels in the mouse model ( p<0.01 ) , indicating kidney injury accompanied by sepsis . </S>", "<S> baicalin decreased bun and cr levels ( p<0.05 ) , and reduced the apoptotic cell percent in the renal tissue ( p<0.05 ) of the clp model . </S>", "<S> it inhibited bax and promoted bcl2 in the renal tissue , which was consistent with cell apoptosis changes.conclusionsbaicalin is capable of suppressing renal cell apoptosis and protecting against aki in pediatric sepsis . </S>", "<S> this study provides a potential adjunctive therapy for treating aki in pediatric sepsis , and further research is necessary to reveal its deeper mechanisms . </S>"], "labels": null, "section_names": ["Background", "Material and methods", "Sample collection", "Animal model", "TUNEL assay", "qRT-PCR", "Western blot", "Statistical analysis", "Results", "Baicalin attenuates AKI in pediatric sepsis patients", "Baicalin improves renal functions in the CLP-induced mouse model", "Baicalin inhibits renal cell apoptosis", "Discussion", "Conclusions"], "sections": [["sepsis is the systemic inflammation caused by infection with bacteria , viruses , fungi , parasites , or toxic products , and has become one of the leading causes of mortality in children . in developed countries ,", "the mortality rate of pediatric sepsis is about 210% and is as high as 50% in developing countries .", "pediatric sepsis usually leads to tachycardia , tachypnea , peripheral vasodilation , fever or hypothermia , and even multiple organ dysfunction syndrome ( mods ) .", "frequently - used applications in pediatric sepsis treatment concentrate on maintaining metabolic , circulatory , and respiratory stabilities , and antimicrobial agents , as well as surgical and adjunctive therapies such as nitric oxide and corticosteroids , which show great benefits in the outcome of pediatric sepsis treatment .", "acute kidney injury ( aki ) is a severe consequence of sepsis and mods , and about 1527% of aki in children is generated from pediatric sepsis .", "the pathophysiology of septic aki is related to the altered global renal blood flow , as well as the comprehensive effects of inflammation , microvasculature blood flow , and bioenergetics factors .", "for example , glomerular endothelial injury during aki progression in a lipopolysaccharide - induced mouse sepsis model is closely related to tumor necrosis factor and receptor 1 . due to the increased risk of mortality caused by septic aki , it is imperative to explore effective therapies for controlling aki in pediatric sepsis .", "baicalin is one of the flavonoid constituents in scutellaria baicalensis georgi , a kind of perennial herb with protective effects against diseases like hydrogen peroxide - induced oxidative stress and cerebral ischemia injury [ 911 ] .", "specifically , baicalin has been revealed to protect against oxidants , anxiety , acute hepatic injury , experimental periodontitis , and sepsis .", "it also alleviates reperfusion - induced damage to the myocardium , the mechanism of which may be associated with its anti - apoptotic roles . moreover", ", a recent study found that baicalin can inhibit inflammation and cell apoptosis , which allows it to protect against ischemia - reperfusion injuries in the kidney .", "however , few studies have investigated the role of baicalin in septic aki or pediatric sepsis .", "this study aimed to uncover the effects of baicalin in treating aki in pediatric sepsis patients .", "the baicalin adjunctive therapy was tested in pediatric sepsis patients , after which the effect was compared based on the renal function assessment from bun and serum creatinine ( cr ) .", "we also performed experiments in the cecal ligation and puncture ( clp)-induced mouse sepsis model to investigate the effects and potential mechanisms of baicalin in septic aki .", "this study lays the foundation for future application of baicalin in adjunctive therapies for aki in pediatric sepsis ."], ["a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 .", "all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) .", "the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure .", "the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients .", "no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) .", "patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions .", "the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital .", "these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital .", "clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report .", "thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin .", "the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity .", "then the cecum was relocated to the abdominal cavity , and the abdomen was closed .", "the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) .", "mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling .", "cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions .", "briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices .", "fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark .", "then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min .", "the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min .", "slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) .", "total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions .", "dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse ,", "1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) . then the synthesized complementary dna ( 20 ng )", "was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) .", "data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control .", "protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions .", "the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes .", "the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c .", "after washing in pbs 5 times , the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature .", "ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals .", "comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) ."], ["a total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 .", "all 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005 , and with aki at the same time according to the criteria of aki proposed by acute kidney injury net ( akin ) .", "the following conditions were excluded : patients with kidney injuries caused by prerenal or postrenal factors , and patients with a history of renal diseases such as acute or chronic renal failure .", "the 50 patients were randomly divided into 2 groups : control and baicalin , each group containing 25 patients .", "no significant difference existed in the age between the 2 groups ( 2.580.17 and 2.460.20 ) .", "patients in the control group received basic treatment , and those in the baicalin group received basic treatment plus oral baicalin ( inner chengzi pharmaceutical , chifeng , china ) according to the manufacturer s instructions .", "the blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital .", "these procedures were approved by our local ethics committee and were performed according to the instructions of the hospital ."], ["clp was performed on male c57bl/6 mice ( spf , age 810 weeks , weight 2226 g ) ( vital river laboratories , beijing , china ) to induce a septic aki model based on the method in a previous report .", "thirty individuals were randomly divided into 3 groups : sham , clp , and clp + baicalin .", "the cecum was ligated at 1 cm to the distal end , and double punctures were made to extrude feces into the abdominal cavity .", "then the cecum was relocated to the abdominal cavity , and the abdomen was closed .", "the mice were resuscitated by intraperitoneal injection of 0.9% saline ( 24 ml / kg ) .", "mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd ) ( pureone biotechnology , shanghai , china ) for 6 days , after which all the mice were anesthetized and killed for blood and renal tissue sampling ."], ["cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling ( tunel ) method with the in situ cell death detection kit , pod ( roche , basel , switzerland ) according to the manufacturer s instructions .", "briefly , the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices .", "fresh tunel mix ( 50 l ) was added to the slices for 1-h incubation at 37c in the dark .", "then , 50 l of converter - pod was added for incubation of 30 min at 37c in the dark , after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min .", "the slices were washed in phosphate - buffered saline ( pbs ) 3 times between steps , each time lasting 5 min .", "slices were then dehydrated , mounted , and observed with an optical microscope ( olympus , tokyo , japan ) ."], ["total rna samples of the mouse renal tissue were extracted by trizol ( invitrogen , carlsbad , ca ) according to the manufacturer s instructions .", "dna contamination was removed by dnase i ( invitrogen ) and then the quality and quantity of rna samples were examined by nanodrop 2000 ( thermo scientific , carlsbad , ca ) . for each mouse ,", "1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase ( invitrogen ) .", "then the synthesized complementary dna ( 20 ng ) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system ( applied biosystems , carlsbad , ca ) with the specific primers for mouse bax ( fw : 5-tgttt gctga tggca acttc-3 and rv : 5-gatgg ttctg atcag ctcgg-3 ) and bcl2 ( fw : 5-gtaga agagg gtgtg acagc-3 and rv : 5-agcac tgact ctggg atcgc-3 ) .", "data were analyzed by the 2 method with gapdh ( fw : 5-tcaac agcaa ctccc actct tcca-3 and rv : 5-accct gttgc tgtag ccgta ttca-3 ) as an internal control ."], ["protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent ( thermo scientific ) and quantified by bio - rad protein assay ( bio - rad , hercules , ca ) according to the manufacturers instructions .", "the protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes .", "the blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 ( ab32503 and ab32124 , abcam , cambridge , uk ) overnight at 4c .", "gapdh ( ab181602 ) was used as an internal control . after washing in pbs 5 times ,", "the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody ( ab6721 ) for 1 h at room temperature .", "ecl plus western blotting substrate ( thermo scientific ) was used to develop positive signals ."], ["comparison between groups was performed with one - way analysis of variance and t test in spss 20 ( ibm , new york , ny ) ."], ["in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies .", "results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) .", "it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy .", "we next performed experiments in the mouse model to analyze the function of baicalin in septic aki .", "mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp . both bun and cr levels", "were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model .", "the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis .", "tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) .", "thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model .", "bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis .", "qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) .", "bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) .", "protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2", "are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model ."], ["in the treatment of 50 pediatric sepsis patients , 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies .", "results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies ( p=0.2842 and 0.2135 , figure 1a , 1b ) , while those of the baicalin group were significantly decreased after baicalin adjunctive therapy ( p=0.0191 and 0.0077 ) .", "it could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved , possibly due to the baicalin adjunctive therapy ."], ["we next performed experiments in the mouse model to analyze the function of baicalin in septic aki .", "mice in the clp group showed significantly elevated bun and cr levels ( p=0.0001 and 0.0013 , figure 2a , 2b ) , suggesting that the kidney injury occurred after clp .", "both bun and cr levels were significantly decreased in the clp + baicalin group ( p=0.0181 and 0.0225 ) , indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model ."], ["the possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis .", "tunel assay showed more apoptotic cells in the clp group compared to the sham group , and baicalin treatment reduced apoptotic cell numbers ( figure 3a ) , with significant differences between groups ( p=0.0087 and 0.0359 , figure 3b ) .", "thus , baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model .", "bax and bcl2 , 2 factors indicating cell apoptosis , were detected at both mrna and protein levels to verify the changes in renal cell apoptosis .", "qrt - pcr results showed that bax mrna level was significantly elevated in the clp group ( p=0.0108 ) and then was down - regulated by baicalin treatment ( p=0.0256 , figure 4a ) .", "bcl2 mrna level showed the opposite changing pattern to bax , with significant differences between groups ( p=0.0004 and 0.0069 , figure 4b ) .", "protein level changes of the 2 factors were consistent with their mrnas , as shown by western blot analysis ( figure 4c ) . since up - regulated bax and down - regulated bcl2", "are usually correlated with promoted cell apoptosis , these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model ."], ["this study performed investigations in pediatric sepsis patients and in a clp - induced mouse sepsis model to reveal the role of baicalin in aki of pediatric sepsis .", "baicalin adjunctive therapy in the patients and treatment in the mouse model decreased bun and cr levels .", "further histological and expression analyses suggested the suppressive function of baicalin in renal cell apoptosis .", "the 50 pediatric patients participating in this study were chosen from pediatric sepsis patients diagnosed with aki , and the mouse model induced by clp was detected with elevated bun and cr levels , which suggested potential kidney injury .", "previous studies using mouse sepsis models used various baicalin treatment methods , including topical application on the mouse skin to analyze the effect of baicalin on epidermis , intraperitoneal injection to investigate the role of baicalin in mammary glands , and baicalin - containing diets to study liver injury , inflammation in the kidney , and lung carcinoma [ 22,2729 ] . in these studies ,", "the dose of baicalin was usually 100200 mg/(kgd ) , and the duration ranged from 3 to 50 days .", "thus , in the present study we chose to perform baicalin treatment in the model mouse via intragastric administration of 200 mg/(kgd ) for 6 days , and the significantly elevated bun and cr levels in the clp group compared to the sham group indicated that this treatment method effectively induced kidney injury in the mouse sepsis model .", "clinical trials with baicalin adjunctive therapy have focused on its protective roles in ulcerative colitis , hepatic fibrosis , and diabetes mellitus [ 3032 ] , and several studies have investigated the potential of baicalin in treating patients with early diabetic nephropathy , in which baicalin inhibits aldose reductase activity and reduces urinary albumin excretion rate and the blood 2-microglobulin .", "the present study further discovered the protective role of baicalin against aki in pediatric sepsis patients , which was reflected in the reduced bun and cr levels .", "moreover , the experiments in the mouse model confirmed that baicalin treatment in a sepsis animal model could improve renal functions .", "these findings show the potential of using baicalin to attenuate aki in pediatric sepsis patients , but the proper dosage and possible complications need to be assessed in future research .", "tunel results showed that baicalin treatment in the mouse model significantly suppressed renal cell apoptosis , and western blot and qrt - pcr analyses indicated consistent changes in the expression of bax and bcl2 .", "a similar mechanism has been reported in mouse mammary glands , where baicalin inhibits cell apoptosis via regulating bax and bcl2 .", "in addition , baicalin inhibits hepatocyte apoptosis in mouse kidney injury induced by concanavalin a ; therefore , suppressing renal cell apoptosis is likely to be one of the mechanisms by which baicalin attenuates septic kidney injury .", "however , the anti - proliferative role of baicalin has also been reported in studies on lung cancer and mouse embryonic stem cells , and it is converted to baicalin by intestinal gut flora to act as a pro - apoptotic and anti - proliferative substance .", "baicalin is capable of promoting neuronal differentiation , and thus may benefit therapies for parkinson and alzheimer diseases .", "we speculate that there are multiple potential mechanisms behind the effects of baicalin in various diseases ; therefore , it is necessary to uncover the detailed mechanisms in order to optimize use of baicalin to control aki in pediatric sepsis patients ."], ["the investigations of baicalin in pediatric sepsis patients and the clp - induced mouse sepsis model reveal the protective role of baicalin against aki in pediatric sepsis .", "baicalin can inhibit renal cell apoptosis , which may be one of its functional mechanisms .", "this study provides a potential adjunctive therapy for treating aki in pediatric sepsis , and future research will concentrate on more detailed mechanisms of baicalin ."]]}
{"article_id": "PMC2902753", "article_text": ["    the main cause of treatment failure and death in cancer patients is metastasis  the formation of secondary tumors in organs distant from the original neoplastic cell tissue .", "adjuvant therapy of proven efficacy is not currently available for cancer patients ; therefore , the search for new targets of therapeutic reagents is required to prevent both proliferation and metastases . during metastasis ,", "it is known that the mode of invasion is one of the markers of the malignancy and prognosis of cancer .", "the receptor for advanced glycation end - products ( rage ) , a multiligand member of the immunoglobulin superfamily of cell surface molecules , interacts with distinct molecules implicated in homeostasis , development , and inflammation .", "rage binding by ligands such as advanced glycation end - products ( ages ) triggers the activation of key cell signaling pathways , thereby reprogramming cellular properties .", "in addition , several reports have suggested that rage is associated with cancer malignancy [ 4 , 5 ] .     ", "the advanced stage of the glycation process ( one of the posttranslational modifications of proteins ) leads to the formation of ages and plays an important role in the pathogenesis of angiopathy in diabetic patients , aging , and neurodegenerative diseases [ 69 ] .", "a growing body of evidence suggests that the interaction of glyceraldehyde - derived ages ( glycer - ages ) , but not glucose - derived ages ( glc - ages ) , with rage elicits oxidative stress generation in numerous types of cells ( vascular wall cells , mesangial cells , schwann cells , and cortical neurons ) , all of which could contribute to the pathological changes seen in diabetic vascular complications of alzheimer 's disease [ 1013 ] .", "we have recently found that glycer - ages stimulated the growth and migration of cultured human melanoma cells and that anti - rage antibodies inhibited the tumor formation and lung metastasis of melanoma cell xenografts and subsequently improved survival in athymic mice .", "however , the effects of glycer - ages on other cancer cells remains poorly understood , and the molecular mechanisms behind their effects have not been clarified .      in the present study , we examined the effects of glycer - ages on cultured human lung adenocarcinoma a549 cells and showed that glycer - ages enhanced their malignancy rather than their proliferation .", "n - acetyl - l - cysteine ( nac ) was purchased from sigma - aldrich ( st . louis , mo , usa ) .", "briefly , 25  mg / ml of bsa ( a0281 , sigma - aldrich ) were incubated at 37c for 7 days under sterile conditions with 0.1  m glyceraldehyde ( ga ; nakalai tesque , kyoto , japan ) and 5  mm diethylenetriamine - pentaacetic acid ( dojindo , kumamoto , japan ) in 0.2  m phosphate buffer ( ph 7.4 ) .", "the modified albumin was then purified by pd-10 column ( ge healthcare , buckinghamshire , england ) chromatography and dialysis against phosphate - buffered saline ( pbs ) .", "control unglycated bsa was incubated under the same conditions except for the absence of glyceraldehyde as a negative control .", "protein concentrations were determined with the dc protein assay reagent ( bio - rad , richmond , ca , usa ) using bsa as a standard .", "the preparations were tested for endotoxin using the pyrotell - t test ( seikagaku bio - business , tokyo , japan ) , but no endotoxin was detected .", "human lung adenocarcinoma a549 and hepatocellular carcinoma hep3b cells were grown in dulbecco 's modified eagle 's medium ( dmem ; sigma - aldrich ) supplemented with 10% fetal bovine serum ( fbs ; equitech - bio , kerrville , tx , usa ) under standard cell culture conditions ( humidified atmosphere , 5% co2 , 37c ) .", "yamagishi , and its mock vector - transfected hep3b cells were maintained in 10% fbs / dmem in the presence of 700  g / ml g418 ( roche , mannheim , germany ) .", "the cells were washed with ice - cold ca and mg free pbs ( pbs ( - ) ) and subjected to lysis buffer ( 1% tritonx-100/nonidet p-40 , 10  mm sodium fluoride , 1  mm sodium orthovanadate , 5  mm sodium pyrophosphate , 2  mm egta , 5  mm edta , and 1 protease inhibitor cocktail ( complete , mini ; roche ) ) .", "subsequently , the cell lysates were incubated on ice for 5 min and centrifuged at 10,000  g for 10  min at 4c .", "their protein concentrations were then measured using the bradford assay ( bio - rad ) .", "cell lysates ( 30  g of proteins / lane ) dissolved in sds sample buffer ( 62.5  mm tris - hcl ( ph 6.8 ) , 2% sds , 10% glycerol , and 0.01% bromophenol blue ) containing 5% 2-mercaptoethanol ( 2-me ) were boiled for 3 min at 95c , separated by sds - page , and then electrotransferred onto pvdf membranes ( millipore , billerica , ma , usa )", ". biotinylated markers ( cell signaling , beverly , ma , usa ) were used as molecular weight markers .", "the membranes were blocked for 1  h using 5% skimmed milk in pbs containing 0.05% polyoxyethylene sorbitan monolaurate ( pbs - t ) . after being washed twice with pbs - t", ", the membranes were incubated with goat anti - rage antibody ( n-16 ) or mouse anti--actin antibody ( santa cruz , santa cruz , ca , usa ) for 1.5  h. subsequently , the membranes were washed twice with pbs - t and incubated with anti - goat igg antibody ( santa cruz ) or anti - mouse ig 's antibody ( biosource , camarillo , ca , usa ) and anti - biotin antibody ( cell signaling ) for 1  h. after being washed a further two times with pbs - t , the immunoreactive proteins were detected with ecl plus western blotting detection reagents ( ge healthcare ) using a luminescent image analyzer ( las-1000uvmini ; fujifilm , tokyo , japan ) .", "the density of the bands was analyzed using a multi gauge version 3.0 ( fujifilm ) .", "the cells ( 710  cells / cm ) were seeded in various plates or culture dishes ( bd biosciences , franklin lakes , nj , usa ) and incubated for 24  h. the control unglycated bsa and glycer - ages treatments were carried out in 0.1% fbs / dmem for 48 and 72  h , and cell viability was determined by the wst-1 assay . after removing the medium , 100  l / well of 10% fbs / dmem and 10  l / well of wst-1 solution ( 5  mm wst-1 , 0.2  mm 1-methoxy-5-methylphenazinium , and 20  mm hepes buffer ( ph 7.4 ) ) ( dojindo ) were added , and the cells were incubated for 2  h. absorbance was measured at 450  nm and 650  nm using a microplate reader ( labsystems multiskan ascent , model no .", "354 ; thermo fisher scientific , kanagawa , japan ) , and the net difference ( a450  a650 ) was used to express cell viability .", "the migration and invasion capacity were evaluated in 24-well transwell chambers and biocoat matrigel invasion chambers ( bd biosciences ) , respectively . in both assays ,", "the upper and lower culture compartments were separated by polyethylene terephthalate filters with a pore size of 8  m .", "ten percent fbs was used as a chemoattractant in the lower chamber compartments . before starting the invasion assay ,", "the matrigel matrix of the chambers was reconstituted by adding serum - free dmem for 2  h. in the migration assay , 1    10 cells in serum - free dmem with control unglycated bsa or glycer - ages ( 100  g / ml ) were added into each of the upper chambers for 20  h. in the invasion assay , 2    10 cells in serum - free dmem with control unglycated bsa or glycer - ages were added to each of the upper chambers for 48  h. then , the nonmigrating or noninvading cells on the upper side of the chamber membranes were removed using cotton swabs .", "the cells that migrated to or invaded the opposite side of the chamber were counted .", "the active form of rac1 was detected with the ez - detect rac1 activation kit ( pierce , rockford , il , usa ) .", "this assay uses the ability of a glutathione s - transferase ( gst ) fusion protein corresponding to the p21-binding domain ( pbd ) of human p21 activated protein kinase 1 ( pak1 ) to specifically bind to the active gtp - bound and not the inactive gdp - bound forms of rac1 and cdc42 .", "cell lysates were incubated with gst - pak1-pbd and swellgel immobilized glutathione discs for 1  h at 4c , leaving an aliquot for measuring the levels of total rac1 .", "the bound proteins were eluted in 2 sds sample buffer containing 5% 2-me for 5  min at 95c and characterized by western blotting using monoclonal anti - rac1 antibody .", "rac1 activity was determined by densitometric quantification of the pulled - down rac1-gtp level and normalizing it to the level of total rac1 detected in the sample lysates .", "total rna was isolated from the cells with isogen ( nippon gene , tokyo , japan ) , and 50  ng of rna were reverse transcribed into cdna with primescript rt reagent kit ( takara , shiga , japan ) using a geneamp pcr system 9700 ( perkin - elmer applied biosystems , foster city , ca , usa ) .", "real - time pcr was performed with sybr premix ex taq using a smart cycler ii system ( takara ) .", "the reaction mixture ( 25  l ) contained 1 sybr premix ex taq , 0.2  m pcr forward primers , 0.2  m pcr reverse primers , and 10  ng of cdna as a template .", "the primers used were as follows : tgf- : 5- gcg tgc taa tgg tgg aaa cc   -3 and 5- cgg agc tct gat gtg ttg aag a -3 , mmp-2 : 5-agt ctg aag agc gtg aag-3 and 5- cca ggt agg agt gag aat g -3 , and -actin : 5- tcc acc tcc agc aga tgt gg -3and 5- gca ttt gcg gtg gac gat -3. all processes were performed according to the manufacturer 's instructions .", "-actin was used as an endogenous control gene in order to normalize target gene expression values .", "the cells were seeded and incubated for 24  h. the control unglycated bsa and glycer - ages treatments were carried out in serum - free dmem for 48  h. the conditioned medium was collected and used with a mmp-2 biotrak activity assay system ( ge healthcare ) .", "all experiments were performed in duplicate and repeated at least two to three times , with each experiment yielding essentially identical results .", "the significance of differences between group means was determined by one - way analysis of variance .", "to investigate whether rage proteins are present in human lung adenocarcinoma a549 cells , we carried out western blot analysis using anti - rage antibody ( n-16 ) .", "rage proteins of different molecular weights were detected in a549 cells ( figure 1 ) . in full length rage", "cdna - transfected human hepatocellular carcinoma hep3b cells , the major band ( 57  kda ) ( indicated by an arrow in figure 1 ) represents the full - length rage protein .", "likewise , the full - length rage protein was also detected in a549 and mock transfected hep3b cells .", "no bands were detected in a neutralization experiment using blocking peptide ( data not shown ) so the other bands may represent deglycosylated rage proteins .", "we examined the effect of glycer - ages on the cell viability of a549 cells .", "cell viability was determined after 48  h incubation with control unglycated bsa or glycer - ages .", "twenty , 50 , and 100  g / ml of glycer - ages significantly decreased cell viability to 83 , 64 , and 54% , respectively , ( figure 2(a ) ) .", "in contrast , control unglycated bsa had no effect . furthermore , glycer - ages significantly attenuated cell proliferation ( figure 2(b ) ) ; however , no cell death was induced by glycer - ages according to cell death detection elisa ( data not shown ) .", "we examined the effect of glycer - ages on cell migration and invasion . in the migration assay ,", "the glycer - ages - treated cells showed significantly ( 2.7 times higher ) greater migration than the control unglycated bsa - treated cells ( figure 3(a ) ) , and in the invasion assay , the glycer - ages - treated cells showed significantly ( 3.3 times higher ) greater invasion than the control unglycated bsa - treated cells ( figure 3(b ) ) . when glycer - ages were added to the lower chamber", ", there was no effect on the results of the invasion assay ( data not shown ) .", "these results suggest that glycer - ages - rage interaction is necessary for cell migration and invasion .", "rho family small gtpases are widely accepted to be key regulators of actin reorganization and motility .", "interestingly , rage signaling has been shown to result in the activation of rac1 [ 1720 ] , and the activation of rage by ages has been shown to induce the generation of reactive oxygen species ( ros ) .", "the glycer - ages - induced rac1 activity was about 2 times higher than that of the control unglycated bsa , and it was suppressed by pretreatment with n - acetyl - l - cysteine ( nac ) , an ros scavenger ( figure 4(a ) ) .", "likewise , the glycer - ages - enhanced migration capacity was also significantly suppressed by pre - treatment with nac ( figure 4(b ) ) .", "thus , glycer - ages - rage interaction enhances migration capacity by activating rac1 via ros generation .", "members of the matrix metalloproteinase ( mmp ) family are widely accepted to be key regulators of tumor invasion [ 2224 ] .", "in particular , mmp-2 and -9 , which are called gelatinases , are key enzymes for degrading type iv collagen and are thought to play critical roles in tumor invasion and metastasis .", "we examined the expression of transforming growth factor- ( tgf- ) and mmp-2 mrna and assessed mmp-2 activity with the mmp-2 biotrak activity assay system .", "neither the mrna expression of tgf- or mmp-2 was significantly increased by the addition of glycer - ages at 24  h ( figures 5(a ) and 5(b ) ) .", "pro - mmp-2 activity was also not significantly altered by the addition of glycer - ages at 48  h ( figure 5(c ) )", ". however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% ( figure 5(d ) ) .", "    rage , which is a multiligand receptor , affects diseases such as cancer and diabetes through its ligands .", "several reports have suggested that rage and amphoterin are closely associated with invasion and metastasis in cancer cells [ 26 , 27 ] .", "ages have been implicated in the development and progression of diabetic angiopathies , including skin dermopathy .", "however , it is still unclear which ages subtypes play a pathogenetic role and which ages receptors mediate the effects of ages on cells .", "we have provided direct immunochemical evidence for the existence of six distinct ages structures within the ages - modified proteins and peptides that circulate in the sera of diabetic patients .", "recently , we found that glycer - ages perform a diverse range of biological activities on vascular wall cells , mesangial cells , schwann cells , and cortical neurons [ 1013 ] .", "we also found that glycer - ages , but not glucose - derived ages ( glc - ages ) , significantly stimulated the growth and migration of human melanoma cells .", "furthermore , the tumor formation of melanoma cells xenografts in athymic mice was prevented by treatment with anti - rage antibody . in tumor bearing - mice ,", "survival rates were prolonged , and spontaneous pulmonary metastases were inhibited by treatment using anti - rage antibody . in addition , glycer - ages were present in the beds of human melanoma tumors , whereas they were hardly detected in normal skin .", "these results suggest that glycer - ages are involved in the growth and invasion of malignant melanoma through their interactions with rage .", "recently , we found that glycer - ages have the strongest binding affinity for rage [ 28 , 29 ] and subsequently elicit oxidative stress generation and evoke vascular inflammation , thereby implicating them in accelerated atherosclerosis in diabetes [ 10 , 11 ] .", "taken together , glycer - ages - rage interactions play an important role in the progression of melanoma cells to malignancy .", "however , the effects of glycer - ages on other cancer cells remain poorly understood , and the molecular mechanisms behind these effects have not been clarified .   in the present study", "rage includes full - length , c - truncated , and n - truncated rage . the rage antibody ( n-16 ) used in this study , which recognizes the n - terminus of rage , detects full - length and c - truncated rage .", "indeed , full - length rage ( 57  kda ) was detected in a549 cells .", "the other bands may have represented the de - glycosylated form or c - truncated rage .", "glycer - ages are associated with cell cytotoxicity , but glc - ages are not [ 21 , 31 , 32 ] . in mesangial cells , intracellular ros produced by glycer - ages were found to induce apoptotic cell death . however , no a549 cell death induced by glycer - ages was observed in this study .", "it is thought that the cytotoxicity of glycer - ages depends on the sensitivity of their target cells to ros .", "furthermore , glycer - ages enhanced the migration and invasion activity of the a549 cells , both of which are prominent features of cancer malignancy .", "glycer - ages induced the invasion of a549 cells across matrigel , indicating that matrix degradation and migration mechanisms had been stimulated in these cells . at the molecular level ,", "glycer - ages - induced phenomena resemble the effects of long - term oxidative stress or tgf-. both oxidative stress and tgf- are key regulators of the malignancy rather than the proliferation of cancer cells [ 3335 ] .", "tgf- strongly induces mmp-2 expression in a549 cells ; however , mrna expression of tgf- was not induced by glycer - ages , and the mrna levels of mmp-2 , which is produced by tgf- , did not change . in long - term oxidative stress , mori et al .", "showed that intracellularly produced ros activated rac1   and enhanced the invasion capacity of tumor cells by activating mmp [ 33 , 3739 ] .", "a recent report showed that sustained exposure of cells to h2o2 , but not one time exposure to h2o2 , increased pro - mmp-2 activation through the induction of membrane type 1-mmp ( mt1-mmp ) expression .", "indeed , our results also showed that glycer - ages enhanced the migration capacity of a549 cells by activating rac1 via ros and increased their invasion capacity by activating mmp-2 .", "however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% .", "it is suggested that the activation of other mmp such as mmp-13 may also participate in the above - mentioned changes , and future studies are necessary to clarify this matter . in conclusion", ", we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation .", "these results suggest that glycer - ages play a critical role in cancer malignancy and are potential targets for therapeutic intervention ."], "abstract_text": ["<S> the receptor for advanced glycation end - products ( rages ) is associated with the malignancy of cancer . </S>", "<S> a recent study has suggested that glyceraldehyde - derived ages ( glycer - ages ) enhanced the malignancy of melanoma cells , but glucose - derived ages did not . however , the effects of glycer - ages on other cancer cells remain poorly understood , and the molecular mechanisms behind the above - mentioned effect have not been clarified . the present paper aimed to examine the effect of glycer - ages on cultured lung cancer a549 cells . </S>", "<S> rage was expressed in a549 cells . </S>", "<S> glycer - ages significantly attenuated cell proliferation . </S>", "<S> furthermore , glycer - ages enhanced the migration capacity of the cells by activating rac1 via ros and also increased their invasion capacity . </S>", "<S> we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation . </S>", "<S> these results suggest that glycer - ages play a critical role in the malignancy of cancer rather than its proliferation and are potential targets for therapeutic intervention . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["    the main cause of treatment failure and death in cancer patients is metastasis  the formation of secondary tumors in organs distant from the original neoplastic cell tissue .", "adjuvant therapy of proven efficacy is not currently available for cancer patients ; therefore , the search for new targets of therapeutic reagents is required to prevent both proliferation and metastases . during metastasis ,", "it is known that the mode of invasion is one of the markers of the malignancy and prognosis of cancer .", "the receptor for advanced glycation end - products ( rage ) , a multiligand member of the immunoglobulin superfamily of cell surface molecules , interacts with distinct molecules implicated in homeostasis , development , and inflammation .", "rage binding by ligands such as advanced glycation end - products ( ages ) triggers the activation of key cell signaling pathways , thereby reprogramming cellular properties .", "in addition , several reports have suggested that rage is associated with cancer malignancy [ 4 , 5 ] .     ", "the advanced stage of the glycation process ( one of the posttranslational modifications of proteins ) leads to the formation of ages and plays an important role in the pathogenesis of angiopathy in diabetic patients , aging , and neurodegenerative diseases [ 69 ] .", "a growing body of evidence suggests that the interaction of glyceraldehyde - derived ages ( glycer - ages ) , but not glucose - derived ages ( glc - ages ) , with rage elicits oxidative stress generation in numerous types of cells ( vascular wall cells , mesangial cells , schwann cells , and cortical neurons ) , all of which could contribute to the pathological changes seen in diabetic vascular complications of alzheimer 's disease [ 1013 ] .", "we have recently found that glycer - ages stimulated the growth and migration of cultured human melanoma cells and that anti - rage antibodies inhibited the tumor formation and lung metastasis of melanoma cell xenografts and subsequently improved survival in athymic mice .", "however , the effects of glycer - ages on other cancer cells remains poorly understood , and the molecular mechanisms behind their effects have not been clarified .      in the present study , we examined the effects of glycer - ages on cultured human lung adenocarcinoma a549 cells and showed that glycer - ages enhanced their malignancy rather than their proliferation ."], ["n - acetyl - l - cysteine ( nac ) was purchased from sigma - aldrich ( st . louis , mo , usa ) .", "briefly , 25  mg / ml of bsa ( a0281 , sigma - aldrich ) were incubated at 37c for 7 days under sterile conditions with 0.1  m glyceraldehyde ( ga ; nakalai tesque , kyoto , japan ) and 5  mm diethylenetriamine - pentaacetic acid ( dojindo , kumamoto , japan ) in 0.2  m phosphate buffer ( ph 7.4 ) .", "the modified albumin was then purified by pd-10 column ( ge healthcare , buckinghamshire , england ) chromatography and dialysis against phosphate - buffered saline ( pbs ) .", "control unglycated bsa was incubated under the same conditions except for the absence of glyceraldehyde as a negative control .", "protein concentrations were determined with the dc protein assay reagent ( bio - rad , richmond , ca , usa ) using bsa as a standard .", "the preparations were tested for endotoxin using the pyrotell - t test ( seikagaku bio - business , tokyo , japan ) , but no endotoxin was detected .", "human lung adenocarcinoma a549 and hepatocellular carcinoma hep3b cells were grown in dulbecco 's modified eagle 's medium ( dmem ; sigma - aldrich ) supplemented with 10% fetal bovine serum ( fbs ; equitech - bio , kerrville , tx , usa ) under standard cell culture conditions ( humidified atmosphere , 5% co2 , 37c ) .", "yamagishi , and its mock vector - transfected hep3b cells were maintained in 10% fbs / dmem in the presence of 700  g / ml g418 ( roche , mannheim , germany ) .", "the cells were washed with ice - cold ca and mg free pbs ( pbs ( - ) ) and subjected to lysis buffer ( 1% tritonx-100/nonidet p-40 , 10  mm sodium fluoride , 1  mm sodium orthovanadate , 5  mm sodium pyrophosphate , 2  mm egta , 5  mm edta , and 1 protease inhibitor cocktail ( complete , mini ; roche ) ) .", "subsequently , the cell lysates were incubated on ice for 5 min and centrifuged at 10,000  g for 10  min at 4c .", "their protein concentrations were then measured using the bradford assay ( bio - rad ) .", "cell lysates ( 30  g of proteins / lane ) dissolved in sds sample buffer ( 62.5  mm tris - hcl ( ph 6.8 ) , 2% sds , 10% glycerol , and 0.01% bromophenol blue ) containing 5% 2-mercaptoethanol ( 2-me ) were boiled for 3 min at 95c , separated by sds - page , and then electrotransferred onto pvdf membranes ( millipore , billerica , ma , usa )", ". biotinylated markers ( cell signaling , beverly , ma , usa ) were used as molecular weight markers .", "the membranes were blocked for 1  h using 5% skimmed milk in pbs containing 0.05% polyoxyethylene sorbitan monolaurate ( pbs - t ) . after being washed twice with pbs - t", ", the membranes were incubated with goat anti - rage antibody ( n-16 ) or mouse anti--actin antibody ( santa cruz , santa cruz , ca , usa ) for 1.5  h. subsequently , the membranes were washed twice with pbs - t and incubated with anti - goat igg antibody ( santa cruz ) or anti - mouse ig 's antibody ( biosource , camarillo , ca , usa ) and anti - biotin antibody ( cell signaling ) for 1  h. after being washed a further two times with pbs - t , the immunoreactive proteins were detected with ecl plus western blotting detection reagents ( ge healthcare ) using a luminescent image analyzer ( las-1000uvmini ; fujifilm , tokyo , japan ) .", "the density of the bands was analyzed using a multi gauge version 3.0 ( fujifilm ) .", "the cells ( 710  cells / cm ) were seeded in various plates or culture dishes ( bd biosciences , franklin lakes , nj , usa ) and incubated for 24  h. the control unglycated bsa and glycer - ages treatments were carried out in 0.1% fbs / dmem for 48 and 72  h , and cell viability was determined by the wst-1 assay . after removing the medium , 100  l / well of 10% fbs / dmem and 10  l / well of wst-1 solution ( 5  mm wst-1 , 0.2  mm 1-methoxy-5-methylphenazinium , and 20  mm hepes buffer ( ph 7.4 ) ) ( dojindo ) were added , and the cells were incubated for 2  h. absorbance was measured at 450  nm and 650  nm using a microplate reader ( labsystems multiskan ascent , model no .", "354 ; thermo fisher scientific , kanagawa , japan ) , and the net difference ( a450  a650 ) was used to express cell viability .", "the migration and invasion capacity were evaluated in 24-well transwell chambers and biocoat matrigel invasion chambers ( bd biosciences ) , respectively . in both assays ,", "the upper and lower culture compartments were separated by polyethylene terephthalate filters with a pore size of 8  m .", "ten percent fbs was used as a chemoattractant in the lower chamber compartments . before starting the invasion assay ,", "the matrigel matrix of the chambers was reconstituted by adding serum - free dmem for 2  h. in the migration assay , 1    10 cells in serum - free dmem with control unglycated bsa or glycer - ages ( 100  g / ml ) were added into each of the upper chambers for 20  h. in the invasion assay , 2    10 cells in serum - free dmem with control unglycated bsa or glycer - ages were added to each of the upper chambers for 48  h. then , the nonmigrating or noninvading cells on the upper side of the chamber membranes were removed using cotton swabs .", "the cells that migrated to or invaded the opposite side of the chamber were counted .", "the active form of rac1 was detected with the ez - detect rac1 activation kit ( pierce , rockford , il , usa ) .", "this assay uses the ability of a glutathione s - transferase ( gst ) fusion protein corresponding to the p21-binding domain ( pbd ) of human p21 activated protein kinase 1 ( pak1 ) to specifically bind to the active gtp - bound and not the inactive gdp - bound forms of rac1 and cdc42 .", "cell lysates were incubated with gst - pak1-pbd and swellgel immobilized glutathione discs for 1  h at 4c , leaving an aliquot for measuring the levels of total rac1 .", "the bound proteins were eluted in 2 sds sample buffer containing 5% 2-me for 5  min at 95c and characterized by western blotting using monoclonal anti - rac1 antibody .", "rac1 activity was determined by densitometric quantification of the pulled - down rac1-gtp level and normalizing it to the level of total rac1 detected in the sample lysates .", "total rna was isolated from the cells with isogen ( nippon gene , tokyo , japan ) , and 50  ng of rna were reverse transcribed into cdna with primescript rt reagent kit ( takara , shiga , japan ) using a geneamp pcr system 9700 ( perkin - elmer applied biosystems , foster city , ca , usa ) .", "real - time pcr was performed with sybr premix ex taq using a smart cycler ii system ( takara ) .", "the reaction mixture ( 25  l ) contained 1 sybr premix ex taq , 0.2  m pcr forward primers , 0.2  m pcr reverse primers , and 10  ng of cdna as a template .", "the primers used were as follows : tgf- : 5- gcg tgc taa tgg tgg aaa cc   -3 and 5- cgg agc tct gat gtg ttg aag a -3 , mmp-2 : 5-agt ctg aag agc gtg aag-3 and 5- cca ggt agg agt gag aat g -3 , and -actin : 5- tcc acc tcc agc aga tgt gg -3and 5- gca ttt gcg gtg gac gat -3. all processes were performed according to the manufacturer 's instructions .", "-actin was used as an endogenous control gene in order to normalize target gene expression values .", "the cells were seeded and incubated for 24  h. the control unglycated bsa and glycer - ages treatments were carried out in serum - free dmem for 48  h. the conditioned medium was collected and used with a mmp-2 biotrak activity assay system ( ge healthcare ) .", "all experiments were performed in duplicate and repeated at least two to three times , with each experiment yielding essentially identical results .", "the significance of differences between group means was determined by one - way analysis of variance ."], ["to investigate whether rage proteins are present in human lung adenocarcinoma a549 cells , we carried out western blot analysis using anti - rage antibody ( n-16 ) .", "rage proteins of different molecular weights were detected in a549 cells ( figure 1 ) . in full length rage", "cdna - transfected human hepatocellular carcinoma hep3b cells , the major band ( 57  kda ) ( indicated by an arrow in figure 1 ) represents the full - length rage protein .", "likewise , the full - length rage protein was also detected in a549 and mock transfected hep3b cells .", "no bands were detected in a neutralization experiment using blocking peptide ( data not shown ) so the other bands may represent deglycosylated rage proteins .", "we examined the effect of glycer - ages on the cell viability of a549 cells .", "cell viability was determined after 48  h incubation with control unglycated bsa or glycer - ages .", "twenty , 50 , and 100  g / ml of glycer - ages significantly decreased cell viability to 83 , 64 , and 54% , respectively , ( figure 2(a ) ) .", "in contrast , control unglycated bsa had no effect . furthermore , glycer - ages significantly attenuated cell proliferation ( figure 2(b ) ) ; however , no cell death was induced by glycer - ages according to cell death detection elisa ( data not shown ) .", "we examined the effect of glycer - ages on cell migration and invasion . in the migration assay ,", "the glycer - ages - treated cells showed significantly ( 2.7 times higher ) greater migration than the control unglycated bsa - treated cells ( figure 3(a ) ) , and in the invasion assay , the glycer - ages - treated cells showed significantly ( 3.3 times higher ) greater invasion than the control unglycated bsa - treated cells ( figure 3(b ) ) . when glycer - ages were added to the lower chamber", ", there was no effect on the results of the invasion assay ( data not shown ) .", "these results suggest that glycer - ages - rage interaction is necessary for cell migration and invasion .", "rho family small gtpases are widely accepted to be key regulators of actin reorganization and motility .", "interestingly , rage signaling has been shown to result in the activation of rac1 [ 1720 ] , and the activation of rage by ages has been shown to induce the generation of reactive oxygen species ( ros ) .", "the glycer - ages - induced rac1 activity was about 2 times higher than that of the control unglycated bsa , and it was suppressed by pretreatment with n - acetyl - l - cysteine ( nac ) , an ros scavenger ( figure 4(a ) ) .", "likewise , the glycer - ages - enhanced migration capacity was also significantly suppressed by pre - treatment with nac ( figure 4(b ) ) .", "thus , glycer - ages - rage interaction enhances migration capacity by activating rac1 via ros generation .", "members of the matrix metalloproteinase ( mmp ) family are widely accepted to be key regulators of tumor invasion [ 2224 ] .", "in particular , mmp-2 and -9 , which are called gelatinases , are key enzymes for degrading type iv collagen and are thought to play critical roles in tumor invasion and metastasis .", "we examined the expression of transforming growth factor- ( tgf- ) and mmp-2 mrna and assessed mmp-2 activity with the mmp-2 biotrak activity assay system .", "neither the mrna expression of tgf- or mmp-2 was significantly increased by the addition of glycer - ages at 24  h ( figures 5(a ) and 5(b ) ) .", "pro - mmp-2 activity was also not significantly altered by the addition of glycer - ages at 48  h ( figure 5(c ) )", ". however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% ( figure 5(d ) ) ."], ["    rage , which is a multiligand receptor , affects diseases such as cancer and diabetes through its ligands .", "several reports have suggested that rage and amphoterin are closely associated with invasion and metastasis in cancer cells [ 26 , 27 ] .", "ages have been implicated in the development and progression of diabetic angiopathies , including skin dermopathy .", "however , it is still unclear which ages subtypes play a pathogenetic role and which ages receptors mediate the effects of ages on cells .", "we have provided direct immunochemical evidence for the existence of six distinct ages structures within the ages - modified proteins and peptides that circulate in the sera of diabetic patients .", "recently , we found that glycer - ages perform a diverse range of biological activities on vascular wall cells , mesangial cells , schwann cells , and cortical neurons [ 1013 ] .", "we also found that glycer - ages , but not glucose - derived ages ( glc - ages ) , significantly stimulated the growth and migration of human melanoma cells .", "furthermore , the tumor formation of melanoma cells xenografts in athymic mice was prevented by treatment with anti - rage antibody . in tumor bearing - mice ,", "survival rates were prolonged , and spontaneous pulmonary metastases were inhibited by treatment using anti - rage antibody . in addition , glycer - ages were present in the beds of human melanoma tumors , whereas they were hardly detected in normal skin .", "these results suggest that glycer - ages are involved in the growth and invasion of malignant melanoma through their interactions with rage .", "recently , we found that glycer - ages have the strongest binding affinity for rage [ 28 , 29 ] and subsequently elicit oxidative stress generation and evoke vascular inflammation , thereby implicating them in accelerated atherosclerosis in diabetes [ 10 , 11 ] .", "taken together , glycer - ages - rage interactions play an important role in the progression of melanoma cells to malignancy .", "however , the effects of glycer - ages on other cancer cells remain poorly understood , and the molecular mechanisms behind these effects have not been clarified .   in the present study", "rage includes full - length , c - truncated , and n - truncated rage . the rage antibody ( n-16 ) used in this study , which recognizes the n - terminus of rage , detects full - length and c - truncated rage .", "indeed , full - length rage ( 57  kda ) was detected in a549 cells .", "the other bands may have represented the de - glycosylated form or c - truncated rage .", "glycer - ages are associated with cell cytotoxicity , but glc - ages are not [ 21 , 31 , 32 ] . in mesangial cells , intracellular ros produced by glycer - ages were found to induce apoptotic cell death . however , no a549 cell death induced by glycer - ages was observed in this study .", "it is thought that the cytotoxicity of glycer - ages depends on the sensitivity of their target cells to ros .", "furthermore , glycer - ages enhanced the migration and invasion activity of the a549 cells , both of which are prominent features of cancer malignancy .", "glycer - ages induced the invasion of a549 cells across matrigel , indicating that matrix degradation and migration mechanisms had been stimulated in these cells . at the molecular level ,", "glycer - ages - induced phenomena resemble the effects of long - term oxidative stress or tgf-. both oxidative stress and tgf- are key regulators of the malignancy rather than the proliferation of cancer cells [ 3335 ] .", "tgf- strongly induces mmp-2 expression in a549 cells ; however , mrna expression of tgf- was not induced by glycer - ages , and the mrna levels of mmp-2 , which is produced by tgf- , did not change . in long - term oxidative stress , mori et al .", "showed that intracellularly produced ros activated rac1   and enhanced the invasion capacity of tumor cells by activating mmp [ 33 , 3739 ] .", "a recent report showed that sustained exposure of cells to h2o2 , but not one time exposure to h2o2 , increased pro - mmp-2 activation through the induction of membrane type 1-mmp ( mt1-mmp ) expression .", "indeed , our results also showed that glycer - ages enhanced the migration capacity of a549 cells by activating rac1 via ros and increased their invasion capacity by activating mmp-2 .", "however , glycer - ages only increased mmp-2 ( the activated form ) activity to 120% .", "it is suggested that the activation of other mmp such as mmp-13 may also participate in the above - mentioned changes , and future studies are necessary to clarify this matter . in conclusion", ", we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation .", "these results suggest that glycer - ages play a critical role in cancer malignancy and are potential targets for therapeutic intervention ."]]}
{"article_id": "PMC3304186", "article_text": ["malaligned implants often complicate the clinical laboratory procedures employed for fabrication of superstructures . due to improper load distribution ,", "a stent is an appliance used for radiographic evaluation of height and width of the available bone during treatment planning for implant placement or during surgical procedures to provide site for optimum implant placement .", "the variable type of stents includes vaccuform or acrylic resin templates adapted over duplicated casts of diagnostic wax - up .", "these include plastic or metal tubes and channels in acrylic resin that dictate the position of implants .", "stents should be made of transparent material , stable and rigid when in position , should cover enough teeth to stabilize its position , and when no teeth are present , stents should extend onto unreflected soft tissue regions . with the use of computed tomography ( ct ) and computer added designing ( cad)/ computer added machining ( cam ) technology", ", it is now possible to construct a surgical implant stent that would allow the clinician to predetermine implant locations virtually and surgically place them without reflecting a tissue flap.[58 ] but the surgical stents prepared with ct and cad / cam technology are very much more cost effective than a conventional surgical stent and require enough time and special center and/or laboratories for their fabrication and later on their correction , if required . in this study", ", we used the conventional surgical stents to evaluate their efficacy in determining the position and diameter of implants .", "the aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter .", "the aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter .", "the study was conducted in the out - patient department of prosthodontics and dental material sciences in collaboration with department of oral and maxillofacial surgery , faculty of dental sciences , csmmu , lucknow .", "patients were treated in this study with the conventional surgical stents ; all the implants were placed to the desired depth as planned in virtual implant planning .", "all the patients ( 45 patients for whom 89 implants were placed at different sites ) [ figures 1 , 2 ] visiting the department of prosthodontics and oral and maxillofacial surgery for implant supported prosthesis placement and who followed the standard inclusion criteria for placement of implants were included in the study .", "first of all , diagnostic cast of the patients was made and acrylic teeth / tooth were waxed - up [ figure 3 ] . then , the impression of that waxed - up cast was taken and poured to reproduce another cast with the missing teeth / tooth replaced by dental stone .", "then , mesiodistal and buccolingual markings were drawn over these teeth / tooth to find a center point on the occlusal surface [ figure 4 ] .", "later on , after applying the separating media over these regions and filling the undercuts with wax , transparent self - curing material was applied at the teeth to be replaced along with some adjacent area antero - posteriorly , which confers the stability to the stent during implant placement [ figure 5 ] .", "the holes were drilled in the stent so formed at the center point for the assessment of the diameter and position of implant and filled with gutta percha [ figure 6 ] .", "this stent was placed in the mouth and radiograph was taken to evaluate the position of gutta percha .", "the efficacy of stents was then evaluated at the time of surgical exposure for implant placement and after the surgery , with the help of radiographs [ figures 7 , 8 ] .", "intraoral view of the patient radiograph of the patients waxed - up missing teeth missing teeth replaced by dental stone in the cast with markings stent made of transparent material stent in place with gutta percha filled in holes post - operative radiograph philip j. described a technique to construct a surgical guide on mounted diagnostic casts .", "he also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis .", "murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning .", "the outer lamina is designed for use in the computed tomography ( ct ) evaluation using radiopaque markers .", "yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker .", "the matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy .", "roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient , when implant - supported restorations are planned . a template that provides radiographic evaluation of the implant site and precise or modified surgical placement", "kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants .", "he also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis .", "murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning .", "the outer lamina is designed for use in the computed tomography ( ct ) evaluation using radiopaque markers .", "yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker .", "the matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy .", "roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient , when implant - supported restorations are planned . a template that provides radiographic evaluation of the implant site and precise or modified surgical placement", "kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants .", "the foremost advantage of the stent used in this study is its surgical ease , simplicity , precise accuracy and low cost .", "it can be fabricated with minimum laboratory procedures which are used in routine dental practice .", "on the other hand , the fixed type screw retained implant stents which are fabricated with the help of ct and cad / cam technology are very costly and require more time and laboratory procedures .", "the stents fabricated with the radiopaque markers provide radiographic as well as clinical ease for optimum implant installation . along with all these advantages", "the drill holes can directly be made through the stent , so the implant surgery may become less traumatic ( with the preservation of soft tissue including the gingival margins of adjacent teeth and the interdental papilla ) with decreased operative time which results in accelerated post - surgical healing , fewer post - operative complications and increased comfort and satisfaction for the patient .", "the possibility of displacement of stent during implant surgery was minimized by extension of the stent antero - posteriorly to cover enough area over the teeth / tooth present adjacent to edentulous area . in cases where no teeth are present ,", "stabilization of stent was achieved by extending it over the unreflected areas like retromolar regions .", "this study shows the extreme accuracy of this conventional surgical stent . if each step of this protocol is followed precisely , it is possible to deliver an optimum implant installation in terms of position and diameter and later on function at a very much reduced rate and time , as taken in ct and cad / cam technology which are the most accepted methods"], "abstract_text": ["<S> aim : demand from implant patients for quality and efficient treatment is increasing . </S>", "<S> fortunately dental implant treatment is evolving with patients expectations.materials and methods : the study comprised of 45 patients for whom 89 implants were placed at different sites . </S>", "<S> efficacy of the stents is evaluated in determining the position and diameter of the implants.conclusion:this study shows the extreme accuracy of this surgical stents in implant installation in terms of position and diameter . </S>"], "labels": null, "section_names": ["I", "Aim of the study", "M", "Review of literature", "D", "C"], "sections": [["osseointegrated implants are a practical alternative to traditional prosthodontics ; however , designing an implant - supported prosthesis with function and esthetics is a challenge .", "malaligned implants often complicate the clinical laboratory procedures employed for fabrication of superstructures . due to improper load distribution ,", "a stent is an appliance used for radiographic evaluation of height and width of the available bone during treatment planning for implant placement or during surgical procedures to provide site for optimum implant placement .", "the variable type of stents includes vaccuform or acrylic resin templates adapted over duplicated casts of diagnostic wax - up .", "these include plastic or metal tubes and channels in acrylic resin that dictate the position of implants .", "stents should be made of transparent material , stable and rigid when in position , should cover enough teeth to stabilize its position , and when no teeth are present , stents should extend onto unreflected soft tissue regions . with the use of computed tomography ( ct ) and computer added designing ( cad)/ computer added machining ( cam ) technology", ", it is now possible to construct a surgical implant stent that would allow the clinician to predetermine implant locations virtually and surgically place them without reflecting a tissue flap.[58 ] but the surgical stents prepared with ct and cad / cam technology are very much more cost effective than a conventional surgical stent and require enough time and special center and/or laboratories for their fabrication and later on their correction , if required . in this study", ", we used the conventional surgical stents to evaluate their efficacy in determining the position and diameter of implants .", "the aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter ."], ["the aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter ."], ["the study was conducted in the out - patient department of prosthodontics and dental material sciences in collaboration with department of oral and maxillofacial surgery , faculty of dental sciences , csmmu , lucknow .", "patients were treated in this study with the conventional surgical stents ; all the implants were placed to the desired depth as planned in virtual implant planning .", "all the patients ( 45 patients for whom 89 implants were placed at different sites ) [ figures 1 , 2 ] visiting the department of prosthodontics and oral and maxillofacial surgery for implant supported prosthesis placement and who followed the standard inclusion criteria for placement of implants were included in the study .", "first of all , diagnostic cast of the patients was made and acrylic teeth / tooth were waxed - up [ figure 3 ] . then , the impression of that waxed - up cast was taken and poured to reproduce another cast with the missing teeth / tooth replaced by dental stone .", "then , mesiodistal and buccolingual markings were drawn over these teeth / tooth to find a center point on the occlusal surface [ figure 4 ] .", "later on , after applying the separating media over these regions and filling the undercuts with wax , transparent self - curing material was applied at the teeth to be replaced along with some adjacent area antero - posteriorly , which confers the stability to the stent during implant placement [ figure 5 ] .", "the holes were drilled in the stent so formed at the center point for the assessment of the diameter and position of implant and filled with gutta percha [ figure 6 ] .", "this stent was placed in the mouth and radiograph was taken to evaluate the position of gutta percha .", "the efficacy of stents was then evaluated at the time of surgical exposure for implant placement and after the surgery , with the help of radiographs [ figures 7 , 8 ] .", "intraoral view of the patient radiograph of the patients waxed - up missing teeth missing teeth replaced by dental stone in the cast with markings stent made of transparent material stent in place with gutta percha filled in holes post - operative radiograph philip j. described a technique to construct a surgical guide on mounted diagnostic casts .", "he also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis .", "murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning .", "the outer lamina is designed for use in the computed tomography ( ct ) evaluation using radiopaque markers .", "yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker .", "the matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy .", "roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient , when implant - supported restorations are planned . a template that provides radiographic evaluation of the implant site and precise or modified surgical placement", "kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants ."], ["he also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis .", "murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning .", "the outer lamina is designed for use in the computed tomography ( ct ) evaluation using radiopaque markers .", "yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker .", "the matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy .", "roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient , when implant - supported restorations are planned . a template that provides radiographic evaluation of the implant site and precise or modified surgical placement", "kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants ."], ["the foremost advantage of the stent used in this study is its surgical ease , simplicity , precise accuracy and low cost .", "it can be fabricated with minimum laboratory procedures which are used in routine dental practice .", "on the other hand , the fixed type screw retained implant stents which are fabricated with the help of ct and cad / cam technology are very costly and require more time and laboratory procedures .", "the stents fabricated with the radiopaque markers provide radiographic as well as clinical ease for optimum implant installation . along with all these advantages", "the drill holes can directly be made through the stent , so the implant surgery may become less traumatic ( with the preservation of soft tissue including the gingival margins of adjacent teeth and the interdental papilla ) with decreased operative time which results in accelerated post - surgical healing , fewer post - operative complications and increased comfort and satisfaction for the patient .", "the possibility of displacement of stent during implant surgery was minimized by extension of the stent antero - posteriorly to cover enough area over the teeth / tooth present adjacent to edentulous area . in cases where no teeth are present ,", "stabilization of stent was achieved by extending it over the unreflected areas like retromolar regions ."], ["this study shows the extreme accuracy of this conventional surgical stent . if each step of this protocol is followed precisely , it is possible to deliver an optimum implant installation in terms of position and diameter and later on function at a very much reduced rate and time , as taken in ct and cad / cam technology which are the most accepted methods"]]}
{"article_id": "PMC4764037", "article_text": ["type 1 diabetes ( t1d ) is characterized by autoimmune destruction of pancreatic -cells , primarily mediated by t lymphocytes , resulting in insulin deficiency and lifelong exogenous insulin dependence ( 1 ) .", "latent autoimmune diabetes in adults ( lada ) is a subtype of autoimmune diabetes , but the progression of autoimmune -cell destruction is slow ; therefore , patients with lada are not insulin dependent at the initial phase ( at least 6 months ) of disease onset", ". clinical manifestation of lada shares the features of classical t1d and type 2 diabetes ( t2d ) ( 2 ) . t1d , lada , and t2d present a spectrum of diabetes .", "they range from patients with classic childhood t1d characterized by autoimmune - mediated destruction of -cells with the lowest c - peptide levels to age - related exacerbation of glucose tolerance with the highest bmi seen in t2d ( 3 ) .", "furthermore , patients with lada have susceptibility genes for both t1d and t2d ( 4 ) , suggesting that lada lies between t1d and t2d .", "b cells infiltrate the islets of young nod mice , the commonly used mouse model for human t1d , and play a role in the initiation of -cell destruction by the autoimmune response ( 5 ) .", "studies have shown that marginal zone b ( mzb ) cells and follicular b ( fob ) cells can effectively activate cd4 t cells and facilitate their proliferation by serving as important antigen - presenting cells , especially as antigen - specific antigen - presenting cells ( 6,7 ) .", "b - cell  targeted therapy has been shown to delay the fall of c - peptide levels in patients with recent - onset t1d ( 8) .", "clinical trials have identified b cells as a possible therapeutic target for the prevention and reversal of t1d .", "b - cell  targeted therapy also has been successfully used in treating other autoimmune diseases , including rheumatoid arthritis ( 9 ) , systemic lupus erythematosus ( 10 ) , and multiple sclerosis ( 11 ) .", "moreover , studies have shown that b cells are important in regulating both innate and adaptive immunity ( 12 ) . a subset of b cells expressing cd19cd5cd1d , which have a regulatory function in infection , inflammation , and autoimmune disease , recently has been identified .", "these regulatory b cells can regulate t - cell responses and inhibit inflammation in an interleukin-10 ( il-10)dependent manner ( 1316 ) .", "the maturation and expansion of b10 cells require il-21 and cd40 signals both in vivo and ex vivo ( 17,18 ) .", "considerable evidence shows that patients with autoimmune diabetes have altered frequencies of circulating immune cells , including t cells ( 19 ) , dendritic cells ( 20 ) , natural killer cells ( 21,22 ) , and neutrophils ( 23 ) .", "although studies have reported no significant change in total b cells in peripheral blood of patients with t1d ( 21,24 ) , little is known about b - cell subpopulations with distinct immune functions that may play a role in the diabetes spectrum of t1d , lada , and t2d .", "we hypothesized that an altered phenotype of b - cell subsets is associated with autoimmune diabetes .", "this study investigated a change of peripheral b - cell subsets in patients with t1d , lada , and t2d compared with healthy control subjects .", "the results show altered frequencies of various b - cell subsets associated with autoimmune diabetes .", "two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1  3.5 years ) , lada ( n = 82 , 6.1  5.9 years ) , or t2d ( n = 95 , 3.0  3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) .", "diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) .", "diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion .", "lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) .", "diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment .", "healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8", "exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease .", "this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol .", "anthropometric and metabolic data of participants data are % ( n ) , mean  sd , and median ( 25th75th percentile ) .", " compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d .", "study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure .", "fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) .", "hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) .", "fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) .", "gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) .", "the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects .", "the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) .", "fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol .", "the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples .", "fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . ,", "dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining .", "1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) .", "the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively .", "the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination .", "b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells .", "c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells .", "anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi .", "all the significant differences between groups are the results of adjusted analyses unless stated otherwise .", "associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations .", "for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software .", "two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1  3.5 years ) , lada ( n = 82 , 6.1  5.9 years ) , or t2d ( n = 95 , 3.0  3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) .", "diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) .", "diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion .", "lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) .", "diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment .", "healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8", "exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease .", "this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol .", "anthropometric and metabolic data of participants data are % ( n ) , mean  sd , and median ( 25th75th percentile ) .", " compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d .", "study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure .", "fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) .", "hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) .", "fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) .", "gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) .", "the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects . the positive samples were tested twice for confirmation .", "the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) .", "fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol .", "the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples .", "fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . ,", "dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining .", "1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) .", "the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively .", "the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination .", "b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells .", "c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells .", "anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi .", "all the significant differences between groups are the results of adjusted analyses unless stated otherwise .", "associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations .", "for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software .", "the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d .", "the percentage of males in the lada group was higher than in the ngt group .", "the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group .", "patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups .", "the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt .", "there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups .", "we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig .", "the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig .", "we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi .", "fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) .", "we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig .", "2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig .", "the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells .", "p values refer to comparison of data after adjustment for age , sex , and bmi .", "* p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig .", "b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig .", "after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig .", "however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d .", "we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells .", "however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level .", "correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test .", "in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d , the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) .", "finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) .", "the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d .", "the percentage of males in the lada group was higher than in the ngt group .", "the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group .", "patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups .", "the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt .", "there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups .", "we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig .", "the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig .", "we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi .", "fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) .", "we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig .", "2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig .", "the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells .", "p values refer to comparison of data after adjustment for age , sex , and bmi .", "* p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig .", "b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig .", "after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig .", "however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d .", "we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells .", "however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level .", "correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test .", "in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d ,", "the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) .", "finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) .", "increasing evidence suggests that b cells are as important as t cells in the immunopathogenesis of autoimmune diabetes .", "most , if not all , diabetogenic t cells are known to be t - helper 1 subsets , but little is known about b - cell subsets in autoimmune diabetes .", "we investigated the phenotype and frequency of various b - cell subsets in the peripheral blood across the clinical spectrum of diabetes .", "the major finding of this study was a distinct alteration of b - cell subsets across the diabetes spectrum and their association with clinical features .", "we found no significant difference in the frequency of cd19 b cells , which is in accordance with the published studies ( 21,30 ) ; however , we did not find a negative correlation between total b cells and age , as previously reported ( 24,31 ) .", "the current study went further than the examination of the frequency of total b cells , focusing on b - cell subsets with distinct immunological functions .", "of note , we found that patients with t1d or lada had an increased frequency of mzb cells but decreased frequency of fob cells compared with control subjects with ngt and patients with t2d . in all groups , we found that fcp was negatively associated with mzb cells but positively associated with fob cells .", "attanavanich and kearney ( 6 ) reported that mzb cells were more effective at activating naive cd4 t cells than fob cells .", "mzb cells in nod mice were also shown to be important antigen - presenting cells for the activation of t cells in pancreatic - draining lymph nodes ( 32 ) . given the negative correlation between mzb cells and fcp , it is possible that mzb cells may promote -cell destruction in patients with t1d or lada .", "decreased frequency of fob cells in the circulation of patients with t1d and lada suggests that some fob cells migrated from the blood to the pancreas or the draining lymph nodes as a result of the local inflammatory response .", "along with the disease progression , more immune cells are known to infiltrate to the islets of both nod mice and patients with t1d ( 5,33 ) , but the patients with t1d in the current study have a relatively long history of diabetes ( 3.1 years ) .", "thus , fob cells appear to be more important in initial rather than established pancreatic -cell autoimmunity ( 34 ) .", "we showed that patients with t2d and lada more frequently had t2-mzp b cells than control subjects with ngt , which associates with parameters of blood pressure , lipidemia , and glycemia but not with hba1c or fcp .", "these observations are consistent with a previous study showing that glycemia and lipidemia are associated with immune cells ( 35 ) . another interesting finding was the decrease of b10 cell frequency in all types of diabetes , being the lowest in t1d .", "whether this phenotype is mediated through a common mechanism or different ones is not clear .", "what is clear is that immune mechanisms are involved and that more studies , especially functional studies , are required .", "although b cells are generally acknowledged for their function in humoral immune response by producing antibodies , they also have been demonstrated to play an immunoregulatory role in the prevention of autoimmune disease by producing il-10 and downregulating t - cell responses ( 13,14,36 ) .", "the observed b10 cell reduction could contribute to the breakdown of self - tolerance that leads to -cell destruction in patients with t1d ( 24 ) or lada .", "antigen - activated il-10producing b cells may selectively inhibit autoreactive t - cell responses to islet - specific antigen to maintain self - tolerance , and hyperglycemic nod mice and patients with t1d lack this population of regulatory b cells ( 37 ) .", "in the clinical trial where b cells in patients with new - onset t1d were depleted , the patients showed a transient remission ( 8) .", "however , blanket targeting of all b cells , as used in the clinical trial and in treating other autoimmune disorders , needs to be improved by selectively deleting pathogenic b cells while preserving regulatory b cells to promote tolerance .", "our preclinical study showed that more regulatory b cells were regenerated after b - cell deletion in an animal model ( 38 ) .", "furthermore , studies have shown that patients with long - standing t2d could have a secondary autoimmune response toward islet -cells ( 39 ) .", "the reduction of b10 regulatory cells might contribute to the dysregulation of immune response in t2d .", "a functional study on il-10producing b cells and further phenotyping will be the goal of future work . in the current study ,", "although we currently can not identify a specific subset that could be used for diagnostic purposes , the results strongly imply that the change of b - cell subtypes is related to the pathogenesis of autoimmune diabetes .", "therefore , it is important to reconsider the approach of b - cell  targeted therapy in patients with t1d and aim to selectively remove pathogenic b cells but promote regulatory b cells . in summary ,", "the current data demonstrate distinct differences in the frequencies of peripheral b - cell subsets in t1d and lada , which are associated with islet function and glycemia .", "the findings support the notion that these immunological alterations are involved in loss of self - tolerance and -cell destruction .", "the findings also suggest that , similar to regulatory t - cell therapy , transfusion of ex vivo expanded autologous b10 cells might open a new and effective therapeutic strategy in treating autoimmune diabetes ."], "abstract_text": ["<S> objectiveb lymphocytes play an important role in the immunopathogenesis of autoimmune diabetes . </S>", "<S> we hypothesized that the altered b - cell subset phenotype is associated with autoimmune diabetes.research design and methodspatients with type 1 diabetes ( t1d ) ( n = 81 ) , latent autoimmune diabetes in adults ( lada ) ( n = 82 ) , or type 2 diabetes ( t2d ) ( n = 95 ) and healthy control subjects ( n = 218 ) with normal glucose tolerance ( ngt ) were recruited . </S>", "<S> we determined the percentage of circulating b - lymphocyte subsets , including cd19+cd23cd21 + ( marginal zone b [ mzb ] ) , cd19+cd23+cd21 ( follicular b [ fob ] ) , and cd19+cd5+cd1dhi ( interleukin-10producing regulatory b [ b10 ] ) cells by flow cytometry.resultspatients with t1d or lada had increased percentages of mzb cells and decreased percentages of fob cells compared with healthy control subjects with ngt and patients with t2d . </S>", "<S> moreover , patients with t1d showed the lowest frequency of b10 cells compared with patients with lada or t2d , whereas healthy control subjects expressed the highest frequency of b10 cells . </S>", "<S> of note , the frequency of mzb cells was negatively associated and the frequency of fob cells was positively associated with fasting c - peptide ( fcp ) . </S>", "<S> the frequency of b10 cells was positively correlated with fcp and negatively correlated with hemoglobin a1c.conclusionsthe data show that patients with t1d or lada express an altered frequency of b - cell subsets , which is associated with islet function and glycemia . </S>", "<S> these findings suggest that b lymphocytes may be involved in loss of self - tolerance and -cell destruction and could be used as a biomarker and potential target for immunological intervention . </S>"], "labels": null, "section_names": ["Introduction", "Research Design and Methods", "Participants", "C-Peptide and HbA", "Islet Autoantibody Assays", "Immunostaining and Flow Cytometry", "Statistical Analysis", "Results", "Anthropometric and Metabolic Data", "Change of B-Cell Subsets in Patients With Diabetes", "B-Cell Subsets Are Associated With Clinical Diabetes", "Conclusions", "Supplementary Material"], "sections": [["type 1 diabetes ( t1d ) is characterized by autoimmune destruction of pancreatic -cells , primarily mediated by t lymphocytes , resulting in insulin deficiency and lifelong exogenous insulin dependence ( 1 ) .", "latent autoimmune diabetes in adults ( lada ) is a subtype of autoimmune diabetes , but the progression of autoimmune -cell destruction is slow ; therefore , patients with lada are not insulin dependent at the initial phase ( at least 6 months ) of disease onset", ". clinical manifestation of lada shares the features of classical t1d and type 2 diabetes ( t2d ) ( 2 ) . t1d , lada , and t2d present a spectrum of diabetes .", "they range from patients with classic childhood t1d characterized by autoimmune - mediated destruction of -cells with the lowest c - peptide levels to age - related exacerbation of glucose tolerance with the highest bmi seen in t2d ( 3 ) .", "furthermore , patients with lada have susceptibility genes for both t1d and t2d ( 4 ) , suggesting that lada lies between t1d and t2d .", "b cells infiltrate the islets of young nod mice , the commonly used mouse model for human t1d , and play a role in the initiation of -cell destruction by the autoimmune response ( 5 ) .", "studies have shown that marginal zone b ( mzb ) cells and follicular b ( fob ) cells can effectively activate cd4 t cells and facilitate their proliferation by serving as important antigen - presenting cells , especially as antigen - specific antigen - presenting cells ( 6,7 ) .", "b - cell  targeted therapy has been shown to delay the fall of c - peptide levels in patients with recent - onset t1d ( 8) .", "clinical trials have identified b cells as a possible therapeutic target for the prevention and reversal of t1d .", "b - cell  targeted therapy also has been successfully used in treating other autoimmune diseases , including rheumatoid arthritis ( 9 ) , systemic lupus erythematosus ( 10 ) , and multiple sclerosis ( 11 ) .", "moreover , studies have shown that b cells are important in regulating both innate and adaptive immunity ( 12 ) . a subset of b cells expressing cd19cd5cd1d , which have a regulatory function in infection , inflammation , and autoimmune disease , recently has been identified .", "these regulatory b cells can regulate t - cell responses and inhibit inflammation in an interleukin-10 ( il-10)dependent manner ( 1316 ) .", "the maturation and expansion of b10 cells require il-21 and cd40 signals both in vivo and ex vivo ( 17,18 ) .", "considerable evidence shows that patients with autoimmune diabetes have altered frequencies of circulating immune cells , including t cells ( 19 ) , dendritic cells ( 20 ) , natural killer cells ( 21,22 ) , and neutrophils ( 23 ) .", "although studies have reported no significant change in total b cells in peripheral blood of patients with t1d ( 21,24 ) , little is known about b - cell subpopulations with distinct immune functions that may play a role in the diabetes spectrum of t1d , lada , and t2d .", "we hypothesized that an altered phenotype of b - cell subsets is associated with autoimmune diabetes .", "this study investigated a change of peripheral b - cell subsets in patients with t1d , lada , and t2d compared with healthy control subjects .", "the results show altered frequencies of various b - cell subsets associated with autoimmune diabetes ."], ["two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1  3.5 years ) , lada ( n = 82 , 6.1  5.9 years ) , or t2d ( n = 95 , 3.0  3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) .", "diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) .", "diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion .", "lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) .", "diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment .", "healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8", "exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease .", "this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol .", "anthropometric and metabolic data of participants data are % ( n ) , mean  sd , and median ( 25th75th percentile ) .", " compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d .", "study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure .", "fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) .", "hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) .", "fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) .", "gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) .", "the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects .", "the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) .", "fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol .", "the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples .", "fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . ,", "dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining .", "1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) .", "the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively .", "the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination .", "b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells .", "c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells .", "anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi .", "all the significant differences between groups are the results of adjusted analyses unless stated otherwise .", "associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations .", "for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software ."], ["two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1  3.5 years ) , lada ( n = 82 , 6.1  5.9 years ) , or t2d ( n = 95 , 3.0  3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) .", "diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) .", "diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion .", "lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) .", "diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment .", "healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8", "exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease .", "this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol .", "anthropometric and metabolic data of participants data are % ( n ) , mean  sd , and median ( 25th75th percentile ) .", " compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d .", "study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure .", "fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) ."], ["hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) .", "fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) ."], ["gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) .", "the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects . the positive samples were tested twice for confirmation .", "the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) ."], ["fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol .", "the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples .", "fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . ,", "dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining .", "1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) .", "the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively .", "the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination .", "b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells .", "c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells ."], ["anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi .", "all the significant differences between groups are the results of adjusted analyses unless stated otherwise .", "associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations .", "for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software ."], ["the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d .", "the percentage of males in the lada group was higher than in the ngt group .", "the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group .", "patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups .", "the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt .", "there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups .", "we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig .", "the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig .", "we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi .", "fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) .", "we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig .", "2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig .", "the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells .", "p values refer to comparison of data after adjustment for age , sex , and bmi .", "* p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig .", "b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig .", "after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig .", "however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d .", "we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells .", "however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level .", "correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test .", "in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d , the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) .", "finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) ."], ["the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d .", "the percentage of males in the lada group was higher than in the ngt group .", "the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group .", "patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups .", "the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt .", "there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups ."], ["we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig .", "the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig .", "we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi .", "fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) .", "we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig .", "2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig .", "the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells .", "p values refer to comparison of data after adjustment for age , sex , and bmi .", "* p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig .", "b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig .", "after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig .", "however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d ."], ["we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells .", "however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level .", "correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test .", "in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d ,", "the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) .", "finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) ."], ["increasing evidence suggests that b cells are as important as t cells in the immunopathogenesis of autoimmune diabetes .", "most , if not all , diabetogenic t cells are known to be t - helper 1 subsets , but little is known about b - cell subsets in autoimmune diabetes .", "we investigated the phenotype and frequency of various b - cell subsets in the peripheral blood across the clinical spectrum of diabetes .", "the major finding of this study was a distinct alteration of b - cell subsets across the diabetes spectrum and their association with clinical features .", "we found no significant difference in the frequency of cd19 b cells , which is in accordance with the published studies ( 21,30 ) ; however , we did not find a negative correlation between total b cells and age , as previously reported ( 24,31 ) .", "the current study went further than the examination of the frequency of total b cells , focusing on b - cell subsets with distinct immunological functions .", "of note , we found that patients with t1d or lada had an increased frequency of mzb cells but decreased frequency of fob cells compared with control subjects with ngt and patients with t2d . in all groups , we found that fcp was negatively associated with mzb cells but positively associated with fob cells .", "attanavanich and kearney ( 6 ) reported that mzb cells were more effective at activating naive cd4 t cells than fob cells .", "mzb cells in nod mice were also shown to be important antigen - presenting cells for the activation of t cells in pancreatic - draining lymph nodes ( 32 ) . given the negative correlation between mzb cells and fcp , it is possible that mzb cells may promote -cell destruction in patients with t1d or lada .", "decreased frequency of fob cells in the circulation of patients with t1d and lada suggests that some fob cells migrated from the blood to the pancreas or the draining lymph nodes as a result of the local inflammatory response .", "along with the disease progression , more immune cells are known to infiltrate to the islets of both nod mice and patients with t1d ( 5,33 ) , but the patients with t1d in the current study have a relatively long history of diabetes ( 3.1 years ) .", "thus , fob cells appear to be more important in initial rather than established pancreatic -cell autoimmunity ( 34 ) .", "we showed that patients with t2d and lada more frequently had t2-mzp b cells than control subjects with ngt , which associates with parameters of blood pressure , lipidemia , and glycemia but not with hba1c or fcp .", "these observations are consistent with a previous study showing that glycemia and lipidemia are associated with immune cells ( 35 ) . another interesting finding was the decrease of b10 cell frequency in all types of diabetes , being the lowest in t1d .", "whether this phenotype is mediated through a common mechanism or different ones is not clear .", "what is clear is that immune mechanisms are involved and that more studies , especially functional studies , are required .", "although b cells are generally acknowledged for their function in humoral immune response by producing antibodies , they also have been demonstrated to play an immunoregulatory role in the prevention of autoimmune disease by producing il-10 and downregulating t - cell responses ( 13,14,36 ) .", "the observed b10 cell reduction could contribute to the breakdown of self - tolerance that leads to -cell destruction in patients with t1d ( 24 ) or lada .", "antigen - activated il-10producing b cells may selectively inhibit autoreactive t - cell responses to islet - specific antigen to maintain self - tolerance , and hyperglycemic nod mice and patients with t1d lack this population of regulatory b cells ( 37 ) .", "in the clinical trial where b cells in patients with new - onset t1d were depleted , the patients showed a transient remission ( 8) .", "however , blanket targeting of all b cells , as used in the clinical trial and in treating other autoimmune disorders , needs to be improved by selectively deleting pathogenic b cells while preserving regulatory b cells to promote tolerance .", "our preclinical study showed that more regulatory b cells were regenerated after b - cell deletion in an animal model ( 38 ) .", "furthermore , studies have shown that patients with long - standing t2d could have a secondary autoimmune response toward islet -cells ( 39 ) .", "the reduction of b10 regulatory cells might contribute to the dysregulation of immune response in t2d .", "a functional study on il-10producing b cells and further phenotyping will be the goal of future work . in the current study ,", "although we currently can not identify a specific subset that could be used for diagnostic purposes , the results strongly imply that the change of b - cell subtypes is related to the pathogenesis of autoimmune diabetes .", "therefore , it is important to reconsider the approach of b - cell  targeted therapy in patients with t1d and aim to selectively remove pathogenic b cells but promote regulatory b cells . in summary ,", "the current data demonstrate distinct differences in the frequencies of peripheral b - cell subsets in t1d and lada , which are associated with islet function and glycemia .", "the findings support the notion that these immunological alterations are involved in loss of self - tolerance and -cell destruction .", "the findings also suggest that , similar to regulatory t - cell therapy , transfusion of ex vivo expanded autologous b10 cells might open a new and effective therapeutic strategy in treating autoimmune diabetes ."], [""]]}
{"article_id": "PMC3897859", "article_text": ["human papillomavirus ( hpv ) , a virus from the papillomavirus family , is capable of infecting humans . about 200 different strains of hpv identified , based on dna homology , have been found to be etiologically linked to cervical , vaginal , vulvar , penile , anal , oral , and plantar infectious lesions and cancers , as well [ 1 , 2 ] .", "the hpv genome , a double - stranded dna molecule , consists of 8 kilobase pairs ( kbp ) of nucleotides , which comprises 3 regions : 6 early open reading frames ( orfs)-e1 , e2 , e4 , e5 , e6 , and e7 ; 2 late orfs - l1 and l2 ; and an upstream regulatory region . a considerable volume of hpv specific information pertaining to its genome , proteome , structure , and disease association is available scattered on the web with trivial annotations", "; however , it is not customized to explore for data analysis , host - pathogen interaction , strain - disease association , drug designing , and sequence analysis , etc .", "therefore , we proposed to develop a comprehensive reserve on hpv with maximum possible inputs and outputs for the end users .", "amongst the existing 200 strains of hpv , 150 have been sequenced as of now , and their data available at the national center for biotechnology information ( ncbi ) .", "genome and proteome information of those viral strains was retrieved from ncbi . besides pubmed , various other online resources and published literature", "were also explored for validating genomic , proteomic , as well as strain and disease - associated information on hpv strains .", "hpv strain - specific information , such as strain name , sequencing status , sequencing centre , ncbi accession i d , associated disease information with references , genome statistics ( gc% , at% , a , t , g , c count , genes , and proteins ) , etc . , were curated from various online resources , and protein parameters ( length , molecular weight , isoelectric point ) were calculated using expasy protparam .", "modeller9v10   and the swiss - model   server were used for protein structure prediction .", "the stereochemistry of each protein was evaluated through procheck   analysis , available at the rcsb validation server ( http://deposit.rcsb.org/validate/ ) , and validated using prosa - web   ( http://prosa.services.came.sbg.ac.at/prosa.php ) .", "human papillomavirus proteome database ( hpvpdb ) , the relational reserve , was developed using microsoft sql server 2005 as the back end .", "html , javascript , and cgi - perl - based web interfaces were employed to execute sql queries .", "the application layer , the web interface , and the backend relational tables were integrated using cgi - perl .", "home , about , tools , search , and advanced search interfaces can be explored to obtain strain- and protein - specific information .", "reserve comprises the strain - specific detailed informationon its name , sequencing status , submission details , date of submission , ncbi ids , disease types and subtypes , type of dna , genome length , molecular weight , nucleotide composition ( a , t , g , c , at , gc content ) , number of genes and proteins , and protein list . a genome map of each strain obtained by geneious 5.4.4 software ( available from http://www.geneious.com/ ) is also integrated in this page .", "users , through an advanced search option , can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome .", "each protein entry comprises protein overview ( name , locus , function , etc . ) , protein sequence information ( amino acid sequences with ncbi accession number with provision for direct protein blast   against ncbi nr database ) , protein parameters ( length , molecular weight , theoretical isoelectric point [ pi ] , amino acid composition , etc . ) , protein structure ( predicted 3d structure by homology modeling viewed by jmol ( available from http://www.jmol.org/ )   with the java platform , ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web .", "hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny .", "the original human papillomaviruses database was developed and hosted by the los alamos national laboratory ( lanl ) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases ( niaid ) . '", "human papillomaviruses : a compilation and analysis of nucleic acid and amino acid sequences ' contains four annual data books of papillomavirus information published in both paper and electronic form ( 1994 , 1995 , 1996 , and 1997 ) but has not been updated since 1997 .", "this contains nucleotide sequences of few hpv strains and other papillomaviruses , amino acid and nucleotide sequence alignments , analysis , related host sequences , and database communication .", "we did not find any structural information in that database . in hpvpdb , along with updated protein sequence information , genome and protein structure information is also provided .", "amongst the existing 200 strains of hpv , 150 have been sequenced as of now , and their data available at the national center for biotechnology information ( ncbi ) .", "genome and proteome information of those viral strains was retrieved from ncbi . besides pubmed , various other online resources and published literature", "were also explored for validating genomic , proteomic , as well as strain and disease - associated information on hpv strains .", "hpv strain - specific information , such as strain name , sequencing status , sequencing centre , ncbi accession i d , associated disease information with references , genome statistics ( gc% , at% , a , t , g , c count , genes , and proteins ) , etc . , were curated from various online resources , and protein parameters ( length , molecular weight , isoelectric point ) were calculated using expasy protparam .", "modeller9v10   and the swiss - model   server were used for protein structure prediction .", "the stereochemistry of each protein was evaluated through procheck   analysis , available at the rcsb validation server ( http://deposit.rcsb.org/validate/ ) , and validated using prosa - web   ( http://prosa.services.came.sbg.ac.at/prosa.php ) .", "human papillomavirus proteome database ( hpvpdb ) , the relational reserve , was developed using microsoft sql server 2005 as the back end .", "html , javascript , and cgi - perl - based web interfaces were employed to execute sql queries .", "the application layer , the web interface , and the backend relational tables were integrated using cgi - perl .", "home , about , tools , search , and advanced search interfaces can be explored to obtain strain- and protein - specific information .", "reserve comprises the strain - specific detailed informationon its name , sequencing status , submission details , date of submission , ncbi ids , disease types and subtypes , type of dna , genome length , molecular weight , nucleotide composition ( a , t , g , c , at , gc content ) , number of genes and proteins , and protein list .", "a genome map of each strain obtained by geneious 5.4.4 software ( available from http://www.geneious.com/ ) is also integrated in this page .", "users , through an advanced search option , can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome .", "each protein entry comprises protein overview ( name , locus , function , etc . ) , protein sequence information ( amino acid sequences with ncbi accession number with provision for direct protein blast   against ncbi nr database ) , protein parameters ( length , molecular weight , theoretical isoelectric point [ pi ] , amino acid composition , etc . ) , protein structure ( predicted 3d structure by homology modeling viewed by jmol ( available from http://www.jmol.org/ )   with the java platform , ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web .", "hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny .", "the original human papillomaviruses database was developed and hosted by the los alamos national laboratory ( lanl ) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases ( niaid ) . '", "human papillomaviruses : a compilation and analysis of nucleic acid and amino acid sequences ' contains four annual data books of papillomavirus information published in both paper and electronic form ( 1994 , 1995 , 1996 , and 1997 ) but has not been updated since 1997 .", "this contains nucleotide sequences of few hpv strains and other papillomaviruses , amino acid and nucleotide sequence alignments , analysis , related host sequences , and database communication .", "we did not find any structural information in that database . in hpvpdb , along with updated protein sequence information , genome and protein structure information is also provided .", "hpvpdb brings together comprehensive information on a total of 1,036 protein sequences and 743 predicted structures .", "the outcome of this study might provide a platform for simultaneous structural comparative analysis of these proteins and help in finding out variations in their structures to explore why different strains of hpv have causative associations with different types of cancers .", "further , this might also help in designing specific drugs or vaccines against specific strains of hpv .", "currently the hpvpdb is updated manually through online resources and available scientific publication review ; however , to sustain the quality , these data are analyzed and checked before incorporation into this reserve .", "meanwhile , to provide regular updates , our team is committed to searching for newly sequenced hpv strains , updating this reserve , and serving the users ."], "abstract_text": ["<S> human papillomavirus ( hpv ) infection is the leading cause of cancer mortality among women worldwide . </S>", "<S> the molecular understanding of hpv proteins has significant connotation for understanding their intrusion in the host and designing novel protein vaccines and anti - viral agents , etc . </S>", "<S> genomic , proteomic , structural , and disease - related information on hpv is available on the web ; yet , with trivial annotations and more so , it is not well customized for data analysis , host - pathogen interaction , strain - disease association , drug designing , and sequence analysis , etc . </S>", "<S> we attempted to design an online reserve with comprehensive information on hpv for the end users desiring the same </S>", "<S> . the human papillomavirus proteome database ( hpvpdb ) domiciles proteomic and genomic information on 150 hpv strains sequenced to date . </S>", "<S> simultaneous easy expandability and retrieval of the strain - specific data , with a provision for sequence analysis and exploration potential of predicted structures , and easy access for curation and annotation through a range of search options at one platform are a few of its important features . </S>", "<S> affluent information in this reserve could be of help for researchers involved in structural virology , cancer research , drug discovery , and vaccine design . </S>"], "labels": null, "section_names": ["Introduction", "Methods and Results", "Data retrieval and curation", "Protein structure prediction and validation", "Reserve architecture and design", "Reserve features", "Conclusion"], "sections": [["human papillomavirus ( hpv ) , a virus from the papillomavirus family , is capable of infecting humans . about 200 different strains of hpv identified , based on dna homology , have been found to be etiologically linked to cervical , vaginal , vulvar , penile , anal , oral , and plantar infectious lesions and cancers , as well [ 1 , 2 ] .", "the hpv genome , a double - stranded dna molecule , consists of 8 kilobase pairs ( kbp ) of nucleotides , which comprises 3 regions : 6 early open reading frames ( orfs)-e1 , e2 , e4 , e5 , e6 , and e7 ; 2 late orfs - l1 and l2 ; and an upstream regulatory region . a considerable volume of hpv specific information pertaining to its genome , proteome , structure , and disease association is available scattered on the web with trivial annotations", "; however , it is not customized to explore for data analysis , host - pathogen interaction , strain - disease association , drug designing , and sequence analysis , etc .", "therefore , we proposed to develop a comprehensive reserve on hpv with maximum possible inputs and outputs for the end users ."], ["amongst the existing 200 strains of hpv , 150 have been sequenced as of now , and their data available at the national center for biotechnology information ( ncbi ) .", "genome and proteome information of those viral strains was retrieved from ncbi . besides pubmed , various other online resources and published literature", "were also explored for validating genomic , proteomic , as well as strain and disease - associated information on hpv strains .", "hpv strain - specific information , such as strain name , sequencing status , sequencing centre , ncbi accession i d , associated disease information with references , genome statistics ( gc% , at% , a , t , g , c count , genes , and proteins ) , etc . , were curated from various online resources , and protein parameters ( length , molecular weight , isoelectric point ) were calculated using expasy protparam .", "modeller9v10   and the swiss - model   server were used for protein structure prediction .", "the stereochemistry of each protein was evaluated through procheck   analysis , available at the rcsb validation server ( http://deposit.rcsb.org/validate/ ) , and validated using prosa - web   ( http://prosa.services.came.sbg.ac.at/prosa.php ) .", "human papillomavirus proteome database ( hpvpdb ) , the relational reserve , was developed using microsoft sql server 2005 as the back end .", "html , javascript , and cgi - perl - based web interfaces were employed to execute sql queries .", "the application layer , the web interface , and the backend relational tables were integrated using cgi - perl .", "home , about , tools , search , and advanced search interfaces can be explored to obtain strain- and protein - specific information .", "reserve comprises the strain - specific detailed informationon its name , sequencing status , submission details , date of submission , ncbi ids , disease types and subtypes , type of dna , genome length , molecular weight , nucleotide composition ( a , t , g , c , at , gc content ) , number of genes and proteins , and protein list . a genome map of each strain obtained by geneious 5.4.4 software ( available from http://www.geneious.com/ ) is also integrated in this page .", "users , through an advanced search option , can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome .", "each protein entry comprises protein overview ( name , locus , function , etc . ) , protein sequence information ( amino acid sequences with ncbi accession number with provision for direct protein blast   against ncbi nr database ) , protein parameters ( length , molecular weight , theoretical isoelectric point [ pi ] , amino acid composition , etc . ) , protein structure ( predicted 3d structure by homology modeling viewed by jmol ( available from http://www.jmol.org/ )   with the java platform , ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web .", "hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny .", "the original human papillomaviruses database was developed and hosted by the los alamos national laboratory ( lanl ) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases ( niaid ) . '", "human papillomaviruses : a compilation and analysis of nucleic acid and amino acid sequences ' contains four annual data books of papillomavirus information published in both paper and electronic form ( 1994 , 1995 , 1996 , and 1997 ) but has not been updated since 1997 .", "this contains nucleotide sequences of few hpv strains and other papillomaviruses , amino acid and nucleotide sequence alignments , analysis , related host sequences , and database communication .", "we did not find any structural information in that database . in hpvpdb , along with updated protein sequence information , genome and protein structure information is also provided ."], ["amongst the existing 200 strains of hpv , 150 have been sequenced as of now , and their data available at the national center for biotechnology information ( ncbi ) .", "genome and proteome information of those viral strains was retrieved from ncbi . besides pubmed , various other online resources and published literature", "were also explored for validating genomic , proteomic , as well as strain and disease - associated information on hpv strains .", "hpv strain - specific information , such as strain name , sequencing status , sequencing centre , ncbi accession i d , associated disease information with references , genome statistics ( gc% , at% , a , t , g , c count , genes , and proteins ) , etc . , were curated from various online resources , and protein parameters ( length , molecular weight , isoelectric point ) were calculated using expasy protparam ."], ["modeller9v10   and the swiss - model   server were used for protein structure prediction .", "the stereochemistry of each protein was evaluated through procheck   analysis , available at the rcsb validation server ( http://deposit.rcsb.org/validate/ ) , and validated using prosa - web   ( http://prosa.services.came.sbg.ac.at/prosa.php ) ."], ["human papillomavirus proteome database ( hpvpdb ) , the relational reserve , was developed using microsoft sql server 2005 as the back end .", "html , javascript , and cgi - perl - based web interfaces were employed to execute sql queries .", "the application layer , the web interface , and the backend relational tables were integrated using cgi - perl ."], ["home , about , tools , search , and advanced search interfaces can be explored to obtain strain- and protein - specific information .", "reserve comprises the strain - specific detailed informationon its name , sequencing status , submission details , date of submission , ncbi ids , disease types and subtypes , type of dna , genome length , molecular weight , nucleotide composition ( a , t , g , c , at , gc content ) , number of genes and proteins , and protein list .", "a genome map of each strain obtained by geneious 5.4.4 software ( available from http://www.geneious.com/ ) is also integrated in this page .", "users , through an advanced search option , can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome .", "each protein entry comprises protein overview ( name , locus , function , etc . ) , protein sequence information ( amino acid sequences with ncbi accession number with provision for direct protein blast   against ncbi nr database ) , protein parameters ( length , molecular weight , theoretical isoelectric point [ pi ] , amino acid composition , etc . ) , protein structure ( predicted 3d structure by homology modeling viewed by jmol ( available from http://www.jmol.org/ )   with the java platform , ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web .", "hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny .", "the original human papillomaviruses database was developed and hosted by the los alamos national laboratory ( lanl ) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases ( niaid ) . '", "human papillomaviruses : a compilation and analysis of nucleic acid and amino acid sequences ' contains four annual data books of papillomavirus information published in both paper and electronic form ( 1994 , 1995 , 1996 , and 1997 ) but has not been updated since 1997 .", "this contains nucleotide sequences of few hpv strains and other papillomaviruses , amino acid and nucleotide sequence alignments , analysis , related host sequences , and database communication .", "we did not find any structural information in that database . in hpvpdb , along with updated protein sequence information , genome and protein structure information is also provided ."], ["hpvpdb brings together comprehensive information on a total of 1,036 protein sequences and 743 predicted structures .", "the outcome of this study might provide a platform for simultaneous structural comparative analysis of these proteins and help in finding out variations in their structures to explore why different strains of hpv have causative associations with different types of cancers .", "further , this might also help in designing specific drugs or vaccines against specific strains of hpv .", "currently the hpvpdb is updated manually through online resources and available scientific publication review ; however , to sustain the quality , these data are analyzed and checked before incorporation into this reserve .", "meanwhile , to provide regular updates , our team is committed to searching for newly sequenced hpv strains , updating this reserve , and serving the users ."]]}
{"article_id": "PMC2850825", "article_text": ["according to numerous estimations , 15% to 29% of patients with cataract have 1.5 diopters ( d ) of refractive astigmatism.1,2 cataract surgeons usually prefer to treat cataract and correct refractive ( spherical and astigmatic ) disorders at the time of the surgery .", "corneal incision ( astigmatic keratotomy ) and peripheral corneal relaxing incisions ( pcris ) were performed to treat these disorders.3,4 the main drawbacks of these approaches are that the outcome depends on multiple factors as the patient s age , the depth and length of the incision , complications related to wound healing , epithelial defects , or induction of dry - eye symptoms .", "these parameters affect the visual outcome in a unpredictable way so the corneal incisions are not considered a reliable method for astigmatism correction .", "toric intraocular lens ( iols ) implantation was introduced in the 1990 s as an option for astigmatism correction in cataract patients .", "initially they presented the disadvantage of postoperative rotation that decreased the visual outcome.58 new toric iol designs ( acrysof   toric iol ; alcon , fort worth , tx ) , approved by the us food and drug administration ( fda ) at the end of 2005 , have been found to be more stable and appear to be the preferred iol for correcting preexisting astigmatism in conjunction with cataract surgery.9,10 the acrysof   iols are available in three options : t3 , t4 , and t5 of astigmatic correction 1.5 , 2.25 , 3.00 d , respectively ( at the iol plane ) .", "the aim of this study is to report the clinical results of acrysof   toric iols implantation for preexisting astigmatism correction and compare the postoperative rotation of the iols one and six months postoperatively .", "this prospective study included eyes that had cataract surgery at the papageorgiou general hospital , thessaloniki , greece , between may 2008 and december 2008 .", "the study was conducted according to the tenets of the declaration of helsinki and patients gave informed consent after the nature and intent of the study had been fully explained to them .", "inclusion criteria were : cataract , age 70 years or younger , and preoperative regular corneal astigmatism greater than 1.00 d. exclusion criteria were : glaucoma , corneal disease , previous corneal or intraocular surgery , macular degeneration or retinopathy , and history of ocular inflammation .", "each patient had a complete ophthalmologic examination , including visual acuity ( va ) , slit - lamp examination , intraocular pressure ( iop ) measurement , and dilated fundus examination .", "topography ( magellan mapper ; nidek technologies , padova , italy ) , automated refractometry ( rk600 ; reichert , inc .", ", depew , ny ) , and ultrasonic immersion biometry ( ocuscan rxp ; alcon ) were performed to determine the appropriate iol spherical power .", "cylindrical power and axis placement to achieve emmetropia were calculated using an online toric iol calculator program ( available from http://www.acrysoforiccalculator.com/ ) .", "the 0180 axis was marked with the patient in a sitting position to avoid cyclotorsion using the nuijts / lane preoperative toric reference corneal marker ( ae-2791tbl ; asico , westmont , il ) ( figure 1 ) .", "intraoperatively , the desired implantation axis was marked using an intra - op toric axis marker ii ( ae-2794 ; asico ) ( figure 2 ) .", "a foldable iol was implanted in the capsular bag through a 2.75 mm limbal incision on 110. the toric iol was injected with a monarch - ii injector ( alcon ) and placed around 1015 off axis before the ophthalmic viscosurgical device ( sodium hyaluronate 1% ) was removed .", "after ophthalmic viscosurgical device removal , the iol was rotated to its final position by exactly aligning the toric reference marks with the limbal implantation axis marks .", "all surgeries were performed by the same experienced surgeon ( it ) using topical anesthesia and a standard divide - and - conquer phacoemulsification technique . because of the intraoperative marker design and the pen mark fading during the operation in several cases , it was difficult to assess the proper alignment of the iol after its placement . because of this difficulty , an image was captured from the real - time streaming recording of the surgery and was assessed blindly by a second operator using a commercially available software ( screen protractor ; iconico , ny , usa ) ( unpublished data ) . to overcome this difficulty we consider that the use of the marker set consisting of beveled degree gauge ( ae-1590 ; asico ) and maloney astigmatism axis marker ( ae-2741 ; asico ) is more appropriate for the whole procedure because it allows maximum visibility .", "the on - screen assessment of the axis remains a useful tool for postoperative evaluation of the iol location ( figure 3 ) .", "measurements of visual acuity ( using the snellen opto - type at a six - meter distance ) , iop , and comprehensive slit lamp examination were performed at one - day and one- and six - month postoperative visits . at the one- and six - month follow - up", ", a digital photograph and corneal topography ( only at the one - month visit ) were obtained to estimate the postoperative rotation of the iol using the software tools mentioned above ( figure 1 ) .", "outcomes of interest included uncorrected va , cylindrical astigmatism power before and after iol implantation , and the possible rotation of the iol one and six months after the operation ( when the initial desired place was at 0 ) .", "absolute values of the rotation used for the analysis after detecting rotation was not under investigation .", "completed data forms were analyzed with microsoft excel 2007 ( microsoft corporation , redmond , wa ) and spss software ( version 16.0 ; spss inc . , chicago , il ) .", "twenty - nine eyes of 19 patients ( mean age 63.03  5.42 years ) were enrolled in this study .", "uncorrected va was found to be 5/10 or more in 26 of 29 eyes ( 89.7% ) and 8/10 or more in 19 of 29 patients ( 65.5% ) .", "preoperative and postoperative corneal topography showed significant reduction of refractive astigmatism in all eyes after the surgery .", "mean power of the astigmatism was 2.38  0.91 d ( range 1.55 d ) preoperatively and 0.64  0.61 d ( range 02.5 d ) postoperatively ( figure 4 ) .", "one month postoperation , the mean toric iol axis rotation was 2.2  1.5 ( range 0.67.8 ) .", "six months postoperation , the mean toric iol axis rotation was 2.7  1.5 ( range 0.98.4 ) .", "the later rotation occurred between one and six months , and was found to be more than one degree ( 1.1 ) only in one eye ( 3% ) and in all other cases was less than this value ( figure 5 ) .", "it is known that one degree of deviation causes 3.3% reduction of the iol cylindrical power .", "calculated reduction of the desired correction ( in diopters ) because of the observed rotation ( mean value , one and two standard deviations ) for the three iol models is shown in table 1 .", "no eye had secondary surgery to reposition the iol axis within the six - month postoperative period .", "the results of our study corroborate previous studies,11 which demonstrates that proper selection and preoperative examination of patients followed by uncomplicated iol implantation of one - piece hydrophobic acrylic toric iols results in acceptable stability and visual outcome .", "in particular , the postoperative rotation of toric iols appears to occur in the early postoperative period ( 1 month ) and remains constant later than six months .", "the lens rotates until a fibronectin and collagen adhesion develops between the iol and the posterior capsule , which prevents any further rotary motion.12 the amount of cylindrical correction reduction due to postoperative rotation was not large enough13 to affect the expected end result in final uncorrected distance va .", "acrysof   one - piece hydrophobic acrylic toric iols implantation shows satisfactory stability , acceptable clinical results , and is an exceptional option for correction of refractive astigmatism ."], "abstract_text": ["<S> purpose : to present clinical results of toric intraocular lens ( iol ) implantation for preexisting astigmatism correction and determine the time of any postoperative rotation.patients and methods : twenty - nine eyes of 19 patients underwent uncomplicated phacoemulsification and were implanted with an acrysof  toric iol . uncorrected visual acuity , residual astigmatism , and postoperative rotation of the iol were estimated one and six months after the operation.results:uncorrected visual acuity was 0.5 in 26 of 29 eyes ( 89.7% ) and 0.8 in 19 of 29 patients ( 65.5% ) . </S>", "<S> the mean toric iol axis rotation was 2.2  1.5 ( range 0.67.8 ) one month postoperation and 2.7  1.5 ( range 0.98.4 ) six months postoperation.conclusion:implantation of one - piece hydrophobic acrylic toric iols appears to have acceptable stability , which encourages visual outcome and emerges as an attractive alternative for correction of refractive astigmatism . </S>"], "labels": null, "section_names": ["Introduction", "Material and methods", "Results", "Discussion", "Conclusion"], "sections": [["according to numerous estimations , 15% to 29% of patients with cataract have 1.5 diopters ( d ) of refractive astigmatism.1,2 cataract surgeons usually prefer to treat cataract and correct refractive ( spherical and astigmatic ) disorders at the time of the surgery .", "corneal incision ( astigmatic keratotomy ) and peripheral corneal relaxing incisions ( pcris ) were performed to treat these disorders.3,4 the main drawbacks of these approaches are that the outcome depends on multiple factors as the patient s age , the depth and length of the incision , complications related to wound healing , epithelial defects , or induction of dry - eye symptoms .", "these parameters affect the visual outcome in a unpredictable way so the corneal incisions are not considered a reliable method for astigmatism correction .", "toric intraocular lens ( iols ) implantation was introduced in the 1990 s as an option for astigmatism correction in cataract patients .", "initially they presented the disadvantage of postoperative rotation that decreased the visual outcome.58 new toric iol designs ( acrysof   toric iol ; alcon , fort worth , tx ) , approved by the us food and drug administration ( fda ) at the end of 2005 , have been found to be more stable and appear to be the preferred iol for correcting preexisting astigmatism in conjunction with cataract surgery.9,10 the acrysof   iols are available in three options : t3 , t4 , and t5 of astigmatic correction 1.5 , 2.25 , 3.00 d , respectively ( at the iol plane ) .", "the aim of this study is to report the clinical results of acrysof   toric iols implantation for preexisting astigmatism correction and compare the postoperative rotation of the iols one and six months postoperatively ."], ["this prospective study included eyes that had cataract surgery at the papageorgiou general hospital , thessaloniki , greece , between may 2008 and december 2008 .", "the study was conducted according to the tenets of the declaration of helsinki and patients gave informed consent after the nature and intent of the study had been fully explained to them .", "inclusion criteria were : cataract , age 70 years or younger , and preoperative regular corneal astigmatism greater than 1.00 d. exclusion criteria were : glaucoma , corneal disease , previous corneal or intraocular surgery , macular degeneration or retinopathy , and history of ocular inflammation .", "each patient had a complete ophthalmologic examination , including visual acuity ( va ) , slit - lamp examination , intraocular pressure ( iop ) measurement , and dilated fundus examination .", "topography ( magellan mapper ; nidek technologies , padova , italy ) , automated refractometry ( rk600 ; reichert , inc .", ", depew , ny ) , and ultrasonic immersion biometry ( ocuscan rxp ; alcon ) were performed to determine the appropriate iol spherical power .", "cylindrical power and axis placement to achieve emmetropia were calculated using an online toric iol calculator program ( available from http://www.acrysoforiccalculator.com/ ) .", "the 0180 axis was marked with the patient in a sitting position to avoid cyclotorsion using the nuijts / lane preoperative toric reference corneal marker ( ae-2791tbl ; asico , westmont , il ) ( figure 1 ) .", "intraoperatively , the desired implantation axis was marked using an intra - op toric axis marker ii ( ae-2794 ; asico ) ( figure 2 ) .", "a foldable iol was implanted in the capsular bag through a 2.75 mm limbal incision on 110. the toric iol was injected with a monarch - ii injector ( alcon ) and placed around 1015 off axis before the ophthalmic viscosurgical device ( sodium hyaluronate 1% ) was removed .", "after ophthalmic viscosurgical device removal , the iol was rotated to its final position by exactly aligning the toric reference marks with the limbal implantation axis marks .", "all surgeries were performed by the same experienced surgeon ( it ) using topical anesthesia and a standard divide - and - conquer phacoemulsification technique . because of the intraoperative marker design and the pen mark fading during the operation in several cases , it was difficult to assess the proper alignment of the iol after its placement . because of this difficulty , an image was captured from the real - time streaming recording of the surgery and was assessed blindly by a second operator using a commercially available software ( screen protractor ; iconico , ny , usa ) ( unpublished data ) . to overcome this difficulty we consider that the use of the marker set consisting of beveled degree gauge ( ae-1590 ; asico ) and maloney astigmatism axis marker ( ae-2741 ; asico ) is more appropriate for the whole procedure because it allows maximum visibility .", "the on - screen assessment of the axis remains a useful tool for postoperative evaluation of the iol location ( figure 3 ) .", "measurements of visual acuity ( using the snellen opto - type at a six - meter distance ) , iop , and comprehensive slit lamp examination were performed at one - day and one- and six - month postoperative visits . at the one- and six - month follow - up", ", a digital photograph and corneal topography ( only at the one - month visit ) were obtained to estimate the postoperative rotation of the iol using the software tools mentioned above ( figure 1 ) .", "outcomes of interest included uncorrected va , cylindrical astigmatism power before and after iol implantation , and the possible rotation of the iol one and six months after the operation ( when the initial desired place was at 0 ) .", "absolute values of the rotation used for the analysis after detecting rotation was not under investigation .", "completed data forms were analyzed with microsoft excel 2007 ( microsoft corporation , redmond , wa ) and spss software ( version 16.0 ; spss inc . , chicago , il ) ."], ["twenty - nine eyes of 19 patients ( mean age 63.03  5.42 years ) were enrolled in this study .", "uncorrected va was found to be 5/10 or more in 26 of 29 eyes ( 89.7% ) and 8/10 or more in 19 of 29 patients ( 65.5% ) .", "preoperative and postoperative corneal topography showed significant reduction of refractive astigmatism in all eyes after the surgery .", "mean power of the astigmatism was 2.38  0.91 d ( range 1.55 d ) preoperatively and 0.64  0.61 d ( range 02.5 d ) postoperatively ( figure 4 ) .", "one month postoperation , the mean toric iol axis rotation was 2.2  1.5 ( range 0.67.8 ) .", "six months postoperation , the mean toric iol axis rotation was 2.7  1.5 ( range 0.98.4 ) .", "the later rotation occurred between one and six months , and was found to be more than one degree ( 1.1 ) only in one eye ( 3% ) and in all other cases was less than this value ( figure 5 ) .", "it is known that one degree of deviation causes 3.3% reduction of the iol cylindrical power .", "calculated reduction of the desired correction ( in diopters ) because of the observed rotation ( mean value , one and two standard deviations ) for the three iol models is shown in table 1 .", "no eye had secondary surgery to reposition the iol axis within the six - month postoperative period ."], ["the results of our study corroborate previous studies,11 which demonstrates that proper selection and preoperative examination of patients followed by uncomplicated iol implantation of one - piece hydrophobic acrylic toric iols results in acceptable stability and visual outcome .", "in particular , the postoperative rotation of toric iols appears to occur in the early postoperative period ( 1 month ) and remains constant later than six months .", "the lens rotates until a fibronectin and collagen adhesion develops between the iol and the posterior capsule , which prevents any further rotary motion.12 the amount of cylindrical correction reduction due to postoperative rotation was not large enough13 to affect the expected end result in final uncorrected distance va ."], ["acrysof   one - piece hydrophobic acrylic toric iols implantation shows satisfactory stability , acceptable clinical results , and is an exceptional option for correction of refractive astigmatism ."]]}
{"article_id": "PMC3363989", "article_text": ["song is a complex , learned motor skill that involves the precise coordination of vocal and respiratory musculature in order to produce highly stereotyped renditions of a memorized song model .", "two pathways contributed to this behavior are identified in the songbird forebrain : the motor pathway that is required throughout life for normal song production , and the anterior forebrain pathway ( afp ) which is necessary for song learning and plasticity [ 13 ] .", "hvc ( used as the proper name ) is the beginning nucleus of both pathways and projects to the robust nucleus of the arcopallium ( ra ) and afp .", "the motor pathway is from hvc to ra , and then ra projects to brainstem nuclei controlling the vocal and respiratory muscles . during song , ra neurons in adult birds generate a highly stereotyped sequence of bursts [ 5 , 6 ] and also receive input from lateral magnocellular nucleus of the anterior nidopallium ( lman ) , the output nucleus of afp .", "notably , recordings from lman neurons projecting to ra revealed highly variable spiking activity across song renditions , suggesting that lman may act as a source of variability .", "these results indicate that synaptic activity in lman - ra synaptic transmission is crucial to song plasticity [ 8 , 9 ] , but the detailed mechanism is unclear . it has been observed that ra received excitatory glutamatergic inputs from hvc and lman .", "the former is mediated by a mixture of n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid- ( ampa- ) type receptors [ 4 , 10 , 11 ] , and the latter is mediated almost exclusively by nmda - type receptors .", "lman - ra synaptic transmission is necessary for the variability of song in adult zebra finch , suggesting that the activity of nmda receptor on the lman - ra synapses might induce the activity pattern change of ra neurons and change the bursts that control the vocal and respiratory muscles .", "the control of gain , which refers to modulation of a neuron 's responsiveness to input , is critically important for normal sensory , cognitive , and motor functions [ 1215 ] .", "gain control is achieved through a divisive process and is observed as a change in the slope of the input - output relationship .", "such gain changes are frequently observed in the responses of cortical neurons and are thought to play an important role in neural computations .", "modulations of gain have been observed in the enhancement of neural responses by attention [ 12 , 16 ] and can also be induced pharmacologically . in a neuron model ,", "nmda receptors caused such membrane potential fluctuations between a hyperpolarized down - state and a depolarized up - state , which may gate the information and gain modulation .", "here , we test the gain change of ra projection neurons after exogenous nmda was applied to selectively activate nmda receptors , using whole - cell current clamp recording in adult male zebra finch slices .", "brain slices were prepared from adult male zebra finches ( taeniopygia guttata ) ( > 90 days old ) obtained from commercial suppliers as previously described [ 19 , 20 ] .", "brains were dissected into ice - cold , oxygenated ( 95% o2 and 5% co2 ) slice solution .", "slice solution consists of ( in mm ) sucrose 248 , kcl 5 , nahco3   28 , glucose 10 , mgso47h2o 1.3 , and nah2po4h2o 1.26 [ 21 , 22 ] .", "coronal brain slices ( 250  m thick ) containing ra were cut with a vibrating microtome ( ma752 , usa ) and collected in artificial cerebrospinal fluid ( acsf ) that had been warmed to 37c and subsequently allowed to cool to room temperature .", "slices were allowed to recover in the holding chamber for at least 1.5  h and were equilibrated to room temperature before recordings were made .", "standard acsf consists of ( in mm ) nacl 125 , nahco3 25 , nah2po4h2o 1.27 , kcl 2.5 , mgso47h2o 1.2 , cacl2   2.0 , and glucose 25 and was adjusted with sucrose to a final osmolarity of 350  mos . for recording ,", "a slice was transferred to a submerge - type chamber where it was continuously exposed to acsf , saturated with 95% o2 and 5% co2 at room temperature ( 2226c ) at the rate of 2.0  ml / min .", "ra and the surrounding tissues were distinguishable under bx51wi microscope connected with a dic - ir video camera ( olympus , japan ) . at high magnification ( 40x ) , ra neurons were visualized and the recordings were made from healthy cells .", "recording pipettes were fabricated from borosilicate glass ( sutter instruments , usa ) using a flaming - brown puller ( model p-97 , sutter instruments , usa ) and were filled with the pipette solution containing ( in mm ) : k meso4   120 , nacl 5 , hepes 10 , egta 2 , mg - atp 2 , and na3-gtp 0.3 ( ph 7.3 - 7.4 , and 340  mos ) .", "the recording pipettes , which had resistances ranging from 3 to 5  m , were positioned using an integrated motorized control system ( sutter instruments , usa ) .", "the potential changes after application of nmda were corrected during the input - output stimulation protocols .", "the series resistance ( rs ) , between 15  m and 30  m , was compensated using the bridge balance .", "all agents were applied by changing the bath perfusate from standard acsf to modified acsf in which various drugs were simply added . unless indicated otherwise ,", "the clampfit 9.2 ( axon instruments , usa ) , mini 6.0 ( synaptosoft inc . , fort lee , nj , usa ) , and originpro 8.0 ( originlab , nothampton , ma , usa ) were used for analysis .", "events with peak amplitude of 50  mv or higher and a rise time of about 0.5  ms were detected automatically , and the results were analyzed with origin pro 8.0 .", "the steady - state spike rate was estimated by counting the number of spikes in 1 second , and the result was plotted versus the intensity of the injected current ( f - i relationship ) .", "the slope of the f - i relationship ( referred to as gain ) was estimated by linear fitting .", "slope parameters were estimated separately for individual neurons and that figures 4 and 5 contain mean slope values averaged for the whole groups of neurons .", "the amplitude of ahp was quantified as the difference between spike threshold and the lowest point of ahp . throughout", "means were compared using paired two - tailed student 's t - test or one - way anova followed by scheffe 's multiple comparison .", "stable whole - cell recordings were obtained in 160  ra projection neurons from 51 adult male zebra finches .", "a neuron with the resting membrane potential < 50  mv and an overshooting action potential was considered viable .", "ra projection neuron was identified by a larger soma , time - dependent inward rectifier induced by hyperpolarizing current and spontaneous regular spike firing or by depolarizing current pulses .", "the input - output relationship was examined by injecting current pulses from 0 to 90  pa with an increment of 10  pa and an interval of 15  s , and they were lasted for 1  s. traces of action potentials in response to 1  s current steps of 10 , 50 , and 90  pa were shown in figure 1(a ) .", "the slopes of the f - i relationships were 246.92  28.00  hz / na before nmda application and increased to 325.49  73.34 ", "furthermore , the input resistances were decreased by 20  m nmda in ra projection neurons ( p < 0.05 , n = 6 ) ( figure 2 ) .", "the afterhyperpolarization ( ahp ) of action potential underlies the modulation of firing frequency . to test the effect of nmda on spike ahp", "comparison of spike shape is shown in figure 3(a ) with 90  pa current injection before and after nmda application . in average", ", there was no significant change in the amplitude of ahp after nmda application ( p > 0.05 , n = 5 ) ( figure 3(b ) ) .", "then we used 50  m dl - apv , a nmda receptor antagonist , to check the selectivity of nmda receptor , and we found that the slope of f - i was not changed by dl - apv alone ( p > 0.05 ) .", "the slope was 255.78  66.69  hz / na and 269.34  56.96  hz / na ( p > 0.05 ) before and after application of 50  m dl - apv ( figure 4 ) , respectively .", "nmda increased the f - i slope 1.32 times ( p < 0.05 ) ; however , the gain modulation induced by nmda was completely abolished by dl - apv ( figure 4 ) .", "previous studies reported that ra projection neurons received glutamatergic and gabaergic inputs [ 23 , 24 ] , which might increase background synaptic activities and modulate the gain of neurons [ 25 , 26 ] . to determine the effect of ampa receptor and gaba receptor on gain modulation by nmda , we examined the effects of nmda in the presence of cnqx , an antagonist for ionotropic glutamate ampa receptors , or / and bicuculline methiodide ( bic ) , a competitive antagonist of gabaa receptors , respectively .", "bic ( 10  m ) accelerated the depolarization by nmda application , but bic alone had little effect on the f - i slope . in the presence of bic ,", "the mean initial slopes were 276.46  59.4 and 332.6  40.02  hz / na before and during application of nmda .", "the f - i slope was increased from 1.32 to 1.39 in the presence of nmda and bic ( p > 0.05 ) ; that is , there is an increase of 105.30% over the nmda treatment ( figure 5 ; p > 0.05 , unpaired t - test ) .", "cnqx ( 10  m ) decreased the membrane potential and abolished the spontaneous spikes of ra projection neurons sometimes but did not affect the slope of f - i relationship . in the presence of cnqx ,", "nmda application hardly changed the f - i slope of ra projection neurons no matter whether bic was present or not ( figure 5 ; p > 0.05 ) .", "these results indicated that the ampa receptor - dependent excitability played a crucial role in the gain modulation by nmda .", "the slope was 0.97 or 1.11 in the presence of cnqx or cnqx and bic , respectively .", "the gain induced by nmda was significantly decreased by cnqx ( p < 0.05 ) .", "in this study , we showed that nmda increased the gain of ra projection neurons in adult zebra finch .", "nmda - induced gain increase was limited to firing frequencies less than 40  hz . the effect of nmda on gain was mediated through nmda receptors and might involve synaptic activities . in songbirds , we found that nmda increased the slope of f - i relationship in ra projection neurons , which was completely blocked by dl - apv , a nmda receptor selective antagonist , suggesting that nmda increased the sensitivity of ra projection neurons .", "the pharmacology of nmda receptors involved in gain modulation has been examined in mammals . in cat", "visual cortex , nmda increased the gain of the contrast - response ( c - r ) curve of the neuron in layers ii / iii and v / vi . in mouse , the thalamocortical neurons in the dorsal lateral geniculate nucleus play a key role in the generation of firing patterns through nmda receptors .", "the neural activity in the afp of songbirds can direct moment - by - moment changes in the primary motor areas responsible for generating song .", "singing - related activity in lman is more variable in pattern across repeated renditions during undirected song than directed song [ 28 , 29 ] .", "reported that the fundamental frequency of the syllable was shifted by stimulating the lman - ra pathway , which is mediated by nmda receptors .", "our results indicated that the activity of nmda receptor changed the sensitivity of ra projection neuron .", "ra , as a premotor nucleus , occupies an important position in the song system , integrating information from both pathways .", "ra projection neurons are responsible for transmitting song information to midbrain and brainstem vocal and respiratory structures . temporal pattern of spike bursts in ra projection neurons is associated with the timing of acoustic features of birdsong .", "precise timing of individual spikes has a close relationship with stereotypic behavior , which suggests that the song is represented in ra by a temporal code [ 5 , 31 ] .", "thus , the modulation of nmda receptors in the activities of ra projection neurons could be very important for the plasticity of birdsong .", "previous studies in slices demonstrated that nmda receptor achieves the nonlinear , multiplicative behavior seen in gain modulation by several mechanisms .", "mel presented results from compartmental modeling studies showing that dendrites containing nmda - type synaptic conductances and voltage - dependent na , k , and ca channels can impose nonlinear interactions between nearby synapses that give rise to approximate multiplications [ 32 , 33 ] . in thalamocortical cell of the rat and cat dlgn , nmda receptors contribute to the burst firing generated by the low - threshold ca potential and depend on the membrane potential range over which this type of firing occurs .", "in substantia nigra dopamine neurons , nmda receptor - mediated synaptic conductance generates transient high - frequency activity by rapidly but transiently overwhelming the conductance 's underlying action potential ahp and/or engaging postsynaptic voltage - dependent ion channels in a manner that overcomes the limiting effects of ahp .", "but our results showed that there was no significant change in spike ahp after nmda application .", "so the gain induced by nmda in ra projection neurons may not be mainly modulated by the change of spike shape , but by synaptic activities .", "the change in membrane potential has been shown to play an important role in the firing pattern regulation by nmda receptor , supporting the idea that depolarization acutely alters firing frequency .", "it has been shown that simple excitability or inhibition alone can induce a multiplicative gain change in the cortical neurons . in this model ,", "one set of synaptic inputs provided purely excitatory drive to a target neuron , and another set of combined excitatory / inhibitory inputs ( or balanced inputs ) provided the modulation . in this setup , changing the firing rates of the balanced inputs produced an approximately multiplicative gain modulation of the response to the purely excitatory drive . here", ", blockade of the excitatory or inhibitory transmission alone had no effect on the f - i slope of ra projection neuron , implying that simple excitability or inhibition alone can not induce a multiplicative gain change .", "however , our results did not exclude a role of spontaneous synaptic activity on gain modulation in vitro , which , due to the loss of synapses in slice , was not sufficient to produce a significant change in gain .  ", "ampa receptors are required for the gain modulation in our study , because blocking excitatory transmission mediated by ampa receptors almost abolished the effect of nmda on gain modulation .", "two reasons might be involved : one is the two states of nmda - base mechanism due to the special anatomical structure of ra .", "ra projection neurons receive inputs from two areas , hvc and lman . both are excitatory glutamatergic but have distinct postsynaptic properties .", "hvc - ra is largely mediated by ampa receptor , whereas lman - ra is almost completely mediated by nmda receptor .", "ampa / nmda receptor composition of different neuronal pathways is the foundation of gating and gain modulation .", "nmda receptor - rich pathway can gate ampa - rich input and increase the gain of a neuron responding to the input of ampa - rich pathway .", "the second is related to the intrinsic properties of nmda receptors , which are uniquely voltage dependent . except binding with neurotransmitter", "blockade of main excitatory inputs mediated by ampa receptor with cnqx hyperpolarized the membrane potential , which caused nmda receptors not activated .", "therefore , the ampa receptor - mediated excitatory transmission is essential for the gain modulation induced by nmda .", "it is well known that the nmda receptor plays a critical role in many physiological and pathological processes , such as learning , memory , neurodegeneration , epilepsy , and ischemia .", "the nmda receptor - mediated synaptic transmission satisfies the associative property of hebbian learning and in fact plays a critical role in its cellular model , long - term synaptic plasticity .", "nevertheless , pharmacological agents which block nmda receptors impair a variety of brain processes , suggesting that nmda receptor transmission also plays an important role beyond long - term memory and participates in shaping the dynamic activity of neural networks . in song system", ", the expression pattern of nmda receptor is related to the development of song learning [ 21 , 3740 ] .", "recent results show that the nmda receptor on the lman - ra synapses generates variability across different song renditions , thereby facilitating reinforced learning of songs . how nmda accomplishes this role is not clear .", "our results showed that nmda application increased the gain of ra projection neurons , in which ampa receptor was necessary .", "the ampa receptor - mediated excitability is derived only from hvc [ 10 , 11 ] .", "therefore , nmda receptor in lman - ra synapse might regulate hvc - ra synaptic transmission , which implicated that nmda receptor is more effective to modulate the syllable online .", "these findings suggest a mechanism by which nmda receptor can selectively modulate behaviorally relevant excitatory inputs ."], "abstract_text": ["<S> the song of zebra finch is stable in life after it was learned successfully . </S>", "<S> vocal plasticity is thought to be a motor exploration that can support continuous learning and optimization of performance . </S>", "<S> the activity of ra , an important pre - motor nucleus in songbird 's brain , influences the song directly . </S>", "<S> this variability in adult birdsong is associated with the activity of nmda receptors in lman - ra synapses , but the detailed mechanism is unclear . </S>", "<S> the control of gain refers to modulation of a neuron 's responsiveness to input and is critically important for normal sensory , cognitive , and motor functions . here , we observed the change of gain in ra projection neurons after exogenous nmda was applied to activate nmda receptors using the whole - cell current clamp recording . </S>", "<S> we found that nmda substantially increased the slope ( gain ) of the firing rate - current relationship in ra projection neurons . </S>", "<S> the ampa receptor - dependent excitability played a crucial role in the modulation of gain by nmda . </S>", "<S> these results suggested that nmda receptors may regulate the dynamics of ra projection neurons by input - output gain . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion"], "sections": [["song is a complex , learned motor skill that involves the precise coordination of vocal and respiratory musculature in order to produce highly stereotyped renditions of a memorized song model .", "two pathways contributed to this behavior are identified in the songbird forebrain : the motor pathway that is required throughout life for normal song production , and the anterior forebrain pathway ( afp ) which is necessary for song learning and plasticity [ 13 ] .", "hvc ( used as the proper name ) is the beginning nucleus of both pathways and projects to the robust nucleus of the arcopallium ( ra ) and afp .", "the motor pathway is from hvc to ra , and then ra projects to brainstem nuclei controlling the vocal and respiratory muscles . during song , ra neurons in adult birds generate a highly stereotyped sequence of bursts [ 5 , 6 ] and also receive input from lateral magnocellular nucleus of the anterior nidopallium ( lman ) , the output nucleus of afp .", "notably , recordings from lman neurons projecting to ra revealed highly variable spiking activity across song renditions , suggesting that lman may act as a source of variability .", "these results indicate that synaptic activity in lman - ra synaptic transmission is crucial to song plasticity [ 8 , 9 ] , but the detailed mechanism is unclear . it has been observed that ra received excitatory glutamatergic inputs from hvc and lman .", "the former is mediated by a mixture of n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid- ( ampa- ) type receptors [ 4 , 10 , 11 ] , and the latter is mediated almost exclusively by nmda - type receptors .", "lman - ra synaptic transmission is necessary for the variability of song in adult zebra finch , suggesting that the activity of nmda receptor on the lman - ra synapses might induce the activity pattern change of ra neurons and change the bursts that control the vocal and respiratory muscles .", "the control of gain , which refers to modulation of a neuron 's responsiveness to input , is critically important for normal sensory , cognitive , and motor functions [ 1215 ] .", "gain control is achieved through a divisive process and is observed as a change in the slope of the input - output relationship .", "such gain changes are frequently observed in the responses of cortical neurons and are thought to play an important role in neural computations .", "modulations of gain have been observed in the enhancement of neural responses by attention [ 12 , 16 ] and can also be induced pharmacologically . in a neuron model ,", "nmda receptors caused such membrane potential fluctuations between a hyperpolarized down - state and a depolarized up - state , which may gate the information and gain modulation .", "here , we test the gain change of ra projection neurons after exogenous nmda was applied to selectively activate nmda receptors , using whole - cell current clamp recording in adult male zebra finch slices ."], ["brain slices were prepared from adult male zebra finches ( taeniopygia guttata ) ( > 90 days old ) obtained from commercial suppliers as previously described [ 19 , 20 ] .", "brains were dissected into ice - cold , oxygenated ( 95% o2 and 5% co2 ) slice solution .", "slice solution consists of ( in mm ) sucrose 248 , kcl 5 , nahco3   28 , glucose 10 , mgso47h2o 1.3 , and nah2po4h2o 1.26 [ 21 , 22 ] .", "coronal brain slices ( 250  m thick ) containing ra were cut with a vibrating microtome ( ma752 , usa ) and collected in artificial cerebrospinal fluid ( acsf ) that had been warmed to 37c and subsequently allowed to cool to room temperature .", "slices were allowed to recover in the holding chamber for at least 1.5  h and were equilibrated to room temperature before recordings were made .", "standard acsf consists of ( in mm ) nacl 125 , nahco3 25 , nah2po4h2o 1.27 , kcl 2.5 , mgso47h2o 1.2 , cacl2   2.0 , and glucose 25 and was adjusted with sucrose to a final osmolarity of 350  mos . for recording ,", "a slice was transferred to a submerge - type chamber where it was continuously exposed to acsf , saturated with 95% o2 and 5% co2 at room temperature ( 2226c ) at the rate of 2.0  ml / min .", "ra and the surrounding tissues were distinguishable under bx51wi microscope connected with a dic - ir video camera ( olympus , japan ) . at high magnification ( 40x ) , ra neurons were visualized and the recordings were made from healthy cells .", "recording pipettes were fabricated from borosilicate glass ( sutter instruments , usa ) using a flaming - brown puller ( model p-97 , sutter instruments , usa ) and were filled with the pipette solution containing ( in mm ) : k meso4   120 , nacl 5 , hepes 10 , egta 2 , mg - atp 2 , and na3-gtp 0.3 ( ph 7.3 - 7.4 , and 340  mos ) .", "the recording pipettes , which had resistances ranging from 3 to 5  m , were positioned using an integrated motorized control system ( sutter instruments , usa ) .", "the potential changes after application of nmda were corrected during the input - output stimulation protocols .", "the series resistance ( rs ) , between 15  m and 30  m , was compensated using the bridge balance .", "all agents were applied by changing the bath perfusate from standard acsf to modified acsf in which various drugs were simply added . unless indicated otherwise ,", "the clampfit 9.2 ( axon instruments , usa ) , mini 6.0 ( synaptosoft inc . , fort lee , nj , usa ) , and originpro 8.0 ( originlab , nothampton , ma , usa ) were used for analysis .", "events with peak amplitude of 50  mv or higher and a rise time of about 0.5  ms were detected automatically , and the results were analyzed with origin pro 8.0 .", "the steady - state spike rate was estimated by counting the number of spikes in 1 second , and the result was plotted versus the intensity of the injected current ( f - i relationship ) .", "the slope of the f - i relationship ( referred to as gain ) was estimated by linear fitting .", "slope parameters were estimated separately for individual neurons and that figures 4 and 5 contain mean slope values averaged for the whole groups of neurons .", "the amplitude of ahp was quantified as the difference between spike threshold and the lowest point of ahp . throughout", "means were compared using paired two - tailed student 's t - test or one - way anova followed by scheffe 's multiple comparison ."], ["stable whole - cell recordings were obtained in 160  ra projection neurons from 51 adult male zebra finches .", "a neuron with the resting membrane potential < 50  mv and an overshooting action potential was considered viable .", "ra projection neuron was identified by a larger soma , time - dependent inward rectifier induced by hyperpolarizing current and spontaneous regular spike firing or by depolarizing current pulses .", "the input - output relationship was examined by injecting current pulses from 0 to 90  pa with an increment of 10  pa and an interval of 15  s , and they were lasted for 1  s. traces of action potentials in response to 1  s current steps of 10 , 50 , and 90  pa were shown in figure 1(a ) .", "the slopes of the f - i relationships were 246.92  28.00  hz / na before nmda application and increased to 325.49  73.34 ", "furthermore , the input resistances were decreased by 20  m nmda in ra projection neurons ( p < 0.05 , n = 6 ) ( figure 2 ) .", "the afterhyperpolarization ( ahp ) of action potential underlies the modulation of firing frequency . to test the effect of nmda on spike ahp", "comparison of spike shape is shown in figure 3(a ) with 90  pa current injection before and after nmda application . in average", ", there was no significant change in the amplitude of ahp after nmda application ( p > 0.05 , n = 5 ) ( figure 3(b ) ) .", "then we used 50  m dl - apv , a nmda receptor antagonist , to check the selectivity of nmda receptor , and we found that the slope of f - i was not changed by dl - apv alone ( p > 0.05 ) .", "the slope was 255.78  66.69  hz / na and 269.34  56.96  hz / na ( p > 0.05 ) before and after application of 50  m dl - apv ( figure 4 ) , respectively .", "nmda increased the f - i slope 1.32 times ( p < 0.05 ) ; however , the gain modulation induced by nmda was completely abolished by dl - apv ( figure 4 ) .", "previous studies reported that ra projection neurons received glutamatergic and gabaergic inputs [ 23 , 24 ] , which might increase background synaptic activities and modulate the gain of neurons [ 25 , 26 ] . to determine the effect of ampa receptor and gaba receptor on gain modulation by nmda , we examined the effects of nmda in the presence of cnqx , an antagonist for ionotropic glutamate ampa receptors , or / and bicuculline methiodide ( bic ) , a competitive antagonist of gabaa receptors , respectively .", "bic ( 10  m ) accelerated the depolarization by nmda application , but bic alone had little effect on the f - i slope . in the presence of bic ,", "the mean initial slopes were 276.46  59.4 and 332.6  40.02  hz / na before and during application of nmda .", "the f - i slope was increased from 1.32 to 1.39 in the presence of nmda and bic ( p > 0.05 ) ; that is , there is an increase of 105.30% over the nmda treatment ( figure 5 ; p > 0.05 , unpaired t - test ) .", "cnqx ( 10  m ) decreased the membrane potential and abolished the spontaneous spikes of ra projection neurons sometimes but did not affect the slope of f - i relationship . in the presence of cnqx ,", "nmda application hardly changed the f - i slope of ra projection neurons no matter whether bic was present or not ( figure 5 ; p > 0.05 ) .", "these results indicated that the ampa receptor - dependent excitability played a crucial role in the gain modulation by nmda .", "the slope was 0.97 or 1.11 in the presence of cnqx or cnqx and bic , respectively .", "the gain induced by nmda was significantly decreased by cnqx ( p < 0.05 ) ."], ["in this study , we showed that nmda increased the gain of ra projection neurons in adult zebra finch .", "nmda - induced gain increase was limited to firing frequencies less than 40  hz . the effect of nmda on gain was mediated through nmda receptors and might involve synaptic activities . in songbirds , we found that nmda increased the slope of f - i relationship in ra projection neurons , which was completely blocked by dl - apv , a nmda receptor selective antagonist , suggesting that nmda increased the sensitivity of ra projection neurons .", "the pharmacology of nmda receptors involved in gain modulation has been examined in mammals . in cat", "visual cortex , nmda increased the gain of the contrast - response ( c - r ) curve of the neuron in layers ii / iii and v / vi . in mouse , the thalamocortical neurons in the dorsal lateral geniculate nucleus play a key role in the generation of firing patterns through nmda receptors .", "the neural activity in the afp of songbirds can direct moment - by - moment changes in the primary motor areas responsible for generating song .", "singing - related activity in lman is more variable in pattern across repeated renditions during undirected song than directed song [ 28 , 29 ] .", "reported that the fundamental frequency of the syllable was shifted by stimulating the lman - ra pathway , which is mediated by nmda receptors .", "our results indicated that the activity of nmda receptor changed the sensitivity of ra projection neuron .", "ra , as a premotor nucleus , occupies an important position in the song system , integrating information from both pathways .", "ra projection neurons are responsible for transmitting song information to midbrain and brainstem vocal and respiratory structures . temporal pattern of spike bursts in ra projection neurons is associated with the timing of acoustic features of birdsong .", "precise timing of individual spikes has a close relationship with stereotypic behavior , which suggests that the song is represented in ra by a temporal code [ 5 , 31 ] .", "thus , the modulation of nmda receptors in the activities of ra projection neurons could be very important for the plasticity of birdsong .", "previous studies in slices demonstrated that nmda receptor achieves the nonlinear , multiplicative behavior seen in gain modulation by several mechanisms .", "mel presented results from compartmental modeling studies showing that dendrites containing nmda - type synaptic conductances and voltage - dependent na , k , and ca channels can impose nonlinear interactions between nearby synapses that give rise to approximate multiplications [ 32 , 33 ] . in thalamocortical cell of the rat and cat dlgn , nmda receptors contribute to the burst firing generated by the low - threshold ca potential and depend on the membrane potential range over which this type of firing occurs .", "in substantia nigra dopamine neurons , nmda receptor - mediated synaptic conductance generates transient high - frequency activity by rapidly but transiently overwhelming the conductance 's underlying action potential ahp and/or engaging postsynaptic voltage - dependent ion channels in a manner that overcomes the limiting effects of ahp .", "but our results showed that there was no significant change in spike ahp after nmda application .", "so the gain induced by nmda in ra projection neurons may not be mainly modulated by the change of spike shape , but by synaptic activities .", "the change in membrane potential has been shown to play an important role in the firing pattern regulation by nmda receptor , supporting the idea that depolarization acutely alters firing frequency .", "it has been shown that simple excitability or inhibition alone can induce a multiplicative gain change in the cortical neurons . in this model ,", "one set of synaptic inputs provided purely excitatory drive to a target neuron , and another set of combined excitatory / inhibitory inputs ( or balanced inputs ) provided the modulation . in this setup , changing the firing rates of the balanced inputs produced an approximately multiplicative gain modulation of the response to the purely excitatory drive . here", ", blockade of the excitatory or inhibitory transmission alone had no effect on the f - i slope of ra projection neuron , implying that simple excitability or inhibition alone can not induce a multiplicative gain change .", "however , our results did not exclude a role of spontaneous synaptic activity on gain modulation in vitro , which , due to the loss of synapses in slice , was not sufficient to produce a significant change in gain .  ", "ampa receptors are required for the gain modulation in our study , because blocking excitatory transmission mediated by ampa receptors almost abolished the effect of nmda on gain modulation .", "two reasons might be involved : one is the two states of nmda - base mechanism due to the special anatomical structure of ra .", "ra projection neurons receive inputs from two areas , hvc and lman . both are excitatory glutamatergic but have distinct postsynaptic properties .", "hvc - ra is largely mediated by ampa receptor , whereas lman - ra is almost completely mediated by nmda receptor .", "ampa / nmda receptor composition of different neuronal pathways is the foundation of gating and gain modulation .", "nmda receptor - rich pathway can gate ampa - rich input and increase the gain of a neuron responding to the input of ampa - rich pathway .", "the second is related to the intrinsic properties of nmda receptors , which are uniquely voltage dependent . except binding with neurotransmitter", "blockade of main excitatory inputs mediated by ampa receptor with cnqx hyperpolarized the membrane potential , which caused nmda receptors not activated .", "therefore , the ampa receptor - mediated excitatory transmission is essential for the gain modulation induced by nmda .", "it is well known that the nmda receptor plays a critical role in many physiological and pathological processes , such as learning , memory , neurodegeneration , epilepsy , and ischemia .", "the nmda receptor - mediated synaptic transmission satisfies the associative property of hebbian learning and in fact plays a critical role in its cellular model , long - term synaptic plasticity .", "nevertheless , pharmacological agents which block nmda receptors impair a variety of brain processes , suggesting that nmda receptor transmission also plays an important role beyond long - term memory and participates in shaping the dynamic activity of neural networks . in song system", ", the expression pattern of nmda receptor is related to the development of song learning [ 21 , 3740 ] .", "recent results show that the nmda receptor on the lman - ra synapses generates variability across different song renditions , thereby facilitating reinforced learning of songs . how nmda accomplishes this role is not clear .", "our results showed that nmda application increased the gain of ra projection neurons , in which ampa receptor was necessary .", "the ampa receptor - mediated excitability is derived only from hvc [ 10 , 11 ] .", "therefore , nmda receptor in lman - ra synapse might regulate hvc - ra synaptic transmission , which implicated that nmda receptor is more effective to modulate the syllable online .", "these findings suggest a mechanism by which nmda receptor can selectively modulate behaviorally relevant excitatory inputs ."]]}
{"article_id": "PMC4189057", "article_text": ["first described by veterinarians , pythiosis is a serious life- and limb - threatening infection endemic to thailand , but rarely seen in the western hemisphere .", "known to infect immunocompetent horses , dogs , and other large animals,2 , 3 \n pythium infection was first reported in humans in 1989 in association with thalassemic hemoglobinopathy.4 , 5 human pythiosis is known to present in one of four clinical entities : subcutaneous , ocular , vascular , and disseminated . here", ", we present a unique case of vascular pythiosis managed with limb - sparing vascular bypass grafts complicated by pseudoaneurysm formation .", "the patient is a 17 year - old jamaican male with a history of paroxysmal nocturnal hemoglobinuria and severe aplastic anemia refractory to horse anti - thymoglobulin treatment , which was transfusion - dependent .", "one month prior to admission at our institution , he went fishing in a fresh water pond in jamaica and his left lower leg was caught in a bush . on the following day , he noticed a small bullae with a black center and later developed severely painful left inguinal swelling .", "he was admitted to a local hospital for the next several weeks , where all diagnostic testing performed was unremarkable .", "his pain persisted despite antibiotic and antifungal therapy , and he was later transferred to our institution for consideration of bone marrow transplant . on admission , he was a febrile with a white blood cell count of 6.2 ( k/l ) and platelet count of 18 ( k/l ) and was complaining of severe left leg pain .", "he had a 9    8  cm soft tissue defect with overlying escar on the posterior aspect of his left leg ( fig .", "1a ) , and his exam was also notable for an exquisitely tender and erythematous left inguinal mass .", "motor and sensory exam were intact in both lower extremities without pain upon passive hip flexion .", "several small , dark macules were noted along the lateral aspect of his left foot ( fig .", "mri showed a t2 bright left groin mass with edema along the fascial planes , and lower extremity angiogram demonstrated occlusion of the left common femoral artery with distal profunda femoris and mid - superficial femoral artery reconstitution ( fig .", "the patient was not a candidate for percutaneous intervention , and was taken to the operating room for exploration .", "visual inspection of the common femoral artery after exposure was significant for several patches of necrotic vessel wall .", "initial attempt with thrombectomy was unsuccessful , as the fogarty catheter actually perforated the anterior wall at the areas of necrosis .", "the patient ultimately received an extra - anatomic left external iliac to superficial femoral artery bypass using a nonreversed greater saphenous vein graft , as the external iliac was the closest available inflow that did not visually appear to be involved with the infection .", "intraoperative wound cultures were positive for pythium insidiosum and the patient was started on a combination of voriconazole , terbinafine , and amphotericin b. given his critical condition , an emergency investigational new drug request was drafted for permission to administer a p. insidiosum antigen vaccine available for compassionate use from the pan american veterinary laboratories .", "the vaccine was given once daily in one - week intervals in conjunction with subcutaneously injected interferon gamma ( ifn- ) , for a total of four weeks .", "three weeks after his initial surgery , the patient developed new sites of pain along the graft site and repeat imaging showed a 2.5  cm pseudoaneurysm at the proximal anastomosis to the external iliac artery with contrast extravasation ( fig .", "he was taken to the operating room for an extra - anatomic left common iliac artery to the previous femoral popliteal bypass graft using a reverse saphenous vein graft .", "the patient later developed a polymicrobial superinfection of his wounds , and his vascular repair continued to break down leading to increased ischemic pain .", "after several multidisciplinary discussions with the family that all medical interventions were exhausted , and that even highly aggressive surgical measures including hip disarticulation or hemipelvectomy could not guarantee source control , conservative measures were initiated and ultimately , the patient expired .", "this patient presented a major diagnostic challenge , and his disease process and subsequent management were previously unknown to our hospital .", "in retrospect , he presented with a classic case of vascular pythiosis , with an antecedent history of exposure to surface water and exam findings of vascular insufficiency , including pain out of proportion to exam , distal emboli , and an ischemic ulcer .", "the correct infectious diagnosis was not made until 1 month after exposure , after a vascular bypass was already performed .", "indeed , in a large multi - institutional study from thailand with a total of 102 cases of pythiosis , 60 patients received a diagnosis of vascular pythiosis , and only the patients who had undergone amputation survived .", "further review of the literature for reports of vascular pythiosis demonstrated that the majority of patients were treated with amputation without any signs of recurrence , and were followed for 5 to 35 months post - operatively.4 , 6 , 8 , 9 in our case , complete source control was not achieved during the first operation since the common femoral artery and the proximal portion of the superficial femoral artery were oversewn and left in place , standard practice for the treatment of peripheral arterial disease .", "incomplete source control eventually led to development of a pseudoaneurysm at the proximal anastomosis and additional surgery .", "while the second repair was cited in previously uninvolved tissue planes , it did not protect against further infection , likely because p. insidiosum is known to invade through proteinase secretion .", "the authors could find no reported cases of confirmed vascular pythiosis treated with limb - sparing procedures.2 , 7 , 11 , 12 medical therapy in the treatment of vascular pythiosis involves antifungal agents , immunotherapy , and iron chelation therapy .", "much of the difficulty in eradicating this organism stems from its complicated phylogenesis;2 , 6 , 14 it shares features with true fungi at the same time it is phylogenetically more closely related to algae and diatomeae . \n", "p. insidiosum develops mycelium like fungi , but its cell walls do not contain chitin and its cytoplasmic membrane lacks ergosterol , both of which are important targets of traditional antifungal drugs . despite this , drugs that affect cellular permeability , including amphotericin b , terbinafine , and the azoles , have been used with some clinical success.2 , 15 more recently , immunotherapy has been used as an adjunct to surgery and antimycotics .", "immunotherapy was first successfully used in humans in 1998 in a patient with a vascular p. insidiosum infection refractory to surgery and antifungals.2 , 16 a p. insidiosum vaccine delivered twice with a two - week interval resulted in a cure 1 year later .", "data collected from clinical trials and other reports estimate vaccine efficacy to be 5560%.2 , 17 the proposed mechanism of action of the p. insidiosum vaccine is to shift host adaptive immune response from a predominantly t helper 2 ( th2 ) response to a t helper 1 ( th1 ) response.2 , 17 \n pythium exoantigens are typically processed by antigen presenting cells ( apcs ) and presented to nave cd4 helper t cells ( th0 ) .", "this induces th0 cells to adopt a th2 phenotype secreting il-4 and il-5 , which trigger many downstream effects , including mast cell and eosinophil migration to the site of infection with subsequent degranulation , releasing inflammatory mediators and causing tissue damage .", "loss of tissue architecture then creates a more favorable environment for these pathogenic hyphae to take hold .", "conversion of a th2 to th1 response would favor secretion of ifn- and il-2 , leading to increased cytotoxic t lymphocyte recruitment and therefore , direct killing of p. insidiosum . in this case , the purpose of injecting ifn- subcutaneously was to augment a th1 response , and co - administration of gm - csf was to increase the efficiency of exoantigen presentation to apcs .", "another treatment modality used to control this infection is iron chelation therapy . given the association between pythium infection in patients with hemoglobinopathies with a long - standing history of blood transfusions4 , 7 and the discovery of a ferrochelatase gene in p. insidiosum ,", "other research efforts have focused on elucidating the role of iron overload in p. insidiosum pathogenesis .", "there is evidence that iron overload from transfusions contributes to derangements in host immunity by impairing lymphocyte function and altering phagocytosis , leading to a more susceptible state for infection6 , 19 and is in fact , a key virulence factor for the survival of p. insidiosum in the host .", "correcting iron overload has been cited as a treatment in infections common in thalassemia , and has also been used in pythiosis . in our patient ,", "in conclusion , this patient 's vascular p. insidiosum infection was refractory to optimized medical treatment , immunotherapy with cytokine injections , and surgery .", "attempted source control with limb salvage necessitating vascular bypass was unsuccessful in treating this case of vascular pythiosis .", "written informed consent was obtained from the patient 's guardian for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request.key learning pointsclinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .", "clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .", "clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency .", "the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .", "clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .", "clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency .", "the most important part in managing vascular pythiosis limb infection is adequate source control with amputation ."], "abstract_text": ["<S> introductionpythiosis is a serious life- and limb - threatening infection endemic to thailand , but rarely seen in the western hemisphere . here </S>", "<S> , we present a unique case of vascular pythiosis initially managed with limb - sparing vascular bypass grafts complicated by a pseudoaneurysm in our repair.presentation of casethe patient is a 17 year - old jamaican male with severe aplastic anemia . </S>", "<S> he sustained a minor injury to his left leg while fishing in jamaica , which evolved to become an exquisitely tender inguinal swelling . </S>", "<S> his physical exam and imaging were significant for arteriovenous fistula with limb ischemia . </S>", "<S> pathology obtained during surgery for an extra - anatomic vascular bypass showed extensive invasion by pythium insidiosum . </S>", "<S> he later developed a pseudoaneurysm at the site of proximal anastomosis and required urgent intervention.discussionthis patient presented with a rare , but classic case of vascular pythiosis , which was unrecognized at the time of presentation . </S>", "<S> a variety of therapeutic modalities have been used to treat this disease , including antibiotics , antifungals , and immunotherapy , but the ultimate management of vascular pythiosis is surgical source control.conclusiona high index of suspicion in susceptible patients is needed for timely diagnosis of vascular pythiosis to achieve optimal source control . </S>"], "labels": null, "section_names": ["Introduction", "Presentation of case", "Discussion", "Conclusion", "Conflict of interest", "Ethical approval", "Key learning points"], "sections": [["first described by veterinarians , pythiosis is a serious life- and limb - threatening infection endemic to thailand , but rarely seen in the western hemisphere .", "known to infect immunocompetent horses , dogs , and other large animals,2 , 3 \n pythium infection was first reported in humans in 1989 in association with thalassemic hemoglobinopathy.4 , 5 human pythiosis is known to present in one of four clinical entities : subcutaneous , ocular , vascular , and disseminated . here", ", we present a unique case of vascular pythiosis managed with limb - sparing vascular bypass grafts complicated by pseudoaneurysm formation ."], ["the patient is a 17 year - old jamaican male with a history of paroxysmal nocturnal hemoglobinuria and severe aplastic anemia refractory to horse anti - thymoglobulin treatment , which was transfusion - dependent .", "one month prior to admission at our institution , he went fishing in a fresh water pond in jamaica and his left lower leg was caught in a bush . on the following day , he noticed a small bullae with a black center and later developed severely painful left inguinal swelling .", "he was admitted to a local hospital for the next several weeks , where all diagnostic testing performed was unremarkable .", "his pain persisted despite antibiotic and antifungal therapy , and he was later transferred to our institution for consideration of bone marrow transplant . on admission , he was a febrile with a white blood cell count of 6.2 ( k/l ) and platelet count of 18 ( k/l ) and was complaining of severe left leg pain .", "he had a 9    8  cm soft tissue defect with overlying escar on the posterior aspect of his left leg ( fig .", "1a ) , and his exam was also notable for an exquisitely tender and erythematous left inguinal mass .", "motor and sensory exam were intact in both lower extremities without pain upon passive hip flexion .", "several small , dark macules were noted along the lateral aspect of his left foot ( fig .", "mri showed a t2 bright left groin mass with edema along the fascial planes , and lower extremity angiogram demonstrated occlusion of the left common femoral artery with distal profunda femoris and mid - superficial femoral artery reconstitution ( fig .", "the patient was not a candidate for percutaneous intervention , and was taken to the operating room for exploration .", "visual inspection of the common femoral artery after exposure was significant for several patches of necrotic vessel wall .", "initial attempt with thrombectomy was unsuccessful , as the fogarty catheter actually perforated the anterior wall at the areas of necrosis .", "the patient ultimately received an extra - anatomic left external iliac to superficial femoral artery bypass using a nonreversed greater saphenous vein graft , as the external iliac was the closest available inflow that did not visually appear to be involved with the infection .", "intraoperative wound cultures were positive for pythium insidiosum and the patient was started on a combination of voriconazole , terbinafine , and amphotericin b. given his critical condition , an emergency investigational new drug request was drafted for permission to administer a p. insidiosum antigen vaccine available for compassionate use from the pan american veterinary laboratories .", "the vaccine was given once daily in one - week intervals in conjunction with subcutaneously injected interferon gamma ( ifn- ) , for a total of four weeks .", "three weeks after his initial surgery , the patient developed new sites of pain along the graft site and repeat imaging showed a 2.5  cm pseudoaneurysm at the proximal anastomosis to the external iliac artery with contrast extravasation ( fig .", "he was taken to the operating room for an extra - anatomic left common iliac artery to the previous femoral popliteal bypass graft using a reverse saphenous vein graft .", "the patient later developed a polymicrobial superinfection of his wounds , and his vascular repair continued to break down leading to increased ischemic pain .", "after several multidisciplinary discussions with the family that all medical interventions were exhausted , and that even highly aggressive surgical measures including hip disarticulation or hemipelvectomy could not guarantee source control , conservative measures were initiated and ultimately , the patient expired ."], ["this patient presented a major diagnostic challenge , and his disease process and subsequent management were previously unknown to our hospital .", "in retrospect , he presented with a classic case of vascular pythiosis , with an antecedent history of exposure to surface water and exam findings of vascular insufficiency , including pain out of proportion to exam , distal emboli , and an ischemic ulcer .", "the correct infectious diagnosis was not made until 1 month after exposure , after a vascular bypass was already performed .", "indeed , in a large multi - institutional study from thailand with a total of 102 cases of pythiosis , 60 patients received a diagnosis of vascular pythiosis , and only the patients who had undergone amputation survived .", "further review of the literature for reports of vascular pythiosis demonstrated that the majority of patients were treated with amputation without any signs of recurrence , and were followed for 5 to 35 months post - operatively.4 , 6 , 8 , 9 in our case , complete source control was not achieved during the first operation since the common femoral artery and the proximal portion of the superficial femoral artery were oversewn and left in place , standard practice for the treatment of peripheral arterial disease .", "incomplete source control eventually led to development of a pseudoaneurysm at the proximal anastomosis and additional surgery .", "while the second repair was cited in previously uninvolved tissue planes , it did not protect against further infection , likely because p. insidiosum is known to invade through proteinase secretion .", "the authors could find no reported cases of confirmed vascular pythiosis treated with limb - sparing procedures.2 , 7 , 11 , 12 medical therapy in the treatment of vascular pythiosis involves antifungal agents , immunotherapy , and iron chelation therapy .", "much of the difficulty in eradicating this organism stems from its complicated phylogenesis;2 , 6 , 14 it shares features with true fungi at the same time it is phylogenetically more closely related to algae and diatomeae . \n", "p. insidiosum develops mycelium like fungi , but its cell walls do not contain chitin and its cytoplasmic membrane lacks ergosterol , both of which are important targets of traditional antifungal drugs . despite this , drugs that affect cellular permeability , including amphotericin b , terbinafine , and the azoles , have been used with some clinical success.2 , 15 more recently , immunotherapy has been used as an adjunct to surgery and antimycotics .", "immunotherapy was first successfully used in humans in 1998 in a patient with a vascular p. insidiosum infection refractory to surgery and antifungals.2 , 16 a p. insidiosum vaccine delivered twice with a two - week interval resulted in a cure 1 year later .", "data collected from clinical trials and other reports estimate vaccine efficacy to be 5560%.2 , 17 the proposed mechanism of action of the p. insidiosum vaccine is to shift host adaptive immune response from a predominantly t helper 2 ( th2 ) response to a t helper 1 ( th1 ) response.2 , 17 \n pythium exoantigens are typically processed by antigen presenting cells ( apcs ) and presented to nave cd4 helper t cells ( th0 ) .", "this induces th0 cells to adopt a th2 phenotype secreting il-4 and il-5 , which trigger many downstream effects , including mast cell and eosinophil migration to the site of infection with subsequent degranulation , releasing inflammatory mediators and causing tissue damage .", "loss of tissue architecture then creates a more favorable environment for these pathogenic hyphae to take hold .", "conversion of a th2 to th1 response would favor secretion of ifn- and il-2 , leading to increased cytotoxic t lymphocyte recruitment and therefore , direct killing of p. insidiosum . in this case , the purpose of injecting ifn- subcutaneously was to augment a th1 response , and co - administration of gm - csf was to increase the efficiency of exoantigen presentation to apcs .", "another treatment modality used to control this infection is iron chelation therapy . given the association between pythium infection in patients with hemoglobinopathies with a long - standing history of blood transfusions4 , 7 and the discovery of a ferrochelatase gene in p. insidiosum ,", "other research efforts have focused on elucidating the role of iron overload in p. insidiosum pathogenesis .", "there is evidence that iron overload from transfusions contributes to derangements in host immunity by impairing lymphocyte function and altering phagocytosis , leading to a more susceptible state for infection6 , 19 and is in fact , a key virulence factor for the survival of p. insidiosum in the host .", "correcting iron overload has been cited as a treatment in infections common in thalassemia , and has also been used in pythiosis . in our patient ,"], ["in conclusion , this patient 's vascular p. insidiosum infection was refractory to optimized medical treatment , immunotherapy with cytokine injections , and surgery .", "attempted source control with limb salvage necessitating vascular bypass was unsuccessful in treating this case of vascular pythiosis ."], [""], ["written informed consent was obtained from the patient 's guardian for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request.key learning pointsclinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .", "clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .", "clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency .", "the most important part in managing vascular pythiosis limb infection is adequate source control with amputation ."], ["clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation .", "clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy , exposure to flat water , and exam findings of vascular insufficiency .", "the most important part in managing vascular pythiosis limb infection is adequate source control with amputation ."]]}
{"article_id": "PMC4778614", "article_text": ["tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model", "( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .", "the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,", "then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12  gy ) at the city of hope ( duarte , ca ) facility .", "subsequently the tissues were incubated for 6  h at 37  c with 5% co2 after irradiation .", "then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna .", "the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .", "the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280  >  2.0 , and rin  > ", "the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) . the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis .", "for this purpose , a quantity of 0.025  mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42  c using the hybbag mixing system with 1   onearray hybridization buffer ( phalanx biotech ) and 0.01  mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure", "the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format .", "the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .", "testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .", "the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values", "were calculated to test the null hypothesis that expression is absent ( called  p - value detected  ) . a p - value detected <  0.05 indicated that the transcript specific to a given probe was truly present or expressed .", "lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,", "rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level .", "three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .", "this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample", "the scattered plots of raw and normalized log2 data were compared using the r function pairs .", "pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <  0.01 .", "scatter plots showed that technical replicates had high repeatability and all the correlation values were >  0.953 .", "our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12  gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .", "we only used genes with |  fold change  |  >  1.5 and p - value  <  0.05 .", "we input gene symbols into david bioinformatics , while throughout we used the default settings .", "the threshold for significance was a benjamini - adjusted p - value  <  0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .", "table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .", "that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .", "also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .", "overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .", "the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "adj p - val  =  benjamini - adjusted p - values . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .", "tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model", "( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .", "the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,", "then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12  gy ) at the city of hope ( duarte , ca ) facility .", "subsequently the tissues were incubated for 6  h at 37  c with 5% co2 after irradiation .", "then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna .", "the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .", "the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280  >  2.0 , and", "the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) .", "the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis . for this purpose ,", "a quantity of 0.025  mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42  c using the hybbag mixing system with 1   onearray hybridization buffer ( phalanx biotech ) and 0.01  mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure ,", "the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format .", "the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .", "testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .", "the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values", "were calculated to test the null hypothesis that expression is absent ( called  p - value detected  ) . a p - value detected <  0.05 indicated that the transcript specific to a given probe was truly present or expressed .", "lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,", "rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level .", "three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .", "this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample", "the scattered plots of raw and normalized log2 data were compared using the r function pairs .", "pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <  0.01 .", "scatter plots showed that technical replicates had high repeatability and all the correlation values were >  0.953 .", "our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12  gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .", "we only used genes with |  fold change  |  >  1.5 and p - value  <  0.05 .", "we input gene symbols into david bioinformatics , while throughout we used the default settings .", "the threshold for significance was a benjamini - adjusted p - value  <  0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .", "table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .", "that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .", "also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .", "overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .", "the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html ."], "abstract_text": ["<S> we evaluated a three - dimensional ( 3d ) human oral cell culture that consisted of two types of cells , oral keratinocytes and fibroblasts as a model of oral mucositis which is a debilitating adverse effect of chemotherapy and radiation treatment . </S>", "<S> the 3d cell culture model was irradiated with 12 or 2  gy , and total rna was collected 6  h after irradiation to compare global gene expression profiles via microarray analysis . here </S>", "<S> we provide detailed methods and analysis on these microarray data , which have been deposited in gene expression omnibus ( geo ) : gse62395 . </S>"], "labels": null, "section_names": ["Direct link to deposited data", "Experimental design, materials and methods", "Tissue culture and irradiation", "RNA isolation and microarray hybridization", "Microarray data pre-processing and statistical analysis", "Microarray data quality control", "Conflict of interest"], "sections": [[""], ["tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model", "( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .", "the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,", "then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12  gy ) at the city of hope ( duarte , ca ) facility .", "subsequently the tissues were incubated for 6  h at 37  c with 5% co2 after irradiation .", "then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna .", "the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .", "the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280  >  2.0 , and rin  > ", "the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) . the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis .", "for this purpose , a quantity of 0.025  mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42  c using the hybbag mixing system with 1   onearray hybridization buffer ( phalanx biotech ) and 0.01  mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure", "the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format .", "the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .", "testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .", "the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values", "were calculated to test the null hypothesis that expression is absent ( called  p - value detected  ) . a p - value detected <  0.05 indicated that the transcript specific to a given probe was truly present or expressed .", "lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,", "rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level .", "three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .", "this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample", "the scattered plots of raw and normalized log2 data were compared using the r function pairs .", "pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <  0.01 .", "scatter plots showed that technical replicates had high repeatability and all the correlation values were >  0.953 .", "our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12  gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .", "we only used genes with |  fold change  |  >  1.5 and p - value  <  0.05 .", "we input gene symbols into david bioinformatics , while throughout we used the default settings .", "the threshold for significance was a benjamini - adjusted p - value  <  0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .", "table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .", "that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .", "also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .", "overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .", "the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "adj p - val  =  benjamini - adjusted p - values . more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html ."], ["tissue culture methods and irradiation procedure have been described in . the three dimensional tissue culture model", "( 3d epioral ) and media were purchased from mattek corporation ( ashland , ma ) .", "the 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . in order to induce differentiation and stratification of the keratinocytes of the 3d cultures ,", "then the tissues were irradiated ( dose of irradiation was 0 , 2 , or 12  gy ) at the city of hope ( duarte , ca ) facility .", "subsequently the tissues were incubated for 6  h at 37  c with 5% co2 after irradiation .", "then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna ."], ["the rneasy plus mini kit ( qiagen , germantown , md ) was used to extract total rna of at least two identically treated tissues .", "the integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer ( agilent technologies , palo alto , ca ) . only rna samples with absorbance ratio , a260/280  >  2.0 , and", "the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase ; the transcription reaction included aminoallyl utp ( aa - dutp ) and the subsequent product was conjugated to cy5 nhs ester ( ge healthcare life sciences , pittsburg , pa ) .", "the human whole genome onearray v4.3 ( phalanx biotech , san diego , ca ) was used to perform the dna microarray analysis . for this purpose ,", "a quantity of 0.025  mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42  c using the hybbag mixing system with 1   onearray hybridization buffer ( phalanx biotech ) and 0.01  mg / ml sheared salmon sperm dna ( promega , madison , wi ) . after the hybridization procedure ,", "the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format ."], ["the data from all the arrays of each experimental set were analyzed using rosetta resolver system ( rosetta biosoftware , usa ) .", "testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver .", "the error - weighted approach ( which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy ) was used to calculate the average expression values . in order to decide whether intensity data was significantly above background , p - values", "were calculated to test the null hypothesis that expression is absent ( called  p - value detected  ) . a p - value detected <  0.05 indicated that the transcript specific to a given probe was truly present or expressed .", "lastly , we also calculated p - values for determining whether genes were differentially expressed . rather than focusing solely on fold changes ,", "rosetta resolver uses error - model - based hypothesis tests , which account for both fold change and expression level ."], ["three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . using the r function boxplot , raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes .", "this analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . for each sample", "the scattered plots of raw and normalized log2 data were compared using the r function pairs .", "pearson correlation tables were used to define the linear relationship of the two variables , raw and normalized data , in the scatter plots . for each technical repeat , we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <  0.01 .", "scatter plots showed that technical replicates had high repeatability and all the correlation values were >  0.953 .", "our article , , emphasizes the differentially expressed genes of tissues that received different amounts of radiation ( i.e. 0 , 2 , or 12  gy ) , while herein we show the profiling of all the transcriptomic data resulting from these treatments .", "we only used genes with |  fold change  |  >  1.5 and p - value  <  0.05 .", "we input gene symbols into david bioinformatics , while throughout we used the default settings .", "the threshold for significance was a benjamini - adjusted p - value  <  0.05 . based on our hypothesis , irradiated samples ( compared to non - irradiated control samples ) would display patterns of gene expression consistent with the physiological effects of irradiation .", "table 2 includes selected enriched categories from our qc enrichment analysis ( table s1 contains all enrichment analysis results ) .", "that is , up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death , cell proliferation , oncogenesis , and ubiquitin conjugation .", "also , down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division .", "overall , these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in .", "the following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html .", "up- and down - regulated genes were analyzed separately for each condition , so there are two separate worksheets listing enrichment analysis results .", "goterm cc  =  gene ontology cellular component , mf  =  molecular function , bp  =  biological process , and up_seq_feature  =  uniprot sequence feature .", "other enriched categories are from the interpro , sp pir , smart , and kegg databases .", "count  =  the number differentially expressed genes annotated with a given enriched term .", "more information on the output from david bioinformatics can be found here : http://david.ncifcrf.gov/content.jsp?file  =  function_annotation.html ."], [""]]}
{"article_id": "PMC3996329", "article_text": ["adolescents in sub - saharan africa face high exposure to human immunodeficiency virus ( hiv ) infection . despite all efforts to prevent the disease , prevalence estimates in sub - saharan unaids ( i.e. , the joint united nations programme on hiv and aids ) priority countries range from 1.3 to 15.6  % among young women and 0.56.5  % among young men aged 1524  years ( unaids , 2011 ) .", "higher prevalence estimates are associated with certain regions ( e.g. , eastern and southern africa ) and subpopulations ( e.g. , adolescent orphaned and/or married girls ; birdthistle et al . , 2008 ; operario , underhill , chuong , & cluver , 2011 ; pascoe et al . , 2010 ) .", "in addition , united nations children s fund ( unicef ) estimated there were 2  million adolescents aged 1019  years living with hiv globally in 2009 ( unicef , 2011 ) .", "kenya and zimbabwe were among countries with the highest numbers of infected adolescents with an estimated 136,000 ( prevalence estimate 1.5  % ) 1 and 104,000 ( prevalence estimate 3.1  % ) adolescents living with hiv , respectively ( unicef , 2011 ) .", "the true measure of effectiveness for such interventions is reduction in hiv and other sexually transmitted infections ( stis ) .", "however , very few adolescent prevention studies have used biomarker data as outcomes ( michielsen et al . , 2010 ) because of concerns about the difficulties in collecting these data in resource poor settings ( jukes , simmons , & bundy , 2008 ) , even when power analyses suggest that moderate intervention effects could be detected .", "behavioral prevention scientists have instead relied on self - reported sexual behavior , which is subject to social desirability bias and likely to differ between study arms ( cowan & pettifor , 2009 ; kirby , obasi , & laris , 2006 ) .", "widespread access to testing and treatment facilitated by the president s emergency plan for aids relief ( pepfar ; http://www.usaid.gov/what-we-do/global-health/hiv-and-aids ) have greatly enhanced opportunities to successfully incorporate biomarker procedures into adolescent hiv prevention research designs , since infected individuals identified through testing offered by studies may access free or subsidized hiv treatment and care ( stringer et al . , 2006 ; wools - kaloustian et al . , 2006 , 2009 )", "moreover , including biomarker outcomes can greatly improve the credibility and legitimacy of hiv prevention research . to address the issue of feasibility , we present two case studies of prevention trials with biomarker data collection among adolescents in kenya and zimbabwe .", "together these studies provide a valuable opportunity to examine the ethical and practical challenges of biomarker procedures within hiv prevention intervention clinical trials in developing countries .", "specifically , we describe the challenges faced by investigators in the two cases in developing and implementing ethical procedures for informed consent , biomarker testing , and disclosure of test results .", "we also present the practical challenges of collecting biological samples in resource - limited countries and compare sample collection by blood spots relative to venipuncture .", "in addition , we examine difficulties with analyzing biological samples when the performance of assays in certain populations is unclear .", "we also provide specific suggestions for further research and development in collecting and analyzing biomarker data for adolescent prevention research .", "the primary goal of hiv prevention among adolescents ( ages 1019  years ) is to reduce new infections and related sexual risk factors [ world health organization ( who ) , 2006 ) .", "although the hiv prevalence of adolescents ages 15 and older is monitored through demographic and health surveys ( dhs)2 in most sub - saharan africa countries , little is known about the prevalence of hiv in adolescents under 15  years .", "moreover , these adolescents are rarely tested for hiv in clinical settings unless they present in poor health or with recurrent infections ( ferrand et al . , 2009 ) .", "although a small number of recent randomized controlled trials have used biomarkers ( baird , mcintosh , & ozler , 2010 ; cowan et al .", "; pronyk et al . , 2006 ; ross et al . , 2007 ) ,", "most adolescent hiv prevention studies have relied on changes in self - reported sexual behavior to assess intervention effectiveness ( see reviews : cowan & pettifor , 2009 ; michielsen et al . ,", "2010 ) , with audio computer - assisted self - interviews intended to improve veracity ( mensch , erulkar , & hewett , 2003 ) .", "however , biomarker comparisons have suggested that self - reported measures have low validity ( cowan et al . , 2002 ; gavin et al . , 2006 ; mensch , hewett , gregory , & helleringer , 2008 ; palen et al . , 2008 ; plummer et al . , 2004 ) .", "for example , in one study , only one of 16 youth with hiv admitted to ever having sex , and four of the 16 also had either chlamydia or gonorrhea ( cowan et al . , 2002 ) .", "inclusion of a highly prevalent and reliable biomarker of sexual activity in conjunction with self - reported sexual behavior can help to improve reliability of this outcome measure .", "hiv infection is not the most useful biomarker of adolescent sexual activity by itself because of the relatively low prevalence among younger adolescents and the potential for transmission through perinatal and non - sexual blood - borne routes ( amornkul et al .", ", 2009 ; ferrand et al . , 2009 ; stover , walker , grassly , & marston , 2006 ) .", "herpes simplex virus type 2 ( hsv-2 ) , the primary cause of genital herpes , is a commonly used biomarker of sexual activity because the presence of hsv-2 antibodies is highly associated with past sexual behavior ( tobian et al . , 2009 ; van de laar et al . , 1998 ) .", "in contrast to other stis such as chlamydia , gonorrhea , syphilis and trichomoniasis , infection with hsv-2 is life - long , and , once established , there is currently no treatment to eliminate it ( geretti , 2006 ; gupta , warren , & wald , 2008 ) .", "hsv-2 prevalence in the adult general population in sub - saharan africa is high , ranging from 30 to 80  % in women and from 10 to 50  % in men , as assessed by detection of hsv-2 antibodies [ amornkul et al . , 2009 ;", ", 2005 ; munjoma et al . , 2010 ; national aids & sti control programme ( nascop ) , republic of kenya , 2009 ; tobian et al . , 2009 ; weiss , 2004 ] .", "prevalence estimates for adolescents are only available through community - based studies and are lower .", "one study in zimbabwe among adolescent females aged 1519  years reported that 12  % of participants tested positive for hsv-2 ( birdthistle et al .", "a study in western kenya found hsv-2 prevalence to be 9  % for females and 4  % for males aged 1314 , and 28  % for females and 17  % for males aged 1519  years ( amornkul et al . , 2009 ) .", "another study conducted in zimbabwe among adolescents whose mean age was 15  years reported a prevalence estimate of 0.2  % , suggesting a later stage of sexual debut ( cowan et al .", "2012 ) evaluated the use of hsv-2 as a biomarker of sexual debut among young people aged 1024  years . following a comprehensive review of the literature", ", the authors concluded that the use of hsv-2 as a biomarker for sexual debut is limited because of its low transmissibility and the fact that not all potential sexual partners are infected with the virus .", "building on these findings , our study describes the challenges of collecting hiv and hsv-2 biological data as biomarkers for sexual activity among sub - saharan african youth in prevention trials .", "we present two clinical trial case studies to examine important issues related to adolescent biomarker data collection in two clinical trials in kenya and zimbabwe ( hallfors et al . ,", "both studies were school - based , cluster randomized trials testing school support ( including payment of school tuition and school uniforms ) as a structural hiv prevention intervention for adolescent orphans . in both studies , we collected blood samples from participants for hiv and hsv-2 testing , but they differed as follows : we collected samples by venipuncture with results disclosed to participants and their guardians in kenya , whereas in zimbabwe , we collected samples by finger pricks for dried blood spots ( dbs ) and results were not disclosed . the kenya sample ( n  =  837 ) comprised young adolescent male ( 52  % ) and female ( 48  % ) orphans in grades 7 ( 61  % ) and 8 ( 39  % ) enrolled in school in siaya district , nyanza province .", "their mean age was 14.9 ( sd 1.5 ; range 1121  years ) ; 22  % reported ever having had sex . among girls ,", "hiv and hsv-2 prevalence was 1  % ( n  =  10 ) and 3  % ( n  =  28 ) , respectively . among those with positive hiv or hsv-2 test results , 70  % ( n  =  7 ) and 64  % ( n  =  18 ) , respectively , reported never having had sex .", "the original sample for the zimbabwe study was orphan 6th grade girls ( n  =  328 ) in manicaland province primary schools ; surveys were administered annually during 20072010 ( hallfors et al . , 2011 ) .", "after securing a project renewal grant , we collected biomarker specimens and a final survey in march through june 2012 , when most participants were 1617  years old . from the original sample , we located 287 ( 88  % ) for the 2012 survey .", "twenty - three percent reported ever having sex ; 18  % had ever been married and/or pregnant .", "hiv and hsv-2 prevalence was 4  % ( n  =  12 ) and 6  % ( n  =  16 ) , respectively . among those with positive hiv or hsv-2 test results , 50  % ( n  =  6 ) and 44  % ( n  =  7 ) , respectively , reported never having had sex .", "although the protection of adult participants in hiv prevention research is well developed ( national institute of mental health collaborative hiv / std prevention trial group , 2007 ) , we found that clear guidance regarding hiv and other sti testing and disclosure is lacking for children and adolescents .", "as an example , our two clinical trials were peer - reviewed by two different nih scientific review panels ; one set of reviewers thought that it was an ethical imperative to disclose hsv-2 as well as hiv results to adolescents , whereas the other was concerned that disclosure of biomarker results might lead to stigma and abuse .", "these divergent opinions led to discrepant protocols for disclosure , with one trial disclosing and the other not disclosing test results to participants .", "we used the national hiv testing and counseling ( htc ) guidelines [ central statistical office ( cso ) & macro international inc .", ", 2007 ; nascop , republic of kenya , 2008 ] for hiv in both countries .", "given the lack of national guidelines for adolescent sti testing in kenya [ ministry of health ( moh ) , republic of kenya , 2006 ) , we developed a protocol in conjunction with the siaya district aids and std control office that closely followed hiv guidelines .", "study protocols were reviewed and approved by irbs in the us , kenya and zimbabwe .", "the kenya study was approved by the ministry of education and siaya district education office ; school head teachers identified orphans and organized meetings in which moi university research collaborators and a district education officer explained the study to students and their caregivers . caregivers and students", "we invited a total of 923 orphans in grades 7 and 8 in 2011 to participate ; 849 ( 92  % ) consented and 837 ( 91  % ) completed both the student survey and biomarker testing .", "following national guidelines , we required kenya study participants under age 18 to have an adult guardian accompany them for hiv rapid testing ( nascop , republic of kenya , 2008 ) . in some instances , however , the guardian was not available because of work , other commitments , or illness .", "in such cases we permitted a relative , teacher or neighbor to stand - in for the guardian if they had a note from the guardian or if research staff were able to speak with the guardian by phone .", "given the sensitive nature of hiv testing , the senior counselor interviewed all unclear or undocumented cases to determine whether or not to proceed with specimen collection and htc .", "we visited clinics several times to give participants the opportunity to return for testing with their guardian or authorized proxy .", "although not required , all participants ages 18 and older were accompanied by an adult for support in case of a positive hiv test result . in accordance with national guidelines", ", we gave referrals to publicly funded hiv care and treatment centers to all adolescents with positive hiv test results , while hiv negative participants were counseled on risk reduction ( nascop , republic of kenya , 2008 ) .", "we disclosed results to both guardians and participants if the latter were under age 18 and to the participants alone if they were 18  years old according to the study protocol . in zimbabwe , with permission from the provincial education officer , the consortium principal investigator and a research associate held meetings at study schools to explain the continuing study to parents / guardians , participants , and key school staff .", "we obtained written parent / guardian consent and participant assent for continued voluntary participation in the study , including separate checkbox consent for survey and blood sample collection .", "guardians raised few concerns , although they did ask why we were not disclosing test results .", "participants who were married or 18  years or older provided their own consent . in deference to local cultural norms and to prevent potential spousal abuse , we prepared a second consent form for husbands to provide consent for their wives participation .", "most of the wives gave their own consent for the survey and did not ask their husbands for consent . in four cases ,", "the husband signed a consent form and in five additional cases , the husband refused and the wife did not take the survey . although asking husbands for permission for their wives study participation is not consistent with us culture , it appears to be a useful protocol accommodation for some young wives in rural zimbabwe .", "we consistently relied on local researchers and key informants to make such culturally sensitive decisions .", "after 4  months , we found 88  % of the original sample who consented to the survey ; 85  % consented to blood specimen collection .", "of those not surveyed , two participants had died in childbirth , 2  % refused , and approximately 10  % of the sample could not be found . of those not consenting to blood specimen collection ( n  =  8) , six were married and refused for religious reasons or husband s objection .", "two were consented for the survey by school officials who refused to provide additional consent for the blood collection , and no guardian was available to give permission .", "hiv testing is technically far more advanced for widespread use in sub - saharan africa than hsv-2 testing .", "rapid hiv antibody tests are now widely used and accepted ( moh , republic of kenya , 2006 ) , and sequential algorithms for retesting have been adopted in kenya and zimbabwe for rapid testing ( cso & macro international inc . , 2007 ; nascop , republic of kenya , 2008 ; who , 2004 ) .", "rapid hiv tests meet high performance standards , sample collection procedures are simple , tests are easy to perform , and interpretation of test results is reasonably clear . in contrast , hsv-2 serologic testing presents some important challenges .", "although a number of hsv-2 type - specific serologic tests are available , only a limited number have been tested among sub - saharan african populations .", "these include the herpeselect hsv-2 enzyme - linked immunosorbent assay ( elisa ; focus diagnostics , cypress , ca ) and kalon hsv-2 elisa ( kalon biological ltd . , guildford , uk ; biraro , mayaud , morrow , grosskurth , & weiss , 2011 ) .", "these tests require skilled laboratory staff , specialized equipment , and a constant source of electricity .", "they are appropriate for use with large batches of samples as in sentinel surveys and are inexpensive , costing about $ 3 per test .", "although the literature reports variable performance of these tests with respect to sensitivity and specificity in sub - saharan populations , kalon is often found to be superior to focus herpeselect ( see biraro et al . , 2011 for a review of the performance of hsv-2 tests in sub - saharan africa ;", "2008 ; ngayo , friedrich , holmes , bukusi , & morrow , 2010 ; smith et al . , 2009 ; van dyck et al . , 2004 ) .", "when using the manufacturer s instructions ( index cutoff value  =  1.1 for both tests ) , kalon tends to have lower sensitivity but higher specificity than focus herpeselect ( delany - moretlwe et al .", ", 2010 ; gamiel et al . , 2008 ; ngayo et al . , 2010 ; smith et al . , 2009 ; van dyck et al . ,", "the low specificity of focus herpeselect is particularly problematic when disclosing results to research participants because of an unacceptably high rate of false positives when prevalence is low ( gamiel et al . , 2008 ; mark et al . , 2008 ; ngayo et al", ", 2010 ; van dyck et al . , 2004 ) . increasing the assay cut - off values is one strategy for improving the performance of both the kalon and focus herpeselect tests .", "a number of studies have shown that increasing the cut - off value for focus herpeselect to 3.5 increases specificity , although sensitivity is necessarily reduced ( delany - moretlwe et al . , 2010 ; smith et al . ,", "other studies similarly improved performance at cut - off values of 3.2 and 3.3 ( delany - moretlwe et al . , 2010 ; gamiel et al . , 2008 ; ngayo et al . ,", "although findings are mixed , increasing the cut - off value for the kalon test to 1.5 has also been shown to increase specificity , with an attendant reduction in sensitivity ( gamiel et al . , 2008 ; ngayo et al .", "in our studies , the kenya laboratory used the kalon assay , and the zimbabwe laboratory used the focus test . at the time of our studies , neither assay had been validated for dbs by the manufacturer in african populations .", "the efficiency of dbs for hsv-2 testing with each assay must be evaluated in specific populations prior to use ( hogrefe , ernst , & su , 2002 ) . because the kalon test had not been validated for hsv-2 testing with dbs samples , specimens in the kenya study were obtained by venipuncture .", "the zimbabwe laboratory had validated the focus test with dbs ( mudzori & mutsogoro , 2006 ) . in both studies", ", we used a modified algorithm to interpret results : samples with initial index values < 0.9 were reported as negative and no further testing was conducted .", "samples with initial index values > 2.5 were defined as high positive and were reported as positive without additional testing .", "samples with initial index values from 1.5 to 2.5 were considered low positive , and those with initial index values between 0.9 and 1.5 were considered equivocal ; all low positives and equivocals were retested in duplicate . if both retest results had index values > 1.5 , the final result was reported as positive . if both retest results were < 0.9 , the final result was negative .", "if retest results were equivocal ( 0.91.5 ) or discordant , the final result was indeterminate .", "although test performance was improved by increasing the cut - off and expanding the range of results for repeat testing , the performance characteristics of tests to detect antibodies to hsv-2 are imperfect . based on a recent systematic review of hsv-2 antibody test performance in sub - saharan africa ( biraro et al . , 2011 ) , sensitivity for the kalon test used in kenya was estimated at 94  % and specificity at 92  % with the higher cut - off value of 1.5 .", "the predictive values of all diagnostic tests are strongly influenced by the prevalence of infection in the population , and in low prevalence situations , even a very good test will have a poor positive predictive value ( ppv ) . using the sensitivity and specificity values for the kalon test under the testing conditions employed in our kenya study ,", "the ppv was estimated at 26  % with a 3  % hsv-2 prevalence .", "that is , we can be confident that about one in four participants with positive test results truly had hsv-2 antibodies present in their blood sample ; however , as many as three in four could have had a false positive test result .", "in kenya , we conducted sample collection and htc primarily in local public health facilities near the schools . eighteen facilities participated .", "in two sites , with permission from the school headmasters and district education officer , we collected blood samples and conducted htc at the school .", "venipuncture was conducted by six skilled phlebotomists trained in htc . to avoid obtaining two separate blood specimens ( finger prick for rapid hiv testing and venipuncture for hsv-2 testing )", "the process , including counseling , blood draw , hiv testing , disclosure of results took on average 20  min .", "the process was longer for participants with discordant results who needed a tie - breaker test and for participants with hiv positive results .", "we offered all participants and their guardians a snack either before or after specimen collection .", "we stored blood samples for hsv-2 testing in labeled vacutainer tubes in a cooler box without ice and transported them to a local laboratory within 24  h for serum processing .", "sera were stored at the local laboratory in a freezer at 20  c until transport on ice weekly to the hsv-2 testing laboratory .", "the logistics of specimen collection , processing and transport for this study were challenging but feasible . in comparison ,", "we did not require guardians to be present because hiv results were not provided to participants .", "sampling and dbs preparation were much less obvious to others nearby , helping to safeguard participant privacy and reduce concerns about witchcraft or other conspiracy theories regarding hiv / aids and blood , which circulate in sub - saharan african communities ( tenkorang , gyimah , maticka - tyndale , & adjei , 2011 ) .", "blood from the finger prick was dropped on five circles on a dbs filter card labeled with the participant s study i d .", "some participants may have been dehydrated or not very well nourished , making it difficult to fill all circles , but adequate samples were collected from all participants .", "specimens were dried , stored with a desiccant , and transported to the testing lab at ambient temperatures .", "dbs samples are easy to store and transport and require no processing or refrigeration ( see table  1 for a summary of the study procedures).table  1comparison of biomarker testing procedures in two research studieskenyazimbabweethical considerationswritten guardian consent if < 18/adolescent assentwritten guardian consent / adolescent assentresults disclosed to guardian and adolescentno results disclosedreferrals for carenot applicablecollecting and analyzing biological samples  methodvenipuncturedried blood spot  hiv testrapid testrapid test  hsv testkalon elisafocus elisafeasibility of collection methods  time20  min3  min  temperature for specimen transportcooler with no iceambienttesting costsreference labreference lab  bench fee , supplies , storage , labor us$ 5,784bench fee , supplies , labor us$ 8,000field site lab  sample processing , labor , supplies , storage us$ 3,611additional fee us$ 480  other supplies us$ 100  transportation us$ 375  total us$ 9,919total us$ 8,480 \n elisa enzyme - linked immunosorbent assay comparison of biomarker testing procedures in two research studies \n elisa enzyme - linked immunosorbent assay", "given the experience we have described , we conclude that it is feasible to obtain hiv and hsv-2 biomarker data for adolescent hiv prevention intervention studies . in particular", "the us pepfar program has accomplished considerable staff training in sub - saharan africa ; it has also stimulated the development of well - equipped laboratories , as well as useful tools for testing and protocols for improving test sensitivity , specificity , and cost ( stringer et al . , 2006 ) .", "in addition , it has stimulated the development of national guidelines ( particularly for adults ) for the disclosure of results . for hiv", "incorporation of hsv-2 serology results poses considerable challenges , particularly if results are disclosed to participants .", "based on our experience , there are two reasons why we do not recommend disclosing hsv-2 serology results to adolescent human subjects in sub - saharan africa .", "first , in populations with low or moderate prevalence of infection , as might be expected for adolescents , the potential for false positive test results is substantial in combination with relatively minor imperfections in test specificity .", "hsv-2 infection is often a silent disease , insofar as only 1025  % of people with antibodies for the condition are aware that they have genital herpes ( fleming et al . , 1997", "; leone , fleming , gilsenan , li , & justus , 2004 ; sizemore , lakeman , whitley , hughes , & hook , 2006 ) .", "adolescents without symptoms may deny ever having had sexual intercourse3 , further complicating the clinical picture and raising concerns about false positives with the potential for psychological and social harm related to participation in the study .", "second , local departments of health and sti authorities in sub - saharan african countries were much less familiar with hsv-2 pathology and treatment than with hiv and some other stis . even in cases where subjects acknowledge sexual exposure or genital herpes symptoms , local health clinics may not be prepared to provide appropriate treatment or care . given these vexing problems , the wisdom of disclosing hsv-2 results to adolescents after testing within a clinical prevention trial is questionable . in particular , decisions about whether to inform adolescent participants in prevention trials about hsv-2 test results should be made only after prevalence data are available and the predictive values of the test can be accurately assessed . in light of the feedback from the nih study section that raised concerns about possible stigma and abuse of hiv - positive participants in zimbabwe , as well as our own concerns ,", "we recommend that more research be conducted to study the consequences of disclosure to adolescents in sub - saharan africa .", "a review of the literature indicates that sub - saharan adolescent prevention studies with biomarker outcomes have taken a variety of approaches to disclosure . in some studies , counselors disclosed participants hiv results immediately after rapid testing ( baird , garfein , mcintosh , & ozler , 2012 ; dalal et al . , 2012 ; medley et al . , 2012 ) .", "in other studies , participants could choose to receive their results after testing , either immediately or a few weeks later ( amornkul et al .", ", 2009 ; jewkes et al . , 2006 ; ross et al . , 2007 ) .", "in still other studies , the research did not provide test results but voluntary counseling and testing ( vct ) was made freely available for study participants ( birdthistle et al .", "none of these studies , however , examined whether adolescents experienced any untoward outcomes with testing and disclosure , whether hiv positive youth enrolled in care or took steps to prevent transmission , whether hiv negative youth benefited from prevention counseling and engaged in subsequent testing , or if adolescents and their guardians felt it was appropriate to learn their status in the context of a research study . from our kenya study , the problem of stigma did not appear to be a major obstacle to adolescent testing and disclosure in our study area .", "perhaps this is because hiv prevalence is among the highest in the country , and few families have been unaffected by hiv / aids .", "moreover , high hiv prevalence has drawn research , education , and hiv counseling and testing campaigns to the area .", "for example , in our kenya clinical trial , more than 50  % of participants ( all in grade 7 and 8) reported that they had previously been tested for hiv , mostly through a home - based testing initiative of the kenya medical research institute ( kemri ) and us centers for disease control and prevention ( cdc ) .", "although our study provided hiv test results to youth , we relied on phlebotomist / nurse counselors to refer youth and their guardians for care , following national guidelines .", "the onus of making contact with the clinic was left up to the individual and was not tracked by the system of care ; and follow up to make sure that hiv positive youth accessed care was well beyond the scope of our study . since it is unknown whether youth experience significant mental trauma and social prejudice , and whether they actually engage in the system of care , we recommend that further research be conducted to examine the perceptions and behaviors of youth  and particularly newly diagnosed youth  after disclosure of their test results to ascertain the consequences of this aspect of research participation and whether it can be improved .", "this is particularly important for adolescents , since results are disclosed in the presence of their guardian .", "regarding procedures for collecting biomarkers , we conclude that finger - sticks and dbs are overwhelmingly superior to venipuncture in sample collection efficiency and reduced burden on participants and community institutions .", "manufacturer validations of dbs for hsv-2 are urgently needed , however , for this procedure to be more widely accessible to researchers . because blood collection for dbs is minimally invasive , this procedure has excellent potential for widespread acceptability and consequent high participation in school and community research sites .", "in addition , rapid hiv testing in sub - saharan africa is typically conducted by finger prick , making this the preferred way for trained african health workers and counselors to participate in research data collection ( who , 2004 ) .", "given the complex decisions required for biomarkers , we recommend that behavioral prevention scientists interested in using biomarker data collaborate from the early planning stages with biomedical scientists who have expert knowledge in hsv-2 , as well as hiv , laboratory tests and testing procedures .", "we found that team members with such expertise provide valuable assistance in selecting qualified in - country laboratories , developing appropriate budgets , protocols , quality assurance and algorithms for interpreting results , and helping research staff to bridge communication with in - country laboratory staff .", "further , we recommend that hsv-2 test kit manufacturers determine optimal cut - off standards for sub - saharan populations , and that researchers who conduct hsv-2 testing explicitly define the testing cutoffs they use in publications .", "there is now a substantial literature documenting validity problems when using manufacturers cutoffs with african populations potentially inflating prevalence findings ( gamiel et al . , 2008 ; ngayo et al . , 2010 ; van dyck et al . , 2004", "this suggests the need for action to improve assay validation practices on the part of manufacturers as well as methodological details from researchers documenting their findings . when conducting collaborative international research , it is important to recognize that alternative ethical systems exist .", "thus , for international research we recommend inclusion of team members who are knowledgeable about local contexts . indeed", ", successful collaborative research partnerships respect local cultures , values and practices ; negotiate effectively within these systems ; and incorporate into study designs ethical practices that are appropriate and sensitive to local cultural contexts ( e.g. , in our case , providing for husband consent for married participants ; christakis , 1992 ; emanuel , wendler , killen , & grady , 2004 ) .", "behavioral prevention scientists traditionally have relied on self - reported sexual behavior survey item measures to evaluate adolescent hiv prevention interventions .", "our experiences conducting research with orphan adolescents in kenya and zimbabwe suggest that collection of hiv and sti test results even in rural , resource - poor settings in sub - saharan africa is a feasible addition to the behavioral research toolkit .", "the use of sti biomarkers can greatly improve the validity of findings from adolescent behavioral intervention trials .", "research is urgently needed to examine the risks and benefits of hiv testing and disclosure of test results in the context of a research study for adolescents . further development and implementation of sti biomarker assessment techniques  particularly pertaining to using dbs  is needed to advance hiv prevention science ."], "abstract_text": ["<S> self - report of sexual behavior among adolescents is notoriously inconsistent , yet such measures are commonly used as outcomes for human immunodeficiency virus ( hiv ) prevention intervention trials . </S>", "<S> there has been a growing interest in the use of hiv and other sexually transmitted disease biomarkers as more valid measures of intervention impact in high hiv prevalence areas , particularly in sub - saharan africa . </S>", "<S> we examine the challenges , benefits , and feasibility of including hiv and herpes simplex virus type 2 ( hsv-2 ) biomarker data , with details about different data collection and disclosure methods from two adolescent prevention trials in kenya and zimbabwe . in kenya , </S>", "<S> whole blood samples were collected using venipuncture ; adult guardians were present during biomarker procedures and test results were disclosed to participants and their guardians . in contrast , in zimbabwe , samples were collected using finger pricks for dried blood spots ( dbs ) ; guardians were not present during biomarker procedures , and results were not disclosed to participants and/or their guardians . in both countries , </S>", "<S> prevalence in the study samples was low . </S>", "<S> although the standard of care for testing for hiv and other sexually transmitted infections includes disclosure in the presence of a guardian for adolescents under age 18 , we conclude that more research about the risks and benefits of disclosure to adolescents in the context of a clinical trial is needed . </S>", "<S> notably , current serological diagnosis for hsv-2 has a low positive predictive value when prevalence is low , resulting in an unacceptable proportion of false positives and serious concerns about disclosing test results to adolescents within a trial . </S>", "<S> we also conclude that the dbs approach is more convenient and efficient than venipuncture for field research , although both approaches are feasible . </S>", "<S> manufacturer validation studies using dbs for hsv-2 , however , are needed for widespread use . </S>"], "labels": null, "section_names": ["Introduction", "Brief Description of Case Studies", "Ethical Considerations Including Consenting Procedures", "Collecting and Analyzing Biological Samples", "Feasibility Comparison of Sample Collection Methods", "Discussion"], "sections": [["adolescents in sub - saharan africa face high exposure to human immunodeficiency virus ( hiv ) infection . despite all efforts to prevent the disease , prevalence estimates in sub - saharan unaids ( i.e. , the joint united nations programme on hiv and aids ) priority countries range from 1.3 to 15.6  % among young women and 0.56.5  % among young men aged 1524  years ( unaids , 2011 ) .", "higher prevalence estimates are associated with certain regions ( e.g. , eastern and southern africa ) and subpopulations ( e.g. , adolescent orphaned and/or married girls ; birdthistle et al . , 2008 ; operario , underhill , chuong , & cluver , 2011 ; pascoe et al . , 2010 ) .", "in addition , united nations children s fund ( unicef ) estimated there were 2  million adolescents aged 1019  years living with hiv globally in 2009 ( unicef , 2011 ) .", "kenya and zimbabwe were among countries with the highest numbers of infected adolescents with an estimated 136,000 ( prevalence estimate 1.5  % ) 1 and 104,000 ( prevalence estimate 3.1  % ) adolescents living with hiv , respectively ( unicef , 2011 ) .", "the true measure of effectiveness for such interventions is reduction in hiv and other sexually transmitted infections ( stis ) .", "however , very few adolescent prevention studies have used biomarker data as outcomes ( michielsen et al . , 2010 ) because of concerns about the difficulties in collecting these data in resource poor settings ( jukes , simmons , & bundy , 2008 ) , even when power analyses suggest that moderate intervention effects could be detected .", "behavioral prevention scientists have instead relied on self - reported sexual behavior , which is subject to social desirability bias and likely to differ between study arms ( cowan & pettifor , 2009 ; kirby , obasi , & laris , 2006 ) .", "widespread access to testing and treatment facilitated by the president s emergency plan for aids relief ( pepfar ; http://www.usaid.gov/what-we-do/global-health/hiv-and-aids ) have greatly enhanced opportunities to successfully incorporate biomarker procedures into adolescent hiv prevention research designs , since infected individuals identified through testing offered by studies may access free or subsidized hiv treatment and care ( stringer et al . , 2006 ; wools - kaloustian et al . , 2006 , 2009 )", "moreover , including biomarker outcomes can greatly improve the credibility and legitimacy of hiv prevention research . to address the issue of feasibility , we present two case studies of prevention trials with biomarker data collection among adolescents in kenya and zimbabwe .", "together these studies provide a valuable opportunity to examine the ethical and practical challenges of biomarker procedures within hiv prevention intervention clinical trials in developing countries .", "specifically , we describe the challenges faced by investigators in the two cases in developing and implementing ethical procedures for informed consent , biomarker testing , and disclosure of test results .", "we also present the practical challenges of collecting biological samples in resource - limited countries and compare sample collection by blood spots relative to venipuncture .", "in addition , we examine difficulties with analyzing biological samples when the performance of assays in certain populations is unclear .", "we also provide specific suggestions for further research and development in collecting and analyzing biomarker data for adolescent prevention research .", "the primary goal of hiv prevention among adolescents ( ages 1019  years ) is to reduce new infections and related sexual risk factors [ world health organization ( who ) , 2006 ) .", "although the hiv prevalence of adolescents ages 15 and older is monitored through demographic and health surveys ( dhs)2 in most sub - saharan africa countries , little is known about the prevalence of hiv in adolescents under 15  years .", "moreover , these adolescents are rarely tested for hiv in clinical settings unless they present in poor health or with recurrent infections ( ferrand et al . , 2009 ) .", "although a small number of recent randomized controlled trials have used biomarkers ( baird , mcintosh , & ozler , 2010 ; cowan et al .", "; pronyk et al . , 2006 ; ross et al . , 2007 ) ,", "most adolescent hiv prevention studies have relied on changes in self - reported sexual behavior to assess intervention effectiveness ( see reviews : cowan & pettifor , 2009 ; michielsen et al . ,", "2010 ) , with audio computer - assisted self - interviews intended to improve veracity ( mensch , erulkar , & hewett , 2003 ) .", "however , biomarker comparisons have suggested that self - reported measures have low validity ( cowan et al . , 2002 ; gavin et al . , 2006 ; mensch , hewett , gregory , & helleringer , 2008 ; palen et al . , 2008 ; plummer et al . , 2004 ) .", "for example , in one study , only one of 16 youth with hiv admitted to ever having sex , and four of the 16 also had either chlamydia or gonorrhea ( cowan et al . , 2002 ) .", "inclusion of a highly prevalent and reliable biomarker of sexual activity in conjunction with self - reported sexual behavior can help to improve reliability of this outcome measure .", "hiv infection is not the most useful biomarker of adolescent sexual activity by itself because of the relatively low prevalence among younger adolescents and the potential for transmission through perinatal and non - sexual blood - borne routes ( amornkul et al .", ", 2009 ; ferrand et al . , 2009 ; stover , walker , grassly , & marston , 2006 ) .", "herpes simplex virus type 2 ( hsv-2 ) , the primary cause of genital herpes , is a commonly used biomarker of sexual activity because the presence of hsv-2 antibodies is highly associated with past sexual behavior ( tobian et al . , 2009 ; van de laar et al . , 1998 ) .", "in contrast to other stis such as chlamydia , gonorrhea , syphilis and trichomoniasis , infection with hsv-2 is life - long , and , once established , there is currently no treatment to eliminate it ( geretti , 2006 ; gupta , warren , & wald , 2008 ) .", "hsv-2 prevalence in the adult general population in sub - saharan africa is high , ranging from 30 to 80  % in women and from 10 to 50  % in men , as assessed by detection of hsv-2 antibodies [ amornkul et al . , 2009 ;", ", 2005 ; munjoma et al . , 2010 ; national aids & sti control programme ( nascop ) , republic of kenya , 2009 ; tobian et al . , 2009 ; weiss , 2004 ] .", "prevalence estimates for adolescents are only available through community - based studies and are lower .", "one study in zimbabwe among adolescent females aged 1519  years reported that 12  % of participants tested positive for hsv-2 ( birdthistle et al .", "a study in western kenya found hsv-2 prevalence to be 9  % for females and 4  % for males aged 1314 , and 28  % for females and 17  % for males aged 1519  years ( amornkul et al . , 2009 ) .", "another study conducted in zimbabwe among adolescents whose mean age was 15  years reported a prevalence estimate of 0.2  % , suggesting a later stage of sexual debut ( cowan et al .", "2012 ) evaluated the use of hsv-2 as a biomarker of sexual debut among young people aged 1024  years . following a comprehensive review of the literature", ", the authors concluded that the use of hsv-2 as a biomarker for sexual debut is limited because of its low transmissibility and the fact that not all potential sexual partners are infected with the virus .", "building on these findings , our study describes the challenges of collecting hiv and hsv-2 biological data as biomarkers for sexual activity among sub - saharan african youth in prevention trials ."], ["we present two clinical trial case studies to examine important issues related to adolescent biomarker data collection in two clinical trials in kenya and zimbabwe ( hallfors et al . ,", "both studies were school - based , cluster randomized trials testing school support ( including payment of school tuition and school uniforms ) as a structural hiv prevention intervention for adolescent orphans . in both studies , we collected blood samples from participants for hiv and hsv-2 testing , but they differed as follows : we collected samples by venipuncture with results disclosed to participants and their guardians in kenya , whereas in zimbabwe , we collected samples by finger pricks for dried blood spots ( dbs ) and results were not disclosed . the kenya sample ( n  =  837 ) comprised young adolescent male ( 52  % ) and female ( 48  % ) orphans in grades 7 ( 61  % ) and 8 ( 39  % ) enrolled in school in siaya district , nyanza province .", "their mean age was 14.9 ( sd 1.5 ; range 1121  years ) ; 22  % reported ever having had sex . among girls ,", "hiv and hsv-2 prevalence was 1  % ( n  =  10 ) and 3  % ( n  =  28 ) , respectively . among those with positive hiv or hsv-2 test results , 70  % ( n  =  7 ) and 64  % ( n  =  18 ) , respectively , reported never having had sex .", "the original sample for the zimbabwe study was orphan 6th grade girls ( n  =  328 ) in manicaland province primary schools ; surveys were administered annually during 20072010 ( hallfors et al . , 2011 ) .", "after securing a project renewal grant , we collected biomarker specimens and a final survey in march through june 2012 , when most participants were 1617  years old . from the original sample , we located 287 ( 88  % ) for the 2012 survey .", "twenty - three percent reported ever having sex ; 18  % had ever been married and/or pregnant .", "hiv and hsv-2 prevalence was 4  % ( n  =  12 ) and 6  % ( n  =  16 ) , respectively . among those with positive hiv or hsv-2 test results , 50  % ( n  =  6 ) and 44  % ( n  =  7 ) , respectively , reported never having had sex ."], ["although the protection of adult participants in hiv prevention research is well developed ( national institute of mental health collaborative hiv / std prevention trial group , 2007 ) , we found that clear guidance regarding hiv and other sti testing and disclosure is lacking for children and adolescents .", "as an example , our two clinical trials were peer - reviewed by two different nih scientific review panels ; one set of reviewers thought that it was an ethical imperative to disclose hsv-2 as well as hiv results to adolescents , whereas the other was concerned that disclosure of biomarker results might lead to stigma and abuse .", "these divergent opinions led to discrepant protocols for disclosure , with one trial disclosing and the other not disclosing test results to participants .", "we used the national hiv testing and counseling ( htc ) guidelines [ central statistical office ( cso ) & macro international inc .", ", 2007 ; nascop , republic of kenya , 2008 ] for hiv in both countries .", "given the lack of national guidelines for adolescent sti testing in kenya [ ministry of health ( moh ) , republic of kenya , 2006 ) , we developed a protocol in conjunction with the siaya district aids and std control office that closely followed hiv guidelines .", "study protocols were reviewed and approved by irbs in the us , kenya and zimbabwe .", "the kenya study was approved by the ministry of education and siaya district education office ; school head teachers identified orphans and organized meetings in which moi university research collaborators and a district education officer explained the study to students and their caregivers . caregivers and students", "we invited a total of 923 orphans in grades 7 and 8 in 2011 to participate ; 849 ( 92  % ) consented and 837 ( 91  % ) completed both the student survey and biomarker testing .", "following national guidelines , we required kenya study participants under age 18 to have an adult guardian accompany them for hiv rapid testing ( nascop , republic of kenya , 2008 ) . in some instances , however , the guardian was not available because of work , other commitments , or illness .", "in such cases we permitted a relative , teacher or neighbor to stand - in for the guardian if they had a note from the guardian or if research staff were able to speak with the guardian by phone .", "given the sensitive nature of hiv testing , the senior counselor interviewed all unclear or undocumented cases to determine whether or not to proceed with specimen collection and htc .", "we visited clinics several times to give participants the opportunity to return for testing with their guardian or authorized proxy .", "although not required , all participants ages 18 and older were accompanied by an adult for support in case of a positive hiv test result . in accordance with national guidelines", ", we gave referrals to publicly funded hiv care and treatment centers to all adolescents with positive hiv test results , while hiv negative participants were counseled on risk reduction ( nascop , republic of kenya , 2008 ) .", "we disclosed results to both guardians and participants if the latter were under age 18 and to the participants alone if they were 18  years old according to the study protocol . in zimbabwe , with permission from the provincial education officer , the consortium principal investigator and a research associate held meetings at study schools to explain the continuing study to parents / guardians , participants , and key school staff .", "we obtained written parent / guardian consent and participant assent for continued voluntary participation in the study , including separate checkbox consent for survey and blood sample collection .", "guardians raised few concerns , although they did ask why we were not disclosing test results .", "participants who were married or 18  years or older provided their own consent . in deference to local cultural norms and to prevent potential spousal abuse , we prepared a second consent form for husbands to provide consent for their wives participation .", "most of the wives gave their own consent for the survey and did not ask their husbands for consent . in four cases ,", "the husband signed a consent form and in five additional cases , the husband refused and the wife did not take the survey . although asking husbands for permission for their wives study participation is not consistent with us culture , it appears to be a useful protocol accommodation for some young wives in rural zimbabwe .", "we consistently relied on local researchers and key informants to make such culturally sensitive decisions .", "after 4  months , we found 88  % of the original sample who consented to the survey ; 85  % consented to blood specimen collection .", "of those not surveyed , two participants had died in childbirth , 2  % refused , and approximately 10  % of the sample could not be found . of those not consenting to blood specimen collection ( n  =  8) , six were married and refused for religious reasons or husband s objection .", "two were consented for the survey by school officials who refused to provide additional consent for the blood collection , and no guardian was available to give permission ."], ["hiv testing is technically far more advanced for widespread use in sub - saharan africa than hsv-2 testing .", "rapid hiv antibody tests are now widely used and accepted ( moh , republic of kenya , 2006 ) , and sequential algorithms for retesting have been adopted in kenya and zimbabwe for rapid testing ( cso & macro international inc . , 2007 ; nascop , republic of kenya , 2008 ; who , 2004 ) .", "rapid hiv tests meet high performance standards , sample collection procedures are simple , tests are easy to perform , and interpretation of test results is reasonably clear . in contrast , hsv-2 serologic testing presents some important challenges .", "although a number of hsv-2 type - specific serologic tests are available , only a limited number have been tested among sub - saharan african populations .", "these include the herpeselect hsv-2 enzyme - linked immunosorbent assay ( elisa ; focus diagnostics , cypress , ca ) and kalon hsv-2 elisa ( kalon biological ltd . , guildford , uk ; biraro , mayaud , morrow , grosskurth , & weiss , 2011 ) .", "these tests require skilled laboratory staff , specialized equipment , and a constant source of electricity .", "they are appropriate for use with large batches of samples as in sentinel surveys and are inexpensive , costing about $ 3 per test .", "although the literature reports variable performance of these tests with respect to sensitivity and specificity in sub - saharan populations , kalon is often found to be superior to focus herpeselect ( see biraro et al . , 2011 for a review of the performance of hsv-2 tests in sub - saharan africa ;", "2008 ; ngayo , friedrich , holmes , bukusi , & morrow , 2010 ; smith et al . , 2009 ; van dyck et al . , 2004 ) .", "when using the manufacturer s instructions ( index cutoff value  =  1.1 for both tests ) , kalon tends to have lower sensitivity but higher specificity than focus herpeselect ( delany - moretlwe et al .", ", 2010 ; gamiel et al . , 2008 ; ngayo et al . , 2010 ; smith et al . , 2009 ; van dyck et al . ,", "the low specificity of focus herpeselect is particularly problematic when disclosing results to research participants because of an unacceptably high rate of false positives when prevalence is low ( gamiel et al . , 2008 ; mark et al . , 2008 ; ngayo et al", ", 2010 ; van dyck et al . , 2004 ) . increasing the assay cut - off values is one strategy for improving the performance of both the kalon and focus herpeselect tests .", "a number of studies have shown that increasing the cut - off value for focus herpeselect to 3.5 increases specificity , although sensitivity is necessarily reduced ( delany - moretlwe et al . , 2010 ; smith et al . ,", "other studies similarly improved performance at cut - off values of 3.2 and 3.3 ( delany - moretlwe et al . , 2010 ; gamiel et al . , 2008 ; ngayo et al . ,", "although findings are mixed , increasing the cut - off value for the kalon test to 1.5 has also been shown to increase specificity , with an attendant reduction in sensitivity ( gamiel et al . , 2008 ; ngayo et al .", "in our studies , the kenya laboratory used the kalon assay , and the zimbabwe laboratory used the focus test . at the time of our studies , neither assay had been validated for dbs by the manufacturer in african populations .", "the efficiency of dbs for hsv-2 testing with each assay must be evaluated in specific populations prior to use ( hogrefe , ernst , & su , 2002 ) . because the kalon test had not been validated for hsv-2 testing with dbs samples , specimens in the kenya study were obtained by venipuncture .", "the zimbabwe laboratory had validated the focus test with dbs ( mudzori & mutsogoro , 2006 ) . in both studies", ", we used a modified algorithm to interpret results : samples with initial index values < 0.9 were reported as negative and no further testing was conducted .", "samples with initial index values > 2.5 were defined as high positive and were reported as positive without additional testing .", "samples with initial index values from 1.5 to 2.5 were considered low positive , and those with initial index values between 0.9 and 1.5 were considered equivocal ; all low positives and equivocals were retested in duplicate . if both retest results had index values > 1.5 , the final result was reported as positive . if both retest results were < 0.9 , the final result was negative .", "if retest results were equivocal ( 0.91.5 ) or discordant , the final result was indeterminate .", "although test performance was improved by increasing the cut - off and expanding the range of results for repeat testing , the performance characteristics of tests to detect antibodies to hsv-2 are imperfect . based on a recent systematic review of hsv-2 antibody test performance in sub - saharan africa ( biraro et al . , 2011 ) , sensitivity for the kalon test used in kenya was estimated at 94  % and specificity at 92  % with the higher cut - off value of 1.5 .", "the predictive values of all diagnostic tests are strongly influenced by the prevalence of infection in the population , and in low prevalence situations , even a very good test will have a poor positive predictive value ( ppv ) . using the sensitivity and specificity values for the kalon test under the testing conditions employed in our kenya study ,", "the ppv was estimated at 26  % with a 3  % hsv-2 prevalence .", "that is , we can be confident that about one in four participants with positive test results truly had hsv-2 antibodies present in their blood sample ; however , as many as three in four could have had a false positive test result ."], ["in kenya , we conducted sample collection and htc primarily in local public health facilities near the schools . eighteen facilities participated .", "in two sites , with permission from the school headmasters and district education officer , we collected blood samples and conducted htc at the school .", "venipuncture was conducted by six skilled phlebotomists trained in htc . to avoid obtaining two separate blood specimens ( finger prick for rapid hiv testing and venipuncture for hsv-2 testing )", "the process , including counseling , blood draw , hiv testing , disclosure of results took on average 20  min .", "the process was longer for participants with discordant results who needed a tie - breaker test and for participants with hiv positive results .", "we offered all participants and their guardians a snack either before or after specimen collection .", "we stored blood samples for hsv-2 testing in labeled vacutainer tubes in a cooler box without ice and transported them to a local laboratory within 24  h for serum processing .", "sera were stored at the local laboratory in a freezer at 20  c until transport on ice weekly to the hsv-2 testing laboratory .", "the logistics of specimen collection , processing and transport for this study were challenging but feasible . in comparison ,", "we did not require guardians to be present because hiv results were not provided to participants .", "sampling and dbs preparation were much less obvious to others nearby , helping to safeguard participant privacy and reduce concerns about witchcraft or other conspiracy theories regarding hiv / aids and blood , which circulate in sub - saharan african communities ( tenkorang , gyimah , maticka - tyndale , & adjei , 2011 ) .", "blood from the finger prick was dropped on five circles on a dbs filter card labeled with the participant s study i d .", "some participants may have been dehydrated or not very well nourished , making it difficult to fill all circles , but adequate samples were collected from all participants .", "specimens were dried , stored with a desiccant , and transported to the testing lab at ambient temperatures .", "dbs samples are easy to store and transport and require no processing or refrigeration ( see table  1 for a summary of the study procedures).table  1comparison of biomarker testing procedures in two research studieskenyazimbabweethical considerationswritten guardian consent if < 18/adolescent assentwritten guardian consent / adolescent assentresults disclosed to guardian and adolescentno results disclosedreferrals for carenot applicablecollecting and analyzing biological samples  methodvenipuncturedried blood spot  hiv testrapid testrapid test  hsv testkalon elisafocus elisafeasibility of collection methods  time20  min3  min  temperature for specimen transportcooler with no iceambienttesting costsreference labreference lab  bench fee , supplies , storage , labor us$ 5,784bench fee , supplies , labor us$ 8,000field site lab  sample processing , labor , supplies , storage us$ 3,611additional fee us$ 480  other supplies us$ 100  transportation us$ 375  total us$ 9,919total us$ 8,480 \n elisa enzyme - linked immunosorbent assay comparison of biomarker testing procedures in two research studies \n elisa enzyme - linked immunosorbent assay"], ["given the experience we have described , we conclude that it is feasible to obtain hiv and hsv-2 biomarker data for adolescent hiv prevention intervention studies . in particular", "the us pepfar program has accomplished considerable staff training in sub - saharan africa ; it has also stimulated the development of well - equipped laboratories , as well as useful tools for testing and protocols for improving test sensitivity , specificity , and cost ( stringer et al . , 2006 ) .", "in addition , it has stimulated the development of national guidelines ( particularly for adults ) for the disclosure of results . for hiv", "incorporation of hsv-2 serology results poses considerable challenges , particularly if results are disclosed to participants .", "based on our experience , there are two reasons why we do not recommend disclosing hsv-2 serology results to adolescent human subjects in sub - saharan africa .", "first , in populations with low or moderate prevalence of infection , as might be expected for adolescents , the potential for false positive test results is substantial in combination with relatively minor imperfections in test specificity .", "hsv-2 infection is often a silent disease , insofar as only 1025  % of people with antibodies for the condition are aware that they have genital herpes ( fleming et al . , 1997", "; leone , fleming , gilsenan , li , & justus , 2004 ; sizemore , lakeman , whitley , hughes , & hook , 2006 ) .", "adolescents without symptoms may deny ever having had sexual intercourse3 , further complicating the clinical picture and raising concerns about false positives with the potential for psychological and social harm related to participation in the study .", "second , local departments of health and sti authorities in sub - saharan african countries were much less familiar with hsv-2 pathology and treatment than with hiv and some other stis . even in cases where subjects acknowledge sexual exposure or genital herpes symptoms , local health clinics may not be prepared to provide appropriate treatment or care . given these vexing problems , the wisdom of disclosing hsv-2 results to adolescents after testing within a clinical prevention trial is questionable . in particular , decisions about whether to inform adolescent participants in prevention trials about hsv-2 test results should be made only after prevalence data are available and the predictive values of the test can be accurately assessed . in light of the feedback from the nih study section that raised concerns about possible stigma and abuse of hiv - positive participants in zimbabwe , as well as our own concerns ,", "we recommend that more research be conducted to study the consequences of disclosure to adolescents in sub - saharan africa .", "a review of the literature indicates that sub - saharan adolescent prevention studies with biomarker outcomes have taken a variety of approaches to disclosure . in some studies , counselors disclosed participants hiv results immediately after rapid testing ( baird , garfein , mcintosh , & ozler , 2012 ; dalal et al . , 2012 ; medley et al . , 2012 ) .", "in other studies , participants could choose to receive their results after testing , either immediately or a few weeks later ( amornkul et al .", ", 2009 ; jewkes et al . , 2006 ; ross et al . , 2007 ) .", "in still other studies , the research did not provide test results but voluntary counseling and testing ( vct ) was made freely available for study participants ( birdthistle et al .", "none of these studies , however , examined whether adolescents experienced any untoward outcomes with testing and disclosure , whether hiv positive youth enrolled in care or took steps to prevent transmission , whether hiv negative youth benefited from prevention counseling and engaged in subsequent testing , or if adolescents and their guardians felt it was appropriate to learn their status in the context of a research study . from our kenya study , the problem of stigma did not appear to be a major obstacle to adolescent testing and disclosure in our study area .", "perhaps this is because hiv prevalence is among the highest in the country , and few families have been unaffected by hiv / aids .", "moreover , high hiv prevalence has drawn research , education , and hiv counseling and testing campaigns to the area .", "for example , in our kenya clinical trial , more than 50  % of participants ( all in grade 7 and 8) reported that they had previously been tested for hiv , mostly through a home - based testing initiative of the kenya medical research institute ( kemri ) and us centers for disease control and prevention ( cdc ) .", "although our study provided hiv test results to youth , we relied on phlebotomist / nurse counselors to refer youth and their guardians for care , following national guidelines .", "the onus of making contact with the clinic was left up to the individual and was not tracked by the system of care ; and follow up to make sure that hiv positive youth accessed care was well beyond the scope of our study . since it is unknown whether youth experience significant mental trauma and social prejudice , and whether they actually engage in the system of care , we recommend that further research be conducted to examine the perceptions and behaviors of youth  and particularly newly diagnosed youth  after disclosure of their test results to ascertain the consequences of this aspect of research participation and whether it can be improved .", "this is particularly important for adolescents , since results are disclosed in the presence of their guardian .", "regarding procedures for collecting biomarkers , we conclude that finger - sticks and dbs are overwhelmingly superior to venipuncture in sample collection efficiency and reduced burden on participants and community institutions .", "manufacturer validations of dbs for hsv-2 are urgently needed , however , for this procedure to be more widely accessible to researchers . because blood collection for dbs is minimally invasive , this procedure has excellent potential for widespread acceptability and consequent high participation in school and community research sites .", "in addition , rapid hiv testing in sub - saharan africa is typically conducted by finger prick , making this the preferred way for trained african health workers and counselors to participate in research data collection ( who , 2004 ) .", "given the complex decisions required for biomarkers , we recommend that behavioral prevention scientists interested in using biomarker data collaborate from the early planning stages with biomedical scientists who have expert knowledge in hsv-2 , as well as hiv , laboratory tests and testing procedures .", "we found that team members with such expertise provide valuable assistance in selecting qualified in - country laboratories , developing appropriate budgets , protocols , quality assurance and algorithms for interpreting results , and helping research staff to bridge communication with in - country laboratory staff .", "further , we recommend that hsv-2 test kit manufacturers determine optimal cut - off standards for sub - saharan populations , and that researchers who conduct hsv-2 testing explicitly define the testing cutoffs they use in publications .", "there is now a substantial literature documenting validity problems when using manufacturers cutoffs with african populations potentially inflating prevalence findings ( gamiel et al . , 2008 ; ngayo et al . , 2010 ; van dyck et al . , 2004", "this suggests the need for action to improve assay validation practices on the part of manufacturers as well as methodological details from researchers documenting their findings . when conducting collaborative international research , it is important to recognize that alternative ethical systems exist .", "thus , for international research we recommend inclusion of team members who are knowledgeable about local contexts . indeed", ", successful collaborative research partnerships respect local cultures , values and practices ; negotiate effectively within these systems ; and incorporate into study designs ethical practices that are appropriate and sensitive to local cultural contexts ( e.g. , in our case , providing for husband consent for married participants ; christakis , 1992 ; emanuel , wendler , killen , & grady , 2004 ) .", "behavioral prevention scientists traditionally have relied on self - reported sexual behavior survey item measures to evaluate adolescent hiv prevention interventions .", "our experiences conducting research with orphan adolescents in kenya and zimbabwe suggest that collection of hiv and sti test results even in rural , resource - poor settings in sub - saharan africa is a feasible addition to the behavioral research toolkit .", "the use of sti biomarkers can greatly improve the validity of findings from adolescent behavioral intervention trials .", "research is urgently needed to examine the risks and benefits of hiv testing and disclosure of test results in the context of a research study for adolescents . further development and implementation of sti biomarker assessment techniques  particularly pertaining to using dbs  is needed to advance hiv prevention science ."]]}
{"article_id": "PMC3322213", "article_text": ["stroke is rare in children , however , a common cause of neurological disease , and it is a major cause of death ranks in the top ten in pediatric period11,15 ) . documented incidence has been reported as 2.5 - 8/100000 children / year12,13 ) .", "medical treatment includes maintaining cerebral perfusion pressure with hyperventilation and osmotherapy , barbiturate coma and in case of thrombotic ischemia , thrombolytic procedure , and anticoagulanttherapy .", "however , if all medical treatment is fail to drop the intracranial pressure ( icp ) and deterioration of patient progressed , alternative treatments , such as surgical decompression could be mandatory .", "the common rationale of decompressive craniectomy with or without duroplasty is to let the volume expansion of the swelling brain to extracranial space via removed skull flap and prevent cerebral herniation and secondary damage of brain parenchyme . in case of traumatic brain injury ,", "however , there has not been a definite evidence or standard guideline for decompressive craniectomy for pediatric patient with non - traumatic acute stroke with uncontrolled refractory high intracranial pressure .", "we will introduce our series about the functional and clinical outcome after decompressive craniectomy to control refractory high icp due to non - traumatic acute stroke in pediatric patient .", "between march 2004 and december 2006 , decompressive - hemicraniectomy and duroplasty was performed in 5 toddlers and preschool children with non - traumatic , malignant refractory high icp .", "any patient have no trauma history , and refractory high icp was diagnosed as clinical evidence of acute stroke , massive infarction or hemorrhage with midline shift , compression of basal cisterns in conventional radiologic examination such as computed tomography ( ct ) and magnetic resonance image ( mri ) and , neurological deterioration consisting of decreased level of consciousness or increasing levels of sedation , or somnolence or stupor compared with baseline status at admission17 ) . and", "all operations were performed by one pediatric neurosurgeon and all patients received treatment in pediatric intensive care unit of one medical center . in all patients , ct or mri", "blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit .", "clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy", "craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone .", "a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression .", "multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin .", "the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later .", "glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge .", "in all patients , ct or mri was obtained immediately , after stabilization of ventilation and hemodynamicsat first .", "blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit .", "clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy", "craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone .", "a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression .", "multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin .", "the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later .", "glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge .", "five pediatric patients were treated with decompressive hemicraniectomy with duroplasty for non - traumatic and refractory intracranial hypertension after unilateral hemispheric stroke .", "four patients were toddler and one patient was pre - school girl . three were boy and two were girl .", "four cases were caused by ischemic stroke , and another one case was hemorrhagic stroke .", "one case of ischemic stroke , patient had atrial septal defect ( asd ) . and including this patient three cases of ischemic stroke patients were taken angiography ; however there was no abnormal findings in angiography . in all patients , conventional laboratory result and special serum anti - body test , including anti - phospholipid antibody were within normal rage . at the time of operation , all patients had a gcs score < 8 ( median 7 , range 6 - 8 ) and all patients hadunilateral mydriasis . surgical decompressive hemicraniectomies were performed at a mean of 12 hours ( range 4 - 19 hours ) . in all cases we had performed supratentorial hemicraniectomies .", "the mean peak icp was 28.6 mm hg ( range 25 - 30 mm hg ) . and the icp of all patients were dropped to normal range within one and half days after operation .", "there were no surgical complications , such as cerebrospinal fluid leak or wound infection , even after cranioplasty .", "one of ischemic stroke patient had got a ventriculoperitoneal shunt operation because of post - stroke hydrocephalus .", "the mean follow - up period was 47.6 months ( 36 - 68 months).gos and pcpcs for 5 patients had been measured to evaluate postoperative neurological outcome .", "based on the regular follow up gos scores , 5 patients had shown satisfied recoveries : 4 had good recoveries ( gos score of 5 ) , and 1 had moderate disabilities ( gos score of 4 ) .", "the pcpcs scores also had revealed contend results : 4 patients received scores of 2 , and only one patient scored of 3 . a 17-month - old girl was transferred to our emergency department with decreased mentality .", "hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side .", "in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd . initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and emergency mri sturdy was performed with mr angiography and diffusion - weighted image .", "but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift .", "after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days .", "a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore .", "the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty .", "the bone flap was secured without any complications . at the end of follow - up ,", "41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus .", "a 17-month - old girl was transferred to our emergency department with decreased mentality . neurological examination revealed a stuporous child without occasional spontaneous eye opening .", "hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side .", "in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd .", "initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and", "but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift .", "after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days .", "a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore .", "the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty .", "the bone flap was secured without any complications . at the end of follow - up ,", "41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus .", "in stroke patient , most common cause of death is due to uncontrolled icp associated with large hemispheric infarctions10,12 ) . when it is present , it is associated with a dramatic increase in mortality .", "in addition , early icp elevation in patients with large hemispheric infarctions is highly concluded with high mortality16 ) .", "it means that the one of most important goal of acute phase stroke treatment is control the icp and prevent a secondary damage due to brain swelling . in spite of many clinical and experimental trials on medical therapeutic method ,", "there has been a widely accepted definite guideline for oxygen saturation , temperature , serum glucose level , blood pressure for adult stroke patient1 ) .", "however , for pediatric stroke patient , even in many of mostly basic and elementary medical guideline has not been established yet14 ) . to prevent and control icp , thrombolytic therapy such as clot lysis has an accepted role in acute stroke treatment . in adult stroke patient group , there are antithrombotic management of adult ischemic stroke protocol and guideline published by the american heart association1 ) , such as intravenous tissue plasminogen activator , intra - arterial thrombolysis and oral administration of aspirin .", "but , for pediatric stroke patient , there is no guideline of this useful treatment method either14 ) . to the best of our knowledge ,", "the first report about the benefits of decompressive hemicraniectomy in pediatric patient was described by carter et al.3 ) at 1997 . however , from this time , only a few report deals with decompressive craniectomy for pediatric patient were published . in adult acute stroke patient group , the effect of decompressive craniectomy also has been established .", "vahedi et al.21 ) reported that in a meta - analysis combining the data of 93 subjects from three small , randomized , controlled trials , mortality for conservative management in adult ischemic stroke patients with early brain edema was 71% as compared with 22% for decompressive surgical intervention . nowadays", ", some case reports and review has been published favorable outcome after decompressive surgery in childhood acute stroke2,17 ) .", "it is not often however in some article , decompressive craniotomy have been regarded as emerging procedure can save life , for pediatric stroke patient6,11 ) , even guideline for craniectomy was introduced11 ) . in point of fact", "widely accepted proper operation timing is immediately after signs of herniation and a midline shift which was defined as a more than equals 5-mm contralateral shift of the midline structures on the preoperative cranial ct scan on septum pellucidum level , compressed basal cisterns , or uncal herniation -- had to be present on cranial ct had been noted .", "recent studies in adult stroke group have shown that this procedure not only reduces mortality but also improves neurological functional outcome .", "this procedure may be life - saving if done early in cases of impending herniation , is associated with good outcomes in pediatric stroke patients9,11 ) .", "maybe if operation was performed earlier , it may minimize the vicious circle of brain swelling , increased icp , ischemia , and infarction12 ) . in our series , four toddlers and one pre - school girl with refractory high icp due to non - traumatic , ischemic and hemorrhagic stroke", "four patient received pcpcs score of 2 ( mild disability ) and only one received that of 3 ( moderate disability ) after 3 years later post operation .", "these results showed that decompressive craniectomy can not only play a rescuer therapeutic role but also guaranty acceptable clinical outcome in the treatment regimen in non - traumatic , refractory high icp in pediatric field . in some articles , decompressive", "aghakhani et al.2 ) report that decompressive craniectomy was performed in pediatric patient with malignant intracranial hypertension due to infectious encephalitis . in adult patient group , there had been not infrequently reported that malignant intracranial hypertension due to encephalitis treated with decompressive craniectomy .", "now , even in case of pediatric patient with infectious disease in central nerve system , decompressive craniectomy could be considered as life - saving - treatment options .", "gordon et al.7 ) report that pediatric stroke can affect not only patient itself , but also his or her whole family .", "over half of stroke survivors have suffer from decrease in quality of life and affects the entire family , and relates to both neurological deficits and psychosocial factors11 ) .", "hence , if all medical treatment is fail to drop the icp in non - traumatic pediatric acute stroke patient , without hesitation , a decompressive craniectomy could be regarded as next step treatment option .", "in this study , we describes the young pediatric patients have undergone decompressivehemicraniectomy for a non - traumatic acute hemispheric stroke .", "it also shows that decompressive hemicraniectomy can be lifesaving and can besafely performed in toddler and pre - school children . and young children can get a survival and relatively independence and family satisfaction.decompressive hemicraniectomy", "should be considered as an alternative therapy for patients with life - threatening brains welling refractory to medical management ."], "abstract_text": ["<S> objectivelife - threatening hemispheric stroke is associated with a high mortality and morbidity . </S>", "<S> decompressive hemicraniectomy has been regarded as an effective treatment option for refractory intracranial hypertension . here , </S>", "<S> we reported the clinical course of 5 children with decompressive craniectomy and duroplasty after non - traumatic refractory intracranial hypertension.methodsfour toddlers and one preschool - girl were included in this study ; there were 3 boys and 2 girls with a mean age of 34.6 months ( range 17 - 80 ) . </S>", "<S> decompressive craniectomy including duroplasty was performed in cases of dilatation of pupil size after intensified standard medical therapy had proven insufficient . </S>", "<S> all children had a pediatric glasgow coma scale score < 8 at pre - operation state . </S>", "<S> the mean time - point of craniectomy after stroke attack was 12 hours ( range 4 - 19).resultsduring the long - term follow - up period ( mean 47.6 months ) , no children died . </S>", "<S> one year later , when we checked their glasgow outcome scale scores , only one toddler received a score of 4 ( moderate disability ) . </S>", "<S> but the others had good recoveries although they had minor physical or mental deficits . </S>", "<S> according to the pediatric cerebral performance category scale , 4 children received a score of 2 ( mild disability).conclusiondespite our small cases , we suggest that decompressive hemicraniectomy and duroplasty is an acceptable and life - saving treatment for refractory intracranial hypertension after unilateral hemispheric stroke in toddlers and preschool children . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Medical treatment", "Surgical protocol", "RESULTS", "Patient presentation", "DISCUSSION", "CONCLUSION"], "sections": [["stroke is rare in children , however , a common cause of neurological disease , and it is a major cause of death ranks in the top ten in pediatric period11,15 ) . documented incidence has been reported as 2.5 - 8/100000 children / year12,13 ) .", "medical treatment includes maintaining cerebral perfusion pressure with hyperventilation and osmotherapy , barbiturate coma and in case of thrombotic ischemia , thrombolytic procedure , and anticoagulanttherapy .", "however , if all medical treatment is fail to drop the intracranial pressure ( icp ) and deterioration of patient progressed , alternative treatments , such as surgical decompression could be mandatory .", "the common rationale of decompressive craniectomy with or without duroplasty is to let the volume expansion of the swelling brain to extracranial space via removed skull flap and prevent cerebral herniation and secondary damage of brain parenchyme . in case of traumatic brain injury ,", "however , there has not been a definite evidence or standard guideline for decompressive craniectomy for pediatric patient with non - traumatic acute stroke with uncontrolled refractory high intracranial pressure .", "we will introduce our series about the functional and clinical outcome after decompressive craniectomy to control refractory high icp due to non - traumatic acute stroke in pediatric patient ."], ["between march 2004 and december 2006 , decompressive - hemicraniectomy and duroplasty was performed in 5 toddlers and preschool children with non - traumatic , malignant refractory high icp .", "any patient have no trauma history , and refractory high icp was diagnosed as clinical evidence of acute stroke , massive infarction or hemorrhage with midline shift , compression of basal cisterns in conventional radiologic examination such as computed tomography ( ct ) and magnetic resonance image ( mri ) and , neurological deterioration consisting of decreased level of consciousness or increasing levels of sedation , or somnolence or stupor compared with baseline status at admission17 ) . and", "all operations were performed by one pediatric neurosurgeon and all patients received treatment in pediatric intensive care unit of one medical center . in all patients , ct or mri", "blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit .", "clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy", "craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone .", "a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression .", "multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin .", "the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later .", "glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge ."], ["in all patients , ct or mri was obtained immediately , after stabilization of ventilation and hemodynamicsat first .", "blood sugar , body temperature , ventilation / oxygenation , blood volume and trans - cutaneous oxygen saturation , arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit .", "clinical and neurologic status was evaluated with the glasgow coma scale ( gcs ) . to reduce icp , hyperosmolar therapy"], ["craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal , parietal , and temporal bone .", "a large skin flap was lifted from the skull with meticulous blood control , multipleburr holes were made , large bone flap removed . in the temporalregion , the craniectomies were extended toward the floor of the middlefossa to maximize decompression .", "multiple openings in the dura were made ; a dural patchwas placed and sutured . in every case , intra - cerebral pressure monitor probe was positioned epidural space of bone margin .", "the bone flaps were stored in a bone bank and then , following improvement of clinical and neurologic status , cranioplasty was performed 3 to 6 months later .", "glasgow outcome scale ( gos)5 ) , and pediatric cerebral performance category scale ( pcpcs)4 ) ( table 2 ) calculated every 6 months after discharge ."], ["five pediatric patients were treated with decompressive hemicraniectomy with duroplasty for non - traumatic and refractory intracranial hypertension after unilateral hemispheric stroke .", "four patients were toddler and one patient was pre - school girl . three were boy and two were girl .", "four cases were caused by ischemic stroke , and another one case was hemorrhagic stroke .", "one case of ischemic stroke , patient had atrial septal defect ( asd ) . and including this patient three cases of ischemic stroke patients were taken angiography ; however there was no abnormal findings in angiography . in all patients , conventional laboratory result and special serum anti - body test , including anti - phospholipid antibody were within normal rage . at the time of operation , all patients had a gcs score < 8 ( median 7 , range 6 - 8 ) and all patients hadunilateral mydriasis . surgical decompressive hemicraniectomies were performed at a mean of 12 hours ( range 4 - 19 hours ) . in all cases we had performed supratentorial hemicraniectomies .", "the mean peak icp was 28.6 mm hg ( range 25 - 30 mm hg ) . and the icp of all patients were dropped to normal range within one and half days after operation .", "there were no surgical complications , such as cerebrospinal fluid leak or wound infection , even after cranioplasty .", "one of ischemic stroke patient had got a ventriculoperitoneal shunt operation because of post - stroke hydrocephalus .", "the mean follow - up period was 47.6 months ( 36 - 68 months).gos and pcpcs for 5 patients had been measured to evaluate postoperative neurological outcome .", "based on the regular follow up gos scores , 5 patients had shown satisfied recoveries : 4 had good recoveries ( gos score of 5 ) , and 1 had moderate disabilities ( gos score of 4 ) .", "the pcpcs scores also had revealed contend results : 4 patients received scores of 2 , and only one patient scored of 3 . a 17-month - old girl was transferred to our emergency department with decreased mentality .", "hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side .", "in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd . initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and emergency mri sturdy was performed with mr angiography and diffusion - weighted image .", "but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift .", "after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days .", "a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore .", "the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty .", "the bone flap was secured without any complications . at the end of follow - up ,", "41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus ."], ["a 17-month - old girl was transferred to our emergency department with decreased mentality . neurological examination revealed a stuporous child without occasional spontaneous eye opening .", "hemiplegia , hyper - reflexia , and babinski 's sign were observed on the left side .", "in general physical examination , she had an ejection systolic murmur . by pediatriccardiologist consultation , patient was diagnosis with asd .", "initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . and", "but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift .", "after surgery , she was treated in pediatric intensive care unit with be kept intubated for 7 days .", "a brain ct at the time of post - operative 7 days revealed sustained brain swelling , but midline shift did not noted anymore .", "the icp dropped to normal gradually within one day after surgery . at the time of postoperative 3-month , she returned for an elective cranioplasty .", "the bone flap was secured without any complications . at the end of follow - up ,", "41 months after surgery , this patient maintained a pcpcs of 2 , and gos of 5 , and she had showed mild left side hemiparesis , mild behavioral disorder and strabismus ."], ["in stroke patient , most common cause of death is due to uncontrolled icp associated with large hemispheric infarctions10,12 ) . when it is present , it is associated with a dramatic increase in mortality .", "in addition , early icp elevation in patients with large hemispheric infarctions is highly concluded with high mortality16 ) .", "it means that the one of most important goal of acute phase stroke treatment is control the icp and prevent a secondary damage due to brain swelling . in spite of many clinical and experimental trials on medical therapeutic method ,", "there has been a widely accepted definite guideline for oxygen saturation , temperature , serum glucose level , blood pressure for adult stroke patient1 ) .", "however , for pediatric stroke patient , even in many of mostly basic and elementary medical guideline has not been established yet14 ) . to prevent and control icp , thrombolytic therapy such as clot lysis has an accepted role in acute stroke treatment . in adult stroke patient group , there are antithrombotic management of adult ischemic stroke protocol and guideline published by the american heart association1 ) , such as intravenous tissue plasminogen activator , intra - arterial thrombolysis and oral administration of aspirin .", "but , for pediatric stroke patient , there is no guideline of this useful treatment method either14 ) . to the best of our knowledge ,", "the first report about the benefits of decompressive hemicraniectomy in pediatric patient was described by carter et al.3 ) at 1997 . however , from this time , only a few report deals with decompressive craniectomy for pediatric patient were published . in adult acute stroke patient group , the effect of decompressive craniectomy also has been established .", "vahedi et al.21 ) reported that in a meta - analysis combining the data of 93 subjects from three small , randomized , controlled trials , mortality for conservative management in adult ischemic stroke patients with early brain edema was 71% as compared with 22% for decompressive surgical intervention . nowadays", ", some case reports and review has been published favorable outcome after decompressive surgery in childhood acute stroke2,17 ) .", "it is not often however in some article , decompressive craniotomy have been regarded as emerging procedure can save life , for pediatric stroke patient6,11 ) , even guideline for craniectomy was introduced11 ) . in point of fact", "widely accepted proper operation timing is immediately after signs of herniation and a midline shift which was defined as a more than equals 5-mm contralateral shift of the midline structures on the preoperative cranial ct scan on septum pellucidum level , compressed basal cisterns , or uncal herniation -- had to be present on cranial ct had been noted .", "recent studies in adult stroke group have shown that this procedure not only reduces mortality but also improves neurological functional outcome .", "this procedure may be life - saving if done early in cases of impending herniation , is associated with good outcomes in pediatric stroke patients9,11 ) .", "maybe if operation was performed earlier , it may minimize the vicious circle of brain swelling , increased icp , ischemia , and infarction12 ) . in our series , four toddlers and one pre - school girl with refractory high icp due to non - traumatic , ischemic and hemorrhagic stroke", "four patient received pcpcs score of 2 ( mild disability ) and only one received that of 3 ( moderate disability ) after 3 years later post operation .", "these results showed that decompressive craniectomy can not only play a rescuer therapeutic role but also guaranty acceptable clinical outcome in the treatment regimen in non - traumatic , refractory high icp in pediatric field . in some articles , decompressive", "aghakhani et al.2 ) report that decompressive craniectomy was performed in pediatric patient with malignant intracranial hypertension due to infectious encephalitis . in adult patient group , there had been not infrequently reported that malignant intracranial hypertension due to encephalitis treated with decompressive craniectomy .", "now , even in case of pediatric patient with infectious disease in central nerve system , decompressive craniectomy could be considered as life - saving - treatment options .", "gordon et al.7 ) report that pediatric stroke can affect not only patient itself , but also his or her whole family .", "over half of stroke survivors have suffer from decrease in quality of life and affects the entire family , and relates to both neurological deficits and psychosocial factors11 ) .", "hence , if all medical treatment is fail to drop the icp in non - traumatic pediatric acute stroke patient , without hesitation , a decompressive craniectomy could be regarded as next step treatment option ."], ["in this study , we describes the young pediatric patients have undergone decompressivehemicraniectomy for a non - traumatic acute hemispheric stroke .", "it also shows that decompressive hemicraniectomy can be lifesaving and can besafely performed in toddler and pre - school children . and young children can get a survival and relatively independence and family satisfaction.decompressive hemicraniectomy", "should be considered as an alternative therapy for patients with life - threatening brains welling refractory to medical management ."]]}
{"article_id": "PMC2752919", "article_text": ["this was a double - blind , randomized , crossover trial in accordance with the declaration of helsinki and good clinical practice .", "the primary objective was to assess albuminuria during different treatments compared with that with placebo ; secondary objectives were to assess effect on 24-h blood pressure , glomerular filtration rate ( gfr ) , biomarkers , and raas components .", "after informed consent , patients attended a screening visit comprising laboratory tests and evaluations of inclusion / exclusion criteria .", "slow - release furosemide in a fixed dose ( mean dose 109 mg/24 h , range 60360 mg/24 h ) was prescribed to prevent blood pressure elevation and fluid retention .", "patients used an electronic blood pressure device ( ua-779 ; a&d instruments , abingdon , u.k . ) to measure home blood pressure throughout the study .", "change in uaer ( percentage ) versus placebo during treatment with 300 mg aliskiren daily , 300 mg irbesartan daily , or the combination ( p < 0.001 vs. placebo for all treatments ) . from a list of prescreened candidates , 41 patients were screened for study participation ( supplementary fig .", "1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-0168/dc1 ) . nine of these were screen failures mainly due to albuminuria levels below the randomization requirement .", "of the 10 randomly assigned patients who left the study before completion , 2 died , 1 was lost to follow - up , 4 had an adverse event that led to exclusion ( diarrhea , severe hypertension , recurrent urinary tract infection , and dizziness ) , and 3 withdrew consent .", "twenty - six patients had the primary end point , albuminuria assessed after randomization , and were included in the final analysis ; the remaining six patients dropped out shortly after random assignment and were not included in the final analysis .", "after washout , patients attended a randomization visit before 2 months of treatment with placebo , 300 mg aliskiren once daily , 300 mg irbesartan daily , or the combination of the two , in random order .", "patients with type 2 diabetes ( world health organization criteria ) aged 3080 years were eligible for randomization with baseline urinary albumin excretion rate ( uaer ) > 100 mg/24 h , hypertension ( baseline office blood pressure > 135/85 mmhg ) , and baseline gfr > 40 ml / min per 1.73 m. exclusion criteria included major cardiovascular disease ( within 6 months ) , heart failure ( new york heart association class ii ", "rather , we used active washout : during the first 14 days of all treatments , every patient received 150 mg aliskiren daily to avoid risk of hypotension or a drastic increase in blood pressure in the switch from placebo to combination treatment or vice versa . during the last 3 days of each treatment", ", patients collected three consecutive 24-h urine samples for assessment of geometric mean uaer . at the last day of each treatment period , patients attended our clinic for assessment of gfr and mounting of standard takeda 24-h blood pressure devices ( tm2421 , version 7 ; a&d medical , tokyo , japan ) .", "measurements were performed every 15 min from 7 to 23 h ( daytime ) and every 30 min from 23 to 7 h ( nighttime ) .", "urinary albumin and creatinine concentrations were determined on a turbidimetric hitachi 912 system ( roche diagnostics , mannheim , germany ) .", "samples for prorenin , plasma renin activity ( pra ) , high - sensitivity pra ( hs - pra ) , immunoreactive plasma renin concentration ( ir - prc ) , angiotensinogen , ang i , ace activity , ang ii , and aldosterone levels were determined after 30 min of supine rest , and the plasma was frozen after centrifugation ( 80c ) .", "raas components and biomarkers were measured at baseline and at the end of treatment periods .", "biomarkers of inflammation , endothelial dysfunction , and cardiovascular risk were measured : high - sensitivity c - reactive protein ( hs - crp ) ( enzyme immunosorbent assay [ eia ] ; dako , glostrup , denmark ) ; serum soluble vascular adhesion molecule-1 and serum soluble intercellular adhesion molecule-1 ( eia ; diaclone , besanon , france ) ; plasma plasminogen activator inhibitor-1 ( hyphen biomed kit ; andresy , france ) ; serum nh2-terminal - probrain natriuretic peptide ( eia kit ; biomedica , wien , austria ) ; fibrinogen ( immunoturbidimetry ) ; and plasma asymmetrical dimethyl arginine ( high - performance liquid chromatography ) .", "total renin concentration , prorenin concentration , plasma angiotensinogen , pra , and biomarkers of inflammation and endothelial dysfunction were measured using methodology described previously ( 4 ) .", "ir - prc was measured with an immunoradiometric kit ( renin iii ; cisbio , gif - sur - yvette , france ) .", "hs - pra was measured by in - house radioimmunoassay of ang i formed during incubation of 25 l plasma and 50 pmol sheep angiotensinogen for 3 h at 37c in a total reaction volume of 100 l .", "the assay was double calibrated against ang i and the international reference preparation of renin , 68/356 , from nibsc ( hertfordshire , u.k . ) .", "plasma ang i and ang ii were measured using in - house radioimmunoassays and ethanol extraction of plasma samples .", "ace activity was determined using a commercial radioenzymatic assay ( ace direct ; bhlmann - laboratories , schnenbuch , switzerland ) .", "randomization was blinded to all investigators , and the study drugs were packed and labeled before delivery to the site .", "it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% .", "this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% .", "the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure", "were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor .", "extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data . a two - sided p value < 0.05 was considered significant", "the correlation between changes in albuminuria and changes in hs - pra or ang ii were assessed by a nonparametric spearman correlation coefficient .", "correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined .", "fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration  gfr ) , and log - transformed levels were compared using a paired t test .", "statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) .", "it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% .", "this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% .", "the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor .", "extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data .", "a two - sided p value < 0.05 was considered significant . the correlation between changes in albuminuria and changes in hs - pra or ang ii", "correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined .", "fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration  gfr ) , and log - transformed levels were compared using a paired t test .", "statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) .", "baseline demographic data are shown in table 1 . primary and secondary objectives were met . during placebo treatment", "geometric mean uaer was 258 ( range 842,361 ) mg / day , mean  sd 24-h blood pressure was 140/73  15/8 mmhg , daytime blood pressure was 144/76  17/9 , and nighttime blood pressure was 130/68  17/9 .", "gfr was 89  27 ml / min per 1.73 m and mean serum creatinine was 88", "demographics of the 32 randomly assigned patients with type 2 diabetes , hypertension , and albuminuria and the 26 included in the final analysis data are means sd , n , or mean ( range ) .", "aliskiren treatment led to a significant reduction in albuminuria by 48% ( 95% ci 2762 ) compared with placebo ( p < 0.001 ) but not significantly different from irbesartan , lowering uaer by 58% ( 4270 ) ( p < 0.001 vs. placebo ) .", "combination treatment reduced albuminuria by 71% ( 5979 ) ( p < 0.001 ) compared with placebo , significantly more than with either monotherapy ( p < 0.001 and p = 0.028 ) .", "the relative difference between aliskiren and combination treatment was 31% . to adjust for treatment - induced changes in gfr and the potential influence on albuminuria reduction , we calculated fractional clearance , which was reduced by 46% versus placebo during aliskiren treatment ( p = 0.021 ) , by 56% versus placebo during irbesartan treatment ( p = 0.002 ) , and by 67% versus placebo during combination treatment ( p = 0.001 ) .", "systolic / diastolic 24-h blood pressure was reduced 3/4 mmhg by aliskiren ( ns / p = 0.009 ) , 12/5 mmhg by irbesartan ( p < 0.001/p = 0.002 ) , and 10/6 mmhg by the combination ( p = 0.001/p < 0.001 ) versus placebo .", "a correlation was found between change in albuminuria and change in 24-h diastolic blood pressure during all treatments ( p = 0.039 ) .", "seated office systolic / diastolic blood pressure was reduced 7/4 mmhg by aliskiren , 6/4 mmhg by irbesartan , and 12/8 mmhg by the combination , all statistically significant compared with placebo , except for diastolic blood pressure during irbesartan treatment .", "gfr was significantly reduced 4.6 ( 95% ci 8.80.3 ) ml / min per 1.73 m by aliskiren ( p = 0.037 ) , 8.0 ( 12.33.6 ) ml / min per 1.73 m by irbesartan ( p < 0.001 ) , and 11.7 ( 15.97.4 ) ml / min per 1.73 m by the combination ( p < 0.001 ) compared with placebo .", "aliskiren significantly reduced hs - pra , ang i , and ang ii by 87 , 75 , and 52% , respectively , compared with placebo ; irbesartan had the opposite effect ( table 2 ) .", "when combined , the activating effect of irbesartan was counteracted by aliskiren , reducing hs - pra , ang i , and ang ii by 88 , 78 , and 56% , respectively , compared with irbesartan monotherapy . whereas combination treatment caused a 1,068% increase in ir - prc versus a 279% increase during aliskiren monotherapy and a 178% increase during irbesartan monotherapy , hs - pra was reduced 47% compared with placebo after combination therapy .", "pra measured by a conventional method was affected similarly to hs - pra , although the changes were smaller ( table 2 ) .", "the renin - specific activity ( renin bioactivity / total renin mass ) was 3 and 4% after aliskiren and combination therapy compared with placebo , respectively , thereby confirming that 9697% of renin was aliskiren bound . during irbesartan monotherapy ,", "changes in raas components and cardiovascular biomarkers versus placebo all treatments were administered once daily .", "adma , asymmetrical dimethyl arginine ; icam-1 , intracellular adhesion molecule-1 ; nt - probnp , nh2-terminal - probrain natriuretic peptide ; pai-1 , plasminogen activator inhibitor-1 ; vcam-1 , vascular adhesion molecule-1 ; vwf , von willebrand factor .", "a significant correlation between reduction in albuminuria and increase in ir - prc was observed for all active treatments combined ( r = 0.597 , p = 0.0001 ) .", "there was a significant correlation between changes in albuminuria and increase in ang ii during irbesartan treatment ( correlation coefficient 0.486 , p = 0.022 ) ; no significant correlations were observed in the other treatment groups .", "hs - crp was reduced 35% from the placebo level with aliskiren ( p = 0.047 ) and 35% with irbesartan ( p = 0.043 ) .", "other statistically significant changes from placebo levels were a 6% reduction in soluble intracellular adhesion molecule-1 ( p = 0.017 ) observed with the combination treatment and a 7% reduction in fibrinogen during aliskiren treatment ( p = 0.037 ) .", "no treatment led to significant changes from placebo levels in any of the other cardiovascular biomarkers ( table 2 ) .", "the most frequent adverse events were urinary tract infection ( four patients , one male ) , pneumonia ( three patients ) , and cough ( three patients ) , occurring during different treatments .", "anemia and hypomagnesemia were detected in two patients during the combination treatment . compared with each monotherapy", ", combination treatment showed an increase in plasma potassium by 0.2 mmol / l ( p = 0.036 ) .", "one patient dropped out during the placebo period after several systolic blood pressure readings > 180 mmhg .", "two patients died before the first measurement of albuminuria after the randomization and were not included in the final end point analysis .", "the first death was that of a 42-year - old obese man ( bmi 42 kg / m ) with a history of ischemic heart disease , myocardial infarction , and hypertension 4 years before study entry .", "the patient experienced sudden cardiac arrest , seemingly after a myocardial infarction during aliskiren treatment .", "the second death was that of a hypertensive , obese , 73-year - old man with diabetes duration of 16 years .", "the deaths were instantly reported to relevant authorities and were not suspected as being related to any of the drugs studied .", "subsequently , home blood pressure measurement frequency was increased from twice weekly to twice daily , and patients were instructed to contact the investigator by direct phone ( available around the clock ) , if any measurement was > 160/100 mmhg .", "extra measurements of sodium , potassium , and creatinine were introduced 3 weeks into each treatment period .", "in this exploratory study , we demonstrated that treatment with 300 mg aliskiren once daily was as efficient in reducing albuminuria as standard therapy with 300 mg irbesartan once daily . when we combined the two treatments at the same doses , the reduction in albuminuria", "was enhanced . the added antiproteinuric effect with combination treatment compared with aliskiren alone was 31% .", "given that the reductions in 24-h systolic blood pressure with aliskiren were unexpectedly small compared with those with placebo relative to 24-h diastolic blood pressure changes and were substantially lower than the office systolic blood pressure measurements in this study , we conducted a thorough review of potential flaws in data collection , storage , device calibration , reporting , and calculation .", "there was no evidence to indicate that the ambulatory data collection , storage , or reporting was flawed , and the unexpected results could be a chance occurrence .", "our study suggested that the combination of aliskiren and irbesartan had an additional raas blocking effect compared with monotherapy because a synergistic increase in ir - prc was observed with the combination , which was related to the antiproteinuric effect , whereas hs - pra was reduced 50% compared with the reduction with placebo .", "as opposed to the aliskiren in the evaluation of proteinuria in diabetes ( avoid ) study ( 5 ) , which showed an additional 20% albuminuria reduction after 24 weeks of treatment with aliskiren compared with placebo added to the maximal recommended dose of losartan and optimal antihypertensive therapy , this is the first study with a head - to - head comparison between aliskiren and irbesartan treatment .", "no other antihypertensive drugs except for furosemide were allowed in our study , thereby offering a clearer picture of the effect of the two compounds used , compared with the avoid study , in which aliskiren was combined with losartan and a mixture of other antihypertensive drugs .", "this study is different with regard to patient population , with a lower mean baseline uaer compared with that in the avoid study .", "in addition , we assessed the effect of renin inhibition on gfr , a measurement that is more precise than the estimated gfr used in the avoid study .", "raas blockade is believed to reduce proteinuria through several different mechanisms : the mean transcapillary hydraulic pressure difference , the glomerular surface area , and the size and charge selectivity of the glomerular filter . in diabetic nephropathy several of these variables", "are abnormal , and raas blockade has been demonstrated to normalize directly measured or estimated glomerular hydraulic pressure ( 79 ) , to reduce the shunt - like defects in the membrane , at least in part ( 10 ) , and to restore the charge - selectivity properties of the glomerular membrane ( 11 ) .", "aliskiren is thought to reduce albuminuria by the same mechanism as during treatment with ace inhibitors or arbs .", "recently , fisher et al . ( 12 ) have shown that aliskiren treatment increases renal plasma flow to a larger extent than the ace inhibitor captopril .", "the increase in renal plasma flow may be a response to angiotensin at1 receptor  dependent reduction of the vascular tone in the efferent arteriole . reduced vascular tone in the efferent glomerular arteriole", "could be responsible for the decrease in intraglomerular pressure , leading to the reduction in albuminuria and gfr as demonstrated in our study .", "although the combination reduced gfr up to 12 ml / min ( 95% ci 15.97.4 ) , we interpreted this as an reversible hemodynamic change and not as an indication of nephrotoxicity ( 13 ) .", "in fact , it has been shown that an early hemodynamic reduction in gfr can translate into long - term renoprotection ( 13 ) .", "when the albuminuria reduction was adjusted during combination treatment for changes in gfr ( fractional clearance ) , it was 11% higher than during irbesartan treatment , a nonsignificant change that was possibly due to a small sample number .", "signs of more effective raas blockade were evident from the synergistic effect of combination treatment on ir - prc .", "this conclusion is based on the fact that renin release into plasma is proportional to the interruption of the permanent negative feedback loop of ang ii on renin secretion ( 14 ) . combining aliskiren with", "irbesartan provided a 12-fold increase in ir - prc , but still with a 50% reduction in hs - pra compared with changes during the placebo period .", "this renin rise could reflect a high degree of intrarenal raas blockade during combination treatment as compared with that for the monotherapies , as has been suggested in nondiabetic patients ( 15 ) .", "the reductions in albuminuria in our study were correlated with the rise in ir - prc , supporting the concept of increased intrarenal raas blockade underlying the additional effects observed during combination treatment .", "compared with other studies of dual raas blockade , the rise in ir - prc is higher in dual raas blockade using aliskiren than in dual raas blockade with an ace inhibitor and an arb ( 5,16,17 ) . such marked increases in renin during aliskiren treatment have been noted before ( 18 ) .", "apart from reflecting more complete ( intrarenal ) raas blockade , they may also be due to the detection of prorenin as renin ( 19 ) or a change in the renin half - life after its binding to aliskiren ( 20 ) .", "pra was measured both by a conventional method and by a new high - sensitivity assay ( hs - pra ) , which is independent of endogenous substrate variation ( 21 ) . because high pra levels confer the risk of cardiovascular disease ( 22 ) , it will be interesting to evaluate long - term effects of direct renin inhibition in the ongoing aliskiren trial in type 2 diabetes using cardio - renal endpoints ( altitude ) , providing data on hard cardiovascular and renal end points ( 23 ) .", "the antihypertensive effect of aliskiren was smaller than that found in previous larger studies ( 16 ) , although the office blood pressure reduction did not differ from that caused by irbesartan .", "the ongoing telmisartan alone and in combination with ramipril global endpoint trial ( ontarget ) investigators ( 24 ) concluded that in a cardiovascular risk population , dual raas blockade with the arb telmisartan and the ace inhibitor ramipril is equivalent in reducing cardiovascular events compared with either as monotherapy , although with more frequent adverse events , including renal adverse events .", "almost 3,000 of the participating 25,260 patients had microalbuminuria at baseline , and substudies of albuminuria effects are expected .", "several short - term studies using dual raas blockade in diabetic nephropathy have shown promising antiproteinuric effects , as reviewed by rossing ( 17 ) , but the largest study so far ( 25 ) did not show additional benefits of a combination of ramipril and irbesartan , compared with ramipril monotherapy , in terms of albuminuria reduction after 20 weeks .", "the size was , however , sufficient to demonstrate the likely beneficial effect of combination therapy with aliskiren and irbesartan , although we evaluated a surrogate end point .", "in addition , the discrepancy between 24 h and office blood pressure readings complicates interpretation of the results .", "studies evaluating mortality and morbidity are ongoing and will provide further information on dual raas blockade with aliskiren . in summary , we demonstrate an antiproteinuric effect of dual raas blockade with aliskiren and irbesartan in combination compared with either treatment alone in patients with type 2 diabetes , hypertension , and albuminuria .", "the synergistic effect on ir - prc illustrates a higher degree of intrarenal raas blockade during combination treatment ."], "abstract_text": ["<S> objectivewe investigated whether the antiproteinuric effect of the direct renin inhibitor aliskiren is comparable to that of irbesartan and the effect of the combination.research design and methodsthis was a double - blind , randomized , crossover trial . </S>", "<S> after a 1-month washout period , 26 patients with type 2 diabetes , hypertension , and albuminuria ( > 100 mg / day ) were randomly assigned to four 2-month treatment periods in random order with placebo , 300 mg aliskiren once daily , 300 mg irbesartan once daily , or the combination using identical doses . </S>", "<S> patients received furosemide in a stable dose throughout the study . </S>", "<S> the primary end point was a change in albuminuria . </S>", "<S> secondary measures included change in 24-h blood pressure and glomerular filtration rate ( gfr).resultsplacebo geometric mean albuminuria was 258 mg / day ( range 842,361 ) , mean  sd 24-h blood pressure was 140/73  15/8 mmhg , and gfr was 89  27 ml / min per 1.73 m2 . </S>", "<S> aliskiren treatment reduced albuminuria by 48% ( 95% ci 2762 ) compared with placebo ( p < 0.001 ) , not significantly different from the 58% ( 4279 ) reduction with irbesartan treatment ( p < 0.001 vs. placebo ) . </S>", "<S> combination treatment reduced albuminuria by 71% ( 5979 ) , more than either monotherapy ( p < 0.001 and p = 0.028 ) . </S>", "<S> fractional clearances of albumin were significantly reduced ( 46 , 56 , and 67% reduction vs. placebo ) . </S>", "<S> twenty - four - hour blood pressure was reduced 3/4 mmhg by aliskiren ( ns / p = 0.009 ) , 12/5 mmhg by irbesartan ( p < 0.001/p = 0.002 ) , and 10/6 mmhg by the combination ( p = 0.001/p < 0.001 ) . </S>", "<S> gfr was significantly reduced 4.6 ( 95% ci 0.38.8 ) ml / min per 1.73 m2 by aliskiren , 8.0 ( 3.612.3 ) ml / min per 1.73 m2 by irbesartan , and 11.7 ( 7.415.9 ) ml / min per 1.73 m2 by the combination.conclusionsthe combination of aliskiren and irbesartan is more antiproteinuric in type 2 diabetic patients with albuminuria than monotherapy . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Statistical analysis", "RESULTS", "CONCLUSIONS", "Supplementary Material"], "sections": [["this was a double - blind , randomized , crossover trial in accordance with the declaration of helsinki and good clinical practice .", "the primary objective was to assess albuminuria during different treatments compared with that with placebo ; secondary objectives were to assess effect on 24-h blood pressure , glomerular filtration rate ( gfr ) , biomarkers , and raas components .", "after informed consent , patients attended a screening visit comprising laboratory tests and evaluations of inclusion / exclusion criteria .", "slow - release furosemide in a fixed dose ( mean dose 109 mg/24 h , range 60360 mg/24 h ) was prescribed to prevent blood pressure elevation and fluid retention .", "patients used an electronic blood pressure device ( ua-779 ; a&d instruments , abingdon , u.k . ) to measure home blood pressure throughout the study .", "change in uaer ( percentage ) versus placebo during treatment with 300 mg aliskiren daily , 300 mg irbesartan daily , or the combination ( p < 0.001 vs. placebo for all treatments ) . from a list of prescreened candidates , 41 patients were screened for study participation ( supplementary fig .", "1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-0168/dc1 ) . nine of these were screen failures mainly due to albuminuria levels below the randomization requirement .", "of the 10 randomly assigned patients who left the study before completion , 2 died , 1 was lost to follow - up , 4 had an adverse event that led to exclusion ( diarrhea , severe hypertension , recurrent urinary tract infection , and dizziness ) , and 3 withdrew consent .", "twenty - six patients had the primary end point , albuminuria assessed after randomization , and were included in the final analysis ; the remaining six patients dropped out shortly after random assignment and were not included in the final analysis .", "after washout , patients attended a randomization visit before 2 months of treatment with placebo , 300 mg aliskiren once daily , 300 mg irbesartan daily , or the combination of the two , in random order .", "patients with type 2 diabetes ( world health organization criteria ) aged 3080 years were eligible for randomization with baseline urinary albumin excretion rate ( uaer ) > 100 mg/24 h , hypertension ( baseline office blood pressure > 135/85 mmhg ) , and baseline gfr > 40 ml / min per 1.73 m. exclusion criteria included major cardiovascular disease ( within 6 months ) , heart failure ( new york heart association class ii ", "rather , we used active washout : during the first 14 days of all treatments , every patient received 150 mg aliskiren daily to avoid risk of hypotension or a drastic increase in blood pressure in the switch from placebo to combination treatment or vice versa . during the last 3 days of each treatment", ", patients collected three consecutive 24-h urine samples for assessment of geometric mean uaer . at the last day of each treatment period , patients attended our clinic for assessment of gfr and mounting of standard takeda 24-h blood pressure devices ( tm2421 , version 7 ; a&d medical , tokyo , japan ) .", "measurements were performed every 15 min from 7 to 23 h ( daytime ) and every 30 min from 23 to 7 h ( nighttime ) .", "urinary albumin and creatinine concentrations were determined on a turbidimetric hitachi 912 system ( roche diagnostics , mannheim , germany ) .", "samples for prorenin , plasma renin activity ( pra ) , high - sensitivity pra ( hs - pra ) , immunoreactive plasma renin concentration ( ir - prc ) , angiotensinogen , ang i , ace activity , ang ii , and aldosterone levels were determined after 30 min of supine rest , and the plasma was frozen after centrifugation ( 80c ) .", "raas components and biomarkers were measured at baseline and at the end of treatment periods .", "biomarkers of inflammation , endothelial dysfunction , and cardiovascular risk were measured : high - sensitivity c - reactive protein ( hs - crp ) ( enzyme immunosorbent assay [ eia ] ; dako , glostrup , denmark ) ; serum soluble vascular adhesion molecule-1 and serum soluble intercellular adhesion molecule-1 ( eia ; diaclone , besanon , france ) ; plasma plasminogen activator inhibitor-1 ( hyphen biomed kit ; andresy , france ) ; serum nh2-terminal - probrain natriuretic peptide ( eia kit ; biomedica , wien , austria ) ; fibrinogen ( immunoturbidimetry ) ; and plasma asymmetrical dimethyl arginine ( high - performance liquid chromatography ) .", "total renin concentration , prorenin concentration , plasma angiotensinogen , pra , and biomarkers of inflammation and endothelial dysfunction were measured using methodology described previously ( 4 ) .", "ir - prc was measured with an immunoradiometric kit ( renin iii ; cisbio , gif - sur - yvette , france ) .", "hs - pra was measured by in - house radioimmunoassay of ang i formed during incubation of 25 l plasma and 50 pmol sheep angiotensinogen for 3 h at 37c in a total reaction volume of 100 l .", "the assay was double calibrated against ang i and the international reference preparation of renin , 68/356 , from nibsc ( hertfordshire , u.k . ) .", "plasma ang i and ang ii were measured using in - house radioimmunoassays and ethanol extraction of plasma samples .", "ace activity was determined using a commercial radioenzymatic assay ( ace direct ; bhlmann - laboratories , schnenbuch , switzerland ) .", "randomization was blinded to all investigators , and the study drugs were packed and labeled before delivery to the site .", "it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% .", "this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% .", "the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure", "were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor .", "extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data . a two - sided p value < 0.05 was considered significant", "the correlation between changes in albuminuria and changes in hs - pra or ang ii were assessed by a nonparametric spearman correlation coefficient .", "correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined .", "fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration  gfr ) , and log - transformed levels were compared using a paired t test .", "statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) ."], ["it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% .", "this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% .", "the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor .", "extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data .", "a two - sided p value < 0.05 was considered significant . the correlation between changes in albuminuria and changes in hs - pra or ang ii", "correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined .", "fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration  gfr ) , and log - transformed levels were compared using a paired t test .", "statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) ."], ["baseline demographic data are shown in table 1 . primary and secondary objectives were met . during placebo treatment", "geometric mean uaer was 258 ( range 842,361 ) mg / day , mean  sd 24-h blood pressure was 140/73  15/8 mmhg , daytime blood pressure was 144/76  17/9 , and nighttime blood pressure was 130/68  17/9 .", "gfr was 89  27 ml / min per 1.73 m and mean serum creatinine was 88", "demographics of the 32 randomly assigned patients with type 2 diabetes , hypertension , and albuminuria and the 26 included in the final analysis data are means sd , n , or mean ( range ) .", "aliskiren treatment led to a significant reduction in albuminuria by 48% ( 95% ci 2762 ) compared with placebo ( p < 0.001 ) but not significantly different from irbesartan , lowering uaer by 58% ( 4270 ) ( p < 0.001 vs. placebo ) .", "combination treatment reduced albuminuria by 71% ( 5979 ) ( p < 0.001 ) compared with placebo , significantly more than with either monotherapy ( p < 0.001 and p = 0.028 ) .", "the relative difference between aliskiren and combination treatment was 31% . to adjust for treatment - induced changes in gfr and the potential influence on albuminuria reduction , we calculated fractional clearance , which was reduced by 46% versus placebo during aliskiren treatment ( p = 0.021 ) , by 56% versus placebo during irbesartan treatment ( p = 0.002 ) , and by 67% versus placebo during combination treatment ( p = 0.001 ) .", "systolic / diastolic 24-h blood pressure was reduced 3/4 mmhg by aliskiren ( ns / p = 0.009 ) , 12/5 mmhg by irbesartan ( p < 0.001/p = 0.002 ) , and 10/6 mmhg by the combination ( p = 0.001/p < 0.001 ) versus placebo .", "a correlation was found between change in albuminuria and change in 24-h diastolic blood pressure during all treatments ( p = 0.039 ) .", "seated office systolic / diastolic blood pressure was reduced 7/4 mmhg by aliskiren , 6/4 mmhg by irbesartan , and 12/8 mmhg by the combination , all statistically significant compared with placebo , except for diastolic blood pressure during irbesartan treatment .", "gfr was significantly reduced 4.6 ( 95% ci 8.80.3 ) ml / min per 1.73 m by aliskiren ( p = 0.037 ) , 8.0 ( 12.33.6 ) ml / min per 1.73 m by irbesartan ( p < 0.001 ) , and 11.7 ( 15.97.4 ) ml / min per 1.73 m by the combination ( p < 0.001 ) compared with placebo .", "aliskiren significantly reduced hs - pra , ang i , and ang ii by 87 , 75 , and 52% , respectively , compared with placebo ; irbesartan had the opposite effect ( table 2 ) .", "when combined , the activating effect of irbesartan was counteracted by aliskiren , reducing hs - pra , ang i , and ang ii by 88 , 78 , and 56% , respectively , compared with irbesartan monotherapy . whereas combination treatment caused a 1,068% increase in ir - prc versus a 279% increase during aliskiren monotherapy and a 178% increase during irbesartan monotherapy , hs - pra was reduced 47% compared with placebo after combination therapy .", "pra measured by a conventional method was affected similarly to hs - pra , although the changes were smaller ( table 2 ) .", "the renin - specific activity ( renin bioactivity / total renin mass ) was 3 and 4% after aliskiren and combination therapy compared with placebo , respectively , thereby confirming that 9697% of renin was aliskiren bound . during irbesartan monotherapy ,", "changes in raas components and cardiovascular biomarkers versus placebo all treatments were administered once daily .", "adma , asymmetrical dimethyl arginine ; icam-1 , intracellular adhesion molecule-1 ; nt - probnp , nh2-terminal - probrain natriuretic peptide ; pai-1 , plasminogen activator inhibitor-1 ; vcam-1 , vascular adhesion molecule-1 ; vwf , von willebrand factor .", "a significant correlation between reduction in albuminuria and increase in ir - prc was observed for all active treatments combined ( r = 0.597 , p = 0.0001 ) .", "there was a significant correlation between changes in albuminuria and increase in ang ii during irbesartan treatment ( correlation coefficient 0.486 , p = 0.022 ) ; no significant correlations were observed in the other treatment groups .", "hs - crp was reduced 35% from the placebo level with aliskiren ( p = 0.047 ) and 35% with irbesartan ( p = 0.043 ) .", "other statistically significant changes from placebo levels were a 6% reduction in soluble intracellular adhesion molecule-1 ( p = 0.017 ) observed with the combination treatment and a 7% reduction in fibrinogen during aliskiren treatment ( p = 0.037 ) .", "no treatment led to significant changes from placebo levels in any of the other cardiovascular biomarkers ( table 2 ) .", "the most frequent adverse events were urinary tract infection ( four patients , one male ) , pneumonia ( three patients ) , and cough ( three patients ) , occurring during different treatments .", "anemia and hypomagnesemia were detected in two patients during the combination treatment . compared with each monotherapy", ", combination treatment showed an increase in plasma potassium by 0.2 mmol / l ( p = 0.036 ) .", "one patient dropped out during the placebo period after several systolic blood pressure readings > 180 mmhg .", "two patients died before the first measurement of albuminuria after the randomization and were not included in the final end point analysis .", "the first death was that of a 42-year - old obese man ( bmi 42 kg / m ) with a history of ischemic heart disease , myocardial infarction , and hypertension 4 years before study entry .", "the patient experienced sudden cardiac arrest , seemingly after a myocardial infarction during aliskiren treatment .", "the second death was that of a hypertensive , obese , 73-year - old man with diabetes duration of 16 years .", "the deaths were instantly reported to relevant authorities and were not suspected as being related to any of the drugs studied .", "subsequently , home blood pressure measurement frequency was increased from twice weekly to twice daily , and patients were instructed to contact the investigator by direct phone ( available around the clock ) , if any measurement was > 160/100 mmhg .", "extra measurements of sodium , potassium , and creatinine were introduced 3 weeks into each treatment period ."], ["in this exploratory study , we demonstrated that treatment with 300 mg aliskiren once daily was as efficient in reducing albuminuria as standard therapy with 300 mg irbesartan once daily . when we combined the two treatments at the same doses , the reduction in albuminuria", "was enhanced . the added antiproteinuric effect with combination treatment compared with aliskiren alone was 31% .", "given that the reductions in 24-h systolic blood pressure with aliskiren were unexpectedly small compared with those with placebo relative to 24-h diastolic blood pressure changes and were substantially lower than the office systolic blood pressure measurements in this study , we conducted a thorough review of potential flaws in data collection , storage , device calibration , reporting , and calculation .", "there was no evidence to indicate that the ambulatory data collection , storage , or reporting was flawed , and the unexpected results could be a chance occurrence .", "our study suggested that the combination of aliskiren and irbesartan had an additional raas blocking effect compared with monotherapy because a synergistic increase in ir - prc was observed with the combination , which was related to the antiproteinuric effect , whereas hs - pra was reduced 50% compared with the reduction with placebo .", "as opposed to the aliskiren in the evaluation of proteinuria in diabetes ( avoid ) study ( 5 ) , which showed an additional 20% albuminuria reduction after 24 weeks of treatment with aliskiren compared with placebo added to the maximal recommended dose of losartan and optimal antihypertensive therapy , this is the first study with a head - to - head comparison between aliskiren and irbesartan treatment .", "no other antihypertensive drugs except for furosemide were allowed in our study , thereby offering a clearer picture of the effect of the two compounds used , compared with the avoid study , in which aliskiren was combined with losartan and a mixture of other antihypertensive drugs .", "this study is different with regard to patient population , with a lower mean baseline uaer compared with that in the avoid study .", "in addition , we assessed the effect of renin inhibition on gfr , a measurement that is more precise than the estimated gfr used in the avoid study .", "raas blockade is believed to reduce proteinuria through several different mechanisms : the mean transcapillary hydraulic pressure difference , the glomerular surface area , and the size and charge selectivity of the glomerular filter . in diabetic nephropathy several of these variables", "are abnormal , and raas blockade has been demonstrated to normalize directly measured or estimated glomerular hydraulic pressure ( 79 ) , to reduce the shunt - like defects in the membrane , at least in part ( 10 ) , and to restore the charge - selectivity properties of the glomerular membrane ( 11 ) .", "aliskiren is thought to reduce albuminuria by the same mechanism as during treatment with ace inhibitors or arbs .", "recently , fisher et al . ( 12 ) have shown that aliskiren treatment increases renal plasma flow to a larger extent than the ace inhibitor captopril .", "the increase in renal plasma flow may be a response to angiotensin at1 receptor  dependent reduction of the vascular tone in the efferent arteriole . reduced vascular tone in the efferent glomerular arteriole", "could be responsible for the decrease in intraglomerular pressure , leading to the reduction in albuminuria and gfr as demonstrated in our study .", "although the combination reduced gfr up to 12 ml / min ( 95% ci 15.97.4 ) , we interpreted this as an reversible hemodynamic change and not as an indication of nephrotoxicity ( 13 ) .", "in fact , it has been shown that an early hemodynamic reduction in gfr can translate into long - term renoprotection ( 13 ) .", "when the albuminuria reduction was adjusted during combination treatment for changes in gfr ( fractional clearance ) , it was 11% higher than during irbesartan treatment , a nonsignificant change that was possibly due to a small sample number .", "signs of more effective raas blockade were evident from the synergistic effect of combination treatment on ir - prc .", "this conclusion is based on the fact that renin release into plasma is proportional to the interruption of the permanent negative feedback loop of ang ii on renin secretion ( 14 ) . combining aliskiren with", "irbesartan provided a 12-fold increase in ir - prc , but still with a 50% reduction in hs - pra compared with changes during the placebo period .", "this renin rise could reflect a high degree of intrarenal raas blockade during combination treatment as compared with that for the monotherapies , as has been suggested in nondiabetic patients ( 15 ) .", "the reductions in albuminuria in our study were correlated with the rise in ir - prc , supporting the concept of increased intrarenal raas blockade underlying the additional effects observed during combination treatment .", "compared with other studies of dual raas blockade , the rise in ir - prc is higher in dual raas blockade using aliskiren than in dual raas blockade with an ace inhibitor and an arb ( 5,16,17 ) . such marked increases in renin during aliskiren treatment have been noted before ( 18 ) .", "apart from reflecting more complete ( intrarenal ) raas blockade , they may also be due to the detection of prorenin as renin ( 19 ) or a change in the renin half - life after its binding to aliskiren ( 20 ) .", "pra was measured both by a conventional method and by a new high - sensitivity assay ( hs - pra ) , which is independent of endogenous substrate variation ( 21 ) . because high pra levels confer the risk of cardiovascular disease ( 22 ) , it will be interesting to evaluate long - term effects of direct renin inhibition in the ongoing aliskiren trial in type 2 diabetes using cardio - renal endpoints ( altitude ) , providing data on hard cardiovascular and renal end points ( 23 ) .", "the antihypertensive effect of aliskiren was smaller than that found in previous larger studies ( 16 ) , although the office blood pressure reduction did not differ from that caused by irbesartan .", "the ongoing telmisartan alone and in combination with ramipril global endpoint trial ( ontarget ) investigators ( 24 ) concluded that in a cardiovascular risk population , dual raas blockade with the arb telmisartan and the ace inhibitor ramipril is equivalent in reducing cardiovascular events compared with either as monotherapy , although with more frequent adverse events , including renal adverse events .", "almost 3,000 of the participating 25,260 patients had microalbuminuria at baseline , and substudies of albuminuria effects are expected .", "several short - term studies using dual raas blockade in diabetic nephropathy have shown promising antiproteinuric effects , as reviewed by rossing ( 17 ) , but the largest study so far ( 25 ) did not show additional benefits of a combination of ramipril and irbesartan , compared with ramipril monotherapy , in terms of albuminuria reduction after 20 weeks .", "the size was , however , sufficient to demonstrate the likely beneficial effect of combination therapy with aliskiren and irbesartan , although we evaluated a surrogate end point .", "in addition , the discrepancy between 24 h and office blood pressure readings complicates interpretation of the results .", "studies evaluating mortality and morbidity are ongoing and will provide further information on dual raas blockade with aliskiren . in summary , we demonstrate an antiproteinuric effect of dual raas blockade with aliskiren and irbesartan in combination compared with either treatment alone in patients with type 2 diabetes , hypertension , and albuminuria .", "the synergistic effect on ir - prc illustrates a higher degree of intrarenal raas blockade during combination treatment ."], [""]]}
{"article_id": "PMC2233875", "article_text": ["nanobiomaterials are characterized by constituent \n particles and/or surface features less than 100  nm \n in at least one dimension \n . \n starting with photolithography and dry etching in the \n 1980 's to high - resolution electron beam lithography and other technologies \n in the 1990 's", ", nanotechnology allows for making surface structures for \n cell engineering and has led to an increasing application in healthcare over the last decades .", "nanolayers are used to enhance the \n surface biocompatibility of polymeric drug delivery systems , \n control the release of substances such as antibiotics or growth factors \n , act as gene - delivery \n vehicles , or serve as robust light emitters for \n cellular labeling and tracking \n [ semiconductor nanocrystals , quantum dots ( qds ) ] \n . \n", "nanotechnology is also applied to modify and improve the surface \n structure in orthopaedic implants to promote their \n osseous integration . however , there are also side effects of nano- and \n microparticles in vivo . \n", "micro- and nanoparticles released by friction of \n articulating partners from artificial joints are a major \n reason for aseptic \n implant loosening in orthopaedic surgery and may lead to severe \n peri - implant \n osteolysis ( particle disease ) \n .", "in addition , \n nanoparticles can induce or promote allergic or inflammatory \n reactions or \n influence hemolysis and blood coagulation \n [ 57 ] .", "although the cytocompatibility of a biomaterial \n is strongly influenced \n by its chemical composition , surface topography plays a \n crucial role for cell - surface \n\n interactions .", "material surface \n properties have been studied intensively , but \n still lack from reliable data about cytocompatibility .", "especially , the \n superordinate principles of cellular responses to \n surfaces with a defined \n topography are not well known and poorly understood . \n because many variables \n influence cellular interactions to surface structures , \n it is difficult to draw \n conclusions and formulate general principles for \n nano- and microstructured \n surfaces .", "this review summarizes recent data of effects by nano- and \n microstructured biomaterials and particles in vitro designed for \n orthopaedic application to get a solid \n framework outlining the critical interactions that govern the \n cytocompatibility . \n because biomaterials in orthopaedics are predominantly \n applied on bone , this review is focussed on \n the interactions of osteoblasts \n  and bone - marrow - derived \n cells with structured biomaterials .", "osteoblasts and osteoclasts are mainly \n responsible for the osteointegration of \n nanostructured biomaterials in orthopaedics . \n", "osteoblasts derive from \n mesenchymal progenitor cells which are localized mainly \n in the bone marrow and \n periosteum .", "they are characterized by cuboidal \n and flat morphology ( diameter about \n 20  m ) , present a large \n amount of rough endoplasmatic reticlum and a large golgi apparatus , \n and are potent to produce osteoid , a collagen i rich matrix \n . \n", "in addition , these mononuclear cells are also responsible \n for osteoid calcification ( hydroxyapatite ) .", "typical marker proteins for \n osteoblasts are cbfa1/runx2 , osteocalcin , osteopontin , osteonectin , bone \n sialoprotein ( bsp ) , osteoprotegerin \n ( opg ) , collagen i , and alkaline phosphates \n ( alp ) .", "figure 1 \n gives a brief summary of the expression \n of several markers during osteoblast differentiation .", "osteocytes act in a paracrine and mechanosensory manner , and \n can activate osetoblasts and osteoclasts . \n the latter cell type derived from the \n hematopoietic line , has multiple nuclei and is \n responsible for bone resorption . \n", "its ruffled border is flanked \n by a sealing zone which facilitates local acidification \n and removal of bony \n matrix such as ca , h3po4 , and \n h2co3 \n by endocytosis .", "osteoclasts express \n high levels of tartrate - resistant acid phosphatase \n ( trap ) and cathepsin k. the \n interaction between osteoblasts and osteoclasts is complex . during differentiation ,", "the ostoblast progenitors express receptor \n activator of nuclear \n factor  ligand ( rankl ) and macrophage colony - stimulating factor \n ( m - csf ) which are strong stimuli for osteoclastogenesis .", "in contrast , \n osteoprotegerin ( opg ) is a potent inhibitor \n of osteoclasts .", "moreover , the \n interactions between osteoblasts and osteoclasts in vivo are regulated \n by several hormones and cytokines , including parathyroid hormone ( pth ) , \n calcitonin , and il-6 .", "it is generally accepted that the \n three - dimensional surface topography \n ( size , shape , surface texture ) is one of \n the most important parameters that influence cellular reactions \n [ 2 , 1119 ] . \n", "although many studies have investigated \n cellular reaction to different surface pattern , \n the significance of macro \n structure studies on bone cell behavior is \n questionable since in vivo adhesion \n structures ( e.g. , cell membranes , \n basement membranes ) are comprised of much \n smaller nanometer scale features [ 20 , 21 ] .", "the immature bone is characterized by an average \n inorganic grain size of \n 1050  nm whereas mature bone has an average \n inorganic grain size of 2050  nm \n ( 25  nm in diameter ) \n . \n", "considering these parameters , modern \n implants for bone application have been \n designed with a smooth surface at the \n nanometer level .", "it was surprising that \n some of these have induced the formation of peri - implant \n fibrous tissue and \n implant loosening in vivo , while other implants with a higher \n degree of roughness showed significant better osteoconductive properties \n [ 2325 ] .", "there are various methods to modify the degree of \n roughness as well as surface energy \n and topography in orthopaedic implants", ". \n typically applied techniques to enhance \n the degree of roughness and promote \n the osteointegrative properties of \n biometals ( e.g. , ti , cocrmo , ss ) are chemical \n etching or anodization and \n \n also sand - blasting , sputter - coating , and machine - tooling .", "the lack of knowledge in cellular reaction to \n nanostructered biomaterials is based \n to a great extent on \n the difficulty in varying surface chemistry and \n topography independently .", "moreover , \n the use of different cell lineages and culture \n conditions makes it difficult to \n compare results from different investigators \n [ 2631 ] \n ( table 1 ) . \n", "there is also a lack of consensus concerning the \n proper representation of implant surface topography \n . \n", "one major misunderstanding is the practice of defining a \n surface by its manufacturing process instead of concisely defining the \n topographic measurements [ 17 , 33 ] . \n considering these limitations for interpretation ,", "the \n following review gives an overview of cellular \n reactions to surface structures \n of different orthopaedic biomaterials .", "the first step after exposure of any biomaterial \n to a biological environment results in the rapid \n adsorption of proteins to its \n surface . \n", "the composition , type , amount , and \n conformation of adsorbed proteins regulate the \n secondary phenomena such as \n cellular adherence and protein exchange \n [ 3537 ] \n and also following cellular reactions such as migration , \n proliferation , and differentiation . \n", "the potency for biomaterials to adsorb \n proteins is influenced by its physiochemical \n\n characteristics such as surface energy or hydrophobicity , \n and is also dependent on \n the local environment ( ph , \n concentration of ions , composition and \n functional groups of proteins , strength \n of solution , temperature ) \n ( vroman effect ) \n ( figures 2 ,   and 3 ) . for inorganic nanocrystals and microstructured \n surfaces", "there are at least two approaches to change \n their hydrophobic surfaces : \n a ligand exchange reaction can replace the original \n hydrophobic surface with \n bifunctional coupling molecules or an inorganic \n coating such as silica ( 1 ) or \n an encapsulation of nanocrystals in an \n  amphiphile organic coating ( 2 ) .", "the first phase of protein adsorption onto a \n biomaterial 's surface is characterized \n by the attachment of small rapidly diffusing \n\n proteins , followed by a progressive replacement by larger \n proteins with a high \n affinity to the substrate . here", ", especially proteins \n with arg - gly - asp ( rgd ) \n containing sequences such as fibronectin or \n vitronectin act as cell receptors \n and have chemotactic or adhesive properties to bone cells . \n", "in addition , these rgd - peptides \n also have a strong effect on matrix maturation \n and biomineralization [ 4648 ] .", "after conditioning of a naked biomaterial by protein adsorption , cells attach rapidly \n on the protein - coated surface . \n besides the influence of proteins , the \n cellular attachement to a nanostructed surface \n", "is also influenced by its physiochemical \n properties , especially by the outer functional groups \n [ 30 , 50 , 51 ] .   ", "schweikl et al .  \n showed on self - assembly monolayers that the \n osteoblast proliferation on hydrocarbon \n chains , terminated by ch3 , \n was as high as on amino groups \n ( nh2 ) \n and hydrophilic oxidized surfaces , but significantly lower \n on fluorocarbon \n ( cf3 ) \n groups .", "mller et al \n .  \n showed that \n 3-aminopropyl triethoxysilane ( apts ) \n presents amine functional groups which allow for \n grafting rgd tripeptides and \n that the rgd - apts hybrid promotes cell adhesion , \n spreading , and cytoskeletal \n organization . here , the zetal potential ( differences in potentials \n between the surface of a tightly \n bounded layer and a diffuse layer ) \n and the interfacial tension ( wettability ) of \n a surface is crucial [ 54 , 55 ] .", "it was demonstrated for cpti surfaces that the contact angle ( ca ) , \n parameter for \n wettability , increases linearly with the average roughness when \n the angles were \n higher than 45 , but decreases linearly with roughness \n when the angle was less \n than 45 . \n", "recent data examining osteoblast \n response to controlled surface chemistries indicate that \n hydrophilic surfaces \n ( high number of polar components ) improve cell attachment \n and matrix synthesis and also the osteogenic \n potency compared to hydrophobic \n surfaces [ 5759 ] . \n", "compared ti alloys and cocr alloys towards protein \n absorptive properties and cell attachment with an \n osteoblast precursor cell \n line .", "they found no significant \n differences between ti alloys and cocr , \n but significantly greater cell adhesion \n rates for the ti implants and concluded that cell \n adhesion is a result of \n higher hydrophilicity of ti alloys . in \n contrast", ", other data showed that a low degree \n of wettability promotes protein \n adhesion and also cellular attachment to a biomaterial \n , \n and mller et al .  \n", "found no direct correlation between the \n wettability of the material surface and the osteoblast attachment and \n proliferation rate .", "also qu et al .  \n found no significant differences of cell attachement \n on various titanium surfaces with different degrees of wettabilities \n ( hydrophobic acid - etched , coarse - blasted large \n grit acid - etched , hydrophilic modified \n acid - etched , and modified coarse - blasted large grit , \n acid - etched \n ) on mg68 cells .", "heating ( oxygen / atm ) or peroxide treatment of biometals \n result in a thicker oxide layer \n and a more hydrophilic surface .", ". \n  showed that \n heat - treated titanium \n surfaces changed the wettability ( more hydrophilic ) \n but does not significantly \n affect the fibronectin and albumin adsorption as well as \n the initial osteoblast \n precursor cell attachment in vitro . \n", "emphasized that the rate \n of protein correlates more with changes in chemical \n composition than \n with changes in wettability in metal surfaces .", "they showed that \n a preheating of ti6al4v specimen does not only lead to a \n thicker oxide layer but also results in \n an enrichment of v and al within \n the surface oxide .", "in contrast , \n post - treatment with butanol after preheating \n reduces the content of v , but not in al , \n and significantly increases the rate \n of fibronectin adsorption up to 2040% \n .", "compared to the cellular attachment phase , the following adhesion \n phase lasts longer and \n involves various proteins and molecules \n ( figure 2 ) . \n as a link between cell and \n biomaterial , the interactions of a surface topography \n and serum proteins are \n crucial for the cytocompatibility of a biomaterial . \n", "especially , the adsorption \n of adhesion proteins , such as fibronectin and vitronectin , from serum \n containing solutions and integrin - mediated signaling has been \n demonstrated to \n mediate cell adhesion and spreading \n . it has been shown that nanotube or nanoparticle surfaces \n created by anodization have \n promoted osteoblast adhesion up to three times \n compared to unanodized ti . \n", "these results were confirmed by the group of webster \n \n and other investigators [ 6871 ] \n who demonstrated that the initial \n attachment of osteoblasts onto the surface of biometals such as cpti , \n ti6al4v , and cocrmo is enhanced by submicron to nanometer \n consistent particles compared \n to metals composed of respective micron particles . \n", "one possible explanation of \n this phenomenon is the higher amount of particle binding sites for \n osteoblast adhesion at the surfaces of nanophase metals \n compared to micron particle size \n metals .", "the theory of enhanced protein and cell binding capacities \n by larger \n surface areas / roughness degrees was also confirmed for porous \n ha materials .", "another example of the significance of surface structures \n for protein binding and \n osteoblast attachment is the helical rosette nanotubes \n ( hrn ) which can build \n self - assembly surface structures .", "it was demonstrated that \n a significant change \n of hrn coverage by heating correlated with the protein - binding \n and osteoblast \n adhesion potency in titanium surfaces [ 73 , \n 74 ] .", "it is evident that not only the surface topography \n influences protein deposition and \n cell adherence but also proteins and cells modify the surface \n properties of a \n defined surface .", "based on a surface analysis of the different \n biometal specimen \n before and after cell cultivation , we showed previously \n \n that a cell attachment and/or protein \n precipitation increase the roughness in polished biomaterials \n ( steel , ti6al4v , \n and cocr ) .", "for porous coated cocr surfaces , we found only slight and no \n relevant changes in roughness whereas cell cultivation onto \n sandblasted ti6al4v \n lead to a strong decrease in specimen roughness . \n", "both , the increase in roughness after \n cell culturing in the different biometals \n and the decrease in roughness of sandblasted ti6al4v could be \n explained by the \n dense cellular growth and accumulation of debris in depth of \n the structured surfaces \n and/or protein deposition as shown by other investigators \n [ 75 , 76 ] .", "in addition , not only the amount but also the type of \n protein adsorption by a \n surface is crucial for cellular adherence and following reactions such as \n migration and differentiation . as an example , ti surfaces ( ra : \n 0.370.01  m ) adsorp \n  fibronectin in higher concentrations \n compared to albumin , and fibronectin - coated ti surface promoted \n more osteoblast attachments \n in comparison to albumin - coated ti surfaces \n . \n", "these results correspond to the data of other authors \n who showed excellent osteoconductive properties after \n fibronectin adsorption \n onto a biomaterials ' surface [ 7880 ] . based on irm and tem analysis ,", "the closest distance \n of cells to a surface ( glass ) was \n found to be approximately 10  nm [ 81 , \n 82 ] .", "historically , results from chicken \n fibroblasts have lead to a classification of three different types of \n separation .", "( 1 ) focal contacts ( fc ) : approximately \n 1015  nm separation from the substrate under the \n peripheral regions of the leading lamellae \n ( appearing black in tem ) .", "fc act as \n an interface between intra and extracellular components and occur linearly \n beneath the associated cytoplasmic stress fibres \n [ 83 , \n 84 ] . \n", "they are tenacious adhesion sites that \n remain attached to the substratum even when cells are forcibly detached , \n indicating their function as anchorage \n structures .", "( 2 ) close contacts : corresponding to approximately \n 30  nm separation ( broader grey areas \n in tem ) .", "( 3 ) greater separation : corresponding to approximately \n 100140  nm ( white \n regions in tem ) .", "it is evident that not only fc appear soon after cellular attachment but also that ( -catenin - positive ) adherence \n junctions occur within 14 hours \n for grooved ti - based substrates . \n", "these observations underline the high \n significance of an early intercellular communication soon \n after adherence to a \n surface .", "the mechanisms of initial cellular adherence to a surface are \n different from long - term adherence as shown by a lack of statistical \n correlation between short - term adhesion ( strength of cell attachment \n and early \n adhesion ) and long - term adhesion ( strength of cell - matrix interface ) \n forces [ 14 , 15 , 86 ] . \n", ". \n \n showed that the cultivation time has an \n influence on the long - term adhesion in biometal surfaces according to \n  td \n ( t ) = atb , \n a being independent of b ( td : time - dependend \n adhesion index , a : surface - dependent parameter , b : substrat - independent \n exponent , 0.5+/-0.03 ) . for polylactides ( plla ) , it was shown on oct-1 osteoblast - like \n cells that cell \n adhesion but not the proliferation could be enhanced by nanoscale \n and microscale \n roughness compared to smooth surfaces .", "in addition , \n there is evidence that fc \n show a dynamic behavior which allows for cellular migration and motility . \n", "linear plla fibres with length scales of 0.52  m , \n constructed by electrospinning , have shown cellular contact \n guidance and enhanced \n osteoblastic differentiation . here , cell morphology revealed that cells \n grown on fibres had smaller projected areas than those on planar \n surfaces .", "also other polymers such as plga have been shown to \n be effective in enhancing osteoblast differentiation in vitro \n .", "diener et al .   demonstrated \n on mg-63 osteoblastic cells \n that fc adhesion was smaller on ti and ss than on collagen - coated glass \n coverslips and that all fc showed a mobility of focal adhesions .", "however , \n anselme et al .   found higher \n adhesions on ti6al4v \n substrates than on noncollagen - covered glass samples , \n and emphasized that substrates \n with various surface compositions \n but with the same surface topography did not \n induce significant differences of adhesion .", "based on the knowledge of protein adsorption and its effects \n on cellular attachment and adherence , a selective surface coating \n of nanostructured \n surfaces with rgd or collagen proteins offer a promising solution \n to improve \n the number of osteoblasts adhered on artificial surfaces \n [ 53 , 95102 ] .", "imprinting \n surfaces technology with deposition of specific \n protein - recognition sites can \n help to promote osteoblastic growth \n and differentiation [ 103106 ] .", "protein - recognition \n can be based on a protein - ligand binding and/or electron donor - acceptor \n interactions or other types of binding forces .", "integrin \n 51 and 5 \n v3 subunits competitively \n bind to rgd - sites of fibronectin [ 107 , \n 108 ] .", "dependent on the \n surface topography and \n chemistry of the \n\n biomaterial , fibronectin \n undergoes changes in structure including \n modulation in functional activity and \n shift in integrin binding capacity .", "based on the data of self - assembled monolayers , \n it was shown that integrin subunits \n show selective binding capacities to different \n terminal groups .", "integrin 51 shows a \n strong affinity to oh and \n nh2 surfaces , whereas \n 51 and \n 5v3 bind also to cooh but show poor binding capacities \n on ch3 \n surfaces [ 109113 ] .", "furthermore , some data show that oh \n and nh2 surfaces can up - regulate \n osteoblast - specific gene expression but also \n matrix mineralization compared \n with cooh and \n ch3 \n functional groups [ 47 , 112 ] .", "cell migration and proliferation is the attachment \n following phase between the cell \n and the material surface .", "it   is evident for designing \n nanostructured implants \n that cells use the nanotopography of a substrate for \n orientation and migration [ 117119 ] .", "although \n it is known that bone cells \n align along defined substrate morphologies ( contact \n guidance ) , the detailed relation between ordered \n nanotopography and cell \n behavior remains unknown in detail . \n", "for the first time , in 1964 it was shown \n that convex surfaces enhance cellular overlap , while \n grooves minimize cellular \n overlap .", "as pre - requisite to reach a defined cell colonization during \n directed tissue formation , structured nanophase surfaces \n lead to a predictable \n osteoblast orientation and migration on these surfaces \n [ 17 , 121 , 122 ] . \n", "interaction between the ecm and associated \n changes in the \n orientation of the cytoskeleton are crucial for cell \n metabolism of cells and \n morphology due to actin - myosin tension structures \n .", "anisotropic topographies ( e.g. , \n topographical grooves , chemically patterned stripes , \n or curved surfaces of a \n fibre ) are potent to exert morphological \n as well as physiochemical \n features on cells at the same time , indicative for the \n complex environmental \n influence on cells .", "focal contacts are important structures for cellular \n adherence onto a surface but may \n also delay migration and mobility of the cells . \n", "it was shown that bone - derived cells ( mg63 cells ) respond to \n a nanoscale roughness by a higher cell thickness and a \n delayed appearance of \n focal contacts . \n", "especially , nanoporous \n ti - oxide surfaces promote cellular \n spreading and induce numerous filopods and osteoblastic differentiation \n [ 124 , \n 125 ] . on", "electrochemically \n microstructured hexagonal \n pattern , mg63-cells go inside \n 30100  m but not in 10  m \n cavities .", "most authors report a \n parallel orientation of cells cultured on \n polished ( smooth ) surfaces [ 57 , 114 , 126 ] \n ( figure 4 ) .", "another method to not only enhance cellular adherence but also to \n promote osteoblastic differentiation and \n biomineralization of biometals is a surface anodization , \n for example , by -glycerophosphate sodium and calcium \n acetate [ 6671 ] .", "cellular adhesion via fc may strengthen the linkage \n between cell and ecm and \n also impair the ability to dynamically remodelling \n the ecm and influence the \n migration rate . for \n collagen - coated coverslips , focal adhesion of mg-63 \n osteoblastic cells moved \n with a speed of 60  nm / min , whereas the speed was \n reduced in ti and more in ss \n surfaces .", "another study \n on nb2o5-coated \n polished cpti samples showed that mc3t3-e1-osteoblast \n migration was fastest on \n smooth surfaces ( ra = 7  nm ) , whereas adhesion strength , \n spreading area , and \n collagen - i synthesis were promoted by intermediate \n roughness ( ra = 15  nm ) .", "however , \n it was surprising that higher degrees of roughness \n ( ra = 40  nm ) were rather \n peaked and reduced the speed of adhesion process in the \n same study . besides the surface properties of a biomaterial , \n the cellular migration rate is \n dependent on the cell type and its differentiation stage . \n", "a higher migration \n rate is associated with a lower level of osteoblast \n differentiation .", "cells with \n a low motility are characterized by a strong formation of fc \n while motile cells \n form less adhesive structures .", "it was found that mature \n osteoblasts spread out \n and form a greater number of fc when settled on smoother surfaces \n .", "although \n cellular spreading is higher on \n smoother surfaces , some data indicate that the \n alp - expression is higher for \n rough isotropic surfaces ( electro - erosion , acid - etching , \n sandblasted ) compared \n to smoother substrates ( machine tooling , polishing ) \n . considering recent publications , there \n is no or only week statistical significance that there \n is a difference between \n the initial number of adherent cells and following proliferation of cells \n cultured onto a biometal or ceramic nano-/microscale surface \n in vitro .", "however , \n some authors emphasize that \n the influence of functional chemical groups for cellular migration and \n proliferation are stronger than general surface \n properties such as wettability . \n", "especially a tio2-layer \n seems to promote \n cellular growth and proliferation on nanostructured \n biometals [ 128 , 129 ] .", "other examples for a promotion of cell - to - bone contact in vitro \n and also in vivo are \n machine - etched ti - surfaces ( e.g. , osteotite ) \n , defined sand - blasted \n implants [ 124 , 125 , 131 ] , and hydroxyapatite ( ha ) coatings , \n for example , by plama - spray techniques [ 132134 ] .", "recent studies investigating the response of adherent \n cells to nanography surfaces \n indicate that different cell phenotypes have different \n levels of sensitivities [ 117 , 135137 ] . here", ", \n osteoblasts react to features as \n low as to the 10 nm dimensions , which is comparable in size to a single \n collagen fibre .", "moreover , the qualitative and quantitative kinetics \n in gene and protein expression is \n strongly influenced by topography and physiochemistry \n of a defined surface .", "microporous \n ha surfaces seem to promote a high number of fc and \n increased levels of alp but \n short actin stress fibres compared to nonmicroporous ha surfaces \n [ 72 , 139 ] . \n", "there is also evidence that ti and ha \n surfaces can activate early intracellular signalling \n pathways as shown by \n expression of relevant molecules such as \n - and 1-integrin , fak , erk followed \n by c - jun and c - fos genes for proliferation and alp for differentiation \n [ 139 , 140 ] . \n", "however , hallgren et al .  \n found no significant histomorphometric \n and biomechanical differences between nanopatterned and control implants . \n", ".  \n showed that microfabricated \n discontinuous - edge surfaces ( des ) , \n repeated open square boxes with a depth of \n 10  m , alter osteoblast adherence and migration \n but enhance cell multilayering , \n matrix deposition and mineralization when compared to smooth controls .", "in contrast to our data , \n anselme et al .   found \n higher proliferation \n rates on ss compared to ti6al4v .", "however , bigerelle et al . \n  demonstrated that neither \n material composition nor surface roughness amplitude influence cell \n proliferation , whereas they found a very significant influence \n on manufacturing \n process and surface topography for long - term adherence \n and proliferation in vitro .", "our in vitro results   confirm the \n well known osteogenic in \n vivo properties of ti implants , which may be based on \n surface factors observed \n on its outer tio2-layer \n [ 143146 ] . \n", "mller et al .  \n demonstrated the ability of osteoblasts \n to grow into an open - porous ti implant ( metal foam ) and li et al", ". \n \n also demonstrated that mc3t3-e1 cells \n attach to and are able to divide well in the inner surface of \n a highly porous \n trabecular ti6al4v implant .", "some in vitro studies demonstrated an \n enhanced total protein and collagen production , \n as well as increased alp \n activity of osteoblasts cultured on nanoparticulate \n metals ( cpti , ti6al4v , and \n cocrmo ) indicating advantages for nanostructured surfaces \n for osteointegration [ 1 , 149 , 150 ] . based on the \n data of redey et al . , \n it can be concluded that the low \n attachment and collagen production rates are related to a low \n wettability of a \n nanosurface .", "nanotextured surfaces of ti surfaces \n prepared by chemical etching \n have upregulated the expression of bsp and \n op . \n as demonstrated by qu et al . , \n the expression of the bone - associated \n genes such as alp , oc , type - i - collagen , osteoprotegerin , and \n glyceraldehyde-3-phosphate - dehydrogenase is promoted \n by modsla ti surfaces . \n some data also suggest that fluoride - modified \n ti surfaces can stimulate \n osteoblastic differentiation compared to \n unmodified titanium surfaces [ 151 , \n 152 ] .", ". \n  showed in their in vitro experiments that nanophase biometals induce \n significantly greater calcium and phosphorus \n deposition by osteoblasts and also allow for \n calcium and phosphorous precipitation from \n culture media without osteoblasts in \n contrast to microphase ti6al4v and cocrmo . \n", "furthermore , the authors found \n advantages in mineral precipitation without osteoblast for \n tial4v but no differences \n in dependency to the type of ti ( wrought , microphase , \n or nanophase ) .", "it was \n evident that the increased calcium and phosphorus \n mineral content correlated to \n greater amounts of underlying aluminium content on \n ti6al4v surfaces . although some data indicate \n that nanostructured ti alloys promote non - cell - mediated ca / po4-mineral \n deposition from culture media compared to cocrmo substrates , \n the greatest cell - dependend calcium and \n phosphorus mineral deposition occurred \n on nanophase cocrmo .", "it is evident that micropattern collagen films or \n scaffolds promote not \n only cellular adhesion but also allow for \n an osteoblastic differentiation and \n biocalcification in vitro [ 153155 ] . \n for ha- and dcpp - coated , ti \n surfaces the ca / p ratio influence the biomineralization \n rate in vitro .", "besides the osteoblast - promoting effects \n of defined substrates and \n surface topographies , some data also allocate \n an inflammatory response induced \n by nano- or microstructured biomaterials . \n", "it was shown in many studies that \n cell - biomaterial interactions can activate macrophages \n which results in the synthesis of proinflammatory \n agents such as tnf , ifn , il-1 and -6 , \n rankl and no [ 157159 ] .", "some data have \n shown proinflammatory effects of different \n biomaterials which increase with the \n degree of surface roughness . \n here ,", "macrophage inflammatory protein-1 , tnf , \n monocyte chemoattractant protein-1 , and \n members of the interleukine and leukotriene family \n play a crucial role in \n biometal - induced inflammations \n [ 160164 ] .", "most studies report about an \n enhanced expression of pro - inflammatory \n cytokines and chemokines by cells \n attached to rougher surfaces .", "some data also \n indicate that anionic and neutral hydrophilic surfaces \n increase macrophage - monocyte apoptosis and reduce macrophage \n fusion to modulate \n inflammatory responses to implanted materials \n .", "however , adverse cellular effects seen with \n metallic implants may \n also be attributed to corrosion products or to \n the separation of metal ions \n ( fe , cr , ni ) which may have a major impact on cellular survival and \n differentiation [ 166168 ] . \n those studies which suggest that a cell - mediated metal ion \n release by biometals that \n  did not affect \n the cell viability or proliferation are \n characterized by short cultivation periods or \n other conditions which limit the \n reliability of data [ 169171 ] . up to date , only few authors report about no significant influence \n of the cellular adherence and expression of osteoblast \n proteins by different \n biometals and surfaces such as alp expression \n [ 172 , \n 173 ] .", "in contrast to the great opportunity enhancing \n biocompatibility and osteogenic potency of surfaces applied on bone by \n nanotechnology , micro- and nanoscaled particles released by friction of \n artificial joints can induce severe \n inflammation and may lead to osteolysis and \n implant failure [ 174 , \n 175 ] \n ( figure 5 , \n table 2 ) . there is a wide \n range in particles size and morphology produced by \n simulators for artificial \n joints .", "particles released from metal - metal ( crcomo alloys ) are \n predominantly chromium oxide particles or cocrmo with \n varying ratios of co and cr .", "they show a round to oval morphology \n and also a substantial number of needle - shaped \n particles were found during the first circles . \n", "emphasize the \n importance of particle size \n as a critical factor in osteoblasts proliferation \n and viability in vitro .", "some data indicate \n that in contrast to ti - surfaces \n nano- and mircoparticles induce an \n inflammatory response although titanium is \n one of the biometals with the highest degree of \n cytocompatibility . as shown by miyanishi et al . \n , \n the release of vegf may play a crucial role in the \n pathogenesis of ti - induced osteolysis .", "some data indicate that \n phagocytosis of ti particles is not a precondition for \n an inflammatory response \n such as a release of tnf or il-6 in \n cultured macrophages . \n", "it is evident that a binding of the macrophage cd11b / cd18 ( macrophage \n mac-1 receptors / receptor of complement cr3bi , \n can also bind to icam-1 and \n icam-2 ) by integrin - specific antibodies \n also increased the release of tnf and il-6 in macrophages . \n", "this finding also suggests \n that the complement system plays a role in the pathogenesis \n of particle - induced \n inflammation , too .", "especially , uhmwpe particles with \n a size range of \n 0.11.0  m have been shown to be most reactive for \n macrophage activation and cytokine \n secretion in bone marrow cells [ 179 , \n 180 ]", ". however , not only the particle size but also the particle volume \n ( number ) is a critical factor for particle - mediated \n release of cytokines by \n macrophages .", "green et al . \n demonstrated for pe \n that the cell - particle ratios of 1 : 100 \n ( size 0.497.2  m ) and 1 : 10 \n ( size : 0.494.2  m ) induced \n significant stronger release \n of tnf and il-1 in macrophages . \n", "the authors conclude that especially \n particles in the phagocytosable size range of \n 0.310  m appear to be the most \n biologically active ones .", "the latter statement was also confirmed for silicon carbide ( sic ) \n particles and biometals such as cpti , \n ti6al4v and uhmwpe [ 184 , 185 ] .", "granchi et al .  \n investigated the in vitro \n effects of al2o3 \n and uhmwpe particles in an \n osteoblast - osteoclast co - culture system . \n", "both particles did not affect either \n cell viability or tnf and gm - csf release , \n whereas il6 release was dependent on \n the particle concentration .", "uhmwpe particles \n increased the release of rankl \n from osteoblasts and induced large amounts \n of multinucleated trap - positive \n giant cells in an osteoblast - osteoclast \n co - culture system .", "also , \n carbon - based particles with low wear \n factors such as p25-cvd showed a high degree \n of cytocompatibility in vitro . howling et al . \n ", "demonstrated \n on fibroblasts and monocytes that p25-cvd \n particles < 100  nm were significantly less cytotoxic \n to both cell types than \n cocr metal wear particles . \n", "while the classical water - suspendable \n nanoc60 nanocrystal is apparently \n cytotoxic to various cell lines , the closely related \n fully hydroxylated , \n c60(oh)24 \n , is nontoxic , \n thus producing no cellular \n response .", "also , \n functionalized single - walled carbon nanotubes are nontoxic \n to cells in culture [ 198200 ] .", "there is evidence that not only particle \n size and chemical content but also the concentration strongly influence \n cellular reactions in vitro .", "wilke \n et al .  \n showed a positive correlation between the \n release of proinflammatory cytokines \n ( il-6 , -1 , and tnf ) and amounts of \n ti6al4v - particles \n ( 10 , \n 10 , 10 , \n and 10 particles / ml ) \n by human bone marrow cells over 2 weeks .", "some in vitro data also indicate that ti particles induce a stronger \n fibroblastic differentiation signal than uhmwpe \n in monocytes and other cells [ 182184 ] .", ".  \n showed that particles \n of high - density polyethylene \n ( hdp ) and ti6al4v induced significantly more \n proinflammatory mediators ( il-1 , \n il-6 , tnf ) and bone resorption compared to \n al2o3 \n and zro2 in vivo . \n", "based on these data , \n it can be assumed that ceramics show a high \n degree of cytocompatibiltiy . for ha especially , particles with a size \n < 53  m inhibit cellular proliferation , \n especially in osteoblasts and lead \n to a decrease in tgf1 and a significant increase in pge2 and ldh \n concentration , but did not influence the tnf \n or alp titer in vitro . \n it could be concluded that larger ha particles may \n be compatible with bone cells while smaller - sized \n ha particles can both activate \n the osteoclasts and decrease the cell \n population of the osteoblasts in vitro .", "numerous variables influence the biocompatibility and osteogenic \n potency of nanostructured biomaterials in \n vitro and in vivo .", "besides the locotypical \n environment in vivo or in vitro , the surface \n structure and the composition of a \n biomaterial affects cellular attachment , \n adherence , proliferation and migration , \n and also \n differentiation and survival of defined cell types . \n here , information about typical \n parameters such as chemical composition , \n surface structure ( topography , geometry , \n roughness , particle size ) , surface energy , \n hydrophobicity , and the degree of \n solubility in aqueous solutions of a biomaterial \n will help to value and grade a \n defined implant concerning its osteblast promoting potency . considering recent publications , we could assume some general \n principles of cytocompatiblity and cell - surface \n interactions in nano- and \n microstructured surfaces .", "( 1 ) wettability of a nanosurface influences significantly \n protein adsorption , which is a prerequisite \n of cellular adherence in serum containing \n solutions .", "( 2 ) nanostructured surfaces enhance the surface area of \n biomaterials and promote cellular adherence . ( 3 ) the chemical outer functional \n groups of a nanosurface significantly influence cellular \n migration , proliferation , and differentiation but direct \n correlations between \n distinct parameters and cell functions are not entirely cleared .", "( 4 ) the formation of fc underly a \n dynamic process and influence the motility and migration of cells . ( 5 ) a higher degree of differentiation \n is corresponding to a decreased cellular motility .", "( 6 ) phagocytable particles with a size \n < 10  m induce the strongest \n cellular response with regard to releasing \n inflammatory cytokines .", "( 7 ) although ti has a high degree of \n cytocompatibility in vitro , phagocytable \n ti particles can induce a fibroblastic \n differentiation ."], "abstract_text": ["<S> cell - surface interactions play a crucial role for biomaterial application in orthopaedics . \n </S>", "<S> it is evident that not only the chemical composition of solid substances influence cellular adherence , \n migration , proliferation and differentiation but also the surface topography of a biomaterial . \n </S>", "<S> the progressive application of nanostructured surfaces in medicine has gained increasing interest \n to improve the cytocompatibility and osteointegration of orthopaedic implants . </S>", "<S> therefore , the \n understanding of cell - surface interactions is of major interest for these substances . in this review </S>", "<S> , \n we elucidate the principle mechanisms of nano- and microscale cell - surface interactions in vitro for \n different cell types onto typical orthopaedic biomaterials such as titanium ( ti ) , \n cobalt - chrome - molybdenum ( cocrmo ) alloys , stainless steel ( ss ) , as well as synthetic polymers \n ( uhmwpe , xlpe , peek , plla ) . </S>", "<S> in addition , effects of nano- and microscaled particles and their \n significance in orthopaedics were reviewed . </S>", "<S> the significance for the cytocompatibility \n of nanobiomaterials is discussed critically . </S>"], "labels": null, "section_names": ["1. INTRODUCTION", "2. BONE CELLS", "3. CYTOCOMPATIBILITY OF MICRO- AND \nNANOSTRUCTURED SURFACES"], "sections": [["nanobiomaterials are characterized by constituent \n particles and/or surface features less than 100  nm \n in at least one dimension \n . \n starting with photolithography and dry etching in the \n 1980 's to high - resolution electron beam lithography and other technologies \n in the 1990 's", ", nanotechnology allows for making surface structures for \n cell engineering and has led to an increasing application in healthcare over the last decades .", "nanolayers are used to enhance the \n surface biocompatibility of polymeric drug delivery systems , \n control the release of substances such as antibiotics or growth factors \n , act as gene - delivery \n vehicles , or serve as robust light emitters for \n cellular labeling and tracking \n [ semiconductor nanocrystals , quantum dots ( qds ) ] \n . \n", "nanotechnology is also applied to modify and improve the surface \n structure in orthopaedic implants to promote their \n osseous integration . however , there are also side effects of nano- and \n microparticles in vivo . \n", "micro- and nanoparticles released by friction of \n articulating partners from artificial joints are a major \n reason for aseptic \n implant loosening in orthopaedic surgery and may lead to severe \n peri - implant \n osteolysis ( particle disease ) \n .", "in addition , \n nanoparticles can induce or promote allergic or inflammatory \n reactions or \n influence hemolysis and blood coagulation \n [ 57 ] .", "although the cytocompatibility of a biomaterial \n is strongly influenced \n by its chemical composition , surface topography plays a \n crucial role for cell - surface \n\n interactions .", "material surface \n properties have been studied intensively , but \n still lack from reliable data about cytocompatibility .", "especially , the \n superordinate principles of cellular responses to \n surfaces with a defined \n topography are not well known and poorly understood . \n because many variables \n influence cellular interactions to surface structures , \n it is difficult to draw \n conclusions and formulate general principles for \n nano- and microstructured \n surfaces .", "this review summarizes recent data of effects by nano- and \n microstructured biomaterials and particles in vitro designed for \n orthopaedic application to get a solid \n framework outlining the critical interactions that govern the \n cytocompatibility . \n because biomaterials in orthopaedics are predominantly \n applied on bone , this review is focussed on \n the interactions of osteoblasts \n  and bone - marrow - derived \n cells with structured biomaterials ."], ["osteoblasts and osteoclasts are mainly \n responsible for the osteointegration of \n nanostructured biomaterials in orthopaedics . \n", "osteoblasts derive from \n mesenchymal progenitor cells which are localized mainly \n in the bone marrow and \n periosteum .", "they are characterized by cuboidal \n and flat morphology ( diameter about \n 20  m ) , present a large \n amount of rough endoplasmatic reticlum and a large golgi apparatus , \n and are potent to produce osteoid , a collagen i rich matrix \n . \n", "in addition , these mononuclear cells are also responsible \n for osteoid calcification ( hydroxyapatite ) .", "typical marker proteins for \n osteoblasts are cbfa1/runx2 , osteocalcin , osteopontin , osteonectin , bone \n sialoprotein ( bsp ) , osteoprotegerin \n ( opg ) , collagen i , and alkaline phosphates \n ( alp ) .", "figure 1 \n gives a brief summary of the expression \n of several markers during osteoblast differentiation .", "osteocytes act in a paracrine and mechanosensory manner , and \n can activate osetoblasts and osteoclasts . \n the latter cell type derived from the \n hematopoietic line , has multiple nuclei and is \n responsible for bone resorption . \n", "its ruffled border is flanked \n by a sealing zone which facilitates local acidification \n and removal of bony \n matrix such as ca , h3po4 , and \n h2co3 \n by endocytosis .", "osteoclasts express \n high levels of tartrate - resistant acid phosphatase \n ( trap ) and cathepsin k. the \n interaction between osteoblasts and osteoclasts is complex . during differentiation ,", "the ostoblast progenitors express receptor \n activator of nuclear \n factor  ligand ( rankl ) and macrophage colony - stimulating factor \n ( m - csf ) which are strong stimuli for osteoclastogenesis .", "in contrast , \n osteoprotegerin ( opg ) is a potent inhibitor \n of osteoclasts .", "moreover , the \n interactions between osteoblasts and osteoclasts in vivo are regulated \n by several hormones and cytokines , including parathyroid hormone ( pth ) , \n calcitonin , and il-6 ."], ["it is generally accepted that the \n three - dimensional surface topography \n ( size , shape , surface texture ) is one of \n the most important parameters that influence cellular reactions \n [ 2 , 1119 ] . \n", "although many studies have investigated \n cellular reaction to different surface pattern , \n the significance of macro \n structure studies on bone cell behavior is \n questionable since in vivo adhesion \n structures ( e.g. , cell membranes , \n basement membranes ) are comprised of much \n smaller nanometer scale features [ 20 , 21 ] .", "the immature bone is characterized by an average \n inorganic grain size of \n 1050  nm whereas mature bone has an average \n inorganic grain size of 2050  nm \n ( 25  nm in diameter ) \n . \n", "considering these parameters , modern \n implants for bone application have been \n designed with a smooth surface at the \n nanometer level .", "it was surprising that \n some of these have induced the formation of peri - implant \n fibrous tissue and \n implant loosening in vivo , while other implants with a higher \n degree of roughness showed significant better osteoconductive properties \n [ 2325 ] .", "there are various methods to modify the degree of \n roughness as well as surface energy \n and topography in orthopaedic implants", ". \n typically applied techniques to enhance \n the degree of roughness and promote \n the osteointegrative properties of \n biometals ( e.g. , ti , cocrmo , ss ) are chemical \n etching or anodization and \n \n also sand - blasting , sputter - coating , and machine - tooling .", "the lack of knowledge in cellular reaction to \n nanostructered biomaterials is based \n to a great extent on \n the difficulty in varying surface chemistry and \n topography independently .", "moreover , \n the use of different cell lineages and culture \n conditions makes it difficult to \n compare results from different investigators \n [ 2631 ] \n ( table 1 ) . \n", "there is also a lack of consensus concerning the \n proper representation of implant surface topography \n . \n", "one major misunderstanding is the practice of defining a \n surface by its manufacturing process instead of concisely defining the \n topographic measurements [ 17 , 33 ] . \n considering these limitations for interpretation ,", "the \n following review gives an overview of cellular \n reactions to surface structures \n of different orthopaedic biomaterials .", "the first step after exposure of any biomaterial \n to a biological environment results in the rapid \n adsorption of proteins to its \n surface . \n", "the composition , type , amount , and \n conformation of adsorbed proteins regulate the \n secondary phenomena such as \n cellular adherence and protein exchange \n [ 3537 ] \n and also following cellular reactions such as migration , \n proliferation , and differentiation . \n", "the potency for biomaterials to adsorb \n proteins is influenced by its physiochemical \n\n characteristics such as surface energy or hydrophobicity , \n and is also dependent on \n the local environment ( ph , \n concentration of ions , composition and \n functional groups of proteins , strength \n of solution , temperature ) \n ( vroman effect ) \n ( figures 2 ,   and 3 ) . for inorganic nanocrystals and microstructured \n surfaces", "there are at least two approaches to change \n their hydrophobic surfaces : \n a ligand exchange reaction can replace the original \n hydrophobic surface with \n bifunctional coupling molecules or an inorganic \n coating such as silica ( 1 ) or \n an encapsulation of nanocrystals in an \n  amphiphile organic coating ( 2 ) .", "the first phase of protein adsorption onto a \n biomaterial 's surface is characterized \n by the attachment of small rapidly diffusing \n\n proteins , followed by a progressive replacement by larger \n proteins with a high \n affinity to the substrate . here", ", especially proteins \n with arg - gly - asp ( rgd ) \n containing sequences such as fibronectin or \n vitronectin act as cell receptors \n and have chemotactic or adhesive properties to bone cells . \n", "in addition , these rgd - peptides \n also have a strong effect on matrix maturation \n and biomineralization [ 4648 ] .", "after conditioning of a naked biomaterial by protein adsorption , cells attach rapidly \n on the protein - coated surface . \n besides the influence of proteins , the \n cellular attachement to a nanostructed surface \n", "is also influenced by its physiochemical \n properties , especially by the outer functional groups \n [ 30 , 50 , 51 ] .   ", "schweikl et al .  \n showed on self - assembly monolayers that the \n osteoblast proliferation on hydrocarbon \n chains , terminated by ch3 , \n was as high as on amino groups \n ( nh2 ) \n and hydrophilic oxidized surfaces , but significantly lower \n on fluorocarbon \n ( cf3 ) \n groups .", "mller et al \n .  \n showed that \n 3-aminopropyl triethoxysilane ( apts ) \n presents amine functional groups which allow for \n grafting rgd tripeptides and \n that the rgd - apts hybrid promotes cell adhesion , \n spreading , and cytoskeletal \n organization . here , the zetal potential ( differences in potentials \n between the surface of a tightly \n bounded layer and a diffuse layer ) \n and the interfacial tension ( wettability ) of \n a surface is crucial [ 54 , 55 ] .", "it was demonstrated for cpti surfaces that the contact angle ( ca ) , \n parameter for \n wettability , increases linearly with the average roughness when \n the angles were \n higher than 45 , but decreases linearly with roughness \n when the angle was less \n than 45 . \n", "recent data examining osteoblast \n response to controlled surface chemistries indicate that \n hydrophilic surfaces \n ( high number of polar components ) improve cell attachment \n and matrix synthesis and also the osteogenic \n potency compared to hydrophobic \n surfaces [ 5759 ] . \n", "compared ti alloys and cocr alloys towards protein \n absorptive properties and cell attachment with an \n osteoblast precursor cell \n line .", "they found no significant \n differences between ti alloys and cocr , \n but significantly greater cell adhesion \n rates for the ti implants and concluded that cell \n adhesion is a result of \n higher hydrophilicity of ti alloys . in \n contrast", ", other data showed that a low degree \n of wettability promotes protein \n adhesion and also cellular attachment to a biomaterial \n , \n and mller et al .  \n", "found no direct correlation between the \n wettability of the material surface and the osteoblast attachment and \n proliferation rate .", "also qu et al .  \n found no significant differences of cell attachement \n on various titanium surfaces with different degrees of wettabilities \n ( hydrophobic acid - etched , coarse - blasted large \n grit acid - etched , hydrophilic modified \n acid - etched , and modified coarse - blasted large grit , \n acid - etched \n ) on mg68 cells .", "heating ( oxygen / atm ) or peroxide treatment of biometals \n result in a thicker oxide layer \n and a more hydrophilic surface .", ". \n  showed that \n heat - treated titanium \n surfaces changed the wettability ( more hydrophilic ) \n but does not significantly \n affect the fibronectin and albumin adsorption as well as \n the initial osteoblast \n precursor cell attachment in vitro . \n", "emphasized that the rate \n of protein correlates more with changes in chemical \n composition than \n with changes in wettability in metal surfaces .", "they showed that \n a preheating of ti6al4v specimen does not only lead to a \n thicker oxide layer but also results in \n an enrichment of v and al within \n the surface oxide .", "in contrast , \n post - treatment with butanol after preheating \n reduces the content of v , but not in al , \n and significantly increases the rate \n of fibronectin adsorption up to 2040% \n .", "compared to the cellular attachment phase , the following adhesion \n phase lasts longer and \n involves various proteins and molecules \n ( figure 2 ) . \n as a link between cell and \n biomaterial , the interactions of a surface topography \n and serum proteins are \n crucial for the cytocompatibility of a biomaterial . \n", "especially , the adsorption \n of adhesion proteins , such as fibronectin and vitronectin , from serum \n containing solutions and integrin - mediated signaling has been \n demonstrated to \n mediate cell adhesion and spreading \n . it has been shown that nanotube or nanoparticle surfaces \n created by anodization have \n promoted osteoblast adhesion up to three times \n compared to unanodized ti . \n", "these results were confirmed by the group of webster \n \n and other investigators [ 6871 ] \n who demonstrated that the initial \n attachment of osteoblasts onto the surface of biometals such as cpti , \n ti6al4v , and cocrmo is enhanced by submicron to nanometer \n consistent particles compared \n to metals composed of respective micron particles . \n", "one possible explanation of \n this phenomenon is the higher amount of particle binding sites for \n osteoblast adhesion at the surfaces of nanophase metals \n compared to micron particle size \n metals .", "the theory of enhanced protein and cell binding capacities \n by larger \n surface areas / roughness degrees was also confirmed for porous \n ha materials .", "another example of the significance of surface structures \n for protein binding and \n osteoblast attachment is the helical rosette nanotubes \n ( hrn ) which can build \n self - assembly surface structures .", "it was demonstrated that \n a significant change \n of hrn coverage by heating correlated with the protein - binding \n and osteoblast \n adhesion potency in titanium surfaces [ 73 , \n 74 ] .", "it is evident that not only the surface topography \n influences protein deposition and \n cell adherence but also proteins and cells modify the surface \n properties of a \n defined surface .", "based on a surface analysis of the different \n biometal specimen \n before and after cell cultivation , we showed previously \n \n that a cell attachment and/or protein \n precipitation increase the roughness in polished biomaterials \n ( steel , ti6al4v , \n and cocr ) .", "for porous coated cocr surfaces , we found only slight and no \n relevant changes in roughness whereas cell cultivation onto \n sandblasted ti6al4v \n lead to a strong decrease in specimen roughness . \n", "both , the increase in roughness after \n cell culturing in the different biometals \n and the decrease in roughness of sandblasted ti6al4v could be \n explained by the \n dense cellular growth and accumulation of debris in depth of \n the structured surfaces \n and/or protein deposition as shown by other investigators \n [ 75 , 76 ] .", "in addition , not only the amount but also the type of \n protein adsorption by a \n surface is crucial for cellular adherence and following reactions such as \n migration and differentiation . as an example , ti surfaces ( ra : \n 0.370.01  m ) adsorp \n  fibronectin in higher concentrations \n compared to albumin , and fibronectin - coated ti surface promoted \n more osteoblast attachments \n in comparison to albumin - coated ti surfaces \n . \n", "these results correspond to the data of other authors \n who showed excellent osteoconductive properties after \n fibronectin adsorption \n onto a biomaterials ' surface [ 7880 ] . based on irm and tem analysis ,", "the closest distance \n of cells to a surface ( glass ) was \n found to be approximately 10  nm [ 81 , \n 82 ] .", "historically , results from chicken \n fibroblasts have lead to a classification of three different types of \n separation .", "( 1 ) focal contacts ( fc ) : approximately \n 1015  nm separation from the substrate under the \n peripheral regions of the leading lamellae \n ( appearing black in tem ) .", "fc act as \n an interface between intra and extracellular components and occur linearly \n beneath the associated cytoplasmic stress fibres \n [ 83 , \n 84 ] . \n", "they are tenacious adhesion sites that \n remain attached to the substratum even when cells are forcibly detached , \n indicating their function as anchorage \n structures .", "( 2 ) close contacts : corresponding to approximately \n 30  nm separation ( broader grey areas \n in tem ) .", "( 3 ) greater separation : corresponding to approximately \n 100140  nm ( white \n regions in tem ) .", "it is evident that not only fc appear soon after cellular attachment but also that ( -catenin - positive ) adherence \n junctions occur within 14 hours \n for grooved ti - based substrates . \n", "these observations underline the high \n significance of an early intercellular communication soon \n after adherence to a \n surface .", "the mechanisms of initial cellular adherence to a surface are \n different from long - term adherence as shown by a lack of statistical \n correlation between short - term adhesion ( strength of cell attachment \n and early \n adhesion ) and long - term adhesion ( strength of cell - matrix interface ) \n forces [ 14 , 15 , 86 ] . \n", ". \n \n showed that the cultivation time has an \n influence on the long - term adhesion in biometal surfaces according to \n  td \n ( t ) = atb , \n a being independent of b ( td : time - dependend \n adhesion index , a : surface - dependent parameter , b : substrat - independent \n exponent , 0.5+/-0.03 ) . for polylactides ( plla ) , it was shown on oct-1 osteoblast - like \n cells that cell \n adhesion but not the proliferation could be enhanced by nanoscale \n and microscale \n roughness compared to smooth surfaces .", "in addition , \n there is evidence that fc \n show a dynamic behavior which allows for cellular migration and motility . \n", "linear plla fibres with length scales of 0.52  m , \n constructed by electrospinning , have shown cellular contact \n guidance and enhanced \n osteoblastic differentiation . here , cell morphology revealed that cells \n grown on fibres had smaller projected areas than those on planar \n surfaces .", "also other polymers such as plga have been shown to \n be effective in enhancing osteoblast differentiation in vitro \n .", "diener et al .   demonstrated \n on mg-63 osteoblastic cells \n that fc adhesion was smaller on ti and ss than on collagen - coated glass \n coverslips and that all fc showed a mobility of focal adhesions .", "however , \n anselme et al .   found higher \n adhesions on ti6al4v \n substrates than on noncollagen - covered glass samples , \n and emphasized that substrates \n with various surface compositions \n but with the same surface topography did not \n induce significant differences of adhesion .", "based on the knowledge of protein adsorption and its effects \n on cellular attachment and adherence , a selective surface coating \n of nanostructured \n surfaces with rgd or collagen proteins offer a promising solution \n to improve \n the number of osteoblasts adhered on artificial surfaces \n [ 53 , 95102 ] .", "imprinting \n surfaces technology with deposition of specific \n protein - recognition sites can \n help to promote osteoblastic growth \n and differentiation [ 103106 ] .", "protein - recognition \n can be based on a protein - ligand binding and/or electron donor - acceptor \n interactions or other types of binding forces .", "integrin \n 51 and 5 \n v3 subunits competitively \n bind to rgd - sites of fibronectin [ 107 , \n 108 ] .", "dependent on the \n surface topography and \n chemistry of the \n\n biomaterial , fibronectin \n undergoes changes in structure including \n modulation in functional activity and \n shift in integrin binding capacity .", "based on the data of self - assembled monolayers , \n it was shown that integrin subunits \n show selective binding capacities to different \n terminal groups .", "integrin 51 shows a \n strong affinity to oh and \n nh2 surfaces , whereas \n 51 and \n 5v3 bind also to cooh but show poor binding capacities \n on ch3 \n surfaces [ 109113 ] .", "furthermore , some data show that oh \n and nh2 surfaces can up - regulate \n osteoblast - specific gene expression but also \n matrix mineralization compared \n with cooh and \n ch3 \n functional groups [ 47 , 112 ] .", "cell migration and proliferation is the attachment \n following phase between the cell \n and the material surface .", "it   is evident for designing \n nanostructured implants \n that cells use the nanotopography of a substrate for \n orientation and migration [ 117119 ] .", "although \n it is known that bone cells \n align along defined substrate morphologies ( contact \n guidance ) , the detailed relation between ordered \n nanotopography and cell \n behavior remains unknown in detail . \n", "for the first time , in 1964 it was shown \n that convex surfaces enhance cellular overlap , while \n grooves minimize cellular \n overlap .", "as pre - requisite to reach a defined cell colonization during \n directed tissue formation , structured nanophase surfaces \n lead to a predictable \n osteoblast orientation and migration on these surfaces \n [ 17 , 121 , 122 ] . \n", "interaction between the ecm and associated \n changes in the \n orientation of the cytoskeleton are crucial for cell \n metabolism of cells and \n morphology due to actin - myosin tension structures \n .", "anisotropic topographies ( e.g. , \n topographical grooves , chemically patterned stripes , \n or curved surfaces of a \n fibre ) are potent to exert morphological \n as well as physiochemical \n features on cells at the same time , indicative for the \n complex environmental \n influence on cells .", "focal contacts are important structures for cellular \n adherence onto a surface but may \n also delay migration and mobility of the cells . \n", "it was shown that bone - derived cells ( mg63 cells ) respond to \n a nanoscale roughness by a higher cell thickness and a \n delayed appearance of \n focal contacts . \n", "especially , nanoporous \n ti - oxide surfaces promote cellular \n spreading and induce numerous filopods and osteoblastic differentiation \n [ 124 , \n 125 ] . on", "electrochemically \n microstructured hexagonal \n pattern , mg63-cells go inside \n 30100  m but not in 10  m \n cavities .", "most authors report a \n parallel orientation of cells cultured on \n polished ( smooth ) surfaces [ 57 , 114 , 126 ] \n ( figure 4 ) .", "another method to not only enhance cellular adherence but also to \n promote osteoblastic differentiation and \n biomineralization of biometals is a surface anodization , \n for example , by -glycerophosphate sodium and calcium \n acetate [ 6671 ] .", "cellular adhesion via fc may strengthen the linkage \n between cell and ecm and \n also impair the ability to dynamically remodelling \n the ecm and influence the \n migration rate . for \n collagen - coated coverslips , focal adhesion of mg-63 \n osteoblastic cells moved \n with a speed of 60  nm / min , whereas the speed was \n reduced in ti and more in ss \n surfaces .", "another study \n on nb2o5-coated \n polished cpti samples showed that mc3t3-e1-osteoblast \n migration was fastest on \n smooth surfaces ( ra = 7  nm ) , whereas adhesion strength , \n spreading area , and \n collagen - i synthesis were promoted by intermediate \n roughness ( ra = 15  nm ) .", "however , \n it was surprising that higher degrees of roughness \n ( ra = 40  nm ) were rather \n peaked and reduced the speed of adhesion process in the \n same study . besides the surface properties of a biomaterial , \n the cellular migration rate is \n dependent on the cell type and its differentiation stage . \n", "a higher migration \n rate is associated with a lower level of osteoblast \n differentiation .", "cells with \n a low motility are characterized by a strong formation of fc \n while motile cells \n form less adhesive structures .", "it was found that mature \n osteoblasts spread out \n and form a greater number of fc when settled on smoother surfaces \n .", "although \n cellular spreading is higher on \n smoother surfaces , some data indicate that the \n alp - expression is higher for \n rough isotropic surfaces ( electro - erosion , acid - etching , \n sandblasted ) compared \n to smoother substrates ( machine tooling , polishing ) \n . considering recent publications , there \n is no or only week statistical significance that there \n is a difference between \n the initial number of adherent cells and following proliferation of cells \n cultured onto a biometal or ceramic nano-/microscale surface \n in vitro .", "however , \n some authors emphasize that \n the influence of functional chemical groups for cellular migration and \n proliferation are stronger than general surface \n properties such as wettability . \n", "especially a tio2-layer \n seems to promote \n cellular growth and proliferation on nanostructured \n biometals [ 128 , 129 ] .", "other examples for a promotion of cell - to - bone contact in vitro \n and also in vivo are \n machine - etched ti - surfaces ( e.g. , osteotite ) \n , defined sand - blasted \n implants [ 124 , 125 , 131 ] , and hydroxyapatite ( ha ) coatings , \n for example , by plama - spray techniques [ 132134 ] .", "recent studies investigating the response of adherent \n cells to nanography surfaces \n indicate that different cell phenotypes have different \n levels of sensitivities [ 117 , 135137 ] . here", ", \n osteoblasts react to features as \n low as to the 10 nm dimensions , which is comparable in size to a single \n collagen fibre .", "moreover , the qualitative and quantitative kinetics \n in gene and protein expression is \n strongly influenced by topography and physiochemistry \n of a defined surface .", "microporous \n ha surfaces seem to promote a high number of fc and \n increased levels of alp but \n short actin stress fibres compared to nonmicroporous ha surfaces \n [ 72 , 139 ] . \n", "there is also evidence that ti and ha \n surfaces can activate early intracellular signalling \n pathways as shown by \n expression of relevant molecules such as \n - and 1-integrin , fak , erk followed \n by c - jun and c - fos genes for proliferation and alp for differentiation \n [ 139 , 140 ] . \n", "however , hallgren et al .  \n found no significant histomorphometric \n and biomechanical differences between nanopatterned and control implants . \n", ".  \n showed that microfabricated \n discontinuous - edge surfaces ( des ) , \n repeated open square boxes with a depth of \n 10  m , alter osteoblast adherence and migration \n but enhance cell multilayering , \n matrix deposition and mineralization when compared to smooth controls .", "in contrast to our data , \n anselme et al .   found \n higher proliferation \n rates on ss compared to ti6al4v .", "however , bigerelle et al . \n  demonstrated that neither \n material composition nor surface roughness amplitude influence cell \n proliferation , whereas they found a very significant influence \n on manufacturing \n process and surface topography for long - term adherence \n and proliferation in vitro .", "our in vitro results   confirm the \n well known osteogenic in \n vivo properties of ti implants , which may be based on \n surface factors observed \n on its outer tio2-layer \n [ 143146 ] . \n", "mller et al .  \n demonstrated the ability of osteoblasts \n to grow into an open - porous ti implant ( metal foam ) and li et al", ". \n \n also demonstrated that mc3t3-e1 cells \n attach to and are able to divide well in the inner surface of \n a highly porous \n trabecular ti6al4v implant .", "some in vitro studies demonstrated an \n enhanced total protein and collagen production , \n as well as increased alp \n activity of osteoblasts cultured on nanoparticulate \n metals ( cpti , ti6al4v , and \n cocrmo ) indicating advantages for nanostructured surfaces \n for osteointegration [ 1 , 149 , 150 ] . based on the \n data of redey et al . , \n it can be concluded that the low \n attachment and collagen production rates are related to a low \n wettability of a \n nanosurface .", "nanotextured surfaces of ti surfaces \n prepared by chemical etching \n have upregulated the expression of bsp and \n op . \n as demonstrated by qu et al . , \n the expression of the bone - associated \n genes such as alp , oc , type - i - collagen , osteoprotegerin , and \n glyceraldehyde-3-phosphate - dehydrogenase is promoted \n by modsla ti surfaces . \n some data also suggest that fluoride - modified \n ti surfaces can stimulate \n osteoblastic differentiation compared to \n unmodified titanium surfaces [ 151 , \n 152 ] .", ". \n  showed in their in vitro experiments that nanophase biometals induce \n significantly greater calcium and phosphorus \n deposition by osteoblasts and also allow for \n calcium and phosphorous precipitation from \n culture media without osteoblasts in \n contrast to microphase ti6al4v and cocrmo . \n", "furthermore , the authors found \n advantages in mineral precipitation without osteoblast for \n tial4v but no differences \n in dependency to the type of ti ( wrought , microphase , \n or nanophase ) .", "it was \n evident that the increased calcium and phosphorus \n mineral content correlated to \n greater amounts of underlying aluminium content on \n ti6al4v surfaces . although some data indicate \n that nanostructured ti alloys promote non - cell - mediated ca / po4-mineral \n deposition from culture media compared to cocrmo substrates , \n the greatest cell - dependend calcium and \n phosphorus mineral deposition occurred \n on nanophase cocrmo .", "it is evident that micropattern collagen films or \n scaffolds promote not \n only cellular adhesion but also allow for \n an osteoblastic differentiation and \n biocalcification in vitro [ 153155 ] . \n for ha- and dcpp - coated , ti \n surfaces the ca / p ratio influence the biomineralization \n rate in vitro .", "besides the osteoblast - promoting effects \n of defined substrates and \n surface topographies , some data also allocate \n an inflammatory response induced \n by nano- or microstructured biomaterials . \n", "it was shown in many studies that \n cell - biomaterial interactions can activate macrophages \n which results in the synthesis of proinflammatory \n agents such as tnf , ifn , il-1 and -6 , \n rankl and no [ 157159 ] .", "some data have \n shown proinflammatory effects of different \n biomaterials which increase with the \n degree of surface roughness . \n here ,", "macrophage inflammatory protein-1 , tnf , \n monocyte chemoattractant protein-1 , and \n members of the interleukine and leukotriene family \n play a crucial role in \n biometal - induced inflammations \n [ 160164 ] .", "most studies report about an \n enhanced expression of pro - inflammatory \n cytokines and chemokines by cells \n attached to rougher surfaces .", "some data also \n indicate that anionic and neutral hydrophilic surfaces \n increase macrophage - monocyte apoptosis and reduce macrophage \n fusion to modulate \n inflammatory responses to implanted materials \n .", "however , adverse cellular effects seen with \n metallic implants may \n also be attributed to corrosion products or to \n the separation of metal ions \n ( fe , cr , ni ) which may have a major impact on cellular survival and \n differentiation [ 166168 ] . \n those studies which suggest that a cell - mediated metal ion \n release by biometals that \n  did not affect \n the cell viability or proliferation are \n characterized by short cultivation periods or \n other conditions which limit the \n reliability of data [ 169171 ] . up to date , only few authors report about no significant influence \n of the cellular adherence and expression of osteoblast \n proteins by different \n biometals and surfaces such as alp expression \n [ 172 , \n 173 ] .", "in contrast to the great opportunity enhancing \n biocompatibility and osteogenic potency of surfaces applied on bone by \n nanotechnology , micro- and nanoscaled particles released by friction of \n artificial joints can induce severe \n inflammation and may lead to osteolysis and \n implant failure [ 174 , \n 175 ] \n ( figure 5 , \n table 2 ) . there is a wide \n range in particles size and morphology produced by \n simulators for artificial \n joints .", "particles released from metal - metal ( crcomo alloys ) are \n predominantly chromium oxide particles or cocrmo with \n varying ratios of co and cr .", "they show a round to oval morphology \n and also a substantial number of needle - shaped \n particles were found during the first circles . \n", "emphasize the \n importance of particle size \n as a critical factor in osteoblasts proliferation \n and viability in vitro .", "some data indicate \n that in contrast to ti - surfaces \n nano- and mircoparticles induce an \n inflammatory response although titanium is \n one of the biometals with the highest degree of \n cytocompatibility . as shown by miyanishi et al . \n , \n the release of vegf may play a crucial role in the \n pathogenesis of ti - induced osteolysis .", "some data indicate that \n phagocytosis of ti particles is not a precondition for \n an inflammatory response \n such as a release of tnf or il-6 in \n cultured macrophages . \n", "it is evident that a binding of the macrophage cd11b / cd18 ( macrophage \n mac-1 receptors / receptor of complement cr3bi , \n can also bind to icam-1 and \n icam-2 ) by integrin - specific antibodies \n also increased the release of tnf and il-6 in macrophages . \n", "this finding also suggests \n that the complement system plays a role in the pathogenesis \n of particle - induced \n inflammation , too .", "especially , uhmwpe particles with \n a size range of \n 0.11.0  m have been shown to be most reactive for \n macrophage activation and cytokine \n secretion in bone marrow cells [ 179 , \n 180 ]", ". however , not only the particle size but also the particle volume \n ( number ) is a critical factor for particle - mediated \n release of cytokines by \n macrophages .", "green et al . \n demonstrated for pe \n that the cell - particle ratios of 1 : 100 \n ( size 0.497.2  m ) and 1 : 10 \n ( size : 0.494.2  m ) induced \n significant stronger release \n of tnf and il-1 in macrophages . \n", "the authors conclude that especially \n particles in the phagocytosable size range of \n 0.310  m appear to be the most \n biologically active ones .", "the latter statement was also confirmed for silicon carbide ( sic ) \n particles and biometals such as cpti , \n ti6al4v and uhmwpe [ 184 , 185 ] .", "granchi et al .  \n investigated the in vitro \n effects of al2o3 \n and uhmwpe particles in an \n osteoblast - osteoclast co - culture system . \n", "both particles did not affect either \n cell viability or tnf and gm - csf release , \n whereas il6 release was dependent on \n the particle concentration .", "uhmwpe particles \n increased the release of rankl \n from osteoblasts and induced large amounts \n of multinucleated trap - positive \n giant cells in an osteoblast - osteoclast \n co - culture system .", "also , \n carbon - based particles with low wear \n factors such as p25-cvd showed a high degree \n of cytocompatibility in vitro . howling et al . \n ", "demonstrated \n on fibroblasts and monocytes that p25-cvd \n particles < 100  nm were significantly less cytotoxic \n to both cell types than \n cocr metal wear particles . \n", "while the classical water - suspendable \n nanoc60 nanocrystal is apparently \n cytotoxic to various cell lines , the closely related \n fully hydroxylated , \n c60(oh)24 \n , is nontoxic , \n thus producing no cellular \n response .", "also , \n functionalized single - walled carbon nanotubes are nontoxic \n to cells in culture [ 198200 ] .", "there is evidence that not only particle \n size and chemical content but also the concentration strongly influence \n cellular reactions in vitro .", "wilke \n et al .  \n showed a positive correlation between the \n release of proinflammatory cytokines \n ( il-6 , -1 , and tnf ) and amounts of \n ti6al4v - particles \n ( 10 , \n 10 , 10 , \n and 10 particles / ml ) \n by human bone marrow cells over 2 weeks .", "some in vitro data also indicate that ti particles induce a stronger \n fibroblastic differentiation signal than uhmwpe \n in monocytes and other cells [ 182184 ] .", ".  \n showed that particles \n of high - density polyethylene \n ( hdp ) and ti6al4v induced significantly more \n proinflammatory mediators ( il-1 , \n il-6 , tnf ) and bone resorption compared to \n al2o3 \n and zro2 in vivo . \n", "based on these data , \n it can be assumed that ceramics show a high \n degree of cytocompatibiltiy . for ha especially , particles with a size \n < 53  m inhibit cellular proliferation , \n especially in osteoblasts and lead \n to a decrease in tgf1 and a significant increase in pge2 and ldh \n concentration , but did not influence the tnf \n or alp titer in vitro . \n it could be concluded that larger ha particles may \n be compatible with bone cells while smaller - sized \n ha particles can both activate \n the osteoclasts and decrease the cell \n population of the osteoblasts in vitro .", "numerous variables influence the biocompatibility and osteogenic \n potency of nanostructured biomaterials in \n vitro and in vivo .", "besides the locotypical \n environment in vivo or in vitro , the surface \n structure and the composition of a \n biomaterial affects cellular attachment , \n adherence , proliferation and migration , \n and also \n differentiation and survival of defined cell types . \n here , information about typical \n parameters such as chemical composition , \n surface structure ( topography , geometry , \n roughness , particle size ) , surface energy , \n hydrophobicity , and the degree of \n solubility in aqueous solutions of a biomaterial \n will help to value and grade a \n defined implant concerning its osteblast promoting potency . considering recent publications , we could assume some general \n principles of cytocompatiblity and cell - surface \n interactions in nano- and \n microstructured surfaces .", "( 1 ) wettability of a nanosurface influences significantly \n protein adsorption , which is a prerequisite \n of cellular adherence in serum containing \n solutions .", "( 2 ) nanostructured surfaces enhance the surface area of \n biomaterials and promote cellular adherence . ( 3 ) the chemical outer functional \n groups of a nanosurface significantly influence cellular \n migration , proliferation , and differentiation but direct \n correlations between \n distinct parameters and cell functions are not entirely cleared .", "( 4 ) the formation of fc underly a \n dynamic process and influence the motility and migration of cells . ( 5 ) a higher degree of differentiation \n is corresponding to a decreased cellular motility .", "( 6 ) phagocytable particles with a size \n < 10  m induce the strongest \n cellular response with regard to releasing \n inflammatory cytokines .", "( 7 ) although ti has a high degree of \n cytocompatibility in vitro , phagocytable \n ti particles can induce a fibroblastic \n differentiation ."]]}
{"article_id": "PMC3272368", "article_text": ["six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) .", "most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries .", "sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping .", "separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) .", "five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) .", "three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) .", "six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) .", "most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries .", "sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping .", "separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) .", "five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) .", "three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) ."], "abstract_text": ["<S> legumes ( fabaceae or leguminosae ) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria , a process that takes place in a specialized structure known as the nodule . </S>", "<S> legumes belong to one of the two main groups of eurosids , the fabidae , which includes most species capable of endosymbiotic nitrogen fixation 1 . </S>", "<S> legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago ( mya ) . </S>", "<S> papilionoids are the largest clade , dating nearly to the origin of legumes and containing most cultivated species 2 . </S>", "<S> medicago truncatula ( mt ) is a long - established model for the study of legume biology . </S>", "<S> here we describe the draft sequence of the mt euchromatin based on a recently completed bac - assembly supplemented with illumina - shotgun sequence , together capturing ~94% of all mt genes . a whole - genome duplication ( wgd ) </S>", "<S> approximately 58 mya played a major role in shaping the mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation . </S>", "<S> subsequent to the wgd , the mt genome experienced higher levels of rearrangement than two other sequenced legumes , glycine max ( gm ) and lotus japonicus ( lj ) . </S>", "<S> mt is a close relative of alfalfa ( m. sativa ) , a widely cultivated crop with limited genomics tools and complex autotetraploid genetics . as such </S>", "<S> , the mt genome sequence provides significant opportunities to expand alfalfa s genomic toolbox . </S>"], "labels": null, "section_names": ["METHODS SUMMARY", "DNA sequencing", "RNA sequencing", "Supplementary Material"], "sections": [["six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) .", "most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries .", "sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping .", "separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) .", "five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) .", "three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) ."], ["six a17 bac and one fosmid library were used to create mt3.5 ( table s1 ) .", "most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries .", "sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping .", "separately , 25 gb of illumina sequence was generated using short ( 375 nt ) inserts plus 2.1 gb from a 5 kb mate - pair library , then assembled using clcbio ( www.clcbio.com ) and soap ( http://soap.genomics.org.cn/ ) ."], ["five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library ( table s12 ) .", "three tissues were used for small rna analysis with ~3 million reads per illumina library ( figures s17-s18 , table s16 , datafile s9 ) ."], [""]]}
{"article_id": "PMC4443416", "article_text": ["surgical treatment of avascular necrosis ( avn ) of head of femur include core decompression , osteotomies , nonvascularized bone grafting , free vascularized fibular grafts , hip resurfacing , bipolar hip arthroplasty ( bha ) and total hip arthroplasty ( tha).123 tha is indicated in the young individual in avn with acetabular involvement ; however , its role is unclear in cases without acetabular involvement.4 bha was initially limited to be used in hip osteoarthritis , nonunions and acute neck femur fractures.56 bateman7 and giliberty8 were first to use bha in ficat stage 3 avn based on the hypothesis that ", "acetabular floor retains a regenerative property , which regenerates bone in the subchondral region , if stimulation in the form of weight bearing is given through an accurately fitted cup  and theorized that preferential motion at inner bearing will decrease the cartilage erosion .", "papers have been published with both satisfactory91011121314 and unsatisfactory results using bha.5151617 the main limitations sighted are migration of the outer cup ( secondary to the acetabular erosion ) and unpredictable pain relief.3514 incidence of groin pain varied from 11.5% to 42%181920 and incidence of acetabular erosion and protrusio is reported to range from 0% to 45%.1819 the activity of the patient and the duration of followup determine the erosion of acetabulum and appearance of symptoms.21 however , the main issue seems to be motion between the outer cup and the acetabulum , a nonconcentric acetabulum and particulate wear.16222324 to decrease this motion between the outer cup and the acetabulum a gentle reaming of the acetabulum with insertion of tight fitting acetabular cup can be done as suggested above by bateman and giliberty.78 this is thought to decrease the incidence of groin pain and the acetabular erosion and also revision secondary to these issues.2526 in this retrospective series , we have assessed the midterm outcome of bha in young adults ( age < 60 ) with ficat stages 3 and 4 avn .", "a retrospective study with a prospective followup was planned . 80 consecutive patients below 60 years of age ( 96 hips ) were operated by the senior author between 1995 and 2010 using bha with tight fitting cup technique .", "all patients were males with a mean age of 42  6.44 years ( range 30 - 59 years ) . out of a total of 96 hips ,", "76 were diagnosed as idiopathic avn , 8 were posttraumatic avn while 12 were post alcohol consumption avn .", "ficat 's classification system was used based on anteroposterior ( ap ) and lateral hip radiograph.26 clinically , the indication for surgery was pain with progressive inability to carry out daily activities .", "patients with radiographs showing no or minimal acetabular involvement were included to undergo the procedure , while cases with advanced osteoarthritis with ostephytosis or protrusio were excluded .", "surgeries were done in the lateral decubitus position using the posterolateral approach under epidural anesthesia .", "the hip joint capsule was excised if diseased . in our study , intraoperatively on gross examination , all the 96 hips ( ficat stage 3 and 4 ) were found to have mild to moderate acetabular involvement as per steinberg et al.2 the cartilage was graded grossly as grade 0 ( normal ) if no abnormalities were observed ; grade i ( mild degeneration ) if there was superficial fibrillation and slight irregularity of the surface ; grade ii ( moderate degeneration ) if there was moderate fibrillation , alteration in color and consistency and thinning of cartilage without complete erosion to bone .", "gentle superficial reaming of the acetabulum was done making the acetabulum concentric for a tight fitting bipolar cup [ figure 1a ] .", "the trial cups should fit snuggly enough so that when the hip is taken through a range of motion , no gross movement should be appreciated between the outer cup and the acetabulum .", "this assures that the prosthesis is tight fitting and movement between the outer cup and the acetabulum is avoided . during the process of making acetabulum concentric , sometimes subchondral bone was exposed . in these cases ,", "pulse lavage of the prepared acetabulum was done to remove all debris before putting the bipolar cup .", "the size of the cup was equal to the size of the tight trial cup chosen .", "we did keep instrumentation for total hip replacement ( thr ) ready intraoperatively ; however , we did not have to do thr in any of our selected cases . ( a ) peroperative photograph showing concentric acetabulum after superficial gentle reaming with the large hand reamer .", "( b and c ) x - ray taken in neutral and abduction showing movement taking place mostly at inner bearing self - centering cup with 28 mm inner head and uncemented stems were used in 60 hips ( zimmer , warsaw , usa , biomet , warsaw , usa ) . in the remaining 36 hips , normocentric hemispherical cups with 26 mm inner head and", "cemented stems were used ( inor , mumbai , india and rch , mumbai , india ) .", "the patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks .", "all patients were called for a final followup for clinical and radiological assessment . the clinical evaluation consisted of recording the details of thigh or groin pain , range of movements at the hip (", "patient was asked to sit cross legged on the bed ) , limb length discrepancy and harris hip score ( hhs ) . the radiological evaluation comprised of looking for subsidence of the stem , recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup .", "ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [ figure 1b and c ] .", "the femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler 's line and center of the outer head , whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs .", "a subgroup analysis was also done , by dividing the cohort according to ficat 's class , to evaluate the effect of acetabular involvement on the outcome of the procedure .", "the patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks .", "all patients were called for a final followup for clinical and radiological assessment . the clinical evaluation consisted of recording the details of thigh or groin pain , range of movements at the hip (", "patient was asked to sit cross legged on the bed ) , limb length discrepancy and harris hip score ( hhs ) .", "the radiological evaluation comprised of looking for subsidence of the stem , recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup .", "ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [ figure 1b and c ] .", "the femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler 's line and center of the outer head , whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs .", "a subgroup analysis was also done , by dividing the cohort according to ficat 's class , to evaluate the effect of acetabular involvement on the outcome of the procedure .", "the mean followup was 7.52  1.80 years ( range , 4 - 16 ) .", "the hhs improved from a preoperative value of 39.33  6.11 ( 30 - 54 ) to 89.12  6.68 ( 74 - 96 ) ( p = 0.0001 , paired t - test ) in the postoperative period . according to hhs grades , the final outcome was excellent in 52 , good in 28 and fair in 16 hips .", "hip and groin pain was reported in four hips ( 5% ) but this did not limit activity in any of the patient [ table 1 ] .", "22 hips had flexion deformity of approximately 3 - 5 with a final range of flexion of 100.2  8.82 ( 85 - 116 ) .", "subsidence of the femoral component was seen in 8 hips ( < 5 mm ) ; however , these hips were asymptomatic . shortening of 1 cm was noted in 10 limbs , and 1.5 cm shortening was noted in 1 limb .", "there was no incidence of hip dislocation or dissociation of the bipolar cup and no case required revision . on ap", "neutral and abduction x - rays there was no appreciable movements noted between the outer cup and the acetabulum in any of the case", ". there was medial migration in 5 hips and proximal migration in 12 hips , but none more than 2 mm and all were asymptomatic .", "comparison of results with various studies on subgroup analysis between the ficat stages 3 and 4 [ table 2 ] it was noted that hips with ficat stage 3 were younger at the time of surgery and also had better final outcome in terms of range of motion .", "the final hhs score was however not statistically different in both groups indicating good results even in cases with acetabular involvement .", "none of the ficat stage 3 patients had any groin pain or femoral subsidence at the final followup while 4 cases of groin pain and 8 cases of femoral subsidence were seen in ficat stage 4 group .", "figures 2 to 5 show case series with final result of four patients in our series .", "comparison between the ficats 3 and 4 subgroups x - ray right hip joint anteroposterior view in a 35 year old male showing ( a ) preoperative radiograph with avascular necrosis right hip secondary to fracture neck femur . also showing implant in situ ( b ) 4 years postoperative radiograph of the same patient treated with bipolar hip arthroplasty .", "( c ) 16 years followup radiograph of the same patient without any loosening or migration and with subchondral sclerosis ( a ) x - ray pelvis with both hip joints anteroposterior view in a 40 year old male showing avascular necrosis ficat stage iii in right hip ( post alcoholic ) ( b ) x - ray of right hip joint anteroposterior view of same patient showing implant in position at 9 years followup without any sign of erosion or migration .", "( c ) clinical photograph of same patient showing cross legged sitting ( a ) x - ray pelvis with both hip joints anteroposterior view in a 65 year old male showing posttraumatic avascular necrosis ficat stage iv ( see non concentric acetabulum ) and implant in situ .", "( b ) x - ray pelvis with both hip joints anteroposterior view of same patient at 9.5 years followup showing well fitting large cup .", "( c and d ) clinical photographs showing range of motion x - ray pelvis with both hip joints anteroposterior view in a 45 year old man with bilateral avascular necrosis of femoral head ( a ) 6 months followup showing right side bipolar arthroplasty left side ficat stage iv avascular necrosis .", "( b ) at 5 years followup showing well fixed implant with large cup well placed in acetabulum", "surface replacement is an option in younger people with avn , but has limited indications and is a demanding procedure with high cost.27 the results published by for surface replacement are not uniform and a longterm followup is lacking.272829 tha is the method of choice for the treatment of advanced avn of the femoral head;53031 however , its overuse may lead to increased wear and need for early revision in young adults.14 various studies have shown that functional utility of tha reduces to 80% at 10 years , 33% at 16 years , subsequently requiring a revision surgery .", "alternate bearing surfaces like metal on metal or ceramic on ceramic decrease the wear rate but have their own set of complications ( metallosis , squeaking)3233343536373839 and long term results are still awaited.40 when compared with bha , conflicting results have been reported .", "chan and shih30 have reported that there was no difference in the incidence of osteolysis , thigh or groin pain , dislocation rates and revision rates between bha and tha .", "they concluded that in young patients with ficat stage 3 avn , bha may be a useful alternative to tha .", "furthermore , bha is less demanding , blood loss is comparatively less5 and revision is easier as compared with the revision of tha as the acetabulum is still intact.41 lee et al.3 have reported 23% outer cup migration rate , 15% gluteal pain and 20% groin pain in bha .", "ito et al.15 have reported 42% radiological failures , 42% incidence of groin pain requiring a revision surgery in 25% hip undergoing bha .", "similar results were shown by cabanela14 and lachiewicz and desman17 with groin pain and acetabular erosion as the two most important reasons for poor results in bha .", "groin pain has been variably attributed to the preservation of diseased joint capsule , to irritation of the subchondral nerve endings of the acetabulum and to the acetabular erosion.1442 in our study , the diseased capsule was always excised to avoid capsular impingement .", "furthermore , as nerve endings in the posterior capsule supply the acetabulum , excising it blocks the nerve supply to the acetabulum and thus helps in relieving pain .", "poor fitting bipolar prostheses can lead to cartilage necrosis giving rise to groin pain and degeneration.3041 acetabular erosion has also been attributed to a fair amount of movement at the outer bearing due to a wide surface of the cup , which was greater than two third of a sphere.4243 reaming in bha technique creates a better fit that reduces movements between the outer cup and the acetabulum41 and ensures that on weight bearing , movements are transmitted to the inner bearing . in effect , this works like a low friction arthroplasty and results in reduced pain and damage to the acetabular bone stock.41 this also explains the low incidence of acetabular migration and low revision rate in our series .", "dudani et al.25 too used the same technique and reported 80% good result at average follow of 7.2 years .", "pellegrini et al.19 have reported a higher risk of revision with acetabular reaming while muraki et al.5 concluded that acetabular reaming increases the tendency of superomedial migration .", "the uniformly good results at the mid term seen in our series may be attributed to the technique and amount ( depth ) of reaming .", "pellegrini et al.19 reamed the acetabulum till a depth when the osseous floor showed punctuate bleeding .", "we have used a more conservative reaming without breaching the integrity of the subchondral bone , and this is probably why we have low incidence and magnitude of medial migration . on the contrary [ figure 2c ]", "the subchondral bone shows sclerosis , asserting the bateman hypothesis regarding the regenerate potential of acetabulum.44 in our series , ten patients showed this sclerosis .", "nagai et al.43 in 12 - 18 years old followup study of nonself centering bateman bipolar endoprosthesis for nontraumatic osteonecrosis of the femoral head concluded that the original bateman endoprosthesis was effective in delaying the need for tha for more than 10 years in ficat stage 3 avn of the femoral head . however , the sample size was very small , with 4 out of 12 patients having some groin pain and study limited to ficat stage iii avn only . in another study tsumura", "et al.45 found no difference in clinical results however incidence of migration in ficat 2 and 3 stage was less when compared with stage 4 . based on this", "they recommended bha only for ficat stages 2 and 3 . in our study , we have used bha for both stages 3 and 4 stages .", "although the final range of motion was better in ficat stage 3 group , the final hhs was similar in both the groups showing effectiveness of bha with tight fitting cup in both ficat 3 and 4 stages .", "thus , even in cases with acetabular involvement this technique can be used with good clinical results and can defer the need for tha .", "bipolar hip arthroplasty using tight fitting cup for avn hip has a low incidence of groin pain , acetabular erosion , and revision in midterm followup .", "this procedure can be used for treatment in young adults with ficat stages 3 and 4 avn of the femoral head to defer a definitive tha .", "further large series with long term followups , multicentric randomized studies and reproducibility of results will be needed to establish this method ."], "abstract_text": ["<S> background : bipolar hip arthroplasty ( bha ) is one of the options for treatment of avascular necrosis ( avn ) of the femoral head . </S>", "<S> acetabular erosion and groin pain are the most allowing for gross motion between the common complications . </S>", "<S> we propose that these complications are secondary to improper acetabular preparation allowing for motion between the bha head and the acetabulum.materials and methods : the current study retrospectively evaluated patientsrecords from case files and also called them for clinical and radiological followup . </S>", "<S> 96 hips with avn of the femoral head treated with bha were included in the study . </S>", "<S> all patients were males with a mean age of 42 years ( range 30 - 59 years ) . in all cases , </S>", "<S> the acetabulum was gently reamed till it became uniformly concentric to achieve tight fitting trial cup . </S>", "<S> clinical followup using harris hip score ( hhs ) and radiological study for cup migration were done at followup.results:the mean followup was 7.52 years ( range 4 - 16 years ) . </S>", "<S> the hhs significantly improved from a preoperative value of 39.3 ( range , 54 - 30 ) to a postoperative value of 89.12 ( range 74 - 96 ) . according to hhs grades , the final outcome was excellent in 52 hips , good in 28 and fair in 16 hips . </S>", "<S> hip and groin pain was reported in four hips ( 5% ) , but did not limit activity . </S>", "<S> subsidence ( less than 5 mm ) of the femoral component was seen in 8 cases . </S>", "<S> subgroup analysis showed patients with ficat stage 3 having better range of motion , but similar hhs as compared to ficat stage 4 patients.conclusion:bipolar hip arthroplasty ( bha ) using tight fitting cup and acetabular reaming in avn hip has a low incidence of groin pain , acetabular erosion and revision in midterm followup . </S>", "<S> good outcome and mid term survival can be achieved irrespective of the ficat stage . </S>"], "labels": null, "section_names": ["I", "M", "Rehabilitation protocol", "Radiological evaluation", "R", "D"], "sections": [["surgical treatment of avascular necrosis ( avn ) of head of femur include core decompression , osteotomies , nonvascularized bone grafting , free vascularized fibular grafts , hip resurfacing , bipolar hip arthroplasty ( bha ) and total hip arthroplasty ( tha).123 tha is indicated in the young individual in avn with acetabular involvement ; however , its role is unclear in cases without acetabular involvement.4 bha was initially limited to be used in hip osteoarthritis , nonunions and acute neck femur fractures.56 bateman7 and giliberty8 were first to use bha in ficat stage 3 avn based on the hypothesis that ", "acetabular floor retains a regenerative property , which regenerates bone in the subchondral region , if stimulation in the form of weight bearing is given through an accurately fitted cup  and theorized that preferential motion at inner bearing will decrease the cartilage erosion .", "papers have been published with both satisfactory91011121314 and unsatisfactory results using bha.5151617 the main limitations sighted are migration of the outer cup ( secondary to the acetabular erosion ) and unpredictable pain relief.3514 incidence of groin pain varied from 11.5% to 42%181920 and incidence of acetabular erosion and protrusio is reported to range from 0% to 45%.1819 the activity of the patient and the duration of followup determine the erosion of acetabulum and appearance of symptoms.21 however , the main issue seems to be motion between the outer cup and the acetabulum , a nonconcentric acetabulum and particulate wear.16222324 to decrease this motion between the outer cup and the acetabulum a gentle reaming of the acetabulum with insertion of tight fitting acetabular cup can be done as suggested above by bateman and giliberty.78 this is thought to decrease the incidence of groin pain and the acetabular erosion and also revision secondary to these issues.2526 in this retrospective series , we have assessed the midterm outcome of bha in young adults ( age < 60 ) with ficat stages 3 and 4 avn ."], ["a retrospective study with a prospective followup was planned . 80 consecutive patients below 60 years of age ( 96 hips ) were operated by the senior author between 1995 and 2010 using bha with tight fitting cup technique .", "all patients were males with a mean age of 42  6.44 years ( range 30 - 59 years ) . out of a total of 96 hips ,", "76 were diagnosed as idiopathic avn , 8 were posttraumatic avn while 12 were post alcohol consumption avn .", "ficat 's classification system was used based on anteroposterior ( ap ) and lateral hip radiograph.26 clinically , the indication for surgery was pain with progressive inability to carry out daily activities .", "patients with radiographs showing no or minimal acetabular involvement were included to undergo the procedure , while cases with advanced osteoarthritis with ostephytosis or protrusio were excluded .", "surgeries were done in the lateral decubitus position using the posterolateral approach under epidural anesthesia .", "the hip joint capsule was excised if diseased . in our study , intraoperatively on gross examination , all the 96 hips ( ficat stage 3 and 4 ) were found to have mild to moderate acetabular involvement as per steinberg et al.2 the cartilage was graded grossly as grade 0 ( normal ) if no abnormalities were observed ; grade i ( mild degeneration ) if there was superficial fibrillation and slight irregularity of the surface ; grade ii ( moderate degeneration ) if there was moderate fibrillation , alteration in color and consistency and thinning of cartilage without complete erosion to bone .", "gentle superficial reaming of the acetabulum was done making the acetabulum concentric for a tight fitting bipolar cup [ figure 1a ] .", "the trial cups should fit snuggly enough so that when the hip is taken through a range of motion , no gross movement should be appreciated between the outer cup and the acetabulum .", "this assures that the prosthesis is tight fitting and movement between the outer cup and the acetabulum is avoided . during the process of making acetabulum concentric , sometimes subchondral bone was exposed . in these cases ,", "pulse lavage of the prepared acetabulum was done to remove all debris before putting the bipolar cup .", "the size of the cup was equal to the size of the tight trial cup chosen .", "we did keep instrumentation for total hip replacement ( thr ) ready intraoperatively ; however , we did not have to do thr in any of our selected cases . ( a ) peroperative photograph showing concentric acetabulum after superficial gentle reaming with the large hand reamer .", "( b and c ) x - ray taken in neutral and abduction showing movement taking place mostly at inner bearing self - centering cup with 28 mm inner head and uncemented stems were used in 60 hips ( zimmer , warsaw , usa , biomet , warsaw , usa ) . in the remaining 36 hips , normocentric hemispherical cups with 26 mm inner head and", "cemented stems were used ( inor , mumbai , india and rch , mumbai , india ) .", "the patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks .", "all patients were called for a final followup for clinical and radiological assessment . the clinical evaluation consisted of recording the details of thigh or groin pain , range of movements at the hip (", "patient was asked to sit cross legged on the bed ) , limb length discrepancy and harris hip score ( hhs ) . the radiological evaluation comprised of looking for subsidence of the stem , recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup .", "ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [ figure 1b and c ] .", "the femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler 's line and center of the outer head , whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs .", "a subgroup analysis was also done , by dividing the cohort according to ficat 's class , to evaluate the effect of acetabular involvement on the outcome of the procedure ."], ["the patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks .", "all patients were called for a final followup for clinical and radiological assessment . the clinical evaluation consisted of recording the details of thigh or groin pain , range of movements at the hip (", "patient was asked to sit cross legged on the bed ) , limb length discrepancy and harris hip score ( hhs ) ."], ["the radiological evaluation comprised of looking for subsidence of the stem , recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup .", "ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [ figure 1b and c ] .", "the femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler 's line and center of the outer head , whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs .", "a subgroup analysis was also done , by dividing the cohort according to ficat 's class , to evaluate the effect of acetabular involvement on the outcome of the procedure ."], ["the mean followup was 7.52  1.80 years ( range , 4 - 16 ) .", "the hhs improved from a preoperative value of 39.33  6.11 ( 30 - 54 ) to 89.12  6.68 ( 74 - 96 ) ( p = 0.0001 , paired t - test ) in the postoperative period . according to hhs grades , the final outcome was excellent in 52 , good in 28 and fair in 16 hips .", "hip and groin pain was reported in four hips ( 5% ) but this did not limit activity in any of the patient [ table 1 ] .", "22 hips had flexion deformity of approximately 3 - 5 with a final range of flexion of 100.2  8.82 ( 85 - 116 ) .", "subsidence of the femoral component was seen in 8 hips ( < 5 mm ) ; however , these hips were asymptomatic . shortening of 1 cm was noted in 10 limbs , and 1.5 cm shortening was noted in 1 limb .", "there was no incidence of hip dislocation or dissociation of the bipolar cup and no case required revision . on ap", "neutral and abduction x - rays there was no appreciable movements noted between the outer cup and the acetabulum in any of the case", ". there was medial migration in 5 hips and proximal migration in 12 hips , but none more than 2 mm and all were asymptomatic .", "comparison of results with various studies on subgroup analysis between the ficat stages 3 and 4 [ table 2 ] it was noted that hips with ficat stage 3 were younger at the time of surgery and also had better final outcome in terms of range of motion .", "the final hhs score was however not statistically different in both groups indicating good results even in cases with acetabular involvement .", "none of the ficat stage 3 patients had any groin pain or femoral subsidence at the final followup while 4 cases of groin pain and 8 cases of femoral subsidence were seen in ficat stage 4 group .", "figures 2 to 5 show case series with final result of four patients in our series .", "comparison between the ficats 3 and 4 subgroups x - ray right hip joint anteroposterior view in a 35 year old male showing ( a ) preoperative radiograph with avascular necrosis right hip secondary to fracture neck femur . also showing implant in situ ( b ) 4 years postoperative radiograph of the same patient treated with bipolar hip arthroplasty .", "( c ) 16 years followup radiograph of the same patient without any loosening or migration and with subchondral sclerosis ( a ) x - ray pelvis with both hip joints anteroposterior view in a 40 year old male showing avascular necrosis ficat stage iii in right hip ( post alcoholic ) ( b ) x - ray of right hip joint anteroposterior view of same patient showing implant in position at 9 years followup without any sign of erosion or migration .", "( c ) clinical photograph of same patient showing cross legged sitting ( a ) x - ray pelvis with both hip joints anteroposterior view in a 65 year old male showing posttraumatic avascular necrosis ficat stage iv ( see non concentric acetabulum ) and implant in situ .", "( b ) x - ray pelvis with both hip joints anteroposterior view of same patient at 9.5 years followup showing well fitting large cup .", "( c and d ) clinical photographs showing range of motion x - ray pelvis with both hip joints anteroposterior view in a 45 year old man with bilateral avascular necrosis of femoral head ( a ) 6 months followup showing right side bipolar arthroplasty left side ficat stage iv avascular necrosis .", "( b ) at 5 years followup showing well fixed implant with large cup well placed in acetabulum"], ["surface replacement is an option in younger people with avn , but has limited indications and is a demanding procedure with high cost.27 the results published by for surface replacement are not uniform and a longterm followup is lacking.272829 tha is the method of choice for the treatment of advanced avn of the femoral head;53031 however , its overuse may lead to increased wear and need for early revision in young adults.14 various studies have shown that functional utility of tha reduces to 80% at 10 years , 33% at 16 years , subsequently requiring a revision surgery .", "alternate bearing surfaces like metal on metal or ceramic on ceramic decrease the wear rate but have their own set of complications ( metallosis , squeaking)3233343536373839 and long term results are still awaited.40 when compared with bha , conflicting results have been reported .", "chan and shih30 have reported that there was no difference in the incidence of osteolysis , thigh or groin pain , dislocation rates and revision rates between bha and tha .", "they concluded that in young patients with ficat stage 3 avn , bha may be a useful alternative to tha .", "furthermore , bha is less demanding , blood loss is comparatively less5 and revision is easier as compared with the revision of tha as the acetabulum is still intact.41 lee et al.3 have reported 23% outer cup migration rate , 15% gluteal pain and 20% groin pain in bha .", "ito et al.15 have reported 42% radiological failures , 42% incidence of groin pain requiring a revision surgery in 25% hip undergoing bha .", "similar results were shown by cabanela14 and lachiewicz and desman17 with groin pain and acetabular erosion as the two most important reasons for poor results in bha .", "groin pain has been variably attributed to the preservation of diseased joint capsule , to irritation of the subchondral nerve endings of the acetabulum and to the acetabular erosion.1442 in our study , the diseased capsule was always excised to avoid capsular impingement .", "furthermore , as nerve endings in the posterior capsule supply the acetabulum , excising it blocks the nerve supply to the acetabulum and thus helps in relieving pain .", "poor fitting bipolar prostheses can lead to cartilage necrosis giving rise to groin pain and degeneration.3041 acetabular erosion has also been attributed to a fair amount of movement at the outer bearing due to a wide surface of the cup , which was greater than two third of a sphere.4243 reaming in bha technique creates a better fit that reduces movements between the outer cup and the acetabulum41 and ensures that on weight bearing , movements are transmitted to the inner bearing . in effect , this works like a low friction arthroplasty and results in reduced pain and damage to the acetabular bone stock.41 this also explains the low incidence of acetabular migration and low revision rate in our series .", "dudani et al.25 too used the same technique and reported 80% good result at average follow of 7.2 years .", "pellegrini et al.19 have reported a higher risk of revision with acetabular reaming while muraki et al.5 concluded that acetabular reaming increases the tendency of superomedial migration .", "the uniformly good results at the mid term seen in our series may be attributed to the technique and amount ( depth ) of reaming .", "pellegrini et al.19 reamed the acetabulum till a depth when the osseous floor showed punctuate bleeding .", "we have used a more conservative reaming without breaching the integrity of the subchondral bone , and this is probably why we have low incidence and magnitude of medial migration . on the contrary [ figure 2c ]", "the subchondral bone shows sclerosis , asserting the bateman hypothesis regarding the regenerate potential of acetabulum.44 in our series , ten patients showed this sclerosis .", "nagai et al.43 in 12 - 18 years old followup study of nonself centering bateman bipolar endoprosthesis for nontraumatic osteonecrosis of the femoral head concluded that the original bateman endoprosthesis was effective in delaying the need for tha for more than 10 years in ficat stage 3 avn of the femoral head . however , the sample size was very small , with 4 out of 12 patients having some groin pain and study limited to ficat stage iii avn only . in another study tsumura", "et al.45 found no difference in clinical results however incidence of migration in ficat 2 and 3 stage was less when compared with stage 4 . based on this", "they recommended bha only for ficat stages 2 and 3 . in our study , we have used bha for both stages 3 and 4 stages .", "although the final range of motion was better in ficat stage 3 group , the final hhs was similar in both the groups showing effectiveness of bha with tight fitting cup in both ficat 3 and 4 stages .", "thus , even in cases with acetabular involvement this technique can be used with good clinical results and can defer the need for tha .", "bipolar hip arthroplasty using tight fitting cup for avn hip has a low incidence of groin pain , acetabular erosion , and revision in midterm followup .", "this procedure can be used for treatment in young adults with ficat stages 3 and 4 avn of the femoral head to defer a definitive tha .", "further large series with long term followups , multicentric randomized studies and reproducibility of results will be needed to establish this method ."]]}
{"article_id": "PMC4071718", "article_text": ["the success of valproic acid ( vpa , 2-propylpentanoic acid ) , a short branched chain fatty acid , for the therapy of absence seizures , partial seizures , tonic - clonic seizures , bipolar disorder , schizoaffective disorder , social phobias , neuropathic pain and for the prophylaxis and treatment of migraine headaches has spurred investigation for its use in the treatment of other conditions .", "treatments for encephalopathy secondary to hyperammonemia as a side - effect of vpa treatment remain empirical and restoration of possible hepatic carnitine deficiency remains controversial .", "while some authors have found a positive correlation between plasma ammonia level and vpa dose or concentration others have found none .", "her elevated vpa levels were associated with elevated ammonia levels as expected of a positive correlation .", "in contrast , later on in the patient 's clinical course her raised venous ammonia levels were found to be slowly down - trending in the presence of sub - therapeutic serum vpa .", "the present case report is about a 25-year - old hispanic female with a history of depression since age of 12 years , bipolar disorder , borderline personality disorder , purging type bulimia and history of suicide attempts at age 12 was admitted to the intensive care unit after attempted suicide by consuming an unknown number of vpa pills .", "this patient had recently started taking vpa ( depakote ) as a mood stabilizer at a dose of 250 mg at bed time about 3 days earlier , after a failed therapy .", "patient was taking paroxetine 20 mg for depression ; this was incremented to 40 mg at the time vpa was started .", "she reported that the change to vpa made her listless ,  lowered  her mood , decreased her motivation , worsened her depression and caused suicidal thoughts .", "she overdoses herself on vpa which she took with alcohol . after ingesting the pills she notified her family who then brought her to the emergency room . on admission ,", "her vitals were stable ( blood pressure : 118/72 mm hg ; temperature : 98.7f ; pulse : 82 beats / min ; oxygen saturation : 99% on room air ) . on initial examination", "she looked disheveled , although drowsy , was oriented to person , place and time .", "the complete blood count and comprehensive metabolic panel that was recorded initially and subsequently were unremarkable [ table 1 ] .", "subsequently , she was administered oral lactulose once and then 1400 mg of levocarnitine orally every 6 h for the next 2 days . her vpa levels which peaked after admission continued to trend down over the next 4 days", ", the ammonia levels also peaked subsequent to admission then showed an initial dip followed by a very slow down - trend [ figure 1a ] .", "the normalized log - linear plot of vpa serum concentration over time showed a down - trending linear first - order kinetics with a calculated t = 15.08 h [ figure 1b ] ( expected t for depakote : 9 - 16 h following oral dosing regimens of 250 - 1000 mg . the serum ammonia normalized log - linear plot in contrast showed two different decay kinetics : initial sharp down - trend for vpa levels greater than 90.9 g / ml but much slower downtrend for vpa levels less than 90.9 g / ml down into the subtherapeutic range of 8.5 g / ml .", "patient continued to complain of lethargy for the next 4 days which subsequently alleviated . at 14 days", "laboratory values of the patient at the time of admission ( a ) valproic acid ( vpa ) and ammonia levels as a function of time ( days ) .", "vertical axes : straight line ( left ) : vpa levels ; dashed line ( right ) : venous ammonia levels .", "( b ) logarithm of normalized vpa and ammonia levels as a function of time in days .", "the normalization for each compound , for levels at each recorded time point ( x ) was to its recorded maximum level ( xmax ) .", "the normalized log - linear plot of vpa showed linear first - order decay kinetics with a calculated t = 15.08 h ( r = 0.994 )", "recognition of symptoms of hyperammonemia remains a challenge especially in psychiatric patients . in a review of 11 case reports of symptomatic hyperammonemia in a psychiatric setting it was noted that acute hyperammonemic encephalopathy in psychiatric patients may present in various ways , sometimes with subtle clinical features .", "the previously reported spectrum of clinical features included : improvement in mood , worsening of psychiatric symptoms , mild complaints of fatigue and delirium . in our patient ,", "hyperammonemia secondary to vpa overdose caused listlessness ,  lowering  of mood , decreased motivation , worsening depression and suicidal ideation .", "we observed presence of vpa in serum caused increased venous ammonia levels , which eventually trended down to normal after its cessation .", "increasing vpa levels in serum was associated with increasing venous ammonia levels initially [ figure 1 ] .", "the peak concentration of ammonia noted was likely a decrement from the real peak based on the time to peak of depakote tablets being 4 h ( the patients initial vpa levels were measured at 2 h after she allegedly consumed the vpa while the next blood draw was 9 h later ) .", "we also made the following observations : ( 1 ) raised venous ammonia levels may remain symptomatic for a significant period of time beyond discontinuation of vpa .", "( 2 ) existence of more than one concurrent mechanism with different decay kinetics in the metabolism of ammonia in presence of high or low concentrations of serum vpa .", "unmasking of an inherent urea cycle metabolic disorder could have amplified the differential decay rates of venous ammonia .", "the possibility of more than one mechanism causing hyperammonia secondary to vpa use is suggested by the different proposed mechanisms of vpa metabolites causing hyperammonemia , these include : ( 1 ) propionic acid inhibiting the urea cycle enzyme carbamoyl phosphate synthetase i ; ( 2 ) valproyl - coa or a closely related compound as a proximate inhibitor of mitochondrial ureagenesis ; ( 3 ) valproyl - carnitine ester causing a relative carnitine deficiency and ( 4 ) 2,4-dien - vpa inhibiting b - oxidation .", "although , we can not speculate on the mechanisms responsible for the different kinetics of decay of ammonia levels in the presence of down - trending vpa levels we can confidently state that non - elevated hepatic enzymes and hyperammonemia despite carnitine replacement indicated that hepatic damage and carnitine deficiency had very little role in causing the slowly down - trending elevated ammonia levels in the presence of subtherapeutic serum vpa levels .", "hyperammonemia and psychiatric disturbance secondary to valproate use in our case showed a score of 9 by a naranjo algorithm , indicating strong causality with the drug and placing it in causality category of  definite . ", "our assessment of a high adverse reaction probability scale was based on the fact that there are previous conclusive reports of hyperammonemia secondary to vpa use with similar psychiatric features as presented in this case report , hyperammonemia occurred only after vpa use and eventually trended down to normal levels with discontinuation of vpa , patients psychiatric disturbance correlated with hyperammonemia , the patient symptomatically improved with drug discontinuation , there are no alternative explanation of hyperammonemia and altered mood in this case , the serum levels of vpa were elevated with concentrations known to be toxic and that toxic effects observed were more severe with high dose and less severe when secondary hyperammonemia decreased .", "this case report suggests that hyperammonemia , which is known to occur in about 50% patients treated with vpa , may have more than one concurrent etiologic mechanism with different decay kinetics which are not related to hepatic damage or carnitine deficiency ."], "abstract_text": ["<S> valproic acid ( vpa ) has successfully been used in the therapy of a number of conditions including absence seizures , partial seizures , tonic - clonic seizures , bipolar disorder , schizoaffective disorder , social phobias , neuropathic pain and migraine headaches . </S>", "<S> there is a high rise in number of cases of toxicity due to overdose of vpa . </S>", "<S> hyperammonemia , a common side - effect of vpa , is caused by several proposed etiologies , reported as having uncertain correlation with vpa dose or concentration . </S>", "<S> we present here a case of a 25-year - old female patient with a past history of psychiatric complaints , presented with elevated serum vpa levels associated with elevated venous ammonia levels subsequent to vpa overdose . later in the presence of sub - therapeutic serum vpa levels </S>", "<S> her venous ammonia levels remained raised and slowly down - trending . </S>", "<S> vpa levels and ammonia levels were found to be normal after 14 days . </S>", "<S> patient was treated with levocarnitine . </S>", "<S> her liver enzymes were never elevated . </S>", "<S> different decay kinetics of venous ammonia in presence of high and low concentrations of vpa indicates that vpa can cause symptomatic hyperammonemia via more than one concurrent etiological mechanism . in this patient , </S>", "<S> the mechanisms causing hyperammonia secondary to vpa use were not related to hepatic damage or carnitine deficiency . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["the success of valproic acid ( vpa , 2-propylpentanoic acid ) , a short branched chain fatty acid , for the therapy of absence seizures , partial seizures , tonic - clonic seizures , bipolar disorder , schizoaffective disorder , social phobias , neuropathic pain and for the prophylaxis and treatment of migraine headaches has spurred investigation for its use in the treatment of other conditions .", "treatments for encephalopathy secondary to hyperammonemia as a side - effect of vpa treatment remain empirical and restoration of possible hepatic carnitine deficiency remains controversial .", "while some authors have found a positive correlation between plasma ammonia level and vpa dose or concentration others have found none .", "her elevated vpa levels were associated with elevated ammonia levels as expected of a positive correlation .", "in contrast , later on in the patient 's clinical course her raised venous ammonia levels were found to be slowly down - trending in the presence of sub - therapeutic serum vpa ."], ["the present case report is about a 25-year - old hispanic female with a history of depression since age of 12 years , bipolar disorder , borderline personality disorder , purging type bulimia and history of suicide attempts at age 12 was admitted to the intensive care unit after attempted suicide by consuming an unknown number of vpa pills .", "this patient had recently started taking vpa ( depakote ) as a mood stabilizer at a dose of 250 mg at bed time about 3 days earlier , after a failed therapy .", "patient was taking paroxetine 20 mg for depression ; this was incremented to 40 mg at the time vpa was started .", "she reported that the change to vpa made her listless ,  lowered  her mood , decreased her motivation , worsened her depression and caused suicidal thoughts .", "she overdoses herself on vpa which she took with alcohol . after ingesting the pills she notified her family who then brought her to the emergency room . on admission ,", "her vitals were stable ( blood pressure : 118/72 mm hg ; temperature : 98.7f ; pulse : 82 beats / min ; oxygen saturation : 99% on room air ) . on initial examination", "she looked disheveled , although drowsy , was oriented to person , place and time .", "the complete blood count and comprehensive metabolic panel that was recorded initially and subsequently were unremarkable [ table 1 ] .", "subsequently , she was administered oral lactulose once and then 1400 mg of levocarnitine orally every 6 h for the next 2 days . her vpa levels which peaked after admission continued to trend down over the next 4 days", ", the ammonia levels also peaked subsequent to admission then showed an initial dip followed by a very slow down - trend [ figure 1a ] .", "the normalized log - linear plot of vpa serum concentration over time showed a down - trending linear first - order kinetics with a calculated t = 15.08 h [ figure 1b ] ( expected t for depakote : 9 - 16 h following oral dosing regimens of 250 - 1000 mg . the serum ammonia normalized log - linear plot in contrast showed two different decay kinetics : initial sharp down - trend for vpa levels greater than 90.9 g / ml but much slower downtrend for vpa levels less than 90.9 g / ml down into the subtherapeutic range of 8.5 g / ml .", "patient continued to complain of lethargy for the next 4 days which subsequently alleviated . at 14 days", "laboratory values of the patient at the time of admission ( a ) valproic acid ( vpa ) and ammonia levels as a function of time ( days ) .", "vertical axes : straight line ( left ) : vpa levels ; dashed line ( right ) : venous ammonia levels .", "( b ) logarithm of normalized vpa and ammonia levels as a function of time in days .", "the normalization for each compound , for levels at each recorded time point ( x ) was to its recorded maximum level ( xmax ) .", "the normalized log - linear plot of vpa showed linear first - order decay kinetics with a calculated t = 15.08 h ( r = 0.994 )"], ["recognition of symptoms of hyperammonemia remains a challenge especially in psychiatric patients . in a review of 11 case reports of symptomatic hyperammonemia in a psychiatric setting it was noted that acute hyperammonemic encephalopathy in psychiatric patients may present in various ways , sometimes with subtle clinical features .", "the previously reported spectrum of clinical features included : improvement in mood , worsening of psychiatric symptoms , mild complaints of fatigue and delirium . in our patient ,", "hyperammonemia secondary to vpa overdose caused listlessness ,  lowering  of mood , decreased motivation , worsening depression and suicidal ideation .", "we observed presence of vpa in serum caused increased venous ammonia levels , which eventually trended down to normal after its cessation .", "increasing vpa levels in serum was associated with increasing venous ammonia levels initially [ figure 1 ] .", "the peak concentration of ammonia noted was likely a decrement from the real peak based on the time to peak of depakote tablets being 4 h ( the patients initial vpa levels were measured at 2 h after she allegedly consumed the vpa while the next blood draw was 9 h later ) .", "we also made the following observations : ( 1 ) raised venous ammonia levels may remain symptomatic for a significant period of time beyond discontinuation of vpa .", "( 2 ) existence of more than one concurrent mechanism with different decay kinetics in the metabolism of ammonia in presence of high or low concentrations of serum vpa .", "unmasking of an inherent urea cycle metabolic disorder could have amplified the differential decay rates of venous ammonia .", "the possibility of more than one mechanism causing hyperammonia secondary to vpa use is suggested by the different proposed mechanisms of vpa metabolites causing hyperammonemia , these include : ( 1 ) propionic acid inhibiting the urea cycle enzyme carbamoyl phosphate synthetase i ; ( 2 ) valproyl - coa or a closely related compound as a proximate inhibitor of mitochondrial ureagenesis ; ( 3 ) valproyl - carnitine ester causing a relative carnitine deficiency and ( 4 ) 2,4-dien - vpa inhibiting b - oxidation .", "although , we can not speculate on the mechanisms responsible for the different kinetics of decay of ammonia levels in the presence of down - trending vpa levels we can confidently state that non - elevated hepatic enzymes and hyperammonemia despite carnitine replacement indicated that hepatic damage and carnitine deficiency had very little role in causing the slowly down - trending elevated ammonia levels in the presence of subtherapeutic serum vpa levels .", "hyperammonemia and psychiatric disturbance secondary to valproate use in our case showed a score of 9 by a naranjo algorithm , indicating strong causality with the drug and placing it in causality category of  definite . ", "our assessment of a high adverse reaction probability scale was based on the fact that there are previous conclusive reports of hyperammonemia secondary to vpa use with similar psychiatric features as presented in this case report , hyperammonemia occurred only after vpa use and eventually trended down to normal levels with discontinuation of vpa , patients psychiatric disturbance correlated with hyperammonemia , the patient symptomatically improved with drug discontinuation , there are no alternative explanation of hyperammonemia and altered mood in this case , the serum levels of vpa were elevated with concentrations known to be toxic and that toxic effects observed were more severe with high dose and less severe when secondary hyperammonemia decreased .", "this case report suggests that hyperammonemia , which is known to occur in about 50% patients treated with vpa , may have more than one concurrent etiologic mechanism with different decay kinetics which are not related to hepatic damage or carnitine deficiency ."]]}
{"article_id": "PMC3339349", "article_text": ["intramammary infection ( mastitis ) is the most common reason for the use of antimicrobials in dairy cows .", "antimicrobials have been used to treat mastitis for more than fifty years , but consensus about the most efficient , safe , and economical treatment is still lacking .", "the concept of evidence - based medicine has been introduced to veterinary medicine   and should apply also to treatment of mastitis . the impact on public health", "the aim of this article is to review current treatments of mastitis during lactation and seek for evidence - based , best practice treatment recommendations for bovine mastitis .", "penetration of substances into milk when administered parenterally or absorption and distribution throughout the udder when infused intramammarily ( imm ) depends on their pharmacokinetic characteristics .", "these are lipid solubility , degree of ionization , extent of binding to serum and udder proteins , and the type of vehicle .", "antimicrobial treatment of dairy cows creates residues into milk , and residue avoidance is an important aspect of mastitis treatment .", "the activity of macrolides , tetracyclines and trimethoprim - sulphonamides has been shown to be reduced in milk . selecting a substance with a low minimum inhibitory concentraton ( mic ) value for the target pathogen", "the antimicrobial should have bactericidal rather than bacteriostatic action , because phagocytosis is impaired in the mammary gland .", "however , activity in vitro does not guarantee efficacy in vivo when treating bovine mastitis .", "antimicrobial resistance amongst mastitis pathogens has not yet emerged as a clinically relevant issue , but geographical regions may differ in this respect .", "the biggest problem is the widespread resistance of staphylococci , particularly staphylococcus aureus , to penicillin g .", "cure rates for mastitis caused by penicillin - resistant strains of s. aureus seem to be inferior to those of mastitis due to penicillin - susceptible strains .", "it is not known if this is due to pharmacologic problems of the drugs used , or virulence factors possibly linked to -lactamase gene of the resistant isolates . using an in vitro -lactamase test for determining resistance to penicillin g of staphylococci before treatment", "coagulase - negative staphylococci tend to be more resistant than s. aureus and easily develop multiresistance .", "mastitis causing streptococci have remained susceptible to penicillin g , but emerging resistance to macrolides and lincosamides has been detected .", "antimicrobial susceptibility of coliform bacteria varies but normally is not a limiting factor for therapy .", "an important question regarding the treatment of mastitis is whether the antimicrobial should accumulate in the milk or in the udder tissue .", "the target site may depend on the causative agent : streptococci are known to remain in the milk compartment , but s. aureus penetrates udder tissue and causes deep infection ( table 1 ) .", "the advantages of this route are high concentrations of the substance achieved in the milk and low consumption of the antimicrobial as the drug is directly infused into the diseased quarter .", "for example , concentration of penicillin g in milk after imm administration is 100 - 1000 times as high as the concentration after systemic ( parenteral ) administration .", "a disadvantage of the imm administration is uneven distribution throughout the udder   and the risk of infecting the quarter when infusing the product via the teat canal .", "efficacy of imm treatment varies according to the causative pathogen , with the best therapeutic response being shown for mastitis caused by streptococci , coagulase - negative staphylococci , and corynebacterium spp .. where to target antimicrobial therapy in clinical mastitis due to different pathogens   the systemic route of administration has been suggested to be more efficient than imm for the treatment of clinical mastitis as antimicrobials theoretically have better penetration of the udder tissue by this route .", "however , it is difficult to attain and maintain therapeutic concentrations in milk or udder tissue following systemic administration .", "many commonly used broad - spectrum antimicrobials such as oxytetracycline , trimethoprim - sulphonamide and ceftiofur , it is difficult to produce and maintain therapeutic concentrations in the milk .", "macrolides would have ideal pharmacokinetics , but clinical studies have failed to demonstrate efficacy when used for the systemic treatment of clinical mastitis . in streptococcal mastitis , spiramycin and tylosin have shown reasonable efficacy .", "one additional problem for the bovine practitioner is that the recommended dosage for many antibiotic preparations for adult cattle may be too low when pharmacological aspects are considered , but residue studies have been carried out using the approved dosages .", "repeated intramuscular injections of large volumes of antibiotics can be irritating and can not be recommended from the animal welfare point of view .", "one substance used for systemic treatment is penicillin g , which as a weak acid penetrates poorly into the mammary gland , however , due to the very low mic values of susceptible organisms , therapeutic concentrations can be achieved in milk .", "penethamate is a more liphophilic penicillin g formulation and diffuses better than penicillin g procaine into milk .", "the efficacy of systemic treatment with penicillin g or penethamate has been shown in clinical trials .", "combinations of penicillin and aminoglycosides should not be used , as there is no scientific evidence demonstrating a better efficacy for the combination   and aminoglycosides are known to produce long - lasting residues .", "the only type of mastitis where systemic treatment would be clearly advantageous may be mastitis caused by s. aureus .", "in severe mastitis due to coliform bacteria , parenteral administration of antimicrobials has been suggested to combat bacteraemia .", "the general benefit of antimicrobial treatment in coliform mastitis has been questioned , but systemic antimicrobial treatment is recommended in cases of severe escherichia coli mastitis with heavy bacterial growth in the udder .", "fluroquinolones and cefquinome have shown efficacy in experimental trials   and ceftiofur in a clinical field trial .", "there is no evidence that administering bactericidal antimicrobials to cows with severe coliform mastitis causes the release of massive amounts of endotoxin .", "finally , the antimicrobial used for systemic treatment of mastitis must be approved for dairy cattle .", "for example , penicillin g procaine or fluoroquinolones are not approved for dairy cattle in the united states .", "treatment of mastitis should be targeted towards the causative bacteria whenever possible , but in acute situations , treatment is initiated based on herd data and personal experience . rapid or on - farm bacteriological diagnosis would facilitate the selection of the most appropriate antimicrobial .", "treatment protocols and drug selection for each farm should be made by veterinarians familiar with the farm .", "the use of on - farm written protocols for mastitis treatment can promote judicious use of antimicrobials . therapeutic response of the cows can be monitored using individual somatic cell count data if available , or using the california mastitis test , and with bacteriological samples in herds with contagious mastitis . in general , the use of narrow - spectrum antimicrobials is preferable ( table 2 ) .", "first choice antimicrobials for treating mastitis caused by streptococci and penicillin - susceptible staphylococci are -lactam antimicrobials , particularly penicillin g. broad - spectrum antimicrobials such as third or fourth generation cephalosporins should not be used as first alternatives for mastitis , as they may increase emergence of broad - spectrum -lactam resistance .", "systemic treatment is recommended in clinical mastitis due to s. aureus and in severe cases of coliform mastitis , preferably in combination with imm treatment . too short", "a duration of standard treatment is probably an important reason for poor cure rates in mastitis therapy .", "a longer treatment improves cure rates , and duration of treatment should generally be extended in mastitis caused by s. aureus and streptococcus uberis .", "clinical mastitis should be treated for at least three days ; this recommended treatment duration is longer than label treatments in many countries .", "all mastitis treatment should be evidence based i.e. , the efficacy of each product and treatment length should be demonstrated by scientific studies .", "treating subclinical mastitis with antimicrobials is generally not economical during lactation because of high treatment costs and poor efficacy . in a study with a large number of subclinical mastitis cases , the overall bacteriological cure rate for antimicrobial treatment was 75% and that for no treatment 68% . the marginal benefit applied for streptococcal mastitis only ; in mastitis due to s. aureus , antimicrobials were equal to no treatment .", "treatment of subclinical mastitis will not affect the incidence of mastitis in the herd unless other preventive measures are taken .", "studies on treating cows based on high somatic cell counts have generally shown that no effect on milk production has been achieved   in herd problems caused by very contagious bacteria such as s. aureus or streptococcus agalactiae treatment of subclinical mastitis is advised ."], "abstract_text": ["<S> treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account . in acute mastitis , where bacteriological diagnosis is not available , treatment should be initiated based on herd data and personal experience . </S>", "<S> rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial . </S>", "<S> treating subclinical mastitis with antimicrobials during lactation is seldom economical , because of high treatment costs and generally poor efficacy . </S>", "<S> all mastitis treatment should be evidence - based , i.e. , the efficacy of each product and treatment length should be demonstrated by scientific studies . </S>", "<S> use of on - farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials . </S>"], "labels": null, "section_names": ["Introduction", "Pharmacokinetic and Pharmacodynamic Considerations", "Intramammary or Systemic Administration?", "Treatment of Clinical Mastitis in Practice", "Subclinical Mastitis"], "sections": [["intramammary infection ( mastitis ) is the most common reason for the use of antimicrobials in dairy cows .", "antimicrobials have been used to treat mastitis for more than fifty years , but consensus about the most efficient , safe , and economical treatment is still lacking .", "the concept of evidence - based medicine has been introduced to veterinary medicine   and should apply also to treatment of mastitis . the impact on public health", "the aim of this article is to review current treatments of mastitis during lactation and seek for evidence - based , best practice treatment recommendations for bovine mastitis ."], ["penetration of substances into milk when administered parenterally or absorption and distribution throughout the udder when infused intramammarily ( imm ) depends on their pharmacokinetic characteristics .", "these are lipid solubility , degree of ionization , extent of binding to serum and udder proteins , and the type of vehicle .", "antimicrobial treatment of dairy cows creates residues into milk , and residue avoidance is an important aspect of mastitis treatment .", "the activity of macrolides , tetracyclines and trimethoprim - sulphonamides has been shown to be reduced in milk . selecting a substance with a low minimum inhibitory concentraton ( mic ) value for the target pathogen", "the antimicrobial should have bactericidal rather than bacteriostatic action , because phagocytosis is impaired in the mammary gland .", "however , activity in vitro does not guarantee efficacy in vivo when treating bovine mastitis .", "antimicrobial resistance amongst mastitis pathogens has not yet emerged as a clinically relevant issue , but geographical regions may differ in this respect .", "the biggest problem is the widespread resistance of staphylococci , particularly staphylococcus aureus , to penicillin g .", "cure rates for mastitis caused by penicillin - resistant strains of s. aureus seem to be inferior to those of mastitis due to penicillin - susceptible strains .", "it is not known if this is due to pharmacologic problems of the drugs used , or virulence factors possibly linked to -lactamase gene of the resistant isolates . using an in vitro -lactamase test for determining resistance to penicillin g of staphylococci before treatment", "coagulase - negative staphylococci tend to be more resistant than s. aureus and easily develop multiresistance .", "mastitis causing streptococci have remained susceptible to penicillin g , but emerging resistance to macrolides and lincosamides has been detected .", "antimicrobial susceptibility of coliform bacteria varies but normally is not a limiting factor for therapy ."], ["an important question regarding the treatment of mastitis is whether the antimicrobial should accumulate in the milk or in the udder tissue .", "the target site may depend on the causative agent : streptococci are known to remain in the milk compartment , but s. aureus penetrates udder tissue and causes deep infection ( table 1 ) .", "the advantages of this route are high concentrations of the substance achieved in the milk and low consumption of the antimicrobial as the drug is directly infused into the diseased quarter .", "for example , concentration of penicillin g in milk after imm administration is 100 - 1000 times as high as the concentration after systemic ( parenteral ) administration .", "a disadvantage of the imm administration is uneven distribution throughout the udder   and the risk of infecting the quarter when infusing the product via the teat canal .", "efficacy of imm treatment varies according to the causative pathogen , with the best therapeutic response being shown for mastitis caused by streptococci , coagulase - negative staphylococci , and corynebacterium spp .. where to target antimicrobial therapy in clinical mastitis due to different pathogens   the systemic route of administration has been suggested to be more efficient than imm for the treatment of clinical mastitis as antimicrobials theoretically have better penetration of the udder tissue by this route .", "however , it is difficult to attain and maintain therapeutic concentrations in milk or udder tissue following systemic administration .", "many commonly used broad - spectrum antimicrobials such as oxytetracycline , trimethoprim - sulphonamide and ceftiofur , it is difficult to produce and maintain therapeutic concentrations in the milk .", "macrolides would have ideal pharmacokinetics , but clinical studies have failed to demonstrate efficacy when used for the systemic treatment of clinical mastitis . in streptococcal mastitis , spiramycin and tylosin have shown reasonable efficacy .", "one additional problem for the bovine practitioner is that the recommended dosage for many antibiotic preparations for adult cattle may be too low when pharmacological aspects are considered , but residue studies have been carried out using the approved dosages .", "repeated intramuscular injections of large volumes of antibiotics can be irritating and can not be recommended from the animal welfare point of view .", "one substance used for systemic treatment is penicillin g , which as a weak acid penetrates poorly into the mammary gland , however , due to the very low mic values of susceptible organisms , therapeutic concentrations can be achieved in milk .", "penethamate is a more liphophilic penicillin g formulation and diffuses better than penicillin g procaine into milk .", "the efficacy of systemic treatment with penicillin g or penethamate has been shown in clinical trials .", "combinations of penicillin and aminoglycosides should not be used , as there is no scientific evidence demonstrating a better efficacy for the combination   and aminoglycosides are known to produce long - lasting residues .", "the only type of mastitis where systemic treatment would be clearly advantageous may be mastitis caused by s. aureus .", "in severe mastitis due to coliform bacteria , parenteral administration of antimicrobials has been suggested to combat bacteraemia .", "the general benefit of antimicrobial treatment in coliform mastitis has been questioned , but systemic antimicrobial treatment is recommended in cases of severe escherichia coli mastitis with heavy bacterial growth in the udder .", "fluroquinolones and cefquinome have shown efficacy in experimental trials   and ceftiofur in a clinical field trial .", "there is no evidence that administering bactericidal antimicrobials to cows with severe coliform mastitis causes the release of massive amounts of endotoxin .", "finally , the antimicrobial used for systemic treatment of mastitis must be approved for dairy cattle .", "for example , penicillin g procaine or fluoroquinolones are not approved for dairy cattle in the united states ."], ["treatment of mastitis should be targeted towards the causative bacteria whenever possible , but in acute situations , treatment is initiated based on herd data and personal experience . rapid or on - farm bacteriological diagnosis would facilitate the selection of the most appropriate antimicrobial .", "treatment protocols and drug selection for each farm should be made by veterinarians familiar with the farm .", "the use of on - farm written protocols for mastitis treatment can promote judicious use of antimicrobials . therapeutic response of the cows can be monitored using individual somatic cell count data if available , or using the california mastitis test , and with bacteriological samples in herds with contagious mastitis . in general , the use of narrow - spectrum antimicrobials is preferable ( table 2 ) .", "first choice antimicrobials for treating mastitis caused by streptococci and penicillin - susceptible staphylococci are -lactam antimicrobials , particularly penicillin g. broad - spectrum antimicrobials such as third or fourth generation cephalosporins should not be used as first alternatives for mastitis , as they may increase emergence of broad - spectrum -lactam resistance .", "systemic treatment is recommended in clinical mastitis due to s. aureus and in severe cases of coliform mastitis , preferably in combination with imm treatment . too short", "a duration of standard treatment is probably an important reason for poor cure rates in mastitis therapy .", "a longer treatment improves cure rates , and duration of treatment should generally be extended in mastitis caused by s. aureus and streptococcus uberis .", "clinical mastitis should be treated for at least three days ; this recommended treatment duration is longer than label treatments in many countries .", "all mastitis treatment should be evidence based i.e. , the efficacy of each product and treatment length should be demonstrated by scientific studies ."], ["treating subclinical mastitis with antimicrobials is generally not economical during lactation because of high treatment costs and poor efficacy . in a study with a large number of subclinical mastitis cases , the overall bacteriological cure rate for antimicrobial treatment was 75% and that for no treatment 68% . the marginal benefit applied for streptococcal mastitis only ; in mastitis due to s. aureus , antimicrobials were equal to no treatment .", "treatment of subclinical mastitis will not affect the incidence of mastitis in the herd unless other preventive measures are taken .", "studies on treating cows based on high somatic cell counts have generally shown that no effect on milk production has been achieved   in herd problems caused by very contagious bacteria such as s. aureus or streptococcus agalactiae treatment of subclinical mastitis is advised ."]]}
{"article_id": "PMC3352396", "article_text": ["approximately 40 million people are infected with the human immunodeficiency virus ( hiv ) worldwide ( unaids 2006 ) .", "infections by this lentivirus from the family retroviridae are characterized by a long asymptomatic period after host immune defenses control the initial infection . during this subsequent chronic phase of infection , hiv slowly diminishes the host", "the virus replicates by hijacking the intracellular molecular machinery to transcribe viral rna , and eventually the productively infected cells die ( fauci 1988 ) . over time , the density of virions in the blood stream increases and the immune system functioning becomes progressively compromised , leaving hiv - infected individuals increasingly susceptible to opportunistic infections .", "being blood - borne , hiv is transmitted via contact with the blood of an infected individual : through transfusions , needle - sharing , sexual contact or from mother to child during childbirth or breast - feeding .", "initially there were concerns that hiv might be vector - borne ( e.g. , transmitted via mosquitoes ) but it has since become widely accepted that such transmission does not occur at any significant level ( bockarie and paru 1996 ) .", "this current belief stems both from epidemiological data and experimental studies that directly examine the potential for hiv transmission via arthropods ( lawrence 1987 ; lifson 1988 ; bockarie and paru 1996 )", ". why is hiv not vector - borne ( throughout this article we use the term  vector synonymously with ", "arthropod ) ? the majority of medical scientists , when asked this question , will offer the following explanations ( bockarie and paru 1996 ) : ( i ) hiv concentrations in the blood are too low during human infection to permit vector transmission ; ( ii ) hiv is unable to survive long enough outside of humans ( or primates ) for vector transmission ; ( iii ) hiv is not able to replicate within arthropod vectors .", "each of these explanations has empirical support ( lifson 1988 ; bockarie and paru 1996 ) and thus all three are good explanations for the lack of vector transmission in hiv .", "if most arthropods pick up very little hiv when feeding on humans , and if the level of hiv in ( or on ) these vectors quickly decays , then no significant vector transmission is expected to occur .", "the above findings provide a satisfying proximate explanation for the lack of vector transmission in hiv .", "given the current characteristics of hiv , these three features of the virus make it unlikely that vector transmission will occur . in this article , we approach this question from an evolutionary perspective . therefore , rather than taking the characteristics of hiv as given , we are interested in understanding why hiv has evolved these particular features and not others instead . for example :", "( i ) why has hiv not evolved a replication strategy that results in viral concentrations high enough to allow vector transmission ? ( ii ) why has hiv not evolved to be more durable and thus to survive longer outside of humans ?", "( iii ) why has hiv not evolved the ability to replicate in arthropod vectors ?", "asking such questions might strike many readers as strange since much of evolutionary biology proceeds by asking why certain features of organisms are as we observe them rather than by asking why some features are absent .", "this is only natural , since it is difficult to decide which , among the infinite number of  missing features , should be the focus of study .", "it is useful to view such questions as falling on a continuum , from questions about the lack of traits that are virtually non - existent in all taxa ( e.g. , why do dogs not have wheeled appendages ) to questions about the lack of traits that are common in some populations or species but not others ( e.g. , why do german shepherds not have curly hair ? ) .", "our contention is that , by asking such questions about the absence of some traits , we can gain deeper insight into biology . to quote philosopher arthur eddington ( 1927 ) ,   the contemplation in natural science of a wider domain than the actual leads to a far better understanding of the actual .  . as in all science , however , not all questions are interesting , and this serves as our primary guide for focusing on some missing features and not others . in particular , we focus on the lack of vector transmission in hiv , because of the profound epidemiological significance of its absence .", "an arthropod - transmissible form of hiv would clearly exacerbate the already devastating impact of the disease , opening up routes of transmission to groups previously at low - risk ( e.g. , children ) .", "therefore , it is worth asking why , from an evolutionary standpoint , this has not occurred ( weiss 2001 ) .", "more to the point , if there are conditions under which hiv could have evolved vector transmission we would do well to understand these , not only from the standpoint of scientific curiosity , but also to prevent such an outcome in the future . in this article , we address the question of why hiv lacks vector transmission , both through a consideration of available empirical data and through the construction of mathematical models . although our results are necessarily speculative , we believe that they shed some light on the evolutionary biology of hiv , and on the evolutionary biology of blood - borne pathogens more generally .", "there are two broad reasons why a trait of interest ( in this case vector - transmission ) might not evolve .", "first , the necessary genetic variation for the trait might arise only very rarely ( if at all ) .", "for instance , the evolution of rna viruses , such as hiv , could be strongly constrained by the size of their genome ( holmes 2003 ) .", "second , the necessary form and strength of natural selection might not be present for the trait to evolve , at least over the timescale under consideration .", "thus , from an evolutionary standpoint , hiv is not vector - borne because either the necessary genetic variation for such transmission has never arisen , or the necessary selective factors that would make such variants increase in frequency over the relevant timescale have not occurred .", "our evolutionary explanation for why hiv is not vector - borne will be sought within these two possibilities . before beginning to consider these explanations", "there are two different processes that might result in vector - transmission , and that are often lumped under this single heading .", "this occurs when the arthropod acts solely as a means of physical transport of viral particles between hosts ( e.g. , having viral particles in and around mouthparts ) .", "this occurs when the virus replicates within the arthropod vector during the time period between feeding events .", "how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ? the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population .", "unfortunately , performing such an assay on all genotypes within the population would be next to impossible .", "furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low . nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue", "if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened .", "below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission :", "a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods .", "such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) .", "for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al .", "1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus .", "most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al .", "this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al . 1993 ) .", "it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) .", "other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods .", "in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al .", "at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al .", "the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al .", "1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) .", "interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) .", "it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al .", "even if this does occur , however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus . biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible .", "( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors .", "evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al .", "1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al .", "furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al .", "in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al .", "1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) .", "it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al .", "alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses .", "the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al .", "there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al .", "1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous .", "thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ?", "on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle .", "this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) .", "alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section", "we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv .", "mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) .", "the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate .", "although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans .", "the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics .", "this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 ) , v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and  is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) .", "note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this . also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) .", "the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people .", "we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) .", "the parameter  is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age .", "our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al . 2007 ) .", "results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption .", "lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age .", "in particular , we will suppose that (a ) has the form where  is the time during the infection at which aids develops .", "this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv . in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia . similarly , evidence suggests that sexual transmission rate ,  , also increases with viremia ( operskalski et al . 1997", "lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of  in untreated patients ( mellors et al .", "with these specifications , our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms .", "suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1  0 ) .", "also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission .", "assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively .", "equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate .", "more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold .", "empirical evidence in support of this functional form for hiv exists ( quinn et al .", ", a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large  ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig .", "1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore ,", "some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission .", "conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig .", "1 ) . sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) .", "b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text .", "parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 ,  = 15 .", "these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs .", "for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans .", "the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1  p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig .", "the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) .", "solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years .", "the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b .", "first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed ", ", we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years .", "also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act .", "more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred .", "unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al .", "1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved . equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv .", "biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however", ", biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve .", "thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost .", "there are at least two biologically plausible mechanisms through which such a cost might arise .", "first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case", ", the cost stems from an evolutionary trade - off between these two transmission routes .", "the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8) \n could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain .", "there is , however , no a priori reason why this would be expected rather than the reverse .", "therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well .", "the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host .", "it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation .", "if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread .", "modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host .", "this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission .", "the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host .", "the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection .", "specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation .", "when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting  be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) .", "the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes .", "first , if rb  ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand , if rb  ra < ba , then vector transmission will never evolve .", "in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve .", "this will be true whenever the mutation rate of the virus is high ( i.e. , large  ) and when the selective advantage of sexual transmission in terms of within - host competition is large .", "the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission .", "how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ?", "the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population .", "unfortunately , performing such an assay on all genotypes within the population would be next to impossible .", "furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low .", "nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue", "if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened .", "below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission :", "a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods .", "such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) .", "for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al .", "1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus .", "most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al .", "this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al .", "it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) .", "other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods .", "in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al .", "at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al .", "the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al .", "1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) .", "interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) .", "it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al .", "1983 ) , although this has been controversial ( kuno 2004 ) . even if this does occur", ", however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus .", "biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible .", "( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors .", "evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al .", "1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al .", "furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al .", "in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al .", "1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) .", "it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al .", "alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses .", "the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al .", "there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al .", "1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous .", "thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ?", "on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle .", "this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) .", "alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section", "we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv .", "mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at", "present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) .", "the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate .", "although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans .", "the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics .", "this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 )", ", v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and  is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) .", "note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this .", "also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) .", "the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people .", "we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) .", "the parameter  is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age .", "our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al .", "results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption .", "lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age . in particular", ", we will suppose that (a ) has the form where  is the time during the infection at which aids develops .", "this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv .", "in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia .", "similarly , evidence suggests that sexual transmission rate ,  , also increases with viremia ( operskalski et al .", "lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of  in untreated patients ( mellors et al . 1997 ; levy 1998 ; raffanti et al .", ", our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms .", "suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1  0 ) .", "also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission .", "assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively", ". equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate .", "more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold .", "empirical evidence in support of this functional form for hiv exists ( quinn et al .", ", a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large  ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig .", "1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore , some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission .", "conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig .", "sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) .", "b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text .", "parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 ,  = 15 .", "these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs . for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans . the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1  p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig .", "the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) .", "solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years .", "the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b .", "first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed ", ", we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years .", "also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act .", "more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred .", "unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al .", "1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved .", "equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv .", "biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however", ", biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve .", "thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost .", "there are at least two biologically plausible mechanisms through which such a cost might arise .", "first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case", ", the cost stems from an evolutionary trade - off between these two transmission routes .", "the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8) \n could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain .", "there is , however , no a priori reason why this would be expected rather than the reverse .", "therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well .", "the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host .", "it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation .", "if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread .", "modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host .", "this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission .", "the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host .", "the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection .", "specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation .", "when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting  be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) . the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes .", "first , if rb  ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand ,", "in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve .", "this will be true whenever the mutation rate of the virus is high ( i.e. , large  ) and when the selective advantage of sexual transmission in terms of within - host competition is large .", "the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission .", "experiments and epidemiological data have unequivocally demonstrated , however , that such vector transmission does not occur at any significant level , and various aspects of hiv biology have been implicated as proximate reasons ( bockarie and paru 1996 ) .", "these reasons do not offer an explanation for why vector transmission has not evolved , however , and as weiss ( 2001 ) points out , we ought to seriously consider whether such evolution might occur in the future ( for a summary , see table 1 ) .", "main conclusions of the study existing data suggest that the lack of mechanical vector transmission in hiv is not due to genetic constraints . while ecological constraints , such as number of vectors and biting rates , may limit vector transmission in certain areas , these constraints", "would likely not explain why hiv has not evolved this form of transmission in areas where vector - borne diseases ( e.g. malaria ) are endemic .", "rather , there must presumably be a reason why such transmission is selectively disadvantageous in hiv .", "effective mechanical vector transmission can be brought about only through the evolution of higher levels of viremia , and this also results in a more rapid onset of aids .", "this reduces the duration over which such strains can be transmitted from an infected human , more than is made up for by the occurrence of vector transmission .", "it also remains possible that insufficient time has elapsed for the evolution of vector transmission to occur , but our calculations suggest that this is not a very compelling possibility . on the other hand , existing data is largely consistent with the hypothesis that biological vector transmission has not evolved in hiv because of genetic constraints . at the same time", ", it is not possible to rule out a selective explanation instead . in particular ,", "if there is a genetic trade - off between efficient replication in humans and replication in arthropod vectors , then a conflict between selection favoring effective replication within humans , and selection favoring arthropod transmission between humans can readily prevent biological transmission from evolving .", "this is particularly likely when the mutation rate of the virus is high , and thus might provide an explanation for the lack of biological vector transmission in all retroviruses .", "our analysis might also be extended to include other forms of transmission , for instance needle transmission ( see bruneau et al .", "several evolutionary consequences of this are possible depending both on the level of viremia required for such transmission to occur and the resulting transmission rate .", "for instance , if needle transmission can be achieved with a lower viremia than sexual transmission , and if this leads to a sufficiently high transmission rate , less virulent strains could be favored .", "conversely , if needle transmission requires a high viremia and leads to a sufficiently high transmission rate , more virulent strains would be favored .", "the only situation in which enhanced needle use could lead to the evolution of vector - borne transmission would be if effective needle transmission requires a viremia close to that of vector - borne transmission , while leading to a much higher transmission rate than vector - borne transmission .", "this way , strains with high viremia could be maintained in the population through needle transmission , and vector - borne transmission would then occur largely as a byproduct .", "our conclusions in this article are necessarily speculative , but such speculation is a necessary part of the initial stages of any research .", "one of our aims is to stimulate future research into the evolutionary biology of hiv transmission . from the results presented here ,", "a number of different directions might be taken to ground these evolutionary ideas more firmly in empirical data .", "one possibility would be to examine more closely mechanical vector transmission in immunodeficiency viruses of other species .", "for example , more data on the epidemiological patterns of siv and its potential for alternative routes of transmission would be enormously useful .", "since siv is believed to be at the evolutionary ancestor of hiv , it would be very interesting to know if the longer evolutionary history it has had with its host has resulted in different transmission patterns .", "to the best of our knowledge , there are no empirical studies testing the potential for vector transmission of siv", ". another fruitful approach might be to conduct artificial selection experiments with hiv in arthropod tissue culture .", "experiments have demonstrated that hiv can not currently replicate significantly in arthropod cells , but no study to our knowledge has attempted to select for the evolution of hiv replication in such cells .", "one could even imagine doing such experiments with both mammalian and arthropod cell cultures to determine of the evolutionary trade - off postulated here actually occurs .", "ultimately , it will require innovative experiments and empirical studies to push the boundaries of our knowledge of hiv , and the use of evolutionary biology as a powerful tool for designing sensible intervention strategies .", "these kinds of studies are beginning to appear for other aspects of hiv biology ( e.g. , see mller et al .", "2006 for an interesting evolutionary analysis of hiv virulence ) but more work on transmission biology would be useful .", "for example , if further empirical research validated the hypothesis presented here , that mechanical vector transmission has not evolved because of its associated mortality costs , this would then have important implications for how we attempt to stop the spread of hiv .", "strategies such as condom use , while beneficial for reducing the extent of sexual transmission , could thereby enhance the relative benefit of vector transmission , potentially resulting in the evolution of this new route of transmission .", "the use of antibiotics against bacterial pathogens has clearly brought home the fact that pathogens can readily evolve the means to circumvent our control measures , and there is no reason to expect things to be any different for other control measures .", "the use of antiviral medication , on the other hand , not only reduces sexual transmission but also the level of viremia , and therefore would presumably not move the selective balance more towards vector transmission .", "it is only by asking these kinds of questions , however , that we will have a chance at preventing adverse future outcomes .", "finally , the question of biological vector transmission addressed here is really a special case of the more general question of the evolution of a pathogen s host range .", "why do some pathogens have a relatively broad taxonomic host range while others are much more conservative ?", "this continues to be an interesting and important question in the evolutionary ecology of parasites ( poulin 2007 ) and there are some theoretical results predicting when we might expect different outcomes ( gandon 2004 ) . from the standpoint of human diseases", "this is also clearly an important question since emerging diseases , such as pandemic influenza , are precisely instances in which a pathogen evolves a different host range .", "a better understanding of the evolutionary biology of parasite host ranges is an important goal for future research ."], "abstract_text": ["<S> abstractmany pathogens of humans are blood borne , including hiv , malaria , hepatitis b and c , west nile virus , dengue , and other viral hemorrhagic fevers . </S>", "<S> although several of these pathogens are transmitted by blood - feeding arthropods , hiv is not . </S>", "<S> a number of properties of hiv and its life cycle have been identified as proximate explanations for the absence of arthropod transmission , but little consideration has been given to why hiv has not evolved this form of transmission . </S>", "<S> we consider the empirical evidence for arthropod transmission , and suggest that mechanical transmission has not evolved in hiv because such strains would induce a faster onset of aids during infection , which would thereby limit their ability to spread . on the other hand , it is not as clear why biological transmission has not occurred . </S>", "<S> available data suggests that a lack of appropriate genetic variation in hiv is one explanation , but it is also possible that a conflict between natural selection occurring within and between infected individuals has prevented its evolution instead . </S>", "<S> we discuss the potential significance of these ideas , and argue that taking such an evolutionary perspective broadens our understanding of infectious diseases and the potential consequences of public health interventions . </S>"], "labels": null, "section_names": ["Introduction", "Why is HIV not vector-borne?", "Genetic variation", "Selection", "Discussion"], "sections": [["approximately 40 million people are infected with the human immunodeficiency virus ( hiv ) worldwide ( unaids 2006 ) .", "infections by this lentivirus from the family retroviridae are characterized by a long asymptomatic period after host immune defenses control the initial infection . during this subsequent chronic phase of infection , hiv slowly diminishes the host", "the virus replicates by hijacking the intracellular molecular machinery to transcribe viral rna , and eventually the productively infected cells die ( fauci 1988 ) . over time , the density of virions in the blood stream increases and the immune system functioning becomes progressively compromised , leaving hiv - infected individuals increasingly susceptible to opportunistic infections .", "being blood - borne , hiv is transmitted via contact with the blood of an infected individual : through transfusions , needle - sharing , sexual contact or from mother to child during childbirth or breast - feeding .", "initially there were concerns that hiv might be vector - borne ( e.g. , transmitted via mosquitoes ) but it has since become widely accepted that such transmission does not occur at any significant level ( bockarie and paru 1996 ) .", "this current belief stems both from epidemiological data and experimental studies that directly examine the potential for hiv transmission via arthropods ( lawrence 1987 ; lifson 1988 ; bockarie and paru 1996 )", ". why is hiv not vector - borne ( throughout this article we use the term  vector synonymously with ", "arthropod ) ? the majority of medical scientists , when asked this question , will offer the following explanations ( bockarie and paru 1996 ) : ( i ) hiv concentrations in the blood are too low during human infection to permit vector transmission ; ( ii ) hiv is unable to survive long enough outside of humans ( or primates ) for vector transmission ; ( iii ) hiv is not able to replicate within arthropod vectors .", "each of these explanations has empirical support ( lifson 1988 ; bockarie and paru 1996 ) and thus all three are good explanations for the lack of vector transmission in hiv .", "if most arthropods pick up very little hiv when feeding on humans , and if the level of hiv in ( or on ) these vectors quickly decays , then no significant vector transmission is expected to occur .", "the above findings provide a satisfying proximate explanation for the lack of vector transmission in hiv .", "given the current characteristics of hiv , these three features of the virus make it unlikely that vector transmission will occur . in this article , we approach this question from an evolutionary perspective . therefore , rather than taking the characteristics of hiv as given , we are interested in understanding why hiv has evolved these particular features and not others instead . for example :", "( i ) why has hiv not evolved a replication strategy that results in viral concentrations high enough to allow vector transmission ? ( ii ) why has hiv not evolved to be more durable and thus to survive longer outside of humans ?", "( iii ) why has hiv not evolved the ability to replicate in arthropod vectors ?", "asking such questions might strike many readers as strange since much of evolutionary biology proceeds by asking why certain features of organisms are as we observe them rather than by asking why some features are absent .", "this is only natural , since it is difficult to decide which , among the infinite number of  missing features , should be the focus of study .", "it is useful to view such questions as falling on a continuum , from questions about the lack of traits that are virtually non - existent in all taxa ( e.g. , why do dogs not have wheeled appendages ) to questions about the lack of traits that are common in some populations or species but not others ( e.g. , why do german shepherds not have curly hair ? ) .", "our contention is that , by asking such questions about the absence of some traits , we can gain deeper insight into biology . to quote philosopher arthur eddington ( 1927 ) ,   the contemplation in natural science of a wider domain than the actual leads to a far better understanding of the actual .  . as in all science , however , not all questions are interesting , and this serves as our primary guide for focusing on some missing features and not others . in particular , we focus on the lack of vector transmission in hiv , because of the profound epidemiological significance of its absence .", "an arthropod - transmissible form of hiv would clearly exacerbate the already devastating impact of the disease , opening up routes of transmission to groups previously at low - risk ( e.g. , children ) .", "therefore , it is worth asking why , from an evolutionary standpoint , this has not occurred ( weiss 2001 ) .", "more to the point , if there are conditions under which hiv could have evolved vector transmission we would do well to understand these , not only from the standpoint of scientific curiosity , but also to prevent such an outcome in the future . in this article , we address the question of why hiv lacks vector transmission , both through a consideration of available empirical data and through the construction of mathematical models . although our results are necessarily speculative , we believe that they shed some light on the evolutionary biology of hiv , and on the evolutionary biology of blood - borne pathogens more generally ."], ["there are two broad reasons why a trait of interest ( in this case vector - transmission ) might not evolve .", "first , the necessary genetic variation for the trait might arise only very rarely ( if at all ) .", "for instance , the evolution of rna viruses , such as hiv , could be strongly constrained by the size of their genome ( holmes 2003 ) .", "second , the necessary form and strength of natural selection might not be present for the trait to evolve , at least over the timescale under consideration .", "thus , from an evolutionary standpoint , hiv is not vector - borne because either the necessary genetic variation for such transmission has never arisen , or the necessary selective factors that would make such variants increase in frequency over the relevant timescale have not occurred .", "our evolutionary explanation for why hiv is not vector - borne will be sought within these two possibilities . before beginning to consider these explanations", "there are two different processes that might result in vector - transmission , and that are often lumped under this single heading .", "this occurs when the arthropod acts solely as a means of physical transport of viral particles between hosts ( e.g. , having viral particles in and around mouthparts ) .", "this occurs when the virus replicates within the arthropod vector during the time period between feeding events .", "how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ? the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population .", "unfortunately , performing such an assay on all genotypes within the population would be next to impossible .", "furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low . nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue", "if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened .", "below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission :", "a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods .", "such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) .", "for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al .", "1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus .", "most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al .", "this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al . 1993 ) .", "it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) .", "other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods .", "in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al .", "at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al .", "the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al .", "1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) .", "interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) .", "it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al .", "even if this does occur , however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus . biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible .", "( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors .", "evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al .", "1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al .", "furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al .", "in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al .", "1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) .", "it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al .", "alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses .", "the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al .", "there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al .", "1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous .", "thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ?", "on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle .", "this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) .", "alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section", "we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv .", "mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) .", "the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate .", "although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans .", "the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics .", "this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 ) , v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and  is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) .", "note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this . also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) .", "the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people .", "we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) .", "the parameter  is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age .", "our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al . 2007 ) .", "results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption .", "lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age .", "in particular , we will suppose that (a ) has the form where  is the time during the infection at which aids develops .", "this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv . in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia . similarly , evidence suggests that sexual transmission rate ,  , also increases with viremia ( operskalski et al . 1997", "lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of  in untreated patients ( mellors et al .", "with these specifications , our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms .", "suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1  0 ) .", "also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission .", "assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively .", "equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate .", "more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold .", "empirical evidence in support of this functional form for hiv exists ( quinn et al .", ", a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large  ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig .", "1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore ,", "some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission .", "conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig .", "1 ) . sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) .", "b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text .", "parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 ,  = 15 .", "these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs .", "for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans .", "the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1  p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig .", "the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) .", "solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years .", "the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b .", "first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed ", ", we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years .", "also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act .", "more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred .", "unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al .", "1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved . equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv .", "biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however", ", biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve .", "thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost .", "there are at least two biologically plausible mechanisms through which such a cost might arise .", "first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case", ", the cost stems from an evolutionary trade - off between these two transmission routes .", "the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8) \n could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain .", "there is , however , no a priori reason why this would be expected rather than the reverse .", "therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well .", "the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host .", "it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation .", "if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread .", "modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host .", "this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission .", "the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host .", "the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection .", "specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation .", "when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting  be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) .", "the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes .", "first , if rb  ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand , if rb  ra < ba , then vector transmission will never evolve .", "in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve .", "this will be true whenever the mutation rate of the virus is high ( i.e. , large  ) and when the selective advantage of sexual transmission in terms of within - host competition is large .", "the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission ."], ["how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ?", "the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population .", "unfortunately , performing such an assay on all genotypes within the population would be next to impossible .", "furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low .", "nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue", "if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened .", "below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission :", "a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods .", "such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) .", "for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al .", "1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus .", "most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al .", "this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al .", "it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) .", "other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods .", "in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al .", "at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al .", "the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al .", "1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) .", "interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) .", "it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al .", "1983 ) , although this has been controversial ( kuno 2004 ) . even if this does occur", ", however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus .", "biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible .", "( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors .", "evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al .", "1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al .", "furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al .", "in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al .", "1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) .", "it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al .", "alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses ."], ["the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al .", "there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al .", "1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous .", "thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ?", "on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle .", "this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) .", "alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section", "we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv .", "mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at", "present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) .", "the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate .", "although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans .", "the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics .", "this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 )", ", v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and  is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) .", "note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this .", "also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) .", "the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people .", "we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) .", "the parameter  is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age .", "our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al .", "results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption .", "lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age . in particular", ", we will suppose that (a ) has the form where  is the time during the infection at which aids develops .", "this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv .", "in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia .", "similarly , evidence suggests that sexual transmission rate ,  , also increases with viremia ( operskalski et al .", "lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of  in untreated patients ( mellors et al . 1997 ; levy 1998 ; raffanti et al .", ", our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms .", "suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1  0 ) .", "also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission .", "assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively", ". equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate .", "more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold .", "empirical evidence in support of this functional form for hiv exists ( quinn et al .", ", a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large  ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig .", "1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore , some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission .", "conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig .", "sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) .", "b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text .", "parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 ,  = 15 .", "these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs . for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans . the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1  p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig .", "the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) .", "solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years .", "the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b .", "first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed ", ", we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years .", "also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act .", "more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred .", "unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al .", "1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved .", "equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv .", "biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however", ", biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve .", "thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost .", "there are at least two biologically plausible mechanisms through which such a cost might arise .", "first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case", ", the cost stems from an evolutionary trade - off between these two transmission routes .", "the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8) \n could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain .", "there is , however , no a priori reason why this would be expected rather than the reverse .", "therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well .", "the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host .", "it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation .", "if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread .", "modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host .", "this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission .", "the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host .", "the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection .", "specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation .", "when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting  be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) . the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes .", "first , if rb  ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand ,", "in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve .", "this will be true whenever the mutation rate of the virus is high ( i.e. , large  ) and when the selective advantage of sexual transmission in terms of within - host competition is large .", "the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission ."], ["experiments and epidemiological data have unequivocally demonstrated , however , that such vector transmission does not occur at any significant level , and various aspects of hiv biology have been implicated as proximate reasons ( bockarie and paru 1996 ) .", "these reasons do not offer an explanation for why vector transmission has not evolved , however , and as weiss ( 2001 ) points out , we ought to seriously consider whether such evolution might occur in the future ( for a summary , see table 1 ) .", "main conclusions of the study existing data suggest that the lack of mechanical vector transmission in hiv is not due to genetic constraints . while ecological constraints , such as number of vectors and biting rates , may limit vector transmission in certain areas , these constraints", "would likely not explain why hiv has not evolved this form of transmission in areas where vector - borne diseases ( e.g. malaria ) are endemic .", "rather , there must presumably be a reason why such transmission is selectively disadvantageous in hiv .", "effective mechanical vector transmission can be brought about only through the evolution of higher levels of viremia , and this also results in a more rapid onset of aids .", "this reduces the duration over which such strains can be transmitted from an infected human , more than is made up for by the occurrence of vector transmission .", "it also remains possible that insufficient time has elapsed for the evolution of vector transmission to occur , but our calculations suggest that this is not a very compelling possibility . on the other hand , existing data is largely consistent with the hypothesis that biological vector transmission has not evolved in hiv because of genetic constraints . at the same time", ", it is not possible to rule out a selective explanation instead . in particular ,", "if there is a genetic trade - off between efficient replication in humans and replication in arthropod vectors , then a conflict between selection favoring effective replication within humans , and selection favoring arthropod transmission between humans can readily prevent biological transmission from evolving .", "this is particularly likely when the mutation rate of the virus is high , and thus might provide an explanation for the lack of biological vector transmission in all retroviruses .", "our analysis might also be extended to include other forms of transmission , for instance needle transmission ( see bruneau et al .", "several evolutionary consequences of this are possible depending both on the level of viremia required for such transmission to occur and the resulting transmission rate .", "for instance , if needle transmission can be achieved with a lower viremia than sexual transmission , and if this leads to a sufficiently high transmission rate , less virulent strains could be favored .", "conversely , if needle transmission requires a high viremia and leads to a sufficiently high transmission rate , more virulent strains would be favored .", "the only situation in which enhanced needle use could lead to the evolution of vector - borne transmission would be if effective needle transmission requires a viremia close to that of vector - borne transmission , while leading to a much higher transmission rate than vector - borne transmission .", "this way , strains with high viremia could be maintained in the population through needle transmission , and vector - borne transmission would then occur largely as a byproduct .", "our conclusions in this article are necessarily speculative , but such speculation is a necessary part of the initial stages of any research .", "one of our aims is to stimulate future research into the evolutionary biology of hiv transmission . from the results presented here ,", "a number of different directions might be taken to ground these evolutionary ideas more firmly in empirical data .", "one possibility would be to examine more closely mechanical vector transmission in immunodeficiency viruses of other species .", "for example , more data on the epidemiological patterns of siv and its potential for alternative routes of transmission would be enormously useful .", "since siv is believed to be at the evolutionary ancestor of hiv , it would be very interesting to know if the longer evolutionary history it has had with its host has resulted in different transmission patterns .", "to the best of our knowledge , there are no empirical studies testing the potential for vector transmission of siv", ". another fruitful approach might be to conduct artificial selection experiments with hiv in arthropod tissue culture .", "experiments have demonstrated that hiv can not currently replicate significantly in arthropod cells , but no study to our knowledge has attempted to select for the evolution of hiv replication in such cells .", "one could even imagine doing such experiments with both mammalian and arthropod cell cultures to determine of the evolutionary trade - off postulated here actually occurs .", "ultimately , it will require innovative experiments and empirical studies to push the boundaries of our knowledge of hiv , and the use of evolutionary biology as a powerful tool for designing sensible intervention strategies .", "these kinds of studies are beginning to appear for other aspects of hiv biology ( e.g. , see mller et al .", "2006 for an interesting evolutionary analysis of hiv virulence ) but more work on transmission biology would be useful .", "for example , if further empirical research validated the hypothesis presented here , that mechanical vector transmission has not evolved because of its associated mortality costs , this would then have important implications for how we attempt to stop the spread of hiv .", "strategies such as condom use , while beneficial for reducing the extent of sexual transmission , could thereby enhance the relative benefit of vector transmission , potentially resulting in the evolution of this new route of transmission .", "the use of antibiotics against bacterial pathogens has clearly brought home the fact that pathogens can readily evolve the means to circumvent our control measures , and there is no reason to expect things to be any different for other control measures .", "the use of antiviral medication , on the other hand , not only reduces sexual transmission but also the level of viremia , and therefore would presumably not move the selective balance more towards vector transmission .", "it is only by asking these kinds of questions , however , that we will have a chance at preventing adverse future outcomes .", "finally , the question of biological vector transmission addressed here is really a special case of the more general question of the evolution of a pathogen s host range .", "why do some pathogens have a relatively broad taxonomic host range while others are much more conservative ?", "this continues to be an interesting and important question in the evolutionary ecology of parasites ( poulin 2007 ) and there are some theoretical results predicting when we might expect different outcomes ( gandon 2004 ) . from the standpoint of human diseases", "this is also clearly an important question since emerging diseases , such as pandemic influenza , are precisely instances in which a pathogen evolves a different host range .", "a better understanding of the evolutionary biology of parasite host ranges is an important goal for future research ."]]}
{"article_id": "PMC3560831", "article_text": ["in the normal kidney , immunohistochemical expression of contractile protein alpha - smooth muscle actin ( asma ) is limited to the vascular smooth muscle cells . in pathological conditions , the expression of asma", "the first experimental publication on this subject , by johnson et al , involved a series of clinical studies on the role of asma in diffuse renal diseases . in 1992", "alpers and colleagues found higher expression of asma in proliferative types of glomerulonephritis gn . in iga nephropathy , asma expression in interstitium is related to damaged renal function and progression of disease .", "the expression of asma in interstitial myofibroblasts helps to differentiate between minimal change disease and the early phase of idiopathic membranous gn . in patients with iga nephropathy ,", "higher mesangial expression of asma predicts a progressive decline in renal function . in focal segmental glomerulosclerosis ,", ", interest in the asma is increasing due to the fact that the asma in cytoplasm of the podocytes is associated with nephrin and thus is indirectly involved in the glomerular filtration barrier .", "asma is analyzed in various types of gn together with markers such as metalloproteinase and nestin in investigation of tissue remodulation and epithelial - mesenchymal transition . to our best knowledge , this is the first morphometrical study of the correlation of asma expression in gn and renal function at the time of biopsy .", "this retrospective study analyzed 142 patients  82 adults and 60 children  who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 .", "patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample", ", pathological diagnoses were classified into 2 categories  proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category", "were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy .", "absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults ,", "absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children ,", "bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey .", "serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low .", "ccr was determined from sc , the patient s height and proportionality constant using the schwartz method .", "the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits .", "asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) .", "paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient .", "tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) .", "hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam .", "positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer .", "computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) .", "a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using  analysis  software ( analysis soft imaging system , usa ) .", "all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium .", "area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) .", "their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor", "the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys ,", "nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc .", "this retrospective study analyzed 142 patients  82 adults and 60 children  who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 .", "patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample", ", pathological diagnoses were classified into 2 categories  proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category", "were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy .", "absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults ,", "absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children ,", "bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey .", "serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low .", "ccr was determined from sc , the patient s height and proportionality constant using the schwartz method .", "the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits .", "asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) .", "paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient .", "tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) .", "hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam .", "positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer .", "computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) .", "a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using  analysis  software ( analysis soft imaging system , usa ) .", "all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium .", "area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) .", "their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor", "the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys ,", "nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc .", "the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) .", "the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) .", "the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults ,", "expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively . in children", ", positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc ,", "the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults ,", "expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) .", "the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) .", "significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) . in children , absolute values of ccr", "negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults", ", there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) .", "children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) .", "no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age .", "the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) .", "the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) .", "the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults ,", "expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively .", "in children , positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc ,", "the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults ,", "expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) .", "the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) .", "significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) .", "in children , absolute values of ccr negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults , there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) .", "children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) .", "no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age .", "studies of asma expression in renal parenchymal diseases began more than 2 decades ago , when it was noticed that a ) damaged glomerular mesangial cells change their immunophenotype expressing asma and b ) asma - positive myofibroblasts start interstitial fibrosis .", "a number of papers were published about asma expression in different types of human gn , its connection to proliferation markers and prognostic impact .", "most authors agree that asma expression in glomeruli was higher in proliferative gn and increases as the disease worsens , which makes asma expression a potential clinical prognostic factor .", "some authors determined the relationship between iga nephropathy , lupus nephritis and other types of gn with expression of asma .", "kim in 2001 found correlation between proliferation marker ki-67 and asma in different types of gn . in this study we focused on the connection of asma expression with impaired renal function in glomerulonephritis , measured at the time of biopsy .", "we did not find correlation with a special type of gn nor proliferation , possibly because of heterogeneity of the sample and the arbitrary method of categorization into proliferative and non - proliferative gn .", "increased expression of asma in the interstitium can be found in different diseases such as proliferative gn , diabetic nephropathy and renal transplant rejection .", "the asma has prognostic value due to the association with interstitial fibrosis , urine protein and sc [ 8,2528 ] .", "asma - positive myofibroblasts are responsible for the increased amount of extracellular matrix and renal fibrosis .", "this study also found higher expression of asma in interstitium of all patients with higher sc .", "greater expression of interstitial asma in children and adults was associated with higher grades of ccr and lower absolute values of ccr .", "we confirm that elevated sc or lower values of ccr are associated with higher expression of asma in interstitium .", "several studies have attempted to predict the development of progressive renal failure , measuring histomorphometric changes in the tubulointerstitial compartment .", "the best correlating parameters of interstitial fibrosis with renal function are the ratio of the accumulation of tgf - beta-1 and its antagonist decorin , interstitial expression of asma , and accumulation of interstitial collagen . according to jiang et al .", ", who analyzed asma production in peritoneal fibroblasts stimulated by tnf - beta-1 , hepatocyte growth factor could be important in blocking postoperative peritoneal adhesion .", "analyzed 133 biopsies of various human renal diseases , and found tubular epithelial cells with mesenchymal phenotype ( vimentin and asma positive ) whose numbers have been associated with the level of sc and degree of interstitial damage .", "analyzed 27 patients with iga nephropathy who had normal ccr at the time of biopsy , and found that expression of asma in mesangium predicts a progressive decline in renal function . in our study ,", "a negative connection of asma expression in glomeruli and sc at the time of biopsy was found , statistically significant in adults and non - significant in children , but with the same trend .", "it is well known that asma expression in the mesangial cell indicates its change to myofibroblastic immunophenotype .", "our presumption is that asma - expressing mesangial cells are capable of higher contraction activity ; this leads to glomerular hyperfiltration as an early adaptive mechanism to glomerular damage .", "in a recent experimental study of membranous glomerulonephritis in rats , osteopontin and asma are expressed together in myofibroblasts in the crescents of damaged glomeruli . in children , the absolute values of ccr", "a possible explanation that children have a better ccr is due to phenotypic modulation of human mesangial cells to more contractile cells , which makes the glomerular function transitory normal or even increased .", "we analyzed association of expression of asma and hypertension , because it is known that glomerular mesangial cells during hypertensive damage express myofibroblast phenotype and expression of asma increases .", "the only significant association that we found was between expression of asma in interstitium and increased blood pressure in children .", "we did not find significant correlation between expression of asma in glomeruli nor interstitium with 24-hour urine protein in our patients .", "this result confirms previous findings that urine protein is primary linked to the changes in permeability of glomerular filtration barrier . nevertheless , according to some studies asma expression in interstitium", "is linked to the degree of proteinuria and sc , and higher interstitial asma expression is an indicator of poor prognosis .", "asma expression in interstitium is associated with sc and ccr and consecutively with decrease of renal function .", "asma expression in glomeruli is associated with lower values of sc in adults , as well as with normal or higher ccr in children .", "this correlation suggests that myofibroblastic phenotypic modulation of glomerular cells has a favorable impact on filtration . when calculated with precise computer - assisted quantitative morphometric technology , renal expression of asma", "the disadvantage of this study is its small sample size with different types of gn ; the next survey should include a much narrower cohort of patients with a specific type of gn , as well as better phenotypic identification of asma - positive glomerular cells ."], "abstract_text": ["<S> summarybackgroundin a healthy kidney , contractile protein alpha - smooth muscle actin ( asma ) is immunohistochemically strongly expressed only in the blood vessels , while in pathological conditions it can be visualized in glomerular mesangial cells and interstitial myofibroblasts . </S>", "<S> the aim of this study was to explore the possible correlation between expression of asma in glomerulonephritis ( gn ) and indicators of renal function.material/methodswe analyzed expression of asma in percutaneous renal biopsy of 142 adult and pediatric patients with gn and its correlation with blood pressure , serum creatinine , creatinine clearance and 24-hour urine protein at the time of biopsy . </S>", "<S> immunoexpression of asma was analyzed quantitatively using computer - assisted morphometric analysis . </S>", "<S> relative surface of asma expression in all glomeruli and interstitium was calculated for each patient.resultsin adults and children , greater expression of asma in interstitium was associated with higher serum creatinine and reduced creatinine clearance . </S>", "<S> conversely , greater asma expression in glomeruli was associated with normal or decreased serum creatinine in adults and increased creatinine clearance in children . in children , correlation was found between high blood pressure and asma expression in interstitium.conclusionswe confirmed that interstitial expression of asma is associated with reduced renal function at time of biopsy . </S>", "<S> the connection of asma expression in glomeruli with lower serum creatinine and normal or increased creatinine clearance suggests a favorable role of this phenotypic change in glomerular filtration rate ; further investigation is needed . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Patients", "Laboratory parameters", "Immunohistochemistry", "Morphometric analysis", "Statistical analysis", "Results", "Renal ASMA expression", "Blood pressure", "Serum creatinine", "Creatinine clearance", "24-hour urine protein", "Discussion", "Conclusions"], "sections": [["in the normal kidney , immunohistochemical expression of contractile protein alpha - smooth muscle actin ( asma ) is limited to the vascular smooth muscle cells . in pathological conditions , the expression of asma", "the first experimental publication on this subject , by johnson et al , involved a series of clinical studies on the role of asma in diffuse renal diseases . in 1992", "alpers and colleagues found higher expression of asma in proliferative types of glomerulonephritis gn . in iga nephropathy , asma expression in interstitium is related to damaged renal function and progression of disease .", "the expression of asma in interstitial myofibroblasts helps to differentiate between minimal change disease and the early phase of idiopathic membranous gn . in patients with iga nephropathy ,", "higher mesangial expression of asma predicts a progressive decline in renal function . in focal segmental glomerulosclerosis ,", ", interest in the asma is increasing due to the fact that the asma in cytoplasm of the podocytes is associated with nephrin and thus is indirectly involved in the glomerular filtration barrier .", "asma is analyzed in various types of gn together with markers such as metalloproteinase and nestin in investigation of tissue remodulation and epithelial - mesenchymal transition . to our best knowledge , this is the first morphometrical study of the correlation of asma expression in gn and renal function at the time of biopsy ."], ["this retrospective study analyzed 142 patients  82 adults and 60 children  who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 .", "patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample", ", pathological diagnoses were classified into 2 categories  proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category", "were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy .", "absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults ,", "absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children ,", "bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey .", "serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low .", "ccr was determined from sc , the patient s height and proportionality constant using the schwartz method .", "the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits .", "asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) .", "paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient .", "tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) .", "hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam .", "positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer .", "computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) .", "a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using  analysis  software ( analysis soft imaging system , usa ) .", "all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium .", "area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) .", "their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor", "the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys ,", "nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc ."], ["this retrospective study analyzed 142 patients  82 adults and 60 children  who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics , university hospital , split , croatia in the period from 1994 to 2007 .", "patients data were collected from the hospital records . due to the small numbers of each type of gn in the sample", ", pathological diagnoses were classified into 2 categories  proliferative glomerulonephritis ( pg ) in 98 cases and non - proliferative glomerulonephritis ( npg ) in 44 cases . in the pg category", "were : 29 iga nephropathy , 24 mesangioproliferative gn , 11 henoch - schenlein purpura , 9 focal segmental gn , 6 rapidly progressive gn , 6 endoproliferative gn and 6 lupus nephritis , 4 membranoproliferative gn and 1 hemolytic uremic syndrome , 1 alport syndrome and 1 churg - strauss syndrome . in the npg category were : 17 membranous gn , 15 focal segmental glomerulosclerosis , 5 minimal change disease , 2 fibrillar gn , 1 thin basement membrane disease , c1q nephropathy , igm nephropathy , amyloidosis and hereditary nephropathy ."], ["absolute values of blood pressure ( bp ) , serum creatinine ( sc ) , creatinine clearance ( ccr ) and 24-hour urine protein , measured within 7 days before the biopsy , were collected from the hospital records . for adults ,", "absolute values were categorized according to the common terminology criteria for adverse events ( ctcae ) . for children ,", "bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey .", "serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories : normal , high and low .", "ccr was determined from sc , the patient s height and proportionality constant using the schwartz method .", "the 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits ."], ["asma expression was analyzed by indirect immunohistochemistry ( envision / hrp system ( dako , denmark ) using mouse monoclonal anti - alpha smooth muscle antibody ( asma / hrp dako , denmark ) .", "paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient .", "tissue sections were incubated with primary asma antibody ( dilution 1:50 ) for 60 min and peroxidase - labeled secondary antibody for 20 min , followed by 10 min incubation with diaminobenzidine substrate - chromogen solution ( dako , denmark ) .", "hematoxylin counter - staining was done ; slides were dehydrated in alcohol gradient , cleared in xylol and mounted with canada balsam .", "positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer ."], ["computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera ( olympus 4.1 zoom ) connected with olympus bx41 microscope ( olympus , japan ) .", "a computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections , using  analysis  software ( analysis soft imaging system , usa ) .", "all foci of asma expression in the interstitium ( including atrophic tubules ) , as well as the perimeter of histological slide , were measured in m at 100 magnification ; their ratio was calculated as a percentage of asma expression in interstitium .", "area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli ( figure 1a ) .", "their ratio was calculated as a percentage of asma expression in glomeruli ( figure 1b ) . in 5 case controls , renal cortex in the highest distance from the tumor", "the area measuring 0.14 mm was defined , and glomeruli and interstitium were analyzed in the same manner . in the control kidneys ,"], ["nonparametric spearman s correlation , mann - whitney test , and analysis of variance ( anova , kruskal - wallis ) were made using graphpad prism statistical software ( graphpad software , inc ."], ["the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) .", "the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) .", "the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults ,", "expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively . in children", ", positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc ,", "the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults ,", "expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) .", "the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) .", "significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) . in children , absolute values of ccr", "negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults", ", there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) .", "children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) .", "no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age ."], ["the expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults ( table 1 ) ."], ["the children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure ( 10.79.4% vs. 3.74.3% , p=0.014 ) ( figure 2 ) .", "the difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure ( 7.94.4% vs. 15.410.6% , p=0.051 ) . in adults ,", "expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure ( 10.714.9% vs. 9.912.6% , p=0.692 ) and ( 13.69.5% vs. 12.97.8% , p=1.00 ) , respectively ."], ["in children , positive correlation was found between asma expression in interstitium and absolute value of sc ( r=0.45 , p=0.002 ) ( figure 3a ) . in categorized sc ,", "the negative correlation of sc and expression of asma in glomeruli was not significant ( r=0.154 , p=0.291 ) . in adults ,", "expression of asma in interstitium was correlated to sc ( r=0.528 , p<0.001 ) ( figure 3b ) .", "the significance was confirmed in categorized sc , where grade iii had higher expression of asma in interstitium ( p=0.0009 ) .", "significant negative correlation was found between expression of asma in glomeruli and sc ( r=0.395 , p=0.002 ) ( figure 3c ) ."], ["in children , absolute values of ccr negatively correlated to expression of asma in interstitium ( r=0.375 , p=0.009 ) and positively to expression of asma in glomeruli ( r=1.00 , p<0.001 ) ( figure 4a , b ) . in adults , there were no significant correlations between asma expression in glomeruli and ccr ( r=0.058 , p=0,643 ) .", "children and adults with ccr grade ii and iii had higher expression of asma in interstitium ( p=0.0152 and p=0.0007 , respectively ) ( figures 5a , b ) ."], ["no significant association between renal expression of asma and 24-hour urine protein was found , regardless of the patient s age ."], ["studies of asma expression in renal parenchymal diseases began more than 2 decades ago , when it was noticed that a ) damaged glomerular mesangial cells change their immunophenotype expressing asma and b ) asma - positive myofibroblasts start interstitial fibrosis .", "a number of papers were published about asma expression in different types of human gn , its connection to proliferation markers and prognostic impact .", "most authors agree that asma expression in glomeruli was higher in proliferative gn and increases as the disease worsens , which makes asma expression a potential clinical prognostic factor .", "some authors determined the relationship between iga nephropathy , lupus nephritis and other types of gn with expression of asma .", "kim in 2001 found correlation between proliferation marker ki-67 and asma in different types of gn . in this study we focused on the connection of asma expression with impaired renal function in glomerulonephritis , measured at the time of biopsy .", "we did not find correlation with a special type of gn nor proliferation , possibly because of heterogeneity of the sample and the arbitrary method of categorization into proliferative and non - proliferative gn .", "increased expression of asma in the interstitium can be found in different diseases such as proliferative gn , diabetic nephropathy and renal transplant rejection .", "the asma has prognostic value due to the association with interstitial fibrosis , urine protein and sc [ 8,2528 ] .", "asma - positive myofibroblasts are responsible for the increased amount of extracellular matrix and renal fibrosis .", "this study also found higher expression of asma in interstitium of all patients with higher sc .", "greater expression of interstitial asma in children and adults was associated with higher grades of ccr and lower absolute values of ccr .", "we confirm that elevated sc or lower values of ccr are associated with higher expression of asma in interstitium .", "several studies have attempted to predict the development of progressive renal failure , measuring histomorphometric changes in the tubulointerstitial compartment .", "the best correlating parameters of interstitial fibrosis with renal function are the ratio of the accumulation of tgf - beta-1 and its antagonist decorin , interstitial expression of asma , and accumulation of interstitial collagen . according to jiang et al .", ", who analyzed asma production in peritoneal fibroblasts stimulated by tnf - beta-1 , hepatocyte growth factor could be important in blocking postoperative peritoneal adhesion .", "analyzed 133 biopsies of various human renal diseases , and found tubular epithelial cells with mesenchymal phenotype ( vimentin and asma positive ) whose numbers have been associated with the level of sc and degree of interstitial damage .", "analyzed 27 patients with iga nephropathy who had normal ccr at the time of biopsy , and found that expression of asma in mesangium predicts a progressive decline in renal function . in our study ,", "a negative connection of asma expression in glomeruli and sc at the time of biopsy was found , statistically significant in adults and non - significant in children , but with the same trend .", "it is well known that asma expression in the mesangial cell indicates its change to myofibroblastic immunophenotype .", "our presumption is that asma - expressing mesangial cells are capable of higher contraction activity ; this leads to glomerular hyperfiltration as an early adaptive mechanism to glomerular damage .", "in a recent experimental study of membranous glomerulonephritis in rats , osteopontin and asma are expressed together in myofibroblasts in the crescents of damaged glomeruli . in children , the absolute values of ccr", "a possible explanation that children have a better ccr is due to phenotypic modulation of human mesangial cells to more contractile cells , which makes the glomerular function transitory normal or even increased .", "we analyzed association of expression of asma and hypertension , because it is known that glomerular mesangial cells during hypertensive damage express myofibroblast phenotype and expression of asma increases .", "the only significant association that we found was between expression of asma in interstitium and increased blood pressure in children .", "we did not find significant correlation between expression of asma in glomeruli nor interstitium with 24-hour urine protein in our patients .", "this result confirms previous findings that urine protein is primary linked to the changes in permeability of glomerular filtration barrier . nevertheless , according to some studies asma expression in interstitium", "is linked to the degree of proteinuria and sc , and higher interstitial asma expression is an indicator of poor prognosis ."], ["asma expression in interstitium is associated with sc and ccr and consecutively with decrease of renal function .", "asma expression in glomeruli is associated with lower values of sc in adults , as well as with normal or higher ccr in children .", "this correlation suggests that myofibroblastic phenotypic modulation of glomerular cells has a favorable impact on filtration . when calculated with precise computer - assisted quantitative morphometric technology , renal expression of asma", "the disadvantage of this study is its small sample size with different types of gn ; the next survey should include a much narrower cohort of patients with a specific type of gn , as well as better phenotypic identification of asma - positive glomerular cells ."]]}
{"article_id": "PMC4782403", "article_text": ["chondroblastoma accounts for < 1% of all primary bone tumors . usually occurring in young adolescents ,", "however , unusual sites of presentation such as patella , talocalcaneal , and temporomandibular joint has been reported .", "since last decade , fine needle aspiration cytology ( fnac ) has evolved as a simple , safe , and noninvasive preoperative tool in the diagnosis of bone tumors .", "it is necessary to differentiate chondroblastoma from other giant cell - rich lesions , as chondroblastoma exhibits predominantly benign behavior , though recurrences and metastasis are on record .", "we report cytomorphology in two cases of chondroblastoma confirmed by histopathology with a brief review of literature .", "a 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months .", "swelling was noted in the upper end of tibia measuring 3 cm  3 cm , and was tender on palpation .", "x ray showed typically lytic , centrally placed , sharply demarcated lesion with sclerotic border in the epiphysis . a 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration .", "local examination revealed swelling at the upper end of humerus measuring 4 cm  4 cm .", "fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case , ultrasound - guided aspiration was done because the lesion was deep - seated .", "smears were highly cellular , polyhedral cells arranged in sheets and small clusters , giving a pebble stone pattern appearance .", "the individual cells were monomorphic with a well - defined cell membrane , glassy cytoplasm at places showing microvacuolation .", "additionally seen was cartilaginous matrix , plenty of scattered osteoclast - type multinucleated giant cells , and bluish granular calcification surrounding individual cells  chicken wire calcification [ figure 1a  c ] .", "( h and e , 40 ) ( b ) cytology smears showing polygonal cells with vacuolization in clusters , with calcification seen in the letilower corner .", "( h and e , 100 ) ( c ) cytology smears showing chicken wire calcification .", "( h and e , 100 ) ( d ) histopathology section showing cartilage , chicken wire calcification , and polygonal cells with vacuolization of cytoplasm .", "( h and e , 40 ) at surgery , curettage and bone grafting was performed .", "grossly , the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage .", "histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm .", "a 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months .", "swelling was noted in the upper end of tibia measuring 3 cm  3 cm , and was tender on palpation .", "x ray showed typically lytic , centrally placed , sharply demarcated lesion with sclerotic border in the epiphysis .", "a 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration .", "local examination revealed swelling at the upper end of humerus measuring 4 cm  4 cm .", "fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case , ultrasound - guided aspiration was done because the lesion was deep - seated .", "smears were highly cellular , polyhedral cells arranged in sheets and small clusters , giving a pebble stone pattern appearance .", "the individual cells were monomorphic with a well - defined cell membrane , glassy cytoplasm at places showing microvacuolation .", "additionally seen was cartilaginous matrix , plenty of scattered osteoclast - type multinucleated giant cells , and bluish granular calcification surrounding individual cells  chicken wire calcification [ figure 1a  c ] .", "( h and e , 40 ) ( b ) cytology smears showing polygonal cells with vacuolization in clusters , with calcification seen in the letilower corner .", "( h and e , 100 ) ( c ) cytology smears showing chicken wire calcification .", "( h and e , 100 ) ( d ) histopathology section showing cartilage , chicken wire calcification , and polygonal cells with vacuolization of cytoplasm .", "( h and e , 40 ) at surgery , curettage and bone grafting was performed .", "grossly , the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage .", "histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm .", "unusual sites of location include pelvis , patella , talus , calcaneum , and temporomandibular joint .", "most of the chondroblastomas are benign in nature requiring simple curettage and bone grafting . however , recurrence is known to occur in 10 - 38% of the cases .", "metastases are unusual but occur in the lungs at the time of recurrence . as in most of the cases of bone tumors ,", "this , compounded with nonconforming radiological features , leads to the tragic delay in diagnosis . though the age of the patient and location of the lesion in the given bone are key pieces of information , accurate and early diagnosis is a must for planning appropriate treatment .", "the past decade has witnessed the impressive use of fnac in the diagnosis of bone tumors . despite this , we have a limited case series describing fnac findings in chondroblastoma", ". fna aspirates of chondroblastoma show mononuclear cells arranged in a dispersed pattern like pebbles .", "these cells have well - defined cell margins , glassy cytoplasm , and nucleus with indentation and grooving .", "apart from associated giant cells and chondroid matrix , the chicken wire calcification that has been described in histopathology sections can be readily appreciated in fnac smears and can be a clue to diagnosis .", "however , errors in the fnac diagnosis of this rare tumor are known to occur , with the major pitfalls being its association with aneurysmal bone cyst ( abc ) leading to a nonrepresentative aspirate and the occurrence of a plethora of giant cell - rich lesions in the differentials .", "in one large series of 12 cases of chondroblastoma , the authors described the cytomorphology of this tumor in detail .", "the diagnosis was missed on fnac , among which , one was associated with abc and in another case an erroneous diagnosis of a giant cell tumor was made .", "the cause of misdiagnosis in one case was due to its association with abc , leading to nonrepresentative aspirate . in the second case , due to the location of the tumor in preauricular region , cartilaginous stroma aspirated", ", in a study of 110 cases of bone lesions attributed the missed diagnosis in two cases of chondroblastoma at fnac to inadequate sampling and misinterpretation as giant cell tumor .", "in giant cell tumor , the mononuclear cells are spindle - shaped and occur in clusters . in chondromyxoid fibroma , the smears are sparsely cellular and lack the mononuclear cell component . instead , stellate cells with pleomorphism are noted .", "aspirates in abc are usually hemorrhagic , scantily cellular , composed of osteoclast giant cells , histiocytes , osteoblasts , and spindle cells .", "in addition to chondroblastoma , abc can occur secondarily in giant cell tumor and osteosarcoma .", "this report reiterates the cytomorphologic findings in chondroblastoma and its differential diagnosis from other giant cell containing lesions of bone .", "the collective application of the knowledge of the cytomorphological features of this rare bone tumor would aid in accurate preoperative diagnosis even in the absence of typical clinical and radiologic presentations ."], "abstract_text": ["<S> chondroblastoma is a rare , giant cell - rich , benign neoplasm of bone . since the past few decades </S>", "<S> fine needle aspiration cytology ( fnac ) has gained momentum in preoperative diagnosis of bone lesions . at cytology , other giant cell - rich tumors and tumorlike lesions such as aneurysmal bone cyst ( abc ) , giant cell tumor , and chondromyxoid fibroma fall under the differential diagnosis of chondroblastoma . due to the difference in the treatment protocol and prognosis , </S>", "<S> preoperative diagnosis is mandatory . </S>", "<S> we describe the cytomorphology in two cases of chondroblastoma diagnosed at fnac and confirmed by histopathology . at cytology , the presence of giant cells , chondroid matrix , mononuclear cells with nuclear indentation , and grooving along with glassy , vacuolated cytoplasm are characteristic of chondroblastoma . </S>", "<S> in addition to this , the presence of chicken wire calcification is a useful clue to the accurate diagnosis of chondroblastoma at fnac . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Case 1", "Case 2", "Discussion", "Conclusion", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["chondroblastoma accounts for < 1% of all primary bone tumors . usually occurring in young adolescents ,", "however , unusual sites of presentation such as patella , talocalcaneal , and temporomandibular joint has been reported .", "since last decade , fine needle aspiration cytology ( fnac ) has evolved as a simple , safe , and noninvasive preoperative tool in the diagnosis of bone tumors .", "it is necessary to differentiate chondroblastoma from other giant cell - rich lesions , as chondroblastoma exhibits predominantly benign behavior , though recurrences and metastasis are on record .", "we report cytomorphology in two cases of chondroblastoma confirmed by histopathology with a brief review of literature ."], ["a 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months .", "swelling was noted in the upper end of tibia measuring 3 cm  3 cm , and was tender on palpation .", "x ray showed typically lytic , centrally placed , sharply demarcated lesion with sclerotic border in the epiphysis . a 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration .", "local examination revealed swelling at the upper end of humerus measuring 4 cm  4 cm .", "fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case , ultrasound - guided aspiration was done because the lesion was deep - seated .", "smears were highly cellular , polyhedral cells arranged in sheets and small clusters , giving a pebble stone pattern appearance .", "the individual cells were monomorphic with a well - defined cell membrane , glassy cytoplasm at places showing microvacuolation .", "additionally seen was cartilaginous matrix , plenty of scattered osteoclast - type multinucleated giant cells , and bluish granular calcification surrounding individual cells  chicken wire calcification [ figure 1a  c ] .", "( h and e , 40 ) ( b ) cytology smears showing polygonal cells with vacuolization in clusters , with calcification seen in the letilower corner .", "( h and e , 100 ) ( c ) cytology smears showing chicken wire calcification .", "( h and e , 100 ) ( d ) histopathology section showing cartilage , chicken wire calcification , and polygonal cells with vacuolization of cytoplasm .", "( h and e , 40 ) at surgery , curettage and bone grafting was performed .", "grossly , the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage .", "histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm ."], ["a 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months .", "swelling was noted in the upper end of tibia measuring 3 cm  3 cm , and was tender on palpation .", "x ray showed typically lytic , centrally placed , sharply demarcated lesion with sclerotic border in the epiphysis ."], ["a 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration .", "local examination revealed swelling at the upper end of humerus measuring 4 cm  4 cm .", "fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case , ultrasound - guided aspiration was done because the lesion was deep - seated .", "smears were highly cellular , polyhedral cells arranged in sheets and small clusters , giving a pebble stone pattern appearance .", "the individual cells were monomorphic with a well - defined cell membrane , glassy cytoplasm at places showing microvacuolation .", "additionally seen was cartilaginous matrix , plenty of scattered osteoclast - type multinucleated giant cells , and bluish granular calcification surrounding individual cells  chicken wire calcification [ figure 1a  c ] .", "( h and e , 40 ) ( b ) cytology smears showing polygonal cells with vacuolization in clusters , with calcification seen in the letilower corner .", "( h and e , 100 ) ( c ) cytology smears showing chicken wire calcification .", "( h and e , 100 ) ( d ) histopathology section showing cartilage , chicken wire calcification , and polygonal cells with vacuolization of cytoplasm .", "( h and e , 40 ) at surgery , curettage and bone grafting was performed .", "grossly , the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage .", "histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm ."], ["unusual sites of location include pelvis , patella , talus , calcaneum , and temporomandibular joint .", "most of the chondroblastomas are benign in nature requiring simple curettage and bone grafting . however , recurrence is known to occur in 10 - 38% of the cases .", "metastases are unusual but occur in the lungs at the time of recurrence . as in most of the cases of bone tumors ,", "this , compounded with nonconforming radiological features , leads to the tragic delay in diagnosis . though the age of the patient and location of the lesion in the given bone are key pieces of information , accurate and early diagnosis is a must for planning appropriate treatment .", "the past decade has witnessed the impressive use of fnac in the diagnosis of bone tumors . despite this , we have a limited case series describing fnac findings in chondroblastoma", ". fna aspirates of chondroblastoma show mononuclear cells arranged in a dispersed pattern like pebbles .", "these cells have well - defined cell margins , glassy cytoplasm , and nucleus with indentation and grooving .", "apart from associated giant cells and chondroid matrix , the chicken wire calcification that has been described in histopathology sections can be readily appreciated in fnac smears and can be a clue to diagnosis .", "however , errors in the fnac diagnosis of this rare tumor are known to occur , with the major pitfalls being its association with aneurysmal bone cyst ( abc ) leading to a nonrepresentative aspirate and the occurrence of a plethora of giant cell - rich lesions in the differentials .", "in one large series of 12 cases of chondroblastoma , the authors described the cytomorphology of this tumor in detail .", "the diagnosis was missed on fnac , among which , one was associated with abc and in another case an erroneous diagnosis of a giant cell tumor was made .", "the cause of misdiagnosis in one case was due to its association with abc , leading to nonrepresentative aspirate . in the second case , due to the location of the tumor in preauricular region , cartilaginous stroma aspirated", ", in a study of 110 cases of bone lesions attributed the missed diagnosis in two cases of chondroblastoma at fnac to inadequate sampling and misinterpretation as giant cell tumor .", "in giant cell tumor , the mononuclear cells are spindle - shaped and occur in clusters . in chondromyxoid fibroma , the smears are sparsely cellular and lack the mononuclear cell component . instead , stellate cells with pleomorphism are noted .", "aspirates in abc are usually hemorrhagic , scantily cellular , composed of osteoclast giant cells , histiocytes , osteoblasts , and spindle cells .", "in addition to chondroblastoma , abc can occur secondarily in giant cell tumor and osteosarcoma ."], ["this report reiterates the cytomorphologic findings in chondroblastoma and its differential diagnosis from other giant cell containing lesions of bone .", "the collective application of the knowledge of the cytomorphological features of this rare bone tumor would aid in accurate preoperative diagnosis even in the absence of typical clinical and radiologic presentations ."], [""], [""]]}
{"article_id": "PMC4211911", "article_text": ["suicide is a grave public health issue with a global mortality rate of 16 per 100,000 individuals.1 suicide is one of the top three leading causes of death , particularly among individuals aged 1544 years , which is a productive age group.1 suicide is a complex event that is influenced by biological , cultural , psychological , and socioeconomic factors .", "more than 90% of suicide victims were diagnosed with psychiatric problems at the time of death , and approximately two thirds had been diagnosed with depression.2,3 depressed patients tend to consult physicians other than psychiatrists , and a variety of medical professionals , including general practitioners , the staff of emergency departments , and non - psychiatric nurses , are key interveners for preventing suicide . among medical staff members , negative attitudes toward suicide such as", "anger could affect nontherapeutic reactions to suicidal individuals.4 furthermore , a belief that suicide is a personal right was negatively related to suicide intervention skills.5 an accurate assessment of the attitudes toward suicide held by medical staff members is relevant .", "previous studies have shown that difficulties with parental bonding during childhood could be a predisposing factor for the onset of many psychiatric conditions , such as anxiety , depressive states , and maladjusted behaviors.68 parental bonding and premorbid personality traits play an important role in shaping the developmental trajectory of an individual , including his / her ability to adjust to stressful events .", "although few studies have assessed the relationship between parental bonding and mental conditions among medical staff members,9,10 no study has evaluated the association between parental bonding and attitudes toward suicide .", "the objective of this study was to investigate whether parental bonding is associated with attitudes toward suicide among medical college students in japan .", "students in their fifth year of medical school at hirosaki university , hirosaki , japan , participated in the study .", "the surveys were distributed to 226 medical students . of the distributed 226 surveys , 160 questionnaires ( 116 males and 44 females )", "the demographic data ( age and sex ) were obtained from self - questionnaires and interviews .", "the data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and the subjects provided written informed consent before participating in the project . the parental bonding instrument ( pbi )", "was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 .", "a total of 12 items measure the dimension of care , and 13 items measure protection , both on a likert scale .", "the care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference .", "the overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy .", "higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective .", "we employed the japanese version of the attitudes toward suicide questionnaire ( atts ) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes , including ", " common occurrence  ,  suicidal expression as mere threat  ,  unjustified behavior  , ", "impulsiveness.12,13 each item , with the exception of items 10 and 28 , was scored on a five point scale from 1 ( strongly agree ) to 5 ( strongly disagree ) .", "items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 ( strongly disagree ) to 5 ( strongly agree ) .", "multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide .", "the data were analyzed using the pasw statistics software program for windows , version 18.0 .", "students in their fifth year of medical school at hirosaki university , hirosaki , japan , participated in the study .", "the surveys were distributed to 226 medical students . of the distributed 226 surveys , 160 questionnaires ( 116 males and 44 females )", "the demographic data ( age and sex ) were obtained from self - questionnaires and interviews .", "the data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and the subjects provided written informed consent before participating in the project .", "the parental bonding instrument ( pbi ) was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 .", "a total of 12 items measure the dimension of care , and 13 items measure protection , both on a likert scale .", "the care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference .", "the overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy .", "higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective .", "we employed the japanese version of the attitudes toward suicide questionnaire ( atts ) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes , including ", " common occurrence  ,  suicidal expression as mere threat  ,  unjustified behavior  , ", "impulsiveness.12,13 each item , with the exception of items 10 and 28 , was scored on a five point scale from 1 ( strongly agree ) to 5 ( strongly disagree ) .", "items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 ( strongly disagree ) to 5 ( strongly agree ) .", "multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide .", "the data were analyzed using the pasw statistics software program for windows , version 18.0 .", "table 1 presents the items included in the six factor atts with their descriptive data .", "paternal care was significantly associated with paternal protection , maternal care , maternal protection , and right to suicide .", "maternal care was significantly associated with maternal protection , right to suicide , and common occurrence .", "right to suicide was significantly associated with common occurrence , unjustified behavior , and preventability / readiness to help .", "table 3 shows the multiple regression analysis of the atts sub - scales with the pbi scores . after adjusting for age and sex ,", "maternal care approached a statistically significant association with the right to suicide . under the same conditions , maternal care", "the present study assessed attitudes toward suicide and their associations with parental bonding among japanese medical college students .", "the majority of the participants in our study agreed that anyone could commit suicide ( 88.8% ) and that suicide is preventable ( 86.3% ) .", "in addition , the multiple regression analysis revealed that participants who reported a higher level of maternal care thought that suicide was a common occurrence and tended to think that people do not have the right to commit suicide .", "domino and takahashi used the suicide opinion questionnaire to investigate the differences in attitudes toward suicide between students in japan and those in the united states , and the results demonstrated that the scores for  right to die  and  normality  were higher in japanese students and that those for  aggression  were higher in american students.14 etzersdorfer et al compared the attitudes towards suicide held by medical students in madras with those in vienna , finding that medical students in madras rejected the right to commit suicide and assisted suicide to a greater degree than the students in vienna.15 in a swedish study to assess whether attitudes differ between students at the beginning and end of their studies , students in the final year of medical school more frequently considered suicide to be an expression of psychiatric disease and thought that people attempting to commit suicide were not responsible for their actions.16 in addition , a study from japan revealed that sympathetic comments increased with the amount of years in school among students while critical comments decreased.17 in japan , mentality on suicide might be more culturally visible and acceptable than other countries . in past years , suicide was regarded as an honorable solution to personal guilt and failure . regarding occupational differences in attitudes toward suicide ,", "onc et al found that medical students and general practitioners held the most permissive attitudes in turkey.18 in addition , they showed that the attitude scores of general practitioners regarding their preparedness to prevent suicide were lower compared to those of other groups . in japan , kodaka et al reported attitudes toward suicide among pharmacists using the atts.13 more than half of pharmacists reported that people understand the wishes of others to commit suicide , and 16.8% of the participants thought that people who mention suicide do not actually take their own lives .", "a 10 year follow - up study of norwegian physicians ( n=631 ) revealed that a low level of maternal care predicted severe depressive symptoms and partially mediated low self - esteem.9 lung et al assessed the psychological effects on 127 health care workers who had cared for suspected severe acute respiratory syndrome ( sars ) patients .", "early maternal attachment and neuroticism were found to heavily affect mental health during life - threatening stress.10 distinct patterns of parental bonding may contribute to the development of different dysfunctional schemas which may influence attitudes toward suicide.19 there are several limitations in this study .", "first , we used pbi to assess the child rearing behavior experienced by our participants .", "although pbi has shown basic reliability for long periods,20 we could not completely rule out possible influences of the current mood state or recall bias in individuals because pbi is an instrument for measuring recalled parental behaviors .", "second , certain parameters were not assessed in this study personality traits , a history of psychiatric disorders , suicidal behavior , and current mood state could affect the attitude towards suicidal behavior . third , because the participants in our study are medical students , we can not generalize these results to general practitioners .", "our study indicated that the majority of participants seriously believe that anyone could commit suicide .", "participants who have experienced a higher level of maternal care thought that suicide occurs commonly and tended to think that people do not have the right to commit suicide .", "although parental bonding predisposes these attitudes toward suicide , educational programs could change caretaking practices.21 to achieve more positive attitudes towards suicidal behavior , promoting the best practices for suicide prevention is needed for medical students ."], "abstract_text": ["<S> backgroundsuicide is a grave public health issue that is responsible for a high mortality rate among individuals aged 1544 years . </S>", "<S> attitudes toward suicide among medical staff members have been associated with appropriate therapeutic responses to suicidal individuals . </S>", "<S> the aim of this study was to examine the effects of parental rearing on attitudes toward suicide among japanese medical college students.methodswe examined the association between parental bonding and attitudes toward suicide in 160 medical college students in japan . </S>", "<S> the parental bonding instrument was used to assess the attitudes and behaviors of parents . </S>", "<S> the attitudes toward suicide were evaluated using the japanese version of the attitudes toward suicide questionnaire.resultsthe mean age of the subjects was 25.24.0 years old . </S>", "<S> the majority of the participants in our study agreed that anyone could commit suicide ( 88.8% ) and that suicide is preventable ( 86.3% ) . after adjusting for age and sex , multivariate regression analysis revealed that maternal care approached a statistically significant association with the  right to suicide  attitude . under the same conditions , </S>", "<S> maternal care was shown to be significantly associated with the  common occurrence  attitude . </S>", "<S> no other significant relationships were observed between parental bonding and attitudes toward suicide.conclusionthis study suggests that a higher level of maternal care ensures that children think that suicide occurs less commonly . </S>", "<S> the promotion of best practices for suicide prevention among medical students is needed . </S>", "<S> child rearing support might be associated with suicide prevention . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Participants", "Procedure", "Statistical analysis", "Results", "Discussion", "Conclusion"], "sections": [["suicide is a grave public health issue with a global mortality rate of 16 per 100,000 individuals.1 suicide is one of the top three leading causes of death , particularly among individuals aged 1544 years , which is a productive age group.1 suicide is a complex event that is influenced by biological , cultural , psychological , and socioeconomic factors .", "more than 90% of suicide victims were diagnosed with psychiatric problems at the time of death , and approximately two thirds had been diagnosed with depression.2,3 depressed patients tend to consult physicians other than psychiatrists , and a variety of medical professionals , including general practitioners , the staff of emergency departments , and non - psychiatric nurses , are key interveners for preventing suicide . among medical staff members , negative attitudes toward suicide such as", "anger could affect nontherapeutic reactions to suicidal individuals.4 furthermore , a belief that suicide is a personal right was negatively related to suicide intervention skills.5 an accurate assessment of the attitudes toward suicide held by medical staff members is relevant .", "previous studies have shown that difficulties with parental bonding during childhood could be a predisposing factor for the onset of many psychiatric conditions , such as anxiety , depressive states , and maladjusted behaviors.68 parental bonding and premorbid personality traits play an important role in shaping the developmental trajectory of an individual , including his / her ability to adjust to stressful events .", "although few studies have assessed the relationship between parental bonding and mental conditions among medical staff members,9,10 no study has evaluated the association between parental bonding and attitudes toward suicide .", "the objective of this study was to investigate whether parental bonding is associated with attitudes toward suicide among medical college students in japan ."], ["students in their fifth year of medical school at hirosaki university , hirosaki , japan , participated in the study .", "the surveys were distributed to 226 medical students . of the distributed 226 surveys , 160 questionnaires ( 116 males and 44 females )", "the demographic data ( age and sex ) were obtained from self - questionnaires and interviews .", "the data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and the subjects provided written informed consent before participating in the project . the parental bonding instrument ( pbi )", "was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 .", "a total of 12 items measure the dimension of care , and 13 items measure protection , both on a likert scale .", "the care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference .", "the overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy .", "higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective .", "we employed the japanese version of the attitudes toward suicide questionnaire ( atts ) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes , including ", " common occurrence  ,  suicidal expression as mere threat  ,  unjustified behavior  , ", "impulsiveness.12,13 each item , with the exception of items 10 and 28 , was scored on a five point scale from 1 ( strongly agree ) to 5 ( strongly disagree ) .", "items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 ( strongly disagree ) to 5 ( strongly agree ) .", "multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide .", "the data were analyzed using the pasw statistics software program for windows , version 18.0 ."], ["students in their fifth year of medical school at hirosaki university , hirosaki , japan , participated in the study .", "the surveys were distributed to 226 medical students . of the distributed 226 surveys , 160 questionnaires ( 116 males and 44 females )", "the demographic data ( age and sex ) were obtained from self - questionnaires and interviews .", "the data collection for this study was approved by the ethics committee of the hirosaki university school of medicine , and the subjects provided written informed consent before participating in the project ."], ["the parental bonding instrument ( pbi ) was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 .", "a total of 12 items measure the dimension of care , and 13 items measure protection , both on a likert scale .", "the care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference .", "the overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy .", "higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective .", "we employed the japanese version of the attitudes toward suicide questionnaire ( atts ) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes , including ", " common occurrence  ,  suicidal expression as mere threat  ,  unjustified behavior  , ", "impulsiveness.12,13 each item , with the exception of items 10 and 28 , was scored on a five point scale from 1 ( strongly agree ) to 5 ( strongly disagree ) .", "items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 ( strongly disagree ) to 5 ( strongly agree ) ."], ["multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide .", "the data were analyzed using the pasw statistics software program for windows , version 18.0 ."], ["table 1 presents the items included in the six factor atts with their descriptive data .", "paternal care was significantly associated with paternal protection , maternal care , maternal protection , and right to suicide .", "maternal care was significantly associated with maternal protection , right to suicide , and common occurrence .", "right to suicide was significantly associated with common occurrence , unjustified behavior , and preventability / readiness to help .", "table 3 shows the multiple regression analysis of the atts sub - scales with the pbi scores . after adjusting for age and sex ,", "maternal care approached a statistically significant association with the right to suicide . under the same conditions , maternal care"], ["the present study assessed attitudes toward suicide and their associations with parental bonding among japanese medical college students .", "the majority of the participants in our study agreed that anyone could commit suicide ( 88.8% ) and that suicide is preventable ( 86.3% ) .", "in addition , the multiple regression analysis revealed that participants who reported a higher level of maternal care thought that suicide was a common occurrence and tended to think that people do not have the right to commit suicide .", "domino and takahashi used the suicide opinion questionnaire to investigate the differences in attitudes toward suicide between students in japan and those in the united states , and the results demonstrated that the scores for  right to die  and  normality  were higher in japanese students and that those for  aggression  were higher in american students.14 etzersdorfer et al compared the attitudes towards suicide held by medical students in madras with those in vienna , finding that medical students in madras rejected the right to commit suicide and assisted suicide to a greater degree than the students in vienna.15 in a swedish study to assess whether attitudes differ between students at the beginning and end of their studies , students in the final year of medical school more frequently considered suicide to be an expression of psychiatric disease and thought that people attempting to commit suicide were not responsible for their actions.16 in addition , a study from japan revealed that sympathetic comments increased with the amount of years in school among students while critical comments decreased.17 in japan , mentality on suicide might be more culturally visible and acceptable than other countries . in past years , suicide was regarded as an honorable solution to personal guilt and failure . regarding occupational differences in attitudes toward suicide ,", "onc et al found that medical students and general practitioners held the most permissive attitudes in turkey.18 in addition , they showed that the attitude scores of general practitioners regarding their preparedness to prevent suicide were lower compared to those of other groups . in japan , kodaka et al reported attitudes toward suicide among pharmacists using the atts.13 more than half of pharmacists reported that people understand the wishes of others to commit suicide , and 16.8% of the participants thought that people who mention suicide do not actually take their own lives .", "a 10 year follow - up study of norwegian physicians ( n=631 ) revealed that a low level of maternal care predicted severe depressive symptoms and partially mediated low self - esteem.9 lung et al assessed the psychological effects on 127 health care workers who had cared for suspected severe acute respiratory syndrome ( sars ) patients .", "early maternal attachment and neuroticism were found to heavily affect mental health during life - threatening stress.10 distinct patterns of parental bonding may contribute to the development of different dysfunctional schemas which may influence attitudes toward suicide.19 there are several limitations in this study .", "first , we used pbi to assess the child rearing behavior experienced by our participants .", "although pbi has shown basic reliability for long periods,20 we could not completely rule out possible influences of the current mood state or recall bias in individuals because pbi is an instrument for measuring recalled parental behaviors .", "second , certain parameters were not assessed in this study personality traits , a history of psychiatric disorders , suicidal behavior , and current mood state could affect the attitude towards suicidal behavior . third , because the participants in our study are medical students , we can not generalize these results to general practitioners ."], ["our study indicated that the majority of participants seriously believe that anyone could commit suicide .", "participants who have experienced a higher level of maternal care thought that suicide occurs commonly and tended to think that people do not have the right to commit suicide .", "although parental bonding predisposes these attitudes toward suicide , educational programs could change caretaking practices.21 to achieve more positive attitudes towards suicidal behavior , promoting the best practices for suicide prevention is needed for medical students ."]]}
{"article_id": "PMC4686224", "article_text": ["copd is characterized by persistent airway obstruction , usually of a progressive nature and associated with an enhanced chronic inflammatory response in airways to noxious particles or gases.1 copd is associated with increased mortality and reduced life expectancy.2 of the 3.8 million mortalities caused by chronic respiratory system diseases in 2010 , 2.9 million were by copd.3 the first consensus report for the diagnosis , management , and prevention of copd was published in 2001 by the global initiative for chronic obstructive lung disease ( gold ) committee , and since then it has been updated several times .", "the major objectives of gold are to increase the awareness of copd and to help the millions of people who suffer from this disease and die prematurely from it or its complications by increasing the standards of care .", "the low adherence to guideline treatment recommendations has been reported in several studies.46 under or overprescription cause the inappropriate treatment of the disease.4,7 undertreatment of copd has also been reported in turkey.8 the bold study in adana , turkey showed that only 12.3% of the adults with copd used medication for their diseases in 2004.9 the chronic diseases and risk factors in turkey survey reported that 46.1% of the patients clinically diagnosed with copd used regular medication in 2011.10 to date , there has been no study on adherence to the gold guidelines for the treatment of copd among pulmonary physicians in turkey . at the time of the study", ", the patients with copd were still treated according to the gold 2010 guideline recommendations .", "therefore , stages and treatments in this study are also classified in accordance with the gold 2010.1 the aim of this study is to investigate the adherence rates to gold 2010 recommendations for stable copd treatment by pulmonary physicians with a national survey in outpatient clinics of turkish pulmonary units from different regions of the country .", "this multi - center , cross - sectional study was conducted with the participation of the pulmonary outpatient clinics of eleven centers ( university hospitals , research and training hospitals , state hospitals , and private hospitals ) from eight cities ( adana , ankara , diyarbakir , erzurum , istanbul , izmir , kocaeli , and samsun ) across turkey .", "sample size estimation has been calculated in order to have the representation of all gold stages , and sequential recruitment of 100 patients was anticipated per center .", "the study was approved by the institutional ethics committee of ankara university school of medicine ( november 21 , 2010 ; # 24,596 ) prior to its initiation and conducted in accordance with the latest version of declaration of helsinki and international conference of harmonization / good clinical practice guideline .", "the inclusion criteria included doctor s diagnosis of copd , age 40 years , available pulmonary function test ( pft ) results from within the past 2 years and copd treatment data from any time within 6 months of the test , and formal written consent .", "patients with a concomitant chronic disease that leads to obstructive ( ie , asthma ) or restrictive ( ie , disseminated bronchiectasis ) changes on pft , with pft results discordant to gold diagnostic criteria ( symptoms of copd , and postbronchodilator spirometric evaluation confirming forced expiratory volume in 1 second ( fev1)/forced vital capacity [ fvc ] < 70% , exclusion of other obstructive lung diseases),1 and those with acute copd exacerbations were excluded from the study analysis . in nonsmoker patients ,", "biomass exposure is considered as one of the major risk factors especially in rural areas , and they were not excluded from the study if they meet the gold spirometric diagnostic criteria .", "patient recruitment has been performed according to the study inclusion and exclusion criteria in each study center .", "patient data were collected from hospital records on standardized forms and following parameters were recorded : any chronic diseases accompanying copd , age at the time of copd diagnosis , spirometry results ( fev1 and fev1/fvc ) and copd treatment recommended within 6 months of this result , and demographic information such as sex , age , location of residence , occupation , level of education , and health insurance status .", "overall , 1,125 patients have been recruited , and 719 patients meeting the gold diagnostic criteria ( fev1/fvc < 70% ) were included in this analysis .", "appropriateness of treatment was established in accordance with the 2010 gold guideline recommendations below : \n in stage i , add short - acting bronchodilator when needed.in stage ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama]).in stages iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 in stage i , add short - acting bronchodilator when needed . in stage", "ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama ] ) . in stages", "iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 all other treatment approaches ( under- or overtreatment ) were evaluated as inappropriate treatment .", "data are reported as mean and sd ; median , minimum , and maximum ; or frequency and percentage .", "monte carlo simulation was used for multigroup comparisons where chi - square test assumptions were not fulfilled .", "bonferroni - corrected mann  whitney u - test was used for subgroup comparisons within numeric variables .", "seven - hundred and nineteen patients ( 614 males , 105 females ) with mean  sd age of 62.99.7 years were included .", "six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% .", "of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv .", "mean  sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv .", "there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) .", "of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity .", "the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) .", "overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group .", "appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage", "i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss .", "the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii ,", "adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) .", "of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations .", "the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) .", "of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics .", "the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage", "iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss .", "the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) .", "of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics .", "the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated .", "inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) .", "the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) .", "only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients ,", "guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment .", "while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd .", "the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively .", "the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 .", "of all the patients , 614 ( 89% ) received treatment containing ics or ics alone .", "the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage", "iv . of the 25 ( 3.5% ) patients who were not on any medication ,", "one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy .", "of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy .", "long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients .", "seven - hundred and nineteen patients ( 614 males , 105 females ) with mean  sd age of 62.99.7 years were included .", "six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% .", "of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv .", "mean  sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv .", "there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) .", "of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity .", "the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) .", "overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group .", "appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage", "i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss .", "the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii ,", "adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) .", "of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations .", "the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) .", "of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics .", "the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage", "iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss .", "the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) .", "of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics .", "the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated .", "inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) .", "the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) .", "only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients ,", "guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment .", "while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd .", "the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively . the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 .", "of all the patients , 614 ( 89% ) received treatment containing ics or ics alone .", "the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage iv . of the 25 ( 3.5% ) patients who were not on any medication , one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy .", "of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy .", "long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients .", "this is the first large - scale real - life study in turkey that reflects the clinical practice of pulmonary specialists through an observational analysis of their adherence to treatment recommendations of gold 2010 guideline .", "the survey was carried out in eleven pulmonary outpatient clinics spread over the national territory .", "our findings show that only 59.5% of overall patients were treated in accordance with the guideline recommendations .", "while inappropriate treatment regimens were more frequently prescribed for stages i ( 93.8% ) and ii ( 85.3% ) , overtreatment in early stages and high rate of ics administration ( 89% ) in all stages of disease resulted in improper therapeutic approach according to gold 2010 recommendations . in more severe disease stages ,", "guideline - adherent therapeutic approaches were detected by wide use of ics addition to one or more long - acting bronchodilators .", "most of the provider s guideline adherence studies have reported the current practices of general practitioners .", "a few recent studies have reported similar rates of adherence to guideline recommendations for copd patients ( 37.9%54.7% ) between pulmonologists in other countries.4,5 stage i patients were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss , while guidelines recommend short - acting bronchodilators as initial therapy . in a study conducted among the pulmonary specialists in italy , corrado and rossi reported that 87.7% of the stage i patients received regular treatment containing long - acting bronchodilators , ics alone , or other treatment combinations of ics plus long - acting bronchodilators.4 in a retrospective database study in the united states , fitch et al reported that only 1% of the stage i patients received ics added to laba and lama , while 6% received ics plus single long - acting bronchodilators , and 76% did not receive any medication in stage i.11 shariff et al reported that 7.7% of the patients in stage i were overtreated and received inappropriate treatment according to the guidelines , in another study on guideline adherence among practitioners and pulmonary specialists.5 in our study , only 14.7% of the patients in stage ii received appropriate therapy , and this was mainly due to overtreatment which was combined long - acting bronchodilators and ics in 77.3% of the patients .", "other reasons for nonadherent therapeutic approaches were single use of ics in 3.4% of the patients and lack of treatment in 3.8% of the patients . other studies found 26%49.2% adherence to gold recommendations in this stage .", "overtreatment was 67.3% in one study,4 and lack of copd treatment has been reported in 54% of the patients in another report for stage ii.11 severe and very severe copd patients were treated by the combined use of one or more long - acting bronchodilators and ics which was compliant to gold recommendations .", "this finding can be explained by the features of this group of patients who are more symptomatic and have more frequent exacerbations .", "similar studies on guideline adherence in different countries also reported frequent use of dual and triple therapies in stages iii and iv.4,5,11 the ics was frequently used at all stages ( 43.8% in stage i , 81.5% in stage ii , 89.3% in stage iii , and 87.4% in stage iv ) mostly as a part of combination therapy .", "this study demonstrates that the pulmonary specialists in turkey have a high tendency to use ics for the treatment of patients with copd . in a study by de miguel - diez et al in spain ,", "34.5% of the patients were prescribed ics , alone or in combination , with a rate of inappropriate use of 18.2%.12 asche et al reported that 33% of the patients received combination treatment containing laba , lama , and/or ics consistent with the current gold guidelines.13 pulmonary specialists in our study mostly preferred to prescribe combination treatments containing ics .", "this might be related to a number of factors : \n inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment.concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist.reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance.preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available.increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use .", "inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment .", "concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist .", "reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance .", "preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available . increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use . in patients treated with regular bronchodilators ( alone or a part of a combination therapy ) ,", "the most frequently used drug was laba ( n=613 ) , followed by lama ( n = 575 ) , t ( n=197 ) . among these results ,", "t use was prescribed for 27.4% of stable copd patients which was remarkably higher than the finding of corrado and rossi that reported 2.2% of patients were using xanthines.4 gold remark on t is that it is effective in copd but , due to its potential toxicity , inhaled bronchodilators are preferred when available.1 in our study , this high rate of t prescription in addition to other inhaled bronchodilators may be explained by the choice of an oral bronchodilator drug even if it has potential toxicity and drug interactions in older patients .", "another finding of this study was that the use of long - acting bronchodilators such as laba and lama alone remains considerably low , and they have been mostly preferred in stages i , ii , and iii .", "the most commonly used bronchodilator in monotherapy was lama ( 4.2% ) , and this might be due to its single - dose use and duration of action of more than 24 hours.14,15 this study has several limitations .", "individual patient data were retrospectively collected through patient records : number of acute exacerbations in the previous year , complete information on comorbidities , adherence to nonpharmacologic interventions were lacking .", "gold guidelines were updated in 2011 to incorporate clinical presentation , including dyspnea as measured by copd assessment test and the modified british medical research council .", "the updated version of the guidelines incorporates clinical presentation with spirometry findings ( gold 2011).16 this study was undertaken between december 2010 and november 2011 .", "study protocol had been designed to evaluate gold 2010 guideline adherence . due to lack of knowledge on dyspnea level ( medical research council scale ) and exacerbation frequency during the last year", "even this new gold classification ( updated in 2011 ) has been changed by considering sypmtoms and exacerbations in addition to spirometric staging , we have not enough data as to the exact superiorities over the old gold classification .", "a very recent analysis showed that gold 2011 shifted the overall copd severity distribution to more severe categories .", "there were nearly three times more copd patients in stage d than in former stage iv ( p<0.05 ) .", "the predictive capacity for survival up to 10 years was significant for both systems ( p<0.01 ) and gold 2007 and 2011 did not differ significantly . in this study ,", "the percent predicted fev1 thresholds of 85% , 55% , and 35% were found better to stage copd severity for mortality , which are similar to the ones used previously in the gold 2010 classification . increasing intensity of treatment of patients with copd according to their gold 2011 reclassification", "is not known to improve health outcomes.17 the classification recommended by the gold committee since 2011 does not seem to be adopted universally throughout europe .", "even if there is a real improvement over the previous classification due to taking into account clinical criteria , quality of life , and exacerbations , criticisms have arisen concerning the choice of certain pathways and therapeutic recommendations not based on prospective studies with a high level of evidence.18 at the national level , we do not have any published data on the adoption of the new gold 2011 classification . according to the previously mentioned obstacles of the new classification , and lack of the knowledge about the frequency of new gold 2011 classification use , instead of the gold 2010 .", "we believe this data on the guideline adherence among pulmonologists may still reflect the current attitude toward the routine approach of the diagnosis and therapies of copd in turkey , and also is concordant with the real life situation around the world .", "turkish thoracic society has published an updated report of copd prevention , diagnosis and treatment in 2014,19 and included in the global alliance against chronic respiratory disease ( gard ) with the ministry of health.20 the improvement of adherence to national and international copd guidelines may be achieved by continuing medical education supported by national thoracic societies . increased awareness of guidelines may influence the standardized care of copd patients , and will have prognostic influences .", "promotion of the compliance to evidence - based medicine by health authorities may have also positive effects on the attitude change in the real - life applications .", "this study showed that the pulmonary specialists in turkey have low rates of adherence to gold guidelines for treatment of stable copd patients .", "major improper approaches were overtreatment in early stages and excessive use of inhaler corticosteroids in all stages of disease .", "new strategies are needed to achieve a standardized approach for the treatment of copd , and thus to improve adherence to guideline recommendations , with particular focus on treatment indications and overtreatment in turkey ."], "abstract_text": ["<S> backgroundlow adherence to global initiative for chronic obstructive lung disease ( gold ) guideline recommendations has been reported worldwide . </S>", "<S> there has been no study on the adherence to gold guidelines for copd treatment in turkey.objectivesto investigate the rates of adherence to gold 2010 guidelines for copd treatment among pulmonologists.designa multi - center , cross - sectional , observational study was carried out in eleven pulmonary outpatient clinics across turkey . </S>", "<S> adherence to gold was evaluated through hospital records . </S>", "<S> demographic and clinical data were recorded.resultsstudy included 719 patients ( mean age : 62.99.7 years ; males 85.4% ) of whom 16 was classified as gold stage i , 238 as ii , 346 as iii , and 119 as iv , and only 59.5% received appropriate treatment . </S>", "<S> rates of guideline adherence varied across gold stages ( i , 6.3% ; ii , 14.7% ; iii , 84.4% ; and iv , 84% ) . </S>", "<S> causes of inappropriate therapies were overtreatment ( stage i , 100% and stage ii , 91.1% ) , undertreatment ( stage iii , 3.3% and stage iv , 10.9% ) and lack of treatment ( stage ii , 3.8% ; stage iii , 2.3% ; and stage iv , 5.9% ) . the most preferred regimen ( 43.4% ) was long - acting 2-agonist - inhaled corticosteroid - long - acting muscarinic antagonist . </S>", "<S> overall , 614 patients ( 89% ) received treatment containing inhaled corticosteroid.conclusionpulmonologists in turkey have low rates of adherence to gold guidelines in copd treatment . </S>", "<S> inappropriateness of therapies was due to overtreatment in early stages and excessive use of inhaled corticosteroid ( ics ) in all disease stages . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Statistical analysis", "Results", "Patient characteristics", "Adherence to GOLD 2010 guideline recommendations for treatment", "Analysis of treatment approaches", "Discussion"], "sections": [["copd is characterized by persistent airway obstruction , usually of a progressive nature and associated with an enhanced chronic inflammatory response in airways to noxious particles or gases.1 copd is associated with increased mortality and reduced life expectancy.2 of the 3.8 million mortalities caused by chronic respiratory system diseases in 2010 , 2.9 million were by copd.3 the first consensus report for the diagnosis , management , and prevention of copd was published in 2001 by the global initiative for chronic obstructive lung disease ( gold ) committee , and since then it has been updated several times .", "the major objectives of gold are to increase the awareness of copd and to help the millions of people who suffer from this disease and die prematurely from it or its complications by increasing the standards of care .", "the low adherence to guideline treatment recommendations has been reported in several studies.46 under or overprescription cause the inappropriate treatment of the disease.4,7 undertreatment of copd has also been reported in turkey.8 the bold study in adana , turkey showed that only 12.3% of the adults with copd used medication for their diseases in 2004.9 the chronic diseases and risk factors in turkey survey reported that 46.1% of the patients clinically diagnosed with copd used regular medication in 2011.10 to date , there has been no study on adherence to the gold guidelines for the treatment of copd among pulmonary physicians in turkey . at the time of the study", ", the patients with copd were still treated according to the gold 2010 guideline recommendations .", "therefore , stages and treatments in this study are also classified in accordance with the gold 2010.1 the aim of this study is to investigate the adherence rates to gold 2010 recommendations for stable copd treatment by pulmonary physicians with a national survey in outpatient clinics of turkish pulmonary units from different regions of the country ."], ["this multi - center , cross - sectional study was conducted with the participation of the pulmonary outpatient clinics of eleven centers ( university hospitals , research and training hospitals , state hospitals , and private hospitals ) from eight cities ( adana , ankara , diyarbakir , erzurum , istanbul , izmir , kocaeli , and samsun ) across turkey .", "sample size estimation has been calculated in order to have the representation of all gold stages , and sequential recruitment of 100 patients was anticipated per center .", "the study was approved by the institutional ethics committee of ankara university school of medicine ( november 21 , 2010 ; # 24,596 ) prior to its initiation and conducted in accordance with the latest version of declaration of helsinki and international conference of harmonization / good clinical practice guideline .", "the inclusion criteria included doctor s diagnosis of copd , age 40 years , available pulmonary function test ( pft ) results from within the past 2 years and copd treatment data from any time within 6 months of the test , and formal written consent .", "patients with a concomitant chronic disease that leads to obstructive ( ie , asthma ) or restrictive ( ie , disseminated bronchiectasis ) changes on pft , with pft results discordant to gold diagnostic criteria ( symptoms of copd , and postbronchodilator spirometric evaluation confirming forced expiratory volume in 1 second ( fev1)/forced vital capacity [ fvc ] < 70% , exclusion of other obstructive lung diseases),1 and those with acute copd exacerbations were excluded from the study analysis . in nonsmoker patients ,", "biomass exposure is considered as one of the major risk factors especially in rural areas , and they were not excluded from the study if they meet the gold spirometric diagnostic criteria .", "patient recruitment has been performed according to the study inclusion and exclusion criteria in each study center .", "patient data were collected from hospital records on standardized forms and following parameters were recorded : any chronic diseases accompanying copd , age at the time of copd diagnosis , spirometry results ( fev1 and fev1/fvc ) and copd treatment recommended within 6 months of this result , and demographic information such as sex , age , location of residence , occupation , level of education , and health insurance status .", "overall , 1,125 patients have been recruited , and 719 patients meeting the gold diagnostic criteria ( fev1/fvc < 70% ) were included in this analysis .", "appropriateness of treatment was established in accordance with the 2010 gold guideline recommendations below : \n in stage i , add short - acting bronchodilator when needed.in stage ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama]).in stages iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 in stage i , add short - acting bronchodilator when needed . in stage", "ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama ] ) . in stages", "iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 all other treatment approaches ( under- or overtreatment ) were evaluated as inappropriate treatment ."], ["data are reported as mean and sd ; median , minimum , and maximum ; or frequency and percentage .", "monte carlo simulation was used for multigroup comparisons where chi - square test assumptions were not fulfilled .", "bonferroni - corrected mann  whitney u - test was used for subgroup comparisons within numeric variables ."], ["seven - hundred and nineteen patients ( 614 males , 105 females ) with mean  sd age of 62.99.7 years were included .", "six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% .", "of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv .", "mean  sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv .", "there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) .", "of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity .", "the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) .", "overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group .", "appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage", "i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss .", "the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii ,", "adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) .", "of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations .", "the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) .", "of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics .", "the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage", "iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss .", "the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) .", "of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics .", "the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated .", "inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) .", "the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) .", "only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients ,", "guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment .", "while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd .", "the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively .", "the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 .", "of all the patients , 614 ( 89% ) received treatment containing ics or ics alone .", "the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage", "iv . of the 25 ( 3.5% ) patients who were not on any medication ,", "one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy .", "of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy .", "long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients ."], ["seven - hundred and nineteen patients ( 614 males , 105 females ) with mean  sd age of 62.99.7 years were included .", "six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% .", "of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv .", "mean  sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv .", "there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) .", "of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity .", "the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) ."], ["overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group .", "appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage", "i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss .", "the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii ,", "adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) .", "of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations .", "the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) .", "of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics .", "the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage", "iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss .", "the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) .", "of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics .", "the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated .", "inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) .", "the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) .", "only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients ,", "guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment .", "while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd ."], ["the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively . the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 .", "of all the patients , 614 ( 89% ) received treatment containing ics or ics alone .", "the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage iv . of the 25 ( 3.5% ) patients who were not on any medication , one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy .", "of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy .", "long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients ."], ["this is the first large - scale real - life study in turkey that reflects the clinical practice of pulmonary specialists through an observational analysis of their adherence to treatment recommendations of gold 2010 guideline .", "the survey was carried out in eleven pulmonary outpatient clinics spread over the national territory .", "our findings show that only 59.5% of overall patients were treated in accordance with the guideline recommendations .", "while inappropriate treatment regimens were more frequently prescribed for stages i ( 93.8% ) and ii ( 85.3% ) , overtreatment in early stages and high rate of ics administration ( 89% ) in all stages of disease resulted in improper therapeutic approach according to gold 2010 recommendations . in more severe disease stages ,", "guideline - adherent therapeutic approaches were detected by wide use of ics addition to one or more long - acting bronchodilators .", "most of the provider s guideline adherence studies have reported the current practices of general practitioners .", "a few recent studies have reported similar rates of adherence to guideline recommendations for copd patients ( 37.9%54.7% ) between pulmonologists in other countries.4,5 stage i patients were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss , while guidelines recommend short - acting bronchodilators as initial therapy . in a study conducted among the pulmonary specialists in italy , corrado and rossi reported that 87.7% of the stage i patients received regular treatment containing long - acting bronchodilators , ics alone , or other treatment combinations of ics plus long - acting bronchodilators.4 in a retrospective database study in the united states , fitch et al reported that only 1% of the stage i patients received ics added to laba and lama , while 6% received ics plus single long - acting bronchodilators , and 76% did not receive any medication in stage i.11 shariff et al reported that 7.7% of the patients in stage i were overtreated and received inappropriate treatment according to the guidelines , in another study on guideline adherence among practitioners and pulmonary specialists.5 in our study , only 14.7% of the patients in stage ii received appropriate therapy , and this was mainly due to overtreatment which was combined long - acting bronchodilators and ics in 77.3% of the patients .", "other reasons for nonadherent therapeutic approaches were single use of ics in 3.4% of the patients and lack of treatment in 3.8% of the patients . other studies found 26%49.2% adherence to gold recommendations in this stage .", "overtreatment was 67.3% in one study,4 and lack of copd treatment has been reported in 54% of the patients in another report for stage ii.11 severe and very severe copd patients were treated by the combined use of one or more long - acting bronchodilators and ics which was compliant to gold recommendations .", "this finding can be explained by the features of this group of patients who are more symptomatic and have more frequent exacerbations .", "similar studies on guideline adherence in different countries also reported frequent use of dual and triple therapies in stages iii and iv.4,5,11 the ics was frequently used at all stages ( 43.8% in stage i , 81.5% in stage ii , 89.3% in stage iii , and 87.4% in stage iv ) mostly as a part of combination therapy .", "this study demonstrates that the pulmonary specialists in turkey have a high tendency to use ics for the treatment of patients with copd . in a study by de miguel - diez et al in spain ,", "34.5% of the patients were prescribed ics , alone or in combination , with a rate of inappropriate use of 18.2%.12 asche et al reported that 33% of the patients received combination treatment containing laba , lama , and/or ics consistent with the current gold guidelines.13 pulmonary specialists in our study mostly preferred to prescribe combination treatments containing ics .", "this might be related to a number of factors : \n inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment.concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist.reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance.preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available.increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use .", "inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment .", "concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist .", "reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance .", "preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available . increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use . in patients treated with regular bronchodilators ( alone or a part of a combination therapy ) ,", "the most frequently used drug was laba ( n=613 ) , followed by lama ( n = 575 ) , t ( n=197 ) . among these results ,", "t use was prescribed for 27.4% of stable copd patients which was remarkably higher than the finding of corrado and rossi that reported 2.2% of patients were using xanthines.4 gold remark on t is that it is effective in copd but , due to its potential toxicity , inhaled bronchodilators are preferred when available.1 in our study , this high rate of t prescription in addition to other inhaled bronchodilators may be explained by the choice of an oral bronchodilator drug even if it has potential toxicity and drug interactions in older patients .", "another finding of this study was that the use of long - acting bronchodilators such as laba and lama alone remains considerably low , and they have been mostly preferred in stages i , ii , and iii .", "the most commonly used bronchodilator in monotherapy was lama ( 4.2% ) , and this might be due to its single - dose use and duration of action of more than 24 hours.14,15 this study has several limitations .", "individual patient data were retrospectively collected through patient records : number of acute exacerbations in the previous year , complete information on comorbidities , adherence to nonpharmacologic interventions were lacking .", "gold guidelines were updated in 2011 to incorporate clinical presentation , including dyspnea as measured by copd assessment test and the modified british medical research council .", "the updated version of the guidelines incorporates clinical presentation with spirometry findings ( gold 2011).16 this study was undertaken between december 2010 and november 2011 .", "study protocol had been designed to evaluate gold 2010 guideline adherence . due to lack of knowledge on dyspnea level ( medical research council scale ) and exacerbation frequency during the last year", "even this new gold classification ( updated in 2011 ) has been changed by considering sypmtoms and exacerbations in addition to spirometric staging , we have not enough data as to the exact superiorities over the old gold classification .", "a very recent analysis showed that gold 2011 shifted the overall copd severity distribution to more severe categories .", "there were nearly three times more copd patients in stage d than in former stage iv ( p<0.05 ) .", "the predictive capacity for survival up to 10 years was significant for both systems ( p<0.01 ) and gold 2007 and 2011 did not differ significantly . in this study ,", "the percent predicted fev1 thresholds of 85% , 55% , and 35% were found better to stage copd severity for mortality , which are similar to the ones used previously in the gold 2010 classification . increasing intensity of treatment of patients with copd according to their gold 2011 reclassification", "is not known to improve health outcomes.17 the classification recommended by the gold committee since 2011 does not seem to be adopted universally throughout europe .", "even if there is a real improvement over the previous classification due to taking into account clinical criteria , quality of life , and exacerbations , criticisms have arisen concerning the choice of certain pathways and therapeutic recommendations not based on prospective studies with a high level of evidence.18 at the national level , we do not have any published data on the adoption of the new gold 2011 classification . according to the previously mentioned obstacles of the new classification , and lack of the knowledge about the frequency of new gold 2011 classification use , instead of the gold 2010 .", "we believe this data on the guideline adherence among pulmonologists may still reflect the current attitude toward the routine approach of the diagnosis and therapies of copd in turkey , and also is concordant with the real life situation around the world .", "turkish thoracic society has published an updated report of copd prevention , diagnosis and treatment in 2014,19 and included in the global alliance against chronic respiratory disease ( gard ) with the ministry of health.20 the improvement of adherence to national and international copd guidelines may be achieved by continuing medical education supported by national thoracic societies . increased awareness of guidelines may influence the standardized care of copd patients , and will have prognostic influences .", "promotion of the compliance to evidence - based medicine by health authorities may have also positive effects on the attitude change in the real - life applications .", "this study showed that the pulmonary specialists in turkey have low rates of adherence to gold guidelines for treatment of stable copd patients .", "major improper approaches were overtreatment in early stages and excessive use of inhaler corticosteroids in all stages of disease .", "new strategies are needed to achieve a standardized approach for the treatment of copd , and thus to improve adherence to guideline recommendations , with particular focus on treatment indications and overtreatment in turkey ."]]}
{"article_id": "PMC5119430", "article_text": ["amphotericin b ( amb ) , an amphoteric polyene macrolide , has been considered one of the main options of antifungal therapy for serious and life - threatening mycotic infections for over 50 years despite the introduction of newer antifungal medications such as the azoles ( e.g. , voriconazole ) and echinocandins ( e.g. , caspofungin ) , which have better safety profiles ( 1 , 2 ) .", "this may be due to the fact that azoles and echinocandins are costly , ineffective against certain pathogenic fungi ( e.g. , candida krusei and candida parapsilosis ) , and not readily available in developing countries such as iran ( 3 ) .", "many acute and chronic adverse reactions have been associated with amb , such as infusion - related reactions ( e.g. , fever , chills , and hypotension ) , normocytic normochromic anemia , cardiac toxicity ( e.g. , ventricular tachycardia and hypertension ) , hepatic toxicity ( e.g. , increase in liver enzymes and bilirubin ) , neurologic toxicity ( e.g. , confusion , delirium , tremor , and seizure ) , and nephrotoxicity ( 4 , 5 ) .", "major features of amb - induced nephrotoxicity include an increased serum creatinine level , a decreased glomerular filtration rate ( gfr ) , urinary potassium wasting , hypokalemia , urinary magnesium wasting , and hypomagnesemia ( 6 ) .", "although it is a common event in clinical settings , different aspects of amb nephrotoxicity have not been studied well or in detail in our population .", "the purpose of this investigation was to specifically assess the frequency , time onset , and possible associated factors of amb nephrotoxicity in hospitalized patients in hematology - oncology wards in the southwest of iran .", "a cross - sectional , observational study was performed over a period of 9 months from august 2015 to april 2016 in 2 hematology - oncology and 1 hematopoietic stem cell transplantation wards of namazi hospital , which is affiliated to shiraz university of medical sciences , shiraz , iran .", "the medical ethics committee of the hospital approved the study , and all patients signed and approved a written informed consent form .", "the inclusion criteria for recruiting patients were as follows : 1 ) age 15 years or older ; 2 ) no documented history of acute kidney injury defined by an increase in serum creatinine  0.3 mg / dl within 48 hours , or an increase in serum creatinine by  1.5 times the baseline in the previous 7 days , or a urine volume < 0.5 ml / kg / h for 6 hours ( 7 ) ; 3 ) no documented history of chronic kidney disease ( clearance creatinine below 60 ml / min/1.73 m calculated by the simplified modification of diet in renal disease equation ) ; 4 ) no documented history of peritoneal or hemodialysis for > 3 months ( 8) ; 5 ) no documented history of having received amb by any administration route in the previous 14 days ; and 6 ) having received any formulation of amb intravenously for at least 1 week .", "they included age , sex , weight , amb dose , duration and indication , duration of amb infusion , type of co - administered medications that may exacerbate or attenuate amb nephrotoxicity , length of hospital stay , and mortality .", "serum urea and creatinine were monitored daily during the course of the amb treatment . according to routine ward practice , serum potassium and magnesium", "were checked daily and once weekly for patients , respectively . because most cases of amb nephrotoxicity occur during the first 2 weeks of treatment ( 9 , 10 ) , urine urea , creatinine , sodium , potassium , and magnesium levels were determined at days 0 , 3 , 5 , 7 , 10 , and 14 of the amb treatment .", "serum as well as urine urea , creatinine , and the aforementioned electrolytes were determined using an auto - analyzer ( shanghai xunda medical instrument , shanghai , china ) .", "the creatinine levels in the serum and urine samples were established using the modified jaffe colorimetric reaction .", "amb nephrotoxicity was defined as an estimated clcr  50% ( calculated by the cockcroft - gault formula ) or an increase in serum creatinine ( double the baseline value ) ( 11 ) .", "either the fractional excretion of sodium > 2% or the fractional excretion of urea > 50% ( in cases of diuretic co - administration ) was considered as acute tubular necrosis ( atn ) ( 12 ) .", "serum level potassium and magnesium below 3 meq / l and 1.2 meq / l were defined as hypokalemia and hypomagnesemia , respectively ( 13 ) .", "a urine potassium - to - creatinine ratio above 13 meq / g was considered renal potassium wasting ( 14 ) .", "the time onset of nephrotoxicity , hypokalemia , and hypomagnesemia after the initiation of amb was also determined .", "daily dose reductions , every other day dosing , discontinuation , or performing dialysis as probable measures of managing amb nephrotoxicity were recorded .", "continuous data were expressed as either mean  standard deviation ( sd ) or mean  standard error ( se ) .", "probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test .", "the fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 .", "the parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests , respectively .", "using the  stepwise  method , the logistic regression analysis with the odds ratio ( or ) and 95% confidence interval ( ci ) were employed to determine the associated factors of amb nephrotoxicity . in the first step , the possible association of each independent variable , namely age , gender , amb cumulative dose , duration of amb infusion , baseline gfr value ,", "mean daily oral / intravenous sodium supplementation , and the co - administration of aminoglycosides , calcineurin inhibitors , vancomycin , acyclovir , loop diuretics , corticosteroids , and sodium bicarbonate , with amb nephrotoxicity ( as the dependent variable ) was assessed separately by univariate analysis . those with p values of less than 0.4", "statistical significance in all analyses was defined by p values < 0.05 , except for the first step of the logistic regression analysis ( p values < 0.4 ) .", "continuous data were expressed as either mean  standard deviation ( sd ) or mean  standard error ( se ) .", "probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test .", "the fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 .", "the parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests , respectively . using the  stepwise  method ,", "the logistic regression analysis with the odds ratio ( or ) and 95% confidence interval ( ci ) were employed to determine the associated factors of amb nephrotoxicity . in the first step , the possible association of each independent variable , namely age , gender , amb cumulative dose , duration of amb infusion", ", baseline gfr value , mean daily oral / intravenous sodium supplementation , and the co - administration of aminoglycosides , calcineurin inhibitors , vancomycin , acyclovir , loop diuretics , corticosteroids , and sodium bicarbonate , with amb nephrotoxicity ( as the dependent variable ) was assessed separately by univariate analysis .", "those with p values of less than 0.4 were then considered together for the multivariate logistic regression analysis .", "statistical significance in all analyses was defined by p values < 0.05 , except for the first step of the logistic regression analysis ( p values < 0.4 ) .", "during the 9-month study period , 90 patients who were scheduled to receive amb were screened .", "a flowchart detailing the reasons patients dropped out of the study is presented in figure 1 .", "the most common admission diagnosis of the patients was acute myeloid leukemia ( 60% ) followed by acute lymphoid leukemia ( 20% ) , hodgkin disease ( 5% ) , and aplastic anemia ( 5% ) .", "prophylaxis of aspergillosis ( 52.5% ) , treatment of aspergillosis ( 17.5% ) , and treatment of mucormycosis ( 12.5% ) were the three most frequent indications of amb administration ( table 1 ) .", "the mean  sd daily doses of conventional and liposomal amb were 28.79  10.84 mg and 212.78  99.33 mg , respectively .", "the mean  sd cumulative dose of conventional amb was 223.79  125.53 mg and 2367.15  2137.89 mg for liposomal amb .", "all courses of either conventional or liposomal amb infusions were administered within 2 - 6 hours .", "potential nephrotoxic medications , namely acyclovir , corticosteroids , calcineurin inhibitors ( cyclosporine ) , vancomycin , loop diuretics ( furosemide ) , and aminoglycosides ( amikacin ) , were given to 38 ( 95% ) , 28 ( 70% ) , 21 ( 52.5% ) , 19 ( 47.5% ) , 5 ( 12.5% ) , and 1 ( 2.5% ) patients , respectively .", "in contrast , spironolactone and sodium bicarbonate as potential nephroprotective agents were administered to 7 ( 17.5% ) and 4 ( 10% ) individuals , respectively .", "no patient received cisplatin , ifosfamide , cyclophosphamide , dopamine , and mannitol during the study period .", "the mean  sd daily oral / intravenous sodium supplementation administered in conjunction with the amb treatment was 124.75  62.15 meq . during the study ,", "11 ( 27.5% ) and 21 ( 52.5% ) patients developed amb nephrotoxicity and atn , respectively .", "the mean  sd onset of amb nephrotoxicity was 6.73  2.36 days . in accordance with the univariate analysis , amb indication ( p = 0.259 ) , vancomycin co - administration ( p = 0.388 ) , duration of liposomal amb infusion ( p = 0.141 ) , and amount of oral / intravenous sodium supplementation ( p = 0.375 )", "based on this model , none of these variables were significantly associated with amb nephrotoxicity ( table 2 ) .", "amb administration was discontinued permanently in 2 individuals who developed nephrotoxicity , and they underwent emergent hemodialysis .", "there was no statistically significant difference in the duration of hospitalization between the patients with and without amb nephrotoxicity ( 27.72  4.81 and 30.33  13.57 days , respectively ; p = 0.541 ) .", "the mortality rate was also comparable between the patients with ( 54.54% ) and without ( 48.27% ) amb nephrotoxicity ( p = 0.723 ) .", "the mean  sd onset of hypokalemia during amb administration was 5.06  3.35 days .", "only one documented episode of hypomagnesemia was detected in the study population during the amb treatment .", "considering the fact that serum magnesium levels were not measured routinely for most of the patients , determining the rate of renal magnesium wasting was not feasible .", "the co - administration of neither loop ( p = 0.642 ) nor potassium - sparing diuretics ( p = 0.211 ) was significantly associated with hypokalemia .", "the mean  se daily amounts of intravenous potassium and magnesium administered during the course of the amb treatment were 52.43  12.13 and 3.51  2.04 meq , respectively .", "the incidence ( 27.5% ) and time onset ( 5.06  3.35 days ) of amb nephrotoxicity in the present study were within the ranges reported in the literature .", "some degree of increase in serum creatinine has been detected within 2 weeks in up to 80% of patients who received amb ( 9 , 10 ) . in one of the most prominent studies in this regard", ", a 9-year retrospective analysis demonstrated that 138 ( 28% ) of 494 adult in - patients experienced some type of nephrotoxicity during amb treatment ( 10 ) . in a 1-year prospective observational study by tavakoli - ardakani et al .", "( 15 ) in the hematology - oncology and stem cell transplantation wards at taleghani hospital in tehran , 9 ( 25.71% ) out of 35 patients developed an increase in serum creatinine and bun during the course of amb treatment .", "this rate was reported to be 27.8% at another referral hematology - oncology and stem cell transplantation center in tehran ( 16 ) . in the adult infectious diseases ward at imam khomeini hospital in tehran , khalili et al .", "( 17 ) demonstrated that 10 ( 76.92% ) of 13 individuals receiving amb alone developed acute kidney injury ( aki ) ; however , the incidence of aki among the patients given amb along with ceftriaxone and/or vancomycin was 86.68% .", "the time onset of amb nephrotoxicity was not generally reported in the aforementioned iranian studies .", "the wide variations in the incidence of amb nephrotoxicity noted above could be due to different study methodologies , clinical settings , relevant risk factors , and definitions .", "notably , 62.5% of the individuals in our study were given liposomal amb , which has a considerably less nephrotoxic formulation .", "this could partially justify the relatively lower rate of amb nephrotoxicity in our cohort than in similar studies in iran . in the present study , amb nephrotoxicity in 45.45% of the affected patients resolved spontaneously without any intervention .", "only 2 patients in our cohort required emergency hemodialysis due to the severity and persistency of amb nephrotoxicity .", "it has been reported that amb nephrotoxicity is predominantly reversible within a few months after discontinuation of treatment ( 18 ) ; however , about 15% of affected patients may require renal replacement therapy such as dialysis ( 19 ) . in the shariati hematology - oncology and stem cell transplantation wards , hayatshahi et al .", "( 16 ) showed that amb nephrotoxicity led to a dose reduction of this agent in 3.7% of cases . in terms of clinical outcomes , mortality and duration of hospitalization were comparable between patients with and without amb nephrotoxicity in the present population .", "in contrast , bates et al.s ( 20 ) study of a large population ( 707 adults ) in the united states reported that acute renal failure due to amb increased the mean length of hospital stay by 8.2 days ( p < 0.0001 ) and increased the total cost of treatment by $ 29,823 .", "the mortality rate was also much higher ( 54% vs. 16% ; p = 0.001 ) .", "differences in the confounding factors ( e.g. , the severity of the underlying disease ) , the type of amb formulation used ( conventional vs. lipid - based ) , and sample size could partially account for these disparities .", "we considered p values greater than 0.05 ( 0.4 ) in the univariate analysis to select all possible variables linked to the dependent variable . among the different demographic , clinical , and paraclinical features , amb indication , vancomycin co - administration , duration of liposomal amb infusion , and amount of oral / intravenous sodium supplementation", "nevertheless , none of these variables were significantly associated with amb nephrotoxicity in the multivariate logistic regression model .", "several large - scale retrospective and prospective studies have identified males , an average daily dose of amb above 35 mg , a cumulative dose of amb greater than 2 - 5 g , dehydration , co - administered diuretics and nephrotoxic agents ( e.g. , aminoglycosides , cyclosporine , foscarnet , cisplatin , and ifosfamide ) or corticosteroids , and baseline renal dysfunction as potential risk factors for amb nephrotoxicity ( 21 - 23 ) .", "inadequate statistical power resulting from the relatively small sample size and the relatively low incidence of amb nephrotoxicity due to the administration of liposomal amb appear to be the main reasons for our findings in this regard .", "it has been suggested that conventional amb should preferably not be administered to patients with two or more of the aforementioned risk factors of amb nephrotoxicity ( 10 , 24 ) .", "notwithstanding , with the introduction of considerably less nephrotoxic formulations of amb into the market , this suggestion does not seem to make sense in clinical practice . during the course of the amb treatment in the current study ,", "hypokalemia and renal potassium wasting developed in 45% and 27.5% of the patients , respectively . in contrast , hypomagnesemia was detected in only 1 patient ( 2.5% ) .", "the incidence of amb - induced hypokalemia in our cohort was lower than that reported in the literature ( 75% - 90% ) ( 25 ) .", "this may be due to the fact that 62.5% of the patients in the present study received liposomal amb .", "several publications have estimated the frequency of amb - induced hypomagnesemia to be between 15% and 100% depending on the dose and formulation of amb ( 26 ) .", "apart from the type of amb formulation and the presence of relevant risk factors , such as the co - administration of loop and potassium - sparing diuretics , the low rate of hypomagnesemia in our population could have been because the patients serum magnesium levels were measured only once a week during the amb treatment in the studied wards .", "severe hypokalemia and hypomagnesemia due to amb can cause metabolic complications , rhabdomyolysis , and life - threatening arrhythmias ( 25 ) ; however , these adverse events were not observed in our cohort at least during the course of the amb treatment . in conclusion ,", "nearly one - third ( 27.5% ) of our cohort developed nephrotoxicity within the first week of amb treatment .", "amb nephrotoxicity resolved spontaneously in about half ( 45.45% ) of the affected patients without any intervention .", "mortality and the duration of hospitalization were comparable between patients with and without amb nephrotoxicity .", "no studied demographic , clinical , and paraclinical features of the study population were significantly associated with amb nephrotoxicity . among the studied electrolyte abnormalities , hypokalemia and renal potassium wasting were the most notable , affecting about one - half and one - third of amb recipients , respectively .", "the co - administration of either loop or potassium - sparing diuretics did not significantly affect electrolyte abnormalities during the course of the amb treatment .", "close monitoring of renal function indexes , including serum creatinine , bun , serum potassium , and magnesium , during amb treatment is highly recommended .", "additionally , implementing approved prophylactic measures , such as saline loading ( 150 meq / day ) before and/or during amb infusion , and exploiting lipid - based formulations of amb ( especially liposomal ) , if available and affordable , should be considered to minimize the possibility of amb nephrotoxicity ."], "abstract_text": ["<S> backgroundnephrotoxicity is the most clinically significant adverse reaction of amphotericin b. different aspects of amphotericin b ( amb ) nephrotoxicity have not been studied well in our population.objectivesthe purpose of this study was to assess the frequency , time onset , and possible associated factors of amb nephrotoxicity in hospitalized patients in hematology - oncology wards in the southwest of iran.patients and methodsa cross - sectional , observational study was performed over a period of 9 months at 2 hematology - oncology and 1 hematopoietic stem cell transplantation wards at namazi hospital . </S>", "<S> patients aged 15 years or older with no documented history of acute kidney injury or chronic kidney disease who were scheduled to receive formulations of amb intravenously for at least 1 week were included . </S>", "<S> the required demographic and clinical data of the patients were recorded . </S>", "<S> urine urea , creatinine , sodium , potassium , and magnesium levels were measured at days 0 , 3 , 5 , 7 , 10 , and 14 of the amb treatment . </S>", "<S> amb nephrotoxicity based on serum creatinine increase , renal potassium wasting , hypokalemia , and hypomagnesemia were determined.resultsamong the 40 patients recruited for the study , 11 ( 27.5% ) patients developed amb nephrotoxicity with a mean  standard deviation onset of 6.73  2.36 days . in 5 patients , </S>", "<S> amb nephrotoxicity resolved spontaneously without any intervention . according to the multivariate logistic regression model </S>", "<S> , none of the studied demographic , clinical , and paraclinical variables were significantly associated with amb nephrotoxicity . </S>", "<S> the duration of hospitalization ( p = 0.541 ) and the mortality rate ( p = 0.723 ) were comparable between the patients with and without amb nephrotoxicity . </S>", "<S> hypokalemia and renal potassium wasting were identified in 45% and 27.5% of the patients during amb treatment , respectively.conclusionsnearly one - third ( 27.5% ) of our cohort developed nephrotoxicity within the first week of amb treatment . </S>", "<S> hypokalemia and renal potassium wasting were more notable , affecting about one - half and one - third of the amb recipients , respectively . </S>"], "labels": null, "section_names": ["1. Background", "2. Objectives", "3. Patients and Methods", "3.1. Statistical Analysis", "4. Results", "5. Discussion"], "sections": [["amphotericin b ( amb ) , an amphoteric polyene macrolide , has been considered one of the main options of antifungal therapy for serious and life - threatening mycotic infections for over 50 years despite the introduction of newer antifungal medications such as the azoles ( e.g. , voriconazole ) and echinocandins ( e.g. , caspofungin ) , which have better safety profiles ( 1 , 2 ) .", "this may be due to the fact that azoles and echinocandins are costly , ineffective against certain pathogenic fungi ( e.g. , candida krusei and candida parapsilosis ) , and not readily available in developing countries such as iran ( 3 ) .", "many acute and chronic adverse reactions have been associated with amb , such as infusion - related reactions ( e.g. , fever , chills , and hypotension ) , normocytic normochromic anemia , cardiac toxicity ( e.g. , ventricular tachycardia and hypertension ) , hepatic toxicity ( e.g. , increase in liver enzymes and bilirubin ) , neurologic toxicity ( e.g. , confusion , delirium , tremor , and seizure ) , and nephrotoxicity ( 4 , 5 ) .", "major features of amb - induced nephrotoxicity include an increased serum creatinine level , a decreased glomerular filtration rate ( gfr ) , urinary potassium wasting , hypokalemia , urinary magnesium wasting , and hypomagnesemia ( 6 ) .", "although it is a common event in clinical settings , different aspects of amb nephrotoxicity have not been studied well or in detail in our population ."], ["the purpose of this investigation was to specifically assess the frequency , time onset , and possible associated factors of amb nephrotoxicity in hospitalized patients in hematology - oncology wards in the southwest of iran ."], ["a cross - sectional , observational study was performed over a period of 9 months from august 2015 to april 2016 in 2 hematology - oncology and 1 hematopoietic stem cell transplantation wards of namazi hospital , which is affiliated to shiraz university of medical sciences , shiraz , iran .", "the medical ethics committee of the hospital approved the study , and all patients signed and approved a written informed consent form .", "the inclusion criteria for recruiting patients were as follows : 1 ) age 15 years or older ; 2 ) no documented history of acute kidney injury defined by an increase in serum creatinine  0.3 mg / dl within 48 hours , or an increase in serum creatinine by  1.5 times the baseline in the previous 7 days , or a urine volume < 0.5 ml / kg / h for 6 hours ( 7 ) ; 3 ) no documented history of chronic kidney disease ( clearance creatinine below 60 ml / min/1.73 m calculated by the simplified modification of diet in renal disease equation ) ; 4 ) no documented history of peritoneal or hemodialysis for > 3 months ( 8) ; 5 ) no documented history of having received amb by any administration route in the previous 14 days ; and 6 ) having received any formulation of amb intravenously for at least 1 week .", "they included age , sex , weight , amb dose , duration and indication , duration of amb infusion , type of co - administered medications that may exacerbate or attenuate amb nephrotoxicity , length of hospital stay , and mortality .", "serum urea and creatinine were monitored daily during the course of the amb treatment . according to routine ward practice , serum potassium and magnesium", "were checked daily and once weekly for patients , respectively . because most cases of amb nephrotoxicity occur during the first 2 weeks of treatment ( 9 , 10 ) , urine urea , creatinine , sodium , potassium , and magnesium levels were determined at days 0 , 3 , 5 , 7 , 10 , and 14 of the amb treatment .", "serum as well as urine urea , creatinine , and the aforementioned electrolytes were determined using an auto - analyzer ( shanghai xunda medical instrument , shanghai , china ) .", "the creatinine levels in the serum and urine samples were established using the modified jaffe colorimetric reaction .", "amb nephrotoxicity was defined as an estimated clcr  50% ( calculated by the cockcroft - gault formula ) or an increase in serum creatinine ( double the baseline value ) ( 11 ) .", "either the fractional excretion of sodium > 2% or the fractional excretion of urea > 50% ( in cases of diuretic co - administration ) was considered as acute tubular necrosis ( atn ) ( 12 ) .", "serum level potassium and magnesium below 3 meq / l and 1.2 meq / l were defined as hypokalemia and hypomagnesemia , respectively ( 13 ) .", "a urine potassium - to - creatinine ratio above 13 meq / g was considered renal potassium wasting ( 14 ) .", "the time onset of nephrotoxicity , hypokalemia , and hypomagnesemia after the initiation of amb was also determined .", "daily dose reductions , every other day dosing , discontinuation , or performing dialysis as probable measures of managing amb nephrotoxicity were recorded .", "continuous data were expressed as either mean  standard deviation ( sd ) or mean  standard error ( se ) .", "probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test .", "the fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 .", "the parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests , respectively .", "using the  stepwise  method , the logistic regression analysis with the odds ratio ( or ) and 95% confidence interval ( ci ) were employed to determine the associated factors of amb nephrotoxicity . in the first step , the possible association of each independent variable , namely age , gender , amb cumulative dose , duration of amb infusion , baseline gfr value ,", "mean daily oral / intravenous sodium supplementation , and the co - administration of aminoglycosides , calcineurin inhibitors , vancomycin , acyclovir , loop diuretics , corticosteroids , and sodium bicarbonate , with amb nephrotoxicity ( as the dependent variable ) was assessed separately by univariate analysis . those with p values of less than 0.4", "statistical significance in all analyses was defined by p values < 0.05 , except for the first step of the logistic regression analysis ( p values < 0.4 ) ."], ["continuous data were expressed as either mean  standard deviation ( sd ) or mean  standard error ( se ) .", "probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test .", "the fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 .", "the parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests , respectively . using the  stepwise  method ,", "the logistic regression analysis with the odds ratio ( or ) and 95% confidence interval ( ci ) were employed to determine the associated factors of amb nephrotoxicity . in the first step , the possible association of each independent variable , namely age , gender , amb cumulative dose , duration of amb infusion", ", baseline gfr value , mean daily oral / intravenous sodium supplementation , and the co - administration of aminoglycosides , calcineurin inhibitors , vancomycin , acyclovir , loop diuretics , corticosteroids , and sodium bicarbonate , with amb nephrotoxicity ( as the dependent variable ) was assessed separately by univariate analysis .", "those with p values of less than 0.4 were then considered together for the multivariate logistic regression analysis .", "statistical significance in all analyses was defined by p values < 0.05 , except for the first step of the logistic regression analysis ( p values < 0.4 ) ."], ["during the 9-month study period , 90 patients who were scheduled to receive amb were screened .", "a flowchart detailing the reasons patients dropped out of the study is presented in figure 1 .", "the most common admission diagnosis of the patients was acute myeloid leukemia ( 60% ) followed by acute lymphoid leukemia ( 20% ) , hodgkin disease ( 5% ) , and aplastic anemia ( 5% ) .", "prophylaxis of aspergillosis ( 52.5% ) , treatment of aspergillosis ( 17.5% ) , and treatment of mucormycosis ( 12.5% ) were the three most frequent indications of amb administration ( table 1 ) .", "the mean  sd daily doses of conventional and liposomal amb were 28.79  10.84 mg and 212.78  99.33 mg , respectively .", "the mean  sd cumulative dose of conventional amb was 223.79  125.53 mg and 2367.15  2137.89 mg for liposomal amb .", "all courses of either conventional or liposomal amb infusions were administered within 2 - 6 hours .", "potential nephrotoxic medications , namely acyclovir , corticosteroids , calcineurin inhibitors ( cyclosporine ) , vancomycin , loop diuretics ( furosemide ) , and aminoglycosides ( amikacin ) , were given to 38 ( 95% ) , 28 ( 70% ) , 21 ( 52.5% ) , 19 ( 47.5% ) , 5 ( 12.5% ) , and 1 ( 2.5% ) patients , respectively .", "in contrast , spironolactone and sodium bicarbonate as potential nephroprotective agents were administered to 7 ( 17.5% ) and 4 ( 10% ) individuals , respectively .", "no patient received cisplatin , ifosfamide , cyclophosphamide , dopamine , and mannitol during the study period .", "the mean  sd daily oral / intravenous sodium supplementation administered in conjunction with the amb treatment was 124.75  62.15 meq . during the study ,", "11 ( 27.5% ) and 21 ( 52.5% ) patients developed amb nephrotoxicity and atn , respectively .", "the mean  sd onset of amb nephrotoxicity was 6.73  2.36 days . in accordance with the univariate analysis , amb indication ( p = 0.259 ) , vancomycin co - administration ( p = 0.388 ) , duration of liposomal amb infusion ( p = 0.141 ) , and amount of oral / intravenous sodium supplementation ( p = 0.375 )", "based on this model , none of these variables were significantly associated with amb nephrotoxicity ( table 2 ) .", "amb administration was discontinued permanently in 2 individuals who developed nephrotoxicity , and they underwent emergent hemodialysis .", "there was no statistically significant difference in the duration of hospitalization between the patients with and without amb nephrotoxicity ( 27.72  4.81 and 30.33  13.57 days , respectively ; p = 0.541 ) .", "the mortality rate was also comparable between the patients with ( 54.54% ) and without ( 48.27% ) amb nephrotoxicity ( p = 0.723 ) .", "the mean  sd onset of hypokalemia during amb administration was 5.06  3.35 days .", "only one documented episode of hypomagnesemia was detected in the study population during the amb treatment .", "considering the fact that serum magnesium levels were not measured routinely for most of the patients , determining the rate of renal magnesium wasting was not feasible .", "the co - administration of neither loop ( p = 0.642 ) nor potassium - sparing diuretics ( p = 0.211 ) was significantly associated with hypokalemia .", "the mean  se daily amounts of intravenous potassium and magnesium administered during the course of the amb treatment were 52.43  12.13 and 3.51  2.04 meq , respectively ."], ["the incidence ( 27.5% ) and time onset ( 5.06  3.35 days ) of amb nephrotoxicity in the present study were within the ranges reported in the literature .", "some degree of increase in serum creatinine has been detected within 2 weeks in up to 80% of patients who received amb ( 9 , 10 ) . in one of the most prominent studies in this regard", ", a 9-year retrospective analysis demonstrated that 138 ( 28% ) of 494 adult in - patients experienced some type of nephrotoxicity during amb treatment ( 10 ) . in a 1-year prospective observational study by tavakoli - ardakani et al .", "( 15 ) in the hematology - oncology and stem cell transplantation wards at taleghani hospital in tehran , 9 ( 25.71% ) out of 35 patients developed an increase in serum creatinine and bun during the course of amb treatment .", "this rate was reported to be 27.8% at another referral hematology - oncology and stem cell transplantation center in tehran ( 16 ) . in the adult infectious diseases ward at imam khomeini hospital in tehran , khalili et al .", "( 17 ) demonstrated that 10 ( 76.92% ) of 13 individuals receiving amb alone developed acute kidney injury ( aki ) ; however , the incidence of aki among the patients given amb along with ceftriaxone and/or vancomycin was 86.68% .", "the time onset of amb nephrotoxicity was not generally reported in the aforementioned iranian studies .", "the wide variations in the incidence of amb nephrotoxicity noted above could be due to different study methodologies , clinical settings , relevant risk factors , and definitions .", "notably , 62.5% of the individuals in our study were given liposomal amb , which has a considerably less nephrotoxic formulation .", "this could partially justify the relatively lower rate of amb nephrotoxicity in our cohort than in similar studies in iran . in the present study , amb nephrotoxicity in 45.45% of the affected patients resolved spontaneously without any intervention .", "only 2 patients in our cohort required emergency hemodialysis due to the severity and persistency of amb nephrotoxicity .", "it has been reported that amb nephrotoxicity is predominantly reversible within a few months after discontinuation of treatment ( 18 ) ; however , about 15% of affected patients may require renal replacement therapy such as dialysis ( 19 ) . in the shariati hematology - oncology and stem cell transplantation wards , hayatshahi et al .", "( 16 ) showed that amb nephrotoxicity led to a dose reduction of this agent in 3.7% of cases . in terms of clinical outcomes , mortality and duration of hospitalization were comparable between patients with and without amb nephrotoxicity in the present population .", "in contrast , bates et al.s ( 20 ) study of a large population ( 707 adults ) in the united states reported that acute renal failure due to amb increased the mean length of hospital stay by 8.2 days ( p < 0.0001 ) and increased the total cost of treatment by $ 29,823 .", "the mortality rate was also much higher ( 54% vs. 16% ; p = 0.001 ) .", "differences in the confounding factors ( e.g. , the severity of the underlying disease ) , the type of amb formulation used ( conventional vs. lipid - based ) , and sample size could partially account for these disparities .", "we considered p values greater than 0.05 ( 0.4 ) in the univariate analysis to select all possible variables linked to the dependent variable . among the different demographic , clinical , and paraclinical features , amb indication , vancomycin co - administration , duration of liposomal amb infusion , and amount of oral / intravenous sodium supplementation", "nevertheless , none of these variables were significantly associated with amb nephrotoxicity in the multivariate logistic regression model .", "several large - scale retrospective and prospective studies have identified males , an average daily dose of amb above 35 mg , a cumulative dose of amb greater than 2 - 5 g , dehydration , co - administered diuretics and nephrotoxic agents ( e.g. , aminoglycosides , cyclosporine , foscarnet , cisplatin , and ifosfamide ) or corticosteroids , and baseline renal dysfunction as potential risk factors for amb nephrotoxicity ( 21 - 23 ) .", "inadequate statistical power resulting from the relatively small sample size and the relatively low incidence of amb nephrotoxicity due to the administration of liposomal amb appear to be the main reasons for our findings in this regard .", "it has been suggested that conventional amb should preferably not be administered to patients with two or more of the aforementioned risk factors of amb nephrotoxicity ( 10 , 24 ) .", "notwithstanding , with the introduction of considerably less nephrotoxic formulations of amb into the market , this suggestion does not seem to make sense in clinical practice . during the course of the amb treatment in the current study ,", "hypokalemia and renal potassium wasting developed in 45% and 27.5% of the patients , respectively . in contrast , hypomagnesemia was detected in only 1 patient ( 2.5% ) .", "the incidence of amb - induced hypokalemia in our cohort was lower than that reported in the literature ( 75% - 90% ) ( 25 ) .", "this may be due to the fact that 62.5% of the patients in the present study received liposomal amb .", "several publications have estimated the frequency of amb - induced hypomagnesemia to be between 15% and 100% depending on the dose and formulation of amb ( 26 ) .", "apart from the type of amb formulation and the presence of relevant risk factors , such as the co - administration of loop and potassium - sparing diuretics , the low rate of hypomagnesemia in our population could have been because the patients serum magnesium levels were measured only once a week during the amb treatment in the studied wards .", "severe hypokalemia and hypomagnesemia due to amb can cause metabolic complications , rhabdomyolysis , and life - threatening arrhythmias ( 25 ) ; however , these adverse events were not observed in our cohort at least during the course of the amb treatment . in conclusion ,", "nearly one - third ( 27.5% ) of our cohort developed nephrotoxicity within the first week of amb treatment .", "amb nephrotoxicity resolved spontaneously in about half ( 45.45% ) of the affected patients without any intervention .", "mortality and the duration of hospitalization were comparable between patients with and without amb nephrotoxicity .", "no studied demographic , clinical , and paraclinical features of the study population were significantly associated with amb nephrotoxicity . among the studied electrolyte abnormalities , hypokalemia and renal potassium wasting were the most notable , affecting about one - half and one - third of amb recipients , respectively .", "the co - administration of either loop or potassium - sparing diuretics did not significantly affect electrolyte abnormalities during the course of the amb treatment .", "close monitoring of renal function indexes , including serum creatinine , bun , serum potassium , and magnesium , during amb treatment is highly recommended .", "additionally , implementing approved prophylactic measures , such as saline loading ( 150 meq / day ) before and/or during amb infusion , and exploiting lipid - based formulations of amb ( especially liposomal ) , if available and affordable , should be considered to minimize the possibility of amb nephrotoxicity ."]]}
{"article_id": "PMC3372779", "article_text": ["o - glycans have been found to play an important role in protein stability and tertiary structure , stabilize protein conformation , modulate the activity of enzymes ( e.g. by reversible attachment of o - linked glcnac to cytoplasmic and nuclear proteins ) and signaling molecules , are essential for a number of recognition processes and are sorting determinants guiding the modified protein in the cell from the place of biosynthesis to its target location [ 13 ] .", "unlike the n - glycans they may be linked to several different amino acid residues ( very often serine or threonine , but also hydroxylysine , tyrosine or hydroxyproline ) of the protein and their occurrence can not be predicted easily by a common consensus sequence .", "extended galnac residues linked to serine or threonine residues ( mucin - type glycans ) are the most frequent and best investigated o - glycans in higher eukaryotes .", "eight core subtype formations showing cell - tissue specific patterns related to their function have been described ( for a review see ) .", "also glcnac--ser / thr without any elongation , a typical feature for nuclear and cytoskeletal proteins existing in a dynamic equilibrium with phosphorylation , and fuc--ser / thr as well as glc--ser , primarily found in epidermal growth factor domains , have been found in eukaryotes .", "a linkage via mannose has been found to be the main o - glycan type in yeasts , but also some mammalian tissues carry this structural feature , which seems to be relevant in some muscular dystrophies , while o - xylosylation is the starting point for the biosynthesis of chondroitin and heparan sulfates , which have roles in development and morphogenesis .", "the o - glycans of nematodes are rather complex with methylation as further modification [ 9 , 10 ] .", "xenopus eggs have o - glycans with up to 10 monosaccharide constituents , often highly fucosylated , with a linkage to the protein via galnac [ 11 , 12 ] .", "even parasites ( helminths and t. cruzi ) display small galnac linked o - glycans . in plants", "o - glycans occur as arabinogalactans linked to hydroxyproline or serine ( gal--hyp / ser ) , or hydroxyproline is glycosylated with short arabinofuranosides ( ara--hyp , ara--hyp ) . here", "we present for the first time an overview on o - glycans of some species of the mollusk branch of the evolutionary tree .", "the glycosylation potential of snails is important in general , because some of them are intermediate hosts of pathogens ( biomphalaria glabrata and lymnaea stagnalis ) or a pest in agriculture", ". a broad knowledge of their glycosylation abilities may show targets for pest control in the near future .", "in the course of the years , n - glycan patterns of several snail species have been presented [ 1417 ] . methylated mannose and galactose residues were found to be frequent constituents . in the present study we determine the main o - glycan structures in snails and characterize a common core - trisaccharide .", "cepaea hortensis , planorbarius corneus , arion lusitanicus , limax maximus and helix pomatia were collected by the authors under the supervision of dr .", "manfred pintar ( department of integrative biology and biodiversity research , institute of zoology , university of natural resources and life sciences , vienna ) in the vicinity of vienna .", "all chemicals purchased were of the highest quality available from sigma or fluka . while water living snails could be dissected right away , land living snails were extensively washed to remove the extraneous mucous components .", "snails were dissected as previously described   and proteins were purified according to   with minor modifications : 1  g of wet tissue was homogenised in 10  ml of chaps - based lysis buffer ( 0.5  % ( w / v ) chaps , 150  mm nacl , 20  mm tris / hcl , 2.5  mm sodium pyrophosphate , 1  mm ethylene - glycol - bis(2-aminoethylether)-n , n , n,n-tetraacetic acid and 1  mm edta , ph  7.5 ) and incubated for 72  h at 4  c .", "snail proteins were then reduced by the addition of 500  l of 10  mm dithiothreitol and incubation at 56  c for 45  min .", "then 500  l of 55  mm iodoacetamide were added and incubated for 30  min at room temperature in the dark .", "the aqueous samples were precipitated with a 4-fold amount of pre - chilled acetone at 20  c for at least 4  h and then centrifuged at 32000  g for 60  min .", "the pellets were resuspended in 0.1  m sodium citrate buffer , ph  4.6 , and incubated with 2 units of -glucosidase from rice [ ec 3.2.1.20 ] at 37  c for 16  h. sugars and polysaccharides were eluted from cation exchange chromatography ( ag 50  w - x2 , biorad , ca ) with 2  % of acetic acid , while proteins were eluted by 1.0  m ammonium acetate , ph  9.0 .", "the protein fraction was again precipitated by acetone followed by a similar precipitation by methanol .", "the dry sample was dissolved in 0.1  m naoh containing 0.8  m nabh3 and incubated at 37  c for 68  h. the sample was carefully neutralised by the addition of 2  m acetic acid , dried in a vacuum centrifuge and washed at least twice with 30  % ( v / v ) methanol .", "the glycans were directly extracted by solid phase extraction on a porous graphitized carbon cartridge ( supelclean envi - carb , 100  mg bed weight , 1  ml column volume or 500  mg bed weight , 6  ml column volume , sigma - aldrich , germany ) according to   by using ammonium formate buffer / acetonitrile for elution   and then on a reversed - phase c18 cartridge ( strata , c18-e , 55  m , 50  mg bed volume , 1  ml column volume , phenomenex , germany ) using 5  % ( v / v ) acetic acid as loading solvent and 25  % ( v / v ) isopropanol in 5  % ( v / v ) acetic acid for elution .", "hplc separation was carried out on a porous graphitized carbon ( pgc ) column ( hypersil - keystone , hypercarb 5   150    3  mm , thermo scientific , austria ) at a flow rate of 0.6  ml / min using 0.3  % ( v / v ) formic acid adjusted to ph  3.1 with ammonium as solvent a and 95  % ( v / v ) acetonitrile in solvent a as solvent b. the elution protocol consisted of 2  min at 100  % ( v / v ) of solvent a followed by a gradient of 28  min till 25  % ( v / v ) of solvent b and a cleaning step of 4  min at 60  % ( v / v ) of solvent b. each fraction was tested by esi - ms for its glycan content .", "single glycan structures were pooled and lyophilised . for monosaccharide analysis alditol acetates and for linkage analysis", "gc analysis was carried out on a vf 5  ms capillary column ( 60  m , 0.25  mm inner diameter ; 0.1  mm film thickness ; varian , darmstadt , germany ) and electron impact ( ei ) mass spectrometry was performed in the positive ion mode using a polaris q instrument ( thermoquest - analytical systems ) . for analysis of small sample amounts with esi - ms an in - gel release method for glycans bound to coomassie", "reduced oligosaccharides were analyzed by pgc - lc - esi - ms on a hypercarb column ( 0.32    150  mm , thermo fisher scientific , austria ) coupled to an ultimate 3000 ( dionex ) capillary hplc and a q - tof ultima ms ( waters ) as described previously [ 21 , 26 ] .", "the interpretation of the ms data was performed with the help of the software tool glycoworkbench .", "arion lusitanicus proteins were isolated and separated from contaminants through several purification steps in order to obtain sufficient amounts ( 12  g ) of pure glycans for further gc - ms analysis ( total monosaccharide and linkage analysis ) .", "the preparation of o - glycans was carried out via -elimination followed by further separation steps . as a final procedure", "the glycans were separated on a pgc ( porous graphitized carbon ) hplc - column by collecting 1-minute fractions which were screened by esi - ms to gain clean single structures . in the course of the -elimination releasing the glycan from the protein backbone , sodium borohydride was used .", "in contrast , for reduction of monosaccharides before gc - ms analysis sodium borodeuteride was employed , which allowed further the discrimination between the sugar released by -elimination and those linked to other sugars previously . in fig", ".  1 monosaccharide constituents of the core trisaccharide representing the main structure found in a. lusitanicus , with a pseudomolecular mass ( [ m+h ] ) of m / z 576.3  da , were determined by gc - ms of the corresponding alditol acetates and identified on the basis of retention times in gc and their characteristic electron impact ( ei ) mass spectra .", "the results revealed the presence of a galnac - residue which was h - reduced , while all the other sugars were h reduced .", "therefore , galnac can be considered as the protein bound sugar . the high amount of 4-o - me - gal indicated that this residue is the one which is mainly involved in the elongation of the glycan .", "minor compounds were identified as contaminations by n - glycans ( glcnac and man ) or storage glycans ( glc and gal ) .", "the lc - esi - ms / ms fragmentation pattern confirmed the occurrence of two methylated hexoses linked to the amino sugar ( fig . ", "1monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination .", "alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms .", "peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced)fig 2lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus . registered ions represent proton adducts [ m+h ] monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination .", "alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms .", "peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced ) lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus .", "registered ions represent proton adducts [ m+h ] moreover , gc - ms analysis of partially methylated alditol acetates was carried out for further confirmation of the compounds and also for linkage determination .", "methylated sugar standards purified by preparative gc were used to elucidate the type of methylated hexose ( man or gal ) based on retention time .", "the selected ion chromatogram of the monosaccharide derivatives obtained from the o - glycan core structure identified 2,3,4,6-tetra - o - methyl - galacitol at 20.66  min and 1,4,5-tri - o - methyl galnac - ol at 33.33  min ( fig", ".  3a ) which could be verified by ei - ms data ( figs . ", "3b and c ) . to determine the position of the naturally occurring methyl group on the galactose", "this allows in the ei - ms fragmentation pattern the discrimination between the naturally occurring and introduced methylgroups in the fragments by a mass difference of 3  da . that way the occurrence of terminal 4-o - methylated galactose", "4 ) . combining these data , the main o - glycan structure of a. lusitanicus was elucidated as a trisaccharide containing two terminal 4-o - methylated galactoses which are 3- and 6-linked to an inner galnac residue ( fig . ", "5).fig 3linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol .", "characteristic primary selected secondary fragment ions are assignedfig 4ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide .", "the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of  glycoworkbench  .", "this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) .", "square  =  galnac , circles  =  gal , me  =  methyl group linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol .", "characteristic primary selected secondary fragment ions are assigned ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide .", "the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of  glycoworkbench  .", "this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) .", "square  =  galnac , circles  =  gal , me  =  methyl group a small amount of an isoform with the same molecular weight , but slightly later retention time in the pgc ( porous graphitized carbon ) selected ion chromatogram ( fig . ", "6c ) , was identified analogously by gc - ms and ei - ms as trisaccharide containing a galnac residue substituted by one 4-o - methylated galactose and one 3-o - methylated mannose ( data not shown).fig 6selected ion chromatograms of partially methylated structures obtained by lc - esi - ms .", "a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) .", "square  =  amino sugar , circle  =  hexose , me  =  methyl group selected ion chromatograms of partially methylated structures obtained by lc - esi - ms . a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) .", "square  =  amino sugar , circle  =  hexose , me  =  methyl group besides the trisaccharide core several other glycan species were found during purification on pgc - column .", "a glycan consisting of the methylated core trisaccharide and one further hexose ( m / z 738.3 [ m+h ] ) was found .", "this additional hexose was an unmethylated gal residue linked alternatively to one of the two arms of the trisaccharide explaining the two isoforms detected by pgc selected ion chromatogram ( fig . ", "the fourth structure ( m / z 914.4 [ m+h ] ) which was obtained in sufficient amounts for gc - ms consisted of the core trisaccharide elongated by an unmethylated hexose on one arm and a methylated hexose on the other one ( fig . ", "the presence of significant amounts of man and 3-o - me - man indicated that these two residues were responsible for the elongation .", "gc - ms analysis using different derivatisation protocols requested a high amount of material and high quality of purification . especially for snail tissues this was a challenge and", "therefore this method could not be carried out for a large number of samples in due time .", "an in - gel -elimination after sds - page followed by a short clean - up ( pgc - cartridge ) procedure was sufficient .", "the shortened protocol also ensured that no weakly bound modifications , such as sulfate or sialic acids for example , got lost . combining our data on snail monosaccharide analysis , data from previous gc - ms methylation analysis   and the fragmentation results of lc - esi - ms / ms analysis of selected snails we could determine the main structures of snail o - glycans and classify them according to their modification of the established core structure into six groups : ( i ) glycans smaller than the core , missing one or both methyl groups or missing one hexose ; ( ii ) the trisaccharide core containing galnac and two 4-o - methylated hexose residues ; ( iii ) the core with additional methylated hexoses ; ( iv ) the core with one or two additional unmethylated hexoses and sometimes one more methylated hexose ; ( v ) glycans containing fucose ; ( vi ) glycans containing one hexnac and up to six unmethylated hexoses .", "for an overview on the structures see table  1.table 1overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans .", "compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex)glycan compositiongroupobserved mass [ m+h]calculated mass [ m+h]arion lusitanicusachatina fulicacepaea hortensisplanorbarius corenusbiomphalaria glabratahelix pomatialimax maximusclea helenahexhexnac(i ) - glycans smaller than the core386.2386.1707000000mehexhexnac400.2400.1850310200hex2hexnac548.2548.22traces000traces000hexmehexhexnac562.3562.2390012182600(mehex)2hexnac(ii ) - core576.3576.2554692612292910089(mehex)3hexnac(iii ) - core with additional methyl hexoses752.3752.325212302700(mehex)4hexnac928.4928.39traces0000000(mehex)5hexnac1104.41104.46traces0000000hex(mehex)2hexnac(iv )  core with additional hexoses and methyl hexoses738.3738.30141617164512011hex2(mehex)2hexnac900.4900.36203720100hex(mehex)3hexnac914.4914.3725505400(mehex)2hexnacdhex(v ) - fucosylated structures722.3722.31220traces1traces00(mehex)3hexnacdhex898.4898.3860000000(mehex)4hexnacdhex1074.41074.44traces0000000hex3hexnac(vi )  hexnac and hexoses710.3710.2700061traces00hex4hexnac872.3872.32000460000hex5hexnac1034.31034.3800030000hex6hexnac1197.01196.43000traces0000 overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans .", "compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex ) analyzing snails in detail , it was obvious that the o - glycan trisaccharide core is an important decoration of snail proteins in all investigated species . in five of the analyzed snails ( a. lusitanicus , a. fulica , h. pomatia , l. maximus , c. helena ) this glycan was the most abundant type of o - glycan .", "b. glabrata and c. hortensis displayed as their main glycan the trisaccharide elongated by one or two unmethylated hexoses , respectively .", "only p. corneus was an exception possessing a large amount of hex4hexnac glycan . whereas the addition of one hexose to the core was relatively frequent , larger structures were minor compounds or were even not present in several species .", "the only detected amino sugar was galnac which provided in all cases the connection to the protein .", "gal and man were present in unmethylated or mono - methylated ( 3-o - me - gal , 4-o - me - gal , 3-o - me - man ) versions .", "fucose , which had been identified as such in previous works [ 23 , 28 ] , occurred only in low amounts and was detected linked to galnac as well as to terminal sugar residues by ms / ms fragmentation .", "during the last decades our knowledge on glycosylation processes - structure of the glycans and the corresponding biochemical pathways including the responsible enzymes - increased enormously , especially for glycans of mammalian origin .", "the glycosylation capabilities of other species were only under investigation if their glycans were for any reason connected with human life ( e.g. some recognition processes of pathogens or allergy on food or plant glycans ) or if they were potent candidates for cell culture systems for the expression of therapeutics ( some insect , yeast and plant cells ) .", "in the meantime more and more invertebrate systems are investigated because of their potential usefulness for biotechnological reasons or to get some deeper insights into the processing pathways of unusual glycan structures .", "previous studies on various invertebrates showed that glycosylation has a lot more varieties than we expected as long as only mammals were investigated , but the data are scattered .", "still the main focus is laid on n - glycans but the interest in o - glycans is growing .", "a number of difficulties arose during snail glycan analysis due to the tough tissue , which was not easy to homogenize and the rigid mucus , which had to be discarded diligently .", "the removal of storage carbohydrates , which might influence monosaccharide analysis and the separation of n- and o - glycans were other struggles to resolve . while the first was done by -glucosidase digestion from rice , the latter was an even more difficult task .", "n - glycosidases ( pngase a and f ) did not remove the n - glycans completely and even mild -elimination conditions seemed to be able to release , at least in part , residual n - glycans which resulted in n - glycan impurities in o - glycan fractions .", "furthermore some n - glycans of snails and digested storage glycans are so small ( 45 sugar residues [ 16 , 17 ] ) that they may co - migrate on hplc with o - glycans", ". therefore only a limited number of glycans could be prepared in terms of sufficient amount and purity for permethylation followed by maldi - tof - ms and gc - ms linkage analysis .", "however , in combination with lc - esi - ms of native glycans which is capable of a high throughput of samples the o - glycan pattern of 8 snail species ( land snails with shell : achatina fulica , cepaea hortensis and helix pomatia ; slugs : arion lusitanicus and limax maximus ; water snails : biomphalaria glabrata , clea helena , and planorbarius corneus ) could be identified .", "four monosaccharide constituents  galnac , gal , man and fuc  are the building blocks of the structures .", "the only further modification is a methylation of the hexoses , resulting in 3-o - me - gal , 4-o - me - gal and 3-o - me - man , which has already been shown for snail n - glycans [ 1417 , 23 ] .", "each o - glycan contains only one amino sugar , which is the protein linked galnac .", "the linkage amino acid was not determined in detail , but due to the easy release by -elimination the common galnac - ser / thr motif is very likely .", "the connection to other amino acids , for example tyrosine , is usually more stable . in snail o - glycans the inner galnac", "is frequently elongated by two 4-o - me - gal residues in ( 13)- and ( 16)-linkage . elongations of this trisaccharide core by one or two hexoses with or without methyl group appeared in most snail species , whereas further elongation or fucosylation are rare events .", "insects , one neighbor of mollusks in the phylogenetic tree , display also internal galnac elongated by one gal in their o - glycan structures , but further elongations and/or branching are frequently done by another amino sugar ( galnac or glcnac ) .", "the main o - glycans of toxocara canis are trisaccharides consisting of galnac , gal or 4-o - gal and 2-o - fuc .", "so far nothing is known about biological functions of snail glycans . for sure these glycans", "are similarly involved in recognition processes as they are in all other species , but especially the significance of the high degree of methylation of hexoses of n- as well as of o - glycans is waiting for elucidation . here", "we enlarge the current knowledge on glycosylation abilities of lower animals by presenting the first o - glycan analysis of snails , representatives of the mollusk phylum ."], "abstract_text": ["<S> the glycosylation abilities of snails deserve attention , because snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites . in analogy to many other host - pathogen relations , the glycosylation of snail proteins may likewise contribute to these host - parasite interactions . here </S>", "<S> we present an overview on the o - glycan structures of 8 different snails ( land and water snails , with or without shell ) : arion lusitanicus , achatina fulica , biomphalaria glabrata , cepaea hortensis , clea helena , helix pomatia , limax maximus and planorbarius corneus . </S>", "<S> the o - glycans were released from the purified snail proteins by -elimination . </S>", "<S> further analysis was carried out by liquid chromatography coupled to electrospray ionization mass spectrometry and  for the main structures  by gas chromatography / mass spectrometry . </S>", "<S> snail o - glycans are built from the four monosaccharide constituents : n - acetylgalactosamine , galactose , mannose and fucose . </S>", "<S> an additional modification is a methylation of the hexoses . </S>", "<S> the common trisaccharide core structure was determined in arion lusitanicus to be n - acetylgalactosamine linked to the protein elongated by two 4-o - methylated galactose residues . </S>", "<S> further elongations by methylated and unmethylated galactose and mannose residues and/or fucose are present . </S>", "<S> the typical snail o - glycan structures are different to those so far described . </S>", "<S> similar to snail n - glycan structures they display methylated hexose residues . </S>"], "labels": null, "section_names": ["Introduction", "Material and methods", "Results", "Discussion and conclusion"], "sections": [["o - glycans have been found to play an important role in protein stability and tertiary structure , stabilize protein conformation , modulate the activity of enzymes ( e.g. by reversible attachment of o - linked glcnac to cytoplasmic and nuclear proteins ) and signaling molecules , are essential for a number of recognition processes and are sorting determinants guiding the modified protein in the cell from the place of biosynthesis to its target location [ 13 ] .", "unlike the n - glycans they may be linked to several different amino acid residues ( very often serine or threonine , but also hydroxylysine , tyrosine or hydroxyproline ) of the protein and their occurrence can not be predicted easily by a common consensus sequence .", "extended galnac residues linked to serine or threonine residues ( mucin - type glycans ) are the most frequent and best investigated o - glycans in higher eukaryotes .", "eight core subtype formations showing cell - tissue specific patterns related to their function have been described ( for a review see ) .", "also glcnac--ser / thr without any elongation , a typical feature for nuclear and cytoskeletal proteins existing in a dynamic equilibrium with phosphorylation , and fuc--ser / thr as well as glc--ser , primarily found in epidermal growth factor domains , have been found in eukaryotes .", "a linkage via mannose has been found to be the main o - glycan type in yeasts , but also some mammalian tissues carry this structural feature , which seems to be relevant in some muscular dystrophies , while o - xylosylation is the starting point for the biosynthesis of chondroitin and heparan sulfates , which have roles in development and morphogenesis .", "the o - glycans of nematodes are rather complex with methylation as further modification [ 9 , 10 ] .", "xenopus eggs have o - glycans with up to 10 monosaccharide constituents , often highly fucosylated , with a linkage to the protein via galnac [ 11 , 12 ] .", "even parasites ( helminths and t. cruzi ) display small galnac linked o - glycans . in plants", "o - glycans occur as arabinogalactans linked to hydroxyproline or serine ( gal--hyp / ser ) , or hydroxyproline is glycosylated with short arabinofuranosides ( ara--hyp , ara--hyp ) . here", "we present for the first time an overview on o - glycans of some species of the mollusk branch of the evolutionary tree .", "the glycosylation potential of snails is important in general , because some of them are intermediate hosts of pathogens ( biomphalaria glabrata and lymnaea stagnalis ) or a pest in agriculture", ". a broad knowledge of their glycosylation abilities may show targets for pest control in the near future .", "in the course of the years , n - glycan patterns of several snail species have been presented [ 1417 ] . methylated mannose and galactose residues were found to be frequent constituents . in the present study we determine the main o - glycan structures in snails and characterize a common core - trisaccharide ."], ["cepaea hortensis , planorbarius corneus , arion lusitanicus , limax maximus and helix pomatia were collected by the authors under the supervision of dr .", "manfred pintar ( department of integrative biology and biodiversity research , institute of zoology , university of natural resources and life sciences , vienna ) in the vicinity of vienna .", "all chemicals purchased were of the highest quality available from sigma or fluka . while water living snails could be dissected right away , land living snails were extensively washed to remove the extraneous mucous components .", "snails were dissected as previously described   and proteins were purified according to   with minor modifications : 1  g of wet tissue was homogenised in 10  ml of chaps - based lysis buffer ( 0.5  % ( w / v ) chaps , 150  mm nacl , 20  mm tris / hcl , 2.5  mm sodium pyrophosphate , 1  mm ethylene - glycol - bis(2-aminoethylether)-n , n , n,n-tetraacetic acid and 1  mm edta , ph  7.5 ) and incubated for 72  h at 4  c .", "snail proteins were then reduced by the addition of 500  l of 10  mm dithiothreitol and incubation at 56  c for 45  min .", "then 500  l of 55  mm iodoacetamide were added and incubated for 30  min at room temperature in the dark .", "the aqueous samples were precipitated with a 4-fold amount of pre - chilled acetone at 20  c for at least 4  h and then centrifuged at 32000  g for 60  min .", "the pellets were resuspended in 0.1  m sodium citrate buffer , ph  4.6 , and incubated with 2 units of -glucosidase from rice [ ec 3.2.1.20 ] at 37  c for 16  h. sugars and polysaccharides were eluted from cation exchange chromatography ( ag 50  w - x2 , biorad , ca ) with 2  % of acetic acid , while proteins were eluted by 1.0  m ammonium acetate , ph  9.0 .", "the protein fraction was again precipitated by acetone followed by a similar precipitation by methanol .", "the dry sample was dissolved in 0.1  m naoh containing 0.8  m nabh3 and incubated at 37  c for 68  h. the sample was carefully neutralised by the addition of 2  m acetic acid , dried in a vacuum centrifuge and washed at least twice with 30  % ( v / v ) methanol .", "the glycans were directly extracted by solid phase extraction on a porous graphitized carbon cartridge ( supelclean envi - carb , 100  mg bed weight , 1  ml column volume or 500  mg bed weight , 6  ml column volume , sigma - aldrich , germany ) according to   by using ammonium formate buffer / acetonitrile for elution   and then on a reversed - phase c18 cartridge ( strata , c18-e , 55  m , 50  mg bed volume , 1  ml column volume , phenomenex , germany ) using 5  % ( v / v ) acetic acid as loading solvent and 25  % ( v / v ) isopropanol in 5  % ( v / v ) acetic acid for elution .", "hplc separation was carried out on a porous graphitized carbon ( pgc ) column ( hypersil - keystone , hypercarb 5   150    3  mm , thermo scientific , austria ) at a flow rate of 0.6  ml / min using 0.3  % ( v / v ) formic acid adjusted to ph  3.1 with ammonium as solvent a and 95  % ( v / v ) acetonitrile in solvent a as solvent b. the elution protocol consisted of 2  min at 100  % ( v / v ) of solvent a followed by a gradient of 28  min till 25  % ( v / v ) of solvent b and a cleaning step of 4  min at 60  % ( v / v ) of solvent b. each fraction was tested by esi - ms for its glycan content .", "single glycan structures were pooled and lyophilised . for monosaccharide analysis alditol acetates and for linkage analysis", "gc analysis was carried out on a vf 5  ms capillary column ( 60  m , 0.25  mm inner diameter ; 0.1  mm film thickness ; varian , darmstadt , germany ) and electron impact ( ei ) mass spectrometry was performed in the positive ion mode using a polaris q instrument ( thermoquest - analytical systems ) . for analysis of small sample amounts with esi - ms an in - gel release method for glycans bound to coomassie", "reduced oligosaccharides were analyzed by pgc - lc - esi - ms on a hypercarb column ( 0.32    150  mm , thermo fisher scientific , austria ) coupled to an ultimate 3000 ( dionex ) capillary hplc and a q - tof ultima ms ( waters ) as described previously [ 21 , 26 ] .", "the interpretation of the ms data was performed with the help of the software tool glycoworkbench ."], ["arion lusitanicus proteins were isolated and separated from contaminants through several purification steps in order to obtain sufficient amounts ( 12  g ) of pure glycans for further gc - ms analysis ( total monosaccharide and linkage analysis ) .", "the preparation of o - glycans was carried out via -elimination followed by further separation steps . as a final procedure", "the glycans were separated on a pgc ( porous graphitized carbon ) hplc - column by collecting 1-minute fractions which were screened by esi - ms to gain clean single structures . in the course of the -elimination releasing the glycan from the protein backbone , sodium borohydride was used .", "in contrast , for reduction of monosaccharides before gc - ms analysis sodium borodeuteride was employed , which allowed further the discrimination between the sugar released by -elimination and those linked to other sugars previously . in fig", ".  1 monosaccharide constituents of the core trisaccharide representing the main structure found in a. lusitanicus , with a pseudomolecular mass ( [ m+h ] ) of m / z 576.3  da , were determined by gc - ms of the corresponding alditol acetates and identified on the basis of retention times in gc and their characteristic electron impact ( ei ) mass spectra .", "the results revealed the presence of a galnac - residue which was h - reduced , while all the other sugars were h reduced .", "therefore , galnac can be considered as the protein bound sugar . the high amount of 4-o - me - gal indicated that this residue is the one which is mainly involved in the elongation of the glycan .", "minor compounds were identified as contaminations by n - glycans ( glcnac and man ) or storage glycans ( glc and gal ) .", "the lc - esi - ms / ms fragmentation pattern confirmed the occurrence of two methylated hexoses linked to the amino sugar ( fig . ", "1monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination .", "alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms .", "peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced)fig 2lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus . registered ions represent proton adducts [ m+h ] monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination .", "alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms .", "peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced ) lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus .", "registered ions represent proton adducts [ m+h ] moreover , gc - ms analysis of partially methylated alditol acetates was carried out for further confirmation of the compounds and also for linkage determination .", "methylated sugar standards purified by preparative gc were used to elucidate the type of methylated hexose ( man or gal ) based on retention time .", "the selected ion chromatogram of the monosaccharide derivatives obtained from the o - glycan core structure identified 2,3,4,6-tetra - o - methyl - galacitol at 20.66  min and 1,4,5-tri - o - methyl galnac - ol at 33.33  min ( fig", ".  3a ) which could be verified by ei - ms data ( figs . ", "3b and c ) . to determine the position of the naturally occurring methyl group on the galactose", "this allows in the ei - ms fragmentation pattern the discrimination between the naturally occurring and introduced methylgroups in the fragments by a mass difference of 3  da . that way the occurrence of terminal 4-o - methylated galactose", "4 ) . combining these data , the main o - glycan structure of a. lusitanicus was elucidated as a trisaccharide containing two terminal 4-o - methylated galactoses which are 3- and 6-linked to an inner galnac residue ( fig . ", "5).fig 3linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol .", "characteristic primary selected secondary fragment ions are assignedfig 4ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide .", "the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of  glycoworkbench  .", "this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) .", "square  =  galnac , circles  =  gal , me  =  methyl group linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol .", "characteristic primary selected secondary fragment ions are assigned ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide .", "the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of  glycoworkbench  .", "this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) .", "square  =  galnac , circles  =  gal , me  =  methyl group a small amount of an isoform with the same molecular weight , but slightly later retention time in the pgc ( porous graphitized carbon ) selected ion chromatogram ( fig . ", "6c ) , was identified analogously by gc - ms and ei - ms as trisaccharide containing a galnac residue substituted by one 4-o - methylated galactose and one 3-o - methylated mannose ( data not shown).fig 6selected ion chromatograms of partially methylated structures obtained by lc - esi - ms .", "a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) .", "square  =  amino sugar , circle  =  hexose , me  =  methyl group selected ion chromatograms of partially methylated structures obtained by lc - esi - ms . a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) .", "square  =  amino sugar , circle  =  hexose , me  =  methyl group besides the trisaccharide core several other glycan species were found during purification on pgc - column .", "a glycan consisting of the methylated core trisaccharide and one further hexose ( m / z 738.3 [ m+h ] ) was found .", "this additional hexose was an unmethylated gal residue linked alternatively to one of the two arms of the trisaccharide explaining the two isoforms detected by pgc selected ion chromatogram ( fig . ", "the fourth structure ( m / z 914.4 [ m+h ] ) which was obtained in sufficient amounts for gc - ms consisted of the core trisaccharide elongated by an unmethylated hexose on one arm and a methylated hexose on the other one ( fig . ", "the presence of significant amounts of man and 3-o - me - man indicated that these two residues were responsible for the elongation .", "gc - ms analysis using different derivatisation protocols requested a high amount of material and high quality of purification . especially for snail tissues this was a challenge and", "therefore this method could not be carried out for a large number of samples in due time .", "an in - gel -elimination after sds - page followed by a short clean - up ( pgc - cartridge ) procedure was sufficient .", "the shortened protocol also ensured that no weakly bound modifications , such as sulfate or sialic acids for example , got lost . combining our data on snail monosaccharide analysis , data from previous gc - ms methylation analysis   and the fragmentation results of lc - esi - ms / ms analysis of selected snails we could determine the main structures of snail o - glycans and classify them according to their modification of the established core structure into six groups : ( i ) glycans smaller than the core , missing one or both methyl groups or missing one hexose ; ( ii ) the trisaccharide core containing galnac and two 4-o - methylated hexose residues ; ( iii ) the core with additional methylated hexoses ; ( iv ) the core with one or two additional unmethylated hexoses and sometimes one more methylated hexose ; ( v ) glycans containing fucose ; ( vi ) glycans containing one hexnac and up to six unmethylated hexoses .", "for an overview on the structures see table  1.table 1overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans .", "compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex)glycan compositiongroupobserved mass [ m+h]calculated mass [ m+h]arion lusitanicusachatina fulicacepaea hortensisplanorbarius corenusbiomphalaria glabratahelix pomatialimax maximusclea helenahexhexnac(i ) - glycans smaller than the core386.2386.1707000000mehexhexnac400.2400.1850310200hex2hexnac548.2548.22traces000traces000hexmehexhexnac562.3562.2390012182600(mehex)2hexnac(ii ) - core576.3576.2554692612292910089(mehex)3hexnac(iii ) - core with additional methyl hexoses752.3752.325212302700(mehex)4hexnac928.4928.39traces0000000(mehex)5hexnac1104.41104.46traces0000000hex(mehex)2hexnac(iv )  core with additional hexoses and methyl hexoses738.3738.30141617164512011hex2(mehex)2hexnac900.4900.36203720100hex(mehex)3hexnac914.4914.3725505400(mehex)2hexnacdhex(v ) - fucosylated structures722.3722.31220traces1traces00(mehex)3hexnacdhex898.4898.3860000000(mehex)4hexnacdhex1074.41074.44traces0000000hex3hexnac(vi )  hexnac and hexoses710.3710.2700061traces00hex4hexnac872.3872.32000460000hex5hexnac1034.31034.3800030000hex6hexnac1197.01196.43000traces0000 overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans .", "compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex ) analyzing snails in detail , it was obvious that the o - glycan trisaccharide core is an important decoration of snail proteins in all investigated species . in five of the analyzed snails ( a. lusitanicus , a. fulica , h. pomatia , l. maximus , c. helena ) this glycan was the most abundant type of o - glycan .", "b. glabrata and c. hortensis displayed as their main glycan the trisaccharide elongated by one or two unmethylated hexoses , respectively .", "only p. corneus was an exception possessing a large amount of hex4hexnac glycan . whereas the addition of one hexose to the core was relatively frequent , larger structures were minor compounds or were even not present in several species .", "the only detected amino sugar was galnac which provided in all cases the connection to the protein .", "gal and man were present in unmethylated or mono - methylated ( 3-o - me - gal , 4-o - me - gal , 3-o - me - man ) versions .", "fucose , which had been identified as such in previous works [ 23 , 28 ] , occurred only in low amounts and was detected linked to galnac as well as to terminal sugar residues by ms / ms fragmentation ."], ["during the last decades our knowledge on glycosylation processes - structure of the glycans and the corresponding biochemical pathways including the responsible enzymes - increased enormously , especially for glycans of mammalian origin .", "the glycosylation capabilities of other species were only under investigation if their glycans were for any reason connected with human life ( e.g. some recognition processes of pathogens or allergy on food or plant glycans ) or if they were potent candidates for cell culture systems for the expression of therapeutics ( some insect , yeast and plant cells ) .", "in the meantime more and more invertebrate systems are investigated because of their potential usefulness for biotechnological reasons or to get some deeper insights into the processing pathways of unusual glycan structures .", "previous studies on various invertebrates showed that glycosylation has a lot more varieties than we expected as long as only mammals were investigated , but the data are scattered .", "still the main focus is laid on n - glycans but the interest in o - glycans is growing .", "a number of difficulties arose during snail glycan analysis due to the tough tissue , which was not easy to homogenize and the rigid mucus , which had to be discarded diligently .", "the removal of storage carbohydrates , which might influence monosaccharide analysis and the separation of n- and o - glycans were other struggles to resolve . while the first was done by -glucosidase digestion from rice , the latter was an even more difficult task .", "n - glycosidases ( pngase a and f ) did not remove the n - glycans completely and even mild -elimination conditions seemed to be able to release , at least in part , residual n - glycans which resulted in n - glycan impurities in o - glycan fractions .", "furthermore some n - glycans of snails and digested storage glycans are so small ( 45 sugar residues [ 16 , 17 ] ) that they may co - migrate on hplc with o - glycans", ". therefore only a limited number of glycans could be prepared in terms of sufficient amount and purity for permethylation followed by maldi - tof - ms and gc - ms linkage analysis .", "however , in combination with lc - esi - ms of native glycans which is capable of a high throughput of samples the o - glycan pattern of 8 snail species ( land snails with shell : achatina fulica , cepaea hortensis and helix pomatia ; slugs : arion lusitanicus and limax maximus ; water snails : biomphalaria glabrata , clea helena , and planorbarius corneus ) could be identified .", "four monosaccharide constituents  galnac , gal , man and fuc  are the building blocks of the structures .", "the only further modification is a methylation of the hexoses , resulting in 3-o - me - gal , 4-o - me - gal and 3-o - me - man , which has already been shown for snail n - glycans [ 1417 , 23 ] .", "each o - glycan contains only one amino sugar , which is the protein linked galnac .", "the linkage amino acid was not determined in detail , but due to the easy release by -elimination the common galnac - ser / thr motif is very likely .", "the connection to other amino acids , for example tyrosine , is usually more stable . in snail o - glycans the inner galnac", "is frequently elongated by two 4-o - me - gal residues in ( 13)- and ( 16)-linkage . elongations of this trisaccharide core by one or two hexoses with or without methyl group appeared in most snail species , whereas further elongation or fucosylation are rare events .", "insects , one neighbor of mollusks in the phylogenetic tree , display also internal galnac elongated by one gal in their o - glycan structures , but further elongations and/or branching are frequently done by another amino sugar ( galnac or glcnac ) .", "the main o - glycans of toxocara canis are trisaccharides consisting of galnac , gal or 4-o - gal and 2-o - fuc .", "so far nothing is known about biological functions of snail glycans . for sure these glycans", "are similarly involved in recognition processes as they are in all other species , but especially the significance of the high degree of methylation of hexoses of n- as well as of o - glycans is waiting for elucidation . here", "we enlarge the current knowledge on glycosylation abilities of lower animals by presenting the first o - glycan analysis of snails , representatives of the mollusk phylum ."]]}
{"article_id": "PMC5292226", "article_text": ["adiponectin ( arcp 30 , adipoq , apm1 or gbp28 ) , secreted by adipose tissue , is a 247-amino acid peptide which was discovered in 1995 ( 1 , 2 ) . the amounts of adiponectin account for 0.01% of total plasma protein .", "the concentration of adiponectin circulating in plasma is very high ( 2 to 20 g / ml ) ( 3 ) .", "plasma levels of adiponectin in the japanese population is about 5 to 10 g / ml ( 4 ) and serum adiponectin is lower in indo - asians when compared with caucasians ( median 3.3 vs. 4.9 g / ml ) ( 5 ) .", "adiponectin has gained particular interest on account of its relation with insulin sensitivity , atherosclerosis , and inflammation ( 6 ) .", "current projections estimate that cardiovascular disease , especially atherosclerosis , will be the leading cause of all disease in 2020 ( 7 ) .", "recent findings suggest that high adiponectin concentration is an independent predictor of mortality in chronic heart failure patients , chronic kidney patients , and elderly patients , who are at high risk for cardiovascular events ( 8 - 12 ) .", "obesity has been proven to be an independent risk factor for cad in both genders , and is a growing health problem in most developed and some developing countries ( 13 - 15 ) . body weight , body mass index ( bmi ) ,", "waist circumference ( wc ) and whr are primary methods to determine obesity . while bmi reflects general obesity , waist circumference and waist  hip ratio are related to central - type obesity , where body fat is primarily located in the abdomen .", "prospective epidemiological studies have revealed that central obesity ( determined by wc and whr ) is more relevant in cad risk compared to general obesity ( determined by bmi ) ( 16 - 18 ) . while whr is commonly used in reflecting central obesity , waist circumference is shown to have a better correlation with abdominal fat localization ( 19 - 22 ) . to our best knowledge , this is the first study performed in kosovo focusing on this particular issue .", "the study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . with the exception of acute coronary syndromes , the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease .", "the study protocol was approved by the ethical committee of the faculty of medicine , university of prishtina , and research was conducted in accordance with declaration of helsinki guidelines . written informed consent", "cad was defined as more than 50% luminal stenosis identified in a minimum of two views , in the case of single - vessel involvement .", "gensini score assesses the severity of coronary artery disease : it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing , 2 for 26 - 50% narrowing , 4 for 51 - 75% , 8 for 76 - 90% , 16 for 91 - 99% and 32 for a completely occluded artery .", "this score is then multiplied by a factor , according to the importance of the coronary artery .", "the multiplication factor for a left main stem ( lms ) lesion is 5 , it is 2.5 for proximal left anterior descending artery ( lad ) and proximal circumflex artery ( cx ) lesions , 1.5 for a mid - lad lesion , and 1 for distal lad , mid / distal cx and right coronary artery lesions .", "gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease ( 23 ) .", "were taken after the patients had removed their shoes and upper garments and had donned an examining gown .", "waist height ratio ( whtr ) is measured by division of weight circumference by their height , always expressed in same units .", "body mass index ( bmi ) was calculated as weight ( kg ) divided by the square of height ( m ) .", "the wc was measured by tape in cm and was also categorized according to the world health organization ( who ) criteria ( 24 ) .", "blood pressure was measured by european society of hypertension - international protocol 2 ( esh - ip2 ) . with the whr ,", "the waist is measured at the narrowest part of the waist , between the lowest rib and iliac crest , and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks .", "the who defines the ratios of > 9.0 in men and > 8.5 in women as one of the decisive benchmarks for metabolic syndrome .", "the 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed .", "the laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program .", "the primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples .", "each sample was assayed in duplicate , and adiponectin level was calculated as the average of the two measurements .", "the continuous variables were primarily analyzed using a t - test , while the binary data were primary analyzed using a chi - squared test .", "the continuous variables are presented in the tables with their means  sd ( standard deviation ) .", "additionally , single ( unadjusted ) and multiple ( adjusted ) binary logistic regressions were carried out with cad group as the dependent variable , and the other significant variables as independent predictors .", "the cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models .", "adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance .", "other adjusting variables included are : diastolic blood pressure ( dbp ) , whr , lvef , gender male , and smoking .", "the study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . with the exception of acute coronary syndromes , the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease .", "the study protocol was approved by the ethical committee of the faculty of medicine , university of prishtina , and research was conducted in accordance with declaration of helsinki guidelines . written informed consent", "cad was defined as more than 50% luminal stenosis identified in a minimum of two views , in the case of single - vessel involvement .", "gensini score assesses the severity of coronary artery disease : it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing , 2 for 26 - 50% narrowing , 4 for 51 - 75% , 8 for 76 - 90% , 16 for 91 - 99% and 32 for a completely occluded artery .", "this score is then multiplied by a factor , according to the importance of the coronary artery .", "the multiplication factor for a left main stem ( lms ) lesion is 5 , it is 2.5 for proximal left anterior descending artery ( lad ) and proximal circumflex artery ( cx ) lesions , 1.5 for a mid - lad lesion , and 1 for distal lad , mid / distal cx and right coronary artery lesions .", "gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease ( 23 ) .", "were taken after the patients had removed their shoes and upper garments and had donned an examining gown .", "waist height ratio ( whtr ) is measured by division of weight circumference by their height , always expressed in same units .", "body mass index ( bmi ) was calculated as weight ( kg ) divided by the square of height ( m ) .", "the wc was measured by tape in cm and was also categorized according to the world health organization ( who ) criteria ( 24 ) .", "blood pressure was measured by european society of hypertension - international protocol 2 ( esh - ip2 ) . with the whr ,", "the waist is measured at the narrowest part of the waist , between the lowest rib and iliac crest , and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks .", "the who defines the ratios of > 9.0 in men and > 8.5 in women as one of the decisive benchmarks for metabolic syndrome .", "the 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed .", "the laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program .", "the primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples .", "each sample was assayed in duplicate , and adiponectin level was calculated as the average of the two measurements .", "the continuous variables were primarily analyzed using a t - test , while the binary data were primary analyzed using a chi - squared test .", "the continuous variables are presented in the tables with their means  sd ( standard deviation ) .", "additionally , single ( unadjusted ) and multiple ( adjusted ) binary logistic regressions were carried out with cad group as the dependent variable , and the other significant variables as independent predictors .", "the cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models .", "adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance .", "other adjusting variables included are : diastolic blood pressure ( dbp ) , whr , lvef , gender male , and smoking .", "of the eighty - two study patients , 41 patients comprised the cad group ( study subject ) and 41 patients were classified as the non - cad group ( control subject ) .", "study group ( mean age 66.76 + 9.12 ) were on the average , 2 years older than the controls ( mean age 64.80  8.30 ) .", "the baseline characteristics of the two groups are depicted in table 1 . according to table 1 , in comparison with the controls ,", "the cad group patients were older and were more likely to be male and smokers .", "the number of male patients in the study group was 29 ( 70.7% ) and in the control group was 17 ( 41.5% ) , which is significantly higher ( p=0.008 ) , also smokers were 27 ( 65.9% ) vs 13 ( 31.7% ) ( p=0.002 ) significantly more numerous in the study group than the controls .", "bmi : basal metabolic index , wc , whtr , whr , systolic blood pressure ( sbp ) , dbp .", "fifteen patients ( 18.2% ) had normal bmi ( mean 23.221.4 ) , 37 patients ( 45% ) were overweight , 30 patients ( 43.4% ) were obese .", "bmi in case group ( mean 28.17 3.32 ) and control ( mean 28.764.68 ) and wc in case ( 96.978.63 ) vs control ( 95.1311.52 ) they were no significant .", "whtr in case ( 1.76 7.56 ) vs control ( 0.570.08 ) , and whr is significant between case and control ( 0.930.06 ) vs ( 0.880.07 ) ( p - value : 0.0001 ) as seen in table 1 .", "cad patients had higher mean gensini scores than non - cad patients ( 53.16  33.46 vs. 1.21 5.66 , p<0.0001 ) , but there was no statistically significant difference detected between cad and non cad for plasma adiponectin levels , presented in table 2 .", "total cholesterol in the populations averaged ( 3.58  1.07 mmol / l ) in cases and ( 3.59  1.19 mmol / l ) in the control g biochemical characteristics of the two groups .", "cad , ast : aspartate aminotransferase , crp : c reactive protein , hdl - c : high density lipoprotein cholesterol , ldl - c : low density lipoprotein cholesterol , lvef . roups .", "baseline adiponectin concentrations correlated significantly in terms of the lipid parameters , positively with hdl cholesterol concentrations ( r= 0.365 , p=0.001 ) and serum triglyceride concentrations were correlated negatively ( r=-0.336 , p=0.001 ) . total cholesterol concentration and ldl cholesterol concentrations", "sixty - two patients ( 75% ) had hypertension , 29 patients ( 35% ) had diabetes , 23 patients ( 28% ) dyslipidemia .", "forty - two patients ( 51% ) had family history of coronary heart disease and sudden non - accidental death or a combination of these", ". correlations of adiponectin with other variables of all patients 14.6% had triple - vessel disease , 18.3% had double - vessel disease and 17% single - vessel disease .", "adiponectin levels show a non - significantly progressive decline as the angiographic severity of coronary artery stenosis increases ( value=0.335 ) as shown in table 4", "cag : coronary angiography , svd : single vessel disease , dvd : double vessel disease , tvd : triple vessel disease . in control group 37 patients ( 45% ) have ( gensini score = 0 ) , 15 patients ( 18% ) had mild disease ( gensini score < 32 ) , 14 patients ( 17% ) had moderate disease ( gensini score= 32 - 58 ) , 16 patients ( 19.5% ) had severe disease ( gensini score 58 ) .", "bmi did not correlate with gensini score by severity of coronary arteries , as seen in table 5 .", "distribution of gensini in different bmi categories significant differences were seen in binary logistic regression models for cad analysis for adiponectin , whr , lvef , dbp , male gender and for smoker .", "whr ( or 14 ; 95% ci 0.94 - 207.60 ; p=0.06 ) , lvef ( or 4.61 ; 95% ci 1.49 - 14.21 ; p=0.01 ) , dbp ( or 2.75 ; 95% 2.75 ( 1.12 - 6.87 ; p=0.03 ) , smoking ( or 4.15 ; 95% ci 1.65 - 10.44 ; p=0.001 ) , male patients ( or 3.41 , 95% ci 1.37 - 8.52 ; p=0.01 ) , all presented in table 6 .", "binary logistic regression models for cad ( unadjusted and adjusted ) , dbp , whr , lvef .", "the present study found that there was a strong association between adiponectin and coronary artery disease .", "these findings provide further evidence for an  adiponectin paradox  in which higher levels of adiponectin may be secreted as a protective or a compensatory response to worsening cardiovascular disease . in our study , we found medium significant positive correlation between gensini and adiponectin ( r= 0.272 , p=0.01 ) .", "this is in compliance with the saphir study , which suggests that the role of adiponectin differs between early atherosclerosis and advanced stage vascular disease ( 25 ) .", "adiponectin protects against the development of disease , once the disease is established , adiponectin concentrations are elevated as a counter - regulatory response to protect against further inflammation and atherosclerosis ( 26 ) .", "findings of positive correlation between adiponectin and culprit lesion necrotic core ( nc ) content is consistent with the hypothesis that in advanced cad , serum adiponectin levels are elevated in a counter - regulatory response ( 27 ) .", "high serum adiponectin levels , instead of being protective , have been shown to be associated with increased mortality and adverse event rates .", "this paradoxical relation is further supported by study that included patients undergoing pci for symptomatic cad ( 28 ) . despite the potential association between heart failure and high plasma adiponectin shown by several clinical studies ,", "other experimental studies have demonstrated a beneficial , protective effect of adiponectin on myocardium . elevated plasma levels of adiponectin among patients with heart failure", "furthermore , several statins are also effective for elevating plasma adiponectin ( 29 , 30 ) .", "there were no significant differences in plasma concentrations of adiponectin between patients with stable angina pectoris ( sap ) and control patients ( 11.3 v 12.8 g / ml ) ( 31 ) .", "our study findings were similar to this study , where plasma concentrations of adiponectin between patients with sap and control group ( 3.45 v 3.49 g / ml ) had no significant differences . in the present study ,", "patients in our sap group had already been receiving some treatment with antianginal and antiatherosclerotic drugs .", "as certain medical treatments may confound interpretation of plasma concentrations of adiponectin measurements and there may not be significant differences in plasma concentrations of adiponectin between patients with sap and control patients .", "another important clinical finding showed that plasma concentrations of adiponectin were considerably increased in older and female patients with cad ( 31 ) .", "as a consequence of their findings they suggested that unstable plaque may stabilize as the patient grows older or that accumulation of adiponectin in atherosclerotic vascular walls may suppress its elimination half - life from plasma , resulting in an increase in plasma concentrations of adiponectin in older patients with cad . although a significant relation between serum adiponectin levels and age was not found in our study , it is known that age has an effect on serum adiponectin levels and that there is a positive relationship between age and serum adiponectin level ( 32 ) .", "including both individuals with and without cardiovascular disease , higher adiponectin has protecting role in those without cardiovascular disease and predictive of worse outcomes in those with existing disease ( 33 ) .", "moreover , it has been suggested that normal and even higher serum adiponectin levels may prevent the development of cardiovascular diseases and complications in healthy individuals ( 34 ) .", "male patients with hypoadiponectinemia ( < 4.0 g / ml ) had a two - fold increase in cad prevalence , independent of well - known cad risk factors ( 35 ) .", "similar to our study in male gender with hypoadiponectinemia ( < 2.0 g / ml ) increase the odds of having cad ( table 6 ) . our current study did not show a significant correlation between bmi and cad ( p - value 0.115 ) , despite the presence of significant association between whr and cad ( p - value= 0,0001 ) .", "this may be explained by the fact that bmi quantifies general adiposity , although person who is overweight or obese is likely to have excess fat but bmi does not give an indication as to how this fat is distributed in the body .", "however , fat distribution is an important determinant of cad independent of bmi and other classic risk factors of cad ( 36 ) .", "welborn and dhaliwal ( 37 ) and srikanthan , seeman , and karlamangla ( 38 ) confirmed , and cited several other investigations that show whr being the superior clinical measurement for predicting all cause and cardiovascular disease mortality .", "welborn and dhaliwal added that the hip circumference indicated a lower risk for body fat accumulation , and thus including it into the waist - to - hip equation enhances the accuracy of this measurement technique .", "relatively small sample of patients that were selected randomly from the general population , may have limited the ability to detect significant relationship between adiponectin and coronary artery disease , thus making it difficult to indicate the results to the general population .", "there is a significant positive correlation between waist - hip ratio and severity of coronary artery disease .", "also , there is a significant positive correlation between adiponectin and gensini score among kosovar patients ."], "abstract_text": ["<S> background : this study aimed to investigate correlation between adiponectin and waist - hip - ratio with severity of coronary artery disease ( cad ) . </S>", "<S> there is uncertainty about the association between circulating concentrations of adiponectin and cad.methods:we enrolled eighty - two consecutive patients undergoing non - urgent coronary angiography for cad survey . according to the angiography results , the patients were divided into two groups in 1:1 ratio patients admitted with a diagnosis of cad and non - cad . </S>", "<S> we conducted hospital based research , involving study group with documented angiographically cad , and control group without evidence of cad . </S>", "<S> angiograms were also quantified for the extent and severity of cad by the gensini scoring system . </S>", "<S> we measured baseline adiponectin levels in stored serum samples of all patients , anthropometric and biochemical risk factors were assessed in both groups.results:the presence of cad was associated with current smoking , male gender , waist  hip ratio ( whr ) and left ventricular ejection fraction ( lvef ) . </S>", "<S> baseline adiponectin concentrations correlated significantly in terms of the lipid parameters , positively with hdl cholesterol concentrations ( r=0.327 , p=0.028 , p<0.05 ) and serum triglyceride concentrations were correlated negatively ( r=-0.513 , p<0.001 ) . no significant difference between median adiponectin levels at baseline was observed between cases and controls.conclusion:there is a significant positive correlation between waist  hip ratio and presence and severity of coronary artery disease . in conclusion , there is a significant positive correlation between adiponectin and gensini score among kosovar patients . </S>"], "labels": null, "section_names": ["1. INTRODUCTION", "2. MATERIALS AND METHODS", "2.1. Study subjects", "2.2. Coronary angiography", "2.3. Anthropometric measurements", "2.4. Laboratory measurements", "2.4. Statistical analysis", "3. RESULTS", "4. DISCUSSION", "5. CONCLUSION"], "sections": [["adiponectin ( arcp 30 , adipoq , apm1 or gbp28 ) , secreted by adipose tissue , is a 247-amino acid peptide which was discovered in 1995 ( 1 , 2 ) . the amounts of adiponectin account for 0.01% of total plasma protein .", "the concentration of adiponectin circulating in plasma is very high ( 2 to 20 g / ml ) ( 3 ) .", "plasma levels of adiponectin in the japanese population is about 5 to 10 g / ml ( 4 ) and serum adiponectin is lower in indo - asians when compared with caucasians ( median 3.3 vs. 4.9 g / ml ) ( 5 ) .", "adiponectin has gained particular interest on account of its relation with insulin sensitivity , atherosclerosis , and inflammation ( 6 ) .", "current projections estimate that cardiovascular disease , especially atherosclerosis , will be the leading cause of all disease in 2020 ( 7 ) .", "recent findings suggest that high adiponectin concentration is an independent predictor of mortality in chronic heart failure patients , chronic kidney patients , and elderly patients , who are at high risk for cardiovascular events ( 8 - 12 ) .", "obesity has been proven to be an independent risk factor for cad in both genders , and is a growing health problem in most developed and some developing countries ( 13 - 15 ) . body weight , body mass index ( bmi ) ,", "waist circumference ( wc ) and whr are primary methods to determine obesity . while bmi reflects general obesity , waist circumference and waist  hip ratio are related to central - type obesity , where body fat is primarily located in the abdomen .", "prospective epidemiological studies have revealed that central obesity ( determined by wc and whr ) is more relevant in cad risk compared to general obesity ( determined by bmi ) ( 16 - 18 ) . while whr is commonly used in reflecting central obesity , waist circumference is shown to have a better correlation with abdominal fat localization ( 19 - 22 ) . to our best knowledge , this is the first study performed in kosovo focusing on this particular issue ."], ["the study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . with the exception of acute coronary syndromes , the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease .", "the study protocol was approved by the ethical committee of the faculty of medicine , university of prishtina , and research was conducted in accordance with declaration of helsinki guidelines . written informed consent", "cad was defined as more than 50% luminal stenosis identified in a minimum of two views , in the case of single - vessel involvement .", "gensini score assesses the severity of coronary artery disease : it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing , 2 for 26 - 50% narrowing , 4 for 51 - 75% , 8 for 76 - 90% , 16 for 91 - 99% and 32 for a completely occluded artery .", "this score is then multiplied by a factor , according to the importance of the coronary artery .", "the multiplication factor for a left main stem ( lms ) lesion is 5 , it is 2.5 for proximal left anterior descending artery ( lad ) and proximal circumflex artery ( cx ) lesions , 1.5 for a mid - lad lesion , and 1 for distal lad , mid / distal cx and right coronary artery lesions .", "gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease ( 23 ) .", "were taken after the patients had removed their shoes and upper garments and had donned an examining gown .", "waist height ratio ( whtr ) is measured by division of weight circumference by their height , always expressed in same units .", "body mass index ( bmi ) was calculated as weight ( kg ) divided by the square of height ( m ) .", "the wc was measured by tape in cm and was also categorized according to the world health organization ( who ) criteria ( 24 ) .", "blood pressure was measured by european society of hypertension - international protocol 2 ( esh - ip2 ) . with the whr ,", "the waist is measured at the narrowest part of the waist , between the lowest rib and iliac crest , and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks .", "the who defines the ratios of > 9.0 in men and > 8.5 in women as one of the decisive benchmarks for metabolic syndrome .", "the 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed .", "the laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program .", "the primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples .", "each sample was assayed in duplicate , and adiponectin level was calculated as the average of the two measurements .", "the continuous variables were primarily analyzed using a t - test , while the binary data were primary analyzed using a chi - squared test .", "the continuous variables are presented in the tables with their means  sd ( standard deviation ) .", "additionally , single ( unadjusted ) and multiple ( adjusted ) binary logistic regressions were carried out with cad group as the dependent variable , and the other significant variables as independent predictors .", "the cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models .", "adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance .", "other adjusting variables included are : diastolic blood pressure ( dbp ) , whr , lvef , gender male , and smoking ."], ["the study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . with the exception of acute coronary syndromes , the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease .", "the study protocol was approved by the ethical committee of the faculty of medicine , university of prishtina , and research was conducted in accordance with declaration of helsinki guidelines . written informed consent"], ["cad was defined as more than 50% luminal stenosis identified in a minimum of two views , in the case of single - vessel involvement .", "gensini score assesses the severity of coronary artery disease : it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing , 2 for 26 - 50% narrowing , 4 for 51 - 75% , 8 for 76 - 90% , 16 for 91 - 99% and 32 for a completely occluded artery .", "this score is then multiplied by a factor , according to the importance of the coronary artery .", "the multiplication factor for a left main stem ( lms ) lesion is 5 , it is 2.5 for proximal left anterior descending artery ( lad ) and proximal circumflex artery ( cx ) lesions , 1.5 for a mid - lad lesion , and 1 for distal lad , mid / distal cx and right coronary artery lesions .", "gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease ( 23 ) ."], ["were taken after the patients had removed their shoes and upper garments and had donned an examining gown .", "waist height ratio ( whtr ) is measured by division of weight circumference by their height , always expressed in same units .", "body mass index ( bmi ) was calculated as weight ( kg ) divided by the square of height ( m ) .", "the wc was measured by tape in cm and was also categorized according to the world health organization ( who ) criteria ( 24 ) .", "blood pressure was measured by european society of hypertension - international protocol 2 ( esh - ip2 ) . with the whr ,", "the waist is measured at the narrowest part of the waist , between the lowest rib and iliac crest , and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks .", "the who defines the ratios of > 9.0 in men and > 8.5 in women as one of the decisive benchmarks for metabolic syndrome ."], ["the 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed .", "the laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program .", "the primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples .", "each sample was assayed in duplicate , and adiponectin level was calculated as the average of the two measurements ."], ["the continuous variables were primarily analyzed using a t - test , while the binary data were primary analyzed using a chi - squared test .", "the continuous variables are presented in the tables with their means  sd ( standard deviation ) .", "additionally , single ( unadjusted ) and multiple ( adjusted ) binary logistic regressions were carried out with cad group as the dependent variable , and the other significant variables as independent predictors .", "the cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models .", "adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance .", "other adjusting variables included are : diastolic blood pressure ( dbp ) , whr , lvef , gender male , and smoking ."], ["of the eighty - two study patients , 41 patients comprised the cad group ( study subject ) and 41 patients were classified as the non - cad group ( control subject ) .", "study group ( mean age 66.76 + 9.12 ) were on the average , 2 years older than the controls ( mean age 64.80  8.30 ) .", "the baseline characteristics of the two groups are depicted in table 1 . according to table 1 , in comparison with the controls ,", "the cad group patients were older and were more likely to be male and smokers .", "the number of male patients in the study group was 29 ( 70.7% ) and in the control group was 17 ( 41.5% ) , which is significantly higher ( p=0.008 ) , also smokers were 27 ( 65.9% ) vs 13 ( 31.7% ) ( p=0.002 ) significantly more numerous in the study group than the controls .", "bmi : basal metabolic index , wc , whtr , whr , systolic blood pressure ( sbp ) , dbp .", "fifteen patients ( 18.2% ) had normal bmi ( mean 23.221.4 ) , 37 patients ( 45% ) were overweight , 30 patients ( 43.4% ) were obese .", "bmi in case group ( mean 28.17 3.32 ) and control ( mean 28.764.68 ) and wc in case ( 96.978.63 ) vs control ( 95.1311.52 ) they were no significant .", "whtr in case ( 1.76 7.56 ) vs control ( 0.570.08 ) , and whr is significant between case and control ( 0.930.06 ) vs ( 0.880.07 ) ( p - value : 0.0001 ) as seen in table 1 .", "cad patients had higher mean gensini scores than non - cad patients ( 53.16  33.46 vs. 1.21 5.66 , p<0.0001 ) , but there was no statistically significant difference detected between cad and non cad for plasma adiponectin levels , presented in table 2 .", "total cholesterol in the populations averaged ( 3.58  1.07 mmol / l ) in cases and ( 3.59  1.19 mmol / l ) in the control g biochemical characteristics of the two groups .", "cad , ast : aspartate aminotransferase , crp : c reactive protein , hdl - c : high density lipoprotein cholesterol , ldl - c : low density lipoprotein cholesterol , lvef . roups .", "baseline adiponectin concentrations correlated significantly in terms of the lipid parameters , positively with hdl cholesterol concentrations ( r= 0.365 , p=0.001 ) and serum triglyceride concentrations were correlated negatively ( r=-0.336 , p=0.001 ) . total cholesterol concentration and ldl cholesterol concentrations", "sixty - two patients ( 75% ) had hypertension , 29 patients ( 35% ) had diabetes , 23 patients ( 28% ) dyslipidemia .", "forty - two patients ( 51% ) had family history of coronary heart disease and sudden non - accidental death or a combination of these", ". correlations of adiponectin with other variables of all patients 14.6% had triple - vessel disease , 18.3% had double - vessel disease and 17% single - vessel disease .", "adiponectin levels show a non - significantly progressive decline as the angiographic severity of coronary artery stenosis increases ( value=0.335 ) as shown in table 4", "cag : coronary angiography , svd : single vessel disease , dvd : double vessel disease , tvd : triple vessel disease . in control group 37 patients ( 45% ) have ( gensini score = 0 ) , 15 patients ( 18% ) had mild disease ( gensini score < 32 ) , 14 patients ( 17% ) had moderate disease ( gensini score= 32 - 58 ) , 16 patients ( 19.5% ) had severe disease ( gensini score 58 ) .", "bmi did not correlate with gensini score by severity of coronary arteries , as seen in table 5 .", "distribution of gensini in different bmi categories significant differences were seen in binary logistic regression models for cad analysis for adiponectin , whr , lvef , dbp , male gender and for smoker .", "whr ( or 14 ; 95% ci 0.94 - 207.60 ; p=0.06 ) , lvef ( or 4.61 ; 95% ci 1.49 - 14.21 ; p=0.01 ) , dbp ( or 2.75 ; 95% 2.75 ( 1.12 - 6.87 ; p=0.03 ) , smoking ( or 4.15 ; 95% ci 1.65 - 10.44 ; p=0.001 ) , male patients ( or 3.41 , 95% ci 1.37 - 8.52 ; p=0.01 ) , all presented in table 6 .", "binary logistic regression models for cad ( unadjusted and adjusted ) , dbp , whr , lvef ."], ["the present study found that there was a strong association between adiponectin and coronary artery disease .", "these findings provide further evidence for an  adiponectin paradox  in which higher levels of adiponectin may be secreted as a protective or a compensatory response to worsening cardiovascular disease . in our study , we found medium significant positive correlation between gensini and adiponectin ( r= 0.272 , p=0.01 ) .", "this is in compliance with the saphir study , which suggests that the role of adiponectin differs between early atherosclerosis and advanced stage vascular disease ( 25 ) .", "adiponectin protects against the development of disease , once the disease is established , adiponectin concentrations are elevated as a counter - regulatory response to protect against further inflammation and atherosclerosis ( 26 ) .", "findings of positive correlation between adiponectin and culprit lesion necrotic core ( nc ) content is consistent with the hypothesis that in advanced cad , serum adiponectin levels are elevated in a counter - regulatory response ( 27 ) .", "high serum adiponectin levels , instead of being protective , have been shown to be associated with increased mortality and adverse event rates .", "this paradoxical relation is further supported by study that included patients undergoing pci for symptomatic cad ( 28 ) . despite the potential association between heart failure and high plasma adiponectin shown by several clinical studies ,", "other experimental studies have demonstrated a beneficial , protective effect of adiponectin on myocardium . elevated plasma levels of adiponectin among patients with heart failure", "furthermore , several statins are also effective for elevating plasma adiponectin ( 29 , 30 ) .", "there were no significant differences in plasma concentrations of adiponectin between patients with stable angina pectoris ( sap ) and control patients ( 11.3 v 12.8 g / ml ) ( 31 ) .", "our study findings were similar to this study , where plasma concentrations of adiponectin between patients with sap and control group ( 3.45 v 3.49 g / ml ) had no significant differences . in the present study ,", "patients in our sap group had already been receiving some treatment with antianginal and antiatherosclerotic drugs .", "as certain medical treatments may confound interpretation of plasma concentrations of adiponectin measurements and there may not be significant differences in plasma concentrations of adiponectin between patients with sap and control patients .", "another important clinical finding showed that plasma concentrations of adiponectin were considerably increased in older and female patients with cad ( 31 ) .", "as a consequence of their findings they suggested that unstable plaque may stabilize as the patient grows older or that accumulation of adiponectin in atherosclerotic vascular walls may suppress its elimination half - life from plasma , resulting in an increase in plasma concentrations of adiponectin in older patients with cad . although a significant relation between serum adiponectin levels and age was not found in our study , it is known that age has an effect on serum adiponectin levels and that there is a positive relationship between age and serum adiponectin level ( 32 ) .", "including both individuals with and without cardiovascular disease , higher adiponectin has protecting role in those without cardiovascular disease and predictive of worse outcomes in those with existing disease ( 33 ) .", "moreover , it has been suggested that normal and even higher serum adiponectin levels may prevent the development of cardiovascular diseases and complications in healthy individuals ( 34 ) .", "male patients with hypoadiponectinemia ( < 4.0 g / ml ) had a two - fold increase in cad prevalence , independent of well - known cad risk factors ( 35 ) .", "similar to our study in male gender with hypoadiponectinemia ( < 2.0 g / ml ) increase the odds of having cad ( table 6 ) . our current study did not show a significant correlation between bmi and cad ( p - value 0.115 ) , despite the presence of significant association between whr and cad ( p - value= 0,0001 ) .", "this may be explained by the fact that bmi quantifies general adiposity , although person who is overweight or obese is likely to have excess fat but bmi does not give an indication as to how this fat is distributed in the body .", "however , fat distribution is an important determinant of cad independent of bmi and other classic risk factors of cad ( 36 ) .", "welborn and dhaliwal ( 37 ) and srikanthan , seeman , and karlamangla ( 38 ) confirmed , and cited several other investigations that show whr being the superior clinical measurement for predicting all cause and cardiovascular disease mortality .", "welborn and dhaliwal added that the hip circumference indicated a lower risk for body fat accumulation , and thus including it into the waist - to - hip equation enhances the accuracy of this measurement technique .", "relatively small sample of patients that were selected randomly from the general population , may have limited the ability to detect significant relationship between adiponectin and coronary artery disease , thus making it difficult to indicate the results to the general population ."], ["there is a significant positive correlation between waist - hip ratio and severity of coronary artery disease .", "also , there is a significant positive correlation between adiponectin and gensini score among kosovar patients ."]]}
{"article_id": "PMC4213880", "article_text": ["neurofibromatosis ( nf ) is an autosomal dominant disease , discovered in 1882 , by the german pathologist friedrich daniel von recklinghausen , characterized by disordered growth of ectodermal tissues , and is part of a group of disorders called phakomatoses ( neurocutaneous syndrome ) .", "neurofibromatosis type - i ( nf-1 ) , also known as von recklinghausen syndrome , is caused by the mutation of a gene on the long arm of chromosome 17 , which encodes a protein known as neurofibromin .", "it is characterized by spots of increased skin pigmentation ( caf au lait spots ) , combined with peripheral nerve tumors and a variety of other dysplastic abnormalities of the skin , nervous system , bones , endocrine organs , and blood vessels .", "the localized form of neurofibromatosis type - i , first described by gammel in 1931 , is very rare .", "crowe et al . proposed the term sectorial neurofibromatosis for this localized form of neurofibromatosis , and miller and sparkes modified the nomenclature to segmental neurofibromatosis ( sn )  the current term for neurofibromas of segmental distribution .", "the commonly affected sites for sn are the thorax and abdomen ( 55% ) , upper extremities ( 20% ) , and lower limb and face ( 10% each ) . only few cases ( less than 10 ) of segmental neurofibromatosis over the face have been described so far .", "a 26-year - old female patient reported to the dental department complaining about the unesthetic appearance of her face , since eight years [ figure 1 ] .", "history revealed that eight years back , the patient underwent surgery in the neck region for a cyst and one to two days after surgery the patient got a swelling on the face .", "the swelling was of the same size since then and there was no pain associated with the swelling .", "the unesthetic appearance of the face on examination , the face of the patient appeared asymmetrical , bilaterally , with a growth on the right side of the face .", "the skin over the right side of her face was thickened and hyperpigmented , with a soft and loose hanging overgrowth .", "there were numerous small and large , sessile and pedunculated nodular growths localized on the right side of her neck , with dark - colored underlying skin .", "the skin over the right cheek and chin area showed coffee - colored pigmentation ( caf - au - lait spots ) [ figure 2 ] .", "nodular growths localized on the right side of the neck with dark colored underlying skin there was a soft , compressible , nontender growth on the lower lip .", "the right ear showed disfigurement and hypertrophy , with thickened cartilage and overlying hyperpigmented skin [ figure 3 ] .", "disfigurement of the ear and hypertrophy with thickened cartilage and overlying hyperpigmented skin intraorally there were numerous soft tissue growths with a sessile base seen on the right buccal mucosa [ figure 4 ] and tongue [ figure 5 ] .", "soft tissue growths with sessile bases on right buccal mucosa soft tissue growths with sessile bases on the tongue laboratory findings revealed a raised erythrocyte sedimentation rate ( esr ) ( 70 mm in the first hour ) , a decreased hemoglobin level ( 7.5 gm% ) , and the enzyme - linked immunosorbent assay ( elisa ) was found to be nonreactive . a panoramic radiograph disclosed asymmetric enlargement of the right side of the mandible , with loss of cortication of the lower border in the midline area , expansion of the body of the mandible causing displacement of the teeth , seen on right side , expansion of the right inferior alveolar nerve canal , enlarged mental foramen , resorption of the roots of the mandibular right teeth , and loss of the right antegonial notch .", "a deficiency was seen on the right side of the maxilla , with crowding of the posterior teeth [ figure 6 ] .", "a panoramic radiograph the right lateral oblique radiograph revealed a deep sigmoid notch , loss of the antegonial notch , an enlarged and rounded coronoid process , with resorption of the mandibular right teeth [ figure 7 ] .", "a right lateral oblique radiograph subsequently , biopsy of one of the nodules was taken and sent for histopathological examination .", "the histopathological slide revealed a non - encapsulated tumor of the dermis with a normal overlying epidermis .", "the tumor consisted of loosely spaced spindle cells and wavy collagenous strands in a clear matrix [ figure 8 ] . a non - encapsulated tumor of the dermis , with a normal overlying epidermis", "these tumors are composed of schwann cells , fibroblasts , mast cells , and vascular components .", "the caf au lait spots are irregularly - shaped , evenly pigmented , brown macules .", "most individuals with neurofibromatosis have six or more spots that are 1.5 cm or greater in diameter . in young children ,", "five or more caf au lait macules , greater than 0.5 cm in diameter , are suggestive of neurofibromatosis .", "less than 1% of the healthy children have three or more such spots , although one or two caf au lait macules are commonly encountered in healthy individuals without disease .", "bone involvement includes pseudoarthrosis of the tibia , bowing of the long bones , and orbital defects .", "treatment of neurofibromatosis is predominantly surgical . when neurofibromas increase in size or cause pain , malignant transformation must be suspected , and excision or biopsy must be performed .", "acoustic neuromas and tumors that cause tinnitus and vertigo must be excised with great caution .", "plastic surgeons may be included in the correction of deformities , especially those of the face . considering the autosomal dominant inheritance pattern of neurofibromatosis , genetic counseling"], "abstract_text": ["<S> segmental neurofibromatosis is a rare disorder , characterized by neurofibromas or caf-au - lait macules limited to one region of the body . </S>", "<S> its occurrence on the face is extremely rare and only few cases of segmental neurofibromatosis over the face have been described so far . </S>", "<S> we present a case of segmental neurofibromatosis involving the buccal mucosa , tongue , cheek , ear , and neck on the right side of the face . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["neurofibromatosis ( nf ) is an autosomal dominant disease , discovered in 1882 , by the german pathologist friedrich daniel von recklinghausen , characterized by disordered growth of ectodermal tissues , and is part of a group of disorders called phakomatoses ( neurocutaneous syndrome ) .", "neurofibromatosis type - i ( nf-1 ) , also known as von recklinghausen syndrome , is caused by the mutation of a gene on the long arm of chromosome 17 , which encodes a protein known as neurofibromin .", "it is characterized by spots of increased skin pigmentation ( caf au lait spots ) , combined with peripheral nerve tumors and a variety of other dysplastic abnormalities of the skin , nervous system , bones , endocrine organs , and blood vessels .", "the localized form of neurofibromatosis type - i , first described by gammel in 1931 , is very rare .", "crowe et al . proposed the term sectorial neurofibromatosis for this localized form of neurofibromatosis , and miller and sparkes modified the nomenclature to segmental neurofibromatosis ( sn )  the current term for neurofibromas of segmental distribution .", "the commonly affected sites for sn are the thorax and abdomen ( 55% ) , upper extremities ( 20% ) , and lower limb and face ( 10% each ) . only few cases ( less than 10 ) of segmental neurofibromatosis over the face have been described so far ."], ["a 26-year - old female patient reported to the dental department complaining about the unesthetic appearance of her face , since eight years [ figure 1 ] .", "history revealed that eight years back , the patient underwent surgery in the neck region for a cyst and one to two days after surgery the patient got a swelling on the face .", "the swelling was of the same size since then and there was no pain associated with the swelling .", "the unesthetic appearance of the face on examination , the face of the patient appeared asymmetrical , bilaterally , with a growth on the right side of the face .", "the skin over the right side of her face was thickened and hyperpigmented , with a soft and loose hanging overgrowth .", "there were numerous small and large , sessile and pedunculated nodular growths localized on the right side of her neck , with dark - colored underlying skin .", "the skin over the right cheek and chin area showed coffee - colored pigmentation ( caf - au - lait spots ) [ figure 2 ] .", "nodular growths localized on the right side of the neck with dark colored underlying skin there was a soft , compressible , nontender growth on the lower lip .", "the right ear showed disfigurement and hypertrophy , with thickened cartilage and overlying hyperpigmented skin [ figure 3 ] .", "disfigurement of the ear and hypertrophy with thickened cartilage and overlying hyperpigmented skin intraorally there were numerous soft tissue growths with a sessile base seen on the right buccal mucosa [ figure 4 ] and tongue [ figure 5 ] .", "soft tissue growths with sessile bases on right buccal mucosa soft tissue growths with sessile bases on the tongue laboratory findings revealed a raised erythrocyte sedimentation rate ( esr ) ( 70 mm in the first hour ) , a decreased hemoglobin level ( 7.5 gm% ) , and the enzyme - linked immunosorbent assay ( elisa ) was found to be nonreactive . a panoramic radiograph disclosed asymmetric enlargement of the right side of the mandible , with loss of cortication of the lower border in the midline area , expansion of the body of the mandible causing displacement of the teeth , seen on right side , expansion of the right inferior alveolar nerve canal , enlarged mental foramen , resorption of the roots of the mandibular right teeth , and loss of the right antegonial notch .", "a deficiency was seen on the right side of the maxilla , with crowding of the posterior teeth [ figure 6 ] .", "a panoramic radiograph the right lateral oblique radiograph revealed a deep sigmoid notch , loss of the antegonial notch , an enlarged and rounded coronoid process , with resorption of the mandibular right teeth [ figure 7 ] .", "a right lateral oblique radiograph subsequently , biopsy of one of the nodules was taken and sent for histopathological examination .", "the histopathological slide revealed a non - encapsulated tumor of the dermis with a normal overlying epidermis .", "the tumor consisted of loosely spaced spindle cells and wavy collagenous strands in a clear matrix [ figure 8 ] . a non - encapsulated tumor of the dermis , with a normal overlying epidermis"], ["these tumors are composed of schwann cells , fibroblasts , mast cells , and vascular components .", "the caf au lait spots are irregularly - shaped , evenly pigmented , brown macules .", "most individuals with neurofibromatosis have six or more spots that are 1.5 cm or greater in diameter . in young children ,", "five or more caf au lait macules , greater than 0.5 cm in diameter , are suggestive of neurofibromatosis .", "less than 1% of the healthy children have three or more such spots , although one or two caf au lait macules are commonly encountered in healthy individuals without disease .", "bone involvement includes pseudoarthrosis of the tibia , bowing of the long bones , and orbital defects .", "treatment of neurofibromatosis is predominantly surgical . when neurofibromas increase in size or cause pain , malignant transformation must be suspected , and excision or biopsy must be performed .", "acoustic neuromas and tumors that cause tinnitus and vertigo must be excised with great caution .", "plastic surgeons may be included in the correction of deformities , especially those of the face . considering the autosomal dominant inheritance pattern of neurofibromatosis , genetic counseling"]]}
{"article_id": "PMC2721933", "article_text": ["the zygomaticomaxillary complex is an important functional and aesthetic landmark of the midface , and it provides prominent cheek structure .", "however , unfortunately it is very vulnerable to injury because of its intrinsically prominent convexity .", "there are four bony attachments between zygoma and other facial bones : a superior attachment to the frontal bone ( frontozygomatic suture line ) , a medial attachment to the maxilla ( zygomaticomaxillary suture line ) , a lateral attachment to the temporal bone ( zygomaticotemporal suture line ) , and a deep attachment to the greater wing of the sphenoid bone ( zygomaticosphenoidal suture line ) . when blunt trauma to the zygomatic complex results in fractures of all four suture lines , it is referred to as a tetrapod fracture .", "closed reduction without surgical incisions has virtually been abandoned because judgment on the adequate reduction of fracture could not been made and it often resulted in disappointing cosmetic results .", "open reduction with surgical incisions has been accomplished through keen 's approach , gillies ' approach , bicoronal scalp flap approach or the more popular dingman 's approach ( 1 ) .", "this procedure has advantages in that it leaves no facial scars and is simple to perform .", "however , the fracture site is not directly visualized and access to the orbit is not possible , which may result in incomplete surgical reduction .", "however , it necessitates a lengthy scalp incision and provides inadequate exposure to the inferior , medial aspect of orbit , and may leave a conspicuous long scalp scar . also ,", "because of its extensive dissection , the temporal branch of the facial nerve may be damaged .", "the dingman 's approach has become the most popular method in repairing zygomatic complex fractures .", "this procedure utilizes a lateral eyebrow incision line and a second incision across the entire lower eyelid in the subciliary line .", "this technique provides good exposure and direct visualization of the frontozygomatic area , inferior orbital rim and orbital floor .", "but , it provides limited exposure of the lateral orbital rim because it consists of two separate facial incisions , and it leaves external cutaneous scars .", "also , there is some risk of lower eyelid ectropion due to cicatrical contracture . since its first description for the removal of herniated orbital fat ,", "there have been subsequent reports demonstrating the efficacy of transconjunctival approach not only for the lower eyelid blepharoplasty but also for the repair of orbital fractures ( 2 - 5 ) .", "mccord and moses ( 6 ) described the addition of the lateral canthotomy incision to gain access to the lateral orbital wall and inferior orbital rim , and nunery ( 1 ) reported a lateral superior cantholysis for the repair of trimalar fractures .", "since then , a few authors ( 7 - 9 ) have described the advantages of this type of incision , but there has been no report that describe the long term results of two - point fixation through the single transconjunctival incision for the treatment of zygoma fracture .", "between march 2002 and january 2006 , a total of 53 patients with zygomatic complex fractures , which were not severely comminuted , were treated with this surgical method .", "included were 48 men and five women , aged 15 to 88 yr ( mean age 33.2 yr ) .", "twenty eight patients ( 52.8% ) had associated blow out fracture , two patients ( 3.7% ) had associated nasal bone and mandible fracture , and one patient ( 1.9% ) had associated nasal bone fracture ( table 1 ) . upon", "knight and north classification , four patients ( 7.5% ) belonged to group i , twenty one patients ( 39.6% ) belonged to group ii , eight patients ( 15% ) belonged to group iv , eleven patients ( 20.7% ) belonged to group v , and nine patients ( 16.9% ) belonged to group vi .", "all patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . if the reduction of zygoma was not complete , an additional small gingivobuccal incision ( less than 2 cm )", "was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . through the small gingivobuccal incision ,", "the follow - up period ranged from 8 to 46 months ( average 24.8 months ) .", "preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments , enophthalmos , diplopia , limited ocular function , scarring and ectropion .", "follow - up radiographs ( waters ' view and zygomatic arch view ) were routinely used to evaluate the adequacy of reduction . under general endotracheal anesthesia , a lateral canthal incision , about 1.0 - 2.0 cm ,", "then , the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum , and the transconjunctival incision is connected to the lateral canthal incision ( fig .", "the lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors .", "dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . through the extended lateral canthal incision ,", "the lateral aspect of the upper lid is retracted superiorly , and the periosteum of lateral orbital rim is exposed . the periosteum is incised and a subperiosteal dissection is done to expose the orbital floor , the inferior orbital rim , and the lateral orbital rim including the zygomaticofrontal suture area ( fig .", ", the dingman 's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done .", "if the reduction of zygoma is not complete or unsatisfactory , an additional small gingivobuccal incision ( less than 2 cm ) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma .", "dingman 's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . in all cases ,", "complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall .", "then , the two - points ( frontozygomatic suture and inferior orbital rim ) are fixated with plates and screws ( fig .", "resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) were used in 35 patients ( 87.5% ) and titanium miniplates ( osteomed , dallas , tx , u.s.a . ) were used in 5 patients ( 12.5% ) . if there is an associated impure blow - out fracture , orbital floor reconstruction with porous polyethylene implant ( medpor ) is performed .", "the periosteum is closed with a 4 - 0 absorbable suture , and the conjunctiva is closed by a 7 - 0 absorbable suture . in case of old patient with lid laxity , the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . in other cases ,", "finally , the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture .", "between march 2002 and january 2006 , a total of 53 patients with zygomatic complex fractures , which were not severely comminuted , were treated with this surgical method .", "included were 48 men and five women , aged 15 to 88 yr ( mean age 33.2 yr ) .", "twenty eight patients ( 52.8% ) had associated blow out fracture , two patients ( 3.7% ) had associated nasal bone and mandible fracture , and one patient ( 1.9% ) had associated nasal bone fracture ( table 1 ) . upon", "knight and north classification , four patients ( 7.5% ) belonged to group i , twenty one patients ( 39.6% ) belonged to group ii , eight patients ( 15% ) belonged to group iv , eleven patients ( 20.7% ) belonged to group v , and nine patients ( 16.9% ) belonged to group vi .", "all patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . if the reduction of zygoma was not complete , an additional small gingivobuccal incision ( less than 2 cm )", "was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . through the small gingivobuccal incision ,", "the follow - up period ranged from 8 to 46 months ( average 24.8 months ) .", "preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments , enophthalmos , diplopia , limited ocular function , scarring and ectropion .", "follow - up radiographs ( waters ' view and zygomatic arch view ) were routinely used to evaluate the adequacy of reduction .", "under general endotracheal anesthesia , a lateral canthal incision , about 1.0 - 2.0 cm , is made down to the periosteum of lateral orbital rim .", "then , the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum , and the transconjunctival incision is connected to the lateral canthal incision ( fig .", "the lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors .", "dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . through the extended lateral canthal incision ,", "the lateral aspect of the upper lid is retracted superiorly , and the periosteum of lateral orbital rim is exposed .", "the periosteum is incised and a subperiosteal dissection is done to expose the orbital floor , the inferior orbital rim , and the lateral orbital rim including the zygomaticofrontal suture area ( fig .", ", the dingman 's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done .", "if the reduction of zygoma is not complete or unsatisfactory , an additional small gingivobuccal incision ( less than 2 cm ) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma .", "dingman 's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . in all cases ,", "complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall .", "then , the two - points ( frontozygomatic suture and inferior orbital rim ) are fixated with plates and screws ( fig .", "resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) were used in 35 patients ( 87.5% ) and titanium miniplates ( osteomed , dallas , tx , u.s.a . ) were used in 5 patients ( 12.5% ) . if there is an associated impure blow - out fracture , orbital floor reconstruction with porous polyethylene implant ( medpor ) is performed .", "the periosteum is closed with a 4 - 0 absorbable suture , and the conjunctiva is closed by a 7 - 0 absorbable suture . in case of old patient with lid laxity , the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . in other cases ,", "finally , the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture .", "during the average 24.8 months of follow up , most patients had functionally and esthetically satisfactory results .", "all patients showed satisfactory facial symmetry , no lateral canthal displacement , and no functional impairment .", "none of them had persistent diplopia , enophthalmos or alteration of visual acuity . upon postoperative radiography ( skull waters view and zygomatic arch view ) ,", "one patient ( 1.9% ) had persistent scleral show and was corrected three months after the repair of zygomatic complex fracture .", "this case was 78 yr - old patient who had lid laxity preoperatively and showed ectropion postoperatively , which may be due to scar contracture . and", "two patients ( 3.7% ) had mild hyperpigmentation at the lateral canthal incision site even after six months postoperatively .", "but most of patients showed hardly visible scar in the lateral canthal area ( fig .", "although subciliary incision with lateral eyebrow incision ( dingman 's approach ) is the most common approach to the zygomatic complex ( 7 , 10 ) , it can leave a visible scar and ectropion of the lower eyelid .", "factors predisposing to an eyelid retraction and ectropion include hematoma , eyelid edema , adhesions of the orbital septum , scar contracture , horizontal laxity of the eyelid margin , weakening of the pretarsal muscle , and wide dissection of the anterior periosteum ( 11 ) . to decrease the complication rates of the subciliary approach , many authors have tried to treat zygoma complex fracture repair using the transconjunctival approach ( 1 , 6 - 9 ) .", "they emphasized the advantage of this approach over the dingman 's approach , in terms of minimal ectropion , hidden scars , and minimal lid lymphedema . in the transconjunctival approach", ", the dissection can be performed in one of two separate planes ; preseptal approach and retroseptal approach .", "some authors ( 12 , 13 ) advocated that the retroseptal approach is preferred because it can be performed easily and the integrity between orbital septum and orbicularis oculi muscle is not disturbed .", "however , the retroseptal approach has increased risk of injury to the inferior oblique muscle and prolapse of orbital fat into the surgical field ( 10 , 11 ) .", "hence , all our patients were treated with the transconjunctival preseptal approach . in the management of the lateral canthal extension site , manson et al .", "( 9 ) advocated that the canthal reattachment is not required in an acute fracture treatment but is necessary in fractures when treatment is delayed , in patients with established canthal deformities , or in patients in whom a simultaneous coronal incision is employed . according to them ,", "the anterior limb of the lateral canthal tendon which is also referred to as the superficial lateral canthal tendon by other authors ( 14 ) , is continuous to the temporal fascia and galea aponeurotica and the diffuse connections of this limb facilitate proper positioning of the canthus after mobilization of the posterior limb ( deep lateral canthal tendon ) .", "the current authors think that in addition to some indications mentioned above , patients of old age with lid laxity also needs lateral canthal reattachments or canthopexy .", "its significance began to be considered following our case of a 78 yr - old patient who developed persistent ectropion when canthal reattachment had not been performed .", "the precise physical stability of the zygoma with respect to the number and location of miniplates applied is not known , because it depends on fracture anatomy , extent , and the amount of displacement .", "some laboratory studies , using human cadaver heads , have been conducted to investigate the stability of fixated zygoma . in 1989 , rinehart et al .", "( 15 ) simulated noncomminuted zygoma fractures in eight human cadaver heads and the stability of fracture fixation was studied for various fixation patterns . in their study ,", "double - miniplate fixation across the zygomaticofrontal and zygomaticomaxillary fracture lines is sufficient to withstand static and oscillating loading similar to physiologic masticatory forces . in 1990 , davidson et al .", "( 16 ) analyzed different methods of internal fixation of simple displaced fractures of the zygoma in an attempt to define the simplest method of achieving postreduction stability . in their report , the three - point fixation ( frontozygomatic suture , inferior orbital rim , and zygomaticomaxillary buttress ) using either miniplates alone or interfragmentary wiring conferred the greatest stability .", "in addition , they proposed that the two - point fixation using miniplate alone conferred a degree of stability comparable to most methods of three - point fixation regardless of the site in which the miniplates were applied .", "we concur with the above assertions and think that two - point miniplate fixation at the infraorbital rim and zygomaticofrontal suture would suffice , provided the comminution of the zygoma is not severe .", "although not the focus of our attention in this study , in case of severely comminuted fracture , three - point fixation through the transconjunctival incision and gingivobuccal incision is preferably performed . to fixate the fracture site and provide stability , we used four hole resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) in most patients , and titanium miniplates ( osteomed , dalllas , tx , u.s.a . ) only when there was a segmental fracture at the infraorbital rim or when requested by the patient due to the lighter financial burden .", "in addition to the inconspicuous scarring , the main advantage of this method is a direct and full visualization of the lateral and inferior orbital wall to be served as a guidance for an accurate reduction , which makes the reduction more successful .", "the only drawback we experienced in our method is a little difficulty in reducing the fracture - displaced zygoma because of limited space and a slightly inadequate vector for the elevator .", "usually a fractured zygoma is displaced and rotated posteriorly and medially , so an anterior - lateral pull is required .", "however , the lateral canthal incision is located medially to the zygoma body and lateral pulling of the zygoma body may be a little difficult . in conclusion", ", we believe that the proposed technique is very useful for the treatment of zygomatic complex fracture which is not severely comminuted , because it provides excellent exposure and postoperative stability of the zygoma with a lower incidence of complications , including visible scarring and ectropion ."], "abstract_text": ["<S> the ultimate goal in treating zygomatic complex fracture is to obtain an accurate , stable reduction while minimizing external scars and functional deformity . </S>", "<S> the present authors present our experiences with a single transconjunctival incision and two - point ( inferior orbital rim and frontozygomatic suture ) fixation in 53 patients with zygomatic complex fracture which were not comminuted . </S>", "<S> all patients had transconjunctival approaches with lateral canthal extensions , and six out of 53 patients also had an additional small ( about less than 2 cm ) gingivobuccal incision to achieve an accurate reduction . </S>", "<S> there were 3 minor complications , and the overall esthetics and functional results were satisfactory with a long term follow - up . </S>", "<S> our method has the following advantages in the reduction of zygomatic complex fracture ; it leaves only an inconspicuous lateral canthal scar . </S>", "<S> in addition , it provides excellent simultaneous visualization of the inferior orbital rim and frontozygomatic suture area . </S>", "<S> hence , two - point fixation through a single incision can be performed with a satisfactory stability . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Patients", "Surgical techniques", "RESULTS", "DISCUSSION"], "sections": [["the zygomaticomaxillary complex is an important functional and aesthetic landmark of the midface , and it provides prominent cheek structure .", "however , unfortunately it is very vulnerable to injury because of its intrinsically prominent convexity .", "there are four bony attachments between zygoma and other facial bones : a superior attachment to the frontal bone ( frontozygomatic suture line ) , a medial attachment to the maxilla ( zygomaticomaxillary suture line ) , a lateral attachment to the temporal bone ( zygomaticotemporal suture line ) , and a deep attachment to the greater wing of the sphenoid bone ( zygomaticosphenoidal suture line ) . when blunt trauma to the zygomatic complex results in fractures of all four suture lines , it is referred to as a tetrapod fracture .", "closed reduction without surgical incisions has virtually been abandoned because judgment on the adequate reduction of fracture could not been made and it often resulted in disappointing cosmetic results .", "open reduction with surgical incisions has been accomplished through keen 's approach , gillies ' approach , bicoronal scalp flap approach or the more popular dingman 's approach ( 1 ) .", "this procedure has advantages in that it leaves no facial scars and is simple to perform .", "however , the fracture site is not directly visualized and access to the orbit is not possible , which may result in incomplete surgical reduction .", "however , it necessitates a lengthy scalp incision and provides inadequate exposure to the inferior , medial aspect of orbit , and may leave a conspicuous long scalp scar . also ,", "because of its extensive dissection , the temporal branch of the facial nerve may be damaged .", "the dingman 's approach has become the most popular method in repairing zygomatic complex fractures .", "this procedure utilizes a lateral eyebrow incision line and a second incision across the entire lower eyelid in the subciliary line .", "this technique provides good exposure and direct visualization of the frontozygomatic area , inferior orbital rim and orbital floor .", "but , it provides limited exposure of the lateral orbital rim because it consists of two separate facial incisions , and it leaves external cutaneous scars .", "also , there is some risk of lower eyelid ectropion due to cicatrical contracture . since its first description for the removal of herniated orbital fat ,", "there have been subsequent reports demonstrating the efficacy of transconjunctival approach not only for the lower eyelid blepharoplasty but also for the repair of orbital fractures ( 2 - 5 ) .", "mccord and moses ( 6 ) described the addition of the lateral canthotomy incision to gain access to the lateral orbital wall and inferior orbital rim , and nunery ( 1 ) reported a lateral superior cantholysis for the repair of trimalar fractures .", "since then , a few authors ( 7 - 9 ) have described the advantages of this type of incision , but there has been no report that describe the long term results of two - point fixation through the single transconjunctival incision for the treatment of zygoma fracture ."], ["between march 2002 and january 2006 , a total of 53 patients with zygomatic complex fractures , which were not severely comminuted , were treated with this surgical method .", "included were 48 men and five women , aged 15 to 88 yr ( mean age 33.2 yr ) .", "twenty eight patients ( 52.8% ) had associated blow out fracture , two patients ( 3.7% ) had associated nasal bone and mandible fracture , and one patient ( 1.9% ) had associated nasal bone fracture ( table 1 ) . upon", "knight and north classification , four patients ( 7.5% ) belonged to group i , twenty one patients ( 39.6% ) belonged to group ii , eight patients ( 15% ) belonged to group iv , eleven patients ( 20.7% ) belonged to group v , and nine patients ( 16.9% ) belonged to group vi .", "all patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . if the reduction of zygoma was not complete , an additional small gingivobuccal incision ( less than 2 cm )", "was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . through the small gingivobuccal incision ,", "the follow - up period ranged from 8 to 46 months ( average 24.8 months ) .", "preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments , enophthalmos , diplopia , limited ocular function , scarring and ectropion .", "follow - up radiographs ( waters ' view and zygomatic arch view ) were routinely used to evaluate the adequacy of reduction . under general endotracheal anesthesia , a lateral canthal incision , about 1.0 - 2.0 cm ,", "then , the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum , and the transconjunctival incision is connected to the lateral canthal incision ( fig .", "the lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors .", "dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . through the extended lateral canthal incision ,", "the lateral aspect of the upper lid is retracted superiorly , and the periosteum of lateral orbital rim is exposed . the periosteum is incised and a subperiosteal dissection is done to expose the orbital floor , the inferior orbital rim , and the lateral orbital rim including the zygomaticofrontal suture area ( fig .", ", the dingman 's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done .", "if the reduction of zygoma is not complete or unsatisfactory , an additional small gingivobuccal incision ( less than 2 cm ) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma .", "dingman 's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . in all cases ,", "complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall .", "then , the two - points ( frontozygomatic suture and inferior orbital rim ) are fixated with plates and screws ( fig .", "resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) were used in 35 patients ( 87.5% ) and titanium miniplates ( osteomed , dallas , tx , u.s.a . ) were used in 5 patients ( 12.5% ) . if there is an associated impure blow - out fracture , orbital floor reconstruction with porous polyethylene implant ( medpor ) is performed .", "the periosteum is closed with a 4 - 0 absorbable suture , and the conjunctiva is closed by a 7 - 0 absorbable suture . in case of old patient with lid laxity , the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . in other cases ,", "finally , the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture ."], ["between march 2002 and january 2006 , a total of 53 patients with zygomatic complex fractures , which were not severely comminuted , were treated with this surgical method .", "included were 48 men and five women , aged 15 to 88 yr ( mean age 33.2 yr ) .", "twenty eight patients ( 52.8% ) had associated blow out fracture , two patients ( 3.7% ) had associated nasal bone and mandible fracture , and one patient ( 1.9% ) had associated nasal bone fracture ( table 1 ) . upon", "knight and north classification , four patients ( 7.5% ) belonged to group i , twenty one patients ( 39.6% ) belonged to group ii , eight patients ( 15% ) belonged to group iv , eleven patients ( 20.7% ) belonged to group v , and nine patients ( 16.9% ) belonged to group vi .", "all patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . if the reduction of zygoma was not complete , an additional small gingivobuccal incision ( less than 2 cm )", "was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . through the small gingivobuccal incision ,", "the follow - up period ranged from 8 to 46 months ( average 24.8 months ) .", "preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments , enophthalmos , diplopia , limited ocular function , scarring and ectropion .", "follow - up radiographs ( waters ' view and zygomatic arch view ) were routinely used to evaluate the adequacy of reduction ."], ["under general endotracheal anesthesia , a lateral canthal incision , about 1.0 - 2.0 cm , is made down to the periosteum of lateral orbital rim .", "then , the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum , and the transconjunctival incision is connected to the lateral canthal incision ( fig .", "the lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors .", "dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . through the extended lateral canthal incision ,", "the lateral aspect of the upper lid is retracted superiorly , and the periosteum of lateral orbital rim is exposed .", "the periosteum is incised and a subperiosteal dissection is done to expose the orbital floor , the inferior orbital rim , and the lateral orbital rim including the zygomaticofrontal suture area ( fig .", ", the dingman 's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done .", "if the reduction of zygoma is not complete or unsatisfactory , an additional small gingivobuccal incision ( less than 2 cm ) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma .", "dingman 's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . in all cases ,", "complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall .", "then , the two - points ( frontozygomatic suture and inferior orbital rim ) are fixated with plates and screws ( fig .", "resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) were used in 35 patients ( 87.5% ) and titanium miniplates ( osteomed , dallas , tx , u.s.a . ) were used in 5 patients ( 12.5% ) . if there is an associated impure blow - out fracture , orbital floor reconstruction with porous polyethylene implant ( medpor ) is performed .", "the periosteum is closed with a 4 - 0 absorbable suture , and the conjunctiva is closed by a 7 - 0 absorbable suture . in case of old patient with lid laxity , the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . in other cases ,", "finally , the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture ."], ["during the average 24.8 months of follow up , most patients had functionally and esthetically satisfactory results .", "all patients showed satisfactory facial symmetry , no lateral canthal displacement , and no functional impairment .", "none of them had persistent diplopia , enophthalmos or alteration of visual acuity . upon postoperative radiography ( skull waters view and zygomatic arch view ) ,", "one patient ( 1.9% ) had persistent scleral show and was corrected three months after the repair of zygomatic complex fracture .", "this case was 78 yr - old patient who had lid laxity preoperatively and showed ectropion postoperatively , which may be due to scar contracture . and", "two patients ( 3.7% ) had mild hyperpigmentation at the lateral canthal incision site even after six months postoperatively .", "but most of patients showed hardly visible scar in the lateral canthal area ( fig ."], ["although subciliary incision with lateral eyebrow incision ( dingman 's approach ) is the most common approach to the zygomatic complex ( 7 , 10 ) , it can leave a visible scar and ectropion of the lower eyelid .", "factors predisposing to an eyelid retraction and ectropion include hematoma , eyelid edema , adhesions of the orbital septum , scar contracture , horizontal laxity of the eyelid margin , weakening of the pretarsal muscle , and wide dissection of the anterior periosteum ( 11 ) . to decrease the complication rates of the subciliary approach , many authors have tried to treat zygoma complex fracture repair using the transconjunctival approach ( 1 , 6 - 9 ) .", "they emphasized the advantage of this approach over the dingman 's approach , in terms of minimal ectropion , hidden scars , and minimal lid lymphedema . in the transconjunctival approach", ", the dissection can be performed in one of two separate planes ; preseptal approach and retroseptal approach .", "some authors ( 12 , 13 ) advocated that the retroseptal approach is preferred because it can be performed easily and the integrity between orbital septum and orbicularis oculi muscle is not disturbed .", "however , the retroseptal approach has increased risk of injury to the inferior oblique muscle and prolapse of orbital fat into the surgical field ( 10 , 11 ) .", "hence , all our patients were treated with the transconjunctival preseptal approach . in the management of the lateral canthal extension site , manson et al .", "( 9 ) advocated that the canthal reattachment is not required in an acute fracture treatment but is necessary in fractures when treatment is delayed , in patients with established canthal deformities , or in patients in whom a simultaneous coronal incision is employed . according to them ,", "the anterior limb of the lateral canthal tendon which is also referred to as the superficial lateral canthal tendon by other authors ( 14 ) , is continuous to the temporal fascia and galea aponeurotica and the diffuse connections of this limb facilitate proper positioning of the canthus after mobilization of the posterior limb ( deep lateral canthal tendon ) .", "the current authors think that in addition to some indications mentioned above , patients of old age with lid laxity also needs lateral canthal reattachments or canthopexy .", "its significance began to be considered following our case of a 78 yr - old patient who developed persistent ectropion when canthal reattachment had not been performed .", "the precise physical stability of the zygoma with respect to the number and location of miniplates applied is not known , because it depends on fracture anatomy , extent , and the amount of displacement .", "some laboratory studies , using human cadaver heads , have been conducted to investigate the stability of fixated zygoma . in 1989 , rinehart et al .", "( 15 ) simulated noncomminuted zygoma fractures in eight human cadaver heads and the stability of fracture fixation was studied for various fixation patterns . in their study ,", "double - miniplate fixation across the zygomaticofrontal and zygomaticomaxillary fracture lines is sufficient to withstand static and oscillating loading similar to physiologic masticatory forces . in 1990 , davidson et al .", "( 16 ) analyzed different methods of internal fixation of simple displaced fractures of the zygoma in an attempt to define the simplest method of achieving postreduction stability . in their report , the three - point fixation ( frontozygomatic suture , inferior orbital rim , and zygomaticomaxillary buttress ) using either miniplates alone or interfragmentary wiring conferred the greatest stability .", "in addition , they proposed that the two - point fixation using miniplate alone conferred a degree of stability comparable to most methods of three - point fixation regardless of the site in which the miniplates were applied .", "we concur with the above assertions and think that two - point miniplate fixation at the infraorbital rim and zygomaticofrontal suture would suffice , provided the comminution of the zygoma is not severe .", "although not the focus of our attention in this study , in case of severely comminuted fracture , three - point fixation through the transconjunctival incision and gingivobuccal incision is preferably performed . to fixate the fracture site and provide stability , we used four hole resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) in most patients , and titanium miniplates ( osteomed , dalllas , tx , u.s.a . ) only when there was a segmental fracture at the infraorbital rim or when requested by the patient due to the lighter financial burden .", "in addition to the inconspicuous scarring , the main advantage of this method is a direct and full visualization of the lateral and inferior orbital wall to be served as a guidance for an accurate reduction , which makes the reduction more successful .", "the only drawback we experienced in our method is a little difficulty in reducing the fracture - displaced zygoma because of limited space and a slightly inadequate vector for the elevator .", "usually a fractured zygoma is displaced and rotated posteriorly and medially , so an anterior - lateral pull is required .", "however , the lateral canthal incision is located medially to the zygoma body and lateral pulling of the zygoma body may be a little difficult . in conclusion", ", we believe that the proposed technique is very useful for the treatment of zygomatic complex fracture which is not severely comminuted , because it provides excellent exposure and postoperative stability of the zygoma with a lower incidence of complications , including visible scarring and ectropion ."]]}
{"article_id": "PMC3892627", "article_text": ["the gram - negative coccobacillus pasteurella multocida is normally found in the oral cavity of dogs and cats . it is a recognised cause of wound infection as a result of bites , licking or scratching .", "bacteraemia and endocarditis are uncommon complications and , from literature reviews , are found to be associated with prior animal trauma , a second source of infection ( e.g. cellulitis ) and high mortality rates .", "we herein report a case in which a pet owner developed p. multocida endocarditis and was subsequently treated via opat ( outpatient parenteral antimicrobial therapy ) service with an excellent clinical outcome .", "species identification of p. multocida was performed using traditional microbiological methods and confirmed with 16s rrna sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) .", "a 38-year - old man was admitted to our hospital with a 2 days history of fever , malaise and cellulitis around a mitrofanoff abdominal catheter .", "multiple comorbidities included marfan syndrome ( with metallic aortic valve inserted sixteen years before ) and congenital urological abnormalities that led to the mitrofanoff appendicovesicostomy and a recent renal transplant , five years and six months before admission respectively .", "he was on steroids and tacrolimus as immunosuppressive therapy . on presentation , the patient was febrile , hypotensive and tachycardic , had a normal leukocyte count ( 5.0910/l ) but raised creactive protein ( crp ) 45", "mg / l with mild anaemia ( hb of 10.1 g / dl from a baseline of 14.6 g / dl ) and renal impairment ( creatinine 163 umol / l and urea 8.1 mmol / l ) .", "the cellulitis around the mitrofanoff abdominal catheter was noted and a swab was taken for culture .", "his chest x - ray did not show any relevant consolidation and abdominal ultrasound noted two small abdominal collections that had already been noted post - transplant and had since decreased in size .", "the patient was documented to be penicillin allergic ( previous skin rash ) , so was initiated on ertapenem iv 1 gr / od . the cellulitis improved and the patient was clinically well within 48 hours of commencing antibiotics .", "the blood culture set ( aerobic and anaerobic bottles ) taken on admission was positive with p. multocida but the abdominal swab grew only mixed anaerobes .", "urine culture was negative and all subsequent blood cultures taken after starting ertapenem were negative after 5 days of incubation .", "transesophageal ( tee ) echocardiography revealed the presence of a vegetation ( 0.57 0.2 cm ) above the atrium of the right coronary artery .", "a previous transthoracic echocardiogram ( tte ) , around one month prior to admission , was reported as negative .", "therefore , the patient was treated following our local protocol for infective endocarditis : he was switched to ceftriaxone iv 2 gr / od , a picc line was inserted in the right arm and he was discharged under the opat ( outpatient parenteral antimicrobial therapy ) service to complete six weeks iv antibiotics as an outpatient . on follow - up , two repeat ttes showed clear aortic valve .", "we reviewed all the previous reported cases of p. multocida endocarditis in the literature through pubmed and cross - references search .", "to our knowledge this is the twenty - first reported case of endocarditis but the first to be reported in a penicillin allergic patient . as with previous cases ,", "the diagnosis of infective endocarditis was made according to duke criteria : one major criterion ( evidence of endocardial involvement ) and three minor criteria ( predisposing heart condition , fever and microbiological evidence with positive blood culture ) .", "laboratory isolation and identification of p. multocida was performed using standard microbiological techniques : api-20ne , api-20e ( biomerieux ) and the phoenix automated system ( bd ) .", "interestingly , both api profiles reported a low confidence value for identification as p. multocida to the species level ( cv 53% ) and the phoenix assay only identified the isolate to genus level as pasteurella spp . thus , confirmation of species identification was performed using 16s rrna sequencing and maldi - tof protein profiling : 16s rrna sequence was performed using pcr parameters detailed in maskell na et al .", "( 2006 ) and we obtained a 584bp fragment sharing 99% sequence homology to that of p. multocida sub species septica .", "maldi - tof profiling was performed using a bruker daltonics microflex platform with recommended -cyano-4-hydroxycinnamic acid matrix under pre - defined conditions as specified by bruker .", "the maldi - tof mass spectra for the clinical isolate was analysed against maldi - tof mass spectra for 3,438 species of bacteria located in the bruker daltonics flexanalysis 3.0 database .", "the clinical isolate was identified as p. multocida with a confidence value of 2.321 indicating highly probable species identification , with no differential bacterial species being reported .", "sensitivities were as follow ( eucast breakpoints ) : penicillin mic ( mg / l ) was 0.19 , ciprofloxacin 0.016 , doxycycline 0.5 , co - trimoxazole 0.032 , ertapenem 0.016 and ceftriaxone < 0.016 , all being susceptible . of note", "the accuracy of previous reports in the literature has been questioned as their standard microbiological description was incomplete and pasteurella species may be misidentified as haemophilus spp .", "they both belong to the pasteurellaceae family and based on the genomic sequence it appears they diverged approximately 270 million years ago .", "are gram - negative coccobacilli generally present in the oral cavity of cats and dogs . to date twenty - one species", "have been identified and the species p. multocida can be further divided into three different subspecies ( subspecies multocida , septicum and gallicida ) .", "the spectrum of infections varies from skin and soft tissue to central nervous system infections .", "ten out of twenty - one ( 47.6% ) endocarditis cases described in the literature had close contact with animals detailed in the clinical history .", "our patient did not have a clear history of bites or scratches but he did have two cats at home and close contact with dogs at his sister 's house .", "in fact , of the cases of p. multocida endocarditis reviewed , two out of twenty - one ( 9.5% ) had cirrhosis as underlying condition .", "penicillin still remains the best antimicrobial agent for the treatment of virtually all forms of pasteurella infection .", "a fluoroquinolone , doxycycline , or co - trimoxazole should be considered for skin and soft tissue infections as an alternative for patients with intolerance to -lactams . there are no clear guidelines about the treatment of p. multocida endocarditis in penicillin - allergic patients .", "the patient was known to be penicillin allergic ( previous rash ) and ciprofloxacin and doxycycline were contraindicated as causing interaction with his anticoagulant treatment .", "we decided then to switch to ceftriaxone as this antibiotic is already vastly used in the treatment of endocarditis caused by other gram - negative organisms and is more cost effective .", "aminoglycosides have moderate to poor in vitro activity against pasteurella and probably should not be used , particularly given the paucity of clinical experience . finally , our patient improved within 48 hours of commencing antimicrobial therapy and surgical intervention was unnecessary . from the literature ,", "only three other cases have been successfully treated with solely antibiotics and valve replacement was mandatory in seventeen out of twenty - one ( 81% ) of cases described .", "clinicians may face various challenges , from achieving the correct species identification to chosing treatment regimens in penicillin - allergic patients . in our case new diagnostic techniques such as 16s rrna sequencing and maldi - tof were demonstrated to be invaluable tools for species confirmation .", "we propose that maldi - tof is a rapid , accurate method for the identification of p. multocida from bacterial cultures and can significantly reduce the time to diagnosis of p. multocida bacteraemia .", "furthermore we have shown that cephalosporins and carbapenems are probably good substitutes for penicillin in case of allergy ."], "abstract_text": ["<S> pasteurella multocida is a rare cause of infective endocarditis with only a few cases described . </S>", "<S> this report involves a 38-year - old penicillin - allergic patient in an immunocompromised state with several co - morbidities . </S>", "<S> two molecular microbiological techniques , 16s rrna sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry were used to confirm the species identification as p. multocida . </S>", "<S> previous reports in the literature are also reviewed . </S>"], "labels": null, "section_names": ["Introduction", "Brief Report", "Discussion", "Conclusions"], "sections": [["the gram - negative coccobacillus pasteurella multocida is normally found in the oral cavity of dogs and cats . it is a recognised cause of wound infection as a result of bites , licking or scratching .", "bacteraemia and endocarditis are uncommon complications and , from literature reviews , are found to be associated with prior animal trauma , a second source of infection ( e.g. cellulitis ) and high mortality rates .", "we herein report a case in which a pet owner developed p. multocida endocarditis and was subsequently treated via opat ( outpatient parenteral antimicrobial therapy ) service with an excellent clinical outcome .", "species identification of p. multocida was performed using traditional microbiological methods and confirmed with 16s rrna sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) ."], ["a 38-year - old man was admitted to our hospital with a 2 days history of fever , malaise and cellulitis around a mitrofanoff abdominal catheter .", "multiple comorbidities included marfan syndrome ( with metallic aortic valve inserted sixteen years before ) and congenital urological abnormalities that led to the mitrofanoff appendicovesicostomy and a recent renal transplant , five years and six months before admission respectively .", "he was on steroids and tacrolimus as immunosuppressive therapy . on presentation , the patient was febrile , hypotensive and tachycardic , had a normal leukocyte count ( 5.0910/l ) but raised creactive protein ( crp ) 45", "mg / l with mild anaemia ( hb of 10.1 g / dl from a baseline of 14.6 g / dl ) and renal impairment ( creatinine 163 umol / l and urea 8.1 mmol / l ) .", "the cellulitis around the mitrofanoff abdominal catheter was noted and a swab was taken for culture .", "his chest x - ray did not show any relevant consolidation and abdominal ultrasound noted two small abdominal collections that had already been noted post - transplant and had since decreased in size .", "the patient was documented to be penicillin allergic ( previous skin rash ) , so was initiated on ertapenem iv 1 gr / od . the cellulitis improved and the patient was clinically well within 48 hours of commencing antibiotics .", "the blood culture set ( aerobic and anaerobic bottles ) taken on admission was positive with p. multocida but the abdominal swab grew only mixed anaerobes .", "urine culture was negative and all subsequent blood cultures taken after starting ertapenem were negative after 5 days of incubation .", "transesophageal ( tee ) echocardiography revealed the presence of a vegetation ( 0.57 0.2 cm ) above the atrium of the right coronary artery .", "a previous transthoracic echocardiogram ( tte ) , around one month prior to admission , was reported as negative .", "therefore , the patient was treated following our local protocol for infective endocarditis : he was switched to ceftriaxone iv 2 gr / od , a picc line was inserted in the right arm and he was discharged under the opat ( outpatient parenteral antimicrobial therapy ) service to complete six weeks iv antibiotics as an outpatient . on follow - up , two repeat ttes showed clear aortic valve ."], ["we reviewed all the previous reported cases of p. multocida endocarditis in the literature through pubmed and cross - references search .", "to our knowledge this is the twenty - first reported case of endocarditis but the first to be reported in a penicillin allergic patient . as with previous cases ,", "the diagnosis of infective endocarditis was made according to duke criteria : one major criterion ( evidence of endocardial involvement ) and three minor criteria ( predisposing heart condition , fever and microbiological evidence with positive blood culture ) .", "laboratory isolation and identification of p. multocida was performed using standard microbiological techniques : api-20ne , api-20e ( biomerieux ) and the phoenix automated system ( bd ) .", "interestingly , both api profiles reported a low confidence value for identification as p. multocida to the species level ( cv 53% ) and the phoenix assay only identified the isolate to genus level as pasteurella spp . thus , confirmation of species identification was performed using 16s rrna sequencing and maldi - tof protein profiling : 16s rrna sequence was performed using pcr parameters detailed in maskell na et al .", "( 2006 ) and we obtained a 584bp fragment sharing 99% sequence homology to that of p. multocida sub species septica .", "maldi - tof profiling was performed using a bruker daltonics microflex platform with recommended -cyano-4-hydroxycinnamic acid matrix under pre - defined conditions as specified by bruker .", "the maldi - tof mass spectra for the clinical isolate was analysed against maldi - tof mass spectra for 3,438 species of bacteria located in the bruker daltonics flexanalysis 3.0 database .", "the clinical isolate was identified as p. multocida with a confidence value of 2.321 indicating highly probable species identification , with no differential bacterial species being reported .", "sensitivities were as follow ( eucast breakpoints ) : penicillin mic ( mg / l ) was 0.19 , ciprofloxacin 0.016 , doxycycline 0.5 , co - trimoxazole 0.032 , ertapenem 0.016 and ceftriaxone < 0.016 , all being susceptible . of note", "the accuracy of previous reports in the literature has been questioned as their standard microbiological description was incomplete and pasteurella species may be misidentified as haemophilus spp .", "they both belong to the pasteurellaceae family and based on the genomic sequence it appears they diverged approximately 270 million years ago .", "are gram - negative coccobacilli generally present in the oral cavity of cats and dogs . to date twenty - one species", "have been identified and the species p. multocida can be further divided into three different subspecies ( subspecies multocida , septicum and gallicida ) .", "the spectrum of infections varies from skin and soft tissue to central nervous system infections .", "ten out of twenty - one ( 47.6% ) endocarditis cases described in the literature had close contact with animals detailed in the clinical history .", "our patient did not have a clear history of bites or scratches but he did have two cats at home and close contact with dogs at his sister 's house .", "in fact , of the cases of p. multocida endocarditis reviewed , two out of twenty - one ( 9.5% ) had cirrhosis as underlying condition .", "penicillin still remains the best antimicrobial agent for the treatment of virtually all forms of pasteurella infection .", "a fluoroquinolone , doxycycline , or co - trimoxazole should be considered for skin and soft tissue infections as an alternative for patients with intolerance to -lactams . there are no clear guidelines about the treatment of p. multocida endocarditis in penicillin - allergic patients .", "the patient was known to be penicillin allergic ( previous rash ) and ciprofloxacin and doxycycline were contraindicated as causing interaction with his anticoagulant treatment .", "we decided then to switch to ceftriaxone as this antibiotic is already vastly used in the treatment of endocarditis caused by other gram - negative organisms and is more cost effective .", "aminoglycosides have moderate to poor in vitro activity against pasteurella and probably should not be used , particularly given the paucity of clinical experience . finally , our patient improved within 48 hours of commencing antimicrobial therapy and surgical intervention was unnecessary . from the literature ,", "only three other cases have been successfully treated with solely antibiotics and valve replacement was mandatory in seventeen out of twenty - one ( 81% ) of cases described ."], ["clinicians may face various challenges , from achieving the correct species identification to chosing treatment regimens in penicillin - allergic patients . in our case new diagnostic techniques such as 16s rrna sequencing and maldi - tof were demonstrated to be invaluable tools for species confirmation .", "we propose that maldi - tof is a rapid , accurate method for the identification of p. multocida from bacterial cultures and can significantly reduce the time to diagnosis of p. multocida bacteraemia .", "furthermore we have shown that cephalosporins and carbapenems are probably good substitutes for penicillin in case of allergy ."]]}
{"article_id": "PMC3974471", "article_text": ["patient aged 64 was admitted to the county hospital urology ward in september 2010 and diagnosed with tumor of the left kidney .", "ultrasonographic exam revealed the structure with a diameter of 45 mm in the central field of the left kidney .", "ct showed a solid focal lesion of the left kidney ( 30 x 40 x 32 mm ) located below the renal stalk and covering the renal pelvis and the paranephric section of the left ureter , apart from that no changes were observed ( figure 1 ) .", "radiological image of the chest did not reveal any pathological changes . in order to make the results of pre  operational diagnosis", "sided ureterorenoscopy was conducted and a urine sample was taken for cytology to exclude epithelial nature of the tumor . after undergoing the above  mentioned test , the patient was qualified for transperitoneal radical nephrectomy . with general anesthesia applied , the left kidney was removed through an incision below the left costal margin . during the operation", ", a significant peripheral infiltration was discovered which hindered access to the renal stalk of the left kidney .", "operative course passed without complications and the patient was discharged in good general condition 8 days after the surgery .", "left kidney tumor in ct exam . on pathological examination ( samples no 12399241239928 ) in all samples", "after detailed hematological work  up which did not show any other tumors , the patient was qualified for radiation treatment and currently remains under permanent oncological supervision .", "medical records and literature mention several cases of malignant kidney lymphoma [ 1 , 2 ] . some authors claim that this entity accounts for 3% of all solid renal tumors among adults .", "lymphomatous renal involvement can have three different causes : most commonly it appears along with the generalized disease and enlarged lymph nodes .", "renal involvement develops without any symptoms then.it may be related to organ transplantation and infection with epstein ", "such cases are rare and difficult to diagnose which may lead to mistakes in treatment . \n ", "such cases are rare and difficult to diagnose which may lead to mistakes in treatment .", "the case presented in the report illustrates diagnostic and therapeutic difficulties that a urologist might encounter during treatment of patients with possible renal tumor .", "results provided by clinical imaging of focal lesions are inconsistent . what must always be taken into account", "is an atypical tumorous process , autoimmunological disorders as well as inflammatory condition . in this particular case", "control appointments in the department of hematology have been scheduled to take place every four months and the prognosis for the patient has been described as good .", "they develop from lymphocytes and , depending on the variety of b and t lymphocytes , those processes can have different clinical outcome .", "some of them are located solely within the lymph nodes or tissues and internal organs , while others are accompanied by leukemia .", "it might seem that when it comes to lymphomas , the pathogens initiating the course of the disease could be both , the epstein  barr viruses which attack the b  lymphocytes , and by htlv1 viruses dwelling in t  lymphocytes and causing a subacute form of leukemia among adults .", "there can also be some co  factors of environmental character : chloro  organic compounds , ionization energy , benzene  like substances , states of immunosuppression and autoimmunological diseases .", "the malignant lymphoma morbidity rate can also be increased by extensive exposition to herbicides , and is even 35 times higher among patients with a kidney transplant .", "it usually dwells within the lymphoid tissue of waldeyer 's ring , spleen and thymus .", "extranodal lymphomas can most often be found in the gastrointestinal tract , other types can be located in skin , bones , eye ", "kidney might be at the stage of involvement which spreads through lymphatic system from the nodes in retro peritoneum .", "if the renal involvement occurs at an advanced stage of the disease , as yet another location of it , it is relatively easy to diagnose .", "once a patient is diagnosed with lymphoma of kidney , further examinations are required in order to rule out a systemic disease .", "the necessary test in such cases include : blood tests , bone marrow biopsy , radiological examination , pathomorphological evaluation and histo  immunological evaluation of enlarged lymph nodes .", "the malignant types , however , build up rapidly , are resistant to treatment and show no signs of improvement within a short period of time .", "how long the patient will live depends on the histopathological diagnosis of the tumor and its stage of development .", "non  hodgkin lymphomas are treated with chemotherapy , radiation , and , in some cases , surgeries . low  malignant , originally extranodal , tumors are usually surgically removed and treated with radiotherapy .", "cell lymphoma associated with chronic inflammation arising in a renal pseudocyst with barely noticeable clinical symptoms .", "publications in english present several cases of spontaneous rupture of the ureter as primary symptom of malignant lymphoma .", "risk of their occurrence increases from 1.2% in 5 years after the operation , to 6.8% in 20 years after the transplantation , which is related to epstein barr virus infections", ". some authors also report on cases of co  existence of inflammatory intestines disease , including the crohn disease , and malignant lymphomas but then changes can most frequently be located in the vicinity of intestines or other adjacent sections of the digestive tract .", "the presented case leads to a conclusion that each focal lesion of unclear etiology found on imagining studies should be treated as a possible malignant lymphoma and ought to be histopathologically verified as quickly as possible ."], "abstract_text": ["<S> we present a 64year  old patient suffering from extranodal malignant lymphoma . </S>", "<S> the patient was admitted to the county hospital urology ward with suspicion of the left kidney tumor . as part of pre  </S>", "<S> operational diagnosis , ultrasonographic examination and computed tomography of kidneys were conducted . </S>", "<S> the results confirmed the initial diagnosis . after undergoing radical surgical treatment , the patient is currently under the care of hematology ward of oncology centre . </S>"], "labels": null, "section_names": ["CASE DESCRIPTION", "DISCUSSION"], "sections": [["patient aged 64 was admitted to the county hospital urology ward in september 2010 and diagnosed with tumor of the left kidney .", "ultrasonographic exam revealed the structure with a diameter of 45 mm in the central field of the left kidney .", "ct showed a solid focal lesion of the left kidney ( 30 x 40 x 32 mm ) located below the renal stalk and covering the renal pelvis and the paranephric section of the left ureter , apart from that no changes were observed ( figure 1 ) .", "radiological image of the chest did not reveal any pathological changes . in order to make the results of pre  operational diagnosis", "sided ureterorenoscopy was conducted and a urine sample was taken for cytology to exclude epithelial nature of the tumor . after undergoing the above  mentioned test , the patient was qualified for transperitoneal radical nephrectomy . with general anesthesia applied , the left kidney was removed through an incision below the left costal margin . during the operation", ", a significant peripheral infiltration was discovered which hindered access to the renal stalk of the left kidney .", "operative course passed without complications and the patient was discharged in good general condition 8 days after the surgery .", "left kidney tumor in ct exam . on pathological examination ( samples no 12399241239928 ) in all samples", "after detailed hematological work  up which did not show any other tumors , the patient was qualified for radiation treatment and currently remains under permanent oncological supervision ."], ["medical records and literature mention several cases of malignant kidney lymphoma [ 1 , 2 ] . some authors claim that this entity accounts for 3% of all solid renal tumors among adults .", "lymphomatous renal involvement can have three different causes : most commonly it appears along with the generalized disease and enlarged lymph nodes .", "renal involvement develops without any symptoms then.it may be related to organ transplantation and infection with epstein ", "such cases are rare and difficult to diagnose which may lead to mistakes in treatment . \n ", "such cases are rare and difficult to diagnose which may lead to mistakes in treatment .", "the case presented in the report illustrates diagnostic and therapeutic difficulties that a urologist might encounter during treatment of patients with possible renal tumor .", "results provided by clinical imaging of focal lesions are inconsistent . what must always be taken into account", "is an atypical tumorous process , autoimmunological disorders as well as inflammatory condition . in this particular case", "control appointments in the department of hematology have been scheduled to take place every four months and the prognosis for the patient has been described as good .", "they develop from lymphocytes and , depending on the variety of b and t lymphocytes , those processes can have different clinical outcome .", "some of them are located solely within the lymph nodes or tissues and internal organs , while others are accompanied by leukemia .", "it might seem that when it comes to lymphomas , the pathogens initiating the course of the disease could be both , the epstein  barr viruses which attack the b  lymphocytes , and by htlv1 viruses dwelling in t  lymphocytes and causing a subacute form of leukemia among adults .", "there can also be some co  factors of environmental character : chloro  organic compounds , ionization energy , benzene  like substances , states of immunosuppression and autoimmunological diseases .", "the malignant lymphoma morbidity rate can also be increased by extensive exposition to herbicides , and is even 35 times higher among patients with a kidney transplant .", "it usually dwells within the lymphoid tissue of waldeyer 's ring , spleen and thymus .", "extranodal lymphomas can most often be found in the gastrointestinal tract , other types can be located in skin , bones , eye ", "kidney might be at the stage of involvement which spreads through lymphatic system from the nodes in retro peritoneum .", "if the renal involvement occurs at an advanced stage of the disease , as yet another location of it , it is relatively easy to diagnose .", "once a patient is diagnosed with lymphoma of kidney , further examinations are required in order to rule out a systemic disease .", "the necessary test in such cases include : blood tests , bone marrow biopsy , radiological examination , pathomorphological evaluation and histo  immunological evaluation of enlarged lymph nodes .", "the malignant types , however , build up rapidly , are resistant to treatment and show no signs of improvement within a short period of time .", "how long the patient will live depends on the histopathological diagnosis of the tumor and its stage of development .", "non  hodgkin lymphomas are treated with chemotherapy , radiation , and , in some cases , surgeries . low  malignant , originally extranodal , tumors are usually surgically removed and treated with radiotherapy .", "cell lymphoma associated with chronic inflammation arising in a renal pseudocyst with barely noticeable clinical symptoms .", "publications in english present several cases of spontaneous rupture of the ureter as primary symptom of malignant lymphoma .", "risk of their occurrence increases from 1.2% in 5 years after the operation , to 6.8% in 20 years after the transplantation , which is related to epstein barr virus infections", ". some authors also report on cases of co  existence of inflammatory intestines disease , including the crohn disease , and malignant lymphomas but then changes can most frequently be located in the vicinity of intestines or other adjacent sections of the digestive tract .", "the presented case leads to a conclusion that each focal lesion of unclear etiology found on imagining studies should be treated as a possible malignant lymphoma and ought to be histopathologically verified as quickly as possible ."]]}
{"article_id": "PMC3388258", "article_text": ["assessment of myocardial viability in patients with coronary artery disease is of great clinical importance , as dysfunctional but viable myocardium has the ability to regain contractility after coronary revascularization with subsequent improvements in cardiac function and prognosis . the perfusable tissue index ( pti )", "can be used as a marker of myocardial viability , having been validated in patients with ischaemic heart disease [ 27 ] .", "however , pti has never been used in clinical practice , primarily due to the complex imaging protocol consisting of dynamic [ o]h2o and [ o]co scans , together with a transmission scan , and the lack of high - quality , clinically usable parametric images .", "recently , however , a method was developed to derive the pti from a single [ o]h2o pet / ct scan .", "given the current potential of clinical [ o]h2o - based perfusion imaging and the rapid growth in the availability of cardiac pet / ct systems , pti viability measurements could become incorporated into clinical practice [ 9 , 10 ] .", "parametric pti images derived from [ o]h2o pet / ct scans , however , have not yet been validated .", "the purpose of this study was to compare these novel parametric pti images with late gadolinium - enhanced cardiovascular magnetic resonance ( lge - cmr ) imaging , an established method for quantifying scar size and a marker of viability .", "the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period .", "all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations .", "patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded .", "the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion .", "[ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner .", "[ o]h2o ( 370  mbq ) was injected as a 5-ml bolus ( 0.8  ml  s ) followed by a 35-ml saline flush ( 2  ml  s ) , simultaneously starting a 6-min list mode emission scan .", "this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5  s , pitch 0.825 , collimation 16    0.625 , acquiring 20  cm in 37  s ) during normal breathing .", "all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed .", "dynamic [ o]h2o images were reconstructed into 22 frames ( 1    10 , 8    5 , 4    10 , 2    15 , 3    20 , 2    30 , 2    60  s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1  cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus .", "these rois were combined into one volume of interest ( voi ) for the ascending aorta .", "a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1  cm from the rv wall to avoid spill - in from myocardial activity .", "both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time  activity curves .", "parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] .", "parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig .  1 ) .", "finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig .", "( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans ,", "a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging .", "image parameters included : slice thickness 5  mm , slice gap 5  mm , temporal resolution < 50  ms , repetition time 3.2  ms , echo time 1.54  ms , flip angle 60 , typical image resolution 1.3    1.6    5.0  mm .", "the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines ,", "stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) .", "contrast images were acquired 10 to 15  min after administration of 0.2  mmol  kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4  ms , tr 9.8  ms , inversion time 250 to 300  ms , typical voxel size 1.3    1.6    5.0  mm ) .", "images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) .", "the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images .", "end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses .", "the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50  % of the maximum hyperenhancement intensity . if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used .", "all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area .", "cmr images were analysed according to the same 16-segment model as used for the parametric pet images .", "finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50  % lge per segment [ 15 , 17 ] .", "continuous variables are presented as means  sd , and categorical data are summarized as frequencies and percentages .", "multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment .", "receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr .", "the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium .", "the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period .", "all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations .", "patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded .", "the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion .", "[ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner .", "[ o]h2o ( 370  mbq ) was injected as a 5-ml bolus ( 0.8  ml  s ) followed by a 35-ml saline flush ( 2  ml  s ) , simultaneously starting a 6-min list mode emission scan .", "this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5  s , pitch 0.825 , collimation 16    0.625 , acquiring 20  cm in 37  s ) during normal breathing .", "all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed .", "dynamic [ o]h2o images were reconstructed into 22 frames ( 1    10 , 8    5 , 4    10 , 2    15 , 3    20 , 2    30 , 2    60  s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1  cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus .", "these rois were combined into one volume of interest ( voi ) for the ascending aorta .", "a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1  cm from the rv wall to avoid spill - in from myocardial activity .", "both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time  activity curves .", "parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] .", "parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig .  1 ) .", "finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig .", "( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease", "cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans ,", "a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging .", "image parameters included : slice thickness 5  mm , slice gap 5  mm , temporal resolution < 50  ms , repetition time 3.2  ms , echo time 1.54  ms , flip angle 60 , typical image resolution 1.3    1.6    5.0  mm .", "the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines ,", "stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) .", "contrast images were acquired 10 to 15  min after administration of 0.2  mmol  kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4  ms , tr 9.8  ms , inversion time 250 to 300  ms , typical voxel size 1.3    1.6    5.0  mm ) .", "images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) .", "the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images .", "end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses .", "the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50  % of the maximum hyperenhancement intensity .", "if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used .", "all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area .", "cmr images were analysed according to the same 16-segment model as used for the parametric pet images .", "finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50  % lge per segment [ 15 , 17 ] .", "continuous variables are presented as means  sd , and categorical data are summarized as frequencies and percentages .", "multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment .", "receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr .", "the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium . a p value of < 0.05 was considered statistically significant .", "lge was seen in the cmr images of 34 patients ( 74  % ) . of the 736 myocardial segments ,", "364 ( 49  % ) showed some degree of lge.table 1patient characteristics ( n  =  46)characteristicvalueage ( years)65    10gender ( male)36 ( 78  % ) previous myocardial infarction34 ( 74  % ) lv end - diastolic volume ( ml)226    65lv end - systolic volume ( ml)135    71lv ejection fraction ( % ) 42    16lv mass ( g)132    39 patient characteristics ( n  =  46 ) pet / ct and cmr data are summarized in table  2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p  <  0.001 by anova ) .", "atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p  <  0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control", "( n  =  372)025 ( n  =  190)2550 ( n  =  83)5075 ( n  =  54)75 ( n  =  37)p value ( anova)atf0.76    0.090.77    0.090.75    0.110.73    0.080.68    0.11**<0.001ptf ( g  ml)0.72    0.080.72    0.090.70    0.090.60    0.08**0.51    0.12***<0.001pti0.91    0.080.89    0.090.89    0.090.77    0.10**0.70 ", "  0.16***<0.001mbf ( ml  g  min)1.02    0.300.91    0.26 * 0.85    0.30 * 0.83    0.24 * 0.67    0.30**<0.001*p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge . segmental pet / ct and lge data * p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge .", "figure  2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig .", "2long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min)fig .", "3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25  % ) segments showing some degree of lge were judged to be nonviable ( lge > 50  % ) . as shown in fig .  4 ,", "the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p  =  0.541 ) .", "mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p  <  0.001 ) .", "optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69  g  ml , 0.80 , and 0.78  ml  min  g with sensitivities of 69  % , 91  % and 72  % , and specificities of 87  % , 73  % and 56  % , respectively .", "figure  3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig .", "4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement", "lge was seen in the cmr images of 34 patients ( 74  % ) . of the 736 myocardial segments ,", "364 ( 49  % ) showed some degree of lge.table 1patient characteristics ( n  =  46)characteristicvalueage ( years)65    10gender ( male)36 ( 78  % ) previous myocardial infarction34 ( 74  % ) lv end - diastolic volume ( ml)226    65lv end - systolic volume ( ml)135    71lv ejection fraction ( % ) 42    16lv mass ( g)132    39 patient characteristics ( n  =  46 )", "pet / ct and cmr data are summarized in table  2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p  <  0.001 by anova ) .", "atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p  <  0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control", "( n  =  372)025 ( n  =  190)2550 ( n  =  83)5075 ( n  =  54)75 ( n  =  37)p value ( anova)atf0.76    0.090.77    0.090.75    0.110.73    0.080.68    0.11**<0.001ptf ( g  ml)0.72    0.080.72    0.090.70    0.090.60    0.08**0.51    0.12***<0.001pti0.91    0.080.89    0.090.89    0.090.77    0.10**0.70    0.16***<0.001mbf ( ml  g  min)1.02    0.300.91    0.26 * 0.85    0.30 * 0.83    0.24 * 0.67    0.30**<0.001*p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge .", "segmental pet / ct and lge data * p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge .", "figure  2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig .", "2long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min)fig .", "3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25  % ) segments showing some degree of lge were judged to be nonviable ( lge > 50  % ) . as shown in fig .  4 ,", "the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p  =  0.541 ) .", "mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p  <  0.001 ) .", "optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69  g  ml , 0.80 , and 0.78  ml  min  g with sensitivities of 69  % , 91  % and 72  % , and specificities of 87  % , 73  % and 56  % , respectively .", "figure  3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig .", "4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement", "the present study was conducted to validate the use of a parametric myocardial viability imaging technique using [ o]h2o pet / ct in patients with ischaemic heart disease .", "viability assessed using parametric ptf and pti imaging was in good agreement with that assessed using lge cmr .", "furthermore , these images and myocardial perfusion imaging are obtained simultaneously allowing both myocardial viability and ischaemia to be evaluated in a single scanning session .", "[ o]h2o is generally considered to be the gold standard for absolute quantification of myocardial perfusion in vivo [ 10 , 18 , 19 ] .", "apart from mbf , [ o]h2o also provides estimates of the extent of myocardium within a roi that is able to exchange water rapidly , i.e. ptf .", "subsequently , ptf can be corrected for partial volume effects by dividing it by its anatomical counterpart atf , resulting in pti .", "it has been shown that both ptf and pti can identify myocardial scarring and thus can act as markers of viability . only recently ,", "however , a method has been developed that enables generation parametric atf , ptf and pti images from a single [ o]h2o pet / ct scan .", "in addition , the traditional ( long ) transmission scan can be replaced by a ( rapid ) low - dose ct scan , thereby shortening the total scanning time substantially .", "now that the method has been shown to produce high - quality parametric images , its implementation into clinical practice needs investigation . using the described parametric imaging approach", ", the present study demonstrated that atf was relatively constant independent of tissue characteristics of the myocardium . only in ( near )", "transmurally infarcted segments was atf significantly reduced , most likely due to wall thinning , as atf is prone to partial volume effects . in contrast , ptf and pti progressively decreased with increasing extent of scarring , as shown by lge cmr .", "these results are in line with those of previous studies in which reductions in both ptf and pti were observed in nonviable scarred myocardium .", "pti in control segments appeared to be somewhat lower than the expected value of unity for normal segments ( 0.91    0.08 ) .", "this phenomenon has previously been observed in cardiomyopathy in both animal experiments and human studies [ 6 , 20 ] .", "slight misalignment between pet and cmr segments may have occurred to additionally account for this reduction in pti .", "furthermore , the presence of interstitial fibrosis in dilated cardiomyopathy may also explain this reduction in ", "normal segments that remains undetected on lge cmr [ 21 , 22 ] . taking lge as a reference , the optimal cut - off values for discriminating between viable and nonviable myocardium", "although this would reduce the data processing time , it would not affect the scanning protocol , as for both parameters a dynamic [ o]h2o pet scan in combination with a low - dose ct scan are required .", "in addition , the sensitivity of pti exceeded that of ptf ( 91  % and 68  % , respectively ) , whereas for specificity the opposite pattern was observed ( 73  % and 78  % , respectively ) .", "although further study is need to determine the cause of this discrepancy , as a potential clinical marker of viability pti may be favoured over ptf to reduce false - negative findings in patients who might benefit from revascularization .", "it is of interest to note that , compared to ptf and pti , mbf performed relatively poorly in distinguishing viable from nonviable tissue , rendering it less suitable for viability imaging .", "previous studies have indicated that the optimal cut - off value for pti is in the range 0.70.9 .", "lge was used as a surrogate end - point of myocardial viability instead of functional recovery of dysfunctional myocardium after revascularization .", "lge has been shown to be a good , but not perfect , marker of myocardial viability [ 15 , 17 ] .", "therefore , the results should be interpreted with caution , and more studies are warranted to establish the value of parametric pti as a viability marker .", "ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease .", "ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease .", "this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited ."], "abstract_text": ["<S> purposethe perfusable tissue index ( pti ) is a marker of myocardial viability . </S>", "<S> recent technological advances have made it possible to generate parametric pti images from a single [ 15o]h2o pet / ct scan . </S>", "<S> the purpose of this study was to validate these parametric pti images.methodsthe study population comprised 46 patients with documented or suspected coronary artery disease who were studied with [ 15o]h2o pet and late gadolinium - enhanced ( lge ) cardiac magnetic resonance imaging ( cmr).resultsof the 736 myocardial segments included , 364 showed some degree of lge . </S>", "<S> pti and perfusable tissue fraction ( ptf ) diminished with increasing lge . </S>", "<S> the areas under the curve of the pti and ptf , used to predict ( near ) transmural lge on cmr , were 0.86 and 0.87 , respectively . </S>", "<S> optimal sensitivity and specificity were 91  % and 73  % for pti and 69  % and 87  % for ptf , respectively.conclusionpti and ptf assessed with a single [ 15o]h2o scan can be utilized as markers of myocardial viability in patients with coronary artery disease . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Study population", "PET/CT image acquisition", "PET/CT image analysis", "CMR image acquisition", "CMR image analysis", "Statistical analysis", "Results", "Baseline characteristics", "PET/CT and CMR parameters", "Predictive values for viability", "Discussion", "Conclusion", "Open Access"], "sections": [["assessment of myocardial viability in patients with coronary artery disease is of great clinical importance , as dysfunctional but viable myocardium has the ability to regain contractility after coronary revascularization with subsequent improvements in cardiac function and prognosis . the perfusable tissue index ( pti )", "can be used as a marker of myocardial viability , having been validated in patients with ischaemic heart disease [ 27 ] .", "however , pti has never been used in clinical practice , primarily due to the complex imaging protocol consisting of dynamic [ o]h2o and [ o]co scans , together with a transmission scan , and the lack of high - quality , clinically usable parametric images .", "recently , however , a method was developed to derive the pti from a single [ o]h2o pet / ct scan .", "given the current potential of clinical [ o]h2o - based perfusion imaging and the rapid growth in the availability of cardiac pet / ct systems , pti viability measurements could become incorporated into clinical practice [ 9 , 10 ] .", "parametric pti images derived from [ o]h2o pet / ct scans , however , have not yet been validated .", "the purpose of this study was to compare these novel parametric pti images with late gadolinium - enhanced cardiovascular magnetic resonance ( lge - cmr ) imaging , an established method for quantifying scar size and a marker of viability ."], ["the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period .", "all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations .", "patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded .", "the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion .", "[ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner .", "[ o]h2o ( 370  mbq ) was injected as a 5-ml bolus ( 0.8  ml  s ) followed by a 35-ml saline flush ( 2  ml  s ) , simultaneously starting a 6-min list mode emission scan .", "this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5  s , pitch 0.825 , collimation 16    0.625 , acquiring 20  cm in 37  s ) during normal breathing .", "all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed .", "dynamic [ o]h2o images were reconstructed into 22 frames ( 1    10 , 8    5 , 4    10 , 2    15 , 3    20 , 2    30 , 2    60  s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1  cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus .", "these rois were combined into one volume of interest ( voi ) for the ascending aorta .", "a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1  cm from the rv wall to avoid spill - in from myocardial activity .", "both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time  activity curves .", "parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] .", "parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig .  1 ) .", "finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig .", "( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans ,", "a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging .", "image parameters included : slice thickness 5  mm , slice gap 5  mm , temporal resolution < 50  ms , repetition time 3.2  ms , echo time 1.54  ms , flip angle 60 , typical image resolution 1.3    1.6    5.0  mm .", "the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines ,", "stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) .", "contrast images were acquired 10 to 15  min after administration of 0.2  mmol  kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4  ms , tr 9.8  ms , inversion time 250 to 300  ms , typical voxel size 1.3    1.6    5.0  mm ) .", "images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) .", "the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images .", "end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses .", "the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50  % of the maximum hyperenhancement intensity . if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used .", "all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area .", "cmr images were analysed according to the same 16-segment model as used for the parametric pet images .", "finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50  % lge per segment [ 15 , 17 ] .", "continuous variables are presented as means  sd , and categorical data are summarized as frequencies and percentages .", "multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment .", "receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr .", "the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium ."], ["the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period .", "all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations .", "patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded .", "the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion ."], ["[ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner .", "[ o]h2o ( 370  mbq ) was injected as a 5-ml bolus ( 0.8  ml  s ) followed by a 35-ml saline flush ( 2  ml  s ) , simultaneously starting a 6-min list mode emission scan .", "this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5  s , pitch 0.825 , collimation 16    0.625 , acquiring 20  cm in 37  s ) during normal breathing .", "all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed .", "dynamic [ o]h2o images were reconstructed into 22 frames ( 1    10 , 8    5 , 4    10 , 2    15 , 3    20 , 2    30 , 2    60  s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1  cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus .", "these rois were combined into one volume of interest ( voi ) for the ascending aorta .", "a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1  cm from the rv wall to avoid spill - in from myocardial activity .", "both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time  activity curves ."], ["parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] .", "parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig .  1 ) .", "finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig .", "( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g  ml ) , ( c ) ptf ( g  ml ) , and ( d ) pti in a patient without coronary artery disease"], ["cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans ,", "a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging .", "image parameters included : slice thickness 5  mm , slice gap 5  mm , temporal resolution < 50  ms , repetition time 3.2  ms , echo time 1.54  ms , flip angle 60 , typical image resolution 1.3    1.6    5.0  mm .", "the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines ,", "stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) .", "contrast images were acquired 10 to 15  min after administration of 0.2  mmol  kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4  ms , tr 9.8  ms , inversion time 250 to 300  ms , typical voxel size 1.3    1.6    5.0  mm ) ."], ["images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) .", "the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images .", "end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses .", "the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50  % of the maximum hyperenhancement intensity .", "if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used .", "all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area .", "cmr images were analysed according to the same 16-segment model as used for the parametric pet images .", "finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50  % lge per segment [ 15 , 17 ] ."], ["continuous variables are presented as means  sd , and categorical data are summarized as frequencies and percentages .", "multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment .", "receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr .", "the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium . a p value of < 0.05 was considered statistically significant ."], ["lge was seen in the cmr images of 34 patients ( 74  % ) . of the 736 myocardial segments ,", "364 ( 49  % ) showed some degree of lge.table 1patient characteristics ( n  =  46)characteristicvalueage ( years)65    10gender ( male)36 ( 78  % ) previous myocardial infarction34 ( 74  % ) lv end - diastolic volume ( ml)226    65lv end - systolic volume ( ml)135    71lv ejection fraction ( % ) 42    16lv mass ( g)132    39 patient characteristics ( n  =  46 ) pet / ct and cmr data are summarized in table  2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p  <  0.001 by anova ) .", "atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p  <  0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control", "( n  =  372)025 ( n  =  190)2550 ( n  =  83)5075 ( n  =  54)75 ( n  =  37)p value ( anova)atf0.76    0.090.77    0.090.75    0.110.73    0.080.68    0.11**<0.001ptf ( g  ml)0.72    0.080.72    0.090.70    0.090.60    0.08**0.51    0.12***<0.001pti0.91    0.080.89    0.090.89    0.090.77    0.10**0.70 ", "  0.16***<0.001mbf ( ml  g  min)1.02    0.300.91    0.26 * 0.85    0.30 * 0.83    0.24 * 0.67    0.30**<0.001*p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge . segmental pet / ct and lge data * p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge .", "figure  2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig .", "2long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min)fig .", "3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25  % ) segments showing some degree of lge were judged to be nonviable ( lge > 50  % ) . as shown in fig .  4 ,", "the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p  =  0.541 ) .", "mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p  <  0.001 ) .", "optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69  g  ml , 0.80 , and 0.78  ml  min  g with sensitivities of 69  % , 91  % and 72  % , and specificities of 87  % , 73  % and 56  % , respectively .", "figure  3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig .", "4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement"], ["lge was seen in the cmr images of 34 patients ( 74  % ) . of the 736 myocardial segments ,", "364 ( 49  % ) showed some degree of lge.table 1patient characteristics ( n  =  46)characteristicvalueage ( years)65    10gender ( male)36 ( 78  % ) previous myocardial infarction34 ( 74  % ) lv end - diastolic volume ( ml)226    65lv end - systolic volume ( ml)135    71lv ejection fraction ( % ) 42    16lv mass ( g)132    39 patient characteristics ( n  =  46 )"], ["pet / ct and cmr data are summarized in table  2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p  <  0.001 by anova ) .", "atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p  <  0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control", "( n  =  372)025 ( n  =  190)2550 ( n  =  83)5075 ( n  =  54)75 ( n  =  37)p value ( anova)atf0.76    0.090.77    0.090.75    0.110.73    0.080.68    0.11**<0.001ptf ( g  ml)0.72    0.080.72    0.090.70    0.090.60    0.08**0.51    0.12***<0.001pti0.91    0.080.89    0.090.89    0.090.77    0.10**0.70    0.16***<0.001mbf ( ml  g  min)1.02    0.300.91    0.26 * 0.85    0.30 * 0.83    0.24 * 0.67    0.30**<0.001*p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge .", "segmental pet / ct and lge data * p  <  0.05 vs. control ; * * p  <  0.05 vs. control , 025  % lge and 2550  % lge ; * * * p  <  0.05 vs. control , 025  % lge , 2550  % lge and 5075  % lge ."], ["figure  2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig .", "2long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min)fig .", "3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a  d ) and polar maps ( e  h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g  ml ) ; c , g pti ; d , h mbf ( ml  g  min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25  % ) segments showing some degree of lge were judged to be nonviable ( lge > 50  % ) . as shown in fig .  4 ,", "the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p  =  0.541 ) .", "mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p  <  0.001 ) .", "optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69  g  ml , 0.80 , and 0.78  ml  min  g with sensitivities of 69  % , 91  % and 72  % , and specificities of 87  % , 73  % and 56  % , respectively .", "figure  3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig .", "4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement"], ["the present study was conducted to validate the use of a parametric myocardial viability imaging technique using [ o]h2o pet / ct in patients with ischaemic heart disease .", "viability assessed using parametric ptf and pti imaging was in good agreement with that assessed using lge cmr .", "furthermore , these images and myocardial perfusion imaging are obtained simultaneously allowing both myocardial viability and ischaemia to be evaluated in a single scanning session .", "[ o]h2o is generally considered to be the gold standard for absolute quantification of myocardial perfusion in vivo [ 10 , 18 , 19 ] .", "apart from mbf , [ o]h2o also provides estimates of the extent of myocardium within a roi that is able to exchange water rapidly , i.e. ptf .", "subsequently , ptf can be corrected for partial volume effects by dividing it by its anatomical counterpart atf , resulting in pti .", "it has been shown that both ptf and pti can identify myocardial scarring and thus can act as markers of viability . only recently ,", "however , a method has been developed that enables generation parametric atf , ptf and pti images from a single [ o]h2o pet / ct scan .", "in addition , the traditional ( long ) transmission scan can be replaced by a ( rapid ) low - dose ct scan , thereby shortening the total scanning time substantially .", "now that the method has been shown to produce high - quality parametric images , its implementation into clinical practice needs investigation . using the described parametric imaging approach", ", the present study demonstrated that atf was relatively constant independent of tissue characteristics of the myocardium . only in ( near )", "transmurally infarcted segments was atf significantly reduced , most likely due to wall thinning , as atf is prone to partial volume effects . in contrast , ptf and pti progressively decreased with increasing extent of scarring , as shown by lge cmr .", "these results are in line with those of previous studies in which reductions in both ptf and pti were observed in nonviable scarred myocardium .", "pti in control segments appeared to be somewhat lower than the expected value of unity for normal segments ( 0.91    0.08 ) .", "this phenomenon has previously been observed in cardiomyopathy in both animal experiments and human studies [ 6 , 20 ] .", "slight misalignment between pet and cmr segments may have occurred to additionally account for this reduction in pti .", "furthermore , the presence of interstitial fibrosis in dilated cardiomyopathy may also explain this reduction in ", "normal segments that remains undetected on lge cmr [ 21 , 22 ] . taking lge as a reference , the optimal cut - off values for discriminating between viable and nonviable myocardium", "although this would reduce the data processing time , it would not affect the scanning protocol , as for both parameters a dynamic [ o]h2o pet scan in combination with a low - dose ct scan are required .", "in addition , the sensitivity of pti exceeded that of ptf ( 91  % and 68  % , respectively ) , whereas for specificity the opposite pattern was observed ( 73  % and 78  % , respectively ) .", "although further study is need to determine the cause of this discrepancy , as a potential clinical marker of viability pti may be favoured over ptf to reduce false - negative findings in patients who might benefit from revascularization .", "it is of interest to note that , compared to ptf and pti , mbf performed relatively poorly in distinguishing viable from nonviable tissue , rendering it less suitable for viability imaging .", "previous studies have indicated that the optimal cut - off value for pti is in the range 0.70.9 .", "lge was used as a surrogate end - point of myocardial viability instead of functional recovery of dysfunctional myocardium after revascularization .", "lge has been shown to be a good , but not perfect , marker of myocardial viability [ 15 , 17 ] .", "therefore , the results should be interpreted with caution , and more studies are warranted to establish the value of parametric pti as a viability marker .", "ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease ."], ["ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease ."], ["this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited ."]]}
{"article_id": "PMC3352477", "article_text": ["while advances in free trade and globalization increase the movement and accelerate the accumulation of invasive species ( lockwood et al .", "2005 ) , it is still unclear how introduced populations can successfully establish . as elton ( 1958 ) pointed out , for every successful invasion that occurs ,  there are enormously more invasions that never happen , or fail quite soon or even after a good many years ( page 109 ) .", "this modern biological paradox can not readily be reconciled , especially in the characteristic case where the founder population is small , as  such populations are definitely in a precarious position ( mayr 1965 ; page 42 ) .", "introductions of populations at low density and/or small size are often faced with inverse density dependent effects , attributed to demographic stochasticity or reduced cooperative interactions ( courchamp et al .", "allee ( 1931 ) first proposed that under these conditions , populations may suffer a decrease in the per - capita rate of increase , from here on referred to as the allee effect . upon arrival in a novel environment , individuals need to overcome a series of challenges in order to reduce the population 's risk of extinction .", "the time period in which this occurs is generally considered the initial establishment phase , and is thought to be a common feature and general pattern of invasion and the process of growth and expansion ( shigesada and kawasaki 1997 ; sakai et al .", "the occurrence of a lag phase that precedes a noticeable increase in population growth and density can result from ecological and/or evolutionary phenomena ( sakai et al .", "small populations that undergo logistic growth slowly increase through the initial phase of the exponential curve , leading to the perception of a time lag .", "where this time lag is more pronounced , populations may be recovering from inverse density dependent effects ( i.e. , allee effects ) .", "individuals may suffer a reduction in fitness at low densities for many reasons ( reviewed by courchamp et al .", "even when the initial population is large , rapid dispersal required for expansion could be suicidal as the population density decreases , thereby enhancing inverse density dependent effects ( lewis and kareiva 1993 ; drake et al .", "component allee effect where the population size and density affects the mean overall fitness or some component of individual fitness , respectively ( stephens et al . 1999 ; gascoigne et al .", "it is often difficult to decipher the exact mechanism that manifests allee effects ( and it is not always the case that component allee effects lead to demographic allee effects ) .", "nevertheless , we focus our attention on population level demographic allee effects with the underlying assumption that a component allee effect led to the demographic allee effect . essentially , it is the case that introduced individuals may be maladapted to small population sizes where their survival and reproductive ability are significantly impacted , and these impacts on individual fitness combine to produce an overall decrease in abundance ( i.e. , demographic allee effects ) . as allee effects impact individual fitness , the underlying traits that influence these effects (", "i.e. , component allee effects ) may be subject to natural selection ( courchamp et al . 2008 ) . whereas propagule pressure is an emerging explanation for the establishment of invasive species ( lockwood et al . 2005 ) , it relies on an obvious relationship between the number and size of introduction events and the probability of success , as safety in numbers helps combat allee effects and stochastic extinction . in the event that propagule size is not large enough to overcome inverse density dependent effects , a population", "traits that may be responsible for reproductive success and survival at small population densities and sizes include mate finding cues ( pheromones and vocal / visual signals ) , dispersal / aggregation behavior , habitat preferences , mating synchronicity , and gamete morphology and performance ( see courchamp et al .", "direct evidence for the evolution of these traits as functional adaptations to allee effects is limited , but we can infer an adaptive evolutionary origin of these traits from studies of sexual selection ( courchamp et al . 2008 ; gascoigne et al .", "an evolutionary response to intensive selection pressure imposed by density dependent survival and reproduction relies on genetic variants for adaptive evolution .", "according to neutral quantitative genetic theory , a loss of genetic variation is expected from population bottlenecks and founder effects ( nei et al .", "however it is not neutral variation that matters , but rather evolvability depends on the variation relevant to selection .", "maintenance or even increases of this ( additive ) genetic variation has been theoretically and empirically observed following a bottleneck or in small founder populations ( bryant et al .", "1986 ; goodnight 1988 ; willis and orr 1993 ; cheverud and routman 1996 ; briggs and goldman 2006 ; turelli and barton 2006 ) .", "additionally , many recent studies suggest that there is actually no significant reduction in genetic diversity in most successful invaders ( lee 2002 ; allendorf and lundquist 2003 ; roman and darling 2007 and references therein ) , and that evolution can occur on contemporary timescales ( reznick and ghalambor 2001 ; carroll et al . 2007 ; kinnison and hairston 2007 ) .", "our purpose here is to explore the feasibility of small populations that may adaptively respond to overcome allee effects in order to establish , given any amount of genetic variation . in this paper", ", we present evidence for the enhanced potential for growth and spread of a small introduced population of organisms faced with allee effects when adaptation occurs .", "we model the growth and spread of this population according to a reaction - diffusion equation , and allow evolution to influence inverse density dependent effects through a genetic subsystem that provides the opportunity for successful invasion when otherwise ( under the current , ecological paradigm ) the population would go extinct .", "the deterministic model that we explore in this paper broadly describes population dynamics with density - mediated growth ( i.e. , an allee effect ) and diffusive dispersal .", "this type of demographic model has been used as a compact and tractable representation of invasion ( e.g. , skellam 1951 ; lewis and kareiva 1993 ) .", "specifically , it has been applied to systems such as introductions of nonindigenous freshwater and marine species through ballast water discharge ( drake et al .", "2005 ; drury et al . 2007 ) . using this approach , drake et al .", "( 2005 ) report acceptable volumes of discharge for various organisms ( with differing reproductive rates ) for a range of invasion risk tolerances . here", ", we consider populations that are introduced below the invasion risk threshold , but nonetheless succeed if evolutionary dynamics are considered in conjunction with the ecological system .", "the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space .", "this equation models the growth of the population ( the first term on the right hand side of eqn .", "1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn .", "1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig .", "growth dynamics of the model population ( a ) , and the solution of eqn .", "( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 .", "trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 .", "there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn .", "the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al .", "1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) .", "figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a )", "there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity .", "1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth ,", "there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) .", "the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect )", ", there exists a unique wave speed of the invasion front that is a result of being ", "pushed from the inside out , as opposed to being  pulled by the leading edge ( lewis and kareiva 1993 )", ". this velocity can be derived through the solution of the pde ( 1 ) :   ( lewis and kareiva 1993 ; murray 1993 ) .", "this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity .", "in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) .", "this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch .", "this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth .", "this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth .", "thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand .", "we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model .", "the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) .", "the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success .", "the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) . in order to incorporate evolutionary factors that may influence invasion success ,", "this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival .", "specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time .", "this quantitative trait influences an organism 's ability to survive and reproduce in a small population .", "the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies .", "therefore , it is important to investigate the possibility of evolution in the analysis of invasions .", "the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn .", "( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance .", "as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) .", "thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) .", "this suggests that the genetic variance (  ) is small ( and constant in this model ) .", "this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale ,  = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection ,", "quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn .", "the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities .", "the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal .", "we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time", ", the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient .", "the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system .", "the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space .", "this equation models the growth of the population ( the first term on the right hand side of eqn .", "1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn .", "1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig .", "growth dynamics of the model population ( a ) , and the solution of eqn .", "( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 .", "trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 .", "there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn .", "the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al .", "1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) .", "figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a )", "there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity .", "1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth ,", "there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) .", "the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth", "versus reduced positive growth from a weak allee effect ) , there exists a unique wave speed of the invasion front that is a result of being ", "pushed from the inside out , as opposed to being  pulled by the leading edge ( lewis and kareiva 1993 )", ". this velocity can be derived through the solution of the pde ( 1 ) :   ( lewis and kareiva 1993 ; murray 1993 ) .", "this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity .", "in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) .", "this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch .", "this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth .", "this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth .", "thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand .", "we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model .", "the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) .", "the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success .", "the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) .", "in order to incorporate evolutionary factors that may influence invasion success , we develop a quantitative genetic subsystem .", "this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival .", "specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time .", "this quantitative trait influences an organism 's ability to survive and reproduce in a small population .", "the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies .", "therefore , it is important to investigate the possibility of evolution in the analysis of invasions .", "the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn .", "( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) .", "thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) .", "this suggests that the genetic variance (  ) is small ( and constant in this model ) .", "this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale ,  = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection ,", "quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn .", "the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities .", "the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal .", "we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time", ", the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient .", "the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system .", "the dynamics of the evolutionary ecology model can be interpreted using the idea of fast and slow timescales ( kondrashov and khibnik 1996 ; webb 2003 ) .", "earlier , we assumed that the genetic variation (  ) was small ( to use mean fitness as a proxy for individual fitness ) , which can subsequently be taken advantage of for our analysis of the coupled evolutionary ecological dynamics . when  = 0 , the situation without evolution , the genetic subsystem is frozen and the population moves towards a stable equilibrium of the ecological subsystem ( carrying capacity or extinction ) depending on its initial density ( greater than or less than the allee threshold respectively ; fig .", "when  > 0 but small , the allee threshold evolves relatively slowly and influences the ecological system . whenever the population is below its carrying capacity ( u = 1 for each spatial coordinate x when space is explicit ) , eqn .", "( 2 ) is negative , and decreases the mean allee threshold ( a ) , as the intensity of selection is density dependent .", "thus , fitness increases as allee effects are suppressed , and selection drives the allee threshold towards zero .", "if the population density is greater than the allee threshold , but still below the carrying capacity , it will progress towards carrying capacity more rapidly than it would without evolution as a decreases ; as the rate at which the population density changes ( eqn .", "1 ) is proportional to the difference between u and a. the ecological dynamics are reversed when the population density is below the allee threshold as the population declines towards extinction , but more slowly than it does without evolution . when u < a , eqn .", "( 1 ) is negative , and the population density approaches extinction more rapidly with a constant ( as the difference between u and a increases ) , than it does with evolution as a decreases ( revealing a more pronounced time lag to extinction ) . during this time lag ,", "as the population slowly declines , the opportunity for evolution to overcome inverse density dependent effects occurs .", "if the rate of evolution is fast enough , the allee threshold can fall below the population density , causing the rate of change of population density to become positive ( where u > a ) and the population grows and can successfully invade .", "the chance that evolution can rescue the population from extinction depends on the relative rates of genetic change in the quantitative trait ( i.e. , allee threshold ) and of population decline ( gomulkiewicz and holt 1995 ) ; hence the amount of genetic variance greatly impacts the ability to adapt and survive .", "a nonspatial example of this process , referred to as evolutionary rescue ( gomulkiewicz and holt 1995 ) , is shown in fig .", "2 , where a population is introduced below the allee threshold . without evolution , the population declines to extinction ( fig . 2a , solid line ) as the allee threshold remains constant ( fig . 2b , solid line ) . when the population can evolve ( fig .", "2 , dotted line ) , it declines at first until it can overcome the magnitude of inverse density dependence , and is then able to successfully establish . as it is difficult to measure the allee effect empirically ( tobin et al .", "2007 ) , we use an extreme value that exaggerates density dependent effects in order to investigate the  worst case scenario ( a = 0.3 , where the population exhibits deterministic decline when its density is less than 30% of its carrying capacity ) . when evolution is included , we used a small value for the genetic variance ,  = 0.02 , in order to remain consistent with fast - slow dynamics , unless otherwise indicated .", "comparison of an invading population introduced at a density below the allee threshold , a = 0.3 ( u = 0.25 , r = 0.6 ) .", "the solid line represents the nonspatial system ( d = 0 ) described by eqns ( 1 ) and ( 2 ) without evolution (  = 0 ) which results in extinction ( a ) and a constant allee threshold ( b ) .", "the dotted line indicates population growth ( a ) when evolution (  = 0.02 ) acts to reduce the allee threshold ( b ) . in general , there is a range of parameter space that permits persistence for a population below the allee threshold in the nonspatial model with evolution ( instead of simple decline to extinction ) .", "as genetic variance increases , we are essentially relaxing the assumption of fast - slow timescales and allow evolution to occur more rapidly .", "3 where initial population densities below the allee threshold require a minimum amount of genetic variance in order to avoid extinction . in this case", ", the rate of reproduction , r , also influences the potential for evolutionary rescue , as it impacts both population growth and rate of evolution ( eqns 1 and 2 , respectively ) .", "as we relax the assumption of fast - slow timescales , the behavior remains qualitatively the same as that described analytically under a strict fast - slow timescale assumption .", "parameter combinations of reproductive rate : r ; genetic variance :  , and initial population density : u , that result in extinction or evolutionary rescue . in this nonspatial scenario ,", "initial population densities greater than the allee threshold ( a = 0.3 ) always succeed , thus the focus is on the parameter space that allows for evolutionary rescue ( i.e. , where the population growth changes from negative to positive ) . as the reproductive rate increases from 0.1 to 1", ", there is less genetic variance needed for a population to evolve to overcome inverse density dependence as increased reproduction will contribute to suppressing allee effects .", "nonetheless , the additional complexities result in qualitatively similar behavior to the nonspatial model . in this case , not only will evolution influence population growth , it affects the wave speed and the critical size threshold , rmin .", "as the population overcomes allee effects with a decreasing allee threshold , the wave speed accelerates and the critical patch size becomes smaller .", "thus , in addition to the initial density of the introduced population and the genetic variance , the initial radius or patch size of the initial invasion area , the ratio of diffusion to reproduction , and gene flow will factor into successful establishment and give rise to a wider range of interactions between the ecology and evolution of this system .", "( 1 ) ( without the evolutionary subsystem ) in one - dimensional space , with an initial population density below the allee threshold , declines to extinction ( fig .", "this is contrasted by the results when the evolutionary subsystem is included . with the initial population density below the allee threshold , fig .", "the same type of rescue occurs for a population that starts near carrying capacity , but occupies an initial spatial size below that which is necessary for a population to successfully establish .", "figure 5a shows a rapidly declining population that goes extinct . under the same circumstance , but where evolution of the allee threshold occurs , fig .", "5b shows the population density at first beginning to shrink and then growing and expanding .", "in addition to the time evolution of population density across space in figs 4 and 5 , the evolution of the mean trait value across space illustrates how gene flow and the density dependent selection gradient influences its rate of change and distribution ( figs 4c and 5c ) . as the intensity of selection is density dependent ( and we assume constant genetic variance ) , locations with smaller populations can evolve the trait value more rapidly compared to other areas where allee effects may not be as strong and experience weaker selection .", "the trait distribution over time , figs 4c and 5c , therefore reflect the population density distribution , but are also influenced by the trait values of the migrants . as individuals disperse out to new locations and push the boundaries of the species range , their trait values are averaged to determine the demographic allee threshold for that spatial coordinate . this demographic allee threshold combines with their local population density to influence individual fitness and population growth ( where the distance between the density and mean trait value is the initial degree of maladaptation ) .", "diffusive dispersal of an introduced population at an initial density ( bold dashed line ) below the initial allee threshold , a = 0.3 ( u = 0.25 , r = 1 , d = 0.1 ) across a linear , one dimensional habitat .", "the population collapses over time to extinction ( a ) where there is no evolution (  = 0 ) , and succeeds ( b ) after an initial decline with evolution (  = 0.02 ) .", "( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) . the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) .", "population density of a diffusion dispersed population across one dimensional space . the initial population density ( bold dashed line )", "is near carrying capacity ( u = 0.95 , a = 0.3 , r = 1 , d = 0.5 ) , but introduced below the minimum radius of area determined to be critical for invasion success .", "( a ) is collapsing to extinction without evolution (  = 0 ) , whereas ( b ) shows success of an invader with evolution (  = 0.02 ) after initial decline .", "( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) .", "the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) .", "we explored when evolutionary rescue occurred across a range of parameter values for the spatially explicit model . according to drake et al .", "( 2005 ) , variability among locations and over time makes it unreasonable to determine precise estimates for the diffusion coefficient , d. we therefore explored a range of values , and present those that best illustrate breadth of behavior .", "the parameter that controls the reproductive rate , r , was also varied substantially , but as the spatial dynamics depend on the ratio of diffusion to rate of reproduction ( resulting in a measure of length ) ; we fixed r and varied d , unless otherwise noted .", "this was justified as the results of the spatial simulations are qualitatively identical for equivalent ratios .", "the effects of the critical patch size , initial population density , ratio of diffusion to growth and genetic variation on evolutionary rescue and population dynamics are shown in fig .", "the ratio of the diffusion coefficient ( d ) to the reproductive rate ( r ) determines whether the population will expand or collapse according to the initial radius of the introduced population .", "the areas under the curves denote combinations of genetic variance and initial population density that result in extinction .", "areas above the curves are combinations of genetic and/or demographic conditions that produce inevitable persistence .", "the parameter space between the vertical dashed lines refers to the different ways population survival is influenced . to the left of the initial allee threshold", ", the initial population density will either go extinct due to density dependent effects ( below the d / r curve ) , or given enough genetic variation , will be evolutionarily rescued ( above the d / r curve ) .", "the area to the right of the initial allee threshold [ and between the dashed lines in ( b ) and ( c ) ] is the case where the initial population density is greater than the allee threshold but due to the initial spatial size and the ratio of diffusion to reproduction , the population may go extinct without sufficient genetic variance ( below the d / r curve ) , otherwise it will evolve to overcome the critical patch size effect . for initial population densities", "thus , the area between the dashed lines in ( b ) and ( c ) truly delineates evolutionary rescue when d / r = 1 .", "the rightmost vertical line moves slightly to the left to the point of intersection of the d / r curve and the x - axis for other values of d / r .", "graphs ( a ) , ( b ) , and ( c ) represent different radii of the linear habitat that the introduced population initially occupies .", "when the size of the initial population is too small ( i.e. , a radius of 1 ) , a population at carrying capacity ( i.e. , u = 1 ) will go extinct without evolution due to the relative effect of diffusion to reproduction ( fig .", "if evolution occurs rapidly enough ( i.e. ,  > 0.02 ) , the population can overcome inverse density dependent effects and compensate for the loss due to diffusion and rebound from low densities . when the initial radius of the population is increased ( fig .", "6b , c ) , the chance of survival and establishment ( growth and expansion ) of populations above or below the allee threshold increases with initial density and genetic variance .", "therefore , the initial radius of the population can significantly impact the likelihood of evolutionary rescue for populations with the same amount of genetic variance .", "this is demonstrated further in fig . 7a , where the rate of recovery ( i.e. , the inverse of the time lag before growth becomes positive and the population reaches carrying capacity ) for a population near carrying capacity depends on its initial size / radius and genetic variance . where size and variance are small", ", rescue never occurs . as these parameters increase , the rate of recovery gradually becomes faster until it essentially plateaus ( although with greater variance and initial radius , the rate of recovery may slow slightly if the initial spatial extent is large enough for the population to experience early growth before diffusion causes decline prior to recovery ) .", "if the population occupies a large enough spatial extent , it will succeed without evolution ( where the genetic variance is zero ) , however the lag time may be more pronounced depending on the ratio of diffusion to reproduction through the tradeoff between growth and spread ( e.g. , if spread is relatively fast compared to reproduction , d / r = 1 ) . the population density may thus initially decline across space until reproduction can sufficiently overcome the loss due to diffusion , and the population can grow to carrying capacity .", "similar to the nonspatial case , a population ( greater than the allee threshold ) above the spatial threshold will grow to carrying capacity more rapidly with evolution than without .", "rate of recovery in terms of the inverse of the time lag before population growth becomes positive , where one  timestep equals 24 iterations of the model . in ( a )", ", the initial population density is near carrying capacity ( u = 0.95 , a = 0.3 , d / r = 1 ) , and the initial radius and genetic variance ,  , varies .", "where the rate of recovery is zero , the population goes extinct as it initially occupies an area smaller than the critical patch size ( in this case , a radius of 1.4 ) or does not have sufficient genetic variance to evolve quickly enough to be rescued prior to extinction . increasing the genetic variance and initial radius will decrease this time lag until the population no longer experiences any negative growth ( in this case , for initial radii  3.8 and   0.036 ; for initial radii > 2.7 , the rate of recovery slows slightly due to early growth followed by a transient decline that precedes ultimate recovery ) .", "when the initial population density varies ( indicating the initial degree of maladaptation where a = 0.3 ) , ( b ) shows the rate of recovery with the initial radius fixed ( as in fig .", "5a where the radius = 1 and d / r = 1 ) . in this case", ", extinction will occur without evolution not only for an initial density below the initial allee threshold , but for any density as the initial radius is below the critical patch size .", "hence , a nonzero rate implies evolutionary rescue and a zero rate means extinction . as shown in fig .", "7b , when evolutionary rescue is possible , the initial level of maladaptation ( a0u0 ) and the genetic variance (  ) also determine the rate at which evolutionary rescue proceeds .", "figure 7b uses parameters ( i.e. , radius and ratio of d to r ) for a population that would decline and go extinct without evolution regardless of the initial density .", "hence , it is clear that the amount of time required for a population to begin growing depends on its initial level of maladaptation ( to both the critical density and spatial thresholds ) and/or genetic variance . as the rate at which this rescue occurs depends on the amount of genetic variance ( eqn .", "2 ) , it may take an extremely long time ( as  0 , the rate of recovery  0 ) for the allee threshold to fall below the population density . in this circumstance , as the population density becomes very close to zero , the rate of change of the allee threshold is greater than that of the population density ( as u  0 , u/t 0 and a/t  -2ra )", ". thus , theoretically , rescue would always occur ( gomulkiewicz and holt 1995 ) . however , to maintain biological realism , when solving this system numerically , we always considered the population extinct when the maximum density ( across space , when diffusion is included ) becomes reasonably close to zero ( i.e. , u = 0.0001 ; we chose this protocol instead of the total population across space due to the diffusion dynamics based on the gaussian dispersal kernel and the  pushed wave front behavior ) .", "overall , the numerical results qualitatively hold for a wide range of dimensional parameter values and initial conditions with and without diffusion and in one- and two - dimensional space .", "results for two - dimensional space are not shown as they are qualitatively similar to the simpler , one dimensional model .", "from these results , it is apparent that adaptations that enable organisms to overcome the negative effects of low densities can allow the population to rebound from a trajectory toward extinction to grow to reach carrying capacity .", "current management strategies ( e.g. , reducing population density or size ) are based on ecological theory ( e.g. drake et al . 2005 ) , but this evolutionary ecology model suggests that adaptive evolution can enable successful establishment and that ecological considerations alone may not be sufficient . under the assumptions of an allee effect and diffusive dispersal , the idea of ecological size thresholds fits well with the ecological evidence that a large founding population is a primary cause of successful establishment ( lockwood et al .", "however , by incorporating evolution , we see that the situation is not quite this simple because ecological size thresholds and genetic variance can interact to determine successful establishment . as", "the ratio of diffusion to reproduction decreases , the spatial constraint on population growth becomes weaker , and less genetic variance is needed to rescue populations with densities below the allee threshold . as the initial spatial radius of introduction increases ,", "furthermore , the rate of this rescue depends on the initial genetic load or maladaptation ( i.e. , how far the population density is from the allee threshold ) , as well as the amount of genetic variance .", "because bottlenecks during founding events do not always result in highly reduced genetic variability , even small founding populations may have sufficient genetic variation to evolve to overcome allee effects and establish , contrary to solely ecologically based predictions .", "additionally , we can draw several general insights about how dispersal impacts selection and evolution of allee effects in an invasion context .", "as species are transported from their native environment into novel habitats or simply disperse on their own , it is clear that the genetic composition of the local population can influence the rate of evolution and adaptation to the new local conditions . given enough genetic diversity , local populations can adapt to their local environment , but dispersal may hinder survival across ecological clines as dispersers tend to be maladapted to the new local environment . essentially , local population persistence depends on the race between the rate of evolution and the degree of maladaptation ( gomulkiewicz and holt 1995 ) . in this case", ", gene flow will play a major role in determining the outcome . as kirkpatrick and barton ( 1997 ) and garca - ramos and kirkpatrick ( 1997 )", "demonstrate , individuals moving from one selection regime from the center of their species range to the periphery can introduce enough maladaptation that the new area becomes a sink environment . on the other hand , holt et al .", "( 2003 , 2004 ) show that immigration can have a positive influence on adaptation to sink environments , in some circumstances .", "resolving the disparity between these perspectives requires understanding what is contributing to the severity of maladaptation and the population 's ability to overcome it . in our model ,", "dispersal impacts survival ecologically through the critical patch size , and genetically , as individuals may move from areas where they are well adapted ( i.e. , the population density is greater than the allee threshold or mean trait value ) to sink regions , where they are maladapted .", "as individuals disperse across space , they may be contributing positively in an ecological sense to the quality of their new local environment ( by increasing the local population density ) .", "however , dispersers are more likely to come from higher density areas where allee effects , and hence selection , are locally weak .", "these dispersers potentially introduce more maladaptation to their new location , because they increase the average phenotype ( allee threshold ) in the new location where density is likely to be lower .", "interestingly , the evolutionary impacts of migration in this model do not dramatically influence the dynamics .", "changes to the local mean phenotype through local selection and simple mixing ( i.e. , diffusion ) actually slightly hastens the evolutionary rescue effect over a model that considers only the impact of local selection .", "as the selection intensity is density dependent and proportional to u 1 for each point in space , the peripheral individuals faced with stronger selection with lower trait values have a small positive influence on the more dense neighboring populations .", "the gradient term accounts for asymmetric gene flow due to differential migration from areas of relatively high population densities .", "however , this term does not alter the overall evolutionary dynamics based on local selection any more than adding the diffusion term , as the negative effects of gene flow and the local rate of evolution ( which is relatively fast , based on the selection intensity ) essentially cancel each other out . in this context ,", "similar to that of holt et al . ( 2003 , 2004 ) , the immigrants simply contribute to the local population density , which helps prevent extinction long enough for evolutionary rescue to occur locally ( i.e. , positive population growth ; note that whereas holt et al .", "( 2003 ) attribute the main effect of immigration to the contribution of variation , this is not the case in our model , as we assume constant genetic variance ) .", "( 2004 ) where immigration has a demographic effect on increasing fitness that can essentially outweigh the ", "the primary determinant of invasion success depends on positive population growth at the center of the introduced range .", "this result comes from the allee effect [ and the solution to the pde ( 1 ) ] by forcing a  pushed travelling wave front ( lewis and kareiva 1993 ) , where the wave speed causes population expansion , contraction , or propagation failure ( i.e. , pinning ; keitt et al", ". 2001 ) . intuitively , aggregation - like behavior emerges based on the strength of the allee effects .", "individuals that disperse too far from the whole are likely to die before they can  pull others in their vicinity . in this", "regard , growth occurs from the inside out , where the population seemingly spills out and overflows to expand its range .", "consequently , in this study , and for biological invasions that exhibit similar dynamics , it is more important to focus on the center of the invader 's range and whether the initial beachhead can survive ( through evolutionary rescue ) , than the fate of peripheral populations at the wave front when determining the importance of evolution on invasion success .", "the overall dynamics are qualitatively similar in the parameter space that allows for evolutionary rescue to occur .", "even though gene flow and spatial structure do not dramatically influence the establishment of an introduced population , additional invasion criteria need to be considered .", "when analyzing the model behavior in a spatially explicit context , there is an ecological tradeoff between growth and spread that affects establishment and the rate of recovery . as previously mentioned , reproduction needs to compensate for the loss due to diffusion . including evolution and suppressing allee effects", ", actually contributes to the acceleration of the wave front ( i.e. , enhancing dispersal speed ) . a population then can more rapidly disperse as it evolves , and may become more of an invasion threat as long as this range expansion does not reduce their density too quickly . whereas this increasing wave speed can lead to a slightly longer lag phase prior to positive population growth , the population will likely be inevitably rescued because this effect primarily influences the dynamics at the periphery and is offset by the reduction in the critical invasion area ( rmin ) .", "although there is no range contraction ( as there is always a positive wave velocity with unbounded expansion due to the parameter values and absence of environmental heterogeneity or range limitations ; filin et al .", "2008 ) , as the critical patch size ( rmin ) becomes smaller with the decreasing allee threshold , rescue occurs more readily at the range center as the critical patch size threshold criteria weakens and the behavior approaches that of the nonspatial model .", "this may seem like an oversimplification of the global dynamics ; however these conclusions are valid in the context of this investigation which focuses on the establishment phase rather than subsequent range expansion and spread . recognizing that evolution can significantly affect the establishment success of invasive species is becoming more widely accepted , influencing the ways in which invasion biologists conduct their research ( see the other articles in this issue ) .", "specifically , adaptations that diminish allee effects and evolutionary responses to density dependence are beginning to emerge as viable explanations for sustaining vulnerable populations at low density and size ( gascoigne et al .", "as it is difficult to conclusively support this claim empirically ( as the origin of the adaptation or the associated cost may be unknown ; courchamp et al . 2008 ; gascoigne et al .", "2009 ) , mathematical models that incorporate evolution and compare the effects of various strategies ( e.g. , mitigating component allee effects ) can help decipher the mechanisms that both limit and facilitate population growth .", "two such models that incorporate adaptations to component mate - finding allee effects compare the efficiency and survival of populations at various densities that attract mates with or without a sexual pheromone ( jonsson et al .", "another study suggests that broadcast spawners that evolve their gamete morphology and performance under sperm limitation ( at low density ) bear a cost of decreased fitness at high density due to hybridization and competition ( levitan 2002 ) .", "in these cases , particular strategies are shown to influence population viability in addition to an associated tradeoff , whereas our investigation provides broad , albeit simplistic , results dealing with generalized demographic allee effects and evolution . in order to understand how the results of this simplistic model extend to more realistic and complex evolutionary scenarios , spatially explicit", ", individually - based stochastic simulation of the introduced populations should be developed to investigate more closely the mechanisms that allow these population level dynamics to emerge .", "in particular , tracking the mean value of a component allee effect is sufficient to illustrate how evolution can overcome inverse density dependence and result in invasion .", "however , this approach may not be sufficient to make the specific quantitative predictions necessary for management of invasive species .", "this is due to the simplifying assumption of constant genetic variance based on mutation - selection balance ( lande 1976 ) .", "complex simulations could relax this assumption and permit genetic variation to change via mutation , selection , and drift , in tandem with the demographic processes in a heterogeneous environment , and explicitly investigate the costs associated with avoiding allee effects .", "hence , future models should incorporate how propagule pressure ( size and frequency of introduction events ) impacts genetic variation and how more realistic genetic architectures contribute to the evolutionary trajectory of invasive species .", "although there is still much more work to be done to elucidate the factors that determine establishment success of founder populations , this theoretical approach has the promise to provide evidence in support of our working hypothesis that adaptive evolution can mitigate allee effects and be an important driver of biological invasions ."], "abstract_text": ["<S> the mechanisms that facilitate success of an invasive species include both ecological and evolutionary processes . investigating the evolutionary dynamics of founder populations can enhance our understanding of patterns of invasiveness and provide insight into management strategies for controlling further establishment of introduced populations . </S>", "<S> our aim is to analyze the evolutionary consequences of ecological processes ( i.e. , propagule pressure and threshold density effects ) that impact successful colonization . </S>", "<S> we address our questions using a spatially - explicit modeling approach that incorporates dispersal , density dependent population growth , and selection . </S>", "<S> our results show that adaptive evolution may occur in small or sparse populations , providing a means of mitigating or avoiding inverse density dependent effects ( i.e. , allee effects ) . </S>", "<S> the rate at which this adaptation occurs is proportional to the amount of genetic variance and is a crucial component in assessing whether natural selection can rescue a population from extinction . </S>", "<S> we provide theoretical evidence for the importance of recognizing evolution in predicting and explaining successful biological invasions . </S>"], "labels": null, "section_names": ["Introduction", "Model description", "The ecological system", "The evolutionary subsystem", "Results", "Discussion"], "sections": [["while advances in free trade and globalization increase the movement and accelerate the accumulation of invasive species ( lockwood et al .", "2005 ) , it is still unclear how introduced populations can successfully establish . as elton ( 1958 ) pointed out , for every successful invasion that occurs ,  there are enormously more invasions that never happen , or fail quite soon or even after a good many years ( page 109 ) .", "this modern biological paradox can not readily be reconciled , especially in the characteristic case where the founder population is small , as  such populations are definitely in a precarious position ( mayr 1965 ; page 42 ) .", "introductions of populations at low density and/or small size are often faced with inverse density dependent effects , attributed to demographic stochasticity or reduced cooperative interactions ( courchamp et al .", "allee ( 1931 ) first proposed that under these conditions , populations may suffer a decrease in the per - capita rate of increase , from here on referred to as the allee effect . upon arrival in a novel environment , individuals need to overcome a series of challenges in order to reduce the population 's risk of extinction .", "the time period in which this occurs is generally considered the initial establishment phase , and is thought to be a common feature and general pattern of invasion and the process of growth and expansion ( shigesada and kawasaki 1997 ; sakai et al .", "the occurrence of a lag phase that precedes a noticeable increase in population growth and density can result from ecological and/or evolutionary phenomena ( sakai et al .", "small populations that undergo logistic growth slowly increase through the initial phase of the exponential curve , leading to the perception of a time lag .", "where this time lag is more pronounced , populations may be recovering from inverse density dependent effects ( i.e. , allee effects ) .", "individuals may suffer a reduction in fitness at low densities for many reasons ( reviewed by courchamp et al .", "even when the initial population is large , rapid dispersal required for expansion could be suicidal as the population density decreases , thereby enhancing inverse density dependent effects ( lewis and kareiva 1993 ; drake et al .", "component allee effect where the population size and density affects the mean overall fitness or some component of individual fitness , respectively ( stephens et al . 1999 ; gascoigne et al .", "it is often difficult to decipher the exact mechanism that manifests allee effects ( and it is not always the case that component allee effects lead to demographic allee effects ) .", "nevertheless , we focus our attention on population level demographic allee effects with the underlying assumption that a component allee effect led to the demographic allee effect . essentially , it is the case that introduced individuals may be maladapted to small population sizes where their survival and reproductive ability are significantly impacted , and these impacts on individual fitness combine to produce an overall decrease in abundance ( i.e. , demographic allee effects ) . as allee effects impact individual fitness , the underlying traits that influence these effects (", "i.e. , component allee effects ) may be subject to natural selection ( courchamp et al . 2008 ) . whereas propagule pressure is an emerging explanation for the establishment of invasive species ( lockwood et al . 2005 ) , it relies on an obvious relationship between the number and size of introduction events and the probability of success , as safety in numbers helps combat allee effects and stochastic extinction . in the event that propagule size is not large enough to overcome inverse density dependent effects , a population", "traits that may be responsible for reproductive success and survival at small population densities and sizes include mate finding cues ( pheromones and vocal / visual signals ) , dispersal / aggregation behavior , habitat preferences , mating synchronicity , and gamete morphology and performance ( see courchamp et al .", "direct evidence for the evolution of these traits as functional adaptations to allee effects is limited , but we can infer an adaptive evolutionary origin of these traits from studies of sexual selection ( courchamp et al . 2008 ; gascoigne et al .", "an evolutionary response to intensive selection pressure imposed by density dependent survival and reproduction relies on genetic variants for adaptive evolution .", "according to neutral quantitative genetic theory , a loss of genetic variation is expected from population bottlenecks and founder effects ( nei et al .", "however it is not neutral variation that matters , but rather evolvability depends on the variation relevant to selection .", "maintenance or even increases of this ( additive ) genetic variation has been theoretically and empirically observed following a bottleneck or in small founder populations ( bryant et al .", "1986 ; goodnight 1988 ; willis and orr 1993 ; cheverud and routman 1996 ; briggs and goldman 2006 ; turelli and barton 2006 ) .", "additionally , many recent studies suggest that there is actually no significant reduction in genetic diversity in most successful invaders ( lee 2002 ; allendorf and lundquist 2003 ; roman and darling 2007 and references therein ) , and that evolution can occur on contemporary timescales ( reznick and ghalambor 2001 ; carroll et al . 2007 ; kinnison and hairston 2007 ) .", "our purpose here is to explore the feasibility of small populations that may adaptively respond to overcome allee effects in order to establish , given any amount of genetic variation . in this paper", ", we present evidence for the enhanced potential for growth and spread of a small introduced population of organisms faced with allee effects when adaptation occurs .", "we model the growth and spread of this population according to a reaction - diffusion equation , and allow evolution to influence inverse density dependent effects through a genetic subsystem that provides the opportunity for successful invasion when otherwise ( under the current , ecological paradigm ) the population would go extinct ."], ["the deterministic model that we explore in this paper broadly describes population dynamics with density - mediated growth ( i.e. , an allee effect ) and diffusive dispersal .", "this type of demographic model has been used as a compact and tractable representation of invasion ( e.g. , skellam 1951 ; lewis and kareiva 1993 ) .", "specifically , it has been applied to systems such as introductions of nonindigenous freshwater and marine species through ballast water discharge ( drake et al .", "2005 ; drury et al . 2007 ) . using this approach , drake et al .", "( 2005 ) report acceptable volumes of discharge for various organisms ( with differing reproductive rates ) for a range of invasion risk tolerances . here", ", we consider populations that are introduced below the invasion risk threshold , but nonetheless succeed if evolutionary dynamics are considered in conjunction with the ecological system .", "the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space .", "this equation models the growth of the population ( the first term on the right hand side of eqn .", "1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn .", "1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig .", "growth dynamics of the model population ( a ) , and the solution of eqn .", "( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 .", "trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 .", "there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn .", "the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al .", "1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) .", "figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a )", "there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity .", "1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth ,", "there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) .", "the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect )", ", there exists a unique wave speed of the invasion front that is a result of being ", "pushed from the inside out , as opposed to being  pulled by the leading edge ( lewis and kareiva 1993 )", ". this velocity can be derived through the solution of the pde ( 1 ) :   ( lewis and kareiva 1993 ; murray 1993 ) .", "this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity .", "in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) .", "this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch .", "this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth .", "this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth .", "thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand .", "we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model .", "the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) .", "the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success .", "the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) . in order to incorporate evolutionary factors that may influence invasion success ,", "this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival .", "specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time .", "this quantitative trait influences an organism 's ability to survive and reproduce in a small population .", "the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies .", "therefore , it is important to investigate the possibility of evolution in the analysis of invasions .", "the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn .", "( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance .", "as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) .", "thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) .", "this suggests that the genetic variance (  ) is small ( and constant in this model ) .", "this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale ,  = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection ,", "quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn .", "the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities .", "the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal .", "we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time", ", the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient .", "the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system ."], ["the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space .", "this equation models the growth of the population ( the first term on the right hand side of eqn .", "1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn .", "1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig .", "growth dynamics of the model population ( a ) , and the solution of eqn .", "( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 .", "trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 .", "there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn .", "the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al .", "1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) .", "figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a )", "there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity .", "1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth ,", "there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) .", "the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth", "versus reduced positive growth from a weak allee effect ) , there exists a unique wave speed of the invasion front that is a result of being ", "pushed from the inside out , as opposed to being  pulled by the leading edge ( lewis and kareiva 1993 )", ". this velocity can be derived through the solution of the pde ( 1 ) :   ( lewis and kareiva 1993 ; murray 1993 ) .", "this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity .", "in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) .", "this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch .", "this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth .", "this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth .", "thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand .", "we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model .", "the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) .", "the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success .", "the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) ."], ["in order to incorporate evolutionary factors that may influence invasion success , we develop a quantitative genetic subsystem .", "this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival .", "specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time .", "this quantitative trait influences an organism 's ability to survive and reproduce in a small population .", "the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies .", "therefore , it is important to investigate the possibility of evolution in the analysis of invasions .", "the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn .", "( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) .", "thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) .", "this suggests that the genetic variance (  ) is small ( and constant in this model ) .", "this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale ,  = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection ,", "quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn .", "the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al .", "1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .", "this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities .", "the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal .", "we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time", ", the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient .", "the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system ."], ["the dynamics of the evolutionary ecology model can be interpreted using the idea of fast and slow timescales ( kondrashov and khibnik 1996 ; webb 2003 ) .", "earlier , we assumed that the genetic variation (  ) was small ( to use mean fitness as a proxy for individual fitness ) , which can subsequently be taken advantage of for our analysis of the coupled evolutionary ecological dynamics . when  = 0 , the situation without evolution , the genetic subsystem is frozen and the population moves towards a stable equilibrium of the ecological subsystem ( carrying capacity or extinction ) depending on its initial density ( greater than or less than the allee threshold respectively ; fig .", "when  > 0 but small , the allee threshold evolves relatively slowly and influences the ecological system . whenever the population is below its carrying capacity ( u = 1 for each spatial coordinate x when space is explicit ) , eqn .", "( 2 ) is negative , and decreases the mean allee threshold ( a ) , as the intensity of selection is density dependent .", "thus , fitness increases as allee effects are suppressed , and selection drives the allee threshold towards zero .", "if the population density is greater than the allee threshold , but still below the carrying capacity , it will progress towards carrying capacity more rapidly than it would without evolution as a decreases ; as the rate at which the population density changes ( eqn .", "1 ) is proportional to the difference between u and a. the ecological dynamics are reversed when the population density is below the allee threshold as the population declines towards extinction , but more slowly than it does without evolution . when u < a , eqn .", "( 1 ) is negative , and the population density approaches extinction more rapidly with a constant ( as the difference between u and a increases ) , than it does with evolution as a decreases ( revealing a more pronounced time lag to extinction ) . during this time lag ,", "as the population slowly declines , the opportunity for evolution to overcome inverse density dependent effects occurs .", "if the rate of evolution is fast enough , the allee threshold can fall below the population density , causing the rate of change of population density to become positive ( where u > a ) and the population grows and can successfully invade .", "the chance that evolution can rescue the population from extinction depends on the relative rates of genetic change in the quantitative trait ( i.e. , allee threshold ) and of population decline ( gomulkiewicz and holt 1995 ) ; hence the amount of genetic variance greatly impacts the ability to adapt and survive .", "a nonspatial example of this process , referred to as evolutionary rescue ( gomulkiewicz and holt 1995 ) , is shown in fig .", "2 , where a population is introduced below the allee threshold . without evolution , the population declines to extinction ( fig . 2a , solid line ) as the allee threshold remains constant ( fig . 2b , solid line ) . when the population can evolve ( fig .", "2 , dotted line ) , it declines at first until it can overcome the magnitude of inverse density dependence , and is then able to successfully establish . as it is difficult to measure the allee effect empirically ( tobin et al .", "2007 ) , we use an extreme value that exaggerates density dependent effects in order to investigate the  worst case scenario ( a = 0.3 , where the population exhibits deterministic decline when its density is less than 30% of its carrying capacity ) . when evolution is included , we used a small value for the genetic variance ,  = 0.02 , in order to remain consistent with fast - slow dynamics , unless otherwise indicated .", "comparison of an invading population introduced at a density below the allee threshold , a = 0.3 ( u = 0.25 , r = 0.6 ) .", "the solid line represents the nonspatial system ( d = 0 ) described by eqns ( 1 ) and ( 2 ) without evolution (  = 0 ) which results in extinction ( a ) and a constant allee threshold ( b ) .", "the dotted line indicates population growth ( a ) when evolution (  = 0.02 ) acts to reduce the allee threshold ( b ) . in general , there is a range of parameter space that permits persistence for a population below the allee threshold in the nonspatial model with evolution ( instead of simple decline to extinction ) .", "as genetic variance increases , we are essentially relaxing the assumption of fast - slow timescales and allow evolution to occur more rapidly .", "3 where initial population densities below the allee threshold require a minimum amount of genetic variance in order to avoid extinction . in this case", ", the rate of reproduction , r , also influences the potential for evolutionary rescue , as it impacts both population growth and rate of evolution ( eqns 1 and 2 , respectively ) .", "as we relax the assumption of fast - slow timescales , the behavior remains qualitatively the same as that described analytically under a strict fast - slow timescale assumption .", "parameter combinations of reproductive rate : r ; genetic variance :  , and initial population density : u , that result in extinction or evolutionary rescue . in this nonspatial scenario ,", "initial population densities greater than the allee threshold ( a = 0.3 ) always succeed , thus the focus is on the parameter space that allows for evolutionary rescue ( i.e. , where the population growth changes from negative to positive ) . as the reproductive rate increases from 0.1 to 1", ", there is less genetic variance needed for a population to evolve to overcome inverse density dependence as increased reproduction will contribute to suppressing allee effects .", "nonetheless , the additional complexities result in qualitatively similar behavior to the nonspatial model . in this case , not only will evolution influence population growth , it affects the wave speed and the critical size threshold , rmin .", "as the population overcomes allee effects with a decreasing allee threshold , the wave speed accelerates and the critical patch size becomes smaller .", "thus , in addition to the initial density of the introduced population and the genetic variance , the initial radius or patch size of the initial invasion area , the ratio of diffusion to reproduction , and gene flow will factor into successful establishment and give rise to a wider range of interactions between the ecology and evolution of this system .", "( 1 ) ( without the evolutionary subsystem ) in one - dimensional space , with an initial population density below the allee threshold , declines to extinction ( fig .", "this is contrasted by the results when the evolutionary subsystem is included . with the initial population density below the allee threshold , fig .", "the same type of rescue occurs for a population that starts near carrying capacity , but occupies an initial spatial size below that which is necessary for a population to successfully establish .", "figure 5a shows a rapidly declining population that goes extinct . under the same circumstance , but where evolution of the allee threshold occurs , fig .", "5b shows the population density at first beginning to shrink and then growing and expanding .", "in addition to the time evolution of population density across space in figs 4 and 5 , the evolution of the mean trait value across space illustrates how gene flow and the density dependent selection gradient influences its rate of change and distribution ( figs 4c and 5c ) . as the intensity of selection is density dependent ( and we assume constant genetic variance ) , locations with smaller populations can evolve the trait value more rapidly compared to other areas where allee effects may not be as strong and experience weaker selection .", "the trait distribution over time , figs 4c and 5c , therefore reflect the population density distribution , but are also influenced by the trait values of the migrants . as individuals disperse out to new locations and push the boundaries of the species range , their trait values are averaged to determine the demographic allee threshold for that spatial coordinate . this demographic allee threshold combines with their local population density to influence individual fitness and population growth ( where the distance between the density and mean trait value is the initial degree of maladaptation ) .", "diffusive dispersal of an introduced population at an initial density ( bold dashed line ) below the initial allee threshold , a = 0.3 ( u = 0.25 , r = 1 , d = 0.1 ) across a linear , one dimensional habitat .", "the population collapses over time to extinction ( a ) where there is no evolution (  = 0 ) , and succeeds ( b ) after an initial decline with evolution (  = 0.02 ) .", "( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) . the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) .", "population density of a diffusion dispersed population across one dimensional space . the initial population density ( bold dashed line )", "is near carrying capacity ( u = 0.95 , a = 0.3 , r = 1 , d = 0.5 ) , but introduced below the minimum radius of area determined to be critical for invasion success .", "( a ) is collapsing to extinction without evolution (  = 0 ) , whereas ( b ) shows success of an invader with evolution (  = 0.02 ) after initial decline .", "( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) .", "the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) .", "we explored when evolutionary rescue occurred across a range of parameter values for the spatially explicit model . according to drake et al .", "( 2005 ) , variability among locations and over time makes it unreasonable to determine precise estimates for the diffusion coefficient , d. we therefore explored a range of values , and present those that best illustrate breadth of behavior .", "the parameter that controls the reproductive rate , r , was also varied substantially , but as the spatial dynamics depend on the ratio of diffusion to rate of reproduction ( resulting in a measure of length ) ; we fixed r and varied d , unless otherwise noted .", "this was justified as the results of the spatial simulations are qualitatively identical for equivalent ratios .", "the effects of the critical patch size , initial population density , ratio of diffusion to growth and genetic variation on evolutionary rescue and population dynamics are shown in fig .", "the ratio of the diffusion coefficient ( d ) to the reproductive rate ( r ) determines whether the population will expand or collapse according to the initial radius of the introduced population .", "the areas under the curves denote combinations of genetic variance and initial population density that result in extinction .", "areas above the curves are combinations of genetic and/or demographic conditions that produce inevitable persistence .", "the parameter space between the vertical dashed lines refers to the different ways population survival is influenced . to the left of the initial allee threshold", ", the initial population density will either go extinct due to density dependent effects ( below the d / r curve ) , or given enough genetic variation , will be evolutionarily rescued ( above the d / r curve ) .", "the area to the right of the initial allee threshold [ and between the dashed lines in ( b ) and ( c ) ] is the case where the initial population density is greater than the allee threshold but due to the initial spatial size and the ratio of diffusion to reproduction , the population may go extinct without sufficient genetic variance ( below the d / r curve ) , otherwise it will evolve to overcome the critical patch size effect . for initial population densities", "thus , the area between the dashed lines in ( b ) and ( c ) truly delineates evolutionary rescue when d / r = 1 .", "the rightmost vertical line moves slightly to the left to the point of intersection of the d / r curve and the x - axis for other values of d / r .", "graphs ( a ) , ( b ) , and ( c ) represent different radii of the linear habitat that the introduced population initially occupies .", "when the size of the initial population is too small ( i.e. , a radius of 1 ) , a population at carrying capacity ( i.e. , u = 1 ) will go extinct without evolution due to the relative effect of diffusion to reproduction ( fig .", "if evolution occurs rapidly enough ( i.e. ,  > 0.02 ) , the population can overcome inverse density dependent effects and compensate for the loss due to diffusion and rebound from low densities . when the initial radius of the population is increased ( fig .", "6b , c ) , the chance of survival and establishment ( growth and expansion ) of populations above or below the allee threshold increases with initial density and genetic variance .", "therefore , the initial radius of the population can significantly impact the likelihood of evolutionary rescue for populations with the same amount of genetic variance .", "this is demonstrated further in fig . 7a , where the rate of recovery ( i.e. , the inverse of the time lag before growth becomes positive and the population reaches carrying capacity ) for a population near carrying capacity depends on its initial size / radius and genetic variance . where size and variance are small", ", rescue never occurs . as these parameters increase , the rate of recovery gradually becomes faster until it essentially plateaus ( although with greater variance and initial radius , the rate of recovery may slow slightly if the initial spatial extent is large enough for the population to experience early growth before diffusion causes decline prior to recovery ) .", "if the population occupies a large enough spatial extent , it will succeed without evolution ( where the genetic variance is zero ) , however the lag time may be more pronounced depending on the ratio of diffusion to reproduction through the tradeoff between growth and spread ( e.g. , if spread is relatively fast compared to reproduction , d / r = 1 ) . the population density may thus initially decline across space until reproduction can sufficiently overcome the loss due to diffusion , and the population can grow to carrying capacity .", "similar to the nonspatial case , a population ( greater than the allee threshold ) above the spatial threshold will grow to carrying capacity more rapidly with evolution than without .", "rate of recovery in terms of the inverse of the time lag before population growth becomes positive , where one  timestep equals 24 iterations of the model . in ( a )", ", the initial population density is near carrying capacity ( u = 0.95 , a = 0.3 , d / r = 1 ) , and the initial radius and genetic variance ,  , varies .", "where the rate of recovery is zero , the population goes extinct as it initially occupies an area smaller than the critical patch size ( in this case , a radius of 1.4 ) or does not have sufficient genetic variance to evolve quickly enough to be rescued prior to extinction . increasing the genetic variance and initial radius will decrease this time lag until the population no longer experiences any negative growth ( in this case , for initial radii  3.8 and   0.036 ; for initial radii > 2.7 , the rate of recovery slows slightly due to early growth followed by a transient decline that precedes ultimate recovery ) .", "when the initial population density varies ( indicating the initial degree of maladaptation where a = 0.3 ) , ( b ) shows the rate of recovery with the initial radius fixed ( as in fig .", "5a where the radius = 1 and d / r = 1 ) . in this case", ", extinction will occur without evolution not only for an initial density below the initial allee threshold , but for any density as the initial radius is below the critical patch size .", "hence , a nonzero rate implies evolutionary rescue and a zero rate means extinction . as shown in fig .", "7b , when evolutionary rescue is possible , the initial level of maladaptation ( a0u0 ) and the genetic variance (  ) also determine the rate at which evolutionary rescue proceeds .", "figure 7b uses parameters ( i.e. , radius and ratio of d to r ) for a population that would decline and go extinct without evolution regardless of the initial density .", "hence , it is clear that the amount of time required for a population to begin growing depends on its initial level of maladaptation ( to both the critical density and spatial thresholds ) and/or genetic variance . as the rate at which this rescue occurs depends on the amount of genetic variance ( eqn .", "2 ) , it may take an extremely long time ( as  0 , the rate of recovery  0 ) for the allee threshold to fall below the population density . in this circumstance , as the population density becomes very close to zero , the rate of change of the allee threshold is greater than that of the population density ( as u  0 , u/t 0 and a/t  -2ra )", ". thus , theoretically , rescue would always occur ( gomulkiewicz and holt 1995 ) . however , to maintain biological realism , when solving this system numerically , we always considered the population extinct when the maximum density ( across space , when diffusion is included ) becomes reasonably close to zero ( i.e. , u = 0.0001 ; we chose this protocol instead of the total population across space due to the diffusion dynamics based on the gaussian dispersal kernel and the  pushed wave front behavior ) .", "overall , the numerical results qualitatively hold for a wide range of dimensional parameter values and initial conditions with and without diffusion and in one- and two - dimensional space .", "results for two - dimensional space are not shown as they are qualitatively similar to the simpler , one dimensional model ."], ["from these results , it is apparent that adaptations that enable organisms to overcome the negative effects of low densities can allow the population to rebound from a trajectory toward extinction to grow to reach carrying capacity .", "current management strategies ( e.g. , reducing population density or size ) are based on ecological theory ( e.g. drake et al . 2005 ) , but this evolutionary ecology model suggests that adaptive evolution can enable successful establishment and that ecological considerations alone may not be sufficient . under the assumptions of an allee effect and diffusive dispersal , the idea of ecological size thresholds fits well with the ecological evidence that a large founding population is a primary cause of successful establishment ( lockwood et al .", "however , by incorporating evolution , we see that the situation is not quite this simple because ecological size thresholds and genetic variance can interact to determine successful establishment . as", "the ratio of diffusion to reproduction decreases , the spatial constraint on population growth becomes weaker , and less genetic variance is needed to rescue populations with densities below the allee threshold . as the initial spatial radius of introduction increases ,", "furthermore , the rate of this rescue depends on the initial genetic load or maladaptation ( i.e. , how far the population density is from the allee threshold ) , as well as the amount of genetic variance .", "because bottlenecks during founding events do not always result in highly reduced genetic variability , even small founding populations may have sufficient genetic variation to evolve to overcome allee effects and establish , contrary to solely ecologically based predictions .", "additionally , we can draw several general insights about how dispersal impacts selection and evolution of allee effects in an invasion context .", "as species are transported from their native environment into novel habitats or simply disperse on their own , it is clear that the genetic composition of the local population can influence the rate of evolution and adaptation to the new local conditions . given enough genetic diversity , local populations can adapt to their local environment , but dispersal may hinder survival across ecological clines as dispersers tend to be maladapted to the new local environment . essentially , local population persistence depends on the race between the rate of evolution and the degree of maladaptation ( gomulkiewicz and holt 1995 ) . in this case", ", gene flow will play a major role in determining the outcome . as kirkpatrick and barton ( 1997 ) and garca - ramos and kirkpatrick ( 1997 )", "demonstrate , individuals moving from one selection regime from the center of their species range to the periphery can introduce enough maladaptation that the new area becomes a sink environment . on the other hand , holt et al .", "( 2003 , 2004 ) show that immigration can have a positive influence on adaptation to sink environments , in some circumstances .", "resolving the disparity between these perspectives requires understanding what is contributing to the severity of maladaptation and the population 's ability to overcome it . in our model ,", "dispersal impacts survival ecologically through the critical patch size , and genetically , as individuals may move from areas where they are well adapted ( i.e. , the population density is greater than the allee threshold or mean trait value ) to sink regions , where they are maladapted .", "as individuals disperse across space , they may be contributing positively in an ecological sense to the quality of their new local environment ( by increasing the local population density ) .", "however , dispersers are more likely to come from higher density areas where allee effects , and hence selection , are locally weak .", "these dispersers potentially introduce more maladaptation to their new location , because they increase the average phenotype ( allee threshold ) in the new location where density is likely to be lower .", "interestingly , the evolutionary impacts of migration in this model do not dramatically influence the dynamics .", "changes to the local mean phenotype through local selection and simple mixing ( i.e. , diffusion ) actually slightly hastens the evolutionary rescue effect over a model that considers only the impact of local selection .", "as the selection intensity is density dependent and proportional to u 1 for each point in space , the peripheral individuals faced with stronger selection with lower trait values have a small positive influence on the more dense neighboring populations .", "the gradient term accounts for asymmetric gene flow due to differential migration from areas of relatively high population densities .", "however , this term does not alter the overall evolutionary dynamics based on local selection any more than adding the diffusion term , as the negative effects of gene flow and the local rate of evolution ( which is relatively fast , based on the selection intensity ) essentially cancel each other out . in this context ,", "similar to that of holt et al . ( 2003 , 2004 ) , the immigrants simply contribute to the local population density , which helps prevent extinction long enough for evolutionary rescue to occur locally ( i.e. , positive population growth ; note that whereas holt et al .", "( 2003 ) attribute the main effect of immigration to the contribution of variation , this is not the case in our model , as we assume constant genetic variance ) .", "( 2004 ) where immigration has a demographic effect on increasing fitness that can essentially outweigh the ", "the primary determinant of invasion success depends on positive population growth at the center of the introduced range .", "this result comes from the allee effect [ and the solution to the pde ( 1 ) ] by forcing a  pushed travelling wave front ( lewis and kareiva 1993 ) , where the wave speed causes population expansion , contraction , or propagation failure ( i.e. , pinning ; keitt et al", ". 2001 ) . intuitively , aggregation - like behavior emerges based on the strength of the allee effects .", "individuals that disperse too far from the whole are likely to die before they can  pull others in their vicinity . in this", "regard , growth occurs from the inside out , where the population seemingly spills out and overflows to expand its range .", "consequently , in this study , and for biological invasions that exhibit similar dynamics , it is more important to focus on the center of the invader 's range and whether the initial beachhead can survive ( through evolutionary rescue ) , than the fate of peripheral populations at the wave front when determining the importance of evolution on invasion success .", "the overall dynamics are qualitatively similar in the parameter space that allows for evolutionary rescue to occur .", "even though gene flow and spatial structure do not dramatically influence the establishment of an introduced population , additional invasion criteria need to be considered .", "when analyzing the model behavior in a spatially explicit context , there is an ecological tradeoff between growth and spread that affects establishment and the rate of recovery . as previously mentioned , reproduction needs to compensate for the loss due to diffusion . including evolution and suppressing allee effects", ", actually contributes to the acceleration of the wave front ( i.e. , enhancing dispersal speed ) . a population then can more rapidly disperse as it evolves , and may become more of an invasion threat as long as this range expansion does not reduce their density too quickly . whereas this increasing wave speed can lead to a slightly longer lag phase prior to positive population growth , the population will likely be inevitably rescued because this effect primarily influences the dynamics at the periphery and is offset by the reduction in the critical invasion area ( rmin ) .", "although there is no range contraction ( as there is always a positive wave velocity with unbounded expansion due to the parameter values and absence of environmental heterogeneity or range limitations ; filin et al .", "2008 ) , as the critical patch size ( rmin ) becomes smaller with the decreasing allee threshold , rescue occurs more readily at the range center as the critical patch size threshold criteria weakens and the behavior approaches that of the nonspatial model .", "this may seem like an oversimplification of the global dynamics ; however these conclusions are valid in the context of this investigation which focuses on the establishment phase rather than subsequent range expansion and spread . recognizing that evolution can significantly affect the establishment success of invasive species is becoming more widely accepted , influencing the ways in which invasion biologists conduct their research ( see the other articles in this issue ) .", "specifically , adaptations that diminish allee effects and evolutionary responses to density dependence are beginning to emerge as viable explanations for sustaining vulnerable populations at low density and size ( gascoigne et al .", "as it is difficult to conclusively support this claim empirically ( as the origin of the adaptation or the associated cost may be unknown ; courchamp et al . 2008 ; gascoigne et al .", "2009 ) , mathematical models that incorporate evolution and compare the effects of various strategies ( e.g. , mitigating component allee effects ) can help decipher the mechanisms that both limit and facilitate population growth .", "two such models that incorporate adaptations to component mate - finding allee effects compare the efficiency and survival of populations at various densities that attract mates with or without a sexual pheromone ( jonsson et al .", "another study suggests that broadcast spawners that evolve their gamete morphology and performance under sperm limitation ( at low density ) bear a cost of decreased fitness at high density due to hybridization and competition ( levitan 2002 ) .", "in these cases , particular strategies are shown to influence population viability in addition to an associated tradeoff , whereas our investigation provides broad , albeit simplistic , results dealing with generalized demographic allee effects and evolution . in order to understand how the results of this simplistic model extend to more realistic and complex evolutionary scenarios , spatially explicit", ", individually - based stochastic simulation of the introduced populations should be developed to investigate more closely the mechanisms that allow these population level dynamics to emerge .", "in particular , tracking the mean value of a component allee effect is sufficient to illustrate how evolution can overcome inverse density dependence and result in invasion .", "however , this approach may not be sufficient to make the specific quantitative predictions necessary for management of invasive species .", "this is due to the simplifying assumption of constant genetic variance based on mutation - selection balance ( lande 1976 ) .", "complex simulations could relax this assumption and permit genetic variation to change via mutation , selection , and drift , in tandem with the demographic processes in a heterogeneous environment , and explicitly investigate the costs associated with avoiding allee effects .", "hence , future models should incorporate how propagule pressure ( size and frequency of introduction events ) impacts genetic variation and how more realistic genetic architectures contribute to the evolutionary trajectory of invasive species .", "although there is still much more work to be done to elucidate the factors that determine establishment success of founder populations , this theoretical approach has the promise to provide evidence in support of our working hypothesis that adaptive evolution can mitigate allee effects and be an important driver of biological invasions ."]]}
{"article_id": "PMC3870114", "article_text": ["genital infection due to chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infections ( stis ) . according to the who estimates , globally 92 million new cases of c. trachomatis infection", "occur each year and about two - thirds of these cases occur in the developing world , where diagnostic and treatment services are scarce [ 2 , 3 ] .", "most epidemiological data on chlamydia trachomatis infection ( cti ) is from industrialized nations and reliable data from the resource poor developing nations is not available where the disease burden is concentrated .", "however , it is important to document laboratory - confirmed incidence and prevalence of cti from the developing world as well .", "the available indian data show a wide variation in ct prevalence with infection rates in indian women ranging from 3.3% to 33% depending on the population sampled [ 413 ] .", "infection with this agent is usually asymptomatic in up to 80% of women which makes diagnosis and detection all the more difficult . left undetected and untreated the infection may evolve into pelvic inflammatory disease and may result in serious sequelae , such as ectopic pregnancy and infertility [ 14 , 15 ] .", "cti in women has also been linked to adverse pregnancy outcomes like recurrent miscarriage and preterm labor and may cause conjunctivitis , nasopharyngitis , and pneumonia in newborns by vertical transmission . because the infection is easily treatable with antibiotics , early detection and treatment of infected individuals", "are the key to prevent adverse sequelae among those infected and reduce c. trachomatis transmission .", "thus , it is important to screen adolescents and sexually active women for cti even if they are asymptomatic [ 17 , 18 ] . but , in the developing countries with the exception of sporadic testing , screening for chlamydia is rarely done .", "epidemiological studies have also shown that untreated genital chlamydia infection can lead to an increased risk for heterosexual acquisition of hiv . hence , screening for cti done in high risk populations can assist in designing hiv risk reduction strategies .", "on the other hand , immunosuppression due to hiv may lead to more aggressive chlamydia disease conditions like pid in hiv seropositive women .", "thus , screening for cti in hiv seropositive women is highly recommended to prevent morbidity associated with the disease and devastating clinical consequences .", "different diagnostic modalities for detection of cti like serology , culture method , elisa for antigen and antibody , direct fluorescence assay ( dfa ) and nucleic acid amplification tests ( naats ) have been used in the last 20 years but none of them are 100% sensitive .", "table 1 shows the prevalence of cti detected by using different diagnostic techniques in new delhi population .", "polymerase chain reaction is an accurate , rapid , and reliable method for the detection of chlamydia trachomatis .", "real - time pcr has increasingly been used and is easier to perform and faster , and since it is performed in a closed system it is less prone to contamination than the conventional pcr . keeping the above background in mind this study was undertaken to generate reliable data regarding prevalence of cti in hiv - infected and hiv - uninfected women by real - time pcr , the most sensitive and specific test available currently for diagnosing genital chlamydia infection .", "the primary objective of this study was to establish the need for screening hiv seropositive women for cti .", "study subjects were recruited as follows : thirty adult hiv seropositive women with symptoms suggestive of rtis ( study group a1),thirty adult hiv seropositive women without symptoms suggestive of rtis ( study group a2),thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis ( control group b1),thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis ( control group b2 ) .", "thirty adult hiv seropositive women with symptoms suggestive of rtis ( study group a1 ) , thirty adult hiv seropositive women without symptoms suggestive of rtis ( study group a2 ) , thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis ( control group b1 ) , thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis ( control group b2 ) .", "all the study subjects were enrolled from the integrated counseling and testing center ( ictc ) for hiv / aids , department of microbiology , maulana azad medical college , which is attached to the lok nayak hospital , new delhi .", "this study was conducted prospectively between july 2010 and january 2011 at the hiv molecular laboratory of the department of microbiology , maulana azad medical college .", "this was a cross - sectional analysis to determine the prevalence of chlamydia trachomatis infection by using real - time pcr in hiv seropositive and seronegative , symptomatic , and asymptomatic women visiting the ictc of our department .", "subjects were enrolled in this study following institutional ethical committee clearance and a written informed consent of all participants . each participant was interviewed using a questionnaire concerning general sociodemographic information , personal details , and clinical symptoms .", "subjects having one or more of the following symptoms were considered symptomatic for rti : vaginal discharge , vesicular and/or nonvesicular genital ulcers , inguinal bubo , lower abdominal pain , genital skin conditions , urinary burning or frequency , dysmenorrhea , menorrhagia , and intermenstrual bleeding .", "vesicular and/or nonvesicular genital ulcers , lower abdominal pain , genital skin conditions , urinary burning or frequency , dysmenorrhea , menorrhagia , and intermenstrual bleeding .", "each study subject then underwent a general physical , per abdomen , per speculum , and per vaginum examination .", "diagnosis of hiv infection was done by following the standard protocol at our ictc that employs pretest and posttest counseling and obtains informed consent before hiv testing .", "three different rapid tests were used to detect hiv-1 and hiv-2 antibodies ( combaids ( span diagnostics ltd . ) , retrocheck hiv ( qualpro diagnostics ) , and tri - line ( rapid diagnostics ) ) following the manufacturer 's instructions .", "one endocervical swab was collected from all participants according to the instructions provided in the specimen collection and transport kit ( amplicor std swab collection and transport set ) for detection of chlamydia trachomatis by real - time pcr .", "genital c. trachomatis infection was diagnosed by using cobas taqman ct test , v2.0 , an in vitro nucleic acid amplification test for the qualitative detection of chlamydia trachomatis dna in female endocervical swab specimens .", "specimens were processed using the amplicor ct / ng specimen preparation kit for manual specimen preparation and the cobas taqman 48 analyzer for automated amplification and detection ( roche diagnostics ) .", "the age and gender profile of all the participants is shown in table 2 . in both the hiv seropositive study group and the hiv seronegative control group", "the mean age of hiv - infected cases was found to be 30.92  5.7 years and in hiv - uninfected controls 28.52  6.9 years . \n", "table 3 shows the presenting complaints of the symptomatic women in both the hiv seropositive study group and the hiv seronegative control group .", "vaginal discharge and lower abdominal pain were the most common presenting complaints amongst the symptomatic participants . \n", "table 4 shows the sti / rti syndromes diagnosed in study subjects on per speculum examination .", "vaginitis was the most common syndrome diagnosed in both the study and the control groups . \n", "table 5 shows the correlation between presence of symptoms and cti in the study and the control groups .", "chlamydia trachomatis infection was more commonly diagnosed in the asymptomatic hiv seropositive study subjects as compared to the symptomatic hiv seropositive study subjects . in our study", "the prevalence of cti was higher in hiv seropositive women as compared to hiv seronegative women ( or 4.214 ; 95% ci 0.45738.865 ) and among the hiv positive asymptomatic as compared to the hiv negative asymptomatic ( or 2.111 ; 95% ci 1.6062.776 ) , although the differences were not found to be statistically significant ( table 6 ) .", "in india stis / rtis and hiv / aids are major public health problems . incidence and prevalence data have a key role in control strategies for hiv and stis .", "comprehensive baseline information on the epidemiology of stis is essential for the design , implementation , and monitoring of successful control programs to reduce their incidence .", "routine surveillance of stis / rtis is not carried out in our country due to the lack of laboratory diagnostic facilities , limited resources , stigma , and discrimination associated with stis and poor attendance of sti patients , especially women , in sexually transmitted disease ( std ) clinics . taking the above facts into consideration ,", "it is meaningful to have genuine laboratory - confirmed data on the incidence / prevalence of rtis / stis in india . as the asymptomatic nature of stis  ", "is well known , there is a need to adopt a specific strategy for the screening of the sexually active population in india to reduce the overall rate of stis , which would , in turn , reduce the risk of hiv infection .", "our study presents an insight into the prevalence data of genital chlamydia infections in hiv - infected and hiv - uninfected women visiting the ictc of new delhi 's largest tertiary care hospital .", "the mean age of the hiv seropositive subjects in our study was 30.92  5.7 and that of the hiv seronegative control group was 28.52  6.9 .", "this compares well with the findings of another study done in baroda , india , to look for the prevalence of rtis in hiv - infected women wherein the mean age for hiv positive women was 30 and that for hiv negative women was 27 .", "the predominant age group in the hiv positive participants in our study was 2635 years .", "this association of chlamydia infection with younger age is consistent with studies from other developing countries [ 32 , 33 ] .", "this finding supports the fact that young sexually active adults should constitute a priority target group in the sti control program .", "however , the findings of our study can not be generalized to all sexually active adults as the study has its limitations of a small sample size and the study participants belonging to a high risk group being recruited from the ictc of our department .", "80% of our hiv positive study participants were married which is quiet similar to what has been reported by another study from sub - saharan africa in which 85.3% of the hiv - infected women were married .", "this finding highlights the importance of concurrently screening and treating spouses / sexual partners to decrease the sti burden in the country but this is challenging due to the lack of knowledge and cooperation from husbands especially in the indian setup . in the present study , vaginal discharge and lower abdominal pain", "were the 2 most frequently reported symptoms by the symptomatic women in both the study and the control groups , confirming the data reported by some previous indian studies [ 22 , 29 ] .", "another notable observation of our study was that vaginitis was the most common clinical finding detected on per speculum examination in both the hiv positive ( 50% ) and the hiv negative groups ( 56.67% ) which is in accordance with the observations of garg et al . and", "balamurugan and bendigeri where a majority of women on clinical examination had vaginitis of 94.6% and 36.9%% , respectively [ 22 , 35 ] .", "the present study also validates the fact that chlamydia infections are usually asymptomatic as 10% of the asymptomatic women were diagnosed with cti while only 3.3% of symptomatic women had lab - confirmed cti in the hiv positive study group .", "once again this emphasizes the importance of routine screening of at risk young sexually active women . in our study", "cti was detected in 6.67% ( 4/60 ) hiv - infected women and in 1.67% ( 1/60 ) hiv - uninfected women by real - time pcr although this difference was not statistically significant but the odds ratio was 4.214 . a study from cuba reported c. trachomatis infection in 10% of hiv - infected cases and 6.6% of hiv - uninfected cases by nested pcr but", "the difference was not statistically significant while the odds ratio was 3.39 which is in concordance with our study .", "studies conducted by natividad - villanueva et al . in usa and seck et al . in senegal reported c. trachomatis infection in 3.33% and 2.1% of hiv - infected females , respectively [ 38 , 39 ] .", "larger studies are required to validate our observation as our study was restricted to a small number of cases .", "this study clearly shows that cti is more prevalent among hiv - infected females ( with or without symptoms of rti ) as compared to hiv - uninfected females .", "our study also stresses the usefulness of screening asymptomatic hiv - infected and hiv - uninfected females for cti by risk assessment and diagnostic testing periodically to prevent the occurrence of adverse outcomes associated with the disease and also to check further spread of infection in the community . for countries like india that still do not currently have an active chlamydia screening program in place ,", "randomized controlled trials are required to delineate the benefits of screening in the sexually active population as our study participants represent a high risk group .", "it is also important that any test adopted in a national screening program be used in the primary care setting by practitioners without the need for expensive training and equipment .", "however , since use of real - time pcr is not feasible in most hospitals in developing countries efforts should be made to develop a simple , cost - effective , sensitive , and specific point of care test to identify and treat women with cti for prevention of sequelae and hiv transmission ."], "abstract_text": ["<S> reproductive tract infection ( rtis)/sexually transmitted infections ( stis ) are recognized as a major public health problem , particularly due to their relationship with hiv infection . </S>", "<S> early detection and treatment of chlamydia trachomatis infection ( cti ) among hiv - infected and hiv - uninfected women may impact heterosexual hiv transmission . </S>", "<S> a total of 120 participants were enrolled : 30 hiv seropositive women with symptoms of rtis , 30 hiv seropositive women without symptoms of rtis , 30 hiv seronegative women with symptoms of rtis , and 30 hiv seronegative women without symptoms of rtis . </S>", "<S> one endocervical swab was collected from all participants and cti was detected by real - time pcr ( cobas taqman ct test , v2.0 ) . </S>", "<S> cti was detected in 4 ( 6.67% ) hiv - infected women and in 1 ( 1.67% ) hiv - uninfected woman ( or 4.214 ; 95% ci 0.45738.865 ) . </S>", "<S> vaginal discharge was present in almost half of hiv - infected and hiv - uninfected women ; lower abdominal pain was present in 11 ( 18.3% ) of hiv - infected and in 9 ( 15% ) of hiv - uninfected women . </S>", "<S> this study showed that cti is more prevalent among hiv - infected females as compared to hiv - uninfected females . </S>", "<S> as the use of real - time pcr is not feasible in most hospitals , efforts should be made to develop a simple , sensitive , and specific test to identify women with cti for prevention of sequelae and hiv transmission . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["genital infection due to chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infections ( stis ) . according to the who estimates , globally 92 million new cases of c. trachomatis infection", "occur each year and about two - thirds of these cases occur in the developing world , where diagnostic and treatment services are scarce [ 2 , 3 ] .", "most epidemiological data on chlamydia trachomatis infection ( cti ) is from industrialized nations and reliable data from the resource poor developing nations is not available where the disease burden is concentrated .", "however , it is important to document laboratory - confirmed incidence and prevalence of cti from the developing world as well .", "the available indian data show a wide variation in ct prevalence with infection rates in indian women ranging from 3.3% to 33% depending on the population sampled [ 413 ] .", "infection with this agent is usually asymptomatic in up to 80% of women which makes diagnosis and detection all the more difficult . left undetected and untreated the infection may evolve into pelvic inflammatory disease and may result in serious sequelae , such as ectopic pregnancy and infertility [ 14 , 15 ] .", "cti in women has also been linked to adverse pregnancy outcomes like recurrent miscarriage and preterm labor and may cause conjunctivitis , nasopharyngitis , and pneumonia in newborns by vertical transmission . because the infection is easily treatable with antibiotics , early detection and treatment of infected individuals", "are the key to prevent adverse sequelae among those infected and reduce c. trachomatis transmission .", "thus , it is important to screen adolescents and sexually active women for cti even if they are asymptomatic [ 17 , 18 ] . but , in the developing countries with the exception of sporadic testing , screening for chlamydia is rarely done .", "epidemiological studies have also shown that untreated genital chlamydia infection can lead to an increased risk for heterosexual acquisition of hiv . hence , screening for cti done in high risk populations can assist in designing hiv risk reduction strategies .", "on the other hand , immunosuppression due to hiv may lead to more aggressive chlamydia disease conditions like pid in hiv seropositive women .", "thus , screening for cti in hiv seropositive women is highly recommended to prevent morbidity associated with the disease and devastating clinical consequences .", "different diagnostic modalities for detection of cti like serology , culture method , elisa for antigen and antibody , direct fluorescence assay ( dfa ) and nucleic acid amplification tests ( naats ) have been used in the last 20 years but none of them are 100% sensitive .", "table 1 shows the prevalence of cti detected by using different diagnostic techniques in new delhi population .", "polymerase chain reaction is an accurate , rapid , and reliable method for the detection of chlamydia trachomatis .", "real - time pcr has increasingly been used and is easier to perform and faster , and since it is performed in a closed system it is less prone to contamination than the conventional pcr . keeping the above background in mind this study was undertaken to generate reliable data regarding prevalence of cti in hiv - infected and hiv - uninfected women by real - time pcr , the most sensitive and specific test available currently for diagnosing genital chlamydia infection .", "the primary objective of this study was to establish the need for screening hiv seropositive women for cti ."], ["study subjects were recruited as follows : thirty adult hiv seropositive women with symptoms suggestive of rtis ( study group a1),thirty adult hiv seropositive women without symptoms suggestive of rtis ( study group a2),thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis ( control group b1),thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis ( control group b2 ) .", "thirty adult hiv seropositive women with symptoms suggestive of rtis ( study group a1 ) , thirty adult hiv seropositive women without symptoms suggestive of rtis ( study group a2 ) , thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis ( control group b1 ) , thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis ( control group b2 ) .", "all the study subjects were enrolled from the integrated counseling and testing center ( ictc ) for hiv / aids , department of microbiology , maulana azad medical college , which is attached to the lok nayak hospital , new delhi .", "this study was conducted prospectively between july 2010 and january 2011 at the hiv molecular laboratory of the department of microbiology , maulana azad medical college .", "this was a cross - sectional analysis to determine the prevalence of chlamydia trachomatis infection by using real - time pcr in hiv seropositive and seronegative , symptomatic , and asymptomatic women visiting the ictc of our department .", "subjects were enrolled in this study following institutional ethical committee clearance and a written informed consent of all participants . each participant was interviewed using a questionnaire concerning general sociodemographic information , personal details , and clinical symptoms .", "subjects having one or more of the following symptoms were considered symptomatic for rti : vaginal discharge , vesicular and/or nonvesicular genital ulcers , inguinal bubo , lower abdominal pain , genital skin conditions , urinary burning or frequency , dysmenorrhea , menorrhagia , and intermenstrual bleeding .", "vesicular and/or nonvesicular genital ulcers , lower abdominal pain , genital skin conditions , urinary burning or frequency , dysmenorrhea , menorrhagia , and intermenstrual bleeding .", "each study subject then underwent a general physical , per abdomen , per speculum , and per vaginum examination .", "diagnosis of hiv infection was done by following the standard protocol at our ictc that employs pretest and posttest counseling and obtains informed consent before hiv testing .", "three different rapid tests were used to detect hiv-1 and hiv-2 antibodies ( combaids ( span diagnostics ltd . ) , retrocheck hiv ( qualpro diagnostics ) , and tri - line ( rapid diagnostics ) ) following the manufacturer 's instructions .", "one endocervical swab was collected from all participants according to the instructions provided in the specimen collection and transport kit ( amplicor std swab collection and transport set ) for detection of chlamydia trachomatis by real - time pcr .", "genital c. trachomatis infection was diagnosed by using cobas taqman ct test , v2.0 , an in vitro nucleic acid amplification test for the qualitative detection of chlamydia trachomatis dna in female endocervical swab specimens .", "specimens were processed using the amplicor ct / ng specimen preparation kit for manual specimen preparation and the cobas taqman 48 analyzer for automated amplification and detection ( roche diagnostics ) ."], ["the age and gender profile of all the participants is shown in table 2 . in both the hiv seropositive study group and the hiv seronegative control group", "the mean age of hiv - infected cases was found to be 30.92  5.7 years and in hiv - uninfected controls 28.52  6.9 years . \n", "table 3 shows the presenting complaints of the symptomatic women in both the hiv seropositive study group and the hiv seronegative control group .", "vaginal discharge and lower abdominal pain were the most common presenting complaints amongst the symptomatic participants . \n", "table 4 shows the sti / rti syndromes diagnosed in study subjects on per speculum examination .", "vaginitis was the most common syndrome diagnosed in both the study and the control groups . \n", "table 5 shows the correlation between presence of symptoms and cti in the study and the control groups .", "chlamydia trachomatis infection was more commonly diagnosed in the asymptomatic hiv seropositive study subjects as compared to the symptomatic hiv seropositive study subjects . in our study", "the prevalence of cti was higher in hiv seropositive women as compared to hiv seronegative women ( or 4.214 ; 95% ci 0.45738.865 ) and among the hiv positive asymptomatic as compared to the hiv negative asymptomatic ( or 2.111 ; 95% ci 1.6062.776 ) , although the differences were not found to be statistically significant ( table 6 ) ."], ["in india stis / rtis and hiv / aids are major public health problems . incidence and prevalence data have a key role in control strategies for hiv and stis .", "comprehensive baseline information on the epidemiology of stis is essential for the design , implementation , and monitoring of successful control programs to reduce their incidence .", "routine surveillance of stis / rtis is not carried out in our country due to the lack of laboratory diagnostic facilities , limited resources , stigma , and discrimination associated with stis and poor attendance of sti patients , especially women , in sexually transmitted disease ( std ) clinics . taking the above facts into consideration ,", "it is meaningful to have genuine laboratory - confirmed data on the incidence / prevalence of rtis / stis in india . as the asymptomatic nature of stis  ", "is well known , there is a need to adopt a specific strategy for the screening of the sexually active population in india to reduce the overall rate of stis , which would , in turn , reduce the risk of hiv infection .", "our study presents an insight into the prevalence data of genital chlamydia infections in hiv - infected and hiv - uninfected women visiting the ictc of new delhi 's largest tertiary care hospital .", "the mean age of the hiv seropositive subjects in our study was 30.92  5.7 and that of the hiv seronegative control group was 28.52  6.9 .", "this compares well with the findings of another study done in baroda , india , to look for the prevalence of rtis in hiv - infected women wherein the mean age for hiv positive women was 30 and that for hiv negative women was 27 .", "the predominant age group in the hiv positive participants in our study was 2635 years .", "this association of chlamydia infection with younger age is consistent with studies from other developing countries [ 32 , 33 ] .", "this finding supports the fact that young sexually active adults should constitute a priority target group in the sti control program .", "however , the findings of our study can not be generalized to all sexually active adults as the study has its limitations of a small sample size and the study participants belonging to a high risk group being recruited from the ictc of our department .", "80% of our hiv positive study participants were married which is quiet similar to what has been reported by another study from sub - saharan africa in which 85.3% of the hiv - infected women were married .", "this finding highlights the importance of concurrently screening and treating spouses / sexual partners to decrease the sti burden in the country but this is challenging due to the lack of knowledge and cooperation from husbands especially in the indian setup . in the present study , vaginal discharge and lower abdominal pain", "were the 2 most frequently reported symptoms by the symptomatic women in both the study and the control groups , confirming the data reported by some previous indian studies [ 22 , 29 ] .", "another notable observation of our study was that vaginitis was the most common clinical finding detected on per speculum examination in both the hiv positive ( 50% ) and the hiv negative groups ( 56.67% ) which is in accordance with the observations of garg et al . and", "balamurugan and bendigeri where a majority of women on clinical examination had vaginitis of 94.6% and 36.9%% , respectively [ 22 , 35 ] .", "the present study also validates the fact that chlamydia infections are usually asymptomatic as 10% of the asymptomatic women were diagnosed with cti while only 3.3% of symptomatic women had lab - confirmed cti in the hiv positive study group .", "once again this emphasizes the importance of routine screening of at risk young sexually active women . in our study", "cti was detected in 6.67% ( 4/60 ) hiv - infected women and in 1.67% ( 1/60 ) hiv - uninfected women by real - time pcr although this difference was not statistically significant but the odds ratio was 4.214 . a study from cuba reported c. trachomatis infection in 10% of hiv - infected cases and 6.6% of hiv - uninfected cases by nested pcr but", "the difference was not statistically significant while the odds ratio was 3.39 which is in concordance with our study .", "studies conducted by natividad - villanueva et al . in usa and seck et al . in senegal reported c. trachomatis infection in 3.33% and 2.1% of hiv - infected females , respectively [ 38 , 39 ] .", "larger studies are required to validate our observation as our study was restricted to a small number of cases ."], ["this study clearly shows that cti is more prevalent among hiv - infected females ( with or without symptoms of rti ) as compared to hiv - uninfected females .", "our study also stresses the usefulness of screening asymptomatic hiv - infected and hiv - uninfected females for cti by risk assessment and diagnostic testing periodically to prevent the occurrence of adverse outcomes associated with the disease and also to check further spread of infection in the community . for countries like india that still do not currently have an active chlamydia screening program in place ,", "randomized controlled trials are required to delineate the benefits of screening in the sexually active population as our study participants represent a high risk group .", "it is also important that any test adopted in a national screening program be used in the primary care setting by practitioners without the need for expensive training and equipment .", "however , since use of real - time pcr is not feasible in most hospitals in developing countries efforts should be made to develop a simple , cost - effective , sensitive , and specific point of care test to identify and treat women with cti for prevention of sequelae and hiv transmission ."]]}
{"article_id": "PMC5293477", "article_text": ["multilevel cervical spondylotic myelopathy ( mcsm ) is a serious disease which can lead to spinal cord dysfunction and a substantial decrease in quality of life . in addition , mcsm requiring multilevel cervical spine surgery can be accompanied by extensive blood loss . as spinal surgery has become increasingly complex , so control of perioperative bleeding has become an important clinical issue for spine surgeons .", "excessive blood loss can lead to a range of comorbidities such as anemia , hypotension , hematoma formation , and inadequate oxygenation of organs , thereby affecting patient outcome .", "excessive blood loss often requires allogeneic blood transfusion , the risks of which are numerous and among which immunological reactions and transmission of viruses are considered the most serious . in addition , hematoma formation within a few millimeters of the spinal canal can cause considerable neurological damage .", "although occurrences of postoperative spinal hematoma formation requiring emergency surgery are rare , it is nevertheless important to control perioperative bleeding to decrease its incidence . tranexamic acid ( txa ) is a synthetic derivative of the amino acid lysine which operates through competitive inhibition of the activation of plasminogen to plasmin by binding specific sites on both plasminogen and plasmin , thereby retarding fibrinolysis , the degradation of blood clots .", "it has been widely used in various medical fields such as cardiac surgery , gynecology , dentistry , urological surgery , and liver transplantation to reduce the perioperative blood loss , as it is relatively inexpensive and has not been cited in the literature as causing any significant untoward side effects . in addition , txa has been reported to reduce blood loss and transfusion requirements during orthopedic surgery , most commonly in knee and hip joint replacement . although the benefits of txa in spinal surgery have been reported", "indeed , there are no studies of txa in cervical laminectomy with lateral mass screw fixation and bone grafting ( clf ) so far .", "clf has been widely used for many years for treating mcsm caused by multilevel cervical spinal cord compression .", "although some authors suggest that laminoplasty is superior to laminectomy with lateral mass screw fixation and bone grafting regarding preserved range of motion , indications for the use of clf are broader than that of laminoplasty and recent studies showed good outcomes for clf . thus , the aim of this study is to evaluate the efficacy and safety of txa in controlling blood loss during clf for the treatment of mcsm .", "a retrospective comparative analysis was performed in patients with mcsm undergoing clf of vertebrae c3 to c6 .", "patients with cirrhosis of the liver , serious cardiac disease , chronic renal failure , cancer , allergy to txa , a history of thromboembolic disease ( deep vein thrombosis , ischemic heart disease , pulmonary embolism , transient ischemic attack , strokes , or subarachnoid hemorrhage ) , bleeding disorders , hypercoagulation status , disseminated intravascular coagulation , pregnancy , combined anterior and posterior spinal fusions , patients receiving antiplatelet and/or anticoagulant therapy at the time of the study , and treatments of vertebrae outside of c3 to c6 were excluded from the study . between november 2014 and april 2016 , 119 clfs fitting the inclusion criteria were performed in our center . each surgeon ( hao , liu , he , wu , wang , zheng , and zhao ) in this study had more than 20 years of experience in spinal surgery .", "the control group consisted of 46 patients who underwent clf for the treatment of mcsm in our institution without being given txa . in both groups", ", we analyzed patient demographic trait ( age at surgery , gender , body weight , height , and body mass index ) and duration of surgery which was defined as the time from the initial incision to the completion of wound closure .", "the quantity of intraoperative and postoperative blood loss and the preoperative and day 1 postoperative hematological data for each patient were obtained in the 2 groups .", "total blood loss was calculated as the sum of intraoperative and postoperative blood loss ( the quantity during the 1st 16  hours ) .", "the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision .", "laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) .", "the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material .", "lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique .", "all patients in the txa group were given a dose of 15  mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100  mg / hour , and continued until wound closure .", "no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug .", "continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value .", "statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables .", "all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) .", "the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision .", "laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) .", "the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material .", "lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique .", "all patients in the txa group were given a dose of 15  mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100  mg / hour , and continued until wound closure .", "no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug .", "continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value .", "statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables .", "all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) .", "there was no statistical difference in demographic values ( patients age , gender , body weight , height , and body mass index ) between the 2 groups ( table 1 ) .", "no statistically significant difference in duration of surgery was observed between the 2 groups , with a mean time of 155.72    15.59 and 153.52    11.91  minutes in the control and txa groups , respectively ( p  =  0.387 ) .", "there was significantly less intraoperative blood loss in the txa group compared to the control ( fig .", "1 ) , at 179.66    81.45 and 269.13    94.68  ml , respectively ( p  <  0.001 ) .", "the txa group had significantly less postoperative blood loss during the 1st 16  hours compared to the control group ( 108.08    44.31 vs 132.83    49.39  ml , p  =  0.005 ) .", "total blood loss in the control group ( 401.96    127.88  ml ) was significantly higher than that in the txa group ( 287.74    115.40  ml , p  <  0.001 ) .", "changes in blood hemoglobin ( hgb ) content and hematocrit ( hct ) were not statistically significantly different between the 2 groups : preoperative hgb :", "138.28    9.12  g / l ( control ) and 140.42    10.05  g / l ( txa ) ; postoperative hgb : 122.35    10.74  g / l ( control ) and 125.21    11.29  g / l ( txa ) ; preoperative hct : 41.78    2.62% ( control ) and 42.07    2.08% ( txa ) ; and postoperative hct : 36.50    3.22% ( control ) and 37.32    2.41% ( txa ) .", "although the hgb and hct values in the txa group were all higher than those of the control group , the differences were not statistically significant ( fig .", "no patient required an allogeneic blood transfusion during or after the surgery in either group and no serious intra- or postoperative complications , for example , dural tear , infection , epidural hematoma formation , deep - vein thrombosis , pulmonary embolism , allergic reaction , renal failure , or cardiopulmonary complications were observed in either group .", "furthermore , no minor side effects associated with the use of txa such as nausea , vomiting , headache , or diarrhea occurred in either of the groups . demographic parameters of patients in each group .", "the txa ( tranexamic acid ) group had significantly less intraoperative , postoperative , and total blood loss compared to the control group . there were no significantly different in blood hgb hct between the control and txa groups .", "hgb  =  hemoglobin , hct  =  content and hematocrit , txa  =  tranexamic acid .", "numerous studies have supported the use of txa in orthopedic surgery . although it has been evaluated in many studies , the safety and efficacy of txa for blood loss reduction in spinal surgery has not been clearly demonstrated , especially for clf .", "clf for the treatment of mcsm has proved to be a successful technique for restoring normal cervical lordotic alignment , recovering neurological function , and decreasing the morbidity of c5 palsy and axial pain .", "the principal disadvantage of this approach is a significant decrease in motion due to the fixation of the target vertebrae .", "laminoplasty allows a better range of neck motion although it becomes more restricted in flexion and extension over time . for patients with mcsm combined with simple instability or correctable kyphosis , clf is a more suitable method than laminoplasty , even if the consequence is neck stiffness .", "fibrinolysis increases transiently when patients undergo surgery , and it has been shown that it contributes to perioperative blood loss during spinal surgery .", "txa inhibits fibrinolysis by blocking the lysine - binding sites of plasminogen , plasmin , and tissue plasminogen activator .", "since fibrinolysis is activated immediately during and after surgery , txa should be administered before surgery begins .", "the half - life of txa is approximately 80 minutes in patients with normal renal function .", "pharmacokinetic evidence suggests a loading dose of 10 to 15  mg / kg followed by a maintenance dose of 1  mg / kg / hour or repeated dosing .", "li et al conducted a meta - analysis of 6 randomized controlled trials and believed that higher txa dosage ( 15 ", "yang et al conducted a meta - analysis of 9 randomized controlled trials which had a similar sample to li 's study .", "the 2 meta - analyses arrived at a similar conclusion although the quantity of blood loss and incidence of blood transfusion were different .", "elwatidy et al used a high single dose ( 30  mg / kg ) of txa and found that it reduced total blood loss and incidence of blood transfusion .", "raksakietisak et al reported that 2 doses of txa ( 15  mg / kg ) reduced perioperative blood loss and incidence of blood transfusion among low - risk adult patients undergoing elective complex thoracolumbar spine surgery .", ", we evaluated the effects of txa with a dose of 15  mg / kg followed by a maintenance dose of 100  mg / hour .", "the txa treatment group lost significantly less blood than that of the control group , including during the intra- and postoperative periods .", "total blood loss in the txa group was 28% less than that of the control group . although the hgb levels and hct in the txa group were higher than that of the control group , the differences were not significant , possibly because the sample size was too small and the perioperative blood loss in clf relatively minor .", "a theoretical anxiety associated with the use of txa is its potential for inducing thromboembolic complications .", "however , many studies have shown that the administration of txa does not increase this risk . in our study , there were no clinical symptoms or signs of thromboembolic events , such as deep - vein thrombosis or symptomatic pulmonary embolism .", "thus , the use of txa in patients undergoing clf should be regarded as safe .", "additionally , we are unsure whether the complexity of the surgery has an effect on the benefit of txa .", "a prospective randomized controlled study would better determine the safety and efficacy of txa during laminectomy with lateral mass screw fixation and bone grafting .", "in this study , blood loss ( both intra- and postoperative ) in the txa group was significantly lower than that in the control group , and no major intraoperative complications occurred .", "our results indicate that the use of intravenous txa is both safe and effective in reducing blood loss in clf .", "prophylactic txa may provide the benefit of limiting excessive blood loss in posterior approach cervical spinal surgery .", "a future prospective randomized controlled trial will provide superior evidence of the efficacy and safety of txa ."], "abstract_text": ["<S> abstractto assess the safety and efficacy of tranexamic acid ( txa ) for decreasing perioperative blood loss in cervical laminectomy with lateral mass screw fixation and bone grafting ( clf ) , in which all surgical procedures are identical.from november 2014 to april 2016 , we performed a retrospective comparative analysis of 119 patients with multilevel cervical spondylotic myelopathy who had undergone a clf from c3 to c6 in our center . </S>", "<S> all surgeries were performed on the patients using a consistent , standard procedure . </S>", "<S> patients were divided into control ( 46 ) and txa ( 73 ) groups according to whether or not they had received txa treatment before and during surgery . </S>", "<S> demographic profiles of patients such as gender , age , body weight , height , and body mass index were collated and differences between the 2 groups compared . </S>", "<S> preoperative and postoperative hematological data in addition to intraoperative and postoperative blood loss were compared between the 2 groups . </S>", "<S> additionally , any complications of txa were also evaluated to assess safety.there was no statistically significant difference in demographic traits between the 2 groups . </S>", "<S> intraoperative blood loss in the txa group ( 179.66    81.45  ml ) was significantly lower than that of the control group ( 269.13    94.68  ml , p  <  0.001 ) , as was postoperative blood loss ( 108.08    44.31 and 132.83    49.39  ml , respectively ; p  =  0.005 ) . </S>", "<S> total blood loss in the txa group ( 287.74    115.40  ml ) was also significantly lower than that of the control group ( 401.96    127.88 , p  <  0.01 ) . </S>", "<S> no major intraoperative complications occurred in any of the cases.txa significantly reduced perioperative blood loss in clf with no major side effects . </S>"], "labels": null, "section_names": ["Introduction", "Material and methods", "Surgical procedure", "Drug dose", "Statistical analysis", "Results", "Discussion", "Conclusions"], "sections": [["multilevel cervical spondylotic myelopathy ( mcsm ) is a serious disease which can lead to spinal cord dysfunction and a substantial decrease in quality of life . in addition , mcsm requiring multilevel cervical spine surgery can be accompanied by extensive blood loss . as spinal surgery has become increasingly complex , so control of perioperative bleeding has become an important clinical issue for spine surgeons .", "excessive blood loss can lead to a range of comorbidities such as anemia , hypotension , hematoma formation , and inadequate oxygenation of organs , thereby affecting patient outcome .", "excessive blood loss often requires allogeneic blood transfusion , the risks of which are numerous and among which immunological reactions and transmission of viruses are considered the most serious . in addition , hematoma formation within a few millimeters of the spinal canal can cause considerable neurological damage .", "although occurrences of postoperative spinal hematoma formation requiring emergency surgery are rare , it is nevertheless important to control perioperative bleeding to decrease its incidence . tranexamic acid ( txa ) is a synthetic derivative of the amino acid lysine which operates through competitive inhibition of the activation of plasminogen to plasmin by binding specific sites on both plasminogen and plasmin , thereby retarding fibrinolysis , the degradation of blood clots .", "it has been widely used in various medical fields such as cardiac surgery , gynecology , dentistry , urological surgery , and liver transplantation to reduce the perioperative blood loss , as it is relatively inexpensive and has not been cited in the literature as causing any significant untoward side effects . in addition , txa has been reported to reduce blood loss and transfusion requirements during orthopedic surgery , most commonly in knee and hip joint replacement . although the benefits of txa in spinal surgery have been reported", "indeed , there are no studies of txa in cervical laminectomy with lateral mass screw fixation and bone grafting ( clf ) so far .", "clf has been widely used for many years for treating mcsm caused by multilevel cervical spinal cord compression .", "although some authors suggest that laminoplasty is superior to laminectomy with lateral mass screw fixation and bone grafting regarding preserved range of motion , indications for the use of clf are broader than that of laminoplasty and recent studies showed good outcomes for clf . thus , the aim of this study is to evaluate the efficacy and safety of txa in controlling blood loss during clf for the treatment of mcsm ."], ["a retrospective comparative analysis was performed in patients with mcsm undergoing clf of vertebrae c3 to c6 .", "patients with cirrhosis of the liver , serious cardiac disease , chronic renal failure , cancer , allergy to txa , a history of thromboembolic disease ( deep vein thrombosis , ischemic heart disease , pulmonary embolism , transient ischemic attack , strokes , or subarachnoid hemorrhage ) , bleeding disorders , hypercoagulation status , disseminated intravascular coagulation , pregnancy , combined anterior and posterior spinal fusions , patients receiving antiplatelet and/or anticoagulant therapy at the time of the study , and treatments of vertebrae outside of c3 to c6 were excluded from the study . between november 2014 and april 2016 , 119 clfs fitting the inclusion criteria were performed in our center . each surgeon ( hao , liu , he , wu , wang , zheng , and zhao ) in this study had more than 20 years of experience in spinal surgery .", "the control group consisted of 46 patients who underwent clf for the treatment of mcsm in our institution without being given txa . in both groups", ", we analyzed patient demographic trait ( age at surgery , gender , body weight , height , and body mass index ) and duration of surgery which was defined as the time from the initial incision to the completion of wound closure .", "the quantity of intraoperative and postoperative blood loss and the preoperative and day 1 postoperative hematological data for each patient were obtained in the 2 groups .", "total blood loss was calculated as the sum of intraoperative and postoperative blood loss ( the quantity during the 1st 16  hours ) .", "the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision .", "laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) .", "the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material .", "lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique .", "all patients in the txa group were given a dose of 15  mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100  mg / hour , and continued until wound closure .", "no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug .", "continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value .", "statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables .", "all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) ."], ["the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision .", "laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) .", "the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material .", "lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique ."], ["all patients in the txa group were given a dose of 15  mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100  mg / hour , and continued until wound closure .", "no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug ."], ["continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value .", "statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables .", "all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) ."], ["there was no statistical difference in demographic values ( patients age , gender , body weight , height , and body mass index ) between the 2 groups ( table 1 ) .", "no statistically significant difference in duration of surgery was observed between the 2 groups , with a mean time of 155.72    15.59 and 153.52    11.91  minutes in the control and txa groups , respectively ( p  =  0.387 ) .", "there was significantly less intraoperative blood loss in the txa group compared to the control ( fig .", "1 ) , at 179.66    81.45 and 269.13    94.68  ml , respectively ( p  <  0.001 ) .", "the txa group had significantly less postoperative blood loss during the 1st 16  hours compared to the control group ( 108.08    44.31 vs 132.83    49.39  ml , p  =  0.005 ) .", "total blood loss in the control group ( 401.96    127.88  ml ) was significantly higher than that in the txa group ( 287.74    115.40  ml , p  <  0.001 ) .", "changes in blood hemoglobin ( hgb ) content and hematocrit ( hct ) were not statistically significantly different between the 2 groups : preoperative hgb :", "138.28    9.12  g / l ( control ) and 140.42    10.05  g / l ( txa ) ; postoperative hgb : 122.35    10.74  g / l ( control ) and 125.21    11.29  g / l ( txa ) ; preoperative hct : 41.78    2.62% ( control ) and 42.07    2.08% ( txa ) ; and postoperative hct : 36.50    3.22% ( control ) and 37.32    2.41% ( txa ) .", "although the hgb and hct values in the txa group were all higher than those of the control group , the differences were not statistically significant ( fig .", "no patient required an allogeneic blood transfusion during or after the surgery in either group and no serious intra- or postoperative complications , for example , dural tear , infection , epidural hematoma formation , deep - vein thrombosis , pulmonary embolism , allergic reaction , renal failure , or cardiopulmonary complications were observed in either group .", "furthermore , no minor side effects associated with the use of txa such as nausea , vomiting , headache , or diarrhea occurred in either of the groups . demographic parameters of patients in each group .", "the txa ( tranexamic acid ) group had significantly less intraoperative , postoperative , and total blood loss compared to the control group . there were no significantly different in blood hgb hct between the control and txa groups .", "hgb  =  hemoglobin , hct  =  content and hematocrit , txa  =  tranexamic acid ."], ["numerous studies have supported the use of txa in orthopedic surgery . although it has been evaluated in many studies , the safety and efficacy of txa for blood loss reduction in spinal surgery has not been clearly demonstrated , especially for clf .", "clf for the treatment of mcsm has proved to be a successful technique for restoring normal cervical lordotic alignment , recovering neurological function , and decreasing the morbidity of c5 palsy and axial pain .", "the principal disadvantage of this approach is a significant decrease in motion due to the fixation of the target vertebrae .", "laminoplasty allows a better range of neck motion although it becomes more restricted in flexion and extension over time . for patients with mcsm combined with simple instability or correctable kyphosis , clf is a more suitable method than laminoplasty , even if the consequence is neck stiffness .", "fibrinolysis increases transiently when patients undergo surgery , and it has been shown that it contributes to perioperative blood loss during spinal surgery .", "txa inhibits fibrinolysis by blocking the lysine - binding sites of plasminogen , plasmin , and tissue plasminogen activator .", "since fibrinolysis is activated immediately during and after surgery , txa should be administered before surgery begins .", "the half - life of txa is approximately 80 minutes in patients with normal renal function .", "pharmacokinetic evidence suggests a loading dose of 10 to 15  mg / kg followed by a maintenance dose of 1  mg / kg / hour or repeated dosing .", "li et al conducted a meta - analysis of 6 randomized controlled trials and believed that higher txa dosage ( 15 ", "yang et al conducted a meta - analysis of 9 randomized controlled trials which had a similar sample to li 's study .", "the 2 meta - analyses arrived at a similar conclusion although the quantity of blood loss and incidence of blood transfusion were different .", "elwatidy et al used a high single dose ( 30  mg / kg ) of txa and found that it reduced total blood loss and incidence of blood transfusion .", "raksakietisak et al reported that 2 doses of txa ( 15  mg / kg ) reduced perioperative blood loss and incidence of blood transfusion among low - risk adult patients undergoing elective complex thoracolumbar spine surgery .", ", we evaluated the effects of txa with a dose of 15  mg / kg followed by a maintenance dose of 100  mg / hour .", "the txa treatment group lost significantly less blood than that of the control group , including during the intra- and postoperative periods .", "total blood loss in the txa group was 28% less than that of the control group . although the hgb levels and hct in the txa group were higher than that of the control group , the differences were not significant , possibly because the sample size was too small and the perioperative blood loss in clf relatively minor .", "a theoretical anxiety associated with the use of txa is its potential for inducing thromboembolic complications .", "however , many studies have shown that the administration of txa does not increase this risk . in our study , there were no clinical symptoms or signs of thromboembolic events , such as deep - vein thrombosis or symptomatic pulmonary embolism .", "thus , the use of txa in patients undergoing clf should be regarded as safe .", "additionally , we are unsure whether the complexity of the surgery has an effect on the benefit of txa .", "a prospective randomized controlled study would better determine the safety and efficacy of txa during laminectomy with lateral mass screw fixation and bone grafting ."], ["in this study , blood loss ( both intra- and postoperative ) in the txa group was significantly lower than that in the control group , and no major intraoperative complications occurred .", "our results indicate that the use of intravenous txa is both safe and effective in reducing blood loss in clf .", "prophylactic txa may provide the benefit of limiting excessive blood loss in posterior approach cervical spinal surgery .", "a future prospective randomized controlled trial will provide superior evidence of the efficacy and safety of txa ."]]}
{"article_id": "PMC3103949", "article_text": ["hip fracture is a moderate musculoskeletal trauma that mainly affects the older population with comorbid conditions .", "the number will increase markedly in coming years due to the ageing of the population .", "comorbidity and the double trauma may dispose them to serious postoperative adverse outcomes and a high mortality dominated by cardiovascular events [ 25 ] .", "myocardial injury may be difficult to diagnose because of impaired communication , limitations of clinical manifestation and non - specific electrocardiographic ( ecg ) changes [ 6 , 7 ] .", "the isoenzyme myocardium - specific creatine kinase ( ck - mb ) is expressed in the myocardium and ck in the skeletal muscle cells .", "these two enzymes have traditionally been analysed in plasma to distinguish myocardial injury and skeletal muscle injury [ 8 , 9 ] .", "troponins have been shown to be more specific and sensitive to cardiac injury [ 10 , 11 ] .", "increased plasma levels of troponin have also been reported in pulmonary embolism , septicaemia and following major orthopaedic and cardiac surgery . in those conditions", "high plasma troponin levels have been associated with severe adverse outcomes and increased mortality [ 7 , 1215 ] . in this study we wanted to test the hypothesis that fatal outcome following hip fracture", "the study was approved by the regional ethics committee and national medical authorities and conducted in accordance with the helsinki declaration .", "a total of 302 consecutive patients over 75  years of age with dislocated hip fractures were enrolled in the study at elverum ( ech ) and buskerud ( bch ) central hospitals during the period 20052009 .", "comorbidity was routinely assessed according to the classification of the american society of anesthesiologists ( asa ) .", "a hemiprosthesis was inserted through a lateral incision and fixed with or without bone cement ( landos titan or landos corail , depuy , warsaw , in , usa ) .", "thromboprophylaxis ( low molecular weight heparins ) was administered routinely preoperatively ( on hospital admission ) with dalteparin 5000  iu s.c .", "blood samples were obtained from an antecubital vein , collected preoperatively ( 1 ) and postoperatively [ within 24  hours ( 0 ) ] and on days one [ 2448  h ( + 1 ) ] and four ( + 4 ) .", "troponin t ( tnt ) and ck - mb were measured by electrochemiluminescence immunoassay ( elica , roche , basel , switzerland and abbott , abbott park , il , usa ) .", "ck was measured by absorption photometry ( roche , basel , switzerland and abbott , abbott park , il , usa ) .", "values for tnt were only measured at the ech study centre ( n  =  146 ) .", "differences between mortality groups were tested using a two - sample t test or chi - square test for continuous or categorical data , respectively .", "non - parametric mann - whitney tests were done for tnt due to skewed distribution . a linear mixed model for repeated measurements with a random intercept and bonferroni adjusted pairwise post hoc comparisons were used to analyse the biochemical markers with respect to mortality and repeated venous blood analyses .", "univariate and stepwise multivariate logistic regression analyses were used to estimate unadjusted and adjusted odds ratios with respect to survival at the three month follow - up .", "results from multivariate logistic regression were based on models including only significant terms from stepwise regression to maximise the number of patients included .", "there were 229 women and 72 men , with a mean age of 84.7 ( sd 5.1 ) and 83.7 ( sd 4.7 ) years , respectively . by three  months , 59 of 302 ( 19.5% ) had died , 62% women and 38% men . \n", "table  1demographic and clinical characteristics of patients at 3-month follow - upcharacteristicsalive ( n  =  243)dead ( n  =  59)p valuesage , years ( mean  sd)84.1 ( 5.1)86.2 ( 4.6)0.004sex  female193 ( 79.4%)37 ( 62.7%)0.005  male50 ( 20.6%)22 ( 37.3%)mobility  not mobile37 ( 15.8%)10 ( 17.2%)0.109  living aid23 ( 9.8%)5 ( 8.6% )  crutches51 ( 21.8%)21 ( 36.2% )  no aid123 ( 52.6%)22 ( 37.9%)asa score  i10 ( 4.2%)0 ( 0.0%)<0.001  ii103 ( 42.9%)9 ( 15.3% )  iii116 ( 48.3%)38 ( 64.4% )  iv11 ( 4.6%)12 ( 20.3%)asa american society of anesthesiologistsminor deviations between total number of patients in categories compared to number alive or dead are due to missing data .", "missing data are assumed to be completely at random demographic and clinical characteristics of patients at 3-month follow - up asa american society of anesthesiologists minor deviations between total number of patients in categories compared to number alive or dead are due to missing data .", "missing data are assumed to be completely at random asa , male sex and age were significantly associated with mortality within three  months ( table  1 ) . within one  day after surgery , the plasma levels of ck and ck - mb increased nearly threefold ( inverse for the ratio ) . on days 1 and + 1 , ck and ck - mb values were significantly higher among those who died compared to those who survived ( p  =  0.001 and p  =  0.031 , respectively ) ( fig .  1 ) .", "on the fourth postoperative day , tnt plasma levels rose twofold in the mortality group and remained unchanged in the alive group .", "the plasma levels were significantly higher ( p  <  0.05 ) in the mortality group at all sampling times except at day 0 ( fig . ", "1mean and 95% confidence intervals for troponin t , creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and ratio of ck - mb to ck before surgery , perioperatively and 1 and 4  days after surgery for 3-month mortality . statistical significance ( p  <  0.05 ) between mortality groups is indicated by an asterisk .", "statistically significant ( p  <  0.05 ) differences between before surgery and during follow - up are indicated by the connecting lines .", "day 1  1  day before surgery , day 0 within 24  h after the operation , day + 1 2448  h postoperatively , day + 4 4  days after surgery mean and 95% confidence intervals for troponin t , creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and ratio of ck - mb to ck before surgery , perioperatively and 1 and 4  days after surgery for 3-month mortality . statistical significance", "( p  <  0.05 ) between mortality groups is indicated by an asterisk . statistically significant ( p  <  0.05 )", "differences between before surgery and during follow - up are indicated by the connecting lines .", "day 1  1  day before surgery , day 0 within 24  h after the operation , day + 1 2448  h postoperatively , day + 4 4  days after surgery the tnt plasma concentrations were split into three equal - sized data subsets , i.e. 0.01 , 0.010.04 and > 0.04 ", "g / l , and the ratios of ck - mb to ck concentrations were split into two equal - sized data subsets , i.e. below and above 0.02 .", "/ l before surgery correlated with three month mortality [ odds ratio ( or ) 10.9 , 95% confidence interval ( ci ) 2.254.0 , p  =  0.003 ] ( table  2 ) .", "stepwise multivariate logistic regression with age , sex , asa category , levels of tnt and ratio of ck - mb to ck concentrations were performed .", "high tnt plasma concentration was associated with increased mortality ( or 6.1 95% ci 623.1 , p  =  0.008 ) at day four after surgery .", "no statistically significant association was found for the ratio of ck - mb to ck during the entire sampling period when adjusted for age , sex and asa ( table  3 ) .", "similar regression analyses were done for ck and ck - mb . on day + 1 , the ck - mb was associated with mortality ( or 1.1 , 95% ci 1.021.2 , p  =  0.012 ) .", "we found that our hypothesis , stating no predictive value from cardio - muscular plasma enzymes with regard to early mortality in patients with hip fracture , was false . \n", "table  2results from univariate logistic regression models predicting 3-month mortality ( death)variablesunadjusted or ( 95% ci)p valuesage1.1 ( 1.01.1)0.004if male2.4 ( 1.34.4)0.006asa3.7 ( 2.26.1)<0.001if ck - mb / ck > 0.02  day 11.6 ( 0.83.4)0.225  day 00.7 ( 0.31.6)0.413  day + 11.5 ( 0.82.9)0.239  day + 41.2 ( 0.62.4)0.620tnt  day 10.011.0 ( reference)0.010.041.5 ( 0.46.0)0.532>0.0411.0 ( 2.254.6)0.0030.011.0 ( reference )  day 00.010.041.5 ( 0.45.4)0.556>0.042.9 ( 0.711.0)0.1270.011.0 ( reference )  day + 10.010.041.6 ( 0.47.4)0.512>0.046.0 ( 1.721.1)0.0050.011.0 ( reference )  day + 40.010.043.3 ( 0.715.0)0.126>0.047.0 ( 1.925.6)0.003or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgerytable  3results from multivariate stepwise logistic regression models predicting 3-month mortality ( death)variablesday 1day 0day + 1day + 4adjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesage1.1 ( 1.01.2)0.0061.1 ( 1.01.2)0.0061.1 ( 1.01.2)0.0011.1 ( 1.01.2)0.027if male2.4 ( 1.24.6)0.0132.4 ( 1.24.60.0132.7 ( 1.45.1)0.002asa3.3 ( 2.05.6)<0.0013.3 ( 2.05.6)<0.001ck - mb / cktnt   0.011.0 ( reference)  0.010.041.9 ( 0.49.4)0.450  > 0.046.1 ( 1.623.1)0.008or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgery results from univariate logistic regression models predicting 3-month mortality ( death ) or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgery results from multivariate stepwise logistic regression models predicting 3-month mortality ( death ) or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgery", "this prospective study on 302 elderly patients with hip fracture disclosed that 19.5% had died within three  months .", "preoperatively obtained basic patient information was shown to be of particular importance to assess the risk of postoperative mortality .", "asa score on comorbidity , male sex and age correlated significantly with three month mortality , also described by other investigators [ 1719 ] .", "autopsy studies have shown that cardiovascular events are the main cause of death after hip fracture surgery [ 20 , 21 ] .", "autopsies are rarely done today and the direct cause of postoperative death has not been possible to establish in this or in other recently conducted studies . in the elderly perioperative myocardial ischaemia", "is often clinically silent , without haemodynamic or notable ecg changes [ 2225 ] . over the years , biochemical plasma markers", "have been used to distinguish myocardial injury from skeletal muscle damage , an approach also adopted in this study .", "potential heart muscle damage was investigated in this study with ck - mb and tnt analyses .", "we found small and inconsistent differences for ck and ck - mb plasma levels between those who died and those who survived .", "the ratio of ck - mb to ck was decreased from hospital admission ( day 1 ) to day 0 and stabilised , indicating that skeletal muscle damage dominated and that any myocardial injury remained undetected .", "this analysis suggests that ck and ck - mb are unspecific enzymes that are not distinct for skeletal and cardiac muscle damage following a hip fracture and are not feasible as prognostic markers of mortality , a finding consistent with other investigators .", "release of troponins into the circulation is considered to specifically reflect cardiac injury . in our study", "we analysed tnt and found that the plasma concentration was significantly higher on the fourth postoperative day in patients that subsequently died compared to those who were alive .", "g / l had a six times higher risk of dying vs those with normal plasma levels .", "this calculation was robust when correction was done for age , sex and comorbidity ( asa score ) .", "these results fit with other reports that showed a second wave of troponin elevation several days after surgery which correlated with postoperative mortality [ 13 , 29 ] . in summary", ", this study showed that basic clinical information on sex , age and comorbidity ( asa score ) and a high postoperative plasma concentration of tnt > 0.04  g", "/ l are robust predictors of three month postoperative mortality in the elderly undergoing hip fracture surgery .", "this information may be of importance for therapeutic and post - hospital health care intervention ."], "abstract_text": ["<S> hip fracture , a moderate musculoskeletal trauma , is associated with a high postoperative mortality . </S>", "<S> most patients are elderly , with comorbid conditions and often with heart disease . </S>", "<S> the objective of this study was to find out if clinical parameters and analyses of specific muscle enzymes could predict three month postoperative mortality . </S>", "<S> a total of 302 patients above 75  years of age with hip fracture were consecutively enrolled . </S>", "<S> baseline information on age , sex and comorbidity assessed with the american society of anesthesiologists ( asa ) score was obtained before surgery . </S>", "<S> creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and troponin t ( tnt ) were analysed from venous blood , collected the day before surgery ( 1 ) and postoperatively , within 24  hours ( 0 ) and on days one ( + 1 ) and four ( + 4 ) . </S>", "<S> the overall three month mortality was 19.5% . </S>", "<S> multivariate analyses showed that age , male sex and comorbidity ( asa ) correlated with mortality ( p  =  0.027 , p  =  0.002 , p  <  0.001 , respectively ) . </S>", "<S> surgery induced a two- to threefold increase of ck and ck - mb but without any correlation with mortality . </S>", "<S> however , high tnt levels > 0.04  g / l correlated significantly with death ( days 1 , + 1 and + 4 , p  =  0.003 , p  =  0.005 and p  =  0.003 , respectively ) . </S>", "<S> multivariate analyses , adjusted for age , sex and asa category , confirmed this correlation ( day + 4 , p  =  0.008 ) . </S>", "<S> thus , in elderly patients with comorbidities undergoing hip fracture surgery information on sex , age , asa category and postoperative laboratory analyses on tnt provide the clinicians with useful information on patients at risk of fatal outcome . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Results", "Discussion"], "sections": [["hip fracture is a moderate musculoskeletal trauma that mainly affects the older population with comorbid conditions .", "the number will increase markedly in coming years due to the ageing of the population .", "comorbidity and the double trauma may dispose them to serious postoperative adverse outcomes and a high mortality dominated by cardiovascular events [ 25 ] .", "myocardial injury may be difficult to diagnose because of impaired communication , limitations of clinical manifestation and non - specific electrocardiographic ( ecg ) changes [ 6 , 7 ] .", "the isoenzyme myocardium - specific creatine kinase ( ck - mb ) is expressed in the myocardium and ck in the skeletal muscle cells .", "these two enzymes have traditionally been analysed in plasma to distinguish myocardial injury and skeletal muscle injury [ 8 , 9 ] .", "troponins have been shown to be more specific and sensitive to cardiac injury [ 10 , 11 ] .", "increased plasma levels of troponin have also been reported in pulmonary embolism , septicaemia and following major orthopaedic and cardiac surgery . in those conditions", "high plasma troponin levels have been associated with severe adverse outcomes and increased mortality [ 7 , 1215 ] . in this study we wanted to test the hypothesis that fatal outcome following hip fracture"], ["the study was approved by the regional ethics committee and national medical authorities and conducted in accordance with the helsinki declaration .", "a total of 302 consecutive patients over 75  years of age with dislocated hip fractures were enrolled in the study at elverum ( ech ) and buskerud ( bch ) central hospitals during the period 20052009 .", "comorbidity was routinely assessed according to the classification of the american society of anesthesiologists ( asa ) .", "a hemiprosthesis was inserted through a lateral incision and fixed with or without bone cement ( landos titan or landos corail , depuy , warsaw , in , usa ) .", "thromboprophylaxis ( low molecular weight heparins ) was administered routinely preoperatively ( on hospital admission ) with dalteparin 5000  iu s.c .", "blood samples were obtained from an antecubital vein , collected preoperatively ( 1 ) and postoperatively [ within 24  hours ( 0 ) ] and on days one [ 2448  h ( + 1 ) ] and four ( + 4 ) .", "troponin t ( tnt ) and ck - mb were measured by electrochemiluminescence immunoassay ( elica , roche , basel , switzerland and abbott , abbott park , il , usa ) .", "ck was measured by absorption photometry ( roche , basel , switzerland and abbott , abbott park , il , usa ) .", "values for tnt were only measured at the ech study centre ( n  =  146 ) .", "differences between mortality groups were tested using a two - sample t test or chi - square test for continuous or categorical data , respectively .", "non - parametric mann - whitney tests were done for tnt due to skewed distribution . a linear mixed model for repeated measurements with a random intercept and bonferroni adjusted pairwise post hoc comparisons were used to analyse the biochemical markers with respect to mortality and repeated venous blood analyses .", "univariate and stepwise multivariate logistic regression analyses were used to estimate unadjusted and adjusted odds ratios with respect to survival at the three month follow - up .", "results from multivariate logistic regression were based on models including only significant terms from stepwise regression to maximise the number of patients included ."], ["there were 229 women and 72 men , with a mean age of 84.7 ( sd 5.1 ) and 83.7 ( sd 4.7 ) years , respectively . by three  months , 59 of 302 ( 19.5% ) had died , 62% women and 38% men . \n", "table  1demographic and clinical characteristics of patients at 3-month follow - upcharacteristicsalive ( n  =  243)dead ( n  =  59)p valuesage , years ( mean  sd)84.1 ( 5.1)86.2 ( 4.6)0.004sex  female193 ( 79.4%)37 ( 62.7%)0.005  male50 ( 20.6%)22 ( 37.3%)mobility  not mobile37 ( 15.8%)10 ( 17.2%)0.109  living aid23 ( 9.8%)5 ( 8.6% )  crutches51 ( 21.8%)21 ( 36.2% )  no aid123 ( 52.6%)22 ( 37.9%)asa score  i10 ( 4.2%)0 ( 0.0%)<0.001  ii103 ( 42.9%)9 ( 15.3% )  iii116 ( 48.3%)38 ( 64.4% )  iv11 ( 4.6%)12 ( 20.3%)asa american society of anesthesiologistsminor deviations between total number of patients in categories compared to number alive or dead are due to missing data .", "missing data are assumed to be completely at random demographic and clinical characteristics of patients at 3-month follow - up asa american society of anesthesiologists minor deviations between total number of patients in categories compared to number alive or dead are due to missing data .", "missing data are assumed to be completely at random asa , male sex and age were significantly associated with mortality within three  months ( table  1 ) . within one  day after surgery , the plasma levels of ck and ck - mb increased nearly threefold ( inverse for the ratio ) . on days 1 and + 1 , ck and ck - mb values were significantly higher among those who died compared to those who survived ( p  =  0.001 and p  =  0.031 , respectively ) ( fig .  1 ) .", "on the fourth postoperative day , tnt plasma levels rose twofold in the mortality group and remained unchanged in the alive group .", "the plasma levels were significantly higher ( p  <  0.05 ) in the mortality group at all sampling times except at day 0 ( fig . ", "1mean and 95% confidence intervals for troponin t , creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and ratio of ck - mb to ck before surgery , perioperatively and 1 and 4  days after surgery for 3-month mortality . statistical significance ( p  <  0.05 ) between mortality groups is indicated by an asterisk .", "statistically significant ( p  <  0.05 ) differences between before surgery and during follow - up are indicated by the connecting lines .", "day 1  1  day before surgery , day 0 within 24  h after the operation , day + 1 2448  h postoperatively , day + 4 4  days after surgery mean and 95% confidence intervals for troponin t , creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and ratio of ck - mb to ck before surgery , perioperatively and 1 and 4  days after surgery for 3-month mortality . statistical significance", "( p  <  0.05 ) between mortality groups is indicated by an asterisk . statistically significant ( p  <  0.05 )", "differences between before surgery and during follow - up are indicated by the connecting lines .", "day 1  1  day before surgery , day 0 within 24  h after the operation , day + 1 2448  h postoperatively , day + 4 4  days after surgery the tnt plasma concentrations were split into three equal - sized data subsets , i.e. 0.01 , 0.010.04 and > 0.04 ", "g / l , and the ratios of ck - mb to ck concentrations were split into two equal - sized data subsets , i.e. below and above 0.02 .", "/ l before surgery correlated with three month mortality [ odds ratio ( or ) 10.9 , 95% confidence interval ( ci ) 2.254.0 , p  =  0.003 ] ( table  2 ) .", "stepwise multivariate logistic regression with age , sex , asa category , levels of tnt and ratio of ck - mb to ck concentrations were performed .", "high tnt plasma concentration was associated with increased mortality ( or 6.1 95% ci 623.1 , p  =  0.008 ) at day four after surgery .", "no statistically significant association was found for the ratio of ck - mb to ck during the entire sampling period when adjusted for age , sex and asa ( table  3 ) .", "similar regression analyses were done for ck and ck - mb . on day + 1 , the ck - mb was associated with mortality ( or 1.1 , 95% ci 1.021.2 , p  =  0.012 ) .", "we found that our hypothesis , stating no predictive value from cardio - muscular plasma enzymes with regard to early mortality in patients with hip fracture , was false . \n", "table  2results from univariate logistic regression models predicting 3-month mortality ( death)variablesunadjusted or ( 95% ci)p valuesage1.1 ( 1.01.1)0.004if male2.4 ( 1.34.4)0.006asa3.7 ( 2.26.1)<0.001if ck - mb / ck > 0.02  day 11.6 ( 0.83.4)0.225  day 00.7 ( 0.31.6)0.413  day + 11.5 ( 0.82.9)0.239  day + 41.2 ( 0.62.4)0.620tnt  day 10.011.0 ( reference)0.010.041.5 ( 0.46.0)0.532>0.0411.0 ( 2.254.6)0.0030.011.0 ( reference )  day 00.010.041.5 ( 0.45.4)0.556>0.042.9 ( 0.711.0)0.1270.011.0 ( reference )  day + 10.010.041.6 ( 0.47.4)0.512>0.046.0 ( 1.721.1)0.0050.011.0 ( reference )  day + 40.010.043.3 ( 0.715.0)0.126>0.047.0 ( 1.925.6)0.003or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgerytable  3results from multivariate stepwise logistic regression models predicting 3-month mortality ( death)variablesday 1day 0day + 1day + 4adjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesage1.1 ( 1.01.2)0.0061.1 ( 1.01.2)0.0061.1 ( 1.01.2)0.0011.1 ( 1.01.2)0.027if male2.4 ( 1.24.6)0.0132.4 ( 1.24.60.0132.7 ( 1.45.1)0.002asa3.3 ( 2.05.6)<0.0013.3 ( 2.05.6)<0.001ck - mb / cktnt   0.011.0 ( reference)  0.010.041.9 ( 0.49.4)0.450  > 0.046.1 ( 1.623.1)0.008or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgery results from univariate logistic regression models predicting 3-month mortality ( death ) or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgery results from multivariate stepwise logistic regression models predicting 3-month mortality ( death ) or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1  day before surgery , day 0 within 24  h after surgery , day + 1  2448  h postoperatively , day + 4 4  days after surgery"], ["this prospective study on 302 elderly patients with hip fracture disclosed that 19.5% had died within three  months .", "preoperatively obtained basic patient information was shown to be of particular importance to assess the risk of postoperative mortality .", "asa score on comorbidity , male sex and age correlated significantly with three month mortality , also described by other investigators [ 1719 ] .", "autopsy studies have shown that cardiovascular events are the main cause of death after hip fracture surgery [ 20 , 21 ] .", "autopsies are rarely done today and the direct cause of postoperative death has not been possible to establish in this or in other recently conducted studies . in the elderly perioperative myocardial ischaemia", "is often clinically silent , without haemodynamic or notable ecg changes [ 2225 ] . over the years , biochemical plasma markers", "have been used to distinguish myocardial injury from skeletal muscle damage , an approach also adopted in this study .", "potential heart muscle damage was investigated in this study with ck - mb and tnt analyses .", "we found small and inconsistent differences for ck and ck - mb plasma levels between those who died and those who survived .", "the ratio of ck - mb to ck was decreased from hospital admission ( day 1 ) to day 0 and stabilised , indicating that skeletal muscle damage dominated and that any myocardial injury remained undetected .", "this analysis suggests that ck and ck - mb are unspecific enzymes that are not distinct for skeletal and cardiac muscle damage following a hip fracture and are not feasible as prognostic markers of mortality , a finding consistent with other investigators .", "release of troponins into the circulation is considered to specifically reflect cardiac injury . in our study", "we analysed tnt and found that the plasma concentration was significantly higher on the fourth postoperative day in patients that subsequently died compared to those who were alive .", "g / l had a six times higher risk of dying vs those with normal plasma levels .", "this calculation was robust when correction was done for age , sex and comorbidity ( asa score ) .", "these results fit with other reports that showed a second wave of troponin elevation several days after surgery which correlated with postoperative mortality [ 13 , 29 ] . in summary", ", this study showed that basic clinical information on sex , age and comorbidity ( asa score ) and a high postoperative plasma concentration of tnt > 0.04  g", "/ l are robust predictors of three month postoperative mortality in the elderly undergoing hip fracture surgery .", "this information may be of importance for therapeutic and post - hospital health care intervention ."]]}
{"article_id": "PMC2992422", "article_text": ["a young man was referred with a chief complaint of blurred vision and pain of the left eye 1 month after herpetic encephalitis in the left frontotemporal lobe .", "the patient had multiple foci of retinitis in the retinal periphery associated with vitritis , blot retinal hemorrhage and retinal arteriolitis .", "the impression of acute retinal necrosis was confirmed by polymerase chain reaction of aqueous humor by detecting herpes simplex virus type 2 ; therefore , the patient received intravenous acyclovir .", "herpetic encephalitis may be a risk factor for acute retinal necrosis . the virus may reach the eye by the trans - axonal route .", "acute retinal necrosis ( arn ) is one of the clinical presentations of herpes simplex virus ( hsv ) .", "this disease may present several years after a primary infection , or it may occur following systemic herpetic infection such as herpetic dermatitis . the prevalence of the disease is equal in both sexes and it occurs in the 5th-7th decade of life .", "american uveitis society criteria for the diagnosis of arn syndrome are : one or more foci of retinal necrosis with discrete borders located in the peripheral retina , rapid progression in the absence of antiviral therapy , circumferential spread , occlusive vasculopathy with arteriolar involvement , prominent vitritis , and anterior chamber inflammation .", "optic neuropathy , scleritis , and pain are supportive but not required . in most patients , the diagnosis is made clinically .", "polymerase chain reaction ( pcr ) analysis of the aqueous humor can detect the cause of arn specifically .", "pcr is very helpful in the detection of the varicella zoster , hsv type 1 and 2 .", "patients with arn due to hsv-1 and varicella zoster virus tend to be older , whereas those with arn due to hsv-2 tend to be younger [ 3 , 4 ] . in this article , we report one case of unilateral arn 1 month after the herpetic encephalitis .", "the patient is a 25-year - old man who was admitted with a chief complaint of decreased vision and pain in left eye for the previous 4 days .", "the patient also complained of perception of floating objects in the visual field of the left eye .", "the patient had no past medical history indicating an immunocompromised state ; he had been admitted to the neurology ward due to herpetic encephalitis 1 month previous to the occurrence of visual symptoms .", "herpetic encephalitis diagnosis was made by brain mri ( fig . 1 ) and pcr analysis of the cerebrospinal fluid .", "he did not report any underlying disease or drug consumption except oral acyclovir 800 mg every 8 h. at physical examination , the right eye was normal . in the left eye ,", "visual acuity was 0.1 , papillary reflex was normal and marcus gunn sign was negative .", "slit lamp examination of the left eye revealed conjunctival hyperemia and ciliary injection , diffuse fine keratic precipitates in the corneal endothelium , 3 + cellular reaction in the anterior chamber , 3 + cellular reaction in the vitreous without snowball or snowbank opacities , or vitreous hemorrhage . in fundoscopic examination , multiple foci of retinitis and retinal necrosis", "2 ) associated with arteriolar involvement ( arteriolitis ) , blot retinal hemorrhage and optic disc hyperemia .", "pcr analysis of aqueous humor revealed hsv-2 , so the patient was admitted with the diagnosis of arn and received intravenous acyclovir for 10 days ( 10 mg / kg / day ) .", "after 48 h of antiviral therapy , a systemic corticosteroid ( prednisolone 1 mg / kg / day ) was introduced and subsequently tapered over several weeks to treat active inflammation .", "the patient also received aspirin to treat an associated hypercoagulable state . with this treatment , no new lesion developed and opacity of the media decreased ; so prophylactic barrier laser photocoagulation in the areas of healthy retina at the posterior borders of the necrotic lesion prevented retinal detachment .", "intravitreal antiviral agents such as ganciclovir and foscarnet were not used in this patient because the disease was controlled by intravenous acyclovir .", "after discharge from hospital , oral acyclovir 400 mg every 5 h was started . during follow - up ,", "the inflammation decreased and the patient had no vitreous traction or retinal detachment or other eye involvement .", "the brain and retinal involvement were on one side ; after 1 month of left frontotemporal lobe involvement of the brain , the retina was involved on the left side .", "some of the studies report arn several years after neonatal and infantile herpetic encephalitis [ 5 , 6 ] ; therefore , herpetic encephalitis may be a risk factor for arn development .", "other articles report that the virus accesses the retina from the brain by the trans - axonal route ; consequently , this virus can cause recurrent episodes of arn [ 8 , 9 ] . these reports may necessitate a prophylaxis with antiviral agents after herpetic encephalitis to prevent arn . in a study by pavsio et al . , the role of long - term prophylaxis with acyclovir in children who had herpetic encephalitis is an important issue .", "prophylaxis could be considered after hsv encephalitis to prevent arn or after arn to prevent second eye involvement .", "acyclovir used as prophylaxis for recurrent genital hsv infection in adults for 5 or more years has been associated with minimal toxicity and the selection of resistant strains has not been demonstrated , but there is little experience with the duration of prophylaxis that would have been necessary to prevent arn in our patient . with this report", "we reemphasize the correlation between these two diseases . on the other hand , arn may be an indication of possible central nervous system involvement and neuroimaging may be necessary in all cases of arn to rule out herpetic encephalitis ."], "abstract_text": ["<S> purposeto report a case of unilateral acute retinal necrosis after herpetic encephalitiscase reporta young man was referred with a chief complaint of blurred vision and pain of the left eye 1 month after herpetic encephalitis in the left frontotemporal lobe . </S>", "<S> the patient had multiple foci of retinitis in the retinal periphery associated with vitritis , blot retinal hemorrhage and retinal arteriolitis . </S>", "<S> the impression of acute retinal necrosis was confirmed by polymerase chain reaction of aqueous humor by detecting herpes simplex virus type 2 ; therefore , the patient received intravenous acyclovir.conclusionherpetic encephalitis may be a risk factor for acute retinal necrosis . </S>", "<S> the virus may reach the eye by the trans - axonal route . </S>", "<S> prophylaxis with acyclovir may be necessary after herpetic encephalitis to prevent acute retinal necrosis . </S>"], "labels": null, "section_names": ["Purpose", "Case Report", "Conclusion", "Introduction", "Case Report", "Discussion"], "sections": [[""], ["a young man was referred with a chief complaint of blurred vision and pain of the left eye 1 month after herpetic encephalitis in the left frontotemporal lobe .", "the patient had multiple foci of retinitis in the retinal periphery associated with vitritis , blot retinal hemorrhage and retinal arteriolitis .", "the impression of acute retinal necrosis was confirmed by polymerase chain reaction of aqueous humor by detecting herpes simplex virus type 2 ; therefore , the patient received intravenous acyclovir ."], ["herpetic encephalitis may be a risk factor for acute retinal necrosis . the virus may reach the eye by the trans - axonal route ."], ["acute retinal necrosis ( arn ) is one of the clinical presentations of herpes simplex virus ( hsv ) .", "this disease may present several years after a primary infection , or it may occur following systemic herpetic infection such as herpetic dermatitis . the prevalence of the disease is equal in both sexes and it occurs in the 5th-7th decade of life .", "american uveitis society criteria for the diagnosis of arn syndrome are : one or more foci of retinal necrosis with discrete borders located in the peripheral retina , rapid progression in the absence of antiviral therapy , circumferential spread , occlusive vasculopathy with arteriolar involvement , prominent vitritis , and anterior chamber inflammation .", "optic neuropathy , scleritis , and pain are supportive but not required . in most patients , the diagnosis is made clinically .", "polymerase chain reaction ( pcr ) analysis of the aqueous humor can detect the cause of arn specifically .", "pcr is very helpful in the detection of the varicella zoster , hsv type 1 and 2 .", "patients with arn due to hsv-1 and varicella zoster virus tend to be older , whereas those with arn due to hsv-2 tend to be younger [ 3 , 4 ] . in this article , we report one case of unilateral arn 1 month after the herpetic encephalitis ."], ["the patient is a 25-year - old man who was admitted with a chief complaint of decreased vision and pain in left eye for the previous 4 days .", "the patient also complained of perception of floating objects in the visual field of the left eye .", "the patient had no past medical history indicating an immunocompromised state ; he had been admitted to the neurology ward due to herpetic encephalitis 1 month previous to the occurrence of visual symptoms .", "herpetic encephalitis diagnosis was made by brain mri ( fig . 1 ) and pcr analysis of the cerebrospinal fluid .", "he did not report any underlying disease or drug consumption except oral acyclovir 800 mg every 8 h. at physical examination , the right eye was normal . in the left eye ,", "visual acuity was 0.1 , papillary reflex was normal and marcus gunn sign was negative .", "slit lamp examination of the left eye revealed conjunctival hyperemia and ciliary injection , diffuse fine keratic precipitates in the corneal endothelium , 3 + cellular reaction in the anterior chamber , 3 + cellular reaction in the vitreous without snowball or snowbank opacities , or vitreous hemorrhage . in fundoscopic examination , multiple foci of retinitis and retinal necrosis", "2 ) associated with arteriolar involvement ( arteriolitis ) , blot retinal hemorrhage and optic disc hyperemia .", "pcr analysis of aqueous humor revealed hsv-2 , so the patient was admitted with the diagnosis of arn and received intravenous acyclovir for 10 days ( 10 mg / kg / day ) .", "after 48 h of antiviral therapy , a systemic corticosteroid ( prednisolone 1 mg / kg / day ) was introduced and subsequently tapered over several weeks to treat active inflammation .", "the patient also received aspirin to treat an associated hypercoagulable state . with this treatment , no new lesion developed and opacity of the media decreased ; so prophylactic barrier laser photocoagulation in the areas of healthy retina at the posterior borders of the necrotic lesion prevented retinal detachment .", "intravitreal antiviral agents such as ganciclovir and foscarnet were not used in this patient because the disease was controlled by intravenous acyclovir .", "after discharge from hospital , oral acyclovir 400 mg every 5 h was started . during follow - up ,", "the inflammation decreased and the patient had no vitreous traction or retinal detachment or other eye involvement ."], ["the brain and retinal involvement were on one side ; after 1 month of left frontotemporal lobe involvement of the brain , the retina was involved on the left side .", "some of the studies report arn several years after neonatal and infantile herpetic encephalitis [ 5 , 6 ] ; therefore , herpetic encephalitis may be a risk factor for arn development .", "other articles report that the virus accesses the retina from the brain by the trans - axonal route ; consequently , this virus can cause recurrent episodes of arn [ 8 , 9 ] . these reports may necessitate a prophylaxis with antiviral agents after herpetic encephalitis to prevent arn . in a study by pavsio et al . , the role of long - term prophylaxis with acyclovir in children who had herpetic encephalitis is an important issue .", "prophylaxis could be considered after hsv encephalitis to prevent arn or after arn to prevent second eye involvement .", "acyclovir used as prophylaxis for recurrent genital hsv infection in adults for 5 or more years has been associated with minimal toxicity and the selection of resistant strains has not been demonstrated , but there is little experience with the duration of prophylaxis that would have been necessary to prevent arn in our patient . with this report", "we reemphasize the correlation between these two diseases . on the other hand , arn may be an indication of possible central nervous system involvement and neuroimaging may be necessary in all cases of arn to rule out herpetic encephalitis ."]]}
{"article_id": "PMC153459", "article_text": ["inspection of the database of clusters of orthologous groups of proteins ( cogs )   revealed only one family of such proteins that is represented in most of the sequenced bacterial , archaeal and eukaryotic genomes .", "the prototype of this family is the rhomboid ( rho ) protein from drosophila melanogaster , a developmental regulator involved in epidermal growth factor ( egf)-dependent signaling pathways [ 2 - 4 ] .", "not only were homologs of rhomboid detected in prokaryotes and eukaryotes , but the pattern of sequence conservation in this family appeared uncharacteristic of nonenzymatic membrane proteins , such as transporters .", "specifically , several polar amino - acid residues are conserved in nearly all members of the rhomboid family , suggesting the possibility of an enzymatic activity . as three of these conserved residues were histidines , it has been hypothesized that rhomboid - family proteins could function as metal - dependent membrane proteases .", "recently , however , it has been shown that rho cleaves a transmembrane helix ( tmh ) in the membrane - bound precursor of the tgf-like growth factor spitz , enabling the released spitz to activate the egf receptor , and that a conserved serine and a conserved histidine in rho are essential for this cleavage .", "thus , it appears that rhomboid - family proteins are a distinct group of intramembrane serine proteases .", "altogether , the genome of drosophila encodes seven rho paralogs ( now designated rho1 - 7 , with the original rhomboid becoming rho-1 ) , at least three of which are involved in distinct egf - dependent pathways , apparently through proteolytic activation of diverse ligands of the egf receptor .", "the newly discovered intramembrane proteolytic activity of rho places the rhomboid family within the framework of regulated intramembrane proteolysis ( rip ) , a new paradigm of signal transduction , which appears to be prominent in all forms of life . under rip , signaling proteins undergo site - specific proteolysis within tmh , resulting in the release of active fragments , which are the actual effectors in signal tranduction cascades . until recently", ", the only characterized cases of rip in eukaryotes involved presenilin-1 , an aspartyl protease , which cleaves a transmembrane helix in type-1 membrane proteins such as amyloid -precursor protein ( app ) , notch and ire1 , and the metalloprotease s2p , which cleaves a tmh in a type-2 transmembrane protein , the sterol - dependent transcription factor srebp .", "notably , s2p has highly conserved bacterial homologs , and the protease domain of presenilins also might be homologous to bacterial and archaeal type iv prepilin peptidases , although , in this case , the sequence similarity is low . in the case of the rhomboid family ,", "the existence of homologs of rho in most prokaryotes is particularly remarkable because animal rho proteins are involved in signaling pathways that are not found outside metazoa , which seems to make functional conservation in prokaryotes a remote possibility .", "the only prokaryotic protein of the rhomboid family that has been characterized experimentally in considerable detail is aara from the bacterium providencia stuartii .", "this protein is involved in the export of a quorum - sensing peptide , a function that , in physiological terms , resembles that of rho , although the signaling molecules , other than rho and aara , are obviously unrelated . in a striking recent development", ", two independent research groups have shown that several bacterial rhomboid - family proteins , including aara , can cleave the egf receptor ligands ( spitz , keren and gurken ) that are normally cleaved by rho paralogs .", "the cleavage depended on the conserved serine and histidine residues   and , moreover , transgenic flies that expressed aara developed a phenotype indistinguishable from that induced by overexpression of rho , whereas rho could substitute for aara in providencia stuartii .", "these unexpected findings demonstrated the conservation of a rip mechanism producing extracellular signals in eukaryotes and prokaryotes .", "eukaryotic rhomboid family proteins seem to show considerable functional variability ; in particular , cross - talk might exist between different rip pathways . a distinct representative of the rhomboid family has been shown to physically interact with presinilins 1 and 2 , and was accordingly named presenilins - associated rhomboid - like protein ( parl ) .", "the yeast ortholog of parl has been suggested to participate in the processing of cytochrome c peroxidase precursor during its import into the mitochondrion .", "the near ubiquity of the rhomboid family among bacteria , archaea and eukaryotes , along with the remarkable functional conservation , suggests that a signaling mechanism mediated by rhomboids might have functioned already in the last common ancestor of all extant life forms , with subsequent loss in several lineages . to address this possibility", "although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives .", "the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) .", "the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) .", "the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho .", "when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases .", "several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below .", "the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function .", "rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances ,", "a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene .", "most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis .", "the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) . only the conserved regions including the tmh and", "short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) .", "the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) .", "neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades .", "instead , the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) .", "the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) .", "the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) .", "the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi", "have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) .", "thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies ,", "several lineage - specific expansions of paralogs   are noticeable , in insects , mammals and plants ( figure 2 ) .", "archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) .", "there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids .", "although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) .", "this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely .", "therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests .", "first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated .", "specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) .", "each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) .", "in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis .", "the tree produced using the mrbayes package   showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) .", "we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method .", "the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) .", "the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) .", "the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) .", "the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) .", "the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids .", "the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont .", "neither can this tree be explained by postulating a small number of lineage - specific gene losses .", "the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events . in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) .", "instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages .", "in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily", "must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages .", "this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts .", "an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs .", "although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria .", "theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying", "the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution .", "a complex luca with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt .", "however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions .", "we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families", "whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical", ", comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument .", "the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms .", "indeed , when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca .", "it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible .", "it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria .", "the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) .", "this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules .", "emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself ,", "the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) .", "the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs   and the archaeo - eukaryotic signal peptide peptidase .", "although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives .", "the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) .", "the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) .", "the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho .", "when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases .", "several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below .", "the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function .", "rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances ,", "a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene .", "most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis .", "the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) .", "only the conserved regions including the tmh and short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) .", "the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) .", "neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades . instead", ", the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) .", "the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) .", "the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) .", "the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi", "have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) .", "thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies ,", "several lineage - specific expansions of paralogs   are noticeable , in insects , mammals and plants ( figure 2 ) .", "archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) .", "there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids .", "although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) .", "this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely .", "therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests .", "first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated .", "specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) .", "each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) .", "in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis .", "the tree produced using the mrbayes package   showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) .", "we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method .", "the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) .", "the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) .", "the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) .", "the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) .", "the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids .", "the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont .", "neither can this tree be explained by postulating a small number of lineage - specific gene losses .", "the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events .", "in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) .", "instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages .", "in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily", "must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages .", "this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts .", "an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs .", "although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria .", "theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution . a complex luca with around 2,000 genes", "is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt .", "however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions .", "we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families", "whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical", ", comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument .", "the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms . indeed ,", "when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca .", "it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible .", "it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria .", "the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) .", "this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules .", "emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself ,", "the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) .", "the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs   and the archaeo - eukaryotic signal peptide peptidase .", "the rhomboid family might be the most widespread and conserved group of integral membrane proteins . in and by itself , this would suggest that this family is part of the gene repertoire of luca .", "however , phylogenetic analysis suggests a different scenario , one of emergence in a bacterial lineage with subsequent multiple , independent hgt events and gene losses . although caution is due in the evolutionary interpretation of phylogenetic trees for large families , particularly when membrane proteins with a relatively small number of conserved positions , such as the rhomboids ,", "are involved , the multiple - hgt scenario seemed to be supported by several methods of tree analysis and statistical tests .", "eukaryotes probably acquired their two major rhomboid subfamilies , rho and parl , as the result of two independent , early hgt events .", "these events , which might have introduced rip as a means of intercellular communication , could have been pivotal in the evolution of eukaryotic multicellularity along the lines discussed previously with regard to the apparent bacterial origin of key components of eukaryotic programmed cell death machinery .", "subsequent evolution of rhomboids in eukaryotes proceeded by lineage - specific expansion of paralogs   followed by diversification through the addition of an extra tmh in different positions relative to the catalytic core , some limited domain accretion ( see figure 2 ) and sequence divergence .", "phylogenetic analysis of the rhomboid family described here carries a general message for studies aimed at the reconstruction of ancestral life forms , particularly luca .", "although most of the ( nearly ) ubiquitous protein families probably do derive from luca , explicit phylogenetic analysis is required to ascertain this in each case .", "the nonredundant ( nr ) protein sequence database at the national center for biotechnology information ( nih , bethesda ) was searched iteratively using the psi - blast program with multiple starting queries .", "psi - blast was normally run with expectation ( e ) value of 0.01 as the cut - off for inclusion of sequences into the position - specific scoring matrix .", "multiple alignments of protein sequences were constructed using the clustalw program   and manually adjusted on the basis of the examination of psi - blast search outputs and the superposition of the predicted tmhs , which were identified using the programs tmpred   and tmap .", "phylogenetic trees were built using the least - squares method   implemented in the fitch program of the phylip package , with subsequent local rearrangement using the protml program of the molphy package to obtain the maximum likelihood tree .", "the reliability of the tree topology was assessed using the rell ( resampling of estimated log - likelihoods ) bootstrap method of molphy , with 10,000 replications .", "alternative placements of selected clades in maximum - likelihood trees were compared by using the rearrangement optimization method ( kishino - hasegawa test ) as implemented in the protml program [ 43 - 45 ] .", "maximum parsimony trees were constructed using the heuristic search option of paup * . in addition , trees were constructed by bayesian inference using the markov chain monte carlo method as implemented in the mrbayes package .", "the complete alignment information , including columns with gaps , was used for the mrbayes analysis .", "constraint trees were imported into paup *   and subjected to neighbor - joining search to generate the phylogenies corresponding to alternative hypotheses .", "these phylogenies were compared using the kh , templeton ( wilcoxon signed - ranks )   and winning - sites ( sign )   tests implemented in paup*.", "l.p . is supported by a grant from the natural sciences and engineering research council of canada .", "the alignment includes the majority of the detected rhomboid family proteins ; some closely related sequences were omitted . only the six conserved ( predicted ) transmembrane helices ( tmh ) and short surrounding regions are shown .", "the boundaries of the predicted tmh are indicated by gray shading and overline and they are numbered 1 - 6 . the number of amino - acid residues in the omitted terminal and internal regions are indicated .", "the consensus shows amino - acid residues present in at least 90% of the aligned sequences ; h stands for hydrophobic residues ( a , c , i , l , v , m , f , y , w in the single - letter amino - acid code ) and s for small residues ( g , a , s , d , n , v ) .", "the proposed catalytic serine ( tmh4 ) and histidine ( tmh6 ) as well as conserved residues in tmh2 with possible ancillary roles in catalysis are highlighted in color .", "the proteins are identified with the gene identification ( gi ) number from the nonredundant database and an abbreviated species name .", "bacterial species are color - coded green , eukaryotic species blue and archaeal species yellow .", "species name abbreviations : aerpe , aeropyrum pernix ; agrtu , agrobacterium tumefaciens ; anoga , anopheles gambiae ; arath , arabidopsis thaliana ; arcfu , archaeoglobus fulgidus ; bacsu , bacillus subtilis ; brume , brucella melitensis ; caeel , caenorhabditis elegans ; caucr , caulobacter crescentus ; chlte , chlorobium tepidum ; cloac , clostridium acetobutilicum ; corgl , corynebacterium glutamicum ; deira , deinococcus radiodurans ; dicdi , dictyostelium discoideum ; drome , drosophila melanogaster ; escco , escherichia coli ; haein , haemophilus influenzae ; halsp , halobacterium sp . ;", "homsa , homo sapiens ; lacla , lactococcus lactis ; lisin , listeria innocua ; metja , methanoccocus jannaschii ; metka , methanopyrus kandleri ; metma , methanosarcina mazei ; meslo , mesorhizobium loti ; mycle , mycobacterium leprae ; myctu , mycobacterium tuberculosis ; neucr , neurospora crassa ; nossp , nostoc sp .", "; prost , providencia stuartii ; pyrab , pyrococcus abyssi ; pyrae , pyrobaculum aerophilum ; ralso , ralstonia solanaraceum ; sacce , saccharomyces cerevisiae ; schpo , schizosaccharomyces pombe ; sinme , sinorhizobium meliloti ; strco , streptomyces coelicolor ; strpn , streptococcus pneumoniae ; sulso , sulfolobus solfataricus ; sulto , sulfolobus tokodaii ; synsp , synechocystis sp . ; theac , thermoplasma acidophilum ; thema , thermotoga maritima ; thete , thermus thermophilus ; vibch , vibrio cholerae ; xanca , xanthomonas campestris ; xylfa , xylella fastidiosa .", "the sequences and their regions used to construct the tree are exactly those shown in figure 1 . the color coding and abbreviations are as in figure 1 .", "the two major eukaryotic subfamilies are denoted as rho and parl ( see text ) and four clusters containing unexpected ( from a phylogenetic viewpoint ) sets of species are denoted 1 - 4 .", "the clades that were investigated in the kh test are denoted a through d. although the tree is shown in a pseudorooted form for convenience , this is an unrooted tree .", "internal nodes with at least 70% rell bootstrap supported are denoted by black circles and nodes with a 50 - 70% support by blue circles .", "the posterior probabilities reported by the mrbayes program are indicated for some key internal branches .", "the rho and parl subfamilies are denoted ; the remaining clusters include prokaryotic rhomboids designated as in figure 2 ( with ' a ' added to the gi number ) . within each cluster ,", "the trees are unrooted , although shown in a pseudorooted form . a hypothetical scenario for the origin and dissemination of the rhomboid family proteases .", "the figure schematically shows the proposed three stages of evolution of the rhomboid family . in ( a ) , the progenitor of the rhomboid family functions as a transporter for a regulatory peptide in some bacterial lineage . in ( b ) , the catalytic site of the intramembrane protease evolves , allowing the switch to rip as the mechanism of the regulatory peptide release . in ( c ) , the emergence of rip is followed by a burst of hgt .", "the transmembrane helices of rhomboid are designated as in figure 1 ; their topology in the membrane is based on that proposed in .", "the catalytic histidine and serine are shown and connected by a dotted line to indicate the proposed charge - relay system of the protease ; possible ancillary catalytic residues are not shown .", "log - likelihood analysis of possible placements of selected branches of maximum likelihood trees for the proteins analyzed * a - d , clades that were subjected to local rearrangements in the tree as indicated in figure 2 and discussed in the text .", "bootstrap probability of the given tree calculated using the rell method ( resampling of estimated log - likelihoods ) .", "statistical comparisons of the best neighbor - joining tree with the hypothesis 1 and hypothesis 2 trees * probability of getting a more extreme test statistic under the null hypothesis of no difference between the two trees ( two - tailed test ) ."], "abstract_text": ["<S> the near - universal presence of the rhomboid family in bacteria , archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor , phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer . </S>"], "labels": null, "section_names": ["Background", "Results and discussion", "Sequence and structural features and phyletic distribution of the rhomboid family", "Phylogeny and evolutionary history of the rhomboid family", "Conclusions", "Materials and methods", "Acknowledgements", "Figures and Tables"], "sections": [["inspection of the database of clusters of orthologous groups of proteins ( cogs )   revealed only one family of such proteins that is represented in most of the sequenced bacterial , archaeal and eukaryotic genomes .", "the prototype of this family is the rhomboid ( rho ) protein from drosophila melanogaster , a developmental regulator involved in epidermal growth factor ( egf)-dependent signaling pathways [ 2 - 4 ] .", "not only were homologs of rhomboid detected in prokaryotes and eukaryotes , but the pattern of sequence conservation in this family appeared uncharacteristic of nonenzymatic membrane proteins , such as transporters .", "specifically , several polar amino - acid residues are conserved in nearly all members of the rhomboid family , suggesting the possibility of an enzymatic activity . as three of these conserved residues were histidines , it has been hypothesized that rhomboid - family proteins could function as metal - dependent membrane proteases .", "recently , however , it has been shown that rho cleaves a transmembrane helix ( tmh ) in the membrane - bound precursor of the tgf-like growth factor spitz , enabling the released spitz to activate the egf receptor , and that a conserved serine and a conserved histidine in rho are essential for this cleavage .", "thus , it appears that rhomboid - family proteins are a distinct group of intramembrane serine proteases .", "altogether , the genome of drosophila encodes seven rho paralogs ( now designated rho1 - 7 , with the original rhomboid becoming rho-1 ) , at least three of which are involved in distinct egf - dependent pathways , apparently through proteolytic activation of diverse ligands of the egf receptor .", "the newly discovered intramembrane proteolytic activity of rho places the rhomboid family within the framework of regulated intramembrane proteolysis ( rip ) , a new paradigm of signal transduction , which appears to be prominent in all forms of life . under rip , signaling proteins undergo site - specific proteolysis within tmh , resulting in the release of active fragments , which are the actual effectors in signal tranduction cascades . until recently", ", the only characterized cases of rip in eukaryotes involved presenilin-1 , an aspartyl protease , which cleaves a transmembrane helix in type-1 membrane proteins such as amyloid -precursor protein ( app ) , notch and ire1 , and the metalloprotease s2p , which cleaves a tmh in a type-2 transmembrane protein , the sterol - dependent transcription factor srebp .", "notably , s2p has highly conserved bacterial homologs , and the protease domain of presenilins also might be homologous to bacterial and archaeal type iv prepilin peptidases , although , in this case , the sequence similarity is low . in the case of the rhomboid family ,", "the existence of homologs of rho in most prokaryotes is particularly remarkable because animal rho proteins are involved in signaling pathways that are not found outside metazoa , which seems to make functional conservation in prokaryotes a remote possibility .", "the only prokaryotic protein of the rhomboid family that has been characterized experimentally in considerable detail is aara from the bacterium providencia stuartii .", "this protein is involved in the export of a quorum - sensing peptide , a function that , in physiological terms , resembles that of rho , although the signaling molecules , other than rho and aara , are obviously unrelated . in a striking recent development", ", two independent research groups have shown that several bacterial rhomboid - family proteins , including aara , can cleave the egf receptor ligands ( spitz , keren and gurken ) that are normally cleaved by rho paralogs .", "the cleavage depended on the conserved serine and histidine residues   and , moreover , transgenic flies that expressed aara developed a phenotype indistinguishable from that induced by overexpression of rho , whereas rho could substitute for aara in providencia stuartii .", "these unexpected findings demonstrated the conservation of a rip mechanism producing extracellular signals in eukaryotes and prokaryotes .", "eukaryotic rhomboid family proteins seem to show considerable functional variability ; in particular , cross - talk might exist between different rip pathways . a distinct representative of the rhomboid family has been shown to physically interact with presinilins 1 and 2 , and was accordingly named presenilins - associated rhomboid - like protein ( parl ) .", "the yeast ortholog of parl has been suggested to participate in the processing of cytochrome c peroxidase precursor during its import into the mitochondrion .", "the near ubiquity of the rhomboid family among bacteria , archaea and eukaryotes , along with the remarkable functional conservation , suggests that a signaling mechanism mediated by rhomboids might have functioned already in the last common ancestor of all extant life forms , with subsequent loss in several lineages . to address this possibility"], ["although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives .", "the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) .", "the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) .", "the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho .", "when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases .", "several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below .", "the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function .", "rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances ,", "a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene .", "most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis .", "the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) . only the conserved regions including the tmh and", "short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) .", "the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) .", "neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades .", "instead , the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) .", "the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) .", "the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) .", "the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi", "have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) .", "thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies ,", "several lineage - specific expansions of paralogs   are noticeable , in insects , mammals and plants ( figure 2 ) .", "archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) .", "there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids .", "although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) .", "this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely .", "therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests .", "first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated .", "specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) .", "each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) .", "in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis .", "the tree produced using the mrbayes package   showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) .", "we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method .", "the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) .", "the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) .", "the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) .", "the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) .", "the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids .", "the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont .", "neither can this tree be explained by postulating a small number of lineage - specific gene losses .", "the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events . in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) .", "instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages .", "in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily", "must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages .", "this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts .", "an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs .", "although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria .", "theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying", "the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution .", "a complex luca with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt .", "however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions .", "we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families", "whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical", ", comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument .", "the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms .", "indeed , when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca .", "it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible .", "it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria .", "the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) .", "this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules .", "emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself ,", "the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) .", "the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs   and the archaeo - eukaryotic signal peptide peptidase ."], ["although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives .", "the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) .", "the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) .", "the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho .", "when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases .", "several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below .", "the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function .", "rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances ,", "a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene .", "most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis ."], ["the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) .", "only the conserved regions including the tmh and short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) .", "the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) .", "neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades . instead", ", the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) .", "the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) .", "the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) .", "the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi", "have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) .", "thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies ,", "several lineage - specific expansions of paralogs   are noticeable , in insects , mammals and plants ( figure 2 ) .", "archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) .", "there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids .", "although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) .", "this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely .", "therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests .", "first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated .", "specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) .", "each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) .", "in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis .", "the tree produced using the mrbayes package   showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) .", "we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method .", "the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) .", "the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) .", "the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) .", "the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) .", "the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids .", "the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont .", "neither can this tree be explained by postulating a small number of lineage - specific gene losses .", "the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events .", "in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) .", "instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages .", "in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily", "must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages .", "this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts .", "an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs .", "although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria .", "theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution . a complex luca with around 2,000 genes", "is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt .", "however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions .", "we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families", "whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical", ", comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument .", "the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms . indeed ,", "when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca .", "it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible .", "it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria .", "the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) .", "this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules .", "emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself ,", "the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) .", "the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs   and the archaeo - eukaryotic signal peptide peptidase ."], ["the rhomboid family might be the most widespread and conserved group of integral membrane proteins . in and by itself , this would suggest that this family is part of the gene repertoire of luca .", "however , phylogenetic analysis suggests a different scenario , one of emergence in a bacterial lineage with subsequent multiple , independent hgt events and gene losses . although caution is due in the evolutionary interpretation of phylogenetic trees for large families , particularly when membrane proteins with a relatively small number of conserved positions , such as the rhomboids ,", "are involved , the multiple - hgt scenario seemed to be supported by several methods of tree analysis and statistical tests .", "eukaryotes probably acquired their two major rhomboid subfamilies , rho and parl , as the result of two independent , early hgt events .", "these events , which might have introduced rip as a means of intercellular communication , could have been pivotal in the evolution of eukaryotic multicellularity along the lines discussed previously with regard to the apparent bacterial origin of key components of eukaryotic programmed cell death machinery .", "subsequent evolution of rhomboids in eukaryotes proceeded by lineage - specific expansion of paralogs   followed by diversification through the addition of an extra tmh in different positions relative to the catalytic core , some limited domain accretion ( see figure 2 ) and sequence divergence .", "phylogenetic analysis of the rhomboid family described here carries a general message for studies aimed at the reconstruction of ancestral life forms , particularly luca .", "although most of the ( nearly ) ubiquitous protein families probably do derive from luca , explicit phylogenetic analysis is required to ascertain this in each case ."], ["the nonredundant ( nr ) protein sequence database at the national center for biotechnology information ( nih , bethesda ) was searched iteratively using the psi - blast program with multiple starting queries .", "psi - blast was normally run with expectation ( e ) value of 0.01 as the cut - off for inclusion of sequences into the position - specific scoring matrix .", "multiple alignments of protein sequences were constructed using the clustalw program   and manually adjusted on the basis of the examination of psi - blast search outputs and the superposition of the predicted tmhs , which were identified using the programs tmpred   and tmap .", "phylogenetic trees were built using the least - squares method   implemented in the fitch program of the phylip package , with subsequent local rearrangement using the protml program of the molphy package to obtain the maximum likelihood tree .", "the reliability of the tree topology was assessed using the rell ( resampling of estimated log - likelihoods ) bootstrap method of molphy , with 10,000 replications .", "alternative placements of selected clades in maximum - likelihood trees were compared by using the rearrangement optimization method ( kishino - hasegawa test ) as implemented in the protml program [ 43 - 45 ] .", "maximum parsimony trees were constructed using the heuristic search option of paup * . in addition , trees were constructed by bayesian inference using the markov chain monte carlo method as implemented in the mrbayes package .", "the complete alignment information , including columns with gaps , was used for the mrbayes analysis .", "constraint trees were imported into paup *   and subjected to neighbor - joining search to generate the phylogenies corresponding to alternative hypotheses .", "these phylogenies were compared using the kh , templeton ( wilcoxon signed - ranks )   and winning - sites ( sign )   tests implemented in paup*."], ["l.p . is supported by a grant from the natural sciences and engineering research council of canada ."], ["the alignment includes the majority of the detected rhomboid family proteins ; some closely related sequences were omitted . only the six conserved ( predicted ) transmembrane helices ( tmh ) and short surrounding regions are shown .", "the boundaries of the predicted tmh are indicated by gray shading and overline and they are numbered 1 - 6 . the number of amino - acid residues in the omitted terminal and internal regions are indicated .", "the consensus shows amino - acid residues present in at least 90% of the aligned sequences ; h stands for hydrophobic residues ( a , c , i , l , v , m , f , y , w in the single - letter amino - acid code ) and s for small residues ( g , a , s , d , n , v ) .", "the proposed catalytic serine ( tmh4 ) and histidine ( tmh6 ) as well as conserved residues in tmh2 with possible ancillary roles in catalysis are highlighted in color .", "the proteins are identified with the gene identification ( gi ) number from the nonredundant database and an abbreviated species name .", "bacterial species are color - coded green , eukaryotic species blue and archaeal species yellow .", "species name abbreviations : aerpe , aeropyrum pernix ; agrtu , agrobacterium tumefaciens ; anoga , anopheles gambiae ; arath , arabidopsis thaliana ; arcfu , archaeoglobus fulgidus ; bacsu , bacillus subtilis ; brume , brucella melitensis ; caeel , caenorhabditis elegans ; caucr , caulobacter crescentus ; chlte , chlorobium tepidum ; cloac , clostridium acetobutilicum ; corgl , corynebacterium glutamicum ; deira , deinococcus radiodurans ; dicdi , dictyostelium discoideum ; drome , drosophila melanogaster ; escco , escherichia coli ; haein , haemophilus influenzae ; halsp , halobacterium sp . ;", "homsa , homo sapiens ; lacla , lactococcus lactis ; lisin , listeria innocua ; metja , methanoccocus jannaschii ; metka , methanopyrus kandleri ; metma , methanosarcina mazei ; meslo , mesorhizobium loti ; mycle , mycobacterium leprae ; myctu , mycobacterium tuberculosis ; neucr , neurospora crassa ; nossp , nostoc sp .", "; prost , providencia stuartii ; pyrab , pyrococcus abyssi ; pyrae , pyrobaculum aerophilum ; ralso , ralstonia solanaraceum ; sacce , saccharomyces cerevisiae ; schpo , schizosaccharomyces pombe ; sinme , sinorhizobium meliloti ; strco , streptomyces coelicolor ; strpn , streptococcus pneumoniae ; sulso , sulfolobus solfataricus ; sulto , sulfolobus tokodaii ; synsp , synechocystis sp . ; theac , thermoplasma acidophilum ; thema , thermotoga maritima ; thete , thermus thermophilus ; vibch , vibrio cholerae ; xanca , xanthomonas campestris ; xylfa , xylella fastidiosa .", "the sequences and their regions used to construct the tree are exactly those shown in figure 1 . the color coding and abbreviations are as in figure 1 .", "the two major eukaryotic subfamilies are denoted as rho and parl ( see text ) and four clusters containing unexpected ( from a phylogenetic viewpoint ) sets of species are denoted 1 - 4 .", "the clades that were investigated in the kh test are denoted a through d. although the tree is shown in a pseudorooted form for convenience , this is an unrooted tree .", "internal nodes with at least 70% rell bootstrap supported are denoted by black circles and nodes with a 50 - 70% support by blue circles .", "the posterior probabilities reported by the mrbayes program are indicated for some key internal branches .", "the rho and parl subfamilies are denoted ; the remaining clusters include prokaryotic rhomboids designated as in figure 2 ( with ' a ' added to the gi number ) . within each cluster ,", "the trees are unrooted , although shown in a pseudorooted form . a hypothetical scenario for the origin and dissemination of the rhomboid family proteases .", "the figure schematically shows the proposed three stages of evolution of the rhomboid family . in ( a ) , the progenitor of the rhomboid family functions as a transporter for a regulatory peptide in some bacterial lineage . in ( b ) , the catalytic site of the intramembrane protease evolves , allowing the switch to rip as the mechanism of the regulatory peptide release . in ( c ) , the emergence of rip is followed by a burst of hgt .", "the transmembrane helices of rhomboid are designated as in figure 1 ; their topology in the membrane is based on that proposed in .", "the catalytic histidine and serine are shown and connected by a dotted line to indicate the proposed charge - relay system of the protease ; possible ancillary catalytic residues are not shown .", "log - likelihood analysis of possible placements of selected branches of maximum likelihood trees for the proteins analyzed * a - d , clades that were subjected to local rearrangements in the tree as indicated in figure 2 and discussed in the text .", "bootstrap probability of the given tree calculated using the rell method ( resampling of estimated log - likelihoods ) .", "statistical comparisons of the best neighbor - joining tree with the hypothesis 1 and hypothesis 2 trees * probability of getting a more extreme test statistic under the null hypothesis of no difference between the two trees ( two - tailed test ) ."]]}
{"article_id": "PMC4817573", "article_text": ["an infliction in the life expectancy figure of patients with sickle cell disease ( scd ) occurred around the middle of the 1970s ( fig .", "this minimal increase in life expectancy coincided with my appointment at thomas jefferson university as the associate director of the newly created adult sickle cell center .", "the number of adults at that time was small and the transition from pediatrics to adult programs was at the age of 18 years .", "the trickle of patients increased gradually and we were faced with adolescent and young adult african american patients who were in a state of confusion .", "stripped from the protective sphere of the pediatric world and the empathy of their pediatric hematologists and the pediatric ancillary staff , they were in a state of fear , anxiety , depression and , worst of all , severe pain .", "the fact that most patients were barely educated , many without a high school degree , unemployed , mediocre health coverage , and dysfunctional family structure conferred a logarithmic dimension to the problem .", "the arrow indicates the infliction point where life expectancy of patients with sickle cell disease began to increase .", "the steady stream of admissions of patients with acute painful vaso - occlusive crises ( vocs ) to the emergency department ( ed ) and hospital were not welcome by most providers , hospital administration , the house and nursing staffs .", "there was subtle resentment of the patients that sometimes extended to the hematologists who showed compassion to the patients . soon labels such as drug addicts , drug - seeking behavior , and hospital hopping and frequent flyer emerged .", "listening to and believing the patients and keeping detailed records of ed and hospital admissions and the analgesics prescribed , revealed that most patients genuinely do not respond to a certain analgesic or a certain dose . increasing the doses of an analgesic or switching to another drug solved the problem in most patients .", "accordingly and with the approval of the institutional review board ( irb ) , i issued an identification wallet - sized , plasticized card that was carried by patients and presented to the provider treating their voc in the ed , hospital or any other medical facility .", "information printed on both sides of the card included : 1 ) demographic data and a recent photograph ; 2 ) hematological data including reticulocyte count ; 3 ) medical data including the type of scd , its complications and co - morbidities if present ; 4 ) all medications being taken by the patient and the recommended treatment of vocs including the name , dose , and the route of administration of the analgesics in question ; and 5 ) my name and contact information for answering questions if needed .", "it was not expensive to issue these cards . a polaroid camera available at that time and a laminator were the only equipments needed to issue these cards .", "later on , information on the card was computerized and a printed copy was given to the patient .", "the patients were very compliant in carrying it as faithfully as they carry their medical cards .", "some providers liked it very much because it facilitated having a concise history about the patients .", "while this controversy was brewing , interesting developments in basic science were in progress to understand the pharmacodynamics and pharmacokinetics of opioids .", "in the 1970s , it was hypothesized that opioids have receptors to bind to and activate in order to relieve pain   by blocking or minimizing the transmission of painful stimuli and raising the pain threshold .", "it did not take long after that to identify opioids as ligands that bind to stereospecific and saturable receptors in the central nervous system and other tissues [ 3 , 4 ] .", "in addition , recent elegant studies [ 6 - 10 ] have revealed a helical structure of the opioid receptors , which forms pockets in which the corresponding opioid ( ligand ) fits snugly ( fig .", "recognition is highly specific , such that only l - isomers of certain opioids exert analgesic activity .", "physiologically , by binding to receptors , opioids initiate a series of biochemical events including activation of g proteins , inhibition of adenylate cyclase , and extrusion of potassium ions , resulting in hyperpolarization of cell membranes [ 14 - 16 ] ; this delays or prevents transmission of painful stimuli .", "thus , the riddle why some patients respond to one opioid but not another had a pathophysiologic explanation .", "( a ) morphine - like molecule ( yellow ) in the deep pocket ( blue ) of the -opioid receptor . ( b )", "-opioid receptors from an intimate pair when crystallized with a ligand ( yellow ) such as morphine . knowing how an opioid molecule ( yellow ) sticks in the pocket of its receptor ( blue )", "parallel to the progress in the pharmacodynamics of opioids mentioned above , a concomitant advance in the pharmacokinetics of opioids was bubbling to the surface .", "phase i involves the cyp enzymes and phase ii metabolism conjugates the drug to hydrophilic substances , such as glucuronic acid , sulfate , glycine , or glutathione .", "morphine , hydromorphone and oxymorphone are metabolized by glucuronidation , whereas the majority of the other opioids are metabolized by the cytochrome p450 isoenzyme system .", "the net effect of an opioid depends on the availability of enzyme(s ) to convert it into metabolites that could be active or inactive .", "briefly , the cyp2d6 genotypes are categorized into phenotypes based on the activity of the variant enzymes .", "ultrarapid metabolizers ( ums ) have greater than normal activity due to duplication or triplication , of active alleles [ 20 - 23 ] , extensive metabolizers ( ems ) have normal enzyme activity , intermediate metabolizers ( ims ) have decreased enzyme activity , and poor metabolizers ( pms ) have absent or little enzyme activity .", "patients who are ums of fentanyl would rapidly convert it into inactive metabolites with minimal or absent analgesic effect requiring increasing the dose of fentanyl .", "on the other hand , patients who are pms of fentanyl would experience prompt relief with relatively small doses of fentanyl but higher doses could be toxic due to the accumulation of unmetabolized fentanyl .", "the cyp3a4 enzyme metabolizes more than 50% of all drugs ; consequently , opioids metabolized by this enzyme have a high risk of drug - drug interactions .", "together , current data on the pharmacodynamics and pharmacokinetics of opioids show great variability of genotypes among patients and extreme variability in individual responses to opioids . determining the pharmacogenetics profile of each patient", "facilitates the choice of drugs that would be efficacious for that patient and avoid those drugs associated with harmful drug - drug interaction .", "this approach in diagnostics and therapeutics ushers in the dawn of a new field for the management of individual patients based on their unique pharmacogenetics , phenotypic and biomarker characteristics .", "this future approach is referred to as personalized medicine or , more recently , precision medicine .", "we hope that this methodology would be approved and sponsored by the insurance companies for patients with scd . in the meantime , listening , believing and respecting the patient with sickle cell pain should be maintained for now as the approach to individualized therapy ."], "abstract_text": ["<S> in the 1970s , sickle cell pain was treated with trial and error approach by increasing or decreasing the dose of an opioid or switching from one analgesic to another . </S>", "<S> this approach was controversial with criticism and doubt about its usefulness . since then , advances in determining the structure of opioid receptors and the role of the cyp450 enzymes in metabolizing opioids revealed that these anatomic and metabolic findings are not the same in all persons , thus explaining the variability in response to opioids among patients . </S>", "<S> thus , the  trial and error approach  has a scientific basis after all . </S>"], "labels": null, "section_names": ["Introduction", "Pharmacology of Opioids", "Conclusion", "Conflict of Interest"], "sections": [["an infliction in the life expectancy figure of patients with sickle cell disease ( scd ) occurred around the middle of the 1970s ( fig .", "this minimal increase in life expectancy coincided with my appointment at thomas jefferson university as the associate director of the newly created adult sickle cell center .", "the number of adults at that time was small and the transition from pediatrics to adult programs was at the age of 18 years .", "the trickle of patients increased gradually and we were faced with adolescent and young adult african american patients who were in a state of confusion .", "stripped from the protective sphere of the pediatric world and the empathy of their pediatric hematologists and the pediatric ancillary staff , they were in a state of fear , anxiety , depression and , worst of all , severe pain .", "the fact that most patients were barely educated , many without a high school degree , unemployed , mediocre health coverage , and dysfunctional family structure conferred a logarithmic dimension to the problem .", "the arrow indicates the infliction point where life expectancy of patients with sickle cell disease began to increase .", "the steady stream of admissions of patients with acute painful vaso - occlusive crises ( vocs ) to the emergency department ( ed ) and hospital were not welcome by most providers , hospital administration , the house and nursing staffs .", "there was subtle resentment of the patients that sometimes extended to the hematologists who showed compassion to the patients . soon labels such as drug addicts , drug - seeking behavior , and hospital hopping and frequent flyer emerged .", "listening to and believing the patients and keeping detailed records of ed and hospital admissions and the analgesics prescribed , revealed that most patients genuinely do not respond to a certain analgesic or a certain dose . increasing the doses of an analgesic or switching to another drug solved the problem in most patients .", "accordingly and with the approval of the institutional review board ( irb ) , i issued an identification wallet - sized , plasticized card that was carried by patients and presented to the provider treating their voc in the ed , hospital or any other medical facility .", "information printed on both sides of the card included : 1 ) demographic data and a recent photograph ; 2 ) hematological data including reticulocyte count ; 3 ) medical data including the type of scd , its complications and co - morbidities if present ; 4 ) all medications being taken by the patient and the recommended treatment of vocs including the name , dose , and the route of administration of the analgesics in question ; and 5 ) my name and contact information for answering questions if needed .", "it was not expensive to issue these cards . a polaroid camera available at that time and a laminator were the only equipments needed to issue these cards .", "later on , information on the card was computerized and a printed copy was given to the patient .", "the patients were very compliant in carrying it as faithfully as they carry their medical cards .", "some providers liked it very much because it facilitated having a concise history about the patients ."], ["while this controversy was brewing , interesting developments in basic science were in progress to understand the pharmacodynamics and pharmacokinetics of opioids .", "in the 1970s , it was hypothesized that opioids have receptors to bind to and activate in order to relieve pain   by blocking or minimizing the transmission of painful stimuli and raising the pain threshold .", "it did not take long after that to identify opioids as ligands that bind to stereospecific and saturable receptors in the central nervous system and other tissues [ 3 , 4 ] .", "in addition , recent elegant studies [ 6 - 10 ] have revealed a helical structure of the opioid receptors , which forms pockets in which the corresponding opioid ( ligand ) fits snugly ( fig .", "recognition is highly specific , such that only l - isomers of certain opioids exert analgesic activity .", "physiologically , by binding to receptors , opioids initiate a series of biochemical events including activation of g proteins , inhibition of adenylate cyclase , and extrusion of potassium ions , resulting in hyperpolarization of cell membranes [ 14 - 16 ] ; this delays or prevents transmission of painful stimuli .", "thus , the riddle why some patients respond to one opioid but not another had a pathophysiologic explanation .", "( a ) morphine - like molecule ( yellow ) in the deep pocket ( blue ) of the -opioid receptor . ( b )", "-opioid receptors from an intimate pair when crystallized with a ligand ( yellow ) such as morphine . knowing how an opioid molecule ( yellow ) sticks in the pocket of its receptor ( blue )", "parallel to the progress in the pharmacodynamics of opioids mentioned above , a concomitant advance in the pharmacokinetics of opioids was bubbling to the surface .", "phase i involves the cyp enzymes and phase ii metabolism conjugates the drug to hydrophilic substances , such as glucuronic acid , sulfate , glycine , or glutathione .", "morphine , hydromorphone and oxymorphone are metabolized by glucuronidation , whereas the majority of the other opioids are metabolized by the cytochrome p450 isoenzyme system .", "the net effect of an opioid depends on the availability of enzyme(s ) to convert it into metabolites that could be active or inactive .", "briefly , the cyp2d6 genotypes are categorized into phenotypes based on the activity of the variant enzymes .", "ultrarapid metabolizers ( ums ) have greater than normal activity due to duplication or triplication , of active alleles [ 20 - 23 ] , extensive metabolizers ( ems ) have normal enzyme activity , intermediate metabolizers ( ims ) have decreased enzyme activity , and poor metabolizers ( pms ) have absent or little enzyme activity .", "patients who are ums of fentanyl would rapidly convert it into inactive metabolites with minimal or absent analgesic effect requiring increasing the dose of fentanyl .", "on the other hand , patients who are pms of fentanyl would experience prompt relief with relatively small doses of fentanyl but higher doses could be toxic due to the accumulation of unmetabolized fentanyl .", "the cyp3a4 enzyme metabolizes more than 50% of all drugs ; consequently , opioids metabolized by this enzyme have a high risk of drug - drug interactions ."], ["together , current data on the pharmacodynamics and pharmacokinetics of opioids show great variability of genotypes among patients and extreme variability in individual responses to opioids . determining the pharmacogenetics profile of each patient", "facilitates the choice of drugs that would be efficacious for that patient and avoid those drugs associated with harmful drug - drug interaction .", "this approach in diagnostics and therapeutics ushers in the dawn of a new field for the management of individual patients based on their unique pharmacogenetics , phenotypic and biomarker characteristics .", "this future approach is referred to as personalized medicine or , more recently , precision medicine .", "we hope that this methodology would be approved and sponsored by the insurance companies for patients with scd . in the meantime , listening , believing and respecting the patient with sickle cell pain should be maintained for now as the approach to individualized therapy ."], [""]]}
{"article_id": "PMC4754549", "article_text": ["the potential of rnai to silence any gene has made it an attractive therapeutic modality .", "however , the main obstacle to rnai in the clinic is delivery . to be effective", ", sirnas must be transported through the body , bind , and be taken up by target cells where they must traverse the plasma membrane and gain access to the cytosolic compartment , where the rnai machinery resides . to be useful ,", "ease of formulation and administration , overall cost , and any associated toxicities are essential considerations .", "dcs are heterogeneous , with subsets defined phenotypically , functionally and by location ( reviewed in ref .", "2 ) . a delivery vehicle that knocks down expression of specific genes in a distinct dc population(s ) would be a valuable tool for targeting diverse diseases including cancers , infectious diseases , autoimmunity and as a vaccine component .", "therefore , a platform that delivers sirnas to specific dc subsets in situ would be useful for inhibiting or activating immune responses .", "lipid nanoparticles ( lnps ) are one of the most advanced platforms for sirna delivery to hepatocytes , and are under clinical evaluation for conditions that require hepatic gene silencing .", "these lnps typically contain ionizable cationic lipids ( pka ~6.5 ) that bind nucleic acids via electrostatic interactions at low ph , but are charge neutral at ph 7.4 . as a well - perfused organ", "furthermore , lnp uptake by hepatocytes is mediated by association with serum apoe leading to efficient uptake via low - density lipoprotein receptors in the liver .", "following cellular uptake of the lnp , the ionization of the lipid within acidic endosomes is thought to promote sirna escape to the cytosol .", "importantly , these lnps are associated with minimal toxicity , including little induction of proinflammatory cytokines following administration of physiologically relevant doses .", "in contrast to the liver , sirna delivery to extra - hepatic cells is challenging . in particular , immune cells such as dcs are relatively resistant to sirna uptake in vitro and in vivo .", "although designed for hepatic gene silencing , the efficacy of these lnps and their derivatives has been assessed for rnai - mediated gene silencing in macrophages ( mos ) and dcs in vitro and in vivo .", "lnp uptake and modest gene silencing was achieved at high doses of lnps ( ~ 5mg / kg ) .", "together with a complementary study , this work demonstrates the feasibility of gene silencing in immune cells using lnp technology . clearly", ", however , lnp formulations must be modified to achieve optimal gene silencing in immune cells .", "enhanced uptake of lnps by primary hepatocytes is mediated by apolipoprotein e binding to the neutral lnps .", "this results in recognition by receptors including the low - density lipoprotein receptor and scavenger receptors largely expressed on hepatocytes .", "the mechanism of uptake strongly suggests that retargeting of these particles to other receptors or other tissues is possible .", "indeed , akinc et al . demonstrated that lnps modified with n - acetylgalactosamine ( galnac ) were retargeted to the asialoglycoprotein receptor ( asgpr ) expressed by hepatocytes in vivo .", "for delivery to nonhepatic cells , we have shown in vitro that anti - transferrin receptor aptamers can be used to redirect similar lnps .", "more recently , liang et al . described a similar approach using aptamer - coated lnps to target osteoblasts in vivo .", "for uptake by immune cells , a recent study coated lnps with a full - length anti - cd4 antibody for sirna delivery to primary cd4 t cells .", "lnp uptake and gene silencing was observed in cd4 t cells in various organs including the spleen , lymph nodes , and blood .", "these studies demonstrate the potential for attaching exogenous ligands to lnps for delivery to hepatic , and importantly nonhepatic primary cells in vivo . drawing from these works", ", we reasoned that this lnp platform could be a useful foundation for the delivery of sirnas to dcs to modulate immune responses . to achieve this", ", we modified lnps using a single chain antibody ( scfv ) specific for the dc receptor dec205 , a c - type lectin expressed at high levels on cd8 dcs .", "dec205 dcs mediate cross - presentation of antigen resulting in modulation of cd8 t - cell responses . therefore , inhibition of gene expression by dec205 dcs is a potentially powerful approach for regulating cd8 t - cell activation . using this approach , we assessed the ability of dec - lnps ( lnps coated with scfv specific for murine dec205 , containing sirnas specific for costimulatory molecules ) to inhibit immune responses .", "injection of dec - lnps resulted in preferential uptake of the dec - lnps by splenic dec205 dcs .", "furthermore , when coadministered with adjuvant , lnps containing sirna targeting cd40 , cd80 , and cd86 reduced expression of these costimulatory molecules to levels similar to those observed in immature dcs .", "most importantly , dcs isolated from mice injected with a low dose ( ~0.6  mg / kg ) of these dec - lnps , were able to suppress a robust mixed lymphocyte reaction ( mlr ) , demonstrating the functional efficacy of this approach .", "interestingly , when we performed experiments using 2 ' fluoro ( 2'f ) modified sirna , a formulation reported to be nonimmunostimulatory and nonimmunogenic , significant immune activation of the targeted dcs was detected . this effect was not observed in assays performed with human peripheral blood mononuclear cells ( pbmcs ) or monocyte - derived dcs ( modcs ) and could be mitigated through selective 2-o - methyl ( 2ome ) modification of the sirna .", "overall , our study demonstrates the functionality of lnps modified for receptor targeting for sirna delivery to dcs .", "additionally our results suggest that specific cell targeting can mediate immunological responses to sirna formulations .", "while care needs to be taken in designing and evaluating targeted approaches using lnps , we found that 2ome modification of the sirna was sufficient to negate immune activation .", "our approach enhances the efficacy of sirna - mediated gene silencing by ~10-fold when compared to nontargeted delivery .", "therefore , this targeted strategy should prove effective for regulating multiple immune - mediated diseases .", "lnps containing sirnas specific either for cd80 , cd86 , cd40 , or a control ( nontarget ) sequence were synthesized by extrusion using a mixture of 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc):dlindma : dspe - peg : cholesterol at a ratio of 15:40:5:40 ( based on refs .", "murine anti - dec205 scfv with a c - terminal cysteine was attached to the lipid dspe - peg by a maleimide group ( figure 1a , b ) . following synthesis , we subjected lnps to quality control to ensure consistency between batches .", "dynamic light scattering was used to determine diameter and polydispersity of lnps , and cryo - electron microscopy confirmed lnp size and their unilamellar structure ( figure 1c , d ) .", "the efficiency of sirna incorporation and scfv binding to dspe - peg was also assessed ( figure 1c , e ) . to show specificity of binding to dec205 ,", "scfv - lnps containing dy547-labeled sirna were cultured with either parental ( dec205 ) or dec205 cho cells . as seen in figure 2a , at 4 c dec205 cells bind dec - lnps approximately fourfold better compared with nontargeted lnps ( nt - lnps : lnps not coated with any scfv ) , or lnps coated with an isotype scfv ( iso - lnps ; cho - dec205 panel ) . when similar assays were performed at 37 c to allow cell uptake , cell staining increased by at least fivefold .", "little uptake was observed when lnps ( targeted or nontargeted ) were incubated with parental cho cells ( cho panel ) .", "similar results were obtained when bone marrow - derived dcs ( bmdcs ) , derived from wild - type mice ( b6 ) were incubated with dec - lnps .", "we note that as dec205 is expressed at lower levels on bmdcs compared with cho - dec205 , less dec - lnp binding and uptake was observed in bmdcs ( ~2-fold enhancement compared with nt- or iso - lnps ; b6 bmdc panel ) . to further confirm target - specific uptake we performed experiments using bmdcs derived from dec205 mice , and no specific cell binding or uptake", "the dec205 antibody is internalized via receptor - mediated endocytosis , and is targeted to late endosomes and lysosomes .", "confocal microscopy confirmed that similar to dec205 antibody , dec - lnps were targeted to lamp-1 late endosomes or lysosomes ( figure 2b ) .", "having demonstrated dec205-mediated uptake of dec - lnps in vitro , we investigated their localization in vivo .", "b6 mice were injected systemically ( intravenous , i.v . ) with fluorescently labeled scfv - lnps .", "as the dec205 receptor is endocytosed when it binds its ligand , we could not use this molecule to identify dec205 dcs .", "therefore , we used cd8 , a protein that is coexpressed on dec205 dcs , as a surrogate marker for tracking these cells .", "more than 50% of cd11ccd8 cells took up dec - lnps , and uptake was largely restricted to the cd8 dc population , with little uptake observed by cd8 dcs ( figure 3a , b ) .", "specificity was demonstrated by comparison of uptake of dec - lnps and iso - lnps .", "significant uptake was only observed in dec205 dcs following dec - lnp injection , and other splenic immune cells took up little dec- or iso - lnps ( figure 3a , c ) . when dec - lnps were injected into dec205 mice ,", "no uptake was detected ( figure 3d ) . to show that dec - lnps were competent for specific gene silencing , we generated dec - lnps containing a sirna specific for cd80 ( having identified the most effective sirna sequence ; see supplementary figure s1 ) and injected into b6 mice .", "cd11cdec205 cells took up similar amounts of lnps following injection with dec - lnp containing either cd80- or control - sirna ( lnp uptake panel , mean fluorescence intensity ( mfi ) : dec - lnp - control = 443 versus dec - lnp - sicd80 = 464 ) . in mice injected with dec - lnp encapsulating cd80-specific sirna , cd80 protein expression approached basal levels , and was reduced by ~2-fold when compared with dec - lnp containing control - sirna ( figure 4a ) .", "a 75% reduction in cd80 mrna was observed in dec205 dcs that had taken up dec - lnps containing cd80-specific sirna ( compared with dec - lnps containing control sirnas ; figure 4b ) .", "we confirmed that gene knockdown was rnai - mediated using 5 race to detect sirna - directed mrna cleavage products ( figure 4c ) .", "sequencing of the polymerase chain reaction ( pcr ) fragment verified that it was derived from cd80 mrna and that the cut site corresponded with nucleotide positions 1011 of the sirna guide strand ( not shown ) .", "interestingly , while the data in figure 4 demonstrate the ability of our dec - lnps to effectively knockdown cd80 expression in dcs in vivo , we observed significant dc activation following injection of dec - lnps containing control sirnas ( figure 4a , right panel ) .", "this observation was surprising considering these experiments were performed using sirnas containing 2f modified rna , previously reported to have reduced immunostimulatory activity . to better assess this effect , we synthesized a series of luciferase - specific control sirnas ( luc ) containing 2oh rna ( unmodified ) , 2f pyrimidines or selected 2ome modifications ( see methods for modification strategy ; based on refs .", "lnps were generated and their ability to stimulate immune responses using multiple assays was examined .", "first , b6 mice were injected with dec - lnps that contained either unmodified , 2f or 2ome modified luc - specific sirnas in the absence of any adjuvant .", "one day later , splenic dcs were analyzed for the expression of costimulatory receptors ( supplementary figure s2a ) .", "as expected , dcs isolated from mice that had received unmodified sirnas demonstrated significant upregulation of cd40 , cd80 , and cd86 .", "consistent with our previous results ( figure 4a ) , animals treated with dec - lnps containing 2f modified sirnas also upregulated these costimulatory molecules . importantly , and similar to work by other groups , we found that 2ome modified sirnas induced minimal immune activation . to attempt to circumvent the need for a time and resource intensive in vivo assay , we wanted to determine whether an in vitro assay could be used to detect immunostimulatory sirnas .", "however , when we cultured b6-derived bmdcs with dec - lnps ( containing cd80-specific sirnas ) no difference between 2oh modified sirna , 2f or 2ome modified sirna was detected ( supplementary figure s2b ) .", "we also used nt - lnps to assess the effects of various sirna formulations on human pbmcs and modcs using standard preclinical assays ( supplementary figure s2c ) .", "pbmcs or modcs were cultured for 24 hours at which time cells were assessed for induction of apoptosis and culture supernatants were tested for the presence of proinflammatory cytokines ( see methods ) .", "apoptosis was not observed under any conditions tested ( data not shown ) . as expected , pbmcs cultured with the highest concentration of lnps containing unmodified sirnas elicited the secretion of several cytokines .", "2f and 2ome luc sirnas induced production of one cytokine ( il8 ) , also at the highest concentration tested .", "no cytokine production was observed following culture of modcs with lnps encapsulating unmodified , 2f or 2ome sirnas .", "taken together , these experiments suggest that the assays tested show marked differences in sensitivity . surprisingly , bmdcs and modcs were the least receptive for detecting immunostimulatory sirnas .", "conversely , dec - lnp injection was useful for the identification of sirnas with immune - activating potential . from these results", ", we used sirnas containing selective 2ome substitutions for all subsequent studies . to target autoimmune diseases , reducing the expression of several costimulatory molecules", "therefore , we next determined the ability of dec - lnps that encapsulated 2ome modified cd86 specific sirnas , to reduce gene expression following i.v . administration ( optimal sirna identified as previously ; supplementary figure s1 ) .", "as the sirnas were 2ome modified , mice were coinjected with lps to stimulate expression of costimulatory molecules .", "analysis of splenic dec205 dcs 1 day following dec - lnp injection showed reduction of cd86 protein to near steady - state levels ( figure 5a ) .", "total rna was isolated from dec205 dcs that had taken up dec - lnps encapsulating either cd86 or control sirnas .", "an approximately 70% reduction in cd86 mrna levels was observed from dcs isolated from mice that received dec - lnps containing cd86 sirna in comparison to dcs derived from control sirna treated mice . to verify knockdown occurred via the rnai pathway ,", "we next tested whether combining sirnas in dec - lnps would induce gene silencing equivalent to that observed with single sirnas .", "a combination of 2ome modified sirnas targeting cd80 , cd86 and cd40 were incorporated into dec - lnps and injected into b6 mice along with adjuvant ( lps ) to activate the dcs .", "reduction of each of the targeted costimulatory molecules was observed , with cd80 and cd86 knocked down to basal levels ( figure 5c ) .", "gene silencing was similar to that achieved following treatment with dec - lnps containing single sirnas ( e.g. , ~50% for cd86 : compare cd86 dec - lnp , figure 5a with mix dec - lnp , figure 5c right panel ) . to determine functional relevance of this knockdown , we used the robust mlr assay .", "b6 mice were injected with dec - lnps encapsulated with either a mix of cd80 , cd86 , and cd40 or control sirnas .", "after 24 hours , splenic cd8 dcs that had taken up dec - lnps were isolated , irradiated , and cultured with splenic t cells derived from a balb / c mouse .", "maximal proliferation was observed when dcs were isolated from mice injected with lps only , or lps plus dec - lnps containing control sirnas . in contrast , proliferation of t cells cultured with dcs derived from mice injected with lps plus dec - lnps containing cd80 , cd86 , and cd40 sirnas was significantly reduced ( figure 5d ) .", "currently , the most advanced lnp platforms use the ionizable cationic lipid dlindma , or its dlin - kc2-dma and dlin - mc3-dma derivatives .", "following systemic administration , these lnps accumulate in first pass organs , i.e. , liver and spleen , making them attractive vehicles for hepatic gene knockdown .", "furthermore , these lnps have been shown to safely and effectively reduce expression of hepatocyte genes in clinical trials .", "however , there is only very limited data demonstrating targeted delivery of potentially clinically relevant lnps to nonhepatic cells .", "we have shown that conjugation of a scfv , specific for the dc receptor dec205 , is sufficient to direct delivery of dlindma - formulated dec - lnps to splenic dcs .", "similar to conventional nt - lnps , dec - lnps incorporated sirnas with high efficiency ( > 80% ) , and were of uniform diameter ( ~100 nmol / l ) .", "we found that coating lnps with only ~50 scfv was sufficient for dc delivery ( figures 1 , 2 , and 3 ) .", "we showed that dec - lnp uptake correlated well with dec205 receptor density : dcs > b cells , t cells and macrophages ( dec205 expression is 1050-fold less than on dcs ) .", "use of dec205 mice further confirmed that uptake occurred via the dec205 receptor ( figures 2a and 3d ) .", "we also showed that the intracellular pathway accessed by dec - lnps was consistent with the dec205 pathway ( figure 2b ) .", "importantly , we demonstrated that targeted delivery resulted in effective rnai - mediated gene silencing of one , or several genes , to essentially basal expression levels ( figures 4 and 5a  c ) .", "this reduction in gene expression was sufficient to inhibit a robust mlr ( figure 5d ) .", "coated lnps formulated with the cationic lipid ddab with full - length dec205 antibody for sirna delivery and demonstrated the ability to reduce expression of the costimulatory molecule cd40 in dec205 dcs . while some gene knockdown was achieved , this lipid is known for its adjuvant qualities , and is being developed for applications that require immune response stimulation .", "the lnps also proved relatively inefficient at sirna loading ( 10% compared with 85% for dlindma lnps ) .", "the sirnas used were also unmodified , which could serve as another possible source of immune stimulation . therefore , the potential for this formulation appears to be limited .", "more recently , ramishetti et al . utilized a full - length antibody targeting cd4 to enhance t - cell uptake and induce sirna - mediated gene silencing using a clinically relevant lnp formulation similar to the one we employed in our work .", "interestingly , both this study and the work from zheng et al . chose to utilize full - length antibodies for lnp targeting , which may limit their utility when considering the immunogenicity of whole antibodies and their rapid clearance from the circulation by fc - mediated uptake by macrophages .", "in fact , in preliminary studies , we coated lnps with full - length dec205 antibody and failed to observe uptake by dec205 dcs . using our dec - lnps", "mg / kg ) comparable to that study which used n - acetylgalactosamine ( galnac)-coated lnps to target hepatocytes via the asialoglycoprotein receptor ( asgpr ) in apoe knockout mice .", "in contrast , when the ability of nt - lnps to silence antigen presenting cells ( apcs ) , including dcs , was investigated a dose of 5  mg / kg was required to achieve partial silencing : ~10-fold higher when compared with our dec - lnps .", "furthermore , the authors used the dlindma derivative , dlin - kc2-dma , which displays improved silencing ability in hepatocytes ( relative ed50 dlin - kc2-dma : 0.1  mg / kg versus dlindma : 1  mg / kg ) . as these lnps were formulated for effective gene silencing in hepatocytes ,", "biodistribution studies show that nt - lnps are found in the spleen , although at ~50-fold less than in the liver . while targeting ligands", "have been shown to have little effect on the overall biodistribution of nanoparticle formulations , they can enhance cell - specific uptake . therefore , while high doses of nt - lnps can confer partial gene silencing in apcs , at low lnp concentrations , we find that a targeted approach is required to achieve knockdown in a specific dc subset", ". injection of low - dose nt - lnps failed to result in detectable uptake / gene silencing .", "as the rnai pathway is resident in the cytoplasm , the intracellular pathway used following receptor ligation is an important consideration .", "we chose the well - characterized dec205 receptor for targeting our lnps . following binding ,", "the internalized receptor is routed to late endosomes and associated cargo gains access to the cytoplasm .", "the dlindma lipid fuses with anionic phospholipids present in the endosomal membrane resulting in release of encapsulated cargo into the cytoplasm .", "however , it is likely that different routes of uptake will affect the efficiency of delivery and subsequent gene silencing . thus , a logical extension to our approach is to determine whether we observe improved gene silencing using other dc specific targeting agents and/or more effective dlindma derivatives .", "we are currently investigating the efficacy of dec - lnps formulated with the dlin - mc3-dma ( ed50 0.03  mg / kg ) .", "having demonstrated that we could efficiently target sirnas to dcs , the lack of toxicity of these lnps and their cargo had to be established .", "we observed significant dc activation following injection of targeted lnps containing unmodified and 2f modified sirnas , and incorporation of 2ome modifications at distinct residues was required to maintain costimulatory molecules analyzed at basal levels ( figure 4a and supplementary figure s2a ) .", "as injecting mice to determine immune stimulation is time and resource intensive , we assessed immune stimulation in murine and human tissue culture systems .", "murine bmdcs were refractory to lnp stimulation suggesting they would not serve as a useful surrogate for gauging immunogenicity of lnps .", "taken together , these assays demonstrate a requirement for in vivo analysis to uncover the stimulatory capacity of the lnps .", "recently , a human whole blood cytokine assay has been described that showed similar activation profiles elicited by lnps when compared with in vivo injection , potentially circumventing a requirement for cumbersome in vivo analyses . for the purposes of inhibiting a multifactorial immune response , such as that observed in graft rejection or autoimmunity , the simultaneous knockdown of several ( co)stimulatory genes", "this approach has been used to demonstrate that combining antibodies targeting cd80 and cd86 can reduce disease severity in experimental autoimmune encephalomyelitis and in antigen - induced arthritis .", "transfer of dcs treated ex vivo with cd40 , cd80 , and cd86 antisense oligonucleotides ( as - odn ) into nod mice delayed type 1 diabetes ( t1d ) onset .", "a follow - up study demonstrated that multiple injections of microspheres containing cd40 , cd80 , and cd86 as - odns were sufficient to prevent t1d , possibly due to reduced expression of the costimulatory molecules by splenic dcs .", "we achieve significant reduction in expression of cd40 , cd80 , and cd86 on splenic dcs using our dec - lnps following systemic delivery , and ongoing studies are addressing the durability of gene knockdown and the potential use of these dec - lnps for treating autoimmune diseases . in summary", ", we have shown that a clinically relevant lnp can be modified for cell - specific delivery of sirnas . in vivo administration", "did not produce overt inflammatory responses , and we achieved significant rnai - mediated gene - specific knockdown at low sirna doses .", "these attributes make this an attractive platform to warrant further investigation as a potential therapeutic agent for various autoimmune diseases .", "furthermore , judicious selection of the ligand coating the lnp opens up the potential for targeting multiple cell types .", "mice . c57bl/6 ( b6 ) and balb / c mice were purchased from taconic ( hudson , ny ) .", "dec205 mice were bred to homozygosity on the b6 background ( dec205 ) , and validated by pcr and flow cytometry .", "mice were housed in the barrier facility at the institute for animal studies , albert einstein college of medicine ( bronx , ny ) according to institutional animal care and use committee ( iacuc ) guidelines .", "the single - chain fragment variable ( scfv ) antibodies constructed for dec205 ( clone nldc145 ) and isotype ( clone gl117 ) were cloned from antibody constructs , as previously described .", "the variable regions of the heavy and light chain sequences were fused with a flexible linker ( g4s)6 separating them and cloned into a pet28a ( emd millipore , billerica , ma ) bacterial expression vector .", "further modifications to the constructs included a strep - tag - ii ( wshpqfek ) at the n - terminus and a 10-residue histidine tag ( h10 ) with a short flexible linker ( g4s ) near the c - terminus .", "a c - terminal unpaired cysteine residue was introduced for scfv conjugation to the lipid dspe - peg - mal .", "the scfv constructs were transformed into bl21 ( de3 ) plyss competent cells ( promega , fitchburg , wi ) .", "the scfv dec205 or isotype were expressed in escherichia coli strain bl21 ( de3 ) plyss as insoluble inclusion bodies . following bacterial growth in luria broth ( lb ) medium and induction of protein expression with isopropyl 1-thio - d - galactopyranoside", "cells were lysed , and insoluble protein pelleted by centrifugation . solubilized inclusion bodies were passed over a ni - nta column and the bound protein was eluted with 8  m guanidine hydrochloride .", "the purified proteins were refolded by rapid dilution in refolding buffer , and purified by size - exclusion chromatography using superdex 200 ( ge healthcare life sciences , pittsburgh , pa ) .", "proteins were buffer - exchanged in phosphate - buffered saline ( pbs ) and concentrated .", "scfvs were tested for endotoxin contamination using a kinetic chromogenic limulous amoebocyte lysate ( lal ) assay ( kinetic - qcl lal assay , lonza , walkersville , md ) .", "the free cysteine engineered onto the n - terminus of the scfv was reduced using tris-(2-carboxyethyl ) phosphine hydrochloride ( life technologies , carlsbad , ca ) . following buffer exchange into pbs", ", the reduced scfv was reacted with a 10-fold molar excess of alexafluor488 ( af488)-c5-maleimide according to manufacturer instructions ( life technologies , carlsbad , ca ) .", "polyacrylamide gel electrophoresis ( sds - page ) and coomassie staining and storm molecular phosphorimager scanning of the stained gel ( ge healthcare , piscataway , nj ) . preparation of lnps .", "lnps were formed using the following lipids at a 15:40:5:40  mol % : dspc ( avanti polar lipids , alabaster , al ) , cholesterol ( chol ; sigma - aldrich , mo ) , 1,2-distearoyl - sn - glycero-3-phosphatidylethanolamine - n-(maleimide-(polyethylene glycol)-2000 ) ( dspe - peg - mal , avanti polar lipids , alabaster , al ) , and 1,2-dilinoleyloxy-3-dimethylaminopropane ( dlindma ) synthesized by the chemical biology core facility at albert einstein college of medicine as previously described .", "a fluorophore conjugated to a lipid , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl ) ( dope - rhodamine b ; avanti polar lipids , alabaster , al ) was incorporated ( 0.1 % ) to monitor lnp delivery .", "lnps were formed using a modified version of the spontaneous vesicle formation by ethanol dilution method .", "the lipid mixture was extruded ( mini - extruder and 1  ml gas - tight syringes , avanti polar lipids , alabaster , al ) sequentially with polycarbonate 200 and 80  nm pore size membranes .", "the extruded lipid mixture was mixed via rapid pipetting and vortexing with sirna , followed by dialysis into pbs .", "for attachment of scfv , lnps were mixed with reduced scfv at room temperature for 1 hour , and free maleimide groups were then quenched with -mercaptoethanol ( me ) .", "tangential flow filtration ( microkros filter module ) with a 500  kda mwco ( spectrum laboratories , rancho dominguez , ca ) was used for buffer exchange into pbs and concentration of lnps .", "lnps were assayed for size and polydispersity by dynamic light scattering ( dynapro plate reader , wyatt technology , santa barbara , ca ) .", "cryo - electron microscopy confirmed lnp size , geometry and lamellarity ( analytical imaging facility ; albert einstein college of medicine , bronx , ny ) .", "sirna incorporation was determined by quant - it ribogreen rna assay ( life technologies , carlsbad , ca ) . to determine scfv conjugation efficiency , lnps were resolved on sds - page gels .", "a 3  kda difference in migration of the scfv was indicative of binding to dspe - peg - mal ( unconjugated scfv ~30  kda , scfv - dspe - peg - mal ~33  kda ) .", "a cholesterol quantification kit ( abcam , cambridge , ma ) was use to determine the total lipid concentration . based on lipid concentration , the surface area per lipid head group ( ~0.6  nm ) , lnp diameter and their unilamellar structure , ~5060 scfvs are conjugated to each lnp under the conditions outlined above .", "sirna synthesis . modified and unmodified individual sirna sequences were synthesized on an expedite 8909 dna synthesizer ( biolytic , fremont , ca ) and purified by standard protocols , as described .", "dylight 547-phosphoramidite was attached to the 5 end of the sense strand for synthesis of fluorescently labeled sirna ( dy547-sirna ; glen research , sterling , va ) .", "sirnas were modified by 2f substitutions at every pyrimidine or by 2ome substitutions at the bolded nucleotides : cd40 sense 5-gaagauuaucccggucauauu-3 ; cd40 anti - sense 5-uaugaccg ; ggauaaucuucuu-3 ; cd80 sense 5-gaauuaccuggcaucaauauu-3 ; cd80 anti - sense 5-uauugaugccagguaauucuu-3 ; cd86 sense 5-caacuggacucuacg acuuuu-3 ; cd86 anti - sense 5-aagucguagaguccaguuguu-3 ; control sense 5-uagcgacuaaacacaucaauu-3 ; control anti - sense 5-uugauguguuuaguc gcuauu-3. luciferase sense 5-gauuauguccgguuauguauu-3 ; luciferase anti - sense 5-uacauaaccggacauaaucuu-3. lnp binding , uptake , and intracellular localization .", "cho cells , cho cells that stably express dec205 ( cho - dec ) , a dec205 b lymphoma line ( a20 cells ) and bmdcs , derived from b6 and dec205 mice , were used to assess lnp binding and uptake .", "cells were grown in complete medium ( dulbecco 's modified eagle 's medium , 5% fbs , 10 mmol / l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 20 mmol / l l - glutamine ) .", "bmdcs were generated from bone marrow derived from the femur and tibia as previously described .", "adherent cells ( a20 , cho ) were harvested using ethylenediaminetetraacetic acid to preserve receptor expression and were incubated at 4 c , 30 minutes with 500 nmol / l scfv - lnps ( containing either 0.1% dope - rhodamine b or cy3-labeled sirnas ) . for uptake ,", "cells were washed with fluorescence - activated cell sorting buffer ( pbs containing 0.5% bsa , 0.1% na n3 , 2 mmol / l edta ) and analyzed by flow cytometry ( lsrii , becton dickinson , franklin lakes , nj ; flowjo , ashland , or ) . to monitor intracellular localization , a20 cells were incubated with scfv - lnps , containing sirnas labeled with dylight 547 ( ge dharmacon , lafayette , co ) , 1 hour , 37  c .", "following one wash with pbs , cells were pipetted onto poly - l - lysine coated coverslips and allowed to adhere .", "adhered cells were fixed with 4% paraformaldehyde , washed with pbs and stained with anti - lamp1 ( clone 1d4b ; ebioscience , san diego , ca ) and anti - eea1 ( clone c45b10 ; cell signaling technology , danvers , ma ) .", "stained cells were mounted with vectashield mounting media containing 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories , burlingame , ca ) and samples were visualized on a leica sp5 aobs confocal microscope ( leica , buffalo grove , il ) .", "image processing ( linear adjustments to brightness and contrast ) was performed on image files using nih imagej software ( bethesda , md ) .", "lnps ( 0.61  mg / kg ) were injected ( retroorbital ( r.o . ) ) into b6 mice and 25  ng ultrapure lps ( invivogen , san diego , ca ) was injected r.o .", "one day later , spleens were harvested , collagenase treated to maximize dc yield , and single cell suspensions subjected to red cells lysis .", "cells were stained with cell surface markers and analyzed by flow cytometry to determine lnp uptake and expression of costimulatory molecules .", "antibodies were used for detection of the following cell surface markers : cd11c , cd8 , dec205 , cd40 , cd80 , cd86 , cd19 , b220 , cd3 , tcr , f4/80 , cd11b , h2k , dcir2 , cd49b , cd45.1 .", "cell viability was monitored using a live / dead exclusion dye , i.e. , live / dead blue ( life technologies , carlsbad , ca ) .", "cells were acquired using an lsrii cytometer and data analyzed using flowjo software . to measure gene knockdown , cd11ccd8 splenocytes that had taken up lnps ( i.e. , sirna - dy547 ) were sorted by flow cytometry ( facs aria iii , becton dickinson , franklin lakes , nj ) into rnaprotect ( qiagen , valencia , ca ) to stabilize rna .", "total rna was isolated using the rneasy rna isolation kit ( qiagen , valencia , ca ) , reverse transcribed using superscript iii ( life technologies , carlsbad , ca ) and random hexamers ( idt , coralville , ia ) , according to manufacturer 's instructions .", "quantitative pcr was performed using platinum taq polymerase ( life technologies , carlsbad , ca ) and sybr green to detect amplified products on an iq5 icycler ( biorad , hercules , ca ) .", "reactions were performed in triplicate with the following primers : u6 forward : 5-ctcgcttcggcagcacatatacta-3 ; u6 reverse : 5-acgaatttgcgtgtcatccttgcg-3 ; cd40 forward : 5-cggctgtgcgcgctatg-3 ; cd40 reverse : 5-ggctgagaattcgcctgagtc-3 ; cd80 forward : 5-ctactctcttatcatcctgggcct-3 ; cd80 reverse : 5-cccggaagcaaagcaggtaatcct-3 ; cd86 forward : 5-gccacccacaggatcaattatcct-3 ; cd86 reverse : 5-aaagagagaggctgttggagatac-3. relative amounts of mrna were normalized to u6 mrna and primer specificity verified by melt curve analysis . to detect rnai - mediated cleavage products ,", "total rna was isolated from cd11ccd8 splenocytes that had taken up lnps , i.e. , sirna - dy547 ( as for gene knockdown by qpcr ) , ligated at the 5 end to an adapter rna oligonucleotide and first strand cdna was synthesized using a gene - specific primer ( gsp ) .", "pcr amplification using an adapter - specific primer and a nested gsp primer will amplify the cleavage product and identify the cut site which corresponds to position 10/11 of the sirna guide strand if mediated via rnai .", "the primers and oligonucleotide sequences used : adapter rna : 5-cgacuggagcacgaggacacugacauggacugaaggaguagaaa-3 ; gsp forward : 5-cgactggagcacgaggacactga-3 ; cd80 gsp reverse : 5-tgcgccgaatcctgccccaa-3 ; cd80 reverse nested : 5-atcaggagggtcttctgggg-3 ; cd86 gsp reverse : 5-agtaactgaagctgtaatctccttccaata-3 ; cd86 reverse nested : 5-ctgtgacattatcttgtgatatctgcatgt-3. analysis of immune stimulation .", "cd11c murine bmdcs ( day 6 ; macs purified , miltenyi biotec , san diego , ca ) were cultured with dec - lnps and monitored for activation ( cd40 , cd80 , cd86 ) 1 day later by flow cytometry . in vivo stimulation", "was assessed by injection of dec - lnps r.o . into b6 mice , followed by analysis of activation markers ( cd40 , cd80 , cd86 ) on splenic dcs after 24 hours by flow cytometry .", "the pbmc fraction was used directly or following purification of cd14 cells ( stemcell technologies , vancouver , british columbia , canada ) , monocytes were cultured for 5 days in the presence of granulocyte - macrophage colony - stimulating factor and il4 to generate modcs .", "nt - lnps ( 5 , 1 , 0.2 , 0.04 , 0.008 mol / l ) were cultured with pbmcs or modcs overnight and cell culture supernatants collected for cytokine analysis ( ifn , il1 , il2 , il4 , il5 , il6 , il7 , il8 , il10 , il12 , il13 , tnf , granulocyte - macrophage colony - stimulating factor ) using a luminex multiplex format ( life technologies carlsbad , ca ) .", "cd11ccd8 splenic dcs were isolated by flow cytometry from b6 mice injected the previous day with dec - lnps containing either a 1:1:1 mix of cd40 , cd80 , and cd86 sirnas or control sirnas .", "splenic t cells from balb / c mice were isolated by macs using a pan t cell isolation kit ii ( miltenyi , san diego , ca ) .", "macs sorted balb / c t cells were carboxyfluorescein succinimidyl ester labeled according to published protocols , and carboxyfluorescein succinimidyl ester - labeled t cells were cocultured with the irradiated b6 dcs .", "three days later , dilution of carboxyfluorescein succinimidyl ester was monitored by flow cytometry to determine t - cell activation ."], "abstract_text": ["<S> due to their ability to knock down the expression of any gene , sirnas have been heralded as ideal candidates for treating a wide variety of diseases , including those involving  </S>", "<S> undruggable  targets . however , the therapeutic potential of sirnas remains severely limited by a lack of effective delivery vehicles . </S>", "<S> recently , lipid nanoparticles ( lnps ) containing ionizable cationic lipids have been developed for hepatic sirna delivery . </S>", "<S> however , their suitability for delivery to other cell types has not been determined . </S>", "<S> we have modified lnps for preferential targeting to dendritic cells ( dcs ) , central regulators of immune responses . to achieve directed delivery , we coated lnps with a single - chain antibody ( scfv ; dec - lnps ) , specific to murine dec205 , which is highly expressed on distinct dc subsets . </S>", "<S> here we show that injection of sirnas encapsulated in dec - lnps are preferentially delivered to dec205 + dcs . </S>", "<S> gene knockdown following uptake of dec - lnps containing sirnas specific for the costimulatory molecules cd40 , cd80 , and cd86 dramatically decreases gene expression levels . </S>", "<S> we demonstrate the functionality of this knockdown with a mixed lymphocyte response ( mlr ) . </S>", "<S> overall , we report that injection of lnps modified to restrict their uptake to a distinct cell population can confer profound gene knockdown , sufficient to inhibit powerful immune responses like the mlr . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Discussion", "Materials and Methods", "Supplementary Material"], "sections": [["the potential of rnai to silence any gene has made it an attractive therapeutic modality .", "however , the main obstacle to rnai in the clinic is delivery . to be effective", ", sirnas must be transported through the body , bind , and be taken up by target cells where they must traverse the plasma membrane and gain access to the cytosolic compartment , where the rnai machinery resides . to be useful ,", "ease of formulation and administration , overall cost , and any associated toxicities are essential considerations .", "dcs are heterogeneous , with subsets defined phenotypically , functionally and by location ( reviewed in ref .", "2 ) . a delivery vehicle that knocks down expression of specific genes in a distinct dc population(s ) would be a valuable tool for targeting diverse diseases including cancers , infectious diseases , autoimmunity and as a vaccine component .", "therefore , a platform that delivers sirnas to specific dc subsets in situ would be useful for inhibiting or activating immune responses .", "lipid nanoparticles ( lnps ) are one of the most advanced platforms for sirna delivery to hepatocytes , and are under clinical evaluation for conditions that require hepatic gene silencing .", "these lnps typically contain ionizable cationic lipids ( pka ~6.5 ) that bind nucleic acids via electrostatic interactions at low ph , but are charge neutral at ph 7.4 . as a well - perfused organ", "furthermore , lnp uptake by hepatocytes is mediated by association with serum apoe leading to efficient uptake via low - density lipoprotein receptors in the liver .", "following cellular uptake of the lnp , the ionization of the lipid within acidic endosomes is thought to promote sirna escape to the cytosol .", "importantly , these lnps are associated with minimal toxicity , including little induction of proinflammatory cytokines following administration of physiologically relevant doses .", "in contrast to the liver , sirna delivery to extra - hepatic cells is challenging . in particular , immune cells such as dcs are relatively resistant to sirna uptake in vitro and in vivo .", "although designed for hepatic gene silencing , the efficacy of these lnps and their derivatives has been assessed for rnai - mediated gene silencing in macrophages ( mos ) and dcs in vitro and in vivo .", "lnp uptake and modest gene silencing was achieved at high doses of lnps ( ~ 5mg / kg ) .", "together with a complementary study , this work demonstrates the feasibility of gene silencing in immune cells using lnp technology . clearly", ", however , lnp formulations must be modified to achieve optimal gene silencing in immune cells .", "enhanced uptake of lnps by primary hepatocytes is mediated by apolipoprotein e binding to the neutral lnps .", "this results in recognition by receptors including the low - density lipoprotein receptor and scavenger receptors largely expressed on hepatocytes .", "the mechanism of uptake strongly suggests that retargeting of these particles to other receptors or other tissues is possible .", "indeed , akinc et al . demonstrated that lnps modified with n - acetylgalactosamine ( galnac ) were retargeted to the asialoglycoprotein receptor ( asgpr ) expressed by hepatocytes in vivo .", "for delivery to nonhepatic cells , we have shown in vitro that anti - transferrin receptor aptamers can be used to redirect similar lnps .", "more recently , liang et al . described a similar approach using aptamer - coated lnps to target osteoblasts in vivo .", "for uptake by immune cells , a recent study coated lnps with a full - length anti - cd4 antibody for sirna delivery to primary cd4 t cells .", "lnp uptake and gene silencing was observed in cd4 t cells in various organs including the spleen , lymph nodes , and blood .", "these studies demonstrate the potential for attaching exogenous ligands to lnps for delivery to hepatic , and importantly nonhepatic primary cells in vivo . drawing from these works", ", we reasoned that this lnp platform could be a useful foundation for the delivery of sirnas to dcs to modulate immune responses . to achieve this", ", we modified lnps using a single chain antibody ( scfv ) specific for the dc receptor dec205 , a c - type lectin expressed at high levels on cd8 dcs .", "dec205 dcs mediate cross - presentation of antigen resulting in modulation of cd8 t - cell responses . therefore , inhibition of gene expression by dec205 dcs is a potentially powerful approach for regulating cd8 t - cell activation . using this approach , we assessed the ability of dec - lnps ( lnps coated with scfv specific for murine dec205 , containing sirnas specific for costimulatory molecules ) to inhibit immune responses .", "injection of dec - lnps resulted in preferential uptake of the dec - lnps by splenic dec205 dcs .", "furthermore , when coadministered with adjuvant , lnps containing sirna targeting cd40 , cd80 , and cd86 reduced expression of these costimulatory molecules to levels similar to those observed in immature dcs .", "most importantly , dcs isolated from mice injected with a low dose ( ~0.6  mg / kg ) of these dec - lnps , were able to suppress a robust mixed lymphocyte reaction ( mlr ) , demonstrating the functional efficacy of this approach .", "interestingly , when we performed experiments using 2 ' fluoro ( 2'f ) modified sirna , a formulation reported to be nonimmunostimulatory and nonimmunogenic , significant immune activation of the targeted dcs was detected . this effect was not observed in assays performed with human peripheral blood mononuclear cells ( pbmcs ) or monocyte - derived dcs ( modcs ) and could be mitigated through selective 2-o - methyl ( 2ome ) modification of the sirna .", "overall , our study demonstrates the functionality of lnps modified for receptor targeting for sirna delivery to dcs .", "additionally our results suggest that specific cell targeting can mediate immunological responses to sirna formulations .", "while care needs to be taken in designing and evaluating targeted approaches using lnps , we found that 2ome modification of the sirna was sufficient to negate immune activation .", "our approach enhances the efficacy of sirna - mediated gene silencing by ~10-fold when compared to nontargeted delivery .", "therefore , this targeted strategy should prove effective for regulating multiple immune - mediated diseases ."], ["lnps containing sirnas specific either for cd80 , cd86 , cd40 , or a control ( nontarget ) sequence were synthesized by extrusion using a mixture of 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc):dlindma : dspe - peg : cholesterol at a ratio of 15:40:5:40 ( based on refs .", "murine anti - dec205 scfv with a c - terminal cysteine was attached to the lipid dspe - peg by a maleimide group ( figure 1a , b ) . following synthesis , we subjected lnps to quality control to ensure consistency between batches .", "dynamic light scattering was used to determine diameter and polydispersity of lnps , and cryo - electron microscopy confirmed lnp size and their unilamellar structure ( figure 1c , d ) .", "the efficiency of sirna incorporation and scfv binding to dspe - peg was also assessed ( figure 1c , e ) . to show specificity of binding to dec205 ,", "scfv - lnps containing dy547-labeled sirna were cultured with either parental ( dec205 ) or dec205 cho cells . as seen in figure 2a , at 4 c dec205 cells bind dec - lnps approximately fourfold better compared with nontargeted lnps ( nt - lnps : lnps not coated with any scfv ) , or lnps coated with an isotype scfv ( iso - lnps ; cho - dec205 panel ) . when similar assays were performed at 37 c to allow cell uptake , cell staining increased by at least fivefold .", "little uptake was observed when lnps ( targeted or nontargeted ) were incubated with parental cho cells ( cho panel ) .", "similar results were obtained when bone marrow - derived dcs ( bmdcs ) , derived from wild - type mice ( b6 ) were incubated with dec - lnps .", "we note that as dec205 is expressed at lower levels on bmdcs compared with cho - dec205 , less dec - lnp binding and uptake was observed in bmdcs ( ~2-fold enhancement compared with nt- or iso - lnps ; b6 bmdc panel ) . to further confirm target - specific uptake we performed experiments using bmdcs derived from dec205 mice , and no specific cell binding or uptake", "the dec205 antibody is internalized via receptor - mediated endocytosis , and is targeted to late endosomes and lysosomes .", "confocal microscopy confirmed that similar to dec205 antibody , dec - lnps were targeted to lamp-1 late endosomes or lysosomes ( figure 2b ) .", "having demonstrated dec205-mediated uptake of dec - lnps in vitro , we investigated their localization in vivo .", "b6 mice were injected systemically ( intravenous , i.v . ) with fluorescently labeled scfv - lnps .", "as the dec205 receptor is endocytosed when it binds its ligand , we could not use this molecule to identify dec205 dcs .", "therefore , we used cd8 , a protein that is coexpressed on dec205 dcs , as a surrogate marker for tracking these cells .", "more than 50% of cd11ccd8 cells took up dec - lnps , and uptake was largely restricted to the cd8 dc population , with little uptake observed by cd8 dcs ( figure 3a , b ) .", "specificity was demonstrated by comparison of uptake of dec - lnps and iso - lnps .", "significant uptake was only observed in dec205 dcs following dec - lnp injection , and other splenic immune cells took up little dec- or iso - lnps ( figure 3a , c ) . when dec - lnps were injected into dec205 mice ,", "no uptake was detected ( figure 3d ) . to show that dec - lnps were competent for specific gene silencing , we generated dec - lnps containing a sirna specific for cd80 ( having identified the most effective sirna sequence ; see supplementary figure s1 ) and injected into b6 mice .", "cd11cdec205 cells took up similar amounts of lnps following injection with dec - lnp containing either cd80- or control - sirna ( lnp uptake panel , mean fluorescence intensity ( mfi ) : dec - lnp - control = 443 versus dec - lnp - sicd80 = 464 ) . in mice injected with dec - lnp encapsulating cd80-specific sirna , cd80 protein expression approached basal levels , and was reduced by ~2-fold when compared with dec - lnp containing control - sirna ( figure 4a ) .", "a 75% reduction in cd80 mrna was observed in dec205 dcs that had taken up dec - lnps containing cd80-specific sirna ( compared with dec - lnps containing control sirnas ; figure 4b ) .", "we confirmed that gene knockdown was rnai - mediated using 5 race to detect sirna - directed mrna cleavage products ( figure 4c ) .", "sequencing of the polymerase chain reaction ( pcr ) fragment verified that it was derived from cd80 mrna and that the cut site corresponded with nucleotide positions 1011 of the sirna guide strand ( not shown ) .", "interestingly , while the data in figure 4 demonstrate the ability of our dec - lnps to effectively knockdown cd80 expression in dcs in vivo , we observed significant dc activation following injection of dec - lnps containing control sirnas ( figure 4a , right panel ) .", "this observation was surprising considering these experiments were performed using sirnas containing 2f modified rna , previously reported to have reduced immunostimulatory activity . to better assess this effect , we synthesized a series of luciferase - specific control sirnas ( luc ) containing 2oh rna ( unmodified ) , 2f pyrimidines or selected 2ome modifications ( see methods for modification strategy ; based on refs .", "lnps were generated and their ability to stimulate immune responses using multiple assays was examined .", "first , b6 mice were injected with dec - lnps that contained either unmodified , 2f or 2ome modified luc - specific sirnas in the absence of any adjuvant .", "one day later , splenic dcs were analyzed for the expression of costimulatory receptors ( supplementary figure s2a ) .", "as expected , dcs isolated from mice that had received unmodified sirnas demonstrated significant upregulation of cd40 , cd80 , and cd86 .", "consistent with our previous results ( figure 4a ) , animals treated with dec - lnps containing 2f modified sirnas also upregulated these costimulatory molecules . importantly , and similar to work by other groups , we found that 2ome modified sirnas induced minimal immune activation . to attempt to circumvent the need for a time and resource intensive in vivo assay , we wanted to determine whether an in vitro assay could be used to detect immunostimulatory sirnas .", "however , when we cultured b6-derived bmdcs with dec - lnps ( containing cd80-specific sirnas ) no difference between 2oh modified sirna , 2f or 2ome modified sirna was detected ( supplementary figure s2b ) .", "we also used nt - lnps to assess the effects of various sirna formulations on human pbmcs and modcs using standard preclinical assays ( supplementary figure s2c ) .", "pbmcs or modcs were cultured for 24 hours at which time cells were assessed for induction of apoptosis and culture supernatants were tested for the presence of proinflammatory cytokines ( see methods ) .", "apoptosis was not observed under any conditions tested ( data not shown ) . as expected , pbmcs cultured with the highest concentration of lnps containing unmodified sirnas elicited the secretion of several cytokines .", "2f and 2ome luc sirnas induced production of one cytokine ( il8 ) , also at the highest concentration tested .", "no cytokine production was observed following culture of modcs with lnps encapsulating unmodified , 2f or 2ome sirnas .", "taken together , these experiments suggest that the assays tested show marked differences in sensitivity . surprisingly , bmdcs and modcs were the least receptive for detecting immunostimulatory sirnas .", "conversely , dec - lnp injection was useful for the identification of sirnas with immune - activating potential . from these results", ", we used sirnas containing selective 2ome substitutions for all subsequent studies . to target autoimmune diseases , reducing the expression of several costimulatory molecules", "therefore , we next determined the ability of dec - lnps that encapsulated 2ome modified cd86 specific sirnas , to reduce gene expression following i.v . administration ( optimal sirna identified as previously ; supplementary figure s1 ) .", "as the sirnas were 2ome modified , mice were coinjected with lps to stimulate expression of costimulatory molecules .", "analysis of splenic dec205 dcs 1 day following dec - lnp injection showed reduction of cd86 protein to near steady - state levels ( figure 5a ) .", "total rna was isolated from dec205 dcs that had taken up dec - lnps encapsulating either cd86 or control sirnas .", "an approximately 70% reduction in cd86 mrna levels was observed from dcs isolated from mice that received dec - lnps containing cd86 sirna in comparison to dcs derived from control sirna treated mice . to verify knockdown occurred via the rnai pathway ,", "we next tested whether combining sirnas in dec - lnps would induce gene silencing equivalent to that observed with single sirnas .", "a combination of 2ome modified sirnas targeting cd80 , cd86 and cd40 were incorporated into dec - lnps and injected into b6 mice along with adjuvant ( lps ) to activate the dcs .", "reduction of each of the targeted costimulatory molecules was observed , with cd80 and cd86 knocked down to basal levels ( figure 5c ) .", "gene silencing was similar to that achieved following treatment with dec - lnps containing single sirnas ( e.g. , ~50% for cd86 : compare cd86 dec - lnp , figure 5a with mix dec - lnp , figure 5c right panel ) . to determine functional relevance of this knockdown , we used the robust mlr assay .", "b6 mice were injected with dec - lnps encapsulated with either a mix of cd80 , cd86 , and cd40 or control sirnas .", "after 24 hours , splenic cd8 dcs that had taken up dec - lnps were isolated , irradiated , and cultured with splenic t cells derived from a balb / c mouse .", "maximal proliferation was observed when dcs were isolated from mice injected with lps only , or lps plus dec - lnps containing control sirnas . in contrast , proliferation of t cells cultured with dcs derived from mice injected with lps plus dec - lnps containing cd80 , cd86 , and cd40 sirnas was significantly reduced ( figure 5d ) ."], ["currently , the most advanced lnp platforms use the ionizable cationic lipid dlindma , or its dlin - kc2-dma and dlin - mc3-dma derivatives .", "following systemic administration , these lnps accumulate in first pass organs , i.e. , liver and spleen , making them attractive vehicles for hepatic gene knockdown .", "furthermore , these lnps have been shown to safely and effectively reduce expression of hepatocyte genes in clinical trials .", "however , there is only very limited data demonstrating targeted delivery of potentially clinically relevant lnps to nonhepatic cells .", "we have shown that conjugation of a scfv , specific for the dc receptor dec205 , is sufficient to direct delivery of dlindma - formulated dec - lnps to splenic dcs .", "similar to conventional nt - lnps , dec - lnps incorporated sirnas with high efficiency ( > 80% ) , and were of uniform diameter ( ~100 nmol / l ) .", "we found that coating lnps with only ~50 scfv was sufficient for dc delivery ( figures 1 , 2 , and 3 ) .", "we showed that dec - lnp uptake correlated well with dec205 receptor density : dcs > b cells , t cells and macrophages ( dec205 expression is 1050-fold less than on dcs ) .", "use of dec205 mice further confirmed that uptake occurred via the dec205 receptor ( figures 2a and 3d ) .", "we also showed that the intracellular pathway accessed by dec - lnps was consistent with the dec205 pathway ( figure 2b ) .", "importantly , we demonstrated that targeted delivery resulted in effective rnai - mediated gene silencing of one , or several genes , to essentially basal expression levels ( figures 4 and 5a  c ) .", "this reduction in gene expression was sufficient to inhibit a robust mlr ( figure 5d ) .", "coated lnps formulated with the cationic lipid ddab with full - length dec205 antibody for sirna delivery and demonstrated the ability to reduce expression of the costimulatory molecule cd40 in dec205 dcs . while some gene knockdown was achieved , this lipid is known for its adjuvant qualities , and is being developed for applications that require immune response stimulation .", "the lnps also proved relatively inefficient at sirna loading ( 10% compared with 85% for dlindma lnps ) .", "the sirnas used were also unmodified , which could serve as another possible source of immune stimulation . therefore , the potential for this formulation appears to be limited .", "more recently , ramishetti et al . utilized a full - length antibody targeting cd4 to enhance t - cell uptake and induce sirna - mediated gene silencing using a clinically relevant lnp formulation similar to the one we employed in our work .", "interestingly , both this study and the work from zheng et al . chose to utilize full - length antibodies for lnp targeting , which may limit their utility when considering the immunogenicity of whole antibodies and their rapid clearance from the circulation by fc - mediated uptake by macrophages .", "in fact , in preliminary studies , we coated lnps with full - length dec205 antibody and failed to observe uptake by dec205 dcs . using our dec - lnps", "mg / kg ) comparable to that study which used n - acetylgalactosamine ( galnac)-coated lnps to target hepatocytes via the asialoglycoprotein receptor ( asgpr ) in apoe knockout mice .", "in contrast , when the ability of nt - lnps to silence antigen presenting cells ( apcs ) , including dcs , was investigated a dose of 5  mg / kg was required to achieve partial silencing : ~10-fold higher when compared with our dec - lnps .", "furthermore , the authors used the dlindma derivative , dlin - kc2-dma , which displays improved silencing ability in hepatocytes ( relative ed50 dlin - kc2-dma : 0.1  mg / kg versus dlindma : 1  mg / kg ) . as these lnps were formulated for effective gene silencing in hepatocytes ,", "biodistribution studies show that nt - lnps are found in the spleen , although at ~50-fold less than in the liver . while targeting ligands", "have been shown to have little effect on the overall biodistribution of nanoparticle formulations , they can enhance cell - specific uptake . therefore , while high doses of nt - lnps can confer partial gene silencing in apcs , at low lnp concentrations , we find that a targeted approach is required to achieve knockdown in a specific dc subset", ". injection of low - dose nt - lnps failed to result in detectable uptake / gene silencing .", "as the rnai pathway is resident in the cytoplasm , the intracellular pathway used following receptor ligation is an important consideration .", "we chose the well - characterized dec205 receptor for targeting our lnps . following binding ,", "the internalized receptor is routed to late endosomes and associated cargo gains access to the cytoplasm .", "the dlindma lipid fuses with anionic phospholipids present in the endosomal membrane resulting in release of encapsulated cargo into the cytoplasm .", "however , it is likely that different routes of uptake will affect the efficiency of delivery and subsequent gene silencing . thus , a logical extension to our approach is to determine whether we observe improved gene silencing using other dc specific targeting agents and/or more effective dlindma derivatives .", "we are currently investigating the efficacy of dec - lnps formulated with the dlin - mc3-dma ( ed50 0.03  mg / kg ) .", "having demonstrated that we could efficiently target sirnas to dcs , the lack of toxicity of these lnps and their cargo had to be established .", "we observed significant dc activation following injection of targeted lnps containing unmodified and 2f modified sirnas , and incorporation of 2ome modifications at distinct residues was required to maintain costimulatory molecules analyzed at basal levels ( figure 4a and supplementary figure s2a ) .", "as injecting mice to determine immune stimulation is time and resource intensive , we assessed immune stimulation in murine and human tissue culture systems .", "murine bmdcs were refractory to lnp stimulation suggesting they would not serve as a useful surrogate for gauging immunogenicity of lnps .", "taken together , these assays demonstrate a requirement for in vivo analysis to uncover the stimulatory capacity of the lnps .", "recently , a human whole blood cytokine assay has been described that showed similar activation profiles elicited by lnps when compared with in vivo injection , potentially circumventing a requirement for cumbersome in vivo analyses . for the purposes of inhibiting a multifactorial immune response , such as that observed in graft rejection or autoimmunity , the simultaneous knockdown of several ( co)stimulatory genes", "this approach has been used to demonstrate that combining antibodies targeting cd80 and cd86 can reduce disease severity in experimental autoimmune encephalomyelitis and in antigen - induced arthritis .", "transfer of dcs treated ex vivo with cd40 , cd80 , and cd86 antisense oligonucleotides ( as - odn ) into nod mice delayed type 1 diabetes ( t1d ) onset .", "a follow - up study demonstrated that multiple injections of microspheres containing cd40 , cd80 , and cd86 as - odns were sufficient to prevent t1d , possibly due to reduced expression of the costimulatory molecules by splenic dcs .", "we achieve significant reduction in expression of cd40 , cd80 , and cd86 on splenic dcs using our dec - lnps following systemic delivery , and ongoing studies are addressing the durability of gene knockdown and the potential use of these dec - lnps for treating autoimmune diseases . in summary", ", we have shown that a clinically relevant lnp can be modified for cell - specific delivery of sirnas . in vivo administration", "did not produce overt inflammatory responses , and we achieved significant rnai - mediated gene - specific knockdown at low sirna doses .", "these attributes make this an attractive platform to warrant further investigation as a potential therapeutic agent for various autoimmune diseases .", "furthermore , judicious selection of the ligand coating the lnp opens up the potential for targeting multiple cell types ."], ["mice . c57bl/6 ( b6 ) and balb / c mice were purchased from taconic ( hudson , ny ) .", "dec205 mice were bred to homozygosity on the b6 background ( dec205 ) , and validated by pcr and flow cytometry .", "mice were housed in the barrier facility at the institute for animal studies , albert einstein college of medicine ( bronx , ny ) according to institutional animal care and use committee ( iacuc ) guidelines .", "the single - chain fragment variable ( scfv ) antibodies constructed for dec205 ( clone nldc145 ) and isotype ( clone gl117 ) were cloned from antibody constructs , as previously described .", "the variable regions of the heavy and light chain sequences were fused with a flexible linker ( g4s)6 separating them and cloned into a pet28a ( emd millipore , billerica , ma ) bacterial expression vector .", "further modifications to the constructs included a strep - tag - ii ( wshpqfek ) at the n - terminus and a 10-residue histidine tag ( h10 ) with a short flexible linker ( g4s ) near the c - terminus .", "a c - terminal unpaired cysteine residue was introduced for scfv conjugation to the lipid dspe - peg - mal .", "the scfv constructs were transformed into bl21 ( de3 ) plyss competent cells ( promega , fitchburg , wi ) .", "the scfv dec205 or isotype were expressed in escherichia coli strain bl21 ( de3 ) plyss as insoluble inclusion bodies . following bacterial growth in luria broth ( lb ) medium and induction of protein expression with isopropyl 1-thio - d - galactopyranoside", "cells were lysed , and insoluble protein pelleted by centrifugation . solubilized inclusion bodies were passed over a ni - nta column and the bound protein was eluted with 8  m guanidine hydrochloride .", "the purified proteins were refolded by rapid dilution in refolding buffer , and purified by size - exclusion chromatography using superdex 200 ( ge healthcare life sciences , pittsburgh , pa ) .", "proteins were buffer - exchanged in phosphate - buffered saline ( pbs ) and concentrated .", "scfvs were tested for endotoxin contamination using a kinetic chromogenic limulous amoebocyte lysate ( lal ) assay ( kinetic - qcl lal assay , lonza , walkersville , md ) .", "the free cysteine engineered onto the n - terminus of the scfv was reduced using tris-(2-carboxyethyl ) phosphine hydrochloride ( life technologies , carlsbad , ca ) . following buffer exchange into pbs", ", the reduced scfv was reacted with a 10-fold molar excess of alexafluor488 ( af488)-c5-maleimide according to manufacturer instructions ( life technologies , carlsbad , ca ) .", "polyacrylamide gel electrophoresis ( sds - page ) and coomassie staining and storm molecular phosphorimager scanning of the stained gel ( ge healthcare , piscataway , nj ) . preparation of lnps .", "lnps were formed using the following lipids at a 15:40:5:40  mol % : dspc ( avanti polar lipids , alabaster , al ) , cholesterol ( chol ; sigma - aldrich , mo ) , 1,2-distearoyl - sn - glycero-3-phosphatidylethanolamine - n-(maleimide-(polyethylene glycol)-2000 ) ( dspe - peg - mal , avanti polar lipids , alabaster , al ) , and 1,2-dilinoleyloxy-3-dimethylaminopropane ( dlindma ) synthesized by the chemical biology core facility at albert einstein college of medicine as previously described .", "a fluorophore conjugated to a lipid , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl ) ( dope - rhodamine b ; avanti polar lipids , alabaster , al ) was incorporated ( 0.1 % ) to monitor lnp delivery .", "lnps were formed using a modified version of the spontaneous vesicle formation by ethanol dilution method .", "the lipid mixture was extruded ( mini - extruder and 1  ml gas - tight syringes , avanti polar lipids , alabaster , al ) sequentially with polycarbonate 200 and 80  nm pore size membranes .", "the extruded lipid mixture was mixed via rapid pipetting and vortexing with sirna , followed by dialysis into pbs .", "for attachment of scfv , lnps were mixed with reduced scfv at room temperature for 1 hour , and free maleimide groups were then quenched with -mercaptoethanol ( me ) .", "tangential flow filtration ( microkros filter module ) with a 500  kda mwco ( spectrum laboratories , rancho dominguez , ca ) was used for buffer exchange into pbs and concentration of lnps .", "lnps were assayed for size and polydispersity by dynamic light scattering ( dynapro plate reader , wyatt technology , santa barbara , ca ) .", "cryo - electron microscopy confirmed lnp size , geometry and lamellarity ( analytical imaging facility ; albert einstein college of medicine , bronx , ny ) .", "sirna incorporation was determined by quant - it ribogreen rna assay ( life technologies , carlsbad , ca ) . to determine scfv conjugation efficiency , lnps were resolved on sds - page gels .", "a 3  kda difference in migration of the scfv was indicative of binding to dspe - peg - mal ( unconjugated scfv ~30  kda , scfv - dspe - peg - mal ~33  kda ) .", "a cholesterol quantification kit ( abcam , cambridge , ma ) was use to determine the total lipid concentration . based on lipid concentration , the surface area per lipid head group ( ~0.6  nm ) , lnp diameter and their unilamellar structure , ~5060 scfvs are conjugated to each lnp under the conditions outlined above .", "sirna synthesis . modified and unmodified individual sirna sequences were synthesized on an expedite 8909 dna synthesizer ( biolytic , fremont , ca ) and purified by standard protocols , as described .", "dylight 547-phosphoramidite was attached to the 5 end of the sense strand for synthesis of fluorescently labeled sirna ( dy547-sirna ; glen research , sterling , va ) .", "sirnas were modified by 2f substitutions at every pyrimidine or by 2ome substitutions at the bolded nucleotides : cd40 sense 5-gaagauuaucccggucauauu-3 ; cd40 anti - sense 5-uaugaccg ; ggauaaucuucuu-3 ; cd80 sense 5-gaauuaccuggcaucaauauu-3 ; cd80 anti - sense 5-uauugaugccagguaauucuu-3 ; cd86 sense 5-caacuggacucuacg acuuuu-3 ; cd86 anti - sense 5-aagucguagaguccaguuguu-3 ; control sense 5-uagcgacuaaacacaucaauu-3 ; control anti - sense 5-uugauguguuuaguc gcuauu-3. luciferase sense 5-gauuauguccgguuauguauu-3 ; luciferase anti - sense 5-uacauaaccggacauaaucuu-3. lnp binding , uptake , and intracellular localization .", "cho cells , cho cells that stably express dec205 ( cho - dec ) , a dec205 b lymphoma line ( a20 cells ) and bmdcs , derived from b6 and dec205 mice , were used to assess lnp binding and uptake .", "cells were grown in complete medium ( dulbecco 's modified eagle 's medium , 5% fbs , 10 mmol / l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 20 mmol / l l - glutamine ) .", "bmdcs were generated from bone marrow derived from the femur and tibia as previously described .", "adherent cells ( a20 , cho ) were harvested using ethylenediaminetetraacetic acid to preserve receptor expression and were incubated at 4 c , 30 minutes with 500 nmol / l scfv - lnps ( containing either 0.1% dope - rhodamine b or cy3-labeled sirnas ) . for uptake ,", "cells were washed with fluorescence - activated cell sorting buffer ( pbs containing 0.5% bsa , 0.1% na n3 , 2 mmol / l edta ) and analyzed by flow cytometry ( lsrii , becton dickinson , franklin lakes , nj ; flowjo , ashland , or ) . to monitor intracellular localization , a20 cells were incubated with scfv - lnps , containing sirnas labeled with dylight 547 ( ge dharmacon , lafayette , co ) , 1 hour , 37  c .", "following one wash with pbs , cells were pipetted onto poly - l - lysine coated coverslips and allowed to adhere .", "adhered cells were fixed with 4% paraformaldehyde , washed with pbs and stained with anti - lamp1 ( clone 1d4b ; ebioscience , san diego , ca ) and anti - eea1 ( clone c45b10 ; cell signaling technology , danvers , ma ) .", "stained cells were mounted with vectashield mounting media containing 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories , burlingame , ca ) and samples were visualized on a leica sp5 aobs confocal microscope ( leica , buffalo grove , il ) .", "image processing ( linear adjustments to brightness and contrast ) was performed on image files using nih imagej software ( bethesda , md ) .", "lnps ( 0.61  mg / kg ) were injected ( retroorbital ( r.o . ) ) into b6 mice and 25  ng ultrapure lps ( invivogen , san diego , ca ) was injected r.o .", "one day later , spleens were harvested , collagenase treated to maximize dc yield , and single cell suspensions subjected to red cells lysis .", "cells were stained with cell surface markers and analyzed by flow cytometry to determine lnp uptake and expression of costimulatory molecules .", "antibodies were used for detection of the following cell surface markers : cd11c , cd8 , dec205 , cd40 , cd80 , cd86 , cd19 , b220 , cd3 , tcr , f4/80 , cd11b , h2k , dcir2 , cd49b , cd45.1 .", "cell viability was monitored using a live / dead exclusion dye , i.e. , live / dead blue ( life technologies , carlsbad , ca ) .", "cells were acquired using an lsrii cytometer and data analyzed using flowjo software . to measure gene knockdown , cd11ccd8 splenocytes that had taken up lnps ( i.e. , sirna - dy547 ) were sorted by flow cytometry ( facs aria iii , becton dickinson , franklin lakes , nj ) into rnaprotect ( qiagen , valencia , ca ) to stabilize rna .", "total rna was isolated using the rneasy rna isolation kit ( qiagen , valencia , ca ) , reverse transcribed using superscript iii ( life technologies , carlsbad , ca ) and random hexamers ( idt , coralville , ia ) , according to manufacturer 's instructions .", "quantitative pcr was performed using platinum taq polymerase ( life technologies , carlsbad , ca ) and sybr green to detect amplified products on an iq5 icycler ( biorad , hercules , ca ) .", "reactions were performed in triplicate with the following primers : u6 forward : 5-ctcgcttcggcagcacatatacta-3 ; u6 reverse : 5-acgaatttgcgtgtcatccttgcg-3 ; cd40 forward : 5-cggctgtgcgcgctatg-3 ; cd40 reverse : 5-ggctgagaattcgcctgagtc-3 ; cd80 forward : 5-ctactctcttatcatcctgggcct-3 ; cd80 reverse : 5-cccggaagcaaagcaggtaatcct-3 ; cd86 forward : 5-gccacccacaggatcaattatcct-3 ; cd86 reverse : 5-aaagagagaggctgttggagatac-3. relative amounts of mrna were normalized to u6 mrna and primer specificity verified by melt curve analysis . to detect rnai - mediated cleavage products ,", "total rna was isolated from cd11ccd8 splenocytes that had taken up lnps , i.e. , sirna - dy547 ( as for gene knockdown by qpcr ) , ligated at the 5 end to an adapter rna oligonucleotide and first strand cdna was synthesized using a gene - specific primer ( gsp ) .", "pcr amplification using an adapter - specific primer and a nested gsp primer will amplify the cleavage product and identify the cut site which corresponds to position 10/11 of the sirna guide strand if mediated via rnai .", "the primers and oligonucleotide sequences used : adapter rna : 5-cgacuggagcacgaggacacugacauggacugaaggaguagaaa-3 ; gsp forward : 5-cgactggagcacgaggacactga-3 ; cd80 gsp reverse : 5-tgcgccgaatcctgccccaa-3 ; cd80 reverse nested : 5-atcaggagggtcttctgggg-3 ; cd86 gsp reverse : 5-agtaactgaagctgtaatctccttccaata-3 ; cd86 reverse nested : 5-ctgtgacattatcttgtgatatctgcatgt-3. analysis of immune stimulation .", "cd11c murine bmdcs ( day 6 ; macs purified , miltenyi biotec , san diego , ca ) were cultured with dec - lnps and monitored for activation ( cd40 , cd80 , cd86 ) 1 day later by flow cytometry . in vivo stimulation", "was assessed by injection of dec - lnps r.o . into b6 mice , followed by analysis of activation markers ( cd40 , cd80 , cd86 ) on splenic dcs after 24 hours by flow cytometry .", "the pbmc fraction was used directly or following purification of cd14 cells ( stemcell technologies , vancouver , british columbia , canada ) , monocytes were cultured for 5 days in the presence of granulocyte - macrophage colony - stimulating factor and il4 to generate modcs .", "nt - lnps ( 5 , 1 , 0.2 , 0.04 , 0.008 mol / l ) were cultured with pbmcs or modcs overnight and cell culture supernatants collected for cytokine analysis ( ifn , il1 , il2 , il4 , il5 , il6 , il7 , il8 , il10 , il12 , il13 , tnf , granulocyte - macrophage colony - stimulating factor ) using a luminex multiplex format ( life technologies carlsbad , ca ) .", "cd11ccd8 splenic dcs were isolated by flow cytometry from b6 mice injected the previous day with dec - lnps containing either a 1:1:1 mix of cd40 , cd80 , and cd86 sirnas or control sirnas .", "splenic t cells from balb / c mice were isolated by macs using a pan t cell isolation kit ii ( miltenyi , san diego , ca ) .", "macs sorted balb / c t cells were carboxyfluorescein succinimidyl ester labeled according to published protocols , and carboxyfluorescein succinimidyl ester - labeled t cells were cocultured with the irradiated b6 dcs .", "three days later , dilution of carboxyfluorescein succinimidyl ester was monitored by flow cytometry to determine t - cell activation ."], [""]]}
{"article_id": "PMC3127317", "article_text": ["sevoflurane does not sensitise the myocardium to arrhythmogenic effects of catecholamines and has the least effect on cardiac conduction system .", "despite all the research demonstrating its safety , adverse cardiac arrhythmias such as nodal rhythm with bradycardia , prolongation of qtc interval , precipitation of torsade de pointes and isorhythmic atrioventricular dissociation ( isravd ) have been reported with sevoflurane anaesthesia .", "a 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy .", "she was a known hypertensive since 4 years , on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily , with no other comorbidities .", "preoperative pulse rate ( pr ) was 60/min and blood pressure ( bp ) was 140/70 mmhg .", "preoperative 12 lead electrocardiograms ( ecg ) showed sinus rhythm with a rate of 60/min .", "diazepam 10 mg was administered orally the night before and 60 minutes before surgery , along with morning dose of metoprolol and amlodipine .", "anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . during induction ,", "sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion ( size 3 ) was accomplished with suxamethonium 100 mg .", "capnograph showed a square wave form . immediately following induction , ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats , then reappeared and recede from qrs complex .", "ecg also showed intermittent electrical alternans [ figure 1 ] and bp dropped to 86/50 mmhg .", "meanwhile , anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen .", "end - tidal carbon dioxide ( etco2 ) was maintained between 35 and 37 mmhg .", "pr ( 40 - 50 beats / min ) and rhythm ( isravd ) remained unaltered throughout the procedure that lasted for 3 hours .", "atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg .", "a discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter .", "hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . at the end of surgery , sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine ( 1.2 mg ) and neostigmine ( 2.5 mg ) .", "few minutes after removal of the lma , rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg .", "metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively .", "patient was observed in intensive care unit for the next 48 hours . throughout the postoperative course , hr and rhythm remained normal .", "real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace .", "the difference in heart rate ( ecg trace ) and pulse rate ( pulse oximetry trace ) is seen due to electrical alternans .", "real - time picture from the monitor shows the inspired and expired concentration of sevoflurane a 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair .", "premedication administered was 7 mg of oral midazolam , 30 minutes before surgery . in the operating room ,", "baseline pr was 88/min with sinus rhythm ; spo2 , 99% on room air and nibp , 96/62 mmhg .", "inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen ( fresh gas flow 5 l / min ) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min .", "inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration .", "the etco2 was between 31 and 33 mmhg , as measured through the angle piece interfaced between the face mask and the breathing circuit . after breathing 8% sevoflurane for 2 minutes", "sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . at that moment , spo2 was 99% and bp was 66/36 mmhg .", "intravenous fentanyl ( 30 g ) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen ( fresh gas flow rate 5 l / min ) .", "a 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy .", "she was a known hypertensive since 4 years , on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily , with no other comorbidities .", "preoperative pulse rate ( pr ) was 60/min and blood pressure ( bp ) was 140/70 mmhg .", "preoperative 12 lead electrocardiograms ( ecg ) showed sinus rhythm with a rate of 60/min .", "diazepam 10 mg was administered orally the night before and 60 minutes before surgery , along with morning dose of metoprolol and amlodipine .", "anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . during induction ,", "sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion ( size 3 ) was accomplished with suxamethonium 100 mg .", "capnograph showed a square wave form . immediately following induction , ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats , then reappeared and recede from qrs complex .", "ecg also showed intermittent electrical alternans [ figure 1 ] and bp dropped to 86/50 mmhg .", "meanwhile , anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen .", "end - tidal carbon dioxide ( etco2 ) was maintained between 35 and 37 mmhg .", "pr ( 40 - 50 beats / min ) and rhythm ( isravd ) remained unaltered throughout the procedure that lasted for 3 hours .", "atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg .", "a discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter .", "hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . at the end of surgery , sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine ( 1.2 mg ) and neostigmine ( 2.5 mg ) .", "few minutes after removal of the lma , rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg .", "metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively .", "patient was observed in intensive care unit for the next 48 hours . throughout the postoperative course , hr and rhythm remained normal .", "real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace .", "the difference in heart rate ( ecg trace ) and pulse rate ( pulse oximetry trace ) is seen due to electrical alternans .", "a 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair .", "premedication administered was 7 mg of oral midazolam , 30 minutes before surgery . in the operating room ,", "baseline pr was 88/min with sinus rhythm ; spo2 , 99% on room air and nibp , 96/62 mmhg .", "inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen ( fresh gas flow 5 l / min ) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min .", "inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration .", "the etco2 was between 31 and 33 mmhg , as measured through the angle piece interfaced between the face mask and the breathing circuit .", "after breathing 8% sevoflurane for 2 minutes , the child did not show any motor response to jaw thrust .", "sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . at that moment , spo2 was 99% and bp was 66/36 mmhg .", "intravenous fentanyl ( 30 g ) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen ( fresh gas flow rate 5 l / min ) .", "american college of cardiology / american heart association recommends using volatile agents during non - cardiac surgery for the maintenance of general anaesthesia in haemodynamically stable patients at risk for myocardial ischemia . anticipating a difficult airway and considering the patient 's cardiac status , and associated hypertension ,", "isravd was recorded during induction of anaesthesia with sevoflurane which reverted back to sinus rhythm after discontinuation of sevoflurane during emergence .", "2 pacemakers ( sinus and av nodes ) fortuitously discharge at the same or nearly similar rate , without antegrade or retrograde conduction across the av node .", "either slowing of the sinus node discharge rate or the emergence of a slightly faster subsidiary pacemaker controlling the ventricles is the common initiating event .", "p waves approaches the narrow qrs complex , disappears within it for several beats , then reappears and recedes from the qrs and this sequence may be repeated .", "the same was observed in the ecg recording in the present case [ figure 1 ] . from the limited number of experimental studies , the development of isravd and the particular electrocardiographic patterns resulting from", "there are dependent upon relative discharge rate of the dominant atrial and subsidiary pacemakers , presence or absence of retrograde conduction , chronotropic response to atrial stretch , baroreceptor - mediated autonomic nervous system output and responsiveness of the sinus node .", "high incidence of nodal rhythm ( 20% ) and bradycardia with use of sevoflurane has been reported in unpremedicated infants .", "the onset of nodal rhythm was significantly earlier in the high - concentration technique than with incremental induction technique .", "on the other hand , nakaigawa et al . demonstrated that up to 2 mac ( minimal alveolar concentration ) sevoflurane does not affect cardiac conduction system significantly . in both our patients , 8% sevoflurane was used for induction that possibly resulted in isravd and junctional rhythm .", "isravd was possibly precipitated by the volatile agent affecting ca release from the sarcoplasmic reticulum , resulting in depression of ca slow inward current in the myocardium . in patients on ca channel and -adrenergic blocker therapy ,", "the arrhythmogenic potential of sevoflurane is likely to be enhanced as ca channel blockers inhibit the entry of ca into the cell or its mobilisation from intracellular stores , while b - adrenergic blockers depresses sinus rate and av node conduction .", "reported a case of intraoperative torsades de pointes ventricular tachycardia and ventricular fibrillation during sevoflurane anaesthesia , which persisted with dc shock but reverted to sinus rhythm after 10 minutes of discontinuation of sevoflurane and after the patient was awakened .", "a discrepancy was observed between the hr and the pr due to intermittent electrical alternans in the ecg .", "it is therefore wise to rely on the pr rather than the hr in such cases to treat bradycardia .", "rarely , sevoflurane - induced suppression of baroreceptor reflex activity might result in desynchronisation between the atria and ventricle causing haemodynamic instability which may require electrical pacing .", "to conclude , sevoflurane in high concentration ( 8% ) should be used with caution in children and in conditions where primary pacemaker activity is suppressed , as in elderly .", "caution should also be exercised with its use in patients on ca channel and b blocker therapy ."], "abstract_text": ["<S> when inhalational induction is indicated , sevoflurane is the most popular choice in both children and adults . </S>", "<S> higher inspired concentrations of sevoflurane can cause adverse cardiac arrhythmias that are seen to disappear once the agent is discontinued . </S>", "<S> we report the occurrence of isorhythmic atrioventricular dissociation and junctional rhythm with absent p waves during sevoflurane anaesthesia in an adult and a child , respectively . </S>"], "labels": null, "section_names": ["Introduction", "Case Reports", "Case 1", "Case 2", "Discussion"], "sections": [["sevoflurane does not sensitise the myocardium to arrhythmogenic effects of catecholamines and has the least effect on cardiac conduction system .", "despite all the research demonstrating its safety , adverse cardiac arrhythmias such as nodal rhythm with bradycardia , prolongation of qtc interval , precipitation of torsade de pointes and isorhythmic atrioventricular dissociation ( isravd ) have been reported with sevoflurane anaesthesia ."], ["a 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy .", "she was a known hypertensive since 4 years , on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily , with no other comorbidities .", "preoperative pulse rate ( pr ) was 60/min and blood pressure ( bp ) was 140/70 mmhg .", "preoperative 12 lead electrocardiograms ( ecg ) showed sinus rhythm with a rate of 60/min .", "diazepam 10 mg was administered orally the night before and 60 minutes before surgery , along with morning dose of metoprolol and amlodipine .", "anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . during induction ,", "sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion ( size 3 ) was accomplished with suxamethonium 100 mg .", "capnograph showed a square wave form . immediately following induction , ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats , then reappeared and recede from qrs complex .", "ecg also showed intermittent electrical alternans [ figure 1 ] and bp dropped to 86/50 mmhg .", "meanwhile , anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen .", "end - tidal carbon dioxide ( etco2 ) was maintained between 35 and 37 mmhg .", "pr ( 40 - 50 beats / min ) and rhythm ( isravd ) remained unaltered throughout the procedure that lasted for 3 hours .", "atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg .", "a discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter .", "hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . at the end of surgery , sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine ( 1.2 mg ) and neostigmine ( 2.5 mg ) .", "few minutes after removal of the lma , rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg .", "metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively .", "patient was observed in intensive care unit for the next 48 hours . throughout the postoperative course , hr and rhythm remained normal .", "real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace .", "the difference in heart rate ( ecg trace ) and pulse rate ( pulse oximetry trace ) is seen due to electrical alternans .", "real - time picture from the monitor shows the inspired and expired concentration of sevoflurane a 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair .", "premedication administered was 7 mg of oral midazolam , 30 minutes before surgery . in the operating room ,", "baseline pr was 88/min with sinus rhythm ; spo2 , 99% on room air and nibp , 96/62 mmhg .", "inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen ( fresh gas flow 5 l / min ) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min .", "inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration .", "the etco2 was between 31 and 33 mmhg , as measured through the angle piece interfaced between the face mask and the breathing circuit . after breathing 8% sevoflurane for 2 minutes", "sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . at that moment , spo2 was 99% and bp was 66/36 mmhg .", "intravenous fentanyl ( 30 g ) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen ( fresh gas flow rate 5 l / min ) ."], ["a 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy .", "she was a known hypertensive since 4 years , on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily , with no other comorbidities .", "preoperative pulse rate ( pr ) was 60/min and blood pressure ( bp ) was 140/70 mmhg .", "preoperative 12 lead electrocardiograms ( ecg ) showed sinus rhythm with a rate of 60/min .", "diazepam 10 mg was administered orally the night before and 60 minutes before surgery , along with morning dose of metoprolol and amlodipine .", "anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . during induction ,", "sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion ( size 3 ) was accomplished with suxamethonium 100 mg .", "capnograph showed a square wave form . immediately following induction , ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats , then reappeared and recede from qrs complex .", "ecg also showed intermittent electrical alternans [ figure 1 ] and bp dropped to 86/50 mmhg .", "meanwhile , anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen .", "end - tidal carbon dioxide ( etco2 ) was maintained between 35 and 37 mmhg .", "pr ( 40 - 50 beats / min ) and rhythm ( isravd ) remained unaltered throughout the procedure that lasted for 3 hours .", "atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg .", "a discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter .", "hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . at the end of surgery , sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine ( 1.2 mg ) and neostigmine ( 2.5 mg ) .", "few minutes after removal of the lma , rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg .", "metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively .", "patient was observed in intensive care unit for the next 48 hours . throughout the postoperative course , hr and rhythm remained normal .", "real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace .", "the difference in heart rate ( ecg trace ) and pulse rate ( pulse oximetry trace ) is seen due to electrical alternans ."], ["a 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair .", "premedication administered was 7 mg of oral midazolam , 30 minutes before surgery . in the operating room ,", "baseline pr was 88/min with sinus rhythm ; spo2 , 99% on room air and nibp , 96/62 mmhg .", "inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen ( fresh gas flow 5 l / min ) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min .", "inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration .", "the etco2 was between 31 and 33 mmhg , as measured through the angle piece interfaced between the face mask and the breathing circuit .", "after breathing 8% sevoflurane for 2 minutes , the child did not show any motor response to jaw thrust .", "sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . at that moment , spo2 was 99% and bp was 66/36 mmhg .", "intravenous fentanyl ( 30 g ) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen ( fresh gas flow rate 5 l / min ) ."], ["american college of cardiology / american heart association recommends using volatile agents during non - cardiac surgery for the maintenance of general anaesthesia in haemodynamically stable patients at risk for myocardial ischemia . anticipating a difficult airway and considering the patient 's cardiac status , and associated hypertension ,", "isravd was recorded during induction of anaesthesia with sevoflurane which reverted back to sinus rhythm after discontinuation of sevoflurane during emergence .", "2 pacemakers ( sinus and av nodes ) fortuitously discharge at the same or nearly similar rate , without antegrade or retrograde conduction across the av node .", "either slowing of the sinus node discharge rate or the emergence of a slightly faster subsidiary pacemaker controlling the ventricles is the common initiating event .", "p waves approaches the narrow qrs complex , disappears within it for several beats , then reappears and recedes from the qrs and this sequence may be repeated .", "the same was observed in the ecg recording in the present case [ figure 1 ] . from the limited number of experimental studies , the development of isravd and the particular electrocardiographic patterns resulting from", "there are dependent upon relative discharge rate of the dominant atrial and subsidiary pacemakers , presence or absence of retrograde conduction , chronotropic response to atrial stretch , baroreceptor - mediated autonomic nervous system output and responsiveness of the sinus node .", "high incidence of nodal rhythm ( 20% ) and bradycardia with use of sevoflurane has been reported in unpremedicated infants .", "the onset of nodal rhythm was significantly earlier in the high - concentration technique than with incremental induction technique .", "on the other hand , nakaigawa et al . demonstrated that up to 2 mac ( minimal alveolar concentration ) sevoflurane does not affect cardiac conduction system significantly . in both our patients , 8% sevoflurane was used for induction that possibly resulted in isravd and junctional rhythm .", "isravd was possibly precipitated by the volatile agent affecting ca release from the sarcoplasmic reticulum , resulting in depression of ca slow inward current in the myocardium . in patients on ca channel and -adrenergic blocker therapy ,", "the arrhythmogenic potential of sevoflurane is likely to be enhanced as ca channel blockers inhibit the entry of ca into the cell or its mobilisation from intracellular stores , while b - adrenergic blockers depresses sinus rate and av node conduction .", "reported a case of intraoperative torsades de pointes ventricular tachycardia and ventricular fibrillation during sevoflurane anaesthesia , which persisted with dc shock but reverted to sinus rhythm after 10 minutes of discontinuation of sevoflurane and after the patient was awakened .", "a discrepancy was observed between the hr and the pr due to intermittent electrical alternans in the ecg .", "it is therefore wise to rely on the pr rather than the hr in such cases to treat bradycardia .", "rarely , sevoflurane - induced suppression of baroreceptor reflex activity might result in desynchronisation between the atria and ventricle causing haemodynamic instability which may require electrical pacing .", "to conclude , sevoflurane in high concentration ( 8% ) should be used with caution in children and in conditions where primary pacemaker activity is suppressed , as in elderly .", "caution should also be exercised with its use in patients on ca channel and b blocker therapy ."]]}
{"article_id": "PMC3964852", "article_text": ["according to the mca , only a compromised ability to make a decision in accordance with certain procedural norms , loosely defined with reference to the ability to  understand , ", "retain and  use or weigh relevant information , or an inability to communicate a decision , properly grounds judgments of incapacity ( s. 3(1 ) ) .", "in addition , the identified problems of decision - making must be attributed to  an impairment of , or disturbance in the functioning of , mind or brain ( s. 2(1 ) ) .", "this law aims to protect the individual 's right to pursue their own ends by adopting a capacity test that focuses on the decision - making process rather than the substance of the patient 's decision .", "influential liberal thinking holds that it is not the place of the law to pursue certain ends  rather , the law should aim to provide a space for individuals to pursue their own ends ( plant , 2011 )  and this position is reflected in english common law relating to mental capacity:[in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences .", "the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind.whether the patient 's decision is consistent with proposed substantive norms in relation to what they should pursue  for example that people should pursue good health , or that they should want to live  is said to be beyond the proper scope of the incapacity test . in adopting this position", ", english law allows that even the most controversial treatment decisions might be explained by the fact that the patient 's evaluation of the situation diverges from a clinical perspective , and perhaps many people 's evaluation of the situation . ", "[ in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences .", "the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind. in practice then , questions of mental capacity might be thought of as boiling down to a question about whether a decision can be explained in terms of the patient 's particular motivating commitments ( their desires , values , projects , ideas about a good life ) ; or whether the decision is properly explained in terms of a problem in decision - making , which is due to a dysfunction of mind or brain . is the patient 's controversial decision understandable in the light of their commitments ? or is the patient 's ability to understand , retain , and use or weigh what the likely costs and benefits of treatment will be for them , currently compromised ? scrutinizing a patient 's commitments in the context of a capacity assessment raises moral concerns because the possibility that a choice can be explained by idiosyncratic commitments plays a significant role in protecting the patient 's right to make controversial decisions .", "perhaps the most straightforward approach to conceiving of commitments , and the one most likely to protect this right , is to say that the person 's commitments are their current commitments  those they recognize at the time of the decision .", "the problem with conceiving of commitments in this way is that it fails to recognise certain problems of practical decision - making to do with motivational coherence over time .", "someone with bipolar disorder , for example , might clearly be choosing or acting in accordance with their current desires during a manic phase , but this perspective fails to capture what seemingly goes wrong with practical decision - making in such cases . as a result , if this understanding of commitments is adopted in assessments of mental capacity the reach of this area of law arguably does not extend as far as it should  a person may be judged to have mental capacity where this seems doubtful .", "psychopathologies characterised by this kind of problem seem better accounted for in a legal structure that conceives of mental capacity , and in particular , commitments , in diachronic terms .", "a diachronic perspective might also help illuminate some other hard cases that are not normally characterised in terms of problems of motivational coherence over time .", "for example , someone with anorexia nervosa may be choosing rationally in accordance with their current commitments  especially a strong desire not to gain weight , or to exert extreme control over what they eat  and so may be judged to have capacity on these grounds . however , an assessment of their evaluative capacities in diachronic terms might yield the conclusion that their decision - making ability is seriously compromised ( craigie , 2011 ) .", "their future self may come to the view that they should have been motivated in ways that they could not recognise or could not act on at the time , and this might be a fairly robust feature of people with a diagnosis of anorexia nervosa .", "this perspective in relation to questions of mental capacity chimes with the suggestion that particularly in psychiatry ,  colloquialisms [ such as ]  you will thank me later  sometimes become the unwritten rules within which the merits of a patient 's consent are assessed ( gostin , 1981 , p. 742 ) .", "such a perspective may also offer a solution in cases where the patient does not have a psychiatric diagnosis .", "mackenzie and watts discuss a case of a woman who was judged to have the capacity to refuse life - saving treatment , and subsequently died , in a situation where she had recently been left by her husband , following the amputation of both her legs .", "they argue that the trauma of these events and her subsequent inability to imagine a life without her partner clouded the woman 's judgment in relation to treatment:the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33", "; for a full description of the case , see halpern , 2012)differences between these cases suggest that taking a diachronic perspective in assessments of mental capacity allows for decision - making to be compromised in at least two ways . in the bipolar disorder and anorexia nervosa cases a diachronic perspective is used to argue that a person 's evaluative capacities may be compromised  the capacities that determine the desires or values that motivate a decision . in the emotional trauma case , which might equally be applied to cases of depression , a diachronic perspective is used to identify a problem in the person 's ability to assess whether what they want or value is achievable . according to jodi halpern 's original analysis of the emotional trauma case , the woman should have been judged to lack capacity on grounds that her beliefs about the future were  unresponsive to evidence , rendering her unable to  think through alternatives ( halpern , 2012 , pp .", " the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33", "; for a full description of the case , see halpern , 2012 ) one problem with adopting a diachronic perspective in assessments of mental capacity is that whether questions of procedural rationality are properly understood in diachronic terms remains a contested issue in the fields of economics , psychology and philosophy .", "the functional element of the mca 's incapacity test concerns problems in meeting certain procedural requirements  problems in the ability to understand , retain , use or weigh relevant information .", "sceptics deny that at least some norms derived from a diachronic perspective fall into this category .", "they hold that such requirements are substantive rather than procedural in nature , so i turn now to this theoretical concern .", "much of the normative and descriptive work on a diachronic perspective in personal decision - making has taken place in the fields of economics and psychology , but these fields have tended to adopt contrasting positions on the normative question .", "economists often treat a person 's weighting of the future as a preference like any other , in a framework that assumes that preferences are not available to rational criticism .", "prudence to refer to taking a diachronic perspective in decision - making:prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect ", "there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said .", " ( nagel , 1970 , p. 28)psychologists , on the other hand , often assume that people are rationally required to give significant weight to the future personal consequences of choices , the strongest version being that people ought to be temporally neutral in their decision - making .", "it is widely accepted within this camp that there are at least some good reasons to give less weight to future consequences , particularly in order to take into account an expected depreciation in their future value .", ". however , foreseeable changes in one 's circumstances can also be relevant :  50 might be much more valuable to a final year law student than  100 will be for them in the following year , when they reasonably expect to be working for a corporate lawyer . on these grounds", "it might be rational for the student to choose  50 now , foregoing  100 in a year 's time .", "likewise , it is argued that uncertainly with respect to future consequences can provide a good reason to choose a certain outcome now , over a more valuable but uncertain outcome in the future ( baron , 1994 , pp .", "i am using the term  temporally neutral to refer to decisions that give equal weight to future and present utility , excepting the above kinds of considerations .", "the pressing question is why people ought to be impartial with respect to all parts of their lives once these kinds of consideration are taken into account . ", "prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect ", "there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said .", " ( nagel , 1970 , p. 28 ) the arguments used to defend this position in the psychological literature tend to take the form that a temporally neutral orientation advances the goals of practical decision - making .", "choosing an outcome merely because it will happen sooner is said to be irrational because if one option  achieves your goals better , then you should choose that one , regardless of when you decide ( baron , 1994 , p. 514 ) . using the classic marshmallow experiments as an example ,", "if a child would rather eat two marshmallows than one , then they should choose to wait and be given two marshmallows sometime later rather than one marshmallow now  because overall this achieves the child 's goals better .", "this kind of argument considers the question of goal satisfaction from a position that is not situated at a particular point in time , and prescribes that people ought to take this perspective in their decision - making :  they should be just as concerned about themselves a year from now as they are about themselves this minute ( p. 516 )", ". individuals should give weight to the implications for their future self , so the argument goes , for their own overall good .", "nagel argues that prudence is a requirement of practical reason , which is secured because the personal consequences of a man 's action concern  his future ( 1970 , p. 42 ; his italics ) .", "according to nagel , this constraint on practical decision - making follows from the person 's awareness that they persist over time .", "it reflects the  individual 's conception of himself as a temporally persistent being : his ability to identify with past and future stages of himself and to regard them as forming a single life .", "failure to be susceptible to prudence entails radical dissociation from one 's future , one 's past , and from oneself as a whole , conceived as a temporally extended individual ( p. 58 ) .", "it is a mistake in practical reasoning because it comes  at the cost of dissociation from one 's temporally extended self ( p. 69 ) .", "the conception of self as extended throughout a human lifetime is powerful in the context of questions of procedural rationality because it offers grounds for claiming that a person ought to give weight to considerations that they may not currently care about .", "it offers an answer to what i have characterised as the economists ' view , that if a person has no interest in the future personal consequences of their choice there is nothing more to be said , rationally speaking .", "applied to the context of treatment decision - making , this kind of perspective is powerful because it offers grounds for calling a patient 's mental capacity into question , with reference to their ability to consider their future and value it appropriately .", "rejections of this kind of rational requirement are therefore often based on a rejection of the idea of the self as something that necessarily extends throughout a lifetime , and derek parfit 's account of personal identity is often used to make this argument ( baron , 1994 , p. 516 ; frederick , 2006 ; maclean , 2006 ) .", "parfit holds that the persistence of a person over time must be understood in terms of their ", "psychological continuity  the connectedness of psychological features such as memories , character , interests and preferences across a lifetime ( parfit , 1983 ; 1984 ) . on this view , whether the future inhabitant of one 's body will be the same person can be a matter of degree . psychological features sometimes change so radically over a human lifetime that a person now will not necessarily be the same person who occupies that body in the future . and", "not giving weight to the interests of a future self is irrational only to the degree that one shares the relevant psychological characteristics with that self .", "more recently , galen strawson has defended a similar position in his rejection of narrative accounts of the self .", "strawson argues that although experiencing oneself as one person throughout a lifetime  what he calls a ", "diachronic self - experience  may be more common , some people experience themselves in what he describes as a more  episodic way . unlike the more diachronic person ,", "the more episodic person has  little or no sense that the self that one is was there in the ( further ) past and will be there in the future , although one is perfectly aware that one has long - term continuity considered as a whole human being ( strawson , 2005 , p. 65 ) .  [ predominantly ] episodic individuals may sometimes connect to charged events in their pasts in such a way that they feel those events happened to them  embarrassing memories are a good example  and anticipate events in their futures in such a way that they think those events are going to happen to them  thoughts of death can be a good example ", "however , the episodic person has no  great or special interest in [ their ] past , and nor do they have  a great deal of concern for [ their ] future ( p. 67 ) .", "strawson holds that even a strongly episodic self is within the normal range of human experience , and that it is normatively on a par with a more diachronic self - experience . on his view", ", theorists such as nagel assert that selves extend over a lifetime , and diachronic norms of procedural rationality are derived from this claim .", "parfit and strawson dispute this assumption , and conclude that disregard for the future personal consequences of a choice may simply reflect an alternative way of living a human life , at least when we consider the question of reasons for action in non - moral terms .", "however , all of the theorists canvassed thus far reason from premises about the extension of self over a lifetime , to conclusions about what reasons individuals have , and it will be argued here that this conception of the relationship between the requirements of practical rationality and the metaphysics of self is the wrong way around . as described above , the kind of requirement in question is often defended  by nagel and others  on the grounds that not giving weight to the future undermines one 's own interests . however , at times a different kind of argument is suggested in nagel 's work .", "the suggestion seems to be that a unified self over a lifetime is not something that is automatically present , but rather something that is generated by giving weight to future personal consequences in decision - making .", "the proposed requirement is justified , not because it deploys the correct conception of self , or because one 's future self will otherwise regret the decision , but because adherence plays a role in bringing the self into being , by enabling the decision - maker  to reach towards something outside himself ( nagel , 1970 , p. 43 ) .", "michael bratman is particularly clear about the role that he proposes giving weight to the future plays in the generation of the self , and how this in turn grounds a diachronic perspective on questions of procedural rationality , so it is his view that i will focus on here .", " self - governing policies  which are integral to temporally extended agency  lie at the core of human agency because it is through these that a person 's identity emerges .", "these processes are essential for understanding what it is for an agent to  take a stand  what it is for an agent to recognise certain desires as one 's own  and it is this feature in particular that distinguishes human agency from the agency of other  purposive creatures ( bratman , 2000 , pp .", "adopting broadly the same approach to personal identity as parfit begins with , bratman suggests that the forward - looking psychological ties that play a role in constituting a unified self over time can be generated through reflective and planning processes : ", "i can help ensure appropriate psychological continuities and connections by sticking with and executing my prior plans and policies , and by monitoring and regulating my motivational structures in favor , say , of my continued commitment to philosophy ", "such processes  play an important role in the constitution and support of continuities and connections characteristic of the identity of the agent over time. ", "indeed, bratman suggests ,  this is what plans and policies are for ( 2000 , p. 47", "by emphasising the role that planning and policy - making play in generating the psychological ties that are proposed to underpin personal identity over time , bratman describes a picture of human agency in which the agent plays an active role . the temporally extended self develops through the agent 's exercise of certain psychological capacities . on this point bratman", "makes a significant departure from parfit and strawson , who seem to hold that the degree to which the relevant psychological ties extend over time is a fixed fact about the individual , from which rational requirements on their action may be derived .", "the claim that engaging in planning and policy - making processes is rationally required is defended primarily on the non - instrumental grounds that these processes play a central role in developing and sustaining the self .", "bratman 's argument takes the form of an answer to the problem that agents who reflect on their first - order desires and form second - order desires do not yet exhibit what is distinctive about human agency , because there is nothing to distinguish these new desires as those that the agent identifies with ; second - order desires seem to be ", "just one more wiggle in the psychic stew ( bratman , 2000 , p. 38 ) .", "the solution that bratman offers is that where an agent stands with respect to a particular first - order desire is established through the exercise of planning and policy - making practices :  self - governing policies might , so to speak , crystallize pressures from various elements of one 's psychic stew into a more decisive attitude that can , in the relevant context , establish where one stands ( 2000 , p. 51 ) .", "these mental processes that underpin temporally extended agency give authority to particular desires , so it is through these that the self  the distinctive feature of human agency  emerges . on this view , a compromised ability to engage in planning and policy - making processes threatens not the interests of the future self , but the very constitution of the self .", "this is what grounds the procedural requirement that one must give weight to the future personal consequences of actions . and this , we might suggest , is what makes the ability to consider and value the future an appropriate consideration to include in a mental capacity test .", "bratman 's account offers a story about the self that fits well with a developmental understanding of personhood that is often appealed to in contemporary medical law and ethics .", "his account of the relationship between temporally extended agency and the self also fits well with the difficulties associated with certain psychopathologies .", "it is no accident , i suggest , that disorders such as dementia , bipolar disorder and depression are associated with seriously compromised capacities for diachronic agency . among many other factors , in a complex picture , problems of diachronic agency surely make a contribution to our sense that these constellations of behavioural and experiential features are appropriate targets for the label of disorder .", "a sense of the self as compromised in the context of problems of diachronic agency is also found in first - person reports of dementia ( addis and tippett , 2004 ; however , see caddell and clare , 2012 ) and depression ( fuchs , 2008 ; ratcliffe , 2012 ) .", "it would seem that the functioning of capacities for diachronic agency is connected to our sense of others as having well - functioning human agency , as well as our subjective sense of selfhood , and for these reasons", "i find it implausible that a strongly episodic self - experience is just a less common way of living a human life .", "strawson defends a view along these lines , claiming that he himself is strongly episodic ( in his sense ) .", "yet he has a successful academic career which could not have been achieved without meeting a wide range of commitments that each extended over considerable periods of time , for example in completing academic qualifications and writing books .", "it seems to me that these achievements evidence considerable skills in diachronic agency . while for strawson a diachronic self - experience is connected to a narrative conception of the self , diachronicity in the sense concerned here", "a life lived in the moment could still be a life lived with diachronic agency , as i will argue at the end of the following section .", "i now turn back to questions of mental capacity and what implications a diachronic perspective such as bratman 's would have .", "the motivation for investigating these theoretical questions about procedural rationality was the idea that a diachronic perspective might enable a mental capacity test such as that adopted in the mca to deal with certain hard cases , particularly in the context of mental disorder , without undermining its value - neutral approach .", "the arguments above suggest that a diachronic perspective on questions of mental capacity is consistent with a procedural understanding of the functional element of the mca 's incapacity test .", "given this , could a diachronic understanding of what it is to have mental capacity help by justifying the inclusion of a requirement such as the ability to consider the future  as a part of what it means to be able to understand , retain , use or weigh information  in assessments of mental capacity ?", "something like this requirement already seems to be assumed in the condition that the patient must understand the consequences of the treatment options .", "the relevant information necessarily concerns the future as well as the present , due to the fact that understanding  the likely effects of deciding one way or the other is a part of this requirement ( mental capacity act 2005 code of practice , s. 4.16 ) .", "for example , a person with a severe learning disability might be judged to lack the capacity to consent to a blood test on grounds that they do not understand that the test is necessary for their future physical well - being .", "the same might be said in a case of a delirious patient who is resisting treatment for a serious injury . in such cases the person 's connection to the world , in the sense of their ability to understand how things are likely to be in the future ,", "what to do is limited in a way that can be compared to john stuart mill 's paradigm case of justified paternalistic intervention , where a person is about to cross a bridge that unbeknownst to them is unsafe", ". the person in the example does not understand that they risk falling into water if they attempt to cross .", "the widely accepted conclusion is that the person is not in a position to assess whether to cross the bridge , and this justifies the use of proportional physical force to stop them from crossing , when this is necessary to inform them about the state of the bridge .", "the same reasoning might be applied in the case of the woman who has recently suffered a serious emotional trauma , on grounds that her ability to imagine a range of future possibilities is severely limited ( halpern , 2012 ) .", "the patient can not currently conceive of alternative futures to the desperately unhappy one she imagines , and perhaps this justifies taking the decision out of her hands until she is seeing things more clearly .", "the problem that returns here is that understanding concerns not simply how things are likely to be , as in the fact that an attempt to cross the bridge is likely to result in a fall into water , or that a certain proportion of people who have both legs amputated learn to walk again using prosthetic limbs .", "it concerns understanding how the relevant state of affairs will be for this particular patient .", "this question about the future necessarily involves an evaluative judgment that , according to a value - neutral approach , only the patient is in a position to make .", "so while it might sometimes be clear that a patient 's ability to consider the future is compromised , for example when someone denies that their condition is life - threatening when it clearly is , in many cases assessing understanding in relation to the future will have no equivalent point of reference .", "in such a context there seems to be no standard against which the functional element of the mental capacity test  the part concerning the patient 's ability to understand , retain , use or weigh information  can be directly assessed .", "therefore , any conclusions drawn about this capacity would seem to be based on the fact that the patient has suffered an emotional trauma . removing a patient 's right to make their own treatment decision under these circumstances may seem justified given what we know about how people who lose limbs adapt , as reflected in self - reported measures of well - being , and given the known psychological effects of emotional trauma", ". however , an appeal to this kind of knowledge in an assessment of mental capacity would involve using generalised observations to draw a conclusion about a particular person 's capacity to assess how things will be for them in the future .", "the legislation is clear that an assessment of decision - making ability should not be based solely on features such as a patient 's diagnosis .", "it must be shown that this particular person 's mental capacities are relevantly impaired , not merely that they belong to a diagnostic category . however", ", this requirement can not be met in the case of this capacity , because there is no way to directly assess it while remaining value neutral in accordance with the aspirations of this area of law . what about the possibility raised at the beginning of this article , that a person 's evaluative capacities might be called into question when viewed from a diachronic perspective ?", "drawing on bratman 's account , planfulenss and self - governing policies are rationally required because of the role they play in developing and sustaining the self .", "caring about the interests of the future inhabitant of one 's body , and having at least some grip on what those interests will be , seem like prerequisites for engaging successfully in these processes . and perhaps this offers part of an explanation about how decision - making can be compromised in the context of disorders such as depression and bipolar disorder .", "very crudely , the idea would be that as part of a much bigger picture the capacities necessary for diachronic agency are not functioning properly and this undermines the constitution of the self .", "i have argued that , understood in this way , these considerations are procedural rather than substantive in nature .", "but would the implementation of these considerations in practice nonetheless come into conflict with the goal of preserving patient autonomy ? assessing a person 's grip on what interests the future inhabitant of their body will have runs into the same difficulties that occurred in the context of assessing understanding in the emotional trauma case . without making substantive assumptions", "judging that a person is likely to lack these capacities in the context of a manic episode may seem justified , because of what we know about the trajectory of bipolar disorder  that fairly reliably people 's commitments during a manic episode radically undermine their overall interests .", "but whether this kind of consideration provides grounds for a judgment of incapacity in english law depends on what evidence is considered appropriate for assessing the functional element of the incapacity test .", "however , as genevra richardson points out ,  existing case law indicates that courts will take a flexible approach to the legal definition [ of capacity ] to enable them to reach their preferred outcome ( richardson , this volume , p. 00 ) .", "this may mean that in practice significant weight is given to a person 's diagnosis .", "however , this solution undermines the protections of patient autonomy conferred by making an individual assessment of functional capacities , rather than diagnosis , the proper grounds for infringements on self - determination .", "a related possibility considered at the beginning of this article was that a diachronic perspective might be used to justify giving weight to a reasonable prediction of what the patient 's future self would want in retrospect , in assessments of mental capacity .", "however , it does not follow from the view offered here , even given the sure knowledge that a patient 's future self would retrospectively endorse a medical intervention , that their present self is irrational in refusing it . in the imagined case there is an unambiguous conflict between what the patient currently wants ( no treatment ) and what the future self will want in retrospect ( treatment ) . using this conflict to doubt the patient 's current capacity to make the treatment decision assumes that the future self 's perspective should be given greater weight in the current self 's decision - making .", "however , there is nothing in the present view that would justify resolving the conflict in this way .", "the proposed account requires that the agent exercise planfulness and self - governing policies in decision - making , which would seem to involve giving equal weight at most , not greater weight , to one 's expected future desires .", "moreover , humans have a well - established time preference for negative experiences to be in the past rather than the present or future , and this makes it easier for the patient to endorse involuntary treatment when it is in the past ( doherty , 2011 ) .", "this would seem to weigh in favour of privileging the perspective of the self who is facing the infringement on liberty . in the absence of any reasons to believe that the future self 's perspective should be privileged", ", this form of justification for doubting mental capacity looks to be question - begging .", "its favouring of the future self 's perspective already assumes that the current self 's decision - making capacity is relevantly impaired .", "favouring the future self 's perspective might seem justified on grounds that , for example , evaluative capacities are often compromised in the context of certain diagnoses or during experiences such as emotional trauma .", "however , at least in theory , this evidence alone does not justify a judgment of incapacity under current english law , for reasons of preserving patient autonomy as argued above .", "finally , i turn to the question of caring about future personal interests , especially in the context of life - threatening choices .", "a central difficulty associated with including a consideration of this kind in questions of mental capacity is the problem of how to assess caring simpliciter , independently of caring about a particular outcome . according to the account offered here , procedural rationality requires that we care about the future personal consequences of choices , but what in the future we should give weight to is left open .", "specifying this in assessments of mental capacity would mean that the test was not value neutral .", "the case of the committed smoker ( exemplified by david hockney ) can be used to illustrate the point .", "one criticism often levelled at smokers is that they are irrational because they do not care sufficiently about the future personal consequences of their smoking .", "however , this criticism assumes that future health , or a long life , is what the person should care about . considering a person 's decision to keep smoking from a value - neutral perspective , which makes no assumptions of this kind ,", "are they being short - sighted ? or do they care about things other than health and long life in a way that makes their decision rational ?", "bratman 's account shows how asking this second question can be consistent with a diachronic perspective on rationality .", "it is clear that the committed smoker is very likely undermining the health of the future inhabitant of his body .", "however , it does not follow that he is not engaging in the psychological processes that underpin temporally extended agency .", "it may be a central feature of the person 's life plan that he should not be overly concerned about living a long life", ". his deep and enduring commitment might be to a hedonistic lifestyle that is incompatible with being concerned about health in old age .", "for such a person , not living fully enough is a more likely source of future regrets than health - endangering behaviours . caring about one 's future might even require , in such a case , life - threatening or health - threatening choices .", "parallel arguments could be made in the case of a decision by a jehovah 's witness to refuse a blood transfusion ; for a political activist 's hunger strike ; for a person who risks their life in an attempt to save a loved one ; and a freediver 's commitment to their dangerous extreme - sports lifestyle .", "these kinds of decision do not seem characterized by a lack of planfulness about the future . rather than undermining the sense of oneself as  a temporally persisting agent", ", decision - making in line with these kinds of commitment looks likely to contribute to the  overlapping webs of cross - temporal connections and continuities that are proposed to play a role in constituting human agency ( bratman 2000 , pp .", "the future self taken into consideration in these cases may be a nearer self than in the case of a person who is invested in living a long life  because the artist who is a committed smoker , for example , might not expect to or want to live a long life ", "nothing in the above account enjoins us to aim for a life of a particular length .", "what it requires is that , at least to some degree , choices be made with a reflectivity about the future , such that insofar as we are agents that agency is temporally extended .", "this perspective allows us to reconcile the ideas that mental capacity requires that we care about the future personal consequences of our choices , and that life - ending decisions can be made with mental capacity .", "however , against this theoretical background , resolving questions about whether a patient 's capacity to care about the future is compromised would seem to require looking beyond their procedural capacities .", "answers to this question will rest on views about whether or not the life - threatening decision is consistent with the person 's commitments , understood from a diachronic perspective .", "these factors are taken into account in an assessment of best interests once it is decided that a patient lacks mental capacity ( mca s. 4(6 ) ) .", "however , if the ability to care about the future is included as part of what it means to have mental capacity , then consideration of a patient 's commitments from a diachronic perspective will also be central to this prior decision .", "it has been argued here that a diachronic perspective on mental capacity is relevant to questions about when an infringement on treatment decision - making liberty is justified . however , including considerations derived from a diachronic perspective in assessments of capacity comes into conflict with central patient autonomy - preserving features of the mca .", "it 's worth reflecting , then , on why a diachronic perspective might be morally relevant in this context .", "i will suggest that the answer lies in a common view about what makes patient autonomy valuable  what makes it the case that for the most part , people 's treatment decisions , especially their refusals , ought to be respected .", "the importance of allowing patients to be self - determining in relation to their own medical care is often justified along lines that it", " protects people 's general capacity to lead their lives out of a distinctive sense of their own character , a sense of what is important to and for them ( dworkin , 1994 , p. 224 ) .", "different theorists argue for the value of patient autonomy on importantly different grounds , but a common theme is that treatment decisions should be respected because of their assumed connection to the self .", "however , in a case where human agency is seriously compromised in the way described by bratman , selfhood is undermined .", "capacities for diachronic agency that play a central role in establishing what 's important for the person  where the person stands  are compromised . and", "in the context of such problems , the importance placed on patient autonomy in liberal societies would seem to give medical professionals a reason to do what they can to restore selfhood , perhaps even if this requires an infringement on personal liberty .", "this might involve overriding a refusal of treatment , for example , though the justification for doing so would only warrant interventions of a particular kind ", "those that are likely to restore the general capacity that the principle of patient autonomy aims to protect .", "this suggests that in some cases , especially in the context of mental illness , the importance placed on patient autonomy in contemporary medical ethics presents the law with a dilemma . on the one hand", ", it seems right that laws which license infringements on decision - making liberty should equally apply to everyone , not just to particular groups ; and that if a patient has mental capacity in relation to a particular treatment decision then they should be free to direct the course of their medical care .", "consequently , it seems we ought to do away with dedicated mental health laws that legalise detention and involuntary treatment without reference to mental capacity only in the context of a mental disorder . on the other hand , the way in which diachronic agency is compromised in some instances  especially in the context of mental disorder", " seems to justify or perhaps even require an infringement on liberty to restore the self .", "these grounds are difficult to take into consideration in assessments of mental capacity without compromising features of the mca that are designed to preserve patient autonomy .", "so while it might be possible to implement the mca in a way that sufficiently accounts for the problems of decision - making and selfhood that are sometimes present in mental disorders , doing so comes at the price of what is seen to be a central virtue of the legislation .", "in this article my focus was requirements of rationality derived from a diachronic perspective , and the question of what kinds of consideration this perspective would enable mental capacity assessments to include . it was an attempt to explore the limits of the mca 's incapacity test , given its value - neutral approach .", "it was argued that considerations derived from a diachronic perspective are relevant to questions of treatment decision - making liberty , and are consistent with a procedural understanding of the functional element of the test .", "however , in practice , appeal to these considerations in assessments of mental capacity undermines the role played by the functional element of the test in preserving patient autonomy . while involuntary treatment without reference to mental capacity is often assumed to undermine the principle of respecting patient autonomy , it was argued here that this is not necessarily so .", "when human agency is seriously compromised , preserving patient autonomy may sometimes require overriding a treatment decision to restore selfhood , on grounds that are not easily accounted for under the mca without compromising the value - neutral aspirations of this area of law .", "in considering a move to abandon dedicated mental health law , legislators must therefore resolve a bind .", "the central motivation for such a move is concern about unjustified infringements on liberty in the context of psychiatric diagnoses .", "the worry is that the protections of patient autonomy in the mca do not currently extend to the treatment of mental disorders , because mental health legislation allows people to be treated for a mental disorder even if they refuse with mental capacity .", "however , considerations derived from a diachronic perspective that seem relevant to when an infringement on decision - making liberty is justified are especially present in mental disorder .", "the inclusion of these considerations seems necessary for adequately caring for those with a mental disorder under universal capacity legislation . yet including these factors in assessments of mental capacity undermines the protections of patient autonomy that supporters of a shift to universal capacity legislation hope to gain ."], "abstract_text": ["<S> calls for the adoption of a universal capacity approach to replace dedicated mental health law are motivated by the idea that the measures designed to protect patient autonomy in legislation such as the mental capacity act 2005 should apply to everyone , including people with a psychiatric diagnosis . in this article </S>", "<S> it is argued that a diachronic perspective on questions of mental capacity is necessary if capacity law is to play this broader role , but that employing this perspective in assessments of capacity undermines central patient autonomy preserving features of the legislation , which presents a moral dilemma . </S>"], "labels": null, "section_names": ["Mental capacity and the ability to consider the future", "Are we rationally required to value the future?", "Including the ability to consider the future in assessments of mental capacity", "A dilemma about what respect for patient autonomy requires", "Conclusion"], "sections": [["according to the mca , only a compromised ability to make a decision in accordance with certain procedural norms , loosely defined with reference to the ability to  understand , ", "retain and  use or weigh relevant information , or an inability to communicate a decision , properly grounds judgments of incapacity ( s. 3(1 ) ) .", "in addition , the identified problems of decision - making must be attributed to  an impairment of , or disturbance in the functioning of , mind or brain ( s. 2(1 ) ) .", "this law aims to protect the individual 's right to pursue their own ends by adopting a capacity test that focuses on the decision - making process rather than the substance of the patient 's decision .", "influential liberal thinking holds that it is not the place of the law to pursue certain ends  rather , the law should aim to provide a space for individuals to pursue their own ends ( plant , 2011 )  and this position is reflected in english common law relating to mental capacity:[in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences .", "the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind.whether the patient 's decision is consistent with proposed substantive norms in relation to what they should pursue  for example that people should pursue good health , or that they should want to live  is said to be beyond the proper scope of the incapacity test . in adopting this position", ", english law allows that even the most controversial treatment decisions might be explained by the fact that the patient 's evaluation of the situation diverges from a clinical perspective , and perhaps many people 's evaluation of the situation . ", "[ in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences .", "the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind. in practice then , questions of mental capacity might be thought of as boiling down to a question about whether a decision can be explained in terms of the patient 's particular motivating commitments ( their desires , values , projects , ideas about a good life ) ; or whether the decision is properly explained in terms of a problem in decision - making , which is due to a dysfunction of mind or brain . is the patient 's controversial decision understandable in the light of their commitments ? or is the patient 's ability to understand , retain , and use or weigh what the likely costs and benefits of treatment will be for them , currently compromised ? scrutinizing a patient 's commitments in the context of a capacity assessment raises moral concerns because the possibility that a choice can be explained by idiosyncratic commitments plays a significant role in protecting the patient 's right to make controversial decisions .", "perhaps the most straightforward approach to conceiving of commitments , and the one most likely to protect this right , is to say that the person 's commitments are their current commitments  those they recognize at the time of the decision .", "the problem with conceiving of commitments in this way is that it fails to recognise certain problems of practical decision - making to do with motivational coherence over time .", "someone with bipolar disorder , for example , might clearly be choosing or acting in accordance with their current desires during a manic phase , but this perspective fails to capture what seemingly goes wrong with practical decision - making in such cases . as a result , if this understanding of commitments is adopted in assessments of mental capacity the reach of this area of law arguably does not extend as far as it should  a person may be judged to have mental capacity where this seems doubtful .", "psychopathologies characterised by this kind of problem seem better accounted for in a legal structure that conceives of mental capacity , and in particular , commitments , in diachronic terms .", "a diachronic perspective might also help illuminate some other hard cases that are not normally characterised in terms of problems of motivational coherence over time .", "for example , someone with anorexia nervosa may be choosing rationally in accordance with their current commitments  especially a strong desire not to gain weight , or to exert extreme control over what they eat  and so may be judged to have capacity on these grounds . however , an assessment of their evaluative capacities in diachronic terms might yield the conclusion that their decision - making ability is seriously compromised ( craigie , 2011 ) .", "their future self may come to the view that they should have been motivated in ways that they could not recognise or could not act on at the time , and this might be a fairly robust feature of people with a diagnosis of anorexia nervosa .", "this perspective in relation to questions of mental capacity chimes with the suggestion that particularly in psychiatry ,  colloquialisms [ such as ]  you will thank me later  sometimes become the unwritten rules within which the merits of a patient 's consent are assessed ( gostin , 1981 , p. 742 ) .", "such a perspective may also offer a solution in cases where the patient does not have a psychiatric diagnosis .", "mackenzie and watts discuss a case of a woman who was judged to have the capacity to refuse life - saving treatment , and subsequently died , in a situation where she had recently been left by her husband , following the amputation of both her legs .", "they argue that the trauma of these events and her subsequent inability to imagine a life without her partner clouded the woman 's judgment in relation to treatment:the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33", "; for a full description of the case , see halpern , 2012)differences between these cases suggest that taking a diachronic perspective in assessments of mental capacity allows for decision - making to be compromised in at least two ways . in the bipolar disorder and anorexia nervosa cases a diachronic perspective is used to argue that a person 's evaluative capacities may be compromised  the capacities that determine the desires or values that motivate a decision . in the emotional trauma case , which might equally be applied to cases of depression , a diachronic perspective is used to identify a problem in the person 's ability to assess whether what they want or value is achievable . according to jodi halpern 's original analysis of the emotional trauma case , the woman should have been judged to lack capacity on grounds that her beliefs about the future were  unresponsive to evidence , rendering her unable to  think through alternatives ( halpern , 2012 , pp .", " the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33", "; for a full description of the case , see halpern , 2012 ) one problem with adopting a diachronic perspective in assessments of mental capacity is that whether questions of procedural rationality are properly understood in diachronic terms remains a contested issue in the fields of economics , psychology and philosophy .", "the functional element of the mca 's incapacity test concerns problems in meeting certain procedural requirements  problems in the ability to understand , retain , use or weigh relevant information .", "sceptics deny that at least some norms derived from a diachronic perspective fall into this category .", "they hold that such requirements are substantive rather than procedural in nature , so i turn now to this theoretical concern ."], ["much of the normative and descriptive work on a diachronic perspective in personal decision - making has taken place in the fields of economics and psychology , but these fields have tended to adopt contrasting positions on the normative question .", "economists often treat a person 's weighting of the future as a preference like any other , in a framework that assumes that preferences are not available to rational criticism .", "prudence to refer to taking a diachronic perspective in decision - making:prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect ", "there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said .", " ( nagel , 1970 , p. 28)psychologists , on the other hand , often assume that people are rationally required to give significant weight to the future personal consequences of choices , the strongest version being that people ought to be temporally neutral in their decision - making .", "it is widely accepted within this camp that there are at least some good reasons to give less weight to future consequences , particularly in order to take into account an expected depreciation in their future value .", ". however , foreseeable changes in one 's circumstances can also be relevant :  50 might be much more valuable to a final year law student than  100 will be for them in the following year , when they reasonably expect to be working for a corporate lawyer . on these grounds", "it might be rational for the student to choose  50 now , foregoing  100 in a year 's time .", "likewise , it is argued that uncertainly with respect to future consequences can provide a good reason to choose a certain outcome now , over a more valuable but uncertain outcome in the future ( baron , 1994 , pp .", "i am using the term  temporally neutral to refer to decisions that give equal weight to future and present utility , excepting the above kinds of considerations .", "the pressing question is why people ought to be impartial with respect to all parts of their lives once these kinds of consideration are taken into account . ", "prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect ", "there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said .", " ( nagel , 1970 , p. 28 ) the arguments used to defend this position in the psychological literature tend to take the form that a temporally neutral orientation advances the goals of practical decision - making .", "choosing an outcome merely because it will happen sooner is said to be irrational because if one option  achieves your goals better , then you should choose that one , regardless of when you decide ( baron , 1994 , p. 514 ) . using the classic marshmallow experiments as an example ,", "if a child would rather eat two marshmallows than one , then they should choose to wait and be given two marshmallows sometime later rather than one marshmallow now  because overall this achieves the child 's goals better .", "this kind of argument considers the question of goal satisfaction from a position that is not situated at a particular point in time , and prescribes that people ought to take this perspective in their decision - making :  they should be just as concerned about themselves a year from now as they are about themselves this minute ( p. 516 )", ". individuals should give weight to the implications for their future self , so the argument goes , for their own overall good .", "nagel argues that prudence is a requirement of practical reason , which is secured because the personal consequences of a man 's action concern  his future ( 1970 , p. 42 ; his italics ) .", "according to nagel , this constraint on practical decision - making follows from the person 's awareness that they persist over time .", "it reflects the  individual 's conception of himself as a temporally persistent being : his ability to identify with past and future stages of himself and to regard them as forming a single life .", "failure to be susceptible to prudence entails radical dissociation from one 's future , one 's past , and from oneself as a whole , conceived as a temporally extended individual ( p. 58 ) .", "it is a mistake in practical reasoning because it comes  at the cost of dissociation from one 's temporally extended self ( p. 69 ) .", "the conception of self as extended throughout a human lifetime is powerful in the context of questions of procedural rationality because it offers grounds for claiming that a person ought to give weight to considerations that they may not currently care about .", "it offers an answer to what i have characterised as the economists ' view , that if a person has no interest in the future personal consequences of their choice there is nothing more to be said , rationally speaking .", "applied to the context of treatment decision - making , this kind of perspective is powerful because it offers grounds for calling a patient 's mental capacity into question , with reference to their ability to consider their future and value it appropriately .", "rejections of this kind of rational requirement are therefore often based on a rejection of the idea of the self as something that necessarily extends throughout a lifetime , and derek parfit 's account of personal identity is often used to make this argument ( baron , 1994 , p. 516 ; frederick , 2006 ; maclean , 2006 ) .", "parfit holds that the persistence of a person over time must be understood in terms of their ", "psychological continuity  the connectedness of psychological features such as memories , character , interests and preferences across a lifetime ( parfit , 1983 ; 1984 ) . on this view , whether the future inhabitant of one 's body will be the same person can be a matter of degree . psychological features sometimes change so radically over a human lifetime that a person now will not necessarily be the same person who occupies that body in the future . and", "not giving weight to the interests of a future self is irrational only to the degree that one shares the relevant psychological characteristics with that self .", "more recently , galen strawson has defended a similar position in his rejection of narrative accounts of the self .", "strawson argues that although experiencing oneself as one person throughout a lifetime  what he calls a ", "diachronic self - experience  may be more common , some people experience themselves in what he describes as a more  episodic way . unlike the more diachronic person ,", "the more episodic person has  little or no sense that the self that one is was there in the ( further ) past and will be there in the future , although one is perfectly aware that one has long - term continuity considered as a whole human being ( strawson , 2005 , p. 65 ) .  [ predominantly ] episodic individuals may sometimes connect to charged events in their pasts in such a way that they feel those events happened to them  embarrassing memories are a good example  and anticipate events in their futures in such a way that they think those events are going to happen to them  thoughts of death can be a good example ", "however , the episodic person has no  great or special interest in [ their ] past , and nor do they have  a great deal of concern for [ their ] future ( p. 67 ) .", "strawson holds that even a strongly episodic self is within the normal range of human experience , and that it is normatively on a par with a more diachronic self - experience . on his view", ", theorists such as nagel assert that selves extend over a lifetime , and diachronic norms of procedural rationality are derived from this claim .", "parfit and strawson dispute this assumption , and conclude that disregard for the future personal consequences of a choice may simply reflect an alternative way of living a human life , at least when we consider the question of reasons for action in non - moral terms .", "however , all of the theorists canvassed thus far reason from premises about the extension of self over a lifetime , to conclusions about what reasons individuals have , and it will be argued here that this conception of the relationship between the requirements of practical rationality and the metaphysics of self is the wrong way around . as described above , the kind of requirement in question is often defended  by nagel and others  on the grounds that not giving weight to the future undermines one 's own interests . however , at times a different kind of argument is suggested in nagel 's work .", "the suggestion seems to be that a unified self over a lifetime is not something that is automatically present , but rather something that is generated by giving weight to future personal consequences in decision - making .", "the proposed requirement is justified , not because it deploys the correct conception of self , or because one 's future self will otherwise regret the decision , but because adherence plays a role in bringing the self into being , by enabling the decision - maker  to reach towards something outside himself ( nagel , 1970 , p. 43 ) .", "michael bratman is particularly clear about the role that he proposes giving weight to the future plays in the generation of the self , and how this in turn grounds a diachronic perspective on questions of procedural rationality , so it is his view that i will focus on here .", " self - governing policies  which are integral to temporally extended agency  lie at the core of human agency because it is through these that a person 's identity emerges .", "these processes are essential for understanding what it is for an agent to  take a stand  what it is for an agent to recognise certain desires as one 's own  and it is this feature in particular that distinguishes human agency from the agency of other  purposive creatures ( bratman , 2000 , pp .", "adopting broadly the same approach to personal identity as parfit begins with , bratman suggests that the forward - looking psychological ties that play a role in constituting a unified self over time can be generated through reflective and planning processes : ", "i can help ensure appropriate psychological continuities and connections by sticking with and executing my prior plans and policies , and by monitoring and regulating my motivational structures in favor , say , of my continued commitment to philosophy ", "such processes  play an important role in the constitution and support of continuities and connections characteristic of the identity of the agent over time. ", "indeed, bratman suggests ,  this is what plans and policies are for ( 2000 , p. 47", "by emphasising the role that planning and policy - making play in generating the psychological ties that are proposed to underpin personal identity over time , bratman describes a picture of human agency in which the agent plays an active role . the temporally extended self develops through the agent 's exercise of certain psychological capacities . on this point bratman", "makes a significant departure from parfit and strawson , who seem to hold that the degree to which the relevant psychological ties extend over time is a fixed fact about the individual , from which rational requirements on their action may be derived .", "the claim that engaging in planning and policy - making processes is rationally required is defended primarily on the non - instrumental grounds that these processes play a central role in developing and sustaining the self .", "bratman 's argument takes the form of an answer to the problem that agents who reflect on their first - order desires and form second - order desires do not yet exhibit what is distinctive about human agency , because there is nothing to distinguish these new desires as those that the agent identifies with ; second - order desires seem to be ", "just one more wiggle in the psychic stew ( bratman , 2000 , p. 38 ) .", "the solution that bratman offers is that where an agent stands with respect to a particular first - order desire is established through the exercise of planning and policy - making practices :  self - governing policies might , so to speak , crystallize pressures from various elements of one 's psychic stew into a more decisive attitude that can , in the relevant context , establish where one stands ( 2000 , p. 51 ) .", "these mental processes that underpin temporally extended agency give authority to particular desires , so it is through these that the self  the distinctive feature of human agency  emerges . on this view , a compromised ability to engage in planning and policy - making processes threatens not the interests of the future self , but the very constitution of the self .", "this is what grounds the procedural requirement that one must give weight to the future personal consequences of actions . and this , we might suggest , is what makes the ability to consider and value the future an appropriate consideration to include in a mental capacity test .", "bratman 's account offers a story about the self that fits well with a developmental understanding of personhood that is often appealed to in contemporary medical law and ethics .", "his account of the relationship between temporally extended agency and the self also fits well with the difficulties associated with certain psychopathologies .", "it is no accident , i suggest , that disorders such as dementia , bipolar disorder and depression are associated with seriously compromised capacities for diachronic agency . among many other factors , in a complex picture , problems of diachronic agency surely make a contribution to our sense that these constellations of behavioural and experiential features are appropriate targets for the label of disorder .", "a sense of the self as compromised in the context of problems of diachronic agency is also found in first - person reports of dementia ( addis and tippett , 2004 ; however , see caddell and clare , 2012 ) and depression ( fuchs , 2008 ; ratcliffe , 2012 ) .", "it would seem that the functioning of capacities for diachronic agency is connected to our sense of others as having well - functioning human agency , as well as our subjective sense of selfhood , and for these reasons", "i find it implausible that a strongly episodic self - experience is just a less common way of living a human life .", "strawson defends a view along these lines , claiming that he himself is strongly episodic ( in his sense ) .", "yet he has a successful academic career which could not have been achieved without meeting a wide range of commitments that each extended over considerable periods of time , for example in completing academic qualifications and writing books .", "it seems to me that these achievements evidence considerable skills in diachronic agency . while for strawson a diachronic self - experience is connected to a narrative conception of the self , diachronicity in the sense concerned here", "a life lived in the moment could still be a life lived with diachronic agency , as i will argue at the end of the following section .", "i now turn back to questions of mental capacity and what implications a diachronic perspective such as bratman 's would have ."], ["the motivation for investigating these theoretical questions about procedural rationality was the idea that a diachronic perspective might enable a mental capacity test such as that adopted in the mca to deal with certain hard cases , particularly in the context of mental disorder , without undermining its value - neutral approach .", "the arguments above suggest that a diachronic perspective on questions of mental capacity is consistent with a procedural understanding of the functional element of the mca 's incapacity test .", "given this , could a diachronic understanding of what it is to have mental capacity help by justifying the inclusion of a requirement such as the ability to consider the future  as a part of what it means to be able to understand , retain , use or weigh information  in assessments of mental capacity ?", "something like this requirement already seems to be assumed in the condition that the patient must understand the consequences of the treatment options .", "the relevant information necessarily concerns the future as well as the present , due to the fact that understanding  the likely effects of deciding one way or the other is a part of this requirement ( mental capacity act 2005 code of practice , s. 4.16 ) .", "for example , a person with a severe learning disability might be judged to lack the capacity to consent to a blood test on grounds that they do not understand that the test is necessary for their future physical well - being .", "the same might be said in a case of a delirious patient who is resisting treatment for a serious injury . in such cases the person 's connection to the world , in the sense of their ability to understand how things are likely to be in the future ,", "what to do is limited in a way that can be compared to john stuart mill 's paradigm case of justified paternalistic intervention , where a person is about to cross a bridge that unbeknownst to them is unsafe", ". the person in the example does not understand that they risk falling into water if they attempt to cross .", "the widely accepted conclusion is that the person is not in a position to assess whether to cross the bridge , and this justifies the use of proportional physical force to stop them from crossing , when this is necessary to inform them about the state of the bridge .", "the same reasoning might be applied in the case of the woman who has recently suffered a serious emotional trauma , on grounds that her ability to imagine a range of future possibilities is severely limited ( halpern , 2012 ) .", "the patient can not currently conceive of alternative futures to the desperately unhappy one she imagines , and perhaps this justifies taking the decision out of her hands until she is seeing things more clearly .", "the problem that returns here is that understanding concerns not simply how things are likely to be , as in the fact that an attempt to cross the bridge is likely to result in a fall into water , or that a certain proportion of people who have both legs amputated learn to walk again using prosthetic limbs .", "it concerns understanding how the relevant state of affairs will be for this particular patient .", "this question about the future necessarily involves an evaluative judgment that , according to a value - neutral approach , only the patient is in a position to make .", "so while it might sometimes be clear that a patient 's ability to consider the future is compromised , for example when someone denies that their condition is life - threatening when it clearly is , in many cases assessing understanding in relation to the future will have no equivalent point of reference .", "in such a context there seems to be no standard against which the functional element of the mental capacity test  the part concerning the patient 's ability to understand , retain , use or weigh information  can be directly assessed .", "therefore , any conclusions drawn about this capacity would seem to be based on the fact that the patient has suffered an emotional trauma . removing a patient 's right to make their own treatment decision under these circumstances may seem justified given what we know about how people who lose limbs adapt , as reflected in self - reported measures of well - being , and given the known psychological effects of emotional trauma", ". however , an appeal to this kind of knowledge in an assessment of mental capacity would involve using generalised observations to draw a conclusion about a particular person 's capacity to assess how things will be for them in the future .", "the legislation is clear that an assessment of decision - making ability should not be based solely on features such as a patient 's diagnosis .", "it must be shown that this particular person 's mental capacities are relevantly impaired , not merely that they belong to a diagnostic category . however", ", this requirement can not be met in the case of this capacity , because there is no way to directly assess it while remaining value neutral in accordance with the aspirations of this area of law . what about the possibility raised at the beginning of this article , that a person 's evaluative capacities might be called into question when viewed from a diachronic perspective ?", "drawing on bratman 's account , planfulenss and self - governing policies are rationally required because of the role they play in developing and sustaining the self .", "caring about the interests of the future inhabitant of one 's body , and having at least some grip on what those interests will be , seem like prerequisites for engaging successfully in these processes . and perhaps this offers part of an explanation about how decision - making can be compromised in the context of disorders such as depression and bipolar disorder .", "very crudely , the idea would be that as part of a much bigger picture the capacities necessary for diachronic agency are not functioning properly and this undermines the constitution of the self .", "i have argued that , understood in this way , these considerations are procedural rather than substantive in nature .", "but would the implementation of these considerations in practice nonetheless come into conflict with the goal of preserving patient autonomy ? assessing a person 's grip on what interests the future inhabitant of their body will have runs into the same difficulties that occurred in the context of assessing understanding in the emotional trauma case . without making substantive assumptions", "judging that a person is likely to lack these capacities in the context of a manic episode may seem justified , because of what we know about the trajectory of bipolar disorder  that fairly reliably people 's commitments during a manic episode radically undermine their overall interests .", "but whether this kind of consideration provides grounds for a judgment of incapacity in english law depends on what evidence is considered appropriate for assessing the functional element of the incapacity test .", "however , as genevra richardson points out ,  existing case law indicates that courts will take a flexible approach to the legal definition [ of capacity ] to enable them to reach their preferred outcome ( richardson , this volume , p. 00 ) .", "this may mean that in practice significant weight is given to a person 's diagnosis .", "however , this solution undermines the protections of patient autonomy conferred by making an individual assessment of functional capacities , rather than diagnosis , the proper grounds for infringements on self - determination .", "a related possibility considered at the beginning of this article was that a diachronic perspective might be used to justify giving weight to a reasonable prediction of what the patient 's future self would want in retrospect , in assessments of mental capacity .", "however , it does not follow from the view offered here , even given the sure knowledge that a patient 's future self would retrospectively endorse a medical intervention , that their present self is irrational in refusing it . in the imagined case there is an unambiguous conflict between what the patient currently wants ( no treatment ) and what the future self will want in retrospect ( treatment ) . using this conflict to doubt the patient 's current capacity to make the treatment decision assumes that the future self 's perspective should be given greater weight in the current self 's decision - making .", "however , there is nothing in the present view that would justify resolving the conflict in this way .", "the proposed account requires that the agent exercise planfulness and self - governing policies in decision - making , which would seem to involve giving equal weight at most , not greater weight , to one 's expected future desires .", "moreover , humans have a well - established time preference for negative experiences to be in the past rather than the present or future , and this makes it easier for the patient to endorse involuntary treatment when it is in the past ( doherty , 2011 ) .", "this would seem to weigh in favour of privileging the perspective of the self who is facing the infringement on liberty . in the absence of any reasons to believe that the future self 's perspective should be privileged", ", this form of justification for doubting mental capacity looks to be question - begging .", "its favouring of the future self 's perspective already assumes that the current self 's decision - making capacity is relevantly impaired .", "favouring the future self 's perspective might seem justified on grounds that , for example , evaluative capacities are often compromised in the context of certain diagnoses or during experiences such as emotional trauma .", "however , at least in theory , this evidence alone does not justify a judgment of incapacity under current english law , for reasons of preserving patient autonomy as argued above .", "finally , i turn to the question of caring about future personal interests , especially in the context of life - threatening choices .", "a central difficulty associated with including a consideration of this kind in questions of mental capacity is the problem of how to assess caring simpliciter , independently of caring about a particular outcome . according to the account offered here , procedural rationality requires that we care about the future personal consequences of choices , but what in the future we should give weight to is left open .", "specifying this in assessments of mental capacity would mean that the test was not value neutral .", "the case of the committed smoker ( exemplified by david hockney ) can be used to illustrate the point .", "one criticism often levelled at smokers is that they are irrational because they do not care sufficiently about the future personal consequences of their smoking .", "however , this criticism assumes that future health , or a long life , is what the person should care about . considering a person 's decision to keep smoking from a value - neutral perspective , which makes no assumptions of this kind ,", "are they being short - sighted ? or do they care about things other than health and long life in a way that makes their decision rational ?", "bratman 's account shows how asking this second question can be consistent with a diachronic perspective on rationality .", "it is clear that the committed smoker is very likely undermining the health of the future inhabitant of his body .", "however , it does not follow that he is not engaging in the psychological processes that underpin temporally extended agency .", "it may be a central feature of the person 's life plan that he should not be overly concerned about living a long life", ". his deep and enduring commitment might be to a hedonistic lifestyle that is incompatible with being concerned about health in old age .", "for such a person , not living fully enough is a more likely source of future regrets than health - endangering behaviours . caring about one 's future might even require , in such a case , life - threatening or health - threatening choices .", "parallel arguments could be made in the case of a decision by a jehovah 's witness to refuse a blood transfusion ; for a political activist 's hunger strike ; for a person who risks their life in an attempt to save a loved one ; and a freediver 's commitment to their dangerous extreme - sports lifestyle .", "these kinds of decision do not seem characterized by a lack of planfulness about the future . rather than undermining the sense of oneself as  a temporally persisting agent", ", decision - making in line with these kinds of commitment looks likely to contribute to the  overlapping webs of cross - temporal connections and continuities that are proposed to play a role in constituting human agency ( bratman 2000 , pp .", "the future self taken into consideration in these cases may be a nearer self than in the case of a person who is invested in living a long life  because the artist who is a committed smoker , for example , might not expect to or want to live a long life ", "nothing in the above account enjoins us to aim for a life of a particular length .", "what it requires is that , at least to some degree , choices be made with a reflectivity about the future , such that insofar as we are agents that agency is temporally extended .", "this perspective allows us to reconcile the ideas that mental capacity requires that we care about the future personal consequences of our choices , and that life - ending decisions can be made with mental capacity .", "however , against this theoretical background , resolving questions about whether a patient 's capacity to care about the future is compromised would seem to require looking beyond their procedural capacities .", "answers to this question will rest on views about whether or not the life - threatening decision is consistent with the person 's commitments , understood from a diachronic perspective .", "these factors are taken into account in an assessment of best interests once it is decided that a patient lacks mental capacity ( mca s. 4(6 ) ) .", "however , if the ability to care about the future is included as part of what it means to have mental capacity , then consideration of a patient 's commitments from a diachronic perspective will also be central to this prior decision ."], ["it has been argued here that a diachronic perspective on mental capacity is relevant to questions about when an infringement on treatment decision - making liberty is justified . however , including considerations derived from a diachronic perspective in assessments of capacity comes into conflict with central patient autonomy - preserving features of the mca .", "it 's worth reflecting , then , on why a diachronic perspective might be morally relevant in this context .", "i will suggest that the answer lies in a common view about what makes patient autonomy valuable  what makes it the case that for the most part , people 's treatment decisions , especially their refusals , ought to be respected .", "the importance of allowing patients to be self - determining in relation to their own medical care is often justified along lines that it", " protects people 's general capacity to lead their lives out of a distinctive sense of their own character , a sense of what is important to and for them ( dworkin , 1994 , p. 224 ) .", "different theorists argue for the value of patient autonomy on importantly different grounds , but a common theme is that treatment decisions should be respected because of their assumed connection to the self .", "however , in a case where human agency is seriously compromised in the way described by bratman , selfhood is undermined .", "capacities for diachronic agency that play a central role in establishing what 's important for the person  where the person stands  are compromised . and", "in the context of such problems , the importance placed on patient autonomy in liberal societies would seem to give medical professionals a reason to do what they can to restore selfhood , perhaps even if this requires an infringement on personal liberty .", "this might involve overriding a refusal of treatment , for example , though the justification for doing so would only warrant interventions of a particular kind ", "those that are likely to restore the general capacity that the principle of patient autonomy aims to protect .", "this suggests that in some cases , especially in the context of mental illness , the importance placed on patient autonomy in contemporary medical ethics presents the law with a dilemma . on the one hand", ", it seems right that laws which license infringements on decision - making liberty should equally apply to everyone , not just to particular groups ; and that if a patient has mental capacity in relation to a particular treatment decision then they should be free to direct the course of their medical care .", "consequently , it seems we ought to do away with dedicated mental health laws that legalise detention and involuntary treatment without reference to mental capacity only in the context of a mental disorder . on the other hand , the way in which diachronic agency is compromised in some instances  especially in the context of mental disorder", " seems to justify or perhaps even require an infringement on liberty to restore the self .", "these grounds are difficult to take into consideration in assessments of mental capacity without compromising features of the mca that are designed to preserve patient autonomy .", "so while it might be possible to implement the mca in a way that sufficiently accounts for the problems of decision - making and selfhood that are sometimes present in mental disorders , doing so comes at the price of what is seen to be a central virtue of the legislation ."], ["in this article my focus was requirements of rationality derived from a diachronic perspective , and the question of what kinds of consideration this perspective would enable mental capacity assessments to include . it was an attempt to explore the limits of the mca 's incapacity test , given its value - neutral approach .", "it was argued that considerations derived from a diachronic perspective are relevant to questions of treatment decision - making liberty , and are consistent with a procedural understanding of the functional element of the test .", "however , in practice , appeal to these considerations in assessments of mental capacity undermines the role played by the functional element of the test in preserving patient autonomy . while involuntary treatment without reference to mental capacity is often assumed to undermine the principle of respecting patient autonomy , it was argued here that this is not necessarily so .", "when human agency is seriously compromised , preserving patient autonomy may sometimes require overriding a treatment decision to restore selfhood , on grounds that are not easily accounted for under the mca without compromising the value - neutral aspirations of this area of law .", "in considering a move to abandon dedicated mental health law , legislators must therefore resolve a bind .", "the central motivation for such a move is concern about unjustified infringements on liberty in the context of psychiatric diagnoses .", "the worry is that the protections of patient autonomy in the mca do not currently extend to the treatment of mental disorders , because mental health legislation allows people to be treated for a mental disorder even if they refuse with mental capacity .", "however , considerations derived from a diachronic perspective that seem relevant to when an infringement on decision - making liberty is justified are especially present in mental disorder .", "the inclusion of these considerations seems necessary for adequately caring for those with a mental disorder under universal capacity legislation . yet including these factors in assessments of mental capacity undermines the protections of patient autonomy that supporters of a shift to universal capacity legislation hope to gain ."]]}
{"article_id": "PMC3821231", "article_text": ["type iii dysbetalipoproteinaemia is a kind of lipid metabolism disorders , caused by apo - e deficiency , which leads to accumulation of chylomicrons and very low density lipoprotein remnants in the plasma.123 lipid metabolism disorders are mostly manifested by xanthomas , which are known as yellowish cholesterol - rich material in large foam cells accumulating in the skins and tendons.14 these yellowish lesions may appear all over the body , on the palm of the hands , sole of the foot , tendons , and even on the eyelids.5 these lesions firstly described with or without hyperlipidaemia in association with monoclonal immunoglobulin . based on this fact , there are three forms of xanthoma : hyperlipaemic xanthoma , normolipaemic xanthoma and necrobiotic xanthogranuloma.6 xanthomatosis is usually associated with hyperlipidaemia , and morbidity and mortality of this condition are related to atherosclerosis and pancreatitis.7 hyperlipaemic xanthoma lesions are more polymorphic and can include tuberous , tendinous , palmar or eruptive xanthoma .", "verruciform xanthoma usually presents as a hyperkeratotic , cauliflower like , verrucous or papillomatous lesion.6 it initially was described as a unique clinicopathologic lesion of the oral mucosa and was subsequently reported in the skin .", "the epidermal - mucosal changes of hyperparakeratosis , neutrophilic infiltrate and dermal - submucosal foam cell reaction are distinctive features of these xanthomas.8 hereby , we present a rare type of xanthomas calling  cauliflower xanthoma  in a 43-year - old man with dysbetalipoproteinaemia .", "a 43-year - old man was presented to outpatient endocrine clinic of tabriz university of medical sciences , tabriz , iran , by his sister for evaluation of skin lesions .", "he had multiple lesions on shoulders and back , lateral and medial part of dorsal surface of the foot , medial and lateral malleus of both feet , and dorsal and palmar surface of both hands .", "siblings of the patient have been followed up in that clinic for clinically diagnosed type - iii dysbetalipoproteinaemia .", "this diagnosis was based on characteristic palmar , eruptive , tuberous and trauma site xanthomas with typical high levels of both total cholesterol and triglycerides in the plasma .", "history of coronary artery or other atherosclerotic disorders were negative in index case and siblings .", "there were not any other complaint , and as a greengrocer , he had an active lifestyle .", "he had a pathology report of skin lesion biopsy that was performed by a dermatologist few months ago , with presence of lipid laden macrophages consistent with diagnosis of xanthoma . fasting serum lipid levels reported as : total cholesterol = 507 mg / dl , triglycerides = 470 mg / dl and high density lipoprotein ( hdl ) cholesterol = 41 mg / dl .", "he had normal fasting blood sugar , and normal thyroid , renal and liver function tests .", "physical examinations revealed numerous xanthomas resembling cauliflower on both knees [ figure 1 ] , unusual eruptive , tuberous xanthomas same as previous lesions , on the lateral malleus of both feet [ figure 2 ] .", "cauliflower xanthomas on the knees and lateral malleus of both lower extremities cauliflower xanthomas on the lateral malleus of lower extremities there were lots of smaller xanthomas on the right shoulder [ figure 3 ] , and cauliflower like xanthomas on the extensor side of the left upper extremity , especially on the elbow [ figure 4 ] . based on available information", ", the patient diagnosed as familial dysbetalipoproteinaemia ( fdl ) and the unique lesions on the lower limb called  cauliflower xanthoma  .", "although siblings of this case had the same disorder , they did nt develop such lesions .", "xanthomas on the shoulder and back of the neck xanthomas on the extensor side of the hand by searching in the medical data bases , we can not find a previous such a lesion . therefore , we decided to present this case without revealing patient 's name , after taking his consent .", "considering the fact that the treatment with high dose statin was effective in correction of lipid abnormality and regression of skin lesions in his three siblings , we wanted to try the treatment procedure in this patient but he refused any intervention .", "fdl , also known as hyperlipoproteinemia type - iii or broad beta disease , is a rare inherited disorder characterised by improper metabolism of certain lipids , specially plasma cholesterol , triglyceride rich chylomicron and very low density lipoproteins ( vldl ) remnants.1 presence or absence of the symptoms of this disease depends on two major risk factors : genetic and diet.910 mutations in the gene for apolipoprotein e ( apo e ) are the main cause of this disease .", "replacement of an arginine by a cysteine in position 158 of the 299-amino acid chain of apo e5 is responsible for the defective binding of chylomicron and vldl remnants to cell receptors .", "thereafter , slower plasma clearance of these particles occurs , and results in the abnormal accumulation of lipids in the body.9 on the other side , diet has an essential role in the development of the disease .", "this means that with standard cholesterol diet , symptoms of fdl will not appear , even in the genetically susceptible person.10 xanthomas can be a symptom of fdl .", "xanthomas may also be the symptoms of a generalised histiocytosis , or a local fat phagocytosing storage process.11 they are yellowish lesions on the skins and tendons , macroscopically .", "these macrophages are filled with lipid droplets , which are dissolved and removed from tissue during histologic processing.8 fdl is mostly diagnosed by combination of clinical and laboratory findings.12 most cases are inherited as autosomal recessive trait .", "men are more susceptible for fdl probably because of protective impact of estrogen in women .", "most of the diagnosed patients were typically young males , with strong family history , characteristic skin lesions , high serum levels of cholesterol and triglycerides and confirming skin histology.113 most of these cases have premature atherosclerosis and other signs of ischaemic disorders.9 the patient in this article was a middle aged man with family history of dysbetalipoproteinaemia and high levels of serum lipids as follows : total cholesterol = 507 mg / dl and triglycerides = 470 mg / dl .", "he had a rare form of xanthoma all over his body , causing social problems for him .", "nicotinic acid , clofibrate , statins or gemfibrozil properly reduce cholesterol and triglycerides in people affected with dysbetalipoproteinaemia.1 the patient mentioned in this article refused receiving any medical intervention .", "he has just been advised to have low lipid diet , fish oil and regular moderate exercise in order to reducing serum lipid levels .", "there should be distinctive monitoring of fdl patients including regular checking of their serum lipids .", "controlling underlying disorders , and reducing excess calories , saturated fat and cholesterol , is the main aim of treating these patients . according to this article", ", cauliflower xanthomas could be a symptom of fdl , and it should be considered as a differential diagnosis while approaching to these lesions ."], "abstract_text": ["<S> familial dysbetalipoproteinaemia ( fdl ) is an inherited disorder in which both cholesterol and triglycerides are elevated in the plasma , pre - disposing the people to coronary artery disease and peripheral vascular disease . </S>", "<S> the disease is mostly manifested by xanthomas , which have variable forms according to lipid amounts in the plasma of the blood . </S>", "<S> hereby , we report a 43-year - old man with fdl , presenting with a rare form of xanthomas calling  cauliflower xanthoma  all over the body . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["type iii dysbetalipoproteinaemia is a kind of lipid metabolism disorders , caused by apo - e deficiency , which leads to accumulation of chylomicrons and very low density lipoprotein remnants in the plasma.123 lipid metabolism disorders are mostly manifested by xanthomas , which are known as yellowish cholesterol - rich material in large foam cells accumulating in the skins and tendons.14 these yellowish lesions may appear all over the body , on the palm of the hands , sole of the foot , tendons , and even on the eyelids.5 these lesions firstly described with or without hyperlipidaemia in association with monoclonal immunoglobulin . based on this fact , there are three forms of xanthoma : hyperlipaemic xanthoma , normolipaemic xanthoma and necrobiotic xanthogranuloma.6 xanthomatosis is usually associated with hyperlipidaemia , and morbidity and mortality of this condition are related to atherosclerosis and pancreatitis.7 hyperlipaemic xanthoma lesions are more polymorphic and can include tuberous , tendinous , palmar or eruptive xanthoma .", "verruciform xanthoma usually presents as a hyperkeratotic , cauliflower like , verrucous or papillomatous lesion.6 it initially was described as a unique clinicopathologic lesion of the oral mucosa and was subsequently reported in the skin .", "the epidermal - mucosal changes of hyperparakeratosis , neutrophilic infiltrate and dermal - submucosal foam cell reaction are distinctive features of these xanthomas.8 hereby , we present a rare type of xanthomas calling  cauliflower xanthoma  in a 43-year - old man with dysbetalipoproteinaemia ."], ["a 43-year - old man was presented to outpatient endocrine clinic of tabriz university of medical sciences , tabriz , iran , by his sister for evaluation of skin lesions .", "he had multiple lesions on shoulders and back , lateral and medial part of dorsal surface of the foot , medial and lateral malleus of both feet , and dorsal and palmar surface of both hands .", "siblings of the patient have been followed up in that clinic for clinically diagnosed type - iii dysbetalipoproteinaemia .", "this diagnosis was based on characteristic palmar , eruptive , tuberous and trauma site xanthomas with typical high levels of both total cholesterol and triglycerides in the plasma .", "history of coronary artery or other atherosclerotic disorders were negative in index case and siblings .", "there were not any other complaint , and as a greengrocer , he had an active lifestyle .", "he had a pathology report of skin lesion biopsy that was performed by a dermatologist few months ago , with presence of lipid laden macrophages consistent with diagnosis of xanthoma . fasting serum lipid levels reported as : total cholesterol = 507 mg / dl , triglycerides = 470 mg / dl and high density lipoprotein ( hdl ) cholesterol = 41 mg / dl .", "he had normal fasting blood sugar , and normal thyroid , renal and liver function tests .", "physical examinations revealed numerous xanthomas resembling cauliflower on both knees [ figure 1 ] , unusual eruptive , tuberous xanthomas same as previous lesions , on the lateral malleus of both feet [ figure 2 ] .", "cauliflower xanthomas on the knees and lateral malleus of both lower extremities cauliflower xanthomas on the lateral malleus of lower extremities there were lots of smaller xanthomas on the right shoulder [ figure 3 ] , and cauliflower like xanthomas on the extensor side of the left upper extremity , especially on the elbow [ figure 4 ] . based on available information", ", the patient diagnosed as familial dysbetalipoproteinaemia ( fdl ) and the unique lesions on the lower limb called  cauliflower xanthoma  .", "although siblings of this case had the same disorder , they did nt develop such lesions .", "xanthomas on the shoulder and back of the neck xanthomas on the extensor side of the hand by searching in the medical data bases , we can not find a previous such a lesion . therefore , we decided to present this case without revealing patient 's name , after taking his consent .", "considering the fact that the treatment with high dose statin was effective in correction of lipid abnormality and regression of skin lesions in his three siblings , we wanted to try the treatment procedure in this patient but he refused any intervention ."], ["fdl , also known as hyperlipoproteinemia type - iii or broad beta disease , is a rare inherited disorder characterised by improper metabolism of certain lipids , specially plasma cholesterol , triglyceride rich chylomicron and very low density lipoproteins ( vldl ) remnants.1 presence or absence of the symptoms of this disease depends on two major risk factors : genetic and diet.910 mutations in the gene for apolipoprotein e ( apo e ) are the main cause of this disease .", "replacement of an arginine by a cysteine in position 158 of the 299-amino acid chain of apo e5 is responsible for the defective binding of chylomicron and vldl remnants to cell receptors .", "thereafter , slower plasma clearance of these particles occurs , and results in the abnormal accumulation of lipids in the body.9 on the other side , diet has an essential role in the development of the disease .", "this means that with standard cholesterol diet , symptoms of fdl will not appear , even in the genetically susceptible person.10 xanthomas can be a symptom of fdl .", "xanthomas may also be the symptoms of a generalised histiocytosis , or a local fat phagocytosing storage process.11 they are yellowish lesions on the skins and tendons , macroscopically .", "these macrophages are filled with lipid droplets , which are dissolved and removed from tissue during histologic processing.8 fdl is mostly diagnosed by combination of clinical and laboratory findings.12 most cases are inherited as autosomal recessive trait .", "men are more susceptible for fdl probably because of protective impact of estrogen in women .", "most of the diagnosed patients were typically young males , with strong family history , characteristic skin lesions , high serum levels of cholesterol and triglycerides and confirming skin histology.113 most of these cases have premature atherosclerosis and other signs of ischaemic disorders.9 the patient in this article was a middle aged man with family history of dysbetalipoproteinaemia and high levels of serum lipids as follows : total cholesterol = 507 mg / dl and triglycerides = 470 mg / dl .", "he had a rare form of xanthoma all over his body , causing social problems for him .", "nicotinic acid , clofibrate , statins or gemfibrozil properly reduce cholesterol and triglycerides in people affected with dysbetalipoproteinaemia.1 the patient mentioned in this article refused receiving any medical intervention .", "he has just been advised to have low lipid diet , fish oil and regular moderate exercise in order to reducing serum lipid levels .", "there should be distinctive monitoring of fdl patients including regular checking of their serum lipids .", "controlling underlying disorders , and reducing excess calories , saturated fat and cholesterol , is the main aim of treating these patients . according to this article", ", cauliflower xanthomas could be a symptom of fdl , and it should be considered as a differential diagnosis while approaching to these lesions ."]]}
{"article_id": "PMC4745858", "article_text": ["exacerbation is an important life - threatening event for patients with copd , and can lead to hospitalization and death.14 patients who suffer frequent and repeated exacerbations within 1 year have a poor prognosis,5 characterized by worsening of health - related quality of life ( hrqol),6,7 a rapid decline in lung function,810 and high mortality.11 frequent exacerbators also carry a high risk of further exacerbation and hospitalization.11,12 however , it has been suggested that japanese patients with copd may have fewer exacerbations , and they also may have a higher proportion of elderly patients , those with emphysema , and those with a lower body mass index in comparison to westerners.1215 the prognosis of japanese patients with copd who suffer frequent and repeated exacerbations is unclear .", "we conducted a 1-year prospective observational trial in a daily - life setting involving 90 japanese patients with copd to investigate whether previous moderate - to - severe exacerbations are associated with future exacerbations in this patient population .", "we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) .", "consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records .", "copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) .", "the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography .", "duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened .", "regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting .", "the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas .", "to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma ", "copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25 baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721 information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians .", "however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ]", "15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities .", "previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events .", "moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year .", "hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation .", "mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators .", "unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms .", "data are expressed as mean  standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline .", "characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey ", "subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan  meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period , the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses .", "medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis .", "statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) .", "we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) .", "consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records .", "copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) .", "the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography .", "duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened .", "regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting .", "the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas .", "to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma ", "copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25", "baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721", "information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians .", "however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ]", "15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities .", "previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events .", "moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year .", "hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation .", "mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators .", "unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms .", "data are expressed as mean  standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline .", "characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey ", "subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan  meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period ,", "the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses .", "medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis .", "statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) .", "ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year .", "the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) . at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) .", "frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) .", "there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) . during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) .", "figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation .", "the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations .", "the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) .", "in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) .", "in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period .", "the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively .", "patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) .", "figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) .", "figure 3b shows the kaplan  meier analysis of the period until the first moderate and severe exacerbations .", "the median ( mean  sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) .", "there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) .", "the median ( mean  sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) .", "univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) .", "however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination .", "the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted , presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) .", "ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year .", "the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) .", "at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) .", "frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) .", "there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) .", "during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) .", "figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation .", "the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations .", "the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) . in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) .", "in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period .", "the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively .", "patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) .", "figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) .", "figure 3b shows the kaplan  meier analysis of the period until the first moderate and severe exacerbations .", "the median ( mean  sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) .", "there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) .", "the median ( mean  sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) .", "univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) .", "however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination . the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted ,", "presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) .", "the frequent - exacerbator phenotype is important to consider in the management of copd patients , who require future exacerbation - related hospitalization associated with high mortality.16 a previous western study suggested that frequent exacerbators tend to have had exacerbations during the previous year , a lower fev1 , more severe hrqol , a history of gerd , and a higher peripheral white blood - cell count in comparison with infrequent exacerbators and nonexacerbators.12 another western study demonstrated that the characteristics of frequent exacerbators include a more severe mmrc - scale grade , lower fev1 predicted , comorbid cardiovascular disease , depression or osteoporosis , and female sex as independent risk factors.32,33 we conducted the present study to observe moderate and severe exacerbations 1 year before and after baseline to investigate the characteristics of japanese copd patients who were frequent exacerbators . based on exacerbations during the previous year , we found that frequent exacerbators were more likely to be female , have a lower body mass index , have a significantly lower fev1 predicted , have a higher mmrc - scale grade ( lower exercise tolerance ) , and have a lower total cat score ( lower hrqol ) .", "we also found that the characteristics of frequent exacerbators were similar between japanese and westerners , except for body mass index , as reported previously.12,16,32 interestingly , univariate analysis did not show that previous frequent exacerbators would become future frequent exacerbators ( table 2 ) .", "indeed , 60% of previous frequent exacerbators did not suffer subsequent exacerbations , whereas conversely 14% of patients who had not previously suffered exacerbations subsequently did so ( figure 2 ) .", "investigation of factors predicting the change in frequency of exacerbations is critically important , as they are still unclear.34 a total of 78 ( 86.7% ) of our 90 patients completed the 1-year prospective study period . among five severe exacerbators , one ( 20% ) died , two ( 40% ) again developed severe exacerbation , and two ( 40% )", "therefore , severe exacerbators appear to have a poor prognosis . however , our study - sample size was small and the observational period short .", "frequent exacerbators had a significantly higher frequency of future exacerbations and a shorter period until the next exacerbation than nonexacerbators , and also had a significantly poorer prognosis than the latter , thus confirming the findings of a previous study.12 previous studies have demonstrated that over half of copd patients have unreported exacerbations .", "such unreported exacerbations are thought to be an important component of hrqol decline.7,35,36 in our present study , to make the conditions for previous and future exacerbations uniform , daily journals for symptoms were not accepted .", "previous studies have demonstrated that the frequency of annual moderate or severe exacerbations per patient in japanese individuals may be lower than that in the usa and europe.13,14,3537 in our study , the mean (  sd [ range ] ) of previous annual total and severe exacerbations were 0.841.48 ( 06 ) and 0.060.23 ( 01 ) exacerbations / year , respectively for all patients ( data not shown ) , whereas those of future total and severe exacerbations were 0.711.08 ( 04 ) and 0.060.28 ( 02 ) exacerbations / year , respectively .", "our data for the frequency of exacerbations seem to indicate a slightly lower incidence in japanese than in westerners.1215,31,32,38 previous japanese reports on the frequency of exacerbations have been scarce.13,14 japanese copd patients may be slightly older and thinner on average than westerners.1215,31,32,38 although the discrepancy between japanese and westerners is still unclear , the difference in the frequency of exacerbations may be associated with different populations of phenotypes with emphysema , locality , and understanding of both the physician and the patient about diseases and exacerbations , such as convenient use of ics , and in definition of exacerbation .", "patients with copd have a wide variety of numerous comorbidities , which are strongly associated with mortality and exacerbation.1,12,26 we assessed baseline comorbidities based on interviews with patients and the physicians diagnosis , and partly through examinations or questionnaires .", "several comorbidities , such as depression21,39 assessed by the cesd questionnaire , acos27,40 based on previous criteria , and congestive status with heart failure and cor pulmonale based on history or medical signs,41 were carefully excluded .", "gerd , although not moderate to severe , was a common major comorbidity , and the proportion of frequent exacerbators with gerd was significantly higher than that of nonexacerbators .", "gerd was an independent , and the highest , risk factor for future frequent exacerbations , although all patients with gerd were receiving proton - pump inhibitors .", "our results confirmed previous reports.12,42 a low fev1 predicted was an independent risk factor for future exacerbations .", "the guidelines1,43 recommend long - term treatment with ics and pneumococcal vaccination for patients with severe and very severe copd , and frequent exacerbations may not be adequately controlled by long - acting bronchodilators . in our study", "regular use of ics , but not pneumococcal vaccination , was an independent risk factor for future frequent exacerbations .", "sixteen ( 17.8% ) of our patients used ics , although none had received ics without long - acting bronchodilators at baseline .", "users of ics included four nonexacerbators with gold stage ii , seven patients with frequent exacerbations , and the remaining five had severe or very severe disease .", "the risk of ics for respiratory infection including pneumonia is a concern.4446 among four nonexacerbators with gold stage ii and users of ics during the prospective observation period , two patients had frequent exacerbations and one had infrequent exacerbations .", "this risk must be weighed against the benefits when prescribing ics to patients with copd .", "first , the study population was small and the observation period rather short for analyzing severe exacerbations and mortality .", "balcells et al demonstrated that female sex was the highest risk factor for exacerbation,33 although in the present study there was no difference in the frequency of exacerbations between males and females .", "both physicians and patients were aware of the severity and frequency of previous exacerbations at baseline .", "we carefully removed severe comorbidities , such as cardiovascular disease and depression , in accordance with previous reports.26,27 however , patients with asthma and acos may have been included , because we did not investigate airway responsiveness and inflammation or serum total immunoglobulin ( ig ) e levels , although we carefully excluded patients with asthma based on symptoms and spirometry", ". however , no patients had received medications for osteoporosis and low peripheral lymphocyte counts , although latent osteoporosis and hiv infections were not tested for .", "fourth , the contents of medicines could not be ethically unified in a clinical setting in japan , and in our study adherence to pharmacological medicines and compliance with inhalation techniques were not assessed or included .", "the effects of respiratory medications , including inhaled medicines , on previous and future exacerbations were investigated .", "the presence of gerd , regular use of ics , and low fev1 may be associated with frequent exacerbations ."], "abstract_text": ["<S> backgroundthe prognosis of japanese patients with copd who suffer repeated exacerbations is unclear , although westerners with such episodes have a poor prognosis.materials and methodswe conducted a 1-year prospective observational trial involving 90 japanese patients with copd : 58 nonexacerbators , 12 infrequent exacerbators , and 20 frequent exacerbators classified on the basis of exacerbation frequency ( zero , one , and two or more exacerbations / year ) , respectively , during the previous year were observed prospectively for 1 year . </S>", "<S> the characteristics of frequent exacerbators , the frequency of exacerbation , and the period until the first event were then compared among the groups.resultsa total of 78 patients completed the study . </S>", "<S> frequent exacerbators had a significantly higher risk of frequent exacerbation in the following year than the case for nonexacerbators ( odds ratio [ 95% confidence interval ] 2.94 [ 1.217.17 ] , p=0.0340 ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ) </S>", "<S> . the mean annual frequency of exacerbations in the following year was significantly ( p=0.0020 ) higher in the frequent exacerbators ( 1.4 exacerbations / year ) than in the nonexacerbators ( 0.4 ) , but not in the infrequent exacerbators ( 0.9 , p>0.05 ) . </S>", "<S> the mean period until the first exacerbation was significantly shorter in the frequent exacerbators than in the infrequent or nonexacerbators ( p=0.0012 ) . </S>", "<S> independent risk factors for future frequent exacerbation included the presence of gastroesophageal reflux disease , more severe airflow obstruction , and use of inhaled corticosteroids.conclusionour present results indicate that japanese copd patients suffering frequent exacerbation have a poor prognosis . </S>", "<S> the characteristics of japanese and western copd patients suffering frequent exacerbation are similar . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Study design", "Diagnosis and spirometric classification of COPD", "Assessment of mMRC scale, total CAT, FSSG scale, and CESD", "Comorbidities", "Frequency and date of mortality, hospitalizations, and exacerbations", "Statistical analysis", "Results", "Study subjects", "Baseline characteristics", "Stability of the frequent and severe exacerbation phenotype in patients who completed the study", "Comparison of annual exacerbation and hospitalization among non-, infrequent, and frequent exacerbators", "Baseline characteristics of patients suffering further exacerbations (one or more/year) and frequent exacerbations (two or more/year)", "Discussion", "Conclusion"], "sections": [["exacerbation is an important life - threatening event for patients with copd , and can lead to hospitalization and death.14 patients who suffer frequent and repeated exacerbations within 1 year have a poor prognosis,5 characterized by worsening of health - related quality of life ( hrqol),6,7 a rapid decline in lung function,810 and high mortality.11 frequent exacerbators also carry a high risk of further exacerbation and hospitalization.11,12 however , it has been suggested that japanese patients with copd may have fewer exacerbations , and they also may have a higher proportion of elderly patients , those with emphysema , and those with a lower body mass index in comparison to westerners.1215 the prognosis of japanese patients with copd who suffer frequent and repeated exacerbations is unclear .", "we conducted a 1-year prospective observational trial in a daily - life setting involving 90 japanese patients with copd to investigate whether previous moderate - to - severe exacerbations are associated with future exacerbations in this patient population ."], ["we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) .", "consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records .", "copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) .", "the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography .", "duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened .", "regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting .", "the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas .", "to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma ", "copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25 baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721 information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians .", "however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ]", "15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities .", "previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events .", "moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year .", "hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation .", "mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators .", "unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms .", "data are expressed as mean  standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline .", "characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey ", "subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan  meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period , the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses .", "medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis .", "statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) ."], ["we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) .", "consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records .", "copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) .", "the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography .", "duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened .", "regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting ."], ["the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas .", "to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma ", "copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25"], ["baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721"], ["information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians .", "however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ]", "15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities ."], ["previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events .", "moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year .", "hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation .", "mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators .", "unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms ."], ["data are expressed as mean  standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline .", "characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey ", "subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan  meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period ,", "the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses .", "medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis .", "statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) ."], ["ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year .", "the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) . at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) .", "frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) .", "there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) . during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) .", "figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation .", "the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations .", "the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) .", "in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) .", "in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period .", "the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively .", "patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) .", "figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) .", "figure 3b shows the kaplan  meier analysis of the period until the first moderate and severe exacerbations .", "the median ( mean  sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) .", "there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) .", "the median ( mean  sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) .", "univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) .", "however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination .", "the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted , presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) ."], ["ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year .", "the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) ."], ["at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) .", "frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) .", "there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) ."], ["during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) .", "figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation .", "the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations .", "the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) . in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) .", "in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period .", "the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively .", "patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) ."], ["figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) .", "figure 3b shows the kaplan  meier analysis of the period until the first moderate and severe exacerbations .", "the median ( mean  sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) .", "there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) .", "the median ( mean  sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) ."], ["univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) .", "however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination . the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted ,", "presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) ."], ["the frequent - exacerbator phenotype is important to consider in the management of copd patients , who require future exacerbation - related hospitalization associated with high mortality.16 a previous western study suggested that frequent exacerbators tend to have had exacerbations during the previous year , a lower fev1 , more severe hrqol , a history of gerd , and a higher peripheral white blood - cell count in comparison with infrequent exacerbators and nonexacerbators.12 another western study demonstrated that the characteristics of frequent exacerbators include a more severe mmrc - scale grade , lower fev1 predicted , comorbid cardiovascular disease , depression or osteoporosis , and female sex as independent risk factors.32,33 we conducted the present study to observe moderate and severe exacerbations 1 year before and after baseline to investigate the characteristics of japanese copd patients who were frequent exacerbators . based on exacerbations during the previous year , we found that frequent exacerbators were more likely to be female , have a lower body mass index , have a significantly lower fev1 predicted , have a higher mmrc - scale grade ( lower exercise tolerance ) , and have a lower total cat score ( lower hrqol ) .", "we also found that the characteristics of frequent exacerbators were similar between japanese and westerners , except for body mass index , as reported previously.12,16,32 interestingly , univariate analysis did not show that previous frequent exacerbators would become future frequent exacerbators ( table 2 ) .", "indeed , 60% of previous frequent exacerbators did not suffer subsequent exacerbations , whereas conversely 14% of patients who had not previously suffered exacerbations subsequently did so ( figure 2 ) .", "investigation of factors predicting the change in frequency of exacerbations is critically important , as they are still unclear.34 a total of 78 ( 86.7% ) of our 90 patients completed the 1-year prospective study period . among five severe exacerbators , one ( 20% ) died , two ( 40% ) again developed severe exacerbation , and two ( 40% )", "therefore , severe exacerbators appear to have a poor prognosis . however , our study - sample size was small and the observational period short .", "frequent exacerbators had a significantly higher frequency of future exacerbations and a shorter period until the next exacerbation than nonexacerbators , and also had a significantly poorer prognosis than the latter , thus confirming the findings of a previous study.12 previous studies have demonstrated that over half of copd patients have unreported exacerbations .", "such unreported exacerbations are thought to be an important component of hrqol decline.7,35,36 in our present study , to make the conditions for previous and future exacerbations uniform , daily journals for symptoms were not accepted .", "previous studies have demonstrated that the frequency of annual moderate or severe exacerbations per patient in japanese individuals may be lower than that in the usa and europe.13,14,3537 in our study , the mean (  sd [ range ] ) of previous annual total and severe exacerbations were 0.841.48 ( 06 ) and 0.060.23 ( 01 ) exacerbations / year , respectively for all patients ( data not shown ) , whereas those of future total and severe exacerbations were 0.711.08 ( 04 ) and 0.060.28 ( 02 ) exacerbations / year , respectively .", "our data for the frequency of exacerbations seem to indicate a slightly lower incidence in japanese than in westerners.1215,31,32,38 previous japanese reports on the frequency of exacerbations have been scarce.13,14 japanese copd patients may be slightly older and thinner on average than westerners.1215,31,32,38 although the discrepancy between japanese and westerners is still unclear , the difference in the frequency of exacerbations may be associated with different populations of phenotypes with emphysema , locality , and understanding of both the physician and the patient about diseases and exacerbations , such as convenient use of ics , and in definition of exacerbation .", "patients with copd have a wide variety of numerous comorbidities , which are strongly associated with mortality and exacerbation.1,12,26 we assessed baseline comorbidities based on interviews with patients and the physicians diagnosis , and partly through examinations or questionnaires .", "several comorbidities , such as depression21,39 assessed by the cesd questionnaire , acos27,40 based on previous criteria , and congestive status with heart failure and cor pulmonale based on history or medical signs,41 were carefully excluded .", "gerd , although not moderate to severe , was a common major comorbidity , and the proportion of frequent exacerbators with gerd was significantly higher than that of nonexacerbators .", "gerd was an independent , and the highest , risk factor for future frequent exacerbations , although all patients with gerd were receiving proton - pump inhibitors .", "our results confirmed previous reports.12,42 a low fev1 predicted was an independent risk factor for future exacerbations .", "the guidelines1,43 recommend long - term treatment with ics and pneumococcal vaccination for patients with severe and very severe copd , and frequent exacerbations may not be adequately controlled by long - acting bronchodilators . in our study", "regular use of ics , but not pneumococcal vaccination , was an independent risk factor for future frequent exacerbations .", "sixteen ( 17.8% ) of our patients used ics , although none had received ics without long - acting bronchodilators at baseline .", "users of ics included four nonexacerbators with gold stage ii , seven patients with frequent exacerbations , and the remaining five had severe or very severe disease .", "the risk of ics for respiratory infection including pneumonia is a concern.4446 among four nonexacerbators with gold stage ii and users of ics during the prospective observation period , two patients had frequent exacerbations and one had infrequent exacerbations .", "this risk must be weighed against the benefits when prescribing ics to patients with copd .", "first , the study population was small and the observation period rather short for analyzing severe exacerbations and mortality .", "balcells et al demonstrated that female sex was the highest risk factor for exacerbation,33 although in the present study there was no difference in the frequency of exacerbations between males and females .", "both physicians and patients were aware of the severity and frequency of previous exacerbations at baseline .", "we carefully removed severe comorbidities , such as cardiovascular disease and depression , in accordance with previous reports.26,27 however , patients with asthma and acos may have been included , because we did not investigate airway responsiveness and inflammation or serum total immunoglobulin ( ig ) e levels , although we carefully excluded patients with asthma based on symptoms and spirometry", ". however , no patients had received medications for osteoporosis and low peripheral lymphocyte counts , although latent osteoporosis and hiv infections were not tested for .", "fourth , the contents of medicines could not be ethically unified in a clinical setting in japan , and in our study adherence to pharmacological medicines and compliance with inhalation techniques were not assessed or included .", "the effects of respiratory medications , including inhaled medicines , on previous and future exacerbations were investigated ."], ["the presence of gerd , regular use of ics , and low fev1 may be associated with frequent exacerbations ."]]}
{"article_id": "PMC4954860", "article_text": ["\n lipoma is a rare tumor but can be found at various sites in the abdominal cavity ( fig .", "2 ) . if it is of a large size , it can compress adjacent tissues or organs and become heterogeneous . in such cases ,", "imaging is incapable of distinguishing it from liposarcoma.lipoblastoma is a type of a benign tumor with embryonic fat .", "it is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . in us , such tumors are echogenic .", "lipoblastomatosis is a form of this tumor characterized by aggressive localized growth.hibernoma is a benign tumor , mainly composed of brown fat , in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive .", "these tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins .", "their characteristic feature is evident flow in color doppler.other benign tumors with a fat component , such as myolipoma , angiomyolipoma and myelolipoma , apart from teratoma ( which is usually benign ) , tend to be homogeneous with echogenicity increased to various degrees ( fig . 3 , 4 ) .", "only gonadal germ cell tumors , such as teratoma and dermoid cyst that arise from several germ layers , frequently have irregular echotexture due to the presence of fluid sebum , hair , soft tissues and bony elements .", "that is why they are encountered in solid , cystic or solid - cystic forms ( fig .", "mature fat tissue can also be found in choristoma which , however , is not a neoplasm .", "lipoma is a rare tumor but can be found at various sites in the abdominal cavity ( fig .", "2 ) . if it is of a large size , it can compress adjacent tissues or organs and become heterogeneous .", "it is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . in us , such tumors are echogenic .", "hibernoma is a benign tumor , mainly composed of brown fat , in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive .", "these tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins .", "other benign tumors with a fat component , such as myolipoma , angiomyolipoma and myelolipoma , apart from teratoma ( which is usually benign ) , tend to be homogeneous with echogenicity increased to various degrees ( fig . 3 , 4 ) .", "only gonadal germ cell tumors , such as teratoma and dermoid cyst that arise from several germ layers , frequently have irregular echotexture due to the presence of fluid sebum , hair , soft tissues and bony elements .", "that is why they are encountered in solid , cystic or solid - cystic forms ( fig .", "mature fat tissue can also be found in choristoma which , however , is not a neoplasm .", "two views show lipoma ( arrows ) as a slightly echogenic lesion in the supraperitoneal fat three lipomas ( l ) in the small bowel mesentery , which in computed tomography showed density ranging from 57 to 74 hounsfield units two views show angiomyolipoma ( l ) located entirely in the adipose capsule of the right kidney .", "an arrow points to the site of regrowth in the form of the beak sign myelolipoma in the right suprarenal field ( arrow ) as a visible hyperechoic mass a heterogeneous mass arising from the retroperitoneal space is a mature teratoma with slight calcifications ( arrows )", "liposarcoma is the only malignancy deriving from adipose tissue that is relatively frequently encountered in the retroperitoneal space ( it constitutes nearly 1/3 of sarcomas at this site and 1015% of all neoplasms of this type ) . according to the who , these tumors can be divided into five histological subtypes depending on the grade of their differentiation ( well - differentiated , dedifferentiated , myxoid , round cell , pleomorphic ) , which is relevant in prognosis .", "its presence can be indicated only by uneven margins and heterogeneous enhancement upon contrast agent administration .", "poorly differentiated subtypes account for even 40% of local relapses , and metastases are observed in 17% of cases . as has already been mentioned , well - differentiated liposarcoma is difficult to distinguish from lipoma also in sonography .", "one of the differential criteria can be tumor 's reaction to compression with a transducer ( fig .", "more aggressive forms of this tumor , however , tend to be unevenly delineated , present heterogeneous echotexture and infiltrate adjacent tissues ( fig .", "myxoid liposarcoma is characterized by a cystic structure and should be distinguished e.g. from lymphangioma , teratoma or cystic mesothelioma .", "no lesion compressibility when pressure is applied with the transducer ( arrow ) heterogeneous retroperitoneal liposarcoma infiltrates into adjacent tissues ( arrows ) mesenteric liposarcoma with pathological vascularity finally , immature teratoma must be mentioned .", "it is a primary germinal tumor that can contain adipose tissue and occurs at similar sites .", "they are characterized by the predominance of a solid component , and the only sign suggesting a malignancy is poorly circumscribed margins . a chance for malignant transformation", "cystic lesions can correspond to mesenteric cysts , lymphatic or vascular angiomatosis and even a cystic form of peritoneal mesothelioma , which is considered a benign lesion ( fig .", "the differential diagnosis of these pathologies should include ovarian cystic tumors , endometrial cysts and cystic tumors growing beyond organs , e.g. of the pancreas , kidneys or liver .", "solid tumors found at these sites include : leiomyoma and rhabdomyoma , angiomas as well as stromal , neurogenic and desmoid tumors ( fig .", "desmoid tumors , also called fibromatosis , are an interesting group of benign tumors that exhibit local aggressiveness and have considerable tendency to recur ( in approximately 50% of cases ) .", "all lesions were hypoechoic with slightly irregular echo patterns , 59% of cases were well - defined and 41% had irregular margins .", "the differential diagnosis should include sarcomas , mainly the two types that are most frequently found in soft tissues : histiocytic fibrosarcoma and liposarcoma , i.e. lesions characterized by higher echogenicity .", "it is of note that desmoid tumors frequently coexist with familial adenomatous polyposis , which is estimated at 918% of cases . of all imaging modalities ,", "some authors recommend punch or even surgical biopsy in the event of doubts . in this case ,", "mesenteric lymphangioma as a multilocular lesion without flow ( arrow ) retroperitoneal neuroblastoma in a 13-year - old ( arrows ) .", "aorta , v  inferior vena cava desmoid , poorly vascularized tumor ( d ) in the small bowel mesentery", "these changes are rarely encountered but they represent a rich spectrum of mainly mesenchymal neoplasms : fibrosarcoma , malignant fibrous histiocytoma , hemangiopericytoma , leiomyosarcoma , rhabdomyosarcoma and gastrointestinal stromal tumors ( fig .", "an epithelioid malignant neoplasm encountered at this site is malignant peritoneal mesothelioma , a lesion that almost exclusively develops in males in the fifth and sixth decade of life .", "it can assume two morphological forms : irregular peritoneal infiltration without ascites ( dry appearance ) ( fig .", "13 ) and nodular thickening of the peritoneum and greater omentum with ascites ( wet appearance ) .", "lesions of this type must be distinguished from papillary serous carcinoma of the peritoneum ( a rare neoplasm found in elderly women ) , abdominal carcinomatosis , in which ascites prevails over implants , and peritoneal tuberculosis , in which there are no tumorous lesions ( but ascites is present , the greater omentum is thickened with preserved smooth surface and the mesenteric lymph nodes are enlarged ) .", "lymphomas , however , are characterized by considerable abdominal lymphadenopathy , rare and slight ascites and no omental involvement .", "rhabdomyosarcoma with rich vascularity in the retroperitoneal space ( arrows ) two views present poorly vascularized malignant mesothelioma of the greater omentum ( m )", "this is the most numerous group with prevailing carcinoma foci , mainly deriving from abdominal organs , such as pancreas , stomach , large bowel or ovaries .", "these lesions spread by direct extension , via the lymphatic system or blood stream and through the peritoneal cavity . their echotexture can vary .", "14 ) and thickened parietal peritoneum ( its normal thickness is up to 1.5 mm ) ( fig .", "echogenic lesions that only slightly differ from the surrounding tissues are the hardest to find ( fig .", "peritoneal sites at which carcinoma implants tend to develop are : the recto - uterine pouch , right iliac fossa , right paracolic gutter and the region of the upper sigmoid mesentery .", "moreover , ovarian carcinoma cells frequently migrate to the supra- and subhepatic area ( fig .", "well - designed prospective studies have proven ultrasound imaging to be highly useful in detecting abdominal carcinomatosis .", "it occurred that lesions in the greater omentum were the easiest to detect ( 91% ) whereas those in the small bowel mesentery were the hardest to find ( 67% ) .", "moreover , ultrasound guidance enables one to obtain diagnostically valuable material for cytological and histological analysis in the vast majority of such cases .", "metastasis of malignant melanoma ( m ) from the interscapular space to the small bowel mesentery peritoneal carcinomatosis of ovarian carcinoma . thickened hypoechoic parietal peritoneum with signs of vascular flow ( arrows ) two views show a hyperechoic implant of ovarian carcinoma in the gastrocolonic ligament ( arrows ) ovarian carcinoma implants in the pouch of morison ( arrows ) another rare neoplasm encountered in fatty bodies of the abdominal cavity is non - hodgkin lymphoma .", "the involvement of the small bowel mesentery , with prominent , large mesenteric lymph nodes , is observed to occur in a half of patients with this neoplasm .", "these lymph nodes , together with the superior mesenteric vessels , create a so - called  sandwich  image ( fig .", "it is usually a small neuroendocrine neoplasm located in the small bowel . sometimes , the first morphological sign of its existence is the presence of mesenteric abnormalities in the form of enlarged lymph nodes or a hypoechoic mass .", "mesenteric thickening or contraction , which can lead to small bowel obstruction , is more rarely a predominant element of the image . any doubts in the assessment of these lesions require verification in computed tomography and , sometimes , magnetic resonance imaging . ultrasound imaging , however , enables precise , percutaneous sampling for cytological and histological analysis , which is worth remembering and applying . ", "sandwich  sign in the small bowel mesentery ( arrows ) created by enlarged lymph nodes surrounding the superior mesenteric vessels  a manifestation of non - hodgkin lymphoma extranodal location of follicular lymphoma in the small bowel mesentery ( arrow ) lesions in the lymph nodes , which are relatively frequently affected by various neoplastic and non - neoplastic processes , have been presented only briefly since these issues are too broad to be included in this review .", "authors do not report any financial or personal connections with other persons or organizations , which might negatively affect the contents of this publication and/or claim authorship rights to this publication ."], "abstract_text": ["<S> this article focuses on various cancerous lesions that are found beyond organs in the intra - abdominal fat and can be visualized with ultrasonography . </S>", "<S> these lesions are divided into five groups . </S>", "<S> the first group includes primary benign tumors containing adipocytes , such as lipoma , lipoblastoma , hibernoma and other lesions with an adipose tissue component , such as myolipoma , angiomyolipoma , myelolipoma and teratoma . </S>", "<S> the second group comprises primary malignant adipocytecontaining tumors , including liposarcoma and immature teratoma . </S>", "<S> the third group contains primary benign tumors without an adipocyte component that are located in intra - abdominal fat . </S>", "<S> this is a numerous group of lesions represented by cystic and solid tumors . </S>", "<S> the fourth group encompasses primary malignant tumors without an adipocyte component that are located in intra - abdominal fat . </S>", "<S> these are rare lesions associated mainly with sarcomas : fibrosarcoma , malignant fibrous histiocytoma , hemangiopericytoma and leiomyosarcoma . </S>", "<S> an epithelioid tumor at this site is mesothelioma . </S>", "<S> the last but not least group includes secondary malignant tumors without an adipocyte component located in intra - abdominal fat . </S>", "<S> this is the most numerous group with prevailing carcinoma foci . for each of these groups , </S>", "<S> the authors present ultrasound features of individual lesions and discuss their differential diagnosis . in the vast majority of cases , </S>", "<S> the material for cytological and histological analysis can be obtained during ultrasound - guided procedures . </S>", "<S> this is the advantage of this imaging modality . </S>"], "labels": null, "section_names": ["Primary benign adipocyte-containing tumors located in intra-abdominal fat", "Liposarcoma", "Primary benign tumors without an adipocyte component located in intra-abdominal fat", "Primary malignant tumors without an adipocyte component located in intra-abdominal fat", "Secondary malignant tumors without an adipocyte component located in intra-abdominal fat", "Conflict of interest"], "sections": [["\n lipoma is a rare tumor but can be found at various sites in the abdominal cavity ( fig .", "2 ) . if it is of a large size , it can compress adjacent tissues or organs and become heterogeneous . in such cases ,", "imaging is incapable of distinguishing it from liposarcoma.lipoblastoma is a type of a benign tumor with embryonic fat .", "it is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . in us , such tumors are echogenic .", "lipoblastomatosis is a form of this tumor characterized by aggressive localized growth.hibernoma is a benign tumor , mainly composed of brown fat , in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive .", "these tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins .", "their characteristic feature is evident flow in color doppler.other benign tumors with a fat component , such as myolipoma , angiomyolipoma and myelolipoma , apart from teratoma ( which is usually benign ) , tend to be homogeneous with echogenicity increased to various degrees ( fig . 3 , 4 ) .", "only gonadal germ cell tumors , such as teratoma and dermoid cyst that arise from several germ layers , frequently have irregular echotexture due to the presence of fluid sebum , hair , soft tissues and bony elements .", "that is why they are encountered in solid , cystic or solid - cystic forms ( fig .", "mature fat tissue can also be found in choristoma which , however , is not a neoplasm .", "lipoma is a rare tumor but can be found at various sites in the abdominal cavity ( fig .", "2 ) . if it is of a large size , it can compress adjacent tissues or organs and become heterogeneous .", "it is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . in us , such tumors are echogenic .", "hibernoma is a benign tumor , mainly composed of brown fat , in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive .", "these tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins .", "other benign tumors with a fat component , such as myolipoma , angiomyolipoma and myelolipoma , apart from teratoma ( which is usually benign ) , tend to be homogeneous with echogenicity increased to various degrees ( fig . 3 , 4 ) .", "only gonadal germ cell tumors , such as teratoma and dermoid cyst that arise from several germ layers , frequently have irregular echotexture due to the presence of fluid sebum , hair , soft tissues and bony elements .", "that is why they are encountered in solid , cystic or solid - cystic forms ( fig .", "mature fat tissue can also be found in choristoma which , however , is not a neoplasm .", "two views show lipoma ( arrows ) as a slightly echogenic lesion in the supraperitoneal fat three lipomas ( l ) in the small bowel mesentery , which in computed tomography showed density ranging from 57 to 74 hounsfield units two views show angiomyolipoma ( l ) located entirely in the adipose capsule of the right kidney .", "an arrow points to the site of regrowth in the form of the beak sign myelolipoma in the right suprarenal field ( arrow ) as a visible hyperechoic mass a heterogeneous mass arising from the retroperitoneal space is a mature teratoma with slight calcifications ( arrows )"], ["liposarcoma is the only malignancy deriving from adipose tissue that is relatively frequently encountered in the retroperitoneal space ( it constitutes nearly 1/3 of sarcomas at this site and 1015% of all neoplasms of this type ) . according to the who , these tumors can be divided into five histological subtypes depending on the grade of their differentiation ( well - differentiated , dedifferentiated , myxoid , round cell , pleomorphic ) , which is relevant in prognosis .", "its presence can be indicated only by uneven margins and heterogeneous enhancement upon contrast agent administration .", "poorly differentiated subtypes account for even 40% of local relapses , and metastases are observed in 17% of cases . as has already been mentioned , well - differentiated liposarcoma is difficult to distinguish from lipoma also in sonography .", "one of the differential criteria can be tumor 's reaction to compression with a transducer ( fig .", "more aggressive forms of this tumor , however , tend to be unevenly delineated , present heterogeneous echotexture and infiltrate adjacent tissues ( fig .", "myxoid liposarcoma is characterized by a cystic structure and should be distinguished e.g. from lymphangioma , teratoma or cystic mesothelioma .", "no lesion compressibility when pressure is applied with the transducer ( arrow ) heterogeneous retroperitoneal liposarcoma infiltrates into adjacent tissues ( arrows ) mesenteric liposarcoma with pathological vascularity finally , immature teratoma must be mentioned .", "it is a primary germinal tumor that can contain adipose tissue and occurs at similar sites .", "they are characterized by the predominance of a solid component , and the only sign suggesting a malignancy is poorly circumscribed margins . a chance for malignant transformation"], ["cystic lesions can correspond to mesenteric cysts , lymphatic or vascular angiomatosis and even a cystic form of peritoneal mesothelioma , which is considered a benign lesion ( fig .", "the differential diagnosis of these pathologies should include ovarian cystic tumors , endometrial cysts and cystic tumors growing beyond organs , e.g. of the pancreas , kidneys or liver .", "solid tumors found at these sites include : leiomyoma and rhabdomyoma , angiomas as well as stromal , neurogenic and desmoid tumors ( fig .", "desmoid tumors , also called fibromatosis , are an interesting group of benign tumors that exhibit local aggressiveness and have considerable tendency to recur ( in approximately 50% of cases ) .", "all lesions were hypoechoic with slightly irregular echo patterns , 59% of cases were well - defined and 41% had irregular margins .", "the differential diagnosis should include sarcomas , mainly the two types that are most frequently found in soft tissues : histiocytic fibrosarcoma and liposarcoma , i.e. lesions characterized by higher echogenicity .", "it is of note that desmoid tumors frequently coexist with familial adenomatous polyposis , which is estimated at 918% of cases . of all imaging modalities ,", "some authors recommend punch or even surgical biopsy in the event of doubts . in this case ,", "mesenteric lymphangioma as a multilocular lesion without flow ( arrow ) retroperitoneal neuroblastoma in a 13-year - old ( arrows ) .", "aorta , v  inferior vena cava desmoid , poorly vascularized tumor ( d ) in the small bowel mesentery"], ["these changes are rarely encountered but they represent a rich spectrum of mainly mesenchymal neoplasms : fibrosarcoma , malignant fibrous histiocytoma , hemangiopericytoma , leiomyosarcoma , rhabdomyosarcoma and gastrointestinal stromal tumors ( fig .", "an epithelioid malignant neoplasm encountered at this site is malignant peritoneal mesothelioma , a lesion that almost exclusively develops in males in the fifth and sixth decade of life .", "it can assume two morphological forms : irregular peritoneal infiltration without ascites ( dry appearance ) ( fig .", "13 ) and nodular thickening of the peritoneum and greater omentum with ascites ( wet appearance ) .", "lesions of this type must be distinguished from papillary serous carcinoma of the peritoneum ( a rare neoplasm found in elderly women ) , abdominal carcinomatosis , in which ascites prevails over implants , and peritoneal tuberculosis , in which there are no tumorous lesions ( but ascites is present , the greater omentum is thickened with preserved smooth surface and the mesenteric lymph nodes are enlarged ) .", "lymphomas , however , are characterized by considerable abdominal lymphadenopathy , rare and slight ascites and no omental involvement .", "rhabdomyosarcoma with rich vascularity in the retroperitoneal space ( arrows ) two views present poorly vascularized malignant mesothelioma of the greater omentum ( m )"], ["this is the most numerous group with prevailing carcinoma foci , mainly deriving from abdominal organs , such as pancreas , stomach , large bowel or ovaries .", "these lesions spread by direct extension , via the lymphatic system or blood stream and through the peritoneal cavity . their echotexture can vary .", "14 ) and thickened parietal peritoneum ( its normal thickness is up to 1.5 mm ) ( fig .", "echogenic lesions that only slightly differ from the surrounding tissues are the hardest to find ( fig .", "peritoneal sites at which carcinoma implants tend to develop are : the recto - uterine pouch , right iliac fossa , right paracolic gutter and the region of the upper sigmoid mesentery .", "moreover , ovarian carcinoma cells frequently migrate to the supra- and subhepatic area ( fig .", "well - designed prospective studies have proven ultrasound imaging to be highly useful in detecting abdominal carcinomatosis .", "it occurred that lesions in the greater omentum were the easiest to detect ( 91% ) whereas those in the small bowel mesentery were the hardest to find ( 67% ) .", "moreover , ultrasound guidance enables one to obtain diagnostically valuable material for cytological and histological analysis in the vast majority of such cases .", "metastasis of malignant melanoma ( m ) from the interscapular space to the small bowel mesentery peritoneal carcinomatosis of ovarian carcinoma . thickened hypoechoic parietal peritoneum with signs of vascular flow ( arrows ) two views show a hyperechoic implant of ovarian carcinoma in the gastrocolonic ligament ( arrows ) ovarian carcinoma implants in the pouch of morison ( arrows ) another rare neoplasm encountered in fatty bodies of the abdominal cavity is non - hodgkin lymphoma .", "the involvement of the small bowel mesentery , with prominent , large mesenteric lymph nodes , is observed to occur in a half of patients with this neoplasm .", "these lymph nodes , together with the superior mesenteric vessels , create a so - called  sandwich  image ( fig .", "it is usually a small neuroendocrine neoplasm located in the small bowel . sometimes , the first morphological sign of its existence is the presence of mesenteric abnormalities in the form of enlarged lymph nodes or a hypoechoic mass .", "mesenteric thickening or contraction , which can lead to small bowel obstruction , is more rarely a predominant element of the image . any doubts in the assessment of these lesions require verification in computed tomography and , sometimes , magnetic resonance imaging . ultrasound imaging , however , enables precise , percutaneous sampling for cytological and histological analysis , which is worth remembering and applying . ", "sandwich  sign in the small bowel mesentery ( arrows ) created by enlarged lymph nodes surrounding the superior mesenteric vessels  a manifestation of non - hodgkin lymphoma extranodal location of follicular lymphoma in the small bowel mesentery ( arrow ) lesions in the lymph nodes , which are relatively frequently affected by various neoplastic and non - neoplastic processes , have been presented only briefly since these issues are too broad to be included in this review ."], ["authors do not report any financial or personal connections with other persons or organizations , which might negatively affect the contents of this publication and/or claim authorship rights to this publication ."]]}
{"article_id": "PMC4334867", "article_text": ["the national cancer institute ( nci ) of the united states of america has proposed a reporting system for thyroid fine needle aspiration ( fna ) cytology , so - called bethesda system , which became an international standard of thyroid cytology [ 1 , 2 ] . following this recommendation , the uk royal college of pathologists ( the uk system ) [ 3 , 4 ] and italian societies of endocrinology and the italian society for anatomic pathology and cytology joint with the italian division of the international academy of pathology ( the italian system ) [ 5 , 6 ] updated their diagnostic schema comparable with the bethesda system . in 2013 ,", "the japan thyroid association ( jta ) published guidelines for clinical practice for the management of thyroid nodules , including its diagnostic system for reporting thyroid fna cytology as shown in table 1 [ 79 ] , using criteria similar to those used in the papanicolaou society recommendation , the bethesda system [ 1 , 2 ] , the uk system [ 3 , 4 ] , and the italian system [ 5 , 6 ] .", "the jta reporting system of thyroid cytology further recommends risk stratification of follicular neoplasms ( fn ) into favor benign ( low risk : lr ) , borderline ( moderate risk : mr ) , and favor malignant ( high risk : hr ) , which was adopted from the practice of high - volume thyroid centers in japan .", "this study examined observer variation in the subclassification of fn among 4 thyroid experts to validate the usefulness and limitations of this characteristic risk stratification of fn recommended in the jta guidelines .", "it is not our purpose to address in detail all the morphological issues of thyroid cytology .", "conventional smear samples of indeterminate diagnosis ( n = 389 ) were selected from files ( n = 3843 ) from the year 2005 at ito hospital , tokyo , japan , by one ( k. kameyama ) of the authors .", "inadequate samples or those with poor preparation and cases under clinical follow - up ( no final histologic diagnosis ) were excluded .", "there were 91 ( 23.4% ) surgically treated patients with thyroid nodule under indeterminate cytological diagnoses .", "these patients visited ito hospital in 2005 and underwent diagnostic surgery or curative surgery in the subsequent years between 2005 and 2008 .", "there were 48 cases of benign diagnoses , including 29 follicular adenomas ( fas ) , 19 adenomatous nodules ( an ) , and 43 malignant diagnoses , including 13 follicular carcinomas ( ftcs ) , 24 papillary carcinomas ( ptcs ) , 1 poorly differentiated carcinoma , 2 c - cell carcinomas , and 3 malignant lymphomas .", "twenty cases of cytological smear samples with follicular patterned lesions were randomly selected by one ( k. kameyama ) of the authors and they were further analyzed for subclassification of fn and diagnoses by 4 reviewers , as shown in table 2 .", "the present study is a reproducibility study undertaken by 4 reviewers ( kennichi kakudo , kaori kameyama , mitsuyoshi hirokawa , and ryohei katoh ) who have special interest in thyroid pathology , all of whom are members of the clinical guideline committee of the jta .", "they were requested to subclassify fn following the new jta reporting system of thyroid cytology as shown in table 1 .", "those 20 cases of smear samples ( one representative smear sample each stained by the papanicolaou method ) were circulated among the 4 reviewers without clinical information .", "the 4 reviewers examined cytological samples independently without exchanging opinions and were unaware of the original cytological diagnoses and final histologic diagnoses . because this study was conceived as an audit of cytology performance and the results are anonymized , institutional ethical committee permission was not required for its conduct .", "informed consent of all 4 reviewers was obtained for this study protocol . for statistical analysis of observer concordance ,  was first introduced as a measure of the level of agreement between pairs of raters and extended to multiple raters , known as composite  [ 11 , 12 ] .", "composite  statistical analysis was performed by using data analysis and statistical software , version 13 ( stata press , college station , texas , usa ) .", "values of  can be interpreted as follows : 0.000.20 , slight or very weak agreement ; 0.210.40 , weak to fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , substantial or good agreement ; and values over 0.81 , optimal or almost perfect agreement .", "there were 8 malignant cases ( 2 ftcs , widely invasive ; 4 ftcs , minimally invasive ; and 2 ptcs ) and 12 benign cases ( 1 an and 11 fas ) .", "all diagnoses made by the 4 reviewers on the 20 follicular pattern lesions are shown in table 2 .", "there were only 6 ( 30% ) cases with a consensus diagnosis among all 4 of the reviewers : 3 in fa and 3 in ftc .", "diagnoses of fn and hr were made in only 7 cases ( 35% ) , including 4 malignant and 3 benign cases .", "composite  statistical analysis clearly demonstrated good concordance for the hr diagnosis in malignant cases (  = 0.7714 ) ( table 3 ) , while this was not found for the other subcategories in both benign and malignant lesions (  < 0.4 ) .", "it is of note that there was no interobserver variation in ftcs , widely invasive ( n = 2 ) , and all 4 reviewers classified the 2 cases into the hr subcategory .", "it is clear that ftcs , widely invasive , have different degrees of cellular abnormality in our study .", "in contrast , there were significant disagreements among other types of malignancy and benign lesions , which clearly confirmed that the subclassification of fn is not a definite diagnosis but risk stratification useful for triage patients .", "concerning ftc , minimally invasive ( n = 4 ) , there was only one concordant case among the 4 reviewers and all reviewers made a mr diagnosis in 1 case .", "there were disagreements between mr and hr in 1 case and between lr and mr in 2 cases .", "as for ptc ( n = 2 ) , the 4 reviewers gave 2 diagnoses each , which included 3 lrs , 3 mrs , 1 hr , and 1 suspicious for ptc .", "however , it is of note that there was no single case in our 20 cases whose subclassification varied among all 3 subcategories . in benign final histologic diagnosis ( n = 12 ) , there were 9 split diagnoses , including 6 cases between lr and mr and 3 cases between mr and hr , but no case between lr and hr .", "the incidence of lr diagnoses by the 4 reviewers in the 12 benign lesions was 31.3% ( 15/48 ) and that of lr diagnoses in the 6 cases of ftc was 16.7% ( 4/24 ) ( p = 0.186 ) .", "the incidence of hr diagnosis in the 12 benign lesions was 10.4% ( 5/48 ) and that of hr in the 6 cases of ftc was 47.1% ( 10/24 ) ( p = 0.002 ) .", "the incidence of malignancy of the hr was high at 60% for reviewer a , 75% for b , 75% for c , and 75% for d ( 6075% ) .", "the incidence of malignancy in the mr was lower than that of hr and was 33.3% for reviewer a , 35.7% for reviewer b , 25% for c , and 30.8% for d ( 2535.7% ) .", "the incidence of malignancy in the lr was lower than that in the hr and was 33.3% for reviewer a , 0% for b , 37.5% for c , and 33.3% for d ( 037.5% ) .", "although the risks of malignancy in the subcategories of fn differed among the 4 reviewers , it is of note that hr cytological diagnosis was significantly correlated with malignant histological diagnosis , and the risk of malignancy ( 6075% ) was significantly higher than that in the other 2 subcategories ( 035.7% ) in our study ( p = 0.002 ) .", "although the standardization of terminology and diagnostic criteria is important for accurate communication among patients , clinical doctors , and cytopathologists [ 110 ] , there are still differences among reporting systems in thyroid cytology as to how to interpret cytological diagnoses and how to decide on the clinical management of patients . in japan , cytopathologists attempted to use an internationally accepted reporting system , but it had to be modified to fit our practice . in japan , all patients with indeterminate cytology undergo further diagnostic procedures , without immediate surgery , to search for higher risk patients who should undergo surgery [ 79 , 1315 ] .", "it is because the majority of thyroid carcinomas in indeterminate cytology are indolent and more conservative approaches , other than immediate diagnostic surgery , usually do not create any harm to the patient with malignancy [ 1620 ] and diagnostic surgery to all patients with indeterminate cytology results in risks of unnecessary surgery to the patients with benign nodules , more than 80% of the patients [ 79 , 1922 ] .", "the proportion of malignancy found at thyroidectomy from patients with indeterminate cytology in this clinical setting will increase in number , and the malignancy rate of patient with indeterminate cytology in our study was calculated as 47.3% as shown in materials and methods .", "takezawa et al . from japan retrospectively analyzed their 1606 cytological samples using bethesda system , although it was written in japanese with english abstract , and they identified 115 ( 7.9% ) cases of aus / flus ( atypia of undetermined significance / follicular lesions of undetermined significance ) and 61 ( 4.2% ) cases of fn / sfn ( follicular neoplasms / suspicious for follicular neoplasms ) .", "the resection rate of their aus / flus nodules was 30.4% ( 35/115 cases ) and its malignancy rate was 88.6% ( 31/35 cases ) , and the resection rate of fn / sfn nodules was 36.0% ( 22/61 cases ) and its malignancy rate was 72.4% ( 16/22 cases ) , which were very different from the ranges reported in most of the literatures from western countries using the bethesda system or the uk system [ 1 , 2 , 2023 ] . as it is clear , further triage of patients with indeterminate nodules reduces resection rates and increases malignancy rates in any diagnostic systems including the bethesda system .", "this clinical management was also true in some other countries [ 2427 ] , as crippa and dina commented in a letter to an editor that thyroid cytology is the most important but not the only important deciding factor and therefore it must be integrated with other diagnostic procedures .", "we propose that future thyroid cytology classification schemes should reconsider clinical managements of indeterminate categories and how to reduce unnecessary surgeries for patients with benign thyroid nodules [ 79 ] .", "the risk stratification of follicular pattern lesions into 3 subcategories ( cellular follicular lesion , fn favor benign , and fn favor malignant ) was suggested by the papanicolaou society in 1996 , but it did not become popular in thyroid cytology worldwide , apart from japan [ 8 , 9 ] . there have been some reports in the literature on risk stratification of fn [ 10 , 2834 ] .", "kelman et al . reported that 31/52 ( 60% ) nodules with nuclear atypia consistent with fn were malignant   and it was 4/9 ( 44.4% ) in fn with atypia by goldstein et al . .", "reported that atypical proliferation was more often malignant than follicular group ( 53% versus 19% ) in their indeterminate category ( tir3/thy3 ) .", "some researchers pointed out that the malignancy rate of fn without atypia is low and assessment later on could be an alternative approach [ 31 , 32 ] and patients with high - risk cytological features such as nuclear overlapping ( crowding ) should be advised to have a surgical intervention [ 3133 ] .", "gerhard and da cunha santos using the papanicolaou society guidelines studied reproducibility between 2 observers in 97 diagnoses .", "they reported a substantial level of diagnostic interobserver (  = 0.71 ) and intraobserver (  = 0.66 ) reproducibility , although interobserver disagreement in the cytological diagnosis occurred in 23 cases ( 24.7% ) and 18 ( 41.7% ) of them were for fn .", "in an interobserver reproducibility study using the uk system , kocjan et al . reported that the  statistic was very poor ( 0.11 ) for the thy3a category and that for thy4 was 0.17 , in contrast to moderate to good agreement for thy1 ( 0.69 ) , thy2 ( 0.55 ) , thy3f ( 0.51 ) , and thy5 ( 0.61 ) .", "the observer variation of fn in our study occurred more often between lr and mr ( 9 cases , 45% ) , followed by between mr and hr ( 5 cases , 25% ) , but it is remarkable to note that none occurred between lr and hr . in other words , discordance is limited to between lr and mr or mr and hr , so we may conclude that the mr subcategory has an essential role in minimizing discordance between lr and hr .", "another choice of subclassification of follicular neoplasms would be two categories ( low cancer risk and high cancer risk )   instead of three categories ( lr , mr , and hr ) , and this modification ( two categories ) is also described as acceptable in the reporting system recommended by the japan thyroid association [ 7 , 8 ] .", "the second conclusion we may draw is that hr in the jta system is a powerful cytological subcategory to be used for the triage of patients for diagnostic surgery because the risk of malignancy of hr is high ( 6075% ) equivalent to suspicious for malignancy category in the bethesda system , with good concordance among the 4 reviewers (  = 0.7714 ) .", "abele and levine reported their rate of indeterminate category to be 5% of 51,000 adequate fnas and they suggested that the national rate of 15% was in large part due to overdiagnosis . this significant difference in ratio of indeterminate categories may be due to experience of thyroid cytology and not patients ' background .", "clary et al . commented in their interobserver variability study that some pathologists make greater use of indeterminate categories such as follicular lesion , favor nonneoplastic or follicular lesion , and favor neoplastic lesion , whereas others show more definitive categorization into benign and neoplastic groups .", "cibas et al . also stated in their report on interobserver variability that cytopathologists with experience of thyroid cytopathology are more likely to make a definitive interpretation ( i.e. , benign or malignant ) .", "this tendency was seen in our present study in which reviewers b and d ( whose indeterminate diagnosis rates are about 15% in their practice ) made mr diagnosis more often ( 70% and 65% , resp . ) and lr diagnosis less frequently ( 10% and 15% , resp . ) than those of reviewers a and c ( whose indeterminate diagnosis rates are about 5% in their practice ) .", "the difference in the prevalence of benign and fn / sfn may explain the different rates of lr ( low risk ) and mr ( moderate risk ) among reviews in our study , because incidence of one category may expand or contract depending on the rates of other categories .", "some fn / sfn lesions with benign pattern would be classified as benign by different authors and the incidence of fn / sfn of bethesda system in recent 7 series varied from 1.5 to 9.7% and that of benign category was between 54 and 77.4% summarized by ohori and schoedel .", "rapid development of molecular analyses on thyroid cytology may lead us possibly in the near future to more accurately identify patients who should be referred to surgery . until that time comes", ", thyroid fna cytology remains a main stay in the management of patients with thyroid nodules integrated with other clinical tests , such as ultrasound image diagnosis . as a conclusion", "the hr subcategory has a high predictive value of malignancy and good agreement among the 4 reviewers which is clinically helpful to triage patients for surgery .", "we believe that patients with cytological diagnosis of hr subcategory of fn in the jta system should be surgically treated especially if other risk factors coexisted . on the other hand , patients with lr or mr category", "therefore , surgical resection rate of indeterminate category is low in japan usually less than 50% , particularly in cases with fn ."], "abstract_text": ["<S> \n background . </S>", "<S> the japan thyroid association recently published guidelines for clinical practice for the management of thyroid nodules , which include a diagnostic system for reporting thyroid fine needle aspiration cytology . </S>", "<S> it is characterized by the subclassification of follicular neoplasms , which is different from other internationally accepted reporting systems . </S>", "<S> materials and methods . </S>", "<S> this study examined observer variability in the subclassification of follicular neoplasms among 4 reviewers using papanicolaou - stained smear samples from 20 surgically treated patients with indeterminate cytology . </S>", "<S> results . </S>", "<S> the favor malignant subcategory had high predictive value of malignancy ( risk of malignancy : 6075% ) and good agreement among the 4 reviewers (  = 0.7714 ) . </S>", "<S> conclusion . </S>", "<S> these results clearly confirmed that the risk stratification of follicular neoplasms , which was adapted from cytology practice of high - volume thyroid centers in japan , can provide clinically helpful information to estimate the risk of malignancy and to triage patients for surgery . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["the national cancer institute ( nci ) of the united states of america has proposed a reporting system for thyroid fine needle aspiration ( fna ) cytology , so - called bethesda system , which became an international standard of thyroid cytology [ 1 , 2 ] . following this recommendation , the uk royal college of pathologists ( the uk system ) [ 3 , 4 ] and italian societies of endocrinology and the italian society for anatomic pathology and cytology joint with the italian division of the international academy of pathology ( the italian system ) [ 5 , 6 ] updated their diagnostic schema comparable with the bethesda system . in 2013 ,", "the japan thyroid association ( jta ) published guidelines for clinical practice for the management of thyroid nodules , including its diagnostic system for reporting thyroid fna cytology as shown in table 1 [ 79 ] , using criteria similar to those used in the papanicolaou society recommendation , the bethesda system [ 1 , 2 ] , the uk system [ 3 , 4 ] , and the italian system [ 5 , 6 ] .", "the jta reporting system of thyroid cytology further recommends risk stratification of follicular neoplasms ( fn ) into favor benign ( low risk : lr ) , borderline ( moderate risk : mr ) , and favor malignant ( high risk : hr ) , which was adopted from the practice of high - volume thyroid centers in japan .", "this study examined observer variation in the subclassification of fn among 4 thyroid experts to validate the usefulness and limitations of this characteristic risk stratification of fn recommended in the jta guidelines .", "it is not our purpose to address in detail all the morphological issues of thyroid cytology ."], ["conventional smear samples of indeterminate diagnosis ( n = 389 ) were selected from files ( n = 3843 ) from the year 2005 at ito hospital , tokyo , japan , by one ( k. kameyama ) of the authors .", "inadequate samples or those with poor preparation and cases under clinical follow - up ( no final histologic diagnosis ) were excluded .", "there were 91 ( 23.4% ) surgically treated patients with thyroid nodule under indeterminate cytological diagnoses .", "these patients visited ito hospital in 2005 and underwent diagnostic surgery or curative surgery in the subsequent years between 2005 and 2008 .", "there were 48 cases of benign diagnoses , including 29 follicular adenomas ( fas ) , 19 adenomatous nodules ( an ) , and 43 malignant diagnoses , including 13 follicular carcinomas ( ftcs ) , 24 papillary carcinomas ( ptcs ) , 1 poorly differentiated carcinoma , 2 c - cell carcinomas , and 3 malignant lymphomas .", "twenty cases of cytological smear samples with follicular patterned lesions were randomly selected by one ( k. kameyama ) of the authors and they were further analyzed for subclassification of fn and diagnoses by 4 reviewers , as shown in table 2 .", "the present study is a reproducibility study undertaken by 4 reviewers ( kennichi kakudo , kaori kameyama , mitsuyoshi hirokawa , and ryohei katoh ) who have special interest in thyroid pathology , all of whom are members of the clinical guideline committee of the jta .", "they were requested to subclassify fn following the new jta reporting system of thyroid cytology as shown in table 1 .", "those 20 cases of smear samples ( one representative smear sample each stained by the papanicolaou method ) were circulated among the 4 reviewers without clinical information .", "the 4 reviewers examined cytological samples independently without exchanging opinions and were unaware of the original cytological diagnoses and final histologic diagnoses . because this study was conceived as an audit of cytology performance and the results are anonymized , institutional ethical committee permission was not required for its conduct .", "informed consent of all 4 reviewers was obtained for this study protocol . for statistical analysis of observer concordance ,  was first introduced as a measure of the level of agreement between pairs of raters and extended to multiple raters , known as composite  [ 11 , 12 ] .", "composite  statistical analysis was performed by using data analysis and statistical software , version 13 ( stata press , college station , texas , usa ) .", "values of  can be interpreted as follows : 0.000.20 , slight or very weak agreement ; 0.210.40 , weak to fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , substantial or good agreement ; and values over 0.81 , optimal or almost perfect agreement ."], ["there were 8 malignant cases ( 2 ftcs , widely invasive ; 4 ftcs , minimally invasive ; and 2 ptcs ) and 12 benign cases ( 1 an and 11 fas ) .", "all diagnoses made by the 4 reviewers on the 20 follicular pattern lesions are shown in table 2 .", "there were only 6 ( 30% ) cases with a consensus diagnosis among all 4 of the reviewers : 3 in fa and 3 in ftc .", "diagnoses of fn and hr were made in only 7 cases ( 35% ) , including 4 malignant and 3 benign cases .", "composite  statistical analysis clearly demonstrated good concordance for the hr diagnosis in malignant cases (  = 0.7714 ) ( table 3 ) , while this was not found for the other subcategories in both benign and malignant lesions (  < 0.4 ) .", "it is of note that there was no interobserver variation in ftcs , widely invasive ( n = 2 ) , and all 4 reviewers classified the 2 cases into the hr subcategory .", "it is clear that ftcs , widely invasive , have different degrees of cellular abnormality in our study .", "in contrast , there were significant disagreements among other types of malignancy and benign lesions , which clearly confirmed that the subclassification of fn is not a definite diagnosis but risk stratification useful for triage patients .", "concerning ftc , minimally invasive ( n = 4 ) , there was only one concordant case among the 4 reviewers and all reviewers made a mr diagnosis in 1 case .", "there were disagreements between mr and hr in 1 case and between lr and mr in 2 cases .", "as for ptc ( n = 2 ) , the 4 reviewers gave 2 diagnoses each , which included 3 lrs , 3 mrs , 1 hr , and 1 suspicious for ptc .", "however , it is of note that there was no single case in our 20 cases whose subclassification varied among all 3 subcategories . in benign final histologic diagnosis ( n = 12 ) , there were 9 split diagnoses , including 6 cases between lr and mr and 3 cases between mr and hr , but no case between lr and hr .", "the incidence of lr diagnoses by the 4 reviewers in the 12 benign lesions was 31.3% ( 15/48 ) and that of lr diagnoses in the 6 cases of ftc was 16.7% ( 4/24 ) ( p = 0.186 ) .", "the incidence of hr diagnosis in the 12 benign lesions was 10.4% ( 5/48 ) and that of hr in the 6 cases of ftc was 47.1% ( 10/24 ) ( p = 0.002 ) .", "the incidence of malignancy of the hr was high at 60% for reviewer a , 75% for b , 75% for c , and 75% for d ( 6075% ) .", "the incidence of malignancy in the mr was lower than that of hr and was 33.3% for reviewer a , 35.7% for reviewer b , 25% for c , and 30.8% for d ( 2535.7% ) .", "the incidence of malignancy in the lr was lower than that in the hr and was 33.3% for reviewer a , 0% for b , 37.5% for c , and 33.3% for d ( 037.5% ) .", "although the risks of malignancy in the subcategories of fn differed among the 4 reviewers , it is of note that hr cytological diagnosis was significantly correlated with malignant histological diagnosis , and the risk of malignancy ( 6075% ) was significantly higher than that in the other 2 subcategories ( 035.7% ) in our study ( p = 0.002 ) ."], ["although the standardization of terminology and diagnostic criteria is important for accurate communication among patients , clinical doctors , and cytopathologists [ 110 ] , there are still differences among reporting systems in thyroid cytology as to how to interpret cytological diagnoses and how to decide on the clinical management of patients . in japan , cytopathologists attempted to use an internationally accepted reporting system , but it had to be modified to fit our practice . in japan , all patients with indeterminate cytology undergo further diagnostic procedures , without immediate surgery , to search for higher risk patients who should undergo surgery [ 79 , 1315 ] .", "it is because the majority of thyroid carcinomas in indeterminate cytology are indolent and more conservative approaches , other than immediate diagnostic surgery , usually do not create any harm to the patient with malignancy [ 1620 ] and diagnostic surgery to all patients with indeterminate cytology results in risks of unnecessary surgery to the patients with benign nodules , more than 80% of the patients [ 79 , 1922 ] .", "the proportion of malignancy found at thyroidectomy from patients with indeterminate cytology in this clinical setting will increase in number , and the malignancy rate of patient with indeterminate cytology in our study was calculated as 47.3% as shown in materials and methods .", "takezawa et al . from japan retrospectively analyzed their 1606 cytological samples using bethesda system , although it was written in japanese with english abstract , and they identified 115 ( 7.9% ) cases of aus / flus ( atypia of undetermined significance / follicular lesions of undetermined significance ) and 61 ( 4.2% ) cases of fn / sfn ( follicular neoplasms / suspicious for follicular neoplasms ) .", "the resection rate of their aus / flus nodules was 30.4% ( 35/115 cases ) and its malignancy rate was 88.6% ( 31/35 cases ) , and the resection rate of fn / sfn nodules was 36.0% ( 22/61 cases ) and its malignancy rate was 72.4% ( 16/22 cases ) , which were very different from the ranges reported in most of the literatures from western countries using the bethesda system or the uk system [ 1 , 2 , 2023 ] . as it is clear , further triage of patients with indeterminate nodules reduces resection rates and increases malignancy rates in any diagnostic systems including the bethesda system .", "this clinical management was also true in some other countries [ 2427 ] , as crippa and dina commented in a letter to an editor that thyroid cytology is the most important but not the only important deciding factor and therefore it must be integrated with other diagnostic procedures .", "we propose that future thyroid cytology classification schemes should reconsider clinical managements of indeterminate categories and how to reduce unnecessary surgeries for patients with benign thyroid nodules [ 79 ] .", "the risk stratification of follicular pattern lesions into 3 subcategories ( cellular follicular lesion , fn favor benign , and fn favor malignant ) was suggested by the papanicolaou society in 1996 , but it did not become popular in thyroid cytology worldwide , apart from japan [ 8 , 9 ] . there have been some reports in the literature on risk stratification of fn [ 10 , 2834 ] .", "kelman et al . reported that 31/52 ( 60% ) nodules with nuclear atypia consistent with fn were malignant   and it was 4/9 ( 44.4% ) in fn with atypia by goldstein et al . .", "reported that atypical proliferation was more often malignant than follicular group ( 53% versus 19% ) in their indeterminate category ( tir3/thy3 ) .", "some researchers pointed out that the malignancy rate of fn without atypia is low and assessment later on could be an alternative approach [ 31 , 32 ] and patients with high - risk cytological features such as nuclear overlapping ( crowding ) should be advised to have a surgical intervention [ 3133 ] .", "gerhard and da cunha santos using the papanicolaou society guidelines studied reproducibility between 2 observers in 97 diagnoses .", "they reported a substantial level of diagnostic interobserver (  = 0.71 ) and intraobserver (  = 0.66 ) reproducibility , although interobserver disagreement in the cytological diagnosis occurred in 23 cases ( 24.7% ) and 18 ( 41.7% ) of them were for fn .", "in an interobserver reproducibility study using the uk system , kocjan et al . reported that the  statistic was very poor ( 0.11 ) for the thy3a category and that for thy4 was 0.17 , in contrast to moderate to good agreement for thy1 ( 0.69 ) , thy2 ( 0.55 ) , thy3f ( 0.51 ) , and thy5 ( 0.61 ) .", "the observer variation of fn in our study occurred more often between lr and mr ( 9 cases , 45% ) , followed by between mr and hr ( 5 cases , 25% ) , but it is remarkable to note that none occurred between lr and hr . in other words , discordance is limited to between lr and mr or mr and hr , so we may conclude that the mr subcategory has an essential role in minimizing discordance between lr and hr .", "another choice of subclassification of follicular neoplasms would be two categories ( low cancer risk and high cancer risk )   instead of three categories ( lr , mr , and hr ) , and this modification ( two categories ) is also described as acceptable in the reporting system recommended by the japan thyroid association [ 7 , 8 ] .", "the second conclusion we may draw is that hr in the jta system is a powerful cytological subcategory to be used for the triage of patients for diagnostic surgery because the risk of malignancy of hr is high ( 6075% ) equivalent to suspicious for malignancy category in the bethesda system , with good concordance among the 4 reviewers (  = 0.7714 ) .", "abele and levine reported their rate of indeterminate category to be 5% of 51,000 adequate fnas and they suggested that the national rate of 15% was in large part due to overdiagnosis . this significant difference in ratio of indeterminate categories may be due to experience of thyroid cytology and not patients ' background .", "clary et al . commented in their interobserver variability study that some pathologists make greater use of indeterminate categories such as follicular lesion , favor nonneoplastic or follicular lesion , and favor neoplastic lesion , whereas others show more definitive categorization into benign and neoplastic groups .", "cibas et al . also stated in their report on interobserver variability that cytopathologists with experience of thyroid cytopathology are more likely to make a definitive interpretation ( i.e. , benign or malignant ) .", "this tendency was seen in our present study in which reviewers b and d ( whose indeterminate diagnosis rates are about 15% in their practice ) made mr diagnosis more often ( 70% and 65% , resp . ) and lr diagnosis less frequently ( 10% and 15% , resp . ) than those of reviewers a and c ( whose indeterminate diagnosis rates are about 5% in their practice ) .", "the difference in the prevalence of benign and fn / sfn may explain the different rates of lr ( low risk ) and mr ( moderate risk ) among reviews in our study , because incidence of one category may expand or contract depending on the rates of other categories .", "some fn / sfn lesions with benign pattern would be classified as benign by different authors and the incidence of fn / sfn of bethesda system in recent 7 series varied from 1.5 to 9.7% and that of benign category was between 54 and 77.4% summarized by ohori and schoedel .", "rapid development of molecular analyses on thyroid cytology may lead us possibly in the near future to more accurately identify patients who should be referred to surgery . until that time comes", ", thyroid fna cytology remains a main stay in the management of patients with thyroid nodules integrated with other clinical tests , such as ultrasound image diagnosis . as a conclusion", "the hr subcategory has a high predictive value of malignancy and good agreement among the 4 reviewers which is clinically helpful to triage patients for surgery .", "we believe that patients with cytological diagnosis of hr subcategory of fn in the jta system should be surgically treated especially if other risk factors coexisted . on the other hand , patients with lr or mr category", "therefore , surgical resection rate of indeterminate category is low in japan usually less than 50% , particularly in cases with fn ."]]}
{"article_id": "PMC3496520", "article_text": ["it is not uncommon to see bleeding symptoms in patients in outpatient or hospital - based practice .", "postpartum hemorrhage complicates pregnancy and accounts for significant morbidity and mortality , particularly in underdeveloped countries.1,2 menorrhagia is a common clinical challenge and is often associated with secondary anemia , excessive fatigue , and a negative effect on health - related quality of life.3 postoperative bleeding is one of the more common complications of surgery .", "trauma is a leading cause of morbidity and mortality in the younger population.4 while bleeding symptoms may be commonly seen by physicians of multiple specialties , it is unclear how frequently these symptoms belie an underlying undiagnosed congenital or acquired bleeding disorder . in the us ,", "the most common congenital bleeding disorders include von willebrand disease , which affects approximately 1% of the population ( males and females equally),5 and hemophilia a and b combined , which affect approximately 20,000 persons ( essentially all males , with rare exception).6 medications can also affect coagulation or platelet function,7 as can certain herbal supplements.8 trauma and surgery can lead to blood loss , and critical reduction in coagulation factors can lead to additional non - surgical bleeding complications ( coagulopathic bleeding).9 the most ubiquitous method for evaluating coagulation is prothrombin time ( pt)/international normalized ratio ( inr ) and activated partial thromboplastin time ( aptt ) .", "typically , they are ordered to monitor anticoagulant therapy ( pt / inr for warfarin , aptt for heparin ) , to evaluate coagulation preoperatively , or in response to hemorrhagic symptoms .", "the pt / inr provides an assessment of the extrinsic ( tissue factor - dependent ) and final common pathways of the coagulation cascade , while the aptt provides an assessment of the intrinsic ( tissue factor - independent ) and final common pathways.10 an example of a potentially life - threatening cause of unexplained recent onset or acute bleeding associated with a prolonged aptt is acquired hemophilia , with an incidence of approximately 1 to 4 per million / year.11 acquired hemophilia primarily affects older adults,12 an ever - growing segment of the population that presents for medical evaluation and care , but may also occur during pregnancy as postpartum hemorrhage or in association with other underlying diseases , including cancer and autoimmune disorders.13 prompt diagnosis is a primary determinant of prognosis in acquired hemophilia14 because initiation of definitive therapy ( ie , hemostatic and immunosuppressive ) is delayed until the diagnosis is made .", "acquired hemophilia - related bleeding does not respond to the typical management algorithms used for hemorrhaging in a patient and , therefore , is associated with high morbidity and mortality .", "severe bleeds occur in up to 90% of patients with acquired hemophilia,11 and the reported overall mortality rate in these patients ranges from 8% to 22%.1517 given the rarity of acquired hemophilia , combined with the general lack of familiarity of nonhematologists with this condition , the diagnosis of acquired hemophilia poses a clinical challenge , even in patients presenting with straightforward bleeding and an isolated , prolonged aptt .", "a survey was conducted of physicians across a number of specialties to identify potential barriers to the effective recognition and management of this rare but important cause of serious bleeding .", "the survey , based on an actual case found to be the result of acquired hemophilia , focused on participants stepwise evaluation and management of a case patient who presented to the hospital with recent - onset bleeding .", "the survey also assessed participants history with regard to consulting hematologists , discovering or diagnosing underlying bleeding disorders , and encountering acquired hemophilia in clinical practice .", "findings pertaining to the interpretation and follow - up of abnormal coagulation studies , enlistment of hematology consultation , and the diagnostic decision process in an actively bleeding case patient were the primary areas assessed in this analysis .", "results were evaluated across specialties to determine any specialty - specific practice trends that might hinder effective recognition and management of an actively bleeding patient with coagulopathy , including acquired hemophilia .", "physicians within the specialties of hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery were randomly sampled from the american medical association physician masterfile .", "physicians who were part of the harris interactive online physician panel were invited via email to participate in the survey , while physicians who were not part of the panel were invited via first class mail .", "the invitation provided a uniform resource locator address and password for one - time use to log on to the survey site , where invitees first encountered questions to determine eligibility to participate in the survey .", "eligible physicians were required to be actively practicing , with additional specialty - specific requirements .", "in particular , hematologists , hematologist / oncologists , general surgeons , and emergency medicine and critical care physicians were required to be practicing at an acute care hospital . for internists and geriatricians ,", "at least 60% of their practice had to consist of patients older than 60 years of age .", "obstetricians / gynecologists were required to have an active obstetrics practice . upon confirmation of eligibility , participants completed the online survey , the average duration of which was approximately 10 minutes .", "each specialty was recruited to a final sample of 50 complete responses to ensure a sufficient number of responses to generate reasonable hypotheses about specialists behavior to test in face - to - face interviews .", "findings from the surveys completed by the physicians specializing in hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , and critical care medicine are presented herein .", "general surgeons and obstetrics and gynecology physicians each completed different surveys from the aforementioned specialties ; the findings of those surveys are not included here .", "survey questions focused on the diagnostic and general therapeutic approach to the management of a case patient ( figure 1 ) whose presentation and clinical course were based on those of a real patient who experienced significant delays in diagnosis despite the involvement of multiple specialists . at each juncture in the case , participants were given lists of actions that included diagnostic tests ( laboratory , radiology , other ) , consultations , and potential treatments addressing bleeding or anemia ( local hemostasis , transfusion ) . since familiarity with the upper limits of normal of the pt / aptt tests was being tested in this study , upper limits of normal were not specified , and aptts were 25% to 30% above the upper limits of normal and set at a value that respective specialties would recognize as abnormal based on their experience with ordering and interpreting this laboratory test .", "the survey did not specify whether laboratory testing , imaging studies , or consultations in this hypothetical case had to be available immediately or locally , and the assumption was that respondents would answer based on their best medical judgment in an ideal practice situation .", "rheumatologists were only asked about an initial emergency department / urgent care presentation because it was expected that if a bleeding disorder developed in one of their patients it would present in an outpatient or emergency department / urgent care setting .", "conversely , critical care specialists started at presentation 2 , since their involvement in a recurrent epistaxis workup would be unlikely .", "vital signs were adjusted to make it more likely that a potential intensive care unit admission would require their evaluation . since emergency medicine physicians would most likely have ordered an imaging study initially to define the patient s retroperitoneal hematoma , we allowed for the option of a general surgery consultation even though the computed tomography findings were never presented .", "hematology / oncology respondents were given the option of ordering additional specialty coagulation laboratory tests ( eg , mixing studies ) . participants were additionally questioned about their experiences diagnosing underlying bleeding disorders and , in the case of nonhematologist specialists , consulting hematologist colleagues .", "conversely , hematology and hematology / oncology specialists were asked about being on the receiving end of consultations , specifically from emergency medicine personnel , and the reasons for those consultations .", "a total of 302 physicians ( 5051 per specialty ) participated in the case - based survey reported herein .", "demographics of the specialty groups were consistent : mean age ranged from 45.6 to 50.6 years , and mean years of practice experience ranged from 14.5 to 19.3 .", "faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies . less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) .", "rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists .", "after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) .", "when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) .", "approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) .", "sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point .", "in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 .", "participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) .", "emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test .", "requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period ,", "the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased .", "internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) .", "in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist .", "the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt .", "emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results . the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a .", "across the majority of specialties , less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home .", "in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility .", "the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital .", "the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation ,", "nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation .", "the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service .", "more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) .", "the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered  acquired hemophilia  ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder .", "hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage .", "table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation .", "the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) ,", "the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt .", "in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason . the highest percentage ( 39% ) of critical care physicians", "the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) .", "the most common response among critical care specialists to the open - ended question ,  what are the circumstances around which you would call for a hematology consult ?  was  an unexplained abnormal lab or bleeding  ( 33 like or similar mentions ) , followed by  any unexplained bleeding disorder ", "conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 .", "the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine .", "the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) .", "faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies .", "less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) .", "rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists .", "after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) .", "when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) .", "approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) .", "sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point .", "in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 .", "participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) .", "emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test .", "requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period ,", "the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased .", "internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) .", "in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist .", "the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt .", "emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results .", "the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . across the majority of specialties ,", "less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home .", "in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital .", "the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation ,", "nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation .", "the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service .", "more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) .", "the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered  acquired hemophilia  ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder .", "hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage .", "table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation .", "the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) , the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt .", "in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason .", "the highest percentage ( 39% ) of critical care physicians had consulted a hematologist at least 5 times for such a patient .", "the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) .", "the most common response among critical care specialists to the open - ended question ,  what are the circumstances around which you would call for a hematology consult ?  was  an unexplained abnormal lab or bleeding  ( 33 like or similar mentions ) , followed by  any unexplained bleeding disorder ", "conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 .", "the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine .", "the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) .", "bleeding is commonly encountered in outpatient and hospital - based medical practice and can have a wide variety of underlying causes .", "while bleeding may be relatively common , most acquired and congenital bleeding disorders are uncommon , and some , such as acquired hemophilia , are rare . nevertheless , the consequences of failure to recognize promptly and treat properly a bleeding disorder may be significant.18 in the case of acquired hemophilia , morbidity and mortality rates are particularly high : severe bleeding is experienced by up to 90% of affected patients , and mortality rates", "are as high as 22%.11 this survey provided a step - wise methodology to tease out specialty - specific patterns of interpretation of clinical data to identify barriers to the diagnosis and treatment of underlying bleeding disorders .", "the sample size obtained across specialties was sufficient to generalize these findings , at least to the point of identifying specific issues for education and development of clinical decision - making pathways .", "when presented with a clinical picture that includes a recent history or symptoms of active bleeding , clinicians typically obtain coagulation times , such as the pt / inr and aptt , as part of the initial diagnostic evaluation .", "proper interpretation of laboratory test results includes recognition of abnormal values and , more important , the potential clinical significance of such results . a common pitfall in the interpretation of coagulation times is failure to appreciate that even mildly abnormal values may represent a serious underlying coagulation deficit .", "another important observation is to identify how an abnormal laboratory value may have changed over time , which can be facilitated by the ability of electronic medical record systems to display data trends . in the absence of iatrogenic causes , even a mildly elevated pt / inr or aptt may be indicative of true coagulopathy and should not be ignored or dismissed , particularly when there is evidence of bleeding , as was the case with this patient , even at initial presentation . after excluding laboratory error ,", "the differential diagnosis of an isolated prolongation of aptt includes heparin effect , lupus anticoagulant , and deficiency of or antibody against an intrinsic pathway factor ( viii , ix , xi , or xii).19,20 a detailed history , focusing on factors such as heparin exposure,19,20 history of thromboembolism ( lupus anticoagulant),21 and prior personal or family history of bleeding,22 may provide diagnostic clues .", "laboratory testing should include a 1:1 mixing test of patient plasma with control plasma to determine whether a prolonged aptt is the result of an intrinsic pathway factor deficiency or an inhibitor that continues to block the activity of the intrinsic system even in the presence of control plasma .", "the majority of inhibitor antibodies identified in this manner will turn out to be lupus anticoagulants .", "although far less common , this is the same diagnostic pathway that leads to the identification of the antifactor viii antibodies associated with acquired hemophilia.23 unlike acquired hemophilia , lupus anticoagulants typically do not present with bleeding , and the abnormal aptt is due to interference with phospholipid - dependent coagulation reactions .", "once an acquired antifactor viii antibody is suspected , confirmatory testing includes measuring factor viii activity , which should be significantly reduced , and the bethesda assay,24 which is used to quantitate antifactor viii antibodies inhibitor activity .", "we found a general lack of appropriate consideration and response to the presenting symptom of bleeding and the prolonged aptt throughout this case study .", "this is consistent with data from the european acquired haemophilia registry ( each-2 ) , which reported a median delay of 3 days between onset of bleeding symptoms and the diagnosis of acquired hemophilia and a median delay of one day between the first abnormal aptt test in those same patients and the established diagnosis.23 in addition , we found that emergency medicine and critical care physicians were reluctant to consider a bleeding disorder as the primary explanation for this patient s clinical presentation .", "the disposition of a patient with active hemorrhage and evidence of coagulopathy should be based on several factors , including the patient s current condition and anticipated clinical course , taking into account the presenting vital signs and evolving laboratory findings . at the time of the patient", "s second presentation , vital signs were notable for mild tachycardia and a pulse pressure at the upper limit of normal , and subsequent laboratory findings indicated a decreasing hemoglobin level and an increasing aptt .", "these findings alone prompted hospital admission , although the exact location ( general floor versus intensive care unit ) of admission may vary , based on the level of monitoring and nurse - to - patient ratios in a particular hospital .", "another important variable is the anticipated potential for clinical deterioration , which is based in large part on clinician appreciation of the seriousness of the diagnosis .", "we found a consistent tendency to consider admission to a general floor bed with the second presentation , even though this was ultimately an unstable , critically ill patient with an undiagnosed bleeding disorder .", "we also found that the physicians who participated in this survey were reluctant to consult a hematologist as they worked through this case scenario , particularly given that options for additional testing ( liver function , disseminated intravascular coagulation ) were not available to evaluate for common causes of coagulopathy .", "relative to their reported historical experience with hematology consultations during an average practice experience of approximately 20 years , this survey finding was somewhat surprising .", "rheumatologists and critical care specialists reported a greater frequency of hematology consultation relative to the other specialties ( table 2 ) .", "we would expect the emergency medicine physicians to be most likely to consult a hematologist , yet 16% of them reported never having consulted a hematologist .", "the highest percentage ( 46% ) had only consulted a hematologist 1 or 2 times , and almost one quarter of geriatricians and internists had never consulted a hematologist , even though these specialists would be expected to first encounter patients with undiagnosed bleeding disorders , including acquired hemophilia .", "one potential reason for not seeking hematology consultation might be the lack of availability of hematology / oncology specialists with expertise in coagulation disorders , including in rural and community hospitals .", "a limitation of this survey was that one can not interpret the thinking behind the responses of the individual participants .", "therefore , 31 qualitative 45-minute interviews were conducted subsequent to the quantitative study and focused particularly on critical care ( n = 7 ) , emergency medicine ( n = 6 ) , hematology / oncology ( n = 4 ) , or hematology ( n = 2 ) physicians to understand the reasoning behind their decisions and depth of knowledge ( unpublished data ) . we found that the physicians focus was generally on finding the source ( location ) of bleeding and not on finding the underlying reason for bleeding .", "this could potentially lead to surgical intervention in the face of an underlying bleeding disorder , with subsequent adverse outcomes . in a series of 67 patients with acquired hemophilia at a single center in bonn , germany ,", "4 of 5 deaths were the result of surgical intervention for bleeding at outside hospitals in the setting of a delayed diagnosis of acquired hemophilia.13 when queried about their experience encountering and/or diagnosing underlying bleeding disorders , particularly acquired hemophilia , more than 85% of physicians in hematology , hematology / oncology , emergency medicine , and critical care medicine reported having ever discovered or diagnosed an underlying bleeding disorder , compared with 65% , 54% , and 47% of rheumatologists , internists , and geriatricians , respectively . while reports of ever specifically having encountered acquired hemophilia were high , it is unclear from this study whether the participants truly understood the diagnosis .", "this seems unlikely , given the rarity of acquired hemophilia , relative to the reported frequency of having encountered it .", "except for hematology and/or oncology ( 77% ) and critical care ( 36% ) specialists , approximately one quarter of surveyed physicians had ever encountered acquired hemophilia .", "although they accounted for the highest percentage of physicians who had ever encountered this condition , nearly one quarter of hematologists had never encountered acquired hemophilia .", "subsequent unpublished data from the aforementioned qualitative research further suggest that , compared with hematology practitioners , specialists in hematology / oncology , who likely practice mostly oncology , might be able to identify  mixing studies  and  inhibitors  but might not fully understand the underlying pathophysiology that constitutes acquired hemophilia , making it hard for them to recognize the condition .", "given the survey findings reflecting the infrequency with which most physicians have encountered these conditions , consultation with a hematologist may facilitate the diagnostic evaluation and proper management of a hemorrhaging patient suspected of having an underlying bleeding diathesis , particularly acquired hemophilia .", "the consulting hematologist can provide specific guidance , leading to the prompt diagnosis and optimal management of an actively bleeding patient with acquired hemophilia , including initiation of immunosuppression , which is usually necessary to eradicate the inhibitor and to prevent additional bleeding episodes .", "however , this requires a level of familiarity and expertise in treating acquired hemophilia and other rare bleeding disorders that is not often seen outside of an academic hematology practice .", "this represents yet another barrier to the effective diagnosis and management of this rare yet serious bleeding diathesis .", "for the hospitalist or intensivist charged with the care of a bleeding patient , immediate stabilization is the initial priority and should take precedence over determination of a specific etiology of bleeding .", "however , in the presence of underlying coagulopathy , particularly acquired hemophilia or other rare disorders , traditional measures of stabilization may not be as effective as expected .", "a prolonged pt / inr or aptt in the absence of iatrogenic causes should never be ignored , even with only minimally prolonged values , and determination of the cause of an abnormal coagulation study should carry at least equal weight to looking for the anatomic site of bleeding . any delay in establishing the diagnosis of a bleeding diathesis such as acquired hemophilia can result in significant morbidity or even death .", "while hospitalists and intensivists should be able to conduct a thorough differential of bleeding and eliminate most common etiologies , consultation with a hematologist ( particularly one with specific expertise in coagulation disorders ) may facilitate the evaluation of coagulopathic patients and subsequent interpretation of diagnostic findings , as well as initiation of appropriate treatment . given the rarity of acquired hemophilia , as exemplified by the findings of this survey", ", physicians must harbor a high index of suspicion to diagnose this condition promptly in patients who present with recent - onset or acute bleeding . given the high morbidity and mortality", "the insights from this survey highlights knowledge and practice gaps that could be the focus of targeted educational initiatives , including diagnostic algorithms , to ensure proper and efficient workup of the abnormal laboratory studies that characterize acquired hemophilia ."], "abstract_text": ["<S> background : bleeding symptoms commonly seen by multiple physician specialties may belie undiagnosed congenital or acquired bleeding disorders . acquired hemophilia is a potentially life - threatening cause of unexplained acute bleeding manifested by an abnormal activated partial thromboplastin time ( aptt ) that does not correct with 1:1 mixing with normal plasma.methods:practicing physicians ( hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery ) completed an online survey based on a hypothetical case scenario.results:excluding surgeons and obstetrician / gynecologist respondents , 302 physicians ( about 50 per specialty ) were presented with an older adult woman complaining of recurrent epistaxis . </S>", "<S> nearly 90% ordered a complete blood count and coagulation studies ( aptt , prothrombin time [ pt]/international normalized ratio [ inr ] ) . despite a prolonged aptt of 42 seconds , </S>", "<S> < 50% of nonhematologists would repeat the aptt , and < 45% would consult a hematologist ; emergency medicine physicians were least likely ( 10% ) and rheumatologists were most likely ( 43% ) to consult . </S>", "<S> after presentation weeks later with bruising and abdominal / back pain , 90% of physicians within each specialty ordered a complete blood count or pt / inr / aptt . despite an aptt of 63 seconds </S>", "<S> , the majority did not repeat the aptt . at this point , </S>", "<S> approximately 75% of internal medicine and geriatric physicians indicated they would consult a hematologist , versus 47% in emergency medicine and 50% in critical care . </S>", "<S> all participants preferred abdominal computed tomography ( 80%84% ) . </S>", "<S> after 12 hours of additional observation , 73% to 94% of respondents consulted a hematologist . </S>", "<S> complete blood count revealed anemia and an aptt twice the upper limit of normal ; emergency medicine physicians remained least likely to request a consult.conclusion:determining the cause of an abnormal coagulation study result should carry equal weight as looking for the site of bleeding and could be facilitated by consultation with a hematologist . </S>", "<S> insight from this survey highlights knowledge and practice gaps that could be the target of focused educational initiatives . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Results", "Initial presentation: recommended diagnostic evaluations and hematology consultation", "Second presentation: recommended diagnostic evaluations and hematology consultation", "Recommendations regarding case patient disposition", "Familiarity with bleeding disorders", "History of hematology consultations", "Discussion", "Conclusion"], "sections": [["it is not uncommon to see bleeding symptoms in patients in outpatient or hospital - based practice .", "postpartum hemorrhage complicates pregnancy and accounts for significant morbidity and mortality , particularly in underdeveloped countries.1,2 menorrhagia is a common clinical challenge and is often associated with secondary anemia , excessive fatigue , and a negative effect on health - related quality of life.3 postoperative bleeding is one of the more common complications of surgery .", "trauma is a leading cause of morbidity and mortality in the younger population.4 while bleeding symptoms may be commonly seen by physicians of multiple specialties , it is unclear how frequently these symptoms belie an underlying undiagnosed congenital or acquired bleeding disorder . in the us ,", "the most common congenital bleeding disorders include von willebrand disease , which affects approximately 1% of the population ( males and females equally),5 and hemophilia a and b combined , which affect approximately 20,000 persons ( essentially all males , with rare exception).6 medications can also affect coagulation or platelet function,7 as can certain herbal supplements.8 trauma and surgery can lead to blood loss , and critical reduction in coagulation factors can lead to additional non - surgical bleeding complications ( coagulopathic bleeding).9 the most ubiquitous method for evaluating coagulation is prothrombin time ( pt)/international normalized ratio ( inr ) and activated partial thromboplastin time ( aptt ) .", "typically , they are ordered to monitor anticoagulant therapy ( pt / inr for warfarin , aptt for heparin ) , to evaluate coagulation preoperatively , or in response to hemorrhagic symptoms .", "the pt / inr provides an assessment of the extrinsic ( tissue factor - dependent ) and final common pathways of the coagulation cascade , while the aptt provides an assessment of the intrinsic ( tissue factor - independent ) and final common pathways.10 an example of a potentially life - threatening cause of unexplained recent onset or acute bleeding associated with a prolonged aptt is acquired hemophilia , with an incidence of approximately 1 to 4 per million / year.11 acquired hemophilia primarily affects older adults,12 an ever - growing segment of the population that presents for medical evaluation and care , but may also occur during pregnancy as postpartum hemorrhage or in association with other underlying diseases , including cancer and autoimmune disorders.13 prompt diagnosis is a primary determinant of prognosis in acquired hemophilia14 because initiation of definitive therapy ( ie , hemostatic and immunosuppressive ) is delayed until the diagnosis is made .", "acquired hemophilia - related bleeding does not respond to the typical management algorithms used for hemorrhaging in a patient and , therefore , is associated with high morbidity and mortality .", "severe bleeds occur in up to 90% of patients with acquired hemophilia,11 and the reported overall mortality rate in these patients ranges from 8% to 22%.1517 given the rarity of acquired hemophilia , combined with the general lack of familiarity of nonhematologists with this condition , the diagnosis of acquired hemophilia poses a clinical challenge , even in patients presenting with straightforward bleeding and an isolated , prolonged aptt .", "a survey was conducted of physicians across a number of specialties to identify potential barriers to the effective recognition and management of this rare but important cause of serious bleeding .", "the survey , based on an actual case found to be the result of acquired hemophilia , focused on participants stepwise evaluation and management of a case patient who presented to the hospital with recent - onset bleeding .", "the survey also assessed participants history with regard to consulting hematologists , discovering or diagnosing underlying bleeding disorders , and encountering acquired hemophilia in clinical practice .", "findings pertaining to the interpretation and follow - up of abnormal coagulation studies , enlistment of hematology consultation , and the diagnostic decision process in an actively bleeding case patient were the primary areas assessed in this analysis .", "results were evaluated across specialties to determine any specialty - specific practice trends that might hinder effective recognition and management of an actively bleeding patient with coagulopathy , including acquired hemophilia ."], ["physicians within the specialties of hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery were randomly sampled from the american medical association physician masterfile .", "physicians who were part of the harris interactive online physician panel were invited via email to participate in the survey , while physicians who were not part of the panel were invited via first class mail .", "the invitation provided a uniform resource locator address and password for one - time use to log on to the survey site , where invitees first encountered questions to determine eligibility to participate in the survey .", "eligible physicians were required to be actively practicing , with additional specialty - specific requirements .", "in particular , hematologists , hematologist / oncologists , general surgeons , and emergency medicine and critical care physicians were required to be practicing at an acute care hospital . for internists and geriatricians ,", "at least 60% of their practice had to consist of patients older than 60 years of age .", "obstetricians / gynecologists were required to have an active obstetrics practice . upon confirmation of eligibility , participants completed the online survey , the average duration of which was approximately 10 minutes .", "each specialty was recruited to a final sample of 50 complete responses to ensure a sufficient number of responses to generate reasonable hypotheses about specialists behavior to test in face - to - face interviews .", "findings from the surveys completed by the physicians specializing in hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , and critical care medicine are presented herein .", "general surgeons and obstetrics and gynecology physicians each completed different surveys from the aforementioned specialties ; the findings of those surveys are not included here .", "survey questions focused on the diagnostic and general therapeutic approach to the management of a case patient ( figure 1 ) whose presentation and clinical course were based on those of a real patient who experienced significant delays in diagnosis despite the involvement of multiple specialists . at each juncture in the case , participants were given lists of actions that included diagnostic tests ( laboratory , radiology , other ) , consultations , and potential treatments addressing bleeding or anemia ( local hemostasis , transfusion ) . since familiarity with the upper limits of normal of the pt / aptt tests was being tested in this study , upper limits of normal were not specified , and aptts were 25% to 30% above the upper limits of normal and set at a value that respective specialties would recognize as abnormal based on their experience with ordering and interpreting this laboratory test .", "the survey did not specify whether laboratory testing , imaging studies , or consultations in this hypothetical case had to be available immediately or locally , and the assumption was that respondents would answer based on their best medical judgment in an ideal practice situation .", "rheumatologists were only asked about an initial emergency department / urgent care presentation because it was expected that if a bleeding disorder developed in one of their patients it would present in an outpatient or emergency department / urgent care setting .", "conversely , critical care specialists started at presentation 2 , since their involvement in a recurrent epistaxis workup would be unlikely .", "vital signs were adjusted to make it more likely that a potential intensive care unit admission would require their evaluation . since emergency medicine physicians would most likely have ordered an imaging study initially to define the patient s retroperitoneal hematoma , we allowed for the option of a general surgery consultation even though the computed tomography findings were never presented .", "hematology / oncology respondents were given the option of ordering additional specialty coagulation laboratory tests ( eg , mixing studies ) . participants were additionally questioned about their experiences diagnosing underlying bleeding disorders and , in the case of nonhematologist specialists , consulting hematologist colleagues .", "conversely , hematology and hematology / oncology specialists were asked about being on the receiving end of consultations , specifically from emergency medicine personnel , and the reasons for those consultations ."], ["a total of 302 physicians ( 5051 per specialty ) participated in the case - based survey reported herein .", "demographics of the specialty groups were consistent : mean age ranged from 45.6 to 50.6 years , and mean years of practice experience ranged from 14.5 to 19.3 .", "faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies . less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) .", "rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists .", "after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) .", "when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) .", "approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) .", "sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point .", "in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 .", "participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) .", "emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test .", "requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period ,", "the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased .", "internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) .", "in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist .", "the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt .", "emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results . the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a .", "across the majority of specialties , less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home .", "in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility .", "the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital .", "the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation ,", "nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation .", "the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service .", "more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) .", "the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered  acquired hemophilia  ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder .", "hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage .", "table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation .", "the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) ,", "the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt .", "in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason . the highest percentage ( 39% ) of critical care physicians", "the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) .", "the most common response among critical care specialists to the open - ended question ,  what are the circumstances around which you would call for a hematology consult ?  was  an unexplained abnormal lab or bleeding  ( 33 like or similar mentions ) , followed by  any unexplained bleeding disorder ", "conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 .", "the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine .", "the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) ."], ["faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies .", "less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) .", "rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists ."], ["after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) .", "when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) .", "approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) .", "sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point .", "in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 .", "participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) .", "emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test .", "requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period ,", "the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased .", "internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) .", "in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist .", "the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt .", "emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results ."], ["the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . across the majority of specialties ,", "less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home .", "in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital .", "the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation ,", "nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation .", "the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service ."], ["more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) .", "the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered  acquired hemophilia  ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder .", "hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage ."], ["table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation .", "the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) , the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt .", "in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason .", "the highest percentage ( 39% ) of critical care physicians had consulted a hematologist at least 5 times for such a patient .", "the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) .", "the most common response among critical care specialists to the open - ended question ,  what are the circumstances around which you would call for a hematology consult ?  was  an unexplained abnormal lab or bleeding  ( 33 like or similar mentions ) , followed by  any unexplained bleeding disorder ", "conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 .", "the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine .", "the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) ."], ["bleeding is commonly encountered in outpatient and hospital - based medical practice and can have a wide variety of underlying causes .", "while bleeding may be relatively common , most acquired and congenital bleeding disorders are uncommon , and some , such as acquired hemophilia , are rare . nevertheless , the consequences of failure to recognize promptly and treat properly a bleeding disorder may be significant.18 in the case of acquired hemophilia , morbidity and mortality rates are particularly high : severe bleeding is experienced by up to 90% of affected patients , and mortality rates", "are as high as 22%.11 this survey provided a step - wise methodology to tease out specialty - specific patterns of interpretation of clinical data to identify barriers to the diagnosis and treatment of underlying bleeding disorders .", "the sample size obtained across specialties was sufficient to generalize these findings , at least to the point of identifying specific issues for education and development of clinical decision - making pathways .", "when presented with a clinical picture that includes a recent history or symptoms of active bleeding , clinicians typically obtain coagulation times , such as the pt / inr and aptt , as part of the initial diagnostic evaluation .", "proper interpretation of laboratory test results includes recognition of abnormal values and , more important , the potential clinical significance of such results . a common pitfall in the interpretation of coagulation times is failure to appreciate that even mildly abnormal values may represent a serious underlying coagulation deficit .", "another important observation is to identify how an abnormal laboratory value may have changed over time , which can be facilitated by the ability of electronic medical record systems to display data trends . in the absence of iatrogenic causes , even a mildly elevated pt / inr or aptt may be indicative of true coagulopathy and should not be ignored or dismissed , particularly when there is evidence of bleeding , as was the case with this patient , even at initial presentation . after excluding laboratory error ,", "the differential diagnosis of an isolated prolongation of aptt includes heparin effect , lupus anticoagulant , and deficiency of or antibody against an intrinsic pathway factor ( viii , ix , xi , or xii).19,20 a detailed history , focusing on factors such as heparin exposure,19,20 history of thromboembolism ( lupus anticoagulant),21 and prior personal or family history of bleeding,22 may provide diagnostic clues .", "laboratory testing should include a 1:1 mixing test of patient plasma with control plasma to determine whether a prolonged aptt is the result of an intrinsic pathway factor deficiency or an inhibitor that continues to block the activity of the intrinsic system even in the presence of control plasma .", "the majority of inhibitor antibodies identified in this manner will turn out to be lupus anticoagulants .", "although far less common , this is the same diagnostic pathway that leads to the identification of the antifactor viii antibodies associated with acquired hemophilia.23 unlike acquired hemophilia , lupus anticoagulants typically do not present with bleeding , and the abnormal aptt is due to interference with phospholipid - dependent coagulation reactions .", "once an acquired antifactor viii antibody is suspected , confirmatory testing includes measuring factor viii activity , which should be significantly reduced , and the bethesda assay,24 which is used to quantitate antifactor viii antibodies inhibitor activity .", "we found a general lack of appropriate consideration and response to the presenting symptom of bleeding and the prolonged aptt throughout this case study .", "this is consistent with data from the european acquired haemophilia registry ( each-2 ) , which reported a median delay of 3 days between onset of bleeding symptoms and the diagnosis of acquired hemophilia and a median delay of one day between the first abnormal aptt test in those same patients and the established diagnosis.23 in addition , we found that emergency medicine and critical care physicians were reluctant to consider a bleeding disorder as the primary explanation for this patient s clinical presentation .", "the disposition of a patient with active hemorrhage and evidence of coagulopathy should be based on several factors , including the patient s current condition and anticipated clinical course , taking into account the presenting vital signs and evolving laboratory findings . at the time of the patient", "s second presentation , vital signs were notable for mild tachycardia and a pulse pressure at the upper limit of normal , and subsequent laboratory findings indicated a decreasing hemoglobin level and an increasing aptt .", "these findings alone prompted hospital admission , although the exact location ( general floor versus intensive care unit ) of admission may vary , based on the level of monitoring and nurse - to - patient ratios in a particular hospital .", "another important variable is the anticipated potential for clinical deterioration , which is based in large part on clinician appreciation of the seriousness of the diagnosis .", "we found a consistent tendency to consider admission to a general floor bed with the second presentation , even though this was ultimately an unstable , critically ill patient with an undiagnosed bleeding disorder .", "we also found that the physicians who participated in this survey were reluctant to consult a hematologist as they worked through this case scenario , particularly given that options for additional testing ( liver function , disseminated intravascular coagulation ) were not available to evaluate for common causes of coagulopathy .", "relative to their reported historical experience with hematology consultations during an average practice experience of approximately 20 years , this survey finding was somewhat surprising .", "rheumatologists and critical care specialists reported a greater frequency of hematology consultation relative to the other specialties ( table 2 ) .", "we would expect the emergency medicine physicians to be most likely to consult a hematologist , yet 16% of them reported never having consulted a hematologist .", "the highest percentage ( 46% ) had only consulted a hematologist 1 or 2 times , and almost one quarter of geriatricians and internists had never consulted a hematologist , even though these specialists would be expected to first encounter patients with undiagnosed bleeding disorders , including acquired hemophilia .", "one potential reason for not seeking hematology consultation might be the lack of availability of hematology / oncology specialists with expertise in coagulation disorders , including in rural and community hospitals .", "a limitation of this survey was that one can not interpret the thinking behind the responses of the individual participants .", "therefore , 31 qualitative 45-minute interviews were conducted subsequent to the quantitative study and focused particularly on critical care ( n = 7 ) , emergency medicine ( n = 6 ) , hematology / oncology ( n = 4 ) , or hematology ( n = 2 ) physicians to understand the reasoning behind their decisions and depth of knowledge ( unpublished data ) . we found that the physicians focus was generally on finding the source ( location ) of bleeding and not on finding the underlying reason for bleeding .", "this could potentially lead to surgical intervention in the face of an underlying bleeding disorder , with subsequent adverse outcomes . in a series of 67 patients with acquired hemophilia at a single center in bonn , germany ,", "4 of 5 deaths were the result of surgical intervention for bleeding at outside hospitals in the setting of a delayed diagnosis of acquired hemophilia.13 when queried about their experience encountering and/or diagnosing underlying bleeding disorders , particularly acquired hemophilia , more than 85% of physicians in hematology , hematology / oncology , emergency medicine , and critical care medicine reported having ever discovered or diagnosed an underlying bleeding disorder , compared with 65% , 54% , and 47% of rheumatologists , internists , and geriatricians , respectively . while reports of ever specifically having encountered acquired hemophilia were high , it is unclear from this study whether the participants truly understood the diagnosis .", "this seems unlikely , given the rarity of acquired hemophilia , relative to the reported frequency of having encountered it .", "except for hematology and/or oncology ( 77% ) and critical care ( 36% ) specialists , approximately one quarter of surveyed physicians had ever encountered acquired hemophilia .", "although they accounted for the highest percentage of physicians who had ever encountered this condition , nearly one quarter of hematologists had never encountered acquired hemophilia .", "subsequent unpublished data from the aforementioned qualitative research further suggest that , compared with hematology practitioners , specialists in hematology / oncology , who likely practice mostly oncology , might be able to identify  mixing studies  and  inhibitors  but might not fully understand the underlying pathophysiology that constitutes acquired hemophilia , making it hard for them to recognize the condition .", "given the survey findings reflecting the infrequency with which most physicians have encountered these conditions , consultation with a hematologist may facilitate the diagnostic evaluation and proper management of a hemorrhaging patient suspected of having an underlying bleeding diathesis , particularly acquired hemophilia .", "the consulting hematologist can provide specific guidance , leading to the prompt diagnosis and optimal management of an actively bleeding patient with acquired hemophilia , including initiation of immunosuppression , which is usually necessary to eradicate the inhibitor and to prevent additional bleeding episodes .", "however , this requires a level of familiarity and expertise in treating acquired hemophilia and other rare bleeding disorders that is not often seen outside of an academic hematology practice .", "this represents yet another barrier to the effective diagnosis and management of this rare yet serious bleeding diathesis ."], ["for the hospitalist or intensivist charged with the care of a bleeding patient , immediate stabilization is the initial priority and should take precedence over determination of a specific etiology of bleeding .", "however , in the presence of underlying coagulopathy , particularly acquired hemophilia or other rare disorders , traditional measures of stabilization may not be as effective as expected .", "a prolonged pt / inr or aptt in the absence of iatrogenic causes should never be ignored , even with only minimally prolonged values , and determination of the cause of an abnormal coagulation study should carry at least equal weight to looking for the anatomic site of bleeding . any delay in establishing the diagnosis of a bleeding diathesis such as acquired hemophilia can result in significant morbidity or even death .", "while hospitalists and intensivists should be able to conduct a thorough differential of bleeding and eliminate most common etiologies , consultation with a hematologist ( particularly one with specific expertise in coagulation disorders ) may facilitate the evaluation of coagulopathic patients and subsequent interpretation of diagnostic findings , as well as initiation of appropriate treatment . given the rarity of acquired hemophilia , as exemplified by the findings of this survey", ", physicians must harbor a high index of suspicion to diagnose this condition promptly in patients who present with recent - onset or acute bleeding . given the high morbidity and mortality", "the insights from this survey highlights knowledge and practice gaps that could be the focus of targeted educational initiatives , including diagnostic algorithms , to ensure proper and efficient workup of the abnormal laboratory studies that characterize acquired hemophilia ."]]}
{"article_id": "PMC3355559", "article_text": ["plant cells need to maintain the functional integrity of their walls during cell morphogenesis and exposure to biotic / abiotic stress .", "the available evidence suggests that a dedicated plant cell wall integrity ( cwi ) maintenance mechanism exists ( wolf et al . , 2011 ) . while our understanding of the mechanisms regulating stress responses and morphogenesis has increased significantly , our knowledge regarding the processes maintaining cwi is still limited . in the last years", "several reviews have been published on the plant cwi maintenance mechanism illustrating the increased interest in this area ( humphrey et al .", ", 2007 ; hematy et al . , 2009 ; ringli , 2010 ; seifert and blaukopf , 2010 ) .", "a recently published review focuses on cwi maintenance during plant cell wall morphogenesis ( wolf et al . , 2011 ) .", "similarities between the yeast and plant cwi maintenance mechanisms have also been reviewed ( hamann and denness , 2011 ) .", "therefore , the available knowledge regarding cwi maintenance during plant development and in yeast will be covered here only briefly to provide a conceptual framework regarding cellular processes involved and to illustrate the degree of functional conservation between species .", "this review will focus on recent developments regarding the role of cwi maintenance during biotic stress responses .", "it will discuss how cwi maintenance could have a previously unrecognized role in the perception of and response to biotic stress .", "while both plant and yeast cells are enveloped by cell walls , certain important differences exist that affect the biological role and function of the plant cwi maintenance mechanism .", "in addition , plant cell walls are structurally and chemically more complex than the yeast cell wall .", "this means that in plant cells the sheer number of cell wall - related signaling events during development and plant environment interaction could disguise the activity of a dedicated plant cwi maintenance mechanism .", "the yeast cwi monitoring and maintenance network is quite complex , providing an indication of the possible complexity of the plant cwi maintenance network . by combining inputs from a turgor pressure sensor ( sln1 ) , mechano - perception ( mid1/cch1 ) , and dedicated cell wall damage ( cwd ) sensors ( wsc1 , 2 , 3 , mid2 , mtl1 ) the yeast cwi maintenance network generates signals that permit highly specific responses to any challenge that impairs the functional integrity of the yeast cell wall .", "the available phenotypic and genetic data also implicate turgor pressure , mechano - perception , and cwd detection in plant cwi maintenance ( hamann et al .", "interestingly , arabidopsis histidine kinase1 ( ahk1 ) and cytokinin receptor1/arabidopsis histidine kinase4 ( cre1/ahk4 ) can at least partially rescue a yeast strain with a loss of function allele in sln1 ( urao et al .", "in addition , expression of the arabidopsis thaliana mid1 complementing activity 1 and 2 ( mca1,2 ) genes rescues a mid1-deficient yeast strain suggesting that they could function as stretch - activated calcium channels ( nakagawa et al .", "no functional homologues for the yeast cwd sensors have been identified in plants . in yeast ,", "the signals generated by the sensors are relayed to the response genes via different signaling cascades involving calcineurin ( mid1/cch1 ) ; rho1/mpk1 ( wsc1 , mid2 ) , and ypd1 ( sln1 ) ( levin , 2005 ) .", "transcription factors mediating the response are skn7 , rlm1 , and swi4/6 ( levin , 2005 ) .", "the response can involve activation of genes required for cell wall biosynthetic processes , remodeling of the cytoskeleton , and cell cycle progression .", "the available data suggest both organizational and functional similarities between the yeast and plant cwi maintenance mechanism while also highlighting how signals from mechanical and chemical sensors regulate jointly the cwd response .", "the plant cell wall is comparable to an exoskeleton surrounding the plant cell and providing both structural support and protection from biotic as well as abiotic stresses .", "it consists of cellulose microfibrils , pectin , hemicelluloses , proteins , and in certain cases lignin ( somerville et al . , 2004 ) .", "plant cell walls are divided into primary ( laid down during cell elongation / differentiation ) and secondary ( formed after cell morphogenesis is concluded ) walls . in parallel ,", "dependent on the presence of certain polysaccharides type i and type ii cell walls are distinguished ( popper et al . , 2011 ) .", "cellulose microfibrils are the main load bearing elements , which are cross - linked to hemicelluloses and ( in vitro ) to pectin ( dick - perez et al . , 2011 ) .", "hemicelluloses and pectin also form direct links creating a matrix in which the microfibrils are embedded like the steel mesh in a concrete wall .", "pectic polysaccharides like homogalacturonan ( hg ) are connected by calcium bridges between dimethyl - esterified parts of the molecules or through borate ester linkages in the case of rhamnogalacturonan ii ( rg ii ) .", "they are targeted by pathogen - derived cell wall degrading enzymes ( polygalacturonases ) , which generate oligogalacturonides ( ogas ) from hg ( hahn et al . , 1981 ; kars et al . , 2005 ) .", "biologically active ogas consist of chains of 915 galacturonic acid ( gala ) monomers and can function as signaling molecules ( see below ) .", "during secondary cell wall formation monolignols ( precursors for lignin ) are secreted into the cell wall space and randomly cross - linked ( vanholme et al . , 2010 ) .", "the cross - linking is dependent on the availability of reactive oxygen species ( ros ) generated by laccases and peroxidases .", "this process reinforces the wall against pathogen infection , waterproofs it , and increases structural integrity ( tronchet et al . , 2010 ;", "reduction of cellulose biosynthesis during primary cell wall formation through genetic or chemical means leads to lignin production ( ellis et al . , 2002 ; hamann et al . ,", "this highlights the ability of the cell to adapt to changes in cell wall composition and provides evidence for the existence of a cwi maintenance mechanism .", "plant cell walls are capable of adjusting their composition and structure in response to pathogen infection ( dong et al . , 2008 ) . a mutation in ( cellulose synthasea3 , cesa3 ) a subunit of the cellulose synthase complex leads to ectopic production of lignin , i.e. , replacement of a missing load bearing cell wall component by another one ( cano - delgado et al", "follow up studies found that inhibition of cellulose biosynthesis during primary cell wall formation either through mutations like constitutive expression of vsp1 ( cev1 ) and ectopic lignification1 ( eli1 ) or inhibitors such as isoxaben results in transcriptional activation of stress response mechanisms ; ectopic production of ethylene ( et ) , salicylic acid ( sa ) , jasmonic acid ( ja ) , callose , and ros as well as changes in cell wall composition and structure ( ellis et al .", ", 2002 ; cano - delgado et al . , 2003 ; manfield et al . , 2004 ; hamann et al . ,", "the experiments also showed that the response to cwd consists of early and late stages reminiscent of the response to pathogen infection ( denness et al . , 2011 ) .", "during the early stage , ros- and calcium - based signaling cascades are required for initiating the response to cwd ( denness et al . , 2011 ) .", "interestingly , 1-aminocyclopropane-1-carboxylic acid ( acc , an et precursor ) and not et itself seems to be acting as signaling substance during the early response to isoxaben with inhibition of cell expansion being an active process and not simply an automatic consequence of cellulose biosynthesis inhibition ( tsang et al . , 2011 ) . during the late stage ,", "responses to cwd - like lignin deposition are initiated and the extent of lignin formation is apparently modulated by a negative feedback loop formed by ros and ja ( denness et al . , 2011 ) .", "a combination of genetic and phenotypic analysis has implicated the nadph oxidases respiratory burst oxidase homologd and f ( rbohd , f ) , the serine / threonine kinase oxidative signal inducible1 ( oxi1 ) , mca1 , the receptor - like kinase ( rlk ) theseus1 ( the1 ) as well as the ja biosynthesis genes allene oxide synthase ( aos ) and jasmonic acid resistant1 ( jar1 ) in the signaling mechanism mediating the response to cwd in arabidopsis seedlings .", "interestingly , the cev1 mutation that affects cellulose biosynthesis during primary cell wall formation also causes enhanced resistance to infection by different powdery mildews ( erysiphe orontii , e. cichoracearum , and oidium lycopersicum ; ellis and turner , 2001 ) .", "a screen for mutants causing resistance to powdery mildew infection provides further evidence of the close relationship between plant cell walls and pathogen resistance ( vogel and somerville , 2000 ) .", "three of the powdery mildew resistance ( pmr ) mutants that have been identified on the molecular level affect genes involved in cell wall biosynthetic processes .", "pmr4 encodes a callose synthase , pmr5 a gene of unknown function required for pectin production and pmr6 a pectate lyase ( vogel et al .", "pmr4 resistance seems to be mediated via hyper - activation of sa signaling , whereas pmr5 and 6 resistance phenotypes are independent of ja , sa , and et signaling .", "mutations in irregularxylem1 ( irx1/cesa8 ) , 3 ( irx3/cesa7 ) , and 5 ( irx5/cesa4 ) impair cellulose biosynthesis during secondary cell wall formation and cause enhanced resistance to the soil borne bacterium ralstonia solanacearum and the necrotrophic fungus plectosphaerella cucumerina ( hernandez - blanco et al . , 2007 ) .", "mutations affecting cellulose biosynthesis during primary cell wall formation ( cesa1 , 3 , 6 ) or other components of the secondary cell wall ( pmr5 , pmr6 ) did not cause enhanced resistance to the same pathogens .", "genetic analysis showed that the enhanced resistance in cesa4 , 7 , 8 is independent of ja , sa , and et - based signaling mechanisms .", "results from expression profiling experiments and genetic analysis using different abscisic acid ( aba)mutants(aba insensitive 1 - 1;2 - 1;aba1 - 6 ) suggest that aba is mediating developmental and pathogen resistance phenotypes caused by the irx mutants .", "however , it remains to be determined if the aba involvement is direct or a secondary effect due to water stress caused by problems with xylem cell wall formation in the mutants .", "to summarize , these results suggest distinct resistance signaling cascades are induced by defects in primary and secondary cell wall formation as well as for different secondary cell wall components .", "they also highlight the direct impact of changes in cell wall composition / structure on the response to pathogen infection .", "the mode of action of the plant cell wall maintenance mechanism is not well understood .", "based on the knowledge from yeast , chemical and physical signals could act as indicators for the functional integrity of the plant cell wall either individually or jointly . by combining these different types of signal", "the plant cell would receive precise information regarding the state of its cell wall and the exact type of cwi impairment occurring .", "physical signals could be generated by stretching of the plasma membrane due to a weakened cell wall that can not resist the high turgor pressure levels within a plant cell or a plasma membrane that is displaced relatively to the cell wall .", "these events could be detected by stretch - activated or mechanosensitive channel proteins that lead to calcium influx into the cytoplasm , indicating cwd .", "sensor candidates could be encoded by members of the mechanosensitive channels of small conductance ( mscs)-like ( msl ) gene family like msl 9 and 10 affect mechano - perception in protoplasts derived from arabidopsis root cells ( haswell et al . , 2008 ) .", "another candidate of interest is the putative stretch - activated calcium channel mca1 is required for cwd - induced lignin deposition .", "interestingly , all isoxaben - induced cwd phenotypes can be suppressed by provision of osmotic support suggesting that changes in turgor pressure due to a weakened cell wall could result in signal generation via turgor pressure sensors ( hamann et al . , 2009 ; denness et al . ,", "2011 ) . while ahk1 and 4/cre1 can function as osmosensors in yeast and have been implicated in abiotic stress responses , no clear evidence exists implicating them in cwd perception in plants ( urao et al . , 1999", "; inoue et al . , 2001 ; tran et al . , 2007 ) .", "in addition , ahk4/cre1 has been shown to function as a cytokinin receptor(inoue et al . , 2001 ) .", "therefore , the question that needs to be resolved at this point is if turgor pressure is a passive element in the process ( generating cell wall fragments due to a weakened cell wall ) or an active component that is being monitored and provides input into the process .", "the plant cell wall contains a large number of components that could generate chemical signals ( ligands ) indicative of cwd or general danger signals .", "the term damage associated molecular patterns ( damps ) has been coined to describe such ligands and the number of possible damps originating in plant cell walls is rather large ( zipfel , 2009 ) . here", "i will focus on the best - characterized group of signals , which are probably ogas .", "they can be generated through degradation of hg by pathogen - derived enzymes ( kars et al .", "ogas have been shown to induce gene expression changes , stomatal closure , production of et , and ros as well as cell wall reinforcement ( denoux et al .", ", 2008 ; ferrari et al . , 2008 ) . a hybrid kinase consisting of the extra cellular domain of wall - associated kinase1 ( wak1 ) and the intracellular domain of elongation factor tu receptor ( efr ) kinase can bind ogas and activate defense responses ( brutus et al . , 2010 )", ". wak1 belongs to a family of five wak genes encoding plasma membrane - localized ser / thr kinases that have been implicated in response to pathogen infection and regulation of cell elongation ( kohorn et al . , 2011 ) .", "the effects of the chimeric wak1 kinase on pathogen resistance suggest that ogas and waks represent an in vivo ligand  receptor pair .", "results from the analysis of a dominant active wak2 allele suggest the cwd signals perceived by waks could be relayed to downstream response genes through mapkinase6 ( mpk6 ) ( kohorn et al .", "interestingly , wak2 has also been implicated in regulation of invertase activity and turgor pressure during cell elongation ( kohorn et al . ,", "2006 ) . however , there is currently no confirmation that waks are actively involved in cwi maintenance . in arabidopsis ,", "more than 600 rlks have been identified and a large number of them have been implicated in developmental and stress response processes ( shiu and bleecker , 2001 ) .", "i will focus here on several kinases that have been implicated in cwd perception and/or pathogen response .", "most of the rlks implicated in cwi maintenance [ the1 , hercules1 ( herk1 ) , feronia ( fer ) ] belong to the catharanthus roseus rlk1 ( crrlk1)-like protein family , which has 17 members in arabidopsis .", "the1 was isolated as a suppressor of the cellulose - deficient cesa6 procuste ( pre ) mutant , which exhibits a hypocotyl elongation defect ( hematy et al . , 2007 ) .", "subsequently it has been shown that the1 is required for cellulose biosynthesis inhibition - induced ros production and lignification in the root elongation zone ( denness et al . , 2011 ) .", "the1 , herk1 , and fer have been implicated in brassinosteroid - induced cell elongation ( guo et al . , 2009 ; deslauriers and larsen , 2010 ) .", "both fer and nortia / mildew resistance locus o 7 ( ml07 ; a seven - transmembrane domain protein involved in powdery mildew resistance ) are required for successful fertilization and resistance to infection by golovinomyces ( syn .", "interestingly , ropgef ( guanine - exchange factors ) proteins have been identified as targets of fer activity ( duan et al . , 2010 ) .", "ropgefs are required for the activation of rho gtpases , which in turn activate nadph oxidases like rbohd / f . these results suggest the same molecular components could mediate cell - cell interaction during development and plant ", "heterotrimeric g - proteins ( g , g , g ) form a highly conserved signaling complex that has been implicated in signal transduction during development and stress responses in mammals , yeast , and plants ( digby et al . , 2006 ; temple and jones , 2007 ) . in arabidopsis , five genes gpa1 ( g ) , agb1 ( g ) , agg1 , 2 , 3 ( g1 , 2 , 3 ) encode the subunits of the complex ( thung et al . , 2011 ) .", "recently , it has been reported that mutations in agg1 , 2 , and agb1 apparently cause enhanced susceptibility to infection with p. cucumerina ( delgado - cerezo et al . , 2011 ) .", "a combination of metabolomic and microarray - based expression profiling studies of the mutants established that the pathogen phenotype is independent of sa , ja , aba , and et signaling cascades .", "interestingly , a large number of cell wall biosynthetic / modifying genes are mis - regulated in agbl and aggl2 plants .", "analysis of cell wall composition / structure in these plants found reduced xylose contents in the mutants compared to wildtype . to summarize ,", "the available evidence supports the notion that the plant cell wall is an integral component contributing to pathogen response mechanisms and illustrates the influence of cell wall defects on infection .", "specific signaling cascades seem to mediate the response to particular cell wall defects , which in turn affect the response to necrotrophic or biotrophic pathogens .", "more importantly the data presented above allow correlation between certain types of cell wall defects , and not only signaling cascades but also resistance phenotypes .", "mutations in cesa4 , 7 , 8 , agg1 , 2 , and agb1 affect resistance to necrotrophs and are independent of phytohormone - based signaling cascades .", "pmr5 , 6 plants exhibit resistance to biotrophs and also do not rely on phytohormone - based signaling cascades .", "pmr4 affects resistance to biotrophs and resistance depends on the integrity of the sa signaling cascade .", "the cell wall composition / structure changes could prevent pathogen colonization simply because the infection machinery of the pathogen is too specialized to breach the chemically modified cell wall .", "another option is that the cell wall mutants cause defects similar to those occurring during infection by particular pathogens , i.e. , simulate infection by necrotrophs or biotrophs .", "this would cause early / constant activation of the cwi maintenance / defense mechanism , which  primes  plant immunity thus making successful infection more difficult .", "the latter would explain both the specificity of the responses observed and dependence on particular signaling mechanisms .", "therefore , studies focusing on the effects of particular cell wall defects on pathogen resistance and the mode of action of the cwi maintenance mechanism could facilitate research into biotic stress response .", "the reason being , that by removing the potentially multiple effects of the pathogen during infection , they reduce the complexity of the interaction and should therefore allow novel insights into the mechanisms responsible for detection of infection and/or physical damage .", "the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S> plant cell walls provide structural support during development and represent together with the cuticle the first line of defense against biotic and abiotic stress . in recent years , evidence has accumulated that a dedicated plant cell wall integrity ( cwi ) maintenance mechanism exists . this mechanism monitors and maintains functional integrity of the cell wall during different biological processes . </S>", "<S> the available data suggest that it may represent a component of the stress response mechanisms underlying biotic and abiotic stress responses , which has not been identified previously as a distinct mechanism . here </S>", "<S> i will review the available evidence regarding the mode of action of the cwi maintenance mechanism and discuss its role in the context of biotic plant stress response mechanisms . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SIMILARITIES AND DIFFERENCES BETWEEN YEAST AND PLANT CELL WALL INTEGRITY MAINTENANCE MECHANISMS", "COMPOSITION AND STRUCTURE OF THE PLANT CELL WALL", "THE PLANT CWI MAINTENANCE MECHANISM AS A COMPONENT OF THE BIOTIC STRESS RESPONSE", "SIGNALING MECHANISMS AND SENSORS IMPLICATED IN PLANT CWD PERCEPTION", "Conflict of Interest Statement"], "sections": [["plant cells need to maintain the functional integrity of their walls during cell morphogenesis and exposure to biotic / abiotic stress .", "the available evidence suggests that a dedicated plant cell wall integrity ( cwi ) maintenance mechanism exists ( wolf et al . , 2011 ) . while our understanding of the mechanisms regulating stress responses and morphogenesis has increased significantly , our knowledge regarding the processes maintaining cwi is still limited . in the last years", "several reviews have been published on the plant cwi maintenance mechanism illustrating the increased interest in this area ( humphrey et al .", ", 2007 ; hematy et al . , 2009 ; ringli , 2010 ; seifert and blaukopf , 2010 ) .", "a recently published review focuses on cwi maintenance during plant cell wall morphogenesis ( wolf et al . , 2011 ) .", "similarities between the yeast and plant cwi maintenance mechanisms have also been reviewed ( hamann and denness , 2011 ) .", "therefore , the available knowledge regarding cwi maintenance during plant development and in yeast will be covered here only briefly to provide a conceptual framework regarding cellular processes involved and to illustrate the degree of functional conservation between species .", "this review will focus on recent developments regarding the role of cwi maintenance during biotic stress responses .", "it will discuss how cwi maintenance could have a previously unrecognized role in the perception of and response to biotic stress ."], ["while both plant and yeast cells are enveloped by cell walls , certain important differences exist that affect the biological role and function of the plant cwi maintenance mechanism .", "in addition , plant cell walls are structurally and chemically more complex than the yeast cell wall .", "this means that in plant cells the sheer number of cell wall - related signaling events during development and plant environment interaction could disguise the activity of a dedicated plant cwi maintenance mechanism .", "the yeast cwi monitoring and maintenance network is quite complex , providing an indication of the possible complexity of the plant cwi maintenance network . by combining inputs from a turgor pressure sensor ( sln1 ) , mechano - perception ( mid1/cch1 ) , and dedicated cell wall damage ( cwd ) sensors ( wsc1 , 2 , 3 , mid2 , mtl1 ) the yeast cwi maintenance network generates signals that permit highly specific responses to any challenge that impairs the functional integrity of the yeast cell wall .", "the available phenotypic and genetic data also implicate turgor pressure , mechano - perception , and cwd detection in plant cwi maintenance ( hamann et al .", "interestingly , arabidopsis histidine kinase1 ( ahk1 ) and cytokinin receptor1/arabidopsis histidine kinase4 ( cre1/ahk4 ) can at least partially rescue a yeast strain with a loss of function allele in sln1 ( urao et al .", "in addition , expression of the arabidopsis thaliana mid1 complementing activity 1 and 2 ( mca1,2 ) genes rescues a mid1-deficient yeast strain suggesting that they could function as stretch - activated calcium channels ( nakagawa et al .", "no functional homologues for the yeast cwd sensors have been identified in plants . in yeast ,", "the signals generated by the sensors are relayed to the response genes via different signaling cascades involving calcineurin ( mid1/cch1 ) ; rho1/mpk1 ( wsc1 , mid2 ) , and ypd1 ( sln1 ) ( levin , 2005 ) .", "transcription factors mediating the response are skn7 , rlm1 , and swi4/6 ( levin , 2005 ) .", "the response can involve activation of genes required for cell wall biosynthetic processes , remodeling of the cytoskeleton , and cell cycle progression .", "the available data suggest both organizational and functional similarities between the yeast and plant cwi maintenance mechanism while also highlighting how signals from mechanical and chemical sensors regulate jointly the cwd response ."], ["the plant cell wall is comparable to an exoskeleton surrounding the plant cell and providing both structural support and protection from biotic as well as abiotic stresses .", "it consists of cellulose microfibrils , pectin , hemicelluloses , proteins , and in certain cases lignin ( somerville et al . , 2004 ) .", "plant cell walls are divided into primary ( laid down during cell elongation / differentiation ) and secondary ( formed after cell morphogenesis is concluded ) walls . in parallel ,", "dependent on the presence of certain polysaccharides type i and type ii cell walls are distinguished ( popper et al . , 2011 ) .", "cellulose microfibrils are the main load bearing elements , which are cross - linked to hemicelluloses and ( in vitro ) to pectin ( dick - perez et al . , 2011 ) .", "hemicelluloses and pectin also form direct links creating a matrix in which the microfibrils are embedded like the steel mesh in a concrete wall .", "pectic polysaccharides like homogalacturonan ( hg ) are connected by calcium bridges between dimethyl - esterified parts of the molecules or through borate ester linkages in the case of rhamnogalacturonan ii ( rg ii ) .", "they are targeted by pathogen - derived cell wall degrading enzymes ( polygalacturonases ) , which generate oligogalacturonides ( ogas ) from hg ( hahn et al . , 1981 ; kars et al . , 2005 ) .", "biologically active ogas consist of chains of 915 galacturonic acid ( gala ) monomers and can function as signaling molecules ( see below ) .", "during secondary cell wall formation monolignols ( precursors for lignin ) are secreted into the cell wall space and randomly cross - linked ( vanholme et al . , 2010 ) .", "the cross - linking is dependent on the availability of reactive oxygen species ( ros ) generated by laccases and peroxidases .", "this process reinforces the wall against pathogen infection , waterproofs it , and increases structural integrity ( tronchet et al . , 2010 ;", "reduction of cellulose biosynthesis during primary cell wall formation through genetic or chemical means leads to lignin production ( ellis et al . , 2002 ; hamann et al . ,", "this highlights the ability of the cell to adapt to changes in cell wall composition and provides evidence for the existence of a cwi maintenance mechanism ."], ["plant cell walls are capable of adjusting their composition and structure in response to pathogen infection ( dong et al . , 2008 ) . a mutation in ( cellulose synthasea3 , cesa3 ) a subunit of the cellulose synthase complex leads to ectopic production of lignin , i.e. , replacement of a missing load bearing cell wall component by another one ( cano - delgado et al", "follow up studies found that inhibition of cellulose biosynthesis during primary cell wall formation either through mutations like constitutive expression of vsp1 ( cev1 ) and ectopic lignification1 ( eli1 ) or inhibitors such as isoxaben results in transcriptional activation of stress response mechanisms ; ectopic production of ethylene ( et ) , salicylic acid ( sa ) , jasmonic acid ( ja ) , callose , and ros as well as changes in cell wall composition and structure ( ellis et al .", ", 2002 ; cano - delgado et al . , 2003 ; manfield et al . , 2004 ; hamann et al . ,", "the experiments also showed that the response to cwd consists of early and late stages reminiscent of the response to pathogen infection ( denness et al . , 2011 ) .", "during the early stage , ros- and calcium - based signaling cascades are required for initiating the response to cwd ( denness et al . , 2011 ) .", "interestingly , 1-aminocyclopropane-1-carboxylic acid ( acc , an et precursor ) and not et itself seems to be acting as signaling substance during the early response to isoxaben with inhibition of cell expansion being an active process and not simply an automatic consequence of cellulose biosynthesis inhibition ( tsang et al . , 2011 ) . during the late stage ,", "responses to cwd - like lignin deposition are initiated and the extent of lignin formation is apparently modulated by a negative feedback loop formed by ros and ja ( denness et al . , 2011 ) .", "a combination of genetic and phenotypic analysis has implicated the nadph oxidases respiratory burst oxidase homologd and f ( rbohd , f ) , the serine / threonine kinase oxidative signal inducible1 ( oxi1 ) , mca1 , the receptor - like kinase ( rlk ) theseus1 ( the1 ) as well as the ja biosynthesis genes allene oxide synthase ( aos ) and jasmonic acid resistant1 ( jar1 ) in the signaling mechanism mediating the response to cwd in arabidopsis seedlings .", "interestingly , the cev1 mutation that affects cellulose biosynthesis during primary cell wall formation also causes enhanced resistance to infection by different powdery mildews ( erysiphe orontii , e. cichoracearum , and oidium lycopersicum ; ellis and turner , 2001 ) .", "a screen for mutants causing resistance to powdery mildew infection provides further evidence of the close relationship between plant cell walls and pathogen resistance ( vogel and somerville , 2000 ) .", "three of the powdery mildew resistance ( pmr ) mutants that have been identified on the molecular level affect genes involved in cell wall biosynthetic processes .", "pmr4 encodes a callose synthase , pmr5 a gene of unknown function required for pectin production and pmr6 a pectate lyase ( vogel et al .", "pmr4 resistance seems to be mediated via hyper - activation of sa signaling , whereas pmr5 and 6 resistance phenotypes are independent of ja , sa , and et signaling .", "mutations in irregularxylem1 ( irx1/cesa8 ) , 3 ( irx3/cesa7 ) , and 5 ( irx5/cesa4 ) impair cellulose biosynthesis during secondary cell wall formation and cause enhanced resistance to the soil borne bacterium ralstonia solanacearum and the necrotrophic fungus plectosphaerella cucumerina ( hernandez - blanco et al . , 2007 ) .", "mutations affecting cellulose biosynthesis during primary cell wall formation ( cesa1 , 3 , 6 ) or other components of the secondary cell wall ( pmr5 , pmr6 ) did not cause enhanced resistance to the same pathogens .", "genetic analysis showed that the enhanced resistance in cesa4 , 7 , 8 is independent of ja , sa , and et - based signaling mechanisms .", "results from expression profiling experiments and genetic analysis using different abscisic acid ( aba)mutants(aba insensitive 1 - 1;2 - 1;aba1 - 6 ) suggest that aba is mediating developmental and pathogen resistance phenotypes caused by the irx mutants .", "however , it remains to be determined if the aba involvement is direct or a secondary effect due to water stress caused by problems with xylem cell wall formation in the mutants .", "to summarize , these results suggest distinct resistance signaling cascades are induced by defects in primary and secondary cell wall formation as well as for different secondary cell wall components .", "they also highlight the direct impact of changes in cell wall composition / structure on the response to pathogen infection ."], ["the mode of action of the plant cell wall maintenance mechanism is not well understood .", "based on the knowledge from yeast , chemical and physical signals could act as indicators for the functional integrity of the plant cell wall either individually or jointly . by combining these different types of signal", "the plant cell would receive precise information regarding the state of its cell wall and the exact type of cwi impairment occurring .", "physical signals could be generated by stretching of the plasma membrane due to a weakened cell wall that can not resist the high turgor pressure levels within a plant cell or a plasma membrane that is displaced relatively to the cell wall .", "these events could be detected by stretch - activated or mechanosensitive channel proteins that lead to calcium influx into the cytoplasm , indicating cwd .", "sensor candidates could be encoded by members of the mechanosensitive channels of small conductance ( mscs)-like ( msl ) gene family like msl 9 and 10 affect mechano - perception in protoplasts derived from arabidopsis root cells ( haswell et al . , 2008 ) .", "another candidate of interest is the putative stretch - activated calcium channel mca1 is required for cwd - induced lignin deposition .", "interestingly , all isoxaben - induced cwd phenotypes can be suppressed by provision of osmotic support suggesting that changes in turgor pressure due to a weakened cell wall could result in signal generation via turgor pressure sensors ( hamann et al . , 2009 ; denness et al . ,", "2011 ) . while ahk1 and 4/cre1 can function as osmosensors in yeast and have been implicated in abiotic stress responses , no clear evidence exists implicating them in cwd perception in plants ( urao et al . , 1999", "; inoue et al . , 2001 ; tran et al . , 2007 ) .", "in addition , ahk4/cre1 has been shown to function as a cytokinin receptor(inoue et al . , 2001 ) .", "therefore , the question that needs to be resolved at this point is if turgor pressure is a passive element in the process ( generating cell wall fragments due to a weakened cell wall ) or an active component that is being monitored and provides input into the process .", "the plant cell wall contains a large number of components that could generate chemical signals ( ligands ) indicative of cwd or general danger signals .", "the term damage associated molecular patterns ( damps ) has been coined to describe such ligands and the number of possible damps originating in plant cell walls is rather large ( zipfel , 2009 ) . here", "i will focus on the best - characterized group of signals , which are probably ogas .", "they can be generated through degradation of hg by pathogen - derived enzymes ( kars et al .", "ogas have been shown to induce gene expression changes , stomatal closure , production of et , and ros as well as cell wall reinforcement ( denoux et al .", ", 2008 ; ferrari et al . , 2008 ) . a hybrid kinase consisting of the extra cellular domain of wall - associated kinase1 ( wak1 ) and the intracellular domain of elongation factor tu receptor ( efr ) kinase can bind ogas and activate defense responses ( brutus et al . , 2010 )", ". wak1 belongs to a family of five wak genes encoding plasma membrane - localized ser / thr kinases that have been implicated in response to pathogen infection and regulation of cell elongation ( kohorn et al . , 2011 ) .", "the effects of the chimeric wak1 kinase on pathogen resistance suggest that ogas and waks represent an in vivo ligand  receptor pair .", "results from the analysis of a dominant active wak2 allele suggest the cwd signals perceived by waks could be relayed to downstream response genes through mapkinase6 ( mpk6 ) ( kohorn et al .", "interestingly , wak2 has also been implicated in regulation of invertase activity and turgor pressure during cell elongation ( kohorn et al . ,", "2006 ) . however , there is currently no confirmation that waks are actively involved in cwi maintenance . in arabidopsis ,", "more than 600 rlks have been identified and a large number of them have been implicated in developmental and stress response processes ( shiu and bleecker , 2001 ) .", "i will focus here on several kinases that have been implicated in cwd perception and/or pathogen response .", "most of the rlks implicated in cwi maintenance [ the1 , hercules1 ( herk1 ) , feronia ( fer ) ] belong to the catharanthus roseus rlk1 ( crrlk1)-like protein family , which has 17 members in arabidopsis .", "the1 was isolated as a suppressor of the cellulose - deficient cesa6 procuste ( pre ) mutant , which exhibits a hypocotyl elongation defect ( hematy et al . , 2007 ) .", "subsequently it has been shown that the1 is required for cellulose biosynthesis inhibition - induced ros production and lignification in the root elongation zone ( denness et al . , 2011 ) .", "the1 , herk1 , and fer have been implicated in brassinosteroid - induced cell elongation ( guo et al . , 2009 ; deslauriers and larsen , 2010 ) .", "both fer and nortia / mildew resistance locus o 7 ( ml07 ; a seven - transmembrane domain protein involved in powdery mildew resistance ) are required for successful fertilization and resistance to infection by golovinomyces ( syn .", "interestingly , ropgef ( guanine - exchange factors ) proteins have been identified as targets of fer activity ( duan et al . , 2010 ) .", "ropgefs are required for the activation of rho gtpases , which in turn activate nadph oxidases like rbohd / f . these results suggest the same molecular components could mediate cell - cell interaction during development and plant ", "heterotrimeric g - proteins ( g , g , g ) form a highly conserved signaling complex that has been implicated in signal transduction during development and stress responses in mammals , yeast , and plants ( digby et al . , 2006 ; temple and jones , 2007 ) . in arabidopsis , five genes gpa1 ( g ) , agb1 ( g ) , agg1 , 2 , 3 ( g1 , 2 , 3 ) encode the subunits of the complex ( thung et al . , 2011 ) .", "recently , it has been reported that mutations in agg1 , 2 , and agb1 apparently cause enhanced susceptibility to infection with p. cucumerina ( delgado - cerezo et al . , 2011 ) .", "a combination of metabolomic and microarray - based expression profiling studies of the mutants established that the pathogen phenotype is independent of sa , ja , aba , and et signaling cascades .", "interestingly , a large number of cell wall biosynthetic / modifying genes are mis - regulated in agbl and aggl2 plants .", "analysis of cell wall composition / structure in these plants found reduced xylose contents in the mutants compared to wildtype . to summarize ,", "the available evidence supports the notion that the plant cell wall is an integral component contributing to pathogen response mechanisms and illustrates the influence of cell wall defects on infection .", "specific signaling cascades seem to mediate the response to particular cell wall defects , which in turn affect the response to necrotrophic or biotrophic pathogens .", "more importantly the data presented above allow correlation between certain types of cell wall defects , and not only signaling cascades but also resistance phenotypes .", "mutations in cesa4 , 7 , 8 , agg1 , 2 , and agb1 affect resistance to necrotrophs and are independent of phytohormone - based signaling cascades .", "pmr5 , 6 plants exhibit resistance to biotrophs and also do not rely on phytohormone - based signaling cascades .", "pmr4 affects resistance to biotrophs and resistance depends on the integrity of the sa signaling cascade .", "the cell wall composition / structure changes could prevent pathogen colonization simply because the infection machinery of the pathogen is too specialized to breach the chemically modified cell wall .", "another option is that the cell wall mutants cause defects similar to those occurring during infection by particular pathogens , i.e. , simulate infection by necrotrophs or biotrophs .", "this would cause early / constant activation of the cwi maintenance / defense mechanism , which  primes  plant immunity thus making successful infection more difficult .", "the latter would explain both the specificity of the responses observed and dependence on particular signaling mechanisms .", "therefore , studies focusing on the effects of particular cell wall defects on pathogen resistance and the mode of action of the cwi maintenance mechanism could facilitate research into biotic stress response .", "the reason being , that by removing the potentially multiple effects of the pathogen during infection , they reduce the complexity of the interaction and should therefore allow novel insights into the mechanisms responsible for detection of infection and/or physical damage ."], ["the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC2698198", "article_text": ["genomic dna hypomethylation has been observed in the peripheral blood mononuclear cells of leukemia patients and in tumor cells of patients with b - cell lymphoproliferative diseases ( 1 ) .", "dna methylation may affect karyotypic stability , may influence euchromatin - heterochromatin interactions , and has been correlated with disease progression ( 2 ) . on the other hand ,", "for example , some patients with lymphomas do not express tumor suppressor genes because the promoters of these genes are methylated .", ", bone marrow produces a large number of abnormal white blood cells , which overwhelm the other types of blood cells , including red blood cells and platelets , thus impairing the production of normal white blood cells . in clinical classification , leukemia can be classified as acute myeloid leukemia ( aml ) and chronic myeloid leukemia ( cml ) .", "cml is a clonal disease of stem cell origin that is characterized by the presence of the philadelphia chromosome ( ph+ ) , which has been named t(9,22)(q34:q11 ) .", "its fusion gene product , bcr - abl , is a constitutively active tyrosine kinase .", "nevertheless , the differential factors through which they produce a different type of leukemia are not yet completely understood . in hematopoietic malignancies ,", "hypermethylation of several genes including e - cadherin , dap kinase , estrogen receptor ( er ) alpha , and p15 are associated with gene inactivation ( 7 - 10 ) .", "genes , such as dab2ip , dlc-1 , h - cadherin , id4 , integrin 4 , runx3 , sfrp1 , and shp1 , has been identified as being implicated in aberrant dna methylation during development of human malignancy ( 11 - 18 ) . in order to gain insight into the differential epigenetic alterations in leukemia , we investigated the methylation statuses at selected locus of these genes in aml and cml patients using a methylation - specific polymerase chain reaction ( msp ) .", "diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml .", "the samples were gathered by the division of hematology / oncology ( department of internal medicine , korea university medical center , seoul , republic of korea ) and analyzed by pathologist . institutional review board approval and informed consent were obtained ( kumc - irb-2006011-p-1 , kumcirb-2006012-p-2 ) . of the aml patients , 17 males and 6 females were included , with ages ranging from 26 to 78 yr , at a median age of 45.61 ( sd , 15.56 ) yr . of the cml patients ,", "11 males and 10 females were included , with ages ranging from 18 to 75 yr ( meansd , 49.9116.53 ) . as controls ,", "22 normal peripheral bloods were obtained from healthy volunteers ( 11 males and 10 females ) ranging from 20 to 78 yr of age ( meansd , 45.3620.64 ) .", "chemical modification was performed as described previously , with minor modifications ( 19 ) . in brief", ", 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37 , followed by the addition of 550 l of 3 m sodium bisulfite ( ph 5.0 ) ( sigma , st .", "louis , mo , u.s.a . ) and 10 mm hydroquinone ( sigma ) , which was brought to a final volume of 600 l .", "the mixtures were incubated at 55 for 16 hr , and the modified dna was then desalted with the wizard clean - up system ( promega corp . ,", "we performed polymerase chain reaction ( pcr ) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences .", "the primers for the unmethylated and methylated dna sequences , pcr product size , and annealing temperature are shown in table 1 .", "the msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software ( www.urogene.org ) .", "the pcr conditions were as follows : initial denaturation and hot start at 95 for 5 min , and cycles consisting of 30 sec at 95 , 30 sec at the annealing temperature , and 30 sec at 72. in addition , sck , a human cholangiocarcinoma cell line , was used as positive control for integrin 4 gene ( 20 ) .", "msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation .", "the msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test ( version 12 ; spss inc . ,", "diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml .", "the samples were gathered by the division of hematology / oncology ( department of internal medicine , korea university medical center , seoul , republic of korea ) and analyzed by pathologist . institutional review board approval and informed consent were obtained ( kumc - irb-2006011-p-1 , kumcirb-2006012-p-2 ) . of the aml patients , 17 males and 6 females were included , with ages ranging from 26 to 78 yr , at a median age of 45.61 ( sd , 15.56 ) yr . of the cml patients ,", "11 males and 10 females were included , with ages ranging from 18 to 75 yr ( meansd , 49.9116.53 ) . as controls ,", "22 normal peripheral bloods were obtained from healthy volunteers ( 11 males and 10 females ) ranging from 20 to 78 yr of age ( meansd , 45.3620.64 ) .", "chemical modification was performed as described previously , with minor modifications ( 19 ) . in brief", ", 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37 , followed by the addition of 550 l of 3 m sodium bisulfite ( ph 5.0 ) ( sigma , st .", "louis , mo , u.s.a . ) and 10 mm hydroquinone ( sigma ) , which was brought to a final volume of 600 l .", "the mixtures were incubated at 55 for 16 hr , and the modified dna was then desalted with the wizard clean - up system ( promega corp . ,", "we performed polymerase chain reaction ( pcr ) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences .", "the primers for the unmethylated and methylated dna sequences , pcr product size , and annealing temperature are shown in table 1 .", "the msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software ( www.urogene.org ) .", "the pcr conditions were as follows : initial denaturation and hot start at 95 for 5 min , and cycles consisting of 30 sec at 95 , 30 sec at the annealing temperature , and 30 sec at 72. in addition , sck , a human cholangiocarcinoma cell line , was used as positive control for integrin 4 gene ( 20 ) .", "msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation .", "the msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test ( version 12 ; spss inc . ,", "the multiple genes were found to be methylated in bone marrow from patients with aml or cml . specifically , the frequencies of promoter hypermethylation at selected locus in the 23 aml samples were : 78.3% ( 18/23 ) for shp1 , 65.2% ( 15/23 ) for id4 and sfrp1 , 26.1% ( 6/23 ) for h - cadherin , 8.7% ( 2/23 ) for dlc-1 , and 4.3% ( 1/23 ) for dab2ip and runx3 .", "the frequencies of dna hypermethylation at selected locus in the 21 cml samples were : 28.6% ( 6/21 ) for shp1 , 19.0% ( 4/21 ) for h - cadherin , 14.3% ( 3/21 ) for id4 , 9.5% for ( 2/21 ) for sfrp1 , and 0% ( 0/21 ) for dab2ip , dlc-1 , integrin 4 , and runx3 ( fig .", "however , promoter hypermethylation of the 22 normal peripheral bloods was observed less frequently ( table 2 ) . there was a statistically significant difference between normal peripheral bloods and aml with respect to the frequencies of methylation of id4 , sfrp1 , and shp1 ( pearson chi - square test ; p<0.0001 , p<0.0001 , and p<0.0001 , respectively ) and between normal peripheral bloods and cml with respect to the frequencies of shp1 methylation ( pearson chi - square test ; p=0.007 )", ". furthermore , there was a statistically significant difference between the dna methylation frequencies of aml patients and cml patients .", "the frequencies of dna methylation of id4 , sfrp1 , and shp1 were higher in aml compared to those in cml ( p=0.001 , p<0.0001 , and p=0.001 , respectively ) ( table 2 ) .", "in contrast , no statistical differences between aml and cml were detected in other genes such as dlc-1 , dab2ip , h - cadherin , integrin 4 , and runx3 .", "promoter methylation results at selected locus of eight genes with msp method were shown in fig .", "the results of the present study demonstrate the substantially increased frequency of promoter hypermethylation in id4 , sfrp1 , and shp1 genes in aml compared to cml .", "leukemia , a heterogeneous group of hematopoietic malignancies that occur worldwide , includes acute and chronic myeloid leukemia . despite many important advances in understanding the different biological and cytogenetic aspects of acute and chronic leukemia ,", "it is understood that some kinds of leukemia present specific cytogenetic alterations ( 21 ) .", "different types of leukemia usually have specific epigenetic modifications that cause the activation of oncogenes and , in particular , the formation of abnormal fusion genes such as aml1-eto ( 22 , 23 ) , a fusion protein resulting from t(8,21 ) translocation that commonly occurs in aml .", "another typical example is the fact that bcr - abl is a constitutively active , cytoplasmic tyrosine kinase that is generated by t(9;22 ) translocation in more than 95% of cml ( 24 ) .", "the central role of epigenetic modification of these genes in leukemia development promoted them as reasonable targets for understanding the differential mechanism between acute and chronic leukemia . in the present study , id4 , sfrp1 , and ship1", "these results suggest that the epigenetic modification of these genes may play specific roles in the development of aml rather than cml and , thus , promoted them as ideal targets for drug development to treat aml . in the future , it is clearly imperative to understand the patho - physiological differential mechanisms of these genes in aml , and to finally explore novel therapeutic strategies . in present study ,", "methylation frequencies of shp1 gene were the high detected in cml as well as aml .", "shp1 is a member of the shp family of proteins , cytoplasmic protein tyrosine phosphatase ( ptp ) , and has been known as a candidate tumor suppressor gene in lymphoma , leukemia and other cancers ( 25 ) .", "also , the hypermethylation of shp1 gene was frequently detected in several human cancers and the reduced expression of the shp1 gene in various types of leukemias and lymphomas mainly occurred by promoter methylation ( 26 - 28 ) . therefore , these results indicate that aberrant dna methylation of shp1 gene may be related to the tumorigenicity of myeloid leukemia .", "the methylation frequencies of the dab2ip , dlc-1 , and runx3 genes were detected at different levels in acute and chronic leukemia patients ; however , the difference was negligible . in some genes like integrin 4 gene ,", "however , hypermethylation of these genes was frequent event in esophageal squamous cell carcinomas , lung cancers , and gastric cancers ( 11 , 12 , 15 , 16 ) .", "thus , dna methylation of these genes may be not related to tumorigenicity of aml / cml in contrast with other cancers . in summary , we have identified that aberrant dna methylation of shp1 is a frequent event in aml and cml . also , the frequencies of dna methylation of several methylation - controlled genes , including id4 , sfrp1 , and shp1 , were higher in aml patients compared to those in cml patients .", "although these results should be confirmed , more comprehensive studies are necessary involving various known risk factors such as smoking and occupational carcinogens , and genetic susceptibilities .", "our results suggest that aberrant dna methylation of shp1 may be related to the tumorigenicity of aml and cml and hypermethylation of id4 , sfrp1 , and shp1 genes may contribute to the pathogenesis mechanism of aml specifically ."], "abstract_text": ["<S> to gain insight into the differential mechanism of gene promoter hypermethylation in acute and chronic leukemia , we identified the methylation status on one part of 5'cpg rich region of 8 genes , dab2ip , dlc-1 , h - cadherin , id4 , integrin 4 , runx3 , sfrp1 , and shp1 in bone marrows from acute myeloid leukemia ( aml ) and chronic myeloid leukemia ( cml ) patients . also , we compared the methylation status of genes in aml and cml using methylation - specific pcr ( msp ) . </S>", "<S> the frequencies of dna methylation of id4 , sfrp1 , and shp1 were higher in aml patients compared to those in cml patients . in contrast , no statistical difference between aml and cml was detected for other genes such as dlc-1 , dab2ip , h - cadherin , integrin 4 , and runx3 . </S>", "<S> taken together , these results suggest that these methylation - controlled genes may have different roles in aml and cml , and thus , may act as a biological marker of aml . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Sample collection", "Sodium bisulfite treatment and MSP", "Statistical analysis", "RESULTS", "DISCUSSION"], "sections": [["genomic dna hypomethylation has been observed in the peripheral blood mononuclear cells of leukemia patients and in tumor cells of patients with b - cell lymphoproliferative diseases ( 1 ) .", "dna methylation may affect karyotypic stability , may influence euchromatin - heterochromatin interactions , and has been correlated with disease progression ( 2 ) . on the other hand ,", "for example , some patients with lymphomas do not express tumor suppressor genes because the promoters of these genes are methylated .", ", bone marrow produces a large number of abnormal white blood cells , which overwhelm the other types of blood cells , including red blood cells and platelets , thus impairing the production of normal white blood cells . in clinical classification , leukemia can be classified as acute myeloid leukemia ( aml ) and chronic myeloid leukemia ( cml ) .", "cml is a clonal disease of stem cell origin that is characterized by the presence of the philadelphia chromosome ( ph+ ) , which has been named t(9,22)(q34:q11 ) .", "its fusion gene product , bcr - abl , is a constitutively active tyrosine kinase .", "nevertheless , the differential factors through which they produce a different type of leukemia are not yet completely understood . in hematopoietic malignancies ,", "hypermethylation of several genes including e - cadherin , dap kinase , estrogen receptor ( er ) alpha , and p15 are associated with gene inactivation ( 7 - 10 ) .", "genes , such as dab2ip , dlc-1 , h - cadherin , id4 , integrin 4 , runx3 , sfrp1 , and shp1 , has been identified as being implicated in aberrant dna methylation during development of human malignancy ( 11 - 18 ) . in order to gain insight into the differential epigenetic alterations in leukemia , we investigated the methylation statuses at selected locus of these genes in aml and cml patients using a methylation - specific polymerase chain reaction ( msp ) ."], ["diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml .", "the samples were gathered by the division of hematology / oncology ( department of internal medicine , korea university medical center , seoul , republic of korea ) and analyzed by pathologist . institutional review board approval and informed consent were obtained ( kumc - irb-2006011-p-1 , kumcirb-2006012-p-2 ) . of the aml patients , 17 males and 6 females were included , with ages ranging from 26 to 78 yr , at a median age of 45.61 ( sd , 15.56 ) yr . of the cml patients ,", "11 males and 10 females were included , with ages ranging from 18 to 75 yr ( meansd , 49.9116.53 ) . as controls ,", "22 normal peripheral bloods were obtained from healthy volunteers ( 11 males and 10 females ) ranging from 20 to 78 yr of age ( meansd , 45.3620.64 ) .", "chemical modification was performed as described previously , with minor modifications ( 19 ) . in brief", ", 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37 , followed by the addition of 550 l of 3 m sodium bisulfite ( ph 5.0 ) ( sigma , st .", "louis , mo , u.s.a . ) and 10 mm hydroquinone ( sigma ) , which was brought to a final volume of 600 l .", "the mixtures were incubated at 55 for 16 hr , and the modified dna was then desalted with the wizard clean - up system ( promega corp . ,", "we performed polymerase chain reaction ( pcr ) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences .", "the primers for the unmethylated and methylated dna sequences , pcr product size , and annealing temperature are shown in table 1 .", "the msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software ( www.urogene.org ) .", "the pcr conditions were as follows : initial denaturation and hot start at 95 for 5 min , and cycles consisting of 30 sec at 95 , 30 sec at the annealing temperature , and 30 sec at 72. in addition , sck , a human cholangiocarcinoma cell line , was used as positive control for integrin 4 gene ( 20 ) .", "msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation .", "the msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test ( version 12 ; spss inc . ,"], ["diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml .", "the samples were gathered by the division of hematology / oncology ( department of internal medicine , korea university medical center , seoul , republic of korea ) and analyzed by pathologist . institutional review board approval and informed consent were obtained ( kumc - irb-2006011-p-1 , kumcirb-2006012-p-2 ) . of the aml patients , 17 males and 6 females were included , with ages ranging from 26 to 78 yr , at a median age of 45.61 ( sd , 15.56 ) yr . of the cml patients ,", "11 males and 10 females were included , with ages ranging from 18 to 75 yr ( meansd , 49.9116.53 ) . as controls ,", "22 normal peripheral bloods were obtained from healthy volunteers ( 11 males and 10 females ) ranging from 20 to 78 yr of age ( meansd , 45.3620.64 ) ."], ["chemical modification was performed as described previously , with minor modifications ( 19 ) . in brief", ", 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37 , followed by the addition of 550 l of 3 m sodium bisulfite ( ph 5.0 ) ( sigma , st .", "louis , mo , u.s.a . ) and 10 mm hydroquinone ( sigma ) , which was brought to a final volume of 600 l .", "the mixtures were incubated at 55 for 16 hr , and the modified dna was then desalted with the wizard clean - up system ( promega corp . ,", "we performed polymerase chain reaction ( pcr ) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences .", "the primers for the unmethylated and methylated dna sequences , pcr product size , and annealing temperature are shown in table 1 .", "the msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software ( www.urogene.org ) .", "the pcr conditions were as follows : initial denaturation and hot start at 95 for 5 min , and cycles consisting of 30 sec at 95 , 30 sec at the annealing temperature , and 30 sec at 72. in addition , sck , a human cholangiocarcinoma cell line , was used as positive control for integrin 4 gene ( 20 ) ."], ["msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation .", "the msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test ( version 12 ; spss inc . ,"], ["the multiple genes were found to be methylated in bone marrow from patients with aml or cml . specifically , the frequencies of promoter hypermethylation at selected locus in the 23 aml samples were : 78.3% ( 18/23 ) for shp1 , 65.2% ( 15/23 ) for id4 and sfrp1 , 26.1% ( 6/23 ) for h - cadherin , 8.7% ( 2/23 ) for dlc-1 , and 4.3% ( 1/23 ) for dab2ip and runx3 .", "the frequencies of dna hypermethylation at selected locus in the 21 cml samples were : 28.6% ( 6/21 ) for shp1 , 19.0% ( 4/21 ) for h - cadherin , 14.3% ( 3/21 ) for id4 , 9.5% for ( 2/21 ) for sfrp1 , and 0% ( 0/21 ) for dab2ip , dlc-1 , integrin 4 , and runx3 ( fig .", "however , promoter hypermethylation of the 22 normal peripheral bloods was observed less frequently ( table 2 ) . there was a statistically significant difference between normal peripheral bloods and aml with respect to the frequencies of methylation of id4 , sfrp1 , and shp1 ( pearson chi - square test ; p<0.0001 , p<0.0001 , and p<0.0001 , respectively ) and between normal peripheral bloods and cml with respect to the frequencies of shp1 methylation ( pearson chi - square test ; p=0.007 )", ". furthermore , there was a statistically significant difference between the dna methylation frequencies of aml patients and cml patients .", "the frequencies of dna methylation of id4 , sfrp1 , and shp1 were higher in aml compared to those in cml ( p=0.001 , p<0.0001 , and p=0.001 , respectively ) ( table 2 ) .", "in contrast , no statistical differences between aml and cml were detected in other genes such as dlc-1 , dab2ip , h - cadherin , integrin 4 , and runx3 .", "promoter methylation results at selected locus of eight genes with msp method were shown in fig ."], ["the results of the present study demonstrate the substantially increased frequency of promoter hypermethylation in id4 , sfrp1 , and shp1 genes in aml compared to cml .", "leukemia , a heterogeneous group of hematopoietic malignancies that occur worldwide , includes acute and chronic myeloid leukemia . despite many important advances in understanding the different biological and cytogenetic aspects of acute and chronic leukemia ,", "it is understood that some kinds of leukemia present specific cytogenetic alterations ( 21 ) .", "different types of leukemia usually have specific epigenetic modifications that cause the activation of oncogenes and , in particular , the formation of abnormal fusion genes such as aml1-eto ( 22 , 23 ) , a fusion protein resulting from t(8,21 ) translocation that commonly occurs in aml .", "another typical example is the fact that bcr - abl is a constitutively active , cytoplasmic tyrosine kinase that is generated by t(9;22 ) translocation in more than 95% of cml ( 24 ) .", "the central role of epigenetic modification of these genes in leukemia development promoted them as reasonable targets for understanding the differential mechanism between acute and chronic leukemia . in the present study , id4 , sfrp1 , and ship1", "these results suggest that the epigenetic modification of these genes may play specific roles in the development of aml rather than cml and , thus , promoted them as ideal targets for drug development to treat aml . in the future , it is clearly imperative to understand the patho - physiological differential mechanisms of these genes in aml , and to finally explore novel therapeutic strategies . in present study ,", "methylation frequencies of shp1 gene were the high detected in cml as well as aml .", "shp1 is a member of the shp family of proteins , cytoplasmic protein tyrosine phosphatase ( ptp ) , and has been known as a candidate tumor suppressor gene in lymphoma , leukemia and other cancers ( 25 ) .", "also , the hypermethylation of shp1 gene was frequently detected in several human cancers and the reduced expression of the shp1 gene in various types of leukemias and lymphomas mainly occurred by promoter methylation ( 26 - 28 ) . therefore , these results indicate that aberrant dna methylation of shp1 gene may be related to the tumorigenicity of myeloid leukemia .", "the methylation frequencies of the dab2ip , dlc-1 , and runx3 genes were detected at different levels in acute and chronic leukemia patients ; however , the difference was negligible . in some genes like integrin 4 gene ,", "however , hypermethylation of these genes was frequent event in esophageal squamous cell carcinomas , lung cancers , and gastric cancers ( 11 , 12 , 15 , 16 ) .", "thus , dna methylation of these genes may be not related to tumorigenicity of aml / cml in contrast with other cancers . in summary , we have identified that aberrant dna methylation of shp1 is a frequent event in aml and cml . also , the frequencies of dna methylation of several methylation - controlled genes , including id4 , sfrp1 , and shp1 , were higher in aml patients compared to those in cml patients .", "although these results should be confirmed , more comprehensive studies are necessary involving various known risk factors such as smoking and occupational carcinogens , and genetic susceptibilities .", "our results suggest that aberrant dna methylation of shp1 may be related to the tumorigenicity of aml and cml and hypermethylation of id4 , sfrp1 , and shp1 genes may contribute to the pathogenesis mechanism of aml specifically ."]]}
{"article_id": "PMC4845415", "article_text": ["acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement .", "vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting .", "a 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism . following attempts at closed reduction under sedation and regional nerve block , eventual open reduction and soft tissue reconstruction was required under general anesthetic . during the open reduction procedure , it was noted that the patella had dislocated into a lateral extra - articular position and rotated around its vertical axis .", "a review of the literature suggests dislocations such as the current presentation , are extremely rare and although have been described to occur with minor trauma , have never been described to occur following a largely atraumatic event . in such cases ,", "closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction .", "acute patellar dislocation is defined as the abrupt disruption in the relationship of the patella within the femoral groove .", "patellar dislocation is a relatively common occurrence and is usually managed in the emergency department with closed manipulation under sedation .", "however , when the patella itself rotates around its vertical axis during dislocation , surgical intervention is usually required .", "a 32-year old gentleman was admitted to our trauma department with a painful , immobile right knee .", "he sustained the injury externally rotating his knee whilst in  slight flexion  while he was moving from the driver s seat to the passenger s seat of his car .", "the first event was 15 years prior , when his knee twisted in flexed position while pushing a car .", "the second event was four years prior , when he sustained an injury while pushing a van , this time with the right knee fully extended . on this occasion ,", "our patient had no other significant past medical or surgical history and explained his right knee was completely asymptomatic prior to this current event . on examination", ", there was an obvious deformity suggesting lateral displacement of the patella with a sulcus in the skin evident over the femoral groove .", "furthermore , the knee was held fixed in 15 degrees of flexion . due to patient positioning difficulties we were unable to obtain true ap and lateral views on x - ray .", "the radiographs demonstrated a laterally displaced and a mal - rotated patella in the vertical plane ( see figs . 1 and 2 ) .", "lateral radiograph of the right knee demonstrating rotation of patella our patient was subsequently taken to theatre where one final attempt of closed reduction was carried out under general anaesthetic and muscle relaxation . ultimately , this failed and an open reduction was performed .", "complete rupture of the medial patellofemoral ligament ( mpfl ) was identified with the patella situated lateral to the lateral femoral condyle , everted by approximately 100 degrees .", "the knee joint was washed out with normal saline and the medial patella retinaculum was repaired . after the repair ,", "post - operative radiographs confirm the patella in a satisfactory position in the antero - posterior and lateral planes ( see figs . 3 and 4 ) .", "the patient was placed in an extension splint for comfort purposes immediately post - operatively .", "early mobilization was encouraged after 5 days and the patient was referred for early physiotherapy . at 3 months follow - up , our patient had no further episodes of dislocation , full range of knee extension and flexion , and normal patella tracking .", "a hypermobility assessment at this stage revealed a beighton score of 2 with extension beyond 10 degrees of both elbows only .", "intraoperative antero - posterior florous copy image following successful reduction lateral radiograph of the right knee following surgery in knee splint", "patellar dislocations can vary widely in their pattern of presentation however vertical axis rotation complicates only a small number of these cases . in 1844 , cooper described the earliest case of patellar dislocation with vertical axis rotation .", "since then , only a handful of case reports describing vertical axis rotation of the patella have been published with the majority describing the location of the patella within the femoral trochlea ( intra - articular ) [ 2 - 11 ] . our case is rare as our patient had a less common variant of extra - articular patellar dislocation complicated by vertical axis rotation and by the fact , that the causative mechanism was largely atraumatic .", "previous descriptions postulate that the prominence of the lateral femoral condyle acts as a pivot point , which can cause the patella to rotate around on its vertical axis . in this case , however , the patient denies any trauma , and instead describes externally rotating the leg whilst extended as the mechanism of injury .", "it is almost certain that the previous dislocations in our patient s case had resulted in significant damage of the mpfl . with the patient reporting his injury taking place with the knee  slightly  flexed , the function of the mpfl in this case as the primary medial restraint during the first 20 degrees of flexion was likely to be inadequate .", "previous authors have described similar cases of extra - articular lateral patellar dislocation with vertical axis rotation and have noted in their subsequent reviews of the limited literature that intra - aritcular dislocations were usually related to far more significant trauma than extra - articular dislocations [ 3 - 10 ] .", "this was also the finding in a previous report highlighting the relative minor force resulting in an extra - articular dislocation .", "we feel this may be an indication of the chronic deficiency in the mpfl likely contributing to cases of extra - articular dislocation . on reviewing the previous cases of extra - articular dislocation in the literature", ", none had described a completely atraumatic mechanism and none had discussed previous dislocations or previous surgical history in their patients .", "detailed imaging can be difficult to obtain in the acute scenario ; however in these cases ct imaging may clarify patella position and demonstrate mechanical engagement of the patella on the lateral femoral condyle .", "in one case report , ct imaging was utilized and confirmed avulsions of the vastusmedialis muscle and medial crus of the patella tendon with resultant impaction of the medial border on the patella onto the lateral femoral condyle . in this case , imaging led to the decision for open reduction being required , minimizing further attempts at closed reduction which could in theory result in further damage .", "it is difficult to be absolutely sure why attempts at closed reduction had failed in our particular case , but this is likely to be multifactorial .", "vertical axis rotation results in impaction of the lateral edge of the patella on the lateral femoral epicondyle which may result in a corresponding femoral defect resulting in a lodging effect . furthermore patient factors , namely obesity and large muscle mass around the knee made manipulation technically difficult in our case .", "open reduction under general anaesthetic is rarely necessary for routine patellar dislocations however there are some obvious advantages .", "these include ; the possibility of direct visualisation of the obvious damage to the soft tissue structures contributing to or resultant from the dislocation . in our case it was likely", "the mpfl had been damaged previously and repair is likely to help in restoring central patella tracking and overall stability . in this case , the patient was noted as having a shallow trochlea which might have accounted for his increased tendency to dislocations .", "direct visual inspection may also help determine patients who are at risk of recurrent dislocations due to factors relating to their bony morphology and allow the surgeon to plan further management accordingly .", "lastly , visualisation of the articulating surfaces allows the surgeon to identify and address any osteochondral defects and corresponding loose bodies which may otherwise have contributed to long term future problems requiring eventual surgical intervention .", "from our experience with this case , difficulty in reducing a dislocated patella should alert surgeons to the possibility of an extra - articular dislocation with possible vertical axis rotation . in these scenarios , multiple attempts at reduction", "we were able to achieve adequate analgesia and sedation using local and regional anaesthesia , without successful reduction of the dislocation .", "although some authors have demonstrated successful closed reduction with the use of local anaesthetic and muscle relaxants , this is not always effective . furthermore , attempted closed reduction under general anaesthesia in our case was also unsuccessful , demonstrating how closed reduction can be futile in certain cases .", "we would recommend a low threshold for open reduction in such cases by a specialist knee surgeon who may assess and reconstruct damaged soft tissue damage in one operation vertical axis rotation is a rare entity complicating patella dislocations . in this rare event", "emergency physicians should be aware of this complication and refer to the relevant specialist for open reduction under general anaesthesia in order to minimize patient discomfort and potential articular surface damage ."], "abstract_text": ["<S> introduction : acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement . vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting.case presentation : a 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism . </S>", "<S> following attempts at closed reduction under sedation and regional nerve block , eventual open reduction and soft tissue reconstruction was required under general anesthetic . during the open reduction procedure , </S>", "<S> it was noted that the patella had dislocated into a lateral extra - articular position and rotated around its vertical axis . </S>", "<S> following patella reduction , the medial patellar retinaculum was repaired . </S>", "<S> examination under anesthetic revealed satisfactory tracking of patella following repair.conclusion:a review of the literature suggests dislocations such as the current presentation , are extremely rare and although have been described to occur with minor trauma , have never been described to occur following a largely atraumatic event . in such cases , </S>", "<S> closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction . </S>", "<S> open reduction is essential in these situations . </S>"], "labels": null, "section_names": ["Introduction:", "Case Presentation:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement .", "vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting ."], ["a 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism . following attempts at closed reduction under sedation and regional nerve block , eventual open reduction and soft tissue reconstruction was required under general anesthetic . during the open reduction procedure , it was noted that the patella had dislocated into a lateral extra - articular position and rotated around its vertical axis ."], ["a review of the literature suggests dislocations such as the current presentation , are extremely rare and although have been described to occur with minor trauma , have never been described to occur following a largely atraumatic event . in such cases ,", "closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction ."], ["acute patellar dislocation is defined as the abrupt disruption in the relationship of the patella within the femoral groove .", "patellar dislocation is a relatively common occurrence and is usually managed in the emergency department with closed manipulation under sedation .", "however , when the patella itself rotates around its vertical axis during dislocation , surgical intervention is usually required ."], ["a 32-year old gentleman was admitted to our trauma department with a painful , immobile right knee .", "he sustained the injury externally rotating his knee whilst in  slight flexion  while he was moving from the driver s seat to the passenger s seat of his car .", "the first event was 15 years prior , when his knee twisted in flexed position while pushing a car .", "the second event was four years prior , when he sustained an injury while pushing a van , this time with the right knee fully extended . on this occasion ,", "our patient had no other significant past medical or surgical history and explained his right knee was completely asymptomatic prior to this current event . on examination", ", there was an obvious deformity suggesting lateral displacement of the patella with a sulcus in the skin evident over the femoral groove .", "furthermore , the knee was held fixed in 15 degrees of flexion . due to patient positioning difficulties we were unable to obtain true ap and lateral views on x - ray .", "the radiographs demonstrated a laterally displaced and a mal - rotated patella in the vertical plane ( see figs . 1 and 2 ) .", "lateral radiograph of the right knee demonstrating rotation of patella our patient was subsequently taken to theatre where one final attempt of closed reduction was carried out under general anaesthetic and muscle relaxation . ultimately , this failed and an open reduction was performed .", "complete rupture of the medial patellofemoral ligament ( mpfl ) was identified with the patella situated lateral to the lateral femoral condyle , everted by approximately 100 degrees .", "the knee joint was washed out with normal saline and the medial patella retinaculum was repaired . after the repair ,", "post - operative radiographs confirm the patella in a satisfactory position in the antero - posterior and lateral planes ( see figs . 3 and 4 ) .", "the patient was placed in an extension splint for comfort purposes immediately post - operatively .", "early mobilization was encouraged after 5 days and the patient was referred for early physiotherapy . at 3 months follow - up , our patient had no further episodes of dislocation , full range of knee extension and flexion , and normal patella tracking .", "a hypermobility assessment at this stage revealed a beighton score of 2 with extension beyond 10 degrees of both elbows only .", "intraoperative antero - posterior florous copy image following successful reduction lateral radiograph of the right knee following surgery in knee splint"], ["patellar dislocations can vary widely in their pattern of presentation however vertical axis rotation complicates only a small number of these cases . in 1844 , cooper described the earliest case of patellar dislocation with vertical axis rotation .", "since then , only a handful of case reports describing vertical axis rotation of the patella have been published with the majority describing the location of the patella within the femoral trochlea ( intra - articular ) [ 2 - 11 ] . our case is rare as our patient had a less common variant of extra - articular patellar dislocation complicated by vertical axis rotation and by the fact , that the causative mechanism was largely atraumatic .", "previous descriptions postulate that the prominence of the lateral femoral condyle acts as a pivot point , which can cause the patella to rotate around on its vertical axis . in this case , however , the patient denies any trauma , and instead describes externally rotating the leg whilst extended as the mechanism of injury .", "it is almost certain that the previous dislocations in our patient s case had resulted in significant damage of the mpfl . with the patient reporting his injury taking place with the knee  slightly  flexed , the function of the mpfl in this case as the primary medial restraint during the first 20 degrees of flexion was likely to be inadequate .", "previous authors have described similar cases of extra - articular lateral patellar dislocation with vertical axis rotation and have noted in their subsequent reviews of the limited literature that intra - aritcular dislocations were usually related to far more significant trauma than extra - articular dislocations [ 3 - 10 ] .", "this was also the finding in a previous report highlighting the relative minor force resulting in an extra - articular dislocation .", "we feel this may be an indication of the chronic deficiency in the mpfl likely contributing to cases of extra - articular dislocation . on reviewing the previous cases of extra - articular dislocation in the literature", ", none had described a completely atraumatic mechanism and none had discussed previous dislocations or previous surgical history in their patients .", "detailed imaging can be difficult to obtain in the acute scenario ; however in these cases ct imaging may clarify patella position and demonstrate mechanical engagement of the patella on the lateral femoral condyle .", "in one case report , ct imaging was utilized and confirmed avulsions of the vastusmedialis muscle and medial crus of the patella tendon with resultant impaction of the medial border on the patella onto the lateral femoral condyle . in this case , imaging led to the decision for open reduction being required , minimizing further attempts at closed reduction which could in theory result in further damage .", "it is difficult to be absolutely sure why attempts at closed reduction had failed in our particular case , but this is likely to be multifactorial .", "vertical axis rotation results in impaction of the lateral edge of the patella on the lateral femoral epicondyle which may result in a corresponding femoral defect resulting in a lodging effect . furthermore patient factors , namely obesity and large muscle mass around the knee made manipulation technically difficult in our case .", "open reduction under general anaesthetic is rarely necessary for routine patellar dislocations however there are some obvious advantages .", "these include ; the possibility of direct visualisation of the obvious damage to the soft tissue structures contributing to or resultant from the dislocation . in our case it was likely", "the mpfl had been damaged previously and repair is likely to help in restoring central patella tracking and overall stability . in this case , the patient was noted as having a shallow trochlea which might have accounted for his increased tendency to dislocations .", "direct visual inspection may also help determine patients who are at risk of recurrent dislocations due to factors relating to their bony morphology and allow the surgeon to plan further management accordingly .", "lastly , visualisation of the articulating surfaces allows the surgeon to identify and address any osteochondral defects and corresponding loose bodies which may otherwise have contributed to long term future problems requiring eventual surgical intervention ."], ["from our experience with this case , difficulty in reducing a dislocated patella should alert surgeons to the possibility of an extra - articular dislocation with possible vertical axis rotation . in these scenarios , multiple attempts at reduction", "we were able to achieve adequate analgesia and sedation using local and regional anaesthesia , without successful reduction of the dislocation .", "although some authors have demonstrated successful closed reduction with the use of local anaesthetic and muscle relaxants , this is not always effective . furthermore , attempted closed reduction under general anaesthesia in our case was also unsuccessful , demonstrating how closed reduction can be futile in certain cases .", "we would recommend a low threshold for open reduction in such cases by a specialist knee surgeon who may assess and reconstruct damaged soft tissue damage in one operation vertical axis rotation is a rare entity complicating patella dislocations . in this rare event", "emergency physicians should be aware of this complication and refer to the relevant specialist for open reduction under general anaesthesia in order to minimize patient discomfort and potential articular surface damage ."]]}
{"article_id": "PMC4204277", "article_text": ["graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution .", "xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) .", "photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz .", "the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . for the isotope - tracer experiment ,", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc ", "graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution .", "xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) .", "photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz .", "the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity .", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc ", "as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors"], "abstract_text": ["<S> a facile one - step microwave - assisted chemical method has been successfully used for the synthesis of cu2o / reduced graphene oxide ( rgo ) composites . </S>", "<S> photocatalytic co2 reduction was then investigated on the junction under ambient conditions . </S>", "<S> the rgo coating dramatically increases cu2o activity for co2 photoreduction to result in a nearly six times higher activity than the optimized cu2o and 50 times higher activity than the cu2o / ruox junction in the 20th hour . </S>", "<S> furthermore , an apparent initial quantum yield of approximately 0.34 % at 400 nm has been achieved by the cu2o / rgo junction for co2 photoreduction . </S>", "<S> the photocurrent of the junction is nearly double that of the blank cu2o photocathode . </S>", "<S> the improved activity together with the enhanced stability of cu2o is attributed to the efficient charge separation and transfer to rgo as well as the protection function of rgo , which was proved by xrd , sem , tem , x - ray photoelectron spectroscopy , photo - electrochemical , photoluminescence , and impedance characterizations . </S>", "<S> this study further presents useful information for other photocatalyst modification for efficient co2 reduction without the need for a noble - metal co - catalyst . </S>"], "labels": null, "section_names": ["Experimental Section", "Synthesis of Cu", "Characterization", "Fabrication of film electrodes and electrochemical measurements", "Photocatalytic activity measurements", "Supporting Information"], "sections": [["graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution .", "xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) .", "photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz .", "the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . for the isotope - tracer experiment ,", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc "], ["graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution ."], ["xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) ."], ["photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz ."], ["the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity .", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc "], ["as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors"]]}
{"article_id": "PMC2836524", "article_text": ["in recent years , the concept of minimal intervention ( mi ) has prevailed in dentistry .", "therefore , the importance of diagnosing caries at an early stage has increased . in conventional procedures ,", "the diagnosis of caries has mainly consisted of visual inspection and tactile assessment with probing .", "however , lussi   reported that the sensitivity of detecting caries was 0.62 by visual inspection and 0.82 by probing .", "in addition , the pressure of probing can damage the demineralized fissure and increase the risk that caries progress [ 3 , 4 ] . to promote mi , diagnosis without a probe has been recommended .", "the laser fluorescence - based caries detection device diagnodent ( kavo , germany ) has been introduced as an alternative . however , no single detection method for caries is sufficient ; therefore , the combination of some detection methods has been recommended [ 57 ] .", "recent improvements in the personal computer have made the process of digital imaging more efficient and convenient .", "if the shape of caries can be quantified , and the relationship between the numerical value and the condition of the lesion can be demonstrated , this information would be helpful to diagnose dental caries .", "the authors of this paper have previously shown that the fractal dimension and proportion of the area of pit - and - fissure discoloration to the area of occlusal surface obtained by digital imaging were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth .", "for assessment of the method as a diagnostic system , the ability of the diagnosis , such as the sensitivity , the specificity , and the accuracy , should be researched in clinical situation .", "the aim of this study was to assess the possibility of the clinical application of the diagnosis of occlusal caries using digital imaging by examining the sensitivity , the specificity , and the accuracy in comparison with the diagnodent values and the dentists ' diagnoses .", "one hundred teeth ( 36 premolars and 64 molars ) with pit - and - fissure discoloration from 19 outpatients were examined at the clinic of oral diagnosis and general dentistry , dental hospital , tokyo medical and dental university .", "the occlusal surface of each tooth was washed with the robinson brush to remove dental plaque without any abrasive paste .", "then , pit - and - fissure discoloration was dried by air and measured three times using diagnodent .", "the mean scores were used as the diagnodent values of the teeth ( dd ) .", "next , the occlusal surface of each tooth was photographed as large as possible with an intraoral digital camera ( penscope , morita , japan ) .", "each image was stored in a personal computer using a video capture interface ( pc - mdvd / u2 , buffalo , japan ) . without knowing the dd ,", "a dentist preliminarily diagnosed each tooth using visual inspection and tactile examination to decide which treatment plan would be appropriate ( preventive or operative ) .", "the clinical diagnosis of preventive treatment for teeth was classified as co. on the other hand , carious lesions requiring operative treatment were removed in a conventional clinical way . if the resulting cavity preparation was limited in the enamel , then the clinical diagnosis was classified as c1 . if the resulting cavity preparation reached the dentin , and sound tissue still remained between the cavity and the pulp chamber , then the clinical diagnosis was classified as c2 .", "five dentists ranging from 3 to 15 years of professional experience examined the teeth after calibration of the criteria of the caries assessment conducted before the study ; the calibration was done as follows ; first , the five dentists examined 30 extracted teeth and decide which treatment plan would be appropriate ( preventive or operative ) . at that time", "then , the teeth were sliced pallarel to the teeth axis and to the depth of lesion for each tooth was determined .", "at last , the dentists discussed to accord the treatment planning for each tooth referring the depth of the lesion .  ", "the digital photographs obtained were processed and analyzed using image analysis software ( image j , nih , usa ) .", "first , each image was converted to an 8-bit gray - scale image , in which the density of grayness of each pixel was linearly scaled from min 0 ( black ) to max 255 ( white ) .", "then , the occlusal surface in the image was isolated from the background using a density histogram of the image , and the area was measured .", "pit - and - fissure discoloration was also isolated from the occlusal surface using the density histogram , and the area was measured .", "the proportion of the area of pit - and - fissure discoloration to the area of the occlusal surface was calculated ( pa ) .", "next , the image of the isolated pit - and - fissure discoloration was converted into a binary image , in which the density of pit - and - fissure discoloration was 0 , and its background was 255 , followed by calculating the fractal dimension of pit - and - fissure discoloration ( fd ) .  ", "differences in fd , pa , and dd between each clinical diagnosis were analyzed using two - way anova and games - howell test to reveal the clinical diagnosis and the effect of the examining dentists .", "the correlation between the clinical diagnosis and each fd , pa , and dd was analyzed using spearman 's correlation coefficient .", "discriminant formulas were obtained using discriminant analysis with the treatment plan ( preventive / operative ) as the objective variable and fd , pa , and dd as explanatory variables .", "sensitivity and specificity were calculated by applying fd , pa , and dd to each discriminant formula .", "the accuracy , ratio of the number of teeth showing accordance between the treatment plan decided by the dentists and the predictive treatment plan decided using the discriminant formula to the number of all the teeth , was also obtained .", "the entire process was approved by the ethics committee of the faculty of dentistry , tokyo medical and dental university ( no . 317 ) .", "fd , pa , and dd values corresponding to each clinical diagnosis are shown in table 1 .", "the two - way anova revealed that the fd , pa , and dd were different among the clinical diagnosis ( p < .01 ) .", "on the other hand , the difference of the examining dentists did not affect the fd , pa , and dd .", "spearman 's correlation coefficients between the clinical diagnosis and each fd , pa , and dd were 0.743 , 0.700 , and 0.652 , respectively ( p < .01 ) .", "there were also significant correlations among fd , pa , and dd ( p < .01 ) . \n", "table 2 shows the discriminant formula , sensitivity , specificity , and accuracy of each explanatory variable .", "based on the discriminant formula of each explanatory variable , the thresholds of fd , pa , and dd between preventive and operative treatments were 1.20 , 0.012 , and 28.8 , respectively .", "the sensitivity of fd was greater than that of pa , dd , and the combination of fd and pa .", "the specificity of pa was greater than that of fd , dd , and the combination of fd and pa .", "the accuracy of the combination of fd and pa was greater than that of fd , pa , and dd .", "previously , it was reported that the fractal dimension and the proportion of the area of pit - and - fissure discoloration to the area of occlusal surface were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth . in this study ,", "the fractal dimensions for c0 , c1 and c2 in the former study were 0.97 , 1.30 , and 1.52 , respectively .", "these results indicate that image analysis of molar pit - and - fissure discoloration was clinically useful for the diagnosis of caries .", "an increase of the proportion of the area of discoloration corresponded to a change of the volume of caries lesion , while an increase of the fractal dimension corresponded to a change of the shape of the lesion caused by caries progression .  ", "a fractal is a geometric shape , possessing characteristics of self - similarity or self - affinity , and widely observed in nature [ 10 , 11 ] .", "recently , fractals have been in the spotlight in the field of medicine , and research has been introduced regarding its use in the field of diagnosis [ 1214 ] .", "for example , a point is described as the zero dimension ; a straight line is described as the first dimension , and a plane is described as the second dimension .", "such decimal dimensions can be obtained by expanding the definition of the dimension as the rate at which the perimeter ( or the surface area ) of an object increases , and the measurement scale is reduced . several ways to measure the fractal dimension have been introduced . in this study", ", the authors used a simple way to determine the fractal dimension called box counting . in this method ,", "a grid of squares is placed over the object , and the number of squares through which any part of the object passes is counted .", "this process is repeated with different grids having different sizes . the number of squares placed over the object versus the length of the side of the square are then plotted on log - log scale . when a regression line is obtained from the plots ,", "the degree of uneven complexity of a boundary or a coast can be quantified using this approach . in this research ,", "the fractal dimension of discoloration increased from 0.8 to 1.6 as the depth of the caries increased , which corresponded to a change in the shape of the discolored area from a point or a line to an area based on the progression of the caries .  ", "generally , the addition of valuables into discriminant formula is one of the ways to improve the accuracy , however , in this study , the accuracy of the combination of fd and pa was similar to that of single fd .", "therefore , further study to find other valuables is needed to improve the accuracy of this method by combination of valuables .", "thus , the accuracy of the diagnosis of occlusal caries using digital images of discolored areas was comparable to that of diagnodent ; therefore , its clinical application as a diagnostic tool is possible .  ", "because this study was clinical , the final diagnosis of each examined tooth was not confirmed by a histological procedure but by a dentist 's clinical examination . as mentioned above ,", ", the results of two - way anova showed that the effect of the examining dentist on the dd , pa , and fd values was not significant .", "therefore , we considered that difference of the diagnosis among the dentists would be small .   in the present study , to examine the possibility of digital imaging in the diagnosis of occlusal caries , diagnodent was used as a comparative pre - existing dental caries detection tool .", "there are other caries detection tools , such as fiber optic transillumination ( foti ) , digital imaging fiber optic transillumination ( difoti ) , quantitative laser or light fluorescence ( qlf ) , and electrical conductive measurements ( ecm ) .", "foti , difoti , and qlf have been tested in vivo , however , the number of clinical studies has still been small [ 15 , 16 ] . on the other hand ,", "a comparatively long time has passed since diagnodent was introduced to the market , and many findings have been reported .", "sheehy et al .   reported that diagnodent had greater sensitivity and specificity than ecm .", "the correlation coefficient between diagnodent readings and the depth and the volume of caries lesions was reported to be 0.47 .", "several researches have pointed out that diagnodent measurements are affected by other factors , such as hypomineralization , plaque , debris , staining and wetness [ 1921 ] , while a high correlation between inter and intraobserver agreements was also mentioned [ 22 , 23 ] .", "we employed diagnodent as a comparison because the diagnosis was provided as a number , the handling was easy , and , moreover , its clinical use has been discussed in other studies .   in the present study ,", "diagnosis using digital images of pit - and - fissure discoloration depended on the statistical relationship between the shape of discoloration and the depth of caries .", "the shape of the discolored area in the occlusal surface is , however , possibly affected by medication history , individual history , and lifestyle .", "consequently , diagnosis using digital images of pit - and - fissure discoloration is rather experimental .", "additionally , the procedure can not be applied to colorless lesions , such as acute caries .", "therefore , diagnosis using digital images of pit - and - fissure discoloration should not be used for definitive diagnosis .", "rather , initial diagnosis , screening such as mass examination would be suitable because of its good sensitivity of fd and , the convenience of the procedure . actually , the core of the diagnostic system is digital imaging processing by computers .", "if computer programing for all procedures was achieved , then screening of hundreds of examinees would be automated after photograph taking , which would make mass examination less time - consuming with low cost . for such automated uses of the computer", ", the process of extracting colors from the image must be improved ; in this study , the threshold for the colored area was decided one by one by an observer 's visual inspection . how to calculate the fractal dimension is also open to discussion .", "we used the box - counting method attached in the image analysis software , image j. the box - counting method is only suitable for self - similar profiles , not for more general , self - affine cases , that is , the fractal dimension using the box - counting method might be the approximate value .", "a special computer program must be developed to measure the fractal dimension more accurately , for example , the minkowski method or the richardson method [ 8 , 10 ] .", "as mentioned above , the shape of the discolored area on an occlusal surface is affected by many factors .", "therefore , the thresholds or the discriminant formulas acquired from this research are not universal .", "further research is needed to determine the discriminant formulas to diagnose caries using the image analysis of molar pit - and - fissure discoloration ."], "abstract_text": ["<S> the aim of this study was to assess the possibility of digital image analysis of pit - and - fissure discoloration in order to diagnose caries . </S>", "<S> digital images showing pit - and - fissure discoloration in 100 teeth of 19 patients were analyzed to obtain the fractal dimension ( fd ) and the proportion of the area of pit - and - fissure discoloration to the area of occlusal surface ( pa ) . </S>", "<S> diagnodent values were measured ( dd ) , and dentists ' diagnoses were also obtained . the sensitivity and specificity of fd , pa , dd , and the combination of fd and pa compared to the dentists ' diagnoses were calculated . </S>", "<S> the sensitivities of fd , pa , dd , and the combination of fd and pa were 0.89 , 0.47 , 0.69 , and 0.86 , respectively , and the specificities were 0.84 , 0.95 , 0.91 , and 0.86 , respectively . </S>", "<S> although further research is needed for the practical use , it is possible to use the analysis of digital images of pit - and - fissure molar discoloration as a diagnostic tool . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["in recent years , the concept of minimal intervention ( mi ) has prevailed in dentistry .", "therefore , the importance of diagnosing caries at an early stage has increased . in conventional procedures ,", "the diagnosis of caries has mainly consisted of visual inspection and tactile assessment with probing .", "however , lussi   reported that the sensitivity of detecting caries was 0.62 by visual inspection and 0.82 by probing .", "in addition , the pressure of probing can damage the demineralized fissure and increase the risk that caries progress [ 3 , 4 ] . to promote mi , diagnosis without a probe has been recommended .", "the laser fluorescence - based caries detection device diagnodent ( kavo , germany ) has been introduced as an alternative . however , no single detection method for caries is sufficient ; therefore , the combination of some detection methods has been recommended [ 57 ] .", "recent improvements in the personal computer have made the process of digital imaging more efficient and convenient .", "if the shape of caries can be quantified , and the relationship between the numerical value and the condition of the lesion can be demonstrated , this information would be helpful to diagnose dental caries .", "the authors of this paper have previously shown that the fractal dimension and proportion of the area of pit - and - fissure discoloration to the area of occlusal surface obtained by digital imaging were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth .", "for assessment of the method as a diagnostic system , the ability of the diagnosis , such as the sensitivity , the specificity , and the accuracy , should be researched in clinical situation .", "the aim of this study was to assess the possibility of the clinical application of the diagnosis of occlusal caries using digital imaging by examining the sensitivity , the specificity , and the accuracy in comparison with the diagnodent values and the dentists ' diagnoses ."], ["one hundred teeth ( 36 premolars and 64 molars ) with pit - and - fissure discoloration from 19 outpatients were examined at the clinic of oral diagnosis and general dentistry , dental hospital , tokyo medical and dental university .", "the occlusal surface of each tooth was washed with the robinson brush to remove dental plaque without any abrasive paste .", "then , pit - and - fissure discoloration was dried by air and measured three times using diagnodent .", "the mean scores were used as the diagnodent values of the teeth ( dd ) .", "next , the occlusal surface of each tooth was photographed as large as possible with an intraoral digital camera ( penscope , morita , japan ) .", "each image was stored in a personal computer using a video capture interface ( pc - mdvd / u2 , buffalo , japan ) . without knowing the dd ,", "a dentist preliminarily diagnosed each tooth using visual inspection and tactile examination to decide which treatment plan would be appropriate ( preventive or operative ) .", "the clinical diagnosis of preventive treatment for teeth was classified as co. on the other hand , carious lesions requiring operative treatment were removed in a conventional clinical way . if the resulting cavity preparation was limited in the enamel , then the clinical diagnosis was classified as c1 . if the resulting cavity preparation reached the dentin , and sound tissue still remained between the cavity and the pulp chamber , then the clinical diagnosis was classified as c2 .", "five dentists ranging from 3 to 15 years of professional experience examined the teeth after calibration of the criteria of the caries assessment conducted before the study ; the calibration was done as follows ; first , the five dentists examined 30 extracted teeth and decide which treatment plan would be appropriate ( preventive or operative ) . at that time", "then , the teeth were sliced pallarel to the teeth axis and to the depth of lesion for each tooth was determined .", "at last , the dentists discussed to accord the treatment planning for each tooth referring the depth of the lesion .  ", "the digital photographs obtained were processed and analyzed using image analysis software ( image j , nih , usa ) .", "first , each image was converted to an 8-bit gray - scale image , in which the density of grayness of each pixel was linearly scaled from min 0 ( black ) to max 255 ( white ) .", "then , the occlusal surface in the image was isolated from the background using a density histogram of the image , and the area was measured .", "pit - and - fissure discoloration was also isolated from the occlusal surface using the density histogram , and the area was measured .", "the proportion of the area of pit - and - fissure discoloration to the area of the occlusal surface was calculated ( pa ) .", "next , the image of the isolated pit - and - fissure discoloration was converted into a binary image , in which the density of pit - and - fissure discoloration was 0 , and its background was 255 , followed by calculating the fractal dimension of pit - and - fissure discoloration ( fd ) .  ", "differences in fd , pa , and dd between each clinical diagnosis were analyzed using two - way anova and games - howell test to reveal the clinical diagnosis and the effect of the examining dentists .", "the correlation between the clinical diagnosis and each fd , pa , and dd was analyzed using spearman 's correlation coefficient .", "discriminant formulas were obtained using discriminant analysis with the treatment plan ( preventive / operative ) as the objective variable and fd , pa , and dd as explanatory variables .", "sensitivity and specificity were calculated by applying fd , pa , and dd to each discriminant formula .", "the accuracy , ratio of the number of teeth showing accordance between the treatment plan decided by the dentists and the predictive treatment plan decided using the discriminant formula to the number of all the teeth , was also obtained .", "the entire process was approved by the ethics committee of the faculty of dentistry , tokyo medical and dental university ( no . 317 ) ."], ["fd , pa , and dd values corresponding to each clinical diagnosis are shown in table 1 .", "the two - way anova revealed that the fd , pa , and dd were different among the clinical diagnosis ( p < .01 ) .", "on the other hand , the difference of the examining dentists did not affect the fd , pa , and dd .", "spearman 's correlation coefficients between the clinical diagnosis and each fd , pa , and dd were 0.743 , 0.700 , and 0.652 , respectively ( p < .01 ) .", "there were also significant correlations among fd , pa , and dd ( p < .01 ) . \n", "table 2 shows the discriminant formula , sensitivity , specificity , and accuracy of each explanatory variable .", "based on the discriminant formula of each explanatory variable , the thresholds of fd , pa , and dd between preventive and operative treatments were 1.20 , 0.012 , and 28.8 , respectively .", "the sensitivity of fd was greater than that of pa , dd , and the combination of fd and pa .", "the specificity of pa was greater than that of fd , dd , and the combination of fd and pa .", "the accuracy of the combination of fd and pa was greater than that of fd , pa , and dd ."], ["previously , it was reported that the fractal dimension and the proportion of the area of pit - and - fissure discoloration to the area of occlusal surface were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth . in this study ,", "the fractal dimensions for c0 , c1 and c2 in the former study were 0.97 , 1.30 , and 1.52 , respectively .", "these results indicate that image analysis of molar pit - and - fissure discoloration was clinically useful for the diagnosis of caries .", "an increase of the proportion of the area of discoloration corresponded to a change of the volume of caries lesion , while an increase of the fractal dimension corresponded to a change of the shape of the lesion caused by caries progression .  ", "a fractal is a geometric shape , possessing characteristics of self - similarity or self - affinity , and widely observed in nature [ 10 , 11 ] .", "recently , fractals have been in the spotlight in the field of medicine , and research has been introduced regarding its use in the field of diagnosis [ 1214 ] .", "for example , a point is described as the zero dimension ; a straight line is described as the first dimension , and a plane is described as the second dimension .", "such decimal dimensions can be obtained by expanding the definition of the dimension as the rate at which the perimeter ( or the surface area ) of an object increases , and the measurement scale is reduced . several ways to measure the fractal dimension have been introduced . in this study", ", the authors used a simple way to determine the fractal dimension called box counting . in this method ,", "a grid of squares is placed over the object , and the number of squares through which any part of the object passes is counted .", "this process is repeated with different grids having different sizes . the number of squares placed over the object versus the length of the side of the square are then plotted on log - log scale . when a regression line is obtained from the plots ,", "the degree of uneven complexity of a boundary or a coast can be quantified using this approach . in this research ,", "the fractal dimension of discoloration increased from 0.8 to 1.6 as the depth of the caries increased , which corresponded to a change in the shape of the discolored area from a point or a line to an area based on the progression of the caries .  ", "generally , the addition of valuables into discriminant formula is one of the ways to improve the accuracy , however , in this study , the accuracy of the combination of fd and pa was similar to that of single fd .", "therefore , further study to find other valuables is needed to improve the accuracy of this method by combination of valuables .", "thus , the accuracy of the diagnosis of occlusal caries using digital images of discolored areas was comparable to that of diagnodent ; therefore , its clinical application as a diagnostic tool is possible .  ", "because this study was clinical , the final diagnosis of each examined tooth was not confirmed by a histological procedure but by a dentist 's clinical examination . as mentioned above ,", ", the results of two - way anova showed that the effect of the examining dentist on the dd , pa , and fd values was not significant .", "therefore , we considered that difference of the diagnosis among the dentists would be small .   in the present study , to examine the possibility of digital imaging in the diagnosis of occlusal caries , diagnodent was used as a comparative pre - existing dental caries detection tool .", "there are other caries detection tools , such as fiber optic transillumination ( foti ) , digital imaging fiber optic transillumination ( difoti ) , quantitative laser or light fluorescence ( qlf ) , and electrical conductive measurements ( ecm ) .", "foti , difoti , and qlf have been tested in vivo , however , the number of clinical studies has still been small [ 15 , 16 ] . on the other hand ,", "a comparatively long time has passed since diagnodent was introduced to the market , and many findings have been reported .", "sheehy et al .   reported that diagnodent had greater sensitivity and specificity than ecm .", "the correlation coefficient between diagnodent readings and the depth and the volume of caries lesions was reported to be 0.47 .", "several researches have pointed out that diagnodent measurements are affected by other factors , such as hypomineralization , plaque , debris , staining and wetness [ 1921 ] , while a high correlation between inter and intraobserver agreements was also mentioned [ 22 , 23 ] .", "we employed diagnodent as a comparison because the diagnosis was provided as a number , the handling was easy , and , moreover , its clinical use has been discussed in other studies .   in the present study ,", "diagnosis using digital images of pit - and - fissure discoloration depended on the statistical relationship between the shape of discoloration and the depth of caries .", "the shape of the discolored area in the occlusal surface is , however , possibly affected by medication history , individual history , and lifestyle .", "consequently , diagnosis using digital images of pit - and - fissure discoloration is rather experimental .", "additionally , the procedure can not be applied to colorless lesions , such as acute caries .", "therefore , diagnosis using digital images of pit - and - fissure discoloration should not be used for definitive diagnosis .", "rather , initial diagnosis , screening such as mass examination would be suitable because of its good sensitivity of fd and , the convenience of the procedure . actually , the core of the diagnostic system is digital imaging processing by computers .", "if computer programing for all procedures was achieved , then screening of hundreds of examinees would be automated after photograph taking , which would make mass examination less time - consuming with low cost . for such automated uses of the computer", ", the process of extracting colors from the image must be improved ; in this study , the threshold for the colored area was decided one by one by an observer 's visual inspection . how to calculate the fractal dimension is also open to discussion .", "we used the box - counting method attached in the image analysis software , image j. the box - counting method is only suitable for self - similar profiles , not for more general , self - affine cases , that is , the fractal dimension using the box - counting method might be the approximate value .", "a special computer program must be developed to measure the fractal dimension more accurately , for example , the minkowski method or the richardson method [ 8 , 10 ] .", "as mentioned above , the shape of the discolored area on an occlusal surface is affected by many factors .", "therefore , the thresholds or the discriminant formulas acquired from this research are not universal .", "further research is needed to determine the discriminant formulas to diagnose caries using the image analysis of molar pit - and - fissure discoloration ."]]}
{"article_id": "PMC5139932", "article_text": ["craniofacial defects are rare , disfiguring facial anomalies , with an incidence of about 1.4 to 4.9 cases per 100,000 live births .", "craniofacial clefts may be caused by failure of the maxillary processes to fuse , external pressure , amniotic bands , oligohydramnios , central disorganization of the neural crest , and hematomas . in 1973 ,", "the median cleft of the upper lip , or tessier 0 class , has a variety of presentations , ranging from a minimal notching of the lip , vermilion , and nose to a wide cleft that divides craniofacial structures .", "tessier 0 clefts result from failure of the two medial nasal processes to fuse at midline .", "median clefts are broadly classified into true and false ; false clefts are due to agenesis of the medial nasal process , while true clefts are due to failure of the medial nasal process to fuse .", "treatment of median clefts depends on the clinical presentation and may vary from simple alignment of orbicularis oris and vermilion , to reconstruction of the cupid 's bow and philtrum for true median clefts , and complex craniofacial procedures in case of false median clefts .", "various techniques have been described to repair mild or moderate true median cleft lips . here", "we report using a pfeifer incision to correct a moderate true median cleft of the upper lip .", "pfeifer incision includes wavy lines , which elongate the incised tissues as the waves are straightened to close in a straight line , and also provide extra tissue for a tension free closure .", "a five - year - old boy with a facial cleft presented to our clinic . on examination", ", there was a median cleft of the upper lip involving the white roll , with no bony involvements .", "the highest- ( points a , a ) , and the deepest - points ( points b , b ) of the white roll were marked on both sides .", "subsequently , a wavy incision line was made starting from the deepest point and extending over the philtrum just above the cleft ( fig .", "a diamond incision was made over the vermilion and the labial mucosa , intra - orally extending just beyond the cleft margin .", "incision was made and the mucosal tissue covering the area medial to the incision site ( sterile zone ) was removed .", "undermining of the orbicularis oris muscle was performed and it was approximated using 4 - 0 vicryl .", "the cupid 's bow was properly aligned , with equal height on both sides ( fig .", "vermilion form was satisfactory and the fullness and continuity of the orbicularis oris were maintained .", "incision marking for pfeifer technique ( a , a : highest points on white roll ; b , b : deepest points on white roll ) postoperative result after suture removal", "a median cleft can also be called midline cleft or vertical cleft through the centre of the upper lip , and is a rare anomaly , the exact developmental origin of which is not clear .", "tessier 0 clefts occur during the third week of gestation due to failure of the two medial nasal processes to close in midline .", "it can occur in isolation or be a part of a syndrome such as orofacial - digital syndrome .", "false clefts are associated with abnormalities of the forebrain and are categorized under the category of holoprosencephaly . in 1937 , veau   categorized median clefts to notch of the lip , median cleft extending to the columella , and defects due to atrophy of the midline facial structures .", "median cleft face syndrome , frontonasal dysplasia , and tessier 0 clefts are various terms used to describe abnormalities associated with true median clefts that are not accompanied by forebrain abnormalities .", "the tessier 0 anomaly may present as a small notch in the soft tissue , or in association with hypertelorism , midline craniofacial osseous defects , and hairline abnormalities .", "various treatment options are present for mild deformities which do not involve the white roll .", "when developing the treatment plan , reconstruction of the cupid 's bow , labial philtrum , vermillion , and buccal mucosa should be kept in mind .", "urata and kawamoto   described using a v - y flap , while weimer et al ,   used a diamond incision to repair these anomalies . da", "silva frietas and colleagues   described a mucosal z transposition technique to treat mild cases . for moderate defects involving the philtrum ,", "these waves are subsequently approximated in a straight line , which help expanding the length and width of the tissue .", "this incision has previously been used to correct other tessier clefts with a high success rate .", "the above - mentioned studies prove the versatility of the pfeifer incision . in our case", "we were able to achieve adequate symmetry of the philtrum and the cupid 's bow , which is important in cases like this .", "the postoperative results were excellent and proper approximation of the orbicularis oris muscle and the vermilion was achieved with a symmetrical cupid 's bow and philtrum .", "the only disadvantage of this technique is that the final closure line is placed directly over the philtrum .", "since the presentation of midline cleft deformities varies widely , each case should be individually considered and treated .", "pfeifer incisions can successfully provide mucosal length , vermilion fullness , and lip symmetry in patients with moderate median cleft lip ."], "abstract_text": ["<S> median cleft is the midline cleft of the lip . </S>", "<S> it develops due to incomplete or failed fusion of the median nasal prominence . </S>", "<S> it can present with minimal deformities such as involvement of the vermilion border , or complex clefting of the midline structures and brain . </S>", "<S> median clefts are broadly classified as true and false clefts . </S>", "<S> this case report describes a rare case of median cleft of the upper lip involving the white roll , which was not associated with any other deformities . </S>", "<S> treatment included reconstruction of the philtrum and the cupid 's bow while maintaining vermilion fullness and continuity , and minimizing scar formation . </S>", "<S> various techniques have been advocated for treatment of this type of median upper lip cleft . here </S>", "<S> we describe a technique using pfeifer incision to correct our patient 's defect . </S>", "<S> pfeifer incision consists of wavy lines and its use has been advocated for correction of various craniofacial abnormalities . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["craniofacial defects are rare , disfiguring facial anomalies , with an incidence of about 1.4 to 4.9 cases per 100,000 live births .", "craniofacial clefts may be caused by failure of the maxillary processes to fuse , external pressure , amniotic bands , oligohydramnios , central disorganization of the neural crest , and hematomas . in 1973 ,", "the median cleft of the upper lip , or tessier 0 class , has a variety of presentations , ranging from a minimal notching of the lip , vermilion , and nose to a wide cleft that divides craniofacial structures .", "tessier 0 clefts result from failure of the two medial nasal processes to fuse at midline .", "median clefts are broadly classified into true and false ; false clefts are due to agenesis of the medial nasal process , while true clefts are due to failure of the medial nasal process to fuse .", "treatment of median clefts depends on the clinical presentation and may vary from simple alignment of orbicularis oris and vermilion , to reconstruction of the cupid 's bow and philtrum for true median clefts , and complex craniofacial procedures in case of false median clefts .", "various techniques have been described to repair mild or moderate true median cleft lips . here", "we report using a pfeifer incision to correct a moderate true median cleft of the upper lip .", "pfeifer incision includes wavy lines , which elongate the incised tissues as the waves are straightened to close in a straight line , and also provide extra tissue for a tension free closure ."], ["a five - year - old boy with a facial cleft presented to our clinic . on examination", ", there was a median cleft of the upper lip involving the white roll , with no bony involvements .", "the highest- ( points a , a ) , and the deepest - points ( points b , b ) of the white roll were marked on both sides .", "subsequently , a wavy incision line was made starting from the deepest point and extending over the philtrum just above the cleft ( fig .", "a diamond incision was made over the vermilion and the labial mucosa , intra - orally extending just beyond the cleft margin .", "incision was made and the mucosal tissue covering the area medial to the incision site ( sterile zone ) was removed .", "undermining of the orbicularis oris muscle was performed and it was approximated using 4 - 0 vicryl .", "the cupid 's bow was properly aligned , with equal height on both sides ( fig .", "vermilion form was satisfactory and the fullness and continuity of the orbicularis oris were maintained .", "incision marking for pfeifer technique ( a , a : highest points on white roll ; b , b : deepest points on white roll ) postoperative result after suture removal"], ["a median cleft can also be called midline cleft or vertical cleft through the centre of the upper lip , and is a rare anomaly , the exact developmental origin of which is not clear .", "tessier 0 clefts occur during the third week of gestation due to failure of the two medial nasal processes to close in midline .", "it can occur in isolation or be a part of a syndrome such as orofacial - digital syndrome .", "false clefts are associated with abnormalities of the forebrain and are categorized under the category of holoprosencephaly . in 1937 , veau   categorized median clefts to notch of the lip , median cleft extending to the columella , and defects due to atrophy of the midline facial structures .", "median cleft face syndrome , frontonasal dysplasia , and tessier 0 clefts are various terms used to describe abnormalities associated with true median clefts that are not accompanied by forebrain abnormalities .", "the tessier 0 anomaly may present as a small notch in the soft tissue , or in association with hypertelorism , midline craniofacial osseous defects , and hairline abnormalities .", "various treatment options are present for mild deformities which do not involve the white roll .", "when developing the treatment plan , reconstruction of the cupid 's bow , labial philtrum , vermillion , and buccal mucosa should be kept in mind .", "urata and kawamoto   described using a v - y flap , while weimer et al ,   used a diamond incision to repair these anomalies . da", "silva frietas and colleagues   described a mucosal z transposition technique to treat mild cases . for moderate defects involving the philtrum ,", "these waves are subsequently approximated in a straight line , which help expanding the length and width of the tissue .", "this incision has previously been used to correct other tessier clefts with a high success rate .", "the above - mentioned studies prove the versatility of the pfeifer incision . in our case", "we were able to achieve adequate symmetry of the philtrum and the cupid 's bow , which is important in cases like this .", "the postoperative results were excellent and proper approximation of the orbicularis oris muscle and the vermilion was achieved with a symmetrical cupid 's bow and philtrum .", "the only disadvantage of this technique is that the final closure line is placed directly over the philtrum ."], ["since the presentation of midline cleft deformities varies widely , each case should be individually considered and treated .", "pfeifer incisions can successfully provide mucosal length , vermilion fullness , and lip symmetry in patients with moderate median cleft lip ."]]}
{"article_id": "PMC4600933", "article_text": ["pseudomonas aeruginosa is an important gram - negative opportunistic pathogen that has the capability to create variable clinical infections , including , but not limited to , wound infections , urinary tract infections ( utis ) , and blood stream infections ( bsi ) , particularly in hospitalized and immunocompromised patients ( 1 , 2 ) .", "pseudomonas aeruginosa was the second most common cause of hospital - acquired pneumonia , as well as the third and fifth common cause of hospital - acquired utis in usa and europe , respectively ( 2 - 4 ) . evidently , p. aeruginosa due to low permeability of its outer membrane in combination with efflux pump overexpression is intrinsically resistant to a variety of antibiotics ( 5 ) .", "expression of different classes of extended - spectrum -lactamas enzymes along with aminoglycoside - modifying enzymes ( aminoglycoside phosphoryltransferases , aminoglycoside acetyltransferases , and aminoglycoside nucleotidyltransferases ) , and mutation are other well - characterized mechanisms for antibiotic resistance in p. aeruginosa ( 6 , 7 ) .", "data extracted from several independent studies in iran showed that p. aeruginosa was responsible for 11%-32.4% of utis , 17.2%-32.4% of pneumonia , 36.7% of bsi , and 47% of wound infections ( 8 - 12 ) . moreover , based on previous studies performed in iran , treatment of p. aeruginosa infections is a major concern for health - care setting due to its high resistant rate to different antibiotics ( 12 , 13 ) .", "since determining bacterial isolates relatedness is essential for understanding the transmission routes , different typing methods that can be divided into two major categories , phenotypic and genotypic methods , have been established ( 14 ) .", "phenotypic methods such as serotyping , pyocin typing , and antimicrobial susceptibility typing depend on the environmental factors , therefore , have low discriminatory power ( 14 , 15 ) .", "genotypic methods have been performed by different typing techniques such as restriction fragment length polymorphism ( rflp ) , pulsed field gel electrophoresis ( pfge ) , multilocus sequence typing ( mlst ) , enterobacterial repetitive intergenic consensus - pcr ( eric - pcr ) , variable number tandem repeat ( vntr ) , dna hybridization and random amplified polymorphic dna ( rapd ) ( 16 - 21 ) .", "although mlst and pfge have more discriminatory power , they are expensive , especially in developing countries ( 22 ) .", "rapd - pcr has the advantage of being fast , reproducible , simple , and low - cost , thus , it is an appropriate approach for primary screening of epidemic strains in large number of isolates ( 23 , 24 ) .", "to our knowledge , limited data are available about genetic diversity of p. aeruginosa recovered from different clinical specimens in isfahan ; the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan , iran .", "this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period", ", a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed .", "primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) .", "the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) .", "resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) .", "the following disks ( mast , uk ) were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) .", "p. aeruginosa standard strain ( atcc 27853 ) was used as the quality control .  ", "optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume .", "dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) .", "electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h .", "we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) .", "separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) .", "rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares ,", "dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands", "we used cut - off value of  80% for determination of potential clonal relatedness ( 31 - 33 ) .", "this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period", ", a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed .", "primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) .", "the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) .", "resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) . the following disks ( mast , uk )", "were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) .", "optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume .", "dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) .", "electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h .", "we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) .", "separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) .", "rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares ,", "dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands", "we used cut - off value of  80% for determination of potential clonal relatedness ( 31 - 33 ) .", "out of 150 samples , 54 ( 36% ) were positive for p. aeruginosa culture .", "distribution of the 54 isolates were as follow , urine 39 ( 72.2 % ) , sputum 7 ( 13% ) , wound 5 ( 9.3% ) , blood 2 ( 3.7% ) , and eye discharge 1 ( 1.9% ) .", "the most frequent involved wards were icu 28 ( 51.9% ) , followed by graft 12 ( 22.2% ) , internal 5 ( 9.3% ) and surgery 9 ( 16.7% ) .", "thirty - six isolates ( 66.7% ) belonged to male patients and 18 ( 33.3% ) belonged to female patients .", "the highest percentage ( 55.6% ) of resistance was observed against ceftazidime and imipenem with 30 of the isolates being resistant ; all isolates were sensitive to polymyxin b ( figure 1 ) .", "twenty - eight ( 51.8% ) isolates tested in the study revealed resistance to all applied antibiotics except for polymyxin b. electrophoresis patterns of rapd - pcr for some isolates of studied p. aeruginosa are shown in figure 2 .", "genetic relatedness of clinical isolates of the detected p. aeruginosa has been carried out using rapd - pcr assay , which showed 55% to 100% similarity ( figure 3 ) .", "the number of bands in rapd typing varied from 2 to 12 , with the length of 150 bp to 4300 bp .", "rapd fingerprinting results of p. aeruginosa based on  80% showed 39 different groups ( figure 3 ) . although the majority of isolates had unique fingerprint , group one composed of 3 isolates ( patients s48 , s29 , s31 ) .", "isolate s29 was fully sensitive to applied antibiotics , whereas isolates s31 and s48 were resistance to applied antibiotics ( figure 3 ) .", "groups 22 , 31 , 35 , and 36 , each consisted of two isolates , were also obtained from the same ward ( figure 3 ) .", "based on 65% similarity level , eight main cluster , with most of the strains belonging to cluster 1 were detected ( figure 3 ) .", "line 1 , 14 dna ladder ( 3000 - 100 bp ) , lines 2 - 13 showed 12 different rapd types .", "abbreviations : amk , amikacin ; azt , aztreonam ; caz , ceftazidime ; cip , ciprofloxacin ; imp , imipenem ; mp , meropenem ; sp , sputum ; uc , urine ; wo , wound .", "constant monitoring of antibiotic susceptibility profiles and genetic relatedness among bacterial infectious agents are essential steps for infection control ( 34 ) . to improve our understanding about p. aeruginosa antibiotic resistance and their distribution , 54 non - duplicate isolates of p. aeruginosa by using kirby - bauer s disk diffusion and rapd - pcr methods", "p. aeruginosa involved in this study were highly resistant to different antibiotic families like , monobactams , cephalosporins , quinolones , and carbapenems ( figure 1 ) .", "our isolates had the highest resistance rate to imipenem and meropenem ( 55.6% ) , which is higher than similar reports from iran ( 11 - 13 ) .", "in addition , increased percentage of resistance to ciprofloxacin and amikacin ( 53.7% and 48.1% , respectively ) were observed in comparison with previous study ( 29% and 17% ) that have been performed in the same hospital ( 35 ) .", "although , in our investigated hospital , carbapenems ( imipenem , meropenem ) along with amikacin are the most currently used antibiotics for the treatment of p. aeruginosa infections , results of this study revealed that prescription of them should be limited . in order to optimize treatment of p. aeruginosa infections in our hospital (", "also it can be mentioned as a limitation of this study ) , determination of resistance to other antibiotics like piperacillin , piperacillin - clavulanic acid , ticarcillin , ticarcillin - clavulanic acid , gentamicin , and cefepime , are essential to be evaluated in future studies .", "the finding of present study corresponds with the data presented by haeili and associates , which showed the lowest percentage of resistance ( 0% ) to polymyxin b ( 11 ) .", "there are reports that pfge and mlst are more reliable methods for establishing clonal relatedness among p. aeruginosa strains , but because of the high cost , their utilization is limited , especially in developing countries ( 36 , 37 ) . allegedly , rapd typing is a valuable and useful method for the evaluation of genetic diversity among p. aeruginosa isolates ( 21 , 23 , 24 ) . out of various primers applied for rapd - pcr ,", "primer 272 was reliable because of its higher discriminatory power and reproducible profiles ( 38 , 39 ) . in the present study , all 54 isolates were typeable by rapd - pcr ; moreover , rapd typing allowed us to reveal 54 unique finger prints among 54 clinical isolates of p. aeruginosa .", "previous studies performed in iran showed different distribution of genotypes . for instance , in the study of salimi et al .", ", by using rapd typing , only 8 different groups among 129 isolates of p. aeruginosa were reported ( 40 ) . in addition , in another study conducted by nanvazadeh et al .", "9 groups among 50 clinical samples of p. aeruginosa were observed ( 41 ) . using limited source for recovering p. aeruginosa", "although in a previous study the correlation between rapd type and p. aeruginosa infection was suggested , our finding is not in agreement with these data ( 42 ) .", "furthermore , our data demonstrated a weak correlation between rapd and antibiotic resistance profiles ; it is supported by the results of other studies ( 34 , 43 ) . in a number of cases ,", "the reason for this diversity may be attributed to the involved different antibiotic resistance mechanisms ( 5 - 7 ) .", "moreover ; results of the independent studies showed that determination of antibiotic resistance pattern as phenotypic method for p. aeruginosa typing has low discriminatory power ( 34 , 43 ) . according to our data ,", "the majority of isolates probably originated from host itself , but cross infection of p. aeruginosa is possible to occur in studied hospital .", "for instance , isolates s48 , s29 , and s31 were recovered from urine of the patients in different wards or isolates 26 and 33 from the same ward and sample had genetic similarity ( based on rapd fingerprinting results ) . before taking any decision", ", we think that clonal relatedness of obtained isolates should be confirmed by more discriminatory methods , such as pfge or mlst . in conclusion ,", "prescription of common antibiotics ( imipenem and amikacin ) in our hospital due to high resistance rate must be restricted . according to our data , considerable genetic diversity exists among isolated p. aeruginosa", "; it is possible that different sources of p. aeruginosa be involved in our hospital that can lead to host colonization . in order to accurately control the infection , other p. aeruginosa isolated from hospital environment"], "abstract_text": ["<S> background : pseudomonas aeruginosa is a well - known opportunistic pathogen , which affects hospitalized patients in different wards due to its natural resistance to drugs.objectives:the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan , iran.materials and methods : out of 150 analyzed samples , 54 p. aeruginosa isolates were recovered and were subjected to antibiotic resistance patterns and genetic diversity determination by kirby - bauer s disk diffusion method and rapd - pcr , respectively.results:the highest percentage of resistance was observed against ceftazidime and imipenem with 30 ( 55.6% ) isolates ; meanwhile all isolates were sensitive to polymyxin b. twenty - eight ( 51.8% ) isolates revealed resistance to all applied antibiotics . </S>", "<S> rapd - pcr ( random amplified polymorphic dna- polymerase chain reaction ) results showed 54 unique genotypes , which were divided into 39 clusters.conclusions:although different source of p. aeruginosa may involve in patient colonization , genetically related strains were isolated from different wards and or the same ward of the hospital . </S>", "<S> our results pointed to the restriction of currently used antibiotics in studied hospital . </S>", "<S> we hope that our results cast light on the control and transmission of the infection in the investigated hospital . </S>"], "labels": null, "section_names": ["1. Background", "2. Objectives", "3. Materials and Methods", "3.1. Pseudomonas aeruginosa Isolation and Identification", "3.2. Antibiotic Susceptibility Test", "3.3. Random Amplified Polymorphic DNA-Genotyping", "4. Results", "5. Discussion"], "sections": [["pseudomonas aeruginosa is an important gram - negative opportunistic pathogen that has the capability to create variable clinical infections , including , but not limited to , wound infections , urinary tract infections ( utis ) , and blood stream infections ( bsi ) , particularly in hospitalized and immunocompromised patients ( 1 , 2 ) .", "pseudomonas aeruginosa was the second most common cause of hospital - acquired pneumonia , as well as the third and fifth common cause of hospital - acquired utis in usa and europe , respectively ( 2 - 4 ) . evidently , p. aeruginosa due to low permeability of its outer membrane in combination with efflux pump overexpression is intrinsically resistant to a variety of antibiotics ( 5 ) .", "expression of different classes of extended - spectrum -lactamas enzymes along with aminoglycoside - modifying enzymes ( aminoglycoside phosphoryltransferases , aminoglycoside acetyltransferases , and aminoglycoside nucleotidyltransferases ) , and mutation are other well - characterized mechanisms for antibiotic resistance in p. aeruginosa ( 6 , 7 ) .", "data extracted from several independent studies in iran showed that p. aeruginosa was responsible for 11%-32.4% of utis , 17.2%-32.4% of pneumonia , 36.7% of bsi , and 47% of wound infections ( 8 - 12 ) . moreover , based on previous studies performed in iran , treatment of p. aeruginosa infections is a major concern for health - care setting due to its high resistant rate to different antibiotics ( 12 , 13 ) .", "since determining bacterial isolates relatedness is essential for understanding the transmission routes , different typing methods that can be divided into two major categories , phenotypic and genotypic methods , have been established ( 14 ) .", "phenotypic methods such as serotyping , pyocin typing , and antimicrobial susceptibility typing depend on the environmental factors , therefore , have low discriminatory power ( 14 , 15 ) .", "genotypic methods have been performed by different typing techniques such as restriction fragment length polymorphism ( rflp ) , pulsed field gel electrophoresis ( pfge ) , multilocus sequence typing ( mlst ) , enterobacterial repetitive intergenic consensus - pcr ( eric - pcr ) , variable number tandem repeat ( vntr ) , dna hybridization and random amplified polymorphic dna ( rapd ) ( 16 - 21 ) .", "although mlst and pfge have more discriminatory power , they are expensive , especially in developing countries ( 22 ) .", "rapd - pcr has the advantage of being fast , reproducible , simple , and low - cost , thus , it is an appropriate approach for primary screening of epidemic strains in large number of isolates ( 23 , 24 ) ."], ["to our knowledge , limited data are available about genetic diversity of p. aeruginosa recovered from different clinical specimens in isfahan ; the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan , iran ."], ["this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period", ", a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed .", "primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) .", "the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) .", "resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) .", "the following disks ( mast , uk ) were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) .", "p. aeruginosa standard strain ( atcc 27853 ) was used as the quality control .  ", "optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume .", "dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) .", "electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h .", "we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) .", "separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) .", "rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares ,", "dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands", "we used cut - off value of  80% for determination of potential clonal relatedness ( 31 - 33 ) ."], ["this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period", ", a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed .", "primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) .", "the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) ."], ["resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) . the following disks ( mast , uk )", "were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) ."], ["optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume .", "dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) .", "electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h .", "we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) .", "separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) .", "rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares ,", "dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands", "we used cut - off value of  80% for determination of potential clonal relatedness ( 31 - 33 ) ."], ["out of 150 samples , 54 ( 36% ) were positive for p. aeruginosa culture .", "distribution of the 54 isolates were as follow , urine 39 ( 72.2 % ) , sputum 7 ( 13% ) , wound 5 ( 9.3% ) , blood 2 ( 3.7% ) , and eye discharge 1 ( 1.9% ) .", "the most frequent involved wards were icu 28 ( 51.9% ) , followed by graft 12 ( 22.2% ) , internal 5 ( 9.3% ) and surgery 9 ( 16.7% ) .", "thirty - six isolates ( 66.7% ) belonged to male patients and 18 ( 33.3% ) belonged to female patients .", "the highest percentage ( 55.6% ) of resistance was observed against ceftazidime and imipenem with 30 of the isolates being resistant ; all isolates were sensitive to polymyxin b ( figure 1 ) .", "twenty - eight ( 51.8% ) isolates tested in the study revealed resistance to all applied antibiotics except for polymyxin b. electrophoresis patterns of rapd - pcr for some isolates of studied p. aeruginosa are shown in figure 2 .", "genetic relatedness of clinical isolates of the detected p. aeruginosa has been carried out using rapd - pcr assay , which showed 55% to 100% similarity ( figure 3 ) .", "the number of bands in rapd typing varied from 2 to 12 , with the length of 150 bp to 4300 bp .", "rapd fingerprinting results of p. aeruginosa based on  80% showed 39 different groups ( figure 3 ) . although the majority of isolates had unique fingerprint , group one composed of 3 isolates ( patients s48 , s29 , s31 ) .", "isolate s29 was fully sensitive to applied antibiotics , whereas isolates s31 and s48 were resistance to applied antibiotics ( figure 3 ) .", "groups 22 , 31 , 35 , and 36 , each consisted of two isolates , were also obtained from the same ward ( figure 3 ) .", "based on 65% similarity level , eight main cluster , with most of the strains belonging to cluster 1 were detected ( figure 3 ) .", "line 1 , 14 dna ladder ( 3000 - 100 bp ) , lines 2 - 13 showed 12 different rapd types .", "abbreviations : amk , amikacin ; azt , aztreonam ; caz , ceftazidime ; cip , ciprofloxacin ; imp , imipenem ; mp , meropenem ; sp , sputum ; uc , urine ; wo , wound ."], ["constant monitoring of antibiotic susceptibility profiles and genetic relatedness among bacterial infectious agents are essential steps for infection control ( 34 ) . to improve our understanding about p. aeruginosa antibiotic resistance and their distribution , 54 non - duplicate isolates of p. aeruginosa by using kirby - bauer s disk diffusion and rapd - pcr methods", "p. aeruginosa involved in this study were highly resistant to different antibiotic families like , monobactams , cephalosporins , quinolones , and carbapenems ( figure 1 ) .", "our isolates had the highest resistance rate to imipenem and meropenem ( 55.6% ) , which is higher than similar reports from iran ( 11 - 13 ) .", "in addition , increased percentage of resistance to ciprofloxacin and amikacin ( 53.7% and 48.1% , respectively ) were observed in comparison with previous study ( 29% and 17% ) that have been performed in the same hospital ( 35 ) .", "although , in our investigated hospital , carbapenems ( imipenem , meropenem ) along with amikacin are the most currently used antibiotics for the treatment of p. aeruginosa infections , results of this study revealed that prescription of them should be limited . in order to optimize treatment of p. aeruginosa infections in our hospital (", "also it can be mentioned as a limitation of this study ) , determination of resistance to other antibiotics like piperacillin , piperacillin - clavulanic acid , ticarcillin , ticarcillin - clavulanic acid , gentamicin , and cefepime , are essential to be evaluated in future studies .", "the finding of present study corresponds with the data presented by haeili and associates , which showed the lowest percentage of resistance ( 0% ) to polymyxin b ( 11 ) .", "there are reports that pfge and mlst are more reliable methods for establishing clonal relatedness among p. aeruginosa strains , but because of the high cost , their utilization is limited , especially in developing countries ( 36 , 37 ) . allegedly , rapd typing is a valuable and useful method for the evaluation of genetic diversity among p. aeruginosa isolates ( 21 , 23 , 24 ) . out of various primers applied for rapd - pcr ,", "primer 272 was reliable because of its higher discriminatory power and reproducible profiles ( 38 , 39 ) . in the present study , all 54 isolates were typeable by rapd - pcr ; moreover , rapd typing allowed us to reveal 54 unique finger prints among 54 clinical isolates of p. aeruginosa .", "previous studies performed in iran showed different distribution of genotypes . for instance , in the study of salimi et al .", ", by using rapd typing , only 8 different groups among 129 isolates of p. aeruginosa were reported ( 40 ) . in addition , in another study conducted by nanvazadeh et al .", "9 groups among 50 clinical samples of p. aeruginosa were observed ( 41 ) . using limited source for recovering p. aeruginosa", "although in a previous study the correlation between rapd type and p. aeruginosa infection was suggested , our finding is not in agreement with these data ( 42 ) .", "furthermore , our data demonstrated a weak correlation between rapd and antibiotic resistance profiles ; it is supported by the results of other studies ( 34 , 43 ) . in a number of cases ,", "the reason for this diversity may be attributed to the involved different antibiotic resistance mechanisms ( 5 - 7 ) .", "moreover ; results of the independent studies showed that determination of antibiotic resistance pattern as phenotypic method for p. aeruginosa typing has low discriminatory power ( 34 , 43 ) . according to our data ,", "the majority of isolates probably originated from host itself , but cross infection of p. aeruginosa is possible to occur in studied hospital .", "for instance , isolates s48 , s29 , and s31 were recovered from urine of the patients in different wards or isolates 26 and 33 from the same ward and sample had genetic similarity ( based on rapd fingerprinting results ) . before taking any decision", ", we think that clonal relatedness of obtained isolates should be confirmed by more discriminatory methods , such as pfge or mlst . in conclusion ,", "prescription of common antibiotics ( imipenem and amikacin ) in our hospital due to high resistance rate must be restricted . according to our data , considerable genetic diversity exists among isolated p. aeruginosa", "; it is possible that different sources of p. aeruginosa be involved in our hospital that can lead to host colonization . in order to accurately control the infection , other p. aeruginosa isolated from hospital environment"]]}
{"article_id": "PMC4144761", "article_text": ["polyarginine peptides \n are known as one of the widely used classes \n of cell - penetrating peptides ( cpps ) and cellular delivery tools .", "it has been reported that the presence of the \n guanidine group in the side chain of arginine plays a key role for \n improved ability of arginine - rich peptides to cross the cell membrane .", "various systematic structural investigations have been performed \n to determine the required number of arginine residues and the length \n of the peptide for the optimization of cellular uptake .", "short polyarginine peptides containing less than six arginine residues \n did not exhibit significant cellular uptake in several previously \n reported investigations .", "thus , the presence of more than six arginine residues in the structure \n of polyarginine peptides is critical for their efficient cell - penetrating \n functions .", "however , several investigations were conducted to \n increase the \n cellular uptake of polyarginines by attaching the fatty acid to the n - terminal of the peptide .", "it has been previously reported \n that the acylation of the n - terminal by fatty acids \n can facilitate the intracellular uptake of polyarginines .", "for instance , katayama et al . synthesized acylated octa - arginines \n and discovered that the introduction of hydrophobic fatty acid enhanced \n the intracellular uptake of octa - arginine peptide and its conjugated \n ubiquitin .", "furthermore , lee et al . designed \n a class of lipopeptides carrying 715 arginine residues . among \n them ,", "myristoylated - hendeca - arginine ( c14r11 ) was found to be the most efficient cell - penetrating peptide . however , the fatty acylated polyarginine peptides \n that contain 715 arginine residues can potentially cause toxicity , \n and they can be easily degraded by proteases . moreover , linear \n peptides carrying l - form are not stable \n in serum and therefore have a limited application for in vivo studies ( figure s2 , supporting information ) . replacing l - form amino acids \n with d - form to improve the peptide stability leads to high \n cost production .", "thus , the synthesis and development of cyclic \n cpps containing short amino acid sequence with less toxicity is desired . herein , we designed acylated cyclic polyarginine peptides ( acpps ) \n containing five arginine residues and investigated their ability as \n cell - penetrating peptides .", "we compared acpps with a corresponding \n acylated linear polyarginine peptide ( alpp ) and a nonacylated cyclic \n polyarginine as controls .", "we hypothesize that the combination of acylation \n and cyclization of short polyarginine peptides having less than six \n arginine residues will increase the intracellular uptake and can generate \n peptides with molecular transporter properties . for convenience , square \n brackets [ ] and parentheses ( )", "phosphopeptide ptyr - glu - glu - ile ( pyeei ) \n is an optimal peptide ligand for binding to the src tyrosine kinase \n sh2 domain . in this study , we used negatively charged fluorescein - labeled \n phosphopeptide f-gpyeei as a model cell - impermeable compound .", "all peptides were synthesized by \n fmoc / tbu solid - phase peptide synthesis strategy either \n manually or using rainin ps3 synthesizer ( protein technologies inc . ) . \n", "manual reactions were carried out in a glass reaction vessel with \n a sintered glass frit by mixing under nitrogen bubbling at room temperature . \n", "fmoc - l - amino acid building blocks , fatty acids , and preloaded \n h - arg(pbf)-2-chlorotrityl resin were used as starting materials .", "2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate \n ( hbtu ) , hydroxybenzotriazole ( hobt ) , and n , n - diisopropylethylamine ( dipea ) in n , n - dimethylformamide \n ( dmf ) were used as coupling and activating reagents , respectively , \n for manual synthesis . in the case of using peptide synthesizer , 0.4 \n m n - methylmorpholine in dmf was used instead of dipea . \n", "piperidine in dmf ( 20% ) was employed to deprotect fmoc group at each \n step . to cleave the linear peptide from the resin , a mixture \n of trifluoroacetic acid ( tfa)/triisoproylsilane ( tis)/water ( 92.5/5/2.5 , \n", "however , in the synthesis of cyclic peptides , the \n side chain protected linear peptides were first cleaved from the resin \n by using a cleavage cocktail , containing 2,2,2-trifluoroethanol ( tfe)/acetic \n acid / dichloromethane ( dcm ) ( 2:1:7 , v / v / v ) .", "cyclization reaction was \n performed by employing a mixture of 1-hydroxy-7-azabenzotriazole ( hoat ) \n and n , n-diisopropylcarbodiimide \n ( dic ) in anhydrous dmf / dcm for 12 h. after solvent evaporation , the \n peptide was deprotected and cleaved from the resin by using a cleavage \n cocktail reagent ", "r  , containing tfa / thioanisole/1,2-ethanedithiol \n ( edt)/anisole ( 90:5:3:2 , v / v / v / v ) for 23 h. the crude peptides \n were precipitated and washed with cold diethyl ether . to purify the \n crude peptides , we used a reversed - phase high pressure liquid chromatography \n ( rp - hplc ) system using shimadzu lc-8a preparative liquid chromatography \n on a phenomenex gemini c18 column ( 10 m , 250  21.2 mm ) \n with a gradient 0100% of acetonitrile ( ch3cn ) containing \n 0.1% tfa ( v / v ) and water containing 0.1% tfa ( v / v ) for 1 h with a \n flow rate at 15.0 ml / min at the wavelength of 214 nm . as a representative \n example , the synthesis of dodecanoyl-[r5 ] is described \n here .", "h - arg(pbf)-2-chlorotrityl resin ( 660 \n mg , 0.35 mmol , 0.53 mmol / g ) was swelled in dmf for 40 min by n2 .", "fmoc - arg(pbf)-oh ( 681 mg , 1.05 mmol , 3 equiv ) was coupled \n to the n - terminal of the resin , using hbtu ( 398 mg , \n 1.05 mmol , 3 equiv ) , hobt ( 142 mg , 1.05 mmol , 3 equiv ) , and dipea \n ( 366 l , 2.1 mmol , 6 equiv ) in dmf ( 15 ml ) by agitating the \n resin for 1 h using n2 .", "after the coupling , the resin was \n washed with dmf , followed by fmoc - deprotection with piperidine in \n dmf ( 20% ) .", "the subsequent three fmoc - arg(pbf)-oh couplings and one \n dde - lys(fmoc)-oh ( 559 mg , 1.05 mmol , 3 equiv ; dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl ) \n coupling was carried out in the same manner , respectively . after removing \n the fmoc group in side chain of lysine , dodecanoic acid (", "210 mg , 1.05 \n mmol , 3 equiv ) was coupled using hbtu , hobt , and dipea .", "then dde protection \n group at n - terminal of peptide was removed by 2% \n hydrazine in dmf , followed by washing with dmf and dcm .", "the side chain \n protected linear peptides were cleaved from the resin by using a cleavage \n cocktail , tfe / acetic acid / dcm ( 2:1:7 , v / v / v ) , for 1 h. the filtrate \n was evaporated , and the residue was dried overnight in a vacuum .", "the \n cyclization was conducted under a dilute condition with anhydrous \n dmf / dcm ( 5:3 , v / v , 250 ml ) , using hoat ( 190 mg , 1.4 mmol , 4 equiv ) \n and dic ( 240 l , 1.54 mmol , 4.4 equiv ) , and stirred for 12 h \n under nitrogen atmosphere .", "after cyclization , the solvent was evaporated , \n and the side chain deprotection was carried out by the addition of \n reagent  r  for 2 h. the crude dodecanoyl-[r5 ] was precipitated and washed with cold diethyl ether and purified \n by preparative rp - hplc system as described above .", "fluorescein - labeled \n peptides were synthesized with the same protocol \n before the attachment of fatty acid .", "we used fmoc-12-aminododecanoic \n acid instead of dodecanoic acid , and after removing fmoc group , 5(6)-carboxyfluorescein \n diisobutyrate ( cfdi ) was attached using 7-azabenzotriazol-1-yl - oxytris(pyrrolidino)phosphonium \n hexafluorophosphate ( pyaop ) , hoat , and dipea .", "as an example , we started \n fluorescein - labeled peptide synthesis in smaller scale with h - arg(pbf)-2-chlorotrityl \n resin ( 208 mg , 0.11 mmol , 0.53 mmol / g ) .", "fmoc - arg(pbf)-oh ( 214 mg , \n 0.33 mmol , 3 equiv ) , dde - lys(fmoc)-oh ( 176 mg , 0.33 mmol , 3 equiv ) , \n and fmoc-12-aminododecanoic acid ( 144 mg , 0.33 mmol , 3 equiv ) were \n used to couple each building block to the resin using hbtu ( 125 mg , \n 0.33 mmol , 3 equiv ) , hobt ( 45 mg , 0.33 mmol , 3 equiv ) , and dipea ( 115 \n l , 0.66 mmol , 6 equiv ) in dmf .", "cfdi ( 170 mg , 0.33 mmol , 3 equiv ) \n was coupled with amino group of 12-aminododecaonic chain using pyaop \n ( 172 mg , 0.33 mmol , 3 equiv ) , hoat ( 45 mg , 0.33 mmol , 3 equiv ) , and \n dipea .", "after removal of dde protecting group with 2% hydrazine in dmf \n and washing with dmf and dcm , side chain protected fluorescein linear \n peptides were cleaved from resin using tfe / acetic acid / dcm ( 2:1:7 , \n v / v / v ) .", "the molecular weights of final products were confirmed by \n an axima performance matrix - assisted laser desorption / ionization - time - of - flight \n ( maldi - tof ) mass spectrometer ( shimadzu corporation ) .", "dodecanoyl-(r6 ) : maldi - tof ( m / z ) c48h96n22o8 calcd . 1108.7781 ; found 1109.7308 [ m + h ] .", "f-dodecanoyl-(r5 ) : maldi - tof ( m / z ) c69h107n23o14 calcd . 1481.8368 ; \n found 1482.7586 [ m + h ] .", "w - dodecanoyl-[r5 ] : maldi - tof ( m / z ) c61h107n25o9 calcd .", "human ovarian adenocarcinoma \n ( sk - ov-3 ) , \n leukemia ( ccrf - cem ) , and embryonic kidney ( hek 293 t ) cells were purchased \n from american type culture collection .", "the sk - ov-3 and hek 293 t cells \n were grown in eagle s minimum essential medium ( emem ) , and \n rpmi-1640 medium ( atcc , manassas , va ) was used for ccrf - cem cells \n in a humidified atmosphere of 5% co2 at 37 c .", "both \n media were supplemented with fetal bovine serum ( fbs , 10% ) and penicillin ", "streptomycin \n solution ( 1% , 10,000 units of penicillin and 10 mg of streptomycin \n in 0.9% nacl ) .", "cytotoxicity \n of peptides was examined \n by mts proliferation assay in two human cancer cell lines ( sk - ov-3 \n and ccrf - cem ) and one human normal cell line ( hek 293 t ) .", "cells were \n seeded into 96-well plates ( sk - ov-3 ( 5  10 cells / well ) , \n ccrf - cem ( 1  10 cells /", "well ) , and hek 293 t ( 1  \n 10 cells / well ) ) .", "then , the cells were incubated with complete \n media ( 100 l ) overnight .", "different concentrations ( 0600 \n m ) of peptide solution ( 10 l ) were added to cells and \n incubated at 37 c with 5% co2 for 24 h. then , celltiter \n 96 aqueous solution ( 20 l ) was added to each well and incubated \n for 14 h. the absorbance was measured at 490 nm using microplate \n reader .", "sk - ov-3 \n cells were grown in 6-well plates ( 2  10 cells / well ) \n with complete emem media 24 h prior to the experiment .", "the fluorescein - labeled \n peptide stock solution ( 1 mm ) was prepared in water and diluted in \n gibco opti - mem i reduced serum medium to obtain the final concentration \n of 5 m .", "the media were removed , and the mixture containing \n fluorescein - labeled peptide solution ( 5 m ) was added .", "after \n 1 h incubation , trypsin  edta solution was added to detach cells \n from the plate s surface and remove cell surface binding peptides . \n", "after 5 min , the complete media ( 2 ml ) were added to neutralize the \n trypsin .", "finally , \n the cells were analyzed by bd facscalibur or facsverse flow cytometer \n using fitc channel .", "5(6)-carboxyfluorescein ( fam ) was used as a negative control . for examination \n of the cellular uptake mechanism of f-dodecanoyl-[r5 ] at low temperature", ", the assay was carried out at 4 c \n to inhibit the energy - dependent cellular uptake pathways .", "the sk - ov-3 \n cells were preincubated at 4 c for 15 min and incubated with \n the fluorescein - labeled peptide for 1 h at 4 c .", "cells were collected \n and analyzed using flow cytometry with the previously described protocol \n above .", "the data collected at 37 c were used as the control . \n to perform the atp - depletion assay ,", "cells were incubated with sodium \n azide ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) for 1 h before \n adding the fluorescein - labeled peptide . during the incubation time \n ( 1 h ) , the fluorescein - labeled bicyclic peptide ( 5 m )", "was prepared \n in the opti - mem i reduced serum medium in the presence of sodium azide \n ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) .", "then , the cells were \n incubated with this solution for 1 h. the following sample preparation \n and flow cytometry analysis protocol was the same as described above .", "sk - ov-3 cells \n were seeded with complete emem on coverslips in 6-well plate ( 1  \n 10 cells / well ) and kept until 50% confluency .", "the media \n were removed , and cells were incubated with f-dodecanoyl-[r5 ] ( 10 m ) and f-dodecanoyl-(r5 ) ( 10 \n m ) in gibco opti - mem i reduced serum medium ( life technologies , \n grand island , ny ) for 1 h at 37 c . then cells were washed with \n 1 phosphate buffered saline with calcium and magnesium ( pbs ) for three times .", "the coverslips were mounted on microscope \n slides , and images were obtained using carl zeiss lsm 700 system with \n a 488 nm argon ion laser excitation and a bp 505530 nm band - pass \n filter .", "sk - ov-3 and ccrf - cem cells were seeded in 6-well plates ( 2  \n 10 cells / well for sk - ov-3 and 1  10cells / well \n for ccrf - cem ) and grown with complete emem media ( rpmi-1640 for ccrf - cem ) \n overnight .", "a mixture of fluorescein - labeled phosphopeptides f-gpyeei \n ( 5 m ) and peptides ( 10 m ) were prepared in opti - mem \n i reduced serum medium at room temperature and incubated for 15 min . \n", "then the cells were incubated with the premixed solution at 37 c \n with 5% co2 for 1 h. the sample preparation for facs analysis \n was carried out by previously mentioned protocol described before . \n in this assay ,", "the \n acylated cyclic polyarginine peptides \n were synthesized by fmoc / tbu solid - phase peptide \n synthesis method .", "fmoc - l - arg(pbf)-oh was coupled on h - arg(pbf)-loaded \n 2-chlorotrityl resin in the presence of hbtu , hobt , and dipea in dmf . \n", "then dde - lys(fmoc)-oh was attached , and a fatty acid was coupled to \n the side chain of lysine .", "dde protecting group was removed by 2% hydrazine \n in dmf , and a cleavage cocktail containing tfe / acetic acid / dcm ( 2:1:7 \n v / v / v ) was used for 1 h to cleave the side - chain protected linear \n peptides from the resin .", "cyclization of linear peptides was carried \n out in the presence of a mixture of hoat and dic in anhydrous dmf / dcm \n for 624 h. the side - chain deprotection of cyclic peptide was \n carried out by a cleavage cocktail reagent ", "r  for 2 \n h. the crude peptides were precipitated and purified with rp - hplc \n as described above .", "as a representative example , the synthesis of \n dodecanoyl-[r5 ] is shown in scheme 1 .", "a corresponding acylated \n linear polyarginine peptide ( alpp ) was \n synthesized for comparative studies with the cyclic peptide ( acpp ) . \n", "moreover , a cyclic polyarginine without fatty acid [ r5 ] \n was also synthesized to investigate the effect of the fatty acid on \n cyclic peptide and its effect on molecular transporting efficiency . \n to investigate whether the peptide alone can enter into cells , fluorescein - labeled \n f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , where f = fluorescein , were synthesized for facs \n and microscopy investigations .", "chemical structures of synthetic peptides \n used in this study ( f , \n fluorescein - labeled ; [ ] , cyclic peptide ; ( ) , linear peptide ) .", "the cytotoxicity \n of all peptides were tested in two different cancer cell lines , adherent \n ( sk - ov-3 ) and nonadherent ( ccrf - cem ) cells , and a normal cell line \n ( hek 293 t ) using mts assay ( figure 2 ) .", "cyclic \n polyarginine [ r5 ] without fatty acid was used to explore \n the effect of n - terminal acylation on cytotoxicity \n and cellular uptake .", "alpp ( dodecanoyl-(r5 ) ) and [ r5 ] peptides showed consistently less cytotoxicity in all three \n cells compared to acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) .", "after 24 h incubation , acpps showed approximately 20% \n toxicity in cells at a concentration of 25 m in ccrf - cem cells . \n", "however , dodecanoyl - linear ( r5 ) and [ r5 ] did \n not exhibit significant cytotoxicity at 25 m and showed less \n than 20% toxicity at the concentration of 100 m . in sk - ov-3 \n cells , dodecanoyl-(r5 ) and [ r5 ] showed more \n than 80% cell viability at the concentration of 25 m .", "in normal \n cells ( hek 293 t ) , all peptides exhibited less than 5% toxicity at \n 25 m .", "this differential behavior of the peptides in normal , \n and cancer cells can be possibly rationalized through the interaction \n between polyarginine peptides and cell membranes .", "the membrane of \n cancer cells holds more negative charges compared to that in normal \n cells because of the presence of anionic lipids such as phosphatidylserine .", "therefore , polyarginine peptides can be interacted \n with cancer cells more effectively compared to normal cells .", "these data indicated \n that acpps are more toxic than alpp and a nonacylated cyclic peptide \n [ r5 ] , especially at concentration of 25 m . \n", "thus , a noncytotoxic concentration of 510 m was used \n in cell - based assays .", "comparison of cytotoxicity between cyclic and linear acylated \n polyarginine \n peptides and nonacylated cyclic peptide [ r5 ] at various \n concentrations against ccrf - cem , sk - ov-3 , and hek 293 t after 24 h. the intracellular uptake studies of fluorescein - labeled \n acylated cyclic and linear pp , f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , was carried out in sk - ov-3 \n cells by using flow cytometry and confocal laser scanning microscopy \n ( clsm ) methods .", "fluorescein ( fam , f ) alone was selected as \n a negative control . as it is shown in figure 3 ,", "the f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) showed approximately 13.7- and 10.3-fold higher cellular \n uptake than that of control 5,6-carboxyfluorescein ( fam ) , respectively , \n in sk - ov-3 cells .", "the cellular uptake of f-dodecanoyl-[r5 ] was confirmed by clsm images ( figure 4 ) . \n", "f-dodecanoyl-[r5 ] showed higher fluorescence intensity \n compared to that of f-dodecanoyl-(r5 ) in sk - ov-3 \n cells .", "therefore , acpp f-dodecanoyl-[r5 ] was found \n to be a more efficient cell - penetrating peptide compared to its linear \n counterpart .", "as shown in figure 4 , the fluorescence \n signal is extended through the whole cells , suggesting that f-dodecanoyl-[r5 ] can get localized in the nucleus as well as cytoplasm .", "comparative \n cellular uptake of f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) ( 5 m ) in sk - ov-3 \n cells ( 1 h ) .", "confocal laser scanning \n microscope image of ( a ) f-dodecanoyl-[r5 ] and ( b ) \n f-dodecanoyl-(r5 ) .", "the peptides \n were incubated for 1 h in sk - ov-3 cells at 10 m concentration .", "the mechanism \n of the cellular internalization of \n f-dodecanoyl-[r5 ] was investigated by a temperature \n control assay at 4 c along with atp depletion assay .", "these two \n assays have been widely used to examine the energy - dependent endocytosis .", "facs results showed that the intracellular uptake \n of f-dodecanoyl-[r5 ] was significantly reduced \n at 4 c , indicating that the mechanism of internalization was \n mainly dependent on the endocytosis pathways ( figure 5 ) .", "furthermore , atp depletion \n assay was performed to investigate receptor - mediated endocytosis . to induce atp depletion ,", "sk - ov-3 cells were \n preincubated with sodium azide ( 10 mm ) and 2-deoxy - d - glucose \n ( 50 mm ) for 1 h prior to the experiment , and a similar concentration \n was maintained during the incubation ( 1 h ) with f-dodecanoyl-[r5 ] .", "the results showed that the cellular uptake of f-acpp \n was inhibited in the presence of sodium azide and 2-deoxy - d - glucose , suggesting that receptor - mediated endocytosis is involved \n for the cellular uptake of f-acpp . in atp depletion assay , \n", "the basic cellular uptake of fam was higher compared to that in temperature \n control assay .", "however , this is evident that the intracellular uptake \n of f-dodecanoyl-[r5 ] was inhibited , and there was \n no significant difference between the cells treated with fam compared \n to those treated with f-dodecanoyl-[r5 ] in temperature \n control and atp depletion assays , suggesting that endocytosis is the \n major pathway for the cellular uptake of f-dodecanoyl-[r5 ] .", "cellular uptake of f-dodecanoyl-[r5 ] ( 5 m ) \n in sk - ov-3 cells in temperature control assay at 37 and 4 c , \n and atp depletion assay with nan3 ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) analyzed by flow cytometry .", "cells with no \n treatment were used as control . the ability \n of acpps as a molecular transporter was evaluated and compared by \n selecting a fluorescein - labeled phosphopeptide , f-gpyeei , \n as a molecular cargo .", "phosphopeptide , pyeei ( ptyr - glu - glu - ile ) is \n an optimal peptide template for the sh2 domain of src tyrosine kinase . \n", "several analogues of this peptide have been synthesized as potent \n ligands for this target . because of the presence of the \n negatively charged amino acid residues in the structure of the peptide \n including phosphorylated tyrosine", "moreover , the internalization of the negatively charged phosphopeptide \n in cancer cells by diffusion is more difficult because cancer cell \n membranes are composed of more negatively charged lipids .", "thus , cellular \n delivery of cell - impermeable negatively charged phosphopeptides is \n significantly challenging .", "we have previously reported different peptide - based \n carriers for the intracellular delivery of negatively charged phosphopeptides \n as model cell - impermeable drugs in several cell lines . in this study ,", "the intracellular uptake \n of f-gpyeei was monitored in the presence and absence of synthetic \n peptides after 1 h incubation by flow cytometry .", "as it is exhibited \n in figure 6 , the acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) delivered the phosphopeptide more \n efficiently compared to alpps , dodecanoyl-(r5 ) and -[r5 ] .", "the intracellular uptake of f-gpyeei in the presence \n of dodecanoyl-[r5 ] and dodecanoyl-[r6 ] was enhanced \n by 3.4- and 5.5-fold higher than the uptake in the absence of acpps . \n", "however , dodecanoyl-(r5 ) and -[r5 ] only improved \n 1.3- and 1.4-fold intracellular uptake , respectively .", "the results \n showed that acylated and cyclized polyarginine peptides can deliver \n the phosphopeptide effectively .", "however , the intracellular uptake \n of the phosphopeptide did not improve significantly in the presence \n of either acylated linear polyarginine peptide or cyclic [ r5 ] .", "these data suggest that a combination of acylation and cyclization \n would improve the molecular transporting efficiency of the polyarginine - based \n peptide ( containing less than six arginines ) for the intracellular \n delivery of a cell - impermeable phosphopeptide .", "this has been previously \n reported that the acylated linear octa - arginine increased the cellular \n uptake of molecular cargoes by just adding fatty acid to the n - terminal of octa - arginine .", "however , we discovered that both cyclization and acylation in a \n short penta - arginine can significantly improve the delivery of a cell - impermeable \n phosphopeptide in sk - ov-3 cells .", "cellular uptake of f-gpyeei ( 5 \n m ) in the presence \n of dodecanoyl-[r5 ] and -[r5 ] , dosecanoyl-(r5 ) , and dodecanoyl-[r6 ] ( 10 m ) in sk - ov-3 \n cell line .", "phosphopeptide delivery efficiency of dodecanoyl-[r5 ] were compared with known cpps ( r7 , crrrrrrr ; \n tat , ygrkkrrqrrr ) .", "the major driving forces for the intracellular delivery are \n presumed \n to be structural rigidity through cyclization of the peptide and the \n interaction of the fatty acid with the cell membrane .", "it has been \n previously reported that the cellular uptake of the peptide can be \n increased due to the structural rigidity by cyclization of arginine - rich \n peptides .", "they proposed that the maximal \n distance between guanidine groups of arginine residue can lead to \n an efficient transduction of arginine - rich peptides .", "our investigations \n showed that dodecanoyl-[r6 ] is able to deliver more efficiently \n by 1.6-fold higher f-gpyeei uptake compared to that of dodecanoyl-[r5 ] . increasing", "the number of positively charged arginine residues \n can enhance the cellular uptake through ionic interactions with the \n negatively charged phosphopeptide and/or phospholipid in the cell \n membrane through ionic interactions .", "however , the higher number of \n arginine residue is not the only responsible element for the efficient \n cellular internalization .", "for example , it has been reported that polyarginine \n containing 11 amino acids ( r11 ) showed higher cellular \n uptake compared to the polyarginine containing 13 amino acids ( r13 ) . at the same time", ", r11 was found to be a more \n potent transporter compared to r9 in prostate cancer cells .", "these investigations showed that an optimal \n number of arginine residues are required for the highest degree of \n functionality .", "however , the greater number of amino acid residues \n in cyclic peptides can decrease the structural rigidity , which lowers \n the ability of the peptide to get into cells .", "dodecanoyl-[r5 ] was also compared with several commonly \n cpps , such as cr7 and tat ( ygrkkrrqrrr ) peptides .", "the acpp \n improved the cellular uptake of the phosphopeptide by 1.4- and 1.8-fold \n higher than those of cr7 and tat , respectively ( figure 6 ) .", "these results revealed that although other peptides \n containing arginine can deliver the molecular cargo , acpp dodecanoyl-[r5 ] with a shorter peptide sequence than cr7 and \n tat can work as a molecular transporter with higher efficiency .", "chemical structures \n of acpps with different length of fatty acid \n chains ( c8 , c12 , and c16 ) . to investigate the effect \n of the chain length on the cell penetration \n potency , we synthesized octanoyl-[r5 ] , dodecanoyl-[r5 ] , and hexadecanoyl-[r5 ] ( figure 7 ) .", "acpps showed less than 20% toxicity in cells at the concentration \n of 25 m ( figure 8a ) .", "the in \n vitro toxicity results showed that increasing the fatty acid \n chain length caused enhanced toxicity in cells as hexadecanoyl-[r5 ] was more cytotoxic than dodecanoyl-[r5 ] and octanoyl-[r5 ] .", "these data indicate that the fatty acid chain length could \n alter the interaction with the cell membrane and disturb the membrane \n integrity . on the basis of the cytotoxicity data ,", "( a ) cytotoxicity \n assay of cyclic polyarginine peptide - fatty acid \n conjugates against sk - ov-3 cells ( 24 h incubation ) .", "( b ) cellular uptake \n of a phosphopeptide , f-gpyeei ( 5 m ) , in the presence \n of peptide - fatty acid conjugates ( 10 m ) in sk - ov-3 cells .", "it has been previously reported that there was a relationship \n between \n the length of fatty acid in polyarginines and their cellular uptake , meaning that the optimal length of fatty acid \n is required for optimal functionality based on the peptide sequence \n and cell type .", "the results exhibited that the cellular uptake of f-gpyeei \n was improved in the order of octanoyl-[r5 ] < dodecanoyl-[r5 ] < hexadecanoyl-[r5 ] ( figure 8b ) .", "the cellular uptake of f-gpyeei in the presence \n of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than \n that in the presence of octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively .", "these data suggest that the length of the \n attached fatty acid chain in the structure significantly influences \n the efficiency of the peptide as a molecular transporter . sixteen - carbon \n chain length ( c16 )", "was found to be an optimized length \n for the intracellular delivery of f-gpyeei in sk - ov-3 cells .", "after acpps were found to act as cpp and molecular \n transporter , a systematic investigation was performed to modify the \n fatty acid to another hydrophobic moiety .", "w4-[r5 ] , \n the whole fatty acid chain was replaced with four tryptophan residues . \n", "the second conjugate w - dodecanoyl-[r5 ] had one more tryptophan \n at the end of the dodecanoyl fatty acid chain .", "w4-[r5 ] enhanced intracellular delivery of f-gpyeei by 4.1-fold \n higher compared to that of dodecanoyl-[r5 ] in sk - ov-3 . \n however , w - dodecanoyl-[r5 ] improved the uptake of f-gpyeei \n by 1.3-fold higher compared to that of w4-[r5 ] in ccrf - cem cells ( figure 9 ) .", "thus , it is not straightforward \n to compare the cellular uptakes in sk - ov-3 and ccrf - cem .", "however , \n the results could be assessed indirectly by relative comparison with \n the cellular uptake by w4-[r5 ] .", "these data suggest \n that the presence of hydrophobic tryptophan moieties could enhance \n the molecular transporting efficiency . in other words ,", "appropriate \n modification of hydrophobic moiety in cpps can increase the drug delivery \n ability of acpp .", "thus , cyclic nature , short length of polyarginine , \n and hydrophobic segments were found to be critical elements to generate \n a peptide with an optimized molecular transporter efficiency .", "cellular uptake \n assay of a phosphopeptide , f-gpyeei ( 5 \n m ) , in the presence of w4-[r5 ] and w - dodecanoyl-[r5 ] ( 10 m ) against sk - ov-3 and ccrf - cem cell lines ( 1 \n h incubation ) .", "cells with no treatment \n were used as control . to evaluate \n whether the presence of peptides can enhance the pharmacological effect \n of a molecular cargo , the antiproliferative activity of doxorubicin \n ( dox )", "was examined in the presence of dodecanoy-[r6 ] ( [ r6]-c12 ) in mcf-7 cells in a time - dependent assay . \n as it is shown in figure 10 ,", "the antiproliferative \n potency of dox ( 5 m ) was enhanced by 7% , 11% , and 14% in the \n presence of [ r6]-c12 ( 5 m ) when compared \n with that of the drug alone after 24 , 48 , and 72 h incubation , respectively . \n", "these results showed that the cell proliferation of mcf-7 cells was \n inhibited in a time - dependent manner suggesting a sustained drug release \n by peptide leading to a higher inhibitory effect .", "the presence of \n the peptide possibly improved the cellular uptake of dox and enhanced \n the antiproliferative activity .", "time - dependent antiproliferative activity \n of dox ( 5 m ) in \n the presence and absence of [ r6]-c12 ( 5 m ) .", "in \n conclusion , acylated cyclic polyarginine peptides were synthesized \n and examined as cpps and potential molecular transporters .", "acpps showed \n higher potency as molecular transporter compared to the corresponding \n linear counterpart and cyclic polyarginine without fatty acid .", "the \n mechanism of the peptide internalization was found to be energy - dependent \n endocytosis .", "cyclization and acylation reactions on the structure \n of the peptide enhanced the intracellular uptake of polyarginine peptides \n although they carry a short length of sequence .", "this intracellular \n delivery property of acpps can be optimized by modifying the length \n of fatty acid chain . to the best of our knowledge ,", "this is the first \n report of cyclic fatty acylated polyarginine peptide as molecular \n transporter of a cell - impermeable phosphopeptide .", "this study provided \n insights about how a combination of the cyclic nature and acylation \n can improve the cell internalization of polyarginines .", "further investigations \n are undergoing to determine whether conjugation of cell - impermeable \n hydrophobic drugs to acylated cyclic polyarginines can be an efficient \n method for designing novel drug delivery systems ."], "abstract_text": ["<S> many \n of the reported arginine - rich cell - penetrating peptides ( cpps ) \n for the enhanced delivery of drugs are linear peptides composed of \n more than seven arginine residues to retain the cell penetration properties . \n herein , we synthesized a class of nine polyarginine peptides containing \n 5 and 6 arginines , namely , r5 and r6 . </S>", "<S> we further \n explored the effect of acylation with long chain fatty acids ( i.e. , \n octanoic acid , dodecanoic acid , and hexadecanoic acid ) and cyclization \n on the cell penetrating properties of the peptides . </S>", "<S> the fluorescence - labeled \n acylated cyclic peptide dodecanoyl-[r5 ] and linear peptide \n dodecanoyl-(r5 ) showed approximately 13.7- and 10.2-fold \n higher cellular uptake than that of control 5,6-carboxyfluorescein , \n respectively . </S>", "<S> the mechanism of the peptide internalization into cells \n was found to be energy - dependent endocytosis . </S>", "<S> dodecanoyl-[r5 ] and dodecanoyl-[r6 ] enhanced the intracellular uptake \n of a fluorescence - labeled cell - impermeable negatively charged phosphopeptide \n ( f-gpyeei ) in human ovarian cancer cells ( sk - ov-3 ) by 3.4-fold \n and 5.5-fold , respectively , as shown by flow cytometry </S>", "<S> . the cellular \n uptake of f-gpyeei in the presence of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than that in the presence of \n octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively . \n </S>", "<S> dodecanoyl-[r5 ] enhanced the cellular uptake of the phosphopeptide \n by 1.42.5-fold higher than the corresponding linear peptide \n dodecanoyl-(r5 ) and those of representative cpps , such \n as hepta - arginine ( cr7 ) and tat peptide . </S>", "<S> these results \n showed that a combination of acylation by long chain fatty acids and \n cyclization on short arginine - containing peptides can improve their \n cell - penetrating property , possibly through efficient interaction \n of rigid positively charged r and hydrophobic dodecanoyl moiety with \n the corresponding residues in the cell membrane phospholipids . </S>"], "labels": null, "section_names": ["Introduction", "Experimental\nSection", "Results and Discussion", "Conclusions"], "sections": [["polyarginine peptides \n are known as one of the widely used classes \n of cell - penetrating peptides ( cpps ) and cellular delivery tools .", "it has been reported that the presence of the \n guanidine group in the side chain of arginine plays a key role for \n improved ability of arginine - rich peptides to cross the cell membrane .", "various systematic structural investigations have been performed \n to determine the required number of arginine residues and the length \n of the peptide for the optimization of cellular uptake .", "short polyarginine peptides containing less than six arginine residues \n did not exhibit significant cellular uptake in several previously \n reported investigations .", "thus , the presence of more than six arginine residues in the structure \n of polyarginine peptides is critical for their efficient cell - penetrating \n functions .", "however , several investigations were conducted to \n increase the \n cellular uptake of polyarginines by attaching the fatty acid to the n - terminal of the peptide .", "it has been previously reported \n that the acylation of the n - terminal by fatty acids \n can facilitate the intracellular uptake of polyarginines .", "for instance , katayama et al . synthesized acylated octa - arginines \n and discovered that the introduction of hydrophobic fatty acid enhanced \n the intracellular uptake of octa - arginine peptide and its conjugated \n ubiquitin .", "furthermore , lee et al . designed \n a class of lipopeptides carrying 715 arginine residues . among \n them ,", "myristoylated - hendeca - arginine ( c14r11 ) was found to be the most efficient cell - penetrating peptide . however , the fatty acylated polyarginine peptides \n that contain 715 arginine residues can potentially cause toxicity , \n and they can be easily degraded by proteases . moreover , linear \n peptides carrying l - form are not stable \n in serum and therefore have a limited application for in vivo studies ( figure s2 , supporting information ) . replacing l - form amino acids \n with d - form to improve the peptide stability leads to high \n cost production .", "thus , the synthesis and development of cyclic \n cpps containing short amino acid sequence with less toxicity is desired . herein , we designed acylated cyclic polyarginine peptides ( acpps ) \n containing five arginine residues and investigated their ability as \n cell - penetrating peptides .", "we compared acpps with a corresponding \n acylated linear polyarginine peptide ( alpp ) and a nonacylated cyclic \n polyarginine as controls .", "we hypothesize that the combination of acylation \n and cyclization of short polyarginine peptides having less than six \n arginine residues will increase the intracellular uptake and can generate \n peptides with molecular transporter properties . for convenience , square \n brackets [ ] and parentheses ( )", "phosphopeptide ptyr - glu - glu - ile ( pyeei ) \n is an optimal peptide ligand for binding to the src tyrosine kinase \n sh2 domain . in this study , we used negatively charged fluorescein - labeled \n phosphopeptide f-gpyeei as a model cell - impermeable compound ."], ["all peptides were synthesized by \n fmoc / tbu solid - phase peptide synthesis strategy either \n manually or using rainin ps3 synthesizer ( protein technologies inc . ) . \n", "manual reactions were carried out in a glass reaction vessel with \n a sintered glass frit by mixing under nitrogen bubbling at room temperature . \n", "fmoc - l - amino acid building blocks , fatty acids , and preloaded \n h - arg(pbf)-2-chlorotrityl resin were used as starting materials .", "2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate \n ( hbtu ) , hydroxybenzotriazole ( hobt ) , and n , n - diisopropylethylamine ( dipea ) in n , n - dimethylformamide \n ( dmf ) were used as coupling and activating reagents , respectively , \n for manual synthesis . in the case of using peptide synthesizer , 0.4 \n m n - methylmorpholine in dmf was used instead of dipea . \n", "piperidine in dmf ( 20% ) was employed to deprotect fmoc group at each \n step . to cleave the linear peptide from the resin , a mixture \n of trifluoroacetic acid ( tfa)/triisoproylsilane ( tis)/water ( 92.5/5/2.5 , \n", "however , in the synthesis of cyclic peptides , the \n side chain protected linear peptides were first cleaved from the resin \n by using a cleavage cocktail , containing 2,2,2-trifluoroethanol ( tfe)/acetic \n acid / dichloromethane ( dcm ) ( 2:1:7 , v / v / v ) .", "cyclization reaction was \n performed by employing a mixture of 1-hydroxy-7-azabenzotriazole ( hoat ) \n and n , n-diisopropylcarbodiimide \n ( dic ) in anhydrous dmf / dcm for 12 h. after solvent evaporation , the \n peptide was deprotected and cleaved from the resin by using a cleavage \n cocktail reagent ", "r  , containing tfa / thioanisole/1,2-ethanedithiol \n ( edt)/anisole ( 90:5:3:2 , v / v / v / v ) for 23 h. the crude peptides \n were precipitated and washed with cold diethyl ether . to purify the \n crude peptides , we used a reversed - phase high pressure liquid chromatography \n ( rp - hplc ) system using shimadzu lc-8a preparative liquid chromatography \n on a phenomenex gemini c18 column ( 10 m , 250  21.2 mm ) \n with a gradient 0100% of acetonitrile ( ch3cn ) containing \n 0.1% tfa ( v / v ) and water containing 0.1% tfa ( v / v ) for 1 h with a \n flow rate at 15.0 ml / min at the wavelength of 214 nm . as a representative \n example , the synthesis of dodecanoyl-[r5 ] is described \n here .", "h - arg(pbf)-2-chlorotrityl resin ( 660 \n mg , 0.35 mmol , 0.53 mmol / g ) was swelled in dmf for 40 min by n2 .", "fmoc - arg(pbf)-oh ( 681 mg , 1.05 mmol , 3 equiv ) was coupled \n to the n - terminal of the resin , using hbtu ( 398 mg , \n 1.05 mmol , 3 equiv ) , hobt ( 142 mg , 1.05 mmol , 3 equiv ) , and dipea \n ( 366 l , 2.1 mmol , 6 equiv ) in dmf ( 15 ml ) by agitating the \n resin for 1 h using n2 .", "after the coupling , the resin was \n washed with dmf , followed by fmoc - deprotection with piperidine in \n dmf ( 20% ) .", "the subsequent three fmoc - arg(pbf)-oh couplings and one \n dde - lys(fmoc)-oh ( 559 mg , 1.05 mmol , 3 equiv ; dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl ) \n coupling was carried out in the same manner , respectively . after removing \n the fmoc group in side chain of lysine , dodecanoic acid (", "210 mg , 1.05 \n mmol , 3 equiv ) was coupled using hbtu , hobt , and dipea .", "then dde protection \n group at n - terminal of peptide was removed by 2% \n hydrazine in dmf , followed by washing with dmf and dcm .", "the side chain \n protected linear peptides were cleaved from the resin by using a cleavage \n cocktail , tfe / acetic acid / dcm ( 2:1:7 , v / v / v ) , for 1 h. the filtrate \n was evaporated , and the residue was dried overnight in a vacuum .", "the \n cyclization was conducted under a dilute condition with anhydrous \n dmf / dcm ( 5:3 , v / v , 250 ml ) , using hoat ( 190 mg , 1.4 mmol , 4 equiv ) \n and dic ( 240 l , 1.54 mmol , 4.4 equiv ) , and stirred for 12 h \n under nitrogen atmosphere .", "after cyclization , the solvent was evaporated , \n and the side chain deprotection was carried out by the addition of \n reagent  r  for 2 h. the crude dodecanoyl-[r5 ] was precipitated and washed with cold diethyl ether and purified \n by preparative rp - hplc system as described above .", "fluorescein - labeled \n peptides were synthesized with the same protocol \n before the attachment of fatty acid .", "we used fmoc-12-aminododecanoic \n acid instead of dodecanoic acid , and after removing fmoc group , 5(6)-carboxyfluorescein \n diisobutyrate ( cfdi ) was attached using 7-azabenzotriazol-1-yl - oxytris(pyrrolidino)phosphonium \n hexafluorophosphate ( pyaop ) , hoat , and dipea .", "as an example , we started \n fluorescein - labeled peptide synthesis in smaller scale with h - arg(pbf)-2-chlorotrityl \n resin ( 208 mg , 0.11 mmol , 0.53 mmol / g ) .", "fmoc - arg(pbf)-oh ( 214 mg , \n 0.33 mmol , 3 equiv ) , dde - lys(fmoc)-oh ( 176 mg , 0.33 mmol , 3 equiv ) , \n and fmoc-12-aminododecanoic acid ( 144 mg , 0.33 mmol , 3 equiv ) were \n used to couple each building block to the resin using hbtu ( 125 mg , \n 0.33 mmol , 3 equiv ) , hobt ( 45 mg , 0.33 mmol , 3 equiv ) , and dipea ( 115 \n l , 0.66 mmol , 6 equiv ) in dmf .", "cfdi ( 170 mg , 0.33 mmol , 3 equiv ) \n was coupled with amino group of 12-aminododecaonic chain using pyaop \n ( 172 mg , 0.33 mmol , 3 equiv ) , hoat ( 45 mg , 0.33 mmol , 3 equiv ) , and \n dipea .", "after removal of dde protecting group with 2% hydrazine in dmf \n and washing with dmf and dcm , side chain protected fluorescein linear \n peptides were cleaved from resin using tfe / acetic acid / dcm ( 2:1:7 , \n v / v / v ) .", "the molecular weights of final products were confirmed by \n an axima performance matrix - assisted laser desorption / ionization - time - of - flight \n ( maldi - tof ) mass spectrometer ( shimadzu corporation ) .", "dodecanoyl-(r6 ) : maldi - tof ( m / z ) c48h96n22o8 calcd . 1108.7781 ; found 1109.7308 [ m + h ] .", "f-dodecanoyl-(r5 ) : maldi - tof ( m / z ) c69h107n23o14 calcd . 1481.8368 ; \n found 1482.7586 [ m + h ] .", "w - dodecanoyl-[r5 ] : maldi - tof ( m / z ) c61h107n25o9 calcd .", "human ovarian adenocarcinoma \n ( sk - ov-3 ) , \n leukemia ( ccrf - cem ) , and embryonic kidney ( hek 293 t ) cells were purchased \n from american type culture collection .", "the sk - ov-3 and hek 293 t cells \n were grown in eagle s minimum essential medium ( emem ) , and \n rpmi-1640 medium ( atcc , manassas , va ) was used for ccrf - cem cells \n in a humidified atmosphere of 5% co2 at 37 c .", "both \n media were supplemented with fetal bovine serum ( fbs , 10% ) and penicillin ", "streptomycin \n solution ( 1% , 10,000 units of penicillin and 10 mg of streptomycin \n in 0.9% nacl ) .", "cytotoxicity \n of peptides was examined \n by mts proliferation assay in two human cancer cell lines ( sk - ov-3 \n and ccrf - cem ) and one human normal cell line ( hek 293 t ) .", "cells were \n seeded into 96-well plates ( sk - ov-3 ( 5  10 cells / well ) , \n ccrf - cem ( 1  10 cells /", "well ) , and hek 293 t ( 1  \n 10 cells / well ) ) .", "then , the cells were incubated with complete \n media ( 100 l ) overnight .", "different concentrations ( 0600 \n m ) of peptide solution ( 10 l ) were added to cells and \n incubated at 37 c with 5% co2 for 24 h. then , celltiter \n 96 aqueous solution ( 20 l ) was added to each well and incubated \n for 14 h. the absorbance was measured at 490 nm using microplate \n reader .", "sk - ov-3 \n cells were grown in 6-well plates ( 2  10 cells / well ) \n with complete emem media 24 h prior to the experiment .", "the fluorescein - labeled \n peptide stock solution ( 1 mm ) was prepared in water and diluted in \n gibco opti - mem i reduced serum medium to obtain the final concentration \n of 5 m .", "the media were removed , and the mixture containing \n fluorescein - labeled peptide solution ( 5 m ) was added .", "after \n 1 h incubation , trypsin  edta solution was added to detach cells \n from the plate s surface and remove cell surface binding peptides . \n", "after 5 min , the complete media ( 2 ml ) were added to neutralize the \n trypsin .", "finally , \n the cells were analyzed by bd facscalibur or facsverse flow cytometer \n using fitc channel .", "5(6)-carboxyfluorescein ( fam ) was used as a negative control . for examination \n of the cellular uptake mechanism of f-dodecanoyl-[r5 ] at low temperature", ", the assay was carried out at 4 c \n to inhibit the energy - dependent cellular uptake pathways .", "the sk - ov-3 \n cells were preincubated at 4 c for 15 min and incubated with \n the fluorescein - labeled peptide for 1 h at 4 c .", "cells were collected \n and analyzed using flow cytometry with the previously described protocol \n above .", "the data collected at 37 c were used as the control . \n to perform the atp - depletion assay ,", "cells were incubated with sodium \n azide ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) for 1 h before \n adding the fluorescein - labeled peptide . during the incubation time \n ( 1 h ) , the fluorescein - labeled bicyclic peptide ( 5 m )", "was prepared \n in the opti - mem i reduced serum medium in the presence of sodium azide \n ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) .", "then , the cells were \n incubated with this solution for 1 h. the following sample preparation \n and flow cytometry analysis protocol was the same as described above .", "sk - ov-3 cells \n were seeded with complete emem on coverslips in 6-well plate ( 1  \n 10 cells / well ) and kept until 50% confluency .", "the media \n were removed , and cells were incubated with f-dodecanoyl-[r5 ] ( 10 m ) and f-dodecanoyl-(r5 ) ( 10 \n m ) in gibco opti - mem i reduced serum medium ( life technologies , \n grand island , ny ) for 1 h at 37 c . then cells were washed with \n 1 phosphate buffered saline with calcium and magnesium ( pbs ) for three times .", "the coverslips were mounted on microscope \n slides , and images were obtained using carl zeiss lsm 700 system with \n a 488 nm argon ion laser excitation and a bp 505530 nm band - pass \n filter .", "sk - ov-3 and ccrf - cem cells were seeded in 6-well plates ( 2  \n 10 cells / well for sk - ov-3 and 1  10cells / well \n for ccrf - cem ) and grown with complete emem media ( rpmi-1640 for ccrf - cem ) \n overnight .", "a mixture of fluorescein - labeled phosphopeptides f-gpyeei \n ( 5 m ) and peptides ( 10 m ) were prepared in opti - mem \n i reduced serum medium at room temperature and incubated for 15 min . \n", "then the cells were incubated with the premixed solution at 37 c \n with 5% co2 for 1 h. the sample preparation for facs analysis \n was carried out by previously mentioned protocol described before . \n in this assay ,"], ["the \n acylated cyclic polyarginine peptides \n were synthesized by fmoc / tbu solid - phase peptide \n synthesis method .", "fmoc - l - arg(pbf)-oh was coupled on h - arg(pbf)-loaded \n 2-chlorotrityl resin in the presence of hbtu , hobt , and dipea in dmf . \n", "then dde - lys(fmoc)-oh was attached , and a fatty acid was coupled to \n the side chain of lysine .", "dde protecting group was removed by 2% hydrazine \n in dmf , and a cleavage cocktail containing tfe / acetic acid / dcm ( 2:1:7 \n v / v / v ) was used for 1 h to cleave the side - chain protected linear \n peptides from the resin .", "cyclization of linear peptides was carried \n out in the presence of a mixture of hoat and dic in anhydrous dmf / dcm \n for 624 h. the side - chain deprotection of cyclic peptide was \n carried out by a cleavage cocktail reagent ", "r  for 2 \n h. the crude peptides were precipitated and purified with rp - hplc \n as described above .", "as a representative example , the synthesis of \n dodecanoyl-[r5 ] is shown in scheme 1 .", "a corresponding acylated \n linear polyarginine peptide ( alpp ) was \n synthesized for comparative studies with the cyclic peptide ( acpp ) . \n", "moreover , a cyclic polyarginine without fatty acid [ r5 ] \n was also synthesized to investigate the effect of the fatty acid on \n cyclic peptide and its effect on molecular transporting efficiency . \n to investigate whether the peptide alone can enter into cells , fluorescein - labeled \n f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , where f = fluorescein , were synthesized for facs \n and microscopy investigations .", "chemical structures of synthetic peptides \n used in this study ( f , \n fluorescein - labeled ; [ ] , cyclic peptide ; ( ) , linear peptide ) .", "the cytotoxicity \n of all peptides were tested in two different cancer cell lines , adherent \n ( sk - ov-3 ) and nonadherent ( ccrf - cem ) cells , and a normal cell line \n ( hek 293 t ) using mts assay ( figure 2 ) .", "cyclic \n polyarginine [ r5 ] without fatty acid was used to explore \n the effect of n - terminal acylation on cytotoxicity \n and cellular uptake .", "alpp ( dodecanoyl-(r5 ) ) and [ r5 ] peptides showed consistently less cytotoxicity in all three \n cells compared to acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) .", "after 24 h incubation , acpps showed approximately 20% \n toxicity in cells at a concentration of 25 m in ccrf - cem cells . \n", "however , dodecanoyl - linear ( r5 ) and [ r5 ] did \n not exhibit significant cytotoxicity at 25 m and showed less \n than 20% toxicity at the concentration of 100 m . in sk - ov-3 \n cells , dodecanoyl-(r5 ) and [ r5 ] showed more \n than 80% cell viability at the concentration of 25 m .", "in normal \n cells ( hek 293 t ) , all peptides exhibited less than 5% toxicity at \n 25 m .", "this differential behavior of the peptides in normal , \n and cancer cells can be possibly rationalized through the interaction \n between polyarginine peptides and cell membranes .", "the membrane of \n cancer cells holds more negative charges compared to that in normal \n cells because of the presence of anionic lipids such as phosphatidylserine .", "therefore , polyarginine peptides can be interacted \n with cancer cells more effectively compared to normal cells .", "these data indicated \n that acpps are more toxic than alpp and a nonacylated cyclic peptide \n [ r5 ] , especially at concentration of 25 m . \n", "thus , a noncytotoxic concentration of 510 m was used \n in cell - based assays .", "comparison of cytotoxicity between cyclic and linear acylated \n polyarginine \n peptides and nonacylated cyclic peptide [ r5 ] at various \n concentrations against ccrf - cem , sk - ov-3 , and hek 293 t after 24 h. the intracellular uptake studies of fluorescein - labeled \n acylated cyclic and linear pp , f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , was carried out in sk - ov-3 \n cells by using flow cytometry and confocal laser scanning microscopy \n ( clsm ) methods .", "fluorescein ( fam , f ) alone was selected as \n a negative control . as it is shown in figure 3 ,", "the f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) showed approximately 13.7- and 10.3-fold higher cellular \n uptake than that of control 5,6-carboxyfluorescein ( fam ) , respectively , \n in sk - ov-3 cells .", "the cellular uptake of f-dodecanoyl-[r5 ] was confirmed by clsm images ( figure 4 ) . \n", "f-dodecanoyl-[r5 ] showed higher fluorescence intensity \n compared to that of f-dodecanoyl-(r5 ) in sk - ov-3 \n cells .", "therefore , acpp f-dodecanoyl-[r5 ] was found \n to be a more efficient cell - penetrating peptide compared to its linear \n counterpart .", "as shown in figure 4 , the fluorescence \n signal is extended through the whole cells , suggesting that f-dodecanoyl-[r5 ] can get localized in the nucleus as well as cytoplasm .", "comparative \n cellular uptake of f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) ( 5 m ) in sk - ov-3 \n cells ( 1 h ) .", "confocal laser scanning \n microscope image of ( a ) f-dodecanoyl-[r5 ] and ( b ) \n f-dodecanoyl-(r5 ) .", "the peptides \n were incubated for 1 h in sk - ov-3 cells at 10 m concentration .", "the mechanism \n of the cellular internalization of \n f-dodecanoyl-[r5 ] was investigated by a temperature \n control assay at 4 c along with atp depletion assay .", "these two \n assays have been widely used to examine the energy - dependent endocytosis .", "facs results showed that the intracellular uptake \n of f-dodecanoyl-[r5 ] was significantly reduced \n at 4 c , indicating that the mechanism of internalization was \n mainly dependent on the endocytosis pathways ( figure 5 ) .", "furthermore , atp depletion \n assay was performed to investigate receptor - mediated endocytosis . to induce atp depletion ,", "sk - ov-3 cells were \n preincubated with sodium azide ( 10 mm ) and 2-deoxy - d - glucose \n ( 50 mm ) for 1 h prior to the experiment , and a similar concentration \n was maintained during the incubation ( 1 h ) with f-dodecanoyl-[r5 ] .", "the results showed that the cellular uptake of f-acpp \n was inhibited in the presence of sodium azide and 2-deoxy - d - glucose , suggesting that receptor - mediated endocytosis is involved \n for the cellular uptake of f-acpp . in atp depletion assay , \n", "the basic cellular uptake of fam was higher compared to that in temperature \n control assay .", "however , this is evident that the intracellular uptake \n of f-dodecanoyl-[r5 ] was inhibited , and there was \n no significant difference between the cells treated with fam compared \n to those treated with f-dodecanoyl-[r5 ] in temperature \n control and atp depletion assays , suggesting that endocytosis is the \n major pathway for the cellular uptake of f-dodecanoyl-[r5 ] .", "cellular uptake of f-dodecanoyl-[r5 ] ( 5 m ) \n in sk - ov-3 cells in temperature control assay at 37 and 4 c , \n and atp depletion assay with nan3 ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) analyzed by flow cytometry .", "cells with no \n treatment were used as control . the ability \n of acpps as a molecular transporter was evaluated and compared by \n selecting a fluorescein - labeled phosphopeptide , f-gpyeei , \n as a molecular cargo .", "phosphopeptide , pyeei ( ptyr - glu - glu - ile ) is \n an optimal peptide template for the sh2 domain of src tyrosine kinase . \n", "several analogues of this peptide have been synthesized as potent \n ligands for this target . because of the presence of the \n negatively charged amino acid residues in the structure of the peptide \n including phosphorylated tyrosine", "moreover , the internalization of the negatively charged phosphopeptide \n in cancer cells by diffusion is more difficult because cancer cell \n membranes are composed of more negatively charged lipids .", "thus , cellular \n delivery of cell - impermeable negatively charged phosphopeptides is \n significantly challenging .", "we have previously reported different peptide - based \n carriers for the intracellular delivery of negatively charged phosphopeptides \n as model cell - impermeable drugs in several cell lines . in this study ,", "the intracellular uptake \n of f-gpyeei was monitored in the presence and absence of synthetic \n peptides after 1 h incubation by flow cytometry .", "as it is exhibited \n in figure 6 , the acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) delivered the phosphopeptide more \n efficiently compared to alpps , dodecanoyl-(r5 ) and -[r5 ] .", "the intracellular uptake of f-gpyeei in the presence \n of dodecanoyl-[r5 ] and dodecanoyl-[r6 ] was enhanced \n by 3.4- and 5.5-fold higher than the uptake in the absence of acpps . \n", "however , dodecanoyl-(r5 ) and -[r5 ] only improved \n 1.3- and 1.4-fold intracellular uptake , respectively .", "the results \n showed that acylated and cyclized polyarginine peptides can deliver \n the phosphopeptide effectively .", "however , the intracellular uptake \n of the phosphopeptide did not improve significantly in the presence \n of either acylated linear polyarginine peptide or cyclic [ r5 ] .", "these data suggest that a combination of acylation and cyclization \n would improve the molecular transporting efficiency of the polyarginine - based \n peptide ( containing less than six arginines ) for the intracellular \n delivery of a cell - impermeable phosphopeptide .", "this has been previously \n reported that the acylated linear octa - arginine increased the cellular \n uptake of molecular cargoes by just adding fatty acid to the n - terminal of octa - arginine .", "however , we discovered that both cyclization and acylation in a \n short penta - arginine can significantly improve the delivery of a cell - impermeable \n phosphopeptide in sk - ov-3 cells .", "cellular uptake of f-gpyeei ( 5 \n m ) in the presence \n of dodecanoyl-[r5 ] and -[r5 ] , dosecanoyl-(r5 ) , and dodecanoyl-[r6 ] ( 10 m ) in sk - ov-3 \n cell line .", "phosphopeptide delivery efficiency of dodecanoyl-[r5 ] were compared with known cpps ( r7 , crrrrrrr ; \n tat , ygrkkrrqrrr ) .", "the major driving forces for the intracellular delivery are \n presumed \n to be structural rigidity through cyclization of the peptide and the \n interaction of the fatty acid with the cell membrane .", "it has been \n previously reported that the cellular uptake of the peptide can be \n increased due to the structural rigidity by cyclization of arginine - rich \n peptides .", "they proposed that the maximal \n distance between guanidine groups of arginine residue can lead to \n an efficient transduction of arginine - rich peptides .", "our investigations \n showed that dodecanoyl-[r6 ] is able to deliver more efficiently \n by 1.6-fold higher f-gpyeei uptake compared to that of dodecanoyl-[r5 ] . increasing", "the number of positively charged arginine residues \n can enhance the cellular uptake through ionic interactions with the \n negatively charged phosphopeptide and/or phospholipid in the cell \n membrane through ionic interactions .", "however , the higher number of \n arginine residue is not the only responsible element for the efficient \n cellular internalization .", "for example , it has been reported that polyarginine \n containing 11 amino acids ( r11 ) showed higher cellular \n uptake compared to the polyarginine containing 13 amino acids ( r13 ) . at the same time", ", r11 was found to be a more \n potent transporter compared to r9 in prostate cancer cells .", "these investigations showed that an optimal \n number of arginine residues are required for the highest degree of \n functionality .", "however , the greater number of amino acid residues \n in cyclic peptides can decrease the structural rigidity , which lowers \n the ability of the peptide to get into cells .", "dodecanoyl-[r5 ] was also compared with several commonly \n cpps , such as cr7 and tat ( ygrkkrrqrrr ) peptides .", "the acpp \n improved the cellular uptake of the phosphopeptide by 1.4- and 1.8-fold \n higher than those of cr7 and tat , respectively ( figure 6 ) .", "these results revealed that although other peptides \n containing arginine can deliver the molecular cargo , acpp dodecanoyl-[r5 ] with a shorter peptide sequence than cr7 and \n tat can work as a molecular transporter with higher efficiency .", "chemical structures \n of acpps with different length of fatty acid \n chains ( c8 , c12 , and c16 ) . to investigate the effect \n of the chain length on the cell penetration \n potency , we synthesized octanoyl-[r5 ] , dodecanoyl-[r5 ] , and hexadecanoyl-[r5 ] ( figure 7 ) .", "acpps showed less than 20% toxicity in cells at the concentration \n of 25 m ( figure 8a ) .", "the in \n vitro toxicity results showed that increasing the fatty acid \n chain length caused enhanced toxicity in cells as hexadecanoyl-[r5 ] was more cytotoxic than dodecanoyl-[r5 ] and octanoyl-[r5 ] .", "these data indicate that the fatty acid chain length could \n alter the interaction with the cell membrane and disturb the membrane \n integrity . on the basis of the cytotoxicity data ,", "( a ) cytotoxicity \n assay of cyclic polyarginine peptide - fatty acid \n conjugates against sk - ov-3 cells ( 24 h incubation ) .", "( b ) cellular uptake \n of a phosphopeptide , f-gpyeei ( 5 m ) , in the presence \n of peptide - fatty acid conjugates ( 10 m ) in sk - ov-3 cells .", "it has been previously reported that there was a relationship \n between \n the length of fatty acid in polyarginines and their cellular uptake , meaning that the optimal length of fatty acid \n is required for optimal functionality based on the peptide sequence \n and cell type .", "the results exhibited that the cellular uptake of f-gpyeei \n was improved in the order of octanoyl-[r5 ] < dodecanoyl-[r5 ] < hexadecanoyl-[r5 ] ( figure 8b ) .", "the cellular uptake of f-gpyeei in the presence \n of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than \n that in the presence of octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively .", "these data suggest that the length of the \n attached fatty acid chain in the structure significantly influences \n the efficiency of the peptide as a molecular transporter . sixteen - carbon \n chain length ( c16 )", "was found to be an optimized length \n for the intracellular delivery of f-gpyeei in sk - ov-3 cells .", "after acpps were found to act as cpp and molecular \n transporter , a systematic investigation was performed to modify the \n fatty acid to another hydrophobic moiety .", "w4-[r5 ] , \n the whole fatty acid chain was replaced with four tryptophan residues . \n", "the second conjugate w - dodecanoyl-[r5 ] had one more tryptophan \n at the end of the dodecanoyl fatty acid chain .", "w4-[r5 ] enhanced intracellular delivery of f-gpyeei by 4.1-fold \n higher compared to that of dodecanoyl-[r5 ] in sk - ov-3 . \n however , w - dodecanoyl-[r5 ] improved the uptake of f-gpyeei \n by 1.3-fold higher compared to that of w4-[r5 ] in ccrf - cem cells ( figure 9 ) .", "thus , it is not straightforward \n to compare the cellular uptakes in sk - ov-3 and ccrf - cem .", "however , \n the results could be assessed indirectly by relative comparison with \n the cellular uptake by w4-[r5 ] .", "these data suggest \n that the presence of hydrophobic tryptophan moieties could enhance \n the molecular transporting efficiency . in other words ,", "appropriate \n modification of hydrophobic moiety in cpps can increase the drug delivery \n ability of acpp .", "thus , cyclic nature , short length of polyarginine , \n and hydrophobic segments were found to be critical elements to generate \n a peptide with an optimized molecular transporter efficiency .", "cellular uptake \n assay of a phosphopeptide , f-gpyeei ( 5 \n m ) , in the presence of w4-[r5 ] and w - dodecanoyl-[r5 ] ( 10 m ) against sk - ov-3 and ccrf - cem cell lines ( 1 \n h incubation ) .", "cells with no treatment \n were used as control . to evaluate \n whether the presence of peptides can enhance the pharmacological effect \n of a molecular cargo , the antiproliferative activity of doxorubicin \n ( dox )", "was examined in the presence of dodecanoy-[r6 ] ( [ r6]-c12 ) in mcf-7 cells in a time - dependent assay . \n as it is shown in figure 10 ,", "the antiproliferative \n potency of dox ( 5 m ) was enhanced by 7% , 11% , and 14% in the \n presence of [ r6]-c12 ( 5 m ) when compared \n with that of the drug alone after 24 , 48 , and 72 h incubation , respectively . \n", "these results showed that the cell proliferation of mcf-7 cells was \n inhibited in a time - dependent manner suggesting a sustained drug release \n by peptide leading to a higher inhibitory effect .", "the presence of \n the peptide possibly improved the cellular uptake of dox and enhanced \n the antiproliferative activity .", "time - dependent antiproliferative activity \n of dox ( 5 m ) in \n the presence and absence of [ r6]-c12 ( 5 m ) ."], ["in \n conclusion , acylated cyclic polyarginine peptides were synthesized \n and examined as cpps and potential molecular transporters .", "acpps showed \n higher potency as molecular transporter compared to the corresponding \n linear counterpart and cyclic polyarginine without fatty acid .", "the \n mechanism of the peptide internalization was found to be energy - dependent \n endocytosis .", "cyclization and acylation reactions on the structure \n of the peptide enhanced the intracellular uptake of polyarginine peptides \n although they carry a short length of sequence .", "this intracellular \n delivery property of acpps can be optimized by modifying the length \n of fatty acid chain . to the best of our knowledge ,", "this is the first \n report of cyclic fatty acylated polyarginine peptide as molecular \n transporter of a cell - impermeable phosphopeptide .", "this study provided \n insights about how a combination of the cyclic nature and acylation \n can improve the cell internalization of polyarginines .", "further investigations \n are undergoing to determine whether conjugation of cell - impermeable \n hydrophobic drugs to acylated cyclic polyarginines can be an efficient \n method for designing novel drug delivery systems ."]]}
{"article_id": "PMC5027039", "article_text": ["as of 2014 , the worldwide prevalence of type 2 diabetes mellitus ( t2 dm ) was estimated to be 9% among adults aged 18 years with great impact on mortality , particularly in low- and middle - income countries ( lmic ) [ 1 , 2 ] . moreover , globally , approximately 25% to 75% of diabetes cases remain undiagnosed [ 3 , 4 ] , until further complications , especially at the macro- and micro - vascular level , manifest clinically . in latin america ,", "an important strategy to prevent or delay t2 dm complications is the early identification of those with undiagnosed diabetes ; yet , universal screening for diabetes at the population level is not practical in resource - limited settings .", "the american diabetes association recommends the use of glucose test as t2 dm screening in people with overweight and obesity as well as in those with other risk factors . as a result", ", risk assessment scores have been developed to address this problem in a simple and inexpensive way .", "most of the available algorithms for diabetes screening have been developed in caucasian [ 79 ] and asian populations [ 1013 ] and very few in other ethnic groups [ 14 , 15 ] . to date , one diabetes risk score has been developed and validated in latin america so far which was derived from one urban area in brazil , thus bearing limited generalizability to the wider region .", "furthermore , it is well established that before adopting existing risk scores as screening tools in different populations and ethnic groups , their performance needs to be evaluated , calibrated , or validated in local settings . as the american diabetes association , the peruvian ministry of health recommends diabetes screening in general population with fasting glucose in adults aged 40 to 70 years with risk factors .", "however , fasting glucose is not always available in primary care settings , especially in semiurban and rural areas . as a result , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method .", "consequently , we aimed to develop a simple laboratory - free risk score to identify people with undiagnosed diabetes and incident diabetes in peru , a latin american country that spans coastal , andean , and rainforest settings . in order to do so", ", this work benefited from two large - scale population - based surveys : the first one , representative at the national level , was used to develop the score , and the second one , a cohort study , was utilized for external validation .", "the national survey of nutritional and biochemical indicators for noncommunicable diseases ( eninbsc in spanish ) , conducted by the peruvian national institute of health , was used to develop our predictive model .", "this was complemented with the cronicas cohort study , whose baseline and longitudinal information was used to validate the risk score .", "the eninbsc is a national population - based survey carried out in peru between august 2004 and april 2005 , designed to estimate the prevalence of hypertension , type 2 diabetes mellitus , and other risk factors for noncommunicable diseases at the national and regional level .", "potential participants were those aged 20 years , habitual residents in the study area , and able to provide consent for their participation in the study .", "pregnant women and those currently breastfeeding were excluded from the study . as per design , the eninbsc sample was stratified according to peru 's five major regions of the country : lima , rest of the coast , urban highlands , rural highlands , and jungle . in each stratum , cluster of blocks", "the cronicas cohort study is an ongoing cardiopulmonary project aimed to estimate the prevalence and incidence of hypertension , diabetes mellitus , and obesity in four different settings in peru that differ in terms of their urbanicity and altitude : pampas de san juan de miraflores , in the highly urbanized lima , puno in the altitude ( 3,825 meters above the sea level ) contributing with rural and urban areas , and tumbes , a semiurban area in the northern coast of peru .", "the study started in september 2010 and a follow - up visit was completed in march 2014 .", "a sex- and age - stratified sample was selected at random for each of the settings and all participants aged 35 years , full time residents in the study area , and able to consent , were enrolled .", "follow - up data used for this analysis was collected , on average , at 30 months after baseline .", "the first one lasted on average 40 minutes and was carried out to apply a face - to - face questionnaire regarding data about household characteristics , demographics , lifestyles behaviors , risk factors , and blood pressure measurements .", "the second visit lasted 30 minutes on average and was planned to have an appropriate period of fasting for blood sampling for glucose , total cholesterol , hdl - cholesterol , and the remaining anthropometric measures ( height , weight , and waist circumference ) using standard procedures .", "similarly , the procedures of the cronicas study has been published elsewhere . in brief , participants responded to a face - to - face questionnaire applied by trained community health workers .", "data collected comprised risk factors for cardiovascular disease based on a modified version of the who step approach questionnaire for surveillance of noncommunicable disease .", "a period of 8 to 12 hours of fasting was required for blood sampling to collect fasting glucose , total cholesterol , and hdl - cholesterol .", "height , weight , and waist circumference were also assessed , and blood pressure was measured in triplicate after five minutes of resting using an automatic monitor ( omron hem-780 ) previously validated in adult 's population . in both studies ,", "diabetes was defined as any of the following conditions : fasting glucose 7.0  mmol / l ( 126  mg / dl ) and/or self - report of physician diagnosis .", "fasting glucose was assessed by an enzymatic colorimetric method ( glucose oxidase god - pap ) in both studies . after excluding individuals without known diabetes , undiagnosed diabetes", "variables included in the analyses were built to guarantee similarities between both studies : sex ; age ( < 55 and 55 years ) ; education ( in years ) ; self - reported smoking ( current versus never / former smoker ) ; alcohol use ( user versus never user ) ; self - reported diabetes in first - degree relatives ( participant 's parents and/or siblings ) ; and levels of physical activity ( low versus moderate / high levels , based on the transport - related domain of the ipaq ) .", "anthropometric measurements included in the analysis were body mass index ( ( bmi ) , < 25 , 2529.9 , and 30  kg / m ) , waist circumference ( < 90 , 9099.9 , and 100  cm ) , waist - to - height ratio ( < 0.50 , 0.500.59 , 0.600.69 , and 0.70 ) , and hypertension ( measured or previously diagnosed ) . a total of 4,206 participants were enrolled in the eninbsc , but only 2,472 were included in this analysis .", "reasons for exclusion were 1,524 because of age < 35 years to make both databases comparable , 129 because of no data about fasting plasma glucose levels being available , and 81 because of known diagnosis of diabetes . in the cronicas study , 3,601 participants were enrolled at baseline but only 2,948 records were analyzed as 465 had no data about glucose levels , and 188 were excluded because of previous diagnosis of diabetes .", "in addition , only data from 2,577 participants was used in the longitudinal assessment of the risk score ( comparison of baseline characteristics among those included and excluded from longitudinal analysis is shown in online supplement : e - table 1 ; see supplementary material available online at http://dx.doi.org/10.1155/2016/8790235 ) .", "initially , population characteristics of both studies were tabulated using proportions in the case of categorical variables and means and standard deviation ( sd ) with numerical variables .", "then , the prevalence and 95% confidence intervals ( 95% ci ) of total diabetes and undiagnosed diabetes were estimated in each study .", "the risk score was derived from data of the eninbsc survey taking into account the multistage sampling strategy of the study .", "each potential risk factor ( i.e. , sex , age , family history of diabetes , etc . ) was assessed in bivariate models using logistic regression and undiagnosed diabetes as the dependent variable .", "then , risk factors with a p value < 0.10 in the bivariate analysis were included in a multiple logistic regression model using stepwise backward elimination with a significance level of 5% . the hosmer - lemeshow goodness - of - fit test was used to assess", "how well the predicted prevalence matched the observed prevalence of undiagnosed diabetes ( i.e. , p values over 0.20 indicate that model fits well ) . as we sought for an easily applicable and implementable algorithm ,", "the risk factors in the final model were each assigned a weighted score by rounding up all regression coefficients in the final model to the nearest integer as in a previous report .", "for the evaluation of the risk score , the area under the receiver operating characteristic ( roc ) curve , sensitivity , specificity , and positive and negative predictive values ( ppv and npv ) were calculated .", "the optimal cut - point was determined using the youden index , a single statistic that captures the performance of a diagnostic test ( i.e. , sensitivity + specificity  1 ) .", "as one of the main aims of a nonlaboratory risk score is to identify people who warrant having a blood test ( i.e. , fasting glucose , glycated haemoglobin , etc . ) , the cut - point with the highest sensitivity was also estimated and described .", "we assessed the performance of our score using bootstrap techniques as well as carrying out an external validation using the cronicas cohort study .", "the resulting 1,000 prediction models were then assessed to estimate the bootstrap auc using the bias - corrected version of the confidence intervals .", "in addition , using baseline data from the cronicas cohort study , validation measures ( auc , sensitivity , specificity , predictive values , and likelihood ratios ) were estimated . to evaluate the performance of our algorithm ,", "the peruvian risk score was compared to previously published models for undiagnosed diabetes including the brazilian risk score , the qingdao score , the indian risk score , the kuwaiti risk score , the patient self - assessment score , and the rotterdam risk score   using the c - statistic .", "finally , using the follow - up data of the cronicas cohort study , the risk score was also evaluated to detect incident cases of t2 dm by excluding those with diabetes diagnosis at baseline .", "analyses were performed using stata 13.0 ( statacorp , college station , tx , usa ) . the protocol and informed consent forms of the eninbsc study were reviewed and approved by the instituto nacional de salud and the centro nacional de alimentacin y nutricin , both part of the ministry of health in lima , peru . in the case of the cronicas cohort study , protocol and consent forms were reviewed and approved by the institutional review boards of the universidad peruana cayetano heredia and the ngo asociacin benfica prisma in lima , peru , and the johns hopkins university in baltimore , usa .", "overall , participants from the cronicas study were 5 years older , reported consuming lower levels of alcohol , and were less physically active than those from the eninbsc survey .", "the overall prevalence of diabetes was 5.1% ( 129/2538 ; 95% ci : 4.2%5.9% ) in the eninbsc survey and 8.7% ( 272/3135 ; 95% ci : 7.7%9.7% ) in the cronicas cohort study 's baseline .", "after excluding those with known diabetes , undiagnosed diabetes was present in 2.0% ( 48/2457 ; 95% ci : 1.4%2.5% ) in the eninbsc survey and in 2.9% ( 85/2948 ; 95% ci : 2.3%3.5% ) in the cronicas cohort study .", "after stepwise backward logistic regression , age , diabetes in first - degree relatives , and waist circumference were independently associated with undiagnosed diabetes ( table 2 ) .", "the hosmer - lemeshow test showed that the final model fitted relatively well ( p = 0.21 ) .", "the peruvian diabetes risk score was constructed based on the coefficients of that final regression model .", "the score gave an auc of 0.73 ( 95% ci : 0.650.78 ) , and the optimal cut - point for undiagnosed diabetes using the youden index was 2 ( figure 1 ) .", "with this cut - point , about 34.8% of participants were categorized as at high risk of diabetes : sensitivity 69.6% , specificity 65.8% , and ppv and npv of 3.9% and 99.1% , respectively . with a cut - point 1", ", 69.8% of participants would be at high risk of diabetes with improved sensitivity ( 93.5% ) but lower specificity ( 30.6% ) .  ", "table 3 shows the performance of the risk score for detecting undiagnosed diabetes at different cut - points .", "when bootstrap was used , the performance of our risk score was similar to the obtained in the development model ( auc = 0.72 ; 95% ci : 0.650.78 ) .", "in addition , when the risk score was evaluated by applying the score to the cronicas cohort study 's population , the auc for undiagnosed diabetes was 0.68 ( 95% ci : 0.620.73 ) . at the suggested cut - point of 2 ,", "42% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 70.2% , 58.9% , 4.8% , and 98.5% , respectively ( table 4 ) . on the other hand , with a cut - point 1 ,", "80% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 94.0% , 20.0% , 3.3% , and 99.1% , respectively . when previous published algorithms for undiagnosed diabetes", "were applied to the cronicas cohort study , the performance of the rotterdam score ( p < 0.001 ) , indian score ( p < 0.001 ) , and qingdao score ( p", "< 0.01 ) was poorer than our score ; however , our algorithm performed similar to the other assessed models , such as the brazilian risk score ( p = 0.93 ) , the kuwaiti score ( p = 0.26 ) , and the patient self - assessment score ( p = 0.74 ) , but having only three variables .", "the performance of this risk score was also assessed to predict incident cases of diabetes using the longitudinal data from the cronicas cohort study .", "one hundred twenty - one new cases of diabetes were found accounting for 6,207 person - years at risk , with an overall incidence of 1.95 ( 95% ci : 1.632.33 ) cases per 100 person - years of risk .", "the auc of the score was 0.66 ( 95% ci : 0.610.71 ) . with a cut - point 2 ,", "42.5% of participants were categorized as at high risk of developing diabetes : sensitivity , specificity , ppv , and npv were 69.4% , 58.9% , 7.8% , and 97.4% , whereas , for a cut - point 1 , the respective values were 79.9% , 91.9% , 20.7% , 5.5% , and 98.1% .", "using a national population - based survey , a simple nonblood based risk score based on age , history of diabetes in first - degree relatives , and waist circumference was built and shown to perform moderately in detecting undiagnosed diabetes when externally validated .", "moreover , the performance of the score was almost similar for detecting incident cases of diabetes in the peruvian population .", "a relatively recent systematic literature search found 23 different blood - free prevalent diabetes risk scores : ten from europe , nine for asian populations , two from the united states , and two from middle east .", "in addition , and not included in the aforementioned review , only one risk score was developed in latin america using brazilian urban population .", "the same systematic review reported that auc for these predictive models was greater in the development studies ( range : 0.65 to 0.88 ) than in the validation studies ( range : 0.63 to 0.80 ) , similar to our findings .", "another systematic review found that several noninvasive algorithms were created using variables such as age , gender , waist circumference and/or bmi , and family history of diabetes in the final model .", "as impracticality due to use of the algorithms was a common barrier to the uptake of risk scores by healthcare staff and individuals , our model , created with three of these more common variables , reached a moderate - to - high sensitivity depending on the used cut - point .", "moreover , two of these variables are easily evaluable during medical appointment or through individual 's self - assessment , and only a measuring tape and no calculations are required to be implemented in clinical practice or at the population level . from a cross - sectional point of view , with a cut - point 2 , from 1000 participants assessed by the peruvian diabetes risk score , a total of 420 would be classified as undiagnosed diabetes with the detection of 20 cases and only 6 will be missing . on the other hand , with a cut - point 1 , from 1000 screened individuals , a total of 804 would be categorized as having undiagnosed diabetes with the detection of 27 cases and only 7 will be missing .", "thus , the reduction of the cut - point of the risk score would increase sensitivity but reducing the specificity and imposing the need of performing a confirmatory test ( i.e. , fasting glucose ) to almost the double of individuals , with the benefit of having only 7 more people diagnosed .", "longitudinally , the same risk score would detect an important number of participants at risk of developing diabetes : 43% of screened individuals would be classified at high risk of diabetes , and of them , 8% would develop diabetes in the next 2.5 years . according to a previous study", ", 17 reports described a noninvasive model to predict the development of diabetes and included a median of six risk predictors , ranging from 2 to 11 .", "although our score did not perform as good as other well - known longitudinal models in the literature such as the findrisc or the aric scores [ 35 , 36 ] , it only included three variables and was built using cross - sectional information .", "in addition , some variables used in the aforementioned studies are difficult to standardize within a country as peru , that is , food portions , physical activity , or sedentarism , limiting therefore its use on a wider scale and in a simple pragmatic fashion .", "our algorithm performed better than the rotterdam , the indian , and the qingdao risk scores in our population , which highlights the need of calibration and/or development of a specific score for different ethnic groups before its adoption .", "as there are ethnic differences in risk factors for diabetes and peru is considered a multiethnic country , it is necessary to create specific scores or recalibrate existing algorithms before applying in specific contexts .", "in addition , with only three variables included , the performance of our predictive model was similar to the other assessed scores included in the analyses .", "taken together , the score developed has the potential to augment , in a pragmatic manner , initial rapid screening for diabetes , especially at various nonspecialized primary healthcare services .", "our findings also demonstrate that approximately 35% of cases of t2 dm ( 39% in the eninbsc survey and 33% in the baseline of the cronicas cohort study ) are not aware of their disease .", "results are similar to those reported in previous studies in our context   and in similar settings in latin america .", "as the developed risk score is simple , it does not require a blood test or laboratory services , and it might be easily implemented in clinical practice .", "moreover , because our score asks for general information in the form of age and diabetes in first - degree relatives and is complemented by a simple anthropometric measure of waist , there is potential for the score to be self - administered . according to our results , any patient aged 55 years and above and having at least one first - degree relative with t2 dm has greater probability of having undiagnosed diabetes but also is at risk of developing diabetes in the future .", "in addition , a greater central obesity , that is , 100  cm or more , independent of the other terms of the score is alone a good predictor of diabetes as reported in previous studies .", "our algorithm included waist circumference instead of body mass index as other risk scores , providing a better indicator of accumulation of visceral fat and metabolic dysfunction in our context .", "recently , the peruvian ministry of health has published the guide of clinical practice for diagnosis , treatment and control of diabetes mellitus in primary care   and only recommends screening in general population with plasma glucose among adults between 40 and 70 years with obesity or overweight as suggested by the american diabetes association . as in other lmic , plasma glucose is not always available in primary care , especially in semiurban and rural areas ; therefore , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method .", "moreover , within the peruvian context , no risk score has been proposed as part of the aforementioned guide .", "thus , our algorithm might fill a gap to facilitate further specialized assessment of high risk individuals for diabetes , an approach that may be of utility to various other countries facing similar challenges .", "the strengths of this study include the use of a national population - based survey , including urban and rural areas across major geographical regions , to develop the peruvian diabetes risk score , as well as its validation using bootstrap but also an independent longitudinal cohort study .", "additionally , it is only based on three variables ensuring its simplicity to be used and implemented . however , the study has also some limitations .", "first , we have utilized fasting plasma glucose as the gold standard for diagnosing diabetes instead of an oral glucose tolerance test ( ogtt ) .", "although the ogtt is more sensitive and specific than the fasting plasma glucose , more cases would have been detected with the overload of glucose ; it is rarely performed as part of the routine clinical practice .", "second , the cronicas cohort study did not include information from the amazon rainforest as did the eninbsc survey . when a sensitivity analysis was performed excluding individuals from the jungle from eninbsc data ,", "in addition , the score was created using a national survey to be applicable to the entire peruvian population .", "third , some variables were not assessed in our logistic regression model such as dietary intake or history of gestational diabetes as such data was not available . as a result", ", some caution should be made when our algorithm is compared to other risk scores .", "fourth , our model is based on the idea of risk stratification instead of individualisation ; for instance , variables were categorized instead of being preserved as numerical .", "nevertheless , the performance of our score did not change when age and waist circumference were treated as numerical variables ( data not shown ) .", "moreover , our idea was to develop a simple and easily applicable score instead of a complex algorithm for predicting undiagnosed and incident diabetes .", "finally , as other diabetes risk scores , the model warrants further scrutiny before it can be used in other populations .", "the peruvian diabetes risk score , built using age , self - reported diabetes in first - degree relatives , and waist circumference , proves to be a simple pragmatic screening tool for undiagnosed and incident cases of diabetes in peru .", "this experience in generating such simple , easy - to - use approaches for the identification of t2 dm can serve to inform other similar lmic efforts who are on early stages of diabetes prevention .", "this tool , due to its simplicity , can facilitate various initiatives oriented to introduce and scale up early preventative and management strategies on a wider scale ."], "abstract_text": ["<S> \n objective . to develop and validate a risk score for detecting cases of undiagnosed diabetes in a resource - constrained country . </S>", "<S> methods . two population - based studies in peruvian population aged 35 years were used in the analysis : the eninbsc survey ( n = 2,472 ) and the cronicas cohort study ( n = 2,945 ) . fasting plasma glucose 7.0  mmol / l was used to diagnose diabetes in both studies . </S>", "<S> coefficients for risk score were derived from the eninbsc data and then the performance was validated using both baseline and follow - up data of the cronicas cohort study . results . </S>", "<S> the prevalence of undiagnosed diabetes was 2.0% in the eninbsc survey and 2.9% in the cronicas cohort study . </S>", "<S> predictors of undiagnosed diabetes were age , diabetes in first - degree relatives , and waist circumference . </S>", "<S> score values ranged from 0 to 4 , with an optimal cutoff 2 and had a moderate performance when applied in the cronicas baseline data ( auc = 0.68 ; 95% ci : 0.620.73 ; sensitivity 70% ; specificity 59% ) . </S>", "<S> when predicting incident cases , the auc was 0.66 ( 95% ci : 0.610.71 ) , with a sensitivity of 69% and specificity of 59% . conclusions . a simple nonblood based risk score based on age , diabetes in first - degree relatives , and waist circumference can be used as a simple screening tool for undiagnosed and incident cases of diabetes in peru . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["as of 2014 , the worldwide prevalence of type 2 diabetes mellitus ( t2 dm ) was estimated to be 9% among adults aged 18 years with great impact on mortality , particularly in low- and middle - income countries ( lmic ) [ 1 , 2 ] . moreover , globally , approximately 25% to 75% of diabetes cases remain undiagnosed [ 3 , 4 ] , until further complications , especially at the macro- and micro - vascular level , manifest clinically . in latin america ,", "an important strategy to prevent or delay t2 dm complications is the early identification of those with undiagnosed diabetes ; yet , universal screening for diabetes at the population level is not practical in resource - limited settings .", "the american diabetes association recommends the use of glucose test as t2 dm screening in people with overweight and obesity as well as in those with other risk factors . as a result", ", risk assessment scores have been developed to address this problem in a simple and inexpensive way .", "most of the available algorithms for diabetes screening have been developed in caucasian [ 79 ] and asian populations [ 1013 ] and very few in other ethnic groups [ 14 , 15 ] . to date , one diabetes risk score has been developed and validated in latin america so far which was derived from one urban area in brazil , thus bearing limited generalizability to the wider region .", "furthermore , it is well established that before adopting existing risk scores as screening tools in different populations and ethnic groups , their performance needs to be evaluated , calibrated , or validated in local settings . as the american diabetes association , the peruvian ministry of health recommends diabetes screening in general population with fasting glucose in adults aged 40 to 70 years with risk factors .", "however , fasting glucose is not always available in primary care settings , especially in semiurban and rural areas . as a result , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method .", "consequently , we aimed to develop a simple laboratory - free risk score to identify people with undiagnosed diabetes and incident diabetes in peru , a latin american country that spans coastal , andean , and rainforest settings . in order to do so", ", this work benefited from two large - scale population - based surveys : the first one , representative at the national level , was used to develop the score , and the second one , a cohort study , was utilized for external validation ."], ["the national survey of nutritional and biochemical indicators for noncommunicable diseases ( eninbsc in spanish ) , conducted by the peruvian national institute of health , was used to develop our predictive model .", "this was complemented with the cronicas cohort study , whose baseline and longitudinal information was used to validate the risk score .", "the eninbsc is a national population - based survey carried out in peru between august 2004 and april 2005 , designed to estimate the prevalence of hypertension , type 2 diabetes mellitus , and other risk factors for noncommunicable diseases at the national and regional level .", "potential participants were those aged 20 years , habitual residents in the study area , and able to provide consent for their participation in the study .", "pregnant women and those currently breastfeeding were excluded from the study . as per design , the eninbsc sample was stratified according to peru 's five major regions of the country : lima , rest of the coast , urban highlands , rural highlands , and jungle . in each stratum , cluster of blocks", "the cronicas cohort study is an ongoing cardiopulmonary project aimed to estimate the prevalence and incidence of hypertension , diabetes mellitus , and obesity in four different settings in peru that differ in terms of their urbanicity and altitude : pampas de san juan de miraflores , in the highly urbanized lima , puno in the altitude ( 3,825 meters above the sea level ) contributing with rural and urban areas , and tumbes , a semiurban area in the northern coast of peru .", "the study started in september 2010 and a follow - up visit was completed in march 2014 .", "a sex- and age - stratified sample was selected at random for each of the settings and all participants aged 35 years , full time residents in the study area , and able to consent , were enrolled .", "follow - up data used for this analysis was collected , on average , at 30 months after baseline .", "the first one lasted on average 40 minutes and was carried out to apply a face - to - face questionnaire regarding data about household characteristics , demographics , lifestyles behaviors , risk factors , and blood pressure measurements .", "the second visit lasted 30 minutes on average and was planned to have an appropriate period of fasting for blood sampling for glucose , total cholesterol , hdl - cholesterol , and the remaining anthropometric measures ( height , weight , and waist circumference ) using standard procedures .", "similarly , the procedures of the cronicas study has been published elsewhere . in brief , participants responded to a face - to - face questionnaire applied by trained community health workers .", "data collected comprised risk factors for cardiovascular disease based on a modified version of the who step approach questionnaire for surveillance of noncommunicable disease .", "a period of 8 to 12 hours of fasting was required for blood sampling to collect fasting glucose , total cholesterol , and hdl - cholesterol .", "height , weight , and waist circumference were also assessed , and blood pressure was measured in triplicate after five minutes of resting using an automatic monitor ( omron hem-780 ) previously validated in adult 's population . in both studies ,", "diabetes was defined as any of the following conditions : fasting glucose 7.0  mmol / l ( 126  mg / dl ) and/or self - report of physician diagnosis .", "fasting glucose was assessed by an enzymatic colorimetric method ( glucose oxidase god - pap ) in both studies . after excluding individuals without known diabetes , undiagnosed diabetes", "variables included in the analyses were built to guarantee similarities between both studies : sex ; age ( < 55 and 55 years ) ; education ( in years ) ; self - reported smoking ( current versus never / former smoker ) ; alcohol use ( user versus never user ) ; self - reported diabetes in first - degree relatives ( participant 's parents and/or siblings ) ; and levels of physical activity ( low versus moderate / high levels , based on the transport - related domain of the ipaq ) .", "anthropometric measurements included in the analysis were body mass index ( ( bmi ) , < 25 , 2529.9 , and 30  kg / m ) , waist circumference ( < 90 , 9099.9 , and 100  cm ) , waist - to - height ratio ( < 0.50 , 0.500.59 , 0.600.69 , and 0.70 ) , and hypertension ( measured or previously diagnosed ) . a total of 4,206 participants were enrolled in the eninbsc , but only 2,472 were included in this analysis .", "reasons for exclusion were 1,524 because of age < 35 years to make both databases comparable , 129 because of no data about fasting plasma glucose levels being available , and 81 because of known diagnosis of diabetes . in the cronicas study , 3,601 participants were enrolled at baseline but only 2,948 records were analyzed as 465 had no data about glucose levels , and 188 were excluded because of previous diagnosis of diabetes .", "in addition , only data from 2,577 participants was used in the longitudinal assessment of the risk score ( comparison of baseline characteristics among those included and excluded from longitudinal analysis is shown in online supplement : e - table 1 ; see supplementary material available online at http://dx.doi.org/10.1155/2016/8790235 ) .", "initially , population characteristics of both studies were tabulated using proportions in the case of categorical variables and means and standard deviation ( sd ) with numerical variables .", "then , the prevalence and 95% confidence intervals ( 95% ci ) of total diabetes and undiagnosed diabetes were estimated in each study .", "the risk score was derived from data of the eninbsc survey taking into account the multistage sampling strategy of the study .", "each potential risk factor ( i.e. , sex , age , family history of diabetes , etc . ) was assessed in bivariate models using logistic regression and undiagnosed diabetes as the dependent variable .", "then , risk factors with a p value < 0.10 in the bivariate analysis were included in a multiple logistic regression model using stepwise backward elimination with a significance level of 5% . the hosmer - lemeshow goodness - of - fit test was used to assess", "how well the predicted prevalence matched the observed prevalence of undiagnosed diabetes ( i.e. , p values over 0.20 indicate that model fits well ) . as we sought for an easily applicable and implementable algorithm ,", "the risk factors in the final model were each assigned a weighted score by rounding up all regression coefficients in the final model to the nearest integer as in a previous report .", "for the evaluation of the risk score , the area under the receiver operating characteristic ( roc ) curve , sensitivity , specificity , and positive and negative predictive values ( ppv and npv ) were calculated .", "the optimal cut - point was determined using the youden index , a single statistic that captures the performance of a diagnostic test ( i.e. , sensitivity + specificity  1 ) .", "as one of the main aims of a nonlaboratory risk score is to identify people who warrant having a blood test ( i.e. , fasting glucose , glycated haemoglobin , etc . ) , the cut - point with the highest sensitivity was also estimated and described .", "we assessed the performance of our score using bootstrap techniques as well as carrying out an external validation using the cronicas cohort study .", "the resulting 1,000 prediction models were then assessed to estimate the bootstrap auc using the bias - corrected version of the confidence intervals .", "in addition , using baseline data from the cronicas cohort study , validation measures ( auc , sensitivity , specificity , predictive values , and likelihood ratios ) were estimated . to evaluate the performance of our algorithm ,", "the peruvian risk score was compared to previously published models for undiagnosed diabetes including the brazilian risk score , the qingdao score , the indian risk score , the kuwaiti risk score , the patient self - assessment score , and the rotterdam risk score   using the c - statistic .", "finally , using the follow - up data of the cronicas cohort study , the risk score was also evaluated to detect incident cases of t2 dm by excluding those with diabetes diagnosis at baseline .", "analyses were performed using stata 13.0 ( statacorp , college station , tx , usa ) . the protocol and informed consent forms of the eninbsc study were reviewed and approved by the instituto nacional de salud and the centro nacional de alimentacin y nutricin , both part of the ministry of health in lima , peru . in the case of the cronicas cohort study , protocol and consent forms were reviewed and approved by the institutional review boards of the universidad peruana cayetano heredia and the ngo asociacin benfica prisma in lima , peru , and the johns hopkins university in baltimore , usa ."], ["overall , participants from the cronicas study were 5 years older , reported consuming lower levels of alcohol , and were less physically active than those from the eninbsc survey .", "the overall prevalence of diabetes was 5.1% ( 129/2538 ; 95% ci : 4.2%5.9% ) in the eninbsc survey and 8.7% ( 272/3135 ; 95% ci : 7.7%9.7% ) in the cronicas cohort study 's baseline .", "after excluding those with known diabetes , undiagnosed diabetes was present in 2.0% ( 48/2457 ; 95% ci : 1.4%2.5% ) in the eninbsc survey and in 2.9% ( 85/2948 ; 95% ci : 2.3%3.5% ) in the cronicas cohort study .", "after stepwise backward logistic regression , age , diabetes in first - degree relatives , and waist circumference were independently associated with undiagnosed diabetes ( table 2 ) .", "the hosmer - lemeshow test showed that the final model fitted relatively well ( p = 0.21 ) .", "the peruvian diabetes risk score was constructed based on the coefficients of that final regression model .", "the score gave an auc of 0.73 ( 95% ci : 0.650.78 ) , and the optimal cut - point for undiagnosed diabetes using the youden index was 2 ( figure 1 ) .", "with this cut - point , about 34.8% of participants were categorized as at high risk of diabetes : sensitivity 69.6% , specificity 65.8% , and ppv and npv of 3.9% and 99.1% , respectively . with a cut - point 1", ", 69.8% of participants would be at high risk of diabetes with improved sensitivity ( 93.5% ) but lower specificity ( 30.6% ) .  ", "table 3 shows the performance of the risk score for detecting undiagnosed diabetes at different cut - points .", "when bootstrap was used , the performance of our risk score was similar to the obtained in the development model ( auc = 0.72 ; 95% ci : 0.650.78 ) .", "in addition , when the risk score was evaluated by applying the score to the cronicas cohort study 's population , the auc for undiagnosed diabetes was 0.68 ( 95% ci : 0.620.73 ) . at the suggested cut - point of 2 ,", "42% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 70.2% , 58.9% , 4.8% , and 98.5% , respectively ( table 4 ) . on the other hand , with a cut - point 1 ,", "80% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 94.0% , 20.0% , 3.3% , and 99.1% , respectively . when previous published algorithms for undiagnosed diabetes", "were applied to the cronicas cohort study , the performance of the rotterdam score ( p < 0.001 ) , indian score ( p < 0.001 ) , and qingdao score ( p", "< 0.01 ) was poorer than our score ; however , our algorithm performed similar to the other assessed models , such as the brazilian risk score ( p = 0.93 ) , the kuwaiti score ( p = 0.26 ) , and the patient self - assessment score ( p = 0.74 ) , but having only three variables .", "the performance of this risk score was also assessed to predict incident cases of diabetes using the longitudinal data from the cronicas cohort study .", "one hundred twenty - one new cases of diabetes were found accounting for 6,207 person - years at risk , with an overall incidence of 1.95 ( 95% ci : 1.632.33 ) cases per 100 person - years of risk .", "the auc of the score was 0.66 ( 95% ci : 0.610.71 ) . with a cut - point 2 ,", "42.5% of participants were categorized as at high risk of developing diabetes : sensitivity , specificity , ppv , and npv were 69.4% , 58.9% , 7.8% , and 97.4% , whereas , for a cut - point 1 , the respective values were 79.9% , 91.9% , 20.7% , 5.5% , and 98.1% ."], ["using a national population - based survey , a simple nonblood based risk score based on age , history of diabetes in first - degree relatives , and waist circumference was built and shown to perform moderately in detecting undiagnosed diabetes when externally validated .", "moreover , the performance of the score was almost similar for detecting incident cases of diabetes in the peruvian population .", "a relatively recent systematic literature search found 23 different blood - free prevalent diabetes risk scores : ten from europe , nine for asian populations , two from the united states , and two from middle east .", "in addition , and not included in the aforementioned review , only one risk score was developed in latin america using brazilian urban population .", "the same systematic review reported that auc for these predictive models was greater in the development studies ( range : 0.65 to 0.88 ) than in the validation studies ( range : 0.63 to 0.80 ) , similar to our findings .", "another systematic review found that several noninvasive algorithms were created using variables such as age , gender , waist circumference and/or bmi , and family history of diabetes in the final model .", "as impracticality due to use of the algorithms was a common barrier to the uptake of risk scores by healthcare staff and individuals , our model , created with three of these more common variables , reached a moderate - to - high sensitivity depending on the used cut - point .", "moreover , two of these variables are easily evaluable during medical appointment or through individual 's self - assessment , and only a measuring tape and no calculations are required to be implemented in clinical practice or at the population level . from a cross - sectional point of view , with a cut - point 2 , from 1000 participants assessed by the peruvian diabetes risk score , a total of 420 would be classified as undiagnosed diabetes with the detection of 20 cases and only 6 will be missing . on the other hand , with a cut - point 1 , from 1000 screened individuals , a total of 804 would be categorized as having undiagnosed diabetes with the detection of 27 cases and only 7 will be missing .", "thus , the reduction of the cut - point of the risk score would increase sensitivity but reducing the specificity and imposing the need of performing a confirmatory test ( i.e. , fasting glucose ) to almost the double of individuals , with the benefit of having only 7 more people diagnosed .", "longitudinally , the same risk score would detect an important number of participants at risk of developing diabetes : 43% of screened individuals would be classified at high risk of diabetes , and of them , 8% would develop diabetes in the next 2.5 years . according to a previous study", ", 17 reports described a noninvasive model to predict the development of diabetes and included a median of six risk predictors , ranging from 2 to 11 .", "although our score did not perform as good as other well - known longitudinal models in the literature such as the findrisc or the aric scores [ 35 , 36 ] , it only included three variables and was built using cross - sectional information .", "in addition , some variables used in the aforementioned studies are difficult to standardize within a country as peru , that is , food portions , physical activity , or sedentarism , limiting therefore its use on a wider scale and in a simple pragmatic fashion .", "our algorithm performed better than the rotterdam , the indian , and the qingdao risk scores in our population , which highlights the need of calibration and/or development of a specific score for different ethnic groups before its adoption .", "as there are ethnic differences in risk factors for diabetes and peru is considered a multiethnic country , it is necessary to create specific scores or recalibrate existing algorithms before applying in specific contexts .", "in addition , with only three variables included , the performance of our predictive model was similar to the other assessed scores included in the analyses .", "taken together , the score developed has the potential to augment , in a pragmatic manner , initial rapid screening for diabetes , especially at various nonspecialized primary healthcare services .", "our findings also demonstrate that approximately 35% of cases of t2 dm ( 39% in the eninbsc survey and 33% in the baseline of the cronicas cohort study ) are not aware of their disease .", "results are similar to those reported in previous studies in our context   and in similar settings in latin america .", "as the developed risk score is simple , it does not require a blood test or laboratory services , and it might be easily implemented in clinical practice .", "moreover , because our score asks for general information in the form of age and diabetes in first - degree relatives and is complemented by a simple anthropometric measure of waist , there is potential for the score to be self - administered . according to our results , any patient aged 55 years and above and having at least one first - degree relative with t2 dm has greater probability of having undiagnosed diabetes but also is at risk of developing diabetes in the future .", "in addition , a greater central obesity , that is , 100  cm or more , independent of the other terms of the score is alone a good predictor of diabetes as reported in previous studies .", "our algorithm included waist circumference instead of body mass index as other risk scores , providing a better indicator of accumulation of visceral fat and metabolic dysfunction in our context .", "recently , the peruvian ministry of health has published the guide of clinical practice for diagnosis , treatment and control of diabetes mellitus in primary care   and only recommends screening in general population with plasma glucose among adults between 40 and 70 years with obesity or overweight as suggested by the american diabetes association . as in other lmic , plasma glucose is not always available in primary care , especially in semiurban and rural areas ; therefore , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method .", "moreover , within the peruvian context , no risk score has been proposed as part of the aforementioned guide .", "thus , our algorithm might fill a gap to facilitate further specialized assessment of high risk individuals for diabetes , an approach that may be of utility to various other countries facing similar challenges .", "the strengths of this study include the use of a national population - based survey , including urban and rural areas across major geographical regions , to develop the peruvian diabetes risk score , as well as its validation using bootstrap but also an independent longitudinal cohort study .", "additionally , it is only based on three variables ensuring its simplicity to be used and implemented . however , the study has also some limitations .", "first , we have utilized fasting plasma glucose as the gold standard for diagnosing diabetes instead of an oral glucose tolerance test ( ogtt ) .", "although the ogtt is more sensitive and specific than the fasting plasma glucose , more cases would have been detected with the overload of glucose ; it is rarely performed as part of the routine clinical practice .", "second , the cronicas cohort study did not include information from the amazon rainforest as did the eninbsc survey . when a sensitivity analysis was performed excluding individuals from the jungle from eninbsc data ,", "in addition , the score was created using a national survey to be applicable to the entire peruvian population .", "third , some variables were not assessed in our logistic regression model such as dietary intake or history of gestational diabetes as such data was not available . as a result", ", some caution should be made when our algorithm is compared to other risk scores .", "fourth , our model is based on the idea of risk stratification instead of individualisation ; for instance , variables were categorized instead of being preserved as numerical .", "nevertheless , the performance of our score did not change when age and waist circumference were treated as numerical variables ( data not shown ) .", "moreover , our idea was to develop a simple and easily applicable score instead of a complex algorithm for predicting undiagnosed and incident diabetes .", "finally , as other diabetes risk scores , the model warrants further scrutiny before it can be used in other populations ."], ["the peruvian diabetes risk score , built using age , self - reported diabetes in first - degree relatives , and waist circumference , proves to be a simple pragmatic screening tool for undiagnosed and incident cases of diabetes in peru .", "this experience in generating such simple , easy - to - use approaches for the identification of t2 dm can serve to inform other similar lmic efforts who are on early stages of diabetes prevention .", "this tool , due to its simplicity , can facilitate various initiatives oriented to introduce and scale up early preventative and management strategies on a wider scale ."]]}
{"article_id": "PMC4632892", "article_text": ["cancer is one of the leading causes of death worldwide and accounted for 7.6 million cases ( 13% of all deaths ) in 2008 ; in 2013 in the united states there are 12,340 estimated new cases and 4,030 deaths by cervical cancer [ american cancer society : cancer facts and figures 2013 ] .", "meanwhile in romania , based on statistical data from 2012 , cervical cancer is ranked third place taking in account the women cancer incidence , after the breast and colorectal cancer .", "cervical cancer develops in a multi - stage process , successively passing from a cervix with a normal epithelium to cervical intraepithelial neoplasia and finally leading to invasive cervical cancer . a relevant epidemiologic risk circumstance for the progress of the cervical cancer is represented by the presence of high risk human papillomavirus infections .", "consequently , considerable importance has been given to the molecular premise which leads to the progression of cervical cancer .", "rnai ( rna interference ) is a natural mechanism able to specific inhibit a target gene .", "the prospective exploitation as a therapeutic has lately been strengthened by the small interfering rna ( sirna ) delivery to human tumours .", "rnai has been demonstrated as a relevant investigation device for particular target genes by using exogenous sirnas .", "sirnas are single - stranded non - coding rnas with 2023 nucleotides length , being able to negatively modulate gene expression profile at post - transcriptional level .", "sirnas represent an encouraging novel type of medicaments for cancer therapy , as a result of targeting the mutated tumour suppressor genes or activated oncogenes .", "many recent investigations have shown that sirna can efficiently inhibit oncogene expression in cancer cells .", "various investigations demonstrated clearly that the deactivation of p53 constitutes a crucial step in carcinogenesis .", "the present study was designed to investigate the potential use of p53 rnai to block p53 expression , as well as the subsequent effect on cell invasion and gene expression on the human cervical cancer cell .", "hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance", ", cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells", "/ well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) .", "a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations .", "siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability .", "we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by .", "the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen .", "all of the samples had a rin ( rna integrity number ) higher than 7.5 .", "the 8 genes selected were assessed using specific primer and upl based on in silico design . the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) . for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system .", "the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step .", "the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene", "the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no .", "hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance", ", cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells", "/ well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) .", "a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations .", "siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability .", "we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by .", "the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen . the rna concentrations and quality were assessed by the nanodrop-1100 agilent 2100 bioanalyzer spectrophotometer .", "all of the samples had a rin ( rna integrity number ) higher than 7.5 .", "the 8 genes selected were assessed using specific primer and upl based on in silico design .", "the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) .", "for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system .", "the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step .", "the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene", "the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no .", "xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels .", "the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells .", "this data can facilitate to comprehend the response to treatment in dynamic . in figure 1", "we can observe a delay and a reduction of the cell migration after the p53sirna treatment .", "taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis .", "relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 .", "after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) .", "xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels .", "the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells .", "this data can facilitate to comprehend the response to treatment in dynamic . in figure 1", "we can observe a delay and a reduction of the cell migration after the p53sirna treatment .", "taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis . relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 .", "after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) .", "although at this moment cervical cancer is considered as a preventable disorder there is a significant risk of disease recurrence causing a persuasive necessity to research new therapeutic targets for this disease management .", "it is now well recognized that the tumour progression of all cancers is characterized by intensified proliferation and invasion rate and diminished in apoptosis . at the same time", "the apoptosis and angiogenesis are interconnected as can be observed from figure 4 , using string.9 database .", "the idea of this study is in agreement with the previous studies which are based on the hypothesis that once mutated , p53 exercised oncogenic role . by using sirna", "the role of the present study is to emphasize the cooperation between oncogenic mechanisms , confirming the crosstalk between apoptotic and angiogenic mechanisms .", "this has a significant therapeutic relevance based on the fact that mutated p53 is related to cancer aggressiveness   or to promoting metastasis . in a similar study", "was observed that , by using sirna targeting p53/p73 , tumoral cells were sensitized to chemotherapy . in a recent study , pinx1 was displayed as a novel target gene of p53 , proposing the suppression of p53/pinx1 pathway as a novel mechanism to enhance the telomerase activity in cervical cancer cells , and leading to reduction of cell proliferation .", "targeting oncogenic signals such as the mutated p53 gene that gains oncogenic role , we observed a downregulation of specific genes involved in apoptosis but also in angiogenesis , connected with a reduction of the invasion rate in the case of p53sirna therapy .", "after 24 hours of treatment with p53sirna we can observe downregulations of important genes involved in apoptosis and angiogenesis , as displayed in figure 2 .", "this study confirmed our hypothesis that p53 plays a central role in carcinogenesis as displayed in figure 3 , as important therapeutic target .", "p53 could promote the convergence of the apoptotic and angiogenic pathways in cervical cancer cells .", "it has been proved that p53 is able to indirectly downregulate vegf gene expression , via the retinoblastoma ( rb ) , in a p21-dependent mode , confirming its function in cell - cycle regulation as displayed by recent studies .", "the complete comprehension of the cross - talk between p53 and angiogenic pathways represents a challenge in cancer therapy [ 2426 ] .", "another study reveals that mutant p53 correlates with overexpression of vegf . in a similar study", "the inhibition of mutant p53 represents a significant therapeutic target , a fact emphasized by walerych as  rebel angel .", "p53 gene knockdown in hela cells plays an important role not only in apoptosis but also in angiogenesis through various mechanisms that regulate tumour cell development and migration .", "the angiogenic phenotype related to p53 targeted by sirna in human cancers is frequently associated with resistance to conventional genotoxic anticancer agents .", "therefore , p53 inhibitions become dependent and rely on angiogenesis for growth and metastasis . the results in the current research", "may also have an important significance outside the context of cervical cancer , by using specific inhibitors for p53 for increasing therapeutic response in a wide range of tumoral pathology ."], "abstract_text": ["<S> background and aimssirnas represent an encouraging novel alternative in cancer therapy as a result of targeting the mutated tumour suppressor genes or activated oncogenes . </S>", "<S> targeting oncogenic signals , as the mutated p53 gene that gains oncogenic role , we observed inhibition of migration , a downregulation of specific genes involved in apoptosis but also in angiogenesis , connected with a reduction in invasion rate in the case of p53sirna therapy.methodsthe study was designed to assess the role of p53 by using rnai ( rna interference ) in hela in vitro cell culture model . therefore cell migration rate was assessed by using xcelligence systems , gene expression for a panel of genes involved in apoptosis and angiogenesis , and validation of gene expression data at protein level.resultson the selected in vitro model p53 sirna therapy was correlated with the reduction of cell migration . </S>", "<S> the downregulation of p53 , pten , tnf , nfkb , bcl-2 , icam-2 , vegf , and fgfb was evidenced as response to p53 inhibition.conclusionrnai may be a valuable technology in order to restore the normal cellular phenotype . </S>", "<S> the results in the current research may also have an important significance outside the context of cervical cancer , by using specific inhibitors for p53 for increasing the therapeutic response in a wide range of tumoral pathology . </S>"], "labels": null, "section_names": ["Background and aims", "Materials and methods", "Cell culture and treatment", "Dynamic monitoring of invasion of HeLa cells using the xCELLigence System", "RNA extraction quality control and qRT-PCR", "qRT-PCR data analysis", "VEGF protein quantification", "Results", "Inhibition of HeLa cell migration after p53 gene knockdown", "qRT-PCR results for main genes involved in apoptosis and angiogenesis", "VEGF protein expression", "Discussion", "Conclusions"], "sections": [["cancer is one of the leading causes of death worldwide and accounted for 7.6 million cases ( 13% of all deaths ) in 2008 ; in 2013 in the united states there are 12,340 estimated new cases and 4,030 deaths by cervical cancer [ american cancer society : cancer facts and figures 2013 ] .", "meanwhile in romania , based on statistical data from 2012 , cervical cancer is ranked third place taking in account the women cancer incidence , after the breast and colorectal cancer .", "cervical cancer develops in a multi - stage process , successively passing from a cervix with a normal epithelium to cervical intraepithelial neoplasia and finally leading to invasive cervical cancer . a relevant epidemiologic risk circumstance for the progress of the cervical cancer is represented by the presence of high risk human papillomavirus infections .", "consequently , considerable importance has been given to the molecular premise which leads to the progression of cervical cancer .", "rnai ( rna interference ) is a natural mechanism able to specific inhibit a target gene .", "the prospective exploitation as a therapeutic has lately been strengthened by the small interfering rna ( sirna ) delivery to human tumours .", "rnai has been demonstrated as a relevant investigation device for particular target genes by using exogenous sirnas .", "sirnas are single - stranded non - coding rnas with 2023 nucleotides length , being able to negatively modulate gene expression profile at post - transcriptional level .", "sirnas represent an encouraging novel type of medicaments for cancer therapy , as a result of targeting the mutated tumour suppressor genes or activated oncogenes .", "many recent investigations have shown that sirna can efficiently inhibit oncogene expression in cancer cells .", "various investigations demonstrated clearly that the deactivation of p53 constitutes a crucial step in carcinogenesis .", "the present study was designed to investigate the potential use of p53 rnai to block p53 expression , as well as the subsequent effect on cell invasion and gene expression on the human cervical cancer cell ."], ["hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance", ", cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells", "/ well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) .", "a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations .", "siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability .", "we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by .", "the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen .", "all of the samples had a rin ( rna integrity number ) higher than 7.5 .", "the 8 genes selected were assessed using specific primer and upl based on in silico design . the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) . for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system .", "the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step .", "the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene", "the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no ."], ["hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance", ", cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells", "/ well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) .", "a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations .", "siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability ."], ["we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by ."], ["the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen . the rna concentrations and quality were assessed by the nanodrop-1100 agilent 2100 bioanalyzer spectrophotometer .", "all of the samples had a rin ( rna integrity number ) higher than 7.5 .", "the 8 genes selected were assessed using specific primer and upl based on in silico design .", "the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) .", "for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system .", "the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step ."], ["the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene"], ["the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no ."], ["xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels .", "the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells .", "this data can facilitate to comprehend the response to treatment in dynamic . in figure 1", "we can observe a delay and a reduction of the cell migration after the p53sirna treatment .", "taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis .", "relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 .", "after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) ."], ["xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels .", "the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells .", "this data can facilitate to comprehend the response to treatment in dynamic . in figure 1", "we can observe a delay and a reduction of the cell migration after the p53sirna treatment ."], ["taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis . relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 ."], ["after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) ."], ["although at this moment cervical cancer is considered as a preventable disorder there is a significant risk of disease recurrence causing a persuasive necessity to research new therapeutic targets for this disease management .", "it is now well recognized that the tumour progression of all cancers is characterized by intensified proliferation and invasion rate and diminished in apoptosis . at the same time", "the apoptosis and angiogenesis are interconnected as can be observed from figure 4 , using string.9 database .", "the idea of this study is in agreement with the previous studies which are based on the hypothesis that once mutated , p53 exercised oncogenic role . by using sirna", "the role of the present study is to emphasize the cooperation between oncogenic mechanisms , confirming the crosstalk between apoptotic and angiogenic mechanisms .", "this has a significant therapeutic relevance based on the fact that mutated p53 is related to cancer aggressiveness   or to promoting metastasis . in a similar study", "was observed that , by using sirna targeting p53/p73 , tumoral cells were sensitized to chemotherapy . in a recent study , pinx1 was displayed as a novel target gene of p53 , proposing the suppression of p53/pinx1 pathway as a novel mechanism to enhance the telomerase activity in cervical cancer cells , and leading to reduction of cell proliferation .", "targeting oncogenic signals such as the mutated p53 gene that gains oncogenic role , we observed a downregulation of specific genes involved in apoptosis but also in angiogenesis , connected with a reduction of the invasion rate in the case of p53sirna therapy .", "after 24 hours of treatment with p53sirna we can observe downregulations of important genes involved in apoptosis and angiogenesis , as displayed in figure 2 .", "this study confirmed our hypothesis that p53 plays a central role in carcinogenesis as displayed in figure 3 , as important therapeutic target .", "p53 could promote the convergence of the apoptotic and angiogenic pathways in cervical cancer cells .", "it has been proved that p53 is able to indirectly downregulate vegf gene expression , via the retinoblastoma ( rb ) , in a p21-dependent mode , confirming its function in cell - cycle regulation as displayed by recent studies .", "the complete comprehension of the cross - talk between p53 and angiogenic pathways represents a challenge in cancer therapy [ 2426 ] .", "another study reveals that mutant p53 correlates with overexpression of vegf . in a similar study", "the inhibition of mutant p53 represents a significant therapeutic target , a fact emphasized by walerych as  rebel angel ."], ["p53 gene knockdown in hela cells plays an important role not only in apoptosis but also in angiogenesis through various mechanisms that regulate tumour cell development and migration .", "the angiogenic phenotype related to p53 targeted by sirna in human cancers is frequently associated with resistance to conventional genotoxic anticancer agents .", "therefore , p53 inhibitions become dependent and rely on angiogenesis for growth and metastasis . the results in the current research", "may also have an important significance outside the context of cervical cancer , by using specific inhibitors for p53 for increasing therapeutic response in a wide range of tumoral pathology ."]]}
{"article_id": "PMC3644743", "article_text": ["atherosclerosis is a coronary heart disease ( chd ) , and is the most common cause of death in the industrialized world .", "high blood pressure ( bp ) , hypercholesterolemia , diabetes mellitus ( dm ) and smoking are considered as the major risk factors for the development of atherosclerosis .", "in addition , increasing age , male sex , family history and genetic factors predispose patients to chd.1 there is some data that have suggested the possible potential role of some infectious agents in the pathogenesis of atherosclerosis.26 the possibility of infectious agents may trigger the process of atherosclerosis by direct or indirect inflammatory effects , especially at younger ages.5 although different studies have been carried out to prove this hypothesis some infections may contribute in the pathogenesis of atherosclerosis ; nothing has ever been conclusively proven . the possible role of some agents such as chlamydophila pneumoniae,6 \n hepatitis c. virus,7 \n human immunodeficiency virus,7 \n hepatitis b virus,8 \n cytomegalovirus,9 \n herpes virus,9 \n epstein - barr virus,10 \n mycobacterium tuberculosis,11 and helicobacter pylori,3 have been considered in the pathogenesis of atherosclerosis . among these pathogens , c. pneumoniae is important , because it could be effectively treated by antibiotics.12 \n c. pneumoniae is an obligatory intracellular organism , and is responsible for at least 10% of community - acquired pneumonias .", "this bacterium has also been associated with pharyngitis , bronchitis , otitis , influenza - like illness , sinusitis and myocarditis.1214 conflicting results have been reported about the relationship between c. pneumonia and atherosclerosis .", "west et al.15 apfalter et al.,16 and zibaeenezhad et al.17 reported no association between c. pneumoniae and atherosclerosis but others found significant association between c. pneumoniae and atherosclerosis.61819 the results of jha et al.,6 dabiri et al.18 and sessa et al.19 support the idea that c. pneumoniae may have a role in the development of atherosclerosis .", "this controversy about the role c. pneumoniae in the pathogenesis of atherosclerosis emphasizes the importance of more research works .", "the aim of this study was to demonstrate whether there is any difference between two group of patients with coronary atherosclerotic plaque and subjects without coronary atherosclerosis for the presence of c. pneumoniae dna by polymerase chain reaction ( pcr ) method .", "this case - control study was carried out on 60 formalin - fixed paraffin - embedded ( ffpe ) coronary artery biopsies in the molecular pathology laboratory , ghaem hospital , mashhad , iran in 2010 .", "coronary artery tissues were selected from another study that was performed previously by our colleagues for determining prevalence of atherosclerotic plaques in autopsy cases with non - cardiac death in northeast iran . in this study , left coronary artery ( lca ) , right coronary artery ( rca ) and left circumflex artery ( lcx ) had been evaluated grossly and microscopically .", "our materials included 30 coronary artery tissues with atherosclerotic plaques that were defined as study group , and 30 coronary artery tissues without atherosclerotic plaques that served as the control group .", "archived slides of the two groups were reviewed by two pathologists for confirmation of diagnosis , tissue adequacy and selection of the best paraffin blocks for dna extraction .", "for all subjects , the weight and height were determined , and the body mass index ( bmi ) was calculated .", "five to seven , 5-m - thick sectionswere cut from each ffpe specimen under sterile conditions , and then the dna was extracted by using proteinase k and non - heating dna extraction method.20 dna concentration was determined by using the thermo scientific nanodrop 2000 spectrophotometer , and specimens with low dna content ( < 20 ng/l ) were excluded from the study .", "pcr was performed for detecting c. pneumoniae dna by using the pcr kits ( dna technology ( jsc ) , pcr kit , moscow , russia ) .", "10 l of pcr master mix and 0.5 l unit of taqpolymerase ( hot start taq dna polymerase ) were added into each paraffin sealed tube and were mixed , then 5 l ( 100 ng ) of dna samples were added ( except for positive and negative controls ) and were spun at 1,000 rpm for 3 - 5 s. the tubes were placed into applied biosystems ( abi ) veriti thermal cycler and pcr was performed with the program of 180 s at 94c for the first step and then 45 cycles was run as follows : 50 s at 94c , 50 s at 64c , 50 s at 72c .", "amplified pcr products were electrophoresed on a 2% agarose gel , stained with ethidium bromide , and photographed under ultraviolet light by gel documentation instrument .", "pcr was interpreted as positive when the dna band corresponds to the band of the positive control ( 254bp ) in addition to the internal control band ( ic:370 bp ) .", "results of pcr in control and atherosclerotic groups were analyzed with spss version 11.5 by a statistician .", "we used fisher 's exact test for comparison of categorical variables and independent sample t - test for continuous variable in patients and control groups .", "the age range in the control group was 20 - 46 years with a mean ( standard deviation ) age of 26.0 ( 6.41 ) years . in the study group ,", "it ranges from 18 to 50 years with a mean ( sd ) age of 32.0 ( 9.46 ) years and the t - test showed no significant difference between the ages of patients in the study and control groups ( p=0.97 ) .", "twenty - five patients ( 83.3% ) were males and five patients ( 16.7% ) were females .", "the male to female ratio was 5:1 and no significant difference was seen between study and control groups for gender ( p = 1.00 ) by fisher 's exact test .", "the mean bmi of the patients in the study and control groups were 24.4 ( 3.77 ) and 23.9 ( 2.62 ) kg / m respectively [ table 1 ] . the frequency of atherosclerotic plaques in the different vessels of heart of the patients in the study is as shown in table 2 . as a result , the possibility of the appearance of advanced atherosclerotic plaques s in lca was higher than the two other vessels .", "concurrent occurrences of different stages of atherosclerosis were seen in our patients . only one vessel involvement with fibro - fatty morphology ( mild )", "was seen in five patients ( 16.7% ) , one vessel with advanced plaque was observed in three patients ( 10.0% ) , seven patients ( 23.3% ) had one vessel with fibro - fatty and one vessel with advanced plaque , two vessels with advanced plaque were noticed in six patients ( 20.0% ) , three vessels with advanced plaque were observed in five patients ( 16.7% ) and four patients ( 13.3% ) had two vessels with advanced plaque and one fibro - fatty morphology .", "comparisons between patients with coronary atherosclerosis and controls for weight , height and body mass index the frequency of atherosclerotic plaques in different vessels of the heart in the study group positive pcr result for c. pneumonia was seen in one ( 3.3% ) sample among the 30 coronary artery tissues with atherosclerosis ( advanced plaque ) , while all the control samples were negative .", "fisher 's exact test showed no significant difference for detection of c. pneumoniae between samples of coronary artery with and without atherosclerotic plaque ( p = 1 ) .", "ischemic heart disease could be observed in the absence of its major risk factors such as increasing age , male gender , hypertension , hyperlipidemia , smoking and dm .", "inflammatory cells are seen during all steps of atherogenesis , and some infectious agents may trigger this inflammation.113 although some studies showed that c. pneumoniae can increase the risk of atherosclerosis , 61819 but we did not find any significant difference in the frequency of c. pneumoniae dna isolation in the coronary arteries with and without atherosclerosis by pcr method .", "we believe that at least two important factors can account for this discrepancy in the results from previous studies : methodology and epidemiology .", "c pneumonia infection can be diagnosed by various methods such as serology with an increase in serum antibodies against the organism , dna detectionby pcr , immunocytochemistry , electron microscopyand c. pneumonia isolation by culture.14 in other studies , the most widely used method for identifying of c. pneumoniae infection was based on antibody detection .", "the frequency of antibody against c. pneumoniae starts to rise in children and is observed approximately in 50% of adolescents.1214 somesero - epidemiologic studies have shown a relationship between c. pneumoniae and atherosclerosis .", "saikku et al.,21 podsiady et al.22 and romano et al.23 detected a higher level of antibody against c. pneumoniae in chd compared to control group but zibaeenezhad et al.,17 romeo et al.,24 and ericson et al.,25 found that the presence of antibodies was unable to predict coronary artery events .", "we used pcr for detection of c. pneumonia ; this assay appears to be more sensitive than cell culture.1426 our finding was in concordance with those of west et al.,15 jantos et al.27 and satpathy et al.28 similarly , reszka et al.,29 studied presence of c. pneumonia dna in aortic vessels , voorend et al.30 in cerebral vessels , and kwon et al.31 in carotid arteries and could not establish any significant difference in pcr results between the study and control groups .", "these findings suggest that serology could detect past as well as present infection , whereas pcr detects current c. pnuemoniae infection . considering the study of west et al.15 and satpathy et al.28 who showed a higher c. pnuemoniae igg antibody in patients with chd compared to control group but could not detect the organism by nested pcr assay in any specimen strengthens our hypothesis .", "we believe that the lack of standardized methods for the detection of c. pnuemoniae infection used in the aforementioned studies is probably one of the important reasons for the discrepancies in their results . c. pnuemoniae is a common human pathogen ; the majority of infected subjects have few or no symptoms .", "the frequency of antibodies to c. pneumoniae is approximately 50% in the northern hemisphere.1432 exposure to c. pneumoniae is probably common in iran .", "moghaddam et al.33 reported 38% seropositivity for c. pneumonia antibodies ( igg and igm ) in healthy subjects by elisa method in tehran , iran .", "studies carried out in iran on the association between c. pneumonia infection , and atherosclerosis revealed conflicting results ; while some studies showed the association between c. pneumonia infection , and atherosclerosis1834 others didnt demonstrate any significant associationbetween c. pneumonia and atherosclerosis.1735 the anatomical localization of vessels may also be a factor influencing the results from previous studies .", "jha et al.6 showed that c. pneumoniae may be associated with chd and coronary artery was more susceptible to c. pneumoniae as compared with carotid artery .", "bahrmand et al.25 and ericson et al.34 examined the relationship of c. pneumoniae infection to the severity of coronary atherosclerosis by pcr and serology respectively .", "they examined coronary artery samples with two different methods , and both revealed a high rate of reactivity to this bacterium in severe atherosclerosis and a much lower rate in mild atherosclerosis ; therefore , they concluded that severity ( stage ) of atherosclerosis can be presumed as an important factor in diagnosis of c. pneumoniae infection .", "our study was performed on 30 archived ffpe coronary artery tissues of patients with atherosclerosis .", "this sample size may be too small for final decision ; therefore , future study with a larger number of case groups and also using fresh specimens are helpful . in conclusion , we did not find any significant association between c. pneumoniae infection and coronary artery atherosclerosis by pcr method .", "further studies involving a greater number of patients and also using more specific methods for detecting living c. pneumonia organisms by culture can be helpful ."], "abstract_text": ["<S> background : atherosclerosis is a coronary heart disease , andis the most common cause of death in the industrialized world . </S>", "<S> some studies suggested that atherosclerosis may be triggered by infectious agents , mostly chlamydophila pneumoniae . </S>", "<S> however , the role of c. pneumoniae in the pathogenesis of coronary atherosclerosis is still controversial.objectives:this study was performed to evaluate whether there is a significant association between coronary artery atherosclerosis and c. pneumoniae by the polymerase chain reaction ( pcr ) method.materials and methods : this case - control study was carried out on formalin - fixed paraffin - embedded tissue biopsies of the coronary arteries obtained from 30 patients with coronary atherosclerosis and 30 subjects without atherosclerosis living in northeast of iran . </S>", "<S> all subjects ' weight and height were determined , and the body mass index was calculated . </S>", "<S> we also reviewed the medical history and previous laboratory reports of patients . </S>", "<S> deoxyribonucleic acid ( dna ) was extracted , and c. pneumonia dna was amplified and detected using pcr assay.results:the age of the patients in the study group was from 18 to 50 years , and the male to female ratio was 5:1 . only oneout of the 30 coronary tissue samples had positive pcr for c. pneumoniae ( 3.3% ) , while it was negative for patients in the control group.conclusions:this study showed that c. pneumoniae infection is not strongly associated with coronary artery atherosclerosis in northeast of iran . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["atherosclerosis is a coronary heart disease ( chd ) , and is the most common cause of death in the industrialized world .", "high blood pressure ( bp ) , hypercholesterolemia , diabetes mellitus ( dm ) and smoking are considered as the major risk factors for the development of atherosclerosis .", "in addition , increasing age , male sex , family history and genetic factors predispose patients to chd.1 there is some data that have suggested the possible potential role of some infectious agents in the pathogenesis of atherosclerosis.26 the possibility of infectious agents may trigger the process of atherosclerosis by direct or indirect inflammatory effects , especially at younger ages.5 although different studies have been carried out to prove this hypothesis some infections may contribute in the pathogenesis of atherosclerosis ; nothing has ever been conclusively proven . the possible role of some agents such as chlamydophila pneumoniae,6 \n hepatitis c. virus,7 \n human immunodeficiency virus,7 \n hepatitis b virus,8 \n cytomegalovirus,9 \n herpes virus,9 \n epstein - barr virus,10 \n mycobacterium tuberculosis,11 and helicobacter pylori,3 have been considered in the pathogenesis of atherosclerosis . among these pathogens , c. pneumoniae is important , because it could be effectively treated by antibiotics.12 \n c. pneumoniae is an obligatory intracellular organism , and is responsible for at least 10% of community - acquired pneumonias .", "this bacterium has also been associated with pharyngitis , bronchitis , otitis , influenza - like illness , sinusitis and myocarditis.1214 conflicting results have been reported about the relationship between c. pneumonia and atherosclerosis .", "west et al.15 apfalter et al.,16 and zibaeenezhad et al.17 reported no association between c. pneumoniae and atherosclerosis but others found significant association between c. pneumoniae and atherosclerosis.61819 the results of jha et al.,6 dabiri et al.18 and sessa et al.19 support the idea that c. pneumoniae may have a role in the development of atherosclerosis .", "this controversy about the role c. pneumoniae in the pathogenesis of atherosclerosis emphasizes the importance of more research works .", "the aim of this study was to demonstrate whether there is any difference between two group of patients with coronary atherosclerotic plaque and subjects without coronary atherosclerosis for the presence of c. pneumoniae dna by polymerase chain reaction ( pcr ) method ."], ["this case - control study was carried out on 60 formalin - fixed paraffin - embedded ( ffpe ) coronary artery biopsies in the molecular pathology laboratory , ghaem hospital , mashhad , iran in 2010 .", "coronary artery tissues were selected from another study that was performed previously by our colleagues for determining prevalence of atherosclerotic plaques in autopsy cases with non - cardiac death in northeast iran . in this study , left coronary artery ( lca ) , right coronary artery ( rca ) and left circumflex artery ( lcx ) had been evaluated grossly and microscopically .", "our materials included 30 coronary artery tissues with atherosclerotic plaques that were defined as study group , and 30 coronary artery tissues without atherosclerotic plaques that served as the control group .", "archived slides of the two groups were reviewed by two pathologists for confirmation of diagnosis , tissue adequacy and selection of the best paraffin blocks for dna extraction .", "for all subjects , the weight and height were determined , and the body mass index ( bmi ) was calculated .", "five to seven , 5-m - thick sectionswere cut from each ffpe specimen under sterile conditions , and then the dna was extracted by using proteinase k and non - heating dna extraction method.20 dna concentration was determined by using the thermo scientific nanodrop 2000 spectrophotometer , and specimens with low dna content ( < 20 ng/l ) were excluded from the study .", "pcr was performed for detecting c. pneumoniae dna by using the pcr kits ( dna technology ( jsc ) , pcr kit , moscow , russia ) .", "10 l of pcr master mix and 0.5 l unit of taqpolymerase ( hot start taq dna polymerase ) were added into each paraffin sealed tube and were mixed , then 5 l ( 100 ng ) of dna samples were added ( except for positive and negative controls ) and were spun at 1,000 rpm for 3 - 5 s. the tubes were placed into applied biosystems ( abi ) veriti thermal cycler and pcr was performed with the program of 180 s at 94c for the first step and then 45 cycles was run as follows : 50 s at 94c , 50 s at 64c , 50 s at 72c .", "amplified pcr products were electrophoresed on a 2% agarose gel , stained with ethidium bromide , and photographed under ultraviolet light by gel documentation instrument .", "pcr was interpreted as positive when the dna band corresponds to the band of the positive control ( 254bp ) in addition to the internal control band ( ic:370 bp ) .", "results of pcr in control and atherosclerotic groups were analyzed with spss version 11.5 by a statistician .", "we used fisher 's exact test for comparison of categorical variables and independent sample t - test for continuous variable in patients and control groups ."], ["the age range in the control group was 20 - 46 years with a mean ( standard deviation ) age of 26.0 ( 6.41 ) years . in the study group ,", "it ranges from 18 to 50 years with a mean ( sd ) age of 32.0 ( 9.46 ) years and the t - test showed no significant difference between the ages of patients in the study and control groups ( p=0.97 ) .", "twenty - five patients ( 83.3% ) were males and five patients ( 16.7% ) were females .", "the male to female ratio was 5:1 and no significant difference was seen between study and control groups for gender ( p = 1.00 ) by fisher 's exact test .", "the mean bmi of the patients in the study and control groups were 24.4 ( 3.77 ) and 23.9 ( 2.62 ) kg / m respectively [ table 1 ] . the frequency of atherosclerotic plaques in the different vessels of heart of the patients in the study is as shown in table 2 . as a result , the possibility of the appearance of advanced atherosclerotic plaques s in lca was higher than the two other vessels .", "concurrent occurrences of different stages of atherosclerosis were seen in our patients . only one vessel involvement with fibro - fatty morphology ( mild )", "was seen in five patients ( 16.7% ) , one vessel with advanced plaque was observed in three patients ( 10.0% ) , seven patients ( 23.3% ) had one vessel with fibro - fatty and one vessel with advanced plaque , two vessels with advanced plaque were noticed in six patients ( 20.0% ) , three vessels with advanced plaque were observed in five patients ( 16.7% ) and four patients ( 13.3% ) had two vessels with advanced plaque and one fibro - fatty morphology .", "comparisons between patients with coronary atherosclerosis and controls for weight , height and body mass index the frequency of atherosclerotic plaques in different vessels of the heart in the study group positive pcr result for c. pneumonia was seen in one ( 3.3% ) sample among the 30 coronary artery tissues with atherosclerosis ( advanced plaque ) , while all the control samples were negative .", "fisher 's exact test showed no significant difference for detection of c. pneumoniae between samples of coronary artery with and without atherosclerotic plaque ( p = 1 ) ."], ["ischemic heart disease could be observed in the absence of its major risk factors such as increasing age , male gender , hypertension , hyperlipidemia , smoking and dm .", "inflammatory cells are seen during all steps of atherogenesis , and some infectious agents may trigger this inflammation.113 although some studies showed that c. pneumoniae can increase the risk of atherosclerosis , 61819 but we did not find any significant difference in the frequency of c. pneumoniae dna isolation in the coronary arteries with and without atherosclerosis by pcr method .", "we believe that at least two important factors can account for this discrepancy in the results from previous studies : methodology and epidemiology .", "c pneumonia infection can be diagnosed by various methods such as serology with an increase in serum antibodies against the organism , dna detectionby pcr , immunocytochemistry , electron microscopyand c. pneumonia isolation by culture.14 in other studies , the most widely used method for identifying of c. pneumoniae infection was based on antibody detection .", "the frequency of antibody against c. pneumoniae starts to rise in children and is observed approximately in 50% of adolescents.1214 somesero - epidemiologic studies have shown a relationship between c. pneumoniae and atherosclerosis .", "saikku et al.,21 podsiady et al.22 and romano et al.23 detected a higher level of antibody against c. pneumoniae in chd compared to control group but zibaeenezhad et al.,17 romeo et al.,24 and ericson et al.,25 found that the presence of antibodies was unable to predict coronary artery events .", "we used pcr for detection of c. pneumonia ; this assay appears to be more sensitive than cell culture.1426 our finding was in concordance with those of west et al.,15 jantos et al.27 and satpathy et al.28 similarly , reszka et al.,29 studied presence of c. pneumonia dna in aortic vessels , voorend et al.30 in cerebral vessels , and kwon et al.31 in carotid arteries and could not establish any significant difference in pcr results between the study and control groups .", "these findings suggest that serology could detect past as well as present infection , whereas pcr detects current c. pnuemoniae infection . considering the study of west et al.15 and satpathy et al.28 who showed a higher c. pnuemoniae igg antibody in patients with chd compared to control group but could not detect the organism by nested pcr assay in any specimen strengthens our hypothesis .", "we believe that the lack of standardized methods for the detection of c. pnuemoniae infection used in the aforementioned studies is probably one of the important reasons for the discrepancies in their results . c. pnuemoniae is a common human pathogen ; the majority of infected subjects have few or no symptoms .", "the frequency of antibodies to c. pneumoniae is approximately 50% in the northern hemisphere.1432 exposure to c. pneumoniae is probably common in iran .", "moghaddam et al.33 reported 38% seropositivity for c. pneumonia antibodies ( igg and igm ) in healthy subjects by elisa method in tehran , iran .", "studies carried out in iran on the association between c. pneumonia infection , and atherosclerosis revealed conflicting results ; while some studies showed the association between c. pneumonia infection , and atherosclerosis1834 others didnt demonstrate any significant associationbetween c. pneumonia and atherosclerosis.1735 the anatomical localization of vessels may also be a factor influencing the results from previous studies .", "jha et al.6 showed that c. pneumoniae may be associated with chd and coronary artery was more susceptible to c. pneumoniae as compared with carotid artery .", "bahrmand et al.25 and ericson et al.34 examined the relationship of c. pneumoniae infection to the severity of coronary atherosclerosis by pcr and serology respectively .", "they examined coronary artery samples with two different methods , and both revealed a high rate of reactivity to this bacterium in severe atherosclerosis and a much lower rate in mild atherosclerosis ; therefore , they concluded that severity ( stage ) of atherosclerosis can be presumed as an important factor in diagnosis of c. pneumoniae infection .", "our study was performed on 30 archived ffpe coronary artery tissues of patients with atherosclerosis .", "this sample size may be too small for final decision ; therefore , future study with a larger number of case groups and also using fresh specimens are helpful . in conclusion , we did not find any significant association between c. pneumoniae infection and coronary artery atherosclerosis by pcr method .", "further studies involving a greater number of patients and also using more specific methods for detecting living c. pneumonia organisms by culture can be helpful ."]]}
{"article_id": "PMC3696216", "article_text": ["confrontation with the consequences of diabetes causes a crisis in physical , mental , and spiritual dimensions . sometimes the spiritual crisis can be tremendous . since spiritual health coordinates different aspects of human life , this study aimed to identify the spiritual health of patients with defects caused by diabetes", "this was a qualitative - phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan .", "outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god .", "the concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and knowing god as cruel .", "the concepts that patients had experienced as contributing factors to the healing process were resorting to imams , god s ordering the disease as a reward , fear of god s punishment , believing in miracles , being closer to god , believing in the mercy of god , returning to religious practice , feeling of enjoying life and knowing that the disease is the atonement of sins .", "with regard to the importance of spiritual and religious care as one of the tasks of nurses , as the key members of health team , they should respect the patients beliefs and values in addition to considering their physical and mental conditions .", "although the mortality rate in diabetic patients due to ketoacidosis and infection is declining , deaths from diabetes complications including diabetic foot complications has increased dramatically . according to some reports , its rate in the first year after amputation is 1340 percent , in the third year", ", 3565 percent and in the fifth year it is 3980 percent , which figures are comparable to death rates in malignancies . in addition , the loss of job and the need for medical and nursing care , reduction in social and family interactions , and lifestyle changes are the major problems that influence the family and socioeconomic status of these patients .", "the absence of the feet and lower limbs can be extremely problematic for these patients and exerts too much energy for walking ; therefore , amputation of higher limbs or development of ulcers may occur .", "ultimately , these patients with amputated legs need long - term hospitalization , rehabilitation , home care and social support .", "organ amputation due to diabetes causes crisis in the physical , mental , and spiritual life of the patients . in this case", ", the spiritual health is the unique element that can harmonize physical , mental and social aspects of the human life and is necessary for coping with the disease . as a matter of fact ,", "when the spiritual health is seriously at risk , a person may experience emotional disturbances such as loneliness , depression and loss of meaning in life .", "the spirit of prayer is a deep human instinct , which is the center of humanity , and where the passion and knowledge to communicate with the world comes from .", "forms and statements of people who pray are different : speaking , listening , waiting and wailing .", "it exposes them to severe stress and they seek different approaches to cope and adapt to life .", "koenig believes that religion creates a positive attitude towards the world and makes the person powerful against unfortunate events in life such as diseases and helps to improve the life with motivation and energy .", "this increases tolerance and acceptance of situations that can not be changed . in many cases of emergency", "in which science is unable to help a person , this issue is of vital importance especially in serious cases .", "the increasing interest of medical scientists to the impact of praying in the treatment of other diseases can be indicative of treatment of some diseases in case of modern medicine failure . on the other hand , medical researchers also acknowledge the importance of medical procedures of traditional and complementary therapies including prayer to treat the diseases .", "even the western scientific community has recently published articles showing that doctors can not be indifferent to the religious beliefs of patients for treatment .", "this study was conducted to understand the spiritual experiences of the patients with organ amputation due to diabetes .", "the aim of phenomenological studies is to understand the phenomena of human experience through the analysis of participants verbal explanations of experiences .", "the study population consisted of patients with defects , caused by diabetes , who had referred to rehabilitation centers in esfahan and zanjan provinces .", "sampling was purposive and the inclusion criteria for participants were the injury caused by diabetes and the willingness to participate in the interview .", "there were 15 participants entered the study ( 8 men and 7 women at 5070 years of age ) .", "all interviews were fully recorded and all participants were given a code based on the interviews .", "in this study , the researcher tried to be cautious with the information collected to be free from any kind of bias and to avoid persons with poor memory and those who could not provide the correct information . in the interviews , the researcher attempted to withdraw his assumptions about the phenomenon under study and thus help to strengthen the data .", "they were asked that  how has your life changed compared to before the disease ?  in response , they described their life experiences with this disability , and how their performance was changed due to the disease . during the interview , the patients focused on cases where the disease led to their functioning .", "when it was necessary to clarify the data in specific areas , the questions were asked in details . finally , participants were asked if they wanted to provide alternative explanations in relationship with their disease .", "the researcher wrote down the participants statements through repeated listening to in - depth interviews .", "after the formation of concepts and ideas from each interview , the next interview was conducted .", "the above process was repeated for each interview ( second stage of colaizzi s method ) .", "after the all interviews , the concepts were formulated in particular subjects and were classified into categories ( third stage ) and eventually all ideas were combined so that a complete description of the phenomenon under the study was derived and presented ( the fourth , fifth and sixth stages ) .", "finally , the concepts were returned to the participants and were analyzed ( the seventh ) . in this study ,", "the criteria for validity and reliability of data were lincoln and guba . in order to make the study believable ,", "the participants and the research partners also reviewed all the data . to achieve reliability and objectivity of the research data , the account access was used . to increase the portability of the present study , the complete research project was introduced .", "there were 15 patients participating in this study , comprised of 7 women and 8 men .", "the age range of participants was between 50 - 75 years with mean age of 59.7 years .", "length of diabetes was between 530 years and the length of organ amputation caused by diabetes also varied from 2 weeks to 11 years with mean of 4.2 years . the education level comprised of uneducated to junior high school .", "regarding employment , 53.3 percent of the participants ( 8 people ) lost their jobs and were unemployed due to organ amputation caused by diabetes .", "about 66.6 percent ( 10 people ) , a leg was amputated below the knee and 26.6 percent of the participants ( 4 people ) both legs were amputated below the knee area . in one patient both legs below the knee and the middle finger were amputated due to diabetes .", "the concepts derived from patients experiences as factors interfering spiritual health in the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and considering god cruel .", "factors promoting the healing process were deemed by god , fear of god punishment , belief in miracles , being closer to god , believing god to be merciful , returning to religious practice , enjoying life and indicating the disease as the atonement of sins .", "most of the participants , encounteringdiabetes - related organ amputatio , said that in such circumstances , intellectual protections , religious sources , and having a strong relationship with a higher power could improve the quality of life , and support the individuals and reduce the severity of symptoms .", "one of the participants expressed his experiences in turning to practice religion as follows : ", "i was deemed to cut my foot to know god , i am sure there is a wisdom behind this , i ve been weak in a prayer .", "i was wavering , but when my leg was cut , i celt much closer to god , i know god took my foot , but after this incident , i feel that god wanted to try me , before this , if my prayer was late , i did not become upset .", "but now , if i do not perform my prayer on time , i will get upset , i feel that i did not do an important job , i become worried with no reason   ( participant 1 ) .", "another participant stated  when my uncle came to visit me , he said the disease is the expiation of sins .", "whenever this is said , my heart becomes calm , but i did not commit a sin to deserve such a punishment . even , i had not bothered anything in my life .", "i do not remember , i wish , everything could be finished in this world   ( participant 3 ) . ", "since my foot ( right foot ) has been cut off , i rub it every day . as i can not bend", "i m afraid ; it may give testimony against me in the doomsday and tells god that i had been overeating because our hands and legs have rights and we must consider this   ( participant 5 ) .  our tenant is a believer who always reads the quran .", "when she saw that i was confused , she said , let me read the koran for you to be calm .", "although i am illiterate and i do not understand anything from the quran , when i hear the peaceful verses of the quran , i forget my sorrows  ", " i participate in the middle of moharram mourning ceremony every year and in mourning ceremonies every thursday , but when my leg was cut , i did not go there anymore because i feel i m too far from god and as a result , these ceremonies can not help  ", "when the wounds of the foot were fine after surgery , at fast ; i performed my prayer in sitting position , but i realized that i do not feel good in just sitting and praying , i just hurt myself   ( participant 15 ) .", "the prayer and vow are likely to enhance the human s tolerance against diseases and problems communication with the source of existence and asking him to help would repair the physical and mental powersof man and relieves a lot of disorders and diseases .", "therefore , the appeal to the imams in patients with organ amputation due to diabetes is high .", "several studies have also shown that when the spiritual health is at a serious risk , prayers in acute cases may face difficulty . in these cases , praying for the patient can be an important spiritual intervention .", "one of the most useful sentences in the form of prayers could be the short ones , asking god for fulfillment of the needs , melting away the fears and giving hopes to the patient and ultimately remembering that god is able to meet the needs of the patients .", "if the patient is depressed , angry or upset or suffering from extreme pain , the expression of praying sentences is like the salt to the wound .", "nurses should pay close attention in the implementation of these provisions , and if the patients are upset , they should leave them alone . in this regard , a research by tracy et al .", "was conducted on two thousand nurses in critical care units in which 55% of nurses reported that patients and families were asking them to pray .", "another study which studied the effects of prayer on blood infection , randomly employed 1691 experimental and 1702 control patients .", "parameters studied included duration of fever in patients , duration of hospitalization and mortality in the hospital .", "the results of this study showed a significant difference between the duration of fever and length of patients stay that was lower in the intervention group compared to the control group .", "nowadays , some of the organizations that evaluate health care systems and are responsible for granting accreditation to them , suggest that the spiritual needs of the patients in health care should be also evaluated .", "finally , the importance of spiritual and religious care , which is today considered as one of nurses tasks , urges them as the key members of health teams to respect patients beliefs and values as well as their physical and mental aspects ."], "abstract_text": ["<S> background : confrontation with the consequences of diabetes causes a crisis in physical , mental , and spiritual dimensions . </S>", "<S> sometimes the spiritual crisis can be tremendous . </S>", "<S> since spiritual health coordinates different aspects of human life , this study aimed to identify the spiritual health of patients with defects caused by diabetes.materials and methods : this was a qualitative - phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan . </S>", "<S> a purposive sample of 15 participants underwent deep interviews . </S>", "<S> colaizzi s method of analysis was used to analyze the data.findings:outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god . </S>", "<S> the concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and knowing god as cruel . </S>", "<S> the concepts that patients had experienced as contributing factors to the healing process were resorting to imams , god s ordering the disease as a reward , fear of god s punishment , believing in miracles , being closer to god , believing in the mercy of god , returning to religious practice , feeling of enjoying life and knowing that the disease is the atonement of sins.conclusions:with regard to the importance of spiritual and religious care as one of the tasks of nurses , as the key members of health team , they should respect the patients beliefs and values in addition to considering their physical and mental conditions . </S>"], "labels": null, "section_names": ["Background:", "Materials and Methods:", "Findings:", "Conclusions:", "I", "M", "F", "D"], "sections": [["confrontation with the consequences of diabetes causes a crisis in physical , mental , and spiritual dimensions . sometimes the spiritual crisis can be tremendous . since spiritual health coordinates different aspects of human life , this study aimed to identify the spiritual health of patients with defects caused by diabetes"], ["this was a qualitative - phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan ."], ["outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god .", "the concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and knowing god as cruel .", "the concepts that patients had experienced as contributing factors to the healing process were resorting to imams , god s ordering the disease as a reward , fear of god s punishment , believing in miracles , being closer to god , believing in the mercy of god , returning to religious practice , feeling of enjoying life and knowing that the disease is the atonement of sins ."], ["with regard to the importance of spiritual and religious care as one of the tasks of nurses , as the key members of health team , they should respect the patients beliefs and values in addition to considering their physical and mental conditions ."], ["although the mortality rate in diabetic patients due to ketoacidosis and infection is declining , deaths from diabetes complications including diabetic foot complications has increased dramatically . according to some reports , its rate in the first year after amputation is 1340 percent , in the third year", ", 3565 percent and in the fifth year it is 3980 percent , which figures are comparable to death rates in malignancies . in addition , the loss of job and the need for medical and nursing care , reduction in social and family interactions , and lifestyle changes are the major problems that influence the family and socioeconomic status of these patients .", "the absence of the feet and lower limbs can be extremely problematic for these patients and exerts too much energy for walking ; therefore , amputation of higher limbs or development of ulcers may occur .", "ultimately , these patients with amputated legs need long - term hospitalization , rehabilitation , home care and social support .", "organ amputation due to diabetes causes crisis in the physical , mental , and spiritual life of the patients . in this case", ", the spiritual health is the unique element that can harmonize physical , mental and social aspects of the human life and is necessary for coping with the disease . as a matter of fact ,", "when the spiritual health is seriously at risk , a person may experience emotional disturbances such as loneliness , depression and loss of meaning in life .", "the spirit of prayer is a deep human instinct , which is the center of humanity , and where the passion and knowledge to communicate with the world comes from .", "forms and statements of people who pray are different : speaking , listening , waiting and wailing .", "it exposes them to severe stress and they seek different approaches to cope and adapt to life .", "koenig believes that religion creates a positive attitude towards the world and makes the person powerful against unfortunate events in life such as diseases and helps to improve the life with motivation and energy .", "this increases tolerance and acceptance of situations that can not be changed . in many cases of emergency", "in which science is unable to help a person , this issue is of vital importance especially in serious cases .", "the increasing interest of medical scientists to the impact of praying in the treatment of other diseases can be indicative of treatment of some diseases in case of modern medicine failure . on the other hand , medical researchers also acknowledge the importance of medical procedures of traditional and complementary therapies including prayer to treat the diseases .", "even the western scientific community has recently published articles showing that doctors can not be indifferent to the religious beliefs of patients for treatment .", "this study was conducted to understand the spiritual experiences of the patients with organ amputation due to diabetes ."], ["the aim of phenomenological studies is to understand the phenomena of human experience through the analysis of participants verbal explanations of experiences .", "the study population consisted of patients with defects , caused by diabetes , who had referred to rehabilitation centers in esfahan and zanjan provinces .", "sampling was purposive and the inclusion criteria for participants were the injury caused by diabetes and the willingness to participate in the interview .", "there were 15 participants entered the study ( 8 men and 7 women at 5070 years of age ) .", "all interviews were fully recorded and all participants were given a code based on the interviews .", "in this study , the researcher tried to be cautious with the information collected to be free from any kind of bias and to avoid persons with poor memory and those who could not provide the correct information . in the interviews , the researcher attempted to withdraw his assumptions about the phenomenon under study and thus help to strengthen the data .", "they were asked that  how has your life changed compared to before the disease ?  in response , they described their life experiences with this disability , and how their performance was changed due to the disease . during the interview , the patients focused on cases where the disease led to their functioning .", "when it was necessary to clarify the data in specific areas , the questions were asked in details . finally , participants were asked if they wanted to provide alternative explanations in relationship with their disease .", "the researcher wrote down the participants statements through repeated listening to in - depth interviews .", "after the formation of concepts and ideas from each interview , the next interview was conducted .", "the above process was repeated for each interview ( second stage of colaizzi s method ) .", "after the all interviews , the concepts were formulated in particular subjects and were classified into categories ( third stage ) and eventually all ideas were combined so that a complete description of the phenomenon under the study was derived and presented ( the fourth , fifth and sixth stages ) .", "finally , the concepts were returned to the participants and were analyzed ( the seventh ) . in this study ,", "the criteria for validity and reliability of data were lincoln and guba . in order to make the study believable ,", "the participants and the research partners also reviewed all the data . to achieve reliability and objectivity of the research data , the account access was used . to increase the portability of the present study , the complete research project was introduced ."], ["there were 15 patients participating in this study , comprised of 7 women and 8 men .", "the age range of participants was between 50 - 75 years with mean age of 59.7 years .", "length of diabetes was between 530 years and the length of organ amputation caused by diabetes also varied from 2 weeks to 11 years with mean of 4.2 years . the education level comprised of uneducated to junior high school .", "regarding employment , 53.3 percent of the participants ( 8 people ) lost their jobs and were unemployed due to organ amputation caused by diabetes .", "about 66.6 percent ( 10 people ) , a leg was amputated below the knee and 26.6 percent of the participants ( 4 people ) both legs were amputated below the knee area . in one patient both legs below the knee and the middle finger were amputated due to diabetes .", "the concepts derived from patients experiences as factors interfering spiritual health in the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and considering god cruel .", "factors promoting the healing process were deemed by god , fear of god punishment , belief in miracles , being closer to god , believing god to be merciful , returning to religious practice , enjoying life and indicating the disease as the atonement of sins .", "most of the participants , encounteringdiabetes - related organ amputatio , said that in such circumstances , intellectual protections , religious sources , and having a strong relationship with a higher power could improve the quality of life , and support the individuals and reduce the severity of symptoms .", "one of the participants expressed his experiences in turning to practice religion as follows : ", "i was deemed to cut my foot to know god , i am sure there is a wisdom behind this , i ve been weak in a prayer .", "i was wavering , but when my leg was cut , i celt much closer to god , i know god took my foot , but after this incident , i feel that god wanted to try me , before this , if my prayer was late , i did not become upset .", "but now , if i do not perform my prayer on time , i will get upset , i feel that i did not do an important job , i become worried with no reason   ( participant 1 ) .", "another participant stated  when my uncle came to visit me , he said the disease is the expiation of sins .", "whenever this is said , my heart becomes calm , but i did not commit a sin to deserve such a punishment . even , i had not bothered anything in my life .", "i do not remember , i wish , everything could be finished in this world   ( participant 3 ) . ", "since my foot ( right foot ) has been cut off , i rub it every day . as i can not bend", "i m afraid ; it may give testimony against me in the doomsday and tells god that i had been overeating because our hands and legs have rights and we must consider this   ( participant 5 ) .  our tenant is a believer who always reads the quran .", "when she saw that i was confused , she said , let me read the koran for you to be calm .", "although i am illiterate and i do not understand anything from the quran , when i hear the peaceful verses of the quran , i forget my sorrows  ", " i participate in the middle of moharram mourning ceremony every year and in mourning ceremonies every thursday , but when my leg was cut , i did not go there anymore because i feel i m too far from god and as a result , these ceremonies can not help  ", "when the wounds of the foot were fine after surgery , at fast ; i performed my prayer in sitting position , but i realized that i do not feel good in just sitting and praying , i just hurt myself   ( participant 15 ) ."], ["the prayer and vow are likely to enhance the human s tolerance against diseases and problems communication with the source of existence and asking him to help would repair the physical and mental powersof man and relieves a lot of disorders and diseases .", "therefore , the appeal to the imams in patients with organ amputation due to diabetes is high .", "several studies have also shown that when the spiritual health is at a serious risk , prayers in acute cases may face difficulty . in these cases , praying for the patient can be an important spiritual intervention .", "one of the most useful sentences in the form of prayers could be the short ones , asking god for fulfillment of the needs , melting away the fears and giving hopes to the patient and ultimately remembering that god is able to meet the needs of the patients .", "if the patient is depressed , angry or upset or suffering from extreme pain , the expression of praying sentences is like the salt to the wound .", "nurses should pay close attention in the implementation of these provisions , and if the patients are upset , they should leave them alone . in this regard , a research by tracy et al .", "was conducted on two thousand nurses in critical care units in which 55% of nurses reported that patients and families were asking them to pray .", "another study which studied the effects of prayer on blood infection , randomly employed 1691 experimental and 1702 control patients .", "parameters studied included duration of fever in patients , duration of hospitalization and mortality in the hospital .", "the results of this study showed a significant difference between the duration of fever and length of patients stay that was lower in the intervention group compared to the control group .", "nowadays , some of the organizations that evaluate health care systems and are responsible for granting accreditation to them , suggest that the spiritual needs of the patients in health care should be also evaluated .", "finally , the importance of spiritual and religious care , which is today considered as one of nurses tasks , urges them as the key members of health teams to respect patients beliefs and values as well as their physical and mental aspects ."]]}
{"article_id": "PMC3432876", "article_text": ["vitamin d deficiency was once thought to exclusively affect bone metabolism , but now there is ample evidence of its role in many other conditions including metabolic syndrome , autoimmune diseases , and cancer .", "vitamin d receptors are recognized to be in numerous extraskeletal tissues , such as pancreas and muscle .", "a systematic review by pittas and colleagues   reported that vitamin d may have a beneficial effect on the action of insulin , either directly or indirectly .", "several observational studies in adults , including the framingham heart study , have reported an inverse association between vitamin d status and insulin resistance .", "however , data from children and adolescents do not consistently report this inverse relationship [ 510 ] . even in studies reporting this association in adolescents and children ,", "further , none of the studies in adolescents have studied the effect of puberty on this association .", "a fall in insulin sensitivity with compensatory increase in insulin secretion has been reported in puberty [ 11 , 12 ] .", "high prevalence of cardiometabolic risk factors , including insulin resistance ( 64.8% of normal weight children had at least one cardiometabolic abnormality ) , even in healthy young children of normal weight , has been reported in indian children and adolescents .", "south - asian adolescents , including those with normal bmi , have a higher prevalence of insulin resistance when compared with age and bmi matched european adolescents [ 14 , 15 ] .", "this ethnic difference in insulin sensitivity was attributed to the higher body fat percentage in south - asian children .", "an alternative hypothesis to explain the ethnic differences in insulin sensitivity is vitamin d deficiency . despite plenty of sunlight available throughout the year", ", a high prevalence of vitamin d deficiency has been reported in all age groups including toddlers , school children , pregnant women and their neonates , and adult males and females residing in rural and urban india .", "vitamin d deficiency has been reported in 8492% children in the age group of 1018 . in view of high prevalence of both ,", "insulin resistance and vitamin d deficiency in asian - indian children and adolescents , we sought to examine the relationship between vitamin d status and insulin resistance .", "we also sought to determine if puberty affected the relationship of vitamin d status and parameters of insulin resistance and sensitivity in asian - indian children and adolescents .", "the study was conducted as a prospective observational study after approval from the ethics committee of the all india institute of medical sciences ( aiims ) , new delhi .", "subjects were selected from the children and adolescents attending the endocrinology outdoor services at aiims for evaluation of obesity .", "the inclusion criteria included children between the ages of 617 years with obesity as per international obesity taskforce ( iotf ) criteria .", "the exclusion criteria included subjects with diagnosed diabetes mellitus or taking metformin or any weight reducing drugs , subjects with any known systemic illness or endocrine or metabolic disorders , known to be associated with obesity , or subjects with symptoms to suggest hypothalamic obesity were excluded from the study .", "blood samples were collected in fasting state ( minimum 9 hours fasting ) , followed by oral glucose tolerance test ( anhydrous glucose = 1.75  gm / kg bodyweight , maximum of 75  gram , dissolved in 250  ml of water , ingested over 5 minutes ) with sample collection at 60 and 120 minutes after glucose ingestion .  ", "height was measured with holtain 's stadiometer ( holtain inc . , crymych , pembs .", "waist circumference was measured ( nonelastic measuring tape ) at the end of normal expiration at the midpoint between the iliac crest and the lower edge of ribs in the midaxillary line .", "blood pressure ( bp ) was measured in the right upper limb in sitting position with a mercury sphygmomanometer after 5-minute rest with an appropriate size cuff .", "total body fat was measured with dual energy x - ray absorptiometry ( dxa , hologic qdr 4500 with pediatric software , hologic inc . ,", "fat mass index ( fmi , fat mass in kg / height in meter ) and fat - free mass index ( ffmi , lean body mass+ bmc)/(height in meter ) were calculated .", "bone mineral area , content ( bmc ) and areal density ( abmd ) at the femoral neck , lumbar spine ( l1 - 5 ) , and forearm were measured , using a hologic qdr 4500a fan beam dxa machine .", "hologic spine phantom ( hologic spine phantom number 21373 ) was scanned daily before subject evaluation .", "the measured phantom bone mineral density was stable throughout the study period at 0.9150.945  gm / cm .", "similarly , whole body phantom ( hologic wb number 1252 ) was also scanned before subject evaluation and remained stable during study period .", "the in vivo precision error for adults was 0.62% for femoral neck abmd , 0.65% for lumbar spine abmd , and 0.77% for forearm abmd .", "however , in view of additional radiation exposure , the reproducibility of these scans was not assessed among children .", "pubertal stage was assessed by a single endocrinologist , based on breast stage and pubic hair development in girls   and genitalia development in boys .", "b - mode ultrasonography was used to measure cimt ( 7.510  mhz probe , philips envisor ultrasound machine ) using standard protocol .  \n insulin resistance ( ir )", "was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels .the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo .area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule . \n  insulin resistance ( ir )", "was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels .", "the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo .", "area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule .  ", "complete blood counts , liver function tests , renal function test , serum calcium ( corrected calcium with serum albumin ) , phosphate , alkaline phosphatase , uric acid , and blood glucose were measured in all subjects with an automated chemistry analyzer ( roche hitachi 912 chemistry analyzer , gmi inc .", "glycosylated hemoglobin ( hba1c ) was measured in whole blood using ion - exchange high performance liquid chromatography ( bio - rad laboratories inc . , ca , us ) .", "serum insulin was measured on an autoanalyzer ( roche elecsys 2010 ) using electrochemiluminometric assay .", "serum total cholesterol ( tc ) , triglyceride ( tg ) , and hdl cholesterol ( hdl - c ) levels were estimated directly with automated analyzer , while ldl cholesterol ( ldl - c ) was estimated by using the friedewald equation .", "vitamin d deficiency was defined as a serum 25(oh)d levels less than 20  ng / ml .", "this was further subdivided to severe , moderate , and mild vitamin d deficiency if serum 25(oh)d levels were < 5  ng / ml , 5<10  ng / ml , and 10<20  ng / ml respectively .", "statistical analysis was carried out using spss ( spss version 11.5 ; spss inc . ,", "continuous variables not showing normal distribution were treated with appropriate log transformations before any parametric analysis .", "student 's t - test was used to determine statistical difference between subjects with serum 25(oh)d < 10  ng / ml or more .", "pearson 's correlation was used to assess association between continuous variables and partial correlations were used to determine relationship after adjusting for relevant covariates .", "stepwise linear regression analysis was used with serum 25(oh)d as dependent variable in the model .", "values for serum 25(oh)d , serum insulin , homa - ir , and matsuda isi were not normally distributed ; therefore , they were logarithmically transformed which yielded normal distribution .", "a total of 62 subjects participated in this cross - sectional study from june 2008 to may 2009 .", "the mean age of subjects was 13.0  3 years ( 35 boys ; 27 girls ) .", "assessment of pubertal status by tanner 's method showed 19 subjects in stage 1 , 11 in stage 2 , 6 in stage 3 , 3 in stage 4 , and 23 subjects in puberty stage 5 .", "no significant difference was observed between boys and girls except higher whr in boys than girls ( 0.93  0.05 versus 0.89  0.05 ; p = 0.013 ) .", "all study subjects were classified as vitamin d deficient ( mean  sd 8.5  4.2  ng / ml ; maximum  minimum : 3.919.2  ng / ml , median = 6.9  ng / ml ) . severe vitamin d deficiency ( < 5  ng / ml ) was seen in 11 subjects ( 17.7% ) while 30 subjects had serum 25(oh)d levels between 5<10  ng / ml ( 48.3% ) .", "serum 25(oh)d equal to or more than 10 but less than 20  ng / ml was present in 21 subjects ( 33.8% ) .", "pearson correlation coefficient analyses showed significant inverse relationship between serum 25(oh)d and total body fat percentage ( r = 0.31 , p = 0.01 ) and serum 25(oh)d and fmi ( r = 0.33 , p = 0.01 , figure 1 ) .", "partial correlation analysis showed persistence of significant correlation between serum 25(oh)d and total body fat percentage ( r = 0.37 , p = 0.005 ) and between serum 25(oh)d and fmi ( r = 0.36 , p = 0.006 ) even after adjustment for age , sex , pubertal stage , and bmi .", "although blood glucose and serum insulin levels showed inverse relationship trends with serum 25(oh)d , but these were not statistically significant ( data not shown ) .", "similarly , no statistically significant correlation was seen between serum 25(oh)d and parameters of insulin sensitivity or resistance .", "fmi showed positive correlation with homa - ir ( r = 0.26 ; p = 0.04 ) , auc insulin ( r = 0.41 ; p = 0.001 ) , auc glucose ( r = 0.31 ; p = 0.018 ) , and negative correlation with wbisi ( r = 0.42 ; r = 0.001 ) .", "however , total body fat did not show any correlation with parameters of glucose and insulin parameters .   on the basis of serum 25(oh)d ,", "subjects were divided into two groups , group a with serum 25(oh)d level < 10  ng / ml ( n = 41 ) and group b with serum 25(oh)d level 10  ng / ml ( n = 21 ) ( an arbitrary cutoff to compare subjects with mild vitamin d deficiency with moderate and severe vitamin d deficiency , 25 ) .", "no statistical significant difference was seen in blood glucose , serum insulin , and auc for both , glucose and insulin between group 1 and group 2 .", "trends for higher insulin resistance ( homa - ir ) and lower insulin sensitivity ( wbisi ) were seen in subjects with lower serum 25(oh)d concentrations but not statistically significant .", "total body fat percentage and fmi were significantly higher in group 1 in comparison to group 2 ( p = 0.015 and 0.007 , resp . ) .", "however , no significant difference was seen in waist and hip circumference and waist hip ratio .", "similarly , ffmi , lipid parameters , or thyroid profile were also not different between the two groups .", "group 1 ( prepubertal group ; tanner stage 1 ) had 19 subjects , group 2 ( peripubertal group ; tanner stage 2 , 3 , and 4 ) had 20 , while group 3 ( postpubertal group ; tanner stage 5 ) had 23 subjects .", "no significant correlation was seen between vitamin d status and any other parameters including total body fat percentage , fmi and parameters of insulin resistance and sensitivity in the prepubertal ( group 1 ) or peripubertal groups ( group 2 ) .", "partial correlations after adjusting for age , sex , and bmi were also nonsignificant for both groups ( data not shown ) . in postpubertal subjects ( group 3 , n = 23 ) , a significant inverse correlation was seen between serum 25(oh)d and homa - ir ( r = 0.41 , p = 0.03 , figure 2 ) .", "this association between serum 25(oh)d and homa - ir persisted even after control for age , sex , and bmi ( r = 0.505 , p = 0.023 ) .", "positive trends of association between serum 25(oh)d and wbisi were also seen , but this was not significant ( r = 0.29 , p = 0.17 ) , even after adjusting for age , sex , and bmi ( r = 0.35 , p = 0.13 ) .", "serum 25(oh)d showed significant inverse correlation with total body fat percentage ( r = 0.58 , p = 0.003 ) and fmi ( r = 0.49 , p = 0.016 ) which improved after adjusting for age sex and bmi ( r = 0.63 , p = 0.002 for total body fat percentage ; r = 0.582 ,  p = 0.007 for fmi ) .", "serum 25(oh)d also showed significant inverse relationship with fasting serum insulin levels ( r = 0.44 , p = 0.034 ) which improved after adjusting for age , sex , and bmi ( r = 0.53 , p = 0.016 ) .", "when prepubertal subjects ( group 1 ) were compared with the prepubescent subjects ( group 2 ) , no significant difference was seen in any of the parameters including blood glucose , serum insulin , homa - ir , and wbisi .", "similarly , when prepubertal subjects were compared with all other subjects ( subjects in group 2 and 3 combined ) , significantly higher insulin sensitivity ( wbisi , mean  sd 6.07  5.7 versus 3.42  2.7 , p < 0.01 ) and lower insulin resistance ( homa - ir , mean  sd 3.68  2.7 versus 5.22  4.0 , p = 0.04 ) were seen in prepubertal subjects .", "this is the first study examining the relationship between serum 25(oh)d levels and parameters of insulin resistance in obese asian - indian children .", "our study confirms a significant inverse relationship between serum 25(oh)d levels and body fat indices as reported in other studies in children [ 6 , 10 ] .", "we demonstrated trends for that higher serum 25(oh)d levels are associated with better insulin sensitivity and less insulin resistance though this correlation did not reach statistical significance .", "one of the main reasons for the absence of significance could be the universal presence of vitamin d deficiency ( < 20  ng / ml ) resulting in very narrow range of serum 25(oh)d .", "we also show , for the first time , that the association between serum 25(oh)d and insulin resistance and sensitivity is influenced by pubertal staging .", "when subjects were stratified by pubertal stage , only subjects in tanner stage 5 exhibited a significant correlation between serum 25(oh)d and homa - ir , while prepubertal subjects did not show statistically significant correlation .", "high prevalence of vitamin d deficiency has been reported across the age range in pediatric , adolescent , and adult in obese individuals [ 5 , 6 , 2631 ] .", "this may be just reflection of very high prevalence of vitamin d deficiency in asian - indians [ 16 , 17 ] .", "however , high prevalence of vitamin d deficiency has been reported in obese children and adolescents from populations where vitamin d deficiency is not that prevalent [ 5 , 6 , 10 ] .", "reis et al . , in a study of 3577 adolescents in the age group of 1219 years , found significantly lower serum 25(oh)d levels in adolescents with bmi > 95th centile in comparison to subjects with bmi < 95th centile ( p < 0.001 ) .", "it has been postulated that increased sequestration of vitamin d in fat tissues , leading to decreased vitamin d bioavailability , low dietary vitamin d intake due to poor nutritional habits , and minimal sun exposure due to sedentary indoor lifestyle are important factors associated with high prevalence in the obese population [ 29 , 32 ] .", "it has also been suggested that vitamin d deficiency is a cause of common obesity and can account for the secular trends in the prevalence of obesity and for individual differences in its onset and severity . the inverse relationship between serum 25(oh)d and insulin resistance in adults", "have been reported in most studies , though some have failed to find this association . in comparison , reports in children and adolescents have shown conflicting results .", "delvin et al . , in a study of 1745 french - canadian children , reported modest but significant negative associations between serum 25(oh)d and homa - ir .", "each 10-nmol / l ( 4  ng / ml ) increase in serum 25(oh)d was associated with lower glycemia and homa - ir .", "did not find significant correlation between serum 25(oh)d levels and insulin , homa - ir and homa - b % . in another study based on nhanes data of 3577 adolescents from 20012004", "showed that serum 25(oh)d levels were inversely associated with plasma glucose concentration ( p = 0.01 ) .", "lenders et al . , in a study of 58 obese adolescents , did not found any correlation between insulin indices and serum 25(oh)d in both , adjusted and unadjusted models . in our study", ", we did not find a statistically significant association between serum 25(oh)d and parameters of insulin sensitivity and resistance .", "however , trends for higher blood glucose and serum insulin levels were seen in subjects with low serum 25(oh)d level .", "no significant difference was seen in insulin resistance and sensitivity between group 1 and group 2 .", "this is in contrast to the observations made by ashraf et al . who reported in 51 african - american obese female adolescents   that higher serum 25(oh)d concentrations were associated with significantly higher wbisi ( p = 0.018 ) but no difference in homa - ir .", "these differences may be due to marked difference in severity of obesity as mean bmi of study subjects was 43.3  9.9 in study by ashraf et al .", ", while it was 29.3  9.8  kg / sqm in our study subjects .", "studies in adult population have shown that parameters of insulin sensitivity and insulin resistance as calculated by hyperinsulinemic euglycemic clamp study ( m value ) correlated better with serum 25(oh)d levels than indirect parameters like homa - ir .", "studies with direct measures of insulin sensitivity like clamp studies are required to further investigate these issues in pediatric and adolescent population .", "the phenomenon of pubertal insulin resistance has been well described in cross - sectional as well as longitudinal studies .", "a longitudinal study of insulin resistance in american children showed that during puberty , insulin sensitivity decreased by 50% , with a compensatory increase in plasma insulin which is independent of the changes in body fat .", "these changes are thought to be mediated by interaction of various hormones during puberty including increased growth hormone secretion .", "no studies have yet reported the relationship between serum 25(oh)d concentrations and insulin sensitivity and resistance in pediatric and adolescent population and the potential influence of puberty except one .", "lenders et al . reported no significant association between serum 25(oh)d and insulin indices , even after adjusting for tanner stage .", "however , our study , for the first time , shows that the relationship between serum 25(oh)d concentrations and insulin resistance is influenced by puberty .", "when subjects with all stages of puberty were analyzed , no significant correlation was found , but when subjects in tanner stage 5 were analyzed alone , significant inverse correlation was seen between serum 25(oh)d concentrations and homa - ir ( r = 0.41 , p = 0.03 ) .", "this association persisted / improved after controlling for age , sex , and bmi ( r = 0.505 , p = 0.023 ) but disappeared when subjects were controlled for fmi ( r =  0.27 , p = 0.24 ) .", "most of the evidence of an association between adiposity and vitamin d status comes from adult studies [ 4 , 33 ] .", "a recently published study   of 382 healthy subjects aged 621  years with dxa scans showed that serum 25(oh)d concentrations were more likely to be lower in those with greater bmi z - scores and fm , but not ffm . when this association was adjusted for possible covariates in the model , it became nonsignificant .", ".   showed that obese adolescents with and without vitamin d deficiency differed according to fm and fm percentage , but not lean mass and this relation of fm percentage to serum 25(oh)d remained significant after potential confounding variables were adjusted for .", "showed that vitamin d deficiency was associated with higher visceral adipose tissue in white and greater subcutaneous adipose tissue in black american children and adolescents . in agreement with these studies ,", "our findings indicate that body fat indices ( total body fat percentage and fmi ) are inversely correlated with serum 25(oh)d level .", "this correlation persisted even after adjustment for possible covariates like age , sex , and bmi .", "the differences in study findings may be explained in part by differences in study design and underlying sample characteristics like bmi and variation in serum 25(oh)d concentrations .", "the major strengths of our study are the use of robust measures of insulin sensitivity and resistance based on ogtt rather than fasting sample and all our study subjects belonged to one ethnic group .", "we have used dxa for assessment of body fat as opposed to several studies which have used bmi as a surrogate for body fat .", "the limitations of our study included , small sample size , presence of vitamin d deficiency in all study subjects which gave us very narrow range of serum 25(oh)d concentrations thereby limiting comparisons .", "absence of pth measurement was another important limitation , since it has been implicated in pathogenesis of insulin resistance and metabolic syndrome .", "another important limitation was inability of dxa technique to differentiate between subcutaneous and visceral adipose tissue . in conclusion", ", our study demonstrates a significant inverse relationship in obese asian - indian children between body fat indices and serum 25(oh)d concentrations which persists even after adjustment of covariates .", "the association between serum 25(oh)d and parameters of insulin sensitivity and resistance is influenced by puberty as a significant association was found between these two variables in postpubertal subjects ( tanner stage 5 ) only which remained significant even after adjustment for covariates . as association is not equal to causation , future longitudinal research studies are required in determining role of puberty on association between serum 25(oh)d and insulin resistance in both , obese as well as subjects with normal bmi ."], "abstract_text": ["<S> to study the effect of puberty on the relationship between serum 25-hydroxyvitamin d ( 25(oh)d ) and parameters of insulin kinetics in obese asian - indian children . </S>", "<S> material and methods . </S>", "<S> the study population included 62 obese asian - indian children and adolescents in the age group of 617 years . </S>", "<S> blood glucose , serum insulin , and serum 25(oh)d were measured . </S>", "<S> total body fat was measured by dual energy x - ray absorptiometry . </S>", "<S> indices of insulin resistance ( homa - ir , auc for insulin ) and sensitivity ( wbisi ) were calculated after oral glucose tolerance test . result . </S>", "<S> a total of 62 subjects ( 35 boys ; mean age = 13.0  3 years ; bmi = 29.3  4.8  kg / sq  m ; 19 subjects in tanner stage 1 , 11 in stage 2 , 6 in stage 3 , 3 in stage 4 , and 23 subjects in tanner stage 5 ) were studied . </S>", "<S> all study subjects were vitamin d deficient with a mean serum 25(oh)d of 8.5  4.2  ng / ml . no significant relationship was observed between serum 25(oh)d and parameters of insulin kinetics in prepubertal children . </S>", "<S> however , a significant inverse correlation was seen between serum 25(oh)d and homair ( r = 0.41 , p = 0.03 ) in postpubertal subjects . </S>", "<S> conclusion . </S>", "<S> the relationship between vitamin d status and parameters of insulin kinetics are affected by puberty . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Statistical Analysis", "4. Results", "5. Discussion"], "sections": [["vitamin d deficiency was once thought to exclusively affect bone metabolism , but now there is ample evidence of its role in many other conditions including metabolic syndrome , autoimmune diseases , and cancer .", "vitamin d receptors are recognized to be in numerous extraskeletal tissues , such as pancreas and muscle .", "a systematic review by pittas and colleagues   reported that vitamin d may have a beneficial effect on the action of insulin , either directly or indirectly .", "several observational studies in adults , including the framingham heart study , have reported an inverse association between vitamin d status and insulin resistance .", "however , data from children and adolescents do not consistently report this inverse relationship [ 510 ] . even in studies reporting this association in adolescents and children ,", "further , none of the studies in adolescents have studied the effect of puberty on this association .", "a fall in insulin sensitivity with compensatory increase in insulin secretion has been reported in puberty [ 11 , 12 ] .", "high prevalence of cardiometabolic risk factors , including insulin resistance ( 64.8% of normal weight children had at least one cardiometabolic abnormality ) , even in healthy young children of normal weight , has been reported in indian children and adolescents .", "south - asian adolescents , including those with normal bmi , have a higher prevalence of insulin resistance when compared with age and bmi matched european adolescents [ 14 , 15 ] .", "this ethnic difference in insulin sensitivity was attributed to the higher body fat percentage in south - asian children .", "an alternative hypothesis to explain the ethnic differences in insulin sensitivity is vitamin d deficiency . despite plenty of sunlight available throughout the year", ", a high prevalence of vitamin d deficiency has been reported in all age groups including toddlers , school children , pregnant women and their neonates , and adult males and females residing in rural and urban india .", "vitamin d deficiency has been reported in 8492% children in the age group of 1018 . in view of high prevalence of both ,", "insulin resistance and vitamin d deficiency in asian - indian children and adolescents , we sought to examine the relationship between vitamin d status and insulin resistance .", "we also sought to determine if puberty affected the relationship of vitamin d status and parameters of insulin resistance and sensitivity in asian - indian children and adolescents ."], ["the study was conducted as a prospective observational study after approval from the ethics committee of the all india institute of medical sciences ( aiims ) , new delhi .", "subjects were selected from the children and adolescents attending the endocrinology outdoor services at aiims for evaluation of obesity .", "the inclusion criteria included children between the ages of 617 years with obesity as per international obesity taskforce ( iotf ) criteria .", "the exclusion criteria included subjects with diagnosed diabetes mellitus or taking metformin or any weight reducing drugs , subjects with any known systemic illness or endocrine or metabolic disorders , known to be associated with obesity , or subjects with symptoms to suggest hypothalamic obesity were excluded from the study .", "blood samples were collected in fasting state ( minimum 9 hours fasting ) , followed by oral glucose tolerance test ( anhydrous glucose = 1.75  gm / kg bodyweight , maximum of 75  gram , dissolved in 250  ml of water , ingested over 5 minutes ) with sample collection at 60 and 120 minutes after glucose ingestion .  ", "height was measured with holtain 's stadiometer ( holtain inc . , crymych , pembs .", "waist circumference was measured ( nonelastic measuring tape ) at the end of normal expiration at the midpoint between the iliac crest and the lower edge of ribs in the midaxillary line .", "blood pressure ( bp ) was measured in the right upper limb in sitting position with a mercury sphygmomanometer after 5-minute rest with an appropriate size cuff .", "total body fat was measured with dual energy x - ray absorptiometry ( dxa , hologic qdr 4500 with pediatric software , hologic inc . ,", "fat mass index ( fmi , fat mass in kg / height in meter ) and fat - free mass index ( ffmi , lean body mass+ bmc)/(height in meter ) were calculated .", "bone mineral area , content ( bmc ) and areal density ( abmd ) at the femoral neck , lumbar spine ( l1 - 5 ) , and forearm were measured , using a hologic qdr 4500a fan beam dxa machine .", "hologic spine phantom ( hologic spine phantom number 21373 ) was scanned daily before subject evaluation .", "the measured phantom bone mineral density was stable throughout the study period at 0.9150.945  gm / cm .", "similarly , whole body phantom ( hologic wb number 1252 ) was also scanned before subject evaluation and remained stable during study period .", "the in vivo precision error for adults was 0.62% for femoral neck abmd , 0.65% for lumbar spine abmd , and 0.77% for forearm abmd .", "however , in view of additional radiation exposure , the reproducibility of these scans was not assessed among children .", "pubertal stage was assessed by a single endocrinologist , based on breast stage and pubic hair development in girls   and genitalia development in boys .", "b - mode ultrasonography was used to measure cimt ( 7.510  mhz probe , philips envisor ultrasound machine ) using standard protocol .  \n insulin resistance ( ir )", "was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels .the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo .area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule . \n  insulin resistance ( ir )", "was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels .", "the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo .", "area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule .  ", "complete blood counts , liver function tests , renal function test , serum calcium ( corrected calcium with serum albumin ) , phosphate , alkaline phosphatase , uric acid , and blood glucose were measured in all subjects with an automated chemistry analyzer ( roche hitachi 912 chemistry analyzer , gmi inc .", "glycosylated hemoglobin ( hba1c ) was measured in whole blood using ion - exchange high performance liquid chromatography ( bio - rad laboratories inc . , ca , us ) .", "serum insulin was measured on an autoanalyzer ( roche elecsys 2010 ) using electrochemiluminometric assay .", "serum total cholesterol ( tc ) , triglyceride ( tg ) , and hdl cholesterol ( hdl - c ) levels were estimated directly with automated analyzer , while ldl cholesterol ( ldl - c ) was estimated by using the friedewald equation .", "vitamin d deficiency was defined as a serum 25(oh)d levels less than 20  ng / ml .", "this was further subdivided to severe , moderate , and mild vitamin d deficiency if serum 25(oh)d levels were < 5  ng / ml , 5<10  ng / ml , and 10<20  ng / ml respectively ."], ["statistical analysis was carried out using spss ( spss version 11.5 ; spss inc . ,", "continuous variables not showing normal distribution were treated with appropriate log transformations before any parametric analysis .", "student 's t - test was used to determine statistical difference between subjects with serum 25(oh)d < 10  ng / ml or more .", "pearson 's correlation was used to assess association between continuous variables and partial correlations were used to determine relationship after adjusting for relevant covariates .", "stepwise linear regression analysis was used with serum 25(oh)d as dependent variable in the model .", "values for serum 25(oh)d , serum insulin , homa - ir , and matsuda isi were not normally distributed ; therefore , they were logarithmically transformed which yielded normal distribution ."], ["a total of 62 subjects participated in this cross - sectional study from june 2008 to may 2009 .", "the mean age of subjects was 13.0  3 years ( 35 boys ; 27 girls ) .", "assessment of pubertal status by tanner 's method showed 19 subjects in stage 1 , 11 in stage 2 , 6 in stage 3 , 3 in stage 4 , and 23 subjects in puberty stage 5 .", "no significant difference was observed between boys and girls except higher whr in boys than girls ( 0.93  0.05 versus 0.89  0.05 ; p = 0.013 ) .", "all study subjects were classified as vitamin d deficient ( mean  sd 8.5  4.2  ng / ml ; maximum  minimum : 3.919.2  ng / ml , median = 6.9  ng / ml ) . severe vitamin d deficiency ( < 5  ng / ml ) was seen in 11 subjects ( 17.7% ) while 30 subjects had serum 25(oh)d levels between 5<10  ng / ml ( 48.3% ) .", "serum 25(oh)d equal to or more than 10 but less than 20  ng / ml was present in 21 subjects ( 33.8% ) .", "pearson correlation coefficient analyses showed significant inverse relationship between serum 25(oh)d and total body fat percentage ( r = 0.31 , p = 0.01 ) and serum 25(oh)d and fmi ( r = 0.33 , p = 0.01 , figure 1 ) .", "partial correlation analysis showed persistence of significant correlation between serum 25(oh)d and total body fat percentage ( r = 0.37 , p = 0.005 ) and between serum 25(oh)d and fmi ( r = 0.36 , p = 0.006 ) even after adjustment for age , sex , pubertal stage , and bmi .", "although blood glucose and serum insulin levels showed inverse relationship trends with serum 25(oh)d , but these were not statistically significant ( data not shown ) .", "similarly , no statistically significant correlation was seen between serum 25(oh)d and parameters of insulin sensitivity or resistance .", "fmi showed positive correlation with homa - ir ( r = 0.26 ; p = 0.04 ) , auc insulin ( r = 0.41 ; p = 0.001 ) , auc glucose ( r = 0.31 ; p = 0.018 ) , and negative correlation with wbisi ( r = 0.42 ; r = 0.001 ) .", "however , total body fat did not show any correlation with parameters of glucose and insulin parameters .   on the basis of serum 25(oh)d ,", "subjects were divided into two groups , group a with serum 25(oh)d level < 10  ng / ml ( n = 41 ) and group b with serum 25(oh)d level 10  ng / ml ( n = 21 ) ( an arbitrary cutoff to compare subjects with mild vitamin d deficiency with moderate and severe vitamin d deficiency , 25 ) .", "no statistical significant difference was seen in blood glucose , serum insulin , and auc for both , glucose and insulin between group 1 and group 2 .", "trends for higher insulin resistance ( homa - ir ) and lower insulin sensitivity ( wbisi ) were seen in subjects with lower serum 25(oh)d concentrations but not statistically significant .", "total body fat percentage and fmi were significantly higher in group 1 in comparison to group 2 ( p = 0.015 and 0.007 , resp . ) .", "however , no significant difference was seen in waist and hip circumference and waist hip ratio .", "similarly , ffmi , lipid parameters , or thyroid profile were also not different between the two groups .", "group 1 ( prepubertal group ; tanner stage 1 ) had 19 subjects , group 2 ( peripubertal group ; tanner stage 2 , 3 , and 4 ) had 20 , while group 3 ( postpubertal group ; tanner stage 5 ) had 23 subjects .", "no significant correlation was seen between vitamin d status and any other parameters including total body fat percentage , fmi and parameters of insulin resistance and sensitivity in the prepubertal ( group 1 ) or peripubertal groups ( group 2 ) .", "partial correlations after adjusting for age , sex , and bmi were also nonsignificant for both groups ( data not shown ) . in postpubertal subjects ( group 3 , n = 23 ) , a significant inverse correlation was seen between serum 25(oh)d and homa - ir ( r = 0.41 , p = 0.03 , figure 2 ) .", "this association between serum 25(oh)d and homa - ir persisted even after control for age , sex , and bmi ( r = 0.505 , p = 0.023 ) .", "positive trends of association between serum 25(oh)d and wbisi were also seen , but this was not significant ( r = 0.29 , p = 0.17 ) , even after adjusting for age , sex , and bmi ( r = 0.35 , p = 0.13 ) .", "serum 25(oh)d showed significant inverse correlation with total body fat percentage ( r = 0.58 , p = 0.003 ) and fmi ( r = 0.49 , p = 0.016 ) which improved after adjusting for age sex and bmi ( r = 0.63 , p = 0.002 for total body fat percentage ; r = 0.582 ,  p = 0.007 for fmi ) .", "serum 25(oh)d also showed significant inverse relationship with fasting serum insulin levels ( r = 0.44 , p = 0.034 ) which improved after adjusting for age , sex , and bmi ( r = 0.53 , p = 0.016 ) .", "when prepubertal subjects ( group 1 ) were compared with the prepubescent subjects ( group 2 ) , no significant difference was seen in any of the parameters including blood glucose , serum insulin , homa - ir , and wbisi .", "similarly , when prepubertal subjects were compared with all other subjects ( subjects in group 2 and 3 combined ) , significantly higher insulin sensitivity ( wbisi , mean  sd 6.07  5.7 versus 3.42  2.7 , p < 0.01 ) and lower insulin resistance ( homa - ir , mean  sd 3.68  2.7 versus 5.22  4.0 , p = 0.04 ) were seen in prepubertal subjects ."], ["this is the first study examining the relationship between serum 25(oh)d levels and parameters of insulin resistance in obese asian - indian children .", "our study confirms a significant inverse relationship between serum 25(oh)d levels and body fat indices as reported in other studies in children [ 6 , 10 ] .", "we demonstrated trends for that higher serum 25(oh)d levels are associated with better insulin sensitivity and less insulin resistance though this correlation did not reach statistical significance .", "one of the main reasons for the absence of significance could be the universal presence of vitamin d deficiency ( < 20  ng / ml ) resulting in very narrow range of serum 25(oh)d .", "we also show , for the first time , that the association between serum 25(oh)d and insulin resistance and sensitivity is influenced by pubertal staging .", "when subjects were stratified by pubertal stage , only subjects in tanner stage 5 exhibited a significant correlation between serum 25(oh)d and homa - ir , while prepubertal subjects did not show statistically significant correlation .", "high prevalence of vitamin d deficiency has been reported across the age range in pediatric , adolescent , and adult in obese individuals [ 5 , 6 , 2631 ] .", "this may be just reflection of very high prevalence of vitamin d deficiency in asian - indians [ 16 , 17 ] .", "however , high prevalence of vitamin d deficiency has been reported in obese children and adolescents from populations where vitamin d deficiency is not that prevalent [ 5 , 6 , 10 ] .", "reis et al . , in a study of 3577 adolescents in the age group of 1219 years , found significantly lower serum 25(oh)d levels in adolescents with bmi > 95th centile in comparison to subjects with bmi < 95th centile ( p < 0.001 ) .", "it has been postulated that increased sequestration of vitamin d in fat tissues , leading to decreased vitamin d bioavailability , low dietary vitamin d intake due to poor nutritional habits , and minimal sun exposure due to sedentary indoor lifestyle are important factors associated with high prevalence in the obese population [ 29 , 32 ] .", "it has also been suggested that vitamin d deficiency is a cause of common obesity and can account for the secular trends in the prevalence of obesity and for individual differences in its onset and severity . the inverse relationship between serum 25(oh)d and insulin resistance in adults", "have been reported in most studies , though some have failed to find this association . in comparison , reports in children and adolescents have shown conflicting results .", "delvin et al . , in a study of 1745 french - canadian children , reported modest but significant negative associations between serum 25(oh)d and homa - ir .", "each 10-nmol / l ( 4  ng / ml ) increase in serum 25(oh)d was associated with lower glycemia and homa - ir .", "did not find significant correlation between serum 25(oh)d levels and insulin , homa - ir and homa - b % . in another study based on nhanes data of 3577 adolescents from 20012004", "showed that serum 25(oh)d levels were inversely associated with plasma glucose concentration ( p = 0.01 ) .", "lenders et al . , in a study of 58 obese adolescents , did not found any correlation between insulin indices and serum 25(oh)d in both , adjusted and unadjusted models . in our study", ", we did not find a statistically significant association between serum 25(oh)d and parameters of insulin sensitivity and resistance .", "however , trends for higher blood glucose and serum insulin levels were seen in subjects with low serum 25(oh)d level .", "no significant difference was seen in insulin resistance and sensitivity between group 1 and group 2 .", "this is in contrast to the observations made by ashraf et al . who reported in 51 african - american obese female adolescents   that higher serum 25(oh)d concentrations were associated with significantly higher wbisi ( p = 0.018 ) but no difference in homa - ir .", "these differences may be due to marked difference in severity of obesity as mean bmi of study subjects was 43.3  9.9 in study by ashraf et al .", ", while it was 29.3  9.8  kg / sqm in our study subjects .", "studies in adult population have shown that parameters of insulin sensitivity and insulin resistance as calculated by hyperinsulinemic euglycemic clamp study ( m value ) correlated better with serum 25(oh)d levels than indirect parameters like homa - ir .", "studies with direct measures of insulin sensitivity like clamp studies are required to further investigate these issues in pediatric and adolescent population .", "the phenomenon of pubertal insulin resistance has been well described in cross - sectional as well as longitudinal studies .", "a longitudinal study of insulin resistance in american children showed that during puberty , insulin sensitivity decreased by 50% , with a compensatory increase in plasma insulin which is independent of the changes in body fat .", "these changes are thought to be mediated by interaction of various hormones during puberty including increased growth hormone secretion .", "no studies have yet reported the relationship between serum 25(oh)d concentrations and insulin sensitivity and resistance in pediatric and adolescent population and the potential influence of puberty except one .", "lenders et al . reported no significant association between serum 25(oh)d and insulin indices , even after adjusting for tanner stage .", "however , our study , for the first time , shows that the relationship between serum 25(oh)d concentrations and insulin resistance is influenced by puberty .", "when subjects with all stages of puberty were analyzed , no significant correlation was found , but when subjects in tanner stage 5 were analyzed alone , significant inverse correlation was seen between serum 25(oh)d concentrations and homa - ir ( r = 0.41 , p = 0.03 ) .", "this association persisted / improved after controlling for age , sex , and bmi ( r = 0.505 , p = 0.023 ) but disappeared when subjects were controlled for fmi ( r =  0.27 , p = 0.24 ) .", "most of the evidence of an association between adiposity and vitamin d status comes from adult studies [ 4 , 33 ] .", "a recently published study   of 382 healthy subjects aged 621  years with dxa scans showed that serum 25(oh)d concentrations were more likely to be lower in those with greater bmi z - scores and fm , but not ffm . when this association was adjusted for possible covariates in the model , it became nonsignificant .", ".   showed that obese adolescents with and without vitamin d deficiency differed according to fm and fm percentage , but not lean mass and this relation of fm percentage to serum 25(oh)d remained significant after potential confounding variables were adjusted for .", "showed that vitamin d deficiency was associated with higher visceral adipose tissue in white and greater subcutaneous adipose tissue in black american children and adolescents . in agreement with these studies ,", "our findings indicate that body fat indices ( total body fat percentage and fmi ) are inversely correlated with serum 25(oh)d level .", "this correlation persisted even after adjustment for possible covariates like age , sex , and bmi .", "the differences in study findings may be explained in part by differences in study design and underlying sample characteristics like bmi and variation in serum 25(oh)d concentrations .", "the major strengths of our study are the use of robust measures of insulin sensitivity and resistance based on ogtt rather than fasting sample and all our study subjects belonged to one ethnic group .", "we have used dxa for assessment of body fat as opposed to several studies which have used bmi as a surrogate for body fat .", "the limitations of our study included , small sample size , presence of vitamin d deficiency in all study subjects which gave us very narrow range of serum 25(oh)d concentrations thereby limiting comparisons .", "absence of pth measurement was another important limitation , since it has been implicated in pathogenesis of insulin resistance and metabolic syndrome .", "another important limitation was inability of dxa technique to differentiate between subcutaneous and visceral adipose tissue . in conclusion", ", our study demonstrates a significant inverse relationship in obese asian - indian children between body fat indices and serum 25(oh)d concentrations which persists even after adjustment of covariates .", "the association between serum 25(oh)d and parameters of insulin sensitivity and resistance is influenced by puberty as a significant association was found between these two variables in postpubertal subjects ( tanner stage 5 ) only which remained significant even after adjustment for covariates . as association is not equal to causation , future longitudinal research studies are required in determining role of puberty on association between serum 25(oh)d and insulin resistance in both , obese as well as subjects with normal bmi ."]]}
{"article_id": "PMC4121494", "article_text": ["a 21-year - old man ( height = 180 cm , weight = 95 kg , body mass index = 29.3 kg / m ) presented to the outpatient department with a long history of frequent and severe snoring . on physical exam ,", "after a diagnosis of obstructive sleep apnea was made , a bilateral tonsillectomy and uvulopalatopharyngoplasty was planned for treatment .", "prior to surgery , the patient had normal laboratory results with hemoglobin ( hb ) of 15.8 g / dl and no systemic or hematological diseases or history of bleeding disorders . on the day of the surgery , the patient received no premedication due to the history of sleep apnea .", "intraoperative monitors included electrocardiogram , non - invasive blood pressure ( bp ) , pulse oximetry ( spo2 ) , and end - tidal co2 .", "pre - anesthetic vital signs were bp 120/82 mmhg , hr 68 bpm , and spo2 96% .", "general anesthesia was induced with remifentanil 0.5 g / kg / min and propofol 180 mg iv at 10:10 am . at loss of eyelash reflex ,", "cisatracurium 16 mg was administered , and the trachea was intubated after a short period of facemask ventilation .", "anesthesia was maintained with sevoflurane 2 vol% in 50% oxygen - enriched air and remifentanil 0.2 g / kg / min .", "the neuromuscular block was antagonized with glycopyrrolate 0.6 mg and pyridostigmine 15 mg iv without neuromuscular monitoring upon the completion of the surgical procedure at 11:25 am .", "postoperatively , a physical examination confirmed the presence of acute bleeding in the left inferior pole of the tonsillectomy site in the post anesthetic care unit .", "the surgeon requested an emergent re - operation under general anesthesia for the control of bleeding .", "a rsi for a second round of general anesthesia was performed with propofol 120 mg , midazolam 5 mg , and rocuronium 100 mg at 11:55 am .", "the surgeon confirmed active bleeding due to an aberrant arterial blood supply in the left inferior pole of the tonsillectomy site .", "after the completion of the surgical procedure , no twitch was shown after train - of - four ( tof ) stimulation ( tof = 0 ) with neuro - stim ( model ns-3cc , houston , tx , usa ) at 12:40 pm . the neuromuscular block was then antagonized with sugammadex 200 mg iv and the rocuronium - induced nmb was completely reversed within 2 min .", "the patient was transferred to the recovery room , and there were no specific findings on physical exam .", "a post - operative laboratory assessment showed hb of 14.6 g / dl ( table 1 ) .", "after 6 h and 20 min , the surgeon again requested emergent anesthesia and re - operation for control of bleeding .", "the surgeon attempted to control bleeding at the ambulatory otolaryngology clinic but was unable to do so .", "when the patient arrived at the operating room ( or ) , vital signs were within normal limits and hb was found to be 13.2 g / dl .", "a third round of general anesthesia was induced with propofol 120 mg and rocuronium 40 mg by the on - call resident at 19:00 pm .", "the neuromuscular block was antagonized with sugammadex 200 mg iv and a tof = 0 at 20:00 pm .", "the patient was again extubated successfully and transferred to the recovery room with no specific findings ( table 1 ) . after 2 h and 35 min ,", "when the patient arrived at the or , vital signs were stable and hb was 11.6 g / dl .", "a fourth induction of general anesthesia was performed with propofol 120 mg and rocuronium 40 mg by the same resident at 22:35 pm . during tof stimulation", ", 1 twitch was seen , and an additional dose of rocuronium 10 mg was given due to bucking 20 min after the induction of anesthesia . at the end of the procedure ,", "the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 at the end of the surgical procedure at 0:20 am .", "extubation was carried out after confirming the recovery of consciousness , regular respiration , and the absence of fade on tof monitoring .", "after extubation , the patient vomited a large amount of blood on the operating table ( approximated to be more than 200 ml ) .", "however , the patient was alert and aware of his surroundings and had regular respirations ( table 1 ) .", "after 20 min , the surgeon requested a re - induction of general anesthesia for additional bleeding control .", "an rsi for a fifth round of general anesthesia was performed with propofol 120 mg and rocuronium 100 mg at 0:40 am .", "the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 after the surgical procedure was completed at 1:30 am .", "approximately 15 min passed after the nmb was antagonized as above , but no evidence of recovery from the rocuronium - induced nmb was seen .", "therefore , we injected a bolus of iv sugammadex 200 mg to reverse the residual nmb .", "once again , the nmb was completely reversed within 2 min , and tracheal extubation was performed in the or uneventfully ( table 1 ) .", "a decision by surgeon was made to perform angiography of the left external carotid artery with subsequent embolization of the bleeding vessel .", "embolization of the left ascending pharyngeal artery was performed with polyvinyl alcohol particle ( 350 m ) and tornado embolization microcoil ( cook , bloomington , usa ) ( 3/2 mm ) ( fig .", "the patient 's postoperative course was uneventful , and hb was found to be 10.5 g / dl .", "pth occurs in approximately 1 in 20 adults ( 5.1% ) , and more than half of patients who bleed are likely to require a procedure of some type to control their hemorrhage .", "aberrant arterial blood supply to the tonsillar region derived from the internal carotid artery or the carotid bulb may be present .", "to control bleeding , electrocauterization , packing of the pharynx , or angiographic embolization of the feeding artery may become necessary .", "the endovascular embolization due to refractory bleeding after tonsillectomy proved to be a valuable treatment method as an effective alternative to surgical intervention .", "it is a safe and permanent treatment option in this potentially life - threatening complication . in a case such as this ,", "the aberrant arterial blood supply in the left inferior pole of tonsillectomy site caused continuous bleeding .", "the anesthesiologist has experienced difficulties , because an experienced surgeon has embarrassed due to repetitive bleeding . in the first induction of anesthesia", ", no problems were encountered with conventional anesthesia , although neuromuscular monitoring was not performed .", "the most common anesthetic used for rsi during the management of children with pth is succinylcholine . since the introduction of sugammadex , rsi with rocuronium followed by reversal with sugammadex allowed for earlier establishment of spontaneous ventilation than with succinylcholine , which has many adverse side events . during the second round of anesthesia , rocuronium 100 mg ( 1 mg / kg )", "was injected for rsi , followed by reversal with sugammadex 200 mg ( 2 mg / kg ) at deep nmb ( tof = 0 ) . during the operation ,", "nmb monitoring was difficult due to problems attaching leads to the arms during the operation .", "tof stimulation was applied at the ulnar nerve using conventional instrumental monitoring rather than quantitative monitoring due to convenience .", "deep nmb was maintained for bleeding control in an attempt to not disturb the procedure . during nmb reversal", ", we found an absence of fade on tof monitoring just 2 min after sugammadex was administered .", "re - administration of 0.6 mg / kg rocuronium could then be given after sugammadex at the recommended waiting time of 6 h . during the third anesthesia induction ,", "an on - call resident used rocuronium 40 mg for muscle relaxation during intubation followed by reversal with sugammadex 200 mg at deep nmb ( tof = 0 ) .", "an absence of fade on tof monitoring was shown 2 min after sugammadex was given . for the fourth round of anesthesia , the same resident used rocuronium 40 mg for intubation 2 h and 35 min after the previous nmb reversal with sugammadex .", "however , we believe this decision was made without considering the recent sugammadex use . when rocuronium is used within a short time interval ( less than 6 h ) after sugammadex administration , the onset time of rocuronium may be prolonged and may also have an unpredictable duration of action .", "we suspect that the additional dose of rocuronium 10 mg due to bucking may be related to the short time interval between sugammadex and rocuronium administration . in situations such as these ,", "the benzylisoquinoline class of medications are recommended instead of re - administering rocuronium within 6 h after sugammadex .", "when considering this case , cisatracurium for muscle relaxation and intubation may have been a better choice for the fourth round anesthesia . however , one drawback of cisatracurium is the slower onset time than rocuronium .", "reversal of the rocuronium - induced nmb with pyridostigmine at a moderate nmb ( tof = 1 ) was uneventful .", "after extubation , the patient vomited more than 200 ml of blood on the operating table ; however , he was alert and showed regular respirations .", "postoperative nausea and vomiting ( ponv ) after an operation for pth is common due to blood swallowed during the procedure .", "therefore , patient awareness and complete reflexes are important , but aspiration must be avoided and monitored closely during the recovery period . however , ponv increases the risk of primary hemorrhage and unexpected postoperative hospital admissions . in this case", "the patient did not aspirate , but did have an increased risk of pth due to ponv .", "for the fifth round of anesthesia , rocuronium 100 mg ( 1 mg / kg ) was injected for rsi after a time interval from sugammadex administration of 4 h and 40 min .", "it has been reported that re - administration of 1.2 mg / kg rocuronium could have been given after sugammadex 2 mg / kg without a waiting time .", "however , neuromuscular monitoring did not show the expected recovery , and the patient had residual paralysis after 15 min .", "it has been shown previously that the reversal of nmb with anticholinesterase drugs may not be complete after a large dose of a neuromuscular blocking agent .", "sugammadex resolved the residual nmb within 2 min , even after reversal with anticholinesterase agents , and is a safe alternative to reversal of nmb induced by steroidal non - depolarizing agents . in this case , deep rocuronium - induced nmb can be reversed successfully with sugammadex at doses of 2 mg / kg and in as little as 2 min .", "re - administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of the nmb agent .", "sugammadex made it possible to perform a rapid , complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase ."], "abstract_text": ["<S> post - tonsillectomy hemorrhage ( pth ) is the most frequent complication of tonsillectomy , and occasionally results in a lethal outcome . </S>", "<S> a 21-year - old man ( height 180 cm , weight 95 kg ) was scheduled for a bilateral tonsillectomy and uvulopalatopharyngoplasty for treatment of obstructive sleep apnea . </S>", "<S> he required 5 rounds of general anesthesia due to recurrent pth . </S>", "<S> the anesthesiologist used sugammadex a total of 3 times to achieve the successful reversal of the deep neuromuscular blockade ( nmb ) induced by rocuronium . </S>", "<S> after sugammadex 2 mg / kg was administered , the nmb was reversed in 2 minutes each time . </S>", "<S> re - administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of neuromuscular blocking agents . </S>", "<S> sugammadex made it possible to perform a rapid , complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 21-year - old man ( height = 180 cm , weight = 95 kg , body mass index = 29.3 kg / m ) presented to the outpatient department with a long history of frequent and severe snoring . on physical exam ,", "after a diagnosis of obstructive sleep apnea was made , a bilateral tonsillectomy and uvulopalatopharyngoplasty was planned for treatment .", "prior to surgery , the patient had normal laboratory results with hemoglobin ( hb ) of 15.8 g / dl and no systemic or hematological diseases or history of bleeding disorders . on the day of the surgery , the patient received no premedication due to the history of sleep apnea .", "intraoperative monitors included electrocardiogram , non - invasive blood pressure ( bp ) , pulse oximetry ( spo2 ) , and end - tidal co2 .", "pre - anesthetic vital signs were bp 120/82 mmhg , hr 68 bpm , and spo2 96% .", "general anesthesia was induced with remifentanil 0.5 g / kg / min and propofol 180 mg iv at 10:10 am . at loss of eyelash reflex ,", "cisatracurium 16 mg was administered , and the trachea was intubated after a short period of facemask ventilation .", "anesthesia was maintained with sevoflurane 2 vol% in 50% oxygen - enriched air and remifentanil 0.2 g / kg / min .", "the neuromuscular block was antagonized with glycopyrrolate 0.6 mg and pyridostigmine 15 mg iv without neuromuscular monitoring upon the completion of the surgical procedure at 11:25 am .", "postoperatively , a physical examination confirmed the presence of acute bleeding in the left inferior pole of the tonsillectomy site in the post anesthetic care unit .", "the surgeon requested an emergent re - operation under general anesthesia for the control of bleeding .", "a rsi for a second round of general anesthesia was performed with propofol 120 mg , midazolam 5 mg , and rocuronium 100 mg at 11:55 am .", "the surgeon confirmed active bleeding due to an aberrant arterial blood supply in the left inferior pole of the tonsillectomy site .", "after the completion of the surgical procedure , no twitch was shown after train - of - four ( tof ) stimulation ( tof = 0 ) with neuro - stim ( model ns-3cc , houston , tx , usa ) at 12:40 pm . the neuromuscular block was then antagonized with sugammadex 200 mg iv and the rocuronium - induced nmb was completely reversed within 2 min .", "the patient was transferred to the recovery room , and there were no specific findings on physical exam .", "a post - operative laboratory assessment showed hb of 14.6 g / dl ( table 1 ) .", "after 6 h and 20 min , the surgeon again requested emergent anesthesia and re - operation for control of bleeding .", "the surgeon attempted to control bleeding at the ambulatory otolaryngology clinic but was unable to do so .", "when the patient arrived at the operating room ( or ) , vital signs were within normal limits and hb was found to be 13.2 g / dl .", "a third round of general anesthesia was induced with propofol 120 mg and rocuronium 40 mg by the on - call resident at 19:00 pm .", "the neuromuscular block was antagonized with sugammadex 200 mg iv and a tof = 0 at 20:00 pm .", "the patient was again extubated successfully and transferred to the recovery room with no specific findings ( table 1 ) . after 2 h and 35 min ,", "when the patient arrived at the or , vital signs were stable and hb was 11.6 g / dl .", "a fourth induction of general anesthesia was performed with propofol 120 mg and rocuronium 40 mg by the same resident at 22:35 pm . during tof stimulation", ", 1 twitch was seen , and an additional dose of rocuronium 10 mg was given due to bucking 20 min after the induction of anesthesia . at the end of the procedure ,", "the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 at the end of the surgical procedure at 0:20 am .", "extubation was carried out after confirming the recovery of consciousness , regular respiration , and the absence of fade on tof monitoring .", "after extubation , the patient vomited a large amount of blood on the operating table ( approximated to be more than 200 ml ) .", "however , the patient was alert and aware of his surroundings and had regular respirations ( table 1 ) .", "after 20 min , the surgeon requested a re - induction of general anesthesia for additional bleeding control .", "an rsi for a fifth round of general anesthesia was performed with propofol 120 mg and rocuronium 100 mg at 0:40 am .", "the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 after the surgical procedure was completed at 1:30 am .", "approximately 15 min passed after the nmb was antagonized as above , but no evidence of recovery from the rocuronium - induced nmb was seen .", "therefore , we injected a bolus of iv sugammadex 200 mg to reverse the residual nmb .", "once again , the nmb was completely reversed within 2 min , and tracheal extubation was performed in the or uneventfully ( table 1 ) .", "a decision by surgeon was made to perform angiography of the left external carotid artery with subsequent embolization of the bleeding vessel .", "embolization of the left ascending pharyngeal artery was performed with polyvinyl alcohol particle ( 350 m ) and tornado embolization microcoil ( cook , bloomington , usa ) ( 3/2 mm ) ( fig .", "the patient 's postoperative course was uneventful , and hb was found to be 10.5 g / dl ."], ["pth occurs in approximately 1 in 20 adults ( 5.1% ) , and more than half of patients who bleed are likely to require a procedure of some type to control their hemorrhage .", "aberrant arterial blood supply to the tonsillar region derived from the internal carotid artery or the carotid bulb may be present .", "to control bleeding , electrocauterization , packing of the pharynx , or angiographic embolization of the feeding artery may become necessary .", "the endovascular embolization due to refractory bleeding after tonsillectomy proved to be a valuable treatment method as an effective alternative to surgical intervention .", "it is a safe and permanent treatment option in this potentially life - threatening complication . in a case such as this ,", "the aberrant arterial blood supply in the left inferior pole of tonsillectomy site caused continuous bleeding .", "the anesthesiologist has experienced difficulties , because an experienced surgeon has embarrassed due to repetitive bleeding . in the first induction of anesthesia", ", no problems were encountered with conventional anesthesia , although neuromuscular monitoring was not performed .", "the most common anesthetic used for rsi during the management of children with pth is succinylcholine . since the introduction of sugammadex , rsi with rocuronium followed by reversal with sugammadex allowed for earlier establishment of spontaneous ventilation than with succinylcholine , which has many adverse side events . during the second round of anesthesia , rocuronium 100 mg ( 1 mg / kg )", "was injected for rsi , followed by reversal with sugammadex 200 mg ( 2 mg / kg ) at deep nmb ( tof = 0 ) . during the operation ,", "nmb monitoring was difficult due to problems attaching leads to the arms during the operation .", "tof stimulation was applied at the ulnar nerve using conventional instrumental monitoring rather than quantitative monitoring due to convenience .", "deep nmb was maintained for bleeding control in an attempt to not disturb the procedure . during nmb reversal", ", we found an absence of fade on tof monitoring just 2 min after sugammadex was administered .", "re - administration of 0.6 mg / kg rocuronium could then be given after sugammadex at the recommended waiting time of 6 h . during the third anesthesia induction ,", "an on - call resident used rocuronium 40 mg for muscle relaxation during intubation followed by reversal with sugammadex 200 mg at deep nmb ( tof = 0 ) .", "an absence of fade on tof monitoring was shown 2 min after sugammadex was given . for the fourth round of anesthesia , the same resident used rocuronium 40 mg for intubation 2 h and 35 min after the previous nmb reversal with sugammadex .", "however , we believe this decision was made without considering the recent sugammadex use . when rocuronium is used within a short time interval ( less than 6 h ) after sugammadex administration , the onset time of rocuronium may be prolonged and may also have an unpredictable duration of action .", "we suspect that the additional dose of rocuronium 10 mg due to bucking may be related to the short time interval between sugammadex and rocuronium administration . in situations such as these ,", "the benzylisoquinoline class of medications are recommended instead of re - administering rocuronium within 6 h after sugammadex .", "when considering this case , cisatracurium for muscle relaxation and intubation may have been a better choice for the fourth round anesthesia . however , one drawback of cisatracurium is the slower onset time than rocuronium .", "reversal of the rocuronium - induced nmb with pyridostigmine at a moderate nmb ( tof = 1 ) was uneventful .", "after extubation , the patient vomited more than 200 ml of blood on the operating table ; however , he was alert and showed regular respirations .", "postoperative nausea and vomiting ( ponv ) after an operation for pth is common due to blood swallowed during the procedure .", "therefore , patient awareness and complete reflexes are important , but aspiration must be avoided and monitored closely during the recovery period . however , ponv increases the risk of primary hemorrhage and unexpected postoperative hospital admissions . in this case", "the patient did not aspirate , but did have an increased risk of pth due to ponv .", "for the fifth round of anesthesia , rocuronium 100 mg ( 1 mg / kg ) was injected for rsi after a time interval from sugammadex administration of 4 h and 40 min .", "it has been reported that re - administration of 1.2 mg / kg rocuronium could have been given after sugammadex 2 mg / kg without a waiting time .", "however , neuromuscular monitoring did not show the expected recovery , and the patient had residual paralysis after 15 min .", "it has been shown previously that the reversal of nmb with anticholinesterase drugs may not be complete after a large dose of a neuromuscular blocking agent .", "sugammadex resolved the residual nmb within 2 min , even after reversal with anticholinesterase agents , and is a safe alternative to reversal of nmb induced by steroidal non - depolarizing agents . in this case , deep rocuronium - induced nmb can be reversed successfully with sugammadex at doses of 2 mg / kg and in as little as 2 min .", "re - administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of the nmb agent .", "sugammadex made it possible to perform a rapid , complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase ."]]}
{"article_id": "PMC4975227", "article_text": ["infertility has been recognized as a public health issue worldwide ( 1 ) leading to an increasing need to the use of assisted reproductive technologies ( art ) , including in vitro fertilization ( ivf ) .", "after the first reported case of ivf in 1978(2 ) art enabled millions of people to have their own children in cases when pregnancy did not occur under natural circumstances .", "700,000 cycles a year in the usa and europe together ( 3,4 ) . despite of evolving intracytoplasmatic sperm injection ( icsi ) technique", "a success rate of 25% and 28% has been reported in 2005(7 ) and 2008(8 ) , respectively .", "nowadays , this rate went up to 32% ( 9 ) , which can not be considered as a significant development .", "earlier clinical protocols preferred multiple embryo transfer , but multiple gestations can result in the increased risk of preterm delivery ( 10 - 16 ) .", "other studies report that multiple gestations also increased the risk of low birth weight cerebral palsy ( 17 ) . in the us alone , preterm births resulting from multiple pregnancies during ivf cause a 1 billion usd extra cost to the social insurance ( 18 ) . in order to exclude the discussed risk factors ,", "it is imperative , however , that accurate and economical methods should be developed to ensure that the most viable euploid embryo is selected for transfer .", "ideally , such tests would be noninvasive , lessening the risks to the embryo and reducing costs and workload in the embryology laboratory ( 19 ) .", "the biggest issue with pre - implantation viability assessment is that due to ethical reasons any assay should be completely non - invasive because no one can predict what kind of interference would be the unwanted result in the later embryonic development .", "the most apparent  and routinely applied - way of the assessment of viability is the morphological evaluation of in vitro fertilized embryos using microscopy .", "there are several morphological features described which could be used for viability assessment purposes , these are dependent on the time spent after fertilization . right after fertilization in the 1-cell embryo the size and symmetry of the two pronuclei can be examined .", "the time of the first cell division is also a good predictor of later implantation potential , as zygotes that divide early tend to develop more frequently to the blastocyst stage .", "criteria as cleavage rate and blastomere shape and symmetry , an adequate trophectoderm layer ( te ) and an inner cell mass ( icm ) is a morphological marker of the later stages ( 5,20 ) .", "not only can the morphology of the fertilized embryo be used for further prediction of implantation potential , but morphological defects of the retrieved oocyte as well . fertilization and pregnancy rate correlates with the grade of cumulus - oocyte complexes , and embryos originating from dysmorphic oocytes show a larger grade of pregnancy loss ( 21 - 23 ) .", "the cleavage stages of morulae and blastocysts or the symmetry and patterns of cell division are also notable and frequently used aspects , and are often examined during the prediction of embryo viability ( 23 ) .", "the biggest issue of morphological assessment is that it is still a highly subjective method ( 20 ) .", "the reason is partly due to the fact that the final decision is made by a clinician , and not by an objective test result , and secondly it is does matter how important are the individual morphological features in the final conclusion ( 24 - 26 ) . to overcome the different practice of laboratories worldwide in 2011 an international consensus ( istanbul consensus ) has been reached on embryo viability assessment ( 27 )", ". the selected morphological markers of respective stage embryos , the weighing of individual features and a scoring system has been set up .", "the limitations due to static time - point observation , is now solved with the use of time - lapse microscopy ( 28,29 ) .", "time - lapse microscopy also enables the observation of dynamics of cytoplasmic movements and cytokinesis , reflecting the functionality of microtubule and actin cytoskeleton , which is critical for proper development . in our laboratory", ", we aimed to improve the success rate of implantation by adopting and further optimizing the istanbul consensus .", "this score has been called as the optimized criteria system ( ocs ) . according to this scoring", ", 3-day old embryos were divided into two subgroups : the subgroup with low blastomere number ( less than 7 ) and with high blastomere number ( 7 or more ) .", "symmetric position of blastomeres indicates the rate of symmetry of holoblastic cleavage along the embryo axis .", "it was classified as good ( full symmetry ) ; fair ( light asymmetry ) ; or poor ( evident asymmetry ) .", "the percent values of fragmentation are based on the ratio of fragmented to total cell numbers . as a further modification to the istanbul consensus ,", "embryos were considered as good if the fragmentation rate was < 15% ( instead of the original 10% ) .", "this shift from 10 to 15% was the result of our observation that a fairly high proportion of the embryos between 10 - 15% appeared to be viable . in summary the optimized criteria system ( ocs )", "highlights 3 modified or new parameters : fragmentation ( with a more permissive criterion of < 15% in the  good  category ) ; symmetry and the blastomere number . in addition , the blastomere size was evaluated according to the original istanbul consensus .", "a scoring - map was created to facilitate the evaluation ( table 1 ) as far as the 5-day old embryos are concerned , we modified the original istanbul consensus for blastocysts by leaving out the hatched stage from the evaluation .", "the istanbul consensus for the 5-day old embryos has a shortcoming , i.e. it does not express the viability of embryos with a single category ( good , fair , poor ) .", "we tried to overcome this by using a scoring map ( table 2 ) . in conclusion", ", we constructed a composite score for day-3 , as well as day-5 old embryos , based on morphological parameters .", "as it is evident from the results , this composite score is sensitive to evaluate viability ( figure 1 )", "another possibility for non - invasive embryo viability assessment is the metabolomic examination of the culture medium surrounding the in vitro fertilized embryo .", "metabolomic , ( proteomic ) profiling of spent embryo culture ( sec ) offers an exceptional , non - invasive opportunity for the assessment of embryo viability ( 30,31 ) .", "the metabolomic profiling ( 32,33 ) of early embryo development might mean the analysis of the total metabolome by following the changes of several selected compounds , metabolomic analysis using unidentified , but significantly differing metabolomic changes , or by the analysis of a limited population of nutrients or end products .", "the common feature in all three concepts is that they are concentrating on the metabolomic alterations caused by differently developing embryos in the culture medium .", "very simple idea is the monitoring of glucose consumption or pyruvate formation , since this would directly indicate the metabolism of the developing embryo and it is an obvious conclusion that a metabolically active embryo would have higher implantation potential .", "some authors reported that the identification of these parameters resulted in successful prediction of embryo implantation potential , but other research groups describe contradictory results ( 34 , 35 ) .", "the amino acid profile of culture media is also used in the prediction of implantation potential , though not exclusively as an independent parameter , rather in combination with morphological features ( 36 ) .", "the detection of unidentified metabolomic changes using near infra - red ( nir ) or raman spectroscopy ( 37 , 38 ) is a very interesting and challenging possibility .", "more complicated is the concept when unknown , new biomarker molecules of embryo viability are searched for , assuming that these biomarkers were secreted by the embryo .", "the difficulty of the concept is that only 4 - 8 cells are present in the culture medium ; thus a very sensitive analytical tool is required .", "mass spectrometry ( ms ) has the potential of specific and sensitive quantification in a wide spectrum of molecular mass ranges and therefore suites well the needs of metabolomic or proteomic fingerprinting and quantification . in parallel to the spreading of mass spectrometry , proteomics is also an emerging field in the understanding of embryo development ( 39,40 ) .", "the analysis of the embryonic secretome ( 41,42 ) provides information of the total transcriptome of the developing embryos .", "mass spectrometry can be used both in targeted and discovery analysis with accurate quantification of identified biomarkers after molecular identification by bottom - up or top - down proteomics using tandem or multiple ms ( 43 - 46 ) . in a recent publication from our laboratory ( 47 ) using liquid chromatography coupled mass spectrometry ( lc - ms ) , a fragment of the human haptoglobin molecule was identified in the culture medium . rather than analyzing the embryonic secretome , the aim this experiment was to use preexisting molecules present in the cell culture media as biomarkers .", "haptoglobin - which was detected in the culture medium - is not a product of the developing embryo ; the polypeptide is a contaminant of the human serum albumin standard used to supplement the culture medium ( 47,48 ) . during the first three days of embryo development the formation of a subunit ( alfa-1 ) of the human haptoglobin molecule was observed .", "this subunit similar to the total haptoglobin molecule was detectable in the blank control medium samples as well .", "the differentiation of the viable and non - viable embryos was done using the observation that compared to blank controls the samples of embryos which later did not resulted in pregnancy contained the alpha-1 subunit in a much larger quantity than the samples of embryos which did ( figure 2 ) .", "clinical statistical analysis of the results revealed that the specificity of the diagnostic test was 64% , while the sensitivity was 100% .", "it is more informative that the positive predictive value of the assay was 51% and  maybe more importantly  the negative predictive value was 100% .", "receiver operating characteristic ( roc ) analysis provides tools to select possibly optimal models and to discard suboptimal ones .", "roc analysis is related in a direct and natural way to cost / benefit analysis of diagnostic decision - making", ". the roc curve of the morphological versus metabolomic approach in relation to the correct prediction of pregnancy outcome is illustrated in figure 3 .", "it is obvious that our biochemical investigation method enables a selection of the embryos by sorting out the non - viable ones .", "the test selected with 100% potential the embryos , which did not lead to successful implantation at all .", "one of the areas of collaboration between clinicians , the clinical laboratory and the research laboratory at the university of pcs is related to the research of infertility . since the clinical background gives the beauty and the medical importance of laboratory research , it was of outstanding importance for us to receive the eflm - abbott diagnostics award for excellence in outcomes research in laboratory medicine ( paris , 2015 ) , the award given to the best published paper ( 47 ) , as judged by an independent panel of experts , which demonstrates improved outcomes arising out of the application or improved utilization of an in - vitro diagnostics test ."], "abstract_text": ["<S> human reproduction is a relatively inefficient process and therefore the number of infertile couples is high . </S>", "<S> assisted reproductive technologies ( art ) have facilitated the birth of over five million children worldwide . </S>", "<S> art , however , superimposes its own relative inefficiency on the preexisting inefficiency of normal reproduction . </S>", "<S> the efficiency ( expressed as pregnancy rate ) is generally not more than 30% . </S>", "<S> modern reproductive medicine is gradually moving from multiple embryo transfer to the transfer of a single embryo , mainly because of obvious and unwanted side effects of multiple embryo transfer ( e.g.  epidemic  multiple pregnancies ) . </S>", "<S> this concept , however , requires a fast , professional selection of the most viable embryo during the first few days of art . </S>", "<S> thus the aim of a modern art is the safe transfer of a healthy , viable , single embryo . </S>", "<S> accurate and rapid methods of quantifying embryo viability are needed to reach this goal . </S>", "<S> methodological advances have the potential to make an important contribution , and there has been a drive to develop alternative non - invasive methods to better meet clinical needs . </S>", "<S> metabolic and genetic profiling of spent embryo culture ( sec ) media should offer an exceptional opportunity for the assessment of embryo viability . </S>", "<S> the current review focuses on the latest non - invasive diagnostic approaches for pre - implantation viability assessment of in vitro fertilized embryos . </S>"], "labels": null, "section_names": ["INTRODUCTION", "THE MORPHOLOGICAL APPROACH", "THE BIOCHEMICAL APPROACH"], "sections": [["infertility has been recognized as a public health issue worldwide ( 1 ) leading to an increasing need to the use of assisted reproductive technologies ( art ) , including in vitro fertilization ( ivf ) .", "after the first reported case of ivf in 1978(2 ) art enabled millions of people to have their own children in cases when pregnancy did not occur under natural circumstances .", "700,000 cycles a year in the usa and europe together ( 3,4 ) . despite of evolving intracytoplasmatic sperm injection ( icsi ) technique", "a success rate of 25% and 28% has been reported in 2005(7 ) and 2008(8 ) , respectively .", "nowadays , this rate went up to 32% ( 9 ) , which can not be considered as a significant development .", "earlier clinical protocols preferred multiple embryo transfer , but multiple gestations can result in the increased risk of preterm delivery ( 10 - 16 ) .", "other studies report that multiple gestations also increased the risk of low birth weight cerebral palsy ( 17 ) . in the us alone , preterm births resulting from multiple pregnancies during ivf cause a 1 billion usd extra cost to the social insurance ( 18 ) . in order to exclude the discussed risk factors ,", "it is imperative , however , that accurate and economical methods should be developed to ensure that the most viable euploid embryo is selected for transfer .", "ideally , such tests would be noninvasive , lessening the risks to the embryo and reducing costs and workload in the embryology laboratory ( 19 ) .", "the biggest issue with pre - implantation viability assessment is that due to ethical reasons any assay should be completely non - invasive because no one can predict what kind of interference would be the unwanted result in the later embryonic development ."], ["the most apparent  and routinely applied - way of the assessment of viability is the morphological evaluation of in vitro fertilized embryos using microscopy .", "there are several morphological features described which could be used for viability assessment purposes , these are dependent on the time spent after fertilization . right after fertilization in the 1-cell embryo the size and symmetry of the two pronuclei can be examined .", "the time of the first cell division is also a good predictor of later implantation potential , as zygotes that divide early tend to develop more frequently to the blastocyst stage .", "criteria as cleavage rate and blastomere shape and symmetry , an adequate trophectoderm layer ( te ) and an inner cell mass ( icm ) is a morphological marker of the later stages ( 5,20 ) .", "not only can the morphology of the fertilized embryo be used for further prediction of implantation potential , but morphological defects of the retrieved oocyte as well . fertilization and pregnancy rate correlates with the grade of cumulus - oocyte complexes , and embryos originating from dysmorphic oocytes show a larger grade of pregnancy loss ( 21 - 23 ) .", "the cleavage stages of morulae and blastocysts or the symmetry and patterns of cell division are also notable and frequently used aspects , and are often examined during the prediction of embryo viability ( 23 ) .", "the biggest issue of morphological assessment is that it is still a highly subjective method ( 20 ) .", "the reason is partly due to the fact that the final decision is made by a clinician , and not by an objective test result , and secondly it is does matter how important are the individual morphological features in the final conclusion ( 24 - 26 ) . to overcome the different practice of laboratories worldwide in 2011 an international consensus ( istanbul consensus ) has been reached on embryo viability assessment ( 27 )", ". the selected morphological markers of respective stage embryos , the weighing of individual features and a scoring system has been set up .", "the limitations due to static time - point observation , is now solved with the use of time - lapse microscopy ( 28,29 ) .", "time - lapse microscopy also enables the observation of dynamics of cytoplasmic movements and cytokinesis , reflecting the functionality of microtubule and actin cytoskeleton , which is critical for proper development . in our laboratory", ", we aimed to improve the success rate of implantation by adopting and further optimizing the istanbul consensus .", "this score has been called as the optimized criteria system ( ocs ) . according to this scoring", ", 3-day old embryos were divided into two subgroups : the subgroup with low blastomere number ( less than 7 ) and with high blastomere number ( 7 or more ) .", "symmetric position of blastomeres indicates the rate of symmetry of holoblastic cleavage along the embryo axis .", "it was classified as good ( full symmetry ) ; fair ( light asymmetry ) ; or poor ( evident asymmetry ) .", "the percent values of fragmentation are based on the ratio of fragmented to total cell numbers . as a further modification to the istanbul consensus ,", "embryos were considered as good if the fragmentation rate was < 15% ( instead of the original 10% ) .", "this shift from 10 to 15% was the result of our observation that a fairly high proportion of the embryos between 10 - 15% appeared to be viable . in summary the optimized criteria system ( ocs )", "highlights 3 modified or new parameters : fragmentation ( with a more permissive criterion of < 15% in the  good  category ) ; symmetry and the blastomere number . in addition , the blastomere size was evaluated according to the original istanbul consensus .", "a scoring - map was created to facilitate the evaluation ( table 1 ) as far as the 5-day old embryos are concerned , we modified the original istanbul consensus for blastocysts by leaving out the hatched stage from the evaluation .", "the istanbul consensus for the 5-day old embryos has a shortcoming , i.e. it does not express the viability of embryos with a single category ( good , fair , poor ) .", "we tried to overcome this by using a scoring map ( table 2 ) . in conclusion", ", we constructed a composite score for day-3 , as well as day-5 old embryos , based on morphological parameters .", "as it is evident from the results , this composite score is sensitive to evaluate viability ( figure 1 )"], ["another possibility for non - invasive embryo viability assessment is the metabolomic examination of the culture medium surrounding the in vitro fertilized embryo .", "metabolomic , ( proteomic ) profiling of spent embryo culture ( sec ) offers an exceptional , non - invasive opportunity for the assessment of embryo viability ( 30,31 ) .", "the metabolomic profiling ( 32,33 ) of early embryo development might mean the analysis of the total metabolome by following the changes of several selected compounds , metabolomic analysis using unidentified , but significantly differing metabolomic changes , or by the analysis of a limited population of nutrients or end products .", "the common feature in all three concepts is that they are concentrating on the metabolomic alterations caused by differently developing embryos in the culture medium .", "very simple idea is the monitoring of glucose consumption or pyruvate formation , since this would directly indicate the metabolism of the developing embryo and it is an obvious conclusion that a metabolically active embryo would have higher implantation potential .", "some authors reported that the identification of these parameters resulted in successful prediction of embryo implantation potential , but other research groups describe contradictory results ( 34 , 35 ) .", "the amino acid profile of culture media is also used in the prediction of implantation potential , though not exclusively as an independent parameter , rather in combination with morphological features ( 36 ) .", "the detection of unidentified metabolomic changes using near infra - red ( nir ) or raman spectroscopy ( 37 , 38 ) is a very interesting and challenging possibility .", "more complicated is the concept when unknown , new biomarker molecules of embryo viability are searched for , assuming that these biomarkers were secreted by the embryo .", "the difficulty of the concept is that only 4 - 8 cells are present in the culture medium ; thus a very sensitive analytical tool is required .", "mass spectrometry ( ms ) has the potential of specific and sensitive quantification in a wide spectrum of molecular mass ranges and therefore suites well the needs of metabolomic or proteomic fingerprinting and quantification . in parallel to the spreading of mass spectrometry , proteomics is also an emerging field in the understanding of embryo development ( 39,40 ) .", "the analysis of the embryonic secretome ( 41,42 ) provides information of the total transcriptome of the developing embryos .", "mass spectrometry can be used both in targeted and discovery analysis with accurate quantification of identified biomarkers after molecular identification by bottom - up or top - down proteomics using tandem or multiple ms ( 43 - 46 ) . in a recent publication from our laboratory ( 47 ) using liquid chromatography coupled mass spectrometry ( lc - ms ) , a fragment of the human haptoglobin molecule was identified in the culture medium . rather than analyzing the embryonic secretome , the aim this experiment was to use preexisting molecules present in the cell culture media as biomarkers .", "haptoglobin - which was detected in the culture medium - is not a product of the developing embryo ; the polypeptide is a contaminant of the human serum albumin standard used to supplement the culture medium ( 47,48 ) . during the first three days of embryo development the formation of a subunit ( alfa-1 ) of the human haptoglobin molecule was observed .", "this subunit similar to the total haptoglobin molecule was detectable in the blank control medium samples as well .", "the differentiation of the viable and non - viable embryos was done using the observation that compared to blank controls the samples of embryos which later did not resulted in pregnancy contained the alpha-1 subunit in a much larger quantity than the samples of embryos which did ( figure 2 ) .", "clinical statistical analysis of the results revealed that the specificity of the diagnostic test was 64% , while the sensitivity was 100% .", "it is more informative that the positive predictive value of the assay was 51% and  maybe more importantly  the negative predictive value was 100% .", "receiver operating characteristic ( roc ) analysis provides tools to select possibly optimal models and to discard suboptimal ones .", "roc analysis is related in a direct and natural way to cost / benefit analysis of diagnostic decision - making", ". the roc curve of the morphological versus metabolomic approach in relation to the correct prediction of pregnancy outcome is illustrated in figure 3 .", "it is obvious that our biochemical investigation method enables a selection of the embryos by sorting out the non - viable ones .", "the test selected with 100% potential the embryos , which did not lead to successful implantation at all .", "one of the areas of collaboration between clinicians , the clinical laboratory and the research laboratory at the university of pcs is related to the research of infertility . since the clinical background gives the beauty and the medical importance of laboratory research , it was of outstanding importance for us to receive the eflm - abbott diagnostics award for excellence in outcomes research in laboratory medicine ( paris , 2015 ) , the award given to the best published paper ( 47 ) , as judged by an independent panel of experts , which demonstrates improved outcomes arising out of the application or improved utilization of an in - vitro diagnostics test ."]]}
{"article_id": "PMC3114341", "article_text": ["this study is part of a larger project examining the possible health effects of lifetime exposure to marine food and pollutants .", "the faroe islands constitute a unique setting at northern latitude in which the residents have an increased exposure to methylmercury and persistent organic pollutants , including polychlorinated biphenyls ( pcb ) , because of their intake of traditional marine food , including pilot whale meat and blubber . to examine subjects aged 7074 years", "all 1,131 faroese citizens born between 2 january 1934 and 31 august 1937 received a letter of invitation , 713 of whom gave consent and were examined , corresponding to a participation rate of 64% , taking into account 14 deceased subjects .", "furthermore , sufficient serum for determination of vitamin d status was not available from 44 subjects ; thus , 668 subjects are included in the current study .", "the participants were asked to fast and arrive before breakfast to undergo blood sampling and a thorough clinical examination .", "all participants traveled to the same examination location and were examined by the same research nurses and a single physician .", "the participants completed current health , past medical history , and lifestyle questionnaires , including summary information on the frequency of fish dinners during the last year .", "a fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor ( precision xceed and abbott precision xtra plus strips ; abbott diabetes care , copenhagen , denmark ) . because plasma has higher water content than whole blood , the blood values", "were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer .", "fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography .", "all hba1c values were given as relative concentration , % ( diabetes control and complications trial , dcct , aligned results ) .", "hba1c % values were converted to international federation of clinical chemistry and laboratory medicine ( ifcc ) units ( millimoles per moles ) by the following equation : hba1c ( dcct ) % = 0.0915  hba1c ( ifcc ) mmol / mol + 2.15 , provided by the laboratory .", "edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay ( autodelfia ; perkinelmer life and analytical sciences , walla oy , turku , finland ) .", "serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction , using a modular p analyzer ( roche diagnostics , indianapolis , in ) .", "diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication ; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . in participants", "not previously diagnosed , type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0", "insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) and calculated as fpi  fpg / 22.5 ( 10 ) , where fpi is fasting plasma insulin and fpg is fasting plasma glucose .", "-cell dysfunction was determined by calculating the homa -index of ( 20  fpi)/(fpg  3.5 ) ( 10 ) .", "after storage at 80c , serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) as described previously ( 11 ) .", "nmol / l , and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l . means ( sd ) were calculated for variables showing normal distribution and median and 2575th percentiles ( interquartile range [ iqr ] ) for those deviating from it .", "when transformations were insufficient to obtain normal distribution , we applied nonparametric spearman s correlation coefficient . when appropriate , multiple regression analysis was carried out .", "a logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes .", "vitamin d status was used as a binary exposure variable , i.e. , dichotomized at above ( sufficient ) or below 50 nmol / l ( deficient ) , and in the simple model , sex was used as an obligate covariate . in the adjusted model , additional covariates", "were included , i.e. , bmi , serum triacylglycerides , serum hdl , pcb exposure ( sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ) , smoking , and month of blood sampling .", "in addition , we examined the association between vitamin d status and risk of having 1 ) newly diagnosed type 2 diabetes or 2 ) a known diagnosis of type 2 diabetes using the same models separately . finally , the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . in the models", ", we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity .", "a value of p < 0.05 ( two - tailed ) was taken to indicate statistical significance .", "stata version 11.0 ( stata corporation , college station , tx ) was used for statistical analyses .", "a fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor ( precision xceed and abbott precision xtra plus strips ; abbott diabetes care , copenhagen , denmark ) . because plasma has higher water content than whole blood , the blood values", "were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer .", "fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography .", "all hba1c values were given as relative concentration , % ( diabetes control and complications trial , dcct , aligned results ) .", "hba1c % values were converted to international federation of clinical chemistry and laboratory medicine ( ifcc ) units ( millimoles per moles ) by the following equation : hba1c ( dcct ) % = 0.0915  hba1c ( ifcc ) mmol / mol + 2.15 , provided by the laboratory .", "edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay ( autodelfia ; perkinelmer life and analytical sciences , walla oy , turku , finland ) .", "serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction , using a modular p analyzer ( roche diagnostics , indianapolis , in ) .", "diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication ; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . in participants", "not previously diagnosed , type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0", "insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) and calculated as fpi  fpg / 22.5 ( 10 ) , where fpi is fasting plasma insulin and fpg is fasting plasma glucose .", "-cell dysfunction was determined by calculating the homa -index of ( 20  fpi)/(fpg  3.5 ) ( 10 ) .", "after storage at 80c , serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) as described previously ( 11 ) .", "nmol / l , and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l .", "means ( sd ) were calculated for variables showing normal distribution and median and 2575th percentiles ( interquartile range [ iqr ] ) for those deviating from it . when transformations were insufficient to obtain normal distribution , we applied nonparametric spearman s correlation coefficient . when appropriate , multiple regression analysis was carried out .", "a logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes .", "control subjects were nondiabetic subjects , i.e. , subjects with fasting blood glucose 6 mmol / l .", "vitamin d status was used as a binary exposure variable , i.e. , dichotomized at above ( sufficient ) or below 50 nmol / l ( deficient ) , and in the simple model , sex was used as an obligate covariate . in the adjusted model , additional covariates were included , i.e. , bmi , serum triacylglycerides , serum hdl , pcb exposure ( sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ) , smoking , and month of blood sampling .", "in addition , we examined the association between vitamin d status and risk of having 1 ) newly diagnosed type 2 diabetes or 2 ) a known diagnosis of type 2 diabetes using the same models separately . finally , the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . in the models", ", we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity . a value of p < 0.05 ( two - tailed ) was taken to indicate statistical significance .", "stata version 11.0 ( stata corporation , college station , tx ) was used for statistical analyses .", "in the current study , the cohort consisted of 327 women and 341 men with a mean age of 72.5 years .", "a total of 158 septuagenarians ( 24% ) had type 2 diabetes , of whom 88 ( 13% ) had been previously diagnosed with the disease and 70 new subjects ( 11% ) were identified in the current study .", "more than half of the subjects had vitamin d deficiency , as suggested by a serum 25(oh)d3 concentration below 50 nmol / l ( median , 47.6 nmol / l ; iqr , 29.864.8 nmol / l ) .", "baseline clinical and biochemical characteristics of 668 faroese septuagenarians by vitamin d status data are means ( sd ) or median ( 2575th percentile ) . *", "hba1c ( ifcc ) mmol / mol can be converted to hba1c ( dcct ) % by the following equation : hba1c ( dcct ) % = 0.0915  hba1c ( ifcc ) mmol / mol + 2.15 ; homa - ir = ( fasting plasma insulin  fasting plasma glucose)/22.5 ; homa-% = ( 20  fasting plasma insulin)/(fasting plasma glucose  3.5 ) ; sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ( 12 ) .", "nmol / l , was associated with an 80% increase in the sex - adjusted odds of having diabetes ( newly diagnosed and previously diagnosed subjects ) compared with sufficient vitamin d status ( odds ratio [ or ] 1.80 [ 95% ci 1.232.64 ] ; p = 0.002 ) .", "after further adjustment for bmi , serum triacylglycerides , serum hdl , pcb exposure , smoking , and month of blood sampling , the association between vitamin d deficiency and type 2 diabetes remained , and the or was only marginally affected ( or 1.67 [ 1.112.50 ] ; p = 0.013 ) . in the separate analyses of previously undiagnosed and diagnosed subjects , we found that the sex - adjusted or for having newly diagnosed type 2 diabetes in the presence of vitamin d deficiency had more than doubled ( or 2.25 [ 95% ci 1.313.85 ] ; p = 0.003 ) .", "after further adjustment for bmi , serum triacylglycerides , serum hdl , pcb exposure , smoking , and month of blood sampling , the association between vitamin d deficiency and newly diagnosed type 2 diabetes remained unchanged ( or 1.99 [ 1.143.48 ] ; p = 0.022 ) , whereas the adjusted or for pre - existing diabetes in vitamin d deficiency was reduced considerably by similar adjustment ( or 1.48 ; p = 0.132 ) ( table 2 ) .", "we performed logistic regression analysis on the impact of fish dinner frequencies on the risk of type 2 diabetes .", "frequent fish intake ( more than twice a week ) did not appear to alter the odds of having type 2 diabetes ( or 1.09 [ 95% ci 0.641.86 ] ; p = 0.744 ) .", "obesity is a risk factor for vitamin d deficiency , and obese subjects had a lower serum 25(oh)d3 concentration than nonobese subjects ( 11 ) .", "furthermore , we have previously demonstrated in this cohort ( 11 ) that women have higher serum 25(oh)d3 concentration and tend to have lower bmi .", "however , we did not find any interaction between vitamin d status and sex ( p = 0.19 ) or vitamin d status and obesity ( p = 0.84 ) on risk of diabetes .", "statistically significant bivariate correlations were observed between serum 25(oh)d3 concentration and several indicators of glucose metabolism , including hba1c ( rs = 0.10 ; p = 0.01 ) , fasting plasma insulin ( rs = 0.10 ; p = 0.01 ) , homa - ir ( rs = 0.10 ; p = 0.01 ) , and homa - b ( r = 0.10 ; p = 0.01 ) , whereas the plasma glucose concentration ( rs = 0.01 ; p = 0.78 ) was not correlated with 25(oh)d3 concentration .", "however , because bmi was strongly associated with vitamin d status ( rs = 0.17 ; p < 0.0001 ) , the observed associations of 25(oh)d3 with fasting plasma insulin and the homa indexes were substantially reduced after adjustment for bmi , sex , smoking status , and serum triacylglyceride concentration , where none of them were statistically significant ( table 3 ) .", "however , hba1c decreased at higher serum 25(oh)d3 concentrations after adjustment (  = 0.026 , p = 0.026 ) .", "excluding subjects with physician - diagnosed type 2 diabetes resulted in a smaller effect that was no longer significant , probably because of reduced variation in hba1c concentration and fewer observations (  = 0.012 , p = 0.112 ) ( table 2 ) .", "finally , multiple regression analysis did not reveal any association between frequency of fish dinners and the hba1c concentration ( results not shown ) .", "unadjusted and adjusted or for type 2 diabetes in elderly faroese aged 7074 years data are or ( 95% ci ) expressed for binary traits .", "mmol / l ; the crude model is adjusted for sex and compares the odds of either overall type 2 diabetes ( all type 2 diabetic subjects ) , newly diagnosed type 2 diabetes ( new subjects ) , or known type 2 diabetes in subjects ( previously known subjects ) with 25(oh)d < 50", "nmol / l ( reference group ) ; the adjusted model includes sex , bmi , total pcb , month of blood sampling , serum triacylglyceride concentration , serum hdl concentration , and smoking status .", "in this cross - sectional analysis of a population - based sample of septuagenarians , we found that the 25(oh)d3 concentration was inversely associated with the risk of newly diagnosed type 2 diabetes after adjustment for bmi and other established risk factors for type 2 diabetes .", "subjects with 25(oh)d3 concentration below 50 nmol / l had the doubled risk of newly diagnosed diabetes compared with those having higher serum concentrations . therefore , our data suggest an inverse association between the serum concentration of 25(oh)d3 and the odds of newly diagnosed type 2 diabetes , thereby suggesting a protective role of vitamin d against the development of the disease , as suggested previously ( 13 ) . in further support of this association , we observed an increasing concentration of hba1c with decreasing 25(oh)d3 concentration , and this association was independent of bmi , serum triacylglycerides , smoking status , and sex .", "vitamin d deficiency showed a much weaker or for type 2 diabetes previously diagnosed , as could perhaps have been anticipated , given the vigorous clinical management of the disease and the longer time interval between disease development and the assessment of vitamin d status .", "the confidence interval for the or for the two groups of type 2 diabetes continues to overlap considerably , and therefore , the slight difference should not be overinterpreted ( table 2 ) .", "linear regression coefficients for the association between 25(oh)d3 and markers of glucose metabolism in elderly faroese including or excluding subjects with known diabetes * numbers vary because of varying missing sample material for each biochemical analysis .", "is considered an indicator of average blood glucose concentrations during the preceding 2 to 3 months and , thus , a long - term marker of glucose homeostasis ( 14 ) .", "abnormalities may be a result of changes in insulin secretion and insulin - stimulated uptake of glucose in muscle and fat tissue . in vitro studies and laboratory animal studies suggest possible mechanisms for the effects of the active form of vitamin d , i.e. , 1,25(oh)2d , on both insulin secretion and insulin sensitivity as reviewed by pittas and dawson - hughes ( 15 ) .", "for instance , in mice expressing a functionally inactive mutant vitamin d receptor , blood glucose concentrations were increased and serum insulin was reduced , as was insulin mrna .", "pancreatic -cells express the vitamin d receptor and the enzyme 1--hydroxylase , thus suggesting that the active form of vitamin d has a functional role in these cells .", "even though the current study can not elucidate the likely mechanisms involved , our results appear highly plausible given the findings in experimental studies .", "increased fat mass adversely influences glucose metabolism , in part by increasing insulin resistance , and is also linked with lower 25(oh)d concentrations in serum ( 1618 ) .", "the results suggest that the negative impact of vitamin d deficiency on hba1c and newly diagnosed type 2 diabetes risk is independent of increased fat mass .", "however , because bmi does not distinguish visceral from subcutaneous fat , some misclassification of adiposity may have occurred , thereby possibly causing some uncertainty in regard to the relative influence of serum 25(oh)d3 and adiposity in diabetes development .", "our study results are comparable with studies on vitamin d status and type 2 diabetes in younger populations ( 4,16,19,20 ) , and the current study extends this association to 70 years of age and older . vitamin d status is known to be lower in the elderly compared with the young as the subcutaneous synthesis of vitamin d decreases with increasing age , due to a reduced concentration of 7-dehydrocholesterol in the skin ( 1 ) and reduced absorption of oral vitamin d ( 8) . hence , if the relationship between vitamin d status and glucose homeostasis is causal , the age - related decline in circulating concentration of 25(oh)d may augment the increase in risk of type 2 diabetes with age .", "in addition , our results suggest that this relationship is not the result of increased fish intake , in agreement with the existing , somewhat equivocal evidence in regard to this effect ( 21,22 ) .", "these differences may be a result of variations in fish intake to inherent differences in the populations examined and the study designs . among the strengths of our study", ", we examined a general population sample with a high prevalence of both vitamin d deficiency ( 11 ) and type 2 diabetes .", "however , the current study is limited by its cross - sectional design and its single measurement of vitamin d status .", "nonetheless , a recent study demonstrated excellent tracking and that the correlation of serum 25(oh)d3 concentrations sampled 14 years apart was similar to that seen for other cardiovascular risk factors and that most subjects with vitamin d deficiency were still deficient 14 years later ( 23 ) .", "patients with type 2 diabetes and elevated hba1c concentrations are at increased risk of cardiovascular disease and total mortality compared with patients with lower hba1c levels ( 24 ) .", "thus , clinical management focuses on glycemic control , including treatment of other modifiable cardiovascular risk factors to reduce the macro- as well as microvascular complications .", "the inverse association between serum 25(oh)d3 and hba1c suggests that vitamin d supplementation could be considered a possible means for supporting glycemic control of type 2 diabetes .", "however , some intervention studies have shown inconclusive results on the effect of vitamin d on hba1c and type 2 diabetes ( 15 ) . for example , the women health s initiative demonstrated that daily supplementation with 10 g of vitamin d3 for a median follow - up time of 7 years did not reduce the risk of type 2 diabetes in postmenopausal women ( 25 ) .", "however , the doses used were probably too low to bring about the necessary concentrations of circulating 25(oh)d3 .", "therefore , further randomized clinical intervention studies are needed to elucidate the impact of vitamin d on the risk of type 2 diabetes . in summary ,", "our study extends previous findings that a high vitamin d status protects against type 2 diabetes in younger subjects to subjects older than 70 years .", "likewise , our findings suggest an inverse association between hba1c and 25(oh)d3 also in the elderly . because our study has a cross - sectional design , formal confirmation requires intervention studies with appropriate doses to elucidate whether vitamin d supplementation could be used to counter the worldwide epidemic of type 2 diabetes . in this", "regard , the need to identify the necessary daily intake of vitamin d should include the possible effect on reducing the risk of type 2 diabetes , with special emphasis on the dietary needs of the elderly ."], "abstract_text": ["<S> objectivevitamin d deficiency is thought to be a risk factor for development of type 2 diabetes , and elderly subjects at northern latitudes may therefore be at particular risk.research design and methodsvitamin d status was assessed from serum concentrations of 25-hydroxyvitamin d3 [ 25(oh)d3 ] in 668 faroese residents aged 7074 years ( 64% of eligible population ) . </S>", "<S> we determined type 2 diabetes prevalence from past medical histories , fasting plasma concentrations of glucose , and/or glycosylated hemoglobin ( hba1c).resultswe observed 70 ( 11% ) new type 2 diabetic subjects , whereas 88 ( 13% ) were previously diagnosed . having vitamin d status </S>", "<S> < 50 nmol / l doubled the risk of newly diagnosed type 2 diabetes after adjustment for bmi , sex , exposure to polychlorinated biphenyls , serum triacylglyceride concentration , serum hdl concentration , smoking status , and month of blood sampling . </S>", "<S> furthermore , the hba1c concentration decreased at higher serum 25(oh)d3 concentrations independent of covariates.conclusionsin elderly subjects , vitamin d sufficiency may provide protection against type 2 diabetes . because the study is cross - sectional , </S>", "<S> intervention studies are needed to elucidate whether vitamin d could be used to prevent development of type 2 diabetes . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Assessment of type 2 diabetes and markers of glucose metabolism", "Vitamin D status", "Statistical analysis", "RESULTS", "CONCLUSIONS"], "sections": [["this study is part of a larger project examining the possible health effects of lifetime exposure to marine food and pollutants .", "the faroe islands constitute a unique setting at northern latitude in which the residents have an increased exposure to methylmercury and persistent organic pollutants , including polychlorinated biphenyls ( pcb ) , because of their intake of traditional marine food , including pilot whale meat and blubber . to examine subjects aged 7074 years", "all 1,131 faroese citizens born between 2 january 1934 and 31 august 1937 received a letter of invitation , 713 of whom gave consent and were examined , corresponding to a participation rate of 64% , taking into account 14 deceased subjects .", "furthermore , sufficient serum for determination of vitamin d status was not available from 44 subjects ; thus , 668 subjects are included in the current study .", "the participants were asked to fast and arrive before breakfast to undergo blood sampling and a thorough clinical examination .", "all participants traveled to the same examination location and were examined by the same research nurses and a single physician .", "the participants completed current health , past medical history , and lifestyle questionnaires , including summary information on the frequency of fish dinners during the last year .", "a fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor ( precision xceed and abbott precision xtra plus strips ; abbott diabetes care , copenhagen , denmark ) . because plasma has higher water content than whole blood , the blood values", "were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer .", "fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography .", "all hba1c values were given as relative concentration , % ( diabetes control and complications trial , dcct , aligned results ) .", "hba1c % values were converted to international federation of clinical chemistry and laboratory medicine ( ifcc ) units ( millimoles per moles ) by the following equation : hba1c ( dcct ) % = 0.0915  hba1c ( ifcc ) mmol / mol + 2.15 , provided by the laboratory .", "edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay ( autodelfia ; perkinelmer life and analytical sciences , walla oy , turku , finland ) .", "serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction , using a modular p analyzer ( roche diagnostics , indianapolis , in ) .", "diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication ; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . in participants", "not previously diagnosed , type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0", "insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) and calculated as fpi  fpg / 22.5 ( 10 ) , where fpi is fasting plasma insulin and fpg is fasting plasma glucose .", "-cell dysfunction was determined by calculating the homa -index of ( 20  fpi)/(fpg  3.5 ) ( 10 ) .", "after storage at 80c , serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) as described previously ( 11 ) .", "nmol / l , and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l . means ( sd ) were calculated for variables showing normal distribution and median and 2575th percentiles ( interquartile range [ iqr ] ) for those deviating from it .", "when transformations were insufficient to obtain normal distribution , we applied nonparametric spearman s correlation coefficient . when appropriate , multiple regression analysis was carried out .", "a logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes .", "vitamin d status was used as a binary exposure variable , i.e. , dichotomized at above ( sufficient ) or below 50 nmol / l ( deficient ) , and in the simple model , sex was used as an obligate covariate . in the adjusted model , additional covariates", "were included , i.e. , bmi , serum triacylglycerides , serum hdl , pcb exposure ( sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ) , smoking , and month of blood sampling .", "in addition , we examined the association between vitamin d status and risk of having 1 ) newly diagnosed type 2 diabetes or 2 ) a known diagnosis of type 2 diabetes using the same models separately . finally , the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . in the models", ", we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity .", "a value of p < 0.05 ( two - tailed ) was taken to indicate statistical significance .", "stata version 11.0 ( stata corporation , college station , tx ) was used for statistical analyses ."], ["a fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor ( precision xceed and abbott precision xtra plus strips ; abbott diabetes care , copenhagen , denmark ) . because plasma has higher water content than whole blood , the blood values", "were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer .", "fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography .", "all hba1c values were given as relative concentration , % ( diabetes control and complications trial , dcct , aligned results ) .", "hba1c % values were converted to international federation of clinical chemistry and laboratory medicine ( ifcc ) units ( millimoles per moles ) by the following equation : hba1c ( dcct ) % = 0.0915  hba1c ( ifcc ) mmol / mol + 2.15 , provided by the laboratory .", "edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay ( autodelfia ; perkinelmer life and analytical sciences , walla oy , turku , finland ) .", "serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction , using a modular p analyzer ( roche diagnostics , indianapolis , in ) .", "diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication ; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . in participants", "not previously diagnosed , type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0", "insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) and calculated as fpi  fpg / 22.5 ( 10 ) , where fpi is fasting plasma insulin and fpg is fasting plasma glucose .", "-cell dysfunction was determined by calculating the homa -index of ( 20  fpi)/(fpg  3.5 ) ( 10 ) ."], ["after storage at 80c , serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) as described previously ( 11 ) .", "nmol / l , and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l ."], ["means ( sd ) were calculated for variables showing normal distribution and median and 2575th percentiles ( interquartile range [ iqr ] ) for those deviating from it . when transformations were insufficient to obtain normal distribution , we applied nonparametric spearman s correlation coefficient . when appropriate , multiple regression analysis was carried out .", "a logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes .", "control subjects were nondiabetic subjects , i.e. , subjects with fasting blood glucose 6 mmol / l .", "vitamin d status was used as a binary exposure variable , i.e. , dichotomized at above ( sufficient ) or below 50 nmol / l ( deficient ) , and in the simple model , sex was used as an obligate covariate . in the adjusted model , additional covariates were included , i.e. , bmi , serum triacylglycerides , serum hdl , pcb exposure ( sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ) , smoking , and month of blood sampling .", "in addition , we examined the association between vitamin d status and risk of having 1 ) newly diagnosed type 2 diabetes or 2 ) a known diagnosis of type 2 diabetes using the same models separately . finally , the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . in the models", ", we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity . a value of p < 0.05 ( two - tailed ) was taken to indicate statistical significance .", "stata version 11.0 ( stata corporation , college station , tx ) was used for statistical analyses ."], ["in the current study , the cohort consisted of 327 women and 341 men with a mean age of 72.5 years .", "a total of 158 septuagenarians ( 24% ) had type 2 diabetes , of whom 88 ( 13% ) had been previously diagnosed with the disease and 70 new subjects ( 11% ) were identified in the current study .", "more than half of the subjects had vitamin d deficiency , as suggested by a serum 25(oh)d3 concentration below 50 nmol / l ( median , 47.6 nmol / l ; iqr , 29.864.8 nmol / l ) .", "baseline clinical and biochemical characteristics of 668 faroese septuagenarians by vitamin d status data are means ( sd ) or median ( 2575th percentile ) . *", "hba1c ( ifcc ) mmol / mol can be converted to hba1c ( dcct ) % by the following equation : hba1c ( dcct ) % = 0.0915  hba1c ( ifcc ) mmol / mol + 2.15 ; homa - ir = ( fasting plasma insulin  fasting plasma glucose)/22.5 ; homa-% = ( 20  fasting plasma insulin)/(fasting plasma glucose  3.5 ) ; sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ( 12 ) .", "nmol / l , was associated with an 80% increase in the sex - adjusted odds of having diabetes ( newly diagnosed and previously diagnosed subjects ) compared with sufficient vitamin d status ( odds ratio [ or ] 1.80 [ 95% ci 1.232.64 ] ; p = 0.002 ) .", "after further adjustment for bmi , serum triacylglycerides , serum hdl , pcb exposure , smoking , and month of blood sampling , the association between vitamin d deficiency and type 2 diabetes remained , and the or was only marginally affected ( or 1.67 [ 1.112.50 ] ; p = 0.013 ) . in the separate analyses of previously undiagnosed and diagnosed subjects , we found that the sex - adjusted or for having newly diagnosed type 2 diabetes in the presence of vitamin d deficiency had more than doubled ( or 2.25 [ 95% ci 1.313.85 ] ; p = 0.003 ) .", "after further adjustment for bmi , serum triacylglycerides , serum hdl , pcb exposure , smoking , and month of blood sampling , the association between vitamin d deficiency and newly diagnosed type 2 diabetes remained unchanged ( or 1.99 [ 1.143.48 ] ; p = 0.022 ) , whereas the adjusted or for pre - existing diabetes in vitamin d deficiency was reduced considerably by similar adjustment ( or 1.48 ; p = 0.132 ) ( table 2 ) .", "we performed logistic regression analysis on the impact of fish dinner frequencies on the risk of type 2 diabetes .", "frequent fish intake ( more than twice a week ) did not appear to alter the odds of having type 2 diabetes ( or 1.09 [ 95% ci 0.641.86 ] ; p = 0.744 ) .", "obesity is a risk factor for vitamin d deficiency , and obese subjects had a lower serum 25(oh)d3 concentration than nonobese subjects ( 11 ) .", "furthermore , we have previously demonstrated in this cohort ( 11 ) that women have higher serum 25(oh)d3 concentration and tend to have lower bmi .", "however , we did not find any interaction between vitamin d status and sex ( p = 0.19 ) or vitamin d status and obesity ( p = 0.84 ) on risk of diabetes .", "statistically significant bivariate correlations were observed between serum 25(oh)d3 concentration and several indicators of glucose metabolism , including hba1c ( rs = 0.10 ; p = 0.01 ) , fasting plasma insulin ( rs = 0.10 ; p = 0.01 ) , homa - ir ( rs = 0.10 ; p = 0.01 ) , and homa - b ( r = 0.10 ; p = 0.01 ) , whereas the plasma glucose concentration ( rs = 0.01 ; p = 0.78 ) was not correlated with 25(oh)d3 concentration .", "however , because bmi was strongly associated with vitamin d status ( rs = 0.17 ; p < 0.0001 ) , the observed associations of 25(oh)d3 with fasting plasma insulin and the homa indexes were substantially reduced after adjustment for bmi , sex , smoking status , and serum triacylglyceride concentration , where none of them were statistically significant ( table 3 ) .", "however , hba1c decreased at higher serum 25(oh)d3 concentrations after adjustment (  = 0.026 , p = 0.026 ) .", "excluding subjects with physician - diagnosed type 2 diabetes resulted in a smaller effect that was no longer significant , probably because of reduced variation in hba1c concentration and fewer observations (  = 0.012 , p = 0.112 ) ( table 2 ) .", "finally , multiple regression analysis did not reveal any association between frequency of fish dinners and the hba1c concentration ( results not shown ) .", "unadjusted and adjusted or for type 2 diabetes in elderly faroese aged 7074 years data are or ( 95% ci ) expressed for binary traits .", "mmol / l ; the crude model is adjusted for sex and compares the odds of either overall type 2 diabetes ( all type 2 diabetic subjects ) , newly diagnosed type 2 diabetes ( new subjects ) , or known type 2 diabetes in subjects ( previously known subjects ) with 25(oh)d < 50", "nmol / l ( reference group ) ; the adjusted model includes sex , bmi , total pcb , month of blood sampling , serum triacylglyceride concentration , serum hdl concentration , and smoking status ."], ["in this cross - sectional analysis of a population - based sample of septuagenarians , we found that the 25(oh)d3 concentration was inversely associated with the risk of newly diagnosed type 2 diabetes after adjustment for bmi and other established risk factors for type 2 diabetes .", "subjects with 25(oh)d3 concentration below 50 nmol / l had the doubled risk of newly diagnosed diabetes compared with those having higher serum concentrations . therefore , our data suggest an inverse association between the serum concentration of 25(oh)d3 and the odds of newly diagnosed type 2 diabetes , thereby suggesting a protective role of vitamin d against the development of the disease , as suggested previously ( 13 ) . in further support of this association , we observed an increasing concentration of hba1c with decreasing 25(oh)d3 concentration , and this association was independent of bmi , serum triacylglycerides , smoking status , and sex .", "vitamin d deficiency showed a much weaker or for type 2 diabetes previously diagnosed , as could perhaps have been anticipated , given the vigorous clinical management of the disease and the longer time interval between disease development and the assessment of vitamin d status .", "the confidence interval for the or for the two groups of type 2 diabetes continues to overlap considerably , and therefore , the slight difference should not be overinterpreted ( table 2 ) .", "linear regression coefficients for the association between 25(oh)d3 and markers of glucose metabolism in elderly faroese including or excluding subjects with known diabetes * numbers vary because of varying missing sample material for each biochemical analysis .", "is considered an indicator of average blood glucose concentrations during the preceding 2 to 3 months and , thus , a long - term marker of glucose homeostasis ( 14 ) .", "abnormalities may be a result of changes in insulin secretion and insulin - stimulated uptake of glucose in muscle and fat tissue . in vitro studies and laboratory animal studies suggest possible mechanisms for the effects of the active form of vitamin d , i.e. , 1,25(oh)2d , on both insulin secretion and insulin sensitivity as reviewed by pittas and dawson - hughes ( 15 ) .", "for instance , in mice expressing a functionally inactive mutant vitamin d receptor , blood glucose concentrations were increased and serum insulin was reduced , as was insulin mrna .", "pancreatic -cells express the vitamin d receptor and the enzyme 1--hydroxylase , thus suggesting that the active form of vitamin d has a functional role in these cells .", "even though the current study can not elucidate the likely mechanisms involved , our results appear highly plausible given the findings in experimental studies .", "increased fat mass adversely influences glucose metabolism , in part by increasing insulin resistance , and is also linked with lower 25(oh)d concentrations in serum ( 1618 ) .", "the results suggest that the negative impact of vitamin d deficiency on hba1c and newly diagnosed type 2 diabetes risk is independent of increased fat mass .", "however , because bmi does not distinguish visceral from subcutaneous fat , some misclassification of adiposity may have occurred , thereby possibly causing some uncertainty in regard to the relative influence of serum 25(oh)d3 and adiposity in diabetes development .", "our study results are comparable with studies on vitamin d status and type 2 diabetes in younger populations ( 4,16,19,20 ) , and the current study extends this association to 70 years of age and older . vitamin d status is known to be lower in the elderly compared with the young as the subcutaneous synthesis of vitamin d decreases with increasing age , due to a reduced concentration of 7-dehydrocholesterol in the skin ( 1 ) and reduced absorption of oral vitamin d ( 8) . hence , if the relationship between vitamin d status and glucose homeostasis is causal , the age - related decline in circulating concentration of 25(oh)d may augment the increase in risk of type 2 diabetes with age .", "in addition , our results suggest that this relationship is not the result of increased fish intake , in agreement with the existing , somewhat equivocal evidence in regard to this effect ( 21,22 ) .", "these differences may be a result of variations in fish intake to inherent differences in the populations examined and the study designs . among the strengths of our study", ", we examined a general population sample with a high prevalence of both vitamin d deficiency ( 11 ) and type 2 diabetes .", "however , the current study is limited by its cross - sectional design and its single measurement of vitamin d status .", "nonetheless , a recent study demonstrated excellent tracking and that the correlation of serum 25(oh)d3 concentrations sampled 14 years apart was similar to that seen for other cardiovascular risk factors and that most subjects with vitamin d deficiency were still deficient 14 years later ( 23 ) .", "patients with type 2 diabetes and elevated hba1c concentrations are at increased risk of cardiovascular disease and total mortality compared with patients with lower hba1c levels ( 24 ) .", "thus , clinical management focuses on glycemic control , including treatment of other modifiable cardiovascular risk factors to reduce the macro- as well as microvascular complications .", "the inverse association between serum 25(oh)d3 and hba1c suggests that vitamin d supplementation could be considered a possible means for supporting glycemic control of type 2 diabetes .", "however , some intervention studies have shown inconclusive results on the effect of vitamin d on hba1c and type 2 diabetes ( 15 ) . for example , the women health s initiative demonstrated that daily supplementation with 10 g of vitamin d3 for a median follow - up time of 7 years did not reduce the risk of type 2 diabetes in postmenopausal women ( 25 ) .", "however , the doses used were probably too low to bring about the necessary concentrations of circulating 25(oh)d3 .", "therefore , further randomized clinical intervention studies are needed to elucidate the impact of vitamin d on the risk of type 2 diabetes . in summary ,", "our study extends previous findings that a high vitamin d status protects against type 2 diabetes in younger subjects to subjects older than 70 years .", "likewise , our findings suggest an inverse association between hba1c and 25(oh)d3 also in the elderly . because our study has a cross - sectional design , formal confirmation requires intervention studies with appropriate doses to elucidate whether vitamin d supplementation could be used to counter the worldwide epidemic of type 2 diabetes . in this", "regard , the need to identify the necessary daily intake of vitamin d should include the possible effect on reducing the risk of type 2 diabetes , with special emphasis on the dietary needs of the elderly ."]]}
{"article_id": "PMC3013730", "article_text": ["leaf senescence is the last phase of plant development and a highly coordinated process regulated by a large number of senescence associated genes ( sags ) ( 12 ) .", "leaf senescence can either be naturally induced during development stages , or stimulated by environmental factors including darkness , nutritional deficiency and various stresses ( 13 ) .", "premature senescence is an important factor leading to the decrease of crop yield and quality , which becomes an increasing concern due to the global climate change in the recent years .", "many advances in the understanding of leaf senescence at the molecular level had been achieved through the identification and characterization of hundreds of sags and senescence - related mutants in arabidopsis thaliana , lycopersicon esculentum and nicotiana tabaccum ( 14 ) .", "microarray expression profiling in arabidopsis revealed that more than 800 genes are up - regulated during the course of leaf senescence ( 5 ) . among them , more than 200 transcription factors , including wrky , nac , mads , myb , bzip and bhlh family members , are involved in the regulation of leaf senescence , indicating that leaf senescence is governed by complex transcriptional regulatory networks .", "molecular and genetic studies of leaf senescence in recent years led to the accumulation of a large volume of scattered information related to sags .", "the construction of a leaf senescence related database with wide - spread collection and systematic annotation of sags may provide a useful resource and a good starting point for the further study of the molecular aspects of leaf senescence .", "some initial efforts to this end had been made ; including the online website of plant senescence network ( sennet ) constructed by thomas and his colleagues ( http://www.sidthomas.net/plant_senescence/ ) and the corresponding senwiki web pages ( http://www.sidthomas.net/senwiki/ ) .", "sennet brings together various community information including meetings , laboratories , websites and useful links related to plant senescence , while senwiki provides general information and knowledge of senescence with texts and images .", "however , a wide - ranging compilation and detailed annotation of known sags that would be of great help and demand to the systemic study of leaf senescence at the molecular level has yet to be done . in this regard ,", "we have developed a leaf senescence database ( lsd ) ( http://www.eplantsenescence.org/ ) by retrieving and integrating information from research papers and public databases . at present", "1145 sags from 21 species were manually curated and categorized into several groups according to their function .", "users can browse the entries in the database to obtain information including literatures , mutants , phenotypes , expression profiles , mirna interactions , orthologs in other plants and cross links to protein domain and family databases .", "users can also search the database easily through the  text search interface with locus names , keywords and author names , etc .", "we have implemented the blast tool kit for sequence similarity search against nucleotide or protein sequences of these sags in the lsd . a major feature of the lsd is the integration of a bioinformatics platform weblab we had developed previously ( 6 ) , which allows users to perform extensive sequence analysis of the sags they are interested in .", "all the sag sequences are freely available for downloading . help information including user guides , a tutorials and faqs are also available online .", "we plan to identify putative sags from completely sequenced genomes and add them into the database in the near future , and improve the user interface based on user feedback , provide more help documents with case studies , add more links to other useful sites , and update the database in time with more leaf senescence related data available .", "we hope that lsd could be a useful resource for the leaf senescence research community , as well as a gateway for the collaborative project we are working with both domestic and international colleagues .", "we made an extensive literature survey and collected approximately 200 leaf senescence related papers published before october 2010 on major plant biology journals including plant cell , plant physiology , plant journal , new phytologist , journal of experimental botany , among others .", "a total of 1145 sags from 21 species including arabidopsis , rice and so on , were identified and manually verified based on genetic , genomic , proteomic , physiological and other experimental evidence . among them ,", "96 and 58 genes were supported by mutational investigation or transgenic over - expression study , respectively ( table 1 ) . \n", "table 1.number of sags and mutants in 21 speciesspeciescommon namesagsmutantstransgenicarabidopsis thalianathale cress9499035oryza sativarice10439medicago truncatulabarrel clover3100brassica napusrape1500lycopersicon esculentumtomato803nicotiana tabacumtobacco503brassica oleraceabroccoli400pisum sativumpea410glycine maxsoybean401sorghum bicolorsorghum400solanum tuberosumpotato303zea maysmaize300hordeum vulgarebarley300astragalus sinicuschinese milk vetch101chenopodium rubrumred goosefoot110festuca pratensis huds.fescue110ipomoea niljapanese morning glory101medicago", "sativaalfalfa101rosa hybridrose100triticum turgidumwheat101triticum aestivumwheat100total2111459658 number of sags and mutants in 21 species the information of 154 leaf senescence related mutants such as name , ecotype and mutagenesis method were also retrieved from literatures .", "expression profiling data were acquired from a classical and systemic research paper ( 5 ) .", "in addition to manual curation , computational approaches were also employed to annotate these sags .", "we predicted the potential mirna targets for the sags using the rnahybrid method ( 7 ) .", "the orthologs of each sag in other plants were retrieved from the online database orthomcl - db ( 8) . finally , putative function domains of sags - encoding proteins were identified with interproscan ( 9 ) .", "to meet the general requirement of data analysis , we integrated the sequence similarity search tool blast ( 10 ) and the sequence analysis platform weblab ( 6 ) into our leaf senescence database .", "users can either retrieve the sequence from lsd , or upload their own sequences to search homologs against different divisions ( gene , mrna , cds and protein ) in the lsd .", "weblab is a web - based bioinformatics platform developed by our center and publicly available worldwide ( http://www.weblab.org.cn/ ) ( 6 ) .", "a user space is provided to save input data and analysis results for registered users .", "users may retrieve sequence data and submit to weblab directly to perform extensive analysis for dna , mrna and protein sequences of the sags they are interested in .", "the lsd database enables users to retrieve and analyze sags through the browse or search page .", "users may browse the entries by clicking the buttons of species , mutants or phenotypes at the main page .", "a tree - like structure was designed for both species and phenotypes , and a table was created for mutants . currently , the major source of sags was from the two model organisms a. thaliana ( 949 entries ) and oryza sativa ( 104 entries ) .", "the phenotypes of all sags were divided into the following groups : ( i ) natural senescence , ( ii ) dark induced senescence , ( iii ) nutrition deficiency induced senescence , ( iv ) stress induced senescence and ( v ) others .", "the text search interface allows users to make queries with three types of data : ( i ) locus name , genbank i d , alias , species and description of genes ; ( ii ) name , type and ecotype of mutants ; and ( iii ) title , author , journal and date of literature papers .", "figure 1 shows the annotation for a typical lsd entry ntl9 , a member of the nac transcription factor family and a membrane - associated gene that mediates osmotic stress signaling in leaf senescence ( 11 ) .", "general information such as locus name , alias , organism , taxonomy was retrieved from the literature .", "functional category , effect and evidence of senescence , as well as a brief description of this gene were manually annotated ( figure 1a ) .", "expression profiles generated from microarray data can be found for most arabidopsis sags ( figure 1c ) .", "we predicted potential mirna targets for some sags and added links to mirbase ( 12 ) for these mirnas ( figure 2a ) .", "orthologs from other plants are listed with links to the orthomcl database ( figure 2b ) .", "putative functional domains of proteins encoded by sags were identified and annotated using the interproscan program ( 9 ) , and matches were displayed with cross links to several protein domain and family databases such as prosite and pfam ( figure 2c ) . \n", "( a ) basic information , ( b ) mutant information and ( c ) expression profile . \n figure 2.computational annotations for the arabidopsis nac transcription factor .", "( a ) mirna targets , ( b ) ortholog groups and ( c ) cross links to other databases . a typical entry in lsd , the arabidopsis nac transcription factor .", "( a ) basic information , ( b ) mutant information and ( c ) expression profile .", "( a ) mirna targets , ( b ) ortholog groups and ( c ) cross links to other databases .", "in addition to sequence similarity search with the blast tool kit implanted in the database , users may also perform extensive analysis for sequence data retrieved from the lsd . for each entry ,", "links to different sequence types ( genomic , mrna , cds and protein ) are provided .", "users can click these links to bring up the corresponding sequence and submit it to weblab for further analysis , such as predicting gene structures , making pairwise or multiple sequence alignment , generating sequence logos , constructing phylogenetic trees , finding sequence motifs , etc .", "we will update the database regularly with more leaf senescence related data available , and predict putative sags from completely sequenced plant genomes in the near future .", "we will improve the user interface with comments and suggestions from the user community and add more documents including case studies to help user to make thorough analysis of sags .", "we hope that lsd can be a platform not only for the domestic and international collaborators we are working with , but also for the research community of leaf senescence worldwide .", "national natural science foundation of china ( 31071160 to j.l . , 30625003 and 30730011 to h.g . ) ; ministry of science and technology of china ( 2009cb119101 to h.g . ) ; ministry of education of china ( ngi2008 - 108 - 3 to j.l .", ", ed20060047 to h.g . ) . funding for open access charge : national laboratory of protein engineering and plant genetic engineering ."], "abstract_text": ["<S> by broad literature survey , we have developed a leaf senescence database ( lsd , http://www.eplantsenescence.org/ ) that contains a total of 1145 senescence associated genes ( sags ) from 21 species . </S>", "<S> these sags were retrieved based on genetic , genomic , proteomic , physiological or other experimental evidence , and were classified into different categories according to their functions in leaf senescence or morphological phenotypes when mutated . </S>", "<S> we made extensive annotations for these sags by both manual and computational approaches , and users can either browse or search the database to obtain information including literatures , mutants , phenotypes , expression profiles , mirna interactions , orthologs in other plants and cross links to other databases . </S>", "<S> we have also integrated a bioinformatics analysis platform weblab into lsd , which allows users to perform extensive sequence analysis of their interested sags . </S>", "<S> the sag sequences in lsd can also be downloaded readily for bulk analysis . </S>", "<S> we believe that the lsd contains the largest number of sags to date and represents the most comprehensive and informative plant senescence - related database , which would facilitate the systems biology research and comparative studies on plant aging . </S>"], "labels": null, "section_names": ["INTRODUCTION", "IDENTIFICATION AND ANNOTATION OF SAGS", "IMPLEMENTATION OF ANALYSIS TOOLS", "BROWSING AND SEARCHING THE DATABASE ENTRIES", "ANALYZING THE SEQUENCE DATA", "FUTURE PLANS", "FUNDING"], "sections": [["leaf senescence is the last phase of plant development and a highly coordinated process regulated by a large number of senescence associated genes ( sags ) ( 12 ) .", "leaf senescence can either be naturally induced during development stages , or stimulated by environmental factors including darkness , nutritional deficiency and various stresses ( 13 ) .", "premature senescence is an important factor leading to the decrease of crop yield and quality , which becomes an increasing concern due to the global climate change in the recent years .", "many advances in the understanding of leaf senescence at the molecular level had been achieved through the identification and characterization of hundreds of sags and senescence - related mutants in arabidopsis thaliana , lycopersicon esculentum and nicotiana tabaccum ( 14 ) .", "microarray expression profiling in arabidopsis revealed that more than 800 genes are up - regulated during the course of leaf senescence ( 5 ) . among them , more than 200 transcription factors , including wrky , nac , mads , myb , bzip and bhlh family members , are involved in the regulation of leaf senescence , indicating that leaf senescence is governed by complex transcriptional regulatory networks .", "molecular and genetic studies of leaf senescence in recent years led to the accumulation of a large volume of scattered information related to sags .", "the construction of a leaf senescence related database with wide - spread collection and systematic annotation of sags may provide a useful resource and a good starting point for the further study of the molecular aspects of leaf senescence .", "some initial efforts to this end had been made ; including the online website of plant senescence network ( sennet ) constructed by thomas and his colleagues ( http://www.sidthomas.net/plant_senescence/ ) and the corresponding senwiki web pages ( http://www.sidthomas.net/senwiki/ ) .", "sennet brings together various community information including meetings , laboratories , websites and useful links related to plant senescence , while senwiki provides general information and knowledge of senescence with texts and images .", "however , a wide - ranging compilation and detailed annotation of known sags that would be of great help and demand to the systemic study of leaf senescence at the molecular level has yet to be done . in this regard ,", "we have developed a leaf senescence database ( lsd ) ( http://www.eplantsenescence.org/ ) by retrieving and integrating information from research papers and public databases . at present", "1145 sags from 21 species were manually curated and categorized into several groups according to their function .", "users can browse the entries in the database to obtain information including literatures , mutants , phenotypes , expression profiles , mirna interactions , orthologs in other plants and cross links to protein domain and family databases .", "users can also search the database easily through the  text search interface with locus names , keywords and author names , etc .", "we have implemented the blast tool kit for sequence similarity search against nucleotide or protein sequences of these sags in the lsd . a major feature of the lsd is the integration of a bioinformatics platform weblab we had developed previously ( 6 ) , which allows users to perform extensive sequence analysis of the sags they are interested in .", "all the sag sequences are freely available for downloading . help information including user guides , a tutorials and faqs are also available online .", "we plan to identify putative sags from completely sequenced genomes and add them into the database in the near future , and improve the user interface based on user feedback , provide more help documents with case studies , add more links to other useful sites , and update the database in time with more leaf senescence related data available .", "we hope that lsd could be a useful resource for the leaf senescence research community , as well as a gateway for the collaborative project we are working with both domestic and international colleagues ."], ["we made an extensive literature survey and collected approximately 200 leaf senescence related papers published before october 2010 on major plant biology journals including plant cell , plant physiology , plant journal , new phytologist , journal of experimental botany , among others .", "a total of 1145 sags from 21 species including arabidopsis , rice and so on , were identified and manually verified based on genetic , genomic , proteomic , physiological and other experimental evidence . among them ,", "96 and 58 genes were supported by mutational investigation or transgenic over - expression study , respectively ( table 1 ) . \n", "table 1.number of sags and mutants in 21 speciesspeciescommon namesagsmutantstransgenicarabidopsis thalianathale cress9499035oryza sativarice10439medicago truncatulabarrel clover3100brassica napusrape1500lycopersicon esculentumtomato803nicotiana tabacumtobacco503brassica oleraceabroccoli400pisum sativumpea410glycine maxsoybean401sorghum bicolorsorghum400solanum tuberosumpotato303zea maysmaize300hordeum vulgarebarley300astragalus sinicuschinese milk vetch101chenopodium rubrumred goosefoot110festuca pratensis huds.fescue110ipomoea niljapanese morning glory101medicago", "sativaalfalfa101rosa hybridrose100triticum turgidumwheat101triticum aestivumwheat100total2111459658 number of sags and mutants in 21 species the information of 154 leaf senescence related mutants such as name , ecotype and mutagenesis method were also retrieved from literatures .", "expression profiling data were acquired from a classical and systemic research paper ( 5 ) .", "in addition to manual curation , computational approaches were also employed to annotate these sags .", "we predicted the potential mirna targets for the sags using the rnahybrid method ( 7 ) .", "the orthologs of each sag in other plants were retrieved from the online database orthomcl - db ( 8) . finally , putative function domains of sags - encoding proteins were identified with interproscan ( 9 ) ."], ["to meet the general requirement of data analysis , we integrated the sequence similarity search tool blast ( 10 ) and the sequence analysis platform weblab ( 6 ) into our leaf senescence database .", "users can either retrieve the sequence from lsd , or upload their own sequences to search homologs against different divisions ( gene , mrna , cds and protein ) in the lsd .", "weblab is a web - based bioinformatics platform developed by our center and publicly available worldwide ( http://www.weblab.org.cn/ ) ( 6 ) .", "a user space is provided to save input data and analysis results for registered users .", "users may retrieve sequence data and submit to weblab directly to perform extensive analysis for dna , mrna and protein sequences of the sags they are interested in ."], ["the lsd database enables users to retrieve and analyze sags through the browse or search page .", "users may browse the entries by clicking the buttons of species , mutants or phenotypes at the main page .", "a tree - like structure was designed for both species and phenotypes , and a table was created for mutants . currently , the major source of sags was from the two model organisms a. thaliana ( 949 entries ) and oryza sativa ( 104 entries ) .", "the phenotypes of all sags were divided into the following groups : ( i ) natural senescence , ( ii ) dark induced senescence , ( iii ) nutrition deficiency induced senescence , ( iv ) stress induced senescence and ( v ) others .", "the text search interface allows users to make queries with three types of data : ( i ) locus name , genbank i d , alias , species and description of genes ; ( ii ) name , type and ecotype of mutants ; and ( iii ) title , author , journal and date of literature papers .", "figure 1 shows the annotation for a typical lsd entry ntl9 , a member of the nac transcription factor family and a membrane - associated gene that mediates osmotic stress signaling in leaf senescence ( 11 ) .", "general information such as locus name , alias , organism , taxonomy was retrieved from the literature .", "functional category , effect and evidence of senescence , as well as a brief description of this gene were manually annotated ( figure 1a ) .", "expression profiles generated from microarray data can be found for most arabidopsis sags ( figure 1c ) .", "we predicted potential mirna targets for some sags and added links to mirbase ( 12 ) for these mirnas ( figure 2a ) .", "orthologs from other plants are listed with links to the orthomcl database ( figure 2b ) .", "putative functional domains of proteins encoded by sags were identified and annotated using the interproscan program ( 9 ) , and matches were displayed with cross links to several protein domain and family databases such as prosite and pfam ( figure 2c ) . \n", "( a ) basic information , ( b ) mutant information and ( c ) expression profile . \n figure 2.computational annotations for the arabidopsis nac transcription factor .", "( a ) mirna targets , ( b ) ortholog groups and ( c ) cross links to other databases . a typical entry in lsd , the arabidopsis nac transcription factor .", "( a ) basic information , ( b ) mutant information and ( c ) expression profile .", "( a ) mirna targets , ( b ) ortholog groups and ( c ) cross links to other databases ."], ["in addition to sequence similarity search with the blast tool kit implanted in the database , users may also perform extensive analysis for sequence data retrieved from the lsd . for each entry ,", "links to different sequence types ( genomic , mrna , cds and protein ) are provided .", "users can click these links to bring up the corresponding sequence and submit it to weblab for further analysis , such as predicting gene structures , making pairwise or multiple sequence alignment , generating sequence logos , constructing phylogenetic trees , finding sequence motifs , etc ."], ["we will update the database regularly with more leaf senescence related data available , and predict putative sags from completely sequenced plant genomes in the near future .", "we will improve the user interface with comments and suggestions from the user community and add more documents including case studies to help user to make thorough analysis of sags .", "we hope that lsd can be a platform not only for the domestic and international collaborators we are working with , but also for the research community of leaf senescence worldwide ."], ["national natural science foundation of china ( 31071160 to j.l . , 30625003 and 30730011 to h.g . ) ; ministry of science and technology of china ( 2009cb119101 to h.g . ) ; ministry of education of china ( ngi2008 - 108 - 3 to j.l .", ", ed20060047 to h.g . ) . funding for open access charge : national laboratory of protein engineering and plant genetic engineering ."]]}
{"article_id": "PMC2518333", "article_text": ["female study participants were recruited from participants in a cross - sectional study assessing bmd in young women with type 1 diabetes and age - matched community control subjects ( 7 ) .", "cases for the cross - sectional study ( baseline ) were recruited from a regional tertiary hospital pediatric diabetes center and from endocrinology practices in western new york . at the time of the initial study , the diabetes center followed over 600 patients aged 221 years with type 1 diabetes .", "female patients aged 1321 years in that clinical center were offered the opportunity to participate in the baseline study ( n = 138 ) .", "recruitment of older women involved contacting former center patients ( aged > 21 years ) , patients from regional endocrinology practices , and affected relatives of subjects screened by our diabetes center for the diabetes prevention trial .", "the comparison group included young women recruited from the same region as the case subjects ; most were classmates or acquaintances of the case subjects or were volunteers who learned of the study through flyers posted at the university and hospital .", "the baseline study enrolled 72 case subjects ( diabetes duration > 2 years ) and 91 control subjects aged 1337 years who were at least 2 years postmenarchal ( 7 ) .", "all type 1 diabetic case subjects and nondiabetic control subjects who participated in the baseline study were recruited 2 years later to assess change in bmd over time .", "contact was maintained with study participants between the baseline and follow - up exam via phone and mail .", "inclusion criteria for the follow - up study were participation in the baseline study , current negative pregnancy test , and signed informed consent . for individuals younger than 18 years of age ,", "exclusion criteria for both the baseline and follow - up study included systemic illness affecting bmd ( other than diabetes ) , other endocrine disorders ( except autoimmune thyroiditis ) , and diagnosis of juvenile osteoporosis or other bone disease .", "the study was approved by the institutional review boards of the women and children 's hospital of buffalo and the university at buffalo . at baseline", ", study participants completed questionnaires relating to personal and family health , lifestyle habits , dietary intake ( food frequency questionnaire ) , and medication intake including use of calcium supplements .", "participants also reported prior fracture history , menstrual history , and demographic information . for participants with type 1 diabetes , information on disease duration , insulin dose schedule ( injections versus continuous subcutaneous insulin infusion ) , and diabetes complications were reported .", "bmi was calculated as weight in kilograms divided by the square of height in meters .", "dexa ( hologic qdr-4500a ; hologic , waltham , ma ) was used to measure bmd in the anterior / posterior ( l14 ) spine , total hip , femoral neck , total forearm , and whole body .", "a single technician performed all baseline scans and 65% of the follow - up scans ; two additional technicians performed the remainder of the follow - up scans ( 27% and 7% , respectively ) .", "coefficients of variation ( cvs ) were determined for measures of all sites for all technicians throughout the study and were all < 1% throughout the study .", "the following biomarkers were measured in nontimed blood samples : serum osteocalcin , igf-1 , igf binding protein ( igfbp)-3 ( by radioimmunoassay ) , and a1c ( by high - performance liquid chromatography with bio - rad variant ; bio - rad , richmond , ca ) .", "random urinary n - teleopeptide levels were measured by enzyme - linked immuno - absorbent assay .", "serum for hormonal assays was frozen at 80c and stored until sent for analysis in batches to esoterix laboratory ( calabasas hills , ca ) .", "a1c ( fresh plasma ) was assayed in a sequential fashion at the women and children 's hospital of buffalo laboratory ( kaleida health ) .", "intra - assay cv was < 3% for n - telopeptide , 6% for igf-1 and osteocalcin , and 13% for igfbp-3 .", "interassay cv was < 7% for n - telopeptide , < 9.7% for igf-1 , 13% for osteocalcin , and < 17% for igfbp-3 ( esoterix laboratories ) .", "data from study participants were analyzed in stratum based on age at initial enrollment ( 7 ) .", "demographic , lifestyle , metabolic characteristics , and bmd were compared between diabetic case subjects and nondiabetic control subjects , stratified by age .", "comparisons were made for baseline differences , differences at follow - up , and percent change between the two time points .", "results were presented as both unadjusted comparisons and comparisons of adjusted means ( adjusted for age , bmi , and oral contraceptive [ oc ] use ) .", "unadjusted differences for continuous variables were examined using student 's t test and for categorical variables using the  test .", "data analyses were performed using sas version 8 ( sas , cary , nc ) .", "post hoc power analysis of follow - up total hip bmd ( adjusted for age , bmi , and oc use ) indicated that our dataset of 63 case subjects ( n = 37 , < 20 years of age ) and 85 control subjects ( n = 36 , < 20 years of age ) gave our study 80% power to detect a difference between case and control subjects with an  of 0.05 and a target effect size of 0.45 in women < 20 years of age and 0.63 in those > 20 years of age .", "table 1 includes baseline and follow - up characteristics of women who participated in this follow - up study . for comparative purposes ,", "the follow - up group included 63 women with type 1 diabetes ( 58.7% < 20 years of age ) and 85 control subjects without diabetes ( 42.4% < 20 years of age ) at ages 1539 years at enrollment .", "of the participants in the baseline study with diabetes , 87.5% ( 63 of 72 ) participated in this follow - up study , and 93.4% ( 85 of 91 ) of control subjects participated in both examinations .", "more subjects from the baseline cohort 20 years of age were lost to follow - up ( 13.8 vs. 3.94% , cohort < 20 years of age ) , with the highest attrition being from individuals with diabetes 20 years of age ( 21.2% ) .", "participants in this follow - up study were predominantly non - hispanic caucasian ( 95% ) and were similar in age at menarche and years since menarche .", "participants 20 years of age with diabetes used oc therapy longer than control subjects ( p  0.05 ) . although few participants were smokers , more women with diabetes reported current smoking ; there were no smokers among the control subjects < 20 years of age . at follow - up ,", "women with diabetes , particularly the older women , continued to have higher bmi than control subjects . weight change ( in percent ) was not significantly different over the follow - up period .", "as reported previously , more patients in the older cohort were using continuous subcutaneous insulin infusion ( p < 0.01 , data not shown ) . while a trend toward lower daily insulin requirement was present in the older cohort ( 0.86  0.42 vs. 0.75  0.22 units  kg  day ) , that difference was not statistically significant .", "subjects 20 years of age were more likely to have diabetes - related complications , although no statistical differences were found .", "table 2 presents bmd values at baseline and follow - up for each body site measured .", "unadjusted means and means adjusted for age , bmi , and oc use are shown stratified by age - group and diabetes diagnosis . in those < 20 years of age", ", both case and control subjects had an increase in bmd over the 2-year interval . the percent increase in bmd from baseline to follow - up was not statistically different between younger subjects and control subjects .", "for all participants in the population 20 years of age , the bmd from baseline to follow - up remained stable .", "significant differences in bmd were identified at the total hip , femoral neck , and whole body in the case subjects 20 years of age compared with the control subjects . as in the previous report , no differences in bmd were seen between groups in the younger cohort . in the adjusted model ( for age , bmi , and oc use ) , bmd values at the total hip ( baseline , p = 0.003 ; follow - up , p = 0.007 ) and femoral neck ( baseline and follow - up , p <", "0.001 ) were significantly lower in older women with diabetes compared with control subjects . after adjustment , the whole - body bmd was no longer significantly different between case and control subjects ; however , both age - groups showed a trend toward lower whole - body bmd in the case subjects .", "further adjustment for dietary intake of calcium and vitamin d as well as physical activity did not alter the bmd results ( data not shown ) .", "when the analysis was restricted to nonsmokers , the adjusted means changed very little . for the older cohort , however ,", "the p values were attenuated for the total hip ( p = 0.017 ) and whole body ( p = 0.215 ) . in nonsmokers", "> 20 years of age , the percent change difference at the total hip became more pronounced between case and control subjects and reached statistical significance ( p = 0.008 ) .", "overall , igf-1 levels were lower for all groups compared with baseline ( table 3 ) .", "this is consistent with a physiologic decrease in igf-1 in postteenage years ( esoterix normative data 11 ) . at the 2-year follow - up , however , the igf-1 level was significantly lower in the younger diabetic subjects than in the control subjects , although still within the normal range .", "igf-1 levels were not statistically different between diabetic participants and control subjects in the older cohort . within the older age - group , igfbp-3 levels were lower in participants with diabetes than in control subjects ( p < 0.05 ) .", "igfbp-3 levels were comparable for control and diabetic subjects in the cohort < 20 years of age .", "serum osteocalcin levels were lower at follow - up for all groups , even with increased bone mineral accrual in the group < 20 years of age ( table 3 ) .", "there was no difference in osteocalcin or n - teleopeptide levels between case and control subjects < 20 years of age ( table 3 ) .", "after adjusting for age and bmi , case subjects had significantly lower osteocalcin levels than control subjects in the population 20 years of age ( p < 0.04 ; table 3 ) .", "in addition , the percent change in osteocalcin level from baseline to follow - up was significantly different in diabetic women 20 years of age compared with control subjects after adjustment ( p < 0.03 ) . markers of bone formation did not correlate with bmd in our subjects .", "metabolic control , as measured by a1c , was poorer in the younger diabetic cohort , as we reported previously ( 7 ) .", "overall metabolic control was stable within a given age stratum between the baseline and follow - up studies .", "no correlation was found between a1c and any of the bmd measurements ( data not presented ) .", "when evaluating for associations between metabolic control and bmd measurements that were statistically lower in case than in control subjects at follow - up ( table 2 ) , the pearson correlation coefficient ( r value ) for a1c and bmd was between 0.121 ( total hip ) and 0.334 ( femoral neck ) ( p > 0.095 , data not shown ) .", "as in our previous study , there was no association found between bmd and diabetes duration , baseline a1c , or years since menarche .", "while longitudinal studies have examined the effect of insulin therapy on bmd in patients with long - standing type 1 diabetes ( 12 ) , few have reported on the natural history of metabolic bone disease in younger women . here", "we present bmd data for a well - characterized cohort of young women with type 1 diabetes at baseline and 2 years later .", "the baseline study demonstrated that the older cohort had significantly lower bmd at the femoral neck and lateral spine than age - matched control subjects ( 7 ) .", "the data presented here demonstrate that case subjects in the older cohort had persistently lower bmd than age - matched healthy control subjects . in this cohort ,", "bmd at the total hip and femoral neck remained lower than in control subjects after adjusting for age , bmi , and oc use , even with no significant decrease in bmd from baseline to follow - up ( table 2 ) .", "similar to our initial study , there was no difference in bmd between women with diabetes aged < 20 years and control subjects at any of the sites measured .", "however , there was a trend toward lower bmd at the total hip and whole body in women with diabetes .", "this difference occurred in the face of bmd increases ( percent change ) at both the total hip and whole body in this population , suggesting that bone mineral accrual is altered in a subtle manner during late adolescence in women with type 1 diabetes .", "individuals with type 2 diabetes have higher than average bmd compared with control subjects ( 13 ) , in part due to the mechanical load of obesity . in our study , women with type 1 diabetes had higher bmi than control subjects .", "however , bmd continued to be lower in older women with type 1 diabetes , even after adjusting for bmi ; thus , overweight status does not confer protection against poor bone mineralization in these young women .", "although the younger cohort had poorer diabetes control than the older cohort , we were again unable to demonstrate an association between bmd measures and metabolic control , as measured by a1c . others have reported similar findings with respect to bmd in children and adolescents with diabetes ( 14 ) .", "a1c measures short - term diabetes control ; perhaps cumulative life - time glycemic control is a better indicator of osteoporotic risk .", "this hypothesis is supported by studies demonstrating that decreased lumbar spine and femoral neck bmd in adults with type 1 diabetes is associated with retinopathy , nephropathy , and peripheral neuropathy , all of which are long - term complications of poor metabolic control ( 1517 ) .", "in addition , our data did not find a correlation between diabetes duration and bmd , further supporting a possible relation to lifetime diabetes control .", "the adverse effects of chronic hyperglycemia related to osteoblast function , osteoclast activity , and formation of advanced glycosylated end products that may impact bone quality as well as bone quantity ( 18 ) are difficult to measure in a systematic fashion .", "the role of insulin as a direct anabolic agent in bone metabolism is unclear ( 19 ) .", "animal models of spontaneous and pharmacologically induced diabetes demonstrate that as insulinopenia develops , there is a suppression of osteoblast markers ( 20 ) .", "it has been postulated that in addition to insulinopenia , relative igf-1 deficiency , whether systemic or local , contributes to low bmd in diabetes .", "igf-1 levels are lower in individuals with type 1 diabetes , and poor glycemic control negatively impacts igf-1 production by the liver . in adults with type 1 diabetes , igf-1 levels were lower in individuals with osteopenia at the femoral neck ( 1 ) .", "additionally , in a study of 127 children ( aged 620 years ) with diabetes , low bone mineral content correlated with low igf-1 levels ( 9 ) . in our study , igf-1 levels of case subjects were significantly lower than those of control subjects only for the younger cohort .", "however , igf-1 levels of subjects with diabetes were lower in the group 20 years of age compared with the group < 20 years of age , and igfbp-3 levels in the older cohort were significantly lower compared with control subjects . given that no biochemical markers of bone metabolism correlated with bmd at the hip or femoral neck in our study , it could be hypothesized that a cumulative history of insulinopenia and low igf-1 levels are better correlates for low bone density .", "our data demonstrate significant differences in bmd at the femoral neck and total hip that are likely to be clinically significant and may explain why women with type 1 diabetes have an increased risk of hip fractures later in life ( 4,5 ) .", "our data also indicate that alterations in bone mineral accrual occur within years of achievement of peak bone mass and that there is not a later period of  catch - up  bone mineralization .", "the most clinically relevant factor is whether a decrease in bmd correlates with fracture risk , which can not be determined in this study . besides bmd , multiple factors impact fracture risk including , but not limited to , age , nutrition status , smoking history , degree of frailty , and fracture history ( 21 ) .", "the well - characterized population , the large percentage of women who participated in the follow - up study , and the standard procedures used in this longitudinal study all represent significant strengths .", "yet , we acknowledge certain limitations . the study population is small , limiting its ability to detect true differences that may still exist .", "study control subjects were community based and may not represent the general population of women .", "finally , the results presented should be interpreted keeping in mind that multiple comparisons were made .", "we are aware of the lack of unanimity of opinion regarding the statistical approach when adjusting the statistical testing for many comparisons ( 22 ) .", "because many of our hypotheses are nested , and not independent , we feel that using a bonferroni approach is overly conservative .", "however , we have interpreted our results with caution and believe they are stronger when considered as a whole rather than as independent statistical tests .", "our findings demonstrate lower bmd in young women with type 1 diabetes compared with control subjects .", "although bone density testing is not routinely performed in young women , these data suggest that screening may be important in young women with type 1 diabetes .", "in addition , these women should be counseled regarding lifestyle interventions that may improve bone health , including adequate intake of calcium and vitamin d , and exercise ."], "abstract_text": ["<S> objective  individuals with type 1 diabetes have decreased bone mineral density ( bmd ) , yet the natural history and pathogenesis of osteopenia are unclear . </S>", "<S> we have previously shown that women with type 1 diabetes ( aged 1335 years ) have lower bmd than community age - matched nondiabetic control subjects . </S>", "<S> we here report 2-year follow - up bmd data in this cohort to determine the natural history of bmd in young women with and without diabetes.research design and methods  bmd was measured by dual - energy x - ray absorptiometry at baseline and 2 years later in 63 women with type 1 diabetes and in 85 age - matched community control subjects . </S>", "<S> a1c , igf-1 , igf binding protein-3 , serum osteocalcin , and urine n - teleopeptide were measured at follow-up.resultsafter adjusting for age , bmi , and oral contraceptive use , bmd at year 2 continued to be lower in women 20 years of age with type 1 diabetes compared with control subjects at the total hip , femoral neck , and whole body . </S>", "<S> lower bmd values were observed in cases < 20 years of age compared with control subjects ; however , the differences were not statistically significant . </S>", "<S> lower bmd did not correlate with diabetes control , growth factors , or metabolic bone markers.conclusionsthis study confirms our previous findings that young women with type 1 diabetes have lower bmd than control subjects and that these differences persist over time , particularly in women 20 years of age . </S>", "<S> persistence of low bmd as well as failure to accrue bone density after age 20 years may contribute to the increased incidence of osteoporotic hip fractures seen in postmenopausal women with type 1 diabetes . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "RESULTS", "CONCLUSIONS"], "sections": [["female study participants were recruited from participants in a cross - sectional study assessing bmd in young women with type 1 diabetes and age - matched community control subjects ( 7 ) .", "cases for the cross - sectional study ( baseline ) were recruited from a regional tertiary hospital pediatric diabetes center and from endocrinology practices in western new york . at the time of the initial study , the diabetes center followed over 600 patients aged 221 years with type 1 diabetes .", "female patients aged 1321 years in that clinical center were offered the opportunity to participate in the baseline study ( n = 138 ) .", "recruitment of older women involved contacting former center patients ( aged > 21 years ) , patients from regional endocrinology practices , and affected relatives of subjects screened by our diabetes center for the diabetes prevention trial .", "the comparison group included young women recruited from the same region as the case subjects ; most were classmates or acquaintances of the case subjects or were volunteers who learned of the study through flyers posted at the university and hospital .", "the baseline study enrolled 72 case subjects ( diabetes duration > 2 years ) and 91 control subjects aged 1337 years who were at least 2 years postmenarchal ( 7 ) .", "all type 1 diabetic case subjects and nondiabetic control subjects who participated in the baseline study were recruited 2 years later to assess change in bmd over time .", "contact was maintained with study participants between the baseline and follow - up exam via phone and mail .", "inclusion criteria for the follow - up study were participation in the baseline study , current negative pregnancy test , and signed informed consent . for individuals younger than 18 years of age ,", "exclusion criteria for both the baseline and follow - up study included systemic illness affecting bmd ( other than diabetes ) , other endocrine disorders ( except autoimmune thyroiditis ) , and diagnosis of juvenile osteoporosis or other bone disease .", "the study was approved by the institutional review boards of the women and children 's hospital of buffalo and the university at buffalo . at baseline", ", study participants completed questionnaires relating to personal and family health , lifestyle habits , dietary intake ( food frequency questionnaire ) , and medication intake including use of calcium supplements .", "participants also reported prior fracture history , menstrual history , and demographic information . for participants with type 1 diabetes , information on disease duration , insulin dose schedule ( injections versus continuous subcutaneous insulin infusion ) , and diabetes complications were reported .", "bmi was calculated as weight in kilograms divided by the square of height in meters .", "dexa ( hologic qdr-4500a ; hologic , waltham , ma ) was used to measure bmd in the anterior / posterior ( l14 ) spine , total hip , femoral neck , total forearm , and whole body .", "a single technician performed all baseline scans and 65% of the follow - up scans ; two additional technicians performed the remainder of the follow - up scans ( 27% and 7% , respectively ) .", "coefficients of variation ( cvs ) were determined for measures of all sites for all technicians throughout the study and were all < 1% throughout the study .", "the following biomarkers were measured in nontimed blood samples : serum osteocalcin , igf-1 , igf binding protein ( igfbp)-3 ( by radioimmunoassay ) , and a1c ( by high - performance liquid chromatography with bio - rad variant ; bio - rad , richmond , ca ) .", "random urinary n - teleopeptide levels were measured by enzyme - linked immuno - absorbent assay .", "serum for hormonal assays was frozen at 80c and stored until sent for analysis in batches to esoterix laboratory ( calabasas hills , ca ) .", "a1c ( fresh plasma ) was assayed in a sequential fashion at the women and children 's hospital of buffalo laboratory ( kaleida health ) .", "intra - assay cv was < 3% for n - telopeptide , 6% for igf-1 and osteocalcin , and 13% for igfbp-3 .", "interassay cv was < 7% for n - telopeptide , < 9.7% for igf-1 , 13% for osteocalcin , and < 17% for igfbp-3 ( esoterix laboratories ) .", "data from study participants were analyzed in stratum based on age at initial enrollment ( 7 ) .", "demographic , lifestyle , metabolic characteristics , and bmd were compared between diabetic case subjects and nondiabetic control subjects , stratified by age .", "comparisons were made for baseline differences , differences at follow - up , and percent change between the two time points .", "results were presented as both unadjusted comparisons and comparisons of adjusted means ( adjusted for age , bmi , and oral contraceptive [ oc ] use ) .", "unadjusted differences for continuous variables were examined using student 's t test and for categorical variables using the  test .", "data analyses were performed using sas version 8 ( sas , cary , nc ) .", "post hoc power analysis of follow - up total hip bmd ( adjusted for age , bmi , and oc use ) indicated that our dataset of 63 case subjects ( n = 37 , < 20 years of age ) and 85 control subjects ( n = 36 , < 20 years of age ) gave our study 80% power to detect a difference between case and control subjects with an  of 0.05 and a target effect size of 0.45 in women < 20 years of age and 0.63 in those > 20 years of age ."], ["table 1 includes baseline and follow - up characteristics of women who participated in this follow - up study . for comparative purposes ,", "the follow - up group included 63 women with type 1 diabetes ( 58.7% < 20 years of age ) and 85 control subjects without diabetes ( 42.4% < 20 years of age ) at ages 1539 years at enrollment .", "of the participants in the baseline study with diabetes , 87.5% ( 63 of 72 ) participated in this follow - up study , and 93.4% ( 85 of 91 ) of control subjects participated in both examinations .", "more subjects from the baseline cohort 20 years of age were lost to follow - up ( 13.8 vs. 3.94% , cohort < 20 years of age ) , with the highest attrition being from individuals with diabetes 20 years of age ( 21.2% ) .", "participants in this follow - up study were predominantly non - hispanic caucasian ( 95% ) and were similar in age at menarche and years since menarche .", "participants 20 years of age with diabetes used oc therapy longer than control subjects ( p  0.05 ) . although few participants were smokers , more women with diabetes reported current smoking ; there were no smokers among the control subjects < 20 years of age . at follow - up ,", "women with diabetes , particularly the older women , continued to have higher bmi than control subjects . weight change ( in percent ) was not significantly different over the follow - up period .", "as reported previously , more patients in the older cohort were using continuous subcutaneous insulin infusion ( p < 0.01 , data not shown ) . while a trend toward lower daily insulin requirement was present in the older cohort ( 0.86  0.42 vs. 0.75  0.22 units  kg  day ) , that difference was not statistically significant .", "subjects 20 years of age were more likely to have diabetes - related complications , although no statistical differences were found .", "table 2 presents bmd values at baseline and follow - up for each body site measured .", "unadjusted means and means adjusted for age , bmi , and oc use are shown stratified by age - group and diabetes diagnosis . in those < 20 years of age", ", both case and control subjects had an increase in bmd over the 2-year interval . the percent increase in bmd from baseline to follow - up was not statistically different between younger subjects and control subjects .", "for all participants in the population 20 years of age , the bmd from baseline to follow - up remained stable .", "significant differences in bmd were identified at the total hip , femoral neck , and whole body in the case subjects 20 years of age compared with the control subjects . as in the previous report , no differences in bmd were seen between groups in the younger cohort . in the adjusted model ( for age , bmi , and oc use ) , bmd values at the total hip ( baseline , p = 0.003 ; follow - up , p = 0.007 ) and femoral neck ( baseline and follow - up , p <", "0.001 ) were significantly lower in older women with diabetes compared with control subjects . after adjustment , the whole - body bmd was no longer significantly different between case and control subjects ; however , both age - groups showed a trend toward lower whole - body bmd in the case subjects .", "further adjustment for dietary intake of calcium and vitamin d as well as physical activity did not alter the bmd results ( data not shown ) .", "when the analysis was restricted to nonsmokers , the adjusted means changed very little . for the older cohort , however ,", "the p values were attenuated for the total hip ( p = 0.017 ) and whole body ( p = 0.215 ) . in nonsmokers", "> 20 years of age , the percent change difference at the total hip became more pronounced between case and control subjects and reached statistical significance ( p = 0.008 ) .", "overall , igf-1 levels were lower for all groups compared with baseline ( table 3 ) .", "this is consistent with a physiologic decrease in igf-1 in postteenage years ( esoterix normative data 11 ) . at the 2-year follow - up , however , the igf-1 level was significantly lower in the younger diabetic subjects than in the control subjects , although still within the normal range .", "igf-1 levels were not statistically different between diabetic participants and control subjects in the older cohort . within the older age - group , igfbp-3 levels were lower in participants with diabetes than in control subjects ( p < 0.05 ) .", "igfbp-3 levels were comparable for control and diabetic subjects in the cohort < 20 years of age .", "serum osteocalcin levels were lower at follow - up for all groups , even with increased bone mineral accrual in the group < 20 years of age ( table 3 ) .", "there was no difference in osteocalcin or n - teleopeptide levels between case and control subjects < 20 years of age ( table 3 ) .", "after adjusting for age and bmi , case subjects had significantly lower osteocalcin levels than control subjects in the population 20 years of age ( p < 0.04 ; table 3 ) .", "in addition , the percent change in osteocalcin level from baseline to follow - up was significantly different in diabetic women 20 years of age compared with control subjects after adjustment ( p < 0.03 ) . markers of bone formation did not correlate with bmd in our subjects .", "metabolic control , as measured by a1c , was poorer in the younger diabetic cohort , as we reported previously ( 7 ) .", "overall metabolic control was stable within a given age stratum between the baseline and follow - up studies .", "no correlation was found between a1c and any of the bmd measurements ( data not presented ) .", "when evaluating for associations between metabolic control and bmd measurements that were statistically lower in case than in control subjects at follow - up ( table 2 ) , the pearson correlation coefficient ( r value ) for a1c and bmd was between 0.121 ( total hip ) and 0.334 ( femoral neck ) ( p > 0.095 , data not shown ) .", "as in our previous study , there was no association found between bmd and diabetes duration , baseline a1c , or years since menarche ."], ["while longitudinal studies have examined the effect of insulin therapy on bmd in patients with long - standing type 1 diabetes ( 12 ) , few have reported on the natural history of metabolic bone disease in younger women . here", "we present bmd data for a well - characterized cohort of young women with type 1 diabetes at baseline and 2 years later .", "the baseline study demonstrated that the older cohort had significantly lower bmd at the femoral neck and lateral spine than age - matched control subjects ( 7 ) .", "the data presented here demonstrate that case subjects in the older cohort had persistently lower bmd than age - matched healthy control subjects . in this cohort ,", "bmd at the total hip and femoral neck remained lower than in control subjects after adjusting for age , bmi , and oc use , even with no significant decrease in bmd from baseline to follow - up ( table 2 ) .", "similar to our initial study , there was no difference in bmd between women with diabetes aged < 20 years and control subjects at any of the sites measured .", "however , there was a trend toward lower bmd at the total hip and whole body in women with diabetes .", "this difference occurred in the face of bmd increases ( percent change ) at both the total hip and whole body in this population , suggesting that bone mineral accrual is altered in a subtle manner during late adolescence in women with type 1 diabetes .", "individuals with type 2 diabetes have higher than average bmd compared with control subjects ( 13 ) , in part due to the mechanical load of obesity . in our study , women with type 1 diabetes had higher bmi than control subjects .", "however , bmd continued to be lower in older women with type 1 diabetes , even after adjusting for bmi ; thus , overweight status does not confer protection against poor bone mineralization in these young women .", "although the younger cohort had poorer diabetes control than the older cohort , we were again unable to demonstrate an association between bmd measures and metabolic control , as measured by a1c . others have reported similar findings with respect to bmd in children and adolescents with diabetes ( 14 ) .", "a1c measures short - term diabetes control ; perhaps cumulative life - time glycemic control is a better indicator of osteoporotic risk .", "this hypothesis is supported by studies demonstrating that decreased lumbar spine and femoral neck bmd in adults with type 1 diabetes is associated with retinopathy , nephropathy , and peripheral neuropathy , all of which are long - term complications of poor metabolic control ( 1517 ) .", "in addition , our data did not find a correlation between diabetes duration and bmd , further supporting a possible relation to lifetime diabetes control .", "the adverse effects of chronic hyperglycemia related to osteoblast function , osteoclast activity , and formation of advanced glycosylated end products that may impact bone quality as well as bone quantity ( 18 ) are difficult to measure in a systematic fashion .", "the role of insulin as a direct anabolic agent in bone metabolism is unclear ( 19 ) .", "animal models of spontaneous and pharmacologically induced diabetes demonstrate that as insulinopenia develops , there is a suppression of osteoblast markers ( 20 ) .", "it has been postulated that in addition to insulinopenia , relative igf-1 deficiency , whether systemic or local , contributes to low bmd in diabetes .", "igf-1 levels are lower in individuals with type 1 diabetes , and poor glycemic control negatively impacts igf-1 production by the liver . in adults with type 1 diabetes , igf-1 levels were lower in individuals with osteopenia at the femoral neck ( 1 ) .", "additionally , in a study of 127 children ( aged 620 years ) with diabetes , low bone mineral content correlated with low igf-1 levels ( 9 ) . in our study , igf-1 levels of case subjects were significantly lower than those of control subjects only for the younger cohort .", "however , igf-1 levels of subjects with diabetes were lower in the group 20 years of age compared with the group < 20 years of age , and igfbp-3 levels in the older cohort were significantly lower compared with control subjects . given that no biochemical markers of bone metabolism correlated with bmd at the hip or femoral neck in our study , it could be hypothesized that a cumulative history of insulinopenia and low igf-1 levels are better correlates for low bone density .", "our data demonstrate significant differences in bmd at the femoral neck and total hip that are likely to be clinically significant and may explain why women with type 1 diabetes have an increased risk of hip fractures later in life ( 4,5 ) .", "our data also indicate that alterations in bone mineral accrual occur within years of achievement of peak bone mass and that there is not a later period of  catch - up  bone mineralization .", "the most clinically relevant factor is whether a decrease in bmd correlates with fracture risk , which can not be determined in this study . besides bmd , multiple factors impact fracture risk including , but not limited to , age , nutrition status , smoking history , degree of frailty , and fracture history ( 21 ) .", "the well - characterized population , the large percentage of women who participated in the follow - up study , and the standard procedures used in this longitudinal study all represent significant strengths .", "yet , we acknowledge certain limitations . the study population is small , limiting its ability to detect true differences that may still exist .", "study control subjects were community based and may not represent the general population of women .", "finally , the results presented should be interpreted keeping in mind that multiple comparisons were made .", "we are aware of the lack of unanimity of opinion regarding the statistical approach when adjusting the statistical testing for many comparisons ( 22 ) .", "because many of our hypotheses are nested , and not independent , we feel that using a bonferroni approach is overly conservative .", "however , we have interpreted our results with caution and believe they are stronger when considered as a whole rather than as independent statistical tests .", "our findings demonstrate lower bmd in young women with type 1 diabetes compared with control subjects .", "although bone density testing is not routinely performed in young women , these data suggest that screening may be important in young women with type 1 diabetes .", "in addition , these women should be counseled regarding lifestyle interventions that may improve bone health , including adequate intake of calcium and vitamin d , and exercise ."]]}
{"article_id": "PMC4663308", "article_text": ["many studies have shown that bracket debonding can cause enamel loss , particularly when the fracture occurs at the enamel - adhesive interface", ". enamel damage may be in the form of enamel cracks , which may propagate during debonding [ 13 ] .", "acid - etching , rinsing , drying and applying resin and bonding agents are the routine steps of conventional orthodontic bracket bonding .", "concurrent with studies on the efficacy of the conventional technique , many studies , aimed at reducing the number of steps necessary for adhesion , have examined new bonding techniques with clinically adequate bond strength but less etching depth [ 56 ] .", "traditional methods of bonding orthodontic brackets to teeth rely on acid etching to achieve adequate retention .", "however , maintaining a sound enamel surface after bracket removal is of primary concern to clinicians .", "bond failure at the bracket - adhesive interface or within the adhesive is generally considered ", "safer  than a fracture at the enamel - adhesive interface ; this is because studies have demonstrated that enamel fracture can occur during debonding . on the other hand ,", "if a considerable amount of adhesive remains on the tooth surface after debonding , more chair time is required to remove the residual adhesive .", "in addition , the process of removing the residual adhesive results in further enamel loss .", "all - purpose or multi - purpose , self - adhesive resin cements are now commercially available in the market .", "they are capable of bonding to different substrates such as enamel , dentin , amalgam , metal and porcelain [ 910 ] .", "these cements require no surface pre - treatment due to their monomer and filler content and initiation technology .", "the phosphoric acid methacrylates react with basic fillers in the luting cement and the hydroxyapatite of the tooth structure [ 1112 ] .", "these self - adhesive cements do not require an  etch and rinse  phase as they are capable of conditioning the tooth surface and simultaneously preparing it for adhesion .", "this not only shortens the clinical application time , but also significantly reduces technique - sensitivity and risk of errors during application or manipulation .", "the aim of the current study was to assess the enamel cracks after debonding of brackets bonded with two different adhesive systems and also to evaluate enamel cracks after removing adhesive remnants and enamel polishing .", "sixty upper central incisors , extracted due to periodontal disease , were selected and stored in normal saline at room temperature until required .", "the study design was approved by the ethics committee of mashhad university of medical sciences . using a stereomicroscope ( blue light industry , waltham , ma , usa ) and digital camera ( exwave had , sony corporation , japan ) a magnified image ( 23.9x )", "the number of enamel cracks and the length and direction of each crack were assessed with the aid of adobe photoshop cs software ( adobe systems incorporate , usa ) ( figure 1 ) .", "enamel cracks before bracket bonding ( a ) , after deboning ( b ) and after polishing ( c ) .", "new cracks are hatched . the number of visible cracks ( i.e. cracks visible to the naked eye ) was also recorded .", "the teeth were then randomly divided into two equal groups . in the first group , the buccal surface of each central incisor", "a standard edgewise twin metal bracket ( dentaurum , pforzheim , germany ) was bonded with transbond xt ( 3 m , usa ) to the center of the labial surface of each tooth . in the second group ,", "the same bracket was bonded with self - adhesive composite cement ( maxcem elite , kerr , usa ) . in both groups ,", "the adhesive was cured for 40 seconds ( 10 seconds on each side of the bracket ) .", "the teeth were then incubated in distilled water at 37c for 24 hours to allow the material to set .", "all the brackets were debonded with rmo i-546 remover pliers ( rmo , germany ) .", "the blades of the pliers were placed at the bracket - adhesive interface and a gentle squeezing force was applied to the pliers by a zwick / z250 at a speed of 0.5 mm / min until bond failure occurred .", "the present method of force application was modified from a diametric compression test for tension that indirectly measures tensile strength .", "this approach was used because it simulates the usual clinical method for applying debonding forces .", "the debonding force recorded by the zwick is not equal to the actual force applied at the bracket adhesive interface because the force was applied at a predetermined distance on the pliers beaks .", "these forces , however , are proportional and can be expressed in the following ratio : the actual force ( f ) equals the measured force ( f ) multiplied by ( a ) divided by ( b ) : f= f ( a / b ) . in this ratio , ( a )", "is the distance from the point of force application to the fulcrum of the pliers and ( b ) is the distance from the point of actual force application to the fulcrum of the pliers .", "deboning force was measured in newton and was then converted to megapascal ( mpa ) .", "after debonding , the buccal surfaces of the teeth in each group were examined by the same stereomicroscope and the magnified images ( 23.9 x ) were taken with the same digital camera . the number of enamel cracks and the length and the direction of each crack were assessed with the aid of the same software .", "a : the distance from the point of force application to the fulcrum of the pliers .", "b : the distance from the point of actual force application to the fulcrum of the pliers the number of visible cracks in each tooth was also recorded ( figure 1 ) .", "the adhesive remnants on each tooth were then assessed with the ari in a 03 scale : 0 indciates no composite left on the tooth ; 1 indicates less than half of the composite left on the tooth , 2 indicates that more than half of the composite is left on the tooth surface and 3 shows that all the composite is left on the tooth surface with a distinct impression of the bracket base .", "the buccal surface of each tooth was polished with 12-blade tungsten carbide bur ( g&h , usa ) under water coolant .", "the enamel surface was examined for the third time in the same way ( figure 1 ) .", "the amount of shear bond strength in each group was compared with independent t - test .", "the enamel cracks in each group were compared before bonding , after debonding and after polishing using repeated measures anova .", "friedman and mann - whitney tests were applied in case of non - normal distribution of data .", "the result of the independent t - test indicated that the mean shear bond strength of maxcem elite ( the self - adhesive composite cement ) was significantly lower than that of transbond xt ( the adhesive composite ) ( p<0.001 ) .", "the mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09 , respectively .", "there were no significant inter - group differences in the number of enamel cracks before debonding .", "there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . in the maxcem elite group , the difference in the number of enamel cracks before and after debonding was statistically significant ( p<0.001 ) .", "the difference , however , was not significant after debonding and after polishing ( p=0.06 ) .", "the number of visible enamel cracks followed a similar pattern . in each group , before and after debonding , a significant intra ", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) however , before and after polishing , the difference was not significant ( table 2 ) .", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) there was no significant difference in the length of enamel cracks between the two groups . in each group , however , a significant increase in the length of enamel cracks was obvious after debonding .", "repeated measures test sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) the mann - whitney - u test showed that the differences were statistically significant among the groups ( p=0.0001 ) ( table 4 ) . the adhesive remnant index ( ari )", "( 0 , no composite left on the tooth ; 1 , less than half of the composite left on the tooth ; 2 , more than half of the composite left on the tooth ; 3 , all the composite left on the tooth with a distinct impression of the bracket base )", "the result of the independent t - test indicated that the mean shear bond strength of maxcem elite ( the self - adhesive composite cement ) was significantly lower than that of transbond xt ( the adhesive composite ) ( p<0.001 ) .", "the mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09 , respectively .", "there were no significant inter - group differences in the number of enamel cracks before debonding .", "there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . in the maxcem elite group , the difference in the number of enamel cracks before and after debonding was statistically significant ( p<0.001 ) .", "the difference , however , was not significant after debonding and after polishing ( p=0.06 ) .", "the number of visible enamel cracks followed a similar pattern . in each group , before and after debonding , a significant intra ", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) however , before and after polishing , the difference was not significant ( table 2 ) .", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test )", "there was no significant difference in the length of enamel cracks between the two groups . in each group , however , a significant increase in the length of enamel cracks was obvious after debonding .", "repeated measures test sd : standard deviation significant difference significant difference between a and b ( wilcoxon test )", "the mann - whitney - u test showed that the differences were statistically significant among the groups ( p=0.0001 ) ( table 4 ) . the adhesive remnant index ( ari ) ( 0 , no composite left on the tooth ; 1 , less than half of the composite left on the tooth ; 2 , more than half of the composite left on the tooth ; 3 , all the composite left on the tooth with a distinct impression of the bracket base )", "the main objective of this study was to evaluate the enamel cracks after debonding brackets , bonded with two different bonding systems followed by polishing .", "the sbs of maxcem elite , a self - adhesive cement , and transbond xt , a conventional adhesive composite , was also compared .", "the number and length of enamel cracks before bonding , after debonding and after polishing were also compared as well as the ari following the removal of orthodontic brackets . before bonding", "zacchrison et al .   showed that debonding of brackets created enamel cracks , which were different in size and direction .", "reported a significant increase in the number and length of enamel cracks after debonding with different pliers .", "stated that 82% of the teeth studied did not show significant increase in the number of enamel cracks after debonding and the teeth showing increased number of cracks had significantly greater mean bond strength . after polishing ,", "the number and length of enamel cracks increased in all groups , although there were no differences among the groups . according to zarrinnia et al .", ", debonding pliers cause resin fracture at the adhesive - bracket interface and using a tungsten carbide bur , accompanied by water coolant , effectively removes all adhesive remnants .", "they showed that the smoothest surface was obtained with the gloss polishing paste . a direct correlation between ari and bond strength", "the current study found significant differences among the ari scores and concluded that , in the transbond xt group , debonding was mostly of cohesive type , in contrast to the adhesive mode in the maxcem elite group .", "reported that self - adhesive cements had satisfactory bond strength to dentin and restorative materials , but that their bond strength to enamel was weak .", "vicente et al .   showed that relyxunicem ( a self - adhesive resin cement ) possessed a significantly lower bond strength than transbond xt .", "reported low bond strength for relyxunicem and suggested that the sbs of this self - adhesive universal cement needs to be increased for its successful use in bonding orthodontic brackets .", ".   showed that one - component adhesives ( relyxunicem and maxcem ) had significantly lower sbs than two- or three - component adhesives .", "bishara et al .   reported that maxcem had significantly lower bond strength than transbond xt . in the current study , maxcem elite ( a new self - adhesive system )", "the mean bond strength in the transbond xt group was significantly greater than that in the maxcem elite group , although the sbs of the latter was 10.291.14 mpa , which is clinically acceptable .", "debonding brackets bonded with maxcem elite resulted in less enamel cracks . removing remnants of adhesives and polishing with a 12-blade tungsten carbide bur did not increase enamel cracks .", "maxcem elite has clinically acceptable bond strength and can be used as a routine adhesive for orthodontic purposes ."], "abstract_text": ["<S> objectives : the objective of this study was to assess the effect of new bonding techniques on enamel surface.materials and methods : sixty upper central incisors were randomly divided into two equal groups . in the first group , </S>", "<S> metal brackets were bonded using transbondxt and , in the second group , the same brackets were bonded with maxcem elite . the shear bond strength ( sbs ) of both agents to enamel was measured and the number and length of enamel cracks before bonding , after debonding and after polishing were compared . </S>", "<S> the number of visible cracks and the adhesive remnant index ( ari ) scores in each group were also measured.results:there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . </S>", "<S> there was no significant difference in the length of enamel cracks between the two groups ; but , in each group , a significant increase in the length of enamel cracks was noticeable after debonding . polishing </S>", "<S> did not cause any statistically significant change in crack length . </S>", "<S> the sbs of maxcem elite was significantly lower than that of transbond xt ( 95% confidence interval).conclusion : maxcem elite offers clinically acceptable bond strength and can thus be used as a routine adhesive for orthodontic purposes since it is less likely to damage the enamel . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "Debonding forces", "Number of Cracks", "Length of cracks", "ARI", "DISCUSSION", "CONCLUSION"], "sections": [["many studies have shown that bracket debonding can cause enamel loss , particularly when the fracture occurs at the enamel - adhesive interface", ". enamel damage may be in the form of enamel cracks , which may propagate during debonding [ 13 ] .", "acid - etching , rinsing , drying and applying resin and bonding agents are the routine steps of conventional orthodontic bracket bonding .", "concurrent with studies on the efficacy of the conventional technique , many studies , aimed at reducing the number of steps necessary for adhesion , have examined new bonding techniques with clinically adequate bond strength but less etching depth [ 56 ] .", "traditional methods of bonding orthodontic brackets to teeth rely on acid etching to achieve adequate retention .", "however , maintaining a sound enamel surface after bracket removal is of primary concern to clinicians .", "bond failure at the bracket - adhesive interface or within the adhesive is generally considered ", "safer  than a fracture at the enamel - adhesive interface ; this is because studies have demonstrated that enamel fracture can occur during debonding . on the other hand ,", "if a considerable amount of adhesive remains on the tooth surface after debonding , more chair time is required to remove the residual adhesive .", "in addition , the process of removing the residual adhesive results in further enamel loss .", "all - purpose or multi - purpose , self - adhesive resin cements are now commercially available in the market .", "they are capable of bonding to different substrates such as enamel , dentin , amalgam , metal and porcelain [ 910 ] .", "these cements require no surface pre - treatment due to their monomer and filler content and initiation technology .", "the phosphoric acid methacrylates react with basic fillers in the luting cement and the hydroxyapatite of the tooth structure [ 1112 ] .", "these self - adhesive cements do not require an  etch and rinse  phase as they are capable of conditioning the tooth surface and simultaneously preparing it for adhesion .", "this not only shortens the clinical application time , but also significantly reduces technique - sensitivity and risk of errors during application or manipulation .", "the aim of the current study was to assess the enamel cracks after debonding of brackets bonded with two different adhesive systems and also to evaluate enamel cracks after removing adhesive remnants and enamel polishing ."], ["sixty upper central incisors , extracted due to periodontal disease , were selected and stored in normal saline at room temperature until required .", "the study design was approved by the ethics committee of mashhad university of medical sciences . using a stereomicroscope ( blue light industry , waltham , ma , usa ) and digital camera ( exwave had , sony corporation , japan ) a magnified image ( 23.9x )", "the number of enamel cracks and the length and direction of each crack were assessed with the aid of adobe photoshop cs software ( adobe systems incorporate , usa ) ( figure 1 ) .", "enamel cracks before bracket bonding ( a ) , after deboning ( b ) and after polishing ( c ) .", "new cracks are hatched . the number of visible cracks ( i.e. cracks visible to the naked eye ) was also recorded .", "the teeth were then randomly divided into two equal groups . in the first group , the buccal surface of each central incisor", "a standard edgewise twin metal bracket ( dentaurum , pforzheim , germany ) was bonded with transbond xt ( 3 m , usa ) to the center of the labial surface of each tooth . in the second group ,", "the same bracket was bonded with self - adhesive composite cement ( maxcem elite , kerr , usa ) . in both groups ,", "the adhesive was cured for 40 seconds ( 10 seconds on each side of the bracket ) .", "the teeth were then incubated in distilled water at 37c for 24 hours to allow the material to set .", "all the brackets were debonded with rmo i-546 remover pliers ( rmo , germany ) .", "the blades of the pliers were placed at the bracket - adhesive interface and a gentle squeezing force was applied to the pliers by a zwick / z250 at a speed of 0.5 mm / min until bond failure occurred .", "the present method of force application was modified from a diametric compression test for tension that indirectly measures tensile strength .", "this approach was used because it simulates the usual clinical method for applying debonding forces .", "the debonding force recorded by the zwick is not equal to the actual force applied at the bracket adhesive interface because the force was applied at a predetermined distance on the pliers beaks .", "these forces , however , are proportional and can be expressed in the following ratio : the actual force ( f ) equals the measured force ( f ) multiplied by ( a ) divided by ( b ) : f= f ( a / b ) . in this ratio , ( a )", "is the distance from the point of force application to the fulcrum of the pliers and ( b ) is the distance from the point of actual force application to the fulcrum of the pliers .", "deboning force was measured in newton and was then converted to megapascal ( mpa ) .", "after debonding , the buccal surfaces of the teeth in each group were examined by the same stereomicroscope and the magnified images ( 23.9 x ) were taken with the same digital camera . the number of enamel cracks and the length and the direction of each crack were assessed with the aid of the same software .", "a : the distance from the point of force application to the fulcrum of the pliers .", "b : the distance from the point of actual force application to the fulcrum of the pliers the number of visible cracks in each tooth was also recorded ( figure 1 ) .", "the adhesive remnants on each tooth were then assessed with the ari in a 03 scale : 0 indciates no composite left on the tooth ; 1 indicates less than half of the composite left on the tooth , 2 indicates that more than half of the composite is left on the tooth surface and 3 shows that all the composite is left on the tooth surface with a distinct impression of the bracket base .", "the buccal surface of each tooth was polished with 12-blade tungsten carbide bur ( g&h , usa ) under water coolant .", "the enamel surface was examined for the third time in the same way ( figure 1 ) .", "the amount of shear bond strength in each group was compared with independent t - test .", "the enamel cracks in each group were compared before bonding , after debonding and after polishing using repeated measures anova .", "friedman and mann - whitney tests were applied in case of non - normal distribution of data ."], ["the result of the independent t - test indicated that the mean shear bond strength of maxcem elite ( the self - adhesive composite cement ) was significantly lower than that of transbond xt ( the adhesive composite ) ( p<0.001 ) .", "the mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09 , respectively .", "there were no significant inter - group differences in the number of enamel cracks before debonding .", "there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . in the maxcem elite group , the difference in the number of enamel cracks before and after debonding was statistically significant ( p<0.001 ) .", "the difference , however , was not significant after debonding and after polishing ( p=0.06 ) .", "the number of visible enamel cracks followed a similar pattern . in each group , before and after debonding , a significant intra ", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) however , before and after polishing , the difference was not significant ( table 2 ) .", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) there was no significant difference in the length of enamel cracks between the two groups . in each group , however , a significant increase in the length of enamel cracks was obvious after debonding .", "repeated measures test sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) the mann - whitney - u test showed that the differences were statistically significant among the groups ( p=0.0001 ) ( table 4 ) . the adhesive remnant index ( ari )", "( 0 , no composite left on the tooth ; 1 , less than half of the composite left on the tooth ; 2 , more than half of the composite left on the tooth ; 3 , all the composite left on the tooth with a distinct impression of the bracket base )"], ["the result of the independent t - test indicated that the mean shear bond strength of maxcem elite ( the self - adhesive composite cement ) was significantly lower than that of transbond xt ( the adhesive composite ) ( p<0.001 ) .", "the mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09 , respectively ."], ["there were no significant inter - group differences in the number of enamel cracks before debonding .", "there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . in the maxcem elite group , the difference in the number of enamel cracks before and after debonding was statistically significant ( p<0.001 ) .", "the difference , however , was not significant after debonding and after polishing ( p=0.06 ) .", "the number of visible enamel cracks followed a similar pattern . in each group , before and after debonding , a significant intra ", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) however , before and after polishing , the difference was not significant ( table 2 ) .", "sd : standard deviation significant difference significant difference between a and b ( wilcoxon test )"], ["there was no significant difference in the length of enamel cracks between the two groups . in each group , however , a significant increase in the length of enamel cracks was obvious after debonding .", "repeated measures test sd : standard deviation significant difference significant difference between a and b ( wilcoxon test )"], ["the mann - whitney - u test showed that the differences were statistically significant among the groups ( p=0.0001 ) ( table 4 ) . the adhesive remnant index ( ari ) ( 0 , no composite left on the tooth ; 1 , less than half of the composite left on the tooth ; 2 , more than half of the composite left on the tooth ; 3 , all the composite left on the tooth with a distinct impression of the bracket base )"], ["the main objective of this study was to evaluate the enamel cracks after debonding brackets , bonded with two different bonding systems followed by polishing .", "the sbs of maxcem elite , a self - adhesive cement , and transbond xt , a conventional adhesive composite , was also compared .", "the number and length of enamel cracks before bonding , after debonding and after polishing were also compared as well as the ari following the removal of orthodontic brackets . before bonding", "zacchrison et al .   showed that debonding of brackets created enamel cracks , which were different in size and direction .", "reported a significant increase in the number and length of enamel cracks after debonding with different pliers .", "stated that 82% of the teeth studied did not show significant increase in the number of enamel cracks after debonding and the teeth showing increased number of cracks had significantly greater mean bond strength . after polishing ,", "the number and length of enamel cracks increased in all groups , although there were no differences among the groups . according to zarrinnia et al .", ", debonding pliers cause resin fracture at the adhesive - bracket interface and using a tungsten carbide bur , accompanied by water coolant , effectively removes all adhesive remnants .", "they showed that the smoothest surface was obtained with the gloss polishing paste . a direct correlation between ari and bond strength", "the current study found significant differences among the ari scores and concluded that , in the transbond xt group , debonding was mostly of cohesive type , in contrast to the adhesive mode in the maxcem elite group .", "reported that self - adhesive cements had satisfactory bond strength to dentin and restorative materials , but that their bond strength to enamel was weak .", "vicente et al .   showed that relyxunicem ( a self - adhesive resin cement ) possessed a significantly lower bond strength than transbond xt .", "reported low bond strength for relyxunicem and suggested that the sbs of this self - adhesive universal cement needs to be increased for its successful use in bonding orthodontic brackets .", ".   showed that one - component adhesives ( relyxunicem and maxcem ) had significantly lower sbs than two- or three - component adhesives .", "bishara et al .   reported that maxcem had significantly lower bond strength than transbond xt . in the current study , maxcem elite ( a new self - adhesive system )", "the mean bond strength in the transbond xt group was significantly greater than that in the maxcem elite group , although the sbs of the latter was 10.291.14 mpa , which is clinically acceptable ."], ["debonding brackets bonded with maxcem elite resulted in less enamel cracks . removing remnants of adhesives and polishing with a 12-blade tungsten carbide bur did not increase enamel cracks .", "maxcem elite has clinically acceptable bond strength and can be used as a routine adhesive for orthodontic purposes ."]]}
{"article_id": "PMC3519246", "article_text": ["sensitive skin syndrome is a common and challenging condition , yet little is known about its underlying pathophysiology .", "sensitive skin is a common term used by patients and clinicians , as well as the cosmetic industry , and represents a complex clinical challenge faced by dermatologists and other skin care professionals .", "patients with sensitive skin often present with subjective complaints that are out of proportion to the objective clinical findings .", "the patients complain of severe facial irritation , burning , and/or stinging after application of cosmetic products and toiletries such as sunscreens and soaps , yet they do not demonstrate the clinical stigmata of scaling , induration , and/or erythema that would be expected in known inflammatory or allergic processes .", "the condition is often worsened by specific climatic conditions and may affect areas other than the face .", "maibach described the cosmetic intolerance syndrome ( cis ) , which probably covers the bulk of the clinical presentation of the sensitive skin syndrome ( sss ) .", "fisher is credited with coining the term status cosmeticus to describe one extreme of the natural history of the cis in which the patient gradually becomes completely intolerant to the application of any cosmetic product .", "fundamentally , sss is a state of hyperreactivity to environmental stimuli , presenting with a clear clinical picture and resulting from a single underlying pathology or a combination of pathologies .", "defined as a self - diagnosed condition , sss is by definition difficult to quantify .", "some of the contradictions between investigators could be explained by flawed methodologies since the scientific community has yet to identify an acceptable objective screening test for sensitive skin .", "however , it is not only the subjective nature of the complaints that make sss difficult to diagnose .", "robinson , by performing patch testing with sodium dodecyl sulfate ( sds ) and looking for intra - individual response patterns , found that there is significant positive correlation between sds and other irritants but noted overall low correlation coefficients .", "he therefore deduced that it is inappropriate to define a subject 's reaction to a chemical based on his or her response to another irritant .", "she skin - tested 58 subjects with history of strong positive sds or lactic acid response .", "sds- or lactic acid - positive group , a reaction to one irritant could not predict a reaction with another . to further complicate matters", "there is evidence to suggest that no correlation exists between the lactic acid stinging test and the response to sds .", "this data resonate the idea that even the reference irritants commonly used in studies of this topic do not correlate with each other and with hyperreactivity tendencies .", "finally , judge tested 22 nonatopic adults with varying doses of sds and found marked inter - individual variation in the response threshold .", "this complexityreinforces the need for a thorough diagnostic algorithm and , specifically , the need to test patients with multiple , repeated and complete ( i.e. all cosmetics applied by the patient at home ) patch testing before making a diagnosis .", "in order to formulate a systematic clinical approach , the medical and cosmetic communities have attempted to characterize the condition .", "lacking an objective screening test , investigators resorted to epidemiological studies using patient surveys . in a large epidemiological study in the uk (", "n = 2316 ) , a staggering 51.4% of women and 38.2% of men self - reported themselves as having sensitive skin . of note ,", "interestingly , atopy did not appear to predict self - perceived sensitivity in the participating women .", "in addition , in the same study self - reports of sss symptoms were statistically over - represented in the  self - reported sensitive cohort compared to the  self - reported nonsensitive cohort .", "this finding validates the link between self - perception of sensitive skin and neurosensory discomfort .", "two recent studies in the us and europe made similar observations . in the us ,", "females were significantly more concerned about sensitive skin than men ; however , no age or ethnic differences were found .", "the european study reported an overall sensitive skin prevalence of 38.4% and found no ethnic differences .", "the european study , once again , demonstrated that people who reported having sensitive skin were significantly more likely to experience sss symptoms . of note ,", "both the american and european studies used large samples ( n = 994 and n = 4506 , respectively ) ; however , they were both limited by a phone survey methodology and the lack of any objective assessment . in light of the increased incidence of self - reported hypersensitivity in females ,", "he compared the patch test responses of 384 patients to sds as the positive control and found increased reactivity in males compared to females .", "lammintausta tested seven males and seven females with sodium lauryl sulfate ( sls ) and then performed visual inspections , transepidermal water loss measurements , and dielectric water content measurements but like other investigators found no reactivity differences between males and females , adding to the overall controversy .", "female self - perception of sensitivity is consistently increased compared to that of males , yet when put through objective reactivity testing the trend is unclear .", "ethnic differences in skin reactivity have been explored through the years , leading to the clinical hypothesis that black skin is less reactive than caucasian skin , which is in turn less reactive than asian skin . however , an epidemiological telephone survey performed in the us ( n = 811 ) revealed similar incidence of self - reported sensitive skin across four major ethnic groups ( afro - americans , asians , euro- american , and hispanics ) . the incidence rate of 52% is comparable to the incidence found in the uk ( see above ) .", "the investigators did , however , find minimally statistically significant ethnic differences in the self - reported triggers and symptoms of sensitivity : afro - americans reacted less to environmental and alcoholic triggers ; hispanics reacted less to alcohol ; asians reacted more to wind , spices , and alcohol ; and euro - american reacted more to wind . in terms of symptomology , statistically significant increase in recurrent itching on the face was noted in the asian population , and fewer euro - american avoided certain cosmetics due to skin reactivity .", "these findings were supported in a critical literature review , which showed that the evidence supporting the ethnic difference hypothesis using objective tests rarely reaches statistical significance and is often biased by subjective endpoints .", "recent data looking into age - related differences in hyperreactivity are showing more consistent results .", "whorl performed patch testing with 34 irritants on 2776 patients and found that young patients were more reactive than old patients .", "robinson 's findings also corroboratedthe trend towards increased reactivity in the young subjects compared to the old subjects .", "the mechanism leading to the sss has been debated in the literature , but the leading hypothesis relates to increased stratum corneum permeability .", "there is a known inverse relationship between corneocyte size and stratum corneum thickness and the permeability of the skin , and changes in this mechanical barrier result in abnormal skin penetration by irritants .", "in addition , low levels of ceramides in the stratum corneum have been linked to increased severity of sls - induced irritant contact dermatitis , pointing toward barrier compromise in sensitive skin .", "seidenari linked self - reported hyperreactivity to decreased skin capacitance ( indicating decreased water content ) , objectively suggesting increased absorption of irritants by the sensitive - skin population . in the same study ,", "increased erythema was objectively measured in hyperreacting subjects suggesting involvement of some baseline vascular component , and leading more recent investigators to propose the involvement of cutaneus sensory innervation in the pathogenesis of sss .", "as with many syndromes , the management scheme presented here emphasizes the diagnostic framework [ table 1 ] .", "the clinical approach to the sss demands a thorough process of eliminating obvious as well as more subtle diagnoses .", "once the diagnosis is made , deriving the appropriate treatment becomes a relatively simple task .", "note that since much of the mechanisms of sss are unknown , the clinician must be comfortable with a certain degree of ambiguity and uncertainty . underlying mechanisms of sensitive skin syndrome it is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss .", "some dermatoses that most clinicians find simple to diagnose may have atypical presentation and as a result lack the  expected morphology .", "eczema , atopic dermatitis , and rosacea are likely the three most common causes of sss related to barrier defect .", "full discussion of these conditions is beyond the scope of this overview but we will highlight some common clinical features .", "careful history , including family history and occupational history , combined with a detailed physical exam will often reveal the diagnosis . in the history", "particular consideration should be given to culprits such as the masking effect of other topical agents applied by the patient .", "the physical exam should include scrutiny of the face and scalp for subtle signs of minor inflammation , which are often masked in sss with underlying endogenous dermatoses . in all of these conditions", "the patients are likely over - exfoliating their skin and thus exacerbating the barrier dysfunction . therefore ,", "following specific treatment to halt the acute pathological process , preventing recurrence with a proper skin care regimen is indicated . in eczema and atopic dermatitis , the careful clinician may resort to short - term ( 2-week ) use of corticosteroids to stop the inflammatory process .", "calcineurin inhibitors are alternatively indicated for delicate areas on the face . in an effort to control histamine release in atopic dermatitis", ", antihistamines can be added and a relatively allergen - free environment should be created . in rosacea , the mainstay of treatment is oral and topical antibiotics . in seborrhoeic dermatitis , azoles are the mainstay of treatment and low potency corticosteroids and emollients can be added acutely to treat the inflammatory process .", "contact dermatitis ( cd ) and photocontact dermatitis ( pcd ) , as well as nonimmune contact urticaria ( nicu ) and immune contact urticaria ( icu ) , are all conditions that may elicit sss symptomology , with transient objective findings on physical exam .", "allergen - free products are now available and should be included as part of further skin care regimen in these patients .", "the main reason why nicu is often a missed diagnosis in patients presenting with the sss is the transient nature of the reaction .", "thus , after testing with small amounts of product on the skin , the patient should be carefully examined using minimal magnification : the lesions will be typically revealed within 20 minutes .", "common trigger agents are fragrances ( e.g. cinnamic aldehyde ) and preservatives such as sorbic and benzoic acids . once triggers", "icu can be demonstrated with open and occluded testing , followed by prick testing with appropriate positive and negative controls if no response is elicited .", "various foods , latex , parabens , and other chemicals have been implicated in the causation of icu .", "however , there are cases in which no clinical clue is provided by the physical exam . in these cases , a 2-week trial of appropriate - strength corticosteroid may solve the mystery by pointing to an eczematous process in the case of symptoms resolution .", "nonetheless , the careful clinician should consider avoiding prolonged use of topical corticosteroids since there is anecdotal clinical experience of topical corticosteroids inducing sss and , at least in the case of barrier dysfunction , earlier and complete steroid tachyphylaxis has been demonstrated .", "if no symptomatic relief is observed , the patient 's sss is likely due to an invisible underlying cause .", "both of these cases defined by maibach , no signs are present or can be elicited on the skin . in objective irritation", "the mechanism is unknown and this diagnosis likely bundles more than one pathological process in it . interestingly ( and clinically frustratingly ) , maibach considers subjective irritation to be the most common cause of cis .", "finally , body dysmorphic disorder ( bdd ) should always be considered by the dermatologist when assessing skin complaints without objective findings .", "a recent cross - sectional study found a prevalence of 14% among patients in a cosmetic dermatology clinic . in the context of sss", ", dermatologists should be aware that facial irritation complaints without any findings on exam are a common presentation in the dysmorphic patients and therefore extra caution is warranted .", "referral to a mental health professional is indicated since these patients are at risk of suicidal behavior .", "maibach notes that patients suspected of having bdd often require a detailed diagnostic process in order to build the trust needed to safely refer them to the mental health professional . table 2 summarizes the steps in the evaluation of a patient suspected to have sss .", "specific algorithms have been developed for the management of sss but they are the result of accumulated clinical experience and lack experimental evidence to support their use . nonetheless , algorithmic thinking may aid in ensuring complete assessment .", "these algorithms include four basic steps : discontinuation , assessment , testing , and slow re - introduction .", "discontinuation of all topical medication and/or products , as well as elimination of activities and clothing resulting in skin friction , should be initiated and may need to last up to 12 months . the patient should then be assessed ( as described above ) for the occurrence of any visible dermatoses . if no clear pathology has emerged , rigorous testing is the next step .", "testing should include patch and photopatch testing of routine allergens as well as of all of the patient 's skin care items .", "specific testing for contact urticaria should be a part of the testing protocol . finally ,", "the next step in management is slow re - introduction of the  minimally necessary skin care products . for females ,", "recommend adding low - allergenic- potential cosmetic products one at a time in the following order : lipstick , face powder , and blush .", "for all products re- introduced , recommend testing by applying the product to a 2-cm area lateral to the eye for five consecutive nights and documenting the positive and/or negative result .", "this exhausting process is not only a rational solution from a clinical management standpoint but may also aid in diagnosing the specific culprit in cases of invisible sss .", "understanding the underlying mechanisms may lead to the development of a well - standardized screening test .", "this will allow clinical studies to rely on objective findings rather than self - reports and this , in turn , may result in more comprehensive and efficient management strategies .", "female self - perception of sensitivity is consistently increased compared to that of males , yet when put through objective reactivity testing the trend is unclear .", "it is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss ."], "abstract_text": ["<S> sensitive skin syndrome ( sss ) is a common and challenging condition , yet little is known about its underlying pathophysiology . </S>", "<S> patients with sss often present with subjective complaints of severe facial irritation , burning , and/or stinging after application of cosmetic products . </S>", "<S> these complaints are out of proportion to the objective clinical findings . defined as a self - diagnosed condition lacking any specific objective findings </S>", "<S> , sss is by definition difficult to quantify and , therefore , the scientific community has yet to identify an acceptable objective screening test . in this overview </S>", "<S> we review recent epidemiological studies , present current thinking on the pathophysiology leading to sss , discuss the challenges sss presents , and recommend a commonsense approach to management . </S>"], "labels": null, "section_names": ["Introduction", "The Sensitive Skin Syndrome - Catch 22", "Epidemiological Data", "Current Thinking on Pathophysiology", "Clinical Approach", "Management", "Conclusion"], "sections": [["sensitive skin syndrome is a common and challenging condition , yet little is known about its underlying pathophysiology .", "sensitive skin is a common term used by patients and clinicians , as well as the cosmetic industry , and represents a complex clinical challenge faced by dermatologists and other skin care professionals .", "patients with sensitive skin often present with subjective complaints that are out of proportion to the objective clinical findings .", "the patients complain of severe facial irritation , burning , and/or stinging after application of cosmetic products and toiletries such as sunscreens and soaps , yet they do not demonstrate the clinical stigmata of scaling , induration , and/or erythema that would be expected in known inflammatory or allergic processes .", "the condition is often worsened by specific climatic conditions and may affect areas other than the face .", "maibach described the cosmetic intolerance syndrome ( cis ) , which probably covers the bulk of the clinical presentation of the sensitive skin syndrome ( sss ) .", "fisher is credited with coining the term status cosmeticus to describe one extreme of the natural history of the cis in which the patient gradually becomes completely intolerant to the application of any cosmetic product .", "fundamentally , sss is a state of hyperreactivity to environmental stimuli , presenting with a clear clinical picture and resulting from a single underlying pathology or a combination of pathologies ."], ["defined as a self - diagnosed condition , sss is by definition difficult to quantify .", "some of the contradictions between investigators could be explained by flawed methodologies since the scientific community has yet to identify an acceptable objective screening test for sensitive skin .", "however , it is not only the subjective nature of the complaints that make sss difficult to diagnose .", "robinson , by performing patch testing with sodium dodecyl sulfate ( sds ) and looking for intra - individual response patterns , found that there is significant positive correlation between sds and other irritants but noted overall low correlation coefficients .", "he therefore deduced that it is inappropriate to define a subject 's reaction to a chemical based on his or her response to another irritant .", "she skin - tested 58 subjects with history of strong positive sds or lactic acid response .", "sds- or lactic acid - positive group , a reaction to one irritant could not predict a reaction with another . to further complicate matters", "there is evidence to suggest that no correlation exists between the lactic acid stinging test and the response to sds .", "this data resonate the idea that even the reference irritants commonly used in studies of this topic do not correlate with each other and with hyperreactivity tendencies .", "finally , judge tested 22 nonatopic adults with varying doses of sds and found marked inter - individual variation in the response threshold .", "this complexityreinforces the need for a thorough diagnostic algorithm and , specifically , the need to test patients with multiple , repeated and complete ( i.e. all cosmetics applied by the patient at home ) patch testing before making a diagnosis ."], ["in order to formulate a systematic clinical approach , the medical and cosmetic communities have attempted to characterize the condition .", "lacking an objective screening test , investigators resorted to epidemiological studies using patient surveys . in a large epidemiological study in the uk (", "n = 2316 ) , a staggering 51.4% of women and 38.2% of men self - reported themselves as having sensitive skin . of note ,", "interestingly , atopy did not appear to predict self - perceived sensitivity in the participating women .", "in addition , in the same study self - reports of sss symptoms were statistically over - represented in the  self - reported sensitive cohort compared to the  self - reported nonsensitive cohort .", "this finding validates the link between self - perception of sensitive skin and neurosensory discomfort .", "two recent studies in the us and europe made similar observations . in the us ,", "females were significantly more concerned about sensitive skin than men ; however , no age or ethnic differences were found .", "the european study reported an overall sensitive skin prevalence of 38.4% and found no ethnic differences .", "the european study , once again , demonstrated that people who reported having sensitive skin were significantly more likely to experience sss symptoms . of note ,", "both the american and european studies used large samples ( n = 994 and n = 4506 , respectively ) ; however , they were both limited by a phone survey methodology and the lack of any objective assessment . in light of the increased incidence of self - reported hypersensitivity in females ,", "he compared the patch test responses of 384 patients to sds as the positive control and found increased reactivity in males compared to females .", "lammintausta tested seven males and seven females with sodium lauryl sulfate ( sls ) and then performed visual inspections , transepidermal water loss measurements , and dielectric water content measurements but like other investigators found no reactivity differences between males and females , adding to the overall controversy .", "female self - perception of sensitivity is consistently increased compared to that of males , yet when put through objective reactivity testing the trend is unclear .", "ethnic differences in skin reactivity have been explored through the years , leading to the clinical hypothesis that black skin is less reactive than caucasian skin , which is in turn less reactive than asian skin . however , an epidemiological telephone survey performed in the us ( n = 811 ) revealed similar incidence of self - reported sensitive skin across four major ethnic groups ( afro - americans , asians , euro- american , and hispanics ) . the incidence rate of 52% is comparable to the incidence found in the uk ( see above ) .", "the investigators did , however , find minimally statistically significant ethnic differences in the self - reported triggers and symptoms of sensitivity : afro - americans reacted less to environmental and alcoholic triggers ; hispanics reacted less to alcohol ; asians reacted more to wind , spices , and alcohol ; and euro - american reacted more to wind . in terms of symptomology , statistically significant increase in recurrent itching on the face was noted in the asian population , and fewer euro - american avoided certain cosmetics due to skin reactivity .", "these findings were supported in a critical literature review , which showed that the evidence supporting the ethnic difference hypothesis using objective tests rarely reaches statistical significance and is often biased by subjective endpoints .", "recent data looking into age - related differences in hyperreactivity are showing more consistent results .", "whorl performed patch testing with 34 irritants on 2776 patients and found that young patients were more reactive than old patients .", "robinson 's findings also corroboratedthe trend towards increased reactivity in the young subjects compared to the old subjects ."], ["the mechanism leading to the sss has been debated in the literature , but the leading hypothesis relates to increased stratum corneum permeability .", "there is a known inverse relationship between corneocyte size and stratum corneum thickness and the permeability of the skin , and changes in this mechanical barrier result in abnormal skin penetration by irritants .", "in addition , low levels of ceramides in the stratum corneum have been linked to increased severity of sls - induced irritant contact dermatitis , pointing toward barrier compromise in sensitive skin .", "seidenari linked self - reported hyperreactivity to decreased skin capacitance ( indicating decreased water content ) , objectively suggesting increased absorption of irritants by the sensitive - skin population . in the same study ,", "increased erythema was objectively measured in hyperreacting subjects suggesting involvement of some baseline vascular component , and leading more recent investigators to propose the involvement of cutaneus sensory innervation in the pathogenesis of sss ."], ["as with many syndromes , the management scheme presented here emphasizes the diagnostic framework [ table 1 ] .", "the clinical approach to the sss demands a thorough process of eliminating obvious as well as more subtle diagnoses .", "once the diagnosis is made , deriving the appropriate treatment becomes a relatively simple task .", "note that since much of the mechanisms of sss are unknown , the clinician must be comfortable with a certain degree of ambiguity and uncertainty . underlying mechanisms of sensitive skin syndrome it is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss .", "some dermatoses that most clinicians find simple to diagnose may have atypical presentation and as a result lack the  expected morphology .", "eczema , atopic dermatitis , and rosacea are likely the three most common causes of sss related to barrier defect .", "full discussion of these conditions is beyond the scope of this overview but we will highlight some common clinical features .", "careful history , including family history and occupational history , combined with a detailed physical exam will often reveal the diagnosis . in the history", "particular consideration should be given to culprits such as the masking effect of other topical agents applied by the patient .", "the physical exam should include scrutiny of the face and scalp for subtle signs of minor inflammation , which are often masked in sss with underlying endogenous dermatoses . in all of these conditions", "the patients are likely over - exfoliating their skin and thus exacerbating the barrier dysfunction . therefore ,", "following specific treatment to halt the acute pathological process , preventing recurrence with a proper skin care regimen is indicated . in eczema and atopic dermatitis , the careful clinician may resort to short - term ( 2-week ) use of corticosteroids to stop the inflammatory process .", "calcineurin inhibitors are alternatively indicated for delicate areas on the face . in an effort to control histamine release in atopic dermatitis", ", antihistamines can be added and a relatively allergen - free environment should be created . in rosacea , the mainstay of treatment is oral and topical antibiotics . in seborrhoeic dermatitis , azoles are the mainstay of treatment and low potency corticosteroids and emollients can be added acutely to treat the inflammatory process .", "contact dermatitis ( cd ) and photocontact dermatitis ( pcd ) , as well as nonimmune contact urticaria ( nicu ) and immune contact urticaria ( icu ) , are all conditions that may elicit sss symptomology , with transient objective findings on physical exam .", "allergen - free products are now available and should be included as part of further skin care regimen in these patients .", "the main reason why nicu is often a missed diagnosis in patients presenting with the sss is the transient nature of the reaction .", "thus , after testing with small amounts of product on the skin , the patient should be carefully examined using minimal magnification : the lesions will be typically revealed within 20 minutes .", "common trigger agents are fragrances ( e.g. cinnamic aldehyde ) and preservatives such as sorbic and benzoic acids . once triggers", "icu can be demonstrated with open and occluded testing , followed by prick testing with appropriate positive and negative controls if no response is elicited .", "various foods , latex , parabens , and other chemicals have been implicated in the causation of icu .", "however , there are cases in which no clinical clue is provided by the physical exam . in these cases , a 2-week trial of appropriate - strength corticosteroid may solve the mystery by pointing to an eczematous process in the case of symptoms resolution .", "nonetheless , the careful clinician should consider avoiding prolonged use of topical corticosteroids since there is anecdotal clinical experience of topical corticosteroids inducing sss and , at least in the case of barrier dysfunction , earlier and complete steroid tachyphylaxis has been demonstrated .", "if no symptomatic relief is observed , the patient 's sss is likely due to an invisible underlying cause .", "both of these cases defined by maibach , no signs are present or can be elicited on the skin . in objective irritation", "the mechanism is unknown and this diagnosis likely bundles more than one pathological process in it . interestingly ( and clinically frustratingly ) , maibach considers subjective irritation to be the most common cause of cis .", "finally , body dysmorphic disorder ( bdd ) should always be considered by the dermatologist when assessing skin complaints without objective findings .", "a recent cross - sectional study found a prevalence of 14% among patients in a cosmetic dermatology clinic . in the context of sss", ", dermatologists should be aware that facial irritation complaints without any findings on exam are a common presentation in the dysmorphic patients and therefore extra caution is warranted .", "referral to a mental health professional is indicated since these patients are at risk of suicidal behavior .", "maibach notes that patients suspected of having bdd often require a detailed diagnostic process in order to build the trust needed to safely refer them to the mental health professional . table 2 summarizes the steps in the evaluation of a patient suspected to have sss ."], ["specific algorithms have been developed for the management of sss but they are the result of accumulated clinical experience and lack experimental evidence to support their use . nonetheless , algorithmic thinking may aid in ensuring complete assessment .", "these algorithms include four basic steps : discontinuation , assessment , testing , and slow re - introduction .", "discontinuation of all topical medication and/or products , as well as elimination of activities and clothing resulting in skin friction , should be initiated and may need to last up to 12 months . the patient should then be assessed ( as described above ) for the occurrence of any visible dermatoses . if no clear pathology has emerged , rigorous testing is the next step .", "testing should include patch and photopatch testing of routine allergens as well as of all of the patient 's skin care items .", "specific testing for contact urticaria should be a part of the testing protocol . finally ,", "the next step in management is slow re - introduction of the  minimally necessary skin care products . for females ,", "recommend adding low - allergenic- potential cosmetic products one at a time in the following order : lipstick , face powder , and blush .", "for all products re- introduced , recommend testing by applying the product to a 2-cm area lateral to the eye for five consecutive nights and documenting the positive and/or negative result .", "this exhausting process is not only a rational solution from a clinical management standpoint but may also aid in diagnosing the specific culprit in cases of invisible sss ."], ["understanding the underlying mechanisms may lead to the development of a well - standardized screening test .", "this will allow clinical studies to rely on objective findings rather than self - reports and this , in turn , may result in more comprehensive and efficient management strategies .", "female self - perception of sensitivity is consistently increased compared to that of males , yet when put through objective reactivity testing the trend is unclear .", "it is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss ."]]}
{"article_id": "PMC4118078", "article_text": ["spontaneous pneumothorax ( sp ) comprises the largest group and is classified into primary spontaneous ( psp ) and secondary spontaneous pneumothorax .", "psp usually occurs in young , tall , thin men , especially smokers , in the absence of an underlying lung disease .", "although , sp is a relatively common condition , bilateral primary spontaneous pneumothorax ( bpsp ) is a very rare clinical condition with an occurrence ranging from 1.3 to 1.9% of all cases of sp .", "herein , we present a case of bpsp in an overweight young male patient , which is an exceptionally rare event .", "an otherwise - healthy , non - smoker , 20-year - old , overweight ( bmi 29.8 kg / m ) greek male patient was presented to the emergency department complaining of low - intensity chest pain and breathlessness of progressive worsening over the preceding 7 days . on admission ,", "blood pressure was 125/70 mmhg , heart rate 100 beats / min , sao2 97% on air and axillary temperature 36.8c .", "initial plain chest x - ray demonstrated bilateral pneumothoraces ( more prominent on the left side ) and no deviation of the trachea ( fig .  1 ) . \n", "plain chest x - rays on admission revealing bilateral pneumothorax . on emergency basis , the patient was managed with bilateral tube thoracostomy through the fifth intercostal spaces . during hospitalization", ", both lungs were gradually expanded ; the use of continuous aspiration was necessary for a couple of days , in order to achieve complete expansion of the left lung ( fig .  2 ) .", "next day the plain x - ray revealed recurrence of pneumothorax on the left side ( fig . ", "the tube was removed after 5 days ; no recurrence took place at this time . during the next 3 days the patient remained under observation ; serial physical examinations and chest x - rays were normal and the patient was discharged home . \n", "recurrence of pneumothorax on the left side after removing the chest tube . during hospitalization a chest computed tomography ( ct ) was performed and revealed the presence of blebs at the apices of both upper lungs ( fig . ", "the patient was referred to a tertiary center for an elective video - assisted thoracoscopic surgery intervention ( vats ) .", "three years after , no recurrence of pneumothorax took place and the patient remains healthy . \n", "psp usually occurs in the absence of any obvious underlying lung disease ; however , blebs and bullae apparently play a role in the pathogenesis of psp , as they are found on ct or during thoracoscopy or thoracotomy in 4879% of these patients .", "diagnosis of bilateral pneumothorax presents difficulties ; while unilateral pneumothorax is relatively easily suspected from the patient 's medical history , physical and radiological findings , the diagnosis of bilateral pneumothorax is usually delayed .", "the presence of equally diminished breath sounds in both sides and the presence of a not deviated trachea often mislead clinicians and delay chest drain insertion which is usually performed after the transaction of chest ct . although sp is a relatively common condition , bpsp is a very rare clinical condition .", "lee et al . , in their study of the 616 patients with 807 episodes of psp , found that the incidence of bpsp was 1.6% ( 13 patients ) . in univariate regression analysis", ", patients with psp compared with patients with bpsp had significantly lower body weight , bmi , higher body height / body weight ratio and higher incidence of bilateral blebs / bullae seen in hrct of the lung .", "however , in multiple regression analysis , only bmi and the presence of bilateral blebs / bullae retained statistically significant importance .", "huang et al . found that only lower bmi and smoking were significantly associated with the formation of bpsp . in their study , the proportion of bilateral blebs / bullae seen in hrct was higher in the bpsp group than that in the psp group ( 63 vs 53% , respectively ) , but the difference was not statistically significant ( p = 0.724 ) .", "the present report describes a case of bpsp in an overweight patient showing that psp is an existing condition in patients with bmi value higher than the normal .", "generally , if an sp affects < 20% of one lung , observation is efficient ; the absorption rate of the air is 1.25% ( 5075 ml / day ) .", "if pneumothorax affects > 20% of the lung or if it increases during observation , chest tube drainage may be required .", "air leakage can be diminished in 5 h and in 48 h in 52 and 82% of patients with tube thoracostomies , respectively . in patients with bpsp , one side", "should always be drained regardless of the extent of pneumothorax , whereas the other side can be managed by simple observation depending on the extent of the air in the pleura space .", "prolonged air leakage is the most common indication for operation in the first episode of pneumothorax . today", "compared with open thoracotomy , this procedure provides visualization of the entire thoracic cavity by video and causes less postoperative pain .", "single - stage bilateral vats procedure for bpsp has been advocated as an elective procedure to avoid subsequent anesthetic and operative procedures and longer hospital stays .", "if any underlying pulmonary disease is detected during surgical treatment , the existence of pleural communications should be investigated and mediastinal pleura should be examined carefully .", "in addition , apical pleurodesis further reduces the risk of recurrence . in our case ,", "the patient was successfully managed initially with a bilateral tube thoracostomy and finally with an elective bilateral vats procedure . in conclusion , bpsp is a rare clinical condition and usually develops in patients with low bmi and bilateral blebs / bullae .", "bpsp in overweight - obese patients is an existing condition and its early diagnosis requires high suspicion index .", "bpsp needs urgent assessment and management , followed by bilateral single - stage vats treatment as a safe and effective procedure ."], "abstract_text": ["<S> bilateral primary spontaneous pneumothorax ( bpsp ) is a rare condition , accounting for 1.6% of spontaneous pneumothoraces . </S>", "<S> patients with primary spontaneous pneumothorax have typically low body weight . </S>", "<S> bpsp in overweight / obese patients is an exceptionally rare event . </S>", "<S> the present report describes a case of an otherwise - healthy 20-year - old male patient with bmi 29.8 kg / m2 presented to our hospital owing to low - intensity chest pain and shortness of breath 7 days after the onset of symptoms . </S>", "<S> plain chest x - ray revealed the presence of bilateral pneumothorax . </S>", "<S> chest tubes were inserted on both sides . during hospitalization , </S>", "<S> a computed tomography demonstrated multiple blebs on the surface of the upper lobes of both lungs . </S>", "<S> hereupon , the patient referred to a tertiary center and underwent elective single - stage minimally invasive bilateral video - assisted thoracoscopic surgery . </S>", "<S> the present case report shows that bpsp in overweight / obese patients is an existing condition . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONFLICT OF INTEREST"], "sections": [["spontaneous pneumothorax ( sp ) comprises the largest group and is classified into primary spontaneous ( psp ) and secondary spontaneous pneumothorax .", "psp usually occurs in young , tall , thin men , especially smokers , in the absence of an underlying lung disease .", "although , sp is a relatively common condition , bilateral primary spontaneous pneumothorax ( bpsp ) is a very rare clinical condition with an occurrence ranging from 1.3 to 1.9% of all cases of sp .", "herein , we present a case of bpsp in an overweight young male patient , which is an exceptionally rare event ."], ["an otherwise - healthy , non - smoker , 20-year - old , overweight ( bmi 29.8 kg / m ) greek male patient was presented to the emergency department complaining of low - intensity chest pain and breathlessness of progressive worsening over the preceding 7 days . on admission ,", "blood pressure was 125/70 mmhg , heart rate 100 beats / min , sao2 97% on air and axillary temperature 36.8c .", "initial plain chest x - ray demonstrated bilateral pneumothoraces ( more prominent on the left side ) and no deviation of the trachea ( fig .  1 ) . \n", "plain chest x - rays on admission revealing bilateral pneumothorax . on emergency basis , the patient was managed with bilateral tube thoracostomy through the fifth intercostal spaces . during hospitalization", ", both lungs were gradually expanded ; the use of continuous aspiration was necessary for a couple of days , in order to achieve complete expansion of the left lung ( fig .  2 ) .", "next day the plain x - ray revealed recurrence of pneumothorax on the left side ( fig . ", "the tube was removed after 5 days ; no recurrence took place at this time . during the next 3 days the patient remained under observation ; serial physical examinations and chest x - rays were normal and the patient was discharged home . \n", "recurrence of pneumothorax on the left side after removing the chest tube . during hospitalization a chest computed tomography ( ct ) was performed and revealed the presence of blebs at the apices of both upper lungs ( fig . ", "the patient was referred to a tertiary center for an elective video - assisted thoracoscopic surgery intervention ( vats ) .", "three years after , no recurrence of pneumothorax took place and the patient remains healthy . \n"], ["psp usually occurs in the absence of any obvious underlying lung disease ; however , blebs and bullae apparently play a role in the pathogenesis of psp , as they are found on ct or during thoracoscopy or thoracotomy in 4879% of these patients .", "diagnosis of bilateral pneumothorax presents difficulties ; while unilateral pneumothorax is relatively easily suspected from the patient 's medical history , physical and radiological findings , the diagnosis of bilateral pneumothorax is usually delayed .", "the presence of equally diminished breath sounds in both sides and the presence of a not deviated trachea often mislead clinicians and delay chest drain insertion which is usually performed after the transaction of chest ct . although sp is a relatively common condition , bpsp is a very rare clinical condition .", "lee et al . , in their study of the 616 patients with 807 episodes of psp , found that the incidence of bpsp was 1.6% ( 13 patients ) . in univariate regression analysis", ", patients with psp compared with patients with bpsp had significantly lower body weight , bmi , higher body height / body weight ratio and higher incidence of bilateral blebs / bullae seen in hrct of the lung .", "however , in multiple regression analysis , only bmi and the presence of bilateral blebs / bullae retained statistically significant importance .", "huang et al . found that only lower bmi and smoking were significantly associated with the formation of bpsp . in their study , the proportion of bilateral blebs / bullae seen in hrct was higher in the bpsp group than that in the psp group ( 63 vs 53% , respectively ) , but the difference was not statistically significant ( p = 0.724 ) .", "the present report describes a case of bpsp in an overweight patient showing that psp is an existing condition in patients with bmi value higher than the normal .", "generally , if an sp affects < 20% of one lung , observation is efficient ; the absorption rate of the air is 1.25% ( 5075 ml / day ) .", "if pneumothorax affects > 20% of the lung or if it increases during observation , chest tube drainage may be required .", "air leakage can be diminished in 5 h and in 48 h in 52 and 82% of patients with tube thoracostomies , respectively . in patients with bpsp , one side", "should always be drained regardless of the extent of pneumothorax , whereas the other side can be managed by simple observation depending on the extent of the air in the pleura space .", "prolonged air leakage is the most common indication for operation in the first episode of pneumothorax . today", "compared with open thoracotomy , this procedure provides visualization of the entire thoracic cavity by video and causes less postoperative pain .", "single - stage bilateral vats procedure for bpsp has been advocated as an elective procedure to avoid subsequent anesthetic and operative procedures and longer hospital stays .", "if any underlying pulmonary disease is detected during surgical treatment , the existence of pleural communications should be investigated and mediastinal pleura should be examined carefully .", "in addition , apical pleurodesis further reduces the risk of recurrence . in our case ,", "the patient was successfully managed initially with a bilateral tube thoracostomy and finally with an elective bilateral vats procedure . in conclusion , bpsp is a rare clinical condition and usually develops in patients with low bmi and bilateral blebs / bullae .", "bpsp in overweight - obese patients is an existing condition and its early diagnosis requires high suspicion index .", "bpsp needs urgent assessment and management , followed by bilateral single - stage vats treatment as a safe and effective procedure ."], [""]]}
{"article_id": "PMC2967835", "article_text": ["familial adenomatous polyposis   account for 1% of colorectal cancers , and provides a model of apc inactivation as an early genetic event for the approximately 80%85% of cancers that develop from sporadic polyps .", "colorectal cancers arising in patients with familial adenomatous polyposis can be largely prevented by polyp surveillance and prophylactic colectomy .", "total proctocolectomy with construction of a conventional ileostomy or ileoanal anastomosis with preparation of an ileal pouch , has various effects on the function of the terminal ileum and the intestinal bacterial flora .", "this may deteriorate cholesterol metabolism , as absorption of cholesterol in duodenum and jejunum requires micellar solubilization with bile acids , fatty acids , monoglycerides , and phospholipids .", "hypothetically , ileal - pouch - anal anastomosis and ileostomy patients might differ with regard to the presence of various fatty acids in feces and their relationship to other reflections of lipoprotein metabolism , but we found no previous study focusing upon this issue .", "dietary fatty acids are incorporated into blood and tissues , and the fatty acid composition in these tissues are often used as biomarkers of fat intake .", "furthermore , the fecal amount and composition of fatty acids reflect fat ingestion , intestinal fatty acid absorption , and the activity of colonic bacteria .", "although some of the discrepancies between studies may be due to the use of different methods to analyze fatty acids , differences in diet , or the fact that assessments have been performed in different body compartments , modifications in the metabolism of fatty acids have been suggested in cancer patients [ 5 , 6 ] .", "it is not clear at what steps in the multistage carcinogenesis process a possible distorted fatty acid metabolism occurs .", "notably , if such alterations occur in the development of carcinogenesis , this may affect the biological functions of essential fatty acids and their derivates .", "very little is known about fatty acid metabolism in familial adenomatous polyposis , although chemoprevention affecting the fatty acid derivates and the cyclooxygenase enzymes is often administered to familial adenomatous polyposis patients .", "deregulation of the cyclooxygenase-2 pathway appears to affect tumorigenesis via a number of distinct mechanisms : promoting tumour maintenance and progression , encouraging metastatic spread , and perhaps even participating in tumour initiation .", "cyclooxygenase-1 and -2 are the rate limiting enzymes in the synthesis of prostaglandins and thromboxanes .", "arachidonic acid is the main substrate for these enzymes , leading to the synthesis of prostaglandins which have growth promoting effects . substituting arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins .", "since cyclooxygenase-2 is a fatty acid metabolising enzyme , the relationships between cyclooxygenase-2 and fatty acid composition of different tissues is of interest .", "colectomized familial adenomatous polyposis patients had a deviant fatty acid profile with high levels of arachidonic acid and docosahexaenoic acid and low levels of linoleic acid and alpha - linolenic acid in serum phospholipids , which is in accordance with studies in patients with other types of cancers [ 5 , 1013 ] .", "in a previous familial adenomatous polyposis study , comparable treatment effects of a cyclooxygenase-2 inhibitor were observed on the fatty acid composition in serum phospholipids and duodenal lesions , presumably and most importantly the nonbeneficial effects involving essential fatty acids .  ", "the results describe the content of fatty acids in feces , and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies , diet , and levels of serum lipoproteins . because the effects of ileostomy construction may differ from that of ileal - pouch - anal anastomosis because of scarcity of the bacterial flora and different surface area of the terminal ileum , we did separate analyses for these two groups .", "previous familial adenomatous polyposis studies have focused upon faecal sterols and bile acids [ 1517 ] , but none has to the best of our knowledge analysed the content of fatty acids in feces , including very long chained fatty acids .", "data from the present study are taken from a randomized double - blind placebo - controlled intervention study with a cyclooxygenase-2 inhibitor [ 12 , 14 , 18 ] .", "the main aim was to compare the effect of rofecoxib treatment on duodenal lesions ( data in preparation ) .", "the present study comprises data from baseline for only the patients operated with ileal - pouch - anal anastomosis and ileostomy ( 77% of total study group ) .", "all of them had been colectomized , and duodenal lesions had been verified by endoscopy and histology .", "rikshospitalet university hospital is a highly specialized university hospital with national responsibilities in the area of complicated treatments , such as follow - ups on the norwegian familial adenomatous polyposis patients .", "inclusion criteria were verified familial adenomatous polyposis , colectomy , 1870 years of age , and documented duodenal lesions graded as spigelman i , ii , or iii and the largest adenoma10  mm .", "exclusion criteria were indications for surgical treatment , suspected or documented intestinal obstruction or stenosis , patients unwilling or unable to adhere to protocol , known cardiac failure requiring medical treatment , and pregnancy .", "as for the adenomas sampled , they were all assessed histologically as mild / moderate dysplasia , all below 10  mm and all flat , located in the descending part of the duodenum .", "samples of feces were transferred to tubes and mixed to make a homogeneous mass of which 100200  ul were used for lipid extraction by adding 50  ul butanol , 100  l h2o , 1  ml methanol , and 1  ml chloroform .", "after 10  min under n2 , the mixture was filtered and the filter washed   with 1  ml chloroform   + 1  ml chloroform / methanol ( 2  :  1 ) + 0.8  ml 0.73% nacl . after mixing for 15  s ,", "the tubes were centrifuged for 30  min at 1000  g. the upper phase was discarded and the lower phase transferred to new tubes and evaporated under n2 at 65c using a heat block .", "then 0.4  ml 0.5  n naoh in methanol was added and the solution kept for 7  min at 100c under nitrogen . after cooling to room temperature , 0.5  ml 12%", "bf3 was added and the solution kept for 10  min at 100c under nitrogen . after cooling to room temperature , 1  ml heptan was added under nitrogen , and mixing for 30  s , 1 ml of saturated nacl solution was added . after mixing ,", "the solution was centrifuged for 10  min at 1000  g , and the upper phase transferred to new tubes .", "finally , the heptan was evaporated and 300  l hexan added before continuing with the gas chromatography procedure referred to earlier .", "dietary intake was assessed by a validated food frequency questionnaire , designed to cover as much of the total habitual diet as possible .", "questions were related to habitual frequency of consumption and the amount of foods eaten during the last year .", "they were asked to identify their habitual choices of edible fats by pointing at specially prepared pictures , in order to increase the validity of the estimate of dietary fat .", "each biopsy was put into a tube containing 600  l trizol and immediately mixed on a mixermill ( retsch gmbh , germany ) .", "gene expression analyses were carried out on a 7900  ht real - time pcr machine from applied biosystems .", "based on the results from running a housekeeping gene test (  taqman human endogenous control plate ,  applied biosystems ) with rna from isolated human leucocytes ( data not shown ) .", "the primers and probes were initially designed as three assays per gene and validated for efficiency and specificity .", "the primers and probes for the cox-1 assay were : forward primer : 5-cttccaggagctcgtagga-3 , probe : 5-agaaggagatggcagcagagttggag-3 , and reverse primer : 5-acgcatcaatgtctccatacaat-3. cox-2 forward primer : 5-tggaacatggaattacccagt-3 , probe : 5-tgttgaatcattcaccaggcaaattgct-3 ,   and reverse primer : 5-tcctaccaccagcaaccct-3. gus forward primer : 5-gaaaatatgtggttggagagctcatt-3 , probe : 5-ccagcactctcgtcggtgactgttca-3 ,   and reverse primer : 5-ccgagtgaagatccccttttta-3.", "tbp forward primer : 5-ctggaaaagttgtattaacaggtgc-3 , probe : 5-agcagaaatttatgaagcatttgaaaacatctaccctatt-3 ,   and reverse primer : 5-cattacgtcgtcttcctgaatc-3.   taqman universal pcr master mix ( applied biosystems ) was added as reaction mix .", "the reaction conditions were initiated by a step of 2  min . at 50c and 10  min . at 95c , followed by 40 cycles of denaturation at 95c for 15 sec . and annealing at 60c for 1  min .", "a combination of cdna from several samples were made and diluted in order to make a dilution curve that was included on each plate .", "the average of the three values for each gene was divided by the average of the corresponding tbp values , generating a normalized value of the gene expression which is a unit less value used to compare the relative amount of mrna for each gene in the different samples .", "patients were informed by a physician , and in addition thoroughly written information was given to all patients .", "the protocol was explained to the subjects that had to give their consent before inclusion .", "was approved by the norwegian health authorities and the regional committee of medical ethics 20/06/2002 ( reference : s-02127 ) .", "nonparametrical statistical methods were chosen , as some of the variables were skewed and the number of observations limited .", "age and fecal content of palmitolic acid , linoleic acid , and arachidonic acid differed between the ileostomy and the ileal - pouch - anal anastomosis patients ( table 1 ) .", "the content of linoleic acid in feces correlated negatively to the proportion of eicosaenoic acid in duodenal biopsies ( r = 0.6 , p < .05 ) .", "the content of arachidonic acid in feces was correlated negatively to the proportions of eicosapentaenoic acid ( r = 0.7 , p = .02 ) and docosahexaenoic acid ( r = 0.7 , p = .008 ) in duodenal biopsies .", "total cholesterol were negatively correlated to the content of palmitic acid ( r = 0.7 , p = .03 ) , palmitoleic acid ( r = 0.7 , p = .02 ) , stearic acid ( r = 0.7 , p = .02 ) , oleic acid ( r = 0.7 , p = .02 ) , linoleic acid ( r = 0.8 , p = .002 ) , and monounsaturated fatty acids ( r = 0.7 , p = .02 ) in feces .", "the levels of triglycerides was negatively correlated to the content of linoleic acid ( r = 0.8 , p = .003 ) and eicosaenoic acid ( r = 0.7 , p = .03 ) in feces .", "no significant correlations were found between the content of different fatty acids in feces and the fatty acid dietary intake ( data not shown ) .   with the exception of the palmitoleic acid / palmitic acid ratio and levels of cyclooxygenase-2 expression in duodenal biopsies ( r = 0.8 , p = .003 ) , no relationships were found between fatty acids in feces and the levels of cyclooxygenase mrna expression ( figure 1 ) .", "significant correlations were found between the content of oleic acid in feces and the proportion of myristic acid ( r = 0.8 , p < .001 ) , oleic acid ( r = 0.5 , p <", ".05 ) , and eicosaenoic acid ( r = 0.5 , p = .03 ) in duodenal biopsies .", "moreover , the content of linoleic acid in feces correlated significantly to the proportion of myristic acid ( r = 0.8 , p < .001 ) in duodenal biopsies , whereas the content of alpha - linolenic acid in feces correlated inversely to eicosaenoic acid ( r = 0.6 , p <", "the content of palmitic acid in feces was positively correlated to levels of ldl - cholesterol ( r = 0.05 , p = .05 ) .", "no significant correlations were found between the content of different fatty acids in feces and the levels of cyclooxygenase mrna expression in duodenal biopsies ( data not shown ) .   several different fatty acids in feces correlated positively to dietary intake of fatty acids , most predominantly monounsaturated fatty acids ( table 2 ) .", "as expected , we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism .", "possibly , our results relate to metabolic differences caused by the different intestinal reconstructions , but the data are not suitable to explain the findings .", "we found no significant relationships between the levels of cyclooxygenase mrna expression in duodenal biopsies and the content of fatty acids in feces , except for the estimates of the content of delta-9-desaturase ( stearoyl - coa desaturase , scd ) activity , namely the ratio of palmitolic acid / palmitic acid [ 19 , 20 ] in the ileostomy group .", "stearoyl - coa desaturase is the central lipogenic enzyme catalyzing in vivo reactions in the synthesis of monounsaturated fatty acids , particularly oleic acid and palmitoleic acid , which are the major monounsaturated fatty acids of membrane phospholipids , triglycerides , wax esters , and cholesteryl esters .", "both delta-9-desaurase and cyclooxygenase-2 are ppar alpha regulated , although this possible link is only a speculation .", "a study in healthy subjects showed that human fecal water contains components can affect both the cyclooxygenase-2 protein level and enzymatic activity .", "one recent study claims that it is possible to detect cyclooxygenase-2 mrna in feces of colorectal cancer patients irrespective of clinical stage .", "notably , increased cyclooxygenase-2 expression in duodenal compared with colonic tissues in familial adenomatous polyposis was recently reported .  ", "the significant negative correlation between content of arachidonic acid in the feces and amounts of omega-3 fatty acids in duodenal biopsies which was found in the ileostomy group is interesting , since substitution of arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins .", "a previous familial adenomatous polyposis study established a positive correlation between the reduction in tissue prostanoid levels and clinical response , as measured by the reduction in size and number of adenomas , when patients were treated with sulindac .", "however , this correlation only became consistently significant when prostanoids were assayed after tissue homogenates were first incubated with arachidonic acid .", "we may suspect transformation of linoleic acid   to arachidonic acid in feces , but data are limited as with regard to feces from colectomized familial adenomatous polyposis subjects .", "however , the dietary assessment showed that the intake of such foods was normal among these familial adenomatous polyposis patients .  ", "dietary controlled intervention studies on ileostomy subjects have shown that dietary manipulation with fat and fiber may modify cholesterol absorption and sterol excretion . in the ileostomy group", ", levels of total cholesterol were negatively correlated to the content of palmitic acid , palmitolic acid , stearic acid , oleic acid , linoleic acid , and monounsaturated fatty acids ( < 18  c atoms ) in feces , which is interesting since the present levels of total cholesterol are lower than those among a healthy reference group . in the ileal - pouch - anal anastomosis group separately , the content of monounsaturated fatty acid in feces was positively correlated to dietary intake of monounsaturated fatty acids in particular . moreover", ", positive correlations were found between the content of oleic acid in feces and the proportion of myristic acid , oleic acid , and eicosaenoic acid in duodenal biopsies .", "the precise role of monounsaturated fatty acids synthesis in cell proliferation and programmed cell death remains unknown .", "the strong correlation of high levels of monounsaturated fatty acids and neoplastic phenotype may suggest that the regulation of stearoyl - coa desaturase must play a role in cancer development .    ", "this was an observational study , and not a metabolic balance study that was designed to investigate the metabolism of fatty acids in familial adenomatous polyposis patients .  ", "although some studies have found that the absorption rate of fatty acids are unaffected by age [ 26 , 27 ] , we can not exclude the possibility that some of the observed differences between the ileal - pouch - anal anastomosis and ileostomy patients might be due to age , rather than type of surgery , since lipoprotein clearance has been shown to be altered by age , and that might affect the relationship between dietary fatty acids and the fatty acid profiles found in tissues .", "moreover , no conclusions can be drawn from the present study because this is an observational study .", ". however , the study addresses the need to learn more about the dietary and lifestyle habits of this subset of at - risk patients .", "the present study includes a number of patients close to the maximum possible number of eligible norwegian familial adenomatous polyposis patients .", "we may , however , suspect that the low sample size and use of a food frequency questionnaire , may have weakened any associations between the content of fatty acids in feces and dietary intake .", "notably , these data do not contradict that the nutrient intake among these patients should at least meet the recommendations for healthy subjects .", "nevertheless , we suggest that these data are compelling enough to suggest that future familial adenomatous polyposis studies should investigate overall fatty acid metabolism , molecular mechanisms relevant to fatty acid metabolism , inflammation , and angiogenesis , in addition to nutrition requirements . in conclusion , we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism .", "we may suggest that fatty acid content in feces is related to dietary intake , serum lipids , and fatty acid composition in duodenal biopsies , even in colectomized familial adenomatous polyposis patients .", "this observational study may represent hypothesis - generating suggestions for future familial adenomatous polyposis studies ."], "abstract_text": ["<S> a few familial adenomatous polyposis studies have focused upon faecal sterols and bile acids but none has analysed the fecal content of fatty acids . </S>", "<S> we report here findings of an observational study on 29 colectomized familial adenomatous polyposis patients that describe the fecal content of fatty acids , and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies , levels of serum lipoproteins , and diet . in the ileostomy group separately ( n = 12 ) , the fecal content of arachidonic acid was correlated negatively to the proportions of eicosapentaenoic acid and docosahexaenoic acid in duodenal biopsies . </S>", "<S> total serum - cholesterol was negatively correlated to the fecal content of saturates and monounsaturates . </S>", "<S> the fecal palmitoleic acid / palmitic acid ratio was positively correlated to the levels of cyclooxygease-2 expression in duodenal biopsies.in the ileal - pouch - anal anastomosis group separately ( n = 17 ) , significant correlations were found between the fecal contents of oleic acid , linoleic acid , and alpha - linolenic acid , and the proportions of myristic acid , oleic acid and eicosaenoic acid in duodenal biopsies . </S>", "<S> dietary monounsaturates were positively correlated to different fecal fatty acids . </S>", "<S> future studies should focus on molecular mechanisms relevant to fatty acid metabolism , inflammation , and angiogenesis , in addition to nutrition . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion"], "sections": [["familial adenomatous polyposis   account for 1% of colorectal cancers , and provides a model of apc inactivation as an early genetic event for the approximately 80%85% of cancers that develop from sporadic polyps .", "colorectal cancers arising in patients with familial adenomatous polyposis can be largely prevented by polyp surveillance and prophylactic colectomy .", "total proctocolectomy with construction of a conventional ileostomy or ileoanal anastomosis with preparation of an ileal pouch , has various effects on the function of the terminal ileum and the intestinal bacterial flora .", "this may deteriorate cholesterol metabolism , as absorption of cholesterol in duodenum and jejunum requires micellar solubilization with bile acids , fatty acids , monoglycerides , and phospholipids .", "hypothetically , ileal - pouch - anal anastomosis and ileostomy patients might differ with regard to the presence of various fatty acids in feces and their relationship to other reflections of lipoprotein metabolism , but we found no previous study focusing upon this issue .", "dietary fatty acids are incorporated into blood and tissues , and the fatty acid composition in these tissues are often used as biomarkers of fat intake .", "furthermore , the fecal amount and composition of fatty acids reflect fat ingestion , intestinal fatty acid absorption , and the activity of colonic bacteria .", "although some of the discrepancies between studies may be due to the use of different methods to analyze fatty acids , differences in diet , or the fact that assessments have been performed in different body compartments , modifications in the metabolism of fatty acids have been suggested in cancer patients [ 5 , 6 ] .", "it is not clear at what steps in the multistage carcinogenesis process a possible distorted fatty acid metabolism occurs .", "notably , if such alterations occur in the development of carcinogenesis , this may affect the biological functions of essential fatty acids and their derivates .", "very little is known about fatty acid metabolism in familial adenomatous polyposis , although chemoprevention affecting the fatty acid derivates and the cyclooxygenase enzymes is often administered to familial adenomatous polyposis patients .", "deregulation of the cyclooxygenase-2 pathway appears to affect tumorigenesis via a number of distinct mechanisms : promoting tumour maintenance and progression , encouraging metastatic spread , and perhaps even participating in tumour initiation .", "cyclooxygenase-1 and -2 are the rate limiting enzymes in the synthesis of prostaglandins and thromboxanes .", "arachidonic acid is the main substrate for these enzymes , leading to the synthesis of prostaglandins which have growth promoting effects . substituting arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins .", "since cyclooxygenase-2 is a fatty acid metabolising enzyme , the relationships between cyclooxygenase-2 and fatty acid composition of different tissues is of interest .", "colectomized familial adenomatous polyposis patients had a deviant fatty acid profile with high levels of arachidonic acid and docosahexaenoic acid and low levels of linoleic acid and alpha - linolenic acid in serum phospholipids , which is in accordance with studies in patients with other types of cancers [ 5 , 1013 ] .", "in a previous familial adenomatous polyposis study , comparable treatment effects of a cyclooxygenase-2 inhibitor were observed on the fatty acid composition in serum phospholipids and duodenal lesions , presumably and most importantly the nonbeneficial effects involving essential fatty acids .  ", "the results describe the content of fatty acids in feces , and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies , diet , and levels of serum lipoproteins . because the effects of ileostomy construction may differ from that of ileal - pouch - anal anastomosis because of scarcity of the bacterial flora and different surface area of the terminal ileum , we did separate analyses for these two groups .", "previous familial adenomatous polyposis studies have focused upon faecal sterols and bile acids [ 1517 ] , but none has to the best of our knowledge analysed the content of fatty acids in feces , including very long chained fatty acids ."], ["data from the present study are taken from a randomized double - blind placebo - controlled intervention study with a cyclooxygenase-2 inhibitor [ 12 , 14 , 18 ] .", "the main aim was to compare the effect of rofecoxib treatment on duodenal lesions ( data in preparation ) .", "the present study comprises data from baseline for only the patients operated with ileal - pouch - anal anastomosis and ileostomy ( 77% of total study group ) .", "all of them had been colectomized , and duodenal lesions had been verified by endoscopy and histology .", "rikshospitalet university hospital is a highly specialized university hospital with national responsibilities in the area of complicated treatments , such as follow - ups on the norwegian familial adenomatous polyposis patients .", "inclusion criteria were verified familial adenomatous polyposis , colectomy , 1870 years of age , and documented duodenal lesions graded as spigelman i , ii , or iii and the largest adenoma10  mm .", "exclusion criteria were indications for surgical treatment , suspected or documented intestinal obstruction or stenosis , patients unwilling or unable to adhere to protocol , known cardiac failure requiring medical treatment , and pregnancy .", "as for the adenomas sampled , they were all assessed histologically as mild / moderate dysplasia , all below 10  mm and all flat , located in the descending part of the duodenum .", "samples of feces were transferred to tubes and mixed to make a homogeneous mass of which 100200  ul were used for lipid extraction by adding 50  ul butanol , 100  l h2o , 1  ml methanol , and 1  ml chloroform .", "after 10  min under n2 , the mixture was filtered and the filter washed   with 1  ml chloroform   + 1  ml chloroform / methanol ( 2  :  1 ) + 0.8  ml 0.73% nacl . after mixing for 15  s ,", "the tubes were centrifuged for 30  min at 1000  g. the upper phase was discarded and the lower phase transferred to new tubes and evaporated under n2 at 65c using a heat block .", "then 0.4  ml 0.5  n naoh in methanol was added and the solution kept for 7  min at 100c under nitrogen . after cooling to room temperature , 0.5  ml 12%", "bf3 was added and the solution kept for 10  min at 100c under nitrogen . after cooling to room temperature , 1  ml heptan was added under nitrogen , and mixing for 30  s , 1 ml of saturated nacl solution was added . after mixing ,", "the solution was centrifuged for 10  min at 1000  g , and the upper phase transferred to new tubes .", "finally , the heptan was evaporated and 300  l hexan added before continuing with the gas chromatography procedure referred to earlier .", "dietary intake was assessed by a validated food frequency questionnaire , designed to cover as much of the total habitual diet as possible .", "questions were related to habitual frequency of consumption and the amount of foods eaten during the last year .", "they were asked to identify their habitual choices of edible fats by pointing at specially prepared pictures , in order to increase the validity of the estimate of dietary fat .", "each biopsy was put into a tube containing 600  l trizol and immediately mixed on a mixermill ( retsch gmbh , germany ) .", "gene expression analyses were carried out on a 7900  ht real - time pcr machine from applied biosystems .", "based on the results from running a housekeeping gene test (  taqman human endogenous control plate ,  applied biosystems ) with rna from isolated human leucocytes ( data not shown ) .", "the primers and probes were initially designed as three assays per gene and validated for efficiency and specificity .", "the primers and probes for the cox-1 assay were : forward primer : 5-cttccaggagctcgtagga-3 , probe : 5-agaaggagatggcagcagagttggag-3 , and reverse primer : 5-acgcatcaatgtctccatacaat-3. cox-2 forward primer : 5-tggaacatggaattacccagt-3 , probe : 5-tgttgaatcattcaccaggcaaattgct-3 ,   and reverse primer : 5-tcctaccaccagcaaccct-3. gus forward primer : 5-gaaaatatgtggttggagagctcatt-3 , probe : 5-ccagcactctcgtcggtgactgttca-3 ,   and reverse primer : 5-ccgagtgaagatccccttttta-3.", "tbp forward primer : 5-ctggaaaagttgtattaacaggtgc-3 , probe : 5-agcagaaatttatgaagcatttgaaaacatctaccctatt-3 ,   and reverse primer : 5-cattacgtcgtcttcctgaatc-3.   taqman universal pcr master mix ( applied biosystems ) was added as reaction mix .", "the reaction conditions were initiated by a step of 2  min . at 50c and 10  min . at 95c , followed by 40 cycles of denaturation at 95c for 15 sec . and annealing at 60c for 1  min .", "a combination of cdna from several samples were made and diluted in order to make a dilution curve that was included on each plate .", "the average of the three values for each gene was divided by the average of the corresponding tbp values , generating a normalized value of the gene expression which is a unit less value used to compare the relative amount of mrna for each gene in the different samples .", "patients were informed by a physician , and in addition thoroughly written information was given to all patients .", "the protocol was explained to the subjects that had to give their consent before inclusion .", "was approved by the norwegian health authorities and the regional committee of medical ethics 20/06/2002 ( reference : s-02127 ) .", "nonparametrical statistical methods were chosen , as some of the variables were skewed and the number of observations limited ."], ["age and fecal content of palmitolic acid , linoleic acid , and arachidonic acid differed between the ileostomy and the ileal - pouch - anal anastomosis patients ( table 1 ) .", "the content of linoleic acid in feces correlated negatively to the proportion of eicosaenoic acid in duodenal biopsies ( r = 0.6 , p < .05 ) .", "the content of arachidonic acid in feces was correlated negatively to the proportions of eicosapentaenoic acid ( r = 0.7 , p = .02 ) and docosahexaenoic acid ( r = 0.7 , p = .008 ) in duodenal biopsies .", "total cholesterol were negatively correlated to the content of palmitic acid ( r = 0.7 , p = .03 ) , palmitoleic acid ( r = 0.7 , p = .02 ) , stearic acid ( r = 0.7 , p = .02 ) , oleic acid ( r = 0.7 , p = .02 ) , linoleic acid ( r = 0.8 , p = .002 ) , and monounsaturated fatty acids ( r = 0.7 , p = .02 ) in feces .", "the levels of triglycerides was negatively correlated to the content of linoleic acid ( r = 0.8 , p = .003 ) and eicosaenoic acid ( r = 0.7 , p = .03 ) in feces .", "no significant correlations were found between the content of different fatty acids in feces and the fatty acid dietary intake ( data not shown ) .   with the exception of the palmitoleic acid / palmitic acid ratio and levels of cyclooxygenase-2 expression in duodenal biopsies ( r = 0.8 , p = .003 ) , no relationships were found between fatty acids in feces and the levels of cyclooxygenase mrna expression ( figure 1 ) .", "significant correlations were found between the content of oleic acid in feces and the proportion of myristic acid ( r = 0.8 , p < .001 ) , oleic acid ( r = 0.5 , p <", ".05 ) , and eicosaenoic acid ( r = 0.5 , p = .03 ) in duodenal biopsies .", "moreover , the content of linoleic acid in feces correlated significantly to the proportion of myristic acid ( r = 0.8 , p < .001 ) in duodenal biopsies , whereas the content of alpha - linolenic acid in feces correlated inversely to eicosaenoic acid ( r = 0.6 , p <", "the content of palmitic acid in feces was positively correlated to levels of ldl - cholesterol ( r = 0.05 , p = .05 ) .", "no significant correlations were found between the content of different fatty acids in feces and the levels of cyclooxygenase mrna expression in duodenal biopsies ( data not shown ) .   several different fatty acids in feces correlated positively to dietary intake of fatty acids , most predominantly monounsaturated fatty acids ( table 2 ) ."], ["as expected , we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism .", "possibly , our results relate to metabolic differences caused by the different intestinal reconstructions , but the data are not suitable to explain the findings .", "we found no significant relationships between the levels of cyclooxygenase mrna expression in duodenal biopsies and the content of fatty acids in feces , except for the estimates of the content of delta-9-desaturase ( stearoyl - coa desaturase , scd ) activity , namely the ratio of palmitolic acid / palmitic acid [ 19 , 20 ] in the ileostomy group .", "stearoyl - coa desaturase is the central lipogenic enzyme catalyzing in vivo reactions in the synthesis of monounsaturated fatty acids , particularly oleic acid and palmitoleic acid , which are the major monounsaturated fatty acids of membrane phospholipids , triglycerides , wax esters , and cholesteryl esters .", "both delta-9-desaurase and cyclooxygenase-2 are ppar alpha regulated , although this possible link is only a speculation .", "a study in healthy subjects showed that human fecal water contains components can affect both the cyclooxygenase-2 protein level and enzymatic activity .", "one recent study claims that it is possible to detect cyclooxygenase-2 mrna in feces of colorectal cancer patients irrespective of clinical stage .", "notably , increased cyclooxygenase-2 expression in duodenal compared with colonic tissues in familial adenomatous polyposis was recently reported .  ", "the significant negative correlation between content of arachidonic acid in the feces and amounts of omega-3 fatty acids in duodenal biopsies which was found in the ileostomy group is interesting , since substitution of arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins .", "a previous familial adenomatous polyposis study established a positive correlation between the reduction in tissue prostanoid levels and clinical response , as measured by the reduction in size and number of adenomas , when patients were treated with sulindac .", "however , this correlation only became consistently significant when prostanoids were assayed after tissue homogenates were first incubated with arachidonic acid .", "we may suspect transformation of linoleic acid   to arachidonic acid in feces , but data are limited as with regard to feces from colectomized familial adenomatous polyposis subjects .", "however , the dietary assessment showed that the intake of such foods was normal among these familial adenomatous polyposis patients .  ", "dietary controlled intervention studies on ileostomy subjects have shown that dietary manipulation with fat and fiber may modify cholesterol absorption and sterol excretion . in the ileostomy group", ", levels of total cholesterol were negatively correlated to the content of palmitic acid , palmitolic acid , stearic acid , oleic acid , linoleic acid , and monounsaturated fatty acids ( < 18  c atoms ) in feces , which is interesting since the present levels of total cholesterol are lower than those among a healthy reference group . in the ileal - pouch - anal anastomosis group separately , the content of monounsaturated fatty acid in feces was positively correlated to dietary intake of monounsaturated fatty acids in particular . moreover", ", positive correlations were found between the content of oleic acid in feces and the proportion of myristic acid , oleic acid , and eicosaenoic acid in duodenal biopsies .", "the precise role of monounsaturated fatty acids synthesis in cell proliferation and programmed cell death remains unknown .", "the strong correlation of high levels of monounsaturated fatty acids and neoplastic phenotype may suggest that the regulation of stearoyl - coa desaturase must play a role in cancer development .    ", "this was an observational study , and not a metabolic balance study that was designed to investigate the metabolism of fatty acids in familial adenomatous polyposis patients .  ", "although some studies have found that the absorption rate of fatty acids are unaffected by age [ 26 , 27 ] , we can not exclude the possibility that some of the observed differences between the ileal - pouch - anal anastomosis and ileostomy patients might be due to age , rather than type of surgery , since lipoprotein clearance has been shown to be altered by age , and that might affect the relationship between dietary fatty acids and the fatty acid profiles found in tissues .", "moreover , no conclusions can be drawn from the present study because this is an observational study .", ". however , the study addresses the need to learn more about the dietary and lifestyle habits of this subset of at - risk patients .", "the present study includes a number of patients close to the maximum possible number of eligible norwegian familial adenomatous polyposis patients .", "we may , however , suspect that the low sample size and use of a food frequency questionnaire , may have weakened any associations between the content of fatty acids in feces and dietary intake .", "notably , these data do not contradict that the nutrient intake among these patients should at least meet the recommendations for healthy subjects .", "nevertheless , we suggest that these data are compelling enough to suggest that future familial adenomatous polyposis studies should investigate overall fatty acid metabolism , molecular mechanisms relevant to fatty acid metabolism , inflammation , and angiogenesis , in addition to nutrition requirements . in conclusion , we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism .", "we may suggest that fatty acid content in feces is related to dietary intake , serum lipids , and fatty acid composition in duodenal biopsies , even in colectomized familial adenomatous polyposis patients .", "this observational study may represent hypothesis - generating suggestions for future familial adenomatous polyposis studies ."]]}
{"article_id": "PMC4327666", "article_text": ["local anesthesia and pain control is one of the most important elements of dentistry , and particularly prosthetic dentistry .", "according to the american dental association , fear of pain is the most important factor preventing patients from visiting their dentists .", "different kinds of fear related to previous clinical experience affect patients ' attitudes to local anesthesia or dentist20 . although local anesthesia techniques do not provide pain - free treatment , this pain is generally tolerable .", "pain can result from the mechanical trauma of needle introduction into the site of injection , or from the sudden distension of the tissues , resulting from a rapid discharge of the contents of the syringe .", "pain can also be caused by the stimulation of the first few drops of the local anaesthetic3,14 .", "contrary to prevalent ideas , needle penetration of tissue is not the primary reason for discomfort .", "volume and pressure of the local anesthetic being injected causes more distress and/or pain . administering local anesthetic injection may not only provoke anxiety in patients , but also in the dentist29 . despite that , administering local anesthesia by injection for pain relief is still the most common method used in dentistry .", "however , there are several ways to alleviate pain before dental procedures begin , or the often painful nature of the injection in local anesthesia .", "transcutaneous electrical nerve stimulation ( tens)24 , use of intraoral lidocaine patch11 and computerized local anesthesia7 ( the wand ) are newly developed methods to alleviate the pain and anxiety of dental patients , as alternatives to conventional local anesthesia .", "the wand ( milestone scientific , livingston , nj ) is a computer - controlled local anesthesia delivery system that drives local anesthetic from a conventional local anesthetic cartridge through plastic microtubing into a plastic handle to which a luer - lok needle is attached . the computer - controlled flow is initiated by exerting pressure on a foot pedal .", "the pump allows administration of local anesthetic at two slow but constant rates , and the computer compensates for variation in resistance to flow4 . during needle insertion , continuous positive pressure delivers an anesthetic drip that precedes the needle . the combination of an anesthetic pathway and controlled flow rate results in a virtually imperceptible injection and rapid onset of profound anesthesia .", "the wand system offers several advantages over conventional syringes , including excellent tactile sensation afforded by a lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues , producing a coring penetration that minimizes needle deflection .", "wand devices automatically deliver the anesthetic more slowly and precisely than a conventional syringe , thereby reducing patient discomfort5,15 .", "anesthetic solution gently pumped into the soft tissue can be absorbed at the same rate by soft tissue in the wand system .", "the theory is that when the needle is advanced slowly , the drops of solution anesthetize the tissue ahead of the needle .", "all techniques of local anesthesia , such as maxillary and mandibular infiltration , mandibular block22 , intraligamentary26 , and anterior middle superior alveolar ( amsa ) injection8 , and anterior superior alveolar ( asa ) injection25 can be performed with the wand system .", "conventionally , maxillary teeth have been anesthetized by administering an infiltration injection on the buccal or labial aspects of the target tooth .", "palatal injections have also been administered by plastic syringe for this objective , but can be painful5,7 .", "the amsa derives its name from the injection 's ability to anesthetize both the anterior and middle superior alveolar nerves57 .", "the middle superior alveolar ( msa ) and asa nerves branch from the infraorbital nerve before they exit from the infraorbital foramen .", "the msa nerve is thought to innervate the maxillary premolars and plays some role in pulpal innervation of the mesiobuccal root of the first molar .", "the anterior , superior alveolar nerve supplies pulpal innervation to the central and lateral incisors and canines18 .", "the plexus where the 2 nerves join is the target site for the amsa injection .", "the 10 maxillary teeth extending from the second premolar to contralateral second premolar , and the associated palatal tissue , can be anesthetised with bilateral amsa injection57 .", "wand research is largely attributable to pediatric dentistry1,2,9 , restorative dentistry7 , endodontics14 , periodontology17,26,27 , maxillofacial surgery16 , comparison of pain perception to the wand and a traditional syringe21,28 , influence of anesthetic flow rate delivered by the wand on pain response to the palatal injection23 .", "the hypothesis of the present study was that the wand anesthesia technique reduces the pain at different phases ( needle insertion , delivery of local anesthetic and tooth preparation ) versus conventional buccal anesthesia with conventional plastic syringe .", "therefore , the study compared pain levels at needle insertion , during delivery of local anesthetic solution and during tooth preparation , for the conventional syringe technique on the buccal or labial aspect on one side , and the amsa technique with wand local anesthesia application on the other side of the maxilla . a 5-point verbal pain rating scale ( vrs)12,17 was used on 16 patients who were under prosthodontic treatment in the ondokuz mayis university clinic", "sixteen adult patients who had partially edentulous states of both left and right maxillary arch , and were to undergo fixed prosthodontic treatment , participated in this study .", "they ranged in age from 27 to 64 , were predominantly women ( 12 women and 4 men ) , and were in a good health .", "patients were not taking any medication that would alter their pain perception , as determined by a written medical health form and oral questioning .", "allergy to lidocaine , mepivacaine , sulphides or articaine , history of significant medical problems , taking central nervous system depressants within 48 hours , pregnancy , or inability to give informed consent , were the exclusion criteria .", "the human subjects review committee of ondokuz mayis university approved this study ( 28.11.2005 dated , and 328 numbered ) , and written , informed consent was obtained from each patient .", "all patients had previously experienced a conventional syringe , no patients had previously received the wand injection , and all patients had at least one tooth absent both on the left and right maxillary arch .", "the amsa technique with the wand for half the maxillary arch and conventional plastic syringe anesthesia on mucobuccal fold ( control group ) for opposite maxillary arch were administered by the same operator .", "the order of anesthesia techniques was randomly selected , and anesthesia procedures and tooth preparations were completed in the same session ( table 1 ) .", "vrs ( 0 no pain , 1 minimal pain , 2 slight pain , 3 moderate pain and 4 severe pain ) .", "the pain level descriptions were as follows : no pain , minimal pain , slight pain , moderate pain and severe pain ( 0 , 1 , 2 , 3 , 4 ) .", "patients were blindfolded with a commonly used sleeping mask so they would not distinguish which anesthetic delivery system was used .", "the wand produces audible beeps as the injection is administered . because the beeping tones can not be turned off with a switch , the sound were produced during both injection methods ( the wand or conventional - control ) so the patients were not aware of the method being used . immediately after needle insertion , delivery of local anesthetic and tooth preparation ,", "patients were asked to remove mask and rate the pain for both conventional and wand injection techniques .", "no topical anesthetic was used because the wand manufacturer 's instructions suggest that topical application is optional .", "this study was conducted according to a split- mouth design , with both injections given to all patients . for each patient ,", "the amsa technique with single wand injection was used on one side for central incisors , lateral incisors , upper canine , first and second premolars , and the traditional plastic aspirating syringe injection was used on the mucobuccal fold of the related tooth on the opposite side", ". the amsa injection site is located at a point that bisects the maxillary first and second premolars , and midway between the crest of the free gingival margin and mid - palatine suture .", "the needle is orientated at a 45 degree angle with the bevel facing the palatal tissue , while rotating the wand needle .", "all injections were given by the same operator who had had two months experience using the wand at the department of prosthodontics , faculty of dentistry , ondokuz mayis university , turkey .", "the ampul form of articaine hydrochloride with adrenalin ( hoechst marion roussel , deutschland , gmbh , germany ) was injected with conventional plastic syringe and 27gauge dental needle .", "the cartridge form of articaine hydrochloride with adrenaline ( hoechst ag , frankfurt , germany ) was administered with the computer - controlled anesthesia device ( the wand , milestone scientific , livingston , nj , usa ) , with 27gauge handpiece and needle ( milestone scientific ) . approximately 0.9 to 1.0 ml local anesthetic was used both for amsa with the wand on one side and control group for each tooth on the other side .", "groups were labelled as follows : group ( c ) , pain perception scores at needle insertion for the conventional technique ; group ( c ) , pain perception scores for local anesthetic delivery by the conventional technique ; group ( c ) , pain perception scores at tooth preparation for the conventional technique ; group ( w ) , pain perception scores at needle insertion by the wand technique ; group ( w ) , pain perception scores for local anesthetic delivery for the wand technique ; and group ( w ) , pain perception scores at tooth preparation for the wand technique .", "power analysis were performed to detect adequate sample size using statistical software ( pass 2008 , ncss , kaysville , ut , usa ) .", "since our variable was an ordinal level , the mann - whitney u test was used to determine the number of times a score from the conventional method is ranked than a score from the wand techniques at 1% significance level .", "statistical tests were performed with the statistical software ( spss version 12.0 ; spss inc , chicago , il , usa ) .", "differences in pain rating were analyzed at needle insertion , delivery of local anesthetic and during tooth preparation for conventional versus the wand techniques .", "sixteen adult patients aged from 27 to 64 ( 12 women and 4 men ) participated in this study .", "median scores , minimum and maximum pain rating values after needle insertion , and delivery of anesthetic and tooth preparation time for both conventional and wand techniques , are shown in table 3 .", "the wand technique registered a highly significantly lower pain level during the needle insertion phase ( z = - 4.064 , p<0.01 ) , with the wand technique having a lower pain level compared to the conventional . during the anesthetic solution delivery phase , the pain level for the wand technique was again highly significantly lower than for the conventional technique ( z = - 3.897 , p<0.01 ) .", "however , there was no difference between the wand and conventional techniques for pain level during tooth preparation ( z = - 0.671 , p>0.05 ) .", "the research hypothesis stated that the wand anesthesia technique reduces the pain at different phases ( needle insertion , delivery of local anesthetic and tooth preparation ) versus conventional buccal anesthesia with conventional plastic syringe .", "the wand anesthesia technique reduces the pain compared the conventional anesthesia for needle insertion and anesthetic solution delivery phases ( p<0.01 ) , with the exception of tooth preparation phase ( p>0.05 ) .", "the majority of literature on the computer - controlled injection system ( the wand ) has dealt with the pain of injection with the computer - assisted injection system , compared to injection using a conventional syringe1,2,8l0,l6,2l,27,28 . in general , the results have been favorable1,8,9,16,23,27 for the computer - assisted injection system , with only 2 studies showing no difference2,28 , and 1 study showing higher pain ratings10 .", "palatal injections are generally considered the most painful injections13 ; wahl , et al.30 showed that palatal injections caused significantly more pain than other intraoral injections .", "the authors of18,30 speculated that palatal injection pain might be mainly the result of pressure .", "the traditional route for administration of anesthesia to the maxillary teeth is supraperiosteal infiltration delivered in the mucobuccal fold near the apex of the tooth to be anesthetized .", "although effective in achieving pulpal anesthesia of the neurovascular bundle by diffusion of anesthetic through the porous maxilla , this approach routinely anesthetizes the surrounding tissues ( lips , surface of the face ) , and also affects muscle activity .", "the patient 's natural repose , as well as active facial expressions are temporarily affected , including an distortion of the smile .", "the patient also experiences the annoying sensation of facial numbness , and the smile line is temporarily invalidated as an esthetic reference for restorative and prosthetic dentistry .", "an alternative to maxillary infiltration is the infraorbital nerve block injection which , if properly performed , achieves anesthesia of the anterior superior alveolar branch of the trigeminal nerve .", "this injection site , however , is also close to muscles of expression , and anesthesia of cutaneous fibers of the infraorbital nerve results in a corresponding loss of sensation in the face and the upper lip29 .", "the palatal approach amsa nerve block can be attempted with a traditional manual syringe , but the precise pressure and volume ratios of the wand are virtually impossible to reproduce5,6 .", "the clinician can anesthetize several teeth in the maxillary arch , extending from the mesiobuccal root of the first molar to the central incisor , by means of a single palatal infiltration .", "in addition to profound pulpal anesthesia , palatal soft tissue anesthesia is realized , which also extends from the maxillary first molar to the central incisor and the associated palatal tissues , without numbness to the lips and face , and without interference with the muscles of facial expression for at least 60 min16,25 . during needle insertion with a coring motion ,", "the drops of solution anesthetize the tissue ahead of the needle when the needle is advanced slowly7,15 . the combination of an anesthetic pathway and controlled flow rate results in virtually imperceptible needle insertion and injection , and rapid onset of profound anesthesia28 .", "the same efficacy of anesthesia was achieved with conventional buccal anesthesia and the palatal approach amsa technique with the wand device25 .", "the current study was in agreement for tooth preparation , but contradictory for needle insertion and delivery of local anesthetic .", "numbness of the lips and face was not observed in the current study for the palatal approach when using the amsa technique with the wand .", "another study using the vrs ( verbal rating scale ) compared computer - controlled and conventional local anesthesia delivery systems for performing scaling and root planing on patients with moderate periodontal disease ; amsa injections with the wand were considered less painful than the conventional syringe17 .", "two further benefits of the amsa injection technique are that it also eliminates the potential for an intravascular injection because no major vessels are encountered in this region of the palate5 , and the palatal bone is porous enough to permit the anesthetic solution to diffuse through the tissues and anesthetize both the anterior and middle branches of the superior alveolar nerve when the injection is deliberately slow and steady . for this reason , the successful deposition of the anesthetic solution through the fibrous tissue", "is said to be accomplished more easily with the computer - controlled delivery device that regulates the pressure and volume ratio of solution delivered5,7 .", "the wand system offers several advantages over conventional syringes , including excellent tactile sensation afforded by the lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues , producing a coring penetration that minimizes needle deflection .", "presumably , the slow rate of anesthetic flow reduces patient discomfort compared with palatal injections administered with a traditional syringe4 . decreasing the total amount of anesthetic and vasoconstrictor necessary for maxillary anesthesia , shorthening the total anesthesia time , and diminishing patient - operator anxiety", "however , the wand 's beeping sounds make the injection seem longer and may make the patient feel uncomfortable .", "other negative aspects of the wand are frequent breakage of the anesthetic cartridge on activation of the unit , and a separate instrument is required for removing the cartridge from the unit10 .", "additionally , 1.4 ml anesthetic solution can be delivered in about 1 min in rapid injection mode , and in 4 min 45 s in slow injection mode9 .", "fukayama , et al.8 examined the efficacy of the amsa anesthesia technique using the wand system to assist with the electrical pulp test .", "electrical pulp stimulation indicated that the lateral incisors , canines , and first and second premolars were more anesthetized than the central incisors and first molars .", "they also stated that anesthetic applied via amsa with the wand has a slow onset and further studies are required to evaluate its effectiveness for this reason .", "however , amsa injection using the wand method seems to both preclude severe injection pain and be very effective for pulpal anesthesia of lateral incisors , canines , and premolars .", "pain scores were higher for amsa technique in the central tooth than in other teeth ( p<0.05 ) in another related study16 .", "in the present study , in two cases of amsa using the wand , pain was at the minimum level ( vrs score 1 ) during tooth preparation of central incisors .", "the reason for this pain rating could be that the central incisors were innervated from the opposite arch , or alternatively , fukayama , et al.8 and lee , et al.16 stated that the effectiveness of the wand is lower in the central incisors and first molar teeth than lateral incisors , canines , and premolars .", "friedman and hochman57 stated that careful needle insertion and slow anesthetic delivery could reduce the sensation of needle insertion .", "primosch and brooks23 showed that slow anesthetic delivery produces significantly lower pain scores than fast anesthetic delivery , and the wand decreases the pain for both slow or fast anesthetic delivery rate , but contrary to the manufacturer 's claims , it did not eliminate pain elicited by palatal injections in some patients . in the present study , a highly significant ( p<0.01 ) difference for pain rating", "was obtained between conventional buccal and amsa using the wand injection for needle insertion and anesthetic solution delivery . however ,", "contrary to the manufacturer 's claims , pain was not eliminated at slow anesthetic flow rate in this study there have been limited prosthodontic or restorative studies for amsa using wand injection . in one study , amsa was bilaterally applied with the wand and 6 anterior maxillary teeth were prepared .", "incisal reduction of these teeth was achieved with reference to the lip , which is not affected by amsa .", "bilaterally , the amsa technique using the wand was repeated for tooth sensitivity during the cementation procedure5,6 . as in previous studies ,", "amsa using the wand was also of benefit for lip guidance in the present study .", "another benefit of palatal amsa injection using the wand is that it reduces the number of injections , and also the amount of anesthetic solution compared to conventional buccal infiltration anesthesia that applies multiple injections to each tooth .", "in addition , more teeth can be anesthetized with a single injection , without numbness of lips and face , compared to the conventional technique .", "the amsa technique administered using the wand in a prosthodontic treatment reduced pain highly significantly more than conventional buccal anesthesia , both in inserting the needle and in the of delivery of local anesthetic . when comparing the anesthesia efficiency of amsa using the wand injection and conventional buccal infiltration techniques , no statistical difference was found .", "although the slow anesthetic flow rate proved superior in pain control , the pain score was not zero for the wand , contrary to the manufacturer 's claim .", "computer - controlled anesthesia using the wand appears advantageous for restorative procedures because more teeth are anesthetized with one palatal injection , and without numbness of lips and face , in contrast to multiple conventional buccal anesthetic injections for each tooth ."], "abstract_text": ["<S> objective : the objective of this study was to compare the pain levels on opposite sides of the maxilla at needle insertion during delivery of local anesthetic solution and tooth preparation for both conventional and anterior middle superior alveolar ( amsa ) technique with the wand computer - controlled local anesthesia application.material and methods : pain scores of 16 patients were evaluated with a 5-point verbal rating scale ( vrs ) and data were analyzed nonparametrically . </S>", "<S> pain differences at needle insertion , during delivery of local anesthetic , and at tooth preparation , for conventional versus the wand technique , were analyzed using the mann - whitney u test ( p=0.01).results : the wand technique had a lower pain level compared to conventional injection for needle insertion ( p<0.01 ) . in the anesthetic delivery phase , pain level for the wand technique was lower ( p<0.01 ) . </S>", "<S> however , there was no difference between the wand and conventional technique for pain level during tooth preparation ( p>0.05).conclusions;the amsa technique using the wand is recommended for prosthodontic treatment because it reduces pain during needle insertion and during delivery of local anaesthetic </S>", "<S> . however , these two techniques have the same pain levels for tooth preparation . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["local anesthesia and pain control is one of the most important elements of dentistry , and particularly prosthetic dentistry .", "according to the american dental association , fear of pain is the most important factor preventing patients from visiting their dentists .", "different kinds of fear related to previous clinical experience affect patients ' attitudes to local anesthesia or dentist20 . although local anesthesia techniques do not provide pain - free treatment , this pain is generally tolerable .", "pain can result from the mechanical trauma of needle introduction into the site of injection , or from the sudden distension of the tissues , resulting from a rapid discharge of the contents of the syringe .", "pain can also be caused by the stimulation of the first few drops of the local anaesthetic3,14 .", "contrary to prevalent ideas , needle penetration of tissue is not the primary reason for discomfort .", "volume and pressure of the local anesthetic being injected causes more distress and/or pain . administering local anesthetic injection may not only provoke anxiety in patients , but also in the dentist29 . despite that , administering local anesthesia by injection for pain relief is still the most common method used in dentistry .", "however , there are several ways to alleviate pain before dental procedures begin , or the often painful nature of the injection in local anesthesia .", "transcutaneous electrical nerve stimulation ( tens)24 , use of intraoral lidocaine patch11 and computerized local anesthesia7 ( the wand ) are newly developed methods to alleviate the pain and anxiety of dental patients , as alternatives to conventional local anesthesia .", "the wand ( milestone scientific , livingston , nj ) is a computer - controlled local anesthesia delivery system that drives local anesthetic from a conventional local anesthetic cartridge through plastic microtubing into a plastic handle to which a luer - lok needle is attached . the computer - controlled flow is initiated by exerting pressure on a foot pedal .", "the pump allows administration of local anesthetic at two slow but constant rates , and the computer compensates for variation in resistance to flow4 . during needle insertion , continuous positive pressure delivers an anesthetic drip that precedes the needle . the combination of an anesthetic pathway and controlled flow rate results in a virtually imperceptible injection and rapid onset of profound anesthesia .", "the wand system offers several advantages over conventional syringes , including excellent tactile sensation afforded by a lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues , producing a coring penetration that minimizes needle deflection .", "wand devices automatically deliver the anesthetic more slowly and precisely than a conventional syringe , thereby reducing patient discomfort5,15 .", "anesthetic solution gently pumped into the soft tissue can be absorbed at the same rate by soft tissue in the wand system .", "the theory is that when the needle is advanced slowly , the drops of solution anesthetize the tissue ahead of the needle .", "all techniques of local anesthesia , such as maxillary and mandibular infiltration , mandibular block22 , intraligamentary26 , and anterior middle superior alveolar ( amsa ) injection8 , and anterior superior alveolar ( asa ) injection25 can be performed with the wand system .", "conventionally , maxillary teeth have been anesthetized by administering an infiltration injection on the buccal or labial aspects of the target tooth .", "palatal injections have also been administered by plastic syringe for this objective , but can be painful5,7 .", "the amsa derives its name from the injection 's ability to anesthetize both the anterior and middle superior alveolar nerves57 .", "the middle superior alveolar ( msa ) and asa nerves branch from the infraorbital nerve before they exit from the infraorbital foramen .", "the msa nerve is thought to innervate the maxillary premolars and plays some role in pulpal innervation of the mesiobuccal root of the first molar .", "the anterior , superior alveolar nerve supplies pulpal innervation to the central and lateral incisors and canines18 .", "the plexus where the 2 nerves join is the target site for the amsa injection .", "the 10 maxillary teeth extending from the second premolar to contralateral second premolar , and the associated palatal tissue , can be anesthetised with bilateral amsa injection57 .", "wand research is largely attributable to pediatric dentistry1,2,9 , restorative dentistry7 , endodontics14 , periodontology17,26,27 , maxillofacial surgery16 , comparison of pain perception to the wand and a traditional syringe21,28 , influence of anesthetic flow rate delivered by the wand on pain response to the palatal injection23 .", "the hypothesis of the present study was that the wand anesthesia technique reduces the pain at different phases ( needle insertion , delivery of local anesthetic and tooth preparation ) versus conventional buccal anesthesia with conventional plastic syringe .", "therefore , the study compared pain levels at needle insertion , during delivery of local anesthetic solution and during tooth preparation , for the conventional syringe technique on the buccal or labial aspect on one side , and the amsa technique with wand local anesthesia application on the other side of the maxilla . a 5-point verbal pain rating scale ( vrs)12,17 was used on 16 patients who were under prosthodontic treatment in the ondokuz mayis university clinic"], ["sixteen adult patients who had partially edentulous states of both left and right maxillary arch , and were to undergo fixed prosthodontic treatment , participated in this study .", "they ranged in age from 27 to 64 , were predominantly women ( 12 women and 4 men ) , and were in a good health .", "patients were not taking any medication that would alter their pain perception , as determined by a written medical health form and oral questioning .", "allergy to lidocaine , mepivacaine , sulphides or articaine , history of significant medical problems , taking central nervous system depressants within 48 hours , pregnancy , or inability to give informed consent , were the exclusion criteria .", "the human subjects review committee of ondokuz mayis university approved this study ( 28.11.2005 dated , and 328 numbered ) , and written , informed consent was obtained from each patient .", "all patients had previously experienced a conventional syringe , no patients had previously received the wand injection , and all patients had at least one tooth absent both on the left and right maxillary arch .", "the amsa technique with the wand for half the maxillary arch and conventional plastic syringe anesthesia on mucobuccal fold ( control group ) for opposite maxillary arch were administered by the same operator .", "the order of anesthesia techniques was randomly selected , and anesthesia procedures and tooth preparations were completed in the same session ( table 1 ) .", "vrs ( 0 no pain , 1 minimal pain , 2 slight pain , 3 moderate pain and 4 severe pain ) .", "the pain level descriptions were as follows : no pain , minimal pain , slight pain , moderate pain and severe pain ( 0 , 1 , 2 , 3 , 4 ) .", "patients were blindfolded with a commonly used sleeping mask so they would not distinguish which anesthetic delivery system was used .", "the wand produces audible beeps as the injection is administered . because the beeping tones can not be turned off with a switch , the sound were produced during both injection methods ( the wand or conventional - control ) so the patients were not aware of the method being used . immediately after needle insertion , delivery of local anesthetic and tooth preparation ,", "patients were asked to remove mask and rate the pain for both conventional and wand injection techniques .", "no topical anesthetic was used because the wand manufacturer 's instructions suggest that topical application is optional .", "this study was conducted according to a split- mouth design , with both injections given to all patients . for each patient ,", "the amsa technique with single wand injection was used on one side for central incisors , lateral incisors , upper canine , first and second premolars , and the traditional plastic aspirating syringe injection was used on the mucobuccal fold of the related tooth on the opposite side", ". the amsa injection site is located at a point that bisects the maxillary first and second premolars , and midway between the crest of the free gingival margin and mid - palatine suture .", "the needle is orientated at a 45 degree angle with the bevel facing the palatal tissue , while rotating the wand needle .", "all injections were given by the same operator who had had two months experience using the wand at the department of prosthodontics , faculty of dentistry , ondokuz mayis university , turkey .", "the ampul form of articaine hydrochloride with adrenalin ( hoechst marion roussel , deutschland , gmbh , germany ) was injected with conventional plastic syringe and 27gauge dental needle .", "the cartridge form of articaine hydrochloride with adrenaline ( hoechst ag , frankfurt , germany ) was administered with the computer - controlled anesthesia device ( the wand , milestone scientific , livingston , nj , usa ) , with 27gauge handpiece and needle ( milestone scientific ) . approximately 0.9 to 1.0 ml local anesthetic was used both for amsa with the wand on one side and control group for each tooth on the other side .", "groups were labelled as follows : group ( c ) , pain perception scores at needle insertion for the conventional technique ; group ( c ) , pain perception scores for local anesthetic delivery by the conventional technique ; group ( c ) , pain perception scores at tooth preparation for the conventional technique ; group ( w ) , pain perception scores at needle insertion by the wand technique ; group ( w ) , pain perception scores for local anesthetic delivery for the wand technique ; and group ( w ) , pain perception scores at tooth preparation for the wand technique .", "power analysis were performed to detect adequate sample size using statistical software ( pass 2008 , ncss , kaysville , ut , usa ) .", "since our variable was an ordinal level , the mann - whitney u test was used to determine the number of times a score from the conventional method is ranked than a score from the wand techniques at 1% significance level .", "statistical tests were performed with the statistical software ( spss version 12.0 ; spss inc , chicago , il , usa ) .", "differences in pain rating were analyzed at needle insertion , delivery of local anesthetic and during tooth preparation for conventional versus the wand techniques ."], ["sixteen adult patients aged from 27 to 64 ( 12 women and 4 men ) participated in this study .", "median scores , minimum and maximum pain rating values after needle insertion , and delivery of anesthetic and tooth preparation time for both conventional and wand techniques , are shown in table 3 .", "the wand technique registered a highly significantly lower pain level during the needle insertion phase ( z = - 4.064 , p<0.01 ) , with the wand technique having a lower pain level compared to the conventional . during the anesthetic solution delivery phase , the pain level for the wand technique was again highly significantly lower than for the conventional technique ( z = - 3.897 , p<0.01 ) .", "however , there was no difference between the wand and conventional techniques for pain level during tooth preparation ( z = - 0.671 , p>0.05 ) ."], ["the research hypothesis stated that the wand anesthesia technique reduces the pain at different phases ( needle insertion , delivery of local anesthetic and tooth preparation ) versus conventional buccal anesthesia with conventional plastic syringe .", "the wand anesthesia technique reduces the pain compared the conventional anesthesia for needle insertion and anesthetic solution delivery phases ( p<0.01 ) , with the exception of tooth preparation phase ( p>0.05 ) .", "the majority of literature on the computer - controlled injection system ( the wand ) has dealt with the pain of injection with the computer - assisted injection system , compared to injection using a conventional syringe1,2,8l0,l6,2l,27,28 . in general , the results have been favorable1,8,9,16,23,27 for the computer - assisted injection system , with only 2 studies showing no difference2,28 , and 1 study showing higher pain ratings10 .", "palatal injections are generally considered the most painful injections13 ; wahl , et al.30 showed that palatal injections caused significantly more pain than other intraoral injections .", "the authors of18,30 speculated that palatal injection pain might be mainly the result of pressure .", "the traditional route for administration of anesthesia to the maxillary teeth is supraperiosteal infiltration delivered in the mucobuccal fold near the apex of the tooth to be anesthetized .", "although effective in achieving pulpal anesthesia of the neurovascular bundle by diffusion of anesthetic through the porous maxilla , this approach routinely anesthetizes the surrounding tissues ( lips , surface of the face ) , and also affects muscle activity .", "the patient 's natural repose , as well as active facial expressions are temporarily affected , including an distortion of the smile .", "the patient also experiences the annoying sensation of facial numbness , and the smile line is temporarily invalidated as an esthetic reference for restorative and prosthetic dentistry .", "an alternative to maxillary infiltration is the infraorbital nerve block injection which , if properly performed , achieves anesthesia of the anterior superior alveolar branch of the trigeminal nerve .", "this injection site , however , is also close to muscles of expression , and anesthesia of cutaneous fibers of the infraorbital nerve results in a corresponding loss of sensation in the face and the upper lip29 .", "the palatal approach amsa nerve block can be attempted with a traditional manual syringe , but the precise pressure and volume ratios of the wand are virtually impossible to reproduce5,6 .", "the clinician can anesthetize several teeth in the maxillary arch , extending from the mesiobuccal root of the first molar to the central incisor , by means of a single palatal infiltration .", "in addition to profound pulpal anesthesia , palatal soft tissue anesthesia is realized , which also extends from the maxillary first molar to the central incisor and the associated palatal tissues , without numbness to the lips and face , and without interference with the muscles of facial expression for at least 60 min16,25 . during needle insertion with a coring motion ,", "the drops of solution anesthetize the tissue ahead of the needle when the needle is advanced slowly7,15 . the combination of an anesthetic pathway and controlled flow rate results in virtually imperceptible needle insertion and injection , and rapid onset of profound anesthesia28 .", "the same efficacy of anesthesia was achieved with conventional buccal anesthesia and the palatal approach amsa technique with the wand device25 .", "the current study was in agreement for tooth preparation , but contradictory for needle insertion and delivery of local anesthetic .", "numbness of the lips and face was not observed in the current study for the palatal approach when using the amsa technique with the wand .", "another study using the vrs ( verbal rating scale ) compared computer - controlled and conventional local anesthesia delivery systems for performing scaling and root planing on patients with moderate periodontal disease ; amsa injections with the wand were considered less painful than the conventional syringe17 .", "two further benefits of the amsa injection technique are that it also eliminates the potential for an intravascular injection because no major vessels are encountered in this region of the palate5 , and the palatal bone is porous enough to permit the anesthetic solution to diffuse through the tissues and anesthetize both the anterior and middle branches of the superior alveolar nerve when the injection is deliberately slow and steady . for this reason , the successful deposition of the anesthetic solution through the fibrous tissue", "is said to be accomplished more easily with the computer - controlled delivery device that regulates the pressure and volume ratio of solution delivered5,7 .", "the wand system offers several advantages over conventional syringes , including excellent tactile sensation afforded by the lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues , producing a coring penetration that minimizes needle deflection .", "presumably , the slow rate of anesthetic flow reduces patient discomfort compared with palatal injections administered with a traditional syringe4 . decreasing the total amount of anesthetic and vasoconstrictor necessary for maxillary anesthesia , shorthening the total anesthesia time , and diminishing patient - operator anxiety", "however , the wand 's beeping sounds make the injection seem longer and may make the patient feel uncomfortable .", "other negative aspects of the wand are frequent breakage of the anesthetic cartridge on activation of the unit , and a separate instrument is required for removing the cartridge from the unit10 .", "additionally , 1.4 ml anesthetic solution can be delivered in about 1 min in rapid injection mode , and in 4 min 45 s in slow injection mode9 .", "fukayama , et al.8 examined the efficacy of the amsa anesthesia technique using the wand system to assist with the electrical pulp test .", "electrical pulp stimulation indicated that the lateral incisors , canines , and first and second premolars were more anesthetized than the central incisors and first molars .", "they also stated that anesthetic applied via amsa with the wand has a slow onset and further studies are required to evaluate its effectiveness for this reason .", "however , amsa injection using the wand method seems to both preclude severe injection pain and be very effective for pulpal anesthesia of lateral incisors , canines , and premolars .", "pain scores were higher for amsa technique in the central tooth than in other teeth ( p<0.05 ) in another related study16 .", "in the present study , in two cases of amsa using the wand , pain was at the minimum level ( vrs score 1 ) during tooth preparation of central incisors .", "the reason for this pain rating could be that the central incisors were innervated from the opposite arch , or alternatively , fukayama , et al.8 and lee , et al.16 stated that the effectiveness of the wand is lower in the central incisors and first molar teeth than lateral incisors , canines , and premolars .", "friedman and hochman57 stated that careful needle insertion and slow anesthetic delivery could reduce the sensation of needle insertion .", "primosch and brooks23 showed that slow anesthetic delivery produces significantly lower pain scores than fast anesthetic delivery , and the wand decreases the pain for both slow or fast anesthetic delivery rate , but contrary to the manufacturer 's claims , it did not eliminate pain elicited by palatal injections in some patients . in the present study , a highly significant ( p<0.01 ) difference for pain rating", "was obtained between conventional buccal and amsa using the wand injection for needle insertion and anesthetic solution delivery . however ,", "contrary to the manufacturer 's claims , pain was not eliminated at slow anesthetic flow rate in this study there have been limited prosthodontic or restorative studies for amsa using wand injection . in one study , amsa was bilaterally applied with the wand and 6 anterior maxillary teeth were prepared .", "incisal reduction of these teeth was achieved with reference to the lip , which is not affected by amsa .", "bilaterally , the amsa technique using the wand was repeated for tooth sensitivity during the cementation procedure5,6 . as in previous studies ,", "amsa using the wand was also of benefit for lip guidance in the present study .", "another benefit of palatal amsa injection using the wand is that it reduces the number of injections , and also the amount of anesthetic solution compared to conventional buccal infiltration anesthesia that applies multiple injections to each tooth .", "in addition , more teeth can be anesthetized with a single injection , without numbness of lips and face , compared to the conventional technique ."], ["the amsa technique administered using the wand in a prosthodontic treatment reduced pain highly significantly more than conventional buccal anesthesia , both in inserting the needle and in the of delivery of local anesthetic . when comparing the anesthesia efficiency of amsa using the wand injection and conventional buccal infiltration techniques , no statistical difference was found .", "although the slow anesthetic flow rate proved superior in pain control , the pain score was not zero for the wand , contrary to the manufacturer 's claim .", "computer - controlled anesthesia using the wand appears advantageous for restorative procedures because more teeth are anesthetized with one palatal injection , and without numbness of lips and face , in contrast to multiple conventional buccal anesthetic injections for each tooth ."]]}
{"article_id": "PMC3462379", "article_text": ["chronic obstructive pulmonary disease ( copd ) is a progressive lung disease which is characterized by airway obstruction and lung parenchyma destruction .", "patients may experience dyspnoea or shortness of breath , a persistent cough with sputum production , decreased exercise tolerance and decreased quality of life [ 1 , 2 ] . globally , copd is the fourth most common cause of death and is predicted by the world health organisation to be a major health concern in the coming decade . in australia ,", "1.2 million people are estimated to have moderate - to - severe copd ( gold stages ii ", "iv ) . however , the true number is potentially much higher as copd is commonly underdiagnosed [ 4 , 5 ] .", "the total cost of living with copd ( including nonmonetary costs ) is estimated to be $ 83  000 aud per person each year [ 6 , 7 ] .  ", "as the severity of copd greatly impacts the cost of care , providing early diagnosis and effective management of the individual is an essential component in reducing its economic impact [ 5 , 810 ] .", "pr is a multidisciplinary approach which incorporates self - management education with exercise training , psychosocial and nutritional support [ 11 , 12 ] .", "it is a fundamental part of managing people with copd and has been shown to be effective in improving exercise tolerance , reducing dyspnoea , improving , quality of life and reducing the severity of acute exacerbations [ 1 , 1117 ] .", "although pr has been shown to be effective in managing symptoms and reducing hospital admissions , poor participation and adherence are a current problem among outpatient programs .", "factors include difficulty travelling to the program 's location [ 1922 ] , inconvenience of hospital attendance , or difficulty accessing programs .", "australia is one of the least densely populated nations ( 2.7 people per square kilometre ) with up to 11.8% of the population residing in outer regional or remote locations .", "conducted a study in kyabram ( rural victoria ) to assess the efficacy of their pr program and noted that patients who lived outside the town centre were more likely to withdraw from pr as the program was conducted within the town hospital .", "this finding is also supported by de angelis and colleagues   who found that transport , travel distance , and related costs were the main reason for nonattendance of a cardiac rehabilitation program .  ", "difficulty in accessing outpatient programs has led to the development of home - based programs .", "it is well established that home - based pr programs are safe and effective [ 13 , 2931 ] and are recommended by many international guidelines and reviews [ 2 , 11 , 12 , 17 ] . given the effectiveness of independent home - based pr programs one could ask what additional benefits supervised remote - based programs would provide .", "the main advantage of supervised remote - based programs is the ability to minimise anxiety in both patient and therapist .", "this benefit has been shown in remote - based cardiac exercise programs where the use of transtelephonic exercise monitoring ( tem ) has been used to relieve patients ' and trainers ' anxiety during exercise [ 3234 ] .", "it is anticipated that a person with copd could exercise to a higher intensity without the risk of undetected adverse events ( e.g. , exercise - induced oxygen desaturation ) .  ", "telerehabilitation is an emerging technology used to facilitate the delivery of rehabilitation services at a distance by combining telecommunication and electronic transmission of health information .", "it has the potential to provide equitable access for people living in rural or remote areas where access to health service is limited .", "for telerehabilitation to be successful , however , there must be accurate assessment and outcome measurement tools available to therapists [ 37 , 38 ] . in pr ,", "the pulse oximeter is a noninvasive device which continuously monitors a person 's peripheral blood oxygen saturation ( spo2 ) and heart rate ( hr ) .", "it is used clinically to measure a person 's exercise intensity and warn of exercise - induced desaturation .", "the concept of remote monitoring of physiological data including spo2 and hr for exercise rehabilitation purposes has received attention in the literature .", "as early as the mid-1980s , physicians were successful at remotely monitoring a patient 's electrocardiogram ( ecg ) through the analogue telephone network during a home - exercise program [ 40 , 41 ] .", "however , the majority of electronic healthcare literature on pr has focused on telemonitoring systems [ 4246 ] or consultations services [ 36 , 47 ] .", "there has been little research using e - health to deliver real - time , live supervision of patients for treatment purposes .", "this study aimed to develop and verify the use of a remote pulse oximetry system on healthy individuals . specifically , it aimed to establish the technical feasibility of transmitting pulse oximetry data remotely , to determine the validity of remote measurements when compared to conventional face to face measurements , and to evaluate the participants ' perception on the usability of the technology .", "it was hypothesised that remote pulse oximetry would not only be technically feasible but would also be valid when compared to conventional units and would be easy for participants to use .", "a favourable result will enable the development of remote pulmonary rehabilitation programs for people living in rural or remote areas .", "this study was reviewed and granted ethical clearance from the appropriate medical research ethics committee at the university of queensland .", "thirty - seven healthy individuals were recruited to participate in a single remote cardiopulmonary exercise session .", "the following exclusion criteria were applied : cardiovascular , respiratory , metabolic , or orthopaedic conditions that would prevent participation in an exercise program , chronic infectious diseases , pregnancy , and mental or physical impairment .  ", "the exercise session was conducted using the ehab telerehabilitation system ( v2.0 , uniquest , brisbane , australia ) which is a computer - based videoconferencing and remote consultation system .", "two ehab devices were used , one with the participant and the other with the therapist .", "the participant and therapist were located in separate rooms . the ehab device with the participant", "was configured to receive data from a bluetooth pulse oximeter module ( onyx ii 9560 , nonin medical inc . , plymouth , mn ) ( figure 1 ) .", "this information was transmitted by the participant device at a frequency of one hertz via an encrypted data channel to an offsite server via an active server page ( asp ) web service .", "the data was subsequently stored in a mysql database on the server . a therapist device retrieved this data via an asp web service to produce a graph of the participant 's heart rate and spo2 recordings in near real time . for this study", "the ehab telerehabilitation system utilised a local wi - fi network connection ; however , the connection speed was throttled to 128  kbit / s to ensure compatibility with the 3g / hsdpa mobile network available in remote areas of australia .  ", "after written consent was obtained , participants were taken to a room with the ehab device , remote pulse oximeter module , headset , and treadmill .", "they were asked to remove any nail polish from their index finger on the nondominant hand or jewellery which may disrupt accurate recordings .", "participants were given an information sheet with instructions on how to affix the pulse oximeter module to the index finger of their nondominant hand and how to establish a connection with a therapist in another room via the ehab device .", "once a videoconference was established , the therapist guided the participant through an exercise session .", "the modified borg score ( mbs )   was used to assess the participant 's exercise intensity . as per current copd exercise protocols", "the session began with a warm - up phase ( mbs 2/10 ) for 5 minutes before gradually progressing to a moderate / somewhat severe intensity ( mbs 4 - 5/10 ) .", "the last 5 minutes consisted of a cool - down phase ( mbs 2/10 ) . if any adverse signs were observed , the exercise session would be ceased immediately .", "interrupted sessions due to technical difficulties were noted , and those participants were asked to repeat the exercise session at a later date .   at the conclusion of the exercise session", ", participants completed a questionnaire about their experience with the remote pulse oximeter module and ehab device by marking on a 100  mm visual analogue scale ( vas ) and providing any open - ended comments .   during each session ,", "the participant ehab device logged heart rate ( hr ) and spo2 values from the pulse oximeter and time stamped the data .", "this data was saved into a database stored locally on the computer ( dataset # 1 ) .", "the participant device also transmitted the data to the remote therapist device where another time stamp was applied to enable the calculation of transmission latency .", "this data was saved into a database stored locally on the therapist device ( dataset # 2 ) .", "the mean data transmission rate was determined by measuring the bandwidth requirement ( kbit / s ) .", "the satisfaction questionnaire rated the comfort , quality , and usability of the ehab device and remote pulse oximetry module .", "the validity of remote pulse oximetry was assessed by comparing the remote measurements ( therapist data ) with conventional face - to - face measurements ( participant data ) .", "these were percentage agreement , mean absolute difference ( mad ) , and the bland and altman 's limits of agreement method [ 49 , 50 ] .", "percentage agreement was calculated as the proportion of data successfully transferred between participant and therapist .", "the limits of agreement method enabled the quantification of the agreement between the two data sets in terms of the clinical significance of the transmission error .", "specifically , it gave the upper and lower bounds which show the 95% confidence interval for the difference between the remote and conventional measurements ( i.e. , mean  1.96  sd ) .", "when applying the limits of agreement method comparing the two datasets , the average of 10 second intervals of data was used .   in terms of clinical significance , the minimum clinically important difference ( mcid ) was used as a threshold value to represent meaningful and worthwhile change .", "remote measurements were classified as valid if the measurement error was lower than the mcid .", "the mcid for hr , however , varies depending on the individual 's exercise tolerance . for the purpose of this study ,", "the manufacturer 's device accuracy of 3 beats per minute ( bpm ) was used as the mcid , which was a conservative estimate .", "data was analysed using statistical package for the social sciences ( v17.0 spss inc . , chicago , il ) . a p value of < 0.05 was considered to be significant , where applicable .", "thirty - seven healthy individuals ( 14 males and 23 females ) completed a total of 15 exercise hours .", "participants had a mean age of 30 years and standard deviation of 15 years ( range : 18 to 62 ) .", "there were 34 successfully completed sessions on first attempt confirming the feasibility of the method , while three had to be repeated due to technical difficulties with the bluetooth or wifi connection .", "the mean ( sd ) transmission latency of the pulse oximetry data ( not the videoconference ) between the computers was 2.94 ( 0.59 ) seconds .", "the mean ( sd ) bandwidth usage for data transmitted was 6.5 ( 1.0 ) kbit / s .", "of the remaining data , 9.0% was found to be omitted from the therapist device , while 11% was found to consist of duplicated numbers .", "the mad and limits of agreement between participant and therapist data are shown in table 1 .  ", "overall , participants rated their experience with the remote pulse oximeter and ehab device highly .", "the mean response to each of the eight questionnaire items is shown in figure 2 .", "positive written comments included the following :  i would be comfortable using [ this device ] at home ", "( n = 4 ) and  [ the ehab was ] simple and easy to use ", "negative comments included worried [ remote pulse oximeter ] could dislodge during vigorous exercise ( n = 5 ) , remote pulse oximeter was uncomfortable ( n = 4 ) , and audio or video quality was  jumpy  ( n = 3 ) .", "however , inconvenience , distance , or availability of pr programs can lead to lack of participation and poor compliance .", "this study demonstrated the feasibility of a remote pulse oximeter for use during a pr program .", "remote measurements of spo2 and hr were valid when compared to conventional face - to - face measurements .", "these findings provide confidence for the development of a remote pr program .   in our study", ", there was an unexpectedly high mean rate of data omission ( 9.0% ) and duplication ( 11% ) between participant and therapist data .", "although these error rates appear comparatively high , they do not necessarily indicate a failure in the data transmission .", "rather , the logging of data points by the participant device appears to not be perfectly synchronized with data retrieval from the therapist device .", "this may be a function of the variable delays that can be present with web service calls which were used in this study to transmit data .", "asynchrony in web service responses may have resulted in either repetition of data or omission of single values . moreover", ", a visual inspection of the data confirms a random distribution of error throughout the data file . despite this asynchrony ,", "the mad analysis revealed a small difference ( spo2 0.04% ; hr 0.21  bpm ) in comparison to the mcid of 4% for spo2 and 3  bpm for hr .", "similarly , the limits of agreement demonstrated a small difference in comparison to the mcid for spo2 and hr .", "the upper and lower bounds for spo2 were 0.67 to 0.67% and for hr were 2.90 to 2.89  bpm .", "both are within the mcid , and this provides further evidence for the validity of remote measurements . unlike mad , which analyses the actual difference , the limits of agreement give a conservative estimation of difference through a 95% confidence interval .", "clinically , it means 95% of the time of the difference due to measurement error will be within the relevant limits for spo2 and hr .", "the mad and limits of agreement values show that remote measurements are sufficiently accurate and valid for real - time monitoring purposes .", "overall , as indicated by questionnaire responses , participants found the remote pulse oximeter easy to use ( mean vas 89/100 ) and felt confident that they would be able to use it at home ( mean vas 87/100 ) .", "this is similar to literature which has found the ehab device user friendly [ 5557 ] .", "however , some participants ( n = 4 ) stated that the remote pulse oximeter was uncomfortable . unlike a conventional pulse oximeter , the remote module has the sensor , display and battery unit all incorporated on the fingertip ( see figure 1 ) .", "this creates additional weight compared to a conventional sensor , and it could be a contributing factor to the feeling of instability experience by these participants .", "future studies should compare the comfort with other remote pulse oximeters that have the display and battery unit located at the wrist .", "furthermore , this needs to be investigated in the copd population as they may be more accustomed to pulse oximetry than healthy adults .", "five participants commented that they were worried the remote pulse oximeter could dislodge during vigorous exercise .", "this could be a result of the weight of the device and high intensity of exercise , where most participants needed to jog or run in order to reach an mbs of 5/10 .  ", "problems with video and audio quality have been identified as key issues in telehealth [ 5860 ] . during testing", ", there were occasional disruptions in audio and video transmissions during the exercise session , and on three occasions , the videoconference unexpectedly exited due to connectivity issues on the wifi network , requiring the call to be reestablished .", "the low frame rate is likely due to the low bandwidth available ( 128  kbit / s ) on the network connection .", "however , it should be noted that the remote pulse oximeter only uses a very small bandwidth ( 6.5  kbit / s ) for data transmission and , therefore , is unlikely to have attributed to the reduced video / audio quality .  ", "there were several limitations of this study ( 1 ) our sample size was small ( n = 37 ) and consisted primarily of young , healthy university students ( mean age 30 years ) .", "our healthy participants did not show any clinical changes ( e.g. , exercise - induced desaturation ) , and this may have affected the magnitude of error in mad and limits of agreement .", "furthermore , the young population may be more confident to use or learn about new technology , and this may have therefore influenced the result of the satisfaction questionnaire .", "( 2 ) this study was conducted within the university clinic . although the participant and therapist were in separate rooms , they were in close proximity to each other , with the therapist on call if any difficulties occurred .", "whist the ehab system was bandwidth throttled to ensure compatibility with the 3g / hsdpa mobile network , other factors such as mobile network coverage , signal strength , and speed were not assessed , and this may have affected the performance and the latency of transmission .  ", "future studies should include a trial of the remote pulse oximetry system in the home environment with a copd population on the 3g / hsdpa mobile telephone network .", "a large sample size is required to more rigorously assess the validity and reliability of data transmissions .", "furthermore , it has been shown that patients demonstrate more positive views of telecare encounters than their healthcare providers .", "a therapist questionnaire is therefore required to assess their satisfaction and confidence in safely conducting a remote exercise session .", "our pilot study demonstrated that remote pulse oximetry was feasible , and the data transmission method produced valid data at the remote end when compared with conventional face - to - face measures .", "although this study was performed on healthy individuals , participants found the ehab device easy to operate and were confident it could be used at home .", "further studies are needed to address the technical feasibility and validity in a home environment and the therapist 's confidence in conducting a safe exercise program ."], "abstract_text": ["<S> pulmonary rehabilitation is an effective treatment for people with chronic obstructive pulmonary disease . </S>", "<S> however , access to these services is limited especially in rural and remote areas . </S>", "<S> telerehabilitation has the potential to deliver pulmonary rehabilitation programs to these communities . </S>", "<S> the aim of this study was threefold : to establish the technical feasibility of transmitting real - time pulse oximetry data , determine the validity of remote measurements compared to conventional face - to - face measures , and evaluate the participants ' perception of the usability of the technology . </S>", "<S> thirty - seven healthy individuals participated in a single remote pulmonary rehabilitation exercise session , conducted using the ehab telerehabilitation system . </S>", "<S> validity was assessed by comparing the participant 's oxygen saturation and heart rate with the data set received at the therapist 's remote location . </S>", "<S> there was an 80% exact agreement between participant and therapist data sets . </S>", "<S> the mean absolute difference and bland and altman 's limits of agreement fell within the minimum clinically important difference for both oxygen saturation and heart rate values . </S>", "<S> participants found the system easy to use and felt confident that they would be able to use it at home . </S>", "<S> remote measurement of pulse oximetry data for a pulmonary rehabilitation exercise session was feasible and valid when compared to conventional face - to - face methods . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["chronic obstructive pulmonary disease ( copd ) is a progressive lung disease which is characterized by airway obstruction and lung parenchyma destruction .", "patients may experience dyspnoea or shortness of breath , a persistent cough with sputum production , decreased exercise tolerance and decreased quality of life [ 1 , 2 ] . globally , copd is the fourth most common cause of death and is predicted by the world health organisation to be a major health concern in the coming decade . in australia ,", "1.2 million people are estimated to have moderate - to - severe copd ( gold stages ii ", "iv ) . however , the true number is potentially much higher as copd is commonly underdiagnosed [ 4 , 5 ] .", "the total cost of living with copd ( including nonmonetary costs ) is estimated to be $ 83  000 aud per person each year [ 6 , 7 ] .  ", "as the severity of copd greatly impacts the cost of care , providing early diagnosis and effective management of the individual is an essential component in reducing its economic impact [ 5 , 810 ] .", "pr is a multidisciplinary approach which incorporates self - management education with exercise training , psychosocial and nutritional support [ 11 , 12 ] .", "it is a fundamental part of managing people with copd and has been shown to be effective in improving exercise tolerance , reducing dyspnoea , improving , quality of life and reducing the severity of acute exacerbations [ 1 , 1117 ] .", "although pr has been shown to be effective in managing symptoms and reducing hospital admissions , poor participation and adherence are a current problem among outpatient programs .", "factors include difficulty travelling to the program 's location [ 1922 ] , inconvenience of hospital attendance , or difficulty accessing programs .", "australia is one of the least densely populated nations ( 2.7 people per square kilometre ) with up to 11.8% of the population residing in outer regional or remote locations .", "conducted a study in kyabram ( rural victoria ) to assess the efficacy of their pr program and noted that patients who lived outside the town centre were more likely to withdraw from pr as the program was conducted within the town hospital .", "this finding is also supported by de angelis and colleagues   who found that transport , travel distance , and related costs were the main reason for nonattendance of a cardiac rehabilitation program .  ", "difficulty in accessing outpatient programs has led to the development of home - based programs .", "it is well established that home - based pr programs are safe and effective [ 13 , 2931 ] and are recommended by many international guidelines and reviews [ 2 , 11 , 12 , 17 ] . given the effectiveness of independent home - based pr programs one could ask what additional benefits supervised remote - based programs would provide .", "the main advantage of supervised remote - based programs is the ability to minimise anxiety in both patient and therapist .", "this benefit has been shown in remote - based cardiac exercise programs where the use of transtelephonic exercise monitoring ( tem ) has been used to relieve patients ' and trainers ' anxiety during exercise [ 3234 ] .", "it is anticipated that a person with copd could exercise to a higher intensity without the risk of undetected adverse events ( e.g. , exercise - induced oxygen desaturation ) .  ", "telerehabilitation is an emerging technology used to facilitate the delivery of rehabilitation services at a distance by combining telecommunication and electronic transmission of health information .", "it has the potential to provide equitable access for people living in rural or remote areas where access to health service is limited .", "for telerehabilitation to be successful , however , there must be accurate assessment and outcome measurement tools available to therapists [ 37 , 38 ] . in pr ,", "the pulse oximeter is a noninvasive device which continuously monitors a person 's peripheral blood oxygen saturation ( spo2 ) and heart rate ( hr ) .", "it is used clinically to measure a person 's exercise intensity and warn of exercise - induced desaturation .", "the concept of remote monitoring of physiological data including spo2 and hr for exercise rehabilitation purposes has received attention in the literature .", "as early as the mid-1980s , physicians were successful at remotely monitoring a patient 's electrocardiogram ( ecg ) through the analogue telephone network during a home - exercise program [ 40 , 41 ] .", "however , the majority of electronic healthcare literature on pr has focused on telemonitoring systems [ 4246 ] or consultations services [ 36 , 47 ] .", "there has been little research using e - health to deliver real - time , live supervision of patients for treatment purposes .", "this study aimed to develop and verify the use of a remote pulse oximetry system on healthy individuals . specifically , it aimed to establish the technical feasibility of transmitting pulse oximetry data remotely , to determine the validity of remote measurements when compared to conventional face to face measurements , and to evaluate the participants ' perception on the usability of the technology .", "it was hypothesised that remote pulse oximetry would not only be technically feasible but would also be valid when compared to conventional units and would be easy for participants to use .", "a favourable result will enable the development of remote pulmonary rehabilitation programs for people living in rural or remote areas ."], ["this study was reviewed and granted ethical clearance from the appropriate medical research ethics committee at the university of queensland .", "thirty - seven healthy individuals were recruited to participate in a single remote cardiopulmonary exercise session .", "the following exclusion criteria were applied : cardiovascular , respiratory , metabolic , or orthopaedic conditions that would prevent participation in an exercise program , chronic infectious diseases , pregnancy , and mental or physical impairment .  ", "the exercise session was conducted using the ehab telerehabilitation system ( v2.0 , uniquest , brisbane , australia ) which is a computer - based videoconferencing and remote consultation system .", "two ehab devices were used , one with the participant and the other with the therapist .", "the participant and therapist were located in separate rooms . the ehab device with the participant", "was configured to receive data from a bluetooth pulse oximeter module ( onyx ii 9560 , nonin medical inc . , plymouth , mn ) ( figure 1 ) .", "this information was transmitted by the participant device at a frequency of one hertz via an encrypted data channel to an offsite server via an active server page ( asp ) web service .", "the data was subsequently stored in a mysql database on the server . a therapist device retrieved this data via an asp web service to produce a graph of the participant 's heart rate and spo2 recordings in near real time . for this study", "the ehab telerehabilitation system utilised a local wi - fi network connection ; however , the connection speed was throttled to 128  kbit / s to ensure compatibility with the 3g / hsdpa mobile network available in remote areas of australia .  ", "after written consent was obtained , participants were taken to a room with the ehab device , remote pulse oximeter module , headset , and treadmill .", "they were asked to remove any nail polish from their index finger on the nondominant hand or jewellery which may disrupt accurate recordings .", "participants were given an information sheet with instructions on how to affix the pulse oximeter module to the index finger of their nondominant hand and how to establish a connection with a therapist in another room via the ehab device .", "once a videoconference was established , the therapist guided the participant through an exercise session .", "the modified borg score ( mbs )   was used to assess the participant 's exercise intensity . as per current copd exercise protocols", "the session began with a warm - up phase ( mbs 2/10 ) for 5 minutes before gradually progressing to a moderate / somewhat severe intensity ( mbs 4 - 5/10 ) .", "the last 5 minutes consisted of a cool - down phase ( mbs 2/10 ) . if any adverse signs were observed , the exercise session would be ceased immediately .", "interrupted sessions due to technical difficulties were noted , and those participants were asked to repeat the exercise session at a later date .   at the conclusion of the exercise session", ", participants completed a questionnaire about their experience with the remote pulse oximeter module and ehab device by marking on a 100  mm visual analogue scale ( vas ) and providing any open - ended comments .   during each session ,", "the participant ehab device logged heart rate ( hr ) and spo2 values from the pulse oximeter and time stamped the data .", "this data was saved into a database stored locally on the computer ( dataset # 1 ) .", "the participant device also transmitted the data to the remote therapist device where another time stamp was applied to enable the calculation of transmission latency .", "this data was saved into a database stored locally on the therapist device ( dataset # 2 ) .", "the mean data transmission rate was determined by measuring the bandwidth requirement ( kbit / s ) .", "the satisfaction questionnaire rated the comfort , quality , and usability of the ehab device and remote pulse oximetry module .", "the validity of remote pulse oximetry was assessed by comparing the remote measurements ( therapist data ) with conventional face - to - face measurements ( participant data ) .", "these were percentage agreement , mean absolute difference ( mad ) , and the bland and altman 's limits of agreement method [ 49 , 50 ] .", "percentage agreement was calculated as the proportion of data successfully transferred between participant and therapist .", "the limits of agreement method enabled the quantification of the agreement between the two data sets in terms of the clinical significance of the transmission error .", "specifically , it gave the upper and lower bounds which show the 95% confidence interval for the difference between the remote and conventional measurements ( i.e. , mean  1.96  sd ) .", "when applying the limits of agreement method comparing the two datasets , the average of 10 second intervals of data was used .   in terms of clinical significance , the minimum clinically important difference ( mcid ) was used as a threshold value to represent meaningful and worthwhile change .", "remote measurements were classified as valid if the measurement error was lower than the mcid .", "the mcid for hr , however , varies depending on the individual 's exercise tolerance . for the purpose of this study ,", "the manufacturer 's device accuracy of 3 beats per minute ( bpm ) was used as the mcid , which was a conservative estimate .", "data was analysed using statistical package for the social sciences ( v17.0 spss inc . , chicago , il ) . a p value of < 0.05 was considered to be significant , where applicable ."], ["thirty - seven healthy individuals ( 14 males and 23 females ) completed a total of 15 exercise hours .", "participants had a mean age of 30 years and standard deviation of 15 years ( range : 18 to 62 ) .", "there were 34 successfully completed sessions on first attempt confirming the feasibility of the method , while three had to be repeated due to technical difficulties with the bluetooth or wifi connection .", "the mean ( sd ) transmission latency of the pulse oximetry data ( not the videoconference ) between the computers was 2.94 ( 0.59 ) seconds .", "the mean ( sd ) bandwidth usage for data transmitted was 6.5 ( 1.0 ) kbit / s .", "of the remaining data , 9.0% was found to be omitted from the therapist device , while 11% was found to consist of duplicated numbers .", "the mad and limits of agreement between participant and therapist data are shown in table 1 .  ", "overall , participants rated their experience with the remote pulse oximeter and ehab device highly .", "the mean response to each of the eight questionnaire items is shown in figure 2 .", "positive written comments included the following :  i would be comfortable using [ this device ] at home ", "( n = 4 ) and  [ the ehab was ] simple and easy to use ", "negative comments included worried [ remote pulse oximeter ] could dislodge during vigorous exercise ( n = 5 ) , remote pulse oximeter was uncomfortable ( n = 4 ) , and audio or video quality was  jumpy  ( n = 3 ) ."], ["however , inconvenience , distance , or availability of pr programs can lead to lack of participation and poor compliance .", "this study demonstrated the feasibility of a remote pulse oximeter for use during a pr program .", "remote measurements of spo2 and hr were valid when compared to conventional face - to - face measurements .", "these findings provide confidence for the development of a remote pr program .   in our study", ", there was an unexpectedly high mean rate of data omission ( 9.0% ) and duplication ( 11% ) between participant and therapist data .", "although these error rates appear comparatively high , they do not necessarily indicate a failure in the data transmission .", "rather , the logging of data points by the participant device appears to not be perfectly synchronized with data retrieval from the therapist device .", "this may be a function of the variable delays that can be present with web service calls which were used in this study to transmit data .", "asynchrony in web service responses may have resulted in either repetition of data or omission of single values . moreover", ", a visual inspection of the data confirms a random distribution of error throughout the data file . despite this asynchrony ,", "the mad analysis revealed a small difference ( spo2 0.04% ; hr 0.21  bpm ) in comparison to the mcid of 4% for spo2 and 3  bpm for hr .", "similarly , the limits of agreement demonstrated a small difference in comparison to the mcid for spo2 and hr .", "the upper and lower bounds for spo2 were 0.67 to 0.67% and for hr were 2.90 to 2.89  bpm .", "both are within the mcid , and this provides further evidence for the validity of remote measurements . unlike mad , which analyses the actual difference , the limits of agreement give a conservative estimation of difference through a 95% confidence interval .", "clinically , it means 95% of the time of the difference due to measurement error will be within the relevant limits for spo2 and hr .", "the mad and limits of agreement values show that remote measurements are sufficiently accurate and valid for real - time monitoring purposes .", "overall , as indicated by questionnaire responses , participants found the remote pulse oximeter easy to use ( mean vas 89/100 ) and felt confident that they would be able to use it at home ( mean vas 87/100 ) .", "this is similar to literature which has found the ehab device user friendly [ 5557 ] .", "however , some participants ( n = 4 ) stated that the remote pulse oximeter was uncomfortable . unlike a conventional pulse oximeter , the remote module has the sensor , display and battery unit all incorporated on the fingertip ( see figure 1 ) .", "this creates additional weight compared to a conventional sensor , and it could be a contributing factor to the feeling of instability experience by these participants .", "future studies should compare the comfort with other remote pulse oximeters that have the display and battery unit located at the wrist .", "furthermore , this needs to be investigated in the copd population as they may be more accustomed to pulse oximetry than healthy adults .", "five participants commented that they were worried the remote pulse oximeter could dislodge during vigorous exercise .", "this could be a result of the weight of the device and high intensity of exercise , where most participants needed to jog or run in order to reach an mbs of 5/10 .  ", "problems with video and audio quality have been identified as key issues in telehealth [ 5860 ] . during testing", ", there were occasional disruptions in audio and video transmissions during the exercise session , and on three occasions , the videoconference unexpectedly exited due to connectivity issues on the wifi network , requiring the call to be reestablished .", "the low frame rate is likely due to the low bandwidth available ( 128  kbit / s ) on the network connection .", "however , it should be noted that the remote pulse oximeter only uses a very small bandwidth ( 6.5  kbit / s ) for data transmission and , therefore , is unlikely to have attributed to the reduced video / audio quality .  ", "there were several limitations of this study ( 1 ) our sample size was small ( n = 37 ) and consisted primarily of young , healthy university students ( mean age 30 years ) .", "our healthy participants did not show any clinical changes ( e.g. , exercise - induced desaturation ) , and this may have affected the magnitude of error in mad and limits of agreement .", "furthermore , the young population may be more confident to use or learn about new technology , and this may have therefore influenced the result of the satisfaction questionnaire .", "( 2 ) this study was conducted within the university clinic . although the participant and therapist were in separate rooms , they were in close proximity to each other , with the therapist on call if any difficulties occurred .", "whist the ehab system was bandwidth throttled to ensure compatibility with the 3g / hsdpa mobile network , other factors such as mobile network coverage , signal strength , and speed were not assessed , and this may have affected the performance and the latency of transmission .  ", "future studies should include a trial of the remote pulse oximetry system in the home environment with a copd population on the 3g / hsdpa mobile telephone network .", "a large sample size is required to more rigorously assess the validity and reliability of data transmissions .", "furthermore , it has been shown that patients demonstrate more positive views of telecare encounters than their healthcare providers .", "a therapist questionnaire is therefore required to assess their satisfaction and confidence in safely conducting a remote exercise session ."], ["our pilot study demonstrated that remote pulse oximetry was feasible , and the data transmission method produced valid data at the remote end when compared with conventional face - to - face measures .", "although this study was performed on healthy individuals , participants found the ehab device easy to operate and were confident it could be used at home .", "further studies are needed to address the technical feasibility and validity in a home environment and the therapist 's confidence in conducting a safe exercise program ."]]}
{"article_id": "PMC3817649", "article_text": ["\n acute mountain sickness ( ams ) occurs during exposure to high altitude ( ha ) and is a clinical syndrome characterised by headache , insomnia , malaise , and gastrointestinal symptoms .", "it is common , developing in 1030% at 25003000 meters   and in up to 60% of those ascending to around 4500 meters .", "it causes significant morbidity and is a challenging clinical condition in remote environments . a biochemical marker of ams , particularly one available as a point - of - care test ( poc ) , could have widespread clinical utility .  ", "the pathophysiology of ams is not clearly understood but involves alterations in fluid balance , endothelial function , vascular permeability , inflammation , and oxidative stress .", "the renal response to ha is an important factor in acclimatization , and ha exposure leads to renal arteriole constriction and relative hypoxia [ 3 , 4 ] . despite the relative renal hypoxia ,", "ngal ( neutrophil gelatinase - associated lipocalin ) is a 25  kda peptide , part of the lipocalin family of small soluble proteins .", "it is produced in a number of human tissues , notably the distal nephron but also in the lung   ngal rises rapidly in the nephron in response to a renal insult and an ngal 150  ng / ml following acute kidney injury ( aki ) is predictive of acute renal failure ( arf ) well before creatinine has risen .", "ngal is also an acute - phase protein , has a role in inflammation [ 8 , 9 ] , and is upregulated in the lung during inflammation [ 5 , 10 , 11 ] .", "ngal is also known to rise in conditions associated with oxidative stress [ 12 , 13 ] , and oxidative stress has been implicated in ams [ 14 , 15 ] .  ", "we therefore hypothesised that ngal would increase at ha secondary to these various stimuli and that the magnitude of any increase might relate to the presence of ams .", "we therefore studied a combined cohort of trekkers from 2 expeditions to ha . in order to clarify the relative contribution of ams , hypoxia or exercise to ngal levels", ", we also studied a cohort pre- and postexercise at near sea level , and a further cohort exposed to acute normobaric hypoxia .", "the potential role of inflammation in stimulating ngal was assessed by the measurement of highly sensitive c - reactive protein ( hscrp ) in a subset of participants .", "all study protocols were approved by the ministry of defence research ethics committee , whitehall , uk , and satisfied the requirements of the declaration of helsinki . in all studies informed ,", "thirty - two subjects participating in a defence medical services ( dms ) trekking expedition ( trek 1 ) in the khumbu region of nepal were studied .", "blood samples were taken from the antecubital fossa at 3 study altitudes : on day 2 at 3400  m , day 6 at 4270  m , and day 10 at 5150  m ( following ascent to everest base camp at 5364  m ) . all samples in this study", "were collected immediately following a day trekking (  posttrek  ) to the study altitude .", "twenty subjects from a further dms expedition ( trek 2 ) to nepal were also studied .", "blood samples were again taken at 3 study altitudes : on day 2 ( 3400  m ) , day 6 ( 4270  m ) , and day 10 ( 5150  m ) ( following ascent to kala patthar ( kp ) , 5643  m ) .", "samples in this study were again collected immediately following a day trekking (  posttrek  ) .", "additional samples were taken at rest in kathmandu ( kat ) at 1300  m and at rest the next morning at the 3 study altitudes .", "as serving members of the military , all subjects were able to fulfil the fitness criteria of their relevant service .", "this broadly includes an age - adjusted ability to run 1.5 miles in under approximately 11 minutes and to perform an age - adjusted number of sits - ups and push - ups within two blocks of 2 minutes .", "fourteen subjects underwent a 3-hour exposure to normobaric hypoxia ( fio2 11.6% , equivalent to 4800  m altitude ) in a hypoxic chamber .", "this exposure included a 5-minute step test ( step height of 25  cm , 1 complete step every 2 seconds ) at 95 minutes .", "a group of 22 subjects had ngal assayed at rest and after exercise at sl in the uk following ascent from sea level to 1085  m over 6 hours ( an equivalent gain in altitude and duration of exercise similar to that experienced on a trekking day in nepal ) .", "two subjects from trek 2 were part of the sl exercise group , but data collection occurred several months apart .  ", "ngal was analysed in the field on a biosite triage point of care monitor ( alere ltd , stockport , uk ) using a triage ngal test kit .", "the triage ngal test is a point - of - care , fluorescence - based immunoassay used which gives a rapid ( 15 minutes ) quantitative measurement of ngal in a range from 60 to 1,300  ng / ml .  ", "oxygen saturation ( digitally on warm hands at rest ) was measured using a nellcor np-20 pulse oximeter ( covidian , ma , usa ) during trek 1 + 2 and in the hypoxic chamber study at the same time as blood samples were taken . during trek 1 + 2 ,", "twice - daily ams scores were assessed using the lake louise score ( lls ) questionnaire .", "the lls allocates a score of 0 to 3 ( symptom not present to severe ) for symptoms of ams ( headache , gastrointestinal symptoms , fatigue / weakness , dizzy / light - headedness , and difficulty sleeping ) .", "a score of 3 or more in the presence of headache is consistent with ams , a score of 6 or more with severe ams .  ", "the commercially available , highly sensitive , immunoturbidimetric assay ( roche diagnostics ) was used to measure crp in trek 2 at the same time points as ngal .", "mg / l and a between - run coefficient of variation between 2.5 and 5.7% . for statistical calculations ,", "the software package spss 14.0 was used . for subjects with a ngal below the limit of detection of the assay ( 60  ng / ml ) ,", "a value of 60  ng / ml was assigned for the purposes of statistical analysis .", "all data were tested for gaussian distribution using the kolmogorov - smirnov test and shapiro wilks statistic .   for the analysis of dependent variables that were normally distributed , changes were tested by student 's paired t - test . for independent variables that were normally distributed ,", "a within - subjects anova was performed to investigate any serial changes in ngal with ascent at rest and post - trek . a two - way mixed anova with either resting or after trek ngal at each study altitude as the within - subjects factor and the presence of ams ( according to the ll score at multiple altitudes ) as the between - subjects factor", "if the mauchly sphericity test was significant , then p values were expressed after multiplication by the greenhouse - geisser epsilon .", "correlation analyses for normally distributed data were performed by calculating the pearson coefficient of correlation .", "a p value < 0.05 ( two - sided ) was considered significant . as the ascent profile and route were closely matched in trek 1 and trek 2 , data were combined and analysed as a whole . taking medication ( acetazolamide , dexamethasone )", "had no apparent effect on ngal values , and therefore these subjects ( n = 11 ) were not excluded from the analysis .", "demographic data for the field study ( trek 1 + 2 ) , the controls , and the hypoxic chamber study are shown in table 1 . in the 22 subjects ascending to 1085  m in the uk", ", there was no significant ( p = 0.084 ) rise in ngal following exercise : resting sl ngal was 64  11 ( ng / ml , mean  sd , range 60104 ) and postexercise ngal was 71  14 ( ng / ml , mean  sd , range 60100 ) . of the 52 subjects ,", "spo2 ( % , mean  sem ) dropped from 97  2 at kat ( 1300  m ) to 84  5 and 79  7 at 4270 and 5150  m , respectively ( p < 0.001 ) .", "there was a moderate inverse correlation between ngal and spo2 at 5150  m ( r = 0.477 , p = 0.001 ) ( figure 1 ) with a weaker inverse correlation between ngal and spo2 at 4270  m ( r = 0.340 , p = 0.019 ) .", "within the subjects , anova demonstrated a significant change in ngal with ascent both at rest ( p = 0.007 ) and after trek ( p = 0.001 ) ( figure 2 ) .", "spo2 ( % , mean  sem ) dropped from 99  0.4 at baseline to a nadir of 79  5 ( p < 0.001 ) . despite an equivalent drop in spo2 to", "that seen in trek 1 + 2 , ngal ( ng / ml , mean  sd , range ) showed no change between baseline and 180 minutes : 63  26 ( 2980 ) versus 67  25 ( 2784 ) , p = 0.538 . in trek 2 , serum creatinine { mol / l , mean  sem , ( range ) , ( p value versus baseline at kat ) } was 78  2 ( 6395 ) at baseline ; at 3400 , 4270 and 5150  m it was 87  3 ( 72120 ) ( p = 0.001 ) ; 84  2 ( 72104 ) ( p < 0.001 ) ; and 94  5 ( 76142 ) ( p < 0.001 ) .", "according to their ll scores at the highest study altitude ( 5150  m ) , there were 23 subjects with no ams , 16 subjects with mild ams , and 7 subjects with severe ams .", "there was a significant difference between ngal depending on the presence or absence of ams at 5150  m ( figure 3 ) with higher values in those with ams and severe ams . a two - way mixed anova revealed a significant change ( p = 0.003 ) in resting ngal with ascent and an interaction with ams at 4270  m ( p = 0.017 ) and 4910  m ( p = 0.002 for change in ngal , p = 0.027 for interaction with ams ) .  ", "hscrp was ( mean  sem , range ) : 1.6  0.4 ( 0.337.53 ) at baseline ; at 3400  m , 4270  m , and 5150  m post - trek : 7  2.9 ( 0.7847.93 ) ( p = 0.002 versus baseline ) ; 25.7  8.1 ( 0.58104 ) ( p < 0.001 versus baseline ) ; 9  3.2 ( 0.5644.62 ) ( p = 0.003 versus baseline ) . at 3400  m , 4270  m , and 5150  m at rest : 6.2  2.9 ( 1.8725.1 ) ( p = 0.001 versus baseline ) ; 21.6  5.7 ( 0.4983.9 ) ( p < 0.001 versus baseline ) ; and 5.8  2.1 ( 0.5426.59 ) ( p = 0.012 versus baseline ) .", "ngal at 5150  m , after trek was moderately correlated with hscrp at 5100  m after exercise ( rho 0.526 , p = 0.036 ) .", "this is the first report to describe an association between ngal and both the presence and severity of ams at ha .", "the significant novel findings are that ngal rises in response to sustained hypobaric hypoxia but not acute normobaric hypoxia or near sl exercise and that this rise is related to ams at 5150  m.   the rise in ngal following trekking ( by day 2 at 3400  m ) was to the levels normally associated with the subsequent development of arf ( > 150  ng / ml ) , but this did not occur .", "although creatinine rose significantly with altitude , the rise was very modest , and we suspect that a combination of factors other than a simple renal insult is responsible for the increase in ngal at ha .  ", "our data suggest an inverse correlation between spo2 and ngal at 5150  m ( and to a lesser extent at 4270  m ) . although no such correlation was found at 3400  m , this may still suggest that prolonged renal hypoxia could be a significant drive to ngal release .", "in addition to renal hypoxia , we suspect that other factors may also contribute to the rise in ngal at ha .   the significantly greater ngal in those with severe or mild ams versus those without at 5150 ", "indeed , ngal is an acute - phase protein   with a role in inflammation [ 8 , 10 , 11 ] .", "exercise stimulates an immune response , and hypoxia is also known to cause a response in immune and endothelial cells with inflammatory markers such as hscrp increasing with ha [ 1922 ] .", "consistent with this , we saw a significantly higher hscrp at all altitudes compared to baseline .", "limited data have suggested hscrp may be associated with ams   but we did not demonstrate any evidence to support this .", "there was a weak correlation between hscrp and ngal at 5150  m but this can not explain the rise in ngal as a whole .  ", "ngal also rises with oxidative stress [ 9 , 12 ] which is increased by exercise , and ha - induced oxidative stress   has been implicated in ams . as such , it is interesting to note that we found a higher ngal following trekking and in those with ams at the highest altitude .   in an attempt to clarify the relative influence of exercise and hypoxia on ngal , we measured ngal before and after exercise of a similar duration ( 6  hrs ) and similar incremental altitude ( 1085  m ) as that experienced daily in nepal and also in a hypoxic chamber . in neither scenario did ngal rise .", "this may reflect inadequate duration or severity of stimulus but may also reflect that the ngal response is not due to exercise or hypoxia alone but is multifactorial involving hypoxia , oxidative stress , an inflammatory response , and other , as yet unidentified , stimuli .", "we also acknowledge limitations such as a lack of serum markers of oxidative stress and a lack of resting ngal data in trek 1 .", "in addition , we did not measure ngal at sl before departure to nepal , although the ngal recorded as a baseline at kat ( 1300  m ) ( 68  ng / ml ) was no different to those recorded at sl in the uk ( 63 and 64  ng / ml ) .", "we also acknowledge the fact that although we measured creatinine in trek 2 , we did not continue to monitor it after the cessation of trekking . as a consequence of creatinine rising more slowly in response to a renal insult than ngal ,", "in conclusion , there are several interesting and novel findings that are worthy of further exploration .", "ngal rises in response to prolonged hypobaric hypoxia ; marked increases in ngal may occur without concomitant arf and the degree of ngal rise at ha is associated with the presence or absence of ams .", "the fact that ngal does not appear to rise secondary to acute normobaric hypoxia or exercise in isolation suggests that the rise at ha and relation with ams may have common pathways , perhaps related to prolonged hypoxia and an inflammatory response . with the huge and increasing popularity of recreational sports undertaken at both moderate and high altitude", "assessment of ngal takes a matter of minutes using poc testing , and its use in identifying ams requires further evaluation ."], "abstract_text": ["<S> acute mountain sickness ( ams ) is a common clinical challenge at high altitude ( ha ) . a point - of - care biochemical marker for ams could have widespread utility . </S>", "<S> neutrophil gelatinase - associated lipocalin ( ngal ) rises in response to renal injury , inflammation and oxidative stress . </S>", "<S> we investigated whether ngal rises with ha and if this rise was related to ams , hypoxia or exercise . </S>", "<S> ngal was assayed in a cohort ( n = 22 ) undertaking 6 hours exercise at near sea - level ( sl ) ; a cohort ( n = 14 ) during 3 hours of normobaric hypoxia ( fio2 11.6% ) and on two trekking expeditions ( n = 52 ) to over 5000  m. ngal did not change with exercise at sl or following normobaric hypoxia . during the trekking expeditions ngal levels ( ng / ml , mean  sd , range ) rose significantly ( p < 0.001 ) from 68  14 ( 60102 ) at 1300  m to 183  107 ( 65519 ) ; 143  66 ( 60315 ) and 150  71 ( 60357 ) at 3400  m , 4270  m and 5150  m respectively . </S>", "<S> at 5150  m there was a significant difference in ngal between those with severe ams ( n = 7 ) , mild ams ( n = 16 ) or no ams ( n = 23 ) : 201  34 versus 171  19 versus 124  12 respectively ( p = 0.009 for severe versus no ams ; p = 0.026 for mild versus no ams ) . in summary , </S>", "<S> ngal rises in response to prolonged hypobaric hypoxia and demonstrates a relationship to the presence and severity of ams . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["\n acute mountain sickness ( ams ) occurs during exposure to high altitude ( ha ) and is a clinical syndrome characterised by headache , insomnia , malaise , and gastrointestinal symptoms .", "it is common , developing in 1030% at 25003000 meters   and in up to 60% of those ascending to around 4500 meters .", "it causes significant morbidity and is a challenging clinical condition in remote environments . a biochemical marker of ams , particularly one available as a point - of - care test ( poc ) , could have widespread clinical utility .  ", "the pathophysiology of ams is not clearly understood but involves alterations in fluid balance , endothelial function , vascular permeability , inflammation , and oxidative stress .", "the renal response to ha is an important factor in acclimatization , and ha exposure leads to renal arteriole constriction and relative hypoxia [ 3 , 4 ] . despite the relative renal hypoxia ,", "ngal ( neutrophil gelatinase - associated lipocalin ) is a 25  kda peptide , part of the lipocalin family of small soluble proteins .", "it is produced in a number of human tissues , notably the distal nephron but also in the lung   ngal rises rapidly in the nephron in response to a renal insult and an ngal 150  ng / ml following acute kidney injury ( aki ) is predictive of acute renal failure ( arf ) well before creatinine has risen .", "ngal is also an acute - phase protein , has a role in inflammation [ 8 , 9 ] , and is upregulated in the lung during inflammation [ 5 , 10 , 11 ] .", "ngal is also known to rise in conditions associated with oxidative stress [ 12 , 13 ] , and oxidative stress has been implicated in ams [ 14 , 15 ] .  ", "we therefore hypothesised that ngal would increase at ha secondary to these various stimuli and that the magnitude of any increase might relate to the presence of ams .", "we therefore studied a combined cohort of trekkers from 2 expeditions to ha . in order to clarify the relative contribution of ams , hypoxia or exercise to ngal levels", ", we also studied a cohort pre- and postexercise at near sea level , and a further cohort exposed to acute normobaric hypoxia .", "the potential role of inflammation in stimulating ngal was assessed by the measurement of highly sensitive c - reactive protein ( hscrp ) in a subset of participants ."], ["all study protocols were approved by the ministry of defence research ethics committee , whitehall , uk , and satisfied the requirements of the declaration of helsinki . in all studies informed ,", "thirty - two subjects participating in a defence medical services ( dms ) trekking expedition ( trek 1 ) in the khumbu region of nepal were studied .", "blood samples were taken from the antecubital fossa at 3 study altitudes : on day 2 at 3400  m , day 6 at 4270  m , and day 10 at 5150  m ( following ascent to everest base camp at 5364  m ) . all samples in this study", "were collected immediately following a day trekking (  posttrek  ) to the study altitude .", "twenty subjects from a further dms expedition ( trek 2 ) to nepal were also studied .", "blood samples were again taken at 3 study altitudes : on day 2 ( 3400  m ) , day 6 ( 4270  m ) , and day 10 ( 5150  m ) ( following ascent to kala patthar ( kp ) , 5643  m ) .", "samples in this study were again collected immediately following a day trekking (  posttrek  ) .", "additional samples were taken at rest in kathmandu ( kat ) at 1300  m and at rest the next morning at the 3 study altitudes .", "as serving members of the military , all subjects were able to fulfil the fitness criteria of their relevant service .", "this broadly includes an age - adjusted ability to run 1.5 miles in under approximately 11 minutes and to perform an age - adjusted number of sits - ups and push - ups within two blocks of 2 minutes .", "fourteen subjects underwent a 3-hour exposure to normobaric hypoxia ( fio2 11.6% , equivalent to 4800  m altitude ) in a hypoxic chamber .", "this exposure included a 5-minute step test ( step height of 25  cm , 1 complete step every 2 seconds ) at 95 minutes .", "a group of 22 subjects had ngal assayed at rest and after exercise at sl in the uk following ascent from sea level to 1085  m over 6 hours ( an equivalent gain in altitude and duration of exercise similar to that experienced on a trekking day in nepal ) .", "two subjects from trek 2 were part of the sl exercise group , but data collection occurred several months apart .  ", "ngal was analysed in the field on a biosite triage point of care monitor ( alere ltd , stockport , uk ) using a triage ngal test kit .", "the triage ngal test is a point - of - care , fluorescence - based immunoassay used which gives a rapid ( 15 minutes ) quantitative measurement of ngal in a range from 60 to 1,300  ng / ml .  ", "oxygen saturation ( digitally on warm hands at rest ) was measured using a nellcor np-20 pulse oximeter ( covidian , ma , usa ) during trek 1 + 2 and in the hypoxic chamber study at the same time as blood samples were taken . during trek 1 + 2 ,", "twice - daily ams scores were assessed using the lake louise score ( lls ) questionnaire .", "the lls allocates a score of 0 to 3 ( symptom not present to severe ) for symptoms of ams ( headache , gastrointestinal symptoms , fatigue / weakness , dizzy / light - headedness , and difficulty sleeping ) .", "a score of 3 or more in the presence of headache is consistent with ams , a score of 6 or more with severe ams .  ", "the commercially available , highly sensitive , immunoturbidimetric assay ( roche diagnostics ) was used to measure crp in trek 2 at the same time points as ngal .", "mg / l and a between - run coefficient of variation between 2.5 and 5.7% . for statistical calculations ,", "the software package spss 14.0 was used . for subjects with a ngal below the limit of detection of the assay ( 60  ng / ml ) ,", "a value of 60  ng / ml was assigned for the purposes of statistical analysis .", "all data were tested for gaussian distribution using the kolmogorov - smirnov test and shapiro wilks statistic .   for the analysis of dependent variables that were normally distributed , changes were tested by student 's paired t - test . for independent variables that were normally distributed ,", "a within - subjects anova was performed to investigate any serial changes in ngal with ascent at rest and post - trek . a two - way mixed anova with either resting or after trek ngal at each study altitude as the within - subjects factor and the presence of ams ( according to the ll score at multiple altitudes ) as the between - subjects factor", "if the mauchly sphericity test was significant , then p values were expressed after multiplication by the greenhouse - geisser epsilon .", "correlation analyses for normally distributed data were performed by calculating the pearson coefficient of correlation .", "a p value < 0.05 ( two - sided ) was considered significant . as the ascent profile and route were closely matched in trek 1 and trek 2 , data were combined and analysed as a whole . taking medication ( acetazolamide , dexamethasone )", "had no apparent effect on ngal values , and therefore these subjects ( n = 11 ) were not excluded from the analysis ."], ["demographic data for the field study ( trek 1 + 2 ) , the controls , and the hypoxic chamber study are shown in table 1 . in the 22 subjects ascending to 1085  m in the uk", ", there was no significant ( p = 0.084 ) rise in ngal following exercise : resting sl ngal was 64  11 ( ng / ml , mean  sd , range 60104 ) and postexercise ngal was 71  14 ( ng / ml , mean  sd , range 60100 ) . of the 52 subjects ,", "spo2 ( % , mean  sem ) dropped from 97  2 at kat ( 1300  m ) to 84  5 and 79  7 at 4270 and 5150  m , respectively ( p < 0.001 ) .", "there was a moderate inverse correlation between ngal and spo2 at 5150  m ( r = 0.477 , p = 0.001 ) ( figure 1 ) with a weaker inverse correlation between ngal and spo2 at 4270  m ( r = 0.340 , p = 0.019 ) .", "within the subjects , anova demonstrated a significant change in ngal with ascent both at rest ( p = 0.007 ) and after trek ( p = 0.001 ) ( figure 2 ) .", "spo2 ( % , mean  sem ) dropped from 99  0.4 at baseline to a nadir of 79  5 ( p < 0.001 ) . despite an equivalent drop in spo2 to", "that seen in trek 1 + 2 , ngal ( ng / ml , mean  sd , range ) showed no change between baseline and 180 minutes : 63  26 ( 2980 ) versus 67  25 ( 2784 ) , p = 0.538 . in trek 2 , serum creatinine { mol / l , mean  sem , ( range ) , ( p value versus baseline at kat ) } was 78  2 ( 6395 ) at baseline ; at 3400 , 4270 and 5150  m it was 87  3 ( 72120 ) ( p = 0.001 ) ; 84  2 ( 72104 ) ( p < 0.001 ) ; and 94  5 ( 76142 ) ( p < 0.001 ) .", "according to their ll scores at the highest study altitude ( 5150  m ) , there were 23 subjects with no ams , 16 subjects with mild ams , and 7 subjects with severe ams .", "there was a significant difference between ngal depending on the presence or absence of ams at 5150  m ( figure 3 ) with higher values in those with ams and severe ams . a two - way mixed anova revealed a significant change ( p = 0.003 ) in resting ngal with ascent and an interaction with ams at 4270  m ( p = 0.017 ) and 4910  m ( p = 0.002 for change in ngal , p = 0.027 for interaction with ams ) .  ", "hscrp was ( mean  sem , range ) : 1.6  0.4 ( 0.337.53 ) at baseline ; at 3400  m , 4270  m , and 5150  m post - trek : 7  2.9 ( 0.7847.93 ) ( p = 0.002 versus baseline ) ; 25.7  8.1 ( 0.58104 ) ( p < 0.001 versus baseline ) ; 9  3.2 ( 0.5644.62 ) ( p = 0.003 versus baseline ) . at 3400  m , 4270  m , and 5150  m at rest : 6.2  2.9 ( 1.8725.1 ) ( p = 0.001 versus baseline ) ; 21.6  5.7 ( 0.4983.9 ) ( p < 0.001 versus baseline ) ; and 5.8  2.1 ( 0.5426.59 ) ( p = 0.012 versus baseline ) .", "ngal at 5150  m , after trek was moderately correlated with hscrp at 5100  m after exercise ( rho 0.526 , p = 0.036 ) ."], ["this is the first report to describe an association between ngal and both the presence and severity of ams at ha .", "the significant novel findings are that ngal rises in response to sustained hypobaric hypoxia but not acute normobaric hypoxia or near sl exercise and that this rise is related to ams at 5150  m.   the rise in ngal following trekking ( by day 2 at 3400  m ) was to the levels normally associated with the subsequent development of arf ( > 150  ng / ml ) , but this did not occur .", "although creatinine rose significantly with altitude , the rise was very modest , and we suspect that a combination of factors other than a simple renal insult is responsible for the increase in ngal at ha .  ", "our data suggest an inverse correlation between spo2 and ngal at 5150  m ( and to a lesser extent at 4270  m ) . although no such correlation was found at 3400  m , this may still suggest that prolonged renal hypoxia could be a significant drive to ngal release .", "in addition to renal hypoxia , we suspect that other factors may also contribute to the rise in ngal at ha .   the significantly greater ngal in those with severe or mild ams versus those without at 5150 ", "indeed , ngal is an acute - phase protein   with a role in inflammation [ 8 , 10 , 11 ] .", "exercise stimulates an immune response , and hypoxia is also known to cause a response in immune and endothelial cells with inflammatory markers such as hscrp increasing with ha [ 1922 ] .", "consistent with this , we saw a significantly higher hscrp at all altitudes compared to baseline .", "limited data have suggested hscrp may be associated with ams   but we did not demonstrate any evidence to support this .", "there was a weak correlation between hscrp and ngal at 5150  m but this can not explain the rise in ngal as a whole .  ", "ngal also rises with oxidative stress [ 9 , 12 ] which is increased by exercise , and ha - induced oxidative stress   has been implicated in ams . as such , it is interesting to note that we found a higher ngal following trekking and in those with ams at the highest altitude .   in an attempt to clarify the relative influence of exercise and hypoxia on ngal , we measured ngal before and after exercise of a similar duration ( 6  hrs ) and similar incremental altitude ( 1085  m ) as that experienced daily in nepal and also in a hypoxic chamber . in neither scenario did ngal rise .", "this may reflect inadequate duration or severity of stimulus but may also reflect that the ngal response is not due to exercise or hypoxia alone but is multifactorial involving hypoxia , oxidative stress , an inflammatory response , and other , as yet unidentified , stimuli .", "we also acknowledge limitations such as a lack of serum markers of oxidative stress and a lack of resting ngal data in trek 1 .", "in addition , we did not measure ngal at sl before departure to nepal , although the ngal recorded as a baseline at kat ( 1300  m ) ( 68  ng / ml ) was no different to those recorded at sl in the uk ( 63 and 64  ng / ml ) .", "we also acknowledge the fact that although we measured creatinine in trek 2 , we did not continue to monitor it after the cessation of trekking . as a consequence of creatinine rising more slowly in response to a renal insult than ngal ,"], ["in conclusion , there are several interesting and novel findings that are worthy of further exploration .", "ngal rises in response to prolonged hypobaric hypoxia ; marked increases in ngal may occur without concomitant arf and the degree of ngal rise at ha is associated with the presence or absence of ams .", "the fact that ngal does not appear to rise secondary to acute normobaric hypoxia or exercise in isolation suggests that the rise at ha and relation with ams may have common pathways , perhaps related to prolonged hypoxia and an inflammatory response . with the huge and increasing popularity of recreational sports undertaken at both moderate and high altitude", "assessment of ngal takes a matter of minutes using poc testing , and its use in identifying ams requires further evaluation ."]]}
{"article_id": "PMC3919176", "article_text": ["when deposited on various \n substrate surfaces , rod - like , -conjugated , \n small organic molecules are well - known for their tendency to form \n highly anisotropic crystal shapes , which are frequently called fibers \n or needles . whereas the epitaxial growth has been studied on various substrates , \n in particular anisotropic surfaces seem favorable to conserve the \n highly anistropic morphology and optical properties , for example , \n polarized emission or adsorption provided by a parallel molecular \n orientation obtained by self - assembly . consequently , \n cu(110 ) , tio2(110 ) and muscovite mica(001 ) are frequently chosen as a proper fundament to study the epitaxial \n growth of rod - like small molecules . in this paper , the epitaxial \n growth of 2,2:6,2-ternaphthalene \n ( nnn ) on muscovite mica(001 ) is reported . as indicated in figure 1a ,", "the molecule is built from three naphthalene \n units , which are linked together by c ", "the coexistence of needle - like structures \n and island - like crystallites is verified .", "structural analysis reveals \n two different crystal orientations . whereas island - like structures \n are built up by upright standing molecules orientated with a ( 001 ) \n contact plane relative to the muscovite mica substrate ( see figure 1b ) ,", "needles consist of nnn molecules with a ( 111 ) \n lying orientation ( see figure 1c ) .", "both crystal \n configurations provide a well - defined azimuthal alignment , which is \n discussed based on force field simulations and a recently reported \n growth model .", "the azimuthal alignment \n of island like structures is explained by  ledge directed  \n", "( b ) a side view of nnn molecules packed in the observed crystal \n structure . each unit cell houses two nnn molecules .", "molecules are \n approximately standing on the s ( 001 ) contact plane , which is indicated \n in blue .", "( c ) a side view along the long molecular axis visualizing \n the edge - on / flat - on herringbone stacking of nnn .", "the blue area represents \n the orientation of the b ( 111 ) contact plane where molecules are aligned \n in almost in lying configuration .", "this all - aromatic compound could be obtained in high yield by coupling \n 2 equiv of 2-naphthaleneboronic acid ( 1 ) with 1 equiv of 2,6-dibromonaphthalene \n ( 2 ) , as described in the supporting information .", "the final product , 2,2:6,2-ternaphthalene \n ( nnn ) , was obtained as a colorless product , which appears to be highly \n insoluble in common solvents and could only be recrystallized from \n 1,2,4-trichlorobenzene ( colorless platelets ) .", "the material was checked \n with gas chromatography and mass spectroscopy and found to be > 99% \n pure before thermal sublimation .", "all samples have \n been fabricated \n on muscovite mica(001 ) substrates ( spi , structure probe , inc . ) .", "muscovite \n mica is a representative of sheet silicate minerals and provides a \n layered structure of aluminum silicate sheets weakly bound by layers \n of potassium ions .", "each layer is characterized by a high symmetry \n direction identified by parallel aligned surface grooves . between \n the individual sheets , the high symmetry direction alternates by 120 \n leading to a periodic  stacking sequence \n along   direction . immediately after \n cleaving , the mica substrates were transferred to the hot wall epitaxy \n ( hwe ) chamber .", "the hwe technique was applied for the deposition \n of the organic material , which allows us to perform the growth process \n close to thermodynamic equilibrium , and in further consequence relatively \n high vapor pressure of the organic deposit in the substrate region \n can be achieved . therefore , the requirements concerning vacuum conditions \n are reduced compared with , for example , molecular beam epitaxy .", "the source material nnn was purified twice by \n thermal sublimation before filling it into the quartz tube of the \n hwe reactor .", "muscovite mica substrates were transferred into the deposition \n chamber via a load lock and subsequently preheated at the deposition \n temperature ( 80 c ) for 30 min to ensure a stable temperature \n during the whole deposition process .", "the deposition was performed \n thereafter under a base pressure of 9  10 mbar .", "optical microscope images \n have been acquired by a nikon labophot 2a microscope in combination \n with a nikon type 115 digital camera .", "scanning force microscopy ( sfm ) \n studies of the deposited organic films were performed using a digital \n instruments dimension 3100 in the tapping mode .", "the 10  10 m images have been acquired at scan speeds of 46 m / s \n using sic tips ( masch , hq : nsc15/al bs ) exhibiting a cone angle \n of 40. nominal values for resonance frequency and tip radius \n are 325 khz and 10 nm , respectively .", "x - ray diffraction ( xrd ) \n measurements were carried out on a philips xpert x - ray diffractometer \n using cr k radiation (  = 2.29  ) and a secondary \n graphite monochromator", ". please note that the monochromator is transparent \n for  , /2 , /3 , etc .", ", so despite the weak intensity \n of the bremsspektrum , it can give clear bragg peaks due to the scatting \n on the single crystalline mica substrate .", "specular scans were performed \n in bragg  brentano configuration by varying the z - component of the scattering vector q. consequently \n it is possible to detect lattice planes that are parallel to the sample \n surface .", "pole \n figures were acquired by measuring at a constant length of q and only varying its direction .", "the unit cell parameters of nnn , \n which were used for analysis , are defined by a = \n 8.148  0.005  , b = 5.978  0.005 \n  , c = 19.45  0.2  , and  \n = 94.6  0.2 describing a monoclinic lattice ( p21/a ) . the \n unit houses two nnn molecules in planar configuration .", "the van der waals ( vdw ) interaction \n between the organic molecule and the dielectric substrate is modeled \n by lennard - jones - type potentials .", "corresponding parameters are taken \n from the universal force field implemented \n in a matlab program .", "the molecules and substrates are assumed to be \n rigid where the internal structure of an isolated nnn molecules is \n determined from the crystal structure .", "simulations were performed for the adsorption of a single nnn molecule \n as well as a crystal stack . by assuming that a single nnn molecule \n prefers to lie flat on the surface , the energy minimization procedure \n is simplified in the following way : we consider only four molecular \n degrees of freedom , the x- , y-", ", \n and z - positions of the molecular center of mass and \n the angle . the angle  defines the azimuthal molecular \n alignment and is probed by rotating the nnn molecule around the z - axis ( surface normal ) . we perform a grid - based optimization \n to search for the best molecular adsorption geometry using a grid \n of 81  81 points for the lateral position .", "the adsorption angle was tested \n with a step size of  = 1. the surface structure \n of the substrate has been assumed to be the same as in the bulk where \n the corrugation is about 0.2  . the substrate surface is assumed \n to be terminated by the tetrahedral layer of muscovite mica .", "for the \n simulation of the 7  2 nnn stack , the molecular packing has \n been deduced from the crystal structure of a ( 111 ) orientated nnn crystallite . due to the presence of flat \n and edge - on molecules ,", "the adsorption distance has been optimized , \n yielding a distance of 1.6  of the lowest h atom of edge - on \n nnn molecules to the substrate surface . because energetic minima are \n significantly narrow for the molecular stack , an angular resolution \n of  = 0.5 has been chosen for the calculations .", "in a first \n step , nnn was deposited by hot wall epitaxy ( hwe ) on muscovite mica(001 ) . \n whereas the substrate temperature was kept constant at 80 c , \n the deposition time was continuously increased .", "scanning force microscopy \n was chosen to study the sample morphology versus deposition time , \n and obtained images ( 10  10 m ) are depicted \n in figure 2 . as the maximum height scale z0 significantly changes with increasing deposition \n time , the corresponding values are indicated for each sample .", "the \n morphology of all samples is dominated by the presence of several \n micrometer long needle like structures that are aligned along multiple \n orientations . as exemplified by the cross - section ( 1 ) ,", "these fibers \n reach height levels up to 200 nm and are characterized by \n similar dimensions in width .", "a more detailed analysis is provided \n in figure 2a , plotting the mean needle height \n versus growth time . as indicated by the solid line", ", the growth rate \n can be approximated by a linear fit , yielding a slope of 17.8 \n  1.2 nmmin .", "interestingly , the fibers \n surface coverage is approximately constant for the whole sample series \n yielding a value of 12.5% .", "the sfm analysis reveals \n that flat islands start to nucleate at the side walls of the fibers \n and continuously fill the substrate surface between the fibers with \n increasing deposition time .", "the latter observation is underlined by \n analyzing the islands surface coverage , which is depicted \n in figure 2b as a function of growth time . \n the solid line , which represents a guide for the eye , indicates an \n asymptotic approach to 85% of the surface area .", "again , a representative \n sample position has been chosen to deduce a cross - section ( 2 ) , which \n is presented in the bottom right of figure 2 . as reported for other rod - like molecules , a steplike morphology with height levels in the range of 1.82 \n nm", "the obtained value \n corresponds to approximately one monolayer of upright standing nnn \n molecules .", "contrarily , the step size of the boundary , defined by the \n islands and the substrate surface , is significantly larger reaching \n values in the range of 15 nm . these steps are further characterized \n by straight extensions , which suggest the formation of well - defined \n crystal facets . scanning force microscopy ( sfm ) images showing the sample \n morphology \n versus deposition time .", "all samples are dominated by needle - like structures . \n with increasing growth time , island - like structures start to nucleate \n at the needle side walls covering continuously the substrate surface \n between the fibers .", "exemplary cross sections for both morphologies \n are indicated in the bottom part of the figure .", "as indicated in part \n a , the height of the fiber like structures linearly increases with \n growth time reaching values of some 100 nm .", "contrarily , the surface \n coverage by needles stays approximately constant ( part b ) .", "step heights \n in the center part of the islands approximately correspond to a monolayer \n of upright standing molecules . in a next step ,", "x - ray diffraction ( xrd ) has been chosen to \n study \n the structural properties of the organic crystallites . in order to \n obtain sufficient diffraction intensity , a sample with 216 nm high \n nnn fibers has been selected .", "figure 3a reports \n the acquired specular xrd diffraction pattern , which is dominated \n by a series of { 00n } diffraction peaks .", "these peaks \n are characteristic for island - shaped crystal morphologies , built up \n by approximately standing nnn molecules , and are consequently abbreviated \n by s - orientations ( q001 = 0.324  ) .", "arrows in the upper part of figure 3a indicate the positions of ( 00.2n ) diffraction \n peaks stemming from the muscovite mica(001 ) substrate .", "additionally , \n a diffraction peak arises at qz = 1.36  , which correlates with ( 111 ) \n orientated nnn crystallites , abbreviated as b orientation .", "the orientation \n is characteristic for a nearly flat lying molecular configuration \n and thus explains the presence of needle - like crystallites as revealed \n by sfm analysis .", "a more detailed analysis is reported in the supporting information . in order to analyze \n the azimuthal alignment of the nnn", "crystallites \n relative to the muscovite mica(001 ) substrate , xrd - pf measurements \n have been performed and are reported in figure 3b . for a profound analysis , xrd - pf have been acquired with a maximum \n sensitivity to diffraction intensities stemming from the scattering \n at { 202 } and { 201 } netplanes and are depicted in the bottom leftand \n right part of figure 3b .", "the diffraction \n intensities show a distinct azimuthal distribution , \n which underlines a well - defined epitaxial relationship to the muscovite \n mica substrate .", "the obtained symmetry of the diffraction intensities \n further underlines the presence of an  terminated muscovite \n mica surface , which is characterized by a mirror plane along the [ 110]mica orientation .", "the orientation of the substrate has been \n determined from diffraction patterns stemming from muscovite mica \n ( 001 ) and are indicated by black solid circles .", "xrd - pf patterns of \n the organic crystallites can be constructed by mirror operation from \n the top hemisphere , sketched by a gray shaded sector . moreover , the \n xrd - pf patterns reveal a 2-fold rotational symmetry , which can be \n understood by an approximately equivalent adsorption energy for 180 \n turned organic crystallites .", "consequently , discrimination between \n both crystal alignments has been omitted and simulated 2-fold symmetric \n diffraction spots are labeled identically .", "based on these geometrical \n considerations , only the diffraction spots of a single quadrant have \n to be analyzed and labeled . ( a ) specular x - ray diffraction ( xrd ) spectrum \n of nnn on muscovite \n mica(001 ) .", "scanning force microscopy images revealed a needle height \n of 216 nm for the chosen sample .", "the spectrum is dominated by a series \n of s ( 001 ) diffractions peaks , which are representative for island - like \n morphologies .", "( b ) xrd pole figure ( xrd - pf ) \n analysis of { 202 } and { 201 } diffraction peaks , providing information \n about the azimuthal crystal orientation . as indicated by the simulated \n pole distribution ( bottom ) ,", "all diffraction spots can be explained \n by the presence of three differently aligned ( 001 ) crystallites , labeled \n as s13 ( red ) .", "the mirror symmetry plane of the \n muscovite mica ( 001 ) surface is indicated by a horizontal line , which \n explains the presence of mirrored s13 * crystallites ( blue ) with a ( 001 ) \n contact plane .", "moreover , xrd - pf reveal a well - defined azimuthal orientation \n of b1 ( 111 ) crystallites ( red circles ) .", "again mirror symmetric \n crystallites b1 * ( 111 ) are indicated by blue symbols .", "diffraction \n intensities from the muscovite mica substrate are indicated by black \n solid filled circles .", "diffraction intensities that are characteristic for s - orientated \n crystallites are located at  = 63 ( 74 ) in the left \n ( right ) xrd - pf , and their azimuthal distribution can be explained \n by the presence of three crystal orientations labeled as s13 .", "consistently , both xrd - pf measurements hint the strongest diffraction \n intensities originating from s1 crystallites .", "diffraction \n spots , which can be attributed to b * ( 111)/b \n ( 111 ) crystallites are expected to appear at   51 \n for both diffraction geometries and are marked by blue / red filled \n circles . as each quadrant reveals the presence of one diffraction \n spot , the azimuthal alignment of b orientated fibers", "again , mirror symmetric crystals \n are labeled as b1 * and are characterized by a ( 111 ) contact plane .", "based on the simulated xrd - pf diffraction peaks , the long needle \n axis ( lna ) and long molecular axis ( lma ) orientations of b orientated \n fibers have been deduced and are presented in figure 4 by solid filled arrows . whereas the lna coincides with the \n [ 110 ] orientation , the lma can be approximated by the alignment \n of [ 101 ] relative to the muscovite mica substrate .", "mirror symmetry \n of the muscovite mica substrate leads to the generation of two energetically \n equivalent crystallites .", "fibers that \n are built up by b1 * ( 111 ) orientated crystallites ( blue ) are aligned with their \n lna ( lma ) 59.5 ( 49 ) relative to the muscovite \n mica substrate s [ 110]mica crystallographic \n orientation .", "contrarily , their mirror symmetric twins ( b1 ) can be constructed by flipping the b1 * crystallites upside down ( red arrows ) , \n azimuthally aligned with their lna ( lma ) 59.5 ( 49 ) \n relative to [ 110]mica . in order to verify \n the lna alignment , which has been constructed based on xrd - pf measurements", ", \n optical microscopy has been chosen , and an image of a representative \n sample area is depicted in figure 4 .", "the sample \n morphology is dominated by fibers that are aligned in a v - shaped , \n herringbone fashion . as indicated by large red and blue arrows , which \n represent the expected b1 and b1 * lna orientations deduced by xrd - pf analysis", "nevertheless , additional needle orientations , which are present in \n minor fraction , can be observed and are marked by small red ( b2 ) and blue ( b1 * ) arrows , respectively . in order to gain better statistics , \n a microscopy image of a larger sample area has been chosen to perform \n a fast fourier transformation ( fft ) and the obtained pattern is depicted \n beside .", "the fft is dominated by two stripes , which are characterized \n by an enclosing angle that perfectly reflects the lna orientation \n of b1 and b*1 crystallites ( indicated by red \n and blue arrows ) . long needle axis ( lna ) ( left ) and long molecular axis \n ( lma ) ( right ) \n orientations of b1 ( red solid filled arrows ) and mirror \n symmetric b1 * ( blue solid filled arrows ) crystallites , deduced by x - ray pole figure \n ( xrd - pf ) measurements . in the outer ring of the lna polar plot , additionally \n the [ 110 ] crystallographic orientations of s13 ( red ) and", "it is visualized that \n the [ 110 ] crystallographic direction of s1 crystallites \n provides the same azimuthal orientation as the lna of b1 fibers .", "below , the obtained lna orientations are verified by the \n observed sample morphology using optical microscopy . beside b1 and b1", "* fibers additionally minor fractions of approximately horizontally \n aligned crystallites are observed ( marked by small red and blue arrows ) . \n by using fast fourier transformation ( fft )", "( depicted beside ) , the \n dominant fraction of b1 and b1 * crystal orientations is further underlined . in a next step", "in particular , the azimuthal alignment \n of their [ 110 ] directions has been deduced and is depicted \n in the outer ring of figure 4 ( lna ) . because \n s1 and s1 * crystallites represent the major fraction , they are indicated \n by large arrows approximately aligned 60 relative to [ 110]mica .", "the azimuthal orientation perfectly coincides with the \n lna of b1 and b1 * crystallites , which already suggests an epitaxial \n relationship of both crystal types . in order to analyze the latter \n observation in more detail ,", "the sfm image , which \n is shown in figure 5 , is dominated by two approximately \n vertically aligned fibers . between \n fibers", ", the presence of an s orientated island can be observed , which \n is terminated in the bottom part of the image by a sharp l - shaped \n boundary .", "the observed boundary shape perfectly correlates with the \n expected angle between the   and [ 110 ] crystallographic \n orientations of an ( 001 ) orientated crystallite .", "the generation \n of the observed crystal shape can be understood by the formation of \n (11n ) and (11n ) side facets , which represent low index planes for n = 1 , 0 , or 1 .", "an extracted cross - section of the observed \n fiber is presented in \n the left part of figure 5 . the observed fiber \n", "is terminated in the right part by a flat plane , which is aligned \n parallel to the substrate at a height of approximately 100 nm .", "contrarily , \n the left side of the fiber shows a constantly decreasing height level . \n", "the slope of the side facet approximately correlates with a 25 nm \n decrease in height along 100 nm of the needle width ( 14 ) . \n", "the lna of b * type crystallites is defined by their [ 110 ] orientation \n and consequently all crystallographic planes (11n ) are aligned parallel to it . because the angular tilt of 13.8 \n between the low index planes ( 110 ) and ( 111 ) \n perfectly correlates with the observed sfm analysis , the theoretically \n expected cross - section of a b1 * fiber has been modeled and is depicted below \n the experimental data . although , the observed steep height decrease \n at the fiber side walls is below the resolution limit of the sfm , \n a termination of the fibers by ( 001 ) and ( 001 ) facets can be \n assumed .", "scanning force microscopy ( sfm ) image \n showing vertically aligned \n fibers and s orientated islands in between after the deposition of \n 60 min nnn at 80 c substrate temperature .", "the island s \n boundaries correlate with the geometrical alignment of   and [ 110 ] \n orientations . the extracted fiber cross - section ( top , left ) can be \n explained by the formation of ( 111 ) , ( 110 ) \n and (001 ) facets . based on the observed crystal shapes , their \n crystallographic orientations relative to each other has been deduced \n and is visualized by a 3d model below .", "the epitaxial relationship \n between fibers and islands is consistent with the structural analysis \n and can be explained by a nucleation of nnn molecules at the fiber \n side walls , also called  ledge directed epitaxy  .", "based on the latter analysis , \n a three - dimensional model of both \n crystal types has been generated and is depicted in the bottom part \n of figure 5 . as indicated by the xrd - pf analysis , \n which is presented in figure 4 ( lna ) , b1 * and s1 * crystallites shared \n the same azimuth for their crystallographic [110 ] orientations . \n in that way", ", the tilt angle of standing nnn molecules within the s - type \n crystallite approximately correlates with the tilt angle of the fiber \n ( 001 ) low energy plane .", "analogous observations were demonstrated for \n 6 t fibers and are explained by the nucleation of islands at the sidewalls \n of already existing needles .", "moreover , the latter \n picture is perfectly consistent with the sfm analysis presented in \n figure 2 , which reports a continuously increasing \n island coverage for longer deposition times .", "such epitaxial alignment \n based on a geometrical fit between nucleating crystallites and already \n existing topographic features on the substrate is called ", "ledge \n directed epitaxy  . based on the latter \n analysis together with xrd - pf data presented in figure 4 ( lna ) , it can be concluded that the minor fraction of s23 crystallites has most likely nucleated at b - type \n fibers , which are orientated approximately 30 tilted relative \n to [ 110]mica of the muscovite mica substrate . \n", "this conclusion is further consistent with the microscopy image , presented \n in figure 4 , revealing the presence of a minor \n fraction of such fibers ( below the detection limit of xrd - pf measurements ) , \n which are subsequently labeled by b2 and b2*.", "in order to understand the observed growth behavior of b1 and b2 fibers on muscovite mica(001 ) , the left part of \n figure 6 depicts a planar nnn molecule in gas \n phase .", "analogous to 6 t , a planar molecule \n is characterized by a mirror plane h , which is aligned in the plane of the naphthalene rings , and a 2-fold \n rotational axis , which is aligned normal to h .", ", it should be assumed that single nnn molecules tend \n to adsorb lying flat on the muscovite mica substrate in order to maximize \n their contact area . in that way ,", "h is orientated parallel to the substrate , and the molecule becomes \n chiral when adsorbed on an arbitrary surface .", "molecules that are intrinsically \n achiral but obtain a form of 2d chirality when adsorbed on a substrate \n surface are also called prochiral .", "analogous \n to 6 t , two mirror symmetric nnn enantiomers ( sketched \n as red and blue molecules ) can adsorb on the muscovite mica surface , \n which can not be brought into congruence by translation and rotation . taking a top view of the molecular stacking at the contact plane \n of b - type crystallites , which is depicted in the right panel of figure 6", ", reveals that ( 111 ) orientated fibers are built \n up by an alternating assembly of only one enantiomer ( red ) and edge - on \n nnn molecules .", "contrarily , their twin crystallites b * ( 111 ) \n are built up by the mirrored molecular configuration ( blue ) only . \n", "the latter observation further explains the consistent choice of a \n red and blue color code for molecules and crystallites . beside a real \n space model for b1 crystallites , which is deduced by xrd - pf \n analysis ,", "the right part of figure 6 further \n includes the proposed geometry of b2 fibers .", "based on a \n growth model that has been deduced for 6 t fibers , it is assumed that two needle orientations , for example , \n b1 and b2 * can originate from one molecular adsorption site .", "the existence \n of these two lna orientations is explained by a mirror symmetric molecular \n stacking during crystal nucleation .", "interestingly , epitaxially grown \n 6 t on muscovite mica showed a comparable fraction of both stacking \n types , which explains the observation of four lna orientations . the \n latter phenomenon is further explained by force field simulations \n for both crystal types , yielding a similar adsorption energy with \n a deviation of some millielectronvolts / molecule .", "contrarily , xrd - pf analysis revealed that nnn fibers are \n dominantly present only in one configuration , which reduces the observed \n lna orientations to two ( see the fft in figure 4 ) .", "consequently , it can be stated that both crystal types seem to \n significantly differ concerning their adsorption energy , which should \n be investigated and underlined by the discussion of force field simulations \n within the next paragraphs .", "molecular symmetry of a planar nnn in gas phase \n and when adsorbed \n flat on an arbitrary surface . due to", "the presence of a mirror symmetry \n plane h parallel to the naphthalene \n rings and a 2-fold rotational axis , aligned normal to it , the nnn \n molecule in gas phase can be described by the c2h point group .", "contrarily , when adsorbed \n flat on a substrate surface , nnn can form two mirror symmetric enantiomers \n ( sketched by red and blue molecules ) , which follow c2 symmetry .", "the right panel depicts a real space model \n of the discussed crystal orientations ( top view ) .", "the molecular alignment \n of nnn within the surface unit cell has been deduced from its bulk \n structure , oriented with a ( 111)/(111 ) contact \n plane for b / b * crystallites .", "the orientation of the long molecular \n axis ( lma ) or long needle axis ( lna ) is indicated by blue or red arrows . \n", "taking a closer look at the molecular stacking at the contact plane \n of b - type crystallites reveals that ( 111 ) orientated crystals are \n alternately assembled by red enantiomers and edge - on nnn molecules . \n", "contrarily , their mirror symmetric twins b * only consist of blue molecular \n configurations . the real space image of b1 ( b1 * ) and b2 * ( b2 )", "the adsorption energy as a function of the long molecular axis \n ( lma ) orientation (  ) is depicted in the top panel in terms \n of a polar plot .", "red and blue arrows indicate the molecular orientation \n deduced by experiments . at the indicated positions , force field simulations \n reveal a broad maximum for the corresponding enantiomer .", "contrarily , \n the adsorption site seems less favorable for the mirror symmetric \n molecule ( e  20 mev ) . a real space \n model that sketches the lateral position for the molecular adsorption \n angle at  = 57", "simulations \n reveal a preferred alignment of the terminating naphthalene units \n in the surface corrugations of the muscovite mica substrate ( indicated \n by horizontal lines ) .", "below , an analogous analysis has been done for \n a 7  2 molecular stack representative for the contact plane \n of a b ( 111 ) crystallite .", "simulations reveal a strongly pronounced \n adsorption maximum at the experimentally observed adsorption angle \n ( red arrow ) and a significantly different adsorption energy for a \n b * crystal with opposite stacking sequence ( blue arrow , e  300 mev / molecule ) . a real space model of the \n optimized adsorption position", "is depicted in the right panel . by comparing \n the alignment of the nnn molecules with the muscovite mica unit cell", ", \n an approximately periodic alignment can be recognized along mica/[110]mica for an / terminated \n surface . in a first step ,", "the optimal adsorption \n energy of a single nnn \n molecule has been deduced based on force - field simulations by selecting \n the most favorable adsorption site for each angle . the angle \n  characterizes the azimuthal alignment of the lma relative \n to mirror symmetry plane of the muscovite mica substrate surface . \n", "the molecules are assumed to adsorb flat on the substrate and the \n adsorption energy , ead , is defined as \n the difference between the energies of the isolated subsystems and \n the energy of the combined system . therefore , maxima in the ead versus  curves evidence the favorable \n adsorption geometries . to increase the readability ,", "ead curves are presented in figure 7 in terms of a polar plot , where ead = e0  ead .", "the parameter e0 represents \n the adsorption energy at the least favorable angle  , yielding \n a value of e0 = 2.49 ev for the \n isolated molecules .", "because nnn molecules can adsorb either in their \n left- or right - handed configuration , simulations have been performed \n for both enantiomers and are color coded by red- and blue - filled curves .", "simulations yield , for both molecular configurations , multiple \n adsorption maxima , which are located for the blue marked enantiomer \n at max , blue = 42 , 57 , 102 , 161 , and 178. \n due to 2-fold rotational symmetry of the nnn molecule , identical values \n are obtained for ead(+180 ) . \n", "moreover , optimized adsorption positions for the red molecular type \n are found at max , red = max , blue due to mirror symmetry of the substrate surface .", "experimentally \n obtained adsorption angles are further indicated by a blue ( red ) arrow \n at  = 49 ( 131 ) .", "both adsorption angles correlate \n with the broadest maxima obtained by simulations and are importantly \n consistent with the simulations of the corresponding enantiomer .", "the \n fact that beside experimentally observed adsorption geometries force \n field calculations also yield additional maxima is attributed to the \n usage of empirical potentials , which in some cases may yield the wrong \n energetic ordering of competing structure solutions .", "nevertheless , it has to be underlined that simulations \n indicate a significant less favorable adsorption site for the mirror \n symmetric molecular configuration ( 20 mev ) .", "in general , adsorption \n energies for both molecular configurations significantly differ , which \n in further consequence leads to a nonequal distribution or even breakup \n of both enantiomers depending on the adsorption angle .", "contrarily , \n simulations as well as experimental data that are reported for 6 t indicate a significantly lower energetic splitting \n between both molecular configurations , which may result from a higher \n symmetry of the molecule .", "the latter statement can be understood by \n the fact that thiophene molecules with an odd ring number , for example , \n quinquethiophene or septithiophene , are characterized by a mirror \n symmetry plane when adsorbed on a surface and consequently do not \n show a prochiral character .", "contrarily , the asymmetric alignment of \n the c  c bond between two naphthalene units of nnn inevitably \n leads to a prochiral behavior when adsorbed on a substrate surface \n and consequently plays an essential role concerning the energetic \n separation of both enantiomers at a defined adsorption angle .", "the experimentally confirmed adsorption position of nnn molecules \n is further depicted in the right part of figure 7 . for both molecules , an adsorption angle  = ", "analogous to calculations for p-6p and 6 t , nnn molecules tend to align their rings in the \n surface corrugations , which are indicated by vertical solid lines . in order to study the adsorption energetics of a b - type crystallite", ", \n a 7  2 molecular stack has been deduced from a ( 111 ) orientated \n crystallite .", "analogous to the force field simulations of an isolated \n molecule , the adsorption energy ead for the stack has been probed depending on the molecular orientation \n  and the adsorption energy at the least favorable adsorption \n angle is given by e0 = 0.41 ev / molecule . \n because the curve calculated for the a b * ( 111 ) \n", "stack follows the same behavior as discussed for a single molecule \n ( mirror symmetric ) , only the results for a b contact plane are depicted \n in order to increase readability .", "interestingly , simulations reveal \n that not only the number of energetically favorable adsorption sites \n decreases but also the angular acceptance , which becomes visible by \n well pronounced peaks .", "simulations further indicate the presence of \n two adsorption maxima , which are located at  = 12 and \n 48. again , the experimentally obtained adsorption angle \n for b1 crystallites is indicated by a red arrow and underlines \n a nearly perfect agreement .", "moreover , it can be recognized that the \n adsorption energy for a b2 crystal ( at + 48 ) becomes \n even more unfavorable than that for a single molecule , which is manifested \n by a much lower value of ead in \n the range of some 100 mev / molecule .", "consequently , force field \n simulations not only reflect the experimental \n observations but also explain the dominant fraction of b1 crystallites by a preferred nucleation of their stacking sequence \n in contrast to b2 crystallites", ". the observed behavior can \n be even better understood by analyzing the real space model of the \n simulated adsorption position at  = 48. besides \n the molecular alignment , also the surface unit cells of the muscovite \n mica and b crystal have been indicated .", "obviously , the unit vector \n [ 110 ] of the nnn crystal stack , which also defines its lna , \n tends to align parallel to one surface unit vector of the muscovite \n mica crystal , which is defined by the mica, ( [ 110]mica, ) orientation for an  \n (  ) terminated surface .", "the epitaxial growth of ternaphtalene \n ( nnn ) on muscovite mica(001 ) has been investigated by combining structural \n ( xrd - pf ) and morphological ( sfm ) methods .", "consistently , both methods \n reveal the formation of s ( 001 ) orientated nnn island - like structures \n which have nucleated at the sidewalls of b ( 111 ) orientated fibers . \n", "it is demonstrated that the latter nnn crystal types tend to align \n along two dominant directions , which leads to the formation of a v - shaped \n sample fiber morphology .", "because the tilt angle of nnn molecules within \n s - orientated crystallites correlates with the tilt angle of the fiber \n side facets , the island nucleation is explained by  ledge directed \n epitaxy  .", "based on this growth model , it can be understood \n that both crystal types provide a well - defined azimuthal orientation \n relative to the muscovite mica substrate . by use of force field \n simulations , the growth of the fibers is further analyzed .", "the epitaxial \n growth of sexithiophene ( 6 t ) on muscovite mica showed the formation \n of four well - defined fiber orientations , which can be explained by \n mirror symmetry of the muscovite mica substrate and two differently \n stacked 6 t crystallites , which can nucleate at a molecular adsorption \n position .", "contrarily , experimental investigations \n indicate that nnn crystallites tend to stack in a single configuration , \n which explains the dominant formation of only two fiber orientations . \n based on force field simulations ,", "the latter observation is further \n investigated and explained by significantly different adsorption energies \n of both crystal types .", "it is further demonstrated that the observed \n behavior results from an interplay of the molecular adsorption and \n lattice match ."], "abstract_text": ["<S> the \n morphology and structure of 2,2:6,2-ternaphthalene \n ( nnn ) deposited on muscovite mica(001 ) substrates was investigated \n by scanning force microscopy ( sfm ) and specular x - ray diffraction \n measurements . </S>", "<S> consistently , both methods reveal the coexistence of \n needle - like structures with a { 111 } contact plane and { 001 } orientated \n island - like crystallites , which are built up by almost upright standing \n nnn molecules . </S>", "<S> both orientations are characterized by a well - defined \n azimuthal alignment relative to the substrate surface , which is analyzed \n by x - ray diffraction pole figure ( xrd - pf ) measurements . </S>", "<S> based on xrd - pf \n and sfm analysis , the azimuthal alignment of { 001 } orientated crystallites \n is explained by ledge - directed epitaxy along the fibers sidewalls . \n </S>", "<S> these fibers are found to orient along two dominant directions , which \n is verified and explained by a doubling of the energetically preferred \n molecular adsorption site by mirror symmetry of the substrate surface . \n </S>", "<S> the experimental findings are confirmed by force - field simulations \n and are discussed based on a recently reported growth model . </S>"], "labels": null, "section_names": ["Introduction", "Experimental Section", "Experimental Results", "Discussion"], "sections": [["when deposited on various \n substrate surfaces , rod - like , -conjugated , \n small organic molecules are well - known for their tendency to form \n highly anisotropic crystal shapes , which are frequently called fibers \n or needles . whereas the epitaxial growth has been studied on various substrates , \n in particular anisotropic surfaces seem favorable to conserve the \n highly anistropic morphology and optical properties , for example , \n polarized emission or adsorption provided by a parallel molecular \n orientation obtained by self - assembly . consequently , \n cu(110 ) , tio2(110 ) and muscovite mica(001 ) are frequently chosen as a proper fundament to study the epitaxial \n growth of rod - like small molecules . in this paper , the epitaxial \n growth of 2,2:6,2-ternaphthalene \n ( nnn ) on muscovite mica(001 ) is reported . as indicated in figure 1a ,", "the molecule is built from three naphthalene \n units , which are linked together by c ", "the coexistence of needle - like structures \n and island - like crystallites is verified .", "structural analysis reveals \n two different crystal orientations . whereas island - like structures \n are built up by upright standing molecules orientated with a ( 001 ) \n contact plane relative to the muscovite mica substrate ( see figure 1b ) ,", "needles consist of nnn molecules with a ( 111 ) \n lying orientation ( see figure 1c ) .", "both crystal \n configurations provide a well - defined azimuthal alignment , which is \n discussed based on force field simulations and a recently reported \n growth model .", "the azimuthal alignment \n of island like structures is explained by  ledge directed  \n", "( b ) a side view of nnn molecules packed in the observed crystal \n structure . each unit cell houses two nnn molecules .", "molecules are \n approximately standing on the s ( 001 ) contact plane , which is indicated \n in blue .", "( c ) a side view along the long molecular axis visualizing \n the edge - on / flat - on herringbone stacking of nnn .", "the blue area represents \n the orientation of the b ( 111 ) contact plane where molecules are aligned \n in almost in lying configuration ."], ["this all - aromatic compound could be obtained in high yield by coupling \n 2 equiv of 2-naphthaleneboronic acid ( 1 ) with 1 equiv of 2,6-dibromonaphthalene \n ( 2 ) , as described in the supporting information .", "the final product , 2,2:6,2-ternaphthalene \n ( nnn ) , was obtained as a colorless product , which appears to be highly \n insoluble in common solvents and could only be recrystallized from \n 1,2,4-trichlorobenzene ( colorless platelets ) .", "the material was checked \n with gas chromatography and mass spectroscopy and found to be > 99% \n pure before thermal sublimation .", "all samples have \n been fabricated \n on muscovite mica(001 ) substrates ( spi , structure probe , inc . ) .", "muscovite \n mica is a representative of sheet silicate minerals and provides a \n layered structure of aluminum silicate sheets weakly bound by layers \n of potassium ions .", "each layer is characterized by a high symmetry \n direction identified by parallel aligned surface grooves . between \n the individual sheets , the high symmetry direction alternates by 120 \n leading to a periodic  stacking sequence \n along   direction . immediately after \n cleaving , the mica substrates were transferred to the hot wall epitaxy \n ( hwe ) chamber .", "the hwe technique was applied for the deposition \n of the organic material , which allows us to perform the growth process \n close to thermodynamic equilibrium , and in further consequence relatively \n high vapor pressure of the organic deposit in the substrate region \n can be achieved . therefore , the requirements concerning vacuum conditions \n are reduced compared with , for example , molecular beam epitaxy .", "the source material nnn was purified twice by \n thermal sublimation before filling it into the quartz tube of the \n hwe reactor .", "muscovite mica substrates were transferred into the deposition \n chamber via a load lock and subsequently preheated at the deposition \n temperature ( 80 c ) for 30 min to ensure a stable temperature \n during the whole deposition process .", "the deposition was performed \n thereafter under a base pressure of 9  10 mbar .", "optical microscope images \n have been acquired by a nikon labophot 2a microscope in combination \n with a nikon type 115 digital camera .", "scanning force microscopy ( sfm ) \n studies of the deposited organic films were performed using a digital \n instruments dimension 3100 in the tapping mode .", "the 10  10 m images have been acquired at scan speeds of 46 m / s \n using sic tips ( masch , hq : nsc15/al bs ) exhibiting a cone angle \n of 40. nominal values for resonance frequency and tip radius \n are 325 khz and 10 nm , respectively .", "x - ray diffraction ( xrd ) \n measurements were carried out on a philips xpert x - ray diffractometer \n using cr k radiation (  = 2.29  ) and a secondary \n graphite monochromator", ". please note that the monochromator is transparent \n for  , /2 , /3 , etc .", ", so despite the weak intensity \n of the bremsspektrum , it can give clear bragg peaks due to the scatting \n on the single crystalline mica substrate .", "specular scans were performed \n in bragg  brentano configuration by varying the z - component of the scattering vector q. consequently \n it is possible to detect lattice planes that are parallel to the sample \n surface .", "pole \n figures were acquired by measuring at a constant length of q and only varying its direction .", "the unit cell parameters of nnn , \n which were used for analysis , are defined by a = \n 8.148  0.005  , b = 5.978  0.005 \n  , c = 19.45  0.2  , and  \n = 94.6  0.2 describing a monoclinic lattice ( p21/a ) . the \n unit houses two nnn molecules in planar configuration .", "the van der waals ( vdw ) interaction \n between the organic molecule and the dielectric substrate is modeled \n by lennard - jones - type potentials .", "corresponding parameters are taken \n from the universal force field implemented \n in a matlab program .", "the molecules and substrates are assumed to be \n rigid where the internal structure of an isolated nnn molecules is \n determined from the crystal structure .", "simulations were performed for the adsorption of a single nnn molecule \n as well as a crystal stack . by assuming that a single nnn molecule \n prefers to lie flat on the surface , the energy minimization procedure \n is simplified in the following way : we consider only four molecular \n degrees of freedom , the x- , y-", ", \n and z - positions of the molecular center of mass and \n the angle . the angle  defines the azimuthal molecular \n alignment and is probed by rotating the nnn molecule around the z - axis ( surface normal ) . we perform a grid - based optimization \n to search for the best molecular adsorption geometry using a grid \n of 81  81 points for the lateral position .", "the adsorption angle was tested \n with a step size of  = 1. the surface structure \n of the substrate has been assumed to be the same as in the bulk where \n the corrugation is about 0.2  . the substrate surface is assumed \n to be terminated by the tetrahedral layer of muscovite mica .", "for the \n simulation of the 7  2 nnn stack , the molecular packing has \n been deduced from the crystal structure of a ( 111 ) orientated nnn crystallite . due to the presence of flat \n and edge - on molecules ,", "the adsorption distance has been optimized , \n yielding a distance of 1.6  of the lowest h atom of edge - on \n nnn molecules to the substrate surface . because energetic minima are \n significantly narrow for the molecular stack , an angular resolution \n of  = 0.5 has been chosen for the calculations ."], ["in a first \n step , nnn was deposited by hot wall epitaxy ( hwe ) on muscovite mica(001 ) . \n whereas the substrate temperature was kept constant at 80 c , \n the deposition time was continuously increased .", "scanning force microscopy \n was chosen to study the sample morphology versus deposition time , \n and obtained images ( 10  10 m ) are depicted \n in figure 2 . as the maximum height scale z0 significantly changes with increasing deposition \n time , the corresponding values are indicated for each sample .", "the \n morphology of all samples is dominated by the presence of several \n micrometer long needle like structures that are aligned along multiple \n orientations . as exemplified by the cross - section ( 1 ) ,", "these fibers \n reach height levels up to 200 nm and are characterized by \n similar dimensions in width .", "a more detailed analysis is provided \n in figure 2a , plotting the mean needle height \n versus growth time . as indicated by the solid line", ", the growth rate \n can be approximated by a linear fit , yielding a slope of 17.8 \n  1.2 nmmin .", "interestingly , the fibers \n surface coverage is approximately constant for the whole sample series \n yielding a value of 12.5% .", "the sfm analysis reveals \n that flat islands start to nucleate at the side walls of the fibers \n and continuously fill the substrate surface between the fibers with \n increasing deposition time .", "the latter observation is underlined by \n analyzing the islands surface coverage , which is depicted \n in figure 2b as a function of growth time . \n the solid line , which represents a guide for the eye , indicates an \n asymptotic approach to 85% of the surface area .", "again , a representative \n sample position has been chosen to deduce a cross - section ( 2 ) , which \n is presented in the bottom right of figure 2 . as reported for other rod - like molecules , a steplike morphology with height levels in the range of 1.82 \n nm", "the obtained value \n corresponds to approximately one monolayer of upright standing nnn \n molecules .", "contrarily , the step size of the boundary , defined by the \n islands and the substrate surface , is significantly larger reaching \n values in the range of 15 nm . these steps are further characterized \n by straight extensions , which suggest the formation of well - defined \n crystal facets . scanning force microscopy ( sfm ) images showing the sample \n morphology \n versus deposition time .", "all samples are dominated by needle - like structures . \n with increasing growth time , island - like structures start to nucleate \n at the needle side walls covering continuously the substrate surface \n between the fibers .", "exemplary cross sections for both morphologies \n are indicated in the bottom part of the figure .", "as indicated in part \n a , the height of the fiber like structures linearly increases with \n growth time reaching values of some 100 nm .", "contrarily , the surface \n coverage by needles stays approximately constant ( part b ) .", "step heights \n in the center part of the islands approximately correspond to a monolayer \n of upright standing molecules . in a next step ,", "x - ray diffraction ( xrd ) has been chosen to \n study \n the structural properties of the organic crystallites . in order to \n obtain sufficient diffraction intensity , a sample with 216 nm high \n nnn fibers has been selected .", "figure 3a reports \n the acquired specular xrd diffraction pattern , which is dominated \n by a series of { 00n } diffraction peaks .", "these peaks \n are characteristic for island - shaped crystal morphologies , built up \n by approximately standing nnn molecules , and are consequently abbreviated \n by s - orientations ( q001 = 0.324  ) .", "arrows in the upper part of figure 3a indicate the positions of ( 00.2n ) diffraction \n peaks stemming from the muscovite mica(001 ) substrate .", "additionally , \n a diffraction peak arises at qz = 1.36  , which correlates with ( 111 ) \n orientated nnn crystallites , abbreviated as b orientation .", "the orientation \n is characteristic for a nearly flat lying molecular configuration \n and thus explains the presence of needle - like crystallites as revealed \n by sfm analysis .", "a more detailed analysis is reported in the supporting information . in order to analyze \n the azimuthal alignment of the nnn", "crystallites \n relative to the muscovite mica(001 ) substrate , xrd - pf measurements \n have been performed and are reported in figure 3b . for a profound analysis , xrd - pf have been acquired with a maximum \n sensitivity to diffraction intensities stemming from the scattering \n at { 202 } and { 201 } netplanes and are depicted in the bottom leftand \n right part of figure 3b .", "the diffraction \n intensities show a distinct azimuthal distribution , \n which underlines a well - defined epitaxial relationship to the muscovite \n mica substrate .", "the obtained symmetry of the diffraction intensities \n further underlines the presence of an  terminated muscovite \n mica surface , which is characterized by a mirror plane along the [ 110]mica orientation .", "the orientation of the substrate has been \n determined from diffraction patterns stemming from muscovite mica \n ( 001 ) and are indicated by black solid circles .", "xrd - pf patterns of \n the organic crystallites can be constructed by mirror operation from \n the top hemisphere , sketched by a gray shaded sector . moreover , the \n xrd - pf patterns reveal a 2-fold rotational symmetry , which can be \n understood by an approximately equivalent adsorption energy for 180 \n turned organic crystallites .", "consequently , discrimination between \n both crystal alignments has been omitted and simulated 2-fold symmetric \n diffraction spots are labeled identically .", "based on these geometrical \n considerations , only the diffraction spots of a single quadrant have \n to be analyzed and labeled . ( a ) specular x - ray diffraction ( xrd ) spectrum \n of nnn on muscovite \n mica(001 ) .", "scanning force microscopy images revealed a needle height \n of 216 nm for the chosen sample .", "the spectrum is dominated by a series \n of s ( 001 ) diffractions peaks , which are representative for island - like \n morphologies .", "( b ) xrd pole figure ( xrd - pf ) \n analysis of { 202 } and { 201 } diffraction peaks , providing information \n about the azimuthal crystal orientation . as indicated by the simulated \n pole distribution ( bottom ) ,", "all diffraction spots can be explained \n by the presence of three differently aligned ( 001 ) crystallites , labeled \n as s13 ( red ) .", "the mirror symmetry plane of the \n muscovite mica ( 001 ) surface is indicated by a horizontal line , which \n explains the presence of mirrored s13 * crystallites ( blue ) with a ( 001 ) \n contact plane .", "moreover , xrd - pf reveal a well - defined azimuthal orientation \n of b1 ( 111 ) crystallites ( red circles ) .", "again mirror symmetric \n crystallites b1 * ( 111 ) are indicated by blue symbols .", "diffraction \n intensities from the muscovite mica substrate are indicated by black \n solid filled circles .", "diffraction intensities that are characteristic for s - orientated \n crystallites are located at  = 63 ( 74 ) in the left \n ( right ) xrd - pf , and their azimuthal distribution can be explained \n by the presence of three crystal orientations labeled as s13 .", "consistently , both xrd - pf measurements hint the strongest diffraction \n intensities originating from s1 crystallites .", "diffraction \n spots , which can be attributed to b * ( 111)/b \n ( 111 ) crystallites are expected to appear at   51 \n for both diffraction geometries and are marked by blue / red filled \n circles . as each quadrant reveals the presence of one diffraction \n spot , the azimuthal alignment of b orientated fibers", "again , mirror symmetric crystals \n are labeled as b1 * and are characterized by a ( 111 ) contact plane .", "based on the simulated xrd - pf diffraction peaks , the long needle \n axis ( lna ) and long molecular axis ( lma ) orientations of b orientated \n fibers have been deduced and are presented in figure 4 by solid filled arrows . whereas the lna coincides with the \n [ 110 ] orientation , the lma can be approximated by the alignment \n of [ 101 ] relative to the muscovite mica substrate .", "mirror symmetry \n of the muscovite mica substrate leads to the generation of two energetically \n equivalent crystallites .", "fibers that \n are built up by b1 * ( 111 ) orientated crystallites ( blue ) are aligned with their \n lna ( lma ) 59.5 ( 49 ) relative to the muscovite \n mica substrate s [ 110]mica crystallographic \n orientation .", "contrarily , their mirror symmetric twins ( b1 ) can be constructed by flipping the b1 * crystallites upside down ( red arrows ) , \n azimuthally aligned with their lna ( lma ) 59.5 ( 49 ) \n relative to [ 110]mica . in order to verify \n the lna alignment , which has been constructed based on xrd - pf measurements", ", \n optical microscopy has been chosen , and an image of a representative \n sample area is depicted in figure 4 .", "the sample \n morphology is dominated by fibers that are aligned in a v - shaped , \n herringbone fashion . as indicated by large red and blue arrows , which \n represent the expected b1 and b1 * lna orientations deduced by xrd - pf analysis", "nevertheless , additional needle orientations , which are present in \n minor fraction , can be observed and are marked by small red ( b2 ) and blue ( b1 * ) arrows , respectively . in order to gain better statistics , \n a microscopy image of a larger sample area has been chosen to perform \n a fast fourier transformation ( fft ) and the obtained pattern is depicted \n beside .", "the fft is dominated by two stripes , which are characterized \n by an enclosing angle that perfectly reflects the lna orientation \n of b1 and b*1 crystallites ( indicated by red \n and blue arrows ) . long needle axis ( lna ) ( left ) and long molecular axis \n ( lma ) ( right ) \n orientations of b1 ( red solid filled arrows ) and mirror \n symmetric b1 * ( blue solid filled arrows ) crystallites , deduced by x - ray pole figure \n ( xrd - pf ) measurements . in the outer ring of the lna polar plot , additionally \n the [ 110 ] crystallographic orientations of s13 ( red ) and", "it is visualized that \n the [ 110 ] crystallographic direction of s1 crystallites \n provides the same azimuthal orientation as the lna of b1 fibers .", "below , the obtained lna orientations are verified by the \n observed sample morphology using optical microscopy . beside b1 and b1", "* fibers additionally minor fractions of approximately horizontally \n aligned crystallites are observed ( marked by small red and blue arrows ) . \n by using fast fourier transformation ( fft )", "( depicted beside ) , the \n dominant fraction of b1 and b1 * crystal orientations is further underlined . in a next step", "in particular , the azimuthal alignment \n of their [ 110 ] directions has been deduced and is depicted \n in the outer ring of figure 4 ( lna ) . because \n s1 and s1 * crystallites represent the major fraction , they are indicated \n by large arrows approximately aligned 60 relative to [ 110]mica .", "the azimuthal orientation perfectly coincides with the \n lna of b1 and b1 * crystallites , which already suggests an epitaxial \n relationship of both crystal types . in order to analyze the latter \n observation in more detail ,", "the sfm image , which \n is shown in figure 5 , is dominated by two approximately \n vertically aligned fibers . between \n fibers", ", the presence of an s orientated island can be observed , which \n is terminated in the bottom part of the image by a sharp l - shaped \n boundary .", "the observed boundary shape perfectly correlates with the \n expected angle between the   and [ 110 ] crystallographic \n orientations of an ( 001 ) orientated crystallite .", "the generation \n of the observed crystal shape can be understood by the formation of \n (11n ) and (11n ) side facets , which represent low index planes for n = 1 , 0 , or 1 .", "an extracted cross - section of the observed \n fiber is presented in \n the left part of figure 5 . the observed fiber \n", "is terminated in the right part by a flat plane , which is aligned \n parallel to the substrate at a height of approximately 100 nm .", "contrarily , \n the left side of the fiber shows a constantly decreasing height level . \n", "the slope of the side facet approximately correlates with a 25 nm \n decrease in height along 100 nm of the needle width ( 14 ) . \n", "the lna of b * type crystallites is defined by their [ 110 ] orientation \n and consequently all crystallographic planes (11n ) are aligned parallel to it . because the angular tilt of 13.8 \n between the low index planes ( 110 ) and ( 111 ) \n perfectly correlates with the observed sfm analysis , the theoretically \n expected cross - section of a b1 * fiber has been modeled and is depicted below \n the experimental data . although , the observed steep height decrease \n at the fiber side walls is below the resolution limit of the sfm , \n a termination of the fibers by ( 001 ) and ( 001 ) facets can be \n assumed .", "scanning force microscopy ( sfm ) image \n showing vertically aligned \n fibers and s orientated islands in between after the deposition of \n 60 min nnn at 80 c substrate temperature .", "the island s \n boundaries correlate with the geometrical alignment of   and [ 110 ] \n orientations . the extracted fiber cross - section ( top , left ) can be \n explained by the formation of ( 111 ) , ( 110 ) \n and (001 ) facets . based on the observed crystal shapes , their \n crystallographic orientations relative to each other has been deduced \n and is visualized by a 3d model below .", "the epitaxial relationship \n between fibers and islands is consistent with the structural analysis \n and can be explained by a nucleation of nnn molecules at the fiber \n side walls , also called  ledge directed epitaxy  .", "based on the latter analysis , \n a three - dimensional model of both \n crystal types has been generated and is depicted in the bottom part \n of figure 5 . as indicated by the xrd - pf analysis , \n which is presented in figure 4 ( lna ) , b1 * and s1 * crystallites shared \n the same azimuth for their crystallographic [110 ] orientations . \n in that way", ", the tilt angle of standing nnn molecules within the s - type \n crystallite approximately correlates with the tilt angle of the fiber \n ( 001 ) low energy plane .", "analogous observations were demonstrated for \n 6 t fibers and are explained by the nucleation of islands at the sidewalls \n of already existing needles .", "moreover , the latter \n picture is perfectly consistent with the sfm analysis presented in \n figure 2 , which reports a continuously increasing \n island coverage for longer deposition times .", "such epitaxial alignment \n based on a geometrical fit between nucleating crystallites and already \n existing topographic features on the substrate is called ", "ledge \n directed epitaxy  . based on the latter \n analysis together with xrd - pf data presented in figure 4 ( lna ) , it can be concluded that the minor fraction of s23 crystallites has most likely nucleated at b - type \n fibers , which are orientated approximately 30 tilted relative \n to [ 110]mica of the muscovite mica substrate . \n", "this conclusion is further consistent with the microscopy image , presented \n in figure 4 , revealing the presence of a minor \n fraction of such fibers ( below the detection limit of xrd - pf measurements ) , \n which are subsequently labeled by b2 and b2*."], ["in order to understand the observed growth behavior of b1 and b2 fibers on muscovite mica(001 ) , the left part of \n figure 6 depicts a planar nnn molecule in gas \n phase .", "analogous to 6 t , a planar molecule \n is characterized by a mirror plane h , which is aligned in the plane of the naphthalene rings , and a 2-fold \n rotational axis , which is aligned normal to h .", ", it should be assumed that single nnn molecules tend \n to adsorb lying flat on the muscovite mica substrate in order to maximize \n their contact area . in that way ,", "h is orientated parallel to the substrate , and the molecule becomes \n chiral when adsorbed on an arbitrary surface .", "molecules that are intrinsically \n achiral but obtain a form of 2d chirality when adsorbed on a substrate \n surface are also called prochiral .", "analogous \n to 6 t , two mirror symmetric nnn enantiomers ( sketched \n as red and blue molecules ) can adsorb on the muscovite mica surface , \n which can not be brought into congruence by translation and rotation . taking a top view of the molecular stacking at the contact plane \n of b - type crystallites , which is depicted in the right panel of figure 6", ", reveals that ( 111 ) orientated fibers are built \n up by an alternating assembly of only one enantiomer ( red ) and edge - on \n nnn molecules .", "contrarily , their twin crystallites b * ( 111 ) \n are built up by the mirrored molecular configuration ( blue ) only . \n", "the latter observation further explains the consistent choice of a \n red and blue color code for molecules and crystallites . beside a real \n space model for b1 crystallites , which is deduced by xrd - pf \n analysis ,", "the right part of figure 6 further \n includes the proposed geometry of b2 fibers .", "based on a \n growth model that has been deduced for 6 t fibers , it is assumed that two needle orientations , for example , \n b1 and b2 * can originate from one molecular adsorption site .", "the existence \n of these two lna orientations is explained by a mirror symmetric molecular \n stacking during crystal nucleation .", "interestingly , epitaxially grown \n 6 t on muscovite mica showed a comparable fraction of both stacking \n types , which explains the observation of four lna orientations . the \n latter phenomenon is further explained by force field simulations \n for both crystal types , yielding a similar adsorption energy with \n a deviation of some millielectronvolts / molecule .", "contrarily , xrd - pf analysis revealed that nnn fibers are \n dominantly present only in one configuration , which reduces the observed \n lna orientations to two ( see the fft in figure 4 ) .", "consequently , it can be stated that both crystal types seem to \n significantly differ concerning their adsorption energy , which should \n be investigated and underlined by the discussion of force field simulations \n within the next paragraphs .", "molecular symmetry of a planar nnn in gas phase \n and when adsorbed \n flat on an arbitrary surface . due to", "the presence of a mirror symmetry \n plane h parallel to the naphthalene \n rings and a 2-fold rotational axis , aligned normal to it , the nnn \n molecule in gas phase can be described by the c2h point group .", "contrarily , when adsorbed \n flat on a substrate surface , nnn can form two mirror symmetric enantiomers \n ( sketched by red and blue molecules ) , which follow c2 symmetry .", "the right panel depicts a real space model \n of the discussed crystal orientations ( top view ) .", "the molecular alignment \n of nnn within the surface unit cell has been deduced from its bulk \n structure , oriented with a ( 111)/(111 ) contact \n plane for b / b * crystallites .", "the orientation of the long molecular \n axis ( lma ) or long needle axis ( lna ) is indicated by blue or red arrows . \n", "taking a closer look at the molecular stacking at the contact plane \n of b - type crystallites reveals that ( 111 ) orientated crystals are \n alternately assembled by red enantiomers and edge - on nnn molecules . \n", "contrarily , their mirror symmetric twins b * only consist of blue molecular \n configurations . the real space image of b1 ( b1 * ) and b2 * ( b2 )", "the adsorption energy as a function of the long molecular axis \n ( lma ) orientation (  ) is depicted in the top panel in terms \n of a polar plot .", "red and blue arrows indicate the molecular orientation \n deduced by experiments . at the indicated positions , force field simulations \n reveal a broad maximum for the corresponding enantiomer .", "contrarily , \n the adsorption site seems less favorable for the mirror symmetric \n molecule ( e  20 mev ) . a real space \n model that sketches the lateral position for the molecular adsorption \n angle at  = 57", "simulations \n reveal a preferred alignment of the terminating naphthalene units \n in the surface corrugations of the muscovite mica substrate ( indicated \n by horizontal lines ) .", "below , an analogous analysis has been done for \n a 7  2 molecular stack representative for the contact plane \n of a b ( 111 ) crystallite .", "simulations reveal a strongly pronounced \n adsorption maximum at the experimentally observed adsorption angle \n ( red arrow ) and a significantly different adsorption energy for a \n b * crystal with opposite stacking sequence ( blue arrow , e  300 mev / molecule ) . a real space model of the \n optimized adsorption position", "is depicted in the right panel . by comparing \n the alignment of the nnn molecules with the muscovite mica unit cell", ", \n an approximately periodic alignment can be recognized along mica/[110]mica for an / terminated \n surface . in a first step ,", "the optimal adsorption \n energy of a single nnn \n molecule has been deduced based on force - field simulations by selecting \n the most favorable adsorption site for each angle . the angle \n  characterizes the azimuthal alignment of the lma relative \n to mirror symmetry plane of the muscovite mica substrate surface . \n", "the molecules are assumed to adsorb flat on the substrate and the \n adsorption energy , ead , is defined as \n the difference between the energies of the isolated subsystems and \n the energy of the combined system . therefore , maxima in the ead versus  curves evidence the favorable \n adsorption geometries . to increase the readability ,", "ead curves are presented in figure 7 in terms of a polar plot , where ead = e0  ead .", "the parameter e0 represents \n the adsorption energy at the least favorable angle  , yielding \n a value of e0 = 2.49 ev for the \n isolated molecules .", "because nnn molecules can adsorb either in their \n left- or right - handed configuration , simulations have been performed \n for both enantiomers and are color coded by red- and blue - filled curves .", "simulations yield , for both molecular configurations , multiple \n adsorption maxima , which are located for the blue marked enantiomer \n at max , blue = 42 , 57 , 102 , 161 , and 178. \n due to 2-fold rotational symmetry of the nnn molecule , identical values \n are obtained for ead(+180 ) . \n", "moreover , optimized adsorption positions for the red molecular type \n are found at max , red = max , blue due to mirror symmetry of the substrate surface .", "experimentally \n obtained adsorption angles are further indicated by a blue ( red ) arrow \n at  = 49 ( 131 ) .", "both adsorption angles correlate \n with the broadest maxima obtained by simulations and are importantly \n consistent with the simulations of the corresponding enantiomer .", "the \n fact that beside experimentally observed adsorption geometries force \n field calculations also yield additional maxima is attributed to the \n usage of empirical potentials , which in some cases may yield the wrong \n energetic ordering of competing structure solutions .", "nevertheless , it has to be underlined that simulations \n indicate a significant less favorable adsorption site for the mirror \n symmetric molecular configuration ( 20 mev ) .", "in general , adsorption \n energies for both molecular configurations significantly differ , which \n in further consequence leads to a nonequal distribution or even breakup \n of both enantiomers depending on the adsorption angle .", "contrarily , \n simulations as well as experimental data that are reported for 6 t indicate a significantly lower energetic splitting \n between both molecular configurations , which may result from a higher \n symmetry of the molecule .", "the latter statement can be understood by \n the fact that thiophene molecules with an odd ring number , for example , \n quinquethiophene or septithiophene , are characterized by a mirror \n symmetry plane when adsorbed on a surface and consequently do not \n show a prochiral character .", "contrarily , the asymmetric alignment of \n the c  c bond between two naphthalene units of nnn inevitably \n leads to a prochiral behavior when adsorbed on a substrate surface \n and consequently plays an essential role concerning the energetic \n separation of both enantiomers at a defined adsorption angle .", "the experimentally confirmed adsorption position of nnn molecules \n is further depicted in the right part of figure 7 . for both molecules , an adsorption angle  = ", "analogous to calculations for p-6p and 6 t , nnn molecules tend to align their rings in the \n surface corrugations , which are indicated by vertical solid lines . in order to study the adsorption energetics of a b - type crystallite", ", \n a 7  2 molecular stack has been deduced from a ( 111 ) orientated \n crystallite .", "analogous to the force field simulations of an isolated \n molecule , the adsorption energy ead for the stack has been probed depending on the molecular orientation \n  and the adsorption energy at the least favorable adsorption \n angle is given by e0 = 0.41 ev / molecule . \n because the curve calculated for the a b * ( 111 ) \n", "stack follows the same behavior as discussed for a single molecule \n ( mirror symmetric ) , only the results for a b contact plane are depicted \n in order to increase readability .", "interestingly , simulations reveal \n that not only the number of energetically favorable adsorption sites \n decreases but also the angular acceptance , which becomes visible by \n well pronounced peaks .", "simulations further indicate the presence of \n two adsorption maxima , which are located at  = 12 and \n 48. again , the experimentally obtained adsorption angle \n for b1 crystallites is indicated by a red arrow and underlines \n a nearly perfect agreement .", "moreover , it can be recognized that the \n adsorption energy for a b2 crystal ( at + 48 ) becomes \n even more unfavorable than that for a single molecule , which is manifested \n by a much lower value of ead in \n the range of some 100 mev / molecule .", "consequently , force field \n simulations not only reflect the experimental \n observations but also explain the dominant fraction of b1 crystallites by a preferred nucleation of their stacking sequence \n in contrast to b2 crystallites", ". the observed behavior can \n be even better understood by analyzing the real space model of the \n simulated adsorption position at  = 48. besides \n the molecular alignment , also the surface unit cells of the muscovite \n mica and b crystal have been indicated .", "obviously , the unit vector \n [ 110 ] of the nnn crystal stack , which also defines its lna , \n tends to align parallel to one surface unit vector of the muscovite \n mica crystal , which is defined by the mica, ( [ 110]mica, ) orientation for an  \n (  ) terminated surface .", "the epitaxial growth of ternaphtalene \n ( nnn ) on muscovite mica(001 ) has been investigated by combining structural \n ( xrd - pf ) and morphological ( sfm ) methods .", "consistently , both methods \n reveal the formation of s ( 001 ) orientated nnn island - like structures \n which have nucleated at the sidewalls of b ( 111 ) orientated fibers . \n", "it is demonstrated that the latter nnn crystal types tend to align \n along two dominant directions , which leads to the formation of a v - shaped \n sample fiber morphology .", "because the tilt angle of nnn molecules within \n s - orientated crystallites correlates with the tilt angle of the fiber \n side facets , the island nucleation is explained by  ledge directed \n epitaxy  .", "based on this growth model , it can be understood \n that both crystal types provide a well - defined azimuthal orientation \n relative to the muscovite mica substrate . by use of force field \n simulations , the growth of the fibers is further analyzed .", "the epitaxial \n growth of sexithiophene ( 6 t ) on muscovite mica showed the formation \n of four well - defined fiber orientations , which can be explained by \n mirror symmetry of the muscovite mica substrate and two differently \n stacked 6 t crystallites , which can nucleate at a molecular adsorption \n position .", "contrarily , experimental investigations \n indicate that nnn crystallites tend to stack in a single configuration , \n which explains the dominant formation of only two fiber orientations . \n based on force field simulations ,", "the latter observation is further \n investigated and explained by significantly different adsorption energies \n of both crystal types .", "it is further demonstrated that the observed \n behavior results from an interplay of the molecular adsorption and \n lattice match ."]]}
{"article_id": "PMC5175095", "article_text": ["a-38-year - old female patient consulted to our emergency outpatient clinic with complaints of fever , sore throat , coughing , shivering , chills , joint , and muscle pain , headache , chest pain continuing for nearly 2 weeks and she was hospitalized to investigate the etiology of fever .", "physical examination findings were as follows : bp 130/80 mm hg , body temperature , 39.8 c ; hr 120/min ; moderately well general physical status with full cooperation , markedly hyperemic pharynx , and tonsils , herpetic rashes around her lips , bilaterally diminished respiratory sounds heard over basal segments and tachycardia .", "her neck rigidity was evaluated however the kernig , and brudzinsky signs could not be elicited .", "laboratory test results were as follows : hb 11 g / dl ( 12.117.2 ) , mcv 80.8 fl ( 82.299 ) , wbc 22.10 k / ul ( 410 ) , neu 90.5% ( 3773% ) , esr 92 mm / s ( 020 ) , procalcitonin 0.8776 ng / ml ( < 0.1 ) , total protein 5.66 g / dl ( 6.48.3 ) , albumin 2.9 g / dl ( 3.55 ) , inr 1.15 ( 11.5 ) , ferritin 2889 ng / ml ( 14150 ) .", "glucose , creatinine , ast , alt , alp , na , k , complete urinalysis , ana , rf , and ena profile were unremarkable .", "on thoracal imagings , bilateral hilar lymph nodes of which the largest being 2 cm in diameter , bilateral pleural effusion , and atelectasic areas in the parenchyma surrounding the pleural effusion were observed ( figure 1 , 2 ) . on abdominal tomograms ,", "no bacterial growth was detected in urine , throat , csf , and blood cultures .", "lumbar punction performed because of suspect neck rigidity did not reveal any abnormal cells in csf .", "cephtriaxone at daily parenteral doses of 2 g ) . during follow - up period , rashes , and swelling on her right , and", "left wrists were detected , so she was consulted to rheumatology clinic , and naproxen sodium therapy was started with the indication of suspect aosd . on physical examination dullness over traube s space", "however her hyperfebrile state persisted , echocardiograms obtained for the differential diagnosis of infective endocarditis .", "posteroanterior chest radiogram costodiaphragmatic sinuses are not distinctly visualized , high - lying diaphragms , and an enlarged mediastinum were observed . biochemical analysis of the fluid drained by thoracenthesis of the patient whose computed tomograms demonstrated bilateral pleural effusion revealed the presence of 2 gm / dl albumin , 3.34 g / dl protein , 483 u / l ldh ( concurrent serum albumin 2.9 gm / dl , total protein 5.99 g / dl , ldh 393 u / l ) .", "antibiotherapy of the patient with pleural effusion ( exudate ) was discontinued , and methylprednisolone treatment at daily doses of 16 mg was initiated .", "after initiation of steroid therapy , she maintained an afebrile state , her joint manifestations regressed , and her complaints decreased during her follow - up period .", "the patient whose pleural effusion completely regressed with steroid therapy discharged to be further followed up by rheumatology outpatient clinic ( figure 3 ) .", "adult onset still s disease is a disease whose diagnosis can be made by excluding infectious , malign , autoimmune , and autoinflammatory diseases , and in consideration of its characteristic clinical , and laboratory findings . for definitive diagnosis , yamaguchi criteria can be used .", "its major criteria consists of higher body temperature at 39c lasting for at least one week , marked nonpruritic macular / maculopapular salmon - colored rashes on the torso or extremities persisting for at least 2 weeks , leukocytosis ( > 10.000/ml ) , and neutrophilia ( > 80% ) .", "minor criteria include sore throat , lymphadenopathy , hepatomegaly or splenomegaly , abnormal hepatic function tests ( increases in especially ast , alt , and ldh levels ) , and ana , and rf negativities . for the establishment of diagnosis , 5 diagnostic criteria ( including at least 2 major criteria ) should be met . in our patient , during approximately 4 weeks up to initiation of steroid therapy , fever exceeding 39c every day , symptoms of arthralgia , marked arthritis of both wrists , leukocytosis , neutrophilia , sore throat , and lymphadenopathies fulfilled the required diagnostic criteria , and pleural exudate seen during the clinical course of the disease was assessed to be associated with the disease . in the adult onset still", "s disease pleural effusion , transient pulmonary infiltration , and pericarditis can be seen in 3040% of the patients .", "affected individuals may complain of mild episodes of coughing , pleuritic chest pain , and shortness of breath .", "even in some patients it may progress to acute respiratory distress syndrome ( ards ) [ 6 , 7 ] . rarely , myocarditis , arrhytmias , heart failure , and cardiac tamponade can be seen .", "myocarditis , and ards are more frequently seen especially in patients with mas . in the differential diagnosis , infectious diseases ( ebv , cmv , hbv , rubella , parvovirus , coxsackie , hiv , subacute bacterial endocarditis , meningococcemia , tuberculosis , syphilis ) ; vasculites ( takayasu arteritis , polyarteritis nodosa , serum disease , thrombotic thrombocytopenic purpura ) ; malign diseases ( leukemia , lymphoma ) , connective tissue diseases ( ra , sle , mixed connective tissue disease ) , granulomatous diseases ( sarcoidosis , crohn disease ) ; autoinflammatory diseases ( kikuchi disease , sweet syndrome ) , and drug hypersensitivity reactions should be considered . in our patient", "serological tests , cultures , and imaging modalities were used for differential diagnosis which led to the establishment of the diagnosis of aosd .", "higher ferritin levels are among the most important laboratory findings of aosd , however markedly lower glycosylated ferritin levels are detected ( < 20% ) .", "increased ferritin level is an indicator of the disease activity , and also a serologic marker of treatment response . in our patient", "ferritin levels may increase as a response of hepatocytes to cytokines as il-18 , and il-1b which may be thought to play a role in the pathogenesis of aosd .", "as the first - line therapy nsaids ( especially endomethacin ) , and corticosteroids are recommended . in cases of severe organ involvement pulsatile high doses of steroids for few days can be used , than daily dose is dropped down to 1 mg / kg . in cases of recurrence or with the intention to decrease steroid dose , methotrexate at a weekly dose of 7.525 mg can be given .", "hydroxychloroquine , sulphasalazine , azothioprine , cyclosporine can be used separately or in combination with methotrexate . in recent years ,", "effectiveness of biological agents ( infliximab , etanercept , anakinra , and tocilizumab ) , and ivig therapies have been reported in the treatment patients with serious organ involvement , and mas .", "still many characteristic features of aosd are not known , and a standard follow - up or a treatment protocol of the disease is not available . in patients who met diagnostic criteria , pleural effusion which may be seen in the clinical course of the disease should be presumably associated with the disease .", "besides regression of pleural effusion can be expected with the treatment of underlying desease , additional locally invasive diagnostic , and/or therapeutic interventions should be avoided ."], "abstract_text": ["<S> adult onset still s disease is a rare systemic inflammatory disorder . at the onset of the disease sore throat , pharyngitis ; which does not respond to antibiotics , one or two times peaking febrile episodes , marked salmon - colored rash on the trunk and extremities , arthralgia , arthritis , myalgia , fatigue , loss of appetite with nausea and weight loss ; hepatosplenomegaly and lymphadenopathy can be seen . among laboratory examinations levels of ferritin and other acute phase reactants </S>", "<S> distinctly rise , and neutrophilic leukocytosis ; ana and rf negativity are detected . </S>", "<S> pleural and pericardial effusions , transient pulmonary infiltration , and rarely myocarditis can be seen during the course of the disease . here </S>", "<S> we report a patient who was examined for fever of unknown origin and diagnosed with adult onset still s disease which is a rare etiology of pleural effusion . </S>"], "labels": null, "section_names": ["CASE REPORT", "DISCUSSION"], "sections": [["a-38-year - old female patient consulted to our emergency outpatient clinic with complaints of fever , sore throat , coughing , shivering , chills , joint , and muscle pain , headache , chest pain continuing for nearly 2 weeks and she was hospitalized to investigate the etiology of fever .", "physical examination findings were as follows : bp 130/80 mm hg , body temperature , 39.8 c ; hr 120/min ; moderately well general physical status with full cooperation , markedly hyperemic pharynx , and tonsils , herpetic rashes around her lips , bilaterally diminished respiratory sounds heard over basal segments and tachycardia .", "her neck rigidity was evaluated however the kernig , and brudzinsky signs could not be elicited .", "laboratory test results were as follows : hb 11 g / dl ( 12.117.2 ) , mcv 80.8 fl ( 82.299 ) , wbc 22.10 k / ul ( 410 ) , neu 90.5% ( 3773% ) , esr 92 mm / s ( 020 ) , procalcitonin 0.8776 ng / ml ( < 0.1 ) , total protein 5.66 g / dl ( 6.48.3 ) , albumin 2.9 g / dl ( 3.55 ) , inr 1.15 ( 11.5 ) , ferritin 2889 ng / ml ( 14150 ) .", "glucose , creatinine , ast , alt , alp , na , k , complete urinalysis , ana , rf , and ena profile were unremarkable .", "on thoracal imagings , bilateral hilar lymph nodes of which the largest being 2 cm in diameter , bilateral pleural effusion , and atelectasic areas in the parenchyma surrounding the pleural effusion were observed ( figure 1 , 2 ) . on abdominal tomograms ,", "no bacterial growth was detected in urine , throat , csf , and blood cultures .", "lumbar punction performed because of suspect neck rigidity did not reveal any abnormal cells in csf .", "cephtriaxone at daily parenteral doses of 2 g ) . during follow - up period , rashes , and swelling on her right , and", "left wrists were detected , so she was consulted to rheumatology clinic , and naproxen sodium therapy was started with the indication of suspect aosd . on physical examination dullness over traube s space", "however her hyperfebrile state persisted , echocardiograms obtained for the differential diagnosis of infective endocarditis .", "posteroanterior chest radiogram costodiaphragmatic sinuses are not distinctly visualized , high - lying diaphragms , and an enlarged mediastinum were observed . biochemical analysis of the fluid drained by thoracenthesis of the patient whose computed tomograms demonstrated bilateral pleural effusion revealed the presence of 2 gm / dl albumin , 3.34 g / dl protein , 483 u / l ldh ( concurrent serum albumin 2.9 gm / dl , total protein 5.99 g / dl , ldh 393 u / l ) .", "antibiotherapy of the patient with pleural effusion ( exudate ) was discontinued , and methylprednisolone treatment at daily doses of 16 mg was initiated .", "after initiation of steroid therapy , she maintained an afebrile state , her joint manifestations regressed , and her complaints decreased during her follow - up period .", "the patient whose pleural effusion completely regressed with steroid therapy discharged to be further followed up by rheumatology outpatient clinic ( figure 3 ) ."], ["adult onset still s disease is a disease whose diagnosis can be made by excluding infectious , malign , autoimmune , and autoinflammatory diseases , and in consideration of its characteristic clinical , and laboratory findings . for definitive diagnosis , yamaguchi criteria can be used .", "its major criteria consists of higher body temperature at 39c lasting for at least one week , marked nonpruritic macular / maculopapular salmon - colored rashes on the torso or extremities persisting for at least 2 weeks , leukocytosis ( > 10.000/ml ) , and neutrophilia ( > 80% ) .", "minor criteria include sore throat , lymphadenopathy , hepatomegaly or splenomegaly , abnormal hepatic function tests ( increases in especially ast , alt , and ldh levels ) , and ana , and rf negativities . for the establishment of diagnosis , 5 diagnostic criteria ( including at least 2 major criteria ) should be met . in our patient , during approximately 4 weeks up to initiation of steroid therapy , fever exceeding 39c every day , symptoms of arthralgia , marked arthritis of both wrists , leukocytosis , neutrophilia , sore throat , and lymphadenopathies fulfilled the required diagnostic criteria , and pleural exudate seen during the clinical course of the disease was assessed to be associated with the disease . in the adult onset still", "s disease pleural effusion , transient pulmonary infiltration , and pericarditis can be seen in 3040% of the patients .", "affected individuals may complain of mild episodes of coughing , pleuritic chest pain , and shortness of breath .", "even in some patients it may progress to acute respiratory distress syndrome ( ards ) [ 6 , 7 ] . rarely , myocarditis , arrhytmias , heart failure , and cardiac tamponade can be seen .", "myocarditis , and ards are more frequently seen especially in patients with mas . in the differential diagnosis , infectious diseases ( ebv , cmv , hbv , rubella , parvovirus , coxsackie , hiv , subacute bacterial endocarditis , meningococcemia , tuberculosis , syphilis ) ; vasculites ( takayasu arteritis , polyarteritis nodosa , serum disease , thrombotic thrombocytopenic purpura ) ; malign diseases ( leukemia , lymphoma ) , connective tissue diseases ( ra , sle , mixed connective tissue disease ) , granulomatous diseases ( sarcoidosis , crohn disease ) ; autoinflammatory diseases ( kikuchi disease , sweet syndrome ) , and drug hypersensitivity reactions should be considered . in our patient", "serological tests , cultures , and imaging modalities were used for differential diagnosis which led to the establishment of the diagnosis of aosd .", "higher ferritin levels are among the most important laboratory findings of aosd , however markedly lower glycosylated ferritin levels are detected ( < 20% ) .", "increased ferritin level is an indicator of the disease activity , and also a serologic marker of treatment response . in our patient", "ferritin levels may increase as a response of hepatocytes to cytokines as il-18 , and il-1b which may be thought to play a role in the pathogenesis of aosd .", "as the first - line therapy nsaids ( especially endomethacin ) , and corticosteroids are recommended . in cases of severe organ involvement pulsatile high doses of steroids for few days can be used , than daily dose is dropped down to 1 mg / kg . in cases of recurrence or with the intention to decrease steroid dose , methotrexate at a weekly dose of 7.525 mg can be given .", "hydroxychloroquine , sulphasalazine , azothioprine , cyclosporine can be used separately or in combination with methotrexate . in recent years ,", "effectiveness of biological agents ( infliximab , etanercept , anakinra , and tocilizumab ) , and ivig therapies have been reported in the treatment patients with serious organ involvement , and mas .", "still many characteristic features of aosd are not known , and a standard follow - up or a treatment protocol of the disease is not available . in patients who met diagnostic criteria , pleural effusion which may be seen in the clinical course of the disease should be presumably associated with the disease .", "besides regression of pleural effusion can be expected with the treatment of underlying desease , additional locally invasive diagnostic , and/or therapeutic interventions should be avoided ."]]}
{"article_id": "PMC3571514", "article_text": ["dual - polymerizing resin cements have been extensively used for placement of indirect restorations and posts .", "the dual - polymerizing materials were developed to compensate for the lack of polymerization in the absence of light and to represent a combination of auto- and light - polymerizing components . in some clinical situations , such as in dark zones at the apical region and during the cementation of indirect restorations , the severe light attenuation results in low degree of conversion ( dc ) , which can compromise the mechanical properties and consequently the longevity of the indirect restorations.14 modifications in the viscosity of the resin cements allow their use in different clinical situations .", "the option for low viscosity versions offer some benefits , such as minor thickness of the pellicle that was formed following the restoration placement .", "the lowest film thickness generates smaller polymerization shrinkage , reducing the possibility of gaps formation and premature marginal leakage.5,6 the difference in the cement formulations that change the viscosity is related to the proportion between resin matrix and filler particle content.7 high volume fraction of fillers can increase the viscosity and the elastic modulus and strength of composites.7,8 low viscosity or flowable resins and resin cements present lower filler loading than regular restorative materials .", "most direct dental restorative composites use bisphenol - a - diglycidylether dimethacrylate ( bis - gma ) , which is considered a very viscous monomer , and when mixed with higher filler loadings , it becomes a nearly solid mass and unusable product .", "vinyl groups ( e.g. , ethylene glycol dimethacrylate ) are added as a thinner or diluent monomer for uncured pastes , which are considered another approach to change the viscosity of resin - based materials .", "the filler loading and the viscosity of composites may interfere in the monomer conversion , since they could restrict the mobility of monomers and the propagation of polymerization reaction.7,911 the restorative resin - based materials must reach a high degree of monomer conversion in order to present better clinical performance and longevity and also to reduce the early failures . for the dual - polymerizing resin cements", ", the self - cure mode should ensure the high level of conversion , especially in the cervical proximal areas , the root canal and in the internal and deep areas of the cavity preparations.1214 several methodologies have been used to analyze the dc of resin - based materials ; however , most of them use fourier transform infrared spectroscopy ( ftir ) . although many researchers have evaluated the polymerization effectiveness to determine the physical and mechanical properties of dental materials,9,15,16 little information is known about the influence of different viscosities of dual - cured resin cements on their physical properties , such as the degree of conversion in situations of self - and dual - polymerization .", "thus , the purpose of this study was to measure the dc of two commercially dual - cured resin cements in different viscosities ( low and high ) when they were light - activated or when the materials were allowed to self - cure solely , after 5 minutes and 24 hours from the mixture of pastes ( base and catalyst ) .", "the hypothesis tested was that curing mode , evaluation time and viscosity would affect the dc of the resin cements .", "two commercial dual - cure resin cements in high and low viscosities were evaluated : variolink ii ( ivoclar vivadent , schaan , liechtenstein ) and nexus 2 ( kerr corp . , orange , ca , usa ) .", "the resin cements consist of two paste components that were equally dispensed and mixed together according to manufacturers instructions . after mixing , resin cements ( n=5 )", "were applied to the horizontal attenuated total reflectance znse crystal at 45 ( fourier transform infrared spectrometer , ft - ir spectrometer 520 , nicolet instrument corp , madson , wi , usa ) at room temperature .", "adhesive tape ( 3 m , sumar , sp , brazil ) was placed on the znse crystal surface to act as a spacer , ensuring standard thickness for all specimens ( 100120 mm ) . a mylar strip ( quimidrol com .", "ltda , joinville , sc , brazil ) was placed over the cement and was pressed flat to spread the material on the crystal surface .", "the spectrum of unpolymerized material was obtained and the cements were either light - cured ( dual - polymerizing mode ) or allowed to self - cure only ( autopolymerizing mode ) .", "each specimen was left on the crystal surface and further spectra were obtained 5 minutes and 24 hours after post mixing . for light - cured groups , a halogen light curing unit ( xl 3000 , 3 m espe , st .", "paul , mn , usa ) was used during 40 seconds (  600 mw / cm ) .", "the light intensity was periodically checked with a radiometer ( curing radiometer , model 100 , kerr corp . , orange , ca , usa ) .", "spectra were obtained between 4000 cm and 750 cm at a resolution of 4 cm .", "monomer conversion was calculated ( % ) according to the changes in the ratio between the absorbance peaks corresponding to the aliphatic ( cc ) ( 1638 cm ) and aromatic ( 1608 cm ) carbon double bonds prior to and 5 minutes and 24 hours after polymerization initiation for both curing modes.10 the aromatic peak is used as an internal reference because the intensity does not change during the polymerization reaction.9,11 the effect of curing mode , viscosity and time intervals on the dc were evaluated for each material .", "three - way repeated measure analysis of variance ( anova ) ( curing mode , viscosities and evaluation time ) was performed and tukey s post - hoc test was used to detect pair wise differences among experimental groups .", "the data was analyzed using the statistical program sas 9.1 ( sas institute , cary , nc , usa ) and all statistical testing was performed at a pre - set alpha of 0.05 ( p<.05 ) .", "the statistical analysis of the data showed significant differences in curing mode , viscosity and evaluation time factors for both resin cements ( p=.01 ) .", "there was significant interaction between factors : the curing mode x evaluation time ( p=.01 ) .", "other double - interactions and the triple - interaction ( curing mode x viscosity x evaluation time ) were not significant ( p>.05 ) .", "the dc of resin cements was higher when the materials were light - cured and when the low - viscosity versions were used than when self - curing mode and the high - viscosity resin cements were tested , respectively ( p<.05 ) .", "the results of this study showed that the curing mode ( auto- and dual - polymerization ) , the viscosity ( low or high ) and the evaluation time ( 5 minutes or 24 hours ) influenced the dc of resin cements .", "thus , the research hypothesis stating that these factors would result in significant changes in the dc was accepted . as the dc is dependent on the monomeric composition and the concentration and type of the photoinitiator in each product,9,17 the statistical analysis comparing the dc between resin cements", "the main factors under study ( curing mode , viscosities and time intervals ) produced similar effects for the resin cements , which presented different formulations regarding the resin matrix composition and type and concentration of filler particles . the variations on the proportion of components of resin matrix and filler particles that produced the low and the high versions caused no difference in behavior between the two cements results.7 a high monomer conversion with cross - linked polymeric network formation is essential for the durability of the resin - based restorations.911 studies have shown the importance of a high dc of resinous materials in order to reach better mechanical properties , since the presence of a high amount of residual methacrylate monomers compromises the hardness , the abrasion and the fracture resistance . moreover", ", the low dc can increase the sorption and the solubility , interfering in the color stability and mass loss of the resin cement.2,4,9,14,1720 the resin cements showed significant difference between the evaluation times , 5 minutes and 24 hours from the polymerization initiation , independently of viscosity or curing mode .", "thus , the polymerization reaction for both resin cements continued after 5 minutes of the mixing of the base and catalyst pastes .", "the continuation of the chemical part of the polymerization reaction ( benzoyl peroxide + tertiary amines ) was primarily responsible for the increase of the dc after 24 hours .", "additionally , the percentage of dc increasing after 24 hours was higher for the self - cure mode than the dual cure mode , which ranged from 38.3 to 42.4% and from 26.6 to 28.4% , respectively . the continuation reaction 24 hours after polymerization initiation resulted in a reduction of absorbance peaks that corresponded to the aliphatic carbon double bonds10 and", "the dual polymerization increased dc , which corroborates with several studies that compared the polymerization modes for resin - based materials.2,11,21,22 for both resin cements used in this study , the direct light - polymerization provided dc that was higher and ranged from 10 to 30% higher than those observed for the self - curing mode . also , it was higher at 5 minutes ( 24 to 30% ) than for 24 hours ( 10 to 12% ) from the polymerization initiation", ". the auto - polymerization may be insufficient to result in an adequate dc for the long lasting restorations .", "clinically , the low viscosity version ensured higher dc , and 24 hours elapsed time after setting of restoration is appropriate for the final appointment , since the higher dc could better support the final oclusal adjustments , finishing , and polishing procedures .", "the direct light - polymerization can be considered a limitation of this study , since the light is applied directly to the resin cement layer only at the periphery of restorations .", "on the other hand , the inner part of indirect restorations is reached by an attenuated light and resin cement may depend only on self - cure activation . regarding the differences in the dc between high and low viscosities , higher mean values were found for low viscosity versions of both resin cements .", "low viscosity materials can be due to lower filler loading and/or higher addition low viscosity diluents monomers .", "the low viscosity monomer allows better mobility and distribution of free radicals inside the resin material , which can increase the polymerization reaction and the monomer conversion.9 the increase of dc promoted by the low viscosity versions in percentage , following the polymerization mode ranged from 0.9 to 3.1% for the dual - polymerizing groups and ranged from 1.5 to 3.9% for the auto - polymerizing mode , which was the highest percentage among the groups from nexus 2 resin cement . for the variolink ii", ", dual - polymerizing groups ranged from 1.4 to 2.8% , while the auto - polymerizing mode ranged from 1.7 to 2.4% .", "the direct light - activation and the low viscosity version were important to provide higher dc for the dual - polymerizing resin cements .", "twenty - four hours after seating the indirect restoration is appropriate for final oclusal adjustments , finishing , and polishing procedures , which are capable of generating stress to the resin cement layer ."], "abstract_text": ["<S> objective : the purpose of this study was to evaluate the effects of the viscosities and curing modes on the degree of conversion ( dc ) of two resin cements.methods:eight experimental groups were evaluated ( n=5 ) , according to the dual - cured resin cements ( nexus 2/variolink ii ) , viscosity ( low and high ) and evaluation time ( 5 minutes and 24 hours ) . </S>", "<S> the resin cements were applied to surface of a horizontal attenuated - total - reflectance unit and were polymerized either with self - cure ( sc ) or light exposure ( xl3000/3 m espe ) for 40 seconds . </S>", "<S> infrared spectra were obtained after 5 minutes and 24 hours ( nicolet 520 ft - ir / thermo scientific inc . ) . </S>", "<S> dc was calculated according to changes in aliphatic - to - aromatic peak ratios pre- and post - curing . </S>", "<S> data ( % ) were analyzed by 3-way repeated measure anova ( curing mode , viscosity and time interval ) and tukey s post - hoc test ( p<.05).results : the dual - polymerizing mode provided higher dc than auto - polymerization . </S>", "<S> the dc mean values increased for both resin cements after 24 hours . </S>", "<S> the low - viscosity resin cements from light - activated or self - cured groups exhibited higher dc than high viscosity version.conclusion:the dc of resin cements was higher for the low viscosity version , following the light - polymerization and when were tested after 24 hours . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS & MATERIALS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["dual - polymerizing resin cements have been extensively used for placement of indirect restorations and posts .", "the dual - polymerizing materials were developed to compensate for the lack of polymerization in the absence of light and to represent a combination of auto- and light - polymerizing components . in some clinical situations , such as in dark zones at the apical region and during the cementation of indirect restorations , the severe light attenuation results in low degree of conversion ( dc ) , which can compromise the mechanical properties and consequently the longevity of the indirect restorations.14 modifications in the viscosity of the resin cements allow their use in different clinical situations .", "the option for low viscosity versions offer some benefits , such as minor thickness of the pellicle that was formed following the restoration placement .", "the lowest film thickness generates smaller polymerization shrinkage , reducing the possibility of gaps formation and premature marginal leakage.5,6 the difference in the cement formulations that change the viscosity is related to the proportion between resin matrix and filler particle content.7 high volume fraction of fillers can increase the viscosity and the elastic modulus and strength of composites.7,8 low viscosity or flowable resins and resin cements present lower filler loading than regular restorative materials .", "most direct dental restorative composites use bisphenol - a - diglycidylether dimethacrylate ( bis - gma ) , which is considered a very viscous monomer , and when mixed with higher filler loadings , it becomes a nearly solid mass and unusable product .", "vinyl groups ( e.g. , ethylene glycol dimethacrylate ) are added as a thinner or diluent monomer for uncured pastes , which are considered another approach to change the viscosity of resin - based materials .", "the filler loading and the viscosity of composites may interfere in the monomer conversion , since they could restrict the mobility of monomers and the propagation of polymerization reaction.7,911 the restorative resin - based materials must reach a high degree of monomer conversion in order to present better clinical performance and longevity and also to reduce the early failures . for the dual - polymerizing resin cements", ", the self - cure mode should ensure the high level of conversion , especially in the cervical proximal areas , the root canal and in the internal and deep areas of the cavity preparations.1214 several methodologies have been used to analyze the dc of resin - based materials ; however , most of them use fourier transform infrared spectroscopy ( ftir ) . although many researchers have evaluated the polymerization effectiveness to determine the physical and mechanical properties of dental materials,9,15,16 little information is known about the influence of different viscosities of dual - cured resin cements on their physical properties , such as the degree of conversion in situations of self - and dual - polymerization .", "thus , the purpose of this study was to measure the dc of two commercially dual - cured resin cements in different viscosities ( low and high ) when they were light - activated or when the materials were allowed to self - cure solely , after 5 minutes and 24 hours from the mixture of pastes ( base and catalyst ) .", "the hypothesis tested was that curing mode , evaluation time and viscosity would affect the dc of the resin cements ."], ["two commercial dual - cure resin cements in high and low viscosities were evaluated : variolink ii ( ivoclar vivadent , schaan , liechtenstein ) and nexus 2 ( kerr corp . , orange , ca , usa ) .", "the resin cements consist of two paste components that were equally dispensed and mixed together according to manufacturers instructions . after mixing , resin cements ( n=5 )", "were applied to the horizontal attenuated total reflectance znse crystal at 45 ( fourier transform infrared spectrometer , ft - ir spectrometer 520 , nicolet instrument corp , madson , wi , usa ) at room temperature .", "adhesive tape ( 3 m , sumar , sp , brazil ) was placed on the znse crystal surface to act as a spacer , ensuring standard thickness for all specimens ( 100120 mm ) . a mylar strip ( quimidrol com .", "ltda , joinville , sc , brazil ) was placed over the cement and was pressed flat to spread the material on the crystal surface .", "the spectrum of unpolymerized material was obtained and the cements were either light - cured ( dual - polymerizing mode ) or allowed to self - cure only ( autopolymerizing mode ) .", "each specimen was left on the crystal surface and further spectra were obtained 5 minutes and 24 hours after post mixing . for light - cured groups , a halogen light curing unit ( xl 3000 , 3 m espe , st .", "paul , mn , usa ) was used during 40 seconds (  600 mw / cm ) .", "the light intensity was periodically checked with a radiometer ( curing radiometer , model 100 , kerr corp . , orange , ca , usa ) .", "spectra were obtained between 4000 cm and 750 cm at a resolution of 4 cm .", "monomer conversion was calculated ( % ) according to the changes in the ratio between the absorbance peaks corresponding to the aliphatic ( cc ) ( 1638 cm ) and aromatic ( 1608 cm ) carbon double bonds prior to and 5 minutes and 24 hours after polymerization initiation for both curing modes.10 the aromatic peak is used as an internal reference because the intensity does not change during the polymerization reaction.9,11 the effect of curing mode , viscosity and time intervals on the dc were evaluated for each material .", "three - way repeated measure analysis of variance ( anova ) ( curing mode , viscosities and evaluation time ) was performed and tukey s post - hoc test was used to detect pair wise differences among experimental groups .", "the data was analyzed using the statistical program sas 9.1 ( sas institute , cary , nc , usa ) and all statistical testing was performed at a pre - set alpha of 0.05 ( p<.05 ) ."], ["the statistical analysis of the data showed significant differences in curing mode , viscosity and evaluation time factors for both resin cements ( p=.01 ) .", "there was significant interaction between factors : the curing mode x evaluation time ( p=.01 ) .", "other double - interactions and the triple - interaction ( curing mode x viscosity x evaluation time ) were not significant ( p>.05 ) .", "the dc of resin cements was higher when the materials were light - cured and when the low - viscosity versions were used than when self - curing mode and the high - viscosity resin cements were tested , respectively ( p<.05 ) ."], ["the results of this study showed that the curing mode ( auto- and dual - polymerization ) , the viscosity ( low or high ) and the evaluation time ( 5 minutes or 24 hours ) influenced the dc of resin cements .", "thus , the research hypothesis stating that these factors would result in significant changes in the dc was accepted . as the dc is dependent on the monomeric composition and the concentration and type of the photoinitiator in each product,9,17 the statistical analysis comparing the dc between resin cements", "the main factors under study ( curing mode , viscosities and time intervals ) produced similar effects for the resin cements , which presented different formulations regarding the resin matrix composition and type and concentration of filler particles . the variations on the proportion of components of resin matrix and filler particles that produced the low and the high versions caused no difference in behavior between the two cements results.7 a high monomer conversion with cross - linked polymeric network formation is essential for the durability of the resin - based restorations.911 studies have shown the importance of a high dc of resinous materials in order to reach better mechanical properties , since the presence of a high amount of residual methacrylate monomers compromises the hardness , the abrasion and the fracture resistance . moreover", ", the low dc can increase the sorption and the solubility , interfering in the color stability and mass loss of the resin cement.2,4,9,14,1720 the resin cements showed significant difference between the evaluation times , 5 minutes and 24 hours from the polymerization initiation , independently of viscosity or curing mode .", "thus , the polymerization reaction for both resin cements continued after 5 minutes of the mixing of the base and catalyst pastes .", "the continuation of the chemical part of the polymerization reaction ( benzoyl peroxide + tertiary amines ) was primarily responsible for the increase of the dc after 24 hours .", "additionally , the percentage of dc increasing after 24 hours was higher for the self - cure mode than the dual cure mode , which ranged from 38.3 to 42.4% and from 26.6 to 28.4% , respectively . the continuation reaction 24 hours after polymerization initiation resulted in a reduction of absorbance peaks that corresponded to the aliphatic carbon double bonds10 and", "the dual polymerization increased dc , which corroborates with several studies that compared the polymerization modes for resin - based materials.2,11,21,22 for both resin cements used in this study , the direct light - polymerization provided dc that was higher and ranged from 10 to 30% higher than those observed for the self - curing mode . also , it was higher at 5 minutes ( 24 to 30% ) than for 24 hours ( 10 to 12% ) from the polymerization initiation", ". the auto - polymerization may be insufficient to result in an adequate dc for the long lasting restorations .", "clinically , the low viscosity version ensured higher dc , and 24 hours elapsed time after setting of restoration is appropriate for the final appointment , since the higher dc could better support the final oclusal adjustments , finishing , and polishing procedures .", "the direct light - polymerization can be considered a limitation of this study , since the light is applied directly to the resin cement layer only at the periphery of restorations .", "on the other hand , the inner part of indirect restorations is reached by an attenuated light and resin cement may depend only on self - cure activation . regarding the differences in the dc between high and low viscosities , higher mean values were found for low viscosity versions of both resin cements .", "low viscosity materials can be due to lower filler loading and/or higher addition low viscosity diluents monomers .", "the low viscosity monomer allows better mobility and distribution of free radicals inside the resin material , which can increase the polymerization reaction and the monomer conversion.9 the increase of dc promoted by the low viscosity versions in percentage , following the polymerization mode ranged from 0.9 to 3.1% for the dual - polymerizing groups and ranged from 1.5 to 3.9% for the auto - polymerizing mode , which was the highest percentage among the groups from nexus 2 resin cement . for the variolink ii", ", dual - polymerizing groups ranged from 1.4 to 2.8% , while the auto - polymerizing mode ranged from 1.7 to 2.4% ."], ["the direct light - activation and the low viscosity version were important to provide higher dc for the dual - polymerizing resin cements .", "twenty - four hours after seating the indirect restoration is appropriate for final oclusal adjustments , finishing , and polishing procedures , which are capable of generating stress to the resin cement layer ."]]}
{"article_id": "PMC1592160", "article_text": ["the task force on child health and maternal health for the united nations millennium development goals reiterated the challenges of achieving the goals in its report  who 's got the power ?", "transforming health systems for women and children .  while the technology and interventions exist to prevent or treat the vast majority of adverse health conditions that affect children and women of reproductive age , many continue to die and even more suffer from ill health [ 2 , 3 ] . the central challenge to achieving these goals is to provide known interventions to the full population and at the right time when most known adverse outcomes can be prevented . despite 15 years of the global  safe motherhood", "maternal and child health is connected to social , economic , and environmental conditions . in resource - poor countries , aside from pre - existing risks for poor maternal and child health outcomes , global changes have introduced new risks to the health of women and pregnancy , including alcohol and tobacco use , over nutrition , and micronutrient malnutrition [ 5 , 6 ] .", "because not all the deficits in achieving adequate prenatal care are likely to be solved in the near future , and global factors that lead to increases in risk can not be reversed , new strategies and policies to improve pregnancy outcome remain a high priority .", "one emerging policy is the role of preconception care as a complement to prenatal care .", "this report highlights examples of national programs and projects indicating that such approaches are feasible and acceptable to the population .", "these case studies on preconception care that were presented at the first national summit on preconception care , atlanta , usa , in 2005 , range from a mature program in hong kong , to emerging programs in korea and belgium , and select program enhancement and recovery efforts in china .", "the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size .", "the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 .", "having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes .", "the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care .", "the service integrates  medical , counseling , and education services for couples planning conception to achieve the following outcomes : \n prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis", "is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", "the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated .", "help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects .", "help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", "at the first visit , the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors .", "this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing .", ", a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment", "is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist .", "regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills .", "the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation .", "this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk .", "the continuous flow of clients for the preconception service reflects public support and trust for the service .", "as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy ,", "an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea .", "the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels .", "education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services .", "service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby .", "the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling .", "the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of", "the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease .", "however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes .", "patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine .", "also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care .", "this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation .", "the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine .", "although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients .", "depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person .", "the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country .", "organization for birth and childhood  ) , is a belgian governmental organization charged with the protection and promotion of health of women and children . given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year .", "this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers .", "an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data .", "the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests .", "the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings .", "the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation .", "the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers .", "the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals .", "these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public .", "the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements .", "medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign .", "medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted", ". the final step will be the evaluation process to assess behavioral changes both within the community and at provider level .", "the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients .", "the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care .", "a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere .", "this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children .", "the unique premarital medical examination system prevalent in china   and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province .", "babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 .", "the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) .", "given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident .", "to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system .", "a feasibility survey in the region had indicated that the majority of the population and public health community were supportive of the concept .", "the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women .", "to facilitate pvct and the antenatal vct and antiretroviral treatment component , 40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions ,", "942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process ,", "training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area .", "an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations .", "the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements .", "information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) .", "the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 .", "our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community .", "a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies .", "given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed . in october 2003 ,", "the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary .", "reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 .", "we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan .", "overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials .", "the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term  voluntary  by women to mean  unnecessary . ", "the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation .", "in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government .", "recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system .", "such recommendations were shared with all stakeholders . following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services .", "the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size .", "the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 .", "having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes .", "the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care .", "the service integrates  medical , counseling , and education services for couples planning conception to achieve the following outcomes : \n prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis", "is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", "the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated .", "help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects .", "help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", ", the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors .", "this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing .", ", a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment", "is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist .", "regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills .", "the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation .", "this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk .", "the continuous flow of clients for the preconception service reflects public support and trust for the service .", "as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy ,", "an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea .", "the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels .", "education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services .", "service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby .", "the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling .", "the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of", "the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease . however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes .", "patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine .", "also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care .", "this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation .", "the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine .", "although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients .", "depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person . since the establishment of preconception services in 2004", "the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country .", "the o.n.e (  office de la naissance et de lenfance  =  organization for birth and childhood  ) , is a belgian governmental organization charged with the protection and promotion of health of women and children .", "given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year .", "this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers .", "an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data .", "the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests .", "the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings .", "the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation .", "the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers .", "the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals .", "these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public .", "the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements .", "medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign .", "medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted", ". the final step will be the evaluation process to assess behavioral changes both within the community and at provider level .", "the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients .", "the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care .", "a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere .", "this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children .", "the unique premarital medical examination system prevalent in china   and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province .", "pilot experience in babu county in guangxi province is outlined here . babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 .", "the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) .", "given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident .", "to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system . a feasibility survey in the region", "had indicated that the majority of the population and public health community were supportive of the concept .", "the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women . to facilitate pvct and the antenatal vct and antiretroviral treatment component ,", "40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions ,", "942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process ,", "training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area .", "an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations .", "the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements .", "information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) .", "the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 .", "our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community .", "a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies .", "given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed .", "in october 2003 , the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary .", "reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 .", "we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan .", "overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials .", "the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term  voluntary  by women to mean  unnecessary . ", "the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation .", "in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government .", "recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system .", "following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services .", "as illustrated by the six examples highlighted above , it is evident that preconception care is not just a conceptual debate but a primary approach used to address various health issues and emerging national challenges . as noted so cogently by atrash et  al .  ", "a healthy baby and a healthy mother are valued hopes and dreams of families and cultural heritages across the world .", "further , in the countries such as south korea and hong kong , and others in similar demographic transition , this ", "although adequate prenatal , obstetric , and primary care services can reduce the infant and maternal mortality in high - mortality - developing countries , such countries are not free from emerging risks to maternal and child outcomes .", "tobacco and alcohol use rates among women in many developing countries have increased . however , as more women than before have access to education and information , are employed , have personal income and decision making power , and delay pregnancy , there are many opportunities to inform them about the need for preconception care and a healthy reproductive life . indeed , even a perfectly organized preconception care system is not likely to address other deficits in the reproductive health care system or in an inadequate prenatal care system in developing countries ; neither will it lead to rapid positive changes in related indicators .", "similar to the arguments in 1990s for and against hiv voluntary counseling services amidst the lack of availability of antiretroviral treatment , or against the attempts to market cell phones where land phones services are inadequate , advocating preconception care as a component of maternal child care services will remain controversial . as such , establishment of preconception care services", "in fact , introduction to preconception care can complement the efforts to improve prenatal care uptake .", "consistent with the conceptual framework of the 2005 bangkok charter on health promotion , preconception care should be seen as a program for the future , a development agenda that is aimed at overall health of the family .", "making preconception care available can have transgenerational impact on some women ; those who are aware of such opportunities and can access such services . our responsibility is to provide that opportunity ."], "abstract_text": ["<S> globally , maternal and child health faces diverse challenges depending on the status of the development of the country . </S>", "<S> some countries have introduced or explored preconception care for various reasons . </S>", "<S> falling birth rates and increasing knowledge about risk factors for adverse pregnancy outcomes led to the introduction of preconception care in hong kong in 1998 , and south korea in 2004 . in hong kong , comprehensive preconception care including laboratory tests </S>", "<S> are provided to over 4000 women each year at a cost of $ 75 per person . in korea , </S>", "<S> about 60% of the women served have known medical risk history , and the challenge is to expand the program capacity to all women who plan pregnancy , and conducting social marketing . </S>", "<S> belgium has established an ad hoc - committee to develop a comprehensive social marketing and professional training strategy for pilot testing preconception care models in the french speaking part of belgium , an area that represents 5 million people and 50,000 births per year using prenatal care and pediatric clinics , gynecological departments , and the genetic centers . in china , </S>", "<S> guangxi province piloted preconceptional hiv testing and counseling among couples who sought the then mandatory premarital medical examination as a component of the three - pronged approach to reduce mother to child transmission of hiv . </S>", "<S> hiv testing rates among couples increased from 38% to 62% over one year period . in october 2003 , </S>", "<S> china changed the legal requirement of premarital medical examination from mandatory to  voluntary .  </S>", "<S> this change was interpreted by most women that the premarital health examination was  unnecessary  and overall premarital health examination rates dropped . </S>", "<S> social marketing efforts piloted in 2004 indicated that 95% of women were willing to pay up to rmb 100 ( us$12 ) for preconception health care services . </S>", "<S> these case studies illustrate programmatic feasibility of preconception care services to address maternal and child health and other public health challenges in developed and emerging economies . </S>"], "labels": null, "section_names": ["Introduction", "Hong Kong: A done deal", "South Korea: An evolving initiative", "Belgium: Baby steps", "China: Preconception care as an HIV/AIDS intervention model", "China: Regaining the old glory", "Conclusions"], "sections": [["the task force on child health and maternal health for the united nations millennium development goals reiterated the challenges of achieving the goals in its report  who 's got the power ?", "transforming health systems for women and children .  while the technology and interventions exist to prevent or treat the vast majority of adverse health conditions that affect children and women of reproductive age , many continue to die and even more suffer from ill health [ 2 , 3 ] . the central challenge to achieving these goals is to provide known interventions to the full population and at the right time when most known adverse outcomes can be prevented . despite 15 years of the global  safe motherhood", "maternal and child health is connected to social , economic , and environmental conditions . in resource - poor countries , aside from pre - existing risks for poor maternal and child health outcomes , global changes have introduced new risks to the health of women and pregnancy , including alcohol and tobacco use , over nutrition , and micronutrient malnutrition [ 5 , 6 ] .", "because not all the deficits in achieving adequate prenatal care are likely to be solved in the near future , and global factors that lead to increases in risk can not be reversed , new strategies and policies to improve pregnancy outcome remain a high priority .", "one emerging policy is the role of preconception care as a complement to prenatal care .", "this report highlights examples of national programs and projects indicating that such approaches are feasible and acceptable to the population .", "these case studies on preconception care that were presented at the first national summit on preconception care , atlanta , usa , in 2005 , range from a mature program in hong kong , to emerging programs in korea and belgium , and select program enhancement and recovery efforts in china .", "the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size .", "the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 .", "having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes .", "the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care .", "the service integrates  medical , counseling , and education services for couples planning conception to achieve the following outcomes : \n prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis", "is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", "the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated .", "help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects .", "help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", "at the first visit , the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors .", "this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing .", ", a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment", "is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist .", "regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills .", "the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation .", "this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk .", "the continuous flow of clients for the preconception service reflects public support and trust for the service .", "as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy ,", "an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea .", "the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels .", "education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services .", "service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby .", "the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling .", "the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of", "the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease .", "however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes .", "patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine .", "also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care .", "this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation .", "the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine .", "although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients .", "depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person .", "the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country .", "organization for birth and childhood  ) , is a belgian governmental organization charged with the protection and promotion of health of women and children . given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year .", "this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers .", "an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data .", "the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests .", "the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings .", "the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation .", "the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers .", "the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals .", "these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public .", "the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements .", "medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign .", "medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted", ". the final step will be the evaluation process to assess behavioral changes both within the community and at provider level .", "the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients .", "the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care .", "a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere .", "this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children .", "the unique premarital medical examination system prevalent in china   and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province .", "babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 .", "the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) .", "given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident .", "to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system .", "a feasibility survey in the region had indicated that the majority of the population and public health community were supportive of the concept .", "the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women .", "to facilitate pvct and the antenatal vct and antiretroviral treatment component , 40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions ,", "942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process ,", "training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area .", "an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations .", "the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements .", "information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) .", "the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 .", "our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community .", "a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies .", "given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed . in october 2003 ,", "the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary .", "reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 .", "we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan .", "overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials .", "the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term  voluntary  by women to mean  unnecessary . ", "the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation .", "in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government .", "recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system .", "such recommendations were shared with all stakeholders . following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services ."], ["the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size .", "the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 .", "having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes .", "the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care .", "the service integrates  medical , counseling , and education services for couples planning conception to achieve the following outcomes : \n prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis", "is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", "the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus .", "thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .", "identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated .", "help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects .", "help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .", ", the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors .", "this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing .", ", a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment", "is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist .", "regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills .", "the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation .", "this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk .", "the continuous flow of clients for the preconception service reflects public support and trust for the service ."], ["as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy ,", "an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea .", "the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels .", "education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services .", "service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby .", "the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling .", "the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of", "the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease . however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes .", "patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine .", "also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care .", "this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation .", "the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine .", "although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients .", "depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person . since the establishment of preconception services in 2004", "the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country ."], ["the o.n.e (  office de la naissance et de lenfance  =  organization for birth and childhood  ) , is a belgian governmental organization charged with the protection and promotion of health of women and children .", "given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year .", "this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers .", "an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data .", "the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests .", "the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings .", "the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation .", "the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers .", "the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals .", "these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public .", "the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements .", "medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign .", "medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted", ". the final step will be the evaluation process to assess behavioral changes both within the community and at provider level .", "the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients .", "the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care ."], ["a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere .", "this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children .", "the unique premarital medical examination system prevalent in china   and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province .", "pilot experience in babu county in guangxi province is outlined here . babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 .", "the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) .", "given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident .", "to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system . a feasibility survey in the region", "had indicated that the majority of the population and public health community were supportive of the concept .", "the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women . to facilitate pvct and the antenatal vct and antiretroviral treatment component ,", "40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions ,", "942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process ,", "training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area .", "an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations .", "the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements .", "information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) .", "the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 .", "our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community .", "a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies .", "given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed ."], ["in october 2003 , the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary .", "reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 .", "we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan .", "overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials .", "the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term  voluntary  by women to mean  unnecessary . ", "the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation .", "in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government .", "recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system .", "following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services ."], ["as illustrated by the six examples highlighted above , it is evident that preconception care is not just a conceptual debate but a primary approach used to address various health issues and emerging national challenges . as noted so cogently by atrash et  al .  ", "a healthy baby and a healthy mother are valued hopes and dreams of families and cultural heritages across the world .", "further , in the countries such as south korea and hong kong , and others in similar demographic transition , this ", "although adequate prenatal , obstetric , and primary care services can reduce the infant and maternal mortality in high - mortality - developing countries , such countries are not free from emerging risks to maternal and child outcomes .", "tobacco and alcohol use rates among women in many developing countries have increased . however , as more women than before have access to education and information , are employed , have personal income and decision making power , and delay pregnancy , there are many opportunities to inform them about the need for preconception care and a healthy reproductive life . indeed , even a perfectly organized preconception care system is not likely to address other deficits in the reproductive health care system or in an inadequate prenatal care system in developing countries ; neither will it lead to rapid positive changes in related indicators .", "similar to the arguments in 1990s for and against hiv voluntary counseling services amidst the lack of availability of antiretroviral treatment , or against the attempts to market cell phones where land phones services are inadequate , advocating preconception care as a component of maternal child care services will remain controversial . as such , establishment of preconception care services", "in fact , introduction to preconception care can complement the efforts to improve prenatal care uptake .", "consistent with the conceptual framework of the 2005 bangkok charter on health promotion , preconception care should be seen as a program for the future , a development agenda that is aimed at overall health of the family .", "making preconception care available can have transgenerational impact on some women ; those who are aware of such opportunities and can access such services . our responsibility is to provide that opportunity ."]]}
{"article_id": "PMC1513055", "article_text": ["pleura is divided into a parietal layer \n which lines the inner aspect of the chest wall and a visceral \n layer which covers the interlobar fissures .", "fluid \n collection within the pleural cavity can be assessed with \n clinical and radiological means .", "when pleural effusion is \n detected , the characteristics of the fluid ( exudate or \n transudate ) must be revealed using thoracocentesis .", "pneumonias \n and congestive heart failure are the most frequently encountered \n causes of transudative and exudative effusions , respectively \n .", "accumulating knowledge about cytokines \n demonstrated their important roles in the pathogenesis of most of \n the infectious diseases .", "interleukins ( ils ) mediate the \n reactions of the our organism against foreign antigens and harmful \n agents . the ils act through autocrine or paracrine rather than \n endocrine pathways . in various infectious diseases significant \n alterations in il concentrations of blood , as well as of body \n fluids , occur .", "although il levels in serum and various body fluids \n have been studied in various diseases , very few researches \n involving il levels in pleural effusions of children have been \n conducted [ 3 , 4 ] .", "it is already acknowledged that \n established criteria used for the differentiation between \n exudative and transudative pleural effusions in adults are not \n valid for children and further studies are required to determine \n diagnostic criteria for children .", "the aim of this study \n is to compare il levels in exudative and transudative pleural \n fluids by measuring il levels in pleural effusions developed \n owing to various etiological factors in childhood .", "we aimed to \n examine whether the changes in pleural fluid protein , glucose , \n and lactate dehydrogenase ( ldh ) parameters , as well as in \n il-1 , il-2 , il-6 , and il-8 levels , were significant in \n differential diagnosis of childhood pleural effusions .", "the study group consisted of 26 patients admitted in the \n department of pediatrics , medical faculty , firat university with a \n diagnosis of pleural effusions .", "thoracal ultrasonograms were \n obtained from patients thought to have pleural effusions in \n consideration of medical history , physical examination , and chest \n x - rays . the presence and the amount of pleural fluids were \n determined and then thoracocentesis was performed .", "all samples \n were obtained by thoracentesis at the   diagnosis time \n ( table 1 , figure 1 ) .", "the first \n samples were centrifuged immediately for the measurement of il \n concentration and the supernatant layers were stored at \n 70c until testing for cytokines .", "the second samples \n were inoculated in aerobic , anaerobic , and lwenstein - jensen \n media and stained with gram , giemsa , and ziehl - nielsen \n dyes .", "the third samples were analysed for protein , glucose , and \n ldh . as indicators of infection ,", "peripheral leukocyte counts \n ( wbc ) , erythrocyte sedimentation rate ( esr ) , c - reactive protein \n ( crp ) measurements , blood culture , and purified protein derivate \n ( ppd ) tests were performed .", "pleural effusions were categorized as exudates and transudates \n according to the clinical and laboratory findings .", "the cutoff values for the differentiation \n between the pleural transudates and exudates were determined as \n follows : protein , 3  g / dl ; ldh , 200  iu / l ; pleural \n fluid / serum protein ratio , 0.5 ; and pleural fluid / serum ldh \n ratio , 0.6 .", "pleural effusions with values above these cutoffs \n were classified as exudates and those below as transudates . \n", "empyematous effusions were characterised by \n the presence of gram - positive bacteria in pleural fluid smears , \n attendant pneumonia with or without bacterial growth in the \n cultures of pleural fluid samples , and typical glucose \n ( < 40 mg / dl ) and/or protein ( > 5  g / dl ) and/or ldh \n ( > 1000 ", "the samples of pleural \n fluids with higher glucose concentrations ( > 40  mg / dl ) whose \n gram - stained smears did not reveal any specific characteristics \n or bacterial growth were considered as parapneumonic effusions . \n", "the patients whose pleural fluid smears revealed afb \n which could be cultured in lwenstein - jensen media and also \n responded favorably to the antituberculostatic treatment were \n acknowledged as cases with tuberculous pleurisy [ 5 , \n 6 , 7 ] .", "pleural fluids preserved at 70c were thawed and \n il-1 , il-2 , il-6 , and il-8 levels in the samples were \n measured using enzyme - linked immunosorbent assay ( elisa ) and \n il-1 , il-2 , il-6 , and il-8 bender medsystems commercial kits .", "the clinical and laboratory characteristics of the patients were \n given as means (  standard deviation ( sd ) ) .", "statistical \n evaluations between different groups were done with kruskal - wallis \n variance analysis and post - hoc tukey - scheffe tests using spss \n 10.0 software programs .", "in our study group pleural effusions were of exudative ( n = 27 , \n 75% ) and transudative ( n = 9 , 25% ) type . the ages of the study \n subjects ranged between 9 months and 14 years .", "twelve \n parapneumonic ( 44% ) , 12 empyematous ( 44% ) , and 3 ( 12% ) \n tuberculous pleural effusions were detected in the exudate group ( table 1 ) .", "a statistically significant difference was found between crp \n values   of the exudate ( 140.2  92.4  mg / dl ) and the \n transudate ( 9.6  3.2  mg / dl ) groups ( p < .001 ) \n ( figure 2 ) .", "protein levels \n in pleural fluids were measured as 5.1  1.2  g / dl and \n 1.6  0.5  g / dl in the exudate and the transudate groups , \n respectively ( p < .001 ) . glucose levels in pleural fluids were \n measured as 53.3  36.6  mg / dl in the exudate group and \n 83.8  14.7  mg / dl in the transudate group ( p < .001 ) \n ( figure 3 ) .", "il-1 levels of pleural fluids in the exudate and the \n transudate groups were detected as 304.1  127.7  pg / ml and \n 70.1  23.4 pg / ml , respectively ( p < .001 ) . \n", "il-1 levels \n were demonstrated as 210  43.5  pg / ml in the parapneumonic \n effusion group , 430.3  69.9 ", "pg / ml in the empyematous \n effusion group , and 175.3  36.3  pg / ml in the tuberculous \n effusion groups .", "statistically significantly higher values were \n found in the empyematous group when compared with the \n parapneumonic and tuberculous pleural effusion groups ( p < .001 ) .", "il-2 concentrations of pleural fluids were ascertained to be at \n undetectable levels in 12 ( 44.4% ) and 176.6  173.6  pg / ml in 15 ( 55.6% ) patients in the exudate group . \n", "statistically significant difference was \n uncovered between the exudate and the transudate groups ( p < .001 ) .", "statistically significant differences were not revealed \n between the empyematous group and the other two groups ( p > .05 ) . \n statistically significantly higher values were recognized in the \n tuberculous pleural effusion group rather than the parapneumonic \n effusion group ( p < .01 ) .", "il-6 levels were detected to be 18589.5  3631.8  pg / ml in \n the exudate group and 65.6  16.0  pg / ml in the transudate \n group ( p < .001 ) .", "the corresponding values were 15295.0  22678.1  pg / ml in the parapneumonic effusion group , \n 20518.0  1410.9 ", "pg / ml in the tuberculous pleural \n effusion group . in the empyematous group ,", "statistically \n significantly higher values were obtained when compared with \n those of the parapneumonic effusion \n group , while the corresponding values detected in the tuberculous \n pleural effusion group were also significantly higher than those \n found in the parapneumonic effusion group ( p < .001 ) .", "pg / ml in the exudate group and 108.8  92.0  pg / ml in the transudate group ( p < .001 ) .", "the \n concentrations of il-8 were 1884.5  366.7 in the \n parapneumonic effusion group , 4789.8  1013.2 ", "pg / ml in the \n empyematous effusion group and 919.0  9454.5  pg / ml in the \n tuberculous pleural effusion group .", "the values were statistically \n significantly higher in the empyematous group when compared with \n the other two groups ( p < .001 ) ( figure 4 ) .", "pleural effusion can be seen due to the intrusion of \n an infectious agent or an irritating foreign substance inside the \n pleural cavity or due a direct access of harmful materials or \n neoplastic cells into the pleural cavity via hematogenous route \n .", "it can be observed following pleural trauma or in \n association with asbestosis related pleural diseases .", "pleural effusions are known to develop \n secondary to trauma , cardiac , renal , collagenous diseases and \n malignancies besides pneumonia [ 9 , \n 10 , \n 11 ] . from \n 61% to 80% of the cases with pleural effusions", "empyema is also an important cause of \n mortality in the developing countries . in the remaining \n 20%39% of the cases ,", "empyema develops secondary to \n trauma , malignancies , and renal diseases [ 5 , \n 12 , 13 , \n 14 , 15 ] . \n in our study group", "many studies have been conducted concerning \n the levels of il-1 , il-2 , il-6 , and il-8 in body fluids in \n various infections .", "although many studies investigating the \n alterations in levels of il according to the exudative or \n transudative nature of pleural effusions in adults have been \n performed , similar studies pertaining to children are relatively \n few [ 16 , 17 , \n 18 ] .", "for that reason many studies have analysed \n il-1 levels in patients with meningitis , sepsis , urinary \n tract infections , and empyema .", "variable results have been obtained \n dependent on the presence of different etiological agents \n [ 17 , 18 ] . in patients with bacterial and tuberculous \n meningitis ,", "il-1 levels of cerebrospinal fluids were found \n to be significantly higher than those with aseptic meningitis and \n in the control group .", "umblical plasma levels of il-1 of \n infected newborns with early - onset sepsis were found to be higher \n than those of the control group .", "il-1 levels in synovial \n fluids were detected to be higher than those in the pediatric \n cases with   suppurative arthritis due to other causes . \n", "silva - mejias et al   have found comparatively higher \n il-1 levels in the empyema group .", "similar to our findings , \n alexandrakis et al   revealed that il-1 levels in \n pleural effusions were higher in the exudate group compared with \n those of the transudate group .", "naito et al   ascertained \n that il-1 levels in pleural effusions were higher than \n those found in bacterial infections due to other causes .", "in \n accordance with the literature in our study , il-1 levels \n were nearly 4.5 times higher in the exudate group .", "il-2 levels in pleural effusions due to malignancies were reported \n to be lower than those with tuberculous pleural effusions \n .", "shimokata et al \n  stated that il-2 levels were \n at undetectable levels in 25% of tuberculous pleural effusions \n and 75% of cancerous pleural effusions . in our study", "il-2 levels \n in pleural fluids were at undetectably lower levels in 44% of \n the patients in the exudate group while the il-2 levels of the \n remaining 56% were assessed as 176.6  33.4  pg / ml . \n", "however all the patients in the transudate group demonstrated \n undetectable il-2 levels . in our study", "a statistically \n significant difference was not present between the empyema group \n and the other two groups .", "however a statistically significant \n difference was detected between the parapneumonic and the \n parapneumonic pleural effusion groups .", "various studies have been performed concerning the \n levels of il-6 which is the mediator and the regulator of \n inflammatory responses in miscellaneous inflammatory \n processes .", "blood il-6 levels were found to be higher in children \n with mycoplasma pneumoniae infections who stayed febrile \n for more than 3 days compared with those having shorter febrile \n episodes .", "they were also \n  statistically significantly higher in pediatric cases \n with urinary tract infections when compared with the control \n groups .", "il-6 levels of cerebrospinal fluid in the patients with \n bacterial meningitis were reported to be significantly higher \n than the aseptic meningitis and in the control groups .", "levels above 100  pg / ml were measured in pediatric cases with \n viral ( 14% ) but especially with bacterial meningitis ( 53% ) . \n", "xirouchaki et al \n and yokoyama et al   reported \n the levels of il-6 in pleural fluids to be significantly higher \n in the exudate group rather than the transudate group .", "they also \n found il-6 concentrations significantly higher in the tuberculous \n group rather than the parapneumonic effusion group . in our study \n not", "only the levels of il-6 were different between the groups \n with parapneumonic and empyematous effusions , but at the same \n time statistically significant differences were found between the \n empyematous and the parapneumonic or tuberculous pleural effusion \n groups .", "alexandrakis et al   measured il-6 levels in \n serum and pleural fluids and ascertained that serum il-6 levels \n did not differ significantly between the exudate and the \n transudate groups .", "however pleural fluid il-6 levels were \n detected to be meaningfully higher in the exudate rather than the \n transudate group . a definite proportion between the serum and \n pleural fluid il-6 concentrations could not be assessed .", "however \n we found il-6 levels in the pleural fluid to be 285 times higher \n than those detected in the exudate group .", "il-8 is the mediator and the regulator of chemotaxis of \n leukocytes in inflammatory processes . in adults", "il-8 levels \n were found to be higher in cases with infectious pleural \n effusions compared with the patients with noninfectious \n  effusions   [ 3 , 26 , \n  27 , 28 , \n  29 , 30 , \n  31 , 32 ] . \n", "ceyhan et al   reported higher levels in \n empyematous / parapneumonic effusion groups rather than the \n tuberculous group .", "however antony et al   detected higher \n levels in cases with parapneumonic effusions unlike those found \n in the group with tuberculous effusions .", "il-8 concentrations in \n the empyematous group were reported to be higher than those \n measured in the parapneumonic group . in accordance with this \n study , in our study the highest levels of il-8 were detected in \n the empyematous , parapneumonic , and tuberculous pleural effusions \n in decreasing order .", "dlugovitzky et al   discovered \n statistically significantly higher il-8 values in the tuberculous \n pleural effusion group in contrast with   findings in the \n parapneumonic pleural effusion group .", "ashitani et al \n and broaddus et al   found higher levels in the empyema \n group compared with the other groups .", "miller and idell \n have detected   significantly higher levels in the exudate group \n rather than the transudate group . in this study ,", "similar to the \n findings of other   investigations , il-8 levels in the exudate \n group were reported to be 28 times higher than those found in the \n transudate group .", "this study have shown that for the differentiation between the \n exudative and transudative pleural effusions , in addition to the \n parametres such as   protein , glucose and ldh , pleural fluid \n il-1 , il-2 , il-6 , and il-8 levels could be used . \n", "etiological factors can be differentiated by determining pleural \n fluid il levels . taking these levels into consideration", "accordingly , a definitive diagnosis , a successful \n treatment and reduction in mortality can be achieved ."], "abstract_text": ["<S> the aim is to examine whether the changes in pleural fluid \n interleukin ( il)-1 , il-2 , il-6 , and il-8 levels were \n significant in differential diagnosis of childhood pleural \n effusions .   </S>", "<S> il-1 , il-2 , il-6 , and il-8 levels in pleural \n fluids of all 36 patients were measured . </S>", "<S> the levels of \n il-1 , il-2 , il-6 , and il-8 in pleural fluids were \n statistically significantly higher in the transudate group \n compared with those of the exudate group . </S>", "<S> the levels of \n il-1 , il-6 , and il-8 were also found to be statistically \n significantly higher in the empyema group compared with both the \n parapneumonic and the tuberculous pleural effusion groups . </S>", "<S> the \n levels of il-2 and il-6 were detected to be statistically \n significantly higher in the tuberculous pleural effusion group in \n comparison with those of the parapneumonic effusion group . \n  </S>", "<S> the results showed that pleural fluids \n il-1 , il-2 , il-6 , and il-8 could be used in pleural fluids \n exudate and transudate distinction . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["pleura is divided into a parietal layer \n which lines the inner aspect of the chest wall and a visceral \n layer which covers the interlobar fissures .", "fluid \n collection within the pleural cavity can be assessed with \n clinical and radiological means .", "when pleural effusion is \n detected , the characteristics of the fluid ( exudate or \n transudate ) must be revealed using thoracocentesis .", "pneumonias \n and congestive heart failure are the most frequently encountered \n causes of transudative and exudative effusions , respectively \n .", "accumulating knowledge about cytokines \n demonstrated their important roles in the pathogenesis of most of \n the infectious diseases .", "interleukins ( ils ) mediate the \n reactions of the our organism against foreign antigens and harmful \n agents . the ils act through autocrine or paracrine rather than \n endocrine pathways . in various infectious diseases significant \n alterations in il concentrations of blood , as well as of body \n fluids , occur .", "although il levels in serum and various body fluids \n have been studied in various diseases , very few researches \n involving il levels in pleural effusions of children have been \n conducted [ 3 , 4 ] .", "it is already acknowledged that \n established criteria used for the differentiation between \n exudative and transudative pleural effusions in adults are not \n valid for children and further studies are required to determine \n diagnostic criteria for children .", "the aim of this study \n is to compare il levels in exudative and transudative pleural \n fluids by measuring il levels in pleural effusions developed \n owing to various etiological factors in childhood .", "we aimed to \n examine whether the changes in pleural fluid protein , glucose , \n and lactate dehydrogenase ( ldh ) parameters , as well as in \n il-1 , il-2 , il-6 , and il-8 levels , were significant in \n differential diagnosis of childhood pleural effusions ."], ["the study group consisted of 26 patients admitted in the \n department of pediatrics , medical faculty , firat university with a \n diagnosis of pleural effusions .", "thoracal ultrasonograms were \n obtained from patients thought to have pleural effusions in \n consideration of medical history , physical examination , and chest \n x - rays . the presence and the amount of pleural fluids were \n determined and then thoracocentesis was performed .", "all samples \n were obtained by thoracentesis at the   diagnosis time \n ( table 1 , figure 1 ) .", "the first \n samples were centrifuged immediately for the measurement of il \n concentration and the supernatant layers were stored at \n 70c until testing for cytokines .", "the second samples \n were inoculated in aerobic , anaerobic , and lwenstein - jensen \n media and stained with gram , giemsa , and ziehl - nielsen \n dyes .", "the third samples were analysed for protein , glucose , and \n ldh . as indicators of infection ,", "peripheral leukocyte counts \n ( wbc ) , erythrocyte sedimentation rate ( esr ) , c - reactive protein \n ( crp ) measurements , blood culture , and purified protein derivate \n ( ppd ) tests were performed .", "pleural effusions were categorized as exudates and transudates \n according to the clinical and laboratory findings .", "the cutoff values for the differentiation \n between the pleural transudates and exudates were determined as \n follows : protein , 3  g / dl ; ldh , 200  iu / l ; pleural \n fluid / serum protein ratio , 0.5 ; and pleural fluid / serum ldh \n ratio , 0.6 .", "pleural effusions with values above these cutoffs \n were classified as exudates and those below as transudates . \n", "empyematous effusions were characterised by \n the presence of gram - positive bacteria in pleural fluid smears , \n attendant pneumonia with or without bacterial growth in the \n cultures of pleural fluid samples , and typical glucose \n ( < 40 mg / dl ) and/or protein ( > 5  g / dl ) and/or ldh \n ( > 1000 ", "the samples of pleural \n fluids with higher glucose concentrations ( > 40  mg / dl ) whose \n gram - stained smears did not reveal any specific characteristics \n or bacterial growth were considered as parapneumonic effusions . \n", "the patients whose pleural fluid smears revealed afb \n which could be cultured in lwenstein - jensen media and also \n responded favorably to the antituberculostatic treatment were \n acknowledged as cases with tuberculous pleurisy [ 5 , \n 6 , 7 ] .", "pleural fluids preserved at 70c were thawed and \n il-1 , il-2 , il-6 , and il-8 levels in the samples were \n measured using enzyme - linked immunosorbent assay ( elisa ) and \n il-1 , il-2 , il-6 , and il-8 bender medsystems commercial kits .", "the clinical and laboratory characteristics of the patients were \n given as means (  standard deviation ( sd ) ) .", "statistical \n evaluations between different groups were done with kruskal - wallis \n variance analysis and post - hoc tukey - scheffe tests using spss \n 10.0 software programs ."], ["in our study group pleural effusions were of exudative ( n = 27 , \n 75% ) and transudative ( n = 9 , 25% ) type . the ages of the study \n subjects ranged between 9 months and 14 years .", "twelve \n parapneumonic ( 44% ) , 12 empyematous ( 44% ) , and 3 ( 12% ) \n tuberculous pleural effusions were detected in the exudate group ( table 1 ) .", "a statistically significant difference was found between crp \n values   of the exudate ( 140.2  92.4  mg / dl ) and the \n transudate ( 9.6  3.2  mg / dl ) groups ( p < .001 ) \n ( figure 2 ) .", "protein levels \n in pleural fluids were measured as 5.1  1.2  g / dl and \n 1.6  0.5  g / dl in the exudate and the transudate groups , \n respectively ( p < .001 ) . glucose levels in pleural fluids were \n measured as 53.3  36.6  mg / dl in the exudate group and \n 83.8  14.7  mg / dl in the transudate group ( p < .001 ) \n ( figure 3 ) .", "il-1 levels of pleural fluids in the exudate and the \n transudate groups were detected as 304.1  127.7  pg / ml and \n 70.1  23.4 pg / ml , respectively ( p < .001 ) . \n", "il-1 levels \n were demonstrated as 210  43.5  pg / ml in the parapneumonic \n effusion group , 430.3  69.9 ", "pg / ml in the empyematous \n effusion group , and 175.3  36.3  pg / ml in the tuberculous \n effusion groups .", "statistically significantly higher values were \n found in the empyematous group when compared with the \n parapneumonic and tuberculous pleural effusion groups ( p < .001 ) .", "il-2 concentrations of pleural fluids were ascertained to be at \n undetectable levels in 12 ( 44.4% ) and 176.6  173.6  pg / ml in 15 ( 55.6% ) patients in the exudate group . \n", "statistically significant difference was \n uncovered between the exudate and the transudate groups ( p < .001 ) .", "statistically significant differences were not revealed \n between the empyematous group and the other two groups ( p > .05 ) . \n statistically significantly higher values were recognized in the \n tuberculous pleural effusion group rather than the parapneumonic \n effusion group ( p < .01 ) .", "il-6 levels were detected to be 18589.5  3631.8  pg / ml in \n the exudate group and 65.6  16.0  pg / ml in the transudate \n group ( p < .001 ) .", "the corresponding values were 15295.0  22678.1  pg / ml in the parapneumonic effusion group , \n 20518.0  1410.9 ", "pg / ml in the tuberculous pleural \n effusion group . in the empyematous group ,", "statistically \n significantly higher values were obtained when compared with \n those of the parapneumonic effusion \n group , while the corresponding values detected in the tuberculous \n pleural effusion group were also significantly higher than those \n found in the parapneumonic effusion group ( p < .001 ) .", "pg / ml in the exudate group and 108.8  92.0  pg / ml in the transudate group ( p < .001 ) .", "the \n concentrations of il-8 were 1884.5  366.7 in the \n parapneumonic effusion group , 4789.8  1013.2 ", "pg / ml in the \n empyematous effusion group and 919.0  9454.5  pg / ml in the \n tuberculous pleural effusion group .", "the values were statistically \n significantly higher in the empyematous group when compared with \n the other two groups ( p < .001 ) ( figure 4 ) ."], ["pleural effusion can be seen due to the intrusion of \n an infectious agent or an irritating foreign substance inside the \n pleural cavity or due a direct access of harmful materials or \n neoplastic cells into the pleural cavity via hematogenous route \n .", "it can be observed following pleural trauma or in \n association with asbestosis related pleural diseases .", "pleural effusions are known to develop \n secondary to trauma , cardiac , renal , collagenous diseases and \n malignancies besides pneumonia [ 9 , \n 10 , \n 11 ] . from \n 61% to 80% of the cases with pleural effusions", "empyema is also an important cause of \n mortality in the developing countries . in the remaining \n 20%39% of the cases ,", "empyema develops secondary to \n trauma , malignancies , and renal diseases [ 5 , \n 12 , 13 , \n 14 , 15 ] . \n in our study group", "many studies have been conducted concerning \n the levels of il-1 , il-2 , il-6 , and il-8 in body fluids in \n various infections .", "although many studies investigating the \n alterations in levels of il according to the exudative or \n transudative nature of pleural effusions in adults have been \n performed , similar studies pertaining to children are relatively \n few [ 16 , 17 , \n 18 ] .", "for that reason many studies have analysed \n il-1 levels in patients with meningitis , sepsis , urinary \n tract infections , and empyema .", "variable results have been obtained \n dependent on the presence of different etiological agents \n [ 17 , 18 ] . in patients with bacterial and tuberculous \n meningitis ,", "il-1 levels of cerebrospinal fluids were found \n to be significantly higher than those with aseptic meningitis and \n in the control group .", "umblical plasma levels of il-1 of \n infected newborns with early - onset sepsis were found to be higher \n than those of the control group .", "il-1 levels in synovial \n fluids were detected to be higher than those in the pediatric \n cases with   suppurative arthritis due to other causes . \n", "silva - mejias et al   have found comparatively higher \n il-1 levels in the empyema group .", "similar to our findings , \n alexandrakis et al   revealed that il-1 levels in \n pleural effusions were higher in the exudate group compared with \n those of the transudate group .", "naito et al   ascertained \n that il-1 levels in pleural effusions were higher than \n those found in bacterial infections due to other causes .", "in \n accordance with the literature in our study , il-1 levels \n were nearly 4.5 times higher in the exudate group .", "il-2 levels in pleural effusions due to malignancies were reported \n to be lower than those with tuberculous pleural effusions \n .", "shimokata et al \n  stated that il-2 levels were \n at undetectable levels in 25% of tuberculous pleural effusions \n and 75% of cancerous pleural effusions . in our study", "il-2 levels \n in pleural fluids were at undetectably lower levels in 44% of \n the patients in the exudate group while the il-2 levels of the \n remaining 56% were assessed as 176.6  33.4  pg / ml . \n", "however all the patients in the transudate group demonstrated \n undetectable il-2 levels . in our study", "a statistically \n significant difference was not present between the empyema group \n and the other two groups .", "however a statistically significant \n difference was detected between the parapneumonic and the \n parapneumonic pleural effusion groups .", "various studies have been performed concerning the \n levels of il-6 which is the mediator and the regulator of \n inflammatory responses in miscellaneous inflammatory \n processes .", "blood il-6 levels were found to be higher in children \n with mycoplasma pneumoniae infections who stayed febrile \n for more than 3 days compared with those having shorter febrile \n episodes .", "they were also \n  statistically significantly higher in pediatric cases \n with urinary tract infections when compared with the control \n groups .", "il-6 levels of cerebrospinal fluid in the patients with \n bacterial meningitis were reported to be significantly higher \n than the aseptic meningitis and in the control groups .", "levels above 100  pg / ml were measured in pediatric cases with \n viral ( 14% ) but especially with bacterial meningitis ( 53% ) . \n", "xirouchaki et al \n and yokoyama et al   reported \n the levels of il-6 in pleural fluids to be significantly higher \n in the exudate group rather than the transudate group .", "they also \n found il-6 concentrations significantly higher in the tuberculous \n group rather than the parapneumonic effusion group . in our study \n not", "only the levels of il-6 were different between the groups \n with parapneumonic and empyematous effusions , but at the same \n time statistically significant differences were found between the \n empyematous and the parapneumonic or tuberculous pleural effusion \n groups .", "alexandrakis et al   measured il-6 levels in \n serum and pleural fluids and ascertained that serum il-6 levels \n did not differ significantly between the exudate and the \n transudate groups .", "however pleural fluid il-6 levels were \n detected to be meaningfully higher in the exudate rather than the \n transudate group . a definite proportion between the serum and \n pleural fluid il-6 concentrations could not be assessed .", "however \n we found il-6 levels in the pleural fluid to be 285 times higher \n than those detected in the exudate group .", "il-8 is the mediator and the regulator of chemotaxis of \n leukocytes in inflammatory processes . in adults", "il-8 levels \n were found to be higher in cases with infectious pleural \n effusions compared with the patients with noninfectious \n  effusions   [ 3 , 26 , \n  27 , 28 , \n  29 , 30 , \n  31 , 32 ] . \n", "ceyhan et al   reported higher levels in \n empyematous / parapneumonic effusion groups rather than the \n tuberculous group .", "however antony et al   detected higher \n levels in cases with parapneumonic effusions unlike those found \n in the group with tuberculous effusions .", "il-8 concentrations in \n the empyematous group were reported to be higher than those \n measured in the parapneumonic group . in accordance with this \n study , in our study the highest levels of il-8 were detected in \n the empyematous , parapneumonic , and tuberculous pleural effusions \n in decreasing order .", "dlugovitzky et al   discovered \n statistically significantly higher il-8 values in the tuberculous \n pleural effusion group in contrast with   findings in the \n parapneumonic pleural effusion group .", "ashitani et al \n and broaddus et al   found higher levels in the empyema \n group compared with the other groups .", "miller and idell \n have detected   significantly higher levels in the exudate group \n rather than the transudate group . in this study ,", "similar to the \n findings of other   investigations , il-8 levels in the exudate \n group were reported to be 28 times higher than those found in the \n transudate group .", "this study have shown that for the differentiation between the \n exudative and transudative pleural effusions , in addition to the \n parametres such as   protein , glucose and ldh , pleural fluid \n il-1 , il-2 , il-6 , and il-8 levels could be used . \n", "etiological factors can be differentiated by determining pleural \n fluid il levels . taking these levels into consideration", "accordingly , a definitive diagnosis , a successful \n treatment and reduction in mortality can be achieved ."]]}
{"article_id": "PMC5040584", "article_text": ["literature analysis showed few articles about this complication , but no publication has described the management of open patella fracture around total knee arthroplasty .", "we report a unique case of an open patellar fracture above a total knee arthroplasty , sustained by a 56-year - old female patient .", "despite the poor outcome of operative management in patellar periprosthetic fracture , this approach should be considered for acute and post traumatic fractures in young patients with a good remaining bone stock .", "the demographic changes with a high rate of aging and active population , as well as the improved life expectancy during the last decades have led to an increase in the number of total knee arthroplasties ( tka ) [ 1 , 2 , 3 , 4 ] , and consequently a raise of post - operative complications . among these complications ,", "periprosthetic fracture after tka is the most frequent cause of post - operative pain , and revision surgery .", "they may occur in the supracondylar region of the femur , followed by the patella , then the proximal tibia [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ] .", "this entity can be very challenging to manage , with unpredictable and poor outcome , especially for displaced fracture or in cases with implant loosening [ 1 , 2 , 3 , 4 , 5 , 8 ] . in this article", ", we report a unique case of an open periprosthetic patellar fracture that was managed with operative treatment .", "it also involves a literature analysis with description of the risk factors and treatment options of this uncommon entity .", "a 56-year - old female underwent a cemented postero - stabilized condylar tka with fixed tibial plateau through an antero - medial approach , in our department .", "she had severe osteo - arthritis of the left knee with a major stiffness ( irreducible flexum at 25 ) .", "initial ( 1 ) and post operative(2 ) knee radiographs before , and after the arthroplasty .", "three weeks later , the patient tripped and fell off with all her standing height striking the anterior aspect of the left knee with hyper flexion .", "she was taken to the emergency room with left knee pain and a large skin wound .", "physical examination showed a large , deep and longitudinal wound measuring 15 cm in length due to a total breakdown of the suture line ( the antero - medial approach performed during the tka ) ( fig . 3 , 4 ) .", "fig 3 . a clinical picture showing the open patellar fracture a clinical picture showing the open patellar fracture .", "lateral knee radiograph showing the patellar fracture above a total knee arthroplasty , without any signs of implants loosening .", "lateral knee radiograph showing the patellar fracture above a total knee arthroplasty , without any signs ofimplants loosening .", "the knee radiographs showed a short oblique fracture at the inferior pole of the patella without any loosening of the implants or dislocation of the knee : this fracture is classified as type i according to ortigueraet d.j .", "she urgently received intravenous antibiotic ( augmentin , gentamicin ) , as well as tetanus vaccination .", "her fracture was temporarily stabilized in a long - leg plaster splint before she was transported to the operating room .", "she underwent wound irrigation , debridement of non - viable soft tissue from the wound , open reduction and fixation with a figure - eight cerclage wiring , without any revision of the implants .", "the wound was closed without any skin tension , after placing an intra - articular drain .", "complementary immobilization with a removable knee pad at full extension , for six weeks was prescribed .", "post operative knee radiographs post operative knee radiographs she received intra venous antibiotics : augmentin for 2 weeks associated with gentamicin during the first five days , as well as subcutaneous heparin .", "she was discharged from the hospital after she was instructed to remain non - weight bearing with a follow - up in 2 weeks .", "at 6 months follow - up , physical examination did not reveal any signs of local or deep infection .", "her knee was pain free and she was able to walk without crutches , but she had a mild stiffness : the knee range of motion was 0/60. no antero - posterior or frontal laxities were noted .", "clinical pictures at last follow - up showing a clean and dry wound ( 7 ) , and knee range of motion ( 8 , 9 ) clinical pictures at last follow - up showing a clean and dry wound ( 7 ) , and knee range of motion ( 8 , 9 ) .", "the patient achieved union of the fracture : knee radiographs showed a healed patellar fracture , with hardware in place .", "since the late 1960s , total knee arthroplasty has been the most effective treatment for knee osteoarthritis , providing good functional outcomes and improvement in the patient s quality of life .", "constant advances in the joint replacement surgery , and especially the total knee arthroplasties have led to an increase in the number of complications following this type of surgery , such as periprosthetic fractures . [ 1 , 2 , 3 , 4 ] this entity is very challenging to treat even for the most skilled and experienced surgeons [ 7 , 8 , 10 ] .", "the femur is the most frequent fracture site , followed by the patella and the tibia [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ] .", "patella fracture after tka can occur after trauma , or result from a stress fracture due to patella osteonecrosis , which is the most common cause of this type of fracture [ 1 , 2 , 3 , 4 , 5 , 8 , 10 , 11 ] .", "chalidis et al ( 2007 ) have reported in a systematic review of a 752 periprosthetic patella fracture that in 88.3% of the cases , the fracture was not associated with a traumatic event , asymptomatic , and discovered during routine follow - up .", "many risk factors of these stress fractures have been described in the previous literature [ 1 , 2 , 3 , 4 , 5 , 7 , 8 , 9 , 10 , 11 ] including : \n poor blood supply for patella after tka especially for antero - medial approach , and in case of lateral release or unwarranted fat pad removal : knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries.mal-alignment of the femoral and tibial implantspatella resurfacing arthroplastiesan excessive ( thickness < 15 mm ) or asymmetric bone resection during patella resurfacingosteonecrosis due to bone cement polymerization : the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature , causing thermal necrosis of the bonethe use of large central fixation peg designs for patellar implant , instead of small peripheral peg design.revision surgeryosteoporosis and bone losshyperflexion of the kneerhumatod arthritis \n  poor blood supply for patella after tka especially for antero - medial approach , and in case of lateral release or unwarranted fat pad removal : knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries . mal - alignment of the femoral and tibial implants patella resurfacing arthroplasties an excessive ( thickness < 15 mm ) or asymmetric bone resection during patella resurfacing osteonecrosis due to bone cement polymerization : the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature , causing thermal necrosis of the bone the use of large central fixation peg designs for patellar implant , instead of small peripheral peg design . osteoporosis and bone loss hyperflexion of the knee open patellar fracture above a total knee arthroplasty is a very uncommon injury , and challenging to treat . to our knowledge ,", "reported a unique case of patellar fracture associated with open knee dislocation after a total knee arthroplasty .", "it was treated with open reduction and internal fixation of the patella with band tension wiring .", "greco et al , reported an open tibial shaft fracture below an ipsilateral tka , managed with intramedullary nailing .", "some factors should be considered to succeed in the management of this type of fracture such as displacement of the fracture , function of the extensor mechanism , stability of the patellar implant , remaining bone stock [ 1 , 2 , 3 , 4 , 5 , 8 , 9 , 10 , 11 , 13 ] .", "it includes immobilization by a brace locked in full extension or a cylinder cast in case of non - operative treatment ; open reduction and internal fixation , implant revision in case of loosening , partial or total patellectomy , repair of the quadriceps tendon or patellar ligament in case of surgical treatment [ 4 , 5 , 7 , 8 ] . the periprosthetic patellar fractures are usually managed by a non - operative treatment especially for type i and ii , because of the high infectious risk of open reduction and internal fixation [ 13 , 14 ] , and consequently a high risk of arthroplasty failure .", "literature analysis showed that conservative or non - operative treatment was more successful for the management of non - displaced patella fracture with stable implant and an intact extension mechanism .", "ortiguera and berry reported good results in 37 out of 38 peri - prosthetic patellar fractures managed with conservative treatment .", "dennis reported a complication rate of 39% of operative treatment ( in a series of thirty one patients who sustained a peri- prosthetic patellar fracture ) .", "chalidis s systematic review has shown that open reduction and internal fixation failed in 92% of the cases . in this particular case , we were forced to opt for an internal fixation with a cerclage wiring after intensive wound irrigation , debridement and soft tissue repair .", "although no conclusion can be established from a single case report , we think that the successful result in our case can be explained by the relative young age of the patient ( 56 years old ) , the good bone stock , the acute and post - traumatic characteristics of the fracture , the early and adequate management necessary to avoid infectious complication . the surgical management may reduce the ability of patella to heal because of the evacuation of peri - fractural hematoma as well as the supplementary disruption of the vascular network around the patella after a knee replacement surgery .", "nevertheless , the high failure rate of the operative treatment in the patellar peri - prosthetic fracture can be explained by : \n the high proportion of stress , atraumatic fracture ( 88% ) due to osteonecrosis which suggests a poor remaining bone stock.the stress fracture , unlike the acute and post traumatic ones , has a higher risk of non-union.the poor outcome of partial or complete patellectomy , which is a type of operative treatment.in our point of view , the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly . \n  the high proportion of stress , atraumatic fracture ( 88% ) due to osteonecrosis which suggests a poor remaining bone stock .", "the stress fracture , unlike the acute and post traumatic ones , has a higher risk of non - union .", "the poor outcome of partial or complete patellectomy , which is a type of operative treatment . in our point of view , the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly .", "peri - prosthetic patella fracture is a very uncommon entity , and challenging to treat , especially the open ones , because of the high risk of infectious complications .", "although several previous studies report a high rate of failure and unpredictable results of open reduction for periprosthetic patellar fracture , this approach ( especially the internal fixation with a strong and dynamic construct ) should be considered in young patients with a good remaining bone stock , who sustained an open or a displaced fracture after a high energy trauma .", "orthopaedic surgeons should consider open reduction and internal fixation as a good management alternative for periprosthetic patellar fracture , in case of a post traumatic and acute fracture in young patients with a good bone stock ."], "abstract_text": ["<S> introduction : periprosthetic patellar fracture after total knee arthroplasty ( tka ) is a rare complication </S>", "<S> . their management can be very challenging with unpredictable results . </S>", "<S> literature analysis showed few articles about this complication , but no publication has described the management of open patella fracture around total knee arthroplasty.case presentation : we report a unique case of an open patellar fracture above a total knee arthroplasty , sustained by a 56-year - old female patient.conclusion:despite the poor outcome of operative management in patellar periprosthetic fracture , this approach should be considered for acute and post traumatic fractures in young patients with a good remaining bone stock . </S>"], "labels": null, "section_names": ["Introduction:", "Case Presentation:", "Conclusion:", "Introduction", "Case Presentation", "Results", "Discussion", "Conclusion"], "sections": [["literature analysis showed few articles about this complication , but no publication has described the management of open patella fracture around total knee arthroplasty ."], ["we report a unique case of an open patellar fracture above a total knee arthroplasty , sustained by a 56-year - old female patient ."], ["despite the poor outcome of operative management in patellar periprosthetic fracture , this approach should be considered for acute and post traumatic fractures in young patients with a good remaining bone stock ."], ["the demographic changes with a high rate of aging and active population , as well as the improved life expectancy during the last decades have led to an increase in the number of total knee arthroplasties ( tka ) [ 1 , 2 , 3 , 4 ] , and consequently a raise of post - operative complications . among these complications ,", "periprosthetic fracture after tka is the most frequent cause of post - operative pain , and revision surgery .", "they may occur in the supracondylar region of the femur , followed by the patella , then the proximal tibia [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ] .", "this entity can be very challenging to manage , with unpredictable and poor outcome , especially for displaced fracture or in cases with implant loosening [ 1 , 2 , 3 , 4 , 5 , 8 ] . in this article", ", we report a unique case of an open periprosthetic patellar fracture that was managed with operative treatment .", "it also involves a literature analysis with description of the risk factors and treatment options of this uncommon entity ."], ["a 56-year - old female underwent a cemented postero - stabilized condylar tka with fixed tibial plateau through an antero - medial approach , in our department .", "she had severe osteo - arthritis of the left knee with a major stiffness ( irreducible flexum at 25 ) .", "initial ( 1 ) and post operative(2 ) knee radiographs before , and after the arthroplasty .", "three weeks later , the patient tripped and fell off with all her standing height striking the anterior aspect of the left knee with hyper flexion .", "she was taken to the emergency room with left knee pain and a large skin wound .", "physical examination showed a large , deep and longitudinal wound measuring 15 cm in length due to a total breakdown of the suture line ( the antero - medial approach performed during the tka ) ( fig . 3 , 4 ) .", "fig 3 . a clinical picture showing the open patellar fracture a clinical picture showing the open patellar fracture .", "lateral knee radiograph showing the patellar fracture above a total knee arthroplasty , without any signs of implants loosening .", "lateral knee radiograph showing the patellar fracture above a total knee arthroplasty , without any signs ofimplants loosening .", "the knee radiographs showed a short oblique fracture at the inferior pole of the patella without any loosening of the implants or dislocation of the knee : this fracture is classified as type i according to ortigueraet d.j .", "she urgently received intravenous antibiotic ( augmentin , gentamicin ) , as well as tetanus vaccination .", "her fracture was temporarily stabilized in a long - leg plaster splint before she was transported to the operating room .", "she underwent wound irrigation , debridement of non - viable soft tissue from the wound , open reduction and fixation with a figure - eight cerclage wiring , without any revision of the implants .", "the wound was closed without any skin tension , after placing an intra - articular drain .", "complementary immobilization with a removable knee pad at full extension , for six weeks was prescribed .", "post operative knee radiographs post operative knee radiographs she received intra venous antibiotics : augmentin for 2 weeks associated with gentamicin during the first five days , as well as subcutaneous heparin .", "she was discharged from the hospital after she was instructed to remain non - weight bearing with a follow - up in 2 weeks ."], ["at 6 months follow - up , physical examination did not reveal any signs of local or deep infection .", "her knee was pain free and she was able to walk without crutches , but she had a mild stiffness : the knee range of motion was 0/60. no antero - posterior or frontal laxities were noted .", "clinical pictures at last follow - up showing a clean and dry wound ( 7 ) , and knee range of motion ( 8 , 9 ) clinical pictures at last follow - up showing a clean and dry wound ( 7 ) , and knee range of motion ( 8 , 9 ) .", "the patient achieved union of the fracture : knee radiographs showed a healed patellar fracture , with hardware in place ."], ["since the late 1960s , total knee arthroplasty has been the most effective treatment for knee osteoarthritis , providing good functional outcomes and improvement in the patient s quality of life .", "constant advances in the joint replacement surgery , and especially the total knee arthroplasties have led to an increase in the number of complications following this type of surgery , such as periprosthetic fractures . [ 1 , 2 , 3 , 4 ] this entity is very challenging to treat even for the most skilled and experienced surgeons [ 7 , 8 , 10 ] .", "the femur is the most frequent fracture site , followed by the patella and the tibia [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ] .", "patella fracture after tka can occur after trauma , or result from a stress fracture due to patella osteonecrosis , which is the most common cause of this type of fracture [ 1 , 2 , 3 , 4 , 5 , 8 , 10 , 11 ] .", "chalidis et al ( 2007 ) have reported in a systematic review of a 752 periprosthetic patella fracture that in 88.3% of the cases , the fracture was not associated with a traumatic event , asymptomatic , and discovered during routine follow - up .", "many risk factors of these stress fractures have been described in the previous literature [ 1 , 2 , 3 , 4 , 5 , 7 , 8 , 9 , 10 , 11 ] including : \n poor blood supply for patella after tka especially for antero - medial approach , and in case of lateral release or unwarranted fat pad removal : knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries.mal-alignment of the femoral and tibial implantspatella resurfacing arthroplastiesan excessive ( thickness < 15 mm ) or asymmetric bone resection during patella resurfacingosteonecrosis due to bone cement polymerization : the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature , causing thermal necrosis of the bonethe use of large central fixation peg designs for patellar implant , instead of small peripheral peg design.revision surgeryosteoporosis and bone losshyperflexion of the kneerhumatod arthritis \n  poor blood supply for patella after tka especially for antero - medial approach , and in case of lateral release or unwarranted fat pad removal : knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries . mal - alignment of the femoral and tibial implants patella resurfacing arthroplasties an excessive ( thickness < 15 mm ) or asymmetric bone resection during patella resurfacing osteonecrosis due to bone cement polymerization : the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature , causing thermal necrosis of the bone the use of large central fixation peg designs for patellar implant , instead of small peripheral peg design . osteoporosis and bone loss hyperflexion of the knee open patellar fracture above a total knee arthroplasty is a very uncommon injury , and challenging to treat . to our knowledge ,", "reported a unique case of patellar fracture associated with open knee dislocation after a total knee arthroplasty .", "it was treated with open reduction and internal fixation of the patella with band tension wiring .", "greco et al , reported an open tibial shaft fracture below an ipsilateral tka , managed with intramedullary nailing .", "some factors should be considered to succeed in the management of this type of fracture such as displacement of the fracture , function of the extensor mechanism , stability of the patellar implant , remaining bone stock [ 1 , 2 , 3 , 4 , 5 , 8 , 9 , 10 , 11 , 13 ] .", "it includes immobilization by a brace locked in full extension or a cylinder cast in case of non - operative treatment ; open reduction and internal fixation , implant revision in case of loosening , partial or total patellectomy , repair of the quadriceps tendon or patellar ligament in case of surgical treatment [ 4 , 5 , 7 , 8 ] . the periprosthetic patellar fractures are usually managed by a non - operative treatment especially for type i and ii , because of the high infectious risk of open reduction and internal fixation [ 13 , 14 ] , and consequently a high risk of arthroplasty failure .", "literature analysis showed that conservative or non - operative treatment was more successful for the management of non - displaced patella fracture with stable implant and an intact extension mechanism .", "ortiguera and berry reported good results in 37 out of 38 peri - prosthetic patellar fractures managed with conservative treatment .", "dennis reported a complication rate of 39% of operative treatment ( in a series of thirty one patients who sustained a peri- prosthetic patellar fracture ) .", "chalidis s systematic review has shown that open reduction and internal fixation failed in 92% of the cases . in this particular case , we were forced to opt for an internal fixation with a cerclage wiring after intensive wound irrigation , debridement and soft tissue repair .", "although no conclusion can be established from a single case report , we think that the successful result in our case can be explained by the relative young age of the patient ( 56 years old ) , the good bone stock , the acute and post - traumatic characteristics of the fracture , the early and adequate management necessary to avoid infectious complication . the surgical management may reduce the ability of patella to heal because of the evacuation of peri - fractural hematoma as well as the supplementary disruption of the vascular network around the patella after a knee replacement surgery .", "nevertheless , the high failure rate of the operative treatment in the patellar peri - prosthetic fracture can be explained by : \n the high proportion of stress , atraumatic fracture ( 88% ) due to osteonecrosis which suggests a poor remaining bone stock.the stress fracture , unlike the acute and post traumatic ones , has a higher risk of non-union.the poor outcome of partial or complete patellectomy , which is a type of operative treatment.in our point of view , the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly . \n  the high proportion of stress , atraumatic fracture ( 88% ) due to osteonecrosis which suggests a poor remaining bone stock .", "the stress fracture , unlike the acute and post traumatic ones , has a higher risk of non - union .", "the poor outcome of partial or complete patellectomy , which is a type of operative treatment . in our point of view , the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly ."], ["peri - prosthetic patella fracture is a very uncommon entity , and challenging to treat , especially the open ones , because of the high risk of infectious complications .", "although several previous studies report a high rate of failure and unpredictable results of open reduction for periprosthetic patellar fracture , this approach ( especially the internal fixation with a strong and dynamic construct ) should be considered in young patients with a good remaining bone stock , who sustained an open or a displaced fracture after a high energy trauma .", "orthopaedic surgeons should consider open reduction and internal fixation as a good management alternative for periprosthetic patellar fracture , in case of a post traumatic and acute fracture in young patients with a good bone stock ."]]}
{"article_id": "PMC4975136", "article_text": ["near - infrared ( nir ) radiation can penetrate the skin and the sclera of the eye .", "the high permeability of nir radiation also allows it to affect tissues deeper within the eye , such as muscles , the lens , and retina .", "nir radiation can induce various biological effects,19 and intensive or long - term exposure to nir radiation is a factor in premature aging . despite the wide prevalence of a variety of ultraviolet ( uv ) blocking materials , such as sunblock", ", sunglasses , glasses , films , and umbrellas , that are useful in protecting our tissue against uv exposure , nir can not be blocked sufficiently.8 consequently , in the absence of suitable protection , nir radiation can induce various kinds of tissue damage and diseases , such as cataracts and photoaging.7,8 the human cornea plays a critical role in refracting light onto the retina and also protects the eye against external agents .", "since the epithelial layer of the cornea provides the first line of defense against environmental insults , the structural integrity of this layer is a key component of corneal function.10 although uv - induced corneal damage has been described in many previous studies,1115 the effects induced by nir radiation on the cornea have not been thoroughly investigated .", "we hypothesized that nir irradiation simulating solar nir radiation that reaches human tissues can induce changes in gene expression . to test this hypothesis , a three - dimensional reconstructed human corneal epithelial model with multilayered ,", "corneal epithelium - like structure was used to simulate the human eye , and we evaluated dna microarray and real - time polymerase chain reaction ( pcr ) analysis results from normal human corneal epithelial cells exposed to water - filtered broad - spectrum nir irradiation to simulate solar nir radiation that reaches the eye .", "nir irradiation was performed with a broadband nir source ( titan ; cutera , brisbane , ca , usa ) .", "the nir device emits an nir spectrum between 1,100 nm and 1,800 nm , with water filtering to remove wavelengths between 1,400 nm and 1,500 nm , and simulates solar nir radiation that reaches the skin of humans on the earth s surface . to avoid thermal effects ,", "the sapphire contact cooling tip was set to a fixed temperature of 20c . in our previous in vitro study , ten rounds at 10 j / cm using continuous energy single irradiation pulses of 4.3 seconds achieved drastic reduction in cell count .", "the three - dimensional reconstructed human corneal epithelial model ( labcyte cornea - model ) prepared from enzymatically digested normal human corneal epithelial tissues was purchased from japan tissue engineering corporation , aichi , japan as an in vitro model of corneal tissue.16 cells were cultured in media ( assay medium ; japan tissue engineering corporation ) , which was changed every 2 days until the cultures reached subconfluence.16 the subconfluent corneal cells were then subcultured with trypsin and seeded on a cell culture insert containing a microporous membrane with a 0.4 m pore size .", "corneal cells were cultivated between the air and liquid interface to form a multilayered , corneal epithelium - like structure .", "total rna from the corneal epithelial cells was extracted in qiazol reagent ( qiagen nv , venlo , the netherlands ) and spin - column purified using a rneasy mini spin column ( qiagen nv ) .", "the rna was extracted from control and irradiated groups , which consisted of six whole individual three - dimensional reconstructed human corneal epithelial models .", "the quantity and quality of the rna samples were then measured using uv absorbance ( nanodrop technologies , wilmington , de , usa ) , and the quality was also assessed using an agilent 2100 bioanalyzer series ii ( agilent technologies , santa clara , ca , usa ) .", "cy3-labeled crna samples were synthesized using the low input quick amp labeling kit ( agilent technologies ) according to the manufacturer s instructions . for each time point , 50 ng of total rna was used to generate first - strand cdna . after the denaturation ( 10 minutes at 65c ) and crna synthesis ( 2 hours at 40c ) steps , the reactions were incubated at 70c for 15 minutes to inactivate the affinity - script enzyme ( agilent technologies ) . for labeling reactions ,", "crna samples were each mixed with 6 l of transcription master mix cocktail containing cy3-ctp and incubated at 40c for 2 hours .", "labeled crna was quantified on a nanodrop nd-1000 ( nanodrop technologies ) , and quality was also checked by agilent 2100 bioanalyzer series ii .", "slides were scanned using the microarray scanner ( agilent technologies ) , and images were processed by the feature extraction software ( agilent technologies ) with background correction .", "data were imported into genespring , and normalization was performed with a 75 percentile shift .", "after normalization and filtering of the raw data output files , log - fold changes in gene expression in the nir - irradiated group compared with the control group were analyzed using the genespring gx 11.0 software ( agilent technologies ) to determine the upregulated and downregulated genes , using a twofold change as the cutoff .", "upregulated and downregulated genes were further clustered into functional gene groups using the ontology analysis function with the genespring gx 11.0 software .", "microarray data were deposited into the minimum information about a microarray experiment ( miame)-compliant gene expression omnibus ( geo ) database ( accession number : gse76500 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse76500 ) .", "quantitative real - time pcr analysis was achieved using the dna engine opticon 2 system ( bio - rad laboratories inc .", "all six whole individual three - dimensional reconstructed human corneal epithelial tissue culture models were used for quantitative real - time pcr analysis .", "the primer sequences of epidermal growth factor receptor ( egfr ) , tubulin tyrosine ligase - like family member 5 ( ttll5 ) , and pq loop repeat containing 2 ( pqlc2 ) are given in table 1 .", "since pqlc was ubiqitously and highly expressed in all samples , pqlc2 was used for normalization .", "the variant - specific primer sequences for the four egfr variants are given in table 2 .", "cdna templates were prepared for each rna sample by reverse transcription reaction using superscript iii ( thermo fisher scientific , waltham , ma , usa ) .", "each quantitative pcr ( 25 l ) contained 1 l of cdna template , 1.5 l of forward and reverse primer ( 10 m each ) , 7.5 l of 2 master mix , and 3.5 l of nuclease - free water .", "the thermal cycling conditions included one cycle at 95c for 5 minutes and 40 cycles at 95c for 10 seconds , 60c for 30 seconds , and 68c for 30 seconds .", "all statistical data are presented as the mean  sem , and a p - value of < 0.05 was considered to be statistically significant .", "nir irradiation was performed with a broadband nir source ( titan ; cutera , brisbane , ca , usa ) .", "the nir device emits an nir spectrum between 1,100 nm and 1,800 nm , with water filtering to remove wavelengths between 1,400 nm and 1,500 nm , and simulates solar nir radiation that reaches the skin of humans on the earth s surface . to avoid thermal effects ,", "the sapphire contact cooling tip was set to a fixed temperature of 20c . in our previous in vitro study , ten rounds at 10 j / cm using continuous energy single irradiation pulses of 4.3 seconds achieved drastic reduction in cell count .", "the three - dimensional reconstructed human corneal epithelial model ( labcyte cornea - model ) prepared from enzymatically digested normal human corneal epithelial tissues was purchased from japan tissue engineering corporation , aichi , japan as an in vitro model of corneal tissue.16 cells were cultured in media ( assay medium ; japan tissue engineering corporation ) , which was changed every 2 days until the cultures reached subconfluence.16 the subconfluent corneal cells were then subcultured with trypsin and seeded on a cell culture insert containing a microporous membrane with a 0.4 m pore size .", "corneal cells were cultivated between the air and liquid interface to form a multilayered , corneal epithelium - like structure .", "total rna from the corneal epithelial cells was extracted in qiazol reagent ( qiagen nv , venlo , the netherlands ) and spin - column purified using a rneasy mini spin column ( qiagen nv ) . the rna was extracted from control and irradiated groups , which consisted of six whole individual three - dimensional reconstructed human corneal epithelial models .", "the quantity and quality of the rna samples were then measured using uv absorbance ( nanodrop technologies , wilmington , de , usa ) , and the quality was also assessed using an agilent 2100 bioanalyzer series ii ( agilent technologies , santa clara , ca , usa ) .", "cy3-labeled crna samples were synthesized using the low input quick amp labeling kit ( agilent technologies ) according to the manufacturer s instructions . for each time point , 50 ng of total rna was used to generate first - strand cdna . after the denaturation ( 10 minutes at 65c ) and crna synthesis ( 2 hours at 40c ) steps , the reactions were incubated at 70c for 15 minutes to inactivate the affinity - script enzyme ( agilent technologies ) . for labeling reactions ,", "crna samples were each mixed with 6 l of transcription master mix cocktail containing cy3-ctp and incubated at 40c for 2 hours .", "labeled crna was quantified on a nanodrop nd-1000 ( nanodrop technologies ) , and quality was also checked by agilent 2100 bioanalyzer series ii .", "slides were scanned using the microarray scanner ( agilent technologies ) , and images were processed by the feature extraction software ( agilent technologies ) with background correction .", "data were imported into genespring , and normalization was performed with a 75 percentile shift .", "after normalization and filtering of the raw data output files , log - fold changes in gene expression in the nir - irradiated group compared with the control group were analyzed using the genespring gx 11.0 software ( agilent technologies ) to determine the upregulated and downregulated genes , using a twofold change as the cutoff .", "upregulated and downregulated genes were further clustered into functional gene groups using the ontology analysis function with the genespring gx 11.0 software .", "microarray data were deposited into the minimum information about a microarray experiment ( miame)-compliant gene expression omnibus ( geo ) database ( accession number : gse76500 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse76500 ) .", "quantitative real - time pcr analysis was achieved using the dna engine opticon 2 system ( bio - rad laboratories inc .", "all six whole individual three - dimensional reconstructed human corneal epithelial tissue culture models were used for quantitative real - time pcr analysis .", "the primer sequences of epidermal growth factor receptor ( egfr ) , tubulin tyrosine ligase - like family member 5 ( ttll5 ) , and pq loop repeat containing 2 ( pqlc2 ) are given in table 1 .", "since pqlc was ubiqitously and highly expressed in all samples , pqlc2 was used for normalization .", "the variant - specific primer sequences for the four egfr variants are given in table 2 .", "cdna templates were prepared for each rna sample by reverse transcription reaction using superscript iii ( thermo fisher scientific , waltham , ma , usa ) .", "each quantitative pcr ( 25 l ) contained 1 l of cdna template , 1.5 l of forward and reverse primer ( 10 m each ) , 7.5 l of 2 master mix , and 3.5 l of nuclease - free water .", "the thermal cycling conditions included one cycle at 95c for 5 minutes and 40 cycles at 95c for 10 seconds , 60c for 30 seconds , and 68c for 30 seconds .", "all statistical data are presented as the mean  sem , and a p - value of < 0.05 was considered to be statistically significant .", "to elucidate changes in genetic expression induced by exposure to nir irradiation that simulates solar nir radiation that reaches human eye tissues , dna microarray analysis was carried out using rna isolated from control and nir - irradiated corneal epithelial cells . using ~6.310 probes , we identified 52 upregulated genes and 98 downregulated genes in the nir - irradiated group compared with the control group .", "gene ontology analysis of these 150 genes identified 41 upregulated genes that encode proteins with receptor - binding properties ( table 3 ) .", "quantitative real - time pcr analysis was then performed for the two genes showing the highest degree of upregulation in the dna microarray analysis : egfr and ttll5 .", "egfr showed a high degree of upregulation ( 19.4-fold ) relative to control cells , although statistically significant upregulation of egfr and ttll5 was not observed ( p=0.5127 and p=0.8273 , respectively ) ( figure 1 ) using the primer sequences listed in table 1 .", "meanwhile , when the expression of egfr variants was assessed in a dna microarray analysis , expression increased by 1.0-fold and 1.3-fold for egfr variants 1 and 3 , respectively . upon inclusion of variant - specific primer sequences for egfr ( table 2 ) , quantitative real - time pcr analysis revealed that statistically significant upregulation of variants 1 and 3 was observed ( p<0.05 ) , whereas there was no statistically significant upregulation of variants 2 and 4 ( p=0.1266 and p=0.5127 , respectively ) ( figure 2 ) .", "to the best of our knowledge , this is the first study that examines changes in genetic expression in three - dimensional reconstructed corneal epithelial tissue after nir irradiation to simulate solar nir radiation that reaches human eye tissues .", "the biological effects of sun and uv exposure have been extensively investigated . however , over half of the solar energy is in the nir range , and most of the uv - blocking materials can not block nir radiation .", "moreover , humans are continuously exposed to artificial nir radiation from sources , such as electrical appliances.17,18 despite the prevalence of nir radiation , the need for protective measures against this type of radiation has received less attention than that of uv . because solar nir radiation is filtered by atmospheric water,19,20 water - filtering in experimental settings is indispensable to provide an accurate simulation of solar nir radiation that reaches human tissues.5 cooling is also recommended when investigating the effects of nir radiation , as nir radiation can increase surface temperatures and induce thermal effects.5 indeed , without water filter or contact cooling , nir irradiation immediately increases the temperature of the superficial layer of culture fluid in a laboratory dish or on skin , as nir radiation is predominantly absorbed by hydrogen bond - containing molecules , such as water and hemoglobin . meanwhile , as the nir radiation penetrates deeper into the target , its energy can dissipate to the point where insufficient amounts of energy reach target cells in the base of culture dishes or deeper tissues in skin samples .", "thus , to accurately investigate the biological effects of solar nir radiation that reaches human tissues , a water - filter that excludes wavelengths between 1,400 nm and 1,500 nm and a cooling system were used in this study.5 a three - dimensional reconstructed human corneal epithelial model was used as an in vitro model of corneal tissue .", "the corneal epithelial model was proven to have a fully differentiated corneal epidermal tissue including all major corneal epithelial cell layers , such as the basal cell layer , the wing layer , and the superficial cell layer .", "cytokeratin-3 is a specific marker of corneal epithelium , and it was expressed in all layers of the corneal epithelial model.16 muc-1 and muc-16 , which compose the transmembrane glycoproteins in the surface of the corneal epithelium , were well expressed in the superficial layer of the corneal epithelial model.16 cells in the corneal epithelium are connected by desmosomes , tight junctions , and adherence junctions.16 claudin-1 , which is a marker of a tight junction , desmograin-3 , which is a marker of desmosome , and e - cadherin , which is a marker of an adherence junction , are localized in the interface between cells at all cell layers , including the superficial layers.16 lamin is an important constituent in the basement membrane at the basal corneal epithelium junction , which is expressed continuously in basal cells of the corneal epithelial model basal layer.16 the basement membrane was smooth like that of the human eye.16 therefore , the corneal epithelial model reproduced many of the characteristics of the native human corneal tissue , and it provides a morphologically relevant means to assess nir effects as an alternative to animal testing .", "the corneal epithelium , like other epithelial barriers in the human body , is continuously subjected to physical , chemical , and biological insults , which can produce a wound and/or loss of barrier functions .", "proper healing of corneal wounds is vital for maintaining a clear , healthy cornea and preserving vision .", "corneal epithelium responds rapidly to injury , wherein wound healing occurs by cells migrating as a sheet to cover the defect and reestablish barrier functions.21 in wounded cornea , the epithelium plays a central role , not only as a key cell type in corneal repair , but also as a growth factor source.22 as in other tissues , several growth factors are suggested to play a role in regulating corneal epithelial function and wound healing.21,23 following nir irradiation that simulates solar radiation , microarray analysis of corneal epithelial cell dna in this study showed that egfr was highly expressed , and statistically significant upregulation of egfr variants 1 and 3 was observed ( p<0.05 ) by quantitative real - time pcr .", "egfr is a prototypic tyrosine kinase receptor that is part of a larger family of erbb receptors .", "erbb2 , erbb3 , and erbb4 share with egfr characteristics that include extracellular ligand - binding sites , intracellular kinase domains , and tyrosine - rich regions .", "egfr activated in response to injury serves as a powerful mediator of corneal epithelial wound healing10 by coordinating multiple extracellular signals generated in response to cell injury.2427 in turn , the levels of egfr and tyrosine kinase activity are major determinant factors for epithelial cell function in tissues and organs.22 echoing this concept is the finding that in some cancer patients , treatment with egfr - specific monoclonal antibodies cetuximab and gefitinib ( an egfr kinase inhibitor ) resulted in ocular abnormalities , including diffuse punctate keratitis and corneal erosion.2830 thus , maintaining a proper level of egfr signaling is critical for corneal homeostasis.22 ttll5 gene has 32 exons with high expression in heart and skeletal muscle and lower expression in many other tissues , including the eye and brain.31,32 it encodes a 1,281-amino acid protein that is localized to the cytoplasm and nucleus.31 ttll5 is thought to be a key initiator of polyglutamylation in -tubulin33 and has been previously reported to be essential for the correct function of sperm flagella.34 ttll5 plays a role in polyglutamylation of primary cilia in vitro.35 notably , genes involved in the polyglutamylation and deglutamylation of -tubulin have been associated with photoreceptor degeneration in mice.35 in this study , ttll5 showed the high degree of upregulation in the dna microarray analysis , whereas statistically significant upregulation of ttll5 was not observed in quantitative real - time pcr analysis .", "further studies are needed to determine if a higher power output or increased treatment frequency may promote significant upregulation of ttll5 .", "the findings here that egfr expression is significantly upregulated following exposure of corneal epithelial cells to nir irradiation support that solar nir radiation that reaches human tissues can induce corneal cell injury .", "although significant egfr expression upregulation occurred after just one treatment at the tested power output , further studies are needed to determine if a higher power output or increased treatment frequency may promote even larger changes in expression .", "furthermore , this was a preliminary study based on a fairly small number of samples , so a large - scale study will be needed .", "nir irradiation simulating solar nir radiation that reaches human tissues induced upregulated expression of egfr in human corneal cells .", "nir radiation represents more than half of solar energy and can not be sufficiently blocked by eyewear . moreover ,", "thus , the results of this study indicate that enhanced protection from and avoidance of both uv and nir radiation should be considered to prevent eye damage ."], "abstract_text": ["<S> background and objectivehumans are increasingly exposed to near - infrared ( nir ) radiation from both natural ( eg , solar ) and artificial ( eg , electrical appliances ) sources . </S>", "<S> although the biological effects of sun and ultraviolet ( uv ) exposure have been extensively investigated , the biological effect of nir radiation is still unclear . </S>", "<S> we previously reported that nir as well as uv induces photoaging and standard uv - blocking materials , such as sunglasses , do not sufficiently block nir . </S>", "<S> the objective of this study was to investigate changes in gene expression in three - dimensional reconstructed corneal epithelial tissue culture exposed to broad - spectrum nir irradiation to simulate solar nir radiation that reaches human tissues.materials and methodsdna microarray and quantitative real - time polymerase chain reaction analysis were used to assess gene expression levels in a three - dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water - filtered broad - spectrum nir irradiation with a contact cooling ( 20c ) . </S>", "<S> the water - filter allowed 1,0001,800 nm wavelengths and excluded 1,4001,500 nm wavelengths.resultsa dna microarray with > 62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold , respectively , after nir irradiation . in particular , epidermal growth factor receptor ( egfr ) was upregulated by 19.4-fold relative to control cells . </S>", "<S> quantitative real - time polymerase chain reaction analysis revealed that two variants of egfr in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 j / cm2 irradiation ( p<0.05).conclusionwe found that nir irradiation induced the upregulated expression of egfr in human corneal cells . since over half of the solar energy reaching the earth is in the nir region , which can not be adequately blocked by eyewear and thus can induce eye damage with intensive or long - term exposure , protection from both uv and nir radiation may prevent changes in gene expression and in turn eye damage . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "NIR irradiation", "Corneal epithelial model", "RNA extraction", "cRNA synthesis, labeling, and purification", "Microarray analysis", "Quantitative real-time PCR analysis", "Statistical analysis", "Results", "Discussion", "Conclusion"], "sections": [["near - infrared ( nir ) radiation can penetrate the skin and the sclera of the eye .", "the high permeability of nir radiation also allows it to affect tissues deeper within the eye , such as muscles , the lens , and retina .", "nir radiation can induce various biological effects,19 and intensive or long - term exposure to nir radiation is a factor in premature aging . despite the wide prevalence of a variety of ultraviolet ( uv ) blocking materials , such as sunblock", ", sunglasses , glasses , films , and umbrellas , that are useful in protecting our tissue against uv exposure , nir can not be blocked sufficiently.8 consequently , in the absence of suitable protection , nir radiation can induce various kinds of tissue damage and diseases , such as cataracts and photoaging.7,8 the human cornea plays a critical role in refracting light onto the retina and also protects the eye against external agents .", "since the epithelial layer of the cornea provides the first line of defense against environmental insults , the structural integrity of this layer is a key component of corneal function.10 although uv - induced corneal damage has been described in many previous studies,1115 the effects induced by nir radiation on the cornea have not been thoroughly investigated .", "we hypothesized that nir irradiation simulating solar nir radiation that reaches human tissues can induce changes in gene expression . to test this hypothesis , a three - dimensional reconstructed human corneal epithelial model with multilayered ,", "corneal epithelium - like structure was used to simulate the human eye , and we evaluated dna microarray and real - time polymerase chain reaction ( pcr ) analysis results from normal human corneal epithelial cells exposed to water - filtered broad - spectrum nir irradiation to simulate solar nir radiation that reaches the eye ."], ["nir irradiation was performed with a broadband nir source ( titan ; cutera , brisbane , ca , usa ) .", "the nir device emits an nir spectrum between 1,100 nm and 1,800 nm , with water filtering to remove wavelengths between 1,400 nm and 1,500 nm , and simulates solar nir radiation that reaches the skin of humans on the earth s surface . to avoid thermal effects ,", "the sapphire contact cooling tip was set to a fixed temperature of 20c . in our previous in vitro study , ten rounds at 10 j / cm using continuous energy single irradiation pulses of 4.3 seconds achieved drastic reduction in cell count .", "the three - dimensional reconstructed human corneal epithelial model ( labcyte cornea - model ) prepared from enzymatically digested normal human corneal epithelial tissues was purchased from japan tissue engineering corporation , aichi , japan as an in vitro model of corneal tissue.16 cells were cultured in media ( assay medium ; japan tissue engineering corporation ) , which was changed every 2 days until the cultures reached subconfluence.16 the subconfluent corneal cells were then subcultured with trypsin and seeded on a cell culture insert containing a microporous membrane with a 0.4 m pore size .", "corneal cells were cultivated between the air and liquid interface to form a multilayered , corneal epithelium - like structure .", "total rna from the corneal epithelial cells was extracted in qiazol reagent ( qiagen nv , venlo , the netherlands ) and spin - column purified using a rneasy mini spin column ( qiagen nv ) .", "the rna was extracted from control and irradiated groups , which consisted of six whole individual three - dimensional reconstructed human corneal epithelial models .", "the quantity and quality of the rna samples were then measured using uv absorbance ( nanodrop technologies , wilmington , de , usa ) , and the quality was also assessed using an agilent 2100 bioanalyzer series ii ( agilent technologies , santa clara , ca , usa ) .", "cy3-labeled crna samples were synthesized using the low input quick amp labeling kit ( agilent technologies ) according to the manufacturer s instructions . for each time point , 50 ng of total rna was used to generate first - strand cdna . after the denaturation ( 10 minutes at 65c ) and crna synthesis ( 2 hours at 40c ) steps , the reactions were incubated at 70c for 15 minutes to inactivate the affinity - script enzyme ( agilent technologies ) . for labeling reactions ,", "crna samples were each mixed with 6 l of transcription master mix cocktail containing cy3-ctp and incubated at 40c for 2 hours .", "labeled crna was quantified on a nanodrop nd-1000 ( nanodrop technologies ) , and quality was also checked by agilent 2100 bioanalyzer series ii .", "slides were scanned using the microarray scanner ( agilent technologies ) , and images were processed by the feature extraction software ( agilent technologies ) with background correction .", "data were imported into genespring , and normalization was performed with a 75 percentile shift .", "after normalization and filtering of the raw data output files , log - fold changes in gene expression in the nir - irradiated group compared with the control group were analyzed using the genespring gx 11.0 software ( agilent technologies ) to determine the upregulated and downregulated genes , using a twofold change as the cutoff .", "upregulated and downregulated genes were further clustered into functional gene groups using the ontology analysis function with the genespring gx 11.0 software .", "microarray data were deposited into the minimum information about a microarray experiment ( miame)-compliant gene expression omnibus ( geo ) database ( accession number : gse76500 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse76500 ) .", "quantitative real - time pcr analysis was achieved using the dna engine opticon 2 system ( bio - rad laboratories inc .", "all six whole individual three - dimensional reconstructed human corneal epithelial tissue culture models were used for quantitative real - time pcr analysis .", "the primer sequences of epidermal growth factor receptor ( egfr ) , tubulin tyrosine ligase - like family member 5 ( ttll5 ) , and pq loop repeat containing 2 ( pqlc2 ) are given in table 1 .", "since pqlc was ubiqitously and highly expressed in all samples , pqlc2 was used for normalization .", "the variant - specific primer sequences for the four egfr variants are given in table 2 .", "cdna templates were prepared for each rna sample by reverse transcription reaction using superscript iii ( thermo fisher scientific , waltham , ma , usa ) .", "each quantitative pcr ( 25 l ) contained 1 l of cdna template , 1.5 l of forward and reverse primer ( 10 m each ) , 7.5 l of 2 master mix , and 3.5 l of nuclease - free water .", "the thermal cycling conditions included one cycle at 95c for 5 minutes and 40 cycles at 95c for 10 seconds , 60c for 30 seconds , and 68c for 30 seconds .", "all statistical data are presented as the mean  sem , and a p - value of < 0.05 was considered to be statistically significant ."], ["nir irradiation was performed with a broadband nir source ( titan ; cutera , brisbane , ca , usa ) .", "the nir device emits an nir spectrum between 1,100 nm and 1,800 nm , with water filtering to remove wavelengths between 1,400 nm and 1,500 nm , and simulates solar nir radiation that reaches the skin of humans on the earth s surface . to avoid thermal effects ,", "the sapphire contact cooling tip was set to a fixed temperature of 20c . in our previous in vitro study , ten rounds at 10 j / cm using continuous energy single irradiation pulses of 4.3 seconds achieved drastic reduction in cell count ."], ["the three - dimensional reconstructed human corneal epithelial model ( labcyte cornea - model ) prepared from enzymatically digested normal human corneal epithelial tissues was purchased from japan tissue engineering corporation , aichi , japan as an in vitro model of corneal tissue.16 cells were cultured in media ( assay medium ; japan tissue engineering corporation ) , which was changed every 2 days until the cultures reached subconfluence.16 the subconfluent corneal cells were then subcultured with trypsin and seeded on a cell culture insert containing a microporous membrane with a 0.4 m pore size .", "corneal cells were cultivated between the air and liquid interface to form a multilayered , corneal epithelium - like structure ."], ["total rna from the corneal epithelial cells was extracted in qiazol reagent ( qiagen nv , venlo , the netherlands ) and spin - column purified using a rneasy mini spin column ( qiagen nv ) . the rna was extracted from control and irradiated groups , which consisted of six whole individual three - dimensional reconstructed human corneal epithelial models .", "the quantity and quality of the rna samples were then measured using uv absorbance ( nanodrop technologies , wilmington , de , usa ) , and the quality was also assessed using an agilent 2100 bioanalyzer series ii ( agilent technologies , santa clara , ca , usa ) ."], ["cy3-labeled crna samples were synthesized using the low input quick amp labeling kit ( agilent technologies ) according to the manufacturer s instructions . for each time point , 50 ng of total rna was used to generate first - strand cdna . after the denaturation ( 10 minutes at 65c ) and crna synthesis ( 2 hours at 40c ) steps , the reactions were incubated at 70c for 15 minutes to inactivate the affinity - script enzyme ( agilent technologies ) . for labeling reactions ,", "crna samples were each mixed with 6 l of transcription master mix cocktail containing cy3-ctp and incubated at 40c for 2 hours .", "labeled crna was quantified on a nanodrop nd-1000 ( nanodrop technologies ) , and quality was also checked by agilent 2100 bioanalyzer series ii ."], ["slides were scanned using the microarray scanner ( agilent technologies ) , and images were processed by the feature extraction software ( agilent technologies ) with background correction .", "data were imported into genespring , and normalization was performed with a 75 percentile shift .", "after normalization and filtering of the raw data output files , log - fold changes in gene expression in the nir - irradiated group compared with the control group were analyzed using the genespring gx 11.0 software ( agilent technologies ) to determine the upregulated and downregulated genes , using a twofold change as the cutoff .", "upregulated and downregulated genes were further clustered into functional gene groups using the ontology analysis function with the genespring gx 11.0 software .", "microarray data were deposited into the minimum information about a microarray experiment ( miame)-compliant gene expression omnibus ( geo ) database ( accession number : gse76500 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse76500 ) ."], ["quantitative real - time pcr analysis was achieved using the dna engine opticon 2 system ( bio - rad laboratories inc .", "all six whole individual three - dimensional reconstructed human corneal epithelial tissue culture models were used for quantitative real - time pcr analysis .", "the primer sequences of epidermal growth factor receptor ( egfr ) , tubulin tyrosine ligase - like family member 5 ( ttll5 ) , and pq loop repeat containing 2 ( pqlc2 ) are given in table 1 .", "since pqlc was ubiqitously and highly expressed in all samples , pqlc2 was used for normalization .", "the variant - specific primer sequences for the four egfr variants are given in table 2 .", "cdna templates were prepared for each rna sample by reverse transcription reaction using superscript iii ( thermo fisher scientific , waltham , ma , usa ) .", "each quantitative pcr ( 25 l ) contained 1 l of cdna template , 1.5 l of forward and reverse primer ( 10 m each ) , 7.5 l of 2 master mix , and 3.5 l of nuclease - free water .", "the thermal cycling conditions included one cycle at 95c for 5 minutes and 40 cycles at 95c for 10 seconds , 60c for 30 seconds , and 68c for 30 seconds ."], ["all statistical data are presented as the mean  sem , and a p - value of < 0.05 was considered to be statistically significant ."], ["to elucidate changes in genetic expression induced by exposure to nir irradiation that simulates solar nir radiation that reaches human eye tissues , dna microarray analysis was carried out using rna isolated from control and nir - irradiated corneal epithelial cells . using ~6.310 probes , we identified 52 upregulated genes and 98 downregulated genes in the nir - irradiated group compared with the control group .", "gene ontology analysis of these 150 genes identified 41 upregulated genes that encode proteins with receptor - binding properties ( table 3 ) .", "quantitative real - time pcr analysis was then performed for the two genes showing the highest degree of upregulation in the dna microarray analysis : egfr and ttll5 .", "egfr showed a high degree of upregulation ( 19.4-fold ) relative to control cells , although statistically significant upregulation of egfr and ttll5 was not observed ( p=0.5127 and p=0.8273 , respectively ) ( figure 1 ) using the primer sequences listed in table 1 .", "meanwhile , when the expression of egfr variants was assessed in a dna microarray analysis , expression increased by 1.0-fold and 1.3-fold for egfr variants 1 and 3 , respectively . upon inclusion of variant - specific primer sequences for egfr ( table 2 ) , quantitative real - time pcr analysis revealed that statistically significant upregulation of variants 1 and 3 was observed ( p<0.05 ) , whereas there was no statistically significant upregulation of variants 2 and 4 ( p=0.1266 and p=0.5127 , respectively ) ( figure 2 ) ."], ["to the best of our knowledge , this is the first study that examines changes in genetic expression in three - dimensional reconstructed corneal epithelial tissue after nir irradiation to simulate solar nir radiation that reaches human eye tissues .", "the biological effects of sun and uv exposure have been extensively investigated . however , over half of the solar energy is in the nir range , and most of the uv - blocking materials can not block nir radiation .", "moreover , humans are continuously exposed to artificial nir radiation from sources , such as electrical appliances.17,18 despite the prevalence of nir radiation , the need for protective measures against this type of radiation has received less attention than that of uv . because solar nir radiation is filtered by atmospheric water,19,20 water - filtering in experimental settings is indispensable to provide an accurate simulation of solar nir radiation that reaches human tissues.5 cooling is also recommended when investigating the effects of nir radiation , as nir radiation can increase surface temperatures and induce thermal effects.5 indeed , without water filter or contact cooling , nir irradiation immediately increases the temperature of the superficial layer of culture fluid in a laboratory dish or on skin , as nir radiation is predominantly absorbed by hydrogen bond - containing molecules , such as water and hemoglobin . meanwhile , as the nir radiation penetrates deeper into the target , its energy can dissipate to the point where insufficient amounts of energy reach target cells in the base of culture dishes or deeper tissues in skin samples .", "thus , to accurately investigate the biological effects of solar nir radiation that reaches human tissues , a water - filter that excludes wavelengths between 1,400 nm and 1,500 nm and a cooling system were used in this study.5 a three - dimensional reconstructed human corneal epithelial model was used as an in vitro model of corneal tissue .", "the corneal epithelial model was proven to have a fully differentiated corneal epidermal tissue including all major corneal epithelial cell layers , such as the basal cell layer , the wing layer , and the superficial cell layer .", "cytokeratin-3 is a specific marker of corneal epithelium , and it was expressed in all layers of the corneal epithelial model.16 muc-1 and muc-16 , which compose the transmembrane glycoproteins in the surface of the corneal epithelium , were well expressed in the superficial layer of the corneal epithelial model.16 cells in the corneal epithelium are connected by desmosomes , tight junctions , and adherence junctions.16 claudin-1 , which is a marker of a tight junction , desmograin-3 , which is a marker of desmosome , and e - cadherin , which is a marker of an adherence junction , are localized in the interface between cells at all cell layers , including the superficial layers.16 lamin is an important constituent in the basement membrane at the basal corneal epithelium junction , which is expressed continuously in basal cells of the corneal epithelial model basal layer.16 the basement membrane was smooth like that of the human eye.16 therefore , the corneal epithelial model reproduced many of the characteristics of the native human corneal tissue , and it provides a morphologically relevant means to assess nir effects as an alternative to animal testing .", "the corneal epithelium , like other epithelial barriers in the human body , is continuously subjected to physical , chemical , and biological insults , which can produce a wound and/or loss of barrier functions .", "proper healing of corneal wounds is vital for maintaining a clear , healthy cornea and preserving vision .", "corneal epithelium responds rapidly to injury , wherein wound healing occurs by cells migrating as a sheet to cover the defect and reestablish barrier functions.21 in wounded cornea , the epithelium plays a central role , not only as a key cell type in corneal repair , but also as a growth factor source.22 as in other tissues , several growth factors are suggested to play a role in regulating corneal epithelial function and wound healing.21,23 following nir irradiation that simulates solar radiation , microarray analysis of corneal epithelial cell dna in this study showed that egfr was highly expressed , and statistically significant upregulation of egfr variants 1 and 3 was observed ( p<0.05 ) by quantitative real - time pcr .", "egfr is a prototypic tyrosine kinase receptor that is part of a larger family of erbb receptors .", "erbb2 , erbb3 , and erbb4 share with egfr characteristics that include extracellular ligand - binding sites , intracellular kinase domains , and tyrosine - rich regions .", "egfr activated in response to injury serves as a powerful mediator of corneal epithelial wound healing10 by coordinating multiple extracellular signals generated in response to cell injury.2427 in turn , the levels of egfr and tyrosine kinase activity are major determinant factors for epithelial cell function in tissues and organs.22 echoing this concept is the finding that in some cancer patients , treatment with egfr - specific monoclonal antibodies cetuximab and gefitinib ( an egfr kinase inhibitor ) resulted in ocular abnormalities , including diffuse punctate keratitis and corneal erosion.2830 thus , maintaining a proper level of egfr signaling is critical for corneal homeostasis.22 ttll5 gene has 32 exons with high expression in heart and skeletal muscle and lower expression in many other tissues , including the eye and brain.31,32 it encodes a 1,281-amino acid protein that is localized to the cytoplasm and nucleus.31 ttll5 is thought to be a key initiator of polyglutamylation in -tubulin33 and has been previously reported to be essential for the correct function of sperm flagella.34 ttll5 plays a role in polyglutamylation of primary cilia in vitro.35 notably , genes involved in the polyglutamylation and deglutamylation of -tubulin have been associated with photoreceptor degeneration in mice.35 in this study , ttll5 showed the high degree of upregulation in the dna microarray analysis , whereas statistically significant upregulation of ttll5 was not observed in quantitative real - time pcr analysis .", "further studies are needed to determine if a higher power output or increased treatment frequency may promote significant upregulation of ttll5 .", "the findings here that egfr expression is significantly upregulated following exposure of corneal epithelial cells to nir irradiation support that solar nir radiation that reaches human tissues can induce corneal cell injury .", "although significant egfr expression upregulation occurred after just one treatment at the tested power output , further studies are needed to determine if a higher power output or increased treatment frequency may promote even larger changes in expression .", "furthermore , this was a preliminary study based on a fairly small number of samples , so a large - scale study will be needed ."], ["nir irradiation simulating solar nir radiation that reaches human tissues induced upregulated expression of egfr in human corneal cells .", "nir radiation represents more than half of solar energy and can not be sufficiently blocked by eyewear . moreover ,", "thus , the results of this study indicate that enhanced protection from and avoidance of both uv and nir radiation should be considered to prevent eye damage ."]]}
{"article_id": "PMC4590913", "article_text": ["hypertension is a common chronic medical condition affecting people in pakistan and the rest of the world .", "it is an important modifiable risk factor for cardiovascular morbidity and mortality , particularly for stroke ( accounting for 51% of all stroke deaths worldwide ) , ischemic heart disease ( 45% of all deaths ) , chronic kidney disease , congestive heart failure , aortic aneurysm , and peripheral arterial disease .", "prevalence of hypertension ( systolic blood pressure > 140  mm hg or diastolic blood pressure > 90  mm hg , or on antihypertensive medications ) in pakistan has increased from 17% in 1980 to 35% in 2008 in adults aged 18 years and older .", "the increasing prevalence of hypertension together with a deficient control makes this one of the frequent conditions that require urgent medical attention .", "the prevalence of uncontrolled hypertension varies around the world , with the lowest prevalence in rural india ( 3.4% in men and 6.8% in women ) and the highest prevalence in poland ( 68.9% in men and 72.5% in women ) .", "however , the control of hypertension was 23% from a community based data in urban population from karachi , pakistan .", "uncontrolled hypertension can progress to hypertensive crisis defined as a systolic blood pressure > 180  mm hg or a diastolic blood pressure > 120  mm hg .", "hypertensive crisis can be further classified as a hypertensive urgency or hypertensive emergency depending on end - organ involvement including cardiac , renal , and neurologic injury . ", "hypertensive urgency  refers to severe hypertension without evidence of new or worsening end - organ injury while  hypertensive emergency  refers to a severe hypertension that is associated with new or progressive end - organ damage .", "hypertensive crises ( 76% urgencies , 24% emergencies ) represented more than one - fourth of all medical urgencies / emergencies .", "hypertensive urgencies frequently present with headache ( 22% ) , epistaxis ( 17% ) , and psychomotor agitation ( 10% ) and hypertensive emergencies frequently present with chest pain ( 27% ) , dyspnea ( 22% ) , and neurological deficit ( 21% ) .", "the reason for uncontrolled hypertension in pakistan is high due to lack of awareness , knowledge , adherence , and attitudes of pakistani patients with hypertension .", "however there is no data on patients with hypertensive crisis from tertiary care hospitals in pakistan .", "additionally the number of patients who complicate towards stroke , myocardial infarction , and chronic kidney disease is also not known .", "hence , it is essential to have figures on prevalence and clinical presentation from our setup . therefore , we conducted this study to determine the prevalence of hypertensive crisis , its management , and its outcome in patients presenting to a tertiary care center in karachi .", "this was a retrospective study conducted at the aga khan university , karachi , pakistan .", "the aga khan university hospital ( akuh ) has 563 beds in operation and provides services to over 50,000 hospitalized patients and to over 600,000 outpatients annually .", "ethical approval from the ethics review committee of the aga khan university ( 1985-med - erc-11 ) was taken for conduct of the study .", "adult inpatients ( > 18  yrs ) presenting to the er who were known hypertensive and had uncontrolled hypertension were included .", "controlled blood pressure was defined as systolic blood pressure ( sbp ) < 140  mm hg or diastolic blood pressure ( dbp ) < 90  mm hg .", "uncontrolled hypertension was defined as sbp > 140  mm hg and dbp > 90  mm hg in both diabetic and nondiabetic patients who were either aware of their problem or under pharmacological treatment .", "the sample was drawn using computerized medical record system international classification of diseases-9-coordination and maintenance ( icd-9-cm ) at health information management system in the hospital .", "patients admitted with primary diagnosis of hypertension crisis , uncontrolled hypertension , hypertensive emergency , and hypertensive urgency were selected through the icd-9-cm ( i-10 : essential ( primary ) hypertension , i-11 : hypertensive heart disease , i-12 : hypertensive renal disease , i-13 : hypertensive heart and renal disease , and i-15 : secondary hypertension ) .", "patients whose medical records did not contain minimal clinical information to allow case classification ( hypertensive urgency or emergency ) were excluded from the study .", "data over a period of 5 years , from year 2005 till year 2010 , was used . a sample of 1336 consecutive patients fulfilling the inclusion criterion was selected .", "all patients gave a general consent on admission ; however informed consent was not taken as data was extracted later through icd-9-cm .", "data on demographics , comorbid conditions , clinical symptoms , blood pressure readings at subsequent time intervals , length of stay , and antihypertensive drug therapy was recorded by trained data collectors .", "a history of physician - diagnosed diabetes mellitus ( dm ) , chronic kidney disease ( ckd ) , ischemic heart disease ( ihd ) , and stroke was noted from the patient 's medical record file .", "dm was defined as fasting plasma glucose 126  mg / dl at a prior visit .", "ckd was defined as rise in serum creatinine of > 1.2  mg / dl for 3 months .", "blood pressure readings , at different time intervals , were recorded from vital sheets for nursing services .", "hypertensive crisis was defined as a systolic blood pressure > 180  mm hg or a diastolic blood pressure > 120  mm hg .", "management of patient was assessed by recording the list of medication from the computer generated pharmacy sheet attached inside the medical record file .", "antihypertensive treatment was divided into two types : medication given per oral and medications given intravenously .", "i values where available were recorded using the patient profile viewer , an online hospital database software .", "mean length of stay was recorded by calculating the time interval between the patient 's admission date / time and discharge date / time from er / ward .", "various complications like myocardial infarction , stroke , aortic dissection , acute renal failure , and pulmonary edema developed during the hospital stay were recorded using the discharge summary notes filled by the primary consultant .", "myocardial infarction was diagnosed when blood levels of sensitive and specific biomarkers such as cardiac troponin or ckmb are increased in the clinical setting of acute myocardial ischemia with electrocardiographic changes . according to who definition ,", "stroke was defined as a rapidly developing clinical sign of focal ( at times global ) disturbance of cerebral function , lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin .", "aortic dissection was referred as the condition when a separation has occurred in aortic wall intima as diagnosed on ct scan , causing blood flow into a new false channel composed of the inner and outer layers of the media .", "acute renal failure was diagnosed when the plasma urea nitrogen ( pun ) or serum creatinine did not stabilize within 72 hours .", "acute pulmonary edema was defined as alveolar or interstitial edema verified by chest x - ray and/or with o2 saturation < 90% on room air prior to treatment accompanied by severe respiratory distress , with crackles over the lungs and orthopnea .", "a minimum sample size of 237 patients was required to estimate a proportion of 19% of patients with hypertensive crises presenting to er , with bound on error of 5% and alpha of 5% .", "mean and standard deviation were used for qualitative variables and frequency and percentage for qualitative variables .", "chi - square test was used to compare categorical variables and student 's t - test was used to compare quantitative variables .", "a total of 73,063 hypertensive patients presented to the er between years 2005 and 2010 out of which a sample of 1336 ( 1.8% ) patients was taken for this study . the prevalence ( % ) of uncontrolled hypertension was 28.9% ( 387 ) .", "prevalence of hypertensive crisis overall was 16.3% ( 218 ) and among those with uncontrolled hypertension is 56.3% ( 218 ) .", "mean age ( sd ) of patients presenting to the er was 56.7 ( 14.7 ) and 175% ( 45.2 ) of patients were male .", "overall , dyslipidemia was the most common comorbidity in patients presenting with uncontrolled hypertension to the er with the prevalence of 43.2% ( 167 ) followed by diabetes mellitus , 36.9% ( 143 ) , and ischemic heart disease , 21.4% ( 83 ) , and 13.9% ( 54 ) of them were smokers .", "the baseline characteristics of patients overall , with hypertensive crisis and without hypertensive crisis , are given in table 1 .", "headache was the most common presenting symptom , 35.7% ( 138 ) , followed by dyspnea , 32.6% ( 126 ) , chest pain , 21.4% ( 83 ) , dizziness , 21.2% ( 82 ) , vomiting , 17.3% ( 67 ) , epistaxis , 5.2% ( 20 ) , and neurologic deficit , 3.6% ( 14 ) .", "on comparison of patients with hypertensive crisis with those with no hypertensive crisis the clinical symptoms that were statistically significant were as follows : headache was present in 42.2% ( 92 ) versus 27.2% ( 46 ) p value = 0.002 and chest pain was present in 17.4% ( 38 ) versus 26.6% ( 45 ) p value = 0.02 . \n", "the mean ( sd ) systolic blood pressure ( sbp ) recorded in patients with hypertensive crisis versus no hypertensive crisis in er was 202 ( 17.971 ) and 158 ( 13.387 ) ( p value  0.001 ) .", "the mean ( sd ) diastolic blood pressure in patients with hypertensive crisis versus no hypertensive crisis in er was 108 ( 17.429 ) mm hg and 87 ( 14.984 ) mm hg , respectively , ( p value  0.001 ) .", "the trend of blood pressure recorded in the er is shown in figures 1 and 2 . \n antihypertensive medications used in management of patients with hypertensive crisis .", "calcium channel blocker was the most widely used oral antihypertensive medication in the er , 35.4% ( 137 ) .", "intravenous ( iv ) nitrate was the most commonly administered iv medication in er , 22.7% ( 88 ) .", "the mean ( sd ) drop in sbp in patients with hypertensive crisis who received intravenous medications versus oral medications was 53.1 ( 29 ) mm hg and 43 ( 27 ) , respectively ( p value = 0.01 ) .", "the mean drop in dbp in patients with hypertensive crisis who received intravenous versus perioral medications was 25.8 ( 19 ) and mm hg and 17.8 ( 22 ) mm hg , respectively ( p value = 0.006 ) .", "comparison of blood pressure trends in patients with and without hypertensive crisis on intravenous or per oral medication is shown in table 2 . the maximum drop in systolic blood pressure and diastolic blood pressure", "was achieved by sodium nitroprusside : 80 ( 15 ) mm hg and 37.5 ( 7.77 ) mm hg .", "types of intravenous medications and drop in sbp and dbp are shown in table 3 .", "the total length of stay ( in days ) for patients with hypertensive crisis was 2.46 ( 0.164 ) whereas the total length of stay ( in days ) for patients without hypertensive crisis was 2.20 ( 0.158 ) .", "overall prevalence ( % ) of complications was 47.7% ( 104 ) in patients with hypertensive crisis .", "acute renal failure was the most common complication with the prevalence ( % ) of 41.3% ( 43 ) followed by myocardial infarction 28.8% ( 30 ) and pulmonary edema 18.26% ( 19 ) .", "stroke accounted for 6.5% ( 12 ) of complications . on comparison of patients with hypertensive crisis and patients without it , myocardial infarction occurred in 7.2% ( 15 ) versus 9% ( 15 ) , stroke occurred in 2.4% ( 5 ) versus 4.2% ( 7 ) , and acute kidney injury occurred in 11.5% ( 24 ) versus 11.4% ( 19 ) patients , respectively .", "on further categorization of patients with hypertensive crisis who received intravenous antihypertensive in er versus not receiving intravenous medication , there was no significant difference in the complications in both groups .", "we have shown in this study on patients presenting to er with hypertensive crisis that the prevalence of hypertensive crisis is high ( 56.3% ) .", "mean length of stay was longer in patients with hypertensive crisis and acute renal failure was the most common complication in these patients .", "the incidence of hypertensive crisis was 47.22% in a study conducted in bosnia and 16% in a study conducted in brazil [ 22 , 23 ] .", "the reasons for high prevalence of patients with hypertensive crisis in our setup are multiple .", "lack of knowledge about control of hypertension and poor compliance to antihypertensive medications is a major issue in pakistan .", "also , lack of proper health infrastructure in public sector leading to inability of the poor population to access healthcare adds onto the severity of hypertension in these patients . in previous studies [ 25 , 26 ] , the most frequent clinical sign of patients presenting to", "the er was headache ( 22% and 42% ) and dizziness was reported among 30% of emergency department patients .", "this study showed ( 36% ) headache as the most common clinical sign followed by dizziness ( 21% ) which supports the previous findings about the characteristics of hypertensive crisis patients .", "complications of hypertensive crisis reported in previous studies conducted in bahrain and italy were acute coronary syndrome ( 32% ) , left ventricular heart failure ( 38% ) , and stroke ( 29.3% )   and cerebral infarction ( 24% ) , pulmonary edema ( 23% ) , and hypertensive encephalopathy ( 16% ) .", "however acute renal failure , myocardial infarction , and pulmonary edema are the most common complications in our setup .", "the reason for high prevalence of acute renal failure in our setup may be the higher number of patients with hypertensive emergency .", "the most common intravenous medication used to control hypertensive crisis in this study was nitrates .", "recommendation for use of intravenous nicardipine was reported by a study conducted in 2005   whereas sodium nitroprusside , because of its direct vasodilator effect and immediate onset of action , was recommended for hypertensive emergency in a subsequent study in 2006 .", "we observed in this study that sodium nitroprusside was the most potent intravenous antihypertensive in dropping blood pressure .", "however it was not the most commonly used drug in this study . in a similar study ,", "intravenous labetalol was reported as the most frequently used antihypertensive medication for emergency department patients presenting with hypertensive urgency .", "this indicates that the use of intravenous nitrate as the most common antihypertensive for management of hypertensive crisis ( apart from use in patients with cardiac cause ) is not completely in accordance with the recommendations .", "however in some studies , oral labetalol , beta blockers , diuretics , ace inhibitors , and calcium channel blockers have been recommended .", "calcium channel blockers have also been found to be the most common antihypertensives used for management of hypertensive in this population and have been found to have good control rates .", "calcium channel is now recommended as a first line antihypertensive in the recent nice guidelines .", "the reduction in systolic and diastolic blood pressure reported in our study shows a smooth decline in order to avoid risks of potential side effects of a much rapid decline .", "it is recommended that the initial reduction in mean arterial pressure ( map ) in case of hypertensive emergency should not be more than 20%25% below the pretreatment blood pressure or that map be reduced within the first 3060 minutes to 110115  mm hg .", "this can be comparable to our study which reported an overall decrease of blood pressure of about 52  mm hg in sbp from intravenous medication from the time of admission until 2 hours after admission in er .", "a rapid decline in blood pressure is associated with acute deterioration in renal function , ischemic , cardiac , or cerebral events , and occasional retinal artery occlusion and acute blindness .", "the goal is to reduce bp by 1015% over a period of 3060 minutes , with the exception of the patient that presents with aortic dissection or acute intracranial bleed .", "however treatment should be individualized to each patient based on the type and extent of end - organ damage , degree of bp elevation , and the specific side effects that each medication could have on a patient 's preexisting comorbidities .", "the strength of this study is that it is the first study from this region to report figures on prevalence of hypertensive crisis , as well as prevalence of complications as a result of hypertensive crisis among patients .", "moreover , this study also reports management of these patients in terms of the treatment received in the er and into the ward and their mean length of stay .", "however there are limitations in this study ; firstly it has limited external validity as the sample is not representative for an entire population .", "it represents population visiting a single - tertiary care hospital and , hence , it is not representative of the entire population of pakistan .", "secondly , compliance issues with the medication regimen have not been taken into consideration ; hence , the medications administered to the patients may be misjudged .", "thirdly , some patients may also have gotten discharged against medical advice ; hence , the optimal length of stay for these patients may not have been achieved .", "fourth , this retrospective study can not strongly give a cause and effect association and we have not reported the hypertensive urgencies and emergencies separately .", "we did not report data separately on hypertensive emergency and urgency and retinopathy could not be examined and reported in all patients .", "the prevalence of hypertensive crisis is high in our subjects . per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in the er and ward ."], "abstract_text": ["<S> \n objectives . hypertension , </S>", "<S> if uncontrolled , can lead to hypertensive crisis . </S>", "<S> we aim to determine the prevalence of hypertensive crisis , its management , and outcome in patients presenting to a tertiary care center in karachi . methods . </S>", "<S> this was a cross - sectional study conducted at the aga khan university , karachi , pakistan . </S>", "<S> adult inpatients ( > 18  yrs ) presenting to the er who were known hypertensive and had uncontrolled hypertension were included . results . out of 1336 patients , 28.6% ( 387 ) had uncontrolled hypertension . </S>", "<S> the prevalence of hypertensive crisis among uncontrolled hypertensive was 56.3% ( 218 ) . per oral calcium channel blocker ; </S>", "<S> 35.4% ( 137 ) and intravenous nitrate ; 22.7% ( 88 ) were the most commonly administered medication in the er . the mean ( sd ) drop in sbp in patients with hypertensive crisis on intravenous treatment was 53.1 ( 29 ) mm  hg and on per oral treatment was 43 ( 27 ) mm  hg . the maximum mean ( sd ) drop in blood pressure was seen by intravenous sodium nitroprusside ; 80 ( 51 ) mm  hg in sbp . </S>", "<S> acute renal failure was the most common complication with a prevalence of 11.5% ( 24 ) . </S>", "<S> conclusion . </S>", "<S> the prevalence of hypertensive crisis is high . per oral calcium channel blocker and intravenous nitrate </S>", "<S> are the most commonly administered medications in our setup . </S>"], "labels": null, "section_names": ["1. Background", "2. Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["hypertension is a common chronic medical condition affecting people in pakistan and the rest of the world .", "it is an important modifiable risk factor for cardiovascular morbidity and mortality , particularly for stroke ( accounting for 51% of all stroke deaths worldwide ) , ischemic heart disease ( 45% of all deaths ) , chronic kidney disease , congestive heart failure , aortic aneurysm , and peripheral arterial disease .", "prevalence of hypertension ( systolic blood pressure > 140  mm hg or diastolic blood pressure > 90  mm hg , or on antihypertensive medications ) in pakistan has increased from 17% in 1980 to 35% in 2008 in adults aged 18 years and older .", "the increasing prevalence of hypertension together with a deficient control makes this one of the frequent conditions that require urgent medical attention .", "the prevalence of uncontrolled hypertension varies around the world , with the lowest prevalence in rural india ( 3.4% in men and 6.8% in women ) and the highest prevalence in poland ( 68.9% in men and 72.5% in women ) .", "however , the control of hypertension was 23% from a community based data in urban population from karachi , pakistan .", "uncontrolled hypertension can progress to hypertensive crisis defined as a systolic blood pressure > 180  mm hg or a diastolic blood pressure > 120  mm hg .", "hypertensive crisis can be further classified as a hypertensive urgency or hypertensive emergency depending on end - organ involvement including cardiac , renal , and neurologic injury . ", "hypertensive urgency  refers to severe hypertension without evidence of new or worsening end - organ injury while  hypertensive emergency  refers to a severe hypertension that is associated with new or progressive end - organ damage .", "hypertensive crises ( 76% urgencies , 24% emergencies ) represented more than one - fourth of all medical urgencies / emergencies .", "hypertensive urgencies frequently present with headache ( 22% ) , epistaxis ( 17% ) , and psychomotor agitation ( 10% ) and hypertensive emergencies frequently present with chest pain ( 27% ) , dyspnea ( 22% ) , and neurological deficit ( 21% ) .", "the reason for uncontrolled hypertension in pakistan is high due to lack of awareness , knowledge , adherence , and attitudes of pakistani patients with hypertension .", "however there is no data on patients with hypertensive crisis from tertiary care hospitals in pakistan .", "additionally the number of patients who complicate towards stroke , myocardial infarction , and chronic kidney disease is also not known .", "hence , it is essential to have figures on prevalence and clinical presentation from our setup . therefore , we conducted this study to determine the prevalence of hypertensive crisis , its management , and its outcome in patients presenting to a tertiary care center in karachi ."], ["this was a retrospective study conducted at the aga khan university , karachi , pakistan .", "the aga khan university hospital ( akuh ) has 563 beds in operation and provides services to over 50,000 hospitalized patients and to over 600,000 outpatients annually .", "ethical approval from the ethics review committee of the aga khan university ( 1985-med - erc-11 ) was taken for conduct of the study .", "adult inpatients ( > 18  yrs ) presenting to the er who were known hypertensive and had uncontrolled hypertension were included .", "controlled blood pressure was defined as systolic blood pressure ( sbp ) < 140  mm hg or diastolic blood pressure ( dbp ) < 90  mm hg .", "uncontrolled hypertension was defined as sbp > 140  mm hg and dbp > 90  mm hg in both diabetic and nondiabetic patients who were either aware of their problem or under pharmacological treatment .", "the sample was drawn using computerized medical record system international classification of diseases-9-coordination and maintenance ( icd-9-cm ) at health information management system in the hospital .", "patients admitted with primary diagnosis of hypertension crisis , uncontrolled hypertension , hypertensive emergency , and hypertensive urgency were selected through the icd-9-cm ( i-10 : essential ( primary ) hypertension , i-11 : hypertensive heart disease , i-12 : hypertensive renal disease , i-13 : hypertensive heart and renal disease , and i-15 : secondary hypertension ) .", "patients whose medical records did not contain minimal clinical information to allow case classification ( hypertensive urgency or emergency ) were excluded from the study .", "data over a period of 5 years , from year 2005 till year 2010 , was used . a sample of 1336 consecutive patients fulfilling the inclusion criterion was selected .", "all patients gave a general consent on admission ; however informed consent was not taken as data was extracted later through icd-9-cm .", "data on demographics , comorbid conditions , clinical symptoms , blood pressure readings at subsequent time intervals , length of stay , and antihypertensive drug therapy was recorded by trained data collectors .", "a history of physician - diagnosed diabetes mellitus ( dm ) , chronic kidney disease ( ckd ) , ischemic heart disease ( ihd ) , and stroke was noted from the patient 's medical record file .", "dm was defined as fasting plasma glucose 126  mg / dl at a prior visit .", "ckd was defined as rise in serum creatinine of > 1.2  mg / dl for 3 months .", "blood pressure readings , at different time intervals , were recorded from vital sheets for nursing services .", "hypertensive crisis was defined as a systolic blood pressure > 180  mm hg or a diastolic blood pressure > 120  mm hg .", "management of patient was assessed by recording the list of medication from the computer generated pharmacy sheet attached inside the medical record file .", "antihypertensive treatment was divided into two types : medication given per oral and medications given intravenously .", "i values where available were recorded using the patient profile viewer , an online hospital database software .", "mean length of stay was recorded by calculating the time interval between the patient 's admission date / time and discharge date / time from er / ward .", "various complications like myocardial infarction , stroke , aortic dissection , acute renal failure , and pulmonary edema developed during the hospital stay were recorded using the discharge summary notes filled by the primary consultant .", "myocardial infarction was diagnosed when blood levels of sensitive and specific biomarkers such as cardiac troponin or ckmb are increased in the clinical setting of acute myocardial ischemia with electrocardiographic changes . according to who definition ,", "stroke was defined as a rapidly developing clinical sign of focal ( at times global ) disturbance of cerebral function , lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin .", "aortic dissection was referred as the condition when a separation has occurred in aortic wall intima as diagnosed on ct scan , causing blood flow into a new false channel composed of the inner and outer layers of the media .", "acute renal failure was diagnosed when the plasma urea nitrogen ( pun ) or serum creatinine did not stabilize within 72 hours .", "acute pulmonary edema was defined as alveolar or interstitial edema verified by chest x - ray and/or with o2 saturation < 90% on room air prior to treatment accompanied by severe respiratory distress , with crackles over the lungs and orthopnea .", "a minimum sample size of 237 patients was required to estimate a proportion of 19% of patients with hypertensive crises presenting to er , with bound on error of 5% and alpha of 5% .", "mean and standard deviation were used for qualitative variables and frequency and percentage for qualitative variables .", "chi - square test was used to compare categorical variables and student 's t - test was used to compare quantitative variables ."], ["a total of 73,063 hypertensive patients presented to the er between years 2005 and 2010 out of which a sample of 1336 ( 1.8% ) patients was taken for this study . the prevalence ( % ) of uncontrolled hypertension was 28.9% ( 387 ) .", "prevalence of hypertensive crisis overall was 16.3% ( 218 ) and among those with uncontrolled hypertension is 56.3% ( 218 ) .", "mean age ( sd ) of patients presenting to the er was 56.7 ( 14.7 ) and 175% ( 45.2 ) of patients were male .", "overall , dyslipidemia was the most common comorbidity in patients presenting with uncontrolled hypertension to the er with the prevalence of 43.2% ( 167 ) followed by diabetes mellitus , 36.9% ( 143 ) , and ischemic heart disease , 21.4% ( 83 ) , and 13.9% ( 54 ) of them were smokers .", "the baseline characteristics of patients overall , with hypertensive crisis and without hypertensive crisis , are given in table 1 .", "headache was the most common presenting symptom , 35.7% ( 138 ) , followed by dyspnea , 32.6% ( 126 ) , chest pain , 21.4% ( 83 ) , dizziness , 21.2% ( 82 ) , vomiting , 17.3% ( 67 ) , epistaxis , 5.2% ( 20 ) , and neurologic deficit , 3.6% ( 14 ) .", "on comparison of patients with hypertensive crisis with those with no hypertensive crisis the clinical symptoms that were statistically significant were as follows : headache was present in 42.2% ( 92 ) versus 27.2% ( 46 ) p value = 0.002 and chest pain was present in 17.4% ( 38 ) versus 26.6% ( 45 ) p value = 0.02 . \n", "the mean ( sd ) systolic blood pressure ( sbp ) recorded in patients with hypertensive crisis versus no hypertensive crisis in er was 202 ( 17.971 ) and 158 ( 13.387 ) ( p value  0.001 ) .", "the mean ( sd ) diastolic blood pressure in patients with hypertensive crisis versus no hypertensive crisis in er was 108 ( 17.429 ) mm hg and 87 ( 14.984 ) mm hg , respectively , ( p value  0.001 ) .", "the trend of blood pressure recorded in the er is shown in figures 1 and 2 . \n antihypertensive medications used in management of patients with hypertensive crisis .", "calcium channel blocker was the most widely used oral antihypertensive medication in the er , 35.4% ( 137 ) .", "intravenous ( iv ) nitrate was the most commonly administered iv medication in er , 22.7% ( 88 ) .", "the mean ( sd ) drop in sbp in patients with hypertensive crisis who received intravenous medications versus oral medications was 53.1 ( 29 ) mm hg and 43 ( 27 ) , respectively ( p value = 0.01 ) .", "the mean drop in dbp in patients with hypertensive crisis who received intravenous versus perioral medications was 25.8 ( 19 ) and mm hg and 17.8 ( 22 ) mm hg , respectively ( p value = 0.006 ) .", "comparison of blood pressure trends in patients with and without hypertensive crisis on intravenous or per oral medication is shown in table 2 . the maximum drop in systolic blood pressure and diastolic blood pressure", "was achieved by sodium nitroprusside : 80 ( 15 ) mm hg and 37.5 ( 7.77 ) mm hg .", "types of intravenous medications and drop in sbp and dbp are shown in table 3 .", "the total length of stay ( in days ) for patients with hypertensive crisis was 2.46 ( 0.164 ) whereas the total length of stay ( in days ) for patients without hypertensive crisis was 2.20 ( 0.158 ) .", "overall prevalence ( % ) of complications was 47.7% ( 104 ) in patients with hypertensive crisis .", "acute renal failure was the most common complication with the prevalence ( % ) of 41.3% ( 43 ) followed by myocardial infarction 28.8% ( 30 ) and pulmonary edema 18.26% ( 19 ) .", "stroke accounted for 6.5% ( 12 ) of complications . on comparison of patients with hypertensive crisis and patients without it , myocardial infarction occurred in 7.2% ( 15 ) versus 9% ( 15 ) , stroke occurred in 2.4% ( 5 ) versus 4.2% ( 7 ) , and acute kidney injury occurred in 11.5% ( 24 ) versus 11.4% ( 19 ) patients , respectively .", "on further categorization of patients with hypertensive crisis who received intravenous antihypertensive in er versus not receiving intravenous medication , there was no significant difference in the complications in both groups ."], ["we have shown in this study on patients presenting to er with hypertensive crisis that the prevalence of hypertensive crisis is high ( 56.3% ) .", "mean length of stay was longer in patients with hypertensive crisis and acute renal failure was the most common complication in these patients .", "the incidence of hypertensive crisis was 47.22% in a study conducted in bosnia and 16% in a study conducted in brazil [ 22 , 23 ] .", "the reasons for high prevalence of patients with hypertensive crisis in our setup are multiple .", "lack of knowledge about control of hypertension and poor compliance to antihypertensive medications is a major issue in pakistan .", "also , lack of proper health infrastructure in public sector leading to inability of the poor population to access healthcare adds onto the severity of hypertension in these patients . in previous studies [ 25 , 26 ] , the most frequent clinical sign of patients presenting to", "the er was headache ( 22% and 42% ) and dizziness was reported among 30% of emergency department patients .", "this study showed ( 36% ) headache as the most common clinical sign followed by dizziness ( 21% ) which supports the previous findings about the characteristics of hypertensive crisis patients .", "complications of hypertensive crisis reported in previous studies conducted in bahrain and italy were acute coronary syndrome ( 32% ) , left ventricular heart failure ( 38% ) , and stroke ( 29.3% )   and cerebral infarction ( 24% ) , pulmonary edema ( 23% ) , and hypertensive encephalopathy ( 16% ) .", "however acute renal failure , myocardial infarction , and pulmonary edema are the most common complications in our setup .", "the reason for high prevalence of acute renal failure in our setup may be the higher number of patients with hypertensive emergency .", "the most common intravenous medication used to control hypertensive crisis in this study was nitrates .", "recommendation for use of intravenous nicardipine was reported by a study conducted in 2005   whereas sodium nitroprusside , because of its direct vasodilator effect and immediate onset of action , was recommended for hypertensive emergency in a subsequent study in 2006 .", "we observed in this study that sodium nitroprusside was the most potent intravenous antihypertensive in dropping blood pressure .", "however it was not the most commonly used drug in this study . in a similar study ,", "intravenous labetalol was reported as the most frequently used antihypertensive medication for emergency department patients presenting with hypertensive urgency .", "this indicates that the use of intravenous nitrate as the most common antihypertensive for management of hypertensive crisis ( apart from use in patients with cardiac cause ) is not completely in accordance with the recommendations .", "however in some studies , oral labetalol , beta blockers , diuretics , ace inhibitors , and calcium channel blockers have been recommended .", "calcium channel blockers have also been found to be the most common antihypertensives used for management of hypertensive in this population and have been found to have good control rates .", "calcium channel is now recommended as a first line antihypertensive in the recent nice guidelines .", "the reduction in systolic and diastolic blood pressure reported in our study shows a smooth decline in order to avoid risks of potential side effects of a much rapid decline .", "it is recommended that the initial reduction in mean arterial pressure ( map ) in case of hypertensive emergency should not be more than 20%25% below the pretreatment blood pressure or that map be reduced within the first 3060 minutes to 110115  mm hg .", "this can be comparable to our study which reported an overall decrease of blood pressure of about 52  mm hg in sbp from intravenous medication from the time of admission until 2 hours after admission in er .", "a rapid decline in blood pressure is associated with acute deterioration in renal function , ischemic , cardiac , or cerebral events , and occasional retinal artery occlusion and acute blindness .", "the goal is to reduce bp by 1015% over a period of 3060 minutes , with the exception of the patient that presents with aortic dissection or acute intracranial bleed .", "however treatment should be individualized to each patient based on the type and extent of end - organ damage , degree of bp elevation , and the specific side effects that each medication could have on a patient 's preexisting comorbidities .", "the strength of this study is that it is the first study from this region to report figures on prevalence of hypertensive crisis , as well as prevalence of complications as a result of hypertensive crisis among patients .", "moreover , this study also reports management of these patients in terms of the treatment received in the er and into the ward and their mean length of stay .", "however there are limitations in this study ; firstly it has limited external validity as the sample is not representative for an entire population .", "it represents population visiting a single - tertiary care hospital and , hence , it is not representative of the entire population of pakistan .", "secondly , compliance issues with the medication regimen have not been taken into consideration ; hence , the medications administered to the patients may be misjudged .", "thirdly , some patients may also have gotten discharged against medical advice ; hence , the optimal length of stay for these patients may not have been achieved .", "fourth , this retrospective study can not strongly give a cause and effect association and we have not reported the hypertensive urgencies and emergencies separately .", "we did not report data separately on hypertensive emergency and urgency and retinopathy could not be examined and reported in all patients ."], ["the prevalence of hypertensive crisis is high in our subjects . per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in the er and ward ."]]}
{"article_id": "PMC4142314", "article_text": ["a basic set of proteins and mrnas are differentially expressed among cell types , temporally and spatially , generating a vast assortment of cell phenotypes and/or activation states within a single tissue .", "outlining this protein / mrna portrait is thus crucial for understanding not only the uniqueness characterizing cells , but especially their distinguished functions .", "this becomes of major relevance when the balance between cell - intrinsic properties and identity cues received and provided by each cell to its neighboring cells then shapes the cell - to - cell cross talk during physiopathological conditions . in the cns , neuroinflammation is the typical consequence of the interchange among different cell types , particularly neurons , astrocytes , oligodendrocytes and microglia , of a variety of cues as neurotransmitters , cytokines , chemokines , toxic metabolites that condition the final protein / mrna profiles of cells , their activation states and functional outcomes [ 2 , 3 ] .", "since neuroinflammation accompanies a large variety of neurodegenerative diseases , there is increasing interest in determining how the different cell phenotypes and cellular interconnectivity might contribute to reduce inflammation and reverse neurodegeneration .", "microglia actively participate to the context - dependent , neuroprotective / neurotoxic molecular network that is triggered during neuroinflammation . among the molecular cues having a key role in this process ,", "extracellular nucleotides are major responsible for intercellular communication and propagation of inflammatory stimuli [ 57 ] .", "this occurs by specific binding to various receptor subtypes , termed ionotropic p2x and metabotropic p2y , which are simultaneously localized on several different cell phenotypes . among these", ", the p2y12 receptor subtype   belonging to the gi class of g protein - coupled receptors is activated by adp .", "two transcript variants apparently encoding the same protein isoform have been identified so far for p2ry12 gene , but the determinants for cell specificity of p2y12 protein expression are still unknown .", "p2y12 is found on the surface mainly , but not exclusively , of blood platelets , where it acts as blood clotting regulator and target for the treatment of thromboembolisms [ 9 , 10 ] . in the nervous system ,", "the tissue- and cellular - selective expression of p2y12 exhibits a pattern throughout white and gray matter consistent with astrocyte expression , although it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna .", "moreover , we previously established in vivo the expression of p2y12 in oligodendrocytes and myelin sheaths of rat cerebral cortex , subcortical areas , and periventricular white matter . this localization is confirmed throughout the corticospinal tract , therefore suggesting high conserved tissue - homogeneity and phenotype - specificity , and a hypothesized role in myelination [ 1215 ] .", "p2y12 is finally observed in brain and spinal cord resident microglia , where it affects activation , chemotaxis [ 1619 ] and neuropathic pain , but it is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] .", "p2y12 expression in primary microglia is variable with postnatal development and shows sexually dimorphic behavior . through the use of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , the aim of the present work is to provide comparative evidence about p2y12 cell specificity in microglia versus oligodendrocytes particularly from the healthy and diseased cns under neuroinflammatory conditions as those sustained during amyotrophic lateral sclerosis ( als ) and multiple sclerosis ( ms ) .", "adult b6.cg-tg ( sod1-g93a)1gur / j mice expressing high copy number of mutant human sod1 with a gly93ala substitution ( sod1-g93a ) were originally obtained from jackson laboratories ( bar harbor , me , usa ) and housed in our indoor animal facility as described in apolloni and collaborators , .", "the animals were euthanized , according to the guidelines for preclinical testing and colony management .", "also neonatal wistar and adult lewis rats ( from charles river laboratories , lc , italy ) were housed in our indoor animal facility .", "animal procedures were performed according to european guidelines for the use of animals in research ( 86/609/cee ) and the requirements of italian laws ( d.l .", "ethical procedures were approved by animal welfare office , department of public health and veterinary , nutrition and food safety , general management of animal care and veterinary drugs of the italian ministry of health .", "all efforts were made to minimize animal suffering and number of animals necessary to produce reliable results .", "microglia primary cultures prepared from mouse cortex as previously described   were about 98% pure , as verified by immunofluorescence with cd11b ( for microglia ) , glial fibrillary acidic protein ( gfap , for astrocytes ) , neuronal nuclei ( neun , for neurons ) , and smi94 ( for oligodendrocytes ) .", "purified cultures of oligodendrocytes were prepared from forebrain of postnatal day 1 - 2 wistar rats , according to minor modifications from a previously described method . after removing the meninges ,", "cortices were minced , digested , and dissociated by passage through 70  m nylon cell strainer ( bd biosciences , europe ) .", "cells were plated in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 20% heat - inactivated fetal bovine serum ( fbs ) , 4  mm glutamine , 1  mm sodium pyruvate , 50  u / ml penicillin , 50  g / ml streptomycin , and 100  g / ml gentamicin in t75 poly - d - lysine - coated flasks , at about 10 million cells / flask .", "the cultures were maintained at 37c in a 5% co2 and 95% air atmosphere for 14 days .", "mixed glial cultures were then shaken at 200  rpm at 37c for 1  h and 98% pure microglia collected from the supernatant of each flask , as verified by immunofluorescence with gfap , neun , neural / glial antigen 2 ( ng2 , for oligodendroglial precursor cells ) , myelin basic protein ( mbp , for mature oligodendrocytes ) , and cd11b .", "after further shaking at 200  rpm and 37c for 1518  h , the detached cell suspension was finally incubated for 1  h at 37c for differential adhesion of contaminating cells .", "the non - adherent cells were filtered through 40  m nylon cell strainer ( bd biosciences ) , spun for 10  min at 100  g , resuspended in oligodendroglial precursor cells medium ( basal chemically defined medium : dmem , 4  mm l - glutamine , 1  mm sodium pyruvate , 0.1% bovine serum albumin ( bsa ) , 50  g  ml apo - transferrin , 5  g ml insulin , 30  nm sodium selenite , 10  nm d - biotin , and 10  nm hydrocortisone ) containing 10  ng  ml platelet derived growth factor - aa ( pdgf - aa ) and 10  ng  ml basic fibroblast growth factor ( bfgf ) , and seeded at the density of 1  10  cells / cm into poly d , l - ornithine - coated plates .", "the cells were then induced to differentiate into mature oligodendrocytes when the basal chemically defined medium was added with 15  nm triiodothyronine , 10  ng  ml ciliary neurotrophic factor ( cntf ) and 0,05  mg 10  ml n - acetyl - l - cysteine ( nac ) , for 47 days .", "a 98% pure population of oligodendrocytes was thus obtained , as verified by immunofluorescence with ng2 , mbp , gfap , neun , and cd11b antibodies .", "organotypic cerebellar slice cultures were prepared with modifications from previously published protocols [ 26 , 27 ] .", "briefly , cerebella were excised from neonatal wistar rat ( 810 days old ) , cut on a mc ilwain tissue chopper ( 400  m ) and parasagittal slices separated into cold hank 's balanced salt solution ( hbss ) .", "two to three slices were plated on each millicell cm culture inserts ( millipore , italy ) and kept in organotypic maintenance medium ( 50% basal medium eagle - bme , 25% hbss , 25% heat - inactivated horse serum , supplemented with 5  mg / ml glucose , 1  mm glutamine , 50  u / ml penicillin , and 50  g / ml streptomycin ) at 37c in a 5% co2 and 95% air atmosphere .", "the medium was changed every 2 days and double immunofluorescence was performed in free floating after 710 days in vitro .", "organotypic cultures were washed three times with pbs , fixed with 4% paraformaldehyde for 1  h , rinsed , and blocked for 1  h in phosphate buffered saline ( pbs)/10% normal donkey serum ( nds)/0.4% triton x-100 .", "primary antibodies were incubated for 24  h in pbs/2% nds/0.4% triton x-100 ( table 1 ) .", "the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1  :  100 , jackson immunoresearch , europe ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1  :  200 , jackson immunoresearch ) . after rinsing ,", "sections were mounted , covered with fluoromount medium ( sigma - aldrich , milan , italy ) and a coverslip , and analyzed by confocal microscopy .", "microglia and oligodendrocytes were washed three times with pbs , fixed with 4% paraformaldehyde for 10  min ( oligodendrocytes ) or 20  min ( microglia ) , washed , permeabilized with 0.050.1% triton x-100 for 10  min , rinsed , and blocked for 30  min in pbs/1% bsa .", "microglia were stained for about 3  h at 37c in 1% pbs / bsa with 5  g / ml cy2-phalloidin ( sigma - aldrich ) , in combination with primary antibodies against p2y12 receptor , as reported in table 1 .", "the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1  :  100 , jackson immunoresearch ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1  :  200 , jackson immunoresearch ) . cells were extensively washed and stained with the nucleic acid blue dye , hoechst 33342 ( 1  :  1000 ) .", "after rinsing , cells were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy .", "animals were anaesthetized by intraperitoneal injection of chloral hydrate ( 500  mg / kg ) and transcardially perfused with pbs followed by 4% paraformaldehyde at ph 7.4 .", "tissue samples ( mice spinal cord and rat brain ) were then postfixed for 1 - 2 days , and cryoprotected in 30% sucrose in pbs at 4c .", "mice spinal cords ( l3l5 ) were cut at 30  m thickness with a frozen microtome .", "sections were mounted on slide glasses and allowed to air - dry ( 1 - 2  h ) .", "a rectangle was then drawn around the sections with a pap pen ( sigma - aldrich ) .", "rat brains were cut at 40  m thickness using a cryostat microtome and sections were processed in free - floating .", "double immunofluorescence analysis was performed after blocking in pbs containing 10% nds and 0.3% triton x-100 for 1  h at room temperature .", "sections were incubated with different combinations of primary antibodies ( table 1 ) , in pbs , 0.3% triton x-100 and 2% nds , for 2448  h at 4c .", "finally , sections were washed with pbs and incubated with appropriate fluorescent - conjugated secondary antibodies for 3  h at room temperature . after rinsing", ", sections were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy .", "tissues supplied by uk multiple sclerosis tissue bank at imperial college , london , were collected postmortem with fully informed consent from both donors and close relatives .", "procedures for retrieval , processing , and storage have gained ethical approval from all appropriate committees .", "the brain tissues analyzed were from 7 neuropathologically confirmed cases of secondary progressive ms ( spms ) .", "analysis was performed also on samples from patients who died due to nonneurological diseases ( healthy ) .", "cerebral hemispheres were fixed with 4% paraformaldehyde for about two weeks , coronally sliced , and blocked .", "individual blocks were cryoprotected in 30% sucrose for one week and frozen by immersion in isopentane precooled on a bed of dry ice .", "cryostat sections ( 3040  m thick ) were either stained with luxol fast blue and cresyl fast violet ( kluver - barrera staining ) , in order to detect white matter lesions and their cellularity , or subjected to immunohistochemistry for mbp , in order to distinguish grey matter lesions .", "sections were processed in free - floating for double immunofluorescence studies , as described in amadio and collaborators .", "immunofluorescence was analyzed by means of a confocal laser scanning microscope ( zeiss , lsm700 , germany ) equipped with four laser lines : 405 , 488 , 561 , and 639  nm .", "the brightness and contrast of the digital images were adjusted using the zen software ( zeiss ) .", "human p2y12 complete cdna was obtained by reverse transcription with enhanced avian rt - pcr kit ( sigma - aldrich ) from total human brain rna ( life technologies , paisley , uk ) .", "the obtained cdna was then cloned into the xhoi and xbai sites of the eukaryotic expression vector cs2 + mt to provide n - terminal 6x c - myc epitope - tagged mammalian expression plasmids , which has been validated by dna sequencing .", "oligos used for amplification were as follows : forward 5gcctcgagatgcaagccgtcgacaacctc3 and reverse 5gctctagagtttacattggagtctcttc designed on human p2y12 mrna ( nm_022788.3 ) .", "human embryonic kidney 293 cells ( hek 293 ) and sloan - kettering neuroblastoma sh - sy5y clone ( sh - sy5y ) cells were grown in dmem supplemented with 10% fbs , 100  unit / ml penicillin , and 100  g / ml streptomycin at 37c in atmosphere containing 5% co2 .", "one day before transfection , hek293 or sh - sy5y cells were plated and transfection of p2y12-cs2 + mt or cs2 + mt empty vector was performed with lipofectamine 2000 ( life technologies ) , according to manufacturer instructions .", "primary rat microglial and oligodendrocyte cells were lysed with trizol ( life technologies ) and total rna was extracted following the manufacturer 's instructions .", "after dnase treatment ( qiagen , hilden , germany ) , equal amount of total rna ( 1  g ) was subjected to retrotranscription by high capacity rna - to - cdna kit ( life technologies ) and 50  ng of each cdna were amplified with rat p2y12 specific primers ( f : 5-gattgataaccattgacc-3 ; r : 5-ggtgagaatcatgttagg-3 ) .", "amplification products ( 10  l of 20 ) were electrophoresed on 2% agarose gel containing ethidium bromide ( 1  g / ml , sigma aldrich ) , photographed under uv light using kodak image station 440cf , with molecular imaging software 4.0.1 . in order to isolate total protein extracts , microglia and oligodendrocytes were harvested with ice - cold ripa buffer ( pbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , and 0.1% sds ) , added with protease inhibitor cocktail ( sigma aldrich ) .", "lysates were kept for 30  min on ice and then centrifuged for 10  min at 14,000  rpm , at 4c .", "snap - frozen blocks from cases ms114 , ms125 , and ms163 supplied by uk multiple sclerosis tissue bank at imperial college in london were homogenized in ripa buffer , added with protease inhibitor cocktail , incubated on ice for 1  h , and centrifuged at 14,000  rpm for 10  min at 4c . rat brain and mouse brain and spinal cord were homogenized and sonicated in ripa buffer , added with protease inhibitor cocktail , kept for 1  h on ice , and centrifuged at 4c for 10  min at 13,000  rpm .", "supernatants were collected and analyzed for protein content by bradford colorimetric assay ( biorad , milan , italy ) .", "analysis of protein components was performed by polyacrylamide gel ( sds - page ) separation ( biorad ) and transfer onto nitrocellulose membranes ( amersham biosciences , cologno monzese , it ) .", "after saturation , blots were probed overnight at 4c , with the specified antibody ( table 1 ) , and finally incubated for 1  h with hrp - conjugated secondary antibodies and detected on x - ray film ( aurogene , rome , italy ) , using ecl advance western blotting detection kit ( amersham biosciences ) .", "p2y12 protein was detected with molecular mass comprised between 40  kda ( as predicted by amino acid sequence ) and 49 - 50  kda ( as predicted by the manufacturer data sheet ) .", "analysis was performed with the statistical software package medcalc ( medcalc software , mariakerke , belgium ) .", "in order to provide wide ranging comparative analysis of p2y12 expression particularly in microglia and oligodendrocytes under neuroinflammatory conditions , we performed immunofluorescence and confocal analysis of receptor expression in primary cortical and organotypic cerebellar cultures , in tissue slices from rat striatum and cerebellum , in spinal cord sections from symptomatic and end stage sod1-g93a als mouse model , finally in autoptic cortical tissue from progressive ms donors .", "p2y12 receptor is formed by two transcript variants that give rise to identical proteins with 342 amino acids , a secondary structure constituted by seven hydrophobic transmembrane domains connected by three extracellular and three intracellular loops , with four extracellular cysteine residues most likely contributing to the nucleotide binding site   ( figure 1(a ) ) .", "the commercially available p2y12 antibodies that we mostly used in our work are raised against the second intracellular loop , and precisely amino acids 125142 ( here named intra1 , intra2 , and intra fl ; see red circle in figure 1(a ) ) , and against the c - terminus , here named c - ter ( see green oval in figure 1(a ) ) ( see also table 1 ) . in order to validate the use of these different antibodies for p2y12 receptor , we compared them on recombinant p2y12 receptor protein obtained by cloning the complete cdna of the human receptor into the eukaryotic expression vector cs2 + mt , to provide the expression of a n - terminal c - myc tagged fusion protein .", "after transfection into sh - sy5y and hek293 cell lines , we analyzed total protein extracts by sds - page and western blotting using c - ter , intra1 , and intra2 antibodies .", "although with different intensities , all the antibodies recognize the myc - p2y12 protein band at the predicted molecular mass of 50  kda .", "these results confirm the specificity of the used antibodies toward denatured recombinant p2y12 receptor ( figure 1(b ) ) . in order to verify the expression of p2y12 mrna in rat primary oligodendrocytes ( ol ) and microglia ( rmg )", ", rt - pcr was performed using specific primers designed on receptor sequence . as shown in figure 1(d ) , rt - pcr on both rmg and ol reveals the presence of the predicted p2y12 mrna band .", "next , we validated the antibodies with protein extracts from dissociated primary cultures or tissues from different species .", "p2y12 protein is specifically recognized in extracts from human cerebral cortex snap frozen tissue , rat and mouse brain , mouse primary microglia ( mmg ) and ol ( figure 1(c ) ) .", "no signals are obtained when the immunoreactions are performed in the presence of p2y12 neutralizing peptides , when available from manufacturer ( data not shown ) . in order to establish cell specificity of p2y12 receptor expression", ", we performed comparative immunofluorescence and confocal analysis in primary dissociated cortical microglia and oligodendrocytes ( figures 2(a ) and 2(b ) ) , as well as organotypic cerebellar cultures ( figure 2(c ) ) .", "p2y12 is strongly recognized by both c - terminus- and second intracellular loop - recognizing antibodies ( red , c - ter , upper left inset ; intra fl , upper right inset ; intra1 , lower right inset and intra2 , lower left inset ) , specifically distinguishing the very heterogeneous morphological features of mouse primary microglia , as shown by double fluorescence and confocal analysis performed with phalloidin ( green ) , a marker for filamentous actin ( figure 2(a ) ) .", "fan - like cells ( insets ) , elongated rod - like cell bodies with short and tiny branches , asymmetrical hairy cells with miniature processes or lamellipodia are simultaneously observed ( c - ter phalloidin hoechst , merged ) .", "likewise , all the p2y12 antibodies ( intra2 , intra1 , c - ter , and intra fl ) recognize the multibranched morphology of rat mature oligodendrocytes ( figure 2(b ) , ol , insets , red ) in primary cultures , as confirmed by double immunofluorescence and confocal analysis with mbp antibody ( ol , intra1-mbp , yellow merged image ) .", "in addition , both intra2 and c - ter antibodies distinguish the ng2-positive rat oligodendrocyte precursor cells ( opc , merged insets ) .", "p2y12 immunoreactivity is confirmed in the ex - vivo system of organotypic rat cerebellar cultures ( figure 2(c ) ) .", "however , differently from mouse microglia and rat oligodendrocyte primary cultures , the second intracellular loop- ( red , intra1 ) and c - terminus - recognizing ( red , c - ter ) antibodies surprisingly immunoreact with different cell phenotypes when the integrity and cytoarchitecture of the tissue is preserved , as in organotypic cultures .", "while intra1 antibody ( red ) exclusively labels mbp - positive fibers ( green ) and highlights myelin structures , c - ter antibody does not immunoreact with myelinated fibers , being present on cells likely resembling microglia ( insets ) , as also confirmed by colocalization with microglial marker cd11b ( data not shown ) .", "results similar to those found with intra1 were also obtained with intra2 antibodies , lots an01/02/04 ( data not shown ) .", "we next verified if the cell type - specific presence of p2y12 receptor observed in organotypic culture , either in myelin structures or in microglia ,  respectively ,   by the use of the second intracellular loop-(intra1 ) or the c - terminus - recognizing ( c - ter ) antibody , is also confirmed in rat cerebellar and striatal slice tissues ( figure 3 ) .", "all the antibodies raised against the second intracellular loop ( red , intra1 , intra2 , intra fl ) identify the abundant presence of p2y12 receptor only on myelinated fibers from both cerebellum ( figures 3(a ) , 3(b ) , and 3(c ) ) and striatum ( figures 3(d ) , 3(e ) , and 3(f ) ) , as confirmed by colocalization of signals obtained with intra fl and mbp antibodies in cerebellum ( + mbp , inset c2 , merged , yellow ) and striatum ( data not shown ) ; colocalization of signals obtained with intra1 and intra2 with mbp antibodies in cerebellum and striatum ( data not shown ) ; immunoreactivity of intra1 , intra2 , and intra fl antibodies for structures identical to those observed in cerebellum ( data not shown ) and striatum with mbp antibody ( see for instance mbp , inset e1 , green ) ; identification by second intracellular loop p2y12 antibodies of structures totally different from both bergmann glia and astrocytes recognized by specific gfap antibody in cerebellum ( inset b1 , green ) , and from microglia identified by specific cd11b antibody in cerebellum ( + cd11b , inset c1 , merged , green ) and striatum ( + cd11b , inset f1 , merged , green ) . of notice ,", "all the 2nd intracellular loop antibodies are able to describe the specific cytoarchitecture of both cerebellum , where radiant and sparse fibers clearly characterize the lobuli , and of striatum , where white matter is instead organized in distinct bundles . as in organotypic cultures ,", "the c - terminus antibody ( red , c - ter ) instead recognizes only microglia in rat cerebellum ( figures 3(g ) , 3(h ) , and 3(i ) , and insets g1 , h1 , i1 ) , striatum ( figures 3(j ) , 3(k ) , and 3(l ) ) and cerebral cortex slices ( data not shown ) , with a signal more uniformly distributed throughout the whole tissue . by comparing the cd11b and c - ter immunolabelling in the striatum", ", we furthermore observe that the mutual intensity of signals is cell - selective within the microglia population , with some cells exclusively positive for c - ter ( arrow ) , and others instead showing different grades of cd11b - c - ter colocalization ( see for instance orange , yellow and greenish cells in the merged field ) .", "regrettably , double immunofluorescence with c - ter and iba1 microglia marker   is not practicable , since both antibodies are raised in the same species .", "however , no colocalization of signals is ever shown between mbp ( green ) and c - ter antibodies ( + mbp , inset j1 , merged ) . in order to verify", "if p2y12 recognized by c - ter antibody exclusively in microglia from rat brain slices could be used as specific microglia marker during neuroinflammation , we validated its use in a typical neuroinflammatory disease such as als and for the first time characterized the presence of p2y12 receptor in sod1-g93a mouse model ( figure 4 ) . by immunofluorescence and confocal analysis on lumbar spinal cord sections ( l3l5 ) of wild - type ( wt ) mice , we first compared the immunoreactive signals obtained with c - ter and specific microglia markers cd11b ( red , recognizing ramified microglia ) and cd68 ( red , recognizing roundish activated microglia ) . in wt mice", ", c - ter ( green ) is abundantly and strongly immunoreacting with the microglia population and colocalizing with the majority of cd11b - positive cells , in both dorsal ( dh ) and ventral ( vh ) horns of spinal cord ( figure 4(a ) , left panel , merged yellow signal ) .", "all cd11b - positive cells share immunoreactivity for c - ter , as proved by the absence of red cd11b signal .", "conversely , not all c - ter - positive cells immunoreact also with cd11b antibody , as proved by the presence of some green c - ter signals .", "in addition , we never observe colocalization with the rare activated cd68-positive ( red ) microglia cells present in wt healthy tissue ( figure 4(a ) , right panel , merged and inset ) . to test if c - ter antibody can further recognize microglia activation during the progression of als , we next examined sod1-g93a spinal cord sections at two different stages of the disease , that is , 20 weeks , a phase when the disease accelerates , and end stage , that is , 23 weeks , when the animals reach the score of 1 accordingly to a behavioral score system . at both stages ,", "sod1-g93a mice show a significant increase in cd68 immunostaining not only when compared to wt mice ( figure 4(a ) ) , but also in vh with respect to dh , and this is even more evident at end stage with respect to 20 weeks ( red , figure 4(b ) ) .", "remarkably , the immunoreactive signal of c - ter ( green , figure 4(b ) ) decreases during disease progression and the effect is pronounced especially in vh , where motor neuron loss , tissue damage , and microglia activation are known to be increased . particularly at 20 weeks", ", c - ter - expressing microglia are still ramified in dh , where few cd68-positive cells are present , but start to disappear in vh , where instead cd68-positive cells are increased . at end stage", ", c - ter staining decreases in dh with respect to 20 weeks and disappears almost completely in vh , concomitantly with a robust increase in cd68 staining . a parallel decrease in c - ter and increase of cd68 immunoreactivities", "is also confirmed by western blot analysis performed on sod1-g93a lumbar spinal cord homogenates at end stage ( figure 4(c ) ) . as in rat organotypic cerebellar cultures ( figure 2(b ) ) and rat cerebellar , striatal , cortical , or mouse spinal cord tissue slices", "( figures 3 and 4 ) , a similar pattern of p2y12 receptor expression is shown in human frontal cortex autoptic tissue by using c - ter antibody that highlights only microglia uniformly distributed throughout the entire healthy tissue ( red ) , but not mbp - positive myelinated structures ( green ) , as detected by the absence of overlapping immunofluorescent signals ( figures 5(a ) , 5(b ) , and 5(c ) , merged and insets a1 , c1 ) . on the contrary , intra1 ( red , figures 5(d ) , 5(e ) , and 5(f ) ) , intra2 , and intra fl ( data not shown ) antibodies recognize exclusively myelinated fibers in tissue from healthy ( insets d1 , e1 , and f1 ) and progressive ms donors ( figures 5(d ) , 5(e ) , and 5(f ) ) , as it is found with rat tissue ( figures 2 and 3 ) .", "moreover , we confirm a decrease of p2y12 receptor signal in proximity to the demyelinating lesion , as detected by loss of mbp - positive fibers ( see asterisks ) .", "no colocalization of signals is shown with mhc ii - positive microglia ( + mhc ii , inset d3 , merged ) .", "importantly , c - ter , intra1 ( red , insets a - f2 ) and intra2 , and intra fl ( data not shown ) antibodies all recognize the presence of p2y12 receptor in integrin ii/3-positive platelets ( green ) contained in the blood vessels of the analyzed tissues . as observed in als mouse spinal cord where p2y12 receptor detected by c - ter antibody is shown to temporally and regionally decrease in microglia as a function of increasing inflammatory damage ( figure 4 ) , we notice that microglia gradually lose immunoreactivity for c - ter antibody ( figure 6 ) in proximity to the demyelinating active cortical lesions of ms expressing augmented positivity for mhc ii [ 3335 ] .", "moreover , by comparing mhc ii and c - ter signals , we recognize four areas (  a  d  ) inside and around the lesion , where microglia express different amount of these proteins . in zone", " a ,  at the edge of the lesion , we observe a predominance of c - ter immunoreactivity ( red ) compared to that of mhc ii ( green ) , as depicted in the merged field by a major occurrence of red signal . in zone  b ,", " closer to the lesion , we notice a prevalence of active mhc ii - positive green cells , but still the presence of few red and yellow signals .", "c  and  d ,  apparently in the core or outside the lesion , respectively , where microglia express either almost exclusively mhc ii protein (  c  ) or p2y12 receptor on ramified microglia (  d  ) .", "the interchange among different cell types of molecular cues that condition the cell specificity and the protein profile of each cell characterizes the morphological and functional heterogeneity in particular of microglia within various cns regions , developmental stages [ 37 , 38 ] and , even more , states of activation during pathological conditions . in the case , for instance , of als , the release of signals from motor neurons apparently denotes one of the earliest phase of the disease , with microglia behaving as an m2 phenotype producing neuroprotective factors to repair motor neurons and preventing them against further injury . as disease rises , motor neurons start releasing  alarm signals ", "that in turn convert microglia from beneficial m2 to cytotoxic m1 phenotype , with consequent release of proinflammatory cytokines .", "for instance , m1 markers are abundantly expressed in normal appearing white matter and throughout active demyelinating ms lesions by activated microglia and macrophages , although in human active ms lesions microglia show an intermediate activation status .", "in addition , m2 microglia appear fundamental to guide oligodendrocyte remyelination in mice , and a switch from m1- to m2-dominant response occurs in microglia and peripherally derived macrophages when remyelination starts . only therapeutic procedures", "that both down - regulate the harmful responses and up - regulate the beneficial responses may hopefully slow pathological progression and provide meaningful hope for treatment . at the same time , the identification of clear markers involved in the m2/m1 microglia transition becomes mandatory for presymptomatic diagnosis , monitoring of disease progression , and efficacy of therapies . under this perspective , and consistently with previous findings establishing the role of purinergic receptors in the pathogenesis of both als and ms [ 14 , 43 ] , our present work serves this aim , by highlighting the gradual loss of p2y12 immunoreactivity as an early marker of neuroinflammation and microglia metamorphosis .", "we have indeed demonstrated here that p2y12 receptor protein identified in primary cultures of both microglia ( figure 2(a ) ) [ 18 , 44 ] and oligodendrocytes ( figure 2(b ) )   by different but p2y12-selective antibodies can be instead recognized in branched microglia exclusively by the use of c - ter antibody ( figures 36 ) .", "this occurs only when the integrity and cytoarchitecture of the tissue is typically preserved in the presence of the least experimental manipulations , that is , in organotypic cultures ( figure 2(c ) ) and tissues slices for instance from rat striatum and cerebellum ( figure 3 ) , mouse spinal cord ( figure 4 ) , and human cerebral cortex ( figures 5 and 6 ) .", "a similar difference in primary cultured cell versus tissue distribution of a protein was previously demonstrated with large - conductance calcium - activated potassium channel expression , in vascular endothelium .", "a first implication emerging from these results is that a reliable evidence about selective p2y12 expression in cells of healthy or neuroinflammatory states is genuine only when cell connectivity and tissue architecture are fully preserved .", "we have indeed shown this , by proving that the antibodies used for p2y12 , recognizing either the c - terminus or the second intracellular loop of the receptor ( figure 1 and table 1 ) and immunolabelling , respectively , microglia or myelinated fibers in the cns , are all still able to immunoreact for instance with platelets ( figure 5 ) , where the receptor was originally described to be present and to have a role in the processes of activation , aggregation [ 4649 ] , primary hemostasis , and arterial thrombosis [ 5055 ] . a possible explanation for the microglia versus oligodendrocyte selectivity of the p2y12 antibodies might be that gi - coupling , and/or quaternary structure , post - transcriptional modifications , and subcellular localization of p2y12 that remain strictly preserved in platelets , are instead divergent in microglia with respect to oligodendrocytes / myelinated fibers . in this case , a cell - specific network of p2y12 oligomeric interactions and/or a distinct subcellular partitioning might simply mask the recognition sites of the different antibodies on p2y12 protein in different cell types . to support this hypothesis", ", we know that in platelets p2y12 indeed resides in subcellular lipid raft structures and its partitioning out from rafts causes for instance inactivation   and that also the presence of another purinergic receptor , the ionotropic p2x3 , in lipid rafts has cell - specific properties shared in cerebellar granule neurons and total brain tissue but not in neuroblastoma cells and dorsal root ganglia   and that the specific antagonist clopidogrel inhibits p2y12 by breaking down the homoligomeric complex to single monomers   and finally , that hetero - oligomerization of p2y12 is demonstrated with p2y1 , p2y2 , p2y13 , and with adenosine a1 , a2a receptors in different cellular backgrounds .", "all these features might very well explain also why it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna   and , moreover , why p2y12 is specifically observed in brain and spinal cord resident microglia but is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] .", "a second implication that emerges from our results is that the morphological metamorphosis that microglia undergo under neuroinflammatory conditions as those triggered during als and ms , can be remarkably highlighted by the progressive reduction of p2y12 immunostaining obtained with c - ter antibody that reacts , also in this case , exclusively with multibranched microglia still present in the tissue ( figures 4 and 6 ) .", "this closely reflects the expression of p2y12 that is robust in the resting / surveillant branched state but dramatically decreased after morphological transition and activation of microglia .", "our observations are also in line with the central role played by p2y12 in branched microglia membrane ruffling and inspection of the environment . on the other hand , they depict a morphological / functional state of microglia that only partially overlaps with cd11b and mhc ii immunoreactivities , which are furthermore known to increase during activation but instead highly contrasts with cd68 immunostaining that is totally absent in ramified microglia . in parallel to our results , these last antibodies actually accentuate the progressive transition of microglia from a lesser ramified shape to a significantly more activated amoeboid phenotype ( figures 4 , 6 , and 7 ) , thus suggesting the dual use of c - ter and cd68 antibodies as markers , respectively , for branched resting / surveillant versus roundish / activated microglia . a reduction of c - ter - p2y12 immunostaining that is concomitant to an increase , for instance , of mhc ii / cd11b / cd68 immunoreactivity , could thus become a feasible approach to detect an increasing neuroinflammatory condition .", "indeed , p2y12 is considered an essential component and primary site at which nucleotides such as adp act to promote directional microglia movement or chemotaxis at early stages of cns injury . in particular ,", "microglia from mice lacking p2y12 exhibit normal baseline motility but are unable to polarize and to elicit directional branch extension and migration toward nucleotides in vitro , or sites of cortical damage in vivo .", "these notions are consistent with our results that fail to describe c - ter - p2y12 immunoreactivity on roundish phagocytizing , or polarized migratory microglia .", "however , we still do not know if reduction / absence of p2y12 c - ter - immunolabelling on activated microglia might be a cause or a consequence of morphological / functional transition or might simply reflect a cell - selective hindrance and lack of access to the immunogenic sites by the antibody .", "anyhow , we can assert that the distinctive recognition of multibranched microglia renders the c - ter antibody a novel and useful tool to discriminate among microglia morphological states , thus making p2y12 receptor a selective and early marker for the ramified phase .", "in parallel to this , we have also proven that all the several antibodies raised against the second intracellular loop of p2y12 ( intra1 , intra2 , and intra fl ) can likely be employed as markers for the presence of ms lesions .", "although with different intensities , they not only recognize the receptor specifically on myelinated fibers of organotypic cultures ( figure 2(c ) ) , tissues slices from rat striatum or cerebellum ( figure 3 ) and human cerebral cortex , but also furthermore highlight the reduction of p2y12 signal that occurs for instance in ms tissue ( figure 5 ) in correlation to the extent of demyelination found in all types of grey matter cortical plaques ( i  iii ) and subcortical white matter . in brief , we have shown here that the presence of p2y12 receptor can be simultaneously identified by the c - terminus and the second intracellular loop antibodies .", "when this occurs , a condition of intact myelinated fibers and branched / surveillant microglia is represented at once that perhaps signifies a ", "for instance in ms , a decrease in the second intracellular loop immunoreactions accompanied by an increase of c - terminus immunoreactivity will possibly depict the loss of myelin and replacement by ramified microglia that often occur in an inactive plaque . on the contrary , a decrease in c - terminus immunolabelling in the abundant presence of second intracellular loop - positive myelinated fibers would indicate an early active plaque where m1/m2 microglia reactivity starts to take place .", "by comparative analysis of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , we have established here that , under experimental conditions of well - preserved cytoarchitecture and tissue integrity , p2y12 receptor expressed by both ramified microglia or oligodendrocytes / myelinated fibers might serve a dual function as specific marker , respectively , of branched / surveillant microglia as well as demyelinating lesions .", "we believe that p2y12 identification and modulation might potentially acquire an important predictive value under neuroinflammatory conditions , as those found in als and ms .", "p2y12 is likely to deserve a key role in the verge of a neuroinflammatory breakdown ."], "abstract_text": ["<S> in the cns , neuroinflammation occurring during pathologies as amyotrophic lateral sclerosis ( als ) and multiple sclerosis ( ms ) is the consequence of an intricate interplay orchestrated by various cell phenotypes . among the molecular cues having a role in this process , extracellular nucleotides are responsible for intercellular communication and propagation of inflammatory stimuli . </S>", "<S> this occurs by binding to several receptor subtypes , defined p2x / p2y , which are widespread in different tissues and simultaneously localized on multiple cells . </S>", "<S> for instance , the metabotropic p2y12 subtype is found in the cns on microglia , affecting activation and chemotaxis , on oligodendrocytes , possessing a hypothesized role in myelination , and on astrocytes . by comparative analysis , </S>", "<S> we have established here that p2y12 receptor immunolabelled by antibodies against c - terminus or second intracellular loop , is , respectively , distributed and modulated under neuroinflammatory conditions on ramified microglia or myelinated fibers , in primary organotypic cerebellar cultures , tissue slices from rat striatum and cerebellum , spinal cord sections from symptomatic / end stage sod1-g93a als mice , and finally autoptic cortical tissue from progressive ms donors . </S>", "<S> we suggest that modulation of p2y12 expression might play a dual role as analytic marker of branched / surveillant microglia and demyelinating lesions , thus potentially acquiring a predictive value under neuroinflammatory conditions as those found in als and ms . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["a basic set of proteins and mrnas are differentially expressed among cell types , temporally and spatially , generating a vast assortment of cell phenotypes and/or activation states within a single tissue .", "outlining this protein / mrna portrait is thus crucial for understanding not only the uniqueness characterizing cells , but especially their distinguished functions .", "this becomes of major relevance when the balance between cell - intrinsic properties and identity cues received and provided by each cell to its neighboring cells then shapes the cell - to - cell cross talk during physiopathological conditions . in the cns , neuroinflammation is the typical consequence of the interchange among different cell types , particularly neurons , astrocytes , oligodendrocytes and microglia , of a variety of cues as neurotransmitters , cytokines , chemokines , toxic metabolites that condition the final protein / mrna profiles of cells , their activation states and functional outcomes [ 2 , 3 ] .", "since neuroinflammation accompanies a large variety of neurodegenerative diseases , there is increasing interest in determining how the different cell phenotypes and cellular interconnectivity might contribute to reduce inflammation and reverse neurodegeneration .", "microglia actively participate to the context - dependent , neuroprotective / neurotoxic molecular network that is triggered during neuroinflammation . among the molecular cues having a key role in this process ,", "extracellular nucleotides are major responsible for intercellular communication and propagation of inflammatory stimuli [ 57 ] .", "this occurs by specific binding to various receptor subtypes , termed ionotropic p2x and metabotropic p2y , which are simultaneously localized on several different cell phenotypes . among these", ", the p2y12 receptor subtype   belonging to the gi class of g protein - coupled receptors is activated by adp .", "two transcript variants apparently encoding the same protein isoform have been identified so far for p2ry12 gene , but the determinants for cell specificity of p2y12 protein expression are still unknown .", "p2y12 is found on the surface mainly , but not exclusively , of blood platelets , where it acts as blood clotting regulator and target for the treatment of thromboembolisms [ 9 , 10 ] . in the nervous system ,", "the tissue- and cellular - selective expression of p2y12 exhibits a pattern throughout white and gray matter consistent with astrocyte expression , although it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna .", "moreover , we previously established in vivo the expression of p2y12 in oligodendrocytes and myelin sheaths of rat cerebral cortex , subcortical areas , and periventricular white matter . this localization is confirmed throughout the corticospinal tract , therefore suggesting high conserved tissue - homogeneity and phenotype - specificity , and a hypothesized role in myelination [ 1215 ] .", "p2y12 is finally observed in brain and spinal cord resident microglia , where it affects activation , chemotaxis [ 1619 ] and neuropathic pain , but it is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] .", "p2y12 expression in primary microglia is variable with postnatal development and shows sexually dimorphic behavior . through the use of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , the aim of the present work is to provide comparative evidence about p2y12 cell specificity in microglia versus oligodendrocytes particularly from the healthy and diseased cns under neuroinflammatory conditions as those sustained during amyotrophic lateral sclerosis ( als ) and multiple sclerosis ( ms ) ."], ["adult b6.cg-tg ( sod1-g93a)1gur / j mice expressing high copy number of mutant human sod1 with a gly93ala substitution ( sod1-g93a ) were originally obtained from jackson laboratories ( bar harbor , me , usa ) and housed in our indoor animal facility as described in apolloni and collaborators , .", "the animals were euthanized , according to the guidelines for preclinical testing and colony management .", "also neonatal wistar and adult lewis rats ( from charles river laboratories , lc , italy ) were housed in our indoor animal facility .", "animal procedures were performed according to european guidelines for the use of animals in research ( 86/609/cee ) and the requirements of italian laws ( d.l .", "ethical procedures were approved by animal welfare office , department of public health and veterinary , nutrition and food safety , general management of animal care and veterinary drugs of the italian ministry of health .", "all efforts were made to minimize animal suffering and number of animals necessary to produce reliable results .", "microglia primary cultures prepared from mouse cortex as previously described   were about 98% pure , as verified by immunofluorescence with cd11b ( for microglia ) , glial fibrillary acidic protein ( gfap , for astrocytes ) , neuronal nuclei ( neun , for neurons ) , and smi94 ( for oligodendrocytes ) .", "purified cultures of oligodendrocytes were prepared from forebrain of postnatal day 1 - 2 wistar rats , according to minor modifications from a previously described method . after removing the meninges ,", "cortices were minced , digested , and dissociated by passage through 70  m nylon cell strainer ( bd biosciences , europe ) .", "cells were plated in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 20% heat - inactivated fetal bovine serum ( fbs ) , 4  mm glutamine , 1  mm sodium pyruvate , 50  u / ml penicillin , 50  g / ml streptomycin , and 100  g / ml gentamicin in t75 poly - d - lysine - coated flasks , at about 10 million cells / flask .", "the cultures were maintained at 37c in a 5% co2 and 95% air atmosphere for 14 days .", "mixed glial cultures were then shaken at 200  rpm at 37c for 1  h and 98% pure microglia collected from the supernatant of each flask , as verified by immunofluorescence with gfap , neun , neural / glial antigen 2 ( ng2 , for oligodendroglial precursor cells ) , myelin basic protein ( mbp , for mature oligodendrocytes ) , and cd11b .", "after further shaking at 200  rpm and 37c for 1518  h , the detached cell suspension was finally incubated for 1  h at 37c for differential adhesion of contaminating cells .", "the non - adherent cells were filtered through 40  m nylon cell strainer ( bd biosciences ) , spun for 10  min at 100  g , resuspended in oligodendroglial precursor cells medium ( basal chemically defined medium : dmem , 4  mm l - glutamine , 1  mm sodium pyruvate , 0.1% bovine serum albumin ( bsa ) , 50  g  ml apo - transferrin , 5  g ml insulin , 30  nm sodium selenite , 10  nm d - biotin , and 10  nm hydrocortisone ) containing 10  ng  ml platelet derived growth factor - aa ( pdgf - aa ) and 10  ng  ml basic fibroblast growth factor ( bfgf ) , and seeded at the density of 1  10  cells / cm into poly d , l - ornithine - coated plates .", "the cells were then induced to differentiate into mature oligodendrocytes when the basal chemically defined medium was added with 15  nm triiodothyronine , 10  ng  ml ciliary neurotrophic factor ( cntf ) and 0,05  mg 10  ml n - acetyl - l - cysteine ( nac ) , for 47 days .", "a 98% pure population of oligodendrocytes was thus obtained , as verified by immunofluorescence with ng2 , mbp , gfap , neun , and cd11b antibodies .", "organotypic cerebellar slice cultures were prepared with modifications from previously published protocols [ 26 , 27 ] .", "briefly , cerebella were excised from neonatal wistar rat ( 810 days old ) , cut on a mc ilwain tissue chopper ( 400  m ) and parasagittal slices separated into cold hank 's balanced salt solution ( hbss ) .", "two to three slices were plated on each millicell cm culture inserts ( millipore , italy ) and kept in organotypic maintenance medium ( 50% basal medium eagle - bme , 25% hbss , 25% heat - inactivated horse serum , supplemented with 5  mg / ml glucose , 1  mm glutamine , 50  u / ml penicillin , and 50  g / ml streptomycin ) at 37c in a 5% co2 and 95% air atmosphere .", "the medium was changed every 2 days and double immunofluorescence was performed in free floating after 710 days in vitro .", "organotypic cultures were washed three times with pbs , fixed with 4% paraformaldehyde for 1  h , rinsed , and blocked for 1  h in phosphate buffered saline ( pbs)/10% normal donkey serum ( nds)/0.4% triton x-100 .", "primary antibodies were incubated for 24  h in pbs/2% nds/0.4% triton x-100 ( table 1 ) .", "the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1  :  100 , jackson immunoresearch , europe ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1  :  200 , jackson immunoresearch ) . after rinsing ,", "sections were mounted , covered with fluoromount medium ( sigma - aldrich , milan , italy ) and a coverslip , and analyzed by confocal microscopy .", "microglia and oligodendrocytes were washed three times with pbs , fixed with 4% paraformaldehyde for 10  min ( oligodendrocytes ) or 20  min ( microglia ) , washed , permeabilized with 0.050.1% triton x-100 for 10  min , rinsed , and blocked for 30  min in pbs/1% bsa .", "microglia were stained for about 3  h at 37c in 1% pbs / bsa with 5  g / ml cy2-phalloidin ( sigma - aldrich ) , in combination with primary antibodies against p2y12 receptor , as reported in table 1 .", "the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1  :  100 , jackson immunoresearch ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1  :  200 , jackson immunoresearch ) . cells were extensively washed and stained with the nucleic acid blue dye , hoechst 33342 ( 1  :  1000 ) .", "after rinsing , cells were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy .", "animals were anaesthetized by intraperitoneal injection of chloral hydrate ( 500  mg / kg ) and transcardially perfused with pbs followed by 4% paraformaldehyde at ph 7.4 .", "tissue samples ( mice spinal cord and rat brain ) were then postfixed for 1 - 2 days , and cryoprotected in 30% sucrose in pbs at 4c .", "mice spinal cords ( l3l5 ) were cut at 30  m thickness with a frozen microtome .", "sections were mounted on slide glasses and allowed to air - dry ( 1 - 2  h ) .", "a rectangle was then drawn around the sections with a pap pen ( sigma - aldrich ) .", "rat brains were cut at 40  m thickness using a cryostat microtome and sections were processed in free - floating .", "double immunofluorescence analysis was performed after blocking in pbs containing 10% nds and 0.3% triton x-100 for 1  h at room temperature .", "sections were incubated with different combinations of primary antibodies ( table 1 ) , in pbs , 0.3% triton x-100 and 2% nds , for 2448  h at 4c .", "finally , sections were washed with pbs and incubated with appropriate fluorescent - conjugated secondary antibodies for 3  h at room temperature . after rinsing", ", sections were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy .", "tissues supplied by uk multiple sclerosis tissue bank at imperial college , london , were collected postmortem with fully informed consent from both donors and close relatives .", "procedures for retrieval , processing , and storage have gained ethical approval from all appropriate committees .", "the brain tissues analyzed were from 7 neuropathologically confirmed cases of secondary progressive ms ( spms ) .", "analysis was performed also on samples from patients who died due to nonneurological diseases ( healthy ) .", "cerebral hemispheres were fixed with 4% paraformaldehyde for about two weeks , coronally sliced , and blocked .", "individual blocks were cryoprotected in 30% sucrose for one week and frozen by immersion in isopentane precooled on a bed of dry ice .", "cryostat sections ( 3040  m thick ) were either stained with luxol fast blue and cresyl fast violet ( kluver - barrera staining ) , in order to detect white matter lesions and their cellularity , or subjected to immunohistochemistry for mbp , in order to distinguish grey matter lesions .", "sections were processed in free - floating for double immunofluorescence studies , as described in amadio and collaborators .", "immunofluorescence was analyzed by means of a confocal laser scanning microscope ( zeiss , lsm700 , germany ) equipped with four laser lines : 405 , 488 , 561 , and 639  nm .", "the brightness and contrast of the digital images were adjusted using the zen software ( zeiss ) .", "human p2y12 complete cdna was obtained by reverse transcription with enhanced avian rt - pcr kit ( sigma - aldrich ) from total human brain rna ( life technologies , paisley , uk ) .", "the obtained cdna was then cloned into the xhoi and xbai sites of the eukaryotic expression vector cs2 + mt to provide n - terminal 6x c - myc epitope - tagged mammalian expression plasmids , which has been validated by dna sequencing .", "oligos used for amplification were as follows : forward 5gcctcgagatgcaagccgtcgacaacctc3 and reverse 5gctctagagtttacattggagtctcttc designed on human p2y12 mrna ( nm_022788.3 ) .", "human embryonic kidney 293 cells ( hek 293 ) and sloan - kettering neuroblastoma sh - sy5y clone ( sh - sy5y ) cells were grown in dmem supplemented with 10% fbs , 100  unit / ml penicillin , and 100  g / ml streptomycin at 37c in atmosphere containing 5% co2 .", "one day before transfection , hek293 or sh - sy5y cells were plated and transfection of p2y12-cs2 + mt or cs2 + mt empty vector was performed with lipofectamine 2000 ( life technologies ) , according to manufacturer instructions .", "primary rat microglial and oligodendrocyte cells were lysed with trizol ( life technologies ) and total rna was extracted following the manufacturer 's instructions .", "after dnase treatment ( qiagen , hilden , germany ) , equal amount of total rna ( 1  g ) was subjected to retrotranscription by high capacity rna - to - cdna kit ( life technologies ) and 50  ng of each cdna were amplified with rat p2y12 specific primers ( f : 5-gattgataaccattgacc-3 ; r : 5-ggtgagaatcatgttagg-3 ) .", "amplification products ( 10  l of 20 ) were electrophoresed on 2% agarose gel containing ethidium bromide ( 1  g / ml , sigma aldrich ) , photographed under uv light using kodak image station 440cf , with molecular imaging software 4.0.1 . in order to isolate total protein extracts , microglia and oligodendrocytes were harvested with ice - cold ripa buffer ( pbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , and 0.1% sds ) , added with protease inhibitor cocktail ( sigma aldrich ) .", "lysates were kept for 30  min on ice and then centrifuged for 10  min at 14,000  rpm , at 4c .", "snap - frozen blocks from cases ms114 , ms125 , and ms163 supplied by uk multiple sclerosis tissue bank at imperial college in london were homogenized in ripa buffer , added with protease inhibitor cocktail , incubated on ice for 1  h , and centrifuged at 14,000  rpm for 10  min at 4c . rat brain and mouse brain and spinal cord were homogenized and sonicated in ripa buffer , added with protease inhibitor cocktail , kept for 1  h on ice , and centrifuged at 4c for 10  min at 13,000  rpm .", "supernatants were collected and analyzed for protein content by bradford colorimetric assay ( biorad , milan , italy ) .", "analysis of protein components was performed by polyacrylamide gel ( sds - page ) separation ( biorad ) and transfer onto nitrocellulose membranes ( amersham biosciences , cologno monzese , it ) .", "after saturation , blots were probed overnight at 4c , with the specified antibody ( table 1 ) , and finally incubated for 1  h with hrp - conjugated secondary antibodies and detected on x - ray film ( aurogene , rome , italy ) , using ecl advance western blotting detection kit ( amersham biosciences ) .", "p2y12 protein was detected with molecular mass comprised between 40  kda ( as predicted by amino acid sequence ) and 49 - 50  kda ( as predicted by the manufacturer data sheet ) .", "analysis was performed with the statistical software package medcalc ( medcalc software , mariakerke , belgium ) ."], ["in order to provide wide ranging comparative analysis of p2y12 expression particularly in microglia and oligodendrocytes under neuroinflammatory conditions , we performed immunofluorescence and confocal analysis of receptor expression in primary cortical and organotypic cerebellar cultures , in tissue slices from rat striatum and cerebellum , in spinal cord sections from symptomatic and end stage sod1-g93a als mouse model , finally in autoptic cortical tissue from progressive ms donors .", "p2y12 receptor is formed by two transcript variants that give rise to identical proteins with 342 amino acids , a secondary structure constituted by seven hydrophobic transmembrane domains connected by three extracellular and three intracellular loops , with four extracellular cysteine residues most likely contributing to the nucleotide binding site   ( figure 1(a ) ) .", "the commercially available p2y12 antibodies that we mostly used in our work are raised against the second intracellular loop , and precisely amino acids 125142 ( here named intra1 , intra2 , and intra fl ; see red circle in figure 1(a ) ) , and against the c - terminus , here named c - ter ( see green oval in figure 1(a ) ) ( see also table 1 ) . in order to validate the use of these different antibodies for p2y12 receptor , we compared them on recombinant p2y12 receptor protein obtained by cloning the complete cdna of the human receptor into the eukaryotic expression vector cs2 + mt , to provide the expression of a n - terminal c - myc tagged fusion protein .", "after transfection into sh - sy5y and hek293 cell lines , we analyzed total protein extracts by sds - page and western blotting using c - ter , intra1 , and intra2 antibodies .", "although with different intensities , all the antibodies recognize the myc - p2y12 protein band at the predicted molecular mass of 50  kda .", "these results confirm the specificity of the used antibodies toward denatured recombinant p2y12 receptor ( figure 1(b ) ) . in order to verify the expression of p2y12 mrna in rat primary oligodendrocytes ( ol ) and microglia ( rmg )", ", rt - pcr was performed using specific primers designed on receptor sequence . as shown in figure 1(d ) , rt - pcr on both rmg and ol reveals the presence of the predicted p2y12 mrna band .", "next , we validated the antibodies with protein extracts from dissociated primary cultures or tissues from different species .", "p2y12 protein is specifically recognized in extracts from human cerebral cortex snap frozen tissue , rat and mouse brain , mouse primary microglia ( mmg ) and ol ( figure 1(c ) ) .", "no signals are obtained when the immunoreactions are performed in the presence of p2y12 neutralizing peptides , when available from manufacturer ( data not shown ) . in order to establish cell specificity of p2y12 receptor expression", ", we performed comparative immunofluorescence and confocal analysis in primary dissociated cortical microglia and oligodendrocytes ( figures 2(a ) and 2(b ) ) , as well as organotypic cerebellar cultures ( figure 2(c ) ) .", "p2y12 is strongly recognized by both c - terminus- and second intracellular loop - recognizing antibodies ( red , c - ter , upper left inset ; intra fl , upper right inset ; intra1 , lower right inset and intra2 , lower left inset ) , specifically distinguishing the very heterogeneous morphological features of mouse primary microglia , as shown by double fluorescence and confocal analysis performed with phalloidin ( green ) , a marker for filamentous actin ( figure 2(a ) ) .", "fan - like cells ( insets ) , elongated rod - like cell bodies with short and tiny branches , asymmetrical hairy cells with miniature processes or lamellipodia are simultaneously observed ( c - ter phalloidin hoechst , merged ) .", "likewise , all the p2y12 antibodies ( intra2 , intra1 , c - ter , and intra fl ) recognize the multibranched morphology of rat mature oligodendrocytes ( figure 2(b ) , ol , insets , red ) in primary cultures , as confirmed by double immunofluorescence and confocal analysis with mbp antibody ( ol , intra1-mbp , yellow merged image ) .", "in addition , both intra2 and c - ter antibodies distinguish the ng2-positive rat oligodendrocyte precursor cells ( opc , merged insets ) .", "p2y12 immunoreactivity is confirmed in the ex - vivo system of organotypic rat cerebellar cultures ( figure 2(c ) ) .", "however , differently from mouse microglia and rat oligodendrocyte primary cultures , the second intracellular loop- ( red , intra1 ) and c - terminus - recognizing ( red , c - ter ) antibodies surprisingly immunoreact with different cell phenotypes when the integrity and cytoarchitecture of the tissue is preserved , as in organotypic cultures .", "while intra1 antibody ( red ) exclusively labels mbp - positive fibers ( green ) and highlights myelin structures , c - ter antibody does not immunoreact with myelinated fibers , being present on cells likely resembling microglia ( insets ) , as also confirmed by colocalization with microglial marker cd11b ( data not shown ) .", "results similar to those found with intra1 were also obtained with intra2 antibodies , lots an01/02/04 ( data not shown ) .", "we next verified if the cell type - specific presence of p2y12 receptor observed in organotypic culture , either in myelin structures or in microglia ,  respectively ,   by the use of the second intracellular loop-(intra1 ) or the c - terminus - recognizing ( c - ter ) antibody , is also confirmed in rat cerebellar and striatal slice tissues ( figure 3 ) .", "all the antibodies raised against the second intracellular loop ( red , intra1 , intra2 , intra fl ) identify the abundant presence of p2y12 receptor only on myelinated fibers from both cerebellum ( figures 3(a ) , 3(b ) , and 3(c ) ) and striatum ( figures 3(d ) , 3(e ) , and 3(f ) ) , as confirmed by colocalization of signals obtained with intra fl and mbp antibodies in cerebellum ( + mbp , inset c2 , merged , yellow ) and striatum ( data not shown ) ; colocalization of signals obtained with intra1 and intra2 with mbp antibodies in cerebellum and striatum ( data not shown ) ; immunoreactivity of intra1 , intra2 , and intra fl antibodies for structures identical to those observed in cerebellum ( data not shown ) and striatum with mbp antibody ( see for instance mbp , inset e1 , green ) ; identification by second intracellular loop p2y12 antibodies of structures totally different from both bergmann glia and astrocytes recognized by specific gfap antibody in cerebellum ( inset b1 , green ) , and from microglia identified by specific cd11b antibody in cerebellum ( + cd11b , inset c1 , merged , green ) and striatum ( + cd11b , inset f1 , merged , green ) . of notice ,", "all the 2nd intracellular loop antibodies are able to describe the specific cytoarchitecture of both cerebellum , where radiant and sparse fibers clearly characterize the lobuli , and of striatum , where white matter is instead organized in distinct bundles . as in organotypic cultures ,", "the c - terminus antibody ( red , c - ter ) instead recognizes only microglia in rat cerebellum ( figures 3(g ) , 3(h ) , and 3(i ) , and insets g1 , h1 , i1 ) , striatum ( figures 3(j ) , 3(k ) , and 3(l ) ) and cerebral cortex slices ( data not shown ) , with a signal more uniformly distributed throughout the whole tissue . by comparing the cd11b and c - ter immunolabelling in the striatum", ", we furthermore observe that the mutual intensity of signals is cell - selective within the microglia population , with some cells exclusively positive for c - ter ( arrow ) , and others instead showing different grades of cd11b - c - ter colocalization ( see for instance orange , yellow and greenish cells in the merged field ) .", "regrettably , double immunofluorescence with c - ter and iba1 microglia marker   is not practicable , since both antibodies are raised in the same species .", "however , no colocalization of signals is ever shown between mbp ( green ) and c - ter antibodies ( + mbp , inset j1 , merged ) . in order to verify", "if p2y12 recognized by c - ter antibody exclusively in microglia from rat brain slices could be used as specific microglia marker during neuroinflammation , we validated its use in a typical neuroinflammatory disease such as als and for the first time characterized the presence of p2y12 receptor in sod1-g93a mouse model ( figure 4 ) . by immunofluorescence and confocal analysis on lumbar spinal cord sections ( l3l5 ) of wild - type ( wt ) mice , we first compared the immunoreactive signals obtained with c - ter and specific microglia markers cd11b ( red , recognizing ramified microglia ) and cd68 ( red , recognizing roundish activated microglia ) . in wt mice", ", c - ter ( green ) is abundantly and strongly immunoreacting with the microglia population and colocalizing with the majority of cd11b - positive cells , in both dorsal ( dh ) and ventral ( vh ) horns of spinal cord ( figure 4(a ) , left panel , merged yellow signal ) .", "all cd11b - positive cells share immunoreactivity for c - ter , as proved by the absence of red cd11b signal .", "conversely , not all c - ter - positive cells immunoreact also with cd11b antibody , as proved by the presence of some green c - ter signals .", "in addition , we never observe colocalization with the rare activated cd68-positive ( red ) microglia cells present in wt healthy tissue ( figure 4(a ) , right panel , merged and inset ) . to test if c - ter antibody can further recognize microglia activation during the progression of als , we next examined sod1-g93a spinal cord sections at two different stages of the disease , that is , 20 weeks , a phase when the disease accelerates , and end stage , that is , 23 weeks , when the animals reach the score of 1 accordingly to a behavioral score system . at both stages ,", "sod1-g93a mice show a significant increase in cd68 immunostaining not only when compared to wt mice ( figure 4(a ) ) , but also in vh with respect to dh , and this is even more evident at end stage with respect to 20 weeks ( red , figure 4(b ) ) .", "remarkably , the immunoreactive signal of c - ter ( green , figure 4(b ) ) decreases during disease progression and the effect is pronounced especially in vh , where motor neuron loss , tissue damage , and microglia activation are known to be increased . particularly at 20 weeks", ", c - ter - expressing microglia are still ramified in dh , where few cd68-positive cells are present , but start to disappear in vh , where instead cd68-positive cells are increased . at end stage", ", c - ter staining decreases in dh with respect to 20 weeks and disappears almost completely in vh , concomitantly with a robust increase in cd68 staining . a parallel decrease in c - ter and increase of cd68 immunoreactivities", "is also confirmed by western blot analysis performed on sod1-g93a lumbar spinal cord homogenates at end stage ( figure 4(c ) ) . as in rat organotypic cerebellar cultures ( figure 2(b ) ) and rat cerebellar , striatal , cortical , or mouse spinal cord tissue slices", "( figures 3 and 4 ) , a similar pattern of p2y12 receptor expression is shown in human frontal cortex autoptic tissue by using c - ter antibody that highlights only microglia uniformly distributed throughout the entire healthy tissue ( red ) , but not mbp - positive myelinated structures ( green ) , as detected by the absence of overlapping immunofluorescent signals ( figures 5(a ) , 5(b ) , and 5(c ) , merged and insets a1 , c1 ) . on the contrary , intra1 ( red , figures 5(d ) , 5(e ) , and 5(f ) ) , intra2 , and intra fl ( data not shown ) antibodies recognize exclusively myelinated fibers in tissue from healthy ( insets d1 , e1 , and f1 ) and progressive ms donors ( figures 5(d ) , 5(e ) , and 5(f ) ) , as it is found with rat tissue ( figures 2 and 3 ) .", "moreover , we confirm a decrease of p2y12 receptor signal in proximity to the demyelinating lesion , as detected by loss of mbp - positive fibers ( see asterisks ) .", "no colocalization of signals is shown with mhc ii - positive microglia ( + mhc ii , inset d3 , merged ) .", "importantly , c - ter , intra1 ( red , insets a - f2 ) and intra2 , and intra fl ( data not shown ) antibodies all recognize the presence of p2y12 receptor in integrin ii/3-positive platelets ( green ) contained in the blood vessels of the analyzed tissues . as observed in als mouse spinal cord where p2y12 receptor detected by c - ter antibody is shown to temporally and regionally decrease in microglia as a function of increasing inflammatory damage ( figure 4 ) , we notice that microglia gradually lose immunoreactivity for c - ter antibody ( figure 6 ) in proximity to the demyelinating active cortical lesions of ms expressing augmented positivity for mhc ii [ 3335 ] .", "moreover , by comparing mhc ii and c - ter signals , we recognize four areas (  a  d  ) inside and around the lesion , where microglia express different amount of these proteins . in zone", " a ,  at the edge of the lesion , we observe a predominance of c - ter immunoreactivity ( red ) compared to that of mhc ii ( green ) , as depicted in the merged field by a major occurrence of red signal . in zone  b ,", " closer to the lesion , we notice a prevalence of active mhc ii - positive green cells , but still the presence of few red and yellow signals .", "c  and  d ,  apparently in the core or outside the lesion , respectively , where microglia express either almost exclusively mhc ii protein (  c  ) or p2y12 receptor on ramified microglia (  d  ) ."], ["the interchange among different cell types of molecular cues that condition the cell specificity and the protein profile of each cell characterizes the morphological and functional heterogeneity in particular of microglia within various cns regions , developmental stages [ 37 , 38 ] and , even more , states of activation during pathological conditions . in the case , for instance , of als , the release of signals from motor neurons apparently denotes one of the earliest phase of the disease , with microglia behaving as an m2 phenotype producing neuroprotective factors to repair motor neurons and preventing them against further injury . as disease rises , motor neurons start releasing  alarm signals ", "that in turn convert microglia from beneficial m2 to cytotoxic m1 phenotype , with consequent release of proinflammatory cytokines .", "for instance , m1 markers are abundantly expressed in normal appearing white matter and throughout active demyelinating ms lesions by activated microglia and macrophages , although in human active ms lesions microglia show an intermediate activation status .", "in addition , m2 microglia appear fundamental to guide oligodendrocyte remyelination in mice , and a switch from m1- to m2-dominant response occurs in microglia and peripherally derived macrophages when remyelination starts . only therapeutic procedures", "that both down - regulate the harmful responses and up - regulate the beneficial responses may hopefully slow pathological progression and provide meaningful hope for treatment . at the same time , the identification of clear markers involved in the m2/m1 microglia transition becomes mandatory for presymptomatic diagnosis , monitoring of disease progression , and efficacy of therapies . under this perspective , and consistently with previous findings establishing the role of purinergic receptors in the pathogenesis of both als and ms [ 14 , 43 ] , our present work serves this aim , by highlighting the gradual loss of p2y12 immunoreactivity as an early marker of neuroinflammation and microglia metamorphosis .", "we have indeed demonstrated here that p2y12 receptor protein identified in primary cultures of both microglia ( figure 2(a ) ) [ 18 , 44 ] and oligodendrocytes ( figure 2(b ) )   by different but p2y12-selective antibodies can be instead recognized in branched microglia exclusively by the use of c - ter antibody ( figures 36 ) .", "this occurs only when the integrity and cytoarchitecture of the tissue is typically preserved in the presence of the least experimental manipulations , that is , in organotypic cultures ( figure 2(c ) ) and tissues slices for instance from rat striatum and cerebellum ( figure 3 ) , mouse spinal cord ( figure 4 ) , and human cerebral cortex ( figures 5 and 6 ) .", "a similar difference in primary cultured cell versus tissue distribution of a protein was previously demonstrated with large - conductance calcium - activated potassium channel expression , in vascular endothelium .", "a first implication emerging from these results is that a reliable evidence about selective p2y12 expression in cells of healthy or neuroinflammatory states is genuine only when cell connectivity and tissue architecture are fully preserved .", "we have indeed shown this , by proving that the antibodies used for p2y12 , recognizing either the c - terminus or the second intracellular loop of the receptor ( figure 1 and table 1 ) and immunolabelling , respectively , microglia or myelinated fibers in the cns , are all still able to immunoreact for instance with platelets ( figure 5 ) , where the receptor was originally described to be present and to have a role in the processes of activation , aggregation [ 4649 ] , primary hemostasis , and arterial thrombosis [ 5055 ] . a possible explanation for the microglia versus oligodendrocyte selectivity of the p2y12 antibodies might be that gi - coupling , and/or quaternary structure , post - transcriptional modifications , and subcellular localization of p2y12 that remain strictly preserved in platelets , are instead divergent in microglia with respect to oligodendrocytes / myelinated fibers . in this case , a cell - specific network of p2y12 oligomeric interactions and/or a distinct subcellular partitioning might simply mask the recognition sites of the different antibodies on p2y12 protein in different cell types . to support this hypothesis", ", we know that in platelets p2y12 indeed resides in subcellular lipid raft structures and its partitioning out from rafts causes for instance inactivation   and that also the presence of another purinergic receptor , the ionotropic p2x3 , in lipid rafts has cell - specific properties shared in cerebellar granule neurons and total brain tissue but not in neuroblastoma cells and dorsal root ganglia   and that the specific antagonist clopidogrel inhibits p2y12 by breaking down the homoligomeric complex to single monomers   and finally , that hetero - oligomerization of p2y12 is demonstrated with p2y1 , p2y2 , p2y13 , and with adenosine a1 , a2a receptors in different cellular backgrounds .", "all these features might very well explain also why it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna   and , moreover , why p2y12 is specifically observed in brain and spinal cord resident microglia but is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] .", "a second implication that emerges from our results is that the morphological metamorphosis that microglia undergo under neuroinflammatory conditions as those triggered during als and ms , can be remarkably highlighted by the progressive reduction of p2y12 immunostaining obtained with c - ter antibody that reacts , also in this case , exclusively with multibranched microglia still present in the tissue ( figures 4 and 6 ) .", "this closely reflects the expression of p2y12 that is robust in the resting / surveillant branched state but dramatically decreased after morphological transition and activation of microglia .", "our observations are also in line with the central role played by p2y12 in branched microglia membrane ruffling and inspection of the environment . on the other hand , they depict a morphological / functional state of microglia that only partially overlaps with cd11b and mhc ii immunoreactivities , which are furthermore known to increase during activation but instead highly contrasts with cd68 immunostaining that is totally absent in ramified microglia . in parallel to our results , these last antibodies actually accentuate the progressive transition of microglia from a lesser ramified shape to a significantly more activated amoeboid phenotype ( figures 4 , 6 , and 7 ) , thus suggesting the dual use of c - ter and cd68 antibodies as markers , respectively , for branched resting / surveillant versus roundish / activated microglia . a reduction of c - ter - p2y12 immunostaining that is concomitant to an increase , for instance , of mhc ii / cd11b / cd68 immunoreactivity , could thus become a feasible approach to detect an increasing neuroinflammatory condition .", "indeed , p2y12 is considered an essential component and primary site at which nucleotides such as adp act to promote directional microglia movement or chemotaxis at early stages of cns injury . in particular ,", "microglia from mice lacking p2y12 exhibit normal baseline motility but are unable to polarize and to elicit directional branch extension and migration toward nucleotides in vitro , or sites of cortical damage in vivo .", "these notions are consistent with our results that fail to describe c - ter - p2y12 immunoreactivity on roundish phagocytizing , or polarized migratory microglia .", "however , we still do not know if reduction / absence of p2y12 c - ter - immunolabelling on activated microglia might be a cause or a consequence of morphological / functional transition or might simply reflect a cell - selective hindrance and lack of access to the immunogenic sites by the antibody .", "anyhow , we can assert that the distinctive recognition of multibranched microglia renders the c - ter antibody a novel and useful tool to discriminate among microglia morphological states , thus making p2y12 receptor a selective and early marker for the ramified phase .", "in parallel to this , we have also proven that all the several antibodies raised against the second intracellular loop of p2y12 ( intra1 , intra2 , and intra fl ) can likely be employed as markers for the presence of ms lesions .", "although with different intensities , they not only recognize the receptor specifically on myelinated fibers of organotypic cultures ( figure 2(c ) ) , tissues slices from rat striatum or cerebellum ( figure 3 ) and human cerebral cortex , but also furthermore highlight the reduction of p2y12 signal that occurs for instance in ms tissue ( figure 5 ) in correlation to the extent of demyelination found in all types of grey matter cortical plaques ( i  iii ) and subcortical white matter . in brief , we have shown here that the presence of p2y12 receptor can be simultaneously identified by the c - terminus and the second intracellular loop antibodies .", "when this occurs , a condition of intact myelinated fibers and branched / surveillant microglia is represented at once that perhaps signifies a ", "for instance in ms , a decrease in the second intracellular loop immunoreactions accompanied by an increase of c - terminus immunoreactivity will possibly depict the loss of myelin and replacement by ramified microglia that often occur in an inactive plaque . on the contrary , a decrease in c - terminus immunolabelling in the abundant presence of second intracellular loop - positive myelinated fibers would indicate an early active plaque where m1/m2 microglia reactivity starts to take place ."], ["by comparative analysis of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , we have established here that , under experimental conditions of well - preserved cytoarchitecture and tissue integrity , p2y12 receptor expressed by both ramified microglia or oligodendrocytes / myelinated fibers might serve a dual function as specific marker , respectively , of branched / surveillant microglia as well as demyelinating lesions .", "we believe that p2y12 identification and modulation might potentially acquire an important predictive value under neuroinflammatory conditions , as those found in als and ms .", "p2y12 is likely to deserve a key role in the verge of a neuroinflammatory breakdown ."]]}
{"article_id": "PMC3556436", "article_text": ["acute kidney injury ( aki ) requiring dialysis is a serious complication in critically ill patients , bringing increased morbidity , mortality , and costs of care [ 14 ] .", "aki requiring dialysis is usually considered the most severe form of kidney injury , and these patients have been conventionally regarded as a relatively  homogenous  group of patients , either when describing epidemiological information or while conducting clinical trials [ 5 , 6 ] .", "however , studies examining interventions in dialysis patients ( e.g. , dialysis modality or frequency have not demonstrated unequivocal survival benefits [ 79 ] .", "it is well recognized that small changes in creatinine ( mild - to - moderate aki ) independently predict mortality [ 10 , 11 ] ; we also recently reported that patients with aki requiring dialysis represent a wider spectrum of severity of kidney injury , contrary to the prevalent notion .", "thus , it can be hypothesized that the degree of elevation of creatinine prior to initiating dialysis may discriminate risk - adjusted mortality , similar to the observations in nondialysis requiring aki .  ", "the acute kidney injury network ( akin ) has issued standard definitions of aki ; currently , in these criteria , aki requiring dialysis is classified as stage iii ( or severe ) aki .", "the consensus panel also proposed that the examination of natural history of severe aki in icu to be one of the high - priority research areas , with a goal that new information may improve our ability in conducting prospective trials of intervention aki [ 14 , 15 ] . to date , there are limited studies that have examined the course or progression of aki after icu admission until the point of dialysis initiation . whether the degree of severity of kidney injury prior to dialysis requirement independently influences mortality risk in aki is not known .   in order to facilitate risk stratification that may be useful for the development of prospective studies , which are geared towards early diagnosis or intervention in severe aki", ", we characterized the spectrum of severity of kidney injury in a cohort of icu patients with aki who required dialysis .", "we specifically tested the association of creatinine elevation during icu stay , starting with icu admission and prior to dialysis initiation , with the risk of hospital mortality .", "we also examined the impact of severity of illness upon icu admission on the mortality risk in these patients .", "we examined a cohort derived from the veterans affairs ( va ) inpatient evaluation center ( ipec ) ; a national quality improvement program which reports risk - adjusted mortality , length of stay , and adherence to evidence - based practices in all va icus , by collecting data electronically from va computer databases .", "an analytic dataset was formed from the ipec retrospective cohort including all patients ( n , 617,927 ) admitted to all 191 va icus in the usa between october 2001 and september 2006 .", "we excluded patients with ( i ) less than three creatinine measurements during the icu stay ( n , 204,963 ) , ( ii ) readmissions to the icu ( n , 50,874 ) , ( iii ) transferred to other hospitals at discharge ( n , 14,219 ) , ( iv ) transplant recipients ( n , 317 ) , and ( v ) those with chronic renal failure defined as prior dialysis ( n , 3,862 patients ) , or international classification of disease-9th  clinical modification ( icd-9-cm ) codes for end - stage renal disease ( esrd ; 1,388 patients 585.6 ) , or with a calculated glomerular filtration rate ( gfr ) < 15  ml / min/1.73  m ( 16,571 ) ( gfr estimated by four variable modified diet in renal disease equation ) ; 296 additional patients who required dialysis in icu but had icd-9 codes for advanced ckd / stage v ckd were also excluded ( based on random q / a , these codes identified esrd status in those who required dialysis ) . thus , 324,999 patients were available for analysis .", "the analyses were reviewed and approved by the university of cincinnati institutional review board and va r&d committees .  ", "briefly , from each hospital database , a customized program identifies patients whose hospitalization included an icu stay , and extracts administrative data from the index hospitalization that includes icd-9-cm codes representing the reason for admission to the icu , length of stay , and all procedure codes . measured values of 11 laboratory tests are extracted to estimate severity of illness from a period of time 7 days prior to icu admission through hospital discharge .", "mortality is defined at death during hospitalization and is validated using the vital status file at the va national database ( austin , tx ) .  ", "aki was present if there was an increment of serum creatinine by > 0.3  mg / dl ( or 1.5 times increase ) during the icu stay relative to the creatinine on icu admission .", "for this analysis , we specifically focused on examining characteristics and outcomes of those aki patients who progressed to new requirement of dialysis ( aki - d ) during icu stay ( part of stage iii aki according to the akin classification system ) .", "dialysis requirement in icu included both intermittent and continuous dialysis , as prescribed by the clinician , and the modality can not be differentiated based on va electronic records that were available .", "we categorized aki - d into four groups based on the change in creatinine level after icu admission but prior to initiation of dialysis : group i had an increase in creatinine ranging from 0.3  mg / dl to < 2-fold increase , group ii an increase in creatinine 2 times baseline but <3 times , group iii 3-fold increase in creatinine , and group iv included those patients with new dialysis requirement in icu but who experienced a creatinine elevation of < 0.3  mg / dl .", "the purpose of this grouping was to determine an equivalent stage of aki that a patient may experience prior to initiating dialysis based on the standard definitions of aki put forth by the akin .", "some patients may have had creatinine values available after dialysis initiation , but these values were not considered for analysis .  ", "the study accounts for differences in patient characteristics using a previously validated logistic regression model that predicts mortality risk for each patient from independent predictors ( age , thirty - one comorbid disease groups ( by icd-9-cm codes ) , eighty - four mutually exclusive admission diagnoses determined from the icd-9-cm codes denoting the reason for admission to the icu , source of admission ( emergency room , outpatient clinics , ward , hospital , operating room , or nursing home ) , and the worst value of each of the 11 laboratory tests measured within 24 hours of icu admission ( sodium , urea , creatinine , bilirubin , albumin , glucose , hematocrit , white blood cell count , pao2 , paco2 , and ph ) .", "diagnosis and comorbid diseases were included in this model as binary variables , most laboratory values and age were included as cubic splines , and paco2 and ph were treated as a categorical interaction term .", "the model has been validated across multiple centers with excellent calibration and discrimination ( c - statistic = 0.88 ; brier 's = 0.06 ) , and comparable with traditional methods of severity of illness assessment ( e.g. , apache ) . from the logistic regression model , a standardized mortality rate for groups", "can be determined ( smr = observed / predicted mortality ; where predicted mortality is estimated by the mortality model described above ) .", "a more detailed account of development and validation of these variables and risk adjustment methods has been published earlier [ 16 , 19 , 20 ] .   ", "univariate comparison of diagnosis categories , comorbid diseases , and their relationship with laboratory variables was tested by chi - square test and kruskal - wallis test .", "a two - step logistic regression model determined the independent contribution of patient groups ( based on creatinine elevation ) to hospital mortality .", "the first step predicted each individual patient 's hospital mortality using the validated method described above .", "independent predictors in the second step included the predicted mortality ( determined from the first step ) and the patient group based on creatinine elevation before dialysis .", "based on the predicted mortality , as determined by the mortality model described above , we also estimated the standardized mortality rate ( observed / expected mortality ) for each of the patient groups under consideration .", "risk estimates were expressed as odds ratios ( or ) and 95% confidence intervals ( 95% ci ) .", "  overall , 21.9% of patients developed aki ( 71,090/324,999 ) of whom 2,744 patients experienced severe aki requiring dialysis .", "561/2744 ( 20.4% ) patients were admitted to icu from the hospital floors , whereas 2,183/2,744 ( 79.6% ) were either direct admission or admissions from emergency rooms .", "the median time for aki - d patients to meet the criteria for aki from the time of admission creatinine measurement was 23.4 hours ( interquartile range , 12.2 , 46.6 ) .", "the median time to initiation of dialysis in icu after icu admission was 96.0 hours ( interquartile range , 33.6 , 231.6 ) ; median time to dialysis initiation from icu admission was 72 hours in group i , 145.1 hours in group ii , 216.0 hours in group iii , and 14.6 hours in group 4 . when examined from the time when patients reached their peak creatinine value to the time of dialysis initiation , the median time was 43.0 , 39.5 , and 67.4 hours in groups i , ii , and iii respectively .  ", "figure 1 shows the percentage of patients initiating dialysis by days after icu admission .  ", "the proportion of patients in each group is shown in figure 2 ; of the 2,744 patients who required new dialysis during icu stay , only about a third of patients ( 31.5% ) experienced greater than 3 times increase in serum creatinine prior to dialysis initiation , and a small proportion of patients ( 11.2% ) required dialysis with little or no elevation in serum creatinine ( likely for fluid - electrolyte / acid - base disturbances ) .  ", "the baseline comorbid and laboratory characteristics of patients who developed aki requiring dialysis , and their univariate comparison across groups i through iv are shown in table 1 .", "group iii patients ( those with > 3 times increase in creatinine prior to dialysis ) had the lowest levels of creatinine or bun on icu admission .", "in contrast patients in other groups , who sustained only milder or no elevation in creatinine before dialysis initiation , had significantly higher creatinine and bun levels on icu admission .  \n", "table 2 shows the frequency of admission diagnosis categories across four subgroups of patients with aki requiring dialysis .", "the frequencies of patients admitted with primary icu admission diagnoses of kidney disorders or electrolyte and metabolic disorders significantly varied across four groups and were 24% and 21% , respectively , in group iv ( < 0.3  mg / dl increase in creatinine before dialysis ) compared with only 1.9% and 2.1% in group iii ( > 3 times increase in creatinine before dialysis ) .", "this suggests that majority of patients in group iv may have been dialyzed for indications other than kidney injury ( e.g. , for electrolyte or metabolic disturbances ) .", "in contrast , cardiothoracic surgery admissions occurred at a higher frequency ( 26% ) among group iii patients ( > 3 times increase in creatinine ) , suggesting that these patients are more likely to have normal renal function on icu admission and hence sustain > 3 times increment in creatinine prior to dialysis requirement .  ", "the degree of creatinine elevation prior to dialysis initiation was associated with increase in odds of death .  ", "table 3 shows odds ratios of mortality across all four groups , with group i as reference group .", "dialysis patients in group ii had greater odds of death ( odds ratio ( or ) , 1.76 , 95% confidence intervals ( 95% ci ) , 1.40 , 2.22 ) ) , those in group iii were more than twice likely to die ( or 2.20 , 95% ci , 1.792.71 ) , whereas patients in group iv were less likely to die compared to group i ( or 0.39 , 95% ci , 0.29 , 0.52 ) .", "the smr ( table 3 ) confirmed a graded association between the degree of creatinine elevation prior to dialysis initiation and mortality risk .", "the relationship between creatinine elevation and risk of death remained qualitatively similar even after stratifying patients by level of renal function on icu admission ( stratified at creatinine level of 1.2  mg / dl ) , but the sample size was relatively small in these subgroups ( data not shown ) .  \n", "figure 3 shows smr associated with creatinine elevation stratified by severity of illness on icu admission .", "dialysis patients in groups ii and iii had a significantly higher smr than those patients in groups i and iv , when predicted mortality was < 10% or 1030% ; smr was not significantly different across all four groups in patients admitted with > 30% predicted mortality on icu admission .", "the present study advances our understanding of natural history of progression of acute kidney injury during icu stay , in those patients who require dialysis .", "the study finds that , in patients with aki requiring dialysis , the risk of mortality is independently associated with the degree of severity of kidney injury sustained during icu stay prior to dialysis initiation and that severity of illness further influences this relationship .  ", "several epidemiological studies have confirmed that icu patients developing aki requiring dialysis experience a high risk of mortality across different clinical settings , including cardiovascular surgery , or in other medical or surgical icus [ 12 , 2123 ] .", "it is also observed that the severity of kidney injury , determined by the degree of creatinine elevation , in non - dialysis requiring aki is associated with a graded increase in mortality risk [ 2 , 10 ] .", "the present study is one of the first reports to show that in patients who developed aki requiring dialysis , the degree of kidney injury prior to dialysis initiation determines mortality risk , after accounting other major predictors of hospital mortality .", "it does so by examining a large , diverse , multicenter cohort that includes all icu settings , with an ability to perform electronic data extraction starting ( including serial creatinine measurements ) from the time of icu admission until the point of dialysis initiation in aki .", "the results indicate that majority of the aki - d patients sustain mild - to - moderate degrees of creatinine elevation ( equivalent of stage i or stage ii aki ) prior to dialysis , whereas less than a third of patients require dialysis after sustaining > 3 times increase in serum creatinine during icu stay ( or equivalent of stage iii aki ) .", "this confirms prior multicenter observations that aki requiring dialysis in icu usually represents a group of patients that sustain acute kidney injury on an underlying renal dysfunction .  ", "there were 11% of patients who required dialysis with < 0.3  mg / dl increase relative to icu admission .", "although the specific indications for dialysis could not be ascertained by electronic data extraction , however , based on the distribution of comorbid conditions and admission diagnoses , it can be suggested that the indications for dialysis in this subgroup may have been for volume overload or electrolyte / metabolic abnormalities rather than the degree of kidney injury .", "these observations are consistent with our prior experience in cardiac surgery settings , where 1015% of cases were dialyzed for reasons related to volume overload or electrolyte disorders and not azotemia / creatinine elevation . in terms of mortality , however , this group was associated with the lowest risk of death among those who required dialysis consistent with the study hypothesis .  ", "prior large multicenter observations provide only a limited insight into the effects of severity kidney injury prior to dialysis initiation on patient outcome .", "somewhat indirect evidence comes from the studies that have reported the impact of  baseline chronic kidney disease ( ckd )  in patients with aki requiring dialysis .", "for example , a third of patients in a multicenter us cohort ( picard cohort ; 618 aki cases , 60% of whom required dialysis ) had stage iv or worse ckd prior to aki . by multivariate", "analysis , baseline ckd status conferred 43% lower adjusted odds of in - hospital mortality ( or , 0.57 , 95% ci , 0.390.84 ) in aki .", "similar observations have been reported in a medicare administrative sample that examined outcomes in aki .", "unadjusted mortality rate in patients that were coded for aki requiring dialysis on an underlying ckd was 22% compared to 30% among those with aki requiring dialysis but without underlying ckd [ 2426 ] .", "our study may offer one of the explanations to the seemingly paradoxical observation that ckd status in aki may confer a  protective  effect : that is , patients with impaired renal function on icu admission are likely to require dialysis after sustaining milder degrees of kidney injury than those admitted with relatively better renal function and thus experience a lower risk of mortality .", "severity of illness on icu admission further modified the relationship between the degree of creatinine elevation and risk - adjusted mortality in aki require dialysis .", "the degree of kidney injury sustained prior to dialysis did not discriminate mortality risk in those patients with very high severity of illness ( > 30% predicted mortality ) on icu admission .", "in contrast , in patients with low - to - moderate severity of illness , it is the degree of injury that determines excess mortality risk rather than dialysis status alone .  ", "first , the data indicates that in half of the patients , dialysis was initiated within 96 hours after icu admission , with ~25% of then requiring it in the first 48 hours .", "thus , in order to target these patients for  early  interventions , we need to develop better predictive models that are based on information derived within 24 hours of icu admission ; similarly , the development of novel biomarkers that may predict dialysis requirement would need to discriminate acute kidney injury from underlying renal dysfunction at the time of icu admission .", "second , it indicates that the efficacy of therapeutic measures in aki requiring dialysis can not be equitably compared without accounting for both severity of illness on admission and degree of kidney injury sustained before dialysis initiation .", "for example , given the same predicted mortality or level of renal function on admission , patients in group i or iv could not be optimally compared with group iii in an intervention trial .", "finally , it also raises the question that whether  early dialysis ,  as indicated by initiation of therapy after a relatively lower magnitude of rise in creatinine , may offer a survival benefit .  ", "the va patient population is predominantly represented by male gender and white race and hence may limit in terms of generalizability of findings to specific demographic subgroups . by way of retrospective design , and lack of standardized criteria to initiate dialysis , the data can not account for differences in dialysis practices across different centers .", "we , however , examine a multicenter , diverse cohort of icu patients that records patient level information that is available for automated extraction by computerized methods .", "our cohort derivation excluded patients with advanced renal dysfunction on icu admission , and based on the present findings it can be expected that a significant proportion of these patients may have required new dialysis in icu .", "we still chose to use this criterion to minimize the likelihood of misclassifying patients with dialysis dependent renal failure prior to icu admission but who were not appropriately coded for it .", "additionally , we considered the degree of creatinine elevation relative to admission creatinine , which may not reflect a true baseline assessment of renal function ; this still allowed us to assess the relationship between magnitude of creatinine elevation and hospital mortality .", "another limitation is that we did not have information on modality or frequency of dialysis and hence could account for those variables in the multivariate analyses .", "the present study relies on the va icu mortality model instead of other traditional methods of measures of severity of illness assessment such as apache criteria .", "the rationale to do so is because the va icu mortality model is derived from an automated electronic data extraction method , which has been validated across multiple va sites with excellent predictive accuracy and calibration ( c - statistic = 0.88 ) and is comparable with other methods .   in summary , within a cohort of patients who develop aki requiring dialysis during icu", ", the mortality risk is independently associated with the degree of creatinine elevation prior to dialysis initiation .", "the mortality risk is lowest in those who experience minimal or no elevation in creatinine and may represent less severe acute illness in patients with ckd , or easily reversible fluid - electrolyte or acid - base disturbances .", "the study also indicates that aki requiring dialysis represents a heterogeneous group of patients , and directly confirms earlier observations that only minority of these cases sustain severe kidney injury prior to dialysis initiation .", "we interpret the data to suggest that prospective studies aimed at examining therapeutic interventions in aki requiring dialysis ( e.g. , timing of dialysis initiation ) would need to consider both the degree of kidney injury sustained prior to dialysis initiation and overall severity of illness , prior to performing equitable comparisons ."], "abstract_text": ["<S> in a multicenter observational cohort of patients - admitted to intensive care units ( icu ) , we assessed whether creatinine elevation prior to dialysis initiation in acute kidney injury ( aki - d ) further discriminates risk - adjusted mortality . </S>", "<S> aki - d was categorized into four groups ( grp ) based on creatinine elevation after icu admission but before dialysis initiation : grp i  >  0.3  mg / dl to < 2-fold increase , grp ii 2 times but <3 times increase , grp iii 3-fold increase in creatinine , and grp iv none or < 0.3  mg / dl increase . standardized mortality rates ( smr ) were calculated by using a validated risk - adjusted mortality model and expressed with 95% confidence intervals ( ci ) . </S>", "<S> 2,744 patients developed aki - d during icu stay ; 36.7% , 20.9% , 31.2% , and 11.2% belonged to groups i , ii , iii , and iv , respectively . </S>", "<S> smr showed a graded increase in grp i , ii , and iii ( 1.40 ( 95% ci , 1.291.42 ) , 1.84 ( 1.662.04 ) , and 2.25 ( 2.072.45 ) ) and was 0.98 ( 0.781.20 ) in grp iv . in icu patients with aki - d , degree of creatinine </S>", "<S> elevation prior to dialysis initiation is independently associated with hospital mortality . </S>", "<S> it is the lowest in those experiencing minor or no elevations in creatinine and may represent reversible fluid - electrolyte disturbances . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion "], "sections": [["acute kidney injury ( aki ) requiring dialysis is a serious complication in critically ill patients , bringing increased morbidity , mortality , and costs of care [ 14 ] .", "aki requiring dialysis is usually considered the most severe form of kidney injury , and these patients have been conventionally regarded as a relatively  homogenous  group of patients , either when describing epidemiological information or while conducting clinical trials [ 5 , 6 ] .", "however , studies examining interventions in dialysis patients ( e.g. , dialysis modality or frequency have not demonstrated unequivocal survival benefits [ 79 ] .", "it is well recognized that small changes in creatinine ( mild - to - moderate aki ) independently predict mortality [ 10 , 11 ] ; we also recently reported that patients with aki requiring dialysis represent a wider spectrum of severity of kidney injury , contrary to the prevalent notion .", "thus , it can be hypothesized that the degree of elevation of creatinine prior to initiating dialysis may discriminate risk - adjusted mortality , similar to the observations in nondialysis requiring aki .  ", "the acute kidney injury network ( akin ) has issued standard definitions of aki ; currently , in these criteria , aki requiring dialysis is classified as stage iii ( or severe ) aki .", "the consensus panel also proposed that the examination of natural history of severe aki in icu to be one of the high - priority research areas , with a goal that new information may improve our ability in conducting prospective trials of intervention aki [ 14 , 15 ] . to date , there are limited studies that have examined the course or progression of aki after icu admission until the point of dialysis initiation . whether the degree of severity of kidney injury prior to dialysis requirement independently influences mortality risk in aki is not known .   in order to facilitate risk stratification that may be useful for the development of prospective studies , which are geared towards early diagnosis or intervention in severe aki", ", we characterized the spectrum of severity of kidney injury in a cohort of icu patients with aki who required dialysis .", "we specifically tested the association of creatinine elevation during icu stay , starting with icu admission and prior to dialysis initiation , with the risk of hospital mortality .", "we also examined the impact of severity of illness upon icu admission on the mortality risk in these patients ."], ["we examined a cohort derived from the veterans affairs ( va ) inpatient evaluation center ( ipec ) ; a national quality improvement program which reports risk - adjusted mortality , length of stay , and adherence to evidence - based practices in all va icus , by collecting data electronically from va computer databases .", "an analytic dataset was formed from the ipec retrospective cohort including all patients ( n , 617,927 ) admitted to all 191 va icus in the usa between october 2001 and september 2006 .", "we excluded patients with ( i ) less than three creatinine measurements during the icu stay ( n , 204,963 ) , ( ii ) readmissions to the icu ( n , 50,874 ) , ( iii ) transferred to other hospitals at discharge ( n , 14,219 ) , ( iv ) transplant recipients ( n , 317 ) , and ( v ) those with chronic renal failure defined as prior dialysis ( n , 3,862 patients ) , or international classification of disease-9th  clinical modification ( icd-9-cm ) codes for end - stage renal disease ( esrd ; 1,388 patients 585.6 ) , or with a calculated glomerular filtration rate ( gfr ) < 15  ml / min/1.73  m ( 16,571 ) ( gfr estimated by four variable modified diet in renal disease equation ) ; 296 additional patients who required dialysis in icu but had icd-9 codes for advanced ckd / stage v ckd were also excluded ( based on random q / a , these codes identified esrd status in those who required dialysis ) . thus , 324,999 patients were available for analysis .", "the analyses were reviewed and approved by the university of cincinnati institutional review board and va r&d committees .  ", "briefly , from each hospital database , a customized program identifies patients whose hospitalization included an icu stay , and extracts administrative data from the index hospitalization that includes icd-9-cm codes representing the reason for admission to the icu , length of stay , and all procedure codes . measured values of 11 laboratory tests are extracted to estimate severity of illness from a period of time 7 days prior to icu admission through hospital discharge .", "mortality is defined at death during hospitalization and is validated using the vital status file at the va national database ( austin , tx ) .  ", "aki was present if there was an increment of serum creatinine by > 0.3  mg / dl ( or 1.5 times increase ) during the icu stay relative to the creatinine on icu admission .", "for this analysis , we specifically focused on examining characteristics and outcomes of those aki patients who progressed to new requirement of dialysis ( aki - d ) during icu stay ( part of stage iii aki according to the akin classification system ) .", "dialysis requirement in icu included both intermittent and continuous dialysis , as prescribed by the clinician , and the modality can not be differentiated based on va electronic records that were available .", "we categorized aki - d into four groups based on the change in creatinine level after icu admission but prior to initiation of dialysis : group i had an increase in creatinine ranging from 0.3  mg / dl to < 2-fold increase , group ii an increase in creatinine 2 times baseline but <3 times , group iii 3-fold increase in creatinine , and group iv included those patients with new dialysis requirement in icu but who experienced a creatinine elevation of < 0.3  mg / dl .", "the purpose of this grouping was to determine an equivalent stage of aki that a patient may experience prior to initiating dialysis based on the standard definitions of aki put forth by the akin .", "some patients may have had creatinine values available after dialysis initiation , but these values were not considered for analysis .  ", "the study accounts for differences in patient characteristics using a previously validated logistic regression model that predicts mortality risk for each patient from independent predictors ( age , thirty - one comorbid disease groups ( by icd-9-cm codes ) , eighty - four mutually exclusive admission diagnoses determined from the icd-9-cm codes denoting the reason for admission to the icu , source of admission ( emergency room , outpatient clinics , ward , hospital , operating room , or nursing home ) , and the worst value of each of the 11 laboratory tests measured within 24 hours of icu admission ( sodium , urea , creatinine , bilirubin , albumin , glucose , hematocrit , white blood cell count , pao2 , paco2 , and ph ) .", "diagnosis and comorbid diseases were included in this model as binary variables , most laboratory values and age were included as cubic splines , and paco2 and ph were treated as a categorical interaction term .", "the model has been validated across multiple centers with excellent calibration and discrimination ( c - statistic = 0.88 ; brier 's = 0.06 ) , and comparable with traditional methods of severity of illness assessment ( e.g. , apache ) . from the logistic regression model , a standardized mortality rate for groups", "can be determined ( smr = observed / predicted mortality ; where predicted mortality is estimated by the mortality model described above ) .", "a more detailed account of development and validation of these variables and risk adjustment methods has been published earlier [ 16 , 19 , 20 ] .   ", "univariate comparison of diagnosis categories , comorbid diseases , and their relationship with laboratory variables was tested by chi - square test and kruskal - wallis test .", "a two - step logistic regression model determined the independent contribution of patient groups ( based on creatinine elevation ) to hospital mortality .", "the first step predicted each individual patient 's hospital mortality using the validated method described above .", "independent predictors in the second step included the predicted mortality ( determined from the first step ) and the patient group based on creatinine elevation before dialysis .", "based on the predicted mortality , as determined by the mortality model described above , we also estimated the standardized mortality rate ( observed / expected mortality ) for each of the patient groups under consideration .", "risk estimates were expressed as odds ratios ( or ) and 95% confidence intervals ( 95% ci ) ."], ["  overall , 21.9% of patients developed aki ( 71,090/324,999 ) of whom 2,744 patients experienced severe aki requiring dialysis .", "561/2744 ( 20.4% ) patients were admitted to icu from the hospital floors , whereas 2,183/2,744 ( 79.6% ) were either direct admission or admissions from emergency rooms .", "the median time for aki - d patients to meet the criteria for aki from the time of admission creatinine measurement was 23.4 hours ( interquartile range , 12.2 , 46.6 ) .", "the median time to initiation of dialysis in icu after icu admission was 96.0 hours ( interquartile range , 33.6 , 231.6 ) ; median time to dialysis initiation from icu admission was 72 hours in group i , 145.1 hours in group ii , 216.0 hours in group iii , and 14.6 hours in group 4 . when examined from the time when patients reached their peak creatinine value to the time of dialysis initiation , the median time was 43.0 , 39.5 , and 67.4 hours in groups i , ii , and iii respectively .  ", "figure 1 shows the percentage of patients initiating dialysis by days after icu admission .  ", "the proportion of patients in each group is shown in figure 2 ; of the 2,744 patients who required new dialysis during icu stay , only about a third of patients ( 31.5% ) experienced greater than 3 times increase in serum creatinine prior to dialysis initiation , and a small proportion of patients ( 11.2% ) required dialysis with little or no elevation in serum creatinine ( likely for fluid - electrolyte / acid - base disturbances ) .  ", "the baseline comorbid and laboratory characteristics of patients who developed aki requiring dialysis , and their univariate comparison across groups i through iv are shown in table 1 .", "group iii patients ( those with > 3 times increase in creatinine prior to dialysis ) had the lowest levels of creatinine or bun on icu admission .", "in contrast patients in other groups , who sustained only milder or no elevation in creatinine before dialysis initiation , had significantly higher creatinine and bun levels on icu admission .  \n", "table 2 shows the frequency of admission diagnosis categories across four subgroups of patients with aki requiring dialysis .", "the frequencies of patients admitted with primary icu admission diagnoses of kidney disorders or electrolyte and metabolic disorders significantly varied across four groups and were 24% and 21% , respectively , in group iv ( < 0.3  mg / dl increase in creatinine before dialysis ) compared with only 1.9% and 2.1% in group iii ( > 3 times increase in creatinine before dialysis ) .", "this suggests that majority of patients in group iv may have been dialyzed for indications other than kidney injury ( e.g. , for electrolyte or metabolic disturbances ) .", "in contrast , cardiothoracic surgery admissions occurred at a higher frequency ( 26% ) among group iii patients ( > 3 times increase in creatinine ) , suggesting that these patients are more likely to have normal renal function on icu admission and hence sustain > 3 times increment in creatinine prior to dialysis requirement .  ", "the degree of creatinine elevation prior to dialysis initiation was associated with increase in odds of death .  ", "table 3 shows odds ratios of mortality across all four groups , with group i as reference group .", "dialysis patients in group ii had greater odds of death ( odds ratio ( or ) , 1.76 , 95% confidence intervals ( 95% ci ) , 1.40 , 2.22 ) ) , those in group iii were more than twice likely to die ( or 2.20 , 95% ci , 1.792.71 ) , whereas patients in group iv were less likely to die compared to group i ( or 0.39 , 95% ci , 0.29 , 0.52 ) .", "the smr ( table 3 ) confirmed a graded association between the degree of creatinine elevation prior to dialysis initiation and mortality risk .", "the relationship between creatinine elevation and risk of death remained qualitatively similar even after stratifying patients by level of renal function on icu admission ( stratified at creatinine level of 1.2  mg / dl ) , but the sample size was relatively small in these subgroups ( data not shown ) .  \n", "figure 3 shows smr associated with creatinine elevation stratified by severity of illness on icu admission .", "dialysis patients in groups ii and iii had a significantly higher smr than those patients in groups i and iv , when predicted mortality was < 10% or 1030% ; smr was not significantly different across all four groups in patients admitted with > 30% predicted mortality on icu admission ."], ["the present study advances our understanding of natural history of progression of acute kidney injury during icu stay , in those patients who require dialysis .", "the study finds that , in patients with aki requiring dialysis , the risk of mortality is independently associated with the degree of severity of kidney injury sustained during icu stay prior to dialysis initiation and that severity of illness further influences this relationship .  ", "several epidemiological studies have confirmed that icu patients developing aki requiring dialysis experience a high risk of mortality across different clinical settings , including cardiovascular surgery , or in other medical or surgical icus [ 12 , 2123 ] .", "it is also observed that the severity of kidney injury , determined by the degree of creatinine elevation , in non - dialysis requiring aki is associated with a graded increase in mortality risk [ 2 , 10 ] .", "the present study is one of the first reports to show that in patients who developed aki requiring dialysis , the degree of kidney injury prior to dialysis initiation determines mortality risk , after accounting other major predictors of hospital mortality .", "it does so by examining a large , diverse , multicenter cohort that includes all icu settings , with an ability to perform electronic data extraction starting ( including serial creatinine measurements ) from the time of icu admission until the point of dialysis initiation in aki .", "the results indicate that majority of the aki - d patients sustain mild - to - moderate degrees of creatinine elevation ( equivalent of stage i or stage ii aki ) prior to dialysis , whereas less than a third of patients require dialysis after sustaining > 3 times increase in serum creatinine during icu stay ( or equivalent of stage iii aki ) .", "this confirms prior multicenter observations that aki requiring dialysis in icu usually represents a group of patients that sustain acute kidney injury on an underlying renal dysfunction .  ", "there were 11% of patients who required dialysis with < 0.3  mg / dl increase relative to icu admission .", "although the specific indications for dialysis could not be ascertained by electronic data extraction , however , based on the distribution of comorbid conditions and admission diagnoses , it can be suggested that the indications for dialysis in this subgroup may have been for volume overload or electrolyte / metabolic abnormalities rather than the degree of kidney injury .", "these observations are consistent with our prior experience in cardiac surgery settings , where 1015% of cases were dialyzed for reasons related to volume overload or electrolyte disorders and not azotemia / creatinine elevation . in terms of mortality , however , this group was associated with the lowest risk of death among those who required dialysis consistent with the study hypothesis .  ", "prior large multicenter observations provide only a limited insight into the effects of severity kidney injury prior to dialysis initiation on patient outcome .", "somewhat indirect evidence comes from the studies that have reported the impact of  baseline chronic kidney disease ( ckd )  in patients with aki requiring dialysis .", "for example , a third of patients in a multicenter us cohort ( picard cohort ; 618 aki cases , 60% of whom required dialysis ) had stage iv or worse ckd prior to aki . by multivariate", "analysis , baseline ckd status conferred 43% lower adjusted odds of in - hospital mortality ( or , 0.57 , 95% ci , 0.390.84 ) in aki .", "similar observations have been reported in a medicare administrative sample that examined outcomes in aki .", "unadjusted mortality rate in patients that were coded for aki requiring dialysis on an underlying ckd was 22% compared to 30% among those with aki requiring dialysis but without underlying ckd [ 2426 ] .", "our study may offer one of the explanations to the seemingly paradoxical observation that ckd status in aki may confer a  protective  effect : that is , patients with impaired renal function on icu admission are likely to require dialysis after sustaining milder degrees of kidney injury than those admitted with relatively better renal function and thus experience a lower risk of mortality .", "severity of illness on icu admission further modified the relationship between the degree of creatinine elevation and risk - adjusted mortality in aki require dialysis .", "the degree of kidney injury sustained prior to dialysis did not discriminate mortality risk in those patients with very high severity of illness ( > 30% predicted mortality ) on icu admission .", "in contrast , in patients with low - to - moderate severity of illness , it is the degree of injury that determines excess mortality risk rather than dialysis status alone .  ", "first , the data indicates that in half of the patients , dialysis was initiated within 96 hours after icu admission , with ~25% of then requiring it in the first 48 hours .", "thus , in order to target these patients for  early  interventions , we need to develop better predictive models that are based on information derived within 24 hours of icu admission ; similarly , the development of novel biomarkers that may predict dialysis requirement would need to discriminate acute kidney injury from underlying renal dysfunction at the time of icu admission .", "second , it indicates that the efficacy of therapeutic measures in aki requiring dialysis can not be equitably compared without accounting for both severity of illness on admission and degree of kidney injury sustained before dialysis initiation .", "for example , given the same predicted mortality or level of renal function on admission , patients in group i or iv could not be optimally compared with group iii in an intervention trial .", "finally , it also raises the question that whether  early dialysis ,  as indicated by initiation of therapy after a relatively lower magnitude of rise in creatinine , may offer a survival benefit .  ", "the va patient population is predominantly represented by male gender and white race and hence may limit in terms of generalizability of findings to specific demographic subgroups . by way of retrospective design , and lack of standardized criteria to initiate dialysis , the data can not account for differences in dialysis practices across different centers .", "we , however , examine a multicenter , diverse cohort of icu patients that records patient level information that is available for automated extraction by computerized methods .", "our cohort derivation excluded patients with advanced renal dysfunction on icu admission , and based on the present findings it can be expected that a significant proportion of these patients may have required new dialysis in icu .", "we still chose to use this criterion to minimize the likelihood of misclassifying patients with dialysis dependent renal failure prior to icu admission but who were not appropriately coded for it .", "additionally , we considered the degree of creatinine elevation relative to admission creatinine , which may not reflect a true baseline assessment of renal function ; this still allowed us to assess the relationship between magnitude of creatinine elevation and hospital mortality .", "another limitation is that we did not have information on modality or frequency of dialysis and hence could account for those variables in the multivariate analyses .", "the present study relies on the va icu mortality model instead of other traditional methods of measures of severity of illness assessment such as apache criteria .", "the rationale to do so is because the va icu mortality model is derived from an automated electronic data extraction method , which has been validated across multiple va sites with excellent predictive accuracy and calibration ( c - statistic = 0.88 ) and is comparable with other methods .   in summary , within a cohort of patients who develop aki requiring dialysis during icu", ", the mortality risk is independently associated with the degree of creatinine elevation prior to dialysis initiation .", "the mortality risk is lowest in those who experience minimal or no elevation in creatinine and may represent less severe acute illness in patients with ckd , or easily reversible fluid - electrolyte or acid - base disturbances .", "the study also indicates that aki requiring dialysis represents a heterogeneous group of patients , and directly confirms earlier observations that only minority of these cases sustain severe kidney injury prior to dialysis initiation .", "we interpret the data to suggest that prospective studies aimed at examining therapeutic interventions in aki requiring dialysis ( e.g. , timing of dialysis initiation ) would need to consider both the degree of kidney injury sustained prior to dialysis initiation and overall severity of illness , prior to performing equitable comparisons ."]]}
{"article_id": "PMC3265212", "article_text": ["many previous studies have revealed that parents , especially mothers , of children with developmental disabilities such as intellectual disabilities ( ids ) , developmental delay , and physical and sensory handicaps are more likely to show signs of psychological distress or depressive symptoms and to exhibit lower well - being than parents of typically developing children [ 15 ] .", "it is generally accepted that caring for a child who has a developmental disability may involve significant and prolonged periods of time and energy .", "since the majority of this increased daily care - giving burden for these children is carried by their parents , they are supposed to have an increased risk for high levels of stress , and thus some cases may be linked to depression [ 7 , 8 ] .", "recently , the mental health needs of adults with i d have also been taken up for discussion .", "studies have revealed that psychiatric disorders are more prevalent in people with i d compared within the general population .", "specialist psychiatric services for people with i d are available in some countries such as the uk and the usa ; however , the provision of high - quality psychiatric services remains a major concern in many countries .", ". evidently , the current psychiatric services in japan are not adequate to meet the complex mental health needs of people with i d appropriately , sensitively , and effectively .", "failure to provide services that meet the needs of these individuals may lead to problems such as high - level caregiver stress and inappropriate therapy .", "the paucity of trained psychiatrists and other allied professionals is a major barrier to the development of this subspecialty in many countries . in japan ,", "high - quality psychiatric services for the parents of children with i d are assumed to be not well established .", "so far , the major portion of resources has been directed to the treatment of and services connected with the children , and not enough attention has been paid to the actual mental condition and needs of the parents . to our knowledge , there have been no reports on a nationwide scale on the mental health and distress of parents having children with developmental disabilities in japan . since the understanding of the parents '", "mental health may be essential for providing more appropriate support , we aimed to obtain a clear perspective on the situation facing the parents through this facility - based nationwide study .", "the current survey included 1,102 institutions and consultation centers for children with developmental disabilities distributing in all prefectures in japan , which were listed in the national register of agencies , organizations , and institutions related to intellectual disabilities   and the directory of support centers for individuals with developmental disabilities .", "in japan , these institutions and centers play a key role in social support networks .", "professional workers in these facilities come into contact with children as their clients , and also with the children 's guardians , mainly with the mothers , on a daily basis .", "the questionnaire was written in japanese and was divided into two sections with a total of 47 multiple choice questions .", "the first section sought information about the characteristics of the facility , such as staff adequacy , the scale of the facility , the health promotion services provided , the ages of their clients , and the clients ' main disabilities , namely , i d , pervasive developmental disorder ( pdd ) , profound intellectual and multiple disabilities ( pimd ) , physical disabilities , and others .", "the second section sought information about the clients ' parents , such as signs of mental health distress and their needs , from the professional caregivers ' point of view .", "also measures taken by the professional caregivers when encountering distressed parents were asked in detail .", "questionnaires were mailed in october 2008 , with an explanatory letter to the person in charge of each institution or consultation center . when agreement was met to participate in this study , one or more service providers in each facility completed the questionnaire based on all sources of information available .", "the questionnaire was to be returned unsigned , using the stamped , self - addressed envelope enclosed . for ethical considerations , the information disclosed in the questionnaire was in a form which made it impossible to identify the facility and clients", "this study was approved and carried out under the supervision of the japan league on developmental disabilities ( jidd ) .", "the statistical analysis including the chi - squared test was performed with the aid of the statistical package for the social sciences 15.0 ( spss japan , inc . ) .", "a two - sided p value of 0.05 or less was considered to be statistically significant . answers to open - ended questions", "a total of 460 out of 1,102 facilities replied within four weeks , the response rate being 41.7% . according to the clients ' main disabilities ,", "each facility was classified into the following categories : i d , pdd , pimd , physical disability group , and others ( table 1 ) .", "the last group was small in number and diverse in character and also included responders who provided incomplete information ; thus , data from this group were omitted from analysis hereafter .", "also in table 1 , the number of clients ( mean persons per facility ) as an index of facility scale , their mean age , and proportion of male gender are shown .", "their average age ( sd ) was 44.7 ( 9.5 ) years old for the i d group , 53.0 ( 7.6 ) for the pdd group , 48.3 ( 8.4 ) for the pimd group , and 55.3 ( 4.9 ) for the physical disability group .", "373 out of the 415 facilities ( about 90% ) experienced cases with difficulties when communicating with the clients ' parents .", "the percentage was high regardless of the children 's disabilities , being concretely highest in the pdd group ( 96.9% ) and lowest in the physical disability group ( 86.7% ) .", "the proportion of such parents , out of the total , was reported to be  almost none  ( 5% or less ) or  few  ( less than one fourth of the clients ) in most of the facilities ( table 2 ) .", "it should be noted that in the i d and pdd groups , about 15% of the facilities responded that  about half  or more of the parents were difficult to communicate with . in all groups ,", "the characteristics of the i d and pdd groups were similar to each other , while the pimd and physical disability groups resembled each other .", "in other words , in the i d and pdd groups , slightly more difficulties were experienced with the fathers compared to in the pimd and physical disability groups , where they experienced more difficulties with other family members such as the grandparents and siblings compared to the i d and pdd groups .", "the primary reason for these difficulties was thought to be the parent 's personal condition , such as poor mental health ( table 2 ) .", "this was especially true for the i d and pdd groups [ (df = 9 ) = 41.9 , p < 0.001 ] . in the pimd and physical disability groups ,", "the parent 's poor socio - economic status and heavy care - giving burden , reflecting the child 's level of disability , were considered to be major factors in approximately one - third of the cases .", "signs of depression or a depressive state were the most common psychiatric disturbances seen in the parents . among the four groups , the pdd group most commonly experienced cases which showed signs of depression or a depressive state in parents .", "the proportion of such parents was approximately 70% of the total [ (df = 12 ) = 65.1 , p < 0.001 ] .", "well - treated cases were only 5% out of the total ; thus , most of these conditions were considered to be not medically well treated .", "for further analysis , we selected 159 cases where symptoms of depression or a depressive state were reported in a descriptive manner . according to these reports ,", "we asked about the time of the onset , that is , whether it was prior to the birth of the child , when the parent first noticed the child 's disability , or after the child had been fully medically diagnosed . apart from the answer ", "unknown  which accounted for approximately half of the cases ,  prior to the birth of the child  was relatively common in the pdd group ( 22.1% ) , whereas ", "after the child had been fully medically diagnosed  was relatively common in the pimd ( 23.5% ) and physical disability groups ( 28.6% ) .", "when a parent suffered from signs of psychiatric disturbances , more than half of the children experienced maltreatment .", "physical neglect was most common in all groups , accounting for about 50% of the cases . in comparison with the other groups , in the pdd group , the child was likely to be emotionally disturbed and confined indoors ( 32.6% ) , in the pimd group , the child was mostly confined indoors ( 33.3% ) , and in the physical disability group , medical care neglect made the utilization of medical and welfare support impossible ( 50% ) [ (df = 12 ) = 27.6 , p = 0.006 ] .", "the leading measures were gathering and disclosure of necessary information , discussion among service providers within the facility about ways to deal with the parent , protection of the child by providing a safe environment , making approaches to other family members for cooperation , intensive collaboration with other organizations such as institutions and consultation centers for better community support , and medical support for the parent provided by specialists such as physicians and psychologists .", "many factors were reported to affect the mental health of parents . the presence or absence of support from other family members (", "i.e. , a spouse ) and the existence ( or not ) of a medical condition in the parent were major factors ( table 5 ) .", "practical use ( or not ) of social support networks , whether the child 's level of disability was severe or not , and parent participation in face - to - face family support groups or not were also thought to be influential factors . in all groups , regardless of the child 's disability , more direct welfare support for the child was considered to be helpful and thus requested to be improved , in order to meet the mental health needs of the parent . besides this , in the physical disability group , family support groups , in the pimd group , support ( including financial support ) for other family members , and in the i d and pdd groups , support for other family members ( mainly siblings and the other parent ) and medical treatment of the parent 's psychiatric disturbance were also emphasized . to fulfill the demanded future role of their facility", ", respondents expressed the importance of more collaboration with other institutions and consultation centers and the necessity for more special training courses targeting support professionals and recognized the urgent demand for more specialists especially psychologists who can support the mental health needs of both the children and their parents .", "in the present study , we surveyed institutions and consultation centers for children with developmental disabilities in japan , which play a central role in the social support networks .", "this facility - based nationwide survey revealed the situation facing the parents of children with i d and other developmental disabilities from the professional caregivers ' point of view .", "almost all of the facilities experienced difficulties in keeping good relations with the clients ' parents , regardless of the children 's disabilities , and the majority of service providers noted that the proportion of such parents , out of the total , was roughly between 5 and 25% , and still increasing .", "the primary reason for these difficulties was thought to be the parent 's condition , such as their mental health and background .", "also , signs of depression or a depressive state were assumed to be the most common psychiatric disturbance seen .", "these results suggested that thorough consideration of the parents ' mental health needs , especially prevention of depressive conditions and support for parents suffering from them , is urgent - demanded task for facilities .", "i d , attention - deficit / hyperactivity disorder ( ad / hd ) , and pdd are common developmental disorders , and the latter two are noted to have substantial genetic components .", "it has been reported that a lifetime prevalence of major mood disorders is higher in parents of children with autism and that the onset for the majority was prior to the birth of their child with autism .", "family history studies of autism consistently uncovered a large subgroup with a high incidence of major mood disorders among family members , suggesting the two entities are related clinically and genetically [ 19 , 20 ] .", "the findings of the present survey must be interpreted allowing for the limitations of the use of unconfirmed data reported by professional caregivers ' who come into contact with children as their clients , and their parents on a day - to - day basis . evidently , the service providers ' perspective on parental mental health was consistent with previous reports from western countries on major mood disorders and pdd .", "in other words , facilities where the clients were diagnosed as having pdd experienced significantly more cases of parents with signs of a depressive state compared to other facilities , and the onset of depression was considered to be prior to the birth of the child in many of the cases . although these professional caregivers were well trained and well experienced , only , one - third were physicians , nurses or psychologists . on account of the limitations of these data ,", "we emphasize confirming the diagnoses of psychiatric disturbances through direct contact with the parents by specialists such as psychiatrists is necessary in future studies , for accurate understanding of the overall problem . in japan", ", there is no official system to check and document the parents ' condition including their mental health status .", "parents are not accustomed to being asked about their own mental health , so there is a tendency to avoid discussion on matters concerning themselves .", "this was the major reason why we asked the professional caregivers about the parents ' condition , not the parents directly , in this study .", "but still we judged it to be worthwhile at this moment to summarize the professional caregivers ' impression on this issue , since the case in japan has never been described in detail despite of its importance . because the majority of burden for daily care of handicapped children is typically carried by mothers , particular concern is raised for their adaptation . in the past studies , the psychosocial outcomes in mothers", "were said to be better predicted by psychosocial factors such as more active social life and family resources .", "recent research focusing on parenting stress illustrated the value of the participation of fathers also .", "greater marital quality predicted lower parenting stress for both mothers and fathers , while greater social support predicted increased parenting efficacy for fathers .", "although the relationship between child characteristics and parental well - being has not reached an invariable agreement , the severity of a child 's disability , intellectual functioning , and so forth are generally considered to be risk factors . since the burden for daily care is mainly carried by the mother , similarly in japan , service providers tend to be in contact with the mother more frequently than other family members", ". this may be partially the reason why service providers experienced difficulties in communicating with mostly the mothers in this study .", "the presence of support from other family members , such as a spouse , is considered to be a major influential factor for the mental health of parents in japan also .", "practical use of social support networks such as participation in face - to - face family support groups , more financial support for the household , more direct welfare support for the child , and medical treatment of the parent 's psychiatric disturbance were reported to be helpful and thus requested to be improved , in order to meet the mental health needs of the parent .", "we would like to emphasize the fact that although the importance of medical treatment for the parents ' psychiatric disturbances was pointed out , it was also realized that these conditions were presently not being medically well treated .", "this shows that current psychiatric services in japan are not adequate to meet the complex mental health needs of the parents appropriately . to resolve this challenging issue ,", "more intensive collaboration between other child - care facilities and organizations , aiming at the mental health needs of the parents , is demanded .", "the paucity of trained psychiatrists and other allied professionals also needs to be addressed , and in order to fulfill this , sufficient discussion and research on the establishment of an effective training system for specialists is warranted . providing direct advice for the parents on the utilization of social support networks is a reliable measure that can be immediately taken .", "training more professionals who can properly deal with the parents ' mental health needs is an urgent matter that must be tackled .", "overall , the present study is the first nationwide survey that examined the professional caregivers ' perspective on the mental health of parents of children with developmental disabilities , and it underscores the importance of understanding and supporting the parents ' mental health needs , which leads to more appropriate support for children with developmental disabilities ."], "abstract_text": ["<S> parents of children with intellectual disabilities and/or physical disabilities are supposed to have an increased risk for parenting stress and psychological distress . </S>", "<S> we as professional caregivers sometimes experience difficulties in keeping good relations or communicating with the parents . </S>", "<S> professional workers in 460 institutions and consultation centers throughout japan answered a questionnaire on their clinical experiences . about 90% of the facilities experienced  distressed parents ,  and the parents ' condition such as mental health seemed to influence this . </S>", "<S> signs of a depressive state were the most common psychiatric disturbances detected , and it was notable in the pervasive developmental disorder group . more welfare support , presence of support groups , support from other family members , and medical treatment of the parents ' problems were considered to be helpful and thus requested to be improved . </S>", "<S> training more professionals who can properly deal with the parents ' mental health needs is an urgent matter that must be tackled . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["many previous studies have revealed that parents , especially mothers , of children with developmental disabilities such as intellectual disabilities ( ids ) , developmental delay , and physical and sensory handicaps are more likely to show signs of psychological distress or depressive symptoms and to exhibit lower well - being than parents of typically developing children [ 15 ] .", "it is generally accepted that caring for a child who has a developmental disability may involve significant and prolonged periods of time and energy .", "since the majority of this increased daily care - giving burden for these children is carried by their parents , they are supposed to have an increased risk for high levels of stress , and thus some cases may be linked to depression [ 7 , 8 ] .", "recently , the mental health needs of adults with i d have also been taken up for discussion .", "studies have revealed that psychiatric disorders are more prevalent in people with i d compared within the general population .", "specialist psychiatric services for people with i d are available in some countries such as the uk and the usa ; however , the provision of high - quality psychiatric services remains a major concern in many countries .", ". evidently , the current psychiatric services in japan are not adequate to meet the complex mental health needs of people with i d appropriately , sensitively , and effectively .", "failure to provide services that meet the needs of these individuals may lead to problems such as high - level caregiver stress and inappropriate therapy .", "the paucity of trained psychiatrists and other allied professionals is a major barrier to the development of this subspecialty in many countries . in japan ,", "high - quality psychiatric services for the parents of children with i d are assumed to be not well established .", "so far , the major portion of resources has been directed to the treatment of and services connected with the children , and not enough attention has been paid to the actual mental condition and needs of the parents . to our knowledge , there have been no reports on a nationwide scale on the mental health and distress of parents having children with developmental disabilities in japan . since the understanding of the parents '", "mental health may be essential for providing more appropriate support , we aimed to obtain a clear perspective on the situation facing the parents through this facility - based nationwide study ."], ["the current survey included 1,102 institutions and consultation centers for children with developmental disabilities distributing in all prefectures in japan , which were listed in the national register of agencies , organizations , and institutions related to intellectual disabilities   and the directory of support centers for individuals with developmental disabilities .", "in japan , these institutions and centers play a key role in social support networks .", "professional workers in these facilities come into contact with children as their clients , and also with the children 's guardians , mainly with the mothers , on a daily basis .", "the questionnaire was written in japanese and was divided into two sections with a total of 47 multiple choice questions .", "the first section sought information about the characteristics of the facility , such as staff adequacy , the scale of the facility , the health promotion services provided , the ages of their clients , and the clients ' main disabilities , namely , i d , pervasive developmental disorder ( pdd ) , profound intellectual and multiple disabilities ( pimd ) , physical disabilities , and others .", "the second section sought information about the clients ' parents , such as signs of mental health distress and their needs , from the professional caregivers ' point of view .", "also measures taken by the professional caregivers when encountering distressed parents were asked in detail .", "questionnaires were mailed in october 2008 , with an explanatory letter to the person in charge of each institution or consultation center . when agreement was met to participate in this study , one or more service providers in each facility completed the questionnaire based on all sources of information available .", "the questionnaire was to be returned unsigned , using the stamped , self - addressed envelope enclosed . for ethical considerations , the information disclosed in the questionnaire was in a form which made it impossible to identify the facility and clients", "this study was approved and carried out under the supervision of the japan league on developmental disabilities ( jidd ) .", "the statistical analysis including the chi - squared test was performed with the aid of the statistical package for the social sciences 15.0 ( spss japan , inc . ) .", "a two - sided p value of 0.05 or less was considered to be statistically significant . answers to open - ended questions"], ["a total of 460 out of 1,102 facilities replied within four weeks , the response rate being 41.7% . according to the clients ' main disabilities ,", "each facility was classified into the following categories : i d , pdd , pimd , physical disability group , and others ( table 1 ) .", "the last group was small in number and diverse in character and also included responders who provided incomplete information ; thus , data from this group were omitted from analysis hereafter .", "also in table 1 , the number of clients ( mean persons per facility ) as an index of facility scale , their mean age , and proportion of male gender are shown .", "their average age ( sd ) was 44.7 ( 9.5 ) years old for the i d group , 53.0 ( 7.6 ) for the pdd group , 48.3 ( 8.4 ) for the pimd group , and 55.3 ( 4.9 ) for the physical disability group .", "373 out of the 415 facilities ( about 90% ) experienced cases with difficulties when communicating with the clients ' parents .", "the percentage was high regardless of the children 's disabilities , being concretely highest in the pdd group ( 96.9% ) and lowest in the physical disability group ( 86.7% ) .", "the proportion of such parents , out of the total , was reported to be  almost none  ( 5% or less ) or  few  ( less than one fourth of the clients ) in most of the facilities ( table 2 ) .", "it should be noted that in the i d and pdd groups , about 15% of the facilities responded that  about half  or more of the parents were difficult to communicate with . in all groups ,", "the characteristics of the i d and pdd groups were similar to each other , while the pimd and physical disability groups resembled each other .", "in other words , in the i d and pdd groups , slightly more difficulties were experienced with the fathers compared to in the pimd and physical disability groups , where they experienced more difficulties with other family members such as the grandparents and siblings compared to the i d and pdd groups .", "the primary reason for these difficulties was thought to be the parent 's personal condition , such as poor mental health ( table 2 ) .", "this was especially true for the i d and pdd groups [ (df = 9 ) = 41.9 , p < 0.001 ] . in the pimd and physical disability groups ,", "the parent 's poor socio - economic status and heavy care - giving burden , reflecting the child 's level of disability , were considered to be major factors in approximately one - third of the cases .", "signs of depression or a depressive state were the most common psychiatric disturbances seen in the parents . among the four groups , the pdd group most commonly experienced cases which showed signs of depression or a depressive state in parents .", "the proportion of such parents was approximately 70% of the total [ (df = 12 ) = 65.1 , p < 0.001 ] .", "well - treated cases were only 5% out of the total ; thus , most of these conditions were considered to be not medically well treated .", "for further analysis , we selected 159 cases where symptoms of depression or a depressive state were reported in a descriptive manner . according to these reports ,", "we asked about the time of the onset , that is , whether it was prior to the birth of the child , when the parent first noticed the child 's disability , or after the child had been fully medically diagnosed . apart from the answer ", "unknown  which accounted for approximately half of the cases ,  prior to the birth of the child  was relatively common in the pdd group ( 22.1% ) , whereas ", "after the child had been fully medically diagnosed  was relatively common in the pimd ( 23.5% ) and physical disability groups ( 28.6% ) .", "when a parent suffered from signs of psychiatric disturbances , more than half of the children experienced maltreatment .", "physical neglect was most common in all groups , accounting for about 50% of the cases . in comparison with the other groups , in the pdd group , the child was likely to be emotionally disturbed and confined indoors ( 32.6% ) , in the pimd group , the child was mostly confined indoors ( 33.3% ) , and in the physical disability group , medical care neglect made the utilization of medical and welfare support impossible ( 50% ) [ (df = 12 ) = 27.6 , p = 0.006 ] .", "the leading measures were gathering and disclosure of necessary information , discussion among service providers within the facility about ways to deal with the parent , protection of the child by providing a safe environment , making approaches to other family members for cooperation , intensive collaboration with other organizations such as institutions and consultation centers for better community support , and medical support for the parent provided by specialists such as physicians and psychologists .", "many factors were reported to affect the mental health of parents . the presence or absence of support from other family members (", "i.e. , a spouse ) and the existence ( or not ) of a medical condition in the parent were major factors ( table 5 ) .", "practical use ( or not ) of social support networks , whether the child 's level of disability was severe or not , and parent participation in face - to - face family support groups or not were also thought to be influential factors . in all groups , regardless of the child 's disability , more direct welfare support for the child was considered to be helpful and thus requested to be improved , in order to meet the mental health needs of the parent . besides this , in the physical disability group , family support groups , in the pimd group , support ( including financial support ) for other family members , and in the i d and pdd groups , support for other family members ( mainly siblings and the other parent ) and medical treatment of the parent 's psychiatric disturbance were also emphasized . to fulfill the demanded future role of their facility", ", respondents expressed the importance of more collaboration with other institutions and consultation centers and the necessity for more special training courses targeting support professionals and recognized the urgent demand for more specialists especially psychologists who can support the mental health needs of both the children and their parents ."], ["in the present study , we surveyed institutions and consultation centers for children with developmental disabilities in japan , which play a central role in the social support networks .", "this facility - based nationwide survey revealed the situation facing the parents of children with i d and other developmental disabilities from the professional caregivers ' point of view .", "almost all of the facilities experienced difficulties in keeping good relations with the clients ' parents , regardless of the children 's disabilities , and the majority of service providers noted that the proportion of such parents , out of the total , was roughly between 5 and 25% , and still increasing .", "the primary reason for these difficulties was thought to be the parent 's condition , such as their mental health and background .", "also , signs of depression or a depressive state were assumed to be the most common psychiatric disturbance seen .", "these results suggested that thorough consideration of the parents ' mental health needs , especially prevention of depressive conditions and support for parents suffering from them , is urgent - demanded task for facilities .", "i d , attention - deficit / hyperactivity disorder ( ad / hd ) , and pdd are common developmental disorders , and the latter two are noted to have substantial genetic components .", "it has been reported that a lifetime prevalence of major mood disorders is higher in parents of children with autism and that the onset for the majority was prior to the birth of their child with autism .", "family history studies of autism consistently uncovered a large subgroup with a high incidence of major mood disorders among family members , suggesting the two entities are related clinically and genetically [ 19 , 20 ] .", "the findings of the present survey must be interpreted allowing for the limitations of the use of unconfirmed data reported by professional caregivers ' who come into contact with children as their clients , and their parents on a day - to - day basis . evidently , the service providers ' perspective on parental mental health was consistent with previous reports from western countries on major mood disorders and pdd .", "in other words , facilities where the clients were diagnosed as having pdd experienced significantly more cases of parents with signs of a depressive state compared to other facilities , and the onset of depression was considered to be prior to the birth of the child in many of the cases . although these professional caregivers were well trained and well experienced , only , one - third were physicians , nurses or psychologists . on account of the limitations of these data ,", "we emphasize confirming the diagnoses of psychiatric disturbances through direct contact with the parents by specialists such as psychiatrists is necessary in future studies , for accurate understanding of the overall problem . in japan", ", there is no official system to check and document the parents ' condition including their mental health status .", "parents are not accustomed to being asked about their own mental health , so there is a tendency to avoid discussion on matters concerning themselves .", "this was the major reason why we asked the professional caregivers about the parents ' condition , not the parents directly , in this study .", "but still we judged it to be worthwhile at this moment to summarize the professional caregivers ' impression on this issue , since the case in japan has never been described in detail despite of its importance . because the majority of burden for daily care of handicapped children is typically carried by mothers , particular concern is raised for their adaptation . in the past studies , the psychosocial outcomes in mothers", "were said to be better predicted by psychosocial factors such as more active social life and family resources .", "recent research focusing on parenting stress illustrated the value of the participation of fathers also .", "greater marital quality predicted lower parenting stress for both mothers and fathers , while greater social support predicted increased parenting efficacy for fathers .", "although the relationship between child characteristics and parental well - being has not reached an invariable agreement , the severity of a child 's disability , intellectual functioning , and so forth are generally considered to be risk factors . since the burden for daily care is mainly carried by the mother , similarly in japan , service providers tend to be in contact with the mother more frequently than other family members", ". this may be partially the reason why service providers experienced difficulties in communicating with mostly the mothers in this study .", "the presence of support from other family members , such as a spouse , is considered to be a major influential factor for the mental health of parents in japan also .", "practical use of social support networks such as participation in face - to - face family support groups , more financial support for the household , more direct welfare support for the child , and medical treatment of the parent 's psychiatric disturbance were reported to be helpful and thus requested to be improved , in order to meet the mental health needs of the parent .", "we would like to emphasize the fact that although the importance of medical treatment for the parents ' psychiatric disturbances was pointed out , it was also realized that these conditions were presently not being medically well treated .", "this shows that current psychiatric services in japan are not adequate to meet the complex mental health needs of the parents appropriately . to resolve this challenging issue ,", "more intensive collaboration between other child - care facilities and organizations , aiming at the mental health needs of the parents , is demanded .", "the paucity of trained psychiatrists and other allied professionals also needs to be addressed , and in order to fulfill this , sufficient discussion and research on the establishment of an effective training system for specialists is warranted . providing direct advice for the parents on the utilization of social support networks is a reliable measure that can be immediately taken .", "training more professionals who can properly deal with the parents ' mental health needs is an urgent matter that must be tackled .", "overall , the present study is the first nationwide survey that examined the professional caregivers ' perspective on the mental health of parents of children with developmental disabilities , and it underscores the importance of understanding and supporting the parents ' mental health needs , which leads to more appropriate support for children with developmental disabilities ."]]}
{"article_id": "PMC2957965", "article_text": ["in may 2001 , 24 cases of s. typhimurium dt160 salmonellosis were reported in the auckland region compared with an average of four sporadic cases each month with this serotype in the previous 7 months .", "raw and undercooked egg consumption was commonly reported by the first 10 case - patients interviewed .", "a case - control study and environmental investigation were undertaken to identify the vehicle of infection and source of the outbreak .", "recognizing the potential for a widely dispersed foodborne outbreak , we expanded the investigation throughout new zealand .", "we defined a case as diarrhea ( 3 loose stools in a 24-hour period ) or vomiting after april 28 , 2001 , with a stool specimen positive for s. typhimurium dt160 .", "patients were excluded if they had a history of contact with another person with culture - confirmed s. typhimurium dt160 infection , or if they had a history of recent overseas travel . each case", "was matched with two controls found from randomly drawn telephone numbers , matching for neighborhood and age ( < 1 , 14 , 514 , > 14 years ) .", "the questionnaire covered symptoms ( patients only ) and contact with other symptomatic persons , bird or animal contact , and food consumption in the 3-day period before onset of illness ( cases and controls ) . parents or guardians were interviewed on behalf of children ages < 12 years .", "stepwise conditional logistic regression analyses were performed , also using sas , to identify the combination of variables that best explained the differences between case - participants and controls .", "samples from the drinking water supply of case - patients with a history of recent consumption of nonreticulated water were collected and tested for coliforms and s. enterica by using standard methods ( 6 ) .", "brands of eggs eaten raw within the incubation period were sampled at random from retail displays at the case - patients purchase site .", "eggshell surfaces and contents were tested with standard methods ( 7 ) . broken or cracked eggs were excluded from analysis .", "salmonella isolates were serotyped by using the kauffman - white scheme ( 8) and s. typhimurium isolates were phage typed by using the laboratory of enteric pathogens method ( 9 ) . from may", "to august 2001 , a total of 170 case - patients meeting the case definition were identified . of these , 119 ( 70% )", "agreed to participate and were enrolled in the study , along with 235 matched controls .", "the median age of case - patients was 8 years ( range 4 months to 90 years ) , and 57% were female .", "the most frequently reported symptoms were diarrhea ( 97% ) , abdominal pain ( 77% ) , excessive tiredness ( 67% ) , and fever ( 66% ) .", "the median duration of illness was 7 days ( range 144 days ) ; 17 ( 15% ) patients were hospitalized , and none died .", "case - patients and controls did not differ significantly according to age , sex , immunosuppressive therapy , treatment to reduce gastric acidity , or use of antibiotics .", "all s. typhimurium dt160 isolates were sensitive to ampicillin , cephalothin , chloramphenicol , ciprofloxacin , co - trimoxazole , gentamicin , streptomycin , sulfonamides , tetracycline , and trimethoprim .", "four represented different levels of contact with other persons with gastrointestinal illness ( i.e. , within 28 days of illness onset ; within 3 days of onset ; within the household ; or outside the household ) . direct handling of dead wild birds , consumption of fast food , and consumption of food at a large gathering , such as at a party or large barbecue , were also significantly associated with illness .", "after stepwise regression , contact with a person with gastrointestinal illness in the 28 days before onset of illness in the case - patient ( adjusted odds ratio [ or ] 2.8 ; 95% confidence interval [ ci ] 1.4 to 5.4 ) , exposure to dead wild birds ( adjusted or 10.5 ; 95% ci 2.3 to 47.5 ) , and consumption of fast food ( adjusted or 1.7 ; 95% ci 1.0 to 2.9 ) had independent significant associations with illness .", "dt , definitive type ; d&v , diarrhea and vomiting ; or , odds ratio ; ci , confidence interval .", "twelve case - patients throughout new zealand indicated that they had drunk water from nonreticulated and untreated water sources .", "seven patients used roof - collected rainwater , and one used rainwater plus water from a dam .", "four of the five patients who had eaten raw eggs could identify the retail brand and outlet of purchase .", "these four patients had purchased six different brands of eggs from seven different retail outlets .", "samples for two brands were positive for s. thompson , both from shell surface washings .", "epidemiologic investigation of an outbreak of s. typhimurium dt160 infection in new from may to august 2001 found that contact with dead wild birds , contact with other persons with gastrointestinal illness , and consumption of fast food were all significantly associated with illness .", "in addition , s. typhimurium dt160 was found in roof - collected rainwater drunk by five patients .", "s. typhimurium dt160 had been previously identified as the cause of large numbers of sparrow deaths in new zealand in 2000 , and analysis by pulsed - field gel electrophoresis ( using the method described by barrett et al", ".   and restriction enzyme xbai ) demonstrated that bird and human isolates in 2000 were indistinguishable ( 4 ) . in our study , information was not collected on exposure to environments contaminated by wild bird feces , such as parks and play areas , a fact that may have underestimated the avian contribution to human illness .", "s. typhimurium dt160 has previously been recognized as a bird pathogen in canada ( 11 ) and in england ( 12 ) . before its emergence in new zealand , the human s. typhimurium", "dt160 infection had only been reported in the context of a 1979 institutional outbreak in the united kingdom , linked to food contamination by sparrow droppings ( 13 ) .", "consumption of undisinfected water has previously been identified as a risk factor for salmonellosis linked to bird transmission ( 14 ) .", "this risk factor was not confirmed by our case - control study , despite the finding of s. typhimurium dt160 in roof - collected rainwater .", "this discrepancy is probably because case - patients and controls were matched by neighborhood , and types of water sources are usually consistent within neighborhoods .", "the association of illness with contact with another person with gastrointestinal illness is likely underestimated because secondary salmonellosis cases were excluded .", "consumption of fast food was associated with illness ; however , no single type of food outlet or food was identified .", "case - patients were equally likely to have eaten food from chain fast - food restaurants as from family - owned fast - food outlets .", "consumption of fast food may have occurred in environments contaminated by bird feces , or the foods themselves may have been contaminated , either during production or by infected foodhandlers ( 15 ) .", "asymptomatic salmonella carriers would not have been excluded from selection as controls , potentially reducing the magnitude of observed associations .", "recall may have been influenced by delays between exposure and interview , although participants were asked to refer to a memory aid ( personal diary or calendar ) .", "the investigation successfully excluded a single common source exposure for this outbreak and instead suggested that multiple exposures contribute to s. typhimurium dt160 infections in new zealand .", "strategies for addressing these exposures include routine treatment of roof - collected rainwater , hygienic disposal of dead birds , and promotion of hand - hygiene protocols and sick foodhandler policies in fast - food outlets .", "the source of this incursion of s. typhimurium dt160 into new zealand remains unknown : bird isolates have been exclusively from nonmigratory birds , s. typhimurium dt160 has not been identified in neighboring countries in the pacific basin , and early case - patients did not have a history of overseas travel ."], "abstract_text": ["<S> an outbreak of human salmonella enterica serotype typhimurium dt160 infection in new zealand was investigated from may to august 2001 . </S>", "<S> handling of dead wild birds , contact with persons with diarrheal illness , and consumption of fast food were associated with infection . </S>", "<S> contaminated roof - collected rainwater was also detected . </S>"], "labels": null, "section_names": ["The Study", "Conclusions"], "sections": [["in may 2001 , 24 cases of s. typhimurium dt160 salmonellosis were reported in the auckland region compared with an average of four sporadic cases each month with this serotype in the previous 7 months .", "raw and undercooked egg consumption was commonly reported by the first 10 case - patients interviewed .", "a case - control study and environmental investigation were undertaken to identify the vehicle of infection and source of the outbreak .", "recognizing the potential for a widely dispersed foodborne outbreak , we expanded the investigation throughout new zealand .", "we defined a case as diarrhea ( 3 loose stools in a 24-hour period ) or vomiting after april 28 , 2001 , with a stool specimen positive for s. typhimurium dt160 .", "patients were excluded if they had a history of contact with another person with culture - confirmed s. typhimurium dt160 infection , or if they had a history of recent overseas travel . each case", "was matched with two controls found from randomly drawn telephone numbers , matching for neighborhood and age ( < 1 , 14 , 514 , > 14 years ) .", "the questionnaire covered symptoms ( patients only ) and contact with other symptomatic persons , bird or animal contact , and food consumption in the 3-day period before onset of illness ( cases and controls ) . parents or guardians were interviewed on behalf of children ages < 12 years .", "stepwise conditional logistic regression analyses were performed , also using sas , to identify the combination of variables that best explained the differences between case - participants and controls .", "samples from the drinking water supply of case - patients with a history of recent consumption of nonreticulated water were collected and tested for coliforms and s. enterica by using standard methods ( 6 ) .", "brands of eggs eaten raw within the incubation period were sampled at random from retail displays at the case - patients purchase site .", "eggshell surfaces and contents were tested with standard methods ( 7 ) . broken or cracked eggs were excluded from analysis .", "salmonella isolates were serotyped by using the kauffman - white scheme ( 8) and s. typhimurium isolates were phage typed by using the laboratory of enteric pathogens method ( 9 ) . from may", "to august 2001 , a total of 170 case - patients meeting the case definition were identified . of these , 119 ( 70% )", "agreed to participate and were enrolled in the study , along with 235 matched controls .", "the median age of case - patients was 8 years ( range 4 months to 90 years ) , and 57% were female .", "the most frequently reported symptoms were diarrhea ( 97% ) , abdominal pain ( 77% ) , excessive tiredness ( 67% ) , and fever ( 66% ) .", "the median duration of illness was 7 days ( range 144 days ) ; 17 ( 15% ) patients were hospitalized , and none died .", "case - patients and controls did not differ significantly according to age , sex , immunosuppressive therapy , treatment to reduce gastric acidity , or use of antibiotics .", "all s. typhimurium dt160 isolates were sensitive to ampicillin , cephalothin , chloramphenicol , ciprofloxacin , co - trimoxazole , gentamicin , streptomycin , sulfonamides , tetracycline , and trimethoprim .", "four represented different levels of contact with other persons with gastrointestinal illness ( i.e. , within 28 days of illness onset ; within 3 days of onset ; within the household ; or outside the household ) . direct handling of dead wild birds , consumption of fast food , and consumption of food at a large gathering , such as at a party or large barbecue , were also significantly associated with illness .", "after stepwise regression , contact with a person with gastrointestinal illness in the 28 days before onset of illness in the case - patient ( adjusted odds ratio [ or ] 2.8 ; 95% confidence interval [ ci ] 1.4 to 5.4 ) , exposure to dead wild birds ( adjusted or 10.5 ; 95% ci 2.3 to 47.5 ) , and consumption of fast food ( adjusted or 1.7 ; 95% ci 1.0 to 2.9 ) had independent significant associations with illness .", "dt , definitive type ; d&v , diarrhea and vomiting ; or , odds ratio ; ci , confidence interval .", "twelve case - patients throughout new zealand indicated that they had drunk water from nonreticulated and untreated water sources .", "seven patients used roof - collected rainwater , and one used rainwater plus water from a dam .", "four of the five patients who had eaten raw eggs could identify the retail brand and outlet of purchase .", "these four patients had purchased six different brands of eggs from seven different retail outlets .", "samples for two brands were positive for s. thompson , both from shell surface washings ."], ["epidemiologic investigation of an outbreak of s. typhimurium dt160 infection in new from may to august 2001 found that contact with dead wild birds , contact with other persons with gastrointestinal illness , and consumption of fast food were all significantly associated with illness .", "in addition , s. typhimurium dt160 was found in roof - collected rainwater drunk by five patients .", "s. typhimurium dt160 had been previously identified as the cause of large numbers of sparrow deaths in new zealand in 2000 , and analysis by pulsed - field gel electrophoresis ( using the method described by barrett et al", ".   and restriction enzyme xbai ) demonstrated that bird and human isolates in 2000 were indistinguishable ( 4 ) . in our study , information was not collected on exposure to environments contaminated by wild bird feces , such as parks and play areas , a fact that may have underestimated the avian contribution to human illness .", "s. typhimurium dt160 has previously been recognized as a bird pathogen in canada ( 11 ) and in england ( 12 ) . before its emergence in new zealand , the human s. typhimurium", "dt160 infection had only been reported in the context of a 1979 institutional outbreak in the united kingdom , linked to food contamination by sparrow droppings ( 13 ) .", "consumption of undisinfected water has previously been identified as a risk factor for salmonellosis linked to bird transmission ( 14 ) .", "this risk factor was not confirmed by our case - control study , despite the finding of s. typhimurium dt160 in roof - collected rainwater .", "this discrepancy is probably because case - patients and controls were matched by neighborhood , and types of water sources are usually consistent within neighborhoods .", "the association of illness with contact with another person with gastrointestinal illness is likely underestimated because secondary salmonellosis cases were excluded .", "consumption of fast food was associated with illness ; however , no single type of food outlet or food was identified .", "case - patients were equally likely to have eaten food from chain fast - food restaurants as from family - owned fast - food outlets .", "consumption of fast food may have occurred in environments contaminated by bird feces , or the foods themselves may have been contaminated , either during production or by infected foodhandlers ( 15 ) .", "asymptomatic salmonella carriers would not have been excluded from selection as controls , potentially reducing the magnitude of observed associations .", "recall may have been influenced by delays between exposure and interview , although participants were asked to refer to a memory aid ( personal diary or calendar ) .", "the investigation successfully excluded a single common source exposure for this outbreak and instead suggested that multiple exposures contribute to s. typhimurium dt160 infections in new zealand .", "strategies for addressing these exposures include routine treatment of roof - collected rainwater , hygienic disposal of dead birds , and promotion of hand - hygiene protocols and sick foodhandler policies in fast - food outlets .", "the source of this incursion of s. typhimurium dt160 into new zealand remains unknown : bird isolates have been exclusively from nonmigratory birds , s. typhimurium dt160 has not been identified in neighboring countries in the pacific basin , and early case - patients did not have a history of overseas travel ."]]}
{"article_id": "PMC3757268", "article_text": ["non - crystallographic symmetry ( ncs ) is common in macromolecular crystals , occurring in about 1/3 or more of structures in the protein data bank [ 2 , 3 , 24 ] . though non - crystallographic symmetry increases the complexity of structure determination , in most cases it yields a distinct advantage in this process because it brings with it information on relationships between density in different parts of the crystal and between coordinates in different parts of the structure [ 4 , 1214 ] .", "these relationships greatly improve density modification and refinement , two key steps in structure determination .", "one is simple examination of a model that has more than one copy of a chain .", "it can be used after molecular replacement has been carried out or after a model has been built .", "another approach is to find symmetry relationships among subsets of atoms in a heavy - atom or anomalously scattering atom substructure [ 11 , 17 ] .", "these symmetry relationships often reflect non - crystallographic symmetry , and the presence of the ncs can be checked by comparison of the density in the resulting electron density map at ncs - related positions and orientations .", "this approach can be used near the beginning of a sad , mad , or other structure determination where a substructure is obtained prior to full structure determination .", "this approach may be difficult to apply if there are only one or two atoms per ncs copy in the substructure and the ncs does not have point - group symmetry .", "this is because the relative orientations of the ncs - related parts of the substructure are not known in this case .", "a third approach that can be applied in cases where proper ncs ( e.g. , a two - fold axis ) is present is to search for locations in a map where nearby points related by proper symmetry have similar density .", "a fourth and very general approach to finding non - crystallographic symmetry is to find patterns of density that are present in more than one part of an electron density map .", "used a step - wise procedure of first finding possible c locations with pattern - matching techniques , classifying the locations with rotation - invariant classifiers , and then finding pairs of these locations that have similar patterns of density surrounding them .", "this method can be fast and effective , but has the potential limitations that the map has to be of high enough quality to locate c positions and that for maximal speed the patterns of density need to be initially represented in a rotation - invariant fashion . here", "we present an approach to finding non - crystallographic symmetry by directly searching for patterns of density that are present in more than one place in a map .", "this approach uses superposition of a cut - out region of the map with the remainder of the map , and therefore can make use of all the local features in a map , not just rotation - invariant ones . in this approach , an fft - based convolution search [ 5 , 16 ]", "is used to find orientations and translations that relate parts of a map to other parts of a map .", "we find that this is a rapid and accurate way to identify ncs , even in a case where the map is poor and the correlation of ncs - related density is low .", "the approach used here to identify non - crystallographic symmetry from an electron density map has three basic parts .", "first , a location in the map is found that is likely to be inside the molecule .", "this is important as it is the local symmetry of the molecule or molecules in the crystal that are of interest , so considering a region inside the molecule is essential for success .", "the method used to identify such a location in the map is similar to a method commonly used to distinguish macromolecule from solvent in a map . for each grid point in the electron density map , the local rms variation in electron density is calculated .", "this is done by simply calculating the standard deviation of the density in the map inside a sphere with radius r ( typically 10   ) , centered at the grid point in question .", "then the grid point in the map with the highest local standard deviation of density is considered the best candidate for a position inside the macromolecule .", "additional points likely to be inside the macromolecule can optionally be found by choosing additional grid points with high local variation in density that are well separated ( typically at least 15   ) from previously chosen locations .", "next , a search for ncs relating the density near the candidate point inside the macromolecule with density elsewhere in the asymmetric unit of the crystal is carried out . to make the calculation rapid", ", the resolution of the map used for this calculation may be chosen to be lower than the resolution of the original map ( typically a resolution of 4   is used )", ". a sphere of density ( typically with a radius of 10   ) is cut out of the map , centered at this position within the molecule .", "this sphere of density is the object that is to be compared with density everywhere in the map .", "possible rotations of this sphere of density are sampled ( typically each rotation differing by about 20 degrees from all others ) . for each rotation ,", "the density is transformed to the new orientation , and an fft - based convolution search [ 5 , 16 ] is used to identify non - crystallographic translations that yield a high correlation of density .", "these rotation / translation pairs that yield high correlation ( typically all those at or above 75  % of the highest value found ) represent a possible set of ncs operators relating the various copies of the molecule in the asymmetric unit of the unit cell .", "additionally , the candidate point used to identify ncs and the ncs - related points in the map may be considered approximate centers of regions where ncs applies . they are used below as the starting point for identification of the region where ncs operators apply .", "once a set of candidate ncs operators is obtained , the correlation of ncs - related density is examined in the original map , and the ncs operators are refined .", "this is done using the same approach as has been applied to ncs operators obtained from heavy - atom positions .", "first the shape and position of the local region that is repeated by ncs ( the asymmetric unit of ncs ) is found .", "then the ncs operators are refined to maximize the correlation of density among these ncs - related regions .", "the region where ncs applies is assumed to contain the point identified in the first step of our procedure that is within the macromolecule .", "a region with arbitrary shape surrounding this point is then chosen by sequential addition of new boundary points such that the local correlation of ncs - related density at each point in the region is above a threshold ( typically 1/4 of the maximum correlation in the region ) .", "the local correlation of density   is calculated using map grid points within a sphere ( normally with the radius used for identification of the solvent boundary , terwilliger 2000b ) , centered at the point in question .", "if the final correlation of ncs - related density ( averaged over all pairs of ncs - related points ) is above a threshold ( typically 0.4 ) then the ncs operators are considered to represent actual ncs in the crystal and are used in later stages of structure determination . in cases where the starting map is very poor ,", "this threshold may be decreased , increasing the sensitivity of the procedure but also increasing the probability of finding ncs where none is present .", "the approach used here to identify non - crystallographic symmetry from an electron density map has three basic parts .", "first , a location in the map is found that is likely to be inside the molecule .", "this is important as it is the local symmetry of the molecule or molecules in the crystal that are of interest , so considering a region inside the molecule is essential for success .", "the method used to identify such a location in the map is similar to a method commonly used to distinguish macromolecule from solvent in a map . for each grid point in the electron density map , the local rms variation in electron density is calculated .", "this is done by simply calculating the standard deviation of the density in the map inside a sphere with radius r ( typically 10   ) , centered at the grid point in question .", "then the grid point in the map with the highest local standard deviation of density is considered the best candidate for a position inside the macromolecule .", "additional points likely to be inside the macromolecule can optionally be found by choosing additional grid points with high local variation in density that are well separated ( typically at least 15   ) from previously chosen locations .", "next , a search for ncs relating the density near the candidate point inside the macromolecule with density elsewhere in the asymmetric unit of the crystal is carried out . to make the calculation rapid ,", "the resolution of the map used for this calculation may be chosen to be lower than the resolution of the original map ( typically a resolution of 4   is used ) .", "a sphere of density ( typically with a radius of 10   ) is cut out of the map , centered at this position within the molecule .", "this sphere of density is the object that is to be compared with density everywhere in the map .", "possible rotations of this sphere of density are sampled ( typically each rotation differing by about 20 degrees from all others ) . for each rotation ,", "the density is transformed to the new orientation , and an fft - based convolution search [ 5 , 16 ] is used to identify non - crystallographic translations that yield a high correlation of density .", "these rotation / translation pairs that yield high correlation ( typically all those at or above 75  % of the highest value found ) represent a possible set of ncs operators relating the various copies of the molecule in the asymmetric unit of the unit cell . additionally , the candidate point used to identify ncs and the ncs - related points in the map may be considered approximate centers of regions where ncs applies .", "they are used below as the starting point for identification of the region where ncs operators apply .", "once a set of candidate ncs operators is obtained , the correlation of ncs - related density is examined in the original map , and the ncs operators are refined .", "this is done using the same approach as has been applied to ncs operators obtained from heavy - atom positions .", "first the shape and position of the local region that is repeated by ncs ( the asymmetric unit of ncs ) is found .", "then the ncs operators are refined to maximize the correlation of density among these ncs - related regions .", "the region where ncs applies is assumed to contain the point identified in the first step of our procedure that is within the macromolecule .", "a region with arbitrary shape surrounding this point is then chosen by sequential addition of new boundary points such that the local correlation of ncs - related density at each point in the region is above a threshold ( typically 1/4 of the maximum correlation in the region ) .", "the local correlation of density   is calculated using map grid points within a sphere ( normally with the radius used for identification of the solvent boundary , terwilliger 2000b ) , centered at the point in question .", "if the final correlation of ncs - related density ( averaged over all pairs of ncs - related points ) is above a threshold ( typically 0.4 ) then the ncs operators are considered to represent actual ncs in the crystal and are used in later stages of structure determination . in cases where the starting map is very poor ,", "this threshold may be decreased , increasing the sensitivity of the procedure but also increasing the probability of finding ncs where none is present .", "we applied our procedure to finding non - crystallographic symmetry to a structure where an electron density map of moderate quality could be obtained using mad phasing .", "this structure ( gere , pdb entry 1fse , ) consists of 6 copies of a protein chain with 74 residues . figure  1a shows a schematic of the protein structure .", "figure  1b , c illustrates the mad - phased electron density map for this structure for corresponding views of chains c and f , respectively , without any density modification or including any information on ncs .", "it may be seen that this map is of moderate quality in the region of chain c and rather poorer quality in the region of chain f.fig .  1 \n a ribbon view of 6 chains of gere in crystal structure pdb entry 1fse .", "b view of experimental mad - phased , non density - modified electron density at a resolution of 2.7   near chain c of gere .", "the high density - variation location identified by phenix.guess_molecular_centers is marked , and circle with radius approximately 10   centered at this point illustrates the region that is to be cut out and compared to other density in the map .", "contours for b  d are at 1.5. c as in b , except view is near chain f of gere , shown in grey .", "d view as in b and c , except that the averaged density based on all 6 ncs - related copies is shown .", "a created with pymol ; b  d created with coot  \n a ribbon view of 6 chains of gere in crystal structure pdb entry 1fse . b view of experimental mad - phased , non density - modified electron density at a resolution of 2.7   near chain c of gere .", "the high density - variation location identified by phenix.guess_molecular_centers is marked , and circle with radius approximately 10   centered at this point illustrates the region that is to be cut out and compared to other density in the map .", "contours for b  d are at 1.5. c as in b , except view is near chain f of gere , shown in grey .", "d view as in b and c , except that the averaged density based on all 6 ncs - related copies is shown .", "a created with pymol ; b  d created with coot   the phenix   tool phenix.find_ncs_from-_density was used to carry out the ncs identification .", "the density map was examined to find locations where the local variation of density was highest using the method phenix.guess_molecular_centers .", "it can be seen that this point is well within the region of the map with protein - like characteristics ( relatively connected and high density ) .", "next , after truncating the resolution of the map to 4   , test density corresponding to a sphere of radius 10   of density centered at the point marked in fig . ", "1b was cut out ( the region inside the circle in fig .  1b , except for the change in resolution of the map ) .", "the density within all 10   spherical regions in the 4   map was then compared to this test density using an fft - based method , sampling all possible rotations of the test density on a grid .", "this density correlation procedure correctly identified all 5 regions that had high similarity to the test density .", "figure  1c shows one of these related regions of density , the one corresponding to chain f of gere , in the same view as fig . ", "it may be seen that within the 10   sphere the density in fig . ", "1b , c are similar , though the density near chain f ( fig . ", "the shape of the density becomes less similar , as expected because the protein chains surrounding the chain located outside the sphere in fig . ", "1b have a different arrangement than those outside the sphere in fig .  1c .", "1c and the other 4 locations within the asymmetric unit of the density in this crystal were then refined to identify the region over which correlation of density existed and to optimize the correlation of density among all copies of this repeated density .", "this process yields as well the transformation matrices describing the relationships among all the ncs - related copies in the asymmetric unit of the crystal .", "the final average pair - wise correlation of density among the 6 copies was 0.53 .", "1b ) the average correlation to other ncs - related regions was 0.56 , while for chain f ( fig . ", "figure  1d shows the average density for all the regions of density that were found to be similar from the same viewpoint as shown in fig . ", "it can be seen that this density has features even more like those expected of a density map of a protein than the individual regions of density ( the tubes of density are better - connected ) .", "the approach described here for finding ncs in a map can be used in a number of situations . in the phenix crystallographic software ,", "the method phenix.find_ncs_from_density is used during structure solution with phenix.autosol   if ncs is expected but can not be found from heavy - atom sites , and it is used during iterative model - building with phenix.autobuild   any time it is expected and has not been identified by any other means .", "the ncs relationships identified in this way can be used as a powerful addition to the information available for density modification [ 4 , 1214 ] .", "these relationships can also be used in model - building , where an incompletely built chain can be built using a more complete ncs - related copy of the same chain as a template [ 9 , 15 , 20 ] .", "further they could potentially be used to place copies of entire chains in low - resolution maps .", "the maps to be analyzed for ncs can be crystallographic maps , maps from electron microscopy , or maps from other sources .", "the approach described here could also be used to find density that is similar in two different electron density maps .", "this last application could be useful in automatically finding relationships between molecules in different crystals when carrying out cross - crystal averaging ."], "abstract_text": ["<S> the internal symmetry of a macromolecule is both an important aspect of its function and a useful feature in obtaining a structure by x - ray crystallography and other techniques . </S>", "<S> a method is presented for finding internal symmetry and other non - crystallographic symmetry in a structure based on patterns of density in a density map for that structure . </S>", "<S> regions in map that are similar are identified by cutting out a sphere of density from a region that has high local variation and using an fft - based correlation search to find other regions that match . </S>", "<S> the relationships among correlated regions are then refined to maximize their correlations and are found to accurately represent non - crystallographic symmetry in the map . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Locating a region that is part of the macromolecule", "Searching for non-crystallographic symmetry", "Evaluating the correlation of NCS-related density", "Results and discussion", "Conclusions"], "sections": [["non - crystallographic symmetry ( ncs ) is common in macromolecular crystals , occurring in about 1/3 or more of structures in the protein data bank [ 2 , 3 , 24 ] . though non - crystallographic symmetry increases the complexity of structure determination , in most cases it yields a distinct advantage in this process because it brings with it information on relationships between density in different parts of the crystal and between coordinates in different parts of the structure [ 4 , 1214 ] .", "these relationships greatly improve density modification and refinement , two key steps in structure determination .", "one is simple examination of a model that has more than one copy of a chain .", "it can be used after molecular replacement has been carried out or after a model has been built .", "another approach is to find symmetry relationships among subsets of atoms in a heavy - atom or anomalously scattering atom substructure [ 11 , 17 ] .", "these symmetry relationships often reflect non - crystallographic symmetry , and the presence of the ncs can be checked by comparison of the density in the resulting electron density map at ncs - related positions and orientations .", "this approach can be used near the beginning of a sad , mad , or other structure determination where a substructure is obtained prior to full structure determination .", "this approach may be difficult to apply if there are only one or two atoms per ncs copy in the substructure and the ncs does not have point - group symmetry .", "this is because the relative orientations of the ncs - related parts of the substructure are not known in this case .", "a third approach that can be applied in cases where proper ncs ( e.g. , a two - fold axis ) is present is to search for locations in a map where nearby points related by proper symmetry have similar density .", "a fourth and very general approach to finding non - crystallographic symmetry is to find patterns of density that are present in more than one part of an electron density map .", "used a step - wise procedure of first finding possible c locations with pattern - matching techniques , classifying the locations with rotation - invariant classifiers , and then finding pairs of these locations that have similar patterns of density surrounding them .", "this method can be fast and effective , but has the potential limitations that the map has to be of high enough quality to locate c positions and that for maximal speed the patterns of density need to be initially represented in a rotation - invariant fashion . here", "we present an approach to finding non - crystallographic symmetry by directly searching for patterns of density that are present in more than one place in a map .", "this approach uses superposition of a cut - out region of the map with the remainder of the map , and therefore can make use of all the local features in a map , not just rotation - invariant ones . in this approach , an fft - based convolution search [ 5 , 16 ]", "is used to find orientations and translations that relate parts of a map to other parts of a map .", "we find that this is a rapid and accurate way to identify ncs , even in a case where the map is poor and the correlation of ncs - related density is low ."], ["the approach used here to identify non - crystallographic symmetry from an electron density map has three basic parts .", "first , a location in the map is found that is likely to be inside the molecule .", "this is important as it is the local symmetry of the molecule or molecules in the crystal that are of interest , so considering a region inside the molecule is essential for success .", "the method used to identify such a location in the map is similar to a method commonly used to distinguish macromolecule from solvent in a map . for each grid point in the electron density map , the local rms variation in electron density is calculated .", "this is done by simply calculating the standard deviation of the density in the map inside a sphere with radius r ( typically 10   ) , centered at the grid point in question .", "then the grid point in the map with the highest local standard deviation of density is considered the best candidate for a position inside the macromolecule .", "additional points likely to be inside the macromolecule can optionally be found by choosing additional grid points with high local variation in density that are well separated ( typically at least 15   ) from previously chosen locations .", "next , a search for ncs relating the density near the candidate point inside the macromolecule with density elsewhere in the asymmetric unit of the crystal is carried out . to make the calculation rapid", ", the resolution of the map used for this calculation may be chosen to be lower than the resolution of the original map ( typically a resolution of 4   is used )", ". a sphere of density ( typically with a radius of 10   ) is cut out of the map , centered at this position within the molecule .", "this sphere of density is the object that is to be compared with density everywhere in the map .", "possible rotations of this sphere of density are sampled ( typically each rotation differing by about 20 degrees from all others ) . for each rotation ,", "the density is transformed to the new orientation , and an fft - based convolution search [ 5 , 16 ] is used to identify non - crystallographic translations that yield a high correlation of density .", "these rotation / translation pairs that yield high correlation ( typically all those at or above 75  % of the highest value found ) represent a possible set of ncs operators relating the various copies of the molecule in the asymmetric unit of the unit cell .", "additionally , the candidate point used to identify ncs and the ncs - related points in the map may be considered approximate centers of regions where ncs applies . they are used below as the starting point for identification of the region where ncs operators apply .", "once a set of candidate ncs operators is obtained , the correlation of ncs - related density is examined in the original map , and the ncs operators are refined .", "this is done using the same approach as has been applied to ncs operators obtained from heavy - atom positions .", "first the shape and position of the local region that is repeated by ncs ( the asymmetric unit of ncs ) is found .", "then the ncs operators are refined to maximize the correlation of density among these ncs - related regions .", "the region where ncs applies is assumed to contain the point identified in the first step of our procedure that is within the macromolecule .", "a region with arbitrary shape surrounding this point is then chosen by sequential addition of new boundary points such that the local correlation of ncs - related density at each point in the region is above a threshold ( typically 1/4 of the maximum correlation in the region ) .", "the local correlation of density   is calculated using map grid points within a sphere ( normally with the radius used for identification of the solvent boundary , terwilliger 2000b ) , centered at the point in question .", "if the final correlation of ncs - related density ( averaged over all pairs of ncs - related points ) is above a threshold ( typically 0.4 ) then the ncs operators are considered to represent actual ncs in the crystal and are used in later stages of structure determination . in cases where the starting map is very poor ,", "this threshold may be decreased , increasing the sensitivity of the procedure but also increasing the probability of finding ncs where none is present ."], ["the approach used here to identify non - crystallographic symmetry from an electron density map has three basic parts .", "first , a location in the map is found that is likely to be inside the molecule .", "this is important as it is the local symmetry of the molecule or molecules in the crystal that are of interest , so considering a region inside the molecule is essential for success .", "the method used to identify such a location in the map is similar to a method commonly used to distinguish macromolecule from solvent in a map . for each grid point in the electron density map , the local rms variation in electron density is calculated .", "this is done by simply calculating the standard deviation of the density in the map inside a sphere with radius r ( typically 10   ) , centered at the grid point in question .", "then the grid point in the map with the highest local standard deviation of density is considered the best candidate for a position inside the macromolecule .", "additional points likely to be inside the macromolecule can optionally be found by choosing additional grid points with high local variation in density that are well separated ( typically at least 15   ) from previously chosen locations ."], ["next , a search for ncs relating the density near the candidate point inside the macromolecule with density elsewhere in the asymmetric unit of the crystal is carried out . to make the calculation rapid ,", "the resolution of the map used for this calculation may be chosen to be lower than the resolution of the original map ( typically a resolution of 4   is used ) .", "a sphere of density ( typically with a radius of 10   ) is cut out of the map , centered at this position within the molecule .", "this sphere of density is the object that is to be compared with density everywhere in the map .", "possible rotations of this sphere of density are sampled ( typically each rotation differing by about 20 degrees from all others ) . for each rotation ,", "the density is transformed to the new orientation , and an fft - based convolution search [ 5 , 16 ] is used to identify non - crystallographic translations that yield a high correlation of density .", "these rotation / translation pairs that yield high correlation ( typically all those at or above 75  % of the highest value found ) represent a possible set of ncs operators relating the various copies of the molecule in the asymmetric unit of the unit cell . additionally , the candidate point used to identify ncs and the ncs - related points in the map may be considered approximate centers of regions where ncs applies .", "they are used below as the starting point for identification of the region where ncs operators apply ."], ["once a set of candidate ncs operators is obtained , the correlation of ncs - related density is examined in the original map , and the ncs operators are refined .", "this is done using the same approach as has been applied to ncs operators obtained from heavy - atom positions .", "first the shape and position of the local region that is repeated by ncs ( the asymmetric unit of ncs ) is found .", "then the ncs operators are refined to maximize the correlation of density among these ncs - related regions .", "the region where ncs applies is assumed to contain the point identified in the first step of our procedure that is within the macromolecule .", "a region with arbitrary shape surrounding this point is then chosen by sequential addition of new boundary points such that the local correlation of ncs - related density at each point in the region is above a threshold ( typically 1/4 of the maximum correlation in the region ) .", "the local correlation of density   is calculated using map grid points within a sphere ( normally with the radius used for identification of the solvent boundary , terwilliger 2000b ) , centered at the point in question .", "if the final correlation of ncs - related density ( averaged over all pairs of ncs - related points ) is above a threshold ( typically 0.4 ) then the ncs operators are considered to represent actual ncs in the crystal and are used in later stages of structure determination . in cases where the starting map is very poor ,", "this threshold may be decreased , increasing the sensitivity of the procedure but also increasing the probability of finding ncs where none is present ."], ["we applied our procedure to finding non - crystallographic symmetry to a structure where an electron density map of moderate quality could be obtained using mad phasing .", "this structure ( gere , pdb entry 1fse , ) consists of 6 copies of a protein chain with 74 residues . figure  1a shows a schematic of the protein structure .", "figure  1b , c illustrates the mad - phased electron density map for this structure for corresponding views of chains c and f , respectively , without any density modification or including any information on ncs .", "it may be seen that this map is of moderate quality in the region of chain c and rather poorer quality in the region of chain f.fig .  1 \n a ribbon view of 6 chains of gere in crystal structure pdb entry 1fse .", "b view of experimental mad - phased , non density - modified electron density at a resolution of 2.7   near chain c of gere .", "the high density - variation location identified by phenix.guess_molecular_centers is marked , and circle with radius approximately 10   centered at this point illustrates the region that is to be cut out and compared to other density in the map .", "contours for b  d are at 1.5. c as in b , except view is near chain f of gere , shown in grey .", "d view as in b and c , except that the averaged density based on all 6 ncs - related copies is shown .", "a created with pymol ; b  d created with coot  \n a ribbon view of 6 chains of gere in crystal structure pdb entry 1fse . b view of experimental mad - phased , non density - modified electron density at a resolution of 2.7   near chain c of gere .", "the high density - variation location identified by phenix.guess_molecular_centers is marked , and circle with radius approximately 10   centered at this point illustrates the region that is to be cut out and compared to other density in the map .", "contours for b  d are at 1.5. c as in b , except view is near chain f of gere , shown in grey .", "d view as in b and c , except that the averaged density based on all 6 ncs - related copies is shown .", "a created with pymol ; b  d created with coot   the phenix   tool phenix.find_ncs_from-_density was used to carry out the ncs identification .", "the density map was examined to find locations where the local variation of density was highest using the method phenix.guess_molecular_centers .", "it can be seen that this point is well within the region of the map with protein - like characteristics ( relatively connected and high density ) .", "next , after truncating the resolution of the map to 4   , test density corresponding to a sphere of radius 10   of density centered at the point marked in fig . ", "1b was cut out ( the region inside the circle in fig .  1b , except for the change in resolution of the map ) .", "the density within all 10   spherical regions in the 4   map was then compared to this test density using an fft - based method , sampling all possible rotations of the test density on a grid .", "this density correlation procedure correctly identified all 5 regions that had high similarity to the test density .", "figure  1c shows one of these related regions of density , the one corresponding to chain f of gere , in the same view as fig . ", "it may be seen that within the 10   sphere the density in fig . ", "1b , c are similar , though the density near chain f ( fig . ", "the shape of the density becomes less similar , as expected because the protein chains surrounding the chain located outside the sphere in fig . ", "1b have a different arrangement than those outside the sphere in fig .  1c .", "1c and the other 4 locations within the asymmetric unit of the density in this crystal were then refined to identify the region over which correlation of density existed and to optimize the correlation of density among all copies of this repeated density .", "this process yields as well the transformation matrices describing the relationships among all the ncs - related copies in the asymmetric unit of the crystal .", "the final average pair - wise correlation of density among the 6 copies was 0.53 .", "1b ) the average correlation to other ncs - related regions was 0.56 , while for chain f ( fig . ", "figure  1d shows the average density for all the regions of density that were found to be similar from the same viewpoint as shown in fig . ", "it can be seen that this density has features even more like those expected of a density map of a protein than the individual regions of density ( the tubes of density are better - connected ) ."], ["the approach described here for finding ncs in a map can be used in a number of situations . in the phenix crystallographic software ,", "the method phenix.find_ncs_from_density is used during structure solution with phenix.autosol   if ncs is expected but can not be found from heavy - atom sites , and it is used during iterative model - building with phenix.autobuild   any time it is expected and has not been identified by any other means .", "the ncs relationships identified in this way can be used as a powerful addition to the information available for density modification [ 4 , 1214 ] .", "these relationships can also be used in model - building , where an incompletely built chain can be built using a more complete ncs - related copy of the same chain as a template [ 9 , 15 , 20 ] .", "further they could potentially be used to place copies of entire chains in low - resolution maps .", "the maps to be analyzed for ncs can be crystallographic maps , maps from electron microscopy , or maps from other sources .", "the approach described here could also be used to find density that is similar in two different electron density maps .", "this last application could be useful in automatically finding relationships between molecules in different crystals when carrying out cross - crystal averaging ."]]}
{"article_id": "PMC2915806", "article_text": ["putting a halt to the rising trend in overweight prevalence has high priority in public health policy all over the developed world .", "prevention , preferably starting at an early age , is considered as the key strategy to achieve this goal , and considerable effort is put into the design of preventive measures .   whereas we know in general terms that overweight is the result of an unbalance between energy intake and energy expenditure , development of effective preventive measures requires a much more specific understanding .   specific dietary habits and ( in)activities associated with overweight risk in specific age groups need to be identified and their quantitative contribution to the overweight prevalence in the population needs to be assessed .", "in addition , we need to know whether the importance of specific behaviors for the development of overweight differs between sub groups in the population .", "it is well established that children from families with a low socio - economic status and children with overweight parents are at increased risk to develop overweight [ 39 ] .", "it is therefore particularly important to assess the role of behavioral risk factors specifically in these children , since they are the priority target groups for interventions to prevent and reduce overweight .  ", "the aim of this study was to identify specific behavioral risk factors for overweight in young children and to assess their quantitative contribution to the prevalence of overweight in the general population and in high risk sub groups . in a large population - based birth cohort , we prospectively investigated associations between dietary habits , screen time and physical activity when the children were 5 and 7 years old and overweight at the age of 8 years .", "we also assessed the role of these behavioral factors separately in high risk sub groups : children of mothers with overweight and children of mothers with low education .", "we estimated the reduction of overweight prevalence that could be achieved if the prevalence of risk factors would be reduced .", "the study population consisted of children who participated in the dutch prevention and incidence of asthma and mite allergy ( piama ) birth cohort study .", "the baseline study population consisted of 4146 pregnant women , recruited from the general population in 1996 - 1997 .", "postal questionnaires , including questions on the child 's lifestyle and health , were sent to the parents during pregnancy , at the child 's ages of 3 and 12 months , and yearly thereafter up to the age of 8 years . at the age of 8 years , the children were invited for a medical examination which included measurement of weight and height .", "the study protocol was approved by the medical ethics committees of the participating institutes , and all parents gave written informed consent . of the baseline population of 4146 pregnant women , 183 ( 5% )", "were lost to follow - up before any data on the child had been collected .", "parental completed questionnaires from age 3 months to 8 years were available for 3934 , 3817 , 3694 , 3563 , 3518 , 3473 , 3373 , and 3269 children , respectively .", "when the medical examination of 8-year - old children was planned , 3668 children ( 93% of 3963 ) were still in the study , 3522 children were invited and 2214 participated ( 63% of those invited ) and had their weight and height measured .", "146 of the 3668 children were not invited for the medical examination , although they were still participating in the study . in the majority of cases", "the reason for this was that their families had moved and were now living too far away from the study centers where the medical examination was conducted .", "complete data on overweight and risk factors were available for 1871 children ( 47% of the 3963 children in the study at birth ) . during the medical examination of the 8-year - old children ,", "weight was measured to 0.1  kg and height to 0.1  cm by trained research staff using calibrated measuring equipment . from the weight and height measurements", "bmi ( body mass index : weight ( kg)/height ( m ) ) was calculated .", " overweight  and  obesity  were defined according to age and gender - specific international standards that use cutoff points equivalent to the 25  kg / m and 30  kg / m cut - offs that are commonly used for adults .  ", "the term  overweight  is used for the total group of children who were overweight , including those who were obese . for physical activity , 3 indicators were selected : time spent walking or cycling to school ( 3 categories ) , membership of a sports club ( yes / no ) , and time spent playing outside ( 3 categories ) .  screen time  ( 4 categories ) included time spent watching television , video 's , or at the computer .", "for dietary intake , 3 indicators were used : fast food consumption , snack consumption , and soft drink consumption .", "data on these exposures were obtained from the questionnaires administered at the ages of 5 and 7 years .", "these questionnaires contained a food frequency questionnaire with 40 different items and 5 answering categories per item ( ranging from  never in the last month  to  on 6 - 7 days per week  ) , which was used to construct the dietary exposure variables . for  fast food consumption ", "a score was constructed based on the consumption frequencies of chips / french fries and of foods like hamburgers and hot dogs . ", "snack consumption  was based on the reported consumption frequencies of 7 different foods / food groups including pie / cake , muffins , candy bars , sweets , chocolate , biscuits , and crisps .", " soft drink consumption  was based on the reported frequencies of consumption of 4 types of soft drinks .", "each of these variables was based on a number of different items , which differed with respect to their energy content . to be able to  add up ", "the different items , total weekly energy intake from each item was calculated based on the reported frequencies , assuming average portion sizes and average energy content per portion .", "data on maternal education and maternal weight and height were obtained from the questionnaire completed when the child was 1 year old .", "maternal bmi was calculated from reported weight and height and overweight and was defined as a bmi 25  kg / m .", "the associations between the exposure variables and overweight at the age of 8 years were analysed by logistic regression , adjusted for gender and birth weight .", "exposure variables were constructed using the data collected at both the age of 5 and at the age of  7  years in order to obtain a more stable estimate of exposure during the years preceding the measurement of weight and height . walking / cycling to school , playing outside and screen time", "were recorded in categories , numbered 1 , 2 , and 3 for walking / cycling to school , 1 , 2 , and 3 for playing outside , and 1 , 2 , 3 , and 4 for screen time ( see table 1 ) .", "the data collected at the ages of 5 and 7 years , were combined by taking the mean of the numbers of the categories reported at the ages of 5 and 7 years respectively .", "this resulted in ordinal variables with 5 categories for walking / cycling to school ( ranging from never at ages 5 and 7 to > 1/2 an hour per day at both ages ) , 5 categories for playing outside ( ranging from < 1  x per week at ages 5 and 7 to > 3  x per week at both ages ) and 7 categories for screen time ( ranging from < 1/2 an hour per day at ages 5 and 7 to > 2 hours per day at both ages ) . after having checked for nonlinearity", "fast food , snack and soft drink consumption at age 57 ( the mean of the consumption scores at age 5 and at age 7 ) , were included in the regression analyses as linear variables , but are shown in categories ( tertiles ) in table 1 .", "overweight ( including obesity ) was used as the outcome measure in the regression analyses .", "the number of obese children was too small for analyses with fully adjusted regression models , and obesity was therefore not used as outcome measure in these analyses . in previous studies in the piama cohort , breast feeding was investigated in relation to overweight risk and was found to be associated with reduced overweight risk   [ 12 , 13 ] . although breastfeeding is not the subject of this study , results on breast feeding are shown in the tables for completeness . in additional analyses ,", "the associations of physical activity and diet with overweight were also assessed separately for the exposures at age 5 and at age 7 .", "thirteen percent of all the data potentially in the study ( 3963 participants  20 variables   =   79260 ) were missing and 2092 children ( 53% ) had a missing value on at least 1 of the variables .", "if data are not  missing completely at random  ( mcar ) , complete case analysis may lead to biased results [ 14 , 15 ] .   in our dataset", "several variables ( including maternal education and breast feeding ) were associated with the probability of a subject having one or more missing values .", "missing data were multiple times imputed , using the  multivariate imputation by chained equations ", "( mice ) procedure   [ 16 , 17 ] , that runs under the statistical program r version 2.5.0   .   for the multiple imputation we used all the data that were used in the analyses , as well as data on the children 's weight and height that were measured and reported by the parents in the yearly questionnaires .", ".    maternal education and maternal overweight could be confounders of the associations studied but could also be factors in causal pathways .", "in addition , the analyses were repeated , stratified for maternal education , maternal overweight and gender .", "differences between the associations observed in the different sub groups were tested by inclusion of interaction terms in the regression models . for the behavioral risk factors that were found", "to be statistically significantly associated with overweight , adjusted population attributable risk percentages ( par% ) were calculated , using the mantel - haenszel approach , as described by benichou .", "\n table 1 shows characteristics of the study population and the prevalence of overweight and obesity in different sub groups .", "children with incomplete data on either risk factors or on measured weight and height at 8 years were compared to the children with complete data , with respect to a number of characteristics assessed during the first year of the study ( data not shown ) .", "children with incomplete data had , compared to children with complete data , a relatively high prevalence of low maternal education ( 27.5% versus 19.4% ) and of no breast feeding ( 19.7% versus 15.9% ) .", "as expected , in the imputed data the prevalence of these characteristics was somewhat higher ( 23.6% and 17.9% , see table 1 ) than in the children with complete data .", "the prevalence of maternal overweight was similar in children with incomplete data and in children with complete data ( 24.1% and 26.1% , resp . ) .", "the prevalence of overweight in the children was almost the same in the imputed data and in the complete cases ( 13.8% and 14.4% , resp . ) and the prevalence of obesity was the same in both datasets ( 2.7% ) . \n", "table 2 shows the associations between diet , physical activity , screen time and overweight . in the analyses adjusted for gender and birth weight only ( model 1 ) , fast food consumption and screen time ,", "sports club membership , active transport to school , playing outside , snack consumption , and soft drink consumption were not associated with overweight . when all risk factors ( including breast feeding ) were included in the same regression model ( model 2 ) ,", "the associations with overweight became somewhat weaker for most factors , indicating some association between the individual risk factors .", "the association of fast food consumption with overweight lost statistical significance in this analysis whereas the association with screen time remained statistically significant .", "surprisingly , an inverse association between snack consumption and overweight was observed .   in additional analyses ,", "the analyses shown in table 2 were repeated with the behavioral risk factors measured at age 5 and ( separately ) with those factors measured at age 7 instead of the variables combining the exposures measured at age 5 and age 7 . for the physical activity indicators and for screen time , results of these analyses were similar to those shown in table 2 , showing associations between screen time and overweight ( aor ( 95% ci ) at age 5  :  1.35 ( 1.191.53 ) and aor ( 95% ci ) at age 7  :  1.22 ( 1.081.39 ) ) but not between the physical activity indicators and overweight .", "snack consumption at age 5 was not associated with overweight at the age of 8 years ( aor ( 95% ci ) : 0.87 ( 0.661.14 ) ) , but there was a significant inverse association between snack consumption at age 7 and overweight at the age of 8 years ( aor ( 95% ci ) 0.70 ( 0.540.91 ) ) .", "low maternal education and especially maternal overweight were strong predictors of childhood overweight ( see table 2 , model 1 ) .", "the association between low maternal education and overweight weakened substantially in the model including the behavioral risk factors ( an almost 25% change in the regression coefficient from 0.50 to 0.38 ) , indicating that these specific factors explain at least part of the excess overweight prevalence in children of mothers with low education ( see table 2 , model 3 ) .", "the association between maternal overweight and overweight in the child weakened only marginally in the model including behavioral risk factors ( 5% change in regression coefficient from 1.07 to 1.02 ) , indicating that the association between maternal overweight and overweight of the child is only to a limited extent mediated by the behavioral factors studied ( see table 2 , model 4 ) .", "the analyses were repeated after stratification for low maternal education , maternal overweight and gender , respectively . in the sub groups with low maternal education and with maternal overweight", "children of mothers with a low level education had a higher prevalence of high fast food and snack intakes , of > 2 hours screen time per day and of not being member of a sport club than children of more highly educated mothers ( see table 3 ) .", "children of overweight mothers had a higher prevalence of high fast food intake and of > 2 hours screen time per day ( at the age of 5 , but not at 7 years ) than children of normal weight mothers ( see table 3 ) .   ", "girls had a slightly higher prevalence of overweight and slightly less screen time than boys ( data not shown ) .", "the results suggest that the associations between screen time and overweight were weaker in children of mothers with overweight or low education than in children of mothers with a normal weight or higher level of education ( table 4 ) .", "interactions between the exposure variables and low maternal education and maternal overweight were not statistically significant , however . for boys and girls these associations were very similar ( data not shown ) .", "of the behavioral risk factors studied , only screen time was statistically significantly associated with overweight . based on the prevalence and adjusted odds ratios for screen time > 1  hr per day ( see table 5 ) , we estimated how much screen time contributed to the total prevalence of overweight .", "table 5 shows the adjusted population attributable risk percentage ( par% ) for a screen time of more than 1 hour per day at age 5 and/or age 7 as compared to less than 1 hour at both ages . of the total overweight prevalence in the low risk sub groups", "an estimated 17% could be attributed to screen time of > 1 hour per day as compared to 10% in the high risk sub groups .", "this means that , if all children would reduce their screen time to less than 1 hour per day , the following reductions in overweight prevalence could be achieved : from 18.3% to 16.4% in children of mothers with low education ; from 12.4% to 10.3% in children of mothers with higher education from 25.0% to 22.4% in children of overweight mothers and from 10.0% to 8.3% in children of normal weight mothers .", "all analyses were conducted in a data set containing only complete cases as well as in the imputed data .", "the odds ratios ( 95% ci ) for screen time , for example , were 1.47 ( 1.241.75 ) and 1.41 ( 1.171.70 ) in model 1 and model 2 , respectively , in the complete case analysis , compared to 1.45 ( 1.261.66 ) and 1.39 ( 1.211.61 ) in the analysis in the imputed data set .", "of 8 different potential behavioral risk factors , only screen time showed consistent and significant associations with overweight . in high risk sub groups of the population ,", "screen time was higher than in low risk sub groups , but the associations between screen time and overweight were weaker ( although not significantly weaker ) in the high risk sub groups .  ", "important strengths of the study were the prospective design , the large study population , the repeated measurements ( at ages 5 and 7 ) of different behavioral risk factors and the availability of measured ( rather than parental reported ) data on weight and height.however , a number of limitations have to be considered .  ", "thirteen percent of our data were missing and 53% of the study population had a missing value on 1 or more of the variables used in the analyses .", "we used multiple imputation to deal with missing data in our study in order to avoid bias due to selective missing of data and to make more efficient use of the available data .", "results of the complete case analysis and the analyses in the imputed data sets were similar .  ", "information on the behavioral risk factors was obtained from parental completed questionnaires and we therefore need to address the possibility of misreporting .", "the questions asked ranged from simple matter - of - fact items ( like sports club membership yes / no ) to items that are more difficult to observe and recall accurately ( like the consumption frequency of a range of different food items ) . we expect that recalling or reporting problems will not have substantially influenced the data on sports club membership or transport to school and that any misreporting on these items will not have been systematically associated with overweight development .", "the data on foods and drinks consumption may well have been influenced by reporting bias .", "many of the foods included in the dietary variables have a bad reputation in relation to overweight , and selective underreporting by parents of children who were developing overweight may have occurred .  ", "another problem with respect to the diet variables is that data were available on the consumption frequencies of food items , but not on portion sizes .", "estimates of the intakes of fast food , snacks and soft drinks and the ranking of children according to their intakes were therefore based on reported consumption frequencies only , and the possibility of misclassification of food intake can not be excluded .", "membership of a sports club and active transportation to school were not associated with overweight in this population .", "the reason that we did not observe the hypothesized associations might be that in young children the duration and intensity of these activities are usually too low to have a sizeable impact on total activity .", "if this would be confirmed in further studies , it may have potentially important implications for the allocation of budgets for the prevention of overweight in this age group . in older children and adolescents active transport to school and sports club membership", "we found some evidence for an inverse relation between playing outside and overweight , but the associations were weak and not statistically significant .", "children who played outside 1  x per week seemed to have a higher overweight risk , but such children were a small minority in the study population .", "screen time was consistently and significantly associated with overweight and the association was largely independent of other lifestyle factors .", "a dose - response relationship was observed when it was included as a categorical variable in the regression analysis .", "our results thus suggest that , among the indicators of active and sedentary behavior that we studied , screen time is the most important risk factor for overweight in the age group studied .", "this result is in agreement with the conclusion of a recent study in which the evidence was summarized for six different strategies to prevent or treat pediatric overweight   .", "our findings on screen time and overweight risk are also in line with the results of a comprehensive meta - analysis on the subject , which observed a statistically significant relationship between tv viewing and body fatness among children and youth   .  ", "we estimated that a reduction in overweight prevalence of up to 2 percentage points could be achieved if all children would reduce their screen time to less than 1 hour per day .", " maximum achievable reduction of prevalence ( marp )  of 1.5 percentage points that was estimated for watching television > 1  hr / day in a study on 5 - 6 year old german children .", "consumption of soft drinks and fast food at ages 57 was not associated with overweight whereas an unexpected inverse association between snack consumption and overweight was observed .", "snack consumption at the age of 5 years was not associated with overweight at age 8 , but snack consumption at age 7 was inversely associated with overweight at age 8 . as indicated previously , these results may have been influenced by selective underreporting of snack consumption by parents of children who were becoming overweight . besides selective underreporting , reverse causation may also have played a role , in the sense that parents of children who were becoming overweight really have reduced their children 's consumption of specific foods and drinks .", "the results on fast food , snack and soft drink intakes seem to indicate that in particular the items included in our snack , variable are the kind of foods that come to parents ' minds when they think of trying to limit excessive weight gain in their child .", "the selective underreporting and reverse causation that are likely to be present in our dietary data , make it impossible to judge from our results how diet influences the development of overweight .", "we hypothesize that these mechanisms might play a role also in other epidemiological studies on diet and overweight . whereas , on theoretical grounds , it is hard to imagine that food intake is unrelated to weight status , observational studies have generally not produced consistent evidence for the expected associations [ 20 , 2327 ] .", "our results do not provide an answer to the question whether consumption of soft drinks , fast food , and snacks causes overweight but seem to indicate that parents may see reduction of snack consumption as a method to counteract the development of overweight .", "further studies into the strategies that parents themselves use to influence their children 's weight gain might provide insights that could be useful for the design of preventive strategies .", "low maternal education and maternal overweight were found to be strongly associated with overweight in the child .", "the association between low maternal education and overweight weakened substantially when the behavioral risk factors were included in the model , indicating that these factors explain part of the association .", "the behavioral risk factors had little influence on the association between maternal overweight and overweight in the child , indicating that other factors , possibly genetic factors , play a more important role in this association .  ", "we repeated the regression analyses in sub groups stratified by maternal weight status and maternal education . whereas in the high risk sub groups , screen time was higher than in low risk sub groups , the associations between screen time and overweight were weaker ( although not statistically significantly ) in these groups , and the population attributable risk percentage was lower than in the low risk sub groups . as the children of mothers with overweight and/or low education are the children at highest risk to develop overweight , this observation is potentially important for the development of preventive strategies and needs to be clarified in future observational as well as intervention studies .", "reduction of screen time should be part of interventions to prevent or reduce overweight in young children and could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risk sub groups .", "our results also suggest however that interventions aimed to promote sports club membership , active transport to school , and playing outside may have little impact on the prevalence of overweight in this age group .", "the possibility that , due to reverse causation , associations between food intake and overweight can not be assessed in observational studies , needs further study ."], "abstract_text": ["<S> \n objective . to prospectively identify behavioral risk factors for childhood overweight and to assess their relevance in high risk sub groups ( children of mothers with overweight or low education ) </S>", "<S> . \n methods . in the piama birth cohort ( n = 3963 ) , </S>", "<S> questionnaire data were obtained at ages 5 and 7 on  screen time  , walking or cycling to school , playing outside , sports club membership , fast food consumption , snack consumption and soft drink consumption . </S>", "<S> weight and height were measured at age 8 years . \n </S>", "<S> results . </S>", "<S> screen time , but none of the other hypothesized behavioral factors , was associated with overweight ( aor 1.4 ( ci : 1.21.6 ) ) . the adjusted population attributable risk fraction for screen time > 1  hr / day was 10% in the high risk and 17% in the low risk sub groups . \n conclusion . </S>", "<S> reduction of screen time to < 1  hr / day could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risks sub groups . \n </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["putting a halt to the rising trend in overweight prevalence has high priority in public health policy all over the developed world .", "prevention , preferably starting at an early age , is considered as the key strategy to achieve this goal , and considerable effort is put into the design of preventive measures .   whereas we know in general terms that overweight is the result of an unbalance between energy intake and energy expenditure , development of effective preventive measures requires a much more specific understanding .   specific dietary habits and ( in)activities associated with overweight risk in specific age groups need to be identified and their quantitative contribution to the overweight prevalence in the population needs to be assessed .", "in addition , we need to know whether the importance of specific behaviors for the development of overweight differs between sub groups in the population .", "it is well established that children from families with a low socio - economic status and children with overweight parents are at increased risk to develop overweight [ 39 ] .", "it is therefore particularly important to assess the role of behavioral risk factors specifically in these children , since they are the priority target groups for interventions to prevent and reduce overweight .  ", "the aim of this study was to identify specific behavioral risk factors for overweight in young children and to assess their quantitative contribution to the prevalence of overweight in the general population and in high risk sub groups . in a large population - based birth cohort , we prospectively investigated associations between dietary habits , screen time and physical activity when the children were 5 and 7 years old and overweight at the age of 8 years .", "we also assessed the role of these behavioral factors separately in high risk sub groups : children of mothers with overweight and children of mothers with low education .", "we estimated the reduction of overweight prevalence that could be achieved if the prevalence of risk factors would be reduced ."], ["the study population consisted of children who participated in the dutch prevention and incidence of asthma and mite allergy ( piama ) birth cohort study .", "the baseline study population consisted of 4146 pregnant women , recruited from the general population in 1996 - 1997 .", "postal questionnaires , including questions on the child 's lifestyle and health , were sent to the parents during pregnancy , at the child 's ages of 3 and 12 months , and yearly thereafter up to the age of 8 years . at the age of 8 years , the children were invited for a medical examination which included measurement of weight and height .", "the study protocol was approved by the medical ethics committees of the participating institutes , and all parents gave written informed consent . of the baseline population of 4146 pregnant women , 183 ( 5% )", "were lost to follow - up before any data on the child had been collected .", "parental completed questionnaires from age 3 months to 8 years were available for 3934 , 3817 , 3694 , 3563 , 3518 , 3473 , 3373 , and 3269 children , respectively .", "when the medical examination of 8-year - old children was planned , 3668 children ( 93% of 3963 ) were still in the study , 3522 children were invited and 2214 participated ( 63% of those invited ) and had their weight and height measured .", "146 of the 3668 children were not invited for the medical examination , although they were still participating in the study . in the majority of cases", "the reason for this was that their families had moved and were now living too far away from the study centers where the medical examination was conducted .", "complete data on overweight and risk factors were available for 1871 children ( 47% of the 3963 children in the study at birth ) . during the medical examination of the 8-year - old children ,", "weight was measured to 0.1  kg and height to 0.1  cm by trained research staff using calibrated measuring equipment . from the weight and height measurements", "bmi ( body mass index : weight ( kg)/height ( m ) ) was calculated .", " overweight  and  obesity  were defined according to age and gender - specific international standards that use cutoff points equivalent to the 25  kg / m and 30  kg / m cut - offs that are commonly used for adults .  ", "the term  overweight  is used for the total group of children who were overweight , including those who were obese . for physical activity , 3 indicators were selected : time spent walking or cycling to school ( 3 categories ) , membership of a sports club ( yes / no ) , and time spent playing outside ( 3 categories ) .  screen time  ( 4 categories ) included time spent watching television , video 's , or at the computer .", "for dietary intake , 3 indicators were used : fast food consumption , snack consumption , and soft drink consumption .", "data on these exposures were obtained from the questionnaires administered at the ages of 5 and 7 years .", "these questionnaires contained a food frequency questionnaire with 40 different items and 5 answering categories per item ( ranging from  never in the last month  to  on 6 - 7 days per week  ) , which was used to construct the dietary exposure variables . for  fast food consumption ", "a score was constructed based on the consumption frequencies of chips / french fries and of foods like hamburgers and hot dogs . ", "snack consumption  was based on the reported consumption frequencies of 7 different foods / food groups including pie / cake , muffins , candy bars , sweets , chocolate , biscuits , and crisps .", " soft drink consumption  was based on the reported frequencies of consumption of 4 types of soft drinks .", "each of these variables was based on a number of different items , which differed with respect to their energy content . to be able to  add up ", "the different items , total weekly energy intake from each item was calculated based on the reported frequencies , assuming average portion sizes and average energy content per portion .", "data on maternal education and maternal weight and height were obtained from the questionnaire completed when the child was 1 year old .", "maternal bmi was calculated from reported weight and height and overweight and was defined as a bmi 25  kg / m .", "the associations between the exposure variables and overweight at the age of 8 years were analysed by logistic regression , adjusted for gender and birth weight .", "exposure variables were constructed using the data collected at both the age of 5 and at the age of  7  years in order to obtain a more stable estimate of exposure during the years preceding the measurement of weight and height . walking / cycling to school , playing outside and screen time", "were recorded in categories , numbered 1 , 2 , and 3 for walking / cycling to school , 1 , 2 , and 3 for playing outside , and 1 , 2 , 3 , and 4 for screen time ( see table 1 ) .", "the data collected at the ages of 5 and 7 years , were combined by taking the mean of the numbers of the categories reported at the ages of 5 and 7 years respectively .", "this resulted in ordinal variables with 5 categories for walking / cycling to school ( ranging from never at ages 5 and 7 to > 1/2 an hour per day at both ages ) , 5 categories for playing outside ( ranging from < 1  x per week at ages 5 and 7 to > 3  x per week at both ages ) and 7 categories for screen time ( ranging from < 1/2 an hour per day at ages 5 and 7 to > 2 hours per day at both ages ) . after having checked for nonlinearity", "fast food , snack and soft drink consumption at age 57 ( the mean of the consumption scores at age 5 and at age 7 ) , were included in the regression analyses as linear variables , but are shown in categories ( tertiles ) in table 1 .", "overweight ( including obesity ) was used as the outcome measure in the regression analyses .", "the number of obese children was too small for analyses with fully adjusted regression models , and obesity was therefore not used as outcome measure in these analyses . in previous studies in the piama cohort , breast feeding was investigated in relation to overweight risk and was found to be associated with reduced overweight risk   [ 12 , 13 ] . although breastfeeding is not the subject of this study , results on breast feeding are shown in the tables for completeness . in additional analyses ,", "the associations of physical activity and diet with overweight were also assessed separately for the exposures at age 5 and at age 7 .", "thirteen percent of all the data potentially in the study ( 3963 participants  20 variables   =   79260 ) were missing and 2092 children ( 53% ) had a missing value on at least 1 of the variables .", "if data are not  missing completely at random  ( mcar ) , complete case analysis may lead to biased results [ 14 , 15 ] .   in our dataset", "several variables ( including maternal education and breast feeding ) were associated with the probability of a subject having one or more missing values .", "missing data were multiple times imputed , using the  multivariate imputation by chained equations ", "( mice ) procedure   [ 16 , 17 ] , that runs under the statistical program r version 2.5.0   .   for the multiple imputation we used all the data that were used in the analyses , as well as data on the children 's weight and height that were measured and reported by the parents in the yearly questionnaires .", ".    maternal education and maternal overweight could be confounders of the associations studied but could also be factors in causal pathways .", "in addition , the analyses were repeated , stratified for maternal education , maternal overweight and gender .", "differences between the associations observed in the different sub groups were tested by inclusion of interaction terms in the regression models . for the behavioral risk factors that were found", "to be statistically significantly associated with overweight , adjusted population attributable risk percentages ( par% ) were calculated , using the mantel - haenszel approach , as described by benichou ."], ["\n table 1 shows characteristics of the study population and the prevalence of overweight and obesity in different sub groups .", "children with incomplete data on either risk factors or on measured weight and height at 8 years were compared to the children with complete data , with respect to a number of characteristics assessed during the first year of the study ( data not shown ) .", "children with incomplete data had , compared to children with complete data , a relatively high prevalence of low maternal education ( 27.5% versus 19.4% ) and of no breast feeding ( 19.7% versus 15.9% ) .", "as expected , in the imputed data the prevalence of these characteristics was somewhat higher ( 23.6% and 17.9% , see table 1 ) than in the children with complete data .", "the prevalence of maternal overweight was similar in children with incomplete data and in children with complete data ( 24.1% and 26.1% , resp . ) .", "the prevalence of overweight in the children was almost the same in the imputed data and in the complete cases ( 13.8% and 14.4% , resp . ) and the prevalence of obesity was the same in both datasets ( 2.7% ) . \n", "table 2 shows the associations between diet , physical activity , screen time and overweight . in the analyses adjusted for gender and birth weight only ( model 1 ) , fast food consumption and screen time ,", "sports club membership , active transport to school , playing outside , snack consumption , and soft drink consumption were not associated with overweight . when all risk factors ( including breast feeding ) were included in the same regression model ( model 2 ) ,", "the associations with overweight became somewhat weaker for most factors , indicating some association between the individual risk factors .", "the association of fast food consumption with overweight lost statistical significance in this analysis whereas the association with screen time remained statistically significant .", "surprisingly , an inverse association between snack consumption and overweight was observed .   in additional analyses ,", "the analyses shown in table 2 were repeated with the behavioral risk factors measured at age 5 and ( separately ) with those factors measured at age 7 instead of the variables combining the exposures measured at age 5 and age 7 . for the physical activity indicators and for screen time , results of these analyses were similar to those shown in table 2 , showing associations between screen time and overweight ( aor ( 95% ci ) at age 5  :  1.35 ( 1.191.53 ) and aor ( 95% ci ) at age 7  :  1.22 ( 1.081.39 ) ) but not between the physical activity indicators and overweight .", "snack consumption at age 5 was not associated with overweight at the age of 8 years ( aor ( 95% ci ) : 0.87 ( 0.661.14 ) ) , but there was a significant inverse association between snack consumption at age 7 and overweight at the age of 8 years ( aor ( 95% ci ) 0.70 ( 0.540.91 ) ) .", "low maternal education and especially maternal overweight were strong predictors of childhood overweight ( see table 2 , model 1 ) .", "the association between low maternal education and overweight weakened substantially in the model including the behavioral risk factors ( an almost 25% change in the regression coefficient from 0.50 to 0.38 ) , indicating that these specific factors explain at least part of the excess overweight prevalence in children of mothers with low education ( see table 2 , model 3 ) .", "the association between maternal overweight and overweight in the child weakened only marginally in the model including behavioral risk factors ( 5% change in regression coefficient from 1.07 to 1.02 ) , indicating that the association between maternal overweight and overweight of the child is only to a limited extent mediated by the behavioral factors studied ( see table 2 , model 4 ) .", "the analyses were repeated after stratification for low maternal education , maternal overweight and gender , respectively . in the sub groups with low maternal education and with maternal overweight", "children of mothers with a low level education had a higher prevalence of high fast food and snack intakes , of > 2 hours screen time per day and of not being member of a sport club than children of more highly educated mothers ( see table 3 ) .", "children of overweight mothers had a higher prevalence of high fast food intake and of > 2 hours screen time per day ( at the age of 5 , but not at 7 years ) than children of normal weight mothers ( see table 3 ) .   ", "girls had a slightly higher prevalence of overweight and slightly less screen time than boys ( data not shown ) .", "the results suggest that the associations between screen time and overweight were weaker in children of mothers with overweight or low education than in children of mothers with a normal weight or higher level of education ( table 4 ) .", "interactions between the exposure variables and low maternal education and maternal overweight were not statistically significant , however . for boys and girls these associations were very similar ( data not shown ) .", "of the behavioral risk factors studied , only screen time was statistically significantly associated with overweight . based on the prevalence and adjusted odds ratios for screen time > 1  hr per day ( see table 5 ) , we estimated how much screen time contributed to the total prevalence of overweight .", "table 5 shows the adjusted population attributable risk percentage ( par% ) for a screen time of more than 1 hour per day at age 5 and/or age 7 as compared to less than 1 hour at both ages . of the total overweight prevalence in the low risk sub groups", "an estimated 17% could be attributed to screen time of > 1 hour per day as compared to 10% in the high risk sub groups .", "this means that , if all children would reduce their screen time to less than 1 hour per day , the following reductions in overweight prevalence could be achieved : from 18.3% to 16.4% in children of mothers with low education ; from 12.4% to 10.3% in children of mothers with higher education from 25.0% to 22.4% in children of overweight mothers and from 10.0% to 8.3% in children of normal weight mothers .", "all analyses were conducted in a data set containing only complete cases as well as in the imputed data .", "the odds ratios ( 95% ci ) for screen time , for example , were 1.47 ( 1.241.75 ) and 1.41 ( 1.171.70 ) in model 1 and model 2 , respectively , in the complete case analysis , compared to 1.45 ( 1.261.66 ) and 1.39 ( 1.211.61 ) in the analysis in the imputed data set ."], ["of 8 different potential behavioral risk factors , only screen time showed consistent and significant associations with overweight . in high risk sub groups of the population ,", "screen time was higher than in low risk sub groups , but the associations between screen time and overweight were weaker ( although not significantly weaker ) in the high risk sub groups .  ", "important strengths of the study were the prospective design , the large study population , the repeated measurements ( at ages 5 and 7 ) of different behavioral risk factors and the availability of measured ( rather than parental reported ) data on weight and height.however , a number of limitations have to be considered .  ", "thirteen percent of our data were missing and 53% of the study population had a missing value on 1 or more of the variables used in the analyses .", "we used multiple imputation to deal with missing data in our study in order to avoid bias due to selective missing of data and to make more efficient use of the available data .", "results of the complete case analysis and the analyses in the imputed data sets were similar .  ", "information on the behavioral risk factors was obtained from parental completed questionnaires and we therefore need to address the possibility of misreporting .", "the questions asked ranged from simple matter - of - fact items ( like sports club membership yes / no ) to items that are more difficult to observe and recall accurately ( like the consumption frequency of a range of different food items ) . we expect that recalling or reporting problems will not have substantially influenced the data on sports club membership or transport to school and that any misreporting on these items will not have been systematically associated with overweight development .", "the data on foods and drinks consumption may well have been influenced by reporting bias .", "many of the foods included in the dietary variables have a bad reputation in relation to overweight , and selective underreporting by parents of children who were developing overweight may have occurred .  ", "another problem with respect to the diet variables is that data were available on the consumption frequencies of food items , but not on portion sizes .", "estimates of the intakes of fast food , snacks and soft drinks and the ranking of children according to their intakes were therefore based on reported consumption frequencies only , and the possibility of misclassification of food intake can not be excluded .", "membership of a sports club and active transportation to school were not associated with overweight in this population .", "the reason that we did not observe the hypothesized associations might be that in young children the duration and intensity of these activities are usually too low to have a sizeable impact on total activity .", "if this would be confirmed in further studies , it may have potentially important implications for the allocation of budgets for the prevention of overweight in this age group . in older children and adolescents active transport to school and sports club membership", "we found some evidence for an inverse relation between playing outside and overweight , but the associations were weak and not statistically significant .", "children who played outside 1  x per week seemed to have a higher overweight risk , but such children were a small minority in the study population .", "screen time was consistently and significantly associated with overweight and the association was largely independent of other lifestyle factors .", "a dose - response relationship was observed when it was included as a categorical variable in the regression analysis .", "our results thus suggest that , among the indicators of active and sedentary behavior that we studied , screen time is the most important risk factor for overweight in the age group studied .", "this result is in agreement with the conclusion of a recent study in which the evidence was summarized for six different strategies to prevent or treat pediatric overweight   .", "our findings on screen time and overweight risk are also in line with the results of a comprehensive meta - analysis on the subject , which observed a statistically significant relationship between tv viewing and body fatness among children and youth   .  ", "we estimated that a reduction in overweight prevalence of up to 2 percentage points could be achieved if all children would reduce their screen time to less than 1 hour per day .", " maximum achievable reduction of prevalence ( marp )  of 1.5 percentage points that was estimated for watching television > 1  hr / day in a study on 5 - 6 year old german children .", "consumption of soft drinks and fast food at ages 57 was not associated with overweight whereas an unexpected inverse association between snack consumption and overweight was observed .", "snack consumption at the age of 5 years was not associated with overweight at age 8 , but snack consumption at age 7 was inversely associated with overweight at age 8 . as indicated previously , these results may have been influenced by selective underreporting of snack consumption by parents of children who were becoming overweight . besides selective underreporting , reverse causation may also have played a role , in the sense that parents of children who were becoming overweight really have reduced their children 's consumption of specific foods and drinks .", "the results on fast food , snack and soft drink intakes seem to indicate that in particular the items included in our snack , variable are the kind of foods that come to parents ' minds when they think of trying to limit excessive weight gain in their child .", "the selective underreporting and reverse causation that are likely to be present in our dietary data , make it impossible to judge from our results how diet influences the development of overweight .", "we hypothesize that these mechanisms might play a role also in other epidemiological studies on diet and overweight . whereas , on theoretical grounds , it is hard to imagine that food intake is unrelated to weight status , observational studies have generally not produced consistent evidence for the expected associations [ 20 , 2327 ] .", "our results do not provide an answer to the question whether consumption of soft drinks , fast food , and snacks causes overweight but seem to indicate that parents may see reduction of snack consumption as a method to counteract the development of overweight .", "further studies into the strategies that parents themselves use to influence their children 's weight gain might provide insights that could be useful for the design of preventive strategies .", "low maternal education and maternal overweight were found to be strongly associated with overweight in the child .", "the association between low maternal education and overweight weakened substantially when the behavioral risk factors were included in the model , indicating that these factors explain part of the association .", "the behavioral risk factors had little influence on the association between maternal overweight and overweight in the child , indicating that other factors , possibly genetic factors , play a more important role in this association .  ", "we repeated the regression analyses in sub groups stratified by maternal weight status and maternal education . whereas in the high risk sub groups , screen time was higher than in low risk sub groups , the associations between screen time and overweight were weaker ( although not statistically significantly ) in these groups , and the population attributable risk percentage was lower than in the low risk sub groups . as the children of mothers with overweight and/or low education are the children at highest risk to develop overweight , this observation is potentially important for the development of preventive strategies and needs to be clarified in future observational as well as intervention studies ."], ["reduction of screen time should be part of interventions to prevent or reduce overweight in young children and could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risk sub groups .", "our results also suggest however that interventions aimed to promote sports club membership , active transport to school , and playing outside may have little impact on the prevalence of overweight in this age group .", "the possibility that , due to reverse causation , associations between food intake and overweight can not be assessed in observational studies , needs further study ."]]}
{"article_id": "PMC3139884", "article_text": ["the protection of water supplies and water distribution infrastructure such as reservoirs is of great concern for those defending against bioterrorism . the us military is concerned because there exist multiple biological warfare agents ( bwas ) that could be intentionally introduced into the water supply which could have serious implications for military operations as well as civilians located within a theater of operations . to protect potable water supplies , the department of defense ( dod ) seeks to develop a rapid field test that would determine if field supplies of water are safe to drink by the warfighter during a military operation .", "the us armed services have fielded biological defense systems that are designed to protect against a large scale attack via bwas dispersed through the air .", "these defensive systems utilize high - efficiency aerosol collectors that impinge the sampled air into a liquid buffer which is tested for bwas using antibody - based handheld assays ( hhas ) .", "civilian first responders use similar versions of these antibody - based hhas for testing suspicious powder samples suspected to contain bwas . depending on which manufacturer of hha", "is being tested , the technology can be low cost , requires no electrical power , and requires minimal to no sample preparation to test a sample .", "it was an hha panel manufactured by the dod that first identified bacillus anthracis in the powder that spilled across a desk in senator daschle 's office in the hart senate office building in 2001 .", "three years later , a dod hha was again used to identify ricin toxin in the mail room of the dirksen senate office building .", "these instances demonstrate that , when hha technologies are used appropriately by an experienced operator trained to understand what the results mean , they can be a useful tool .", "additionally , hhas have proven fast and effective in the laboratory for the detection of francisella tularensis   and biotoxins such as staphylococcal enterotoxin b ( seb )   and microcystins .   the dod has initiated an effort called the joint chemical biological and radiological agent water monitor ( jcbrawm ) program to develop a system which will allow rapid field testing of drinking water supplies used by military personnel .", "the effectiveness of hhas in detecting biological agents in the air and in powders has resulted in a desire to test these assays to gauge their effectiveness to protect drinking water supplies used by us warfighters . in the present study , hhas from multiple manufacturers were evaluated for their effectiveness at detecting two specific targets , seb and ricin , in multiple water matrices .", "the hhas were not modified in any way to optimize for the detection of toxins within water supplies despite the fact that they were initially developed to test for concentrated agent collected from high - efficiency aerosol collectors and impinged into a phosphate buffered saline ( pbs ) solution rather than to test unbuffered water supplies .  ", "the ability of the hhas to detect seb and ricin in four different water matrices , representative of waters used by different branches of the armed forces , was evaluated .", "the four different water matrices were formulated tap water , formulated tap water with 1  mg / l free available chlorine ( fac ) , water treated by reverse osmosis ( ro ) with 1  mg / l fac , and ro water with 2  mg / l total residual bromine . suggested health effect levels as used by the us military for ricin and seb are 455  ng / ml and 4.55  ng / ml , respectively .", "these minimal health effect levels were derived using the tri service standards for field water and based on an average consumption of 10 liters per day for seven days for a 70  kg service member . therefore , detection of ricin and seb in water at or below the suggested health effect level is desired .", "both singleplex ( one - line ) and multiplex ( two - line and two - dot ) hha models produced by two manufacturers were evaluated in the present study for the detection of ricin toxin and seb [ 5 , 6 ] .", "active seb ( toxin technologies , sarasota , fla , usa ) and active ricin whole toxin ( vector laboratories , burlington , on , canada ) were obtained from the dod 's critical reagents program ( crp ) for use in the present study .", "briefly , approximately 500  ml of astm type i deionized water was added to a one - liter volumetric flask to which each stock solution was added to achieve the desired final concentration and the total volume was brought to one liter with deionized water .", "after 1520 minutes , the ph was assessed and adjusted if needed ( with minimal volume change ) to 7.67.8 .", "once prepared , synthetic tap water was stored at 4c with a shelf life of one week .  ", "briefly , one gram of calcium hypochlorite was added to twenty milliliters of astm type i deionized water and shaken vigorously for one to two minutes .", "briefly , approximately five microliters of the chlorine stock solution ( supernatant , not solids ) was added to 100  ml of the formulated tap water or ro water , which was prepared by running local tap water through a commercial ro unit .", "the fac was measured using a hach dr 2500 spectrophotometer per manufacturer 's instructions , and the fac level was adjusted as needed to achieve the desired fac level of 1 ", "chlorinated water was considered stable for only one day and therefore made fresh daily just prior to use .", "briefly , 25  ml of bromine resin was transferred to a 500  ml , 0.20  m filter unit ( corning life science , lowell , mass , usa ) .", "total residual bromine in the collected water was measured using a hach dr 2500 spectrophotometer per manufacturer 's instructions and adjusted as needed to achieve the desired final concentration of 2  mg / l .", "each water type was prepared fresh daily as described above and spiked separately with each target at desired concentrations .", "after spiking , 100  l of sample was loaded onto the sample well of the hha .", "the hha was allowed to develop for 15 minutes prior to reading the result by eye .", "each challenge concentration was tested a minimum of two independent times with a minimum of three handheld assays per concentration .", "limits of detection ( lod ) were determined for each hha type in each water matrix and defined as the minimum concentration of toxin that could be detected by eye .", "samples for circular dichroism ( cd ) were prepared by spiking ro water containing 1  mg / l fac with seb to a final concentration of 2  g / ml . control samples received no chlorine .", "after 24 hours , sodium thiosulfate ( 0.005% w / v final concentration ; fisher scientific catalog number s446 ) was added to all samples to quench any remaining chlorine prior to analysis by cd .", "cd experiments employed a jasco model j-810 spectropolarimeter ( jasco analytical instruments , easton , md , usa ) equipped with a ptc-423s peltier thermoelectric temperature control system .", "cd spectra were obtained for seb at 25c from 260 to 190  nm with a data pitch of 0.5  nm , a band width of 1  nm , a response time of 1  sec , and a scanning speed of 10  nm / min .", "the cd was carried out on 2  g / ml samples in 1  cm quartz cuvettes and required long - time signal averaging .", "the concentration of seb was verified for a representative stock solution by uv absorption at 277  nm using an e for seb of 14 .", "for each sample , the blank - subtracted cd curve for 100 averaged scans was converted to molar ellipticity using a molecular weight of 28,366 daltons and fit with the secondary structure estimation application in jasco 's spectra manager ( version 1.53.01 ) software to obtain the fraction of helix , beta sheet , turn , and random coil secondary structure .", "the percentages reported here were obtained using the reed reference set for the secondary structure calculations .  ", "samples for sds - page were prepared by spiking ro water containing 1  mg / l fac with seb to a final concentration of 2  g / ml . control samples received no chlorine .", "after one minute , one hour , and 24 hours , sodium thiosulfate ( 0.005% w / v final concentration ) was added to all samples to quench any remaining chlorine prior to analysis by sds - page . reducing gel eletrophoresis", "was carried out using the mini - protean 2 cell and 1020% tris - hcl pager gold precast gels from lonza ( rockland , me , usa ) .", "fifty nanograms of seb samples and 1  :  20 diluted mark12 unstained standard ( invitrogen ) were prepared in sds sample buffer , followed by heating at 95c for 5 minutes prior to loading .", "running buffer was prepared by dilution from 10x tris / glycine / sds buffer ( biorad ) .", "following electrophoresis , the gel was fixed with 50% methanol and 7% acetic acid ( both v / v ) for a total of 30 minutes .", "the rapid staining protocol with sypro ruby protein stain ( invitrogen ) was used for the detection of seb .", "the gel was washed with a solution of 10% methanol and 7% acetic acid ( both v / v ) for 30 minutes and imaged and documented with uv light using syngene ( frederick , md , usa ) .", "all hhas were evaluated for their performance in formulated tap water , formulated tap water with chlorine , ro water with chlorine , and ro water with bromine for the detection of ricin and seb .", "each water type was spiked with multiple concentrations of each toxin and loaded onto hhas for testing until the lod was determined for each hha model .", "it was determined that ricin could be detected down to approximately 1  ng / ml for most hha models in both formulated tap water and formulated tap with chlorine , while exhibiting only slightly higher lods in ro with chlorine for most models . however , in ro with bromine , the lod for ricin increased to 3,000  ng / ml for all models tested . for seb , lods in formulated tap water were low , ranging from 0.9  ng / ml to 12.5  ng / ml . however , in both chlorinated and brominated water , lods increased to 2,500  ng / ml for singleplex hhas and even higher to 5,000 or 10,000  ng / ml for multiplex models . in all cases", "detection below the suggested health effect level was desired ; however , seb was not detected below the suggested health effect level in chlorinated and brominated water and ricin was not detected below that level in brominated water .", "due to the increased detection limits observed in chlorinated and brominated water , cd spectroscopy , which assesses the three - dimensional fold of a protein , was performed on samples of untreated seb and seb exposed to chlorine in an effort to evaluate the basis for increased lods ( figure 1 ) . for each sample", ", the individual cd scans were compared prior to averaging to ascertain the structural stability of the sample .", "untreated seb at 2  g / ml was determined to contain 14.3% helix , 34.50% sheet , 17.6% turn , and 33.6% random coil ( rmsd < 0.1% ) .", "the cd spectrum of seb treated with 1  mg / l fac was stable over the time of experimentation .", "the curve - fitting calculation for chlorine - treated seb resulted in 25.9% helix , 0.0% sheet , 0.0% turn , and 74.1 random coil ( rmsd < 0.1% ) .   sds - page was also performed to ascertain the overall effect of chemical reaction with chlorine on the molecular weight of seb .", "for the treated and untreated seb , 1.5  g of seb was loaded per well . as shown in figure 2 , for untreated seb ,", "the seb was visible on the gel as a band with a molecular weight of approximately 28  kda for all time points tested . for chlorine - treated seb ,", "no band was visible at the same molecular weight as untreated seb for any of the time points tested .", "ricin and seb are considered potential biological threat agents as both can cause illness either by inhalation or ingestion .", "seb could potentially be used to attack low - volume water supplies ; however , due to the fact that ricin is less toxic by oral ingestion , it may be a less likely threat to drinking water .", "however , detection of both agents in water is still a concern and technologies capable of detection in water must be investigated .", "performance of hhas for air and surfaces is well known ; however , the use of hhas for detection of agents in water is less recognized .", "therefore , the application of hhas for detection in water was assessed in this study . in formulated tap water , detection of seb and ricin", "was achieved at or below the suggested health effect levels for each model of hha in almost every case .", "however , the presence of disinfectants proved problematic with regard to detecting either seb or ricin using all models of hhas .", "the increased lods observed when disinfectants were present are believed to be due to the degradation of the target or toxins by chlorine and bromine .", "therefore , it is likely that protein toxins would also be affected by these strong oxidants .", "in fact , sodium hypochlorite is known to completely inactivate seb and ricin toxin [ 11 , 12 ] .", "therefore , when exposed to chlorine or bromine , protein toxins could become damaged or denatured causing antibodies present in an antibody - based assay , such as hhas , to no longer bind to the altered antigen as effectively .", "this decrease in binding efficiency could yield higher lods for the target , such as those observed in the present evaluation .   to address this issue , cd spectroscopy and sds - page", "the cd results for untreated seb were similar to those obtained by singh et al .", "for seb at 0.5 - 0.6  mg / ml in 10  mm sodium phosphate .", "the cd results for chlorine - treated seb indicate a significant change in three - dimensional fold resulting in less well - defined structure for the chlorine - treated seb relative to the untreated seb . based on curve - fitting calculations , the percentage of random coil more than doubled upon treatment with chlorine .", "the secondary structure percentages obtained from the cd curve - fitting routine may well represent an average of multiple seb - based species in solution , which would be consistent with the sds - page results .", "the absence of bands for the chlorine - treated seb could occur if the seb was cleaved into smaller peptides that diffuse out of the gel or if multiple higher molecular weight species are formed , with no single species being concentrated enough to be visualized on the gel . in either of these cases , chiral components would be present in the sample and would lead to a measureable cd spectrum . the cd and", "page results together suggest a major change in seb structure that may result in multiple seb - based antigens , the majority of which are less easily recognized and not bound as effectively as untreated seb by the antibody present on the membrane of the assay", ". the decreased binding capacity for the seb - based antigen(s ) would account for the increased detection limits seen for seb in chlorinated water .", "these results support the idea that increased lods in disinfected water are caused by disruption in antigen - antibody binding due to degradation of the antigen .  ", "in addition to the detection studies reported herein , currently studies are being performed to address whether both ricin and seb remain active , or toxic , after exposure to chlorine and bromine . however , regardless of whether activity remains , the ability to detect the presence of each toxin in disinfected water supplies is important .", "presence of these agents could indicate a potential compromise or attack on the water supply , thereby revealing security risks as well as potential health risks .", "additionally , it was noted in the present study that chlorine did not impact detection levels of ricin as significantly as detection levels of seb under the test conditions employed .", "additional exposure time may be required in order for ricin detection to be affected in the same manner as that observed for seb in chlorinated water .", "this could be due to a difference in protein structure or stability in chlorinated water between the two toxins .", "further tests allowing for prolonged incubation of ricin in chlorinated water prior to detection could address this issue ."], "abstract_text": ["<S> development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation . </S>", "<S> the present study sought to assess the effectiveness of handheld assays ( hhas ) in detecting ricin and staphylococcal enterotoxin b ( seb ) in water . </S>", "<S> performance of hhas was evaluated in formulated tap water with and without chlorine , reverse osmosis water ( ro ) with chlorine , and ro with bromine . </S>", "<S> each matrix was prepared , spiked with ricin or seb at multiple concentrations , and then loaded onto hhas . </S>", "<S> hhas were allowed to develop and then read visually . </S>", "<S> limits of detection ( lod ) were determined for all hhas in each water type . </S>", "<S> both ricin and seb were detected by hhas in formulated tap water at or below the suggested health effect levels of 455  ng / ml and 4.55  ng / ml , respectively . </S>", "<S> however , in brominated or chlorinated waters , lods for seb increased to approximately 2,500  ng / ml . lods for ricin increased in chlorinated water , but still remained below the suggested health effect level . in brominated water , the lod for ricin increased to approximately 2,500  ng / ml . in conclusion , the hhas tested were less effective at detecting ricin and seb in disinfected water , as currently configured . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["the protection of water supplies and water distribution infrastructure such as reservoirs is of great concern for those defending against bioterrorism . the us military is concerned because there exist multiple biological warfare agents ( bwas ) that could be intentionally introduced into the water supply which could have serious implications for military operations as well as civilians located within a theater of operations . to protect potable water supplies , the department of defense ( dod ) seeks to develop a rapid field test that would determine if field supplies of water are safe to drink by the warfighter during a military operation .", "the us armed services have fielded biological defense systems that are designed to protect against a large scale attack via bwas dispersed through the air .", "these defensive systems utilize high - efficiency aerosol collectors that impinge the sampled air into a liquid buffer which is tested for bwas using antibody - based handheld assays ( hhas ) .", "civilian first responders use similar versions of these antibody - based hhas for testing suspicious powder samples suspected to contain bwas . depending on which manufacturer of hha", "is being tested , the technology can be low cost , requires no electrical power , and requires minimal to no sample preparation to test a sample .", "it was an hha panel manufactured by the dod that first identified bacillus anthracis in the powder that spilled across a desk in senator daschle 's office in the hart senate office building in 2001 .", "three years later , a dod hha was again used to identify ricin toxin in the mail room of the dirksen senate office building .", "these instances demonstrate that , when hha technologies are used appropriately by an experienced operator trained to understand what the results mean , they can be a useful tool .", "additionally , hhas have proven fast and effective in the laboratory for the detection of francisella tularensis   and biotoxins such as staphylococcal enterotoxin b ( seb )   and microcystins .   the dod has initiated an effort called the joint chemical biological and radiological agent water monitor ( jcbrawm ) program to develop a system which will allow rapid field testing of drinking water supplies used by military personnel .", "the effectiveness of hhas in detecting biological agents in the air and in powders has resulted in a desire to test these assays to gauge their effectiveness to protect drinking water supplies used by us warfighters . in the present study , hhas from multiple manufacturers were evaluated for their effectiveness at detecting two specific targets , seb and ricin , in multiple water matrices .", "the hhas were not modified in any way to optimize for the detection of toxins within water supplies despite the fact that they were initially developed to test for concentrated agent collected from high - efficiency aerosol collectors and impinged into a phosphate buffered saline ( pbs ) solution rather than to test unbuffered water supplies .  ", "the ability of the hhas to detect seb and ricin in four different water matrices , representative of waters used by different branches of the armed forces , was evaluated .", "the four different water matrices were formulated tap water , formulated tap water with 1  mg / l free available chlorine ( fac ) , water treated by reverse osmosis ( ro ) with 1  mg / l fac , and ro water with 2  mg / l total residual bromine . suggested health effect levels as used by the us military for ricin and seb are 455  ng / ml and 4.55  ng / ml , respectively .", "these minimal health effect levels were derived using the tri service standards for field water and based on an average consumption of 10 liters per day for seven days for a 70  kg service member . therefore , detection of ricin and seb in water at or below the suggested health effect level is desired ."], ["both singleplex ( one - line ) and multiplex ( two - line and two - dot ) hha models produced by two manufacturers were evaluated in the present study for the detection of ricin toxin and seb [ 5 , 6 ] .", "active seb ( toxin technologies , sarasota , fla , usa ) and active ricin whole toxin ( vector laboratories , burlington , on , canada ) were obtained from the dod 's critical reagents program ( crp ) for use in the present study .", "briefly , approximately 500  ml of astm type i deionized water was added to a one - liter volumetric flask to which each stock solution was added to achieve the desired final concentration and the total volume was brought to one liter with deionized water .", "after 1520 minutes , the ph was assessed and adjusted if needed ( with minimal volume change ) to 7.67.8 .", "once prepared , synthetic tap water was stored at 4c with a shelf life of one week .  ", "briefly , one gram of calcium hypochlorite was added to twenty milliliters of astm type i deionized water and shaken vigorously for one to two minutes .", "briefly , approximately five microliters of the chlorine stock solution ( supernatant , not solids ) was added to 100  ml of the formulated tap water or ro water , which was prepared by running local tap water through a commercial ro unit .", "the fac was measured using a hach dr 2500 spectrophotometer per manufacturer 's instructions , and the fac level was adjusted as needed to achieve the desired fac level of 1 ", "chlorinated water was considered stable for only one day and therefore made fresh daily just prior to use .", "briefly , 25  ml of bromine resin was transferred to a 500  ml , 0.20  m filter unit ( corning life science , lowell , mass , usa ) .", "total residual bromine in the collected water was measured using a hach dr 2500 spectrophotometer per manufacturer 's instructions and adjusted as needed to achieve the desired final concentration of 2  mg / l .", "each water type was prepared fresh daily as described above and spiked separately with each target at desired concentrations .", "after spiking , 100  l of sample was loaded onto the sample well of the hha .", "the hha was allowed to develop for 15 minutes prior to reading the result by eye .", "each challenge concentration was tested a minimum of two independent times with a minimum of three handheld assays per concentration .", "limits of detection ( lod ) were determined for each hha type in each water matrix and defined as the minimum concentration of toxin that could be detected by eye .", "samples for circular dichroism ( cd ) were prepared by spiking ro water containing 1  mg / l fac with seb to a final concentration of 2  g / ml . control samples received no chlorine .", "after 24 hours , sodium thiosulfate ( 0.005% w / v final concentration ; fisher scientific catalog number s446 ) was added to all samples to quench any remaining chlorine prior to analysis by cd .", "cd experiments employed a jasco model j-810 spectropolarimeter ( jasco analytical instruments , easton , md , usa ) equipped with a ptc-423s peltier thermoelectric temperature control system .", "cd spectra were obtained for seb at 25c from 260 to 190  nm with a data pitch of 0.5  nm , a band width of 1  nm , a response time of 1  sec , and a scanning speed of 10  nm / min .", "the cd was carried out on 2  g / ml samples in 1  cm quartz cuvettes and required long - time signal averaging .", "the concentration of seb was verified for a representative stock solution by uv absorption at 277  nm using an e for seb of 14 .", "for each sample , the blank - subtracted cd curve for 100 averaged scans was converted to molar ellipticity using a molecular weight of 28,366 daltons and fit with the secondary structure estimation application in jasco 's spectra manager ( version 1.53.01 ) software to obtain the fraction of helix , beta sheet , turn , and random coil secondary structure .", "the percentages reported here were obtained using the reed reference set for the secondary structure calculations .  ", "samples for sds - page were prepared by spiking ro water containing 1  mg / l fac with seb to a final concentration of 2  g / ml . control samples received no chlorine .", "after one minute , one hour , and 24 hours , sodium thiosulfate ( 0.005% w / v final concentration ) was added to all samples to quench any remaining chlorine prior to analysis by sds - page . reducing gel eletrophoresis", "was carried out using the mini - protean 2 cell and 1020% tris - hcl pager gold precast gels from lonza ( rockland , me , usa ) .", "fifty nanograms of seb samples and 1  :  20 diluted mark12 unstained standard ( invitrogen ) were prepared in sds sample buffer , followed by heating at 95c for 5 minutes prior to loading .", "running buffer was prepared by dilution from 10x tris / glycine / sds buffer ( biorad ) .", "following electrophoresis , the gel was fixed with 50% methanol and 7% acetic acid ( both v / v ) for a total of 30 minutes .", "the rapid staining protocol with sypro ruby protein stain ( invitrogen ) was used for the detection of seb .", "the gel was washed with a solution of 10% methanol and 7% acetic acid ( both v / v ) for 30 minutes and imaged and documented with uv light using syngene ( frederick , md , usa ) ."], ["all hhas were evaluated for their performance in formulated tap water , formulated tap water with chlorine , ro water with chlorine , and ro water with bromine for the detection of ricin and seb .", "each water type was spiked with multiple concentrations of each toxin and loaded onto hhas for testing until the lod was determined for each hha model .", "it was determined that ricin could be detected down to approximately 1  ng / ml for most hha models in both formulated tap water and formulated tap with chlorine , while exhibiting only slightly higher lods in ro with chlorine for most models . however , in ro with bromine , the lod for ricin increased to 3,000  ng / ml for all models tested . for seb , lods in formulated tap water were low , ranging from 0.9  ng / ml to 12.5  ng / ml . however , in both chlorinated and brominated water , lods increased to 2,500  ng / ml for singleplex hhas and even higher to 5,000 or 10,000  ng / ml for multiplex models . in all cases", "detection below the suggested health effect level was desired ; however , seb was not detected below the suggested health effect level in chlorinated and brominated water and ricin was not detected below that level in brominated water .", "due to the increased detection limits observed in chlorinated and brominated water , cd spectroscopy , which assesses the three - dimensional fold of a protein , was performed on samples of untreated seb and seb exposed to chlorine in an effort to evaluate the basis for increased lods ( figure 1 ) . for each sample", ", the individual cd scans were compared prior to averaging to ascertain the structural stability of the sample .", "untreated seb at 2  g / ml was determined to contain 14.3% helix , 34.50% sheet , 17.6% turn , and 33.6% random coil ( rmsd < 0.1% ) .", "the cd spectrum of seb treated with 1  mg / l fac was stable over the time of experimentation .", "the curve - fitting calculation for chlorine - treated seb resulted in 25.9% helix , 0.0% sheet , 0.0% turn , and 74.1 random coil ( rmsd < 0.1% ) .   sds - page was also performed to ascertain the overall effect of chemical reaction with chlorine on the molecular weight of seb .", "for the treated and untreated seb , 1.5  g of seb was loaded per well . as shown in figure 2 , for untreated seb ,", "the seb was visible on the gel as a band with a molecular weight of approximately 28  kda for all time points tested . for chlorine - treated seb ,", "no band was visible at the same molecular weight as untreated seb for any of the time points tested ."], ["ricin and seb are considered potential biological threat agents as both can cause illness either by inhalation or ingestion .", "seb could potentially be used to attack low - volume water supplies ; however , due to the fact that ricin is less toxic by oral ingestion , it may be a less likely threat to drinking water .", "however , detection of both agents in water is still a concern and technologies capable of detection in water must be investigated .", "performance of hhas for air and surfaces is well known ; however , the use of hhas for detection of agents in water is less recognized .", "therefore , the application of hhas for detection in water was assessed in this study . in formulated tap water , detection of seb and ricin", "was achieved at or below the suggested health effect levels for each model of hha in almost every case .", "however , the presence of disinfectants proved problematic with regard to detecting either seb or ricin using all models of hhas .", "the increased lods observed when disinfectants were present are believed to be due to the degradation of the target or toxins by chlorine and bromine .", "therefore , it is likely that protein toxins would also be affected by these strong oxidants .", "in fact , sodium hypochlorite is known to completely inactivate seb and ricin toxin [ 11 , 12 ] .", "therefore , when exposed to chlorine or bromine , protein toxins could become damaged or denatured causing antibodies present in an antibody - based assay , such as hhas , to no longer bind to the altered antigen as effectively .", "this decrease in binding efficiency could yield higher lods for the target , such as those observed in the present evaluation .   to address this issue , cd spectroscopy and sds - page", "the cd results for untreated seb were similar to those obtained by singh et al .", "for seb at 0.5 - 0.6  mg / ml in 10  mm sodium phosphate .", "the cd results for chlorine - treated seb indicate a significant change in three - dimensional fold resulting in less well - defined structure for the chlorine - treated seb relative to the untreated seb . based on curve - fitting calculations , the percentage of random coil more than doubled upon treatment with chlorine .", "the secondary structure percentages obtained from the cd curve - fitting routine may well represent an average of multiple seb - based species in solution , which would be consistent with the sds - page results .", "the absence of bands for the chlorine - treated seb could occur if the seb was cleaved into smaller peptides that diffuse out of the gel or if multiple higher molecular weight species are formed , with no single species being concentrated enough to be visualized on the gel . in either of these cases , chiral components would be present in the sample and would lead to a measureable cd spectrum . the cd and", "page results together suggest a major change in seb structure that may result in multiple seb - based antigens , the majority of which are less easily recognized and not bound as effectively as untreated seb by the antibody present on the membrane of the assay", ". the decreased binding capacity for the seb - based antigen(s ) would account for the increased detection limits seen for seb in chlorinated water .", "these results support the idea that increased lods in disinfected water are caused by disruption in antigen - antibody binding due to degradation of the antigen .  ", "in addition to the detection studies reported herein , currently studies are being performed to address whether both ricin and seb remain active , or toxic , after exposure to chlorine and bromine . however , regardless of whether activity remains , the ability to detect the presence of each toxin in disinfected water supplies is important .", "presence of these agents could indicate a potential compromise or attack on the water supply , thereby revealing security risks as well as potential health risks .", "additionally , it was noted in the present study that chlorine did not impact detection levels of ricin as significantly as detection levels of seb under the test conditions employed .", "additional exposure time may be required in order for ricin detection to be affected in the same manner as that observed for seb in chlorinated water .", "this could be due to a difference in protein structure or stability in chlorinated water between the two toxins .", "further tests allowing for prolonged incubation of ricin in chlorinated water prior to detection could address this issue ."]]}
{"article_id": "PMC4772913", "article_text": ["osteosarcoma is a common primary malignant bone tumor with complicated pathogenesis and frequent distal metastasis , thus causing higher mortality rates , especially in the teenagers .", "epidemiological surveys revealed that nearly 40% of osteosarcoma patients died from misdiagnosis and/or early tumor metastasis , especially toward the lung tissue [ 24 ] .", "therefore , it is of critical importance to find a simple and effective biological marker for osteosarcoma , in order to make early diagnosis .", "current studies have revealed the role of gene mutation in the proliferation of tumor cells .", "cell apoptosis - related genes , including vascular epithelial growth factor ( vegf ) and microrna ( mir)-96 , were found to be closely correlated with the occurrence and prognosis of osteosarcoma .", "p15 , a novel tumor - suppressor gene , has been reported to play an important regulatory role in the progression of various malignant tumors , such as pulmonary cancer , nasopharynx cancer , and kidney cancer .", "the fragment loss , point mutation , or methylation of p15 gene may all lead to its abnormal tumor - suppressing function , thus facilitating the malignancy and metastasis of tumor cells .", "previous studies have been performed on the pathogenesis and related mechanisms of pulmonary metastasis of osteosarcoma , but the involvement of p15 gene has not been reported . as it is of critical importance for early diagnosis of osteosarcoma to discover a simple but effective biological tumor marker , this study aimed to describe the mechanism of p15 gene mutation in the occurrence and lung metastasis of osteosarcoma in a rat model , as previously described .", "this study may provide further evidence for developing methods in clinical diagnosis of malignant osteosarcoma .", "a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) .", "the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group .", "rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university .", "after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues .", "the tumor volume was approximated as : ( short diameter  long diameter)/2 , as reported elsewhere .", "the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration .", "we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature .", "pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody .", "after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times ,", "3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min .", "dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting .", "sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields", "we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) .", "peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above .", "after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents .", "total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) ,", "pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) .", "the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min .", "agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) .", "spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison", "a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) .", "the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group .", "rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university .", "after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues .", "morphological changes were observed , along with the measurement of tumor size . the tumor volume was approximated as : ( short diameter  long diameter)/2 , as reported elsewhere .", "the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration .", "we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature .", "pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody .", "after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times , 3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min .", "dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting .", "sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields", "we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) .", "peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above .", "after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents .", "total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) ,", "pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) .", "the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min .", "agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) .", "spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison", "one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites .", "as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) .", "abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week", ", the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues .", "on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly .", "a longitudinal dissection of the tumor revealed white and hard tissues with centralized necrosis . before tumor cell inoculation", "after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time .", "on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) .", "in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points", "further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) .", "one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites .", "as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) .", "abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week", ", the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues .", "on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly .", "after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time .", "on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) .", "in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points", "further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) .", "due to its unfavorable prognosis , early diagnosis and treatment is of critical importance for preventing metastasis of osteosarcoma lesions .", "the clinical features of osteosarcoma mainly include bone pain , hypercalcinemia , and osteolysis metastasis .", "the complication of other organ / tissues , such as the lung , may indicate the metastasis of tumors .", "the proliferation of primary tumor lesions causes the detachment of tumor cells , which penetrate vessel walls to enter the general lymph or blood circulation system for the invasion of distal tissues , where tumor  seeds ", "investigation of the mechanisms underlying the progression and metastasis of osteosarcoma is a popular research topic .", "recent studies revealed complicated genetic processes regulating the tumor metastasis , as a  hot - spot  located in chromosome 9p21 - 22 region affects gene fragment loss or transcriptional abnormalities in various malignant tumors [ 1719 ] .", "p15 gene , as a representative gene in this region , has been shown to have mutants and expression down - regulation in kidney and bladder cancer . in approximately 23% of cancer cells of", "it has been indicated that single - base mutation occurs in 67 , 119 , and 135 codons of p15 gene . due to the methylation of p15 gene ,", "hematopoietic progenitor cells lose the regulation of tgf--mediated growth inhibition , which in turn increased the incidence of malignant bone tumors .", "recent evidence suggests the complexity of the abnormal regulation of p15 gene involved in bone tumor metastasis .", "however , the relationship between p15 gene and lung metastasis of osteosarcoma remains to be determined .", "this study utilized umr-106 cell line , which is a classical osteosarcoma cell line induced by radioactive phosphorous isotope ( 32-p ) , to generate an osteosarcoma rat model by subcutaneous injection .", "the observation of tumor size and pulmonary tissue damage , along with p15 gene expression at different time points after the injection , showed normal tissue structures and almost no tumor cells in the control group . after the inoculation ,", "the overall survival rate of rats was 83.3% , in which about 48% of cases had pulmonary metastasis , suggesting the effectiveness and usefulness of this model .", "the tumor cell expression maintained at minimal level with lower mutation rate of p15 gene at 1 week after the inoculation . at 26 weeks , however , abnormal tumor - like structures began to occur in the pulmonary tissues in the model group , along with elevated p15 gene mutation rates over time .", "the tumor - positive cell percentage in pulmonary tissues increased from 10% to more than 72% at the final time point ( 6 weeks ) , suggesting the occurrence of lung metastasis , as consistent with previous reports .", "other studies demonstrated the suppressed incidence of esophagus cancer and pharynx squamous carcinoma by the inhibition of p15 gene methylation .", "furthermore , p15 expression level was higher in tongue squamous carcinoma without lymph node metastasis , compared to those patients with metastasis , who also had unfavorable prognosis .", "all these studies point to the importance of p15 gene in inhibiting tumor growth and invasion , both of which are consistent with our results in osteosarcoma .", "the proliferation of cells is highly dependent on the complete cell cycle , whose disruption or dysfunction may directly affect the tumor growth . p15 gene , as an inhibitory factor in cell - cycle - dependent kinase ( cdk ) , exerts its function mainly on g1 phase of the cell cycle via modulating the n - terminal protein expression of multiple tumor - suppressing factors , thereby inhibiting the abnormal growth of various malignant tumor cells .", "the correlation between p15 gene mutation in peripheral blood cells and the growth curve of osteosarcoma lesion suggests the invasion of tumor cells via blood circulation into distal sites , in addition to the facilitating role of mutant p15 in the pulmonary metastasis of osteosarcoma .", "our results indicate the close correlation between p15 gene mutation and growth of osteosarcoma , and the potentially direct involvement of p15 gene in pulmonary metastasis .", "our results suggest the potency of p15 mutant genes as a novel biological marker in early diagnosis of osteosarcoma in clinics , although its detailed molecular mechanism in progression and pulmonary metastasis require further study . molecular biology research is still required to elucidate the mechanism of mutation location of p15 gene , as well as its role in the metastasis of osteosarcoma to the lung ."], "abstract_text": ["<S> backgroundas a type of primary malignant bone tumor , osteosarcoma has high incidence and poor prognosis , and is predisposed for pulmonary metastasis . </S>", "<S> the abnormal expression of p15 gene directly participates in the invasion of various cancers . </S>", "<S> therefore , this study investigated the gene mutation of p15 in both primary lesion and pulmonary metastasis lesion of osteosarcoma in a rat model , in an attempt to elucidate the value of p15 gene as a biological marker.material/methodsa total of 60 sd rats were randomly divided into 2 groups . </S>", "<S> model rats had injection of osteosarcoma umr-106 cells ( 5106 ) inoculated underneath the right forelimb skin , while control rats received saline injection instead . </S>", "<S> six rats were sacrificed after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation . </S>", "<S> tissue samples from inoculation sites and lungs were extracted for measuring the tumor size . </S>", "<S> sp immunohistochemical ( ihc ) staining was used to detect the positive expression rate , while p15 gene mutation was detected by pcr method.resultswith the elongation of inoculation time , tumor size was significantly increased ( p<0.05 ) . </S>", "<S> the positive expression rates in both primary and pulmonary metastasis lesions were also significantly elevated ( p<0.05 ) . the occurrence rate of p15 gene mutation in model rats was significantly elevated and showed a correlation with the tumor formation ( r=0.998 , p<0.05).conclusionsthe p15 gene mutation was significantly correlated with osteosarcoma formation and metastasis towards the pulmonary tissue , suggesting its potency as a novel biological marker for early diagnosis of osteosarcoma . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Animal model", "SP-immunohistochemical (IHC) staining", "PCR for P15 gene mutation", "Statistical analysis", "Results", "Tumor size of osteosarcoma lesion", "Tumor cell percentage of osteosarcoma and pulmonary metastatic lesions by SP-IHC", "P15 gene mutation in rat peripheral blood cells", "Discussion", "Conclusions"], "sections": [["osteosarcoma is a common primary malignant bone tumor with complicated pathogenesis and frequent distal metastasis , thus causing higher mortality rates , especially in the teenagers .", "epidemiological surveys revealed that nearly 40% of osteosarcoma patients died from misdiagnosis and/or early tumor metastasis , especially toward the lung tissue [ 24 ] .", "therefore , it is of critical importance to find a simple and effective biological marker for osteosarcoma , in order to make early diagnosis .", "current studies have revealed the role of gene mutation in the proliferation of tumor cells .", "cell apoptosis - related genes , including vascular epithelial growth factor ( vegf ) and microrna ( mir)-96 , were found to be closely correlated with the occurrence and prognosis of osteosarcoma .", "p15 , a novel tumor - suppressor gene , has been reported to play an important regulatory role in the progression of various malignant tumors , such as pulmonary cancer , nasopharynx cancer , and kidney cancer .", "the fragment loss , point mutation , or methylation of p15 gene may all lead to its abnormal tumor - suppressing function , thus facilitating the malignancy and metastasis of tumor cells .", "previous studies have been performed on the pathogenesis and related mechanisms of pulmonary metastasis of osteosarcoma , but the involvement of p15 gene has not been reported . as it is of critical importance for early diagnosis of osteosarcoma to discover a simple but effective biological tumor marker , this study aimed to describe the mechanism of p15 gene mutation in the occurrence and lung metastasis of osteosarcoma in a rat model , as previously described .", "this study may provide further evidence for developing methods in clinical diagnosis of malignant osteosarcoma ."], ["a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) .", "the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group .", "rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university .", "after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues .", "the tumor volume was approximated as : ( short diameter  long diameter)/2 , as reported elsewhere .", "the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration .", "we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature .", "pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody .", "after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times ,", "3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min .", "dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting .", "sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields", "we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) .", "peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above .", "after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents .", "total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) ,", "pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) .", "the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min .", "agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) .", "spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison"], ["a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) .", "the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group .", "rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university ."], ["after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues .", "morphological changes were observed , along with the measurement of tumor size . the tumor volume was approximated as : ( short diameter  long diameter)/2 , as reported elsewhere .", "the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration .", "we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature .", "pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody .", "after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times , 3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min .", "dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting .", "sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields", "we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) ."], ["peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above .", "after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents .", "total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) ,", "pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) .", "the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min .", "agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) ."], ["spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison"], ["one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites .", "as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) .", "abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week", ", the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues .", "on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly .", "a longitudinal dissection of the tumor revealed white and hard tissues with centralized necrosis . before tumor cell inoculation", "after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time .", "on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) .", "in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points", "further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) ."], ["one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites .", "as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) .", "abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week", ", the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues .", "on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly ."], ["after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time .", "on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) ."], ["in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points", "further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) ."], ["due to its unfavorable prognosis , early diagnosis and treatment is of critical importance for preventing metastasis of osteosarcoma lesions .", "the clinical features of osteosarcoma mainly include bone pain , hypercalcinemia , and osteolysis metastasis .", "the complication of other organ / tissues , such as the lung , may indicate the metastasis of tumors .", "the proliferation of primary tumor lesions causes the detachment of tumor cells , which penetrate vessel walls to enter the general lymph or blood circulation system for the invasion of distal tissues , where tumor  seeds ", "investigation of the mechanisms underlying the progression and metastasis of osteosarcoma is a popular research topic .", "recent studies revealed complicated genetic processes regulating the tumor metastasis , as a  hot - spot  located in chromosome 9p21 - 22 region affects gene fragment loss or transcriptional abnormalities in various malignant tumors [ 1719 ] .", "p15 gene , as a representative gene in this region , has been shown to have mutants and expression down - regulation in kidney and bladder cancer . in approximately 23% of cancer cells of", "it has been indicated that single - base mutation occurs in 67 , 119 , and 135 codons of p15 gene . due to the methylation of p15 gene ,", "hematopoietic progenitor cells lose the regulation of tgf--mediated growth inhibition , which in turn increased the incidence of malignant bone tumors .", "recent evidence suggests the complexity of the abnormal regulation of p15 gene involved in bone tumor metastasis .", "however , the relationship between p15 gene and lung metastasis of osteosarcoma remains to be determined .", "this study utilized umr-106 cell line , which is a classical osteosarcoma cell line induced by radioactive phosphorous isotope ( 32-p ) , to generate an osteosarcoma rat model by subcutaneous injection .", "the observation of tumor size and pulmonary tissue damage , along with p15 gene expression at different time points after the injection , showed normal tissue structures and almost no tumor cells in the control group . after the inoculation ,", "the overall survival rate of rats was 83.3% , in which about 48% of cases had pulmonary metastasis , suggesting the effectiveness and usefulness of this model .", "the tumor cell expression maintained at minimal level with lower mutation rate of p15 gene at 1 week after the inoculation . at 26 weeks , however , abnormal tumor - like structures began to occur in the pulmonary tissues in the model group , along with elevated p15 gene mutation rates over time .", "the tumor - positive cell percentage in pulmonary tissues increased from 10% to more than 72% at the final time point ( 6 weeks ) , suggesting the occurrence of lung metastasis , as consistent with previous reports .", "other studies demonstrated the suppressed incidence of esophagus cancer and pharynx squamous carcinoma by the inhibition of p15 gene methylation .", "furthermore , p15 expression level was higher in tongue squamous carcinoma without lymph node metastasis , compared to those patients with metastasis , who also had unfavorable prognosis .", "all these studies point to the importance of p15 gene in inhibiting tumor growth and invasion , both of which are consistent with our results in osteosarcoma .", "the proliferation of cells is highly dependent on the complete cell cycle , whose disruption or dysfunction may directly affect the tumor growth . p15 gene , as an inhibitory factor in cell - cycle - dependent kinase ( cdk ) , exerts its function mainly on g1 phase of the cell cycle via modulating the n - terminal protein expression of multiple tumor - suppressing factors , thereby inhibiting the abnormal growth of various malignant tumor cells .", "the correlation between p15 gene mutation in peripheral blood cells and the growth curve of osteosarcoma lesion suggests the invasion of tumor cells via blood circulation into distal sites , in addition to the facilitating role of mutant p15 in the pulmonary metastasis of osteosarcoma ."], ["our results indicate the close correlation between p15 gene mutation and growth of osteosarcoma , and the potentially direct involvement of p15 gene in pulmonary metastasis .", "our results suggest the potency of p15 mutant genes as a novel biological marker in early diagnosis of osteosarcoma in clinics , although its detailed molecular mechanism in progression and pulmonary metastasis require further study . molecular biology research is still required to elucidate the mechanism of mutation location of p15 gene , as well as its role in the metastasis of osteosarcoma to the lung ."]]}
{"article_id": "PMC4212291", "article_text": ["the term was used by hallopeau in 1889 and is derived from the greek ; thrix - hair , tillein - pull out and mania - madness .", "scalp is the most common site for pulling hair although other hair bearing areas may be involved . in less severe forms", "diagnosis is mainly by history and clinical examination , nevertheless , sometimes it is very difficult to differentiate it from other causes of noncicatricial alopecia .", "trichoscopy , dermoscopy hair and scalp , is a noninvasive technique for differential diagnosis of various hair and scalp diseases . here", "authors evaluated trichoscopic patterns in ttm and authors believe that these patterns are specific to ttm , which can aide in early diagnosis of this chronic condition .", "this study was carried out on 10 patients attending department of dermatology in a tertiary hospital attached to s. nijalingappa medical college at bagalkot , south india between january 2014 and july 2014 .", "ten patients with clinical features of ttm were subjected for a complete history and dermatological examination .", "demographic data such as age and gender and clinical variables in terms of site of lesions and disease duration were documented .", "dermlite 3 dermoscope ( 10 magnifications ) with both polarized and nonpolarized lights was employed in the study .", "sony camera ( digital , 14 mega pixels ) was attached to save the images .", "initially , ultrasound gel was applied either on the faceplate of dermoscopy or on the skin lesions and then lesions were observed through the eyepiece of dermoscopy .", "although polarized dermoscopy was employed , ultrasound gel was applied for clarity of images and to lessen distortions associated with light .", "this study was carried out on 10 patients attending department of dermatology in a tertiary hospital attached to s. nijalingappa medical college at bagalkot , south india between january 2014 and july 2014 .", "ten patients with clinical features of ttm were subjected for a complete history and dermatological examination .", "demographic data such as age and gender and clinical variables in terms of site of lesions and disease duration were documented .", "dermlite 3 dermoscope ( 10 magnifications ) with both polarized and nonpolarized lights was employed in the study . however , only polarized light version was used in our study .", "sony camera ( digital , 14 mega pixels ) was attached to save the images .", "initially , ultrasound gel was applied either on the faceplate of dermoscopy or on the skin lesions and then lesions were observed through the eyepiece of dermoscopy .", "although polarized dermoscopy was employed , ultrasound gel was applied for clarity of images and to lessen distortions associated with light .", "mean age of the patients was 34 years ( minimum age 13 years and maximum age 55 years ) .", "mean duration of disease was 19 months ( minimum 2 months and maximum 36 months ) .", "most common symptom was patchy loss of hair over the scalp ( 100% ) especially in the frontal area [ figure 1 ] and one patient ( 10% ) had tonsure pattern of hair loss .", "four patients agreed to knowingly pulling of hair , and one patient ( 10% ) gave a history of eating them ( trichophagia ) .", "the most common trichoscopic pattern observed in all patients was decreased hair density and hairs broken at different lengths ( 100% ) [ figure 2 ] .", "excoriations are also seen on the forehead trichoscopy showing decreased hair density , trichoptilosis ( yellow arrows ) , tulip hairs ( red arrows ) and perifollicular whitish areas ( red stars ) short hair with trichoptilosis (  split ends  ) [ figures 25 ] , irregular coiled hairs [ figure 3 ] and upright re - growing hairs were demonstrated in 8 patients ( 80% ) each .", "the novel diagnostic signs like black dots and flame hair [ figure 4 ] , v - sign [ figure 3 ] , follicular hemorrhages [ figure 5 ] were observed in 3 patients ( 30% ) each .", "tulip hair and hair powder [ figure 6 ] patterns were observed in 1 ( 10% ) patient each .", "all patients ( 100% ) had the noninflammatory alopecia with follicles having distorted and collapsed inner root sheath [ figure 7 ] in histopathology .", "trichoscopy showing coiled hairs ( red arrows ) and v - hairs or v - sign ( yellow arrows ) trichoscopy showing flame hairs ( red arrows ) and black dots ( yellow arrows ) trichoscopy showing decreased hair density , follicular hemorrhage ( black arrow ) and trichoptilosis ( red arrows ) trichoscopy showing tulip hairs ( red arrows ) and hair powder ( yellow arrows ) histopathology showing diffuse , noninflammatory alopecia , missing hair shafts ( black stars ) and inward collapse of outer root sheath ( black arrows ) .", "few unaffected follicles are also seen ( yellow arrows ) ( h and e , 4 ) histopathology showing an irregular , deformed and pigmented shaft characteristic of trichomalacia ( h and e , 40 )", "trichotillomania is a chronic impulse control disorder characterized by repetitive hair pulling resulting in alopecia . in young children , it is usually a habit that resolves spontaneously or with minimal treatment . in older age groups , adolescents and adults ,", "seen predominantly in females and evidence of some form of psychological or behavioral stress is often apparent .", "the favorite site is the easily reached fronto - parietal region of the scalp followed by eyelashes , eyebrows , pubic hair , body hair and facial hair .", "this results in patchy loss of hair , often in a bizarre or angular pattern , in which the hairs are twisted and broken at various distances from the clinically normal scalp .", "a hairball , trichobezoar , is a rare accompaniment of ttm in those who also eat the plucked hair ( trichophagia ) . in our study ,", "most of the patients belonged to adolescent , and middle age group and most of them were females .", "all of them presented with patchy hair loss over the scalp , none of the patients gave a history of hair loss in other areas .", "only one patient had a history of eating of pulled hairs , but there were no signs and symptoms of trichobezoar in her .", "the diagnosis of ttm is often difficult as other hair loss diseases appear similar in clinical manifestations .", "this difficulty is encountered especially with alopecia areata ( aa ) . as ttm often coexists with aa", "trichoscopy is a novel diagnostic technique , both simple and noninvasive , can be used as a handy bed side tool for diagnosing common hair and scalp disorder .", "several common trichoscopy features of ttm were identified . decreased hair density , hairs broken at different lengths , short hairs with trichoptilosis ( \" split ends \" ) are described in the literature as diagnostic of ttm .", "authors observed these findings in all the patients , and this is due to irregular and repetitive pulling of hairs leading to damage to the cuticle . irregular coiled hairs and upright re - growing hairs are also reported to be seen in ttm . in this study , these findings were demonstrated in 80% of patients . coiled hair results from hair shaft fracture and coiling of the remaining proximal part which is fixed to the scalp .", "black dots are believed to be remnants of hair shafts arising from tapering hairs , broken hairs , and bent hairs . in a study conducted by shim et al .", "rakowska et al . observed that black dots tend to be uniform in size and shape in aa , whereas in ttm and tinea capitis they are variable in diameter and round , oval , irregular in shape .", "it presents as a red dot corresponding to follicular ostia capped or stuffed with the blood clot and suggests a history of traumatic forced plucking .", "recently , flame hairs , v - sign , tulip hairs , and hair powder are described , and authors claim these signs are specific only for ttm .", "flame hairs are semi - transparent , wavy and cone - shaped hair residues , which develop as a result of severe mechanical hair pulling and shredding .", "v - sign is created when two or more hairs emerge from one follicular ostium , which are pulled simultaneously and break at the same length above scalp surface .", "when hair shafts are almost totally damaged by mechanical manipulation , only a sprinkled hair residue is visible .", "this finding is referred to as  hair powder .  in the present study , these trichoscopic patterns were demonstrated in variable frequency . in aa , the common trichoscopic findings are yellow dots , uniform black dots , broken hair , trichoptilosis , upright re - growing hair , and vellus hair .", "as some of the trichoscopic features are overlapping in ttm and aa , histopathology plays a corroborative role in definitive diagnosis .", "the histopathological features of ttm include empty follicles , incomplete disrupted follicular anatomy , trichomalacia , and pigment casts without significant inflammation .", "collapsed inner root sheath and missing hair shaft indicate the extraction of the hair follicle due to forcible hair pulling . partially extracted and distorted root sheath material with fragments of pigment can be identified as ", "the germinative follicular epithelium may produce abnormally formed , diminished and distorted hair shaft with irregular pigmentation as a result of trauma .", "a new trichoscopic pattern was observed by the authors in one patient , in the form of perifollicular whitish areas [ figure 2 ] .", "authors believe these whitish areas represent hyperkeratosis that is due to perifollicular damage as a result of repeated hair pulling", "trichotillomania is often chronic and difficult to treat . patients may attempt to disguise the condition due to its social implications . hence", "trichoscopy , being a noninvasive and in vivo diagnostic technique , can be utilized in this condition as it plays a vital role in the diagnosis of this condition by demonstrating specific trichoscopic patterns .", "further studies on trichoscopic patterns correlating with duration of disease and histopathology of ttm are suggested ."], "abstract_text": ["<S> introduction : trichotillomania ( ttm ) is characterized by patchy alopecia of hair bearing areas . this is because of compulsive urge to pull the hair . </S>", "<S> scalp is the most common site for pulling hair . </S>", "<S> usually , patients may have only small areas of baldness , in severe forms , tonsure pattern of baldness is observed . </S>", "<S> diagnosis is by history and clinical examination . </S>", "<S> however , it is difficult to differentiate from other causes of noncicatricial alopecia . here , authors observed trichoscopic patterns and evaluated their importance in the diagnosis of ttm.materials and methods : this study was conducted in s. nijalingappa medical college , bagalkot , from january 2014 to july 2014 . </S>", "<S> ten patients with clinically suspected ttm were included in the study . </S>", "<S> informed consent was taken and ethical clearance was obtained . </S>", "<S> dermlite3 dermoscope was used with sony camera attachment to save the images . </S>", "<S> histopathological examination was conducted in all the patients to confirm the diagnosis.results:ten patients were included in the study . </S>", "<S> mean age of the patients was 34 years . </S>", "<S> most common symptom was patchy loss of hair in the frontal area ( 100% ) . </S>", "<S> common trichoscopic feature was decreased hair density and broken hairs . </S>", "<S> trichoptilosis ( split ends ) and irregular coiled hairs were seen in 80% patients . </S>", "<S> novel diagnostic signs like black dots , flame hair , v - sign , follicular hemorrhages , were seen in 30% each . </S>", "<S> tulip hair and hair powder were observed in 10% of patients . </S>", "<S> all patients had the noninflammatory alopecia with distorted and collapsed inner root sheath in histopathology.conclusion:trichotillomania is often chronic and difficult to treat . hence </S>", "<S> , early diagnosis and treatment is necessary . </S>", "<S> authors believe that the trichoscopy plays a vital role in the diagnosis of this condition by demonstrating specific trichoscopic patterns . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Patients", "Trichoscopic examination", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["the term was used by hallopeau in 1889 and is derived from the greek ; thrix - hair , tillein - pull out and mania - madness .", "scalp is the most common site for pulling hair although other hair bearing areas may be involved . in less severe forms", "diagnosis is mainly by history and clinical examination , nevertheless , sometimes it is very difficult to differentiate it from other causes of noncicatricial alopecia .", "trichoscopy , dermoscopy hair and scalp , is a noninvasive technique for differential diagnosis of various hair and scalp diseases . here", "authors evaluated trichoscopic patterns in ttm and authors believe that these patterns are specific to ttm , which can aide in early diagnosis of this chronic condition ."], ["this study was carried out on 10 patients attending department of dermatology in a tertiary hospital attached to s. nijalingappa medical college at bagalkot , south india between january 2014 and july 2014 .", "ten patients with clinical features of ttm were subjected for a complete history and dermatological examination .", "demographic data such as age and gender and clinical variables in terms of site of lesions and disease duration were documented .", "dermlite 3 dermoscope ( 10 magnifications ) with both polarized and nonpolarized lights was employed in the study .", "sony camera ( digital , 14 mega pixels ) was attached to save the images .", "initially , ultrasound gel was applied either on the faceplate of dermoscopy or on the skin lesions and then lesions were observed through the eyepiece of dermoscopy .", "although polarized dermoscopy was employed , ultrasound gel was applied for clarity of images and to lessen distortions associated with light ."], ["this study was carried out on 10 patients attending department of dermatology in a tertiary hospital attached to s. nijalingappa medical college at bagalkot , south india between january 2014 and july 2014 .", "ten patients with clinical features of ttm were subjected for a complete history and dermatological examination .", "demographic data such as age and gender and clinical variables in terms of site of lesions and disease duration were documented ."], ["dermlite 3 dermoscope ( 10 magnifications ) with both polarized and nonpolarized lights was employed in the study . however , only polarized light version was used in our study .", "sony camera ( digital , 14 mega pixels ) was attached to save the images .", "initially , ultrasound gel was applied either on the faceplate of dermoscopy or on the skin lesions and then lesions were observed through the eyepiece of dermoscopy .", "although polarized dermoscopy was employed , ultrasound gel was applied for clarity of images and to lessen distortions associated with light ."], ["mean age of the patients was 34 years ( minimum age 13 years and maximum age 55 years ) .", "mean duration of disease was 19 months ( minimum 2 months and maximum 36 months ) .", "most common symptom was patchy loss of hair over the scalp ( 100% ) especially in the frontal area [ figure 1 ] and one patient ( 10% ) had tonsure pattern of hair loss .", "four patients agreed to knowingly pulling of hair , and one patient ( 10% ) gave a history of eating them ( trichophagia ) .", "the most common trichoscopic pattern observed in all patients was decreased hair density and hairs broken at different lengths ( 100% ) [ figure 2 ] .", "excoriations are also seen on the forehead trichoscopy showing decreased hair density , trichoptilosis ( yellow arrows ) , tulip hairs ( red arrows ) and perifollicular whitish areas ( red stars ) short hair with trichoptilosis (  split ends  ) [ figures 25 ] , irregular coiled hairs [ figure 3 ] and upright re - growing hairs were demonstrated in 8 patients ( 80% ) each .", "the novel diagnostic signs like black dots and flame hair [ figure 4 ] , v - sign [ figure 3 ] , follicular hemorrhages [ figure 5 ] were observed in 3 patients ( 30% ) each .", "tulip hair and hair powder [ figure 6 ] patterns were observed in 1 ( 10% ) patient each .", "all patients ( 100% ) had the noninflammatory alopecia with follicles having distorted and collapsed inner root sheath [ figure 7 ] in histopathology .", "trichoscopy showing coiled hairs ( red arrows ) and v - hairs or v - sign ( yellow arrows ) trichoscopy showing flame hairs ( red arrows ) and black dots ( yellow arrows ) trichoscopy showing decreased hair density , follicular hemorrhage ( black arrow ) and trichoptilosis ( red arrows ) trichoscopy showing tulip hairs ( red arrows ) and hair powder ( yellow arrows ) histopathology showing diffuse , noninflammatory alopecia , missing hair shafts ( black stars ) and inward collapse of outer root sheath ( black arrows ) .", "few unaffected follicles are also seen ( yellow arrows ) ( h and e , 4 ) histopathology showing an irregular , deformed and pigmented shaft characteristic of trichomalacia ( h and e , 40 )"], ["trichotillomania is a chronic impulse control disorder characterized by repetitive hair pulling resulting in alopecia . in young children , it is usually a habit that resolves spontaneously or with minimal treatment . in older age groups , adolescents and adults ,", "seen predominantly in females and evidence of some form of psychological or behavioral stress is often apparent .", "the favorite site is the easily reached fronto - parietal region of the scalp followed by eyelashes , eyebrows , pubic hair , body hair and facial hair .", "this results in patchy loss of hair , often in a bizarre or angular pattern , in which the hairs are twisted and broken at various distances from the clinically normal scalp .", "a hairball , trichobezoar , is a rare accompaniment of ttm in those who also eat the plucked hair ( trichophagia ) . in our study ,", "most of the patients belonged to adolescent , and middle age group and most of them were females .", "all of them presented with patchy hair loss over the scalp , none of the patients gave a history of hair loss in other areas .", "only one patient had a history of eating of pulled hairs , but there were no signs and symptoms of trichobezoar in her .", "the diagnosis of ttm is often difficult as other hair loss diseases appear similar in clinical manifestations .", "this difficulty is encountered especially with alopecia areata ( aa ) . as ttm often coexists with aa", "trichoscopy is a novel diagnostic technique , both simple and noninvasive , can be used as a handy bed side tool for diagnosing common hair and scalp disorder .", "several common trichoscopy features of ttm were identified . decreased hair density , hairs broken at different lengths , short hairs with trichoptilosis ( \" split ends \" ) are described in the literature as diagnostic of ttm .", "authors observed these findings in all the patients , and this is due to irregular and repetitive pulling of hairs leading to damage to the cuticle . irregular coiled hairs and upright re - growing hairs are also reported to be seen in ttm . in this study , these findings were demonstrated in 80% of patients . coiled hair results from hair shaft fracture and coiling of the remaining proximal part which is fixed to the scalp .", "black dots are believed to be remnants of hair shafts arising from tapering hairs , broken hairs , and bent hairs . in a study conducted by shim et al .", "rakowska et al . observed that black dots tend to be uniform in size and shape in aa , whereas in ttm and tinea capitis they are variable in diameter and round , oval , irregular in shape .", "it presents as a red dot corresponding to follicular ostia capped or stuffed with the blood clot and suggests a history of traumatic forced plucking .", "recently , flame hairs , v - sign , tulip hairs , and hair powder are described , and authors claim these signs are specific only for ttm .", "flame hairs are semi - transparent , wavy and cone - shaped hair residues , which develop as a result of severe mechanical hair pulling and shredding .", "v - sign is created when two or more hairs emerge from one follicular ostium , which are pulled simultaneously and break at the same length above scalp surface .", "when hair shafts are almost totally damaged by mechanical manipulation , only a sprinkled hair residue is visible .", "this finding is referred to as  hair powder .  in the present study , these trichoscopic patterns were demonstrated in variable frequency . in aa , the common trichoscopic findings are yellow dots , uniform black dots , broken hair , trichoptilosis , upright re - growing hair , and vellus hair .", "as some of the trichoscopic features are overlapping in ttm and aa , histopathology plays a corroborative role in definitive diagnosis .", "the histopathological features of ttm include empty follicles , incomplete disrupted follicular anatomy , trichomalacia , and pigment casts without significant inflammation .", "collapsed inner root sheath and missing hair shaft indicate the extraction of the hair follicle due to forcible hair pulling . partially extracted and distorted root sheath material with fragments of pigment can be identified as ", "the germinative follicular epithelium may produce abnormally formed , diminished and distorted hair shaft with irregular pigmentation as a result of trauma .", "a new trichoscopic pattern was observed by the authors in one patient , in the form of perifollicular whitish areas [ figure 2 ] .", "authors believe these whitish areas represent hyperkeratosis that is due to perifollicular damage as a result of repeated hair pulling"], ["trichotillomania is often chronic and difficult to treat . patients may attempt to disguise the condition due to its social implications . hence", "trichoscopy , being a noninvasive and in vivo diagnostic technique , can be utilized in this condition as it plays a vital role in the diagnosis of this condition by demonstrating specific trichoscopic patterns .", "further studies on trichoscopic patterns correlating with duration of disease and histopathology of ttm are suggested ."]]}
{"article_id": "PMC2779291", "article_text": ["the prevalence of syphilis in the normal population has decreased significantly since 1977 in korea ( 1 , 2 ) . however ,", "in western countries the incidence of syphilis has risen , mainly in the 20 - 24 age group and its clinical manifestations have been various ( 3 ) .", "sexual activity has become more acceptable in our culture recently , initiating various forms of sex , and as a result , many different clinical conditions of sexually transmitted disease ( std ) have appeared .", "it is usually asymptomatic and less frequently presents as proctitis , ulcer and pseudotumors ( 3 , 4 ) .", "therefore , it is difficult to diagnose and physicians occasionally might prescribe inappropriate treatments ( 5 , 6 ) . in this paper", "we describe a case of primary rectal syphilis which was suspected to be rectal cancer .", "he complained of bowel habit change with tenesmus , mucous discharge , anal pain and intermittent presence of blood in stool .", "he had a 2 cm sized , firm , non - tender and indurated nodule on the left side of the inguinal area and multiple condyloma acuminatum around the anus .", "sigmoidoscopy in our gastrointestinal endoscopy unit showed two indurated masses of 1.5 to 2 cm on the posterior wall of the middle and lower rectum ( fig .", "the masses had a coin like appearance , and each of them had a slightly depressed and ulcerated surface .", "the vdrl quantitative test was 1:64 and the fta - abs igm and igg test was positive . a human immunodeficiency virus ( hiv )", "the patient was treated with intramuscular penicillin g benzathine ( 2,400,000 iu im/1 week , thrice ) .", "follow - up sigmoidoscopy after 1 month showed nearly complete regression of the chancre ( fig .", "a repeat biopsy was done and the histologic result showed focal lymphoid hyperplasia . a vdrl quantitative test after 3 months was 1:1 .", "most physicians have a tendency to consider rectal ulceration as a neoplasm ( 7 , 8) . similarly in our case symptoms and signs suggested rectal neoplasia and the clinical history did not reveal homosexuality .", "however , after the vdrl test the patient confessed that he had been raped by a homosexual male . therefore , when rectal syphilis is suspected ( i.e. , perianal condyloma acuminatum associated with inguinal lymphadenopathy ) the history of sexual intercourse , especially rectal intercourse , has to be taken .", "a dark - field examination is recommended by many studies ( 3 ) . in our case , however , a dark - field examination was not performed , because rectal chancre was highly suggested by the clinical history , anorectal lesions , pathologic findings and positive serologic test . additionally , like other syphilis cases benzathine penicillin therapy induced a rapid regression of the rectal lesions .", "we believe when a positive serologic syphilis test is associated with ulcerative lesion , additional laboratory tests are not necessary .", "inguinal lymphadenopathy need not require an initial biopsy when evidence of regional infection is present . usually after antibiotic therapy of at least 4 weeks", ", most physicians might decide whether a biopsy is necessary . in this case , however , the physician did not have experience recognizing rectal syphilis , so he immediately obtained a biopsy specimen from the inguinal lesion .", ". in western countries the incidence of rectal syphilis is rising , especially in groups of active homosexual males .", "bassi et al . emphasized in his case report that endoscopists should keep in mind the rising incidence of syphilis ( 3 ) .", "therefore an initial physician should encourage his / her patient to inform him / her of the patient 's sexual history ."], "abstract_text": ["<S> a 30-yr - old man was referred for suspicious rectal cancer because of ulcerated lesions in the rectum and a palpable mass in left inguinal area . </S>", "<S> sigmoidoscopy showed two indurated masses and histologic evaluation of biopsy revealed obliterative endarteritis with heavy plasma cell infiltration . both venereal disease research laboratories ( vdrl ) and </S>", "<S> fluorescent treponemal antibody absorption ( fta - abs ) tests were positive . </S>", "<S> after injection of penicillin g benzathine for 3 weeks , the rectal chancre and the palpable mass disappeared . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["the prevalence of syphilis in the normal population has decreased significantly since 1977 in korea ( 1 , 2 ) . however ,", "in western countries the incidence of syphilis has risen , mainly in the 20 - 24 age group and its clinical manifestations have been various ( 3 ) .", "sexual activity has become more acceptable in our culture recently , initiating various forms of sex , and as a result , many different clinical conditions of sexually transmitted disease ( std ) have appeared .", "it is usually asymptomatic and less frequently presents as proctitis , ulcer and pseudotumors ( 3 , 4 ) .", "therefore , it is difficult to diagnose and physicians occasionally might prescribe inappropriate treatments ( 5 , 6 ) . in this paper", "we describe a case of primary rectal syphilis which was suspected to be rectal cancer ."], ["he complained of bowel habit change with tenesmus , mucous discharge , anal pain and intermittent presence of blood in stool .", "he had a 2 cm sized , firm , non - tender and indurated nodule on the left side of the inguinal area and multiple condyloma acuminatum around the anus .", "sigmoidoscopy in our gastrointestinal endoscopy unit showed two indurated masses of 1.5 to 2 cm on the posterior wall of the middle and lower rectum ( fig .", "the masses had a coin like appearance , and each of them had a slightly depressed and ulcerated surface .", "the vdrl quantitative test was 1:64 and the fta - abs igm and igg test was positive . a human immunodeficiency virus ( hiv )", "the patient was treated with intramuscular penicillin g benzathine ( 2,400,000 iu im/1 week , thrice ) .", "follow - up sigmoidoscopy after 1 month showed nearly complete regression of the chancre ( fig .", "a repeat biopsy was done and the histologic result showed focal lymphoid hyperplasia . a vdrl quantitative test after 3 months was 1:1 ."], ["most physicians have a tendency to consider rectal ulceration as a neoplasm ( 7 , 8) . similarly in our case symptoms and signs suggested rectal neoplasia and the clinical history did not reveal homosexuality .", "however , after the vdrl test the patient confessed that he had been raped by a homosexual male . therefore , when rectal syphilis is suspected ( i.e. , perianal condyloma acuminatum associated with inguinal lymphadenopathy ) the history of sexual intercourse , especially rectal intercourse , has to be taken .", "a dark - field examination is recommended by many studies ( 3 ) . in our case , however , a dark - field examination was not performed , because rectal chancre was highly suggested by the clinical history , anorectal lesions , pathologic findings and positive serologic test . additionally , like other syphilis cases benzathine penicillin therapy induced a rapid regression of the rectal lesions .", "we believe when a positive serologic syphilis test is associated with ulcerative lesion , additional laboratory tests are not necessary .", "inguinal lymphadenopathy need not require an initial biopsy when evidence of regional infection is present . usually after antibiotic therapy of at least 4 weeks", ", most physicians might decide whether a biopsy is necessary . in this case , however , the physician did not have experience recognizing rectal syphilis , so he immediately obtained a biopsy specimen from the inguinal lesion .", ". in western countries the incidence of rectal syphilis is rising , especially in groups of active homosexual males .", "bassi et al . emphasized in his case report that endoscopists should keep in mind the rising incidence of syphilis ( 3 ) .", "therefore an initial physician should encourage his / her patient to inform him / her of the patient 's sexual history ."]]}
{"article_id": "PMC4823619", "article_text": ["the protective effects of vagus nerve during ischemia / reperfusion ( i / r ) have been suggested ( 1 ) .", "vagus nerve stimulation decreases the inflammation and the injuries resulted from i / r through affecting neutrophils while vagotomy increases these processes ( 1 ) .", "moreover , it has been observed that stimulation of vagus nerve can cause protective effects on gastric injury through releasing prostaglandins , nitric oxide ( no ) and calcitonin gene related peptide ( cgrp ) ( 2 ) .", "it is probable that vagus nerve exerts its gastric protective effects through releasing the aforementioned mediators .", "also , it has been reported that cholinergic activity of the efferent vagus nerve can participate in immunity modulation ( 3 ) .", "another study has shown that nicotine in monocytes not only decreases the production of pre - inflammatory cytokines , but also changes the response to il-10 , an anti - inflammatory cytokine ( 4 , 5 ) .", "cho et al have shown that alternative stimulation of cervical vagus nerve increases intra - gastric pressure and also causes bleeding ulcers in mucosal glands of stomach , and these effects can be prevented by atropine administration or sub - diaphragmatic vagotomy ( 6 ) .", "it has been reported that gastric vagotomy is effective in treating gastric and duodenal ulcers ( 7 ) .", "melatonin as a neurohormone is basically produced in the pineal gland and also in epiphysis , gastrointestinal ( gi ) mucosa and other organs .", "melatonin has several physiological activities such as controlling circadian rhythm , sleep induction , regulation of seasonal reproduction , and improvement of immunity .", "the most important effect of melatonin is an anti - oxidant effect that protects living organisms against oxidative stress ( 11 ) . in several studies ,", "the ability of melatonin in decreasing molecular injury has been shown during i / r .", "melatonin prevents myocardial infarction , necrotic cell death and renal abnormality after i / r ( 8 , 12 ) , reduces brain edema in ischemia ( 12 ) , and prevents acute gastric ulcers resulted from stress .", "these effects can be done through direct reactive oxygen species / reactive nitrogen species ( ros / rns ) detoxification , increase in anti - oxidative enzymes , and the prevention of electron leakage in mitochondrial inner membrane ( 11 , 13 ) .", "melatonin has a role in expression and function of nicotinic acetylcholine receptors and increases the efficacy of beta bungarotoxin - sensitive acetylcholine receptors ( 14 ) .", "it has been recognized that intra - cerebral injection of melatonin is effective in prevention of acid and pepsin secretion through cholinergic activity ( 15 ) .", "it has been identified that 5ht3 and 5ht2 receptors are involved in stimulatory effects of melatonin in releasing pancreatic enzymes ( 11 ) .", "protective effects of melatonin are not only due to antioxidant activity , but it also activates capsaicin - sensitive afferent fibers ( 1 , 16 ) .", "melatonin may have a potential impact on the treatment of peptic ulcer via significant increase in ghrelin expression ( 17 ) .", "melatonin increases the release of pancreatic amylase and proteins through vagus nerve ( 18 ) .", "the effects of melatonin on pancreatic enzymes are reversed by vagotomy and administration of capsaicin ( 18 , 19 ) .", "intra - lumen administration of melatonin increases plasma cholecystokinin ( cck ) and antioxidants ( 11 , 19 , 20 ) .", "it has been shown that sensory nerve fibers are involved in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer ( 16 ) .", "it has been reported that vagus nerve increases the secretion of bicarbonate from duodenum mucosa through an increase in melatonin secretion ( 21 ) .", "vagal pathways are the most important regulatory pathways in the gastrointestinal tract which is one of the most important sources of melatonin production . in previous studies ,", "both protective and harmful effects of the stimulation of vagus nerve in digestive system have been reported .", "the role of sensory neurons in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer has been reported .", "there is no study about the combined effects of melatonin administration and intervention of vagus nerve during gastric i / r injury .", "probably , vagus nerve and melatonin have interactions in their protective effects during gastric i / r events", "so , the present study was performed to examine this interaction during gastric i / r .", "this study was performed in 42 male wistar rats weighing 180 - 220 g. animals were kept at room temperature 20 - 22  c and 12 hr-12 hr light - dark cycle .", "animals had free access to water and food and were randomly divided into 6 groups of 7 rats .", "rats were fasted for 12 hr prior to the experiment but had access to water .", "experimental groups were : 1 ) base+i / r+vehicle ; 2 ) base+i / r+melatonin ; 3 ) vagotomy+i / r+vehicle ; 4 ) vagotomy+i / r+melatonin ; 5)vagus nerve stimulation+i / r+vehicle ; 6)vagus nerve stimulation+i / r+ melatonin . base , means condition that neither vagus nerve was stimulated nor vagotomy was performed .", "animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) . after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) .", "after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec .", "stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram", "gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve .", "then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk .", "occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) .", "melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) .", "melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) .", "one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) .", "mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation .", "optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) .", "glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein .", "superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) .", "catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) .", "the h & e  stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks .", "the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece .", "comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test .", "animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) .", "after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) .", "after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec .", "stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram", "gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve .", "then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk .", "occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) .", "melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) .", "melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) .", "one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) .", "mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation .", "optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) .", "glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein .", "superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) .", "catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) .", "the h & e  stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks .", "the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece .", "comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test .", "mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) .", "/ r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) .", "/ r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) .", "a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) .", "# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle mda level in the study groups is presented in figure 2 .", "as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) .", "moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) .", "/ r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i", "/ r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) .", "mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) .", "* p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle catalase activity in the study groups are shown in figure 3 .", "catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) .", "a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) .", "/ r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group . #", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle figure 4 shows glutathione peroxidase activity in the studied groups .", "a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) .", "/ r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i", "this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) .", "level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . #", "b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 .", "as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity", "/ r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group .", "this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) .", "superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle", "mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) .", "/ r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) .", "/ r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) .", "a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) .", "# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle", "as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) .", "moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) .", "/ r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i", "/ r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) .", "mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) .", "* p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . #", "a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle", "catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) .", "a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) .", "/ r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group .", "# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle", "a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) .", "/ r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i", "this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) .", "level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle", "sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 .", "as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity", "/ r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group .", "this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) .", "superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle", "the present study was performed to investigate the protective effect of interaction between melatonin and vagus nerve during gastric i / r .", "the results of this study showed that melatonin administration decreased neutrophils infiltration and mda level and increased sod , cat and gpx activities in gastric tissue . also , vagus stimulation increased neutrophils infiltration and mda level in gastric tissue while melatonin administration along with vagus stimulation decreased the effect of vagus stimulation in gastric tissue .", "finally , vagotomy prevented some of the protective effects of melatonin during gastric i / r . according to the results of this study", ", melatonin can reduce gastritis during i / r , probably through decreasing the activity of oxidant enzymes and increasing the activity of anti - oxidant enzymes such as sod , cat and gpx .", "it has also been demonstrated that melatonin decreases mad level ( 27 - 29 ) .", "other probable mechanisms underlying the protective effect of melatonin include an increase in gastric microcirculation ( 1 ) , a reduction in acid and pepsin secretions ( 15 ) , a reduction in production of inflammatory cytokines ( 16 ) and an increase in the expression of anti - oxidant gene ( 17 , 30 - 32 ) .", "moreover , in our study , a stimulation of vagus nerve increased gastritis which was associated with an increase in mda level while vagotomy after i / r was ineffective .", "it is probable that the stimulation of vagus nerve following gastric i / r aggravated the condition and increased the activity of oxidant enzymes .", "since vagotomy in basal condition did not have any effect , it seems that the control of gastritis and the activity of oxidant and anti - oxidant enzymes are not under the control of vagus nerve in basal conditions .", "it has been demonstrated that the stimulation of vagus nerve increases output of gastric juice and acid ( 33 ) , and decreases the activity of anti - oxidant enzymes like sod ( 33 ) . on the other hand , vagotomy showed healing effects on duodenal ulcers ( 7 ) similar to the effect of atropine ( 34 ) . in a study ,", "diaphragmatic vagotomy did not change the activity of catalase in the intestine ( 35 ) which was similar to the result of the present study .", "the results of some studies are not consistent with our data . for example , it has been identified that vagal afferent fibers decrease gastrointestinal tract inflammation through nicotinic receptors leading to a decrease in inflammatory cytokines ( 16 ) .", "cholinergic agonists such as neostigmine decrease superoxide anion and inflammation in i / r ( 36 ) .", "elevation of acetylcholine transferase activity and decrease in acetylcholine esterase inhibitors increase gpx ( 35 , 37 ) .", "treatment with acetylcholine esterase inhibitors increases catalase half - life ( 38 ) . in the present study , melatonin administration affected the outcomes in i / r+vagus stimulation group .", "melatonin decreased gastritis and mda level , and increased sod and gpx activity , but not in vagotomy group .", "these findings show that protective effects of melatonin are probably mediated through vagus nerve . also , interaction between melatonin and vagus stimulation is suggested since these show a synergic effect in relation with anti - oxidant factors . regarding this probable interaction ,", "it is suggested that melatonin exerts its gastric protective effect through stimulation of gi tract neurons leading to cgrp release ( 1 ) .", "intra - cerebral injection of melatonin inhibits acid and pepsin secretions through vagus cholinergic activity ( 15 ) .", "melatonin increases the release of amylase and pancreas proteins through vagus nerve ( 16 ) .", "effect of melatonin administration accompanied with vagotomy has not suggested in the release of amylase(18 ) .", "studies have shown that intra - lumen administration of melatonin or its precursor induces the release of pancreatic enzymes , while this effect is not shown in isolated pancreas . moreover ,", "these effects of melatonin are reversed by vagotomy and capsaicin administration ( 11 , 18 , 19 ) .", "it has been reported that melatonin effect is mediated indirectly through the release of cck followed by vagovagal reflex ( 11 , 19 , 39 ) .", "it has been shown that intra - lumen administration of melatonin increases plasma cck level ( 11 , 19 ) and cck acts through stimulation of vagal afferent fibers in gi tract ( 40 , 41 ) .", "it has been reported that vagotomy in combination with melatonin administration inhibits the release of pancreas proteins ( 7 , 11 , 16 ) .", "it has been reported that melatonin increases the expression and activity of ach receptors , and synergistic effects are induced during vagus stimulation ( 42 ) . according to a study , injection of phenylephrine causes melatonin release from mucosa enterochromaffin cells in the presence of normal vagus nerve and sympathetic paths ( 39 ) .", "melatonin increases the release of bicarbonate from gastric mucosa ( 39 ) and decreases the release of inflammatory cytokines of gi through increasing vagus activity ( 16 ) .", "it is suggested that melatonin is neuroprotective in gastric i / r probably by decreasing gastritis and mda and increasing the activities of cat , sod and gpx .", "these effects of melatonin are probably mediated by vagus nerve . moreover , in gastric i / r , melatonin can reverse harmful effects of vagus stimulation .", "it is suggested that further studies are required to find the mechanisms involved in this interaction ."], "abstract_text": ["<S> objectives : vagal pathways in gastrointestinal tract are the most important pathways that regulate ischemia / reperfusion ( i / r ) . </S>", "<S> gastrointestinal tract is one of the important sources of melatonin production . </S>", "<S> the aim of this study was to investigate probable protective effect of the interaction between vagus nerve and melatonin after i / r.materials and methods : this study was performed in male rats that were divided into six groups . </S>", "<S> cervical vagus nerve was cut bilaterally after induction of i / r and the right one was stimulated by stimulator . </S>", "<S> melatonin or vehicle was injected intraperitoneally . </S>", "<S> the stomach was removed for histopathological and biochemical investigations.results:a significant decrease in infiltration of gastric neutrophils and malondialdehyde ( mda ) level after i / r was induced by melatonin and was disappeared after vagotomy . </S>", "<S> the stimulation of vagus nerve significantly enhanced these effects of melatonin . </S>", "<S> however , a stimulation of vagus nerve alone increased neutrophils infiltration and mda level . </S>", "<S> melatonin significantly increased the activities of catalase , glutathione peroxidase ( gpx ) , superoxide dismutases ( sod ) . </S>", "<S> unlike stimulation of vagus nerve , vagotomy decreased these effects of melatonin.conclusion:according to these results , it is probable that protective effects of melatonin after i / r may be mediated by vagus nerve . therefore , there is an interaction between melatonin and vagus nerve in their protective effects . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Vagus nerve stimulation", "Ischemia/reperfusion", "Measuring malondialdehyde (MDA) level, and glutathione peroxides (GPX), catalase and superoxide dismutase (SOD) activities", "Infiltration of neutrophils", "Statistical analysis", "Results", "Infiltration of neutrophils", "MDA level", "Catalase activity", "Glutathione peroxidase (GPX) activity", "Superoxide dismutase (SOD) activity", "Discussion", "Conclusion", "Conflict interest"], "sections": [["the protective effects of vagus nerve during ischemia / reperfusion ( i / r ) have been suggested ( 1 ) .", "vagus nerve stimulation decreases the inflammation and the injuries resulted from i / r through affecting neutrophils while vagotomy increases these processes ( 1 ) .", "moreover , it has been observed that stimulation of vagus nerve can cause protective effects on gastric injury through releasing prostaglandins , nitric oxide ( no ) and calcitonin gene related peptide ( cgrp ) ( 2 ) .", "it is probable that vagus nerve exerts its gastric protective effects through releasing the aforementioned mediators .", "also , it has been reported that cholinergic activity of the efferent vagus nerve can participate in immunity modulation ( 3 ) .", "another study has shown that nicotine in monocytes not only decreases the production of pre - inflammatory cytokines , but also changes the response to il-10 , an anti - inflammatory cytokine ( 4 , 5 ) .", "cho et al have shown that alternative stimulation of cervical vagus nerve increases intra - gastric pressure and also causes bleeding ulcers in mucosal glands of stomach , and these effects can be prevented by atropine administration or sub - diaphragmatic vagotomy ( 6 ) .", "it has been reported that gastric vagotomy is effective in treating gastric and duodenal ulcers ( 7 ) .", "melatonin as a neurohormone is basically produced in the pineal gland and also in epiphysis , gastrointestinal ( gi ) mucosa and other organs .", "melatonin has several physiological activities such as controlling circadian rhythm , sleep induction , regulation of seasonal reproduction , and improvement of immunity .", "the most important effect of melatonin is an anti - oxidant effect that protects living organisms against oxidative stress ( 11 ) . in several studies ,", "the ability of melatonin in decreasing molecular injury has been shown during i / r .", "melatonin prevents myocardial infarction , necrotic cell death and renal abnormality after i / r ( 8 , 12 ) , reduces brain edema in ischemia ( 12 ) , and prevents acute gastric ulcers resulted from stress .", "these effects can be done through direct reactive oxygen species / reactive nitrogen species ( ros / rns ) detoxification , increase in anti - oxidative enzymes , and the prevention of electron leakage in mitochondrial inner membrane ( 11 , 13 ) .", "melatonin has a role in expression and function of nicotinic acetylcholine receptors and increases the efficacy of beta bungarotoxin - sensitive acetylcholine receptors ( 14 ) .", "it has been recognized that intra - cerebral injection of melatonin is effective in prevention of acid and pepsin secretion through cholinergic activity ( 15 ) .", "it has been identified that 5ht3 and 5ht2 receptors are involved in stimulatory effects of melatonin in releasing pancreatic enzymes ( 11 ) .", "protective effects of melatonin are not only due to antioxidant activity , but it also activates capsaicin - sensitive afferent fibers ( 1 , 16 ) .", "melatonin may have a potential impact on the treatment of peptic ulcer via significant increase in ghrelin expression ( 17 ) .", "melatonin increases the release of pancreatic amylase and proteins through vagus nerve ( 18 ) .", "the effects of melatonin on pancreatic enzymes are reversed by vagotomy and administration of capsaicin ( 18 , 19 ) .", "intra - lumen administration of melatonin increases plasma cholecystokinin ( cck ) and antioxidants ( 11 , 19 , 20 ) .", "it has been shown that sensory nerve fibers are involved in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer ( 16 ) .", "it has been reported that vagus nerve increases the secretion of bicarbonate from duodenum mucosa through an increase in melatonin secretion ( 21 ) .", "vagal pathways are the most important regulatory pathways in the gastrointestinal tract which is one of the most important sources of melatonin production . in previous studies ,", "both protective and harmful effects of the stimulation of vagus nerve in digestive system have been reported .", "the role of sensory neurons in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer has been reported .", "there is no study about the combined effects of melatonin administration and intervention of vagus nerve during gastric i / r injury .", "probably , vagus nerve and melatonin have interactions in their protective effects during gastric i / r events", "so , the present study was performed to examine this interaction during gastric i / r ."], ["this study was performed in 42 male wistar rats weighing 180 - 220 g. animals were kept at room temperature 20 - 22  c and 12 hr-12 hr light - dark cycle .", "animals had free access to water and food and were randomly divided into 6 groups of 7 rats .", "rats were fasted for 12 hr prior to the experiment but had access to water .", "experimental groups were : 1 ) base+i / r+vehicle ; 2 ) base+i / r+melatonin ; 3 ) vagotomy+i / r+vehicle ; 4 ) vagotomy+i / r+melatonin ; 5)vagus nerve stimulation+i / r+vehicle ; 6)vagus nerve stimulation+i / r+ melatonin . base , means condition that neither vagus nerve was stimulated nor vagotomy was performed .", "animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) . after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) .", "after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec .", "stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram", "gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve .", "then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk .", "occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) .", "melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) .", "melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) .", "one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) .", "mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation .", "optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) .", "glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein .", "superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) .", "catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) .", "the h & e  stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks .", "the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece .", "comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test ."], ["animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) .", "after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) .", "after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec .", "stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram"], ["gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve .", "then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk .", "occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) .", "melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) .", "melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) ."], ["one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) .", "mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation .", "optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) .", "glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein .", "superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) .", "catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) ."], ["the h & e  stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks .", "the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece ."], ["comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test ."], ["mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) .", "/ r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) .", "/ r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) .", "a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) .", "# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle mda level in the study groups is presented in figure 2 .", "as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) .", "moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) .", "/ r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i", "/ r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) .", "mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) .", "* p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle catalase activity in the study groups are shown in figure 3 .", "catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) .", "a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) .", "/ r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group . #", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle figure 4 shows glutathione peroxidase activity in the studied groups .", "a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) .", "/ r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i", "this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) .", "level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . #", "b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 .", "as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity", "/ r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group .", "this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) .", "superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle"], ["mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) .", "/ r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) .", "/ r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) .", "a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) .", "# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle"], ["as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) .", "moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) .", "/ r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i", "/ r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) .", "mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) .", "* p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . #", "a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle"], ["catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) .", "a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) .", "/ r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .", "catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group .", "# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle"], ["a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) .", "/ r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i", "this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) .", "level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle"], ["sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 .", "as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity", "/ r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group .", "this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) .", "superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .", "base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle"], ["the present study was performed to investigate the protective effect of interaction between melatonin and vagus nerve during gastric i / r .", "the results of this study showed that melatonin administration decreased neutrophils infiltration and mda level and increased sod , cat and gpx activities in gastric tissue . also , vagus stimulation increased neutrophils infiltration and mda level in gastric tissue while melatonin administration along with vagus stimulation decreased the effect of vagus stimulation in gastric tissue .", "finally , vagotomy prevented some of the protective effects of melatonin during gastric i / r . according to the results of this study", ", melatonin can reduce gastritis during i / r , probably through decreasing the activity of oxidant enzymes and increasing the activity of anti - oxidant enzymes such as sod , cat and gpx .", "it has also been demonstrated that melatonin decreases mad level ( 27 - 29 ) .", "other probable mechanisms underlying the protective effect of melatonin include an increase in gastric microcirculation ( 1 ) , a reduction in acid and pepsin secretions ( 15 ) , a reduction in production of inflammatory cytokines ( 16 ) and an increase in the expression of anti - oxidant gene ( 17 , 30 - 32 ) .", "moreover , in our study , a stimulation of vagus nerve increased gastritis which was associated with an increase in mda level while vagotomy after i / r was ineffective .", "it is probable that the stimulation of vagus nerve following gastric i / r aggravated the condition and increased the activity of oxidant enzymes .", "since vagotomy in basal condition did not have any effect , it seems that the control of gastritis and the activity of oxidant and anti - oxidant enzymes are not under the control of vagus nerve in basal conditions .", "it has been demonstrated that the stimulation of vagus nerve increases output of gastric juice and acid ( 33 ) , and decreases the activity of anti - oxidant enzymes like sod ( 33 ) . on the other hand , vagotomy showed healing effects on duodenal ulcers ( 7 ) similar to the effect of atropine ( 34 ) . in a study ,", "diaphragmatic vagotomy did not change the activity of catalase in the intestine ( 35 ) which was similar to the result of the present study .", "the results of some studies are not consistent with our data . for example , it has been identified that vagal afferent fibers decrease gastrointestinal tract inflammation through nicotinic receptors leading to a decrease in inflammatory cytokines ( 16 ) .", "cholinergic agonists such as neostigmine decrease superoxide anion and inflammation in i / r ( 36 ) .", "elevation of acetylcholine transferase activity and decrease in acetylcholine esterase inhibitors increase gpx ( 35 , 37 ) .", "treatment with acetylcholine esterase inhibitors increases catalase half - life ( 38 ) . in the present study , melatonin administration affected the outcomes in i / r+vagus stimulation group .", "melatonin decreased gastritis and mda level , and increased sod and gpx activity , but not in vagotomy group .", "these findings show that protective effects of melatonin are probably mediated through vagus nerve . also , interaction between melatonin and vagus stimulation is suggested since these show a synergic effect in relation with anti - oxidant factors . regarding this probable interaction ,", "it is suggested that melatonin exerts its gastric protective effect through stimulation of gi tract neurons leading to cgrp release ( 1 ) .", "intra - cerebral injection of melatonin inhibits acid and pepsin secretions through vagus cholinergic activity ( 15 ) .", "melatonin increases the release of amylase and pancreas proteins through vagus nerve ( 16 ) .", "effect of melatonin administration accompanied with vagotomy has not suggested in the release of amylase(18 ) .", "studies have shown that intra - lumen administration of melatonin or its precursor induces the release of pancreatic enzymes , while this effect is not shown in isolated pancreas . moreover ,", "these effects of melatonin are reversed by vagotomy and capsaicin administration ( 11 , 18 , 19 ) .", "it has been reported that melatonin effect is mediated indirectly through the release of cck followed by vagovagal reflex ( 11 , 19 , 39 ) .", "it has been shown that intra - lumen administration of melatonin increases plasma cck level ( 11 , 19 ) and cck acts through stimulation of vagal afferent fibers in gi tract ( 40 , 41 ) .", "it has been reported that vagotomy in combination with melatonin administration inhibits the release of pancreas proteins ( 7 , 11 , 16 ) .", "it has been reported that melatonin increases the expression and activity of ach receptors , and synergistic effects are induced during vagus stimulation ( 42 ) . according to a study , injection of phenylephrine causes melatonin release from mucosa enterochromaffin cells in the presence of normal vagus nerve and sympathetic paths ( 39 ) .", "melatonin increases the release of bicarbonate from gastric mucosa ( 39 ) and decreases the release of inflammatory cytokines of gi through increasing vagus activity ( 16 ) ."], ["it is suggested that melatonin is neuroprotective in gastric i / r probably by decreasing gastritis and mda and increasing the activities of cat , sod and gpx .", "these effects of melatonin are probably mediated by vagus nerve . moreover , in gastric i / r , melatonin can reverse harmful effects of vagus stimulation .", "it is suggested that further studies are required to find the mechanisms involved in this interaction ."], [""]]}
{"article_id": "PMC3279550", "article_text": ["a stratified , multistage probability cluster sampling design was used in this survey ( 13 ) . based on socioeconomic characteristics", ", in the first stage of sampling , 22 counties were randomly selected from each of the 6 regions in china . in the second stage ,", "three townships were randomly selected from each of the selected counties . from each of the townships ,", "2 residential villages were randomly selected ; and 90 households were then randomly sampled from each village for physical examination .", "one - third of the households were selected to participate in the dietary survey and blood draw .", "for the present study , we used residents who were living in rural areas and were born between october 1 , 1952 , and september 30 , 1964 , as our analytic population . to minimize misclassification of the exposure periods , subjects who were born between october 1 , 1958 , and", "september 30 , 1959 , and between october 1 , 1961 , and september 30 , 1962 , were excluded since the exact dates of the start and the end of the chinese famine were not available and not the same across regions .", "flow chart on the sampling method in each region * of the 2002 china national nutrition and health survey . *", "the mainland of china is classified into 6 regions defined by the chinese bureau of statistics according to their socioeconomic development .", "they are metropolis , general city , type i rural site , type ii rural site , type iii rural site , and type iv rural site .", "subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood  exposed cohort .", "subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort .", "subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts .", "mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood ", "exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively .", "the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) .", "the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 ) .", "an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas .", "we split all five cohorts into severely affected famine areas and less severely affected famine areas .", "this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences .", "all subjects were invited for blood collection after an 10 to 14 h overnight fast .", "the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine .", "fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation .", "every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) .", "we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) .", "mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes .", "in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes .", "dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the  mismatch  between fetal nutrition and adult nutrition .", "the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) .", "briefly , four dietary patterns were derived through cluster analysis , which were labeled as  green water ,   yellow earth ,   new affluence ,  and  western adopter .   green water  and  yellow earth ", "patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study", ", we combined the clusters of  green water  and  yellow earth  as the traditional dietary pattern , and we combined the clusters of  new affluence  and  western adopter  as the affluent / western pattern .", "current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable .", "the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status .", "we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) .", "the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants .", "we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample .", "survey weights were derived from the 2000 china national population census and associated administrative data .", "mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) .", "risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model . interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model .", "analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood .", "the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine .", "in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes .", "subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood  exposed cohort .", "subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort .", "subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts .", "mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood  exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively .", "the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) .", "the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 )", ". an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas .", "we split all five cohorts into severely affected famine areas and less severely affected famine areas .", "this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences .", "all subjects were invited for blood collection after an 10 to 14 h overnight fast .", "the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine .", "fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation .", "every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) .", "we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) .", "mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes .", "in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes .", "dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the  mismatch  between fetal nutrition and adult nutrition .", "the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) .", "briefly , four dietary patterns were derived through cluster analysis , which were labeled as  green water ,   yellow earth ,   new affluence ,  and  western adopter .   green water  and  yellow earth ", "patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study , we combined the clusters of  green water  and  yellow earth  as the traditional dietary pattern , and we combined the clusters of  new affluence  and  western adopter  as the affluent / western pattern .", "current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable .", "the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status .", "we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) .", "the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants .", "we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample .", "survey weights were derived from the 2000 china national population census and associated administrative data .", "mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) .", "risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model .", "interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model .", "analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood .", "the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine .", "in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes .", "basic characteristics of the study population are shown in table 1 . in our main study population", "( n = 7,874 ) , 1,005 ( 12.8% ) subjects had been exposed to the chinese famine during fetal life , and 4,915 ( 62.4% ) subjects had been exposed during childhood . as compared with the nonexposed individuals , fetal - exposed subjects were 0.9 cm shorter as adults , and childhood - exposed subjects were 1.5 cm shorter ( table 1 ) .", "the prevalence of hyperglycemia among adults in the nonexposed , fetal - exposed , early childhood , mid childhood , and late childhood  exposed birth cohorts was 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively .", "basic characteristics of study population according to chinese famine exposure * * data are adjusted means ( se ) .", "adjusted factors included sex , educational level , family history of diabetes ( only for glucose ) , current smoking , alcohol use , and physical activity level .", "compared with the nonexposed cohort , p < 0.05 . in severely affected famine areas ,", "fpg concentration was significantly higher in the fetal - exposed cohort than in the nonexposed cohort with a mean difference of 0.20", "no significant difference was observed in the less severely affected famine areas ( p for interaction = 0.001 , table 2 ) .", "compared with nonexposed subjects , fpg was higher in the late childhood  exposed cohort in both the severely affected famine areas and less severely affected famine areas .", "a significant interaction between the exposed cohort and areas was found only for the fetal - exposed cohort ( table 2 ) .", "concentrations of fasting plasma glucose and prevalence rates of hyperglycemia and type 2 diabetes by birth cohort and severity of the chinese famine area * data are adjusted means ( se ) for fasting plasma glucose , sex standard prevalence , and odds ratio for hyperglycemia and diabetes .", "adjusted factors included sex , education level , family history of diabetes , and current smoking , alcohol use , and physical activity level .", "subjects exposed to famine during fetal life in severely affected famine areas had a higher prevalence of hyperglycemia than the nonexposed cohort .", "the odds ratios were significantly different between the severe and less severe famine areas ( table 2 ) , suggesting a stronger famine effect in the severely affected famine areas . compared with the nonexposed cohort , subjects in the late childhood  exposed cohort had a higher risk of hyperglycemia in both severely and less severely affected famine areas , but the odds ratios were not significantly different between the severe and less severe famine areas ( table 2 ) .", "a significantly higher prevalence of type 2 diabetes was observed among subjects exposed in late childhood as compared with the nonexposed cohort ( table 1 ) .", "however , table 2 shows that after stratification of this group by severity of famine exposure , no significant difference of type 2 diabetes risk was observed anymore between different famine cohorts .", "stratified analyses by dietary pattern , economic status , and bmi for severely affected famine areas are shown in fig .", "figure 2a1 shows that the prevalence of hyperglycemia was highest ( 18.9% ) in subjects in the fetal - exposed cohort and who consumed an affluent / western diet .", "as compared with the relatively nonexposed cohort , the odds ratio of hyperglycemia in the fetal - exposed cohort was 7.63 ( 95% ci : 2.4124.1 , p = 0.0005 ) for those who had an affluent / western dietary pattern , and 2.34 ( 95% ci : 0.826.70 , p = 0.112 ) for those with a traditional dietary pattern .", "prevalence of hyperglycemia among birth cohorts according to early life famine exposure and later life dietary patterns ( a1 and b1 ) , socioeconomic status ( a2 and b2 ) , and bmi ( a3 and b3 ) in severely ( column a ) and less severely affected famine areas ( column b ) .", "figure 2a2 shows that as compared with nonexposed subjects , the odds ratio of hyperglycemia in the fetal - exposed cohort was 6.20 ( 95% ci : 2.0818.5 , p = 0.001 ) in subjects with a higher adult economic status , and 1.68 ( 95% ci : 0.505.71 , p = 0.404 ) in subjects with a lower adult economic status .", "figure 2a3 shows that overweight subjects in the fetal - exposed cohort had the highest prevalence of hyperglycemia ( 13.9% ) .", "however , the risks of hyperglycemia were largely comparable in these two groups ; the odds ratio of hyperglycemia in the fetal - exposed cohort was 3.71 ( 95% ci : 1.1312.2 , p = 0.031 ) in overweight subjects and 4.37 ( 95% ci : 1.1516.5 , p = 0.030 ) in normal weight subjects , respectively , compared with the nonexposed cohort .", "similar analyses were performed in subjects exposed to less severely affected famine areas during fetal life and childhood ( fig .", "2 , right column , graphs b1 , b2 , and b3 ) , but did not show consistent associations . when we defined the severely affected famine areas as those with an excess death rate 100% , the prevalence of hyperglycemia among the fetal - exposed cohort in severely affected famine areas increased to 8.1% , but this did not change the associations between fetal exposure to famine and risk of hyperglycemia in adulthood .", "in addition , neither using subjects who were born between october 1 , 1962 , and september 30 , 1968 , as a nonexposed cohort nor excluding subjects with a family history of diabetes materially changed the associations ( table 3 )", ". prevalence rate of hyperglycemia by birth cohorts and severity of famine areas : sensitivity analyses all odds ratios used the nonexposed cohort as the reference cohort . adjusted factors include sex , educational level , family history of diabetes , and current smoking , alcohol use , and physical activity level in 2002 .", "in this study of a large sample of chinese adults , we found a significant association between severe famine exposure during the fetal period and an increased risk of hyperglycemia in adulthood .", "this association was stronger in subjects with a western dietary pattern or higher economic status in adulthood .", "several mechanisms might explain the associations between fetal famine exposure and risk of diabetes in later life .", "exposure to extreme starvation in rats led to poor development of pancreatic -cell mass and function and insulin resistance , which might persist in later life ( 18 ) .", "a poor intrauterine environment may also reduce skeletal muscle development ( 19 ) , which may subsequently lead to insulin resistance in peripheral tissues ( 20 ) .", "it has also been suggested that stress suffering from fetal famine exposure could change the setpoint of the hypothalamic - pituitary - adrenal ( hpa ) axis , which could result in long - term changes in secretion of neuroendocrine mediators of the stress response , and predispose to cardiovascular and metabolic disease in later life ( 21,22 ) . to our knowledge ,", "thus far three studies have assessed the associations of exposure to famine with measures of glucose intolerance .", "these studies were performed in the netherlands ( the dutch famine study ) ( 4,5 ) , russia ( the leningrad siege study ) ( 6 ) , and china ( our chinese famine study ) .", "the dutch famine study reported higher 2-h glucose and insulin levels among subjects who were exposed to famine during fetal life ( 4,5 ) , but this association was not observed in the leningrad siege study ( 6).the inconsistent results might be due to differences in postnatal environmental life exposures . although the dutch population rapidly developed into a wealthy and rich population after the famine , the leningrad people remained relatively poor . in our study , we observed that fetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood , which was exacerbated by an unhealthy adult diet and higher economic status .", "our results support the hypothesis that exposure to a nutritionally rich environment modifies the association between fetal famine exposure and disease in later life ( 1,20,23 ) .", "the association between fetal famine exposure and hyperglycemia was stronger in participants with an affluent / western dietary pattern .", "these subjects were , to a large extent , less poor and more highly educated ( 15 ) , and they have benefited most from dramatically enhanced economic opportunities and have broken away from traditional chinese food patterns ( 15 ) .", "their diet is characterized by a high intake of meat , eggs , dairy , sugary beverages , edible oils , and a low vegetable use ( 15 ) . apparently , this nutrition ", "rich  environment did not match the fetal starvation environment that people of fetal exposed cohort experienced , which in turn increased the risk of hyperglycemia in later life ( 1,20,23 ) .", "our study used annual mean income as the cutoff to categorize economic status ( 2,000 chinese yuan / person / year ) .", "subjects in the lower economic group might consume mostly traditional plant foods with little meat .", "therefore , the discrepancy between the nutritional environment in adulthood and fetal undernutrition conditions may be less evident for those with a higher economic status . in other words", ", there was probably greater  mismatch  between in utero and adulthood environments in the higher economic group , which triggered an increased prevalence of hyperglycemia in the fetal - exposed cohort .", "similar results were described in the dutch famine study ( 4 ) , showing that 2-h glucose concentrations were especially high among people exposed to the famine during fetal life and who became obese in later life . however , the relative risk of hyperglycemia in overweight subjects was not different from that in normal weight subjects", ". this may be partly due to the increased prevalence of hyperglycemia in the nonexposed cohort in overweight subset .", "these results therefore indicate that both improving fetal nutritional environmental and controlling bmi in later life are important for prevention of a disturbed glucose metabolism .", "childhood nutritional status , particularly during infancy , is another key factor in influencing the propensity to develop disease in adulthood ( 23 ) .", "animal studies have shown that postnatal caloric restriction might hamper -cell development ( 24 ) and might disturb glucose metabolism in later life in rats ( 25 ) .", "our study found significantly increased fpg in the early childhood  exposed cohort in the severely affected famine areas , but no significant differences in fpg in the less severely affected famine areas .", "we also observed a higher risk of hyperglycemia among subjects exposed in late childhood in both severely and less severely affected famine areas .", "these results suggest that famine exposure during childhood may increase the risk of hyperglycemia in later life .", "however , we can not exclude a potential cohort effect , such as aging ( 26 ) .", "similar risks of hyperglycemia among subjects exposed during childhood in both famine - exposed areas and nonfamine - exposed areas suggest rather a cohort ( older age ) effect than a famine effect .", "however , since almost all rural regions in china were affected by the famine during 19591961 , no valid nonfamine - exposed cohort comprising subjects born in the same time period was available .", "thus , the association between childhood exposure to famine and risk of hyperglycemia needs to be studied in more detail .", "first , we assumed that the residents we investigated at the time of the survey were born in the same province and in a similar rural area .", "however , severe restrictions on migration and relocation in china made our sample quite stable .", "migration with permanent resident permission still needed to be approved by authorities on a case - by - case basis in china .", "according to the 2000 china national population census , 2.68% of the rural population lived in provinces other than the provinces of their birthplaces ( 27 ) .", "our study sample was based on the residence registration system ; only subjects with permanent resident permission in local areas were involved in our study .", "therefore , we do not expect that intraprovince migration leading to measurement error in the coding of birth place is a major concern in our results ( 12 ) .", "second , subjects in our fetal - exposed cohort may have actually experienced severe famine during both the fetal period and the infancy period because the famine lasted approximately 3 years .", "it was therefore difficult to distinguish whether the fetal period or the infancy period was more important .", "however , the early childhood cohort also included subjects exposed to famine in infancy , which did not have a substantial influence on the risk of hyperglycemia .", "thus , our results indicate that the fetal period should be considered as the primary critical period .", "third , our subjects who experienced severe famine in the fetal period were in their early 40s in 2002 , and the cases of type 2 diabetes were few .", "the small numbers may partly explain why we did not observed significant associations with the risk of type 2 diabetes .", "we used the excess death rate as an indirect measure of famine exposure . with this method", ", we could not distinguish death due to famine from death due to unfavorable weather conditions or infections .", "we also did not have reliable information about individual food availability during the famine period .", "therefore , from our data , we can not conclude that the higher risk of hyperglycemia among subjects exposed to famine is exclusively due to malnutrition in early life . however , nutrition deficiency was highly prevalent during the chinese famine .", "china 's grain output declined by 15% in 1959 and in the following 2 years , and its food supply plunged further to 70% of its 1958 level ( 8) . as almost all foods were delivered through communal kitchens at that time , no social groups were spared from the effects of the famine ( 9 ) .", "however , since the famine effect on glucose intolerance did not depend on birth size in the dutch famine study ( 4 ) , we do not consider the lack of information about individual birth outcomes as a major limitation . in conclusion", ", we found that exposure to severe famine in fetal life increased the risk of hyperglycemia in adulthood .", "the  mismatched nutrition postnatal environment  represented by a western dietary pattern and improved economic status further increased susceptibility to hyperglycemia in those who experienced fetal exposure to famine . together with previous studies", ", our study emphasizes that early life environment is critical for the risk of hyperglycemia in adult life ."], "abstract_text": ["<S> objectiveearly developmental adaptations in response to undernutrition may play an essential role in susceptibility to type 2 diabetes , particularly for those experiencing a  mismatched rich nutritional environment  in later life . </S>", "<S> we examined the associations of exposure to the chinese famine ( 19591961 ) during fetal life and childhood with the risk of hyperglycemia and type 2 diabetes in adulthood.research design and methodswe used the data for 7,874 rural adults born between 1954 and 1964 in selected communities from the cross - sectional 2002 china national nutrition and health survey . </S>", "<S> hyperglycemia was defined as fasting plasma glucose 6.1 mmol / l and/or 2-h plasma glucose 7.8 </S>", "<S> mmol / l and/or a previous clinical diagnosis of type 2 diabetes.resultsprevalences of hyperglycemia among adults in nonexposed , fetal exposed , early - childhood , mid - childhood , and late - childhood exposed cohorts were 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively . in severely affected famine areas , </S>", "<S> fetal - exposed subjects had an increased risk of hyperglycemia compared with nonexposed subjects ( odds ratio = 3.92 ; 95% ci : 1.649.39 ; p = 0.002 ) ; this difference was not observed in less severely affected famine areas ( odds ratio = 0.57 ; 95% ci : 0.251.31 ; p = 0.185 ) . </S>", "<S> the odds ratios were significantly different between groups from the severe and less severe famine areas ( p for interaction = 0.001 ) . in severely affected famine areas , fetal - exposed subjects who followed an affluent / western dietary pattern ( odds ratios = 7.63 ; 95% ci : 2.4124.1 ; p = 0.0005 ) or who had a higher economic status in later life experienced a substantially elevated risk of hyperglycemia ( odds ratios = 6.20 ; 95% ci : 2.0818.5 ; p = 0.001).conclusionsfetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood . </S>", "<S> this association appears to be exacerbated by a nutritionally rich environment in later life . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Famine cohorts and areas.", "Assessments of blood glucose and type 2 diabetes.", "Stratification factors.", "Statistical analyses.", "RESULTS", "DISCUSSION"], "sections": [["the 2002 cnnhs is a nationally representative cross - sectional study on nutrition and chronic diseases .", "a stratified , multistage probability cluster sampling design was used in this survey ( 13 ) . based on socioeconomic characteristics", ", in the first stage of sampling , 22 counties were randomly selected from each of the 6 regions in china . in the second stage ,", "three townships were randomly selected from each of the selected counties . from each of the townships ,", "2 residential villages were randomly selected ; and 90 households were then randomly sampled from each village for physical examination .", "one - third of the households were selected to participate in the dietary survey and blood draw .", "for the present study , we used residents who were living in rural areas and were born between october 1 , 1952 , and september 30 , 1964 , as our analytic population . to minimize misclassification of the exposure periods , subjects who were born between october 1 , 1958 , and", "september 30 , 1959 , and between october 1 , 1961 , and september 30 , 1962 , were excluded since the exact dates of the start and the end of the chinese famine were not available and not the same across regions .", "flow chart on the sampling method in each region * of the 2002 china national nutrition and health survey . *", "the mainland of china is classified into 6 regions defined by the chinese bureau of statistics according to their socioeconomic development .", "they are metropolis , general city , type i rural site , type ii rural site , type iii rural site , and type iv rural site .", "subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood  exposed cohort .", "subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort .", "subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts .", "mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood ", "exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively .", "the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) .", "the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 ) .", "an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas .", "we split all five cohorts into severely affected famine areas and less severely affected famine areas .", "this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences .", "all subjects were invited for blood collection after an 10 to 14 h overnight fast .", "the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine .", "fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation .", "every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) .", "we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) .", "mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes .", "in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes .", "dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the  mismatch  between fetal nutrition and adult nutrition .", "the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) .", "briefly , four dietary patterns were derived through cluster analysis , which were labeled as  green water ,   yellow earth ,   new affluence ,  and  western adopter .   green water  and  yellow earth ", "patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study", ", we combined the clusters of  green water  and  yellow earth  as the traditional dietary pattern , and we combined the clusters of  new affluence  and  western adopter  as the affluent / western pattern .", "current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable .", "the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status .", "we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) .", "the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants .", "we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample .", "survey weights were derived from the 2000 china national population census and associated administrative data .", "mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) .", "risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model . interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model .", "analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood .", "the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine .", "in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes ."], ["subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood  exposed cohort .", "subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort .", "subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts .", "mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood  exposed cohort , mid childhood  exposed cohort , and late childhood  exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively .", "the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) .", "the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 )", ". an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas .", "we split all five cohorts into severely affected famine areas and less severely affected famine areas .", "this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences ."], ["all subjects were invited for blood collection after an 10 to 14 h overnight fast .", "the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine .", "fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation .", "every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) .", "we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) .", "mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes .", "in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes ."], ["dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the  mismatch  between fetal nutrition and adult nutrition .", "the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) .", "briefly , four dietary patterns were derived through cluster analysis , which were labeled as  green water ,   yellow earth ,   new affluence ,  and  western adopter .   green water  and  yellow earth ", "patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study , we combined the clusters of  green water  and  yellow earth  as the traditional dietary pattern , and we combined the clusters of  new affluence  and  western adopter  as the affluent / western pattern .", "current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable .", "the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status .", "we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) .", "the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants ."], ["we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample .", "survey weights were derived from the 2000 china national population census and associated administrative data .", "mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) .", "risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model .", "interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model .", "analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood .", "the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine .", "in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes ."], ["basic characteristics of the study population are shown in table 1 . in our main study population", "( n = 7,874 ) , 1,005 ( 12.8% ) subjects had been exposed to the chinese famine during fetal life , and 4,915 ( 62.4% ) subjects had been exposed during childhood . as compared with the nonexposed individuals , fetal - exposed subjects were 0.9 cm shorter as adults , and childhood - exposed subjects were 1.5 cm shorter ( table 1 ) .", "the prevalence of hyperglycemia among adults in the nonexposed , fetal - exposed , early childhood , mid childhood , and late childhood  exposed birth cohorts was 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively .", "basic characteristics of study population according to chinese famine exposure * * data are adjusted means ( se ) .", "adjusted factors included sex , educational level , family history of diabetes ( only for glucose ) , current smoking , alcohol use , and physical activity level .", "compared with the nonexposed cohort , p < 0.05 . in severely affected famine areas ,", "fpg concentration was significantly higher in the fetal - exposed cohort than in the nonexposed cohort with a mean difference of 0.20", "no significant difference was observed in the less severely affected famine areas ( p for interaction = 0.001 , table 2 ) .", "compared with nonexposed subjects , fpg was higher in the late childhood  exposed cohort in both the severely affected famine areas and less severely affected famine areas .", "a significant interaction between the exposed cohort and areas was found only for the fetal - exposed cohort ( table 2 ) .", "concentrations of fasting plasma glucose and prevalence rates of hyperglycemia and type 2 diabetes by birth cohort and severity of the chinese famine area * data are adjusted means ( se ) for fasting plasma glucose , sex standard prevalence , and odds ratio for hyperglycemia and diabetes .", "adjusted factors included sex , education level , family history of diabetes , and current smoking , alcohol use , and physical activity level .", "subjects exposed to famine during fetal life in severely affected famine areas had a higher prevalence of hyperglycemia than the nonexposed cohort .", "the odds ratios were significantly different between the severe and less severe famine areas ( table 2 ) , suggesting a stronger famine effect in the severely affected famine areas . compared with the nonexposed cohort , subjects in the late childhood  exposed cohort had a higher risk of hyperglycemia in both severely and less severely affected famine areas , but the odds ratios were not significantly different between the severe and less severe famine areas ( table 2 ) .", "a significantly higher prevalence of type 2 diabetes was observed among subjects exposed in late childhood as compared with the nonexposed cohort ( table 1 ) .", "however , table 2 shows that after stratification of this group by severity of famine exposure , no significant difference of type 2 diabetes risk was observed anymore between different famine cohorts .", "stratified analyses by dietary pattern , economic status , and bmi for severely affected famine areas are shown in fig .", "figure 2a1 shows that the prevalence of hyperglycemia was highest ( 18.9% ) in subjects in the fetal - exposed cohort and who consumed an affluent / western diet .", "as compared with the relatively nonexposed cohort , the odds ratio of hyperglycemia in the fetal - exposed cohort was 7.63 ( 95% ci : 2.4124.1 , p = 0.0005 ) for those who had an affluent / western dietary pattern , and 2.34 ( 95% ci : 0.826.70 , p = 0.112 ) for those with a traditional dietary pattern .", "prevalence of hyperglycemia among birth cohorts according to early life famine exposure and later life dietary patterns ( a1 and b1 ) , socioeconomic status ( a2 and b2 ) , and bmi ( a3 and b3 ) in severely ( column a ) and less severely affected famine areas ( column b ) .", "figure 2a2 shows that as compared with nonexposed subjects , the odds ratio of hyperglycemia in the fetal - exposed cohort was 6.20 ( 95% ci : 2.0818.5 , p = 0.001 ) in subjects with a higher adult economic status , and 1.68 ( 95% ci : 0.505.71 , p = 0.404 ) in subjects with a lower adult economic status .", "figure 2a3 shows that overweight subjects in the fetal - exposed cohort had the highest prevalence of hyperglycemia ( 13.9% ) .", "however , the risks of hyperglycemia were largely comparable in these two groups ; the odds ratio of hyperglycemia in the fetal - exposed cohort was 3.71 ( 95% ci : 1.1312.2 , p = 0.031 ) in overweight subjects and 4.37 ( 95% ci : 1.1516.5 , p = 0.030 ) in normal weight subjects , respectively , compared with the nonexposed cohort .", "similar analyses were performed in subjects exposed to less severely affected famine areas during fetal life and childhood ( fig .", "2 , right column , graphs b1 , b2 , and b3 ) , but did not show consistent associations . when we defined the severely affected famine areas as those with an excess death rate 100% , the prevalence of hyperglycemia among the fetal - exposed cohort in severely affected famine areas increased to 8.1% , but this did not change the associations between fetal exposure to famine and risk of hyperglycemia in adulthood .", "in addition , neither using subjects who were born between october 1 , 1962 , and september 30 , 1968 , as a nonexposed cohort nor excluding subjects with a family history of diabetes materially changed the associations ( table 3 )", ". prevalence rate of hyperglycemia by birth cohorts and severity of famine areas : sensitivity analyses all odds ratios used the nonexposed cohort as the reference cohort . adjusted factors include sex , educational level , family history of diabetes , and current smoking , alcohol use , and physical activity level in 2002 ."], ["in this study of a large sample of chinese adults , we found a significant association between severe famine exposure during the fetal period and an increased risk of hyperglycemia in adulthood .", "this association was stronger in subjects with a western dietary pattern or higher economic status in adulthood .", "several mechanisms might explain the associations between fetal famine exposure and risk of diabetes in later life .", "exposure to extreme starvation in rats led to poor development of pancreatic -cell mass and function and insulin resistance , which might persist in later life ( 18 ) .", "a poor intrauterine environment may also reduce skeletal muscle development ( 19 ) , which may subsequently lead to insulin resistance in peripheral tissues ( 20 ) .", "it has also been suggested that stress suffering from fetal famine exposure could change the setpoint of the hypothalamic - pituitary - adrenal ( hpa ) axis , which could result in long - term changes in secretion of neuroendocrine mediators of the stress response , and predispose to cardiovascular and metabolic disease in later life ( 21,22 ) . to our knowledge ,", "thus far three studies have assessed the associations of exposure to famine with measures of glucose intolerance .", "these studies were performed in the netherlands ( the dutch famine study ) ( 4,5 ) , russia ( the leningrad siege study ) ( 6 ) , and china ( our chinese famine study ) .", "the dutch famine study reported higher 2-h glucose and insulin levels among subjects who were exposed to famine during fetal life ( 4,5 ) , but this association was not observed in the leningrad siege study ( 6).the inconsistent results might be due to differences in postnatal environmental life exposures . although the dutch population rapidly developed into a wealthy and rich population after the famine , the leningrad people remained relatively poor . in our study , we observed that fetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood , which was exacerbated by an unhealthy adult diet and higher economic status .", "our results support the hypothesis that exposure to a nutritionally rich environment modifies the association between fetal famine exposure and disease in later life ( 1,20,23 ) .", "the association between fetal famine exposure and hyperglycemia was stronger in participants with an affluent / western dietary pattern .", "these subjects were , to a large extent , less poor and more highly educated ( 15 ) , and they have benefited most from dramatically enhanced economic opportunities and have broken away from traditional chinese food patterns ( 15 ) .", "their diet is characterized by a high intake of meat , eggs , dairy , sugary beverages , edible oils , and a low vegetable use ( 15 ) . apparently , this nutrition ", "rich  environment did not match the fetal starvation environment that people of fetal exposed cohort experienced , which in turn increased the risk of hyperglycemia in later life ( 1,20,23 ) .", "our study used annual mean income as the cutoff to categorize economic status ( 2,000 chinese yuan / person / year ) .", "subjects in the lower economic group might consume mostly traditional plant foods with little meat .", "therefore , the discrepancy between the nutritional environment in adulthood and fetal undernutrition conditions may be less evident for those with a higher economic status . in other words", ", there was probably greater  mismatch  between in utero and adulthood environments in the higher economic group , which triggered an increased prevalence of hyperglycemia in the fetal - exposed cohort .", "similar results were described in the dutch famine study ( 4 ) , showing that 2-h glucose concentrations were especially high among people exposed to the famine during fetal life and who became obese in later life . however , the relative risk of hyperglycemia in overweight subjects was not different from that in normal weight subjects", ". this may be partly due to the increased prevalence of hyperglycemia in the nonexposed cohort in overweight subset .", "these results therefore indicate that both improving fetal nutritional environmental and controlling bmi in later life are important for prevention of a disturbed glucose metabolism .", "childhood nutritional status , particularly during infancy , is another key factor in influencing the propensity to develop disease in adulthood ( 23 ) .", "animal studies have shown that postnatal caloric restriction might hamper -cell development ( 24 ) and might disturb glucose metabolism in later life in rats ( 25 ) .", "our study found significantly increased fpg in the early childhood  exposed cohort in the severely affected famine areas , but no significant differences in fpg in the less severely affected famine areas .", "we also observed a higher risk of hyperglycemia among subjects exposed in late childhood in both severely and less severely affected famine areas .", "these results suggest that famine exposure during childhood may increase the risk of hyperglycemia in later life .", "however , we can not exclude a potential cohort effect , such as aging ( 26 ) .", "similar risks of hyperglycemia among subjects exposed during childhood in both famine - exposed areas and nonfamine - exposed areas suggest rather a cohort ( older age ) effect than a famine effect .", "however , since almost all rural regions in china were affected by the famine during 19591961 , no valid nonfamine - exposed cohort comprising subjects born in the same time period was available .", "thus , the association between childhood exposure to famine and risk of hyperglycemia needs to be studied in more detail .", "first , we assumed that the residents we investigated at the time of the survey were born in the same province and in a similar rural area .", "however , severe restrictions on migration and relocation in china made our sample quite stable .", "migration with permanent resident permission still needed to be approved by authorities on a case - by - case basis in china .", "according to the 2000 china national population census , 2.68% of the rural population lived in provinces other than the provinces of their birthplaces ( 27 ) .", "our study sample was based on the residence registration system ; only subjects with permanent resident permission in local areas were involved in our study .", "therefore , we do not expect that intraprovince migration leading to measurement error in the coding of birth place is a major concern in our results ( 12 ) .", "second , subjects in our fetal - exposed cohort may have actually experienced severe famine during both the fetal period and the infancy period because the famine lasted approximately 3 years .", "it was therefore difficult to distinguish whether the fetal period or the infancy period was more important .", "however , the early childhood cohort also included subjects exposed to famine in infancy , which did not have a substantial influence on the risk of hyperglycemia .", "thus , our results indicate that the fetal period should be considered as the primary critical period .", "third , our subjects who experienced severe famine in the fetal period were in their early 40s in 2002 , and the cases of type 2 diabetes were few .", "the small numbers may partly explain why we did not observed significant associations with the risk of type 2 diabetes .", "we used the excess death rate as an indirect measure of famine exposure . with this method", ", we could not distinguish death due to famine from death due to unfavorable weather conditions or infections .", "we also did not have reliable information about individual food availability during the famine period .", "therefore , from our data , we can not conclude that the higher risk of hyperglycemia among subjects exposed to famine is exclusively due to malnutrition in early life . however , nutrition deficiency was highly prevalent during the chinese famine .", "china 's grain output declined by 15% in 1959 and in the following 2 years , and its food supply plunged further to 70% of its 1958 level ( 8) . as almost all foods were delivered through communal kitchens at that time , no social groups were spared from the effects of the famine ( 9 ) .", "however , since the famine effect on glucose intolerance did not depend on birth size in the dutch famine study ( 4 ) , we do not consider the lack of information about individual birth outcomes as a major limitation . in conclusion", ", we found that exposure to severe famine in fetal life increased the risk of hyperglycemia in adulthood .", "the  mismatched nutrition postnatal environment  represented by a western dietary pattern and improved economic status further increased susceptibility to hyperglycemia in those who experienced fetal exposure to famine . together with previous studies", ", our study emphasizes that early life environment is critical for the risk of hyperglycemia in adult life ."]]}
{"article_id": "PMC3692593", "article_text": ["with the goal of discovering the genes that contribute to the risk of common diseases , numerous susceptibility loci have been identified using linkage analysis . despite replication of many of these linkages in a second sample ,", "some exceptions include tbc1d1 , identified as the gene responsible for the obesity linkage on chromosome 4p15 - 14 , and hoxb13 , identified as the gene responsible for the prostate cancer linkage on chromosome 17q21 - 22 .", "however , for many other common disease linkage signals , the underlying causal genes and variants await discovery .", "the expectation that a single gene accounts for a linkage peak may contribute to the difficulty in identifying causal genes . on the contrary", "attributed a triglyceride linkage on chromosome 7q36 to variants in five genes and christians et al .", "found that fine - mapping caused a single quantitative trait locus ( qtl ) for body size in mice to resolve first into three qtls , then one of those to split into two .", "furthermore , the clustering of causal genes may have stymied the multi - group effort to identify type 2 diabetes ( t2d ) genes on chromosome1q ; two strong associations present in populations of european ancestry failed replication and confirmation in other ethnic groups .", "these examples suggest that abandoned linkage findings might yet reveal susceptibility genes if reappraised while considering the possibility of multiple causal genes . as for other common diseases ,", "the challenge is even greater in african american ( aa ) populations where both prevalence and genetic diversity are higher , and pathophysiology may differ .", "genome wide case - control association studies ( gwass ) have identified single nucleotide polymorphisms ( snps ) with small effects on t2d risk , but few of the associated snps , identified primarily in european ancestry populations , are replicated in aas and few snps have been identified in aa populations . in general , the widespread rejection of family studies in favor of gwass has failed to produce the promised prognostic and diagnostic variants for t2d . as a resource for the discovery of genes related to t2d and its complications , the american diabetes association established the genetics of niddm ( gennid ) study . from 1993 to 2003", "we have used this resource to increase understanding of the genetics of t2d by applying linkage and family - based association analysis to the african american ( aa ) subset of the gennid sample .", "genome - wide linkage analysis using genotypes on 5,914 snps identified chromosomal regions that potentially harbor risk genes for t2d and age of t2d diagnosis ( aod ) .", "the strongest signal for t2d occurred on chromosome 2 at 95121 megabases ( mb ) , with weaker support for aod in the same region and for t2d at 6895 mb .", "both t2d and aod also showed linkage on chromosome 13 at 1930 mb , but linkage was limited to t2d on chromosome 7 at 5079 mb and to aod on chromosome 18 at 3165 mb .", "other analyses inferred pleiotropy with triglyceride in the chromosome 2p region and pleiotropy with obesity in the chromosome 13 region . herein we present linkage and association analyses on the aa subset of the gennid sample using genotypes on 9,203 fine - mapping snps added to the genome scan snps used previously .", "our first goal was to more precisely localize the t2d susceptibility genes in each of the five chromosomal regions identified in the genome scan ( chromosome 2 at 6895 and 95121 mb , chromosome 7 at 5079 mb , chromosome 13 at 1930 mb , and chromosome 18 at 3165 mb ) .", "our second goal was to test for association of the gene - based fine - mapping snps with t2d and aod in order to identify genes related to t2d in aas .", "the gennid study ascertained families through sibling pairs each with a t2d diagnosis . during phase 1 ,", "extended family members were also studied ; one site ascertained aas . during phase 2 , data collection beyond the sibling pair was limited to parents , or , if parents were unavailable , unaffected siblings ; five sites ascertained aas . during phase 3 , only affected sibling pairs and trios were studied ; 5 additional sites ascertained aas . in total , 1,496 aa members of 580 pedigrees were studied at 10 sites .", "data cleaning and re - evaluation of t2d diagnoses by current criteria reduced the analysis sample to 1,344 members of 524 pedigrees ; for this study , we selected an informative subset of 1,077 members of 415 pedigrees .", "t2d diagnoses , originally following national diabetes data group criteria , were re - evaluated using current criteria prior to all analyses ; 84 affected and 13 unaffected sample members were re - assigned as unknown .", "body mass index ( bmi ) was computed from height and weight obtained from physical examination .", "we selected a subset of the sample for fine - mapping by excluding unaffected individuals below age 53 , the age by which onset had occurred in 75% of cases , and then any individual , either affected or unaffected , who consequently had no family members in the sample .", "this sample subset maintained significant lod scores in each of the linkage regions and produced power over 80% to detect association .", "power to produce a p - value of 0.00001 was estimated from simulation of 1000 replicates of a snp with minor allele frequency 0.4 and heterozygous effect a 10% increase in penetrance at age 50 for t2d or 3 year increase in aod . for fine - map genotyping , we used snagger ( http://snagger.sourceforge.net/ ) to select tagsnps within genes in the linkage regions on chromosomes 2 , 7 , 13 , and 18 . using hapmap phase 1 and 2 ( http://hapmap.ncbi.nlm.nih.gov ) ,", "variants in the region were tagged at linkage disequilibrium ( ld ) r  0.7 .", "each snp selected had minor allele frequency > 0.1 and illumina design score > 0.4 ; snp pairs had minimum spacing of 60 base pairs ( bp ) .", "the genetic map locations of the snps in centimorgans ( cm ) were obtained from the rutgers combined linkage - physical map of the human genome ( http://compgen.rutgers.edu/maps ) ; locations of snps not mapped directly were estimated using physical positions from ncbi dbsnp build 123 in a smoothing calculation ( conway institute bioinformatics service ; http://integrin.ucd.ie ) .", "genotyping errors were identified using pedcheck and merlin ; we zeroed 4978 genotypes of 710 snps , 74 identified by pedcheck and 4904 by merlin .", "genotypes for 24 snps , available on less than 95% of the sample following data cleaning , were retained after confirmation of minimal effects on the results .", "multi - point identity by descent ( ibd ) probabilities were computed at each cm using merlin , treating as haplotypes snp sets with pairwise ld r > 0.7 .", "we identified 279 , 175 , 133 , and 259 snp sets on chromosomes 2 , 7 , 13 , and 18 , respectively .", "likelihood analysis , as implemented in jpap , was used for univariate linkage analysis of unadjusted t2d ( ut2d ) , t2d adjusted for bmi ( bt2d ) , and aod and for bivariate association analysis of ut2d and aod .", "aod was adjusted for gender and modeled as a normal density with mean , standard deviation , and gender effect as parameters .", "t2d risk was modeled to account for aod in affected pedigree members , while allowing for censored observations with age , gender , and bmi ( for bt2d only ) effects and penetrance as parameters . for each trait ,", "additional parameters included heritability , a quantitative trait locus ( qtl ) effect in linkage analysis , and an additive snp effect in association analysis .", "we applied variance components linkage analysis using the univariate model for each trait in conjunction with the ibd probabilities . at each cm across the 5 regions , all parameters were estimated for aod while only heritability and qtl effect were estimated for ut2d and bt2d with all other parameters for those traits fixed at estimates obtained upon maximizing the likelihood while correcting the likelihood for the ascertainment of each pedigree through an affected sib pair .", "the lod score at each cm was computed as the common logarithm of the ratio of the maximized likelihoods with the qtl effect estimated to the maximized likelihood with the qtl effect set to zero .", "we assessed each fine - mapping snp for association with ut2d and aod by coding the snp genotype as an additive covariate and testing its effect on both traits in a bivariate model .", "p values were determined using a 2 degree of freedom  statistic computed as twice the natural logarithm of the ratio of the maximized likelihood with covariate effects estimated for both t2d and aod to the maximized likelihood with both effects set equal to zero .", "we controlled for multiple testing separately within each of the 5 linkage regions by specifying a false discovery rate of 0.05 accounting for 1291 , 1780 , 1819 , 1336 , and 2977 fine - mapping snps for chromosomes 2p , 2q , 7 , 13 , and 18 , respectively .", "we tested for an independent effect of each secondary associated snp , conditional on the snp within the same gene that attained the highest significance , by comparing the maximized likelihood estimating t2d and aod covariate effects for both snps simultaneously to the maximized likelihood estimating t2d and aod covariate effects for the most significant snp alone .", "p < 0.05 after bonferroni correction for the number of secondary snps in the gene supported the independence of the secondary snp .", "we genotyped 9,203 snps within five regions encompassing a total of 133 cm and 128 mb ( table 1 ) and merged them with 244 ( 36 duplicates ) genome scan snps .", "spacing between snps averaged 0.0145 cm ( 13,910 bp ) and ranged from 0.0 to 5.3 cm and from 26 bp to 6 mb . to comply with the sample size limitations of the cidr genotyping platform , we selected a subset of the original linkage sample to be informative for both linkage and association .", "the genotyped subset included 1077 members of 415 pedigrees that ranged from 2 to 11 members ( 1 to 8 were genotyped ) . in this subset ,", "aod and bmi differed little from the complete sample , but the unaffected sample members , selected for a minimum age of 53 years , were older ( table 2 ) .", "we previously identified 5 linkage peaks within four chromosomal regions from autosomal scans of t2d and aod ; the chromosome 2 region contained 2 peaks separated by 30 mb and the centromere .", "using updated genetic map positions for the genome scan snps , we repeated the linkage analyses on the smaller sample : the genome scan lod scores for all 5 peaks ( table 3 ) generally agreed with our published lod scores for bt2d and aod and for ut2d . upon adding the fine - mapping snps , linkage evidence remained in all 5 regions , but strengthened only on chromosome 2 ( table 3 ) . on chromosome 18 , only aod supported linkage , although ut2d provided weak support ( lod = 2.62 ) upon elimination of sample members with bmi > 45", ". the most remarkable effect of fine - mapping was the splitting of the lod score curves into multiple peaks in all of the regions except on chromosome 18 .", "additional evidence that multiple susceptibility genes contribute to the linkage signals derived from the identification of associated snps that reside in multiple genes within each of the 5 regions , including on chromosome 18 ( table 4 ) .", "the number of associated snps ranged from 4 on chromosome 13 to 17 on chromosome 2q ; the number of associated genes ranged from 2 on chromosome 13 to 9 on chromosome 7 .", "although 20 of the 27 associated genes were identified through a single snp , 5 genes were identified through 2 or 3 snps and arhgap25 and dpp10 were identified by 8 and 10 , respectively", ". however , six associated snp pairs exceeded ld r  0.7 : arhgap25 ( rs6714065 and rs7605681 ) , ctnna2 ( rs968820 and rs1368915 ) , dpp10 ( rs843417 and rs1823267 , rs10204212 and rs13432035 , rs4848376 and rs11694256 ) , and hip1 ( rs1179625 and rs1179622 ) . nevertheless , conditional association analysis supported the independence of two or more snps in arhgap25 , polr1b , dpp10 , and mtus2 ( supplementary table s1 ) .", "the fine - mapping of five chromosomal regions allowed us not only to confirm our genome scan linkages , but also to infer the presence of multiple t2d susceptibility genes in each of the regions .", "two types of evidence supported the presence of multiple susceptibility genes underlying the linkage peaks .", "first , except chromosome 18 , each lod score peak split into at least two peaks when fine - mapping snps were added to the linkage analysis .", "second , in every region including chromosome 18 , two or more potential susceptibility genes were identified through the association with t2d and aod of fine - mapping snps residing in those genes .", "in fact , martin et al . attributed many failures to identify causal variants to the incorrect assumption that a single or a limited number of variants are responsible for a linkage signal .", "as evidence , they identified variants in five genes that fully accounted for a linkage peak for plasma triglyceride level", ". if small effects are typical of common disease susceptibility genes , their detection through linkage analysis may be limited to locations where the genes cluster .", "however , fine - mapping using next generation sequencing may provide sufficient power necessary to detect even isolated genes . although chromosome 18 failed to show t2d linkage and chromosome 7 failed to show aod linkage", ", we nevertheless expect that the susceptibility genes residing in all regions both increase t2d risk and decrease aod .", "variation between regions in the information available undoubtedly affects the relative strength of the corresponding lod scores for t2d or aod . as evidence , the elimination of t2d cases with bmi > 45 kg / m revealed a weak t2d linkage on chromosome 18 and fine - mapping revealed aod linkages on chromosomes 2p and 2q .", "the lod scores are generally lower for aod than for t2d as expected since aod reflects onset age imprecisely and the familial correlation of aod may partially result from temporal clustering of t2d diagnoses among relatives . nevertheless , aod proved sufficiently accurate to produce linkage evidence in 4 of the chromosomal regions .", "few of the associated genes have previously been reported to associate with t2d or related traits .", "the exceptions include grb10 with t2d , \n nedd4l with diabetic nephropathy , and lipg with lipid metabolism .", "novel candidates reported herein include a cytokine ( il36b ) and genes involved in lipid metabolism ( acoxl ) and cell - cell and cell - matrix adhesions ( magi2 , cldn4 , ctnna2 ) .", "interestingly , the candidates also included genes involved in williams - beuren syndrome ( wbscr28 , wbscr17 , cldn4 ) , whose sufferers have a high prevalence of diabetes and pre - diabetes .", "dpp10 is related to dpp4 , whose inhibitors are oral anti - hyperglycemics used in t2d therapy , and dpp6 , which contributes to a triglyceride linkage , however , unlike dpp4 , ddp10 lacks serine protease / dipeptidyl peptidase activity .", "support for the functionality of these genes derived from evidence that their expression levels are affected by our associated snps or proxies with high ld .", "we tested 14 snps in the 5 genes for which expression levels from transformed lymphocytes were measured on a subset of 160 sample members ; nominal significance was obtained for one : p = 0.0148 for an effect of rs7579103 on arhgap2 expression levels .", "in other populations and various tissues , expression levels of arhgap2 , aff3 , polr1b , hip1 , lipg , and nedd4l showed evidence of an effect of an associated snp or its proxy ( p<1105 ) . for further insight into the nature of the associations , we haplotyped sets of 1530 fine - mapping snps , each encompassing two or more of the associated snps reported in table 4 .", "for arhgap25 , two haplotypes associated with t2d : the first extended across 4 of the associated snps and contained the risk alleles for all 4 ; the second extended across 6 associated snps and contained the risk alleles for all 6 , but differed from the first haplotype for alleles at other snps .", "we propose that each haplotype harbors a distinct causal variant , despite sharing associations with the same risk alleles , demonstrating the complexity of disease associations .", "the advantages include the opportunity to exploit information on local ancestry to localize disease susceptibility genes .", "as with any such analysis , confirmation of these findings awaits replication in another sample . in summary , linkage and association analysis using genotypes on an additional 9,203 fine - mapping snps added to five chromosomal regions confirmed each linkage and identified potential susceptibility genes in each of the regions ."], "abstract_text": ["<S> we previously localized type 2 diabetes ( t2d ) susceptibility genes to five chromosomal regions through a genome - wide linkage scan of t2d and age of diagnosis ( aod ) in the african american subset of the gennid sample . </S>", "<S> to follow up these findings , we repeated the linkage and association analysis using genotypes on an additional 9,203 fine - mapping snps selected to tag genes under the linkage peaks . in each of the five regions , we confirmed linkage and inferred the presence two or more susceptibility genes . the evidence of multiple susceptibility genes comprised : 1 ) multiple linkage peaks in four of the five regions ; 2 ) association of t2d and aod with snps within 2 or more genes in every region . </S>", "<S> the associated genes included 3 previously reported to associate with t2d or related traits ( grb10 , nedd4l , lipg ) , and 24 novel candidate genes including genes in lipid metabolism ( acoxl ) and cell - cell and cell - matrix adhesion ( magi2 , cldn4 , ctnna2 ) . </S>"], "labels": null, "section_names": ["Introduction", "Subjects and Methods", "Results", "Discussion", "Supplementary Material"], "sections": [["with the goal of discovering the genes that contribute to the risk of common diseases , numerous susceptibility loci have been identified using linkage analysis . despite replication of many of these linkages in a second sample ,", "some exceptions include tbc1d1 , identified as the gene responsible for the obesity linkage on chromosome 4p15 - 14 , and hoxb13 , identified as the gene responsible for the prostate cancer linkage on chromosome 17q21 - 22 .", "however , for many other common disease linkage signals , the underlying causal genes and variants await discovery .", "the expectation that a single gene accounts for a linkage peak may contribute to the difficulty in identifying causal genes . on the contrary", "attributed a triglyceride linkage on chromosome 7q36 to variants in five genes and christians et al .", "found that fine - mapping caused a single quantitative trait locus ( qtl ) for body size in mice to resolve first into three qtls , then one of those to split into two .", "furthermore , the clustering of causal genes may have stymied the multi - group effort to identify type 2 diabetes ( t2d ) genes on chromosome1q ; two strong associations present in populations of european ancestry failed replication and confirmation in other ethnic groups .", "these examples suggest that abandoned linkage findings might yet reveal susceptibility genes if reappraised while considering the possibility of multiple causal genes . as for other common diseases ,", "the challenge is even greater in african american ( aa ) populations where both prevalence and genetic diversity are higher , and pathophysiology may differ .", "genome wide case - control association studies ( gwass ) have identified single nucleotide polymorphisms ( snps ) with small effects on t2d risk , but few of the associated snps , identified primarily in european ancestry populations , are replicated in aas and few snps have been identified in aa populations . in general , the widespread rejection of family studies in favor of gwass has failed to produce the promised prognostic and diagnostic variants for t2d . as a resource for the discovery of genes related to t2d and its complications , the american diabetes association established the genetics of niddm ( gennid ) study . from 1993 to 2003", "we have used this resource to increase understanding of the genetics of t2d by applying linkage and family - based association analysis to the african american ( aa ) subset of the gennid sample .", "genome - wide linkage analysis using genotypes on 5,914 snps identified chromosomal regions that potentially harbor risk genes for t2d and age of t2d diagnosis ( aod ) .", "the strongest signal for t2d occurred on chromosome 2 at 95121 megabases ( mb ) , with weaker support for aod in the same region and for t2d at 6895 mb .", "both t2d and aod also showed linkage on chromosome 13 at 1930 mb , but linkage was limited to t2d on chromosome 7 at 5079 mb and to aod on chromosome 18 at 3165 mb .", "other analyses inferred pleiotropy with triglyceride in the chromosome 2p region and pleiotropy with obesity in the chromosome 13 region . herein we present linkage and association analyses on the aa subset of the gennid sample using genotypes on 9,203 fine - mapping snps added to the genome scan snps used previously .", "our first goal was to more precisely localize the t2d susceptibility genes in each of the five chromosomal regions identified in the genome scan ( chromosome 2 at 6895 and 95121 mb , chromosome 7 at 5079 mb , chromosome 13 at 1930 mb , and chromosome 18 at 3165 mb ) .", "our second goal was to test for association of the gene - based fine - mapping snps with t2d and aod in order to identify genes related to t2d in aas ."], ["the gennid study ascertained families through sibling pairs each with a t2d diagnosis . during phase 1 ,", "extended family members were also studied ; one site ascertained aas . during phase 2 , data collection beyond the sibling pair was limited to parents , or , if parents were unavailable , unaffected siblings ; five sites ascertained aas . during phase 3 , only affected sibling pairs and trios were studied ; 5 additional sites ascertained aas . in total , 1,496 aa members of 580 pedigrees were studied at 10 sites .", "data cleaning and re - evaluation of t2d diagnoses by current criteria reduced the analysis sample to 1,344 members of 524 pedigrees ; for this study , we selected an informative subset of 1,077 members of 415 pedigrees .", "t2d diagnoses , originally following national diabetes data group criteria , were re - evaluated using current criteria prior to all analyses ; 84 affected and 13 unaffected sample members were re - assigned as unknown .", "body mass index ( bmi ) was computed from height and weight obtained from physical examination .", "we selected a subset of the sample for fine - mapping by excluding unaffected individuals below age 53 , the age by which onset had occurred in 75% of cases , and then any individual , either affected or unaffected , who consequently had no family members in the sample .", "this sample subset maintained significant lod scores in each of the linkage regions and produced power over 80% to detect association .", "power to produce a p - value of 0.00001 was estimated from simulation of 1000 replicates of a snp with minor allele frequency 0.4 and heterozygous effect a 10% increase in penetrance at age 50 for t2d or 3 year increase in aod . for fine - map genotyping , we used snagger ( http://snagger.sourceforge.net/ ) to select tagsnps within genes in the linkage regions on chromosomes 2 , 7 , 13 , and 18 . using hapmap phase 1 and 2 ( http://hapmap.ncbi.nlm.nih.gov ) ,", "variants in the region were tagged at linkage disequilibrium ( ld ) r  0.7 .", "each snp selected had minor allele frequency > 0.1 and illumina design score > 0.4 ; snp pairs had minimum spacing of 60 base pairs ( bp ) .", "the genetic map locations of the snps in centimorgans ( cm ) were obtained from the rutgers combined linkage - physical map of the human genome ( http://compgen.rutgers.edu/maps ) ; locations of snps not mapped directly were estimated using physical positions from ncbi dbsnp build 123 in a smoothing calculation ( conway institute bioinformatics service ; http://integrin.ucd.ie ) .", "genotyping errors were identified using pedcheck and merlin ; we zeroed 4978 genotypes of 710 snps , 74 identified by pedcheck and 4904 by merlin .", "genotypes for 24 snps , available on less than 95% of the sample following data cleaning , were retained after confirmation of minimal effects on the results .", "multi - point identity by descent ( ibd ) probabilities were computed at each cm using merlin , treating as haplotypes snp sets with pairwise ld r > 0.7 .", "we identified 279 , 175 , 133 , and 259 snp sets on chromosomes 2 , 7 , 13 , and 18 , respectively .", "likelihood analysis , as implemented in jpap , was used for univariate linkage analysis of unadjusted t2d ( ut2d ) , t2d adjusted for bmi ( bt2d ) , and aod and for bivariate association analysis of ut2d and aod .", "aod was adjusted for gender and modeled as a normal density with mean , standard deviation , and gender effect as parameters .", "t2d risk was modeled to account for aod in affected pedigree members , while allowing for censored observations with age , gender , and bmi ( for bt2d only ) effects and penetrance as parameters . for each trait ,", "additional parameters included heritability , a quantitative trait locus ( qtl ) effect in linkage analysis , and an additive snp effect in association analysis .", "we applied variance components linkage analysis using the univariate model for each trait in conjunction with the ibd probabilities . at each cm across the 5 regions , all parameters were estimated for aod while only heritability and qtl effect were estimated for ut2d and bt2d with all other parameters for those traits fixed at estimates obtained upon maximizing the likelihood while correcting the likelihood for the ascertainment of each pedigree through an affected sib pair .", "the lod score at each cm was computed as the common logarithm of the ratio of the maximized likelihoods with the qtl effect estimated to the maximized likelihood with the qtl effect set to zero .", "we assessed each fine - mapping snp for association with ut2d and aod by coding the snp genotype as an additive covariate and testing its effect on both traits in a bivariate model .", "p values were determined using a 2 degree of freedom  statistic computed as twice the natural logarithm of the ratio of the maximized likelihood with covariate effects estimated for both t2d and aod to the maximized likelihood with both effects set equal to zero .", "we controlled for multiple testing separately within each of the 5 linkage regions by specifying a false discovery rate of 0.05 accounting for 1291 , 1780 , 1819 , 1336 , and 2977 fine - mapping snps for chromosomes 2p , 2q , 7 , 13 , and 18 , respectively .", "we tested for an independent effect of each secondary associated snp , conditional on the snp within the same gene that attained the highest significance , by comparing the maximized likelihood estimating t2d and aod covariate effects for both snps simultaneously to the maximized likelihood estimating t2d and aod covariate effects for the most significant snp alone .", "p < 0.05 after bonferroni correction for the number of secondary snps in the gene supported the independence of the secondary snp ."], ["we genotyped 9,203 snps within five regions encompassing a total of 133 cm and 128 mb ( table 1 ) and merged them with 244 ( 36 duplicates ) genome scan snps .", "spacing between snps averaged 0.0145 cm ( 13,910 bp ) and ranged from 0.0 to 5.3 cm and from 26 bp to 6 mb . to comply with the sample size limitations of the cidr genotyping platform , we selected a subset of the original linkage sample to be informative for both linkage and association .", "the genotyped subset included 1077 members of 415 pedigrees that ranged from 2 to 11 members ( 1 to 8 were genotyped ) . in this subset ,", "aod and bmi differed little from the complete sample , but the unaffected sample members , selected for a minimum age of 53 years , were older ( table 2 ) .", "we previously identified 5 linkage peaks within four chromosomal regions from autosomal scans of t2d and aod ; the chromosome 2 region contained 2 peaks separated by 30 mb and the centromere .", "using updated genetic map positions for the genome scan snps , we repeated the linkage analyses on the smaller sample : the genome scan lod scores for all 5 peaks ( table 3 ) generally agreed with our published lod scores for bt2d and aod and for ut2d . upon adding the fine - mapping snps , linkage evidence remained in all 5 regions , but strengthened only on chromosome 2 ( table 3 ) . on chromosome 18 , only aod supported linkage , although ut2d provided weak support ( lod = 2.62 ) upon elimination of sample members with bmi > 45", ". the most remarkable effect of fine - mapping was the splitting of the lod score curves into multiple peaks in all of the regions except on chromosome 18 .", "additional evidence that multiple susceptibility genes contribute to the linkage signals derived from the identification of associated snps that reside in multiple genes within each of the 5 regions , including on chromosome 18 ( table 4 ) .", "the number of associated snps ranged from 4 on chromosome 13 to 17 on chromosome 2q ; the number of associated genes ranged from 2 on chromosome 13 to 9 on chromosome 7 .", "although 20 of the 27 associated genes were identified through a single snp , 5 genes were identified through 2 or 3 snps and arhgap25 and dpp10 were identified by 8 and 10 , respectively", ". however , six associated snp pairs exceeded ld r  0.7 : arhgap25 ( rs6714065 and rs7605681 ) , ctnna2 ( rs968820 and rs1368915 ) , dpp10 ( rs843417 and rs1823267 , rs10204212 and rs13432035 , rs4848376 and rs11694256 ) , and hip1 ( rs1179625 and rs1179622 ) . nevertheless , conditional association analysis supported the independence of two or more snps in arhgap25 , polr1b , dpp10 , and mtus2 ( supplementary table s1 ) ."], ["the fine - mapping of five chromosomal regions allowed us not only to confirm our genome scan linkages , but also to infer the presence of multiple t2d susceptibility genes in each of the regions .", "two types of evidence supported the presence of multiple susceptibility genes underlying the linkage peaks .", "first , except chromosome 18 , each lod score peak split into at least two peaks when fine - mapping snps were added to the linkage analysis .", "second , in every region including chromosome 18 , two or more potential susceptibility genes were identified through the association with t2d and aod of fine - mapping snps residing in those genes .", "in fact , martin et al . attributed many failures to identify causal variants to the incorrect assumption that a single or a limited number of variants are responsible for a linkage signal .", "as evidence , they identified variants in five genes that fully accounted for a linkage peak for plasma triglyceride level", ". if small effects are typical of common disease susceptibility genes , their detection through linkage analysis may be limited to locations where the genes cluster .", "however , fine - mapping using next generation sequencing may provide sufficient power necessary to detect even isolated genes . although chromosome 18 failed to show t2d linkage and chromosome 7 failed to show aod linkage", ", we nevertheless expect that the susceptibility genes residing in all regions both increase t2d risk and decrease aod .", "variation between regions in the information available undoubtedly affects the relative strength of the corresponding lod scores for t2d or aod . as evidence , the elimination of t2d cases with bmi > 45 kg / m revealed a weak t2d linkage on chromosome 18 and fine - mapping revealed aod linkages on chromosomes 2p and 2q .", "the lod scores are generally lower for aod than for t2d as expected since aod reflects onset age imprecisely and the familial correlation of aod may partially result from temporal clustering of t2d diagnoses among relatives . nevertheless , aod proved sufficiently accurate to produce linkage evidence in 4 of the chromosomal regions .", "few of the associated genes have previously been reported to associate with t2d or related traits .", "the exceptions include grb10 with t2d , \n nedd4l with diabetic nephropathy , and lipg with lipid metabolism .", "novel candidates reported herein include a cytokine ( il36b ) and genes involved in lipid metabolism ( acoxl ) and cell - cell and cell - matrix adhesions ( magi2 , cldn4 , ctnna2 ) .", "interestingly , the candidates also included genes involved in williams - beuren syndrome ( wbscr28 , wbscr17 , cldn4 ) , whose sufferers have a high prevalence of diabetes and pre - diabetes .", "dpp10 is related to dpp4 , whose inhibitors are oral anti - hyperglycemics used in t2d therapy , and dpp6 , which contributes to a triglyceride linkage , however , unlike dpp4 , ddp10 lacks serine protease / dipeptidyl peptidase activity .", "support for the functionality of these genes derived from evidence that their expression levels are affected by our associated snps or proxies with high ld .", "we tested 14 snps in the 5 genes for which expression levels from transformed lymphocytes were measured on a subset of 160 sample members ; nominal significance was obtained for one : p = 0.0148 for an effect of rs7579103 on arhgap2 expression levels .", "in other populations and various tissues , expression levels of arhgap2 , aff3 , polr1b , hip1 , lipg , and nedd4l showed evidence of an effect of an associated snp or its proxy ( p<1105 ) . for further insight into the nature of the associations , we haplotyped sets of 1530 fine - mapping snps , each encompassing two or more of the associated snps reported in table 4 .", "for arhgap25 , two haplotypes associated with t2d : the first extended across 4 of the associated snps and contained the risk alleles for all 4 ; the second extended across 6 associated snps and contained the risk alleles for all 6 , but differed from the first haplotype for alleles at other snps .", "we propose that each haplotype harbors a distinct causal variant , despite sharing associations with the same risk alleles , demonstrating the complexity of disease associations .", "the advantages include the opportunity to exploit information on local ancestry to localize disease susceptibility genes .", "as with any such analysis , confirmation of these findings awaits replication in another sample . in summary , linkage and association analysis using genotypes on an additional 9,203 fine - mapping snps added to five chromosomal regions confirmed each linkage and identified potential susceptibility genes in each of the regions ."], [""]]}
{"article_id": "PMC4893437", "article_text": ["the circadian system organizes the different biological functions in 24 h , such as sleep / activity , temperature , heart rate , glucose level , cortisol production , and oxidative stress . in mammals ,", "this system is organized by a central clock localized in the suprachiasmatic nucleus of the hypothalamus ( scn ) and in a series of peripheral oscillators such as the liver , lung , adrenal gland , fibroblast cells , and others tissues [ 2 , 4 ] .", "the peripheral oscillators are synchronized every day via nervous or humoral signals , and the most important humoral signal is the melatonin hormone , secreted by the pineal gland during the dark hours , and its impairment is associated with different disorders such as insomnia , cardiovascular disease , and cancer .", "the molecular clock is organized by transcriptional / translational feedback loop of clock genes named clock , bmal1 , per13 , and cry1 - 2 . at the molecular level ,", "the complex clock - bmal1 stimulates the expression of negative regulators per13 and cry1 - 2 , and their protein inhibits the effect of the heterodimer clock / bmal1 [ 6 , 7 ] .", "moreover , the molecular clock has different modulators which give fine tuning of output signals such as rev - erb , a negative regulator of bmal-1 expression [ 79 ] , sirt1 , a regulator of clock - mediated acetylase activity [ 10 , 11 ] , and pgc1 , a stimulator of bmal1 expression [ 12 , 13 ] ( see figure 1 ) .", "this system provides an output signal to genes such as hexokinase , dbp [ 1517 ] , vegf , steroidogenic enzymes star , 3-hsd , and wee-1 , giving a circadian oscillation of physiological functions such as metabolism , angiogenesis , cortisol production [ 2 , 19 ] , and cellular proliferation .", "the molecular clock can be modified by environmental changes and our modern lifestyle , resulting in physiological alteration and risk of pathologies ; for example , during the monkey 's pregnancy , the light exposition during night hours induces a lower body temperature and absence of circadian rhythm of temperature in the newborn . in humans ,", "different reports showed that the alterations of the circadian system increased the risks of cancer [ 2123 ] , preeclampsia , diabetes [ 3 , 25 ] , and mood disorder .", "curiously , a large quantity of polymorphisms has been detected in clock genes , which can be influencing the development of diseases through different physiological systems .", "this review will primarily focus on the hypothesis which states that the variation of genetic components of the circadian system , similar to environmental changes , can affect several physiological systems and produce an elevation of the risk of developing a disease . in recent years", ", there has been a significant increase in available information from polymorphic variations and epigenetic modification over clock genes and their risk of diseases .", "the report of health from the united states ( 2014 ) showed about 10% of population have a poor health associated with pathologies such as obesity ( 35.5% ) , hypercholesterolemia ( 30% ) , diabetes ( 32% ) , and cancer ( 6.4% ) . curiously , despite its low frequency in population , cancer is the second cause of death in the united states .", "the above pathologies add the infertility , which affects about 10.9% of women in the united states   and 17% of women in other developed countries . during short - stay in the hospital ,", "mood disorder and psychosis are the principal causer of hospital stay during 2014 and represent the third cause of morbidity worldwide .", "this present research reports the last single nucleotide polymorphism ( snp ) associated with an elevated risk of developing principal pathologies worldwide such as mood disorder , infertility , cancer , metabolism and diabetes   and addictions , and a description of usefulness for a more detailed study in other pathologies .", "in mammals , the circadian system is a supraphysiological system that regulates biological functions every 24 h   such as glucose homeostasis , temperature , blood pressure , tone in the coronary artery , and heart rate .", "the bmal1/clock complex target genes are related to the circadian expression of metabolic pathways as was observed by hatanaka et al .   through high resolution", "the author detected a circadian expression of ( i ) glucose metabolism - related genes such as glut2 , por , pck1 , and gys2 and ( ii ) cholesterol metabolism - related genes such as cyp2a4 , cyp2a5 , cyp4a14 , cyp7a1 , and cyp2c55 ; and considering the very important role these systems have in metabolism , any alteration of them can have negative health effects .", "today there are several metabolic problems affecting the population , such as obesity , hyperglycemia , dyslipidemia , and hypertension .", "the cooccurrence of three or more metabolic disorders , including obesity , is defined as  metabolic syndrome  , showing a prevalence in the united states during 2009 - 2010 of about 22% of adults ( 21.4626.15 ) . the insulin resistance or type", "2 diabetes is one of the most important metabolic diseases currently affecting about 366 million people , causing more than 3.8 million deaths , and showing a rise in the number of affected people as observed in the united states population during 19992010 .", "several factors could be increasing the risk of death by diabetes , such as dyslipidemia , hypertension , and obesity , all of which are driving to an elevated risk of cardiovascular disease .", "a variant of this pathology can also develop during the pregnancy ( gestational diabetes mellitus ) and is characterized by glucose intolerance   in the mother and adverse consequences for her offspring , such as an increase in blood pressure , body mass index ( bmi ) , and body fat and a decrease in hdl levels . at the molecular level ,", "both diseases have in common the impairment of signal transduction of insulin receptors such as pi3kinase / akt and ras / map kinases [ 42 , 45 , 46 ] , leading to metabolic dysregulations   such as inhibitions of glycogen synthesis , impaired translocation of glut4 to plasmatic membrane , or antilipolytic effects of insulin in white adipose tissue [ 39 , 46 ] .", "meta - analyses from 448 articles reporting shift work and health consequence have detected a strong relationship between shift work , a potent chronodisruptor , and diabetes mellitus type 2 ( odd ratio about 1.42 times higher than day workers ) .", "however , this is not the only chronodisruption which we are exposed too . in animal models exposed to light / dark patterns similar to shift work , the length of the working day and changes of feeding times induce a modification of the liver weight and plasmatic glucose and a modification of the circadian profile for glucose , insulin , and triglyceride .", "these findings add to the observation in the circadian disruption in mutant animals , showing alterations of cholesterol metabolism , abolition of the circadian production of glycerol , free fatty acids , and impaired expression of rate - limiting lipolytic enzymes such as lipase , all of which add to increased weight gain , adipocyte hypertrophy , high level of glucose , glucose intolerance , and hypersecretion of insulin [ 4952 ] .", "polymorphic variations of clock genes can be incrementing the risks of developing a disease similar to chronodisruption by environmental changes as it has been detected in human metabolic disorders ( see table 1 ) .", "for example , the genotyping of clock genes in 346 greek pregnant women and their risk of diabetes was performed , detecting that the polymorphisms of bmal1 rs7950226 and rs11022775 are associated with gestational diabetes mellitus ( p = 0.025 , or = 1.46 and p = 4.455e  06 , or = 2.64 , resp . ) , while the study of the haplotype analysis of rs7950226/rs11022775 showed a major frequency in women with gestational diabetes mellitus ( p = 0.0069 , or = 6.96 ) .", "similarly , a study performed in subjects from the united kingdom and pakistan reported that cry1 and cry2 polymorphisms rs2292912 and rs12315175 , respectively , are associated with diabetes ( p = 0.015 and 0.008 , resp . ) . moreover", ", the variant rs12315175 for cry2 has a tendency to elevate the risk at about 5% compared to the variation of cry1 ( or = 1.05 and 0.95 , resp . ) .", "also , the authors did not find associations between diabetes and bmal1 polymorphisms rs7950226 and rs11022775 , as occurs in gestational diabetes mellitus .", "genotyping of 19,000 adults from northern sweden for variants rs8192440 for cry1 and rs11605924 for cry2 is associated with higher level of glucose concentrations at 2 hours ( p = 0.06 and p = 0.005 , resp . ) .", "another study genotyping 1304 individuals from 424 british families , containing at least one patient with diabetes type 2 ( diabetes in families study collection ) , demonstrates the relation between bmal1 polymorphisms rs7950226 and rs11022775 and diabetes ( p = 0.002 ) , reinforcing what was previously described .", "similar relations are observed for bmal2 polymorphism rs7958822 in obese men and women ( or 2.2 and 2.7 , resp . )   and the deletion / insertion of 54 base pair sequences of five repeat alleles on per3 gene ( rs57875989 ) [ 57 , 58 ] .", "in contrast , the per2 polymorphism rs7602358 is associated with a protection from type 2 diabetes in the uk population , which suggests that not all polymorphisms are negative for health . at the level of dyslipidemia", ", an interesting correlation has been detected between small dense ldl level and polymorphism of clock genes . in individuals carrying the polymorphism", "associated with the clock gene ( rs1801260 ) , the genotype tt or tc showed a major level of small dense ldl , which leads to increased triglycerides and increased risk of cardiovascular and obesity diseases , similar to the  metabolic syndrome  . likewise , genotyping in the japanese population identified clock gene polymorphism rs1801260 associated with higher odds ratio ( or ; 1.5 ) of type 2 diabetes ; in contrast , a multicenter study detected the clock polymorphism rs4580704 is associated with prevention of diabetes and cardiovascular disease .", "the haplotypes of rs10002541 and rs4864546 from clock ( cg and tg variations ) are associated with abdominal obesity in chinese population ( or 0.74 and 1.70 , resp . )   and polymorphism rs4864546 is associated with low level of hdl / apolipoprotein a1 ratio in spain .", "moreover , clock polymorphisms rs12649507 and rs3749474 are associated with higher intake of polyunsaturated fatty acid   and fat intake , which can modify the body mass index ( bmi ) .", "this is an antecedent which , bearing in mind the ideas above , suggests the genetic components of the circadian system as a critical factor in the development of metabolic diseases .", "in humans , infertility affects about 9% of couples , and about one - third of infertility cases associated with idiopathic male infertility are multifactorial , with 50% due to genetic abnormalities .", "the circadian system is important during reproduction and development [ 89 , 90 ] ; for example , it is involved in the timing of the lh surge [ 90 , 91 ] , stimulation of ovulation [ 90 , 92 ] , and regulating the level of steroidogenic acute regulatory protein ( star ) expression and is critical for cholesterol translocation inside mitochondria and sperm count .", "the genetic factors of clock genes are implicated in the pathogenesis of infertility ( see table 2 ) , as it occurs in male partners of infertile couples .", "the genotyping of male partners detected a correlation between single nucleotide polymorphism of clock rs11932595 , rs6811520 , and 6850524 , with infertility ( p < 0.05 and or ranged between 1.4 and 1.9 ) .", "similarly , the analysis of bmal1 polymorphism rs4757144 in slovenian and serbian caucasian men showed a significant correlation with infertility ( p = 0.047 ) , suggesting that clock genes clock and bmal1 contribute to successful fertilization .", "mood or affective disorders are important causes of morbidity , where we can highlight depressive pathologies and hypomaniac and maniac disorders as major diseases affecting the population [ 29 , 66 ] , and it can be highlighted that depressive pathologies are the third largest source of morbidity in the world .", "curiously , a potent correlation has been detected between mood disorders , sleep / activity , and the circadian system   suggesting that the circadian system can be modulating the neuronal activity . during pregnancy ,", "the prevalence of mental illnesses in women is about 8% , suggesting an imbalance between physiology of sleep and/or the circadian system .", "this can be observed in the onset of a mental disease as has been observed in the impaired circadian production of melatonin during the pregnancy of depressed women ( approximately 34 weeks of gestation ) , which shows an advance onset time of melatonin production of about 40 minutes and a minor production during the dark hours [ 97 , 98 ] .", "moreover , workers who do shift work ( circadian disruption ) showed decreased alertness , cognitive functions , mood , social and work activities , and health [ 26 , 99 ] , which are associated with an impaired circadian system via melatonin suppression , all of which leads to the appearance of a mood disorder .", "for this reason , we can say that the circadian system may contribute to the risk of developing a mood disorder and the genetic component of clock genes can be involved in the development of mood pathologies   ( see table 2 ) .", "at the genetic component level of the circadian system , a study of 744 people who carry polymorphisms rs2291739 and rs11171856 from tim , a member of the clock gene family which interacts with per1 - 2 proteins [ 100 , 101 ] , showed that they have an elevated risk of developing mood disorders ranging between 19 and 23% ( or 1.19 and 1.23 , resp . ) .", "moreover , gene variants of positive regulator clock have also been associated with mood disorders .", "people who carry the variants rs1801260 and rs11932595 showed a major risk of developing a bipolar disorder ranging between 45 and 37% , respectively , in comparison to a patients without polymorphism ( or 1.45 and 1.37 , resp . ) . in the same study", ", it was also observed that the polymorphisms rs2291739 and rs11171856 from tim gene are associated with unipolar disease , with a risk ranging between 37 and 40% in comparison to a patient without polymorphism ( or 1.37 and 1.40 , resp . ) .", "/ rs11022779g / rs1122780 t ( haplotype ) are associated with mood disorders   and bipolar disease [ 29 , 66 ] .", "it has also been seen that the bipolar pathologies are associated with other genetic variations of bmal1 gene ( rs4757144 , rs1982350 , and rs1481892 ) and the negative regulator per3 ( rs2859387 ) .", "curiously , a study performed an indian families reported the bmal1 polymorphisms rs2279287 are associated with seasonal affective disorder .", "in addition , the gene variant of cry2 gene ( rs4132063 )   and deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 )   may also be contributing to mood disorders via increased risk of developing bipolar disease .", "for example , a study in south india showed the prevalence of five repeat homozygotes from rs57875989 is associated with bipolar disease ( or 1.72 ) but not with schizophrenia . at the level of depressive pathologies , a study performed in china compared 485 subjects ( control ) to 105 patients suffering from a depression disorder .", "the genotyping for clock genes showed that the variants of cry1 ( rs2287161 ) and cry2 ( rs10838524 ) are correlated to the depression disease with an odds ratio of 1.75 ( p = 0.012 ) , which suggests that a patient with the allele has 1.75 times more risk of developing depression .", "moreover the authors detected a single nucleotide polymorphism for tef gene , the variant rs738499 , is associated with 2.22 times more risk of development of depression ( odds ratio of 2.22 .", "finally , in a study performed in young adults , the risk of excessive intake of alcohol is minor when the polymorphism of per2 gene rs56013859 is present , suggesting a possible protector role for allelic variation of clock genes in addictions .", "in fact , this suggests that some polymorphisms are not negative for our health and it is necessary to study them in greater depth .", "cellular proliferation is a critical event for the survival and restitution of tissues of all living things and the circadian system is capable of delivering temporary information to the cell cycle [ 102104 ] .", "however , an uncontrolled cellular proliferation and an excessive tissue growth are observed by an altered cellular cycle during cancer , a pathology that showed a higher incidence in patients exposed to an impaired circadian system such as a shiftwork [ 106 , 107 ] . the cell cycle is a finely regulated process from a cell that is capable of generating multiple cells through a series of cell divisions , including four critical and successive steps named g1 phase ( growth phase 1 ) , s phase ( synthesis ) , g2 ( growth phase 2 ) , and m phase ( mitosis ) . during the cell cycle , cyclin - dependent kinases ( cdk )", "are critical for the transition between different stages , for example , cdc2 plus cyclin b protein kinase form the complex cdk1 which regulates g2/m transition [ 108 , 109 ] . during dna replication", ", there are a number of controls or checkpoints that are critical in the cell cycle when dna damage is detected : the activation of the rad protein , which induces the action of cds 1 proteins , is triggered , as well as chk1 which are involved in cell cycle arrest via action of wee-1 and mik-1 proteins .", "however , the efficiency of this process changes depending on the time of the day when the lesion occurred .", "a lesion in the liver , occurring during the last light hours , induced a massive entry to m phase compared to a lesion which occurs early in the morning , which shows that the hour of surgery and the circadian system are critical for liver regeneration .", "similarly , oral mucosa is a highly proliferative tissue and it showed a circadian expression of clock genes bmal1 , per1 , and cry1 and thymidylate synthase activity , critical for dna synthesis during phase s. curiously , the peak of per1 mrna precedes the peak of thymidylate synthase activity , which suggests that the circadian system modulates the cellular proliferation in mucosa .", "wee-1 gene regulates cellular proliferation , and its promoter has three conserved sequence cacgtg ( e - box ) critical for the circadian expression of wee-1 .", "its protein is capable of inactivating the cdc2-cyclin b complex via phosphorylation which inhibits transition between g2/m .", "curiously , knock - down of bmal1 in carcinoma cells of the colon ( c26 cells ) , fibroblast cells ( l929 cell ) , and intestine epithelial cells ( iecs ) produces cellular proliferation in vitro and increments the size of tumor cells injected subcutaneously , via the inhibition of apoptosis and the reduction of the time transition between g2/m , reduction of p53 and wee-1 expression .", "moreover , the knock - out for clock genes bmal1 y per2 in mice previously exposed to gamma radiation caused in mice hyperplasic growth and development of lymphoma , hepatic carcinomas , ovary tumors , and osteosarcomas via reduction of p53 expression .", "a precedent that reinforces the idea that clock genes can be modulators of cellular cycle via wee-1 and p53 proteins . at the genetic component level", ", different studies reveal the importance of the polymorphic variant of clock genes ( see table 3 ) and how the chrono - type modulates the risk of cancer , such as observed in breast cancer , in which the risk is more elevated in premenopausal women ( or , 2.43 and 2.55 ; resp . ) .", "similarly , women showed evening or night preference such as shown in the case of a study performed on norwegian nurses working in night shift which revealed two polymorphisms associated with breast cancer .", "the risk is incremented when women spend more time working during night hours , but this risk is higher when women have the alleles for bmal1 rs2290035 ( or 1.91 ) , rs969485 ( or 1.64 ) , and rs3903529 ( or 2.77 ) or variant rs3750420 ( or 1.6 ) of roreb gene .", "similarly , an incremented risk is associated with breast cancer in women from france when they have the allele rs11932595 in clock gene ( or = 0.74 ) or in connecticut ( usa ) when they have the polymorphisms rs7698022 ( or 1.34 ) and rs1048004 ( or , 1.43 ) .", "curiously , when norwegian nurses are exposed to shift work during four nights , they showed three times more risk ( or 2.75 ) of developing breast cancer when they were carrying clock polymorphism rs11133373 , which suggests that the disruption of endogenous circadian rhythm by polymorphism associated with clock genes increases the risk of cancer .", "moreover , clock gene expression is induced in breast tissue from patients with breast cancer ; this expression is associated with hypomethylation of clock gene .", "the silencing of clock gene lowered when women carry polymorphisms associated with cancer such as rs10448004 and rs7698022 , which suggests that clock gene is a critical protagonist of cancer development . at level of colorectal cancer", ", a strong correlation is detected between cancer and the genetic component of the circadian system .", "for example , a polymorphism in the clock gene ( rs1801260 ) showed a major prevalence in a cancer patient ( p < 0.0001 , or = 1.78 for c - allele ) compared to a control patient .", "similarly , a screening performed in south carolina , usa , showed that a variation by deletion / insertion of a 54 base pair sequence on per3 gene ( rs57875989 ) is associated with a higher risk of colorectal adenoma formation with odds ratio within 2.15.1 .", "moreover , a population study performed on the residents of king county , washington ( usa ) , detected six different types of polymorphism , which are significantly associated with the risk and aggressiveness of prostate cancer .", "these genetics variants are rs1012477 for per3 ( or 1.3 ) , rs7602358 for per2 ( or 1.24 ) , and rs2289591 in per1 ( or 1.7 ) .", "similarly , a genotyping of a patient with prostate cancer showed a strong correlation between cry1 polymorphisms rs7297614 , rs1921126 , and rs12315175 and fatal prostate cancer ( or mean within 1.52 )   and a study conducted in china showed that the cry2 variant rs1401417 and the deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 ) were associated with a major risk of developing prostate cancer ( or ; 1.7 and 1.3 , resp . ) . a study", "performed in brazilian patients with pulmonary cancer showed a strong correlation of per3 polymorphism rs228644 and the risk of cancer ( or 1.99 ) .", "moreover , the authors reported the ancestral haplotypes for per3 rs228729 , rs228727 , rs707467 , rs228644 , and rs10462020 are associated with a higher cancer frequency ; similarly , a meta - analysis performed by literature search showed that the variant insertion / deletion of per3 rs57875989 is associated with an increase of cancer susceptibility in about 17%   or 70% . in a similar way", ", a soft risk is associated with breast cancer in premenopausal women from india , which suggests there is a relation between per3 and cellular proliferation .", "the frequency analysis of 1,538 breast cancer cases and 1,605 controls in china for clock gene variants showed the strong associations between three snps in circadian clock genes and the risk of developing breast cancer .", "the variants for cry1 rs1056560 are correlated to cancer , which elevate the risk in about 11% ( or 1.11 ) ; per2 rs934945 in about 15% ( or 1.15 ) ; and clock rs3805151 in about 35% ( or 1.35 ) .", "in contrast , tim protection for breast cancer development is detected in patients that carry the c - allele of rs7302060 ( or , 0.54 ) .", "the g allele of rs2291738 and the c - allele of rs7302060 are associated with reduced risk of breast cancer among estrogen receptor (  ) or progesterone receptor (  ) positive breast cancer cases ( or , 0.46 and 0.36 , resp . ) .", "the exogenous expression of human clock in cell lines of colorectal carcinoma induces the cellular proliferation in about 28% .", "in contrast , knock - down of endogenous clock gene expression inhibits the cell proliferation in about 34% . moreover", ", exogenous clock inhibits the apoptosis ( 42% reduction ) via inhibition on apoptosis associated proteins expression of bax and bid and the increase of phosphorylation of akt . in vivo experiments by xenograft transplant of colorectal carcinoma cell line transduced with clock", "increase the tumor volume and tumor weight in about 61% and a 91% , respectively .", "however , the pharmacological inhibition of cry in human breast cancer by treatment with pharmacological agent ks15   inhibits the proliferation and cell viability by stimulation of wee-1 expression   and stimulates the activity of heterodimer complex bmal1:clock [ 112 , 113 ] .", "curiously , two polymorphisms for clock gene cry2 , rs11038689 and rs1401417 , have a protective action over the mammary cancer , which suggests that not all polymorphisms associated with clock genes are negative for our health .", "non - hodgkin 's lymphoma is characterized by lymphoproliferation and advanced clinical stages , invasion to other tissues , and death .", "the estimated deaths from non - hodgkin 's lymphoma in the united states amounted to 19,790 during 2015 .", "analysis of cry2 variants showed the polymorphisms rs11038689 , rs7123390 , and rs1401417 increase the risk of lymphoma ( or , 2.34 ; 2.40 and 2.97 , resp . ) .", "moreover , a minor association of clock gene variants and glioma is detected for per1 rs2585405 , clock rs11133391 , and cry1 rs12315175 ( or , 1.16 ; 1.08 and 1.02 , resp . ) .", "genotyping of han chinese patients diagnosed with primary hepatocellular carcinoma showed an association between single snps of per3 rs228669 and cry1 rs3809236 with odds ratio of 1.41 and 1.26 , respectively .", "these precedents reinforce the idea that clock genes can be modulators of cellular cycle and that modulates the risk of cancer .", "the circadian system is a supraphysiological system which modulates different physiological systems , and any alteration of this can have a negative impact on human health .", "different chronodisruptors have been described in literature such as light / dark pattern and inhibition of melatonin production as occurs in shifts work .", "however , other factors can be contributing on a minor scale , such as mealtimes or a genetic component .", "the relevance of the genetic variation of clock genes and how it can interact with the environment is unknown .", "but it has been described that the genetic component in the population predisposes the development of different pathologies such as diabetes , dyslipidemias , obesity , mood disorders , and addiction , all of which suggest the importance of this system to our health .", "however , it is also necessary to say that the genetic component could be protecting our health such as in the case of polymorphisms associated with per2 and cry2 genes in diabetes , alcohol intake , and cancer . these findings will need to be implemented and evaluated at the genetic interaction level and also the way in which the environment factors trigger the expression of these pathologies will be examined .", "finally , prospective studies are necessary to assess the predictive potential of these markers and to implement early treatment with consequent cost reduction for the health system ."], "abstract_text": ["<S> the circadian system is a supraphysiological system that modulates different biological functions such as metabolism , sleep - wake , cellular proliferation , and body temperature . </S>", "<S> different chronodisruptors have been identified , such as shift work , feeding time , long days , and stress . </S>", "<S> the environmental changes and our modern lifestyle can alter the circadian system and increase the risk of developing pathologies such as cancer , preeclampsia , diabetes , and mood disorder . </S>", "<S> this system is organized by transcriptional / tranductional feedback loops of clock genes clock , bmal1 , per13 , and cry1 - 2 . </S>", "<S> how molecular components of the clock are able to influence the development of diseases and their risk relation with genetic components of polymorphism of clock genes is unknown . </S>", "<S> this research describes different genetic variations in the population and how these are associated with risk of cancer , metabolic diseases such as diabetes , obesity , and dyslipidemias , and also mood disorders such as depression , bipolar disease , excessive alcohol intake , and infertility . </S>", "<S> finally , these findings will need to be implemented and evaluated at the level of genetic interaction and how the environment factors trigger the expression of these pathologies will be examined . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Clock Genes and Metabolic Disorder", "3. Clock Genes and Infertility", "4. Clock Genes, Mood Disorder, and Others", "5. Clock Genes and Cancer", "6. Conclusion"], "sections": [["the circadian system organizes the different biological functions in 24 h , such as sleep / activity , temperature , heart rate , glucose level , cortisol production , and oxidative stress . in mammals ,", "this system is organized by a central clock localized in the suprachiasmatic nucleus of the hypothalamus ( scn ) and in a series of peripheral oscillators such as the liver , lung , adrenal gland , fibroblast cells , and others tissues [ 2 , 4 ] .", "the peripheral oscillators are synchronized every day via nervous or humoral signals , and the most important humoral signal is the melatonin hormone , secreted by the pineal gland during the dark hours , and its impairment is associated with different disorders such as insomnia , cardiovascular disease , and cancer .", "the molecular clock is organized by transcriptional / translational feedback loop of clock genes named clock , bmal1 , per13 , and cry1 - 2 . at the molecular level ,", "the complex clock - bmal1 stimulates the expression of negative regulators per13 and cry1 - 2 , and their protein inhibits the effect of the heterodimer clock / bmal1 [ 6 , 7 ] .", "moreover , the molecular clock has different modulators which give fine tuning of output signals such as rev - erb , a negative regulator of bmal-1 expression [ 79 ] , sirt1 , a regulator of clock - mediated acetylase activity [ 10 , 11 ] , and pgc1 , a stimulator of bmal1 expression [ 12 , 13 ] ( see figure 1 ) .", "this system provides an output signal to genes such as hexokinase , dbp [ 1517 ] , vegf , steroidogenic enzymes star , 3-hsd , and wee-1 , giving a circadian oscillation of physiological functions such as metabolism , angiogenesis , cortisol production [ 2 , 19 ] , and cellular proliferation .", "the molecular clock can be modified by environmental changes and our modern lifestyle , resulting in physiological alteration and risk of pathologies ; for example , during the monkey 's pregnancy , the light exposition during night hours induces a lower body temperature and absence of circadian rhythm of temperature in the newborn . in humans ,", "different reports showed that the alterations of the circadian system increased the risks of cancer [ 2123 ] , preeclampsia , diabetes [ 3 , 25 ] , and mood disorder .", "curiously , a large quantity of polymorphisms has been detected in clock genes , which can be influencing the development of diseases through different physiological systems .", "this review will primarily focus on the hypothesis which states that the variation of genetic components of the circadian system , similar to environmental changes , can affect several physiological systems and produce an elevation of the risk of developing a disease . in recent years", ", there has been a significant increase in available information from polymorphic variations and epigenetic modification over clock genes and their risk of diseases .", "the report of health from the united states ( 2014 ) showed about 10% of population have a poor health associated with pathologies such as obesity ( 35.5% ) , hypercholesterolemia ( 30% ) , diabetes ( 32% ) , and cancer ( 6.4% ) . curiously , despite its low frequency in population , cancer is the second cause of death in the united states .", "the above pathologies add the infertility , which affects about 10.9% of women in the united states   and 17% of women in other developed countries . during short - stay in the hospital ,", "mood disorder and psychosis are the principal causer of hospital stay during 2014 and represent the third cause of morbidity worldwide .", "this present research reports the last single nucleotide polymorphism ( snp ) associated with an elevated risk of developing principal pathologies worldwide such as mood disorder , infertility , cancer , metabolism and diabetes   and addictions , and a description of usefulness for a more detailed study in other pathologies ."], ["in mammals , the circadian system is a supraphysiological system that regulates biological functions every 24 h   such as glucose homeostasis , temperature , blood pressure , tone in the coronary artery , and heart rate .", "the bmal1/clock complex target genes are related to the circadian expression of metabolic pathways as was observed by hatanaka et al .   through high resolution", "the author detected a circadian expression of ( i ) glucose metabolism - related genes such as glut2 , por , pck1 , and gys2 and ( ii ) cholesterol metabolism - related genes such as cyp2a4 , cyp2a5 , cyp4a14 , cyp7a1 , and cyp2c55 ; and considering the very important role these systems have in metabolism , any alteration of them can have negative health effects .", "today there are several metabolic problems affecting the population , such as obesity , hyperglycemia , dyslipidemia , and hypertension .", "the cooccurrence of three or more metabolic disorders , including obesity , is defined as  metabolic syndrome  , showing a prevalence in the united states during 2009 - 2010 of about 22% of adults ( 21.4626.15 ) . the insulin resistance or type", "2 diabetes is one of the most important metabolic diseases currently affecting about 366 million people , causing more than 3.8 million deaths , and showing a rise in the number of affected people as observed in the united states population during 19992010 .", "several factors could be increasing the risk of death by diabetes , such as dyslipidemia , hypertension , and obesity , all of which are driving to an elevated risk of cardiovascular disease .", "a variant of this pathology can also develop during the pregnancy ( gestational diabetes mellitus ) and is characterized by glucose intolerance   in the mother and adverse consequences for her offspring , such as an increase in blood pressure , body mass index ( bmi ) , and body fat and a decrease in hdl levels . at the molecular level ,", "both diseases have in common the impairment of signal transduction of insulin receptors such as pi3kinase / akt and ras / map kinases [ 42 , 45 , 46 ] , leading to metabolic dysregulations   such as inhibitions of glycogen synthesis , impaired translocation of glut4 to plasmatic membrane , or antilipolytic effects of insulin in white adipose tissue [ 39 , 46 ] .", "meta - analyses from 448 articles reporting shift work and health consequence have detected a strong relationship between shift work , a potent chronodisruptor , and diabetes mellitus type 2 ( odd ratio about 1.42 times higher than day workers ) .", "however , this is not the only chronodisruption which we are exposed too . in animal models exposed to light / dark patterns similar to shift work , the length of the working day and changes of feeding times induce a modification of the liver weight and plasmatic glucose and a modification of the circadian profile for glucose , insulin , and triglyceride .", "these findings add to the observation in the circadian disruption in mutant animals , showing alterations of cholesterol metabolism , abolition of the circadian production of glycerol , free fatty acids , and impaired expression of rate - limiting lipolytic enzymes such as lipase , all of which add to increased weight gain , adipocyte hypertrophy , high level of glucose , glucose intolerance , and hypersecretion of insulin [ 4952 ] .", "polymorphic variations of clock genes can be incrementing the risks of developing a disease similar to chronodisruption by environmental changes as it has been detected in human metabolic disorders ( see table 1 ) .", "for example , the genotyping of clock genes in 346 greek pregnant women and their risk of diabetes was performed , detecting that the polymorphisms of bmal1 rs7950226 and rs11022775 are associated with gestational diabetes mellitus ( p = 0.025 , or = 1.46 and p = 4.455e  06 , or = 2.64 , resp . ) , while the study of the haplotype analysis of rs7950226/rs11022775 showed a major frequency in women with gestational diabetes mellitus ( p = 0.0069 , or = 6.96 ) .", "similarly , a study performed in subjects from the united kingdom and pakistan reported that cry1 and cry2 polymorphisms rs2292912 and rs12315175 , respectively , are associated with diabetes ( p = 0.015 and 0.008 , resp . ) . moreover", ", the variant rs12315175 for cry2 has a tendency to elevate the risk at about 5% compared to the variation of cry1 ( or = 1.05 and 0.95 , resp . ) .", "also , the authors did not find associations between diabetes and bmal1 polymorphisms rs7950226 and rs11022775 , as occurs in gestational diabetes mellitus .", "genotyping of 19,000 adults from northern sweden for variants rs8192440 for cry1 and rs11605924 for cry2 is associated with higher level of glucose concentrations at 2 hours ( p = 0.06 and p = 0.005 , resp . ) .", "another study genotyping 1304 individuals from 424 british families , containing at least one patient with diabetes type 2 ( diabetes in families study collection ) , demonstrates the relation between bmal1 polymorphisms rs7950226 and rs11022775 and diabetes ( p = 0.002 ) , reinforcing what was previously described .", "similar relations are observed for bmal2 polymorphism rs7958822 in obese men and women ( or 2.2 and 2.7 , resp . )   and the deletion / insertion of 54 base pair sequences of five repeat alleles on per3 gene ( rs57875989 ) [ 57 , 58 ] .", "in contrast , the per2 polymorphism rs7602358 is associated with a protection from type 2 diabetes in the uk population , which suggests that not all polymorphisms are negative for health . at the level of dyslipidemia", ", an interesting correlation has been detected between small dense ldl level and polymorphism of clock genes . in individuals carrying the polymorphism", "associated with the clock gene ( rs1801260 ) , the genotype tt or tc showed a major level of small dense ldl , which leads to increased triglycerides and increased risk of cardiovascular and obesity diseases , similar to the  metabolic syndrome  . likewise , genotyping in the japanese population identified clock gene polymorphism rs1801260 associated with higher odds ratio ( or ; 1.5 ) of type 2 diabetes ; in contrast , a multicenter study detected the clock polymorphism rs4580704 is associated with prevention of diabetes and cardiovascular disease .", "the haplotypes of rs10002541 and rs4864546 from clock ( cg and tg variations ) are associated with abdominal obesity in chinese population ( or 0.74 and 1.70 , resp . )   and polymorphism rs4864546 is associated with low level of hdl / apolipoprotein a1 ratio in spain .", "moreover , clock polymorphisms rs12649507 and rs3749474 are associated with higher intake of polyunsaturated fatty acid   and fat intake , which can modify the body mass index ( bmi ) .", "this is an antecedent which , bearing in mind the ideas above , suggests the genetic components of the circadian system as a critical factor in the development of metabolic diseases ."], ["in humans , infertility affects about 9% of couples , and about one - third of infertility cases associated with idiopathic male infertility are multifactorial , with 50% due to genetic abnormalities .", "the circadian system is important during reproduction and development [ 89 , 90 ] ; for example , it is involved in the timing of the lh surge [ 90 , 91 ] , stimulation of ovulation [ 90 , 92 ] , and regulating the level of steroidogenic acute regulatory protein ( star ) expression and is critical for cholesterol translocation inside mitochondria and sperm count .", "the genetic factors of clock genes are implicated in the pathogenesis of infertility ( see table 2 ) , as it occurs in male partners of infertile couples .", "the genotyping of male partners detected a correlation between single nucleotide polymorphism of clock rs11932595 , rs6811520 , and 6850524 , with infertility ( p < 0.05 and or ranged between 1.4 and 1.9 ) .", "similarly , the analysis of bmal1 polymorphism rs4757144 in slovenian and serbian caucasian men showed a significant correlation with infertility ( p = 0.047 ) , suggesting that clock genes clock and bmal1 contribute to successful fertilization ."], ["mood or affective disorders are important causes of morbidity , where we can highlight depressive pathologies and hypomaniac and maniac disorders as major diseases affecting the population [ 29 , 66 ] , and it can be highlighted that depressive pathologies are the third largest source of morbidity in the world .", "curiously , a potent correlation has been detected between mood disorders , sleep / activity , and the circadian system   suggesting that the circadian system can be modulating the neuronal activity . during pregnancy ,", "the prevalence of mental illnesses in women is about 8% , suggesting an imbalance between physiology of sleep and/or the circadian system .", "this can be observed in the onset of a mental disease as has been observed in the impaired circadian production of melatonin during the pregnancy of depressed women ( approximately 34 weeks of gestation ) , which shows an advance onset time of melatonin production of about 40 minutes and a minor production during the dark hours [ 97 , 98 ] .", "moreover , workers who do shift work ( circadian disruption ) showed decreased alertness , cognitive functions , mood , social and work activities , and health [ 26 , 99 ] , which are associated with an impaired circadian system via melatonin suppression , all of which leads to the appearance of a mood disorder .", "for this reason , we can say that the circadian system may contribute to the risk of developing a mood disorder and the genetic component of clock genes can be involved in the development of mood pathologies   ( see table 2 ) .", "at the genetic component level of the circadian system , a study of 744 people who carry polymorphisms rs2291739 and rs11171856 from tim , a member of the clock gene family which interacts with per1 - 2 proteins [ 100 , 101 ] , showed that they have an elevated risk of developing mood disorders ranging between 19 and 23% ( or 1.19 and 1.23 , resp . ) .", "moreover , gene variants of positive regulator clock have also been associated with mood disorders .", "people who carry the variants rs1801260 and rs11932595 showed a major risk of developing a bipolar disorder ranging between 45 and 37% , respectively , in comparison to a patients without polymorphism ( or 1.45 and 1.37 , resp . ) . in the same study", ", it was also observed that the polymorphisms rs2291739 and rs11171856 from tim gene are associated with unipolar disease , with a risk ranging between 37 and 40% in comparison to a patient without polymorphism ( or 1.37 and 1.40 , resp . ) .", "/ rs11022779g / rs1122780 t ( haplotype ) are associated with mood disorders   and bipolar disease [ 29 , 66 ] .", "it has also been seen that the bipolar pathologies are associated with other genetic variations of bmal1 gene ( rs4757144 , rs1982350 , and rs1481892 ) and the negative regulator per3 ( rs2859387 ) .", "curiously , a study performed an indian families reported the bmal1 polymorphisms rs2279287 are associated with seasonal affective disorder .", "in addition , the gene variant of cry2 gene ( rs4132063 )   and deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 )   may also be contributing to mood disorders via increased risk of developing bipolar disease .", "for example , a study in south india showed the prevalence of five repeat homozygotes from rs57875989 is associated with bipolar disease ( or 1.72 ) but not with schizophrenia . at the level of depressive pathologies , a study performed in china compared 485 subjects ( control ) to 105 patients suffering from a depression disorder .", "the genotyping for clock genes showed that the variants of cry1 ( rs2287161 ) and cry2 ( rs10838524 ) are correlated to the depression disease with an odds ratio of 1.75 ( p = 0.012 ) , which suggests that a patient with the allele has 1.75 times more risk of developing depression .", "moreover the authors detected a single nucleotide polymorphism for tef gene , the variant rs738499 , is associated with 2.22 times more risk of development of depression ( odds ratio of 2.22 .", "finally , in a study performed in young adults , the risk of excessive intake of alcohol is minor when the polymorphism of per2 gene rs56013859 is present , suggesting a possible protector role for allelic variation of clock genes in addictions .", "in fact , this suggests that some polymorphisms are not negative for our health and it is necessary to study them in greater depth ."], ["cellular proliferation is a critical event for the survival and restitution of tissues of all living things and the circadian system is capable of delivering temporary information to the cell cycle [ 102104 ] .", "however , an uncontrolled cellular proliferation and an excessive tissue growth are observed by an altered cellular cycle during cancer , a pathology that showed a higher incidence in patients exposed to an impaired circadian system such as a shiftwork [ 106 , 107 ] . the cell cycle is a finely regulated process from a cell that is capable of generating multiple cells through a series of cell divisions , including four critical and successive steps named g1 phase ( growth phase 1 ) , s phase ( synthesis ) , g2 ( growth phase 2 ) , and m phase ( mitosis ) . during the cell cycle , cyclin - dependent kinases ( cdk )", "are critical for the transition between different stages , for example , cdc2 plus cyclin b protein kinase form the complex cdk1 which regulates g2/m transition [ 108 , 109 ] . during dna replication", ", there are a number of controls or checkpoints that are critical in the cell cycle when dna damage is detected : the activation of the rad protein , which induces the action of cds 1 proteins , is triggered , as well as chk1 which are involved in cell cycle arrest via action of wee-1 and mik-1 proteins .", "however , the efficiency of this process changes depending on the time of the day when the lesion occurred .", "a lesion in the liver , occurring during the last light hours , induced a massive entry to m phase compared to a lesion which occurs early in the morning , which shows that the hour of surgery and the circadian system are critical for liver regeneration .", "similarly , oral mucosa is a highly proliferative tissue and it showed a circadian expression of clock genes bmal1 , per1 , and cry1 and thymidylate synthase activity , critical for dna synthesis during phase s. curiously , the peak of per1 mrna precedes the peak of thymidylate synthase activity , which suggests that the circadian system modulates the cellular proliferation in mucosa .", "wee-1 gene regulates cellular proliferation , and its promoter has three conserved sequence cacgtg ( e - box ) critical for the circadian expression of wee-1 .", "its protein is capable of inactivating the cdc2-cyclin b complex via phosphorylation which inhibits transition between g2/m .", "curiously , knock - down of bmal1 in carcinoma cells of the colon ( c26 cells ) , fibroblast cells ( l929 cell ) , and intestine epithelial cells ( iecs ) produces cellular proliferation in vitro and increments the size of tumor cells injected subcutaneously , via the inhibition of apoptosis and the reduction of the time transition between g2/m , reduction of p53 and wee-1 expression .", "moreover , the knock - out for clock genes bmal1 y per2 in mice previously exposed to gamma radiation caused in mice hyperplasic growth and development of lymphoma , hepatic carcinomas , ovary tumors , and osteosarcomas via reduction of p53 expression .", "a precedent that reinforces the idea that clock genes can be modulators of cellular cycle via wee-1 and p53 proteins . at the genetic component level", ", different studies reveal the importance of the polymorphic variant of clock genes ( see table 3 ) and how the chrono - type modulates the risk of cancer , such as observed in breast cancer , in which the risk is more elevated in premenopausal women ( or , 2.43 and 2.55 ; resp . ) .", "similarly , women showed evening or night preference such as shown in the case of a study performed on norwegian nurses working in night shift which revealed two polymorphisms associated with breast cancer .", "the risk is incremented when women spend more time working during night hours , but this risk is higher when women have the alleles for bmal1 rs2290035 ( or 1.91 ) , rs969485 ( or 1.64 ) , and rs3903529 ( or 2.77 ) or variant rs3750420 ( or 1.6 ) of roreb gene .", "similarly , an incremented risk is associated with breast cancer in women from france when they have the allele rs11932595 in clock gene ( or = 0.74 ) or in connecticut ( usa ) when they have the polymorphisms rs7698022 ( or 1.34 ) and rs1048004 ( or , 1.43 ) .", "curiously , when norwegian nurses are exposed to shift work during four nights , they showed three times more risk ( or 2.75 ) of developing breast cancer when they were carrying clock polymorphism rs11133373 , which suggests that the disruption of endogenous circadian rhythm by polymorphism associated with clock genes increases the risk of cancer .", "moreover , clock gene expression is induced in breast tissue from patients with breast cancer ; this expression is associated with hypomethylation of clock gene .", "the silencing of clock gene lowered when women carry polymorphisms associated with cancer such as rs10448004 and rs7698022 , which suggests that clock gene is a critical protagonist of cancer development . at level of colorectal cancer", ", a strong correlation is detected between cancer and the genetic component of the circadian system .", "for example , a polymorphism in the clock gene ( rs1801260 ) showed a major prevalence in a cancer patient ( p < 0.0001 , or = 1.78 for c - allele ) compared to a control patient .", "similarly , a screening performed in south carolina , usa , showed that a variation by deletion / insertion of a 54 base pair sequence on per3 gene ( rs57875989 ) is associated with a higher risk of colorectal adenoma formation with odds ratio within 2.15.1 .", "moreover , a population study performed on the residents of king county , washington ( usa ) , detected six different types of polymorphism , which are significantly associated with the risk and aggressiveness of prostate cancer .", "these genetics variants are rs1012477 for per3 ( or 1.3 ) , rs7602358 for per2 ( or 1.24 ) , and rs2289591 in per1 ( or 1.7 ) .", "similarly , a genotyping of a patient with prostate cancer showed a strong correlation between cry1 polymorphisms rs7297614 , rs1921126 , and rs12315175 and fatal prostate cancer ( or mean within 1.52 )   and a study conducted in china showed that the cry2 variant rs1401417 and the deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 ) were associated with a major risk of developing prostate cancer ( or ; 1.7 and 1.3 , resp . ) . a study", "performed in brazilian patients with pulmonary cancer showed a strong correlation of per3 polymorphism rs228644 and the risk of cancer ( or 1.99 ) .", "moreover , the authors reported the ancestral haplotypes for per3 rs228729 , rs228727 , rs707467 , rs228644 , and rs10462020 are associated with a higher cancer frequency ; similarly , a meta - analysis performed by literature search showed that the variant insertion / deletion of per3 rs57875989 is associated with an increase of cancer susceptibility in about 17%   or 70% . in a similar way", ", a soft risk is associated with breast cancer in premenopausal women from india , which suggests there is a relation between per3 and cellular proliferation .", "the frequency analysis of 1,538 breast cancer cases and 1,605 controls in china for clock gene variants showed the strong associations between three snps in circadian clock genes and the risk of developing breast cancer .", "the variants for cry1 rs1056560 are correlated to cancer , which elevate the risk in about 11% ( or 1.11 ) ; per2 rs934945 in about 15% ( or 1.15 ) ; and clock rs3805151 in about 35% ( or 1.35 ) .", "in contrast , tim protection for breast cancer development is detected in patients that carry the c - allele of rs7302060 ( or , 0.54 ) .", "the g allele of rs2291738 and the c - allele of rs7302060 are associated with reduced risk of breast cancer among estrogen receptor (  ) or progesterone receptor (  ) positive breast cancer cases ( or , 0.46 and 0.36 , resp . ) .", "the exogenous expression of human clock in cell lines of colorectal carcinoma induces the cellular proliferation in about 28% .", "in contrast , knock - down of endogenous clock gene expression inhibits the cell proliferation in about 34% . moreover", ", exogenous clock inhibits the apoptosis ( 42% reduction ) via inhibition on apoptosis associated proteins expression of bax and bid and the increase of phosphorylation of akt . in vivo experiments by xenograft transplant of colorectal carcinoma cell line transduced with clock", "increase the tumor volume and tumor weight in about 61% and a 91% , respectively .", "however , the pharmacological inhibition of cry in human breast cancer by treatment with pharmacological agent ks15   inhibits the proliferation and cell viability by stimulation of wee-1 expression   and stimulates the activity of heterodimer complex bmal1:clock [ 112 , 113 ] .", "curiously , two polymorphisms for clock gene cry2 , rs11038689 and rs1401417 , have a protective action over the mammary cancer , which suggests that not all polymorphisms associated with clock genes are negative for our health .", "non - hodgkin 's lymphoma is characterized by lymphoproliferation and advanced clinical stages , invasion to other tissues , and death .", "the estimated deaths from non - hodgkin 's lymphoma in the united states amounted to 19,790 during 2015 .", "analysis of cry2 variants showed the polymorphisms rs11038689 , rs7123390 , and rs1401417 increase the risk of lymphoma ( or , 2.34 ; 2.40 and 2.97 , resp . ) .", "moreover , a minor association of clock gene variants and glioma is detected for per1 rs2585405 , clock rs11133391 , and cry1 rs12315175 ( or , 1.16 ; 1.08 and 1.02 , resp . ) .", "genotyping of han chinese patients diagnosed with primary hepatocellular carcinoma showed an association between single snps of per3 rs228669 and cry1 rs3809236 with odds ratio of 1.41 and 1.26 , respectively .", "these precedents reinforce the idea that clock genes can be modulators of cellular cycle and that modulates the risk of cancer ."], ["the circadian system is a supraphysiological system which modulates different physiological systems , and any alteration of this can have a negative impact on human health .", "different chronodisruptors have been described in literature such as light / dark pattern and inhibition of melatonin production as occurs in shifts work .", "however , other factors can be contributing on a minor scale , such as mealtimes or a genetic component .", "the relevance of the genetic variation of clock genes and how it can interact with the environment is unknown .", "but it has been described that the genetic component in the population predisposes the development of different pathologies such as diabetes , dyslipidemias , obesity , mood disorders , and addiction , all of which suggest the importance of this system to our health .", "however , it is also necessary to say that the genetic component could be protecting our health such as in the case of polymorphisms associated with per2 and cry2 genes in diabetes , alcohol intake , and cancer . these findings will need to be implemented and evaluated at the genetic interaction level and also the way in which the environment factors trigger the expression of these pathologies will be examined .", "finally , prospective studies are necessary to assess the predictive potential of these markers and to implement early treatment with consequent cost reduction for the health system ."]]}
{"article_id": "PMC3132988", "article_text": ["cellular adaptation requires biochemical processes including post - translational mechanisms to modify existing proteins . catalyzed by opposing kinases and phosphatases , reversible phosphorylation of serine , threonine , and tyrosine residues is now appreciated as a fundamental regulatory mechanism with the majority of phosphorylation ( > 99% ) occurring on serine and threonine residues [ 1 , 2 ] . due to their untapped therapeutic potential ,", "protein phosphatases have been identified as promising targets for xenobiotic manipulation through rational drug design ( reviewed in [ 36 ] ) . in particular ,", "the ubiquitously expressed protein phosphatase 2a ( pp2a ) has been proposed as a target for the treatment of a number of pathologies ranging from neurodegenerative diseases such as alzheimer disease to a variety of neoplasias [ 79 ] .      ", "compared to other members of the phosphoprotein phosphatase ( ppp ) superfamily of serine / threonine phosphatases , a detailed understanding of the mechanism by which pp2a recognizes substrates and mediates site - specific dephosphorylation remains to be developed . sequence and structural homology of the catalytic subunits of ppp family members", "has revealed a conserved catalytic mechanism in which a divalent metal cation activates a water molecule to hydrolyze phospho - serine / threonine without the formation of a phosphoenzyme intermediate ( reviewed in [ 1012 ] ) . despite a shared catalytic mechanism , substrate specificity within the ppp family", "for example , the ppp calcineurin ( also known as protein phosphatase 2b ) has been shown to interact with two consensus sequences , pxixit and lxvp , found on nonsubstrate - interacting proteins and target substrates ( reviewed in [ 11 , 13 ] ) . for protein phosphatase-1 ( pp1 ) , substrate specificity is conferred by incorporation of pp1-interacting proteins via a conserved docking motif with a general consensus sequence of rvxf ( reviewed in [ 11 , 12 ] ) . at present , consensus sequences in pp2a substrates have not been identified .", "this review will focus on our emerging understanding of pp2a substrate specificity , which appears to involve additive effects of multiple discrete interactions .          ", "pp2a is a highly conserved serine / threonine phosphatase which , depending on the tissue of origin and cell type , may account for up to 1% of cellular protein and the majority of serine / threonine phosphatase activity .", "the physiological functions of pp2a have been implicated in all facets of cellular existence ( reviewed in ) .", "further , pp2a functions as a critical tumor suppressor whose interruption leads to proliferative diseases .", "the heterotrimeric holoenzyme is composed of a catalytic subunit ( c ) a scaffold subunit ( a ) and one member of four families of regulatory subunits ( b ) ( figure 1 ) .", "the diversity of pp2a heterotrimers is achieved through expression of two c subunits , two a subunits and approximately fifteen b subunits in vertebrates .", "the b subunits are derived from four diverse gene families ( b , b , b , and b ) that have little sequence similarity between families but maintain high sequence similarity within families .", "the b family ( b55 , pr55 , ppp2r2 ) of regulatory subunits consists of four genes (  ,  ,  ,  ) , the b - family ( b56 , pr61 , ppp2r5 ) is comprised of five isoforms (  ,  ,  ,  ,  ) , the b family ( pr72 , ppp2r3 ) includes three isoforms ( /pr72/130 , /pr59 , /pr48 ) , and the b family ( pr93/pr110 ) is made up of three proteins ( sg2na , striatin , and mmob1 ) .", "there is some controversy as to whether the b family members , most notably sg2na , are bona fide pp2a regulatory subunits that always associate with the ac dimer or whether they are merely regulated by association with the pp2a dimer .", "given the large number of pp2a subunits , it is thought that each cell expresses a dozen or more distinct holoenzyme complexes which act on a diverse array of substrates .", "pp2a holoenzyme diversity has been the subject of several excellent papers [ 14 , 15 , 18 ] . like pp1 , regulatory subunit incorporation is thought to dictate the substrate specificity of the pp2a complex , however , only recently have molecular studies begun to develop insight into the mechanism by which the regulatory subunit acts .", "the results from recent studies suggests a multitiered mechanism wherein pp2a substrate specificity arises from ( 1 ) subcellular localization of pp2a defined by the b subunit , ( 2 ) selective holoenzyme assembly by posttranslational modification , ( 3 ) interaction with specific endogenous inhibitors , ( 4 ) interactions between the b subunit and phosphosubstrates at sites distant from the active site , and ( 5 ) b - subunit residues which infiltrate the catalytic cleft of the c subunit .", "this paper will provide a summary of these studies and how the understanding of the determinants of pp2a substrate specificity has advanced .", "the heterotrimeric holoenzyme is targeted to discrete subcellular locales dictated in part by which b - regulatory subunit is incorporated .", "the localization imparted by the b - regulatory subunit dictates the spatial sphere of influence of the holoenzyme complex for potential substrates .", "this mechanism of targeting pp2a activity is highlighted by extensive studies of the b family of regulatory subunits .", "for instance , the b family regulatory subunits target the holoenzyme to different cellular compartments in the brain . specifically , b and b are primarily cytosolic where as the b-regulatory subunit associates with a detergent - resistant protein fraction consistent with an interaction at the cytoskeleton", ".    similar diversity has been observed in the b family of regulatory subunits . a c - terminal nuclear export signal common to b , b , and b which , when these regulatory subunits are incorporated into the pp2a holoenzyme , results in cytoplasmic localization of the heterotrimer . b and b isoforms lack a similar sequence and are found primarily in the nucleus . in cardiomyocytes , b interacts with the protein ankyrin - b through its c - terminus which leads to localization at the cardiac m - line .", "b on the other hand has been shown to target the holoenzyme complex to subnuclear structures in cardiomyocytes where pp2a / b may regulate gene expression .", "loss of proper subcellular targeting of pp2a has been implicated in the biogenesis and aggressive phenotype of neoplastic growths .", "specifically , a truncated form of b ( b ) , has been isolated from a melanoma cell line wherein the pp2a/b complex is targeted to the trans - golgi network , blunts p53 responsiveness , contributes to genetic instability and increases metastatic motility [ 2427 ] .    ", "this was first observed in a member of the b family , b. two neuron - specific isoforms of b , b1 , and b2 , are generated by the use of an alternative 5 exon which results in the production of a divergent n - terminal extension on b2 [ 28 , 29 ] .", "the n - terminal extension of b2 directs the holoenzyme complex to the outer mitochondrial membrane ( omm ) by targeting the mitochondrial translocase complex and forming an abortive complex resistant to import into the mitochondrial matrix [ 28 , 30 ] .", "the omm - directed pp2a / b2 complex promotes fragmentation of the mitochondria reticulum and increases cell susceptibility to proapoptotic insults through an unknown mechanism .", "alternative splicing of b - regulatory subunits directing subcellular localization of the pp2a holoenzyme has also been observed in both the b and b families .", "two b isoforms differ by the inclusion or exclusion an n - terminal nuclear localization signal leading to isoform - specific nuclear or cytoplasmic localization .", "the murine - specific b family member , b , is also subject to alternative splicing leading to isoform - specific nuclear or cytoplasmic localization through an as yet unidentified mechanism .", "for example , the interaction between pp2a / b and shugoshin during meiosis is crucial for spatial and temporal regulation of sister chromatid disjunction . during meiosis , the cohesin complexes , which link the arms of bivalent chromosomes and the centromeres of sister chromatids ,", "must be released in a stepwise fashion by the protease separase ; the cohesin complex is firstly hydrolyzed along the arms of the bivalent chromosomes for completion of anaphase i and secondly at the centromeres of sister chromatids for completion of anaphase ii . during anaphase", "i , the centromeric cohesin complex is protected from separase - dependent proteolysis by shugoshin [ 34 , 35 ] .", "shugoshin recruits pp2a / b to the centromere which likely results in dephosphorylation of the cohesin complex leading to protection of the cohesin complex from separase - dependent proteolysis [ 36 , 37 ] . through cocrystallization ,", "xu and colleagues , have revealed that dimeric human shugoshin 1 interacts with pp2a / b through a coiled - coil region across a broad composite surface of the c and b subunits .", "incorporation of specific regulatory subunits is influenced by reversible posttranslational modification of the c subunit .", "many groups have shown that the c - terminus of the c subunit is modified through phosphorylation and methylation on y307 and l309 , respectively [ 3946 ] .", "phosphorylation of y307 is catalyzed by src kinase and is likely opposed by pp2a - catalyzed autodephosphorylation of this phosphotyrosine .", "phosphorylation of y307 selectively inhibits recruitment of the b family and some b family members to the dimeric ac complex whereas b recruitment is not effected .   ", "methylation of the c subunit at the c - terminal l309 is catalyzed by the protein phosphatase methyltransferase ( ppm1 ) and is opposed by the phosphatase methylesterase ( pme-1 ) [ 4751 ] .", "reversible methylation of pp2a is absolutely critical as knocking - out pme-1 in mice changes the phosphoproteome and results in early perinatal lethality .", "further , methylation of the c subunit is a dynamic process which plays a role in cellular response to acute stimuli .", "the recruitment of the b subunit to the ac dimer has been postulated to require methylation of the c subunit for some of the b - subunit families .", "however , conflicting results have been reported that may reflect differences in experimental design and will be discussed further .", "studies wherein pp2a / b is isolated from intact cells have revealed that methylation of the c subunit at l309 is required for incorporation of the b family of regulatory subunits into the holoenzyme complex [ 39 , 40 , 42 , 44 , 54 , 55 ] .", "conversely , in vitro assembly of the pp2a / b holoenzyme complex does not require methylation of the c subunit for incorporation of the b family of regulatory subunits [ 56 , 57 ] .", "similarly controversial , the requirement for c subunit l309 methylation was observed to be study - specific in in vitro pp2a / b timer formation [ 58 , 59 ] .", "methylation was dispensable for isolation of the pp2a / b holoenzyme complex from intact cells .", "the role of methylation of the c subunit in recruitment of b and b families of regulatory subunits is less controversial with methylation of the c subunit being dispensable [ 44 , 54 ] . for more information ,", "posttranslational modifications which influence formation of the pp2a holoenzyme complex also occur on the b subunit .", "pp2a / b negatively regulates the erk map kinase signal transduction pathway . through formation of a ternary complex of the early response gene product iex-1 , b1 , and erk ,", "erk mediates its own disinhibition by phosphorylation of b1 on s327 leading to b1 disassociation from the pp2a holoenzyme .", "since s327 is conserved among b - subunit family members , it is likely that other b subunits are regulated similarly .", "additionally , b is likely phosphorylated on s167 to disrupt the b subunit from the ac dimer in early mitotic stages .", "however , pp2a / b activity is necessary to resolve the mitotic spindles and conclude mitosis ; therefore , autodephosphorylation may occur on b to allow efficient pp2a / b heterotrimer formation and cell cycle progression .", "thus , phosphorylation of the b - regulatory subunits also influences holoenzyme assembly and , therefore , substrate specificity .", "phosphorylation of the b subunit of heterotrimeric pp2a also potentiates the catalytic activity of the holoenzyme complex . in response to activation of d1 dopamine receptors on striatal neurons ,", "camp - dependent protein kinase a ( pka ) phosphorylates    b at s566 increasing activity of the pp2a/ b holoenzyme towards dopamine- and camp - regulated neuronal phosphoprotein ( darpp-32 ) [ 64 , 65 ] .", "dephosphorylation of t75 on darpp-32 by pp2a / b disinhibits pka - mediated phosphorylation of darpp-32 at t34 which converts darpp-32 into a potent pp1 inhibitor leading to changes in neuronal signaling .", "this circuit acts to attenuate phospho - t75 inhibition of t34 phosphorylation of darpp-32 . this circuit has been shown to be differentially regulated by psychomotor stimulants and antipsychotics acting on different striatal neuron subpopulations .   ", "while assembly of the many trimeric pp2a holoenzymes directs cellular localization and substrate specificity , further regulation is afforded through binding of specific protein inhibitors of pp2a .", "one such inhibitor set ( i2/taf-1 ) is upregulated during the progression of chronic myelogenous leukemia through bcr / abl activity and results in decreased pp2a activity .", "an additional pp2a inhibitor is the protein cip2a ( cancerous inhibitor of pp2a ) , overexpression of which is associated with several human malignancies .", "cip2a associates with c - myc to protect its phosphorylated s62 from pp2a - directed activity stabilizing the c - myc protein and allowing it to promote oncogenesis .   ", "recently an interplay between the drosophila serine / threonine kinase greatwall ( gwl ) and pp2a / b was observed during mitotic entry in two separate studies [ 69 , 70 ] .", "pp2a / b activity prevents mitotic entry by maintaining cdc25 in a dephosphorylated and inactive state .", "gwl reverses this inhibition through the phosphorylation of s67 of both -endosulfine ( ensa ) and cyclic adenosine monophosphate-(camp- ) regulated phosphoprotein-19 ( arpp-19 ) .", "phosphorylation converts ensa and arpp-19 into very specific inhibitors of pp2a / b activity and produces activation of cdc25 leading to cell cycle progression .", "similar cell cycle regulatory activity has been observed with the mammalian ortholog of gwl , mastl ; however , the mastl - pp2a interaction has yet to be characterized .", "once targeted to specific subcellular locales , the pp2a holoenzyme must recruit and dephosphorylate target substrates .", "recent structural studies have begun to suggest the mechanism by which the regulatory subunit of pp2a mediates initial binding to target substrates .", "other -propeller proteins have been shown to bind ligands in the central depression on the top surface of the toroid .", "crystallization of pp2a / b revealed a cluster of acidic residues in this depression that is available to recruit potential substrates containing a basic motif . in this same study ,", "the acidic central depression of b was experimentally confirmed to bind the microtubule - associated protein tau , an established pp2a / b substrate .", "several conserved aspartate and glutamate residues in b engage in weak , electrostatic interactions across a large basic portion of tau and support dephosphorylation of tau at multiple sites through cycles of binding and unbinding .    structurally divergent b family members may recruit substrates in a similar fashion as the b family .", "the crystal structure of pp2a / b shows that the b subunit contains 18 stacked -helices which adopt 8 huntingtin elongation a subunit tor- ( heat- ) like repeats [ 58 , 59 ] .", "a portion of these heat - like repeats interact with the a subunit of the holoenzyme to mediate regulatory subunit incorporation into the holoenzyme complex . like the b subunit", ", an acidic patch is exposed in the b family of regulatory subunits and may mediate protein - protein interactions and substrate recruitment .", "structural studies of pp2a have revealed a conserved loop in the b family of regulatory subunits which infiltrates the catalytic core of the holoenzyme [ 58 , 59 ] ( figure 2(a ) ) . at the tip of this loop", "is a conserved glutamate residue , e153 ( b numbering ) , which contacts through its carbonyl oxygen the catalytic subunit and through its carboxyl group a cocrystallized microcystin molecule in the active site .", "mutational analysis revealed that e153 is an absolute requirement for efficient dephosphorylation of tyrosine hydroxylase ( th ) , a known pp2a / b substrate , as well as other as yet unidentified cellular substrates of this pp2a holoenzyme .", "further , it was determined that e153 of b interacts with r37 and r38 of th to mediate dephosphorylation of both s31 and s40 on th .", "positively charged residues in the vicinity of target phospho - serine / threonine residues could represent a consensus sequence for b - subunit - mediated dephosphorylation .", "further , the infiltrating loop is likely a conformationally dynamic structure which is not sterically hindered by surrounding portions of the a , c , or b subunits .", "since r37/38 are important for dephosphorylation of both upstream ( s40 ) and downstream ( s31 ) sites , it appears that the orientation of phosphopeptides relative to the catalytic cleft is not constrained by additional interactions .", "collectively , the above observations support a model in which the sites of interaction between the substrate and the b regulatory subunit that are distant and near the active site together control substrate specificity .", "first , the interaction occurring at sites distant from the active site increases the local substrate concentration . following this initial substrate recruitment ,", "although divergent in its sequence , an analogous structure from the -propeller fold of the b family of regulatory subunits extends to the catalytic core of the holoenzyme   ( figure 2(b ) ) .", "this loop places conserved residues of the b - family subunits very near the holoenzyme active site . unpublished observations generated in our lab suggests that of these loop residues k87 of b2 may play a similar role as e153 of b in site - specific dephosphorylation of target substrates ; however , further characterization of this substrate specificity loop is required .", "pp2a is a ubiquitous protein phosphatase responsible for the dephosphorylation of many different intracellular targets . the diverse repertoire of potential substrates for pp2a is imparted by the incorporation of one of fifteen unique b - regulatory subunits .", "recent studies have increased our understanding of the mechanisms by which the b subunit imparts specificity to the holoenzyme complex . through selective incorporation of the b", "- regulatory subunit , the holoenzyme complex is recruited to discrete subcellular locales which define the sphere of influence for the phosphatase .", "secondly , interactions between endogenous inhibitors and specific pp2a heterotrimers further restrict phosphatase activity . as shown for the b family , regulatory subunits mediate low - affinity interactions with substrates to increase the local concentration of substrates . through a flexible substrate selectivity loop which contacts the catalytic subunit", ", interactions between the regulatory subunit and phosphosubstrate may mediate multiple nearby dephosphorylation events . with the current structural information available for", "the pp2a complexes , future high - resolution studies will further define the molecular mechanism of pp2a substrate specificity . as general ,", "inhibitors of pp2a are either clinically irrelevant or toxic , as in the case of the small molecule inhibitor microcystin , novel methods to increase the specificity of pp2a inhibition or activation must be developed .", "a clearer understanding of the pp2a substrate specificity mechanisms will serve as the foundation for rational drug design of selective inhibitors and activators of specific pp2a holoenzyme complexes ."], "abstract_text": ["<S> protein phosphatase 2a- ( pp2a- ) catalyzed dephosphorylation of target substrate proteins is widespread and critical for cellular function . </S>", "<S> pp2a is predominantly found as a heterotrimeric complex of a catalytic subunit ( c ) , a scaffolding subunit ( a ) , and one member of 4 families of regulatory subunits ( b ) . </S>", "<S> substrate specificity of the holoenzyme complex is determined by the subcellular locale the complex is confined to , selective incorporation of the b subunit , interactions with endogenous inhibitory proteins , and specific intermolecular interactions between pp2a and target substrates . here </S>", "<S> , we discuss recent studies that have advanced our understanding of the molecular determinants for pp2a substrate specificity . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Mechanisms of Substrate Specificity", "3. Conclusion"], "sections": [["cellular adaptation requires biochemical processes including post - translational mechanisms to modify existing proteins . catalyzed by opposing kinases and phosphatases , reversible phosphorylation of serine , threonine , and tyrosine residues is now appreciated as a fundamental regulatory mechanism with the majority of phosphorylation ( > 99% ) occurring on serine and threonine residues [ 1 , 2 ] . due to their untapped therapeutic potential ,", "protein phosphatases have been identified as promising targets for xenobiotic manipulation through rational drug design ( reviewed in [ 36 ] ) . in particular ,", "the ubiquitously expressed protein phosphatase 2a ( pp2a ) has been proposed as a target for the treatment of a number of pathologies ranging from neurodegenerative diseases such as alzheimer disease to a variety of neoplasias [ 79 ] .      ", "compared to other members of the phosphoprotein phosphatase ( ppp ) superfamily of serine / threonine phosphatases , a detailed understanding of the mechanism by which pp2a recognizes substrates and mediates site - specific dephosphorylation remains to be developed . sequence and structural homology of the catalytic subunits of ppp family members", "has revealed a conserved catalytic mechanism in which a divalent metal cation activates a water molecule to hydrolyze phospho - serine / threonine without the formation of a phosphoenzyme intermediate ( reviewed in [ 1012 ] ) . despite a shared catalytic mechanism , substrate specificity within the ppp family", "for example , the ppp calcineurin ( also known as protein phosphatase 2b ) has been shown to interact with two consensus sequences , pxixit and lxvp , found on nonsubstrate - interacting proteins and target substrates ( reviewed in [ 11 , 13 ] ) . for protein phosphatase-1 ( pp1 ) , substrate specificity is conferred by incorporation of pp1-interacting proteins via a conserved docking motif with a general consensus sequence of rvxf ( reviewed in [ 11 , 12 ] ) . at present , consensus sequences in pp2a substrates have not been identified .", "this review will focus on our emerging understanding of pp2a substrate specificity , which appears to involve additive effects of multiple discrete interactions .          ", "pp2a is a highly conserved serine / threonine phosphatase which , depending on the tissue of origin and cell type , may account for up to 1% of cellular protein and the majority of serine / threonine phosphatase activity .", "the physiological functions of pp2a have been implicated in all facets of cellular existence ( reviewed in ) .", "further , pp2a functions as a critical tumor suppressor whose interruption leads to proliferative diseases .", "the heterotrimeric holoenzyme is composed of a catalytic subunit ( c ) a scaffold subunit ( a ) and one member of four families of regulatory subunits ( b ) ( figure 1 ) .", "the diversity of pp2a heterotrimers is achieved through expression of two c subunits , two a subunits and approximately fifteen b subunits in vertebrates .", "the b subunits are derived from four diverse gene families ( b , b , b , and b ) that have little sequence similarity between families but maintain high sequence similarity within families .", "the b family ( b55 , pr55 , ppp2r2 ) of regulatory subunits consists of four genes (  ,  ,  ,  ) , the b - family ( b56 , pr61 , ppp2r5 ) is comprised of five isoforms (  ,  ,  ,  ,  ) , the b family ( pr72 , ppp2r3 ) includes three isoforms ( /pr72/130 , /pr59 , /pr48 ) , and the b family ( pr93/pr110 ) is made up of three proteins ( sg2na , striatin , and mmob1 ) .", "there is some controversy as to whether the b family members , most notably sg2na , are bona fide pp2a regulatory subunits that always associate with the ac dimer or whether they are merely regulated by association with the pp2a dimer .", "given the large number of pp2a subunits , it is thought that each cell expresses a dozen or more distinct holoenzyme complexes which act on a diverse array of substrates .", "pp2a holoenzyme diversity has been the subject of several excellent papers [ 14 , 15 , 18 ] . like pp1 , regulatory subunit incorporation is thought to dictate the substrate specificity of the pp2a complex , however , only recently have molecular studies begun to develop insight into the mechanism by which the regulatory subunit acts .", "the results from recent studies suggests a multitiered mechanism wherein pp2a substrate specificity arises from ( 1 ) subcellular localization of pp2a defined by the b subunit , ( 2 ) selective holoenzyme assembly by posttranslational modification , ( 3 ) interaction with specific endogenous inhibitors , ( 4 ) interactions between the b subunit and phosphosubstrates at sites distant from the active site , and ( 5 ) b - subunit residues which infiltrate the catalytic cleft of the c subunit .", "this paper will provide a summary of these studies and how the understanding of the determinants of pp2a substrate specificity has advanced ."], ["the heterotrimeric holoenzyme is targeted to discrete subcellular locales dictated in part by which b - regulatory subunit is incorporated .", "the localization imparted by the b - regulatory subunit dictates the spatial sphere of influence of the holoenzyme complex for potential substrates .", "this mechanism of targeting pp2a activity is highlighted by extensive studies of the b family of regulatory subunits .", "for instance , the b family regulatory subunits target the holoenzyme to different cellular compartments in the brain . specifically , b and b are primarily cytosolic where as the b-regulatory subunit associates with a detergent - resistant protein fraction consistent with an interaction at the cytoskeleton", ".    similar diversity has been observed in the b family of regulatory subunits . a c - terminal nuclear export signal common to b , b , and b which , when these regulatory subunits are incorporated into the pp2a holoenzyme , results in cytoplasmic localization of the heterotrimer . b and b isoforms lack a similar sequence and are found primarily in the nucleus . in cardiomyocytes , b interacts with the protein ankyrin - b through its c - terminus which leads to localization at the cardiac m - line .", "b on the other hand has been shown to target the holoenzyme complex to subnuclear structures in cardiomyocytes where pp2a / b may regulate gene expression .", "loss of proper subcellular targeting of pp2a has been implicated in the biogenesis and aggressive phenotype of neoplastic growths .", "specifically , a truncated form of b ( b ) , has been isolated from a melanoma cell line wherein the pp2a/b complex is targeted to the trans - golgi network , blunts p53 responsiveness , contributes to genetic instability and increases metastatic motility [ 2427 ] .    ", "this was first observed in a member of the b family , b. two neuron - specific isoforms of b , b1 , and b2 , are generated by the use of an alternative 5 exon which results in the production of a divergent n - terminal extension on b2 [ 28 , 29 ] .", "the n - terminal extension of b2 directs the holoenzyme complex to the outer mitochondrial membrane ( omm ) by targeting the mitochondrial translocase complex and forming an abortive complex resistant to import into the mitochondrial matrix [ 28 , 30 ] .", "the omm - directed pp2a / b2 complex promotes fragmentation of the mitochondria reticulum and increases cell susceptibility to proapoptotic insults through an unknown mechanism .", "alternative splicing of b - regulatory subunits directing subcellular localization of the pp2a holoenzyme has also been observed in both the b and b families .", "two b isoforms differ by the inclusion or exclusion an n - terminal nuclear localization signal leading to isoform - specific nuclear or cytoplasmic localization .", "the murine - specific b family member , b , is also subject to alternative splicing leading to isoform - specific nuclear or cytoplasmic localization through an as yet unidentified mechanism .", "for example , the interaction between pp2a / b and shugoshin during meiosis is crucial for spatial and temporal regulation of sister chromatid disjunction . during meiosis , the cohesin complexes , which link the arms of bivalent chromosomes and the centromeres of sister chromatids ,", "must be released in a stepwise fashion by the protease separase ; the cohesin complex is firstly hydrolyzed along the arms of the bivalent chromosomes for completion of anaphase i and secondly at the centromeres of sister chromatids for completion of anaphase ii . during anaphase", "i , the centromeric cohesin complex is protected from separase - dependent proteolysis by shugoshin [ 34 , 35 ] .", "shugoshin recruits pp2a / b to the centromere which likely results in dephosphorylation of the cohesin complex leading to protection of the cohesin complex from separase - dependent proteolysis [ 36 , 37 ] . through cocrystallization ,", "xu and colleagues , have revealed that dimeric human shugoshin 1 interacts with pp2a / b through a coiled - coil region across a broad composite surface of the c and b subunits .", "incorporation of specific regulatory subunits is influenced by reversible posttranslational modification of the c subunit .", "many groups have shown that the c - terminus of the c subunit is modified through phosphorylation and methylation on y307 and l309 , respectively [ 3946 ] .", "phosphorylation of y307 is catalyzed by src kinase and is likely opposed by pp2a - catalyzed autodephosphorylation of this phosphotyrosine .", "phosphorylation of y307 selectively inhibits recruitment of the b family and some b family members to the dimeric ac complex whereas b recruitment is not effected .   ", "methylation of the c subunit at the c - terminal l309 is catalyzed by the protein phosphatase methyltransferase ( ppm1 ) and is opposed by the phosphatase methylesterase ( pme-1 ) [ 4751 ] .", "reversible methylation of pp2a is absolutely critical as knocking - out pme-1 in mice changes the phosphoproteome and results in early perinatal lethality .", "further , methylation of the c subunit is a dynamic process which plays a role in cellular response to acute stimuli .", "the recruitment of the b subunit to the ac dimer has been postulated to require methylation of the c subunit for some of the b - subunit families .", "however , conflicting results have been reported that may reflect differences in experimental design and will be discussed further .", "studies wherein pp2a / b is isolated from intact cells have revealed that methylation of the c subunit at l309 is required for incorporation of the b family of regulatory subunits into the holoenzyme complex [ 39 , 40 , 42 , 44 , 54 , 55 ] .", "conversely , in vitro assembly of the pp2a / b holoenzyme complex does not require methylation of the c subunit for incorporation of the b family of regulatory subunits [ 56 , 57 ] .", "similarly controversial , the requirement for c subunit l309 methylation was observed to be study - specific in in vitro pp2a / b timer formation [ 58 , 59 ] .", "methylation was dispensable for isolation of the pp2a / b holoenzyme complex from intact cells .", "the role of methylation of the c subunit in recruitment of b and b families of regulatory subunits is less controversial with methylation of the c subunit being dispensable [ 44 , 54 ] . for more information ,", "posttranslational modifications which influence formation of the pp2a holoenzyme complex also occur on the b subunit .", "pp2a / b negatively regulates the erk map kinase signal transduction pathway . through formation of a ternary complex of the early response gene product iex-1 , b1 , and erk ,", "erk mediates its own disinhibition by phosphorylation of b1 on s327 leading to b1 disassociation from the pp2a holoenzyme .", "since s327 is conserved among b - subunit family members , it is likely that other b subunits are regulated similarly .", "additionally , b is likely phosphorylated on s167 to disrupt the b subunit from the ac dimer in early mitotic stages .", "however , pp2a / b activity is necessary to resolve the mitotic spindles and conclude mitosis ; therefore , autodephosphorylation may occur on b to allow efficient pp2a / b heterotrimer formation and cell cycle progression .", "thus , phosphorylation of the b - regulatory subunits also influences holoenzyme assembly and , therefore , substrate specificity .", "phosphorylation of the b subunit of heterotrimeric pp2a also potentiates the catalytic activity of the holoenzyme complex . in response to activation of d1 dopamine receptors on striatal neurons ,", "camp - dependent protein kinase a ( pka ) phosphorylates    b at s566 increasing activity of the pp2a/ b holoenzyme towards dopamine- and camp - regulated neuronal phosphoprotein ( darpp-32 ) [ 64 , 65 ] .", "dephosphorylation of t75 on darpp-32 by pp2a / b disinhibits pka - mediated phosphorylation of darpp-32 at t34 which converts darpp-32 into a potent pp1 inhibitor leading to changes in neuronal signaling .", "this circuit acts to attenuate phospho - t75 inhibition of t34 phosphorylation of darpp-32 . this circuit has been shown to be differentially regulated by psychomotor stimulants and antipsychotics acting on different striatal neuron subpopulations .   ", "while assembly of the many trimeric pp2a holoenzymes directs cellular localization and substrate specificity , further regulation is afforded through binding of specific protein inhibitors of pp2a .", "one such inhibitor set ( i2/taf-1 ) is upregulated during the progression of chronic myelogenous leukemia through bcr / abl activity and results in decreased pp2a activity .", "an additional pp2a inhibitor is the protein cip2a ( cancerous inhibitor of pp2a ) , overexpression of which is associated with several human malignancies .", "cip2a associates with c - myc to protect its phosphorylated s62 from pp2a - directed activity stabilizing the c - myc protein and allowing it to promote oncogenesis .   ", "recently an interplay between the drosophila serine / threonine kinase greatwall ( gwl ) and pp2a / b was observed during mitotic entry in two separate studies [ 69 , 70 ] .", "pp2a / b activity prevents mitotic entry by maintaining cdc25 in a dephosphorylated and inactive state .", "gwl reverses this inhibition through the phosphorylation of s67 of both -endosulfine ( ensa ) and cyclic adenosine monophosphate-(camp- ) regulated phosphoprotein-19 ( arpp-19 ) .", "phosphorylation converts ensa and arpp-19 into very specific inhibitors of pp2a / b activity and produces activation of cdc25 leading to cell cycle progression .", "similar cell cycle regulatory activity has been observed with the mammalian ortholog of gwl , mastl ; however , the mastl - pp2a interaction has yet to be characterized .", "once targeted to specific subcellular locales , the pp2a holoenzyme must recruit and dephosphorylate target substrates .", "recent structural studies have begun to suggest the mechanism by which the regulatory subunit of pp2a mediates initial binding to target substrates .", "other -propeller proteins have been shown to bind ligands in the central depression on the top surface of the toroid .", "crystallization of pp2a / b revealed a cluster of acidic residues in this depression that is available to recruit potential substrates containing a basic motif . in this same study ,", "the acidic central depression of b was experimentally confirmed to bind the microtubule - associated protein tau , an established pp2a / b substrate .", "several conserved aspartate and glutamate residues in b engage in weak , electrostatic interactions across a large basic portion of tau and support dephosphorylation of tau at multiple sites through cycles of binding and unbinding .    structurally divergent b family members may recruit substrates in a similar fashion as the b family .", "the crystal structure of pp2a / b shows that the b subunit contains 18 stacked -helices which adopt 8 huntingtin elongation a subunit tor- ( heat- ) like repeats [ 58 , 59 ] .", "a portion of these heat - like repeats interact with the a subunit of the holoenzyme to mediate regulatory subunit incorporation into the holoenzyme complex . like the b subunit", ", an acidic patch is exposed in the b family of regulatory subunits and may mediate protein - protein interactions and substrate recruitment .", "structural studies of pp2a have revealed a conserved loop in the b family of regulatory subunits which infiltrates the catalytic core of the holoenzyme [ 58 , 59 ] ( figure 2(a ) ) . at the tip of this loop", "is a conserved glutamate residue , e153 ( b numbering ) , which contacts through its carbonyl oxygen the catalytic subunit and through its carboxyl group a cocrystallized microcystin molecule in the active site .", "mutational analysis revealed that e153 is an absolute requirement for efficient dephosphorylation of tyrosine hydroxylase ( th ) , a known pp2a / b substrate , as well as other as yet unidentified cellular substrates of this pp2a holoenzyme .", "further , it was determined that e153 of b interacts with r37 and r38 of th to mediate dephosphorylation of both s31 and s40 on th .", "positively charged residues in the vicinity of target phospho - serine / threonine residues could represent a consensus sequence for b - subunit - mediated dephosphorylation .", "further , the infiltrating loop is likely a conformationally dynamic structure which is not sterically hindered by surrounding portions of the a , c , or b subunits .", "since r37/38 are important for dephosphorylation of both upstream ( s40 ) and downstream ( s31 ) sites , it appears that the orientation of phosphopeptides relative to the catalytic cleft is not constrained by additional interactions .", "collectively , the above observations support a model in which the sites of interaction between the substrate and the b regulatory subunit that are distant and near the active site together control substrate specificity .", "first , the interaction occurring at sites distant from the active site increases the local substrate concentration . following this initial substrate recruitment ,", "although divergent in its sequence , an analogous structure from the -propeller fold of the b family of regulatory subunits extends to the catalytic core of the holoenzyme   ( figure 2(b ) ) .", "this loop places conserved residues of the b - family subunits very near the holoenzyme active site . unpublished observations generated in our lab suggests that of these loop residues k87 of b2 may play a similar role as e153 of b in site - specific dephosphorylation of target substrates ; however , further characterization of this substrate specificity loop is required ."], ["pp2a is a ubiquitous protein phosphatase responsible for the dephosphorylation of many different intracellular targets . the diverse repertoire of potential substrates for pp2a is imparted by the incorporation of one of fifteen unique b - regulatory subunits .", "recent studies have increased our understanding of the mechanisms by which the b subunit imparts specificity to the holoenzyme complex . through selective incorporation of the b", "- regulatory subunit , the holoenzyme complex is recruited to discrete subcellular locales which define the sphere of influence for the phosphatase .", "secondly , interactions between endogenous inhibitors and specific pp2a heterotrimers further restrict phosphatase activity . as shown for the b family , regulatory subunits mediate low - affinity interactions with substrates to increase the local concentration of substrates . through a flexible substrate selectivity loop which contacts the catalytic subunit", ", interactions between the regulatory subunit and phosphosubstrate may mediate multiple nearby dephosphorylation events . with the current structural information available for", "the pp2a complexes , future high - resolution studies will further define the molecular mechanism of pp2a substrate specificity . as general ,", "inhibitors of pp2a are either clinically irrelevant or toxic , as in the case of the small molecule inhibitor microcystin , novel methods to increase the specificity of pp2a inhibition or activation must be developed .", "a clearer understanding of the pp2a substrate specificity mechanisms will serve as the foundation for rational drug design of selective inhibitors and activators of specific pp2a holoenzyme complexes ."]]}
{"article_id": "PMC3316147", "article_text": ["connectivity is key to understanding activity in neural systems ( sporns et al . , 2000 ) .", "network connectivity in science and in engineering fields as diverse as mechanics , communication technology , public health , geography and town planning , is studied mathematically using the concepts of graph theory ( bollobas , 1998 ) .", "recently , graph theory is being applied to brain connectivity ( sporns and zwi , 2004 ; bullmore and sporns , 2009 ) and its pathologies in alzheimer 's disease ( stam , 2004 ; stam et al . , 2007 ) , brain tumors ( bartolomei et al . , 2006 ) , epilepsy ( ponten et al . , 2007 ) and , in particular , schizophrenia ( bleuler , 1911/1950 ; friston and frith , 1995 ; andreasen , 1999 ; micheloyannis et al . , 2006 ;", "applying graph - theoretic concepts to the brain sheds new light on the basic principles of integration and segregation underlying adaptive cognitive processes , and on their disruption in maladaptive states .", "schizophrenia has been understood as a cognitive disorder ( bleuler , 1911/1950 ) based on the breakdown of large - scale cortico - cerebellar - thalamic - cortical ( andreasen , 1999 ) or prefronto - temporal circuits ( friston and frith , 1995 ; goldman - rakic and selemon , 1997 ) , or more generally the inability to integrate neural processes in different brain areas , a syndrome termed dysconnectivity ( stephan et al .", "this condition may pare down , in particular , the input to pyramidal cells of the dorsolateral prefrontal and temporal cortex ( garey et al .", "these cells are glutamatergic and receive projections from the thalamus and widespread cortical areas , and hence are likely to be involved in higher - level cognition .", "reduced connectivity may thus lead to fragmentation , a loss of coherence in cognitive activity .", "graph theory enables us to model the loss of connectivity in simulated neuronal networks and predict the time course of fragmentation . on the face of it ,", "percolation plays an important role in the evolution , growth , and maintenance of a large variety of natural , technological , and social systems ( ben avraham and havlin , 2000 ) .", "it refers to the probability of existence of a path between every pair of nodes in a graph , or equivalently , the graph being  connected ", "whilst many previous studies have examined cortical network connectivity in schizophrenia and other disorders , none to our knowledge have employed the concept of percolation , an issue that we presently redress .", "the percolation function is the cumulative density function ( cdf ) of percolation as a function of connectivity .", "it is possible , in principle , to measure the percolation function in living neural tissue , by using progressive lesioning , for instance through the administration of inhibitory neurotransmitters ( breskin et al . ,", "observations on human brain functional connectivity may be compared to the theoretical percolation function for random networks ( erds and rnyi , 1959 ) .", "the percolation function cp(n ) of a random graph of n vertices and e edges is given by cp(n ) = e the presence of a critical threshold motivates us to revisit the notion of cortical dysconnectivity as a sudden breakdown of percolation .", "there are , however , reasons to assume that the percolation threshold is neither the first , nor the most predominant , critical transition in the development of schizophrenia : brains are not random networks . in both the structural ( sporns and zwi , 2004 ) and functional ( salvador et al .", "2006 ) domains , the hallmarks of brain organization include local clustering as expressed in high values for the clustering coefficient ( cc ) , high global connectedness as specified by a short characteristic path length ( cpl ) ( watts and strogatz , 1998 ) , and modularity ( murre and sturdy , 1995)a combination characteristic of modular small - world networks ( he et al . , 2009 ) .", "graph - theoretical studies ( murre and sturdy , 1995 ; watts and strogatz , 1998 ) showed that small - world and modular networks can secure global connectivity with a small number of connections . for brains configured as modular small - worlds , a few connections will suffice to ensure percolation .", "most likely , therefore , percolation is not the crucial bottleneck for brain pathologies such as schizophrenia .", "we will propose as an alternative theoretical possibility that , instead , brain pathologies are associated with a breakdown in the local organization . in schizophrenia patients ,", "functional connectivity in scalp eeg channels appears to reflect a loss of clustering after correcting for differences in the density of functional connections ( micheloyannis et al .", "the question , therefore , arises , whether fragmentation can be understood as a critical breakdown in the ability of the brain to establish and maintain a modular small - world functional architecture .", "here we show by numerical simulations that in neural activity networks , with loss of connectivity a self - organizing small - world neural network can not sustain its local clustering , well before global connectivity breaks down .", "the iterated logistic map f(x)= 1  ax is unimodal on [ 1;1 ]  [ 1;1 ] and capable of both periodic and chaotic behavior depending on its control parameter a. in this study , we construct networks of coupled logistic maps , all with parameter a = 1.7 such that the dynamics of a single unit are chaotic under iteration of a randomly chosen initial activation value . a unit x is coupled with coupling strength  = 0.4 to any number mi of other units in the network such that its activation value xn + 1 at iteration n + 1 depends on the activation value of itself and all adjacent units at iteration n : \n\n in this equation , b(i ) is the set of units adjacent to unit i , mi is the number of units adjacent to unit i. the coupling strength  is divided by mi , and has a compensation term ( 1   ) to make sure that logistic map of an individual unit retains its mapping [ 1:1 ]  [ 1:1 ] , and thus functions properly for any numbers of adjacent units . a network of this type can be used to study the buildup and breakdown of modularity resulting from hebbian adaptive structural self - organization .", "it implements a simple rewiring rule based on synchronization of chaotic activity and rewires at most one connection per iteration , carefully keeping the network 's total number of connections constant throughout the process .", "a single iteration of the network consists of four steps , to be repeated several times after an initial random inception : \n initialize the network .", "randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] .", "though values of v and e are chosen such that a network has a high probability of being connected , this is not required.update units .", "synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1).select pivot and candidate .", "randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's .", "then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant .", "if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped.iteration completed .", "randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] .", "though values of v and e are chosen such that a network has a high probability of being connected , this is not required .", "synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1 ) .", "randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's .", "then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant .", "if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped .", "networks implementing these iterative steps exhibit development from an initial random configuration to modular small - world configurations ( gong and van leeuwen , 2003 ; van den berg and van leeuwen , 2004 ; rubinov et al . , 2009b )", "but as it turns out , both the consistent build - up of connective modularity on one hand , or the loss of structural coherence due to functional fragmentation on the other , are a result of changing dynamic activity depending critically on the number of connections in the network .", "the influence of these numbers shows a close relationship to the percolation function of random graphs .", "a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) .", "this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring ,", "does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) .", "the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity .", "these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al .", ", 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) .", "adaptive rewiring operates on this activity according to the general hebbian principle of  what fires together wires together  ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity", ", connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time", "the network gradually assumes a modular , small - world structure ( figure 1 ) .", "adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected", "receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500,  ,1000 vertices and numbers of edges e that differ by small steps of 20 .", "for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs .", "the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 .", "meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure .", "crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al .", "attractors  in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) .", "when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes", ". this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest .", "for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease .", "self - organization from random to small - world critically in a network of 700 vertices .", "the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges .", "we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices .", "figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4 \n cp(n ) .", "parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] .", "the behavior of these parameters across network sizes was not monotonic ( figure 4 ) .", "parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) .", "note : anchor point arand(n ) =   n  ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection .", "aswn(n ) : anchor point for the small - world networks fitted according to figure 4 .", "marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) .", "a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) .", "this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring ,", "does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) .", "the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity .", "these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al .", ", 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) .", "adaptive rewiring operates on this activity according to the general hebbian principle of  what fires together wires together  ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity", ", connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time", "the network gradually assumes a modular , small - world structure ( figure 1 ) .", "adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected", "receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500,  ,1000 vertices and numbers of edges e that differ by small steps of 20 .", "for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs .", "the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 .", "meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure .", "crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al .", "attractors  in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) .", "when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes", ". this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest .", "for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease .", "self - organization from random to small - world critically in a network of 700 vertices .", "the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges .", "we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices .", "figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4 \n cp(n ) .", "parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] .", "the behavior of these parameters across network sizes was not monotonic ( figure 4 ) .", "parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) .", "note : anchor point arand(n ) =   n  ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection .", "aswn(n ) : anchor point for the small - world networks fitted according to figure 4 .", "marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) .", "we propose that important insights into cortical activity and architecture can be obtained by modeling the activity - dependent rewiring of neural connections during development ( gong and van leeuwen , 2003 ; rubinov et al . , 2009b ) .", "in our model , network connections evolve in accordance with the principle that the structure rewires in adaptation to spontaneous , on - going activity .", "this evolution , however , is only guaranteed if there are sufficiently many connections available .", "if connectivity is reduced below this number , the structure shifts toward randomness ; in particular , local clustering is reduced .", "andreasen ( 1999 ) and friston and frith ( 1995 ) considered schizophrenia as fragmentation , understood as the breakdown of integration between widely distributed brain areas ( stephan et al . , 2006 , 2009 ) .", "this breakdown can be associated with the loss of connectivity ( zalesky et al . , 2011 ) , in particular of input to layer 3 pyramidal cells , an effect which is well - documented ( e.g. , garey et al .", "( 2011 ) observed widespread impairment in structural connectivity in schizophrenic patients , involving medial frontal , parietal / occipital and left frontal cortex .", "it should be observed that the loss of connectivity that may lead to the onset of schizophrenia can be relatively subtle . across the population ,", "inputs to layer 3 pyramidal cells are substantially reduced during late adolescence , the typical period for the onset of schizophrenia ( bourgeois et al . , 1994 ) .", "given that brain connectivity is costly , it may well be that in normals , its density hovers just above the critical level ( the anchor point in figure 4 ) , but in early schizophrenia it may fall just below this point . the graph - theoretical concept of percolation tells us that a small decline in connectivity can lead to a sudden breakdown of global network coherence .", "based on our results , however , we argue that fragmentation in brain pathologies such as schizophrenia may be considered theoretically as a breakdown in the local connectivity structure , prior to the loss of global coherence . the number minimally needed to secure local modularity , and hence to prevent it from shifting toward randomness in structure , is systematically related to , and greater than , that needed to secure global connectivity , even if the system has fallen into entirely random connectivity .", "this result is of potential importance for understanding the pathophysiological processes that give rise to this disorder .", "the loss of local clustering in our model is in accordance with observations in schizophrenic patients by micheloyannis et al .", "et al . ( 2006 ) , the clinical group also showed longer path lengths than the controls , whereas in rubinov et al .", "nevertheless , we might attribute the discrepancy to the fact that in both studies comparisons were made , for statistical reasons , between networks that were thresholded to have identical connectivity .", "whereas the above - mentioned effects of clustering remain relatively unaffected by threshold setting , the differences in path length rapidly disappear for lower thresholds ( figure 1 in micheloyannis et al . , 2006 ) .", "( 2003b ) reported that although there were no significant increases in the occurrence of nonlinear interdependence between pairs of electrodes in schizophrenia , there was an increase in the co - occurrence in multiple ( widespread ) instances of nonlinear interdependence .", "this means that a relatively large number of global connections will have survived thresholding in rubinov 's study , leading to their observation of path length shortening .", "it can not be concluded from rubinov 's study , therefore , that global connectivity is stronger in schizophrenics than in normals ; it could , however , be concluded that the global connectivity becomes stronger in schizophrenia relatively to their local connectivity .", "such a conclusion would entirely be in accordance with the modularity breakdown observed in our model .", "along the lines set out here , a shift in the balance from local to global connectivity is perfectly consistent with an overall loss of connectivity in early schizophrenia .", "( 2003 ) introduced the notion of  overbindingthe formation of excessive connections that are effectively random  and , as such , do not enable distinguishing external from internal sources , thus providing conditions favorable for phenomena such as hallucination . a possible objection to our findings is the specific choice of our rewiring algorithm .", "note , however , that in the present paper we sought to establish the principled possibility using the simplest possible model , rather than to establish the empirical validity through the most realistic model possible", ". note that , as a consequence , the model contains only generic dynamical and adaptive principles .", "we have discussed elsewhere the robustness of this model ( gong and van leeuwen , 2003 , 2004 ; van den berg and van leeuwen , 2004 ; kwok et al .", "an important limitation is that the model inevitably makes over - simplifying assumptions . in particular", "inter - modular connections are physically of longer range than intra - modular ones and , therefore , have a higher metabolic cost and a greater vulnerability .", "preliminary analysis of models with more realistic constraints does not appear , however , to affect our conclusions .", "clearly , a more differentiated model is needed to address empirical datasets such as ( rubinov et al . , 2009a ) , an important goal of future work .", "however , it should also be noted that uncovering universal principles  such as those reported here  has the advantage of being ", "detail invariantthat is , robust across a range of potential constraints , whereas findings arising in detailed models may not be robust to changes in those details .", "we observed universal scaling behavior in adaptive self - organization of clustered small - world networks : the connectivity needed for these network properties to emerge under hebbian rewiring scales with a universal power  = 1.17 to the percolation function in random networks .", "note , first , that  > 1 might have been expected , given that the requirement to observe clustering and small - world structure are constraints additional to percolation .", "what is surprising is that these requirements are met with alpha very close to unity ; near - linear scaling implies that these additional constraints can be realized with great efficiency . in terms of kolmogorov - complexity , small - worlds are compressible , whereas almost every possible network of n nodes and e edges ( or equivalently a bit string of length l = n(n  1 ) with e ones and l - e zeros ) will be incompressible ( li and vitnyi , 1993 ) . in this perspective , the ubiquity of small - world structure in real - world networks is quite astonishing : within human brains ( sporns and zwi , 2004 ; stam , 2004 ; eguluz et al .", "bartolomei et al . , 2006 ; micheloyannis et al . , 2006 ; ponten et al .", ", 2007 ; rubinov et al . , 2009a ; bassett et al . , 2010 ) , as well as between them : networks of scientific co - authorship ( newman , 2001 ) , collaborating movie actors ( watts and strogatz , 1998 ; amaral et al . , 2000 ) , social networks in general ( wasserman and faust , 1994 ) . here", "we showed how such a network could arise with minimal connectivity close to random network percolation what is the reason for the universality of the scaling exponent ?", "we may wonder whether the same exponent found in other domains , could help us understand the principle .", "a study of class graphs in open - source , object - oriented software systems ranging from simple paint programs , peer - to - peer downloaders , racing games , database management software to a complete operating system , showed that the number of links between classes scales to the number of classes with an exact power = 1.17 .", "the authors found that class graphs are small - world networks at the critical threshold for the breakdown of modularity , which happens when developments to the system are widely dispersed and affect many unrelated classes in apparently distant modules ( valverde and sol , 2007 ) .", "the similarity of this finding to ours supports the view that the scaling exponent reflects a general feature in the emergence and breakdown of modular network structure .", "the study of self - organizing modular small - world networks casts a new perspective on psychiatric illnesses characterized by disorganized cognition , such as schizophrenia , of which the expression has been attributed to fragmentation  a  subtle but pernicious disconnection  ( friston , 1996 , p. 644 ) . rather than a breakdown in global connectivity", ", we propose that fragmentation is to be understood as a failure to organize the functional connectivity of the brain into a modular small - world structure .", "this is in accordance with the observed  random shift  in schizophrenic ( micheloyannis et al . , 2006 ;", "random networks are considered extremely uneconomical ; in terms of cable length , an optimal configuration combines local modules with a limited number of large - scale connections ( murre and sturdy , 1995 ) . even though our model does not consider distance , in terms of network topology it is still the case that information travels efficiently both within locally connected circuits of modular small - world graphs and between their circuits , which makes these networks efficient for transport or communication ( latora and marchiori , 2001 ; bassett et al . , 2010 ) .", "the scaling observations tell us that fragmentation is a result of a breakdown in local , rather than global structure . with progressive loss of connectivity ,", "ultimately , it may not matter which connections are lost first , the result may be a cascade of changes that lead to the network falling apart . for diagnosis , however , a proper understanding of the early stages of the disease is crucial ; loss of modularity might offer a new perspective on the origins of the disease .", "one simulation consists of one network of v units and e connections , which is randomly initialized and then iterated exactly 4,000,000 times , simultaneously rearranging its connections and activity patterns , according to the adaptive rewiring scenario .", "the smallest simulation we adopt has v = 300 units and e = 2400 connections . during iteration ,", "its cpl and its cc are taken every 1000th iteration ( 1000 , 2000 ,  , 40,00,000 ) resulting in a 4000 point record , with a value for cc and a value for cpl at each point .", "although the speed of convergence depends on the size of a network , 4 , 000 , 000 iterations prove to be enough to clearly discern asymptotic behavior for all simulations used in this investigation ( figure 3 ) .", "evolution under adaptive rewiring of maximum , minimum , and average cluster coefficient and characteristic path length .", "( a ) the values of minimum , maximum and average cc for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040,  ,10,000 ] after extensive adaptive rewiring . note that beyond 9000 edges , cc - values tighten to a narrow range , indicating strong and consistent clustering behavior .", "( b ) the values of minimum , maximum , and average cpl for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040,  ,10,000 ] after extensive adaptive rewiring . beyond 9000 edges", "thus , for 700 vertices , at least 9000 edges are needed for adaptive rewiring to converge to small - world structure . from the 4000 point record ,", "the maximum , minimum , and average values for both cpl and cc are calculated from the last 2000 points . for statistical robustness", ", we do any single simulation five times , and average the five values over this simulation - quintuple , resulting in a maximum , minimum , and average cc and cpl for the ( v = 300 , e = 2400 ) network .", "we then start a new quintuple of simulations , increasing the number of connections e by 20 , generating five networks with v = 300 units and e = 2420 connections , and calculate the maximum , minimum , and average cc and cpl values from these five new simulations .", "we keep starting new quintuples , repeatedly increasing e by 20 , until e = 3300 and the batch of 300-quintuples is complete . from the entire batch , the six cc and cpl values of every ( v ,", "e ) are taken to graph the asymptotic clustering and path - length behavior of networks of 300 units as it depends on the numbers of connections ( figure 4 ) .", "gray lines represent minimal , maximal and average observed values for clustering coefficient , the dotted line is the predicted clustering coefficient , ccpred , a linear function of the percolation function cp(n ) of a random graph of n vertices : ccpred = k3 + k4  cp(n ) fitted with parameters k3 and k4 to the minimum observed clustering ; the arrow indicates its anchor point aswn(n ) with the corresponding number of edges in parentheses .", "this process is then repeated for a batch of quintuples of networks with 400 units and numbers of connections 3600 , 3620 ,  , 5000 ( see figure 4 , top - right box ) .", "we continue doing this for batches of networks with 500 , 600 , 700 , 800 , 900 , and 1000 units , with connections increasing by 20 , showing asymptotic clustering and path - length behavior depending on connectivity for networks of different sizes ( figure 4 ) .", "note that although for 300 units , connections ranged from e = 2400 to e = 3300 , these numbers are different for larger networks .", "for each of the eight batches , a phase transition was witnessed for both the cc and the cpl .", "to pin down the exact location of the steepest inclination in the phase transition of the cc ( its center , or  anchor point  ) , the percolation function for classic random graphs was function - matched to the minimum cc of every batch .", "the minimum cluster coefficient was chosen over the average and the maximum cluster coefficient because it has the steepest inclination , which facilitates the fitting best .", "macquardt an iterative curve - fitting algorithm which operates by minimizing the summed squares of the residuals , in this case the difference between minimal cc - values of n - edge simulation quintuples on the one hand , and the ( erds and rnyi , 1959 ) percolation function 's value for n edges on the other .", "macquardt algorithm is sensitive to local minima which makes it inefficient when using completely random initial values .", "initial parameters were hand - guessed separately for each of the eight subgraphs in figure 4 , after which the algorithm was ran until convergence beyond the program 's six - digit resolution , a procedure that was repeated three times with small differences in the hand - guessed initial parameters .", "the final values did not differ within the program 's six - digit resolution over the three repetitions , and convergence was very fast ( typically well before 100 iterations ) .", "the fits show significant deviations from the data curve , due to intrinsic fluctuations in the data .", "we , therefore , considered reliable the estimates of the scaling power and other model parameters .", "even more reliable estimates could , in principle , be obtained by scaling up the network size to 2000 , 5000 , and 10,000 vertices , resources permitting , as computation time and data grow nonlinearly with network size .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S> psychiatric illnesses characterized by disorganized cognition , such as schizophrenia , have been described in terms of fragmentation and hence understood as reduction in functional brain connectivity , particularly in prefrontal and parietal areas . however , as graph theory shows , relatively small numbers of nonlocal connections are sufficient to ensure global coherence in the modular small - world network structure of the brain . </S>", "<S> we reconsider fragmentation in this perspective . </S>", "<S> computational studies have shown that for a given level of connectivity in a model of coupled nonlinear oscillators , modular small - world networks evolve from an initially random organization . here </S>", "<S> we demonstrate that with decreasing connectivity , the probability of evolving into a modular small - world network breaks down at a critical point , which scales to the percolation function of random networks with a universal exponent of  = 1.17 . </S>", "<S> thus , according to the model , local modularity systematically breaks down before there is loss of global coherence in network connectivity . </S>", "<S> we , therefore , propose that fragmentation may involve , at least in its initial stages , the inability of a dynamically evolving network to sustain a modular small - world structure . </S>", "<S> the result is in a shift in the balance in schizophrenia from local to global functional connectivity . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Evolving networks", "Percolation and self-organization in small-worlds", "Discussion", "Data", "Conflict of interest statement"], "sections": [["connectivity is key to understanding activity in neural systems ( sporns et al . , 2000 ) .", "network connectivity in science and in engineering fields as diverse as mechanics , communication technology , public health , geography and town planning , is studied mathematically using the concepts of graph theory ( bollobas , 1998 ) .", "recently , graph theory is being applied to brain connectivity ( sporns and zwi , 2004 ; bullmore and sporns , 2009 ) and its pathologies in alzheimer 's disease ( stam , 2004 ; stam et al . , 2007 ) , brain tumors ( bartolomei et al . , 2006 ) , epilepsy ( ponten et al . , 2007 ) and , in particular , schizophrenia ( bleuler , 1911/1950 ; friston and frith , 1995 ; andreasen , 1999 ; micheloyannis et al . , 2006 ;", "applying graph - theoretic concepts to the brain sheds new light on the basic principles of integration and segregation underlying adaptive cognitive processes , and on their disruption in maladaptive states .", "schizophrenia has been understood as a cognitive disorder ( bleuler , 1911/1950 ) based on the breakdown of large - scale cortico - cerebellar - thalamic - cortical ( andreasen , 1999 ) or prefronto - temporal circuits ( friston and frith , 1995 ; goldman - rakic and selemon , 1997 ) , or more generally the inability to integrate neural processes in different brain areas , a syndrome termed dysconnectivity ( stephan et al .", "this condition may pare down , in particular , the input to pyramidal cells of the dorsolateral prefrontal and temporal cortex ( garey et al .", "these cells are glutamatergic and receive projections from the thalamus and widespread cortical areas , and hence are likely to be involved in higher - level cognition .", "reduced connectivity may thus lead to fragmentation , a loss of coherence in cognitive activity .", "graph theory enables us to model the loss of connectivity in simulated neuronal networks and predict the time course of fragmentation . on the face of it ,", "percolation plays an important role in the evolution , growth , and maintenance of a large variety of natural , technological , and social systems ( ben avraham and havlin , 2000 ) .", "it refers to the probability of existence of a path between every pair of nodes in a graph , or equivalently , the graph being  connected ", "whilst many previous studies have examined cortical network connectivity in schizophrenia and other disorders , none to our knowledge have employed the concept of percolation , an issue that we presently redress .", "the percolation function is the cumulative density function ( cdf ) of percolation as a function of connectivity .", "it is possible , in principle , to measure the percolation function in living neural tissue , by using progressive lesioning , for instance through the administration of inhibitory neurotransmitters ( breskin et al . ,", "observations on human brain functional connectivity may be compared to the theoretical percolation function for random networks ( erds and rnyi , 1959 ) .", "the percolation function cp(n ) of a random graph of n vertices and e edges is given by cp(n ) = e the presence of a critical threshold motivates us to revisit the notion of cortical dysconnectivity as a sudden breakdown of percolation .", "there are , however , reasons to assume that the percolation threshold is neither the first , nor the most predominant , critical transition in the development of schizophrenia : brains are not random networks . in both the structural ( sporns and zwi , 2004 ) and functional ( salvador et al .", "2006 ) domains , the hallmarks of brain organization include local clustering as expressed in high values for the clustering coefficient ( cc ) , high global connectedness as specified by a short characteristic path length ( cpl ) ( watts and strogatz , 1998 ) , and modularity ( murre and sturdy , 1995)a combination characteristic of modular small - world networks ( he et al . , 2009 ) .", "graph - theoretical studies ( murre and sturdy , 1995 ; watts and strogatz , 1998 ) showed that small - world and modular networks can secure global connectivity with a small number of connections . for brains configured as modular small - worlds , a few connections will suffice to ensure percolation .", "most likely , therefore , percolation is not the crucial bottleneck for brain pathologies such as schizophrenia .", "we will propose as an alternative theoretical possibility that , instead , brain pathologies are associated with a breakdown in the local organization . in schizophrenia patients ,", "functional connectivity in scalp eeg channels appears to reflect a loss of clustering after correcting for differences in the density of functional connections ( micheloyannis et al .", "the question , therefore , arises , whether fragmentation can be understood as a critical breakdown in the ability of the brain to establish and maintain a modular small - world functional architecture .", "here we show by numerical simulations that in neural activity networks , with loss of connectivity a self - organizing small - world neural network can not sustain its local clustering , well before global connectivity breaks down ."], ["the iterated logistic map f(x)= 1  ax is unimodal on [ 1;1 ]  [ 1;1 ] and capable of both periodic and chaotic behavior depending on its control parameter a. in this study , we construct networks of coupled logistic maps , all with parameter a = 1.7 such that the dynamics of a single unit are chaotic under iteration of a randomly chosen initial activation value . a unit x is coupled with coupling strength  = 0.4 to any number mi of other units in the network such that its activation value xn + 1 at iteration n + 1 depends on the activation value of itself and all adjacent units at iteration n : \n\n in this equation , b(i ) is the set of units adjacent to unit i , mi is the number of units adjacent to unit i. the coupling strength  is divided by mi , and has a compensation term ( 1   ) to make sure that logistic map of an individual unit retains its mapping [ 1:1 ]  [ 1:1 ] , and thus functions properly for any numbers of adjacent units . a network of this type can be used to study the buildup and breakdown of modularity resulting from hebbian adaptive structural self - organization .", "it implements a simple rewiring rule based on synchronization of chaotic activity and rewires at most one connection per iteration , carefully keeping the network 's total number of connections constant throughout the process .", "a single iteration of the network consists of four steps , to be repeated several times after an initial random inception : \n initialize the network .", "randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] .", "though values of v and e are chosen such that a network has a high probability of being connected , this is not required.update units .", "synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1).select pivot and candidate .", "randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's .", "then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant .", "if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped.iteration completed .", "randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] .", "though values of v and e are chosen such that a network has a high probability of being connected , this is not required .", "synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1 ) .", "randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's .", "then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant .", "if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped .", "networks implementing these iterative steps exhibit development from an initial random configuration to modular small - world configurations ( gong and van leeuwen , 2003 ; van den berg and van leeuwen , 2004 ; rubinov et al . , 2009b )", "but as it turns out , both the consistent build - up of connective modularity on one hand , or the loss of structural coherence due to functional fragmentation on the other , are a result of changing dynamic activity depending critically on the number of connections in the network .", "the influence of these numbers shows a close relationship to the percolation function of random graphs ."], ["a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) .", "this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring ,", "does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) .", "the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity .", "these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al .", ", 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) .", "adaptive rewiring operates on this activity according to the general hebbian principle of  what fires together wires together  ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity", ", connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time", "the network gradually assumes a modular , small - world structure ( figure 1 ) .", "adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected", "receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500,  ,1000 vertices and numbers of edges e that differ by small steps of 20 .", "for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs .", "the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 .", "meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure .", "crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al .", "attractors  in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) .", "when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes", ". this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest .", "for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease .", "self - organization from random to small - world critically in a network of 700 vertices .", "the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges .", "we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices .", "figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4 \n cp(n ) .", "parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] .", "the behavior of these parameters across network sizes was not monotonic ( figure 4 ) .", "parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) .", "note : anchor point arand(n ) =   n  ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection .", "aswn(n ) : anchor point for the small - world networks fitted according to figure 4 .", "marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) ."], ["a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) .", "this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring ,", "does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) .", "the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity .", "these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al .", ", 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) .", "adaptive rewiring operates on this activity according to the general hebbian principle of  what fires together wires together  ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity", ", connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time", "the network gradually assumes a modular , small - world structure ( figure 1 ) .", "adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected", "receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500,  ,1000 vertices and numbers of edges e that differ by small steps of 20 .", "for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs .", "the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 .", "meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure .", "crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al .", "attractors  in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) .", "when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes", ". this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest .", "for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease .", "self - organization from random to small - world critically in a network of 700 vertices .", "the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges ."], ["we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices .", "figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4 \n cp(n ) .", "parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] .", "the behavior of these parameters across network sizes was not monotonic ( figure 4 ) .", "parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) .", "note : anchor point arand(n ) =   n  ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection .", "aswn(n ) : anchor point for the small - world networks fitted according to figure 4 .", "marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) ."], ["we propose that important insights into cortical activity and architecture can be obtained by modeling the activity - dependent rewiring of neural connections during development ( gong and van leeuwen , 2003 ; rubinov et al . , 2009b ) .", "in our model , network connections evolve in accordance with the principle that the structure rewires in adaptation to spontaneous , on - going activity .", "this evolution , however , is only guaranteed if there are sufficiently many connections available .", "if connectivity is reduced below this number , the structure shifts toward randomness ; in particular , local clustering is reduced .", "andreasen ( 1999 ) and friston and frith ( 1995 ) considered schizophrenia as fragmentation , understood as the breakdown of integration between widely distributed brain areas ( stephan et al . , 2006 , 2009 ) .", "this breakdown can be associated with the loss of connectivity ( zalesky et al . , 2011 ) , in particular of input to layer 3 pyramidal cells , an effect which is well - documented ( e.g. , garey et al .", "( 2011 ) observed widespread impairment in structural connectivity in schizophrenic patients , involving medial frontal , parietal / occipital and left frontal cortex .", "it should be observed that the loss of connectivity that may lead to the onset of schizophrenia can be relatively subtle . across the population ,", "inputs to layer 3 pyramidal cells are substantially reduced during late adolescence , the typical period for the onset of schizophrenia ( bourgeois et al . , 1994 ) .", "given that brain connectivity is costly , it may well be that in normals , its density hovers just above the critical level ( the anchor point in figure 4 ) , but in early schizophrenia it may fall just below this point . the graph - theoretical concept of percolation tells us that a small decline in connectivity can lead to a sudden breakdown of global network coherence .", "based on our results , however , we argue that fragmentation in brain pathologies such as schizophrenia may be considered theoretically as a breakdown in the local connectivity structure , prior to the loss of global coherence . the number minimally needed to secure local modularity , and hence to prevent it from shifting toward randomness in structure , is systematically related to , and greater than , that needed to secure global connectivity , even if the system has fallen into entirely random connectivity .", "this result is of potential importance for understanding the pathophysiological processes that give rise to this disorder .", "the loss of local clustering in our model is in accordance with observations in schizophrenic patients by micheloyannis et al .", "et al . ( 2006 ) , the clinical group also showed longer path lengths than the controls , whereas in rubinov et al .", "nevertheless , we might attribute the discrepancy to the fact that in both studies comparisons were made , for statistical reasons , between networks that were thresholded to have identical connectivity .", "whereas the above - mentioned effects of clustering remain relatively unaffected by threshold setting , the differences in path length rapidly disappear for lower thresholds ( figure 1 in micheloyannis et al . , 2006 ) .", "( 2003b ) reported that although there were no significant increases in the occurrence of nonlinear interdependence between pairs of electrodes in schizophrenia , there was an increase in the co - occurrence in multiple ( widespread ) instances of nonlinear interdependence .", "this means that a relatively large number of global connections will have survived thresholding in rubinov 's study , leading to their observation of path length shortening .", "it can not be concluded from rubinov 's study , therefore , that global connectivity is stronger in schizophrenics than in normals ; it could , however , be concluded that the global connectivity becomes stronger in schizophrenia relatively to their local connectivity .", "such a conclusion would entirely be in accordance with the modularity breakdown observed in our model .", "along the lines set out here , a shift in the balance from local to global connectivity is perfectly consistent with an overall loss of connectivity in early schizophrenia .", "( 2003 ) introduced the notion of  overbindingthe formation of excessive connections that are effectively random  and , as such , do not enable distinguishing external from internal sources , thus providing conditions favorable for phenomena such as hallucination . a possible objection to our findings is the specific choice of our rewiring algorithm .", "note , however , that in the present paper we sought to establish the principled possibility using the simplest possible model , rather than to establish the empirical validity through the most realistic model possible", ". note that , as a consequence , the model contains only generic dynamical and adaptive principles .", "we have discussed elsewhere the robustness of this model ( gong and van leeuwen , 2003 , 2004 ; van den berg and van leeuwen , 2004 ; kwok et al .", "an important limitation is that the model inevitably makes over - simplifying assumptions . in particular", "inter - modular connections are physically of longer range than intra - modular ones and , therefore , have a higher metabolic cost and a greater vulnerability .", "preliminary analysis of models with more realistic constraints does not appear , however , to affect our conclusions .", "clearly , a more differentiated model is needed to address empirical datasets such as ( rubinov et al . , 2009a ) , an important goal of future work .", "however , it should also be noted that uncovering universal principles  such as those reported here  has the advantage of being ", "detail invariantthat is , robust across a range of potential constraints , whereas findings arising in detailed models may not be robust to changes in those details .", "we observed universal scaling behavior in adaptive self - organization of clustered small - world networks : the connectivity needed for these network properties to emerge under hebbian rewiring scales with a universal power  = 1.17 to the percolation function in random networks .", "note , first , that  > 1 might have been expected , given that the requirement to observe clustering and small - world structure are constraints additional to percolation .", "what is surprising is that these requirements are met with alpha very close to unity ; near - linear scaling implies that these additional constraints can be realized with great efficiency . in terms of kolmogorov - complexity , small - worlds are compressible , whereas almost every possible network of n nodes and e edges ( or equivalently a bit string of length l = n(n  1 ) with e ones and l - e zeros ) will be incompressible ( li and vitnyi , 1993 ) . in this perspective , the ubiquity of small - world structure in real - world networks is quite astonishing : within human brains ( sporns and zwi , 2004 ; stam , 2004 ; eguluz et al .", "bartolomei et al . , 2006 ; micheloyannis et al . , 2006 ; ponten et al .", ", 2007 ; rubinov et al . , 2009a ; bassett et al . , 2010 ) , as well as between them : networks of scientific co - authorship ( newman , 2001 ) , collaborating movie actors ( watts and strogatz , 1998 ; amaral et al . , 2000 ) , social networks in general ( wasserman and faust , 1994 ) . here", "we showed how such a network could arise with minimal connectivity close to random network percolation what is the reason for the universality of the scaling exponent ?", "we may wonder whether the same exponent found in other domains , could help us understand the principle .", "a study of class graphs in open - source , object - oriented software systems ranging from simple paint programs , peer - to - peer downloaders , racing games , database management software to a complete operating system , showed that the number of links between classes scales to the number of classes with an exact power = 1.17 .", "the authors found that class graphs are small - world networks at the critical threshold for the breakdown of modularity , which happens when developments to the system are widely dispersed and affect many unrelated classes in apparently distant modules ( valverde and sol , 2007 ) .", "the similarity of this finding to ours supports the view that the scaling exponent reflects a general feature in the emergence and breakdown of modular network structure .", "the study of self - organizing modular small - world networks casts a new perspective on psychiatric illnesses characterized by disorganized cognition , such as schizophrenia , of which the expression has been attributed to fragmentation  a  subtle but pernicious disconnection  ( friston , 1996 , p. 644 ) . rather than a breakdown in global connectivity", ", we propose that fragmentation is to be understood as a failure to organize the functional connectivity of the brain into a modular small - world structure .", "this is in accordance with the observed  random shift  in schizophrenic ( micheloyannis et al . , 2006 ;", "random networks are considered extremely uneconomical ; in terms of cable length , an optimal configuration combines local modules with a limited number of large - scale connections ( murre and sturdy , 1995 ) . even though our model does not consider distance , in terms of network topology it is still the case that information travels efficiently both within locally connected circuits of modular small - world graphs and between their circuits , which makes these networks efficient for transport or communication ( latora and marchiori , 2001 ; bassett et al . , 2010 ) .", "the scaling observations tell us that fragmentation is a result of a breakdown in local , rather than global structure . with progressive loss of connectivity ,", "ultimately , it may not matter which connections are lost first , the result may be a cascade of changes that lead to the network falling apart . for diagnosis , however , a proper understanding of the early stages of the disease is crucial ; loss of modularity might offer a new perspective on the origins of the disease ."], ["one simulation consists of one network of v units and e connections , which is randomly initialized and then iterated exactly 4,000,000 times , simultaneously rearranging its connections and activity patterns , according to the adaptive rewiring scenario .", "the smallest simulation we adopt has v = 300 units and e = 2400 connections . during iteration ,", "its cpl and its cc are taken every 1000th iteration ( 1000 , 2000 ,  , 40,00,000 ) resulting in a 4000 point record , with a value for cc and a value for cpl at each point .", "although the speed of convergence depends on the size of a network , 4 , 000 , 000 iterations prove to be enough to clearly discern asymptotic behavior for all simulations used in this investigation ( figure 3 ) .", "evolution under adaptive rewiring of maximum , minimum , and average cluster coefficient and characteristic path length .", "( a ) the values of minimum , maximum and average cc for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040,  ,10,000 ] after extensive adaptive rewiring . note that beyond 9000 edges , cc - values tighten to a narrow range , indicating strong and consistent clustering behavior .", "( b ) the values of minimum , maximum , and average cpl for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040,  ,10,000 ] after extensive adaptive rewiring . beyond 9000 edges", "thus , for 700 vertices , at least 9000 edges are needed for adaptive rewiring to converge to small - world structure . from the 4000 point record ,", "the maximum , minimum , and average values for both cpl and cc are calculated from the last 2000 points . for statistical robustness", ", we do any single simulation five times , and average the five values over this simulation - quintuple , resulting in a maximum , minimum , and average cc and cpl for the ( v = 300 , e = 2400 ) network .", "we then start a new quintuple of simulations , increasing the number of connections e by 20 , generating five networks with v = 300 units and e = 2420 connections , and calculate the maximum , minimum , and average cc and cpl values from these five new simulations .", "we keep starting new quintuples , repeatedly increasing e by 20 , until e = 3300 and the batch of 300-quintuples is complete . from the entire batch , the six cc and cpl values of every ( v ,", "e ) are taken to graph the asymptotic clustering and path - length behavior of networks of 300 units as it depends on the numbers of connections ( figure 4 ) .", "gray lines represent minimal , maximal and average observed values for clustering coefficient , the dotted line is the predicted clustering coefficient , ccpred , a linear function of the percolation function cp(n ) of a random graph of n vertices : ccpred = k3 + k4  cp(n ) fitted with parameters k3 and k4 to the minimum observed clustering ; the arrow indicates its anchor point aswn(n ) with the corresponding number of edges in parentheses .", "this process is then repeated for a batch of quintuples of networks with 400 units and numbers of connections 3600 , 3620 ,  , 5000 ( see figure 4 , top - right box ) .", "we continue doing this for batches of networks with 500 , 600 , 700 , 800 , 900 , and 1000 units , with connections increasing by 20 , showing asymptotic clustering and path - length behavior depending on connectivity for networks of different sizes ( figure 4 ) .", "note that although for 300 units , connections ranged from e = 2400 to e = 3300 , these numbers are different for larger networks .", "for each of the eight batches , a phase transition was witnessed for both the cc and the cpl .", "to pin down the exact location of the steepest inclination in the phase transition of the cc ( its center , or  anchor point  ) , the percolation function for classic random graphs was function - matched to the minimum cc of every batch .", "the minimum cluster coefficient was chosen over the average and the maximum cluster coefficient because it has the steepest inclination , which facilitates the fitting best .", "macquardt an iterative curve - fitting algorithm which operates by minimizing the summed squares of the residuals , in this case the difference between minimal cc - values of n - edge simulation quintuples on the one hand , and the ( erds and rnyi , 1959 ) percolation function 's value for n edges on the other .", "macquardt algorithm is sensitive to local minima which makes it inefficient when using completely random initial values .", "initial parameters were hand - guessed separately for each of the eight subgraphs in figure 4 , after which the algorithm was ran until convergence beyond the program 's six - digit resolution , a procedure that was repeated three times with small differences in the hand - guessed initial parameters .", "the final values did not differ within the program 's six - digit resolution over the three repetitions , and convergence was very fast ( typically well before 100 iterations ) .", "the fits show significant deviations from the data curve , due to intrinsic fluctuations in the data .", "we , therefore , considered reliable the estimates of the scaling power and other model parameters .", "even more reliable estimates could , in principle , be obtained by scaling up the network size to 2000 , 5000 , and 10,000 vertices , resources permitting , as computation time and data grow nonlinearly with network size .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], ["the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC2771838", "article_text": ["dysphagia is the subjective awareness of difficulty in the passage of solids or liquids from the oropharynx to the stomach .", "dysphagia can be classified into an oropharyngeal or an esophageal location , and it is caused by neuromuscular motility disorders and mechanical obstruction.1 ) mechanical dysphagia is associated with intrinsic or extrinsic compression , resulting in progressive intolerance to solids . the term , dysphagia aortica , has been used to describe difficulty in swallowing caused by external compression from an ectatic , tortuous , or aneurysmal aorta as a result of age - related degeneration.2 ) dysphagia aortica is classically seen in elderly women with short stature who have hypertension and kyphosis.3 ) we report herein a patient with dysphagia associated with an aortic aneurysm .", "an 86-year - old woman presented with worsening nausea and vomiting . because of her progressive dysphagia to solids for the last 6 months , she had ingested only semisolids and liquids .", "three days before seeking evaluation at our hospital , she had difficulty in swallowing liquids , along with nausea and vomiting .", "the medical history revealed that she had been diagnosed with primary hypertension , an ascending aortic aneurysm , congestive heart failure , moderate aortic regurgitation , and moderate mitral regurgitation 6 years previously . because of old age", "she had undergone vertebroplasty due to multiple compression fractures of the thoracic and lumbar vertebrae 5 years ago . on admission to the hospital", "the blood pressure was 130/90 mmhg , the pulse rate was 64 beats / min , the respiratory rate was 28 breath / min , the body temperature was 36.0 , the height was 1.43 m , and the body weight was 37 kg .", "the physical examination showed a diastolic murmur at the right upper sternal border and a pansystolic ejection murmur at the left lower sternal border .", "the laboratory findings were as follows : the white blood cell ( wbc ) count was 5,100/mm , the hemoglobin was 11.8 g / dl , the platelet count was 151,000/mm , the blood urea nitrogen ( bun ) was 39.8 mg / dl , the creatinine was 1.5 mg / dl , the total protein was 5.8 g / dl , the albumin was 3.2 g / dl , the lactate dehydrogenase ( ldh ) was 546", "her chest radiograph revealed blunting signs at both costophrenic angles , cardiomegaly with a cardio - thoracic ratio of 0.8 , and an enlarged , tortuous aorta ( fig .", "1 ) . computed tomography ( ct ) of the chest demonstrated an enlarged , tortuous aorta ( fig .", "the diameters of the ascending thoracic aorta , the descending thoracic aorta , and the proximal abdominal aorta were 7 cm , 6 cm , and 5.3 cm , respectively .", "the upper gastrointestinal barium study revealed marked extrinsic compression of the distal esophagus just above the esophagogastric junction ( fig .", "we concluded that the symptoms and the results of the imaging studies were consistent with dysphagia aortica .", "although we recommended surgical correction of the aortic aneurysm or percutaneous endoscopic gastrostomy , the patient declined any invasive procedures and she was transferred to a nursing home on the 12 hospital day .", "the esophagus normally begins on the right side of the thoracic aorta and then descends .", "then , the esophagus lies on the left side of the aorta and penetrates the diaphragm through the diaphragmatic histus.4 ) the aging process and the accompanying degenerative changes with the loss of elasticity causes a dilated , elongated , and distorted aorta , which may result in a so - called reverse c- or reverse s - shaped aorta.3 ) as a result , the esophagus is pushed and compressed by the aorta against the cardiac chambers , which are anterior in location.5 ) there is no gold standard diagnostic procedure for dysphagia aortica .", "the association of suggestive symptoms , such as progressive intolerance to solids with concomitant weight loss along with the results of imaging and other diagnostic studies provide a high index of suspicion.6 ) the diagnostic work - up includes radiologic , endoscopic , and manometric studies . on a standard chest radiography and ct scan , the enlargement of the aortic arch and the tortuous dilated aorta can be observed .", "a barium swallow test may show partial esophageal obstruction and pulsatile movement of the barium synchronous with aortic pulsation.7 ) endoscopy reveals pulsatile extrinsic compression and stenosis of the lower esophagus with proximal dilatation .", "esophageal manometry may demonstrate a localized high pressure band with superimposed pounding that is synchronous with the cardiac pulsation.8 ) however , the typical findings of dysphagia aortica can be inconsistent .", "the radiographic findings are often inconclusive because a dilated , tortuous thoracic aorta is frequently encountered in elderly patients with dysphagia .", "although a ct scan is useful for evaluating not only the aortic lumen , but also the aortic wall,9 ) occasionally this is of no value in assessing compression of the esophagus by the aorta.2 ) false negative results are common for barium swallow studies .", "in addition , the classical manometric features suggestive of dysphagia aortica also occur in normal subjects.8 ) the differential diagnosis of dysphagia aortica includes various common structural and neuromuscular abnormalities.1 ) gastroesophageal reflux disease and motility disorders are common causes of dysphagia .", "the co - existence of these conditions and the lack of sensitivity and specificity of the usual diagnostic tests make it difficult to diagnose dysphagia aortica with certainty .", "wilkinson et al.2 ) demonstrated that a video solid bolus swallow test could be useful in determining the manometric findings that are suspicious for dysphagia aortica when the standard evaluation fails .", "mild cases may be treated conservatively , such as avoiding sticky solids and feeding on a liquid diet .", "the surgical procedures include transposition of the distal esophagus , separation of the distal esophagus from the aorta , esophagomyotomy , division of the right crus of the diaphragm , aortic resection , and repair of an aneurysm . for patients who are not candidates for surgery , insertion of a feeding tube via percutaneous endoscopic gastrostomy ( peg )", "although we did not perform esophageal manometry , the patient 's symptoms and imaging studies were consistent with the classic findings of dysphagia aortica .", "although the patient declined any invasive procedures , a peg for a feeding tube might have been helpful for nutritional support .", "dysphagia aortica is an uncommon type of dysphagia that is caused by extrinsic mechanical compression .", "it should be differentiated from other causes of dysphagia , such as gastroesophageal reflux disease or motility disorders , because dysphagia aortica often requires surgical intervention that can significantly reduce the morbidity and these interventions can be curative in some situations ."], "abstract_text": ["<S> dysphagia aortica is difficulty in swallowing caused by extrinsic compression of the esophagus due to an ectatic , tortuous , or aneurysmatic atherosclerotic thoracic aorta . </S>", "<S> this condition is very uncommon , and it is usually associated with old age , women with short stature , hypertension , and kyphosis . </S>", "<S> we report herein a case involving a patient with dysphagia who had an aortic aneurysm . </S>"], "labels": null, "section_names": ["Introduction", "Case", "Discussion"], "sections": [["dysphagia is the subjective awareness of difficulty in the passage of solids or liquids from the oropharynx to the stomach .", "dysphagia can be classified into an oropharyngeal or an esophageal location , and it is caused by neuromuscular motility disorders and mechanical obstruction.1 ) mechanical dysphagia is associated with intrinsic or extrinsic compression , resulting in progressive intolerance to solids . the term , dysphagia aortica , has been used to describe difficulty in swallowing caused by external compression from an ectatic , tortuous , or aneurysmal aorta as a result of age - related degeneration.2 ) dysphagia aortica is classically seen in elderly women with short stature who have hypertension and kyphosis.3 ) we report herein a patient with dysphagia associated with an aortic aneurysm ."], ["an 86-year - old woman presented with worsening nausea and vomiting . because of her progressive dysphagia to solids for the last 6 months , she had ingested only semisolids and liquids .", "three days before seeking evaluation at our hospital , she had difficulty in swallowing liquids , along with nausea and vomiting .", "the medical history revealed that she had been diagnosed with primary hypertension , an ascending aortic aneurysm , congestive heart failure , moderate aortic regurgitation , and moderate mitral regurgitation 6 years previously . because of old age", "she had undergone vertebroplasty due to multiple compression fractures of the thoracic and lumbar vertebrae 5 years ago . on admission to the hospital", "the blood pressure was 130/90 mmhg , the pulse rate was 64 beats / min , the respiratory rate was 28 breath / min , the body temperature was 36.0 , the height was 1.43 m , and the body weight was 37 kg .", "the physical examination showed a diastolic murmur at the right upper sternal border and a pansystolic ejection murmur at the left lower sternal border .", "the laboratory findings were as follows : the white blood cell ( wbc ) count was 5,100/mm , the hemoglobin was 11.8 g / dl , the platelet count was 151,000/mm , the blood urea nitrogen ( bun ) was 39.8 mg / dl , the creatinine was 1.5 mg / dl , the total protein was 5.8 g / dl , the albumin was 3.2 g / dl , the lactate dehydrogenase ( ldh ) was 546", "her chest radiograph revealed blunting signs at both costophrenic angles , cardiomegaly with a cardio - thoracic ratio of 0.8 , and an enlarged , tortuous aorta ( fig .", "1 ) . computed tomography ( ct ) of the chest demonstrated an enlarged , tortuous aorta ( fig .", "the diameters of the ascending thoracic aorta , the descending thoracic aorta , and the proximal abdominal aorta were 7 cm , 6 cm , and 5.3 cm , respectively .", "the upper gastrointestinal barium study revealed marked extrinsic compression of the distal esophagus just above the esophagogastric junction ( fig .", "we concluded that the symptoms and the results of the imaging studies were consistent with dysphagia aortica .", "although we recommended surgical correction of the aortic aneurysm or percutaneous endoscopic gastrostomy , the patient declined any invasive procedures and she was transferred to a nursing home on the 12 hospital day ."], ["the esophagus normally begins on the right side of the thoracic aorta and then descends .", "then , the esophagus lies on the left side of the aorta and penetrates the diaphragm through the diaphragmatic histus.4 ) the aging process and the accompanying degenerative changes with the loss of elasticity causes a dilated , elongated , and distorted aorta , which may result in a so - called reverse c- or reverse s - shaped aorta.3 ) as a result , the esophagus is pushed and compressed by the aorta against the cardiac chambers , which are anterior in location.5 ) there is no gold standard diagnostic procedure for dysphagia aortica .", "the association of suggestive symptoms , such as progressive intolerance to solids with concomitant weight loss along with the results of imaging and other diagnostic studies provide a high index of suspicion.6 ) the diagnostic work - up includes radiologic , endoscopic , and manometric studies . on a standard chest radiography and ct scan , the enlargement of the aortic arch and the tortuous dilated aorta can be observed .", "a barium swallow test may show partial esophageal obstruction and pulsatile movement of the barium synchronous with aortic pulsation.7 ) endoscopy reveals pulsatile extrinsic compression and stenosis of the lower esophagus with proximal dilatation .", "esophageal manometry may demonstrate a localized high pressure band with superimposed pounding that is synchronous with the cardiac pulsation.8 ) however , the typical findings of dysphagia aortica can be inconsistent .", "the radiographic findings are often inconclusive because a dilated , tortuous thoracic aorta is frequently encountered in elderly patients with dysphagia .", "although a ct scan is useful for evaluating not only the aortic lumen , but also the aortic wall,9 ) occasionally this is of no value in assessing compression of the esophagus by the aorta.2 ) false negative results are common for barium swallow studies .", "in addition , the classical manometric features suggestive of dysphagia aortica also occur in normal subjects.8 ) the differential diagnosis of dysphagia aortica includes various common structural and neuromuscular abnormalities.1 ) gastroesophageal reflux disease and motility disorders are common causes of dysphagia .", "the co - existence of these conditions and the lack of sensitivity and specificity of the usual diagnostic tests make it difficult to diagnose dysphagia aortica with certainty .", "wilkinson et al.2 ) demonstrated that a video solid bolus swallow test could be useful in determining the manometric findings that are suspicious for dysphagia aortica when the standard evaluation fails .", "mild cases may be treated conservatively , such as avoiding sticky solids and feeding on a liquid diet .", "the surgical procedures include transposition of the distal esophagus , separation of the distal esophagus from the aorta , esophagomyotomy , division of the right crus of the diaphragm , aortic resection , and repair of an aneurysm . for patients who are not candidates for surgery , insertion of a feeding tube via percutaneous endoscopic gastrostomy ( peg )", "although we did not perform esophageal manometry , the patient 's symptoms and imaging studies were consistent with the classic findings of dysphagia aortica .", "although the patient declined any invasive procedures , a peg for a feeding tube might have been helpful for nutritional support .", "dysphagia aortica is an uncommon type of dysphagia that is caused by extrinsic mechanical compression .", "it should be differentiated from other causes of dysphagia , such as gastroesophageal reflux disease or motility disorders , because dysphagia aortica often requires surgical intervention that can significantly reduce the morbidity and these interventions can be curative in some situations ."]]}
{"article_id": "PMC4854663", "article_text": ["from now on , molecular study regarding female sexuality has focused on female sexual hormone , like estrogen receptor ( er ) and er derivatives.12 but flibanserin is an agent of 5-hydroxytryptamin ( 5-ht ) type 1a receptor and an antagonist of 5-ht type 2a and thus a new molecular entity.3 flibanserin was originally developed as antidepressant drug . in clinical stage 2a as antidepressant , flibanserin could not prove its effectiveness , but almost no sexual function disorder was reported in subjects . for this reason , the arizona sexual experiences scale ( asex ) was used for making a comparison of the sexual function effectiveness of flibanserin on antidepressant vs. placebo proven in stage 2b studies among four depression programs.4 stage 2b studies have failed to prove its general effectiveness on depression , but in answering this question \" how intense is your sexual desire ? \" in the asex , flibanserin turned out to be more excellent in both placebo and active - comparator .", "since then , drug development has changed its directivity toward being a potential medicine for treating hypoactive sexual desire disorder ( hsdd ) .", "hsdd is chara or absence of sexual fantasy and sexual desire associated with personal pain in the 4th edition of the diagnostic and statistical manual - text revision ( dsm - iv - tr).5 determining lack or absence is done by clinicians by considering the factors influencing sexual activitie such as age and contexts of personal life .", "sexual function disorder is not explained well by other axis 1 disorders ( except other sexual function disorders ) and also it is not that it is caused only by direct physiological effects of materials ( i.e. , drug overuse , drug ) or overall medical status .", "the dsm-5 completed in 2013 addresses new illness called as female sexual interest / arousal disorder ( fsiad ) , which has both properties of hsdd and another illness of dsm - iv known as female sexual arousal disorder .", "flibanserin underwent new drug application ( nda ) as original drug in 2009 . the two central phase 3 studies ( violet6 , daisy7 ) all used e - diary and did not show big statistical improvement compared to placebo in the pre - specified co - primary efficacy endpoint that assessed daily sexual desire . in both violet6 and daisy7 studies , flibanserin showed an increase in satisfying sexual events ( sse ) and female sexual function index ( fsfi ) and reduction in female sexual distress scale - revised ( fsds - r ) , but did not show a meaningful increase in e - diary scores compared to placebo . in both studies ,", "the most commonly reported adverse effects in women prescribed with flibanserin include drowsiness ( 11.8% ) , vertigo ( 10.5% ) , and fatigue ( 10.3%).67 these studies show a statistically significant improvement in secondary end points that measured sexual desire with other tools known as fsfi compared to placebo .", "applicants told that the effectiveness of flibanserin against sexual desire is better explained with fsfi , but most advisory committees did not agree to alteration in the e - diary evaluation method set as the one designed to evaluate sexual desire .", "the safety consciousness expressed by the advisory committees includes adverse effects such as fatigue and drowsiness as well as drug - drug interactions ( ddis ) and flibanserinalcohol interactions .", "another central stage 3 study is begonia,8 which set sse , fsfi , fsds - r as major evaluation methods rather than using e - diary in the existing viole and daisy studies and reported that flibanserin shows a statistically significant increase in sse and fsfi and reduction in fsds - r compared to placebo .", "however , many problems with safety were brought up in the second submission , too .", "vertigo , drowsiness , and vomiting are the most commonly reported adverse effects , and these occurred 2% to 3% , respectively if using placebo during stage 3 placebo - control premenopausal hsdd , whereas occurred close to 11% in subjects who used flibanserin as 100 mg every night at bedtime .", "events matching central nervous system depression ( fatigue , drowsiness , and vertigo ) occurred close to 21% in subjects who used flibanserin as 100 mg every night at bedtime and this proportion is three times higher than in placebo group .", "flibanserin is very difficult to endure if taking in combination with fluconazole and strong cytochrome p450 3a4 ( cyp3a4 ) suppressant and this combination may increase the risk of swoon and symptomatic low blood pressure . and", "ethanol administered in combination with flibanserin greatly increases the risk of drowsiness , fatigue , orthostatic hypotension , and swoon .", "additionally , these studies were limited to general healthy women who did not take benzodiazepines , sleep aid , narcotics and many other drugs .", "the effectiveness was not so big in general , and considering these concerns , fda refused to approve it again and recommended that additional safety studies are required . according to the latest fda review , there is no data indicating new efficacy . instead , flibanserin sponsors submitted additional safety data next day including research data proving that there is no obstacle to driving , data comparing the commercialized products and the side effects profile of these products , and analysis clarifying the potential effects of alcohol on side effects.9 despite the trustfulness of highly influential studies , fda product review is not a fundamental comparison , in fact , and so comparison of safety between individual products may be misunderstood . in particular ,", "alcohol interaction study was conducted in 25 healthy volunteers as a sample , among which only two were female .", "since the refusal from fda in 2013 , a civic group called as even the score was formed to support what was called as \" gender equality \" in the manner approaching sexual function disorder treatment.10 this group insisted that there is no treatment for women although even 26 items for male sexual function disorder were already approved .", "this argument was rejected by fda on the ground that there are no approved products for low sexual desire in men and the 26 items for treatment contain various mixtures of testosterone . despite the fact that flibanserin is supported by consumer advocacy groups and consequently not the first product supported by pharmaceutical companies , fda 's argument on gender bias while regulating is worthy of notice in that the range of making efforts against this argument is from campaigns via social media even to letters from the members of the national assembly.11 another noteworthy feature in application for flibanserin to fda is use of the findings on sexual desire reported by patients as primary efficacy variable for approval .", "sexual desire can be seen by those who experience it and the results reported by patients can be measured without confounding this concept from others .", "the results reported by patients have become more important in studies and other drugs that have such results as primary end point have been approved ( although none were based on sexual desire ) . among all new molecular entities and biological permit applications approved by fda from 2006 to 2010 ,", "gnanasakthy et al.12 found that among which , 17% ( 20 in 116 ) used the results reported by acknowledged patients as primary outcome in approval labels .", "the complex element in evaluating flibanserin lies in recalling the frequency of desire and extent of intensity and then measuring with fsfi index four weeks after the daily records of the most intense desires when applying for the primary desire end point first time .", "fda raised some concerns about optimizing fsfi as desire assessment tool , including the validity of contents and the period of recall , but the recent advisory committees did not focus on changing the primary end points or consulting the validity of fsfi .", "the final concern with flibanserin is associated with its use in clinical settings and this was an important issue for the committees . despite little reliable estimate of hsdd prevalence", ", this product will be used obviously in women in a broader sense than had been studied so far like others , many will not satisfy the official diagnostic criteria of hsdd , and many will increase the risk of adverse effects or be administering other drugs .", "the concerns with its use without labeling were reemphasized again by speakers who argued that they need flibanserin , but at the same time , it was reported that those who were diagnosed with cancer or in menopause should be excluded from treatment with flibanserin by labeling when sold .", "on june 4 , 2015 , fda called a committee meeting to review the efficacy and safety of flibanserin , and after the meeting , the committee approved flibanserin under the condition that risk management tools should be enforced mandatorily . despite the presence of many problems with flibanserin ,", "fda finally approved it under the condition that risk evaluation and mitigation strategies ( rems ) should be enforced for flibanserin . in the future", ", flibanserin is expected to gradually increase in amount with changes from off - label to on - label .", "if prescription with flibanserin is determined as necessary , before using it , it is important to check prescription criterion , possible adverse effects after prescription , and precautions during administration . to get a prescription with flibanserin ,", "the first criteria is premenopausal women aged 18 year or more who met the stages of reproductive aging workshop ( straw ) criteria .", "the straw criteria are that she has a regular menstrual cycle for 21 to 35 days in the previous twelve months and a normal level of follicle stimulating hormone ( fsh ) .", "in addition , it should be prescribed if primary diagnosis is hsdd according to dsm - iv - tr criteria or if there is any secondary sexual arousal disorder or female disposition disorder accompanied by hsdd . besides , its prescription is likely to be considered if the fsds - r ( range , 0 - 52 ) score13 used in clinical studies is 15 points or higher . in addition , in case that sexual desire disorder comes from disease , psychiatric problems , and interpersonal problems or in case that it does so from medications or other substances taken in conjunction , these cases are not the diseases for which medicine is efficacious . besides ,", "postmenopausal women and sexual desire decline disorder male patients are not adapted during use and i t is not intended for reinforcing sexuality.1415", "the adverse effects most common in prescribing flibanserin are reported as vertigo , fatigue , vomiting , insomnia , and mouth dryness .", "precautions in prescription include avoiding the use of other substances associated with cyp3a4 and cytochrome p450 2c19 ( cyp2c19 ) which are associated with drug metabolism , for example , administration with ketoconazole as cyp3a4 inhibitor and grapefruit juice and fluconazole as cyp3a4 or cyp2c19 inhibitor , which are likely to increase the risk of orthostatic hypotension side effects .", "furthermore , selective serotonin reuptake inhibitors and selective norepinephrine reuptake inhibitors ( ssri / snri ) may cause anxiety , drowsiness , fatigue , insomnia , and vertigo , if used . so carefulness is required .", "alcohol is contraindication because if administered with alcohol , it is highly likely to cause central nervous system depression ( fatigue , drowsiness , and vertigo ) , low blood pressure , and swoon .", "hormonal contraceptives are known as weak cyp3a4 inhibitors , and likely to cause side effects such as vertigo , drowsiness , and fatigue if coadministered with flibanserin .", "triptans is used for treating migraine as agonist of 5-ht type 1b and 1d receptor and may cause side effects of drowsiness if administered with flibanserin .", "in addition , if flibanserin is administered to patients with decline in liver failure , increase in concentration of drug in blood is observed and thus it 's considered that adverse effects are likely to occur . besides , taking it with herb extracts and digesters and gastroprotective drugs requiring no doctor prescription is likely to cause the risk of adverse effects and the use of flibanserin to women who are breast - feeding is contraindication .", "as we discussed above , flibanserin was refused to approve by fda in 2009 for its adverse effects and discordance of its efficacy from different evaluation methods .", "most common side effects were central nervous system depression , like fatigue , drowsiness , and vertigo .", "ddis with fluconazole , ethanol , and many other drugs were mainly discussed and fda refused to approve it again recommending additional safety data . with the additional profile and analysis which can prove their safety compared with other products ,", "on june 4 , 2015 , fda finally approved it on the grounds that rems should be enforced , and soon we can get flibanserin on - label under the strict prescription .", "flibanserin can be prescribed to premenopausal women aged 18 years or higher who has a regular menstrual cycle , but if liver function fails , if breast - feeding , and if drinking alcohol , the use of flibanserin is contraindication .", "also , its administration with fluconazole , ketoconazole , ssri / snri , triptans , hormonal contraceptives , herb extracts , and several other drugs requiring no doctor prescription is prohibited because it is likely to increase the risk of adverse effects .", "we are waiting for patients ' reaction , considering the cost and effectiveness , in countries where flibanserin will be on sale , and more agents like unicenta ( uncnt ) and melsmon , which proved as having a efficacy of menopausal symptoms , have to be studied as treating for female sexual dysfunction.16"], "abstract_text": ["<S> there have been several products developed for male sexual dysfunction . </S>", "<S> however , developing agents for female sexual dysfunction is lagging behind for various reasons . </S>", "<S> sildenafil citrate ( viagra ) and tadalafil ( cialis ) , which have been prescribed for male sexual function disorders , are known to act on vessels.[1 ] on the other hand , flibanserin is thought to act on brain . </S>", "<S> flibanserin has been approved by u. s. food and drug administration ( fda ) for treatment of hypoactive sexual desire disorder ( hsdd ) of premenopausal women in 2015 , and is expected to be released in south korea soon . authors wrote this article to acknowledge flibanserin to sexologists for females or physicians for menopausal medicine , so that this agent can be safely used for females who have hsdd . </S>"], "labels": null, "section_names": ["Introduction of Flibanserin", "Diagnostic Criteria of HSDD", "Clinical Studies of Flibanserin", "Precautions in Prescription of Flibanserin", "Conclusion"], "sections": [["from now on , molecular study regarding female sexuality has focused on female sexual hormone , like estrogen receptor ( er ) and er derivatives.12 but flibanserin is an agent of 5-hydroxytryptamin ( 5-ht ) type 1a receptor and an antagonist of 5-ht type 2a and thus a new molecular entity.3 flibanserin was originally developed as antidepressant drug . in clinical stage 2a as antidepressant , flibanserin could not prove its effectiveness , but almost no sexual function disorder was reported in subjects . for this reason , the arizona sexual experiences scale ( asex ) was used for making a comparison of the sexual function effectiveness of flibanserin on antidepressant vs. placebo proven in stage 2b studies among four depression programs.4 stage 2b studies have failed to prove its general effectiveness on depression , but in answering this question \" how intense is your sexual desire ? \" in the asex , flibanserin turned out to be more excellent in both placebo and active - comparator .", "since then , drug development has changed its directivity toward being a potential medicine for treating hypoactive sexual desire disorder ( hsdd ) ."], ["hsdd is chara or absence of sexual fantasy and sexual desire associated with personal pain in the 4th edition of the diagnostic and statistical manual - text revision ( dsm - iv - tr).5 determining lack or absence is done by clinicians by considering the factors influencing sexual activitie such as age and contexts of personal life .", "sexual function disorder is not explained well by other axis 1 disorders ( except other sexual function disorders ) and also it is not that it is caused only by direct physiological effects of materials ( i.e. , drug overuse , drug ) or overall medical status .", "the dsm-5 completed in 2013 addresses new illness called as female sexual interest / arousal disorder ( fsiad ) , which has both properties of hsdd and another illness of dsm - iv known as female sexual arousal disorder ."], ["flibanserin underwent new drug application ( nda ) as original drug in 2009 . the two central phase 3 studies ( violet6 , daisy7 ) all used e - diary and did not show big statistical improvement compared to placebo in the pre - specified co - primary efficacy endpoint that assessed daily sexual desire . in both violet6 and daisy7 studies , flibanserin showed an increase in satisfying sexual events ( sse ) and female sexual function index ( fsfi ) and reduction in female sexual distress scale - revised ( fsds - r ) , but did not show a meaningful increase in e - diary scores compared to placebo . in both studies ,", "the most commonly reported adverse effects in women prescribed with flibanserin include drowsiness ( 11.8% ) , vertigo ( 10.5% ) , and fatigue ( 10.3%).67 these studies show a statistically significant improvement in secondary end points that measured sexual desire with other tools known as fsfi compared to placebo .", "applicants told that the effectiveness of flibanserin against sexual desire is better explained with fsfi , but most advisory committees did not agree to alteration in the e - diary evaluation method set as the one designed to evaluate sexual desire .", "the safety consciousness expressed by the advisory committees includes adverse effects such as fatigue and drowsiness as well as drug - drug interactions ( ddis ) and flibanserinalcohol interactions .", "another central stage 3 study is begonia,8 which set sse , fsfi , fsds - r as major evaluation methods rather than using e - diary in the existing viole and daisy studies and reported that flibanserin shows a statistically significant increase in sse and fsfi and reduction in fsds - r compared to placebo .", "however , many problems with safety were brought up in the second submission , too .", "vertigo , drowsiness , and vomiting are the most commonly reported adverse effects , and these occurred 2% to 3% , respectively if using placebo during stage 3 placebo - control premenopausal hsdd , whereas occurred close to 11% in subjects who used flibanserin as 100 mg every night at bedtime .", "events matching central nervous system depression ( fatigue , drowsiness , and vertigo ) occurred close to 21% in subjects who used flibanserin as 100 mg every night at bedtime and this proportion is three times higher than in placebo group .", "flibanserin is very difficult to endure if taking in combination with fluconazole and strong cytochrome p450 3a4 ( cyp3a4 ) suppressant and this combination may increase the risk of swoon and symptomatic low blood pressure . and", "ethanol administered in combination with flibanserin greatly increases the risk of drowsiness , fatigue , orthostatic hypotension , and swoon .", "additionally , these studies were limited to general healthy women who did not take benzodiazepines , sleep aid , narcotics and many other drugs .", "the effectiveness was not so big in general , and considering these concerns , fda refused to approve it again and recommended that additional safety studies are required . according to the latest fda review , there is no data indicating new efficacy . instead , flibanserin sponsors submitted additional safety data next day including research data proving that there is no obstacle to driving , data comparing the commercialized products and the side effects profile of these products , and analysis clarifying the potential effects of alcohol on side effects.9 despite the trustfulness of highly influential studies , fda product review is not a fundamental comparison , in fact , and so comparison of safety between individual products may be misunderstood . in particular ,", "alcohol interaction study was conducted in 25 healthy volunteers as a sample , among which only two were female .", "since the refusal from fda in 2013 , a civic group called as even the score was formed to support what was called as \" gender equality \" in the manner approaching sexual function disorder treatment.10 this group insisted that there is no treatment for women although even 26 items for male sexual function disorder were already approved .", "this argument was rejected by fda on the ground that there are no approved products for low sexual desire in men and the 26 items for treatment contain various mixtures of testosterone . despite the fact that flibanserin is supported by consumer advocacy groups and consequently not the first product supported by pharmaceutical companies , fda 's argument on gender bias while regulating is worthy of notice in that the range of making efforts against this argument is from campaigns via social media even to letters from the members of the national assembly.11 another noteworthy feature in application for flibanserin to fda is use of the findings on sexual desire reported by patients as primary efficacy variable for approval .", "sexual desire can be seen by those who experience it and the results reported by patients can be measured without confounding this concept from others .", "the results reported by patients have become more important in studies and other drugs that have such results as primary end point have been approved ( although none were based on sexual desire ) . among all new molecular entities and biological permit applications approved by fda from 2006 to 2010 ,", "gnanasakthy et al.12 found that among which , 17% ( 20 in 116 ) used the results reported by acknowledged patients as primary outcome in approval labels .", "the complex element in evaluating flibanserin lies in recalling the frequency of desire and extent of intensity and then measuring with fsfi index four weeks after the daily records of the most intense desires when applying for the primary desire end point first time .", "fda raised some concerns about optimizing fsfi as desire assessment tool , including the validity of contents and the period of recall , but the recent advisory committees did not focus on changing the primary end points or consulting the validity of fsfi .", "the final concern with flibanserin is associated with its use in clinical settings and this was an important issue for the committees . despite little reliable estimate of hsdd prevalence", ", this product will be used obviously in women in a broader sense than had been studied so far like others , many will not satisfy the official diagnostic criteria of hsdd , and many will increase the risk of adverse effects or be administering other drugs .", "the concerns with its use without labeling were reemphasized again by speakers who argued that they need flibanserin , but at the same time , it was reported that those who were diagnosed with cancer or in menopause should be excluded from treatment with flibanserin by labeling when sold .", "on june 4 , 2015 , fda called a committee meeting to review the efficacy and safety of flibanserin , and after the meeting , the committee approved flibanserin under the condition that risk management tools should be enforced mandatorily . despite the presence of many problems with flibanserin ,", "fda finally approved it under the condition that risk evaluation and mitigation strategies ( rems ) should be enforced for flibanserin . in the future", ", flibanserin is expected to gradually increase in amount with changes from off - label to on - label .", "if prescription with flibanserin is determined as necessary , before using it , it is important to check prescription criterion , possible adverse effects after prescription , and precautions during administration . to get a prescription with flibanserin ,", "the first criteria is premenopausal women aged 18 year or more who met the stages of reproductive aging workshop ( straw ) criteria .", "the straw criteria are that she has a regular menstrual cycle for 21 to 35 days in the previous twelve months and a normal level of follicle stimulating hormone ( fsh ) .", "in addition , it should be prescribed if primary diagnosis is hsdd according to dsm - iv - tr criteria or if there is any secondary sexual arousal disorder or female disposition disorder accompanied by hsdd . besides , its prescription is likely to be considered if the fsds - r ( range , 0 - 52 ) score13 used in clinical studies is 15 points or higher . in addition , in case that sexual desire disorder comes from disease , psychiatric problems , and interpersonal problems or in case that it does so from medications or other substances taken in conjunction , these cases are not the diseases for which medicine is efficacious . besides ,", "postmenopausal women and sexual desire decline disorder male patients are not adapted during use and i t is not intended for reinforcing sexuality.1415"], ["the adverse effects most common in prescribing flibanserin are reported as vertigo , fatigue , vomiting , insomnia , and mouth dryness .", "precautions in prescription include avoiding the use of other substances associated with cyp3a4 and cytochrome p450 2c19 ( cyp2c19 ) which are associated with drug metabolism , for example , administration with ketoconazole as cyp3a4 inhibitor and grapefruit juice and fluconazole as cyp3a4 or cyp2c19 inhibitor , which are likely to increase the risk of orthostatic hypotension side effects .", "furthermore , selective serotonin reuptake inhibitors and selective norepinephrine reuptake inhibitors ( ssri / snri ) may cause anxiety , drowsiness , fatigue , insomnia , and vertigo , if used . so carefulness is required .", "alcohol is contraindication because if administered with alcohol , it is highly likely to cause central nervous system depression ( fatigue , drowsiness , and vertigo ) , low blood pressure , and swoon .", "hormonal contraceptives are known as weak cyp3a4 inhibitors , and likely to cause side effects such as vertigo , drowsiness , and fatigue if coadministered with flibanserin .", "triptans is used for treating migraine as agonist of 5-ht type 1b and 1d receptor and may cause side effects of drowsiness if administered with flibanserin .", "in addition , if flibanserin is administered to patients with decline in liver failure , increase in concentration of drug in blood is observed and thus it 's considered that adverse effects are likely to occur . besides , taking it with herb extracts and digesters and gastroprotective drugs requiring no doctor prescription is likely to cause the risk of adverse effects and the use of flibanserin to women who are breast - feeding is contraindication ."], ["as we discussed above , flibanserin was refused to approve by fda in 2009 for its adverse effects and discordance of its efficacy from different evaluation methods .", "most common side effects were central nervous system depression , like fatigue , drowsiness , and vertigo .", "ddis with fluconazole , ethanol , and many other drugs were mainly discussed and fda refused to approve it again recommending additional safety data . with the additional profile and analysis which can prove their safety compared with other products ,", "on june 4 , 2015 , fda finally approved it on the grounds that rems should be enforced , and soon we can get flibanserin on - label under the strict prescription .", "flibanserin can be prescribed to premenopausal women aged 18 years or higher who has a regular menstrual cycle , but if liver function fails , if breast - feeding , and if drinking alcohol , the use of flibanserin is contraindication .", "also , its administration with fluconazole , ketoconazole , ssri / snri , triptans , hormonal contraceptives , herb extracts , and several other drugs requiring no doctor prescription is prohibited because it is likely to increase the risk of adverse effects .", "we are waiting for patients ' reaction , considering the cost and effectiveness , in countries where flibanserin will be on sale , and more agents like unicenta ( uncnt ) and melsmon , which proved as having a efficacy of menopausal symptoms , have to be studied as treating for female sexual dysfunction.16"]]}
{"article_id": "PMC3555381", "article_text": ["kcm is itself very rare and distinct type of keratoacanthoma which usually occurs over extremities and scalp is unusual site for development of lesion .", "treatment is difficult as lesion of kcm present with large raised , rolled borders with peripheral extension .", "keratoacanthoma ( ka ) is a rapidly evolving tumor , composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated .", "it is relatively common , especially in whites occurring in middle age while being uncommon in dark - skinned .", ". it presents as firm , rounded , flesh - colored or reddish papule ; with a rapid growth phase becoming 10 - 20 mm and then spontaneous healing taking place over three months .", "there are three rare clinical variants of solitary ka , namely giant ka , keratoacanthoma centrifugum marginatum ( kcm ) and subungual ka . in kcm , lesions are large , reaching upto 20 cms .", "the most common locations are dorsa of hands and legs , lesions on scalp being rare .", "we report a rare case of kcm occurring on the scalp which is an unusual site .", "a 62-year - old male , watchman by occupation presented with asymptomatic raised lesion on scalp since one year .", "lesion had developed de novo , as a pea sized lesion with gradual increase to cover entire vertex .", "cutaneous examination revealed a single , irregular , 12  15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater [ figure 1 ] . on palpation , the plaque was firm , non - tender , non - indurated and not attached to underlying structure .", "ultrasonography of lesion showed ill defined hypo echoic mass involving epidermis , dermis and subcutaneous tissue .", "a single , irregular , 12  15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater .", "deep punch biopsy revealed exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base .", ". marked epidermal proliferation with crater formation with horn pearls and inflammatory infiltrate at the base of the lesion was suggestive of a fully developed lesion of kcm .", "complete surgical excision with grafting was done successfully with dramatic improvement with no recurrence at 9 months [ figure 5 ] .", "exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base epidermis forming buttress over the pseudhorn cyst dense nodular lymphocytic infiltrate in the upper dermis surgical excision with grafting", "the etiology is multifactorial that includes chronic ultraviolet ray exposure , smoking , contact with chemical carcinogens like pitch , mineral oil , tar , trauma and vaccination .", "the role of human papilloma virus remains inconclusive but in one study , hpv type 6 and 11 were detected within the lesion .", "it can be localized to any region of the body but is more frequently seen on dorsum of hands and legs .", "progressive peripheral extension with a raised rolled - out margin and atrophy at the centre is a characteristic feature of kcm .", "the margin of the lesion showing multiple comedonal orifices giving rise to a cribriform pattern may represent a unique phenomenon of kcm .", "it is hypothesized that this typical appearance may arise as a result of sequential involvement of multiple adjacent hair follicles in a centrifugal fashion .", "kcm does not show tendency for spontaneous regression , a feature also seen in giant ka , which grows rapidly reaching a size of 5 cm or more and occurring commonly on nose and eyelids .", "kcm is differentiated from giant ka by absence of downward vertical spread and destruction of underlying tissue .", "nevertheless , in giant ka spontaneous involution takes place after several months , often accompanied by detachment of a large keratotic plaque .", "the other differential diagnoses include squamous cell carcinoma , lupus vulgaris , botryomycosis , blastomycosis - like pyoderma and pseudoepitheliomatous hyperplasia , hypertrophic lupus erythematosus , atypical mycobacterial infections , or deep fungal infections .", "there are reports that kcm has been treated successfully with oral retinoids ( acitretin , etretinate , or isotretinoin 0.5 - 1 mg / kg / day ) which should be given until complete clearance of lesion .", "surgical intervention is a preferred mode of therapy keeping in mind that a wide excision is performed to prevent subsequent recurrence .", "other treatment modalities successfully used include topical 5-fluorouracil , intralesional injections of interferon alpha , methotrexate , or bleomycin , or mohs micrographic surgery . in kcm , lesions are usually too large for excisional biopsy for diagnosis , but certain characteristic features like clinical behavior , appearance of lesions , histological features ( hyperkeratosis and hyperplasia ) , and cytological features ( tumor cells with eosinophilic and glassy cytoplasm , few mitoses ) help clinch the correct diagnosis .", "first time from india we are reporting case of kcm presenting over scalp which is successfully treated with surgical excision without any recurrence ."], "abstract_text": ["<S> keratoacanthoma ( ka ) is a rapidly evolving tumor , composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated . </S>", "<S> it is relatively uncommon in dark - skinned and occurs in middle aged individuals . </S>", "<S> males are three times more affected than females . </S>", "<S> it presents as firm , rounded , flesh - colored or reddish papule ; with a rapid growth phase followed by spontaneous healing over three months . </S>", "<S> two types of ka exist i.e. , solitary and multiple . </S>", "<S> there are three rare clinical variants of solitary ka , namely giant ka , keratoacanthoma centrifugum marginatum ( kcm ) and subungual ka . in kcm , lesions are large , reaching upto 20cms . </S>", "<S> there is peripheral extension with raised , rolled border and atrophy in the center . </S>", "<S> there is no tendency toward spontaneous involution . </S>", "<S> the most common locations are dorsa of hands and legs , lesions on scalp being rare . </S>", "<S> a rare case of kcm occurring on scalp which is an unusual site is reported . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["kcm is itself very rare and distinct type of keratoacanthoma which usually occurs over extremities and scalp is unusual site for development of lesion .", "treatment is difficult as lesion of kcm present with large raised , rolled borders with peripheral extension .", "keratoacanthoma ( ka ) is a rapidly evolving tumor , composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated .", "it is relatively common , especially in whites occurring in middle age while being uncommon in dark - skinned .", ". it presents as firm , rounded , flesh - colored or reddish papule ; with a rapid growth phase becoming 10 - 20 mm and then spontaneous healing taking place over three months .", "there are three rare clinical variants of solitary ka , namely giant ka , keratoacanthoma centrifugum marginatum ( kcm ) and subungual ka . in kcm , lesions are large , reaching upto 20 cms .", "the most common locations are dorsa of hands and legs , lesions on scalp being rare .", "we report a rare case of kcm occurring on the scalp which is an unusual site ."], ["a 62-year - old male , watchman by occupation presented with asymptomatic raised lesion on scalp since one year .", "lesion had developed de novo , as a pea sized lesion with gradual increase to cover entire vertex .", "cutaneous examination revealed a single , irregular , 12  15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater [ figure 1 ] . on palpation , the plaque was firm , non - tender , non - indurated and not attached to underlying structure .", "ultrasonography of lesion showed ill defined hypo echoic mass involving epidermis , dermis and subcutaneous tissue .", "a single , irregular , 12  15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater .", "deep punch biopsy revealed exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base .", ". marked epidermal proliferation with crater formation with horn pearls and inflammatory infiltrate at the base of the lesion was suggestive of a fully developed lesion of kcm .", "complete surgical excision with grafting was done successfully with dramatic improvement with no recurrence at 9 months [ figure 5 ] .", "exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base epidermis forming buttress over the pseudhorn cyst dense nodular lymphocytic infiltrate in the upper dermis surgical excision with grafting"], ["the etiology is multifactorial that includes chronic ultraviolet ray exposure , smoking , contact with chemical carcinogens like pitch , mineral oil , tar , trauma and vaccination .", "the role of human papilloma virus remains inconclusive but in one study , hpv type 6 and 11 were detected within the lesion .", "it can be localized to any region of the body but is more frequently seen on dorsum of hands and legs .", "progressive peripheral extension with a raised rolled - out margin and atrophy at the centre is a characteristic feature of kcm .", "the margin of the lesion showing multiple comedonal orifices giving rise to a cribriform pattern may represent a unique phenomenon of kcm .", "it is hypothesized that this typical appearance may arise as a result of sequential involvement of multiple adjacent hair follicles in a centrifugal fashion .", "kcm does not show tendency for spontaneous regression , a feature also seen in giant ka , which grows rapidly reaching a size of 5 cm or more and occurring commonly on nose and eyelids .", "kcm is differentiated from giant ka by absence of downward vertical spread and destruction of underlying tissue .", "nevertheless , in giant ka spontaneous involution takes place after several months , often accompanied by detachment of a large keratotic plaque .", "the other differential diagnoses include squamous cell carcinoma , lupus vulgaris , botryomycosis , blastomycosis - like pyoderma and pseudoepitheliomatous hyperplasia , hypertrophic lupus erythematosus , atypical mycobacterial infections , or deep fungal infections .", "there are reports that kcm has been treated successfully with oral retinoids ( acitretin , etretinate , or isotretinoin 0.5 - 1 mg / kg / day ) which should be given until complete clearance of lesion .", "surgical intervention is a preferred mode of therapy keeping in mind that a wide excision is performed to prevent subsequent recurrence .", "other treatment modalities successfully used include topical 5-fluorouracil , intralesional injections of interferon alpha , methotrexate , or bleomycin , or mohs micrographic surgery . in kcm , lesions are usually too large for excisional biopsy for diagnosis , but certain characteristic features like clinical behavior , appearance of lesions , histological features ( hyperkeratosis and hyperplasia ) , and cytological features ( tumor cells with eosinophilic and glassy cytoplasm , few mitoses ) help clinch the correct diagnosis .", "first time from india we are reporting case of kcm presenting over scalp which is successfully treated with surgical excision without any recurrence ."]]}
{"article_id": "PMC4508471", "article_text": ["hydroxyapatite biomaterials are materials that are very widely used in several health purposes , including as a source of calcium for the manufacture of toothpaste and as an important material in the formation / bone repair .", "the chemical properties of hydroxyapatite are bioactive and compatible with the adjacent bone and teeth .", "hydroxyapatite is a calcium phosphate ceramic that is totally biocompatible and nontoxic and becomes an integral part of living bone and teeth tissue [ 18 ] .", "the raw material for the production of hydroxyapatite biomaterial is very easily available and abundant in indonesia .", "the production process was easy and the cost is also relatively inexpensive if done on a large scale . among", "the abundant raw materials are the shells of crabs , which are one of indonesia 's main export commodities .", "export of crabs commodity by indonesia amounted to 604.215625.000 tons / year without the shell form , while domestic consumption is expected to be so much more , as in makassar , carrying 292.5 tons exported in the form of crab without shell with the main export destination being singapore .", "if the mass of crab shells 2550% of the total mass , it can be estimated that in 2012 produced the shells of crabs around 151,053.75 to 302,107.5 tons in indonesia and 73.125 to 146.25 tons in makassar ( a region of indonesia ) , there was just from crabs production were exported .", "this value is of course even more if the crab 's consumptions in the country are also taken into account .", "this suggests the existence of crab shells is abundant in indonesia , including in makassar .", "as known in indonesia , the shells of crabs have not been used , so it will only be a waste disturbing environment [ 7 , 8 ] .", "crabs shells containing calcium carbonate ( caco3 ) are very abundant ; amount 4070% , varies according to the species .", "calcium carbonate can be further processed into calcium hydroxyapatite [ ca10(po4)6(oh)2 ] [ 4 , 5 ] . according to strassler ,", "hydroxyapatite is one of the active ingredient materials that are widely used in toothpaste products for protection against teeth demineralization [ 1113 ] .", "the crystals structure of hydroxyapatite will be better by using cao as a precursor of calcium .", "however , the use of these compounds also produces carbonate apatite [ ca10(po4)6co3 ] in a fairly large percentage .", "this is because the calcination process can not completely eliminate carbon dioxide ( co2 ) in caco3 so that there can be reaction with the precursor phosphate .", "however , the carbonate apatite heating at a temperature of 700900c for 25 hours , followed by washing using distilled water , the carbonate apatite can be hydroxyapatite [ 1113 ] . the instrument were used in this research were glass apparatus , ohaus analytical balance , petri dish , porcelain cup , buchner flask , buchner funnel , vacuum pump sargent - welch co. model 1400 , magnetic stirrer , magnetic bar , hotplate idealife , ph meter , furnace thermolyse 6000-barnstead , dessicator , thermometers , spnisosfd oven , stopwatch , shimadzu x - ray diffractometer ( xrd ) model 6000 , x - ray fluorescence ( xrf ) shimadzu , fourier transform infra - red ( ftir ) prestige-21 shimadzu , scanning electron microscopy combined with the ability to generate localized chemical information ( sem - edxa ) variant , and uv - vis shimadzu model 6105 .", "this is because the carbonate ion compounds are able to inhibit the crystallization process ca10(po4)6(oh)2 so that the results will be dominated by an amorphous phase .", "if the caco3 burned at temperature calcinations , decomposition reaction of caco3 into cao will occur and co2 emissions are dominant and will be issued as a result of the combustion reaction [ 1315 ] .", "waste of crabs shells portunus pelagicus species was taken from an exporter crabs company in industry areal , makassar city , south sulawesi province , indonesia .", "wahidin hospital , and ( nh4)2hpo4 was obtained from fluka chemical , ch3cooh glacial 100% , ch3coona3h2o from merck , ( nh4)6mo7o244h2o from aldrich , nh4vo3 from aldrich , hno3 and naf from fluka chem . , ( nh4)2c2o4 , distilled water , aluminum foil , and whatman filter paper numbers 40 and 42 . the instruments were used in this research that glass as commonly used in laboratories , ohaus analytical balance , petri dish , porcelain cup , buchner flask , buchner funnel , vacuum pump sargent - welch co. model 1400 , magnetic stirrer , magnetic bar , hotplate idealife , ph meter , furnace thermolyse 6000 barnstead , dessicator , thermometers , oven spnisosfd , stopwatch , shimadzu x - ray diffractometer ( xrd ) model 6000 , x - ray fluorescence ( xrf ) shimadzu , fourier transform infrared ( ftir ) prestige-21 shimadzu , scanning electron microscopy combined with the ability to generate localized chemical information ( sem - edxa ) variant , and uv - vis shimadzu model 6105 .", "crabs shells portunus pelagicus waste was cleaned with distilled water and dried at room temperature . furthermore , to transform crabs shells into caco3 and then into cao , calcinations were performed on the samples at 1000c temperature for 5 hours at a rate of temperature rise 5c / minute .", "calcium oxide ( cao ) obtaining dominated as result of calcinations and then made suspensions in 100  ml of distilled water with a calcium concentration of 0.3  m. the suspensions reacted by dropwise with a 100  ml 0.2  m of ( nh4)2hpo4 solution through the coprecipitation method , at temperatures around 40c while the solution was stirred for 25 hours .", "the precipitation allowed stand overnight or 24 hours at room temperature , and the precipitate is filtered with a whatman filter paper number 40 and dried at 110c for 5 hours .", "the pure hydroxyapatite obtained by sintering to the dried precipitate at various temperatures of 500900c for 4 hours [ 16 , 17 ] .", "the characterization of the compounds was performed by using x - ray diffraction ( xrd ) , ftir , and sem - edxa .", "the proven in vitro demineralization of teeth was conducted through evaluating the concentration of phosphate in solution by using the uv - vis spectroscopy .", "the effectiveness of hydroxyapatite to protection against teeth demineralization was tested in acetate buffer ph 5.0 solutions , with 1 m of acetic acid concentration with the addition of hydroxyapatite in varying concentrations and immersion time .", "each of 5 beakers was filled with 300  ml of acetate buffer ph 5.0 with acetic acid concentration of 1  m. an acetate buffer was left without adding anything as a comparison .", "an acetate buffer was then added 10  ppm of naf left without addition of ca10(po4)6(oh)2 .", "other three pieces beaker of acetate buffer containing 10  ppm of naf are adding of ca10(po4)6(oh)2 with variation concentration of 25  ppm , 50  ppm , and 100  ppm .", "the immersed times of tooth samples in solution are 3 , 6 , 9 , 24 , and 48 hours , respectively .", "furthermore , the phosphate concentration in each solution was measured by using uv - vis spectrophotometer with the wavelength for phosphate ( maks ) being 432  nm .", "the calcinations aim to eliminate the organic component in the shells of crabs and convert caco3 compound which is the dominant compound in the shells of crabs into cao through the elimination of co2 in gas form . characterizing the calcinations results", "the results can be seen from the diffraction pattern of the crab shells before and after calcinations at a temperature of 1000c for 5 hours ( figure 1 ) . in this figure", "can be observed changes in the diffraction pattern of crab shells , where the change in the diffraction pattern is due to the chemical change from mixture of caco3 with organic matter to pure cao .", "the appearance of the sharper peaks in figure 1 after calcinations ( b ) is a result of the crystallinity of the cao .", "identification by ft - ir as shown in figure 2 showed there is a reduction process of -co3 groups and some of ir - spectra were missing after calcinations .", "the elimination of -co3 groups and organic components can also be seen from the data of mass reduction of sample calcinations .", "the determinations of calcium contained in sample was conducted by using x - ray fluorescence , where obtained calcium is 66.62% after calcinations .", "these results are then used to calculate the stoichiometry in determining the number of results calcinations which is needed to react with ( nh4)2hpo4 as the precursor phosphate .", "the precipitation reactions aiming to produce ca10(po4)6(oh)2 used phosphate , ( nh4)2hpo4 , as the precursor and then reacted with cao as calcinations results .", "side results ca10(po4)6co3 also occur as a product of reaction of ( nh4)2hpo4 and caco3 presence in the calcinations results .", "dried precipitate further sintered on temperature variations of 4001000c for 2 hours ; it is intended to determine the optimum temperature where the ca10(po4)6(oh)2 is formed , furthermore characterized by using xrd , ft - ir , and sem - edxa .", "the diffractograms of each sintering results compounds indicate that the temperature is closely related to the formation of crystals .", "this is due to the nature of the vibrating atoms moving faster in higher temperatures .", "the optimum temperature formation of hydroxyapatite was determined by calculation of the probability of the sample phase from the xrd results analysis according to jcpds standard data , which , jcpds ; 24 - 0033 is standard data for ca10(po4)6(oh)2 ; 09 - 0169 for -ca3(po4)2 ; 29 - 0359 for -ca3(po4)2 , 35 - 0180 -ca3(po4)2 ; 35 - 0180 for ca10(po4)6co3(oh)2 and 19 - 0272 standard data for ca10(po4)6co3(oh)2 .", "figures 3 and 4 showed that the temperature is closely associated with the formation of hydroxyapatite phase . in the both graphs it can be seen that the maximum intensity of the phase formation of hydroxyapatite has been found by sintering at temperature of 800c , it means the optimum temperature of hydroxyapatite formation is 800c , and then this result will be used for another application . the formations of hydroxyapatite phase had been dominated at 800c confirmed by percentage probability sample phase ( figure 5 ) , in which the percentage of hydroxyapatite phase formed around 46.61% , while phases of -ca3(po4)2 and phase -ca3(po4)2 are 17.76% and 19.37% , respectively .", "however , also there is still presence of a phase ca10(po4)6co3 and ca10(po4)6co3(oh)2 with a range of 11.84% and 4.43% , respectively , which indicates the presence of carbonates .", "the x - ray diffractograms of ca10(po4)6(oh)2 synthesized at a 800c temperature sintering can be seen in figure 6 , where the peaks of ha are symbolized by the peak of the crystal ca10(po4)6(oh)2 , while peak -tkf is symbolized crystalline peaks of -ca3(po4)2 , the symbol -tkf for crystalline -ca3(po4)2 , aka for ca10(po4)6co3 , and akb for ca10(po4)6co3(oh)2 .", "the highest intensity peak at 31.2572  deg corresponding to crystalline -ca3(po4)2 is seen , the second highest peak intensity is 31.7783 , and the third highest peak intensity is 28.0565 crystals suitable for ca10(po4)6(oh)2 .", "ftir results showed that the sintering temperature variation affects the absorption band shapes which generally all sintering results showed absorption band of -oh , absorption band 1 , 2 , 3 , and 4 of po4 , and co3 groups .", "infrared spectra in figure 7 show the -oh groups at 633  cm which are characteristic of hydroxyapatite   appearing on the sintering temperatures of 4001000c .", "additionally spectrum also showed higher sintering temperature causing the sharper peaks phosphate group ( po4 ) because the nature of the vibrating atoms moves faster at higher temperatures .", "the ft - ir spectra of sample were sintered at a temperature of 800c ( figure 8) showing that hydroxyapatite is the dominant compound formed .", "the stretching frequencies of po4 group are indicated by 1120.64  cm , 1091.71  cm , 1043.49  cm ( 3 ) , 993.34  cm , 877.61  cm ( 1 ) , 603.72  cm , 565.14  cm , ( 4 ) , and 370.33  cm ( 2 ) . and", "a sharp spectrum in the area of 3570.24  cm indicates the presence of free -oh and 3427.51  cm indicating -oh bounded , and this is indicating that the dominant compound is ca10(po4)6(oh)2 . while area of 1654.92  cm , 1458.18  cm , and 1421.54  cm indicates the presence of carbonate groups ( co3 ) it can be identified as ca10(po4)6co3 and ca10(po4)6co3(oh)2 which has not been transformed into ca10(po4)6(oh)2 during the sintering process .", "the results of characterization by sem - edxa shown in figure 9(a ) show that the size of hydroxyapatite formed from the synthesis tends to be small and only a few are large .", "while figure 9(b ) shows that the surface of hydroxyapatite is smooth and nonporous , this shows that hydroxyapatite which has synthesis from crab shells can function well as inhibiting tooth demineralization . while in figure 10 the edxa spectrum shows the composition of the synthesis yield", "was dominated by oxygen ( o ) up to 59.52% , calcium ( ca ) up to 23.76% , and phosphorus ( p ) up to 13.32% .", "demineralization of teeth is a process of decomposition of the crystal of ca10(po4)6(oh)2 due to the acidic conditions by releasing ca and po4 ions .", "demineralization of tooth causing increased levels of ca and po4 in saliva in direct contact with the tooth . in vitro", ", the rate of tooth demineralization can be observed through the concentrations of ca and po4 ions in solutions where the tooth was soaked each unit of time .", "therefore , increase of po4 ions concentrations in solution a soaked gear can be one of indicators to measure the rate of tooth demineralization . \n", "figure 11 shows the relationship between the soaking time of teeth versus the increase of the ion po4 levels in solution where the tooth was soaked as well ; it appears that with the increasing addition of ca10(po4)6(oh)2 into the acetate buffer equals to the rate of demineralization decrease .", "it is can be altered by the amount of po4 ions in solutions ; however the addition of ca10(po4)6(oh)2 ions showed lower amount of po4 ions compared to solutions without the addition of ca10(po4)6(oh)2 .", "this proves that the ca10(po4)6(oh)2 were synthesized from the crab shell effective for protection against tooth demineralization .", "the decrease in the rate of tooth demineralization with the addition of ca10(po4)6(oh)2 can also be observed through analyzing the tooth mass reduction in the fifth variation of acetate buffer solution as shown in table 1", ". the greater concentrations of ca10(po4)6(oh)2 in the acetate buffer solution were teeth immersed exhibit the smaller mass of teeth in the solution . \n", "based on these results it is concluded as follows.waste of shells crabs ( portunus pelagicus ) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste.the optimum temperature formation of ca10(po4)6(oh)2 is at 800c.ca10(po4)6(oh)2 were synthesized from waste of shell crabs ( portunus pelagicus ) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution , the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution .", "waste of shells crabs ( portunus pelagicus ) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste .", "ca10(po4)6(oh)2 were synthesized from waste of shell crabs ( portunus pelagicus ) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution , the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution ."], "abstract_text": ["<S> crab 's shells of portunus pelagicus species were used as raw materials for synthesis of hydroxyapatite were used for protection against demineralization of teeth . </S>", "<S> calcination was conducted to crab 's shells of portunus pelagicus at temperature of 1000c for 5 hours . </S>", "<S> the results of calcination was reacted with ( nh4)2hpo4 , then dried at 110c for 5 hours . </S>", "<S> sintering was conducted to results of precipitated dried with temperature variations 4001000c for a hour each variation of temperature then characterized by x - ray diffractometer and ftir in order to obtain the optimum formation temperature of hydroxyapatite is 800c . the hydroxyapatite is then tested its effectiveness in protection against tooth demineralization using acetate buffer ph 5.0 with 1  m acetic acid concentration with the addition of hydroxyapatite and time variation of immersion . </S>", "<S> the results showed that the rate of tooth demineralization in acetate buffer decreased significantly with the provision of hydroxyapatite into a solution where the addition of the magnitude of hydroxyapatite is greater decrease in the rate of tooth demineralization . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Experimental", "3. Results and Discussion", "4. Conclusion"], "sections": [["hydroxyapatite biomaterials are materials that are very widely used in several health purposes , including as a source of calcium for the manufacture of toothpaste and as an important material in the formation / bone repair .", "the chemical properties of hydroxyapatite are bioactive and compatible with the adjacent bone and teeth .", "hydroxyapatite is a calcium phosphate ceramic that is totally biocompatible and nontoxic and becomes an integral part of living bone and teeth tissue [ 18 ] .", "the raw material for the production of hydroxyapatite biomaterial is very easily available and abundant in indonesia .", "the production process was easy and the cost is also relatively inexpensive if done on a large scale . among", "the abundant raw materials are the shells of crabs , which are one of indonesia 's main export commodities .", "export of crabs commodity by indonesia amounted to 604.215625.000 tons / year without the shell form , while domestic consumption is expected to be so much more , as in makassar , carrying 292.5 tons exported in the form of crab without shell with the main export destination being singapore .", "if the mass of crab shells 2550% of the total mass , it can be estimated that in 2012 produced the shells of crabs around 151,053.75 to 302,107.5 tons in indonesia and 73.125 to 146.25 tons in makassar ( a region of indonesia ) , there was just from crabs production were exported .", "this value is of course even more if the crab 's consumptions in the country are also taken into account .", "this suggests the existence of crab shells is abundant in indonesia , including in makassar .", "as known in indonesia , the shells of crabs have not been used , so it will only be a waste disturbing environment [ 7 , 8 ] .", "crabs shells containing calcium carbonate ( caco3 ) are very abundant ; amount 4070% , varies according to the species .", "calcium carbonate can be further processed into calcium hydroxyapatite [ ca10(po4)6(oh)2 ] [ 4 , 5 ] . according to strassler ,", "hydroxyapatite is one of the active ingredient materials that are widely used in toothpaste products for protection against teeth demineralization [ 1113 ] .", "the crystals structure of hydroxyapatite will be better by using cao as a precursor of calcium .", "however , the use of these compounds also produces carbonate apatite [ ca10(po4)6co3 ] in a fairly large percentage .", "this is because the calcination process can not completely eliminate carbon dioxide ( co2 ) in caco3 so that there can be reaction with the precursor phosphate .", "however , the carbonate apatite heating at a temperature of 700900c for 25 hours , followed by washing using distilled water , the carbonate apatite can be hydroxyapatite [ 1113 ] . the instrument were used in this research were glass apparatus , ohaus analytical balance , petri dish , porcelain cup , buchner flask , buchner funnel , vacuum pump sargent - welch co. model 1400 , magnetic stirrer , magnetic bar , hotplate idealife , ph meter , furnace thermolyse 6000-barnstead , dessicator , thermometers , spnisosfd oven , stopwatch , shimadzu x - ray diffractometer ( xrd ) model 6000 , x - ray fluorescence ( xrf ) shimadzu , fourier transform infra - red ( ftir ) prestige-21 shimadzu , scanning electron microscopy combined with the ability to generate localized chemical information ( sem - edxa ) variant , and uv - vis shimadzu model 6105 .", "this is because the carbonate ion compounds are able to inhibit the crystallization process ca10(po4)6(oh)2 so that the results will be dominated by an amorphous phase .", "if the caco3 burned at temperature calcinations , decomposition reaction of caco3 into cao will occur and co2 emissions are dominant and will be issued as a result of the combustion reaction [ 1315 ] ."], ["waste of crabs shells portunus pelagicus species was taken from an exporter crabs company in industry areal , makassar city , south sulawesi province , indonesia .", "wahidin hospital , and ( nh4)2hpo4 was obtained from fluka chemical , ch3cooh glacial 100% , ch3coona3h2o from merck , ( nh4)6mo7o244h2o from aldrich , nh4vo3 from aldrich , hno3 and naf from fluka chem . , ( nh4)2c2o4 , distilled water , aluminum foil , and whatman filter paper numbers 40 and 42 . the instruments were used in this research that glass as commonly used in laboratories , ohaus analytical balance , petri dish , porcelain cup , buchner flask , buchner funnel , vacuum pump sargent - welch co. model 1400 , magnetic stirrer , magnetic bar , hotplate idealife , ph meter , furnace thermolyse 6000 barnstead , dessicator , thermometers , oven spnisosfd , stopwatch , shimadzu x - ray diffractometer ( xrd ) model 6000 , x - ray fluorescence ( xrf ) shimadzu , fourier transform infrared ( ftir ) prestige-21 shimadzu , scanning electron microscopy combined with the ability to generate localized chemical information ( sem - edxa ) variant , and uv - vis shimadzu model 6105 .", "crabs shells portunus pelagicus waste was cleaned with distilled water and dried at room temperature . furthermore , to transform crabs shells into caco3 and then into cao , calcinations were performed on the samples at 1000c temperature for 5 hours at a rate of temperature rise 5c / minute .", "calcium oxide ( cao ) obtaining dominated as result of calcinations and then made suspensions in 100  ml of distilled water with a calcium concentration of 0.3  m. the suspensions reacted by dropwise with a 100  ml 0.2  m of ( nh4)2hpo4 solution through the coprecipitation method , at temperatures around 40c while the solution was stirred for 25 hours .", "the precipitation allowed stand overnight or 24 hours at room temperature , and the precipitate is filtered with a whatman filter paper number 40 and dried at 110c for 5 hours .", "the pure hydroxyapatite obtained by sintering to the dried precipitate at various temperatures of 500900c for 4 hours [ 16 , 17 ] .", "the characterization of the compounds was performed by using x - ray diffraction ( xrd ) , ftir , and sem - edxa .", "the proven in vitro demineralization of teeth was conducted through evaluating the concentration of phosphate in solution by using the uv - vis spectroscopy .", "the effectiveness of hydroxyapatite to protection against teeth demineralization was tested in acetate buffer ph 5.0 solutions , with 1 m of acetic acid concentration with the addition of hydroxyapatite in varying concentrations and immersion time .", "each of 5 beakers was filled with 300  ml of acetate buffer ph 5.0 with acetic acid concentration of 1  m. an acetate buffer was left without adding anything as a comparison .", "an acetate buffer was then added 10  ppm of naf left without addition of ca10(po4)6(oh)2 .", "other three pieces beaker of acetate buffer containing 10  ppm of naf are adding of ca10(po4)6(oh)2 with variation concentration of 25  ppm , 50  ppm , and 100  ppm .", "the immersed times of tooth samples in solution are 3 , 6 , 9 , 24 , and 48 hours , respectively .", "furthermore , the phosphate concentration in each solution was measured by using uv - vis spectrophotometer with the wavelength for phosphate ( maks ) being 432  nm ."], ["the calcinations aim to eliminate the organic component in the shells of crabs and convert caco3 compound which is the dominant compound in the shells of crabs into cao through the elimination of co2 in gas form . characterizing the calcinations results", "the results can be seen from the diffraction pattern of the crab shells before and after calcinations at a temperature of 1000c for 5 hours ( figure 1 ) . in this figure", "can be observed changes in the diffraction pattern of crab shells , where the change in the diffraction pattern is due to the chemical change from mixture of caco3 with organic matter to pure cao .", "the appearance of the sharper peaks in figure 1 after calcinations ( b ) is a result of the crystallinity of the cao .", "identification by ft - ir as shown in figure 2 showed there is a reduction process of -co3 groups and some of ir - spectra were missing after calcinations .", "the elimination of -co3 groups and organic components can also be seen from the data of mass reduction of sample calcinations .", "the determinations of calcium contained in sample was conducted by using x - ray fluorescence , where obtained calcium is 66.62% after calcinations .", "these results are then used to calculate the stoichiometry in determining the number of results calcinations which is needed to react with ( nh4)2hpo4 as the precursor phosphate .", "the precipitation reactions aiming to produce ca10(po4)6(oh)2 used phosphate , ( nh4)2hpo4 , as the precursor and then reacted with cao as calcinations results .", "side results ca10(po4)6co3 also occur as a product of reaction of ( nh4)2hpo4 and caco3 presence in the calcinations results .", "dried precipitate further sintered on temperature variations of 4001000c for 2 hours ; it is intended to determine the optimum temperature where the ca10(po4)6(oh)2 is formed , furthermore characterized by using xrd , ft - ir , and sem - edxa .", "the diffractograms of each sintering results compounds indicate that the temperature is closely related to the formation of crystals .", "this is due to the nature of the vibrating atoms moving faster in higher temperatures .", "the optimum temperature formation of hydroxyapatite was determined by calculation of the probability of the sample phase from the xrd results analysis according to jcpds standard data , which , jcpds ; 24 - 0033 is standard data for ca10(po4)6(oh)2 ; 09 - 0169 for -ca3(po4)2 ; 29 - 0359 for -ca3(po4)2 , 35 - 0180 -ca3(po4)2 ; 35 - 0180 for ca10(po4)6co3(oh)2 and 19 - 0272 standard data for ca10(po4)6co3(oh)2 .", "figures 3 and 4 showed that the temperature is closely associated with the formation of hydroxyapatite phase . in the both graphs it can be seen that the maximum intensity of the phase formation of hydroxyapatite has been found by sintering at temperature of 800c , it means the optimum temperature of hydroxyapatite formation is 800c , and then this result will be used for another application . the formations of hydroxyapatite phase had been dominated at 800c confirmed by percentage probability sample phase ( figure 5 ) , in which the percentage of hydroxyapatite phase formed around 46.61% , while phases of -ca3(po4)2 and phase -ca3(po4)2 are 17.76% and 19.37% , respectively .", "however , also there is still presence of a phase ca10(po4)6co3 and ca10(po4)6co3(oh)2 with a range of 11.84% and 4.43% , respectively , which indicates the presence of carbonates .", "the x - ray diffractograms of ca10(po4)6(oh)2 synthesized at a 800c temperature sintering can be seen in figure 6 , where the peaks of ha are symbolized by the peak of the crystal ca10(po4)6(oh)2 , while peak -tkf is symbolized crystalline peaks of -ca3(po4)2 , the symbol -tkf for crystalline -ca3(po4)2 , aka for ca10(po4)6co3 , and akb for ca10(po4)6co3(oh)2 .", "the highest intensity peak at 31.2572  deg corresponding to crystalline -ca3(po4)2 is seen , the second highest peak intensity is 31.7783 , and the third highest peak intensity is 28.0565 crystals suitable for ca10(po4)6(oh)2 .", "ftir results showed that the sintering temperature variation affects the absorption band shapes which generally all sintering results showed absorption band of -oh , absorption band 1 , 2 , 3 , and 4 of po4 , and co3 groups .", "infrared spectra in figure 7 show the -oh groups at 633  cm which are characteristic of hydroxyapatite   appearing on the sintering temperatures of 4001000c .", "additionally spectrum also showed higher sintering temperature causing the sharper peaks phosphate group ( po4 ) because the nature of the vibrating atoms moves faster at higher temperatures .", "the ft - ir spectra of sample were sintered at a temperature of 800c ( figure 8) showing that hydroxyapatite is the dominant compound formed .", "the stretching frequencies of po4 group are indicated by 1120.64  cm , 1091.71  cm , 1043.49  cm ( 3 ) , 993.34  cm , 877.61  cm ( 1 ) , 603.72  cm , 565.14  cm , ( 4 ) , and 370.33  cm ( 2 ) . and", "a sharp spectrum in the area of 3570.24  cm indicates the presence of free -oh and 3427.51  cm indicating -oh bounded , and this is indicating that the dominant compound is ca10(po4)6(oh)2 . while area of 1654.92  cm , 1458.18  cm , and 1421.54  cm indicates the presence of carbonate groups ( co3 ) it can be identified as ca10(po4)6co3 and ca10(po4)6co3(oh)2 which has not been transformed into ca10(po4)6(oh)2 during the sintering process .", "the results of characterization by sem - edxa shown in figure 9(a ) show that the size of hydroxyapatite formed from the synthesis tends to be small and only a few are large .", "while figure 9(b ) shows that the surface of hydroxyapatite is smooth and nonporous , this shows that hydroxyapatite which has synthesis from crab shells can function well as inhibiting tooth demineralization . while in figure 10 the edxa spectrum shows the composition of the synthesis yield", "was dominated by oxygen ( o ) up to 59.52% , calcium ( ca ) up to 23.76% , and phosphorus ( p ) up to 13.32% .", "demineralization of teeth is a process of decomposition of the crystal of ca10(po4)6(oh)2 due to the acidic conditions by releasing ca and po4 ions .", "demineralization of tooth causing increased levels of ca and po4 in saliva in direct contact with the tooth . in vitro", ", the rate of tooth demineralization can be observed through the concentrations of ca and po4 ions in solutions where the tooth was soaked each unit of time .", "therefore , increase of po4 ions concentrations in solution a soaked gear can be one of indicators to measure the rate of tooth demineralization . \n", "figure 11 shows the relationship between the soaking time of teeth versus the increase of the ion po4 levels in solution where the tooth was soaked as well ; it appears that with the increasing addition of ca10(po4)6(oh)2 into the acetate buffer equals to the rate of demineralization decrease .", "it is can be altered by the amount of po4 ions in solutions ; however the addition of ca10(po4)6(oh)2 ions showed lower amount of po4 ions compared to solutions without the addition of ca10(po4)6(oh)2 .", "this proves that the ca10(po4)6(oh)2 were synthesized from the crab shell effective for protection against tooth demineralization .", "the decrease in the rate of tooth demineralization with the addition of ca10(po4)6(oh)2 can also be observed through analyzing the tooth mass reduction in the fifth variation of acetate buffer solution as shown in table 1", ". the greater concentrations of ca10(po4)6(oh)2 in the acetate buffer solution were teeth immersed exhibit the smaller mass of teeth in the solution . \n"], ["based on these results it is concluded as follows.waste of shells crabs ( portunus pelagicus ) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste.the optimum temperature formation of ca10(po4)6(oh)2 is at 800c.ca10(po4)6(oh)2 were synthesized from waste of shell crabs ( portunus pelagicus ) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution , the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution .", "waste of shells crabs ( portunus pelagicus ) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste .", "ca10(po4)6(oh)2 were synthesized from waste of shell crabs ( portunus pelagicus ) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution , the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution ."]]}
{"article_id": "PMC4616145", "article_text": ["brain injury is caused by intrinsic or extrinsic factors and it can result in various \n disabilities such as motor , sensory , behavioral , or cognitive dysfunction depending on the \n area of the brain lesion1 . cognitive \n impairment due to brain injury is an important factor affecting patients independent \n functions and participation in activities2 , interfering with their return to daily living and work3 .", "it can also influence motivation and the \n ability to participate in rehabilitation programs and interfere with a return to the \n community .", "therefore , for successful rehabilitation , accurate and comprehensive cognitive \n assessment and treatment are required4 . for cognitive rehabilitation of patients with brain injury , traditional treatment and \n computer - based cognitive therapy", "( vr ) technology is \n gaining recognition as a useful tool for cognitive research , evaluation , and \n rehabilitation5 .", "vr systems allow users \n to interact in various sensory environments and to obtain real - time feedback on their \n performance using computer technology6 . \n", "the virtual environment offered via vr technology makes it possible for patients to \n participate in activities in settings and environments similar to those encountered in real \n life7 , 8 .", "in addition , vr tools can be used to record accurate measurements \n of the subject s performance9 and to \n deliver greater therapeutic stimulation to users5 .", "vr has been used as a \n tool to diagnose cognitive impairment and as a vehicle to provide new treatments5 .", "although the use of vr in cognitive \n rehabilitation has been increasing , few systematic reviews have investigated the use of vr \n programs in cognitive rehabilitation and the overall effect of these programs on cognition . \n therefore , this systematic review investigated the different types of vr programs used for \n cognitive evaluation and interventions for patients with brain injury .", "studies using vr \n programs for cognitive intervention were reviewed according to pico ( patient , intervention , \n comparison , and outcome ) methods .", "( virtual reality or virtual or game based virtual reality or computer based virtual \n reality ) and ( stroke or cerebral vascular accident or hemiplegia or brain injury or \n traumatic brain injury ) and ( cognition or cognitive or memory or attention or executive \n function ) .", " inclusion criteria were : ( 1 ) subjects over the age of 19  years with brain \n injury ; ( 2 ) articles written in english ; and ( 3 ) studies that used vr in cognitive \n rehabilitation .", "exclusion criteria were : ( 1 ) subjects who were animals or children ; ( 2 ) \n review articles ; and ( 3 ) 2d computer - based cognitive rehabilitation .", "randomized controlled trials ( rcts ) and crossover studies were scored on the physiotherapy \n evidence database ( pedro ) scale12 .", "two \n authors independently assessed the methodological quality of the included studies , and \n disagreements were resolved by reaching consensus .", "memory \n assessment was the most common study topic , followed by assessments of executive function \n and attention .", "a variety of vr programs were used ( table 1table 1.analysis of studies of cognitive assessment using vrauthor , yearassessment areatype of vrbrooks et al .", "three were rcts24,25,26 , one was a crossover \n study27 , and one was a case report28 . excepting the case report , four studies \n were assessed using the pedro scale .", "two studies24 , \n 25 scored 4 , and the remaining two \n studies scored 326 and 127 , respectively .", "table 2table 2.analysis of studies with cognitive interventions using vrstudypatientinterventioncomparisonoutcomenmean agetype of vrintensityoutcome measure(s)findingsakinwuntan et al .", ", 20102469eg 55 cg 54stisim drive system60  min / day , 3 times / week , total 5 weeksnon - computer based cognitive therapyufov testsignificant within group improvements but no significant \n difference between two groupscaglio et al .", ", 201228124midtown madness 290  min / day , 3 times / week , total 5 weeks - fdst , bdst , ravlt - ir , dr , rbmt - ir , dr , corsi s block tapping \n test , corsi s supraspan test - ir , dr , tmt - a , b , phonemic fluency mmse , adassignificant improvements in ravlt - ir and corsis supraspan - ir , \n drgrealy et al . ,", "1999271332.38nonimm - ersive vr exercise bicycle25  min / day , 3 times / week , total 4 weeksno treatmentfdst , bdst , digit symbol test , tmt - a & b , auditory learning \n test , velt , vilt , logical memory , complex figure testeg showed significant improvements in digit symbol test , velt , \n vilt compared to cgjacoby et al .", ", 20132512eg 27.83 cg 30.67irex45  min / day , 34 times / week , total 3 weeksconventional otmet - sv , efpteg showed significant improvements in all outcome measures \n compared to the cgkim et al . , 20112628eg 66.5 cg 62.0irex30  min / day , 5 times / week , total 4 weekscomputer based cognitive therapyk - mmse , tol , vcpt , acpt , word - color test , fdst , bdst , fvst , bvst , \n vilt , velt , tmt - asignificant difference between experimental group and control \n group in vcpt and bvsteg : experimental group , cg : control group , met - sv : multiple errands test - simplified \n version , efpt : executive function performance test , fdst : forward digit span test , \n bdst : backward digit span test , tmt - a : trail making test - type a , tmt - b : trail making \n test - type b , velt : verbal learning test , vilt : visual learning test , ufov : useful \n field of view test , k - mmse : korean version of the mini - mental status examination , tol : \n tower of london test , vcpt : visual continuous performance test , acpt : auditory \n continuous performance test , fvst : forward visual span test , bvst : backward visual \n span test , ravlt : rey auditory verbal learning test , ir : immediate recall , dr : delayed \n recall , rbmt - the rivermead behavioural memory test , adas : alzheimer s disease \n assessment scale presents the characteristics of the five studies .", "flowchart of the article search and study selection eg : experimental group , cg : control group , met - sv : multiple errands test - simplified \n version , efpt : executive function performance test , fdst : forward digit span test , \n bdst : backward digit span test , tmt - a : trail making test - type a , tmt - b : trail making \n test - type b , velt : verbal learning test , vilt : visual learning test , ufov : useful \n field of view test , k - mmse : korean version of the mini - mental status examination , tol : \n tower of london test , vcpt : visual continuous performance test , acpt : auditory \n continuous performance test , fvst : forward visual span test , bvst : backward visual \n span test , ravlt : rey auditory verbal learning test , ir : immediate recall , dr : delayed \n recall , rbmt - the rivermead behavioural memory test , adas : alzheimer s disease \n assessment scale", "in this review , the types of vr programs that have been used in cognitive evaluations of \n patients with brain injury were identified and studies of cognitive interventions were \n reviewed according to pico methods . in the included studies , the vr programs could \n distinguish the cognitive disability of patients in comparisons with healthy subjects .", "thus , \n vr could be used as a new assessment method of the cognitive function of patients with brain \n injury .", "therefore , in contrast to conventional cognitive \n assessments , vr programs can provide consistently accurate measurements of cognitive \n function .", "however , some methodological problems were found in the reviewed articles . in most \n of the studies ,", "the vr tool used was not compared with a standardized assessment tool , and \n the inter - rater reliability was not measured .", "the five studies of cognitive therapy using vr all reported positive effects . in the \n assessment of cognitive function", ", the vr interventions resulted in improvements in the areas \n of memory and attention but not executive function .", "ben - yishay et al.29 stated that to effectively raise cognitive function , \n normal attention is needed .", "if the ability to concentrate on external information is \n impaired , memory , problem - solving skills , and appropriate behavior may be difficult .", "thus , \n they suggested that the impairment of attention due to brain injury may interfere with the \n recovery of other cognitive functions , such as memory , executive function , and planning .", "the \n results of this systematic review indicate that the cognitive improvement of attention using \n vr programs will have a positive impact on the recovery of general cognitive function .", "the \n advantage of cognitive rehabilitation using vr is that it provides a variety of environments \n similar to those encountered in real life30 .", "the results of this review suggest that patients are more \n motivated in virtual environments than they are in conventional settings .", "therefore , vr \n programs can be expected to lead to an improvement in cognitive function . in vr \n interventions", ", patients can be treated in a safe environment compared to real settings . in \n addition", ", vr programs can be tailored to the type of injury and easily adjusted to the level \n of cognitive disability , the complexity of a task , the reaction conditions , and the \n characteristics and patterns of feedback30 .", "as vr systems are constantly evolving and becoming smaller and \n more easily adjustable , they can be expected to provide specialized therapy in new settings , \n such as patients homes or clinics .", "these advantages of vr systems can benefit patients who \n find it difficult to visit health care organizations5 .", "the results of this systematic review suggest that vr is an effective cognitive therapy for \n patients with brain injury compared to control therapy .", ", there was a \n significant risk of bias with regard to allocation concealment and blinding .", "given the \n heterogeneity of the included studies , the ability to draw conclusions is limited . \n", "well - designed rcts and blind studies will be needed to provide evidence of the benefits of \n vr on cognitive function ."], "abstract_text": ["<S> [ purpose ] the purpose of this review was to investigate various types of vr programs and \n their use in cognitive evaluations and interventions for patients with brain injury . \n [ subjects and methods ] pubmed , cochrane , and otseeker electronic databases were searched \n with the search terms . at of 350 titles and abstracts </S>", "<S> were retrieved , and 17 articles were \n selected for this review . selected articles were assessed on the level of evidence using \n the physiotherapy evidence database ( pedro ) scale . </S>", "<S> [ results ] articles assessing the impact \n of cognitive impairments in memory were most commonly found , and vr interventions elicited \n positive effects in patients with brain injury . [ conclusion ] vr can be considered a new \n tool for cognitive rehabilitation after brain injury . </S>", "<S> vr interventions also have a number \n of advantages , e.g. cost - effectiveness , compared to other interventions . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["brain injury is caused by intrinsic or extrinsic factors and it can result in various \n disabilities such as motor , sensory , behavioral , or cognitive dysfunction depending on the \n area of the brain lesion1 . cognitive \n impairment due to brain injury is an important factor affecting patients independent \n functions and participation in activities2 , interfering with their return to daily living and work3 .", "it can also influence motivation and the \n ability to participate in rehabilitation programs and interfere with a return to the \n community .", "therefore , for successful rehabilitation , accurate and comprehensive cognitive \n assessment and treatment are required4 . for cognitive rehabilitation of patients with brain injury , traditional treatment and \n computer - based cognitive therapy", "( vr ) technology is \n gaining recognition as a useful tool for cognitive research , evaluation , and \n rehabilitation5 .", "vr systems allow users \n to interact in various sensory environments and to obtain real - time feedback on their \n performance using computer technology6 . \n", "the virtual environment offered via vr technology makes it possible for patients to \n participate in activities in settings and environments similar to those encountered in real \n life7 , 8 .", "in addition , vr tools can be used to record accurate measurements \n of the subject s performance9 and to \n deliver greater therapeutic stimulation to users5 .", "vr has been used as a \n tool to diagnose cognitive impairment and as a vehicle to provide new treatments5 .", "although the use of vr in cognitive \n rehabilitation has been increasing , few systematic reviews have investigated the use of vr \n programs in cognitive rehabilitation and the overall effect of these programs on cognition . \n therefore , this systematic review investigated the different types of vr programs used for \n cognitive evaluation and interventions for patients with brain injury .", "studies using vr \n programs for cognitive intervention were reviewed according to pico ( patient , intervention , \n comparison , and outcome ) methods ."], ["( virtual reality or virtual or game based virtual reality or computer based virtual \n reality ) and ( stroke or cerebral vascular accident or hemiplegia or brain injury or \n traumatic brain injury ) and ( cognition or cognitive or memory or attention or executive \n function ) .", " inclusion criteria were : ( 1 ) subjects over the age of 19  years with brain \n injury ; ( 2 ) articles written in english ; and ( 3 ) studies that used vr in cognitive \n rehabilitation .", "exclusion criteria were : ( 1 ) subjects who were animals or children ; ( 2 ) \n review articles ; and ( 3 ) 2d computer - based cognitive rehabilitation .", "randomized controlled trials ( rcts ) and crossover studies were scored on the physiotherapy \n evidence database ( pedro ) scale12 .", "two \n authors independently assessed the methodological quality of the included studies , and \n disagreements were resolved by reaching consensus ."], ["memory \n assessment was the most common study topic , followed by assessments of executive function \n and attention .", "a variety of vr programs were used ( table 1table 1.analysis of studies of cognitive assessment using vrauthor , yearassessment areatype of vrbrooks et al .", "three were rcts24,25,26 , one was a crossover \n study27 , and one was a case report28 . excepting the case report , four studies \n were assessed using the pedro scale .", "two studies24 , \n 25 scored 4 , and the remaining two \n studies scored 326 and 127 , respectively .", "table 2table 2.analysis of studies with cognitive interventions using vrstudypatientinterventioncomparisonoutcomenmean agetype of vrintensityoutcome measure(s)findingsakinwuntan et al .", ", 20102469eg 55 cg 54stisim drive system60  min / day , 3 times / week , total 5 weeksnon - computer based cognitive therapyufov testsignificant within group improvements but no significant \n difference between two groupscaglio et al .", ", 201228124midtown madness 290  min / day , 3 times / week , total 5 weeks - fdst , bdst , ravlt - ir , dr , rbmt - ir , dr , corsi s block tapping \n test , corsi s supraspan test - ir , dr , tmt - a , b , phonemic fluency mmse , adassignificant improvements in ravlt - ir and corsis supraspan - ir , \n drgrealy et al . ,", "1999271332.38nonimm - ersive vr exercise bicycle25  min / day , 3 times / week , total 4 weeksno treatmentfdst , bdst , digit symbol test , tmt - a & b , auditory learning \n test , velt , vilt , logical memory , complex figure testeg showed significant improvements in digit symbol test , velt , \n vilt compared to cgjacoby et al .", ", 20132512eg 27.83 cg 30.67irex45  min / day , 34 times / week , total 3 weeksconventional otmet - sv , efpteg showed significant improvements in all outcome measures \n compared to the cgkim et al . , 20112628eg 66.5 cg 62.0irex30  min / day , 5 times / week , total 4 weekscomputer based cognitive therapyk - mmse , tol , vcpt , acpt , word - color test , fdst , bdst , fvst , bvst , \n vilt , velt , tmt - asignificant difference between experimental group and control \n group in vcpt and bvsteg : experimental group , cg : control group , met - sv : multiple errands test - simplified \n version , efpt : executive function performance test , fdst : forward digit span test , \n bdst : backward digit span test , tmt - a : trail making test - type a , tmt - b : trail making \n test - type b , velt : verbal learning test , vilt : visual learning test , ufov : useful \n field of view test , k - mmse : korean version of the mini - mental status examination , tol : \n tower of london test , vcpt : visual continuous performance test , acpt : auditory \n continuous performance test , fvst : forward visual span test , bvst : backward visual \n span test , ravlt : rey auditory verbal learning test , ir : immediate recall , dr : delayed \n recall , rbmt - the rivermead behavioural memory test , adas : alzheimer s disease \n assessment scale presents the characteristics of the five studies .", "flowchart of the article search and study selection eg : experimental group , cg : control group , met - sv : multiple errands test - simplified \n version , efpt : executive function performance test , fdst : forward digit span test , \n bdst : backward digit span test , tmt - a : trail making test - type a , tmt - b : trail making \n test - type b , velt : verbal learning test , vilt : visual learning test , ufov : useful \n field of view test , k - mmse : korean version of the mini - mental status examination , tol : \n tower of london test , vcpt : visual continuous performance test , acpt : auditory \n continuous performance test , fvst : forward visual span test , bvst : backward visual \n span test , ravlt : rey auditory verbal learning test , ir : immediate recall , dr : delayed \n recall , rbmt - the rivermead behavioural memory test , adas : alzheimer s disease \n assessment scale"], ["in this review , the types of vr programs that have been used in cognitive evaluations of \n patients with brain injury were identified and studies of cognitive interventions were \n reviewed according to pico methods . in the included studies , the vr programs could \n distinguish the cognitive disability of patients in comparisons with healthy subjects .", "thus , \n vr could be used as a new assessment method of the cognitive function of patients with brain \n injury .", "therefore , in contrast to conventional cognitive \n assessments , vr programs can provide consistently accurate measurements of cognitive \n function .", "however , some methodological problems were found in the reviewed articles . in most \n of the studies ,", "the vr tool used was not compared with a standardized assessment tool , and \n the inter - rater reliability was not measured .", "the five studies of cognitive therapy using vr all reported positive effects . in the \n assessment of cognitive function", ", the vr interventions resulted in improvements in the areas \n of memory and attention but not executive function .", "ben - yishay et al.29 stated that to effectively raise cognitive function , \n normal attention is needed .", "if the ability to concentrate on external information is \n impaired , memory , problem - solving skills , and appropriate behavior may be difficult .", "thus , \n they suggested that the impairment of attention due to brain injury may interfere with the \n recovery of other cognitive functions , such as memory , executive function , and planning .", "the \n results of this systematic review indicate that the cognitive improvement of attention using \n vr programs will have a positive impact on the recovery of general cognitive function .", "the \n advantage of cognitive rehabilitation using vr is that it provides a variety of environments \n similar to those encountered in real life30 .", "the results of this review suggest that patients are more \n motivated in virtual environments than they are in conventional settings .", "therefore , vr \n programs can be expected to lead to an improvement in cognitive function . in vr \n interventions", ", patients can be treated in a safe environment compared to real settings . in \n addition", ", vr programs can be tailored to the type of injury and easily adjusted to the level \n of cognitive disability , the complexity of a task , the reaction conditions , and the \n characteristics and patterns of feedback30 .", "as vr systems are constantly evolving and becoming smaller and \n more easily adjustable , they can be expected to provide specialized therapy in new settings , \n such as patients homes or clinics .", "these advantages of vr systems can benefit patients who \n find it difficult to visit health care organizations5 .", "the results of this systematic review suggest that vr is an effective cognitive therapy for \n patients with brain injury compared to control therapy .", ", there was a \n significant risk of bias with regard to allocation concealment and blinding .", "given the \n heterogeneity of the included studies , the ability to draw conclusions is limited . \n", "well - designed rcts and blind studies will be needed to provide evidence of the benefits of \n vr on cognitive function ."]]}
{"article_id": "PMC3547305", "article_text": ["despite a considerable progress in malaria control in iran over the past few years that led to significant reduction of cases , the disease still remains a major health problem in south and southeastern parts of the country .", "2008 , afsharpad et al . 2012 ) and insecticide resistance of anopheles vectors ( enayati et al .", "2012 ) are aggravated by continuous influx of imported cases , mostly with plasmodium falciparum , from neighboring countries of afghanistan and pakistan ( zakeri et al .", "the latest checklists of iranian mosquitoes include 28 anopheles species , identified mostly on the basis of morphological features , and a few by dna - based approaches ( azari - hamidian 2007 ) .", "maculipennis are known to be responsible for transmission of malaria in the country ( manouchehri et al .", "some important malaria vectors in iran are assumed to be members of species complexes or species groups , which are often difficult to distinguish morphologically .", "application of dna - based approaches has resolved some cryptic species in iranian complex species including an .", "fluviatilis , however , the identity of some members are still doubtful or were refuted later ( azari - hamidian 2007 , naddaf et al .", "this , to great extent , is due to introduction of molecular markers such as its2 and 28s - d3 genes for discriminating the members of this complex species in india ( manonmani et al .", "biology , variation in behaviors , and role of this species in malaria transmission in different geographical areas of iran has been extensively reviewed by others ( eshghi et al .", "1976 , edalat 19971998 , hanafi - bojd et al . 2012 ) . in an early study comparison of its2 sequence of iranian specimens from various localities in south and southeastern", "iran revealed only species y , which is presumably species t ( naddaf et al . 2003 ) , however , rapd - pcr analysis of same specimens revealed two distinct patterns , separating representatives of fars province from other areas ( naddaf et al .", "analysis of 28s - d3 gene from same populations corroborated rapd results , fars province specimens showed to be identical to species u in india , while individuals from other areas exhibited heterozygocity at the only base pair position that identifies species u and t ( naddaf et al . 2010 ) .", "in addition , in a separate study based on 28s - d3 analysis , species t and species u were reported from jiroft of fars province and chabahar of sistan va baluchestan province , respectively ( mehravaran et al . 2011 ) .", "the aim of this study was to evaluate cytochrome oxidase i ( coi ) gene alongside 28s - d3 as a diagnostic tool for identification of an .", "coi gene sequences have been extensively used for population studies and resolving evolutionary relationship among closely related species groups of insects ( lunt et al . 1996 ) and", "anopheline mosquitoes ( krzywinski and besansky 2003 ) . variations in this fragment have been exploited as dna barcodes for identifications of culicidae mosquitoes including an .", "fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces .", "the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al .", "all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al .", "the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al .", "each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna .", "pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany .", "the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis .", "the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al .", "the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 .", "fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces .", "the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al .", "all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al .", "the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al . (", "each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna .", "pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany .", "the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis .", "the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al .", "the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 .", "all the dna specimens from fars province yield only a product of approximately 375 bp length indicative of species u , whereas specimens from hormozgan , kerman and sistan va baluchestan provinces amplified two bands of 375 bp and 128 bp length .", "phylogenetic analysis using coi gene grouped individuals from fars province in two distinct clades separate from other iranian individuals representing populations of hormozgan , kerman , and sistan va baluchestan ( fig .", "the mean distance between iranian and indian groups was 1.66% , while the value between fars group and the group comprising other iranian members was 2.06% .", "the indian group exhibited the same distance ( 2.06% ) with fars group . the highest distance ( 3.09% ) among iranian individuals was between specimens 930 from fars province and 398 from sistan va baluchestan province .", "six individuals belonging to two clades ( 87 , 97 , 655 , and 928 ) and ( 92 ) from fars province showed 100% identity .", "in addition , ten individuals from other geographical areas including four from koveh ( 170 , 172 , 173 , and 186 ) and one from minab ( 121 ) in hormozgan province , two from abchekan ( 392393 ) in sistan va baluchestan province , and three from kahnouj in kerman province ( 815 , 836 , and 839 ) were 100% identical .", "accurate identification of malaria vectors is not only one of the most basic requisite for success of malaria control programs , but also has become an intriguing issue for understanding speciation process and evolution of anopheles mosquitoes . in absence of cytotaxonomic evidence , rapd - pcr methodology and variation in 28s - d3 gene have resolved two potential sibling species in an .", "we report further evidence for the occurrence of these two sibling species by coi analysis of mitochondrial dna from the same specimens .", "anopheles fluviatilis james is a complex of cryptic species ; cytotaxonomic studies of polythene chromosomes has revealed three reproductively isolated species in india known as s , t , and u ( subbarao et al . 1994 ) .", "application of the first dna - based method using its2 gene identified two putative species of x and y that are presumably equivalent to species s and t , respectively ( manonmani et al .", "later , a complete as - pcr assay based on variations of 28s - d3 gene against chromosomally examined specimens identified all the members of the complex ( singh et al .", "fluvialitis mosquitoes of iran . however , the same specimens displayed variations in 28s - d3 gene ; the individuals from fars exhibited similarity with species u in india whereas individuals from others areas showed heterozygocity at the single nucleotide position that identifies species u and t. the identity of an .", "fluviatilis complex in iran became complicated as the heterozygocity in 28-d3 was not reflected in its2 fragment and individuals from fars province exhibited dual identity of t and u based on its2 and 28s genes , respectively ( chen et al .", "fluviatilis sibling species were not addressed in their study , however , k2p genetic distances between different species of culicidae were reported to be > 2% . in our study , the mean distance between iranian and indian populations was 1.66% , whereas the value between fars group and the group comprising other iranian individuals was 2.06% .", "the distance between most individuals from fars province and individuals from other areas was > 2% .", "two individuals ( 9192 ) from fars province exhibited almost equivalent distance with all other members including those ( 930 , 926 , 928 , 655 , 87 , and 97 ) from the same province .", "jf966741 , unpublished ) that showed a high divergence from other sequences and appeared as an out group beyond an .", "the results of present study were almost concordant with earlier results obtained by rapd - pcr methodology and 28s - d3 analysis ( naddaf et al .", "this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an .", "fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species .", "this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an .", "fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species ."], "abstract_text": ["<S> background : anopheles fluviatilis , one of the major malaria vectors in iran , is assumed to be a complex of sibling species . </S>", "<S> the aim of this study was to evaluate cytochrome oxidase i ( coi ) gene alongside 28s - d3 as a diagnostic tool for identification of an . </S>", "<S> fluviatilis sibling species in iran.methods:dna sample belonging to 24 an . </S>", "<S> fluviatilis mosquitoes from different geographical areas in south and southeastern iran were used for amplification of coi gene followed by sequencing . </S>", "<S> the 474475 bp coi sequences obtained in this study were aligned with 59 similar sequences of an . </S>", "<S> fluviatilis and a sequence of anopheles minimus , as out group , from genbank database . </S>", "<S> the distances between group and individual sequences were calculated and phylogenetic tree for obtained sequences was generated by using kimura two parameter ( k2p ) model of neighbor - joining method.results:phylogenetic analysis using coi gene grouped members of fars province ( central iran ) in two distinct clades separate from other iranian members representing hormozgan , kerman , and sistan va baluchestan provinces . </S>", "<S> the mean distance between iranian and indian individuals was 1.66% , whereas the value between fars province individuals and the group comprising individuals from other areas of iran was 2.06%.conclusion : presence of 2.06% mean distance between individuals from fars province and those from other areas of iran is indicative of at least two sibling species in an . </S>", "<S> fluviatilis mosquitoes of iran . </S>", "<S> this finding confirms earlier results based on rapd - pcr and 28s - d3 analysis . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Mosquitoes DNA", "PCR and sequencing of DNA", "Results", "Discussion", "Conclusion"], "sections": [["despite a considerable progress in malaria control in iran over the past few years that led to significant reduction of cases , the disease still remains a major health problem in south and southeastern parts of the country .", "2008 , afsharpad et al . 2012 ) and insecticide resistance of anopheles vectors ( enayati et al .", "2012 ) are aggravated by continuous influx of imported cases , mostly with plasmodium falciparum , from neighboring countries of afghanistan and pakistan ( zakeri et al .", "the latest checklists of iranian mosquitoes include 28 anopheles species , identified mostly on the basis of morphological features , and a few by dna - based approaches ( azari - hamidian 2007 ) .", "maculipennis are known to be responsible for transmission of malaria in the country ( manouchehri et al .", "some important malaria vectors in iran are assumed to be members of species complexes or species groups , which are often difficult to distinguish morphologically .", "application of dna - based approaches has resolved some cryptic species in iranian complex species including an .", "fluviatilis , however , the identity of some members are still doubtful or were refuted later ( azari - hamidian 2007 , naddaf et al .", "this , to great extent , is due to introduction of molecular markers such as its2 and 28s - d3 genes for discriminating the members of this complex species in india ( manonmani et al .", "biology , variation in behaviors , and role of this species in malaria transmission in different geographical areas of iran has been extensively reviewed by others ( eshghi et al .", "1976 , edalat 19971998 , hanafi - bojd et al . 2012 ) . in an early study comparison of its2 sequence of iranian specimens from various localities in south and southeastern", "iran revealed only species y , which is presumably species t ( naddaf et al . 2003 ) , however , rapd - pcr analysis of same specimens revealed two distinct patterns , separating representatives of fars province from other areas ( naddaf et al .", "analysis of 28s - d3 gene from same populations corroborated rapd results , fars province specimens showed to be identical to species u in india , while individuals from other areas exhibited heterozygocity at the only base pair position that identifies species u and t ( naddaf et al . 2010 ) .", "in addition , in a separate study based on 28s - d3 analysis , species t and species u were reported from jiroft of fars province and chabahar of sistan va baluchestan province , respectively ( mehravaran et al . 2011 ) .", "the aim of this study was to evaluate cytochrome oxidase i ( coi ) gene alongside 28s - d3 as a diagnostic tool for identification of an .", "coi gene sequences have been extensively used for population studies and resolving evolutionary relationship among closely related species groups of insects ( lunt et al . 1996 ) and", "anopheline mosquitoes ( krzywinski and besansky 2003 ) . variations in this fragment have been exploited as dna barcodes for identifications of culicidae mosquitoes including an ."], ["fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces .", "the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al .", "all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al .", "the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al .", "each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna .", "pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany .", "the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis .", "the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al .", "the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 ."], ["fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces .", "the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al ."], ["all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al .", "the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al . (", "each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna .", "pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany .", "the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis .", "the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al .", "the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 ."], ["all the dna specimens from fars province yield only a product of approximately 375 bp length indicative of species u , whereas specimens from hormozgan , kerman and sistan va baluchestan provinces amplified two bands of 375 bp and 128 bp length .", "phylogenetic analysis using coi gene grouped individuals from fars province in two distinct clades separate from other iranian individuals representing populations of hormozgan , kerman , and sistan va baluchestan ( fig .", "the mean distance between iranian and indian groups was 1.66% , while the value between fars group and the group comprising other iranian members was 2.06% .", "the indian group exhibited the same distance ( 2.06% ) with fars group . the highest distance ( 3.09% ) among iranian individuals was between specimens 930 from fars province and 398 from sistan va baluchestan province .", "six individuals belonging to two clades ( 87 , 97 , 655 , and 928 ) and ( 92 ) from fars province showed 100% identity .", "in addition , ten individuals from other geographical areas including four from koveh ( 170 , 172 , 173 , and 186 ) and one from minab ( 121 ) in hormozgan province , two from abchekan ( 392393 ) in sistan va baluchestan province , and three from kahnouj in kerman province ( 815 , 836 , and 839 ) were 100% identical ."], ["accurate identification of malaria vectors is not only one of the most basic requisite for success of malaria control programs , but also has become an intriguing issue for understanding speciation process and evolution of anopheles mosquitoes . in absence of cytotaxonomic evidence , rapd - pcr methodology and variation in 28s - d3 gene have resolved two potential sibling species in an .", "we report further evidence for the occurrence of these two sibling species by coi analysis of mitochondrial dna from the same specimens .", "anopheles fluviatilis james is a complex of cryptic species ; cytotaxonomic studies of polythene chromosomes has revealed three reproductively isolated species in india known as s , t , and u ( subbarao et al . 1994 ) .", "application of the first dna - based method using its2 gene identified two putative species of x and y that are presumably equivalent to species s and t , respectively ( manonmani et al .", "later , a complete as - pcr assay based on variations of 28s - d3 gene against chromosomally examined specimens identified all the members of the complex ( singh et al .", "fluvialitis mosquitoes of iran . however , the same specimens displayed variations in 28s - d3 gene ; the individuals from fars exhibited similarity with species u in india whereas individuals from others areas showed heterozygocity at the single nucleotide position that identifies species u and t. the identity of an .", "fluviatilis complex in iran became complicated as the heterozygocity in 28-d3 was not reflected in its2 fragment and individuals from fars province exhibited dual identity of t and u based on its2 and 28s genes , respectively ( chen et al .", "fluviatilis sibling species were not addressed in their study , however , k2p genetic distances between different species of culicidae were reported to be > 2% . in our study , the mean distance between iranian and indian populations was 1.66% , whereas the value between fars group and the group comprising other iranian individuals was 2.06% .", "the distance between most individuals from fars province and individuals from other areas was > 2% .", "two individuals ( 9192 ) from fars province exhibited almost equivalent distance with all other members including those ( 930 , 926 , 928 , 655 , 87 , and 97 ) from the same province .", "jf966741 , unpublished ) that showed a high divergence from other sequences and appeared as an out group beyond an .", "the results of present study were almost concordant with earlier results obtained by rapd - pcr methodology and 28s - d3 analysis ( naddaf et al .", "this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an .", "fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species ."], ["this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an .", "fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species ."]]}
{"article_id": "PMC2988961", "article_text": ["they are found most commonly in the cranial and peripheral nerves , and occurrence in the omentum is very rare .", "however , there have been some cases reported to develop serious complications and , if there was malignancy , to cause metastasis or recurrence .", "we presented a case of schwannoma originating from the great omentum , including histological and immunohistological studies .", "a 55-year - old man was referred to our department for the treatment of a tumor detected close to the stomach by ultrasound . medical examination including", "ultrasound had been regularly performed to follow up his gallbladder stone for the past 6 years .", "although he had no complaints and symptoms , the tumor had been increasing in size over one year .", "abdominal and endoscopic ultrasound showed a 2.0  1.3 cm cystic mass lateral to the wall of the stomach .", "its component included an 11.8  5.7 mm elevated lesion ( fig . 1 , fig", "computed tomography scan of the abdomen and pelvis showed a 2.6  1.9 cm cystic mass , which was slightly enhanced , and a gallbladder stone ( fig .", "3 ) . magnetic resonance imaging demonstrated a hyperintense mass on t2-weighted image . a distance from the stomach wall", "laboratory tests , including tumor makers , were normal . under a perioperative diagnosis of cystic tumor in the abdomen and cholelithiasis", ", we performed laparoscopic resection with vessel sealing system , which revealed a 2.0 cm mass arising from the great omentum , not adherent to other organs .", "grossly , the tumor was configured by a well - encapsulated round mass measuring 30  18  15 mm in diameter ( fig .", "histologically , the spindle - shaped cells were arranged in interlacing bundles and fascicles , together with varying numbers of tumor cells containing various amounts of light brown or grayish pigment ( fig .", "these alternate with looser antoni b tissue , which is comprised of cells showing clear , vacuolated cytoplasm due to lipid accumulation .", "immunohistochemically , most of the neoplastic cells reacted moderately to nse , ncam and s-100 protein ( fig .", "schwannomas are classified as one kind of the peripheral nerve sheath tumors , of which schwannnoma and neurofibroma are the most frequent . currently , the most precise determination of the tumor 's cell type is established by its immunohistochemical profile , ultrastructural features , or both . a tumor composed of cells with distinctly schwannian characteristics", "schwannomas are known to mainly arise from the peripheral and caranial nerves , the extremities and the retroperitoneum .", "they are seldom found in the abdomen , especially the extragastrointestinal tract , of which solitaly schwannoma of the great omentum is an extremely rare tumor . in 303 schwannomas reported by das gupta", ", one case originated from the retroperitoneum , others from the central nervous system and peripheral nerve . in another review of the literature , stout and", "of these , 37 were in the stomach , 3 in the small bowel , and none in the omentum and the abdominal cavity . previously published cases of schwannoma in the omentum were collected from a computerized medical literature search ( pubmed ) . only 6 cases of schwannoma from the great omentum have been observed   ( table 1 ) .", "we can see that more cases of schwannoma from the lesser omentum have been recorded than from the great omentum .", "it is mentioned that the lesser omentum contains a small amount of nerves in almost equal distribution , the great omentum has a paucity of nervous tissue and anatomically consists of fat and lymphatic tissue .", "we found a case of malignant schwannoma arising from the omentum which demonstrated peritoneal metastasis , and another reported case of the small intestine emphasized poor prognosis since only 2 of 24 patients survived for more than 5 years .", "benign schwannomas are also reported to increase in size and to eventually cause complications by compressing other organs or by causing bleeding in or outside the gastrointestinal tract .", "pigmented schwannoma is another type of schwannoma , usually arising from the sympathetic nervous system .", "additionally abdominal schwannomas cause diagnostic problems because clinical symptoms are uncharacteristic or misleading even if the tumor is large .", "ct imaging typically showed a low attenuation mass , peripheral enhancement and cystic degeneration pattern .", "mri disclosed schwannoma of low signal intensity on t1-weighted image and high signal intensity on t2-weighted image . in spite of these characteristic , it is difficult to exclude other abdominal tumors , leiomyomas , lymphomas and unspecified sarcomas etc . while previous cases confirm that the tumor may attain a considerable size , bigger than 5 cm or producing symptoms , our case of a schwannoma which was small and asymptomatic is hardly diagnosed .", "however this tumor showing progression in size is thought to have potential to behave very aggressively despite benign histological features , which stresses the need of more information on this type of tumor and diagnosis .", "consequently , histological analysis of the surgical specimen is necessary for a correct diagnosis , and common treatment for schwannoma is surgery .", "therefore laparoscopic resection seemed to be the most adequate method to diagnose and rule out malignant tumor as a minimally invasive surgery .", "the majority of tumors can be safely resected , the surgeon being careful of the dividing feeder vessels because schwannomas are well known as hypervascular tumors . to our knowledge", "there are no case reports documenting other treatments , including chemotherapy for schwannomas in the abdominal cavity ."], "abstract_text": ["<S> schwannoma in the abdomen is an uncommon neoplasm that occurs most frequently in the cranial and peripheral nerves ; it is extremely rare in the great omentum and only 6 cases of schwannoma of the great omentum have been observed previously . </S>", "<S> we report the case of a schwannoma found in the great omentum of a 55-year - old man who was treated with laparoscopic surgery . </S>", "<S> though it was difficult to diagnose preoperatively , the tumor showed malignant potential by rapidly increasing in size . </S>", "<S> histologically it was configured by a well - encapsulated round mass measuring 30  18  15 mm in diameter . </S>", "<S> immunohistochemically most of the neoplastic cells reacted moderately to nse , ncam and s-100 protein . </S>", "<S> we document the clinicopathological study of a schwannoma of the great omentum , followed by a review of the literature . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["they are found most commonly in the cranial and peripheral nerves , and occurrence in the omentum is very rare .", "however , there have been some cases reported to develop serious complications and , if there was malignancy , to cause metastasis or recurrence .", "we presented a case of schwannoma originating from the great omentum , including histological and immunohistological studies ."], ["a 55-year - old man was referred to our department for the treatment of a tumor detected close to the stomach by ultrasound . medical examination including", "ultrasound had been regularly performed to follow up his gallbladder stone for the past 6 years .", "although he had no complaints and symptoms , the tumor had been increasing in size over one year .", "abdominal and endoscopic ultrasound showed a 2.0  1.3 cm cystic mass lateral to the wall of the stomach .", "its component included an 11.8  5.7 mm elevated lesion ( fig . 1 , fig", "computed tomography scan of the abdomen and pelvis showed a 2.6  1.9 cm cystic mass , which was slightly enhanced , and a gallbladder stone ( fig .", "3 ) . magnetic resonance imaging demonstrated a hyperintense mass on t2-weighted image . a distance from the stomach wall", "laboratory tests , including tumor makers , were normal . under a perioperative diagnosis of cystic tumor in the abdomen and cholelithiasis", ", we performed laparoscopic resection with vessel sealing system , which revealed a 2.0 cm mass arising from the great omentum , not adherent to other organs .", "grossly , the tumor was configured by a well - encapsulated round mass measuring 30  18  15 mm in diameter ( fig .", "histologically , the spindle - shaped cells were arranged in interlacing bundles and fascicles , together with varying numbers of tumor cells containing various amounts of light brown or grayish pigment ( fig .", "these alternate with looser antoni b tissue , which is comprised of cells showing clear , vacuolated cytoplasm due to lipid accumulation .", "immunohistochemically , most of the neoplastic cells reacted moderately to nse , ncam and s-100 protein ( fig ."], ["schwannomas are classified as one kind of the peripheral nerve sheath tumors , of which schwannnoma and neurofibroma are the most frequent . currently , the most precise determination of the tumor 's cell type is established by its immunohistochemical profile , ultrastructural features , or both . a tumor composed of cells with distinctly schwannian characteristics", "schwannomas are known to mainly arise from the peripheral and caranial nerves , the extremities and the retroperitoneum .", "they are seldom found in the abdomen , especially the extragastrointestinal tract , of which solitaly schwannoma of the great omentum is an extremely rare tumor . in 303 schwannomas reported by das gupta", ", one case originated from the retroperitoneum , others from the central nervous system and peripheral nerve . in another review of the literature , stout and", "of these , 37 were in the stomach , 3 in the small bowel , and none in the omentum and the abdominal cavity . previously published cases of schwannoma in the omentum were collected from a computerized medical literature search ( pubmed ) . only 6 cases of schwannoma from the great omentum have been observed   ( table 1 ) .", "we can see that more cases of schwannoma from the lesser omentum have been recorded than from the great omentum .", "it is mentioned that the lesser omentum contains a small amount of nerves in almost equal distribution , the great omentum has a paucity of nervous tissue and anatomically consists of fat and lymphatic tissue .", "we found a case of malignant schwannoma arising from the omentum which demonstrated peritoneal metastasis , and another reported case of the small intestine emphasized poor prognosis since only 2 of 24 patients survived for more than 5 years .", "benign schwannomas are also reported to increase in size and to eventually cause complications by compressing other organs or by causing bleeding in or outside the gastrointestinal tract .", "pigmented schwannoma is another type of schwannoma , usually arising from the sympathetic nervous system .", "additionally abdominal schwannomas cause diagnostic problems because clinical symptoms are uncharacteristic or misleading even if the tumor is large .", "ct imaging typically showed a low attenuation mass , peripheral enhancement and cystic degeneration pattern .", "mri disclosed schwannoma of low signal intensity on t1-weighted image and high signal intensity on t2-weighted image . in spite of these characteristic , it is difficult to exclude other abdominal tumors , leiomyomas , lymphomas and unspecified sarcomas etc . while previous cases confirm that the tumor may attain a considerable size , bigger than 5 cm or producing symptoms , our case of a schwannoma which was small and asymptomatic is hardly diagnosed .", "however this tumor showing progression in size is thought to have potential to behave very aggressively despite benign histological features , which stresses the need of more information on this type of tumor and diagnosis .", "consequently , histological analysis of the surgical specimen is necessary for a correct diagnosis , and common treatment for schwannoma is surgery .", "therefore laparoscopic resection seemed to be the most adequate method to diagnose and rule out malignant tumor as a minimally invasive surgery .", "the majority of tumors can be safely resected , the surgeon being careful of the dividing feeder vessels because schwannomas are well known as hypervascular tumors . to our knowledge", "there are no case reports documenting other treatments , including chemotherapy for schwannomas in the abdominal cavity ."]]}
{"article_id": "PMC3137987", "article_text": ["coronary artery disease ( cad ) is a major public health problem worldwide and the single largest cause of mortality in the united states , responsible for one of every six deaths ( aha heart disease and stroke statistics , 2010 ) .", "cad is caused by atherosclerosis , which is an inflammatory disease that involves multiple cell types , including circulating cells and cells in the vessel wall . despite advances in risk factor management on an epidemiological level ,", "various blood markers associated with increased risk for death and cardiovascular endpoints have been identified , but currently very few have been shown to have a diagnostic impact or important clinical implications that would affect patient management .  ", "therefore , there is a great need for innovative biomarkers that can assess risk for cad , assess activity of the atherosclerotic process , and guide evaluation of therapy .   ", "several recent studies have suggested that circulating micrornas could be useful as biomarkers for various human disease states , including cancer , acute myocardial infarction [ 47 ] , heart failure , and chronic vascular disease [ 8 , 9 ] .  ", "micrornas ( mirnas ) are a recently recognized class of short ( 1925  nt ) , single - stranded , noncoding rnas that regulate an array of cellular functions through the degradation and translational repression of mrnas that contain complementary sequences", ".   more than 1000 human mirnas have been identified , and , in tissues , mirnas regulate the expression of genes involved in critical cellular processes , including differentiation , growth , proliferation , and apoptosis .", "importantly , mirnas are now regarded as rheostats that fine - tune expression of proteins involved in just about every process in human cells .   ", "mirnas have been found in tissues , whole blood , serum , plasma , and other body fluids in a stable form that is protected from endogenous rnase activity [ 3 , 12 ] .", "although the biological function of mirna is yet to be fully understood , tissue levels of specific mirnas have been shown to correlate with pathological development of different diseases .", "mirnas function as managers in gene regulatory networks , and they are distinct from other biomarkers because they have a pathogenic role in the disease process and are not merely byproducts of the disease state .  ", "thus , mirna expression signatures in tissues and blood have a potential role in the diagnosis , prognosis , and assessment of therapy . in this study", ", we sought to compare mirna expression in whole blood of patients with angiographically significant cad to that of healthy aged - matched controls .  ", "we performed an initial exploratory microarray analysis in 5 cases and controls and then further examined the most highly expressed mirnas in an additional 15 cases and controls .", "study participants were recruited as part of the emory cardiology biobank , consisting of 3492 consecutive patients enrolled prior to undergoing elective or emergent cardiac catheterization across three emory healthcare sites , between 2003 and 2008 .  ", "patients aged 2090 years were interviewed to collect demographic characteristics , medical history , and lifestyle habits .  ", "risk factor prevalence was determined by physician diagnosis and/or treatment for hypertension , hyperlipidemia , and diabetes .   ", "coronary angiograms were evaluated independently by two operators , who made visual estimation of luminal narrowing in multiple segments based on a modified form of the aha / acc classification of the coronary tree .   using these data ,", "significant cad was defined as at least one major epicardial vessel with > 50% stenosis , assessed by quantitative coronary angiography . additionally , we collected whole blood samples from patients with 2 risk factors for cad , but did not have angiographically significant cad .", "the study was approved by the institutional review board at emory university , atlanta , ga , usa .   all subjects provided written informed consent at the time of enrollment .", "the healthy subject cohort was a random sample of employees from emory university who were fully employed , productive people .", "these subjects were aged 18 and older , not taking medications , and had not been hospitalized for at least one year prior to participation .", "venous blood samples were drawn via antecubial venipuncture into paxgene blood rna tubes and stored at 20c within 24 hours before rna extraction .", "mirna was isolated using the preanalytix paxgene mirna isolation kit ( qiagen ) according to the manufacturer 's protocol .", "mirna microarray analysis was performed by asuragen , inc . , which uses the affymetrix manufactured genechip mirna arrays .", "once labeled , the mirna targets were hybridized overnight onto the microarrays following which the arrays were washed and stained using streptavidin - phycoerythrin ( sape ) .", "expression analysis was performed for all small rnas for homo sapiens ( e.g. , mirna , small nuclear rnas like snorna and scarna ) and separately for sanger mirna registry content ( mirbase 11.0 , http://microrna.sanger.ac.uk/ ) for homo sapiens .", "mirna reverse transcription was performed using the taqman microrna reverse transcription kit ( applied biosystems ) at 16c for 30 min , 42c for 30  min , and denaturation of the enzyme at 85c for 5  min .  ", "the rt reaction was performed at 37c for 1 hour followed by 5  min at 95c .", "taqman microrna assays ( applied biosystems ) were performed using the 7500 fast real - time pcr system at the 9600 emulation run mode .", "ct values were converted into copy numbers ( copy no . = 2 ) and normalized to rnu44 .", "unpaired student 's t - tests were used to compare data . a p value < .05 ( two sided ) was considered significant . for analysis of microarray expression data ,", "a two- sample t - test was carried out for every gene , followed by multiplicity correction to control the false discovery rate ( fdr ) at  .05 .", "we initially performed expression profiling of all small rnas ( 1770 genes , figure 1 ) in the whole blood samples .  ", "three mirnas passed the fdr cutoff of 0.05 : mir-584 ( 7.9-fold higher in healthy versus cad patients , p = .000103 , t - test ) , mir-542 - 5p ( 3.9-fold higher in healthy versus cad pts , p = .000168 , t - test ) , and mir-187 * ( 2.77-fold higher in healthy versus cad pts , p = 8.1  10 , t - test ) .  ", "however , signal intensities for mir-542 - 5p and mir-187 * were very low and not detected in most samples .  ", "separately , we performed an analysis of sanger registry mirnas ( 848 for homo sapiens ) in the whole blood samples , but none of the mirnas passed the fdr cutoff of 0.05 .  ", "ten mirnas passed a fdr cutoff of 0.10 , but only one ( mir-129 - 5p , 1.57 fold higher in healthy patients , p = .000044 ) of these mirnas had consistently detectable signal across the 10 blood samples .  ", "although we were able to detect some differences in whole blood mirna levels between healthy subjects and cad patients ( mir-584 , in particular ) , our microarray data suggest that , similar to other reports , levels of mirnas in the blood are low and microarrays may lack the sensitivity to adequately identify mirnas that might serve as vascular disease biomarkers .  ", "interestingly , among the mirnas that tended to show consistent differences between healthy and cad blood , mirna expression was generally higher in the blood of healthy subjects , a finding previously observed by others .", "next , we performed qrt - pcr on rna isolated from whole blood of another 10 patients with angiographically significant cad and 15 healthy subjects .", "we evaluated the levels of mirnas that , based on our microarray data , were highly expressed in blood and tended to have different levels of expression between the two groups .  ", "this analysis included mir-150 , mir-584 , mir-21 , mir-24 , mir-126 , mir-92a , mir-34a , mir-19a , mir-145 , mir-155 , mir-222 , mir-378 , mir-29a , mir-30e-5p , mir-342 , and mir-181d . among these we found that mir-19a , mir-584 , mir-155 , mir-222 , mir-145 , mir-29a , mir-378 , mir-342 , mir-181d , mir-150 , and mir-30e-5p were significantly downregulated in the blood of patients with cad compared to healthy subjects ( figure 2 ) .  ", "furthermore , we also assessed expression of these 11 mirnas in a cohort of patients who had 2 risk factors for cad , but did not have angiographically significant cad .  ", "we found that there was no difference in whole blood mirna expression of this latter group compared to that in patients with significant cad ( not shown ) , suggesting that these mirnas are markers for vascular inflammation rather than markers specific for significant cad .", "there has been one report that mirna expression in blood may be influenced by medications .   indeed , one of the mirnas that we found to be downregulated in the blood of patients with cad is mir-155 , which is known to target the at1 receptor .  ", "therefore , we assessed the impact of medications on expression of mir-155 , mir-145 , mir-181d , mir-222 , mir-19a , mir-342 , mir-29a , mir-30e-5p , and mir-378 .  ", "we compared the mirna expression levels in the blood of patients with angiographically significant cad or 2 risk factors for cad but not on acei or arb to that in blood of similar patients who were taking at least one of medications .  ", "interestingly , levels of mir-155 , mir-19a , mir-145 , mir-222 , mir-342 , mir-30e-5p , and mir-378 ( figure 3 ) were significantly decreased in patients taking acei or arb compared to those who were not , suggesting that these medications may directly modulate expression of these mirnas , or they may influence inflammatory factors that otherwise regulate their expression .", "importantly , we did not find that statin use had a significant effect on the levels of mirnas identified in this study ( not shown ) .", "several recent studies have indicated that there is a potential role for circulating mirna levels as valuable biomarkers for different disease processes , including cancer , cardiomyopathy , and acute myocardial infarction .   in this study , we wanted to address the hypothesis that mirna expression levels in blood could predict the presence of significant coronary artery disease in human subjects .  ", "we identified 11 mirnas whose expression was significantly downregulated in patients with angiographic evidence of significant atherosclerosis compared to healthy subjects that were matched for age and gender .  ", "in addition , our data suggest that inhibition of the renin angiotensin system by acei or arb use further influences mirna expression in blood .", "this study confirms previous reports showing differences in circulating mirna levels of patients with cad compared to those of healthy subjects [ 8 , 16 ] .", "our study differs from the other studies in the content of the group of mirnas that were downregulated in patients with cad .", "we performed this analysis using the paxgene blood rna system because it provides a way to stabilize rna immediately after sample collection and facilitates storage of the samples for a relatively long period of time without compromising rna integrity [ 17 , 18 ] .  ", "importantly , mrna profiling of whole blood or peripheral mononuclear cells has been previously applied to cardiovascular disease [ 19 , 20 ] , and a relationship has been identified between mrna levels in whole blood and extent of coronary artery disease .  ", "our study extends this work by providing insight into the whole blood expression of mirnas that are potentially involved in regulating these cad genes . in our qrt - pcr analysis of whole blood samples from cad patients", ", we found similar changes in expression of mir-155 and mir-145 as what has been reported previously .", "however , we failed to detect changes in other mirnas that were described in this previous report , namely mir-126 and mir-92a .  ", "first , we studied whole blood samples , whereas plasma was studied previously .   thus our mirna profile", "likely reflects intracellular and extracellular mirnas levels in contrast to exclusively extracellular mirnas that would be detected in plasma .  ", "another difference in our study is that we normalized mirna expression to expression of rnu44 , a small nucleolar rna that , based on our microarray analysis , we found to be highly and consistently expressed across blood samples from cad patients and controls .   ", "this study confirmed the observation made by others regarding the difficulty of using microarray analysis to profile mirna expression in blood samples .   undoubtedly , this is in part due to the limitation of a relatively small sample size for our microarray analysis , as well as reduced sensitivity of the microarray method compared to qrt - pcr .  ", "in addition , principal component analysis of our data suggested that two of the mirna profiles for healthy subjects were actually similar to that of the cad patients , suggesting that these individuals may in fact have subclinical vascular inflammation .  ", ", we believe that careful selection of patients and well - matched control subjects from a larger group of well - characterized individuals reduced some of the variability in our qrt - pcr analysis", ". we can only speculate as to why expression of circulated mirnas is generally decreased in patients with cad .  ", "it has been hypothesized that levels of circulating mirnas are decreased in vascular disease because they have been taken up into atherosclerotic lesions .  ", "the levels of circulating mirnas may be affected by multiple factors , including transcription , processing and stability of the mirnas within circulating cells , as well as the ability of these cells to release mirnas into the plasma .  ", "circulating mirnas may be delivered to cells in the heart or blood vessels through microvesicles , exosomes , or apoptotic bodies .   because our study assessed mirna expression in whole blood ,", "our findings are likely more reflective of changes in mirna transcription or processing rather than release from the circulating cells .   ", "it remains to be determined whether downregulation of mirnas in cad patients is directly involved in inflammation or a compensatory response to this process .  ", "based on our observed changes in mirna expression in response to acei / arb therapy , we believe that circulating mirna levels reflect a compensatory response to inflammation .   further experimental studies are necessary to explore the mechanisms by which cad and therapies affect tissue versus circulating mirna levels . in summary ,", "the present study provides insight into whole blood levels of mirnas in patients with cad compared to healthy subjects and demonstrates their potential utility as biomarkers for vascular disease .  ", "validation of the changes in mirna expression observed here in larger studies will be a necessary step to confirm their candidacy as biomarkers and therapeutic targets .", "we believe that further elucidation of the role that these mirnas play in the pathogenesis and progression of chronic cad will contribute to our understanding of the disease process and lead to new therapeutic and preventative strategies ."], "abstract_text": ["<S> coronary artery disease ( cad ) is the largest killer of males and females in the united states . </S>", "<S> there is a need to develop innovative diagnostic markers for this disease . </S>", "<S> micrornas ( mirnas ) are a class of noncoding rnas that posttranscriptionally regulate the expression of genes involved in important cellular processes , and we hypothesized that the mirna expression profile would be altered in whole blood samples of patients with cad . </S>", "<S> we performed a microarray analysis on rna from the blood of 5 male subjects with cad and 5 healthy subjects ( mean age 53 years ) . </S>", "<S> subsequently , we performed qrt - pcr analysis of mirna expression in whole blood of another 10 patients with cad and 15 healthy subjects . </S>", "<S> we identified 11 mirnas that were significantly downregulated in cad subjects ( p < .05 ) . </S>", "<S> furthermore , we found an association between acei / arb use and downregulation of several mirnas that was independent of the presence of significant cad . in conclusion , we have identified a distinct mirna signature in whole blood that discriminates cad patients from healthy subjects . </S>", "<S> importantly , medication use may significantly alter mirna expression . </S>", "<S> these findings may have significant implications for identifying and managing individuals that either have cad or are at risk of developing the disease . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["coronary artery disease ( cad ) is a major public health problem worldwide and the single largest cause of mortality in the united states , responsible for one of every six deaths ( aha heart disease and stroke statistics , 2010 ) .", "cad is caused by atherosclerosis , which is an inflammatory disease that involves multiple cell types , including circulating cells and cells in the vessel wall . despite advances in risk factor management on an epidemiological level ,", "various blood markers associated with increased risk for death and cardiovascular endpoints have been identified , but currently very few have been shown to have a diagnostic impact or important clinical implications that would affect patient management .  ", "therefore , there is a great need for innovative biomarkers that can assess risk for cad , assess activity of the atherosclerotic process , and guide evaluation of therapy .   ", "several recent studies have suggested that circulating micrornas could be useful as biomarkers for various human disease states , including cancer , acute myocardial infarction [ 47 ] , heart failure , and chronic vascular disease [ 8 , 9 ] .  ", "micrornas ( mirnas ) are a recently recognized class of short ( 1925  nt ) , single - stranded , noncoding rnas that regulate an array of cellular functions through the degradation and translational repression of mrnas that contain complementary sequences", ".   more than 1000 human mirnas have been identified , and , in tissues , mirnas regulate the expression of genes involved in critical cellular processes , including differentiation , growth , proliferation , and apoptosis .", "importantly , mirnas are now regarded as rheostats that fine - tune expression of proteins involved in just about every process in human cells .   ", "mirnas have been found in tissues , whole blood , serum , plasma , and other body fluids in a stable form that is protected from endogenous rnase activity [ 3 , 12 ] .", "although the biological function of mirna is yet to be fully understood , tissue levels of specific mirnas have been shown to correlate with pathological development of different diseases .", "mirnas function as managers in gene regulatory networks , and they are distinct from other biomarkers because they have a pathogenic role in the disease process and are not merely byproducts of the disease state .  ", "thus , mirna expression signatures in tissues and blood have a potential role in the diagnosis , prognosis , and assessment of therapy . in this study", ", we sought to compare mirna expression in whole blood of patients with angiographically significant cad to that of healthy aged - matched controls .  ", "we performed an initial exploratory microarray analysis in 5 cases and controls and then further examined the most highly expressed mirnas in an additional 15 cases and controls ."], ["study participants were recruited as part of the emory cardiology biobank , consisting of 3492 consecutive patients enrolled prior to undergoing elective or emergent cardiac catheterization across three emory healthcare sites , between 2003 and 2008 .  ", "patients aged 2090 years were interviewed to collect demographic characteristics , medical history , and lifestyle habits .  ", "risk factor prevalence was determined by physician diagnosis and/or treatment for hypertension , hyperlipidemia , and diabetes .   ", "coronary angiograms were evaluated independently by two operators , who made visual estimation of luminal narrowing in multiple segments based on a modified form of the aha / acc classification of the coronary tree .   using these data ,", "significant cad was defined as at least one major epicardial vessel with > 50% stenosis , assessed by quantitative coronary angiography . additionally , we collected whole blood samples from patients with 2 risk factors for cad , but did not have angiographically significant cad .", "the study was approved by the institutional review board at emory university , atlanta , ga , usa .   all subjects provided written informed consent at the time of enrollment .", "the healthy subject cohort was a random sample of employees from emory university who were fully employed , productive people .", "these subjects were aged 18 and older , not taking medications , and had not been hospitalized for at least one year prior to participation .", "venous blood samples were drawn via antecubial venipuncture into paxgene blood rna tubes and stored at 20c within 24 hours before rna extraction .", "mirna was isolated using the preanalytix paxgene mirna isolation kit ( qiagen ) according to the manufacturer 's protocol .", "mirna microarray analysis was performed by asuragen , inc . , which uses the affymetrix manufactured genechip mirna arrays .", "once labeled , the mirna targets were hybridized overnight onto the microarrays following which the arrays were washed and stained using streptavidin - phycoerythrin ( sape ) .", "expression analysis was performed for all small rnas for homo sapiens ( e.g. , mirna , small nuclear rnas like snorna and scarna ) and separately for sanger mirna registry content ( mirbase 11.0 , http://microrna.sanger.ac.uk/ ) for homo sapiens .", "mirna reverse transcription was performed using the taqman microrna reverse transcription kit ( applied biosystems ) at 16c for 30 min , 42c for 30  min , and denaturation of the enzyme at 85c for 5  min .  ", "the rt reaction was performed at 37c for 1 hour followed by 5  min at 95c .", "taqman microrna assays ( applied biosystems ) were performed using the 7500 fast real - time pcr system at the 9600 emulation run mode .", "ct values were converted into copy numbers ( copy no . = 2 ) and normalized to rnu44 .", "unpaired student 's t - tests were used to compare data . a p value < .05 ( two sided ) was considered significant . for analysis of microarray expression data ,", "a two- sample t - test was carried out for every gene , followed by multiplicity correction to control the false discovery rate ( fdr ) at  .05 ."], ["we initially performed expression profiling of all small rnas ( 1770 genes , figure 1 ) in the whole blood samples .  ", "three mirnas passed the fdr cutoff of 0.05 : mir-584 ( 7.9-fold higher in healthy versus cad patients , p = .000103 , t - test ) , mir-542 - 5p ( 3.9-fold higher in healthy versus cad pts , p = .000168 , t - test ) , and mir-187 * ( 2.77-fold higher in healthy versus cad pts , p = 8.1  10 , t - test ) .  ", "however , signal intensities for mir-542 - 5p and mir-187 * were very low and not detected in most samples .  ", "separately , we performed an analysis of sanger registry mirnas ( 848 for homo sapiens ) in the whole blood samples , but none of the mirnas passed the fdr cutoff of 0.05 .  ", "ten mirnas passed a fdr cutoff of 0.10 , but only one ( mir-129 - 5p , 1.57 fold higher in healthy patients , p = .000044 ) of these mirnas had consistently detectable signal across the 10 blood samples .  ", "although we were able to detect some differences in whole blood mirna levels between healthy subjects and cad patients ( mir-584 , in particular ) , our microarray data suggest that , similar to other reports , levels of mirnas in the blood are low and microarrays may lack the sensitivity to adequately identify mirnas that might serve as vascular disease biomarkers .  ", "interestingly , among the mirnas that tended to show consistent differences between healthy and cad blood , mirna expression was generally higher in the blood of healthy subjects , a finding previously observed by others .", "next , we performed qrt - pcr on rna isolated from whole blood of another 10 patients with angiographically significant cad and 15 healthy subjects .", "we evaluated the levels of mirnas that , based on our microarray data , were highly expressed in blood and tended to have different levels of expression between the two groups .  ", "this analysis included mir-150 , mir-584 , mir-21 , mir-24 , mir-126 , mir-92a , mir-34a , mir-19a , mir-145 , mir-155 , mir-222 , mir-378 , mir-29a , mir-30e-5p , mir-342 , and mir-181d . among these we found that mir-19a , mir-584 , mir-155 , mir-222 , mir-145 , mir-29a , mir-378 , mir-342 , mir-181d , mir-150 , and mir-30e-5p were significantly downregulated in the blood of patients with cad compared to healthy subjects ( figure 2 ) .  ", "furthermore , we also assessed expression of these 11 mirnas in a cohort of patients who had 2 risk factors for cad , but did not have angiographically significant cad .  ", "we found that there was no difference in whole blood mirna expression of this latter group compared to that in patients with significant cad ( not shown ) , suggesting that these mirnas are markers for vascular inflammation rather than markers specific for significant cad .", "there has been one report that mirna expression in blood may be influenced by medications .   indeed , one of the mirnas that we found to be downregulated in the blood of patients with cad is mir-155 , which is known to target the at1 receptor .  ", "therefore , we assessed the impact of medications on expression of mir-155 , mir-145 , mir-181d , mir-222 , mir-19a , mir-342 , mir-29a , mir-30e-5p , and mir-378 .  ", "we compared the mirna expression levels in the blood of patients with angiographically significant cad or 2 risk factors for cad but not on acei or arb to that in blood of similar patients who were taking at least one of medications .  ", "interestingly , levels of mir-155 , mir-19a , mir-145 , mir-222 , mir-342 , mir-30e-5p , and mir-378 ( figure 3 ) were significantly decreased in patients taking acei or arb compared to those who were not , suggesting that these medications may directly modulate expression of these mirnas , or they may influence inflammatory factors that otherwise regulate their expression .", "importantly , we did not find that statin use had a significant effect on the levels of mirnas identified in this study ( not shown ) ."], ["several recent studies have indicated that there is a potential role for circulating mirna levels as valuable biomarkers for different disease processes , including cancer , cardiomyopathy , and acute myocardial infarction .   in this study , we wanted to address the hypothesis that mirna expression levels in blood could predict the presence of significant coronary artery disease in human subjects .  ", "we identified 11 mirnas whose expression was significantly downregulated in patients with angiographic evidence of significant atherosclerosis compared to healthy subjects that were matched for age and gender .  ", "in addition , our data suggest that inhibition of the renin angiotensin system by acei or arb use further influences mirna expression in blood .", "this study confirms previous reports showing differences in circulating mirna levels of patients with cad compared to those of healthy subjects [ 8 , 16 ] .", "our study differs from the other studies in the content of the group of mirnas that were downregulated in patients with cad .", "we performed this analysis using the paxgene blood rna system because it provides a way to stabilize rna immediately after sample collection and facilitates storage of the samples for a relatively long period of time without compromising rna integrity [ 17 , 18 ] .  ", "importantly , mrna profiling of whole blood or peripheral mononuclear cells has been previously applied to cardiovascular disease [ 19 , 20 ] , and a relationship has been identified between mrna levels in whole blood and extent of coronary artery disease .  ", "our study extends this work by providing insight into the whole blood expression of mirnas that are potentially involved in regulating these cad genes . in our qrt - pcr analysis of whole blood samples from cad patients", ", we found similar changes in expression of mir-155 and mir-145 as what has been reported previously .", "however , we failed to detect changes in other mirnas that were described in this previous report , namely mir-126 and mir-92a .  ", "first , we studied whole blood samples , whereas plasma was studied previously .   thus our mirna profile", "likely reflects intracellular and extracellular mirnas levels in contrast to exclusively extracellular mirnas that would be detected in plasma .  ", "another difference in our study is that we normalized mirna expression to expression of rnu44 , a small nucleolar rna that , based on our microarray analysis , we found to be highly and consistently expressed across blood samples from cad patients and controls .   ", "this study confirmed the observation made by others regarding the difficulty of using microarray analysis to profile mirna expression in blood samples .   undoubtedly , this is in part due to the limitation of a relatively small sample size for our microarray analysis , as well as reduced sensitivity of the microarray method compared to qrt - pcr .  ", "in addition , principal component analysis of our data suggested that two of the mirna profiles for healthy subjects were actually similar to that of the cad patients , suggesting that these individuals may in fact have subclinical vascular inflammation .  ", ", we believe that careful selection of patients and well - matched control subjects from a larger group of well - characterized individuals reduced some of the variability in our qrt - pcr analysis", ". we can only speculate as to why expression of circulated mirnas is generally decreased in patients with cad .  ", "it has been hypothesized that levels of circulating mirnas are decreased in vascular disease because they have been taken up into atherosclerotic lesions .  ", "the levels of circulating mirnas may be affected by multiple factors , including transcription , processing and stability of the mirnas within circulating cells , as well as the ability of these cells to release mirnas into the plasma .  ", "circulating mirnas may be delivered to cells in the heart or blood vessels through microvesicles , exosomes , or apoptotic bodies .   because our study assessed mirna expression in whole blood ,", "our findings are likely more reflective of changes in mirna transcription or processing rather than release from the circulating cells .   ", "it remains to be determined whether downregulation of mirnas in cad patients is directly involved in inflammation or a compensatory response to this process .  ", "based on our observed changes in mirna expression in response to acei / arb therapy , we believe that circulating mirna levels reflect a compensatory response to inflammation .   further experimental studies are necessary to explore the mechanisms by which cad and therapies affect tissue versus circulating mirna levels . in summary ,", "the present study provides insight into whole blood levels of mirnas in patients with cad compared to healthy subjects and demonstrates their potential utility as biomarkers for vascular disease .  ", "validation of the changes in mirna expression observed here in larger studies will be a necessary step to confirm their candidacy as biomarkers and therapeutic targets .", "we believe that further elucidation of the role that these mirnas play in the pathogenesis and progression of chronic cad will contribute to our understanding of the disease process and lead to new therapeutic and preventative strategies ."]]}
{"article_id": "PMC355903", "article_text": ["among eusocial insects , paper wasps ( hymenoptera : vespidae : polistinae ) are notable for the diversity of their nest architecture ( jeanne , 1975 ; wenzel , 1991 ) .", "many swarm - founding polistine wasps , including most species of the neotropical tribe epiponini , construct envelopes around their brood combs .", "comparative and experimental studies suggest that , among other functions , nest envelopes reduce rates of predation and parasitism on wasp brood ( london and jeanne , 1998 ; smith et al . , 2001 ) .", "however , little is known about the adaptive significance of variation in the materials that wasps use to construct nest envelopes ( hansell 1984 ; wenzel , 1991 ; cole et al . , 2001 ) .", "polybia emaciata is unusual among the polistine wasps because it uses mud , rather than wood pulp or plant fibers , as the main raw material for nest construction ( schremmer , 1984 ) .", "the genus polybia includes 56 described species ; of these , only the other three species in the subgenus pedothoeca , and one species in the subgenus furnariana , share the derived trait of mud nest construction ( richards , 1978 ; jeanne , 1991 ; cooper , 1993 ) .", "no other swarm - founding polistine wasps are known to build primarily mud nests , although small amounts of inorganic material are sometimes incorporated into the nest paper of other species ( wenzel , 1991 ) .", "the nest of p. emaciata is otherwise typical of the genus ( jeanne , 1975 ; wenzel , 1991 ) , being phragmocyttarus ( comprising horizontal layers of comb that are conjoined and enclosed by a continuous envelope ) with a single small entrance hole near the bottom of the nest .", "numbers of adults in mature colonies are relatively small for the genus , ranging from fewer than 100 up to about 500 adults ( richards , 1978 ; chadab , 1979 ; strassmann et al . , 1992 ) .", "polybia emaciata nests are hard and much more durable than most paper nests of similar size and shape ( rau , 1933 ; richards , 1978 ) .", "for example , skutch ( 1971 ) noted that nests of this species in costa rica were unlike paper nests in that they persisted for several years .", "the high durability of p. emaciata construction material suggests that the nest itself may provide greater brood protection than paper nests .", "if so , we hypothesized that the wasps ' defensive responses to vertebrate attacks have changed to take advantage of nest durability .", "we predicted that the nests ' protective properties reduced the need for behavioral defenses by p. emaciata workers , relative to paper - nesting polybia species with similar colony and nest sizes .", "we tested this prediction by disturbing p. emaciata colonies , and comparing the workers ' responses to those of simultaneously observed paper - nesting p. occidentalis colonies in the same location .", "chadab ( 1979 ) surveyed the defensive responses of swarm - founding paper wasps to vertebrate - like disturbances , including tapping on the nest and/or on the supporting vegetation . the most frequently observed response was for dozens to hundreds of workers to rush out onto the nest envelope , sometimes adopting aposematic postures ( see also o'donnell et al . ,", "response was seen in the genera angiopolybia , charterginus , leipomeles , parachartergus , polybia , pseudopolybia , and synoeca , and accords with our observations of what appears to be typical epiponine defensive behavior .", "weaker exit - nest responses ( either requiring several taps , or involving fewer wasps ) were seen in species of leipomeles , metapolybia , and protopolybia .", "interspecific variation in mature colony size was not a good predictor of the type of defensive response , as species with colony sizes smaller and larger than p. emaciata performed nest exiting defensive behavior .", "an attack response often coincides with , or quickly follows , the nest exit response and involves workers flying from the nest , striking the intruder , and attempting to sting ( hermann and blum , 1981 ) .", "when paper - using polybia nests are tapped , some of the workers on the nest surface may immediately perform attacks , while large numbers of workers rush out of the nest entrance and stand in alert posture on the envelope ( jeanne , 1981 ; jeanne et al . , 1992 ) .", "further tapping elicits additional attacks . breathing on the nest surface or into the nest results in immediate attack by several to dozens of workers .", "we discuss the possible adaptive significance of deviations from the typical nest exit that have been documented in several paper wasp species , including p. emaciata .", "this study was conducted in and near the town of gamboa , panama province , republic of panama ( 0907n 7942w ; 50 m elevation ) .", "observations and manipulations were conducted during the wet season from 1 august to 15 october 1990 and from 3 to 5 july 1998 .", "surveys of eusocial wasp colonies during these periods indicated that p. emaciata was among the most abundant swarm - founding wasps in terms of nest density ( s.o'd . , unpublished data ) .", "most of the data presented were collected from four colonies of p. emaciata and five colonies of p. occidentalis that had been moved approximately 0.5 km to 1 km from their natural nesting sites to artificial supports to facilitate observation .", "all subject colonies were moved into a small area ( < 0.25 ha ) , with similar amounts of tree cover ( one p. occidentalis colony was additionally sheltered by building eaves ) .", "all colonies were in place at least 2 weeks before observations on defense behavior were initiated , and all continued to forage and to raise brood ( visible through the nest entrance in the lower combs ) throughout the observation period .", "the p. emaciata observation colonies had nests ranging in size from 9 cm long to 22 cm long .", "the numbers of adult wasps and colony age probably varied positively with nest size , though we did not measure these variables .", "we also collected data opportunistically from p. emaciata colonies in situ in 1990 and again in 1998 .", "these nests were located in the gamboa area on thin tree branches from 1.5 m to 5 m above the ground .", "we disturbed p. emaciata and p. occidentalis colonies in an effort to evoke defensive behavior , using stimuli that elicited nest exit and attack responses in costa rican p. occidentalis ( s.o'd . and", "we disturbed the polybia nests by first tapping on the side of the outer envelope near the middle of the nest for 30 sec while wearing a full bee suit , and we noted the responses of on - nest and in - nest workers .", "if there was no attack response to tapping , we blew into the nest entrance for 30 sec .", "each polybia observation colony was disturbed 10 times over the course of the study period in 1990 .", "the in situ nests of p. emaciata were tested once each in a similar fashion .", "we used a non - parametric wilcoxon test for species differences in the probability of immediate attack responses using the four p. emaciata and five p. occidentalis observation colonies .", "trials were pooled within colonies and each colony was used as a single data point in the analysis .", "all observation and in situ polybia colonies contained brood ( larvae and/or pupae were visible in the lower combs ) when they were tested . based on forager traffic and on the numbers of wasps exiting the nests during attacks", "polybia emaciata only rarely ( 5 occasions out of 40 trials on the observation nests and 2 occasions out of > 15 trials on in situ nests ) exhibited a typical polybia attack response within 30 sec when the nest was tapped .", "polybia emaciata workers never rushed out of the nest and onto the nest envelope . in all trials where the wasps did not attack in response to the initial mechanical disturbance ( within the first 30 sec ) ,", "some of the workers on the nest surface flew off the nest and departed ; the remainder rapidly entered the nest .", "after the initial disturbance , the nest surface and the lowest layer of comb ( partially visible through the nest entrance , fig .", "breathing into the nest entrance did not elicit rapid ( within 10 sec ) attack responses .", "after continued disturbance within a trial ( breath and tapping ) , p. emaciata workers often attacked violently . in approximately 75% of trials ,", "several dozen individuals rapidly exited the nest , took flight , and struck and attempted to sting the observer 's bee veil .", "the p. emaciata nests were not abandoned following these attacks , and workers later returned to the nests .", "in contrast , all 50 nest - tapping trials with p. occidentalis resulted in immediate ( within 1 sec ) exit - nest and attack responses .", "the species difference in the per - colony probability of nest exit and attack within 30 sec of disturbance was significant ( fig .", "2 ; wilcoxon 2-tailed test , z=2.60 , p<0.01 ) . breathing in or on p. occidentalis nests always elicited immediate attack responses , therefore , habituation to tapping and breathing stimuli was not evident over the course of the study in p. occidentalis .", "the four p. emaciata observation nests suffered no damage while exposed to nearly daily heavy rains for 3 weeks ; four of the p. occidentalis observation nests in the same location were visibly damaged by rain during the same period . even during heavy downpours", ", the mud nests repelled water much longer than nearby p. occidentalis nests . during one rain shower", ", the mud nests did not appear waterlogged until 15 min of rainfall had elapsed , while p. occidentalis nests stopped shedding water and became waterlogged after < 5 min .", "the nest fell 2 m to the ground , with only minor damage to its envelope , and the nest was still occupied by adult wasps when it was collected within 12 h of falling .", "rather than exiting and attacking within a few sec of vibrational disturbance , p. emaciata workers usually either departed or fled into the nest interior . unlike many other paper wasps ( s.o'd . and r.l.j . , personal observation ) , p. emaciata failed to respond aggressively to human breath , even when we blew directly into the nest entrance .", "polybia emaciata behavioral responses to nest disturbance were unusual among their congeners , and among paper - nesting epiponini with similar colony sizes ( s.o'd and r.l.j . personal observation ; chadab , 1979 ) . however , a few species of paper wasps in other genera exhibit similar responses to human disturbance , which chadab ( 1979 ) labeled ", "richards ( 1978 ) collected a p. emaciata colony in brazil , and the wasps did not exit the nest until 1.5 h after it was collected .", "residents of costa rica from several rural communities reported collecting and moving active p. emaciata colonies without being stung , by placing a finger over the nest entrance and plucking the nest from its attachment point .", "chadab ( 1979 ) noted that one colony of p. emaciata in ecuador had  durable  nest paper and exhibited defensive retreat responses like those that we recorded ; two other p. emaciata colonies had  fragile  nests and performed exit - nest and attack behavior when jarred .", "we did not observe qualitative variation in p. emaciata nest durability in panama ( > 50 colonies observed ; not all were tested for defensive responses ) .", "predation on swarm - founding wasp nests by bats ( jeanne , 1970a ) , birds ( skutch , 1971 ; windsor , 1976 ) , and primates ( vecht , 1967 ) has been documented .", "we hypothesize that the unusual defensive response of p. emaciata is a behavioral adaptation to its use of mud in nest construction , which may make the nest more resistant than paper to entry by vertebrate predators .", "it appears that the wasps initially rely on the nest itself , rather than on exit and attack behavior , to thwart vertebrate predators .", "we suggest that this represents a special type of architectural defense ( hermann and blum , 1981 ) , where the defensive behavior of a species has been modified to exploit the properties of its nest material", "skutch ( 1959 ) observed a red throated caracara ( daptrius americanus ) removing combs from a p. emaciata nest and feeding on the brood in costa rica .", "our subject nests often responded with attack behavior after extended disturbances , suggesting that attack can be effective for this species .", "when attack did come it was sudden and massive , suggesting that it was coordinated by an alarm pheromone , as has been demonstrated for p. occidentalis ( jeanne , 1981 ) .", "although army ants are among swarm - founding wasps ' most frequent predators in wet tropical habitats , preliminary tests on the reaction to eciton army ants did not suggest that p. emaciata 's mud nest is effective in resisting these predators ( chadab , 1979 ; s.o'd . personal observation ) .", "the use of mud as a nesting material may have evolved in response to predation pressure , particularly by vertebrate enemies .", "similar advantages against hornet ( vespa ) predation may have favored the evolution of mud construction in hover wasps of the genus liostenogaster ( hansell 1984 ; turillazzi 1991 ) . however , mud construction appears to provide other benefits .", "our observations suggest that p. emaciata mud nests were more water repellent , and more resistant to mechanical damage , than similarly sized paper nests .", "even if increases in general durability were the original selective advantage driving the evolution of mud construction , the workers ' defensive behavior has apparently been secondarily modified .", "there is variability in average colony size and in the details of nest architecture among species within the mud - nesting polybia subgenus pedothoeca , and in the independently evolved mud - nester p. furnaria ( richards , 1978 ; carpenter et al . , 2000 ) .", "of special value to our understanding of the evolutionary significance of the mud - nesting habit will be field studies on the defensive behaviors of the other mud - nesting polybia species .", "in particular , such studies could establish whether retreat behavior in response to vertebrate disturbance always accompanies the use of mud in nest building .", "useful comparisons of defensive behavior could also be made to epiponine paper - nesters with relatively strong nest paper , such as epipona and chartergus .", "retreat behavior in response to vertebrate disturbance is not restricted to mud - nesting species of epiponini .", "similar responses to human approach or nest vibration have been observed in paper- and secretion - nesting species ( protopolybia emortualis , protopolybia exigua , and protopolybia ( formerly pseudochartergus ) fuscatus ) and in cavity - nesting agelaia cajennensis ( jeanne , 1970b ; chadab , 1979 ; carpenter et al . , 2001 ) .", "paper - nesting polybia jurenei colonies retreated when their nest tree was rubbed , but exited and attacked when the substrate was jarred ( s. o'd .", "these patterns suggest that retreat behavior has evolved convergently in several lineages of epiponini , perhaps in response to particular types of predators .", "photograph of a mud nest of polybia emaciata collected july 1998 in gamboa , panama .", "box plots showing the range of probabilities of response to nest disturbance with attack within 30 sec by polybia emaciata and p. occidentalis colonies ."], "abstract_text": ["<S> the swarm - founding wasp polybia emaciata is unusual among eusocial vespidae because it uses mud , rather than wood pulp , as its primary nest construction material . </S>", "<S> polybia emaciata nests are more durable than similarly sized paper nests . </S>", "<S> we tested the hypothesis that the defensive behavior of this wasp may have been modified to take advantage of their strong nests in defense against vertebrate attacks . </S>", "<S> we simulated vertebrate disturbances by tapping on , and breathing in , p. emaciata nests and similarly sized p. occidentalis paper nests in the same location at the same time . </S>", "<S> polybia emaciata responses to disturbance were qualitatively different from those of p. occidentalis . </S>", "<S> the latter exit the nest and attack , while p. emaciata workers typically fled or entered the nest , attacking only after repeated and extended disturbances . </S>", "<S> we conclude that durable nest material may permit predator avoidance behavior in p. emaciata . </S>", "<S> we compare the defensive responses of p. emaciata workers with those of other swarm - founding vespidae , and discuss several selective forces that could cause the evolution of species variation in nest defense behavior . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Figures and Tables"], "sections": [["among eusocial insects , paper wasps ( hymenoptera : vespidae : polistinae ) are notable for the diversity of their nest architecture ( jeanne , 1975 ; wenzel , 1991 ) .", "many swarm - founding polistine wasps , including most species of the neotropical tribe epiponini , construct envelopes around their brood combs .", "comparative and experimental studies suggest that , among other functions , nest envelopes reduce rates of predation and parasitism on wasp brood ( london and jeanne , 1998 ; smith et al . , 2001 ) .", "however , little is known about the adaptive significance of variation in the materials that wasps use to construct nest envelopes ( hansell 1984 ; wenzel , 1991 ; cole et al . , 2001 ) .", "polybia emaciata is unusual among the polistine wasps because it uses mud , rather than wood pulp or plant fibers , as the main raw material for nest construction ( schremmer , 1984 ) .", "the genus polybia includes 56 described species ; of these , only the other three species in the subgenus pedothoeca , and one species in the subgenus furnariana , share the derived trait of mud nest construction ( richards , 1978 ; jeanne , 1991 ; cooper , 1993 ) .", "no other swarm - founding polistine wasps are known to build primarily mud nests , although small amounts of inorganic material are sometimes incorporated into the nest paper of other species ( wenzel , 1991 ) .", "the nest of p. emaciata is otherwise typical of the genus ( jeanne , 1975 ; wenzel , 1991 ) , being phragmocyttarus ( comprising horizontal layers of comb that are conjoined and enclosed by a continuous envelope ) with a single small entrance hole near the bottom of the nest .", "numbers of adults in mature colonies are relatively small for the genus , ranging from fewer than 100 up to about 500 adults ( richards , 1978 ; chadab , 1979 ; strassmann et al . , 1992 ) .", "polybia emaciata nests are hard and much more durable than most paper nests of similar size and shape ( rau , 1933 ; richards , 1978 ) .", "for example , skutch ( 1971 ) noted that nests of this species in costa rica were unlike paper nests in that they persisted for several years .", "the high durability of p. emaciata construction material suggests that the nest itself may provide greater brood protection than paper nests .", "if so , we hypothesized that the wasps ' defensive responses to vertebrate attacks have changed to take advantage of nest durability .", "we predicted that the nests ' protective properties reduced the need for behavioral defenses by p. emaciata workers , relative to paper - nesting polybia species with similar colony and nest sizes .", "we tested this prediction by disturbing p. emaciata colonies , and comparing the workers ' responses to those of simultaneously observed paper - nesting p. occidentalis colonies in the same location .", "chadab ( 1979 ) surveyed the defensive responses of swarm - founding paper wasps to vertebrate - like disturbances , including tapping on the nest and/or on the supporting vegetation . the most frequently observed response was for dozens to hundreds of workers to rush out onto the nest envelope , sometimes adopting aposematic postures ( see also o'donnell et al . ,", "response was seen in the genera angiopolybia , charterginus , leipomeles , parachartergus , polybia , pseudopolybia , and synoeca , and accords with our observations of what appears to be typical epiponine defensive behavior .", "weaker exit - nest responses ( either requiring several taps , or involving fewer wasps ) were seen in species of leipomeles , metapolybia , and protopolybia .", "interspecific variation in mature colony size was not a good predictor of the type of defensive response , as species with colony sizes smaller and larger than p. emaciata performed nest exiting defensive behavior .", "an attack response often coincides with , or quickly follows , the nest exit response and involves workers flying from the nest , striking the intruder , and attempting to sting ( hermann and blum , 1981 ) .", "when paper - using polybia nests are tapped , some of the workers on the nest surface may immediately perform attacks , while large numbers of workers rush out of the nest entrance and stand in alert posture on the envelope ( jeanne , 1981 ; jeanne et al . , 1992 ) .", "further tapping elicits additional attacks . breathing on the nest surface or into the nest results in immediate attack by several to dozens of workers .", "we discuss the possible adaptive significance of deviations from the typical nest exit that have been documented in several paper wasp species , including p. emaciata ."], ["this study was conducted in and near the town of gamboa , panama province , republic of panama ( 0907n 7942w ; 50 m elevation ) .", "observations and manipulations were conducted during the wet season from 1 august to 15 october 1990 and from 3 to 5 july 1998 .", "surveys of eusocial wasp colonies during these periods indicated that p. emaciata was among the most abundant swarm - founding wasps in terms of nest density ( s.o'd . , unpublished data ) .", "most of the data presented were collected from four colonies of p. emaciata and five colonies of p. occidentalis that had been moved approximately 0.5 km to 1 km from their natural nesting sites to artificial supports to facilitate observation .", "all subject colonies were moved into a small area ( < 0.25 ha ) , with similar amounts of tree cover ( one p. occidentalis colony was additionally sheltered by building eaves ) .", "all colonies were in place at least 2 weeks before observations on defense behavior were initiated , and all continued to forage and to raise brood ( visible through the nest entrance in the lower combs ) throughout the observation period .", "the p. emaciata observation colonies had nests ranging in size from 9 cm long to 22 cm long .", "the numbers of adult wasps and colony age probably varied positively with nest size , though we did not measure these variables .", "we also collected data opportunistically from p. emaciata colonies in situ in 1990 and again in 1998 .", "these nests were located in the gamboa area on thin tree branches from 1.5 m to 5 m above the ground .", "we disturbed p. emaciata and p. occidentalis colonies in an effort to evoke defensive behavior , using stimuli that elicited nest exit and attack responses in costa rican p. occidentalis ( s.o'd . and", "we disturbed the polybia nests by first tapping on the side of the outer envelope near the middle of the nest for 30 sec while wearing a full bee suit , and we noted the responses of on - nest and in - nest workers .", "if there was no attack response to tapping , we blew into the nest entrance for 30 sec .", "each polybia observation colony was disturbed 10 times over the course of the study period in 1990 .", "the in situ nests of p. emaciata were tested once each in a similar fashion .", "we used a non - parametric wilcoxon test for species differences in the probability of immediate attack responses using the four p. emaciata and five p. occidentalis observation colonies .", "trials were pooled within colonies and each colony was used as a single data point in the analysis ."], ["all observation and in situ polybia colonies contained brood ( larvae and/or pupae were visible in the lower combs ) when they were tested . based on forager traffic and on the numbers of wasps exiting the nests during attacks", "polybia emaciata only rarely ( 5 occasions out of 40 trials on the observation nests and 2 occasions out of > 15 trials on in situ nests ) exhibited a typical polybia attack response within 30 sec when the nest was tapped .", "polybia emaciata workers never rushed out of the nest and onto the nest envelope . in all trials where the wasps did not attack in response to the initial mechanical disturbance ( within the first 30 sec ) ,", "some of the workers on the nest surface flew off the nest and departed ; the remainder rapidly entered the nest .", "after the initial disturbance , the nest surface and the lowest layer of comb ( partially visible through the nest entrance , fig .", "breathing into the nest entrance did not elicit rapid ( within 10 sec ) attack responses .", "after continued disturbance within a trial ( breath and tapping ) , p. emaciata workers often attacked violently . in approximately 75% of trials ,", "several dozen individuals rapidly exited the nest , took flight , and struck and attempted to sting the observer 's bee veil .", "the p. emaciata nests were not abandoned following these attacks , and workers later returned to the nests .", "in contrast , all 50 nest - tapping trials with p. occidentalis resulted in immediate ( within 1 sec ) exit - nest and attack responses .", "the species difference in the per - colony probability of nest exit and attack within 30 sec of disturbance was significant ( fig .", "2 ; wilcoxon 2-tailed test , z=2.60 , p<0.01 ) . breathing in or on p. occidentalis nests always elicited immediate attack responses , therefore , habituation to tapping and breathing stimuli was not evident over the course of the study in p. occidentalis .", "the four p. emaciata observation nests suffered no damage while exposed to nearly daily heavy rains for 3 weeks ; four of the p. occidentalis observation nests in the same location were visibly damaged by rain during the same period . even during heavy downpours", ", the mud nests repelled water much longer than nearby p. occidentalis nests . during one rain shower", ", the mud nests did not appear waterlogged until 15 min of rainfall had elapsed , while p. occidentalis nests stopped shedding water and became waterlogged after < 5 min .", "the nest fell 2 m to the ground , with only minor damage to its envelope , and the nest was still occupied by adult wasps when it was collected within 12 h of falling ."], ["rather than exiting and attacking within a few sec of vibrational disturbance , p. emaciata workers usually either departed or fled into the nest interior . unlike many other paper wasps ( s.o'd . and r.l.j . , personal observation ) , p. emaciata failed to respond aggressively to human breath , even when we blew directly into the nest entrance .", "polybia emaciata behavioral responses to nest disturbance were unusual among their congeners , and among paper - nesting epiponini with similar colony sizes ( s.o'd and r.l.j . personal observation ; chadab , 1979 ) . however , a few species of paper wasps in other genera exhibit similar responses to human disturbance , which chadab ( 1979 ) labeled ", "richards ( 1978 ) collected a p. emaciata colony in brazil , and the wasps did not exit the nest until 1.5 h after it was collected .", "residents of costa rica from several rural communities reported collecting and moving active p. emaciata colonies without being stung , by placing a finger over the nest entrance and plucking the nest from its attachment point .", "chadab ( 1979 ) noted that one colony of p. emaciata in ecuador had  durable  nest paper and exhibited defensive retreat responses like those that we recorded ; two other p. emaciata colonies had  fragile  nests and performed exit - nest and attack behavior when jarred .", "we did not observe qualitative variation in p. emaciata nest durability in panama ( > 50 colonies observed ; not all were tested for defensive responses ) .", "predation on swarm - founding wasp nests by bats ( jeanne , 1970a ) , birds ( skutch , 1971 ; windsor , 1976 ) , and primates ( vecht , 1967 ) has been documented .", "we hypothesize that the unusual defensive response of p. emaciata is a behavioral adaptation to its use of mud in nest construction , which may make the nest more resistant than paper to entry by vertebrate predators .", "it appears that the wasps initially rely on the nest itself , rather than on exit and attack behavior , to thwart vertebrate predators .", "we suggest that this represents a special type of architectural defense ( hermann and blum , 1981 ) , where the defensive behavior of a species has been modified to exploit the properties of its nest material", "skutch ( 1959 ) observed a red throated caracara ( daptrius americanus ) removing combs from a p. emaciata nest and feeding on the brood in costa rica .", "our subject nests often responded with attack behavior after extended disturbances , suggesting that attack can be effective for this species .", "when attack did come it was sudden and massive , suggesting that it was coordinated by an alarm pheromone , as has been demonstrated for p. occidentalis ( jeanne , 1981 ) .", "although army ants are among swarm - founding wasps ' most frequent predators in wet tropical habitats , preliminary tests on the reaction to eciton army ants did not suggest that p. emaciata 's mud nest is effective in resisting these predators ( chadab , 1979 ; s.o'd . personal observation ) .", "the use of mud as a nesting material may have evolved in response to predation pressure , particularly by vertebrate enemies .", "similar advantages against hornet ( vespa ) predation may have favored the evolution of mud construction in hover wasps of the genus liostenogaster ( hansell 1984 ; turillazzi 1991 ) . however , mud construction appears to provide other benefits .", "our observations suggest that p. emaciata mud nests were more water repellent , and more resistant to mechanical damage , than similarly sized paper nests .", "even if increases in general durability were the original selective advantage driving the evolution of mud construction , the workers ' defensive behavior has apparently been secondarily modified .", "there is variability in average colony size and in the details of nest architecture among species within the mud - nesting polybia subgenus pedothoeca , and in the independently evolved mud - nester p. furnaria ( richards , 1978 ; carpenter et al . , 2000 ) .", "of special value to our understanding of the evolutionary significance of the mud - nesting habit will be field studies on the defensive behaviors of the other mud - nesting polybia species .", "in particular , such studies could establish whether retreat behavior in response to vertebrate disturbance always accompanies the use of mud in nest building .", "useful comparisons of defensive behavior could also be made to epiponine paper - nesters with relatively strong nest paper , such as epipona and chartergus .", "retreat behavior in response to vertebrate disturbance is not restricted to mud - nesting species of epiponini .", "similar responses to human approach or nest vibration have been observed in paper- and secretion - nesting species ( protopolybia emortualis , protopolybia exigua , and protopolybia ( formerly pseudochartergus ) fuscatus ) and in cavity - nesting agelaia cajennensis ( jeanne , 1970b ; chadab , 1979 ; carpenter et al . , 2001 ) .", "paper - nesting polybia jurenei colonies retreated when their nest tree was rubbed , but exited and attacked when the substrate was jarred ( s. o'd .", "these patterns suggest that retreat behavior has evolved convergently in several lineages of epiponini , perhaps in response to particular types of predators ."], ["photograph of a mud nest of polybia emaciata collected july 1998 in gamboa , panama .", "box plots showing the range of probabilities of response to nest disturbance with attack within 30 sec by polybia emaciata and p. occidentalis colonies ."]]}
{"article_id": "PMC3303893", "article_text": ["human gliomas represent 50% to 60% of all intracranial tumors . according to the world health organization ( who ) guidelines ,", "gliomas are histologically classified into four grades : pilocytic astrocytoma ( grade i ) , low - grade diffuse astrocytoma ( grade ii ) , anaplastic astrocytoma ( grade iii ) , and glioblastoma multiforme ( gbm , grade iv ) . both diagnostic technologies and therapeutic strategies", "the 5-year survival rates of low - grade ( grade i~ii ) and high - grade ( grade iii~iv ) glioma patients in china are 75.4% and 18.2% , respectively .", "especially , the median survival time for patients with gbm is still only 12 months . indeed ,", "early diagnosis and prolonging survival in glioma patients remains a great challenge for clinicians in the field of neurooncology .", "there have been several prognostic factors for glioma patients , such as age , preoperative duration of symptoms , karnofsky performance status ( kps ) score , histologic grade , tumor necrosis , surgical resection extent , use of postoperative radiation therapy , and , probably , adjuvant chemotherapy .", "however , these clinical parameters can not completely account for the observed variation in survival because of the heterogeneity of glioma patients .", "thus , there is an urgent need to further investigate the molecular mechanisms of glioma and to identify the effective prognostic indicators for survival prediction.the dna - base excision repair ( ber ) pathway is responsible for the repair of exogenous and endogenous alkylating and oxidative dna damage , which may lead to carcinogenesis , cell death , and aging if left unrepaired", "this pathway involves the recognition and removal of damaged bases by a dna glycosylase , followed by incision of the resulting abasic ( ap ) site by ap endonuclease , dna synthesis by polymerase , and strand ligation by dna ligase .", "thus , the ber pathway is an important candidate for intervention into the cellular responses to dna change .", "n - methylpurine - dna glycosylase ( mpg ) as a dna repair enzyme is a main component in the ber pathway . in previous study aimed at understanding the significance of initiating lesions removed by the ber pathway , kaina et al .   identified the over - expression of the human mpg in chinese hamster ovary cells . in the n - alkylpurine repair process , mpg is responsible for the glycolytic removal of the modified base , which leads to the formation of apurinic sites .", "although n - alkylpurines have not been found to be directly mutagenic , apurinic sites left by this repair process can block replication and lead to mutation .", "mpg also participates in the repair of 8-hydroxyguanine and hypoxanthine . because of the potential role of dna base lesions in mutagenesis and carcinogenesis ,", "a number of studies have been performed to investigate the association of mpg with various human cancers .", "( 1998 )   detected the increased mpg gene and protein expression in the breast cancer cells versus normal breast epithelial cells by northern analysis , southern blots , immunofluorescence , immunohistochemistry , and western blot analysis . in 2001 , sohn et al .  ", "reported that the expression of mpg was increased in high - risk hpv - infected cervical neoplasias and the intracellular distribution of mpg protein was altered , suggesting a role of mpg in carcinogenesis . in an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change ,", "many researchers have been interested in the effects of mpg in tumor sensitivity to the clinical chemotherapeutic agents . as their results , mpg - overexpressing ovarian cancer , osteosarcoma , and", "breast cancer   cells are significantly more sensitive to the clinical chemotherapeutic agents , suggesting that the overexpression of mpg may be a possible gene therapy approach to sensitize tumor cells to the cell - killing effects of chemotherapeutic alkylating agents . the biological mechanism behind the increase of sensitivity to the chemotherapeutic agents in mpg overexpressing cell lines may be that the balance between glycosylase activity , leading to apurinic sites and formation of strand breaks , and subsequent excision repair processes may play an important role in determining cellular cytotoxicity and resistance to alkylating agents . of our interests , we focus on the involvement of mpg in human gliomas .", "recent study has demonstrated that mpg mrna expression was higher in astrocytic tumor tissues than in brain tissues adjacent to tumor , and mpg protein localization in immunohistochemical study was detected only in the nucleus of all tumor tissues , suggesting an mpg 's role in human astrocytic tumors and raise the possibility that the altered mpg expression and intracellular localization could be associated with astrocytic tumorigenesis .", "tang et al .   further demonstrated that mpg overexpression , together with the inhibition of ber , could sensitize glioma cells to the alkylating agent .", "however , little is known about the expression level of mpg in human gliomas with different clinical grades and its prognostic significance . to address this problem", ", we used quantitative real - time pcr , immunohistochemistry assay , and western blot analysis to investigate the expression pattern of mpg gene and protein in glioma specimens and normal control brain tissues .", "next , we analyzed the relationship between mpg expression and the glioma stage as well as the survival of patients .", "this study was approved by the research ethics committee of the institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r . china . written informed consent", "a total of 128 formalin - fixed , paraffin - embedded specimens of gliomas resected between 2000 and 2010 were retrieved from the archives of the pathology department of tangdu hospital , fourth military medical university , p.r .", "all the slides were reevaluated according to who classifications   by two pathologists , with differences resolved by careful discussion .", "a total of 76 males and 52 females ( 1.46  :  1 ) were enrolled in this study , and the median age was 42 years ( range , 1271 ) .", "thirty - two of the 128 gliomas were classified as low - grade [ 18 pilocytic astrocytomas ( who i ) and 14 diffuse astrocytomas ( who ii ) ] , and 96 were classified as high - grade gliomas [ 38 anaplasia astrocytomas ( who iii ) , and 58 primary glioblastomas ( who iv ) ] .", "the clinicopathological features , and the treatment strategies of all the patients were indicated in table 1 .", "paraffin and snap - frozen sections of nonneoplastic brain tissues from 10 patients with intractable epilepsy were also included as controls .", "five - year followup was performed , and all patients had complete followup until death .", "overall survival time was calculated from the date of the initial surgical operation to death .", "patients , who died of diseases not directly related to their gliomas or due to unexpected events , were excluded from this study .", "in addition , 20 glioma specimens [ 5 pilocytic astrocytomas ( who i ) , 3 diffuse astrocytomas ( who ii ) , 3 anaplasia astrocytomas ( who iii ) , and 9 primary glioblastomas ( who iv ) ] were snap - frozen in liquid nitrogen and stored at 80c following surgery for mrna and protein isolation . total rna purified from all 20 glioma tissues and 10 control brain tissues", "real - time monitoring of polymerase chain reactions ( pcrs ) was performed using the abi 7900ht ( idaho technology , idaho falls , i d , usa ) .", "cdna was synthesized using random primers and oligo 18dt and superscript ii reverse transcriptase ( invitrogen ) .", "gene expression was determined using the sybr green i dye ( biogene ) , which binds preferentially to double - stranded dna , and 10  ng of template .", "fluorescence signals , which are proportional to the concentration of the pcr product , are measured at the end of each cycle and immediately displayed on a computer screen , permitting real - time monitoring of the pcr .", "the reaction is characterized by the point during cycling when amplification of pcr products is first detected , rather than the amount of pcr product accumulated after a fixed number of cycles . the higher the starting quantity of the template , the earlier a significant increase in fluorescence is observed .", "the threshold cycle is defined as the fractional cycle number at which fluorescence passes a fixed threshold above the baseline .", "the primers 5-gtc cta gtc cgg cga ctt cc-3 and 5-ctt gtc tgg gca ggc cct ttg c-3 were used to amplify 603-bp transcripts of mpg , and the primers 5-ggt ggc ttt tag gat ggc aag3 and 5-act gga acg gtg aag gtg aca g3 were used to amplify 161-bp transcripts of -actin .", "the pcr profile consisted of an initial melting step of 1  min at 94c , followed by 38 cycles of 15  s at 94c , 15  s at 56c , and 45  s at 72c , and a final elongation step of 10  min at 72c .  ", "fluorescence data were converted into cycle threshold measurements using the sds system software and exported to microsoft excel .", "thermal dissociation plots were examined for biphasic melting curves , indicative of whether primer dimers or other nonspecific products could be contributing to the amplification signal .", "twenty glioma tissues and 10 control brain tissues were homogenized in lysis buffer [ pbs , 1% nonidet p-40 ( np-40 ) , 0.5% sodium deoxycholate , 0.1% sodium dodecyl sulfate ( sds ) , 100  ug / ml aprotinin , 100  g / ml phenylmethylsulfonyl fluoride ( pmsf ) , sodium orthovanadate ] at 4c throughout all procedures , and sonicated for 70  s , then add 300  g pmsf per gram of tissue and incubate on ice for 30  min , followed by centrifugation at 15,000  rpm for 20  min at 4c .", "the protein content was determined according to bradford 's method , with bovine serum albumin used as a standard .", "protein samples ( 30  g ) were boiled with 2  sample buffer containing 5%  -mercaptoethanol for 5  min , separated by size on 15% polyacrylamide gel under sds denaturing conditions , and transferred to a nitrocellulose membrane at 90  v for 2  h. the nitrocellulose membranes were stained with ponceau s to assess the efficiency of transfer .", "nonspecific binding was blocked by incubation in block buffer ( 5% nonfat dry milk , 0.05% tween-20 , 1  tris - cl - buffered saline ) overnight at 4c , the membranes were hybridized with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) , then incubated with a horseradish peroxidase - labeled goat anti - mouse igg ( 1  :  500 ) .", "the bound secondary antibody was detected by enhanced chemiluminescence ( amersham life science , little chalfont , uk ) .", "positive immunoreactive bands were quantified densitometrically ( leica q500iw image analysis system ) and expressed as ratio of mpg to -actin in optical density units .  ", "immunohistochemical assay was performed using the conventional immunoperoxidase technique according to the protocol of the department of neurosurgery , institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r .", "briefly , following peroxidase blocking with 0.3% h2o2/methanol for 30  min , specimens were blocked with phosphate - buffered saline ( pbs ) containing 5% normal horse serum ( vector laboratories inc . ,", "all incubations with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) at 1  :  500 dilution were carried out overnight at 4c .", "then the specimens were briefly washed in pbs and incubated at room temperature with the anti - mouse antibody and avidin - biotin peroxidase ( vector laboratories inc . ,", "the specimens were then washed in pbs and color developed by diaminobenzidine solution ( dako corporation , carpinteria , ca , usa ) . after washing with water , specimens were counterstained with meyer 's hematoxylin ( sigma chemical co. , st louis , mo , usa ) .", "nonneoplastic brain tissues were used as control tissues , and nonimmune igg was also used as negative control antibody for immunohistochemical staining .", "immunostaining was scored by two independent experienced pathologists , who were blinded to the clinicopathologic parameters and clinical outcomes of the patients .", "5% scored 0 , 625% scored 1 , 2650% scored 2 , 5175% scored 3 , > 75% scored 4 ; ( 2 ) intensity of stain : colorless scored 0 , pallide - flavens scored 1 , yellow scored 2 , brown scored 3 .", "the staining score was stratified as  ( 0 score , absent ) , + ( 14 score , weak ) , + + ( 58 score , moderate ) , and + + + ( 912 score , strong ) according to the proportion and intensity of positively stained cancer cells .", "all computations were carried out using the software of spss version13.0 for windows ( spss inc , il , usa ) .", "randomized block design anova was used to analyze the statistical difference among different tissue types . in the analysis of glioma morbidity for all patients", ", we used the kaplan - meier estimator and univariate cox regression analysis to assess the marginal effect of each factor .", "a spearman 's analysis was carried out to analyze the correlation between mpg mrna and protein expression levels .", "the gene expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was detected by real - time pcr . as shown in figure 2 ,", "the expression levels of mpg gene in human glioma tissues were conspicuously higher than those in control brain tissues ( p < 0.001 ) .", "interestingly , mpg gene expression increased with the advancement of who grades from i to iv ( p < 0.001 ) .", "the protein expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was also detected by western blot analysis .", "as the results , the expression levels of mpg protein tended to increase from the glioma to the normal tissue ( figures 3(a ) and 3(c ) ) .", "mpg expression was highest in grade iv and lowest in grade i ( figures 3(b ) and 3(c ) ) , indicating a close correlation of mpg protein expression with who grade .", "there was a significant positive correlation between the expression of mpg gene and protein expression levels from the same glioma tissues ( rs = 0.82 , p < 0.001 ) .", "the expression and the subcellular localization of mpg protein in 10 nonneoplastic brain and 128 glioma tissues were detected by immunohistochemistry assay .", "as shown in figure 4 , the positive staining for mpg was mainly observed in the nuclei of tumor cells in glioma tissues , which was consistent with the previous study of wang et al .  ", "the representative photographs were shown in figures 4(a ) and 4(b ) . among the glioma specimens , 102 ( 79.7% ) glioma specimens", "were positively stained for mpg , whereas its immunoreactivities in nonneoplastic brain sections ranged from undetectable to low ( figure 4(c ) ) .", "additionally , the immunostaining of mpg in nonneoplastic brain tissues is below detection levels of both real - time pcr and western blot . according to the statistical analysis ,", "the expression level of mpg protein in glioma tissues was significantly higher ( p < 0.001 ) than that in nonneoplastic brain tissues , which was consistent with the results of western blot analysis .  ", "in addition , mpg expression was not significantly affected by the gender and age ( both p > 0.05 ) of the patients .", "in contrast , the mpg expression was the closely correlated with who grade , as well as kps .", "the expression levels of mpg protein in glioma tissues with higher who grade ( table 1 ; p = 0.002 ) and lower kps ( table 1 ; p = 0.01 ) were significantly higher than those with lower who grade and higher kps .", "moreover , we reviewed clinical information of these mpg - positive or -negative glioma patients . during the follow - up period ,", "100 of the 128 glioma patients ( 78.1% ) had died ( 24 from the mpg - negative group and 76 from the mpg - positive group ) . as determined by the log - rank test ,", "the survival rate of patients with positive mpg staining was lower than those without mpg staining ( p < 0.001 ; figure 5(a ) ) .", "the median survival time of patients with negative expression of mpg could not be estimated by statistical analysis because all patients survived better than the overall median level , and those patients with strong positive ( + + + ) , moderate positive ( + + ) , and weak positive ( + ) of mpg were 9.1  1.5 months , 12.3  1.1 months , and 22.6  2.2 months ( log - rank test : p < 0.001 ) .", "furthermore , figure 5(b ) shows the postoperative survival curve of patients with glioma and mpg expression after adjusting for age , gender , who grade , and kps . by multivariate analysis ( table 2 ) ,", "the loss of mpg expression was a significant ( p < 0.001 ) and independent prognostic indicator for patients with glioma besides age , who grade and kps .", "the cox - proportional hazards model showed that mpg over - expression was associated with poor overall survival .", "because of the dismal survival in the patients with glioma , especially in gbm patients , there is an urgent need to find novel specific and effective prognostic markers in order to apply individualized treatments to specifically target the pathophysiologic and molecular properties of glioma patients . in the current study", ", we investigated the expression of mpg in 128 cases of human glioma and compared the expression with tumor grade and survival rates of patients .", "our data demonstrated that mpg gene and protein were both increased in glioma compared to nonneoplastic brain tissue .", "we found an increased trend of both mpg protein level and gene level from who grade i to who grade iv glioma .", "these results suggest that the transcriptional and translational activation of human mpg might participate in the tumorigenesis and progression of glioma .", "dna in all somatic and germ cells in the body are continuously exposed to exogenous and endogenous alkylating and oxidative agents that result in damage to dna .", "if dna damage was not repaired , it may lead to genetic mutation , chromosome aberration , aging , and carcinogenesis .", "the lack of precise dna repair activities may lead to defective embryogenesis , tissue - specific dysfunction , hypersensitivity to dna - damaging agents , premature senescence , genetic instability , and elevated cancer rates .", "the repair of dna bases damaged by alkylation is initiated in mammalian cells by mpg , as an alkyladenine dna glycosylase .", "the majority of repair that is initiated by mpg occurs via short - patch ber , a mechanism whereby only one nucleotide is replaced .", "the majority of base damage induced by methylation is removed by mpg , including the principal adducts 3-mea and 7-meg .", "it has been reported that the expression of mpg could be induced by viral damage and a variety of dna - damaging agents .", "for example , the increased expression of mpg gene was studied in breast cancer , cervical neoplasia , and thymic carcinoma in sv 40 t - antigen - expressing transgenic mice . as previous investigation , mammalian cells actively regulated dna repair enzymes and genes during cell proliferation .", "dna repair enzymes are expressed in the cell cycle in a defined temporal pattern relative to the induction of dna replication . in brain cells that are not undergoing dna replication ,", "kim et al .   indicated that mpg expression in the brain was relative high in 1 week after birth , and the level remained low in day 400 mature adults , suggesting that brain tissue is terminally differentiated and nonproliferating tissues .", "it has been demonstrated that dna repair enzymes play an important role in the carcinogenesis of several cancers .", "of our interests , mpg was overexpressed in breast cancer up to 24-fold as compared to normal primary breast epithelium , suggesting the role of mpg in breast carcinogenesis . in astrocytomas ,", "kim et al .   in 2003 reported that mpg mrna level was significantly higher than that in tumor - adjacent brain tissues . with the similar results", ", we in this study also found the upregulation of mpg protein and gene in glioma tissues .", "additionally , we provide evidence that mpg protein is found in nuclear localization of the glioma tissues , which was consistent with the results of kim et al . .", "the nuclear localization of mpg in tumor cells of glioma tissues may be suggestive of a role of mpg in the regulation of cell division and cell cycle .", "furthermore , the most important finding of this study was the correlation of mpg expression and survival rates of patients .", "kaplan - meier and multivariate analysis both showed a significantly worse overall survival for patients whose tumors had high mpg levels , indicating that high mpg protein level is a marker of poor prognosis for patients with gliomas .", "this is the first investigation to demonstrate the prognostic value of mpg in human cancers .", "our data showed the over - expression of mpg gene and protein in human gliomas and also suggested for the first time that mpg be an unfavorable independent prognostic indicator for glioma patients ."], "abstract_text": ["<S> \n aim . to examine the expression of n - methylpurine - dna glycosylase \n ( mpg ) gene and protein in glioma samples with different who grades \n and its association with patients ' survival </S>", "<S> . methods . \n </S>", "<S> immunohistochemistry assay , quantitative real - time pcr and western \n blot analysis were carried out to investigate the expression of \n mpg gene and protein in 128 glioma and 10 non - neoplastic brain \n tissues . </S>", "<S> results . </S>", "<S> mpg gene expression level in glioma tissues was \n significantly higher than that in non - neoplastic brain tissues \n ( p < 0.001 ) . </S>", "<S> immunohistochemistry also showed that mpg protein was \n over - expressed in glioma tissues , which was consistent with the \n resutls of western blot analysis . </S>", "<S> additionally , the expression \n levels of mpg gene and protein both increase from grade i to grade \n iv glioma according to the results of real - time pcr , \n immunohistochemistry and western blot analysis . </S>", "<S> moreover , the \n survival rate of mpg - positive patients was significantly lower \n than that of mpg - negative patients ( p < 0.001 ) . </S>", "<S> we further confirmed \n that the over - expression of mpg was a significant and independent \n prognostic indicator in glioma by multivariate analysis ( p < 0.001 ) . \n </S>", "<S> conclusions . </S>", "<S> our data showed the over - expression of mpg gene and \n protein in human gliomas , and also suggested for the first time \n that mpg be an unfavorable independent prognostic indicator for \n glioma patients . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["human gliomas represent 50% to 60% of all intracranial tumors . according to the world health organization ( who ) guidelines ,", "gliomas are histologically classified into four grades : pilocytic astrocytoma ( grade i ) , low - grade diffuse astrocytoma ( grade ii ) , anaplastic astrocytoma ( grade iii ) , and glioblastoma multiforme ( gbm , grade iv ) . both diagnostic technologies and therapeutic strategies", "the 5-year survival rates of low - grade ( grade i~ii ) and high - grade ( grade iii~iv ) glioma patients in china are 75.4% and 18.2% , respectively .", "especially , the median survival time for patients with gbm is still only 12 months . indeed ,", "early diagnosis and prolonging survival in glioma patients remains a great challenge for clinicians in the field of neurooncology .", "there have been several prognostic factors for glioma patients , such as age , preoperative duration of symptoms , karnofsky performance status ( kps ) score , histologic grade , tumor necrosis , surgical resection extent , use of postoperative radiation therapy , and , probably , adjuvant chemotherapy .", "however , these clinical parameters can not completely account for the observed variation in survival because of the heterogeneity of glioma patients .", "thus , there is an urgent need to further investigate the molecular mechanisms of glioma and to identify the effective prognostic indicators for survival prediction.the dna - base excision repair ( ber ) pathway is responsible for the repair of exogenous and endogenous alkylating and oxidative dna damage , which may lead to carcinogenesis , cell death , and aging if left unrepaired", "this pathway involves the recognition and removal of damaged bases by a dna glycosylase , followed by incision of the resulting abasic ( ap ) site by ap endonuclease , dna synthesis by polymerase , and strand ligation by dna ligase .", "thus , the ber pathway is an important candidate for intervention into the cellular responses to dna change .", "n - methylpurine - dna glycosylase ( mpg ) as a dna repair enzyme is a main component in the ber pathway . in previous study aimed at understanding the significance of initiating lesions removed by the ber pathway , kaina et al .   identified the over - expression of the human mpg in chinese hamster ovary cells . in the n - alkylpurine repair process , mpg is responsible for the glycolytic removal of the modified base , which leads to the formation of apurinic sites .", "although n - alkylpurines have not been found to be directly mutagenic , apurinic sites left by this repair process can block replication and lead to mutation .", "mpg also participates in the repair of 8-hydroxyguanine and hypoxanthine . because of the potential role of dna base lesions in mutagenesis and carcinogenesis ,", "a number of studies have been performed to investigate the association of mpg with various human cancers .", "( 1998 )   detected the increased mpg gene and protein expression in the breast cancer cells versus normal breast epithelial cells by northern analysis , southern blots , immunofluorescence , immunohistochemistry , and western blot analysis . in 2001 , sohn et al .  ", "reported that the expression of mpg was increased in high - risk hpv - infected cervical neoplasias and the intracellular distribution of mpg protein was altered , suggesting a role of mpg in carcinogenesis . in an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change ,", "many researchers have been interested in the effects of mpg in tumor sensitivity to the clinical chemotherapeutic agents . as their results , mpg - overexpressing ovarian cancer , osteosarcoma , and", "breast cancer   cells are significantly more sensitive to the clinical chemotherapeutic agents , suggesting that the overexpression of mpg may be a possible gene therapy approach to sensitize tumor cells to the cell - killing effects of chemotherapeutic alkylating agents . the biological mechanism behind the increase of sensitivity to the chemotherapeutic agents in mpg overexpressing cell lines may be that the balance between glycosylase activity , leading to apurinic sites and formation of strand breaks , and subsequent excision repair processes may play an important role in determining cellular cytotoxicity and resistance to alkylating agents . of our interests , we focus on the involvement of mpg in human gliomas .", "recent study has demonstrated that mpg mrna expression was higher in astrocytic tumor tissues than in brain tissues adjacent to tumor , and mpg protein localization in immunohistochemical study was detected only in the nucleus of all tumor tissues , suggesting an mpg 's role in human astrocytic tumors and raise the possibility that the altered mpg expression and intracellular localization could be associated with astrocytic tumorigenesis .", "tang et al .   further demonstrated that mpg overexpression , together with the inhibition of ber , could sensitize glioma cells to the alkylating agent .", "however , little is known about the expression level of mpg in human gliomas with different clinical grades and its prognostic significance . to address this problem", ", we used quantitative real - time pcr , immunohistochemistry assay , and western blot analysis to investigate the expression pattern of mpg gene and protein in glioma specimens and normal control brain tissues .", "next , we analyzed the relationship between mpg expression and the glioma stage as well as the survival of patients ."], ["this study was approved by the research ethics committee of the institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r . china . written informed consent", "a total of 128 formalin - fixed , paraffin - embedded specimens of gliomas resected between 2000 and 2010 were retrieved from the archives of the pathology department of tangdu hospital , fourth military medical university , p.r .", "all the slides were reevaluated according to who classifications   by two pathologists , with differences resolved by careful discussion .", "a total of 76 males and 52 females ( 1.46  :  1 ) were enrolled in this study , and the median age was 42 years ( range , 1271 ) .", "thirty - two of the 128 gliomas were classified as low - grade [ 18 pilocytic astrocytomas ( who i ) and 14 diffuse astrocytomas ( who ii ) ] , and 96 were classified as high - grade gliomas [ 38 anaplasia astrocytomas ( who iii ) , and 58 primary glioblastomas ( who iv ) ] .", "the clinicopathological features , and the treatment strategies of all the patients were indicated in table 1 .", "paraffin and snap - frozen sections of nonneoplastic brain tissues from 10 patients with intractable epilepsy were also included as controls .", "five - year followup was performed , and all patients had complete followup until death .", "overall survival time was calculated from the date of the initial surgical operation to death .", "patients , who died of diseases not directly related to their gliomas or due to unexpected events , were excluded from this study .", "in addition , 20 glioma specimens [ 5 pilocytic astrocytomas ( who i ) , 3 diffuse astrocytomas ( who ii ) , 3 anaplasia astrocytomas ( who iii ) , and 9 primary glioblastomas ( who iv ) ] were snap - frozen in liquid nitrogen and stored at 80c following surgery for mrna and protein isolation . total rna purified from all 20 glioma tissues and 10 control brain tissues", "real - time monitoring of polymerase chain reactions ( pcrs ) was performed using the abi 7900ht ( idaho technology , idaho falls , i d , usa ) .", "cdna was synthesized using random primers and oligo 18dt and superscript ii reverse transcriptase ( invitrogen ) .", "gene expression was determined using the sybr green i dye ( biogene ) , which binds preferentially to double - stranded dna , and 10  ng of template .", "fluorescence signals , which are proportional to the concentration of the pcr product , are measured at the end of each cycle and immediately displayed on a computer screen , permitting real - time monitoring of the pcr .", "the reaction is characterized by the point during cycling when amplification of pcr products is first detected , rather than the amount of pcr product accumulated after a fixed number of cycles . the higher the starting quantity of the template , the earlier a significant increase in fluorescence is observed .", "the threshold cycle is defined as the fractional cycle number at which fluorescence passes a fixed threshold above the baseline .", "the primers 5-gtc cta gtc cgg cga ctt cc-3 and 5-ctt gtc tgg gca ggc cct ttg c-3 were used to amplify 603-bp transcripts of mpg , and the primers 5-ggt ggc ttt tag gat ggc aag3 and 5-act gga acg gtg aag gtg aca g3 were used to amplify 161-bp transcripts of -actin .", "the pcr profile consisted of an initial melting step of 1  min at 94c , followed by 38 cycles of 15  s at 94c , 15  s at 56c , and 45  s at 72c , and a final elongation step of 10  min at 72c .  ", "fluorescence data were converted into cycle threshold measurements using the sds system software and exported to microsoft excel .", "thermal dissociation plots were examined for biphasic melting curves , indicative of whether primer dimers or other nonspecific products could be contributing to the amplification signal .", "twenty glioma tissues and 10 control brain tissues were homogenized in lysis buffer [ pbs , 1% nonidet p-40 ( np-40 ) , 0.5% sodium deoxycholate , 0.1% sodium dodecyl sulfate ( sds ) , 100  ug / ml aprotinin , 100  g / ml phenylmethylsulfonyl fluoride ( pmsf ) , sodium orthovanadate ] at 4c throughout all procedures , and sonicated for 70  s , then add 300  g pmsf per gram of tissue and incubate on ice for 30  min , followed by centrifugation at 15,000  rpm for 20  min at 4c .", "the protein content was determined according to bradford 's method , with bovine serum albumin used as a standard .", "protein samples ( 30  g ) were boiled with 2  sample buffer containing 5%  -mercaptoethanol for 5  min , separated by size on 15% polyacrylamide gel under sds denaturing conditions , and transferred to a nitrocellulose membrane at 90  v for 2  h. the nitrocellulose membranes were stained with ponceau s to assess the efficiency of transfer .", "nonspecific binding was blocked by incubation in block buffer ( 5% nonfat dry milk , 0.05% tween-20 , 1  tris - cl - buffered saline ) overnight at 4c , the membranes were hybridized with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) , then incubated with a horseradish peroxidase - labeled goat anti - mouse igg ( 1  :  500 ) .", "the bound secondary antibody was detected by enhanced chemiluminescence ( amersham life science , little chalfont , uk ) .", "positive immunoreactive bands were quantified densitometrically ( leica q500iw image analysis system ) and expressed as ratio of mpg to -actin in optical density units .  ", "immunohistochemical assay was performed using the conventional immunoperoxidase technique according to the protocol of the department of neurosurgery , institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r .", "briefly , following peroxidase blocking with 0.3% h2o2/methanol for 30  min , specimens were blocked with phosphate - buffered saline ( pbs ) containing 5% normal horse serum ( vector laboratories inc . ,", "all incubations with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) at 1  :  500 dilution were carried out overnight at 4c .", "then the specimens were briefly washed in pbs and incubated at room temperature with the anti - mouse antibody and avidin - biotin peroxidase ( vector laboratories inc . ,", "the specimens were then washed in pbs and color developed by diaminobenzidine solution ( dako corporation , carpinteria , ca , usa ) . after washing with water , specimens were counterstained with meyer 's hematoxylin ( sigma chemical co. , st louis , mo , usa ) .", "nonneoplastic brain tissues were used as control tissues , and nonimmune igg was also used as negative control antibody for immunohistochemical staining .", "immunostaining was scored by two independent experienced pathologists , who were blinded to the clinicopathologic parameters and clinical outcomes of the patients .", "5% scored 0 , 625% scored 1 , 2650% scored 2 , 5175% scored 3 , > 75% scored 4 ; ( 2 ) intensity of stain : colorless scored 0 , pallide - flavens scored 1 , yellow scored 2 , brown scored 3 .", "the staining score was stratified as  ( 0 score , absent ) , + ( 14 score , weak ) , + + ( 58 score , moderate ) , and + + + ( 912 score , strong ) according to the proportion and intensity of positively stained cancer cells .", "all computations were carried out using the software of spss version13.0 for windows ( spss inc , il , usa ) .", "randomized block design anova was used to analyze the statistical difference among different tissue types . in the analysis of glioma morbidity for all patients", ", we used the kaplan - meier estimator and univariate cox regression analysis to assess the marginal effect of each factor .", "a spearman 's analysis was carried out to analyze the correlation between mpg mrna and protein expression levels ."], ["the gene expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was detected by real - time pcr . as shown in figure 2 ,", "the expression levels of mpg gene in human glioma tissues were conspicuously higher than those in control brain tissues ( p < 0.001 ) .", "interestingly , mpg gene expression increased with the advancement of who grades from i to iv ( p < 0.001 ) .", "the protein expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was also detected by western blot analysis .", "as the results , the expression levels of mpg protein tended to increase from the glioma to the normal tissue ( figures 3(a ) and 3(c ) ) .", "mpg expression was highest in grade iv and lowest in grade i ( figures 3(b ) and 3(c ) ) , indicating a close correlation of mpg protein expression with who grade .", "there was a significant positive correlation between the expression of mpg gene and protein expression levels from the same glioma tissues ( rs = 0.82 , p < 0.001 ) .", "the expression and the subcellular localization of mpg protein in 10 nonneoplastic brain and 128 glioma tissues were detected by immunohistochemistry assay .", "as shown in figure 4 , the positive staining for mpg was mainly observed in the nuclei of tumor cells in glioma tissues , which was consistent with the previous study of wang et al .  ", "the representative photographs were shown in figures 4(a ) and 4(b ) . among the glioma specimens , 102 ( 79.7% ) glioma specimens", "were positively stained for mpg , whereas its immunoreactivities in nonneoplastic brain sections ranged from undetectable to low ( figure 4(c ) ) .", "additionally , the immunostaining of mpg in nonneoplastic brain tissues is below detection levels of both real - time pcr and western blot . according to the statistical analysis ,", "the expression level of mpg protein in glioma tissues was significantly higher ( p < 0.001 ) than that in nonneoplastic brain tissues , which was consistent with the results of western blot analysis .  ", "in addition , mpg expression was not significantly affected by the gender and age ( both p > 0.05 ) of the patients .", "in contrast , the mpg expression was the closely correlated with who grade , as well as kps .", "the expression levels of mpg protein in glioma tissues with higher who grade ( table 1 ; p = 0.002 ) and lower kps ( table 1 ; p = 0.01 ) were significantly higher than those with lower who grade and higher kps .", "moreover , we reviewed clinical information of these mpg - positive or -negative glioma patients . during the follow - up period ,", "100 of the 128 glioma patients ( 78.1% ) had died ( 24 from the mpg - negative group and 76 from the mpg - positive group ) . as determined by the log - rank test ,", "the survival rate of patients with positive mpg staining was lower than those without mpg staining ( p < 0.001 ; figure 5(a ) ) .", "the median survival time of patients with negative expression of mpg could not be estimated by statistical analysis because all patients survived better than the overall median level , and those patients with strong positive ( + + + ) , moderate positive ( + + ) , and weak positive ( + ) of mpg were 9.1  1.5 months , 12.3  1.1 months , and 22.6  2.2 months ( log - rank test : p < 0.001 ) .", "furthermore , figure 5(b ) shows the postoperative survival curve of patients with glioma and mpg expression after adjusting for age , gender , who grade , and kps . by multivariate analysis ( table 2 ) ,", "the loss of mpg expression was a significant ( p < 0.001 ) and independent prognostic indicator for patients with glioma besides age , who grade and kps .", "the cox - proportional hazards model showed that mpg over - expression was associated with poor overall survival ."], ["because of the dismal survival in the patients with glioma , especially in gbm patients , there is an urgent need to find novel specific and effective prognostic markers in order to apply individualized treatments to specifically target the pathophysiologic and molecular properties of glioma patients . in the current study", ", we investigated the expression of mpg in 128 cases of human glioma and compared the expression with tumor grade and survival rates of patients .", "our data demonstrated that mpg gene and protein were both increased in glioma compared to nonneoplastic brain tissue .", "we found an increased trend of both mpg protein level and gene level from who grade i to who grade iv glioma .", "these results suggest that the transcriptional and translational activation of human mpg might participate in the tumorigenesis and progression of glioma .", "dna in all somatic and germ cells in the body are continuously exposed to exogenous and endogenous alkylating and oxidative agents that result in damage to dna .", "if dna damage was not repaired , it may lead to genetic mutation , chromosome aberration , aging , and carcinogenesis .", "the lack of precise dna repair activities may lead to defective embryogenesis , tissue - specific dysfunction , hypersensitivity to dna - damaging agents , premature senescence , genetic instability , and elevated cancer rates .", "the repair of dna bases damaged by alkylation is initiated in mammalian cells by mpg , as an alkyladenine dna glycosylase .", "the majority of repair that is initiated by mpg occurs via short - patch ber , a mechanism whereby only one nucleotide is replaced .", "the majority of base damage induced by methylation is removed by mpg , including the principal adducts 3-mea and 7-meg .", "it has been reported that the expression of mpg could be induced by viral damage and a variety of dna - damaging agents .", "for example , the increased expression of mpg gene was studied in breast cancer , cervical neoplasia , and thymic carcinoma in sv 40 t - antigen - expressing transgenic mice . as previous investigation , mammalian cells actively regulated dna repair enzymes and genes during cell proliferation .", "dna repair enzymes are expressed in the cell cycle in a defined temporal pattern relative to the induction of dna replication . in brain cells that are not undergoing dna replication ,", "kim et al .   indicated that mpg expression in the brain was relative high in 1 week after birth , and the level remained low in day 400 mature adults , suggesting that brain tissue is terminally differentiated and nonproliferating tissues .", "it has been demonstrated that dna repair enzymes play an important role in the carcinogenesis of several cancers .", "of our interests , mpg was overexpressed in breast cancer up to 24-fold as compared to normal primary breast epithelium , suggesting the role of mpg in breast carcinogenesis . in astrocytomas ,", "kim et al .   in 2003 reported that mpg mrna level was significantly higher than that in tumor - adjacent brain tissues . with the similar results", ", we in this study also found the upregulation of mpg protein and gene in glioma tissues .", "additionally , we provide evidence that mpg protein is found in nuclear localization of the glioma tissues , which was consistent with the results of kim et al . .", "the nuclear localization of mpg in tumor cells of glioma tissues may be suggestive of a role of mpg in the regulation of cell division and cell cycle .", "furthermore , the most important finding of this study was the correlation of mpg expression and survival rates of patients .", "kaplan - meier and multivariate analysis both showed a significantly worse overall survival for patients whose tumors had high mpg levels , indicating that high mpg protein level is a marker of poor prognosis for patients with gliomas .", "this is the first investigation to demonstrate the prognostic value of mpg in human cancers .", "our data showed the over - expression of mpg gene and protein in human gliomas and also suggested for the first time that mpg be an unfavorable independent prognostic indicator for glioma patients ."]]}
{"article_id": "PMC3901970", "article_text": ["the prevalence of overweight and obesity among children and adolescents has widely increased worldwide [ 1 , 2 ] , making it one of the most common chronic disorders in this age group and in adulthood .  ", "the use of body mass index ( bmi ) for age to define being overweight and obese in children and adolescents is well established for both clinical and public health applications , because of their feasibility under clinical settings and in epidemiological studies [ 3 , 4 ] . in children and adolescents ,", "the natural increases in bmi that occur with age necessitate the use of age - sex - specific thresholds .", "the most widely used growth charts are the centers for disease control and prevention ( cdc-2000 ) , the international task force ( iotf ) , and the 2007 growth references for 5 to 19 year olds produced by the world health organization ( who-2007 ) .   the cdc-2000 growth charts were developed to evaluate the nutritional status of us children and were originated from five cross - sectional representative surveys carried out in the us between 1963 and 1994 .", "these growth charts are routinely applied to identify children and adolescents with a bmi greater than the 85th or 95th percentiles following the advice of the us expert committee on childhood obesity .", "however , the appropriateness of an american dataset for defining overweight in young people from other countries is questionable .", "the iotf reference also uses age - sex - specific bmi percentiles , and overweight and obesity definition corresponds to an adult bmi of 25 and 30  kg / m , respectively , and reflects values in children tracking to overweight and obesity in adults .", "this reference is based on six large international cross - sectional representative datasets , identifying the bmi values that extrapolate to childhood .  ", "the who-2007 growth references were created to replace the national center for health statistics ( nchs ) references [ 10 , 11 ] .", "this reference was constructed using data from the 1977 nchs / who growth reference ( 1 to 24 years old ) merged with data from the 2006 who child growth standards for preschool children ( under 5 years of age ) using state - of - the - art statistical methods .", "although valuable information has been appearing in the literature or online , such as works from the health behaviour of school - aged children study which is mainly related to social determinants of health and well - being among young people , no systematic review has been conducted to understand the worldwide magnitude of the overweight and obesity problem among the adolescent population .", "thus , the objective of this study was to systematically review the literature regarding the prevalence of overweight and obesity in adolescents ( 1019 years old ) of both sexes published in the past 12 academic years ( 19992011 ) .", "a systematic literature search was performed which ended on june 7 , 2012 ( see figure 1 ) .", "the literature search was conducted in medline and scopus using the following mesh terms :  overweight  ;  obesity  ;  prevalence  ;  adolescent  . in total , 2537 articles were selected .", "we also reviewed the data on the prevalence of childhood overweight and obesity on the international obesity task force website at http://www.iaso.org / iotf/. to find the articles included in this review , the following inclusion criteria were used : ( 1 ) cross - sectional studies conducted in the last 12 years ( 19992011)when the original study did not report the survey year , it was not included ; ( 2 ) national and regional representative samples , but articles published on the prevalence of overweight in towns , urban , or rural areas in a country were excluded ; ( 3 ) weight and height objectively measured ; ( 4 ) results presented by sex ; ( 5 ) and for both overweight and obesity prevalence ; ( 6 ) the definition of overweight and obesity using the ( i ) cdc-2000 , ( ii ) iotf , and ( iii ) who-2007   growth references ; and ( 7 ) studies written in english , spanish , italian , or portuguese .", "moreover , if there were more than one national or regional study in the same country , the most recent one was included in the prevalence tables ( except for usa   and canada , countries in which the most recent data was not included in the tables due to differences in the representativeness of the samples   and the impossibility to calculate a single prevalence of overweight and obesity for adolescents ' boys and girls ; however , no differences in prevalence were observed between studies as it has been indicated in the discussion ) .", "the final number of articles included in this review was 39 articles related to overweight and obesity and also a study on the latest statistics on the prevalence of overweight and obesity in south africa .", "potentially relevant papers were selected by ( 1 ) screening the titles ; ( 2 ) screening the abstracts , and ( 3 ) if abstracts were not available or did not provide sufficient data , the entire article was retrieved and screened to determine whether it met the inclusion criteria .", "full - text articles were assessed by 2 authors ( m. m. bibiloni and j. a. tur ) .", "any matter of doubt was discussed by at least two of the reviewers ( m. m. bibiloni , a. pons , and j. a. tur ) .", "a total of thirty - nine articles and a national health report were eligible according to the inclusion criteria established for this review .  ", "table 1 presents a description of the forty studies selected for this review including the continent and the country where it was performed ( and region for not national studies ) , year of publishing , total number of participants in the study , number of adolescents , age range , proportion of girls , and number and definition for overweight and obesity classification used .", "nationally representative data were obtained in twenty - five countries ( including northern ireland ) [ 1539 ] , and ten countries were represented only by regional data [ 40 , 42 , 44 , 45 , 47 , 5054 ] . \n", "table 2 shows overweight and obesity prevalence from the twenty - five national studies ( one of them including data from northern ireland ) that were included in this review according to the continent and the country where it was performed , year of survey , study population , age range , criteria used for classifying overweight and obesity used , and along with total data by sex .", "there were thirty - two different prevalence levels described in the included articles , because five countries presented data using at least two different criteria for overweight and obesity classification [ 18 , 23 , 27 , 36 , 39 ] .", "the iotf cut - off was used to classify overweight and obesity in twenty - three of the twenty - five national studies considered in the present review .", "in general , the prevalence of overweight plus obesity was higher in america [ 1820 ] , oceania [ 38 , 39 ] and europe [ 3037 ] and lower in africa [ 1517 ] and certain parts of asia [ 2129 ] ( in china   and iran   the total prevalence was less than 10% by the iotf cut - offs ) .", "overall , about 30% of american adolescents and 22%25% of european adolescents ( excepting the czech republic and italian adolescents ' which showed a prevalence of 13.7% and 17.9% , resp . ) were overweight or obese . among oceanian adolescents the prevalence ranged from 23.2% in australia in 2004 to 34.2% in new zealand in 2007 .", "in africa , the overall prevalence of overweight and obesity was lower than 20% . among asian adolescents there was a broad range of overweight plus obesity . using", "iotf cut - off , the prevalence of being overweight or obese for asian boys and girls ranged from 5.2% in china in 2002 to 36.4% in bahrain in 2000 . \n", "specific prevalence from all the geographic regions was included in this review from three countries : south africa ( nine provinces ) , usa ( fifty two states ) , and italy ( five regions ) . in europe , data from islands of greece ( crete )   and italy ( sicily and sardinia ) [ 48 , 49 ] and spain ( balearic islands ' archipelago ; and the grand canary island ) were also included . on the other hand , regional but not national data was found for eleven countries ( italy , brazil , india , jordan , denmark , france , hungary , poland , spain [ 51 , 52 ] , switzerland , and turkey ) .", "the iotf cut - off was used to classify overweight and obesity in fourteen of the eighteen selected studies that included regional data . in one study", ", data was presented using only the who-2007 growth charts and in two studies using only the cdc-2000 growth reference [ 20 , 43 ] . in south africa and usa , substantial geographic variations in adolescent", "overweight and obesity prevalence varied in south africa from 13.5% in limpopo to 25.5% in kwazulu - natal . in 2007 , overweight and obesity varied in usa from 23.1% in utah and minnesota to 44.5% in mississippi . in 2002 ,", "the prevalence of overweight and obesity in southern italy and italian islands was higher among boys .", "in southern italy , the overweight prevalence among girls also was higher than in the other geographic regions .", "comparison between the islands from greece ( crete ) , italy ( sicily and sardinia ) , and spain ( balearic islands and grand canary island ) which were included in this review showed that crete had the highest prevalence of overweight and obesity  despite data were presented using different definition . in spain", ", using the iotf cut - off ( data not shown for balearic islands but given by authors ) , the prevalence of overweight plus obesity was higher in the grand canary island ( 29.1% ) than in the balearic islands ( 24.7% ) .  ", "according to national data , the prevalence of overweight among boys was 10% higher than girls in nine countries ( canada , qatar , taiwan , cyprus , czech republic , germany , greece , italy , australia , denmark , and hungary ) and among girls 10% higher than boys in seven of the twenty - five countries ( south africa , seychelles , tunisia , mexico , bahrain , saudi arabia , and sweden ) .", "the obesity prevalence was 10% higher among boys in seventeen countries ( canada , usa , china , iran , israel , qatar , saudi arabia , taiwan , cyprus , czech republic , germany , greece , italy , portugal , sweden , australia , new zealand , denmark , and hungary ) and 10% higher among girls in four of the twenty - five countries ( south africa , seychelles , tunisia , and bahrain ) .", "the aim of this study was to review systematically the literature on overweight and obesity prevalence among adolescents worldwide .", "thirty - nine articles and one national health report that met the inclusion criteria were considered .", "the overweight and obesity prevalence in the included studies ranged widely . in sixteen of the twenty - three countries with national representative data using the iotf cut - off , overweight and obesity prevalence", "higher than 20% were found , five countries showed prevalence above 30% , and just in two countries prevalence was lower than 10% .", "regarding national data , when prevalence was analysed according to sex , it was observed that boys showed a higher prevalence of overweight in almost half of the countries and a higher prevalence of obesity in almost all countries .", "these results are consistent with previous studies that pointed out a high prevalence of abdominal obesity among boys", ". differences of prevalence of overweight and obesity between genders have been related to geopolitical and cultural conditions .", "eight articles compared data between 1980s and/or 1990s with 2000s [ 16 , 19 , 20 , 22 , 28 , 32 , 37 , 50 ] and pointed out an increased prevalence of overweight and obesity in both sexes over this period . however , among australian adolescents   the overweight and obesity prevalence increased significantly among boys but not among girls over the period 19972004 . in the australian national children 's nutrition and physical activity survey 2007 ( ncnpas07 ) , 25% of boys and 30% of girls aged 9- to 13-year - olds and 25% of boys and", "23% of girls aged 14- to 16-year - olds were overweight or obese using the iotf criteria .", "a comparison of the 1985 , 1995 , and 2007 australian national surveys of 7- to 15-year - olds indicated that australian children are changing body shape to a more central fat distribution . in usa ,", "overweight and obesity prevalence increased by 3% and 18% among usa girls over this period .", "however , a cross - sectional analyses of a representative sample ( n = 4111 ) of the usa child and adolescent population ( birth through 19 years of age ) with data from the national health and nutrition examination survey 2009 - 10 ( nhanes ) indicated a prevalence of overweight and obesity among adolescents aged 12 through 19 years of 15.2% and 18.4% , respectively . analyses of trends in obesity prevalence for the last two nhanes surveys ( 2007 - 08 and 2009 - 10 ) indicated that the prevalence of obesity in children and adolescents has not changed in 2009 - 10 compared with 2007 - 08 . on the other hand , since 2004 the overweight and obesity trends were stabilized or decreased among german adolescents . in usa , substantial geographic disparities in adolescent overweight and obesity were found , with an apparent shift toward higher prevalence in 2007 for several states .", "reported that children in northern europe countries generally tended to have lower overweight and obesity prevalence ( 1020% ) than in southern europe ( 2035% ) .", "also within the same country , the prevalence and trends of overweight and obesity may not be homogeneous according to different geographic regions . in italy ,", "a north - south gradient in overweight and obesity prevalence among boys but also in overweight prevalence among girls was also reported .", "a higher prevalence of overweight and obesity has been reported in southern spain in both children   and adults .", "it is important to note that the choice of a reference and a cut - off point will determine the absolute prevalence of overweight and obesity and its trends , and hence different information will be obtained from the papers .", "argued that the reference they published , supported by the iotf , is less arbitrary and more international than others and recommended its use in international comparisons .", "lately , monasta et al .   suggested that the iotf reference and cut - offs could be preferable to identify overweight and obesity both at individual and population levels because they are at least based on a crude association with ill and health later in life , namely , the definition of overweight and obesity at age 18 years .", "however , the iotf cut - offs have been not recommended for clinical use when assessing an individual child 's growth [ 9 , 6264 ] .", "furthermore , recent findings suggested that a universal bmi classification system for childhood and adolescent overweight and obesity may not correspond to a comparable level of body fatness in all populations .", "the prevalence estimates may not accurately characterize the population groups most at risk of health disadvantages because the correlation of bmi with adiposity is highly variable and dependent on ethnic group [ 9 , 60 , 65 , 66 ] .", "the comparisons of overweight and obesity prevalence need interpretation with caution due to the difference in survey sampling methods , sample sizes , age range of subjects , quality of data in terms of height and weight measurement , and whether national programmes or strategies to tackle overweight and obesity are in place . even within the same country", ", the prevalence and trends of overweight and obesity may not be homogenous in view of different ethnicities , geographic regions , and socioeconomic status . only articles in english , spanish , italian , and portuguese were included in this review .", "the results of this review allow the following conclusions : ( 1 ) overweight and obesity prevalence is high ; ( 2 ) obesity is higher among boys , although it is not clear which sex has a higher proportion of adolescents with overweight ; ( 3 ) despite that there is no consensus about criteria to be used to classify adolescents as overweighed or obese , the most frequently used was the iotf reference .", "however , the international reference charts for monitoring the secular trends in childhood obesity need to be continually refined and evaluated . the results of this study would contribute to guiding health planners and administrators to develop proper tools for adolescent obesity management ."], "abstract_text": ["<S> \n objective . to review the extant literature on the prevalence of overweight and obesity in adolescents ( 1019 years old ) of both sexes </S>", "<S> . \n design . </S>", "<S> the search was carried out using medline and scopus considering articles published from the establishment of the databanks until june 7 , 2012 . </S>", "<S> data on the prevalence of children being overweight and obese from the international obesity task force ( iotf ) website was also reviewed . only original articles and </S>", "<S> one national health report were considered . </S>", "<S> forty studies met the inclusion criteria . </S>", "<S> results . </S>", "<S> twenty - five of these studies were nationally representative , and ten countries were represented only by regional data . conclusions . </S>", "<S> the prevalence of overweight and obesity among adolescents worldwide is high , and obesity is higher among boys . </S>", "<S> the iotf criterion is the most frequently used method to classify adolescents as overweighed or obese in public health research . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Limitations of the Study", "6. Conclusions"], "sections": [["the prevalence of overweight and obesity among children and adolescents has widely increased worldwide [ 1 , 2 ] , making it one of the most common chronic disorders in this age group and in adulthood .  ", "the use of body mass index ( bmi ) for age to define being overweight and obese in children and adolescents is well established for both clinical and public health applications , because of their feasibility under clinical settings and in epidemiological studies [ 3 , 4 ] . in children and adolescents ,", "the natural increases in bmi that occur with age necessitate the use of age - sex - specific thresholds .", "the most widely used growth charts are the centers for disease control and prevention ( cdc-2000 ) , the international task force ( iotf ) , and the 2007 growth references for 5 to 19 year olds produced by the world health organization ( who-2007 ) .   the cdc-2000 growth charts were developed to evaluate the nutritional status of us children and were originated from five cross - sectional representative surveys carried out in the us between 1963 and 1994 .", "these growth charts are routinely applied to identify children and adolescents with a bmi greater than the 85th or 95th percentiles following the advice of the us expert committee on childhood obesity .", "however , the appropriateness of an american dataset for defining overweight in young people from other countries is questionable .", "the iotf reference also uses age - sex - specific bmi percentiles , and overweight and obesity definition corresponds to an adult bmi of 25 and 30  kg / m , respectively , and reflects values in children tracking to overweight and obesity in adults .", "this reference is based on six large international cross - sectional representative datasets , identifying the bmi values that extrapolate to childhood .  ", "the who-2007 growth references were created to replace the national center for health statistics ( nchs ) references [ 10 , 11 ] .", "this reference was constructed using data from the 1977 nchs / who growth reference ( 1 to 24 years old ) merged with data from the 2006 who child growth standards for preschool children ( under 5 years of age ) using state - of - the - art statistical methods .", "although valuable information has been appearing in the literature or online , such as works from the health behaviour of school - aged children study which is mainly related to social determinants of health and well - being among young people , no systematic review has been conducted to understand the worldwide magnitude of the overweight and obesity problem among the adolescent population .", "thus , the objective of this study was to systematically review the literature regarding the prevalence of overweight and obesity in adolescents ( 1019 years old ) of both sexes published in the past 12 academic years ( 19992011 ) ."], ["a systematic literature search was performed which ended on june 7 , 2012 ( see figure 1 ) .", "the literature search was conducted in medline and scopus using the following mesh terms :  overweight  ;  obesity  ;  prevalence  ;  adolescent  . in total , 2537 articles were selected .", "we also reviewed the data on the prevalence of childhood overweight and obesity on the international obesity task force website at http://www.iaso.org / iotf/. to find the articles included in this review , the following inclusion criteria were used : ( 1 ) cross - sectional studies conducted in the last 12 years ( 19992011)when the original study did not report the survey year , it was not included ; ( 2 ) national and regional representative samples , but articles published on the prevalence of overweight in towns , urban , or rural areas in a country were excluded ; ( 3 ) weight and height objectively measured ; ( 4 ) results presented by sex ; ( 5 ) and for both overweight and obesity prevalence ; ( 6 ) the definition of overweight and obesity using the ( i ) cdc-2000 , ( ii ) iotf , and ( iii ) who-2007   growth references ; and ( 7 ) studies written in english , spanish , italian , or portuguese .", "moreover , if there were more than one national or regional study in the same country , the most recent one was included in the prevalence tables ( except for usa   and canada , countries in which the most recent data was not included in the tables due to differences in the representativeness of the samples   and the impossibility to calculate a single prevalence of overweight and obesity for adolescents ' boys and girls ; however , no differences in prevalence were observed between studies as it has been indicated in the discussion ) .", "the final number of articles included in this review was 39 articles related to overweight and obesity and also a study on the latest statistics on the prevalence of overweight and obesity in south africa .", "potentially relevant papers were selected by ( 1 ) screening the titles ; ( 2 ) screening the abstracts , and ( 3 ) if abstracts were not available or did not provide sufficient data , the entire article was retrieved and screened to determine whether it met the inclusion criteria .", "full - text articles were assessed by 2 authors ( m. m. bibiloni and j. a. tur ) .", "any matter of doubt was discussed by at least two of the reviewers ( m. m. bibiloni , a. pons , and j. a. tur ) ."], ["a total of thirty - nine articles and a national health report were eligible according to the inclusion criteria established for this review .  ", "table 1 presents a description of the forty studies selected for this review including the continent and the country where it was performed ( and region for not national studies ) , year of publishing , total number of participants in the study , number of adolescents , age range , proportion of girls , and number and definition for overweight and obesity classification used .", "nationally representative data were obtained in twenty - five countries ( including northern ireland ) [ 1539 ] , and ten countries were represented only by regional data [ 40 , 42 , 44 , 45 , 47 , 5054 ] . \n", "table 2 shows overweight and obesity prevalence from the twenty - five national studies ( one of them including data from northern ireland ) that were included in this review according to the continent and the country where it was performed , year of survey , study population , age range , criteria used for classifying overweight and obesity used , and along with total data by sex .", "there were thirty - two different prevalence levels described in the included articles , because five countries presented data using at least two different criteria for overweight and obesity classification [ 18 , 23 , 27 , 36 , 39 ] .", "the iotf cut - off was used to classify overweight and obesity in twenty - three of the twenty - five national studies considered in the present review .", "in general , the prevalence of overweight plus obesity was higher in america [ 1820 ] , oceania [ 38 , 39 ] and europe [ 3037 ] and lower in africa [ 1517 ] and certain parts of asia [ 2129 ] ( in china   and iran   the total prevalence was less than 10% by the iotf cut - offs ) .", "overall , about 30% of american adolescents and 22%25% of european adolescents ( excepting the czech republic and italian adolescents ' which showed a prevalence of 13.7% and 17.9% , resp . ) were overweight or obese . among oceanian adolescents the prevalence ranged from 23.2% in australia in 2004 to 34.2% in new zealand in 2007 .", "in africa , the overall prevalence of overweight and obesity was lower than 20% . among asian adolescents there was a broad range of overweight plus obesity . using", "iotf cut - off , the prevalence of being overweight or obese for asian boys and girls ranged from 5.2% in china in 2002 to 36.4% in bahrain in 2000 . \n", "specific prevalence from all the geographic regions was included in this review from three countries : south africa ( nine provinces ) , usa ( fifty two states ) , and italy ( five regions ) . in europe , data from islands of greece ( crete )   and italy ( sicily and sardinia ) [ 48 , 49 ] and spain ( balearic islands ' archipelago ; and the grand canary island ) were also included . on the other hand , regional but not national data was found for eleven countries ( italy , brazil , india , jordan , denmark , france , hungary , poland , spain [ 51 , 52 ] , switzerland , and turkey ) .", "the iotf cut - off was used to classify overweight and obesity in fourteen of the eighteen selected studies that included regional data . in one study", ", data was presented using only the who-2007 growth charts and in two studies using only the cdc-2000 growth reference [ 20 , 43 ] . in south africa and usa , substantial geographic variations in adolescent", "overweight and obesity prevalence varied in south africa from 13.5% in limpopo to 25.5% in kwazulu - natal . in 2007 , overweight and obesity varied in usa from 23.1% in utah and minnesota to 44.5% in mississippi . in 2002 ,", "the prevalence of overweight and obesity in southern italy and italian islands was higher among boys .", "in southern italy , the overweight prevalence among girls also was higher than in the other geographic regions .", "comparison between the islands from greece ( crete ) , italy ( sicily and sardinia ) , and spain ( balearic islands and grand canary island ) which were included in this review showed that crete had the highest prevalence of overweight and obesity  despite data were presented using different definition . in spain", ", using the iotf cut - off ( data not shown for balearic islands but given by authors ) , the prevalence of overweight plus obesity was higher in the grand canary island ( 29.1% ) than in the balearic islands ( 24.7% ) .  ", "according to national data , the prevalence of overweight among boys was 10% higher than girls in nine countries ( canada , qatar , taiwan , cyprus , czech republic , germany , greece , italy , australia , denmark , and hungary ) and among girls 10% higher than boys in seven of the twenty - five countries ( south africa , seychelles , tunisia , mexico , bahrain , saudi arabia , and sweden ) .", "the obesity prevalence was 10% higher among boys in seventeen countries ( canada , usa , china , iran , israel , qatar , saudi arabia , taiwan , cyprus , czech republic , germany , greece , italy , portugal , sweden , australia , new zealand , denmark , and hungary ) and 10% higher among girls in four of the twenty - five countries ( south africa , seychelles , tunisia , and bahrain ) ."], ["the aim of this study was to review systematically the literature on overweight and obesity prevalence among adolescents worldwide .", "thirty - nine articles and one national health report that met the inclusion criteria were considered .", "the overweight and obesity prevalence in the included studies ranged widely . in sixteen of the twenty - three countries with national representative data using the iotf cut - off , overweight and obesity prevalence", "higher than 20% were found , five countries showed prevalence above 30% , and just in two countries prevalence was lower than 10% .", "regarding national data , when prevalence was analysed according to sex , it was observed that boys showed a higher prevalence of overweight in almost half of the countries and a higher prevalence of obesity in almost all countries .", "these results are consistent with previous studies that pointed out a high prevalence of abdominal obesity among boys", ". differences of prevalence of overweight and obesity between genders have been related to geopolitical and cultural conditions .", "eight articles compared data between 1980s and/or 1990s with 2000s [ 16 , 19 , 20 , 22 , 28 , 32 , 37 , 50 ] and pointed out an increased prevalence of overweight and obesity in both sexes over this period . however , among australian adolescents   the overweight and obesity prevalence increased significantly among boys but not among girls over the period 19972004 . in the australian national children 's nutrition and physical activity survey 2007 ( ncnpas07 ) , 25% of boys and 30% of girls aged 9- to 13-year - olds and 25% of boys and", "23% of girls aged 14- to 16-year - olds were overweight or obese using the iotf criteria .", "a comparison of the 1985 , 1995 , and 2007 australian national surveys of 7- to 15-year - olds indicated that australian children are changing body shape to a more central fat distribution . in usa ,", "overweight and obesity prevalence increased by 3% and 18% among usa girls over this period .", "however , a cross - sectional analyses of a representative sample ( n = 4111 ) of the usa child and adolescent population ( birth through 19 years of age ) with data from the national health and nutrition examination survey 2009 - 10 ( nhanes ) indicated a prevalence of overweight and obesity among adolescents aged 12 through 19 years of 15.2% and 18.4% , respectively . analyses of trends in obesity prevalence for the last two nhanes surveys ( 2007 - 08 and 2009 - 10 ) indicated that the prevalence of obesity in children and adolescents has not changed in 2009 - 10 compared with 2007 - 08 . on the other hand , since 2004 the overweight and obesity trends were stabilized or decreased among german adolescents . in usa , substantial geographic disparities in adolescent overweight and obesity were found , with an apparent shift toward higher prevalence in 2007 for several states .", "reported that children in northern europe countries generally tended to have lower overweight and obesity prevalence ( 1020% ) than in southern europe ( 2035% ) .", "also within the same country , the prevalence and trends of overweight and obesity may not be homogeneous according to different geographic regions . in italy ,", "a north - south gradient in overweight and obesity prevalence among boys but also in overweight prevalence among girls was also reported .", "a higher prevalence of overweight and obesity has been reported in southern spain in both children   and adults .", "it is important to note that the choice of a reference and a cut - off point will determine the absolute prevalence of overweight and obesity and its trends , and hence different information will be obtained from the papers .", "argued that the reference they published , supported by the iotf , is less arbitrary and more international than others and recommended its use in international comparisons .", "lately , monasta et al .   suggested that the iotf reference and cut - offs could be preferable to identify overweight and obesity both at individual and population levels because they are at least based on a crude association with ill and health later in life , namely , the definition of overweight and obesity at age 18 years .", "however , the iotf cut - offs have been not recommended for clinical use when assessing an individual child 's growth [ 9 , 6264 ] .", "furthermore , recent findings suggested that a universal bmi classification system for childhood and adolescent overweight and obesity may not correspond to a comparable level of body fatness in all populations .", "the prevalence estimates may not accurately characterize the population groups most at risk of health disadvantages because the correlation of bmi with adiposity is highly variable and dependent on ethnic group [ 9 , 60 , 65 , 66 ] ."], ["the comparisons of overweight and obesity prevalence need interpretation with caution due to the difference in survey sampling methods , sample sizes , age range of subjects , quality of data in terms of height and weight measurement , and whether national programmes or strategies to tackle overweight and obesity are in place . even within the same country", ", the prevalence and trends of overweight and obesity may not be homogenous in view of different ethnicities , geographic regions , and socioeconomic status . only articles in english , spanish , italian , and portuguese were included in this review ."], ["the results of this review allow the following conclusions : ( 1 ) overweight and obesity prevalence is high ; ( 2 ) obesity is higher among boys , although it is not clear which sex has a higher proportion of adolescents with overweight ; ( 3 ) despite that there is no consensus about criteria to be used to classify adolescents as overweighed or obese , the most frequently used was the iotf reference .", "however , the international reference charts for monitoring the secular trends in childhood obesity need to be continually refined and evaluated . the results of this study would contribute to guiding health planners and administrators to develop proper tools for adolescent obesity management ."]]}
{"article_id": "PMC5089965", "article_text": ["\n sacubitril / valsartan is a combination of a neprilysin inhibitor and an angiotensin ii receptor blocker , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure.a patient previously stable on atorvastatin developed severe rhabdomyolysis and an elevation of transaminases within 26  days of initiation of sacubitril / valsartan . \n", "rhabdomyolysis is a syndrome characterized by skeletal muscle cell damage , leading to elevated creatine kinase ( ck ) , lactate dehydrogenase ( ldh ) , and aspartate aminotranferase ( ast ) , which can result in severe sequelae , including renal failure , cardiac arrhythmia , hyperthermia and death .", "associated elevations in transaminases may be seen , even in the absence of significant liver injury .", "clinically , rhabdomyolysis classically presents with muscle pain , weakness and dark , red or tea - colored urine ; however , less than 10% of patients will demonstrate all three components of this triad .", "rhabdomyolysis may be triggered by hereditary and/or acquired mechanisms , with approximately 75% of initial episodes being a result of acquired causes . in particular , a large number of prescription drugs and drugs of abuse can cause rhabdomyolysis , with statins being one of the top three prescription drugs most commonly responsible for rhabdomyolysis for this syndrome .", "myopathy and myositis have been reported in conjunction with all of the currently available statins and are considered to be a class effect of these medications .", "in addition , it has been recognized that statin - associated muscle complaints are dose - related and drug  drug interactions that cause an elevation of statin levels have been identified .", "subgroups of patients at greater risk of statin - associated muscle pathology have been identified and include age > 70  years , impaired renal function , and impaired hepatic function .", "entresto is a combination of sacubitril , a neprilysin inhibitor , and valsartan , an angiotensin ii receptor blocker ( arb ) , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure ( new york heart association [ nyha ] class ii ", "sacubitril / valsartan is usually administered in conjunction with other heart failure drug regimens instead of an angiotensin - converting enzyme inhibitor ( acei ) or another arb .", "the commonly occurring ( 5% ) adverse reactions reported are hypotension , hyperkalemia , cough , dizziness , and renal failure [ 6 , 7 ] .", "sacubitril / valsartan has not been associated with rhabdomyolysis and is commonly used in patients who are also receiving statin therapy .", "a 63-year - old guyanese woman with hypertension , hyperlipidemia , congestive heart failure ( chf ) , atrial fibrillation , and peripheral vascular disease presented to her primary care provider s office with a complaint of an episode of weakness at home associated with general malaise .", "she was receiving atorvastatin 40  mg daily , carvedilol 25  mg twice daily , digoxin 0.125  mg daily , furosemide 40  mg daily , spironolactone 25  mg daily , rivaroxaban 15  mg daily , and sacubitril / valsartan 24/26  mg twice daily .", "she was initiated on sacubitril / valsartan 22  days before her clinical presentation and had been on all of her other concurrent medications for more than 1  year . on the review of systems ,", "the patient reported generalized weakness , pain in her knees , and an ill - defined stinging pain in the skin of her arms and legs .", "her physical examination was non - focal , with no muscle weakness or tenderness of either the skin or muscles noted .", "laboratory tests performed on presentation were significant for elevated ast / alanine aminotransferase ( alt ) of 351  u / l/131 ", "be having a reaction to her statin therapy prescribed for hyperlipidemia and therefore the atorvastatin was withheld .", "she was scheduled to return in 4  days for repeat laboratory testing and re - evaluation .", "when the patient returned in 4  days , she complained of dark urine , generalized weakness , and increased muscle and skin pain throughout her body .", "she was then found to have rhabdomyolysis , with ck at 58,349  u / l ( > 50 the upper limit of normal ) .", "sacubitril / valsartan was stopped at this time as it was the only new medication in her regimen .", "the ck level decreased steadily and her symptoms resolved over the course of a 5-day hospitalization ( fig .  1 ) .", "her renal function was preserved throughout and her bilirubin and transaminases briefly increased and also subsequently steadily decreased ( figs .  2 , 3 ) .", "barr virus , hepatitis a , herpes simplex virus , hiv , and cytomegalovirus ( cmv ) .", "barr virus , and herpes simplex virus , and had negative hepatitis a and b , hiv , hsv . and cmv tests.fig . ", "ast aspartate aminotransferase , alt alanine aminotransferase \n  creatine kinase values during and post hospitalization .", "ast aspartate aminotransferase , alt alanine aminotransferase by 23  days after her initial presentation , the patient no longer had any muscle or skin pain and her ck and liver function test ( lft ) levels were completely normalized .", "the patient has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46  days , and her bilirubin and transaminase levels also returned to normal ( figs .  2 , 3 ) .", "one week later , the patient was started on an alternate statin at a low dose ( rosuvastatin 5  mg ) and the ck levels and lfts have remained stable .", "this patient had been on a variety of statins for more than 7  years , including atorvastatin , simvastatin , and rosuvastatin .", "her baseline ck and lfts were normal prior to the initiation of sacubitril / valsartan .", "she had previously experienced two transient elevations in ck , to a maximum of 1608 ", "u / l , while hospitalized for an implantable defibrillator infection and explanation complicated by an acute chf exacerbation and a recurrent chf exacerbation in the 3  years prior to the current admission .", "her ck levels returned to normal after her acute illnesses , without change in her lfts . in this case , the immediate cause of her rhabdomyolysis was unknown , but the new medication in her profile seemed to be the likely trigger . due to the severity of her rhabdomyolysis", "the naranjo adverse drug reaction probability scale score was 5 , making it probable that the adverse drug reaction was precipitated by sacubitril / valsartan .", "the drug interaction probability scale score was 3 , consistent with a possible interaction as a cause for the reaction , with sacubitril / valsartan as the precipitant drug and atorvastatin as the object drug .", "of note , one of the concurrent medications , carvedilol , is a p - glycoprotein inhibitor that may increase the serum concentration of atorvastatin , a p - glycoprotein substrate ; however , the patient was stable on this combination for 7  years before sacubitril / valsartan was initiated .", "none of the other medications taken by this patient are known to cause rhabdomyolysis or an elevation in transaminases .", "a literature search regarding a drug interaction between sacubitril and statins yielded no reports of rhabdomyolysis with statins .", "as per a recent study , coadministration of sacubitril / valsartan with atorvastatin led to a twofold increase in the maximum concentration ( c \n max ) of atorvastatin and its metabolites .", "in addition , the us and canadian package inserts of sacubitril / valsartan cite in vitro data showing sacubitril inhibiting organic anion transporting polypeptides ( oatp ) 1b1 and 1b3 , resulting in increased c \n max and area under the curve ( auc ) of atorvastatin with coadministration of sacubitril / valsartan [ 6 , 7 ] . the canadian version of the product insert further states that sacubitril / valsartan may increase the systemic exposure of oatp1b1 and oatp1b3 substrates , such as statins .", "sacubitril / valsartan increases the c \n max of atorvastatin and its metabolites by up to twofold , and auc by up to 1.3-fold .", "the canadian insert recommends that sacubitril / valsartan must be coadministered cautiously with statins , especially simvastatin , a sensitive oatp1b1/1b3 substrate , and a decrease in dose of simvastatin and atorvastatin may be considered when coadministered with sacubitril / valsartan .", "the interaction between atorvastatin and sacubitril is mentioned in the us product package insert as a theoretical possibility .", "there is currently no recommendation to increase monitoring or empirically decrease the dose of any statin on initiation of sacubitril / valsartan .", "a number of medications are known to inhibit oatps and have been shown to increase statin plasma concentrations .", "many single nucleotide polymorphisms ( snps ) have been found within the slco1b1 and/or slco1b3 genes encoding oatp1b1 and oatp1b3 proteins , respectively , and were shown to effect the pharmacokinetics and/or pharmacodynamics of statins [ 12 , 13 ] .", "the clinical consequences of the drug interactions may vary based on the genetic variation in oatp - encoding genes and inhibition of oatp function .", "the slco1b1 * 1b haplotype appears to be associated with enhanced hepatic uptake and reduced plasma concentrations of oatp1b1 substrates .", "discontinuation of the statin alone did not result in a decrease in the patient s lfts .", "it was not until sacubitril / valsartan was also discontinued , and treatment for rhabdomyolysis was initiated , that the patient s symptoms began to improve and her ck and lfts returned to baseline .", "this could be due to either a direct effect of sacubitril / valsartan or the increased serum concentration of atorvastatin , as described in the literature .", "because of the above - stated pharmacodynamics , we believe that the more likely mechanism is the increased serum concentration of atorvastatin . currently , there is no recommendation regarding the initiation or continuation of statins in patients with nyha class ii ", "iv heart failure as there is insufficient information on which to base recommendations for or against statin treatment . in these patients , the choice of statin dose , and using a statin for cardiovascular risk reduction benefit , must be weighed against adverse effects , drug  drug interactions , precautions and contraindications to statin therapy .", "we would have liked to have arranged slcob testing for the patient but were unable to .", "further research will be useful in identifying patients at risk for this adverse reaction , and will likely include genetic testing .", "this case of severe rhabdomyolysis should stimulate further investigation of the potential negative effects of the initiation of sacubitril / valsartan in patients receiving statin therapy .", "since sacubitril / valsartan is targeted for patients with heart failure , we predict that this would comprise a large number of the candidates for treatment .", "we recommend consideration of obtaining baseline ck levels and lfts and close observation of patients . in this case", ", rhabdomyolysis developed within 3  weeks of initiation of sacubitril / valsartan and evaluation within a shorter time frame may be appropriate .", "future reports of this reaction may help to define high - risk patients , but it should be noted that our patient had no known high - risk characteristics for the development of rhabdomyolysis , except for prior mild elevations of ck during hospitalizations for chf exacerbations .", "we encourage the reporting of any similar reactions to the us fda , the manufacturer , and the medical community .", "eve s. faber , madhavi gavini , ronald ramirez and richard sadovsky declare that they have no conflicts of interest that are directly relevant to the content of this report .", "a copy of the written consent may be requested for review from the corresponding author ."], "abstract_text": ["<S> a 63-year - old woman previously stable on a regimen of atorvastatin 40  mg daily , carvedilol 25  mg twice daily , digoxin 0.125  mg daily , furosemide 40  mg daily , spironolactone 25  mg daily , rivaroxaban 15  mg daily , and enalapril 20  mg twice daily for heart failure developed rhabdomyolysis 26  days after enalapril was stopped and sacubitril / valsartan ( entresto ) started . </S>", "<S> the patient received sacubitril / valsartan at 24/26  mg twice daily for heart failure ; however , after 26  days she developed muscle and skin pain . </S>", "<S> investigations revealed elevated creatine kinase and liver function tests , and rhabdomyolysis with raised transaminases was diagnosed . </S>", "<S> sacubitril / valsartan and atorvastatin were discontinued and the patient was hydrated . </S>", "<S> she returned to baseline in 23  days and has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46  days . </S>", "<S> a naranjo assessment score of 5 was obtained , indicating a probable relationship between the patient s rhabdomyolysis and her use of sacubitril / valsartan . </S>", "<S> the drug interaction probability scale score was 3 , consistent with a possible interaction as a cause for the reaction , with sacubitril / valsartan as the precipitant drug and atorvastatin as the object drug . </S>"], "labels": null, "section_names": ["Key Points", "Introduction", "Case Report", "Discussion", "Conclusions", "Funding", "Conflict of interest", "Consent"], "sections": [["\n sacubitril / valsartan is a combination of a neprilysin inhibitor and an angiotensin ii receptor blocker , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure.a patient previously stable on atorvastatin developed severe rhabdomyolysis and an elevation of transaminases within 26  days of initiation of sacubitril / valsartan . \n"], ["rhabdomyolysis is a syndrome characterized by skeletal muscle cell damage , leading to elevated creatine kinase ( ck ) , lactate dehydrogenase ( ldh ) , and aspartate aminotranferase ( ast ) , which can result in severe sequelae , including renal failure , cardiac arrhythmia , hyperthermia and death .", "associated elevations in transaminases may be seen , even in the absence of significant liver injury .", "clinically , rhabdomyolysis classically presents with muscle pain , weakness and dark , red or tea - colored urine ; however , less than 10% of patients will demonstrate all three components of this triad .", "rhabdomyolysis may be triggered by hereditary and/or acquired mechanisms , with approximately 75% of initial episodes being a result of acquired causes . in particular , a large number of prescription drugs and drugs of abuse can cause rhabdomyolysis , with statins being one of the top three prescription drugs most commonly responsible for rhabdomyolysis for this syndrome .", "myopathy and myositis have been reported in conjunction with all of the currently available statins and are considered to be a class effect of these medications .", "in addition , it has been recognized that statin - associated muscle complaints are dose - related and drug  drug interactions that cause an elevation of statin levels have been identified .", "subgroups of patients at greater risk of statin - associated muscle pathology have been identified and include age > 70  years , impaired renal function , and impaired hepatic function .", "entresto is a combination of sacubitril , a neprilysin inhibitor , and valsartan , an angiotensin ii receptor blocker ( arb ) , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure ( new york heart association [ nyha ] class ii ", "sacubitril / valsartan is usually administered in conjunction with other heart failure drug regimens instead of an angiotensin - converting enzyme inhibitor ( acei ) or another arb .", "the commonly occurring ( 5% ) adverse reactions reported are hypotension , hyperkalemia , cough , dizziness , and renal failure [ 6 , 7 ] .", "sacubitril / valsartan has not been associated with rhabdomyolysis and is commonly used in patients who are also receiving statin therapy ."], ["a 63-year - old guyanese woman with hypertension , hyperlipidemia , congestive heart failure ( chf ) , atrial fibrillation , and peripheral vascular disease presented to her primary care provider s office with a complaint of an episode of weakness at home associated with general malaise .", "she was receiving atorvastatin 40  mg daily , carvedilol 25  mg twice daily , digoxin 0.125  mg daily , furosemide 40  mg daily , spironolactone 25  mg daily , rivaroxaban 15  mg daily , and sacubitril / valsartan 24/26  mg twice daily .", "she was initiated on sacubitril / valsartan 22  days before her clinical presentation and had been on all of her other concurrent medications for more than 1  year . on the review of systems ,", "the patient reported generalized weakness , pain in her knees , and an ill - defined stinging pain in the skin of her arms and legs .", "her physical examination was non - focal , with no muscle weakness or tenderness of either the skin or muscles noted .", "laboratory tests performed on presentation were significant for elevated ast / alanine aminotransferase ( alt ) of 351  u / l/131 ", "be having a reaction to her statin therapy prescribed for hyperlipidemia and therefore the atorvastatin was withheld .", "she was scheduled to return in 4  days for repeat laboratory testing and re - evaluation .", "when the patient returned in 4  days , she complained of dark urine , generalized weakness , and increased muscle and skin pain throughout her body .", "she was then found to have rhabdomyolysis , with ck at 58,349  u / l ( > 50 the upper limit of normal ) .", "sacubitril / valsartan was stopped at this time as it was the only new medication in her regimen .", "the ck level decreased steadily and her symptoms resolved over the course of a 5-day hospitalization ( fig .  1 ) .", "her renal function was preserved throughout and her bilirubin and transaminases briefly increased and also subsequently steadily decreased ( figs .  2 , 3 ) .", "barr virus , hepatitis a , herpes simplex virus , hiv , and cytomegalovirus ( cmv ) .", "barr virus , and herpes simplex virus , and had negative hepatitis a and b , hiv , hsv . and cmv tests.fig . ", "ast aspartate aminotransferase , alt alanine aminotransferase \n  creatine kinase values during and post hospitalization .", "ast aspartate aminotransferase , alt alanine aminotransferase by 23  days after her initial presentation , the patient no longer had any muscle or skin pain and her ck and liver function test ( lft ) levels were completely normalized .", "the patient has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46  days , and her bilirubin and transaminase levels also returned to normal ( figs .  2 , 3 ) .", "one week later , the patient was started on an alternate statin at a low dose ( rosuvastatin 5  mg ) and the ck levels and lfts have remained stable ."], ["this patient had been on a variety of statins for more than 7  years , including atorvastatin , simvastatin , and rosuvastatin .", "her baseline ck and lfts were normal prior to the initiation of sacubitril / valsartan .", "she had previously experienced two transient elevations in ck , to a maximum of 1608 ", "u / l , while hospitalized for an implantable defibrillator infection and explanation complicated by an acute chf exacerbation and a recurrent chf exacerbation in the 3  years prior to the current admission .", "her ck levels returned to normal after her acute illnesses , without change in her lfts . in this case , the immediate cause of her rhabdomyolysis was unknown , but the new medication in her profile seemed to be the likely trigger . due to the severity of her rhabdomyolysis", "the naranjo adverse drug reaction probability scale score was 5 , making it probable that the adverse drug reaction was precipitated by sacubitril / valsartan .", "the drug interaction probability scale score was 3 , consistent with a possible interaction as a cause for the reaction , with sacubitril / valsartan as the precipitant drug and atorvastatin as the object drug .", "of note , one of the concurrent medications , carvedilol , is a p - glycoprotein inhibitor that may increase the serum concentration of atorvastatin , a p - glycoprotein substrate ; however , the patient was stable on this combination for 7  years before sacubitril / valsartan was initiated .", "none of the other medications taken by this patient are known to cause rhabdomyolysis or an elevation in transaminases .", "a literature search regarding a drug interaction between sacubitril and statins yielded no reports of rhabdomyolysis with statins .", "as per a recent study , coadministration of sacubitril / valsartan with atorvastatin led to a twofold increase in the maximum concentration ( c \n max ) of atorvastatin and its metabolites .", "in addition , the us and canadian package inserts of sacubitril / valsartan cite in vitro data showing sacubitril inhibiting organic anion transporting polypeptides ( oatp ) 1b1 and 1b3 , resulting in increased c \n max and area under the curve ( auc ) of atorvastatin with coadministration of sacubitril / valsartan [ 6 , 7 ] . the canadian version of the product insert further states that sacubitril / valsartan may increase the systemic exposure of oatp1b1 and oatp1b3 substrates , such as statins .", "sacubitril / valsartan increases the c \n max of atorvastatin and its metabolites by up to twofold , and auc by up to 1.3-fold .", "the canadian insert recommends that sacubitril / valsartan must be coadministered cautiously with statins , especially simvastatin , a sensitive oatp1b1/1b3 substrate , and a decrease in dose of simvastatin and atorvastatin may be considered when coadministered with sacubitril / valsartan .", "the interaction between atorvastatin and sacubitril is mentioned in the us product package insert as a theoretical possibility .", "there is currently no recommendation to increase monitoring or empirically decrease the dose of any statin on initiation of sacubitril / valsartan .", "a number of medications are known to inhibit oatps and have been shown to increase statin plasma concentrations .", "many single nucleotide polymorphisms ( snps ) have been found within the slco1b1 and/or slco1b3 genes encoding oatp1b1 and oatp1b3 proteins , respectively , and were shown to effect the pharmacokinetics and/or pharmacodynamics of statins [ 12 , 13 ] .", "the clinical consequences of the drug interactions may vary based on the genetic variation in oatp - encoding genes and inhibition of oatp function .", "the slco1b1 * 1b haplotype appears to be associated with enhanced hepatic uptake and reduced plasma concentrations of oatp1b1 substrates .", "discontinuation of the statin alone did not result in a decrease in the patient s lfts .", "it was not until sacubitril / valsartan was also discontinued , and treatment for rhabdomyolysis was initiated , that the patient s symptoms began to improve and her ck and lfts returned to baseline .", "this could be due to either a direct effect of sacubitril / valsartan or the increased serum concentration of atorvastatin , as described in the literature .", "because of the above - stated pharmacodynamics , we believe that the more likely mechanism is the increased serum concentration of atorvastatin . currently , there is no recommendation regarding the initiation or continuation of statins in patients with nyha class ii ", "iv heart failure as there is insufficient information on which to base recommendations for or against statin treatment . in these patients , the choice of statin dose , and using a statin for cardiovascular risk reduction benefit , must be weighed against adverse effects , drug  drug interactions , precautions and contraindications to statin therapy .", "we would have liked to have arranged slcob testing for the patient but were unable to .", "further research will be useful in identifying patients at risk for this adverse reaction , and will likely include genetic testing ."], ["this case of severe rhabdomyolysis should stimulate further investigation of the potential negative effects of the initiation of sacubitril / valsartan in patients receiving statin therapy .", "since sacubitril / valsartan is targeted for patients with heart failure , we predict that this would comprise a large number of the candidates for treatment .", "we recommend consideration of obtaining baseline ck levels and lfts and close observation of patients . in this case", ", rhabdomyolysis developed within 3  weeks of initiation of sacubitril / valsartan and evaluation within a shorter time frame may be appropriate .", "future reports of this reaction may help to define high - risk patients , but it should be noted that our patient had no known high - risk characteristics for the development of rhabdomyolysis , except for prior mild elevations of ck during hospitalizations for chf exacerbations .", "we encourage the reporting of any similar reactions to the us fda , the manufacturer , and the medical community ."], [""], ["eve s. faber , madhavi gavini , ronald ramirez and richard sadovsky declare that they have no conflicts of interest that are directly relevant to the content of this report ."], ["a copy of the written consent may be requested for review from the corresponding author ."]]}
{"article_id": "PMC4999398", "article_text": ["the nlrp3 inflammasome , a member of the nlr family , is a key player in the production of uric acid - mediated il-1 and is an important cytoplasmic protein complex involved in gouty inflammation ( 12 ) .", "although the precise pathogenic mechanism of gout has not been clearly determined , several crucial proteins such as the purinergic receptor p2x ligand - gated ion channel 7 ( p2x7r ) ( 345 ) and caspase activation and recruitment domain 8 ( card8 ) ( 67 ) proteins are known to be responsible for the pathogenesis of gout .", "recent single - nucleotide polymorphism ( snp ) studies suggested that genetic alternations of several target molecules such as card8 and p2x7r contribute to the process of nlrp3 inflammasome activation .", "genetic variants of card8 were identified to play a role in the pathogenesis of a variety of inflammatory diseases , such as rheumatoid arthritis ( ra ) ( 8) and inflammatory bowel disease ( ibd ) ( 9 ) .", "chen et al . ( 6 ) demonstrated a significantly different genotypic distribution of the card8 rs2043211 polymorphism between gout and control patients in the chinese population .", "another candidate gene involved in gouty inflammation might be p2x7r , whose protein product binds with atp and induces the efflux of k ions through the p2x7r channel from cells , finally triggering activation of the nlrp3 inflammasome ( 34 ) . a loss - of - function ( 1513 a > c ) snp of the p2x7r gene affects atp - induced cellular functions such as apoptotic cell death ( 10 ) and il-1 release ( 11 ) . more evidence to support the intimate relationship of p2x7r gene polymorphisms with autoimmune diseases are the demonstrated links between p2x7r polymorphisms and ra and systemic lupus erythematosus ( sle ) ( 1213 ) . however , there are no available data linking p2x7r snps with gout . here", ", we investigated the association of p2x7r rs3751142 and card8 rs2043211 polymorphisms with the susceptibility and clinical manifestations of gout in the male korean population .", "a total of 242 male gout patients fulfilled the preliminary criteria for classification of primary gout proposed by the american college of rheumatology ( 14 ) and a total of 280 healthy male controls were consecutively enrolled in this study .", "clinical and laboratory variables , including age at the time of study , disease onset age , body mass index ( bmi ) , disease duration , and serum uric acid level were identified by individual interviews with each patient and medical record review .", "medications including corticosteroids , non - steroidal anti - inflammatory drugs ( nsaids ) , colchicine , allopurinol , and benzbromarone that were used for gout treatment within one month of the study onset were evaluated through medical record reviews .", "the study protocol was reviewed and approved by the institutional review board / ethics committee at each medical center that participated in this study .", "assay reagents for rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were designed by applied biosystems ( applied biosystems , foster city , ca , usa ) .", "the reagents consisted of taqman mgb probes ( fam and vic dye - labeled ) .", "the reaction in a 10 l total volume was optimized at 0.125 l 40x reagents , 5 l 2x taqman genotyping master mix ( applied biosystems ) and 2 l 50 ng genomic dna .", "pcr conditions included one cycle at 95c for 10 minutes followed by 40 cycles at 95c for 15 seconds and 60c for 1 minute .", "the pcr was performed using the abi plus ( applied biosystems ) system and the samples were read and analyzed using abi plus ( applied biosystems ) software .", "the reference sequence was based on the sequence of human chromosome 12 , 12q24 for the p2rx7 gene , and human chromosome 19 , 19q13.33 for the card8 gene .", "snp , single nucleotide polymorphism ; p2x7r , purinergic receptor p2x ligand - gated ion channel 7 ; card8 , caspase activation and recruitment domain 8 .", "clinical parameters are described as the median with interquartile range ( iqr ) or the number with percent ( % ) .", "the hardy - weinberg equilibrium for gout patients and controls was assessed using a web - based calculator ( http://www.had2know.com/academics/hardy-weinberg-equilibrium-calculator-2-alleles.html ) ( 15 ) .", "the genotypic frequency of each gene was calculated and assessed using the chi - square and fisher s exact tests , if appropriate .", "both kolmogorov - smirnov and shapiro - wilk analyses were tested to identify normality of data distribution , which did not show a normal distribution .", "the kruskal - wallis test was used to compare parametric parameters such as age , onset age , disease duration , bmi , and serum uric acid among the three genotypes of each gene . logistic regression analysis", "adjusted with age was applied to test whether the combined p2x7r rs3751142 and card8 rs2043211 genotype influenced the risk of gout development compared to those in controls .", "the results are described as the odds ratio ( or ) with a 95% confidence interval ( ci ) .", "all patients provided written informed consent , and the protocol of this study was approved by the institutional review board ( irb ) of daegu catholic university medical center ( irb no .", "the authors assert that all procedures contributing to this work comply with the helsinki declaration of 1975 and its later amendments .", "a total of 242 male gout patients fulfilled the preliminary criteria for classification of primary gout proposed by the american college of rheumatology ( 14 ) and a total of 280 healthy male controls were consecutively enrolled in this study .", "clinical and laboratory variables , including age at the time of study , disease onset age , body mass index ( bmi ) , disease duration , and serum uric acid level were identified by individual interviews with each patient and medical record review .", "medications including corticosteroids , non - steroidal anti - inflammatory drugs ( nsaids ) , colchicine , allopurinol , and benzbromarone that were used for gout treatment within one month of the study onset were evaluated through medical record reviews .", "the study protocol was reviewed and approved by the institutional review board / ethics committee at each medical center that participated in this study .", "assay reagents for rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were designed by applied biosystems ( applied biosystems , foster city , ca , usa ) .", "the reagents consisted of taqman mgb probes ( fam and vic dye - labeled ) .", "the reaction in a 10 l total volume was optimized at 0.125 l 40x reagents , 5 l 2x taqman genotyping master mix ( applied biosystems ) and 2 l 50 ng genomic dna .", "pcr conditions included one cycle at 95c for 10 minutes followed by 40 cycles at 95c for 15 seconds and 60c for 1 minute .", "the pcr was performed using the abi plus ( applied biosystems ) system and the samples were read and analyzed using abi plus ( applied biosystems ) software .", "the reference sequence was based on the sequence of human chromosome 12 , 12q24 for the p2rx7 gene , and human chromosome 19 , 19q13.33 for the card8 gene .", "snp , single nucleotide polymorphism ; p2x7r , purinergic receptor p2x ligand - gated ion channel 7 ; card8 , caspase activation and recruitment domain 8 .", "clinical parameters are described as the median with interquartile range ( iqr ) or the number with percent ( % ) . the hardy - weinberg equilibrium for gout patients and controls", "the genotypic frequency of each gene was calculated and assessed using the chi - square and fisher s exact tests , if appropriate .", "both kolmogorov - smirnov and shapiro - wilk analyses were tested to identify normality of data distribution , which did not show a normal distribution .", "the kruskal - wallis test was used to compare parametric parameters such as age , onset age , disease duration , bmi , and serum uric acid among the three genotypes of each gene . logistic regression analysis", "adjusted with age was applied to test whether the combined p2x7r rs3751142 and card8 rs2043211 genotype influenced the risk of gout development compared to those in controls .", "the results are described as the odds ratio ( or ) with a 95% confidence interval ( ci ) .", "statistical analyses were performed using ibm spss statistics 19.0 software ( ibm corp . , armonk , ny , usa ) .", "all patients provided written informed consent , and the protocol of this study was approved by the institutional review board ( irb ) of daegu catholic university medical center ( irb no .", "the authors assert that all procedures contributing to this work comply with the helsinki declaration of 1975 and its later amendments .", "the mean age was 55.0 ( iqr 45.063.0 ) years for gout patients and 45.0 ( iqr 41.053.0 ) for controls , which was significantly different between two groups ( p < 0.001 ) .", "data were expressed as with interquartile range ( iqr ) for continuous variables and number ( % ) for categorical variables .", "no genotypic deviations of rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in card8 based on the hardy - weinberg equilibrium were noted for gout or control patients ( p > 0.05 ) .", "table 3 shows that a frequency difference of alleles and genotypes for rs3751142(c > a ) and rs2043211(a >", "t ) was not found between gout or control patients . under the recessive comparison model ( cc vs. ca", "/ aa in p2x7r and aa vs. at / tt in card8 ) , genotypic frequency differences between the two groups were also not detected at each snp ( p > 0.05 of both snps ) .", "there were no differences of clinical variables according to genotypic distribution of p2x7r rs3751142 and card8 rs2043211 . in a comparison model sub - analysis for onset age", "(  30 vs. > 30 years ) and serum uric acid level (  7.0 vs. > 7.0 mg / l ) , genotypic differences of each clinical parameter were not observed at the two snps ( data not shown ) .", "rs3751142 ( c > a ) in p2x7r gene and rs2043211(a > t ) in card8 gene . a pair - wise comparison of p2x7r rs3751142 and card8 rs2043211 genotype combinations showed that a trend toward higher risk of gout development was identified in subjects who were heterozygous for the ca p2x7r rs3751142 genotype and recessive homozygous for the tt card8 rs2043211 genotype compared to the cc / aa combination ( p = 0.056 , or = 2.618 , 95% ci 0.975 - 7.031 ) ( table 5 ) .", "the statistical analysis was performed using logistic regression analysis after adjustment with age . or , odds ratio ; ci , confidence interval .", "the main objective of this study was to investigate whether genetic variability in the p2x7r rs3751142 and card8 rs2043211 genes was involved in the process of nlrp3 inflammasome activation , which might influence susceptibility for the development of gout .", "this study identified that card8 rs2043211 was not associated with increased gout risk , which is in contrast to the known difference of genetic distribution of this snp in the chinese and european gout population ( 67 ) . as far as we know , this study is the first to determine genetic association between the p2x7r snp and gout patients , revealing that p2x7r polymorphism did not increase the risk of gout in our study population .", "the nlrp3 inflammasome is a cytoplasmic protein complex that activates caspase-1 , inducing the maturation and secretion of il-1 , which is associated with gout pathogenesis ( 12 ) .", "the mechanisms by which signaling pathways activate the nlrp3 inflammasome are not clearly determined ; however , it is known that cytoplasmic k efflux from the cell through the p2x7r channel in response to danger signals allows for nlrp3 inflammasome activation ( 34 ) .", "similarly , msu crystals , atp , and asbestos are known to induce inflammasome activation by decreasing intracellular k levels ( 16 ) .", "the p2x7r gene is markedly polymorphic and includes at least 12 non - synonymous snps in the coding region ( 17 ) .", "p2x7r snps that encode glu496ala or ala348thr substitutions are most commonly found to be associated with il-1 secretion capacity ( 1117 ) .", "the loss - of - function ( 1513 a > c ) snp of p2x7r gene induced lower atp - mediated k ion efflux , leading to reduced il-1 levels in monocytes ( 11 ) .", "( 18 ) recently suggested the possibility that p2x7r is a key regulator for the production of il-1 by msu crystals during acute gouty arthritis .", "human myeloid leukemic kg-1 cells contain several snps of the p2x7r gene ( 19 ) . among them , kg-1 cells with rs3751142 a revealed neutral effect on p2x7r function . however , there is no evidence as to whether the p2x7r polymorphism has a potent influence on the development of gout . in our data ,", "a minor allele frequency ( maf , a allele ) of p2x7r rs3751142 in gout and control patients was 0.132 and 0.161 , respectively , which are more compatible to those in the dbsnp rs3751142 ( http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?locusid05027 ) .", "this study found that p2x7r rs3751142 was not a genetic risk factor associated with gout susceptibility .", "the activation of the nlrp3 inflammasome , which is comprised of multiple cytoplasmic proteins including nlrp3 , an asc protein , and caspase-1 , is completed through the binding of the pyd of nlrp3 with the pyd of asc , which is followed by the recruitment of procaspase-1 by linkages of each card within the two proteins .", "card8 was shown to be a negative regulator of nuclear factor-b ( nf-b ) and caspase-1 activation ( 2021 ) , which implicates a role in the suppression of nlrp3 inflammasome activation .", "the rs2043211 snp in exon 5 of the card8 gene is a nonsense mutation resulting in the production of a truncated protein .", "although the functional role of polymorphic card8 within the nlrp3 inflammasome is complicated , genetic variants of card8 rs2043211 contribute to an enhanced inflammatory response in inflammatory diseases such as ra and ibd through increased expression of il-1 ( 8922 ) .", "mckinney et al . ( 7 ) demonstrated that the prevalence of card8 rs2043211 was different between gout and control patients in the european and polynesian populations ( or 1.11 , p = 0.023 and or 1.15 , p = 0.078 , respectively ) .", "another case - control study showed that the maf ( a allele ) card8 gene was shown to have an increasing trend relative to the t allele in the chinese male population ( or = 0.084 , p = 0.08 ) ( 6 ) .", "however , our study revealed no association of card8 rs2043211 with the presence of gout in korean study population .", "the frequency of maf ( t allele ) is at 0.457 in gout and 0.452 in control patients , which is similar to the frequency in the new zealand polynesian ( 0.499 in gout and 0.439 in control ) and chinese ( 0.498 in gout and 0.463 in control ) patients , whereas a lower frequency of maf was noted in the european population ( 7 ) .", "based on conflicting data for card8 rs2043211 between these three studies , the functional role of the card8 gene in gout susceptibility has not been clearly determined .", "therefore , the distinct role of the card8 gene in the pathogenesis of gout should be considered in a diverse ethnic study population .", "first , the lack of completeness for clinical information of control subjects is an important weakness in our study .", "this study used genomic dnas provided by the biobank of wonkwang university hospital , korea as control group samples .", "we were just able to identify a part of demographic data including gender and age .", "second , this study did not perform functional analysis for genotypes in each target gene . as shown disparity of genetic susceptibility of gout in several ethnic groups ( 67 ) , it is important to identify functional performance of each gene to understand the role of p2x7r and card8 snps in our study population .", "it is noted that the p2x7r and card8 proteins are closely involved in activation of the nlrp3 inflammasome and il-1 expression , which could explain their role in the pathogenesis of gout ( 12 ) .", "our study demonstrated that the p2x7r rs3751142 and card8 rs2043211 genetic variants were not implicated in the development of gout in the male korean population .", "however , we found that in a pair - wise comparison of the ca / tt p2x7r and card8 genotype combination was shown to have an increased trend for the risk of gout ( or = 2.618 , p = 0.056 ) . in conclusion , this study provides evidence that the interaction of msu crystal - mediated p2x7r with card8 may be at least in part responsible for the pathogenesis of gout ."], "abstract_text": ["<S> the aim of this study was to determine the association between p2x7r rs3751142 and card8 rs2043211 polymorphisms and gout susceptibility in male korean subjects . </S>", "<S> this study enrolled a total of 242 male patients with gout and 280 healthy controls . </S>", "<S> the polymorphisms of two individual genes including rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were assessed using taq - man analysis . </S>", "<S> statistical analyses were performed using the chi - square test , kruskal - wallis test , and logistic regression analyses . a difference in genotypic frequency of the p2x7r rs3751142 and card8 rs2043211 genes </S>", "<S> was not detected between gout and control patients . </S>", "<S> clinical parameters including age , onset age , disease duration , body mass index , and serum uric acid levels were not different among the three genotypes for either p2x7r or card8 ( p > 0.05 for all ) . a pair - wise comparison of p2x7r rs3751142 and card8 rs2043211 genotype combinations revealed that subjects with the ca p2x7r rs3751142 genotype and the tt card8 rs2043211 genotype had a trend toward a higher risk of gout compared to the cc / aa combination ( p = 0.056 , or = 2.618 , 95% ci 0.975 - 7.031 ) . in conclusion , this study revealed that genetic variability of the p2x7r rs3751142 and card8 rs2043211 genes might , in part , be associated with susceptibility for gout . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Subjects and collection of clinical information", "Genotyping", "Statistical analysis", "Ethics statement", "RESULTS", "DISCUSSION"], "sections": [["the nlrp3 inflammasome , a member of the nlr family , is a key player in the production of uric acid - mediated il-1 and is an important cytoplasmic protein complex involved in gouty inflammation ( 12 ) .", "although the precise pathogenic mechanism of gout has not been clearly determined , several crucial proteins such as the purinergic receptor p2x ligand - gated ion channel 7 ( p2x7r ) ( 345 ) and caspase activation and recruitment domain 8 ( card8 ) ( 67 ) proteins are known to be responsible for the pathogenesis of gout .", "recent single - nucleotide polymorphism ( snp ) studies suggested that genetic alternations of several target molecules such as card8 and p2x7r contribute to the process of nlrp3 inflammasome activation .", "genetic variants of card8 were identified to play a role in the pathogenesis of a variety of inflammatory diseases , such as rheumatoid arthritis ( ra ) ( 8) and inflammatory bowel disease ( ibd ) ( 9 ) .", "chen et al . ( 6 ) demonstrated a significantly different genotypic distribution of the card8 rs2043211 polymorphism between gout and control patients in the chinese population .", "another candidate gene involved in gouty inflammation might be p2x7r , whose protein product binds with atp and induces the efflux of k ions through the p2x7r channel from cells , finally triggering activation of the nlrp3 inflammasome ( 34 ) . a loss - of - function ( 1513 a > c ) snp of the p2x7r gene affects atp - induced cellular functions such as apoptotic cell death ( 10 ) and il-1 release ( 11 ) . more evidence to support the intimate relationship of p2x7r gene polymorphisms with autoimmune diseases are the demonstrated links between p2x7r polymorphisms and ra and systemic lupus erythematosus ( sle ) ( 1213 ) . however , there are no available data linking p2x7r snps with gout . here", ", we investigated the association of p2x7r rs3751142 and card8 rs2043211 polymorphisms with the susceptibility and clinical manifestations of gout in the male korean population ."], ["a total of 242 male gout patients fulfilled the preliminary criteria for classification of primary gout proposed by the american college of rheumatology ( 14 ) and a total of 280 healthy male controls were consecutively enrolled in this study .", "clinical and laboratory variables , including age at the time of study , disease onset age , body mass index ( bmi ) , disease duration , and serum uric acid level were identified by individual interviews with each patient and medical record review .", "medications including corticosteroids , non - steroidal anti - inflammatory drugs ( nsaids ) , colchicine , allopurinol , and benzbromarone that were used for gout treatment within one month of the study onset were evaluated through medical record reviews .", "the study protocol was reviewed and approved by the institutional review board / ethics committee at each medical center that participated in this study .", "assay reagents for rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were designed by applied biosystems ( applied biosystems , foster city , ca , usa ) .", "the reagents consisted of taqman mgb probes ( fam and vic dye - labeled ) .", "the reaction in a 10 l total volume was optimized at 0.125 l 40x reagents , 5 l 2x taqman genotyping master mix ( applied biosystems ) and 2 l 50 ng genomic dna .", "pcr conditions included one cycle at 95c for 10 minutes followed by 40 cycles at 95c for 15 seconds and 60c for 1 minute .", "the pcr was performed using the abi plus ( applied biosystems ) system and the samples were read and analyzed using abi plus ( applied biosystems ) software .", "the reference sequence was based on the sequence of human chromosome 12 , 12q24 for the p2rx7 gene , and human chromosome 19 , 19q13.33 for the card8 gene .", "snp , single nucleotide polymorphism ; p2x7r , purinergic receptor p2x ligand - gated ion channel 7 ; card8 , caspase activation and recruitment domain 8 .", "clinical parameters are described as the median with interquartile range ( iqr ) or the number with percent ( % ) .", "the hardy - weinberg equilibrium for gout patients and controls was assessed using a web - based calculator ( http://www.had2know.com/academics/hardy-weinberg-equilibrium-calculator-2-alleles.html ) ( 15 ) .", "the genotypic frequency of each gene was calculated and assessed using the chi - square and fisher s exact tests , if appropriate .", "both kolmogorov - smirnov and shapiro - wilk analyses were tested to identify normality of data distribution , which did not show a normal distribution .", "the kruskal - wallis test was used to compare parametric parameters such as age , onset age , disease duration , bmi , and serum uric acid among the three genotypes of each gene . logistic regression analysis", "adjusted with age was applied to test whether the combined p2x7r rs3751142 and card8 rs2043211 genotype influenced the risk of gout development compared to those in controls .", "the results are described as the odds ratio ( or ) with a 95% confidence interval ( ci ) .", "all patients provided written informed consent , and the protocol of this study was approved by the institutional review board ( irb ) of daegu catholic university medical center ( irb no .", "the authors assert that all procedures contributing to this work comply with the helsinki declaration of 1975 and its later amendments ."], ["a total of 242 male gout patients fulfilled the preliminary criteria for classification of primary gout proposed by the american college of rheumatology ( 14 ) and a total of 280 healthy male controls were consecutively enrolled in this study .", "clinical and laboratory variables , including age at the time of study , disease onset age , body mass index ( bmi ) , disease duration , and serum uric acid level were identified by individual interviews with each patient and medical record review .", "medications including corticosteroids , non - steroidal anti - inflammatory drugs ( nsaids ) , colchicine , allopurinol , and benzbromarone that were used for gout treatment within one month of the study onset were evaluated through medical record reviews .", "the study protocol was reviewed and approved by the institutional review board / ethics committee at each medical center that participated in this study ."], ["assay reagents for rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were designed by applied biosystems ( applied biosystems , foster city , ca , usa ) .", "the reagents consisted of taqman mgb probes ( fam and vic dye - labeled ) .", "the reaction in a 10 l total volume was optimized at 0.125 l 40x reagents , 5 l 2x taqman genotyping master mix ( applied biosystems ) and 2 l 50 ng genomic dna .", "pcr conditions included one cycle at 95c for 10 minutes followed by 40 cycles at 95c for 15 seconds and 60c for 1 minute .", "the pcr was performed using the abi plus ( applied biosystems ) system and the samples were read and analyzed using abi plus ( applied biosystems ) software .", "the reference sequence was based on the sequence of human chromosome 12 , 12q24 for the p2rx7 gene , and human chromosome 19 , 19q13.33 for the card8 gene .", "snp , single nucleotide polymorphism ; p2x7r , purinergic receptor p2x ligand - gated ion channel 7 ; card8 , caspase activation and recruitment domain 8 ."], ["clinical parameters are described as the median with interquartile range ( iqr ) or the number with percent ( % ) . the hardy - weinberg equilibrium for gout patients and controls", "the genotypic frequency of each gene was calculated and assessed using the chi - square and fisher s exact tests , if appropriate .", "both kolmogorov - smirnov and shapiro - wilk analyses were tested to identify normality of data distribution , which did not show a normal distribution .", "the kruskal - wallis test was used to compare parametric parameters such as age , onset age , disease duration , bmi , and serum uric acid among the three genotypes of each gene . logistic regression analysis", "adjusted with age was applied to test whether the combined p2x7r rs3751142 and card8 rs2043211 genotype influenced the risk of gout development compared to those in controls .", "the results are described as the odds ratio ( or ) with a 95% confidence interval ( ci ) .", "statistical analyses were performed using ibm spss statistics 19.0 software ( ibm corp . , armonk , ny , usa ) ."], ["all patients provided written informed consent , and the protocol of this study was approved by the institutional review board ( irb ) of daegu catholic university medical center ( irb no .", "the authors assert that all procedures contributing to this work comply with the helsinki declaration of 1975 and its later amendments ."], ["the mean age was 55.0 ( iqr 45.063.0 ) years for gout patients and 45.0 ( iqr 41.053.0 ) for controls , which was significantly different between two groups ( p < 0.001 ) .", "data were expressed as with interquartile range ( iqr ) for continuous variables and number ( % ) for categorical variables .", "no genotypic deviations of rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in card8 based on the hardy - weinberg equilibrium were noted for gout or control patients ( p > 0.05 ) .", "table 3 shows that a frequency difference of alleles and genotypes for rs3751142(c > a ) and rs2043211(a >", "t ) was not found between gout or control patients . under the recessive comparison model ( cc vs. ca", "/ aa in p2x7r and aa vs. at / tt in card8 ) , genotypic frequency differences between the two groups were also not detected at each snp ( p > 0.05 of both snps ) .", "there were no differences of clinical variables according to genotypic distribution of p2x7r rs3751142 and card8 rs2043211 . in a comparison model sub - analysis for onset age", "(  30 vs. > 30 years ) and serum uric acid level (  7.0 vs. > 7.0 mg / l ) , genotypic differences of each clinical parameter were not observed at the two snps ( data not shown ) .", "rs3751142 ( c > a ) in p2x7r gene and rs2043211(a > t ) in card8 gene . a pair - wise comparison of p2x7r rs3751142 and card8 rs2043211 genotype combinations showed that a trend toward higher risk of gout development was identified in subjects who were heterozygous for the ca p2x7r rs3751142 genotype and recessive homozygous for the tt card8 rs2043211 genotype compared to the cc / aa combination ( p = 0.056 , or = 2.618 , 95% ci 0.975 - 7.031 ) ( table 5 ) .", "the statistical analysis was performed using logistic regression analysis after adjustment with age . or , odds ratio ; ci , confidence interval ."], ["the main objective of this study was to investigate whether genetic variability in the p2x7r rs3751142 and card8 rs2043211 genes was involved in the process of nlrp3 inflammasome activation , which might influence susceptibility for the development of gout .", "this study identified that card8 rs2043211 was not associated with increased gout risk , which is in contrast to the known difference of genetic distribution of this snp in the chinese and european gout population ( 67 ) . as far as we know , this study is the first to determine genetic association between the p2x7r snp and gout patients , revealing that p2x7r polymorphism did not increase the risk of gout in our study population .", "the nlrp3 inflammasome is a cytoplasmic protein complex that activates caspase-1 , inducing the maturation and secretion of il-1 , which is associated with gout pathogenesis ( 12 ) .", "the mechanisms by which signaling pathways activate the nlrp3 inflammasome are not clearly determined ; however , it is known that cytoplasmic k efflux from the cell through the p2x7r channel in response to danger signals allows for nlrp3 inflammasome activation ( 34 ) .", "similarly , msu crystals , atp , and asbestos are known to induce inflammasome activation by decreasing intracellular k levels ( 16 ) .", "the p2x7r gene is markedly polymorphic and includes at least 12 non - synonymous snps in the coding region ( 17 ) .", "p2x7r snps that encode glu496ala or ala348thr substitutions are most commonly found to be associated with il-1 secretion capacity ( 1117 ) .", "the loss - of - function ( 1513 a > c ) snp of p2x7r gene induced lower atp - mediated k ion efflux , leading to reduced il-1 levels in monocytes ( 11 ) .", "( 18 ) recently suggested the possibility that p2x7r is a key regulator for the production of il-1 by msu crystals during acute gouty arthritis .", "human myeloid leukemic kg-1 cells contain several snps of the p2x7r gene ( 19 ) . among them , kg-1 cells with rs3751142 a revealed neutral effect on p2x7r function . however , there is no evidence as to whether the p2x7r polymorphism has a potent influence on the development of gout . in our data ,", "a minor allele frequency ( maf , a allele ) of p2x7r rs3751142 in gout and control patients was 0.132 and 0.161 , respectively , which are more compatible to those in the dbsnp rs3751142 ( http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?locusid05027 ) .", "this study found that p2x7r rs3751142 was not a genetic risk factor associated with gout susceptibility .", "the activation of the nlrp3 inflammasome , which is comprised of multiple cytoplasmic proteins including nlrp3 , an asc protein , and caspase-1 , is completed through the binding of the pyd of nlrp3 with the pyd of asc , which is followed by the recruitment of procaspase-1 by linkages of each card within the two proteins .", "card8 was shown to be a negative regulator of nuclear factor-b ( nf-b ) and caspase-1 activation ( 2021 ) , which implicates a role in the suppression of nlrp3 inflammasome activation .", "the rs2043211 snp in exon 5 of the card8 gene is a nonsense mutation resulting in the production of a truncated protein .", "although the functional role of polymorphic card8 within the nlrp3 inflammasome is complicated , genetic variants of card8 rs2043211 contribute to an enhanced inflammatory response in inflammatory diseases such as ra and ibd through increased expression of il-1 ( 8922 ) .", "mckinney et al . ( 7 ) demonstrated that the prevalence of card8 rs2043211 was different between gout and control patients in the european and polynesian populations ( or 1.11 , p = 0.023 and or 1.15 , p = 0.078 , respectively ) .", "another case - control study showed that the maf ( a allele ) card8 gene was shown to have an increasing trend relative to the t allele in the chinese male population ( or = 0.084 , p = 0.08 ) ( 6 ) .", "however , our study revealed no association of card8 rs2043211 with the presence of gout in korean study population .", "the frequency of maf ( t allele ) is at 0.457 in gout and 0.452 in control patients , which is similar to the frequency in the new zealand polynesian ( 0.499 in gout and 0.439 in control ) and chinese ( 0.498 in gout and 0.463 in control ) patients , whereas a lower frequency of maf was noted in the european population ( 7 ) .", "based on conflicting data for card8 rs2043211 between these three studies , the functional role of the card8 gene in gout susceptibility has not been clearly determined .", "therefore , the distinct role of the card8 gene in the pathogenesis of gout should be considered in a diverse ethnic study population .", "first , the lack of completeness for clinical information of control subjects is an important weakness in our study .", "this study used genomic dnas provided by the biobank of wonkwang university hospital , korea as control group samples .", "we were just able to identify a part of demographic data including gender and age .", "second , this study did not perform functional analysis for genotypes in each target gene . as shown disparity of genetic susceptibility of gout in several ethnic groups ( 67 ) , it is important to identify functional performance of each gene to understand the role of p2x7r and card8 snps in our study population .", "it is noted that the p2x7r and card8 proteins are closely involved in activation of the nlrp3 inflammasome and il-1 expression , which could explain their role in the pathogenesis of gout ( 12 ) .", "our study demonstrated that the p2x7r rs3751142 and card8 rs2043211 genetic variants were not implicated in the development of gout in the male korean population .", "however , we found that in a pair - wise comparison of the ca / tt p2x7r and card8 genotype combination was shown to have an increased trend for the risk of gout ( or = 2.618 , p = 0.056 ) . in conclusion , this study provides evidence that the interaction of msu crystal - mediated p2x7r with card8 may be at least in part responsible for the pathogenesis of gout ."]]}
{"article_id": "PMC3354430", "article_text": ["isolated involvement of the appendix in crohn 's disease is reported to be 0.2% to 1.8% , and is usually associated with ileocaecal crohn 's disease in 25% of ileal and 50% of caecal disease .", "while appendicitis in a patient who was previously diagnosed to have ileocaecal crohn 's may be managed with appendicectomy and ileocaecal resection , appendicectomy alone when performed for appendicitis in a patient with unsuspected ileocaecal crohn 's disease could lead to postoperative complications including enterocutaneous fistula .", "a young female patient who underwent appendicectomy elsewhere for acute appendicitis presented to us with a persistent enterocutaneous fistula of 6 weeks duration .", "she had complained of general ill health and occasional altered bowel habits for 6 months prior to the acute appendicitis presentation .", "our investigations , including a ct scan , suggested the possibility of ileocaecal crohn 's disease .", "she underwent excision of the enterocutaneous fistula and ileocaecal resection , and histopathology of the resected specimen confirmed crohn 's disease . in the postoperative period she received mesasalazine .", "when last seen 2 years later during her regular follow - up , she was found to be in good health .", "the possibility of ileocaecal crohn 's disease should be considered in patients presenting with unexplained postoperative enterocutaneous fistula following appendicectomy .", "a high index of clinical suspicion is required to make a prompt diagnosis and institute appropriate further treatment in form of ileocaecal resection .", "crohn 's disease is a chronic inflammatory condition that may involve the entire gi tract .", "histological features include granuloma , lymphoid aggregates , fissures and ulcers extending into muscularis propria , and transmural inflammation .", "crohn 's disease has been reported to be the cause of acute appendicitis in 0.2% to 1.8% of all the appendicectomies in some series .", "operations described in the literature for crohn 's disease of the appendix include appendicectomy with caecectomy and ileocaecal resection .", "however , patients who undergo appendectomy alone are at the risk of complications including enterocutaneous fistula in 34% to 58% of the cases", "a 24-year - old unmarried female patient presented to us with a persistent fistula in the right iliac fossa following an appendicectomy done in another hospital 6 weeks previously . on further questioning , she informed us that she had presented to this hospital with a 2-week history of abdominal pain , confined to the right lower abdomen .", "she complained of general ill health and occasional altered bowel habits for 6 months prior to this acute episode .", "an enquiry with the doctors who had initially seen her revealed that at the time of presentation for acute appendicitis she was quite unwell with fever , tachycardia , and dehydration .", "her abdomen was tender all over with guarding , more pronounced over the right lower half .", "an x - ray of her abdomen showed a few fluid levels and an ultrasound scan revealed free fluid in the abdomen with an associated right iliac fossa mass .", "a diagnosis of pelvic peritonitis secondary to acute appendicitis was made by the physicians , and she was taken up for surgery after adequate resuscitation .", "there was pus in the abdomen with an appendicular mass , and the appendix appeared oedematous , thickened , and congested .", "the abdomen was closed after placing a drain , which was removed on the 3 post - operative day . in spite of antibiotic administration", "she was managed conservatively with antibiotics and anti - inflammatory medication and eventually improved , except for a persistent purulent discharge from the wound .", "she then decided to seek our opinion and was admitted under our care for further investigation and management .", "the appendicular histopathology was retrieved from the previous hospital and revealed transmural inflammation with granulomas suggestive of crohn 's disease .", "a computerized tomography ( ct ) scan of the abdomen carried out in our hospital showed pericaecal collection with an inflammatory mass in front of the caecum ( fig .", "the abdomen was surgically explored through the previous incision after excising the fistula leading into the caecum . an inflammatory mass associated with the caecum was noted .", "the appendicular stump had not healed , and was draining into a cavity which was communicating with the wound , indicating a complex enterocutaneous fistula . a limited right hemicolectomy was carried out and continuity established with a primary anastomosis of macroscopically - appearing healthy bowel of the ascending colon and terminal ileum .", "a specimen of the resected caecum revealed the cobblestone appearance of the mucosa , strongly suggesting the possibility of underlying caecal crohn 's disease ( fig .", "the histology was reported as inflammatory bowel disease ( ibd ) consistent with crohn 's disease ( fig .", "she was referred to a gastroenterologist and was being treated with mesasalazine and a regular annual colonoscopy .", "when last seen at two years post - surgery she continued to remain in good health . limited right hemicolectomy specimen showing the cobble stone appearance of the mucosa typical of crohn 's disease .", "crohn 's disease may involve the appendix by extension from the terminal ileum or the caecum and present as an acute or sub - acute appendicitis[15 ] .", "crohn 's disease limited to the appendix usually affects young adults in their 20s and 30s , although it is not limited to this age group and can occur at any age .", "the clinical presentation is variable , with acute right iliac fossa pain suggestive of acute appendicitis in about 85% of patients , and chronic pain and a palpable mass in the right iliac fossa in about 25% of patients .", "when the preoperative diagnosis is acute appendicitis , crohn 's disease should be suspected when an atypical or protracted clinical course is present in a patient prior to the appendicitis , particularly in areas where crohn 's disease is prevalent .", "crohn 's disease is diagnosed by a combination of clinical manifestations and radiological findings including barium studies , showing string strictures , fissures , and fistula in the ileum and caecum .", "recently , gray scale sonography and color doppler - flow features have been used in diagnosing crohn 's disease . when the disease is located atypically in the appendix ,", "macroscopically , the appendix will be seen to be enlarged , swollen and adherent to the surrounding structures , these findings being secondary to chronic inflammation .", "histologically , the disease is characterized by transmural inflammation with thickening of the appendiceal wall , epitheloid granulomas , lymphoid aggregates , and mucosal ulceration .", "other histological features are multinucleated giant langherhan 's cells , crypt abscess , neural hyperplasia and lymphangiectasia .", "differential diagnosis includes the presence of foreign bodies and diverticulitis of the appendix , which also give rise to chronic granulomatous inflammation with induration and fibrosis of the appendiceal wall , and granulomatous disease of unknown aetiology such as appendiceal sarcoidosis , which is rare and often accompanies systemic disease .", "differential diagnosis should also include infectious diseases such as tuberculosis , actinomycosis and yersenia infection .", "a rare fungal infection such as histoplasmosis or blastomycosis , or a parasitic infestation such as schistosomiasis or enterobius vermicularis infestation can elicit an appendiceal granulomatous reaction .", "the diagnostic difficulty arises when a patient undergoes an appendicectomy for suspected acute appendicitis and the surgeon unexpectedly encounters ileocaecal pathology whose nature is difficult to ascertain in an emergency situation . in view of this , a definitive treatment in the form of ileocaecal resection for conditions like crohn 's disease may be difficult to carry out . while a frozen section may be helpful to differentiate some of these conditions it may not always be feasible in an emergency situation . if crohn 's disease of the appendix is suspected and is limited to the appendix then appendicectomy alone is a routine surgical procedure with very low intraoperative or postoperative mortality and a low rate of fistula formation.the postoperative enterocutaneous fistula incidence rate in crohn 's disease restricted to appendix alone has been reported to be 3.5% , whereas in patients with crohn 's disease of the ileocaecal segment , as in our patient , the postoperative enterocutaneous rate rises to 34% to 58% .", "weston et al reported that the majority of the patients whose first presentation of crohn 's disease simulates appendicitis and who undergo appendicectomy alone leaving the ileocaecal segment in place to be treated medically , returned postoperatively within 3 years with symptoms . a significant percentage ( 38% ) of these patients returned within one year and most of them ( 77% ) were frequently ill with their disease in the interim period .", "patients who had an ileocolic resection at the time of the initial operation did not require early reoperation , did not develop short bowel syndrome , and did not have significant postoperative complications .", "in the postoperative period these patients receive medical therapy to keep the disease quiescent such as salazopyrine , 5-asa , prednisilone , and azothioprine.crohn's disease - related fistula have also been treated with infliximab with some success . despite improvements in medical therapy , between 70% to 90% of the patients with crohn 's disease will eventually require surgical intervention , and approximately half of these will require additional operations . because of the high risk of reoperations and a relatively young age at the time of first operation , a minimally invasive approach has recently been used to carry out the ileocolic resections .", "a recent meta - analysis of laparoscopic ileocolic resection has revealed a shorter hospital stay compared to open resection , while the morbidity rates were equal and conversion rates were acceptable .", "the average interval between surgery and recurrence after appendicectomy for crohn 's disease of the appendix is 4 years , after which some claim the recurrence rate to be almost nil .", "however , others have reported a recurrence rate of 10% over an 8-year follow - up and is much higher in patients with crohn 's disease of the appendix associated with ileocaecal crohn 's disease .", "most authors recommend periodic surveillance visits with radiological and endoscopic examination of the small intestinal and colon for a period of at least 3 years to promptly detect recurrence .", "however , others claim that patients with crohn 's disease should be followed for 10 years , and if disease free by then are considered cured .", "crohn 's disease of the appendix is usually associated with crohn 's disease of the ileum and caecum .", "appendicectomy will suffice in those who present subacutely , are diagnosed preoperatively by clinical signs , and radiographic evidence shows that the disease is restricted to the appendix alone .", "however , in the presence of ileocaecal involvement , resection of the ileocaecal segment may be required to prevent postoperative complications of enterocutaneous fistula . while patients who develop enterocutaneous fistula post - appendicectomy in an unsuspected crohn 's disease case have been treated with infliximab with some success , the majority of the patients required ileocaecal resection . moreover ,"], "abstract_text": ["<S> context : isolated involvement of the appendix in crohn 's disease is reported to be 0.2% to 1.8% , and is usually associated with ileocaecal crohn 's disease in 25% of ileal and 50% of caecal disease . </S>", "<S> while appendicitis in a patient who was previously diagnosed to have ileocaecal crohn 's may be managed with appendicectomy and ileocaecal resection , appendicectomy alone when performed for appendicitis in a patient with unsuspected ileocaecal crohn 's disease could lead to postoperative complications including enterocutaneous fistula.case report : a young female patient who underwent appendicectomy elsewhere for acute appendicitis presented to us with a persistent enterocutaneous fistula of 6 weeks duration . </S>", "<S> she had complained of general ill health and occasional altered bowel habits for 6 months prior to the acute appendicitis presentation . </S>", "<S> our investigations , including a ct scan , suggested the possibility of ileocaecal crohn 's disease . </S>", "<S> she underwent excision of the enterocutaneous fistula and ileocaecal resection , and histopathology of the resected specimen confirmed crohn 's disease . in the postoperative period she received mesasalazine . when last seen 2 years later during her regular follow - up , she was found to be in good health.conclusion:the possibility of ileocaecal crohn 's disease </S>", "<S> should be considered in patients presenting with unexplained postoperative enterocutaneous fistula following appendicectomy . </S>", "<S> a high index of clinical suspicion is required to make a prompt diagnosis and institute appropriate further treatment in form of ileocaecal resection . </S>"], "labels": null, "section_names": ["Context:", "Case Report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["isolated involvement of the appendix in crohn 's disease is reported to be 0.2% to 1.8% , and is usually associated with ileocaecal crohn 's disease in 25% of ileal and 50% of caecal disease .", "while appendicitis in a patient who was previously diagnosed to have ileocaecal crohn 's may be managed with appendicectomy and ileocaecal resection , appendicectomy alone when performed for appendicitis in a patient with unsuspected ileocaecal crohn 's disease could lead to postoperative complications including enterocutaneous fistula ."], ["a young female patient who underwent appendicectomy elsewhere for acute appendicitis presented to us with a persistent enterocutaneous fistula of 6 weeks duration .", "she had complained of general ill health and occasional altered bowel habits for 6 months prior to the acute appendicitis presentation .", "our investigations , including a ct scan , suggested the possibility of ileocaecal crohn 's disease .", "she underwent excision of the enterocutaneous fistula and ileocaecal resection , and histopathology of the resected specimen confirmed crohn 's disease . in the postoperative period she received mesasalazine .", "when last seen 2 years later during her regular follow - up , she was found to be in good health ."], ["the possibility of ileocaecal crohn 's disease should be considered in patients presenting with unexplained postoperative enterocutaneous fistula following appendicectomy .", "a high index of clinical suspicion is required to make a prompt diagnosis and institute appropriate further treatment in form of ileocaecal resection ."], ["crohn 's disease is a chronic inflammatory condition that may involve the entire gi tract .", "histological features include granuloma , lymphoid aggregates , fissures and ulcers extending into muscularis propria , and transmural inflammation .", "crohn 's disease has been reported to be the cause of acute appendicitis in 0.2% to 1.8% of all the appendicectomies in some series .", "operations described in the literature for crohn 's disease of the appendix include appendicectomy with caecectomy and ileocaecal resection .", "however , patients who undergo appendectomy alone are at the risk of complications including enterocutaneous fistula in 34% to 58% of the cases"], ["a 24-year - old unmarried female patient presented to us with a persistent fistula in the right iliac fossa following an appendicectomy done in another hospital 6 weeks previously . on further questioning , she informed us that she had presented to this hospital with a 2-week history of abdominal pain , confined to the right lower abdomen .", "she complained of general ill health and occasional altered bowel habits for 6 months prior to this acute episode .", "an enquiry with the doctors who had initially seen her revealed that at the time of presentation for acute appendicitis she was quite unwell with fever , tachycardia , and dehydration .", "her abdomen was tender all over with guarding , more pronounced over the right lower half .", "an x - ray of her abdomen showed a few fluid levels and an ultrasound scan revealed free fluid in the abdomen with an associated right iliac fossa mass .", "a diagnosis of pelvic peritonitis secondary to acute appendicitis was made by the physicians , and she was taken up for surgery after adequate resuscitation .", "there was pus in the abdomen with an appendicular mass , and the appendix appeared oedematous , thickened , and congested .", "the abdomen was closed after placing a drain , which was removed on the 3 post - operative day . in spite of antibiotic administration", "she was managed conservatively with antibiotics and anti - inflammatory medication and eventually improved , except for a persistent purulent discharge from the wound .", "she then decided to seek our opinion and was admitted under our care for further investigation and management .", "the appendicular histopathology was retrieved from the previous hospital and revealed transmural inflammation with granulomas suggestive of crohn 's disease .", "a computerized tomography ( ct ) scan of the abdomen carried out in our hospital showed pericaecal collection with an inflammatory mass in front of the caecum ( fig .", "the abdomen was surgically explored through the previous incision after excising the fistula leading into the caecum . an inflammatory mass associated with the caecum was noted .", "the appendicular stump had not healed , and was draining into a cavity which was communicating with the wound , indicating a complex enterocutaneous fistula . a limited right hemicolectomy was carried out and continuity established with a primary anastomosis of macroscopically - appearing healthy bowel of the ascending colon and terminal ileum .", "a specimen of the resected caecum revealed the cobblestone appearance of the mucosa , strongly suggesting the possibility of underlying caecal crohn 's disease ( fig .", "the histology was reported as inflammatory bowel disease ( ibd ) consistent with crohn 's disease ( fig .", "she was referred to a gastroenterologist and was being treated with mesasalazine and a regular annual colonoscopy .", "when last seen at two years post - surgery she continued to remain in good health . limited right hemicolectomy specimen showing the cobble stone appearance of the mucosa typical of crohn 's disease ."], ["crohn 's disease may involve the appendix by extension from the terminal ileum or the caecum and present as an acute or sub - acute appendicitis[15 ] .", "crohn 's disease limited to the appendix usually affects young adults in their 20s and 30s , although it is not limited to this age group and can occur at any age .", "the clinical presentation is variable , with acute right iliac fossa pain suggestive of acute appendicitis in about 85% of patients , and chronic pain and a palpable mass in the right iliac fossa in about 25% of patients .", "when the preoperative diagnosis is acute appendicitis , crohn 's disease should be suspected when an atypical or protracted clinical course is present in a patient prior to the appendicitis , particularly in areas where crohn 's disease is prevalent .", "crohn 's disease is diagnosed by a combination of clinical manifestations and radiological findings including barium studies , showing string strictures , fissures , and fistula in the ileum and caecum .", "recently , gray scale sonography and color doppler - flow features have been used in diagnosing crohn 's disease . when the disease is located atypically in the appendix ,", "macroscopically , the appendix will be seen to be enlarged , swollen and adherent to the surrounding structures , these findings being secondary to chronic inflammation .", "histologically , the disease is characterized by transmural inflammation with thickening of the appendiceal wall , epitheloid granulomas , lymphoid aggregates , and mucosal ulceration .", "other histological features are multinucleated giant langherhan 's cells , crypt abscess , neural hyperplasia and lymphangiectasia .", "differential diagnosis includes the presence of foreign bodies and diverticulitis of the appendix , which also give rise to chronic granulomatous inflammation with induration and fibrosis of the appendiceal wall , and granulomatous disease of unknown aetiology such as appendiceal sarcoidosis , which is rare and often accompanies systemic disease .", "differential diagnosis should also include infectious diseases such as tuberculosis , actinomycosis and yersenia infection .", "a rare fungal infection such as histoplasmosis or blastomycosis , or a parasitic infestation such as schistosomiasis or enterobius vermicularis infestation can elicit an appendiceal granulomatous reaction .", "the diagnostic difficulty arises when a patient undergoes an appendicectomy for suspected acute appendicitis and the surgeon unexpectedly encounters ileocaecal pathology whose nature is difficult to ascertain in an emergency situation . in view of this , a definitive treatment in the form of ileocaecal resection for conditions like crohn 's disease may be difficult to carry out . while a frozen section may be helpful to differentiate some of these conditions it may not always be feasible in an emergency situation . if crohn 's disease of the appendix is suspected and is limited to the appendix then appendicectomy alone is a routine surgical procedure with very low intraoperative or postoperative mortality and a low rate of fistula formation.the postoperative enterocutaneous fistula incidence rate in crohn 's disease restricted to appendix alone has been reported to be 3.5% , whereas in patients with crohn 's disease of the ileocaecal segment , as in our patient , the postoperative enterocutaneous rate rises to 34% to 58% .", "weston et al reported that the majority of the patients whose first presentation of crohn 's disease simulates appendicitis and who undergo appendicectomy alone leaving the ileocaecal segment in place to be treated medically , returned postoperatively within 3 years with symptoms . a significant percentage ( 38% ) of these patients returned within one year and most of them ( 77% ) were frequently ill with their disease in the interim period .", "patients who had an ileocolic resection at the time of the initial operation did not require early reoperation , did not develop short bowel syndrome , and did not have significant postoperative complications .", "in the postoperative period these patients receive medical therapy to keep the disease quiescent such as salazopyrine , 5-asa , prednisilone , and azothioprine.crohn's disease - related fistula have also been treated with infliximab with some success . despite improvements in medical therapy , between 70% to 90% of the patients with crohn 's disease will eventually require surgical intervention , and approximately half of these will require additional operations . because of the high risk of reoperations and a relatively young age at the time of first operation , a minimally invasive approach has recently been used to carry out the ileocolic resections .", "a recent meta - analysis of laparoscopic ileocolic resection has revealed a shorter hospital stay compared to open resection , while the morbidity rates were equal and conversion rates were acceptable .", "the average interval between surgery and recurrence after appendicectomy for crohn 's disease of the appendix is 4 years , after which some claim the recurrence rate to be almost nil .", "however , others have reported a recurrence rate of 10% over an 8-year follow - up and is much higher in patients with crohn 's disease of the appendix associated with ileocaecal crohn 's disease .", "most authors recommend periodic surveillance visits with radiological and endoscopic examination of the small intestinal and colon for a period of at least 3 years to promptly detect recurrence .", "however , others claim that patients with crohn 's disease should be followed for 10 years , and if disease free by then are considered cured ."], ["crohn 's disease of the appendix is usually associated with crohn 's disease of the ileum and caecum .", "appendicectomy will suffice in those who present subacutely , are diagnosed preoperatively by clinical signs , and radiographic evidence shows that the disease is restricted to the appendix alone .", "however , in the presence of ileocaecal involvement , resection of the ileocaecal segment may be required to prevent postoperative complications of enterocutaneous fistula . while patients who develop enterocutaneous fistula post - appendicectomy in an unsuspected crohn 's disease case have been treated with infliximab with some success , the majority of the patients required ileocaecal resection . moreover ,"]]}
{"article_id": "PMC3169089", "article_text": ["the random sample comprises 267 caucasian preadolescents ( 121 girls , 146 boys ) and 330 caucasian adolescents ( 164 girls , 166 boys ) of a population belonging to the swedish part of the european youth heart study ( eyhs ) .", "the eyhs is a cross - sectional school - based study of risk factors for future cardiovascular disease among preadolescents ( 910 years old ) and adolescents ( 1516 years old ) .", "the mean ages in the swedish sample for preadolescents and adolescents were 9.6 years and 15.6 years , respectively .", "height , weight , and birth weight were measured by internationally accepted standardised procedures ( 22 ) .", "body mass index ( bmi ) was calculated as weight / height2 ( kg / m ) .", "identification of sexual maturity was assessed according to tanner and whitehouse 1976 . a researcher of the same gender as the child recorded the pubertal stage after brief observation . on account of ethical reasons", "the direction of one school preferred not to take part in the assessment of sexual maturity .", "fifty subjects did therefore not participate in this assessment ( 3 preadolescent girls , 7 preadolescent boys , 25 adolescent girls , and 15 adolescent boys ) .", "the consumption of milk was assessed by an interviewer - mediated 24-hour recall . in preadolescents ,", "a qualitative food record completed the day before the interview with the help of parents served as a checklist for the data obtained during the recall .", "dietary data were processed by stormats ( version 4.02 , rudans lttdata , sweden ) and analysed using the swedish national food database ( version 99.1 ) . as part of a broad - ranging questionnaire , parents of the participants were asked if their child had a chronic illness or adhered to a special diet . since under - nourishment is practically non - existent in a nutritionally replete population such as sweden and milk intake accounts in part for daily energy intake , the bmi was taken as proxy for energy intake .", "furthermore , bmi is traditionally used to validate energy intake data ( 23 ) . for the genetic analysis of lp and lnp ,", "genomic dna was isolated from edta whole blood samples from the individuals with the qiaamp dna blood mini kit spin procedure .", "the dna fragment spanning the -13910-c / t polymorphic site was amplified using a biotinylated forward - primer ( 5 gggctggcaatacagataagata-3 ) and an unbiotinylated reverse - primer ( 5 agcagggctcaaagaacaatcta-3 ) .", "the applied sequencing primer was : 5-ctttgaggccaggg-3. sequencing was performed using a psq96 snp reagent kit and a psq 96ma system ( pyrosequencing ab ) psq 96ma 2.0.1 software .", "the procedure has been previously described in detail ( 24 , 25 ) . for the determination of socio - economic status ( ses ) we used the dichotomous variable , below versus above the mean income level in the catchment areas of the sample ( below or above the mean in their municipality ) on account of the relatively equal distribution of income in sweden ( 26 ) . for four subjects , data for ses", "student 's t - test was used to determine differences in milk intake ( g / d ) between lp and lnp subjects .", "milk intake ( g / d ) was somewhat skewed to the right and therefore split in quintiles of milk intake .", "stepwise backward multiple linear regression analysis was performed in order to study the relationship between milk intake in quintiles and body height ( cm ) after adjustments for sex , birth weight ( g ) , father 's and mother 's height ( cm ) , bmi , tanner stage ( 15 ) , and ses .", "the study was approved by the research ethics committees of rebro county council and huddinge university hospital .", "parents and 15-year - olds gave specific written informed consent to participate in the study .", "the variables included in the backward multivariate regression model were : milk intake , lct c > t-13910 polymorphism ( lp = ct , tt vs. lnp = cc ) , sex , birth weight , father 's and mother 's height , bmi , tanner stage , and ses .", "characteristics of preadolescents and adolescents by quintile of milk intake are given in table 2 .", "basic characteristics of covariates to body height in the study population of swedish preadolescents and adolescents variables associated to body height by quintiles of milk intake fifty - six ( 9% ) of the subjects of the whole sample were lnp . where lnp may restrict the intake of milk in these subjects and", "can thereby have an effect on the dependent variable , body height as well as the exposure variable , i.e. milk intake .", "milk intake tended to be lower in lnp compared to lp but the difference did not reach statistical significance ( p=0.067 ) .", "the studied exposure variables in this model were milk intake in quintiles and the lct c > t-13910 polymorphism , and the outcome variable was body height .", "the model explained 90% of the observed variance of body height in preadolescents and adolescents ( adjusted r \n = 0.89 ) .", "non - significant variables were removed in a stepwise manner by elimination when p(f )  0.10 .", "model 1 included the variables : milk intake in quintiles , lct-13910 c > t polymorphism , sex , birth weight , parents height , tanner stage , bmi , and ses . in a first step", "the variable bmi was eliminated and in a second step ses ( table 3 ) .", "milk intake ( g / d ) in quintiles remained significantly different ( =0.46 ; 95% ci : 0.040 , 0.87 and p=0.032 ) in the final model .", "in addition , the lct c > t-13910 polymorphism ( lp vs. lnp ) , remained significant in the final model ( =2.05 ; 95% ci : 0.18 , 3.92 and p=0.032 ) , showing a positive association of lp with height .", "main effect by quintiles of milk intake and lct-13910 c > t ( exposure variables ) on body height in swedish preadolescents and adolescents ( n=542 ) ; stepwise backward multiple linear regression models were used included variables in model 1 : birth weight , sex , mother 's and father 's height .", "bmi , tanner stage , ses excluded variable : bmi ( p[f ]  0.10 ) . excluded variables : ses ( p[f ]  0.10 ) .", "we found that milk intake and lct c > t-13910 polymorphism , known to modulate milk consumption , were significant contributors to the observed variance of body height in the studied population .", "we primarily did not expect to find our hypothesis confirmed in a nutritionally replete country with already one of the highest intakes of milk and dairy products per capita in the world .", "the lct c > t-13910 polymorphism might contribute with a small effect to phenotypic variation in height .", "as far as we know no genome wide association studies or meta - analysis have been performed previously addressing the question whether lp or lnp affects body height .", "increasing evidence suggests that cow 's milk consumption exerts a positive effect on longitudinal growth in preadolescents and adolescents , for instance , by affecting the igf - i - axis ( 8 , 11 , 2730 ) .", "but not all studies show a positive effect of milk on height , especially in nordic countries , where average milk intake per capita traditionally is high ( 31 ) .", "the lct c > t-13910 polymorphism , known to modulate milk intake ( 32 , 33 ) , remained significant in the final model . by including the lct c >", "t-13910 polymorphism in our model , we subtract for hidden heritability in an already predominantly lp population ( 18 ) . in a recent prospective study , evidence of an association between increased infant size at birth and cow milk consumption during pregnancy", "it is known that lp subjects consume on average more milk than lnp subjects . where lp might thus lead to an increased susceptibility towards already prenatally programmed increased body sizes , mediated by the higher capacity of lp mothers to consume milk and dairy products .", "our study accounts for parental height as regards milk intake and its potential effects on body height and growth in preadolescents and adolescents .", "despite the circumstance that body height is a highly heritable complex trait ; corrections for parental height continue to be an exception , also shown in recent studies ( 31 ) .", "even twin growth studies have been performed without consideration of parental height ( 35 ) .", "it has been demonstrated that at the current stage a simple prediction based on phenotype of relatives obviously outperforms sophisticated genomic predictions as regards body height ( 36 )", ". limitations of this study are the sample size , and the limits inherent to cross - sectional studies as regards causal inference .", "cow 's milk is an evolutionary food constructed to promote growth and development in calves and appears to affect growth in human beings also ( 13 , 14 , 28 ) .", "milk has become a normative food for preadolescents and adolescents beyond weaning age , even in asian populations and developing countries , which traditionally did not have home fare based on domesticated dairy animals ( 8 , 37 ) .", "the long - term effects of this changed nutritional normative are yet poorly understood , even in western countries .", "we conclude that actual milk intake and the genetic lp trait is positively associated with body height in preadolescents and adolescents .", "nevertheless , higher milk consumption in childhood might exert both negative and positive effects , since greater height has been associated with higher risk of some cancers ( 29 , 3841 ) .", "this study was supported by grants from rebro lns landstings forskningskommitt and by nyckelfonden , rebro , sweden ."], "abstract_text": ["<S> backgroundbody height is a classic polygenic trait . about 80%90% of height </S>", "<S> is inherited and 10%20% owed to environmental factors , of which the most important ones are nutrition and diseases in preadolescents and adolescents.objectivethe aim of this study was to explore potential relations between the lct ( lactase ) c > t-13910 polymorphism , milk consumption , and body height in a sample of swedish preadolescents and adolescents.designin a cross - sectional study , using a random sample of preadolescents and adolescents ( n = 597 ) , dietary intakes were determined . </S>", "<S> anthropometric measurements including sexual maturity ( tanner stage ) and birth weight were assessed . parental body height and socio - economic status ( ses ) were obtained by questionnaires . </S>", "<S> genotyping for the lct c > t-13910 polymorphism that renders individuals lactase persistent ( lp ) or lactase non - persistent ( lnp ) was performed by dna sequencing . </S>", "<S> stepwise backward multivariate linear regression was used.resultsmilk consumption was significantly and positively associated with body height (  = 0.45 ; 95% ci : 0.040 , 0.87 , p = 0.032 ) . </S>", "<S> adjustments were performed for sex , parental height , birth weight , body mass index ( bmi ) , ses , and tanner stage . </S>", "<S> this model explains 90% of the observed variance of body height ( adjusted r2 = 0.89 ) . </S>", "<S> the presence of the -13910 t allele was positively associated with body height (  = 2.05 ; 95% ci : 0.18 , 3.92 , p = 0.032).conclusionsmilk consumption is positively associated with body height in preadolescents and adolescents . we show for the first time that a nutrigenetic variant might be able to explain in part phenotypic variation of body height in preadolescents and adolescents . due to the small sample size </S>", "<S> further studies are needed . </S>"], "labels": null, "section_names": ["Methods", "Results", "Discussion", "Conflict of interest and funding"], "sections": [["the random sample comprises 267 caucasian preadolescents ( 121 girls , 146 boys ) and 330 caucasian adolescents ( 164 girls , 166 boys ) of a population belonging to the swedish part of the european youth heart study ( eyhs ) .", "the eyhs is a cross - sectional school - based study of risk factors for future cardiovascular disease among preadolescents ( 910 years old ) and adolescents ( 1516 years old ) .", "the mean ages in the swedish sample for preadolescents and adolescents were 9.6 years and 15.6 years , respectively .", "height , weight , and birth weight were measured by internationally accepted standardised procedures ( 22 ) .", "body mass index ( bmi ) was calculated as weight / height2 ( kg / m ) .", "identification of sexual maturity was assessed according to tanner and whitehouse 1976 . a researcher of the same gender as the child recorded the pubertal stage after brief observation . on account of ethical reasons", "the direction of one school preferred not to take part in the assessment of sexual maturity .", "fifty subjects did therefore not participate in this assessment ( 3 preadolescent girls , 7 preadolescent boys , 25 adolescent girls , and 15 adolescent boys ) .", "the consumption of milk was assessed by an interviewer - mediated 24-hour recall . in preadolescents ,", "a qualitative food record completed the day before the interview with the help of parents served as a checklist for the data obtained during the recall .", "dietary data were processed by stormats ( version 4.02 , rudans lttdata , sweden ) and analysed using the swedish national food database ( version 99.1 ) . as part of a broad - ranging questionnaire , parents of the participants were asked if their child had a chronic illness or adhered to a special diet . since under - nourishment is practically non - existent in a nutritionally replete population such as sweden and milk intake accounts in part for daily energy intake , the bmi was taken as proxy for energy intake .", "furthermore , bmi is traditionally used to validate energy intake data ( 23 ) . for the genetic analysis of lp and lnp ,", "genomic dna was isolated from edta whole blood samples from the individuals with the qiaamp dna blood mini kit spin procedure .", "the dna fragment spanning the -13910-c / t polymorphic site was amplified using a biotinylated forward - primer ( 5 gggctggcaatacagataagata-3 ) and an unbiotinylated reverse - primer ( 5 agcagggctcaaagaacaatcta-3 ) .", "the applied sequencing primer was : 5-ctttgaggccaggg-3. sequencing was performed using a psq96 snp reagent kit and a psq 96ma system ( pyrosequencing ab ) psq 96ma 2.0.1 software .", "the procedure has been previously described in detail ( 24 , 25 ) . for the determination of socio - economic status ( ses ) we used the dichotomous variable , below versus above the mean income level in the catchment areas of the sample ( below or above the mean in their municipality ) on account of the relatively equal distribution of income in sweden ( 26 ) . for four subjects , data for ses", "student 's t - test was used to determine differences in milk intake ( g / d ) between lp and lnp subjects .", "milk intake ( g / d ) was somewhat skewed to the right and therefore split in quintiles of milk intake .", "stepwise backward multiple linear regression analysis was performed in order to study the relationship between milk intake in quintiles and body height ( cm ) after adjustments for sex , birth weight ( g ) , father 's and mother 's height ( cm ) , bmi , tanner stage ( 15 ) , and ses .", "the study was approved by the research ethics committees of rebro county council and huddinge university hospital .", "parents and 15-year - olds gave specific written informed consent to participate in the study ."], ["the variables included in the backward multivariate regression model were : milk intake , lct c > t-13910 polymorphism ( lp = ct , tt vs. lnp = cc ) , sex , birth weight , father 's and mother 's height , bmi , tanner stage , and ses .", "characteristics of preadolescents and adolescents by quintile of milk intake are given in table 2 .", "basic characteristics of covariates to body height in the study population of swedish preadolescents and adolescents variables associated to body height by quintiles of milk intake fifty - six ( 9% ) of the subjects of the whole sample were lnp . where lnp may restrict the intake of milk in these subjects and", "can thereby have an effect on the dependent variable , body height as well as the exposure variable , i.e. milk intake .", "milk intake tended to be lower in lnp compared to lp but the difference did not reach statistical significance ( p=0.067 ) .", "the studied exposure variables in this model were milk intake in quintiles and the lct c > t-13910 polymorphism , and the outcome variable was body height .", "the model explained 90% of the observed variance of body height in preadolescents and adolescents ( adjusted r \n = 0.89 ) .", "non - significant variables were removed in a stepwise manner by elimination when p(f )  0.10 .", "model 1 included the variables : milk intake in quintiles , lct-13910 c > t polymorphism , sex , birth weight , parents height , tanner stage , bmi , and ses . in a first step", "the variable bmi was eliminated and in a second step ses ( table 3 ) .", "milk intake ( g / d ) in quintiles remained significantly different ( =0.46 ; 95% ci : 0.040 , 0.87 and p=0.032 ) in the final model .", "in addition , the lct c > t-13910 polymorphism ( lp vs. lnp ) , remained significant in the final model ( =2.05 ; 95% ci : 0.18 , 3.92 and p=0.032 ) , showing a positive association of lp with height .", "main effect by quintiles of milk intake and lct-13910 c > t ( exposure variables ) on body height in swedish preadolescents and adolescents ( n=542 ) ; stepwise backward multiple linear regression models were used included variables in model 1 : birth weight , sex , mother 's and father 's height .", "bmi , tanner stage , ses excluded variable : bmi ( p[f ]  0.10 ) . excluded variables : ses ( p[f ]  0.10 ) ."], ["we found that milk intake and lct c > t-13910 polymorphism , known to modulate milk consumption , were significant contributors to the observed variance of body height in the studied population .", "we primarily did not expect to find our hypothesis confirmed in a nutritionally replete country with already one of the highest intakes of milk and dairy products per capita in the world .", "the lct c > t-13910 polymorphism might contribute with a small effect to phenotypic variation in height .", "as far as we know no genome wide association studies or meta - analysis have been performed previously addressing the question whether lp or lnp affects body height .", "increasing evidence suggests that cow 's milk consumption exerts a positive effect on longitudinal growth in preadolescents and adolescents , for instance , by affecting the igf - i - axis ( 8 , 11 , 2730 ) .", "but not all studies show a positive effect of milk on height , especially in nordic countries , where average milk intake per capita traditionally is high ( 31 ) .", "the lct c > t-13910 polymorphism , known to modulate milk intake ( 32 , 33 ) , remained significant in the final model . by including the lct c >", "t-13910 polymorphism in our model , we subtract for hidden heritability in an already predominantly lp population ( 18 ) . in a recent prospective study , evidence of an association between increased infant size at birth and cow milk consumption during pregnancy", "it is known that lp subjects consume on average more milk than lnp subjects . where lp might thus lead to an increased susceptibility towards already prenatally programmed increased body sizes , mediated by the higher capacity of lp mothers to consume milk and dairy products .", "our study accounts for parental height as regards milk intake and its potential effects on body height and growth in preadolescents and adolescents .", "despite the circumstance that body height is a highly heritable complex trait ; corrections for parental height continue to be an exception , also shown in recent studies ( 31 ) .", "even twin growth studies have been performed without consideration of parental height ( 35 ) .", "it has been demonstrated that at the current stage a simple prediction based on phenotype of relatives obviously outperforms sophisticated genomic predictions as regards body height ( 36 )", ". limitations of this study are the sample size , and the limits inherent to cross - sectional studies as regards causal inference .", "cow 's milk is an evolutionary food constructed to promote growth and development in calves and appears to affect growth in human beings also ( 13 , 14 , 28 ) .", "milk has become a normative food for preadolescents and adolescents beyond weaning age , even in asian populations and developing countries , which traditionally did not have home fare based on domesticated dairy animals ( 8 , 37 ) .", "the long - term effects of this changed nutritional normative are yet poorly understood , even in western countries .", "we conclude that actual milk intake and the genetic lp trait is positively associated with body height in preadolescents and adolescents .", "nevertheless , higher milk consumption in childhood might exert both negative and positive effects , since greater height has been associated with higher risk of some cancers ( 29 , 3841 ) ."], ["this study was supported by grants from rebro lns landstings forskningskommitt and by nyckelfonden , rebro , sweden ."]]}
{"article_id": "PMC3317101", "article_text": ["primary hyperparathyroidism ( php ) is a hypercalcemic disease stemming from an abnormal increase in parathyroid hormone ( pth ) secretion by one or more parathyroid glands .", "the hallmark of this condition is the presence of high levels of calcium and high or inappropriate levels of pth .", "primary hyperparathyroidism is more common in women than in men and increases with aging in both genders .", "the goal of parathyroidectomy is the excision of the abnormal parathyroid gland(s ) , preserving the normal ones in order to achieve and maintain a postoperative normocalcemic state .", "success rates for surgical treatment depend on the skill and experience of the surgeon in finding and recognizing the pathologic changes and excising the correct amount of hyperfunctioning parathyroid tissue .", "the surgical treatment of php has undergone substantive changes since the first successful parathyroidectomy was performed by felix mandl in 1925 .", "it is now expected that the vast majority of patients will be cured during initial surgical exploration at a low probability of morbidity . the conventional time - honored operation employing general endotracheal anesthesia and bilateral cervical exploration is safe and effective when performed by experienced surgeons .", "however , recent technical innovations , including improved preoperative localization and availability of rapid intraoperative pth assays ( iopth ) , have yielded focused approaches with excellent outcomes .", "sporadic primary hyperparathyroidism is caused by a single enlarged parathyroid gland ( parathyroid adenoma ) in approximately 85% of the cases , whereas multigland hyperplasia occurs in 15% and parathyroid carcinoma is found in less than 1% of patients . unlike the previous dogma that required surgical identification of both enlarged and normal parathyroid glands , the current paradigm in many centers is to identify and excise the incident enlarged gland and to confirm operative cure , employing a rapid intraoperative pth assay . due to the relatively short half - life of pth ( 4 - 5  min ) ,", "a dramatic drop in circulating hormone can be detected once the abnormally secreting gland or glands have been removed .", "a curative drop in pth allows the surgeon to terminate the operation and obviate additional exploration , whereas failure of the pth levels to demonstrate an adequate decrement asks for additional exploration because of the presence of presumed additional hypersecreting gland(s ) .", "the aim of the present study was to evaluate our 10-year experience in employing a rapid intraoperative pth assay for php .", "this is a prospective study on a cohort of operated patients treated at a university referral center .", "this investigation was approved by the unifesp / epm ethics committee . from june 2000 to april 2011 , 96 surgeries for php were performed at hospital so paulo unifesp / epm , so paulo , brazil .", "these 96 procedures were performed in 91 previous unexplored patients who had at least 6 months of postoperative follow - up with enough reported data to be eligible for the study .", "the php diagnoses were established in the presence of high levels of calcium and high or inappropriate levels of pth .", "all patients had their age , gender , symptoms ( bone and kidney ) , preoperative localization tests , serum ionized calcium ( ica ) , serum total calcium and intact pth recorded before parathyroidectomy , as well as iopth dosages .", "the laboratorial tests were repeated 1 month , 6 months , and in 1-year increments after surgery .", "a series of 91 consecutive patients with primary hyperparathyroidism underwent parathyroidectomy guided by intraoperative pth at federal university of so paulo , brazil , from june 2000 to april 2011 . focused parathyroidectomy guided by intraoperative pth was the initial procedure when preoperative localization tests were positive and when there were no suspicion of malignant disease .", "a bilateral cervical exploration guided by intraoperative pth was performed when preoperative localization tests were negative .", "a baseline peripheral venous blood sample was obtained just after anesthesia induction as well as 10 minutes after the abnormal parathyroid tissue removal .", "the intraoperative criterion used to predict successful parathyroidectomy was a decrease in the intact pth levels exceeding 50% from the preincision hormone level .", "if this criterion was met , surgical exploration of the neck would be completed and the incision closed .", "otherwise , further surgical exploration of the neck would have to be carried on .  ", "intraoperative pth was measured using elecsys pth immunoassay ( elecsys 1010 system , roche , mannheim , germany ) .", "the test is an immunometric assay based on monoclonal antibodies , magnetic particles as solid phase , and ruthenium complex as chemiluminescent label .", "total time to perform the assay is 9 minutes ; reference values are 1065  pg / ml . to validate the rapid pth assay , 170 samples from the study", "the iopth accuracy calculation was based on the following definitions : a true - positive ( tp ) result of iopth was defined as the correct prediction of postoperative normal calcium levels for at least 6 months ; true negative ( tn ) was the correct prediction of incomplete excision by either resection of an additional gland(s ) or operative failure ; false positive ( fp ) was the incorrect prediction of normocalcemia with subsequent postoperative persistent hypercalcemia and high pth levels ; false negative ( fn ) was the incorrect prediction of incomplete excision followed by postoperative normocalcemia .", "persistent disease was considered when serum calcium and pth levels remained above normal range just after surgery .", "recurrence was defined when , after reaching normocalcemic levels , serum calcium and intact pth measurements start to rise to abnormal values at least 6 months after surgery .  ", "serum ionized calcium ( 1.151.32  mmol / l ) , serum total calcium ( 8.510.5  mg / dl ) and intact pth ( 1167  pg / ml ) , were measured using standard automatic assays .", "the demographic , clinical , and biochemical aspects of 91 patients with php are shown in table 1 . among all cohort of 91 patients , 69 ( 75.8% ) had solitary adenoma , 10 ( 11.0% ) had multiple endocrine neoplasia type 1 ( men1 ) , 6 ( 6.6% ) had double adenomas , 4 ( 4.4% ) had carcinomas , and 2 ( 2.2% ) patients are still waiting for remedial surgery for multiglandular disease ( mgd ) , 1 of whom had recurrence disease after 10 years of follow - up and the other persistent disease after a false positive iopth ( table 2 ) .", "we had 85 ( 93.4% ) successful parathyroidectomies 6 ( 6.6% ) failed parathyroidectomies in 91 previous unexplored patients , and 5 ( 100% ) successful remedial surgeries . among the 85 successful patients ,", "69 ( 81.2% ) had solitary adenoma , 3 ( 3.5% ) had carcinoma , 4 ( 4.7% ) had double adenomas , 8 ( 9.4% ) had men1 , and 1 ( 1.2% ) still has mgd ( table 2 ) .", "the mean decay of iopth in the successful group of patients was 80.5% ( 34.3% to 96.0% ) . in these 85 patients", ", we had 80 tp results ( 67 adenomas , 3 carcinomas , 8 men1 and 2 recurrences ", "we also had 3 cases of double adenoma in which the iopth resulted in a tn value , requiring additional exploration to prevent persistent disease ( table 2 ) .", "there were only 2 fn results among the successful group patients ( table 4 ) .", "the mean decay of iopth in the 80 patients with tp results was 81.7% , with a minimum drop of 55.0% ( 55.0% to 96.0% ) .", "operative failure of the initial surgery occurred in 6 patients : 2 double adenoma , 2 men1 , 1 carcinoma , and 1 mgd who is still waiting for remedial surgery ( table 5 ) .", "both cases of double adenoma had a tn decay of iopth , but the second adenoma wasnot found during the initial operation . with the aid of new localization exams ,", "successful remedial operations were performed with an iopth tp result . the patient with parathyroid carcinoma developed hypercalcemia and high levels of pth two months after surgery . in search of distant metastasis , this patient underwent a pulmonary computerized tomography and multiple pulmonary nodules were found .", "for this reason , she was classified as iopth false - positive . in both men1 patients ,", "one patient already had men1 diagnosis at the time of initial surgery and we failed to find the fourth gland .", "the other patient was first operated as a solitary adenoma and men1 diagnosis surfaced just in the follow - up .", "there was only one case in the operative failure patients ; the iopth had an fp result ( table 4 ) .", "the surgical treatment for primary php has undergone some changes in recent years , evolving from the standard bilateral neck exploration technique to a less time - consuming procedure of unilateral neck exploration . taking into account the increasing number of asymptomatic and/or oligosymptomatic primary hyperparathyroidism diagnosis in the recent years , the need for a safe and less time - consuming procedure with low perioperative morbidity is clear .", "preoperative parathyroid localization imaging study and iopth are essential components of the focused parathyroidectomy that allow the excision of all abnormal parathyroid glands without the examination of the normally secreting glands .", "the clinical utility of rapid iopth measurements in parathyroidectomy was first reported in 1988 using a modified intact pth irma assay . since then", ", rapid assays have been developed by means of radioactive [ 4 , 11 , 12 ] as well as nonradioactive formats [ 12 , 13 ] .", "the predominance of a solitary adenoma disease in 85% to 96% of cases of php and the short half - life of intact pth ( 184 ) of only 1.4 to 4 minutes [ 1420 ] combined with the remaining suppressed normal parathyroid glands after removal of all hyperfunctioning tissue   allows the measurement of iopth to evaluate its decline rates .", "several studies have demonstrated the utility of iopth monitoring in the treatment of single - gland primary hyperparathyroidism [ 3 , 8 , 2127 ] .", "most experts agree that iopth assay is the most useful intraoperative adjunct to assist the surgeon in php surgical treatment [ 28 , 29 ] .", "it is worth highlighting the useful employment of preoperative localization imaging study in conjunction with iopth : the former points out where the surgeon should start exploration from , and the latter assures that hypersecretory parathyroid tissue removal was accomplished . the current usual criteria for iopth measurement describe a decrease of 50% or over from either the baseline ( preincision )   or the highest preincision or preexcision   value within 10 minutes following hyperfunctioning parathyroid resection , pointing out surgical cure and predicting normocalcemia .", "by resorting to those criteria , high accuracy in intraoperative prediction of cure is achieved .", "mostly for practice and cost reasons , we have used just two samples as our criteria since we started making use of the iopth : the preincision and 10 minutes postexcision of hyperfunctioning parathyroid .", "our series of 96 php consecutive surgeries over 10 years reflects a complex tertiary referral center and has its limitations .", "most of these patients were referred specifically due to their severe signs / symptoms and comorbid medical conditions , which are related to their high bone and kidney disease and the 4.4% incidence of carcinoma .", "furthermore , all patients were first evaluated by an endocrinologist group specialized in osteometabolic disease who elected the patients for surgical treatment , reflecting the 11% incidence of men1 and the absence of familial hypocalciuric hypercalcemia case in our surgical series .", "the php diagnose has not been made in a routine practice in most brazilian centers , and many patients have their diagnoses made just after severe signs / symptoms .", "we have noticed an increase in the percentage of asymptomatic patients over the years in the number of php cases , but probably such increase has not been enough to raise the number of our small solitary adenomas ( 78.3% ) .", "we get an inferior number of successful parathyroidectomy ( 93.4% ) in previous unexplored patients compared to some large series [ 9 , 32 ] , but it can be considered a satisfactory result , taking into account the high number of patients with carcinoma and men1 in our series . on the other hand , we have obtained good results ( 100% ) in 5 successful second surgical explorations so far : first , we removed a second adenoma in three patients ( two persistent and one recurrent disease ) ; second , we were able to remove a not found fourth gland in a men1 patient , and finally , we removed three glands in a men1 patient previous operated as an sporadic php . in our series of 91 patients ,", "iopth had true results in 87 patients ( 95.6% ) . however , considering our 96 surgical procedures ( 5 remedial surgeries ) , iopth was able to obviate or to ask for additional exploration because of the presence of presumed additional hypersecreting gland(s ) in 92 ( 95.8% ) procedures .", "the iopth had a mean decay of 81.7% in the tp patients , and the minimum drop in our series that results in patient cure ( operative success ) was 55.0% .", "one of these cases occurred in a ruptured parathyroid cystic adenoma that may have resulted in a substantial elevation of the hormone levels after the preincision sample , and it should be related to an inadequate iopth decay .", "the second patient had the others three parathyroid glands identified in normal conditions . based on such scenario ,", "the iopth decay was 50.9% , dropping from 216  pg / dl to 106  pg / dl .", "as observed by other authors , the fp cases are usually represented by a marginal pth level decrease with a final pth level above the normal range .", "for these reasons , stricter criteria for the iopth dynamics have been suggested , such as the return of the 10-minute pth to within normal range .", "however , these stricter criteria were estimated to increase the operative success only by 0.3% , with significant increase in the false negative results , bringing on more unnecessary bilateral neck dissection [ 9 , 33 ] . using these stricter criteria in our patients", "would have brought about 21 ( 24.7% ) unnecessary bilateral dissections , and just one additional diagnose .", "we classified a patient with parathyroid carcinoma that revealed metastatic pulmonary disease two months after surgery as an fp result because she certainly had metastatic disease at the surgery and iopth ( 84.1% decay ) failed to predict the presence of presumed additional hypersecreting tissue .", "however , it can be controversial to suppose that iopth has failed in this patient , once iopth was not defined to predict distant disease .", "all 4 patients with parathyroid carcinoma had very suggestive signals of malignant disease at presentation ( very high pth levels , severe hypercalcemia and palpable neck mass ) and were not elected to the focused approach .", "they were submitted to an  en bloc  tumor resection , removing the parathyroid tumor , the ipsilateral thyroid lobe , and the lymph nodes related to a central neck dissection . during the follow - up period , 2 ( 2.1% )", "one patient removed a second adenoma and had a successful remedial surgery with iopth decay of 78.4% .", "the second patient recurrence occurred almost 10 years after initial surgery and is still waiting for remedial surgery .", "published a large series of 1,650 php patients and found 5 ( 0.3% ) recurrent cases .", "one of our series limitations is the absence of vitamin d dosages , one important factor that should be related to the genesis of php recurrence .", "the iopth revealed to be an important technological adjunct in the current parathyroid surgery for php ."], "abstract_text": ["<S> \n introduction . primary hyperparathyroidism ( php ) is characteristically determined by high levels of calcium and high or inappropriate levels of parathyroid hormone ( pth ) . </S>", "<S> technological advances have dramatically changed the surgical technique over the years once intraoperative parathyroid hormone ( iopth ) assay had allowed for focused approaches . objective . to evaluate our 10-year experience in employing a rapid intraoperative pth assay for php . </S>", "<S> methods . a prospective cohort of 91 php - operated patients in a tertiary institution in so paulo , brazil , from june 2000 to april 2011 . results . </S>", "<S> we had 85 ( 93.4% ) successful parathyroidectomies , 6 ( 6.6% ) failed parathyroidectomies in 91 previous unexplored patients , and 5 ( 100% ) successful remedial surgeries . </S>", "<S> the iopth was true - positive in 88.5% , true - negative in 7.3% , false - positive in 2.1% , and false - negative in 2.1% of the procedures . </S>", "<S> iopth was able to obviate additional exploration or to ask for additional exploration in 92 ( 95.8% ) procedures . conclusion . </S>", "<S> the iopth revealed to be an important technological adjunct in the current parathyroid surgery for php . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Patients and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["primary hyperparathyroidism ( php ) is a hypercalcemic disease stemming from an abnormal increase in parathyroid hormone ( pth ) secretion by one or more parathyroid glands .", "the hallmark of this condition is the presence of high levels of calcium and high or inappropriate levels of pth .", "primary hyperparathyroidism is more common in women than in men and increases with aging in both genders .", "the goal of parathyroidectomy is the excision of the abnormal parathyroid gland(s ) , preserving the normal ones in order to achieve and maintain a postoperative normocalcemic state .", "success rates for surgical treatment depend on the skill and experience of the surgeon in finding and recognizing the pathologic changes and excising the correct amount of hyperfunctioning parathyroid tissue .", "the surgical treatment of php has undergone substantive changes since the first successful parathyroidectomy was performed by felix mandl in 1925 .", "it is now expected that the vast majority of patients will be cured during initial surgical exploration at a low probability of morbidity . the conventional time - honored operation employing general endotracheal anesthesia and bilateral cervical exploration is safe and effective when performed by experienced surgeons .", "however , recent technical innovations , including improved preoperative localization and availability of rapid intraoperative pth assays ( iopth ) , have yielded focused approaches with excellent outcomes .", "sporadic primary hyperparathyroidism is caused by a single enlarged parathyroid gland ( parathyroid adenoma ) in approximately 85% of the cases , whereas multigland hyperplasia occurs in 15% and parathyroid carcinoma is found in less than 1% of patients . unlike the previous dogma that required surgical identification of both enlarged and normal parathyroid glands , the current paradigm in many centers is to identify and excise the incident enlarged gland and to confirm operative cure , employing a rapid intraoperative pth assay . due to the relatively short half - life of pth ( 4 - 5  min ) ,", "a dramatic drop in circulating hormone can be detected once the abnormally secreting gland or glands have been removed .", "a curative drop in pth allows the surgeon to terminate the operation and obviate additional exploration , whereas failure of the pth levels to demonstrate an adequate decrement asks for additional exploration because of the presence of presumed additional hypersecreting gland(s ) .", "the aim of the present study was to evaluate our 10-year experience in employing a rapid intraoperative pth assay for php ."], ["this is a prospective study on a cohort of operated patients treated at a university referral center .", "this investigation was approved by the unifesp / epm ethics committee . from june 2000 to april 2011 , 96 surgeries for php were performed at hospital so paulo unifesp / epm , so paulo , brazil .", "these 96 procedures were performed in 91 previous unexplored patients who had at least 6 months of postoperative follow - up with enough reported data to be eligible for the study .", "the php diagnoses were established in the presence of high levels of calcium and high or inappropriate levels of pth .", "all patients had their age , gender , symptoms ( bone and kidney ) , preoperative localization tests , serum ionized calcium ( ica ) , serum total calcium and intact pth recorded before parathyroidectomy , as well as iopth dosages .", "the laboratorial tests were repeated 1 month , 6 months , and in 1-year increments after surgery .", "a series of 91 consecutive patients with primary hyperparathyroidism underwent parathyroidectomy guided by intraoperative pth at federal university of so paulo , brazil , from june 2000 to april 2011 . focused parathyroidectomy guided by intraoperative pth was the initial procedure when preoperative localization tests were positive and when there were no suspicion of malignant disease .", "a bilateral cervical exploration guided by intraoperative pth was performed when preoperative localization tests were negative .", "a baseline peripheral venous blood sample was obtained just after anesthesia induction as well as 10 minutes after the abnormal parathyroid tissue removal .", "the intraoperative criterion used to predict successful parathyroidectomy was a decrease in the intact pth levels exceeding 50% from the preincision hormone level .", "if this criterion was met , surgical exploration of the neck would be completed and the incision closed .", "otherwise , further surgical exploration of the neck would have to be carried on .  ", "intraoperative pth was measured using elecsys pth immunoassay ( elecsys 1010 system , roche , mannheim , germany ) .", "the test is an immunometric assay based on monoclonal antibodies , magnetic particles as solid phase , and ruthenium complex as chemiluminescent label .", "total time to perform the assay is 9 minutes ; reference values are 1065  pg / ml . to validate the rapid pth assay , 170 samples from the study", "the iopth accuracy calculation was based on the following definitions : a true - positive ( tp ) result of iopth was defined as the correct prediction of postoperative normal calcium levels for at least 6 months ; true negative ( tn ) was the correct prediction of incomplete excision by either resection of an additional gland(s ) or operative failure ; false positive ( fp ) was the incorrect prediction of normocalcemia with subsequent postoperative persistent hypercalcemia and high pth levels ; false negative ( fn ) was the incorrect prediction of incomplete excision followed by postoperative normocalcemia .", "persistent disease was considered when serum calcium and pth levels remained above normal range just after surgery .", "recurrence was defined when , after reaching normocalcemic levels , serum calcium and intact pth measurements start to rise to abnormal values at least 6 months after surgery .  ", "serum ionized calcium ( 1.151.32  mmol / l ) , serum total calcium ( 8.510.5  mg / dl ) and intact pth ( 1167  pg / ml ) , were measured using standard automatic assays ."], ["the demographic , clinical , and biochemical aspects of 91 patients with php are shown in table 1 . among all cohort of 91 patients , 69 ( 75.8% ) had solitary adenoma , 10 ( 11.0% ) had multiple endocrine neoplasia type 1 ( men1 ) , 6 ( 6.6% ) had double adenomas , 4 ( 4.4% ) had carcinomas , and 2 ( 2.2% ) patients are still waiting for remedial surgery for multiglandular disease ( mgd ) , 1 of whom had recurrence disease after 10 years of follow - up and the other persistent disease after a false positive iopth ( table 2 ) .", "we had 85 ( 93.4% ) successful parathyroidectomies 6 ( 6.6% ) failed parathyroidectomies in 91 previous unexplored patients , and 5 ( 100% ) successful remedial surgeries . among the 85 successful patients ,", "69 ( 81.2% ) had solitary adenoma , 3 ( 3.5% ) had carcinoma , 4 ( 4.7% ) had double adenomas , 8 ( 9.4% ) had men1 , and 1 ( 1.2% ) still has mgd ( table 2 ) .", "the mean decay of iopth in the successful group of patients was 80.5% ( 34.3% to 96.0% ) . in these 85 patients", ", we had 80 tp results ( 67 adenomas , 3 carcinomas , 8 men1 and 2 recurrences ", "we also had 3 cases of double adenoma in which the iopth resulted in a tn value , requiring additional exploration to prevent persistent disease ( table 2 ) .", "there were only 2 fn results among the successful group patients ( table 4 ) .", "the mean decay of iopth in the 80 patients with tp results was 81.7% , with a minimum drop of 55.0% ( 55.0% to 96.0% ) .", "operative failure of the initial surgery occurred in 6 patients : 2 double adenoma , 2 men1 , 1 carcinoma , and 1 mgd who is still waiting for remedial surgery ( table 5 ) .", "both cases of double adenoma had a tn decay of iopth , but the second adenoma wasnot found during the initial operation . with the aid of new localization exams ,", "successful remedial operations were performed with an iopth tp result . the patient with parathyroid carcinoma developed hypercalcemia and high levels of pth two months after surgery . in search of distant metastasis , this patient underwent a pulmonary computerized tomography and multiple pulmonary nodules were found .", "for this reason , she was classified as iopth false - positive . in both men1 patients ,", "one patient already had men1 diagnosis at the time of initial surgery and we failed to find the fourth gland .", "the other patient was first operated as a solitary adenoma and men1 diagnosis surfaced just in the follow - up .", "there was only one case in the operative failure patients ; the iopth had an fp result ( table 4 ) ."], ["the surgical treatment for primary php has undergone some changes in recent years , evolving from the standard bilateral neck exploration technique to a less time - consuming procedure of unilateral neck exploration . taking into account the increasing number of asymptomatic and/or oligosymptomatic primary hyperparathyroidism diagnosis in the recent years , the need for a safe and less time - consuming procedure with low perioperative morbidity is clear .", "preoperative parathyroid localization imaging study and iopth are essential components of the focused parathyroidectomy that allow the excision of all abnormal parathyroid glands without the examination of the normally secreting glands .", "the clinical utility of rapid iopth measurements in parathyroidectomy was first reported in 1988 using a modified intact pth irma assay . since then", ", rapid assays have been developed by means of radioactive [ 4 , 11 , 12 ] as well as nonradioactive formats [ 12 , 13 ] .", "the predominance of a solitary adenoma disease in 85% to 96% of cases of php and the short half - life of intact pth ( 184 ) of only 1.4 to 4 minutes [ 1420 ] combined with the remaining suppressed normal parathyroid glands after removal of all hyperfunctioning tissue   allows the measurement of iopth to evaluate its decline rates .", "several studies have demonstrated the utility of iopth monitoring in the treatment of single - gland primary hyperparathyroidism [ 3 , 8 , 2127 ] .", "most experts agree that iopth assay is the most useful intraoperative adjunct to assist the surgeon in php surgical treatment [ 28 , 29 ] .", "it is worth highlighting the useful employment of preoperative localization imaging study in conjunction with iopth : the former points out where the surgeon should start exploration from , and the latter assures that hypersecretory parathyroid tissue removal was accomplished . the current usual criteria for iopth measurement describe a decrease of 50% or over from either the baseline ( preincision )   or the highest preincision or preexcision   value within 10 minutes following hyperfunctioning parathyroid resection , pointing out surgical cure and predicting normocalcemia .", "by resorting to those criteria , high accuracy in intraoperative prediction of cure is achieved .", "mostly for practice and cost reasons , we have used just two samples as our criteria since we started making use of the iopth : the preincision and 10 minutes postexcision of hyperfunctioning parathyroid .", "our series of 96 php consecutive surgeries over 10 years reflects a complex tertiary referral center and has its limitations .", "most of these patients were referred specifically due to their severe signs / symptoms and comorbid medical conditions , which are related to their high bone and kidney disease and the 4.4% incidence of carcinoma .", "furthermore , all patients were first evaluated by an endocrinologist group specialized in osteometabolic disease who elected the patients for surgical treatment , reflecting the 11% incidence of men1 and the absence of familial hypocalciuric hypercalcemia case in our surgical series .", "the php diagnose has not been made in a routine practice in most brazilian centers , and many patients have their diagnoses made just after severe signs / symptoms .", "we have noticed an increase in the percentage of asymptomatic patients over the years in the number of php cases , but probably such increase has not been enough to raise the number of our small solitary adenomas ( 78.3% ) .", "we get an inferior number of successful parathyroidectomy ( 93.4% ) in previous unexplored patients compared to some large series [ 9 , 32 ] , but it can be considered a satisfactory result , taking into account the high number of patients with carcinoma and men1 in our series . on the other hand , we have obtained good results ( 100% ) in 5 successful second surgical explorations so far : first , we removed a second adenoma in three patients ( two persistent and one recurrent disease ) ; second , we were able to remove a not found fourth gland in a men1 patient , and finally , we removed three glands in a men1 patient previous operated as an sporadic php . in our series of 91 patients ,", "iopth had true results in 87 patients ( 95.6% ) . however , considering our 96 surgical procedures ( 5 remedial surgeries ) , iopth was able to obviate or to ask for additional exploration because of the presence of presumed additional hypersecreting gland(s ) in 92 ( 95.8% ) procedures .", "the iopth had a mean decay of 81.7% in the tp patients , and the minimum drop in our series that results in patient cure ( operative success ) was 55.0% .", "one of these cases occurred in a ruptured parathyroid cystic adenoma that may have resulted in a substantial elevation of the hormone levels after the preincision sample , and it should be related to an inadequate iopth decay .", "the second patient had the others three parathyroid glands identified in normal conditions . based on such scenario ,", "the iopth decay was 50.9% , dropping from 216  pg / dl to 106  pg / dl .", "as observed by other authors , the fp cases are usually represented by a marginal pth level decrease with a final pth level above the normal range .", "for these reasons , stricter criteria for the iopth dynamics have been suggested , such as the return of the 10-minute pth to within normal range .", "however , these stricter criteria were estimated to increase the operative success only by 0.3% , with significant increase in the false negative results , bringing on more unnecessary bilateral neck dissection [ 9 , 33 ] . using these stricter criteria in our patients", "would have brought about 21 ( 24.7% ) unnecessary bilateral dissections , and just one additional diagnose .", "we classified a patient with parathyroid carcinoma that revealed metastatic pulmonary disease two months after surgery as an fp result because she certainly had metastatic disease at the surgery and iopth ( 84.1% decay ) failed to predict the presence of presumed additional hypersecreting tissue .", "however , it can be controversial to suppose that iopth has failed in this patient , once iopth was not defined to predict distant disease .", "all 4 patients with parathyroid carcinoma had very suggestive signals of malignant disease at presentation ( very high pth levels , severe hypercalcemia and palpable neck mass ) and were not elected to the focused approach .", "they were submitted to an  en bloc  tumor resection , removing the parathyroid tumor , the ipsilateral thyroid lobe , and the lymph nodes related to a central neck dissection . during the follow - up period , 2 ( 2.1% )", "one patient removed a second adenoma and had a successful remedial surgery with iopth decay of 78.4% .", "the second patient recurrence occurred almost 10 years after initial surgery and is still waiting for remedial surgery .", "published a large series of 1,650 php patients and found 5 ( 0.3% ) recurrent cases .", "one of our series limitations is the absence of vitamin d dosages , one important factor that should be related to the genesis of php recurrence ."], ["the iopth revealed to be an important technological adjunct in the current parathyroid surgery for php ."]]}
{"article_id": "PMC4876731", "article_text": ["the prevalence of diabetes in the u.s . has increased ( 1 ) , and cardiovascular ( cv ) complications remain the major cause of morbidity and mortality in persons with type 2 diabetes , contributing substantially to increased health care costs ( 2 ) . as individuals attempt to manage their diabetes , there is increasing recognition that comorbid behavioral problems , including both depressive symptoms and stress , are associated with poor glycemic control , poor lifestyle behaviors , and increased health services utilization ( 310 ) .", "we ( 11 ) and others ( 3,4 ) have shown that these behavioral challenges are associated with inadequate medication adherence , which may be associated with adverse outcomes .", "similarly , poor meta - bolic control may worsen depressive symptoms ( 5 ) , and the relationship appears to be bidirectional ( 8) . the prevalence of these comorbidities and their potential to worsen disease management have led some to develop interventions designed to address comorbid depressive symptoms or stress in persons with diabetes ( 1214 ) .", "while many studies have documented the cross - sectional presence of these comorbidities and the effect on glycemic control in subjects with diabetes , only a limited number of studies have examined the potential impact on cv outcomes .", "most of these studies have been limited to patients with depression recruited from established health care settings , and the true population - level impact is unknown .", "likewise , there has not been adequate comparison of individuals with and without diabetes who have concurrent depressive symptoms and/or elevated levels of perceived stress to examine the unique consequences of behavioral and medical comorbidity .", "the objective of the present report was to examine the association between the presence of depressive symptoms and/or increased levels of perceived stress , determined at baseline , and risk of incident cv events over 5 years of follow - up in a major national cohort study that used population - based sampling methods , collecting data in the home both in subjects with and without diabetes .", "the description of the population for this analysis has previously been reported ( 15 ) . in short ,", "the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the  stroke belt ", "it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .", "( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .", "the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .", "the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .", "the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .", "the study also included an in - home visit by a trained health professional for data collection .", "participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .", "the present analysis included all members of the cohort who had complete follow - up data .", "a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .", "height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .", "all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .", "atrial fibrillation was determined to be present if there was a self - reported history ( asked as  has a doctor or other health professional ever told you that you had atrial fibrillation ?  ) or if characteristic findings were present on the ecg as defined in the protocol .", "left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .", "hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) .", "diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin  each obtained during the home visit .", "the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .", "the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .", "the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .", "these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .", "subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .", "perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .", "it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .", "subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores .", "the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .", "living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .", "a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease .", "medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,", "each event was adjudicated via medical record review by a neurologist - led medical review team .", "stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .", "after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .", "specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .", "participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index .", "for information surrounding the circumstances of participant death , proxies or next of kin were interviewed . additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports", "were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) .", "data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .", "additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .", "also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .", "smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker .", "the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .", "follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .", "the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) . the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .", "age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .", "these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non  stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) .", "the description of the population for this analysis has previously been reported ( 15 ) . in short ,", "the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the  stroke belt ", "it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .", "( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .", "the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .", "the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .", "the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .", "the study also included an in - home visit by a trained health professional for data collection .", "participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .", "the present analysis included all members of the cohort who had complete follow - up data .", "a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .", "height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .", "all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .", "atrial fibrillation was determined to be present if there was a self - reported history ( asked as  has a doctor or other health professional ever told you that you had atrial fibrillation ?  ) or if characteristic findings were present on the ecg as defined in the protocol .", "left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .", "hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) .", "diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin  each obtained during the home visit .", "the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .", "the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .", "the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .", "these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .", "subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .", "perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .", "it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .", "subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores .", "the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .", "living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .", "a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease . for each positive response , the date and time of each event were recorded .", "medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,", "each event was adjudicated via medical record review by a neurologist - led medical review team .", "stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .", "after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .", "specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .", "participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index . for information surrounding the circumstances of participant death , proxies or next of kin", "additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) .", "data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .", "additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .", "also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .", "smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker .", "the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .", "follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .", "the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) .", "the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .", "age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and", "subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .", "these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non  stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) .", "the current study included a total of 22,003 subjects , of whom 42% were black , 58% female , and 56% living in the southeastern u.s . stroke belt . among all subjects at baseline , 18.6% subjects ( n = 4,090 ) had diabetes , 10% ( n = 2,202 ) reported increased depressive symptoms , and 28% ( n = 6,132 ) reported elevated levels of stress .", "subjects with diabetes were more likely to report either elevated depressive symptoms or stress or both than were subjects without diabetes ( 36.8% vs. 29.5% ; p < 0.001 ) .", "detailed demographic characteristics for the study group are given in table 1 , separated into those with and without diabetes and stratified by the presence of behavioral comorbidities . among subjects with diabetes ,", "those reporting either increased depressive symptoms or stress or the combination were more likely to be women , black , live in the stroke belt , and have limited income .", "subjects reporting either elevated depressive symptoms or stress or both had a higher prevalence of elevated baseline hs - crp values . across each category of behavioral comorbidity ( i.e. , none , either elevated depressive symptoms or stress , both elevated ) , subjects with diabetes had a higher prevalence of elevated baseline hs - crp values , higher mean systolic bp , and greater prevalence of statin use than those without diabetes .", "baseline characteristics of regards participants with or without diabetes , by depressive symptom / stress category * p value for comparison of subjects with diabetes vs. subjects with no diabetes .", "table 2 provides age - adjusted incidence rates per 1,000 person - years of follow - up for each of the three independent cv outcomes , broken out by the presence of behavioral comorbidities in those with and without diabetes . with respect to individual cv outcomes ,", "the pattern of age - adjusted incidence rates was approximately twofold higher in those with diabetes than in those without diabetes across the range of comorbid behavioral illness . among individuals with diabetes and one or both behavioral comorbidities , age - adjusted incidence rates were highest for acute chd , followed by cv death , and stroke . among those without diabetes but with one or more behavioral comorbidities , incidence rates were again highest for acute chd but were followed by stroke and cv death .", "event numbers , age - adjusted incidence rates per 1,000 person - years , and crude and adjusted hrs for stroke , acute chd , and cv disease death for individuals with stress or depressive symptoms and with stress and depressive symptoms compared with those with neither , for individuals with and without diabetes crude model adjusted for depressive symptom and stress category .", "model 1 adjusted for crude model plus demographic factors ( race , sex , age , region ) .", "model 2 adjusted for crude model plus demographic factors plus social and economic factors ( income , education , health insurance ) .", "full model adjusted for crude model plus demographic factors , social and economic factors , and risk factors ( bmi , total cholesterol , hs - crp , atrial fibrillation , left ventricular hypertrophy , systolic blood pressure , statin use , current cigarette smoking ) .", "table 2 also provides the results of a series of crude and progressively adjusted hazard ratio ( hr ) models for each of the three independent cv outcomes , demonstrating the progressive impact of comorbidity on acute chd and cv death among subjects with diabetes , relative to those without diabetes , even in fully adjusted models that accounted for demographic , socioeconomic , and cv risk factors . unlike the pattern for acute chd and cv death ,", "the magnitude of the hrs for stroke was very similar in subjects with one versus both behavioral comorbidities and diabetes .", "figure 1 illustrates this significant and progressive pattern of worsening fully adjusted hrs for cv death among those with behavioral comorbidities , relative to those with no behavioral comorbidities , in subjects with diabetes and in comparison with those who do not have diabetes . among individuals with diabetes , the presence of a single behavioral comorbidity  either increased depressive symptoms or stress  increased the risk of cv death by 53% relative to individuals with diabetes but without either behavioral comorbidity . among individuals with diabetes , the presence of both behavioral comorbidities  increased depressive symptoms and stress ", "increased the risk of cv death by 115% relative to individuals with neither behavioral comorbidity , even after adjustment for a wide range of demographic and cv risk factors .", "while there was a pattern of increasing risk of cv death among those with one or both behavioral comorbidities ( vs. neither ) in subjects without diabetes , the differences were substantially smaller ( 12% and 27% increases , respectively ) and were not statistically significant .", "consequences of comorbid diabetes and elevated depressive symptoms and/or stress on cv death during follow - up among participants in the regards study .", "this article joins a growing body of literature that demonstrates a compelling pattern of augmented risk for cv events or cv death associated with comorbid behavioral illness among individuals with diabetes that far exceeds that observed in individuals with these same illnesses but without diabetes .", "while several studies have shown that formally diagnosed depression and/or depressive symptoms in subjects who are patients in an existing health care system are associated with incident chd , this is among the first studies to compare the impact of these behavioral comorbidities on cv outcomes and mortality in a population - based sample of subjects with versus without diabetes in the same study .", "it is the first study to compare the effects of both depressive symptoms and stress together and separately in subjects with versus without diabetes , obtained by population - based sampling rather than recruitment in established health care settings .", "further , the regards study has a large sample size ; includes oversampling of african americans in the stroke belt region , allowing important racial comparisons ; and also includes rich data obtained in the subject s home environment .", "the study is novel in that , as a community - sampled study , it provides more precise estimates of the population burden and racial disparities associated with these comorbidities and their cv consequences than other studies in which patients were recruited in health care settings .", "the study provides a much larger sample of african americans and illustrates the greater prevalence of comorbidities among african american women in particular , suggesting the need for more careful screening .", "further , cv outcomes and cv death were carefully adjudicated events in the current study ; by contrast , no adjudication process occurred in many other studies .", "our findings suggest that the adverse consequences observed in individuals with behavioral comorbidities and diabetes are best understood in a larger context or spectrum of linkages between physical and behavioral illness and subsequent outcomes .", "elevated levels of stress and depressive symptoms , alone and together , were associated with a pattern of increased hrs for acute coronary disease and cv death in adjusted models both in subjects with and in subjects without diabetes , achieving statistical significance in those with diabetes .", "together this pattern suggests the potential for progressive impact of single and multiple behavioral comorbidities on adverse cv outcomes and mortality that is of greatest concern in subjects with comorbid diabetes .", "there was a significant pattern of increasing hrs for acute chd and cv death in those with diabetes and with one or both behavioral comorbidities ( none , stress or depressive symptoms alone , or both together ) .", "while diabetes has long been known to increase cv outcomes , our findings suggest that , relative to those individuals with diabetes who have no behavioral comorbidity , those with either depressive symptoms or elevated levels of stress as well as those with both together have progressively increased risks for acute chd events and cv mortality during 5 years of follow - up , even when other risk factors are controlled for .", "in particular , behavioral comorbidities such as stress or depressive symptoms are not usually screened for in busy primary care practices , and many patients are reluctant to share these symptoms with busy primary care providers .", "further , many individuals are reluctant to seek care from mental health providers . among the demographic group in this study shown to have the highest prevalence of comorbid behavioral disease and diabetes ( i.e. , black females with limited income in the southeastern u.s . ) , there may also be distrust of the traditional health care delivery system . as a result , these comorbidities may go unrecognized and unmanaged .", "this study therefore provides strong evidence for adverse consequences associated with these comorbidities and suggests the need for more careful research to identify optimal screening and treatment strategies that work in busy primary care practices or other community settings .", "interestingly , while the presence of any behavioral comorbidity resulted in important increases in the hr for stroke , the increased hrs for one versus both behavioral comorbidities in subjects with diabetes were the same in fully adjusted models .", "this may suggest that a different mechanism is operative in the increased relationship of behavioral comorbidities and stroke .", "these findings build on and expand an established literature that links the cross - sectional and longitudinal presence of stress , depressive symptoms , and/or established depression with cv outcomes and/or mortality including in individuals with comorbid diabetes ( 10,2832 ) .", "because our study measured these symptoms at baseline and then followed patients longitudinally , it suggests the potential for the chronic influence of these behavioral comorbidities in subjects with diabetes to across time lead to adverse cv outcomes .", "indeed , the chronic nature of depressive symptoms and stress in individuals with type 2 diabetes has been described by fisher et al .", "( 10,33 ) , who called for a more careful understanding of the conjoint physical and emotional burden in these patients .", "further , these authors join our findings in calling for integrating the screening and management of the emotional side of diabetes into regular diabetes care . in addition , hamer et al .", "( 34 ) demonstrated an association between stress and cv events with hr of 1.5 and with the suggestion that behavioral processes explained the largest proportion of the variance in the hazards model .", "this raises an important question regarding the mechanism(s ) through which behavioral comorbidities result in increased cv events and/or cv death .", "an early review by plante ( 35 ) suggested that stress may lead to depressive symptoms that are subsequently associated with accelerated atherosclerosis , endothelial dysfunction , inflammatory reactions , and interstitial disturbances that may predispose the subject to premature cv disease or death . in the current study ,", "hs - crp levels were elevated in subjects with diabetes relative to those without diabetes , but among subjects with diabetes , there was a clearly progressive pattern of higher hs - crp levels among those with either versus both behavioral comorbidities .", "these elevated hs - crp levels may suggest a pattern of worsening atherosclerotic disease in subjects with diabetes with one or both behavioral comorbidities relative to those with no comorbidity . a recent article by pizzi et al .", "( 36 ) supports this and shows that depressive symptoms are associated with progressive longitudinal increases in carotid intima - media thickness relative to those who did not report these symptoms .", "this progressive pattern of atherosclerosis associated with these symptoms or potentially other behavioral problems might be expected to be more common in subjects with diabetes and therefore contribute to adverse cv outcomes .", "behavioral comorbidities may also interfere with self - care behaviors . among individuals with diabetes , a study by bonnet et al .", "( 37 ) showed that those with anxiety or depressive symptoms were less likely to engage in healthy behaviors including physical activity , proper eating behaviors , and avoidance of smoking than were those without anxiety or depressive symptoms .", "similarly , a meta - analysis by gonzalez et al . ( 3 ) that included studies of individuals with depressive symptoms showed that those with depressive symptoms or depression were more likely to be nonadherent with medications as well as with the larger treatment regimen .", "( 38 ) suggest a stronger relationship between diabetes - related distress and glycemic control than between depressive symptoms and glycemic control , and recent data by this group ( 39 ) show that specific reductions in regimen - related distress can result in improvements in medication adherence , physical activity , and glycemic control , all of which may influence cv risk .", "the scope of the implications from these important associations with cv outcomes should be considered in the context of the recent second diabetes attitudes , wishes and needs ( dawn2 ) study , which shows that these behavioral comorbidities know no geographic or ethnic boundaries and are highly prevalent in subjects with diabetes in multiple countries around the world ( 6 ) .", "this suggests the need for additional research to understand the mechanisms by which these behavioral comorbidities are associated with cv outcomes in subjects with diabetes as well as to understand innovative intervention strategies that will not only improve the behavioral comorbidities but will also decrease the risk for cv outcomes .", "clearly , more research is needed to understand whether the effective treatment of depressive symptoms and stress can lower cvd events in individuals with diabetes . and ,", "as noted above , more work needs to be done to establish appropriate screening strategies for behavioral comorbidities that can be effectively disseminated in busy primary care settings where most individuals with diabetes are managed .", "this study is a prospective cohort study and not a randomized clinical trial , and the incident outcomes should be understood as associations with baseline characteristics without clear evidence of causality .", "by design , included only non - hispanic white and black subjects ; extrapolation to other ethnic groups or to other nations should not be undertaken .", "the current study did not include a measure of diabetes duration , which may have influenced the risk of cv events .", "the study included data on a large number of cv risk factors that were used to help minimize the influence of confounders ; however , there may have been other unmeasured risk factors that were associated with adverse cv outcomes .", "the presence of elevated stress or depressive symptoms in community - dwelling subjects with diabetes was associated with an increased risk for acute chd or cv death .", "moreover , subjects with diabetes who reported both stress and depressive symptoms together had the greatest risk for acute chd and cv death .", "the presence of either or both behavioral comorbidities in subjects with diabetes was also associated with increased hrs for stroke .", "elevated stress and/or depressive symptoms in subjects without diabetes resulted in increased hrs in fully adjusted models that were much smaller in magnitude and not statistically significant .", "these findings demonstrate the persistent disparities and negative cv impact of these comorbidities at the population level and suggest the need for more careful integration of behavioral screening and management in primary care settings , where most patients with type 2 diabetes are managed ."], "abstract_text": ["<S> objectiveto evaluate the impact of comorbid depressive symptoms and/or stress on adverse cardiovascular ( cv ) outcomes in individuals with diabetes compared with those without diabetes.research design and methodsinvestigators examined the relationship between baseline depressive symptoms and/or stress in adults with and without diabetes and physician - adjudicated incident cv outcomes including stroke , myocardial infarction / acute coronary heart disease , and cv death over a median follow - up of 5.95 years in the national regards cohort study.resultssubjects included 22,003 adults ( 4,090 with diabetes ) ( mean age 64 years , 58% female , 42% black , and 56% living in the southeastern  stroke belt  ) . </S>", "<S> elevated stress and/or depressive symptoms were more common in subjects with diabetes ( 36.8% vs. 29.5% ; p < 0.001 ) </S>", "<S> . in fully adjusted models , reporting either elevated stress or depressive symptoms was associated with a significantly increased incidence of stroke ( hr 1.57 [ 95% ci 1.05 , 2.33 ] vs. 1.01 [ 0.79 , 1.30 ] ) and cv death ( 1.53 [ 1.08 , 2.17 ] vs. 1.12 [ 0.90 , 1.38 ] ) in subjects with diabetes but not in those without diabetes . the combination of both elevated stress and depressive symptoms in subjects with diabetes was associated with a higher incidence of cv death ( 2.15 [ 1.33 , 3.47 ] ) than either behavioral comorbidity alone ( 1.53 [ 1.08 , 2.17 ] ) and higher than in those with both elevated stress and depressive symptoms but without diabetes ( 1.27 [ 0.86 , 1.88]).conclusionscomorbid stress and/or depressive symptoms are common in individuals with diabetes and together are associated with progressively increased risks for adverse cv outcomes . </S>"], "labels": null, "section_names": ["Introduction", "Research Design and Methods", "Study Design, Cohort Description, and Medication Information", "Physical Examination and Laboratory Measures", "Diabetes, Depressive Symptoms, and Stress Measures", "CV Outcome Measures and Death", "Measurement of Potential Confounders", "Analysis", "Results", "Conclusions"], "sections": [["the prevalence of diabetes in the u.s . has increased ( 1 ) , and cardiovascular ( cv ) complications remain the major cause of morbidity and mortality in persons with type 2 diabetes , contributing substantially to increased health care costs ( 2 ) . as individuals attempt to manage their diabetes , there is increasing recognition that comorbid behavioral problems , including both depressive symptoms and stress , are associated with poor glycemic control , poor lifestyle behaviors , and increased health services utilization ( 310 ) .", "we ( 11 ) and others ( 3,4 ) have shown that these behavioral challenges are associated with inadequate medication adherence , which may be associated with adverse outcomes .", "similarly , poor meta - bolic control may worsen depressive symptoms ( 5 ) , and the relationship appears to be bidirectional ( 8) . the prevalence of these comorbidities and their potential to worsen disease management have led some to develop interventions designed to address comorbid depressive symptoms or stress in persons with diabetes ( 1214 ) .", "while many studies have documented the cross - sectional presence of these comorbidities and the effect on glycemic control in subjects with diabetes , only a limited number of studies have examined the potential impact on cv outcomes .", "most of these studies have been limited to patients with depression recruited from established health care settings , and the true population - level impact is unknown .", "likewise , there has not been adequate comparison of individuals with and without diabetes who have concurrent depressive symptoms and/or elevated levels of perceived stress to examine the unique consequences of behavioral and medical comorbidity .", "the objective of the present report was to examine the association between the presence of depressive symptoms and/or increased levels of perceived stress , determined at baseline , and risk of incident cv events over 5 years of follow - up in a major national cohort study that used population - based sampling methods , collecting data in the home both in subjects with and without diabetes ."], ["the description of the population for this analysis has previously been reported ( 15 ) . in short ,", "the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the  stroke belt ", "it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .", "( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .", "the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .", "the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .", "the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .", "the study also included an in - home visit by a trained health professional for data collection .", "participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .", "the present analysis included all members of the cohort who had complete follow - up data .", "a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .", "height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .", "all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .", "atrial fibrillation was determined to be present if there was a self - reported history ( asked as  has a doctor or other health professional ever told you that you had atrial fibrillation ?  ) or if characteristic findings were present on the ecg as defined in the protocol .", "left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .", "hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) .", "diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin  each obtained during the home visit .", "the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .", "the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .", "the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .", "these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .", "subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .", "perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .", "it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .", "subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores .", "the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .", "living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .", "a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease .", "medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,", "each event was adjudicated via medical record review by a neurologist - led medical review team .", "stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .", "after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .", "specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .", "participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index .", "for information surrounding the circumstances of participant death , proxies or next of kin were interviewed . additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports", "were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) .", "data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .", "additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .", "also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .", "smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker .", "the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .", "follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .", "the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) . the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .", "age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .", "these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non  stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) ."], ["the description of the population for this analysis has previously been reported ( 15 ) . in short ,", "the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the  stroke belt ", "it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .", "( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .", "the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .", "the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .", "the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .", "the study also included an in - home visit by a trained health professional for data collection .", "participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .", "the present analysis included all members of the cohort who had complete follow - up data ."], ["a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .", "height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .", "all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .", "atrial fibrillation was determined to be present if there was a self - reported history ( asked as  has a doctor or other health professional ever told you that you had atrial fibrillation ?  ) or if characteristic findings were present on the ecg as defined in the protocol .", "left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .", "hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) ."], ["diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin  each obtained during the home visit .", "the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .", "the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .", "the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .", "these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .", "subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .", "perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .", "it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .", "subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores ."], ["the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .", "living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .", "a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease . for each positive response , the date and time of each event were recorded .", "medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,", "each event was adjudicated via medical record review by a neurologist - led medical review team .", "stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .", "after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .", "specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .", "participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index . for information surrounding the circumstances of participant death , proxies or next of kin", "additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) ."], ["data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .", "additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .", "also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .", "smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker ."], ["the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .", "follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .", "the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) .", "the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .", "age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and", "subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .", "these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non  stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) ."], ["the current study included a total of 22,003 subjects , of whom 42% were black , 58% female , and 56% living in the southeastern u.s . stroke belt . among all subjects at baseline , 18.6% subjects ( n = 4,090 ) had diabetes , 10% ( n = 2,202 ) reported increased depressive symptoms , and 28% ( n = 6,132 ) reported elevated levels of stress .", "subjects with diabetes were more likely to report either elevated depressive symptoms or stress or both than were subjects without diabetes ( 36.8% vs. 29.5% ; p < 0.001 ) .", "detailed demographic characteristics for the study group are given in table 1 , separated into those with and without diabetes and stratified by the presence of behavioral comorbidities . among subjects with diabetes ,", "those reporting either increased depressive symptoms or stress or the combination were more likely to be women , black , live in the stroke belt , and have limited income .", "subjects reporting either elevated depressive symptoms or stress or both had a higher prevalence of elevated baseline hs - crp values . across each category of behavioral comorbidity ( i.e. , none , either elevated depressive symptoms or stress , both elevated ) , subjects with diabetes had a higher prevalence of elevated baseline hs - crp values , higher mean systolic bp , and greater prevalence of statin use than those without diabetes .", "baseline characteristics of regards participants with or without diabetes , by depressive symptom / stress category * p value for comparison of subjects with diabetes vs. subjects with no diabetes .", "table 2 provides age - adjusted incidence rates per 1,000 person - years of follow - up for each of the three independent cv outcomes , broken out by the presence of behavioral comorbidities in those with and without diabetes . with respect to individual cv outcomes ,", "the pattern of age - adjusted incidence rates was approximately twofold higher in those with diabetes than in those without diabetes across the range of comorbid behavioral illness . among individuals with diabetes and one or both behavioral comorbidities , age - adjusted incidence rates were highest for acute chd , followed by cv death , and stroke . among those without diabetes but with one or more behavioral comorbidities , incidence rates were again highest for acute chd but were followed by stroke and cv death .", "event numbers , age - adjusted incidence rates per 1,000 person - years , and crude and adjusted hrs for stroke , acute chd , and cv disease death for individuals with stress or depressive symptoms and with stress and depressive symptoms compared with those with neither , for individuals with and without diabetes crude model adjusted for depressive symptom and stress category .", "model 1 adjusted for crude model plus demographic factors ( race , sex , age , region ) .", "model 2 adjusted for crude model plus demographic factors plus social and economic factors ( income , education , health insurance ) .", "full model adjusted for crude model plus demographic factors , social and economic factors , and risk factors ( bmi , total cholesterol , hs - crp , atrial fibrillation , left ventricular hypertrophy , systolic blood pressure , statin use , current cigarette smoking ) .", "table 2 also provides the results of a series of crude and progressively adjusted hazard ratio ( hr ) models for each of the three independent cv outcomes , demonstrating the progressive impact of comorbidity on acute chd and cv death among subjects with diabetes , relative to those without diabetes , even in fully adjusted models that accounted for demographic , socioeconomic , and cv risk factors . unlike the pattern for acute chd and cv death ,", "the magnitude of the hrs for stroke was very similar in subjects with one versus both behavioral comorbidities and diabetes .", "figure 1 illustrates this significant and progressive pattern of worsening fully adjusted hrs for cv death among those with behavioral comorbidities , relative to those with no behavioral comorbidities , in subjects with diabetes and in comparison with those who do not have diabetes . among individuals with diabetes , the presence of a single behavioral comorbidity  either increased depressive symptoms or stress  increased the risk of cv death by 53% relative to individuals with diabetes but without either behavioral comorbidity . among individuals with diabetes , the presence of both behavioral comorbidities  increased depressive symptoms and stress ", "increased the risk of cv death by 115% relative to individuals with neither behavioral comorbidity , even after adjustment for a wide range of demographic and cv risk factors .", "while there was a pattern of increasing risk of cv death among those with one or both behavioral comorbidities ( vs. neither ) in subjects without diabetes , the differences were substantially smaller ( 12% and 27% increases , respectively ) and were not statistically significant .", "consequences of comorbid diabetes and elevated depressive symptoms and/or stress on cv death during follow - up among participants in the regards study ."], ["this article joins a growing body of literature that demonstrates a compelling pattern of augmented risk for cv events or cv death associated with comorbid behavioral illness among individuals with diabetes that far exceeds that observed in individuals with these same illnesses but without diabetes .", "while several studies have shown that formally diagnosed depression and/or depressive symptoms in subjects who are patients in an existing health care system are associated with incident chd , this is among the first studies to compare the impact of these behavioral comorbidities on cv outcomes and mortality in a population - based sample of subjects with versus without diabetes in the same study .", "it is the first study to compare the effects of both depressive symptoms and stress together and separately in subjects with versus without diabetes , obtained by population - based sampling rather than recruitment in established health care settings .", "further , the regards study has a large sample size ; includes oversampling of african americans in the stroke belt region , allowing important racial comparisons ; and also includes rich data obtained in the subject s home environment .", "the study is novel in that , as a community - sampled study , it provides more precise estimates of the population burden and racial disparities associated with these comorbidities and their cv consequences than other studies in which patients were recruited in health care settings .", "the study provides a much larger sample of african americans and illustrates the greater prevalence of comorbidities among african american women in particular , suggesting the need for more careful screening .", "further , cv outcomes and cv death were carefully adjudicated events in the current study ; by contrast , no adjudication process occurred in many other studies .", "our findings suggest that the adverse consequences observed in individuals with behavioral comorbidities and diabetes are best understood in a larger context or spectrum of linkages between physical and behavioral illness and subsequent outcomes .", "elevated levels of stress and depressive symptoms , alone and together , were associated with a pattern of increased hrs for acute coronary disease and cv death in adjusted models both in subjects with and in subjects without diabetes , achieving statistical significance in those with diabetes .", "together this pattern suggests the potential for progressive impact of single and multiple behavioral comorbidities on adverse cv outcomes and mortality that is of greatest concern in subjects with comorbid diabetes .", "there was a significant pattern of increasing hrs for acute chd and cv death in those with diabetes and with one or both behavioral comorbidities ( none , stress or depressive symptoms alone , or both together ) .", "while diabetes has long been known to increase cv outcomes , our findings suggest that , relative to those individuals with diabetes who have no behavioral comorbidity , those with either depressive symptoms or elevated levels of stress as well as those with both together have progressively increased risks for acute chd events and cv mortality during 5 years of follow - up , even when other risk factors are controlled for .", "in particular , behavioral comorbidities such as stress or depressive symptoms are not usually screened for in busy primary care practices , and many patients are reluctant to share these symptoms with busy primary care providers .", "further , many individuals are reluctant to seek care from mental health providers . among the demographic group in this study shown to have the highest prevalence of comorbid behavioral disease and diabetes ( i.e. , black females with limited income in the southeastern u.s . ) , there may also be distrust of the traditional health care delivery system . as a result , these comorbidities may go unrecognized and unmanaged .", "this study therefore provides strong evidence for adverse consequences associated with these comorbidities and suggests the need for more careful research to identify optimal screening and treatment strategies that work in busy primary care practices or other community settings .", "interestingly , while the presence of any behavioral comorbidity resulted in important increases in the hr for stroke , the increased hrs for one versus both behavioral comorbidities in subjects with diabetes were the same in fully adjusted models .", "this may suggest that a different mechanism is operative in the increased relationship of behavioral comorbidities and stroke .", "these findings build on and expand an established literature that links the cross - sectional and longitudinal presence of stress , depressive symptoms , and/or established depression with cv outcomes and/or mortality including in individuals with comorbid diabetes ( 10,2832 ) .", "because our study measured these symptoms at baseline and then followed patients longitudinally , it suggests the potential for the chronic influence of these behavioral comorbidities in subjects with diabetes to across time lead to adverse cv outcomes .", "indeed , the chronic nature of depressive symptoms and stress in individuals with type 2 diabetes has been described by fisher et al .", "( 10,33 ) , who called for a more careful understanding of the conjoint physical and emotional burden in these patients .", "further , these authors join our findings in calling for integrating the screening and management of the emotional side of diabetes into regular diabetes care . in addition , hamer et al .", "( 34 ) demonstrated an association between stress and cv events with hr of 1.5 and with the suggestion that behavioral processes explained the largest proportion of the variance in the hazards model .", "this raises an important question regarding the mechanism(s ) through which behavioral comorbidities result in increased cv events and/or cv death .", "an early review by plante ( 35 ) suggested that stress may lead to depressive symptoms that are subsequently associated with accelerated atherosclerosis , endothelial dysfunction , inflammatory reactions , and interstitial disturbances that may predispose the subject to premature cv disease or death . in the current study ,", "hs - crp levels were elevated in subjects with diabetes relative to those without diabetes , but among subjects with diabetes , there was a clearly progressive pattern of higher hs - crp levels among those with either versus both behavioral comorbidities .", "these elevated hs - crp levels may suggest a pattern of worsening atherosclerotic disease in subjects with diabetes with one or both behavioral comorbidities relative to those with no comorbidity . a recent article by pizzi et al .", "( 36 ) supports this and shows that depressive symptoms are associated with progressive longitudinal increases in carotid intima - media thickness relative to those who did not report these symptoms .", "this progressive pattern of atherosclerosis associated with these symptoms or potentially other behavioral problems might be expected to be more common in subjects with diabetes and therefore contribute to adverse cv outcomes .", "behavioral comorbidities may also interfere with self - care behaviors . among individuals with diabetes , a study by bonnet et al .", "( 37 ) showed that those with anxiety or depressive symptoms were less likely to engage in healthy behaviors including physical activity , proper eating behaviors , and avoidance of smoking than were those without anxiety or depressive symptoms .", "similarly , a meta - analysis by gonzalez et al . ( 3 ) that included studies of individuals with depressive symptoms showed that those with depressive symptoms or depression were more likely to be nonadherent with medications as well as with the larger treatment regimen .", "( 38 ) suggest a stronger relationship between diabetes - related distress and glycemic control than between depressive symptoms and glycemic control , and recent data by this group ( 39 ) show that specific reductions in regimen - related distress can result in improvements in medication adherence , physical activity , and glycemic control , all of which may influence cv risk .", "the scope of the implications from these important associations with cv outcomes should be considered in the context of the recent second diabetes attitudes , wishes and needs ( dawn2 ) study , which shows that these behavioral comorbidities know no geographic or ethnic boundaries and are highly prevalent in subjects with diabetes in multiple countries around the world ( 6 ) .", "this suggests the need for additional research to understand the mechanisms by which these behavioral comorbidities are associated with cv outcomes in subjects with diabetes as well as to understand innovative intervention strategies that will not only improve the behavioral comorbidities but will also decrease the risk for cv outcomes .", "clearly , more research is needed to understand whether the effective treatment of depressive symptoms and stress can lower cvd events in individuals with diabetes . and ,", "as noted above , more work needs to be done to establish appropriate screening strategies for behavioral comorbidities that can be effectively disseminated in busy primary care settings where most individuals with diabetes are managed .", "this study is a prospective cohort study and not a randomized clinical trial , and the incident outcomes should be understood as associations with baseline characteristics without clear evidence of causality .", "by design , included only non - hispanic white and black subjects ; extrapolation to other ethnic groups or to other nations should not be undertaken .", "the current study did not include a measure of diabetes duration , which may have influenced the risk of cv events .", "the study included data on a large number of cv risk factors that were used to help minimize the influence of confounders ; however , there may have been other unmeasured risk factors that were associated with adverse cv outcomes .", "the presence of elevated stress or depressive symptoms in community - dwelling subjects with diabetes was associated with an increased risk for acute chd or cv death .", "moreover , subjects with diabetes who reported both stress and depressive symptoms together had the greatest risk for acute chd and cv death .", "the presence of either or both behavioral comorbidities in subjects with diabetes was also associated with increased hrs for stroke .", "elevated stress and/or depressive symptoms in subjects without diabetes resulted in increased hrs in fully adjusted models that were much smaller in magnitude and not statistically significant .", "these findings demonstrate the persistent disparities and negative cv impact of these comorbidities at the population level and suggest the need for more careful integration of behavioral screening and management in primary care settings , where most patients with type 2 diabetes are managed ."]]}
{"article_id": "PMC4330666", "article_text": ["antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn .", "membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation .", "a 48-year - old man presented with marked proteinuria , hypoalbuminemia , and renal dysfunction with positive results for myeloperoxidase ( mpo ) and anca .", "renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on immunofluorescent staining .", "after one - month follow - up , antibody level and renal function did not improve .", "coexistence of mn with mpo - anca crescentic gn is very rare and should be managed aggressively .", "membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation .", "approximately 75% of mn represent as primary disease and the rest results from secondary causes , most commonly systemic lupus nephritis ( sle ) , infections such as hepatitis b or c viruses , malignancy , or drugs .", "antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn ( 1 - 3 ) .", "we report a rare case of mn with myeloperoxidase ( mpo)-anca - associated crescentic gn in a 48 year - old - man who was admitted to our institute .", "a 48-year - old man presented with intermittent puffiness of face and edema of the feet for two months .", "he did not have fever , hematuria , or breathlessness . on examination , he had bilateral pitting pedal edema ( + + ) , pulse rate of 98 per minute , and blood pressure of 140/96 mm hg .", "cardiovascular and respiratory examinations were unremarkable . on investigation the following laboratory results were reported : hemoglobin , 6.1 gm / dl ; white blood cell ( wbc ) count , 5.6  10/l ; platelet count , 2.11  10/l ; blood urea nitrogen , 35 mmol / l ; serum creatinine , 807 mol / l ; random blood sugar , 5.33 mmol / l ; total serum protein , 500 g / l ; serum albumin , 31", "g / l ; serum sodium , 132  mmol / l ; serum potassium , 4.54   mmol / l ; and serum cholesterol , 5.28 mmol / l .", "urine analysis showed 3 + albumin with 35 to 40/hpf of red blood cells and 8 to10/hpf of wbc .", "results of viral screening for human immunodeficiency virus , hepatitis b and hepatitis c viruses were negative .", "serum mpo - anca level was 220 u / ml ( normal range , 1 - 5 ) .", "serum anti - nuclear antibody ( ana ) , the levels of serum complements c3 and c4 were in normal limits .", "chest radiograph revealed normal findings and renal ultrasonography showed right kidney dimension of 8.6  3.4 cm and left kidney dimension of 9.0  4.5 cm , with increased echogenicity and maintained corticomedullary differentiation .", "renal biopsy was performed and after paraffin embedding , 3-m - thick sections were prepared and stained by hematoxylin and eosin ( h and e ) , periodic acid schiff , jone s silver methenamine , and gomori s trichrome stains .", "histopathologic examination ( figures 1 and 2 ) showed a single core of renal tissue containing 14 glomeruli with surrounding tubules and vessels .", "immunofluorescence ( if ) studies ( figure 3 ) showed fine granular fluorescence ( + 3/4 ) across 80% to 90% of glomerular capillary walls on staining with anti - human igg .", "no fluorescence was revealed on staining with anti - human iga , c3 , c1q , fibrinogen , and igm antisera .", "he was diagnosed as a case of mpo - anca - associated crescentic gn with mn .", "he was treated with intravenous methylprednisolone ( 500 mg / d ) for three days , followed by intravenous cyclophosphamide ( 500 mg ) and oral prednisolone ( 0.5 mg / kg / d ) with antihypertensive drugs .", "after two - month follow - up , his serum creatinine was 389 mol / l , urine albumin was 3 + with 5 to 7/hpf of rbcs .", "his renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on if staining .", "coexistence of anca - gn with mn is very rare ( 1 , 4 - 7 ) .", "surindran et al . detected coexistence of anti - phospholipase a2 receptor antibody ( pla2r)-positive mn and anca - gn ( 9 ) .", "ram et al . reported mn superimposed on churg - strauss syndrome ( 10 ) . the combination of anti gbm - gn and mn are also reported ( 11 , 12 ) .", "nasr et al . and nayak et al . reported that mn preceded the development of anti gbm in 50% of cases ( 1 , 13 ) .", "morizane et al . have reported a case of mpo - anca - associated gn , which was superimposed on type-3 mpgn .", "mn can be classified into primary and secondary forms according to igg subclass . in primary mn , igg4 is positive and in secondary mn , igg1 and igg4 .", "speculated that mpo - anca - associated gn might cause secondary mn as both igg1 and igg4 were present in their case ( 4 ) .", "the membranous lupus nephritis ( ln ) with focal or diffuse ln may have crescents with mn .", "such mixed patterns of ln have endocapillary proliferation and concomitant subendothelial immune deposits with full - house staining by if .", "nasr et al . reviewed kidney biopsies from january 2000 to february 2008 and concluded that coexistence of mn and anca - associated gn would be coincidental ( 1 ) . the primary mn is associated with hla - dq1 and pla2r , suggesting genetic predisposition ( 9 , 15 ) .", "anca - associated gn is also associated with environmental triggers such as drugs and infections ( 9 ) .", "when patient presents as rapidly progressive glomerulonephritis with marked proteinuria , further investigations to rule out possibility of mn with anca associated crescentic gn should be performed and the patient should be managed aggressively ."], "abstract_text": ["<S> introduction : antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn . </S>", "<S> membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation.case presentation : a 48-year - old man presented with marked proteinuria , hypoalbuminemia , and renal dysfunction with positive results for myeloperoxidase ( mpo ) and anca . </S>", "<S> renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on immunofluorescent staining . </S>", "<S> the condition was diagnosed as mn with mpo - anca - associated crescentic gn . </S>", "<S> he was treated with intravenous methylprednisolone and cyclophosphamide . </S>", "<S> after one - month follow - up , antibody level and renal function did not improve.conclusions:coexistence of mn with mpo - anca crescentic gn is very rare and should be managed aggressively . </S>"], "labels": null, "section_names": ["Introduction:", "Case Presentation:", "Conclusions:", "1. Introduction", "2. Case Presentation", "3. Discussion"], "sections": [["antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn .", "membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation ."], ["a 48-year - old man presented with marked proteinuria , hypoalbuminemia , and renal dysfunction with positive results for myeloperoxidase ( mpo ) and anca .", "renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on immunofluorescent staining .", "after one - month follow - up , antibody level and renal function did not improve ."], ["coexistence of mn with mpo - anca crescentic gn is very rare and should be managed aggressively ."], ["membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation .", "approximately 75% of mn represent as primary disease and the rest results from secondary causes , most commonly systemic lupus nephritis ( sle ) , infections such as hepatitis b or c viruses , malignancy , or drugs .", "antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn ( 1 - 3 ) .", "we report a rare case of mn with myeloperoxidase ( mpo)-anca - associated crescentic gn in a 48 year - old - man who was admitted to our institute ."], ["a 48-year - old man presented with intermittent puffiness of face and edema of the feet for two months .", "he did not have fever , hematuria , or breathlessness . on examination , he had bilateral pitting pedal edema ( + + ) , pulse rate of 98 per minute , and blood pressure of 140/96 mm hg .", "cardiovascular and respiratory examinations were unremarkable . on investigation the following laboratory results were reported : hemoglobin , 6.1 gm / dl ; white blood cell ( wbc ) count , 5.6  10/l ; platelet count , 2.11  10/l ; blood urea nitrogen , 35 mmol / l ; serum creatinine , 807 mol / l ; random blood sugar , 5.33 mmol / l ; total serum protein , 500 g / l ; serum albumin , 31", "g / l ; serum sodium , 132  mmol / l ; serum potassium , 4.54   mmol / l ; and serum cholesterol , 5.28 mmol / l .", "urine analysis showed 3 + albumin with 35 to 40/hpf of red blood cells and 8 to10/hpf of wbc .", "results of viral screening for human immunodeficiency virus , hepatitis b and hepatitis c viruses were negative .", "serum mpo - anca level was 220 u / ml ( normal range , 1 - 5 ) .", "serum anti - nuclear antibody ( ana ) , the levels of serum complements c3 and c4 were in normal limits .", "chest radiograph revealed normal findings and renal ultrasonography showed right kidney dimension of 8.6  3.4 cm and left kidney dimension of 9.0  4.5 cm , with increased echogenicity and maintained corticomedullary differentiation .", "renal biopsy was performed and after paraffin embedding , 3-m - thick sections were prepared and stained by hematoxylin and eosin ( h and e ) , periodic acid schiff , jone s silver methenamine , and gomori s trichrome stains .", "histopathologic examination ( figures 1 and 2 ) showed a single core of renal tissue containing 14 glomeruli with surrounding tubules and vessels .", "immunofluorescence ( if ) studies ( figure 3 ) showed fine granular fluorescence ( + 3/4 ) across 80% to 90% of glomerular capillary walls on staining with anti - human igg .", "no fluorescence was revealed on staining with anti - human iga , c3 , c1q , fibrinogen , and igm antisera .", "he was diagnosed as a case of mpo - anca - associated crescentic gn with mn .", "he was treated with intravenous methylprednisolone ( 500 mg / d ) for three days , followed by intravenous cyclophosphamide ( 500 mg ) and oral prednisolone ( 0.5 mg / kg / d ) with antihypertensive drugs .", "after two - month follow - up , his serum creatinine was 389 mol / l , urine albumin was 3 + with 5 to 7/hpf of rbcs ."], ["his renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on if staining .", "coexistence of anca - gn with mn is very rare ( 1 , 4 - 7 ) .", "surindran et al . detected coexistence of anti - phospholipase a2 receptor antibody ( pla2r)-positive mn and anca - gn ( 9 ) .", "ram et al . reported mn superimposed on churg - strauss syndrome ( 10 ) . the combination of anti gbm - gn and mn are also reported ( 11 , 12 ) .", "nasr et al . and nayak et al . reported that mn preceded the development of anti gbm in 50% of cases ( 1 , 13 ) .", "morizane et al . have reported a case of mpo - anca - associated gn , which was superimposed on type-3 mpgn .", "mn can be classified into primary and secondary forms according to igg subclass . in primary mn , igg4 is positive and in secondary mn , igg1 and igg4 .", "speculated that mpo - anca - associated gn might cause secondary mn as both igg1 and igg4 were present in their case ( 4 ) .", "the membranous lupus nephritis ( ln ) with focal or diffuse ln may have crescents with mn .", "such mixed patterns of ln have endocapillary proliferation and concomitant subendothelial immune deposits with full - house staining by if .", "nasr et al . reviewed kidney biopsies from january 2000 to february 2008 and concluded that coexistence of mn and anca - associated gn would be coincidental ( 1 ) . the primary mn is associated with hla - dq1 and pla2r , suggesting genetic predisposition ( 9 , 15 ) .", "anca - associated gn is also associated with environmental triggers such as drugs and infections ( 9 ) .", "when patient presents as rapidly progressive glomerulonephritis with marked proteinuria , further investigations to rule out possibility of mn with anca associated crescentic gn should be performed and the patient should be managed aggressively ."]]}
{"article_id": "PMC3259484", "article_text": ["although enhanced cardiovascular reactivity is generally associated with future development of hypertension and other cardiovascular events [ 15 ] , there are studies that have failed to show any relationship between reactivity to stress and future elevation of blood pressure [ 1 , 612 ] .", "emerging evidence suggest that some stress tests may be better predictors of future cardiovascular events than other stressors [ 4 , 5 , 10 ] .", "for example , blood pressure response to arithmetic and star tracing stress tests predicted high blood pressure while reactivity to cold pressor stress test did not [ 13 , 14 ] .", "furthermore , metanalysis of studies that assessed mental stress tests and hypertension development revealed variable success of mental tests in predicting hypertension . among the different types of mental stressors , cognitive mental stressors were more consistent in predicting hypertension compared to emotion evoking , interview , and public speaking stressors .", "the inconsistencies in prediction do not appear to be explained by differences in the type ( mental , physical , or psychophysical ) of stress tests for there is inconsistent predictability even among the types of stressors .", "another possible explanation for the inconsistencies in reactivity prediction of adverse cardiovascular outcomes is the interaction of psychosocial factors with cardiovascular responses to acute laboratory stressors .", "metanalysis of over 700 studies revealed that chronic ( trait ) anxiety is associated with decreased cardiovascular reactivity .", "in contrast , a study of young european population revealed that acute ( state ) anxiety was associated with significantly increased reactivity to cold pressor test but not mental stress test .", "these observations , when taken together , suggest that individuals may be inaccurately identified as hyperresponsive if anxiety is not considered as a confounder in the reactivity response to acute laboratory stress tests .", "consequently , inaccurate assessment of increased reactivity due to the interaction of anxiety with acute stressors may explain the inconsistent reports of increased risk of hypertension with increased reactivity . this study sought to investigate whether ( 1 ) anxiety determined the blood pressure response to stress tests and ( 2 ) anxiety differentially influenced blood pressure response to anger recall and cold pressor stress tests in african americans .", "we chose to study african americans for several reasons : ( 1 ) this group is characterized as hyperresponsive to stress [ 7 , 1720 ] , ( 2 ) several reports have failed to find increased reactivity in this population [ 4 , 8 , 2123 ] , and ( 3 ) psychosocial factors , including anxiety , are significantly associated with blood pressure in this population [ 2429 ] .", "we report that state ( in the moment ) anxiety was significantly associated with blood pressure response to both stressors ( anger recall and cold pressor stress tests ) in this population .", "these results support the idea that identification of hyperresponders to acute stress tests among african americans must take into account anxiety levels before determining whether an individual has increased reactivity to acute stress and/or that anxiety may play an important role along with reactivity response in hypertension development .", "however , our results do not support the idea that anxiety differentially impacts reactivity response to psychological , psychophysical , and physical stressors .", "a sample of 179 ( 116 males , 63 females ) participants of african descent were recruited to the study .", "all study procedures and materials were approved by and in compliance with the north carolina central university institutional review board .", "eligibility criteria for entry were ( 1 ) be 18 to 65 years old   ( 2 ) being a student or employee at north carolina central university or living in the surrounding regions of durham , orange and wake counties , ( 3 ) having no diagnosed cardiovascular disease ( self - reported ) , and ( 4 ) not taking any hypertensive medication .", "these regions of durham , orange , and wake counties make up the north carolina triangle region that is in the stroke belt ( e.g. , a geographic region with a higher occurrence of stroke ) . of these 179 ,", "only 50 are reported in the current report ; these were selected based on the type of mental stress used .", "the 50 participants reported here met the following criteria : ( 1 ) completed both the trait anxiety scale and the state anxiety scale , and ( 2 ) were between the ages of 18 and 40 years old .", "study participants were scheduled at either 9  am or 1  pm for the three - hour study protocol . after receiving informed consent , trained staff measured blood pressure by sphygmomanometer method with", "a ge dinamap pro 100 automatic model and a cuff size appropriate for the body size .", "each participant was allowed five minutes to sit quietly before taking the first resting parameters .", "the dinamap was set to assess systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) at one - minute intervals for the resting measurements as well as during the two acute stressor tasks .", "following resting blood pressure and heart rate measurements , participants were administered the cold pressor test , consisting of submersion of the hand in ice cold water for three minutes followed by a five - minute recovery period .", "a psychological stressor , anger recall , was given only after blood pressures and heart rate returned to baseline resting values .", "anger recall stress consisted of 5 minutes of contemplating an event that evoked anger , 5-minute discussion about the event , and 5-minute recovery period .", "cardiovascular reactivity was calculated as the difference between the average baseline prestressor blood pressure and the average change in blood pressure over the 5-minute stress period . the study protocol consisted of state anxiety assessment , resting bp measurement , second resting bp measurement , cold pressor stressor , third resting bp measurement , anger recall stressor , trait anxiety assessment , recording medical history , body mass index measurement , and completing a demographics questionnaire .", "state anxiety is defined as an acute response to a threatening or challenging situation , while trait anxiety is defined as a stable and enduring tendency to be anxious .", "each item is rated on a four - point scale ( 1 = almost never , 4 = almost always ) . items from each subscale are summed to create a total state anxiety score and a total trait anxiety score .", "higher scores on the state anxiety subscale indicate greater anxiety at the present time ; higher scores on the trait anxiety subscale indicate greater anxiety , in general .", "the state anxiety subscale has an alpha coefficient of  .87 , and the trait anxiety has an alpha coefficient of  .88 , indicating good ( since  .80 or greater ) internal consistency in this sample .", "scoring of the psychosocial scales spielberger state trait anxiety inventory utilized scoring protocols documented in prior research as indicated above and were confirmed with factor analysis .", "mean , standard deviation , standard error , median , and quartile calculations provide data reductions for sbp , dbp , and other clinical measures with multiple measurements .", "regression models , goodness of fit , multivariate parameter estimates , and confidence intervals were evaluated for each stressors impact on sbp , and dbp .", "two participants did not complete the cold pressor stressor ;   thus , the sample size is 48 for the cold pressor cardiovascular reactivity and 50 for the anger recall cardiovascular reactivity .", "the african american study samples are relatively young ( median age of twenty - one years ) with normal bmi ( median bmi was 26.2  kg / m ; normal bmi is 2530  kg / m ) and waist circumference normal values of less than 102 for males and 88  cm for females [ 3335 ] .", "this group also had normal cholesterol ( less than 200  mg / dl ) , triglycerides ( less than 150  mg / dl ) , glucose ( less than 126  mg / dl ) , and insulin ( < 10  miu ) levels .", "this group was normotensive with median systolic ( sbp ) and diastolic ( dbp ) blood pressures of 114 and 70  mmhg , respectively .", "a summary of the cardiovascular reactivity responses to ar and cp is shown in table 2 .", "cardiovascular reactivity was defined as the change in cardiovascular parameters ( sbp , dbp , mean arterial pressure ( map ) , and heart rate ( hr ) ) following the induction of a stress stimulus compared to baseline cardiovascular parameters .", "both the cp stressor and the ar stressor produced significant rises in sbp , dbp , and hr .", "all of the values returned to baseline during the recovery period except for sbp during the cp recovery period .", "repeated measures anova and the multiple comparison test , student - newman - keuls test , verified that the two stressor tasks ( cp and   ar ) produced statistically significant increases ( p < 0.0001 ) in the cardiovascular parameters in comparison to the resting measurements .", "we examined pearson 's correlations of state anxiety , trait anxiety , age , bmi , and resting cardiovascular measures with the cardiovascular reactivity parameters .", "trait anxiety had a statistically significant , positive correlation with state anxiety ( n = 50 ; pearson 's r = 0.47 ; p < 0.001 , two - tailed pearson 's correlation ) .", "state anxiety had statistically significant , positive correlations with cp reactivity response for sbp ( average change ; n = 48 ; pearson 's r = 0.37 ; p = 0.01 ) , dbp ( average change ; n = 48 ; pearson 's r = 0.40 ; p = 0.005 ) .", "similarly , state anxiety was highly correlated with the ar reactivity response for sbp ( n = 50 ; pearson 's r = 0.34 ; p = 0.015 ) and dbp ( n = 50 ; pearson 's r = 0.35 ; p = 0.013 ) .", "specifically , state anxiety was significantly associated with hr change to cp ( average change ; n = 48 ; pearson 's r = 0.37 ; p = 0.009 ) but was not related to hr changes to ar ( n = 50 ; pearson 's r = 0.14 ; p = 0.30 ) .", "trait anxiety had a nonsignificant correlation with sbp ( n = 48 ; pearson 's r = 0.23 ; p = 0.12 ) but a positive , significant correlation for dbp ( n = 48 ; pearson 's r = 0.34 ; p = 0.02 ) reactivity response to cp . as for ar ,", "trait anxiety was positively and significantly correlated with sbp ( n = 50 ; pearson 's r = 0.35 ; p = 0.012 ) but was not significantly correlated with dbp ( n = 50 ; pearson 's r = 0.23 ; p = 0.11 ) .", "age , bmi , and resting cardiovascular measures had nonsignificant correlations with both cp reactivity and ar reactivity as measured by sbp , dbp , and hr changes .", "resting sbp was only associated with map changes with cp and ar stress tests ( pearson 's r = 0.33 ; p < 0.02 and r = 0.38 ; p < 0.01 , resp . ) .  ", "the following variables were evaluated in the stepwise procedure : state anxiety , trait anxiety , resting cardiovascular measures , body mass index , and age .", "the variable selection results of the stepwise algorithm suggested the use of state anxiety for a parsimonious model of both cold pressor cardiovascular reactivity as well as anger recall cardiovascular reactivity .", "our next step was to evaluate regression models to predict cardiovascular reactivity with state anxiety as the independent variable .", "the model for predicting the cp increase in sbp ( see table 3 ) had an r of 0.14 and state anxiety as a significant parameter ( p = 0.009 ) .", "state anxiety was also a significant independent variable ( p = 0.005 ) in the model of the cp change in dbp ( r = 0.16 ) .", "similar results were found with the model for predicting the change in cardiovascular reactivity for the ar stressor as shown in table 4 .", "state anxiety was a significant parameter for the change in sbp ( p = 0.015 , r = 0.12 ) and dbp ( p = 0.013 , r = 0.12 ) .", "each model was scrutinized to verify adherence to the assumptions of regression modeling ( linear relationship between independent variables and dependent variables , homoscedasticity of the errors , and errors are independent and normally distributed ) .", "shapiro - wilke statistics did not reject the null hypothesis of normal distribution of the residuals .", "this study investigated whether anxiety differentially affects cardiovascular reactivity to cold pressor and anger recall stress tests in a sample of young , healthy , community dwelling african american adults , a population prone to develop cardiovascular disease .", "importantly , we show that the state ( at the moment ) anxiety was significantly associated sbp and dbp responses to both cold pressor and anger recall laboratory stress tests in this population .", "in contrast to blood pressure , state anxiety differentially predicted hr response to cp but not ar . on the other hand ,", "chronic ( trait ) anxiety was not a significant predictor of reactivity in our statistical models .", "we interpret these results to mean that the state of anxiety at the time of the stressor must be considered when assessing cardiovascular reactivity to laboratory stress tests .", "failure to consider state anxiety as a confounder of reactivity responses may lead to misidentifying some individuals as hyperresponders when compared to others .", "misidentification of individuals may contribute in part to the inconsistent findings of increased reactivity in african americans and to the inconsistent prediction of hypertension in those with increased vascular reactivity .", "alternatively , these results can be interpreted to mean that the interaction of state anxiety and cardiovascular reactivity may be important determinants of hypertension development in african americans .", "anxiety , chronic anxiety in particular , has been linked to the development of disease .", "contrary to what would be expected , chronic anxiety has been shown to be negatively associated with cardiovascular reactivity .", "although chronic anxiety and cardiovascular reactivity associations have been studied , few studies have investigated the role of state anxiety in determining blood pressure response to stress .", "white coat  response and is effective in predicting ambulatory evening systolic blood pressure in young black males .", "studies that have investigated the effect of state anxiety on reactivity did not include african americans [ 16 , 37 ] .", "our study provides evidence that young healthy african americans who are anxious prior to the stress tests are likely to have higher blood pressure responses to the stress .", "thus , variability in hyperresponsiveness response to laboratory stress tests in african americans may be due in part to a failure to consider state anxiety as a confounder .", "although many studies have shown that enhanced cardiovascular reactivity predicts hypertension development and other cardiovascular events [ 15 ] , there are several studies that failed to show any relationship between reactivity to stress and hypertension development [ 1 , 612 ] .", "the inconsistency may be a consequence of the type of stressors used [ 4 , 5 , 38 , 39 ] and the interaction of psychosocial factors with blood pressure response to the stressor [ 4042 ] .", "psychological stress tests may be better than cold pressor stress tests at predicting future cardiovascular events [ 4 , 5 , 22 , 43 , 44 ] .", "this differential effect of stressor type and hypertension development may be a consequence of differential psychosocial factor interaction with stressors . in a study of a european population ,", "psychosocial factors appear to have a greater impact on reactivity to cold pressor than reactivity to mental stress .", "our study compares the impact of anxiety on reactivity to cold pressor and anger recall in african americans .", "another explanation for the inconsistent findings of increased risk of hypertension development with increased reactivity is the interaction of psychosocial factors with cardiovascular reactivity to promote hypertension development .", "for example , studies show that hostility , depression , and anger contribute to increased reactivity [ 40 , 45 , 46 ] .", "psychosocial factors also are associated with increased incidence of cardiovascular events [ 4750 ] as well as the development of cardiovascular disease [ 51 , 52 ] .", "we show that the psychosocial factor , anxiety , can influence the reactivity response to acute stress .", "however , because the study design was cross - sectional , it could not be determined whether state anxiety interaction with cardiovascular response to acute stress predicts hypertension development .", "this study shows that the current state of anxiety was significantly associated with blood pressure response to laboratory stress tests .", "consequently , anxiety levels should be assessed when using acute laboratory stress tests for identification of those with increased cardiovascular reactivity .", "further studies are needed to determine whether reactivity normalized to anxiety increases the accuracy in identifying hyperresponders and , subsequently , predicting future hypertension development .", "these findings need to be validated in a larger cross sectional population of african americans .", "additionally , our study design did not allow us to determine whether the impact of anxiety on blood pressure response to acute stressor is unique to anxiety or whether other psychosocial factors similarly influence blood pressure response to acute stress in our cohort .", "another limitation of the study is that adrenergic system activation was not measured ; consequently , it could not be determined if the two stressors differentially activated the beta or alpha - adrenergic receptor pathways .", "this information would be helpful in future longitudinal studies that will address how activation of the alpha and beta adrenergic receptors pathways ultimately leads to hypertension development and the attending cardiovascular disease . a longitudinal study design will also help to address the question of whether inclusion of anxiety enhances the ability of increased reactivity to predict future elevations of blood pressure ."], "abstract_text": ["<S> although several studies have shown that enhanced cardiovascular reactivity can predict hypertension development in african americans , these findings have not been consistent among all studies examining reactivity and hypertension susceptibility . </S>", "<S> this inconsistency may be explained by the influence of anxiety ( state and trait ) on the blood pressure response to stress . </S>", "<S> therefore , this study sought to determine whether anxiety is associated with blood pressure response to cold pressor ( cp ) and anger recall ( ar ) stress tests in young healthy african americans . </S>", "<S> modeling using state and trait anxiety revealed that state anxiety predicts systolic ( sbp ) and diastolic blood pressure dbp response to cp and ar ( p  0.02 ) . </S>", "<S> interestingly , state anxiety predicted heart rate changes only to cp ( p < 0.01 ; p = 0.3 for ar ) . </S>", "<S> although   trait anxiety was associated with sbp response to ar and not cp , it was not a significant predictor of reactivity in our models . we conclude that anxiety levels may contribute to the variable blood pressure response to acute stressors and , therefore , should be assessed when performing cardiovascular reactivity measures . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Statistics", "4. Results", "5. Discussion", "6. Limitations"], "sections": [["although enhanced cardiovascular reactivity is generally associated with future development of hypertension and other cardiovascular events [ 15 ] , there are studies that have failed to show any relationship between reactivity to stress and future elevation of blood pressure [ 1 , 612 ] .", "emerging evidence suggest that some stress tests may be better predictors of future cardiovascular events than other stressors [ 4 , 5 , 10 ] .", "for example , blood pressure response to arithmetic and star tracing stress tests predicted high blood pressure while reactivity to cold pressor stress test did not [ 13 , 14 ] .", "furthermore , metanalysis of studies that assessed mental stress tests and hypertension development revealed variable success of mental tests in predicting hypertension . among the different types of mental stressors , cognitive mental stressors were more consistent in predicting hypertension compared to emotion evoking , interview , and public speaking stressors .", "the inconsistencies in prediction do not appear to be explained by differences in the type ( mental , physical , or psychophysical ) of stress tests for there is inconsistent predictability even among the types of stressors .", "another possible explanation for the inconsistencies in reactivity prediction of adverse cardiovascular outcomes is the interaction of psychosocial factors with cardiovascular responses to acute laboratory stressors .", "metanalysis of over 700 studies revealed that chronic ( trait ) anxiety is associated with decreased cardiovascular reactivity .", "in contrast , a study of young european population revealed that acute ( state ) anxiety was associated with significantly increased reactivity to cold pressor test but not mental stress test .", "these observations , when taken together , suggest that individuals may be inaccurately identified as hyperresponsive if anxiety is not considered as a confounder in the reactivity response to acute laboratory stress tests .", "consequently , inaccurate assessment of increased reactivity due to the interaction of anxiety with acute stressors may explain the inconsistent reports of increased risk of hypertension with increased reactivity . this study sought to investigate whether ( 1 ) anxiety determined the blood pressure response to stress tests and ( 2 ) anxiety differentially influenced blood pressure response to anger recall and cold pressor stress tests in african americans .", "we chose to study african americans for several reasons : ( 1 ) this group is characterized as hyperresponsive to stress [ 7 , 1720 ] , ( 2 ) several reports have failed to find increased reactivity in this population [ 4 , 8 , 2123 ] , and ( 3 ) psychosocial factors , including anxiety , are significantly associated with blood pressure in this population [ 2429 ] .", "we report that state ( in the moment ) anxiety was significantly associated with blood pressure response to both stressors ( anger recall and cold pressor stress tests ) in this population .", "these results support the idea that identification of hyperresponders to acute stress tests among african americans must take into account anxiety levels before determining whether an individual has increased reactivity to acute stress and/or that anxiety may play an important role along with reactivity response in hypertension development .", "however , our results do not support the idea that anxiety differentially impacts reactivity response to psychological , psychophysical , and physical stressors ."], ["a sample of 179 ( 116 males , 63 females ) participants of african descent were recruited to the study .", "all study procedures and materials were approved by and in compliance with the north carolina central university institutional review board .", "eligibility criteria for entry were ( 1 ) be 18 to 65 years old   ( 2 ) being a student or employee at north carolina central university or living in the surrounding regions of durham , orange and wake counties , ( 3 ) having no diagnosed cardiovascular disease ( self - reported ) , and ( 4 ) not taking any hypertensive medication .", "these regions of durham , orange , and wake counties make up the north carolina triangle region that is in the stroke belt ( e.g. , a geographic region with a higher occurrence of stroke ) . of these 179 ,", "only 50 are reported in the current report ; these were selected based on the type of mental stress used .", "the 50 participants reported here met the following criteria : ( 1 ) completed both the trait anxiety scale and the state anxiety scale , and ( 2 ) were between the ages of 18 and 40 years old .", "study participants were scheduled at either 9  am or 1  pm for the three - hour study protocol . after receiving informed consent , trained staff measured blood pressure by sphygmomanometer method with", "a ge dinamap pro 100 automatic model and a cuff size appropriate for the body size .", "each participant was allowed five minutes to sit quietly before taking the first resting parameters .", "the dinamap was set to assess systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) at one - minute intervals for the resting measurements as well as during the two acute stressor tasks .", "following resting blood pressure and heart rate measurements , participants were administered the cold pressor test , consisting of submersion of the hand in ice cold water for three minutes followed by a five - minute recovery period .", "a psychological stressor , anger recall , was given only after blood pressures and heart rate returned to baseline resting values .", "anger recall stress consisted of 5 minutes of contemplating an event that evoked anger , 5-minute discussion about the event , and 5-minute recovery period .", "cardiovascular reactivity was calculated as the difference between the average baseline prestressor blood pressure and the average change in blood pressure over the 5-minute stress period . the study protocol consisted of state anxiety assessment , resting bp measurement , second resting bp measurement , cold pressor stressor , third resting bp measurement , anger recall stressor , trait anxiety assessment , recording medical history , body mass index measurement , and completing a demographics questionnaire .", "state anxiety is defined as an acute response to a threatening or challenging situation , while trait anxiety is defined as a stable and enduring tendency to be anxious .", "each item is rated on a four - point scale ( 1 = almost never , 4 = almost always ) . items from each subscale are summed to create a total state anxiety score and a total trait anxiety score .", "higher scores on the state anxiety subscale indicate greater anxiety at the present time ; higher scores on the trait anxiety subscale indicate greater anxiety , in general .", "the state anxiety subscale has an alpha coefficient of  .87 , and the trait anxiety has an alpha coefficient of  .88 , indicating good ( since  .80 or greater ) internal consistency in this sample ."], ["scoring of the psychosocial scales spielberger state trait anxiety inventory utilized scoring protocols documented in prior research as indicated above and were confirmed with factor analysis .", "mean , standard deviation , standard error , median , and quartile calculations provide data reductions for sbp , dbp , and other clinical measures with multiple measurements .", "regression models , goodness of fit , multivariate parameter estimates , and confidence intervals were evaluated for each stressors impact on sbp , and dbp .", "two participants did not complete the cold pressor stressor ;   thus , the sample size is 48 for the cold pressor cardiovascular reactivity and 50 for the anger recall cardiovascular reactivity ."], ["the african american study samples are relatively young ( median age of twenty - one years ) with normal bmi ( median bmi was 26.2  kg / m ; normal bmi is 2530  kg / m ) and waist circumference normal values of less than 102 for males and 88  cm for females [ 3335 ] .", "this group also had normal cholesterol ( less than 200  mg / dl ) , triglycerides ( less than 150  mg / dl ) , glucose ( less than 126  mg / dl ) , and insulin ( < 10  miu ) levels .", "this group was normotensive with median systolic ( sbp ) and diastolic ( dbp ) blood pressures of 114 and 70  mmhg , respectively .", "a summary of the cardiovascular reactivity responses to ar and cp is shown in table 2 .", "cardiovascular reactivity was defined as the change in cardiovascular parameters ( sbp , dbp , mean arterial pressure ( map ) , and heart rate ( hr ) ) following the induction of a stress stimulus compared to baseline cardiovascular parameters .", "both the cp stressor and the ar stressor produced significant rises in sbp , dbp , and hr .", "all of the values returned to baseline during the recovery period except for sbp during the cp recovery period .", "repeated measures anova and the multiple comparison test , student - newman - keuls test , verified that the two stressor tasks ( cp and   ar ) produced statistically significant increases ( p < 0.0001 ) in the cardiovascular parameters in comparison to the resting measurements .", "we examined pearson 's correlations of state anxiety , trait anxiety , age , bmi , and resting cardiovascular measures with the cardiovascular reactivity parameters .", "trait anxiety had a statistically significant , positive correlation with state anxiety ( n = 50 ; pearson 's r = 0.47 ; p < 0.001 , two - tailed pearson 's correlation ) .", "state anxiety had statistically significant , positive correlations with cp reactivity response for sbp ( average change ; n = 48 ; pearson 's r = 0.37 ; p = 0.01 ) , dbp ( average change ; n = 48 ; pearson 's r = 0.40 ; p = 0.005 ) .", "similarly , state anxiety was highly correlated with the ar reactivity response for sbp ( n = 50 ; pearson 's r = 0.34 ; p = 0.015 ) and dbp ( n = 50 ; pearson 's r = 0.35 ; p = 0.013 ) .", "specifically , state anxiety was significantly associated with hr change to cp ( average change ; n = 48 ; pearson 's r = 0.37 ; p = 0.009 ) but was not related to hr changes to ar ( n = 50 ; pearson 's r = 0.14 ; p = 0.30 ) .", "trait anxiety had a nonsignificant correlation with sbp ( n = 48 ; pearson 's r = 0.23 ; p = 0.12 ) but a positive , significant correlation for dbp ( n = 48 ; pearson 's r = 0.34 ; p = 0.02 ) reactivity response to cp . as for ar ,", "trait anxiety was positively and significantly correlated with sbp ( n = 50 ; pearson 's r = 0.35 ; p = 0.012 ) but was not significantly correlated with dbp ( n = 50 ; pearson 's r = 0.23 ; p = 0.11 ) .", "age , bmi , and resting cardiovascular measures had nonsignificant correlations with both cp reactivity and ar reactivity as measured by sbp , dbp , and hr changes .", "resting sbp was only associated with map changes with cp and ar stress tests ( pearson 's r = 0.33 ; p < 0.02 and r = 0.38 ; p < 0.01 , resp . ) .  ", "the following variables were evaluated in the stepwise procedure : state anxiety , trait anxiety , resting cardiovascular measures , body mass index , and age .", "the variable selection results of the stepwise algorithm suggested the use of state anxiety for a parsimonious model of both cold pressor cardiovascular reactivity as well as anger recall cardiovascular reactivity .", "our next step was to evaluate regression models to predict cardiovascular reactivity with state anxiety as the independent variable .", "the model for predicting the cp increase in sbp ( see table 3 ) had an r of 0.14 and state anxiety as a significant parameter ( p = 0.009 ) .", "state anxiety was also a significant independent variable ( p = 0.005 ) in the model of the cp change in dbp ( r = 0.16 ) .", "similar results were found with the model for predicting the change in cardiovascular reactivity for the ar stressor as shown in table 4 .", "state anxiety was a significant parameter for the change in sbp ( p = 0.015 , r = 0.12 ) and dbp ( p = 0.013 , r = 0.12 ) .", "each model was scrutinized to verify adherence to the assumptions of regression modeling ( linear relationship between independent variables and dependent variables , homoscedasticity of the errors , and errors are independent and normally distributed ) .", "shapiro - wilke statistics did not reject the null hypothesis of normal distribution of the residuals ."], ["this study investigated whether anxiety differentially affects cardiovascular reactivity to cold pressor and anger recall stress tests in a sample of young , healthy , community dwelling african american adults , a population prone to develop cardiovascular disease .", "importantly , we show that the state ( at the moment ) anxiety was significantly associated sbp and dbp responses to both cold pressor and anger recall laboratory stress tests in this population .", "in contrast to blood pressure , state anxiety differentially predicted hr response to cp but not ar . on the other hand ,", "chronic ( trait ) anxiety was not a significant predictor of reactivity in our statistical models .", "we interpret these results to mean that the state of anxiety at the time of the stressor must be considered when assessing cardiovascular reactivity to laboratory stress tests .", "failure to consider state anxiety as a confounder of reactivity responses may lead to misidentifying some individuals as hyperresponders when compared to others .", "misidentification of individuals may contribute in part to the inconsistent findings of increased reactivity in african americans and to the inconsistent prediction of hypertension in those with increased vascular reactivity .", "alternatively , these results can be interpreted to mean that the interaction of state anxiety and cardiovascular reactivity may be important determinants of hypertension development in african americans .", "anxiety , chronic anxiety in particular , has been linked to the development of disease .", "contrary to what would be expected , chronic anxiety has been shown to be negatively associated with cardiovascular reactivity .", "although chronic anxiety and cardiovascular reactivity associations have been studied , few studies have investigated the role of state anxiety in determining blood pressure response to stress .", "white coat  response and is effective in predicting ambulatory evening systolic blood pressure in young black males .", "studies that have investigated the effect of state anxiety on reactivity did not include african americans [ 16 , 37 ] .", "our study provides evidence that young healthy african americans who are anxious prior to the stress tests are likely to have higher blood pressure responses to the stress .", "thus , variability in hyperresponsiveness response to laboratory stress tests in african americans may be due in part to a failure to consider state anxiety as a confounder .", "although many studies have shown that enhanced cardiovascular reactivity predicts hypertension development and other cardiovascular events [ 15 ] , there are several studies that failed to show any relationship between reactivity to stress and hypertension development [ 1 , 612 ] .", "the inconsistency may be a consequence of the type of stressors used [ 4 , 5 , 38 , 39 ] and the interaction of psychosocial factors with blood pressure response to the stressor [ 4042 ] .", "psychological stress tests may be better than cold pressor stress tests at predicting future cardiovascular events [ 4 , 5 , 22 , 43 , 44 ] .", "this differential effect of stressor type and hypertension development may be a consequence of differential psychosocial factor interaction with stressors . in a study of a european population ,", "psychosocial factors appear to have a greater impact on reactivity to cold pressor than reactivity to mental stress .", "our study compares the impact of anxiety on reactivity to cold pressor and anger recall in african americans .", "another explanation for the inconsistent findings of increased risk of hypertension development with increased reactivity is the interaction of psychosocial factors with cardiovascular reactivity to promote hypertension development .", "for example , studies show that hostility , depression , and anger contribute to increased reactivity [ 40 , 45 , 46 ] .", "psychosocial factors also are associated with increased incidence of cardiovascular events [ 4750 ] as well as the development of cardiovascular disease [ 51 , 52 ] .", "we show that the psychosocial factor , anxiety , can influence the reactivity response to acute stress .", "however , because the study design was cross - sectional , it could not be determined whether state anxiety interaction with cardiovascular response to acute stress predicts hypertension development .", "this study shows that the current state of anxiety was significantly associated with blood pressure response to laboratory stress tests .", "consequently , anxiety levels should be assessed when using acute laboratory stress tests for identification of those with increased cardiovascular reactivity .", "further studies are needed to determine whether reactivity normalized to anxiety increases the accuracy in identifying hyperresponders and , subsequently , predicting future hypertension development ."], ["these findings need to be validated in a larger cross sectional population of african americans .", "additionally , our study design did not allow us to determine whether the impact of anxiety on blood pressure response to acute stressor is unique to anxiety or whether other psychosocial factors similarly influence blood pressure response to acute stress in our cohort .", "another limitation of the study is that adrenergic system activation was not measured ; consequently , it could not be determined if the two stressors differentially activated the beta or alpha - adrenergic receptor pathways .", "this information would be helpful in future longitudinal studies that will address how activation of the alpha and beta adrenergic receptors pathways ultimately leads to hypertension development and the attending cardiovascular disease . a longitudinal study design will also help to address the question of whether inclusion of anxiety enhances the ability of increased reactivity to predict future elevations of blood pressure ."]]}
{"article_id": "PMC4681224", "article_text": ["sweet 's syndrome ( ss ) presenting in a localized and symmetric pattern is a very rare phenomenon . there have , however , been , a few case reports for the same .", "ss per se was first reported in 1964 by robert douglas sweet . though classically presenting as tender pseudovesicles", ", ss may demonstrate numerous topographic variations , which could be quite confusing for the diagnosing dermatologist .", "a 45-year - old farmer from nepal presented to the department of dermatology with complaints of an abrupt onset of an asymptomatic cutaneous eruption involving the neck and both the forearms since the past 5 days .", "the lesions were smaller earlier which had progressively increased in size over the past 5 days to reach the current status .", "there were no associated constitutional symptoms and no history of contact with cattle or sheep .", ", papules and plaques of varying sizes with a striking central umbilication were identified involving the forearms and neck that gave an illusion of vesiculation [ figures 13 ] .", "some of the lesions over the neck had a crust overlying the depressed center [ figures 4 and 5 ] .", "gram staining of the tissue obtained from one of the papules after needle extirpation was inconclusive .", "a skin biopsy from these lesions showed absence of epidermal changes with a dense inflammatory neutrophilic dermal infiltrate along with dermal edema as the hallmark finding , without evidence of vasculitis [ figures 68 ] .", "laboratory parameters revealed a hemoglobin level of 14 g / dl , a total white blood cell count of 13,000 cells / mm with 89% neutrophils , and a normal peripheral smear .", "his renal and hepatic profiles were normal , and imaging studies of the abdomen and pelvis were insignificant .", "the patient was diagnosed as ss and started on 20 mg of prednisolone once daily for 2 weeks and 100 mg of dapsone once at bedtime for 2 weeks .", "fleshy pseudovesicles with central umbilication seen on the right forearm of our patient over the left forearm , a plaque with the characteristic central umbilication seen , closely mimicking the lesions encountered in molluscum contagiosum . furthermore , to note here is the symmetric pattern of lesion arrangement molluscoid pseudovesicles over the nape of the neck juicy plaques with a central depression and an overlying crust seen on the right side of the neck similar lesions on the left side of the neck as seen in figure 4 . to note here again ,", "is the striking symmetric distribution of the nuchal lesions section of the plaque on the right forearm demonstrating dermal edema and a dense dermal inflammatory infiltrate ( h and e , 10 ) same section showing a dense inflammatory infiltrate composed of neutrophils around the blood vessels and also diffusely distributed ( h and e , 20 ) absence of vasculitis on microscopy ( h and e , 40 )", "sweet 's syndrome is the prototypical neutrophilic dermatosis , generally characterized by fever , an increase in the neutrophilic count along with tender pseudovesicles distributed over the body in an asymmetric manner .", "etiologically , three types of ss have been described , namely the idiopathic , paraneoplastic , and the drug - induced type of ss . as mentioned earlier ,", "there have , however , been reports of the occurrence of ss localized to specific sites of the body though , some of which have presented with very unusual morphologic patterns , as shown by sommer et al . who described palmoplantar pustulosis as the sole finding in a ss patient , verma et al . demonstrating the occurrence of ss confined to the photo exposed sites alone , verma who elucidated a rare recurrent bullous eruption localized to the flexural aspect of both the forearms in a ss patient and brechtel et al . who reported a localized facial presentation of ss manifesting as an inflammatory lesion with a central depression superimposed with several pustules on the surface of the plaque .", "a variant of ss referred to as neutrophilic dermatosis of the dorsal hands ( nddh ) is a specific entity confined only to the dorsa of the hands and has been extensively reviewed in literature by galaria et al . , takahama and kanbe , cook et al .", "nddh usually presents as edematous and violaceous plaques and papulonodules on the radial aspect of the dorsal surface of both hands .", "our patient presented with molluscoid pseudovesicles distributed over the neck and forearms only , which is a very rare morphologic pattern witnessed in ss and to the best of our knowledge has not been previously described .", "second , our patient lacked the usual constitutional symptoms that herald ss , which made the diagnosis a challenge .", "third , as no systemic association with ss could be identified in our patient , he was further sub - classified as a case of idiopathic ss , which again is a rarity in male patients , who usually present with the paraneoplastic or the drug - induced variant of ss .", "the hallmark finding in our patient was the central depression in all the pseudovesicles examined .", "the other possibilities , we kept in mind after clinically examining our patient , were , atypical erythema multiforme , histioid leprosy , milker 's nodule , cowpox , and ecthyma contagiosum , but these were eventually ruled out .", "hence , to conclude , we see that ss is a dermatosis that could be a great mimicker just like leprosy and syphilis and therefore when faced with diagnostic dilemmas in these kinds of atypical presentations , it would be worthwhile considering ss as a differential diagnosis . \n", "idiopathic sweet 's syndrome ( ss ) may even occur in males though highly uncommonthe absence of constitutional symptoms does not necessarily rule out the diagnosis of ssthe plaques encountered in ss could even be asymptomatic.molluscoid lesions seen in ss is a very rare morphologic pattern encountered and to the best of our knowledge is being reported for the 1 time in literaturelocalized patterns of ss are not so rare as previously thought . \n ", "idiopathic sweet 's syndrome ( ss ) may even occur in males though highly uncommon the absence of constitutional symptoms does not necessarily rule out the diagnosis of ss the plaques encountered in ss could even be asymptomatic .", "molluscoid lesions seen in ss is a very rare morphologic pattern encountered and to the best of our knowledge is being reported for the 1 time in literature localized patterns of ss are not so rare as previously thought ."], "abstract_text": ["<S> sweet 's syndrome is a well - known entity in the field of dermatology . </S>", "<S> it has presented itself in myriad forms , well - recorded in literature . </S>", "<S> our patient presented with a sudden eruption of nontender molluscoid pseudovesicles distributed over the neck and forearms alone . a complete diagnostic work up for the same did not give any clue regarding an underlying systemic ailment , responsible for the dermatosis . </S>", "<S> this case is being presented because of this extremely rare morphology of gomm - button disease . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["sweet 's syndrome ( ss ) presenting in a localized and symmetric pattern is a very rare phenomenon . there have , however , been , a few case reports for the same .", "ss per se was first reported in 1964 by robert douglas sweet . though classically presenting as tender pseudovesicles", ", ss may demonstrate numerous topographic variations , which could be quite confusing for the diagnosing dermatologist ."], ["a 45-year - old farmer from nepal presented to the department of dermatology with complaints of an abrupt onset of an asymptomatic cutaneous eruption involving the neck and both the forearms since the past 5 days .", "the lesions were smaller earlier which had progressively increased in size over the past 5 days to reach the current status .", "there were no associated constitutional symptoms and no history of contact with cattle or sheep .", ", papules and plaques of varying sizes with a striking central umbilication were identified involving the forearms and neck that gave an illusion of vesiculation [ figures 13 ] .", "some of the lesions over the neck had a crust overlying the depressed center [ figures 4 and 5 ] .", "gram staining of the tissue obtained from one of the papules after needle extirpation was inconclusive .", "a skin biopsy from these lesions showed absence of epidermal changes with a dense inflammatory neutrophilic dermal infiltrate along with dermal edema as the hallmark finding , without evidence of vasculitis [ figures 68 ] .", "laboratory parameters revealed a hemoglobin level of 14 g / dl , a total white blood cell count of 13,000 cells / mm with 89% neutrophils , and a normal peripheral smear .", "his renal and hepatic profiles were normal , and imaging studies of the abdomen and pelvis were insignificant .", "the patient was diagnosed as ss and started on 20 mg of prednisolone once daily for 2 weeks and 100 mg of dapsone once at bedtime for 2 weeks .", "fleshy pseudovesicles with central umbilication seen on the right forearm of our patient over the left forearm , a plaque with the characteristic central umbilication seen , closely mimicking the lesions encountered in molluscum contagiosum . furthermore , to note here is the symmetric pattern of lesion arrangement molluscoid pseudovesicles over the nape of the neck juicy plaques with a central depression and an overlying crust seen on the right side of the neck similar lesions on the left side of the neck as seen in figure 4 . to note here again ,", "is the striking symmetric distribution of the nuchal lesions section of the plaque on the right forearm demonstrating dermal edema and a dense dermal inflammatory infiltrate ( h and e , 10 ) same section showing a dense inflammatory infiltrate composed of neutrophils around the blood vessels and also diffusely distributed ( h and e , 20 ) absence of vasculitis on microscopy ( h and e , 40 )"], ["sweet 's syndrome is the prototypical neutrophilic dermatosis , generally characterized by fever , an increase in the neutrophilic count along with tender pseudovesicles distributed over the body in an asymmetric manner .", "etiologically , three types of ss have been described , namely the idiopathic , paraneoplastic , and the drug - induced type of ss . as mentioned earlier ,", "there have , however , been reports of the occurrence of ss localized to specific sites of the body though , some of which have presented with very unusual morphologic patterns , as shown by sommer et al . who described palmoplantar pustulosis as the sole finding in a ss patient , verma et al . demonstrating the occurrence of ss confined to the photo exposed sites alone , verma who elucidated a rare recurrent bullous eruption localized to the flexural aspect of both the forearms in a ss patient and brechtel et al . who reported a localized facial presentation of ss manifesting as an inflammatory lesion with a central depression superimposed with several pustules on the surface of the plaque .", "a variant of ss referred to as neutrophilic dermatosis of the dorsal hands ( nddh ) is a specific entity confined only to the dorsa of the hands and has been extensively reviewed in literature by galaria et al . , takahama and kanbe , cook et al .", "nddh usually presents as edematous and violaceous plaques and papulonodules on the radial aspect of the dorsal surface of both hands .", "our patient presented with molluscoid pseudovesicles distributed over the neck and forearms only , which is a very rare morphologic pattern witnessed in ss and to the best of our knowledge has not been previously described .", "second , our patient lacked the usual constitutional symptoms that herald ss , which made the diagnosis a challenge .", "third , as no systemic association with ss could be identified in our patient , he was further sub - classified as a case of idiopathic ss , which again is a rarity in male patients , who usually present with the paraneoplastic or the drug - induced variant of ss .", "the hallmark finding in our patient was the central depression in all the pseudovesicles examined .", "the other possibilities , we kept in mind after clinically examining our patient , were , atypical erythema multiforme , histioid leprosy , milker 's nodule , cowpox , and ecthyma contagiosum , but these were eventually ruled out .", "hence , to conclude , we see that ss is a dermatosis that could be a great mimicker just like leprosy and syphilis and therefore when faced with diagnostic dilemmas in these kinds of atypical presentations , it would be worthwhile considering ss as a differential diagnosis . \n", "idiopathic sweet 's syndrome ( ss ) may even occur in males though highly uncommonthe absence of constitutional symptoms does not necessarily rule out the diagnosis of ssthe plaques encountered in ss could even be asymptomatic.molluscoid lesions seen in ss is a very rare morphologic pattern encountered and to the best of our knowledge is being reported for the 1 time in literaturelocalized patterns of ss are not so rare as previously thought . \n ", "idiopathic sweet 's syndrome ( ss ) may even occur in males though highly uncommon the absence of constitutional symptoms does not necessarily rule out the diagnosis of ss the plaques encountered in ss could even be asymptomatic .", "molluscoid lesions seen in ss is a very rare morphologic pattern encountered and to the best of our knowledge is being reported for the 1 time in literature localized patterns of ss are not so rare as previously thought ."]]}
{"article_id": "PMC4745498", "article_text": ["in order to prevent and control microbial proliferation in industrial settings , cleaning and disinfection plans are applied on a regular basis [ 1 , 2 ] . in food processing plants ,", "the control of microbial contamination generally involves clean - in - place ( cip ) procedures which consist of running alternated cycles of detergent and disinfectant solutions with water rinses in high turbulence regimes through the plant and pipeline circuits without dismantling or opening the equipment [ 25 ] .  ", "biocides are currently used in industrial processes as the most significant countermeasure to control microbial growth and proliferation .", "industry moved progressively towards the use of surfactants that are less toxic and more biodegradable .", "surfactants are classified according to the ionic physiognomies of their hydrophilic group as anionic , cationic , nonionic , and zwitterionic [ 6 , 8 ] .", "quaternary ammonium compounds ( qacs ) are cationic surfactants that are commonly used because of their hard - surface cleaning , odor removal and antimicrobial properties . besides killing bacteria , the chemical nature of qacs can cause modifications on the properties of abiotic surfaces , decreasing their tension and therefore preventing attachment of microorganisms .", "the antimicrobial mode of action of cationic surfactants is proposed by some authors as a sequence of events : attraction by the negatively charged cell surface ; adsorption to the cell wall through the hydrophobic headgroup ; reaction with the lipids and proteins that compose the cytoplasmic membrane ; and cell penetration and interaction with intracellular constituents [ 10 , 11 ] .", "thus , qacs damage the outer layers of bacteria , thereby promoting the release of intracellular constituents .", "antimicrobial efficacy tests require planning of an adequate strategy and should include all the parameters found in real settings .", "aspects such as the proper contact time under known water hardness and conditions of high or low soil content should be considered . for an effective cleaning and disinfection plan", ", the choice of the disinfectant must follow specific criteria such as compatibility with the surfaces to be disinfected , economic constraints , safety in the workplace , toxicological safety , and biological degradability .", "it should , most of all , target the type of bacteria and the type of soiling .", "in fact , disinfectants can be seriously affected by the presence of organic matter .  ", "interfering substances have been studied in the last years and included in cleaning and disinfection plans regulated by the authorities such as the european standard en-1276 .", "however , most of these studies only address the effects of bovine serum albumin ( bsa ) and water hardness [ 9 , 14 , 15 , 1921 ] .", "evaluated the bactericidal activity of disinfectants referred in the german veterinary society guidelines as references for testing disinfectants used in dairy and food industries . in order to simulate the conditions found in practice , they used low fat milk as an organic load and reported the significance in choosing an appropriate disinfectant since the inclusion of a challenging substance ( organic material ) is important to access the proper bactericidal activity .", "bessems   demonstrated that a qac tested on three microorganisms ( pseudomonas aeruginosa , staphylococcus aureus , and candida albicans ) had a similar killing rate in the absence of interfering substances and after the inclusion of 17  dh water hardness , a strong reduction of the killing activity was found for the gram - negative bacteria", "assessed the effect of dried yeast and human serum on the activity of benzalkonium chloride and concluded that the bactericidal activity of the qac was inhibited by solutions of both interfering substances .", "this work provides information on the influence of potential interfering substances ( bovine serum albumin ", "ha ) on the antimicrobial activity of two qacs ( benzalkonium chloride and cetyltrimethyl ammonium bromide ) against bacillus cereus and pseudomonas fluorescens , as they are two major contaminants in the food industry , particularly the dairy industry , and are a known cause of produce spoilage and foodborne illnesses [ 2 , 2226 ] .", "some of the interfering substances used throughout the experiments are proposed in the european standard en-1276   as potential interfering agents in disinfection while the others are extracellular polymeric substances ( eps ) from the biofilm matrix that have an important role in antimicrobial resistance .", "the bacteria used in this work were pseudomonas fluorescens atcc  13525 and a bacillus cereus strain , isolated from a disinfectant solution and identified by 16s rrna gene sequencing .", "bacterial strains were grown at a temperature of 30  2c and ph 7 , with glucose as the main carbon source .", "culture medium consisted of 5  gl glucose , 2.5  gl peptone , and 1.25  gl yeast extract in phosphate buffer ( pb ) ( ph 7 , 0.025  m ) . a bacterial suspension was prepared by inoculation of a single colony grown on solid medium into a 1  l flask containing 250  ml of sterile nutrient medium .", "this bacterial suspension was incubated overnight at the given temperature with agitation ( 120  rpm ) .  ", "the qacs used throughout the experiments were benzalkonium chloride ( bac ) and cetyltrimethyl ammonium bromide ( ctab ) ( sigma , portugal ) .", "preliminary studies with a concentration range between 0 and 5000  mgl were initially made . in order to ascertain the behaviour of bacteria to the qac ,", "the selected concentrations for further studies were 3 , 5 , 10 , 20 , and 35  mgl .", "the qacs were used individually and in combination ( both chemicals were combined in equal volumes and concentrations ) .  ", "ha ( acros organics , fisher chemical , portugal ) , and yeast extract  ye ( merck , portugal ) .", "after the growth period , the suspensions were centrifuged ( 3999  g , 5 minutes ) , washed two times , and resuspended in pb to a final cell density of approximately 1  10  cellsml . in the case of the consortium ,", "both bacterial suspensions were washed two times resuspended in pb to a final cell density of approximately 1  10  cellsml , and combined in equal volumes to obtain the same cell concentrations of the single species tests .", "afterwards , all bacterial suspensions were exposed to several concentrations of qac for a period of 30 minutes .", "the effects of the chemicals were evaluated by the assessment of the oxygen uptake rate due to glucose oxidation , according to simes et al . .  ", "to investigate the influence of interfering substances on the antimicrobial efficacy , the same procedure was followed with the addition of 300  mgl of bsa , alg , ye , or ha to the bacterial suspension , simulating low concentrations of interfering substances according to the european standard en-1276 . three independent experiments , each with duplicate samples , were performed for each condition tested .", "the methodology was performed according to johnston et al .   for a period of 10 minutes .", "bac and ctab were chemically neutralized by a sterile solution of ( w / v ) 0.1% peptone , 0.5% tween 80 , 0.1% sodium thiosulphate , and 0.07% lecithin dissolved in pb .", "control experiments were performed to ascertain the effects of the 10-minute exposure to the neutralization solution , and no effects were detected on the respiratory activity of b. cereus and p. fluorescens ( data not shown ) .", "after the neutralization step , the bacterial suspensions were centrifuged ( 3999  g , 5  min ) and resuspended in the same volume of pb .", "the respiratory activity was ascertained by measuring oxygen uptake rates in a biological oxygen monitor ( yellow springs instruments 5300a ) .", "demonstrated that this procedure is more adequate and rapid than the assessment of colony forming units to characterize the antimicrobial activity of biocides against heterotrophic aerobic bacteria .", "samples were placed in the temperature - controlled vessel of the biological oxygen monitor ( t = 25  1c ) each containing a dissolved oxygen probe connected to a dissolved oxygen meter . before measuring", ", the samples were aerated for 10 minutes to ensure oxygen saturation ( [ o2 ] = 8.6  mgl ) .", "the vessel was closed , and the decrease of oxygen concentration was monitored over time .", "the initial linear decrease corresponds to the endogenous respiration rate . to determine the oxygen uptake due to substrate oxidation , 12.5  l of a 5  gl glucose solution was added to each vessel .", "the slope of the initial linear decrease in dissolved oxygen , after glucose injection , corresponds to the total respiration rate .", "the difference between these two rates is the oxygen uptake rate due to glucose oxidation .", "the inactivation was calculated using metabolic activities according to the following equation : \n ( 1)%  inactivation=(mcmt)mc100 , \n where mc is the metabolic activity of the control experiments ( without antimicrobial exposure ) and mt is the metabolic activity of the bacterial solutions exposed to the antimicrobial .", "if % inactivation > 0 there was inactivation of the microorganisms whereas if % inactivation < 0 there was metabolic potentiation .  ", "the mbc for each situation was determined as the lowest concentration of qac or qac combination where no respiratory activity was detected . for each parameter", "the statistical significance of the results was evaluated using the wilcoxon test ( confidence level  95% ) to investigate whether the differences between the resulting experimental values could be considered significant .", "the antibacterial activity of bac , ctab , and their combination was investigated in the absence and in the presence of four selected interfering substances . in the absence of interfering substances bac caused the inactivation of b. cereus at 10  mgl , p. fluorescens at 35  mgl , and the consortium at 20  mgl .", "ctab at 20  mgl completely inactivated b. cereus and at 35  mgl inactivated the total population of p. fluorescens and the consortium .", "the combination of both qacs was synergistic in the inactivation of b. cereus ( total inactivation with 3  mgl ) and indifferent for p. fluorescens ( 35  mgl ) and the bacterial consortium ( 35  mgl ) .", "the inclusion of the selected interfering substances influenced the antimicrobial activity of the qacs to some extent ( figures 13 ) .", "the inactivation of b. cereus ( figure 1 ) was not affected by the presence of any interfering substances ( p > 0.05 ) , except with ha .", "the antimicrobial action of the qacs against p. fluorescens ( figure 2 ) was not significantly influenced by the presence of most potential interfering substances ( p > 0.05 ) , except for ha where interference was observed ( p < 0.05 ) .", "the antimicrobial activity of the qacs against the bacterial consortium ( figure 3 ) was affected by the presence of interfering substances .", "ha reduced significantly the activity of ctab at higher concentrations ( p < 0.05 ) .", "bsa and ye resulted in a significant reduction of the activity of the combination of qacs ( p < 0.05 ) .", "linear correlations were determined to assess the relationship between qac concentrations and the inactivation data .", "the effect of increasing qac concentration on bacterial inactivation shows that there are strong linear correlations ( r > 0.850 ) for the control assays , with the exception of b. cereus ( this bacterium was inactivated with low qac concentrations ) .", "the most extreme cases are the treatments with ctab to p. fluorescens with alg as an interfering substance ( r = 0.771 ) and the bacterial consortium in the presence of ye ( r = 0.738 ) .", "likewise , this decrease of linear correlation factors was found for p. fluorescens and for the consortium exposed to ha where the lowest correlation factor was 0.153 , which was obtained for p. fluorescens treated with ctab .", "this phenomenon only happened when the qacs were used on p. fluorescens and the bacterial consortium in the presence of ye and ha .", "the most significant cases of oxygen uptake rate increase were verified for p. fluorescens exposed to bac ( 5 to 35  mgl ) and ctab ( 3 to 35  mgl ) in the presence of ha and combination of qacs ( 3 to 10  mgl ) in the presence of ye .", "a similar metabolic behaviour was found for the bacterial consortium exposed to bac ( 3 to 35  mgl ) and ctab ( 5 and 10  mgl ) for ha and qac combination ( 3 to 20  mgl ) with ye .", "the mbc values for the different conditions tested ( single and combined qacs , in the absence and presence of potential disinfection interfering substances ) are shown in table 1 .", "the presence of bsa increased the mbc of the combination of qacs for b. cereus ( 3 to 5  mgl ) and the consortium .", "alg increased the mbc of bac for the consortium ( 20 to over 35  mgl ) and qacs combination ( 3 to 5  mgl ) for b. cereus .", "ye increased the mbc of bac for b. cereus ( 10 to 20  mgl ) and qac combination ( 3 to 5  mgl ) .", "the mbc values for the consortium of cells increased in the presence of ye ( bac20 to 35  mgl , ctab35 to over 35  mgl , and qac combination35 to over 35  mgl ) .", "ha increased the mbc for all the scenarios , except of ctab when applied to b. cereus ( in this situation the mbc was reduced ) .", "the mbc was reduced in other situations such as , for b. cereus , in the presence of alg when using bac and ctab ( 10 to 5  mgl and 20 to 5  mgl , resp . ) and in the presence of ye when using ctab ( 20 to 3  mgl ) .", "fluorescens inactivation by ctab was reduced by bsa ( 35 to 20  mgl ) .", "alg also reduced the antimicrobial activity of the combination of qacs against the bacterial consortium ( 35 to 20  mgl ) .", "it is assumed that the organic material can potentially interfere with the antimicrobial agents by chemical and/or ionic interactions [ 15 , 33 ] .", "therefore , it is necessary to know the role of each potential interfering substance in the antimicrobial activity in order to develop effective disinfection strategies .", "the interfering substances tested are commonly found as residuals in the food industry ( from food products and from microbial contaminants , biofilms ) [ 18 , 27 ] .   in this study", ", higher inactivation rates were verified for b. cereus in comparison to p. fluorescens at the same qac concentration .", "in fact , when b. cereus and p. fluorescens are combined in a 1  :  1 bacterial suspension , it is expected that the first is more affected than the second .", "b. cereus is more susceptible due to the fact that it is a gram - positive bacterium that lacks an outer membrane , which typically provides increased protection to gram - negative bacteria .", "this fact is corroborated by previous reports which stated that gram - positive bacteria are more susceptible to cationic surfactants than gram - negative bacteria [ 34 , 35 ] .", "bsa was already studied as an interfering substance in disinfection practices [ 9 , 14 , 1921 , 36 ] .", "the negative effect of bsa on the action of biocides against p. fluorescens was demonstrated by simes et al .", "p. fluorescens treatment with ctab with the addition of 3  gl of bsa resulted in a 10-fold increase on the mbc of this qac [ 9 , 21 ] . in the present study ,", "the efficacy of the combination of qacs against b. cereus and the cell consortium was also reduced .", "this effect of bsa as an antimicrobial quencher is apparently due to the strong ability of qacs to react with proteins .", "proteins can precipitate in the form of their anions , in this way , the negative - charged protein ions will cling to the positively charged molecules of the cationic compounds .", "ctab is a biocide that targets the membrane and has a strong affinity for proteins .", "bac is composed of a positively charged hydrophobic headgroup which clings to opposite charged surfaces [ 8 , 37 ] .", "studied the effect of the alkyl chain of bac binding to bsa and dried yeast .", "their conclusions were that bac is often inactivated by organic matter , either by adsorption to the bacterial surface or by adsorption to the organic matter in general .", "these authors also suggested that the reduction in the activity of bac was probably related to more than one physical property of the compounds like the chain length ( longer chains result in more adsorption to the bacterial surface ) .  ", "alg is a common constituent of the extracellular polymeric substances of the biofilm matrix [ 3840 ] .", "a function frequently attributed to eps is their general protective effect on biofilm microorganisms against adverse conditions .", "the eps matrix delays or prevents antimicrobials from reaching target microorganisms within the biofilm by diffusion limitation and/or chemical interaction with the extracellular proteins and polysaccharides [ 32 , 41 ] . in this study , alg either potentiated or hindered the antimicrobial activity of the selected qacs .", "the presence of this interfering substance was not obvious on the inactivation of p. fluorescens .", "on the other hand , the inactivation of b. cereus by bac and ctab and the consortium by the combination of qacs was easier in the presence of this interfering substance .", "the bacterial consortium treatments with bac and b. cereus with the combination of qacs were hampered by the presence of alg .", "davies et al . found that the production of alg was triggered by membrane perturbation induced by ethanol stress , nitrogen limitation , attachment to surfaces , or even high oxygen tension [ 42 , 43 ] .", "this substance is suggested as one of the main biofilm resistance vectors either by reacting with the antimicrobials or by hindering antimicrobials diffusion to the cells .", "the antimicrobial interference caused by alg is apparently due to electrostatic interactions between the anionic alg and the cationic - selected qacs .  ", "the presence of ye as interfering substance resulted in three different outcomes on the antimicrobial activity of the qacs : ( 1 ) no effect / indifference , ( 2 ) the respiratory activity reduced , and ( 3 ) the respiratory activity potentiated .", "this interfering substance worked mainly as a hinderer of the antimicrobial activity by increasing the mbc of b. cereus in all cases except for ctab , of p. fluorescens with the combination of qacs , and of the consortium of cells with ctab and the combination of qacs .", "ye is listed in the european standard en-1276 as an interfering substance native to the brewery industry .", "the constituents of ye are very similar to the components of the bacterial cells , thus , it is expected that the antimicrobial agents that target the bacterial cells are also drawn to ye . in a similar study by jon et al .  ", "it was shown that the presence of dried yeast decreased the biocidal effectiveness of bac .  ", "humic substances are found ubiquitously in the environment and can be found in the biofilm matrix [ 2 , 46 ] .", "ha reduced the antimicrobial activity of the qacs in most of the cases , although in some cases it promoted the respiratory activity ( potentiation ) .", "the presence of these compounds had the strongest effect compared to the remaining interfering substances .", "studied the sorption mechanisms of anionic and cationic surfactants to natural soils concluding that the dominant sorption mechanism of surfactants to clay is cation exchange .", "koopal et al .   also verified the formation of complexes ha - cationic surfactant .", "respiratory activity potentiation was verified with the addition of ha to p. fluorescens and ye to the bacterial consortium .", "it is known that ha participates in cellular metabolism processes such as growth , respiration , photosynthesis , and nitrogen fixation . on the other hand , ha were proposed to replace synthetic surfactants such as sds , tween 80 , and triton x-100 in industrial applications such as textile dying or washing .", "it is therefore possible that the inclusion of humic substances in a solution of qacs may interfere with the chemical characteristics of the solution .", "the resultant mixture , with an apparent reduced antimicrobial efficacy , seems to potentiate the respiratory activity of the bacteria , particularly of p. fluorescens .", "as qacs are membrane active agents , their use at sublethal concentrations could improve membrane permeability and consequently the nutrient influx , without compromising the bacterial viability . also , there is the hypothesis that the potentially interfering agents could be used as nutrients .", "in fact , it was found that the growth rates of anaerobic and aerobic microorganisms increased when humic substances were added , which stimulated enzyme activity [ 52 , 53 ] . in a similar way ,", "ha are likely to be used for growth in the same way as ye ; these might be broken down to smaller molecules that can be used by cells as a carbon   or nitrogen sources .", "the antimicrobial activity of the tested qacs was enhanced in some cases , where the interfering substances were present .", "this is an unexpected result due to the recognized potential of alg , bsa , ha , and ye to interfere with disinfection .", "this effect is probably due to the low concentration of interfering substances tested that caused both respiratory activity reduction and potentiation .", "ethylenediamine tetraacetate ( edta ) was reported as early as 1965 to increase the biocidal effects of bac and chlorhexidine diacetate on pseudomonas aeruginosa .", "reported that chitosan ( a polysaccharide ) potentiated the antimicrobial action of sodium benzoate on spoilage yeasts . in dairy plants", ", disinfection is potentiated by pre - washes with alkali or enzyme - based cleaning agents .", "most of these cases were observed for b. cereus ( four occurrences ) , one was observed for p. fluorescens , and another one was observed for the consortium of cells .", "the mbc was improved by more than 50% in the cases of b. cereus and less than 30% for p. fluorescens and the consortium of cells . to our knowledge", "there are no reported cases of antimicrobial agents potentiation by bsa , ye , or alg .", "although the exact chemical structure of ha has not yet been determined , ha could be chemically similar to the tested qacs , presenting a positive hydrophilic head and a hydrophobic tail . with this structure ha could act as detergents in conditions such as those observed in the treatment of b. cereus with ctab .", "the present work shows that increasing qacs concentrations lead to an increase in antimicrobial effectiveness .", "this is valid mainly when the qacs were applied in the absence of interfering substances .", "this means that disinfection was concentration dependent , as found for most of the antimicrobial chemicals .", "however , the linear dependency of inactivation versus concentration is not verified for most of the tests where interfering substances were added .", "this result evidences that the mathematical modelling of disinfection strategies requires a case - to - case analysis when interfering substances are present .", "the overall results demonstrate that a disinfection process in the presence of the selected interfering substances can reduce the effectiveness of bac , ctab , and their combination .", "the bacteria were inactivated equally by all qacs , although in the absence of interfering substances ctab was the most efficient solution .", "p. fluorescens was the bacterium with the highest resistance to inactivation , followed by the bacterial consortium .", "the tested interfering substances , referred in the european standard 1276 ( bsa and ye ) , and known eps constituents related with biofilm resistance ( alg ) resulted in mild interferences on the activity of the qacs .", "ha were the interfering substance that resulted in the most severe effect by reducing the activity of qacs , causing , in some circumstances , significant respiratory activity potentiation ."], "abstract_text": ["<S> standard cleaning processes may not remove all the soiling typically found in food industry , such as carbohydrates , fats , or proteins . </S>", "<S> contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants . </S>", "<S> the influence of alginic acid , bovine serum albumin , yeast extract , and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against bacillus cereus vegetative cells and pseudomonas fluorescens . </S>", "<S> the bacteria ( single and consortium ) were exposed to surfactants ( single and combined ) in the absence and presence of potential disinfection interfering substances . </S>", "<S> the antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation . </S>", "<S> the tested surfactants were efficient against both bacteria ( single and consortium ) with minimum bactericidal concentrations ranging from 3 to 35  mgl1 . </S>", "<S> the strongest effect was caused by humic acids that severely quenched antimicrobial action , increasing the minimum bactericidal concentration of the surfactants on p. fluorescens and the consortium . </S>", "<S> the inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity . </S>", "<S> this study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies . </S>"], "labels": null, "section_names": ["1. Introduction ", "2. Materials and Methods", "3. Results ", "4. Discussion ", "5. Conclusions"], "sections": [["in order to prevent and control microbial proliferation in industrial settings , cleaning and disinfection plans are applied on a regular basis [ 1 , 2 ] . in food processing plants ,", "the control of microbial contamination generally involves clean - in - place ( cip ) procedures which consist of running alternated cycles of detergent and disinfectant solutions with water rinses in high turbulence regimes through the plant and pipeline circuits without dismantling or opening the equipment [ 25 ] .  ", "biocides are currently used in industrial processes as the most significant countermeasure to control microbial growth and proliferation .", "industry moved progressively towards the use of surfactants that are less toxic and more biodegradable .", "surfactants are classified according to the ionic physiognomies of their hydrophilic group as anionic , cationic , nonionic , and zwitterionic [ 6 , 8 ] .", "quaternary ammonium compounds ( qacs ) are cationic surfactants that are commonly used because of their hard - surface cleaning , odor removal and antimicrobial properties . besides killing bacteria , the chemical nature of qacs can cause modifications on the properties of abiotic surfaces , decreasing their tension and therefore preventing attachment of microorganisms .", "the antimicrobial mode of action of cationic surfactants is proposed by some authors as a sequence of events : attraction by the negatively charged cell surface ; adsorption to the cell wall through the hydrophobic headgroup ; reaction with the lipids and proteins that compose the cytoplasmic membrane ; and cell penetration and interaction with intracellular constituents [ 10 , 11 ] .", "thus , qacs damage the outer layers of bacteria , thereby promoting the release of intracellular constituents .", "antimicrobial efficacy tests require planning of an adequate strategy and should include all the parameters found in real settings .", "aspects such as the proper contact time under known water hardness and conditions of high or low soil content should be considered . for an effective cleaning and disinfection plan", ", the choice of the disinfectant must follow specific criteria such as compatibility with the surfaces to be disinfected , economic constraints , safety in the workplace , toxicological safety , and biological degradability .", "it should , most of all , target the type of bacteria and the type of soiling .", "in fact , disinfectants can be seriously affected by the presence of organic matter .  ", "interfering substances have been studied in the last years and included in cleaning and disinfection plans regulated by the authorities such as the european standard en-1276 .", "however , most of these studies only address the effects of bovine serum albumin ( bsa ) and water hardness [ 9 , 14 , 15 , 1921 ] .", "evaluated the bactericidal activity of disinfectants referred in the german veterinary society guidelines as references for testing disinfectants used in dairy and food industries . in order to simulate the conditions found in practice , they used low fat milk as an organic load and reported the significance in choosing an appropriate disinfectant since the inclusion of a challenging substance ( organic material ) is important to access the proper bactericidal activity .", "bessems   demonstrated that a qac tested on three microorganisms ( pseudomonas aeruginosa , staphylococcus aureus , and candida albicans ) had a similar killing rate in the absence of interfering substances and after the inclusion of 17  dh water hardness , a strong reduction of the killing activity was found for the gram - negative bacteria", "assessed the effect of dried yeast and human serum on the activity of benzalkonium chloride and concluded that the bactericidal activity of the qac was inhibited by solutions of both interfering substances .", "this work provides information on the influence of potential interfering substances ( bovine serum albumin ", "ha ) on the antimicrobial activity of two qacs ( benzalkonium chloride and cetyltrimethyl ammonium bromide ) against bacillus cereus and pseudomonas fluorescens , as they are two major contaminants in the food industry , particularly the dairy industry , and are a known cause of produce spoilage and foodborne illnesses [ 2 , 2226 ] .", "some of the interfering substances used throughout the experiments are proposed in the european standard en-1276   as potential interfering agents in disinfection while the others are extracellular polymeric substances ( eps ) from the biofilm matrix that have an important role in antimicrobial resistance ."], ["the bacteria used in this work were pseudomonas fluorescens atcc  13525 and a bacillus cereus strain , isolated from a disinfectant solution and identified by 16s rrna gene sequencing .", "bacterial strains were grown at a temperature of 30  2c and ph 7 , with glucose as the main carbon source .", "culture medium consisted of 5  gl glucose , 2.5  gl peptone , and 1.25  gl yeast extract in phosphate buffer ( pb ) ( ph 7 , 0.025  m ) . a bacterial suspension was prepared by inoculation of a single colony grown on solid medium into a 1  l flask containing 250  ml of sterile nutrient medium .", "this bacterial suspension was incubated overnight at the given temperature with agitation ( 120  rpm ) .  ", "the qacs used throughout the experiments were benzalkonium chloride ( bac ) and cetyltrimethyl ammonium bromide ( ctab ) ( sigma , portugal ) .", "preliminary studies with a concentration range between 0 and 5000  mgl were initially made . in order to ascertain the behaviour of bacteria to the qac ,", "the selected concentrations for further studies were 3 , 5 , 10 , 20 , and 35  mgl .", "the qacs were used individually and in combination ( both chemicals were combined in equal volumes and concentrations ) .  ", "ha ( acros organics , fisher chemical , portugal ) , and yeast extract  ye ( merck , portugal ) .", "after the growth period , the suspensions were centrifuged ( 3999  g , 5 minutes ) , washed two times , and resuspended in pb to a final cell density of approximately 1  10  cellsml . in the case of the consortium ,", "both bacterial suspensions were washed two times resuspended in pb to a final cell density of approximately 1  10  cellsml , and combined in equal volumes to obtain the same cell concentrations of the single species tests .", "afterwards , all bacterial suspensions were exposed to several concentrations of qac for a period of 30 minutes .", "the effects of the chemicals were evaluated by the assessment of the oxygen uptake rate due to glucose oxidation , according to simes et al . .  ", "to investigate the influence of interfering substances on the antimicrobial efficacy , the same procedure was followed with the addition of 300  mgl of bsa , alg , ye , or ha to the bacterial suspension , simulating low concentrations of interfering substances according to the european standard en-1276 . three independent experiments , each with duplicate samples , were performed for each condition tested .", "the methodology was performed according to johnston et al .   for a period of 10 minutes .", "bac and ctab were chemically neutralized by a sterile solution of ( w / v ) 0.1% peptone , 0.5% tween 80 , 0.1% sodium thiosulphate , and 0.07% lecithin dissolved in pb .", "control experiments were performed to ascertain the effects of the 10-minute exposure to the neutralization solution , and no effects were detected on the respiratory activity of b. cereus and p. fluorescens ( data not shown ) .", "after the neutralization step , the bacterial suspensions were centrifuged ( 3999  g , 5  min ) and resuspended in the same volume of pb .", "the respiratory activity was ascertained by measuring oxygen uptake rates in a biological oxygen monitor ( yellow springs instruments 5300a ) .", "demonstrated that this procedure is more adequate and rapid than the assessment of colony forming units to characterize the antimicrobial activity of biocides against heterotrophic aerobic bacteria .", "samples were placed in the temperature - controlled vessel of the biological oxygen monitor ( t = 25  1c ) each containing a dissolved oxygen probe connected to a dissolved oxygen meter . before measuring", ", the samples were aerated for 10 minutes to ensure oxygen saturation ( [ o2 ] = 8.6  mgl ) .", "the vessel was closed , and the decrease of oxygen concentration was monitored over time .", "the initial linear decrease corresponds to the endogenous respiration rate . to determine the oxygen uptake due to substrate oxidation , 12.5  l of a 5  gl glucose solution was added to each vessel .", "the slope of the initial linear decrease in dissolved oxygen , after glucose injection , corresponds to the total respiration rate .", "the difference between these two rates is the oxygen uptake rate due to glucose oxidation .", "the inactivation was calculated using metabolic activities according to the following equation : \n ( 1)%  inactivation=(mcmt)mc100 , \n where mc is the metabolic activity of the control experiments ( without antimicrobial exposure ) and mt is the metabolic activity of the bacterial solutions exposed to the antimicrobial .", "if % inactivation > 0 there was inactivation of the microorganisms whereas if % inactivation < 0 there was metabolic potentiation .  ", "the mbc for each situation was determined as the lowest concentration of qac or qac combination where no respiratory activity was detected . for each parameter", "the statistical significance of the results was evaluated using the wilcoxon test ( confidence level  95% ) to investigate whether the differences between the resulting experimental values could be considered significant ."], ["the antibacterial activity of bac , ctab , and their combination was investigated in the absence and in the presence of four selected interfering substances . in the absence of interfering substances bac caused the inactivation of b. cereus at 10  mgl , p. fluorescens at 35  mgl , and the consortium at 20  mgl .", "ctab at 20  mgl completely inactivated b. cereus and at 35  mgl inactivated the total population of p. fluorescens and the consortium .", "the combination of both qacs was synergistic in the inactivation of b. cereus ( total inactivation with 3  mgl ) and indifferent for p. fluorescens ( 35  mgl ) and the bacterial consortium ( 35  mgl ) .", "the inclusion of the selected interfering substances influenced the antimicrobial activity of the qacs to some extent ( figures 13 ) .", "the inactivation of b. cereus ( figure 1 ) was not affected by the presence of any interfering substances ( p > 0.05 ) , except with ha .", "the antimicrobial action of the qacs against p. fluorescens ( figure 2 ) was not significantly influenced by the presence of most potential interfering substances ( p > 0.05 ) , except for ha where interference was observed ( p < 0.05 ) .", "the antimicrobial activity of the qacs against the bacterial consortium ( figure 3 ) was affected by the presence of interfering substances .", "ha reduced significantly the activity of ctab at higher concentrations ( p < 0.05 ) .", "bsa and ye resulted in a significant reduction of the activity of the combination of qacs ( p < 0.05 ) .", "linear correlations were determined to assess the relationship between qac concentrations and the inactivation data .", "the effect of increasing qac concentration on bacterial inactivation shows that there are strong linear correlations ( r > 0.850 ) for the control assays , with the exception of b. cereus ( this bacterium was inactivated with low qac concentrations ) .", "the most extreme cases are the treatments with ctab to p. fluorescens with alg as an interfering substance ( r = 0.771 ) and the bacterial consortium in the presence of ye ( r = 0.738 ) .", "likewise , this decrease of linear correlation factors was found for p. fluorescens and for the consortium exposed to ha where the lowest correlation factor was 0.153 , which was obtained for p. fluorescens treated with ctab .", "this phenomenon only happened when the qacs were used on p. fluorescens and the bacterial consortium in the presence of ye and ha .", "the most significant cases of oxygen uptake rate increase were verified for p. fluorescens exposed to bac ( 5 to 35  mgl ) and ctab ( 3 to 35  mgl ) in the presence of ha and combination of qacs ( 3 to 10  mgl ) in the presence of ye .", "a similar metabolic behaviour was found for the bacterial consortium exposed to bac ( 3 to 35  mgl ) and ctab ( 5 and 10  mgl ) for ha and qac combination ( 3 to 20  mgl ) with ye .", "the mbc values for the different conditions tested ( single and combined qacs , in the absence and presence of potential disinfection interfering substances ) are shown in table 1 .", "the presence of bsa increased the mbc of the combination of qacs for b. cereus ( 3 to 5  mgl ) and the consortium .", "alg increased the mbc of bac for the consortium ( 20 to over 35  mgl ) and qacs combination ( 3 to 5  mgl ) for b. cereus .", "ye increased the mbc of bac for b. cereus ( 10 to 20  mgl ) and qac combination ( 3 to 5  mgl ) .", "the mbc values for the consortium of cells increased in the presence of ye ( bac20 to 35  mgl , ctab35 to over 35  mgl , and qac combination35 to over 35  mgl ) .", "ha increased the mbc for all the scenarios , except of ctab when applied to b. cereus ( in this situation the mbc was reduced ) .", "the mbc was reduced in other situations such as , for b. cereus , in the presence of alg when using bac and ctab ( 10 to 5  mgl and 20 to 5  mgl , resp . ) and in the presence of ye when using ctab ( 20 to 3  mgl ) .", "fluorescens inactivation by ctab was reduced by bsa ( 35 to 20  mgl ) .", "alg also reduced the antimicrobial activity of the combination of qacs against the bacterial consortium ( 35 to 20  mgl ) ."], ["it is assumed that the organic material can potentially interfere with the antimicrobial agents by chemical and/or ionic interactions [ 15 , 33 ] .", "therefore , it is necessary to know the role of each potential interfering substance in the antimicrobial activity in order to develop effective disinfection strategies .", "the interfering substances tested are commonly found as residuals in the food industry ( from food products and from microbial contaminants , biofilms ) [ 18 , 27 ] .   in this study", ", higher inactivation rates were verified for b. cereus in comparison to p. fluorescens at the same qac concentration .", "in fact , when b. cereus and p. fluorescens are combined in a 1  :  1 bacterial suspension , it is expected that the first is more affected than the second .", "b. cereus is more susceptible due to the fact that it is a gram - positive bacterium that lacks an outer membrane , which typically provides increased protection to gram - negative bacteria .", "this fact is corroborated by previous reports which stated that gram - positive bacteria are more susceptible to cationic surfactants than gram - negative bacteria [ 34 , 35 ] .", "bsa was already studied as an interfering substance in disinfection practices [ 9 , 14 , 1921 , 36 ] .", "the negative effect of bsa on the action of biocides against p. fluorescens was demonstrated by simes et al .", "p. fluorescens treatment with ctab with the addition of 3  gl of bsa resulted in a 10-fold increase on the mbc of this qac [ 9 , 21 ] . in the present study ,", "the efficacy of the combination of qacs against b. cereus and the cell consortium was also reduced .", "this effect of bsa as an antimicrobial quencher is apparently due to the strong ability of qacs to react with proteins .", "proteins can precipitate in the form of their anions , in this way , the negative - charged protein ions will cling to the positively charged molecules of the cationic compounds .", "ctab is a biocide that targets the membrane and has a strong affinity for proteins .", "bac is composed of a positively charged hydrophobic headgroup which clings to opposite charged surfaces [ 8 , 37 ] .", "studied the effect of the alkyl chain of bac binding to bsa and dried yeast .", "their conclusions were that bac is often inactivated by organic matter , either by adsorption to the bacterial surface or by adsorption to the organic matter in general .", "these authors also suggested that the reduction in the activity of bac was probably related to more than one physical property of the compounds like the chain length ( longer chains result in more adsorption to the bacterial surface ) .  ", "alg is a common constituent of the extracellular polymeric substances of the biofilm matrix [ 3840 ] .", "a function frequently attributed to eps is their general protective effect on biofilm microorganisms against adverse conditions .", "the eps matrix delays or prevents antimicrobials from reaching target microorganisms within the biofilm by diffusion limitation and/or chemical interaction with the extracellular proteins and polysaccharides [ 32 , 41 ] . in this study , alg either potentiated or hindered the antimicrobial activity of the selected qacs .", "the presence of this interfering substance was not obvious on the inactivation of p. fluorescens .", "on the other hand , the inactivation of b. cereus by bac and ctab and the consortium by the combination of qacs was easier in the presence of this interfering substance .", "the bacterial consortium treatments with bac and b. cereus with the combination of qacs were hampered by the presence of alg .", "davies et al . found that the production of alg was triggered by membrane perturbation induced by ethanol stress , nitrogen limitation , attachment to surfaces , or even high oxygen tension [ 42 , 43 ] .", "this substance is suggested as one of the main biofilm resistance vectors either by reacting with the antimicrobials or by hindering antimicrobials diffusion to the cells .", "the antimicrobial interference caused by alg is apparently due to electrostatic interactions between the anionic alg and the cationic - selected qacs .  ", "the presence of ye as interfering substance resulted in three different outcomes on the antimicrobial activity of the qacs : ( 1 ) no effect / indifference , ( 2 ) the respiratory activity reduced , and ( 3 ) the respiratory activity potentiated .", "this interfering substance worked mainly as a hinderer of the antimicrobial activity by increasing the mbc of b. cereus in all cases except for ctab , of p. fluorescens with the combination of qacs , and of the consortium of cells with ctab and the combination of qacs .", "ye is listed in the european standard en-1276 as an interfering substance native to the brewery industry .", "the constituents of ye are very similar to the components of the bacterial cells , thus , it is expected that the antimicrobial agents that target the bacterial cells are also drawn to ye . in a similar study by jon et al .  ", "it was shown that the presence of dried yeast decreased the biocidal effectiveness of bac .  ", "humic substances are found ubiquitously in the environment and can be found in the biofilm matrix [ 2 , 46 ] .", "ha reduced the antimicrobial activity of the qacs in most of the cases , although in some cases it promoted the respiratory activity ( potentiation ) .", "the presence of these compounds had the strongest effect compared to the remaining interfering substances .", "studied the sorption mechanisms of anionic and cationic surfactants to natural soils concluding that the dominant sorption mechanism of surfactants to clay is cation exchange .", "koopal et al .   also verified the formation of complexes ha - cationic surfactant .", "respiratory activity potentiation was verified with the addition of ha to p. fluorescens and ye to the bacterial consortium .", "it is known that ha participates in cellular metabolism processes such as growth , respiration , photosynthesis , and nitrogen fixation . on the other hand , ha were proposed to replace synthetic surfactants such as sds , tween 80 , and triton x-100 in industrial applications such as textile dying or washing .", "it is therefore possible that the inclusion of humic substances in a solution of qacs may interfere with the chemical characteristics of the solution .", "the resultant mixture , with an apparent reduced antimicrobial efficacy , seems to potentiate the respiratory activity of the bacteria , particularly of p. fluorescens .", "as qacs are membrane active agents , their use at sublethal concentrations could improve membrane permeability and consequently the nutrient influx , without compromising the bacterial viability . also , there is the hypothesis that the potentially interfering agents could be used as nutrients .", "in fact , it was found that the growth rates of anaerobic and aerobic microorganisms increased when humic substances were added , which stimulated enzyme activity [ 52 , 53 ] . in a similar way ,", "ha are likely to be used for growth in the same way as ye ; these might be broken down to smaller molecules that can be used by cells as a carbon   or nitrogen sources .", "the antimicrobial activity of the tested qacs was enhanced in some cases , where the interfering substances were present .", "this is an unexpected result due to the recognized potential of alg , bsa , ha , and ye to interfere with disinfection .", "this effect is probably due to the low concentration of interfering substances tested that caused both respiratory activity reduction and potentiation .", "ethylenediamine tetraacetate ( edta ) was reported as early as 1965 to increase the biocidal effects of bac and chlorhexidine diacetate on pseudomonas aeruginosa .", "reported that chitosan ( a polysaccharide ) potentiated the antimicrobial action of sodium benzoate on spoilage yeasts . in dairy plants", ", disinfection is potentiated by pre - washes with alkali or enzyme - based cleaning agents .", "most of these cases were observed for b. cereus ( four occurrences ) , one was observed for p. fluorescens , and another one was observed for the consortium of cells .", "the mbc was improved by more than 50% in the cases of b. cereus and less than 30% for p. fluorescens and the consortium of cells . to our knowledge", "there are no reported cases of antimicrobial agents potentiation by bsa , ye , or alg .", "although the exact chemical structure of ha has not yet been determined , ha could be chemically similar to the tested qacs , presenting a positive hydrophilic head and a hydrophobic tail . with this structure ha could act as detergents in conditions such as those observed in the treatment of b. cereus with ctab .", "the present work shows that increasing qacs concentrations lead to an increase in antimicrobial effectiveness .", "this is valid mainly when the qacs were applied in the absence of interfering substances .", "this means that disinfection was concentration dependent , as found for most of the antimicrobial chemicals .", "however , the linear dependency of inactivation versus concentration is not verified for most of the tests where interfering substances were added .", "this result evidences that the mathematical modelling of disinfection strategies requires a case - to - case analysis when interfering substances are present ."], ["the overall results demonstrate that a disinfection process in the presence of the selected interfering substances can reduce the effectiveness of bac , ctab , and their combination .", "the bacteria were inactivated equally by all qacs , although in the absence of interfering substances ctab was the most efficient solution .", "p. fluorescens was the bacterium with the highest resistance to inactivation , followed by the bacterial consortium .", "the tested interfering substances , referred in the european standard 1276 ( bsa and ye ) , and known eps constituents related with biofilm resistance ( alg ) resulted in mild interferences on the activity of the qacs .", "ha were the interfering substance that resulted in the most severe effect by reducing the activity of qacs , causing , in some circumstances , significant respiratory activity potentiation ."]]}
{"article_id": "PMC4180206", "article_text": ["social scientists who explore factors mediating and moderating the relationships between social stressors and mental health , including drinking outcomes , have highlighted modes of coping [ 1 , 2 ] . these studies have explored behaviors which protect people from being psychologically harmed   and cognitive appraisals which influence behaviors such as problem - focused coping   or using alcohol to self - medicate distress .", "however , studies have not considered the characteristics of the stressful situation itself that may make certain coping strategies more or less effective . in particular ,", "psychiatric epidemiologic studies have tended to emphasize microlevel stressors ( e.g. , stressors in individuals ' role domains ) and , until recently , have ignored the linkages between macrolevel social forces and the daily stressors in people 's lives [ 79 ] .", "however , macrolevel social conditions can affect the magnitude of stressors experienced in people 's lives and the extent to which they experience  cumulative adversity  .", "this paper focuses on coping with the fallout from one type of macrolevel social stressor : the recent great recession .", "this economic downturn constituted the most severe economic crisis in the united states since the great depression   and had persisting economic effects ( e.g. , job loss , less desirable working conditions , loss of home , loss of retirement savings , lack of health care access , and social isolation ) which have been linked with deleterious drinking outcomes .", "a key issue involving the effectiveness of alternative modes of coping with stressors derived from macrolevel social forces and protecting against deleterious drinking outcomes is the question of whether individual modes of coping outside of the political realm emphasized in the overall coping literature are most efficacious . or , alternatively , do stressors engendered by macrolevel social forces require unique forms of coping , that encompass the political realm ?", "with respect to coping strategies employed in the wake of the great recession , politically oriented coping strategies might be of particular relevance due to the impact of governmental decision - making on the state of the economy .", "politically oriented coping strategies might include political activism oriented to altering economic policies or support for campaigns by politicians offering solutions to economically based hardships . scholarly work on the occupy wall street movement , for example , documents involvement by individuals sharing their own economic struggles   and collectively gathering to protest their precarious economic situation and uncertain economic future .", "earlier work also suggests the salience of activities oriented toward changing politically based social realities such as through the act of voting   or by collectively challenging community - level decisions such as school closings .", "by contrast , the traditional coping literature has emphasized individual , nonpolitical modes of coping , such as emotional acceptance of the stressful situation , blaming one 's self for the situation , or taking individual actions such as looking for a job if unemployed .", "while sociologists have recently accorded greater attention to the prevalence and alcohol - related consequences of macrolevel stressors , they have yet to address the extent to which politically oriented modes of coping may be the most efficacious ways to address problems stemming from macrolevel social forces or events . prior to the more recent focus on macrolevel stressors , kaplan and liu   embraced the idea of collective coping as one means for individuals who maintained stigmatized personal identities to challenge and transform conventional socionormative systems through participation in social movements .", "subsequently , thoits   more explicitly addressed collective coping within the context of the stress paradigm involving acute and chronic stressors not limited to the specific area of stigmatized statuses .", "she argued that individuals who find themselves in problematic situations can deliberately work to transform the meaning of their experiences and they can additionally use these experiences  as a basis for helping or effecting changes in the lives of others ", "she proposed the concept of  transformatory coping  to include engagement in collective activist activities with others who share similar problems .", "for example , parents of autistic children have lobbied governments for social services perceived to aid in their children 's development . in sum , ( 1 ) collective coping tactics ( such as politically oriented coping ) represent an unmeasured dimension of coping behaviors beyond that represented in the coping literature to date and ( 2 ) collective coping tactics may demonstrate a stronger association between stressors , particularly those stemming from macrolevel social forces , and deleterious drinking outcomes compared to the use of modes of coping previously emphasized in the literature on coping .", "the present study extends previous work by empirically addressing the extent to which politically oriented coping activities engaged in as a response to a macrolevel social stressor , the great recession , are protective against alcohol - related outcomes compared with coping strategies focused outside of the political realm .", "we hypothesize that politically oriented coping will be more protective against economic stressors linked with the great recession than nonpolitical modes of coping and will uniquely account for some portion of the associations between economy - related stressors and drinking outcomes .", "further , we also examine whether politically oriented coping and coping outside of the political realm are more protective for men versus women in the face of macrolevel engendered stressors such as those involving the economic fallout from the great recession .", "there is consistent evidence that women are more likely to use support - based coping strategies ( e.g. , seeking support from others such as partner family and friends ) in response to stress in contrast to men and some indication that avoidant coping techniques are associated with greater alcohol consumption among men but not women [ 1 , 2022 ] .", "( in contrast , parity by gender in the use of individual active coping strategies and their significance for mental health outcomes is generally reported [ 1 , 21 ] . ) however , whether there is a corresponding propensity for men and women to differ in the use of politically oriented coping to offset the alcohol - related effects of economy - related stressors is less certain .", "earlier research tended to argue that women were less politically interested , informed , and efficacious compared to men .", "more recent work has shown that women and men differ in particular modes of participation ; women are more likely to vote and engage in individual political actions such as signing petitions or donating money , whereas men are more likely to be engaged in collective forms of action such as group protest activities .", "thus , we hypothesize that there will be no overall differences in the extent to which women and men manifest politically oriented coping or in the effect that politically oriented coping has on drinking outcomes . following a transactional model of stress", "[ 4 , 25 ] , we model coping as a mediator of the relationship between the stressor ( i.e. , stressful consequences of the great recession ) and the stress response ( i.e. , drinking outcomes ) .", "data were derived from a study conducted in the united states between june , 2010 , and january , 2011 , that was undertaken in order to understand life change consequences of the major downturn in the economy known as the great recession .", "respondents were selected by a random digit dial ( rdd ) phone survey of the continental united states , and those who consented to participate in the study were mailed questionnaires .", "eligible respondents were selected from the households using the troldahl - carter - bryant method of respondent selection which involves the means to randomly select a respondent from all eligible household members .", "respondents were told during the phone screen that a $ 50 american express gift card would be sent to the eligible respondent if he or she completed the questionnaire .", "respondents were mailed an initial survey , a postcard reminder to nonresponders , and a second questionnaire if they still had not responded .", "figure 1 encompasses a flow chart characterizing each stage in the data collection process through the completion of the questionnaires .", "65.9% ( n = 663 ) of the respondents completing the screening calls returned the questionnaire .", "the telephone screening cooperation rate and the mail survey response rate were each calculated using the conservative aapor response rate formula 3 .", "the overall survey response rate is the product of the phone screening cooperation rate ( 35.5% ) and the mail questionnaire return response rate ( 65.9% ) or 16.8% .", "we acknowledge that this response rate is less than ideal and further address this issue in the discussion of study limitations and note other indicators of the representativeness of the final sample .", "selection weights were calculated for each of the cases to weight for the different probability of selection for each case .", "poststratification weights were calculated for the dataset to ensure that the distribution of sample cases on important demographic variables ( age , race / ethnicity , and gender ) conformed to the distribution of these variables in census bureau 's 2008 united states population estimates .", "it should be noted that estimates of alcohol consumption for the present sample did appear to conform to national estimates preweighting .", "for example , the average number of drinks consumed in the past month on days when one drank for the present sample is 2.16 , versus the estimated average of 2.10 reported by the centers for disease control and prevention 's behavioral risk factor surveillance system for 2010   ( by gender , the averages are 2.35 for men and 1.89 for women in the present sample versus 2.43 for men and 1.81 for women in the cdc estimates ; by race / ethnicity , the averages are 2.16 for non - hispanic whites , 2.08 for asians , 2.12 for african americans , and 2.72 for hispanics in the present sample versus 2.26 for non - hispanic whites , 2.41 for asians , 2.19 for african americans , and 2.68 for hispanics in the cdc data ) .", "it should additionally be noted that the respondents included in this sample reported an overall higher level of education than the general population based on 2008 census estimates .", "analysis of variance revealed no significant variation by education in either the outcomes or support coping , avoidant coping , or politically oriented coping .", "respondents with less than a high school degree are found to be marginally less likely ( p < 0.10 ) to use active coping strategies compared to those with a college degree or postcollege training . given this discrepancy as well as the fact that education is generally protective against problem drinking", "predictor variables are economy - related stressors , coping strategies enacted outside of the political realm ( i.e. , active coping , support coping , and avoidant coping ) , politically oriented coping , and gender . the sociodemographic characteristics of age , education , and race / ethnicity are controlled in all analyses . to assess past - month drinking patterns , we use the quantity - frequency - variability index ( qfv ) developed by cahalan et al . .", "frequency of drinking is measured as a count of the days on which alcohol was consumed in the past 30 days , and quantity of drinking is measured as the average number of drinks consumed on those days .", "variability is calculated by the greatest number of drinks consumed on any one day in the past 30 days .", "scores are calculated by multiplying responses to the quantity , frequency , and variability questions (  = 0.87 ) . as with the current data", ", this index tends to approximate a continuous scale , and ample evidence supports its use as such ( see fitzgerald and mulford   for a review ) .", "our measure of problematic drinking is the 10-item bmast (  = 0.74 ) , which is a count measure of difficulties related to alcohol use over the past year .", "respondents were asked to indicate  yes  or  no  in response to 10 items such as having an accident , losing a close friend , spouse , or loved one , being hospitalized , having trouble at work , and soliciting professional help because of one 's drinking . the bmast is one of the most widely used tools for assessing alcohol dependence and problems .", "it correlates strongly with the full - length mast and evidence of its reliability and validity is widely available [ 31 , 32 ] .", "moreover , we are not using the bmast as a diagnostic tool for depicting  problem  versus", " nonproblem  drinking , but as it is intended , as suggestive of different degrees of problematic drinking .", "the measure of economy - related stressors is the life change consequences of the great recession ( lccgr ) instrument .", "this construct was developed on the basis of qualitative analyses of transcripts derived from focus groups involving both genders and diverse racial / ethnic groups .", "the items fall into seven categories : home ownership problems , such as difficulties in mortgage payments , difficulties in paying property taxes , or a drop in credit rating ; undesirable living situation , including having to live in a less desired location to save money or having gas and electricity or heat shut off due to an inability to pay bills ; problematic employment situation , including a pay - cut , furlough days , and increased feelings of competition with fellow employees ; unemployment or underemployment ; inadequate health insurance , including lack of medical or dental coverage , decreased quality of coverage , and inability to obtain coverage ; social role constraints , such as dissolution of spouse / partner relationship , decreased social life , and increased social isolation due to finances ; and inadequate sick time , including inadequate sick days and having to work despite poor health .", "consistent with common practice , each score for this measure is a straight count of the number of stressors reported .", "three dimensions of nonpolitical modes of coping are assessed : active coping , support coping , and avoidant coping .", "these measures of coping are derived from subscales of the brief cope instrument   and have previously been validated as stand - alone indices in community samples [ 3436 ] .", "participants in the present study were asked whether they have used these coping strategies in response to the economic recession .", "confirmatory factor analysis of the present data supports the inclusion of these items as three separate coping indices , consistent with prior research .", "active coping (  = 0.84 ) includes eight items measuring acceptance , positive reframing , and planning and taking action in response to the economic recession .", "support coping (  = 0.81 ) includes four items measuring use of emotional and instrumental support ( i.e. , receiving emotional support and getting advice and help from other people ) .", "avoidant coping (  = 0.75 ) includes 10 items measuring self - distraction , behavioral disengagement , self - denial , blame , and a tendency to vent about or make fun of the situation . all items were rated on a four - point likert - type scale ranging from 0 ( i did not do this at all ) to 3 ( i did this a lot ) . the measure of politically oriented coping", "is assessed by a four - item instrument (  = 0.79 ) drawn from the summed responses ( i.e. , not at all , a little , some , quite a bit , and a lot ) to four statements asking how often respondents , in response to the economic recession , have been ( 1 )  engaging in political activities such as signing petitions , leading or participating in rallies or marches , or writing to political representatives ;  ( 2 )  organizing with others to challenge politicians currently in office ;  ( 3 )  voting in elections to support politicians who share your political beliefs ;  and ( 4 )  participating in groups trying to influence the policies of the government at the local , state , or national level . ", "the questions used to construct this index were derived from analyses of focus group transcripts ( see   for details about these focus groups ) .", "confirmatory factor analysis reveals that these items load on a single factor , supporting their inclusion as one index .", "education is a categorical variable based on the educational attainment categories of ( 1 ) less than high school ( n = 45 ) ; ( 2 ) high school graduate ( n = 350 ) ; ( 3 ) college graduate ( n = 110 ) ; and ( 4 ) postcollege training ( n = 150 ) . race / ethnicity is a dummy variable including non - hispanic whites ( n = 436 ) , african americans ( n = 80 ) , hispanics ( n = 91 ) , asians ( n = 29 ) , and individuals who identify as an  other  race / ethnicity ( n = 17 ) . in all analyses ,", "non - hispanic whites serve as the reference category . after examining bivariate correlations in order to assess the basic patterns of association among key study variables , we performed structural equation modeling ( sem ) using mplus software   to examine the predictive significance of economy - related stressors for coping outside of the political realm and politically oriented coping tactics and the two drinking outcomes considered ( i.e. , past - month drinking and problematic drinking ) , net of the sociodemographic control variables .", "we considered the potential for nonpolitical coping and politically oriented coping tactics to mediate the associations between economy - related stressors and each of the outcomes assessed in two models .", "the first model tested for associations between economic stressors and the drinking - related outcomes .", "the second model adds nonpolitical and politically oriented coping tactics to test the full mediation model .", "this latter model assesses all of the direct and indirect paths between economic stressors and the drinking outcomes considered through the nonpolitical and politically oriented coping tactics investigated .", "we formally tested for mediation using the procedures described by muthen and muthen   for mplus software , which apply the tests described by mackinnon et al . .", "finally , because the associations between social stress and drinking are found to vary by gender [ 33 , 39 ] , we examined whether any observed mediating effects of the coping strategies investigated vary by gender . for these tests ,", "separate equations include the interaction term for gender by each coping strategy in the path models linking coping with drinking outcomes .", "it is noteworthy that stressors related to the economy are associated with each of the alcohol - related outcomes and all of the coping resources considered : economy - related stressors are associated with more alcohol consumption and problematic drinking , as well as higher levels of active coping , support coping , avoidant coping , and politically oriented coping .", "it is also noteworthy that only two of the coping strategies assessed are associated with the drinking outcomes .", "avoidant coping and politically oriented coping are associated with both alcohol consumption and problematic drinking , but in opposite directions .", "that is , greater avoidant coping is associated with greater alcohol consumption and problematic drinking , whereas greater politically oriented coping is associated with less alcohol consumption and problematic drinking .", "the lack of correlation between active coping and support coping and each of the drinking outcomes , respectively , provides some indication that not all of the coping strategies may be useful in understanding the associations between economic stressors and drinking - related outcomes . additionally , the possibility that coping resources may vary by gender is not strongly supported by the pattern of correlations reported , with the exception that women reported significantly more emotional support than men . however , and consistent with previous research , women are found to drink less and less problematically .", "the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes are further elaborated upon in the structural equation model .", "estimation of the first model ( figure 2 ) , including only economic stressors , the drinking - related outcomes , and sociodemographic controls , produces a just identified model and , as such , meaningful fit statistics are not provided .", "the standardized path coefficients demonstrate that economic stressors and each of the drinking - related outcomes considered are significantly and positively related .", "net of the sociodemographic controls , greater economic strain is associated with greater alcohol consumption over the past month (  = 0.094 , s.e .", "= 0.004 , and p < 0.01 ) and a higher incidence of problematic drinking over the past year (  = 0.181 , s.e .", "sem analysis testing the second model , of the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes considered , is presented as figure 3 . in figure 3 ,", "solid lines indicate effects that are statistically significant , and dashed lines indicate effects that are not statistically significant .", "the model fit criteria provided by hu and bentler   ( cfi > 0.95 ; rmsea < 0.06 ; srmr < 0.08 ) are used to assess the measurement model . based on these criteria , there is consistent evidence of good fit for this model ( cfi = 0.982 ; rmsea = 0.046 ;", "this model demonstrates that economic stress is associated with higher levels of each of the coping resources considered .", "that is , greater economic strain appears to predict a greater propensity to use both maladaptive coping tactics ( i.e. , avoidant coping ) and adaptive coping tactics ( i.e. , active coping , support coping , and politically oriented coping ) .", "avoidant coping is associated with higher levels of past - month drinking and problematic drinking , whereas politically oriented coping is associated with less alcohol consumption and problematic drinking .", "formal mediation tests next reveal that the effects of economy - related stressors on the alcohol - related outcomes assessed are partly explained by variation in avoidant coping and politically oriented coping .", "significant indirect effects are found for the pathways from economy - related stress to both past - month drinking and problematic drinking . of the total effect of economy - related stress on past - month drinking ( 0.071 ) ,", "0.047 is accounted for by the indirect effect of economy - related stress through avoidant coping and 0.024 is explained by the indirect influence of politically oriented coping .", "a similar pattern emerges with respect to the relationship between economy - related stressors and problematic drinking .", "the indirect effects of avoidant coping account for 0.046 of the total effect of economic stressors on problematic drinking ( 0.098 ) and politically oriented coping explains 0.032 .", "taken together , these findings indicate that both adaptive and maladaptive coping strategies come to bear on drinking patterns associated with economic strain . on the one hand , economic strain appears to be associated with drinking more and more problematically , in the extent to which it is associated with a tendency to engage in avoidant coping strategies .", "but , then , again , economic strain is also associated with greater politically oriented coping , which is protective against alcohol use and misuse .", "moderation tests next determine whether the mediating effects of the coping strategies investigated vary by gender .", "however , the mediating effect of avoidant coping for the economy - related stressor problematic drinking association differs for men and women in this sample . this effect is displayed in figure 4 , which presents the predicted pattern of gender contrasts in the effects of avoidant coping on problematic drinking based on the mean , plus and minus two standard deviation values of avoidant coping ( as displayed in table 1 ) .", "as shown in figure 4 , the mediating effects of avoidant coping differ for men and women because the relationship between avoidant coping and problematic drinking is significantly less strong for women compared to men (  = 0.319 , s.e .", "thus , the observation that economic strain is associated with greater problematic drinking in the extent to which it is associated with avoidant coping strategies appears to be more pronounced among men than among women .", "the findings from this study support a broadened conceptualization of modes of coping in stress paradigm - oriented research to encompass politically oriented coping , in addition to modes of coping outside of the political realm which have predominated in studies of the coping - related moderators or mediators of the associations between social stressors and drinking outcomes .", "moreover , given our focus on economic stressors deriving , at least in part , from the macrolevel social forces producing the great recession , we suggest that politically oriented coping is particularly salient as a mode of behavioral adaptation in relation to societally engendered stressors as opposed to stressors that are less affected by macrolevel social forces .", "while this study specifically addressed deleterious drinking consequences of the recent great recession , social scientists have also delineated broader social - structural and political forces occurring over the last three decades which have led to pervasive job insecurity across all sectors of the us workforce and the erosion in the standard of living for most of the population [ 4143 ] .", "these phenomena include globalization and the outsourcing of work , the downsizing of corporate entities , the shift from secure semiskilled industrial jobs which paid a living wage to low wage service sector jobs , an increase in contingent workers with lower pay , and lack of job security and fringe benefits . thus , politically oriented coping encompassing the goal of changing governmental policies ( e.g. , rallying for changes in the tax structure influencing the distribution of wealth throughout society , fighting for government stimulus policies oriented toward job creation , or rallying support for raising the government - mandated minimum wage ) may prove to be a more efficacious mode of coping with economic stressors and more protective against deleterious drinking outcomes compared to nonpolitically oriented active coping activities such as looking for a job when unemployed , especially if adequate numbers of jobs relative to demand do not exist and a large proportion of jobs that do exist pay less than a living wage .", "consistent with our gender - linked hypothesis , our data showed that males and females did not differ either in the use of politically oriented coping or in the extent to which politically oriented coping was protective in relation to drinking outcomes .", "however , our data showed that male but not female avoidant coping significantly predicted problem drinking .", "thus , with regard to nonpolitically oriented coping with economic stressors , males clearly utilized a coping mode that was maladaptive .", "this finding might be seen as congruent with early male socialization patterns which have been viewed as fostering men 's sense of self - importance rather than connectedness in social relationships   insofar as men 's avoidance in dealing with economic problems may affect both themselves and others close to them .", "alternatively , avoidant coping may predict problem drinking in men but not women to the extent to which males are socialized to drink more heavily than women .", "our findings should be viewed within the context of the methodological limitations of this study .", "the politically oriented coping measure which we developed for this study represents an initial attempt to operationalize this concept .", "however , it is limited to 4 items in contrast to the much longer nonpolitically oriented coping instrument and does not differentiate politically oriented coping tactics into discrete modes , similar to the measurement of nonpolitically oriented coping .", "although factor analysis supported the inclusion of the indicators of politically oriented coping as a single measure , additional work on the concept of politically oriented coping would be useful , with a differentiation between alternative types of politically oriented coping .", "first , the differentiation between individual modes of political action ( e.g. , voting , signing petitions , and donating money ) and collective political activities ( participation and leadership roles in different types of political action groups ) would be useful .", "secondly , some types of collective coping could be viewed as relatively more adaptive versus maladaptive .", "for example , the tea party social movement was motivated by both economic and cultural concerns [ 45 , 46 ] .", "however , one might differentiate between the tea party articulation of problem - focused social policies such as its belief in the need to diminish the size of government and racially oriented collective venting , such as screams of  kill him  by audience members in response to sarah palin 's critique of barak obama at rallies during the 2008 presidential election .", "second , this study utilized cross - sectional data and , thus , likely provides only a snapshot of the complex processes linking economy - related stressors , coping strategies , and drinking outcomes . while our theoretical framework postulated that exposure to economic stressors coupled with particular modes of coping leads to problematic drinking", ", it is possible that problem drinking coupled with maladaptive coping might make one prone to experiencing economic stressors such as losing one 's job .", "moreover , we lack data regarding alcohol consumption prior to exposure to the stressors focused on in this study and the extent to which alcohol may have been used as a coping mechanism . thus ,", "further longitudinal studies are necessary to more clearly delineate the causal directions of the relationships between economic stressors , modes of coping , and drinking outcomes .", "third , the study methodology encompassed random - digit - dialing for recruiting the sample , thus only reaching individuals with landline telephone numbers .", "consequently , individuals relying on cell phones only , along with households without access to any telephone , were not included in this study .", "this potential noncoverage error is a source of concern because comparisons of our data with the us population revealed that the sample underrepresented african americans , latinos , and younger ( < age 40 ) and less - educated ( high school or less ) persons .", "however , our data were weighted to reflect the demographics of the overall population , and we compared weighted and unweighted estimates of each of our dependent variables to determine if nonresponse and/or noncoverage may have introduced serious bias into one or more of them . in each instance , we found that the weighted values of each measure fell well within 1  sd of the unweighted values , suggesting that the distributions of our key measures were not appreciably influenced by the underrepresentation of particular demographic groups .", "despite the noted limitations , this study extends prior research on the moderators and mediators of the social stressor - drinking outcome relationships to broaden notions of coping to include politically oriented coping .", "future studies incorporating this mode of coping may more clearly elucidate the political dynamics involved in both the macrolevel production of social stressors and the deleterious alcohol - related consequences of these stressors .", "this line of research would also have implications for the treatment of alcohol - related problems .", "in particular , considerations of more adaptive modes of coping might go beyond recommending individual behaviors such as job seeking by unemployed individuals to also suggest politically oriented coping to collectively try to influence the social conditions such as unemployment levels that may give rise to the propensity to self - medicate distress through the use of alcohol ."], "abstract_text": ["<S> research derived from the stress paradigm suggests that certain types of coping ( e.g. , problem - focused coping instead of behavioral disengagement ) are protective against problem - related drinking to deal with social stressors . </S>", "<S> going beyond the typical focus in the coping literature , we hypothesize that stressors engendered by macrolevel social forces may require coping actions within the political realm in contrast to modes of coping focused outside of the political realm . </S>", "<S> a united states sample of 663 respondents completed a mail survey in 2010 , including measures of stressful consequences of the great recession , drinking patterns and problems , modes of coping encompassed in the brief cope instrument , and politically oriented coping . </S>", "<S> structural equation modeling examined whether modes of coping mediated the links between stressors and drinking outcomes . </S>", "<S> a substantial portion of the associations between stressors and drinking was explained by modes of coping . </S>", "<S> politically oriented coping was protective against problem drinking for both genders . </S>", "<S> future studies should further explore politically oriented coping in addition to modes of coping outside of the political realm when studying the relationships between macrolevel social stressors and deleterious drinking outcomes . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods ", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["social scientists who explore factors mediating and moderating the relationships between social stressors and mental health , including drinking outcomes , have highlighted modes of coping [ 1 , 2 ] . these studies have explored behaviors which protect people from being psychologically harmed   and cognitive appraisals which influence behaviors such as problem - focused coping   or using alcohol to self - medicate distress .", "however , studies have not considered the characteristics of the stressful situation itself that may make certain coping strategies more or less effective . in particular ,", "psychiatric epidemiologic studies have tended to emphasize microlevel stressors ( e.g. , stressors in individuals ' role domains ) and , until recently , have ignored the linkages between macrolevel social forces and the daily stressors in people 's lives [ 79 ] .", "however , macrolevel social conditions can affect the magnitude of stressors experienced in people 's lives and the extent to which they experience  cumulative adversity  .", "this paper focuses on coping with the fallout from one type of macrolevel social stressor : the recent great recession .", "this economic downturn constituted the most severe economic crisis in the united states since the great depression   and had persisting economic effects ( e.g. , job loss , less desirable working conditions , loss of home , loss of retirement savings , lack of health care access , and social isolation ) which have been linked with deleterious drinking outcomes .", "a key issue involving the effectiveness of alternative modes of coping with stressors derived from macrolevel social forces and protecting against deleterious drinking outcomes is the question of whether individual modes of coping outside of the political realm emphasized in the overall coping literature are most efficacious . or , alternatively , do stressors engendered by macrolevel social forces require unique forms of coping , that encompass the political realm ?", "with respect to coping strategies employed in the wake of the great recession , politically oriented coping strategies might be of particular relevance due to the impact of governmental decision - making on the state of the economy .", "politically oriented coping strategies might include political activism oriented to altering economic policies or support for campaigns by politicians offering solutions to economically based hardships . scholarly work on the occupy wall street movement , for example , documents involvement by individuals sharing their own economic struggles   and collectively gathering to protest their precarious economic situation and uncertain economic future .", "earlier work also suggests the salience of activities oriented toward changing politically based social realities such as through the act of voting   or by collectively challenging community - level decisions such as school closings .", "by contrast , the traditional coping literature has emphasized individual , nonpolitical modes of coping , such as emotional acceptance of the stressful situation , blaming one 's self for the situation , or taking individual actions such as looking for a job if unemployed .", "while sociologists have recently accorded greater attention to the prevalence and alcohol - related consequences of macrolevel stressors , they have yet to address the extent to which politically oriented modes of coping may be the most efficacious ways to address problems stemming from macrolevel social forces or events . prior to the more recent focus on macrolevel stressors , kaplan and liu   embraced the idea of collective coping as one means for individuals who maintained stigmatized personal identities to challenge and transform conventional socionormative systems through participation in social movements .", "subsequently , thoits   more explicitly addressed collective coping within the context of the stress paradigm involving acute and chronic stressors not limited to the specific area of stigmatized statuses .", "she argued that individuals who find themselves in problematic situations can deliberately work to transform the meaning of their experiences and they can additionally use these experiences  as a basis for helping or effecting changes in the lives of others ", "she proposed the concept of  transformatory coping  to include engagement in collective activist activities with others who share similar problems .", "for example , parents of autistic children have lobbied governments for social services perceived to aid in their children 's development . in sum , ( 1 ) collective coping tactics ( such as politically oriented coping ) represent an unmeasured dimension of coping behaviors beyond that represented in the coping literature to date and ( 2 ) collective coping tactics may demonstrate a stronger association between stressors , particularly those stemming from macrolevel social forces , and deleterious drinking outcomes compared to the use of modes of coping previously emphasized in the literature on coping .", "the present study extends previous work by empirically addressing the extent to which politically oriented coping activities engaged in as a response to a macrolevel social stressor , the great recession , are protective against alcohol - related outcomes compared with coping strategies focused outside of the political realm .", "we hypothesize that politically oriented coping will be more protective against economic stressors linked with the great recession than nonpolitical modes of coping and will uniquely account for some portion of the associations between economy - related stressors and drinking outcomes .", "further , we also examine whether politically oriented coping and coping outside of the political realm are more protective for men versus women in the face of macrolevel engendered stressors such as those involving the economic fallout from the great recession .", "there is consistent evidence that women are more likely to use support - based coping strategies ( e.g. , seeking support from others such as partner family and friends ) in response to stress in contrast to men and some indication that avoidant coping techniques are associated with greater alcohol consumption among men but not women [ 1 , 2022 ] .", "( in contrast , parity by gender in the use of individual active coping strategies and their significance for mental health outcomes is generally reported [ 1 , 21 ] . ) however , whether there is a corresponding propensity for men and women to differ in the use of politically oriented coping to offset the alcohol - related effects of economy - related stressors is less certain .", "earlier research tended to argue that women were less politically interested , informed , and efficacious compared to men .", "more recent work has shown that women and men differ in particular modes of participation ; women are more likely to vote and engage in individual political actions such as signing petitions or donating money , whereas men are more likely to be engaged in collective forms of action such as group protest activities .", "thus , we hypothesize that there will be no overall differences in the extent to which women and men manifest politically oriented coping or in the effect that politically oriented coping has on drinking outcomes . following a transactional model of stress", "[ 4 , 25 ] , we model coping as a mediator of the relationship between the stressor ( i.e. , stressful consequences of the great recession ) and the stress response ( i.e. , drinking outcomes ) ."], ["data were derived from a study conducted in the united states between june , 2010 , and january , 2011 , that was undertaken in order to understand life change consequences of the major downturn in the economy known as the great recession .", "respondents were selected by a random digit dial ( rdd ) phone survey of the continental united states , and those who consented to participate in the study were mailed questionnaires .", "eligible respondents were selected from the households using the troldahl - carter - bryant method of respondent selection which involves the means to randomly select a respondent from all eligible household members .", "respondents were told during the phone screen that a $ 50 american express gift card would be sent to the eligible respondent if he or she completed the questionnaire .", "respondents were mailed an initial survey , a postcard reminder to nonresponders , and a second questionnaire if they still had not responded .", "figure 1 encompasses a flow chart characterizing each stage in the data collection process through the completion of the questionnaires .", "65.9% ( n = 663 ) of the respondents completing the screening calls returned the questionnaire .", "the telephone screening cooperation rate and the mail survey response rate were each calculated using the conservative aapor response rate formula 3 .", "the overall survey response rate is the product of the phone screening cooperation rate ( 35.5% ) and the mail questionnaire return response rate ( 65.9% ) or 16.8% .", "we acknowledge that this response rate is less than ideal and further address this issue in the discussion of study limitations and note other indicators of the representativeness of the final sample .", "selection weights were calculated for each of the cases to weight for the different probability of selection for each case .", "poststratification weights were calculated for the dataset to ensure that the distribution of sample cases on important demographic variables ( age , race / ethnicity , and gender ) conformed to the distribution of these variables in census bureau 's 2008 united states population estimates .", "it should be noted that estimates of alcohol consumption for the present sample did appear to conform to national estimates preweighting .", "for example , the average number of drinks consumed in the past month on days when one drank for the present sample is 2.16 , versus the estimated average of 2.10 reported by the centers for disease control and prevention 's behavioral risk factor surveillance system for 2010   ( by gender , the averages are 2.35 for men and 1.89 for women in the present sample versus 2.43 for men and 1.81 for women in the cdc estimates ; by race / ethnicity , the averages are 2.16 for non - hispanic whites , 2.08 for asians , 2.12 for african americans , and 2.72 for hispanics in the present sample versus 2.26 for non - hispanic whites , 2.41 for asians , 2.19 for african americans , and 2.68 for hispanics in the cdc data ) .", "it should additionally be noted that the respondents included in this sample reported an overall higher level of education than the general population based on 2008 census estimates .", "analysis of variance revealed no significant variation by education in either the outcomes or support coping , avoidant coping , or politically oriented coping .", "respondents with less than a high school degree are found to be marginally less likely ( p < 0.10 ) to use active coping strategies compared to those with a college degree or postcollege training . given this discrepancy as well as the fact that education is generally protective against problem drinking", "predictor variables are economy - related stressors , coping strategies enacted outside of the political realm ( i.e. , active coping , support coping , and avoidant coping ) , politically oriented coping , and gender . the sociodemographic characteristics of age , education , and race / ethnicity are controlled in all analyses . to assess past - month drinking patterns , we use the quantity - frequency - variability index ( qfv ) developed by cahalan et al . .", "frequency of drinking is measured as a count of the days on which alcohol was consumed in the past 30 days , and quantity of drinking is measured as the average number of drinks consumed on those days .", "variability is calculated by the greatest number of drinks consumed on any one day in the past 30 days .", "scores are calculated by multiplying responses to the quantity , frequency , and variability questions (  = 0.87 ) . as with the current data", ", this index tends to approximate a continuous scale , and ample evidence supports its use as such ( see fitzgerald and mulford   for a review ) .", "our measure of problematic drinking is the 10-item bmast (  = 0.74 ) , which is a count measure of difficulties related to alcohol use over the past year .", "respondents were asked to indicate  yes  or  no  in response to 10 items such as having an accident , losing a close friend , spouse , or loved one , being hospitalized , having trouble at work , and soliciting professional help because of one 's drinking . the bmast is one of the most widely used tools for assessing alcohol dependence and problems .", "it correlates strongly with the full - length mast and evidence of its reliability and validity is widely available [ 31 , 32 ] .", "moreover , we are not using the bmast as a diagnostic tool for depicting  problem  versus", " nonproblem  drinking , but as it is intended , as suggestive of different degrees of problematic drinking .", "the measure of economy - related stressors is the life change consequences of the great recession ( lccgr ) instrument .", "this construct was developed on the basis of qualitative analyses of transcripts derived from focus groups involving both genders and diverse racial / ethnic groups .", "the items fall into seven categories : home ownership problems , such as difficulties in mortgage payments , difficulties in paying property taxes , or a drop in credit rating ; undesirable living situation , including having to live in a less desired location to save money or having gas and electricity or heat shut off due to an inability to pay bills ; problematic employment situation , including a pay - cut , furlough days , and increased feelings of competition with fellow employees ; unemployment or underemployment ; inadequate health insurance , including lack of medical or dental coverage , decreased quality of coverage , and inability to obtain coverage ; social role constraints , such as dissolution of spouse / partner relationship , decreased social life , and increased social isolation due to finances ; and inadequate sick time , including inadequate sick days and having to work despite poor health .", "consistent with common practice , each score for this measure is a straight count of the number of stressors reported .", "three dimensions of nonpolitical modes of coping are assessed : active coping , support coping , and avoidant coping .", "these measures of coping are derived from subscales of the brief cope instrument   and have previously been validated as stand - alone indices in community samples [ 3436 ] .", "participants in the present study were asked whether they have used these coping strategies in response to the economic recession .", "confirmatory factor analysis of the present data supports the inclusion of these items as three separate coping indices , consistent with prior research .", "active coping (  = 0.84 ) includes eight items measuring acceptance , positive reframing , and planning and taking action in response to the economic recession .", "support coping (  = 0.81 ) includes four items measuring use of emotional and instrumental support ( i.e. , receiving emotional support and getting advice and help from other people ) .", "avoidant coping (  = 0.75 ) includes 10 items measuring self - distraction , behavioral disengagement , self - denial , blame , and a tendency to vent about or make fun of the situation . all items were rated on a four - point likert - type scale ranging from 0 ( i did not do this at all ) to 3 ( i did this a lot ) . the measure of politically oriented coping", "is assessed by a four - item instrument (  = 0.79 ) drawn from the summed responses ( i.e. , not at all , a little , some , quite a bit , and a lot ) to four statements asking how often respondents , in response to the economic recession , have been ( 1 )  engaging in political activities such as signing petitions , leading or participating in rallies or marches , or writing to political representatives ;  ( 2 )  organizing with others to challenge politicians currently in office ;  ( 3 )  voting in elections to support politicians who share your political beliefs ;  and ( 4 )  participating in groups trying to influence the policies of the government at the local , state , or national level . ", "the questions used to construct this index were derived from analyses of focus group transcripts ( see   for details about these focus groups ) .", "confirmatory factor analysis reveals that these items load on a single factor , supporting their inclusion as one index .", "education is a categorical variable based on the educational attainment categories of ( 1 ) less than high school ( n = 45 ) ; ( 2 ) high school graduate ( n = 350 ) ; ( 3 ) college graduate ( n = 110 ) ; and ( 4 ) postcollege training ( n = 150 ) . race / ethnicity is a dummy variable including non - hispanic whites ( n = 436 ) , african americans ( n = 80 ) , hispanics ( n = 91 ) , asians ( n = 29 ) , and individuals who identify as an  other  race / ethnicity ( n = 17 ) . in all analyses ,", "non - hispanic whites serve as the reference category . after examining bivariate correlations in order to assess the basic patterns of association among key study variables , we performed structural equation modeling ( sem ) using mplus software   to examine the predictive significance of economy - related stressors for coping outside of the political realm and politically oriented coping tactics and the two drinking outcomes considered ( i.e. , past - month drinking and problematic drinking ) , net of the sociodemographic control variables .", "we considered the potential for nonpolitical coping and politically oriented coping tactics to mediate the associations between economy - related stressors and each of the outcomes assessed in two models .", "the first model tested for associations between economic stressors and the drinking - related outcomes .", "the second model adds nonpolitical and politically oriented coping tactics to test the full mediation model .", "this latter model assesses all of the direct and indirect paths between economic stressors and the drinking outcomes considered through the nonpolitical and politically oriented coping tactics investigated .", "we formally tested for mediation using the procedures described by muthen and muthen   for mplus software , which apply the tests described by mackinnon et al . .", "finally , because the associations between social stress and drinking are found to vary by gender [ 33 , 39 ] , we examined whether any observed mediating effects of the coping strategies investigated vary by gender . for these tests ,", "separate equations include the interaction term for gender by each coping strategy in the path models linking coping with drinking outcomes ."], ["it is noteworthy that stressors related to the economy are associated with each of the alcohol - related outcomes and all of the coping resources considered : economy - related stressors are associated with more alcohol consumption and problematic drinking , as well as higher levels of active coping , support coping , avoidant coping , and politically oriented coping .", "it is also noteworthy that only two of the coping strategies assessed are associated with the drinking outcomes .", "avoidant coping and politically oriented coping are associated with both alcohol consumption and problematic drinking , but in opposite directions .", "that is , greater avoidant coping is associated with greater alcohol consumption and problematic drinking , whereas greater politically oriented coping is associated with less alcohol consumption and problematic drinking .", "the lack of correlation between active coping and support coping and each of the drinking outcomes , respectively , provides some indication that not all of the coping strategies may be useful in understanding the associations between economic stressors and drinking - related outcomes . additionally , the possibility that coping resources may vary by gender is not strongly supported by the pattern of correlations reported , with the exception that women reported significantly more emotional support than men . however , and consistent with previous research , women are found to drink less and less problematically .", "the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes are further elaborated upon in the structural equation model .", "estimation of the first model ( figure 2 ) , including only economic stressors , the drinking - related outcomes , and sociodemographic controls , produces a just identified model and , as such , meaningful fit statistics are not provided .", "the standardized path coefficients demonstrate that economic stressors and each of the drinking - related outcomes considered are significantly and positively related .", "net of the sociodemographic controls , greater economic strain is associated with greater alcohol consumption over the past month (  = 0.094 , s.e .", "= 0.004 , and p < 0.01 ) and a higher incidence of problematic drinking over the past year (  = 0.181 , s.e .", "sem analysis testing the second model , of the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes considered , is presented as figure 3 . in figure 3 ,", "solid lines indicate effects that are statistically significant , and dashed lines indicate effects that are not statistically significant .", "the model fit criteria provided by hu and bentler   ( cfi > 0.95 ; rmsea < 0.06 ; srmr < 0.08 ) are used to assess the measurement model . based on these criteria , there is consistent evidence of good fit for this model ( cfi = 0.982 ; rmsea = 0.046 ;", "this model demonstrates that economic stress is associated with higher levels of each of the coping resources considered .", "that is , greater economic strain appears to predict a greater propensity to use both maladaptive coping tactics ( i.e. , avoidant coping ) and adaptive coping tactics ( i.e. , active coping , support coping , and politically oriented coping ) .", "avoidant coping is associated with higher levels of past - month drinking and problematic drinking , whereas politically oriented coping is associated with less alcohol consumption and problematic drinking .", "formal mediation tests next reveal that the effects of economy - related stressors on the alcohol - related outcomes assessed are partly explained by variation in avoidant coping and politically oriented coping .", "significant indirect effects are found for the pathways from economy - related stress to both past - month drinking and problematic drinking . of the total effect of economy - related stress on past - month drinking ( 0.071 ) ,", "0.047 is accounted for by the indirect effect of economy - related stress through avoidant coping and 0.024 is explained by the indirect influence of politically oriented coping .", "a similar pattern emerges with respect to the relationship between economy - related stressors and problematic drinking .", "the indirect effects of avoidant coping account for 0.046 of the total effect of economic stressors on problematic drinking ( 0.098 ) and politically oriented coping explains 0.032 .", "taken together , these findings indicate that both adaptive and maladaptive coping strategies come to bear on drinking patterns associated with economic strain . on the one hand , economic strain appears to be associated with drinking more and more problematically , in the extent to which it is associated with a tendency to engage in avoidant coping strategies .", "but , then , again , economic strain is also associated with greater politically oriented coping , which is protective against alcohol use and misuse .", "moderation tests next determine whether the mediating effects of the coping strategies investigated vary by gender .", "however , the mediating effect of avoidant coping for the economy - related stressor problematic drinking association differs for men and women in this sample . this effect is displayed in figure 4 , which presents the predicted pattern of gender contrasts in the effects of avoidant coping on problematic drinking based on the mean , plus and minus two standard deviation values of avoidant coping ( as displayed in table 1 ) .", "as shown in figure 4 , the mediating effects of avoidant coping differ for men and women because the relationship between avoidant coping and problematic drinking is significantly less strong for women compared to men (  = 0.319 , s.e .", "thus , the observation that economic strain is associated with greater problematic drinking in the extent to which it is associated with avoidant coping strategies appears to be more pronounced among men than among women ."], ["the findings from this study support a broadened conceptualization of modes of coping in stress paradigm - oriented research to encompass politically oriented coping , in addition to modes of coping outside of the political realm which have predominated in studies of the coping - related moderators or mediators of the associations between social stressors and drinking outcomes .", "moreover , given our focus on economic stressors deriving , at least in part , from the macrolevel social forces producing the great recession , we suggest that politically oriented coping is particularly salient as a mode of behavioral adaptation in relation to societally engendered stressors as opposed to stressors that are less affected by macrolevel social forces .", "while this study specifically addressed deleterious drinking consequences of the recent great recession , social scientists have also delineated broader social - structural and political forces occurring over the last three decades which have led to pervasive job insecurity across all sectors of the us workforce and the erosion in the standard of living for most of the population [ 4143 ] .", "these phenomena include globalization and the outsourcing of work , the downsizing of corporate entities , the shift from secure semiskilled industrial jobs which paid a living wage to low wage service sector jobs , an increase in contingent workers with lower pay , and lack of job security and fringe benefits . thus , politically oriented coping encompassing the goal of changing governmental policies ( e.g. , rallying for changes in the tax structure influencing the distribution of wealth throughout society , fighting for government stimulus policies oriented toward job creation , or rallying support for raising the government - mandated minimum wage ) may prove to be a more efficacious mode of coping with economic stressors and more protective against deleterious drinking outcomes compared to nonpolitically oriented active coping activities such as looking for a job when unemployed , especially if adequate numbers of jobs relative to demand do not exist and a large proportion of jobs that do exist pay less than a living wage .", "consistent with our gender - linked hypothesis , our data showed that males and females did not differ either in the use of politically oriented coping or in the extent to which politically oriented coping was protective in relation to drinking outcomes .", "however , our data showed that male but not female avoidant coping significantly predicted problem drinking .", "thus , with regard to nonpolitically oriented coping with economic stressors , males clearly utilized a coping mode that was maladaptive .", "this finding might be seen as congruent with early male socialization patterns which have been viewed as fostering men 's sense of self - importance rather than connectedness in social relationships   insofar as men 's avoidance in dealing with economic problems may affect both themselves and others close to them .", "alternatively , avoidant coping may predict problem drinking in men but not women to the extent to which males are socialized to drink more heavily than women .", "our findings should be viewed within the context of the methodological limitations of this study .", "the politically oriented coping measure which we developed for this study represents an initial attempt to operationalize this concept .", "however , it is limited to 4 items in contrast to the much longer nonpolitically oriented coping instrument and does not differentiate politically oriented coping tactics into discrete modes , similar to the measurement of nonpolitically oriented coping .", "although factor analysis supported the inclusion of the indicators of politically oriented coping as a single measure , additional work on the concept of politically oriented coping would be useful , with a differentiation between alternative types of politically oriented coping .", "first , the differentiation between individual modes of political action ( e.g. , voting , signing petitions , and donating money ) and collective political activities ( participation and leadership roles in different types of political action groups ) would be useful .", "secondly , some types of collective coping could be viewed as relatively more adaptive versus maladaptive .", "for example , the tea party social movement was motivated by both economic and cultural concerns [ 45 , 46 ] .", "however , one might differentiate between the tea party articulation of problem - focused social policies such as its belief in the need to diminish the size of government and racially oriented collective venting , such as screams of  kill him  by audience members in response to sarah palin 's critique of barak obama at rallies during the 2008 presidential election .", "second , this study utilized cross - sectional data and , thus , likely provides only a snapshot of the complex processes linking economy - related stressors , coping strategies , and drinking outcomes . while our theoretical framework postulated that exposure to economic stressors coupled with particular modes of coping leads to problematic drinking", ", it is possible that problem drinking coupled with maladaptive coping might make one prone to experiencing economic stressors such as losing one 's job .", "moreover , we lack data regarding alcohol consumption prior to exposure to the stressors focused on in this study and the extent to which alcohol may have been used as a coping mechanism . thus ,", "further longitudinal studies are necessary to more clearly delineate the causal directions of the relationships between economic stressors , modes of coping , and drinking outcomes .", "third , the study methodology encompassed random - digit - dialing for recruiting the sample , thus only reaching individuals with landline telephone numbers .", "consequently , individuals relying on cell phones only , along with households without access to any telephone , were not included in this study .", "this potential noncoverage error is a source of concern because comparisons of our data with the us population revealed that the sample underrepresented african americans , latinos , and younger ( < age 40 ) and less - educated ( high school or less ) persons .", "however , our data were weighted to reflect the demographics of the overall population , and we compared weighted and unweighted estimates of each of our dependent variables to determine if nonresponse and/or noncoverage may have introduced serious bias into one or more of them . in each instance , we found that the weighted values of each measure fell well within 1  sd of the unweighted values , suggesting that the distributions of our key measures were not appreciably influenced by the underrepresentation of particular demographic groups ."], ["despite the noted limitations , this study extends prior research on the moderators and mediators of the social stressor - drinking outcome relationships to broaden notions of coping to include politically oriented coping .", "future studies incorporating this mode of coping may more clearly elucidate the political dynamics involved in both the macrolevel production of social stressors and the deleterious alcohol - related consequences of these stressors .", "this line of research would also have implications for the treatment of alcohol - related problems .", "in particular , considerations of more adaptive modes of coping might go beyond recommending individual behaviors such as job seeking by unemployed individuals to also suggest politically oriented coping to collectively try to influence the social conditions such as unemployment levels that may give rise to the propensity to self - medicate distress through the use of alcohol ."]]}
{"article_id": "PMC4327281", "article_text": ["the development of titanium fixtures has brought several benefits for the rehabilitation of edentulous patients . when biological and mechanical principles are respected", ", this treatment modality may successfully restore the functional and esthetic impairments caused by tooth loss3 . in spite of the significant evolution of a number of implant systems , implant design and features , such as those related to the mechanical behavior of implant - supported prostheses ,", "dental prostheses do fail during function mainly due to abutment and prosthesis screw loosening and/or fracture .", "in addition , it has been reported that abutment screw loosening is only surpassed by loss of osseointegration as the main cause of failure on implant - supported restorations , as shown in longitudinal follow - up studies5 . when two metal surfaces are in contact , adhesion and friction forces do limit the movement between them .", "an applied method aimed to reduce this friction and improve adhesion consists of interposing a lubricating film between these surfaces .", "a metal with low shear strength , such as pure gold , may act as a dry lubricant .", "when compared to screws without gold coating , it has been found that gold - coated abutment screws subjected to torques of 12 , 20 , and 32 ncm aiming a 0.0064 mm opening of the implant - abutment interface presented 26 , 24 , and 24% of preload increase , respectively7 . in another study , it was found that when a torque force is applied to an abutment screw , a significant part of this force is lost due to friction between the contact points of metal surfaces , inhibiting the rotation of the screw .", "thus , decreasing of friction between metallic surfaces may increase the screw rotation and , consequently , the preload .", "the rotation of gold - coated abutment screws placed with torque forces of 12 , 20 , and 32 ncm increased 73 , 76 , and 62% , respectively , when compared to titanium abutment screws2 .", "the purpose of this study was to evaluate by strain gauges the preload and torque removal values on three abutment screws ( gold , titanium , and titanium with surface treatment screws ) applying an equivalent torque force ( 30.070.28 ncm ) .", "three machined self - tapping external hex titanium screws with 4 mm in diameter and 15 mm in length were used in this investigation ( master screw , conexo sistemas de prtese ltda .", "three transmucosal cera one type abutments ( 2.0 mm height , cera one , conexo sistemas de prtese ltda . ,", "reference 045022 , batch # 5080915121 ) , designed for cemented single standing implant - supported restorations were also used .", "the transmucosal abutments were attached to the fixtures using 10 gold screws ( conexo , sistemas de prtese ltda .", ", reference 121022 , batch # 5073147 ) ( figure 1 a a ) , 10 titanium screws ( conexo sistemas de prtese ltda .", ", reference 121024 , batch # 5063223 ) ( figure 1a b ) , and 10 surface treated titanium screws ( ti - tite , conexo sistemas de prtese ltda . , reference 121026 , batch # 5063035 ) ( figure 1a c ) . a load cell adapted from a model described in the literature", "this load cell presented a central void for fixture fixation in its upper portion and a horizontal plate for fixation of four strain gauges on its lower portion ( figure 1 b ) .", "the abutments were placed on the upper plate of the cell ( figure 1 c ) in such way that the contact between abutments and fixtures was free of any interference .", "therefore , when the abutment was fixed to the fixture by the abutment screw , tensions generated by this fixation pulled the unit against the abutment , producing a deformation in the lower plate connected to the strain gauges .", "next , the force ( preload ) generated on the abutment screws was captured by the strain gauges in volts ( v ) and later converted to newton ( n ) .", "the aforementioned sample was arranged into 3 different groups according to the characteristics of each abutment screw : group a was formed by gold screws , group b was formed by titanium screws , and group c was formed by surface - treated titanium screws . to determine fixation and removal torques ,", "a digital torquemeter was used ( torqueleader , model tsd150 , type i , class e , part # 117317 ) , previously calibrated according with iso 6789:2003 ( e ) standard . a square hand wrench ( 1.27 mm diameter , conexo ,", ", reference 062300 , batch # 5091632 ) was attached to the torquemeter to allow proper connection between torquemeter and screws ( figure 1 d ) . following the proper assembly of fixture and abutment on group a", ", the gold screw was then attached to the fixture with an applied torque force of 30.070.28 ncm , being repeated afterwards for groups b and c. then , the initial preload value for the abutment screws was determined ( in volts ) and all screws were kept in their positions for 5 minutes for preload stabilization , according to the literature9 . during this the preload stabilization period ,", "preload values were obtained after 1 , 2 , 3 , 4 , and 5 minutes after fixation , and thereafter a mean value was calculated .", "after this evaluation , the abutment screw was removed using a digital torquemeter and the maximum value of reverse torque force required for screw removal was recorded in ncm .", "next , the same screw was fixed again another 4 times , obtaining the preload and torque removal mean values .", "nine other screws from group a as well as 10 other screws from b and c groups were subjected to the same procedure described above .", "the values of fixation torque forces of the 3 groups were analyzed statistically by to one - way analysis of variance ( anova ) .", "comparison of preload values within groups was possible using the mean of 6 measurements ( 0 to 5 minutes ) obtained at the first abutment fixation , using two - way anova .", "one - way anova was used to compare the mean preload values among the groups .", "tukey 's test was used for multiple comparisons among the groups . for all statistical tests ,", "all fixations for each screw ( n=5 ) were considered for evaluation of the torque removal to obtain an average value , and the statistical analysis was performed similarly to the preload data .", "two - way anova was used to evaluate the mean torque removal among the groups , and one - way anova to evaluate the differences between screws .", "tukey 's multiple comparison test was used for individual comparisons among the groups . for all statistical tests ,", "the mean fixation torque force when all screws were considered was 30.070.28 ncm ( c.i .", "there were no statistically significant differences ( p=0.1244 ) among the groups regarding the fixation torque .", "the gold screws presented the highest preload values and the titanium screws the lowest values ( figure 2 ) .", "the mean preload values of the gold screws was 131.728.98 n , and presented statistically significant differences ( p<0.01 ) compared to groups", "b and c. screw # 3 and screw # 1 presented the highest ( 140.48 n ) and the lowest ( 117.73 n ) preload values , respectively . the titanium screws presented statistically significant differences ( p<0.01 ) ( mean 37.035.69 n ) .", "the highest preload value was obtained for screw # 7 ( 49.68 n ) , while screw # 3 presented the lowest value ( 25.30 n ) . in the group of surface - treated titanium screws ,", "the mean preload value was 97.784.68 n. statistically significant differences ( p<0.01 ) were also observed in this group , ( maximum : 104.09 n for screw # 4 ; minimum : 90.28 n for screw # 2 ) . the removal torque presented statistically significant differences among the analyzed materials ( p<0.001 ) ( figure 3 ) .", "the gold screws presented a mean removal torque of 17.641.12 ncm , i.e. they did not shown statistically significant differences ( p=0.3713 ) .", "titanium screws presented a mean removal torque of 18.751.89 ncm , and showed statistically significant differences ( p<0.001 ) .", "the group of surface - treated titanium screws present statistically significant difference ( p=0.004 ) ( mean : 16.431.33 ncm ) .", "the results of this study are similar to those of previous investigations4,8 , which found statistically higher preload values for gold fixation screws in comparison to other tested materials , in spite of using different methodologies and materials .", "a maximum preload value of 666.4 n for gold screws and 458.2 n for titanium screws was reported8 .", "in addition , the finds of the present study suggest that the evaluated surface treatment of titanium screws was effective , since these screws presented higher preload values than conventional titanium screws .", "most previously published reports in the dental literature do not specify the removal torque of abutment screws .", "the effect of different cyclic loads has been evaluated using a 32 ncm torque for fixation of the screws , which was repeated after 10 minutes to avoid contact relaxation6 .", "several manufacturers have suggested this clinical approach to decrease screw loosening . in the present study , to allow a direct comparison between results of preload and torque removal for the studied groups , all screws received a similar fixation torque of30.070.28 ncm , despite the manufacturer 's recommendations of using 35 ncm torque for the ti - tite screws .", "this was performed to eliminate a possible bias caused by elastic deformation that might occur in titanium screws during fixation , which might not only quantitatively but qualitatively influence the study outcomes and impair comparison among groups .", "this approach allowed that , in this investigation , only the screw was left as a study variable .", "another relevant issue is that up to 10% of the initial preload may be lost to smooth contact surfaces ( embedment relaxation ) , rather than elongation stresses .", "however , it has been previously observed that when the same screw is fixed several times , its preload values increased4 .", "comparison to different results obtained in other studies might be a difficult task due to variations of tested products and variables that may influence the produced preload value , such as the elastic modulus of the screws , opposing joint surfaces , abutment design , friction coefficient , lubrication , rate of the applied torque and the adaptation between the fixture hexagon and the abutment2 .", "the findings of the present study suggest a trend for greater preload values verified for gold screws , followed by surface - treated titanium screws and titanium screws , confirming the outcomes of previous investigations4,8 . finite element analysis has been used to evaluate preloading through elongation of the abutment screw .", "a study evaluating the preload of two different systems of the same implant manufacturer in function , found that the optimal preload produced should be 75% of the screw yield stress , and also that the preload value increases dramatically as the friction coefficient between implant and screw decreases7 .", "as loosening of implant / abutment joint causes clinical problems , another question that may be raised is whether the torque values recommended by the manufacturers should be increased in order to obtain greater longevity of screw tightening .", "the preload produced by gold or titanium fixation screws can be evaluated through the measurement of elongation stresses .", "it has been found that the stresses were 60% lower than the strengths against the torques applied according to the manufacturer 's instructions4 . on the other hand", ", it has been reported that gold or titanium screws could support higher torques than those indicated by the manufacturers without presenting plastic deformations , although it has been recommended elsewhere that the stresses should not exceed 65% of the screw 's fracture strength1 .", "although the results of this study showed that gold screws have a clear superiority of the produced preload , application of cyclic loads would be required for a closer simulation of the masticatory function on implanted - supported restorations .", "it may be concluded : 1 . gold should be the material of choice for abutment fixation screws , since it produced the highest preload values , followed by surface - treated titanium screws and conventional titanium screws ; 2 .", "titanium screws presented the highest torque removal values , followed by gold screws , and surface - treated titanium screws ."], "abstract_text": ["<S> several authors still consider the mechanical problems of fracture and component loosening as the main causes of failure of implant - supported restorations . </S>", "<S> the purpose of this in vitro study was to compare the preload of three types of screw for transmucosal abutment attachment used in single implant - supported prosthesis through strain gauge and removal torque measurements . </S>", "<S> three external hex fixtures were used , and each received a transmucosal abutment ( cera one ) , which was fixed to the implant with its respective screw : group a- gold screw , group b- titanium screw and group c- surface - treated titanium screw ( ti - tite ) . </S>", "<S> ten screws of each type were attached applying a 30.070.28 ncm torque force and maintained in position for 5 minutes . </S>", "<S> after this , the preload values were measured using strain gauges and a measurement cell . </S>", "<S> gold screws presented higher preload values ( 131.728.98 n ) , followed by surface - treated titanium screws ( 97.784.68 n ) and titanium screws ( 37.035.69 n ) . </S>", "<S> anova ( p<0.05 ) and tukey 's test ( p<0.05 ) were applied . </S>", "<S> statistically significant differences were found among the groups for both preload and removal torque values . in conclusion </S>", "<S> , gold screws may be indicated to achieve superior longevity of the abutment - implant connection and , consequently , prosthetic restoration due to greater preload values yielded . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["the development of titanium fixtures has brought several benefits for the rehabilitation of edentulous patients . when biological and mechanical principles are respected", ", this treatment modality may successfully restore the functional and esthetic impairments caused by tooth loss3 . in spite of the significant evolution of a number of implant systems , implant design and features , such as those related to the mechanical behavior of implant - supported prostheses ,", "dental prostheses do fail during function mainly due to abutment and prosthesis screw loosening and/or fracture .", "in addition , it has been reported that abutment screw loosening is only surpassed by loss of osseointegration as the main cause of failure on implant - supported restorations , as shown in longitudinal follow - up studies5 . when two metal surfaces are in contact , adhesion and friction forces do limit the movement between them .", "an applied method aimed to reduce this friction and improve adhesion consists of interposing a lubricating film between these surfaces .", "a metal with low shear strength , such as pure gold , may act as a dry lubricant .", "when compared to screws without gold coating , it has been found that gold - coated abutment screws subjected to torques of 12 , 20 , and 32 ncm aiming a 0.0064 mm opening of the implant - abutment interface presented 26 , 24 , and 24% of preload increase , respectively7 . in another study , it was found that when a torque force is applied to an abutment screw , a significant part of this force is lost due to friction between the contact points of metal surfaces , inhibiting the rotation of the screw .", "thus , decreasing of friction between metallic surfaces may increase the screw rotation and , consequently , the preload .", "the rotation of gold - coated abutment screws placed with torque forces of 12 , 20 , and 32 ncm increased 73 , 76 , and 62% , respectively , when compared to titanium abutment screws2 .", "the purpose of this study was to evaluate by strain gauges the preload and torque removal values on three abutment screws ( gold , titanium , and titanium with surface treatment screws ) applying an equivalent torque force ( 30.070.28 ncm ) ."], ["three machined self - tapping external hex titanium screws with 4 mm in diameter and 15 mm in length were used in this investigation ( master screw , conexo sistemas de prtese ltda .", "three transmucosal cera one type abutments ( 2.0 mm height , cera one , conexo sistemas de prtese ltda . ,", "reference 045022 , batch # 5080915121 ) , designed for cemented single standing implant - supported restorations were also used .", "the transmucosal abutments were attached to the fixtures using 10 gold screws ( conexo , sistemas de prtese ltda .", ", reference 121022 , batch # 5073147 ) ( figure 1 a a ) , 10 titanium screws ( conexo sistemas de prtese ltda .", ", reference 121024 , batch # 5063223 ) ( figure 1a b ) , and 10 surface treated titanium screws ( ti - tite , conexo sistemas de prtese ltda . , reference 121026 , batch # 5063035 ) ( figure 1a c ) . a load cell adapted from a model described in the literature", "this load cell presented a central void for fixture fixation in its upper portion and a horizontal plate for fixation of four strain gauges on its lower portion ( figure 1 b ) .", "the abutments were placed on the upper plate of the cell ( figure 1 c ) in such way that the contact between abutments and fixtures was free of any interference .", "therefore , when the abutment was fixed to the fixture by the abutment screw , tensions generated by this fixation pulled the unit against the abutment , producing a deformation in the lower plate connected to the strain gauges .", "next , the force ( preload ) generated on the abutment screws was captured by the strain gauges in volts ( v ) and later converted to newton ( n ) .", "the aforementioned sample was arranged into 3 different groups according to the characteristics of each abutment screw : group a was formed by gold screws , group b was formed by titanium screws , and group c was formed by surface - treated titanium screws . to determine fixation and removal torques ,", "a digital torquemeter was used ( torqueleader , model tsd150 , type i , class e , part # 117317 ) , previously calibrated according with iso 6789:2003 ( e ) standard . a square hand wrench ( 1.27 mm diameter , conexo ,", ", reference 062300 , batch # 5091632 ) was attached to the torquemeter to allow proper connection between torquemeter and screws ( figure 1 d ) . following the proper assembly of fixture and abutment on group a", ", the gold screw was then attached to the fixture with an applied torque force of 30.070.28 ncm , being repeated afterwards for groups b and c. then , the initial preload value for the abutment screws was determined ( in volts ) and all screws were kept in their positions for 5 minutes for preload stabilization , according to the literature9 . during this the preload stabilization period ,", "preload values were obtained after 1 , 2 , 3 , 4 , and 5 minutes after fixation , and thereafter a mean value was calculated .", "after this evaluation , the abutment screw was removed using a digital torquemeter and the maximum value of reverse torque force required for screw removal was recorded in ncm .", "next , the same screw was fixed again another 4 times , obtaining the preload and torque removal mean values .", "nine other screws from group a as well as 10 other screws from b and c groups were subjected to the same procedure described above .", "the values of fixation torque forces of the 3 groups were analyzed statistically by to one - way analysis of variance ( anova ) .", "comparison of preload values within groups was possible using the mean of 6 measurements ( 0 to 5 minutes ) obtained at the first abutment fixation , using two - way anova .", "one - way anova was used to compare the mean preload values among the groups .", "tukey 's test was used for multiple comparisons among the groups . for all statistical tests ,", "all fixations for each screw ( n=5 ) were considered for evaluation of the torque removal to obtain an average value , and the statistical analysis was performed similarly to the preload data .", "two - way anova was used to evaluate the mean torque removal among the groups , and one - way anova to evaluate the differences between screws .", "tukey 's multiple comparison test was used for individual comparisons among the groups . for all statistical tests ,"], ["the mean fixation torque force when all screws were considered was 30.070.28 ncm ( c.i .", "there were no statistically significant differences ( p=0.1244 ) among the groups regarding the fixation torque .", "the gold screws presented the highest preload values and the titanium screws the lowest values ( figure 2 ) .", "the mean preload values of the gold screws was 131.728.98 n , and presented statistically significant differences ( p<0.01 ) compared to groups", "b and c. screw # 3 and screw # 1 presented the highest ( 140.48 n ) and the lowest ( 117.73 n ) preload values , respectively . the titanium screws presented statistically significant differences ( p<0.01 ) ( mean 37.035.69 n ) .", "the highest preload value was obtained for screw # 7 ( 49.68 n ) , while screw # 3 presented the lowest value ( 25.30 n ) . in the group of surface - treated titanium screws ,", "the mean preload value was 97.784.68 n. statistically significant differences ( p<0.01 ) were also observed in this group , ( maximum : 104.09 n for screw # 4 ; minimum : 90.28 n for screw # 2 ) . the removal torque presented statistically significant differences among the analyzed materials ( p<0.001 ) ( figure 3 ) .", "the gold screws presented a mean removal torque of 17.641.12 ncm , i.e. they did not shown statistically significant differences ( p=0.3713 ) .", "titanium screws presented a mean removal torque of 18.751.89 ncm , and showed statistically significant differences ( p<0.001 ) .", "the group of surface - treated titanium screws present statistically significant difference ( p=0.004 ) ( mean : 16.431.33 ncm ) ."], ["the results of this study are similar to those of previous investigations4,8 , which found statistically higher preload values for gold fixation screws in comparison to other tested materials , in spite of using different methodologies and materials .", "a maximum preload value of 666.4 n for gold screws and 458.2 n for titanium screws was reported8 .", "in addition , the finds of the present study suggest that the evaluated surface treatment of titanium screws was effective , since these screws presented higher preload values than conventional titanium screws .", "most previously published reports in the dental literature do not specify the removal torque of abutment screws .", "the effect of different cyclic loads has been evaluated using a 32 ncm torque for fixation of the screws , which was repeated after 10 minutes to avoid contact relaxation6 .", "several manufacturers have suggested this clinical approach to decrease screw loosening . in the present study , to allow a direct comparison between results of preload and torque removal for the studied groups , all screws received a similar fixation torque of30.070.28 ncm , despite the manufacturer 's recommendations of using 35 ncm torque for the ti - tite screws .", "this was performed to eliminate a possible bias caused by elastic deformation that might occur in titanium screws during fixation , which might not only quantitatively but qualitatively influence the study outcomes and impair comparison among groups .", "this approach allowed that , in this investigation , only the screw was left as a study variable .", "another relevant issue is that up to 10% of the initial preload may be lost to smooth contact surfaces ( embedment relaxation ) , rather than elongation stresses .", "however , it has been previously observed that when the same screw is fixed several times , its preload values increased4 .", "comparison to different results obtained in other studies might be a difficult task due to variations of tested products and variables that may influence the produced preload value , such as the elastic modulus of the screws , opposing joint surfaces , abutment design , friction coefficient , lubrication , rate of the applied torque and the adaptation between the fixture hexagon and the abutment2 .", "the findings of the present study suggest a trend for greater preload values verified for gold screws , followed by surface - treated titanium screws and titanium screws , confirming the outcomes of previous investigations4,8 . finite element analysis has been used to evaluate preloading through elongation of the abutment screw .", "a study evaluating the preload of two different systems of the same implant manufacturer in function , found that the optimal preload produced should be 75% of the screw yield stress , and also that the preload value increases dramatically as the friction coefficient between implant and screw decreases7 .", "as loosening of implant / abutment joint causes clinical problems , another question that may be raised is whether the torque values recommended by the manufacturers should be increased in order to obtain greater longevity of screw tightening .", "the preload produced by gold or titanium fixation screws can be evaluated through the measurement of elongation stresses .", "it has been found that the stresses were 60% lower than the strengths against the torques applied according to the manufacturer 's instructions4 . on the other hand", ", it has been reported that gold or titanium screws could support higher torques than those indicated by the manufacturers without presenting plastic deformations , although it has been recommended elsewhere that the stresses should not exceed 65% of the screw 's fracture strength1 .", "although the results of this study showed that gold screws have a clear superiority of the produced preload , application of cyclic loads would be required for a closer simulation of the masticatory function on implanted - supported restorations ."], ["it may be concluded : 1 . gold should be the material of choice for abutment fixation screws , since it produced the highest preload values , followed by surface - treated titanium screws and conventional titanium screws ; 2 .", "titanium screws presented the highest torque removal values , followed by gold screws , and surface - treated titanium screws ."]]}
{"article_id": "PMC3311057", "article_text": ["the central nervous system ( cns ) represents an important target for hiv infection during multiple stages of the disease : early , after invasion of the host , since the virus rapidly enters the cns , which then constantly acts as a viral reservoir ; lately , subverting its function and causing peripheral neuropathies and neurocognitive disorders ; and lastly , during the final stage of neuroaids , triggering opportunistic cns infections , cancers , and dementia ( tardieu and boutet , 2002 ) .", "highly active antiretroviral therapy ( haart ) , a combination of drugs that inhibits enzymes essential for hiv replication , can reduce the viremia and the onset of opportunistic infections in most patients , and prolong the survival ( see table 1 ) .", "although haart has reduced the incidence of clinical signs of neurological disease in hiv - infected individuals , autopsy studies suggest that there has been no corresponding decline in the incidence of inflammatory lesions in the cns .", "haart currently in use in the clinical practice . among the limits of the current treatments", "the most noticeable is the inability to eradicate hiv - infected cells , both , limiting the time frame in which haart initiated after exposure to hiv can prevent infection , and allowing replication - competent virus that persists in infected cells to emerge rapidly after the cessation of haart . according to those considerations", ", there is evidence that haart is less effective in lowering virus replication in the cns than in the blood ( gisolf et al . , 2000 ) ; and haart resistant viruses are more often found in the cerebrospinal fluid ( csf ) than in biological samples ( cinque et al . ,", "the principal challenges in drug development for cns  hiv infections are : ( 1 ) understanding the pathways for viral entry , replication , reservoirs , and glial activation in order to identify new targets for treatment , ( 2 ) developing selective therapeutics agents for those targets , or shaping effective strategies to deliver them within the cns , and finally but not to underestimate ( 3 ) collaborating with pharmaceutical companies to test promising drugs in clinical trials , and eventually exploit them in clinical practice .", "hiv is known to productively replicate in macrophages and microglia of the cns , but it also infects , via a cd4-independent pathway , macroglial cells such as astrocytes and most - importantly brain microvascular endothelial cells , which represent the major component of the blood brain barrier ( bbb ; bissel and wiley , 2004 ) . although it is generally accepted that neurons do not become infected by hiv , recent reports have shown that neuronal progenitor brain cells can be infected by various viral strains ( i.e. , hiv-1 ) , eventually playing a role as viral reservoirs ( lawrence et al . , 2004 ) . moreover , one of the important features of cns infection is that its viral populations can diverge from those of plasma : indeed , genetic compartmentalization of csf hiv-1 infection has been nicely demonstrated in a series of studies using the heteroduplex tracking essay to compare blood and csf viral populations cross - sectionally and longitudinally ( ritola et al . , 2005 ) ; therefore even if during acute infection the two population are identical , during chronic infection and especially in cases of hiv encephalitis they genetically and functionally diverge , and may justify the failure of antiretroviral therapy ( strain et al . , 2005 ) .", "identification of viral and cellular mechanisms in the cns that control virus replication after acute infection , maintain latency during the asymptomatic stage , and mediate resurgence of virus replication and development of hiv - associated encephalitis during late infection , is critical .", "the dynamic signaling pathways that are activated by virus replication and active / infiltrating macrophages and lymphocytes and the cytokines , chemokines , and neurotoxic products they elaborate may interfere with the homeostatic signaling pathways that maintain normal quiescent brain function . to understand the correlates in terms of neuroprotection , neuroinflammation , and neurodegeneration processes", "many preclinical models have been developed , such as : hiv transgenic rat ( i.e. , tg rat from reid et al . , 2001 ) , or mice ( i.e. , gp 120 tg mice from toggas et al . , 1994 ) , but also those obtained by injection of hiv proteins ( i.e. , gp 120 from sundar et al . , 1991 ; or tat and bansal et al . , 2000 ) , or hiv - infected macrophages into the brain of mice ( anderson et al . , 2003 )", "the most studied signaling pathways include , among others , the gsk3 , cdk5 , erk , notch , p38 , and jnk cascades .", "for instance , it is noteworthy that many mitogen - activated protein kinases are pivotal during acute , asymptomatic , and terminal infection : some of them seems to promote cell growth and differentiation in response to initial infection ( i.e. , erk ) , whereas others are associated to growth arrest , apoptosis , and oncogenic transformation during aids .", "cns infection : in contrast to hiv - infected cd4 + t cells , infection in brain macrophages and microglial cell lines leads to an extended life span and elevated survival against apoptotic stresses ( chugh et al . , 2007 ) , and only during terminal infection ,", "when there is resurgent virus replication , the balance favors neuronal death ( i.e. , via activation of jnk and p38 ) reflecting a failure to maintain homeostasis in the cns ( barber et al . , 2004 ) with the consequent motor and cognitive impairments .", "many strategies are currently under evaluation to improve hiv treatment , but a relatively small number are primarily dedicated to the alleviation of neurological complaints or the control of neuroaids , and unfortunately more than 98% of drug candidates for cns disorders never make it to the clinic ( pardridge , 2003 ) .", "herein are a couple of examples picturing how researches , initially conducted for neoplastic , neurodegenerative , or epileptic disorders , on specific pathways and delivery systems , might be adapted and applied to the field of cns  hiv infection ( see figure 1 ) .", "neutralization of vif on microvascular endothelial cells ( ifn and ifn ) and subsequent inhibition of viral entry into the cns ; 3 .", "inhibition of hiv replication in macrophages and viral spread into the cns ( nk-1-agonists ) ; 4 .", "inhibition of cytoprotective effect exerted by tat proteins over microglial cells which act as cns viral reservoirs ( pi3k and akt inhibitors ) ; 5 .", "protection of neural integrity and plasticity along with prevention of neurotoxicity and neuronal death ( glutamate receptors- , gsk3- , jnk- , and p38-inhibitors ) . discovered in 1898 ,", "mitoguazone ( mgbm ) had been investigated but abandoned as an anti - cancer drug , recently it was found to cause selective loss of cd16 + hiv - infected cells , specifically by blocking osteopontin needed to transform a circulating macrophage into a cns / resident one .", "mgbg although ineffective in reducing hiv viremia , does appear to shut down viral evolution by selectively killing recent migrants macrophages rather than resident cells , and to be safe over the long - term treatment in aids patients ( jeymohan et al . , 2009 ) .", "the manipulation of ifn and ifn apobec pathways could prevent hiv invasion of cns because of the potent intrinsic immunity exerted by the expression of apobec3 g gene in microvascular endothelial cells ( argyris et al . , 2007 ) .", "this gene code for proteins possessing cytidine deaminase activity and allow the bbb to actively neutralize the effects of viral infectivity factors ( vif ) , which are essentials for viral entry and spread into the cns ( jeong et al . , 2008 ) .", "nk-1 , also known as substance p , was the first neuropeptide discovered in 1931 , it is widely distributed in the central and peripheral nervous system where it functions as a potent mediator of immunoregulation .", "agonists to nk-1 receptor potently inhibit both in vitro and in vivo hiv replication in human macrophages , apparently down regulating ccr5 ( jeymohan et al . , 2009 ) .", "initially recognized , and extensively studied as a promising target for anti - cancer therapies because of its role during tumorigenesis , the pi3k / akt cell survival pathway became a target for anti - hiv treatment after the observation that pi3k inhibitors ( such as wortmannin and ly294002 ) or akt inhibitors ( such as miltefosine ) , both able to cross the bbb , contrast the cytoprotective effect exerted by tat proteins over macrophage / microglial cells , and make them again susceptible to cell death following an apoptotic stimulus ( chugh et al .", "most interestingly pi3k / akt inhibitors proved to be effective without harming uninfected cells and therefore experimental data as far available support their possible use for hiv - therapy and targeting of long - lived viral reservoirs .", "nanoformulated haart essentially manipulates the immune system for therapeutic purposes by using synthetic or biological nanoengineered drug carriers .", "those nanoformulated drugs have been proven to be efficient delivery vehicles for a wide range of therapeutic agents , and can be easily injected in the blood circulation or virtually into any site of the brain with minimal invasiveness .", "major concerns to this therapeutic approach are the choice of drug administration ( systemic or local ) and consequently the residence in the systemic blood circulation , or in the brain tissue adjacent to topic injection , the drug loading efficiency , and the burst release effect .", "furthermore , many other question marks are still to be answered , or even formulated .", "concerning synthetic carriers , hyaluronic acid , a linear polysaccharide composed of alternating d - glucuronic acid and n - acetyl - d - glucosamine units , because of its immunoneutrality , has been proposed as a biocompatible and biodegradable biomaterial for tissue engineering , and in drug delivery systems into the cns : particularly appealing would seem the opportunity to produce drug - incorporated hyaluronic acid nanoparticles ( jeong et al . , 2008 ) . as well monocytes , because of their abilities to move drugs inside the cells have advantages as a depot for haart , but also they might improve compliance as drugs can be released from cells for periods up to weeks or longer .", "nevertheless , further application of biological nanoengineered drug carriers will require optimization of nanoparticles uptake , deeper knowledge of their kinetics across the bbbs , and more sophisticated imaging systems to track migration of monocyte  macrophages to the brain and target the delivery of specific therapeutics ( jeymohan et al . , 2009 ) . beside compounds aimed at control the virus ,", "others try to treat the effects of the underlying neuroinflammation and neurodegeneration mechanisms , and might provide a beneficial supplement to haart .", "neurotoxicity is mainly mediated through glutamate production , which is triggered by nmda subtypes of glutamate receptors , and correlated to caspase activation and subsequent neuronal apoptosis ( erdmann et al . ,", "2006 ) ; accordingly , pharmacologic inhibition of glutaminase directly correlate to neuronal survival ( tian et al . , 2008 ) .", "those insights support the hypothesis that inhibition of glutamate production by blocking mitochondrial glutaminase activity may clinically prevent neurotoxicity during hiv-1 infection . taken together the gsk3 , cdk5 , jnk , and p38 signaling pathways are important regulators of neurotoxicity , being responsible for synaptic and dendritic damage to pyramidal neurons , loss of immunoreactive neurons , and demyelinization .", "gsk3 inhibitors ( i.e. , lithium , valproic acid ) as well as cdk5 inhibitors ( i.e. , roscovitine ) seems to protect neuronal integrity and plasticity ( crews et al . , 2009 ) , while jnk- and p38-inhibitors ( i.e. , minocycline ) may prevent neuronal death ( lin et al .", ", 2001 ; bozyczko - coyne et al . , 2002 ) each of those target holds promise for the development of treatment strategies to ameliorated the neuropathological effects exerted by hiv proteins ( tat , vif , etc . ) .", "discovered in 1898 , mitoguazone ( mgbm ) had been investigated but abandoned as an anti - cancer drug , recently it was found to cause selective loss of cd16 + hiv - infected cells , specifically by blocking osteopontin needed to transform a circulating macrophage into a cns / resident one .", "mgbg although ineffective in reducing hiv viremia , does appear to shut down viral evolution by selectively killing recent migrants macrophages rather than resident cells , and to be safe over the long - term treatment in aids patients ( jeymohan et al . , 2009 ) .", "the manipulation of ifn and ifn apobec pathways could prevent hiv invasion of cns because of the potent intrinsic immunity exerted by the expression of apobec3 g gene in microvascular endothelial cells ( argyris et al . , 2007 ) .", "this gene code for proteins possessing cytidine deaminase activity and allow the bbb to actively neutralize the effects of viral infectivity factors ( vif ) , which are essentials for viral entry and spread into the cns ( jeong et al .", "nk-1 , also known as substance p , was the first neuropeptide discovered in 1931 , it is widely distributed in the central and peripheral nervous system where it functions as a potent mediator of immunoregulation .", "agonists to nk-1 receptor potently inhibit both in vitro and in vivo hiv replication in human macrophages , apparently down regulating ccr5 ( jeymohan et al . , 2009 ) .", "initially recognized , and extensively studied as a promising target for anti - cancer therapies because of its role during tumorigenesis , the pi3k / akt cell survival pathway became a target for anti - hiv treatment after the observation that pi3k inhibitors ( such as wortmannin and ly294002 ) or akt inhibitors ( such as miltefosine ) , both able to cross the bbb , contrast the cytoprotective effect exerted by tat proteins over macrophage / microglial cells , and make them again susceptible to cell death following an apoptotic stimulus ( chugh et al .", "most interestingly pi3k / akt inhibitors proved to be effective without harming uninfected cells and therefore experimental data as far available support their possible use for hiv - therapy and targeting of long - lived viral reservoirs .", "nanoformulated haart essentially manipulates the immune system for therapeutic purposes by using synthetic or biological nanoengineered drug carriers .", "those nanoformulated drugs have been proven to be efficient delivery vehicles for a wide range of therapeutic agents , and can be easily injected in the blood circulation or virtually into any site of the brain with minimal invasiveness .", "major concerns to this therapeutic approach are the choice of drug administration ( systemic or local ) and consequently the residence in the systemic blood circulation , or in the brain tissue adjacent to topic injection , the drug loading efficiency , and the burst release effect . furthermore , many other question marks are still to be answered , or even formulated . concerning synthetic carriers , hyaluronic acid , a linear polysaccharide composed of alternating d", "- glucuronic acid and n - acetyl - d - glucosamine units , because of its immunoneutrality , has been proposed as a biocompatible and biodegradable biomaterial for tissue engineering , and in drug delivery systems into the cns : particularly appealing would seem the opportunity to produce drug - incorporated hyaluronic acid nanoparticles ( jeong et al . , 2008 ) .", "as well monocytes , because of their abilities to move drugs inside the cells have advantages as a depot for haart , but also they might improve compliance as drugs can be released from cells for periods up to weeks or longer . nevertheless , further application of biological nanoengineered drug carriers will require optimization of nanoparticles uptake , deeper knowledge of their kinetics across the bbbs , and more sophisticated imaging systems to track migration of monocyte  macrophages to the brain and target the delivery of specific therapeutics ( jeymohan et al . , 2009 ) .", "beside compounds aimed at control the virus , others try to treat the effects of the underlying neuroinflammation and neurodegeneration mechanisms , and might provide a beneficial supplement to haart .", "neurotoxicity is mainly mediated through glutamate production , which is triggered by nmda subtypes of glutamate receptors , and correlated to caspase activation and subsequent neuronal apoptosis ( erdmann et al . , 2006 ) ; accordingly , pharmacologic inhibition of glutaminase directly correlate to neuronal survival ( tian et al . , 2008 ) .", "those insights support the hypothesis that inhibition of glutamate production by blocking mitochondrial glutaminase activity may clinically prevent neurotoxicity during hiv-1 infection . taken together the gsk3 , cdk5 , jnk , and p38 signaling pathways are important regulators of neurotoxicity , being responsible for synaptic and dendritic damage to pyramidal neurons , loss of immunoreactive neurons , and demyelinization .", "gsk3 inhibitors ( i.e. , lithium , valproic acid ) as well as cdk5 inhibitors ( i.e. , roscovitine ) seems to protect neuronal integrity and plasticity ( crews et al . , 2009 ) , while jnk- and p38-inhibitors ( i.e. , minocycline ) may prevent neuronal death ( lin et al . , 2001 ; bozyczko - coyne et al . , 2002 ) each of those target holds promise for the development of treatment strategies to ameliorated the neuropathological effects exerted by hiv proteins ( tat , vif , etc . )", "combinations of drugs work better and longer than seen previously , patients benefit demonstrably from the magnitude and duration of viral suppression , and require more and more symptomatic treatment of neuroaids cognitive impairment .", "however haart is very demanding for patients , non - adherence can lead to therapeutic failure , and in addition drug resistance is a continuing problem ; therefore , the need for improved drug and delivery systems , but also an interest for adaptive treatment strategies and mathematical modeling to manage structured treatment interruptions ( rosenberg et al . , 2007 ) , have been outlined by many researchers . to this", "regard a thigh collaboration with industry is strictly required and introduces a new set of challenges such as : ( 1 ) circulation of promising ideas , often just developed with university grants , ( 2 ) protection of intellectual property and patent rights , ( 3 ) fund - raising opportunities for start - ups during preclinical researches on novel compounds , until ( 4 ) identification of potential blockbusters appealing to big pharma companies for introduction in the market and their clinical exploitation .", "finally , current criteria for initiation of haart do not consider cns outcomes as a benchmark of efficacy , thus establishing cns - based criteria for initiation of protocols should be the ultimate goal not only for therapeutic but also for neuroprotection strategies ; as recently stated by jeymohan et al .", "( 2009 ) on behalf of the neuroaids research participants these priorities should be seen as a template for future growth of the field and its long - term impact .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S> the central nervous system ( cns ) represents an important target for hiv infection during multiple stages of the disease : early , after invasion of the host , acting as a viral reservoir ; lately , subverting its function and causing peripheral neuropathies and neurocognitive disorders ; and lastly , during the final stage of neuroaids , triggering opportunistic infections , cancers , and dementia . </S>", "<S> highly active antiretroviral therapy , a combination of drugs that inhibits enzymes essential for hiv replication , can reduce the viremia and the onset of opportunistic infections in most patients , and prolong the survival . among the limits of the current treatments </S>", "<S> the most noticeable is the inability to eradicate hiv - infected cells , both , limiting the time frame in which antiretroviral therapies initiated after exposure to hiv can prevent infection , and allowing replication - competent virus that persists in infected cells to emerge rapidly after the cessation of treatments . </S>", "<S> many strategies are currently under evaluation to improve hiv treatment , unfortunately more than 98% of drug candidates for cns disorders never make it to the clinic ; here in we report how nanoformulated strategies might be adapted and applied to the field of cns  hiv infection . </S>"], "labels": null, "section_names": ["Backgroung", "Current Understanding of CNSHIV Infection", "Developing Strategies", "Innovatively targeted pathways", "Nanoformulated HAART", "Neuroprotective strategy", "Conclusion", "Conflict of Interest Statement"], "sections": [["the central nervous system ( cns ) represents an important target for hiv infection during multiple stages of the disease : early , after invasion of the host , since the virus rapidly enters the cns , which then constantly acts as a viral reservoir ; lately , subverting its function and causing peripheral neuropathies and neurocognitive disorders ; and lastly , during the final stage of neuroaids , triggering opportunistic cns infections , cancers , and dementia ( tardieu and boutet , 2002 ) .", "highly active antiretroviral therapy ( haart ) , a combination of drugs that inhibits enzymes essential for hiv replication , can reduce the viremia and the onset of opportunistic infections in most patients , and prolong the survival ( see table 1 ) .", "although haart has reduced the incidence of clinical signs of neurological disease in hiv - infected individuals , autopsy studies suggest that there has been no corresponding decline in the incidence of inflammatory lesions in the cns .", "haart currently in use in the clinical practice . among the limits of the current treatments", "the most noticeable is the inability to eradicate hiv - infected cells , both , limiting the time frame in which haart initiated after exposure to hiv can prevent infection , and allowing replication - competent virus that persists in infected cells to emerge rapidly after the cessation of haart . according to those considerations", ", there is evidence that haart is less effective in lowering virus replication in the cns than in the blood ( gisolf et al . , 2000 ) ; and haart resistant viruses are more often found in the cerebrospinal fluid ( csf ) than in biological samples ( cinque et al . ,", "the principal challenges in drug development for cns  hiv infections are : ( 1 ) understanding the pathways for viral entry , replication , reservoirs , and glial activation in order to identify new targets for treatment , ( 2 ) developing selective therapeutics agents for those targets , or shaping effective strategies to deliver them within the cns , and finally but not to underestimate ( 3 ) collaborating with pharmaceutical companies to test promising drugs in clinical trials , and eventually exploit them in clinical practice ."], ["hiv is known to productively replicate in macrophages and microglia of the cns , but it also infects , via a cd4-independent pathway , macroglial cells such as astrocytes and most - importantly brain microvascular endothelial cells , which represent the major component of the blood brain barrier ( bbb ; bissel and wiley , 2004 ) . although it is generally accepted that neurons do not become infected by hiv , recent reports have shown that neuronal progenitor brain cells can be infected by various viral strains ( i.e. , hiv-1 ) , eventually playing a role as viral reservoirs ( lawrence et al . , 2004 ) . moreover , one of the important features of cns infection is that its viral populations can diverge from those of plasma : indeed , genetic compartmentalization of csf hiv-1 infection has been nicely demonstrated in a series of studies using the heteroduplex tracking essay to compare blood and csf viral populations cross - sectionally and longitudinally ( ritola et al . , 2005 ) ; therefore even if during acute infection the two population are identical , during chronic infection and especially in cases of hiv encephalitis they genetically and functionally diverge , and may justify the failure of antiretroviral therapy ( strain et al . , 2005 ) .", "identification of viral and cellular mechanisms in the cns that control virus replication after acute infection , maintain latency during the asymptomatic stage , and mediate resurgence of virus replication and development of hiv - associated encephalitis during late infection , is critical .", "the dynamic signaling pathways that are activated by virus replication and active / infiltrating macrophages and lymphocytes and the cytokines , chemokines , and neurotoxic products they elaborate may interfere with the homeostatic signaling pathways that maintain normal quiescent brain function . to understand the correlates in terms of neuroprotection , neuroinflammation , and neurodegeneration processes", "many preclinical models have been developed , such as : hiv transgenic rat ( i.e. , tg rat from reid et al . , 2001 ) , or mice ( i.e. , gp 120 tg mice from toggas et al . , 1994 ) , but also those obtained by injection of hiv proteins ( i.e. , gp 120 from sundar et al . , 1991 ; or tat and bansal et al . , 2000 ) , or hiv - infected macrophages into the brain of mice ( anderson et al . , 2003 )", "the most studied signaling pathways include , among others , the gsk3 , cdk5 , erk , notch , p38 , and jnk cascades .", "for instance , it is noteworthy that many mitogen - activated protein kinases are pivotal during acute , asymptomatic , and terminal infection : some of them seems to promote cell growth and differentiation in response to initial infection ( i.e. , erk ) , whereas others are associated to growth arrest , apoptosis , and oncogenic transformation during aids .", "cns infection : in contrast to hiv - infected cd4 + t cells , infection in brain macrophages and microglial cell lines leads to an extended life span and elevated survival against apoptotic stresses ( chugh et al . , 2007 ) , and only during terminal infection ,", "when there is resurgent virus replication , the balance favors neuronal death ( i.e. , via activation of jnk and p38 ) reflecting a failure to maintain homeostasis in the cns ( barber et al . , 2004 ) with the consequent motor and cognitive impairments ."], ["many strategies are currently under evaluation to improve hiv treatment , but a relatively small number are primarily dedicated to the alleviation of neurological complaints or the control of neuroaids , and unfortunately more than 98% of drug candidates for cns disorders never make it to the clinic ( pardridge , 2003 ) .", "herein are a couple of examples picturing how researches , initially conducted for neoplastic , neurodegenerative , or epileptic disorders , on specific pathways and delivery systems , might be adapted and applied to the field of cns  hiv infection ( see figure 1 ) .", "neutralization of vif on microvascular endothelial cells ( ifn and ifn ) and subsequent inhibition of viral entry into the cns ; 3 .", "inhibition of hiv replication in macrophages and viral spread into the cns ( nk-1-agonists ) ; 4 .", "inhibition of cytoprotective effect exerted by tat proteins over microglial cells which act as cns viral reservoirs ( pi3k and akt inhibitors ) ; 5 .", "protection of neural integrity and plasticity along with prevention of neurotoxicity and neuronal death ( glutamate receptors- , gsk3- , jnk- , and p38-inhibitors ) . discovered in 1898 ,", "mitoguazone ( mgbm ) had been investigated but abandoned as an anti - cancer drug , recently it was found to cause selective loss of cd16 + hiv - infected cells , specifically by blocking osteopontin needed to transform a circulating macrophage into a cns / resident one .", "mgbg although ineffective in reducing hiv viremia , does appear to shut down viral evolution by selectively killing recent migrants macrophages rather than resident cells , and to be safe over the long - term treatment in aids patients ( jeymohan et al . , 2009 ) .", "the manipulation of ifn and ifn apobec pathways could prevent hiv invasion of cns because of the potent intrinsic immunity exerted by the expression of apobec3 g gene in microvascular endothelial cells ( argyris et al . , 2007 ) .", "this gene code for proteins possessing cytidine deaminase activity and allow the bbb to actively neutralize the effects of viral infectivity factors ( vif ) , which are essentials for viral entry and spread into the cns ( jeong et al . , 2008 ) .", "nk-1 , also known as substance p , was the first neuropeptide discovered in 1931 , it is widely distributed in the central and peripheral nervous system where it functions as a potent mediator of immunoregulation .", "agonists to nk-1 receptor potently inhibit both in vitro and in vivo hiv replication in human macrophages , apparently down regulating ccr5 ( jeymohan et al . , 2009 ) .", "initially recognized , and extensively studied as a promising target for anti - cancer therapies because of its role during tumorigenesis , the pi3k / akt cell survival pathway became a target for anti - hiv treatment after the observation that pi3k inhibitors ( such as wortmannin and ly294002 ) or akt inhibitors ( such as miltefosine ) , both able to cross the bbb , contrast the cytoprotective effect exerted by tat proteins over macrophage / microglial cells , and make them again susceptible to cell death following an apoptotic stimulus ( chugh et al .", "most interestingly pi3k / akt inhibitors proved to be effective without harming uninfected cells and therefore experimental data as far available support their possible use for hiv - therapy and targeting of long - lived viral reservoirs .", "nanoformulated haart essentially manipulates the immune system for therapeutic purposes by using synthetic or biological nanoengineered drug carriers .", "those nanoformulated drugs have been proven to be efficient delivery vehicles for a wide range of therapeutic agents , and can be easily injected in the blood circulation or virtually into any site of the brain with minimal invasiveness .", "major concerns to this therapeutic approach are the choice of drug administration ( systemic or local ) and consequently the residence in the systemic blood circulation , or in the brain tissue adjacent to topic injection , the drug loading efficiency , and the burst release effect .", "furthermore , many other question marks are still to be answered , or even formulated .", "concerning synthetic carriers , hyaluronic acid , a linear polysaccharide composed of alternating d - glucuronic acid and n - acetyl - d - glucosamine units , because of its immunoneutrality , has been proposed as a biocompatible and biodegradable biomaterial for tissue engineering , and in drug delivery systems into the cns : particularly appealing would seem the opportunity to produce drug - incorporated hyaluronic acid nanoparticles ( jeong et al . , 2008 ) . as well monocytes , because of their abilities to move drugs inside the cells have advantages as a depot for haart , but also they might improve compliance as drugs can be released from cells for periods up to weeks or longer .", "nevertheless , further application of biological nanoengineered drug carriers will require optimization of nanoparticles uptake , deeper knowledge of their kinetics across the bbbs , and more sophisticated imaging systems to track migration of monocyte  macrophages to the brain and target the delivery of specific therapeutics ( jeymohan et al . , 2009 ) . beside compounds aimed at control the virus ,", "others try to treat the effects of the underlying neuroinflammation and neurodegeneration mechanisms , and might provide a beneficial supplement to haart .", "neurotoxicity is mainly mediated through glutamate production , which is triggered by nmda subtypes of glutamate receptors , and correlated to caspase activation and subsequent neuronal apoptosis ( erdmann et al . ,", "2006 ) ; accordingly , pharmacologic inhibition of glutaminase directly correlate to neuronal survival ( tian et al . , 2008 ) .", "those insights support the hypothesis that inhibition of glutamate production by blocking mitochondrial glutaminase activity may clinically prevent neurotoxicity during hiv-1 infection . taken together the gsk3 , cdk5 , jnk , and p38 signaling pathways are important regulators of neurotoxicity , being responsible for synaptic and dendritic damage to pyramidal neurons , loss of immunoreactive neurons , and demyelinization .", "gsk3 inhibitors ( i.e. , lithium , valproic acid ) as well as cdk5 inhibitors ( i.e. , roscovitine ) seems to protect neuronal integrity and plasticity ( crews et al . , 2009 ) , while jnk- and p38-inhibitors ( i.e. , minocycline ) may prevent neuronal death ( lin et al .", ", 2001 ; bozyczko - coyne et al . , 2002 ) each of those target holds promise for the development of treatment strategies to ameliorated the neuropathological effects exerted by hiv proteins ( tat , vif , etc . ) ."], ["discovered in 1898 , mitoguazone ( mgbm ) had been investigated but abandoned as an anti - cancer drug , recently it was found to cause selective loss of cd16 + hiv - infected cells , specifically by blocking osteopontin needed to transform a circulating macrophage into a cns / resident one .", "mgbg although ineffective in reducing hiv viremia , does appear to shut down viral evolution by selectively killing recent migrants macrophages rather than resident cells , and to be safe over the long - term treatment in aids patients ( jeymohan et al . , 2009 ) .", "the manipulation of ifn and ifn apobec pathways could prevent hiv invasion of cns because of the potent intrinsic immunity exerted by the expression of apobec3 g gene in microvascular endothelial cells ( argyris et al . , 2007 ) .", "this gene code for proteins possessing cytidine deaminase activity and allow the bbb to actively neutralize the effects of viral infectivity factors ( vif ) , which are essentials for viral entry and spread into the cns ( jeong et al .", "nk-1 , also known as substance p , was the first neuropeptide discovered in 1931 , it is widely distributed in the central and peripheral nervous system where it functions as a potent mediator of immunoregulation .", "agonists to nk-1 receptor potently inhibit both in vitro and in vivo hiv replication in human macrophages , apparently down regulating ccr5 ( jeymohan et al . , 2009 ) .", "initially recognized , and extensively studied as a promising target for anti - cancer therapies because of its role during tumorigenesis , the pi3k / akt cell survival pathway became a target for anti - hiv treatment after the observation that pi3k inhibitors ( such as wortmannin and ly294002 ) or akt inhibitors ( such as miltefosine ) , both able to cross the bbb , contrast the cytoprotective effect exerted by tat proteins over macrophage / microglial cells , and make them again susceptible to cell death following an apoptotic stimulus ( chugh et al .", "most interestingly pi3k / akt inhibitors proved to be effective without harming uninfected cells and therefore experimental data as far available support their possible use for hiv - therapy and targeting of long - lived viral reservoirs ."], ["nanoformulated haart essentially manipulates the immune system for therapeutic purposes by using synthetic or biological nanoengineered drug carriers .", "those nanoformulated drugs have been proven to be efficient delivery vehicles for a wide range of therapeutic agents , and can be easily injected in the blood circulation or virtually into any site of the brain with minimal invasiveness .", "major concerns to this therapeutic approach are the choice of drug administration ( systemic or local ) and consequently the residence in the systemic blood circulation , or in the brain tissue adjacent to topic injection , the drug loading efficiency , and the burst release effect . furthermore , many other question marks are still to be answered , or even formulated . concerning synthetic carriers , hyaluronic acid , a linear polysaccharide composed of alternating d", "- glucuronic acid and n - acetyl - d - glucosamine units , because of its immunoneutrality , has been proposed as a biocompatible and biodegradable biomaterial for tissue engineering , and in drug delivery systems into the cns : particularly appealing would seem the opportunity to produce drug - incorporated hyaluronic acid nanoparticles ( jeong et al . , 2008 ) .", "as well monocytes , because of their abilities to move drugs inside the cells have advantages as a depot for haart , but also they might improve compliance as drugs can be released from cells for periods up to weeks or longer . nevertheless , further application of biological nanoengineered drug carriers will require optimization of nanoparticles uptake , deeper knowledge of their kinetics across the bbbs , and more sophisticated imaging systems to track migration of monocyte  macrophages to the brain and target the delivery of specific therapeutics ( jeymohan et al . , 2009 ) ."], ["beside compounds aimed at control the virus , others try to treat the effects of the underlying neuroinflammation and neurodegeneration mechanisms , and might provide a beneficial supplement to haart .", "neurotoxicity is mainly mediated through glutamate production , which is triggered by nmda subtypes of glutamate receptors , and correlated to caspase activation and subsequent neuronal apoptosis ( erdmann et al . , 2006 ) ; accordingly , pharmacologic inhibition of glutaminase directly correlate to neuronal survival ( tian et al . , 2008 ) .", "those insights support the hypothesis that inhibition of glutamate production by blocking mitochondrial glutaminase activity may clinically prevent neurotoxicity during hiv-1 infection . taken together the gsk3 , cdk5 , jnk , and p38 signaling pathways are important regulators of neurotoxicity , being responsible for synaptic and dendritic damage to pyramidal neurons , loss of immunoreactive neurons , and demyelinization .", "gsk3 inhibitors ( i.e. , lithium , valproic acid ) as well as cdk5 inhibitors ( i.e. , roscovitine ) seems to protect neuronal integrity and plasticity ( crews et al . , 2009 ) , while jnk- and p38-inhibitors ( i.e. , minocycline ) may prevent neuronal death ( lin et al . , 2001 ; bozyczko - coyne et al . , 2002 ) each of those target holds promise for the development of treatment strategies to ameliorated the neuropathological effects exerted by hiv proteins ( tat , vif , etc . )"], ["combinations of drugs work better and longer than seen previously , patients benefit demonstrably from the magnitude and duration of viral suppression , and require more and more symptomatic treatment of neuroaids cognitive impairment .", "however haart is very demanding for patients , non - adherence can lead to therapeutic failure , and in addition drug resistance is a continuing problem ; therefore , the need for improved drug and delivery systems , but also an interest for adaptive treatment strategies and mathematical modeling to manage structured treatment interruptions ( rosenberg et al . , 2007 ) , have been outlined by many researchers . to this", "regard a thigh collaboration with industry is strictly required and introduces a new set of challenges such as : ( 1 ) circulation of promising ideas , often just developed with university grants , ( 2 ) protection of intellectual property and patent rights , ( 3 ) fund - raising opportunities for start - ups during preclinical researches on novel compounds , until ( 4 ) identification of potential blockbusters appealing to big pharma companies for introduction in the market and their clinical exploitation .", "finally , current criteria for initiation of haart do not consider cns outcomes as a benchmark of efficacy , thus establishing cns - based criteria for initiation of protocols should be the ultimate goal not only for therapeutic but also for neuroprotection strategies ; as recently stated by jeymohan et al .", "( 2009 ) on behalf of the neuroaids research participants these priorities should be seen as a template for future growth of the field and its long - term impact ."], ["the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC3138923", "article_text": ["colorectal adenocarcinoma is the fourth most frequently diagnosed malignancy in korea , accounting for 12% of newly diagnosed cancer cases . according to the ministry of health and welfare , the incidence of colorectal cancer has increased in both males and females during the past 2 decades . although surgery is potentially curative , approximately one third of all newly diagnosed patients present with inoperable metastatic disease . palliative chemotherapy is more effective than the best supportive care for improving overall survival ( os ) as well as quality of life in advanced colorectal cancer . while significant advances have been made in recent years , cure is rarely possible in advanced colorectal cancer ( acc ) , making further improvements in therapy imperative . for more than three decades ,", "the treatment options for acc have been almost exclusively based on 5-fluorouracil ( 5-fu ) and folinic acid ( fa ) . until the early 1990s", ", 5-fu , often modulated by fa , was the single effective chemotherapy available , but only led to meaningful responses in a small minority of treated patients .", "the recent integration of oxaliplatin and irinotecan for the management of acc patients has extended median os in a meaningful way .", "both drugs have been shown to have synergistic effects with 5-fu and fa in colorectal cancer cell lines . since irinotecan and oxaliplatin", "have been used for acc treatment , the efficacy of palliative chemotherapy for acc has considerably improved .", "first - line treatment with irinotecan and either bolus or infusional 5-fu / fa , namely irinotecan / bolus fluorouracil / lv or 5-fluorouracil , folinic acid plus irinotecan ( folfiri ) , significantly improved outcomes as compared with 5-fu / fa . similarly , a combination of oxaliplatin and infusional 5-fu / fa , known as 5-fluorouracil , folinic acid plus oxaliplatin ( folfox ) , significantly increased the response rate and the time to progression compared with 5-fu / fa . these results established 5-fu - based chemotherapy , in combination with either irinotecan or oxaliplatin , as standard first - line chemotherapy regimens for patients with acc .", "furthermore , capecitabine has been shown to exhibit antitumor activity against acc both as a single agent   and in combination with oxaliplatin [ 12 - 14 ] , as well as with irinotecan .", "in korea , capecitabine was approved in january 2006 as a partner with oxaliplatin or irinotecan for the treatment of acc . a decision regarding chemotherapy regimens for", "factors considered include the extent of disease , potential toxicities , especially for those with impaired oral intake or with decreased performance status , activity of chemotherapy , and the patient preference .", "we therefore decided to evaluate various combination chemotherapy regimens as first - line chemotherapy for acc .", "this report describes a single - center , retrospective study . between january 2006 and december 2007 ,", "a total of 537 acc patients were treated with first - line chemotherapy for advanced disease at samsung medical center ( seoul , korea ) .", "fifty - nine patients were excluded because they were treated with single - agent chemotherapy .", "the criteria for case inclusion were as follows ; 1 ) histologically confirmed diagnosis of adenocarcinoma arising from colon or rectum , 2 ) no prior chemotherapy or radiotherapy except for adjuvant use , 3 ) advanced ( metastatic and/or recurrent ) disease , 4 ) availability of clinical data at the beginning of therapy and follow - up .", "we attempted to exclude patients who were enrolled in clinical trials to ensure the choice of chemotherapy regimen was solely at the discretion of the treating physician .", "all the data were prospectively recorded and only the survival data was updated at the time of analysis . written informed consent was given by all patients prior to receiving chemotherapy , according to institutional guidelines .", "the patients were required to have a life expectancy of 12 weeks of more ; age of at least 18 years or older ; eastern cooperative oncology group ( ecog ) performance status of 2 or lower ; adequate hematologic ( neutrophil count>1,500/mm and platelet count>100,000/mm ) , hepatic ( serum total bilirubin level<1.5 mg / dl and aspartate aminotransferase / alanine aminotransferase<3upper limit normal ) and renal ( serum creatinine level<1.5 mg / dl ) functions .", "our department guidelines define combination chemotherapy regimens as follows : in folfox or folfiri , fa is given at the dose of 200 mg / m followed by bolus 5-fu 400 mg", "/ m on day 1 , and a 46-hour continuous infusion of 5-fu 2,400 mg / m on days 1 and 2 .", "oxaliplatin 85 mg / m or irinotecan 180 mg / m was administered on day 1 as a 2-hour infusion , concurrent with fa .", "in capecitabine plus oxaliplatin ( xelox ) and capecitabine plus irinotecan ( xeliri ) , oxaliplatin 130 mg / m or irinotecan 240 mg / m was given on day 1 in combination with oral capecitabine 1,000 mg / m twice daily on days 1 to 14 .", "folfox and folfiri were repeated every 2 weeks , whereas xelox and xeliri were repeated every 3 weeks until disease progression or unacceptable toxicity occurred , or until a patient chose to discontinue treatment .", "the dosages for subsequent cycles were adjusted according to the toxic effects that developed during the preceding cycle .", "the prophylactic use of hematopoietic growth factors was not allowed during treatment , except for patients with febrile neutropenia or grade 4 myelosuppression at the treating physician 's discretion .", "after this combination chemotherapy had failed , second - line chemotherapy was recommended to all the patients if their performance status was preserved . according to department policies ,", "all tumor measurements were assessed every 2 or 3 cycles of chemotherapy by using an abdominopelvic computed tomography ( ct ) scan and other tests that were initially used to stage the tumor .", "tumor response and progression were evaluated according to the response evaluation criteria for solid tumors ( recist ) .", "the primary endpoint of this retrospective study was progression - free survival ( pfs ) .", "the date of disease progression or death from causes other than acc was used in calculating pfs .", "pfs and os were estimated according to the kaplan - meier method and the statistical significance of differences in survival curves between groups was tested with a log - rank test .", "multivariate models were used for exploratory purposes to examine the impact of each regimen on the outcomes of chemotherapy .", "covariates included were age ( below vs.median ) , gender , previous adjuvant therapy , an ecog performance status ( 0 - 1 vs.2 ) , number of involved sites ( 1 vs.2 ) , metastases ( liver , peritoneum , and lung ) , baseline chemistry profiles ( serum albumin , alkaline phosphatase , and bilirubin ) , combination with bevacizumab , and chemotherapy regimens .", "laboratory parameters were initially recorded as continuous variables and later dichotomized according to the median value of each variable ( below vs.median ) .", "between january 2006 and december 2007 , a total of 478 acc patients were treated with combination chemotherapy in a first - line setting : folfox ( n=172 ) , folfiri ( n=95 ) , xelox ( n=155 ) , and xeliri ( n=56 ) . in 29 patients , chemotherapy involved bevacizumab .", ". sixty percent of patients were male , and 33 patients ( 7% ) had an ecog performance status of 2 or more .", "more than half of the patients ( n=263 ) had received surgery for curative intent , and 206 patients received adjuvant chemotherapy or chemoradiotherapy .", "approximately one - third of the patients had two or more metastatic disease sites , mostly involving liver and abdominal lymph nodes .", "we noted that more folfiri or xeliri patients had received adjuvant therapy involving oxaliplatin than folfox or xelox patients .", "after first - line failure , second - line chemotherapy was administered for more than half of the patients ( n=290 ) .", "median follow - up duration was 40.6 months ( 95% confidence interval [ ci ] , 39.3 to 41.8 months ) . at the time of data collection , 449 ( 89% )", "median chemotherapy durations for folfox , folfiri , xelox , and xeliri were 4.9 , 4.5 , 5.7 and 5.4 months , respectively .", "we found no statistically significant difference among chemotherapy durations for these regimens ( log - rank p=0.19 ) .", "the most common reason for discontinuation of treatment was disease progression . in 172 patients receiving folfox , two - thirds of patients", "discontinued therapy due to disease progression ( n=118 ) , physician 's recommendation or patient withdrawal ( n=33 ) , or toxicity ( n=21 ) .", "folfiri patients discontinued therapy due to progression ( n=60 ) , physician 's recommendation or patient withdrawal ( n=13 ) , toxicity ( n=20 ) , or unknown causes ( n=2 ) .", "similarly , disease progression was the most common cause of therapy discontinuation in 101 xelox patients and 34 xeliri patients .", "we found no relevant difference in the occurrence of overall grade 3 or 4 toxicities among regimens ( table 2 ) . in brief , the main difference in grade 3 or 4 toxicities with folfiri or xeliri was diarrhea ( 21% for folfiri and 27% for xeliri vs. 11% for folfox and 9% for xelox ; p=0.02 ) , and with folfox or xelox the difference involved peripheral neuropathy ( 9% for folfox and 7% for xelox vs. 2% for folfiri and xeliri ; p<0.01 ) .", "although patients who were treated with irinotecan - containing chemotherapy more frequently experienced grade 3 or 4 leukopenia ( 13% vs. 10% ; p=0.09 ) than those treated with oxaliplatin - containing regimens , we found no significant difference in the incidence of febrile neutropenia ( 5% in each arm ) .", "in addition , grade 2 or more hand - foot syndrome was more frequently observed in xelox and xeliri ( 14% and 12% , respectively ) than in folfox and folfiri ( 6% and 8% , respectively ) . in the current retrospective analysis ,", "one death occurred in the midst of treatment with folfox , with no clinical evidence of progression having been demonstrated .", "another patient died of respiratory failure shortly after completion of the second folfox cycle , in which the possibility of disease progression could not be completely excluded .", "the patient , initially presenting with multiple pulmonary nodules and bilateral pleural effusion , complained about increasing dyspnea and suffered a sudden respiratory arrest .", "five other deaths occurred in patients while receiving folfiri or xelox , and were attributed to neutropenic sepsis .", "of a total of 478 patients , 61 could not be evaluated for responses because of the absence of any measurable lesions or early discontinuation of therapy .", "objective responses to chemotherapy were noted in 200 patients ( response rate , 48% ; 95% ci , 43 to 53% ) ; 42% for folfox , 49% for folfiri , 55% for xelox , and 51% for xeliri . the difference in the response rates for each regimen was not statistically significant ( p=0.09 ) .", "patients who had a poor performance status ( 2 in ecog scale ) were significantly less likely to respond to first - line chemotherapy ( 24% vs. 50% ; p<0.01 ) compared to those with an ecog performance status of 0 or 1 .", "response rate was not significantly influenced by age , gender , prior treatments , baseline laboratory parameters , metastatic sites , or chemotherapy regimen .", "the estimated median pfs for all patients was 6.8 months ( 95% ci , 6.3 to 7.3 months ) , and the median os was not yet reached .", "pfs was shorter , although statistically insignificant ( p=0.12 ) , in patients receiving folfox or folfiri ( 6.2 months and 6.0 months , respectively ) than those receiving xelox ( 7.9 months ) or xeliri ( 7.1 months ) .", "one - year pfs rates for folfox , folfiri , xelox , and xeliri were 24% , 21% , 32% , and 32% , respectively ( fig .", "1 ) . a multivariate regression model revealed that pfs was significantly affected by performance status ( hazard ratio , 0.39 ; 95% ci , 0.28 to 0.57 ; p<0.01 ) .", "we also tested whether the pfs was modified by interaction between the effect of baseline clinical parameters and the chemotherapy regimens ; the first - level interaction term between these variables was entered into a separate multivariate model .", "however , we found no interaction between any first - line chemotherapy regimens for each clinical parameter .", "despite recent advances in the treatment of acc , patients treated with first - line chemotherapy have a median os rarely exceeding 24 months . in the current analysis ,", "our observation that pfs was slightly shorter with folfox or folfiri than with xelox or xeliri is possibly related to negative prognostic factors influencing the choice of intravenous chemotherapeutic agents instead of oral agents .", "similarly , the choice of a chemotherapeutic agent depends on the ones previously used in the adjuvant setting .", "although this study is retrospective in nature , the results provide a piece of evidence that patients with acc may derive an indisputable benefit from fluoropyrimidine - based combination chemotherapy .", "the decision to use specific chemotherapeutic agent(s ) in patients with acc should be determined by their relative merits on a case - by - case basis .", "the evolution of treatment using irinotecan and oxaliplatin in combination with 5-fu regimens has resulted in a significant prolongation of life in patients with acc .", "since both drugs have demonstrated similar activity in first- and second - line settings , the optimal chemotherapy regimen and dosing schedule in patients with acc remains undecided .", "randomized trials comparing first - line folfox with folfiri indicated equivalent activity and moderate toxicity differences between these two regimens , suggesting that use of either oxaliplatin or irinotecan is reasonable . in another phase iii study in which patients crossed over to the alternative regimen upon progression while on their first - line regimen , the overall pfs on first - line therapy was 8.1 months for folfox and 8.5 months for folfiri . in a combined analysis of seven phase iii trials in acc , grothey et al .  ", "reported that the strategy of making these three active drugs ( fluoropyrimidine , oxaliplatin , and irinotecan ) available to patients with acc could maximize os .", "in addition to efficacy data , patient convenience and satisfaction are assuming increased importance in cancer management .", "oral administration has the advantage of permitting convenient , patient - orientated therapy , providing the patient with a degree of independence and improved quality of life , while avoiding complications associated with intravenous drug administration .", "chemotherapy regimens involving oral capecitabine showed similar safety and efficacy profiles compared with 5-fu - based regimens .", "oral capecitabine was proved to have a similar quality of life profile to that of 5-fu , but seemed to be more convenient in terms of administration .", "in korea , a capecitabine - containing regimen was approved in january 2006 as the first - line treatment of acc .", "data from the current retrospective study suggest that patients are highly compliant with capecitabine , when given in combination with either oxaliplatin or irinotecan .", "patients treated with capecitabine experienced a higher incidence of clinically relevant hand - foot syndrome , but this toxic effect was predictable and manageable .", "an improved understanding of the molecular pathogenesis of cancer has lead to the development of novel agents designed to target critical cellular pathways .", "one of the most successful novel approaches in the treatment of acc involves therapeutic agents that inhibit the neovascularization process of growing tumors .", "bevacizumab is a recombinant humanized anti - vascular endothelial growth factor monoclonal antibody that is being clinically evaluated in many tumor types , including acc .", "because bevacizumab was only recently made available in korea as first - line chemotherapy , only 29 patients received bevacizumab .", "this study confirms that both oxaliplatin- and irinotecan - containing combination chemotherapy are effective for the first - line treatment of acc .", "capecitabine clearly represents an effective and well - tolerated oral alternative to 5-fu . while the current study was primarily focused on cytotoxic therapies ,", "the integration of targeted agents , including bevacizumab , is a novel standard option to further improve outcomes for patients with acc ."], "abstract_text": ["<S> purposefluoropyrimidine - based combination chemotherapy , in combination with either oxaliplatin or irinotecan , has demonstrated efficacy and tolerability in treatment of advanced colorectal cancer ( acc).materials and methodsbetween january 2006 and december 2007 , a total of 478 acc patients were treated with combination chemotherapy in first - line settings . </S>", "<S> combination therapies included : 5-fluorouracil , folinic acid plus oxaliplatin ( folfox , n=172 ) , 5-fluorouracil , folinic acid plus irinotecan ( folfiri , n=95 ) , capecitabine plus oxaliplatin ( xelox , n=155 ) , and capecitabine plus irinotecan ( xeliri , n=56 ) . </S>", "<S> folfox and folfiri were repeated every 2 weeks , whereas xelox and xeliri were repeated every 3 weeks until occurrence of disease progression or unacceptable toxicity , or until a patient chose to discontinue treatment.resultsthe median age was 58 years ( range , 19 to 84 years ) and the median chemotherapy durations for folfox , folfiri , xelox , and xeliri were 4.9 , 4.5 , 5.7 , and 5.4 months , respectively . </S>", "<S> combination chemotherapy regimens were generally well tolerated . </S>", "<S> the estimated median progression - free - survival ( pfs ) for all patients was 6.8 months ( 95% confidence interval , 6.3 to 7.3 months ) . </S>", "<S> no statistically significant difference in pfs was found among regimens used as first - line chemotherapy . </S>", "<S> sixty percent ( n=290 ) of patients received second or further lines of therapy after failure.conclusionfluoropyrimidine-based combination chemotherapy regimens appear to be equally active and tolerable as first - line therapy for acc . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion"], "sections": [["colorectal adenocarcinoma is the fourth most frequently diagnosed malignancy in korea , accounting for 12% of newly diagnosed cancer cases . according to the ministry of health and welfare , the incidence of colorectal cancer has increased in both males and females during the past 2 decades . although surgery is potentially curative , approximately one third of all newly diagnosed patients present with inoperable metastatic disease . palliative chemotherapy is more effective than the best supportive care for improving overall survival ( os ) as well as quality of life in advanced colorectal cancer . while significant advances have been made in recent years , cure is rarely possible in advanced colorectal cancer ( acc ) , making further improvements in therapy imperative . for more than three decades ,", "the treatment options for acc have been almost exclusively based on 5-fluorouracil ( 5-fu ) and folinic acid ( fa ) . until the early 1990s", ", 5-fu , often modulated by fa , was the single effective chemotherapy available , but only led to meaningful responses in a small minority of treated patients .", "the recent integration of oxaliplatin and irinotecan for the management of acc patients has extended median os in a meaningful way .", "both drugs have been shown to have synergistic effects with 5-fu and fa in colorectal cancer cell lines . since irinotecan and oxaliplatin", "have been used for acc treatment , the efficacy of palliative chemotherapy for acc has considerably improved .", "first - line treatment with irinotecan and either bolus or infusional 5-fu / fa , namely irinotecan / bolus fluorouracil / lv or 5-fluorouracil , folinic acid plus irinotecan ( folfiri ) , significantly improved outcomes as compared with 5-fu / fa . similarly , a combination of oxaliplatin and infusional 5-fu / fa , known as 5-fluorouracil , folinic acid plus oxaliplatin ( folfox ) , significantly increased the response rate and the time to progression compared with 5-fu / fa . these results established 5-fu - based chemotherapy , in combination with either irinotecan or oxaliplatin , as standard first - line chemotherapy regimens for patients with acc .", "furthermore , capecitabine has been shown to exhibit antitumor activity against acc both as a single agent   and in combination with oxaliplatin [ 12 - 14 ] , as well as with irinotecan .", "in korea , capecitabine was approved in january 2006 as a partner with oxaliplatin or irinotecan for the treatment of acc . a decision regarding chemotherapy regimens for", "factors considered include the extent of disease , potential toxicities , especially for those with impaired oral intake or with decreased performance status , activity of chemotherapy , and the patient preference .", "we therefore decided to evaluate various combination chemotherapy regimens as first - line chemotherapy for acc ."], ["this report describes a single - center , retrospective study . between january 2006 and december 2007 ,", "a total of 537 acc patients were treated with first - line chemotherapy for advanced disease at samsung medical center ( seoul , korea ) .", "fifty - nine patients were excluded because they were treated with single - agent chemotherapy .", "the criteria for case inclusion were as follows ; 1 ) histologically confirmed diagnosis of adenocarcinoma arising from colon or rectum , 2 ) no prior chemotherapy or radiotherapy except for adjuvant use , 3 ) advanced ( metastatic and/or recurrent ) disease , 4 ) availability of clinical data at the beginning of therapy and follow - up .", "we attempted to exclude patients who were enrolled in clinical trials to ensure the choice of chemotherapy regimen was solely at the discretion of the treating physician .", "all the data were prospectively recorded and only the survival data was updated at the time of analysis . written informed consent was given by all patients prior to receiving chemotherapy , according to institutional guidelines .", "the patients were required to have a life expectancy of 12 weeks of more ; age of at least 18 years or older ; eastern cooperative oncology group ( ecog ) performance status of 2 or lower ; adequate hematologic ( neutrophil count>1,500/mm and platelet count>100,000/mm ) , hepatic ( serum total bilirubin level<1.5 mg / dl and aspartate aminotransferase / alanine aminotransferase<3upper limit normal ) and renal ( serum creatinine level<1.5 mg / dl ) functions .", "our department guidelines define combination chemotherapy regimens as follows : in folfox or folfiri , fa is given at the dose of 200 mg / m followed by bolus 5-fu 400 mg", "/ m on day 1 , and a 46-hour continuous infusion of 5-fu 2,400 mg / m on days 1 and 2 .", "oxaliplatin 85 mg / m or irinotecan 180 mg / m was administered on day 1 as a 2-hour infusion , concurrent with fa .", "in capecitabine plus oxaliplatin ( xelox ) and capecitabine plus irinotecan ( xeliri ) , oxaliplatin 130 mg / m or irinotecan 240 mg / m was given on day 1 in combination with oral capecitabine 1,000 mg / m twice daily on days 1 to 14 .", "folfox and folfiri were repeated every 2 weeks , whereas xelox and xeliri were repeated every 3 weeks until disease progression or unacceptable toxicity occurred , or until a patient chose to discontinue treatment .", "the dosages for subsequent cycles were adjusted according to the toxic effects that developed during the preceding cycle .", "the prophylactic use of hematopoietic growth factors was not allowed during treatment , except for patients with febrile neutropenia or grade 4 myelosuppression at the treating physician 's discretion .", "after this combination chemotherapy had failed , second - line chemotherapy was recommended to all the patients if their performance status was preserved . according to department policies ,", "all tumor measurements were assessed every 2 or 3 cycles of chemotherapy by using an abdominopelvic computed tomography ( ct ) scan and other tests that were initially used to stage the tumor .", "tumor response and progression were evaluated according to the response evaluation criteria for solid tumors ( recist ) .", "the primary endpoint of this retrospective study was progression - free survival ( pfs ) .", "the date of disease progression or death from causes other than acc was used in calculating pfs .", "pfs and os were estimated according to the kaplan - meier method and the statistical significance of differences in survival curves between groups was tested with a log - rank test .", "multivariate models were used for exploratory purposes to examine the impact of each regimen on the outcomes of chemotherapy .", "covariates included were age ( below vs.median ) , gender , previous adjuvant therapy , an ecog performance status ( 0 - 1 vs.2 ) , number of involved sites ( 1 vs.2 ) , metastases ( liver , peritoneum , and lung ) , baseline chemistry profiles ( serum albumin , alkaline phosphatase , and bilirubin ) , combination with bevacizumab , and chemotherapy regimens .", "laboratory parameters were initially recorded as continuous variables and later dichotomized according to the median value of each variable ( below vs.median ) ."], ["between january 2006 and december 2007 , a total of 478 acc patients were treated with combination chemotherapy in a first - line setting : folfox ( n=172 ) , folfiri ( n=95 ) , xelox ( n=155 ) , and xeliri ( n=56 ) . in 29 patients , chemotherapy involved bevacizumab .", ". sixty percent of patients were male , and 33 patients ( 7% ) had an ecog performance status of 2 or more .", "more than half of the patients ( n=263 ) had received surgery for curative intent , and 206 patients received adjuvant chemotherapy or chemoradiotherapy .", "approximately one - third of the patients had two or more metastatic disease sites , mostly involving liver and abdominal lymph nodes .", "we noted that more folfiri or xeliri patients had received adjuvant therapy involving oxaliplatin than folfox or xelox patients .", "after first - line failure , second - line chemotherapy was administered for more than half of the patients ( n=290 ) .", "median follow - up duration was 40.6 months ( 95% confidence interval [ ci ] , 39.3 to 41.8 months ) . at the time of data collection , 449 ( 89% )", "median chemotherapy durations for folfox , folfiri , xelox , and xeliri were 4.9 , 4.5 , 5.7 and 5.4 months , respectively .", "we found no statistically significant difference among chemotherapy durations for these regimens ( log - rank p=0.19 ) .", "the most common reason for discontinuation of treatment was disease progression . in 172 patients receiving folfox , two - thirds of patients", "discontinued therapy due to disease progression ( n=118 ) , physician 's recommendation or patient withdrawal ( n=33 ) , or toxicity ( n=21 ) .", "folfiri patients discontinued therapy due to progression ( n=60 ) , physician 's recommendation or patient withdrawal ( n=13 ) , toxicity ( n=20 ) , or unknown causes ( n=2 ) .", "similarly , disease progression was the most common cause of therapy discontinuation in 101 xelox patients and 34 xeliri patients .", "we found no relevant difference in the occurrence of overall grade 3 or 4 toxicities among regimens ( table 2 ) . in brief , the main difference in grade 3 or 4 toxicities with folfiri or xeliri was diarrhea ( 21% for folfiri and 27% for xeliri vs. 11% for folfox and 9% for xelox ; p=0.02 ) , and with folfox or xelox the difference involved peripheral neuropathy ( 9% for folfox and 7% for xelox vs. 2% for folfiri and xeliri ; p<0.01 ) .", "although patients who were treated with irinotecan - containing chemotherapy more frequently experienced grade 3 or 4 leukopenia ( 13% vs. 10% ; p=0.09 ) than those treated with oxaliplatin - containing regimens , we found no significant difference in the incidence of febrile neutropenia ( 5% in each arm ) .", "in addition , grade 2 or more hand - foot syndrome was more frequently observed in xelox and xeliri ( 14% and 12% , respectively ) than in folfox and folfiri ( 6% and 8% , respectively ) . in the current retrospective analysis ,", "one death occurred in the midst of treatment with folfox , with no clinical evidence of progression having been demonstrated .", "another patient died of respiratory failure shortly after completion of the second folfox cycle , in which the possibility of disease progression could not be completely excluded .", "the patient , initially presenting with multiple pulmonary nodules and bilateral pleural effusion , complained about increasing dyspnea and suffered a sudden respiratory arrest .", "five other deaths occurred in patients while receiving folfiri or xelox , and were attributed to neutropenic sepsis .", "of a total of 478 patients , 61 could not be evaluated for responses because of the absence of any measurable lesions or early discontinuation of therapy .", "objective responses to chemotherapy were noted in 200 patients ( response rate , 48% ; 95% ci , 43 to 53% ) ; 42% for folfox , 49% for folfiri , 55% for xelox , and 51% for xeliri . the difference in the response rates for each regimen was not statistically significant ( p=0.09 ) .", "patients who had a poor performance status ( 2 in ecog scale ) were significantly less likely to respond to first - line chemotherapy ( 24% vs. 50% ; p<0.01 ) compared to those with an ecog performance status of 0 or 1 .", "response rate was not significantly influenced by age , gender , prior treatments , baseline laboratory parameters , metastatic sites , or chemotherapy regimen .", "the estimated median pfs for all patients was 6.8 months ( 95% ci , 6.3 to 7.3 months ) , and the median os was not yet reached .", "pfs was shorter , although statistically insignificant ( p=0.12 ) , in patients receiving folfox or folfiri ( 6.2 months and 6.0 months , respectively ) than those receiving xelox ( 7.9 months ) or xeliri ( 7.1 months ) .", "one - year pfs rates for folfox , folfiri , xelox , and xeliri were 24% , 21% , 32% , and 32% , respectively ( fig .", "1 ) . a multivariate regression model revealed that pfs was significantly affected by performance status ( hazard ratio , 0.39 ; 95% ci , 0.28 to 0.57 ; p<0.01 ) .", "we also tested whether the pfs was modified by interaction between the effect of baseline clinical parameters and the chemotherapy regimens ; the first - level interaction term between these variables was entered into a separate multivariate model .", "however , we found no interaction between any first - line chemotherapy regimens for each clinical parameter ."], ["despite recent advances in the treatment of acc , patients treated with first - line chemotherapy have a median os rarely exceeding 24 months . in the current analysis ,", "our observation that pfs was slightly shorter with folfox or folfiri than with xelox or xeliri is possibly related to negative prognostic factors influencing the choice of intravenous chemotherapeutic agents instead of oral agents .", "similarly , the choice of a chemotherapeutic agent depends on the ones previously used in the adjuvant setting .", "although this study is retrospective in nature , the results provide a piece of evidence that patients with acc may derive an indisputable benefit from fluoropyrimidine - based combination chemotherapy .", "the decision to use specific chemotherapeutic agent(s ) in patients with acc should be determined by their relative merits on a case - by - case basis .", "the evolution of treatment using irinotecan and oxaliplatin in combination with 5-fu regimens has resulted in a significant prolongation of life in patients with acc .", "since both drugs have demonstrated similar activity in first- and second - line settings , the optimal chemotherapy regimen and dosing schedule in patients with acc remains undecided .", "randomized trials comparing first - line folfox with folfiri indicated equivalent activity and moderate toxicity differences between these two regimens , suggesting that use of either oxaliplatin or irinotecan is reasonable . in another phase iii study in which patients crossed over to the alternative regimen upon progression while on their first - line regimen , the overall pfs on first - line therapy was 8.1 months for folfox and 8.5 months for folfiri . in a combined analysis of seven phase iii trials in acc , grothey et al .  ", "reported that the strategy of making these three active drugs ( fluoropyrimidine , oxaliplatin , and irinotecan ) available to patients with acc could maximize os .", "in addition to efficacy data , patient convenience and satisfaction are assuming increased importance in cancer management .", "oral administration has the advantage of permitting convenient , patient - orientated therapy , providing the patient with a degree of independence and improved quality of life , while avoiding complications associated with intravenous drug administration .", "chemotherapy regimens involving oral capecitabine showed similar safety and efficacy profiles compared with 5-fu - based regimens .", "oral capecitabine was proved to have a similar quality of life profile to that of 5-fu , but seemed to be more convenient in terms of administration .", "in korea , a capecitabine - containing regimen was approved in january 2006 as the first - line treatment of acc .", "data from the current retrospective study suggest that patients are highly compliant with capecitabine , when given in combination with either oxaliplatin or irinotecan .", "patients treated with capecitabine experienced a higher incidence of clinically relevant hand - foot syndrome , but this toxic effect was predictable and manageable .", "an improved understanding of the molecular pathogenesis of cancer has lead to the development of novel agents designed to target critical cellular pathways .", "one of the most successful novel approaches in the treatment of acc involves therapeutic agents that inhibit the neovascularization process of growing tumors .", "bevacizumab is a recombinant humanized anti - vascular endothelial growth factor monoclonal antibody that is being clinically evaluated in many tumor types , including acc .", "because bevacizumab was only recently made available in korea as first - line chemotherapy , only 29 patients received bevacizumab ."], ["this study confirms that both oxaliplatin- and irinotecan - containing combination chemotherapy are effective for the first - line treatment of acc .", "capecitabine clearly represents an effective and well - tolerated oral alternative to 5-fu . while the current study was primarily focused on cytotoxic therapies ,", "the integration of targeted agents , including bevacizumab , is a novel standard option to further improve outcomes for patients with acc ."]]}
{"article_id": "PMC4432382", "article_text": ["lymphomatoid granulomatosis ( lyg ) is a rare angiocentric and angiodestructive lymphoproliferative disease with granulomatous reaction involving the lungs most frequently , however , it may also involve the kidneys , skin and especially the central nervous system4,6,9,11 ) .", "lyg is characterized by an infiltration of atypical lymphocytoid and plasmocytoid cells , with granulomatous inflammation in an angiocentric and angiodestructive polymorphic cellular infiltrate6,8 ) .", "therefore the purpose of this study is to submit the first report about the case with thoracic spinal lyg after diagnosis with b - precursor childhood acute lymphoblastic leukemia ( all ) .", "after surgical decompression with biopsy on the spinal lesion , diagnosis of grade ii lyg was made and rituximab was administered .", "the patient is in good neurological condition without recurrence at the 6-year follow - up .", "a 4-year - old girl was diagnosed with b - precursor all with the involvement of kidneys and pancreas in november , 2004 .", "treatment was initiated according to the pediatric oncology group ( pog ) protocol 900612 ) .", "on april 12 , 2006 , the patient was admitted to pediatric clinic with a complaint of intermittent fever for 3 days . on april 19 , 2006 , 7 days after the admission , the patient developed a gait disturbance .", "neurological examination revealed paraparesis of grade 3 on medical research council ( mrc ) muscle strength grading scale1 ) with increased deep tendon reflex in both ankles , knee jerks , and bilateral positive babinski sign , but her senses were intact .", "cerebrospinal fluid ( csf ) findings were as follows : elevation of protein ( 243.3mg / dl ) , and decrease of glucose ( 44mg / dl ) .", "the condition was suspected to be an acute inflammatory demyelinating polyneuropathy and was treated with intravenous immunoglobulin ( 400 mg / kg / day ) for 5 days .", "however , her paraparesis was aggravated to grade 1 on mrc muscle strength grading scale from april 26 , 2006 , 14 days after the admission , and spinal mri was performed .", "t2 weighted mri demonstrated heterogeneous high signal intensity infiltration on t4 vertebral body and subligamental spreading soft tissue mass on t3 - 5 epidural space from t4 posterior vertebral body , causing spinal cord compression ( fig .", "1 ) . on april 27 , 2006 , laminectomy of t3 , 4 with open biopsy were performed .", "the epidural mass was encircling the spinal cord mainly on ventral surface and was slightly grey to yellow , solid and compact nature .", "a histopathologic examination showed an angiocentric distribution of polymorphous lymphoid infiltrate , with extensive necrosis .", "immunohistoche mical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells ( fig .", "hybridization revealed positivity for epstein - barr virus ( ebv ) encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg .", "one - week post - operative neurologic examination showed paraparesis grade 3 on mrc scale , and bare independent walking but as she still had gait impairment , she received rehabilitation .", "after four weeks of the surgery , she restored her normal state and could walk quite well . at that time", ", there were no pulmonary lesions , so we suspected the spinal lesion as the primary involvement site .", "two weeks after , chest and abdomen ct revealed multiple low density nodular mass on the liver and the lower lobe of the left lung .", "histopathologically , lesions were not blastic in appearance and in immunophenotype , and the lesions were negative for cd10 , tdt , and cd22 , so we ruled out the relapse of all .", "she was treated with rituximab ( 275mg / m / day , 4 times per week ) for 4 weeks and intravenous high dose methylprednisolone ( 30mg / kg per day for 3 days , 20mg / kg per day for 2 days and 10mg / kg per day for 2 days ) .", "chest and abdomen ct showed marked improvement of the lesions 3 months after the treatment compared with the lesions in the images obtained before the treatment .", "a follow - up mri study performed 4 months after the operation documented no evidence of residual mass or recurrence ( fig .", "3 ) . chest and abdomen ct performed 1 year after the operation showed no demonstrable focal lesion in lung field and mediastinum and unremarkable abdominal organs .", "her weakness improved and she was able to walk independently and had no gait impairment .", "but unfortunately we failed to take additional follow - up of mri for the patient , as she was terrified to take mri and full sedation was not possible .", "moreover the parents of the patient did not agree with the idea of taking mri one more time because she was in good state at that time . to date ,", "lyg is a rare lymphoproliferative disease characterized by angiocentric and angiodestructive ebv - positive b - cell proliferation of uncertain malignant potential , which might be associated with exuberant reactive t cell infiltration2,9 ) .", "lyg affects men more than women with ratio 2 to 1 and presents mainly in adults between the fourth and sixth decades of life , although patients of all ages can be affected .", "its occurrence in children is quite rare4 ) . there were three cases of lymphomatoid granulomatosis with onset after all .", "two of these patients died of the disease , only one obtained benefit from acyclovir therapy7 ) .", "lyg usually affects lungs primarily , and it may also present several significant extrapulmonary manifestations , affecting skin , kidney , spleen , and the central nervous system .", "involvement of the spine has been rarely reported . to my best knowledge , this is the first report of thoracic spinal lyg presenting with unique progressive myelopathy .", "however , numerous granulomatous lesions in the central nervous system and lungs are highly suggestive of lyg .", "lyg with central nervous system involvement , observed in approximately 30% of the affected patients , indicates poor prognosis4,9,11 ) .", "there was only one case report about lyg with spinal involvement . in that case ,", "lyg involve cervical spinal cord , and the patient submitted four cycles of adjuvant cyclophosphamide , doxorubicin , vincristine and prednisone ( chop ) .", "but the patient died 5 months after the treatment due to systemic progression of the disease7 ) .", "a grading system of lyg is based on the number of atypical lymphocytes , ebv - positive b - cells and amount of necrosis5 ) .", "pathophysiologically , grade i lesions consist of a polymorphous angiocentric and angiodestructive infiltrate of lymphocytes , plasma cells , and histocytes , with or without eosinophils .", "large lymphoid cells or immunoblasts were less than five in high power field ( 400 ) or absent . if large lymphoid cells or immunoblasts were present , there would be no cellular atypia and minimal - to - absent necrosis .", "grade ii lesions consisted of large lymphoid cells with less than twenty ebv - positive cells in high power field with some cellular atypia in small lymphoid cells , and necrosis was more common .", "grade iii lesions , angiocentric lymphomas , consisted of large atypical cells with more than twenty ebv positive cells in high power field with the marked cytologic atypia in both small and large lymphoid cells4,13 ) . to date , no standard treatment has been established for lyg and treatments approach to systemic lyg include drugs acting on the immune system(corticosteroids , interferon alfa-2b ,", "cyclosporine a , anti - cd20 monoclonal antibody - rituximab ) , mono- or combined chemotherapy , radiotherapy , and autologous stem cell transplantation .", "several treatments with rituximab have been reported , with or without chemotherapy or radiotherapy3,9,10,15 ) .", "rituximab was approved for the treatment of recurrent , poorly differentiated refractory or follicular cd20 positive b - cell non - hodgkin 's lymphoma14 ) . in this case , immunohistochemical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells .", "in situ hybridization revealed positive ebv encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg .", "the patient experienced rapid worsening of neurological symptoms and absence of systemic involvement on pre - operative laboratory findings and radiological imaging , and she therefore was treated with surgical decompression and biopsy on the t3 - 5 epidural lesion and improved neurologic symptoms .", "surgical decompression can improve neurological morbidity of the patient . however , appropriate chemotherapy is highly needed as systemic progression of lyg make poor prognosis .", "the patient responded to rituximab therapy well without adverse effects , and her condition remained stable for 6 years .", "therefore it was suggestive that decompression and rituximab is suitable as the initial therapy for symptomatic spinal lyg .", "this case described an unusual occurrence of thoracic spinal lyg in a 4-year - old girl .", "childhood lyg is a rare disease but should be considered in the nodular pulmonary infiltrates and central nervous system manifestations , especially in the past diagnosis of childhood acute lymphoblastic leukemia .", "awareness of the features of lyg can lead to earlier diagnosis and prompt appropriate therapy ."], "abstract_text": ["<S> lymphomatoid granulomatosis ( lyg ) is a lymphoproliferative disease involving the lungs most frequently ; however , it may also involve the kidneys , skin and especially the central nervous system . </S>", "<S> unique initial presentation of spinal involvement is extremely rare and epidural lesion of thoracic spine has not been reported . </S>", "<S> the prognosis for lyg has been reported to be poor , and there currently exists no satisfactory established treatment protocol . the purpose of this study is to report a case of successful treatment with surgery and rituximab combination therapy in thoracic spinal lyg . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["lymphomatoid granulomatosis ( lyg ) is a rare angiocentric and angiodestructive lymphoproliferative disease with granulomatous reaction involving the lungs most frequently , however , it may also involve the kidneys , skin and especially the central nervous system4,6,9,11 ) .", "lyg is characterized by an infiltration of atypical lymphocytoid and plasmocytoid cells , with granulomatous inflammation in an angiocentric and angiodestructive polymorphic cellular infiltrate6,8 ) .", "therefore the purpose of this study is to submit the first report about the case with thoracic spinal lyg after diagnosis with b - precursor childhood acute lymphoblastic leukemia ( all ) .", "after surgical decompression with biopsy on the spinal lesion , diagnosis of grade ii lyg was made and rituximab was administered .", "the patient is in good neurological condition without recurrence at the 6-year follow - up ."], ["a 4-year - old girl was diagnosed with b - precursor all with the involvement of kidneys and pancreas in november , 2004 .", "treatment was initiated according to the pediatric oncology group ( pog ) protocol 900612 ) .", "on april 12 , 2006 , the patient was admitted to pediatric clinic with a complaint of intermittent fever for 3 days . on april 19 , 2006 , 7 days after the admission , the patient developed a gait disturbance .", "neurological examination revealed paraparesis of grade 3 on medical research council ( mrc ) muscle strength grading scale1 ) with increased deep tendon reflex in both ankles , knee jerks , and bilateral positive babinski sign , but her senses were intact .", "cerebrospinal fluid ( csf ) findings were as follows : elevation of protein ( 243.3mg / dl ) , and decrease of glucose ( 44mg / dl ) .", "the condition was suspected to be an acute inflammatory demyelinating polyneuropathy and was treated with intravenous immunoglobulin ( 400 mg / kg / day ) for 5 days .", "however , her paraparesis was aggravated to grade 1 on mrc muscle strength grading scale from april 26 , 2006 , 14 days after the admission , and spinal mri was performed .", "t2 weighted mri demonstrated heterogeneous high signal intensity infiltration on t4 vertebral body and subligamental spreading soft tissue mass on t3 - 5 epidural space from t4 posterior vertebral body , causing spinal cord compression ( fig .", "1 ) . on april 27 , 2006 , laminectomy of t3 , 4 with open biopsy were performed .", "the epidural mass was encircling the spinal cord mainly on ventral surface and was slightly grey to yellow , solid and compact nature .", "a histopathologic examination showed an angiocentric distribution of polymorphous lymphoid infiltrate , with extensive necrosis .", "immunohistoche mical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells ( fig .", "hybridization revealed positivity for epstein - barr virus ( ebv ) encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg .", "one - week post - operative neurologic examination showed paraparesis grade 3 on mrc scale , and bare independent walking but as she still had gait impairment , she received rehabilitation .", "after four weeks of the surgery , she restored her normal state and could walk quite well . at that time", ", there were no pulmonary lesions , so we suspected the spinal lesion as the primary involvement site .", "two weeks after , chest and abdomen ct revealed multiple low density nodular mass on the liver and the lower lobe of the left lung .", "histopathologically , lesions were not blastic in appearance and in immunophenotype , and the lesions were negative for cd10 , tdt , and cd22 , so we ruled out the relapse of all .", "she was treated with rituximab ( 275mg / m / day , 4 times per week ) for 4 weeks and intravenous high dose methylprednisolone ( 30mg / kg per day for 3 days , 20mg / kg per day for 2 days and 10mg / kg per day for 2 days ) .", "chest and abdomen ct showed marked improvement of the lesions 3 months after the treatment compared with the lesions in the images obtained before the treatment .", "a follow - up mri study performed 4 months after the operation documented no evidence of residual mass or recurrence ( fig .", "3 ) . chest and abdomen ct performed 1 year after the operation showed no demonstrable focal lesion in lung field and mediastinum and unremarkable abdominal organs .", "her weakness improved and she was able to walk independently and had no gait impairment .", "but unfortunately we failed to take additional follow - up of mri for the patient , as she was terrified to take mri and full sedation was not possible .", "moreover the parents of the patient did not agree with the idea of taking mri one more time because she was in good state at that time . to date ,"], ["lyg is a rare lymphoproliferative disease characterized by angiocentric and angiodestructive ebv - positive b - cell proliferation of uncertain malignant potential , which might be associated with exuberant reactive t cell infiltration2,9 ) .", "lyg affects men more than women with ratio 2 to 1 and presents mainly in adults between the fourth and sixth decades of life , although patients of all ages can be affected .", "its occurrence in children is quite rare4 ) . there were three cases of lymphomatoid granulomatosis with onset after all .", "two of these patients died of the disease , only one obtained benefit from acyclovir therapy7 ) .", "lyg usually affects lungs primarily , and it may also present several significant extrapulmonary manifestations , affecting skin , kidney , spleen , and the central nervous system .", "involvement of the spine has been rarely reported . to my best knowledge , this is the first report of thoracic spinal lyg presenting with unique progressive myelopathy .", "however , numerous granulomatous lesions in the central nervous system and lungs are highly suggestive of lyg .", "lyg with central nervous system involvement , observed in approximately 30% of the affected patients , indicates poor prognosis4,9,11 ) .", "there was only one case report about lyg with spinal involvement . in that case ,", "lyg involve cervical spinal cord , and the patient submitted four cycles of adjuvant cyclophosphamide , doxorubicin , vincristine and prednisone ( chop ) .", "but the patient died 5 months after the treatment due to systemic progression of the disease7 ) .", "a grading system of lyg is based on the number of atypical lymphocytes , ebv - positive b - cells and amount of necrosis5 ) .", "pathophysiologically , grade i lesions consist of a polymorphous angiocentric and angiodestructive infiltrate of lymphocytes , plasma cells , and histocytes , with or without eosinophils .", "large lymphoid cells or immunoblasts were less than five in high power field ( 400 ) or absent . if large lymphoid cells or immunoblasts were present , there would be no cellular atypia and minimal - to - absent necrosis .", "grade ii lesions consisted of large lymphoid cells with less than twenty ebv - positive cells in high power field with some cellular atypia in small lymphoid cells , and necrosis was more common .", "grade iii lesions , angiocentric lymphomas , consisted of large atypical cells with more than twenty ebv positive cells in high power field with the marked cytologic atypia in both small and large lymphoid cells4,13 ) . to date , no standard treatment has been established for lyg and treatments approach to systemic lyg include drugs acting on the immune system(corticosteroids , interferon alfa-2b ,", "cyclosporine a , anti - cd20 monoclonal antibody - rituximab ) , mono- or combined chemotherapy , radiotherapy , and autologous stem cell transplantation .", "several treatments with rituximab have been reported , with or without chemotherapy or radiotherapy3,9,10,15 ) .", "rituximab was approved for the treatment of recurrent , poorly differentiated refractory or follicular cd20 positive b - cell non - hodgkin 's lymphoma14 ) . in this case , immunohistochemical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells .", "in situ hybridization revealed positive ebv encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg .", "the patient experienced rapid worsening of neurological symptoms and absence of systemic involvement on pre - operative laboratory findings and radiological imaging , and she therefore was treated with surgical decompression and biopsy on the t3 - 5 epidural lesion and improved neurologic symptoms .", "surgical decompression can improve neurological morbidity of the patient . however , appropriate chemotherapy is highly needed as systemic progression of lyg make poor prognosis .", "the patient responded to rituximab therapy well without adverse effects , and her condition remained stable for 6 years .", "therefore it was suggestive that decompression and rituximab is suitable as the initial therapy for symptomatic spinal lyg ."], ["this case described an unusual occurrence of thoracic spinal lyg in a 4-year - old girl .", "childhood lyg is a rare disease but should be considered in the nodular pulmonary infiltrates and central nervous system manifestations , especially in the past diagnosis of childhood acute lymphoblastic leukemia .", "awareness of the features of lyg can lead to earlier diagnosis and prompt appropriate therapy ."]]}
{"article_id": "PMC4897483", "article_text": ["kidney transplant recipients not only have numerous comorbidities associated with renal failure , but also often have additional problems due to chronic immunosuppression [ 13 ] .", "the surgical management of colorectal cancer is risky to the patient in terms of the surgical procedure and the interruption of immunosuppression . in general , these patients are more susceptible to perioperative complications . although open colorectal resection has been practiced for many years in kidney recipient patients , the minimally invasive procedure has been developed and is gaining popularity .", "multiple randomized controlled trials have proven that minimally invasive surgery yields better short - term outcomes than open colorectal surgery in terms of reduced intraoperative bleeding , postoperative pain , and hospital stay , as well as a lower incidence of infection and respiratory complications , with equivalent long - term outcomes [ 4 , 5 ] .", "however , selected high - risk patients may benefit from minimally invasive colorectal resection due to reduced morbidity .", "the aim of this study was to prove the safety and feasibility of laparoscopic and robotic colorectal resection in kidney transplant recipients by evaluating the technical protocol and the short- and long - term outcomes .", "this retrospective review of all kidney transplant recipients diagnosed with colon or rectal cancer who received laparoscopic or robotic resection was conducted between may 2007 and august 2012 at yonsei university college of medicine , south korea .", "data were analyzed using the spss statistical program ( version 18 for windows ; spss inc . ,", "all patients received 4 liters of golytely ( peg-3350 and electrolytes for oral solution , braintree laboratories , inc . ,", "antibiotics and a stress dose of steroids of 100  mg were given on induction of anesthesia and continued postoperatively with tapering dose of steroid .", "a main concern in laparoscopic and robotic colorectal resection is accurate and safe port placement to avoid injury to the transplanted kidney .", "after periumbilical port insertion using an open or closed technique , the abdomen was insufflated with medical co2 to 1214  mmhg pressure .", "the kidney was shifted posterolaterally to provide adequate space for the working ports ( figure 1 ) .", "next , depending on the type of procedure , two right- or left - sided trocars under direct visualization were easily placed . for robotic trocar positioning", ", we used a hybrid technique by which the colon was mobilized laparoscopically and the robot was docked between the legs of the patients and used mainly for pelvic dissection .", "robotic port insertion depended on the location of the kidney : for the right kidney , arms 2 and 3 were on the left and arm 1 was on the right ; for the left kidney , arms 1 and 3 were on the right and arm 2 was on the left .", "the procedure used colonic mobilization and followed total mesorectal excision principles as required for pelvic dissection .", "we started medial to lateral mobilization of the colon 2  cm distal to the sacral promontory going upward toward the inferior mesenteric artery where most of it is ligated at its origin .", "then , lateral dissection followed including splenic flexure mobilization . finally , we dissect the rectum starting posteriorly going deep to the levator ani muscles carefully to avoid hypogastric nerve injury .", "then , we dissect laterally and anteriorly where we should avoid injury to prostrate and neurovascular puddle . usually , we perform a rectal irrigation before transection .", "the specimen extracted from the left iliac fossa assistant port and duple stabling technique was used for bowel anastomosis . regarding the right colectomy cases , medial to lateral approach", "our preferred method is applying complete mesocolic principles where vessel ligation is performed at its origin .", "ten of these recipients were diagnosed with colon or rectal cancer and underwent minimally invasive surgical resection .", "five ( 50% ) patients were males ; the mean patient age was 56.8 ( range , 4772 ) years , and the mean body mass index was 22.4 ( range 20.526.4 )  kg / m .", "nine ( 90% ) patients had hypertension , three ( 30% ) had diabetes mellitus , one patient had a history of hepatitis , and one had a history of chronic pulmonary tuberculosis .", "the american society of anesthesiologists scores were ii in 60% of patients and i and iii in 20% of patients each .", "all rectal patients were evaluated by computed tomography ( ct ) scan and a colonoscopy .", "in addition , magnetic resonance imaging ( mri ) was used for further evaluation of the patients with rectal cancer .", "preoperative stages were i and ii in 40% of the patients and iii in 10% of the patients . no tumor invasion or extension beyond the mesorectal fat with a maximum stage", "preoperative chemoradiation therapy was administered to one patient with low - rectal cancer with a preoperative stage iii ( t3n1  m0 ) .", "four ( 40% ) patients underwent an anterior and a low anterior resection , two ( 20% ) underwent a covering ileostomy and a right hemicolectomy , and one patient ( 10% ) underwent a left hemicolectomy .", "most ( 90% ) procedures were performed laparoscopically and 10% were conducted robotically ( table 1 ) .", "pre- and postoperative laboratory blood findings were evaluated every other day to minimize intra- and postoperative complications ( table 2 ) .", "the mean carcinoembryonic antigen level was 3.66  2.53 ( range 0.729.18 )  ng / ml and the mean white blood cell count was 7 , 315  3,842 ( range 2,99016,520 )  mm .", "three ( 30% ) patients were anemic , with a mean hematocrit of 34.65  7.72% ( range 26.753.4% ) and a mean hemoglobin concentration of 11.38  2.54 ( range 8.917.4 )  g / dl .", "mean preoperative urea and creatinine levels were 27.7  23.62 ( range 11.489.4 )  mg / dl and 1.43  0.98 ( range 0.593.94 )  mg / dl , respectively .", "most of the immunosuppressive medications used pre- and postoperatively were cyclosporine or tacrolimus combined with low dose of steroid ranging between 5  mg to 12  mg . no change in immunosuppressive", "the mean postoperative hemoglobin concentration was 10.63  2.45 ( range 7.616.1 )  g / dl and no patient required a blood transfusion .", "the mean postoperative leukocyte count ( 10,655  2,219 ( range 7,53014,220 )  mm ) was slightly but significantly higher than the preoperative value ( p = 0.032 ) , and no severe leukocytosis or leukopenia was seen .", "mean postoperative urea and creatinine levels were 18.35  17.8 ( range 11.460.7 )  mg / dl and 1.40  0.76 ( range 0.723.09 )  mg / dl , respectively .", "the mean interval between kidney transplantation and surgery was 11.7 ( range 1.322 ) years .", "the mean estimated blood loss was 30  53.74 ( range 0150 )  ml .", "we rarely found adhesions ; most observed were minimal and were noted when the peritoneum was opened accidentally during the kidney transplant or another procedure .", "no significant difference was observed between pre- and postoperative kidney function parameters , and no organ rejection was reported .", "the mean timing of the first oral intake was 3.8  5.81 ( range 27 ) days postoperatively .", "postoperative stages were i in 20% of the patients , ii in 50% of the patients , and iii in 30% of the patients ( table 3 ) .", "one patient underwent adjuvant chemoradiation ( xeloda ( capecitabine ) + radiation ) and four patients underwent adjuvant chemotherapy alone ( fluorouracil , leucovorin , folic acid , oxaliplatin ( folfox ) in three cases , and 5-fluorouracil + leucovorin in one case ) . over a median follow - up period of 31.4  21.57 ( range 263 ) months , no mortality occurred .", "one patient showed liver metastasis 1 year postoperatively , which was treated by radiofrequency ablation and no further recurrence till the present date of our study . in six patients who were followed for a mean of 43.5  9.84 ( range 3263 ) months ,", "the 3-year disease - free rate was 83.3% and the 3-year overall survival rate was 100% ( table 3 ) .", "the risk of colorectal neoplasia is high among renal transplant recipients and is associated with the duration of immunosuppression , regardless of age .", "the reported incidence of colorectal cancer in kidney transplant recipients has ranged from 0 to 0.9% .", "kim et al .   found that the rate of right colon cancer was significantly higher in transplant recipients than in the general cancer population ( 35.2% versus 15.4% ) , whereas the rate of rectal cancer was significantly lower in transplant recipients ( 33.5% versus 46.5% ; p = 0.031 ) .", "also reported that the risk of colorectal cancer was twofold higher in the first posttransplantation year than in the general population , and an additional 2.2-fold higher after the third posttransplantation year .", "in contrast , saidi et al .   found no increased risk of colorectal cancer among transplant recipients compared with the general population and noted the need for further evaluation of this issue .", "parnaby et al .   reported that three population - based studies showed increases of up to twofold in the incidence of colonic but not rectal cancer in renal transplant recipients and that two population - based studies showed threefold and 10-fold increases in the incidence of anal cancer in these patients .", "patients on immunosuppressive medications or who have undergone organ transplantation with comorbidities have increased morbidity ( stress - related complications ) and mortality and a prolonged postoperative recovery period , after major open surgery , including colorectal surgery [ 1215 ] .", "he concluded that the nonspecific immune response appeared to be less affected by laparoscopic surgery compared with open surgery , while specific cell - mediated immunity was equally affected .", "laparoscopic colorectal resection is less physically stressful and offers short - term advantages compared with open resection . technically ,", "selection of the port insertion site is the most important step in a laparoscopic or robotic procedure .", "after the establishment of pneumoperitoneum , the kidney is moved inferolaterally to allow port placement .", "our hospital is one of the leading hospitals in robotic surgery in korea . as we reported in our results , that one case was performed by robotic method . in that case", ", we used a hybrid technique where the colonic mobilization was completed laparoscopically and the da vinci robot was used only for pelvic dissection .", "we found that the robotic port position of the 3rd arm should be positioned on the opposite side of the transplanted kidney because of its close proximity with the anterior superior iliac spine where the kidney is located even after pneumoperitoneum .", "however , any port position even in totally robotic technique could be safe as long as the ports are placed away from transplanted kidney .", "we found no or minimal adhesion during the procedures , which may have been related to peritoneum opening during retroperitoneal dissection in the kidney transplant procedure .", "adhesions from previous surgeries , possibly related to the use of steroids , were not an issue .", "only one case series of laparoscopic - assisted colectomy in three kidney transplant recipients has been published . in that series , the average time since transplantation was 8 ( range 610 ) years and no patient experienced organ rejection . all patients had colon cancer , and all allografts were contralateral to colon resection .", "the mean operative time was 103 ( range 100105 ) min and no complications occurred .", "the average hospital stay was 5 ( range 47 ) days and the mean follow - up time was 17 ( range 340 ) months .", "our series was larger than that described above , and it included patients with colonic and rectal cancer .", "moreover , we included patients who underwent laparoscopic and robotic surgery . in our series ,", "the mean interval between kidney transplantation and diagnosis of colorectal cancer ( 11.7 years ) was slightly longer .", "our mean operative time was 192.5  53.87 ( range 77263 ) min , due to the longer operative time ( 263  min ) of the robotic procedure ; the mean operative time for laparoscopic procedures was 184.6  50.74  min .", "we observed similarly intact renal function and no rejection during the follow - up period .", "two patients in our series had minor complications ( wound seroma and chyloperitoneum ) that were managed conservatively .", "the leukocyte count is normally raised after any major surgical procedure , but no severe leukocytosis or leukopenia occurred in our patients indicating infection or rejection .", "no change in the form or dosage of immunosuppressive medications was required before surgery , and medications were resumed postoperatively under monitoring by the transplant team . in general , these patients are more susceptible to perioperative complications .", "immunosuppression can lead to a higher infection rate and surgical stress can increase immunosuppression , a phenomenon that is more pronounced following open ( versus laparoscopic ) surgical procedures [ 12 , 14 , 15 ] .", "our follow - up period ( mean 31.4  21.57 months ) was longer than that previously reported .", "in addition , the mean follow - up period was 43.5  9.84 months for the six patients in whom long - term outcomes were assessed .", "no mortality occurred ; the 3-year disease - free rate was 83.3% , and the 3-year overall survival rate was 100% . in conclusion , minimally invasive surgery is feasible in kidney transplant recipients , who can benefit from fewer wound - related problems .", "technical proficiency , experience , and the use of a multidisciplinary team approach including an oncologist and transplant team can result in a successful procedure and ensure the safety of the transplanted kidney .", "therefore , minimally invasive colorectal procedures could be considered safe alternatives to open colorectal resection in selected kidney transplant patients ."], "abstract_text": ["<S> \n\n aim . to prove the safety and feasibility of minimally invasive ( laparoscopic and robotic ) colorectal resection in kidney recipients by evaluating the technical protocol and reviewing short- and long - term outcomes . methods . between may 2007 and august 2012 , </S>", "<S> a retrospective review of ten kidney transplant patients diagnosed with colorectal cancer was evaluated for technical tips , short- and long - term outcomes . </S>", "<S> results . </S>", "<S> the mean patients ' age was 56.8  9.91 years and 50% of them were male . </S>", "<S> anterior and low anterior resections were performed in 40% of the patients each ; 20% and 10% of the patients underwent right and left hemicolectomy , respectively . </S>", "<S> most ( 90% ) procedures were performed laparoscopically and 10% were performed robotically . no open conversions . </S>", "<S> mean operating time was 192.5  15  min , blood loss was 30  50  ml , and mean hospital stay was 9.7  5.5 days . </S>", "<S> two ( 20% ) patients had postoperative complications : wound seroma and chyloperitoneum . over a mean follow - up period of 31.4  21.57 months , </S>", "<S> no mortality or kidney rejection occurred . among the six patients followed up for a mean of 43.5  9.84 months , 83.3% were 3-year disease - free and the overall survival rate was 100% . conclusion . </S>", "<S> minimally invasive colorectal resection is likely to be safe and feasible , with fewer complications and acceptable short- and long - term outcomes , in kidney transplant recipients . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Aim", "3. Methods and Materials", "4. Results", "5. Discussion"], "sections": [["kidney transplant recipients not only have numerous comorbidities associated with renal failure , but also often have additional problems due to chronic immunosuppression [ 13 ] .", "the surgical management of colorectal cancer is risky to the patient in terms of the surgical procedure and the interruption of immunosuppression . in general , these patients are more susceptible to perioperative complications . although open colorectal resection has been practiced for many years in kidney recipient patients , the minimally invasive procedure has been developed and is gaining popularity .", "multiple randomized controlled trials have proven that minimally invasive surgery yields better short - term outcomes than open colorectal surgery in terms of reduced intraoperative bleeding , postoperative pain , and hospital stay , as well as a lower incidence of infection and respiratory complications , with equivalent long - term outcomes [ 4 , 5 ] .", "however , selected high - risk patients may benefit from minimally invasive colorectal resection due to reduced morbidity ."], ["the aim of this study was to prove the safety and feasibility of laparoscopic and robotic colorectal resection in kidney transplant recipients by evaluating the technical protocol and the short- and long - term outcomes ."], ["this retrospective review of all kidney transplant recipients diagnosed with colon or rectal cancer who received laparoscopic or robotic resection was conducted between may 2007 and august 2012 at yonsei university college of medicine , south korea .", "data were analyzed using the spss statistical program ( version 18 for windows ; spss inc . ,", "all patients received 4 liters of golytely ( peg-3350 and electrolytes for oral solution , braintree laboratories , inc . ,", "antibiotics and a stress dose of steroids of 100  mg were given on induction of anesthesia and continued postoperatively with tapering dose of steroid .", "a main concern in laparoscopic and robotic colorectal resection is accurate and safe port placement to avoid injury to the transplanted kidney .", "after periumbilical port insertion using an open or closed technique , the abdomen was insufflated with medical co2 to 1214  mmhg pressure .", "the kidney was shifted posterolaterally to provide adequate space for the working ports ( figure 1 ) .", "next , depending on the type of procedure , two right- or left - sided trocars under direct visualization were easily placed . for robotic trocar positioning", ", we used a hybrid technique by which the colon was mobilized laparoscopically and the robot was docked between the legs of the patients and used mainly for pelvic dissection .", "robotic port insertion depended on the location of the kidney : for the right kidney , arms 2 and 3 were on the left and arm 1 was on the right ; for the left kidney , arms 1 and 3 were on the right and arm 2 was on the left .", "the procedure used colonic mobilization and followed total mesorectal excision principles as required for pelvic dissection .", "we started medial to lateral mobilization of the colon 2  cm distal to the sacral promontory going upward toward the inferior mesenteric artery where most of it is ligated at its origin .", "then , lateral dissection followed including splenic flexure mobilization . finally , we dissect the rectum starting posteriorly going deep to the levator ani muscles carefully to avoid hypogastric nerve injury .", "then , we dissect laterally and anteriorly where we should avoid injury to prostrate and neurovascular puddle . usually , we perform a rectal irrigation before transection .", "the specimen extracted from the left iliac fossa assistant port and duple stabling technique was used for bowel anastomosis . regarding the right colectomy cases , medial to lateral approach", "our preferred method is applying complete mesocolic principles where vessel ligation is performed at its origin ."], ["ten of these recipients were diagnosed with colon or rectal cancer and underwent minimally invasive surgical resection .", "five ( 50% ) patients were males ; the mean patient age was 56.8 ( range , 4772 ) years , and the mean body mass index was 22.4 ( range 20.526.4 )  kg / m .", "nine ( 90% ) patients had hypertension , three ( 30% ) had diabetes mellitus , one patient had a history of hepatitis , and one had a history of chronic pulmonary tuberculosis .", "the american society of anesthesiologists scores were ii in 60% of patients and i and iii in 20% of patients each .", "all rectal patients were evaluated by computed tomography ( ct ) scan and a colonoscopy .", "in addition , magnetic resonance imaging ( mri ) was used for further evaluation of the patients with rectal cancer .", "preoperative stages were i and ii in 40% of the patients and iii in 10% of the patients . no tumor invasion or extension beyond the mesorectal fat with a maximum stage", "preoperative chemoradiation therapy was administered to one patient with low - rectal cancer with a preoperative stage iii ( t3n1  m0 ) .", "four ( 40% ) patients underwent an anterior and a low anterior resection , two ( 20% ) underwent a covering ileostomy and a right hemicolectomy , and one patient ( 10% ) underwent a left hemicolectomy .", "most ( 90% ) procedures were performed laparoscopically and 10% were conducted robotically ( table 1 ) .", "pre- and postoperative laboratory blood findings were evaluated every other day to minimize intra- and postoperative complications ( table 2 ) .", "the mean carcinoembryonic antigen level was 3.66  2.53 ( range 0.729.18 )  ng / ml and the mean white blood cell count was 7 , 315  3,842 ( range 2,99016,520 )  mm .", "three ( 30% ) patients were anemic , with a mean hematocrit of 34.65  7.72% ( range 26.753.4% ) and a mean hemoglobin concentration of 11.38  2.54 ( range 8.917.4 )  g / dl .", "mean preoperative urea and creatinine levels were 27.7  23.62 ( range 11.489.4 )  mg / dl and 1.43  0.98 ( range 0.593.94 )  mg / dl , respectively .", "most of the immunosuppressive medications used pre- and postoperatively were cyclosporine or tacrolimus combined with low dose of steroid ranging between 5  mg to 12  mg . no change in immunosuppressive", "the mean postoperative hemoglobin concentration was 10.63  2.45 ( range 7.616.1 )  g / dl and no patient required a blood transfusion .", "the mean postoperative leukocyte count ( 10,655  2,219 ( range 7,53014,220 )  mm ) was slightly but significantly higher than the preoperative value ( p = 0.032 ) , and no severe leukocytosis or leukopenia was seen .", "mean postoperative urea and creatinine levels were 18.35  17.8 ( range 11.460.7 )  mg / dl and 1.40  0.76 ( range 0.723.09 )  mg / dl , respectively .", "the mean interval between kidney transplantation and surgery was 11.7 ( range 1.322 ) years .", "the mean estimated blood loss was 30  53.74 ( range 0150 )  ml .", "we rarely found adhesions ; most observed were minimal and were noted when the peritoneum was opened accidentally during the kidney transplant or another procedure .", "no significant difference was observed between pre- and postoperative kidney function parameters , and no organ rejection was reported .", "the mean timing of the first oral intake was 3.8  5.81 ( range 27 ) days postoperatively .", "postoperative stages were i in 20% of the patients , ii in 50% of the patients , and iii in 30% of the patients ( table 3 ) .", "one patient underwent adjuvant chemoradiation ( xeloda ( capecitabine ) + radiation ) and four patients underwent adjuvant chemotherapy alone ( fluorouracil , leucovorin , folic acid , oxaliplatin ( folfox ) in three cases , and 5-fluorouracil + leucovorin in one case ) . over a median follow - up period of 31.4  21.57 ( range 263 ) months , no mortality occurred .", "one patient showed liver metastasis 1 year postoperatively , which was treated by radiofrequency ablation and no further recurrence till the present date of our study . in six patients who were followed for a mean of 43.5  9.84 ( range 3263 ) months ,", "the 3-year disease - free rate was 83.3% and the 3-year overall survival rate was 100% ( table 3 ) ."], ["the risk of colorectal neoplasia is high among renal transplant recipients and is associated with the duration of immunosuppression , regardless of age .", "the reported incidence of colorectal cancer in kidney transplant recipients has ranged from 0 to 0.9% .", "kim et al .   found that the rate of right colon cancer was significantly higher in transplant recipients than in the general cancer population ( 35.2% versus 15.4% ) , whereas the rate of rectal cancer was significantly lower in transplant recipients ( 33.5% versus 46.5% ; p = 0.031 ) .", "also reported that the risk of colorectal cancer was twofold higher in the first posttransplantation year than in the general population , and an additional 2.2-fold higher after the third posttransplantation year .", "in contrast , saidi et al .   found no increased risk of colorectal cancer among transplant recipients compared with the general population and noted the need for further evaluation of this issue .", "parnaby et al .   reported that three population - based studies showed increases of up to twofold in the incidence of colonic but not rectal cancer in renal transplant recipients and that two population - based studies showed threefold and 10-fold increases in the incidence of anal cancer in these patients .", "patients on immunosuppressive medications or who have undergone organ transplantation with comorbidities have increased morbidity ( stress - related complications ) and mortality and a prolonged postoperative recovery period , after major open surgery , including colorectal surgery [ 1215 ] .", "he concluded that the nonspecific immune response appeared to be less affected by laparoscopic surgery compared with open surgery , while specific cell - mediated immunity was equally affected .", "laparoscopic colorectal resection is less physically stressful and offers short - term advantages compared with open resection . technically ,", "selection of the port insertion site is the most important step in a laparoscopic or robotic procedure .", "after the establishment of pneumoperitoneum , the kidney is moved inferolaterally to allow port placement .", "our hospital is one of the leading hospitals in robotic surgery in korea . as we reported in our results , that one case was performed by robotic method . in that case", ", we used a hybrid technique where the colonic mobilization was completed laparoscopically and the da vinci robot was used only for pelvic dissection .", "we found that the robotic port position of the 3rd arm should be positioned on the opposite side of the transplanted kidney because of its close proximity with the anterior superior iliac spine where the kidney is located even after pneumoperitoneum .", "however , any port position even in totally robotic technique could be safe as long as the ports are placed away from transplanted kidney .", "we found no or minimal adhesion during the procedures , which may have been related to peritoneum opening during retroperitoneal dissection in the kidney transplant procedure .", "adhesions from previous surgeries , possibly related to the use of steroids , were not an issue .", "only one case series of laparoscopic - assisted colectomy in three kidney transplant recipients has been published . in that series , the average time since transplantation was 8 ( range 610 ) years and no patient experienced organ rejection . all patients had colon cancer , and all allografts were contralateral to colon resection .", "the mean operative time was 103 ( range 100105 ) min and no complications occurred .", "the average hospital stay was 5 ( range 47 ) days and the mean follow - up time was 17 ( range 340 ) months .", "our series was larger than that described above , and it included patients with colonic and rectal cancer .", "moreover , we included patients who underwent laparoscopic and robotic surgery . in our series ,", "the mean interval between kidney transplantation and diagnosis of colorectal cancer ( 11.7 years ) was slightly longer .", "our mean operative time was 192.5  53.87 ( range 77263 ) min , due to the longer operative time ( 263  min ) of the robotic procedure ; the mean operative time for laparoscopic procedures was 184.6  50.74  min .", "we observed similarly intact renal function and no rejection during the follow - up period .", "two patients in our series had minor complications ( wound seroma and chyloperitoneum ) that were managed conservatively .", "the leukocyte count is normally raised after any major surgical procedure , but no severe leukocytosis or leukopenia occurred in our patients indicating infection or rejection .", "no change in the form or dosage of immunosuppressive medications was required before surgery , and medications were resumed postoperatively under monitoring by the transplant team . in general , these patients are more susceptible to perioperative complications .", "immunosuppression can lead to a higher infection rate and surgical stress can increase immunosuppression , a phenomenon that is more pronounced following open ( versus laparoscopic ) surgical procedures [ 12 , 14 , 15 ] .", "our follow - up period ( mean 31.4  21.57 months ) was longer than that previously reported .", "in addition , the mean follow - up period was 43.5  9.84 months for the six patients in whom long - term outcomes were assessed .", "no mortality occurred ; the 3-year disease - free rate was 83.3% , and the 3-year overall survival rate was 100% . in conclusion , minimally invasive surgery is feasible in kidney transplant recipients , who can benefit from fewer wound - related problems .", "technical proficiency , experience , and the use of a multidisciplinary team approach including an oncologist and transplant team can result in a successful procedure and ensure the safety of the transplanted kidney .", "therefore , minimally invasive colorectal procedures could be considered safe alternatives to open colorectal resection in selected kidney transplant patients ."]]}
{"article_id": "PMC2988921", "article_text": ["acute occlusion of the superior mesenteric artery ( sma ) causes extensive intestinal necrosis due to the difficulty of early diagnosis , resulting in poor prognosis , with a high postoperative mortality rate of 65.2% .", "recent reports indicate that selective thrombolytic therapy with intraarterial infusion of urokinase is effective for acute sma occlusion diagnosed early after onset .", "early thrombolytic therapy can not always induce complete thrombolysis , and even if intestinal necrosis is avoided by administration of initial thrombolytic therapy , indications for additional thrombolytic therapy or the method for monitoring intestinal viability during subsequent follow - up have not been established . in this report , we present a case of acute sma occlusion diagnosed early after onset that was successfully treated by sequential and intermittent thrombolytic therapy by intraarterial urokinase infusion with angiographic evaluation of blood flow , thereby avoiding intestinal resection .", "an 82-year - old woman with a past history of atrial fibrillation was admitted to our hospital , complaining of acute epigastric pain and stool with fresh blood .", "physical examination was as follows : blood pressure 140/80 , heart rate 70/min , o2 saturation 97% ( room air ) , body temperature 37.2c .", "the abdomen was flat and soft , with no apparent tenderness or sign of peritoneal irritation .", "laboratory data on admission revealed an elevated wbc count and mildly elevated hepatic enzyme values ( wbc 15,400/mm , crp 0.05 mg / dl , ldh 502 iu / l , ast 57 iu / l , alt 21 iu / l , cpk 113", "colonoscopy revealed mildly erosive , edematous mucosa , but no obvious bleeding from the ascending colon to the terminal ileum .", "abdominal enhanced ct showed a filling defect in the proximal portion of the sma main trunk , which led to the diagnosis of acute sma occlusion ( fig .", ", selective sma angiography was performed , showing complete occlusion of the sma around the first jejunal artery branch .", "subsequently , intraarterial bolus infusion of urokinase ( 600,000 iu ) with thrombus suction was performed . as a result ,", "however , the thrombus remained , and intramural blood flow of the affected intestine was not visualized , suggesting possible necrosis of the affected intestine ( fig .", "a mild ischemic change was observed at the intestinal wall from the jejunum 40 cm distal from the treitz ligament to the ascending colon , but no apparent necrosis .", "blood flow in the mesentery was well palpable at the central portion of the sma , while peripheral blood flow was not palpable .", "the operation was completed without intestinal resection or direct removal of the thrombus from the sma .", "24 h after the laparotomy , a second angiography was performed via the catheter that remained in place after first angiography , because clinical signs such as abdominal pain suggested the progression of intestinal ischemia .", "peripheral blood flow was well visualized , and no signs of intestinal necrosis were observed .", "thereafter , thrombolysis with urokinase infusion ( 240,000 iu ) and suction of the residual thrombus were performed .", "in addition , 36 h after the laparotomy , a third angiography and thrombolysis ( urokinase 240,000 iu ) were performed , showing improvement in peripheral blood flow via the gradual development of collateral blood flow .", "follow - up carefully monitored the possible onset of shower embolism by the residual thrombus .", "intestinal blood flow was well visualized on ct angiography performed 48 h after laparotomy ( 60 h after the first thrombolytic therapy ) .", "the patient began to take food by mouth on the 6th day after laparotomy and was discharged from our hospital 35 days after laparotomy .", "early diagnosis and treatment contribute to the improvement of therapeutic results for acute sma occlusion . a delay in diagnosis results in an extremely high mortality rate due to extensive intestinal necrosis ; however , successful resection of necrotic intestine causes short bowel syndrome , severely impairing quality of life .", "recent reports indicate that enhanced ct is feasible for early diagnosis of acute sma occlusion .", "when acute sma occlusion is suspected from symptom , clinical history and physical findings , enhanced ct plays a role not only in identifying the causal portion , but also in determining the subsequent treatment plan .", "early thrombolytic therapy is effective for acute sma occlusion in avoiding extensive intestinal resection . since the first report by jamieson et al .", ", reports that support the usefulness of selective thrombolytic therapy via a catheter for angiography have increased .", "the  golden hour , the time during which the viability of the ischemic intestine can be preserved , varies , dependent on the portion and extent of occlusion . muneoka et al .  ", "studied 23 cases of acute sma occlusion treated by selective thrombolytic therapy in japan , and concluded that the golden hour is 5 h for occlusion of the sma main trunk and 12 h for the occlusion of the distal sma .", "the goal of thrombolytic therapy is complete thrombolysis , but administration over 48 h increases the risk of complication .", "easy bleeding due to fibrinolysis promoted by urokinase and shower emboli induced by the release of residual thrombi are known to be the major complications associated with thrombolytic therapy with urokinase .", "careful monitoring during 24 h after the completion of thrombolytic therapy is essential , and emergency laparotomy is mandatory if intestinal necrosis is suspected .", "urokinase has no influence during surgery as its half - life in blood is only 16 min . in the present case ,", "the thrombus was detected in the sma main trunk by angiography performed 5 h from onset .", "therefore , judging that we were within the golden hour , we added urokinase infusion and thrombus suction via the angiography catheter .", "however , the thrombus remained left , and visualization of intramural blood flow was poor , therefore , we selected exploratory laparotomy . on the other hand , when no apparent intestinal necrosis is observed during exploratory laparotomy , a clinical consensus concerning the indication of additional thrombolytic therapy or optimal methods for evaluation of intestinal blood flow or viability in the subsequent follow - up is not yet established .", "even if intestinal necrosis can be avoided , intestinal ischemia would cause perforation or stricture of the affected intestine . from 1985 to 2007 , 45 cases of thrombolytic therapy for acute sma occlusion were reported in japan .", "complete thrombolysis was successful in 28 cases ( 63.6% ) , and 17 cases ( 36.4% ) received laparotomy following thrombolytic therapy . among those 17 patients ,", "intestinal resection was performed in 5 cases ( 11.1% ) due to intestinal necrosis ; the remaining 12 cases ( 26.7% ) without intestinal necrosis at laparotomy were thereafter free from intestinal necrosis .", "of those 12 patients , 2 received angiographic reevaluation of blood flow and subsequent second - look operation , 3 received embolectomy , and 6 were carefully observed by laboratory examination and physical findings without any invasive treatments . in the present case , we performed thrombolytic therapy with angiographic evaluation of blood flow , sequentially and intermittently , after exploratory laparotomy , which enabled us not only to accurately evaluate intestinal blood flow or viability , but also to avoid a second - look operation . in conclusion ,", "sequential and intermittent thrombolytic therapy with meticulous angiographic evaluation of blood flow is effective in the early stage of acute sma . in particular , it is much less invasive than strategies described in previous reports and enables accurate evaluation of intestinal blood flow or viability over the time course ."], "abstract_text": ["<S> acute occlusion of the superior mesenteric artery ( sma ) causes extensive bowel necrosis , resulting in a poor prognosis with an extremely high mortality rate . </S>", "<S> an 82-year - old woman was admitted to our hospital with the complaint of abdominal pain . </S>", "<S> she was diagnosed as having acute sma occlusion by enhanced ct . </S>", "<S> five hours from onset , the first thrombolytic therapy with urokinase was performed , but failed to complete thrombolysis and recanalization of peripheral blood flow . </S>", "<S> an exploratory laparotomy following the first thrombolytic therapy showed a mild ischemic change in the affected intestine and mesentery , but no sign of necrosis . </S>", "<S> after the laparotomy , local thrombolytic therapy with angiographic evaluation of blood flow at 24 , 36 and 48 h from the first thrombolysis was performed . as a result , </S>", "<S> the residual thrombus disappeared and all branches of the sma became well visualized . </S>", "<S> the patient was discharged well without a second - look operation or major bowel resection . </S>", "<S> sequential intermittent thrombolytic therapy with meticulous angiographic evaluation of blood flow is effective for early - stage acute sma occlusion . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["acute occlusion of the superior mesenteric artery ( sma ) causes extensive intestinal necrosis due to the difficulty of early diagnosis , resulting in poor prognosis , with a high postoperative mortality rate of 65.2% .", "recent reports indicate that selective thrombolytic therapy with intraarterial infusion of urokinase is effective for acute sma occlusion diagnosed early after onset .", "early thrombolytic therapy can not always induce complete thrombolysis , and even if intestinal necrosis is avoided by administration of initial thrombolytic therapy , indications for additional thrombolytic therapy or the method for monitoring intestinal viability during subsequent follow - up have not been established . in this report , we present a case of acute sma occlusion diagnosed early after onset that was successfully treated by sequential and intermittent thrombolytic therapy by intraarterial urokinase infusion with angiographic evaluation of blood flow , thereby avoiding intestinal resection ."], ["an 82-year - old woman with a past history of atrial fibrillation was admitted to our hospital , complaining of acute epigastric pain and stool with fresh blood .", "physical examination was as follows : blood pressure 140/80 , heart rate 70/min , o2 saturation 97% ( room air ) , body temperature 37.2c .", "the abdomen was flat and soft , with no apparent tenderness or sign of peritoneal irritation .", "laboratory data on admission revealed an elevated wbc count and mildly elevated hepatic enzyme values ( wbc 15,400/mm , crp 0.05 mg / dl , ldh 502 iu / l , ast 57 iu / l , alt 21 iu / l , cpk 113", "colonoscopy revealed mildly erosive , edematous mucosa , but no obvious bleeding from the ascending colon to the terminal ileum .", "abdominal enhanced ct showed a filling defect in the proximal portion of the sma main trunk , which led to the diagnosis of acute sma occlusion ( fig .", ", selective sma angiography was performed , showing complete occlusion of the sma around the first jejunal artery branch .", "subsequently , intraarterial bolus infusion of urokinase ( 600,000 iu ) with thrombus suction was performed . as a result ,", "however , the thrombus remained , and intramural blood flow of the affected intestine was not visualized , suggesting possible necrosis of the affected intestine ( fig .", "a mild ischemic change was observed at the intestinal wall from the jejunum 40 cm distal from the treitz ligament to the ascending colon , but no apparent necrosis .", "blood flow in the mesentery was well palpable at the central portion of the sma , while peripheral blood flow was not palpable .", "the operation was completed without intestinal resection or direct removal of the thrombus from the sma .", "24 h after the laparotomy , a second angiography was performed via the catheter that remained in place after first angiography , because clinical signs such as abdominal pain suggested the progression of intestinal ischemia .", "peripheral blood flow was well visualized , and no signs of intestinal necrosis were observed .", "thereafter , thrombolysis with urokinase infusion ( 240,000 iu ) and suction of the residual thrombus were performed .", "in addition , 36 h after the laparotomy , a third angiography and thrombolysis ( urokinase 240,000 iu ) were performed , showing improvement in peripheral blood flow via the gradual development of collateral blood flow .", "follow - up carefully monitored the possible onset of shower embolism by the residual thrombus .", "intestinal blood flow was well visualized on ct angiography performed 48 h after laparotomy ( 60 h after the first thrombolytic therapy ) .", "the patient began to take food by mouth on the 6th day after laparotomy and was discharged from our hospital 35 days after laparotomy ."], ["early diagnosis and treatment contribute to the improvement of therapeutic results for acute sma occlusion . a delay in diagnosis results in an extremely high mortality rate due to extensive intestinal necrosis ; however , successful resection of necrotic intestine causes short bowel syndrome , severely impairing quality of life .", "recent reports indicate that enhanced ct is feasible for early diagnosis of acute sma occlusion .", "when acute sma occlusion is suspected from symptom , clinical history and physical findings , enhanced ct plays a role not only in identifying the causal portion , but also in determining the subsequent treatment plan .", "early thrombolytic therapy is effective for acute sma occlusion in avoiding extensive intestinal resection . since the first report by jamieson et al .", ", reports that support the usefulness of selective thrombolytic therapy via a catheter for angiography have increased .", "the  golden hour , the time during which the viability of the ischemic intestine can be preserved , varies , dependent on the portion and extent of occlusion . muneoka et al .  ", "studied 23 cases of acute sma occlusion treated by selective thrombolytic therapy in japan , and concluded that the golden hour is 5 h for occlusion of the sma main trunk and 12 h for the occlusion of the distal sma .", "the goal of thrombolytic therapy is complete thrombolysis , but administration over 48 h increases the risk of complication .", "easy bleeding due to fibrinolysis promoted by urokinase and shower emboli induced by the release of residual thrombi are known to be the major complications associated with thrombolytic therapy with urokinase .", "careful monitoring during 24 h after the completion of thrombolytic therapy is essential , and emergency laparotomy is mandatory if intestinal necrosis is suspected .", "urokinase has no influence during surgery as its half - life in blood is only 16 min . in the present case ,", "the thrombus was detected in the sma main trunk by angiography performed 5 h from onset .", "therefore , judging that we were within the golden hour , we added urokinase infusion and thrombus suction via the angiography catheter .", "however , the thrombus remained left , and visualization of intramural blood flow was poor , therefore , we selected exploratory laparotomy . on the other hand , when no apparent intestinal necrosis is observed during exploratory laparotomy , a clinical consensus concerning the indication of additional thrombolytic therapy or optimal methods for evaluation of intestinal blood flow or viability in the subsequent follow - up is not yet established .", "even if intestinal necrosis can be avoided , intestinal ischemia would cause perforation or stricture of the affected intestine . from 1985 to 2007 , 45 cases of thrombolytic therapy for acute sma occlusion were reported in japan .", "complete thrombolysis was successful in 28 cases ( 63.6% ) , and 17 cases ( 36.4% ) received laparotomy following thrombolytic therapy . among those 17 patients ,", "intestinal resection was performed in 5 cases ( 11.1% ) due to intestinal necrosis ; the remaining 12 cases ( 26.7% ) without intestinal necrosis at laparotomy were thereafter free from intestinal necrosis .", "of those 12 patients , 2 received angiographic reevaluation of blood flow and subsequent second - look operation , 3 received embolectomy , and 6 were carefully observed by laboratory examination and physical findings without any invasive treatments . in the present case , we performed thrombolytic therapy with angiographic evaluation of blood flow , sequentially and intermittently , after exploratory laparotomy , which enabled us not only to accurately evaluate intestinal blood flow or viability , but also to avoid a second - look operation . in conclusion ,", "sequential and intermittent thrombolytic therapy with meticulous angiographic evaluation of blood flow is effective in the early stage of acute sma . in particular , it is much less invasive than strategies described in previous reports and enables accurate evaluation of intestinal blood flow or viability over the time course ."]]}
{"article_id": "PMC4993619", "article_text": ["advances in imaging technology have continued , and recently there has been increasing interest in the use of standing whole - body stereoradiography ( sr ) when evaluating spinal deformity .", "the purported advantages of sr include lack of magnification , lack of parallax distortion , simultaneous two - dimensional ( anteroposterior and lateral ) high - quality imaging of the entire skeleton with lower radiation exposure , as well as capabilities for three - dimensional image reconstruction.2 however , both arms must be forward flexed at the shoulders during sr imaging to reduce obstruction by arm overlapping the thoracic and lumbar regions , and a previous study has found arm positioning , particularly arms flexed with hands touching the clavicle , may change cervical sagittal alignment during xr imaging .", "previous studies have demonstrated that arm positioning is an important factor during xr imaging , changing sagittal alignment parameters of the thoracic and lumbar spine.3 \n 4 \n 5 \n 6 \n 7 \n 8 in fact , park et al found that lateral whole - spine xrs with hands positioned touching the clavicle were associated with a decrease in t1 slope ( t1-s ) , posterior translation of the head , hypolordotic cervical spine , and downward gazing when compared with standing lateral cervical xrs with arms relaxed on either side of the body.9 \n  to our knowledge , no study to date has compared cervical sagittal alignment parameters obtained from standing lateral cervical xrs versus lateral whole - body srs .", "the purpose of this study is to evaluate the correlation and reliability of cervical sagittal alignment parameters measured from lateral cervical xrs compared with lateral whole - body eos srs ( eos imaging , paris , france ) .", "we hypothesized that the difference in arm position and in the trajectory of the radiation beam between standard cervical xr and sr would result in a difference for all cervical sagittal alignment parameters .", "we retrospectively evaluated adult patients with a primary diagnosis of cervical deformity treated by a single surgeon between january 2010 and december 2014 with both lateral cervical xrs and lateral srs obtained within 1  week of each other . at our institution ,", "lateral cervical xrs are routinely obtained with the patient standing , with the arms in a relaxed neutral position , hanging at the side of the body ; this image is compared with srs obtained with eos imaging , which are acquired with patients standing and arms forward flexed at the shoulders .", "cervical sagittal alignment measurements were performed using a picture archiving and communications system , with each xr measured side by side on the same computer monitor to allow equal magnification / resolution of the cervical region from the lateral whole - body sr compared with the lateral cervical xr ( fig .", "were measured by two independent spine surgeon reviewers on two separate occasions , using the following cervical sagittal alignment parameters : c2c7 sagittal cobb angle ( sca ) , c2c7 sagittal vertical axis ( sva ) , c1c7 translational distance ( c1c7 ) , t1-s , neck tilt ( nt ) , and thoracic inlet angle ( tia ) ( fig .", "cobb angle was measured by drawing the angle between a line parallel to the inferior end plate of c2 and a line parallel to the inferior end plate of c7.10 positive values demonstrate kyphotic alignment and negative values , lordotic alignment .", "the distance between the vertical plumb line from the center of c2 and the vertical line from the posterosuperior corner of c7 was measured for c2c7 sva .", "translational distance was defined by the distance from anterior tubercle of c1 to the vertical line from the posteroinferior corner of c7 to evaluate the horizontal translation of the head position.10 t1-s was defined by the angle between the line parallel to the upper end plate of t1 and the horizontal line.11 nt is the angle formed by the line from the mid - t1 upper end plate to the cranial - most aspect of the midsternum and the vertical line from the cranial - most aspect of the midsternum .", "tia was then calculated by the formula tia  =  t1 slope  +  nt.12 \n  the measuring parameters from cervical xr and sr were analyzed using r package  irr . ", "the intrarater reliability was quantified by intraclass correlation coefficient ( icc ) ( 3 , 1 ) , termed by shrout and fleiss.13 this type of icc is appropriate when study patients are a random sample of the underlying patient population , and a test and retest are only two occasions of interest .", "only two variance components are involved : the variance of study subjects and the variance of random errors .", "this type of icc is appropriate when both study patients and raters are a random sample from their respectively underlying populations .", "three variance components are involved : the variance of the study subjects , the variance of the raters , and the variance of random errors .", "icc values of more than 0.75 represent excellent reliability , values between 0.4 and 0.75 represent fair to good reliability , and value less than 0.4 represent poor reliability.14 the relationships between the parameters obtained from xrs and those from srs were compared by correlation analysis ( pearson correlation coefficient ) and paired t test .", "a total of 35 ( 12 male , 23 female ) patients , with mean age of 59 years old , were included in the study .", "intrarater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr .", "c1c7 demonstrated the highest intrarater icc in both groups ( 0.994 xr , 0.995 sr ) , with nt having the lowest intrarater icc in both groups ( 0.799 xr , 0.791 sr ; table 1 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "note : 95% ci is the 95% upper and lower boundaries for the confidence interval of icc .", "icc ( 3 , 1 ) : two random sample components model , two tails .", "interrater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , except nt and tia parameters , which had fair reliability .", "interrater icc was highest for sva ( 0.990 ) in the xr group and c1c7 ( 0.978 ) in the sr group , with nt having the lowest interrater icc in both groups ( 0.465 xr , 0.414 sr ; table 2 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is 95% upper and lower boundaries for the confidence interval of icc", ". icc ( 2 , 1 ) : three random sample components model , two tails .", "we found excellent correlations between xr and sr measurements for most cervical sagittal alignment parameters , with sca , sva , and c1c7 having r  >  0.90 .", "t1-s and tia were between 0.75 and 0.90 , and only nt had an r  <  0.70 .", "we found a significant difference between the groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p  <  0.001 ) and c1c7 ( 1.02 cm lower , p  <  0.001 ) .", "there were no differences between the groups for the other cervical sagittal alignment parameters ( sca , t1-s , nt , and tia ; tables 3 and 4 , fig .", "icc values of cervical alignment parameters between lateral cervical radiograph and lateral whole - body stereoradiography .", "abbreviations : icc , intraclass correlation coefficient ; nt , neck tilt ; sca , sagittal cobb angle ; sva , sagittal vertical axis ; td , translational distance ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "intrarater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr .", "c1c7 demonstrated the highest intrarater icc in both groups ( 0.994 xr , 0.995 sr ) , with nt having the lowest intrarater icc in both groups ( 0.799 xr , 0.791 sr ; table 1 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is the 95% upper and lower boundaries for the confidence interval of icc .", "icc ( 3 , 1 ) : two random sample components model , two tails .", "interrater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , except nt and tia parameters , which had fair reliability .", "interrater icc was highest for sva ( 0.990 ) in the xr group and c1c7 ( 0.978 ) in the sr group , with nt having the lowest interrater icc in both groups ( 0.465 xr , 0.414 sr ; table 2 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is 95% upper and lower boundaries for the confidence interval of icc .", "icc ( 2 , 1 ) : three random sample components model , two tails .", "we found excellent correlations between xr and sr measurements for most cervical sagittal alignment parameters , with sca , sva , and c1c7 having r  >  0.90 .", "t1-s and tia were between 0.75 and 0.90 , and only nt had an r  <  0.70 . we found a significant difference between the groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p  <  0.001 ) and c1c7 ( 1.02 cm lower , p  <  0.001 ) .", "there were no differences between the groups for the other cervical sagittal alignment parameters ( sca , t1-s , nt , and tia ; tables 3 and 4 , fig .", "icc values of cervical alignment parameters between lateral cervical radiograph and lateral whole - body stereoradiography .", "abbreviations : icc , intraclass correlation coefficient ; nt , neck tilt ; sca , sagittal cobb angle ; sva , sagittal vertical axis ; td , translational distance ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "cervical sagittal imbalance remains a complex problem,9 \n 15 and optimal imaging to assess cervical sagittal alignment parameters is important during patient evaluation and particularly preoperative planning .", "when cervical sagittal imbalance or deformity is identified , additional standing full - length spine xrs are recommended to assess whether other regions of the spine may also have deformity and contribute to global sagittal imbalance .", "however , obtaining several separate xrs is often time - consuming with repeated radiation exposure .", "recent advances in imaging techniques include full - body sr , which allows low - dose , high - quality imaging of the entire spinal column and pelvis , as well as assessing for concomitant length discrepancy / deformity of the lower extremities and joints ( hips , knees , ankles).16 despite the purported advantages of sr , the difference in arm positioning compared with the standing lateral cervical xr and the effect on cervical sagittal alignment parameters is not completely understood .", "our study found most cervical sagittal alignment parameters ( sca , sva , and c1c7 ) measured from lateral cervical xrs and lateral whole - body eos srs had excellent correlation , with excellent intra- and interrater reliability in both groups .", "our results regarding sca are similar to a previous report by vidal et al that also found excellent intra- ( icc  =  0.847 to 0.955 ) and interrater ( icc  =  0.846 to 0.954 ) reliability for sca measured on sr imaging , both in the upper cervical spine ( c1c3 ) and lower cervical spine ( c3c7).14 however , this study was performed in patients with adolescent idiopathic scoliosis , and although global sagittal alignment using the c7 plumb line was also found to have excellent reliability , other cervical sagittal alignment parameters were not evaluated .", "intra- and interrater reliability were lowest for nt and tia , which had fair reliability .", "we postulate the lower reliability for nt and tia was due to poor visualization of the sternum on both imaging techniques , and they may be suboptimal cervical sagittal alignment parameters in the setting of spinal deformity . similarly , there was only fair correlation between the xr and sr groups for nt , which further emphasizes that nt may not be an ideal cervical sagittal alignment parameter .", "the most important finding from our study was the significant difference in mean values for both sva and c1c7 between the xr and sr groups .", "we found the sr group had a mean sva that was 0.68 cm lower and mean c1c7 that was 1.02 cm lower than the xr group , although there was excellent correlation with r  >  0.900 for both measurements .", "the most plausible explanation for our findings is the difference in arm positioning during xr and sr imaging , with forward arm flexion during sr causing the head position to shift posteriorly .", "our findings are similar to a previous study by park et al , who evaluated cervical sagittal alignment parameters in 101 asymptomatic adults and found that lateral whole - spine xrs with arms flexed in the hand - touching - clavicle position also resulted in posterior head translation compared with lateral cervical xrs.9 however , the study also found the arm - flexed position in the whole - spine xrs resulted in significantly lower t1-s , cervical hypolordosis , and downward gaze .", "our study did not find differences in t1-s or sca ; however , this discrepancy may be explained by the smaller sample size of our study , as well as the difference in studied population between symptomatic adults with cervical deformity versus asymptomatic adults .", "we postulate that patients with symptomatic cervical deformity have degenerative changes with less flexibility of the subaxial spine . also", ", the forward flexion arm position for sr is not as exaggerated as the whole - spine lateral xr , which requires additional muscle activation / strain with elbow and wrist flexion to touch the clavicles . weaknesses of our study include the retrospective design and the potential for selection bias based on nonconsecutive sampling of patients .", "also , changes in pain or worsening deformity over time may have resulted in differences in cervical sagittal alignment parameters ; however , we attempted to reduce this confounding variable by including only patients with xr and sr images obtained less than 1  week apart . also , our study may have had insufficient sample size to avoid type ii error for various cervical sagittal alignment parameters .", "based on the findings of our study , we believe sr is a reliable method for determining most cervical sagittal alignment parameters and a viable alternative to lateral cervical xr despite differences in arm position during imaging . however , measurements requiring visualization of the sternum such as nt and tia may not be reliable and do not appear to be optimal cervical sagittal alignment parameters .", "also , sr results in slight posterior head translation and subsequently lower sva and c17 translational distance compared with lateral cervical xr and may underestimate regional cervical sagittal alignment parameters in patients with cervical deformity or sagittal imbalance .", "the lower radiation exposure using single sr has to be weighed against its higher cost compared with xr ."], "abstract_text": ["<S> study design \n retrospective analysis.objective \n to evaluate the correlation and reliability of cervical sagittal alignment parameters obtained from lateral cervical radiographs ( xrs ) compared with lateral whole - body stereoradiographs ( srs).methods \n we evaluated adults with cervical deformity using both lateral xrs and lateral srs obtained within 1  week of each other between 2010 and 2014 . </S>", "<S> xr and sr images were measured by two independent spine surgeons using the following sagittal alignment parameters : c2c7 sagittal cobb angle ( sca ) , c2c7 sagittal vertical axis ( sva ) , c1c7 translational distance ( c17 ) , t1 slope ( t1-s ) , neck tilt ( nt ) , and thoracic inlet angle ( tia ) . </S>", "<S> pearson correlation and paired t test were used for statistical analysis , with intra- and interrater reliability analyzed using intraclass correlation coefficient ( icc).results \n a total of 35 patients were included in the study . </S>", "<S> we found excellent intrarater reliability for all sagittal alignment parameters in both the xr and sr groups with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr . </S>", "<S> interrater reliability was also excellent for all parameters except nt and tia , which had fair reliability . </S>", "<S> we also found excellent correlations between xr and sr measurements for most sagittal alignment parameters ; sca , sva , and c1c7 had r  >  0.90 , and only nt had r  <  0.70 . </S>", "<S> there was a significant difference between groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p  <  0.001 ) and c1c7 ( 1.02 cm lower , p  <  0.001 ) . </S>", "<S> there were no differences between groups for sca , t1-s , nt , and tia.conclusion \n whole - body stereoradiography appears to be a viable alternative for measuring cervical sagittal alignment parameters compared with standard radiography . </S>", "<S> xr and sr demonstrated excellent correlation for most sagittal alignment parameters except nt . </S>", "<S> however , sr had significantly lower average sva and c1c7 measurements than xr </S>", "<S> . the lower radiation exposure using single sr has to be weighed against its higher cost compared with xr . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Intrarater Reliability", "Interrater Reliability", "Correlation Analysis XR versus SR", "Discussion", "Conclusion"], "sections": [["cervical sagittal alignment is an important component of global spinal alignment , allowing optimized energy expenditure during upright human posture by positioning the head over the pelvis and maintaining appropriate horizontal gaze.1 cervical radiographs ( xrs ) are most commonly performed in the standing position with the arms relaxed on either side of the body , which is considered the most physiologic position during standing .", "advances in imaging technology have continued , and recently there has been increasing interest in the use of standing whole - body stereoradiography ( sr ) when evaluating spinal deformity .", "the purported advantages of sr include lack of magnification , lack of parallax distortion , simultaneous two - dimensional ( anteroposterior and lateral ) high - quality imaging of the entire skeleton with lower radiation exposure , as well as capabilities for three - dimensional image reconstruction.2 however , both arms must be forward flexed at the shoulders during sr imaging to reduce obstruction by arm overlapping the thoracic and lumbar regions , and a previous study has found arm positioning , particularly arms flexed with hands touching the clavicle , may change cervical sagittal alignment during xr imaging .", "previous studies have demonstrated that arm positioning is an important factor during xr imaging , changing sagittal alignment parameters of the thoracic and lumbar spine.3 \n 4 \n 5 \n 6 \n 7 \n 8 in fact , park et al found that lateral whole - spine xrs with hands positioned touching the clavicle were associated with a decrease in t1 slope ( t1-s ) , posterior translation of the head , hypolordotic cervical spine , and downward gazing when compared with standing lateral cervical xrs with arms relaxed on either side of the body.9 \n  to our knowledge , no study to date has compared cervical sagittal alignment parameters obtained from standing lateral cervical xrs versus lateral whole - body srs .", "the purpose of this study is to evaluate the correlation and reliability of cervical sagittal alignment parameters measured from lateral cervical xrs compared with lateral whole - body eos srs ( eos imaging , paris , france ) .", "we hypothesized that the difference in arm position and in the trajectory of the radiation beam between standard cervical xr and sr would result in a difference for all cervical sagittal alignment parameters ."], ["we retrospectively evaluated adult patients with a primary diagnosis of cervical deformity treated by a single surgeon between january 2010 and december 2014 with both lateral cervical xrs and lateral srs obtained within 1  week of each other . at our institution ,", "lateral cervical xrs are routinely obtained with the patient standing , with the arms in a relaxed neutral position , hanging at the side of the body ; this image is compared with srs obtained with eos imaging , which are acquired with patients standing and arms forward flexed at the shoulders .", "cervical sagittal alignment measurements were performed using a picture archiving and communications system , with each xr measured side by side on the same computer monitor to allow equal magnification / resolution of the cervical region from the lateral whole - body sr compared with the lateral cervical xr ( fig .", "were measured by two independent spine surgeon reviewers on two separate occasions , using the following cervical sagittal alignment parameters : c2c7 sagittal cobb angle ( sca ) , c2c7 sagittal vertical axis ( sva ) , c1c7 translational distance ( c1c7 ) , t1-s , neck tilt ( nt ) , and thoracic inlet angle ( tia ) ( fig .", "cobb angle was measured by drawing the angle between a line parallel to the inferior end plate of c2 and a line parallel to the inferior end plate of c7.10 positive values demonstrate kyphotic alignment and negative values , lordotic alignment .", "the distance between the vertical plumb line from the center of c2 and the vertical line from the posterosuperior corner of c7 was measured for c2c7 sva .", "translational distance was defined by the distance from anterior tubercle of c1 to the vertical line from the posteroinferior corner of c7 to evaluate the horizontal translation of the head position.10 t1-s was defined by the angle between the line parallel to the upper end plate of t1 and the horizontal line.11 nt is the angle formed by the line from the mid - t1 upper end plate to the cranial - most aspect of the midsternum and the vertical line from the cranial - most aspect of the midsternum .", "tia was then calculated by the formula tia  =  t1 slope  +  nt.12 \n  the measuring parameters from cervical xr and sr were analyzed using r package  irr . ", "the intrarater reliability was quantified by intraclass correlation coefficient ( icc ) ( 3 , 1 ) , termed by shrout and fleiss.13 this type of icc is appropriate when study patients are a random sample of the underlying patient population , and a test and retest are only two occasions of interest .", "only two variance components are involved : the variance of study subjects and the variance of random errors .", "this type of icc is appropriate when both study patients and raters are a random sample from their respectively underlying populations .", "three variance components are involved : the variance of the study subjects , the variance of the raters , and the variance of random errors .", "icc values of more than 0.75 represent excellent reliability , values between 0.4 and 0.75 represent fair to good reliability , and value less than 0.4 represent poor reliability.14 the relationships between the parameters obtained from xrs and those from srs were compared by correlation analysis ( pearson correlation coefficient ) and paired t test ."], ["a total of 35 ( 12 male , 23 female ) patients , with mean age of 59 years old , were included in the study .", "intrarater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr .", "c1c7 demonstrated the highest intrarater icc in both groups ( 0.994 xr , 0.995 sr ) , with nt having the lowest intrarater icc in both groups ( 0.799 xr , 0.791 sr ; table 1 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "note : 95% ci is the 95% upper and lower boundaries for the confidence interval of icc .", "icc ( 3 , 1 ) : two random sample components model , two tails .", "interrater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , except nt and tia parameters , which had fair reliability .", "interrater icc was highest for sva ( 0.990 ) in the xr group and c1c7 ( 0.978 ) in the sr group , with nt having the lowest interrater icc in both groups ( 0.465 xr , 0.414 sr ; table 2 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is 95% upper and lower boundaries for the confidence interval of icc", ". icc ( 2 , 1 ) : three random sample components model , two tails .", "we found excellent correlations between xr and sr measurements for most cervical sagittal alignment parameters , with sca , sva , and c1c7 having r  >  0.90 .", "t1-s and tia were between 0.75 and 0.90 , and only nt had an r  <  0.70 .", "we found a significant difference between the groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p  <  0.001 ) and c1c7 ( 1.02 cm lower , p  <  0.001 ) .", "there were no differences between the groups for the other cervical sagittal alignment parameters ( sca , t1-s , nt , and tia ; tables 3 and 4 , fig .", "icc values of cervical alignment parameters between lateral cervical radiograph and lateral whole - body stereoradiography .", "abbreviations : icc , intraclass correlation coefficient ; nt , neck tilt ; sca , sagittal cobb angle ; sva , sagittal vertical axis ; td , translational distance ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle ."], ["intrarater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr .", "c1c7 demonstrated the highest intrarater icc in both groups ( 0.994 xr , 0.995 sr ) , with nt having the lowest intrarater icc in both groups ( 0.799 xr , 0.791 sr ; table 1 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is the 95% upper and lower boundaries for the confidence interval of icc .", "icc ( 3 , 1 ) : two random sample components model , two tails ."], ["interrater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , except nt and tia parameters , which had fair reliability .", "interrater icc was highest for sva ( 0.990 ) in the xr group and c1c7 ( 0.978 ) in the sr group , with nt having the lowest interrater icc in both groups ( 0.465 xr , 0.414 sr ; table 2 ) .", "abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is 95% upper and lower boundaries for the confidence interval of icc .", "icc ( 2 , 1 ) : three random sample components model , two tails ."], ["we found excellent correlations between xr and sr measurements for most cervical sagittal alignment parameters , with sca , sva , and c1c7 having r  >  0.90 .", "t1-s and tia were between 0.75 and 0.90 , and only nt had an r  <  0.70 . we found a significant difference between the groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p  <  0.001 ) and c1c7 ( 1.02 cm lower , p  <  0.001 ) .", "there were no differences between the groups for the other cervical sagittal alignment parameters ( sca , t1-s , nt , and tia ; tables 3 and 4 , fig .", "icc values of cervical alignment parameters between lateral cervical radiograph and lateral whole - body stereoradiography .", "abbreviations : icc , intraclass correlation coefficient ; nt , neck tilt ; sca , sagittal cobb angle ; sva , sagittal vertical axis ; td , translational distance ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle .", "abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle ."], ["cervical sagittal imbalance remains a complex problem,9 \n 15 and optimal imaging to assess cervical sagittal alignment parameters is important during patient evaluation and particularly preoperative planning .", "when cervical sagittal imbalance or deformity is identified , additional standing full - length spine xrs are recommended to assess whether other regions of the spine may also have deformity and contribute to global sagittal imbalance .", "however , obtaining several separate xrs is often time - consuming with repeated radiation exposure .", "recent advances in imaging techniques include full - body sr , which allows low - dose , high - quality imaging of the entire spinal column and pelvis , as well as assessing for concomitant length discrepancy / deformity of the lower extremities and joints ( hips , knees , ankles).16 despite the purported advantages of sr , the difference in arm positioning compared with the standing lateral cervical xr and the effect on cervical sagittal alignment parameters is not completely understood .", "our study found most cervical sagittal alignment parameters ( sca , sva , and c1c7 ) measured from lateral cervical xrs and lateral whole - body eos srs had excellent correlation , with excellent intra- and interrater reliability in both groups .", "our results regarding sca are similar to a previous report by vidal et al that also found excellent intra- ( icc  =  0.847 to 0.955 ) and interrater ( icc  =  0.846 to 0.954 ) reliability for sca measured on sr imaging , both in the upper cervical spine ( c1c3 ) and lower cervical spine ( c3c7).14 however , this study was performed in patients with adolescent idiopathic scoliosis , and although global sagittal alignment using the c7 plumb line was also found to have excellent reliability , other cervical sagittal alignment parameters were not evaluated .", "intra- and interrater reliability were lowest for nt and tia , which had fair reliability .", "we postulate the lower reliability for nt and tia was due to poor visualization of the sternum on both imaging techniques , and they may be suboptimal cervical sagittal alignment parameters in the setting of spinal deformity . similarly , there was only fair correlation between the xr and sr groups for nt , which further emphasizes that nt may not be an ideal cervical sagittal alignment parameter .", "the most important finding from our study was the significant difference in mean values for both sva and c1c7 between the xr and sr groups .", "we found the sr group had a mean sva that was 0.68 cm lower and mean c1c7 that was 1.02 cm lower than the xr group , although there was excellent correlation with r  >  0.900 for both measurements .", "the most plausible explanation for our findings is the difference in arm positioning during xr and sr imaging , with forward arm flexion during sr causing the head position to shift posteriorly .", "our findings are similar to a previous study by park et al , who evaluated cervical sagittal alignment parameters in 101 asymptomatic adults and found that lateral whole - spine xrs with arms flexed in the hand - touching - clavicle position also resulted in posterior head translation compared with lateral cervical xrs.9 however , the study also found the arm - flexed position in the whole - spine xrs resulted in significantly lower t1-s , cervical hypolordosis , and downward gaze .", "our study did not find differences in t1-s or sca ; however , this discrepancy may be explained by the smaller sample size of our study , as well as the difference in studied population between symptomatic adults with cervical deformity versus asymptomatic adults .", "we postulate that patients with symptomatic cervical deformity have degenerative changes with less flexibility of the subaxial spine . also", ", the forward flexion arm position for sr is not as exaggerated as the whole - spine lateral xr , which requires additional muscle activation / strain with elbow and wrist flexion to touch the clavicles . weaknesses of our study include the retrospective design and the potential for selection bias based on nonconsecutive sampling of patients .", "also , changes in pain or worsening deformity over time may have resulted in differences in cervical sagittal alignment parameters ; however , we attempted to reduce this confounding variable by including only patients with xr and sr images obtained less than 1  week apart . also , our study may have had insufficient sample size to avoid type ii error for various cervical sagittal alignment parameters ."], ["based on the findings of our study , we believe sr is a reliable method for determining most cervical sagittal alignment parameters and a viable alternative to lateral cervical xr despite differences in arm position during imaging . however , measurements requiring visualization of the sternum such as nt and tia may not be reliable and do not appear to be optimal cervical sagittal alignment parameters .", "also , sr results in slight posterior head translation and subsequently lower sva and c17 translational distance compared with lateral cervical xr and may underestimate regional cervical sagittal alignment parameters in patients with cervical deformity or sagittal imbalance .", "the lower radiation exposure using single sr has to be weighed against its higher cost compared with xr ."]]}
{"article_id": "PMC2943111", "article_text": ["nucleotide excision repair ( ner ) plays a central role in preserving the genome of prokaryotes and eukaryotes .  ", "this versatile repair system removes structurally and chemically diverse bulky dna lesions , including those induced by exposure to uv light and environmental chemical carcinogens [ 1 , 2 ] .  ", "the vital importance of this mechanism is demonstrated by several human ner - deficiency syndromes including xeroderma pigmentosum ( xp ) , cockayne syndrome ( cs ) , and trichothiodystrophy ( ttd ) .  ", "xp , for example , is characterized by high photosensitivity , hyperpigmentation , premature skin ageing , and proneness to developing skin cancer .  ", "furthermore , the capacity of the ner pathway is important in cancer chemotherapy : ner diminishes   the efficacy of chemotherapeutic agents such as cisplatin , which act via the formation of bulky dna adducts .", "a better understanding of the mechanisms of recognition of dna lesions by the ner system may lead to the design of improved chemotherapeutic drugs that can modulate the repair response .  ", "recent findings reveal that polymorphisms in human ner repair genes have an impact on the repair of dna lesions and cancer   susceptibility [ 6 , 7 ] , as well as on chemotherapeutic efficacy .   ", "the eukaryotic ner pathway is a biologically complicated process and consists of two sub - pathways with different substrate specificity :   global genome ner ( gg - ner ) [ 9 , 10 ] and transcription - coupled repair ( tcr ) [ 1114 ] .  ", "both sub - pathways consist of ordered multistep processes , which differ in the early steps , when the dna lesions are recognized , but converge in the later steps .   in gg - ner ,", "the focus of our present interest , the whole genome is scanned for bulky lesions to initiate the repair process .", "two independent complexes , one involving the xpc / hr23b / centrin 2 proteins [ 1517 ] and the other involving the ddb1/ddb2 heterodimer [ 1821 ] , have been implicated in the early steps of base - damage recognition during ner .  ", "by contrast , the tcr sub - pathway is activated by a stalled rna polymerase during transcription .  ", "once the lesion is detected , the two sub - pathways proceed in an essentially identical manner to excise it :   the multisubunit transcription factor .", "tfiih , containing helicases xpb , and xpd , is recruited to the lesion site , followed by xpa , the single - strand dna binding protein rpa , and the two nucleases xpg and xpf - ercc1 .  ", "once assembled , a 2432 oligonucleotide stretch containing the lesion is excised from the damaged strand .", "finally , gap resynthesis by dna polymerases  ,  , and    and ligation by dna ligase i complete the ner process .   ", "one remarkable characteristic of the ner pathway is its ability to excise an astounding variety of chemically and structurally diverse lesions , and the rates of repair can vary over several orders of magnitude .  ", "however , the differences in the structural and thermodynamic properties of the lesions that control the diverse ner efficiencies have remained elusive .   it has been suggested that the ner factors do not recognize the lesion itself , but rather the local distortions and destabilizations in the dna that are associated with it [ 2430 ] .   a number of different properties of damaged dna that elicit the ner response have been proposed .  ", "these   include disruption of watson - crick hydrogen bonding [ 24 , 31 ] , kinks in the damaged dna ,   thermodynamic destabilization [ 24 , 29 , 33 ] , diminished base stacking [ 34 , 35 ] , local conformational flexibility , and flipped - out bases in the unmodified complementary strand [ 3740 ] .   a crystal structure of   yeast rad4/rad23", ", the homolog of the human ner recognition factor xpc / hr23b , bound to dna containing a cyclobutane pyrimidine dimer , shows that rad4/rad23 inserts a -hairpin through the dna duplex and expels two mismatched thymines in the undamaged strand out of the duplex to bind with the enzyme   ( pdb i d : 2qsg ) .  ", "this structure suggests that lesions which thermodynamically destabilize the dna duplex and facilitate the flipping of base pairs and the intrusion of the beta - hairpin are good substrates to the ner machinery :   the more locally destabilized the lesion , the better it is repaired .   ", "the modulation of ner susceptibility for the same lesion by neighboring base sequence context , is however , a relatively unexplored area .  ", "if a lesion is better repaired in one sequence context than the other , a lesion - induced mutational hotspot could result .   in order to elucidate the relationship between ner efficiency and base sequence - governed dna distortion and destabilization induced by a bulky dna adduct , we have employed as a model system the major lesion derived from the cancer - causing compound benzo[a]pyrene ( b[a]p ) .  ", "b[a]p is the most well - studied member in a family of ubiquitous environmental pollutants known as polycyclic aromatic hydrocarbons .", "the tumorigenic metabolite of b[a]p   is the diol epoxide r7 , t8-dihydroxy - t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ( b[a]pde ) .  ", "this intermediate reacts with dna and rna ; the most abundantly stable adduct produced in mammalian cells [ 4446 ] is the 10s ( + ) -trans - anti - b[a]p - n - dg adduct ( [ g * ] ) ( figure 1(a ) ) , the focus of our work .   this adduct , unless removed by dna repair mechanisms , is highly mutagenic [ 48 , 49 ] .", "we have investigated the identical 10s ( + ) -trans - anti - b[a]p - n - dg adduct in the six sequence contexts shown in figure 1(b ) , utilizing an array of approaches :   ner in human hela cell extracts , ligation and polyacrylamide gel electrophoresis techniques to assess bending properties of the modified duplexes , and structural studies utilizing high resolution nmr methods as well as unrestrained molecular dynamics ( md ) simulations .  ", "the position of the b[a]p ring system in the b - dna minor groove , directed 5 along the modified strand , was first determined by nmr in the 5-  c[g*]c - i  sequence in 1992 , but sequence - governed structural details as well as dynamic properties remained to be elucidated .   one important motivation   for our work   was to explore the role of nearby guanine amino groups on the structural properties and ner susceptibilities of these duplexes .  ", "the key difference in these duplexes is the presence and positioning of guanines flanking the [ g * ] , either immediately adjacent to the lesion or beyond :   the b[a]p rings compete for space   with the bulky amino group of guanine on the minor groove side of b - dna , which we anticipated would differentially impact the structures of the damaged duplexes in a sequence context - dependent manner .   a further motivation was to explore the role of differing sequence contexts beyond the lesion that vary in intrinsic flexibility .  ", "we hypothesized that subtle but critical structural effects governed by sequence context would manifest themselves by impacting ner efficiencies .   ", "our results determined that sequence context could cause an up to four - fold difference in relative ner susceptibility , with even distant neighbors influencing ner .  ", "locally disturbed watson - crick hydrogen bonding and flexible bending are two key sequence - governed structural distortions caused by this lesion that the ner machinery appears to recognize with different efficiencies .   more generally , different lesions in varied sequence contexts will cause different kinds of distortions ; thus , the extent of the local thermodynamic destabilization will also vary ; we hypothesize that it is the extent and type of destabilization that determines the relative ner efficiency .", "\n the 5-  c[g * ]  , 5-  g[g * ] c  , and 5-  i[g * ]  sequences .  ", "high resolution nmr solution studies have shown that the bulky aromatic b[a]p residue is positioned in the minor groove on the 5-side of [ g * ]   in the 5-  c[g*]g  and 5-  g[g*]c  duplexes ( figure 2 ) .   however , there are sequence - governed differences in some of the structural features .  ", "duplex ,   nmr studies revealed that the c  :  g base pair on the 5-side of [ g * ] is severely disturbed .  ", "in the case of the sequence - isomer 5-  g[g*]c  duplex , this perturbance is not observed .  ", "on the other hand , analyses of md simulations [ 51 , 52 ] based on the nmr data revealed significant   unwinding near the lesion site combined with an anomalously enlarged roll ( figure 3 ) , not observed in the 5-  c[g*]g  duplex .  ", "duplex , which is a manifestation of a kink    that is highly flexible .  ", "this flexible bend is caused on a molecular level by the severe untwisting and enlarged roll determined by md from the nmr data : dna bending is largely caused by increased roll , which is correlated with untwisting [ 5557 ] .  ", "the underlying structural reasons for the disturbed watson - crick hydrogen bond in the 5- ", ", the bulky amino group on g20 ( figure 3 ) , which is partner to the c on the 5 side of [ g * ] , is sterically crowded by the b[a]p ring system since both are on the minor groove side , and hence this c5  :  g20 base pair is episodically denatured ( figure 3(a ) ) ;   for the 5-  g[g*]c  case , the b[a]p rings crowd the g6 amino group , and in this case the crowding is relieved by the severe untwisting accompanied by the increased roll , which produces the flexible bend observed by gel electrophoresis", ".    investigations with the 5-  i[g*]c  sequence context substantiated the critical role of the guanine amino group since   ", "i  ( figure 1(b ) ) lacks this group :   the gel electrophoretic manifestation of a flexible bend was abolished .", "the nmr data showed conformational heterogeneity in minor groove conformations , and the md simulations showed episodic denaturation of one of the two hydrogen bonds at the i : c base pair , explaining the heterogeneity .", "the repair efficiency relative to 5-  c[g*]c - i  , the standard sequence utilized in many nmr and ner studies [ 53 , 58 ] , is 4.1  0.2 , 1.7  0.2 and 1.3  0.2 for the 5-  c[g*]g  , 5-  g[g*]c  and 5-  i[g*]c  duplexes , respectively ( figure 4 ) .  ", "duplex , dynamic episodic denaturation of watson  crick base pairing flanking the lesion on the 5-side   correlates with the greatest ner susceptibility while the flexible bend in 5-  g[g*]c  is a less pronounced ner recognition signal , and the disturbance to one hydrogen bond in the 5-  i[g*]c  case provides a still lesser signal [ 52 , 53 ] in this series .  \n", "the 5-  c[g * ] c - i  and 5-  t[g * ] t - ii  sequence contexts . the 5-  c[g*]c - ii  and 5-  t[g*]t - ii ", "while a single , well - defined minor groove adduct conformation is observed in 5-  c[g*]c  duplexes , in the 5-  t[g*]t - ii  sequence context , the minor groove - aligned adduct conformation is heterogeneous .   furthermore , polyacrylamide gel electrophoresis studies showed that the adduct induces a rigid bend in the 5-c[g*]c - ii ", "sequence context , the lesion induces a highly flexible bend [ 59 , 60 ] .  ", "also , the 5-  t[g*]t - ii  11-mer duplex has a lower thermal melting point than the 11-mer 5- ", "duplex   ( the exact difference depends on sequence length )    as expected from the thermodynamic properties of t  :  a and c  :  g watson - crick base pairs   [ 62 , 63 ] .  ", "molecular insights on these experimental observations   were provided by md simulations for the 5-  t[g*]t - ii  and 5-  c[g*]c - ii  duplexes .", "consistent with the conformational heterogeneity observed in the nmr studies   , it was found that the 5-  t[g*]t - ii  duplex is much more dynamic than the 5-  c[g*]c - ii  duplex : the highly dynamic base pair on the 5-side of the lesion exhibits episodic denaturation of one of the two watson - crick hydrogen bonds , in agreement with the partial rupturing of this base pair observed by the nmr methods ; also , the 5-  t[g*]t - ii  duplex shows somewhat increased and more dynamic roll and untwisting compared to the 5-  c[g*]c - ii ", "duplex , consistent with the flexible bend observed only for the 5-  t[g*]t - ii  case ; in addition , the b[a]p ring system exhibits greater mobility and the duplex groove dimensions are more variable .  ", "the differences are accounted for by a coupled series of properties : the intrinsically weaker stacking of t - g compared to c - g steps allows for greater flexibility in the 5-  t[g*]t - ii ", "duplex ;   the weaker t  :  a pair , with only two hydrogen bonds , compared to the c  :  g pair , with three bonds , provides enhanced flexibility ; moreover , the absence of guanine amino groups adjacent to the [ g * ] in the 5-  t[g*]t - ii  case allows for greater mobility for the b[a]p ring system .", "overall , the greater flexibility of the 5-  t[g*]t - ii  sequence is attributable to the absence of the guanine amino group .   the rates of incision in the human hela cell assay relative to 5-  c[g*]c - i ", "is   2.4  0.2 and 1.6  0.2 for   the 5-  t[g*]t - ii  and the 5-  c[g*]c - ii  duplexes , respectively , corresponding to a 1.5  0.2-fold higher - repair efficiency for the 5-  t[g*]t - ii ", "case is consistent with the overall enhanced dynamics manifested in various structural properties , notably watson - crick hydrogen bonding and bending .", "the  5-  c[g*]c - i   and  5-  c[g*]c - ii ", "sequences ( figure 1(b ) ) differ in the   sequences   beyond the nearest neighbors to [ g * ] .    ", "since different sequence steps are known to be differentially flexible [ 57 , 65 ] , we hypothesized that the same minor groove lesion [ 50 , 64 ] with different distant neighbors would be differentially repaired .  ", "polyacrylamide gel electrophoresis and self - ligation circularization experiments revealed that the 5-  c[g*]c - ii ", "duplex is more bent and suggested that it has more torsional flexibility than the 5-  c[g*]c - i  duplex .  ", "the key role is played by the unique -c3-a4-c5- segment in the 5-  c[g*]c - ii  duplex .    ", "duplex originates from the guanine amino group at the c3  :  g20 pair ( figure 5 ) .  ", "this amino group acts as a wedge to open the minor groove ; facilitated by the highly deformable local -c3-a4- base step , the amino group allows the b[a]p ring system to better bury its hydrophobic surface within the groove walls .", "this produces a yet more enlarged minor groove which is coupled with more local untwisting and more enlarged and flexible roll ,   causing the greater bend   in 5-  c[g*]c - ii    ( figure 5 ) .   ", "the ner efficiencies are 1.6  0.2 times greater in the 5-  c[g*]c - ii  than in the 5-  c[g*]c - i  sequence context   showing that distant neighbors to [ g * ] modulate the ner susceptibility .   the greater ner susceptibility for the 5-  c[g*]c - ii ", "duplex is explained by its greater bending with enhanced flexibility : the intrinsic minor groove enlargement caused by both the guanine amino groups [ 55 , 68 ] and the great flexibility of pyrimidine - purine steps , including the c - a step [ 57 , 6972 ]   allow the b[a]p moiety ( figure 5 )   to more favorably position itself , but at the expense of the greater bend that makes it more repair - susceptible .", "we have carried out a series of studies with the same 10s ( + ) -trans - anti - b[a]p - n - dg lesion in a number of sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups .  ", "additionally , we have considered differences in intrinsic flexibility of sequences flanking the lesion .   these are model systems for gaining understanding of ner lesion recognition factors .  ", "we have obtained molecular structural data by nmr and md simulations , bending properties from gel electrophoresis studies , and ner data from human hela cell extracts for all of our investigated sequence contexts ( figure 1(b ) ) .  ", "figure 4 summarizes our key findings and enables us to infer a hierarchy of ner recognition signals for the series of sequences and the single lesion we explored .  ", "we point out here that a variety of structural disturbances are found in each case , which are correlated .", "examples include impaired watson - crick pairing that is accompanied by diminished base stacking , and dna bending towards the major groove , that is induced by a minor groove lesion and is accompanied by minor groove enlargement .  ", "our present model system suggests that disturbed watson - crick base pairing is a better recognition signal than a flexible bend , and that these can act in concert to provide an enhanced signal : for example , for 5-  t[g*]t - ii  one episodically ruptured watson - crick hydrogen bond combined with the flexible bend results in better repair than   just one disturbed hydrogen bond as in 5-  i[g*]c  , or the flexible bend alone in 5-  g[g*]c    ( figure 4 ) .   for our system , steric hindrance between the minor groove - aligned lesion and nearby guanine amino groups , if present , determines the exact nature of the disturbances , depending on exactly where the guanine amino groups are situated .  ", "the intrinsic flexibility of the specific base steps also plays an important role in causing the differential disturbances .  ", "both the nearest neighbor and the more distant neighbor sequence contexts have an impact .   more globally , different lesions may cause different types of distortions depending on the specific nature of the lesion and its sequence context .", "however , regardless of exactly what these distortions are , we hypothesize that they must provide a local thermodynamic destabilization signal for repair to ensue , and the greater the extent of destabilization , the better the repair", ".   the destabilization would facilitate the strand separation , base - flipping , and -hairpin insertion by the xpc / hr23b recognition factor [ 41 , 73 ] needed to initiate ner .   in this way ,", "the ner machinery would excise a large variety of lesions with different efficiencies , by recognizing the thermodynamic impact of the lesions rather than the lesions themselves [ 24 , 29 , 41 , 73 ] .    lesions that resist ner present a great hazard , as they survive to the replication step and produce a mutagenic outcome ; such ner - resistant lesions provide an important opportunity for gaining further understanding of the mechanism utilized by the ner apparatus to recognize different lesions ."], "abstract_text": ["<S> nucleotide excision repair ( ner ) plays a critical role in maintaining the integrity of the genome when damaged by bulky dna lesions , since inefficient repair can cause mutations and human diseases notably cancer .   </S>", "<S> the structural properties of dna lesions that determine their relative susceptibilities to ner are therefore of great interest .   as a model system </S>", "<S> , we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene ( b[a]p ) , \n                   10s ( + ) -trans - anti - b[a]p - n2-dg in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups .   </S>", "<S> we have obtained molecular structural data by nmr and md simulations , bending properties from gel electrophoresis studies , and ner data obtained from human hela cell extracts for our six investigated sequence contexts .   </S>", "<S> this model system suggests that disturbed watson - crick base pairing is a better recognition signal than a flexible bend , and that these can act in concert to provide an enhanced signal . </S>", "<S> steric hinderance between the minor groove - aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances .   both nearest neighbor and more distant neighbor sequence contexts have an impact .   </S>", "<S> regardless of the exact distortions , we hypothesize that they provide a local thermodynamic destabilization signal for repair . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Nearest Neighbor Base Sequence Context Impacts NER of the  ", "3.  Distant Neighbor Base  Sequence Context Affects NER of the ", "4. Understanding Repairability Differences: the Degree of Local Thermodynamic Destabilization Is a Unifying Hypothesis"], "sections": [["nucleotide excision repair ( ner ) plays a central role in preserving the genome of prokaryotes and eukaryotes .  ", "this versatile repair system removes structurally and chemically diverse bulky dna lesions , including those induced by exposure to uv light and environmental chemical carcinogens [ 1 , 2 ] .  ", "the vital importance of this mechanism is demonstrated by several human ner - deficiency syndromes including xeroderma pigmentosum ( xp ) , cockayne syndrome ( cs ) , and trichothiodystrophy ( ttd ) .  ", "xp , for example , is characterized by high photosensitivity , hyperpigmentation , premature skin ageing , and proneness to developing skin cancer .  ", "furthermore , the capacity of the ner pathway is important in cancer chemotherapy : ner diminishes   the efficacy of chemotherapeutic agents such as cisplatin , which act via the formation of bulky dna adducts .", "a better understanding of the mechanisms of recognition of dna lesions by the ner system may lead to the design of improved chemotherapeutic drugs that can modulate the repair response .  ", "recent findings reveal that polymorphisms in human ner repair genes have an impact on the repair of dna lesions and cancer   susceptibility [ 6 , 7 ] , as well as on chemotherapeutic efficacy .   ", "the eukaryotic ner pathway is a biologically complicated process and consists of two sub - pathways with different substrate specificity :   global genome ner ( gg - ner ) [ 9 , 10 ] and transcription - coupled repair ( tcr ) [ 1114 ] .  ", "both sub - pathways consist of ordered multistep processes , which differ in the early steps , when the dna lesions are recognized , but converge in the later steps .   in gg - ner ,", "the focus of our present interest , the whole genome is scanned for bulky lesions to initiate the repair process .", "two independent complexes , one involving the xpc / hr23b / centrin 2 proteins [ 1517 ] and the other involving the ddb1/ddb2 heterodimer [ 1821 ] , have been implicated in the early steps of base - damage recognition during ner .  ", "by contrast , the tcr sub - pathway is activated by a stalled rna polymerase during transcription .  ", "once the lesion is detected , the two sub - pathways proceed in an essentially identical manner to excise it :   the multisubunit transcription factor .", "tfiih , containing helicases xpb , and xpd , is recruited to the lesion site , followed by xpa , the single - strand dna binding protein rpa , and the two nucleases xpg and xpf - ercc1 .  ", "once assembled , a 2432 oligonucleotide stretch containing the lesion is excised from the damaged strand .", "finally , gap resynthesis by dna polymerases  ,  , and    and ligation by dna ligase i complete the ner process .   ", "one remarkable characteristic of the ner pathway is its ability to excise an astounding variety of chemically and structurally diverse lesions , and the rates of repair can vary over several orders of magnitude .  ", "however , the differences in the structural and thermodynamic properties of the lesions that control the diverse ner efficiencies have remained elusive .   it has been suggested that the ner factors do not recognize the lesion itself , but rather the local distortions and destabilizations in the dna that are associated with it [ 2430 ] .   a number of different properties of damaged dna that elicit the ner response have been proposed .  ", "these   include disruption of watson - crick hydrogen bonding [ 24 , 31 ] , kinks in the damaged dna ,   thermodynamic destabilization [ 24 , 29 , 33 ] , diminished base stacking [ 34 , 35 ] , local conformational flexibility , and flipped - out bases in the unmodified complementary strand [ 3740 ] .   a crystal structure of   yeast rad4/rad23", ", the homolog of the human ner recognition factor xpc / hr23b , bound to dna containing a cyclobutane pyrimidine dimer , shows that rad4/rad23 inserts a -hairpin through the dna duplex and expels two mismatched thymines in the undamaged strand out of the duplex to bind with the enzyme   ( pdb i d : 2qsg ) .  ", "this structure suggests that lesions which thermodynamically destabilize the dna duplex and facilitate the flipping of base pairs and the intrusion of the beta - hairpin are good substrates to the ner machinery :   the more locally destabilized the lesion , the better it is repaired .   ", "the modulation of ner susceptibility for the same lesion by neighboring base sequence context , is however , a relatively unexplored area .  ", "if a lesion is better repaired in one sequence context than the other , a lesion - induced mutational hotspot could result .   in order to elucidate the relationship between ner efficiency and base sequence - governed dna distortion and destabilization induced by a bulky dna adduct , we have employed as a model system the major lesion derived from the cancer - causing compound benzo[a]pyrene ( b[a]p ) .  ", "b[a]p is the most well - studied member in a family of ubiquitous environmental pollutants known as polycyclic aromatic hydrocarbons .", "the tumorigenic metabolite of b[a]p   is the diol epoxide r7 , t8-dihydroxy - t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ( b[a]pde ) .  ", "this intermediate reacts with dna and rna ; the most abundantly stable adduct produced in mammalian cells [ 4446 ] is the 10s ( + ) -trans - anti - b[a]p - n - dg adduct ( [ g * ] ) ( figure 1(a ) ) , the focus of our work .   this adduct , unless removed by dna repair mechanisms , is highly mutagenic [ 48 , 49 ] .", "we have investigated the identical 10s ( + ) -trans - anti - b[a]p - n - dg adduct in the six sequence contexts shown in figure 1(b ) , utilizing an array of approaches :   ner in human hela cell extracts , ligation and polyacrylamide gel electrophoresis techniques to assess bending properties of the modified duplexes , and structural studies utilizing high resolution nmr methods as well as unrestrained molecular dynamics ( md ) simulations .  ", "the position of the b[a]p ring system in the b - dna minor groove , directed 5 along the modified strand , was first determined by nmr in the 5-  c[g*]c - i  sequence in 1992 , but sequence - governed structural details as well as dynamic properties remained to be elucidated .   one important motivation   for our work   was to explore the role of nearby guanine amino groups on the structural properties and ner susceptibilities of these duplexes .  ", "the key difference in these duplexes is the presence and positioning of guanines flanking the [ g * ] , either immediately adjacent to the lesion or beyond :   the b[a]p rings compete for space   with the bulky amino group of guanine on the minor groove side of b - dna , which we anticipated would differentially impact the structures of the damaged duplexes in a sequence context - dependent manner .   a further motivation was to explore the role of differing sequence contexts beyond the lesion that vary in intrinsic flexibility .  ", "we hypothesized that subtle but critical structural effects governed by sequence context would manifest themselves by impacting ner efficiencies .   ", "our results determined that sequence context could cause an up to four - fold difference in relative ner susceptibility , with even distant neighbors influencing ner .  ", "locally disturbed watson - crick hydrogen bonding and flexible bending are two key sequence - governed structural distortions caused by this lesion that the ner machinery appears to recognize with different efficiencies .   more generally , different lesions in varied sequence contexts will cause different kinds of distortions ; thus , the extent of the local thermodynamic destabilization will also vary ; we hypothesize that it is the extent and type of destabilization that determines the relative ner efficiency ."], ["\n the 5-  c[g * ]  , 5-  g[g * ] c  , and 5-  i[g * ]  sequences .  ", "high resolution nmr solution studies have shown that the bulky aromatic b[a]p residue is positioned in the minor groove on the 5-side of [ g * ]   in the 5-  c[g*]g  and 5-  g[g*]c  duplexes ( figure 2 ) .   however , there are sequence - governed differences in some of the structural features .  ", "duplex ,   nmr studies revealed that the c  :  g base pair on the 5-side of [ g * ] is severely disturbed .  ", "in the case of the sequence - isomer 5-  g[g*]c  duplex , this perturbance is not observed .  ", "on the other hand , analyses of md simulations [ 51 , 52 ] based on the nmr data revealed significant   unwinding near the lesion site combined with an anomalously enlarged roll ( figure 3 ) , not observed in the 5-  c[g*]g  duplex .  ", "duplex , which is a manifestation of a kink    that is highly flexible .  ", "this flexible bend is caused on a molecular level by the severe untwisting and enlarged roll determined by md from the nmr data : dna bending is largely caused by increased roll , which is correlated with untwisting [ 5557 ] .  ", "the underlying structural reasons for the disturbed watson - crick hydrogen bond in the 5- ", ", the bulky amino group on g20 ( figure 3 ) , which is partner to the c on the 5 side of [ g * ] , is sterically crowded by the b[a]p ring system since both are on the minor groove side , and hence this c5  :  g20 base pair is episodically denatured ( figure 3(a ) ) ;   for the 5-  g[g*]c  case , the b[a]p rings crowd the g6 amino group , and in this case the crowding is relieved by the severe untwisting accompanied by the increased roll , which produces the flexible bend observed by gel electrophoresis", ".    investigations with the 5-  i[g*]c  sequence context substantiated the critical role of the guanine amino group since   ", "i  ( figure 1(b ) ) lacks this group :   the gel electrophoretic manifestation of a flexible bend was abolished .", "the nmr data showed conformational heterogeneity in minor groove conformations , and the md simulations showed episodic denaturation of one of the two hydrogen bonds at the i : c base pair , explaining the heterogeneity .", "the repair efficiency relative to 5-  c[g*]c - i  , the standard sequence utilized in many nmr and ner studies [ 53 , 58 ] , is 4.1  0.2 , 1.7  0.2 and 1.3  0.2 for the 5-  c[g*]g  , 5-  g[g*]c  and 5-  i[g*]c  duplexes , respectively ( figure 4 ) .  ", "duplex , dynamic episodic denaturation of watson  crick base pairing flanking the lesion on the 5-side   correlates with the greatest ner susceptibility while the flexible bend in 5-  g[g*]c  is a less pronounced ner recognition signal , and the disturbance to one hydrogen bond in the 5-  i[g*]c  case provides a still lesser signal [ 52 , 53 ] in this series .  \n", "the 5-  c[g * ] c - i  and 5-  t[g * ] t - ii  sequence contexts . the 5-  c[g*]c - ii  and 5-  t[g*]t - ii ", "while a single , well - defined minor groove adduct conformation is observed in 5-  c[g*]c  duplexes , in the 5-  t[g*]t - ii  sequence context , the minor groove - aligned adduct conformation is heterogeneous .   furthermore , polyacrylamide gel electrophoresis studies showed that the adduct induces a rigid bend in the 5-c[g*]c - ii ", "sequence context , the lesion induces a highly flexible bend [ 59 , 60 ] .  ", "also , the 5-  t[g*]t - ii  11-mer duplex has a lower thermal melting point than the 11-mer 5- ", "duplex   ( the exact difference depends on sequence length )    as expected from the thermodynamic properties of t  :  a and c  :  g watson - crick base pairs   [ 62 , 63 ] .  ", "molecular insights on these experimental observations   were provided by md simulations for the 5-  t[g*]t - ii  and 5-  c[g*]c - ii  duplexes .", "consistent with the conformational heterogeneity observed in the nmr studies   , it was found that the 5-  t[g*]t - ii  duplex is much more dynamic than the 5-  c[g*]c - ii  duplex : the highly dynamic base pair on the 5-side of the lesion exhibits episodic denaturation of one of the two watson - crick hydrogen bonds , in agreement with the partial rupturing of this base pair observed by the nmr methods ; also , the 5-  t[g*]t - ii  duplex shows somewhat increased and more dynamic roll and untwisting compared to the 5-  c[g*]c - ii ", "duplex , consistent with the flexible bend observed only for the 5-  t[g*]t - ii  case ; in addition , the b[a]p ring system exhibits greater mobility and the duplex groove dimensions are more variable .  ", "the differences are accounted for by a coupled series of properties : the intrinsically weaker stacking of t - g compared to c - g steps allows for greater flexibility in the 5-  t[g*]t - ii ", "duplex ;   the weaker t  :  a pair , with only two hydrogen bonds , compared to the c  :  g pair , with three bonds , provides enhanced flexibility ; moreover , the absence of guanine amino groups adjacent to the [ g * ] in the 5-  t[g*]t - ii  case allows for greater mobility for the b[a]p ring system .", "overall , the greater flexibility of the 5-  t[g*]t - ii  sequence is attributable to the absence of the guanine amino group .   the rates of incision in the human hela cell assay relative to 5-  c[g*]c - i ", "is   2.4  0.2 and 1.6  0.2 for   the 5-  t[g*]t - ii  and the 5-  c[g*]c - ii  duplexes , respectively , corresponding to a 1.5  0.2-fold higher - repair efficiency for the 5-  t[g*]t - ii ", "case is consistent with the overall enhanced dynamics manifested in various structural properties , notably watson - crick hydrogen bonding and bending ."], ["the  5-  c[g*]c - i   and  5-  c[g*]c - ii ", "sequences ( figure 1(b ) ) differ in the   sequences   beyond the nearest neighbors to [ g * ] .    ", "since different sequence steps are known to be differentially flexible [ 57 , 65 ] , we hypothesized that the same minor groove lesion [ 50 , 64 ] with different distant neighbors would be differentially repaired .  ", "polyacrylamide gel electrophoresis and self - ligation circularization experiments revealed that the 5-  c[g*]c - ii ", "duplex is more bent and suggested that it has more torsional flexibility than the 5-  c[g*]c - i  duplex .  ", "the key role is played by the unique -c3-a4-c5- segment in the 5-  c[g*]c - ii  duplex .    ", "duplex originates from the guanine amino group at the c3  :  g20 pair ( figure 5 ) .  ", "this amino group acts as a wedge to open the minor groove ; facilitated by the highly deformable local -c3-a4- base step , the amino group allows the b[a]p ring system to better bury its hydrophobic surface within the groove walls .", "this produces a yet more enlarged minor groove which is coupled with more local untwisting and more enlarged and flexible roll ,   causing the greater bend   in 5-  c[g*]c - ii    ( figure 5 ) .   ", "the ner efficiencies are 1.6  0.2 times greater in the 5-  c[g*]c - ii  than in the 5-  c[g*]c - i  sequence context   showing that distant neighbors to [ g * ] modulate the ner susceptibility .   the greater ner susceptibility for the 5-  c[g*]c - ii ", "duplex is explained by its greater bending with enhanced flexibility : the intrinsic minor groove enlargement caused by both the guanine amino groups [ 55 , 68 ] and the great flexibility of pyrimidine - purine steps , including the c - a step [ 57 , 6972 ]   allow the b[a]p moiety ( figure 5 )   to more favorably position itself , but at the expense of the greater bend that makes it more repair - susceptible ."], ["we have carried out a series of studies with the same 10s ( + ) -trans - anti - b[a]p - n - dg lesion in a number of sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups .  ", "additionally , we have considered differences in intrinsic flexibility of sequences flanking the lesion .   these are model systems for gaining understanding of ner lesion recognition factors .  ", "we have obtained molecular structural data by nmr and md simulations , bending properties from gel electrophoresis studies , and ner data from human hela cell extracts for all of our investigated sequence contexts ( figure 1(b ) ) .  ", "figure 4 summarizes our key findings and enables us to infer a hierarchy of ner recognition signals for the series of sequences and the single lesion we explored .  ", "we point out here that a variety of structural disturbances are found in each case , which are correlated .", "examples include impaired watson - crick pairing that is accompanied by diminished base stacking , and dna bending towards the major groove , that is induced by a minor groove lesion and is accompanied by minor groove enlargement .  ", "our present model system suggests that disturbed watson - crick base pairing is a better recognition signal than a flexible bend , and that these can act in concert to provide an enhanced signal : for example , for 5-  t[g*]t - ii  one episodically ruptured watson - crick hydrogen bond combined with the flexible bend results in better repair than   just one disturbed hydrogen bond as in 5-  i[g*]c  , or the flexible bend alone in 5-  g[g*]c    ( figure 4 ) .   for our system , steric hindrance between the minor groove - aligned lesion and nearby guanine amino groups , if present , determines the exact nature of the disturbances , depending on exactly where the guanine amino groups are situated .  ", "the intrinsic flexibility of the specific base steps also plays an important role in causing the differential disturbances .  ", "both the nearest neighbor and the more distant neighbor sequence contexts have an impact .   more globally , different lesions may cause different types of distortions depending on the specific nature of the lesion and its sequence context .", "however , regardless of exactly what these distortions are , we hypothesize that they must provide a local thermodynamic destabilization signal for repair to ensue , and the greater the extent of destabilization , the better the repair", ".   the destabilization would facilitate the strand separation , base - flipping , and -hairpin insertion by the xpc / hr23b recognition factor [ 41 , 73 ] needed to initiate ner .   in this way ,", "the ner machinery would excise a large variety of lesions with different efficiencies , by recognizing the thermodynamic impact of the lesions rather than the lesions themselves [ 24 , 29 , 41 , 73 ] .    lesions that resist ner present a great hazard , as they survive to the replication step and produce a mutagenic outcome ; such ner - resistant lesions provide an important opportunity for gaining further understanding of the mechanism utilized by the ner apparatus to recognize different lesions ."]]}
{"article_id": "PMC2688912", "article_text": ["differences in life span between males and females are commonly observed across many species . for example , where the heterogametic sex ( xy sex chromosomes ) is male , as in humans and drosophila , females tend to live longer than males .", "similarly , in caenorhabditis elegans , where the hermaphrodite has two x chromosomes ( xx ) and the male has one ( xo ) , the hermaphrodite tends to live longer .", "in contrast , in most bird species , where the heterogametic sex is female ( zw sex chromosomes ) , males tend to live longer than females .", "genetic and environmental interventions that affect life span tend to have a greater effect in one sex than the other .", "for example , reduced insulin / insulin - like growth factor 1 ( igf-1 ) signaling and dietary restriction tend to increase life span more in females than males in drosophila and mammals , whereas mild stress tends to increase life span more in males than in females , at least in drosophila .", "quantitative genetic analyses have revealed a different genetic architecture of life span in males versus females .", "for example , quantitative trait loci ( qtls ) that affect life span are often sex - specific or sex - biased in drosophila , mice and humans , and studies over the past few years show strikingly different effects of inbreeding in male versus female insects .", "two recent studies in bmc evolutionary biology on the effects of inbreeding in a seed beetle ( bilde et al . ) and in drosophila ( vermeulen et al . ) , respectively , provide additional insight into the genetic factors involved .", "taken together , all these data suggest that the genetic differences between males and females have a significant effect upon aging and life span .", "several possible and potentially overlapping genetic mechanisms have been suggested to explain differences in life span between genders , including asymmetric inheritance of sex chromosomes , differences in physiology , maternal effects , and sex - specific selective pressures .", "for example , the asymmetric inheritance of the sex chromosomes , such that males inherit a single x chromosome in flies , c. elegans and humans , means that in males any x chromosome recessive mutant phenotype will be expressed ( the ' unprotected x ' model ) , whereas in females the presence of the second x chromosome means that there is likely to be a wild - type copy of the gene present , and the recessive phenotype will not be expressed .", "these deleterious recessive mutations could lead to decreased life span , affecting males more than females .", "consistent with this idea , inbreeding ( which will tend to make recessive mutations homozygous ) has been found to cause decreased life span in drosophila , mice and several other species ( called inbreeding depression of life span ) . however , several other studies , including that of vermeulen et al .", "( and see references therein ) have failed to detect inbreeding depression of adult life span , or found effects that varied depending upon the particular strain , sex , or environmental conditions .", "for example , vermeulen et al . mapped a recessive qtl on the second chromosome of drosophila that causes a temperature sensitive reduction in life span in inbred males but not females .", "asymmetric inheritance of mitochondrial genomes and other cytoplasmic genomes is another possible contributor to sex - specific differences in life span . given that the mitochondrial genome is inherited maternally in drosophila and humans", ", natural selection can not act to optimize mitochondrial function or nuclear - mitochondrial genetic interactions in the male genetic background .", "this might result in suboptimal mitochondrial function in males and reduced life span in males relative to females .", "the maternal effect may also contribute to differences in life span between males and female .", "in many species , the mother makes a large contribution of gene products to the egg or embryo , and this has been shown to affect life span in a gender - specific way in certain species . because the mother contributes these materials equally to eggs that will develop as either male or female", ", the genetic differences between male and female zygotes must underlie aspects of the sex - specific effects of maternal products on life span .", "one possibility is that because maternal - effect gene products are being produced by a female genome , they may be more optimized for female offspring , thereby contributing to the reduced life span often observed in males .", "consistent with this idea , maternal effects on life span are greater in males than females for certain species such as the seed beetle .", "finding mechanisms that explain the difference in life span between males and females is hindered by our lack of understanding of the basic mechanisms of aging and underlying causes of mortality .", "life span appears to be limited by the accumulation of irreversible damage , probably including oxidative damage to macromolecules , mutations , and loss of epigenetic regulation , as well as more acute and dynamic modifiers of mortality rates , perhaps including the efficiency of detoxification and excretion .", "mechanistic explanations often involve the concept of tradeoffs , that is , the allocation of energy or other ' resources ' to functions such as reproduction and behavior , at the expense of somatic maintenance pathways required for optimal longevity . in several recent studies , however , it was shown that life span can be increased by dietary restriction or altered insulin / igf-1 signaling without a detectable decrease in reproduction or overall metabolism , and conversely , reproduction can be increased in old female flies with no detectable cost for life span .", "seed beetles ( figure 1 ) could be a particularly powerful model in which to look for trade - offs between somatic maintenance required for optimal life span and other traits such as fecundity .", "the adult is ' facultatively aphagous ' and does not require food or drink , but can rely on nutrient stores accumulated during development .", "have examined the effects of inbreeding on male and female life span in the species callosobruchus maculatus .", "they found that inbreeding reduced fitness of both males and females , as indicated by reduced total reproductive output .", "as expected , female life span was decreased by extreme inbreeding , but surprisingly , male life span was increased .", "previous studies of seed beetles by fox et al .   had found a large maternal effect on life span of males but not females .", "however , the bilde et al . study included an elegant control for maternal effects , in that animals with varying amounts of inbreeding had mothers of the same genotype , thus separating the effects of cytoplasmic factors such as mitochondria and maternally contributed gene products from the effects of inbreeding .", "of course , this result does not rule out an important role for maternal products in modulating life span , but it does show that they are not the direct targets of the observed inbreeding effects .", "one possible explanation for the decrease in female life span is that inbreeding led to homozygosity of recessive alleles that are deleterious for female lifespan , providing support for the unprotected x hypothesis .", "however , this hypothesis can not account for the increase in life span observed in males .", "suggest that the increase in male life span might be due to changes in energy - intensive behaviors , such as a reduction in courtship or aggression , thereby leading to longer life span .", "( a ) male and ( b ) female . the sex specific coloration of the posterior abdominal plate ( pygidium ) is shown .", "the squares are 1 mm . from beanbeetles.org . photographs by lawrence blumer , reproduced with permission .", "sex - specific differences in genetic architecture could contribute to the observed differences in life span and the effects of inbreeding .", "for example , a recent study examined how evolution shapes variation in transcript abundance in male and female drosophila , and sex - specific differences in the mode of transcriptome inheritance were identified . in males ,", "variation in gene expression was found to be due mostly to additive interactions of alleles , whereas in females , gene expression variation was found to be due mostly to non - additive ( epistatic ) interactions between alleles ; a substantial x - chromosome effect was shown to underlie these differences .", "similarly , in the seed beetle , loci affecting life span exhibited more non - additive interactions ( dominance ) in females than in males .", "given that additive variation responds to selection more quickly , because additive variation does not involve interactions of multiple loci , sex - specific differences in selection could underlie aspects of sexual dimorphism in life span observed in many species .", "sex - specific selective pressures that result in higher male reproductive fitness may contribute to sexual dimorphism of life span .", "for example , costly male - biased metabolism or behaviors , such as aggression or specific courtship behaviors , might be positively selected for , but could result in decreased life - span in males relative to females .", "future studies may be directed toward further study of the underlying differences in genetic architecture between males and females , in particular , testing the idea that deleterious alleles affecting life span may be more exposed to selection in males than in females due to reduced non - additive effects in males , thereby reducing inbreeding load for male - specific deleterious alleles in the population .", "it will be particularly interesting to ask if the increased life span observed in highly inbred male seed beetles by bilde et al .  ", "can be found to correlate with a reduction in specific costly aspects of metabolism , or behaviors such as locomotion and aggression .", "we thank sergey nuzhdin for helpful discussions . this work was supported by grants from the department of health and human services to jt ( 1r01ag011833 ) and to ma ( 1r01gm073039 ) ."], "abstract_text": ["<S> several possible and potentially overlapping genetic mechanisms have been suggested to explain differences in life span between males and females . </S>", "<S> two recent papers in bmc evolutionary biology on the effects of inbreeding provide additional insight into the genetic architecture underlying life span differences between genders in two different insects . </S>"], "labels": null, "section_names": ["None", "Asymmetric inheritance of sex chromosomes and maternal effects", "Aging mechanisms and trade-offs", "Sex-specific genetic architectures", "Acknowledgements"], "sections": [["differences in life span between males and females are commonly observed across many species . for example , where the heterogametic sex ( xy sex chromosomes ) is male , as in humans and drosophila , females tend to live longer than males .", "similarly , in caenorhabditis elegans , where the hermaphrodite has two x chromosomes ( xx ) and the male has one ( xo ) , the hermaphrodite tends to live longer .", "in contrast , in most bird species , where the heterogametic sex is female ( zw sex chromosomes ) , males tend to live longer than females .", "genetic and environmental interventions that affect life span tend to have a greater effect in one sex than the other .", "for example , reduced insulin / insulin - like growth factor 1 ( igf-1 ) signaling and dietary restriction tend to increase life span more in females than males in drosophila and mammals , whereas mild stress tends to increase life span more in males than in females , at least in drosophila .", "quantitative genetic analyses have revealed a different genetic architecture of life span in males versus females .", "for example , quantitative trait loci ( qtls ) that affect life span are often sex - specific or sex - biased in drosophila , mice and humans , and studies over the past few years show strikingly different effects of inbreeding in male versus female insects .", "two recent studies in bmc evolutionary biology on the effects of inbreeding in a seed beetle ( bilde et al . ) and in drosophila ( vermeulen et al . ) , respectively , provide additional insight into the genetic factors involved .", "taken together , all these data suggest that the genetic differences between males and females have a significant effect upon aging and life span ."], ["several possible and potentially overlapping genetic mechanisms have been suggested to explain differences in life span between genders , including asymmetric inheritance of sex chromosomes , differences in physiology , maternal effects , and sex - specific selective pressures .", "for example , the asymmetric inheritance of the sex chromosomes , such that males inherit a single x chromosome in flies , c. elegans and humans , means that in males any x chromosome recessive mutant phenotype will be expressed ( the ' unprotected x ' model ) , whereas in females the presence of the second x chromosome means that there is likely to be a wild - type copy of the gene present , and the recessive phenotype will not be expressed .", "these deleterious recessive mutations could lead to decreased life span , affecting males more than females .", "consistent with this idea , inbreeding ( which will tend to make recessive mutations homozygous ) has been found to cause decreased life span in drosophila , mice and several other species ( called inbreeding depression of life span ) . however , several other studies , including that of vermeulen et al .", "( and see references therein ) have failed to detect inbreeding depression of adult life span , or found effects that varied depending upon the particular strain , sex , or environmental conditions .", "for example , vermeulen et al . mapped a recessive qtl on the second chromosome of drosophila that causes a temperature sensitive reduction in life span in inbred males but not females .", "asymmetric inheritance of mitochondrial genomes and other cytoplasmic genomes is another possible contributor to sex - specific differences in life span . given that the mitochondrial genome is inherited maternally in drosophila and humans", ", natural selection can not act to optimize mitochondrial function or nuclear - mitochondrial genetic interactions in the male genetic background .", "this might result in suboptimal mitochondrial function in males and reduced life span in males relative to females .", "the maternal effect may also contribute to differences in life span between males and female .", "in many species , the mother makes a large contribution of gene products to the egg or embryo , and this has been shown to affect life span in a gender - specific way in certain species . because the mother contributes these materials equally to eggs that will develop as either male or female", ", the genetic differences between male and female zygotes must underlie aspects of the sex - specific effects of maternal products on life span .", "one possibility is that because maternal - effect gene products are being produced by a female genome , they may be more optimized for female offspring , thereby contributing to the reduced life span often observed in males .", "consistent with this idea , maternal effects on life span are greater in males than females for certain species such as the seed beetle ."], ["finding mechanisms that explain the difference in life span between males and females is hindered by our lack of understanding of the basic mechanisms of aging and underlying causes of mortality .", "life span appears to be limited by the accumulation of irreversible damage , probably including oxidative damage to macromolecules , mutations , and loss of epigenetic regulation , as well as more acute and dynamic modifiers of mortality rates , perhaps including the efficiency of detoxification and excretion .", "mechanistic explanations often involve the concept of tradeoffs , that is , the allocation of energy or other ' resources ' to functions such as reproduction and behavior , at the expense of somatic maintenance pathways required for optimal longevity . in several recent studies , however , it was shown that life span can be increased by dietary restriction or altered insulin / igf-1 signaling without a detectable decrease in reproduction or overall metabolism , and conversely , reproduction can be increased in old female flies with no detectable cost for life span .", "seed beetles ( figure 1 ) could be a particularly powerful model in which to look for trade - offs between somatic maintenance required for optimal life span and other traits such as fecundity .", "the adult is ' facultatively aphagous ' and does not require food or drink , but can rely on nutrient stores accumulated during development .", "have examined the effects of inbreeding on male and female life span in the species callosobruchus maculatus .", "they found that inbreeding reduced fitness of both males and females , as indicated by reduced total reproductive output .", "as expected , female life span was decreased by extreme inbreeding , but surprisingly , male life span was increased .", "previous studies of seed beetles by fox et al .   had found a large maternal effect on life span of males but not females .", "however , the bilde et al . study included an elegant control for maternal effects , in that animals with varying amounts of inbreeding had mothers of the same genotype , thus separating the effects of cytoplasmic factors such as mitochondria and maternally contributed gene products from the effects of inbreeding .", "of course , this result does not rule out an important role for maternal products in modulating life span , but it does show that they are not the direct targets of the observed inbreeding effects .", "one possible explanation for the decrease in female life span is that inbreeding led to homozygosity of recessive alleles that are deleterious for female lifespan , providing support for the unprotected x hypothesis .", "however , this hypothesis can not account for the increase in life span observed in males .", "suggest that the increase in male life span might be due to changes in energy - intensive behaviors , such as a reduction in courtship or aggression , thereby leading to longer life span .", "( a ) male and ( b ) female . the sex specific coloration of the posterior abdominal plate ( pygidium ) is shown .", "the squares are 1 mm . from beanbeetles.org . photographs by lawrence blumer , reproduced with permission ."], ["sex - specific differences in genetic architecture could contribute to the observed differences in life span and the effects of inbreeding .", "for example , a recent study examined how evolution shapes variation in transcript abundance in male and female drosophila , and sex - specific differences in the mode of transcriptome inheritance were identified . in males ,", "variation in gene expression was found to be due mostly to additive interactions of alleles , whereas in females , gene expression variation was found to be due mostly to non - additive ( epistatic ) interactions between alleles ; a substantial x - chromosome effect was shown to underlie these differences .", "similarly , in the seed beetle , loci affecting life span exhibited more non - additive interactions ( dominance ) in females than in males .", "given that additive variation responds to selection more quickly , because additive variation does not involve interactions of multiple loci , sex - specific differences in selection could underlie aspects of sexual dimorphism in life span observed in many species .", "sex - specific selective pressures that result in higher male reproductive fitness may contribute to sexual dimorphism of life span .", "for example , costly male - biased metabolism or behaviors , such as aggression or specific courtship behaviors , might be positively selected for , but could result in decreased life - span in males relative to females .", "future studies may be directed toward further study of the underlying differences in genetic architecture between males and females , in particular , testing the idea that deleterious alleles affecting life span may be more exposed to selection in males than in females due to reduced non - additive effects in males , thereby reducing inbreeding load for male - specific deleterious alleles in the population .", "it will be particularly interesting to ask if the increased life span observed in highly inbred male seed beetles by bilde et al .  ", "can be found to correlate with a reduction in specific costly aspects of metabolism , or behaviors such as locomotion and aggression ."], ["we thank sergey nuzhdin for helpful discussions . this work was supported by grants from the department of health and human services to jt ( 1r01ag011833 ) and to ma ( 1r01gm073039 ) ."]]}
{"article_id": "PMC3141151", "article_text": ["sepsis can be difficult to distinguish from non - infectious conditions in critically ill or comatose patients in the early stages ; and diagnosis , treatment and outcomes greatly differ between patients with and without sepsis .", "positive bacteriological cultures , including blood cultures , may not be available before 24 to 48 hours ; interpretation of local colonization may be ambiguous ; and traditional markers of infection , such as body temperature and white blood cell ( wbc ) count , may not be specific . furthermore , there are concerns about possible blood culture  negative clinical sepsis , particularly in the setting of increased prophylactic and empirical antibiotic use .", "conversely , differentiating true infection from contamination after growth of common skin commensals in blood cultures , poses a diagnostic problem . since early identification of infections and", "sepsis is crucial for patient management , an effective marker specific for bacterial infection is very useful in the critical care settings .", "there are several markers of sepsis , like c - reactive protein , serum procalcitonin ( pct ) , il-6 , il-8 , lactate , etc . , of", "pct has been proposed as an indicator of the presence of infection and as a useful marker of the severity of sepsis .", "the present study was an attempt to assess the usefulness of serum pct as a marker of sepsis in critically ill patients , using the semi - quantitative , rapid immunochromatographic kit , in apollo hospitals , bangalore .", "this study was carried out from july 2007 to june 2008 in the department of microbiology , apollo hospitals , bangalore .", "the study included patients from the intensive care unit ( icu ) with suspected sepsis .", "sepsis was confirmed clinically and by positive blood culture ( bact / alert system ) .", "( sepsis , severe sepsis , septic shock ) or no sepsis based on the accp ( american college of chest physicians ) recommendations .", "serum pct was assayed semi - quantitatively by rapid immunochromatographic technique using a commercially available test kit ( pct - q , b.r.a.h.m.s .", "the result was independently read by two microbiologists to minimize reading bias and interpreted as per the manufacturer 's recommendations : \n i)pct > 10 ng / ml : severe bacterial sepsis or septic shockii)pct 2 to 10 ng / ml : severe systemic inflammatory response , most likely due to sepsis unless other causes are knowniii)pct 0.5 to 2 ng / ml : a systemic infection can not be excludediv)pct < 0.5 ng / ml : local bacterial infection possible ; sepsis unlikely \n  pct > 10 ng / ml : severe bacterial sepsis or septic shock pct 2 to 10 ng / ml : severe systemic inflammatory response , most likely due to sepsis unless other causes are known pct 0.5 to 2 ng / ml : a systemic infection can not be excluded pct < 0.5 ng / ml : local bacterial infection possible ; sepsis unlikely the clinical condition , signs and symptoms of sepsis , antibiotics used , blood culture and final outcome of patients were recorded for all patients .", "sensitivity and specificity of the pct assay were analyzed using two different cut - off values for pct ( 0.5 ng / ml and 2 ng / ml ) ; sensitivity being pct positives / total no .", "of sepsis cases  100 and specificity being pct positives / total no . of cases without sepsis", "statistical analysis was carried out using chi - square test to evaluate the correlation between pct levels and sepsis .", "sensitivity and specificity of the pct assay were analyzed using two different cut - off values for pct ( 0.5 ng / ml and 2 ng / ml ) ; sensitivity being pct positives / total no . of sepsis cases", "statistical analysis was carried out using chi - square test to evaluate the correlation between pct levels and sepsis .", "patient ages ranged from 18 to 84 years [ male - female ratio , 28:12 ] . among these patients ,", "pct above 10 ng / ml was seen in 12 patients ; 2 - 10 ng / ml , in 7 patients ; 0.5 - 2 ng / ml , in 3 patients ; and < 0.5 ng / ml , in 18 patients [ table 1 ] .", "serum procalcitonin values in the 40 patients with suspected sepsis among the 12 patients with pct > 10 ng / ml , 1 patient in shock did not have any signs of sepsis or infection and recovered with inotropic support only ( no antibiotics were required ; cardiogenic shock )", ". of the 11 patients with clinically diagnosed sepsis , 8 yielded positive blood cultures subsequently ( 2 patients with septic shock had staphylococcus aureus bacteremia ) ; 1 patient had rickettsial disease with a typical clinical presentation ( fever , skin rashes , altered sensorium ) , which was confirmed by a positive weil felix reaction which showed elevated titres and a rise in titre within a week , and responded well to doxycycline and supportive therapy ; and 2 patients with negative blood cultures were on empiric antimicrobial therapy .", "four patients in this group expired due to complications of sepsis ( mortality , 33.3% ) .", "one of the patients with renal transplant and with features of sepsis had a serum pct > 10 ng / ml ( day 1 ) .", "he responded to parenteral meropenem and teicoplanin , and his subsequent serum pct levels reduced to < 0.5 ng / ml on day 3 and day 5 .", "all 7 patients with pct of 2 - 10 ng / ml had some form of sepsis ( sepsis , 4 patients ; severe sepsis , 2 ; and septic shock , 1 patient ) .", "four of them yielded positive blood cultures ; 1 patient had parasitemia ; and 2 malignancy patients with febrile neutropenia who were on empiric antimicrobial therapy had negative blood cultures .", "of the 3 patients with pct of 0.5 - 2 ng / ml , 1 patient had clinical evidence of sepsis secondary to culture - proven pseudomonas bronchopneumonia , though his blood culture did not yield any organism .", "he responded well to a combination therapy of cefoperazone - sulbactam and amikacin and was stable at discharge .", "the other 2 patients had no evidence of infection ( congestive cardiac failure and unstable angina ) , and 1 of them expired due to cardiac complications .", "of the 18 patients with pct < 0.5 ng / ml , only 2 patients with bronchopneumonia had evidence of sepsis .", "among 3 other patients with infections , 1 patient had lobar pneumonia but without sepsis ; 1 , pyrexia of unknown origin ( possibly a viral infection ) ; and 1 , probable viral encephalitis but no signs of sepsis .", "two patients ( post - myomectomy hypertrophic obstructive cardiomyopathy and acute myocardial infarction ) with positive blood cultures ( s. epidermidis and s. haemolyticus ) were concluded to have cardiogenic shock and not septic shock , as they responded well to inotropic support . in 1 of them , antibiotics were given additionally , considering his postoperative status ( single - vessel stenting done 3 days prior to pct testing ) . in this cohort , in a patient with a differential diagnosis of food botulism , a low pct assisted in excluding an infective aetiology , and the patient was subsequently diagnosed as having acute oculobulbar syndrome . in 8 out of the 9 fatal cases ,", "serum pct cut - offs of 0.5 ng / ml and 2 ng / ml were analyzed separately for their sensitivity and specificity as biomarkers for sepsis among the 21 patients with sepsis and 19 patients without sepsis .", "there was a statistically significant correlation with the presence of sepsis determined using either pct 0.5 ng / ml or 2 ng / ml ( p<0.0001 in both cases ) . using 0.5 ng / ml or more as cut - off , 19 of the 21 patients with sepsis could be detected , but 3 out of the 19 patients without sepsis showed pct above 0.5 ng / ml ( sensitivity , 90% , i.e. , 19/19 + 2 ; and specificity , 84% , i.e. , 16/16 + 3 ) .", "using pct cut - off of 2 ng / ml or more as a marker of sepsis , 18 of the 21 patients with sepsis could be detected by pct , and only 1 out of the 19 patients without sepsis showed pct above 2 ng / ml ( sensitivity , 85.7% , i.e. , 18/18 + 3 ; and specificity , 94.7% , i.e. , 18/18 + 1 ) [ table 2 ] .", "relation of serum pct levels with sepsis , number of patients ( blood cultures ; mortality )", "the aim of this study was to assess the usefulness of serum pct as a marker of sepsis in critically ill patients in our hospital .", "early identification of infection and sepsis in critically ill patients is a challenge for clinicians .", "serum pct has been found to be a very good indicator of sepsis syndrome . to the best of our knowledge", "these include studies on its use in determining bacterial sepsis in children with febrile neutropenia , its use as a marker of renal parenchymal infection and its use as a marker of the severity of acute pancreatitis.[68 ] most studies using pct for interpretation of bacterial infection and sepsis have used the quantitative pct assay ( immunoluminometric method ) .", "several studies have achieved high sensitivity and modest specificity with a cut - off of 1 to 1.2 ng / ml .", "others have used 2 ng / ml as the diagnostic threshold for infection and sepsis .", "various studies have found the pct values in sepsis ranging from 0.5 to 3.5 ng / ml ; in severe sepsis , 6.2 - 9.1 ng / ml ; and in septic shock , 12.8 to 38.5 ng / ml . on the other hand , semi - quantitative pct assay is a rapid immunochromatographic assay and is easy to perform .", "the method shows good sensitivity and specificity in diagnosing bacterial sepsis at pct levels of 2 ng / ml.[1315 ] using pct levels above 2 ng / ml as the cut - off to indicate sepsis syndrome , the present study showed sensitivity of 86% and specificity of 95% . using a cut - off of above 0.5 ng / ml revealed higher sensitivity but with a reduction in the specificity to 84% .", "hence serum pct of 2 ng / ml or more using the rapid immunochromatographic method is an effective marker of sepsis and may help in aggressive management of such patients along the lines of sepsis . despite improved methods , blood cultures are not always positive in clinically confirmed sepsis .", "presumed bacterial cause of fever can not be detected in 60% to 80% of patients with suspected primary and secondary bloodstream infections . in this study , excluding the 2 cases of malaria and rickettsial fever , the sensitivity of blood culture was 63% ( 12 of 19 ) ; and in all these patients , serum pct was above 2 ng / ml . differentiating true infection from contamination or probable skin commensals in blood cultures poses a diagnostic problem .", "the 2 blood cultures yielding coagulase - negative staphylococci were probable skin commensals , as both patients did not have any infective foci and both responded well with inotropes alone . in both these patients ,", "the serum pct was less than 2 ng / ml . falsely high pct is known to occur in cardiogenic shock , major surgery , accidental trauma , pancreatitis and burn patients . of patients with cardiogenic shock , 1 patient had pct above 10 ng / ml , 2 had pct less than 0.5 ng / ml and 1 patient with coexisting sepsis had pct of 2 - 10 ng / ml .", "pct levels are significantly higher in patients with septic shock than in those with cardiogenic shock , and hence a cut - off of above 10 ng / ml is recommended to discriminate between septic and cardiogenic shocks .", "serum pct increases early after trauma and peaks within 48 to 72 hours post - trauma and declines thereafter in the absence of infection or sepsis . in the present study , out of 13 patients with major surgery or trauma , 4 patients had surgery or trauma with in 72 hours of suspected sepsis and pct testing ; 3 of them with pct levels below 0.5", "ng / ml did not have sepsis , and 1 patient had septic shock ( pct > 10 ng / ml ) .", "in contrast , initial falsely low pct levels , typically seen during the early course or localized state of infection , often show a gradual increase during follow - up measurements after 6 - 24 hours .", "reduction in concentration of pct has been described in response to antibiotic administration and may be used to detect antibiotic responsiveness . in the present study ,", "serial serum pct levels were not estimated for most patients . however , one of the patients ( post - renal transplant status ) from this cohort for whom 3 serum pct levels were performed showed a marked decrease in the pct levels in response to the antibiotics administered .", "this is in concordance with the several clinical - management protocols , like the  pct - guided antimicrobial stewardship ", ", that have been used to either withhold or continue antibiotics based on the pct levels .", "a limitation of the present study is that serial measurements of pct were not done for most patients .", "more indian studies evaluating pct on a larger number of patients with suspected sepsis are needed to conclusively corroborate the findings of this study , and such studies will be useful in an age of overuse of antibiotics and emerging antibiotic resistance .", "another limitation of the study was that c - reactive protein ( crp ) levels were not estimated for most patients .", "however , most studies have found superiority of pct in comparison to crp as a marker of infection and sepsis .", "the pct assay was found to be a promising biomarker of sepsis in this small exploratory study and provided valuable and early information before the culture results were available .", "the semi - quantitative , rapid assay revealed moderate sensitivity ( 86% ) and high specificity ( 95% ) at a cut - off above 2 ng / ml .", "pct assay might assist in avoiding unwarranted antibiotic usage in critically ill patients who present with symptoms similar to those in infective conditions .", "however , the assay result must be interpreted in association with the clinical findings and other laboratory parameters ."], "abstract_text": ["<S> sepsis can sometimes be difficult to substantiate , and its distinction from non - infectious conditions in critically ill patients is often a challenge . </S>", "<S> serum procalcitonin ( pct ) assay is one of the biomarkers of sepsis . </S>", "<S> the present study was aimed to assess the usefulness of pct assay in critically ill patients with suspected sepsis . </S>", "<S> the study included 40 patients from the intensive care unit with suspected sepsis . </S>", "<S> sepsis was confirmed clinically and/or by positive blood culture . </S>", "<S> serum pct was assayed semi - quantitatively by rapid immunochromatographic technique ( within 2 hours of sample receipt ) . among 40 critically ill patients , 21 </S>", "<S> had clinically confirmed sepsis . </S>", "<S> there were 12 patients with serum pct 10 ng / ml ( 8 , blood culture positive ; 1 , rickettsia ; 2 , post - antibiotic blood culture sterile ; and 1 , non - sepsis ) ; 7 patients with pct 2 - 10 ng / ml ( 4 , blood culture positive ; 1 , falciparum malaria ; 2 , post - antibiotic blood culture sterile ) ; 3 patients with pct of 0.5 to 2 ng / ml ( sepsis in 1 patient ) ; and 18 patients with pct < 0.5 ng / ml ( sepsis in 2 patients ) . patients with pct  2 ng / ml had statistically significant correlation with the presence of sepsis ( p<0.0001 ) . </S>", "<S> the pct assay revealed moderate sensitivity ( 86% ) and high specificity ( 95% ) at a cut - off  2 ng / ml . </S>", "<S> the pct assay was found to be a useful biomarker of sepsis in this study . the assay could be performed and reported rapidly and provided valuable information before availability of culture results . </S>", "<S> this might assist in avoiding unwarranted antibiotic usage . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Statistic analysis", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["sepsis can be difficult to distinguish from non - infectious conditions in critically ill or comatose patients in the early stages ; and diagnosis , treatment and outcomes greatly differ between patients with and without sepsis .", "positive bacteriological cultures , including blood cultures , may not be available before 24 to 48 hours ; interpretation of local colonization may be ambiguous ; and traditional markers of infection , such as body temperature and white blood cell ( wbc ) count , may not be specific . furthermore , there are concerns about possible blood culture  negative clinical sepsis , particularly in the setting of increased prophylactic and empirical antibiotic use .", "conversely , differentiating true infection from contamination after growth of common skin commensals in blood cultures , poses a diagnostic problem . since early identification of infections and", "sepsis is crucial for patient management , an effective marker specific for bacterial infection is very useful in the critical care settings .", "there are several markers of sepsis , like c - reactive protein , serum procalcitonin ( pct ) , il-6 , il-8 , lactate , etc . , of", "pct has been proposed as an indicator of the presence of infection and as a useful marker of the severity of sepsis .", "the present study was an attempt to assess the usefulness of serum pct as a marker of sepsis in critically ill patients , using the semi - quantitative , rapid immunochromatographic kit , in apollo hospitals , bangalore ."], ["this study was carried out from july 2007 to june 2008 in the department of microbiology , apollo hospitals , bangalore .", "the study included patients from the intensive care unit ( icu ) with suspected sepsis .", "sepsis was confirmed clinically and by positive blood culture ( bact / alert system ) .", "( sepsis , severe sepsis , septic shock ) or no sepsis based on the accp ( american college of chest physicians ) recommendations .", "serum pct was assayed semi - quantitatively by rapid immunochromatographic technique using a commercially available test kit ( pct - q , b.r.a.h.m.s .", "the result was independently read by two microbiologists to minimize reading bias and interpreted as per the manufacturer 's recommendations : \n i)pct > 10 ng / ml : severe bacterial sepsis or septic shockii)pct 2 to 10 ng / ml : severe systemic inflammatory response , most likely due to sepsis unless other causes are knowniii)pct 0.5 to 2 ng / ml : a systemic infection can not be excludediv)pct < 0.5 ng / ml : local bacterial infection possible ; sepsis unlikely \n  pct > 10 ng / ml : severe bacterial sepsis or septic shock pct 2 to 10 ng / ml : severe systemic inflammatory response , most likely due to sepsis unless other causes are known pct 0.5 to 2 ng / ml : a systemic infection can not be excluded pct < 0.5 ng / ml : local bacterial infection possible ; sepsis unlikely the clinical condition , signs and symptoms of sepsis , antibiotics used , blood culture and final outcome of patients were recorded for all patients .", "sensitivity and specificity of the pct assay were analyzed using two different cut - off values for pct ( 0.5 ng / ml and 2 ng / ml ) ; sensitivity being pct positives / total no .", "of sepsis cases  100 and specificity being pct positives / total no . of cases without sepsis", "statistical analysis was carried out using chi - square test to evaluate the correlation between pct levels and sepsis ."], ["sensitivity and specificity of the pct assay were analyzed using two different cut - off values for pct ( 0.5 ng / ml and 2 ng / ml ) ; sensitivity being pct positives / total no . of sepsis cases", "statistical analysis was carried out using chi - square test to evaluate the correlation between pct levels and sepsis ."], ["patient ages ranged from 18 to 84 years [ male - female ratio , 28:12 ] . among these patients ,", "pct above 10 ng / ml was seen in 12 patients ; 2 - 10 ng / ml , in 7 patients ; 0.5 - 2 ng / ml , in 3 patients ; and < 0.5 ng / ml , in 18 patients [ table 1 ] .", "serum procalcitonin values in the 40 patients with suspected sepsis among the 12 patients with pct > 10 ng / ml , 1 patient in shock did not have any signs of sepsis or infection and recovered with inotropic support only ( no antibiotics were required ; cardiogenic shock )", ". of the 11 patients with clinically diagnosed sepsis , 8 yielded positive blood cultures subsequently ( 2 patients with septic shock had staphylococcus aureus bacteremia ) ; 1 patient had rickettsial disease with a typical clinical presentation ( fever , skin rashes , altered sensorium ) , which was confirmed by a positive weil felix reaction which showed elevated titres and a rise in titre within a week , and responded well to doxycycline and supportive therapy ; and 2 patients with negative blood cultures were on empiric antimicrobial therapy .", "four patients in this group expired due to complications of sepsis ( mortality , 33.3% ) .", "one of the patients with renal transplant and with features of sepsis had a serum pct > 10 ng / ml ( day 1 ) .", "he responded to parenteral meropenem and teicoplanin , and his subsequent serum pct levels reduced to < 0.5 ng / ml on day 3 and day 5 .", "all 7 patients with pct of 2 - 10 ng / ml had some form of sepsis ( sepsis , 4 patients ; severe sepsis , 2 ; and septic shock , 1 patient ) .", "four of them yielded positive blood cultures ; 1 patient had parasitemia ; and 2 malignancy patients with febrile neutropenia who were on empiric antimicrobial therapy had negative blood cultures .", "of the 3 patients with pct of 0.5 - 2 ng / ml , 1 patient had clinical evidence of sepsis secondary to culture - proven pseudomonas bronchopneumonia , though his blood culture did not yield any organism .", "he responded well to a combination therapy of cefoperazone - sulbactam and amikacin and was stable at discharge .", "the other 2 patients had no evidence of infection ( congestive cardiac failure and unstable angina ) , and 1 of them expired due to cardiac complications .", "of the 18 patients with pct < 0.5 ng / ml , only 2 patients with bronchopneumonia had evidence of sepsis .", "among 3 other patients with infections , 1 patient had lobar pneumonia but without sepsis ; 1 , pyrexia of unknown origin ( possibly a viral infection ) ; and 1 , probable viral encephalitis but no signs of sepsis .", "two patients ( post - myomectomy hypertrophic obstructive cardiomyopathy and acute myocardial infarction ) with positive blood cultures ( s. epidermidis and s. haemolyticus ) were concluded to have cardiogenic shock and not septic shock , as they responded well to inotropic support . in 1 of them , antibiotics were given additionally , considering his postoperative status ( single - vessel stenting done 3 days prior to pct testing ) . in this cohort , in a patient with a differential diagnosis of food botulism , a low pct assisted in excluding an infective aetiology , and the patient was subsequently diagnosed as having acute oculobulbar syndrome . in 8 out of the 9 fatal cases ,", "serum pct cut - offs of 0.5 ng / ml and 2 ng / ml were analyzed separately for their sensitivity and specificity as biomarkers for sepsis among the 21 patients with sepsis and 19 patients without sepsis .", "there was a statistically significant correlation with the presence of sepsis determined using either pct 0.5 ng / ml or 2 ng / ml ( p<0.0001 in both cases ) . using 0.5 ng / ml or more as cut - off , 19 of the 21 patients with sepsis could be detected , but 3 out of the 19 patients without sepsis showed pct above 0.5 ng / ml ( sensitivity , 90% , i.e. , 19/19 + 2 ; and specificity , 84% , i.e. , 16/16 + 3 ) .", "using pct cut - off of 2 ng / ml or more as a marker of sepsis , 18 of the 21 patients with sepsis could be detected by pct , and only 1 out of the 19 patients without sepsis showed pct above 2 ng / ml ( sensitivity , 85.7% , i.e. , 18/18 + 3 ; and specificity , 94.7% , i.e. , 18/18 + 1 ) [ table 2 ] .", "relation of serum pct levels with sepsis , number of patients ( blood cultures ; mortality )"], ["the aim of this study was to assess the usefulness of serum pct as a marker of sepsis in critically ill patients in our hospital .", "early identification of infection and sepsis in critically ill patients is a challenge for clinicians .", "serum pct has been found to be a very good indicator of sepsis syndrome . to the best of our knowledge", "these include studies on its use in determining bacterial sepsis in children with febrile neutropenia , its use as a marker of renal parenchymal infection and its use as a marker of the severity of acute pancreatitis.[68 ] most studies using pct for interpretation of bacterial infection and sepsis have used the quantitative pct assay ( immunoluminometric method ) .", "several studies have achieved high sensitivity and modest specificity with a cut - off of 1 to 1.2 ng / ml .", "others have used 2 ng / ml as the diagnostic threshold for infection and sepsis .", "various studies have found the pct values in sepsis ranging from 0.5 to 3.5 ng / ml ; in severe sepsis , 6.2 - 9.1 ng / ml ; and in septic shock , 12.8 to 38.5 ng / ml . on the other hand , semi - quantitative pct assay is a rapid immunochromatographic assay and is easy to perform .", "the method shows good sensitivity and specificity in diagnosing bacterial sepsis at pct levels of 2 ng / ml.[1315 ] using pct levels above 2 ng / ml as the cut - off to indicate sepsis syndrome , the present study showed sensitivity of 86% and specificity of 95% . using a cut - off of above 0.5 ng / ml revealed higher sensitivity but with a reduction in the specificity to 84% .", "hence serum pct of 2 ng / ml or more using the rapid immunochromatographic method is an effective marker of sepsis and may help in aggressive management of such patients along the lines of sepsis . despite improved methods , blood cultures are not always positive in clinically confirmed sepsis .", "presumed bacterial cause of fever can not be detected in 60% to 80% of patients with suspected primary and secondary bloodstream infections . in this study , excluding the 2 cases of malaria and rickettsial fever , the sensitivity of blood culture was 63% ( 12 of 19 ) ; and in all these patients , serum pct was above 2 ng / ml . differentiating true infection from contamination or probable skin commensals in blood cultures poses a diagnostic problem .", "the 2 blood cultures yielding coagulase - negative staphylococci were probable skin commensals , as both patients did not have any infective foci and both responded well with inotropes alone . in both these patients ,", "the serum pct was less than 2 ng / ml . falsely high pct is known to occur in cardiogenic shock , major surgery , accidental trauma , pancreatitis and burn patients . of patients with cardiogenic shock , 1 patient had pct above 10 ng / ml , 2 had pct less than 0.5 ng / ml and 1 patient with coexisting sepsis had pct of 2 - 10 ng / ml .", "pct levels are significantly higher in patients with septic shock than in those with cardiogenic shock , and hence a cut - off of above 10 ng / ml is recommended to discriminate between septic and cardiogenic shocks .", "serum pct increases early after trauma and peaks within 48 to 72 hours post - trauma and declines thereafter in the absence of infection or sepsis . in the present study , out of 13 patients with major surgery or trauma , 4 patients had surgery or trauma with in 72 hours of suspected sepsis and pct testing ; 3 of them with pct levels below 0.5", "ng / ml did not have sepsis , and 1 patient had septic shock ( pct > 10 ng / ml ) .", "in contrast , initial falsely low pct levels , typically seen during the early course or localized state of infection , often show a gradual increase during follow - up measurements after 6 - 24 hours .", "reduction in concentration of pct has been described in response to antibiotic administration and may be used to detect antibiotic responsiveness . in the present study ,", "serial serum pct levels were not estimated for most patients . however , one of the patients ( post - renal transplant status ) from this cohort for whom 3 serum pct levels were performed showed a marked decrease in the pct levels in response to the antibiotics administered .", "this is in concordance with the several clinical - management protocols , like the  pct - guided antimicrobial stewardship ", ", that have been used to either withhold or continue antibiotics based on the pct levels .", "a limitation of the present study is that serial measurements of pct were not done for most patients .", "more indian studies evaluating pct on a larger number of patients with suspected sepsis are needed to conclusively corroborate the findings of this study , and such studies will be useful in an age of overuse of antibiotics and emerging antibiotic resistance .", "another limitation of the study was that c - reactive protein ( crp ) levels were not estimated for most patients .", "however , most studies have found superiority of pct in comparison to crp as a marker of infection and sepsis ."], ["the pct assay was found to be a promising biomarker of sepsis in this small exploratory study and provided valuable and early information before the culture results were available .", "the semi - quantitative , rapid assay revealed moderate sensitivity ( 86% ) and high specificity ( 95% ) at a cut - off above 2 ng / ml .", "pct assay might assist in avoiding unwarranted antibiotic usage in critically ill patients who present with symptoms similar to those in infective conditions .", "however , the assay result must be interpreted in association with the clinical findings and other laboratory parameters ."]]}
{"article_id": "PMC2671454", "article_text": ["we used monthly reports of slide - confirmed malaria and annual census  based population data from 434 counties ( municpios ) in the brazilian amazon region for 19961999 , during which no coordinated national malaria interventions occurred ( 12 ) . to study the relationship of reported malaria cases to climate , we used monthly precipitation and temperature from the cru ts 2.1 gridded climate data set for selected states ( 13 ) ( figure 1 ) . to consider how the precipitation  malaria relationship depends on surface water conditions ,", "including the extent of open water and wetlands , we used 100 m  100 m maps from the jers-1 synthetic aperture radar satellite and calculated the percentage of maximum inundatable open water and wetland coverage for each county ( figure 2 , panel a ) ( 14 ) . in this region ,", "monthly temperatures were between 24.6c and 29.4c ( well within the range for optimal malaria transmission ) for 95% of the observations ( 18,416 of 19,364 ) included in the analysis ( temperature relationships not shown ) .", "malaria incidence per 1,000 population ( black lines ) and mean monthly precipitation ( blue lines ) during la nia ( orange bars ) and el nio ( red bars ) events for the states of a ) amazonas , b ) mato grosso , and c ) roraima .", "a ) percentage of wetlands in amazon basin counties ( shades of blue ) , counties without wetlands data ( yellow ) , and counties with < 80 total malaria cases ( gray ) .", "wetland colors correspond to percentage wetland values in panel d. b ) risk ratios for malaria incidence for 1 sd ( 14 cm ) change in monthly precipitation ( january 1996december 1999 ) , plotted at each county seat of government ; c ) spatially smoothed risk ratios for 14-cm changes in monthly precipitation . in both panels ,", "red shaded squares show reduced risk for 14-cm increase in monthly precipitation ; blue shaded squares show increased risk for malaria with increased precipitation .", "d ) boxplot of risk ratios for malaria incidence for 14-cm changes in monthly precipitation , by percentage wetland cover .", "if the lag and the rainfall coefficient vary across regions , meaningful geographic comparisons would be difficult to achieve because neither the lag nor coefficient have consistent meanings across models . to interpret results , either the coefficient must be fixed and the lags varied ( difficult to do ) or the lags must be fixed and the coefficients varied ( relatively easy to do ) .", "the aim is to describe the variable patterns of malaria incidence and precipitation , not create a highly predictive model .", "we chose to fix the lag and vary the coefficients . to assess the association between malaria incidence and precipitation data", ", we estimated the rate ratio of malaria incidence associated with 1 sd - increase in monthly precipitation ( 14 cm ) for each county by using the following poisson regression model , which includes a flexible temporal trend represented as a natural cubic spline with 6 degrees of freedom ( figure 2 , panel b ) : malariait  poisson(it ) log it = log popit + i + iprecipit + fi(t ) i  normal(g(lati , loni ) ,  ) the ( estimated ) regression coefficients from the county - specific models were then modeled as a spatially smooth surface , a thin - plate spline .", "degrees of freedom for the thin - plate spline were selected using generalized cross - validation ( figure 2 , panel c ) .", "the relationships between precipitation and malaria incidence in the amazon basin are spatially varied and change signs , depending on the region .", "positive correlations between monthly precipitation and malaria incidence ( rate ratios > 1 ) occur in the upland regions of the southwest and central amazon basin , whereas negative correlations between precipitation and incidence ( rate ratios < 1 ) occur in the north , largely along the main waterways of the amazon river and the major wetland regions of the basin ( figure 2 ) . for", "a 14-cm increase in monthly rainfall , the malaria rate can double in the upland area , yet decrease by up to 80% along the main amazon channel .", "the p values of the precipitation coefficient are 0.00020.0009 along the main waterways and 0.0040.10 in uplands areas .", "we hypothesize that this reversal of the malaria  precipitation relationship from positive to negative is related to the extent of open water and wetlands in the basin .", "mosquito habitats in wetlands or along large rivers may wash out or become too deep during months with high precipitation , but in areas with fewer wetlands , mosquito habitats are limited by precipitation . to test this hypothesis ,", "we compared the malaria  precipitation association for 338 counties that reported > 80 cases of malaria over the 48 months against the estimated percentage of open water and wetland cover for each county ( figure 2 , panel d ) .", "as expected , the precipitation - linked risk for malaria fell as the percentage of wetland in each county increased , but the risk for malaria varied in counties with low percentages of wetlands .", "the central - east region had the lowest level of malaria incidence , which may explain why this region also lacked a malaria - precipitation relationship .", "studies have proposed that flooding created new pools of water suitable for mosquito larvae as the water levels slowly receded from alluvial forests along the rio branco river in roraima and the maroni river on the frontiers of suriname and french guiana ( 6,15 ) .", "our results suggest that monthly precipitation along the amazon basin can have both strong positive and negative associations with malaria incidence .", "further research is needed to address the limitations of our study , including its short time frame and the crude countywide approximation of percentage wetlands as an exposure .", "the quality and reliability of the health data were concerns , but we verified that the distribution of null reporting was unbiased temporally and spatially . also , our study did not quantify increasing malaria incidence in response to increasing or decreasing precipitation or the impact of lag factors . instead , we focused on the seasonality of these patterns until longer data series of malaria incidence and climate data are available .", "our evidence suggests that precipitation drives malaria risk in the amazon basin , but the relationship varies in the uplands ( more precipitation , more / less malaria ) and is negative in areas dominated by wetlands and large rivers ( more precipitation , less malaria ) .", "our findings show the need to account for local landscape characteristics , especially the extent of wetlands and open water , in regional to global projections of the effects of climate change on malaria .", "better understanding the impact of climate and landscape on malaria will improve our ability to assess health risks ."], "abstract_text": ["<S> climate changes are altering patterns of temperature and precipitation , potentially affecting regions of malaria transmission . </S>", "<S> we show that areas of the amazon basin with few wetlands show a variable relationship between precipitation and malaria , while areas with extensive wetlands show a negative relationship with malaria incidence . </S>"], "labels": null, "section_names": ["The Study", "Conclusions"], "sections": [["we used monthly reports of slide - confirmed malaria and annual census  based population data from 434 counties ( municpios ) in the brazilian amazon region for 19961999 , during which no coordinated national malaria interventions occurred ( 12 ) . to study the relationship of reported malaria cases to climate , we used monthly precipitation and temperature from the cru ts 2.1 gridded climate data set for selected states ( 13 ) ( figure 1 ) . to consider how the precipitation  malaria relationship depends on surface water conditions ,", "including the extent of open water and wetlands , we used 100 m  100 m maps from the jers-1 synthetic aperture radar satellite and calculated the percentage of maximum inundatable open water and wetland coverage for each county ( figure 2 , panel a ) ( 14 ) . in this region ,", "monthly temperatures were between 24.6c and 29.4c ( well within the range for optimal malaria transmission ) for 95% of the observations ( 18,416 of 19,364 ) included in the analysis ( temperature relationships not shown ) .", "malaria incidence per 1,000 population ( black lines ) and mean monthly precipitation ( blue lines ) during la nia ( orange bars ) and el nio ( red bars ) events for the states of a ) amazonas , b ) mato grosso , and c ) roraima .", "a ) percentage of wetlands in amazon basin counties ( shades of blue ) , counties without wetlands data ( yellow ) , and counties with < 80 total malaria cases ( gray ) .", "wetland colors correspond to percentage wetland values in panel d. b ) risk ratios for malaria incidence for 1 sd ( 14 cm ) change in monthly precipitation ( january 1996december 1999 ) , plotted at each county seat of government ; c ) spatially smoothed risk ratios for 14-cm changes in monthly precipitation . in both panels ,", "red shaded squares show reduced risk for 14-cm increase in monthly precipitation ; blue shaded squares show increased risk for malaria with increased precipitation .", "d ) boxplot of risk ratios for malaria incidence for 14-cm changes in monthly precipitation , by percentage wetland cover .", "if the lag and the rainfall coefficient vary across regions , meaningful geographic comparisons would be difficult to achieve because neither the lag nor coefficient have consistent meanings across models . to interpret results , either the coefficient must be fixed and the lags varied ( difficult to do ) or the lags must be fixed and the coefficients varied ( relatively easy to do ) .", "the aim is to describe the variable patterns of malaria incidence and precipitation , not create a highly predictive model .", "we chose to fix the lag and vary the coefficients . to assess the association between malaria incidence and precipitation data", ", we estimated the rate ratio of malaria incidence associated with 1 sd - increase in monthly precipitation ( 14 cm ) for each county by using the following poisson regression model , which includes a flexible temporal trend represented as a natural cubic spline with 6 degrees of freedom ( figure 2 , panel b ) : malariait  poisson(it ) log it = log popit + i + iprecipit + fi(t ) i  normal(g(lati , loni ) ,  ) the ( estimated ) regression coefficients from the county - specific models were then modeled as a spatially smooth surface , a thin - plate spline .", "degrees of freedom for the thin - plate spline were selected using generalized cross - validation ( figure 2 , panel c ) .", "the relationships between precipitation and malaria incidence in the amazon basin are spatially varied and change signs , depending on the region .", "positive correlations between monthly precipitation and malaria incidence ( rate ratios > 1 ) occur in the upland regions of the southwest and central amazon basin , whereas negative correlations between precipitation and incidence ( rate ratios < 1 ) occur in the north , largely along the main waterways of the amazon river and the major wetland regions of the basin ( figure 2 ) . for", "a 14-cm increase in monthly rainfall , the malaria rate can double in the upland area , yet decrease by up to 80% along the main amazon channel .", "the p values of the precipitation coefficient are 0.00020.0009 along the main waterways and 0.0040.10 in uplands areas .", "we hypothesize that this reversal of the malaria  precipitation relationship from positive to negative is related to the extent of open water and wetlands in the basin .", "mosquito habitats in wetlands or along large rivers may wash out or become too deep during months with high precipitation , but in areas with fewer wetlands , mosquito habitats are limited by precipitation . to test this hypothesis ,", "we compared the malaria  precipitation association for 338 counties that reported > 80 cases of malaria over the 48 months against the estimated percentage of open water and wetland cover for each county ( figure 2 , panel d ) .", "as expected , the precipitation - linked risk for malaria fell as the percentage of wetland in each county increased , but the risk for malaria varied in counties with low percentages of wetlands .", "the central - east region had the lowest level of malaria incidence , which may explain why this region also lacked a malaria - precipitation relationship ."], ["studies have proposed that flooding created new pools of water suitable for mosquito larvae as the water levels slowly receded from alluvial forests along the rio branco river in roraima and the maroni river on the frontiers of suriname and french guiana ( 6,15 ) .", "our results suggest that monthly precipitation along the amazon basin can have both strong positive and negative associations with malaria incidence .", "further research is needed to address the limitations of our study , including its short time frame and the crude countywide approximation of percentage wetlands as an exposure .", "the quality and reliability of the health data were concerns , but we verified that the distribution of null reporting was unbiased temporally and spatially . also , our study did not quantify increasing malaria incidence in response to increasing or decreasing precipitation or the impact of lag factors . instead , we focused on the seasonality of these patterns until longer data series of malaria incidence and climate data are available .", "our evidence suggests that precipitation drives malaria risk in the amazon basin , but the relationship varies in the uplands ( more precipitation , more / less malaria ) and is negative in areas dominated by wetlands and large rivers ( more precipitation , less malaria ) .", "our findings show the need to account for local landscape characteristics , especially the extent of wetlands and open water , in regional to global projections of the effects of climate change on malaria .", "better understanding the impact of climate and landscape on malaria will improve our ability to assess health risks ."]]}
{"article_id": "PMC5097962", "article_text": ["the clavicle is a rare site for bone tumours . according to the world health organisation", ", the giant cell tumor is an aggressive potentially malignant lesion which means that its evolution based on histological features is unpredictable .", "sites commonly affected by giant cell tumour are proximal tibia , distal femur and distal radius .", "the oncologic patterns of clavicle resemble that of flat bones   and not other long bones . among tumors of clavicle ,", "a 60  year old man presented to our department with pain and swelling over lateral end of left clavicle ( fig . 1 , fig .", "the pain was insidious in onset , non radiating and had no diurnal variations and was aggravated on shoulder movements and relieved on taking medications .", "we got a plain radiograph which revealed which an expansile radiolucent lesion arising from lateral end of left clavicle ( fig .", "3 ) . the swelling demonstrated geographic type destruction without any soft tissue component or periosteal reaction .", "4 ) . to aid in the diagnosis fine needle aspiration cytology was done which revealed a predominantly cellular lesion having sheets of plump , oval mononuclear cells with mild pleomorphism .", "the cells had moderate cytoplasm , oval to elongated nucleus with moderate anisokaryocytosis with irregular nuclear membrane . amongst these cells , many multinucleated giant cells were also which were distributed evenly .", "the differential diagnosis which were kept in mind are aneurysmal bone cyst , non ossifying fibroma , eosinophilic granuloma and tuberculous osteomyelitis .", "since the clavicle does not necessary require reconstruction and the patient was a retired school teacher , not engaged in any physical work so surgical resection of the tumor was planned .", "after proper investigations and pre anaesthetic clearance , a wide excision of the mass along with 3  cm of the healthy tissue was done ( fig .", "the excised mass was sent for histopathological examination which also confirmed it to be a giant cell tumor .", "the range of motion of the left shoulder was normal and post operatively there was no neurovascular deficict .", "the patient was happy with the surgical outcome and at 1  year follow up there was no evidence of recurrence or metastasis .", ". primary bone tumors of the clavicle are more likely to be malignant than benign ,   and amongst these tumors which occur in clavicle , giant cell tumor is a rare entity .", "the differential diagnosis of giant cell tumor of clavicle which pose a diagnostic challenge both for the surgeon and the histopathologist are primary aneurysmal bone cyst , non ossifying fibroma.eosiniphilic granuloma and tuberculous osteomyelitis .", "giant cell tumor is basically a cellular lesion made up of sheets of plump mononuclear cells with mild pleomorphism .", "the cells have moderate amount of cytoplasm , oval to elongated nuceuswith moderate anisokaryosis with irregular nuclear membrane and 01 nucleolus . amongst these cells", "are multinucleated giant cells distributed in a regular fashion.no collagen formation , no new bone formation or no necrosis is usually seen .", "giant cells in abc are smaller as compared to giant cell tumor and their arrangement is loose with collagen formation .", "histologically it has histiocytes loaded with lipid and hemosiderin and spindle cells arranged in storiform or whorled pattern and there is presence of collagen fibres .", "eosinophilic granuloma has diagnostic langerhans cells and also it has large number of leucocytes , fibroblasts , plasma cells and lymphocytes .", "curettage remains the main stay of treatment for giant cell tumors but for giant cell tumors occurring in expendable bones like distal ulna , iliac wing or proximal fibula , en bloc resection is performed without any reconstruction . after extensive search of literature", "it was found that there are very few case reports describing giant cell tumors of clavicle , , . due to the paucity of the available literature", "no definite treatment guidelines are available on the management of giant cell tumor of clavicle .", "some authors   reported not so favourable outcomes after total claviculectomy due to pain , loss of muscle strength and dropping of shoulder . while some authors established that total or subtotal excision of clavicle", "was rarely associated with loss of function . based on their reports we also performed partial claviculectomy and at one year follow up , patient was well satisfied with", "the clavicle is a rare site for bone tumors and shares its oncological behavior with that of flat bones rather than long bones .", "we have reported this case to emphasize the fact any expansile lytic lesion occurring in the lateral end of clavicle should be taken seriously and the diagnosis can be easily missed both clinically and radiographically if the clinician is not aware of the wide array of differential diagnosis which range from an infectious etiology like tuberulous osteomyelitis to a neoplastic etiology like giant cell tumour .", "there seems to be a difference in opinion regarding functional outcome after claviculectomy for tumour like lesions of clavicle and our case report further highlights the fact the claviculectomy without any reconstruction seems to be a good option with no disability noted in long term .", "since it is a single case report involving a single subject and we are not reporting the first case of this type in the literature , hence no approval was taken from the relevant ethics committee but written informed consent was taken from the patient to publish his details and clinical photographs .", "written informed consent was obtained from the patient for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request .", "ak , kk and js contributed to the development of protocol and edited the manuscript  ."], "abstract_text": ["<S> highlightsgiant cell tumour of clavicle is a rare entity.the differntial diagnosis of giant cell tumour of clavicle which pose a diagnostic challenege gor both surgeon and histopathologist are aneurysmal bone cyst , non ossifying fibroma , tubercular osteomyelitis and eosniophilic granuloma.claviculecomy is a good option with no disability noted at long term . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion", "Conclusion", "Conflict of interest", "Funding", "Ethical approval", "Consent", "Authors contributions", "Guarantor"], "sections": [["the clavicle is a rare site for bone tumours . according to the world health organisation", ", the giant cell tumor is an aggressive potentially malignant lesion which means that its evolution based on histological features is unpredictable .", "sites commonly affected by giant cell tumour are proximal tibia , distal femur and distal radius .", "the oncologic patterns of clavicle resemble that of flat bones   and not other long bones . among tumors of clavicle ,"], ["a 60  year old man presented to our department with pain and swelling over lateral end of left clavicle ( fig . 1 , fig .", "the pain was insidious in onset , non radiating and had no diurnal variations and was aggravated on shoulder movements and relieved on taking medications .", "we got a plain radiograph which revealed which an expansile radiolucent lesion arising from lateral end of left clavicle ( fig .", "3 ) . the swelling demonstrated geographic type destruction without any soft tissue component or periosteal reaction .", "4 ) . to aid in the diagnosis fine needle aspiration cytology was done which revealed a predominantly cellular lesion having sheets of plump , oval mononuclear cells with mild pleomorphism .", "the cells had moderate cytoplasm , oval to elongated nucleus with moderate anisokaryocytosis with irregular nuclear membrane . amongst these cells , many multinucleated giant cells were also which were distributed evenly .", "the differential diagnosis which were kept in mind are aneurysmal bone cyst , non ossifying fibroma , eosinophilic granuloma and tuberculous osteomyelitis .", "since the clavicle does not necessary require reconstruction and the patient was a retired school teacher , not engaged in any physical work so surgical resection of the tumor was planned .", "after proper investigations and pre anaesthetic clearance , a wide excision of the mass along with 3  cm of the healthy tissue was done ( fig .", "the excised mass was sent for histopathological examination which also confirmed it to be a giant cell tumor .", "the range of motion of the left shoulder was normal and post operatively there was no neurovascular deficict .", "the patient was happy with the surgical outcome and at 1  year follow up there was no evidence of recurrence or metastasis ."], [". primary bone tumors of the clavicle are more likely to be malignant than benign ,   and amongst these tumors which occur in clavicle , giant cell tumor is a rare entity .", "the differential diagnosis of giant cell tumor of clavicle which pose a diagnostic challenge both for the surgeon and the histopathologist are primary aneurysmal bone cyst , non ossifying fibroma.eosiniphilic granuloma and tuberculous osteomyelitis .", "giant cell tumor is basically a cellular lesion made up of sheets of plump mononuclear cells with mild pleomorphism .", "the cells have moderate amount of cytoplasm , oval to elongated nuceuswith moderate anisokaryosis with irregular nuclear membrane and 01 nucleolus . amongst these cells", "are multinucleated giant cells distributed in a regular fashion.no collagen formation , no new bone formation or no necrosis is usually seen .", "giant cells in abc are smaller as compared to giant cell tumor and their arrangement is loose with collagen formation .", "histologically it has histiocytes loaded with lipid and hemosiderin and spindle cells arranged in storiform or whorled pattern and there is presence of collagen fibres .", "eosinophilic granuloma has diagnostic langerhans cells and also it has large number of leucocytes , fibroblasts , plasma cells and lymphocytes .", "curettage remains the main stay of treatment for giant cell tumors but for giant cell tumors occurring in expendable bones like distal ulna , iliac wing or proximal fibula , en bloc resection is performed without any reconstruction . after extensive search of literature", "it was found that there are very few case reports describing giant cell tumors of clavicle , , . due to the paucity of the available literature", "no definite treatment guidelines are available on the management of giant cell tumor of clavicle .", "some authors   reported not so favourable outcomes after total claviculectomy due to pain , loss of muscle strength and dropping of shoulder . while some authors established that total or subtotal excision of clavicle", "was rarely associated with loss of function . based on their reports we also performed partial claviculectomy and at one year follow up , patient was well satisfied with"], ["the clavicle is a rare site for bone tumors and shares its oncological behavior with that of flat bones rather than long bones .", "we have reported this case to emphasize the fact any expansile lytic lesion occurring in the lateral end of clavicle should be taken seriously and the diagnosis can be easily missed both clinically and radiographically if the clinician is not aware of the wide array of differential diagnosis which range from an infectious etiology like tuberulous osteomyelitis to a neoplastic etiology like giant cell tumour .", "there seems to be a difference in opinion regarding functional outcome after claviculectomy for tumour like lesions of clavicle and our case report further highlights the fact the claviculectomy without any reconstruction seems to be a good option with no disability noted in long term ."], [""], [""], ["since it is a single case report involving a single subject and we are not reporting the first case of this type in the literature , hence no approval was taken from the relevant ethics committee but written informed consent was taken from the patient to publish his details and clinical photographs ."], ["written informed consent was obtained from the patient for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request ."], ["ak , kk and js contributed to the development of protocol and edited the manuscript  ."], [""]]}
{"article_id": "PMC5215459", "article_text": ["pathological gambling is defined in the current classification system of the world health organization ( 1992 ) ( icd10 ) as an impulse control disorder ( icd ) which causes excessive , uncontrollable gambling despite financial losses and social problems , while the latest version of the diagnostic and statistical manual ( dsm5 ) of the american psychiatric association ( 2013 ) grouped pathological gambling together with substancerelated and addictive disorders and renamed it to gambling disorder . despite this aetiological debate , in parkinson 's patients", "it has been observed that pathological gambling occurs more frequently ( 3.46.1% ) than in the general population ( 0.252% ) , alongside with icds , such as binge eating , so called hypersexuality and compulsive shopping ( cox et  al . , 2005 ; avanzi et  al . , 2006 ; grosset et  al . , 2006 ; voon et  al . , 2006 ;", "bondolfi et  al . , 2008 ; weintraub et  al . , 2010 ; santangelo et  al . ,", "the aetiology of the development of pathological gambling in parkinson 's disease is still unclear , however , research suggests an association with dopamine replacement therapy , specifically with dopamine agonists ( voon et  al . , 2006 ;", "weintraub et  al . , 2006 ; gallagher et  al . , 2007 ) .", "this review summarizes evidence in this field of research attempting to reveal the relationship between parkinson therapy and pathological gambling , discusses the reasons why some patients react on them differently than others , what the relevant risk factors are and considers how impulsivity may contribute to the development of gambling symptoms .", "( 2007 ) found that these patients are younger , earned a higher score in tests investigating noveltyseeking and impulsive behaviour , and were more likely to have a personal or family history of alcohol abuse .", "being male and smoking in the past also seem to be risk factors ( gallagher et  al .", ", 2007 ; valena et  al . , 2013 ) . in this respect , pathological gambling with and without", "parkinson 's disease is rather similar : young age , male sex , impulsivity , noveltyseeking , smoking and alcoholism are also considered risk factors for pathological gambling in the general population ( johansson et  al . , 2009 ) .", "observing the progress of their disease , in comparison to other parkinson 's patients , those who later develop pathological gambling tend to have an earlier onset of the illness and also suffer more frequently from manic or hypomanic episodes during the onperiod of dopaminergic medication ( voon et  al . , 2007 ) .", "even in the first case reports about parkinson 's patients developing pathological gambling , a clear correlation has been observed with the initiation or dose escalation of dopaminergic medication ( molina et  al . , 2000 ;", "seedat et  al . , 2000 ) . in further studies comparing the effect of different parkinson 's therapies , dopamine agonists emerged as the medication with the strongest association with the development of pathological gambling ( voon et  al . , 2006 ; weintraub et  al . , 2006 , 2010 ;", "some studies claim that pramipexole could have the largest effect ( dodd et  al . , 2005 ) .", "other studies systematically comparing different dopamine agonists have found no significant difference between each of them ( weintraub et  al . , 2006 ;", "recent research also shows a strong effect of aripiprazole , prescribed for the treatment of mood disorders and schizophrenia , with stronger gamblingrelated cognition in comparison to other dopamine agonists ( grallbronnec et  al . , 2016 ) .", "levodopa seems to play a less important role as only a few patients developed pathological gambling under levodopa monotherapy ( dodd et  al .", ", 2005 ; voon et  al . , 2006 ; gallagher et  al . , 2007 ) , however , studies suggest that additionally prescribed levodopa raises the risk of the development of pathological gambling and icds ( dodd et  al . ,", "particularly high doses and longterm use of levodopa and shortacting dopamine agonists are also associated with dopamine dysregulation syndrome and punding , that is , stereotypic behaviour ( gallagher et  al . , 2007 ) .", "further , subthalamic nucleus deep brain stimulation ( stn dbs ) has a controversial role in the development of pathological gambling in parkinson 's disease .", "it has been observed that after the initiation of stn dbs therapy , gambling symptoms resolved ( ardouin et  al . , 2006 ; bandini et  al .", "these results could be explained by the significant reduction in the dosage of dopamine agonist medications .", "however , in some individual cases , pathological gambling and/or impulsive behaviour only developed after stn dbs surgery ( funkiewiez et  al .", ", 2003 ; contarino et  al . , 2007 ; smeding et  al . , 2007 ;", "2011 ) ; although in these cases the symptoms resolved spontaneously or after the change in stimulation parameters and further reduction in dopaminergic therapy . this could be associated with the stimulation of the limbic subregion of the stn which has been shown to affect neurotransmission in the limbic basal gangliathalamocortical circuitry ( winter et  al . , 2008 ) .", "evidence also shows that patients are more impulsive after activating stn dbs ( frank et  al .", "2009 ) . as impulsivity is considered as a risk factor for developing pathological gambling in parkinson patients ( voon et  al . , 2007 ) , the contentious effects of stn dbs raise questions about the role of impulsivity in the development of gambling behaviour in general ( table  1 ) .", "main results of studies on the association of pathological gambling with parkinson 's disease therapy bis , barratt impulsivity scale ; da , dopamine agonists ; icd , impulse control disorder ; igt , iowa gambling task ; ledd , ldopa equivalent daily dose ; pd , parkinson 's disease ; pg , pathological gambling ; stn dbs , subthalamic nucleus deep brain stimulation .", "the fact that not all parkinson 's patients develop medicationassociated impulse control disorders or pathological gambling and that most of the patients solely developed pathological gambling under dopaminergic medication suggests an underlying genetic vulnerability mechanism ( voon et  al . ,", "2006 ) . to analyse the genetic susceptibility of parkinson 's patients with pathological gambling , several genes have been examined that are relevant for the function of the mesolimbic reward system . the most obvious genes to investigate are the dopamine receptor genes , which could be affected by dopaminergic medications .", "some studies suggest that a certain mutation of the drd2 gene ( taq1a ) is more frequent in pathological gamblers than in the general population ( lobo et  al . , 2010 ) .", "this variation in the gene may be connected to a lower density of d2receptors in the striatum ( thompson et  al . , 1997 ) and to impulsivity ( eisenberg et  al . , 2007 ) , while the literature is inconclusive regarding its potential role in alcohol addiction ( heinz et  al . , 1996 ; heinz & goldman , 2000 ; munaf et  al . , 2007", "however , no significant difference was found between the frequency of this mutation between parkinson 's patients with and without pathological gambling ( lee et  al . , 2009 ) .", "interestingly , recent case reports suggest that not only dopamine agonists but also dopamine antagonists acting on the d2 receptors can trigger pathological gambling ( grtsch et  al . , 2015 ) , which underlines the role of this receptor . on the other hand ,", "the homozygote genotype of a single nucleotide mutation ( p.s9 g ) of the drd3 gene has been shown to have a higher frequency in pathological gamblers with parkinson 's disease ( lee et  al . , 2009 ) .", "this mutation is not associated with increased risk for pathological gambling in the general population ( lobo et  al . , 2010 ) .", "however , the heterozygote genotype of this mutation has been reported to be linked to impulsivity ( retz et  al . , 2003 ; limosin et  al . ,", "this mutation was also associated with decreased response rate to pramipexole in parkinson 's patients ( liu et  al . , 2009 ) , which could result in higher prescribed dosage .", "according to our current knowledge , there has not been any study performed yet to assess the relationship between drd4 mutations and pathological gambling in parkinson 's patients .", "however , the number of tandem repeats of a 48bp region in the drd4 gene is associated with pathological gambling , substance abuse and impulsivity , with discordant results of what number of repeats is relevant ( de castro et  al . , 1997 ; comings et  al . , 1999", "healthy subjects with this genotype also presented an increased gambling behaviour after receiving ldopa ( eisenegger et  al . , 2010 ) .", "another neurotransmitter system that has been shown to be affected in patients with pathological gambling is the serotoninergic system .", "( 1999 ) have observed a significantly higher frequency of the short ( s ) allele of the promoter region of the serotonin transporter gene , 5httlpr , in male pathological gamblers compared to the general population .", "the s allele of 5httlpr has also been associated with increased risk of developing depression under stress ( karg et  al . , 2011 ) , some aspects of impulsivity ( sakado et  al . ,", "2003 ) , impulsive aggression and increased activity in the amygdala after negative affective visual stimuli ( heinz et  al . , 2011 ) .", "an association between this mutation and pathological gambling has indeed been observed in patients with parkinson 's disease ( lee et  al . , 2009 ) .", "another mutation that may be associated with pathological gambling in parkinson 's patients is the mutation of grin2b ( lee et  al . , 2009 ) .", "grin2b is a gene from the 2b subunit of the nmda receptor , which is mainly expressed in the hippocampus , the striatum and also the cortex ( loftis & janowsky , 2003 ) .", "the variation found to be more frequent in parkinson 's patients with pathological gambling is a single nucleotide polymorphism .", "its specific role in the development of pathological gambling in parkinson 's disease is unclear , as this variation does not cause an amino acid change ( c.366c > g ) .", "furthermore , it was also found to be associated with schizophrenia ( li & he , 2007 ) , as a different polymorphism of grin2b has been associated with obsessive compulsive disorder ( arnold et  al . , 2004 ) .", "( 2011 ) found a different single nucleotide polymorphism of the grin2b gene to be related with risky decisionmaking , which might be considered as impulsive behaviour and therefore explain a link to pg in parkinson 's disease .", "these research findings suggest that an underlying genetic susceptibility might facilitate the development of pathological gambling in parkinson 's patients .", "however , some studies are inconsistent and there are some differences between pathological gamblers with and without parkinson 's disease .", "altogether , these results and the observed connection to dopaminergic medication described above suggest that the vulnerability of parkinson patients towards pathological gambling may be triggered by dopamine agonists .", "several studies have compared neuronal activation patterns of parkinson 's patients with and without pathological gambling . summarizing the results ,", "differences have been found in the activity of regions associated with the mesolimbic reward system , mainly in the orbitofrontal cortex ( ofc ) and the ventral striatum ( cilia et  al . , 2008 ;", "( 2008 ) compared the blood perfusion of different brain regions in parkinson 's patients with pathological gambling with patients who only have parkinson 's disease and a control group in a spect imaging study in a resting condition .", "they have observed a generally increased blood flow in the ofc , hippocampus , parahippocampal gyrus , amygdala , ventral striatum and cuneus on the right hemisphere and in the insulae on both sides in parkinson 's patients with pathological gambling compared to both other groups .", "( 2013 ) studied the function of the subthalamic nucleus by capturing local field potentials ( lfp ) in parkinson 's patients with and without pathological gambling on medication during an economic task .", "the lfps were recorded with the aid of stn dbs electrodes that were implanted 4  days prior to the experiment .", "the economic task included nonconflict and conflict decisions with stimuli pairs with the same probability vs. stimuli pairs with different probabilities of winning money . in conflict situations , risky choices could result in a higher reward , however , the task was overall designed to reward more nonrisky choices .", "the results showed that during the economic decisionmaking task , lowfrequency oscillations synchronize in the subthalamic nucleus .", "this synchronization was stronger during highconflict situations in comparison to lowconflict situations in patients with pathological gambling .", "patients without pathological gambling showed no differences in the synchronization of lowfrequency oscillations during conflict or nonconflict situations .", "the results of this experiment underline the role of the subthalamic nucleus in decisionmaking and might also explain why symptoms of pathological gambling resolve in some parkinson patients after stn dbs surgery .", "however , the results do not explain why patients usually only improve after months of stn dbs therapy .", "some studies focused more on the dopaminergic system and several differences were found between pathological gamblers with parkinson 's disease and parkinson 's patients without gambling .", "the turnover of monoamines , including dopamine , in the ofc was found to be higher ( joutsa et  al . , 2012 ) ,", "further the dopamine release during gambling tasks was found to be significantly increased in pathological gamblers ( steeves et  al . , 2009 ) .", "these results suggest that the vulnerability to gambling problems is partly mediated by increased dopaminergic neurotransmisson the ofc and the ventral striatum .", "pathological gambling in these patients may be caused by dopamine agonists in the mesolimbic dopaminergic system , particularly in the ventral striatum , which is less affected by the disease than the dorsal striatum . as dopamine agonist therapy seems to have a very strong association with the development of pathological gambling ( voon et  al .", "2010 ) , imaging studies have been conducted to further understand the effect of this medication .", "dopamine agonists have been shown to effect reward processing ; patients on this medication have a diminished reaction in the ofc after negative prediction errors compared to patients on levodopa therapy or off medication ( van eimeren et  al . , 2009 ) , suggesting a decreased learning effect after punishment .", "parkinson 's patients with and without pathological gambling or compulsive shopping were compared in a prediction learning task on or off dopamine agonists .", "patients with pathological gambling were faster and better at learning and had a higher activity in the ventral striatum and the ofc during rewardrelated learning while on medication . on the contrary , while learning through loss , the activity of these areas was lower in this group of patients than in the group with parkinson 's disease only under the same circumstances .", "( 2012 ) suggest that these patients have an impaired activation of d2 and d3autoreceptors caused by tonic stimulation through dopamine agonists . through the absence of negative feedback ,", "these findings could be used to propose that dopamine agonists cause a higher vulnerability to pathological gambling due to impaired learning processes . as a consequence of the impaired negative feedback", ", the dopamine concentration would not decrease to the previous level after a rewardrelated dopamine release .", "the high level of dopamine could also blunt the drop of dopamine concentration after punishment .", "imaging studies with nonparkinson patients with pathological gambling also showed differences in the activation of the mesolimbic rewards system , however , the results are not consistent .", "some studies showed a reduced activation of the prefrontal cortex and ventral striatum during loss and gain anticipation as well ( balodis et  al .", "2012 ) , others showed higher activity in the striatum during gain anticipation ( romanczukseiferth et  al . , 2015 ) .", "the activity of the prefrontal cortex and the ventral striatum also seems to be diminished after successful loss avoidance compared to healthy control subjects ( romanczukseiferth et  al .", "neuronal activity during loss and gain anticipation and loss avoidance have not been researched yet in parkinson 's patients with pathological gambling .", "as described above , impulsive behaviour is considered to be a general risk factor for developing pathological gambling in patients with parkinson 's disease ( voon et  al .", ", 2007 ; johansson et  al . , 2009 ) . however , there are studies that indicate a more specific connection : frank et  al .", "( 2007 ) compared two groups of patients with parkinson 's disease with a control group , assessing their learning ability in a probabilistic prediction task and their performance in a conflictbased decision task .", "one of the groups of parkinson 's patients was treated with dopaminergic medication , the other group with stn dbs and lowdose dopaminergic therapy .", "the first group 's performance was compared on and off medication , the second group 's performance on and off stn dbs without changing the dosage of their medication .", "the results in the prediction task in the group taking dopaminergic medication only were similar to the findings of voon et  al .", "( 2010 ) described above , that is , the learning ability of patients from negative outcome was impaired on medication .", "the activation of deep brain stimulation showed no effect on the learning ability of the patients , neither after reward nor after punishment . on the other hand , in the conflictbased decision task , patients with activated stn dbs responded faster in high rather than in lowconflict conditions , while off deep brain stimulation , their response was slower during highconflict situations .", "dopaminergic medication did not affect the difference in decisionmaking speed in high vs. lowconflict conditions .", "this result is supported by other experiments assessing patients with stn dbs clinically with the barratt impulsiveness scale ( hlbig et  al . , 2009 ) and the simon task ( wylie et  al . , 2010 ) . if high impulsivity can promote pathological gambling , as suggested by the results of frank et  al .", "( 2007 ) , there should be a higher risk for parkinson 's patients treated with stn dbs .", "however , there are only individual cases of patients developing pathological gambling after initiation of deep brain stimulation ( smeding et  al . , 2007 ) , with no clear way of interpretation , because dopaminergic medication had also been changed postoperatively .", "( 2009 ) found a higher frequency of impulse control disorders ( icds ) in parkinson 's patients treated with stn dbs .", "however , the difference in prevalence of icds to the patient group only receiving drug therapy was not significant and it was not described when these patients developed icds and how long they had already received dbs therapy .", "this information is relevant , as the recovery from icds after the initiation of dbs therapy can take up to 4  years and in some cases the symptoms initially worsen after the therapy ( ardouin et  al . , 2006 ) .", "the effects on impulsive and compulsive behaviour of stn dbs can also depend on the localization of the electrodes .", "the stimulation of the limbic subregion of the stn or the stimulation of adjacent structures can change the neurotransmission in limbic brain regions ( winter et  al . , 2008 ) .", "these findings question the causal relationship between high impulsivity and pathological gambling in parkinson 's patients .", "altogether , more research is needed for clarification of the effects of stn dbs . on the other hand , the results of those studies comparing the effect of stn dbs and dopamine agonist medication support the theory that parkinson 's patients with pathological gambling show impaired learning mechanisms modulated by dopamine agonists .", "therefore , alterations of reward and punishment processing seem to play a prominent role in the development of pathological gambling in parkinson 's patients .", "several genetic and neurofunctional findings suggest that individual differences in dopaminergic neurotransmission in the ventral striatum and associated brain areas contribute to pathological gambling in parkinson 's disease , and indicate complex interactions between such risk factors . taken together ,", "altered learning processes in parkinson 's patients with pathological gambling appear to include increased baseline blood perfusion of mesolimbic brain areas , increased activation by reward and reduced activation by punishment in those brain areas , which are implicated in reinforcement learning ( schultz , 2002 ) , impulsivity ( horn et  al . , 2003 ) , addiction ( kalivas & volkow , 2005 ) and pathological gambling ( romanczukseiferth et  al . , 2015 )", ". however , most of the studies performed in parkinson 's patients with pathological gambling are retrospective or crosssectional research , which makes the analysis of potentially causal factors more difficult .", "for example , in crosssectional studies , impulsivity seems to be an important risk factor ( voon et  al . , 2007 ) ; however , these findings are not fully consistent with the results of experimental studies on the effects of dbs of the stn . prospective or longitudinal studies could broaden the perspective on the role of potential risk factors , that is , impulsivity or impaired learning . despite the obstacles in conducting such studies , the results of this research can play a crucial role in understanding the development of pathological gambling and icds not only in parkinson 's patients but also in the general population ."], "abstract_text": ["<S> abstractthe incidence of pathological gambling in parkinson 's patients is significantly greater than in the general population . </S>", "<S> a correlation has been observed between dopamine agonist medication and the development of pathological gambling . </S>", "<S> however , scientists conjecture that the affected patients have underlying risk factors . studies analysing parkinson 's patients have detected that patients who developed pathological gambling are younger , score higher on noveltyseeking tests , are more impulsive and are more likely to have a personal or family history of alcohol addiction . </S>", "<S> in addition , some genetic variations have been associated with the susceptibility of developing pathological gambling , which include mutations of drd3 , 5httlpr and grin2b . </S>", "<S> studies focusing on neurofunctional discrepancies between parkinson 's patients with and without pathological gambling have found increased functional activation and dopamine release in regions associated with the mesolimbic reward system . </S>", "<S> furthermore , there is also evidence showing increased processing of reward and decreased activation elicited by punishment , suggesting altered learning processes . </S>", "<S> furthermore , the role of deep brain stimulation of the nucleus subthalamicus ( stn dbs ) is controversial . in most parkinson 's patients , </S>", "<S> pathological gambling resolved after the initiation of the stn dbs , which might be explained by discontinuation or decrease in dopamine agonist medication . </S>", "<S> however , it has been also shown that some patients are more impulsive while the stn dbs is activated . </S>", "<S> these differences may depend on the dbs localization in the more limbic or motor part of the stn and their regulative effects on impulsivity . </S>", "<S> further research is needed to clarify susceptibility factors for the development of pathological gambling in parkinson 's patients . </S>"], "labels": null, "section_names": ["Introduction", "Risk factors", "Association with Parkinson's disease therapy", "Genetic predisposition", "Neurofunctional alterations", "The role of impulsivity", "Conclusions", "Conflict of interests"], "sections": [["pathological gambling is defined in the current classification system of the world health organization ( 1992 ) ( icd10 ) as an impulse control disorder ( icd ) which causes excessive , uncontrollable gambling despite financial losses and social problems , while the latest version of the diagnostic and statistical manual ( dsm5 ) of the american psychiatric association ( 2013 ) grouped pathological gambling together with substancerelated and addictive disorders and renamed it to gambling disorder . despite this aetiological debate , in parkinson 's patients", "it has been observed that pathological gambling occurs more frequently ( 3.46.1% ) than in the general population ( 0.252% ) , alongside with icds , such as binge eating , so called hypersexuality and compulsive shopping ( cox et  al . , 2005 ; avanzi et  al . , 2006 ; grosset et  al . , 2006 ; voon et  al . , 2006 ;", "bondolfi et  al . , 2008 ; weintraub et  al . , 2010 ; santangelo et  al . ,", "the aetiology of the development of pathological gambling in parkinson 's disease is still unclear , however , research suggests an association with dopamine replacement therapy , specifically with dopamine agonists ( voon et  al . , 2006 ;", "weintraub et  al . , 2006 ; gallagher et  al . , 2007 ) .", "this review summarizes evidence in this field of research attempting to reveal the relationship between parkinson therapy and pathological gambling , discusses the reasons why some patients react on them differently than others , what the relevant risk factors are and considers how impulsivity may contribute to the development of gambling symptoms ."], ["( 2007 ) found that these patients are younger , earned a higher score in tests investigating noveltyseeking and impulsive behaviour , and were more likely to have a personal or family history of alcohol abuse .", "being male and smoking in the past also seem to be risk factors ( gallagher et  al .", ", 2007 ; valena et  al . , 2013 ) . in this respect , pathological gambling with and without", "parkinson 's disease is rather similar : young age , male sex , impulsivity , noveltyseeking , smoking and alcoholism are also considered risk factors for pathological gambling in the general population ( johansson et  al . , 2009 ) .", "observing the progress of their disease , in comparison to other parkinson 's patients , those who later develop pathological gambling tend to have an earlier onset of the illness and also suffer more frequently from manic or hypomanic episodes during the onperiod of dopaminergic medication ( voon et  al . , 2007 ) ."], ["even in the first case reports about parkinson 's patients developing pathological gambling , a clear correlation has been observed with the initiation or dose escalation of dopaminergic medication ( molina et  al . , 2000 ;", "seedat et  al . , 2000 ) . in further studies comparing the effect of different parkinson 's therapies , dopamine agonists emerged as the medication with the strongest association with the development of pathological gambling ( voon et  al . , 2006 ; weintraub et  al . , 2006 , 2010 ;", "some studies claim that pramipexole could have the largest effect ( dodd et  al . , 2005 ) .", "other studies systematically comparing different dopamine agonists have found no significant difference between each of them ( weintraub et  al . , 2006 ;", "recent research also shows a strong effect of aripiprazole , prescribed for the treatment of mood disorders and schizophrenia , with stronger gamblingrelated cognition in comparison to other dopamine agonists ( grallbronnec et  al . , 2016 ) .", "levodopa seems to play a less important role as only a few patients developed pathological gambling under levodopa monotherapy ( dodd et  al .", ", 2005 ; voon et  al . , 2006 ; gallagher et  al . , 2007 ) , however , studies suggest that additionally prescribed levodopa raises the risk of the development of pathological gambling and icds ( dodd et  al . ,", "particularly high doses and longterm use of levodopa and shortacting dopamine agonists are also associated with dopamine dysregulation syndrome and punding , that is , stereotypic behaviour ( gallagher et  al . , 2007 ) .", "further , subthalamic nucleus deep brain stimulation ( stn dbs ) has a controversial role in the development of pathological gambling in parkinson 's disease .", "it has been observed that after the initiation of stn dbs therapy , gambling symptoms resolved ( ardouin et  al . , 2006 ; bandini et  al .", "these results could be explained by the significant reduction in the dosage of dopamine agonist medications .", "however , in some individual cases , pathological gambling and/or impulsive behaviour only developed after stn dbs surgery ( funkiewiez et  al .", ", 2003 ; contarino et  al . , 2007 ; smeding et  al . , 2007 ;", "2011 ) ; although in these cases the symptoms resolved spontaneously or after the change in stimulation parameters and further reduction in dopaminergic therapy . this could be associated with the stimulation of the limbic subregion of the stn which has been shown to affect neurotransmission in the limbic basal gangliathalamocortical circuitry ( winter et  al . , 2008 ) .", "evidence also shows that patients are more impulsive after activating stn dbs ( frank et  al .", "2009 ) . as impulsivity is considered as a risk factor for developing pathological gambling in parkinson patients ( voon et  al . , 2007 ) , the contentious effects of stn dbs raise questions about the role of impulsivity in the development of gambling behaviour in general ( table  1 ) .", "main results of studies on the association of pathological gambling with parkinson 's disease therapy bis , barratt impulsivity scale ; da , dopamine agonists ; icd , impulse control disorder ; igt , iowa gambling task ; ledd , ldopa equivalent daily dose ; pd , parkinson 's disease ; pg , pathological gambling ; stn dbs , subthalamic nucleus deep brain stimulation ."], ["the fact that not all parkinson 's patients develop medicationassociated impulse control disorders or pathological gambling and that most of the patients solely developed pathological gambling under dopaminergic medication suggests an underlying genetic vulnerability mechanism ( voon et  al . ,", "2006 ) . to analyse the genetic susceptibility of parkinson 's patients with pathological gambling , several genes have been examined that are relevant for the function of the mesolimbic reward system . the most obvious genes to investigate are the dopamine receptor genes , which could be affected by dopaminergic medications .", "some studies suggest that a certain mutation of the drd2 gene ( taq1a ) is more frequent in pathological gamblers than in the general population ( lobo et  al . , 2010 ) .", "this variation in the gene may be connected to a lower density of d2receptors in the striatum ( thompson et  al . , 1997 ) and to impulsivity ( eisenberg et  al . , 2007 ) , while the literature is inconclusive regarding its potential role in alcohol addiction ( heinz et  al . , 1996 ; heinz & goldman , 2000 ; munaf et  al . , 2007", "however , no significant difference was found between the frequency of this mutation between parkinson 's patients with and without pathological gambling ( lee et  al . , 2009 ) .", "interestingly , recent case reports suggest that not only dopamine agonists but also dopamine antagonists acting on the d2 receptors can trigger pathological gambling ( grtsch et  al . , 2015 ) , which underlines the role of this receptor . on the other hand ,", "the homozygote genotype of a single nucleotide mutation ( p.s9 g ) of the drd3 gene has been shown to have a higher frequency in pathological gamblers with parkinson 's disease ( lee et  al . , 2009 ) .", "this mutation is not associated with increased risk for pathological gambling in the general population ( lobo et  al . , 2010 ) .", "however , the heterozygote genotype of this mutation has been reported to be linked to impulsivity ( retz et  al . , 2003 ; limosin et  al . ,", "this mutation was also associated with decreased response rate to pramipexole in parkinson 's patients ( liu et  al . , 2009 ) , which could result in higher prescribed dosage .", "according to our current knowledge , there has not been any study performed yet to assess the relationship between drd4 mutations and pathological gambling in parkinson 's patients .", "however , the number of tandem repeats of a 48bp region in the drd4 gene is associated with pathological gambling , substance abuse and impulsivity , with discordant results of what number of repeats is relevant ( de castro et  al . , 1997 ; comings et  al . , 1999", "healthy subjects with this genotype also presented an increased gambling behaviour after receiving ldopa ( eisenegger et  al . , 2010 ) .", "another neurotransmitter system that has been shown to be affected in patients with pathological gambling is the serotoninergic system .", "( 1999 ) have observed a significantly higher frequency of the short ( s ) allele of the promoter region of the serotonin transporter gene , 5httlpr , in male pathological gamblers compared to the general population .", "the s allele of 5httlpr has also been associated with increased risk of developing depression under stress ( karg et  al . , 2011 ) , some aspects of impulsivity ( sakado et  al . ,", "2003 ) , impulsive aggression and increased activity in the amygdala after negative affective visual stimuli ( heinz et  al . , 2011 ) .", "an association between this mutation and pathological gambling has indeed been observed in patients with parkinson 's disease ( lee et  al . , 2009 ) .", "another mutation that may be associated with pathological gambling in parkinson 's patients is the mutation of grin2b ( lee et  al . , 2009 ) .", "grin2b is a gene from the 2b subunit of the nmda receptor , which is mainly expressed in the hippocampus , the striatum and also the cortex ( loftis & janowsky , 2003 ) .", "the variation found to be more frequent in parkinson 's patients with pathological gambling is a single nucleotide polymorphism .", "its specific role in the development of pathological gambling in parkinson 's disease is unclear , as this variation does not cause an amino acid change ( c.366c > g ) .", "furthermore , it was also found to be associated with schizophrenia ( li & he , 2007 ) , as a different polymorphism of grin2b has been associated with obsessive compulsive disorder ( arnold et  al . , 2004 ) .", "( 2011 ) found a different single nucleotide polymorphism of the grin2b gene to be related with risky decisionmaking , which might be considered as impulsive behaviour and therefore explain a link to pg in parkinson 's disease .", "these research findings suggest that an underlying genetic susceptibility might facilitate the development of pathological gambling in parkinson 's patients .", "however , some studies are inconsistent and there are some differences between pathological gamblers with and without parkinson 's disease .", "altogether , these results and the observed connection to dopaminergic medication described above suggest that the vulnerability of parkinson patients towards pathological gambling may be triggered by dopamine agonists ."], ["several studies have compared neuronal activation patterns of parkinson 's patients with and without pathological gambling . summarizing the results ,", "differences have been found in the activity of regions associated with the mesolimbic reward system , mainly in the orbitofrontal cortex ( ofc ) and the ventral striatum ( cilia et  al . , 2008 ;", "( 2008 ) compared the blood perfusion of different brain regions in parkinson 's patients with pathological gambling with patients who only have parkinson 's disease and a control group in a spect imaging study in a resting condition .", "they have observed a generally increased blood flow in the ofc , hippocampus , parahippocampal gyrus , amygdala , ventral striatum and cuneus on the right hemisphere and in the insulae on both sides in parkinson 's patients with pathological gambling compared to both other groups .", "( 2013 ) studied the function of the subthalamic nucleus by capturing local field potentials ( lfp ) in parkinson 's patients with and without pathological gambling on medication during an economic task .", "the lfps were recorded with the aid of stn dbs electrodes that were implanted 4  days prior to the experiment .", "the economic task included nonconflict and conflict decisions with stimuli pairs with the same probability vs. stimuli pairs with different probabilities of winning money . in conflict situations , risky choices could result in a higher reward , however , the task was overall designed to reward more nonrisky choices .", "the results showed that during the economic decisionmaking task , lowfrequency oscillations synchronize in the subthalamic nucleus .", "this synchronization was stronger during highconflict situations in comparison to lowconflict situations in patients with pathological gambling .", "patients without pathological gambling showed no differences in the synchronization of lowfrequency oscillations during conflict or nonconflict situations .", "the results of this experiment underline the role of the subthalamic nucleus in decisionmaking and might also explain why symptoms of pathological gambling resolve in some parkinson patients after stn dbs surgery .", "however , the results do not explain why patients usually only improve after months of stn dbs therapy .", "some studies focused more on the dopaminergic system and several differences were found between pathological gamblers with parkinson 's disease and parkinson 's patients without gambling .", "the turnover of monoamines , including dopamine , in the ofc was found to be higher ( joutsa et  al . , 2012 ) ,", "further the dopamine release during gambling tasks was found to be significantly increased in pathological gamblers ( steeves et  al . , 2009 ) .", "these results suggest that the vulnerability to gambling problems is partly mediated by increased dopaminergic neurotransmisson the ofc and the ventral striatum .", "pathological gambling in these patients may be caused by dopamine agonists in the mesolimbic dopaminergic system , particularly in the ventral striatum , which is less affected by the disease than the dorsal striatum . as dopamine agonist therapy seems to have a very strong association with the development of pathological gambling ( voon et  al .", "2010 ) , imaging studies have been conducted to further understand the effect of this medication .", "dopamine agonists have been shown to effect reward processing ; patients on this medication have a diminished reaction in the ofc after negative prediction errors compared to patients on levodopa therapy or off medication ( van eimeren et  al . , 2009 ) , suggesting a decreased learning effect after punishment .", "parkinson 's patients with and without pathological gambling or compulsive shopping were compared in a prediction learning task on or off dopamine agonists .", "patients with pathological gambling were faster and better at learning and had a higher activity in the ventral striatum and the ofc during rewardrelated learning while on medication . on the contrary , while learning through loss , the activity of these areas was lower in this group of patients than in the group with parkinson 's disease only under the same circumstances .", "( 2012 ) suggest that these patients have an impaired activation of d2 and d3autoreceptors caused by tonic stimulation through dopamine agonists . through the absence of negative feedback ,", "these findings could be used to propose that dopamine agonists cause a higher vulnerability to pathological gambling due to impaired learning processes . as a consequence of the impaired negative feedback", ", the dopamine concentration would not decrease to the previous level after a rewardrelated dopamine release .", "the high level of dopamine could also blunt the drop of dopamine concentration after punishment .", "imaging studies with nonparkinson patients with pathological gambling also showed differences in the activation of the mesolimbic rewards system , however , the results are not consistent .", "some studies showed a reduced activation of the prefrontal cortex and ventral striatum during loss and gain anticipation as well ( balodis et  al .", "2012 ) , others showed higher activity in the striatum during gain anticipation ( romanczukseiferth et  al . , 2015 ) .", "the activity of the prefrontal cortex and the ventral striatum also seems to be diminished after successful loss avoidance compared to healthy control subjects ( romanczukseiferth et  al .", "neuronal activity during loss and gain anticipation and loss avoidance have not been researched yet in parkinson 's patients with pathological gambling ."], ["as described above , impulsive behaviour is considered to be a general risk factor for developing pathological gambling in patients with parkinson 's disease ( voon et  al .", ", 2007 ; johansson et  al . , 2009 ) . however , there are studies that indicate a more specific connection : frank et  al .", "( 2007 ) compared two groups of patients with parkinson 's disease with a control group , assessing their learning ability in a probabilistic prediction task and their performance in a conflictbased decision task .", "one of the groups of parkinson 's patients was treated with dopaminergic medication , the other group with stn dbs and lowdose dopaminergic therapy .", "the first group 's performance was compared on and off medication , the second group 's performance on and off stn dbs without changing the dosage of their medication .", "the results in the prediction task in the group taking dopaminergic medication only were similar to the findings of voon et  al .", "( 2010 ) described above , that is , the learning ability of patients from negative outcome was impaired on medication .", "the activation of deep brain stimulation showed no effect on the learning ability of the patients , neither after reward nor after punishment . on the other hand , in the conflictbased decision task , patients with activated stn dbs responded faster in high rather than in lowconflict conditions , while off deep brain stimulation , their response was slower during highconflict situations .", "dopaminergic medication did not affect the difference in decisionmaking speed in high vs. lowconflict conditions .", "this result is supported by other experiments assessing patients with stn dbs clinically with the barratt impulsiveness scale ( hlbig et  al . , 2009 ) and the simon task ( wylie et  al . , 2010 ) . if high impulsivity can promote pathological gambling , as suggested by the results of frank et  al .", "( 2007 ) , there should be a higher risk for parkinson 's patients treated with stn dbs .", "however , there are only individual cases of patients developing pathological gambling after initiation of deep brain stimulation ( smeding et  al . , 2007 ) , with no clear way of interpretation , because dopaminergic medication had also been changed postoperatively .", "( 2009 ) found a higher frequency of impulse control disorders ( icds ) in parkinson 's patients treated with stn dbs .", "however , the difference in prevalence of icds to the patient group only receiving drug therapy was not significant and it was not described when these patients developed icds and how long they had already received dbs therapy .", "this information is relevant , as the recovery from icds after the initiation of dbs therapy can take up to 4  years and in some cases the symptoms initially worsen after the therapy ( ardouin et  al . , 2006 ) .", "the effects on impulsive and compulsive behaviour of stn dbs can also depend on the localization of the electrodes .", "the stimulation of the limbic subregion of the stn or the stimulation of adjacent structures can change the neurotransmission in limbic brain regions ( winter et  al . , 2008 ) .", "these findings question the causal relationship between high impulsivity and pathological gambling in parkinson 's patients .", "altogether , more research is needed for clarification of the effects of stn dbs . on the other hand , the results of those studies comparing the effect of stn dbs and dopamine agonist medication support the theory that parkinson 's patients with pathological gambling show impaired learning mechanisms modulated by dopamine agonists .", "therefore , alterations of reward and punishment processing seem to play a prominent role in the development of pathological gambling in parkinson 's patients ."], ["several genetic and neurofunctional findings suggest that individual differences in dopaminergic neurotransmission in the ventral striatum and associated brain areas contribute to pathological gambling in parkinson 's disease , and indicate complex interactions between such risk factors . taken together ,", "altered learning processes in parkinson 's patients with pathological gambling appear to include increased baseline blood perfusion of mesolimbic brain areas , increased activation by reward and reduced activation by punishment in those brain areas , which are implicated in reinforcement learning ( schultz , 2002 ) , impulsivity ( horn et  al . , 2003 ) , addiction ( kalivas & volkow , 2005 ) and pathological gambling ( romanczukseiferth et  al . , 2015 )", ". however , most of the studies performed in parkinson 's patients with pathological gambling are retrospective or crosssectional research , which makes the analysis of potentially causal factors more difficult .", "for example , in crosssectional studies , impulsivity seems to be an important risk factor ( voon et  al . , 2007 ) ; however , these findings are not fully consistent with the results of experimental studies on the effects of dbs of the stn . prospective or longitudinal studies could broaden the perspective on the role of potential risk factors , that is , impulsivity or impaired learning . despite the obstacles in conducting such studies , the results of this research can play a crucial role in understanding the development of pathological gambling and icds not only in parkinson 's patients but also in the general population ."], [""]]}
{"article_id": "PMC5328611", "article_text": ["suicide is one of the leading causes of death throughout the world ; it ranks among the top 10 causes of death for individuals of all ages and is the leading cause of death in males under 35 years old.1,2 a decrease in the concentrations of serotonin metabolites ( in particular the serotonin 5-hydroxyindoleacetic ) in patients with suicide behavior ( suicide attempt and suicide ) suggests that the serotonergic system is associated with suicide behavior.3,4 studies have consistently proposed that genes of the serotonergic system could play an important role in suicide.5,6 for example , in sh - sy5y cells , the administration of human interferon - alpha modifies the htr2c mrna editing , and this could be a mechanism of drug - induced depression or suicidal side effects in humans.7,8 it has also been proposed that the serotonin receptor 2c ( htr2c ) gene9,10 contributes to suicide behavior.11 the htr2c belongs to the family of 5-ht2 receptors , which has three members : 5-ht2a , 5-ht2b , and 5-ht2c.1 the gene that encodes the htr2c is located on the long arm of the x chromosome at position 24 ; and it contains six exons and five introns , spanning at least 230 kb.12 one of the most common variants of the htr2c is the cys23ser ( rs6318 ) , which is a functional polymorphism ( c / g at position 68 ) that results in the substitution of cysteine for serine at position 23 of the n - terminal extracellular domain.2,13,14 in the last few years , several authors have reported an association between the htr2c polymorphism and suicidal behavior.15 furthermore , in analyses performed by gender , a negative association has been observed between the htr2c polymorphism and suicidal behavior.14,16 other polymorphisms of htr2c that also have been studied in suicide behavior are rs547536 , rs2192372 , rs2428707 , and rs4272555;14 however , no association has been established in several populations . therefore , the association between the htr2c gene remains controversial , given that the available case ", "control studies have obtained both positive and negative findings.1315,1620 in addition , to date , the relationship between the htr2c gene and suicide behavior in the mexican population has not yet been studied . as the htr2c gene is located on chromosome x ,", "the objective of this work was to analyze the association by gender between five genetic variants of htr2c gene , rs547536 , rs2192372 , rs6318 , rs2428707 , and rs4272555 , and suicide attempt in a mexican population .", "a total of 187 unrelated patients , of whom 110 ( 58.8% ) were females and 77 ( 41.2% ) were males who had attempted suicide , were included in this study .", "we defined suicide attempt as a self - harm behavior with at least some intent to end one s life .", "the subjects were recruited from the outpatient service of the general hospital of comalcalco in the state of tabasco , mexico .", "we also included 223 healthy subjects as controls ( 146 females [ 65.5% ] , 77 males [ 34.5% ] ) who were recruited from the blood donor center at the same hospital .", "we want to emphasize that all the individuals in this study were exclusively from comalcalco and had parents and grandparents of mexican origin so as to reduce ethnic variation and stratification effects .", "we considered as exclusion criteria patients who had an organic disorder or self - injury behavior , patients who had concomitant diagnoses of mental retardation or drug dependence , and patients with somatic or neurological illnesses that impaired psychiatric evaluation .", "all the individuals ( suicide patients and healthy subjects ) were evaluated by trained psychiatrists or clinical psychologists with a master s degree as the minimum education level . for all of them , the lifetime diagnoses were determined using the diagnostic and statistical manual of mental disorders  fourth edition ( dsm - iv ) criteria following appropriate interviews ( structured clinical interview for dsm - iv [ scid ] i and ii).21 the suicide patients were classified in one of the following psychiatric diagnoses : schizophrenia spectrum disorders n=32 ( 17.1% ) , mood disorders n=59 ( 31.5% ) , stress - related disorders n=73 ( 39.0% ) , and substance - related disorders 23 ( 12.4% ) . written informed consent was obtained from all subjects after the procedures had been fully explained to them .", "the study was approved by the local ethics and research committee of divisin acadmica multidisciplinaria de comalcalco ( damc)-universidad jurez autnoma de tabasco ( ujat ) ( ujat - damc-2012 - 02 ) .", "the study was performed in compliance with the ethical standards of the 1964 declaration of helsinki .", "genomic dna was isolated from whole blood samples using standard procedures that have been previously reported.9,22 genotyping of the htr2c variants rs547536 , rs2192372 , rs4272555 , rs6318 , and rs2428707 was performed using the taqman ( applied biosystems inc , foster city , ca , usa ) snp genotyping assay obtained from applied biosystems .", "we selected five single - nucleotide polymorphisms ( snps ) of the htr2c gene found in the promotor and introns of the gene ( table 1 ) .", "first , the hardy  weinberg equilibrium for the htr2c variants was determined using pearson s goodness of fit  test . for female controls", ", the genotype frequencies of rs6318 , rs2428707 , and rs4272555 did not deviate from hardy  weinberg equilibrium ( p>0.05 ) ; however , the genotype frequencies of rs547536 and rs2192372 did deviate from hardy  weinberg equilibrium ( p<0.05 ) .", "random blind duplicates of up 30% of the samples were used as a quality control measure , and we obtained consistent results .", "then ,  and fisher s exact tests were used to compare the genotype and allele frequencies between cases and controls .", "finally , the haploview 4.2 ( broad institute , cambridge , ma , usa)23 was employed to calculate the linkage disequilibrium ( ld ) of the markers .", "all the individuals ( suicide patients and healthy subjects ) were evaluated by trained psychiatrists or clinical psychologists with a master s degree as the minimum education level . for all of them , the lifetime diagnoses were determined using the diagnostic and statistical manual of mental disorders  fourth edition ( dsm - iv ) criteria following appropriate interviews ( structured clinical interview for dsm - iv [ scid ] i and ii).21 the suicide patients were classified in one of the following psychiatric diagnoses : schizophrenia spectrum disorders n=32 ( 17.1% ) , mood disorders n=59 ( 31.5% ) , stress - related disorders n=73 ( 39.0% ) , and substance - related disorders 23 ( 12.4% ) .", "written informed consent was obtained from all subjects after the procedures had been fully explained to them .", "the study was approved by the local ethics and research committee of divisin acadmica multidisciplinaria de comalcalco ( damc)-universidad jurez autnoma de tabasco ( ujat ) ( ujat - damc-2012 - 02 ) .", "the study was performed in compliance with the ethical standards of the 1964 declaration of helsinki .", "genomic dna was isolated from whole blood samples using standard procedures that have been previously reported.9,22 genotyping of the htr2c variants rs547536 , rs2192372 , rs4272555 , rs6318 , and rs2428707 was performed using the taqman ( applied biosystems inc , foster city , ca , usa ) snp genotyping assay obtained from applied biosystems .", "we selected five single - nucleotide polymorphisms ( snps ) of the htr2c gene found in the promotor and introns of the gene ( table 1 ) .", "first , the hardy  weinberg equilibrium for the htr2c variants was determined using pearson s goodness of fit  test . for female controls ,", "the genotype frequencies of rs6318 , rs2428707 , and rs4272555 did not deviate from hardy  weinberg equilibrium ( p>0.05 ) ; however , the genotype frequencies of rs547536 and rs2192372 did deviate from hardy  weinberg equilibrium ( p<0.05 ) .", "random blind duplicates of up 30% of the samples were used as a quality control measure , and we obtained consistent results .", "then ,  and fisher s exact tests were used to compare the genotype and allele frequencies between cases and controls .", "finally , the haploview 4.2 ( broad institute , cambridge , ma , usa)23 was employed to calculate the linkage disequilibrium ( ld ) of the markers .", "the demographic characteristics of the cases ( mean age : 25.96 years , standard deviation [ sd ] : 9.26 range : 1456 years ) and comparison group ( mean age : 31.95 years , sd : 13.03 range : 1461 years ) in this mexican population are presented in table 2 .", "table 3 shows the genotype and allele frequencies of the htr2c variants rs547536 , rs21923372 , rs6318 , rs2428707 , and rs4272555 in the suicide attempters and control groups .", "the htr2c variants rs4272555 and rs2428707 were significantly associated with suicide attempt in this mexican population . in the female group ,", "the g allele of the snp rs2428707 was associated with an increased risk of suicide attempt ( p=0.01 , odd ratio [ or ] = 3.68 , 95% confidence inrterval [ ci ] : 1.2410.90 ) , while the c allele of the snp rs4272555 was associated with a decreased risk of suicide attempt ( p=0.01 , or = 0.26 , 95% ci : 0.090.79 ) .", "the distributions of the htr2c variants rs547536 , rs21923372 , and rs6318 were not significantly different between the subjects with suicidal behavior and the control group ( table 3 ) .", "subsequently , for the snps that showed a significant association , we summarized the evidence and performed a meta - analysis .", "the results of the meta - analysis ( supplementary materials ) showed a nonsignificant association for the rs2428707 ( fixed effects : or : 1.21 , 95% ci : 0.801.82 , p(q)=0.03 ) and for the rs4272555 ( fixed effects : or : 0.56 , 95% ci : 0.161.94 , p(q)=0.03 ) ( figures s1s4 and tables s1 and s2 ) .", "the ld ( linkage disequilibrium ) in this mexican population was measured for all markers examined in this case  control study ( rs547536 , rs2192372 , rs6318 , rs4272555 , and rs2428707 ) .", "we conducted a haplotype analysis of the same five markers and did not find a significant association between suicide attempt and any of the examined haplotypes ( p>0.01 , table 4 ) .", "the demographic characteristics of the cases ( mean age : 25.96 years , standard deviation [ sd ] : 9.26 range : 1456 years ) and comparison group ( mean age : 31.95 years , sd : 13.03 range : 1461 years ) in this mexican population are presented in table 2 .", "table 3 shows the genotype and allele frequencies of the htr2c variants rs547536 , rs21923372 , rs6318 , rs2428707 , and rs4272555 in the suicide attempters and control groups .", "the htr2c variants rs4272555 and rs2428707 were significantly associated with suicide attempt in this mexican population . in the female group ,", "the g allele of the snp rs2428707 was associated with an increased risk of suicide attempt ( p=0.01 , odd ratio [ or ] = 3.68 , 95% confidence inrterval [ ci ] : 1.2410.90 ) , while the c allele of the snp rs4272555 was associated with a decreased risk of suicide attempt ( p=0.01 , or = 0.26 , 95% ci : 0.090.79 ) .", "the distributions of the htr2c variants rs547536 , rs21923372 , and rs6318 were not significantly different between the subjects with suicidal behavior and the control group ( table 3 ) .", "subsequently , for the snps that showed a significant association , we summarized the evidence and performed a meta - analysis .", "the results of the meta - analysis ( supplementary materials ) showed a nonsignificant association for the rs2428707 ( fixed effects : or : 1.21 , 95% ci : 0.801.82 , p(q)=0.03 ) and for the rs4272555 ( fixed effects : or : 0.56 , 95% ci : 0.161.94 , p(q)=0.03 ) ( figures s1s4 and tables s1 and s2 ) .", "the ld ( linkage disequilibrium ) in this mexican population was measured for all markers examined in this case  control study ( rs547536 , rs2192372 , rs6318 , rs4272555 , and rs2428707 ) .", "we conducted a haplotype analysis of the same five markers and did not find a significant association between suicide attempt and any of the examined haplotypes ( p>0.01 , table 4 ) .", "the aim of the present study was to assess the association between the htr2c variants rs547536 , rs2192372 , rs6318 , rs2428707 , and rs4272555 and suicide attempt in a mexican population .", "to our knowledge , this is the first association analysis between htr2c polymorphisms and suicidal behavior performed in a latin american population .", "we found that the g allele of rs2428707 was associated with an increased risk of suicidal behavior in females ; however , this is not similar to the results of previous studies.14,20 this discrepancy could be due to differences of the sample sizes used .", "the number of control subjects used in those previous studies and our study were similar ; however , the number of cases in our study were fewer than what the german and italian populations used in previous studies.14,20 another possible explanation for this discrepancy is the heterogeneity between the studies . in our study", ", the absence of the a allele of rs2428707 and the t allele of rs4272555 in the female group , could be ethnic - dependent because the allelic and genotypic distributions we observed were different from those reported in the literature.14,20 third , the  positive results  of this study are modest because we did not exclude the presence of false positives , even when the results were corrected by bonferroni s . for major evidence , we performed a meta - analysis that included studies that have been reported up to date and observed a no - association .", "however , we considered that the low number of studies , the low number of samples , and the presence of heterogeneity in the analysis reduced the statistical power to detect a possible association between the htr2c polymorphisms and suicidal behavior . in consequence , more studies that analyze this association are needed .", "our analysis also showed an opposite effect for the rs4272555 and suicide ; we observed that in females , the c allele of the rs4272555 is a protective allele against suicidal behavior .", "it is fundamental to consider these findings with caution for the following reasons : first , the ld analysis indicated stronger linkage disequilibrium for rs2428707 and rs4272555 than for the other htr2c variants .", "second , the small sample size of our study may have resulted in a weaker statistical association than other studies with a larger study population .", "therefore , it is necessary to analyze other populations and increase the sample size , without the presence of the heterogeneity , in order to have conclusive results .", "the htr2c cys23ser ( rs6318 ) polymorphism is perhaps the most extensively studied for a possible association with suicidal behavior11,13,14,1720 and self - injury.24 the present study did not find an association between the htr2c cys23ser ( rs6318 ) and suicidal behavior .", "however , this finding is in accordance with previous studies14,20 performed in caucasian and chinese populations.18,19 in contrast , in a recent study by karanovic et al,11 an association between the c allele of htr2c rs6318 and suicide attempt was observed .", "karanovi et al11 included 165 suicide attempters and 188 controls , which is similar to what we used in the present study ; however , their controls were subjects with psychiatric diseases other than suicidal behavior and our controls were subjects without any psychiatric disorders .", "hence , these findings need to be replicated before any strong conclusions can be drawn .", "second , although we perform a bonferroni correction and performed a meta - analysis , the small sample size and the small number of the studies ( respectively ) do not ensure the absence of false positives . finally , we did not analyze the clinical features of suicide attempters that may predispose them to this behavior .", "our results suggest an association between the rs2428707 and rs4272555 polymorphisms of htr2c in mexican females with suicidal ideation .", "future research must examine the potential mediating effects of the htr2c gene variants to highlight the importance of the serotonin system in suicidal behavior , with special focus on the receptor gene htr2c .", "or and forest plot for the meta - analysis of rs2428707 . note : g allele vs a allele with heterogeneity .", "note : g allele vs a allele with heterogeneity . or and forest plot for the meta - analysis of rs4272555 .", "funnel plot indicating publication bias for studies included in the meta - analysis of rs4272555 .", "descriptive characteristics of two studies analyzing the role of the htr2c gene variant rs2428707 in suicide attempt descriptive characteristics of two studies in the meta - analysis of the htr2c gene variant rs4272555 in suicide attempt"], "abstract_text": ["<S> backgroundthe aim of this case  control study was to explore the association by gender between the htr2c gene variants and suicidal behavior in a mexican population.subjects and methodsa total of 183 suicide attempters and 208 healthy volunteers were included in this study . </S>", "<S> we genotyped five polymorphisms of htr2c ( rs547536 , rs2192372 , rs4272555 , rs6318 , and rs2428707 ) , then measured the association by genotype , allele , and haplotype.resultsin the female group , we found an association between two polymorphisms of the htr2c ( rs4272555 and rs2428707 ) and suicide attempts . </S>", "<S> the c allele of the single - nucleotide polymorphism ( snp ) rs4272555 was associated with a decreased risk of suicide attempt ( p=0.01 , odds ratio = 0.26 , 95% confidence interval : 0.090.79 ) , whereas the g allele of the snp rs2428707 was associated with an increased risk of suicide attempt ( p=0.01 , odds ratio = 3.68 , 95% confidence interval : 1.2410.90 ) . </S>", "<S> no significant association was observed between the other polymorphisms studied ( rs547536 , rs2192372 , rs6318 ) or haplotypes with suicide attempts.conclusionthese findings suggest a possible risk factor of the htr2c gene in the pathology of suicidal behavior in mexican population . more studies are necessary to confirm this association . </S>"], "labels": null, "section_names": ["Introduction", "Subjects and methods", "Clinical evaluation", "Ethics statement", "Genotype assays", "Statistical analysis", "Results", "SNP association analysis", "Haplotype association analysis", "Discussion", "Conclusion", "Supplementary materials"], "sections": [["suicide is one of the leading causes of death throughout the world ; it ranks among the top 10 causes of death for individuals of all ages and is the leading cause of death in males under 35 years old.1,2 a decrease in the concentrations of serotonin metabolites ( in particular the serotonin 5-hydroxyindoleacetic ) in patients with suicide behavior ( suicide attempt and suicide ) suggests that the serotonergic system is associated with suicide behavior.3,4 studies have consistently proposed that genes of the serotonergic system could play an important role in suicide.5,6 for example , in sh - sy5y cells , the administration of human interferon - alpha modifies the htr2c mrna editing , and this could be a mechanism of drug - induced depression or suicidal side effects in humans.7,8 it has also been proposed that the serotonin receptor 2c ( htr2c ) gene9,10 contributes to suicide behavior.11 the htr2c belongs to the family of 5-ht2 receptors , which has three members : 5-ht2a , 5-ht2b , and 5-ht2c.1 the gene that encodes the htr2c is located on the long arm of the x chromosome at position 24 ; and it contains six exons and five introns , spanning at least 230 kb.12 one of the most common variants of the htr2c is the cys23ser ( rs6318 ) , which is a functional polymorphism ( c / g at position 68 ) that results in the substitution of cysteine for serine at position 23 of the n - terminal extracellular domain.2,13,14 in the last few years , several authors have reported an association between the htr2c polymorphism and suicidal behavior.15 furthermore , in analyses performed by gender , a negative association has been observed between the htr2c polymorphism and suicidal behavior.14,16 other polymorphisms of htr2c that also have been studied in suicide behavior are rs547536 , rs2192372 , rs2428707 , and rs4272555;14 however , no association has been established in several populations . therefore , the association between the htr2c gene remains controversial , given that the available case ", "control studies have obtained both positive and negative findings.1315,1620 in addition , to date , the relationship between the htr2c gene and suicide behavior in the mexican population has not yet been studied . as the htr2c gene is located on chromosome x ,", "the objective of this work was to analyze the association by gender between five genetic variants of htr2c gene , rs547536 , rs2192372 , rs6318 , rs2428707 , and rs4272555 , and suicide attempt in a mexican population ."], ["a total of 187 unrelated patients , of whom 110 ( 58.8% ) were females and 77 ( 41.2% ) were males who had attempted suicide , were included in this study .", "we defined suicide attempt as a self - harm behavior with at least some intent to end one s life .", "the subjects were recruited from the outpatient service of the general hospital of comalcalco in the state of tabasco , mexico .", "we also included 223 healthy subjects as controls ( 146 females [ 65.5% ] , 77 males [ 34.5% ] ) who were recruited from the blood donor center at the same hospital .", "we want to emphasize that all the individuals in this study were exclusively from comalcalco and had parents and grandparents of mexican origin so as to reduce ethnic variation and stratification effects .", "we considered as exclusion criteria patients who had an organic disorder or self - injury behavior , patients who had concomitant diagnoses of mental retardation or drug dependence , and patients with somatic or neurological illnesses that impaired psychiatric evaluation .", "all the individuals ( suicide patients and healthy subjects ) were evaluated by trained psychiatrists or clinical psychologists with a master s degree as the minimum education level . for all of them , the lifetime diagnoses were determined using the diagnostic and statistical manual of mental disorders  fourth edition ( dsm - iv ) criteria following appropriate interviews ( structured clinical interview for dsm - iv [ scid ] i and ii).21 the suicide patients were classified in one of the following psychiatric diagnoses : schizophrenia spectrum disorders n=32 ( 17.1% ) , mood disorders n=59 ( 31.5% ) , stress - related disorders n=73 ( 39.0% ) , and substance - related disorders 23 ( 12.4% ) . written informed consent was obtained from all subjects after the procedures had been fully explained to them .", "the study was approved by the local ethics and research committee of divisin acadmica multidisciplinaria de comalcalco ( damc)-universidad jurez autnoma de tabasco ( ujat ) ( ujat - damc-2012 - 02 ) .", "the study was performed in compliance with the ethical standards of the 1964 declaration of helsinki .", "genomic dna was isolated from whole blood samples using standard procedures that have been previously reported.9,22 genotyping of the htr2c variants rs547536 , rs2192372 , rs4272555 , rs6318 , and rs2428707 was performed using the taqman ( applied biosystems inc , foster city , ca , usa ) snp genotyping assay obtained from applied biosystems .", "we selected five single - nucleotide polymorphisms ( snps ) of the htr2c gene found in the promotor and introns of the gene ( table 1 ) .", "first , the hardy  weinberg equilibrium for the htr2c variants was determined using pearson s goodness of fit  test . for female controls", ", the genotype frequencies of rs6318 , rs2428707 , and rs4272555 did not deviate from hardy  weinberg equilibrium ( p>0.05 ) ; however , the genotype frequencies of rs547536 and rs2192372 did deviate from hardy  weinberg equilibrium ( p<0.05 ) .", "random blind duplicates of up 30% of the samples were used as a quality control measure , and we obtained consistent results .", "then ,  and fisher s exact tests were used to compare the genotype and allele frequencies between cases and controls .", "finally , the haploview 4.2 ( broad institute , cambridge , ma , usa)23 was employed to calculate the linkage disequilibrium ( ld ) of the markers ."], ["all the individuals ( suicide patients and healthy subjects ) were evaluated by trained psychiatrists or clinical psychologists with a master s degree as the minimum education level . for all of them , the lifetime diagnoses were determined using the diagnostic and statistical manual of mental disorders  fourth edition ( dsm - iv ) criteria following appropriate interviews ( structured clinical interview for dsm - iv [ scid ] i and ii).21 the suicide patients were classified in one of the following psychiatric diagnoses : schizophrenia spectrum disorders n=32 ( 17.1% ) , mood disorders n=59 ( 31.5% ) , stress - related disorders n=73 ( 39.0% ) , and substance - related disorders 23 ( 12.4% ) ."], ["written informed consent was obtained from all subjects after the procedures had been fully explained to them .", "the study was approved by the local ethics and research committee of divisin acadmica multidisciplinaria de comalcalco ( damc)-universidad jurez autnoma de tabasco ( ujat ) ( ujat - damc-2012 - 02 ) .", "the study was performed in compliance with the ethical standards of the 1964 declaration of helsinki ."], ["genomic dna was isolated from whole blood samples using standard procedures that have been previously reported.9,22 genotyping of the htr2c variants rs547536 , rs2192372 , rs4272555 , rs6318 , and rs2428707 was performed using the taqman ( applied biosystems inc , foster city , ca , usa ) snp genotyping assay obtained from applied biosystems .", "we selected five single - nucleotide polymorphisms ( snps ) of the htr2c gene found in the promotor and introns of the gene ( table 1 ) ."], ["first , the hardy  weinberg equilibrium for the htr2c variants was determined using pearson s goodness of fit  test . for female controls ,", "the genotype frequencies of rs6318 , rs2428707 , and rs4272555 did not deviate from hardy  weinberg equilibrium ( p>0.05 ) ; however , the genotype frequencies of rs547536 and rs2192372 did deviate from hardy  weinberg equilibrium ( p<0.05 ) .", "random blind duplicates of up 30% of the samples were used as a quality control measure , and we obtained consistent results .", "then ,  and fisher s exact tests were used to compare the genotype and allele frequencies between cases and controls .", "finally , the haploview 4.2 ( broad institute , cambridge , ma , usa)23 was employed to calculate the linkage disequilibrium ( ld ) of the markers ."], ["the demographic characteristics of the cases ( mean age : 25.96 years , standard deviation [ sd ] : 9.26 range : 1456 years ) and comparison group ( mean age : 31.95 years , sd : 13.03 range : 1461 years ) in this mexican population are presented in table 2 .", "table 3 shows the genotype and allele frequencies of the htr2c variants rs547536 , rs21923372 , rs6318 , rs2428707 , and rs4272555 in the suicide attempters and control groups .", "the htr2c variants rs4272555 and rs2428707 were significantly associated with suicide attempt in this mexican population . in the female group ,", "the g allele of the snp rs2428707 was associated with an increased risk of suicide attempt ( p=0.01 , odd ratio [ or ] = 3.68 , 95% confidence inrterval [ ci ] : 1.2410.90 ) , while the c allele of the snp rs4272555 was associated with a decreased risk of suicide attempt ( p=0.01 , or = 0.26 , 95% ci : 0.090.79 ) .", "the distributions of the htr2c variants rs547536 , rs21923372 , and rs6318 were not significantly different between the subjects with suicidal behavior and the control group ( table 3 ) .", "subsequently , for the snps that showed a significant association , we summarized the evidence and performed a meta - analysis .", "the results of the meta - analysis ( supplementary materials ) showed a nonsignificant association for the rs2428707 ( fixed effects : or : 1.21 , 95% ci : 0.801.82 , p(q)=0.03 ) and for the rs4272555 ( fixed effects : or : 0.56 , 95% ci : 0.161.94 , p(q)=0.03 ) ( figures s1s4 and tables s1 and s2 ) .", "the ld ( linkage disequilibrium ) in this mexican population was measured for all markers examined in this case  control study ( rs547536 , rs2192372 , rs6318 , rs4272555 , and rs2428707 ) .", "we conducted a haplotype analysis of the same five markers and did not find a significant association between suicide attempt and any of the examined haplotypes ( p>0.01 , table 4 ) ."], ["the demographic characteristics of the cases ( mean age : 25.96 years , standard deviation [ sd ] : 9.26 range : 1456 years ) and comparison group ( mean age : 31.95 years , sd : 13.03 range : 1461 years ) in this mexican population are presented in table 2 .", "table 3 shows the genotype and allele frequencies of the htr2c variants rs547536 , rs21923372 , rs6318 , rs2428707 , and rs4272555 in the suicide attempters and control groups .", "the htr2c variants rs4272555 and rs2428707 were significantly associated with suicide attempt in this mexican population . in the female group ,", "the g allele of the snp rs2428707 was associated with an increased risk of suicide attempt ( p=0.01 , odd ratio [ or ] = 3.68 , 95% confidence inrterval [ ci ] : 1.2410.90 ) , while the c allele of the snp rs4272555 was associated with a decreased risk of suicide attempt ( p=0.01 , or = 0.26 , 95% ci : 0.090.79 ) .", "the distributions of the htr2c variants rs547536 , rs21923372 , and rs6318 were not significantly different between the subjects with suicidal behavior and the control group ( table 3 ) .", "subsequently , for the snps that showed a significant association , we summarized the evidence and performed a meta - analysis .", "the results of the meta - analysis ( supplementary materials ) showed a nonsignificant association for the rs2428707 ( fixed effects : or : 1.21 , 95% ci : 0.801.82 , p(q)=0.03 ) and for the rs4272555 ( fixed effects : or : 0.56 , 95% ci : 0.161.94 , p(q)=0.03 ) ( figures s1s4 and tables s1 and s2 ) ."], ["the ld ( linkage disequilibrium ) in this mexican population was measured for all markers examined in this case  control study ( rs547536 , rs2192372 , rs6318 , rs4272555 , and rs2428707 ) .", "we conducted a haplotype analysis of the same five markers and did not find a significant association between suicide attempt and any of the examined haplotypes ( p>0.01 , table 4 ) ."], ["the aim of the present study was to assess the association between the htr2c variants rs547536 , rs2192372 , rs6318 , rs2428707 , and rs4272555 and suicide attempt in a mexican population .", "to our knowledge , this is the first association analysis between htr2c polymorphisms and suicidal behavior performed in a latin american population .", "we found that the g allele of rs2428707 was associated with an increased risk of suicidal behavior in females ; however , this is not similar to the results of previous studies.14,20 this discrepancy could be due to differences of the sample sizes used .", "the number of control subjects used in those previous studies and our study were similar ; however , the number of cases in our study were fewer than what the german and italian populations used in previous studies.14,20 another possible explanation for this discrepancy is the heterogeneity between the studies . in our study", ", the absence of the a allele of rs2428707 and the t allele of rs4272555 in the female group , could be ethnic - dependent because the allelic and genotypic distributions we observed were different from those reported in the literature.14,20 third , the  positive results  of this study are modest because we did not exclude the presence of false positives , even when the results were corrected by bonferroni s . for major evidence , we performed a meta - analysis that included studies that have been reported up to date and observed a no - association .", "however , we considered that the low number of studies , the low number of samples , and the presence of heterogeneity in the analysis reduced the statistical power to detect a possible association between the htr2c polymorphisms and suicidal behavior . in consequence , more studies that analyze this association are needed .", "our analysis also showed an opposite effect for the rs4272555 and suicide ; we observed that in females , the c allele of the rs4272555 is a protective allele against suicidal behavior .", "it is fundamental to consider these findings with caution for the following reasons : first , the ld analysis indicated stronger linkage disequilibrium for rs2428707 and rs4272555 than for the other htr2c variants .", "second , the small sample size of our study may have resulted in a weaker statistical association than other studies with a larger study population .", "therefore , it is necessary to analyze other populations and increase the sample size , without the presence of the heterogeneity , in order to have conclusive results .", "the htr2c cys23ser ( rs6318 ) polymorphism is perhaps the most extensively studied for a possible association with suicidal behavior11,13,14,1720 and self - injury.24 the present study did not find an association between the htr2c cys23ser ( rs6318 ) and suicidal behavior .", "however , this finding is in accordance with previous studies14,20 performed in caucasian and chinese populations.18,19 in contrast , in a recent study by karanovic et al,11 an association between the c allele of htr2c rs6318 and suicide attempt was observed .", "karanovi et al11 included 165 suicide attempters and 188 controls , which is similar to what we used in the present study ; however , their controls were subjects with psychiatric diseases other than suicidal behavior and our controls were subjects without any psychiatric disorders .", "hence , these findings need to be replicated before any strong conclusions can be drawn .", "second , although we perform a bonferroni correction and performed a meta - analysis , the small sample size and the small number of the studies ( respectively ) do not ensure the absence of false positives . finally , we did not analyze the clinical features of suicide attempters that may predispose them to this behavior ."], ["our results suggest an association between the rs2428707 and rs4272555 polymorphisms of htr2c in mexican females with suicidal ideation .", "future research must examine the potential mediating effects of the htr2c gene variants to highlight the importance of the serotonin system in suicidal behavior , with special focus on the receptor gene htr2c ."], ["or and forest plot for the meta - analysis of rs2428707 . note : g allele vs a allele with heterogeneity .", "note : g allele vs a allele with heterogeneity . or and forest plot for the meta - analysis of rs4272555 .", "funnel plot indicating publication bias for studies included in the meta - analysis of rs4272555 .", "descriptive characteristics of two studies analyzing the role of the htr2c gene variant rs2428707 in suicide attempt descriptive characteristics of two studies in the meta - analysis of the htr2c gene variant rs4272555 in suicide attempt"]]}
{"article_id": "PMC3017954", "article_text": ["in the right amounts , boron is an essential nutrient for animals , plants , and fungi [ 13 ] .", "however , at high concentrations boric acid ( ba ) becomes an effective poison that is widely used for the killing of diverse organisms ranging from bacteria to rodents . in medicine", "while the molecular details of ba action on yeast remain unclear , it was recently shown that ba interferes with morphogenesis , to the effect that it inhibits the transition from the yeast to the hyphal form of the pathogenic yeast c. albicans .", "because the ability to switch to hyphal growth is an important virulence factor in c. albicans , suppression of such elongated growth by ba may in part explain its therapeutic effect .", "the present study was undertaken to assess the effect of ba on morphogenesis and cell wall synthesis in yeast , using the well - established model organism saccharomyces cerevisiae as a study subject .  ", "in s. cerevisiae , morphogenesis and cell wall synthesis depend on the correct assembly of cytoskeletal proteins . to guide cell wall synthesis during cytokinesis , a ring of septin filaments forms during the g1 phase of the cell cycle and", "is subsequently completed into a contractile actomyosin ring ( car ) by the addition of myosin and actin , among other proteins [ 8 , 9 ] .", "to complete abscission , the last phase of cytokinesis , the cells first separate mother and daughter cells with a chitin primary septum .", "the deposition of glucan and mannoprotein - rich cell wall material on the mother and daughter side of the primary septum later completes the trilaminar septum that can be observed under normal culture conditions . a disturbance in the assembly of the septation apparatus  the cohesive functional unit that constructs the primary septum  leads to the formation of highly aberrant septa [ 10 , 11 ] .", "the septa formed under these conditions do not allow for the separation of cells after cytokinesis , leading to the formation of chains and clumps of misshaped cells .", "based on the observation that ba causes such clumping and chain formation in s. cerevisiae , the present study was initiated to assess the influence of ba on the function of the septation apparatus .", "strain yms415 ( chs3::ha - his3 ) was constructed by the short flanking homology method .", "the chs3::3ha - his3 fragment was amplified by pcr from plasmid pfa6a-3ha - his3mx6 with primers 5-tattctcaatcggaaggaggaaagtgactccttcgttgcaggtggaggtggaggtggaggtggacggatccccgggttaattaa-3 and 5-tcaacttgtaagtatcacagtaaaaatattttcatactgtgaattcgagctcgtttaaac-3. integration of the fragment was verified by pcr and western blotting .", "transformants showed a wild - type like distribution of chitin in calcofluor white stained preparations , demonstrating full functionality of the chs3p - ha fusion protein .", "a visual representation of ba sensitivity was obtained by serially diluting an overnight culture of yeast grown in ypd and spotting 5  l of cell dilutions on ypd plates with the indicated concentrations of ba .", "viability staining of yeast cultures was performed by incubating cells in 0.2  mg / ml methylene blue in 50  mm kh2po4 for 5 minutes at rt . in order to visualize the distribution of chitin and glucan in the cell wall ,", "cells were washed and incubated for 5  minutes in 0.01% calcofluor white or 0.5% aniline blue , respectively .", "fluorescence was observed with a standard diamidino-2-phenylindol ( dapi ) filter set ( zeiss ) .", "fifty ml yeast cultures at a titer of 1510  cells / ml in ypd were fixed by the addition of formaldehyde to a final concentration of 4% and incubated for 10  minutes at room temperature .", "cells were pelleted and incubated for 1  hour in phosphate buffered saline ( pbs ; 137  mm nacl , 2.7  mm kcl , 10  mm na2hpo4 , 1.76  mm kh2po4 , ph 7.4 ) with 4% formaldehyde . after 2 washes in pbs ,", "cells were suspended in 100  l pbs with 10  l of an alexa 568-phalloidin solution ( 6.6  m in methanol ; invitrogen ) and 1  l of 1  mg / ml calcofluor white .", "cells were incubated for 1  hour in the dark and washed twice with pbs .", "cells were then pelleted and taken up in 50  l prolong antifade reagent ( invitrogen ) before mounting on slides .", "gfp - tagged cdc3p and myo1p were observed with the gfp - filter set ( zeiss ) in cells transformed with prs316cdc3gfp   and pms55 .", "analysis of slt2p phosphorylation and total slt2p by western blotting followed established protocols [ 19 , 20 ] . as a loading control , an antibody directed against tub4p ( goat -tubulin yk18 , santa cruz biotechnology ) was used at 1/1,000 dilution .", "for the determination of chs3p - ha , cell membranes were isolated according to orlean .", "one volume of mercaptoethanol - free 2x sample buffer with 2% sds ( bio - rad ) was added to the protein extracts and samples were loaded on 6% sds polyacrylamide gels without boiling .", "antibodies used were a 1/5,000 dilution of anti - ha/1/5,000 dilution of goat antimouse hrp ( santa cruz biotechnology ) .", "membranes were stained in 0.02% coomassie blue r250 in 40% methanol , 5% acetic acid to serve as loading controls . for the determination of enzyme activities ,", "a 40  l glucan synthase assay contained 20  l of membrane suspension at a protein concentration of 1  mg ml , 5 mm c - udp - glucose ( activity 1  10  cpm  mmol ) , 75  mm tris - cl ph 7.5 , 25  mm kf , and  when indicated  with 20  m gtp--s for the determination of maximal gs activity .", "the reaction was incubated for 1  hour at 30c and then stopped by adding 1  ml 10% trichloroacetic acid ( tca ) .", "reaction mixtures were filtered through a type a / e glass fiber filter ( pall ) .", "filters were washed twice with 1  ml 10% tca and four times with 70% ethanol .", "filters were dried and taken up in 10  ml cytoscint es scintillation fluid ( icn ) , and activity was recorded in a scintillation counter .", "chitin synthesis was determined as described by choi and cabib . to measure chitin synthase 2 and 3 activities ,", "experiments were performed in the chs1 deletion strain ecy46 - 1 - 8d . the chs1 deletion was found to have no influence on ba sensitivity ( figure 1(a ) ) .", "the chitin synthase assay mixture contained 20 l of membrane suspension at 1  mg protein ml , 5  l of 0.5 m tris / cl ph 7.8 , 5  l of 20  mm cobalt acetate , 5  l of 10  mm c - udp - glcnac ( 5000 c.p.m . ", "l ) , and 2  l of trypsin solutions ( sigma ) at concentrations from 0.25 to 2.0  mg ml in a total volume of 46  l . for determination of chitin synthase 3 activity", ", 5  l of water were substituted with 5  l of 50  mm nickel acetate .", "proteolysis was stopped after incubating for 15  minutes at 30c by adding 2  l of a soybean trypsin inhibitor solution at 1.5x the concentration of the trypsin solution .", "chitin synthesis was initiated by adding 2  l of 0.8  m glcnac . after incubating for 60  minutes at 30c , chitin synthesis", "reaction mixtures were filtered , washed , and assayed in cytoscint fluid as described above .", "assays containing cobalt only show activities of chitin synthases 2 and 3 while cobalt / nickel assays show the activity of chitin synthase 3 .", "vital staining of strains yph499 and ecy46 - 1 - 8d with 0.05% methylene blue showed that ba concentrations between 0.1 and 0.4% do not severely reduce cell viability ( figure 1(a ) ) . in the examined range around the minimal inhibitory concentration of 0.31% ,", "ba thus functions as a fungistatic agent that slows down proliferation but does not kill cells .", "note that the decline in viability in chs1 deletion strain ecy46 - 1 - 8d parallels the decline in wildtype viability .", "due to the previously reported lysis of daughter cells in chs1 mutants , strain ecy46 - 1 - 8d shows a higher fraction of dead cells in all of the examined samples .    ", "clumping and chain formation of cells occurs at ba concentrations above 0.2% , with the most striking effect observed at 0.4% ( figure 1(b ) ) .", "this clumping is the hallmark of a cytokinesis defect that causes daughter cells to remain attached to mother cells .", "a spot test of by4742 cells on ypd plates showed that no growth occurs at ba concentrations above 0.5%although viable cells can be retrieved even after 10 days of incubation under these conditions ( data not shown ) .", "staining of chitin and glucan in walls of cells grown with 0.4% ba revealed a buildup of cell wall material at bud necks , particularly in cell chains ( figure 2 ) . in order to characterize the cytokinesis defect in ba - treated cells ,", "the localization of key morphogenetic proteins at the bud neck was examined by fluorescence microscopy .", "it was found that ba influences the localization of the septin cdc3 , the cytokinetic myosin myo1p , and the ring of filamentous actin that form sequentially in preparation for cytokinesis .", "increasing concentrations of ba leads to the formation of cdc3gfp rings that are disorganized , uneven , and not centered at the bud neck .", "moreover , ba causes the formation of cdc3gfp patches at sites other than the buck neck ( figure 3 ) .  ", "imaging of myo1gfp and actin shows that the formation of the car is impaired in a manner similar to the disturbance in septin ring organization .", "increasing ba concentrations lowers the fraction of cells with myo1gfp rings at the bud neck from 49  5% at 0% to 25  11% at 0.2% to 11  11% at 0.4% .", "in addition , myo1gfp rings formed under the influence of ba are often irregular in shape and at high concentrations ( 0.4% ) ectopic localization of myo1gfp patches at sites other than the bud neck is common ( figure 4(a ) ) .", "ba also impairs the assembly of the actin ring at the bud neck , which is the last component to be incorporated into the car before contraction starts ( figure 4(b ) ) .", "actin rings formed under the influence of ba become blurry , faint , and irregular in thickness . under these conditions", ", it can be observed that some actin rings fail to localize to the narrowest point of the bud neck . in some cases , ba - treated cells", "due to the low abundance of cells with actin rings in culture ( <  2% in controls ) , quantification of rings yielded no statistically significant data .", "cultures of cells grown with 0.3% and 0.4% ba were examined by electron microscopy using a protocol that allowed for the visualization of chitin ( figure 5 ) .", "the analysis of ba - treated cells showed massive abnormalities in the cell wall , particularly in the area of the septum , worse at 0.4% than at 0.3% . at these concentrations ,", "ba prevented the construction of a single , straight chitin septum to separate mother and daughter cells .", "instead , the cells synthesized protuberances extending far into the cytosol and large irregular septa , often at ectopic locations at the cell periphery . in order to assess ba - induced cell wall integrity signaling , the amounts and the phosphorylation status of the signaling kinase slt2p were determined by western blotting ( figure 6(a ) ) .", "phosphorylation of slt2p increases with escalating concentrations of ba , suggesting that ba induces a cell wall integrity stress response .", "the dramatic increase in phosphorylation of slt2p is accompanied by a weak increase in protein expression .", "furthermore , it was determined that a slt2 deletion mutant is sensitive to ba ( figure 6(b ) ) , suggesting that slt2p - signaling improves ba resistance by causing a transcriptional response to ba stress .", "western blotting showed a nonlinear correlation between ba concentration and abundance of chs3p - ha ( figure 7(a ) ) .", "while at ba concentrations of 0.1 and 0.2% the amount of chs3p - ha decreases , there is an increase of chs3p - ha at 0.4% ba .", "the increase of chs3p - ha parallels the increase of chitin - rich septa visible in calcofluor white - stained cultures .  ", "the multiple posttranslational and protein targeting processes involved in chs3p and chs2p activation   necessitate enzymatic determination of chitin synthase activities in addition to detection of protein amounts .  ", "figure 7(b ) shows that ba - induced changes in chitin synthase activities mirror the changes in chs3p - ha amount ( figure 7(a ) ) and bud neck thickness ( figure 2 ) .", "while at 0.1% and 0.2% ba decreases chitin synthase activities , presence of 0.3% and 0.4% ba increases chitin synthesis . at 0.4% ba ,", "a measurement of glucan synthase activity in the membranes of strain yph499 showed a constant decline with increasing ba concentrations ( figure 7(b ) ) .", "neither maximal nor physiological ( without addition of gtp-s ) glucan synthase activity showed an increase at high concentrations of ba .", "living organisms are constantly exposed to boron , a mineral that is abundant in soil and water .", "boron is a weak acid and , at physiological ph , is present mostly as boric acid ( h3bo3 ; ba ) . for each organism", ", there is an optimal ba concentration . too little ba causes symptoms of deficiency while too much ba has a poorly defined cytotoxic effect .", "particularly the toxic effects of boron overload have led to a variety of applications for ba and related compounds , ranging from pest control to the treatment of vaginal yeast infections .", ". the biological function of ba might be due to its reactivity with cis - hydroxyl groups on carbohydrate molecules .", "examples for boron - dependent reactions include plant hormone - sensitive nadh oxidase activity , the crosslinking of plant cell wall carbohydrates , and the mineralization of bone .  ", "the molecular effects of boron lead to developmental defects in animals , particularly in the formation of the skeleton [ 30 , 31 ] .", "these teratogenic effects of boron might be caused by direct or indirect inhibition of histone deacetylase activity   and a shift in hox gene expression .  ", "however , none of the above observations about ba toxicity serve to explain its effect on yeast  an organism that does not undergo genomic imprinting or hox - dependent development .", "the only published data about the ba effect on yeast come from a recent study of the yeast c. albicans that suggested that ba impairs oxidative metabolism . while this observation in itself is interesting and deserves further study", ", the authors also show that ba directly or indirectly influences the morphology of c. albicans .", "the present study expands on this observation by showing that ba disturbs morphogenesis in the model yeast saccharomyces cerevisiae , particularly during cytokinesis .     ", "the data presented here show that ba impairs the formation of the primary septum in s. cerevisiae  a defect that can be explained by incorrect assembly of the cytoskeleton at the bud neck .", "the molecular machinery that constructs the chitin cell wall between mother and daughter cells , the septation apparatus , consists of chitin synthase 2 , and a contractile actomyosin ring ( car ) .", "the septation apparatus is assembled sequentially from myosin , chitin synthase 2 and actin on a scaffold of septins at the bud neck [ 8 , 9 ] .", "should the assembly of a functional septation apparatus fail , the cell is unable to construct an orderly primary septum by chitin synthase 2 and is forced to divide by depositing large amounts of chitin - rich cell wall material at the bud neck .", "the chitin in these so - called default septa is provided by chitin synthase 3 [ 10 , 33 , 34 ] .", "since these irregular septum structures are resistant to degradation by chitinase , cells remain connected after cytokinesis and form cell chains and clumps . in s. cerevisiae", ". a septin assembly that erroneously localizes to a site other than the bud neck will direct the formation of a septum - like structure at the respective location .    ", "our data show that in s. cerevisiae increasing concentrations of ba leads to an irregular assembly of the septin scaffold , an inability to position the myo1 ring , and a failure to correctly assemble an actin ring at the bud neck .", "the aberrant localization and irregular appearance of the septin cdc3gfp in ba - treated cells is an important key to understanding the septation defect .", "we propose that ba causes problems with the assembly of the septin scaffold which later impair the localization and function of the car , resulting in the formation of highly irregular cell wall structures .  ", "like other threats to the integrity of the cell wall , septation defects trigger cell wall integrity signaling through the protein kinase c ( pkc ) pathway . under these conditions , cell wall integrity signaling leads to hyperphosphorylation of the pkc downstream effector slt2p [ 3638 ] , accompanied with a much weaker increase in slt2p amount .", "this will ultimately lead to the activation of cell wall repair enzymes , most notably chitin synthase 3 [ 36 , 40 ] .", "the present study shows that ba activates yeast cell wall integrity signaling pathways as evidenced by ba concentration - dependent phosphorylation of slt2p .", "presumably in response to cell wall integrity signaling , cells increase the activity of the cell wall repair enzyme chitin synthase 3 in a ba concentration - dependent manner .", "a decline at low concentrations followed by an increase at higher doses  should be interpreted based on the impact boric acid on growth .", "we propose that at concentrations below the mic where the impact on growth is measurable but weak , boric acid stress reduces cell wall synthesis activity along with other metabolic activities . once", "boric acid stress exceeds a tolerable limit  at concentrations above the mic  the cell responds forcefully by induction of stress survival mechanisms such as chitin synthase 3-mediated cell wall reinforcement .     it is worth noting that the activity of the chitin septum - forming chitin synthase 2 also increases during ba exposure .", "it is evident that the increase in chitin synthase 2 activity correlates well with the increased number and size of chitin septa in ba - treated cells .", "however , since the regulation of chitin synthase 2 activity is not well understood , we dare not hazard an explanation for this phenomenon .     the data presented in this study show that in s. cerevisiae ba disturbs the localization of the contractile actomyosin ring secondary to causing irregularities in the septin scaffold at the bud neck .", "in agreement with the reviewed literature we propose that the aberrant localization of the septins ultimately impedes the formation of the primary septum , which leads to the synthesis of thick , chitin - rich default septa .", "in addition , the localization of septins at sites other than the bud neck explains the synthesis of cell wall protuberances that should be interpreted as incomplete ectopic septa .", "furthermore , our data show that a ba - induced septation defect , just like other septation defects , triggers cell wall integrity signaling through the pkc1-slt2 pathway and results in increased chitin synthase 3 activity ."], "abstract_text": ["<S> boric acid ( ba ) has broad antimicrobial activity that makes it a popular treatment for yeast vaginitis in complementary and alternative medicine . in the model yeast s. cerevisiae </S>", "<S> , ba disturbs the cytoskeleton at the bud neck and impairs the assembly of the septation apparatus . </S>", "<S> ba treatment causes cells to form irregular septa and leads to the synthesis of irregular cell wall protuberances that extend far into the cytoplasm . </S>", "<S> the thick , chitin - rich septa that are formed during ba exposure prevent separation of cells after abscission and cause the formation of cell chains and clumps . as a response to the ba insult , </S>", "<S> cells signal cell wall stress through the slt2p pathway and increase chitin synthesis , presumably to repair cell wall damage . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["in the right amounts , boron is an essential nutrient for animals , plants , and fungi [ 13 ] .", "however , at high concentrations boric acid ( ba ) becomes an effective poison that is widely used for the killing of diverse organisms ranging from bacteria to rodents . in medicine", "while the molecular details of ba action on yeast remain unclear , it was recently shown that ba interferes with morphogenesis , to the effect that it inhibits the transition from the yeast to the hyphal form of the pathogenic yeast c. albicans .", "because the ability to switch to hyphal growth is an important virulence factor in c. albicans , suppression of such elongated growth by ba may in part explain its therapeutic effect .", "the present study was undertaken to assess the effect of ba on morphogenesis and cell wall synthesis in yeast , using the well - established model organism saccharomyces cerevisiae as a study subject .  ", "in s. cerevisiae , morphogenesis and cell wall synthesis depend on the correct assembly of cytoskeletal proteins . to guide cell wall synthesis during cytokinesis , a ring of septin filaments forms during the g1 phase of the cell cycle and", "is subsequently completed into a contractile actomyosin ring ( car ) by the addition of myosin and actin , among other proteins [ 8 , 9 ] .", "to complete abscission , the last phase of cytokinesis , the cells first separate mother and daughter cells with a chitin primary septum .", "the deposition of glucan and mannoprotein - rich cell wall material on the mother and daughter side of the primary septum later completes the trilaminar septum that can be observed under normal culture conditions . a disturbance in the assembly of the septation apparatus  the cohesive functional unit that constructs the primary septum  leads to the formation of highly aberrant septa [ 10 , 11 ] .", "the septa formed under these conditions do not allow for the separation of cells after cytokinesis , leading to the formation of chains and clumps of misshaped cells .", "based on the observation that ba causes such clumping and chain formation in s. cerevisiae , the present study was initiated to assess the influence of ba on the function of the septation apparatus ."], ["strain yms415 ( chs3::ha - his3 ) was constructed by the short flanking homology method .", "the chs3::3ha - his3 fragment was amplified by pcr from plasmid pfa6a-3ha - his3mx6 with primers 5-tattctcaatcggaaggaggaaagtgactccttcgttgcaggtggaggtggaggtggaggtggacggatccccgggttaattaa-3 and 5-tcaacttgtaagtatcacagtaaaaatattttcatactgtgaattcgagctcgtttaaac-3. integration of the fragment was verified by pcr and western blotting .", "transformants showed a wild - type like distribution of chitin in calcofluor white stained preparations , demonstrating full functionality of the chs3p - ha fusion protein .", "a visual representation of ba sensitivity was obtained by serially diluting an overnight culture of yeast grown in ypd and spotting 5  l of cell dilutions on ypd plates with the indicated concentrations of ba .", "viability staining of yeast cultures was performed by incubating cells in 0.2  mg / ml methylene blue in 50  mm kh2po4 for 5 minutes at rt . in order to visualize the distribution of chitin and glucan in the cell wall ,", "cells were washed and incubated for 5  minutes in 0.01% calcofluor white or 0.5% aniline blue , respectively .", "fluorescence was observed with a standard diamidino-2-phenylindol ( dapi ) filter set ( zeiss ) .", "fifty ml yeast cultures at a titer of 1510  cells / ml in ypd were fixed by the addition of formaldehyde to a final concentration of 4% and incubated for 10  minutes at room temperature .", "cells were pelleted and incubated for 1  hour in phosphate buffered saline ( pbs ; 137  mm nacl , 2.7  mm kcl , 10  mm na2hpo4 , 1.76  mm kh2po4 , ph 7.4 ) with 4% formaldehyde . after 2 washes in pbs ,", "cells were suspended in 100  l pbs with 10  l of an alexa 568-phalloidin solution ( 6.6  m in methanol ; invitrogen ) and 1  l of 1  mg / ml calcofluor white .", "cells were incubated for 1  hour in the dark and washed twice with pbs .", "cells were then pelleted and taken up in 50  l prolong antifade reagent ( invitrogen ) before mounting on slides .", "gfp - tagged cdc3p and myo1p were observed with the gfp - filter set ( zeiss ) in cells transformed with prs316cdc3gfp   and pms55 .", "analysis of slt2p phosphorylation and total slt2p by western blotting followed established protocols [ 19 , 20 ] . as a loading control , an antibody directed against tub4p ( goat -tubulin yk18 , santa cruz biotechnology ) was used at 1/1,000 dilution .", "for the determination of chs3p - ha , cell membranes were isolated according to orlean .", "one volume of mercaptoethanol - free 2x sample buffer with 2% sds ( bio - rad ) was added to the protein extracts and samples were loaded on 6% sds polyacrylamide gels without boiling .", "antibodies used were a 1/5,000 dilution of anti - ha/1/5,000 dilution of goat antimouse hrp ( santa cruz biotechnology ) .", "membranes were stained in 0.02% coomassie blue r250 in 40% methanol , 5% acetic acid to serve as loading controls . for the determination of enzyme activities ,", "a 40  l glucan synthase assay contained 20  l of membrane suspension at a protein concentration of 1  mg ml , 5 mm c - udp - glucose ( activity 1  10  cpm  mmol ) , 75  mm tris - cl ph 7.5 , 25  mm kf , and  when indicated  with 20  m gtp--s for the determination of maximal gs activity .", "the reaction was incubated for 1  hour at 30c and then stopped by adding 1  ml 10% trichloroacetic acid ( tca ) .", "reaction mixtures were filtered through a type a / e glass fiber filter ( pall ) .", "filters were washed twice with 1  ml 10% tca and four times with 70% ethanol .", "filters were dried and taken up in 10  ml cytoscint es scintillation fluid ( icn ) , and activity was recorded in a scintillation counter .", "chitin synthesis was determined as described by choi and cabib . to measure chitin synthase 2 and 3 activities ,", "experiments were performed in the chs1 deletion strain ecy46 - 1 - 8d . the chs1 deletion was found to have no influence on ba sensitivity ( figure 1(a ) ) .", "the chitin synthase assay mixture contained 20 l of membrane suspension at 1  mg protein ml , 5  l of 0.5 m tris / cl ph 7.8 , 5  l of 20  mm cobalt acetate , 5  l of 10  mm c - udp - glcnac ( 5000 c.p.m . ", "l ) , and 2  l of trypsin solutions ( sigma ) at concentrations from 0.25 to 2.0  mg ml in a total volume of 46  l . for determination of chitin synthase 3 activity", ", 5  l of water were substituted with 5  l of 50  mm nickel acetate .", "proteolysis was stopped after incubating for 15  minutes at 30c by adding 2  l of a soybean trypsin inhibitor solution at 1.5x the concentration of the trypsin solution .", "chitin synthesis was initiated by adding 2  l of 0.8  m glcnac . after incubating for 60  minutes at 30c , chitin synthesis", "reaction mixtures were filtered , washed , and assayed in cytoscint fluid as described above .", "assays containing cobalt only show activities of chitin synthases 2 and 3 while cobalt / nickel assays show the activity of chitin synthase 3 ."], ["vital staining of strains yph499 and ecy46 - 1 - 8d with 0.05% methylene blue showed that ba concentrations between 0.1 and 0.4% do not severely reduce cell viability ( figure 1(a ) ) . in the examined range around the minimal inhibitory concentration of 0.31% ,", "ba thus functions as a fungistatic agent that slows down proliferation but does not kill cells .", "note that the decline in viability in chs1 deletion strain ecy46 - 1 - 8d parallels the decline in wildtype viability .", "due to the previously reported lysis of daughter cells in chs1 mutants , strain ecy46 - 1 - 8d shows a higher fraction of dead cells in all of the examined samples .    ", "clumping and chain formation of cells occurs at ba concentrations above 0.2% , with the most striking effect observed at 0.4% ( figure 1(b ) ) .", "this clumping is the hallmark of a cytokinesis defect that causes daughter cells to remain attached to mother cells .", "a spot test of by4742 cells on ypd plates showed that no growth occurs at ba concentrations above 0.5%although viable cells can be retrieved even after 10 days of incubation under these conditions ( data not shown ) .", "staining of chitin and glucan in walls of cells grown with 0.4% ba revealed a buildup of cell wall material at bud necks , particularly in cell chains ( figure 2 ) . in order to characterize the cytokinesis defect in ba - treated cells ,", "the localization of key morphogenetic proteins at the bud neck was examined by fluorescence microscopy .", "it was found that ba influences the localization of the septin cdc3 , the cytokinetic myosin myo1p , and the ring of filamentous actin that form sequentially in preparation for cytokinesis .", "increasing concentrations of ba leads to the formation of cdc3gfp rings that are disorganized , uneven , and not centered at the bud neck .", "moreover , ba causes the formation of cdc3gfp patches at sites other than the buck neck ( figure 3 ) .  ", "imaging of myo1gfp and actin shows that the formation of the car is impaired in a manner similar to the disturbance in septin ring organization .", "increasing ba concentrations lowers the fraction of cells with myo1gfp rings at the bud neck from 49  5% at 0% to 25  11% at 0.2% to 11  11% at 0.4% .", "in addition , myo1gfp rings formed under the influence of ba are often irregular in shape and at high concentrations ( 0.4% ) ectopic localization of myo1gfp patches at sites other than the bud neck is common ( figure 4(a ) ) .", "ba also impairs the assembly of the actin ring at the bud neck , which is the last component to be incorporated into the car before contraction starts ( figure 4(b ) ) .", "actin rings formed under the influence of ba become blurry , faint , and irregular in thickness . under these conditions", ", it can be observed that some actin rings fail to localize to the narrowest point of the bud neck . in some cases , ba - treated cells", "due to the low abundance of cells with actin rings in culture ( <  2% in controls ) , quantification of rings yielded no statistically significant data .", "cultures of cells grown with 0.3% and 0.4% ba were examined by electron microscopy using a protocol that allowed for the visualization of chitin ( figure 5 ) .", "the analysis of ba - treated cells showed massive abnormalities in the cell wall , particularly in the area of the septum , worse at 0.4% than at 0.3% . at these concentrations ,", "ba prevented the construction of a single , straight chitin septum to separate mother and daughter cells .", "instead , the cells synthesized protuberances extending far into the cytosol and large irregular septa , often at ectopic locations at the cell periphery . in order to assess ba - induced cell wall integrity signaling , the amounts and the phosphorylation status of the signaling kinase slt2p were determined by western blotting ( figure 6(a ) ) .", "phosphorylation of slt2p increases with escalating concentrations of ba , suggesting that ba induces a cell wall integrity stress response .", "the dramatic increase in phosphorylation of slt2p is accompanied by a weak increase in protein expression .", "furthermore , it was determined that a slt2 deletion mutant is sensitive to ba ( figure 6(b ) ) , suggesting that slt2p - signaling improves ba resistance by causing a transcriptional response to ba stress .", "western blotting showed a nonlinear correlation between ba concentration and abundance of chs3p - ha ( figure 7(a ) ) .", "while at ba concentrations of 0.1 and 0.2% the amount of chs3p - ha decreases , there is an increase of chs3p - ha at 0.4% ba .", "the increase of chs3p - ha parallels the increase of chitin - rich septa visible in calcofluor white - stained cultures .  ", "the multiple posttranslational and protein targeting processes involved in chs3p and chs2p activation   necessitate enzymatic determination of chitin synthase activities in addition to detection of protein amounts .  ", "figure 7(b ) shows that ba - induced changes in chitin synthase activities mirror the changes in chs3p - ha amount ( figure 7(a ) ) and bud neck thickness ( figure 2 ) .", "while at 0.1% and 0.2% ba decreases chitin synthase activities , presence of 0.3% and 0.4% ba increases chitin synthesis . at 0.4% ba ,", "a measurement of glucan synthase activity in the membranes of strain yph499 showed a constant decline with increasing ba concentrations ( figure 7(b ) ) .", "neither maximal nor physiological ( without addition of gtp-s ) glucan synthase activity showed an increase at high concentrations of ba ."], ["living organisms are constantly exposed to boron , a mineral that is abundant in soil and water .", "boron is a weak acid and , at physiological ph , is present mostly as boric acid ( h3bo3 ; ba ) . for each organism", ", there is an optimal ba concentration . too little ba causes symptoms of deficiency while too much ba has a poorly defined cytotoxic effect .", "particularly the toxic effects of boron overload have led to a variety of applications for ba and related compounds , ranging from pest control to the treatment of vaginal yeast infections .", ". the biological function of ba might be due to its reactivity with cis - hydroxyl groups on carbohydrate molecules .", "examples for boron - dependent reactions include plant hormone - sensitive nadh oxidase activity , the crosslinking of plant cell wall carbohydrates , and the mineralization of bone .  ", "the molecular effects of boron lead to developmental defects in animals , particularly in the formation of the skeleton [ 30 , 31 ] .", "these teratogenic effects of boron might be caused by direct or indirect inhibition of histone deacetylase activity   and a shift in hox gene expression .  ", "however , none of the above observations about ba toxicity serve to explain its effect on yeast  an organism that does not undergo genomic imprinting or hox - dependent development .", "the only published data about the ba effect on yeast come from a recent study of the yeast c. albicans that suggested that ba impairs oxidative metabolism . while this observation in itself is interesting and deserves further study", ", the authors also show that ba directly or indirectly influences the morphology of c. albicans .", "the present study expands on this observation by showing that ba disturbs morphogenesis in the model yeast saccharomyces cerevisiae , particularly during cytokinesis .     ", "the data presented here show that ba impairs the formation of the primary septum in s. cerevisiae  a defect that can be explained by incorrect assembly of the cytoskeleton at the bud neck .", "the molecular machinery that constructs the chitin cell wall between mother and daughter cells , the septation apparatus , consists of chitin synthase 2 , and a contractile actomyosin ring ( car ) .", "the septation apparatus is assembled sequentially from myosin , chitin synthase 2 and actin on a scaffold of septins at the bud neck [ 8 , 9 ] .", "should the assembly of a functional septation apparatus fail , the cell is unable to construct an orderly primary septum by chitin synthase 2 and is forced to divide by depositing large amounts of chitin - rich cell wall material at the bud neck .", "the chitin in these so - called default septa is provided by chitin synthase 3 [ 10 , 33 , 34 ] .", "since these irregular septum structures are resistant to degradation by chitinase , cells remain connected after cytokinesis and form cell chains and clumps . in s. cerevisiae", ". a septin assembly that erroneously localizes to a site other than the bud neck will direct the formation of a septum - like structure at the respective location .    ", "our data show that in s. cerevisiae increasing concentrations of ba leads to an irregular assembly of the septin scaffold , an inability to position the myo1 ring , and a failure to correctly assemble an actin ring at the bud neck .", "the aberrant localization and irregular appearance of the septin cdc3gfp in ba - treated cells is an important key to understanding the septation defect .", "we propose that ba causes problems with the assembly of the septin scaffold which later impair the localization and function of the car , resulting in the formation of highly irregular cell wall structures .  ", "like other threats to the integrity of the cell wall , septation defects trigger cell wall integrity signaling through the protein kinase c ( pkc ) pathway . under these conditions , cell wall integrity signaling leads to hyperphosphorylation of the pkc downstream effector slt2p [ 3638 ] , accompanied with a much weaker increase in slt2p amount .", "this will ultimately lead to the activation of cell wall repair enzymes , most notably chitin synthase 3 [ 36 , 40 ] .", "the present study shows that ba activates yeast cell wall integrity signaling pathways as evidenced by ba concentration - dependent phosphorylation of slt2p .", "presumably in response to cell wall integrity signaling , cells increase the activity of the cell wall repair enzyme chitin synthase 3 in a ba concentration - dependent manner .", "a decline at low concentrations followed by an increase at higher doses  should be interpreted based on the impact boric acid on growth .", "we propose that at concentrations below the mic where the impact on growth is measurable but weak , boric acid stress reduces cell wall synthesis activity along with other metabolic activities . once", "boric acid stress exceeds a tolerable limit  at concentrations above the mic  the cell responds forcefully by induction of stress survival mechanisms such as chitin synthase 3-mediated cell wall reinforcement .     it is worth noting that the activity of the chitin septum - forming chitin synthase 2 also increases during ba exposure .", "it is evident that the increase in chitin synthase 2 activity correlates well with the increased number and size of chitin septa in ba - treated cells .", "however , since the regulation of chitin synthase 2 activity is not well understood , we dare not hazard an explanation for this phenomenon .     the data presented in this study show that in s. cerevisiae ba disturbs the localization of the contractile actomyosin ring secondary to causing irregularities in the septin scaffold at the bud neck .", "in agreement with the reviewed literature we propose that the aberrant localization of the septins ultimately impedes the formation of the primary septum , which leads to the synthesis of thick , chitin - rich default septa .", "in addition , the localization of septins at sites other than the bud neck explains the synthesis of cell wall protuberances that should be interpreted as incomplete ectopic septa .", "furthermore , our data show that a ba - induced septation defect , just like other septation defects , triggers cell wall integrity signaling through the pkc1-slt2 pathway and results in increased chitin synthase 3 activity ."]]}
{"article_id": "PMC4116581", "article_text": ["the pylorus , duodenal c - loop , and ileocecal valve are the three physiological narrowings in the gastrointestinal tract , and most of the swallowed indigestible foreign bodies pass through it without complications . however , foreign bodies such as a toothbrush can not pass out of the stomach , and the gastrointestinal tract should get rid of these objects as soon as possible to avoid pressure necrosis and gastrointestinal perforation .", "although these objects are extracted either by endoscopy or laparoscopic gastrostomy , we devised an innovative technique by using pneumatic gastric insufflation and extracted the toothbrush by a tiny gastrotomy under local anesthesia .", "a 35-year - old male presented in our hospital at m.m . institute of medical sciences and research ( mmimsr ) , mullana , ambala , haryana , india in may 2013 ; he had accidentally swallowed a toothbrush two months back and there was a history of epigastric discomfort especially after meals . however , the vital signs were within normal limits and the abdomen was soft and nontender .", "x - ray of the abdomen suggested the presence of a foreign body and a computed tomography ( ct ) scan was done which confirmed a toothbrush lying in the stomach [ figures 1 , 2 ] .", "an upper gastrointestinal endoscopy was done which revealed the toothbrush in the stomach with its head toward the gastroduodenal junction .", "biopsy forceps were used to deliver the toothbrush by holding its bristles [ figure 3 ] .", "the endoscope was kept inside to insufflate and distend the stomach and a minilaparotomy with gastrotomy of 1.5 - 2 cm was performed through the midline under local anesthesia and the toothbrush was successfully removed .", "x - ray of the abdomen suggesting the presence of the toothbrush in the abdomen computed tomography ( ct ) scan of the abdomen showing the presence of the toothbrush in the tomach endoscopic picture of toothbrush with biopsy forceps in situ to retrieve it from the stomach", "in the stomach , 80 - 90% of foreign bodies pass uneventfully through the gastrointestinal tract without complications .", "however , objects longer than 10 cm like a toothbrush can not negotiate the duodenal c - loop due to its fixed position in the retroperitoneum , and these must be removed as soon as possible to avoid pressure necrosis and gastric perforation .", "more than 40 cases of toothbrush ingestion have been reported in the literature till date .", "however , extreme caution and experience of the endoscopist is required for such procedures . in failed cases of endoscopic removal ,", "however , we devised a simple technique of minilaparotomy and gastrotomy under local anesthesia for removing such foreign bodies . in this technique ,", "the stomach is distended with the help of air insufflation through the endoscope and a small incision is made in the midepigasrium under local anesthesia and the foreign object can be removed directly under the vision of the endoscope .", "a swallowed toothbrush is a rare occurrence and it never passes through the gastrointestinal tract spontaneously .", "early removal of the toothbrush is critical for reducing morbidity and mortality . in cases where endoscopic removal fails ,", "endoscopy still remains an aid in performing surgical gastrotomy for delivering such complex foreign bodies under local anesthesia ."], "abstract_text": ["<S> most of the ingested foreign bodies pass uneventfully through the gastrointestinal tract . however , long and rigid foreign bodies are associated with an increased risk of gastrointestinal impaction , perforation , and bleeding . spontaneous passage of a toothbrush has not been reported till date and the technique of its removal is a curiosity for surgeons . </S>", "<S> endoscopy is a recommended technique for the removal of such complex foreign bodies . </S>", "<S> however , if it fails , the foreign body can be removed successfully with a laparoscopic gastrotomy . </S>", "<S> we devised an innovative technique by using pneumatic gastric insufflation and extracted the toothbrush by a tiny gastrotomy under local anesthesia . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["the pylorus , duodenal c - loop , and ileocecal valve are the three physiological narrowings in the gastrointestinal tract , and most of the swallowed indigestible foreign bodies pass through it without complications . however , foreign bodies such as a toothbrush can not pass out of the stomach , and the gastrointestinal tract should get rid of these objects as soon as possible to avoid pressure necrosis and gastrointestinal perforation .", "although these objects are extracted either by endoscopy or laparoscopic gastrostomy , we devised an innovative technique by using pneumatic gastric insufflation and extracted the toothbrush by a tiny gastrotomy under local anesthesia ."], ["a 35-year - old male presented in our hospital at m.m . institute of medical sciences and research ( mmimsr ) , mullana , ambala , haryana , india in may 2013 ; he had accidentally swallowed a toothbrush two months back and there was a history of epigastric discomfort especially after meals . however , the vital signs were within normal limits and the abdomen was soft and nontender .", "x - ray of the abdomen suggested the presence of a foreign body and a computed tomography ( ct ) scan was done which confirmed a toothbrush lying in the stomach [ figures 1 , 2 ] .", "an upper gastrointestinal endoscopy was done which revealed the toothbrush in the stomach with its head toward the gastroduodenal junction .", "biopsy forceps were used to deliver the toothbrush by holding its bristles [ figure 3 ] .", "the endoscope was kept inside to insufflate and distend the stomach and a minilaparotomy with gastrotomy of 1.5 - 2 cm was performed through the midline under local anesthesia and the toothbrush was successfully removed .", "x - ray of the abdomen suggesting the presence of the toothbrush in the abdomen computed tomography ( ct ) scan of the abdomen showing the presence of the toothbrush in the tomach endoscopic picture of toothbrush with biopsy forceps in situ to retrieve it from the stomach"], ["in the stomach , 80 - 90% of foreign bodies pass uneventfully through the gastrointestinal tract without complications .", "however , objects longer than 10 cm like a toothbrush can not negotiate the duodenal c - loop due to its fixed position in the retroperitoneum , and these must be removed as soon as possible to avoid pressure necrosis and gastric perforation .", "more than 40 cases of toothbrush ingestion have been reported in the literature till date .", "however , extreme caution and experience of the endoscopist is required for such procedures . in failed cases of endoscopic removal ,", "however , we devised a simple technique of minilaparotomy and gastrotomy under local anesthesia for removing such foreign bodies . in this technique ,", "the stomach is distended with the help of air insufflation through the endoscope and a small incision is made in the midepigasrium under local anesthesia and the foreign object can be removed directly under the vision of the endoscope ."], ["a swallowed toothbrush is a rare occurrence and it never passes through the gastrointestinal tract spontaneously .", "early removal of the toothbrush is critical for reducing morbidity and mortality . in cases where endoscopic removal fails ,", "endoscopy still remains an aid in performing surgical gastrotomy for delivering such complex foreign bodies under local anesthesia ."]]}
{"article_id": "PMC5433638", "article_text": ["in a contemporary health - care environment characterized by rapidly changing developments and relentlessly increasing knowledge , professional nurses need to develop critical thinking ( ct ) skills that will provide them with expertise in flexible , individualized , situation - specific problem - solving .", "ct has been defined as  a nonlinear and cycled process that allows people to make decisions on what to believe and what to do within a given context .", " according to kostovich et al . , the ability to think critically is an essential attribute for today 's nurses , and the development of this skill in nursing students requires multiple approaches and techniques .", "use of the nursing process is an essential element to cultivate ct and judgment skills in nursing students .", "the nursing process is a nonlinear , dynamic activity , which focuses on the multifaceted aspects of a patient , their family , and the environment .", "nurses and nursing students must develop and write nursing care plans to provide and organize nursing interventions based upon identified patients needs .", "however , some researchers have reported that the current linear nursing care plan format does not meet the educational needs of students to develop ct skills and visualize the interconnectedness of patient clinical data .", "in addition , nurse educators feel that the nursing care plans developed by students are not case sensitive and need in - depth comprehension of each client 's physical , psychological , social , and spiritual health . in the authors program ,", "the traditional linear format nursing care plan used in the first five semesters was found not to meet the needs of sixth - semester nursing students .", "these students need to be able to quickly conceptualize the plan of care in a functional format as they graduate and enter the practice arena , where they will be expected to care for multiple patients simultaneously and to rapidly use the nursing process to develop a nursing plan of care based upon priority needs of these patients .", "concept map development is an alternative to using care plans as a method to document a plan of care based on evidenced - based practices .", ", learners assimilate new concepts into their existing cognitive structure . when new concepts are integrated by identifying relationships with concepts already possessed , learning becomes meaningful .", "hence , concept maps provide a format to visualize physiological , pathological , and psychological relationships and interactions in a concrete fashion .", "visualization of patient care priorities through a holistic view of the patient can be achieved through concept mapping .", "in addition , the minimal use of text or words makes it easy to scan for a word , phrase , or the general idea that is being explored .", "some studies have explored the short - term effects of concept map teaching on students ct and found positive effects in clinical or classroom teaching .", "concept mapping has also been described in an online course on  distance education to adults  in nursing to assess thinking processes of the students where it was found that they helped the students to self - assess their own thinking processes , thus facilitating reflective practice .", "it has also identified by gerdeman et al . to improve clinical judgment skills in nursing students . however , contrary to previous positive results , some other studies reported contradictory results , for example in a quasi - experimental study , wheeler and collins found that the experimental group scored significantly higher on the overall analysis and evaluation scores in the pre and posttest comparisons , but no significant differences between the groups were found .", "yeh and chen also found no significant differences of ct scores between their experimental and control groups , and one longitudinal study on concept mapping , conducted in a united states medical school with 1 year medical students , found no significant differences between ct scores in the pre- and post - tests .", "possible explanations for the findings of these studies may stem from several factors , such as measurement error , instrumentation , the curriculum and various definition of ct adopted by the studies . on the other hand , some studies indicate that ct skill of nursing students in different countries had been different .", "in iran , it can be stated that although ct is important in clinical judgments and decisions but during the training period , have had no significant development therefore the traditional education system needs evolution and revision in order to realize training purposes in line with fostering creative and efficient students . with regard to the need for finding best way to promote nursing students ct and the limited evidence of comparing concept mapping with traditional nursing care plan in clinical setting", ", this study was performed to compare the effect of concept mapping with traditional nursing care plan on nursing students ct in clinical pediatric course .", "a before - after experimental design with the control group was used to compare the effect of concept mapping and traditional linear nursing care planning on baccalaureate nursing student 's ct skills at shahrekord university of medical sciences in shahrekord , iran .", "all 60 students from the nursing faculty of the shahrekord university of medical sciences , shahrekord , iran , who had enrolled in pediatric nursing clinical course , and in sixth - semester of their study in the year 2011 , were invited to participate in this study .", "students were randomly divided into six equal groups of 10 students ; three groups as the control group and the other three groups as an experimental group . to prevent contact between the control and experimental groups , they take the pediatric clinical course in sequential time order . during the course", ", students cared for patients from an assigned pediatric setting . because students were not able to have prior contact with their patients , they began developing their nursing care plans on the 1 day of their clinical experience .", "they were asked to create a nursing care plan for a child they were caring for , which required gathering information on complete patient histories , relevant medications , treatments and medical diagnoses .", "on the 2 day of clinical experience , clinical instructor who has the experience of concept mapping development taught the students in the experimental group how to create concept maps .", "the students were informed in advance that a concept map should display each nursing diagnosis and more importantly , its relationship to the patient 's data , pathophysiology of the disease and interventions in a holistic view .", "students were required to analyze assessment data and identify nursing diagnoses , relevant pathophysiology and interventions related to those diagnoses .", "they used diagrams , color , and shapes to code the parts of the nursing process .", "they also had 5 days after the clinical experience to reflect on and evaluate the effectiveness of their nursing care before the concept maps were due .", "each student in the experimental group created nine concept maps during the course . during clinical group discussions", "this activity provided all students with the opportunity to think out aloud about the accuracy and completeness of the nursing diagnoses , goals , nursing interventions and relationship depicted on their concept maps .", "a sample concept map demonstrated by student students in the control group received traditional clinical teaching to create linear nursing care plans on a blank sheet .", "the students were asked to read and analyze the patient profile data , formulate initial problems , prioritize nursing diagnoses and identify the relationship between nursing diagnoses , then develop and implement interventions to solve the problems , and to evaluate the effectiveness of their nursing care .", "linear care plans were in text format without using any shapes , propositions or arrow to illustrate the interconnectedness between the data , nursing diagnosis and interventions .", "they also had the opportunity to discuss their care plans in group discussion with guidance provided by the teacher .", "nobody in the control group asked about concept mapping showing that control groups did not contaminate with the intervention by experimental groups . a well - known , validated and reliable tool was selected for this study .", "ct skills were measured using the california critical thinking skill test ( cctst ) with subscales of analysis ( 9 items ) , evaluation ( 14 items ) , inference ( 11 items ) , and two types of reasoning ( deductive reasoning and inductive reasoning included 16 and 14 items respectively ) .", "we selected this scale because it was tested for reliability and validity in iranian nursing students .", "khalili and hossein zadeh reported that this scale in comparison with the other measuring tools of ct is more comprehensive .", "the reliability coefficient of subscales after factor analysis in their study was in the range of 62 - 67% .", "( l4 items ) note that a kr-20 range of 0.650.75 for this type of instrument is acceptable .", "this instrument contains 34 items in multiple - choice format , 19 four - option multiple - choice items and 15 five - option multiple - choice items .", "the range of possible total scores was 034 , with each subscale having a possible score range of 012 .", "an overall score of < 11 indicated a lack of ct skills , a score of 1125 indicated average ct skills , and a score above 25 indicated strong ct skills . for a given ct skill , a subscale score of 4 or less indicated weakness , whereas a subscale score of 7 or above indicated strength .", "data were collected before and after the program in a clinical setting at the beginning and end of clinical education .", "agreement to use and evaluate concept mapping as a teaching strategy in clinical courses was obtained from the nursing department of shahrekord university of medical sciences , shahrekord , iran .", "information about the study was given to every participant to assure the protection of human rights by the clinical teacher .", "participants in the experimental group were informed that they would be free to change to the traditional teaching strategy at any time without effects on their course evaluation . to ensure students in the control group were not disadvantaged , after completing the study , they were taught concept mapping during a 1-day workshop .", "chicago , il , usa ) software . in all analyses , p - 0.05 was considered as statistically significant .", "we used independent and paired t - tests to compare mean scores between the experimental and control groups at pre- and post - tests .", "a before - after experimental design with the control group was used to compare the effect of concept mapping and traditional linear nursing care planning on baccalaureate nursing student 's ct skills at shahrekord university of medical sciences in shahrekord , iran .", "all 60 students from the nursing faculty of the shahrekord university of medical sciences , shahrekord , iran , who had enrolled in pediatric nursing clinical course , and in sixth - semester of their study in the year 2011 , were invited to participate in this study .", "students were randomly divided into six equal groups of 10 students ; three groups as the control group and the other three groups as an experimental group .", "to prevent contact between the control and experimental groups , they take the pediatric clinical course in sequential time order . during the course , students cared for patients from an assigned pediatric setting . because students were not able to have prior contact with their patients , they began developing their nursing care plans on the 1 day of their clinical experience .", "they were asked to create a nursing care plan for a child they were caring for , which required gathering information on complete patient histories , relevant medications , treatments and medical diagnoses .", "on the 2 day of clinical experience , clinical instructor who has the experience of concept mapping development taught the students in the experimental group how to create concept maps .", "the students were informed in advance that a concept map should display each nursing diagnosis and more importantly , its relationship to the patient 's data , pathophysiology of the disease and interventions in a holistic view .", "students were required to analyze assessment data and identify nursing diagnoses , relevant pathophysiology and interventions related to those diagnoses .", "they used diagrams , color , and shapes to code the parts of the nursing process .", "they also had 5 days after the clinical experience to reflect on and evaluate the effectiveness of their nursing care before the concept maps were due .", "each student in the experimental group created nine concept maps during the course . during clinical group discussions", "this activity provided all students with the opportunity to think out aloud about the accuracy and completeness of the nursing diagnoses , goals , nursing interventions and relationship depicted on their concept maps .", "a sample concept map demonstrated by student students in the control group received traditional clinical teaching to create linear nursing care plans on a blank sheet .", "the students were asked to read and analyze the patient profile data , formulate initial problems , prioritize nursing diagnoses and identify the relationship between nursing diagnoses , then develop and implement interventions to solve the problems , and to evaluate the effectiveness of their nursing care .", "linear care plans were in text format without using any shapes , propositions or arrow to illustrate the interconnectedness between the data , nursing diagnosis and interventions .", "they also had the opportunity to discuss their care plans in group discussion with guidance provided by the teacher .", "nobody in the control group asked about concept mapping showing that control groups did not contaminate with the intervention by experimental groups .", "ct skills were measured using the california critical thinking skill test ( cctst ) with subscales of analysis ( 9 items ) , evaluation ( 14 items ) , inference ( 11 items ) , and two types of reasoning ( deductive reasoning and inductive reasoning included 16 and 14 items respectively ) .", "we selected this scale because it was tested for reliability and validity in iranian nursing students .", "khalili and hossein zadeh reported that this scale in comparison with the other measuring tools of ct is more comprehensive .", "the reliability coefficient of subscales after factor analysis in their study was in the range of 62 - 67% .", "( l4 items ) note that a kr-20 range of 0.650.75 for this type of instrument is acceptable .", "this instrument contains 34 items in multiple - choice format , 19 four - option multiple - choice items and 15 five - option multiple - choice items .", "the range of possible total scores was 034 , with each subscale having a possible score range of 012 .", "an overall score of < 11 indicated a lack of ct skills , a score of 1125 indicated average ct skills , and a score above 25 indicated strong ct skills . for a given ct skill , a subscale score of 4 or less indicated weakness , whereas a subscale score of 7 or above indicated strength .", "data were collected before and after the program in a clinical setting at the beginning and end of clinical education .", "agreement to use and evaluate concept mapping as a teaching strategy in clinical courses was obtained from the nursing department of shahrekord university of medical sciences , shahrekord , iran .", "information about the study was given to every participant to assure the protection of human rights by the clinical teacher .", "participants in the experimental group were informed that they would be free to change to the traditional teaching strategy at any time without effects on their course evaluation . to ensure students in the control group were not disadvantaged , after completing the study , they were taught concept mapping during a 1-day workshop .", "data analysis was performed using statistical package for the social sciences ( spssinc . , chicago , il , usa ) software . in all analyses , p - 0.05", "we used independent and paired t - tests to compare mean scores between the experimental and control groups at pre- and post - tests .", "almost all students were 21 years old except one who was 22 years old ( range : 2122 ) .", "the statistical analysis showed that two groups did not appreciably differ in age or sex .", "there was no significant difference between the groups overall , and in all subscales before the program [ table 1 ] .", "paired t - test showed significant improvement in overall and all subscales of cctst from pretest to posttest in both groups ( p < 0.001 ) [ tables 2 and 3 ] but t - test demonstrates that improvement in students ct skills in the experimental group was significantly greater than that in the control group after the program ( p < 0.001 ) [ table 4 ] .", "student 's ct skills on the pretest comparison of overall and subscales of cctst - a in the experimental group comparison of overall and subscales of cctst - a in the control group student s ct skills on the posttest", "this study was developed to examine whether concept mapping helped students develop better ct skills than the traditional method in an undergraduate pediatric clinical course . in the current study", ", it was found that either concept map or traditional linear nursing care plan could promote ct skills in nursing students from lacking to an average ct standard .", "however , similar to findings of other prior studies which observed improvements to nurses ct abilities following a concept mapping teaching strategy , the present study provided evidence to indicate that the effects of concept mapping were greater than those of traditional linear nursing care plans , with greater improvements to students overall and all subscales of ct skills as indicated by cctst scores . considering that the test of ct is based on the problem - solving process and the nursing process in the defined problem - solving stages , the use of which could improve ct skills .", "the ct skills overall describe overall strength in using reasoning to form reflective judgments about what to believe or what to do and includes core reasoning skills such as analysis , inference , evaluation , induction , and deduction .", "analytical reasoning skills enable people to identify the elements of a situation and determine how these parts interact . in the nursing process , students identify bio - psycho - social aspects of patients health and determine how these parts interact . in our study , students tried to understand the relationship between patient data , nursing process , interactions and connections between sign and symptoms , diagnostic data and medications as they developed traditional care plans or concept maps .", "although both care plans and concept maps could reinforce the students analytical reasoning skills , concept mapping had a greater effect . according to cook et al . , the linear format of the nursing care plan is based upon the nursing process that does not always allow for a holistic picture of patient needs and does not allow for visualization of the interrelatedness of patient data .", "in contrast , concept maps provide a format to visualize physiological , pathophysiological and psychological relationships and interactions in a concrete fashion , which is more effective to support quality analysis .", "schuster also suggested that visualization of patient care priorities through a holistic view of the patient can be achieved through concept mapping .", "nirmala and shakuntala also supported that the cross - links in concept maps are useful for correlating patients diagnoses , symptoms , treatment and interventions and then to thinking critically in clinical decision - making . according to facione et al .", "inductive reasoning helps in isolating the cause of an ailment or arriving at a theory to explain the relationship between symptoms .", "evaluative reasoning skills enable students to assess the credibility of resources of information and the claims they make . in our study , after the 5 weeks program , the concept mapping participants demonstrated greater ct abilities to assess the credibility of , and relationships between statements and to provide reasoning according to evidence and using deduction , comprehend , express and clarify meaning and to perform inductive reasoning tasks .", "similar to some previous studies ( yeh and chen ) , our study found that the experimental group had significantly higher scores of inference and deduction than those of the control group in the posttest . when constructing concept maps , students need to draw logical conclusions from factual knowledge or promises known through a process of inference such as identifying the major concepts , determining the relationship between concepts and making propositions using cross - links before coming to conclusions . in summary , converting to a concept map format allow for visualization of all aspects of patient clinical data , physical assessment , disease process and the relationship between this information , facilitating ct in nursing students in a clinical area .", "previous researches on the ct of nursing students in iranian faculties showed that learning in the educational system takes place at the initial cognitive levels , and higher levels such as analysis or synthesis and evaluation are less addressed .", "in fact , less attention is paid to the growth of the ct power according to kermansaravi et al .", "there are main and serious obstacles in the development of ct , one of which is the predominant use of traditional teaching methods in the current education system which is preventing from the development of decision making and troubleshooting ( or problem - solving ) skills in the learners and as a result limits the opportunities for students ct . considering that the test of ct is based on the problem - solving process and the nursing process is the defined problem - solving stages , the use of which is one - way emphasized in nursing education programs to the growth of ct .", "our study suggested that preparing nursing care using the concept map is more effective in promoting ct in nursing students than traditional linear nursing care plans .", "as with all studies with small samples , generalization of findings must be made cautiously .", "however , this study adds to the growing body of knowledge that suggests concept mapping improves students abilities to see patterns and relationships .", "although this study identified some short - term effects of the concept mapping program , its long - term effects remain unknown .", "the sample set came from only one university in iran limiting the feasibility of generalization of results to other universities and countries .", "future investigations should include multisite evaluations in a range of geographic locales with larger sample sizes .", "as with all studies with small samples , generalization of findings must be made cautiously . however , this study adds to the growing body of knowledge that suggests concept mapping improves students abilities to see patterns and relationships .", "although this study identified some short - term effects of the concept mapping program , its long - term effects remain unknown .", "the sample set came from only one university in iran limiting the feasibility of generalization of results to other universities and countries .", "future investigations should include multisite evaluations in a range of geographic locales with larger sample sizes .", "when presented with complex health - care situations and the need to process vast amounts of information , clinical nurses must be in ownership of ct skills in order to make appropriate professional judgments and clinical decision making .", "results from the present study support the application of concept mapping as a clinical teaching strategy to promote the development of ct skills .", "concept mapping , in comparison with the traditional linear nursing care plan , resulted in greater improvements in all ct skills .", "however , the 5 weeks program demonstrated only short - term effects , therefore , further longitudinal studies are suggested ."], "abstract_text": ["<S> introduction : concept map is a useful cognitive tool for enhancing a student 's critical thinking ( ct ) by encouraging students to process information deeply for understanding . however , the evidence regarding its effectiveness on nursing students ct is contradictory . </S>", "<S> this paper compares the effectiveness of concept mapping and traditional linear nursing care planning on students ct.methods:an experimental design was used to examine the ct of 60 baccalaureate students who participated in pediatric clinical nursing course in the shahrekord university of medical sciences , shahrekord , iran in 2013.results:participants were randomly divided into six equal groups of each 10 student , of which three groups were the control group , and the others were the experimental group . </S>", "<S> the control group completed nine traditional linear nursing care plans , whereas experimental group completed nine concept maps during the course . </S>", "<S> both groups showed significant improvement in overall and all subscales of the california ct skill test from pretest to posttest ( p < 0.001 ) , but t - test demonstrated that improvement in students ct skills in the experimental group was significantly greater than in the control group after the program ( p < 0.001).conclusions : our findings support that concept mapping can be used as a clinical teaching - learning activity to promote ct in nursing students . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Design", "Setting and sample", "Intervention procedure", "Critical thinking measurement", "Ethical review", "Data analysis", "RESULTS", "DISCUSSION", "Limitations", "CONCLUSIONS", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["in a contemporary health - care environment characterized by rapidly changing developments and relentlessly increasing knowledge , professional nurses need to develop critical thinking ( ct ) skills that will provide them with expertise in flexible , individualized , situation - specific problem - solving .", "ct has been defined as  a nonlinear and cycled process that allows people to make decisions on what to believe and what to do within a given context .", " according to kostovich et al . , the ability to think critically is an essential attribute for today 's nurses , and the development of this skill in nursing students requires multiple approaches and techniques .", "use of the nursing process is an essential element to cultivate ct and judgment skills in nursing students .", "the nursing process is a nonlinear , dynamic activity , which focuses on the multifaceted aspects of a patient , their family , and the environment .", "nurses and nursing students must develop and write nursing care plans to provide and organize nursing interventions based upon identified patients needs .", "however , some researchers have reported that the current linear nursing care plan format does not meet the educational needs of students to develop ct skills and visualize the interconnectedness of patient clinical data .", "in addition , nurse educators feel that the nursing care plans developed by students are not case sensitive and need in - depth comprehension of each client 's physical , psychological , social , and spiritual health . in the authors program ,", "the traditional linear format nursing care plan used in the first five semesters was found not to meet the needs of sixth - semester nursing students .", "these students need to be able to quickly conceptualize the plan of care in a functional format as they graduate and enter the practice arena , where they will be expected to care for multiple patients simultaneously and to rapidly use the nursing process to develop a nursing plan of care based upon priority needs of these patients .", "concept map development is an alternative to using care plans as a method to document a plan of care based on evidenced - based practices .", ", learners assimilate new concepts into their existing cognitive structure . when new concepts are integrated by identifying relationships with concepts already possessed , learning becomes meaningful .", "hence , concept maps provide a format to visualize physiological , pathological , and psychological relationships and interactions in a concrete fashion .", "visualization of patient care priorities through a holistic view of the patient can be achieved through concept mapping .", "in addition , the minimal use of text or words makes it easy to scan for a word , phrase , or the general idea that is being explored .", "some studies have explored the short - term effects of concept map teaching on students ct and found positive effects in clinical or classroom teaching .", "concept mapping has also been described in an online course on  distance education to adults  in nursing to assess thinking processes of the students where it was found that they helped the students to self - assess their own thinking processes , thus facilitating reflective practice .", "it has also identified by gerdeman et al . to improve clinical judgment skills in nursing students . however , contrary to previous positive results , some other studies reported contradictory results , for example in a quasi - experimental study , wheeler and collins found that the experimental group scored significantly higher on the overall analysis and evaluation scores in the pre and posttest comparisons , but no significant differences between the groups were found .", "yeh and chen also found no significant differences of ct scores between their experimental and control groups , and one longitudinal study on concept mapping , conducted in a united states medical school with 1 year medical students , found no significant differences between ct scores in the pre- and post - tests .", "possible explanations for the findings of these studies may stem from several factors , such as measurement error , instrumentation , the curriculum and various definition of ct adopted by the studies . on the other hand , some studies indicate that ct skill of nursing students in different countries had been different .", "in iran , it can be stated that although ct is important in clinical judgments and decisions but during the training period , have had no significant development therefore the traditional education system needs evolution and revision in order to realize training purposes in line with fostering creative and efficient students . with regard to the need for finding best way to promote nursing students ct and the limited evidence of comparing concept mapping with traditional nursing care plan in clinical setting", ", this study was performed to compare the effect of concept mapping with traditional nursing care plan on nursing students ct in clinical pediatric course ."], ["a before - after experimental design with the control group was used to compare the effect of concept mapping and traditional linear nursing care planning on baccalaureate nursing student 's ct skills at shahrekord university of medical sciences in shahrekord , iran .", "all 60 students from the nursing faculty of the shahrekord university of medical sciences , shahrekord , iran , who had enrolled in pediatric nursing clinical course , and in sixth - semester of their study in the year 2011 , were invited to participate in this study .", "students were randomly divided into six equal groups of 10 students ; three groups as the control group and the other three groups as an experimental group . to prevent contact between the control and experimental groups , they take the pediatric clinical course in sequential time order . during the course", ", students cared for patients from an assigned pediatric setting . because students were not able to have prior contact with their patients , they began developing their nursing care plans on the 1 day of their clinical experience .", "they were asked to create a nursing care plan for a child they were caring for , which required gathering information on complete patient histories , relevant medications , treatments and medical diagnoses .", "on the 2 day of clinical experience , clinical instructor who has the experience of concept mapping development taught the students in the experimental group how to create concept maps .", "the students were informed in advance that a concept map should display each nursing diagnosis and more importantly , its relationship to the patient 's data , pathophysiology of the disease and interventions in a holistic view .", "students were required to analyze assessment data and identify nursing diagnoses , relevant pathophysiology and interventions related to those diagnoses .", "they used diagrams , color , and shapes to code the parts of the nursing process .", "they also had 5 days after the clinical experience to reflect on and evaluate the effectiveness of their nursing care before the concept maps were due .", "each student in the experimental group created nine concept maps during the course . during clinical group discussions", "this activity provided all students with the opportunity to think out aloud about the accuracy and completeness of the nursing diagnoses , goals , nursing interventions and relationship depicted on their concept maps .", "a sample concept map demonstrated by student students in the control group received traditional clinical teaching to create linear nursing care plans on a blank sheet .", "the students were asked to read and analyze the patient profile data , formulate initial problems , prioritize nursing diagnoses and identify the relationship between nursing diagnoses , then develop and implement interventions to solve the problems , and to evaluate the effectiveness of their nursing care .", "linear care plans were in text format without using any shapes , propositions or arrow to illustrate the interconnectedness between the data , nursing diagnosis and interventions .", "they also had the opportunity to discuss their care plans in group discussion with guidance provided by the teacher .", "nobody in the control group asked about concept mapping showing that control groups did not contaminate with the intervention by experimental groups . a well - known , validated and reliable tool was selected for this study .", "ct skills were measured using the california critical thinking skill test ( cctst ) with subscales of analysis ( 9 items ) , evaluation ( 14 items ) , inference ( 11 items ) , and two types of reasoning ( deductive reasoning and inductive reasoning included 16 and 14 items respectively ) .", "we selected this scale because it was tested for reliability and validity in iranian nursing students .", "khalili and hossein zadeh reported that this scale in comparison with the other measuring tools of ct is more comprehensive .", "the reliability coefficient of subscales after factor analysis in their study was in the range of 62 - 67% .", "( l4 items ) note that a kr-20 range of 0.650.75 for this type of instrument is acceptable .", "this instrument contains 34 items in multiple - choice format , 19 four - option multiple - choice items and 15 five - option multiple - choice items .", "the range of possible total scores was 034 , with each subscale having a possible score range of 012 .", "an overall score of < 11 indicated a lack of ct skills , a score of 1125 indicated average ct skills , and a score above 25 indicated strong ct skills . for a given ct skill , a subscale score of 4 or less indicated weakness , whereas a subscale score of 7 or above indicated strength .", "data were collected before and after the program in a clinical setting at the beginning and end of clinical education .", "agreement to use and evaluate concept mapping as a teaching strategy in clinical courses was obtained from the nursing department of shahrekord university of medical sciences , shahrekord , iran .", "information about the study was given to every participant to assure the protection of human rights by the clinical teacher .", "participants in the experimental group were informed that they would be free to change to the traditional teaching strategy at any time without effects on their course evaluation . to ensure students in the control group were not disadvantaged , after completing the study , they were taught concept mapping during a 1-day workshop .", "chicago , il , usa ) software . in all analyses , p - 0.05 was considered as statistically significant .", "we used independent and paired t - tests to compare mean scores between the experimental and control groups at pre- and post - tests ."], ["a before - after experimental design with the control group was used to compare the effect of concept mapping and traditional linear nursing care planning on baccalaureate nursing student 's ct skills at shahrekord university of medical sciences in shahrekord , iran ."], ["all 60 students from the nursing faculty of the shahrekord university of medical sciences , shahrekord , iran , who had enrolled in pediatric nursing clinical course , and in sixth - semester of their study in the year 2011 , were invited to participate in this study .", "students were randomly divided into six equal groups of 10 students ; three groups as the control group and the other three groups as an experimental group .", "to prevent contact between the control and experimental groups , they take the pediatric clinical course in sequential time order . during the course , students cared for patients from an assigned pediatric setting . because students were not able to have prior contact with their patients , they began developing their nursing care plans on the 1 day of their clinical experience .", "they were asked to create a nursing care plan for a child they were caring for , which required gathering information on complete patient histories , relevant medications , treatments and medical diagnoses ."], ["on the 2 day of clinical experience , clinical instructor who has the experience of concept mapping development taught the students in the experimental group how to create concept maps .", "the students were informed in advance that a concept map should display each nursing diagnosis and more importantly , its relationship to the patient 's data , pathophysiology of the disease and interventions in a holistic view .", "students were required to analyze assessment data and identify nursing diagnoses , relevant pathophysiology and interventions related to those diagnoses .", "they used diagrams , color , and shapes to code the parts of the nursing process .", "they also had 5 days after the clinical experience to reflect on and evaluate the effectiveness of their nursing care before the concept maps were due .", "each student in the experimental group created nine concept maps during the course . during clinical group discussions", "this activity provided all students with the opportunity to think out aloud about the accuracy and completeness of the nursing diagnoses , goals , nursing interventions and relationship depicted on their concept maps .", "a sample concept map demonstrated by student students in the control group received traditional clinical teaching to create linear nursing care plans on a blank sheet .", "the students were asked to read and analyze the patient profile data , formulate initial problems , prioritize nursing diagnoses and identify the relationship between nursing diagnoses , then develop and implement interventions to solve the problems , and to evaluate the effectiveness of their nursing care .", "linear care plans were in text format without using any shapes , propositions or arrow to illustrate the interconnectedness between the data , nursing diagnosis and interventions .", "they also had the opportunity to discuss their care plans in group discussion with guidance provided by the teacher .", "nobody in the control group asked about concept mapping showing that control groups did not contaminate with the intervention by experimental groups ."], ["ct skills were measured using the california critical thinking skill test ( cctst ) with subscales of analysis ( 9 items ) , evaluation ( 14 items ) , inference ( 11 items ) , and two types of reasoning ( deductive reasoning and inductive reasoning included 16 and 14 items respectively ) .", "we selected this scale because it was tested for reliability and validity in iranian nursing students .", "khalili and hossein zadeh reported that this scale in comparison with the other measuring tools of ct is more comprehensive .", "the reliability coefficient of subscales after factor analysis in their study was in the range of 62 - 67% .", "( l4 items ) note that a kr-20 range of 0.650.75 for this type of instrument is acceptable .", "this instrument contains 34 items in multiple - choice format , 19 four - option multiple - choice items and 15 five - option multiple - choice items .", "the range of possible total scores was 034 , with each subscale having a possible score range of 012 .", "an overall score of < 11 indicated a lack of ct skills , a score of 1125 indicated average ct skills , and a score above 25 indicated strong ct skills . for a given ct skill , a subscale score of 4 or less indicated weakness , whereas a subscale score of 7 or above indicated strength .", "data were collected before and after the program in a clinical setting at the beginning and end of clinical education ."], ["agreement to use and evaluate concept mapping as a teaching strategy in clinical courses was obtained from the nursing department of shahrekord university of medical sciences , shahrekord , iran .", "information about the study was given to every participant to assure the protection of human rights by the clinical teacher .", "participants in the experimental group were informed that they would be free to change to the traditional teaching strategy at any time without effects on their course evaluation . to ensure students in the control group were not disadvantaged , after completing the study , they were taught concept mapping during a 1-day workshop ."], ["data analysis was performed using statistical package for the social sciences ( spssinc . , chicago , il , usa ) software . in all analyses , p - 0.05", "we used independent and paired t - tests to compare mean scores between the experimental and control groups at pre- and post - tests ."], ["almost all students were 21 years old except one who was 22 years old ( range : 2122 ) .", "the statistical analysis showed that two groups did not appreciably differ in age or sex .", "there was no significant difference between the groups overall , and in all subscales before the program [ table 1 ] .", "paired t - test showed significant improvement in overall and all subscales of cctst from pretest to posttest in both groups ( p < 0.001 ) [ tables 2 and 3 ] but t - test demonstrates that improvement in students ct skills in the experimental group was significantly greater than that in the control group after the program ( p < 0.001 ) [ table 4 ] .", "student 's ct skills on the pretest comparison of overall and subscales of cctst - a in the experimental group comparison of overall and subscales of cctst - a in the control group student s ct skills on the posttest"], ["this study was developed to examine whether concept mapping helped students develop better ct skills than the traditional method in an undergraduate pediatric clinical course . in the current study", ", it was found that either concept map or traditional linear nursing care plan could promote ct skills in nursing students from lacking to an average ct standard .", "however , similar to findings of other prior studies which observed improvements to nurses ct abilities following a concept mapping teaching strategy , the present study provided evidence to indicate that the effects of concept mapping were greater than those of traditional linear nursing care plans , with greater improvements to students overall and all subscales of ct skills as indicated by cctst scores . considering that the test of ct is based on the problem - solving process and the nursing process in the defined problem - solving stages , the use of which could improve ct skills .", "the ct skills overall describe overall strength in using reasoning to form reflective judgments about what to believe or what to do and includes core reasoning skills such as analysis , inference , evaluation , induction , and deduction .", "analytical reasoning skills enable people to identify the elements of a situation and determine how these parts interact . in the nursing process , students identify bio - psycho - social aspects of patients health and determine how these parts interact . in our study , students tried to understand the relationship between patient data , nursing process , interactions and connections between sign and symptoms , diagnostic data and medications as they developed traditional care plans or concept maps .", "although both care plans and concept maps could reinforce the students analytical reasoning skills , concept mapping had a greater effect . according to cook et al . , the linear format of the nursing care plan is based upon the nursing process that does not always allow for a holistic picture of patient needs and does not allow for visualization of the interrelatedness of patient data .", "in contrast , concept maps provide a format to visualize physiological , pathophysiological and psychological relationships and interactions in a concrete fashion , which is more effective to support quality analysis .", "schuster also suggested that visualization of patient care priorities through a holistic view of the patient can be achieved through concept mapping .", "nirmala and shakuntala also supported that the cross - links in concept maps are useful for correlating patients diagnoses , symptoms , treatment and interventions and then to thinking critically in clinical decision - making . according to facione et al .", "inductive reasoning helps in isolating the cause of an ailment or arriving at a theory to explain the relationship between symptoms .", "evaluative reasoning skills enable students to assess the credibility of resources of information and the claims they make . in our study , after the 5 weeks program , the concept mapping participants demonstrated greater ct abilities to assess the credibility of , and relationships between statements and to provide reasoning according to evidence and using deduction , comprehend , express and clarify meaning and to perform inductive reasoning tasks .", "similar to some previous studies ( yeh and chen ) , our study found that the experimental group had significantly higher scores of inference and deduction than those of the control group in the posttest . when constructing concept maps , students need to draw logical conclusions from factual knowledge or promises known through a process of inference such as identifying the major concepts , determining the relationship between concepts and making propositions using cross - links before coming to conclusions . in summary , converting to a concept map format allow for visualization of all aspects of patient clinical data , physical assessment , disease process and the relationship between this information , facilitating ct in nursing students in a clinical area .", "previous researches on the ct of nursing students in iranian faculties showed that learning in the educational system takes place at the initial cognitive levels , and higher levels such as analysis or synthesis and evaluation are less addressed .", "in fact , less attention is paid to the growth of the ct power according to kermansaravi et al .", "there are main and serious obstacles in the development of ct , one of which is the predominant use of traditional teaching methods in the current education system which is preventing from the development of decision making and troubleshooting ( or problem - solving ) skills in the learners and as a result limits the opportunities for students ct . considering that the test of ct is based on the problem - solving process and the nursing process is the defined problem - solving stages , the use of which is one - way emphasized in nursing education programs to the growth of ct .", "our study suggested that preparing nursing care using the concept map is more effective in promoting ct in nursing students than traditional linear nursing care plans .", "as with all studies with small samples , generalization of findings must be made cautiously .", "however , this study adds to the growing body of knowledge that suggests concept mapping improves students abilities to see patterns and relationships .", "although this study identified some short - term effects of the concept mapping program , its long - term effects remain unknown .", "the sample set came from only one university in iran limiting the feasibility of generalization of results to other universities and countries .", "future investigations should include multisite evaluations in a range of geographic locales with larger sample sizes ."], ["as with all studies with small samples , generalization of findings must be made cautiously . however , this study adds to the growing body of knowledge that suggests concept mapping improves students abilities to see patterns and relationships .", "although this study identified some short - term effects of the concept mapping program , its long - term effects remain unknown .", "the sample set came from only one university in iran limiting the feasibility of generalization of results to other universities and countries .", "future investigations should include multisite evaluations in a range of geographic locales with larger sample sizes ."], ["when presented with complex health - care situations and the need to process vast amounts of information , clinical nurses must be in ownership of ct skills in order to make appropriate professional judgments and clinical decision making .", "results from the present study support the application of concept mapping as a clinical teaching strategy to promote the development of ct skills .", "concept mapping , in comparison with the traditional linear nursing care plan , resulted in greater improvements in all ct skills .", "however , the 5 weeks program demonstrated only short - term effects , therefore , further longitudinal studies are suggested ."], [""], [""]]}
{"article_id": "PMC4210413", "article_text": ["these include the \n intrinsic flexor pollicis brevis , flexor pollicis longus , lumbricalis , flexor brevis , and \n flexor longus muscles1 .", "toe - gripping \n strength is measured in the sitting position with the trunk vertical , the hip and knee \n joints at 90 , and the ankle joint in the neutral position1,2,3 .", "low toe - gripping strength is a risk factor of falls for the \n elderly , and several studies have reported that toe - gripping strength is lower in subjects \n with a history of falls than in those with no history of falls1,2,3,4 .", "furthermore , toe - gripping \n strength can be increased by training5 , \n which can decrease the risk of falls1 . \n", "souma et al.6 studied the % iemg of \n several crural muscles ( the soleus muscle , medial head of the gastrocnemius muscle , and \n tibialis anterior ) during toe - gripping in 3 different ankle joint positions10 of plantar \n flexion and 0 and 10 of dorsiflexion  and reported that the crural muscles help the ankle \n joint by co - contracting during toe - gripping . in another study ,", "nakae et al.7 calculated the % iemg of the same muscles \n during toe - gripping with subjects seated on the edge of a seat or standing , and reported \n that the % iemg of the medial gastrocnemius muscle was significantly lower when subjects were \n seated than when they were standing .", "however , as both studies focused on the activity of the \n crural muscles , the activity of the femoral muscles during toe - gripping remains unclear .", "we \n believe it is important to elucidate the contribution of femoral muscles to toe - gripping \n strength . in the present study , we investigated femoral muscle activity during toe - gripping to \n examine the role of the femoral muscles in toe - gripping strength .", "their \n average ( mean  sd ) age , height , and body weight were 20.6  1.0  years , 159.4  6.3  cm , and \n 51.8  5.8  kg , respectively .", "this study was approved by the ethics committee for human \n research of tohoku fukushi university , and written informed consent was received from all of \n the subjects after the purpose of the study had been explained to them .", "toe - gripping strength of the dominant toe and emg activity of the ipsilateral thigh were \n synchronously recorded to assess the activity of the rectus femoris and biceps femoris \n muscles .", "toe - gripping strength was measured using a toe - gripping dynamometer ( t.k.k.3360 ; \n takei co , ltd , niigata , japan ) . as described by uritani8", ", the subjects were instructed to sit with their trunk in the \n vertical position , hip and knee joints at 90 , and ankle joints in the neutral position .", "after a sufficient \n number of training trials and adequate rest , toe - gripping strength was measured twice and \n the maximal force was used in the analysis . for all subjects ,", "the right toe was dominant \n ( defined as the toe used to kick a ball ) . to measure the maximum voluntary contraction ( mvc ) activity of the rectus femoris muscles , \n as described by kai9 ,", "each subject was \n instructed to sit on a chair with the hip and knee joints at 90 , and to exert maximal \n isometric force of knee extension while resisting an opposing force applied by the examiner . \n", "the emg was recorded for 3 seconds while each subject exerted maximal force of knee \n extension . to measure the mvc of the biceps femoris muscle ,", "each subject was instructed to \n generate maximal isometric force of knee flexion while resisting an opposing force applied \n by the examiner .", "the emg was recorded for 3 seconds while each subject exerted maximal force \n of knee flexion .", "after confirmation of adequate skin preparation ( skin resistance of less \n than 5 k ) , three bipolar lead electrodes ( de-2.1 , delsys , inc . , boston , usa ) were attached \n to the skin over the rectus femoris and the long head of the biceps femoris , as described by \n peroto10 . for measurement of the rectus \n", "femoris muscle , the electrode was attached at the midpoint between the superior edge of the \n patella and the anterior superior iliac spine . for measurement of the long head of the \n biceps femoris ,", "the electrode was attached at the midpoint between the head of the fibula \n and ischial tuberosity .", "the emg signal was collected using ml846 power lab 4/26 ( sample : 1,000  hz ; ad instruments \n co. , ltd . ) and transferred to a personal computer .", "the bandwidth was 20500  hz . the emg \n signal segment selected and integrated ( integrated electromyogram : iemg ) for analysis was \n the middle 1  s of the entire 3-s duration of continuous maximal toe - gripping strength .", "the iemg was analyzed using lab chart pro v7.3.5 ( ad instruments co. , ltd . ) and normalized \n to the iemg of each muscle s mvc .", "repeated mann - whitney u tests were used to compare the % iemg of the \n rectus and biceps femoris muscles .", "relationships between the % iemg of the rectus and biceps \n femoris muscles were statistically analyzed using spearman s correlation coefficient .", "the average ( mean  sd ) toe - gripping strength was 20.9  3.6  kg .", "the average ( mean  sd ) \n % iemg values of the rectus and biceps femoris muscles were 7.0%  6.2% and 25.6%  15.9% , \n respectively .", "the % iemg of the biceps femoris was significantly higher than that of the \n rectus femoris ( p < 0.01 , table 1table 1.comparison of the % iemg values of the rectus femoris and biceps femoris muscles \n during toe - gripping actionrectus femorisbiceps femorismedian ( range :  min  max)median ( range :  min  max)%iemg4.8 * ( 1.521.1)27.2 ( 3.068.1)mann - whitney u test * : p < 0.05 ) .", "mann - whitney u test * : p < 0.05 using spearman s correlation coefficient analysis , a significant positive correlation was \n found between the % iemg of the rectus femoris and that of the biceps femoris ( r = 0.548 , p \n", "in the present study , we found that the % iemg of the biceps femoris was significantly \n higher than that of the rectus femoris .", "moreover , a significant positive correlation was \n found between the % iemg of these two muscles .", "these results suggest that femoral muscles \n co - contract during toe - gripping action , and thus possibly contribute to knee joint \n stability .", "we found that toe - gripping action stimulates femoral muscle activity and that the % iemg of \n the biceps femoris was significantly higher than that of the rectus femoris .", "sato et \n al.11 reported that during \n toe - gripping , the tibialis anterior and soleus muscles are activated first , followed by the \n gradual activation of the medial head of the gastrocnemius muscle .", "the biceps femoris \n initiates knee flexion , and the medial head of the gastrocnemius is a synergist for knee \n flexion .", "additionally , active tension of the biceps femoris decreases during toe - gripping in \n knee flexion .", "this may be because the length of the biceps femoris is shorter during active \n tension than during resting tension .", "we think that the activation of the biceps femoris \n during toe - gripping was caused by increasing motor unit recruitment and synchronization of \n the impulse to compensate for decreasing activity of the biceps femoris . in the present study", ", we found a significant positive correlation between the % iemg of the \n rectus femoris and that of the biceps femoris .", "this suggests that the % iemg of the rectus \n femoris increases with increases in the % iemg of the biceps femoris .", "toe - gripping strength was measured using a closed kinetic chain movement , and therefore , \n the likelihood of movement in the directions of flexion or extension of the knee joint is \n less . however , because the gastrocnemius also acts on knee joint flexion during exertion of \n toe - gripping strength , the knee joint is readily displaced in the direction of flexion . to \n prevent this", ", it is thought that the knee joint is immobilized by contraction of the rectus \n femoris , that is , we think the rectus femoris during exertion of toe - gripping strength acts \n in order to control the action of knee joint flexion by the gastrocnemius .", "first , we were unable to avoid the common problems that \n negatively affect surface emg recording , such as skin resistance , artifacts , and the effects \n of proximal muscles .", "second , as only healthy young women participated in this study , it is \n difficult to extend our findings to the general population . in future studies", ", we need to \n consider this aspect , and involve healthy young men and individuals of different age \n groups ."], "abstract_text": ["<S> [ purpose ] in the present study , we investigated femoral muscle activity during \n toe - gripping , and the role of the femoral muscles in toe - gripping strength . </S>", "<S> [ subjects ] \n fourteen healthy young women were selected . </S>", "<S> [ methods ] we measured the maximum voluntary \n contraction of the rectus femoris and long head of the biceps femoris muscles . </S>", "<S> we then \n calculated the percent integrated emg ( % iemg ) during the toe - gripping action . </S>", "<S> [ results ] we \n found that the % iemg of the biceps femoris was significantly higher than that of the \n rectus femoris . </S>", "<S> moreover , a significant positive correlation was found between the % iemg \n of the rectus femoris and that of the biceps femoris . </S>", "<S> [ conclusion ] these results suggest \n that femoral muscles co - contract during the toe - gripping action , and thus possibly \n contribute to knee joint stability . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["these include the \n intrinsic flexor pollicis brevis , flexor pollicis longus , lumbricalis , flexor brevis , and \n flexor longus muscles1 .", "toe - gripping \n strength is measured in the sitting position with the trunk vertical , the hip and knee \n joints at 90 , and the ankle joint in the neutral position1,2,3 .", "low toe - gripping strength is a risk factor of falls for the \n elderly , and several studies have reported that toe - gripping strength is lower in subjects \n with a history of falls than in those with no history of falls1,2,3,4 .", "furthermore , toe - gripping \n strength can be increased by training5 , \n which can decrease the risk of falls1 . \n", "souma et al.6 studied the % iemg of \n several crural muscles ( the soleus muscle , medial head of the gastrocnemius muscle , and \n tibialis anterior ) during toe - gripping in 3 different ankle joint positions10 of plantar \n flexion and 0 and 10 of dorsiflexion  and reported that the crural muscles help the ankle \n joint by co - contracting during toe - gripping . in another study ,", "nakae et al.7 calculated the % iemg of the same muscles \n during toe - gripping with subjects seated on the edge of a seat or standing , and reported \n that the % iemg of the medial gastrocnemius muscle was significantly lower when subjects were \n seated than when they were standing .", "however , as both studies focused on the activity of the \n crural muscles , the activity of the femoral muscles during toe - gripping remains unclear .", "we \n believe it is important to elucidate the contribution of femoral muscles to toe - gripping \n strength . in the present study , we investigated femoral muscle activity during toe - gripping to \n examine the role of the femoral muscles in toe - gripping strength ."], ["their \n average ( mean  sd ) age , height , and body weight were 20.6  1.0  years , 159.4  6.3  cm , and \n 51.8  5.8  kg , respectively .", "this study was approved by the ethics committee for human \n research of tohoku fukushi university , and written informed consent was received from all of \n the subjects after the purpose of the study had been explained to them .", "toe - gripping strength of the dominant toe and emg activity of the ipsilateral thigh were \n synchronously recorded to assess the activity of the rectus femoris and biceps femoris \n muscles .", "toe - gripping strength was measured using a toe - gripping dynamometer ( t.k.k.3360 ; \n takei co , ltd , niigata , japan ) . as described by uritani8", ", the subjects were instructed to sit with their trunk in the \n vertical position , hip and knee joints at 90 , and ankle joints in the neutral position .", "after a sufficient \n number of training trials and adequate rest , toe - gripping strength was measured twice and \n the maximal force was used in the analysis . for all subjects ,", "the right toe was dominant \n ( defined as the toe used to kick a ball ) . to measure the maximum voluntary contraction ( mvc ) activity of the rectus femoris muscles , \n as described by kai9 ,", "each subject was \n instructed to sit on a chair with the hip and knee joints at 90 , and to exert maximal \n isometric force of knee extension while resisting an opposing force applied by the examiner . \n", "the emg was recorded for 3 seconds while each subject exerted maximal force of knee \n extension . to measure the mvc of the biceps femoris muscle ,", "each subject was instructed to \n generate maximal isometric force of knee flexion while resisting an opposing force applied \n by the examiner .", "the emg was recorded for 3 seconds while each subject exerted maximal force \n of knee flexion .", "after confirmation of adequate skin preparation ( skin resistance of less \n than 5 k ) , three bipolar lead electrodes ( de-2.1 , delsys , inc . , boston , usa ) were attached \n to the skin over the rectus femoris and the long head of the biceps femoris , as described by \n peroto10 . for measurement of the rectus \n", "femoris muscle , the electrode was attached at the midpoint between the superior edge of the \n patella and the anterior superior iliac spine . for measurement of the long head of the \n biceps femoris ,", "the electrode was attached at the midpoint between the head of the fibula \n and ischial tuberosity .", "the emg signal was collected using ml846 power lab 4/26 ( sample : 1,000  hz ; ad instruments \n co. , ltd . ) and transferred to a personal computer .", "the bandwidth was 20500  hz . the emg \n signal segment selected and integrated ( integrated electromyogram : iemg ) for analysis was \n the middle 1  s of the entire 3-s duration of continuous maximal toe - gripping strength .", "the iemg was analyzed using lab chart pro v7.3.5 ( ad instruments co. , ltd . ) and normalized \n to the iemg of each muscle s mvc .", "repeated mann - whitney u tests were used to compare the % iemg of the \n rectus and biceps femoris muscles .", "relationships between the % iemg of the rectus and biceps \n femoris muscles were statistically analyzed using spearman s correlation coefficient ."], ["the average ( mean  sd ) toe - gripping strength was 20.9  3.6  kg .", "the average ( mean  sd ) \n % iemg values of the rectus and biceps femoris muscles were 7.0%  6.2% and 25.6%  15.9% , \n respectively .", "the % iemg of the biceps femoris was significantly higher than that of the \n rectus femoris ( p < 0.01 , table 1table 1.comparison of the % iemg values of the rectus femoris and biceps femoris muscles \n during toe - gripping actionrectus femorisbiceps femorismedian ( range :  min  max)median ( range :  min  max)%iemg4.8 * ( 1.521.1)27.2 ( 3.068.1)mann - whitney u test * : p < 0.05 ) .", "mann - whitney u test * : p < 0.05 using spearman s correlation coefficient analysis , a significant positive correlation was \n found between the % iemg of the rectus femoris and that of the biceps femoris ( r = 0.548 , p \n"], ["in the present study , we found that the % iemg of the biceps femoris was significantly \n higher than that of the rectus femoris .", "moreover , a significant positive correlation was \n found between the % iemg of these two muscles .", "these results suggest that femoral muscles \n co - contract during toe - gripping action , and thus possibly contribute to knee joint \n stability .", "we found that toe - gripping action stimulates femoral muscle activity and that the % iemg of \n the biceps femoris was significantly higher than that of the rectus femoris .", "sato et \n al.11 reported that during \n toe - gripping , the tibialis anterior and soleus muscles are activated first , followed by the \n gradual activation of the medial head of the gastrocnemius muscle .", "the biceps femoris \n initiates knee flexion , and the medial head of the gastrocnemius is a synergist for knee \n flexion .", "additionally , active tension of the biceps femoris decreases during toe - gripping in \n knee flexion .", "this may be because the length of the biceps femoris is shorter during active \n tension than during resting tension .", "we think that the activation of the biceps femoris \n during toe - gripping was caused by increasing motor unit recruitment and synchronization of \n the impulse to compensate for decreasing activity of the biceps femoris . in the present study", ", we found a significant positive correlation between the % iemg of the \n rectus femoris and that of the biceps femoris .", "this suggests that the % iemg of the rectus \n femoris increases with increases in the % iemg of the biceps femoris .", "toe - gripping strength was measured using a closed kinetic chain movement , and therefore , \n the likelihood of movement in the directions of flexion or extension of the knee joint is \n less . however , because the gastrocnemius also acts on knee joint flexion during exertion of \n toe - gripping strength , the knee joint is readily displaced in the direction of flexion . to \n prevent this", ", it is thought that the knee joint is immobilized by contraction of the rectus \n femoris , that is , we think the rectus femoris during exertion of toe - gripping strength acts \n in order to control the action of knee joint flexion by the gastrocnemius .", "first , we were unable to avoid the common problems that \n negatively affect surface emg recording , such as skin resistance , artifacts , and the effects \n of proximal muscles .", "second , as only healthy young women participated in this study , it is \n difficult to extend our findings to the general population . in future studies", ", we need to \n consider this aspect , and involve healthy young men and individuals of different age \n groups ."]]}
{"article_id": "PMC4437290", "article_text": ["the majority of our likes and dislikes we acquire throughout the lifespan are the product of learning .", "one of the most important ways through which stimuli acquire affective meaning is the change of valence that results from pairing one stimulus ( cs ) with a positive or negative affective stimulus ( ucs ) . as a result", "this effect is called ( associative ) evaluative conditioning and has been demonstrated in humans with a large variety of procedures and stimuli ( e.g. , [ 24 ] ; for a meta - analysis see ) .   in dementia severely impaired explicit memory", "nonetheless , patients with dementia might still be able to implicitly learn affective reactions through the process of evaluative conditioning .", "however , to the best of our knowledge , no study applied this approach in dementia patients .", "another classical paradigm that has already been used to investigate conditioning of affective reactions in this population is fear conditioning .", "indeed , two studies indicate that fear conditioning is impaired in dementia patients [ 6 , 7 ] .", "even though fear conditioning is impaired , evaluative conditioning might still be possible in dementia patients .", "some researchers have argued that while on a procedural level evaluative conditioning is similar to fear conditioning , the underlying processes might be different .", "it was hypothesized that fear conditioning is an instance of signal learning ; it is learned that the ucs is going to appear after the presentation of the cs .", "evaluative conditioning , on the other hand , only involves a reference to the ucs without expectation of its occurrence .", "thus , explicit knowledge about the cs - ucs relation seems crucial in fear conditioning while evaluative conditioning can be demonstrated in the absence of contingency awareness [ 1012 ] . since there could be variables that play a different role in these forms of learning ; dementia patients might show intact evaluative conditioning despite impaired fear conditioning .   indeed ,", "some studies indicate that dementia patients might retain the capacity to acquire affective reactions [ 13 , 14 ] .", "blessing et al .   demonstrated that dementia patients ' affective reactions can be influenced by pairing faces with fictional biographical content that characterized the depicted persons in terms of either positive or negative traits .", "pictures were rated before and at two different time points after the presentation of fictional biographical content with respect to valence and arousal .", "patients changed their ratings of pictures according to the biographical information presented , but did not recognize pictures above chance level or recall biographical information .", "these findings were replicated and extended in a subsequent study . the paradigm used by blessing et al . [", "however , there are two main differences : ( 1 ) in standard evaluative conditioning paradigms the subjects are not explicitly informed about the interrelation between stimuli in the learning phase . in the paradigm used by blessing et al .", "[ 13 , 14 ] the paring of the cs and ucs is made explicit and thus , the procedure can not be described as a simple cooccurrence of stimuli ; ( 2 ) in contrast to the approach of blessing et al .", "[ 13 , 14 ] the ucs and cs in evaluative conditioning paradigms belong to the same type of stimuli ( e.g. , faces ) .", "hence , in the present study we addressed the question if affective evaluations of dementia patients can be manipulated using a standard evaluative conditioning paradigm .", "\n participants . demographical data and clinical characteristics of both groups are listed in table 1 .", "the study included 15 dementia patients ( diagnosed as alzheimer 's disease ( n = 10 ) or mixed dementia ( n = 5 ) ) .", "patients were outpatients in the memory clinic of the psychiatric clinic of muensterlingen at the time of testing .", "all patients were diagnosed by a multidisciplinary team of the hospital ward using icd 10 criteria .", "all patients had received medical attendance including magnetic resonance imaging and specific screening blood tests , in order to exclude syphilis , diabetes , thyroid disorders , and vitamin b12 and folic acid deficiency .  ", "they reported that they had no known cns diseases , contact with toxic substances , or substance abuse .", "test stimuli were 10 pictures of neutral ( i.e. , displaying no facial expression of emotion ) unfamiliar faces ( 6 female : 3 young , 3 old ; 4 male : 2 young , 2 old ) and one picture of a happy young , female adult as well as one picture of a happy old , male adult selected from the productive aging laboratory face database . however , based on affective valence ratings of these pictures in other studies ( dann hier referenz ) , some of the included faces were also rated as positive or negative .", "the happy faces were added to increase the variance in subjective liking between pictures . \n emotional ratings .", "the sam was designed to assess subjective ratings of participants ' emotional responses and minimize the influence of language and culture on ratings .", "the sam valence rating scale has been successfully used in previous studies with dementia patients [ 13 , 14 ] . using the paper - pencil version of this instrument , participants rated the stimuli as to their emotional valence ( range : 19 ) .  ", "similar to other studies investigating evaluative conditioning we used a cover story in order to minimize demand effects .", "participants were told that the aim of the experiment was to examine the relationship between mood and subjective affective evaluation . in line with this cover", "story participants rated their mood on a five - point likert scale ( very good / good / normal / bad / very bad ) .", "subsequently , the pictures were presented to participants one after the other and rated with respect to valence using the sam rating scale .", "pictures were presented in four different pseudorandom sequences . after the valence rating an individual , unequivocal preference order of all stimuli was established .", "the pictures that received identical valence ratings were again presented to participants who then had to decide which of the pictures they preferred and the respective picture was put aside .", "again , the participants had to choose which of the remaining pictures with identical valence ratings they preferred and so on .  ", "the most preferred stimulus out of the 12 faces was used as the liked ( l ) stimulus and the stimulus with the lowest ranking was used as the disliked ( d ) stimulus .", "the four stimuli ranked 5th , 6th , 7th , and 8th were used as neutral ( n ) stimuli .", "the experimenter entered these six individual l , d , and n stimuli in a computer program ( presentations 11.3 ) that automatically formed three stimuli pairs ( i.e. , neutral - liked , neutral - disliked , and neutral - neutral ) by randomly assigning the neutral stimuli .", "participants were then seated about 50  cm from the computer screen and instructed to look at the pictures that would appear on the screen .", "each picture from the different pairs ( i.e. , ( n - l ) , ( n - d ) or (", "n - n ) ) was presented 10 times in the center of the computer screen .", "the duration of each stimulus presentation was 1 second and the interstimulus interval ( isi ; i.e. , onset of the first stimulus of a pair to onset of the second stimulus of a pair ) was 4 seconds .", "the inter - trial interval ( iti ; i.e. , onset of the first stimulus of the previous trial to onset of the first stimulus of the next trial ) was 13 seconds .", "after the presentation of stimuli on the computer screen , participants were again asked to rate the n , d , and l stimuli on the sam valence rating scale .", "the three relevant n stimuli ( i.e. , the first stimulus of each pair ) were placed one after the other in front of participants together with the three stimuli second in the pairs ( l , d , n ) .", "participants were asked to indicate which of these three stimuli followed the currently presented n stimulus .", "the experimenter registered the answer on a response sheet .   a repeated measures analyses of variance ( anovas )", "was conducted for the valence ratings of the relevant n stimuli ( i.e. , the first stimulus of each pair ) .", "a separate analysis was performed for ratings of stimuli paired with l and d pictures .", "type of stimulus pair and measurement point ( i.e. , valence rating pre and post presentation ) were used as within - participant factors and group was included as between subject factor . in the case of significant group differences a separate analysis was performed for both groups .", "the repeated measures anova revealed no main effect of type of stimulus pair ( f(2,26 ) = 2.42 ; p > .109 ) but an interaction between type of stimulus pair and time ( f(2,26 ) = 7.354 ; p > .003 ; see table 2 ) .", "this interaction was due to the fact that there was no influence of the type of stimulus pair at baseline ( f(2,27 ) = 0.296 ; p > .746 ) , but a significant effect after conditioning in the direction of the experimental manipulation ( f(2,27 ) = 5.460 ; p < .010 ) .", "no interaction between type of stimulus pair and group ( f(2,26 ) = 3.023 ; p > .066 ) or stimulus pair , time , and group ( f(2,26 ) = 2.798 ; p > .079 ) appeared .", "anova indicated no main effect of time ( f(1,27 ) = 0.78 ; p > .385 ) .  ", "as expected , we found a grater rating change over time for stimuli paired with l and d pictures than stimuli paired with n pictures ( see table 2 ) .", "we conducted a separate analysis using only pictures paired with l and d pictures to further investigate the influence of the experimental manipulation .", "the repeated measures anova revealed a main effect of type of stimulus pair ( f(1,27 ) = 4962 ; p > .034 ) along with an interaction between type of stimulus pair and time ( f(1,26 ) = 15.237 ; p > .001 ; see table 2 ) .", "we found no significant interaction between type of stimulus pair and group ( f(2,26 ) = 2.894 ; p > .10 ) but a significant interactions between stimulus pair , time , and group ( f(1,26 ) = 5.784 ; p < .023 ) .", "anova indicated no main effect of time ( f(1,27 ) = 0.296 ; p > .591 ) .  ", "because of the significant interaction between stimulus pair , time , and group , indicating group differences , we performed a separate analysis for each group .", "the repeated measures anova for valence ratings of pictures paired with l and d stimuli of dementia patients revealed a significant main effect of type of stimulus pair ( f(1,14 ) = 16.000 ; p >", ".001 ) along with an interaction between type of stimulus pair and time ( f(1,14 ) = 29.007 ; p > .001 ) .", "we found no main effect of time ( f(1,14 ) = 0.121 ; p > .733 ) .  ", "the repeated measures anova for ratings of controls indicated no main effect of type of stimulus pair ( f(1,13 ) = .087 ; p > .773 ) and no interaction between type of stimulus pair and time ( f(1,13 ) = .832 ; p > .378 ) .", "again , anova indicated no main effect of time ( f(1,13 ) = 0.188 ; p > .671 ) .   in the group of dementia patients , 24.4% of the relevant n stimuli", "were assigned to the correct stimulus second in the pairs in the forced choice test , which did not differ from chance level ( i.e. , 33.3% ; p > .14 ) . in the control group ,", "31% relevant n stimuli were assigned to the correct stimulus second in the pairs in the forced choice test , which did not differ from chance level ( i.e. , 33.3% ; p > .37 ) .", "the main aim of the present study was to investigate if affective evaluations of dementia patients can specifically be influenced through a standard evaluative conditioning paradigm .", "as hypothesized , dementia patients changed their valence ratings of unfamiliar and previously neutral faces according to its pairing with either a liked or disliked face stimulus . generally , the neutral pictures that were paired with a liked stimulus were rated higher and the neutral picture that was paired with the disliked stimulus was rated lower on the valence dimension after our evaluative conditioning intervention by dementia patients .", "thus , results indicate that ratings of initially neutral stimuli can be influenced in the according direction through simple time - near presentation ( i.e. , pairing ) with both a liked as well as a disliked stimuli", ". it does not seem surprising that our forced choice recognition test indicated no contingency awareness as this is based on the functionality of explicit memory that is severely impaired in dementia patients .   however , dementia patients significantly differed from controls in the effect of the experimental manipulation .", "the negative finding in the control group could indicate that our paradigm did not produce evaluative conditioning effects .", "consequently , the detected influence in the ad group could be accounted for by demand effects or other nonevaluative conditioning effects .", "another explanation could be that there was only a small effect in the control group that could not be detected due to small sample size .", "however , the sample size was only slightly smaller than that of the ad group .", "we looked at the data on the level of individual subjects and found that 11 of 14 subjects in the control group changed their ratings in the expected direction or showed no rating change .", "the negative result in the control group was due to two participants who showed a strong rating change in the opposite direction .", "post hoc analysis indicated a significant influence of the experimental manipulation in the control group after exclusion of these two outliers ( p < .028 ) .", "thus , it seems possible that evaluative conditioning effects in the control group were masked by the influence of outliers in our small sample .", "the rating changes of outliers could have been motivated by reactance , as observed in other studies investigating evaluative conditioning .", "our result could also indicate that evaluative conditioning effects are stronger in dementia patients than in healthy elderly controls .", "a possible reason for stronger effects could be lower attentional resources in dementia patients due to disease - related cognitive impairments .", "some studies indicate that attentional resources have a negative impact on affective learning through evaluative conditioning ( e.g. [ 19 , 20 ] ) and accordingly it seems possible that dementia patients show stronger effects . however , there are conflicting findings concerning the influence of attentional resources on evaluative conditioning ( e.g. , )", ".   the observed positive change of the valence rating in the neutral stimulus that was paired with another neutral stimulus could be a result of the mere exposure effect .", "this effect describes the preference for stimuli that have been previously presented over novel stimuli .", "preference changes due to the mere exposure effect have also been demonstrated in dementia patients [ 2325 ] . in line with this explanation", "are findings from other studies investigating evaluative conditioning that report a similar trend of positive changes in evaluations of neutral pictures paired with other neutral pictures over time .  ", "the results suggest that dementia patients changed their ratings according to the experimental manipulation without contingency awareness , since pairings were not identified above chance level in the forced choice recognition test .", "in fact , participants of the control group performed better than dementia patients in the recognition test , yet their results did not differ from chance level as well .", "the results of dementia patients are in line with findings demonstrating evaluative conditioning in healthy participants using subliminally presented stimuli [ 2729 ] .", "could show that the individual amount of the evaluative conditioning effect is not related to the number of pairings ( i.e. , neutral stimulus - affective stimulus ) participants were aware of .", "on the other hand , findings of a recent meta analysis suggest that contingency awareness is an important moderator in evaluative conditioning .   however , a critical limitation of the present study is our assessment of contingency awareness using a forced choice recognition test .", "following the recent discussion of gawronski and walther , it seems possible that we measured contingency memory rather than contingency awareness .", "subjects may have realised the respective pairings during conditioning but may not have been able to remember these pairings explicitly when the recognition test was applied .", "this may have been the case especially in dementia patients because of severe memory deficits .", "subjects may have remembered statistical probabilities that could not be assessed using a forced choice test .", "thus , it is also possible that contingency awareness differed between dementia patients and controls in our study and that contingency awareness influenced the results . to prohibit contingency awareness , very long time intervals on", "short isi could facilitate the detection of contingencies in the combinations of pictures and would enhance contingency awareness .  ", "however , this result has to be considered preliminary , since it is unclear what prevented rating changes in the expected direction in the control group in our paradigm .", "nevertheless , the results seem to be in contrast to previously reported impaired fear conditioning . as discussed above it", "is still unresolved whether evaluative conditioning is a form of pavlovian conditioning or if there are variables that are unique to evaluative conditioning .", "thus , the results of our study support the notion that different processes are involved in evaluative conditioning and fear conditioning .", "a relevant difference could be the dependence of pavlovian conditioning on contingency awareness in contrast to evaluative conditioning as discussed above .", "another reason for conflicting findings in fear conditioning versus evaluative conditioning studies could be the use of different dependent measures . fear conditioning studies primarily use skin conductance as a dependent measure , whereas evaluative conditioning studies rely on behavioural measures .", "this explanation is supported by recent findings from our lab revealing that changes of affective evaluations were not related to skin conductance responses but to heart rate response in dementia patients in a face - emotion association paradigm . accordingly", ", there might be a specific impairment in dementia patients with respect to skin conductance response .   on a neurophysiological level , the underpinnings of evaluative conditioning are not well understood .", "some studies indicate that temporal regions and specifically the amygdala are involved in both fear conditioning [ 3235 ] and evaluative conditioning .", "however , there is also evidence suggesting that the functionality of the amygdaloid nuclear complex may not be crucial for the occurrence of evaluative conditioning . hence , preserved evaluative conditioning in dementia patients would be in line with findings demonstrating that despite of impaired fear conditioning evaluative conditioning is preserved in persons with unilateral damage to the amygdaloid nuclear complex .", "similarly , tranel and damasio   report the case of a patient with bilateral damage to the entire medial lobe who could learn connections between unfamiliar persons and affective valence they displayed , despite severely impaired explicit memory .   in summary ,", "results of our study suggest that dementia patient ' affective evaluations of neutral stimuli can be changed through pairing with liked or disliked stimuli .", "however , caution is warranted since it is not unambiguous what caused these rating changes in our study .", "future research should focus on preserved learning processes in dementia patients since they are of great importance for nonpharmacological therapeutic interventions ."], "abstract_text": ["<S> we present results of a study investigating evaluative learning in dementia patients with a classic evaluative conditioning paradigm . </S>", "<S> picture pairs of three unfamiliar faces with liked , disliked , or neutral faces , that were rated prior to the presentation , were presented 10 times each to a group of dementia patients ( n = 15 ) and healthy controls ( n = 14 ) in random order . </S>", "<S> valence ratings of all faces were assessed before and after presentation . </S>", "<S> in contrast to controls , dementia patients changed their valence ratings of unfamiliar faces according to their pairing with either a liked or disliked face , although they were not able to explicitly assign the picture pairs after the presentation . </S>", "<S> our finding suggests preserved evaluative conditioning in dementia patients . </S>", "<S> however , the result has to be considered preliminary , as it is unclear which factors prevented the predicted rating changes in the expected direction in the control group . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Design and Methods", "3. Results", "4. Discussion"], "sections": [["the majority of our likes and dislikes we acquire throughout the lifespan are the product of learning .", "one of the most important ways through which stimuli acquire affective meaning is the change of valence that results from pairing one stimulus ( cs ) with a positive or negative affective stimulus ( ucs ) . as a result", "this effect is called ( associative ) evaluative conditioning and has been demonstrated in humans with a large variety of procedures and stimuli ( e.g. , [ 24 ] ; for a meta - analysis see ) .   in dementia severely impaired explicit memory", "nonetheless , patients with dementia might still be able to implicitly learn affective reactions through the process of evaluative conditioning .", "however , to the best of our knowledge , no study applied this approach in dementia patients .", "another classical paradigm that has already been used to investigate conditioning of affective reactions in this population is fear conditioning .", "indeed , two studies indicate that fear conditioning is impaired in dementia patients [ 6 , 7 ] .", "even though fear conditioning is impaired , evaluative conditioning might still be possible in dementia patients .", "some researchers have argued that while on a procedural level evaluative conditioning is similar to fear conditioning , the underlying processes might be different .", "it was hypothesized that fear conditioning is an instance of signal learning ; it is learned that the ucs is going to appear after the presentation of the cs .", "evaluative conditioning , on the other hand , only involves a reference to the ucs without expectation of its occurrence .", "thus , explicit knowledge about the cs - ucs relation seems crucial in fear conditioning while evaluative conditioning can be demonstrated in the absence of contingency awareness [ 1012 ] . since there could be variables that play a different role in these forms of learning ; dementia patients might show intact evaluative conditioning despite impaired fear conditioning .   indeed ,", "some studies indicate that dementia patients might retain the capacity to acquire affective reactions [ 13 , 14 ] .", "blessing et al .   demonstrated that dementia patients ' affective reactions can be influenced by pairing faces with fictional biographical content that characterized the depicted persons in terms of either positive or negative traits .", "pictures were rated before and at two different time points after the presentation of fictional biographical content with respect to valence and arousal .", "patients changed their ratings of pictures according to the biographical information presented , but did not recognize pictures above chance level or recall biographical information .", "these findings were replicated and extended in a subsequent study . the paradigm used by blessing et al . [", "however , there are two main differences : ( 1 ) in standard evaluative conditioning paradigms the subjects are not explicitly informed about the interrelation between stimuli in the learning phase . in the paradigm used by blessing et al .", "[ 13 , 14 ] the paring of the cs and ucs is made explicit and thus , the procedure can not be described as a simple cooccurrence of stimuli ; ( 2 ) in contrast to the approach of blessing et al .", "[ 13 , 14 ] the ucs and cs in evaluative conditioning paradigms belong to the same type of stimuli ( e.g. , faces ) .", "hence , in the present study we addressed the question if affective evaluations of dementia patients can be manipulated using a standard evaluative conditioning paradigm ."], ["\n participants . demographical data and clinical characteristics of both groups are listed in table 1 .", "the study included 15 dementia patients ( diagnosed as alzheimer 's disease ( n = 10 ) or mixed dementia ( n = 5 ) ) .", "patients were outpatients in the memory clinic of the psychiatric clinic of muensterlingen at the time of testing .", "all patients were diagnosed by a multidisciplinary team of the hospital ward using icd 10 criteria .", "all patients had received medical attendance including magnetic resonance imaging and specific screening blood tests , in order to exclude syphilis , diabetes , thyroid disorders , and vitamin b12 and folic acid deficiency .  ", "they reported that they had no known cns diseases , contact with toxic substances , or substance abuse .", "test stimuli were 10 pictures of neutral ( i.e. , displaying no facial expression of emotion ) unfamiliar faces ( 6 female : 3 young , 3 old ; 4 male : 2 young , 2 old ) and one picture of a happy young , female adult as well as one picture of a happy old , male adult selected from the productive aging laboratory face database . however , based on affective valence ratings of these pictures in other studies ( dann hier referenz ) , some of the included faces were also rated as positive or negative .", "the happy faces were added to increase the variance in subjective liking between pictures . \n emotional ratings .", "the sam was designed to assess subjective ratings of participants ' emotional responses and minimize the influence of language and culture on ratings .", "the sam valence rating scale has been successfully used in previous studies with dementia patients [ 13 , 14 ] . using the paper - pencil version of this instrument , participants rated the stimuli as to their emotional valence ( range : 19 ) .  ", "similar to other studies investigating evaluative conditioning we used a cover story in order to minimize demand effects .", "participants were told that the aim of the experiment was to examine the relationship between mood and subjective affective evaluation . in line with this cover", "story participants rated their mood on a five - point likert scale ( very good / good / normal / bad / very bad ) .", "subsequently , the pictures were presented to participants one after the other and rated with respect to valence using the sam rating scale .", "pictures were presented in four different pseudorandom sequences . after the valence rating an individual , unequivocal preference order of all stimuli was established .", "the pictures that received identical valence ratings were again presented to participants who then had to decide which of the pictures they preferred and the respective picture was put aside .", "again , the participants had to choose which of the remaining pictures with identical valence ratings they preferred and so on .  ", "the most preferred stimulus out of the 12 faces was used as the liked ( l ) stimulus and the stimulus with the lowest ranking was used as the disliked ( d ) stimulus .", "the four stimuli ranked 5th , 6th , 7th , and 8th were used as neutral ( n ) stimuli .", "the experimenter entered these six individual l , d , and n stimuli in a computer program ( presentations 11.3 ) that automatically formed three stimuli pairs ( i.e. , neutral - liked , neutral - disliked , and neutral - neutral ) by randomly assigning the neutral stimuli .", "participants were then seated about 50  cm from the computer screen and instructed to look at the pictures that would appear on the screen .", "each picture from the different pairs ( i.e. , ( n - l ) , ( n - d ) or (", "n - n ) ) was presented 10 times in the center of the computer screen .", "the duration of each stimulus presentation was 1 second and the interstimulus interval ( isi ; i.e. , onset of the first stimulus of a pair to onset of the second stimulus of a pair ) was 4 seconds .", "the inter - trial interval ( iti ; i.e. , onset of the first stimulus of the previous trial to onset of the first stimulus of the next trial ) was 13 seconds .", "after the presentation of stimuli on the computer screen , participants were again asked to rate the n , d , and l stimuli on the sam valence rating scale .", "the three relevant n stimuli ( i.e. , the first stimulus of each pair ) were placed one after the other in front of participants together with the three stimuli second in the pairs ( l , d , n ) .", "participants were asked to indicate which of these three stimuli followed the currently presented n stimulus .", "the experimenter registered the answer on a response sheet .   a repeated measures analyses of variance ( anovas )", "was conducted for the valence ratings of the relevant n stimuli ( i.e. , the first stimulus of each pair ) .", "a separate analysis was performed for ratings of stimuli paired with l and d pictures .", "type of stimulus pair and measurement point ( i.e. , valence rating pre and post presentation ) were used as within - participant factors and group was included as between subject factor . in the case of significant group differences a separate analysis was performed for both groups ."], ["the repeated measures anova revealed no main effect of type of stimulus pair ( f(2,26 ) = 2.42 ; p > .109 ) but an interaction between type of stimulus pair and time ( f(2,26 ) = 7.354 ; p > .003 ; see table 2 ) .", "this interaction was due to the fact that there was no influence of the type of stimulus pair at baseline ( f(2,27 ) = 0.296 ; p > .746 ) , but a significant effect after conditioning in the direction of the experimental manipulation ( f(2,27 ) = 5.460 ; p < .010 ) .", "no interaction between type of stimulus pair and group ( f(2,26 ) = 3.023 ; p > .066 ) or stimulus pair , time , and group ( f(2,26 ) = 2.798 ; p > .079 ) appeared .", "anova indicated no main effect of time ( f(1,27 ) = 0.78 ; p > .385 ) .  ", "as expected , we found a grater rating change over time for stimuli paired with l and d pictures than stimuli paired with n pictures ( see table 2 ) .", "we conducted a separate analysis using only pictures paired with l and d pictures to further investigate the influence of the experimental manipulation .", "the repeated measures anova revealed a main effect of type of stimulus pair ( f(1,27 ) = 4962 ; p > .034 ) along with an interaction between type of stimulus pair and time ( f(1,26 ) = 15.237 ; p > .001 ; see table 2 ) .", "we found no significant interaction between type of stimulus pair and group ( f(2,26 ) = 2.894 ; p > .10 ) but a significant interactions between stimulus pair , time , and group ( f(1,26 ) = 5.784 ; p < .023 ) .", "anova indicated no main effect of time ( f(1,27 ) = 0.296 ; p > .591 ) .  ", "because of the significant interaction between stimulus pair , time , and group , indicating group differences , we performed a separate analysis for each group .", "the repeated measures anova for valence ratings of pictures paired with l and d stimuli of dementia patients revealed a significant main effect of type of stimulus pair ( f(1,14 ) = 16.000 ; p >", ".001 ) along with an interaction between type of stimulus pair and time ( f(1,14 ) = 29.007 ; p > .001 ) .", "we found no main effect of time ( f(1,14 ) = 0.121 ; p > .733 ) .  ", "the repeated measures anova for ratings of controls indicated no main effect of type of stimulus pair ( f(1,13 ) = .087 ; p > .773 ) and no interaction between type of stimulus pair and time ( f(1,13 ) = .832 ; p > .378 ) .", "again , anova indicated no main effect of time ( f(1,13 ) = 0.188 ; p > .671 ) .   in the group of dementia patients , 24.4% of the relevant n stimuli", "were assigned to the correct stimulus second in the pairs in the forced choice test , which did not differ from chance level ( i.e. , 33.3% ; p > .14 ) . in the control group ,", "31% relevant n stimuli were assigned to the correct stimulus second in the pairs in the forced choice test , which did not differ from chance level ( i.e. , 33.3% ; p > .37 ) ."], ["the main aim of the present study was to investigate if affective evaluations of dementia patients can specifically be influenced through a standard evaluative conditioning paradigm .", "as hypothesized , dementia patients changed their valence ratings of unfamiliar and previously neutral faces according to its pairing with either a liked or disliked face stimulus . generally , the neutral pictures that were paired with a liked stimulus were rated higher and the neutral picture that was paired with the disliked stimulus was rated lower on the valence dimension after our evaluative conditioning intervention by dementia patients .", "thus , results indicate that ratings of initially neutral stimuli can be influenced in the according direction through simple time - near presentation ( i.e. , pairing ) with both a liked as well as a disliked stimuli", ". it does not seem surprising that our forced choice recognition test indicated no contingency awareness as this is based on the functionality of explicit memory that is severely impaired in dementia patients .   however , dementia patients significantly differed from controls in the effect of the experimental manipulation .", "the negative finding in the control group could indicate that our paradigm did not produce evaluative conditioning effects .", "consequently , the detected influence in the ad group could be accounted for by demand effects or other nonevaluative conditioning effects .", "another explanation could be that there was only a small effect in the control group that could not be detected due to small sample size .", "however , the sample size was only slightly smaller than that of the ad group .", "we looked at the data on the level of individual subjects and found that 11 of 14 subjects in the control group changed their ratings in the expected direction or showed no rating change .", "the negative result in the control group was due to two participants who showed a strong rating change in the opposite direction .", "post hoc analysis indicated a significant influence of the experimental manipulation in the control group after exclusion of these two outliers ( p < .028 ) .", "thus , it seems possible that evaluative conditioning effects in the control group were masked by the influence of outliers in our small sample .", "the rating changes of outliers could have been motivated by reactance , as observed in other studies investigating evaluative conditioning .", "our result could also indicate that evaluative conditioning effects are stronger in dementia patients than in healthy elderly controls .", "a possible reason for stronger effects could be lower attentional resources in dementia patients due to disease - related cognitive impairments .", "some studies indicate that attentional resources have a negative impact on affective learning through evaluative conditioning ( e.g. [ 19 , 20 ] ) and accordingly it seems possible that dementia patients show stronger effects . however , there are conflicting findings concerning the influence of attentional resources on evaluative conditioning ( e.g. , )", ".   the observed positive change of the valence rating in the neutral stimulus that was paired with another neutral stimulus could be a result of the mere exposure effect .", "this effect describes the preference for stimuli that have been previously presented over novel stimuli .", "preference changes due to the mere exposure effect have also been demonstrated in dementia patients [ 2325 ] . in line with this explanation", "are findings from other studies investigating evaluative conditioning that report a similar trend of positive changes in evaluations of neutral pictures paired with other neutral pictures over time .  ", "the results suggest that dementia patients changed their ratings according to the experimental manipulation without contingency awareness , since pairings were not identified above chance level in the forced choice recognition test .", "in fact , participants of the control group performed better than dementia patients in the recognition test , yet their results did not differ from chance level as well .", "the results of dementia patients are in line with findings demonstrating evaluative conditioning in healthy participants using subliminally presented stimuli [ 2729 ] .", "could show that the individual amount of the evaluative conditioning effect is not related to the number of pairings ( i.e. , neutral stimulus - affective stimulus ) participants were aware of .", "on the other hand , findings of a recent meta analysis suggest that contingency awareness is an important moderator in evaluative conditioning .   however , a critical limitation of the present study is our assessment of contingency awareness using a forced choice recognition test .", "following the recent discussion of gawronski and walther , it seems possible that we measured contingency memory rather than contingency awareness .", "subjects may have realised the respective pairings during conditioning but may not have been able to remember these pairings explicitly when the recognition test was applied .", "this may have been the case especially in dementia patients because of severe memory deficits .", "subjects may have remembered statistical probabilities that could not be assessed using a forced choice test .", "thus , it is also possible that contingency awareness differed between dementia patients and controls in our study and that contingency awareness influenced the results . to prohibit contingency awareness , very long time intervals on", "short isi could facilitate the detection of contingencies in the combinations of pictures and would enhance contingency awareness .  ", "however , this result has to be considered preliminary , since it is unclear what prevented rating changes in the expected direction in the control group in our paradigm .", "nevertheless , the results seem to be in contrast to previously reported impaired fear conditioning . as discussed above it", "is still unresolved whether evaluative conditioning is a form of pavlovian conditioning or if there are variables that are unique to evaluative conditioning .", "thus , the results of our study support the notion that different processes are involved in evaluative conditioning and fear conditioning .", "a relevant difference could be the dependence of pavlovian conditioning on contingency awareness in contrast to evaluative conditioning as discussed above .", "another reason for conflicting findings in fear conditioning versus evaluative conditioning studies could be the use of different dependent measures . fear conditioning studies primarily use skin conductance as a dependent measure , whereas evaluative conditioning studies rely on behavioural measures .", "this explanation is supported by recent findings from our lab revealing that changes of affective evaluations were not related to skin conductance responses but to heart rate response in dementia patients in a face - emotion association paradigm . accordingly", ", there might be a specific impairment in dementia patients with respect to skin conductance response .   on a neurophysiological level , the underpinnings of evaluative conditioning are not well understood .", "some studies indicate that temporal regions and specifically the amygdala are involved in both fear conditioning [ 3235 ] and evaluative conditioning .", "however , there is also evidence suggesting that the functionality of the amygdaloid nuclear complex may not be crucial for the occurrence of evaluative conditioning . hence , preserved evaluative conditioning in dementia patients would be in line with findings demonstrating that despite of impaired fear conditioning evaluative conditioning is preserved in persons with unilateral damage to the amygdaloid nuclear complex .", "similarly , tranel and damasio   report the case of a patient with bilateral damage to the entire medial lobe who could learn connections between unfamiliar persons and affective valence they displayed , despite severely impaired explicit memory .   in summary ,", "results of our study suggest that dementia patient ' affective evaluations of neutral stimuli can be changed through pairing with liked or disliked stimuli .", "however , caution is warranted since it is not unambiguous what caused these rating changes in our study .", "future research should focus on preserved learning processes in dementia patients since they are of great importance for nonpharmacological therapeutic interventions ."]]}
{"article_id": "PMC3825974", "article_text": ["para - aortic and pelvic lymphadenectomy is often performed in the treatment of ovarian or testicular cancer .", "internal herniation of a small bowel behind the external iliac artery after lymphadenectomy is a very rare complication to this procedure and to our knowledge only reported twice in the literature . in the first paper published in 1978", "the author described how they managed their patient with laparotomy and resection of a perforated small bowel .", "the hernia orifice was closed by using a free peritoneal graft harvested from the under surface of the anterior abdominal wall .", "this patient had to undergo several surgical procedures to re - establish blood flow to the extremity because of trombosis of the iliac artery .", "as there are few similar cases reported worldwide , there is no consensus or guidelines available .", "we did not close the orifice . because of the skeletonized vessel we were afraid of compromising the vessel if we tried to close the defect . at follow - up after 10 months", "a 56-year - old woman underwent in 2008 prophylactic bilateral laparoscopic salpingoophrectomy because of a mutation in brca1 gene .", "the histological result revealed a serous papillary adenocarcinoma in the right ovary and on its surface . therefore a restaging operation was performed .", "she underwent laparotomy with total abdominal hysterectomy , omentectomy , appendectomy , with a radical retroperitoneal lymphadenectomy which involved  en bloc  dissection and removal of the para - aortic and iliac lymphe nodes .", "there was no metastasis in the biopsies taken from the rectum wall and the laparoscopy trocar ports as well as the 46 harvested lymphe nodes .", "the patient did not receive any radiation therapy . in 2012 , 4 years later , she was admitted to hospital with a 2 days history of severe abdominal pain , vomiting and the inability to pass gas or stools for the last few days .", "the abdomen was distended , diffused tender and tympanitic , without guarding or rebound tenderness .", "her groin examination was normal without any sign of herniation through the femoral or inguinal canals .", "blood pressure 140/90  mmhg , pulse 71 beats / minute and a temperature of 37.9  c .", "our patient received 90  ml of iomeprol 350  mgl / ml i.v . and this was sufficient to see the vessels .", "the ct scan demonstrated transition zone both where the loop entered behind the external iliac vessel and where the intestine passed out behind the vessel as a closed loop .", "the small bowel was dilated both proximal to , as well as inside the closed loop ( figs . 2 and 3 ) .", "the patient received a prophylactic antibiotics composed of metronidazole 1.5  g and doxycycline 400  mg i.v .", "we established pneumoperitoneum with a pressure of 12  mmhg and inserted two other working ports of 12  mm and one of 5  mm .", "we examined the non dilated bowel from the ileocoecal junction , approximately 200  cm from the ilecoecal junction the small bowel made a closed loop underneath the left external iliac artery ( picture 1 ) .", "the small bowel was strangulated by the external iliac vessel . by gentle manipulation we reduced the herniated small bowel ( picture 2 ) that soon recovered .", "the blue color and venous congestion were fading out and we noticed peristaltic movement of the previously herniated bowel we therefore did not perform any bowel resection . the defect underneath the artery was about 23  cm ( picture 3 ) in diameter .", "we found it risky to try to close the orifice and decided to leave it unrepaired .", "we noticed pulsation on the iliac vessel after the small bowel reduction and we evaluated the pulsation of the dorsalis pedis artery and the posterior tibial artery during the operation .", "the abdomen was exsufflated and the fascia defects in the 12  mm ports were sutured and the skin closed with stapler .", "the patient was observed in the intensive care unit until the next morning , with focus on her leg pulse and color .", "dalteparin 5000 ie was given subcutaneously 6  h after surgery and continued for the first 24  h after surgery .", "the postoperative course was uneventful and she was discharged from hospital the day following operation .", "due to limited peritoneum in the area of the hernia defect and the skeletonized external iliac artery , we did not do any direct suture of the orifice as this might have compromised the artery and/or the vein . due to previous omentectomy ,", "an alternative might have been to harvest a free peritoneum graft to cover the defect as described in the two other cases .", "another option was to use a mesh covered with an oxidized regenerated cellulose or expanded polytetrafluoroethylene ( eptfe ) that minimize bowel adhesions .", "a longer follow - up of the patient is needed to clearly conclude if this simple procedure has been sufficient .", "if the patient later experience any sign of recurrence and need another operation we will have to close the defect .", "a challenge would have been if the bowel could not be reduced due to long standing obstruction .", "in such a case a bowel resection and/or a vascular surgical procedure might have been necessary .", "the small bowel in ileus might be fragile , gangrenous or necrotic and there is probably a higher risk of bowel perforation performing a laparoscopic approach .", "we do not routinely use verres needle in laparoscopy , we prefer to use visiport as an optical trocar or an open hasson approach that is another safe alternative to minimize the chance of bowel perforation on entrance into the abdominal cavity .", "strangulated internal hernia behind the iliac vessel is a rare entity only reported twice in the literature .", "this is the first case in the literature where laparoscopically reduction of the internal herniated bowel underneath the iliac vessel is performed .", "a quick recovery with no complications , short hospital stay and minimal discomfort postoperatively made our approach an acceptable surgical option for this patient .", "written informed consent was obtained from the patient for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request ."], "abstract_text": ["<S> introductioninternal herniation of a small bowel behind pelvic vessels is a rare complication seen after pelvic lymphadenectomy.presentation of casea 56-year - old woman was operated due to a gynecological cancer . </S>", "<S> 4 years thereafter she presented with a 2 days history of abdominal pain and vomiting . </S>", "<S> clinical and radiological findings indicated a small bowel obstruction . </S>", "<S> a loop of small bowel had herniated behind the left external iliac artery . using laparoscopic technique </S>", "<S> the herniated bowel was reduced . due to limited peritoneum around the area and skeletonized vessel , we decided not to do any repair of the hernia orifice </S>", "<S> . the postoperative recovery was uneventful , bowel activity returned to normal and she was discharged the next day.follow-up was done at 1 month and the latest at 10 months . </S>", "<S> she did nt experience pain or discomfort after the operation.discussiondue to limited peritoneum around the skeletonized vessel , we decided to leave the hernia orifice unrepaired . </S>", "<S> we found it hazardous to do any direct suture of the orifice or use a free peritoneal graft to repair the defect as the fibrosis and inflammatory process might have compromised the artery or the vein . </S>", "<S> a longer follow - up of the patient is needed to clearly conclude if this simple procedure has been sufficient . </S>", "<S> we agreed that if the patient would experience any sign of recurrence and need another operation we would close the defect at that time.conclusion4 years after pelvic lymphadenectomy a small bowel herniation behind an external iliac artery occurred . </S>", "<S> the patient was successfully treated with reduction of the small bowel using laparoscopic technique . a quick recovery with minimal discomfort and no sign of recurrence after 10 months made our approach an acceptable surgical option . </S>"], "labels": null, "section_names": ["Introduction", "Presentation of case", "Discussion", "Conclusion", "Conflicts of interest", "Funding", "Ethical approval statement"], "sections": [["para - aortic and pelvic lymphadenectomy is often performed in the treatment of ovarian or testicular cancer .", "internal herniation of a small bowel behind the external iliac artery after lymphadenectomy is a very rare complication to this procedure and to our knowledge only reported twice in the literature . in the first paper published in 1978", "the author described how they managed their patient with laparotomy and resection of a perforated small bowel .", "the hernia orifice was closed by using a free peritoneal graft harvested from the under surface of the anterior abdominal wall .", "this patient had to undergo several surgical procedures to re - establish blood flow to the extremity because of trombosis of the iliac artery .", "as there are few similar cases reported worldwide , there is no consensus or guidelines available .", "we did not close the orifice . because of the skeletonized vessel we were afraid of compromising the vessel if we tried to close the defect . at follow - up after 10 months"], ["a 56-year - old woman underwent in 2008 prophylactic bilateral laparoscopic salpingoophrectomy because of a mutation in brca1 gene .", "the histological result revealed a serous papillary adenocarcinoma in the right ovary and on its surface . therefore a restaging operation was performed .", "she underwent laparotomy with total abdominal hysterectomy , omentectomy , appendectomy , with a radical retroperitoneal lymphadenectomy which involved  en bloc  dissection and removal of the para - aortic and iliac lymphe nodes .", "there was no metastasis in the biopsies taken from the rectum wall and the laparoscopy trocar ports as well as the 46 harvested lymphe nodes .", "the patient did not receive any radiation therapy . in 2012 , 4 years later , she was admitted to hospital with a 2 days history of severe abdominal pain , vomiting and the inability to pass gas or stools for the last few days .", "the abdomen was distended , diffused tender and tympanitic , without guarding or rebound tenderness .", "her groin examination was normal without any sign of herniation through the femoral or inguinal canals .", "blood pressure 140/90  mmhg , pulse 71 beats / minute and a temperature of 37.9  c .", "our patient received 90  ml of iomeprol 350  mgl / ml i.v . and this was sufficient to see the vessels .", "the ct scan demonstrated transition zone both where the loop entered behind the external iliac vessel and where the intestine passed out behind the vessel as a closed loop .", "the small bowel was dilated both proximal to , as well as inside the closed loop ( figs . 2 and 3 ) .", "the patient received a prophylactic antibiotics composed of metronidazole 1.5  g and doxycycline 400  mg i.v .", "we established pneumoperitoneum with a pressure of 12  mmhg and inserted two other working ports of 12  mm and one of 5  mm .", "we examined the non dilated bowel from the ileocoecal junction , approximately 200  cm from the ilecoecal junction the small bowel made a closed loop underneath the left external iliac artery ( picture 1 ) .", "the small bowel was strangulated by the external iliac vessel . by gentle manipulation we reduced the herniated small bowel ( picture 2 ) that soon recovered .", "the blue color and venous congestion were fading out and we noticed peristaltic movement of the previously herniated bowel we therefore did not perform any bowel resection . the defect underneath the artery was about 23  cm ( picture 3 ) in diameter .", "we found it risky to try to close the orifice and decided to leave it unrepaired .", "we noticed pulsation on the iliac vessel after the small bowel reduction and we evaluated the pulsation of the dorsalis pedis artery and the posterior tibial artery during the operation .", "the abdomen was exsufflated and the fascia defects in the 12  mm ports were sutured and the skin closed with stapler .", "the patient was observed in the intensive care unit until the next morning , with focus on her leg pulse and color .", "dalteparin 5000 ie was given subcutaneously 6  h after surgery and continued for the first 24  h after surgery .", "the postoperative course was uneventful and she was discharged from hospital the day following operation ."], ["due to limited peritoneum in the area of the hernia defect and the skeletonized external iliac artery , we did not do any direct suture of the orifice as this might have compromised the artery and/or the vein . due to previous omentectomy ,", "an alternative might have been to harvest a free peritoneum graft to cover the defect as described in the two other cases .", "another option was to use a mesh covered with an oxidized regenerated cellulose or expanded polytetrafluoroethylene ( eptfe ) that minimize bowel adhesions .", "a longer follow - up of the patient is needed to clearly conclude if this simple procedure has been sufficient .", "if the patient later experience any sign of recurrence and need another operation we will have to close the defect .", "a challenge would have been if the bowel could not be reduced due to long standing obstruction .", "in such a case a bowel resection and/or a vascular surgical procedure might have been necessary .", "the small bowel in ileus might be fragile , gangrenous or necrotic and there is probably a higher risk of bowel perforation performing a laparoscopic approach .", "we do not routinely use verres needle in laparoscopy , we prefer to use visiport as an optical trocar or an open hasson approach that is another safe alternative to minimize the chance of bowel perforation on entrance into the abdominal cavity ."], ["strangulated internal hernia behind the iliac vessel is a rare entity only reported twice in the literature .", "this is the first case in the literature where laparoscopically reduction of the internal herniated bowel underneath the iliac vessel is performed .", "a quick recovery with no complications , short hospital stay and minimal discomfort postoperatively made our approach an acceptable surgical option for this patient ."], [""], [""], ["written informed consent was obtained from the patient for publication of this case report and accompanying images .", "a copy of the written consent is available for review by the editor - in - chief of this journal on request ."]]}
{"article_id": "PMC3255473", "article_text": ["tumor - associated cell cycle defects , manifesting in unscheduled proliferation , and the associated genomic and chromosomal instabilities are mediated by misregulation of cyclin - dependent kinases ( cdks ) .", "because of the main role in the division cycle , cdks have been recognized as targets for anticancer therapy .", "many small - molecule organic compounds , which have been identified as cdk modulators , are currently in preclinical or clinical development . however , no cdk inhibitors have gained marketing approval , despite 20 years of scientific investigation .", "several studies have shown synergism when cdk inhibitors were combined with organic ( e.g. , doxorubicin , paclitaxel ) and inorganic ( e.g. , cisplatin , carboplatin ) cytotoxic drugs .", "the first publications have appeared recently and include fe , cu , and pt complexes with cdk inhibitors derived from 6-benzylaminopurine , metal - based indolo-[3,2-d]benzazepines ( paullones ; ga , cu , ru , and os ) , and indolo-[3,2-c]quinolines ( ru , os ) . another class of compounds potentially suitable for targeted metal - based chemotherapy is that of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines .", "these have been documented recently as potent cdk1 inhibitors with antiproliferative activity in hela ( cervical carcinoma ) , hct116 ( colon carcinoma ) , and a375 ( melanoma ) human cancer cell lines .", "comparison of cdk1 inhibitory activity with the inhibiting activity in four other protein kinases ( vegf - r2 , her2 , aurora - a , and ret ) revealed selectivity for cdk1 . structural modifications consisting of a replacement of both bicyclic rings in 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines by monocycles while retaining the imidazolyl pyrazole core have been proposed in order to obtain inhibitors with improved pharmacokinetic and solubility properties .", "the most promising were suggested to be 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( see chart 1 ) .", "the inspection of substitution patterns on the benzimidazole moiety and structure  activity relationships revealed that a methoxymethyl group in position 7b ( 4b ) is favorable for cdk1 inhibiting potency .", "the role of the pyrazole nh group is also of note , since its methylation led to a significant reduction of cdk1 activity .", "the effect of various heteroaryl groups in position 5a was also remarkable for the development of more - effective cdk inhibitors and antiproliferative agents .", "our previous experience with metal - based indolo-[3,2-d]benzazepines prompted the use of the half - sandwich metal - arene moiety as a suitable scaffold to which 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines may be attached .", "organometallic compounds [ m(-arene)(yz)x ] ( where m = ru , os ) exhibit promising anticancer activity and are the focus of attention for several groups .", "these compounds have shown activity toward classic ( dna ) and nonclassic ( e.g. , cdks ) targets in anticancer chemotherapy .", "herein , we report ( i ) the modified synthetic approach to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( recall chart 1 : l1 , x = h , y = h ; l2 , x = br ; y = h ; l3 , x = br ; y = ch2och3 ) ; ( ii ) the synthesis and characterization of a new family of organoruthenium(ii ) ( 11a , 12a , 13a ) and osmium(ii ) ( 11b , 12b , 13b ) complexes of the general formula [ mcl(-p - cymene)l]cl , where l = 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( chart 1 ) ; ( iii ) stabilization of the 7b tautomer of methoxymethyl - substituted l3 by metal coordination as well as ( iv ) nmr spectroscopic characterization of two tautomers 7b - l3 and 4b-l3 in a metal - free state ; and ( v ) cell cycle effects , as well as the antiproliferative and cdk inhibitory activities of both metal - free ligands and organometallic complexes .", "1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) was obtained via the oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base , followed by acidification with 37% hcl .", "solvents [ toluene , ethanol ( etoh ) , tetrahydrofuran ( thf ) , diethyl ether ( et2o ) ] were dried using standard procedures .", "3-methyl-1h - pyrazolo[3,4-b]pyridine ( 10.9 g , 0.08 mol ) and anhydrous sodium acetate ( 10.21 g , 0.13 mol ) were suspended in glacial acetic acid ( 42 ml ) .", "bromine ( 20.42 g , 0.13 mol ) was added , and the resulting mixture was stirred at room temperature for 22.5 h and then at 110115 c for 2.53 h. afterward , water ( 300350 ml ) was added and the mixture was stirred at room temperature .", "the formed light yellow precipitate was filtered off and dried in vacuo at 4050 c .", "yield : 17 g. the raw product was used without further purification in the next step .", "purification by column chromatography afforded a white powder ( sio2 , etoac , rf = 0.79 ; 12.5 g , 72.6% yield ) .", "esi - ms in meoh ( positive ) : m / z 213 [ m+h ] , 235 [ m+na ] , 253 [ m+k ] ; ( negative ) : m / z 211 [ m  h ] .", "h nmr ( 500.32 mhz , meoh - d4 ) :  8.55 ( d , 1h , j = 2.16 hz , ch ) , 8.43 ( d , 1h , j = 2.15 hz , ch ) , 2.56 ( s , 3h , ch3 ) ppm .", "h nmr ( 500.32 mhz , dmso - d6 ) :  13.42 ( brs , 1h , nh ) , 8.54 ( d , 1h , j = 2.19 hz , ch ) , 8.53 ( d , 1h , j = 2.18 hz , ch ) , 2.49 ( s , 3h , ch3 ) ppm .", "colorless crystals of 20.5h2o suitable for x - ray diffraction ( xrd ) study were grown in etoac ( see figure s1 in the supporting information ) . to sodium hydroxide ( 9.54 g , 0.24 mol ) in water ( 150 ml ) was added the raw product 2 ( 7.1 g , 0.03 mol ) . after a dropwise addition of kmno4 ( 16.98 g , 0.11 mol ) in water ( 300 ml ) at 100 c over 2 h , the reaction mixture was further heated for 1 h. mno2 was filtered off from the hot reaction mixture and washed with hot water .", "400 ml , and the yellow solution was acidified to ph 2 , using concentrated hcl .", "yield : 2.28 g. the crude hydrochloride of 3 was used without further purification in the next step .", "esi - ms in meoh ( positive ) : m / z 243 [ m+h ] , 265 [ m+na ] , 287 [ m+2na  h ] ; ( negative ) : m / z 241 [ m  h ] .", "h nmr ( 500.32 mhz , meoh - d4 ) :  8.69 ( d , 1h , j = 2.22 hz , ch ) , 8.68 ( d , 1h , j = 2.22 hz , ch ) ppm .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.65 ( s , 1h , nh ) , 13.45 ( brs , 1h , nh ) , 8.72 ( d , 1h , j = 2.23 hz , ch ) , 8.58 ( d , 1h , j = 2.25 hz , ch ) ppm .", "nah ( 1.75 g , 0.07 mol ) was suspended in dry thf ( 200 ml ) . a solution of 2-amino-3-nitrobenzyl alcohol ( 5.23 g , 0.03 mol ) in dry thf ( 100 ml )", "was added dropwise at 0 c and the mixture was stirred at the same temperature for 15 min . after a dropwise addition of mei ( 11.5 g , 0.08 mol ) ,", "stirring was continued at room temperature for 3 h. a saturated aqueous solution of nahco3 ( 300 ml ) and meoh ( 300 ml ) then were added .", "the organic phase was dried over na2so4 , filtered , and evaporated to yield a red oil ( 4.85 g ) .", "the raw product was purified by column chromatography ( sio2 , etoac , or etoac / hexane 1/1 , first fraction , a red - orange oil crystallized to form a red solid at 4 c ; yield : 3.68 g , 65% ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  8.00 ( d , 1h , j = 8.71 hz ,", "c6h3 ) , 7.09 ( br , 2h , nh2 ) , 6.67 ( t , 1h , j = 8.45 hz , c6h3 ) , 4.48 ( s , 2h , ch2 ) , 3.31 ( s , 3h , ch3 ) ppm .", "a mixture of 4 ( 1.4 g , 0.008 mol ) and 10% pd / c ( 0.18 g ) in dry etoh ( 55 ml ) was stirred under hydrogen atmosphere at room temperature for 1824 h. the catalyst was removed by filtration through gf-3-filter under argon and washed with dry etoh ( 5070 ml ) .", "the filtrate was evaporated in vacuo to give a light - orange solid ( 1.17 g , 100% yield ) , which was used immediately in the next step .", "h nmr ( 500.32 mhz , dmso - d6 ) :  6.52 ( dd , 1h , j = 1.87 hz , j = 7.25 , c6h3 ) , 6.426.36 ( m , 2h , c6h3 ) , 4.48 ( br , 2h , nh2 ) , 4.31 ( br , 4h , nh2+ch2 ) , 3.24 ( s , 3h , ch3 ) ppm .", "n , n-carbonyldiimidazole ( cdi , 4.16 g , 0.026 mol ) was added in small portions to 1 ( 2.34 g ) in dry dmf ( 12 ml ) .", "the mixture was stirred at room temperature for 2024 h. water ( 5 ml ) then was added and the suspension was stirred until all co2 was ceased .", "the white precipitate was filtered off , washed with water ( 35 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .", "esi - ms in methanol ( positive ) : m / z 214 [ m+h ] , 237 [ m+na ] ; ( negative ) : m / z 212 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.95 ( brs , 1h , nh ) , 8.91 ( s , 1h , chim ) , 8.74 ( dd , 1h , j = 1.61 hz , j = 4.51 hz , chpy ) , 8.63 ( dd , 1h , j = 1.59 hz , j = 8.12 hz , chpy ) , 8.14 ( t , 1h , j = 1.23 hz , chim ) , 7.51 ( dd , 1h , j = 4.46 hz , j = 8.06 hz , chpy ) , 7.20 ( s , 1h , chim ) ppm .", "n , n-carbonyldiimidazole ( cdi , 16.2 g , 0.1 mol ) was added in small portions to crude 3 ( 11.58 g ) in dry dmf ( 70 ml ) .", "the mixture was stirred at room temperature for 2024 h. water ( 10 ml ) then was added and the suspension was stirred until all co2 was ceased .", "the white precipitate was filtered off , washed with water ( 1015 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .", "esi - ms in methanol ( positive ) : m / z 315 [ m+na ] ; ( negative ) : m / z 291 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.95 ( brs , 1h , nh ) , 8.89 ( s , 1h , chim ) , 8.83 ( d , 1h , j = 2.12 hz , chpy ) , 8.75 ( d , 1h , j = 2.07 hz , chpy ) , 8.13 ( s , 1h , chim ) , 7.20 ( s , 1h , chim ) ppm .", "the solution of 1,2-diaminobenzene ( 0.5 g , 4.67 mmol ) in dry dmf ( 2 ml ) was added to the suspension of 6 ( 0.94 g , 4.41 mmol ) in dry dmf ( 13 ml ) , and this reaction mixture was heated under argon at 85 c for 5 h. dmf then was evaporated in vacuo at 50 c and water ( 1012 ml ) was added .", "the light - yellow precipitate was filtered off and dried in vacuo at 50 c .", "yield : 0.86 g. the raw product was used without purification in the next step .", "esi - ms in methanol ( positive ) : m / z 255 [ m+h ] , 277 [ m+na ] ; ( negative ) : m / z 253 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.30 ( brs , 1h , nhpz ) , 9.73 ( brs , 1h , conh ) , 8.64 ( dd , 1h , j = 1.59 hz , j = 4.44 hz , chpy ) , 8.56 ( dd , 1h , j = 1.53 hz , j = 8.02 hz , chpy ) , 7.397.36 ( m , 2h , chpy+chbz ) , 6.97 ( td , 1h , j = 1.31 hz , j = 7.73 hz , chbz ) , 6.82 ( dd , 1h , j = 1.2 hz , j = 7.89 hz , chbz ) , 6.64 ( td , 1h , j = 1.2 hz , j = 7.67 hz , chbz ) , 4.93 ( s , 2h , nh2 ) ppm .", "( 10 ml ) was added to the suspension of 7 ( 2.28 g , 7.8 mmol ) in dry dmf ( 10 ml ) , and this mixture was heated under argon at 85 c for 7 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .", "yield : 2.2 g. the raw product was used without purification in the next step .", "esi - ms in methanol ( positive ) : m / z 333 [ m+h ] , 355 [ m+na ] ; ( negative ) : m / z 331 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.54 ( brs , 1h , nhpz ) , 9.80 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.26 hz , chpy ) , 8.69 ( d , 1h , j = 2.22 hz , chpy ) , 7.34 ( dd , 1h , j = 0.98 hz , j = 7.88 hz , chbz ) , 6.99 ( td , 1h , j = 1.43 hz , j = 8.03 hz , chbz ) , 6.82 ( dd , 1h , j = 1.22 hz , j = 7.96 hz , chbz ) , 6.64 ( td , 1h , j = 1.19 hz , j = 7.73 hz , chbz ) , 4.94 ( s , 2h , nh2 ) ppm .", "a mixture of 5 ( 1.17 g , 7.69 mmol ) and 7 ( 2.1 g , 7.2 mmol ) in dry dmf ( 45 ml ) was heated under argon at 85 c for 20 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .", "yield : 2.3 g. the product was used without further purification in the next step .", "esi - ms in methanol ( positive ) : m / z 399 [ m+na ] ; ( negative ) : m / z 375 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.54 ( brs , 1h , nhpz ) , 9.85 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.13 hz , chpy ) , 8.68 ( d , 1h , j = 2.05 hz , chpy ) , 7.29 ( d , 1h , j = 7.56 hz , chbz ) , 7.03 ( d , 1h , j = 7.29 hz , chbz ) , 6.65 ( t , 1h , j = 7.79 hz , chbz ) , 4.78 ( brs , 2h , nh2 ) , 4.42 ( s , 2h , ch2 ) , 3.29 ( s , 3h , ch3 ) ppm . the raw product 8 ( 0.86 g ) was heated in a glacial acetic acid ( 10 ml ) at 125 c for 2.5 h. the solvent was evaporated under reduced pressure and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 4/1 , 25 ml ) , filtered off , and dried in vacuo to give l1 as a white powder ( 0.4 g ) .", "the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac , rf = 0.58 ) to give an additional amount of l1 ( 0.22 g ) .", "calcd for 110.15h2o0.1etoac ( mr = 246.76 g / mol ) : c , 65.22 ; h , 4.13 ; n , 28.38 . found : c , 65.56 ; h , 3.90 ; n , 28.39 .", "esi - ms in methanol ( positive ) : m / z 237 [ m+h ] , 259 [ m+na ] ; ( negative ) : m / z 235 [ m  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 232 ( 25618 ) , 275 ( 15198 ) , 324 ( 22333 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.19 ( brs , 1h , h1a ) , 13.11 ( brs , 1h , h1b ) , 8.85 ( dd , 1h , j = 1.5 hz ,", "j = 8.1 hz , h4a ) , 8.66 ( dd , 1h , j = 1.5 hz , j = 4.5 hz , h6a ) , 7.75 ( d , 1h , j", "= 7.3 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.7 hz , h4b or h7b ) , 7.39 ( dd , 1h , j = 4.5 hz , j = 8.0 hz , h5a ) , 7.24 ( m , 2h , h5b+h6b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  153.13 ( c8a ) , 150.22 ( c6a ) , 146.99 ( c2b ) , 144.34 ( c8b or c9b ) , 136.06 ( c3a ) , 134.68 ( c8b or c9b ) , 131.82 ( c4a ) , 123.35 ( c5b or c6b ) , 122.11 ( c5b or c6b ) , 119.44 ( c4b or c7b ) , 118.65 ( c5a ) , 113.33 ( c9a ) , 112.01 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) :  166.2 ( n1a ) , 121.3 ( n1b ) ppm . the raw product 9 ( 2.2 g ) was heated in a glacial acetic acid ( 30 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 7/1 , 50 ml ) , filtered off and dried in vacuo to give l2 as a white powder ( 1.15 g ) .", "the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac / hexane 2/1 , rf = 0.6 ) to give an additional amount of the product ( 0.27 g ) . yield : 1.42 g , 58% based on 7 .", "calcd for 120.25h2o0.04etoac ( mr = 322.17 g / mol ) : c , 49.06 ; h , 2.76 ; n , 21.74 .", "esi - ms in methanol ( positive ) : m / z 315 [ m+h ] , 337 [ m+na ] ; ( negative ) : m / z 313 [ m  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 234 ( 28154 ) , 282 ( 17628 ) , 335 ( 17198 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.43 ( brs , 1h , h1a ) , 13.19 ( brs , 1h , h1b ) , 8.99 ( d , 1h , j = 2.2 hz , h4a ) , 8.75 ( d , 1h , j = 2.3 hz , h6a ) , 7.77 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.26 ( m , 2h , h5b+h6b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  151.49 ( c8a ) , 150.66 ( c6a ) , 146.42 ( c2b ) , 144.24 ( c8b or c9b ) , 135.61 ( c3a ) , 134.67 ( c8b or c9b ) , 133.35 ( c4a ) , 123.54 ( c5b or c6b ) , 122.27 ( c5b or c6b ) , 119.55 ( c4b or c7b ) , 114.87 ( c5a or c9a ) , 113.49 ( c5a or c9a ) , 112.09 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) :  167.5 ( n1a ) , 121.3 ( n1b ) ppm .", "the raw product 10 ( 2.3 g ) was heated in a glacial acetic acid ( 40 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue dried in vacuo at 50 c . after washing with ch2cl2 ( 30 ml ) , ch2cl2/meoh ( 2/1 , 57 ml ) the gray product was purified by column chromatography ( sio2 , etoac , rf = 0.68 ) to give a white powder ( 0.9 g ) . the filtrates were evaporated and the remaining solid was purified by column chromatography to give an additional amount of the product ( 0.3 g ) .", "calcd for 13 : c , 50.29 ; h , 3.38 ; n , 19.55 . found : c , 50.03 ; h , 3.19 ; n , 19.19 .", "esi - ms in methanol ( positive ) : m / z 381 [ m+na ] ; ( negative ) : m / z 357 [ m  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 235 ( 29776 ) , 282 ( 17544 ) , 337 ( 16958 ) .", "nmr characterization of 7b - l3 and 4b-l3 tautomers ( 1/1.3 ) in dmso - d6 : h nmr ( 500.32 mhz , dmso - d6 ) , 7b - l3 :  14.47 ( brs , 1h , h1a ) , 13.25 ( brs , 1h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.72 ( dd , 1h , j = 1.8 hz ,", "j = 6.8 hz , h4b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.80 ( s , 2h , h10b ) , 3.37 ( s , 3h , h11b ) ppm .", "h nmr ( 500.32 mhz , dmso - d6 ) , 4b-l3 :  14.43 ( brs , 1.3h , h1a ) , 13.22 ( brs , 1.3h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.47 ( dd , 1.3h , j = 1.2 hz ,", "j = 7.7 hz , h7b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.96 ( s , 2.6h , h10b ) , 3.44 ( s , 3.9h , h11b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) , 7b - l3 :  151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.70 ( c2b ) , 144.46 ( c9b ) , 135.62 ( c3a+c3a ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 133.29 ( c8b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.95 ( c7b ) , 122.16 ( c5b or c6b ) , 118.97 ( c4b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 70.49 ( c10b ) , 57.95 ( c11b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) , 4b-l3 :  151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.13 ( c2b ) , 142.50 ( c9b ) , 135.62 ( c3a+c3a ) , 134.42 ( c8b ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 129.38 ( c4b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.16 ( c5b or c6b ) , 120.81 ( c5b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 111.17 ( c7b ) , 69.90 ( c10b ) , 58.35 ( c11b ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  167.6 ( n1a+n1a ) , 121.4 ( n1b+n1b ) ppm .", "a mixture of l1 ( 54.7 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was evaporated up to ca .", "calcd for 11ah2o ( mr = 559.45 g / mol ) : c , 49.38 ; h , 4.50 ; n , 12.52 ; cl , 12.67 .", "found : c , 49.68 ; h , 4.25 ; n , 12.11 ; cl , 12.26 .", "esi - ms in methanol ( positive ) : m / z 470 [ m  hcl  cl ] , 507 [ m  cl ] , 528 [ m  hcl+na ] ; ( negative ) : m / z 468 [ m2hcl  h ] , 505 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 251 ( 16335 ) , 299 ( 26663 ) , sh 347 ( 16012 ) .", "vis ( h2o ) , max , nm (  , m cm ) : sh 245 ( 10728 ) , 294 ( 18597 ) , 360 ( 9666 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.91 ( brs , 1h , h1b ) , 9.17 ( d , 1h , j = 7.7 hz , h4a ) , 8.82 ( d , 1h , j = 3.8 hz , h6a ) , 8.11 ( d , 1h , j = 7.1 hz , h4b ) , 7.84 ( d , 1h , j = 8.5 hz , h7b ) , 7.58 ( m , 3h , h5a+h5b+h6b ) , 6.43 ( m , 2h , h2c+h6c ) , 6.31 ( d , 1h , j = 5.3 hz , h3c or h5c ) , 6.12 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 2.54 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.21 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.6 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.6 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  153.97 ( c8a ) , 150.45 ( c6a ) , 146.52 ( c2b ) , 141.37 ( c9b ) , 134.70 ( c8b ) , 134.64 ( c3a ) , 131.48 ( c4a ) , 125.39 ( c5b or c6b ) , 124.92 ( c5b or c6b ) , 119.56 ( c5a ) , 117.85 ( c4b ) , 113.96 ( c7b ) , 111.54 ( c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.73 ( c2c or c6c ) , 82.15 ( c3c or c5c ) , 80.84 ( c3c or c5c ) , 31.12 ( c7c ) , 22.19 ( c8c+c9c ) , 19.17 ( c10c ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  128.7 ( n1b ) ppm .", "a mixture of l1 ( 42.5 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 11bh2o ( mr = 648.61 g / mol ) : c , 42.59 ; h , 3.88 ; n , 10.79 . found : c , 42.56 ; h , 3.57 ; n , 10.97 .", "esi - ms in methanol ( positive ) : m / z [ m  hcl  cl ] , 596 [ m  cl ] , 618 [ m  hcl+na ] ; ( negative ) : m / z 595 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 252 ( 17048 ) , 299 ( 20228 ) , 343 ( 19879 ) .", "h nmr ( 500.32 mhz , meoh - d4 ) :  8.99 ( dd , 1h , j = 1.3 hz ,", "j = 8.1 hz , h4a ) , 8.77 ( dd , 1h , j = 1.4 hz ,", "j = 4.8 hz , h6a ) , 7.96 ( dd , 1h , j = 2.0 hz , j = 6.4 hz , h4b ) , 7.80 ( dd , 1h , j", "= 1.9 hz , j = 6.1 hz , h7b ) , 7.647.59 ( m , 3h , h5a+h5b+h6b ) , 6.66 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.59 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.43 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.43 ( sep , 1h , j = 6.9 hz , h7c ) , 2.38 ( s , 3h , h10c ) , 0.96 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.92 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , meoh - d4 ) :  153.25 ( c8a ) , 148.91 ( c2b ) , 147.42 ( c6a ) , 140.49 ( c9b ) , 135.15 ( c3a ) , 134.25 ( c8b ) , 132.07 ( c4a ) , 125.47 ( c5b or c6b ) , 124.81 ( c5b or c6b ) , 118.48 ( c5a ) , 116.89 ( c4b ) , 112.98 ( c7b ) , 112.92 ( c9a ) , 97.71 ( c4c ) , 94.88 ( c1c ) , 76.44 ( c2c or c6c ) , 74.99 ( c2c or c6c ) , 71.93 ( c3c or c5c ) , 70.44 ( c2c or c6c ) , 31.37 ( c7c ) , 21.35 ( c8c or c9c ) , 21.09 ( c8c or c9c ) , 17.84 ( c10c ) ppm .", "crystals of 11b4h2o suitable for xrd study have been obtained from a solution of 11b in ethanol .", "a mixture of l2 ( 73.2 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 12ah2o ( mr = 638.35 g / mol ) : c , 43.28 ; h , 3.79 ; n , 10.97 .", "esi - ms in methanol ( positive ) : m / z 548 [ m  hcl  cl ] , 608 [ m  hcl+na ] ; ( negative ) : m / z 549 [ m2hcl  h ] , 582 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 254 ( 17778 ) , 303 ( 29953 ) , 351 ( 17387 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.34 ( brs , 1h , h1b ) , 9.21 ( s , 1h , h4a ) , 8.73 ( s , 1h , h6a ) , 8.06 ( d , 1h , j = 7.6 hz , h4b ) , 7.79 ( d , 1h , j = 8.5 hz , h7b ) , 7.52 ( m , 2h , h5b+h6b ) , 6.32 ( d , 2h , j = 5.8 hz , h2c+h6c ) , 6.22 ( d , 1h , j = 6.2 hz , h3c or h5c ) , 6.03 ( d , 1h , j = 6.1 hz , h3c or h5c ) , 2.52 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.18 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.8 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  154.04 ( c8a ) , 151.24 ( c6a ) , 146.57 ( c2b ) , 141.37 ( c9b ) , 134.63 ( c8b ) , 133.43 ( c3a ) , 131.82 ( c4a ) , 125.33 ( c5b or c6b ) , 124.88 ( c5b or c6b ) , 117.78 ( c4b ) , 114.72 ( c5a or c9a ) , 113.85 ( c7b ) , 112.48 ( c5a or c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.69 ( c2c or c6c ) , 82.16 ( c3c or c5c ) , 80.76 ( c3c or c5c ) , 31.09 ( c7c ) , 22.20 ( c8c or c9c ) , 22.17 ( c8c or c9c ) , 19.16 ( c10c ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  127.3 ( n1b ) ppm .", "a mixture of l2 ( 56.4 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 12bh2o ( mr = 727.51 g / mol ) : c , 37.97 ; h , 3.33 ; n , 9.63 . found : c , 37.82 ; h , 3.02 ; n , 9.33 .", "esi - ms in methanol ( positive ) : m / z 638 [ m  hcl  cl ] , 696 [ m  hcl+na ] ; ( negative ) : m / z 672 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 253 ( 21638 ) , 302 ( 30505 ) , 357 ( 21813 ) .", "h nmr ( 500.32 mhz , meoh - d4 ) :  9.08 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.1 hz , h6a ) , 7.96 ( dd , 1h , j = 2.1 hz ,", "j = 6 hz , h4b ) , 7.80 ( dd , 1h , j = 2.1 hz ,", "j = 6.1 hz , h7b ) , 7.63 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.63 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.48 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.22 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.42 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.95 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , meoh - d4 ) :  153.04 ( c8a ) , 152.49 ( c6a ) , 148.15 ( c2b ) , 140.47 ( c9b ) , 134.58 ( c3a ) , 134.19 ( c8b ) , 131.13 ( c4a ) , 125.79 ( c5b or c6b ) , 125.05 ( c5b or c6b ) , 116.99 ( c4b ) , 115.39 ( c5a or c9a ) , 113.09 ( c7b ) , 112.11 ( c5a or c9a ) , 98.54 ( c4c ) , 95.52 ( c1c ) , 75.73 ( c2c or c6c ) , 75.25 ( c2c or c6c ) , 71.87 ( c3c or c5c ) , 69.99 ( c3c or c5c ) , 31.41 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.84 ( c10c ) ppm .", "crystals of 12b and 12b2ch3oh2h2o suitable for xrd study have been obtained from a solution of 12b in methanol .", "a mixture of l3 ( 64 mg , 0.18 mmol ) and [ rucl2(-p - cymene)]2 ( 50 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 13a1.5h2o ( mr = 691.41 g / mol ) : c , 43.43 ; h , 4.23 ; n , 10.13 ; cl , 10.26 .", "found : c , 43.81 ; h , 4.24 ; n , 10.11 ; cl , 10.57 .", "esi - ms in methanol ( positive ) : m / z 592 [ m  hcl  cl ] , 650 [ m  hcl+na ] ; ( negative ) : m / z 591 [ m2hcl  h ] , 628 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , mcm ) : sh 252 ( 18744 ) , 306 ( 28529 ) , 354 ( 16889 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  13.88 ( brs , 1h , h1b ) , 9.41 ( s , 1h , h4a ) , 8.68 ( s , 1h , h6a ) , 7.99 ( d , 1h , j = 7.7 hz , h4b ) , 7.49 ( m , 2h , h5b+h6b ) , 6.29 ( m , 2h , h2c+h6c ) , 6.20 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.00 ( d , 1h , j = 5.9 hz , h3c or h5c ) , 4.88 ( s , 2h , h10b ) , 3.39 ( s , 3h , h11b ) , 2.48 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.17 ( s , 3h , h10c ) , 0.93 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.87 ( d , 3h , j = 6.8 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  155.35 ( c8a ) , 150.27 ( c6a ) , 147.43 ( c2b ) , 141.64 ( c9b ) , 133.07 ( c8b ) , 132.68 ( c3a ) , 131.68 ( c4a ) , 124.87 ( c5b or c6b ) , 124.59 ( c5b or c6b ) , 124.53 ( c7b ) , 117.18 ( c4b ) , 114.23 ( c5a or c9a ) , 112.78 ( c5a or c9a ) , 103.74 ( c4c ) , 103.09 ( c1c ) , 85.38 ( c2c or c6c ) , 83.68 ( c2c or c6c ) , 82.13 ( c3c or c5c ) , 81.05 ( c3c or c5c ) , 70.19 ( c10b ) , 58.03 ( c11b ) , 31.04 ( c7c ) , 22.22 ( c8c or c9c ) , 22.09 ( c8c or c9c ) , 19.13 ( c10c ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  122.9 ( n1b ) ppm .", "the red xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) were obtained from etoh / et2o and an etoh solution of 13a ( long crystallization ) .", "the yellow crystals of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) suitable for xrd study have been obtained from methanolic solution of 13a ( short crystallization ) .", "h nmr ( 13c , 500.32 mhz , dmso - d6 ) :  13.43 ( s , 1h ) , 9.18 ( d , 1h , j = 2.1 hz ) , 8.48 ( d , 1h , j = 2.2 hz ) , 7.94 ( d , 1h , j = 8.2 hz ) , 7.45 ( t , 1h , j = 7.8 hz ) , 7.39 ( d , 1h , j = 7.2 hz ) , 6.16 ( d , 1h , j = 6.0 hz ) , 6.13 ( d , 1h , j = 6.1 hz ) , 6.07 ( d , 1h , j = 5.9 hz ) , 5.89 ( d , 1h , j = 6.4 hz ) , 4.85 ( s , 2h ) , 4.04 ( s , 3h ) , 2.14 ( s , 3h ) , 0.93 ( d , 3h , j = 6.8 hz ) , 0.86 ( d , 3h , j = 6.9", "a mixture of l3 ( 59 mg , 0.17 mmol ) and [ oscl2(-p - cymene)]2 ( 60 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 3 h. ethanol was evaporated up to ca .", "calcd for 13b : c , 39.85 ; h , 3.48 ; n , 9.29 . found : c , 39.60 ; h , 3.32 ; n , 9.20 .", "esi - ms in methanol ( positive ) : m / z 682 [ m  hcl  cl ] , 704 [ m2hcl+na ] ; ( negative ) : m / z 680 [ m2hcl  h ] , 716 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 256 ( 19825 ) , 303 ( 24634 ) , 357 ( 18850 ) .", "h nmr ( 500.32 mhz , meoh - d4 ) :  9.33 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.2 hz , h6a ) , 7.91 ( dd , 1h , j = 1.2 hz ,", "j = 7.8 hz , h4b ) , 7.647.58 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.62 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.47 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 4.94 ( q , 2h , j = 12.4 hz , h10b ) , 3.49 ( s , 3h , h11b ) , 2.41 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm . c nmr ( 125.81 mhz , meoh - d4 ) :  153.18 ( c8a ) , 152.16 ( c6a ) , 148.36 ( c2b ) , 140.82 ( c9b ) , 134.35 ( c3a ) , 132.68 ( c8b ) , 131.68 ( c4a ) , 125.63 ( c5b or c6b ) , 124.85 ( c5b or c6b ) , 124.51 ( c7b ) , 116.72 ( c4b ) , 115.24 ( c5a or c9a ) , 112.26 ( c5a or c9a ) , 98.75 ( c4c ) , 95.50 ( c1c ) , 75.98 ( c2c or c6c ) , 75.34 ( c2c or c6c ) , 71.82 ( c3c or c5c ) , 70.29 ( c10b ) , 70.04 ( c3c or c5c ) , 57.17 ( c11b ) , 31.39 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.85 ( c10c ) ppm .", "the yellow crystals of [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75 ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) suitable for xrd study have been obtained from a methanolic solution of 13b .", "elemental analyses ( c , h , n , cl ) were performed by the microanalytical service of the institute of physical chemistry , university of vienna .", "electrospray ionization mass spectrometry ( esi - ms ) was carried out with an esquire 3000 instrument ( bruker daltonics , bremen , germany ) , using solutions of compounds in methanol .", "vis spectra were recorded on a lambda 20 uv  vis spectrophotometer ( perkin  elmer ) , using samples dissolved in methanol ( l1l3 , 11a , 12a , 13a , 11b , 12b , 13b ) and water ( 11a ) over 24 or 48 h , correspondingly . the one - dimensional ( h , c , n ) and two - dimensional spectra ( n , h hsqc , c , h hsqc , c , h hmbc , h , h cosy , h , h tocsy , h , h noesy ( l3 , 12a ) , h , h roesy ( l3 , 11a , 12a , 13a , 13b ) ) were recorded with a bruker model dpx500 ( ultrashield magnet ) system in dmso - d6 ( l1l3 , 11a , 12a , 13a ) , meoh - d4 ( 11b , 12b , 13b ) , d2o ( h nmr , 11a ) , and d2o / dmso - d6 ( h nmr , 12a ) , using standard pulse programs at 500.32 ( h ) , 125.81 ( c ) and 50.70 ( n ) mhz .", "h signals are referenced relative to the solvent signals ( dmso - d6 at 2.51 , meoh - d4 at 3.33 ppm ) .", "single crystals were positioned at distances of 35 , 40 , 40 , 40 , 40 , 40 , and 35 mm from the detector , and 1556 , 1131 , 518 , 1911 , 1176 , 1978 , and 1039 frames were measured , each for 30 , 60 , 30 , 20 , 70 , 60 , and 30 s over 1 scan width for 20.5h2o , 11b4h2o , 12b , 12b2ch3oh2h2o , 13c , 13dch3oh , and 13e0.75ch3oh0.25h2o , correspondingly .", "crystal data , data collection parameters , and structure refinement details are given in table 1 .", "the structures were solved by direct methods and refined by full - matrix least - squares techniques .", "refinement of the structure of 12b revealed that the chloride counteranion occupies two statistically disordered positions with site occupation factor ( sof ) values of 0.57:0.43 , whereas , in 13c , the methoxymethylene group was found to be disordered over two positions with sof values of 0.80:0.20 .", "similarly , the methoxymethylene group of one crystallographically independent complex in 13dch3oh or in both crystallographically independent complexes in 13e0.75ch3oh0.25h2o is disordered with sof values of 0.35:0.65 and 0.60:0.40 , 0.80:0.20 , correspondingly . the disorder was resolved with constrained anisotropic displacement parameters and restrained bond distances using eadp and sadi instructions of shelx97 , respectively .", "structure solution was achieved with shelxs-97 and refinement with shelxl-97 , and graphics were produced with ortep-3 .", "wr2 = { [w(fo  fc)]/[w(fo)]}. gof = { [w(fo  fc)]/(n  p ) } , where n is the number of reflections and p is the total number of parameters refined .", "a549 ( non - small cell lung carcinoma , human ) and sw480 ( colon carcinoma , human ) cells were kindly provided by brigitte marian ( institute of cancer research , department of medicine i , medical university of vienna , austria ) .", "ch1 cells ( ovarian cancer , human ) were a gift from lloyd r. kelland ( crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . ) .", "cells were grown in 75-cm culture flasks ( iwaki ) in a complete medium ( i.e. , minimum essential medium supplemented with 10% heat - inactivated fetal bovine serum , 1 mm sodium pyruvate , 4 mm l - glutamine , and 1% nonessential amino acids from 100 stock ) as adherent monolayer cultures .", "cultures were grown at 37 c under a humidified atmosphere containing 5% co2 and 95% air .", "antiproliferative activity in vitro was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide , fluka ) . for this purpose", ", cells were harvested from culture flasks by the use of trypsin and seeded in complete medium ( 100 l / well ) into 96-well plates ( iwaki ) in densities of 4  10 ( a549 ) , 1.5  10 ( ch1 ) , and 2.5  10 ( sw480 ) viable cells per well .", "test compounds were dissolved in dmso first , appropriately diluted in complete medium , and instantly added to the plates ( 100 l / well ) , where the dmso content did not exceed 0.4% and 1% for the ligands and complexes , respectively . after exposure for 96 h , the medium was replaced with 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) plus 20 l / well mtt solution in phosphate - buffered saline ( 5 mg / ml ) , followed by incubation for 4 h. subsequently , the medium / mtt mixture was removed , and the formazan product formed by viable cells was dissolved in dmso ( 150 l / well ) .", "optical densities were measured with a microplate reader ( tecan spectra classic ) at 550 nm ( and a reference wavelength of 690 nm ) to yield relative quantities of viable cells as percentages of untreated controls , and 50% inhibitory concentrations ( ic50 ) were interpolated from concentration  effect curves .", "calculations are based on at least three independent experiments with triplicates for each concentration level . to study the effects on the cell cycle of exponentially growing ch1 cells by flow cytometric analysis of their relative dna content", ", cells were harvested from culture flasks , seeded in complete medium into 90-mm petri dishes ( 1  10 cells / dish ) and , after recovery for 24 h , exposed to various concentrations of the test compounds for 24 h. for this purpose , test compounds were diluted from dmso stocks with complete medium ( see above ) such that the effective dmso content did not exceed 0.5% . after exposure , treated and control cells were collected by scratching , washed with pbs , and stained with 5 g / ml propidium iodide overnight .", "their fluorescence was measured with a facs calibur instrument ( becton dickinson ) , and the obtained histograms were analyzed with cell quest pro software ( becton dickinson ) .", "at least two independent experiments were performed for each setting , and 2.5 or 3.0  10 cells were measured per sample .", "the cdk - inhibitory capacities of test compounds were studied by a radiochemical assay using recombinant cdk1/cyclin b and cdk2/cyclin e isolated from sf21 insect cells and histone h1 as the substrate for phosphorylation , as described by marko et al .", "briefly , mops - buffered assay mixtures containing the test compound ( with a maximum of 1% dmso ) , the respective kinase / cyclin complex , histone h1 , and 0.4 ci ( -p)atp per sample were incubated for 10 min at 30 c .", "aliquots of the solution were spotted onto phosphocellulose squares , which had been washed three times with 0.75% phosphoric acid , followed by acetone .", "the dried squares were measured in scintillation vials by -counting ( perkin  elmer tri - carb 2800tr ; quanta smart software ) .", "results were obtained in duplicate in at least two independent experiments , and ic50 values were calculated by interpolation .", "several routes to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been proposed by johnson & johnson pharmaceutical research & development l.l.c .", "the first one was developed for 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety and involved sulfur - induced benzimidazole ring formation via the treatment of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde with 1,2-diaminobenzene .", "the poor reproducibility of this synthesis prompted the exploration of an alternative way , via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid , which was used for the preparation of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with a substituted benzimidazole moiety .", "the patented route to 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) consists of four steps ( see scheme 1 , steps i  iv ) : oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base with subsequent acidification with h2so4 , esterification of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) in the presence of h2so4 in methanol , bromination of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in an acoh / acona mixture , and hydrolysis of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in the presence of naoh , followed by acidification with hcl .", "reagents and conditions : ( i ) kmno4 , naoh , 3 h , 100 c , h2so4 ; ( ii ) methanol , h2so4 , reflux , 68 h , nahco3 , 42% ( i + ii ) ; ( iii ) br2 , acoh , acona , 115 c , overnight , chromatographic purification , 43% ; ( iv ) naoh , meoh , reflux , 4 h , hcl , 100% ; ( v ) br2 , acoh , acona , 115 c , 2.53 h ; and ( vi ) kmno4 , naoh , 3 h , 100 c , hcl , 2435% ( v + vi ) .", "we performed the synthesis of 3 in two steps ( see scheme 1 , steps v and vi ) : bromination of 3-methyl-1h - pyrazolo[3,4-b]pyridine in acoh / acona mixture and oxidation of crude 5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) by kmno4 in a basic medium , followed by acidification with 37% hcl , with an overall yield of 2435% .", "5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) is a known compound , the synthesis of which is well - documented . for the benzimidazole ring formation , 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene", "the reported synthesis of 1-methoxymethyl-2,3-diaminobenzene ( 5 ) from 2,3-diaminobenzyl alcohol afforded the desired product in 34% yield ( see scheme 2 , step i ) .", "however , the instability and low yield of diamines , as well as the necessity to purify the desired ether using column chromatography , stimulated the search for a more - convenient procedure that was subsequently proposed : etherification of 2-amino-3-nitrobenzyl alcohol , followed by the reduction of 1-methoxymethyl-2-amino-3-nitrobenzene ( 4 ) with 10% pd / c in ethanol under a hydrogen atmosphere afforded 5 in 6575% yield ( see scheme 2 , steps ii and iii ) .", "reagents and conditions : ( i ) nah , mei , dry thf , 0 c , 30 min , room temperature , overnight , chromatographic purification , 34% yield ; ( ii ) nah , mei , dry thf , 0 c , 15 min , room temperature , 3 h , chromatographic purification , 6575% yield ; ( iii ) 10% pd / c , ethanol , h2 , room temperature , 1824 h , 100% yield .", "patented benzimidazole ring formation by cyclization of the 3-carboxyl group of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) with substituted 1,2-diaminobenzenes was realized via amide formation using coupling reagents , followed by treatment with glacial acetic acid .", "hatu ( n , n , n,n-tetramethyl - o-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate ) was used as a coupling reagent .", "three 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( where x = h , br ; and y = h , ch2och3 ) have been synthesized in this work ( see chart 1 ) : 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l1 ) is a new compound that we have prepared as a model ligand for coordination to metals ; 3-(1h - benzimidazol-2-yl)-5-bromo-1h - pyrazolo[3,4-b]pyridine ( l2 ) was previously known as boc - protected compound prepared via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde;5-bromo-3-(4-methoxymethyl-1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l3 ) was synthesized via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) and patented as a potential cdk inhibitor and antiproliferative agent . for the synthesis of l1l3 , we used n , n-carbonyldiimidazole ( cdi ) as an amide - coupling reagent , because it is relatively inexpensive and the side products , carbon dioxide and imidazole , could be easily removed from the reaction mixture .", "cdi - mediated amidation of acids 1 and 3 was performed as shown in scheme 3 ( steps i and ii ) . in the first step ,", "the acyl - imidazolides 6 and 7 were obtained in dry dmf at room temperature and isolated as white solids .", "the yields based on 3-methyl-1h - pyrazolo[3,4-b]pyridine are : 2529% ( 6 ) and 1316% ( 7 ) . in the second step , monoacylation of phenylenediamines ( 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene ( 5 ) ) using acyl - imidazolides was performed in dmf at 8085 c .", "reagents and conditions : ( i ) cdi , dry dmf , room temperature , 2024 h ; ( ii ) 1,2-diaminobenzene or 5 , dry dmf , 8085 c ; 520 h ; and ( iii ) glacial acoh , 120125 c , 2.54.5 h , chromatographic purification .", "amides 810 were used without further purification in ring closure reactions in a glacial acetic acid at 120125 c and afforded the desired 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines : l1 ( 5560% ) , l2 ( 5861% ) , and l3 ( 4451% ) , based on 6 and 7 ( see scheme 3 , step iii ) .", "the reported synthesis of l3 is a one - pot approach for amide formation using hatu with 57% yield , followed by cyclization under acidic conditions ( acetic acid ) with 87% yield .", "thus , the ligands l1 , l2 , and l3 have been prepared in 7 , 8 , and 11 steps , correspondingly .", "finally , the ligands l1l3 were reacted with [ mcl2(-p - cymene)]2 ( where m = ru , os ) in a 2:1 molar ratio in dry ethanol at room temperature to give [ mcl(-arene)(l)]cl complexes ( 11a , 11b , 12a , 12b , 13a , 13b ) in quantitative yields .", "crystallization of [ rucl(-p - cymene)(l3)]cl ( 13a ) in etoh or etoh / et2o resulted in xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) , while the crystallization of 13a in methanol afforded crystals of composition [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) .", "the osmium(ii ) analogue 13e0.75ch3oh0.25h2o was obtained via the crystallization of 13b in methanol . the full assignment of proton , carbon , and nitrogen resonances for l1l3 , 11a , 11b , 12a", ", 12b , 13a , and 13b is quoted in tables s1s3 in the supporting information .", "3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety ( l1 , l2 ) display one set of signals .", "l1 is characterized by three doublets of doublets for h4a , h5a , h6a ( 8.85 , 7.39 , 8.66 ppm , correspondingly ) of pyrazolopyridine moiety , two doublets for h4b , h7b ( 7.54 and 7.75 ppm ) , the overlapped h5b , h6b proton signals in one multiplet ( 7.24 ppm ) for a benzimidazole moiety and two singlet resonances for h1a and h1b ( for atom numbering , see chart 1 ) . the substitution of h5a by bromine ( l2 ) results in reduced multiplicity for the h4a and h6a signals ( two doublets at 8.99 and 8.75 ppm ) , whereas the benzimidazole moiety spectrum remains almost unchanged .", "two singlets were attributed to nh protons and related to pyrazolopyridine ( h1a , at 14.19 ( l1 ) , 14.43 ( l2 ) ppm ) and benzimidazole ( h1b , at 13.11 ( l1 ) , 13.19 ( l2 ) ppm ) moieties .", "nmr spectra for 11a , 11b , 12a , and 12b , where l1 and l2 coordinate as bidentate ligands via n2a and n3b with the formation of a stable five - membered chelate cycle , show one set of signals .", "there was no evidence for monodentate or tridentate coordination of ligands with the formation of dinuclear or polynuclear complexes .", "coordination of l1 and l2 to ruthenium(ii ) made it possible to assign the two doublets to proton resonances of h4b and h7b of the benzimidazole moiety . in the h ,", "h roesy plots , one of them has cross - peaks with a ch cymene ring and the nearest to them is h4b ( e.g. , at 8.11 ( 11a ) , 8.06 ( 12a ) ppm ) .", "a singlet at 14.91 ( 11a ) , 14.34 ( 12a ) ppm was assigned to nh proton and showed no couplings with other atoms .", "the nitrogen resonance shift at 128.7 ( 11a ) , 127.3 ( 12a ) ppm is closer to the benzimidazole nh chemical shift in metal - free ligands ( 121.3 ( l1 , l2 ) ppm ) and , therefore , was assigned as n1b ( see table s3 in the supporting information ) .", "the h1b resonance shows a significant shift by 1.8 and 1.15 ppm for 11a and 12a , respectively , upon ligand coordination ( l1 and l2 ) to the metal(ii)-arene moiety .", "the nearest to the metal binding site pyrazolopyridine proton h1a was not detected for 11a , 11b , 12a , and 12b , and the proton resonance of h1b was also not seen for 11b or 12b .", "l3 displays two sets of signals originated from 7b - l3 and 4b-l3 tautomers ( see chart 1 ) . the signals of pyrazolopyridine moieties are partially overlapped ( e.g. , h1a ( h1a ) at 14.47 and 14.43 ppm , h4a ( h4a ) at 8.99 and 8.98 ppm , h6a ( h6a ) at 8.75 ppm ) , whereas the signals of the benzimidazole moieties are better resolved ( see table s1 in the supporting information ) .", "according to the h , h roesy plot , one of the ch2 groups at 4.80 ppm couples with the nh proton at 13.25 ppm , indicating their attribution to the 7b - l3 tautomer , whereas another nh proton at 13.22 ppm gives a cross - peak with h7b at 7.47 ppm and belongs to 4b-l3 tautomer ( figure 1 ) .", "tautomers 7b - l3 and 4b-l3 are present in solution in 1:1.3 molar ratio and give , for the substituted benzimidazole moiety , two singlets ( h1b at 13.22 ppm , h1b at 13.25 ppm ) , two doublets of doublets ( h7b at 7.47 ppm , h4b at 7.72 ppm ) , one multiplet ( h5b , h6b , h5b , h6b at 7.287.21 ppm ) , two ch2 ( 4.80 ( h10b ) , 4.96 ( h10b ) ppm ) and two ch3 singlets at 3.37 ( h11b ) and 3.44 ( h11b ) ppm .", "the absence of cross - peaks between h7b and h4b in the h , h cosy plot supports their relation to the two different molecules . the binding site in 4b-l3 tautomer is sterically shielded by a methoxymethyl group .", "consistent with this nmr spectra display , only one set of signals is shown for 13a and 13b with the coordination of the 7b - l3 tautomer to ruthenium(ii ) and osmium(ii ) via n2a and n3b ( see scheme 4 ) .", "the preference for coordination of the 7b - l3 tautomer is also confirmed by h , h roesy plots : h4b ( 7.99 ( 13a ) , 7.91 ( 13b ) ppm ) has couplings with ch protons of cymene ring .", "the cross - peak between h10b at 4.88 ppm ( 13a ) and nh at 13.88 ppm ( 13a ) enabled the assignment of this singlet to a benzimidazole moiety ( h1b ) .", "in addition , the chemical shifts for the c atoms of the benzimidazole moiety ( c4b , c7b , c5b , c6b ) of the coordinated ligand and metal - free 7b - l3 tautomer are very similar ( see table s2 in the supporting information ) .", "the nitrogen resonance at 122.9 ppm ( 13a ) compares well to the benzimidazole nh chemical shift in metal - free l3 at 121.4 ppm . as for 11a , 11b , 12a , and", "12b , the nearest to the coordination place pyrazolopyridine proton h1a resonance was not detected .", "according to the h , h roesy plots of 11a , 12a , 13a , and 13b only ch cymene ring protons have couplings with the nearest h4b benzene ring proton , suggesting that the isopropyl or methyl groups are further away from the h4b proton .", "the results of the xrd studies of [ oscl(-p - cymene)(l1)]cl4h2o ( 11b4h2o ) , [ oscl(-p - cymene)(l2)]cl ( 12b ) , [ oscl(-p - cymene)(l2)]cl2ch3oh2h2o ( 12b2ch3oh2h2o ) , [ rucl(-p - cymene)(l3h ) ] ( 13c ) , [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) and [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) are shown in figures 2 , figure s2 in the supporting information , and figures 36 , respectively .", "all complexes have a typical  three - legged piano - stool  geometry of ruthenium(ii ) and osmium(ii ) arene complexes , with an  -bound p - cymene ring forming the seat and three other donor atoms ( two nitrogens n1 and n5 of the corresponding 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine and one chlorido ligand ) as the legs of the stool . selected bond distances and angles are quoted in the figure captions .", "all complexes crystallize as racemates , because of the presence of the stereogenic metal center .", "fragment of the crystal structure of 11b4h2o showing nitrogen atoms n3 and n4 , which act as proton donors in intermolecular hydrogen bonding interactions n3hcl2 [ n3cl2 3.067(7 )  , n3hcl2 177.3 ] and n4ho3 [ n4o3 2.671(9 )  , n4ho3 170.9 ] ; thermal ellipsoids have been drawn at 50% probability level .", "selected bond lengths and angles : os  cl1 , 2.421(2 )  ; os ", "c(arene)av , 2.198(32 )  ; c1n2 , 1.349(11 )  ; n2n1 , 1.366(10 )  ; n1c6 , 1.357(11 )  ; c6c7 , 1.428(12 )  ; c7n5 , 1.345(11 )  ; n5c13 , 1.391(11 )  ; n1os  n5 , 75.6(3) ; n1os  cl1 , 85.5(2) ; n5os  cl1 , 83.4(2). the bidentate 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which can have different substituents in positions 5a and 7b , reveal different acid  base properties .", "it can act as a neutral organic ligand , with nitrogen atoms n2 and n4 as proton donors involved in intermolecular hydrogen bonding interactions n4ho1(x + 1 , y + 1 , z + 2 ) [ n4o1 , 2.730(4 )  ; n4ho1 , 176.1 ] and n2ho3(x + 2 , y + 1 , z + 1 [ n2o3 , 2.697(4 )  ; n4ho1 , 172.3 ] with one methanol and one water molecule , respectively , as is the case for 12b2ch3oh2h2o ( figure 3 ) or n2hcl2(cl2x ) and n4hcl1(x , y , z + 2 ) [ n4cl1 , 3.213(9 )  ; n4ho1 , 162.91 ] in 12b ( see figure s2 in the supporting information ) , correspondingly .", "the ligand can be protonated at n3 ( n3hcl2 ) and deprotonated at n2 [ n2h  o4 ( x + 1 , y + 1 , z + 1 ) with n2o4 2.923(9 )  , n2h", " o4 138.7 ] ( overall charge zero ) with atom n4 as a proton donor to one of the four co - crystallized water molecules n4ho3 , as occurs in 11b4h2o ( see figure 2 ) .", "ortep plot of the structure of the cation [ oscl(-p - cymene)(l2 ) ] in 12b2ch3oh2h2o .", "thermal ellipsoids have been drawn at the 50% probability level . selected bond lengths and angles : os  cl1 , 2.4043(1 )  ; os ", "c(arene)av , 2.197(26 )  ; c1n2 , 1.361(5 )  ; n2n1 , 1.344(4 )  ; n1c6 , 1.336(5 )  ; c6c7 , 1.447(6 )  ; c7n5 , 1.335(5 )  ; n5c13 , 1.380(5 )  ; n1os  n5 , 74.73(13) , n1os  cl1 , 84.29(10) ; and n5os  cl1 , 83.25(10). in 13c , the ligand was found to be deprotonated at n2 , acting as a proton acceptor in the intermolecular hydrogen bonding interaction n2h  n4 ( i denotes symmetry transformations x , y + 1.5 , z + 0.5 ) used to generate equivalent atoms ) [ n2n4 , 2.896(7 )  ; n2h  n4 , 155.6 ] ( see figure 4 ) . ortep plot of the complex [ rucl(-p - cymene)(l3h ) ] ( 13c ) .", "thermal ellipsoids have been drawn at 30% probability level . selected bond lengths and angles : ru  cl , 2.399(2 )  ; ru  n1 ,", "c(arene)av , 2.186(31 )  ; c1n2 , 1.366(8 )  ; n2n1 , 1.353(7 )  ; n1c6 , 1.362(7 )  ; c6c7 , 1.426(9 )  ; c7n5 , 1.335(8 )  ; n5c13 , 1.398(8 )  ; n1ru  n5 , 75.9(2) ; n1ru  cl , 87.35(17) ; and n5ru  cl , 85.73(17). complexes 13dch3oh and 13e0.75ch3oh0.25h2o crystallized both in the centrosymmetric triclinic space group p1. the asymmetric unit in both consists of a neutral complex [ mcl(-p - cymene)(l3h ) ] and a complex cation [ mcl(-p - cymene)(l3 ) ] ( m = ru or os ) , a chloride counteranion and co - crystallized solvent ( methanol or methanol / water )", ". deprotonation of the organic ligand in [ mcl(-p - cymene)(l3h ) ] is corroborated by the presence of hydrogen bonding of the type n2ah  n2b ( x + 1 , y + 1 , z + 1 ) in the crystal structure of the ruthenium complex 13dch3oh ( figure 5 ) and a similar interaction n2bh  n2a ( x , y + 1 , z ) [ n2bn2a , 2.810(9 )  ; n2bh  n2a , 170.1 ] in the crystal of the osmium analogue 13e0.75ch3oh0.25h2o . in figure 6 the structure of [ oscl(-p - cymene)(l3h ) ]", "is shown . in both structures , the atoms n4a and n4b act as proton donors in strong hydrogen bonding interactions to the chloride counteranion , namely , n4ah ", "cl2 ( x + 1 , y + 2 , z + 1 ) [ n4cl2 , 3.275(6 )  ; n4ah  cl2 , 176.9 ] , n4bh ", "cl2 [ n4bcl2 , 3.211(5 )  ; n4bh  cl2 , 176.0 ] ( 13dch3oh ) and n4ah  cl2 ( x + 1 , y + 1 , z + 1 ) [ n4acl2 , 3.273(8 )  ; n4ah  cl2 , 177.0 ] , n4bh ", "cl2 [ n4bcl2 , 3.204(7 )  ; n4bh  cl2 , 177.0 ] ( 13e0.75ch3oh0.25h2o ) .", "fragment of the crystal structure of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) showing the intermolecular hydrogen bonding n2a  hn2b [ n2an2b ( x + 1 , y + 1 , z + 1 ) , 2.814(7 )  ; n2a  hn2b , 163.3 ] . selected bond lengths and angles : ru1a  cl1a , 2.3952(17 )  ; ru1a ", "c(arene)av , 2.194(33 )  ; c1a  n2a , 1.358(8 )  ; n2a  n1a , 1.354(7 )  ; n1a  c6a , 1.350(8 )  ; c6a  c7a , 1.441(9 )  ; c7a  n5a , 1.335(8 )  ; n5a  c13a , 1.381(8 )  ; n1a  ru1a  n5a , 75.6(2) ; n1a ", "ru1a  cl1a , 84.45(15) ; and n5a  ru1a  cl1a , 85.05(15). ortep plot of [ oscl(-p - cymene)(l3h ) ] in [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) .", "selected bond lengths and angles : os1a  cl1a , 2.402(2 )  ; os1a  n1a , 2.068(7 )  ; os1a  n5a , 2.086(7 )  ; os1a  c(arene)av , 2.192(25 )  ; c1a  n2a , 1.349(10 )  ; n2a  n1a 1.369(9 )  ; n1a  c6a , 1.361(10 )  ; c6a  c7a , 1.431(11 )  ; c7a  n5a , 1.339(10 )  ; n5a  c13a , 1.398(11 )  ; n1a  os1a  n5a , 75.1(3) ; n1a ", "os1a  cl1a , 83.56(18) ; and n5a  os1a  cl1a , 84.2(2). ligands l1l3 , as well as the corresponding ruthenium(ii ) ( 11a13a ) and osmium(ii ) ( 11b13b ) arene complexes , were studied with regard to their capacity of inhibiting cell growth in vitro in the human cancer cell lines ch1 ( ovarian carcinoma ) , sw480 ( colon carcinoma ) , and a549 ( non - small cell lung cancer ) , yielding the ic50 values listed in table 2 .", "all compounds show the strongest effect in the generally chemosensitive ch1 cells , whereas the more chemoresistant a549 cells are also less affected by the compounds investigated here .", "> l2 > l1 , indicating that bromination ( l2 , l3 ) and , even more so , the double substitution ( bromination and an additional replacement of h by a methoxymethyl group ( l3 ) ) are advantageous , with regard to activity .", "these structure  activity relationships are also reflected in the rank orders of the corresponding ruthenium and osmium complexes for the ruthenium complexes , 13a > 12a > 11a , and for the osmium complexes , 13b > 12b > 11b .", "however , the ic50 values are shifted to higher concentrations ( see figure 7 ) .", "the differences between the ruthenium and osmium analogues are mostly small ( ic50 values differ only up to a factor of 2.4 ) , with the osmium complexes being at least as active as their ruthenium counterparts .", "ch1 denotes ovarian cancer , human ; sw480 denotes colon carcinoma , human ; and a549 denotes non - small - cell lung carcinoma , human .", "50% inhibitory concentrations ( mean value  standard deviation from at least three independent experiments ) obtained by the mtt assay ( 96-h exposure ) . concentration", " effect curves of each ligand , compared to the corresponding ruthenium and osmium complexes , in ch1 ovarian cancer cells ( mtt assay , 96 h exposure ) : ( a ) l1 , 11a , 11b ; ( b ) l2 , 12a , 12b ; and ( c ) l3 , 13a , 13b . both the presence of substituents in the ligands and complexation result in a marked shift of antiproliferative activity . since 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been reported to be potent cdk inhibitors , we expected the investigated ligands and complexes to induce cell cycle perturbations . to confirm this assumption in a sensitive cell line ,", "exponentially growing ch1 cells were treated with the compounds for 24 h , stained with propidium iodide , and analyzed for their dna content by fluorescence - activated cell sorting ( facs ) .", "surprisingly , the metal - free ligands l1l3 turned out to exert only modest effects on cell cycle distribution , with a slight increase of the g2/m fraction from 32% in the untreated control to 53% by 20 m l2 as the strongest effect observed in this setting ( higher concentrations of this compound led to disintegration of cells already after 24 h ) .", "however , the less cytotoxic ruthenium complexes 12a and 13a , both bearing substituted ligands ( l2 and l3 correspondingly ) , cause a more pronounced g2/m phase arrest , as reflected by an increase of this cell fraction to 65% at 80 m and 59% at 40 m , respectively , and a concomitant decrease of the g0/g1 fraction to 21% and 24% ( compared to 42% in controls ) , whereas ruthenium complex 11a bearing an unsubstituted ligand l1 is devoid of activity on the cell cycle . on the other hand", ", the osmium complexes do not generally show stronger effects on the cell cycle than the metal - free ligands , perhaps with the exception of 13b , which induces an increase of the g2/m fraction up to 53% at 80 m , accompanied by a decline of the g0/g1 fraction to 31% ( see figure 8) .", "concentration - dependent effects of metal - free ligands ( top ) , and the corresponding ruthenium ( middle ) and osmium complexes ( bottom ) , on the cell cycle distribution of ch1 cells ( (  ) g0/g1 , (  ) s , and (  ) g2/m phase ) .", "although the lack of generally pronounced cell cycle effects does not argue for a strong role of cdk inhibition in the mechanism of action of the investigated compounds , inhibitory potencies were studied in cell - free experiments with two recombinant cdk / cyclin complexes , by means of the histone h1 kinase assay .", "results reveal that all compounds are capable of inhibiting kinase activities in a concentration - dependent manner , being more effective on cdk2/cyclin e than on cdk1/cyclin b ( see figure 9 ) .", "in contrast to the observed cell cycle effects , but in accordance with antiproliferative activities , cdk - inhibitory potency of the metal - free ligands is consistently higher than that of the metal complexes . in particular , only l1l3 effectively inhibit cdk2/cyclin e in low concentrations ( 1 m or 10 m ) , whereas all complexes require higher concentrations to exert > 50% inhibitory effects . as in the mtt assay ,", "differences between the effects of corresponding ruthenium and osmium complexes are minor , compared to the differences from those of the metal - free ligands .", "concentration - dependent effects of metal - free ligands ( l1l3 ) as well as corresponding ruthenium ( 11a13a ) and osmium complexes ( 11b13b ) , on kinase activities of recombinant cdk1/cyclin b ( top ) and cdk2/cyclin e ( bottom ) .", "the known multistep synthesis of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which we have modified , essentially afforded three organic compounds ( l1l3 ) that possess cdk - inhibiting properties .", "these were used as bidentate ligands , and six novel organometallic complexes of the general formula [ mcl(-p - cymene)(l)]cl , where m = ru ( 11a , 12a , 13a ) or os ( 11b , 12b , 13b ) and l = l1l3 , have been synthesized and comprehensively characterized using spectroscopic and x - ray diffraction methods .", "complexation of l1l3 with ruthenium or osmium yielded compounds with increased solubility in the biological medium , yet lowered antiproliferative activity in human cancer cell lines .", "modulation of biological activities by substitution at the ligands can be accomplished in the metal - free molecules and their metal complexes in a similar way .", "the known cdk - inhibitory activity of the ligands could be confirmed in cell - free experiments , in particular in cdk2/cyclin e , and their stronger effects on cdk activity parallel their higher capacity of inhibiting cancer cell growth in vitro , compared to their metal complexes .", "nevertheless , the lack of pronounced effects on the cell cycle of chemosensitive ovarian cancer cells argues against a major role for inhibition of cell growth , at least in this setting ."], "abstract_text": ["<S> six organometallic complexes of the general formula [ miicl(6-p - cymene)(l)]cl , where m = ru ( 11a , 12a , 13a ) or os ( 11b , 12b , 13b ) and l = 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) have been synthesized . </S>", "<S> the latter are known as potential cyclin - dependent kinase ( cdk ) inhibitors . </S>", "<S> all compounds have been comprehensively characterized by elemental analysis , one- and two - dimensional nmr spectroscopy , uv  vis spectroscopy , esi mass spectrometry , and x - ray crystallography ( 11b and 12b ) . </S>", "<S> the multistep synthesis of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) , which was reported by other researchers , has been modified by us essentially ( e.g. , the synthesis of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) via 5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) ; the synthesis of 1-methoxymethyl-2,3-diaminobenzene ( 5 ) by avoiding the use of unstable 2,3-diaminobenzyl alcohol ; and the activation of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acids ( 1 , 3 ) through the use of an inexpensive coupling reagent , n , n-carbonyldiimidazole ( cdi ) ) . </S>", "<S> stabilization of the 7b tautomer of methoxymethyl - substituted l3 by coordination to a metal(ii ) center , as well as the nmr spectroscopic characterization of two tautomers 7b - l3 and 4b-l3 in a metal - free state are described . </S>", "<S> structure  </S>", "<S> activity relationships with regard to cytotoxicity and cell cycle effects in human cancer cells , as well as cdk inhibitory activity , are also reported . </S>"], "labels": null, "section_names": ["Introduction", "Experimental Section", "Results and Discussion", "Conclusion"], "sections": [["tumor - associated cell cycle defects , manifesting in unscheduled proliferation , and the associated genomic and chromosomal instabilities are mediated by misregulation of cyclin - dependent kinases ( cdks ) .", "because of the main role in the division cycle , cdks have been recognized as targets for anticancer therapy .", "many small - molecule organic compounds , which have been identified as cdk modulators , are currently in preclinical or clinical development . however , no cdk inhibitors have gained marketing approval , despite 20 years of scientific investigation .", "several studies have shown synergism when cdk inhibitors were combined with organic ( e.g. , doxorubicin , paclitaxel ) and inorganic ( e.g. , cisplatin , carboplatin ) cytotoxic drugs .", "the first publications have appeared recently and include fe , cu , and pt complexes with cdk inhibitors derived from 6-benzylaminopurine , metal - based indolo-[3,2-d]benzazepines ( paullones ; ga , cu , ru , and os ) , and indolo-[3,2-c]quinolines ( ru , os ) . another class of compounds potentially suitable for targeted metal - based chemotherapy is that of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines .", "these have been documented recently as potent cdk1 inhibitors with antiproliferative activity in hela ( cervical carcinoma ) , hct116 ( colon carcinoma ) , and a375 ( melanoma ) human cancer cell lines .", "comparison of cdk1 inhibitory activity with the inhibiting activity in four other protein kinases ( vegf - r2 , her2 , aurora - a , and ret ) revealed selectivity for cdk1 . structural modifications consisting of a replacement of both bicyclic rings in 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines by monocycles while retaining the imidazolyl pyrazole core have been proposed in order to obtain inhibitors with improved pharmacokinetic and solubility properties .", "the most promising were suggested to be 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( see chart 1 ) .", "the inspection of substitution patterns on the benzimidazole moiety and structure  activity relationships revealed that a methoxymethyl group in position 7b ( 4b ) is favorable for cdk1 inhibiting potency .", "the role of the pyrazole nh group is also of note , since its methylation led to a significant reduction of cdk1 activity .", "the effect of various heteroaryl groups in position 5a was also remarkable for the development of more - effective cdk inhibitors and antiproliferative agents .", "our previous experience with metal - based indolo-[3,2-d]benzazepines prompted the use of the half - sandwich metal - arene moiety as a suitable scaffold to which 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines may be attached .", "organometallic compounds [ m(-arene)(yz)x ] ( where m = ru , os ) exhibit promising anticancer activity and are the focus of attention for several groups .", "these compounds have shown activity toward classic ( dna ) and nonclassic ( e.g. , cdks ) targets in anticancer chemotherapy .", "herein , we report ( i ) the modified synthetic approach to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( recall chart 1 : l1 , x = h , y = h ; l2 , x = br ; y = h ; l3 , x = br ; y = ch2och3 ) ; ( ii ) the synthesis and characterization of a new family of organoruthenium(ii ) ( 11a , 12a , 13a ) and osmium(ii ) ( 11b , 12b , 13b ) complexes of the general formula [ mcl(-p - cymene)l]cl , where l = 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( chart 1 ) ; ( iii ) stabilization of the 7b tautomer of methoxymethyl - substituted l3 by metal coordination as well as ( iv ) nmr spectroscopic characterization of two tautomers 7b - l3 and 4b-l3 in a metal - free state ; and ( v ) cell cycle effects , as well as the antiproliferative and cdk inhibitory activities of both metal - free ligands and organometallic complexes ."], ["1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) was obtained via the oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base , followed by acidification with 37% hcl .", "solvents [ toluene , ethanol ( etoh ) , tetrahydrofuran ( thf ) , diethyl ether ( et2o ) ] were dried using standard procedures .", "3-methyl-1h - pyrazolo[3,4-b]pyridine ( 10.9 g , 0.08 mol ) and anhydrous sodium acetate ( 10.21 g , 0.13 mol ) were suspended in glacial acetic acid ( 42 ml ) .", "bromine ( 20.42 g , 0.13 mol ) was added , and the resulting mixture was stirred at room temperature for 22.5 h and then at 110115 c for 2.53 h. afterward , water ( 300350 ml ) was added and the mixture was stirred at room temperature .", "the formed light yellow precipitate was filtered off and dried in vacuo at 4050 c .", "yield : 17 g. the raw product was used without further purification in the next step .", "purification by column chromatography afforded a white powder ( sio2 , etoac , rf = 0.79 ; 12.5 g , 72.6% yield ) .", "esi - ms in meoh ( positive ) : m / z 213 [ m+h ] , 235 [ m+na ] , 253 [ m+k ] ; ( negative ) : m / z 211 [ m  h ] .", "h nmr ( 500.32 mhz , meoh - d4 ) :  8.55 ( d , 1h , j = 2.16 hz , ch ) , 8.43 ( d , 1h , j = 2.15 hz , ch ) , 2.56 ( s , 3h , ch3 ) ppm .", "h nmr ( 500.32 mhz , dmso - d6 ) :  13.42 ( brs , 1h , nh ) , 8.54 ( d , 1h , j = 2.19 hz , ch ) , 8.53 ( d , 1h , j = 2.18 hz , ch ) , 2.49 ( s , 3h , ch3 ) ppm .", "colorless crystals of 20.5h2o suitable for x - ray diffraction ( xrd ) study were grown in etoac ( see figure s1 in the supporting information ) . to sodium hydroxide ( 9.54 g , 0.24 mol ) in water ( 150 ml ) was added the raw product 2 ( 7.1 g , 0.03 mol ) . after a dropwise addition of kmno4 ( 16.98 g , 0.11 mol ) in water ( 300 ml ) at 100 c over 2 h , the reaction mixture was further heated for 1 h. mno2 was filtered off from the hot reaction mixture and washed with hot water .", "400 ml , and the yellow solution was acidified to ph 2 , using concentrated hcl .", "yield : 2.28 g. the crude hydrochloride of 3 was used without further purification in the next step .", "esi - ms in meoh ( positive ) : m / z 243 [ m+h ] , 265 [ m+na ] , 287 [ m+2na  h ] ; ( negative ) : m / z 241 [ m  h ] .", "h nmr ( 500.32 mhz , meoh - d4 ) :  8.69 ( d , 1h , j = 2.22 hz , ch ) , 8.68 ( d , 1h , j = 2.22 hz , ch ) ppm .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.65 ( s , 1h , nh ) , 13.45 ( brs , 1h , nh ) , 8.72 ( d , 1h , j = 2.23 hz , ch ) , 8.58 ( d , 1h , j = 2.25 hz , ch ) ppm .", "nah ( 1.75 g , 0.07 mol ) was suspended in dry thf ( 200 ml ) . a solution of 2-amino-3-nitrobenzyl alcohol ( 5.23 g , 0.03 mol ) in dry thf ( 100 ml )", "was added dropwise at 0 c and the mixture was stirred at the same temperature for 15 min . after a dropwise addition of mei ( 11.5 g , 0.08 mol ) ,", "stirring was continued at room temperature for 3 h. a saturated aqueous solution of nahco3 ( 300 ml ) and meoh ( 300 ml ) then were added .", "the organic phase was dried over na2so4 , filtered , and evaporated to yield a red oil ( 4.85 g ) .", "the raw product was purified by column chromatography ( sio2 , etoac , or etoac / hexane 1/1 , first fraction , a red - orange oil crystallized to form a red solid at 4 c ; yield : 3.68 g , 65% ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  8.00 ( d , 1h , j = 8.71 hz ,", "c6h3 ) , 7.09 ( br , 2h , nh2 ) , 6.67 ( t , 1h , j = 8.45 hz , c6h3 ) , 4.48 ( s , 2h , ch2 ) , 3.31 ( s , 3h , ch3 ) ppm .", "a mixture of 4 ( 1.4 g , 0.008 mol ) and 10% pd / c ( 0.18 g ) in dry etoh ( 55 ml ) was stirred under hydrogen atmosphere at room temperature for 1824 h. the catalyst was removed by filtration through gf-3-filter under argon and washed with dry etoh ( 5070 ml ) .", "the filtrate was evaporated in vacuo to give a light - orange solid ( 1.17 g , 100% yield ) , which was used immediately in the next step .", "h nmr ( 500.32 mhz , dmso - d6 ) :  6.52 ( dd , 1h , j = 1.87 hz , j = 7.25 , c6h3 ) , 6.426.36 ( m , 2h , c6h3 ) , 4.48 ( br , 2h , nh2 ) , 4.31 ( br , 4h , nh2+ch2 ) , 3.24 ( s , 3h , ch3 ) ppm .", "n , n-carbonyldiimidazole ( cdi , 4.16 g , 0.026 mol ) was added in small portions to 1 ( 2.34 g ) in dry dmf ( 12 ml ) .", "the mixture was stirred at room temperature for 2024 h. water ( 5 ml ) then was added and the suspension was stirred until all co2 was ceased .", "the white precipitate was filtered off , washed with water ( 35 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .", "esi - ms in methanol ( positive ) : m / z 214 [ m+h ] , 237 [ m+na ] ; ( negative ) : m / z 212 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.95 ( brs , 1h , nh ) , 8.91 ( s , 1h , chim ) , 8.74 ( dd , 1h , j = 1.61 hz , j = 4.51 hz , chpy ) , 8.63 ( dd , 1h , j = 1.59 hz , j = 8.12 hz , chpy ) , 8.14 ( t , 1h , j = 1.23 hz , chim ) , 7.51 ( dd , 1h , j = 4.46 hz , j = 8.06 hz , chpy ) , 7.20 ( s , 1h , chim ) ppm .", "n , n-carbonyldiimidazole ( cdi , 16.2 g , 0.1 mol ) was added in small portions to crude 3 ( 11.58 g ) in dry dmf ( 70 ml ) .", "the mixture was stirred at room temperature for 2024 h. water ( 10 ml ) then was added and the suspension was stirred until all co2 was ceased .", "the white precipitate was filtered off , washed with water ( 1015 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .", "esi - ms in methanol ( positive ) : m / z 315 [ m+na ] ; ( negative ) : m / z 291 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.95 ( brs , 1h , nh ) , 8.89 ( s , 1h , chim ) , 8.83 ( d , 1h , j = 2.12 hz , chpy ) , 8.75 ( d , 1h , j = 2.07 hz , chpy ) , 8.13 ( s , 1h , chim ) , 7.20 ( s , 1h , chim ) ppm .", "the solution of 1,2-diaminobenzene ( 0.5 g , 4.67 mmol ) in dry dmf ( 2 ml ) was added to the suspension of 6 ( 0.94 g , 4.41 mmol ) in dry dmf ( 13 ml ) , and this reaction mixture was heated under argon at 85 c for 5 h. dmf then was evaporated in vacuo at 50 c and water ( 1012 ml ) was added .", "the light - yellow precipitate was filtered off and dried in vacuo at 50 c .", "yield : 0.86 g. the raw product was used without purification in the next step .", "esi - ms in methanol ( positive ) : m / z 255 [ m+h ] , 277 [ m+na ] ; ( negative ) : m / z 253 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.30 ( brs , 1h , nhpz ) , 9.73 ( brs , 1h , conh ) , 8.64 ( dd , 1h , j = 1.59 hz , j = 4.44 hz , chpy ) , 8.56 ( dd , 1h , j = 1.53 hz , j = 8.02 hz , chpy ) , 7.397.36 ( m , 2h , chpy+chbz ) , 6.97 ( td , 1h , j = 1.31 hz , j = 7.73 hz , chbz ) , 6.82 ( dd , 1h , j = 1.2 hz , j = 7.89 hz , chbz ) , 6.64 ( td , 1h , j = 1.2 hz , j = 7.67 hz , chbz ) , 4.93 ( s , 2h , nh2 ) ppm .", "( 10 ml ) was added to the suspension of 7 ( 2.28 g , 7.8 mmol ) in dry dmf ( 10 ml ) , and this mixture was heated under argon at 85 c for 7 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .", "yield : 2.2 g. the raw product was used without purification in the next step .", "esi - ms in methanol ( positive ) : m / z 333 [ m+h ] , 355 [ m+na ] ; ( negative ) : m / z 331 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.54 ( brs , 1h , nhpz ) , 9.80 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.26 hz , chpy ) , 8.69 ( d , 1h , j = 2.22 hz , chpy ) , 7.34 ( dd , 1h , j = 0.98 hz , j = 7.88 hz , chbz ) , 6.99 ( td , 1h , j = 1.43 hz , j = 8.03 hz , chbz ) , 6.82 ( dd , 1h , j = 1.22 hz , j = 7.96 hz , chbz ) , 6.64 ( td , 1h , j = 1.19 hz , j = 7.73 hz , chbz ) , 4.94 ( s , 2h , nh2 ) ppm .", "a mixture of 5 ( 1.17 g , 7.69 mmol ) and 7 ( 2.1 g , 7.2 mmol ) in dry dmf ( 45 ml ) was heated under argon at 85 c for 20 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .", "yield : 2.3 g. the product was used without further purification in the next step .", "esi - ms in methanol ( positive ) : m / z 399 [ m+na ] ; ( negative ) : m / z 375 [ m  h ] .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.54 ( brs , 1h , nhpz ) , 9.85 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.13 hz , chpy ) , 8.68 ( d , 1h , j = 2.05 hz , chpy ) , 7.29 ( d , 1h , j = 7.56 hz , chbz ) , 7.03 ( d , 1h , j = 7.29 hz , chbz ) , 6.65 ( t , 1h , j = 7.79 hz , chbz ) , 4.78 ( brs , 2h , nh2 ) , 4.42 ( s , 2h , ch2 ) , 3.29 ( s , 3h , ch3 ) ppm . the raw product 8 ( 0.86 g ) was heated in a glacial acetic acid ( 10 ml ) at 125 c for 2.5 h. the solvent was evaporated under reduced pressure and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 4/1 , 25 ml ) , filtered off , and dried in vacuo to give l1 as a white powder ( 0.4 g ) .", "the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac , rf = 0.58 ) to give an additional amount of l1 ( 0.22 g ) .", "calcd for 110.15h2o0.1etoac ( mr = 246.76 g / mol ) : c , 65.22 ; h , 4.13 ; n , 28.38 . found : c , 65.56 ; h , 3.90 ; n , 28.39 .", "esi - ms in methanol ( positive ) : m / z 237 [ m+h ] , 259 [ m+na ] ; ( negative ) : m / z 235 [ m  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 232 ( 25618 ) , 275 ( 15198 ) , 324 ( 22333 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.19 ( brs , 1h , h1a ) , 13.11 ( brs , 1h , h1b ) , 8.85 ( dd , 1h , j = 1.5 hz ,", "j = 8.1 hz , h4a ) , 8.66 ( dd , 1h , j = 1.5 hz , j = 4.5 hz , h6a ) , 7.75 ( d , 1h , j", "= 7.3 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.7 hz , h4b or h7b ) , 7.39 ( dd , 1h , j = 4.5 hz , j = 8.0 hz , h5a ) , 7.24 ( m , 2h , h5b+h6b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  153.13 ( c8a ) , 150.22 ( c6a ) , 146.99 ( c2b ) , 144.34 ( c8b or c9b ) , 136.06 ( c3a ) , 134.68 ( c8b or c9b ) , 131.82 ( c4a ) , 123.35 ( c5b or c6b ) , 122.11 ( c5b or c6b ) , 119.44 ( c4b or c7b ) , 118.65 ( c5a ) , 113.33 ( c9a ) , 112.01 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) :  166.2 ( n1a ) , 121.3 ( n1b ) ppm . the raw product 9 ( 2.2 g ) was heated in a glacial acetic acid ( 30 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 7/1 , 50 ml ) , filtered off and dried in vacuo to give l2 as a white powder ( 1.15 g ) .", "the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac / hexane 2/1 , rf = 0.6 ) to give an additional amount of the product ( 0.27 g ) . yield : 1.42 g , 58% based on 7 .", "calcd for 120.25h2o0.04etoac ( mr = 322.17 g / mol ) : c , 49.06 ; h , 2.76 ; n , 21.74 .", "esi - ms in methanol ( positive ) : m / z 315 [ m+h ] , 337 [ m+na ] ; ( negative ) : m / z 313 [ m  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 234 ( 28154 ) , 282 ( 17628 ) , 335 ( 17198 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.43 ( brs , 1h , h1a ) , 13.19 ( brs , 1h , h1b ) , 8.99 ( d , 1h , j = 2.2 hz , h4a ) , 8.75 ( d , 1h , j = 2.3 hz , h6a ) , 7.77 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.26 ( m , 2h , h5b+h6b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  151.49 ( c8a ) , 150.66 ( c6a ) , 146.42 ( c2b ) , 144.24 ( c8b or c9b ) , 135.61 ( c3a ) , 134.67 ( c8b or c9b ) , 133.35 ( c4a ) , 123.54 ( c5b or c6b ) , 122.27 ( c5b or c6b ) , 119.55 ( c4b or c7b ) , 114.87 ( c5a or c9a ) , 113.49 ( c5a or c9a ) , 112.09 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) :  167.5 ( n1a ) , 121.3 ( n1b ) ppm .", "the raw product 10 ( 2.3 g ) was heated in a glacial acetic acid ( 40 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue dried in vacuo at 50 c . after washing with ch2cl2 ( 30 ml ) , ch2cl2/meoh ( 2/1 , 57 ml ) the gray product was purified by column chromatography ( sio2 , etoac , rf = 0.68 ) to give a white powder ( 0.9 g ) . the filtrates were evaporated and the remaining solid was purified by column chromatography to give an additional amount of the product ( 0.3 g ) .", "calcd for 13 : c , 50.29 ; h , 3.38 ; n , 19.55 . found : c , 50.03 ; h , 3.19 ; n , 19.19 .", "esi - ms in methanol ( positive ) : m / z 381 [ m+na ] ; ( negative ) : m / z 357 [ m  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 235 ( 29776 ) , 282 ( 17544 ) , 337 ( 16958 ) .", "nmr characterization of 7b - l3 and 4b-l3 tautomers ( 1/1.3 ) in dmso - d6 : h nmr ( 500.32 mhz , dmso - d6 ) , 7b - l3 :  14.47 ( brs , 1h , h1a ) , 13.25 ( brs , 1h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.72 ( dd , 1h , j = 1.8 hz ,", "j = 6.8 hz , h4b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.80 ( s , 2h , h10b ) , 3.37 ( s , 3h , h11b ) ppm .", "h nmr ( 500.32 mhz , dmso - d6 ) , 4b-l3 :  14.43 ( brs , 1.3h , h1a ) , 13.22 ( brs , 1.3h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.47 ( dd , 1.3h , j = 1.2 hz ,", "j = 7.7 hz , h7b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.96 ( s , 2.6h , h10b ) , 3.44 ( s , 3.9h , h11b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) , 7b - l3 :  151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.70 ( c2b ) , 144.46 ( c9b ) , 135.62 ( c3a+c3a ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 133.29 ( c8b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.95 ( c7b ) , 122.16 ( c5b or c6b ) , 118.97 ( c4b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 70.49 ( c10b ) , 57.95 ( c11b ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) , 4b-l3 :  151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.13 ( c2b ) , 142.50 ( c9b ) , 135.62 ( c3a+c3a ) , 134.42 ( c8b ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 129.38 ( c4b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.16 ( c5b or c6b ) , 120.81 ( c5b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 111.17 ( c7b ) , 69.90 ( c10b ) , 58.35 ( c11b ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  167.6 ( n1a+n1a ) , 121.4 ( n1b+n1b ) ppm .", "a mixture of l1 ( 54.7 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was evaporated up to ca .", "calcd for 11ah2o ( mr = 559.45 g / mol ) : c , 49.38 ; h , 4.50 ; n , 12.52 ; cl , 12.67 .", "found : c , 49.68 ; h , 4.25 ; n , 12.11 ; cl , 12.26 .", "esi - ms in methanol ( positive ) : m / z 470 [ m  hcl  cl ] , 507 [ m  cl ] , 528 [ m  hcl+na ] ; ( negative ) : m / z 468 [ m2hcl  h ] , 505 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : 251 ( 16335 ) , 299 ( 26663 ) , sh 347 ( 16012 ) .", "vis ( h2o ) , max , nm (  , m cm ) : sh 245 ( 10728 ) , 294 ( 18597 ) , 360 ( 9666 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.91 ( brs , 1h , h1b ) , 9.17 ( d , 1h , j = 7.7 hz , h4a ) , 8.82 ( d , 1h , j = 3.8 hz , h6a ) , 8.11 ( d , 1h , j = 7.1 hz , h4b ) , 7.84 ( d , 1h , j = 8.5 hz , h7b ) , 7.58 ( m , 3h , h5a+h5b+h6b ) , 6.43 ( m , 2h , h2c+h6c ) , 6.31 ( d , 1h , j = 5.3 hz , h3c or h5c ) , 6.12 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 2.54 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.21 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.6 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.6 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  153.97 ( c8a ) , 150.45 ( c6a ) , 146.52 ( c2b ) , 141.37 ( c9b ) , 134.70 ( c8b ) , 134.64 ( c3a ) , 131.48 ( c4a ) , 125.39 ( c5b or c6b ) , 124.92 ( c5b or c6b ) , 119.56 ( c5a ) , 117.85 ( c4b ) , 113.96 ( c7b ) , 111.54 ( c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.73 ( c2c or c6c ) , 82.15 ( c3c or c5c ) , 80.84 ( c3c or c5c ) , 31.12 ( c7c ) , 22.19 ( c8c+c9c ) , 19.17 ( c10c ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  128.7 ( n1b ) ppm .", "a mixture of l1 ( 42.5 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 11bh2o ( mr = 648.61 g / mol ) : c , 42.59 ; h , 3.88 ; n , 10.79 . found : c , 42.56 ; h , 3.57 ; n , 10.97 .", "esi - ms in methanol ( positive ) : m / z [ m  hcl  cl ] , 596 [ m  cl ] , 618 [ m  hcl+na ] ; ( negative ) : m / z 595 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 252 ( 17048 ) , 299 ( 20228 ) , 343 ( 19879 ) .", "h nmr ( 500.32 mhz , meoh - d4 ) :  8.99 ( dd , 1h , j = 1.3 hz ,", "j = 8.1 hz , h4a ) , 8.77 ( dd , 1h , j = 1.4 hz ,", "j = 4.8 hz , h6a ) , 7.96 ( dd , 1h , j = 2.0 hz , j = 6.4 hz , h4b ) , 7.80 ( dd , 1h , j", "= 1.9 hz , j = 6.1 hz , h7b ) , 7.647.59 ( m , 3h , h5a+h5b+h6b ) , 6.66 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.59 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.43 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.43 ( sep , 1h , j = 6.9 hz , h7c ) , 2.38 ( s , 3h , h10c ) , 0.96 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.92 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , meoh - d4 ) :  153.25 ( c8a ) , 148.91 ( c2b ) , 147.42 ( c6a ) , 140.49 ( c9b ) , 135.15 ( c3a ) , 134.25 ( c8b ) , 132.07 ( c4a ) , 125.47 ( c5b or c6b ) , 124.81 ( c5b or c6b ) , 118.48 ( c5a ) , 116.89 ( c4b ) , 112.98 ( c7b ) , 112.92 ( c9a ) , 97.71 ( c4c ) , 94.88 ( c1c ) , 76.44 ( c2c or c6c ) , 74.99 ( c2c or c6c ) , 71.93 ( c3c or c5c ) , 70.44 ( c2c or c6c ) , 31.37 ( c7c ) , 21.35 ( c8c or c9c ) , 21.09 ( c8c or c9c ) , 17.84 ( c10c ) ppm .", "crystals of 11b4h2o suitable for xrd study have been obtained from a solution of 11b in ethanol .", "a mixture of l2 ( 73.2 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 12ah2o ( mr = 638.35 g / mol ) : c , 43.28 ; h , 3.79 ; n , 10.97 .", "esi - ms in methanol ( positive ) : m / z 548 [ m  hcl  cl ] , 608 [ m  hcl+na ] ; ( negative ) : m / z 549 [ m2hcl  h ] , 582 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 254 ( 17778 ) , 303 ( 29953 ) , 351 ( 17387 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  14.34 ( brs , 1h , h1b ) , 9.21 ( s , 1h , h4a ) , 8.73 ( s , 1h , h6a ) , 8.06 ( d , 1h , j = 7.6 hz , h4b ) , 7.79 ( d , 1h , j = 8.5 hz , h7b ) , 7.52 ( m , 2h , h5b+h6b ) , 6.32 ( d , 2h , j = 5.8 hz , h2c+h6c ) , 6.22 ( d , 1h , j = 6.2 hz , h3c or h5c ) , 6.03 ( d , 1h , j = 6.1 hz , h3c or h5c ) , 2.52 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.18 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.8 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  154.04 ( c8a ) , 151.24 ( c6a ) , 146.57 ( c2b ) , 141.37 ( c9b ) , 134.63 ( c8b ) , 133.43 ( c3a ) , 131.82 ( c4a ) , 125.33 ( c5b or c6b ) , 124.88 ( c5b or c6b ) , 117.78 ( c4b ) , 114.72 ( c5a or c9a ) , 113.85 ( c7b ) , 112.48 ( c5a or c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.69 ( c2c or c6c ) , 82.16 ( c3c or c5c ) , 80.76 ( c3c or c5c ) , 31.09 ( c7c ) , 22.20 ( c8c or c9c ) , 22.17 ( c8c or c9c ) , 19.16 ( c10c ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  127.3 ( n1b ) ppm .", "a mixture of l2 ( 56.4 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 12bh2o ( mr = 727.51 g / mol ) : c , 37.97 ; h , 3.33 ; n , 9.63 . found : c , 37.82 ; h , 3.02 ; n , 9.33 .", "esi - ms in methanol ( positive ) : m / z 638 [ m  hcl  cl ] , 696 [ m  hcl+na ] ; ( negative ) : m / z 672 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 253 ( 21638 ) , 302 ( 30505 ) , 357 ( 21813 ) .", "h nmr ( 500.32 mhz , meoh - d4 ) :  9.08 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.1 hz , h6a ) , 7.96 ( dd , 1h , j = 2.1 hz ,", "j = 6 hz , h4b ) , 7.80 ( dd , 1h , j = 2.1 hz ,", "j = 6.1 hz , h7b ) , 7.63 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.63 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.48 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.22 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.42 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.95 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , meoh - d4 ) :  153.04 ( c8a ) , 152.49 ( c6a ) , 148.15 ( c2b ) , 140.47 ( c9b ) , 134.58 ( c3a ) , 134.19 ( c8b ) , 131.13 ( c4a ) , 125.79 ( c5b or c6b ) , 125.05 ( c5b or c6b ) , 116.99 ( c4b ) , 115.39 ( c5a or c9a ) , 113.09 ( c7b ) , 112.11 ( c5a or c9a ) , 98.54 ( c4c ) , 95.52 ( c1c ) , 75.73 ( c2c or c6c ) , 75.25 ( c2c or c6c ) , 71.87 ( c3c or c5c ) , 69.99 ( c3c or c5c ) , 31.41 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.84 ( c10c ) ppm .", "crystals of 12b and 12b2ch3oh2h2o suitable for xrd study have been obtained from a solution of 12b in methanol .", "a mixture of l3 ( 64 mg , 0.18 mmol ) and [ rucl2(-p - cymene)]2 ( 50 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .", "calcd for 13a1.5h2o ( mr = 691.41 g / mol ) : c , 43.43 ; h , 4.23 ; n , 10.13 ; cl , 10.26 .", "found : c , 43.81 ; h , 4.24 ; n , 10.11 ; cl , 10.57 .", "esi - ms in methanol ( positive ) : m / z 592 [ m  hcl  cl ] , 650 [ m  hcl+na ] ; ( negative ) : m / z 591 [ m2hcl  h ] , 628 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , mcm ) : sh 252 ( 18744 ) , 306 ( 28529 ) , 354 ( 16889 ) .", "h nmr ( 500.32 mhz , dmso - d6 ) :  13.88 ( brs , 1h , h1b ) , 9.41 ( s , 1h , h4a ) , 8.68 ( s , 1h , h6a ) , 7.99 ( d , 1h , j = 7.7 hz , h4b ) , 7.49 ( m , 2h , h5b+h6b ) , 6.29 ( m , 2h , h2c+h6c ) , 6.20 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.00 ( d , 1h , j = 5.9 hz , h3c or h5c ) , 4.88 ( s , 2h , h10b ) , 3.39 ( s , 3h , h11b ) , 2.48 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.17 ( s , 3h , h10c ) , 0.93 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.87 ( d , 3h , j = 6.8 hz , h8c or h9c ) ppm .", "c nmr ( 125.81 mhz , dmso - d6 ) :  155.35 ( c8a ) , 150.27 ( c6a ) , 147.43 ( c2b ) , 141.64 ( c9b ) , 133.07 ( c8b ) , 132.68 ( c3a ) , 131.68 ( c4a ) , 124.87 ( c5b or c6b ) , 124.59 ( c5b or c6b ) , 124.53 ( c7b ) , 117.18 ( c4b ) , 114.23 ( c5a or c9a ) , 112.78 ( c5a or c9a ) , 103.74 ( c4c ) , 103.09 ( c1c ) , 85.38 ( c2c or c6c ) , 83.68 ( c2c or c6c ) , 82.13 ( c3c or c5c ) , 81.05 ( c3c or c5c ) , 70.19 ( c10b ) , 58.03 ( c11b ) , 31.04 ( c7c ) , 22.22 ( c8c or c9c ) , 22.09 ( c8c or c9c ) , 19.13 ( c10c ) ppm .", "n nmr ( 50.70 mhz , dmso - d6 ) :  122.9 ( n1b ) ppm .", "the red xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) were obtained from etoh / et2o and an etoh solution of 13a ( long crystallization ) .", "the yellow crystals of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) suitable for xrd study have been obtained from methanolic solution of 13a ( short crystallization ) .", "h nmr ( 13c , 500.32 mhz , dmso - d6 ) :  13.43 ( s , 1h ) , 9.18 ( d , 1h , j = 2.1 hz ) , 8.48 ( d , 1h , j = 2.2 hz ) , 7.94 ( d , 1h , j = 8.2 hz ) , 7.45 ( t , 1h , j = 7.8 hz ) , 7.39 ( d , 1h , j = 7.2 hz ) , 6.16 ( d , 1h , j = 6.0 hz ) , 6.13 ( d , 1h , j = 6.1 hz ) , 6.07 ( d , 1h , j = 5.9 hz ) , 5.89 ( d , 1h , j = 6.4 hz ) , 4.85 ( s , 2h ) , 4.04 ( s , 3h ) , 2.14 ( s , 3h ) , 0.93 ( d , 3h , j = 6.8 hz ) , 0.86 ( d , 3h , j = 6.9", "a mixture of l3 ( 59 mg , 0.17 mmol ) and [ oscl2(-p - cymene)]2 ( 60 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 3 h. ethanol was evaporated up to ca .", "calcd for 13b : c , 39.85 ; h , 3.48 ; n , 9.29 . found : c , 39.60 ; h , 3.32 ; n , 9.20 .", "esi - ms in methanol ( positive ) : m / z 682 [ m  hcl  cl ] , 704 [ m2hcl+na ] ; ( negative ) : m / z 680 [ m2hcl  h ] , 716 [ m  hcl  h ] .", "vis ( methanol ) , max , nm (  , m cm ) : sh 256 ( 19825 ) , 303 ( 24634 ) , 357 ( 18850 ) .", "h nmr ( 500.32 mhz , meoh - d4 ) :  9.33 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.2 hz , h6a ) , 7.91 ( dd , 1h , j = 1.2 hz ,", "j = 7.8 hz , h4b ) , 7.647.58 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.62 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.47 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 4.94 ( q , 2h , j = 12.4 hz , h10b ) , 3.49 ( s , 3h , h11b ) , 2.41 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm . c nmr ( 125.81 mhz , meoh - d4 ) :  153.18 ( c8a ) , 152.16 ( c6a ) , 148.36 ( c2b ) , 140.82 ( c9b ) , 134.35 ( c3a ) , 132.68 ( c8b ) , 131.68 ( c4a ) , 125.63 ( c5b or c6b ) , 124.85 ( c5b or c6b ) , 124.51 ( c7b ) , 116.72 ( c4b ) , 115.24 ( c5a or c9a ) , 112.26 ( c5a or c9a ) , 98.75 ( c4c ) , 95.50 ( c1c ) , 75.98 ( c2c or c6c ) , 75.34 ( c2c or c6c ) , 71.82 ( c3c or c5c ) , 70.29 ( c10b ) , 70.04 ( c3c or c5c ) , 57.17 ( c11b ) , 31.39 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.85 ( c10c ) ppm .", "the yellow crystals of [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75 ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) suitable for xrd study have been obtained from a methanolic solution of 13b .", "elemental analyses ( c , h , n , cl ) were performed by the microanalytical service of the institute of physical chemistry , university of vienna .", "electrospray ionization mass spectrometry ( esi - ms ) was carried out with an esquire 3000 instrument ( bruker daltonics , bremen , germany ) , using solutions of compounds in methanol .", "vis spectra were recorded on a lambda 20 uv  vis spectrophotometer ( perkin  elmer ) , using samples dissolved in methanol ( l1l3 , 11a , 12a , 13a , 11b , 12b , 13b ) and water ( 11a ) over 24 or 48 h , correspondingly . the one - dimensional ( h , c , n ) and two - dimensional spectra ( n , h hsqc , c , h hsqc , c , h hmbc , h , h cosy , h , h tocsy , h , h noesy ( l3 , 12a ) , h , h roesy ( l3 , 11a , 12a , 13a , 13b ) ) were recorded with a bruker model dpx500 ( ultrashield magnet ) system in dmso - d6 ( l1l3 , 11a , 12a , 13a ) , meoh - d4 ( 11b , 12b , 13b ) , d2o ( h nmr , 11a ) , and d2o / dmso - d6 ( h nmr , 12a ) , using standard pulse programs at 500.32 ( h ) , 125.81 ( c ) and 50.70 ( n ) mhz .", "h signals are referenced relative to the solvent signals ( dmso - d6 at 2.51 , meoh - d4 at 3.33 ppm ) .", "single crystals were positioned at distances of 35 , 40 , 40 , 40 , 40 , 40 , and 35 mm from the detector , and 1556 , 1131 , 518 , 1911 , 1176 , 1978 , and 1039 frames were measured , each for 30 , 60 , 30 , 20 , 70 , 60 , and 30 s over 1 scan width for 20.5h2o , 11b4h2o , 12b , 12b2ch3oh2h2o , 13c , 13dch3oh , and 13e0.75ch3oh0.25h2o , correspondingly .", "crystal data , data collection parameters , and structure refinement details are given in table 1 .", "the structures were solved by direct methods and refined by full - matrix least - squares techniques .", "refinement of the structure of 12b revealed that the chloride counteranion occupies two statistically disordered positions with site occupation factor ( sof ) values of 0.57:0.43 , whereas , in 13c , the methoxymethylene group was found to be disordered over two positions with sof values of 0.80:0.20 .", "similarly , the methoxymethylene group of one crystallographically independent complex in 13dch3oh or in both crystallographically independent complexes in 13e0.75ch3oh0.25h2o is disordered with sof values of 0.35:0.65 and 0.60:0.40 , 0.80:0.20 , correspondingly . the disorder was resolved with constrained anisotropic displacement parameters and restrained bond distances using eadp and sadi instructions of shelx97 , respectively .", "structure solution was achieved with shelxs-97 and refinement with shelxl-97 , and graphics were produced with ortep-3 .", "wr2 = { [w(fo  fc)]/[w(fo)]}. gof = { [w(fo  fc)]/(n  p ) } , where n is the number of reflections and p is the total number of parameters refined .", "a549 ( non - small cell lung carcinoma , human ) and sw480 ( colon carcinoma , human ) cells were kindly provided by brigitte marian ( institute of cancer research , department of medicine i , medical university of vienna , austria ) .", "ch1 cells ( ovarian cancer , human ) were a gift from lloyd r. kelland ( crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . ) .", "cells were grown in 75-cm culture flasks ( iwaki ) in a complete medium ( i.e. , minimum essential medium supplemented with 10% heat - inactivated fetal bovine serum , 1 mm sodium pyruvate , 4 mm l - glutamine , and 1% nonessential amino acids from 100 stock ) as adherent monolayer cultures .", "cultures were grown at 37 c under a humidified atmosphere containing 5% co2 and 95% air .", "antiproliferative activity in vitro was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide , fluka ) . for this purpose", ", cells were harvested from culture flasks by the use of trypsin and seeded in complete medium ( 100 l / well ) into 96-well plates ( iwaki ) in densities of 4  10 ( a549 ) , 1.5  10 ( ch1 ) , and 2.5  10 ( sw480 ) viable cells per well .", "test compounds were dissolved in dmso first , appropriately diluted in complete medium , and instantly added to the plates ( 100 l / well ) , where the dmso content did not exceed 0.4% and 1% for the ligands and complexes , respectively . after exposure for 96 h , the medium was replaced with 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) plus 20 l / well mtt solution in phosphate - buffered saline ( 5 mg / ml ) , followed by incubation for 4 h. subsequently , the medium / mtt mixture was removed , and the formazan product formed by viable cells was dissolved in dmso ( 150 l / well ) .", "optical densities were measured with a microplate reader ( tecan spectra classic ) at 550 nm ( and a reference wavelength of 690 nm ) to yield relative quantities of viable cells as percentages of untreated controls , and 50% inhibitory concentrations ( ic50 ) were interpolated from concentration  effect curves .", "calculations are based on at least three independent experiments with triplicates for each concentration level . to study the effects on the cell cycle of exponentially growing ch1 cells by flow cytometric analysis of their relative dna content", ", cells were harvested from culture flasks , seeded in complete medium into 90-mm petri dishes ( 1  10 cells / dish ) and , after recovery for 24 h , exposed to various concentrations of the test compounds for 24 h. for this purpose , test compounds were diluted from dmso stocks with complete medium ( see above ) such that the effective dmso content did not exceed 0.5% . after exposure , treated and control cells were collected by scratching , washed with pbs , and stained with 5 g / ml propidium iodide overnight .", "their fluorescence was measured with a facs calibur instrument ( becton dickinson ) , and the obtained histograms were analyzed with cell quest pro software ( becton dickinson ) .", "at least two independent experiments were performed for each setting , and 2.5 or 3.0  10 cells were measured per sample .", "the cdk - inhibitory capacities of test compounds were studied by a radiochemical assay using recombinant cdk1/cyclin b and cdk2/cyclin e isolated from sf21 insect cells and histone h1 as the substrate for phosphorylation , as described by marko et al .", "briefly , mops - buffered assay mixtures containing the test compound ( with a maximum of 1% dmso ) , the respective kinase / cyclin complex , histone h1 , and 0.4 ci ( -p)atp per sample were incubated for 10 min at 30 c .", "aliquots of the solution were spotted onto phosphocellulose squares , which had been washed three times with 0.75% phosphoric acid , followed by acetone .", "the dried squares were measured in scintillation vials by -counting ( perkin  elmer tri - carb 2800tr ; quanta smart software ) .", "results were obtained in duplicate in at least two independent experiments , and ic50 values were calculated by interpolation ."], ["several routes to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been proposed by johnson & johnson pharmaceutical research & development l.l.c .", "the first one was developed for 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety and involved sulfur - induced benzimidazole ring formation via the treatment of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde with 1,2-diaminobenzene .", "the poor reproducibility of this synthesis prompted the exploration of an alternative way , via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid , which was used for the preparation of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with a substituted benzimidazole moiety .", "the patented route to 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) consists of four steps ( see scheme 1 , steps i  iv ) : oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base with subsequent acidification with h2so4 , esterification of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) in the presence of h2so4 in methanol , bromination of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in an acoh / acona mixture , and hydrolysis of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in the presence of naoh , followed by acidification with hcl .", "reagents and conditions : ( i ) kmno4 , naoh , 3 h , 100 c , h2so4 ; ( ii ) methanol , h2so4 , reflux , 68 h , nahco3 , 42% ( i + ii ) ; ( iii ) br2 , acoh , acona , 115 c , overnight , chromatographic purification , 43% ; ( iv ) naoh , meoh , reflux , 4 h , hcl , 100% ; ( v ) br2 , acoh , acona , 115 c , 2.53 h ; and ( vi ) kmno4 , naoh , 3 h , 100 c , hcl , 2435% ( v + vi ) .", "we performed the synthesis of 3 in two steps ( see scheme 1 , steps v and vi ) : bromination of 3-methyl-1h - pyrazolo[3,4-b]pyridine in acoh / acona mixture and oxidation of crude 5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) by kmno4 in a basic medium , followed by acidification with 37% hcl , with an overall yield of 2435% .", "5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) is a known compound , the synthesis of which is well - documented . for the benzimidazole ring formation , 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene", "the reported synthesis of 1-methoxymethyl-2,3-diaminobenzene ( 5 ) from 2,3-diaminobenzyl alcohol afforded the desired product in 34% yield ( see scheme 2 , step i ) .", "however , the instability and low yield of diamines , as well as the necessity to purify the desired ether using column chromatography , stimulated the search for a more - convenient procedure that was subsequently proposed : etherification of 2-amino-3-nitrobenzyl alcohol , followed by the reduction of 1-methoxymethyl-2-amino-3-nitrobenzene ( 4 ) with 10% pd / c in ethanol under a hydrogen atmosphere afforded 5 in 6575% yield ( see scheme 2 , steps ii and iii ) .", "reagents and conditions : ( i ) nah , mei , dry thf , 0 c , 30 min , room temperature , overnight , chromatographic purification , 34% yield ; ( ii ) nah , mei , dry thf , 0 c , 15 min , room temperature , 3 h , chromatographic purification , 6575% yield ; ( iii ) 10% pd / c , ethanol , h2 , room temperature , 1824 h , 100% yield .", "patented benzimidazole ring formation by cyclization of the 3-carboxyl group of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) with substituted 1,2-diaminobenzenes was realized via amide formation using coupling reagents , followed by treatment with glacial acetic acid .", "hatu ( n , n , n,n-tetramethyl - o-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate ) was used as a coupling reagent .", "three 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( where x = h , br ; and y = h , ch2och3 ) have been synthesized in this work ( see chart 1 ) : 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l1 ) is a new compound that we have prepared as a model ligand for coordination to metals ; 3-(1h - benzimidazol-2-yl)-5-bromo-1h - pyrazolo[3,4-b]pyridine ( l2 ) was previously known as boc - protected compound prepared via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde;5-bromo-3-(4-methoxymethyl-1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l3 ) was synthesized via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) and patented as a potential cdk inhibitor and antiproliferative agent . for the synthesis of l1l3 , we used n , n-carbonyldiimidazole ( cdi ) as an amide - coupling reagent , because it is relatively inexpensive and the side products , carbon dioxide and imidazole , could be easily removed from the reaction mixture .", "cdi - mediated amidation of acids 1 and 3 was performed as shown in scheme 3 ( steps i and ii ) . in the first step ,", "the acyl - imidazolides 6 and 7 were obtained in dry dmf at room temperature and isolated as white solids .", "the yields based on 3-methyl-1h - pyrazolo[3,4-b]pyridine are : 2529% ( 6 ) and 1316% ( 7 ) . in the second step , monoacylation of phenylenediamines ( 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene ( 5 ) ) using acyl - imidazolides was performed in dmf at 8085 c .", "reagents and conditions : ( i ) cdi , dry dmf , room temperature , 2024 h ; ( ii ) 1,2-diaminobenzene or 5 , dry dmf , 8085 c ; 520 h ; and ( iii ) glacial acoh , 120125 c , 2.54.5 h , chromatographic purification .", "amides 810 were used without further purification in ring closure reactions in a glacial acetic acid at 120125 c and afforded the desired 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines : l1 ( 5560% ) , l2 ( 5861% ) , and l3 ( 4451% ) , based on 6 and 7 ( see scheme 3 , step iii ) .", "the reported synthesis of l3 is a one - pot approach for amide formation using hatu with 57% yield , followed by cyclization under acidic conditions ( acetic acid ) with 87% yield .", "thus , the ligands l1 , l2 , and l3 have been prepared in 7 , 8 , and 11 steps , correspondingly .", "finally , the ligands l1l3 were reacted with [ mcl2(-p - cymene)]2 ( where m = ru , os ) in a 2:1 molar ratio in dry ethanol at room temperature to give [ mcl(-arene)(l)]cl complexes ( 11a , 11b , 12a , 12b , 13a , 13b ) in quantitative yields .", "crystallization of [ rucl(-p - cymene)(l3)]cl ( 13a ) in etoh or etoh / et2o resulted in xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) , while the crystallization of 13a in methanol afforded crystals of composition [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) .", "the osmium(ii ) analogue 13e0.75ch3oh0.25h2o was obtained via the crystallization of 13b in methanol . the full assignment of proton , carbon , and nitrogen resonances for l1l3 , 11a , 11b , 12a", ", 12b , 13a , and 13b is quoted in tables s1s3 in the supporting information .", "3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety ( l1 , l2 ) display one set of signals .", "l1 is characterized by three doublets of doublets for h4a , h5a , h6a ( 8.85 , 7.39 , 8.66 ppm , correspondingly ) of pyrazolopyridine moiety , two doublets for h4b , h7b ( 7.54 and 7.75 ppm ) , the overlapped h5b , h6b proton signals in one multiplet ( 7.24 ppm ) for a benzimidazole moiety and two singlet resonances for h1a and h1b ( for atom numbering , see chart 1 ) . the substitution of h5a by bromine ( l2 ) results in reduced multiplicity for the h4a and h6a signals ( two doublets at 8.99 and 8.75 ppm ) , whereas the benzimidazole moiety spectrum remains almost unchanged .", "two singlets were attributed to nh protons and related to pyrazolopyridine ( h1a , at 14.19 ( l1 ) , 14.43 ( l2 ) ppm ) and benzimidazole ( h1b , at 13.11 ( l1 ) , 13.19 ( l2 ) ppm ) moieties .", "nmr spectra for 11a , 11b , 12a , and 12b , where l1 and l2 coordinate as bidentate ligands via n2a and n3b with the formation of a stable five - membered chelate cycle , show one set of signals .", "there was no evidence for monodentate or tridentate coordination of ligands with the formation of dinuclear or polynuclear complexes .", "coordination of l1 and l2 to ruthenium(ii ) made it possible to assign the two doublets to proton resonances of h4b and h7b of the benzimidazole moiety . in the h ,", "h roesy plots , one of them has cross - peaks with a ch cymene ring and the nearest to them is h4b ( e.g. , at 8.11 ( 11a ) , 8.06 ( 12a ) ppm ) .", "a singlet at 14.91 ( 11a ) , 14.34 ( 12a ) ppm was assigned to nh proton and showed no couplings with other atoms .", "the nitrogen resonance shift at 128.7 ( 11a ) , 127.3 ( 12a ) ppm is closer to the benzimidazole nh chemical shift in metal - free ligands ( 121.3 ( l1 , l2 ) ppm ) and , therefore , was assigned as n1b ( see table s3 in the supporting information ) .", "the h1b resonance shows a significant shift by 1.8 and 1.15 ppm for 11a and 12a , respectively , upon ligand coordination ( l1 and l2 ) to the metal(ii)-arene moiety .", "the nearest to the metal binding site pyrazolopyridine proton h1a was not detected for 11a , 11b , 12a , and 12b , and the proton resonance of h1b was also not seen for 11b or 12b .", "l3 displays two sets of signals originated from 7b - l3 and 4b-l3 tautomers ( see chart 1 ) . the signals of pyrazolopyridine moieties are partially overlapped ( e.g. , h1a ( h1a ) at 14.47 and 14.43 ppm , h4a ( h4a ) at 8.99 and 8.98 ppm , h6a ( h6a ) at 8.75 ppm ) , whereas the signals of the benzimidazole moieties are better resolved ( see table s1 in the supporting information ) .", "according to the h , h roesy plot , one of the ch2 groups at 4.80 ppm couples with the nh proton at 13.25 ppm , indicating their attribution to the 7b - l3 tautomer , whereas another nh proton at 13.22 ppm gives a cross - peak with h7b at 7.47 ppm and belongs to 4b-l3 tautomer ( figure 1 ) .", "tautomers 7b - l3 and 4b-l3 are present in solution in 1:1.3 molar ratio and give , for the substituted benzimidazole moiety , two singlets ( h1b at 13.22 ppm , h1b at 13.25 ppm ) , two doublets of doublets ( h7b at 7.47 ppm , h4b at 7.72 ppm ) , one multiplet ( h5b , h6b , h5b , h6b at 7.287.21 ppm ) , two ch2 ( 4.80 ( h10b ) , 4.96 ( h10b ) ppm ) and two ch3 singlets at 3.37 ( h11b ) and 3.44 ( h11b ) ppm .", "the absence of cross - peaks between h7b and h4b in the h , h cosy plot supports their relation to the two different molecules . the binding site in 4b-l3 tautomer is sterically shielded by a methoxymethyl group .", "consistent with this nmr spectra display , only one set of signals is shown for 13a and 13b with the coordination of the 7b - l3 tautomer to ruthenium(ii ) and osmium(ii ) via n2a and n3b ( see scheme 4 ) .", "the preference for coordination of the 7b - l3 tautomer is also confirmed by h , h roesy plots : h4b ( 7.99 ( 13a ) , 7.91 ( 13b ) ppm ) has couplings with ch protons of cymene ring .", "the cross - peak between h10b at 4.88 ppm ( 13a ) and nh at 13.88 ppm ( 13a ) enabled the assignment of this singlet to a benzimidazole moiety ( h1b ) .", "in addition , the chemical shifts for the c atoms of the benzimidazole moiety ( c4b , c7b , c5b , c6b ) of the coordinated ligand and metal - free 7b - l3 tautomer are very similar ( see table s2 in the supporting information ) .", "the nitrogen resonance at 122.9 ppm ( 13a ) compares well to the benzimidazole nh chemical shift in metal - free l3 at 121.4 ppm . as for 11a , 11b , 12a , and", "12b , the nearest to the coordination place pyrazolopyridine proton h1a resonance was not detected .", "according to the h , h roesy plots of 11a , 12a , 13a , and 13b only ch cymene ring protons have couplings with the nearest h4b benzene ring proton , suggesting that the isopropyl or methyl groups are further away from the h4b proton .", "the results of the xrd studies of [ oscl(-p - cymene)(l1)]cl4h2o ( 11b4h2o ) , [ oscl(-p - cymene)(l2)]cl ( 12b ) , [ oscl(-p - cymene)(l2)]cl2ch3oh2h2o ( 12b2ch3oh2h2o ) , [ rucl(-p - cymene)(l3h ) ] ( 13c ) , [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) and [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) are shown in figures 2 , figure s2 in the supporting information , and figures 36 , respectively .", "all complexes have a typical  three - legged piano - stool  geometry of ruthenium(ii ) and osmium(ii ) arene complexes , with an  -bound p - cymene ring forming the seat and three other donor atoms ( two nitrogens n1 and n5 of the corresponding 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine and one chlorido ligand ) as the legs of the stool . selected bond distances and angles are quoted in the figure captions .", "all complexes crystallize as racemates , because of the presence of the stereogenic metal center .", "fragment of the crystal structure of 11b4h2o showing nitrogen atoms n3 and n4 , which act as proton donors in intermolecular hydrogen bonding interactions n3hcl2 [ n3cl2 3.067(7 )  , n3hcl2 177.3 ] and n4ho3 [ n4o3 2.671(9 )  , n4ho3 170.9 ] ; thermal ellipsoids have been drawn at 50% probability level .", "selected bond lengths and angles : os  cl1 , 2.421(2 )  ; os ", "c(arene)av , 2.198(32 )  ; c1n2 , 1.349(11 )  ; n2n1 , 1.366(10 )  ; n1c6 , 1.357(11 )  ; c6c7 , 1.428(12 )  ; c7n5 , 1.345(11 )  ; n5c13 , 1.391(11 )  ; n1os  n5 , 75.6(3) ; n1os  cl1 , 85.5(2) ; n5os  cl1 , 83.4(2). the bidentate 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which can have different substituents in positions 5a and 7b , reveal different acid  base properties .", "it can act as a neutral organic ligand , with nitrogen atoms n2 and n4 as proton donors involved in intermolecular hydrogen bonding interactions n4ho1(x + 1 , y + 1 , z + 2 ) [ n4o1 , 2.730(4 )  ; n4ho1 , 176.1 ] and n2ho3(x + 2 , y + 1 , z + 1 [ n2o3 , 2.697(4 )  ; n4ho1 , 172.3 ] with one methanol and one water molecule , respectively , as is the case for 12b2ch3oh2h2o ( figure 3 ) or n2hcl2(cl2x ) and n4hcl1(x , y , z + 2 ) [ n4cl1 , 3.213(9 )  ; n4ho1 , 162.91 ] in 12b ( see figure s2 in the supporting information ) , correspondingly .", "the ligand can be protonated at n3 ( n3hcl2 ) and deprotonated at n2 [ n2h  o4 ( x + 1 , y + 1 , z + 1 ) with n2o4 2.923(9 )  , n2h", " o4 138.7 ] ( overall charge zero ) with atom n4 as a proton donor to one of the four co - crystallized water molecules n4ho3 , as occurs in 11b4h2o ( see figure 2 ) .", "ortep plot of the structure of the cation [ oscl(-p - cymene)(l2 ) ] in 12b2ch3oh2h2o .", "thermal ellipsoids have been drawn at the 50% probability level . selected bond lengths and angles : os  cl1 , 2.4043(1 )  ; os ", "c(arene)av , 2.197(26 )  ; c1n2 , 1.361(5 )  ; n2n1 , 1.344(4 )  ; n1c6 , 1.336(5 )  ; c6c7 , 1.447(6 )  ; c7n5 , 1.335(5 )  ; n5c13 , 1.380(5 )  ; n1os  n5 , 74.73(13) , n1os  cl1 , 84.29(10) ; and n5os  cl1 , 83.25(10). in 13c , the ligand was found to be deprotonated at n2 , acting as a proton acceptor in the intermolecular hydrogen bonding interaction n2h  n4 ( i denotes symmetry transformations x , y + 1.5 , z + 0.5 ) used to generate equivalent atoms ) [ n2n4 , 2.896(7 )  ; n2h  n4 , 155.6 ] ( see figure 4 ) . ortep plot of the complex [ rucl(-p - cymene)(l3h ) ] ( 13c ) .", "thermal ellipsoids have been drawn at 30% probability level . selected bond lengths and angles : ru  cl , 2.399(2 )  ; ru  n1 ,", "c(arene)av , 2.186(31 )  ; c1n2 , 1.366(8 )  ; n2n1 , 1.353(7 )  ; n1c6 , 1.362(7 )  ; c6c7 , 1.426(9 )  ; c7n5 , 1.335(8 )  ; n5c13 , 1.398(8 )  ; n1ru  n5 , 75.9(2) ; n1ru  cl , 87.35(17) ; and n5ru  cl , 85.73(17). complexes 13dch3oh and 13e0.75ch3oh0.25h2o crystallized both in the centrosymmetric triclinic space group p1. the asymmetric unit in both consists of a neutral complex [ mcl(-p - cymene)(l3h ) ] and a complex cation [ mcl(-p - cymene)(l3 ) ] ( m = ru or os ) , a chloride counteranion and co - crystallized solvent ( methanol or methanol / water )", ". deprotonation of the organic ligand in [ mcl(-p - cymene)(l3h ) ] is corroborated by the presence of hydrogen bonding of the type n2ah  n2b ( x + 1 , y + 1 , z + 1 ) in the crystal structure of the ruthenium complex 13dch3oh ( figure 5 ) and a similar interaction n2bh  n2a ( x , y + 1 , z ) [ n2bn2a , 2.810(9 )  ; n2bh  n2a , 170.1 ] in the crystal of the osmium analogue 13e0.75ch3oh0.25h2o . in figure 6 the structure of [ oscl(-p - cymene)(l3h ) ]", "is shown . in both structures , the atoms n4a and n4b act as proton donors in strong hydrogen bonding interactions to the chloride counteranion , namely , n4ah ", "cl2 ( x + 1 , y + 2 , z + 1 ) [ n4cl2 , 3.275(6 )  ; n4ah  cl2 , 176.9 ] , n4bh ", "cl2 [ n4bcl2 , 3.211(5 )  ; n4bh  cl2 , 176.0 ] ( 13dch3oh ) and n4ah  cl2 ( x + 1 , y + 1 , z + 1 ) [ n4acl2 , 3.273(8 )  ; n4ah  cl2 , 177.0 ] , n4bh ", "cl2 [ n4bcl2 , 3.204(7 )  ; n4bh  cl2 , 177.0 ] ( 13e0.75ch3oh0.25h2o ) .", "fragment of the crystal structure of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) showing the intermolecular hydrogen bonding n2a  hn2b [ n2an2b ( x + 1 , y + 1 , z + 1 ) , 2.814(7 )  ; n2a  hn2b , 163.3 ] . selected bond lengths and angles : ru1a  cl1a , 2.3952(17 )  ; ru1a ", "c(arene)av , 2.194(33 )  ; c1a  n2a , 1.358(8 )  ; n2a  n1a , 1.354(7 )  ; n1a  c6a , 1.350(8 )  ; c6a  c7a , 1.441(9 )  ; c7a  n5a , 1.335(8 )  ; n5a  c13a , 1.381(8 )  ; n1a  ru1a  n5a , 75.6(2) ; n1a ", "ru1a  cl1a , 84.45(15) ; and n5a  ru1a  cl1a , 85.05(15). ortep plot of [ oscl(-p - cymene)(l3h ) ] in [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) .", "selected bond lengths and angles : os1a  cl1a , 2.402(2 )  ; os1a  n1a , 2.068(7 )  ; os1a  n5a , 2.086(7 )  ; os1a  c(arene)av , 2.192(25 )  ; c1a  n2a , 1.349(10 )  ; n2a  n1a 1.369(9 )  ; n1a  c6a , 1.361(10 )  ; c6a  c7a , 1.431(11 )  ; c7a  n5a , 1.339(10 )  ; n5a  c13a , 1.398(11 )  ; n1a  os1a  n5a , 75.1(3) ; n1a ", "os1a  cl1a , 83.56(18) ; and n5a  os1a  cl1a , 84.2(2). ligands l1l3 , as well as the corresponding ruthenium(ii ) ( 11a13a ) and osmium(ii ) ( 11b13b ) arene complexes , were studied with regard to their capacity of inhibiting cell growth in vitro in the human cancer cell lines ch1 ( ovarian carcinoma ) , sw480 ( colon carcinoma ) , and a549 ( non - small cell lung cancer ) , yielding the ic50 values listed in table 2 .", "all compounds show the strongest effect in the generally chemosensitive ch1 cells , whereas the more chemoresistant a549 cells are also less affected by the compounds investigated here .", "> l2 > l1 , indicating that bromination ( l2 , l3 ) and , even more so , the double substitution ( bromination and an additional replacement of h by a methoxymethyl group ( l3 ) ) are advantageous , with regard to activity .", "these structure  activity relationships are also reflected in the rank orders of the corresponding ruthenium and osmium complexes for the ruthenium complexes , 13a > 12a > 11a , and for the osmium complexes , 13b > 12b > 11b .", "however , the ic50 values are shifted to higher concentrations ( see figure 7 ) .", "the differences between the ruthenium and osmium analogues are mostly small ( ic50 values differ only up to a factor of 2.4 ) , with the osmium complexes being at least as active as their ruthenium counterparts .", "ch1 denotes ovarian cancer , human ; sw480 denotes colon carcinoma , human ; and a549 denotes non - small - cell lung carcinoma , human .", "50% inhibitory concentrations ( mean value  standard deviation from at least three independent experiments ) obtained by the mtt assay ( 96-h exposure ) . concentration", " effect curves of each ligand , compared to the corresponding ruthenium and osmium complexes , in ch1 ovarian cancer cells ( mtt assay , 96 h exposure ) : ( a ) l1 , 11a , 11b ; ( b ) l2 , 12a , 12b ; and ( c ) l3 , 13a , 13b . both the presence of substituents in the ligands and complexation result in a marked shift of antiproliferative activity . since 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been reported to be potent cdk inhibitors , we expected the investigated ligands and complexes to induce cell cycle perturbations . to confirm this assumption in a sensitive cell line ,", "exponentially growing ch1 cells were treated with the compounds for 24 h , stained with propidium iodide , and analyzed for their dna content by fluorescence - activated cell sorting ( facs ) .", "surprisingly , the metal - free ligands l1l3 turned out to exert only modest effects on cell cycle distribution , with a slight increase of the g2/m fraction from 32% in the untreated control to 53% by 20 m l2 as the strongest effect observed in this setting ( higher concentrations of this compound led to disintegration of cells already after 24 h ) .", "however , the less cytotoxic ruthenium complexes 12a and 13a , both bearing substituted ligands ( l2 and l3 correspondingly ) , cause a more pronounced g2/m phase arrest , as reflected by an increase of this cell fraction to 65% at 80 m and 59% at 40 m , respectively , and a concomitant decrease of the g0/g1 fraction to 21% and 24% ( compared to 42% in controls ) , whereas ruthenium complex 11a bearing an unsubstituted ligand l1 is devoid of activity on the cell cycle . on the other hand", ", the osmium complexes do not generally show stronger effects on the cell cycle than the metal - free ligands , perhaps with the exception of 13b , which induces an increase of the g2/m fraction up to 53% at 80 m , accompanied by a decline of the g0/g1 fraction to 31% ( see figure 8) .", "concentration - dependent effects of metal - free ligands ( top ) , and the corresponding ruthenium ( middle ) and osmium complexes ( bottom ) , on the cell cycle distribution of ch1 cells ( (  ) g0/g1 , (  ) s , and (  ) g2/m phase ) .", "although the lack of generally pronounced cell cycle effects does not argue for a strong role of cdk inhibition in the mechanism of action of the investigated compounds , inhibitory potencies were studied in cell - free experiments with two recombinant cdk / cyclin complexes , by means of the histone h1 kinase assay .", "results reveal that all compounds are capable of inhibiting kinase activities in a concentration - dependent manner , being more effective on cdk2/cyclin e than on cdk1/cyclin b ( see figure 9 ) .", "in contrast to the observed cell cycle effects , but in accordance with antiproliferative activities , cdk - inhibitory potency of the metal - free ligands is consistently higher than that of the metal complexes . in particular , only l1l3 effectively inhibit cdk2/cyclin e in low concentrations ( 1 m or 10 m ) , whereas all complexes require higher concentrations to exert > 50% inhibitory effects . as in the mtt assay ,", "differences between the effects of corresponding ruthenium and osmium complexes are minor , compared to the differences from those of the metal - free ligands .", "concentration - dependent effects of metal - free ligands ( l1l3 ) as well as corresponding ruthenium ( 11a13a ) and osmium complexes ( 11b13b ) , on kinase activities of recombinant cdk1/cyclin b ( top ) and cdk2/cyclin e ( bottom ) ."], ["the known multistep synthesis of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which we have modified , essentially afforded three organic compounds ( l1l3 ) that possess cdk - inhibiting properties .", "these were used as bidentate ligands , and six novel organometallic complexes of the general formula [ mcl(-p - cymene)(l)]cl , where m = ru ( 11a , 12a , 13a ) or os ( 11b , 12b , 13b ) and l = l1l3 , have been synthesized and comprehensively characterized using spectroscopic and x - ray diffraction methods .", "complexation of l1l3 with ruthenium or osmium yielded compounds with increased solubility in the biological medium , yet lowered antiproliferative activity in human cancer cell lines .", "modulation of biological activities by substitution at the ligands can be accomplished in the metal - free molecules and their metal complexes in a similar way .", "the known cdk - inhibitory activity of the ligands could be confirmed in cell - free experiments , in particular in cdk2/cyclin e , and their stronger effects on cdk activity parallel their higher capacity of inhibiting cancer cell growth in vitro , compared to their metal complexes .", "nevertheless , the lack of pronounced effects on the cell cycle of chemosensitive ovarian cancer cells argues against a major role for inhibition of cell growth , at least in this setting ."]]}
{"article_id": "PMC3830247", "article_text": ["fibro - osseous lesions of the jaws represent a diverse group of entities which include developmental ( hamartomatous ) lesions , reactive or dysplastic processes and neoplasms .", "benign fibro - osseous lesions of the head and neck region are uncommon and represent a wide range of tumors sharing some histopathological features ; which comprise fibrous dysplasia , ossifying fibroma ( of ) , and cement - osseous dysplasia .", "of can be further divided into conventional and juvenile forms ( juvenile ossifying fibroma ( jof ) ) .", "jof has to be distinguished from a larger group of ofs on the basis of the age of the patient , site of involvement and clinical behaviour .", "according to the world health organization ( who ) classification of odontogenic tumors 2005 , jof is further subdivided into psammomatoid jof ( psjof ) and trabecular jof ( trjof ) . benjamins in 1938 first reported psjof as an  osteoid fibroma with atypical ossification of the frontal sinus ", ", golgi in 1949 called it as  psammomatoid ossifying fibroma  , and johnson in 1952 coined the term  juvenile active ossifying fibroma  . according to who classification of odontogenic tumors 2005 , it was named as", "the term juvenile psammomatoid ossifying fibroma is now used to designate the neoplasm of the craniofacial skeleton of young age with well - defined clinicopathological features .", "the pathogenesis of these jaw lesions are related to the abnormal development of basal generative mechanism that is essential for root formation .", "this paper highlights a rare entity of psjof involving the maxillary sinus and nasal cavity on the left side .", "a 20-year - old female patient reported with a painless progressive swelling in the left cheek region and difficulty in breathing since 1 year .", "past medical history revealed that the patient underwent surgery in a private dental clinic 5 years back for the impacted tooth 23 which was associated with pathology .", "the swelling was 6  4 cm in size approximately extending anteroposteriorly from ala of the nose to 5 cm from tragus of the ear on the left side .", "nasal polyp was seen in the left nostril with obliteration of left ala of the nose [ figure 1 ] .", "nasal polyp seen in left nostril with obliteration of left ala of nose intraoral examination presented a swelling extending anteroposteriorly from the distal aspect of 21 to mesial aspect of 26 ; medially till the mid palatine raphe and laterally the buccal vestibule was obliterated and the swelling extended from 21 to 26 .", "paranasal sinus ( pns ) x - ray revealed haziness in the left maxillary sinus . computed tomography ( ct ) scan confirmed well - defined mixed radiolucent and radiopaque areas with calcifications extending superoinferiorly from infraorbital rim to alveolus and anteroposteriorly from the nasal septum to post zygomatic buttress on left side [ figure 2 ] .", "axial computed tomography revealing well - defined radiolucent and radiopaque areas with calcifications histopathological examination of incisional biopsy revealed connective tissue stroma with numerous spherical / irregular ossifications interspersed with cellular fibrous tissue [ figure 3 ] .", "the ossifications showed peripheral brush border surrounded by an eosinophilic rimming [ figure 4 ] .", "haemorrhagic areas were also seen . the constellation of clinical , radiological , and histopathological features of this lesion supported an interpretation of psammomatoid variant of jof .", "photomicrograph of hematoxylin and eosin ( h&e ) stained section ( 100 ) showing numerous spherical and irregular calcifications interspersed with fibrous tissue photomicrograph of h&e stained section ( 400 ) showing peripheral brush border surrounded by eosinophilic rimming under general anesthesia , a weber - ferguson incision was given to expose the complete lesion .", "subtotal maxillectomy was performed and the tumor mass along with nasal and ethmoidal polyps were removed with the help of a chisel and mallet .", "after complete removal of the mass , borders were carefully osteotomized to avoid the chances of recurrence [ figure 5 ] . the excised specimen was sent for histopathological examination [ figure 6 ] and the diagnosis of psjof was confirmed .", "jof are benign , potentially aggressive fibro - osseous lesions of the craniofacial bones . the word ", "psjof is a unique variant of jof that has a predilection for orbit and pnss accounting for about 72% followed by calvarium 11% , maxilla 10% , and mandible 7% .", "the ethmoidal sinuses are most commonly involved , followed by the frontal sinuses , the maxillary sinuses , and the sphenoid sinus .", "both the variants of jof show a predilection for males . the swelling in this case", "is associated with maxilla involving maxillary sinus and nasal cavity in a 20-year - old female patient .", "table 1 summarizes the clinical presentation of case series of psjof reviewed in the literature .", "the patients may develop exopthalmoses , bulbar displacement , and nasal obstruction . in the present case because of the extension of the tumor into the nasal cavity , nasal obstruction was present on the left side .", "radiographically , psjof can be radiolucent , radiopaque , or mixed depending upon cystic changes and the degree of calcification .", "sclerotic changes are evident in the lesion which may show a ground - glass appearance .", "histologically , psjof shows highly cellular fibrous stroma often with whorled pattern containing closely packed spherical ossicles resembling psammoma bodies .", "these ossicles are round to oval in shape which have a basophilic center and peripheral pink rim showing some radiating fibers which corroborates with the microscopic features provided in the present case .", "psjof should be differentiated from extracranial meningioma with psammoma bodies , which demonstrates epithelial membrane antigen ( ema ) positivity and even the psammomatoid ossicles in psjof are clearly different from spherical true psammoma bodies .", "other differential diagnosis include fibrous dysplasia , osteoma , cementoblastoma , well - differentiated osteosarcoma , psammomatous extracranial meningioma [ table 2 ] .", "differential diagnosis the clinical management of smaller lesions is simple excision with surrounding marginal bone , whereas larger lesions warrant more aggressive surgical management .", "prognosis is good with a recurrence rate of about 30 - 58% . no malignant transformation has been documented . even though these lesions tend to locally invade , there were no cases of metastasis being reported .", "therefore , it is very important to correlate the clinical , radiographical , and histopathological findings for proper treatment .", "early detection and complete surgical excision of the lesion followed by long - term follow - up is necessary for proper clinical management ."], "abstract_text": ["<S> juvenile ossifying fibroma ( jof ) is an uncommon , benign , bone - forming neoplasm with an aggressive local growth that is distinguished from other fibro - osseous lesions primarily by its age of onset , clinical presentation and aggressive behaviour . </S>", "<S> jof is considered as a variant of the ossifying fibroma ( of ) and the former includes psammomatoid jof ( psjof ) and trabecular jof ( trjof ) . </S>", "<S> both variants involve the craniofacial bones with the trabecular variant being more common in the jaws and the psammomatoid variant being more common in the craniofacial skeleton . </S>", "<S> psjof is an unique variant of jof that has a predilection for the sinonasal tract and the orbit particularly centered on the periorbital , frontal , and ethmoid bones . </S>", "<S> we report a rare case of massive psjof involving the maxillary sinus in a 20-year - old female . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["fibro - osseous lesions of the jaws represent a diverse group of entities which include developmental ( hamartomatous ) lesions , reactive or dysplastic processes and neoplasms .", "benign fibro - osseous lesions of the head and neck region are uncommon and represent a wide range of tumors sharing some histopathological features ; which comprise fibrous dysplasia , ossifying fibroma ( of ) , and cement - osseous dysplasia .", "of can be further divided into conventional and juvenile forms ( juvenile ossifying fibroma ( jof ) ) .", "jof has to be distinguished from a larger group of ofs on the basis of the age of the patient , site of involvement and clinical behaviour .", "according to the world health organization ( who ) classification of odontogenic tumors 2005 , jof is further subdivided into psammomatoid jof ( psjof ) and trabecular jof ( trjof ) . benjamins in 1938 first reported psjof as an  osteoid fibroma with atypical ossification of the frontal sinus ", ", golgi in 1949 called it as  psammomatoid ossifying fibroma  , and johnson in 1952 coined the term  juvenile active ossifying fibroma  . according to who classification of odontogenic tumors 2005 , it was named as", "the term juvenile psammomatoid ossifying fibroma is now used to designate the neoplasm of the craniofacial skeleton of young age with well - defined clinicopathological features .", "the pathogenesis of these jaw lesions are related to the abnormal development of basal generative mechanism that is essential for root formation .", "this paper highlights a rare entity of psjof involving the maxillary sinus and nasal cavity on the left side ."], ["a 20-year - old female patient reported with a painless progressive swelling in the left cheek region and difficulty in breathing since 1 year .", "past medical history revealed that the patient underwent surgery in a private dental clinic 5 years back for the impacted tooth 23 which was associated with pathology .", "the swelling was 6  4 cm in size approximately extending anteroposteriorly from ala of the nose to 5 cm from tragus of the ear on the left side .", "nasal polyp was seen in the left nostril with obliteration of left ala of the nose [ figure 1 ] .", "nasal polyp seen in left nostril with obliteration of left ala of nose intraoral examination presented a swelling extending anteroposteriorly from the distal aspect of 21 to mesial aspect of 26 ; medially till the mid palatine raphe and laterally the buccal vestibule was obliterated and the swelling extended from 21 to 26 .", "paranasal sinus ( pns ) x - ray revealed haziness in the left maxillary sinus . computed tomography ( ct ) scan confirmed well - defined mixed radiolucent and radiopaque areas with calcifications extending superoinferiorly from infraorbital rim to alveolus and anteroposteriorly from the nasal septum to post zygomatic buttress on left side [ figure 2 ] .", "axial computed tomography revealing well - defined radiolucent and radiopaque areas with calcifications histopathological examination of incisional biopsy revealed connective tissue stroma with numerous spherical / irregular ossifications interspersed with cellular fibrous tissue [ figure 3 ] .", "the ossifications showed peripheral brush border surrounded by an eosinophilic rimming [ figure 4 ] .", "haemorrhagic areas were also seen . the constellation of clinical , radiological , and histopathological features of this lesion supported an interpretation of psammomatoid variant of jof .", "photomicrograph of hematoxylin and eosin ( h&e ) stained section ( 100 ) showing numerous spherical and irregular calcifications interspersed with fibrous tissue photomicrograph of h&e stained section ( 400 ) showing peripheral brush border surrounded by eosinophilic rimming under general anesthesia , a weber - ferguson incision was given to expose the complete lesion .", "subtotal maxillectomy was performed and the tumor mass along with nasal and ethmoidal polyps were removed with the help of a chisel and mallet .", "after complete removal of the mass , borders were carefully osteotomized to avoid the chances of recurrence [ figure 5 ] . the excised specimen was sent for histopathological examination [ figure 6 ] and the diagnosis of psjof was confirmed ."], ["jof are benign , potentially aggressive fibro - osseous lesions of the craniofacial bones . the word ", "psjof is a unique variant of jof that has a predilection for orbit and pnss accounting for about 72% followed by calvarium 11% , maxilla 10% , and mandible 7% .", "the ethmoidal sinuses are most commonly involved , followed by the frontal sinuses , the maxillary sinuses , and the sphenoid sinus .", "both the variants of jof show a predilection for males . the swelling in this case", "is associated with maxilla involving maxillary sinus and nasal cavity in a 20-year - old female patient .", "table 1 summarizes the clinical presentation of case series of psjof reviewed in the literature .", "the patients may develop exopthalmoses , bulbar displacement , and nasal obstruction . in the present case because of the extension of the tumor into the nasal cavity , nasal obstruction was present on the left side .", "radiographically , psjof can be radiolucent , radiopaque , or mixed depending upon cystic changes and the degree of calcification .", "sclerotic changes are evident in the lesion which may show a ground - glass appearance .", "histologically , psjof shows highly cellular fibrous stroma often with whorled pattern containing closely packed spherical ossicles resembling psammoma bodies .", "these ossicles are round to oval in shape which have a basophilic center and peripheral pink rim showing some radiating fibers which corroborates with the microscopic features provided in the present case .", "psjof should be differentiated from extracranial meningioma with psammoma bodies , which demonstrates epithelial membrane antigen ( ema ) positivity and even the psammomatoid ossicles in psjof are clearly different from spherical true psammoma bodies .", "other differential diagnosis include fibrous dysplasia , osteoma , cementoblastoma , well - differentiated osteosarcoma , psammomatous extracranial meningioma [ table 2 ] .", "differential diagnosis the clinical management of smaller lesions is simple excision with surrounding marginal bone , whereas larger lesions warrant more aggressive surgical management .", "prognosis is good with a recurrence rate of about 30 - 58% . no malignant transformation has been documented . even though these lesions tend to locally invade , there were no cases of metastasis being reported ."], ["therefore , it is very important to correlate the clinical , radiographical , and histopathological findings for proper treatment .", "early detection and complete surgical excision of the lesion followed by long - term follow - up is necessary for proper clinical management ."]]}
{"article_id": "PMC4499929", "article_text": ["endoscopic methods are among the most important and frequently employed means of treating pancreatic pseudocysts .", "when possible an endoscopic approach should generally be attempted in all patients with pseudocysts , and for pancreatic fluid collections endoscopic transmural drainage is the treatment of choice .", "if the cyst contains more than 2/3 solid material a self - expandable stent should be selected to ensure effective drainage .", "an additional advantage of the self - expandable metal stent is that the necrotic tissue contained within the cyst can be actively removed using an endoscope .", "this article discusses the unusual case of a stent that became dislodged and migrated into the omental bursa . following this migration some time", "was allowed to elapse , and the gastrotomy closed completely . since a simple endoscopic retrieval was no longer possible , a combined laparoscopic - endoscopic technique ( rendezvous technique ) was used instead , and the stent was successfully retrieved .", "in 2013 , a 49-year - old male smoker with a history of diabetes mellitus and arterial hypertension developed severe necrotizing pancreatitis .", "imaging revealed paralytic ileus along with an expanding cystic formation near the liver hilus and pancreatic head with compression of the duodenum .", "a pseudocyst near the left abdominal wall with enormous but stable proportions was also found to be compressing the left colon transversum ( 14 cm ) . due to the extreme thinness of the wall", "areas of necrosis in the corpus area developed , necessitating transgastral cystotomy and placement of a wall stent ( niti - s nagi stent - taewoong - medical co , seoul , south korea , 14 mm wide , 22 mm long ) .", "twenty - four hours later , however , the stent became dislodged and migrated into the omental bursa .", "an attempt at endoscopic removal the following day was unsuccessful , making the insertion of a second stent ( nagi fully covered taewoong,16 mm wide , 22 mm long ) necessary .", "endoscopic retrograde cholangiopancreatography ( ercp ) was then performed , along with papilotomy and insertion of an 8.5 fr .", "two weeks later , after significant clinical improvement and drainage of necrotic tissue , the second transgastric stent was removed with no complications . for technical reasons ,", "retrieval of the migrated stent was not possible . at follow - up gastroscopy 4 weeks later the gastric wall had completely closed and hence that endoscopic localization of the stent was no longer possible . in november 2013", "the pseudocysts had regressed significantly , and a computed tomography of the abdomen showed the migrated stent in the omental bursa with no direct contact to the gastric wall . in february 2014", ", we decided to attempt a laparoscopic - endoscopic retrieval of the displaced wall stent . in the first step ,", "intraoperative endoscopy demonstrated normal gastric mucosa so that a purely macroscopic localization of the stent was not possible [ figure 1 ] .", "after localization of the stent via image converter we made a targeted 3 cm incision into the posterior gastric antrum using an endo knife ( erbe , germany ) . due to the location of the stent deep within the omental bursa neither optical nor palpatory contact could be made , and consequently , endoscopic retrieval was not an option . in the next step ,", "first , electrocoagulation was used to make three incisions in the anterior wall of the greater gastric curvature ( two in antrum , one in fundus ) . then , 3 ( applied medical kii , usa ) 5 mm trocars were inserted [ figure 1 ] .", "dissection within the omental bursa was performed using two dissectors , inserted through an incision that had been made endoscopically .", "after localization of the stent and some blunt dissection it was possible to carefully dislodge the stent into the gastric lumen [ figure 2 ] .", "after further inspection , the transgastral trocars were removed , and the gastric wall incisions were closed with lahodny stitches ( ethicon , usa ) .", "the postoperative clinical course was without complications , and the patient was discharged on the 6 postoperative day . at follow - up 4 weeks later , no complications were found .", "the gastric wall has closed , and the stent can not be localized ( a ) .", "three applied kii 5 mm trocars in the stomach ( b ) blunt dissection near the omental bursa after incision and retrieval of the stent ( a ) .", "currently , endoscopic ultrasound - guided drainage is a safe and effective means of treating pancreatic fluid collections . compared with surgical or percutaneous drainage it is less invasive and results in lower mortality , costs , and hospitalization times .", "drainage - related complications either may be related directly to the procedure or can occur in relation to the stents themselves .", "the stent should not , however , be removed before complete involution of the pseudocyst or before the passage of at least 2 months . on average ,", "15% of stents will become accidentally dislocated , but efforts to develop technically superior stents continue .", "tllez - vila et al . , for example , report on fully covered self - expandable metal stents with an innovative anti - migration system .", "describe a stent - in - stent combination which employs the mechanical advantages of both plastic stents and fully covered self - expandable metal stents .", "although these new solutions will help reduce the rate of migration , they will not eliminate it completely . in most cases of stent migration endoscopic retrieval", "can be performed without difficulty . in cases of extraluminal loss , however , a surgical approach is often necessary , and a minimally invasive exploration of the abdominal cavity is the approach of choice .", "retrieval from the retroperitoneal space may pose a more significant challenge due to the proximity of the large vessels .", "when endoscopic retrieval fails a combined laparoscopic - endoscopic approach offers an alternative to open surgery .", "the choice of technique is likely best predicated by individual patient presentation and local expertise . with this endoscopy - laparoscopy combination ( rendezvous technique )"], "abstract_text": ["<S> endoscopic drainage is a widely used treatment for pancreatic pseudocysts . </S>", "<S> drainage - related complications may be related directly to the procedure or may occur later as stents migrate or erode into adjacent structures . </S>", "<S> migration of a self - expanding metal stent into peritoneal cavity and incorporation in the omental bursa is rare . when endoscopic retrieval fails a combined laparoscopic - endoscopic ( rendezvous technique ) approach offers an alternative to open surgery . </S>", "<S> we report a case of successful gastroscopic - transgastric laparoscopic removal of a stent that was dislocated into the omental bursa after a  year observation period . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["endoscopic methods are among the most important and frequently employed means of treating pancreatic pseudocysts .", "when possible an endoscopic approach should generally be attempted in all patients with pseudocysts , and for pancreatic fluid collections endoscopic transmural drainage is the treatment of choice .", "if the cyst contains more than 2/3 solid material a self - expandable stent should be selected to ensure effective drainage .", "an additional advantage of the self - expandable metal stent is that the necrotic tissue contained within the cyst can be actively removed using an endoscope .", "this article discusses the unusual case of a stent that became dislodged and migrated into the omental bursa . following this migration some time", "was allowed to elapse , and the gastrotomy closed completely . since a simple endoscopic retrieval was no longer possible , a combined laparoscopic - endoscopic technique ( rendezvous technique ) was used instead , and the stent was successfully retrieved ."], ["in 2013 , a 49-year - old male smoker with a history of diabetes mellitus and arterial hypertension developed severe necrotizing pancreatitis .", "imaging revealed paralytic ileus along with an expanding cystic formation near the liver hilus and pancreatic head with compression of the duodenum .", "a pseudocyst near the left abdominal wall with enormous but stable proportions was also found to be compressing the left colon transversum ( 14 cm ) . due to the extreme thinness of the wall", "areas of necrosis in the corpus area developed , necessitating transgastral cystotomy and placement of a wall stent ( niti - s nagi stent - taewoong - medical co , seoul , south korea , 14 mm wide , 22 mm long ) .", "twenty - four hours later , however , the stent became dislodged and migrated into the omental bursa .", "an attempt at endoscopic removal the following day was unsuccessful , making the insertion of a second stent ( nagi fully covered taewoong,16 mm wide , 22 mm long ) necessary .", "endoscopic retrograde cholangiopancreatography ( ercp ) was then performed , along with papilotomy and insertion of an 8.5 fr .", "two weeks later , after significant clinical improvement and drainage of necrotic tissue , the second transgastric stent was removed with no complications . for technical reasons ,", "retrieval of the migrated stent was not possible . at follow - up gastroscopy 4 weeks later the gastric wall had completely closed and hence that endoscopic localization of the stent was no longer possible . in november 2013", "the pseudocysts had regressed significantly , and a computed tomography of the abdomen showed the migrated stent in the omental bursa with no direct contact to the gastric wall . in february 2014", ", we decided to attempt a laparoscopic - endoscopic retrieval of the displaced wall stent . in the first step ,", "intraoperative endoscopy demonstrated normal gastric mucosa so that a purely macroscopic localization of the stent was not possible [ figure 1 ] .", "after localization of the stent via image converter we made a targeted 3 cm incision into the posterior gastric antrum using an endo knife ( erbe , germany ) . due to the location of the stent deep within the omental bursa neither optical nor palpatory contact could be made , and consequently , endoscopic retrieval was not an option . in the next step ,", "first , electrocoagulation was used to make three incisions in the anterior wall of the greater gastric curvature ( two in antrum , one in fundus ) . then , 3 ( applied medical kii , usa ) 5 mm trocars were inserted [ figure 1 ] .", "dissection within the omental bursa was performed using two dissectors , inserted through an incision that had been made endoscopically .", "after localization of the stent and some blunt dissection it was possible to carefully dislodge the stent into the gastric lumen [ figure 2 ] .", "after further inspection , the transgastral trocars were removed , and the gastric wall incisions were closed with lahodny stitches ( ethicon , usa ) .", "the postoperative clinical course was without complications , and the patient was discharged on the 6 postoperative day . at follow - up 4 weeks later , no complications were found .", "the gastric wall has closed , and the stent can not be localized ( a ) .", "three applied kii 5 mm trocars in the stomach ( b ) blunt dissection near the omental bursa after incision and retrieval of the stent ( a ) ."], ["currently , endoscopic ultrasound - guided drainage is a safe and effective means of treating pancreatic fluid collections . compared with surgical or percutaneous drainage it is less invasive and results in lower mortality , costs , and hospitalization times .", "drainage - related complications either may be related directly to the procedure or can occur in relation to the stents themselves .", "the stent should not , however , be removed before complete involution of the pseudocyst or before the passage of at least 2 months . on average ,", "15% of stents will become accidentally dislocated , but efforts to develop technically superior stents continue .", "tllez - vila et al . , for example , report on fully covered self - expandable metal stents with an innovative anti - migration system .", "describe a stent - in - stent combination which employs the mechanical advantages of both plastic stents and fully covered self - expandable metal stents .", "although these new solutions will help reduce the rate of migration , they will not eliminate it completely . in most cases of stent migration endoscopic retrieval", "can be performed without difficulty . in cases of extraluminal loss , however , a surgical approach is often necessary , and a minimally invasive exploration of the abdominal cavity is the approach of choice .", "retrieval from the retroperitoneal space may pose a more significant challenge due to the proximity of the large vessels .", "when endoscopic retrieval fails a combined laparoscopic - endoscopic approach offers an alternative to open surgery .", "the choice of technique is likely best predicated by individual patient presentation and local expertise . with this endoscopy - laparoscopy combination ( rendezvous technique )"]]}
{"article_id": "PMC3997246", "article_text": ["adenomyoepithelioma has been described as a rare benign neoplasm that occurs almost exclusively in the breast . in the breast", "it is defined as a neoplasm composed of two structures , namely , tubules limited by an inner epithelial layer of duct - like cells and an outer layer with mostly clear myoepithelial cells .", "apart from the breast , adenomyoepitheliomas have been described in the salivary glands and in the lung . in the skin ,", "these neoplasms seem to be exceedingly rare , with only few reports of adenomyoepitheliomas published to date [ 47 ] .", "we present the case of a 53-year - old woman with a clinically benign nodular cutaneous lesion that revealed histopathologic and immunohistochemical features of adenomyoepithelioma .", "a 53-year - old woman presented to her dermatologist with a 3 cm asymptomatic nodule on the left forearm .", "the biopsy specimen consisted of a 2.5  1.9  1.5 cm tan ellipse of skin and contained a hard yellow nodule measuring 1.5 cm in greatest dimension .", "examination of the sections at scanning magnification revealed beneath an intact epidermis a zone of fibrosis within the upper part of the dermis ( figure 1 a , b ) . beneath the scar", ", there was a large , lobulated neoplasm , which for the most part was surrounded by a compressed fibrous pseudocapsule .", "each of the aggregates of neoplastic cells varied in sizes and shapes and some were large and nodular ( figure 1a  c ) . at higher magnification , most of the cells that constituted the lesion displayed myoepithelial differentiation with polygonal and  plasmacytoid  features ( figure 2 ) .", "the cells presented sometimes as solid sheets , but also as cords and solitary units . in places glandular and ductal structures were evident ; in some areas apocrine - type secretion within glandular structures was found ( figure 2 a  c ) .", "f ) . in some areas cells were present within a myxoid stroma ( figure 2 g ", "foci of cells with pleomorphic nuclei and mitotic figures were identified ( figure 2 k ", "the myoepithelial cellular component of the neoplasm stained for s100 protein and was negative for cytokeratin and carcinoembryonic antigen ( cea ) expression .", "we describe a cutaneous neoplasm composed of myoepithelial cells and a focal epithelial and glandular component .", "the s100 positivity indicates myoepithelial cells , while epithelial - glandular cells are negative for s100 protein expression .", "based on findings by conventional microscopy and immunohistochemistry , this neoplasm shows myoepithelial differentiation and focal epithelial lined tubules with features of apocrine secretion , findings that are consistent with the diagnosis of primary adenomyoepithelioma of the skin .", "while various adnexal neoplasms with a myoepithelial cellular component have been described in the skin , adenomyoepithelioma of the skin seems to be extremely rare .", "what are the criteria that distinguish adenomyoepithelioma from other benign cutaneous neoplasms with myoepithelial differentiation ? while myoepithelioma is defined as a benign neoplasm consisting exclusively of myoepithelial cells embedded in a myxoid stroma , adenomyoepithelioma shows in addition to myoepithelial cells a second component displaying various degrees of epithelial - ductal differentiation .", "chondroid syringoma ,  at the other end of the spectrum is a benign adnexal neoplasm that , in addition to apocrine epithelium , manifests various degrees of follicular and/or sebaceous differentiation .", "clinically , myoepitheliomas occur usually in children and young adults and are located on the extremities , while mixed apocrine tumors affect older individuals and are usually found on the face .", "the few cases of cutaneous adenomyoepitheliomas that have been described to date , including the present case , were described in older patients and were located on the extremities and on the trunk .", "pleomorphic adenoma in the breast is regarded as the analogue to mixed apocrine tumor in the skin , while adenomyoepithelioma of the breast is defined as a neoplasm with nodular aggregations of clear myoepithelial cells that surround epithelial lined tubules .", "occasionally , the myoepithelial component predominates and loses the close association with epithelial structures . in the skin", ", the predominance of myoepithelial cells in relation to ductal epithelial structures lacking features of follicular and/or sebaceous differentiation separates this neoplasm from mixed apocrine tumor .", "therefore , myoepithelioma , adenomyoepithelioma and mixed apocrine tumor lie in the spectrum of neoplasms with pure myoepithelial differentiation at one end and apocrine - sebaceous - follicular differentiation at the other .", "the most important question for the patient is the biologic potential of such a lesion . while the chronic course in our patient and the majority of the histopathologic features suggest a benign neoplasm , foci of cells having pleomorphic nuclei and mitotic figures were present .", "both findings could indicate the potential for locally aggressive behavior and/or metastasis , as has been rarely described in adenomyoepitheliomas of the breast .", "therefore , adenomyoepitheliomas of the skin should be completely excised , as has been recommended in the reported case ."], "abstract_text": ["<S> analogous to adenomyoepitheliomas of the breast , cutaneous adenomyoepithelioma is composed of two components , one being myoepithelial , the other ductal epithelial , and it lies in the spectrum of neoplasms having a  pure  myoepithelioma at one end and a mixed apocrine tumor at the other . </S>", "<S> we present the case of a 53-year - old woman with a 3 cm nodular lesion on her left forearm that had been present for many years . </S>", "<S> histopathologic examination revealed a large lobulated neoplasm surrounded by a compressed fibrous pseudocapsule . </S>", "<S> most of the cells that constituted the lesion displayed myoepithelial differentiation arranged in solid sheets , cords , and solitary units . </S>", "<S> glandular and ductal structures with features of apocrine differentiation composed the second part of the neoplasm . the myoepithelial cellular component of the neoplasm stained for s100 protein and was negative for cytokeratin and carcinoembryonic antigen ( cea ) . based on findings by conventional microscopy and immunohistochemistry , </S>", "<S> the neoplasm was classified as primary adenomyoepithelioma of the skin . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Conclusions"], "sections": [["adenomyoepithelioma has been described as a rare benign neoplasm that occurs almost exclusively in the breast . in the breast", "it is defined as a neoplasm composed of two structures , namely , tubules limited by an inner epithelial layer of duct - like cells and an outer layer with mostly clear myoepithelial cells .", "apart from the breast , adenomyoepitheliomas have been described in the salivary glands and in the lung . in the skin ,", "these neoplasms seem to be exceedingly rare , with only few reports of adenomyoepitheliomas published to date [ 47 ] .", "we present the case of a 53-year - old woman with a clinically benign nodular cutaneous lesion that revealed histopathologic and immunohistochemical features of adenomyoepithelioma ."], ["a 53-year - old woman presented to her dermatologist with a 3 cm asymptomatic nodule on the left forearm .", "the biopsy specimen consisted of a 2.5  1.9  1.5 cm tan ellipse of skin and contained a hard yellow nodule measuring 1.5 cm in greatest dimension .", "examination of the sections at scanning magnification revealed beneath an intact epidermis a zone of fibrosis within the upper part of the dermis ( figure 1 a , b ) . beneath the scar", ", there was a large , lobulated neoplasm , which for the most part was surrounded by a compressed fibrous pseudocapsule .", "each of the aggregates of neoplastic cells varied in sizes and shapes and some were large and nodular ( figure 1a  c ) . at higher magnification , most of the cells that constituted the lesion displayed myoepithelial differentiation with polygonal and  plasmacytoid  features ( figure 2 ) .", "the cells presented sometimes as solid sheets , but also as cords and solitary units . in places glandular and ductal structures were evident ; in some areas apocrine - type secretion within glandular structures was found ( figure 2 a  c ) .", "f ) . in some areas cells were present within a myxoid stroma ( figure 2 g ", "foci of cells with pleomorphic nuclei and mitotic figures were identified ( figure 2 k ", "the myoepithelial cellular component of the neoplasm stained for s100 protein and was negative for cytokeratin and carcinoembryonic antigen ( cea ) expression ."], ["we describe a cutaneous neoplasm composed of myoepithelial cells and a focal epithelial and glandular component .", "the s100 positivity indicates myoepithelial cells , while epithelial - glandular cells are negative for s100 protein expression .", "based on findings by conventional microscopy and immunohistochemistry , this neoplasm shows myoepithelial differentiation and focal epithelial lined tubules with features of apocrine secretion , findings that are consistent with the diagnosis of primary adenomyoepithelioma of the skin .", "while various adnexal neoplasms with a myoepithelial cellular component have been described in the skin , adenomyoepithelioma of the skin seems to be extremely rare .", "what are the criteria that distinguish adenomyoepithelioma from other benign cutaneous neoplasms with myoepithelial differentiation ? while myoepithelioma is defined as a benign neoplasm consisting exclusively of myoepithelial cells embedded in a myxoid stroma , adenomyoepithelioma shows in addition to myoepithelial cells a second component displaying various degrees of epithelial - ductal differentiation .", "chondroid syringoma ,  at the other end of the spectrum is a benign adnexal neoplasm that , in addition to apocrine epithelium , manifests various degrees of follicular and/or sebaceous differentiation .", "clinically , myoepitheliomas occur usually in children and young adults and are located on the extremities , while mixed apocrine tumors affect older individuals and are usually found on the face .", "the few cases of cutaneous adenomyoepitheliomas that have been described to date , including the present case , were described in older patients and were located on the extremities and on the trunk .", "pleomorphic adenoma in the breast is regarded as the analogue to mixed apocrine tumor in the skin , while adenomyoepithelioma of the breast is defined as a neoplasm with nodular aggregations of clear myoepithelial cells that surround epithelial lined tubules .", "occasionally , the myoepithelial component predominates and loses the close association with epithelial structures . in the skin", ", the predominance of myoepithelial cells in relation to ductal epithelial structures lacking features of follicular and/or sebaceous differentiation separates this neoplasm from mixed apocrine tumor .", "therefore , myoepithelioma , adenomyoepithelioma and mixed apocrine tumor lie in the spectrum of neoplasms with pure myoepithelial differentiation at one end and apocrine - sebaceous - follicular differentiation at the other .", "the most important question for the patient is the biologic potential of such a lesion . while the chronic course in our patient and the majority of the histopathologic features suggest a benign neoplasm , foci of cells having pleomorphic nuclei and mitotic figures were present .", "both findings could indicate the potential for locally aggressive behavior and/or metastasis , as has been rarely described in adenomyoepitheliomas of the breast .", "therefore , adenomyoepitheliomas of the skin should be completely excised , as has been recommended in the reported case ."]]}
{"article_id": "PMC3616698", "article_text": ["although the human genome sequence was released a decade ago , the role of functional noncoding rnas ( ncrnas ) is much less understood compared with their coding counterparts .", "several previous studies have demonstrated that the human genome is pervasively transcribed ( 14 ) , but thoroughly cataloging all the rna species ( especially ncrna ) is challenging .", "undiscovered ncrnas might be rare , transient or beyond the detection limits of conventional approaches .", "furthermore , ncrnas also tend to be idiosyncratic to species and tissues ( 5,6 ) .", "nevertheless , advances in rna - seq have provided a new method of surveying the whole transcriptome to an unprecedented degree .", "recent genome - wide studies revealed tens of thousands of novel transcripts , the majority of which were long noncoding rnas ( lncrnas , > 200 nt ) ( 49 ) .", "although a few dozen lncrnas have been characterized to some extent and are reported to have critical roles in diverse cellular and disease development processes ( 6,1014 ) , the biogenesis and function of most lncrnas remain unclear .", "accurate and quantitative assessment of coding potential is the first step toward comprehensive annotation of newly discovered transcripts .", "until now , prediction of coding potential heavily relied on sequence alignment , either pairwise homology search for protein evidence such as that used in the coding - potential calculator ( cpc ) and portrait methods ( 15,16 ) or multiple alignments to calculate the phylogenetic conservation score such as that used in the phylogenetic codon substitution frequencies ( phylocsf ) and rnacode methods ( 17,18 ) .", "alignment - based approaches are particularly useful for highly conserved protein - coding genes and , to a lesser extent , short genes encoding housekeeping or regulatory rnas ( e.g. snrnas , snorna , transfer rna ) .", "however , these approaches can not immediately apply to all the novel transcripts because of several intrinsic limitations .", "first , most newly discovered transcripts are lncrnas , which tend to be lineage specific and less conserved ( 5,6 ) .", "for example , only 29 of 550 lncrnas identified from zebrafish had detectable sequence similarity with putative mammalian orthologs ( 6 ) , and only 993 of 8195 human lncrnas have orthologous transcripts in other species ( 5 ) .", "second , considerable fractions of lncrnas are overlapped with either the sense or antisense strand of protein - coding genes .", "these lncrnas can not be correctly classified by homology searching because they would have significant matches to protein - coding genes ( 3,8,19 ) .", "third , the reliability of alignment - based approaches largely depends on the quality of alignments ( 20 ) .", "this is problematic because most widely used multiple - sequence alignment tools use heuristics and do not guarantee optimal alignments .", "for instance , cpc and phylocsf took 2 days to evaluate the coding potential of 14 353 lncrnas identified by cabili et al .", "this problem is getting more attention as massive - scale rna sequencing is increasingly being performed .", "consequently , a more accurate , robust and faster method that does not rely on sequence alignment is needed to distinguish ncrnas , especially lncrnas , from protein - coding genes . here , we present coding - potential assessment tool ( cpat ) , an alignment - free program , which uses logistic regression to distinguish between coding and noncoding transcripts on the basis of four sequence features .", "cpat is highly accurate ( 0.967 ) and extremely efficient ( 10 000 times faster than cpc and phylocsf , and 50 times faster than portrait ) .", "cpat needs only the sequence or coordinate file as input , and it is straightforward to use .", "we expanded the availability of cpat to a larger scientific audience via a web interface , which allows users to submit sequences and receive the prediction results back almost instantaneously ( http://lilab.research.bcm.edu/cpat/index.php ) .", "coding - potential prediction is essentially a binary decision problem , which makes logistic regression a suitable approach . as an alignment - free method , all selected features ( predictor variables )", "the first feature was the maximum length of the open reading frame ( orf ) .", "orf length is one of the most fundamental features used to distinguish ncrna from messenger rna because a long putative orf is unlikely to be observed by random chance in noncoding sequences . despite the simplicity , orf length has high concordance with more sophisticated discrimination methods and remains the primary criterion in almost all coding - potential prediction methods ( 21 ) .", "the second feature was orf coverage defined as the ratio of orf to transcript lengths .", "this feature also has good classification power , and it is highly complementary to , and independent of , the orf length ( supplementary figures s1 and s3 ) . some large bona fide ncrnas may contain putative long orfs by random chance ( 5 ) , and thus can not be classified correctly by orf length alone .", "fortunately , those large ncrnas usually have much lower orf coverage than protein - coding rnas ( figure 1b ) . \n", "figure 1.score distribution between coding ( red ) and noncoding ( blue ) transcripts for the four linguistic features selected to build the logistic regression model ; training data set containing 10 000 coding and 10 000 noncoding transcripts were used .", "score distribution between coding ( red ) and noncoding ( blue ) transcripts for the four linguistic features selected to build the logistic regression model ; training data set containing 10 000 coding and 10 000 noncoding transcripts were used .", "the third feature we used was the fickett testcode score ( termed  fickett score hereafter ) , which is a simple linguistic feature that distinguishes protein - coding rna and ncrna according to the combinational effect of nucleotide composition and codon usage bias ( 22 ) .", "briefly , the fickett score is obtained by computing four position values and four composition values ( nucleotide content ) from the dna sequence .", "the position value reflects the degree to which each base is favored in one codon position versus another .", "for example , position value of a ( apos ) is calculated as follows : \n\n  cpos , gpos and tpos are determined in the same way .", "these eight values are then converted into probabilities ( p ) of coding using a lookup table provided in the original article .", "each probability is multiplied by a weight ( w ) for the respective base , where the value of w reflects the percentage of time each parameter alone successfully predicts coding or noncoding function for the sequences of known function .", "finally , the fickett score is calculated as follows : \n\n  the fickett score is independent of the orf , and when the test region is 200 nt in length ( which includes most lncrna ) , this feature alone can achieve 94% sensitivity and 97% specificity , with ", "the fourth feature we used was hexamer usage bias ( termed  hexamer score hereafter ) .", "this may be the most discriminating feature because of the dependence between adjacent amino acids in proteins ( 23 ) .", "the hexamer score can be computed in numerous ways ; here , we used a log - likelihood ratio to measure differential hexamer usage between coding and noncoding sequences . for a given dna sequence , we calculated the probability of the sequence under the model of coding dna and under the model of noncoding dna , and then we took the logarithm of the ratio of these probabilities as the score of coding potential .", "we used f ( hi ) ( i = 0 , 1 ,  , 4095 ) and f ( hi ) ( i = 0 , 1 ,  , 4095 ) to represent in - frame hexamer frequency , calculated from coding and noncoding training data sets ( described below ) , respectively .", "for a given hexamer sequence s = h1 , h2 ,  , hm , \n\n  hexamer score determines the relative degree of hexamer usage bias in a particular sequence .", "positive values indicate a coding sequence , whereas negative values indicate a noncoding sequence . we build a logistic regression model using these four linguistic features as predictor variables .", "a  test was used to evaluate whether our logit model with predictors fits the training data significantly better than the null model , which had only an intercept .", "we built a high - confidence training data set to measure the prediction performance of our logit model .", "this data set contained 10 000 protein - coding transcripts selected from the refseq database ; all transcripts had high - quality protein sequences annotated by the consensus coding sequence project .", "we evaluate the model with a 10-fold cross - validation and measured its sensitivity , specificity , accuracy , precision and area under the curve ( auc ) characteristics .", "the receiver operating characteristic ( roc ) curve and precision  recall ( pr ) curve were generated using rocr package ( 24 ) .", "we also built a nonparametric two - graph roc curve for selecting the optimal cpat score threshold that maximizes the sensitivity and specificity of cpat while minimizing misclassifications .", "we built an independent test data set to compare the performance of cpat with that of cpc , phylocsf and portrait .", "this test set composed of 4000 high - quality protein - coding genes ( refseq annotated ) and 4000 lncrnas from a human lncrna catalog ( 5 ) .", "assuming that all 4000 lncrnas are truly noncoding sequences , we could compute the sensitivity , specificity , accuracy and precision of the algorithms to measure their performance .", "those 528 genes were equally assigned to the true - negative and false - positive categories .", "the abbreviations in the equations below are as follows : fn , false negative ; fp , false positive ; tn , true negative ; tp , true positive \n\n\n", "all four selected features were concordantly higher in coding transcripts and lower in noncoding transcripts ( figure 1 ) .", "we plotted three major features ( orf size , fickett score and hexamer score ) in a three - dimensional space to evaluate their combinatorial effect ( figure 2 ) .", "coding and noncoding transcripts in our training data set were grouped into two distinct clusters , indicating good concordance between features .", "the  test p value was < .001 ( = 23 548.44 ; degrees of freedom = 4 ) , indicating that the logit model as a whole fits significantly better than the null model .", "ten - fold cross - validation showed that cpat could achieve very high accuracy , with an auc of 0.9927 ( figure 3a ) .", "we also provide the pr curve because the roc curve can be misleading when the test data are largely skewed ( figure 3b ) .", "we use nonparametric two - graph roc curves to determine an optimal cpat score threshold that maximizes the discriminatory power ( figure 3c and d ) . according to figure 3d ,", "a score threshold of 0.364 gave the highest sensitivity and specificity ( 0.966 for both ) for human data . \n", "figure 2.three-dimensional plot shows combinatorial effects of fickett score , hexamer score and orf size on 10 000 coding genes ( red dots ) and 10 000 noncoding genes ( blue dots ) . \n", "dashed curves represent the 10-fold cross - validation ; solid curves represent the averaged curve from 10 validation runs .", "( d ) two - graph roc curve is used to determine the optimum cutoff value .", "three - dimensional plot shows combinatorial effects of fickett score , hexamer score and orf size on 10 000 coding genes ( red dots ) and 10 000 noncoding genes ( blue dots ) .", "dashed curves represent the 10-fold cross - validation ; solid curves represent the averaged curve from 10 validation runs .", "( d ) two - graph roc curve is used to determine the optimum cutoff value .", "we compared the performance of cpat with that of cpc , phylocsf and portrait ( protein - independent support vector machine model ) using an independent test data set composed of 4000 coding genes and 4000 noncoding genes .", "a multiple alignment of 45 vertebrate genomes , including that of human , was downloaded from the ucsc ( university of california , santa cruz ) genome browser and was used as the input alignment for phylocsf . in general , cpat ( sensitivity : 0.96 , specificity : 0.97 ) had greater classification power compared with all other programs ( figure 4 ; supplementary tables s1 and s2 ) .", "although cpc had the highest sensitivity ( 0.99 ) , it suffered from poor specificity ( 0.74 ) .", "one possible explanation is that a significant proportion of ncrnas has a certain degree of sequence similarity to protein - coding genes .", "phylocsf had the least sensitivity ( 0.90 ) and the lowest specificity ( 0.63 ) .", "part of the reason for these outcomes is that nonconserved transcripts can not be processed by phylocsf .", "if we consider those 528 nonconserved transcripts as noncoding , the specificity increased from 0.63 to 0.69 , and the sensitivity remained unchanged .", "cpat achieved highest overall accuracy ( 0.97 ) when compared with cpc ( 0.87 ) , phylocsf ( 0.76 ) and portrait ( 0.92 ) .", "cpat s excellent discriminatory power was further demonstrated by the greatest separation between the score distributions of coding and noncoding sequences ( figure 5 ) . unlike cpc , phylocsf and portrait , choosing a smaller cpat score threshold to increase the sensitivity will not sacrifice too much specificity . \n", "figure 4.performance comparison between cpat , cpc , phylocsf and portrait using roc curves . \n", "figure 5.cumulative curves of coding - potential assessment score for ( a ) cpat , ( b ) portrait , ( c ) cpc and ( d ) phylocsf .", "cumulative curves of coding - potential assessment score for ( a ) cpat , ( b ) portrait , ( c ) cpc and ( d ) phylocsf .", "one could argue that phylocsf underperformed in this study because we used whole transcripts for testing rather than consecutive protein - coding exons and intergenic regions as used in its original article ( 17 ) . to address this issue , we compiled another single - exon test data set consisting of 184 protein - coding and 278 noncoding transcripts . the test results with this data set indicated that cpat ( sensitivity : 0.962 , specificity : 0.842 ) still outperformed phylocsf ( sensitivity : 0.832 , specificity : 0.588 , supplementary figure s2 ) .", "( 25 ) , phylocsf ( sensitivity 0.91 , specificity 0.99 ) has better performance than cpat ( sensitivity 0.50 , specificity 0.98 ) .", "this is reasonable because lncrnas in our test data set are poorly conserved , whereas lncrnas in lin et al .", "test data set are highly conserved because they are taken from multiple - sequence alignments of three closely related drosophila species .", "hence , we argue that phylocsf works better if the transcripts are highly conserved , which are rare to find in lncrnas ( 5,6 ) .", "this also highlights the achilles heel of the alignment - based methods for detecting lncrnas .", "in contrast , the dramatic decrease in cpat s sensitivity is due to the lack of orf information in lin et al .", "test data set , which is largely composed of individual exons , and not exon - length complete transcripts .", "this , however , will not limit the application scope of cpat because most full - length transcripts can be constructed at the current sequencing depth ( 8) .", "we measured the computational speed of cpat , cpc and phylocsf on a sample of 200 sequences randomly selected from the test data set .", "cpat took 0.67 s to process the data , and it was four orders of magnitudes faster than both cpc [ 11 945 s ( 3.3 h ) ] and phylocsf [ 11 737 s ( 3.3 h ) ] . furthermore ,", "computational time for the phylocsf did not include the time spent preparing multiple - alignment files for analysis .", "portrait was significantly faster than cpc and phylocsf , and therefore all 8000 test genes were used to evaluate its speed : cpat took 23.83 s to process the test set , and it was 48 times faster than portrait [ 1146.30 s ( 19 min ) ] .", "a number of linguistic features characterizing coding rna sequences have been developed over the past 30 years .", "these include maximum orf size , dinucleotide usage , codon usage bias , hexamer usage bias , nucleotide composition bias between codon positions and imperfect periodicity in base occurrences ( 23,26 ) . among these features , we selected orf features ( size and coverage ) because of their discriminatory power and ease of calculation ( 21 ) . in - frame hexamer score", "was selected because it has the highest prediction accuracy ( average of sensitivity and specificity ) as evaluated by fickett and tung in 1992 ( 23 ) .", "fickett score was selected because it simultaneously captures the compositional bias and position asymmetry , which are orthogonal to the orf features .", "supplementary figure s3 shows the performance of these individual features as well as the combined feature set .", "the combined feature set has very high sensitivity and specificity ( > 0.966 ) , leaving very little room for further improvement .", "annotation of genomes has always been a challenging task for biologists , and these efforts have been accelerated by deep transcriptome sequencing .", "distinguishing between protein - coding and noncoding sequences is the first and arguably the most crucial step in genome annotation .", "detecting the coding - potential of these transcripts via alignment - based software is intractable .", "we developed cpat , a highly accurate alignment - free method , which uses a logistic regression model to discriminate between coding and noncoding transcripts using pure linguistic features . compared with other tools ,", "cpat is more robust , markedly faster and more convenient to use . taken together , cpat is able to accurately assess the coding potential of tens of thousands of transcripts in real - time , and will be a valuable tool for the rapidly growing rna - seq community .", "source code was implemented in c and python and is freely available at : http://code.google.com / p / cpat/. the web server was implemented in php , mysql and apache , with support for all major browsers : http://lilab.research.bcm.edu/cpat/index.php .", "supplementary data are available at nar online : supplementary tables 1 and 2 and supplementary figures 13 .", "department of defense prostate cancer program [ pc094421 to w.l . ] ; the cancer prevention and research institute of texas [ rp110471-c3 to w.l . ] ; the center for individualized medicine ( cim ) at mayo clinic ( to j.p.k . ) . funding for open access charge : cancer prevention and research institute of texas [ rp110471-c3 to w.l . ] ."], "abstract_text": ["<S> thousands of novel transcripts have been identified using deep transcriptome sequencing . </S>", "<S> this discovery of large and  </S>", "<S> hidden transcriptome rejuvenates the demand for methods that can rapidly distinguish between coding and noncoding rna . here </S>", "<S> , we present a novel alignment - free method , coding potential assessment tool ( cpat ) , which rapidly recognizes coding and noncoding transcripts from a large pool of candidates . to this end </S>", "<S> , cpat uses a logistic regression model built with four sequence features : open reading frame size , open reading frame coverage , fickett testcode statistic and hexamer usage bias . </S>", "<S> cpat software outperformed ( sensitivity : 0.96 , specificity : 0.97 ) other state - of - the - art alignment - based software such as coding - potential calculator ( sensitivity : 0.99 , specificity : 0.74 ) and phylo codon substitution frequencies ( sensitivity : 0.90 , specificity : 0.63 ) . </S>", "<S> in addition to high accuracy , cpat is approximately four orders of magnitude faster than coding - potential calculator and phylo codon substitution frequencies , enabling its users to process thousands of transcripts within seconds . </S>", "<S> the software accepts input sequences in either fasta- or bed - formatted data files . </S>", "<S> we also developed a web interface for cpat that allows users to submit sequences and receive the prediction results almost instantly . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "AVAILABILITY AND IMPLEMENTATION", "SUPPLEMENTARY DATA", "FUNDING", "Supplementary Material"], "sections": [["although the human genome sequence was released a decade ago , the role of functional noncoding rnas ( ncrnas ) is much less understood compared with their coding counterparts .", "several previous studies have demonstrated that the human genome is pervasively transcribed ( 14 ) , but thoroughly cataloging all the rna species ( especially ncrna ) is challenging .", "undiscovered ncrnas might be rare , transient or beyond the detection limits of conventional approaches .", "furthermore , ncrnas also tend to be idiosyncratic to species and tissues ( 5,6 ) .", "nevertheless , advances in rna - seq have provided a new method of surveying the whole transcriptome to an unprecedented degree .", "recent genome - wide studies revealed tens of thousands of novel transcripts , the majority of which were long noncoding rnas ( lncrnas , > 200 nt ) ( 49 ) .", "although a few dozen lncrnas have been characterized to some extent and are reported to have critical roles in diverse cellular and disease development processes ( 6,1014 ) , the biogenesis and function of most lncrnas remain unclear .", "accurate and quantitative assessment of coding potential is the first step toward comprehensive annotation of newly discovered transcripts .", "until now , prediction of coding potential heavily relied on sequence alignment , either pairwise homology search for protein evidence such as that used in the coding - potential calculator ( cpc ) and portrait methods ( 15,16 ) or multiple alignments to calculate the phylogenetic conservation score such as that used in the phylogenetic codon substitution frequencies ( phylocsf ) and rnacode methods ( 17,18 ) .", "alignment - based approaches are particularly useful for highly conserved protein - coding genes and , to a lesser extent , short genes encoding housekeeping or regulatory rnas ( e.g. snrnas , snorna , transfer rna ) .", "however , these approaches can not immediately apply to all the novel transcripts because of several intrinsic limitations .", "first , most newly discovered transcripts are lncrnas , which tend to be lineage specific and less conserved ( 5,6 ) .", "for example , only 29 of 550 lncrnas identified from zebrafish had detectable sequence similarity with putative mammalian orthologs ( 6 ) , and only 993 of 8195 human lncrnas have orthologous transcripts in other species ( 5 ) .", "second , considerable fractions of lncrnas are overlapped with either the sense or antisense strand of protein - coding genes .", "these lncrnas can not be correctly classified by homology searching because they would have significant matches to protein - coding genes ( 3,8,19 ) .", "third , the reliability of alignment - based approaches largely depends on the quality of alignments ( 20 ) .", "this is problematic because most widely used multiple - sequence alignment tools use heuristics and do not guarantee optimal alignments .", "for instance , cpc and phylocsf took 2 days to evaluate the coding potential of 14 353 lncrnas identified by cabili et al .", "this problem is getting more attention as massive - scale rna sequencing is increasingly being performed .", "consequently , a more accurate , robust and faster method that does not rely on sequence alignment is needed to distinguish ncrnas , especially lncrnas , from protein - coding genes . here , we present coding - potential assessment tool ( cpat ) , an alignment - free program , which uses logistic regression to distinguish between coding and noncoding transcripts on the basis of four sequence features .", "cpat is highly accurate ( 0.967 ) and extremely efficient ( 10 000 times faster than cpc and phylocsf , and 50 times faster than portrait ) .", "cpat needs only the sequence or coordinate file as input , and it is straightforward to use .", "we expanded the availability of cpat to a larger scientific audience via a web interface , which allows users to submit sequences and receive the prediction results back almost instantaneously ( http://lilab.research.bcm.edu/cpat/index.php ) ."], ["coding - potential prediction is essentially a binary decision problem , which makes logistic regression a suitable approach . as an alignment - free method , all selected features ( predictor variables )", "the first feature was the maximum length of the open reading frame ( orf ) .", "orf length is one of the most fundamental features used to distinguish ncrna from messenger rna because a long putative orf is unlikely to be observed by random chance in noncoding sequences . despite the simplicity , orf length has high concordance with more sophisticated discrimination methods and remains the primary criterion in almost all coding - potential prediction methods ( 21 ) .", "the second feature was orf coverage defined as the ratio of orf to transcript lengths .", "this feature also has good classification power , and it is highly complementary to , and independent of , the orf length ( supplementary figures s1 and s3 ) . some large bona fide ncrnas may contain putative long orfs by random chance ( 5 ) , and thus can not be classified correctly by orf length alone .", "fortunately , those large ncrnas usually have much lower orf coverage than protein - coding rnas ( figure 1b ) . \n", "figure 1.score distribution between coding ( red ) and noncoding ( blue ) transcripts for the four linguistic features selected to build the logistic regression model ; training data set containing 10 000 coding and 10 000 noncoding transcripts were used .", "score distribution between coding ( red ) and noncoding ( blue ) transcripts for the four linguistic features selected to build the logistic regression model ; training data set containing 10 000 coding and 10 000 noncoding transcripts were used .", "the third feature we used was the fickett testcode score ( termed  fickett score hereafter ) , which is a simple linguistic feature that distinguishes protein - coding rna and ncrna according to the combinational effect of nucleotide composition and codon usage bias ( 22 ) .", "briefly , the fickett score is obtained by computing four position values and four composition values ( nucleotide content ) from the dna sequence .", "the position value reflects the degree to which each base is favored in one codon position versus another .", "for example , position value of a ( apos ) is calculated as follows : \n\n  cpos , gpos and tpos are determined in the same way .", "these eight values are then converted into probabilities ( p ) of coding using a lookup table provided in the original article .", "each probability is multiplied by a weight ( w ) for the respective base , where the value of w reflects the percentage of time each parameter alone successfully predicts coding or noncoding function for the sequences of known function .", "finally , the fickett score is calculated as follows : \n\n  the fickett score is independent of the orf , and when the test region is 200 nt in length ( which includes most lncrna ) , this feature alone can achieve 94% sensitivity and 97% specificity , with ", "the fourth feature we used was hexamer usage bias ( termed  hexamer score hereafter ) .", "this may be the most discriminating feature because of the dependence between adjacent amino acids in proteins ( 23 ) .", "the hexamer score can be computed in numerous ways ; here , we used a log - likelihood ratio to measure differential hexamer usage between coding and noncoding sequences . for a given dna sequence , we calculated the probability of the sequence under the model of coding dna and under the model of noncoding dna , and then we took the logarithm of the ratio of these probabilities as the score of coding potential .", "we used f ( hi ) ( i = 0 , 1 ,  , 4095 ) and f ( hi ) ( i = 0 , 1 ,  , 4095 ) to represent in - frame hexamer frequency , calculated from coding and noncoding training data sets ( described below ) , respectively .", "for a given hexamer sequence s = h1 , h2 ,  , hm , \n\n  hexamer score determines the relative degree of hexamer usage bias in a particular sequence .", "positive values indicate a coding sequence , whereas negative values indicate a noncoding sequence . we build a logistic regression model using these four linguistic features as predictor variables .", "a  test was used to evaluate whether our logit model with predictors fits the training data significantly better than the null model , which had only an intercept .", "we built a high - confidence training data set to measure the prediction performance of our logit model .", "this data set contained 10 000 protein - coding transcripts selected from the refseq database ; all transcripts had high - quality protein sequences annotated by the consensus coding sequence project .", "we evaluate the model with a 10-fold cross - validation and measured its sensitivity , specificity , accuracy , precision and area under the curve ( auc ) characteristics .", "the receiver operating characteristic ( roc ) curve and precision  recall ( pr ) curve were generated using rocr package ( 24 ) .", "we also built a nonparametric two - graph roc curve for selecting the optimal cpat score threshold that maximizes the sensitivity and specificity of cpat while minimizing misclassifications .", "we built an independent test data set to compare the performance of cpat with that of cpc , phylocsf and portrait .", "this test set composed of 4000 high - quality protein - coding genes ( refseq annotated ) and 4000 lncrnas from a human lncrna catalog ( 5 ) .", "assuming that all 4000 lncrnas are truly noncoding sequences , we could compute the sensitivity , specificity , accuracy and precision of the algorithms to measure their performance .", "those 528 genes were equally assigned to the true - negative and false - positive categories .", "the abbreviations in the equations below are as follows : fn , false negative ; fp , false positive ; tn , true negative ; tp , true positive \n\n\n"], ["all four selected features were concordantly higher in coding transcripts and lower in noncoding transcripts ( figure 1 ) .", "we plotted three major features ( orf size , fickett score and hexamer score ) in a three - dimensional space to evaluate their combinatorial effect ( figure 2 ) .", "coding and noncoding transcripts in our training data set were grouped into two distinct clusters , indicating good concordance between features .", "the  test p value was < .001 ( = 23 548.44 ; degrees of freedom = 4 ) , indicating that the logit model as a whole fits significantly better than the null model .", "ten - fold cross - validation showed that cpat could achieve very high accuracy , with an auc of 0.9927 ( figure 3a ) .", "we also provide the pr curve because the roc curve can be misleading when the test data are largely skewed ( figure 3b ) .", "we use nonparametric two - graph roc curves to determine an optimal cpat score threshold that maximizes the discriminatory power ( figure 3c and d ) . according to figure 3d ,", "a score threshold of 0.364 gave the highest sensitivity and specificity ( 0.966 for both ) for human data . \n", "figure 2.three-dimensional plot shows combinatorial effects of fickett score , hexamer score and orf size on 10 000 coding genes ( red dots ) and 10 000 noncoding genes ( blue dots ) . \n", "dashed curves represent the 10-fold cross - validation ; solid curves represent the averaged curve from 10 validation runs .", "( d ) two - graph roc curve is used to determine the optimum cutoff value .", "three - dimensional plot shows combinatorial effects of fickett score , hexamer score and orf size on 10 000 coding genes ( red dots ) and 10 000 noncoding genes ( blue dots ) .", "dashed curves represent the 10-fold cross - validation ; solid curves represent the averaged curve from 10 validation runs .", "( d ) two - graph roc curve is used to determine the optimum cutoff value .", "we compared the performance of cpat with that of cpc , phylocsf and portrait ( protein - independent support vector machine model ) using an independent test data set composed of 4000 coding genes and 4000 noncoding genes .", "a multiple alignment of 45 vertebrate genomes , including that of human , was downloaded from the ucsc ( university of california , santa cruz ) genome browser and was used as the input alignment for phylocsf . in general , cpat ( sensitivity : 0.96 , specificity : 0.97 ) had greater classification power compared with all other programs ( figure 4 ; supplementary tables s1 and s2 ) .", "although cpc had the highest sensitivity ( 0.99 ) , it suffered from poor specificity ( 0.74 ) .", "one possible explanation is that a significant proportion of ncrnas has a certain degree of sequence similarity to protein - coding genes .", "phylocsf had the least sensitivity ( 0.90 ) and the lowest specificity ( 0.63 ) .", "part of the reason for these outcomes is that nonconserved transcripts can not be processed by phylocsf .", "if we consider those 528 nonconserved transcripts as noncoding , the specificity increased from 0.63 to 0.69 , and the sensitivity remained unchanged .", "cpat achieved highest overall accuracy ( 0.97 ) when compared with cpc ( 0.87 ) , phylocsf ( 0.76 ) and portrait ( 0.92 ) .", "cpat s excellent discriminatory power was further demonstrated by the greatest separation between the score distributions of coding and noncoding sequences ( figure 5 ) . unlike cpc , phylocsf and portrait , choosing a smaller cpat score threshold to increase the sensitivity will not sacrifice too much specificity . \n", "figure 4.performance comparison between cpat , cpc , phylocsf and portrait using roc curves . \n", "figure 5.cumulative curves of coding - potential assessment score for ( a ) cpat , ( b ) portrait , ( c ) cpc and ( d ) phylocsf .", "cumulative curves of coding - potential assessment score for ( a ) cpat , ( b ) portrait , ( c ) cpc and ( d ) phylocsf .", "one could argue that phylocsf underperformed in this study because we used whole transcripts for testing rather than consecutive protein - coding exons and intergenic regions as used in its original article ( 17 ) . to address this issue , we compiled another single - exon test data set consisting of 184 protein - coding and 278 noncoding transcripts . the test results with this data set indicated that cpat ( sensitivity : 0.962 , specificity : 0.842 ) still outperformed phylocsf ( sensitivity : 0.832 , specificity : 0.588 , supplementary figure s2 ) .", "( 25 ) , phylocsf ( sensitivity 0.91 , specificity 0.99 ) has better performance than cpat ( sensitivity 0.50 , specificity 0.98 ) .", "this is reasonable because lncrnas in our test data set are poorly conserved , whereas lncrnas in lin et al .", "test data set are highly conserved because they are taken from multiple - sequence alignments of three closely related drosophila species .", "hence , we argue that phylocsf works better if the transcripts are highly conserved , which are rare to find in lncrnas ( 5,6 ) .", "this also highlights the achilles heel of the alignment - based methods for detecting lncrnas .", "in contrast , the dramatic decrease in cpat s sensitivity is due to the lack of orf information in lin et al .", "test data set , which is largely composed of individual exons , and not exon - length complete transcripts .", "this , however , will not limit the application scope of cpat because most full - length transcripts can be constructed at the current sequencing depth ( 8) .", "we measured the computational speed of cpat , cpc and phylocsf on a sample of 200 sequences randomly selected from the test data set .", "cpat took 0.67 s to process the data , and it was four orders of magnitudes faster than both cpc [ 11 945 s ( 3.3 h ) ] and phylocsf [ 11 737 s ( 3.3 h ) ] . furthermore ,", "computational time for the phylocsf did not include the time spent preparing multiple - alignment files for analysis .", "portrait was significantly faster than cpc and phylocsf , and therefore all 8000 test genes were used to evaluate its speed : cpat took 23.83 s to process the test set , and it was 48 times faster than portrait [ 1146.30 s ( 19 min ) ] ."], ["a number of linguistic features characterizing coding rna sequences have been developed over the past 30 years .", "these include maximum orf size , dinucleotide usage , codon usage bias , hexamer usage bias , nucleotide composition bias between codon positions and imperfect periodicity in base occurrences ( 23,26 ) . among these features , we selected orf features ( size and coverage ) because of their discriminatory power and ease of calculation ( 21 ) . in - frame hexamer score", "was selected because it has the highest prediction accuracy ( average of sensitivity and specificity ) as evaluated by fickett and tung in 1992 ( 23 ) .", "fickett score was selected because it simultaneously captures the compositional bias and position asymmetry , which are orthogonal to the orf features .", "supplementary figure s3 shows the performance of these individual features as well as the combined feature set .", "the combined feature set has very high sensitivity and specificity ( > 0.966 ) , leaving very little room for further improvement .", "annotation of genomes has always been a challenging task for biologists , and these efforts have been accelerated by deep transcriptome sequencing .", "distinguishing between protein - coding and noncoding sequences is the first and arguably the most crucial step in genome annotation .", "detecting the coding - potential of these transcripts via alignment - based software is intractable .", "we developed cpat , a highly accurate alignment - free method , which uses a logistic regression model to discriminate between coding and noncoding transcripts using pure linguistic features . compared with other tools ,", "cpat is more robust , markedly faster and more convenient to use . taken together , cpat is able to accurately assess the coding potential of tens of thousands of transcripts in real - time , and will be a valuable tool for the rapidly growing rna - seq community ."], ["source code was implemented in c and python and is freely available at : http://code.google.com / p / cpat/. the web server was implemented in php , mysql and apache , with support for all major browsers : http://lilab.research.bcm.edu/cpat/index.php ."], ["supplementary data are available at nar online : supplementary tables 1 and 2 and supplementary figures 13 ."], ["department of defense prostate cancer program [ pc094421 to w.l . ] ; the cancer prevention and research institute of texas [ rp110471-c3 to w.l . ] ; the center for individualized medicine ( cim ) at mayo clinic ( to j.p.k . ) . funding for open access charge : cancer prevention and research institute of texas [ rp110471-c3 to w.l . ] ."], [""]]}
{"article_id": "PMC3656010", "article_text": ["beyond its role of cellular energy currency and phosphate donor , atp plays a potent signaling role through its extracellular release and activation of cell surface purinergic receptors .", "fast responses to atp are mediated through activation of p2x receptors , a family ( p2x1p2x7 ) of atp - activated ligand - gated ion channels and metabotropic effects through the activation of p2y receptors ( p2y12 , p2y4 , p2y6 , p2y1114 ) which couple to heterotrimeric g proteins . human leukocytes including monocytes , mast cells , neutrophils and central microglia express a diverse repertoire of p2y receptors though p2x1 , p2x4 and p2x7 are the dominant p2x subtypes present .", "activation of purinergic receptors in leukocytes is coupled to the production and secretion of cytokines and other pro - inflammatory molecules including prostaglandin e2 .", "purinergic receptors are associated with inflammation , with some receptors inhibited either directly ( p2y12 ) or indirectly through the activity of anti - thrombotic , in the case of platelet p2y12 , or anti - inflammatory agents , in the case of the action of statins on monocyte p2x4 .", "atp can act as a non - peptide damage - associated molecular pattern ( damp ) release from injured cells and tissues . in this fashion", "the release of cellular atp is unregulated and released due to cell lysis or puncture .", "cell surface purinergic receptors are activated by this atp damp signal which serves to initiate an inflammatory response and promote wound healing .", "however , atp can be released from cells physiologically and act as a critical signal for painful , inflammatory processes .", "do release other nucleotides including utp and udp - sugars but our focus here is atp release . mechanisms of atp release during physiological processes remain diverse and controversial .", "investigation into how cellular stress stimulates atp release in non - leukocytes suggests roles for connexin and pannexin hemichannels , maxi- and volume - regulated anion channels and efflux through the p2x7 receptor , though release routes and signal transduction mechanism underlying atp release in leukocytes remain poorly defined .", "in neutrophils atp is released in response to activation of fmlp receptor by bacterially derived n - formylmethionine .", "the precise release mechanism is unclear but occurs at the leading edge of migrating neutrophils .", "released atp and its subsequent metabolism to adenosine at the cell surface activate p2y2 and a3 receptors serving to direct cell orientation and promote migration in response to chemotactic signals .", "bacterially derived lipopolysaccharide can stimulate central microglia to secrete atp which in - turn activates neighboring astrocytes and modulates excitatory neurotransmission .", "this raises the possibility that atp can act in a feed forward loop possibly amplifying responses to itself or other external cues which couple to atp secretion .", "furthermore , such constitutive secretion leads to activation of cell surface gq - coupled p2y receptors which elevate intracellular calcium levels through release of calcium .", "this mode of constitutive secretion generates a constant pericellular atp cloud or  halo  which appears to be important in regulating intracellular calcium homeostasis following p2y receptor activation . in cells of hematopoietic lineage such as monocyte / macrophage , nk killer and mast cells , secretory lysosomes have evolved as bifunctional organelles which combine classical degradative properties with secretion .", "the mechanism of lysosomal atp transport remains undefined though a nucleotide transporter ( v - nut ) has been characterized for other atp containing vesicles .", "the signal transduction coupling external cues such as cytokines , chemokines and bacterially derived molecules to atp release is poorly defined .", "what are the release machines in leukocytes and do they differ from other cell types ? it is expected that therapeutic intervention in agonist stimulated atp release is a potentially novel route to pharmacological modulation of innate immune responses but also in chronic inflammatory disease where normal inflammatory responses are heighten and act deleteriously ."], "abstract_text": ["<S> release and reception of extracellular atp by leukocytes plays a critical role in immune responses to infection , injury and cardiovascular disease . </S>", "<S> leukocytes of both the innate , adaptive immune and central nervous system express a repertoire of cell surface receptors for atp ( p2x and p2y receptors ) and its metabolites . </S>", "<S> atp acts as a damage - associated molecule pattern ( damp ) released by injured or dying cells . </S>", "<S> detection of released atp by neighboring leukocytes initiates inflammation and wound healing . </S>", "<S> however , recent evidence from our group and others suggests atp release by leukocytes themselves serves to regulate homeostatic mechanisms and coordinate responses to external pro - inflammatory cues . </S>", "<S> examples include the homeostatic control of intracellular calcium and regulation of migratory guidance during chemotactic response to external cues . </S>", "<S> though there has been some progress in elucidating atp release mechanisms of some mammalian cells types , release conduits and coupling signal transduction machinery remain larger elusive for leukocytes . </S>", "<S> our recent studies suggest a role for secretory lysosomes in releasing atp in monocytes . though poorly defined , </S>", "<S> targeting atp release mechanisms in leukocytes have great anti - inflammatory potential . </S>"], "labels": null, "section_names": ["Purinergic Signaling in Blood Cells and Leukocytes", "ATP Release Mechanisms in Leukocytes", "Agonist Stimulated ATP Release", "Lysosomes as ATP Release Machines", "Future Investigation"], "sections": [["beyond its role of cellular energy currency and phosphate donor , atp plays a potent signaling role through its extracellular release and activation of cell surface purinergic receptors .", "fast responses to atp are mediated through activation of p2x receptors , a family ( p2x1p2x7 ) of atp - activated ligand - gated ion channels and metabotropic effects through the activation of p2y receptors ( p2y12 , p2y4 , p2y6 , p2y1114 ) which couple to heterotrimeric g proteins . human leukocytes including monocytes , mast cells , neutrophils and central microglia express a diverse repertoire of p2y receptors though p2x1 , p2x4 and p2x7 are the dominant p2x subtypes present .", "activation of purinergic receptors in leukocytes is coupled to the production and secretion of cytokines and other pro - inflammatory molecules including prostaglandin e2 .", "purinergic receptors are associated with inflammation , with some receptors inhibited either directly ( p2y12 ) or indirectly through the activity of anti - thrombotic , in the case of platelet p2y12 , or anti - inflammatory agents , in the case of the action of statins on monocyte p2x4 .", "atp can act as a non - peptide damage - associated molecular pattern ( damp ) release from injured cells and tissues . in this fashion", "the release of cellular atp is unregulated and released due to cell lysis or puncture .", "cell surface purinergic receptors are activated by this atp damp signal which serves to initiate an inflammatory response and promote wound healing .", "however , atp can be released from cells physiologically and act as a critical signal for painful , inflammatory processes .", "do release other nucleotides including utp and udp - sugars but our focus here is atp release . mechanisms of atp release during physiological processes remain diverse and controversial ."], ["investigation into how cellular stress stimulates atp release in non - leukocytes suggests roles for connexin and pannexin hemichannels , maxi- and volume - regulated anion channels and efflux through the p2x7 receptor , though release routes and signal transduction mechanism underlying atp release in leukocytes remain poorly defined ."], ["in neutrophils atp is released in response to activation of fmlp receptor by bacterially derived n - formylmethionine .", "the precise release mechanism is unclear but occurs at the leading edge of migrating neutrophils .", "released atp and its subsequent metabolism to adenosine at the cell surface activate p2y2 and a3 receptors serving to direct cell orientation and promote migration in response to chemotactic signals .", "bacterially derived lipopolysaccharide can stimulate central microglia to secrete atp which in - turn activates neighboring astrocytes and modulates excitatory neurotransmission .", "this raises the possibility that atp can act in a feed forward loop possibly amplifying responses to itself or other external cues which couple to atp secretion ."], ["furthermore , such constitutive secretion leads to activation of cell surface gq - coupled p2y receptors which elevate intracellular calcium levels through release of calcium .", "this mode of constitutive secretion generates a constant pericellular atp cloud or  halo  which appears to be important in regulating intracellular calcium homeostasis following p2y receptor activation . in cells of hematopoietic lineage such as monocyte / macrophage , nk killer and mast cells , secretory lysosomes have evolved as bifunctional organelles which combine classical degradative properties with secretion .", "the mechanism of lysosomal atp transport remains undefined though a nucleotide transporter ( v - nut ) has been characterized for other atp containing vesicles ."], ["the signal transduction coupling external cues such as cytokines , chemokines and bacterially derived molecules to atp release is poorly defined .", "what are the release machines in leukocytes and do they differ from other cell types ? it is expected that therapeutic intervention in agonist stimulated atp release is a potentially novel route to pharmacological modulation of innate immune responses but also in chronic inflammatory disease where normal inflammatory responses are heighten and act deleteriously ."]]}
{"article_id": "PMC3503369", "article_text": ["paraoxonases were originally discovered as enzymes hydrolyzing exogenous toxic organophosphate compounds such as insecticide paraoxon .", "there are three members of paraoxonases family currently known : paraoxonase 1 ( pon1 ) , paraoxonase 2 ( pon2 ) , and paraoxonase 3 ( pon3 ) , which are encoded by three separate genes on the same chromosome 7 ( human ) or chromosome 6 ( mouse ) .", "all three human members of the family are 70% identical at nucleotide level and 60% identical at the amino acid level .", "studies on enzymatic activity of paraoxonases revealed esterase and lactonase / lactonizing activities in addition to organophosphatase activity . despite of the large number of compounds that can be hydrolyzed by paraoxonase ,", "in many cases arylesterase activity is measured to access pon1 level in biological samples . based on kinetic parameters of paraoxonases toward different substrates", "hydrolysis of homocysteine thiolactone by pon1 is considered to be protective against coronary artery disease ( cad ) .", "after the introduction of the oxidative stress hypothesis of atherosclerosis and the discovery of antioxidant effect of high - density lipoprotein ( hdl ) , pon1 attracted significant interest as a protein that is responsible for the most of antioxidant properties of hdl .", "purified pon1 protects hdl and low - density lipoprotein ( ldl ) from oxidation catalyzed by copper ions .", "pon1 inhibits copper - induced hdl oxidation by prolonging oxidation lag phase , and reduces peroxide and aldehyde content in oxidized hdl .", "remarkably , pon1 inhibitors pd-11612 , pd-65950 , pd-92770 , and pd-113487 lessen this antioxidative effect of pon1 .", "pon1 is especially effective in decomposition of linoleate hydroperoxides . also , mass - spectrometry analysis of biologically active fraction of oxidized2-arachidonoyl - sn - glycero-3-phosphorylcholine ( ox - papc ) underwent pon1 treatment showed degradation of these oxidized phospholipids by pon1 .", "the exact antioxidant mechanism of pon1 is not known yet , although it is known that protection is not caused by chelating of copper ions or because of potential lipid transfer from ldl to hdl .", "existence of an enzymatic mechanism is supported by the observation that heat inactivation of purified pon1 abolishes its antioxidant effect .", "some in vitro data suggest that antioxidant activity might be related to other components of purified pon1 preparations .", "however , experiments with pon1 deficient mouse provide strong evidences that pon1 is required to enable hdl antioxidant properties .", "hdl from pon1 knockout animals were more prone to oxidation and were less efficient in the protection of ldl from oxidation in co - cultured cell model of the artery wall compared with hdl from control mice .", "also , transfection of pon1-deficient peritoneal macrophages ( isolated from pon1 knockout mice ) with human pon1 decreased level of peroxides , lowered release of superoxide , and increased intracellular level of reduced glutathione , key observations are summarized in table 1 .", "summary of key facts on pon1 expression , activity , and effects in in vitro and in vivo studies", "the first pon1 messenger ribonucleic acid ( mrna)ionanalysis in different rabbit tissues was performed by northern blot , and revealed pon1 mrna expression predominately in liver .", "polymerase chain reaction ( pcr ) amplification using a panel of first - strand complementary deoxyribonucleic acid ( cdnas ) from 24 tissues detected pon1 expression in kidney and colon beside liver and fetal liver expression .", "biopsies showed pon1 mrna and protein expression in human but not in mouse gastrointestinal tract .", "deletion analysis in cultured cells revealed that cell type specific expression in liver and kidney is determined within first 200 bp of promoter area .", "there are several transcription factors and pathways that regulate pon1 expression [ figure 1 ] .", "all processes occur in liver , and bile acid -stimulated synthesis of fibroblast growth factor 19 ( fgf-19 ; or fgf-15 in mouse ) might be additionally occur in ileum .", "signals from pma ( phorbol 12-myristate 13-acetate ) and high glucose activate transcription factor sp1 through protein kinases c ( pkc ) and p44/ p42 mitogen - activated protein kinases ( mapk ) a ubiquitous mammalian transcription factor specificity protein 1(sp1 ) plays an essential role in regulation of pon1 expression .", "high glucose level activates protein kinase c ( pkc ) , which activates sp1 , and stimulate pon1 transcription in human hepatoma cell lines hepg2 and huh7 . a potent pkc activator phorbol 12-myristate 13-acetate ( pma ) also stimulates pon1 transcription in hepg2 through activation of sp1 .", "two members of pkc family are involved , pkc ( zeta ) and pkc- ( alpha ) activation .", "statins ( pitavastatin , simvastatin , or atorvastatin ) stimulate pon1 transcription through sp1 activation as well , however , they activate another kinase , p44/p42 mitogen - activated protein ( map ) kinase , as was observed for pitavastatin in huh7 cells . also , pivostatin - stimulated p44/p42 mitogen - activated protein kinase ( mapk ) activates sterol regulatory element binding protein 2 ( srebp-2 ) , which contributes to transcriptional activation of pon1 .", "dietary polyphenols , such as resveratrol , aspirin and its hydrolysis product salicylate , and artificial ligands of aryl hydrocarbon receptor ( ahr ) , such as 3-methylcholanthrene , activate ahr and stimulate pon1 transcription activation in mouse liver and hepg2 cell line.[2426 ] c - jun is another transcription factors that is involved in pon1 expression .", "the activity of c - jun is regulated by c - jun n - terminal kinase ( jnk ) .", "phosphorylated c - jun in a complex with c - fos or some other transcription factors forms an active ap-1 complex that usually promotes transcription of target genes .", "thus , berberine ( benzyl tetrahydroxyquinoline ) , a cholesterol lowering alkaloid , activates jnk , c - jun and stimulates pon1 transcription in human hepatoma cell lines .", "however , stimulation of jnk / c - jun pathway by bile acids leads to opposite effect .", "bile acids such as taurocholate , cholic acid , or chenodeoxycholic acid inhibit pon1 expression in liver of c57bl/6j mice , and in hepg2 and huh7 .", "the inhibition of pon1 expression starts with activation of farnesoid x receptor ( fxr ) by bile acids that promote expression of fibroblast growth factor 19 in human ( fgf-19 ) or fgf-15 in mouse .", "the growth factor activates fibroblast growth factor receptor 4 ( fgfr4 ) followed by activation of jnk , and phosphorylation of c - jun .", "opposite to activation of c - jun by berberine , fxr / fgf-19/fgfr4/jnk / c - jun pathway results in suppression of pon1 transcription .", "it is likely that pon1 stays associated with endoplasmic reticulum through its hydrophobic n - terminus until it is released from the hepatocytes .", "the mechanism of pon1 secretion is not well investigated . a study on cultured hepatocytes and transfected chinese hamster ovary ( cho ) cells , which do not naturally express pon1 and apoa proteins ,", "when pon1 protein is synthesized , it accumulates on plasma membrane and slowly dissociate into extracellular medium .", "dissociation is promoted by hdl , very - low - density lipoprotein ( vldl ) , and in much lesser extent by protein - free phospholipids particles or apoa - i protein .", "pon1 binds to hdl through interaction of hydrophobic n - terminus to phospholipids , and through pon1-apoa interaction .", "two major principal hdl proteins , that is , apoa - i and/or apoa - ii which differ in their properties are linked to atherosclerosis modulation . thus , transgenic mice with hdl consisting of both human apoa - i and apoa - ii developed 15-fold greater lesions compared with mice with hdl containing solely human apoa - i . in vitro study of reconstituted hdl demonstrated that apoa - i containing hdl stabilizes pon1 binding as compared with protein - free hdl particles , and apoa - ii in opposite destabilizes the hdl ", "the paraoxonase and arylesterase activities of pon1 do not depend on the apolipoprotein content of hdl .", "however , apoa - i containing hdl significantly increases lactonase activity of pon1 , promotes inhibition of ldl oxidation , and stimulates macrophage cholesterol efflux compared with apoa - ii containing hdl .", "a study of human hdl isolated from apoa - i deficient patients revealed that pon1 is still associated with hdl in the absence of apoa-1 , however , the pon1hdl complex is less stable , and pon1 loses activity faster than in normal controls .", "a higher content of human apoa - ii protein in mouse hdl suppresses pon1 binding to hdl compared with hdl with lower human apoa - ii content .", "hdl high in human apoa - ii binds less pon1 protein , possesses less pon1 activity , and is impaired in protection of ldl from oxidation .", "transgenic mouse with higher human apoa - ii content in hdl are more susceptible to atherosclerosis compared with transgenic animals with lower apoa - ii level in hdl and control animal .", "hdl - associated pon1 can be successfully transferred to cultured cells in vitro and deliver protection from oxidative stress and bacterial substrate of pon1 n-3-oxo - dodecanoyl - l - homoserine lactone . beside hdl", "while liver is the major tissue of pon1 gene expression , lipoprotein - assisted circulation of pon1 in plasma delivers the enzyme to multiple tissues that do not express pon1 themselves .", "immunohistochemical staining of rat tissues revealed pon1 presence in liver , kidney , in endothelial lining of lung and brain .", "more recent study with mouse tissue detected pon1 protein in hepatocytes , adipocytes , chondrocytes , skeletal and cardiac muscle , kidney , spermatozoa , and epithelial cells of skin , stomach , intestine , trachea , bronchiole , and eye lens .", "studies did not find co - localization of pon1 with apoa - i protein that might mean local pon1 expression or different sources of apoa - i , an integral component of hdl .", "immunostaining of healthy human aorta shows a low level and granular distribution of pon1 in smooth muscle cells .", "western blot confirmed presence of intact and degraded pon1 in media of healthy aorta . with the development of atherosclerosis , pon1", "increasing accumulation of pon1 with the progression of the atherosclerosis can be seen in the intimal part of aorta as well .", "aorta with advanced atherosclerosis lesions shows massive pon1 accumulation . a more recent study attributed an increased pon1 immunostaining in human atherosclerotic arteries to macrophages accumulated in lesions .", "high  fat , high - cholesterol ( atherogenic ) diet leads to fast development of atherosclerosis in c57bl/6j mouse strain with concomitant decrease in liver pon1 expression . at the same time pon1 expression and its protective effects", "macrophages from this mouse did not protect ldl from oxidation by other cells in vitro , and pon1 knockout mice developed atherosclerosis faster than control mice on high fat , high cholesterol diet . to study the effect of pon1 knockout further ,", ". plasma lipid profile of these double knockout mice was similar to apoe-/- control animals with slightly decreased level of intermediate density lipoprotein ( idl ) and ldl , and increased level of lysophosphatidylcholine and oxidized phospholipids in idl and ldl .", "the rates of ldl oxidation and clearance were higher in pon1-/- , apoe-/- mice than in control mice as was determined by injection of human ldl .", "expression of genes responding to increase of oxldl , such as heme oxygenase-1 , peroxisome proliferator - activated receptor gamma , scavenger receptor type a , cd36 , and macrosialin , was upregulated in liver of pon1-/- , apoe-/- mice compared with apoe-/- mice .", "involvement of pon in inflammation and oxidative stress were detected in vascular wall of pon-/- animals .", "expression of mrna of essential cell adhesion molecules p - selectin and icam1 was upregulated in aortic wall .", "if a deficiency of pon1 leads to inflammation and oxidative stress , then increases in pon1 function could be beneficial .", "a transgenic mouse mpon1 was developed in 2001 , with 5-fold higher level of mouse pon1 protein and corresponding increase in arylesterase activity .", "pon1 was associated with hdl , and protected hdl from oxidation by copper ions as it was assessed by lipid hydroperoxide formation assay , and by retaining oxidation - sensitive activity of lecithin : cholesterol acyltransferase ( lcat ) . a decrease in atherosclerotic lesion size and improved oxidation status of aorta and", "peritoneal macrophages in pon1 overexpressing transgenic mouse on apoe-/- background have been compared with apoe-/- control have been reported . in another transgenic mouse model , pon1-tg , which overexpresses human pon1 gene of 55l/192q genotype , plasma level of arylesterase activity was twice higher in pon1-tg mouse compared with control mouse when animals were fed normal chow .", "the excess of pon1 level resulted in better retaining of its activity after feeding animals with atherogenic diet .", "pon1 activity decreased to about three - fourth ( 3/4 ) of original level after 15 weeks of atherogenic diet feeding to pon1-tg mice , and to less than one - half ( 1/2 ) of original level in control animals .", "thus , arylesterase activity mediated by pon1 was almost 4-fold higher in pon1-tg mice compared with control mice after feeding atherogenic diet .", "aortic lesion areas in pon1-tg animals were about half of the size of areas of the control mice after feeding atherogenic diet .", "apoe-/- , pon1-tg genotype mice fed normal chow similarly developed lesions slower , compared with apoe-/- controls ; however , the difference in lesion area was just 22% .", "plasma arylesterase activity of apoe-/- , pon1-tg mice was 2.5-fold higher than that of apoe-/- animals , however , there is no difference between their lipid profile . a decreased inflammatory status of aortas in apoe-/- , pon1-tg mice compared with apoe-/- mice was detected by examination of the expression level of mcp-1 cytokine gene .", "hdl isolated from pon1-tg mouse plasma or apoe-/- , pon1-tg mouse plasma provided better protection against ldl oxidation , than hdl isolated from control animals . beside pon1 studies on transgenic mouse , a transient overexpression of pon1 gene in mouse", "adenovirus - mediated delivery of human pon1 gene ( 55 m , 192q genotype ) in double knockout leptin-/- ( ob / ob ) , ldlr-/- mouse resulted in more than 4-fold increase of pon1 activity on day 7 with gradual decrease to the control level by day 21 .", "mice were injected with adenovirus at the age of 18 weeks when the progression of atherosclerosis occurs in a fast rate .", "six weeks later , cross - sections of the aortic root were analyzed by immunohistochemistry .", "plaque size , volume of macrophages , concentration of oxldl was significantly lower in pon1 expressing animals .", "the titer of anti - oxldl antibodies was lower in plasma of pon1 overexpressing mice as well .", "while an early pon1 intervention proved to have significant athero - protective effect , another study was performed to assess whether transient pon1 overexpression can improve vascular function in advanced atherosclerosis .", "similar to the previous study , 55m/192q variant of human pon1 gene was delivered in 18 month old apoe-/- mouse fed normal chow using adenovirus vector .", "serum pon1 activity was increased 10-fold by day 7 and 2.5-fold on the day 21 in pon1 overexpressing animals compared with controls . after 3 weeks , lesions were measured and no changes in their size were found . however , vasomotor function was improved by transient overexpression of pon1 .", "thus , phenylephrine - constricted segments of the aortas relaxed significantly better in response to endothelium dependent agonists ach , adenosine-5-triphosphate ( atp ) , and uridine-5-triphosphate ( utp ) observations on pon1 role in animal models is summarized in table 2 .", "meta - analysis of 47 studies with 9853 cad and 11,408 control subjects published in 2012 confirmed association of lower plasma pon1 activity with increased cad risk ( 19% lower pon1 activity , p < 0.00001 ) .", "another meta - analysis published in 2012 included 43 studies with total 20,629 subjects showed similar association of pon1 activity and cad with standardized mean difference ( smd ) of -0.78 ( p < 0.001 ) for cad subjects compared with controls .", "slightly weaker association was between arylesterase activity of pon1 and cad with smd of -0.50 ( p < 0.001 ) .", "both meta - analyses observed increased risk of cad in subjects with lower pon1 regardless of age and ethnicity ; tables 3 and 4 summarizes the role of pon1 in human studies .", "summary of allele differences of pon1 in vitro , and conclusions from pon1 human studies recent human studies on the role of pon1 in cvd some alleles of polymorphous genes can be causative factors in development of diseases and reliable predictors .", "there are at least five known polymorphisms in promoter region of pon1 : -909/-907 ( c or g ) , -832/-824 ( a or g ) , -162/-160 ( a or g ) , -126 ( c or g ) , and -108/-107 ( c or t ) . also , there are two polymorphisms in the pon1 coding region , q192r ( aka rs662 or 575a > g ) and l55 m ( aka rs854560 or 163 t > a ) , and several polymorphisms in 3 untranslated region of the gene .", "pon1 protein level in human serum varies more than 13-fold , and variations in the sequence of promoter region of pon1 gene might cause difference in its expression .", "two - fold differences in expression level between alleles were observed in cell culture reporter gene assay for three polymorphisms : -909/-907 ( c or g ) , -162/-160 ( a or g ) , and -108/-107 ( c or t ) .", "polymorphisms -162/-160 ( a or g ) is a putative binding site for transcription factor nf - i , and -108/-107 ( c or t ) is a putative binding site for sp1 .", "no effect on expression level in the reporter gene assay was observed for -832/-824 ( a or g ) , -162/-160 ( a or g ) polymorphisms .", "gene expression and enzyme activity studies on pon1 polymorphisms in protein coding region q192r and l55 m resulted in several observations .", "first , messenger rna of allele 55 m appeared to be more stable than 55l as determined by pcr amplification and restriction analysis of cdna synthesized from heterozygous liver samples .", "l55 m heterozygous liver had roughly twice more mrna of 55 m allele compared with 55l . that might results in difference in protein expression levels between alleles and ultimately in pon1 activity and protection , although it was not assessed in the study .", "q192r and l55 m alleles differ in their enzymatic activities . in a study of serum of 279 healthy human subjects", "activity of 55 m homozygotes were always lower than 55l / m heterozygotes or 55l homozygotes .", "qq , qr , and rr phenotypes can be reliably determined by assaying pon1 catalytic activities toward two substrates : diazoxon and paraoxon .", "pon1 of qq genotype exhibits relatively high activity toward diazoxon and relatively low activity toward paraoxon .", "rr genotype , opposite to qq , exhibits low activity toward diazoxon and high activity toward paraoxon .", "192r pon1 hydrolyzes homocysteine thiolactone faster than 192q pon1 . q192r and l55 m pon1 enzymes differ in their protection to ldl from oxidation in vitro using ldl oxidation assay with copper in the presence of hdl .", "protection by pon1 decreases in the order of genotypes qq > qr > rr with almost no antioxidant activity in rr genotype , and about a half of qq activity in qr phenotype .", "the antioxidant activity of pon1 decreased in the series mm > lm > ll , with the activity of ll genotype about a half of activity of mm genotype .", "multiple clinical studies were performed to expose whether pon1 polymorphism may contribute to cad and other diseases .", "currently , analyses of association of pon1 polymorphism with atherosclerosis - related diseases determined just one reliable association , an association of q192r polymorphism with ischemic stroke .", "meta - analysis of 22 studies have been published earlier up to mid of 2009 , totaling 7384 ischemic stroke subjects and 11,074 controls revealed odds ratio of 1.10 for g ( 192r ) allele ( 95% ci : 1.041.17 ) .", "another meta - analysis study that was published in 2010 and summarized results of 11 studies confirmed that 192r allele confers significant risk of ischemic stroke ( odds ratio = 1.25 , 95% ci : 1.07,1.46 , p = 0.006 ) .", "surprisingly , this risk is confined to caucasian subjects , but there is no significant association of 192r allele of pon1 and ischemic stroke in east asian population .", "association of pon1 polymorphism q192r with cad was examined in extensive meta - analysis of studies published before 2011 . per - allele", "odds ratio for cad for 192r was 1.11 ( 95% ci : 1.05 , 1.17 ) based on all studies regardless of their size .", "however , analysis suggested that small studies were less reliable , perhaps because of small studies bias .", "no significant association of 192r allele with cad was observed when 10 larger studies with more than 500 cases each were analyzed .", "similar conclusions regarding small study bias and the absence of reliable association of q192r polymorphism with cad were concluded in two other independent massive meta - analysis studies published in 2004 ; also , no association was found between cad and l55 m or -108/-107 ( c or t ) polymorphisms of pon1 gene .", "meta - analysis for l55 m and -108/-107 ( c or t ) pon1 polymorphisms determined per - allele odds ratios for cad as 0.94 ( 95% ci : 0.88,1.00 ) for 55 m and 1.02 ( 95% ci : 0.911.15 ) for -108/-107 c , respectively .", "no significant association of l55 m or -108/-107 ( c or t ) pon1 polymorphisms with cad were observed in an earlier meta - analysis study .", "no association between l55 m ( rs854560 ) polymorphism and ischemic stroke was found in meta - analysis of 16 studies published before mid-2009 totaling 5518 ischemic stroke subjects and 8951 controls .", "similarly , a meta - analysis published in 2010 for 10 studies did not find significant association 55l allele with stroke regardless on the stroke type , age of patients , and ethnicity . resent human studies generally support the conclusions of meta - analyses [ table 4 ] .", "a novel pon1 polymorphism associated with atherosclerosis was recently revealed , snp rs854563(a / g ) . in conclusion ,", "pon1 antioxidant and anti - inflammatory effect is extensively examined in vitro , in cell culture and animal models .", "human studies confirm protective role of pon1 in cad and another atherosclerosis - related disease , ischemic stroke .", "although the current knowledge of pon1 provides valuable insights on the function and role of pon1 , yet mechanism of pon1 action is still not well investigated .", "transient overexpression of pon1 in mouse demonstrated beneficial effects of pon1 beyond its antiatherogenic properties .", "further research of pon1 could potentially lead to new clinical strategies in prevention and treatment of cardiovascular diseases ."], "abstract_text": ["<S> paraoxonase 1 ( pon1 ) is a hydrolytic enzyme with wide range of substrates , and capability to protect against lipid oxidation . despite of the large number of compounds that can be hydrolyzed by paraoxonase , </S>", "<S> the biologically relevant substrates are still not clearly determined . </S>", "<S> there is a massive in vitro and in vivo data to demonstrate the beneficial effects of pon1 in several atherosclerosis - related processes . </S>", "<S> the enzyme is primarily expressed in liver ; however , it is also localized in other tissues . </S>", "<S> pon1 attracted significant interest as a protein that is responsible for the most of antioxidant properties of high - density lipoprotein ( hdl ) . </S>", "<S> several bioactive molecules such as dietary polyphenols , aspirin and its hydrolysis product salicylate , are known to stimulate pon1 transcription activation in mouse liver and hepg2 cell line . </S>", "<S> studies on the activity , function , and genetic makeup have revealed a protective role of pon1 . </S>", "<S> some striking data were obtained in pon1 gene knockout and pon1 transgenic mouse models and in human studies . </S>", "<S> the goal of this review is to assess the current understanding of pon1 expression , enzymatic and antioxidant activity , and its atheroprotective effects . </S>", "<S> results from in vivo and in vitro basic studies ; and from human studies on the association of pon1 with coronary artery disease ( cad ) and ischemic stroke will be discussed . </S>"], "labels": null, "section_names": ["PON1 Enzymatic Activity and Antioxidant Effects", "PON1 Expression and Tissue Distribution", "Atheroprotective Effects of PON1 in Animal Models", "PON1 Serum Activity, Polymorphism and CAD in Human"], "sections": [["paraoxonases were originally discovered as enzymes hydrolyzing exogenous toxic organophosphate compounds such as insecticide paraoxon .", "there are three members of paraoxonases family currently known : paraoxonase 1 ( pon1 ) , paraoxonase 2 ( pon2 ) , and paraoxonase 3 ( pon3 ) , which are encoded by three separate genes on the same chromosome 7 ( human ) or chromosome 6 ( mouse ) .", "all three human members of the family are 70% identical at nucleotide level and 60% identical at the amino acid level .", "studies on enzymatic activity of paraoxonases revealed esterase and lactonase / lactonizing activities in addition to organophosphatase activity . despite of the large number of compounds that can be hydrolyzed by paraoxonase ,", "in many cases arylesterase activity is measured to access pon1 level in biological samples . based on kinetic parameters of paraoxonases toward different substrates", "hydrolysis of homocysteine thiolactone by pon1 is considered to be protective against coronary artery disease ( cad ) .", "after the introduction of the oxidative stress hypothesis of atherosclerosis and the discovery of antioxidant effect of high - density lipoprotein ( hdl ) , pon1 attracted significant interest as a protein that is responsible for the most of antioxidant properties of hdl .", "purified pon1 protects hdl and low - density lipoprotein ( ldl ) from oxidation catalyzed by copper ions .", "pon1 inhibits copper - induced hdl oxidation by prolonging oxidation lag phase , and reduces peroxide and aldehyde content in oxidized hdl .", "remarkably , pon1 inhibitors pd-11612 , pd-65950 , pd-92770 , and pd-113487 lessen this antioxidative effect of pon1 .", "pon1 is especially effective in decomposition of linoleate hydroperoxides . also , mass - spectrometry analysis of biologically active fraction of oxidized2-arachidonoyl - sn - glycero-3-phosphorylcholine ( ox - papc ) underwent pon1 treatment showed degradation of these oxidized phospholipids by pon1 .", "the exact antioxidant mechanism of pon1 is not known yet , although it is known that protection is not caused by chelating of copper ions or because of potential lipid transfer from ldl to hdl .", "existence of an enzymatic mechanism is supported by the observation that heat inactivation of purified pon1 abolishes its antioxidant effect .", "some in vitro data suggest that antioxidant activity might be related to other components of purified pon1 preparations .", "however , experiments with pon1 deficient mouse provide strong evidences that pon1 is required to enable hdl antioxidant properties .", "hdl from pon1 knockout animals were more prone to oxidation and were less efficient in the protection of ldl from oxidation in co - cultured cell model of the artery wall compared with hdl from control mice .", "also , transfection of pon1-deficient peritoneal macrophages ( isolated from pon1 knockout mice ) with human pon1 decreased level of peroxides , lowered release of superoxide , and increased intracellular level of reduced glutathione , key observations are summarized in table 1 .", "summary of key facts on pon1 expression , activity , and effects in in vitro and in vivo studies"], ["the first pon1 messenger ribonucleic acid ( mrna)ionanalysis in different rabbit tissues was performed by northern blot , and revealed pon1 mrna expression predominately in liver .", "polymerase chain reaction ( pcr ) amplification using a panel of first - strand complementary deoxyribonucleic acid ( cdnas ) from 24 tissues detected pon1 expression in kidney and colon beside liver and fetal liver expression .", "biopsies showed pon1 mrna and protein expression in human but not in mouse gastrointestinal tract .", "deletion analysis in cultured cells revealed that cell type specific expression in liver and kidney is determined within first 200 bp of promoter area .", "there are several transcription factors and pathways that regulate pon1 expression [ figure 1 ] .", "all processes occur in liver , and bile acid -stimulated synthesis of fibroblast growth factor 19 ( fgf-19 ; or fgf-15 in mouse ) might be additionally occur in ileum .", "signals from pma ( phorbol 12-myristate 13-acetate ) and high glucose activate transcription factor sp1 through protein kinases c ( pkc ) and p44/ p42 mitogen - activated protein kinases ( mapk ) a ubiquitous mammalian transcription factor specificity protein 1(sp1 ) plays an essential role in regulation of pon1 expression .", "high glucose level activates protein kinase c ( pkc ) , which activates sp1 , and stimulate pon1 transcription in human hepatoma cell lines hepg2 and huh7 . a potent pkc activator phorbol 12-myristate 13-acetate ( pma ) also stimulates pon1 transcription in hepg2 through activation of sp1 .", "two members of pkc family are involved , pkc ( zeta ) and pkc- ( alpha ) activation .", "statins ( pitavastatin , simvastatin , or atorvastatin ) stimulate pon1 transcription through sp1 activation as well , however , they activate another kinase , p44/p42 mitogen - activated protein ( map ) kinase , as was observed for pitavastatin in huh7 cells . also , pivostatin - stimulated p44/p42 mitogen - activated protein kinase ( mapk ) activates sterol regulatory element binding protein 2 ( srebp-2 ) , which contributes to transcriptional activation of pon1 .", "dietary polyphenols , such as resveratrol , aspirin and its hydrolysis product salicylate , and artificial ligands of aryl hydrocarbon receptor ( ahr ) , such as 3-methylcholanthrene , activate ahr and stimulate pon1 transcription activation in mouse liver and hepg2 cell line.[2426 ] c - jun is another transcription factors that is involved in pon1 expression .", "the activity of c - jun is regulated by c - jun n - terminal kinase ( jnk ) .", "phosphorylated c - jun in a complex with c - fos or some other transcription factors forms an active ap-1 complex that usually promotes transcription of target genes .", "thus , berberine ( benzyl tetrahydroxyquinoline ) , a cholesterol lowering alkaloid , activates jnk , c - jun and stimulates pon1 transcription in human hepatoma cell lines .", "however , stimulation of jnk / c - jun pathway by bile acids leads to opposite effect .", "bile acids such as taurocholate , cholic acid , or chenodeoxycholic acid inhibit pon1 expression in liver of c57bl/6j mice , and in hepg2 and huh7 .", "the inhibition of pon1 expression starts with activation of farnesoid x receptor ( fxr ) by bile acids that promote expression of fibroblast growth factor 19 in human ( fgf-19 ) or fgf-15 in mouse .", "the growth factor activates fibroblast growth factor receptor 4 ( fgfr4 ) followed by activation of jnk , and phosphorylation of c - jun .", "opposite to activation of c - jun by berberine , fxr / fgf-19/fgfr4/jnk / c - jun pathway results in suppression of pon1 transcription .", "it is likely that pon1 stays associated with endoplasmic reticulum through its hydrophobic n - terminus until it is released from the hepatocytes .", "the mechanism of pon1 secretion is not well investigated . a study on cultured hepatocytes and transfected chinese hamster ovary ( cho ) cells , which do not naturally express pon1 and apoa proteins ,", "when pon1 protein is synthesized , it accumulates on plasma membrane and slowly dissociate into extracellular medium .", "dissociation is promoted by hdl , very - low - density lipoprotein ( vldl ) , and in much lesser extent by protein - free phospholipids particles or apoa - i protein .", "pon1 binds to hdl through interaction of hydrophobic n - terminus to phospholipids , and through pon1-apoa interaction .", "two major principal hdl proteins , that is , apoa - i and/or apoa - ii which differ in their properties are linked to atherosclerosis modulation . thus , transgenic mice with hdl consisting of both human apoa - i and apoa - ii developed 15-fold greater lesions compared with mice with hdl containing solely human apoa - i . in vitro study of reconstituted hdl demonstrated that apoa - i containing hdl stabilizes pon1 binding as compared with protein - free hdl particles , and apoa - ii in opposite destabilizes the hdl ", "the paraoxonase and arylesterase activities of pon1 do not depend on the apolipoprotein content of hdl .", "however , apoa - i containing hdl significantly increases lactonase activity of pon1 , promotes inhibition of ldl oxidation , and stimulates macrophage cholesterol efflux compared with apoa - ii containing hdl .", "a study of human hdl isolated from apoa - i deficient patients revealed that pon1 is still associated with hdl in the absence of apoa-1 , however , the pon1hdl complex is less stable , and pon1 loses activity faster than in normal controls .", "a higher content of human apoa - ii protein in mouse hdl suppresses pon1 binding to hdl compared with hdl with lower human apoa - ii content .", "hdl high in human apoa - ii binds less pon1 protein , possesses less pon1 activity , and is impaired in protection of ldl from oxidation .", "transgenic mouse with higher human apoa - ii content in hdl are more susceptible to atherosclerosis compared with transgenic animals with lower apoa - ii level in hdl and control animal .", "hdl - associated pon1 can be successfully transferred to cultured cells in vitro and deliver protection from oxidative stress and bacterial substrate of pon1 n-3-oxo - dodecanoyl - l - homoserine lactone . beside hdl", "while liver is the major tissue of pon1 gene expression , lipoprotein - assisted circulation of pon1 in plasma delivers the enzyme to multiple tissues that do not express pon1 themselves .", "immunohistochemical staining of rat tissues revealed pon1 presence in liver , kidney , in endothelial lining of lung and brain .", "more recent study with mouse tissue detected pon1 protein in hepatocytes , adipocytes , chondrocytes , skeletal and cardiac muscle , kidney , spermatozoa , and epithelial cells of skin , stomach , intestine , trachea , bronchiole , and eye lens .", "studies did not find co - localization of pon1 with apoa - i protein that might mean local pon1 expression or different sources of apoa - i , an integral component of hdl .", "immunostaining of healthy human aorta shows a low level and granular distribution of pon1 in smooth muscle cells .", "western blot confirmed presence of intact and degraded pon1 in media of healthy aorta . with the development of atherosclerosis , pon1", "increasing accumulation of pon1 with the progression of the atherosclerosis can be seen in the intimal part of aorta as well .", "aorta with advanced atherosclerosis lesions shows massive pon1 accumulation . a more recent study attributed an increased pon1 immunostaining in human atherosclerotic arteries to macrophages accumulated in lesions ."], ["high  fat , high - cholesterol ( atherogenic ) diet leads to fast development of atherosclerosis in c57bl/6j mouse strain with concomitant decrease in liver pon1 expression . at the same time pon1 expression and its protective effects", "macrophages from this mouse did not protect ldl from oxidation by other cells in vitro , and pon1 knockout mice developed atherosclerosis faster than control mice on high fat , high cholesterol diet . to study the effect of pon1 knockout further ,", ". plasma lipid profile of these double knockout mice was similar to apoe-/- control animals with slightly decreased level of intermediate density lipoprotein ( idl ) and ldl , and increased level of lysophosphatidylcholine and oxidized phospholipids in idl and ldl .", "the rates of ldl oxidation and clearance were higher in pon1-/- , apoe-/- mice than in control mice as was determined by injection of human ldl .", "expression of genes responding to increase of oxldl , such as heme oxygenase-1 , peroxisome proliferator - activated receptor gamma , scavenger receptor type a , cd36 , and macrosialin , was upregulated in liver of pon1-/- , apoe-/- mice compared with apoe-/- mice .", "involvement of pon in inflammation and oxidative stress were detected in vascular wall of pon-/- animals .", "expression of mrna of essential cell adhesion molecules p - selectin and icam1 was upregulated in aortic wall .", "if a deficiency of pon1 leads to inflammation and oxidative stress , then increases in pon1 function could be beneficial .", "a transgenic mouse mpon1 was developed in 2001 , with 5-fold higher level of mouse pon1 protein and corresponding increase in arylesterase activity .", "pon1 was associated with hdl , and protected hdl from oxidation by copper ions as it was assessed by lipid hydroperoxide formation assay , and by retaining oxidation - sensitive activity of lecithin : cholesterol acyltransferase ( lcat ) . a decrease in atherosclerotic lesion size and improved oxidation status of aorta and", "peritoneal macrophages in pon1 overexpressing transgenic mouse on apoe-/- background have been compared with apoe-/- control have been reported . in another transgenic mouse model , pon1-tg , which overexpresses human pon1 gene of 55l/192q genotype , plasma level of arylesterase activity was twice higher in pon1-tg mouse compared with control mouse when animals were fed normal chow .", "the excess of pon1 level resulted in better retaining of its activity after feeding animals with atherogenic diet .", "pon1 activity decreased to about three - fourth ( 3/4 ) of original level after 15 weeks of atherogenic diet feeding to pon1-tg mice , and to less than one - half ( 1/2 ) of original level in control animals .", "thus , arylesterase activity mediated by pon1 was almost 4-fold higher in pon1-tg mice compared with control mice after feeding atherogenic diet .", "aortic lesion areas in pon1-tg animals were about half of the size of areas of the control mice after feeding atherogenic diet .", "apoe-/- , pon1-tg genotype mice fed normal chow similarly developed lesions slower , compared with apoe-/- controls ; however , the difference in lesion area was just 22% .", "plasma arylesterase activity of apoe-/- , pon1-tg mice was 2.5-fold higher than that of apoe-/- animals , however , there is no difference between their lipid profile . a decreased inflammatory status of aortas in apoe-/- , pon1-tg mice compared with apoe-/- mice was detected by examination of the expression level of mcp-1 cytokine gene .", "hdl isolated from pon1-tg mouse plasma or apoe-/- , pon1-tg mouse plasma provided better protection against ldl oxidation , than hdl isolated from control animals . beside pon1 studies on transgenic mouse , a transient overexpression of pon1 gene in mouse", "adenovirus - mediated delivery of human pon1 gene ( 55 m , 192q genotype ) in double knockout leptin-/- ( ob / ob ) , ldlr-/- mouse resulted in more than 4-fold increase of pon1 activity on day 7 with gradual decrease to the control level by day 21 .", "mice were injected with adenovirus at the age of 18 weeks when the progression of atherosclerosis occurs in a fast rate .", "six weeks later , cross - sections of the aortic root were analyzed by immunohistochemistry .", "plaque size , volume of macrophages , concentration of oxldl was significantly lower in pon1 expressing animals .", "the titer of anti - oxldl antibodies was lower in plasma of pon1 overexpressing mice as well .", "while an early pon1 intervention proved to have significant athero - protective effect , another study was performed to assess whether transient pon1 overexpression can improve vascular function in advanced atherosclerosis .", "similar to the previous study , 55m/192q variant of human pon1 gene was delivered in 18 month old apoe-/- mouse fed normal chow using adenovirus vector .", "serum pon1 activity was increased 10-fold by day 7 and 2.5-fold on the day 21 in pon1 overexpressing animals compared with controls . after 3 weeks , lesions were measured and no changes in their size were found . however , vasomotor function was improved by transient overexpression of pon1 .", "thus , phenylephrine - constricted segments of the aortas relaxed significantly better in response to endothelium dependent agonists ach , adenosine-5-triphosphate ( atp ) , and uridine-5-triphosphate ( utp ) observations on pon1 role in animal models is summarized in table 2 ."], ["meta - analysis of 47 studies with 9853 cad and 11,408 control subjects published in 2012 confirmed association of lower plasma pon1 activity with increased cad risk ( 19% lower pon1 activity , p < 0.00001 ) .", "another meta - analysis published in 2012 included 43 studies with total 20,629 subjects showed similar association of pon1 activity and cad with standardized mean difference ( smd ) of -0.78 ( p < 0.001 ) for cad subjects compared with controls .", "slightly weaker association was between arylesterase activity of pon1 and cad with smd of -0.50 ( p < 0.001 ) .", "both meta - analyses observed increased risk of cad in subjects with lower pon1 regardless of age and ethnicity ; tables 3 and 4 summarizes the role of pon1 in human studies .", "summary of allele differences of pon1 in vitro , and conclusions from pon1 human studies recent human studies on the role of pon1 in cvd some alleles of polymorphous genes can be causative factors in development of diseases and reliable predictors .", "there are at least five known polymorphisms in promoter region of pon1 : -909/-907 ( c or g ) , -832/-824 ( a or g ) , -162/-160 ( a or g ) , -126 ( c or g ) , and -108/-107 ( c or t ) . also , there are two polymorphisms in the pon1 coding region , q192r ( aka rs662 or 575a > g ) and l55 m ( aka rs854560 or 163 t > a ) , and several polymorphisms in 3 untranslated region of the gene .", "pon1 protein level in human serum varies more than 13-fold , and variations in the sequence of promoter region of pon1 gene might cause difference in its expression .", "two - fold differences in expression level between alleles were observed in cell culture reporter gene assay for three polymorphisms : -909/-907 ( c or g ) , -162/-160 ( a or g ) , and -108/-107 ( c or t ) .", "polymorphisms -162/-160 ( a or g ) is a putative binding site for transcription factor nf - i , and -108/-107 ( c or t ) is a putative binding site for sp1 .", "no effect on expression level in the reporter gene assay was observed for -832/-824 ( a or g ) , -162/-160 ( a or g ) polymorphisms .", "gene expression and enzyme activity studies on pon1 polymorphisms in protein coding region q192r and l55 m resulted in several observations .", "first , messenger rna of allele 55 m appeared to be more stable than 55l as determined by pcr amplification and restriction analysis of cdna synthesized from heterozygous liver samples .", "l55 m heterozygous liver had roughly twice more mrna of 55 m allele compared with 55l . that might results in difference in protein expression levels between alleles and ultimately in pon1 activity and protection , although it was not assessed in the study .", "q192r and l55 m alleles differ in their enzymatic activities . in a study of serum of 279 healthy human subjects", "activity of 55 m homozygotes were always lower than 55l / m heterozygotes or 55l homozygotes .", "qq , qr , and rr phenotypes can be reliably determined by assaying pon1 catalytic activities toward two substrates : diazoxon and paraoxon .", "pon1 of qq genotype exhibits relatively high activity toward diazoxon and relatively low activity toward paraoxon .", "rr genotype , opposite to qq , exhibits low activity toward diazoxon and high activity toward paraoxon .", "192r pon1 hydrolyzes homocysteine thiolactone faster than 192q pon1 . q192r and l55 m pon1 enzymes differ in their protection to ldl from oxidation in vitro using ldl oxidation assay with copper in the presence of hdl .", "protection by pon1 decreases in the order of genotypes qq > qr > rr with almost no antioxidant activity in rr genotype , and about a half of qq activity in qr phenotype .", "the antioxidant activity of pon1 decreased in the series mm > lm > ll , with the activity of ll genotype about a half of activity of mm genotype .", "multiple clinical studies were performed to expose whether pon1 polymorphism may contribute to cad and other diseases .", "currently , analyses of association of pon1 polymorphism with atherosclerosis - related diseases determined just one reliable association , an association of q192r polymorphism with ischemic stroke .", "meta - analysis of 22 studies have been published earlier up to mid of 2009 , totaling 7384 ischemic stroke subjects and 11,074 controls revealed odds ratio of 1.10 for g ( 192r ) allele ( 95% ci : 1.041.17 ) .", "another meta - analysis study that was published in 2010 and summarized results of 11 studies confirmed that 192r allele confers significant risk of ischemic stroke ( odds ratio = 1.25 , 95% ci : 1.07,1.46 , p = 0.006 ) .", "surprisingly , this risk is confined to caucasian subjects , but there is no significant association of 192r allele of pon1 and ischemic stroke in east asian population .", "association of pon1 polymorphism q192r with cad was examined in extensive meta - analysis of studies published before 2011 . per - allele", "odds ratio for cad for 192r was 1.11 ( 95% ci : 1.05 , 1.17 ) based on all studies regardless of their size .", "however , analysis suggested that small studies were less reliable , perhaps because of small studies bias .", "no significant association of 192r allele with cad was observed when 10 larger studies with more than 500 cases each were analyzed .", "similar conclusions regarding small study bias and the absence of reliable association of q192r polymorphism with cad were concluded in two other independent massive meta - analysis studies published in 2004 ; also , no association was found between cad and l55 m or -108/-107 ( c or t ) polymorphisms of pon1 gene .", "meta - analysis for l55 m and -108/-107 ( c or t ) pon1 polymorphisms determined per - allele odds ratios for cad as 0.94 ( 95% ci : 0.88,1.00 ) for 55 m and 1.02 ( 95% ci : 0.911.15 ) for -108/-107 c , respectively .", "no significant association of l55 m or -108/-107 ( c or t ) pon1 polymorphisms with cad were observed in an earlier meta - analysis study .", "no association between l55 m ( rs854560 ) polymorphism and ischemic stroke was found in meta - analysis of 16 studies published before mid-2009 totaling 5518 ischemic stroke subjects and 8951 controls .", "similarly , a meta - analysis published in 2010 for 10 studies did not find significant association 55l allele with stroke regardless on the stroke type , age of patients , and ethnicity . resent human studies generally support the conclusions of meta - analyses [ table 4 ] .", "a novel pon1 polymorphism associated with atherosclerosis was recently revealed , snp rs854563(a / g ) . in conclusion ,", "pon1 antioxidant and anti - inflammatory effect is extensively examined in vitro , in cell culture and animal models .", "human studies confirm protective role of pon1 in cad and another atherosclerosis - related disease , ischemic stroke .", "although the current knowledge of pon1 provides valuable insights on the function and role of pon1 , yet mechanism of pon1 action is still not well investigated .", "transient overexpression of pon1 in mouse demonstrated beneficial effects of pon1 beyond its antiatherogenic properties .", "further research of pon1 could potentially lead to new clinical strategies in prevention and treatment of cardiovascular diseases ."]]}
{"article_id": "PMC4295945", "article_text": ["uterine adenomyosis is a common gynecologic disorder characterized by the presence and growth of heterotopic endometrial or endometrium - like structures in the myometrium , with adjacent smooth muscle hyperplasia and can lead to dysmenorrhea and infertility .", "the ectopic endometrial tissue induces hypertrophy and hyperplasia of the surrounding myometrium , resulting in diffuse globular enlargement of the uterus analogous to the concentric enlargement of the pregnant uterus .", "the presenting symptoms include a soft and diffusely enlarged uterus with menorrhagia ( 40%50% ) , dysmenorrhea ( 10%30% ) , metrorrhagia ( 10%12% ) , dyspareunia and dyschezia .", "the advised treatment for severe adenomyosis is total hysterectomy , but for patients wishing to preserve their uterus , a minimally invasive alternative procedure , uterine artery embolization ( uae ) , can be performed .", "uae is a nonsurgical alternative for patients with menorrhagia , symptomatic adenomyosis , or symptomatic uterine fibroids .", "although uterine infarction is a relatively common occurrence after uae for the treatment of fibroids or adenomyosis , uterine infarction after ivf - et in a patient with adenomyosis is very rare . to our knowledge", ", this is the first report of uterine infarction after ivf - et in a patient with uterine adenomyosis in korea .", "she had suffered severe dysmenorrhea , menorrhagia , and pelvic pain due to severe adenomyosis which was diagnosed following a pelvic magnetic resonance imaging ( mri ) and clinical examination three years earlier .", "the patient had undergone three unsuccessful cycles of frozen - thawed embryo transfer at an external infertility clinic , due to primary infertility , during the period from 4 - 7 months before hospital admission .", "the patient was admitted to hospital for treatment of ovarian hyperstimulation syndrome ( ohss ) arising after controlled ovarian hyperstimulation about four months before the initiation of embryo transfer .", "a thyroid function test on january 2013 indicated a euthyroid state : triiodothyronine 166 ng / dl ( range , 80 - 200 ng / dl ) , thyroid stimulating hormone 0.17 iu / ml ( range , 0.4 - 4.1 iu / ml ) , free thyroxine 1.21 ng / dl ( range , 0.80 - 1.90 mg / dl ) .", "the patient underwent another cycle of ivf at a local clinic in february 2013 , during which she received follitropin- 150 iu from menstrual cycle day 3 to day 12 and chorionic gonadotropin 250 g at menstrual cycle day 13 .", "ovum pick - up was performed about four weeks before she present to the emergency department and embryo transfer three days later .", "the patient received intramural progesterone 50 mg and was prescribed a vaginal progesterone gel ( crinone ) for daily use for 11 days after the embryo transfer .", "the patient presented to hospital with vaginal bleeding , fever and abdominal pain on eleven days after embryo transfer , and received antibiotic treatment followed by supportive care at a local clinic for three days .", "she visited our outpatient clinic due to vaginal bleeding and lower abdominal pain on sixteen days after embryo transfer and was admitted to hospital .", "c - reactive protein ( crp ) was elevated to 7.52 mg / dl and intravenous antibiotic treatment was given .", "biochemical abortion was diagnosed on nineteen days following embryo transfer after serum -hcg decreased from 161 to 63 mlu / ml in two days .", "the patient was discharged after symptom improvement , but presented with vaginal bleeding and lower abdominal pain , at a local clinic two days later , where she received hydration .", "the last day of that month , she presented to the emergency department with high fever ( 39 ) and , lower abdominal pain .", "peripheral blood cell analysis indicated a : white blood cell count of 17.510/l , an elevated crp of 24.3 mg / dl , a hemoglobin level of 7.6 g / dl and a hematocrit level of 24.5% .", "computed tomography at the emergency department revealed a wedge - shaped low attenuation area in the anterior uterine corpus .", "focal uterine infarction was suspected , and the pelvic mri revealed a wedge - shaped , irregularly marginated , non - enhancing area in the anterior uterine wall and the presence of preserved myometrium between the endometrial cavity and the inner margin of the necrotic myometrium ( figure 1 ) .", "blood markers investigated to establish the cause of infarction , including antiphospholipid immunoglobulin g ( igg ) and igm , protein c activation , protein s , and plasminogen , were all within normal ranges .", "five days after hospital admission , pelvic pain improved and crp decreased to 6.08 mg / dl , and the patient was discharged .", "two months later , pelvic mri showed interval regression of the previously noted possible focal uterine infarction site with a remarkably improved blood flow ( figure 2 ) .", "adenomyosis uteri is a pathological entity characterized by the presence of endometrial glands and stroma embedded within the myometrium , but without apparent contact with the endo - myometrial junction .", "uterine adenomyosis is relatively frequent , affecting 1% of females ; its diagnosis is more often made in multiparous patients , in their fourth and fifth decade of life , and for this reason , infertility is not frequently concurrent with adenomyosis .", "however , given the trend for first pregnancies to be delayed until women are in their thirties or forties , adenomyosis uteri becoming a more frequently considered diagnosis", ". pregnancy resulting from assisted reproductive technology ( art ) is possible in patients with ademoyosis ; however , spontaneous abortion , including biochemical abortion , still occurs in these patients .", "uterine infarction after ivf - et in a patient with adenomyosis has not previously been reported .", "the objective of uae is to initiate tumor infarction resulting in substantial reduction of the uterine and tumor volumes .", "first , when undergoing ivf - et , the risk of thrombosis is increased , as in the cases of pregnancy and hyperthyroidism , with which the patient also presented . the precise mechanisms by which the ohss and exogenous hormonal stimulation used in art induce thromboembolic events are still uncertain . however , vascular endothelial growth factor secreted during ohss , high estradiol concentrations , and blood hyperviscosity play a prominent part in inducing a prothrombotic state .", "reported that the overall venous thrombosis incidence rate during ivf pregnancies was three times higher than in reference pregnancies .", "an elevated crp can activate the coagulation system , following which microthrombosis formation , in the uterine myometrium , could cause necrosis of the myometrium and lead to focal uterine infarction .", "second , the disruption of the endometrial - myometrial interface also disrupts the organization of myometrial muscle fibers and may also disrupt normal contractility of the subendometrium .", "uterine adenomyosis with abnormal contractility and the subsequent abortion caused massive vaginal bleeding which may have led to uterine ischemia , and the resulting uterine infarction .", "clinical hyperthyroidism is generally accompanied by a hypercoagulable state , which may lead to microthrombosis of the uterine myometrium .", "last , fever and the elevated crp suggested that infection could have complicated the ischemic necrosis after uterine infarction .", "however , there were no clinical features such as rapid tumor growth , extrauterine extension or metastases , leading to the conclusion that the possibility of uterine sarcoma was very small .", "a follow - up mri three months later showed only traces of uterine infarction lesion and adenomyosis . in conclusion , we experienced a rare case of uterine adenomyosis that developed into uterine infarction after ivf - et and biochemical pregnancy .", "this case demonstrates that uterine infarction should be considered in the differential diagnosis of acute abdominal pain , vaginal bleeding and infectious signs in women with biochemical abortion after ivf - et , with a history of adenomyosis and hyperthyroidism .", "the insertion of a hormonal intrauterine device could be considered another option for treating the symptoms of adenomyosis in cases presenting with focal uterine infarction dysmenorrhea and menorrhagia .", "in addition , the surgical management of focal uterine infarction , may be considered in the management of uterine infarction after ivf - et in patients with uterine adenomyosis if future fertility is not desired .", "finally , it is important to note that the previous infarction site might be a potential risk site for uterine rupture in a future pregnancy ."], "abstract_text": ["<S> adenomyosis is a common gynecological disorder characterized by the presence of endometrial glands and stroma deep within the myometrium associated with myometrial hypertrophy and hyperplasia . </S>", "<S> focal uterine infarction after ivf - et in a patient with adenomyosis following biochemical pregnancy has not been previously reported , although it occurs after uterine artery embolization in order to control symptoms caused by fibroids or adenomyosis . </S>", "<S> we report a case of a nulliparous woman who had uterine adenomyosis presenting with fever , pelvic pain and biochemical abortion after undergoing an ivf - et procedure and the detection of a slightly elevated serum hcg . </S>", "<S> focal uterine infarction was suspected after a pelvic magnetic resonance imaging demonstrated preserved myometrium between the endometrial cavity and inner margin of the necrotic myometrium . </S>", "<S> this case demonstrates that focal uterine infarction should be considered in the differential diagnosis of acute abdominal pain , vaginal bleeding and infectious signs in women experiencing biochemical abortion after an ivf - et procedure . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["uterine adenomyosis is a common gynecologic disorder characterized by the presence and growth of heterotopic endometrial or endometrium - like structures in the myometrium , with adjacent smooth muscle hyperplasia and can lead to dysmenorrhea and infertility .", "the ectopic endometrial tissue induces hypertrophy and hyperplasia of the surrounding myometrium , resulting in diffuse globular enlargement of the uterus analogous to the concentric enlargement of the pregnant uterus .", "the presenting symptoms include a soft and diffusely enlarged uterus with menorrhagia ( 40%50% ) , dysmenorrhea ( 10%30% ) , metrorrhagia ( 10%12% ) , dyspareunia and dyschezia .", "the advised treatment for severe adenomyosis is total hysterectomy , but for patients wishing to preserve their uterus , a minimally invasive alternative procedure , uterine artery embolization ( uae ) , can be performed .", "uae is a nonsurgical alternative for patients with menorrhagia , symptomatic adenomyosis , or symptomatic uterine fibroids .", "although uterine infarction is a relatively common occurrence after uae for the treatment of fibroids or adenomyosis , uterine infarction after ivf - et in a patient with adenomyosis is very rare . to our knowledge", ", this is the first report of uterine infarction after ivf - et in a patient with uterine adenomyosis in korea ."], ["she had suffered severe dysmenorrhea , menorrhagia , and pelvic pain due to severe adenomyosis which was diagnosed following a pelvic magnetic resonance imaging ( mri ) and clinical examination three years earlier .", "the patient had undergone three unsuccessful cycles of frozen - thawed embryo transfer at an external infertility clinic , due to primary infertility , during the period from 4 - 7 months before hospital admission .", "the patient was admitted to hospital for treatment of ovarian hyperstimulation syndrome ( ohss ) arising after controlled ovarian hyperstimulation about four months before the initiation of embryo transfer .", "a thyroid function test on january 2013 indicated a euthyroid state : triiodothyronine 166 ng / dl ( range , 80 - 200 ng / dl ) , thyroid stimulating hormone 0.17 iu / ml ( range , 0.4 - 4.1 iu / ml ) , free thyroxine 1.21 ng / dl ( range , 0.80 - 1.90 mg / dl ) .", "the patient underwent another cycle of ivf at a local clinic in february 2013 , during which she received follitropin- 150 iu from menstrual cycle day 3 to day 12 and chorionic gonadotropin 250 g at menstrual cycle day 13 .", "ovum pick - up was performed about four weeks before she present to the emergency department and embryo transfer three days later .", "the patient received intramural progesterone 50 mg and was prescribed a vaginal progesterone gel ( crinone ) for daily use for 11 days after the embryo transfer .", "the patient presented to hospital with vaginal bleeding , fever and abdominal pain on eleven days after embryo transfer , and received antibiotic treatment followed by supportive care at a local clinic for three days .", "she visited our outpatient clinic due to vaginal bleeding and lower abdominal pain on sixteen days after embryo transfer and was admitted to hospital .", "c - reactive protein ( crp ) was elevated to 7.52 mg / dl and intravenous antibiotic treatment was given .", "biochemical abortion was diagnosed on nineteen days following embryo transfer after serum -hcg decreased from 161 to 63 mlu / ml in two days .", "the patient was discharged after symptom improvement , but presented with vaginal bleeding and lower abdominal pain , at a local clinic two days later , where she received hydration .", "the last day of that month , she presented to the emergency department with high fever ( 39 ) and , lower abdominal pain .", "peripheral blood cell analysis indicated a : white blood cell count of 17.510/l , an elevated crp of 24.3 mg / dl , a hemoglobin level of 7.6 g / dl and a hematocrit level of 24.5% .", "computed tomography at the emergency department revealed a wedge - shaped low attenuation area in the anterior uterine corpus .", "focal uterine infarction was suspected , and the pelvic mri revealed a wedge - shaped , irregularly marginated , non - enhancing area in the anterior uterine wall and the presence of preserved myometrium between the endometrial cavity and the inner margin of the necrotic myometrium ( figure 1 ) .", "blood markers investigated to establish the cause of infarction , including antiphospholipid immunoglobulin g ( igg ) and igm , protein c activation , protein s , and plasminogen , were all within normal ranges .", "five days after hospital admission , pelvic pain improved and crp decreased to 6.08 mg / dl , and the patient was discharged .", "two months later , pelvic mri showed interval regression of the previously noted possible focal uterine infarction site with a remarkably improved blood flow ( figure 2 ) ."], ["adenomyosis uteri is a pathological entity characterized by the presence of endometrial glands and stroma embedded within the myometrium , but without apparent contact with the endo - myometrial junction .", "uterine adenomyosis is relatively frequent , affecting 1% of females ; its diagnosis is more often made in multiparous patients , in their fourth and fifth decade of life , and for this reason , infertility is not frequently concurrent with adenomyosis .", "however , given the trend for first pregnancies to be delayed until women are in their thirties or forties , adenomyosis uteri becoming a more frequently considered diagnosis", ". pregnancy resulting from assisted reproductive technology ( art ) is possible in patients with ademoyosis ; however , spontaneous abortion , including biochemical abortion , still occurs in these patients .", "uterine infarction after ivf - et in a patient with adenomyosis has not previously been reported .", "the objective of uae is to initiate tumor infarction resulting in substantial reduction of the uterine and tumor volumes .", "first , when undergoing ivf - et , the risk of thrombosis is increased , as in the cases of pregnancy and hyperthyroidism , with which the patient also presented . the precise mechanisms by which the ohss and exogenous hormonal stimulation used in art induce thromboembolic events are still uncertain . however , vascular endothelial growth factor secreted during ohss , high estradiol concentrations , and blood hyperviscosity play a prominent part in inducing a prothrombotic state .", "reported that the overall venous thrombosis incidence rate during ivf pregnancies was three times higher than in reference pregnancies .", "an elevated crp can activate the coagulation system , following which microthrombosis formation , in the uterine myometrium , could cause necrosis of the myometrium and lead to focal uterine infarction .", "second , the disruption of the endometrial - myometrial interface also disrupts the organization of myometrial muscle fibers and may also disrupt normal contractility of the subendometrium .", "uterine adenomyosis with abnormal contractility and the subsequent abortion caused massive vaginal bleeding which may have led to uterine ischemia , and the resulting uterine infarction .", "clinical hyperthyroidism is generally accompanied by a hypercoagulable state , which may lead to microthrombosis of the uterine myometrium .", "last , fever and the elevated crp suggested that infection could have complicated the ischemic necrosis after uterine infarction .", "however , there were no clinical features such as rapid tumor growth , extrauterine extension or metastases , leading to the conclusion that the possibility of uterine sarcoma was very small .", "a follow - up mri three months later showed only traces of uterine infarction lesion and adenomyosis . in conclusion , we experienced a rare case of uterine adenomyosis that developed into uterine infarction after ivf - et and biochemical pregnancy .", "this case demonstrates that uterine infarction should be considered in the differential diagnosis of acute abdominal pain , vaginal bleeding and infectious signs in women with biochemical abortion after ivf - et , with a history of adenomyosis and hyperthyroidism .", "the insertion of a hormonal intrauterine device could be considered another option for treating the symptoms of adenomyosis in cases presenting with focal uterine infarction dysmenorrhea and menorrhagia .", "in addition , the surgical management of focal uterine infarction , may be considered in the management of uterine infarction after ivf - et in patients with uterine adenomyosis if future fertility is not desired .", "finally , it is important to note that the previous infarction site might be a potential risk site for uterine rupture in a future pregnancy ."]]}
{"article_id": "PMC3440953", "article_text": ["to optimize the benefits of minimally invasive procedures , surgeons have attempted to reduce the overall abdominal wall trauma by decreasing either the size of the ports or the number of trocars . in these efforts", ", transumbilical single - port surgery uses an umbilical single incision technique to access the peritoneal cavity and target organs . owing to the nature of umbilicus , single - port laparoscopy through the umbilicus offers an exciting opportunity to perform laparoscopic surgery with no visible scar . however , transumbilical single - port laparoscopy is not a new concept in gynecologic surgery [ 15 ] . in 1969 ,", "wheeless and thompson first published the technique and the results of a large series of laparoscopic tubal ligations using single - trocar laparoscopy .", "later , wheeless reported a large series of one - incision tubal ligation . additionally , in 1991 , the first laparoscopic total abdominal hysterectomy with bilateral salpingooophorectomy ( bso ) using only a single incision was reported by pelosi and pelosi iii .", "one year later , four supracervical hysterectomies with bso for benign uterine disease were reported by the same authors [ 15 ] .", "although single - port surgery enhances cosmetic benefits and reduces postoperative pain and morbidity , use of this technique was not widespread due to technical difficulties .", "however , with advances in instrumental and surgical skills , the technical difficulties associated with this surgical procedure have been overcome considerably [ 615 ] .", "particularly , single - port surgery is ideal for laparoscopic - assisted vaginal hysterectomy ( lavh ) because the vagina of woman can be considered as an additional route for surgery ; thus , uterine manipulators can be applied through the vagina [ 1117 ] . unlike uterine repair following myomectomy or bowel reanastomosis after bowel resection", "this is because the vaginal stump can be repaired not by laparoscopy , but through the vagina . in this study", ", we report our initial 100 cases observations of spa - lavh ( with or without bilateral salpingooophrectomy ( bso ) ) using a homemade , single - port , three - channel system .", "a retrospective medical records review was performed for the initial 100 patients who underwent spa - lavh at eun hospital . between march 2010 and september 2011 ,", "100 patients had undergone spa - lavh for nonmalignant gynecological diseases , including uterine leiomyoma ( 25 cases ) , adenomyosis ( 19 cases ) , adenomyosis coexisting leiomyoma ( 41 cases ) , preinvasive lesion of cervix coexisting adenomyosis or leiomyoma ( 7 cases ) , ovarian huge cyst ( 5 cases ) , endometrial hyperplasia ( 2 cases ) , and tuboovarian abscess ( 1 case ) . past abdominopelvic surgery , body mass index ( bmi ) , and the size of the uterus were not considered as exclusion criteria .", "the following parameters were determined in the present observational study : age , parity , bmi , surgical history , indication for surgery , operative time ( from incision to final umbilical closure ) , largest dimension of the uterus , weight of the extirpated uterus ( as pathology report ) , hemoglobin change ( from before surgery to postoperative day 1 ) , and perioperative and postoperative complications .", "we used homemade , single - port , three - channel system using the alexis wound retractor ( applied medical , rancho santa margarita , ca , usa ) , surgical glove , two 10  mm trocars , and one 5  mm trocar [ 7 , 16 , 17 ] .", "after partial eversion of the umbilicus , a curved semilunar skin incision was performed at the hidden lateral aspect of the umbilical crater .", "the incision was c - shaped and followed the natural curve of the inferior lateral aspect of the umbilical crater near the base .", "after skin incision , a rectus fasciotomy and peritoneal incision were performed by direct cut - down technique .", "an approximately 1.5 2  cm - sized skin incision was sufficient to install the three - channel , single - port system , because of the elasticity of the skin and the tissue beneath it , which can be dissected as long as required [ 16 , 17 ] .", "as shown in figure 1(a ) , the fascial edges were tagged with suture for traction prior to port system installation ; this was useful for fascial closure at the end of the procedure .", "the alexis wound retractor consists of a proximal ring , distal ring , and connecting retractable sleeve .", "as shown in figure 1(a ) , the distal ring was loaded within the intraperitoneal space and tightly turned inside out of the proximal ring ( rolled up manner ) , creating an effective seal and a wider opening of the single - port incision by connecting retractable sleeve between the distal and proximal rings .", "once fixed in the opening site , it laterally retracted the sides of the wound opening .", "subsequently , as shown in figure 1 , a sterile surgical glove was placed over the proximal ring and fixed tightly to prevent leakage of carbon dioxide gas .", "three trocars were inserted through the surgical glove with cut edges of the distal fingertips and tied with an elastic string .", "the elastic nature of the glove enabled to achieve an airtight seal , which maintained the pneumoperitoneum .", "the multiple truncated fingers of the glove functioned as a multiport for surgical instruments [ 16 , 17 ] .", "a limited range of motion was closely related to the bulkiest portion of the trocar head and instrumental grip ( external handle ) extracorporeally overlapping . as shown in figure 1(b ) , the length of the instruments was the same , but the lengths of the truncated glove digits varied . however , varying the height of the trocar head may minimize clashing of the bulkiest portion of the trocar head and instrumental grip ( the external handle ) extracorporeally overlapping .", "all the surgical procedures were performed as a standard lavh ( with or without bso ) technique using conventional nonarticulated rigid laparoscopic instruments and the ligasure system ( valleylab , boulder , co , usa ) . as has been established earlier , exploration of pelvis , coagulation and cut of ligaments and vessel above the uterine vessel , and bladder mobilization were undertaken in laparoscopic phase .", "ligation of uterine vessel , cardinal and uterosacral ligament , extirpation of uterus , and vaginal stump closure were undertaken in the vaginal phase .", "all procedures were successfully completed through the single - port system and vagina without the need for extraumbilical puncture or conversion to laparotomy .", "as shown in table 1 , the mean  standard deviation ( sd ) of patient age , parity , and bmi was 48.2  6.5  years , 2.3  1.0 , 25.4  3.3  kg / m , respectively .", "thirty - three patients had a past history of abdominopelvic surgery , such as a caesarean section , laparoscopic tubal ligation , appendectomy , ovarian cystectomy , or salpingooophrectomy . among these patients , six had a history of caesarean sections , five had a history of repeat caesarean sections , and five had a history of three caesarean sections .", "seven patients needed 2 - 3 units of packed red blood cell transfusion due to chronic anemia or intraoperative hemorrhage .", "the mean  sd of time to installation of the transumbilical single - port system was 7.3    1.5  min .", "the mean  sd of total operative time , largest dimension of the uterus , and weight of the uterus were 73.1  24.6  min , 10.5    2.1  cm , and 300.8  192.5  gram , respectively .", "the operative time between laparoscopic phase and vaginal phase was similar but depended on pelvic pathology .", "the median decline in the hemoglobin level from before surgery to postoperative day 1 was 1.8  0.9  g / dl .", "bladder injury occurred in one patient who had a history of three caesarean sections but was repaired through intraoperative laparoscopic suture .", "the postoperative course was uneventful in most patients , but three had a transient paralytic ileus , and five had pelvic hematoma , all of whom recovered following conservative managements .", "no port - related complications were noted , and the cosmetic results and patient satisfaction were excellent .", "spa - lavh is a technically safe and feasible procedure , and the homemade single - port system offers reliable and cost - effective access for single - port surgery .", "as mentioned earlier , lavh is most ideal for single - port surgery because the vagina of woman can be considered as an additional route for surgery ; thus , uterine manipulators can be applied through the vagina . unlike uterine repair after myomectomy", "this is because the vaginal stump can be repaired not by laparoscopy , but through the vagina .", "thus , spa - lavh is safe , and the procedure can be learned by skillful surgeons over a short period of time , because a considerable portion of the procedure can be performed through the vagina .", "the homemade three - channel , single - port system using a surgical glove and an alexis wound retractor offers reliable , flexible , and cost - effective access for single - port procedures , and the system can be applicable in nonarticulated , rigid , conventional laparoscopic instruments [ 16 , 17 ] . limitations of single - port surgery include the loss of instrumental triangulation , reduced operative working space , reduced laparoscopic visualization , and instrumental crowding and clashing .", "these limitations act as hurdles for some reconstructive procedures , such as repair after myomectomy .", "however , the reconstructive procedure can be performed with instrumental advancement , such as the use of articulated instruments [ 615 ] .", "our observations show that a history of abdominopelvic surgery is not a contraindication for single - port surgery ; however , central obesity is problematic to secure a route for the single - port system through a small intraumbilical incision .", "procedural difficulties resulting from previous abdominopelvic surgery are not because of the single - port surgery itself , but owing to abdominopelvic conditions [ 10 , 1517 ] .", "a linear correlation existed between the operation time and an extirpated uterine weight of > 400  g , because more time was needed for uterine fragmentation for extirpation through the vagina ; however , no linear correlation existed between the operation time and a uterus weight of < 400  g. for pelvic adhesion , such as in previous pelvic surgery or endometriosis , additional operation time is required for adhesiolysis .", "it is not a case - control study , and pain score , hospital stay , cost effectiveness , and return to work were not considered because of the retrospective nature of the study .", "additional clinical data and long - term followup may be needed to address port - related complications ."], "abstract_text": ["<S> \n objectives . to present our initial experiences with laparoscopically assisted vaginal hysterectomy performed using homemade transumbilical single - port system . materials and methods . </S>", "<S> we reviewed the medical records of one hundred patients who underwent single - port access laparoscopically assisted vaginal hysterectomy ( spa - lavh ) . </S>", "<S> spa - lavh was performed with homemade single port system and conventional rigid laparoscopic instruments . </S>", "<S> results . </S>", "<S> all procedures were successfully completed through the single - port system and vagina without need for extraumbilical puncture or conversion to laparotomy . </S>", "<S> the median patient age was 48.2  6.5 years . </S>", "<S> thirty - three patients had history of past abdominopelvic surgery . the median total operative time , </S>", "<S> largest dimension of the uterus , and weight of the uterus were 73.1  24.6  min , 10.5  2.1  cm , and 300.8  192.5  gram , respectively . </S>", "<S> the median decline in the hemoglobin from before surgery to postoperative day 1 was 1.8  0.9  g / dl . </S>", "<S> bladder injury in occurred one patient who was repaired through intraoperative laparoscopic suture . </S>", "<S> the postoperative course was uneventful in most patients except for three who had a transient paralytic ileus , five who had pelvic hematoma , but they were recovered following conservative managements . </S>", "<S> no port - related complications were noted , and the cosmetic results were excellent . </S>", "<S> conclusions . </S>", "<S> spa - lavh is technically safe procedure , and the homemade single - port system offers reliable access for single - port surgery . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Conclusion", "5. Discussion"], "sections": [["to optimize the benefits of minimally invasive procedures , surgeons have attempted to reduce the overall abdominal wall trauma by decreasing either the size of the ports or the number of trocars . in these efforts", ", transumbilical single - port surgery uses an umbilical single incision technique to access the peritoneal cavity and target organs . owing to the nature of umbilicus , single - port laparoscopy through the umbilicus offers an exciting opportunity to perform laparoscopic surgery with no visible scar . however , transumbilical single - port laparoscopy is not a new concept in gynecologic surgery [ 15 ] . in 1969 ,", "wheeless and thompson first published the technique and the results of a large series of laparoscopic tubal ligations using single - trocar laparoscopy .", "later , wheeless reported a large series of one - incision tubal ligation . additionally , in 1991 , the first laparoscopic total abdominal hysterectomy with bilateral salpingooophorectomy ( bso ) using only a single incision was reported by pelosi and pelosi iii .", "one year later , four supracervical hysterectomies with bso for benign uterine disease were reported by the same authors [ 15 ] .", "although single - port surgery enhances cosmetic benefits and reduces postoperative pain and morbidity , use of this technique was not widespread due to technical difficulties .", "however , with advances in instrumental and surgical skills , the technical difficulties associated with this surgical procedure have been overcome considerably [ 615 ] .", "particularly , single - port surgery is ideal for laparoscopic - assisted vaginal hysterectomy ( lavh ) because the vagina of woman can be considered as an additional route for surgery ; thus , uterine manipulators can be applied through the vagina [ 1117 ] . unlike uterine repair following myomectomy or bowel reanastomosis after bowel resection", "this is because the vaginal stump can be repaired not by laparoscopy , but through the vagina . in this study", ", we report our initial 100 cases observations of spa - lavh ( with or without bilateral salpingooophrectomy ( bso ) ) using a homemade , single - port , three - channel system ."], ["a retrospective medical records review was performed for the initial 100 patients who underwent spa - lavh at eun hospital . between march 2010 and september 2011 ,", "100 patients had undergone spa - lavh for nonmalignant gynecological diseases , including uterine leiomyoma ( 25 cases ) , adenomyosis ( 19 cases ) , adenomyosis coexisting leiomyoma ( 41 cases ) , preinvasive lesion of cervix coexisting adenomyosis or leiomyoma ( 7 cases ) , ovarian huge cyst ( 5 cases ) , endometrial hyperplasia ( 2 cases ) , and tuboovarian abscess ( 1 case ) . past abdominopelvic surgery , body mass index ( bmi ) , and the size of the uterus were not considered as exclusion criteria .", "the following parameters were determined in the present observational study : age , parity , bmi , surgical history , indication for surgery , operative time ( from incision to final umbilical closure ) , largest dimension of the uterus , weight of the extirpated uterus ( as pathology report ) , hemoglobin change ( from before surgery to postoperative day 1 ) , and perioperative and postoperative complications .", "we used homemade , single - port , three - channel system using the alexis wound retractor ( applied medical , rancho santa margarita , ca , usa ) , surgical glove , two 10  mm trocars , and one 5  mm trocar [ 7 , 16 , 17 ] .", "after partial eversion of the umbilicus , a curved semilunar skin incision was performed at the hidden lateral aspect of the umbilical crater .", "the incision was c - shaped and followed the natural curve of the inferior lateral aspect of the umbilical crater near the base .", "after skin incision , a rectus fasciotomy and peritoneal incision were performed by direct cut - down technique .", "an approximately 1.5 2  cm - sized skin incision was sufficient to install the three - channel , single - port system , because of the elasticity of the skin and the tissue beneath it , which can be dissected as long as required [ 16 , 17 ] .", "as shown in figure 1(a ) , the fascial edges were tagged with suture for traction prior to port system installation ; this was useful for fascial closure at the end of the procedure .", "the alexis wound retractor consists of a proximal ring , distal ring , and connecting retractable sleeve .", "as shown in figure 1(a ) , the distal ring was loaded within the intraperitoneal space and tightly turned inside out of the proximal ring ( rolled up manner ) , creating an effective seal and a wider opening of the single - port incision by connecting retractable sleeve between the distal and proximal rings .", "once fixed in the opening site , it laterally retracted the sides of the wound opening .", "subsequently , as shown in figure 1 , a sterile surgical glove was placed over the proximal ring and fixed tightly to prevent leakage of carbon dioxide gas .", "three trocars were inserted through the surgical glove with cut edges of the distal fingertips and tied with an elastic string .", "the elastic nature of the glove enabled to achieve an airtight seal , which maintained the pneumoperitoneum .", "the multiple truncated fingers of the glove functioned as a multiport for surgical instruments [ 16 , 17 ] .", "a limited range of motion was closely related to the bulkiest portion of the trocar head and instrumental grip ( external handle ) extracorporeally overlapping . as shown in figure 1(b ) , the length of the instruments was the same , but the lengths of the truncated glove digits varied . however , varying the height of the trocar head may minimize clashing of the bulkiest portion of the trocar head and instrumental grip ( the external handle ) extracorporeally overlapping .", "all the surgical procedures were performed as a standard lavh ( with or without bso ) technique using conventional nonarticulated rigid laparoscopic instruments and the ligasure system ( valleylab , boulder , co , usa ) . as has been established earlier , exploration of pelvis , coagulation and cut of ligaments and vessel above the uterine vessel , and bladder mobilization were undertaken in laparoscopic phase .", "ligation of uterine vessel , cardinal and uterosacral ligament , extirpation of uterus , and vaginal stump closure were undertaken in the vaginal phase ."], ["all procedures were successfully completed through the single - port system and vagina without the need for extraumbilical puncture or conversion to laparotomy .", "as shown in table 1 , the mean  standard deviation ( sd ) of patient age , parity , and bmi was 48.2  6.5  years , 2.3  1.0 , 25.4  3.3  kg / m , respectively .", "thirty - three patients had a past history of abdominopelvic surgery , such as a caesarean section , laparoscopic tubal ligation , appendectomy , ovarian cystectomy , or salpingooophrectomy . among these patients , six had a history of caesarean sections , five had a history of repeat caesarean sections , and five had a history of three caesarean sections .", "seven patients needed 2 - 3 units of packed red blood cell transfusion due to chronic anemia or intraoperative hemorrhage .", "the mean  sd of time to installation of the transumbilical single - port system was 7.3    1.5  min .", "the mean  sd of total operative time , largest dimension of the uterus , and weight of the uterus were 73.1  24.6  min , 10.5    2.1  cm , and 300.8  192.5  gram , respectively .", "the operative time between laparoscopic phase and vaginal phase was similar but depended on pelvic pathology .", "the median decline in the hemoglobin level from before surgery to postoperative day 1 was 1.8  0.9  g / dl .", "bladder injury occurred in one patient who had a history of three caesarean sections but was repaired through intraoperative laparoscopic suture .", "the postoperative course was uneventful in most patients , but three had a transient paralytic ileus , and five had pelvic hematoma , all of whom recovered following conservative managements .", "no port - related complications were noted , and the cosmetic results and patient satisfaction were excellent ."], ["spa - lavh is a technically safe and feasible procedure , and the homemade single - port system offers reliable and cost - effective access for single - port surgery ."], ["as mentioned earlier , lavh is most ideal for single - port surgery because the vagina of woman can be considered as an additional route for surgery ; thus , uterine manipulators can be applied through the vagina . unlike uterine repair after myomectomy", "this is because the vaginal stump can be repaired not by laparoscopy , but through the vagina .", "thus , spa - lavh is safe , and the procedure can be learned by skillful surgeons over a short period of time , because a considerable portion of the procedure can be performed through the vagina .", "the homemade three - channel , single - port system using a surgical glove and an alexis wound retractor offers reliable , flexible , and cost - effective access for single - port procedures , and the system can be applicable in nonarticulated , rigid , conventional laparoscopic instruments [ 16 , 17 ] . limitations of single - port surgery include the loss of instrumental triangulation , reduced operative working space , reduced laparoscopic visualization , and instrumental crowding and clashing .", "these limitations act as hurdles for some reconstructive procedures , such as repair after myomectomy .", "however , the reconstructive procedure can be performed with instrumental advancement , such as the use of articulated instruments [ 615 ] .", "our observations show that a history of abdominopelvic surgery is not a contraindication for single - port surgery ; however , central obesity is problematic to secure a route for the single - port system through a small intraumbilical incision .", "procedural difficulties resulting from previous abdominopelvic surgery are not because of the single - port surgery itself , but owing to abdominopelvic conditions [ 10 , 1517 ] .", "a linear correlation existed between the operation time and an extirpated uterine weight of > 400  g , because more time was needed for uterine fragmentation for extirpation through the vagina ; however , no linear correlation existed between the operation time and a uterus weight of < 400  g. for pelvic adhesion , such as in previous pelvic surgery or endometriosis , additional operation time is required for adhesiolysis .", "it is not a case - control study , and pain score , hospital stay , cost effectiveness , and return to work were not considered because of the retrospective nature of the study .", "additional clinical data and long - term followup may be needed to address port - related complications ."]]}
{"article_id": "PMC4127542", "article_text": ["drug eruptions are common , affecting 23% of all hospitalized patients , and complications associated with medications make up the largest proportion of adverse events seen in hospitals .", "drug eruptions can be difficult to diagnose , particularly causality , as even biopsies are nonspecific and patients are often on more than one potentially offending medication .", "we present 3 cases that highlight these difficulties and the broad spectrum of drug eruptions in the setting of lymphoma patients receiving multidrug regimens including bendamustine .", "a 60-year - old caucasian female presented with a 6-month history of constitutional symptoms , dyspnea on exertion , lymphadenopathy and pleural effusions and was diagnosed with stage iv marginal zone lymphoma with bone marrow involvement .", "four to six weeks prior to admission , she had developed a progressive generalized erythematous rash and edema involving her trunk and extremities .", "these findings were characterized as consistent with drug eruption , although no new drugs were identified in her history .", "the decision was made to treat with bendamustine and rituxan as an inpatient due to nonresolving malignant pleural effusion .", "her initial rash on presentation was thought to be due to possible lymphomatous involvement and lymphedema as it improved over the 23 days following chemotherapy . on day 7", "she developed a bullous rash involving 6070% of her skin as well as mucosa ( fig .", "a new biopsy revealed vacuolar interface dermatitis with necrosis of keratinocytes in the epidermis , with the underlying dermis displaying a superficial perivascular lymphocytic inflammatory infiltrate with eosinophils ( fig . 2 ; fig .", "3 ) . she began to deteriorate rapidly with a clinical course further complicated by the development of severe hepatic and renal failure .", "a diagnosis of dress syndrome was made . despite a high dose of steroids , the rash spread , eventually involving 100% of her body surface area , including her eyelids , conjunctiva , oral and vaginal mucus membranes . despite the use of filgrastim and broad spectrum antibiotics", "the second case was a 66-year - old male diagnosed in 2009 with chronic lymphocytic leukemia associated with the deletion of chromosome 13 . in 2012 , his lymphocyte count reached 253,000 , and he developed autoimmune hemolytic anemia .", "he initially received one cycle of intravenous immunoglobulin , cyclophosphamide , vincristine and prednisone ( without rituxan ) .", "the initial cycle was again given without rituxan due to the high burden of the disease .", "he received antibiotic therapy with cefepime , fluconazole , azithromycin and levofloxacin during two separate hospitalizations .", "two days following the second admission ( day 7 following bendamustine ) , he developed a generalized erythematous rash with desquamation that did not resolve with the discontinuation of cefepime or the administration of rituxan .", "his rash slowly resolved . a second episode of nonneutropenic fever , hypotension and acute renal failure occurred with the second cycle of bendamustine and rituxan 2 months later .", "this resolved as well . given the timing of the reaction , it was questioned that the offending drug was bendamustine in both events .", "the third patient was a 59-year - old female diagnosed with stage ivb splenic marginal zone lymphoma after presenting with constitutional symptoms and urticaria .", "she was treated with splenectomy and systemic chemotherapy consisting of bendamustine and rituxan . prior to the third cycle ,", "the eruption worsened 2 weeks later as the lesions became intensely pruritic , erythematous and ulcerative . despite the treatment with antibiotics and corticosteroids , she progressed over the course of 1 week to a tense bullous rash covering the extremities .", "a 60-year - old caucasian female presented with a 6-month history of constitutional symptoms , dyspnea on exertion , lymphadenopathy and pleural effusions and was diagnosed with stage iv marginal zone lymphoma with bone marrow involvement .", "four to six weeks prior to admission , she had developed a progressive generalized erythematous rash and edema involving her trunk and extremities .", "these findings were characterized as consistent with drug eruption , although no new drugs were identified in her history .", "the decision was made to treat with bendamustine and rituxan as an inpatient due to nonresolving malignant pleural effusion .", "her initial rash on presentation was thought to be due to possible lymphomatous involvement and lymphedema as it improved over the 23 days following chemotherapy . on day 7", "she developed a bullous rash involving 6070% of her skin as well as mucosa ( fig .", "a new biopsy revealed vacuolar interface dermatitis with necrosis of keratinocytes in the epidermis , with the underlying dermis displaying a superficial perivascular lymphocytic inflammatory infiltrate with eosinophils ( fig . 2 ; fig .", "3 ) . she began to deteriorate rapidly with a clinical course further complicated by the development of severe hepatic and renal failure .", "a diagnosis of dress syndrome was made . despite a high dose of steroids , the rash spread , eventually involving 100% of her body surface area , including her eyelids , conjunctiva , oral and vaginal mucus membranes . despite the use of filgrastim and broad spectrum antibiotics", "the second case was a 66-year - old male diagnosed in 2009 with chronic lymphocytic leukemia associated with the deletion of chromosome 13 . in 2012 , his lymphocyte count reached 253,000 , and he developed autoimmune hemolytic anemia .", "he initially received one cycle of intravenous immunoglobulin , cyclophosphamide , vincristine and prednisone ( without rituxan ) .", "the initial cycle was again given without rituxan due to the high burden of the disease .", "he received antibiotic therapy with cefepime , fluconazole , azithromycin and levofloxacin during two separate hospitalizations .", "two days following the second admission ( day 7 following bendamustine ) , he developed a generalized erythematous rash with desquamation that did not resolve with the discontinuation of cefepime or the administration of rituxan .", "his rash slowly resolved . a second episode of nonneutropenic fever , hypotension and acute renal failure occurred with the second cycle of bendamustine and rituxan 2 months later .", "this resolved as well . given the timing of the reaction , it was questioned that the offending drug was bendamustine in both events .", "the third patient was a 59-year - old female diagnosed with stage ivb splenic marginal zone lymphoma after presenting with constitutional symptoms and urticaria .", "prior to the third cycle , the patient developed a mild pruritic eruption with raised plaques .", "the eruption worsened 2 weeks later as the lesions became intensely pruritic , erythematous and ulcerative . despite the treatment with antibiotics and corticosteroids , she progressed over the course of 1 week to a tense bullous rash covering the extremities .", "severe drug eruptions associated with high morbidity and mortality include stevens - johnson syndrome and toxic epidermal necrolysis .", "these syndromes are often confused and may represent a continuum of each other , with toxic epidermal necrolysis having > 30% of epidermal detachment and stevens - johnson syndrome having only 10% or less", "morbidity and mortality from these conditions stem from their complications including massive fluid losses , electrolyte imbalances , infections and diffuse interstitial pneumonitis , leading to acute respiratory distress syndrome .", "dress syndrome represents another severe drug eruption with a 10% mortality rate , typically in the setting of hepatic failure .", "the term dress syndrome was later coined in 1996 as a syndrome specifically describing a drug eruption with internal organ involvement and hematologic abnormalities , particularly eosinophilia or atypical lymphocytosis .", "dress syndrome can be difficult to diagnose , as onset is typically 26 weeks after the drug was administered but is more probable when associated with hypereosinophilia , liver involvement , fever and lymphadenopathy .", "etiology , as with other severe drug eruptions , is unclear but postulated to be associated with human herpes virus reactivation .", "many drugs have been implicated in dress syndrome ; however , the ones most often reported are allopurinol , penicillins , sulfonamides and antipsychotics .", "severity and likelihood of the diagnosis of dress as well as other severe cutaneous drug reactions are scored using the regiscar in europe .", "the mainstay of treatment remains the removal of the offending drug and supportive care with the use of steroids remaining controversial in their effectiveness [ 2 , 5 ] .", "bendamustine was first synthesized in 1963 as a chemotherapeutic agent with both antimetabolite and alkylating properties .", "since that time , bendamustine has demonstrated significant efficacy in many lymphoma subtypes , particularly indolent lymphomas resistant to other alkylators . in 2008 ,", "it was approved by the fda for the treatment of rituximab - refractory chronic lymphocytic leukemia and indolent b - cell non - hodgkin lymphomas .", "its side effect profile has been shown to be well tolerated , with the most common side effects being cytopenias and fatigue .", "the fda prescribing guidelines state that skin reactions have been reported with bendamustine , most often in combination with another agent ( rituximab or allopurinol ) , so that true causality can not be determined .", "however , these reactions have been reported to be severe and progressive with several deaths mentioned .", "most described rashes develop following the first administration of bendamustine and generally worsen in severity over the following days .", "one patient with b - cell prolymphocytic leukemia developed rash with palpable purpura and hemorrhagic plaques while thrombocytopenic .", "this rash was preceded by a severe generalized maculopapular rash with most bendamustine skin reactions .", "a biopsy revealed a diffuse eosinophilic infiltrate . in a case series of 16 patients with chronic lymphocytic leukemia or follicular lymphoma , over 50% of the patients developed an erythematous maculopapular rash .", "another case report described a severe desquamating rash involving > 75% of the skin as well as oral mucosa .", "a biopsy revealed interface dermatitis with a lymphohistiocytic infiltrate . in our case of dress syndrome ,", "this led to the question of the offending agent ; however , the patient did not have a reasonable alternate medication exposure .", "we also postulate that both the patient 's lymphoma and bendamustine itself may have induced a quicker response based on already reduced immunoglobulin levels and a rapid reduction in b - cell lymphocytes .", "this initiating factor in the mechanism of dress was proposed by criado et al .   and has been cited as the reason for the long latency in cases .", "case 2 represents the typical scenario of a drug exanthem where the causative agent is in question .", "rash developed following bendamustine , but the patient was also exposed to antibiotics that are known to cause dermatologic complications .", "however , unlike most reported cases , rituximab was not a questionable cause , as it was not administered with the bendamustine initially .", "case 3 also represents a more atypical presentation than initially reported , with a reaction after the second cycle rather than after initial exposure to the drug . however , no other causative agent was found ."], "abstract_text": ["<S> cutaneous drug reactions make up the largest proportion of adverse events in the medical field . </S>", "<S> causality , in particular , is difficult to determine , and therefore , preventing recurrent reactions can be challenging . </S>", "<S> bendamustine was initially thought to be a well - tolerated chemotherapy agent with few side effects aside from bone marrow suppression . </S>", "<S> however , the incidence of cutaneous reactions reported is rising . </S>", "<S> we describe three such reactions in relation to bendamustine administration in hopes of adding to the awareness of such side effects . </S>"], "labels": null, "section_names": ["Introduction", "Case Presentations", "Case 1: Drug Reaction with Eosinophilia and Systemic Symptoms Syndrome", "Case 2: Stevens-Johnson Syndrome", "Case 3: Bullous Pemphigoid", "Discussion"], "sections": [["drug eruptions are common , affecting 23% of all hospitalized patients , and complications associated with medications make up the largest proportion of adverse events seen in hospitals .", "drug eruptions can be difficult to diagnose , particularly causality , as even biopsies are nonspecific and patients are often on more than one potentially offending medication .", "we present 3 cases that highlight these difficulties and the broad spectrum of drug eruptions in the setting of lymphoma patients receiving multidrug regimens including bendamustine ."], ["a 60-year - old caucasian female presented with a 6-month history of constitutional symptoms , dyspnea on exertion , lymphadenopathy and pleural effusions and was diagnosed with stage iv marginal zone lymphoma with bone marrow involvement .", "four to six weeks prior to admission , she had developed a progressive generalized erythematous rash and edema involving her trunk and extremities .", "these findings were characterized as consistent with drug eruption , although no new drugs were identified in her history .", "the decision was made to treat with bendamustine and rituxan as an inpatient due to nonresolving malignant pleural effusion .", "her initial rash on presentation was thought to be due to possible lymphomatous involvement and lymphedema as it improved over the 23 days following chemotherapy . on day 7", "she developed a bullous rash involving 6070% of her skin as well as mucosa ( fig .", "a new biopsy revealed vacuolar interface dermatitis with necrosis of keratinocytes in the epidermis , with the underlying dermis displaying a superficial perivascular lymphocytic inflammatory infiltrate with eosinophils ( fig . 2 ; fig .", "3 ) . she began to deteriorate rapidly with a clinical course further complicated by the development of severe hepatic and renal failure .", "a diagnosis of dress syndrome was made . despite a high dose of steroids , the rash spread , eventually involving 100% of her body surface area , including her eyelids , conjunctiva , oral and vaginal mucus membranes . despite the use of filgrastim and broad spectrum antibiotics", "the second case was a 66-year - old male diagnosed in 2009 with chronic lymphocytic leukemia associated with the deletion of chromosome 13 . in 2012 , his lymphocyte count reached 253,000 , and he developed autoimmune hemolytic anemia .", "he initially received one cycle of intravenous immunoglobulin , cyclophosphamide , vincristine and prednisone ( without rituxan ) .", "the initial cycle was again given without rituxan due to the high burden of the disease .", "he received antibiotic therapy with cefepime , fluconazole , azithromycin and levofloxacin during two separate hospitalizations .", "two days following the second admission ( day 7 following bendamustine ) , he developed a generalized erythematous rash with desquamation that did not resolve with the discontinuation of cefepime or the administration of rituxan .", "his rash slowly resolved . a second episode of nonneutropenic fever , hypotension and acute renal failure occurred with the second cycle of bendamustine and rituxan 2 months later .", "this resolved as well . given the timing of the reaction , it was questioned that the offending drug was bendamustine in both events .", "the third patient was a 59-year - old female diagnosed with stage ivb splenic marginal zone lymphoma after presenting with constitutional symptoms and urticaria .", "she was treated with splenectomy and systemic chemotherapy consisting of bendamustine and rituxan . prior to the third cycle ,", "the eruption worsened 2 weeks later as the lesions became intensely pruritic , erythematous and ulcerative . despite the treatment with antibiotics and corticosteroids , she progressed over the course of 1 week to a tense bullous rash covering the extremities ."], ["a 60-year - old caucasian female presented with a 6-month history of constitutional symptoms , dyspnea on exertion , lymphadenopathy and pleural effusions and was diagnosed with stage iv marginal zone lymphoma with bone marrow involvement .", "four to six weeks prior to admission , she had developed a progressive generalized erythematous rash and edema involving her trunk and extremities .", "these findings were characterized as consistent with drug eruption , although no new drugs were identified in her history .", "the decision was made to treat with bendamustine and rituxan as an inpatient due to nonresolving malignant pleural effusion .", "her initial rash on presentation was thought to be due to possible lymphomatous involvement and lymphedema as it improved over the 23 days following chemotherapy . on day 7", "she developed a bullous rash involving 6070% of her skin as well as mucosa ( fig .", "a new biopsy revealed vacuolar interface dermatitis with necrosis of keratinocytes in the epidermis , with the underlying dermis displaying a superficial perivascular lymphocytic inflammatory infiltrate with eosinophils ( fig . 2 ; fig .", "3 ) . she began to deteriorate rapidly with a clinical course further complicated by the development of severe hepatic and renal failure .", "a diagnosis of dress syndrome was made . despite a high dose of steroids , the rash spread , eventually involving 100% of her body surface area , including her eyelids , conjunctiva , oral and vaginal mucus membranes . despite the use of filgrastim and broad spectrum antibiotics"], ["the second case was a 66-year - old male diagnosed in 2009 with chronic lymphocytic leukemia associated with the deletion of chromosome 13 . in 2012 , his lymphocyte count reached 253,000 , and he developed autoimmune hemolytic anemia .", "he initially received one cycle of intravenous immunoglobulin , cyclophosphamide , vincristine and prednisone ( without rituxan ) .", "the initial cycle was again given without rituxan due to the high burden of the disease .", "he received antibiotic therapy with cefepime , fluconazole , azithromycin and levofloxacin during two separate hospitalizations .", "two days following the second admission ( day 7 following bendamustine ) , he developed a generalized erythematous rash with desquamation that did not resolve with the discontinuation of cefepime or the administration of rituxan .", "his rash slowly resolved . a second episode of nonneutropenic fever , hypotension and acute renal failure occurred with the second cycle of bendamustine and rituxan 2 months later .", "this resolved as well . given the timing of the reaction , it was questioned that the offending drug was bendamustine in both events ."], ["the third patient was a 59-year - old female diagnosed with stage ivb splenic marginal zone lymphoma after presenting with constitutional symptoms and urticaria .", "prior to the third cycle , the patient developed a mild pruritic eruption with raised plaques .", "the eruption worsened 2 weeks later as the lesions became intensely pruritic , erythematous and ulcerative . despite the treatment with antibiotics and corticosteroids , she progressed over the course of 1 week to a tense bullous rash covering the extremities ."], ["severe drug eruptions associated with high morbidity and mortality include stevens - johnson syndrome and toxic epidermal necrolysis .", "these syndromes are often confused and may represent a continuum of each other , with toxic epidermal necrolysis having > 30% of epidermal detachment and stevens - johnson syndrome having only 10% or less", "morbidity and mortality from these conditions stem from their complications including massive fluid losses , electrolyte imbalances , infections and diffuse interstitial pneumonitis , leading to acute respiratory distress syndrome .", "dress syndrome represents another severe drug eruption with a 10% mortality rate , typically in the setting of hepatic failure .", "the term dress syndrome was later coined in 1996 as a syndrome specifically describing a drug eruption with internal organ involvement and hematologic abnormalities , particularly eosinophilia or atypical lymphocytosis .", "dress syndrome can be difficult to diagnose , as onset is typically 26 weeks after the drug was administered but is more probable when associated with hypereosinophilia , liver involvement , fever and lymphadenopathy .", "etiology , as with other severe drug eruptions , is unclear but postulated to be associated with human herpes virus reactivation .", "many drugs have been implicated in dress syndrome ; however , the ones most often reported are allopurinol , penicillins , sulfonamides and antipsychotics .", "severity and likelihood of the diagnosis of dress as well as other severe cutaneous drug reactions are scored using the regiscar in europe .", "the mainstay of treatment remains the removal of the offending drug and supportive care with the use of steroids remaining controversial in their effectiveness [ 2 , 5 ] .", "bendamustine was first synthesized in 1963 as a chemotherapeutic agent with both antimetabolite and alkylating properties .", "since that time , bendamustine has demonstrated significant efficacy in many lymphoma subtypes , particularly indolent lymphomas resistant to other alkylators . in 2008 ,", "it was approved by the fda for the treatment of rituximab - refractory chronic lymphocytic leukemia and indolent b - cell non - hodgkin lymphomas .", "its side effect profile has been shown to be well tolerated , with the most common side effects being cytopenias and fatigue .", "the fda prescribing guidelines state that skin reactions have been reported with bendamustine , most often in combination with another agent ( rituximab or allopurinol ) , so that true causality can not be determined .", "however , these reactions have been reported to be severe and progressive with several deaths mentioned .", "most described rashes develop following the first administration of bendamustine and generally worsen in severity over the following days .", "one patient with b - cell prolymphocytic leukemia developed rash with palpable purpura and hemorrhagic plaques while thrombocytopenic .", "this rash was preceded by a severe generalized maculopapular rash with most bendamustine skin reactions .", "a biopsy revealed a diffuse eosinophilic infiltrate . in a case series of 16 patients with chronic lymphocytic leukemia or follicular lymphoma , over 50% of the patients developed an erythematous maculopapular rash .", "another case report described a severe desquamating rash involving > 75% of the skin as well as oral mucosa .", "a biopsy revealed interface dermatitis with a lymphohistiocytic infiltrate . in our case of dress syndrome ,", "this led to the question of the offending agent ; however , the patient did not have a reasonable alternate medication exposure .", "we also postulate that both the patient 's lymphoma and bendamustine itself may have induced a quicker response based on already reduced immunoglobulin levels and a rapid reduction in b - cell lymphocytes .", "this initiating factor in the mechanism of dress was proposed by criado et al .   and has been cited as the reason for the long latency in cases .", "case 2 represents the typical scenario of a drug exanthem where the causative agent is in question .", "rash developed following bendamustine , but the patient was also exposed to antibiotics that are known to cause dermatologic complications .", "however , unlike most reported cases , rituximab was not a questionable cause , as it was not administered with the bendamustine initially .", "case 3 also represents a more atypical presentation than initially reported , with a reaction after the second cycle rather than after initial exposure to the drug . however , no other causative agent was found ."]]}
{"article_id": "PMC3452276", "article_text": ["although indomethacin is usually well tolerated , some patients developed gastrointestinal side effects , specially those patients who require long term therapy", ". therefore , the use of cyclo - oxygenase-2 specific inhibitors could reduce secondary effects and they are essentially equipotent to indomethacin in vitro and in vivo .", "indeed , some reports in the literature indicate the usefulness of the cox-2 inhibitors in the treatment of indomethacin - responsive headaches [ 38 ] .", "we report four cases of hemicrania continua and a patient suffering chronic paroxysmal hemicrania completely responsive to cox-2 inhibitors .", "a 42  years - old woman had sudden onset of short - lived pains on the left side of her head .", "she suffers for a month an intense headache with short attacks ( about 510  min ) but high frequency ( 1018 attacks per day ) .", "we put her on indomethacin 25  mg 3  times a day , and in 24  h her hemicranial pain completely disappears . because of legs oedema we discontinued indomethacin and 1  week later the pain returned .", "a 56  years - old man present with an 8  month history of a continuous left - sided headache strictly unilateral .", "the pain was moderate in intensity but fluctuating ( between 2 and 7/10 vas ) .", "the pain exacerbations were associated with lacrimation but not phono , photofobia , nausea or vomiting .", "an mri was normal and indomethacin 25  mg 3  times a day was started with completely recovery .", "two - month later he started to have gastric symptoms , indomethacin was discontinued and the hemicrania return .", "a 78  years - old woman with 1  year history of continuous and strictly left - sided headache , fluctuating in intensity ( 39 in vas ) and accompanied by conjuntival injection and nasal congestion during exacerbation time .", "with indomethacin 50  mg 3  times a day the hemicrania disappear , however , she developed disabling subjective tinnitus 1  month later .", "we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 18  month later .", "a 64  years - old woman with 3  years history of right - sided headache , fluctuating in intensity ( 28 in vas ) .", "with indomethacin 25  mg 3  times a day the hemicrania disappear , however , the patient suffer gastrointestinal disturbances", ". we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 10  month later . a 76  years - old man with 6  month history of continuous left - sided headache and facial pain .", "after 2  weeks on indometacina he complaint about pyrosis , that persists after omeprazol 40  mg per day .", "we decided to discontinue indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 6  month later .", "a 42  years - old woman had sudden onset of short - lived pains on the left side of her head .", "she suffers for a month an intense headache with short attacks ( about 510  min ) but high frequency ( 1018 attacks per day ) .", "we put her on indomethacin 25  mg 3  times a day , and in 24  h her hemicranial pain completely disappears . because of legs oedema we discontinued indomethacin and 1  week later the pain returned .", "a 56  years - old man present with an 8  month history of a continuous left - sided headache strictly unilateral .", "the pain was moderate in intensity but fluctuating ( between 2 and 7/10 vas ) .", "the pain exacerbations were associated with lacrimation but not phono , photofobia , nausea or vomiting .", "an mri was normal and indomethacin 25  mg 3  times a day was started with completely recovery .", "two - month later he started to have gastric symptoms , indomethacin was discontinued and the hemicrania return .", "with celecoxib 200  mg twice a day the hemicrania disappears . after 10  month", "a 78  years - old woman with 1  year history of continuous and strictly left - sided headache , fluctuating in intensity ( 39 in vas ) and accompanied by conjuntival injection and nasal congestion during exacerbation time .", "with indomethacin 50  mg 3  times a day the hemicrania disappear , however , she developed disabling subjective tinnitus 1  month later .", "we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 18  month later .", "a 64  years - old woman with 3  years history of right - sided headache , fluctuating in intensity ( 28 in vas ) .", "with indomethacin 25  mg 3  times a day the hemicrania disappear , however , the patient suffer gastrointestinal disturbances .", "we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 10  month later .", "a 76  years - old man with 6  month history of continuous left - sided headache and facial pain .", "after 2  weeks on indometacina he complaint about pyrosis , that persists after omeprazol 40  mg per day .", "we decided to discontinue indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 6  month later .", "we present five patients suffering indomethacin - responsive headache case 1 suffering from paroxysmal hemicranias and cases from 2 to 5 from hemicranias continua ( table  1 ) .", "all of them have absolutely response to indomethacin , but the presence of adverse reactions or intolerability let us discontinued and introduce celecoxib with completely recovery of the symptoms.table  1clinical characteristics of the patientscaseagegenderheadache typesidedoses ( mg)time in month142fhpleft20012256mhcleft40010378fhcleft40018464fhcright40010576mhcleft4006all doses correspond to celecoxibf female , m male , hc hemicrania continua , hp hemicrania paroxistica clinical characteristics of the patients all doses correspond to celecoxib f female , m male , hc hemicrania continua , hp hemicrania paroxistica indomethacin is consider the first - choice drug for the treatment of  indomethacin - responsive headaches  , and the headache resolution is consider as a diagnosis criteria in some of theses headaches .", "although other drugs have been reported as useful treatment , most of them are anecdotic or single cases [ 915 ] .", "other anti - inflammatory drugs that have been demonstrated as alternative drugs , in particular piroxicam , however , their efficacy is lower when compared with indomethacin .", "the pathophysiology of theses  indomethacin - response headache  are still unknown , but the cyclo - oxygenase ( cox)-2 should be implicated in the pathogenesis .", "celecoxib have been previously reported to be effective in the treatment of other indomethacin - responsive headache [ 311 ] , however , there was no absolutely response in all patients .", "anyhow , in our experience celecoxib is a good option treatment for patients suffering from hemicrania continua or chronic paroxysmal hemicranea that presents indomethacin adverse effects ."], "abstract_text": ["<S> paroxysmal hemicrania and hemicrania continua are both indomethacin - responsive headaches . </S>", "<S> although indomethacin use to be well tolerated , some patients developed gastrointestinal side effects . </S>", "<S> we report four cases of hemicrania continua and a patient suffering chronic paroxysmal hemicrania completely responsive to celecoxib . in our experience celecoxib </S>", "<S> is a good option treatment for patients suffering from hemicrania continua or chronic paroxysmal hemicranea that presents indomethacin adverse effects . </S>"], "labels": null, "section_names": ["Introduction", "Case reports", "Case 1", "Case 2", "Case 3", "Case 4", "Case 5", "Discussion"], "sections": [["although indomethacin is usually well tolerated , some patients developed gastrointestinal side effects , specially those patients who require long term therapy", ". therefore , the use of cyclo - oxygenase-2 specific inhibitors could reduce secondary effects and they are essentially equipotent to indomethacin in vitro and in vivo .", "indeed , some reports in the literature indicate the usefulness of the cox-2 inhibitors in the treatment of indomethacin - responsive headaches [ 38 ] .", "we report four cases of hemicrania continua and a patient suffering chronic paroxysmal hemicrania completely responsive to cox-2 inhibitors ."], ["a 42  years - old woman had sudden onset of short - lived pains on the left side of her head .", "she suffers for a month an intense headache with short attacks ( about 510  min ) but high frequency ( 1018 attacks per day ) .", "we put her on indomethacin 25  mg 3  times a day , and in 24  h her hemicranial pain completely disappears . because of legs oedema we discontinued indomethacin and 1  week later the pain returned .", "a 56  years - old man present with an 8  month history of a continuous left - sided headache strictly unilateral .", "the pain was moderate in intensity but fluctuating ( between 2 and 7/10 vas ) .", "the pain exacerbations were associated with lacrimation but not phono , photofobia , nausea or vomiting .", "an mri was normal and indomethacin 25  mg 3  times a day was started with completely recovery .", "two - month later he started to have gastric symptoms , indomethacin was discontinued and the hemicrania return .", "a 78  years - old woman with 1  year history of continuous and strictly left - sided headache , fluctuating in intensity ( 39 in vas ) and accompanied by conjuntival injection and nasal congestion during exacerbation time .", "with indomethacin 50  mg 3  times a day the hemicrania disappear , however , she developed disabling subjective tinnitus 1  month later .", "we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 18  month later .", "a 64  years - old woman with 3  years history of right - sided headache , fluctuating in intensity ( 28 in vas ) .", "with indomethacin 25  mg 3  times a day the hemicrania disappear , however , the patient suffer gastrointestinal disturbances", ". we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 10  month later . a 76  years - old man with 6  month history of continuous left - sided headache and facial pain .", "after 2  weeks on indometacina he complaint about pyrosis , that persists after omeprazol 40  mg per day .", "we decided to discontinue indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 6  month later ."], ["a 42  years - old woman had sudden onset of short - lived pains on the left side of her head .", "she suffers for a month an intense headache with short attacks ( about 510  min ) but high frequency ( 1018 attacks per day ) .", "we put her on indomethacin 25  mg 3  times a day , and in 24  h her hemicranial pain completely disappears . because of legs oedema we discontinued indomethacin and 1  week later the pain returned ."], ["a 56  years - old man present with an 8  month history of a continuous left - sided headache strictly unilateral .", "the pain was moderate in intensity but fluctuating ( between 2 and 7/10 vas ) .", "the pain exacerbations were associated with lacrimation but not phono , photofobia , nausea or vomiting .", "an mri was normal and indomethacin 25  mg 3  times a day was started with completely recovery .", "two - month later he started to have gastric symptoms , indomethacin was discontinued and the hemicrania return .", "with celecoxib 200  mg twice a day the hemicrania disappears . after 10  month"], ["a 78  years - old woman with 1  year history of continuous and strictly left - sided headache , fluctuating in intensity ( 39 in vas ) and accompanied by conjuntival injection and nasal congestion during exacerbation time .", "with indomethacin 50  mg 3  times a day the hemicrania disappear , however , she developed disabling subjective tinnitus 1  month later .", "we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 18  month later ."], ["a 64  years - old woman with 3  years history of right - sided headache , fluctuating in intensity ( 28 in vas ) .", "with indomethacin 25  mg 3  times a day the hemicrania disappear , however , the patient suffer gastrointestinal disturbances .", "we discontinued indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 10  month later ."], ["a 76  years - old man with 6  month history of continuous left - sided headache and facial pain .", "after 2  weeks on indometacina he complaint about pyrosis , that persists after omeprazol 40  mg per day .", "we decided to discontinue indomethacin and introduced celecoxib 200  mg twice a day with completely recovery that persists 6  month later ."], ["we present five patients suffering indomethacin - responsive headache case 1 suffering from paroxysmal hemicranias and cases from 2 to 5 from hemicranias continua ( table  1 ) .", "all of them have absolutely response to indomethacin , but the presence of adverse reactions or intolerability let us discontinued and introduce celecoxib with completely recovery of the symptoms.table  1clinical characteristics of the patientscaseagegenderheadache typesidedoses ( mg)time in month142fhpleft20012256mhcleft40010378fhcleft40018464fhcright40010576mhcleft4006all doses correspond to celecoxibf female , m male , hc hemicrania continua , hp hemicrania paroxistica clinical characteristics of the patients all doses correspond to celecoxib f female , m male , hc hemicrania continua , hp hemicrania paroxistica indomethacin is consider the first - choice drug for the treatment of  indomethacin - responsive headaches  , and the headache resolution is consider as a diagnosis criteria in some of theses headaches .", "although other drugs have been reported as useful treatment , most of them are anecdotic or single cases [ 915 ] .", "other anti - inflammatory drugs that have been demonstrated as alternative drugs , in particular piroxicam , however , their efficacy is lower when compared with indomethacin .", "the pathophysiology of theses  indomethacin - response headache  are still unknown , but the cyclo - oxygenase ( cox)-2 should be implicated in the pathogenesis .", "celecoxib have been previously reported to be effective in the treatment of other indomethacin - responsive headache [ 311 ] , however , there was no absolutely response in all patients .", "anyhow , in our experience celecoxib is a good option treatment for patients suffering from hemicrania continua or chronic paroxysmal hemicranea that presents indomethacin adverse effects ."]]}
{"article_id": "PMC4993942", "article_text": ["according to early treatment of diabetic retinopathy study ( etdrs ) , protocol panretinal photocoagulation ( prp ) can prevent severe visual loss in proliferative diabetic retinopathy ( pdr ) .", "several studies have shown histopathologic changes in choroid of diabetic patients [ 24 ] . meanwhile , using panretinal photocoagulation ( prp ) , as was suggested beneficially by diabetic retinopathy study for proliferative diabetic retinopathy , has gotten general acceptance . with the advent of enhanced depth imaging optical coherence tomography ( edi - oct ) , it is now possible to visualize and measure choroidal changes easily .", "to the best of our knowledge , no other study has compared these two laser modalities in changing choroidal or central retinal thickness . in this study , we have evaluated the subfoveal choroidal , central retinal , and peripapillary retinal nerve fiber layer ( rnfl ) thickness changes before and after prp using edi - oct and we compare the green and red laser in this regard .", "this clinical trial was approved by the institutional review board / ethics committee . written informed consents were obtained from all participants .", "exclusion criteria were advanced proliferative diabetic retinopathy , a history of any laser treatment ( panretinal or focal laser photocoagulation ) , any history of intravitreal drug injection , ocular surgery , significant media opacity , myopia , or hyperopia with refractive error of more than 3 diopters .", "primary objective was to determine subfoveal choroidal and retinal thickness and peripapillary nerve fiber layer thickness changes 6 weeks after red versus prp laser . a complete ophthalmic examination including best corrected visual acuity ( bcva ) using snellen chart , goldmann applanation tonometry , and dilated indirect ophthalmoscopy was conducted .", "enhanced depth imaging optical coherence tomography ( edi - oct ) mapping was performed using sd - oct ( spectralis , heidelberg engineering , heidelberg , germany ) .", "a total number of 1600 to 2000 spots per eye were applied to ablate retina using ellex integre pro scan laser photocoagulator ( 82 gilbert street , adelaide , sa 5000 , australia ) .", "the order of treated areas was as follows : nasal , inferior , superior , and then temporal retina .", "power setting was chosen to have a mild gray - white burn ( grade 2 ) according to etdrs guidelines .", "laser setting parameters did not differ significantly between two groups ( mean power 303  mwatts in red and 290  mwatts in green laser group , p value : 0.14 ; mean laser spots 1789 spots in red and 1869 spots in green laser group , p value : 0.13 ) .", "one eye of all patients was randomly assigned to red laser and the other eye to green laser .", "spectral domain optical coherence tomography images were obtained using spectralis oct ( heidelberg engineering , heidelberg , germany ) to measure central retinal thickness and enhanced depth imaging ( edi ) mode to measure subfoveal choroidal thickness .", "bcva measurement and an oct scan were performed at baseline and 6 weeks after completion of prp .", "subfoveal choroidal thickness was measured using apparatus measurement tool at baseline and 6 weeks after prp .", "choroidal segmentation was done manually , moving the reference lines from the retinal borders to the choroidal borders .", "the internal limiting membrane line was moved to the base of the retinal pigment epithelium .", "also retinal nerve fiber layer ( rnfl ) thickness analysis was done at baseline and 6 weeks after treatment .", "data were analyzed using spss software ( version 16 , spss , inc . ) .", "a paired sample t - test was used to compare macula and choroid thickness before and after treatment .", "independent sample t - test was performed to compare macula and choroid thickness changes from baseline between two groups .", "the mean visual acuity of patients was 0.32  0.28  logmar and 0.27  0.23  logmar in red laser group and green laser group , respectively .", "the mean subfoveal ct increased significantly in each group at 6 weeks follow - up ( in red laser group , 202.14  24.5 micron at baseline and 211.7  26.6 micron at 6 weeks , p value < 0.00 , showing 4.86% ct increase compared to baseline ) ( in green group , 201.9  21.13 micron at baseline and 208.9  25.0 at 6 weeks , p  value <", "there was not a significant difference between red and green laser in terms of choroidal thickness changes from baseline ( p value 0.184 ) .", "in red laser group , the mean central retinal thickness was 271.6  30.33 micron at baseline and 298.5  62.14 micron at week 6 of follow - up ( p value 0.03 ) .", "in green laser group , the mean central retinal thickness was 267.0  36.9 micron at baseline and 306.4  73.3 micron at week 6 of follow - up , ( p value 0.000 for both groups ) .", "there was no significant difference between macular thickness increase between two groups ( p value : 0.404 ) ( table 2 ) .", "there was no significant association between choroidal thickness change and retinal thickness change in each group ( p values : 0.051 and 0.52 , resp . ) .", "the mean peripapillary rnfl thickness was 100.5  20.1 micron in red laser group at baseline which increased to 108.2  23.1 micron at 6 weeks ( p value 0.000 ) ( 7.8% as compared to baseline ) .", "in green laser group , it was 103.5  19.8 micron at baseline which increased to 112.7  22.9 in week 6 of follow - up ( p value 0.000 ) ( 9.2% as compared to baseline ) .", "rnfl change between two groups was not significant ( p value : 0.762 ) ( table 2 ) .", "in this study , in eyes with proliferative diabetic retinopathy and without significant macular edema , the mean subfoveal choroidal , central retinal , and peripapillary rnfl thickness increased significantly after both red and green argon laser treatments .", "this is in accordance with some of previously published studies that confirm this finding [ 69 ] .", "takahashi et al . measured changes in choroidal blood flow in the foveal area one month after prp in patients with severe diabetic retinopathy and no macular edema using a laser - doppler flowmetry .", "they reported that prp increases both the choroidal blood flow and the choroidal blood volume .", "changes in subfoveal choroidal thickness did not correlate with changes in macular thickness in their study .", "first , it has been attributed to redistribution of choroidal blood flow [ 1014 ] .", "this leads to redistribution of blood in other untreated areas , especially the macula . following blood flow increase ,", "this transient inflammation probably causes release of cytokines and leads to increase of blood flow and choroidal thickness [ 18 , 19 ] .", "mean ct increase in both groups in this study is similar to what have been reported before in other trials [ 710 ] .", "it is noteworthy that , in some other trials , the measured subfoveal ct showed decreased thickness after prp in short term follow - up .", "they have hypothesized that the reason may be due to the destruction of choroidal vasculature after thermal damage of prp [ 20 , 21 ] .", "also , we found that central macular thickness and rnfl thickness increased significantly in both groups ; this is also similar to what has been found in other studies [ 2024 ] .", "this finding is similar to what has been reported before about the increase of retinal thickness after prp and it was attributed to the release of proinflammatory cytokines or hypoxia - induced macular edema [ 2024 ] .", "also , we did not find any correlation between changes of choroidal thickness and central retinal thickness .", "one of the novel analyses done in this study is the comparison between red and green laser in changing retinal , choroidal , and rnfl thickness .", "although all three measured variables showed significant increase after treatment in each eye , intereye comparisons were not statistically significant considering central retinal , subfoveal choroidal , and rnfl thickness changes ( p values : 0.404 , 0.184 , and 0.762 , resp . ) that may indicate no considerable difference between red and green laser in this regard . to the best of our knowledge , there is only one report by ghassemi et al .   that compared the difference between red and green laser in pdr patients .", "they reported significant increase of rnfl thickness after prp without any difference between red and green laser .", "one of the advantages of this study is selection of both eyes of one patient which can exclude the effect of systemic factors on retinal , choroidal , and rnfl thickness .", "this study has some limitations including low sample size and short follow - up time .", "nonetheless , this study showed significant increase of subfoveal choroidal , foveal retinal , and peripapillary rnfl thickness after red and green laser prp and no significant difference between these two lasers in aforementioned measurements ."], "abstract_text": ["<S> \n purpose . to compare subfoveal choroidal , central retinal , and peripapillary nerve fiber layer ( rnfl ) thickness after panretinal photocoagulation ( prp ) with red and green laser in diabetic patients </S>", "<S> . study design . </S>", "<S> randomized clinical trial . methods . </S>", "<S> a total of 50 patients with bilateral proliferative diabetic retinopathy and no diabetic macular edema underwent prp . </S>", "<S> one eye was randomly assigned to red or green laser . </S>", "<S> subfoveal choroidal , central retinal , and rnfl thicknesses were evaluated at baseline and 6 weeks after treatment . </S>", "<S> results . </S>", "<S> the mean subfoveal choroidal , central retinal , and peripapillary nerve fiber layer ( rnfl ) thickness increased significantly in each eye 6 weeks after prp ( p values in red laser group : < 0.01 , 0.03 , and < 0.01 , resp . , and in green laser group < 0.01 , < 0.01 , and < 0.01 ) </S>", "<S> . there was no difference between red and green laser considering subfoveal choroidal , central retinal , and peripapillary nerve fiber layer ( rnfl ) thickness increase after prp ( p values : 0.184 , 0.404 , and 0.726 , resp . ) . </S>", "<S> conclusion . </S>", "<S> both red and green lasers increased mean subfoveal choroidal , central retinal , and peripapillary nerve fiber layer ( rnfl ) thickness significantly 6 weeks after prp , but there is no difference between these two modalities in this regard . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["according to early treatment of diabetic retinopathy study ( etdrs ) , protocol panretinal photocoagulation ( prp ) can prevent severe visual loss in proliferative diabetic retinopathy ( pdr ) .", "several studies have shown histopathologic changes in choroid of diabetic patients [ 24 ] . meanwhile , using panretinal photocoagulation ( prp ) , as was suggested beneficially by diabetic retinopathy study for proliferative diabetic retinopathy , has gotten general acceptance . with the advent of enhanced depth imaging optical coherence tomography ( edi - oct ) , it is now possible to visualize and measure choroidal changes easily .", "to the best of our knowledge , no other study has compared these two laser modalities in changing choroidal or central retinal thickness . in this study , we have evaluated the subfoveal choroidal , central retinal , and peripapillary retinal nerve fiber layer ( rnfl ) thickness changes before and after prp using edi - oct and we compare the green and red laser in this regard ."], ["this clinical trial was approved by the institutional review board / ethics committee . written informed consents were obtained from all participants .", "exclusion criteria were advanced proliferative diabetic retinopathy , a history of any laser treatment ( panretinal or focal laser photocoagulation ) , any history of intravitreal drug injection , ocular surgery , significant media opacity , myopia , or hyperopia with refractive error of more than 3 diopters .", "primary objective was to determine subfoveal choroidal and retinal thickness and peripapillary nerve fiber layer thickness changes 6 weeks after red versus prp laser . a complete ophthalmic examination including best corrected visual acuity ( bcva ) using snellen chart , goldmann applanation tonometry , and dilated indirect ophthalmoscopy was conducted .", "enhanced depth imaging optical coherence tomography ( edi - oct ) mapping was performed using sd - oct ( spectralis , heidelberg engineering , heidelberg , germany ) .", "a total number of 1600 to 2000 spots per eye were applied to ablate retina using ellex integre pro scan laser photocoagulator ( 82 gilbert street , adelaide , sa 5000 , australia ) .", "the order of treated areas was as follows : nasal , inferior , superior , and then temporal retina .", "power setting was chosen to have a mild gray - white burn ( grade 2 ) according to etdrs guidelines .", "laser setting parameters did not differ significantly between two groups ( mean power 303  mwatts in red and 290  mwatts in green laser group , p value : 0.14 ; mean laser spots 1789 spots in red and 1869 spots in green laser group , p value : 0.13 ) .", "one eye of all patients was randomly assigned to red laser and the other eye to green laser .", "spectral domain optical coherence tomography images were obtained using spectralis oct ( heidelberg engineering , heidelberg , germany ) to measure central retinal thickness and enhanced depth imaging ( edi ) mode to measure subfoveal choroidal thickness .", "bcva measurement and an oct scan were performed at baseline and 6 weeks after completion of prp .", "subfoveal choroidal thickness was measured using apparatus measurement tool at baseline and 6 weeks after prp .", "choroidal segmentation was done manually , moving the reference lines from the retinal borders to the choroidal borders .", "the internal limiting membrane line was moved to the base of the retinal pigment epithelium .", "also retinal nerve fiber layer ( rnfl ) thickness analysis was done at baseline and 6 weeks after treatment .", "data were analyzed using spss software ( version 16 , spss , inc . ) .", "a paired sample t - test was used to compare macula and choroid thickness before and after treatment .", "independent sample t - test was performed to compare macula and choroid thickness changes from baseline between two groups ."], ["the mean visual acuity of patients was 0.32  0.28  logmar and 0.27  0.23  logmar in red laser group and green laser group , respectively .", "the mean subfoveal ct increased significantly in each group at 6 weeks follow - up ( in red laser group , 202.14  24.5 micron at baseline and 211.7  26.6 micron at 6 weeks , p value < 0.00 , showing 4.86% ct increase compared to baseline ) ( in green group , 201.9  21.13 micron at baseline and 208.9  25.0 at 6 weeks , p  value <", "there was not a significant difference between red and green laser in terms of choroidal thickness changes from baseline ( p value 0.184 ) .", "in red laser group , the mean central retinal thickness was 271.6  30.33 micron at baseline and 298.5  62.14 micron at week 6 of follow - up ( p value 0.03 ) .", "in green laser group , the mean central retinal thickness was 267.0  36.9 micron at baseline and 306.4  73.3 micron at week 6 of follow - up , ( p value 0.000 for both groups ) .", "there was no significant difference between macular thickness increase between two groups ( p value : 0.404 ) ( table 2 ) .", "there was no significant association between choroidal thickness change and retinal thickness change in each group ( p values : 0.051 and 0.52 , resp . ) .", "the mean peripapillary rnfl thickness was 100.5  20.1 micron in red laser group at baseline which increased to 108.2  23.1 micron at 6 weeks ( p value 0.000 ) ( 7.8% as compared to baseline ) .", "in green laser group , it was 103.5  19.8 micron at baseline which increased to 112.7  22.9 in week 6 of follow - up ( p value 0.000 ) ( 9.2% as compared to baseline ) .", "rnfl change between two groups was not significant ( p value : 0.762 ) ( table 2 ) ."], ["in this study , in eyes with proliferative diabetic retinopathy and without significant macular edema , the mean subfoveal choroidal , central retinal , and peripapillary rnfl thickness increased significantly after both red and green argon laser treatments .", "this is in accordance with some of previously published studies that confirm this finding [ 69 ] .", "takahashi et al . measured changes in choroidal blood flow in the foveal area one month after prp in patients with severe diabetic retinopathy and no macular edema using a laser - doppler flowmetry .", "they reported that prp increases both the choroidal blood flow and the choroidal blood volume .", "changes in subfoveal choroidal thickness did not correlate with changes in macular thickness in their study .", "first , it has been attributed to redistribution of choroidal blood flow [ 1014 ] .", "this leads to redistribution of blood in other untreated areas , especially the macula . following blood flow increase ,", "this transient inflammation probably causes release of cytokines and leads to increase of blood flow and choroidal thickness [ 18 , 19 ] .", "mean ct increase in both groups in this study is similar to what have been reported before in other trials [ 710 ] .", "it is noteworthy that , in some other trials , the measured subfoveal ct showed decreased thickness after prp in short term follow - up .", "they have hypothesized that the reason may be due to the destruction of choroidal vasculature after thermal damage of prp [ 20 , 21 ] .", "also , we found that central macular thickness and rnfl thickness increased significantly in both groups ; this is also similar to what has been found in other studies [ 2024 ] .", "this finding is similar to what has been reported before about the increase of retinal thickness after prp and it was attributed to the release of proinflammatory cytokines or hypoxia - induced macular edema [ 2024 ] .", "also , we did not find any correlation between changes of choroidal thickness and central retinal thickness .", "one of the novel analyses done in this study is the comparison between red and green laser in changing retinal , choroidal , and rnfl thickness .", "although all three measured variables showed significant increase after treatment in each eye , intereye comparisons were not statistically significant considering central retinal , subfoveal choroidal , and rnfl thickness changes ( p values : 0.404 , 0.184 , and 0.762 , resp . ) that may indicate no considerable difference between red and green laser in this regard . to the best of our knowledge , there is only one report by ghassemi et al .   that compared the difference between red and green laser in pdr patients .", "they reported significant increase of rnfl thickness after prp without any difference between red and green laser .", "one of the advantages of this study is selection of both eyes of one patient which can exclude the effect of systemic factors on retinal , choroidal , and rnfl thickness .", "this study has some limitations including low sample size and short follow - up time .", "nonetheless , this study showed significant increase of subfoveal choroidal , foveal retinal , and peripapillary rnfl thickness after red and green laser prp and no significant difference between these two lasers in aforementioned measurements ."]]}
{"article_id": "PMC3278202", "article_text": ["the prognosis for patients with non - small cell lung cancer ( nsclc ) deteriorates with advancing disease stage , and only about 1% of patients with metastatic nsclc are alive after 5 years.1 the most appropriate treatment for these patients is palliative , systemic , platinum - based chemotherapy in first line.25 however , platinum - based , first - line treatment is limited to 46 cycles due to cumulative toxicity and a plateau in effectiveness . following discontinuation of chemotherapy , most patients will experience disease progression within 23 months.6,7 previous guidelines have recommended withholding second - line treatment until disease progression.2,3 however , cappuzzo et al suggested that 30%50% of patients were not receiving second - line treatment due to rapid disease progression and decreasing performance status.8 hence , there is a need for therapies that prolong the clinical benefits of first - line treatment .", "bevacizumab has been approved for use in patients with metastatic non - squamous nsclc in combination with platinum - based chemotherapy until disease progression.9 however , there remains an unmet need for further treatment options that extend survival in patients with advanced or metastatic nsclc .", "an alternative treatment strategy that can be used to prolong duration of first - line treatment and extend survival in metastatic nsclc is first - line maintenance therapy .", "maintenance therapy is defined as the prolongation of treatment duration or administration of additional treatment at the end of a defined number of initial chemotherapy cycles , after maximum tumor response has been achieved ( this may be complete response , partial response , or stable disease ) .", "erlotinib ( tarceva , f hoffmann - la roche ltd , basel , switzerland ) is one of only two treatments approved for use as first - line maintenance therapy by the european medicines agency,10 the other being pemetrexed.11 the randomized , multicenter , phase iii saturn ( sequential tarceva in unresectable nsclc ) study compared first - line maintenance therapy with either erlotinib or placebo ( n = 487 ) following four cycles of platinum - based chemotherapy in patients with metastatic nsclc.8 patients who had not experienced disease progression after initial chemotherapy were randomized 1:1 to receive erlotinib 150 mg / day orally + best supportive care or placebo + best supportive care until disease progression , unacceptable toxicity , or death.8 erlotinib therapy significantly improved progression - free survival ( hazard ratio [ hr ] : 0.71 ; 95% confidence interval [ ci ] : 0.620.82 ; p < 0.0001 ) and overall survival ( hr : 0.81 , 95% ci : 0.700.95 ; p = 0.0088 ) in the intent - to - treat population ( n = 438)8 and in a subpopulation of patients ( n = 252 ) with stable disease following initial first - line chemotherapy ( progression - free survival hr : 0.68 , 95% ci : 0.560.83 ; p < 0.0001 ; overall survival hr : 0.72 , 95% ci : 0.590.89 ; p = 0.00198 ) .", "the survival benefits observed following erlotinib maintenance therapy in patients with mutated epidermal growth factor receptor and patients with wild - type epidermal growth factor receptor were also achieved without significantly compromising tolerability or health - related quality of life .", "the objective of this analysis was to estimate the cost - effectiveness of erlotinib versus best supportive care when used as first - line maintenance therapy for patients with locally advanced or metastatic nsclc and stable disease following first - line therapy in three european countries , ie , france , germany , and italy .", "to estimate the incremental cost - effectiveness of erlotinib , an economic decision model was developed using patient data on progression - free survival and overall survival for erlotinib and best supportive care as first - line maintenance therapy from the saturn trial.8 an area under the curve ( or partitioned survival ) model was developed consisting of three health states , ie , progression - free survival , progression , and death ( figure 1 ) .", "survival data from the trial were used to follow patients from the progression - free survival to the progression and death states , and allowed for extrapolation of the data beyond the trial period .", "the model uses the stable disease population , as indicated in the european union label,12 to calculate efficacy and the same dose as was used in the trial , ie , erlotinib 150 mg / day orally + best supportive care or placebo + best supportive care .", "the area under the curve model works by assuming that , at any discrete time point , the difference between the proportion of patients in overall survival and the proportion of patients in progression - free survival determines the proportion of patients who have experienced disease progression . at the start of the analysis", ", it was assumed that all patients were in the progression - free survival health state .", "a half - cycle correction is used to account for events that occur during each monthly cycle .", "the time horizon of 5 years can be considered to be a lifetime perspective in this patient population ( after 5 years , virtually all patients will have died ) .", "the model was developed from the perspective of the national health care payer in three european countries , ie , france , germany , and italy ; therefore , indirect costs , including travel costs , and costs to other public agencies were not included .", "probabilistic sensitivity analyses were performed to investigate the impact of changes in the key input parameters and assumptions on the results of the base - case analysis .", "distributions around the following parameters were used to reflect uncertainty in the model ( ie , the probability of erlotinib being cost - effective ) : estimates for the parametric progression - free survival and overall survival functions , and cost and frequency of adverse events .", "adverse events were selected for inclusion in the model by grade of severity and frequency of occurrence by event and treatment arm , according to the following rules : all treatment - related adverse events grade 34 , and adverse events that occurred from the start of the study and 28 days after the last cycle of first - line treatment inclusively .", "the frequency of each event was further categorized into the number of patients experiencing at least one adverse event .", "these values were used for calculating the average number of each adverse event per patient ( eg , probability of a specified adverse event per patient  average number of episodes of adverse event per patient ) .", "the model does not include post - progression - free survival treatments ( following disease progression ) once maintenance therapy is stopped , because it was not designed to incorporate this information .", "therefore , second - line and further - line treatment costs , capturing the various treatment strategies used following progression , were not accounted for within the model .", "this was due to the variation in second - line treatment observed in the saturn study ; uncontrolled patients received multiple therapies after disease progression , which included taxanes ( eg , docetaxel ) , antimetabolites ( eg , pemetrexed ) , anti - neoplastic agents , epidermal growth factor receptor tyrosine kinase inhibitors ( eg , erlotinib ) , and platinum compounds .", "in addition , insufficient data were reported in the saturn study on second - line treatment dosing and duration .", "survival curves were fitted parametrically to the kaplan  meier progression - free survival and overall survival curves to facilitate extrapolation beyond the clinical trial period .", "the gamma function13,14 provided the best fit for overall survival , whereas for progression - free survival the gompertz distribution14 was shown to be the best fit for the stable disease population .", "therefore , these functions were used in all analyses to estimate and extrapolate progression - free survival and overall survival beyond the clinical trial period .", "the cost of erlotinib was incorporated into the model using the country - specific list price of a pack of 30  150 mg tablets (  2130.00 ,  2928.24 , and  1864.57 for france , germany , and italy , respectively ) and was applied in the base - case analysis for the average treatment duration during the clinical trial ( table 1 ) . as per the study protocol", ", patients received a single daily dose of erlotinib 150 mg orally . in order to calculate the monthly cost of erlotinib , the cost per 150 mg tablet (  71.00 ,  83.63 , and  49.10 , respectively ) was multiplied by the average number of days per month ( 30.4 days , table 1 ) . to estimate the resource use associated with administration of erlotinib", ", the appropriate reference costs associated with administration of oral medication in the pharmacy were used ( table 1 ) .", "administration costs were obtained from france,15 germany,16 and italy.17 country - specific estimates of the most likely minimum and maximum costs of treatment - related adverse events were employed to propagate the gamma distribution to express uncertainty in the cost of treating the event.18 the cost values were then applied to the frequency of adverse events.1921", "an area under the curve ( or partitioned survival ) model was developed consisting of three health states , ie , progression - free survival , progression , and death ( figure 1 ) . survival data from the trial were used to follow patients from the progression - free survival to the progression and death states , and allowed for extrapolation of the data beyond the trial period .", "the model uses the stable disease population , as indicated in the european union label,12 to calculate efficacy and the same dose as was used in the trial , ie , erlotinib 150 mg / day orally + best supportive care or placebo + best supportive care .", "the area under the curve model works by assuming that , at any discrete time point , the difference between the proportion of patients in overall survival and the proportion of patients in progression - free survival determines the proportion of patients who have experienced disease progression . at the start of the analysis", ", it was assumed that all patients were in the progression - free survival health state .", "a half - cycle correction is used to account for events that occur during each monthly cycle .", "the time horizon of 5 years can be considered to be a lifetime perspective in this patient population ( after 5 years , virtually all patients will have died ) .", "the model was developed from the perspective of the national health care payer in three european countries , ie , france , germany , and italy ; therefore , indirect costs , including travel costs , and costs to other public agencies were not included .", "probabilistic sensitivity analyses were performed to investigate the impact of changes in the key input parameters and assumptions on the results of the base - case analysis .", "distributions around the following parameters were used to reflect uncertainty in the model ( ie , the probability of erlotinib being cost - effective ) : estimates for the parametric progression - free survival and overall survival functions , and cost and frequency of adverse events .", "adverse events were selected for inclusion in the model by grade of severity and frequency of occurrence by event and treatment arm , according to the following rules : all treatment - related adverse events grade 34 , and adverse events that occurred from the start of the study and 28 days after the last cycle of first - line treatment inclusively .", "the frequency of each event was further categorized into the number of patients experiencing at least one adverse event .", "these values were used for calculating the average number of each adverse event per patient ( eg , probability of a specified adverse event per patient  average number of episodes of adverse event per patient ) .", "the model does not include post - progression - free survival treatments ( following disease progression ) once maintenance therapy is stopped , because it was not designed to incorporate this information .", "therefore , second - line and further - line treatment costs , capturing the various treatment strategies used following progression , were not accounted for within the model .", "this was due to the variation in second - line treatment observed in the saturn study ; uncontrolled patients received multiple therapies after disease progression , which included taxanes ( eg , docetaxel ) , antimetabolites ( eg , pemetrexed ) , anti - neoplastic agents , epidermal growth factor receptor tyrosine kinase inhibitors ( eg , erlotinib ) , and platinum compounds .", "in addition , insufficient data were reported in the saturn study on second - line treatment dosing and duration .", "survival curves were fitted parametrically to the kaplan  meier progression - free survival and overall survival curves to facilitate extrapolation beyond the clinical trial period .", "the gamma function13,14 provided the best fit for overall survival , whereas for progression - free survival the gompertz distribution14 was shown to be the best fit for the stable disease population .", "therefore , these functions were used in all analyses to estimate and extrapolate progression - free survival and overall survival beyond the clinical trial period .", "the cost of erlotinib was incorporated into the model using the country - specific list price of a pack of 30  150 mg tablets (  2130.00 ,  2928.24 , and  1864.57 for france , germany , and italy , respectively ) and was applied in the base - case analysis for the average treatment duration during the clinical trial ( table 1 ) . as per the study protocol , patients received a single daily dose of erlotinib 150 mg orally . in order to calculate the monthly cost of erlotinib , the cost per 150 mg tablet (  71.00 ,  83.63 , and  49.10 , respectively ) was multiplied by the average number of days per month ( 30.4 days , table 1 ) . to estimate the resource use associated with administration of erlotinib ,", "the appropriate reference costs associated with administration of oral medication in the pharmacy were used ( table 1 ) .", "administration costs were obtained from france,15 germany,16 and italy.17 country - specific estimates of the most likely minimum and maximum costs of treatment - related adverse events were employed to propagate the gamma distribution to express uncertainty in the cost of treating the event.18 the cost values were then applied to the frequency of adverse events.1921", "comparison of the total average costs of alternative therapies for first - line maintenance shows that erlotinib was more costly than best supportive care in all three countries .", "treatment with erlotinib in first - line maintenance resulted in a mean life expectancy of 1.39 years ( 16.7 months ) in all countries , compared with a mean 1.11 years ( 13.3 months ) with best supportive care , which represents 0.28 life - years ( 3.4 life - months ) gained with erlotinib versus best supportive care .", "results of the base - case and probabilistic analyses , presented as mean values and corresponding 95% ci for the latter are shown in table 2 . in the base - case analysis ,", "the cost per life - year gained was  39,783 ,  46,931 , and  27,885 in france , germany , and italy , respectively . in the probabilistic analysis , the cost per life - year gained was  39,214 ,  46,816 , and  27,864 , respectively .", "the variation in cost per life - year gained between countries was due to the difference in the total monthly cost of erlotinib , which comprises administration costs , acquisition costs , and costs of adverse events ( table 3).2225 a cost - effectiveness acceptability curve for each country , generated from the probabilistic analysis , is presented in figure 2 .", "these curves show that there is a 50% chance that erlotinib would be cost - effective in first - line maintenance with a willingness to pay approximately  50,000 in france and germany and  40,000 in italy .", "treatment of advanced or metastatic nsclc is often considered costly ; however , the value of a new treatment should incorporate the clinical benefit it provides compared with current treatment options , and the incremental cost of funding the new therapy versus the unmet need . in view of the current poor survival outcomes in metastatic nsclc", ", it is of interest to consider the cost per life - year gained with a new therapy .", "an increase in health care expenditure in cancer care makes cost - effectiveness analysis an important tool for national health care payers.26 this is the first analysis of its kind to present a cross - market analysis of cost per life - year gained for first - line maintenance therapy in patients with locally advanced or metastatic nsclc . in each country , erlotinib resulted in a survival gain when compared with best supportive care .", "the difference between highest and lowest cost per life - year gained across the three countries was  19,000 for both base - case and probabilistic results .", "figure 2 demonstrates that the chance of erlotinib being cost - effective versus best supportive care in first - line maintenance is relatively stable across the three countries , although slightly higher in italy given the same willingness to pay .", "a strength of the model is its transparency , given that the discrete health states closely match the end points measured in the clinical trial .", "the methods of extrapolation are also similar to those commonly suggested by the health economic community,18 as well as the modeling methods widely reported in peer - reviewed publications . despite the strengths of the model , it is important to recognize any associated limitations .", "the model was developed to estimate the cost - effectiveness of treatment in the first - line maintenance setting only .", "this was due to a variety of second - line and further - line treatments reported in the saturn trial and insufficient data reported on treatment dosing and duration .", "pemetrexed , a multitargeted antifolate chemotherapeutic agent , is the only other therapy approved by the european medicines agency for use in first - line maintenance for metastatic nsclc ( in patients with non - squamous disease).11 in the jmen trial,27 pemetrexed was associated with similar efficacy benefits versus best supportive care ( progression - free survival hr : 0.50 , 95% ci : 0.420.61 ; p < 0.0001)27 as erlotinib versus best supportive care in the saturn trial . in a population - matched , indirect comparison analysis comparing erlotinib and pemetrexed as first - line maintenance therapy in metastatic nsclc", ", both treatments were found to be similarly efficacious ( table 3).23 an economic analysis of pemetrexed versus best supportive care in the us market showed that the cost per life - year gained using pemetrexed first - line maintenance therapy versus best supportive care in patients with advanced , non - squamous nsclc was us$122,371 (  86,869).22 the analysis suggests that pemetrexed is not cost - effective compared with best supportive care , given the cost - effectiveness ratios for medication typically reimbursed in the us , while our analysis shows that erlotinib is cost - effective when compared with best supportive care ( table 3 ) .", "however , it is difficult to compare the cost - effectiveness of erlotinib and pemetrexed using the results of these three analyses .", "pemetrexed was only efficacious in a non - squamous population and erlotinib was efficacious in both the intent - to - treat and stable disease populations .", "patient distributions of key characteristics between the saturn and jmen studies were not balanced . the adenocarcinoma population varied by 3% , the number of patients with stage iiib and iv disease varied by 8% between the two trials for both disease stages , and eastern cooperative oncology group performance status varied by 9% for both 0 and 1 .", "patients in the jmen trial had a better prognosis at baseline.8,27 furthermore , drug costs in the us and europe are different .", "if a cost - effectiveness analysis of erlotinib versus pemetrexed were to be performed , the comparable efficacy of erlotinib and pemetrexed derived from the indirect comparison analysis23 would make drug costs the key driver .", "monthly costs of erlotinib and pemetrexed comprising administration , acquisition , and adverse event costs ( table 3 ) derived from a manuscript in press28 ( published in part at a recent scientific conference24,25 ) show erlotinib has lower monthly per - patient treatment costs . based on these costs , a cost - effectiveness analysis of erlotinib versus pemetrexed", "might be expected to show that erlotinib is as efficacious as pemetrexed but less costly .", "however , the results would need to be interpreted with caution , given the imbalance in the key characteristics of patient populations between the saturn and jmen trials.8,27 given that erlotinib is cost - effective versus best supportive care based on this economic analysis , and has efficacy similar to that of pemetrexed at a lower cost , it could become the new standard of care in first - line maintenance therapy in locally advanced and metastatic nsclc .", "in addition to the clinical similarity and economic advantages of erlotinib over pemetrexed , erlotinib may also have other advantages due to a lower incidence of adverse events and an oral , as opposed to intravenous , formulation.8 oral treatment is often considered more convenient and is not usually associated with any administration costs .", "erlotinib is a cost - effective treatment option versus best supportive care when used as first - line maintenance therapy for locally advanced or metastatic nsclc in patients with stable disease .", "this study suggests that , in conjunction with toxicity and efficacy data , economic analyses are useful in defining the best maintenance strategy for patients with locally advanced or metastatic nsclc ."], "abstract_text": ["<S> backgroundplatinum - doublet , first - line treatment of locally advanced or metastatic non - small cell lung cancer ( nsclc ) is limited to 46 cycles . </S>", "<S> an alternative strategy used to prolong the duration of first - line treatment and extend survival in metastatic nsclc is first - line maintenance therapy . </S>", "<S> erlotinib was approved for first - line maintenance in a stable disease population following results from a randomized , controlled phase iii trial comparing erlotinib with best supportive care . </S>", "<S> we aimed to estimate the incremental cost - effectiveness of erlotinib 150 mg / day versus best supportive care when used as first - line maintenance therapy for patients with locally advanced or metastatic nsclc and stable disease.methodsan economic decision model was developed using patient - level data for progression - free survival and overall survival from the saturn ( sequential tarceva in unresectable nsclc ) study . </S>", "<S> an area under the curve model was developed ; all patients entered the model in the progression - free survival health state and , after each month , moved to progression or death . </S>", "<S> a time horizon of 5 years was used . </S>", "<S> the model was conducted from the perspective of national health care payers in france , germany , and italy . </S>", "<S> probabilistic sensitivity analyses were performed.resultstreatment with erlotinib in first - line maintenance resulted in a mean life expectancy of 1.39 years in all countries , compared with a mean 1.11 years with best supportive care , which represents 0.28 life - years ( 3.4 life - months ) gained with erlotinib versus best supportive care . in the base - case analysis , </S>", "<S> the cost per life - year gained was  39,783 ,  46,931 , and  27,885 in france , germany , and italy , respectively.conclusionerlotinib is a cost - effective treatment option when used as first - line maintenance therapy for locally advanced or metastatic nsclc . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Model structure", "Model inputs", "Results", "Discussion", "Conclusion"], "sections": [["the prognosis for patients with non - small cell lung cancer ( nsclc ) deteriorates with advancing disease stage , and only about 1% of patients with metastatic nsclc are alive after 5 years.1 the most appropriate treatment for these patients is palliative , systemic , platinum - based chemotherapy in first line.25 however , platinum - based , first - line treatment is limited to 46 cycles due to cumulative toxicity and a plateau in effectiveness . following discontinuation of chemotherapy , most patients will experience disease progression within 23 months.6,7 previous guidelines have recommended withholding second - line treatment until disease progression.2,3 however , cappuzzo et al suggested that 30%50% of patients were not receiving second - line treatment due to rapid disease progression and decreasing performance status.8 hence , there is a need for therapies that prolong the clinical benefits of first - line treatment .", "bevacizumab has been approved for use in patients with metastatic non - squamous nsclc in combination with platinum - based chemotherapy until disease progression.9 however , there remains an unmet need for further treatment options that extend survival in patients with advanced or metastatic nsclc .", "an alternative treatment strategy that can be used to prolong duration of first - line treatment and extend survival in metastatic nsclc is first - line maintenance therapy .", "maintenance therapy is defined as the prolongation of treatment duration or administration of additional treatment at the end of a defined number of initial chemotherapy cycles , after maximum tumor response has been achieved ( this may be complete response , partial response , or stable disease ) .", "erlotinib ( tarceva , f hoffmann - la roche ltd , basel , switzerland ) is one of only two treatments approved for use as first - line maintenance therapy by the european medicines agency,10 the other being pemetrexed.11 the randomized , multicenter , phase iii saturn ( sequential tarceva in unresectable nsclc ) study compared first - line maintenance therapy with either erlotinib or placebo ( n = 487 ) following four cycles of platinum - based chemotherapy in patients with metastatic nsclc.8 patients who had not experienced disease progression after initial chemotherapy were randomized 1:1 to receive erlotinib 150 mg / day orally + best supportive care or placebo + best supportive care until disease progression , unacceptable toxicity , or death.8 erlotinib therapy significantly improved progression - free survival ( hazard ratio [ hr ] : 0.71 ; 95% confidence interval [ ci ] : 0.620.82 ; p < 0.0001 ) and overall survival ( hr : 0.81 , 95% ci : 0.700.95 ; p = 0.0088 ) in the intent - to - treat population ( n = 438)8 and in a subpopulation of patients ( n = 252 ) with stable disease following initial first - line chemotherapy ( progression - free survival hr : 0.68 , 95% ci : 0.560.83 ; p < 0.0001 ; overall survival hr : 0.72 , 95% ci : 0.590.89 ; p = 0.00198 ) .", "the survival benefits observed following erlotinib maintenance therapy in patients with mutated epidermal growth factor receptor and patients with wild - type epidermal growth factor receptor were also achieved without significantly compromising tolerability or health - related quality of life .", "the objective of this analysis was to estimate the cost - effectiveness of erlotinib versus best supportive care when used as first - line maintenance therapy for patients with locally advanced or metastatic nsclc and stable disease following first - line therapy in three european countries , ie , france , germany , and italy ."], ["to estimate the incremental cost - effectiveness of erlotinib , an economic decision model was developed using patient data on progression - free survival and overall survival for erlotinib and best supportive care as first - line maintenance therapy from the saturn trial.8 an area under the curve ( or partitioned survival ) model was developed consisting of three health states , ie , progression - free survival , progression , and death ( figure 1 ) .", "survival data from the trial were used to follow patients from the progression - free survival to the progression and death states , and allowed for extrapolation of the data beyond the trial period .", "the model uses the stable disease population , as indicated in the european union label,12 to calculate efficacy and the same dose as was used in the trial , ie , erlotinib 150 mg / day orally + best supportive care or placebo + best supportive care .", "the area under the curve model works by assuming that , at any discrete time point , the difference between the proportion of patients in overall survival and the proportion of patients in progression - free survival determines the proportion of patients who have experienced disease progression . at the start of the analysis", ", it was assumed that all patients were in the progression - free survival health state .", "a half - cycle correction is used to account for events that occur during each monthly cycle .", "the time horizon of 5 years can be considered to be a lifetime perspective in this patient population ( after 5 years , virtually all patients will have died ) .", "the model was developed from the perspective of the national health care payer in three european countries , ie , france , germany , and italy ; therefore , indirect costs , including travel costs , and costs to other public agencies were not included .", "probabilistic sensitivity analyses were performed to investigate the impact of changes in the key input parameters and assumptions on the results of the base - case analysis .", "distributions around the following parameters were used to reflect uncertainty in the model ( ie , the probability of erlotinib being cost - effective ) : estimates for the parametric progression - free survival and overall survival functions , and cost and frequency of adverse events .", "adverse events were selected for inclusion in the model by grade of severity and frequency of occurrence by event and treatment arm , according to the following rules : all treatment - related adverse events grade 34 , and adverse events that occurred from the start of the study and 28 days after the last cycle of first - line treatment inclusively .", "the frequency of each event was further categorized into the number of patients experiencing at least one adverse event .", "these values were used for calculating the average number of each adverse event per patient ( eg , probability of a specified adverse event per patient  average number of episodes of adverse event per patient ) .", "the model does not include post - progression - free survival treatments ( following disease progression ) once maintenance therapy is stopped , because it was not designed to incorporate this information .", "therefore , second - line and further - line treatment costs , capturing the various treatment strategies used following progression , were not accounted for within the model .", "this was due to the variation in second - line treatment observed in the saturn study ; uncontrolled patients received multiple therapies after disease progression , which included taxanes ( eg , docetaxel ) , antimetabolites ( eg , pemetrexed ) , anti - neoplastic agents , epidermal growth factor receptor tyrosine kinase inhibitors ( eg , erlotinib ) , and platinum compounds .", "in addition , insufficient data were reported in the saturn study on second - line treatment dosing and duration .", "survival curves were fitted parametrically to the kaplan  meier progression - free survival and overall survival curves to facilitate extrapolation beyond the clinical trial period .", "the gamma function13,14 provided the best fit for overall survival , whereas for progression - free survival the gompertz distribution14 was shown to be the best fit for the stable disease population .", "therefore , these functions were used in all analyses to estimate and extrapolate progression - free survival and overall survival beyond the clinical trial period .", "the cost of erlotinib was incorporated into the model using the country - specific list price of a pack of 30  150 mg tablets (  2130.00 ,  2928.24 , and  1864.57 for france , germany , and italy , respectively ) and was applied in the base - case analysis for the average treatment duration during the clinical trial ( table 1 ) . as per the study protocol", ", patients received a single daily dose of erlotinib 150 mg orally . in order to calculate the monthly cost of erlotinib , the cost per 150 mg tablet (  71.00 ,  83.63 , and  49.10 , respectively ) was multiplied by the average number of days per month ( 30.4 days , table 1 ) . to estimate the resource use associated with administration of erlotinib", ", the appropriate reference costs associated with administration of oral medication in the pharmacy were used ( table 1 ) .", "administration costs were obtained from france,15 germany,16 and italy.17 country - specific estimates of the most likely minimum and maximum costs of treatment - related adverse events were employed to propagate the gamma distribution to express uncertainty in the cost of treating the event.18 the cost values were then applied to the frequency of adverse events.1921"], ["an area under the curve ( or partitioned survival ) model was developed consisting of three health states , ie , progression - free survival , progression , and death ( figure 1 ) . survival data from the trial were used to follow patients from the progression - free survival to the progression and death states , and allowed for extrapolation of the data beyond the trial period .", "the model uses the stable disease population , as indicated in the european union label,12 to calculate efficacy and the same dose as was used in the trial , ie , erlotinib 150 mg / day orally + best supportive care or placebo + best supportive care .", "the area under the curve model works by assuming that , at any discrete time point , the difference between the proportion of patients in overall survival and the proportion of patients in progression - free survival determines the proportion of patients who have experienced disease progression . at the start of the analysis", ", it was assumed that all patients were in the progression - free survival health state .", "a half - cycle correction is used to account for events that occur during each monthly cycle .", "the time horizon of 5 years can be considered to be a lifetime perspective in this patient population ( after 5 years , virtually all patients will have died ) .", "the model was developed from the perspective of the national health care payer in three european countries , ie , france , germany , and italy ; therefore , indirect costs , including travel costs , and costs to other public agencies were not included .", "probabilistic sensitivity analyses were performed to investigate the impact of changes in the key input parameters and assumptions on the results of the base - case analysis .", "distributions around the following parameters were used to reflect uncertainty in the model ( ie , the probability of erlotinib being cost - effective ) : estimates for the parametric progression - free survival and overall survival functions , and cost and frequency of adverse events .", "adverse events were selected for inclusion in the model by grade of severity and frequency of occurrence by event and treatment arm , according to the following rules : all treatment - related adverse events grade 34 , and adverse events that occurred from the start of the study and 28 days after the last cycle of first - line treatment inclusively .", "the frequency of each event was further categorized into the number of patients experiencing at least one adverse event .", "these values were used for calculating the average number of each adverse event per patient ( eg , probability of a specified adverse event per patient  average number of episodes of adverse event per patient ) .", "the model does not include post - progression - free survival treatments ( following disease progression ) once maintenance therapy is stopped , because it was not designed to incorporate this information .", "therefore , second - line and further - line treatment costs , capturing the various treatment strategies used following progression , were not accounted for within the model .", "this was due to the variation in second - line treatment observed in the saturn study ; uncontrolled patients received multiple therapies after disease progression , which included taxanes ( eg , docetaxel ) , antimetabolites ( eg , pemetrexed ) , anti - neoplastic agents , epidermal growth factor receptor tyrosine kinase inhibitors ( eg , erlotinib ) , and platinum compounds .", "in addition , insufficient data were reported in the saturn study on second - line treatment dosing and duration ."], ["survival curves were fitted parametrically to the kaplan  meier progression - free survival and overall survival curves to facilitate extrapolation beyond the clinical trial period .", "the gamma function13,14 provided the best fit for overall survival , whereas for progression - free survival the gompertz distribution14 was shown to be the best fit for the stable disease population .", "therefore , these functions were used in all analyses to estimate and extrapolate progression - free survival and overall survival beyond the clinical trial period .", "the cost of erlotinib was incorporated into the model using the country - specific list price of a pack of 30  150 mg tablets (  2130.00 ,  2928.24 , and  1864.57 for france , germany , and italy , respectively ) and was applied in the base - case analysis for the average treatment duration during the clinical trial ( table 1 ) . as per the study protocol , patients received a single daily dose of erlotinib 150 mg orally . in order to calculate the monthly cost of erlotinib , the cost per 150 mg tablet (  71.00 ,  83.63 , and  49.10 , respectively ) was multiplied by the average number of days per month ( 30.4 days , table 1 ) . to estimate the resource use associated with administration of erlotinib ,", "the appropriate reference costs associated with administration of oral medication in the pharmacy were used ( table 1 ) .", "administration costs were obtained from france,15 germany,16 and italy.17 country - specific estimates of the most likely minimum and maximum costs of treatment - related adverse events were employed to propagate the gamma distribution to express uncertainty in the cost of treating the event.18 the cost values were then applied to the frequency of adverse events.1921"], ["comparison of the total average costs of alternative therapies for first - line maintenance shows that erlotinib was more costly than best supportive care in all three countries .", "treatment with erlotinib in first - line maintenance resulted in a mean life expectancy of 1.39 years ( 16.7 months ) in all countries , compared with a mean 1.11 years ( 13.3 months ) with best supportive care , which represents 0.28 life - years ( 3.4 life - months ) gained with erlotinib versus best supportive care .", "results of the base - case and probabilistic analyses , presented as mean values and corresponding 95% ci for the latter are shown in table 2 . in the base - case analysis ,", "the cost per life - year gained was  39,783 ,  46,931 , and  27,885 in france , germany , and italy , respectively . in the probabilistic analysis , the cost per life - year gained was  39,214 ,  46,816 , and  27,864 , respectively .", "the variation in cost per life - year gained between countries was due to the difference in the total monthly cost of erlotinib , which comprises administration costs , acquisition costs , and costs of adverse events ( table 3).2225 a cost - effectiveness acceptability curve for each country , generated from the probabilistic analysis , is presented in figure 2 .", "these curves show that there is a 50% chance that erlotinib would be cost - effective in first - line maintenance with a willingness to pay approximately  50,000 in france and germany and  40,000 in italy ."], ["treatment of advanced or metastatic nsclc is often considered costly ; however , the value of a new treatment should incorporate the clinical benefit it provides compared with current treatment options , and the incremental cost of funding the new therapy versus the unmet need . in view of the current poor survival outcomes in metastatic nsclc", ", it is of interest to consider the cost per life - year gained with a new therapy .", "an increase in health care expenditure in cancer care makes cost - effectiveness analysis an important tool for national health care payers.26 this is the first analysis of its kind to present a cross - market analysis of cost per life - year gained for first - line maintenance therapy in patients with locally advanced or metastatic nsclc . in each country , erlotinib resulted in a survival gain when compared with best supportive care .", "the difference between highest and lowest cost per life - year gained across the three countries was  19,000 for both base - case and probabilistic results .", "figure 2 demonstrates that the chance of erlotinib being cost - effective versus best supportive care in first - line maintenance is relatively stable across the three countries , although slightly higher in italy given the same willingness to pay .", "a strength of the model is its transparency , given that the discrete health states closely match the end points measured in the clinical trial .", "the methods of extrapolation are also similar to those commonly suggested by the health economic community,18 as well as the modeling methods widely reported in peer - reviewed publications . despite the strengths of the model , it is important to recognize any associated limitations .", "the model was developed to estimate the cost - effectiveness of treatment in the first - line maintenance setting only .", "this was due to a variety of second - line and further - line treatments reported in the saturn trial and insufficient data reported on treatment dosing and duration .", "pemetrexed , a multitargeted antifolate chemotherapeutic agent , is the only other therapy approved by the european medicines agency for use in first - line maintenance for metastatic nsclc ( in patients with non - squamous disease).11 in the jmen trial,27 pemetrexed was associated with similar efficacy benefits versus best supportive care ( progression - free survival hr : 0.50 , 95% ci : 0.420.61 ; p < 0.0001)27 as erlotinib versus best supportive care in the saturn trial . in a population - matched , indirect comparison analysis comparing erlotinib and pemetrexed as first - line maintenance therapy in metastatic nsclc", ", both treatments were found to be similarly efficacious ( table 3).23 an economic analysis of pemetrexed versus best supportive care in the us market showed that the cost per life - year gained using pemetrexed first - line maintenance therapy versus best supportive care in patients with advanced , non - squamous nsclc was us$122,371 (  86,869).22 the analysis suggests that pemetrexed is not cost - effective compared with best supportive care , given the cost - effectiveness ratios for medication typically reimbursed in the us , while our analysis shows that erlotinib is cost - effective when compared with best supportive care ( table 3 ) .", "however , it is difficult to compare the cost - effectiveness of erlotinib and pemetrexed using the results of these three analyses .", "pemetrexed was only efficacious in a non - squamous population and erlotinib was efficacious in both the intent - to - treat and stable disease populations .", "patient distributions of key characteristics between the saturn and jmen studies were not balanced . the adenocarcinoma population varied by 3% , the number of patients with stage iiib and iv disease varied by 8% between the two trials for both disease stages , and eastern cooperative oncology group performance status varied by 9% for both 0 and 1 .", "patients in the jmen trial had a better prognosis at baseline.8,27 furthermore , drug costs in the us and europe are different .", "if a cost - effectiveness analysis of erlotinib versus pemetrexed were to be performed , the comparable efficacy of erlotinib and pemetrexed derived from the indirect comparison analysis23 would make drug costs the key driver .", "monthly costs of erlotinib and pemetrexed comprising administration , acquisition , and adverse event costs ( table 3 ) derived from a manuscript in press28 ( published in part at a recent scientific conference24,25 ) show erlotinib has lower monthly per - patient treatment costs . based on these costs , a cost - effectiveness analysis of erlotinib versus pemetrexed", "might be expected to show that erlotinib is as efficacious as pemetrexed but less costly .", "however , the results would need to be interpreted with caution , given the imbalance in the key characteristics of patient populations between the saturn and jmen trials.8,27 given that erlotinib is cost - effective versus best supportive care based on this economic analysis , and has efficacy similar to that of pemetrexed at a lower cost , it could become the new standard of care in first - line maintenance therapy in locally advanced and metastatic nsclc .", "in addition to the clinical similarity and economic advantages of erlotinib over pemetrexed , erlotinib may also have other advantages due to a lower incidence of adverse events and an oral , as opposed to intravenous , formulation.8 oral treatment is often considered more convenient and is not usually associated with any administration costs ."], ["erlotinib is a cost - effective treatment option versus best supportive care when used as first - line maintenance therapy for locally advanced or metastatic nsclc in patients with stable disease .", "this study suggests that , in conjunction with toxicity and efficacy data , economic analyses are useful in defining the best maintenance strategy for patients with locally advanced or metastatic nsclc ."]]}
{"article_id": "PMC4090994", "article_text": ["spinal anaesthesia has been widely used for caesarean section ( cs ) deliveries because of greater maternal safety , foetal benefits , higher parental satisfaction , and consumer demand . however , to address the problem of limited duration of action and to improve the quality of analgesia , various adjuvants are added intrathecally with local anaesthetics ( la ) .", "the adjuvants gained widespread popularity as they reduce the amount of la and thus the incidence of side - effects .", "clonidine , a selective partial agonist for alpha-2 adrenoreceptors , is an attractive alternative to commonly used opioids , and is known to increase both sensory and motor block of la .", "several studies have shown that clonidine also has antihyperalgesic effect and thus reduces the post - operative analgesic requirement .", "commonly , adjuvants are mixed with la in a single syringe before injecting the drugs intrathecally .", "mixing of these drugs changes the density of both drugs , thus affecting their spread in the cerebrospinal fluid ( csf ) .", "density is known to influence the spread of la , but the effect of adjuvant solution density on its movement in the csf has not been studied extensively .", "therefore , we hypothesised that if we administer la and the adjuvants separately , it may minimise the effect of the changes in densities and also hence their actions . thus , in this study we aimed to compare block characteristics , intra - operative haemodynamics and post - operative pain relief in parturients undergoing cs under subarachnoid block ( sab ) , after administering hyperbaric bupivacaine ( hb ) and clonidine as a mixture in single syringe and sequentially in two syringes .", "after approval by the institutional ethical committee and written informed consent , sixty parturients with singleton pregnancy , american society of anaesthesiologist ( asa ) i and ii physical status , scheduled for elective cs under sab , were enrolled in this single - blind prospective randomised controlled trial .", "patients having multiple pregnancy , intrauterine deaths or known foetal anomaly , severe pregnancy induced hypertension , contraindication to sab , patients on cardiovascular medications and those having history of hypersensitivity to clonidine and la were excluded from the study . using a sealed envelope technique ,", "( n = 30 ) received hyperbaric bupivacaine ( 0.5% ) 10 mg ( 2 ml ) and clonidine 75 mcg ( 0.5 ml ) as a mixture .", "group b ( n = 30 ) received clonidine 75 mcg ( 0.5 ml ) followed by hyperbaric bupivacaine ( 0.5% ) 10 mg ( 2 ml ) in different syringes . for our study", ", the two drugs used were sourced from same company to avoid manufacturer 's difference .", "hyperbaric bupivacaine used was heavy anawint and clonidine used was cloneont manufactured by neon laboratories limited , mumbai .", "patients were kept fasting overnight and antacid prophylaxis with oral ranitidine 150 mg at night and on the morning prior to surgery were given .", "the patients were familiarised with the concept of visual analogue scale ( vas ) for pain assessment with 0 = no pain and 10 = worst possible pain . in the operating room ,", "monitor for heart rate ( hr ) , non - invasive blood pressure , electrocardiography and oxygen saturation ( spo2 ) was connected and baseline parameters were recorded . after establishing 18 gauge venous cannula , patients were pre - loaded with 15 ml / kg of lactated ringer 's solution 15 - 20 min before spinal block . under all aseptic precautions sab was administered with 23 g quincke spinal needle through mid - line approach in sitting position .", "intrathecal ( it ) drug was injected in l3-l4 interspace over 30 s ( including the time for change of syringe in sequential administration ) .", "after the block was performed , the patients were made supine with 15-20 left displacement of uterus until birth of baby by keeping a wedge under the right buttock .", "fluid therapy was maintained with lactated ringer 's solution 10 ml / kg / h .", "an experienced anaesthesiologist who was unaware of the drug given evaluated the spinal block and other physiological parameters .", "haemodynamic parameters such as hr , systolic arterial pressure ( sap ) , diastolic arterial pressure ( dap ) were monitored at every 2 min ( min ) for the first 20 min and then every 5 min subsequently until 75 min or until completion of surgery . any episode of hypotension and bradycardia in 24 h was noted .", "hypotension ( decrease in sap below 90 mmhg or a fall in blood pressure by > 20% of baseline values ) was treated with a rapid infusion of crystalloids ( 200 ml ) and a bolus of ephedrine 5 mg intravenous ( i.v ) was administered if hypotension persisted .", "bradycardia ( hr < 50 beats / min ) was treated with injection atropine 10 mcg / kg i.v .", "the onset of sensory block was assessed by loss of pin prick sensation along the mid clavicular line bilaterally .", "dermatomal level was tested every 2 min after sab until level was stabilised for four consecutive readings .", "the time from it injection to highest sensory level ( maximum block height ) was noted .", "furthermore , level was tested every 30 min until regression from highest level to t10 dermatome was noted .", "degree of motor block was assessed by modified bromage scale as follows ; i  free movement of legs and feet ; ii  just able to flex knees with free movement of feet ; iii ", "unable to flex knees but with free movement of feet ; iv  unable to move legs and feet .", "time to achieve complete motor block ( modified bromage iv ) and its regression to modified bromage i was noted .", "respiration was monitored and respiratory depression was defined as respiratory rate < 10 breaths / min or spo2 < 92% ; oxygen was then supplemented through nasal prongs at 4 l / min .", "sedation score was assessed at the same interval as sensory block until 2 h post - operatively by ramsay sedation score ( rss ) as : level 1  awake , anxious , agitated , restlessness ; level 2  awake , tranquil , co - operative ; level 3  responds to commands ; level 4  asleep , brisk response to stimuli ; level 5 n asleep , sluggish response to stimuli ; level 6  asleep , no response to stimuli .", "intra - operative pain was checked and expressed as vas , whenever the parturients complained of any discomfort or pain .", "duration of effective analgesia was defined as time from it injection till vas was 3 , when rescue analgesia in the form of injection i.v diclofinac sodium 75 mg was administered .", "patients complaining of nausea or having any episode of vomiting were given injection ondansetron 0.15 mg / kg i.v .", ", new - born 's apgar scores were determined by a paediatrician not otherwise involved in the study at 1 , 5 , and 10 min .", "post - operatively any incidence of bradycardia , hypotension , nausea / vomiting , prolonged sedation reported by the recruited post - operative care unit staff was taken into account and managed accordingly .", "the parturients were also interviewed for post - dural puncture headache ( pdph ) , backache , and examined for any neurological deficit .", "power analysis suggested that a sample size of 30 patients / group was required to achieve a power of 80% and a level significance of 0.05 to be able to detect a difference between the groups , based on the assumption that an increase in the mean duration of analgesia by 60 min in sequential group .", "interpretation of the data was carried out and analysed using software microsoft excel and spss version 19 .", "data is represented as mean  standard deviation for continuous data and frequency ( percentage % ) or median ( range ) for non - parametric ( categorical ) data .", "the proportion of adverse effects was compared using chi - square test and level of sedation was compared with the help of wilcoxon test .", "the p < 0.05 was considered significant and p < 0.01 was considered highly significant .", "demographic data in terms of age , height , weight , asa physical status and duration of surgery were comparable in both groups [ table 1 ] .", "the onset time of sensory and motor block and also the highest level of block achieved ( t4 ) were comparable in both groups [ table 2 ] . mean time to reach maximal cephalad sensory block height was significantly less in group b ( 3.21  0.13 min ) than in group m ( 4.43  0.26 min ) and the total duration of analgesia lasted significantly longer in group b ( 474.3  20.79 min ) as compared to group m ( 337  18.22 min ) ( p = 0.000 ) .", "complete motor blockade was achieved earlier in group b ( 4.75  0.40 ) than in group m ( 5.84  0.36 ) ( p = 0.000 ) .", "the resolution time of motor block back to modified bromage i was significantly prolonged in group b ( 292.23  15.24 min ) than in group m ( 189.50  16.31 min ) [ table 2 ] .", "characteristics of sensory and motor block haemodynamic parameters showed that the lowest values of the hr were after 45 min of the administration of sab , but none of the patients had bradycardia [ figure 1 ] .", "there was a significant fall in sap at 2 min and 4 min after administration of sab in both groups [ figure 2 ] .", "a significant fall in dap was seen at 2 , 4 , 6 , and 8 min of administration of sab .", "there was an overall trend of fall in sap and dap in both groups , except during the time intervals of 20 and 25 min ( during delivery of baby ) where there was rise in both sap and dap [ figures 2 and 3 ] .", "the falling trend of arterial blood pressures was more in the group b than in group m. heart rates at different time intervals systolic blood pressure at different time intervals diastolic blood pressure at different time intervals there was no noticeable fall in spo2 in both groups .", "it was observed that only one patient in group m and 3 in group b had sedation score of 4 according to rss .", "intra - operative incidence of hypotension , bradycardia , respiratory depression , nausea / vomiting , dry mouth and additional analgesic requirement are comparable in both groups [ figure 4 ] .", "newborn 's well - being , assessed by the apgar scoring system was observed in both groups .", "the median value was 8 at 1 min , 9 at 5 min and 10 at 10 min in both groups .", "activation of post - synaptic alpha-2 receptors in the substantia gelatinosa of the spinal cord is the presumed mechanism by which clonidine produces analgesia .", "these receptors are located on primary afferent terminals ( both at peripheral and spinal endings ) , on neurons in the superficial lamina of the spinal cord , and within several brainstem nuclei implicated in analgesia , supporting the possibility of analgesic action at peripheral , spinal , and brainstem sites .", "various authors have used different doses of it clonidine ranging from 15 mcg to 300 mcg along with local anaesthetics .", "kaabachi et al . , in their study used 2 mcg / kg of it clonidine and reported extended duration of post - operative analgesia , but with moderate side - effects .", "sethi et al . , used 70 mcg of clonidine and found a significant decrease in mean arterial pressure and hr in clonidine group , but no therapeutic intervention was required for either . since marked decrease in blood pressure", "is observed only with intermediate doses of spinal clonidine ( 150 mcg ) and relative haemodynamic stability is maintained after larger doses ( 300 - 400 mcg ) , we were interested to test a dose of lower range of efficacy . hence , we selected a 75 mcg of preservative free clonidine as an adjuvant for spinal anaesthesia in cs .", "patients scheduled for cs were chosen for the study because it is a well - known fact that visceral discomfort and pain is common occurence in cs under sab .", "the observations and results obtained in the study are based on the assumption that the original densities of hyperbaric bupivacaine and clonidine are lost when they are premixed in a syringe thus , they exert suboptimal actions when compared to their it administration in a sequential manner .", "the above assumption is supported by the work of desai et al . who studied the same effect by adding opioids to la solution intrathecally .", "the densities of the drugs that we used ( hb and clonidine ) were 1.0260 and 0.9930 , respectively . the density of the mixture of 2 ml ( 10 mg ) of hyperbaric bupivacaine and 0.5 ml ( 75 mcg ) clonidine was also estimated and it was found to be 1.0189 . in our study , we observed that the mean onset time of sensory and motor block was similar in both groups .", "however , onset of sensory block does not get any better after a particular dose as supported by a study done by heo et al . who did not report any difference in onset time even after using 150 mcg clonidine .", "the time to reach maximum sensory block height and maximum motor block was significantly less in group b ( sequential drugs ) than in group m ( mixed drugs ) in this study .", "this difference might have existed because of the preferential cephalad spread of clonidine when we administered it through a separate syringe , owing to its hypobaric nature which is lost when the drugs are premixed .", "desai et al . also observed that the time to reach highest level of block was less when morphine and fentanyl were administered sequentially with hb than when given as a mixture . in our study , we found that the mean time taken for sensory block to regress to t10 level was significantly longer in group b ( 240.67  18.47 min ) than in group m ( 153.83  13.11 min ) .", "similarly , the mean duration of analgesia lasted significantly longer in group b ( 474.33  20.79 min ) than in group m ( 337  18.22 min ) , depicting significant prolongation of analgesic effect in the group receiving drugs in a sequential fashion", ". this difference might be due to the fact that injecting clonidine and bupivacaine as a mixture dilutes clonidine and receptor occupancy might decrease leading to less pronounced effect .", "however , if clonidine is administered separately , we expect a greater spread and therefore formation of stronger bonds with the receptor leading to a denser and prolonged block . according to desai et al .", ", dextrose in a hb solution slow the movement of morphine molecules in the csf , reducing the exposure of supraspinal centres to morphine .", "clonidine also being hypobaric drug , acting on both spinal and supraspinal receptors , might exhibit similar properties .", "gray et al . observed that duration of analgesia is increased when it morphine is administered with normal saline ( hypobaric ) than with dextrose saline ( hyperbaric ) .", "clonidine decreases hr by a presynaptic mediated inhibition of nor epinephrine release and by a direct depression of atrioventricular nodal conduction after systemic absorption .", "the maximum fall in the hr when compared to the baseline was 28% in group b , whereas it was only 13% in group m which was statistically significant ( p < 0.001 ) .", "this fall in hr was more pronounced after about 40 - 60 min of administration of sab and toward end of the surgery .", "however , in our study none of the patients had bradycardia . a significant fall in arterial blood pressure after sab", "the fall from baseline sap and dap in group m was 15% and 18% and in group b was 19% and 20% , respectively .", "haemodynamic effects of clonidine after neuraxial or systemic administration begin within 30 min , reach maximum within 1 - 2 h , and last approximately 6 - 8 h after a single injection .", "we observed hypotension in 13% patients in group m and 16% patient in group b. hypotention was managed by i.v fluids and vasopressors were needed for only 1% and 3% parturients in groups m and b , respectively which was comparable in both groups , suggesting that the clonidine groups did not have a higher predisposition for the development of hypotension if administered sequentially . in our study , the level of sedation provided by it clonidine ( rss 2 and 3 ) was not only acceptable , but also beneficial owing to its anxiolytic role .", "none of the patients needed any additional analgesics during the intra - operative period . in line with our observations , benhamou et al . found that when it clonidine was administered with hb , none of patients required additional analgesics to obtain an adequate sensory block .", "none of the patients complained of dry mouth . in the post - operative period one patient each in group m and b developed pdph , which was managed conservatively .", "there was no incidence of hypotension , bradycardia and nausea / vomiting , neurological deficit , prolonged sedation in the post - operative period .", "neves et al . also concluded that addition of it clonidine did not adversely affect the neonatal outcome in terms of apgar scores .", "the limitation of our study was that we measured the densities of solutions in vitro ; but , we could not measure the densities when injected into the csf .", "sequential administration of clonidine reduces the time to achieve complete sensory and motor block and significantly prolongs the total duration of analgesia .", "addition of clonidine to hyperbaric bupivacaine provided a dense surgical anaesthesia irrespective of the technique of administration .", "however , we noticed that sequential technique did not increase the level of sedation and incidence of hypotension or bradycardia as compared to the administration of drugs as mixture ."], "abstract_text": ["<S> background and aims : mixing adjuvants with hyperbaric bupivacaine in a single syringe before injecting the drugs intrathecally is an age old practice . in doing so , the density of the hyperbaric solution and also of the adjuvant drugs may be altered , thus affecting the spread of drugs . administering local anaesthetic and the adjuvants </S>", "<S> separately may minimise the effect of the changes in densities . </S>", "<S> we aimed to compare block characteristics , intraoperative haemodynamics and post - operative pain relief in parturients undergoing caesarean section ( cs ) after administering hyperbaric bupivacaine and clonidine intrathecally as a mixture and sequentially.methods:in this single - blind prospective randomised controlled study at a tertiary care centre from 2010 to 12 , 60 full - term parturients scheduled for elective css were divided into two groups on the basis of technique of intrathecal drug administration . </S>", "<S> group m received mixture of clonidine ( 75 mcg ) and hyperbaric bupivacaine 0.5% ( 10 mg ) intrathecally , whereas group b received clonidine ( 75 mcg ) followed by hyperbaric bupivacaine 0.5% ( 10 mg ) through separate syringes . </S>", "<S> observational descriptive statistics , analysis of variance test , wilcoxon test and chi - square test were used as applicable.results:duration of analgesia was significantly longer in group b ( 474.33  20.79 min ) in which the drug was given sequentially than in group m ( 337  18.22 min ) . </S>", "<S> furthermore , the time to achieve highest sensory block and complete motor block was significantly less in group b without any major haemodynamic instability and neonatal outcome.conclusions:when clonidine and hyperbaric bupivacaine were administered in a sequential manner , block characteristics improved significantly compared to the administration of the mixture of the two drugs . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["spinal anaesthesia has been widely used for caesarean section ( cs ) deliveries because of greater maternal safety , foetal benefits , higher parental satisfaction , and consumer demand . however , to address the problem of limited duration of action and to improve the quality of analgesia , various adjuvants are added intrathecally with local anaesthetics ( la ) .", "the adjuvants gained widespread popularity as they reduce the amount of la and thus the incidence of side - effects .", "clonidine , a selective partial agonist for alpha-2 adrenoreceptors , is an attractive alternative to commonly used opioids , and is known to increase both sensory and motor block of la .", "several studies have shown that clonidine also has antihyperalgesic effect and thus reduces the post - operative analgesic requirement .", "commonly , adjuvants are mixed with la in a single syringe before injecting the drugs intrathecally .", "mixing of these drugs changes the density of both drugs , thus affecting their spread in the cerebrospinal fluid ( csf ) .", "density is known to influence the spread of la , but the effect of adjuvant solution density on its movement in the csf has not been studied extensively .", "therefore , we hypothesised that if we administer la and the adjuvants separately , it may minimise the effect of the changes in densities and also hence their actions . thus , in this study we aimed to compare block characteristics , intra - operative haemodynamics and post - operative pain relief in parturients undergoing cs under subarachnoid block ( sab ) , after administering hyperbaric bupivacaine ( hb ) and clonidine as a mixture in single syringe and sequentially in two syringes ."], ["after approval by the institutional ethical committee and written informed consent , sixty parturients with singleton pregnancy , american society of anaesthesiologist ( asa ) i and ii physical status , scheduled for elective cs under sab , were enrolled in this single - blind prospective randomised controlled trial .", "patients having multiple pregnancy , intrauterine deaths or known foetal anomaly , severe pregnancy induced hypertension , contraindication to sab , patients on cardiovascular medications and those having history of hypersensitivity to clonidine and la were excluded from the study . using a sealed envelope technique ,", "( n = 30 ) received hyperbaric bupivacaine ( 0.5% ) 10 mg ( 2 ml ) and clonidine 75 mcg ( 0.5 ml ) as a mixture .", "group b ( n = 30 ) received clonidine 75 mcg ( 0.5 ml ) followed by hyperbaric bupivacaine ( 0.5% ) 10 mg ( 2 ml ) in different syringes . for our study", ", the two drugs used were sourced from same company to avoid manufacturer 's difference .", "hyperbaric bupivacaine used was heavy anawint and clonidine used was cloneont manufactured by neon laboratories limited , mumbai .", "patients were kept fasting overnight and antacid prophylaxis with oral ranitidine 150 mg at night and on the morning prior to surgery were given .", "the patients were familiarised with the concept of visual analogue scale ( vas ) for pain assessment with 0 = no pain and 10 = worst possible pain . in the operating room ,", "monitor for heart rate ( hr ) , non - invasive blood pressure , electrocardiography and oxygen saturation ( spo2 ) was connected and baseline parameters were recorded . after establishing 18 gauge venous cannula , patients were pre - loaded with 15 ml / kg of lactated ringer 's solution 15 - 20 min before spinal block . under all aseptic precautions sab was administered with 23 g quincke spinal needle through mid - line approach in sitting position .", "intrathecal ( it ) drug was injected in l3-l4 interspace over 30 s ( including the time for change of syringe in sequential administration ) .", "after the block was performed , the patients were made supine with 15-20 left displacement of uterus until birth of baby by keeping a wedge under the right buttock .", "fluid therapy was maintained with lactated ringer 's solution 10 ml / kg / h .", "an experienced anaesthesiologist who was unaware of the drug given evaluated the spinal block and other physiological parameters .", "haemodynamic parameters such as hr , systolic arterial pressure ( sap ) , diastolic arterial pressure ( dap ) were monitored at every 2 min ( min ) for the first 20 min and then every 5 min subsequently until 75 min or until completion of surgery . any episode of hypotension and bradycardia in 24 h was noted .", "hypotension ( decrease in sap below 90 mmhg or a fall in blood pressure by > 20% of baseline values ) was treated with a rapid infusion of crystalloids ( 200 ml ) and a bolus of ephedrine 5 mg intravenous ( i.v ) was administered if hypotension persisted .", "bradycardia ( hr < 50 beats / min ) was treated with injection atropine 10 mcg / kg i.v .", "the onset of sensory block was assessed by loss of pin prick sensation along the mid clavicular line bilaterally .", "dermatomal level was tested every 2 min after sab until level was stabilised for four consecutive readings .", "the time from it injection to highest sensory level ( maximum block height ) was noted .", "furthermore , level was tested every 30 min until regression from highest level to t10 dermatome was noted .", "degree of motor block was assessed by modified bromage scale as follows ; i  free movement of legs and feet ; ii  just able to flex knees with free movement of feet ; iii ", "unable to flex knees but with free movement of feet ; iv  unable to move legs and feet .", "time to achieve complete motor block ( modified bromage iv ) and its regression to modified bromage i was noted .", "respiration was monitored and respiratory depression was defined as respiratory rate < 10 breaths / min or spo2 < 92% ; oxygen was then supplemented through nasal prongs at 4 l / min .", "sedation score was assessed at the same interval as sensory block until 2 h post - operatively by ramsay sedation score ( rss ) as : level 1  awake , anxious , agitated , restlessness ; level 2  awake , tranquil , co - operative ; level 3  responds to commands ; level 4  asleep , brisk response to stimuli ; level 5 n asleep , sluggish response to stimuli ; level 6  asleep , no response to stimuli .", "intra - operative pain was checked and expressed as vas , whenever the parturients complained of any discomfort or pain .", "duration of effective analgesia was defined as time from it injection till vas was 3 , when rescue analgesia in the form of injection i.v diclofinac sodium 75 mg was administered .", "patients complaining of nausea or having any episode of vomiting were given injection ondansetron 0.15 mg / kg i.v .", ", new - born 's apgar scores were determined by a paediatrician not otherwise involved in the study at 1 , 5 , and 10 min .", "post - operatively any incidence of bradycardia , hypotension , nausea / vomiting , prolonged sedation reported by the recruited post - operative care unit staff was taken into account and managed accordingly .", "the parturients were also interviewed for post - dural puncture headache ( pdph ) , backache , and examined for any neurological deficit .", "power analysis suggested that a sample size of 30 patients / group was required to achieve a power of 80% and a level significance of 0.05 to be able to detect a difference between the groups , based on the assumption that an increase in the mean duration of analgesia by 60 min in sequential group .", "interpretation of the data was carried out and analysed using software microsoft excel and spss version 19 .", "data is represented as mean  standard deviation for continuous data and frequency ( percentage % ) or median ( range ) for non - parametric ( categorical ) data .", "the proportion of adverse effects was compared using chi - square test and level of sedation was compared with the help of wilcoxon test .", "the p < 0.05 was considered significant and p < 0.01 was considered highly significant ."], ["demographic data in terms of age , height , weight , asa physical status and duration of surgery were comparable in both groups [ table 1 ] .", "the onset time of sensory and motor block and also the highest level of block achieved ( t4 ) were comparable in both groups [ table 2 ] . mean time to reach maximal cephalad sensory block height was significantly less in group b ( 3.21  0.13 min ) than in group m ( 4.43  0.26 min ) and the total duration of analgesia lasted significantly longer in group b ( 474.3  20.79 min ) as compared to group m ( 337  18.22 min ) ( p = 0.000 ) .", "complete motor blockade was achieved earlier in group b ( 4.75  0.40 ) than in group m ( 5.84  0.36 ) ( p = 0.000 ) .", "the resolution time of motor block back to modified bromage i was significantly prolonged in group b ( 292.23  15.24 min ) than in group m ( 189.50  16.31 min ) [ table 2 ] .", "characteristics of sensory and motor block haemodynamic parameters showed that the lowest values of the hr were after 45 min of the administration of sab , but none of the patients had bradycardia [ figure 1 ] .", "there was a significant fall in sap at 2 min and 4 min after administration of sab in both groups [ figure 2 ] .", "a significant fall in dap was seen at 2 , 4 , 6 , and 8 min of administration of sab .", "there was an overall trend of fall in sap and dap in both groups , except during the time intervals of 20 and 25 min ( during delivery of baby ) where there was rise in both sap and dap [ figures 2 and 3 ] .", "the falling trend of arterial blood pressures was more in the group b than in group m. heart rates at different time intervals systolic blood pressure at different time intervals diastolic blood pressure at different time intervals there was no noticeable fall in spo2 in both groups .", "it was observed that only one patient in group m and 3 in group b had sedation score of 4 according to rss .", "intra - operative incidence of hypotension , bradycardia , respiratory depression , nausea / vomiting , dry mouth and additional analgesic requirement are comparable in both groups [ figure 4 ] .", "newborn 's well - being , assessed by the apgar scoring system was observed in both groups .", "the median value was 8 at 1 min , 9 at 5 min and 10 at 10 min in both groups ."], ["activation of post - synaptic alpha-2 receptors in the substantia gelatinosa of the spinal cord is the presumed mechanism by which clonidine produces analgesia .", "these receptors are located on primary afferent terminals ( both at peripheral and spinal endings ) , on neurons in the superficial lamina of the spinal cord , and within several brainstem nuclei implicated in analgesia , supporting the possibility of analgesic action at peripheral , spinal , and brainstem sites .", "various authors have used different doses of it clonidine ranging from 15 mcg to 300 mcg along with local anaesthetics .", "kaabachi et al . , in their study used 2 mcg / kg of it clonidine and reported extended duration of post - operative analgesia , but with moderate side - effects .", "sethi et al . , used 70 mcg of clonidine and found a significant decrease in mean arterial pressure and hr in clonidine group , but no therapeutic intervention was required for either . since marked decrease in blood pressure", "is observed only with intermediate doses of spinal clonidine ( 150 mcg ) and relative haemodynamic stability is maintained after larger doses ( 300 - 400 mcg ) , we were interested to test a dose of lower range of efficacy . hence , we selected a 75 mcg of preservative free clonidine as an adjuvant for spinal anaesthesia in cs .", "patients scheduled for cs were chosen for the study because it is a well - known fact that visceral discomfort and pain is common occurence in cs under sab .", "the observations and results obtained in the study are based on the assumption that the original densities of hyperbaric bupivacaine and clonidine are lost when they are premixed in a syringe thus , they exert suboptimal actions when compared to their it administration in a sequential manner .", "the above assumption is supported by the work of desai et al . who studied the same effect by adding opioids to la solution intrathecally .", "the densities of the drugs that we used ( hb and clonidine ) were 1.0260 and 0.9930 , respectively . the density of the mixture of 2 ml ( 10 mg ) of hyperbaric bupivacaine and 0.5 ml ( 75 mcg ) clonidine was also estimated and it was found to be 1.0189 . in our study , we observed that the mean onset time of sensory and motor block was similar in both groups .", "however , onset of sensory block does not get any better after a particular dose as supported by a study done by heo et al . who did not report any difference in onset time even after using 150 mcg clonidine .", "the time to reach maximum sensory block height and maximum motor block was significantly less in group b ( sequential drugs ) than in group m ( mixed drugs ) in this study .", "this difference might have existed because of the preferential cephalad spread of clonidine when we administered it through a separate syringe , owing to its hypobaric nature which is lost when the drugs are premixed .", "desai et al . also observed that the time to reach highest level of block was less when morphine and fentanyl were administered sequentially with hb than when given as a mixture . in our study , we found that the mean time taken for sensory block to regress to t10 level was significantly longer in group b ( 240.67  18.47 min ) than in group m ( 153.83  13.11 min ) .", "similarly , the mean duration of analgesia lasted significantly longer in group b ( 474.33  20.79 min ) than in group m ( 337  18.22 min ) , depicting significant prolongation of analgesic effect in the group receiving drugs in a sequential fashion", ". this difference might be due to the fact that injecting clonidine and bupivacaine as a mixture dilutes clonidine and receptor occupancy might decrease leading to less pronounced effect .", "however , if clonidine is administered separately , we expect a greater spread and therefore formation of stronger bonds with the receptor leading to a denser and prolonged block . according to desai et al .", ", dextrose in a hb solution slow the movement of morphine molecules in the csf , reducing the exposure of supraspinal centres to morphine .", "clonidine also being hypobaric drug , acting on both spinal and supraspinal receptors , might exhibit similar properties .", "gray et al . observed that duration of analgesia is increased when it morphine is administered with normal saline ( hypobaric ) than with dextrose saline ( hyperbaric ) .", "clonidine decreases hr by a presynaptic mediated inhibition of nor epinephrine release and by a direct depression of atrioventricular nodal conduction after systemic absorption .", "the maximum fall in the hr when compared to the baseline was 28% in group b , whereas it was only 13% in group m which was statistically significant ( p < 0.001 ) .", "this fall in hr was more pronounced after about 40 - 60 min of administration of sab and toward end of the surgery .", "however , in our study none of the patients had bradycardia . a significant fall in arterial blood pressure after sab", "the fall from baseline sap and dap in group m was 15% and 18% and in group b was 19% and 20% , respectively .", "haemodynamic effects of clonidine after neuraxial or systemic administration begin within 30 min , reach maximum within 1 - 2 h , and last approximately 6 - 8 h after a single injection .", "we observed hypotension in 13% patients in group m and 16% patient in group b. hypotention was managed by i.v fluids and vasopressors were needed for only 1% and 3% parturients in groups m and b , respectively which was comparable in both groups , suggesting that the clonidine groups did not have a higher predisposition for the development of hypotension if administered sequentially . in our study , the level of sedation provided by it clonidine ( rss 2 and 3 ) was not only acceptable , but also beneficial owing to its anxiolytic role .", "none of the patients needed any additional analgesics during the intra - operative period . in line with our observations , benhamou et al . found that when it clonidine was administered with hb , none of patients required additional analgesics to obtain an adequate sensory block .", "none of the patients complained of dry mouth . in the post - operative period one patient each in group m and b developed pdph , which was managed conservatively .", "there was no incidence of hypotension , bradycardia and nausea / vomiting , neurological deficit , prolonged sedation in the post - operative period .", "neves et al . also concluded that addition of it clonidine did not adversely affect the neonatal outcome in terms of apgar scores .", "the limitation of our study was that we measured the densities of solutions in vitro ; but , we could not measure the densities when injected into the csf ."], ["sequential administration of clonidine reduces the time to achieve complete sensory and motor block and significantly prolongs the total duration of analgesia .", "addition of clonidine to hyperbaric bupivacaine provided a dense surgical anaesthesia irrespective of the technique of administration .", "however , we noticed that sequential technique did not increase the level of sedation and incidence of hypotension or bradycardia as compared to the administration of drugs as mixture ."]]}
{"article_id": "PMC4897561", "article_text": ["in 2014 , there will be 63,920 new cases of kidney and renal pelvis cancer diagnoses resulting in approximately 13,860 deaths .", "renal cell carcinoma ( rcc ) is one of the most lethal genitourinary cancers and comprises 85% of these new diagnoses .", "the incidence of rcc has steadily increased over the past few decades due to incidentally discovered small renal masses ( srm ) on radiographic imaging resulting in stage migration to clinical t1a disease from more advanced disease .", "numerous treatment options exist for clinical t1a disease , including total nephrectomy , partial nephrectomy , radiofrequency ablation , cryoablation , and high - intensity focused ultrasound ( hifu ) . in 2009 ,", "the american urologic association ( aua ) published the  guideline for the clinical stage 1 renal mass  which concluded that partial nephrectomy is the standard of care for clinical t1a disease suggesting that the open approach is preferred over minimally invasive techniques .", "in experienced hands , laparoscopic partial nephrectomy ( lpn ) is associated with similar oncologic outcomes to open partial nephrectomy ( opn ) with the added benefit of decreased narcotic use , decreased hospital stay , and earlier convalescence [ 57 ] .", "robotic partial nephrectomy ( rpn ) and lpn have also been compared with similar outcomes , if not superior rpn outcomes , in the hands of an experienced minimally invasive surgeon .", "most studies report the outcomes of either ( a ) single surgeon and single and/or dual approach or ( b ) multicenter , multisurgeon , and dual approach .", "to our knowledge , there have been no reports of a single surgeon 's experience with each of the three modalities .", "our hypothesis is that when performed by a single surgeon , open , laparoscopic , and robotic partial nephrectomy could differ in postoperative complication rates and overall kidney function .", "thus , we report the perioperative outcomes of 106 consecutive patients treated with partial nephrectomy by a single surgeon using all three surgical approaches  opn , lpn , and rpn .", "after obtaining institutional review board approval , a retrospective chart review of a prospectively collected kidney cancer database was performed . from august 2006 to february 2012 , a single surgeon ( rm ) with minimally invasive and urologic oncology fellowship training performed 106 consecutive partial nephrectomies using any of the three available surgical modalities  opn , lpn , or rpn .", "the decision for the approach was multifactorial and was based on patient and tumor characteristics .", "initially in the practice , lpn was offered to patients with tumors of favorable size and location .", "opn was reserved for patients who were uninephric , had compromised kidney function , or had large tumors in an unfavorable location . in early 2008 , rpn gradually replaced lpn as the preferred minimally invasive approach .", "preoperative variables and demographics analyzed included age , gender , body mass index ( bmi ) , american society of anesthesiologist ( asa ) score , glomerular filtration rate ( gfr ) , hematocrit ( within 30 days of surgery ) , tumor laterality , and tumor size .", "operative variables analyzed included operative time , estimated blood loss ( ebl ) , complications , pathology ( benign , malignant ) , and margin status .", "postoperative variables included length of stay , discharge hematocrit , transfusion , gfr , 3-month gfr , and 30-day complications .", "the indication for partial nephrectomy was an enhancing renal mass found on cross - sectional abdominal imaging .", "pathology was reviewed by a dedicated genitourinary pathologist for margin status and tumor type . \n", "briefly , patients were positioned in a modified 45-degree flank and an incision was made for access to the retroperitoneum .", "the kidney was dissected free of attachments in the retroperitoneal space and the hilum was identified .", "intravenous mannitol ( 12.5  g ) administration and renal hypothermia ( cold ischemia ) were utilized prior to hilar clamping in all cases .", "the hilum was clamped using the satinsky vascular clamp and the resection of the renal lesion was performed with sharp dissection .", "the collecting system was closed primarily , and individual vessels were identified and controlled using nonabsorbable monofilament suture .", "argon beam coagulation achieved hemostasis prior to reapproximation of the renal parenchyma with surgicel ( ethicon inc , somerville , nj ) bolsters . in all cases ,", "the patient was positioned in a modified flank configuration with a total of four ports .", "once the hilum and mass were identified , mannitol ( 12.5  g ) was administered and the hilum was controlled using a laparoscopic vascular clamp ( warm ischemia ) .", "surgicel ( ethicon inc , somerville , nj ) and floseal ( biosurgery , deerfield , il ) were used for hemostasis and the tumor bed was repaired in a running fashion based on the depth of the lesion .", "the parenchyma was reapproximated with 2.0  v lock suture , surgicel ( ethicon inc , somerville , nj ) bolsters , and secured with hem - o - lok clips . in superficial tumors penetrating less than 1  cm into kidney parenchyma , clamping and/or repair of the base of resection was not performed . \n", "all patients underwent warm ischemia , which was achieved using either laparoscopic bulldog clamps or a vascular clamp .", "similar to the lpn technique , the resection bed was closed in a running fashion using an absorbable suture and the defect was approximated using a surgicel ( ethicon inc , somerville , nj ) bolster in most cases . the statistical package for the social sciences v.19 ( spss inc .", "all p values were one - sided , and p < 0.05 was considered statistically significant .", "categorical variables were compared using a chi - square test and continuous variables were compared using one - way anova .", "among the 106 patients in the study , 23 patients ( 22% ) underwent opn , 48 patients ( 45% ) underwent lpn , and 35 patients ( 33% ) underwent rpn .", "there was no difference in age , gender , bmi , asa score tumor laterality , or tumor size between the three groups ( table 1 ) .", "there was a statistical trend towards lower preoperative hematocrit for opn patients compared to the minimally invasive patients ( opn40  4% , lpn43  4% , rpn42  4% , p = 0.09 ) .", "preoperative gfr was 71  28  ml / min for opn patients , 76  25  ml / min for lpn patients , and 96  37  ml / min for rpn patients ( p = 0.004 ) .", "there was no difference in intraoperative complication rate , tumor pathology , or margin status between the opn , ldn , and rpn patients ( table 2 ) .", "operative time was the shortest in the opn group ( opn163  35  min , lpn227  53  min , rpn244  53  min , p < 0.0001 ) .", "ebl ( opn367  286  ml , lpn163  179  ml , rpn191  173  ml , p <", "all patients who had opn had the kidney cooled for an average time of 8 minutes before excision of the tumor .", "there were no differences in length of stay , blood transfusion , discharge gfr , or 3-month gfr for the three cohorts of patients ( table 3 ) .", "discharge hematocrit was significantly lower for opn patients ( 30  3% ) compared to lpn ( 36  4% ) and rpn ( 35  4% ) patients ( p < 0.0001 ) . upon follow - up", ", the opn group had the highest incidence of 30-day complications ( 30% ) , while the rpn group had the lowest ( 14% , p = 0.008 ) .", "the opn complications included clavien i ( n = 2 ) , clavien ii ( n = 1 ) , clavien iii ( n = 3 ) , and clavien iv ( n = 1 ) . the lpn complications included clavien i ( n = 4 ) , clavien ii ( n", "the rpn complications included clavien i ( n = 1 ) , clavien ii ( n = 1 ) , and clavien iii ( n = 3 ) .", "radical nephrectomy ( rn ) has been the mainstay of treatment for clinical stage i tumors resulting in excellent cancer specific survival , local tumor control , and progression free survival ; however , reports have highlighted a negative impact on renal function and chronic kidney disease ( ckd ) associated with rn .", "this effect was acknowledged in the 2009 aua guideline for the management of small renal masses which concluded that opn should be considered standard of care and that minimally invasive options , such as lpn and rpn , be considered as second line treatment modalities .", "furthermore , recent studies have established that partial nephrectomy ( pn ) provides similar oncological outcomes when compared to rn [ 1214 ] .", "the current study is the first , to our knowledge , which compares all three modalities of pn ( opn , lpn , and rpn ) by a single surgeon .", "multi - institutional studies may represent dissimilar patient cohorts that introduce selection bias and may invoke concerns regarding surgeon variability .", ", there was no randomization or objective criteria for the pn approach ; rather , the trend was for more minimally invasive surgery as the surgeon developed a comfort level with the lpn and rpn approach .", "this is evident by the fact that tumor characteristics for each group are comparable ( laterality , size , pathology , and margin status ) .", "previous single surgeon studies have analyzed rpn versus lpn [ 11 , 1519 ] with the consensus being that rpn offers a shorter learning curve to minimally invasive pn and comparable warm ischemia time , ebl , and length of stay .", "two common criticisms associated with rpn include ( i ) the cost of the robot and ( ii ) increased surgical time .", "although it is difficult to mitigate the cost associated with rpn compared to lpn and opn , the oft - held conception that procedures take significantly longer may be a misconception after achieving the learning curve associated with rpn .", "concluded that rpn achieved similar operative times to lpn after only 5 procedures and the current study corroborates similar operative times between the two approaches ( 244  53 versus 227  53  min ) . in the opn cohort", ", there was a 30-day complication rate of 30% , which was statistically significant compared to the lpn ( 17% ) and rpn ( 14% ) groups , and higher than that reported by other authors .", "gill et al . reported a 16% and 13% postoperative complication rate in their initial comparison of lpn and opn , respectively .", "bhayani and waxman and winfield reported postoperative complication rates of 16% and 18.2% , respectively , in each of their single - surgeon experiences with lpn [ 21 , 22 ] .", "a recent report , which pooled data from 4 institutions considered centers - of - excellence , reported a postoperative complication rate of 14.4% in patients who underwent rpn .", "most complications were clavien grade i / ii and were managed conservatively and affirm the safety of rpn in experienced hands .", "the complication rate was lowest in the rpn group at 14% and is similar to other reports , with ranges of 1020% [ 8 , 15 , 24 ] .", "the primary limitation of the study is the inherent selection bias and patient confounders associated with a retrospective study .", "surgical confounders were minimized as only operations performed by a single surgeon were analyzed . secondly , renorrhaphy differed between the opn and minimally invasive cohorts along with the use of renal cooling in the opn group compared to warm ischemia time for the minimally invasive approaches .", "nephron sparing surgery has emerged as the optimal surgical approach for t1a and in experienced hands t1b kidney masses . by removing surgeon as a confounding variable ,", "although not currently the gold standard for partial nephrectomy , robotic surgery continues to emerge as the future for renal surgery secondary .", "rpn offers less blood loss and fewer postoperative complications than opn and overcomes the technical difficulties of lpn ."], "abstract_text": ["<S> \n objective . to report the perioperative outcomes of patients treated with partial nephrectomy by a single surgeon using three surgical modalities  </S>", "<S> open , laparoscopic , and robotic . methods . between august 2006 and february 2012 </S>", "<S> , 106 consecutive patients underwent open partial nephrectomy ( opn ) ( n = 23 ) , laparoscopic partial nephrectomy ( lpn ) ( n = 48 ) , and robotic partial nephrectomy ( rpn ) ( n = 35 ) by a single surgeon . </S>", "<S> clinical variables , operative parameters , and renal functional outcomes were analyzed . </S>", "<S> results . </S>", "<S> preoperative patient characteristics were similar except for baseline glomerular filtration rate ( gfr ) , which was highest in the rpn group ( p = 0.004 ) . </S>", "<S> surgery time was longest in the rpn group ( 244 minutes ) and shortest in the opn group ( 163 minutes , p < 0.0001 ) . </S>", "<S> patients who had opn had the highest incidence of 30-day complications ( 30% ) , while the rpn approach had the lowest ( 14% , p = 0.008 ) . conclusions . when performed by a single surgeon , robotic partial nephrectomy appears to be associated with fewer complications than both open and laparoscopic partial nephrectomy . </S>", "<S> kidney function was not affected by surgical approach . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["in 2014 , there will be 63,920 new cases of kidney and renal pelvis cancer diagnoses resulting in approximately 13,860 deaths .", "renal cell carcinoma ( rcc ) is one of the most lethal genitourinary cancers and comprises 85% of these new diagnoses .", "the incidence of rcc has steadily increased over the past few decades due to incidentally discovered small renal masses ( srm ) on radiographic imaging resulting in stage migration to clinical t1a disease from more advanced disease .", "numerous treatment options exist for clinical t1a disease , including total nephrectomy , partial nephrectomy , radiofrequency ablation , cryoablation , and high - intensity focused ultrasound ( hifu ) . in 2009 ,", "the american urologic association ( aua ) published the  guideline for the clinical stage 1 renal mass  which concluded that partial nephrectomy is the standard of care for clinical t1a disease suggesting that the open approach is preferred over minimally invasive techniques .", "in experienced hands , laparoscopic partial nephrectomy ( lpn ) is associated with similar oncologic outcomes to open partial nephrectomy ( opn ) with the added benefit of decreased narcotic use , decreased hospital stay , and earlier convalescence [ 57 ] .", "robotic partial nephrectomy ( rpn ) and lpn have also been compared with similar outcomes , if not superior rpn outcomes , in the hands of an experienced minimally invasive surgeon .", "most studies report the outcomes of either ( a ) single surgeon and single and/or dual approach or ( b ) multicenter , multisurgeon , and dual approach .", "to our knowledge , there have been no reports of a single surgeon 's experience with each of the three modalities .", "our hypothesis is that when performed by a single surgeon , open , laparoscopic , and robotic partial nephrectomy could differ in postoperative complication rates and overall kidney function .", "thus , we report the perioperative outcomes of 106 consecutive patients treated with partial nephrectomy by a single surgeon using all three surgical approaches  opn , lpn , and rpn ."], ["after obtaining institutional review board approval , a retrospective chart review of a prospectively collected kidney cancer database was performed . from august 2006 to february 2012 , a single surgeon ( rm ) with minimally invasive and urologic oncology fellowship training performed 106 consecutive partial nephrectomies using any of the three available surgical modalities  opn , lpn , or rpn .", "the decision for the approach was multifactorial and was based on patient and tumor characteristics .", "initially in the practice , lpn was offered to patients with tumors of favorable size and location .", "opn was reserved for patients who were uninephric , had compromised kidney function , or had large tumors in an unfavorable location . in early 2008 , rpn gradually replaced lpn as the preferred minimally invasive approach .", "preoperative variables and demographics analyzed included age , gender , body mass index ( bmi ) , american society of anesthesiologist ( asa ) score , glomerular filtration rate ( gfr ) , hematocrit ( within 30 days of surgery ) , tumor laterality , and tumor size .", "operative variables analyzed included operative time , estimated blood loss ( ebl ) , complications , pathology ( benign , malignant ) , and margin status .", "postoperative variables included length of stay , discharge hematocrit , transfusion , gfr , 3-month gfr , and 30-day complications .", "the indication for partial nephrectomy was an enhancing renal mass found on cross - sectional abdominal imaging .", "pathology was reviewed by a dedicated genitourinary pathologist for margin status and tumor type . \n", "briefly , patients were positioned in a modified 45-degree flank and an incision was made for access to the retroperitoneum .", "the kidney was dissected free of attachments in the retroperitoneal space and the hilum was identified .", "intravenous mannitol ( 12.5  g ) administration and renal hypothermia ( cold ischemia ) were utilized prior to hilar clamping in all cases .", "the hilum was clamped using the satinsky vascular clamp and the resection of the renal lesion was performed with sharp dissection .", "the collecting system was closed primarily , and individual vessels were identified and controlled using nonabsorbable monofilament suture .", "argon beam coagulation achieved hemostasis prior to reapproximation of the renal parenchyma with surgicel ( ethicon inc , somerville , nj ) bolsters . in all cases ,", "the patient was positioned in a modified flank configuration with a total of four ports .", "once the hilum and mass were identified , mannitol ( 12.5  g ) was administered and the hilum was controlled using a laparoscopic vascular clamp ( warm ischemia ) .", "surgicel ( ethicon inc , somerville , nj ) and floseal ( biosurgery , deerfield , il ) were used for hemostasis and the tumor bed was repaired in a running fashion based on the depth of the lesion .", "the parenchyma was reapproximated with 2.0  v lock suture , surgicel ( ethicon inc , somerville , nj ) bolsters , and secured with hem - o - lok clips . in superficial tumors penetrating less than 1  cm into kidney parenchyma , clamping and/or repair of the base of resection was not performed . \n", "all patients underwent warm ischemia , which was achieved using either laparoscopic bulldog clamps or a vascular clamp .", "similar to the lpn technique , the resection bed was closed in a running fashion using an absorbable suture and the defect was approximated using a surgicel ( ethicon inc , somerville , nj ) bolster in most cases . the statistical package for the social sciences v.19 ( spss inc .", "all p values were one - sided , and p < 0.05 was considered statistically significant .", "categorical variables were compared using a chi - square test and continuous variables were compared using one - way anova ."], ["among the 106 patients in the study , 23 patients ( 22% ) underwent opn , 48 patients ( 45% ) underwent lpn , and 35 patients ( 33% ) underwent rpn .", "there was no difference in age , gender , bmi , asa score tumor laterality , or tumor size between the three groups ( table 1 ) .", "there was a statistical trend towards lower preoperative hematocrit for opn patients compared to the minimally invasive patients ( opn40  4% , lpn43  4% , rpn42  4% , p = 0.09 ) .", "preoperative gfr was 71  28  ml / min for opn patients , 76  25  ml / min for lpn patients , and 96  37  ml / min for rpn patients ( p = 0.004 ) .", "there was no difference in intraoperative complication rate , tumor pathology , or margin status between the opn , ldn , and rpn patients ( table 2 ) .", "operative time was the shortest in the opn group ( opn163  35  min , lpn227  53  min , rpn244  53  min , p < 0.0001 ) .", "ebl ( opn367  286  ml , lpn163  179  ml , rpn191  173  ml , p <", "all patients who had opn had the kidney cooled for an average time of 8 minutes before excision of the tumor .", "there were no differences in length of stay , blood transfusion , discharge gfr , or 3-month gfr for the three cohorts of patients ( table 3 ) .", "discharge hematocrit was significantly lower for opn patients ( 30  3% ) compared to lpn ( 36  4% ) and rpn ( 35  4% ) patients ( p < 0.0001 ) . upon follow - up", ", the opn group had the highest incidence of 30-day complications ( 30% ) , while the rpn group had the lowest ( 14% , p = 0.008 ) .", "the opn complications included clavien i ( n = 2 ) , clavien ii ( n = 1 ) , clavien iii ( n = 3 ) , and clavien iv ( n = 1 ) . the lpn complications included clavien i ( n = 4 ) , clavien ii ( n", "the rpn complications included clavien i ( n = 1 ) , clavien ii ( n = 1 ) , and clavien iii ( n = 3 ) ."], ["radical nephrectomy ( rn ) has been the mainstay of treatment for clinical stage i tumors resulting in excellent cancer specific survival , local tumor control , and progression free survival ; however , reports have highlighted a negative impact on renal function and chronic kidney disease ( ckd ) associated with rn .", "this effect was acknowledged in the 2009 aua guideline for the management of small renal masses which concluded that opn should be considered standard of care and that minimally invasive options , such as lpn and rpn , be considered as second line treatment modalities .", "furthermore , recent studies have established that partial nephrectomy ( pn ) provides similar oncological outcomes when compared to rn [ 1214 ] .", "the current study is the first , to our knowledge , which compares all three modalities of pn ( opn , lpn , and rpn ) by a single surgeon .", "multi - institutional studies may represent dissimilar patient cohorts that introduce selection bias and may invoke concerns regarding surgeon variability .", ", there was no randomization or objective criteria for the pn approach ; rather , the trend was for more minimally invasive surgery as the surgeon developed a comfort level with the lpn and rpn approach .", "this is evident by the fact that tumor characteristics for each group are comparable ( laterality , size , pathology , and margin status ) .", "previous single surgeon studies have analyzed rpn versus lpn [ 11 , 1519 ] with the consensus being that rpn offers a shorter learning curve to minimally invasive pn and comparable warm ischemia time , ebl , and length of stay .", "two common criticisms associated with rpn include ( i ) the cost of the robot and ( ii ) increased surgical time .", "although it is difficult to mitigate the cost associated with rpn compared to lpn and opn , the oft - held conception that procedures take significantly longer may be a misconception after achieving the learning curve associated with rpn .", "concluded that rpn achieved similar operative times to lpn after only 5 procedures and the current study corroborates similar operative times between the two approaches ( 244  53 versus 227  53  min ) . in the opn cohort", ", there was a 30-day complication rate of 30% , which was statistically significant compared to the lpn ( 17% ) and rpn ( 14% ) groups , and higher than that reported by other authors .", "gill et al . reported a 16% and 13% postoperative complication rate in their initial comparison of lpn and opn , respectively .", "bhayani and waxman and winfield reported postoperative complication rates of 16% and 18.2% , respectively , in each of their single - surgeon experiences with lpn [ 21 , 22 ] .", "a recent report , which pooled data from 4 institutions considered centers - of - excellence , reported a postoperative complication rate of 14.4% in patients who underwent rpn .", "most complications were clavien grade i / ii and were managed conservatively and affirm the safety of rpn in experienced hands .", "the complication rate was lowest in the rpn group at 14% and is similar to other reports , with ranges of 1020% [ 8 , 15 , 24 ] .", "the primary limitation of the study is the inherent selection bias and patient confounders associated with a retrospective study .", "surgical confounders were minimized as only operations performed by a single surgeon were analyzed . secondly , renorrhaphy differed between the opn and minimally invasive cohorts along with the use of renal cooling in the opn group compared to warm ischemia time for the minimally invasive approaches ."], ["nephron sparing surgery has emerged as the optimal surgical approach for t1a and in experienced hands t1b kidney masses . by removing surgeon as a confounding variable ,", "although not currently the gold standard for partial nephrectomy , robotic surgery continues to emerge as the future for renal surgery secondary .", "rpn offers less blood loss and fewer postoperative complications than opn and overcomes the technical difficulties of lpn ."]]}
{"article_id": "PMC5338956", "article_text": ["job syndrome is one of the hyper immunoglobulin e ( ige ) syndrome ( hies ) conditions characterized by signal transducer and activator of transcription 3 ( stat3 ) signaling mutations .", "patients with job syndrome have characteristic clinical features in association with mutations in stat3 signaling .", "these include marked elevation in serum ige and susceptibility to staphylococcal infections of the pulmonary tract and skin.1 one of the hallmark features of the disease includes the formation of abscesses . on the basis of the elevation of serum ige levels", ", it was hypothesized that there would be a lower incidence of clinical anaphylaxis among pediatric job syndrome patients .", "an electronic medical record database , including both inpatient and outpatient records , was utilized .", "the database from a large urban children s medical center was searched from january 1 , 2009 to december 31 , 2014 , using the international classification of diseases , ninth revision ( icd-9 ) codes assigned to each diagnosis , job syndrome , hies , and anaphylaxis .", "the icd-9 codes used corresponding to each diagnosis were as follows : asthma , 493.00493.99 ; job syndrome , 288.1 ; and anaphylactic shock , 995.61 ( peanut ) and 995.67 ( milk ) .", "the anonymous data generated for anaphylactic shock , asthma with anaphylactic shock , and job syndrome / hies were used to identify patients .", "the total number of patients was identified with 1 ) job syndrome / hies ; 2 ) asthma with anaphylactic shock ; 3 ) anaphylactic shock with and without peanut allergy , and with and without milk allergy ; and 4 ) job syndrome / hies with anaphylactic shock .", "the database search was conducted with the medical center health information support and did not involve personal health information analysis .", "the study was exempt from irb review because there was no use of personal health information , as discussed with the university of california irb office .", "the total number of patients with job syndrome / hies identified in the database was 18 . among the 22,890 patients evaluated with asthma", "of the 614 total anaphylactic patient cases identified , 286 ( 46.5% ) had anaphylaxis with no determined specific cause .", "there were 250 cases ( 40.7% ) of peanut allergen - induced anaphylaxis and 78 cases ( 12.7% ) were because of milk products .", "some patients may have been coded for one or more causes of shock . despite the elevation of ige associated with hies / job syndrome", "a one- and two - tailed t - test showed that there was no statistically significant difference when comparing asthma and anaphylaxis and hies with anaphylaxis .", "a one- and two - tailed t - test showed that there was no statistically significant difference when comparing asthma and anaphylaxis and hies with anaphylaxis .", "this study shows that there were no identified cases of anaphy - laxis among the job syndrome / hies population studied .", "this low incidence is in contrast to established higher rates of ana - phylaxis among atopic conditions associated with high levels of serum ige .", "the incidence of anaphylaxis among children and adolescents has been reported as between 0.05% and 2%.2 this study identified > 600 cases of anaphylaxis , during the same time period at the same medical center , among the non - hies pediatric population .", "the association between allergic disease and autosomal - dominant ( ad ) hies was studied in a murine model .", "these mutations result in a very elevated serum ige level , usually without any evidence of atopic disease .", "the authors found that in a murine model of mast cell degranulation , mast cell - induced anaphylaxis was blunted in the mutant mice .", "the animals treated with a c188 - 9 stat3 inhibitor demonstrated altered physiologic responses . the responses measured included body temperature and survival .", "those treated with the inhibitor had longer survival and less change in body temperature.3 in a cellular model described in the same citation , human umbilical vein cells from ad hies patients or control cells treated with a stat3 inhibitor demonstrated aberrant responses to mast cell mediators , such as histamine or platelet - activating factor .", "the cells from two newborns with hies were less permeable than wild - type cells when exposed to mast cell mediators.3 these findings suggest that stat3 mutations confer resistance to mast cell degranulation . a clinical study of food allergies and adult hies conducted a clinical evaluation of food allergies in a cohort of 71 patients with ad hies compared with healthy control subjects ( n=41 ) and atopic subjects ( n=65 ) .", "the results indicated that fewer patients with ad hies developed food allergies and anaphy - laxis than atopic patients with marked serum ige elevation , eczema , and no stat3 mutations .", "the study reported that fewer patients with ad hies had anaphylaxis to a food allergen than atopic controls ( 8.5% vs. 33.3% ) .", "the ad hies patients did demonstrate that they had specific ige to food allergens . when comparing peanut anaphylaxis between the groups , there were no cases of peanut - induced anaphylaxis found in the patients with hies.4 the authors concluded that stat3 mutations may play a role in protecting the individual from food allergies as a trigger for anaphylaxis . in studying the activation of a key regulatory cell ,", "basophils from hies patients were less sensitive to ige cross - linking than basophils from non - allergic subjects.4 this study demonstrates that among the pediatric cases identified , there have been no cases of anaphylaxis among the hies patients .", "this is in contrast to a large number of children without hies who did have anaphylaxis due to peanut or milk allergens .", "this study finds support for a potential protective effect of stat3 mutations in preventing anaphylaxis and demonstrates this finding in a pediatric population .", "further studies are warranted to identify the molecular and cellular pathways involved in reducing vascular permeability during mast cell degranulation among children with hies ."], "abstract_text": ["<S> patients with autosomal - dominant ( ad ) hyper immunoglobulin e ( ige ) syndrome ( hies ) or job syndrome develop frequent dermatologic and pulmonary infections . </S>", "<S> as patients have an extreme elevation of ige levels , this database analysis study sought to study the association between ad hies , job syndrome , and anaphylaxis . </S>", "<S> hies is a heterogeneous group of immune disorders characterized by extremely elevated levels of serum ige . </S>", "<S> although the molecular defects and clinical phenotypes found in association with job syndrome are well characterized , the association with severe allergic reactions and anaphylaxis is a subject of ongoing investigation . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Statistical analysis", "Discussion"], "sections": [["job syndrome is one of the hyper immunoglobulin e ( ige ) syndrome ( hies ) conditions characterized by signal transducer and activator of transcription 3 ( stat3 ) signaling mutations .", "patients with job syndrome have characteristic clinical features in association with mutations in stat3 signaling .", "these include marked elevation in serum ige and susceptibility to staphylococcal infections of the pulmonary tract and skin.1 one of the hallmark features of the disease includes the formation of abscesses . on the basis of the elevation of serum ige levels", ", it was hypothesized that there would be a lower incidence of clinical anaphylaxis among pediatric job syndrome patients ."], ["an electronic medical record database , including both inpatient and outpatient records , was utilized .", "the database from a large urban children s medical center was searched from january 1 , 2009 to december 31 , 2014 , using the international classification of diseases , ninth revision ( icd-9 ) codes assigned to each diagnosis , job syndrome , hies , and anaphylaxis .", "the icd-9 codes used corresponding to each diagnosis were as follows : asthma , 493.00493.99 ; job syndrome , 288.1 ; and anaphylactic shock , 995.61 ( peanut ) and 995.67 ( milk ) .", "the anonymous data generated for anaphylactic shock , asthma with anaphylactic shock , and job syndrome / hies were used to identify patients .", "the total number of patients was identified with 1 ) job syndrome / hies ; 2 ) asthma with anaphylactic shock ; 3 ) anaphylactic shock with and without peanut allergy , and with and without milk allergy ; and 4 ) job syndrome / hies with anaphylactic shock .", "the database search was conducted with the medical center health information support and did not involve personal health information analysis .", "the study was exempt from irb review because there was no use of personal health information , as discussed with the university of california irb office ."], ["the total number of patients with job syndrome / hies identified in the database was 18 . among the 22,890 patients evaluated with asthma", "of the 614 total anaphylactic patient cases identified , 286 ( 46.5% ) had anaphylaxis with no determined specific cause .", "there were 250 cases ( 40.7% ) of peanut allergen - induced anaphylaxis and 78 cases ( 12.7% ) were because of milk products .", "some patients may have been coded for one or more causes of shock . despite the elevation of ige associated with hies / job syndrome", "a one- and two - tailed t - test showed that there was no statistically significant difference when comparing asthma and anaphylaxis and hies with anaphylaxis ."], ["a one- and two - tailed t - test showed that there was no statistically significant difference when comparing asthma and anaphylaxis and hies with anaphylaxis ."], ["this study shows that there were no identified cases of anaphy - laxis among the job syndrome / hies population studied .", "this low incidence is in contrast to established higher rates of ana - phylaxis among atopic conditions associated with high levels of serum ige .", "the incidence of anaphylaxis among children and adolescents has been reported as between 0.05% and 2%.2 this study identified > 600 cases of anaphylaxis , during the same time period at the same medical center , among the non - hies pediatric population .", "the association between allergic disease and autosomal - dominant ( ad ) hies was studied in a murine model .", "these mutations result in a very elevated serum ige level , usually without any evidence of atopic disease .", "the authors found that in a murine model of mast cell degranulation , mast cell - induced anaphylaxis was blunted in the mutant mice .", "the animals treated with a c188 - 9 stat3 inhibitor demonstrated altered physiologic responses . the responses measured included body temperature and survival .", "those treated with the inhibitor had longer survival and less change in body temperature.3 in a cellular model described in the same citation , human umbilical vein cells from ad hies patients or control cells treated with a stat3 inhibitor demonstrated aberrant responses to mast cell mediators , such as histamine or platelet - activating factor .", "the cells from two newborns with hies were less permeable than wild - type cells when exposed to mast cell mediators.3 these findings suggest that stat3 mutations confer resistance to mast cell degranulation . a clinical study of food allergies and adult hies conducted a clinical evaluation of food allergies in a cohort of 71 patients with ad hies compared with healthy control subjects ( n=41 ) and atopic subjects ( n=65 ) .", "the results indicated that fewer patients with ad hies developed food allergies and anaphy - laxis than atopic patients with marked serum ige elevation , eczema , and no stat3 mutations .", "the study reported that fewer patients with ad hies had anaphylaxis to a food allergen than atopic controls ( 8.5% vs. 33.3% ) .", "the ad hies patients did demonstrate that they had specific ige to food allergens . when comparing peanut anaphylaxis between the groups , there were no cases of peanut - induced anaphylaxis found in the patients with hies.4 the authors concluded that stat3 mutations may play a role in protecting the individual from food allergies as a trigger for anaphylaxis . in studying the activation of a key regulatory cell ,", "basophils from hies patients were less sensitive to ige cross - linking than basophils from non - allergic subjects.4 this study demonstrates that among the pediatric cases identified , there have been no cases of anaphylaxis among the hies patients .", "this is in contrast to a large number of children without hies who did have anaphylaxis due to peanut or milk allergens .", "this study finds support for a potential protective effect of stat3 mutations in preventing anaphylaxis and demonstrates this finding in a pediatric population .", "further studies are warranted to identify the molecular and cellular pathways involved in reducing vascular permeability during mast cell degranulation among children with hies ."]]}
{"article_id": "PMC3306939", "article_text": ["\n leishmania parasites are heteroxenous kinetoplastid protozoan organisms , which undergo complete differentiation upon a cycle of proliferation / differentiation in the midgut of phlebotomine sand flies followed by the transmission of infective metacyclic promastigotes [ 1 , 2 ] to mammalian hosts during the insect blood meal .", "once infecting mammalian hosts , these organisms , from free - living protozoa , become obligate intracellular parasites , residing and proliferating inside phagolysosomes of mononuclear phagocytic cells as amastigote forms .   in humans , leishmania parasites can cause a broad spectrum of clinical manifestations from mild , self - resolving skin diseases to potentially fatal , disseminated visceral diseases .", "the outcome of the infection is dependent on multiple , interdependent factors , such as vector species , parasite species and strain , genetic background , and immunological status of the host .", "there are two main groups of parasites , stratified upon the clinical outcome of the infection : the ones capable of causing tegumentar and the ones capable of causing visceral diseases . in both cases ,", "disease is initiated by the bite of an infected sand fly , followed by the generation of a skin lesion , mainly caused by the inflammatory response induced on that site . in some cases the disease", "is confined to the skin or mucosal tissues , and is termed cutaneous ( cl ) or muco - cutaneous ( mcl ) leishmaniasis , respectively .", "in addition , diffuse cutaneous leishmaniasis ( dcl ) occurs when the parasite disseminates causing the appearance of multiple skin lesions , in distal sites relative to the transmission site . in a similar way , in visceral leishmaniasis", ", there is parasite dissemination through blood and lymphatic vessels from the initial lesion site .", "however , these parasites establish in organs that comprise important populations of mononuclear phagocytes , such as bone marrow , spleen , and liver . among the clinical manifestations observed in humans with the tegumentary disease , diffuse and mucocutaneous leishmaniasis", "most patients were found in the south and central america , associated with l. amazonensis infection for dcl and l. braziliensis infection for mcl .", "diffuse cutaneous leishmaniasis ( dcl ) is a rare clinical manifestation and is characterized by the appearance of several nonulcerated nodular skin lesions , uncontrolled parasite proliferation , an inefficient cellular immune response against parasite antigens , and resistance to most therapeutic strategies [ 5 , 6 ] .", "the intense parasitism in the dcl lesions reflects the functional state of macrophages , which are considered permissive .", "the deficient macrophage activation in dcl hinders the elimination of leishmania resulting in a disorganized inflammatory process , unable to control the infection .", "the determinants of dcl are multifactorial and may be associated with both immunologic and genetic events of the patient and the pathogenic factors related to the parasite and vector .", "the participation of factors associated with the parasite has been shown by some authors although it is a point that still remains to be further explored . in this context", ", the exhibition of markers of apoptosis by the parasite could be a contributing factor during host - parasite interactions as a possible immunosuppressive mechanism of dcl .", "experimental infection models with leishmania parasites have been extensively used as a tool to study immune responses , especially regarding t - cell differentiation [ 8 , 9 ] .", "this is due to the fact that inbred mice strains demonstrate specific patterns of susceptibility and resistance to the disease [ 9 , 10 ] which correlate with the immune response built by these animals . the classical experimental model that generated this knowledge was infection with l. major parasites .", "c57bl/6 mice infected with this parasite develop a th1 cd4 t - cell response , which is highly effective to activate leishmanicidal and inflammatory mechanisms in macrophages , leading to intracellular parasite destruction . in this case ,", "nevertheless , latent parasites remain in the infected tissue , providing antigens to maintain a protective immune response that prevent reinfections . on the other hand ,", "balb / c mice infected with the same parasite species and strain developed a th2 cd4 t - cell response , which is not efficient to promote macrophage classical activation , leading to progressive disease .", "at the cellular level , this difference is mainly due to the activation of a population of cells that express a highly restricted t - cell receptor , v4 v8 , which recognizes the lack ( leishmania homologue of receptors for activated kinase ) antigen and rapidly produces il-4 , necessary to deviate the immune response towards th2 .", "currently , it is clear that the proposed model of susceptibility and resistance to leishmania infection is quite reproducible when working with some specific strains of l. major though , for other strains and/or species , the picture is relatively more complex . indeed ,", "effective macrophage activation is the key to control the infection ; however , the phenotype displayed by t cells in different situations is not as polarized as observed in the classical model .", "actually , there are several papers that suggest that most correlations between cd4 t - cell response and disease development are not straightforward .", "balb / c il-4 receptor knock - out ( ko ) mice remained susceptible to l. major infection when infected with lv39 strain , which seems to be due to an increased production of il-10 by t cells .", "c57bl/6 mice infected with a l. major strain , isolated from a patient with nonhealing lesions , still developed a th1 response but displayed a progressive disease .", "in addition , when infected with the ir173 strain of l. major , cd4 t cells from both balb / c and the resistant mice strain b10.d2 produce il-4 very rapidly .", "other factors such as infection route , number of parasites inoculated , and type of infection ( needle versus sand fly inoculation ) are crucial to determine the type of response elicited ( reviewed in ) .", "the complexity of the interactions that determines the clinical and immunological outcome of the disease is much less known , and apparently much more multifactorial in other infection systems , such as the ones that involve l. amazonensis infection .", "experimental infection with l. amazonensis parasites leads to progressive disease and uncontrolled lesion development in all inbred mouse strains , including those ones that are highly resistant to l. major infection .", "however , there is a gradient of disease severity , ranging from balb / c mice , which develop a very fast lesion , that ulcerate , generating extensive areas of necrotic tissue , to c3h.hen mice that still develop nonhealing lesions , however , displaying slow progression rates [ 16 , 17 ] .", "nonetheless , the phenotype displayed by different mouse strains does not correlate with dichotomic th1/th2 responses . actually , in the analyzed mouse strains such as balb / c , c57bl/6 , and c3h.hen , it was possible to observe cd4 t cells capable of producing different types of cytokines such as th2 cytokines ( il-4 , il-5 , and il-13 ) , th1 cytokines ( ifn , and tnf ) , and regulatory cytokines ( tgf and il-10 ) , which characterizes an unpolarized cellular response [ 1820 ] .", "targeted deletion of the il4 or il10 gene [ 21 , 22 ] causes minimal effects on lesion development and parasite tissue loads as well as treatment of infected mice with ifn   or il-12 .", "interestingly , l. amazonensis promastigotes and , especially amastigotes , are able to get through the innate immune response almost unnoticeable . as mentioned before , the main host cell for leishmania proliferation in the mammalian host is the macrophage , which is , together with dendritic cells ( dcs ) , the main antigen presenting cells of the innate immune response .", "when compared to l. braziliensis parasites , for example , l. amazonensis parasites are much less capable of triggering the expression of cd40 and cd80 , both costimulatory molecules for t - cell activation , and the production of il-12p40 . actually , amastigote infection is able to downregulate the expression of mhc class ii molecules , which , during macrophage infection , is depending on sequestering these molecules inside the parasitophorous vacuole for degradation [ 26 , 27 ] . during the first week of infection in c57bl/6 mice", ", chemokines such as ccl5 , ccl3 , ccl2 , ccl4 , and ccl11 as well as their receptors , are not upregulated when compared to l. major infection , both at the lesion site and draining lymph node .", "additionally , amastigote infection downregulates several intracellular pathways that lead to dc activation such as stat 1 , stat 3 and erk 1/2 phosphorylation and the expression of the interferon - responsive elements irf8 and 1 , suggesting a global inhibition of inflammatory responses of these cells .", "the most well - characterized ligand for amastigote recognition and internalization in macrophages is the opsonizing antibodies produced throughout infection .", "triggering of fc receptors on the host cells lead to il-10 production and has a pathogenic role .", "these events are necessary to evade the early immune response culminating with the ineffective t - cell response observed in most cases . in parallel to l. major infection in balb / c mice , where the oligoclonal v4 v8 cd4 t - cell population is necessary to the development of susceptibility in the host and is considered as pathogenic t cells , in l. amazonensis infection , t cells , in a general way , seem to be highly pathogenic .", "first of all , there is no clonal or oligoclonal t - cell population involved , since there is no predominance of a single or a group of v chains expressed on t cells that respond to l. amazonensis infection .", "however , the disruption of cd4 t cell effector functions , as observed in recombinase - activating gene ko mice ( rag ko ) , mhc class ii transactivator ko mice ( ciita ko ) and nude mice leads to transient resistance to l. amazonensis infection , measured by lesion development .", "in addition , the adoptive transfer of regulatory t cells also restrains pathogenic effector t cells , diminishing lesion size and parasite tissue loads .", "the mechanisms underlying pathogenic role of t cells for the disease need to be determined .", "phosphatidylserine ( ps ) is a structural phospholipid present in virtually all membranes and cell types . in normal cells", "these molecules face the cytoplasmic leaflet of the plasma membrane , whereas during apoptotic cell death these molecules translocate to the outer surface .", "once outside the cell , ps becomes one of the ligands recognized by surrounding phagocytes to clear dying cells .", "ps is the most characterized tickling   ligand of apoptotic cells , which means that ps provides the signals for the phagocyte to activate immunosuppressive and anti - inflammatory mechanisms .", "ps recognition is mandatory to prevent the establishment of a response to the self - antigens engulfed by these cells during apoptotic cell clearance and to avoid triggering inflammatory responses , especially during the embryogenesis , when massive amounts of apoptotic cells are generated and therefore , cleared [ 3537 ] , but also in adults to prevent inflammatory immunopathologies .", "the intracellular events , receptors , and soluble factors involved in this mechanism are still being deciphered and are not the focus of this discussion .", "however , the effects of ps recognition in macrophages and dcs have a direct impact in immune responses .", "apoptotic cells actively induce the production of the anti - inflammatory cytokines tgf , pge2 , and paf   and actively inhibit the production of tnf and il-1 , even upon lps challenge [ 35 , 38 ] .", "recognition of apoptotic cells also decreases the expression of several activation markers and costimulatory molecules by both human and murine dcs [ 39 , 40 ] and regulates the expression of cytokines involved with t - cell differentiation at the transcriptional level [ 41 , 42 ] . at the single cell level , dcs that ingested an apoptotic cell and bacteria at the same time are able to discern between them and only present bacterial antigens", "this is possible because the generation of peptide - mhc class ii complexes is controlled by toll - like receptors ( tlrs ) in a strictly phagosome autonomous manner . since apoptotic cells do not trigger tlr activation , the generation of stable complexes is inhibited or abrogated .", "all these effects are fundamental to maintain homeostasis and comprehend the last step of the efferocytosis   or apoptotic cell clearance .", "however , it seems that intracellular parasites elegantly make use of these mechanisms to establish in the host [ 4547 ] .", "furthermore , some parasites mimic the features of apoptotic cells to avoid host immune response , as discussed in the next section .", "one of the most common pcd phenotypes is phosphatidylserine ( ps ) exposure , which can be observed upon chemotherapy , starvation , and heat shock conditions in several unicellular organisms [ 4851 ] or is actively displayed in normal conditions .", "our group observed that lesion - derived amastigotes of l. amazonensis actively expose high levels of ps , and by blocking this molecule there is a drastic decrease in the ability of these parasites to infect and establish in the macrophages .", "these parasites are viable and capable of differentiating into promastigote forms in vitro ( unpublished data ) and inside the sand fly vector   and to infect macrophages and mice [ 52 , 54 ] and did not display other markers of pcd .", "ps exposure on amastigotes of l. amazonensis occurs in virtually 100% of the parasites ; however , the amount of ps molecules depends on the infected host .", "parasites obtained from balb / c mice expose higher amounts of ps than the ones obtained from c57bl/6 mice .", "this observation demonstrates that the amount of ps at the surface of the amastigotes has a positive correlation with the severity of the disease and suggests that the host is able to modulate this phenotype of the parasite .", "following our description several other groups demonstrated the role of ps exposure and recognition in different infection models .", "blood and cell - derived trypomastigotes of trypanosoma cruzi are able to expose ps , in contrast with epimastigotes , which are not . in addition , infection with ps - exposing trypomastigote forms induces smad nuclear translocation and inducible nitric oxide synthase inhibition ( inos ) , suggesting an autocrine modulation of the host cell dependent on tgf .", "it is interesting to note that , among all t. cruzi parasite stages , only the ones that are infective for mammalian cells evolve the ability to expose ps , suggesting the presence of an evolutionary link between ps exposure and the ability to infect host cells .", "similarly , toxoplasma gondii peritoneal tachyzoites expose ps at their surface , and the recognition of this molecule seems to be necessary to downmodulate inos expression and activity upon macrophage infection .", "more recently , several papers have demonstrated the role of exposed ps molecules for the infection by enveloped viral particles .", "for human immunodeficiency virus-1 ( hiv-1 ) , ps at the viral envelope is a cofactor for monocyte infection ; in vaccinia virus infection , ps recognition modulates the activity of proteins involved in cytoskeleton reorganization such as p21-activated kinase ( pak ) and the small rho gtpase rac , leading to increased macropinocytic activity and uptake of viral particles .", "in addition , ps exposure by tumor cell , microvesicles shed by transformed cells , or endothelial cells in the intratumor environment seems to be involved in different events in tumor development , maintenance , and metastasis [ 59 , 60 ] .", "this knowledge stimulated some researchers to evaluate the efficacy of anti - ps antibodies to treat viral and tumoral diseases .", "actually the results so far are promising . in murine models of lassa fever ( pichinde virus ) or murine", "cytomegaloviruses the treatment efficacy was very high , reaching complete cure ( total absence of detectable viral loads ) in combination with available antiaviral drugs . for experimental tumoral disease , lung cancer , pancreatic tumors , and glioblastomas", "were efficiently treated , decreasing tumor growth and metastasis in some cases or potentiating the effect of chemo- and radio - therapies [ 6264 ] .", "our group has been committed to study the role of ps exposure on the surface of different isolates of l. amazonensis .", "we worked with the hypothesis that l. amazonensis isolates from dcl patients would have higher ps exposure compared with localized cutaneous leishmaniasis ( lcl ) , and this would contribute to macrophage deactivation , favoring parasite replication . for this , we compared ps exposure in l. amazonensis isolates from dcl clinical cases in the active phase of the disease , reported in maranho state in brazil , to those isolated from lcl patients of clinical cases from bahia .", "the results indicate that the isolates obtained from dcl patients indeed displayed more ps than isolates from lcl patients at early times postinfection .", "in addition , isolates from dcl patients were more infective than the ones obtained from lcl patients ( frana - costa et al .", "the other hand , independent of parasite strain analyzed , the parameters of infectivity correlated positively with the exposure of ps in the parasites .", "these data suggest that in human infections the pattern observed in mice when comparing balb / c versus c57bl/6 mice is maintained .", "however , it is necessary to investigate the mechanisms by which the recognition of ps on the surface of the isolates of l. amazonensis deactivate the macrophage response .", "particularly , it would be necessary to evaluate whether freshly isolated parasites display this phenotype to validate our analysis made on amastigotes derived from macrophages infected in vitro with cultured promastigote parasites isolated from human lesions .", "we believe that understanding the dynamics of ps expression , along with identification of the mechanisms involved in the immunosuppression of dcl patients , can result in therapeutic targets for intervention in the immunopathogenesis of this chronic and severe form of leishmaniasis .   in a similar way we are interested in the immunomodulatory mechanisms underlying ps exposure in different inbred mice strains . for that we are currently evaluating these mechanisms during balb / c infection , which induces high levels of ps exposure on intracellular amastigotes . we observed that ps exposure is intrinsic to the intracellular parasite and", "however , these levels are dramatically increased when infected macrophages are in the presence of previously primed t cells or their soluble products .", "we confirmed these results by infecting balb / c nude mice where we observed that the amastigotes obtained from these mice display minimal levels of ps , which are completely restored if we adoptively transfer primed cd4 t cells to nude mice ( wanderley et al .", "interestingly , these data indicate that one possible role for the previously reported pathogenic t cells   would be to induce ps exposure on intracellular amastigotes and , therefore , contributing to the generation of highly infective parasites .", "the t - cell - dependent ps exposure on amastigotes seems to be dependent on the induction of inos expression on host macrophages , and parasite survival is dependent on the concomitant induction of arginase 1 expression ( wanderley et al .", "we propose that high levels of ps exposure are induced by parasite stress delivered by inos activity .", "in this case , it is still unknown whether ps exposure on amastigotes is indeed a phenotype triggered by pcd or a specific process involving modulation of ps translocation . under ps - inducting conditions ,", "unpublished results ) , that is the enzyme necessary to produce ornithine , the precursor of polyamines . in this situation", ", polyamines could protect the parasite from the inos - dependent stress , stimulating parasite growth [ 66 , 67 ] and increasing dna stabilization [ 68 , 69 ] .", "we understand that the unique characteristics of the t - cell response to l. amazonensis infection contribute to the generation of a perfect environment to stimulate and maintain increased levels of ps on the surface of intracellular parasites .", "probably the balance observed in infected balb / c mice , when disrupted , leads to the differences observed among different mouse strains . in figure 1", "we summarized our hypothesis regarding the t - cell - dependent modulation of ps exposure on intracellular amastigotes of l. amazonensis .", "the observation of ps exposure as a strategy to evade the immune system and persist in the mammalian host , made initially in the experimental model of l. amazonensis infection , was a breakthrough since it stimulated different groups around the world to look for the possibilities for basic and applied research on the field .", "our group is still studying the immunological , cellular , and molecular mechanisms underlying control of ps exposure in parasites and the effects of its recognition by parasitized cells and organisms .", "we believe that this could be a major strategy in different systems where avoidance from immune surveillance is necessary to establish a disease ."], "abstract_text": ["<S> \n leishmania amazonensis parasites cause progressive disease in most inbred mouse strains and are associated with the development of diffuse cutaneous leishmaniasis in humans . the poor activation of an effective cellular response is correlated with the ability of these parasites to infect mononuclear phagocytic cells without triggering their activation or actively suppressing innate responses of these cells . </S>", "<S> here we discuss the possible role of phosphatidylserine exposure by these parasites as a main regulator of the mechanism underlying subversion of the immune system at different steps during the infection . </S>"], "labels": null, "section_names": ["1. ", "2. Immune Response", "3. Phosphatidylserine Exposure", "4. Final Remarks"], "sections": [["\n leishmania parasites are heteroxenous kinetoplastid protozoan organisms , which undergo complete differentiation upon a cycle of proliferation / differentiation in the midgut of phlebotomine sand flies followed by the transmission of infective metacyclic promastigotes [ 1 , 2 ] to mammalian hosts during the insect blood meal .", "once infecting mammalian hosts , these organisms , from free - living protozoa , become obligate intracellular parasites , residing and proliferating inside phagolysosomes of mononuclear phagocytic cells as amastigote forms .   in humans , leishmania parasites can cause a broad spectrum of clinical manifestations from mild , self - resolving skin diseases to potentially fatal , disseminated visceral diseases .", "the outcome of the infection is dependent on multiple , interdependent factors , such as vector species , parasite species and strain , genetic background , and immunological status of the host .", "there are two main groups of parasites , stratified upon the clinical outcome of the infection : the ones capable of causing tegumentar and the ones capable of causing visceral diseases . in both cases ,", "disease is initiated by the bite of an infected sand fly , followed by the generation of a skin lesion , mainly caused by the inflammatory response induced on that site . in some cases the disease", "is confined to the skin or mucosal tissues , and is termed cutaneous ( cl ) or muco - cutaneous ( mcl ) leishmaniasis , respectively .", "in addition , diffuse cutaneous leishmaniasis ( dcl ) occurs when the parasite disseminates causing the appearance of multiple skin lesions , in distal sites relative to the transmission site . in a similar way , in visceral leishmaniasis", ", there is parasite dissemination through blood and lymphatic vessels from the initial lesion site .", "however , these parasites establish in organs that comprise important populations of mononuclear phagocytes , such as bone marrow , spleen , and liver . among the clinical manifestations observed in humans with the tegumentary disease , diffuse and mucocutaneous leishmaniasis", "most patients were found in the south and central america , associated with l. amazonensis infection for dcl and l. braziliensis infection for mcl .", "diffuse cutaneous leishmaniasis ( dcl ) is a rare clinical manifestation and is characterized by the appearance of several nonulcerated nodular skin lesions , uncontrolled parasite proliferation , an inefficient cellular immune response against parasite antigens , and resistance to most therapeutic strategies [ 5 , 6 ] .", "the intense parasitism in the dcl lesions reflects the functional state of macrophages , which are considered permissive .", "the deficient macrophage activation in dcl hinders the elimination of leishmania resulting in a disorganized inflammatory process , unable to control the infection .", "the determinants of dcl are multifactorial and may be associated with both immunologic and genetic events of the patient and the pathogenic factors related to the parasite and vector .", "the participation of factors associated with the parasite has been shown by some authors although it is a point that still remains to be further explored . in this context", ", the exhibition of markers of apoptosis by the parasite could be a contributing factor during host - parasite interactions as a possible immunosuppressive mechanism of dcl ."], ["experimental infection models with leishmania parasites have been extensively used as a tool to study immune responses , especially regarding t - cell differentiation [ 8 , 9 ] .", "this is due to the fact that inbred mice strains demonstrate specific patterns of susceptibility and resistance to the disease [ 9 , 10 ] which correlate with the immune response built by these animals . the classical experimental model that generated this knowledge was infection with l. major parasites .", "c57bl/6 mice infected with this parasite develop a th1 cd4 t - cell response , which is highly effective to activate leishmanicidal and inflammatory mechanisms in macrophages , leading to intracellular parasite destruction . in this case ,", "nevertheless , latent parasites remain in the infected tissue , providing antigens to maintain a protective immune response that prevent reinfections . on the other hand ,", "balb / c mice infected with the same parasite species and strain developed a th2 cd4 t - cell response , which is not efficient to promote macrophage classical activation , leading to progressive disease .", "at the cellular level , this difference is mainly due to the activation of a population of cells that express a highly restricted t - cell receptor , v4 v8 , which recognizes the lack ( leishmania homologue of receptors for activated kinase ) antigen and rapidly produces il-4 , necessary to deviate the immune response towards th2 .", "currently , it is clear that the proposed model of susceptibility and resistance to leishmania infection is quite reproducible when working with some specific strains of l. major though , for other strains and/or species , the picture is relatively more complex . indeed ,", "effective macrophage activation is the key to control the infection ; however , the phenotype displayed by t cells in different situations is not as polarized as observed in the classical model .", "actually , there are several papers that suggest that most correlations between cd4 t - cell response and disease development are not straightforward .", "balb / c il-4 receptor knock - out ( ko ) mice remained susceptible to l. major infection when infected with lv39 strain , which seems to be due to an increased production of il-10 by t cells .", "c57bl/6 mice infected with a l. major strain , isolated from a patient with nonhealing lesions , still developed a th1 response but displayed a progressive disease .", "in addition , when infected with the ir173 strain of l. major , cd4 t cells from both balb / c and the resistant mice strain b10.d2 produce il-4 very rapidly .", "other factors such as infection route , number of parasites inoculated , and type of infection ( needle versus sand fly inoculation ) are crucial to determine the type of response elicited ( reviewed in ) .", "the complexity of the interactions that determines the clinical and immunological outcome of the disease is much less known , and apparently much more multifactorial in other infection systems , such as the ones that involve l. amazonensis infection .", "experimental infection with l. amazonensis parasites leads to progressive disease and uncontrolled lesion development in all inbred mouse strains , including those ones that are highly resistant to l. major infection .", "however , there is a gradient of disease severity , ranging from balb / c mice , which develop a very fast lesion , that ulcerate , generating extensive areas of necrotic tissue , to c3h.hen mice that still develop nonhealing lesions , however , displaying slow progression rates [ 16 , 17 ] .", "nonetheless , the phenotype displayed by different mouse strains does not correlate with dichotomic th1/th2 responses . actually , in the analyzed mouse strains such as balb / c , c57bl/6 , and c3h.hen , it was possible to observe cd4 t cells capable of producing different types of cytokines such as th2 cytokines ( il-4 , il-5 , and il-13 ) , th1 cytokines ( ifn , and tnf ) , and regulatory cytokines ( tgf and il-10 ) , which characterizes an unpolarized cellular response [ 1820 ] .", "targeted deletion of the il4 or il10 gene [ 21 , 22 ] causes minimal effects on lesion development and parasite tissue loads as well as treatment of infected mice with ifn   or il-12 .", "interestingly , l. amazonensis promastigotes and , especially amastigotes , are able to get through the innate immune response almost unnoticeable . as mentioned before , the main host cell for leishmania proliferation in the mammalian host is the macrophage , which is , together with dendritic cells ( dcs ) , the main antigen presenting cells of the innate immune response .", "when compared to l. braziliensis parasites , for example , l. amazonensis parasites are much less capable of triggering the expression of cd40 and cd80 , both costimulatory molecules for t - cell activation , and the production of il-12p40 . actually , amastigote infection is able to downregulate the expression of mhc class ii molecules , which , during macrophage infection , is depending on sequestering these molecules inside the parasitophorous vacuole for degradation [ 26 , 27 ] . during the first week of infection in c57bl/6 mice", ", chemokines such as ccl5 , ccl3 , ccl2 , ccl4 , and ccl11 as well as their receptors , are not upregulated when compared to l. major infection , both at the lesion site and draining lymph node .", "additionally , amastigote infection downregulates several intracellular pathways that lead to dc activation such as stat 1 , stat 3 and erk 1/2 phosphorylation and the expression of the interferon - responsive elements irf8 and 1 , suggesting a global inhibition of inflammatory responses of these cells .", "the most well - characterized ligand for amastigote recognition and internalization in macrophages is the opsonizing antibodies produced throughout infection .", "triggering of fc receptors on the host cells lead to il-10 production and has a pathogenic role .", "these events are necessary to evade the early immune response culminating with the ineffective t - cell response observed in most cases . in parallel to l. major infection in balb / c mice , where the oligoclonal v4 v8 cd4 t - cell population is necessary to the development of susceptibility in the host and is considered as pathogenic t cells , in l. amazonensis infection , t cells , in a general way , seem to be highly pathogenic .", "first of all , there is no clonal or oligoclonal t - cell population involved , since there is no predominance of a single or a group of v chains expressed on t cells that respond to l. amazonensis infection .", "however , the disruption of cd4 t cell effector functions , as observed in recombinase - activating gene ko mice ( rag ko ) , mhc class ii transactivator ko mice ( ciita ko ) and nude mice leads to transient resistance to l. amazonensis infection , measured by lesion development .", "in addition , the adoptive transfer of regulatory t cells also restrains pathogenic effector t cells , diminishing lesion size and parasite tissue loads .", "the mechanisms underlying pathogenic role of t cells for the disease need to be determined ."], ["phosphatidylserine ( ps ) is a structural phospholipid present in virtually all membranes and cell types . in normal cells", "these molecules face the cytoplasmic leaflet of the plasma membrane , whereas during apoptotic cell death these molecules translocate to the outer surface .", "once outside the cell , ps becomes one of the ligands recognized by surrounding phagocytes to clear dying cells .", "ps is the most characterized tickling   ligand of apoptotic cells , which means that ps provides the signals for the phagocyte to activate immunosuppressive and anti - inflammatory mechanisms .", "ps recognition is mandatory to prevent the establishment of a response to the self - antigens engulfed by these cells during apoptotic cell clearance and to avoid triggering inflammatory responses , especially during the embryogenesis , when massive amounts of apoptotic cells are generated and therefore , cleared [ 3537 ] , but also in adults to prevent inflammatory immunopathologies .", "the intracellular events , receptors , and soluble factors involved in this mechanism are still being deciphered and are not the focus of this discussion .", "however , the effects of ps recognition in macrophages and dcs have a direct impact in immune responses .", "apoptotic cells actively induce the production of the anti - inflammatory cytokines tgf , pge2 , and paf   and actively inhibit the production of tnf and il-1 , even upon lps challenge [ 35 , 38 ] .", "recognition of apoptotic cells also decreases the expression of several activation markers and costimulatory molecules by both human and murine dcs [ 39 , 40 ] and regulates the expression of cytokines involved with t - cell differentiation at the transcriptional level [ 41 , 42 ] . at the single cell level , dcs that ingested an apoptotic cell and bacteria at the same time are able to discern between them and only present bacterial antigens", "this is possible because the generation of peptide - mhc class ii complexes is controlled by toll - like receptors ( tlrs ) in a strictly phagosome autonomous manner . since apoptotic cells do not trigger tlr activation , the generation of stable complexes is inhibited or abrogated .", "all these effects are fundamental to maintain homeostasis and comprehend the last step of the efferocytosis   or apoptotic cell clearance .", "however , it seems that intracellular parasites elegantly make use of these mechanisms to establish in the host [ 4547 ] .", "furthermore , some parasites mimic the features of apoptotic cells to avoid host immune response , as discussed in the next section .", "one of the most common pcd phenotypes is phosphatidylserine ( ps ) exposure , which can be observed upon chemotherapy , starvation , and heat shock conditions in several unicellular organisms [ 4851 ] or is actively displayed in normal conditions .", "our group observed that lesion - derived amastigotes of l. amazonensis actively expose high levels of ps , and by blocking this molecule there is a drastic decrease in the ability of these parasites to infect and establish in the macrophages .", "these parasites are viable and capable of differentiating into promastigote forms in vitro ( unpublished data ) and inside the sand fly vector   and to infect macrophages and mice [ 52 , 54 ] and did not display other markers of pcd .", "ps exposure on amastigotes of l. amazonensis occurs in virtually 100% of the parasites ; however , the amount of ps molecules depends on the infected host .", "parasites obtained from balb / c mice expose higher amounts of ps than the ones obtained from c57bl/6 mice .", "this observation demonstrates that the amount of ps at the surface of the amastigotes has a positive correlation with the severity of the disease and suggests that the host is able to modulate this phenotype of the parasite .", "following our description several other groups demonstrated the role of ps exposure and recognition in different infection models .", "blood and cell - derived trypomastigotes of trypanosoma cruzi are able to expose ps , in contrast with epimastigotes , which are not . in addition , infection with ps - exposing trypomastigote forms induces smad nuclear translocation and inducible nitric oxide synthase inhibition ( inos ) , suggesting an autocrine modulation of the host cell dependent on tgf .", "it is interesting to note that , among all t. cruzi parasite stages , only the ones that are infective for mammalian cells evolve the ability to expose ps , suggesting the presence of an evolutionary link between ps exposure and the ability to infect host cells .", "similarly , toxoplasma gondii peritoneal tachyzoites expose ps at their surface , and the recognition of this molecule seems to be necessary to downmodulate inos expression and activity upon macrophage infection .", "more recently , several papers have demonstrated the role of exposed ps molecules for the infection by enveloped viral particles .", "for human immunodeficiency virus-1 ( hiv-1 ) , ps at the viral envelope is a cofactor for monocyte infection ; in vaccinia virus infection , ps recognition modulates the activity of proteins involved in cytoskeleton reorganization such as p21-activated kinase ( pak ) and the small rho gtpase rac , leading to increased macropinocytic activity and uptake of viral particles .", "in addition , ps exposure by tumor cell , microvesicles shed by transformed cells , or endothelial cells in the intratumor environment seems to be involved in different events in tumor development , maintenance , and metastasis [ 59 , 60 ] .", "this knowledge stimulated some researchers to evaluate the efficacy of anti - ps antibodies to treat viral and tumoral diseases .", "actually the results so far are promising . in murine models of lassa fever ( pichinde virus ) or murine", "cytomegaloviruses the treatment efficacy was very high , reaching complete cure ( total absence of detectable viral loads ) in combination with available antiaviral drugs . for experimental tumoral disease , lung cancer , pancreatic tumors , and glioblastomas", "were efficiently treated , decreasing tumor growth and metastasis in some cases or potentiating the effect of chemo- and radio - therapies [ 6264 ] .", "our group has been committed to study the role of ps exposure on the surface of different isolates of l. amazonensis .", "we worked with the hypothesis that l. amazonensis isolates from dcl patients would have higher ps exposure compared with localized cutaneous leishmaniasis ( lcl ) , and this would contribute to macrophage deactivation , favoring parasite replication . for this , we compared ps exposure in l. amazonensis isolates from dcl clinical cases in the active phase of the disease , reported in maranho state in brazil , to those isolated from lcl patients of clinical cases from bahia .", "the results indicate that the isolates obtained from dcl patients indeed displayed more ps than isolates from lcl patients at early times postinfection .", "in addition , isolates from dcl patients were more infective than the ones obtained from lcl patients ( frana - costa et al .", "the other hand , independent of parasite strain analyzed , the parameters of infectivity correlated positively with the exposure of ps in the parasites .", "these data suggest that in human infections the pattern observed in mice when comparing balb / c versus c57bl/6 mice is maintained .", "however , it is necessary to investigate the mechanisms by which the recognition of ps on the surface of the isolates of l. amazonensis deactivate the macrophage response .", "particularly , it would be necessary to evaluate whether freshly isolated parasites display this phenotype to validate our analysis made on amastigotes derived from macrophages infected in vitro with cultured promastigote parasites isolated from human lesions .", "we believe that understanding the dynamics of ps expression , along with identification of the mechanisms involved in the immunosuppression of dcl patients , can result in therapeutic targets for intervention in the immunopathogenesis of this chronic and severe form of leishmaniasis .   in a similar way we are interested in the immunomodulatory mechanisms underlying ps exposure in different inbred mice strains . for that we are currently evaluating these mechanisms during balb / c infection , which induces high levels of ps exposure on intracellular amastigotes . we observed that ps exposure is intrinsic to the intracellular parasite and", "however , these levels are dramatically increased when infected macrophages are in the presence of previously primed t cells or their soluble products .", "we confirmed these results by infecting balb / c nude mice where we observed that the amastigotes obtained from these mice display minimal levels of ps , which are completely restored if we adoptively transfer primed cd4 t cells to nude mice ( wanderley et al .", "interestingly , these data indicate that one possible role for the previously reported pathogenic t cells   would be to induce ps exposure on intracellular amastigotes and , therefore , contributing to the generation of highly infective parasites .", "the t - cell - dependent ps exposure on amastigotes seems to be dependent on the induction of inos expression on host macrophages , and parasite survival is dependent on the concomitant induction of arginase 1 expression ( wanderley et al .", "we propose that high levels of ps exposure are induced by parasite stress delivered by inos activity .", "in this case , it is still unknown whether ps exposure on amastigotes is indeed a phenotype triggered by pcd or a specific process involving modulation of ps translocation . under ps - inducting conditions ,", "unpublished results ) , that is the enzyme necessary to produce ornithine , the precursor of polyamines . in this situation", ", polyamines could protect the parasite from the inos - dependent stress , stimulating parasite growth [ 66 , 67 ] and increasing dna stabilization [ 68 , 69 ] .", "we understand that the unique characteristics of the t - cell response to l. amazonensis infection contribute to the generation of a perfect environment to stimulate and maintain increased levels of ps on the surface of intracellular parasites .", "probably the balance observed in infected balb / c mice , when disrupted , leads to the differences observed among different mouse strains . in figure 1", "we summarized our hypothesis regarding the t - cell - dependent modulation of ps exposure on intracellular amastigotes of l. amazonensis ."], ["the observation of ps exposure as a strategy to evade the immune system and persist in the mammalian host , made initially in the experimental model of l. amazonensis infection , was a breakthrough since it stimulated different groups around the world to look for the possibilities for basic and applied research on the field .", "our group is still studying the immunological , cellular , and molecular mechanisms underlying control of ps exposure in parasites and the effects of its recognition by parasitized cells and organisms .", "we believe that this could be a major strategy in different systems where avoidance from immune surveillance is necessary to establish a disease ."]]}
{"article_id": "PMC3713951", "article_text": ["despite of presentation of medical ethics as a new science in academic teaching , the ethical concepts have been alongside the medicine , and its antiquity back to the medicine history .", "for instance , literatures such as hippocratic oath letter , liturgy of ibn maymun , and shirazi ethics ordinance are the old literatures in which the principles such as the necessity of patient preference defender on the physician and observing the principle of confidentiality have been emphasized . however , in the past literature using  physician s commitment and pledge ", "human being is a creature with physical , mental and spiritual dimensions which has rights during the health and illness .", "patient rights are the very expectations he has from the health care services and must encompass his physical , mental , spiritual and social needs which are manifested as criteria , standards , rules and laws ( 4 , 5 ) .", "emphasis on patient rights in the health care services particularly maintains patient dignity as a rank of a human , and is considered important especially when patient s vulnerability easily expose him to the violations and weaknesses of the health care system ( 6 ) .", "considering that health is the most important existence aspect of every person and according to the article 29 of the constitution , providing health is the most important commitment of the government of islamic republic of iran ; because of this , in 2002 , for the first time the patients right charter was developed in iran and was notified by department of health , treatment and medical education ( 7 )", ". the patients right charter of iran was approved by health policy council with a new and comprehensive viewpoint aimed to clarify the rights of the health services recipients and observance of moral standards in the treatment and medical fields in november 26th , and in december 1st it was communicated to the relevant centers ( 1 , 8 , 9 ) . in this charter patient satisfaction is considered as one of the characteristics of hospital effectiveness ( 9 ) .", "today , the issues related to the quality of health care services , attention to the patients as customers and accomplishing their satisfaction are the main priorities and are of high importance .", "one of the important factors in patient satisfaction is regarding their demands and observing their rights and providing care along with respect ( 6 ) .", "awareness of the patients rights and observing them accomplishes more satisfaction of the patient , physician and other medical team and hospital staff and will lead to the spread of good morals among patients and medical team ; so eventually the moral status of all the individuals such as patients and medical team will be upgraded , but otherwise provided not observing these rights , it would lead to distrust to health care team . if there is no trust between medical staff and patients , it would lead to damages and losses for the patient and the medical team .", "furthermore , it would lead to terrible and unpleasant occurrences which are difficult to compensate and would be followed by the legal prosecution ( 5 , 10 ) . protecting the patient rights by the nurses", "only will be possible when they have gained necessary knowledge about it and suitable conditions be provided for respecting these rights ( 11 ) .", "appropriate care and observing patient rights require nurses knowledge which would be possible through different ways such as side studies , retraining courses , and academic courses during education ( 5 ) . in the studied", "researches about the nurses level of knowledge from patient rights , several aspects were mentioned .", "nasiriani et al ( 5 ) and houshmand et al ( 11 ) reported good nurses level of knowledge in yazd hospitals and teaching public wards of tehran hospitals respectively .", "observed moderate and parsinia et al obtained weak nurses knowledge in tehran and karaj hospitals respectively ( 10 ) .", "the clinical researchers in different wards of teaching hospitals have reported that the patients rights have been ignored or have not been considered seriously due to shortage of nursing staff , high number of patients in the hospitals , psychological pressures and etc .", "taken together , we decided to evaluate the nurses knowledge about patients rights in one of the teaching hospitals of tehran city .", "this was a descriptive cross - sectional study in which the nurses knowledge about patient rights was determined .", "the study samples consisted of 156 nurses who have been selected randomly from one of the teaching hospitals of tehran .", "the data collection instrument was a two - part questionnaire ; the first part included the demographic information ( age , gender , marital status , work experience , educational level , etc . ) ; the second part consisted of 10 questions according to the 10 section of patients rights charter of iran ( 11 ) .", "this part determined the following areas about patient rights : right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers .", "right to receive respectful and quick treatment and care regardless of cultural and racial factors .", "right to know about the probable complications , treatment options and participate in the ultimate treatment choosing .", "right to make a decision about the presence of those who are not directly involved in the treatment process .", "right to announce personal satisfaction from ending the treatment and referring to other centers , and right to preserve privacy and being ensured about confidentiality of all of the medical information .", "the answers of the study subjects to the questions were quantified by measuring a three - score scale ;  good  ( 3 scores ) ,  moderate  ( 2 scores ) and  weak  ( 1 scores ) .", "the maximum score in the questionnaire was 30 scores which were considered 2130 as high knowledge , 1020 as moderate knowledge and less than 10 as weak knowledge .", "the validity was done using content validity i.e. the questionnaire was given to 10 faculty members of universities of tehran and after collecting the comments , the relevant comments were applied .", "the validated questionnaire was given to 10 eligible study subjects for reliability and in both stages the questionnaire was completed with a 10-day interval and the required correlation of the first and second answers and confidence was obtained r = 0.90 and finally these people were excluded from the study population .", "the questionnaires were completed during two weeks by direct referring of the researcher to the wards . in order to observe ethics , confidentiality and integrity ,", "the mentioned questionnaires were anonymous and in all stages of the study the information were collected confidential and were kept by the researcher .", "the review of the demographic variables indicated that the majority of the nurses were females ( 76.28% ) with mean age of 34.31  7.3 .", "most of them ( 91.2% ) had bachelor degree and were married ( 62.82% ) . in terms of employment and work experience , almost half of nurses ( 47.43% ) were contractual , 46.79% had 610 years of work experiences , 30.99% never passed any course about  patient rights  and 30.99% of them had the simultaneous experience in public and private sectors .", "no association between the variables of gender , age , degree and marital status and nurses knowledge about patient rights was found .", "there was no significant difference between them in terms of work section , work experience and simultaneous work in the public and private sectors , however there was a significant difference between their level of knowledge about patient rights and simultaneous work in the public and private hospitals and work experience ( table 1 ) .", "the findings of the study about the nurses level of knowledge in different areas indicated that the highest level of knowledge ( 95.51% ) was in the area of  right to preserve privacy and being ensured about confidentiality of all medical information  and the lowest level was  right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers  .", "observing patients rights is the most important ethical issue in a hospital which should absolutely be considered . regarding patients rights and respecting them are two main factors for patients care .", "observing patients rights means the accountability of all health care staff to the patients at the time of treatment and care giving ( 12,13 ) .", "promoting patients rights is a multidimensional issue and in order to achieve it , comprehensive efforts should be done .", "world health organization has offered some strategies such as active participation of health care recipients and providers policy making and extending educational programs for health care providers and entire community ( 11 ) .", "the findings of the study showed that nurses level of knowledge about patients rights in terms of the variables such as type of the hospital , work experience , degree , age and gender was good , moderate and weak in 58.33% , 39.10% , and 2.56% of them respectively . in the studies conducted in karaj hospitals ( 10 ) and the public wards of teaching hospitals of tehran ( 11 ) also the nurses level of knowledge reported as weak .", "today patients are more aware of their rights and physicians responsibilities and commitments and also hospitals managements regarding their rights ( 14 ) and they are more insisting on their principle rights ( 2 ) . therefore it is highly needed that nurses with low level of knowledge , and the other health care providers be aware of patients right charter and increase their knowledge .", "determined the reasons of the nurses low level of knowledge as lack of institutionalization and regulation of the rights ( 12 ) ; lack of adequate time for studying and researching due to various obstacles such as poor economic conditions ; lack of positive vision in selecting nursing profession ; tough job conditions in the hospitals such as large number of the patients versus staff shortages , and lack of necessary facilities such as adequate and suitable libraries ( 5 ) .", "the results indicated that there was a significant and direct association between level of knowledge and simultaneous jobs in the public and private hospitals ( p=0.01 ) i.e. the nurses who provided services simultaneously in the public and private hospitals had higher level of knowledge .", "the reason might be due to regulations and rules which are performed more in the private hospitals compared to the public hospitals i.e. no implementation of patients right charter which may be followed by punishment .", "the findings of the present study indicated that in the area of  right to preserve privacy and being ensured about confidentiality of all the medical information  , the nurses had the highest level of knowledge . in the patients", "right charter codified in 2009 it says observance of the principle of confidentiality is necessary about all the information related to the patient except for the cases the law excluded ( 1 ) . in a comparative study about the patients right charter in the selected countries with iran in 2007 , it was indicated that regulations of iran for the right of the confidentiality of the patient information and medical records is similar to the other countries such as hungary , hong kong , new zealand , united states , south africa , european union and lithuania ( 14 ) . unlike the above results , in canada", "it was indicated that 84% of the medical staff did not have enough knowledge about commitments and obligations of the law about confidentiality of the information and privacy of the patients information ( 15 ) .", "our findings indicated that there was a direct and significant association between level of knowledge and work experience ( p= 0.008 ) and the level of knowledge of the study subjects from patients rights charter was increased by increasing work experience which was in accordance with the studies of nasiriani et al . and", "in addition , the study indicated that there was no association between level of knowledge and degree and increasing degree had no effect on the level of knowledge . in a study in karaj", "it was mentioned that the level of knowledge of the technicians was higher in comparison with supervisors and matrons and the most important reasons were sense of job security , professional status and disregardness of matrons and supervisors to the important tasks ( 10 ) .", "the findings indicated that nurses in the area of  right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers  had the lowest level of knowledge .", "the patients right charter codified in 2009 states that information must be given to the patient at the right time and appropriate condition by considering individual characteristics such as language , degree and perception ; however the patient has the right to access all the information recorded in his / her clinical records ( 16 , 17 ) . in the patients right charter of some countries such as canada and united states unlike the other countries such as iran , hong kong , new zealand , africa , european union , lithuania ,", " right to ask for explanation about the costs  has been discussed ( 14 ) .", "the results indicated that 66% of the nurses was aware of the  right to access to the health care providers during hospitalization  , instead of 90% of turkish nurses ( 17 ) . nowadays ethical and legal concepts such as patient rights are included in the educational curriculum of greek and turkey ( 18 , 19 ) .", "according to the considerable gap between development and realization of the patient rights ( 20 ) and the increased knowledge , claims and demands of the patients and also the results of this study , in order to observe patients right the following solutions are suggested : - encouraging the nurses to consider patients right charter seriously . - establishing and empowering the sanction of patients right charter - more emphasizing on concepts of professional ethics and patient rights in teaching nursing students - attending expertise meetings with presence of beneficiaries for evaluating barriers and presenting strategies in order to implement the patients right charter as soon as possible - providing periodic educational programs for health care providers and patients about patient rights", "generally , since 41.6% of the nurses of the study had not a proper level of knowledge about patient rights and considering the fact that awareness and knowledge can be the base of nurses performance and also patients increasingly are getting informed about their rights , the implementation of patients right charter is highly recommended .", "holding educational programs , seminars , workshops and academic panels for nurses , and nursing students help overcome difficulties ."], "abstract_text": ["<S> patients rights observance is one of the effective measures of patients satisfaction of health care services . </S>", "<S> we performed this study at the aim of evaluation of nurses awareness of patients rights in a teaching hospital in tehran.this cross - sectional study was conducted in 2010 . in this study 156 </S>", "<S> nurses were randomly selected . </S>", "<S> two - part questionnaire was used for data collection . </S>", "<S> the validity and reliability of questionnaire was determined and then it was distributed between subjects . </S>", "<S> the data were analyzed by spss version 15 using descriptive and inferential statistics . </S>", "<S> our results showed that % 58.33 , % 39.10 and % 2.56 of nurses have good , medium , and poor levels of awareness respectively . </S>", "<S> we observed a significant relationship between nurses awareness and work experience ( p=0.008 ) and concurrent work in public and private hospitals ( p=0.01 ) . </S>", "<S> the most of the nurses ( % 95.51 ) were aware of  right to privacy protection and ensure confidentiality of information  and the least of them ( % 33.97 ) were aware of  right to receiving necessary information about the health care providers , the rate of tariff and insurance coverage.according to our survey it is concluded that implementation of patients right charter in this hospital is accompanied by some limitations which necessitates promotion of the nurses awareness about patients rights . </S>", "<S> taken together in order to enhance nurses awareness special measures and strategies should be considered . </S>"], "labels": null, "section_names": ["Introduction", "Method", "Results", "Discussion", "Conclusion"], "sections": [["despite of presentation of medical ethics as a new science in academic teaching , the ethical concepts have been alongside the medicine , and its antiquity back to the medicine history .", "for instance , literatures such as hippocratic oath letter , liturgy of ibn maymun , and shirazi ethics ordinance are the old literatures in which the principles such as the necessity of patient preference defender on the physician and observing the principle of confidentiality have been emphasized . however , in the past literature using  physician s commitment and pledge ", "human being is a creature with physical , mental and spiritual dimensions which has rights during the health and illness .", "patient rights are the very expectations he has from the health care services and must encompass his physical , mental , spiritual and social needs which are manifested as criteria , standards , rules and laws ( 4 , 5 ) .", "emphasis on patient rights in the health care services particularly maintains patient dignity as a rank of a human , and is considered important especially when patient s vulnerability easily expose him to the violations and weaknesses of the health care system ( 6 ) .", "considering that health is the most important existence aspect of every person and according to the article 29 of the constitution , providing health is the most important commitment of the government of islamic republic of iran ; because of this , in 2002 , for the first time the patients right charter was developed in iran and was notified by department of health , treatment and medical education ( 7 )", ". the patients right charter of iran was approved by health policy council with a new and comprehensive viewpoint aimed to clarify the rights of the health services recipients and observance of moral standards in the treatment and medical fields in november 26th , and in december 1st it was communicated to the relevant centers ( 1 , 8 , 9 ) . in this charter patient satisfaction is considered as one of the characteristics of hospital effectiveness ( 9 ) .", "today , the issues related to the quality of health care services , attention to the patients as customers and accomplishing their satisfaction are the main priorities and are of high importance .", "one of the important factors in patient satisfaction is regarding their demands and observing their rights and providing care along with respect ( 6 ) .", "awareness of the patients rights and observing them accomplishes more satisfaction of the patient , physician and other medical team and hospital staff and will lead to the spread of good morals among patients and medical team ; so eventually the moral status of all the individuals such as patients and medical team will be upgraded , but otherwise provided not observing these rights , it would lead to distrust to health care team . if there is no trust between medical staff and patients , it would lead to damages and losses for the patient and the medical team .", "furthermore , it would lead to terrible and unpleasant occurrences which are difficult to compensate and would be followed by the legal prosecution ( 5 , 10 ) . protecting the patient rights by the nurses", "only will be possible when they have gained necessary knowledge about it and suitable conditions be provided for respecting these rights ( 11 ) .", "appropriate care and observing patient rights require nurses knowledge which would be possible through different ways such as side studies , retraining courses , and academic courses during education ( 5 ) . in the studied", "researches about the nurses level of knowledge from patient rights , several aspects were mentioned .", "nasiriani et al ( 5 ) and houshmand et al ( 11 ) reported good nurses level of knowledge in yazd hospitals and teaching public wards of tehran hospitals respectively .", "observed moderate and parsinia et al obtained weak nurses knowledge in tehran and karaj hospitals respectively ( 10 ) .", "the clinical researchers in different wards of teaching hospitals have reported that the patients rights have been ignored or have not been considered seriously due to shortage of nursing staff , high number of patients in the hospitals , psychological pressures and etc .", "taken together , we decided to evaluate the nurses knowledge about patients rights in one of the teaching hospitals of tehran city ."], ["this was a descriptive cross - sectional study in which the nurses knowledge about patient rights was determined .", "the study samples consisted of 156 nurses who have been selected randomly from one of the teaching hospitals of tehran .", "the data collection instrument was a two - part questionnaire ; the first part included the demographic information ( age , gender , marital status , work experience , educational level , etc . ) ; the second part consisted of 10 questions according to the 10 section of patients rights charter of iran ( 11 ) .", "this part determined the following areas about patient rights : right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers .", "right to receive respectful and quick treatment and care regardless of cultural and racial factors .", "right to know about the probable complications , treatment options and participate in the ultimate treatment choosing .", "right to make a decision about the presence of those who are not directly involved in the treatment process .", "right to announce personal satisfaction from ending the treatment and referring to other centers , and right to preserve privacy and being ensured about confidentiality of all of the medical information .", "the answers of the study subjects to the questions were quantified by measuring a three - score scale ;  good  ( 3 scores ) ,  moderate  ( 2 scores ) and  weak  ( 1 scores ) .", "the maximum score in the questionnaire was 30 scores which were considered 2130 as high knowledge , 1020 as moderate knowledge and less than 10 as weak knowledge .", "the validity was done using content validity i.e. the questionnaire was given to 10 faculty members of universities of tehran and after collecting the comments , the relevant comments were applied .", "the validated questionnaire was given to 10 eligible study subjects for reliability and in both stages the questionnaire was completed with a 10-day interval and the required correlation of the first and second answers and confidence was obtained r = 0.90 and finally these people were excluded from the study population .", "the questionnaires were completed during two weeks by direct referring of the researcher to the wards . in order to observe ethics , confidentiality and integrity ,", "the mentioned questionnaires were anonymous and in all stages of the study the information were collected confidential and were kept by the researcher ."], ["the review of the demographic variables indicated that the majority of the nurses were females ( 76.28% ) with mean age of 34.31  7.3 .", "most of them ( 91.2% ) had bachelor degree and were married ( 62.82% ) . in terms of employment and work experience , almost half of nurses ( 47.43% ) were contractual , 46.79% had 610 years of work experiences , 30.99% never passed any course about  patient rights  and 30.99% of them had the simultaneous experience in public and private sectors .", "no association between the variables of gender , age , degree and marital status and nurses knowledge about patient rights was found .", "there was no significant difference between them in terms of work section , work experience and simultaneous work in the public and private sectors , however there was a significant difference between their level of knowledge about patient rights and simultaneous work in the public and private hospitals and work experience ( table 1 ) .", "the findings of the study about the nurses level of knowledge in different areas indicated that the highest level of knowledge ( 95.51% ) was in the area of  right to preserve privacy and being ensured about confidentiality of all medical information  and the lowest level was  right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers  ."], ["observing patients rights is the most important ethical issue in a hospital which should absolutely be considered . regarding patients rights and respecting them are two main factors for patients care .", "observing patients rights means the accountability of all health care staff to the patients at the time of treatment and care giving ( 12,13 ) .", "promoting patients rights is a multidimensional issue and in order to achieve it , comprehensive efforts should be done .", "world health organization has offered some strategies such as active participation of health care recipients and providers policy making and extending educational programs for health care providers and entire community ( 11 ) .", "the findings of the study showed that nurses level of knowledge about patients rights in terms of the variables such as type of the hospital , work experience , degree , age and gender was good , moderate and weak in 58.33% , 39.10% , and 2.56% of them respectively . in the studies conducted in karaj hospitals ( 10 ) and the public wards of teaching hospitals of tehran ( 11 ) also the nurses level of knowledge reported as weak .", "today patients are more aware of their rights and physicians responsibilities and commitments and also hospitals managements regarding their rights ( 14 ) and they are more insisting on their principle rights ( 2 ) . therefore it is highly needed that nurses with low level of knowledge , and the other health care providers be aware of patients right charter and increase their knowledge .", "determined the reasons of the nurses low level of knowledge as lack of institutionalization and regulation of the rights ( 12 ) ; lack of adequate time for studying and researching due to various obstacles such as poor economic conditions ; lack of positive vision in selecting nursing profession ; tough job conditions in the hospitals such as large number of the patients versus staff shortages , and lack of necessary facilities such as adequate and suitable libraries ( 5 ) .", "the results indicated that there was a significant and direct association between level of knowledge and simultaneous jobs in the public and private hospitals ( p=0.01 ) i.e. the nurses who provided services simultaneously in the public and private hospitals had higher level of knowledge .", "the reason might be due to regulations and rules which are performed more in the private hospitals compared to the public hospitals i.e. no implementation of patients right charter which may be followed by punishment .", "the findings of the present study indicated that in the area of  right to preserve privacy and being ensured about confidentiality of all the medical information  , the nurses had the highest level of knowledge . in the patients", "right charter codified in 2009 it says observance of the principle of confidentiality is necessary about all the information related to the patient except for the cases the law excluded ( 1 ) . in a comparative study about the patients right charter in the selected countries with iran in 2007 , it was indicated that regulations of iran for the right of the confidentiality of the patient information and medical records is similar to the other countries such as hungary , hong kong , new zealand , united states , south africa , european union and lithuania ( 14 ) . unlike the above results , in canada", "it was indicated that 84% of the medical staff did not have enough knowledge about commitments and obligations of the law about confidentiality of the information and privacy of the patients information ( 15 ) .", "our findings indicated that there was a direct and significant association between level of knowledge and work experience ( p= 0.008 ) and the level of knowledge of the study subjects from patients rights charter was increased by increasing work experience which was in accordance with the studies of nasiriani et al . and", "in addition , the study indicated that there was no association between level of knowledge and degree and increasing degree had no effect on the level of knowledge . in a study in karaj", "it was mentioned that the level of knowledge of the technicians was higher in comparison with supervisors and matrons and the most important reasons were sense of job security , professional status and disregardness of matrons and supervisors to the important tasks ( 10 ) .", "the findings indicated that nurses in the area of  right to receive essential information about health care providers , rate of tariff , target insurance coverage if sent to the other medical centers  had the lowest level of knowledge .", "the patients right charter codified in 2009 states that information must be given to the patient at the right time and appropriate condition by considering individual characteristics such as language , degree and perception ; however the patient has the right to access all the information recorded in his / her clinical records ( 16 , 17 ) . in the patients right charter of some countries such as canada and united states unlike the other countries such as iran , hong kong , new zealand , africa , european union , lithuania ,", " right to ask for explanation about the costs  has been discussed ( 14 ) .", "the results indicated that 66% of the nurses was aware of the  right to access to the health care providers during hospitalization  , instead of 90% of turkish nurses ( 17 ) . nowadays ethical and legal concepts such as patient rights are included in the educational curriculum of greek and turkey ( 18 , 19 ) .", "according to the considerable gap between development and realization of the patient rights ( 20 ) and the increased knowledge , claims and demands of the patients and also the results of this study , in order to observe patients right the following solutions are suggested : - encouraging the nurses to consider patients right charter seriously . - establishing and empowering the sanction of patients right charter - more emphasizing on concepts of professional ethics and patient rights in teaching nursing students - attending expertise meetings with presence of beneficiaries for evaluating barriers and presenting strategies in order to implement the patients right charter as soon as possible - providing periodic educational programs for health care providers and patients about patient rights"], ["generally , since 41.6% of the nurses of the study had not a proper level of knowledge about patient rights and considering the fact that awareness and knowledge can be the base of nurses performance and also patients increasingly are getting informed about their rights , the implementation of patients right charter is highly recommended .", "holding educational programs , seminars , workshops and academic panels for nurses , and nursing students help overcome difficulties ."]]}
{"article_id": "PMC2750235", "article_text": ["all subjects were consecutively recruited from the greater philadelphia area from 20062009 at the children 's hospital of philadelphia .", "our study cohort consisted of 5,465 singleton children of european ancestry with recorded birth weight information .", "all of these participants had their blood drawn in an 8-ml ethylenediamine tetraacetic acid blood collection tube and subsequently dna extracted for genotyping .", "this study was approved by the institutional review board of the children 's hospital of philadelphia .", "parental informed consent was given for each study participant for both the blood collection and subsequent genotyping .", "we performed high - throughput genome - wide single nucleotide polymorphism ( snp ) genotyping using the illumina infinium ii humanhap550 or human 610 beadchip technology ( illumina , san diego , ca ) at the children 's hospital of philadelphia 's center for applied genomics , as described previously ( 23 ) .", "the snps analyzed survived the filtering of the genome - wide dataset for snps with call rates < 95% , minor allele frequency < 1% , missing rate per person > 2% , and hardy - weinberg equilibrium p < 10 .", "most loci described from gwa studies published to date have been found using either the affymetrix or illumina platform . in the event", "a locus was reported using both the illumina and affymetrix arrays , we used the snps present on the illumina array . in the event of a signal", "only being described on the affymetrix array , we either already had that snp on our illumina array or identified and used the best surrogate snp available based on the ceu hapmap ( supplementary table 1 , available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0506/dc1 ) .", "we used two snps at the cdkal1 ( rs4712523 and rs7756992 ; r = 0.677 ) , hhex - ide ( rs1111875 and rs7923837 ; r = 0.698 ) , and pparg ( rs17793693 and rs6802898 ; r = 0.011 ) loci as the association with type 2 diabetes , taken from various gwa studies that reported various snps that were in imperfect linkage disequilibrium with each other .", "in addition , rs4712523 is a proxy ( r = 1 ) for rs10946398 , which was previously associated with birth weight . from our database", ", we eliminated outliers with birth weight < 1 or > 8 kg , i.e. , those individuals not within the credible range for birth weight at term , to avoid the potential consequences of error or mendelian causes of extreme birth weight .", "each birth weight value was adjusted for each sex separately then expressed as a z score .", "all statistical analyses were carried out using the software package plink v. 1.05 ( 24 ) .", "ethnicity for our cohort was derived using the multidimensional scaling feature within plink . by treating birth weight as a quantitative trait ( treated as a z score after correcting for sex ) , association analysis for each snp", "was carried out using linear regression analysis with the snp included as an independent variable ( coded as 0 , 1 , and 2 ) . with 5,465 subjects , the powers to detect 0.2 , 0.3 , 0.4 , 0.5 , 0.6 , 0.8 , and 1% variation at the p = 0.002 level ( i.e. , the corrected p value for the number of tests ) were 47.4 , 74.6 , 90.0 , 96.6 , 98.9 , 100 , and 100% , respectively .", "all subjects were consecutively recruited from the greater philadelphia area from 20062009 at the children 's hospital of philadelphia .", "our study cohort consisted of 5,465 singleton children of european ancestry with recorded birth weight information .", "all of these participants had their blood drawn in an 8-ml ethylenediamine tetraacetic acid blood collection tube and subsequently dna extracted for genotyping .", "this study was approved by the institutional review board of the children 's hospital of philadelphia .", "parental informed consent was given for each study participant for both the blood collection and subsequent genotyping .", "all subjects were consecutively recruited from the greater philadelphia area from 20062009 at the children 's hospital of philadelphia .", "our study cohort consisted of 5,465 singleton children of european ancestry with recorded birth weight information .", "all of these participants had their blood drawn in an 8-ml ethylenediamine tetraacetic acid blood collection tube and subsequently dna extracted for genotyping .", "this study was approved by the institutional review board of the children 's hospital of philadelphia .", "parental informed consent was given for each study participant for both the blood collection and subsequent genotyping .", "we performed high - throughput genome - wide single nucleotide polymorphism ( snp ) genotyping using the illumina infinium ii humanhap550 or human 610 beadchip technology ( illumina , san diego , ca ) at the children 's hospital of philadelphia 's center for applied genomics , as described previously ( 23 ) .", "the snps analyzed survived the filtering of the genome - wide dataset for snps with call rates < 95% , minor allele frequency < 1% , missing rate per person > 2% , and hardy - weinberg equilibrium p < 10 .", "most loci described from gwa studies published to date have been found using either the affymetrix or illumina platform . in the event", "a locus was reported using both the illumina and affymetrix arrays , we used the snps present on the illumina array . in the event of a signal", "only being described on the affymetrix array , we either already had that snp on our illumina array or identified and used the best surrogate snp available based on the ceu hapmap ( supplementary table 1 , available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0506/dc1 ) .", "we used two snps at the cdkal1 ( rs4712523 and rs7756992 ; r = 0.677 ) , hhex - ide ( rs1111875 and rs7923837 ; r = 0.698 ) , and pparg ( rs17793693 and rs6802898 ; r = 0.011 ) loci as the association with type 2 diabetes , taken from various gwa studies that reported various snps that were in imperfect linkage disequilibrium with each other .", "in addition , rs4712523 is a proxy ( r = 1 ) for rs10946398 , which was previously associated with birth weight .", "we performed high - throughput genome - wide single nucleotide polymorphism ( snp ) genotyping using the illumina infinium ii humanhap550 or human 610 beadchip technology ( illumina , san diego , ca ) at the children 's hospital of philadelphia 's center for applied genomics , as described previously ( 23 ) .", "the snps analyzed survived the filtering of the genome - wide dataset for snps with call rates < 95% , minor allele frequency < 1% , missing rate per person > 2% , and hardy - weinberg equilibrium p < 10 .", "most loci described from gwa studies published to date have been found using either the affymetrix or illumina platform . in the event", "a locus was reported using both the illumina and affymetrix arrays , we used the snps present on the illumina array . in the event of a signal", "only being described on the affymetrix array , we either already had that snp on our illumina array or identified and used the best surrogate snp available based on the ceu hapmap ( supplementary table 1 , available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0506/dc1 ) .", "we used two snps at the cdkal1 ( rs4712523 and rs7756992 ; r = 0.677 ) , hhex - ide ( rs1111875 and rs7923837 ; r = 0.698 ) , and pparg ( rs17793693 and rs6802898 ; r = 0.011 ) loci as the association with type 2 diabetes , taken from various gwa studies that reported various snps that were in imperfect linkage disequilibrium with each other .", "in addition , rs4712523 is a proxy ( r = 1 ) for rs10946398 , which was previously associated with birth weight .", "from our database , we eliminated outliers with birth weight < 1 or > 8 kg , i.e. , those individuals not within the credible range for birth weight at term , to avoid the potential consequences of error or mendelian causes of extreme birth weight .", "each birth weight value was adjusted for each sex separately then expressed as a z score .", "from our database , we eliminated outliers with birth weight < 1 or > 8 kg , i.e. , those individuals not within the credible range for birth weight at term , to avoid the potential consequences of error or mendelian causes of extreme birth weight .", "each birth weight value was adjusted for each sex separately then expressed as a z score .", "all statistical analyses were carried out using the software package plink v. 1.05 ( 24 ) .", "ethnicity for our cohort was derived using the multidimensional scaling feature within plink . by treating birth weight as a quantitative trait ( treated as a z score after correcting for sex ) , association analysis for each snp", "was carried out using linear regression analysis with the snp included as an independent variable ( coded as 0 , 1 , and 2 ) . with 5,465 subjects , the powers to detect 0.2 , 0.3 , 0.4 , 0.5 , 0.6 , 0.8 , and 1% variation at the p = 0.002 level ( i.e. , the corrected p value for the number of tests ) were 47.4 , 74.6 , 90.0 , 96.6 , 98.9 , 100 , and 100% , respectively .", "in our initial analysis , 12 snps corresponding to the 9 type 2 diabetes loci previously studied in the context of birth weight were investigated in our cohort , namely , tcf7l2 , hhex - ide , pparg , kcnj11 , slc30a8 , igf2bp2 , cdkal1 , cdkn2a/2b , and jazf1 ( 4,6 ) ( table 1 ) .", "quantitative association results for previously studied type 2 diabetes risk alleles with birth weight in the european american cohort ( n = 5,465 ) , sorted by chromosomal location the direction of effect is shown for the minor allele in each case . *", "major allele previously reported to be associated with type 2 diabetes ; * * p  0.002 .", " , regression coefficient for the test snp ; bp , base pair position ; maf , minor allele frequency ; n , number of subjects tested ; p , two - sided trend test p value ; r , value in linear regression ; t , test statistic . as a result ,", "we observed strong association with rs7756992 ( p = 8 10 ) at the cdkal1 locus with low birth weight ; this snp yielded strongest association to type 2 diabetes in an icelandic gwa study carried out on the illumina humanhap500 platform ( 21 ) .", "snps rs10946398 or rs7754840 at the same locus have been reported to be most strongly associated with type 2 diabetes from gwa studies on the affymetrix platform or the illumina humanhap300 beadchip ( 16,18,19 ) ; however , using a perfect surrogate , rs4712523 ( r = 1 ) , we only observed nominally significant association ( p = 0.01 ) .", "it should be noted that rs10946398 and rs7756992 are far from being in perfect linkage disequilibrium ( r = 0.677 ) , thus the inclusion of both in this current study . unlike previous reports", ", we did not observe association between rs1111875 at the hhex - ide locus and this trait ( 6 ) . in line with previous reports", ", we also did not observe association between birth weight and tcf7l2 , pparg , kcnj11 , slc30a8 , igf2bp2 , cdkn2a/2b , or jazf1 ( 4,6,10 ) .", "furthermore , we did not observe any significant association with risk alleles at other type 2 diabetes loci after correction for multiple testing for all 23 snps ( threshold p  0.002 ) ( supplementary table 2 ) .", "we detected nominal association with rs1387153 ( p = 0.02 ) at the mtnr1b locus ; however , the corresponding type 2 diabetes risk allele was tracking with higher birth weight .", "we also analyzed male and female subjects separately , but the effect of each locus on birth weight did not vary by sex ( supplementary tables 3 and 4 ) .", "from this interim analysis of our ongoing gwa study of birth weight in a european american cohort , it is clear that the cdkal1 locus , which was uncovered in gwa analyses of type 2 diabetes , is strongly associated with birth weight in our study population .", "this result clearly supports a previous report that came to a similar conclusion ( 6 ) .", "however , the study by freathy et al . used a different snp , namely , rs10946398 , which was not present on our illumina beadchip ; we used a perfect surrogate , i.e. , rs4712523 ( r = 1 ) , that only yielded nominal significance ( p = 0.01 ) .", "although they did not report for rs7756992 , we found that it gave us the strongest association ( p = 8 10 ) and was selected for this study because it yielded the strongest association to type 2 diabetes in an icelandic gwa study ( 21 ) .", "secondly , we did not observe association between hhex - ide and birth weight , which is in contrast with what had been described previously ( 6 ) .", "we acknowledge that our cohort is smaller than the original report ( 5,465 vs. 19,200 individuals ) ; indeed , this association was not observed ( p < 0.05 ) in the similarly sized 1958 birth cohort ( 6 ) .", "the lack of available covariate data , such as gestational age , was also a limitation of this study .", "therefore , it is possible that with a larger cohort with additional covariate data we may observe the association of this locus with birth weight ; however , it could also indicate that hhex - ide has a less pronounced impact on birth weight than cdkal1 .", "consistent with the existing literature , we did not find any evidence of association between birth weight and tcf7l2 , pparg , kcnj11 , slc30a8 , igf2bp2 , cdkn2a/2b , or jazf1 ( 4,6,10 ) . given the monogenic precedent for opposing effects of maternal and fetal genotype ( 25 ) , it is possible that effects of common type 2 diabetes alleles could be masked by this phenomenon .", "it has been shown that cdkal1 is expressed in the rat pancreatic -cell line ins-1 ( 21 ) .", "homozygous carriers of the risk allele have been shown to have a 22% lower corrected insulin response than individuals who are wild - type carriers .", "it has been suggested that cdkal1 might influence the secretion of insulin by interacting with cdk5 ( 21 ) .", "our data contributes another piece of evidence supporting the hypothesis , namely , that the same genotype conferring lower birth weight can also confer higher type 2 diabetes risk later in life .", "cdkal1 was first described in the context of type 2 diabetes in both european caucasians and in han chinese ( 21 ) ; as such , it would be interesting to examine whether the association of cdkal1 with lower birth weight also stands in this and other ethnicities , such as african americans and hispanics . in conclusion", ", we strongly confirm that the established type 2 diabetes locus cdkal1 also influences birth weight .", "however , we do not observe such association with tcf7l2 , hhex - ide , cdkn2a/2b , or jazf1 .", "in addition , of all the other established type 2 diabetes loci to date , we do not observe a convincing role for them in the determination of birth weight ."], "abstract_text": ["<S> objectivea number of studies have found that reduced birth weight is associated with type 2 diabetes later in life ; however , the underlying mechanism for this correlation remains unresolved . </S>", "<S> recently , association has been demonstrated between low birth weight and single nucleotide polymorphisms ( snps ) at the cdkal1 and hhex - ide loci , regions that were previously implicated in the pathogenesis of type 2 diabetes . in order to investigate whether type 2 diabetes risk  conferring alleles associate with low birth weight in our caucasian childhood cohort , we examined the effects of 20 such loci on this trait.research design and methodsusing data from an ongoing genome - wide association study in our cohort of 5,465 caucasian children with recorded birth weights , we investigated the association of the previously reported type 2 diabetes  associated variation at 20 loci including tcf7l2 , hhex - ide , pparg , kcnj11 , slc30a8 , igf2bp2 , cdkal1 , cdkn2a/2b , and jazf1 with birth weight.resultsour </S>", "<S> data show that the minor allele of rs7756992 ( p = 8  105 ) at the cdkal1 locus is strongly associated with lower birth weight , whereas a perfect surrogate for variation previously implicated for the trait at the same locus only yielded nominally significant association ( p = 0.01 ; r2 rs7756992 = 0.677 ) . </S>", "<S> however , association was not detected with any of the other type 2 diabetes loci studied.conclusionswe observe association between lower birth weight and type 2 diabetes risk  conferring alleles at the cdkal1 locus . </S>", "<S> our data show that the same genetic locus that has been identified as a marker for type 2 diabetes in previous studies also influences birth weight . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Research subjects", "European American Pediatric cohort from Philadelphia.", "Genotyping", "Illumina Infinium assay.", "Analysis", "Normalization of birth weight data.", "Association.", "RESULTS", "DISCUSSION"], "sections": [["all subjects were consecutively recruited from the greater philadelphia area from 20062009 at the children 's hospital of philadelphia .", "our study cohort consisted of 5,465 singleton children of european ancestry with recorded birth weight information .", "all of these participants had their blood drawn in an 8-ml ethylenediamine tetraacetic acid blood collection tube and subsequently dna extracted for genotyping .", "this study was approved by the institutional review board of the children 's hospital of philadelphia .", "parental informed consent was given for each study participant for both the blood collection and subsequent genotyping .", "we performed high - throughput genome - wide single nucleotide polymorphism ( snp ) genotyping using the illumina infinium ii humanhap550 or human 610 beadchip technology ( illumina , san diego , ca ) at the children 's hospital of philadelphia 's center for applied genomics , as described previously ( 23 ) .", "the snps analyzed survived the filtering of the genome - wide dataset for snps with call rates < 95% , minor allele frequency < 1% , missing rate per person > 2% , and hardy - weinberg equilibrium p < 10 .", "most loci described from gwa studies published to date have been found using either the affymetrix or illumina platform . in the event", "a locus was reported using both the illumina and affymetrix arrays , we used the snps present on the illumina array . in the event of a signal", "only being described on the affymetrix array , we either already had that snp on our illumina array or identified and used the best surrogate snp available based on the ceu hapmap ( supplementary table 1 , available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0506/dc1 ) .", "we used two snps at the cdkal1 ( rs4712523 and rs7756992 ; r = 0.677 ) , hhex - ide ( rs1111875 and rs7923837 ; r = 0.698 ) , and pparg ( rs17793693 and rs6802898 ; r = 0.011 ) loci as the association with type 2 diabetes , taken from various gwa studies that reported various snps that were in imperfect linkage disequilibrium with each other .", "in addition , rs4712523 is a proxy ( r = 1 ) for rs10946398 , which was previously associated with birth weight . from our database", ", we eliminated outliers with birth weight < 1 or > 8 kg , i.e. , those individuals not within the credible range for birth weight at term , to avoid the potential consequences of error or mendelian causes of extreme birth weight .", "each birth weight value was adjusted for each sex separately then expressed as a z score .", "all statistical analyses were carried out using the software package plink v. 1.05 ( 24 ) .", "ethnicity for our cohort was derived using the multidimensional scaling feature within plink . by treating birth weight as a quantitative trait ( treated as a z score after correcting for sex ) , association analysis for each snp", "was carried out using linear regression analysis with the snp included as an independent variable ( coded as 0 , 1 , and 2 ) . with 5,465 subjects , the powers to detect 0.2 , 0.3 , 0.4 , 0.5 , 0.6 , 0.8 , and 1% variation at the p = 0.002 level ( i.e. , the corrected p value for the number of tests ) were 47.4 , 74.6 , 90.0 , 96.6 , 98.9 , 100 , and 100% , respectively ."], ["all subjects were consecutively recruited from the greater philadelphia area from 20062009 at the children 's hospital of philadelphia .", "our study cohort consisted of 5,465 singleton children of european ancestry with recorded birth weight information .", "all of these participants had their blood drawn in an 8-ml ethylenediamine tetraacetic acid blood collection tube and subsequently dna extracted for genotyping .", "this study was approved by the institutional review board of the children 's hospital of philadelphia .", "parental informed consent was given for each study participant for both the blood collection and subsequent genotyping ."], ["all subjects were consecutively recruited from the greater philadelphia area from 20062009 at the children 's hospital of philadelphia .", "our study cohort consisted of 5,465 singleton children of european ancestry with recorded birth weight information .", "all of these participants had their blood drawn in an 8-ml ethylenediamine tetraacetic acid blood collection tube and subsequently dna extracted for genotyping .", "this study was approved by the institutional review board of the children 's hospital of philadelphia .", "parental informed consent was given for each study participant for both the blood collection and subsequent genotyping ."], ["we performed high - throughput genome - wide single nucleotide polymorphism ( snp ) genotyping using the illumina infinium ii humanhap550 or human 610 beadchip technology ( illumina , san diego , ca ) at the children 's hospital of philadelphia 's center for applied genomics , as described previously ( 23 ) .", "the snps analyzed survived the filtering of the genome - wide dataset for snps with call rates < 95% , minor allele frequency < 1% , missing rate per person > 2% , and hardy - weinberg equilibrium p < 10 .", "most loci described from gwa studies published to date have been found using either the affymetrix or illumina platform . in the event", "a locus was reported using both the illumina and affymetrix arrays , we used the snps present on the illumina array . in the event of a signal", "only being described on the affymetrix array , we either already had that snp on our illumina array or identified and used the best surrogate snp available based on the ceu hapmap ( supplementary table 1 , available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0506/dc1 ) .", "we used two snps at the cdkal1 ( rs4712523 and rs7756992 ; r = 0.677 ) , hhex - ide ( rs1111875 and rs7923837 ; r = 0.698 ) , and pparg ( rs17793693 and rs6802898 ; r = 0.011 ) loci as the association with type 2 diabetes , taken from various gwa studies that reported various snps that were in imperfect linkage disequilibrium with each other .", "in addition , rs4712523 is a proxy ( r = 1 ) for rs10946398 , which was previously associated with birth weight ."], ["we performed high - throughput genome - wide single nucleotide polymorphism ( snp ) genotyping using the illumina infinium ii humanhap550 or human 610 beadchip technology ( illumina , san diego , ca ) at the children 's hospital of philadelphia 's center for applied genomics , as described previously ( 23 ) .", "the snps analyzed survived the filtering of the genome - wide dataset for snps with call rates < 95% , minor allele frequency < 1% , missing rate per person > 2% , and hardy - weinberg equilibrium p < 10 .", "most loci described from gwa studies published to date have been found using either the affymetrix or illumina platform . in the event", "a locus was reported using both the illumina and affymetrix arrays , we used the snps present on the illumina array . in the event of a signal", "only being described on the affymetrix array , we either already had that snp on our illumina array or identified and used the best surrogate snp available based on the ceu hapmap ( supplementary table 1 , available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0506/dc1 ) .", "we used two snps at the cdkal1 ( rs4712523 and rs7756992 ; r = 0.677 ) , hhex - ide ( rs1111875 and rs7923837 ; r = 0.698 ) , and pparg ( rs17793693 and rs6802898 ; r = 0.011 ) loci as the association with type 2 diabetes , taken from various gwa studies that reported various snps that were in imperfect linkage disequilibrium with each other .", "in addition , rs4712523 is a proxy ( r = 1 ) for rs10946398 , which was previously associated with birth weight ."], ["from our database , we eliminated outliers with birth weight < 1 or > 8 kg , i.e. , those individuals not within the credible range for birth weight at term , to avoid the potential consequences of error or mendelian causes of extreme birth weight .", "each birth weight value was adjusted for each sex separately then expressed as a z score ."], ["from our database , we eliminated outliers with birth weight < 1 or > 8 kg , i.e. , those individuals not within the credible range for birth weight at term , to avoid the potential consequences of error or mendelian causes of extreme birth weight .", "each birth weight value was adjusted for each sex separately then expressed as a z score ."], ["all statistical analyses were carried out using the software package plink v. 1.05 ( 24 ) .", "ethnicity for our cohort was derived using the multidimensional scaling feature within plink . by treating birth weight as a quantitative trait ( treated as a z score after correcting for sex ) , association analysis for each snp", "was carried out using linear regression analysis with the snp included as an independent variable ( coded as 0 , 1 , and 2 ) . with 5,465 subjects , the powers to detect 0.2 , 0.3 , 0.4 , 0.5 , 0.6 , 0.8 , and 1% variation at the p = 0.002 level ( i.e. , the corrected p value for the number of tests ) were 47.4 , 74.6 , 90.0 , 96.6 , 98.9 , 100 , and 100% , respectively ."], ["in our initial analysis , 12 snps corresponding to the 9 type 2 diabetes loci previously studied in the context of birth weight were investigated in our cohort , namely , tcf7l2 , hhex - ide , pparg , kcnj11 , slc30a8 , igf2bp2 , cdkal1 , cdkn2a/2b , and jazf1 ( 4,6 ) ( table 1 ) .", "quantitative association results for previously studied type 2 diabetes risk alleles with birth weight in the european american cohort ( n = 5,465 ) , sorted by chromosomal location the direction of effect is shown for the minor allele in each case . *", "major allele previously reported to be associated with type 2 diabetes ; * * p  0.002 .", " , regression coefficient for the test snp ; bp , base pair position ; maf , minor allele frequency ; n , number of subjects tested ; p , two - sided trend test p value ; r , value in linear regression ; t , test statistic . as a result ,", "we observed strong association with rs7756992 ( p = 8 10 ) at the cdkal1 locus with low birth weight ; this snp yielded strongest association to type 2 diabetes in an icelandic gwa study carried out on the illumina humanhap500 platform ( 21 ) .", "snps rs10946398 or rs7754840 at the same locus have been reported to be most strongly associated with type 2 diabetes from gwa studies on the affymetrix platform or the illumina humanhap300 beadchip ( 16,18,19 ) ; however , using a perfect surrogate , rs4712523 ( r = 1 ) , we only observed nominally significant association ( p = 0.01 ) .", "it should be noted that rs10946398 and rs7756992 are far from being in perfect linkage disequilibrium ( r = 0.677 ) , thus the inclusion of both in this current study . unlike previous reports", ", we did not observe association between rs1111875 at the hhex - ide locus and this trait ( 6 ) . in line with previous reports", ", we also did not observe association between birth weight and tcf7l2 , pparg , kcnj11 , slc30a8 , igf2bp2 , cdkn2a/2b , or jazf1 ( 4,6,10 ) .", "furthermore , we did not observe any significant association with risk alleles at other type 2 diabetes loci after correction for multiple testing for all 23 snps ( threshold p  0.002 ) ( supplementary table 2 ) .", "we detected nominal association with rs1387153 ( p = 0.02 ) at the mtnr1b locus ; however , the corresponding type 2 diabetes risk allele was tracking with higher birth weight .", "we also analyzed male and female subjects separately , but the effect of each locus on birth weight did not vary by sex ( supplementary tables 3 and 4 ) ."], ["from this interim analysis of our ongoing gwa study of birth weight in a european american cohort , it is clear that the cdkal1 locus , which was uncovered in gwa analyses of type 2 diabetes , is strongly associated with birth weight in our study population .", "this result clearly supports a previous report that came to a similar conclusion ( 6 ) .", "however , the study by freathy et al . used a different snp , namely , rs10946398 , which was not present on our illumina beadchip ; we used a perfect surrogate , i.e. , rs4712523 ( r = 1 ) , that only yielded nominal significance ( p = 0.01 ) .", "although they did not report for rs7756992 , we found that it gave us the strongest association ( p = 8 10 ) and was selected for this study because it yielded the strongest association to type 2 diabetes in an icelandic gwa study ( 21 ) .", "secondly , we did not observe association between hhex - ide and birth weight , which is in contrast with what had been described previously ( 6 ) .", "we acknowledge that our cohort is smaller than the original report ( 5,465 vs. 19,200 individuals ) ; indeed , this association was not observed ( p < 0.05 ) in the similarly sized 1958 birth cohort ( 6 ) .", "the lack of available covariate data , such as gestational age , was also a limitation of this study .", "therefore , it is possible that with a larger cohort with additional covariate data we may observe the association of this locus with birth weight ; however , it could also indicate that hhex - ide has a less pronounced impact on birth weight than cdkal1 .", "consistent with the existing literature , we did not find any evidence of association between birth weight and tcf7l2 , pparg , kcnj11 , slc30a8 , igf2bp2 , cdkn2a/2b , or jazf1 ( 4,6,10 ) . given the monogenic precedent for opposing effects of maternal and fetal genotype ( 25 ) , it is possible that effects of common type 2 diabetes alleles could be masked by this phenomenon .", "it has been shown that cdkal1 is expressed in the rat pancreatic -cell line ins-1 ( 21 ) .", "homozygous carriers of the risk allele have been shown to have a 22% lower corrected insulin response than individuals who are wild - type carriers .", "it has been suggested that cdkal1 might influence the secretion of insulin by interacting with cdk5 ( 21 ) .", "our data contributes another piece of evidence supporting the hypothesis , namely , that the same genotype conferring lower birth weight can also confer higher type 2 diabetes risk later in life .", "cdkal1 was first described in the context of type 2 diabetes in both european caucasians and in han chinese ( 21 ) ; as such , it would be interesting to examine whether the association of cdkal1 with lower birth weight also stands in this and other ethnicities , such as african americans and hispanics . in conclusion", ", we strongly confirm that the established type 2 diabetes locus cdkal1 also influences birth weight .", "however , we do not observe such association with tcf7l2 , hhex - ide , cdkn2a/2b , or jazf1 .", "in addition , of all the other established type 2 diabetes loci to date , we do not observe a convincing role for them in the determination of birth weight ."]]}
{"article_id": "PMC4054060", "article_text": ["the most often reported reasons have been dehydration of dentin , removal of tooth structure during root canal treatment , prolonged use of high concentrations of irrigation solutions , and excessive pressure during obturation . in the literature", "there are several studies in which the fracture resistance of endodontically treated teeth were evaluated or the techniques for reinforcing of these teeth were described .", "it is important to examine systems in an in vitro model prior to in vivo use in order to identify treatment or materials that might improve clinical performances .", "extracted human teeth are also widely used for in vitro studies in fracture resistance tests . however standardization among the extracted teeth should be performed in order not to affect the study 's results .", "standardization of the roots is one of the important steps in the study in which fracture resistance is evaluated .", "if roots were not distributed among the groups equally , these variables could have affected the results of the studies . in many studies , the mesiodistal ( md ) and buccolingual ( bl ) dimensions and the lengths of the roots", "researchers also attempt to choose the same type of teeth in order to standardize the specimens . in spite of these standardization attempts", ", it has been discussed that the standard deviations within the groups were rather high , rendering the results meaningless , and prompting studies using a larger number of specimens .", "the aim of this study was to determine how physical ( weight , volume , and density ) and morphological ( md and bl dimensions ) properties affect the fracture resistance of roots , and which criteria are important for standardization in fracture resistance evaluated studies .", "the null hypothesis was that different physical properties of roots would not affect the fracture resistance of endodontically treated roots .", "seventy - five human canine teeth extracted for periodontal reasons with completed apices and similar lengths were used in this study .", "mesiodistal ( md ) and buccolingual ( bl ) radiographs were taken of the specimens to evaluate the anatomical structures of the teeth .", "the teeth with internal or external resorption , those which had two or more root canals , and those with calcification were discarded .", "the teeth were examined under a stereomicroscope to discard specimens with cracks and craze lines .", "soft tissues and calculus were removed mechanically from the root surfaces using a periodontal scaler .", "specimens were decoronated with a diamond disc under a water coolant to obtain a standardized root length of 16 mm .", "was carried out , weight , volume [ figure 1 ] , and density [ figure 1 ] were calculated with ", "precisa xb 220a precision balance ( precisa , gravimetrics ag , dietikon , switzerland ) which had a capacity of 220 g , and a readability of 0.0001 g. weight change in distilled water is equal to root volume , because density of distilled water is 1 g / cm .", "md and bl dimensions at 16 mm ( the coronal end of the root ) from the apex of the each root were recorded with root number . a size 10 k - file ( dentsply , maillefer , ballaigues , switzerland ) was inserted into the canal until it was visible at the apical foramen , and the working length was determined to be 1 mm short of this position .", "root canal shaping procedures were performed with protaper universal rotary files ( dentsply maillefer , ballaigues , switzerland ) , and apical region was prepared to size # 30 ( f3 ) in all specimens .", "the canal was irrigated with 2 ml freshly prepared 5% naocl solution with a 27-gauge needle after each file .", "a final rinse with 5 ml 17% edta for 1 min , followed by rinsing with 5 ml 5% naocl for 1 min was applied for smear layer removal .", "the specimens were dried with paper points and filled with gutta - percha and ah plus sealer ( dentsply detrey , kontanz , germany ) using cold lateral compaction .", "calculation of root volume and density using a precise balance proper wax stencils were molded and the roots were mounted into acyclic resin at an angle of 45 degrees to its long axis , leaving 6 mm of each root exposed [ figure 2 ] . a universal testing machine ( instron corp .", "vertical force was loaded with a speed of 1 mm / min until fracture occurred .", "the results indicated that volume ( r = 0.427 , p < 0.001 ) and weight ( r = 0.394 , p", "< 0.001 ) of the roots had a more significant effect than bl ( r = 0.197 , p = 0.085 ) , md ( r = 0.239 , p = 0.037 ) dimension which implying that volume or weight had greater importance regarding the prediction of fracture strength [ table 1 ] .", "physical and morphological properties of the samples along with pearson correlation coefficients and p values .", "when extracted human teeth are used for evaluating the fracture resistance of roots , there is a potential for large uncontrollable variations to affect strength . for that reason , all controllable factors should be standardized as much as possible .", "many researchers who have carried out this type of study - performed standardization , according to root length , md and bl dimensions . however , many of these studies admitted very high standard deviations as normal .", "thus , this study aimed to control factors such as weight , volume , and density , which can affect the fracture resistance of teeth in order to contribute to the standardization process . in the current study , the volumes or weights of root specimens have more statistically significant value than density , md , and bl dimension . in previous studies , however , these variations did not take into account when the standardization of groups was performed .", "future studies should be standardized to inhibit high standard deviations . during the study design ,", "standardization is generally achieved based on the lengths , and md and bl dimensions of roots with referrals to other studies in the literature . however", ", this study has shown that the physical properties of teeth can affect the resistance of roots .", "thus , it is important to determine the alternative properties of roots which could be useful for the standardization process in future investigations .", "this is the first study in the literature to conclude that  the volume or weight of root as the most important determining factors in root fracture. within the limitations of this study , it can be concluded that when forming groups to evaluate fracture resistance , after the root length is standardized , the roots should be equally distributed according to their volumes or weights , rather than their md and bl dimensions , as these dimensions can not closely simulate the entire strength of the root as much as the volume or weight ."], "abstract_text": ["<S> objective : the aim of this study was to determine how physical and morphological properties affect the fracture resistance of roots , and which criteria are important for root specimen standardization in fracture resistance studies.materials and methods : seventy - five freshly extracted human canine teeth were selected . </S>", "<S> crowns were sectioned from the cement  enamel junction and the root lengths were set at 16 mm </S>", "<S> . then they were prepared up to protaper f3 file . </S>", "<S> each root was numbered and data were obtained by measuring mesiodistal and buccolingual dimensions , volume , weight , and density . </S>", "<S> tests for fracture strength were performed using an instron testing machine ( instron corp . </S>", "<S> ma , usa ) . </S>", "<S> the force was applied axially , angled at 45 degrees with a constant speed of 1 mm / min . for each sample , </S>", "<S> the force at the time of fracture was recorded in newtons . </S>", "<S> results were evaluated statistically using linear regression analysis.results:volume and weight of the roots had more effect than mesiodistal or buccolingual dimensions on root fracture resistance.conclusions:in root fracture resistance studies , volume or weight of the roots must be standardized when distributing roots to groups . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["the most often reported reasons have been dehydration of dentin , removal of tooth structure during root canal treatment , prolonged use of high concentrations of irrigation solutions , and excessive pressure during obturation . in the literature", "there are several studies in which the fracture resistance of endodontically treated teeth were evaluated or the techniques for reinforcing of these teeth were described .", "it is important to examine systems in an in vitro model prior to in vivo use in order to identify treatment or materials that might improve clinical performances .", "extracted human teeth are also widely used for in vitro studies in fracture resistance tests . however standardization among the extracted teeth should be performed in order not to affect the study 's results .", "standardization of the roots is one of the important steps in the study in which fracture resistance is evaluated .", "if roots were not distributed among the groups equally , these variables could have affected the results of the studies . in many studies , the mesiodistal ( md ) and buccolingual ( bl ) dimensions and the lengths of the roots", "researchers also attempt to choose the same type of teeth in order to standardize the specimens . in spite of these standardization attempts", ", it has been discussed that the standard deviations within the groups were rather high , rendering the results meaningless , and prompting studies using a larger number of specimens .", "the aim of this study was to determine how physical ( weight , volume , and density ) and morphological ( md and bl dimensions ) properties affect the fracture resistance of roots , and which criteria are important for standardization in fracture resistance evaluated studies .", "the null hypothesis was that different physical properties of roots would not affect the fracture resistance of endodontically treated roots ."], ["seventy - five human canine teeth extracted for periodontal reasons with completed apices and similar lengths were used in this study .", "mesiodistal ( md ) and buccolingual ( bl ) radiographs were taken of the specimens to evaluate the anatomical structures of the teeth .", "the teeth with internal or external resorption , those which had two or more root canals , and those with calcification were discarded .", "the teeth were examined under a stereomicroscope to discard specimens with cracks and craze lines .", "soft tissues and calculus were removed mechanically from the root surfaces using a periodontal scaler .", "specimens were decoronated with a diamond disc under a water coolant to obtain a standardized root length of 16 mm .", "was carried out , weight , volume [ figure 1 ] , and density [ figure 1 ] were calculated with ", "precisa xb 220a precision balance ( precisa , gravimetrics ag , dietikon , switzerland ) which had a capacity of 220 g , and a readability of 0.0001 g. weight change in distilled water is equal to root volume , because density of distilled water is 1 g / cm .", "md and bl dimensions at 16 mm ( the coronal end of the root ) from the apex of the each root were recorded with root number . a size 10 k - file ( dentsply , maillefer , ballaigues , switzerland ) was inserted into the canal until it was visible at the apical foramen , and the working length was determined to be 1 mm short of this position .", "root canal shaping procedures were performed with protaper universal rotary files ( dentsply maillefer , ballaigues , switzerland ) , and apical region was prepared to size # 30 ( f3 ) in all specimens .", "the canal was irrigated with 2 ml freshly prepared 5% naocl solution with a 27-gauge needle after each file .", "a final rinse with 5 ml 17% edta for 1 min , followed by rinsing with 5 ml 5% naocl for 1 min was applied for smear layer removal .", "the specimens were dried with paper points and filled with gutta - percha and ah plus sealer ( dentsply detrey , kontanz , germany ) using cold lateral compaction .", "calculation of root volume and density using a precise balance proper wax stencils were molded and the roots were mounted into acyclic resin at an angle of 45 degrees to its long axis , leaving 6 mm of each root exposed [ figure 2 ] . a universal testing machine ( instron corp .", "vertical force was loaded with a speed of 1 mm / min until fracture occurred ."], ["the results indicated that volume ( r = 0.427 , p < 0.001 ) and weight ( r = 0.394 , p", "< 0.001 ) of the roots had a more significant effect than bl ( r = 0.197 , p = 0.085 ) , md ( r = 0.239 , p = 0.037 ) dimension which implying that volume or weight had greater importance regarding the prediction of fracture strength [ table 1 ] .", "physical and morphological properties of the samples along with pearson correlation coefficients and p values ."], ["when extracted human teeth are used for evaluating the fracture resistance of roots , there is a potential for large uncontrollable variations to affect strength . for that reason , all controllable factors should be standardized as much as possible .", "many researchers who have carried out this type of study - performed standardization , according to root length , md and bl dimensions . however , many of these studies admitted very high standard deviations as normal .", "thus , this study aimed to control factors such as weight , volume , and density , which can affect the fracture resistance of teeth in order to contribute to the standardization process . in the current study , the volumes or weights of root specimens have more statistically significant value than density , md , and bl dimension . in previous studies , however , these variations did not take into account when the standardization of groups was performed .", "future studies should be standardized to inhibit high standard deviations . during the study design ,", "standardization is generally achieved based on the lengths , and md and bl dimensions of roots with referrals to other studies in the literature . however", ", this study has shown that the physical properties of teeth can affect the resistance of roots .", "thus , it is important to determine the alternative properties of roots which could be useful for the standardization process in future investigations ."], ["this is the first study in the literature to conclude that  the volume or weight of root as the most important determining factors in root fracture. within the limitations of this study , it can be concluded that when forming groups to evaluate fracture resistance , after the root length is standardized , the roots should be equally distributed according to their volumes or weights , rather than their md and bl dimensions , as these dimensions can not closely simulate the entire strength of the root as much as the volume or weight ."]]}
{"article_id": "PMC4576138", "article_text": ["intensive insulin therapy using basal - bolus insulin regimen is the standard therapy for patients with type 1 diabetes mellitus . by mimicking the endogenous insulin secretion profile in healthy subjects ,", "it has been shown to improve glycemic control and reduce the risk of long - term complications compared with conventional insulin therapy [ 1 , 2 ] .", "unfortunately , many patients with type 1 diabetes can not achieve the target glycemic control , and insulin therapy leaves room for improvement .", "thus , the efficacy of basal insulin is especially important in this group of patients .", "importantly , in some type 1 diabetes patients with severe loss of endogenous insulin secretion capacity , once - daily injection of basal insulin does not always cover the basal effect of insulin over the 24-hour period [ 35 ] .", "in addition , the intraday and day - to - day variability in insulin agents could sometimes be an obstacle for optimized titration of insulin and a cause of increased frequency of hypoglycemia . given that increased frequency of injection and large fluctuations in blood glucose could be a burden in such patients , any improvement in the efficacy of basal insulin agents should be appreciated .", "insulin degludec is a new ultra - long - acting basal insulin that forms soluble multihexamers at the subcutaneous injection site from which insulin monomers are slowly and continuously absorbed into the circulation , leading to a peakless action profile over 42 hours .", "consistent with this pharmacological action , begin basal - bolus type 1 trial   showed that the rate of nocturnal - confirmed hypoglycemia was 25% lower with insulin degludec than with insulin glargine .", "in addition , it was reported that the day - to - day variability in plasma glucose in type 1 diabetes patients treated with insulin degludec was lower compared to insulin glargine . taking these unique actions of insulin degludec into consideration , switching from twice - daily injections of basal insulin to once - daily injection of insulin degludec", "could provide great benefit to patients with type 1 diabetes . in this study , to evaluate the efficacy of insulin degludec as a basal insulin for basal - bolus regimen for japanese patients with type 1 diabetes mellitus who are treated with twice - daily basal insulin injection therapy , we investigated glycemic control , daily , and day - to - day variability in plasma glucose using continuous glucose monitoring before and after switching to once - daily insulin degludec injection in 22 patients with type 1 diabetes .", "we recruited 24 eligible japanese patients ( 8 males and 16 females ) with type 1 diabetes who visited the outpatient clinic of juntendo university hospital between july 2013 and january 2014 .", "patients who satisfied the following conditions were included : ( 1 ) treated with basal - bolus insulin regimen with twice - daily injections by insulin glargine or detemir and ( 2 ) aged more than 20 and less than 80 years . also , patients were excluded if they ( 1 ) had serious liver disease ( ast and/or alt > 100 ", "iu / l ) , ( 2 ) had serious kidney disease ( serum creatinine > 2.0  mg / dl ) , ( 3 ) had untreated severe diabetic retinopathy , ( 4 ) had adrenal or pituitary insufficiency , ( 5 ) had other conditions considered by the attending physician to be contraindicated to inclusion in the study , or ( 6 ) were pregnant or breastfeeding women .", "this trial was conducted in accordance with the declaration of helsinki , and the protocol was approved by the human ethics committee of juntendo university .", "all patients provided a written informed consent prior to trial initiation . in this prospective , single - center , single - arm , open - label , 12-week study , we compared the effects of switching from twice - daily basal insulin to once - daily insulin degludec on glycemic control , daily , and day - to - day variability in plasma glucose .  ", "figure 1 shows the patient enrolment process . at baseline before switching , the following laboratory tests were performed in each patient : fasting plasma glucose ( fpg ) , plasma c - peptide , hba1c , and glycosylated albumin .", "plasma c - peptide assay was performed using ultrasensitive c - peptide elisa kit ( mercodia , uppsala , sweden ) for precise determination of intrinsic basal insulin level .", "then , cgm and 7-point self - measured blood glucose ( smbg ) profiles ( before and 2 hours after meals and bedtime ) were obtained .", "after that , the patient was switched from twice - daily basal insulin to once - daily insulin degludec , which involved 10% reduction in insulin dosage without any change in the rapid acting insulin therapy .", "insulin degludec was administered once - daily at bedtime . at 4 weeks after switching , the same fasting laboratory tests , cgm and 7-point smbg , were repeated .", "after 4 weeks , the basal insulin dose was adjusted for each individual patient based on self - measured fbg levels taken before breakfast .", "the dose of insulin degludec was decreased by 1 unit if fbg was  80  mg / dl over three consecutive days just before the hospital visit .", "then , the increase of the dose of basal insulin or titration of rapid acting insulin was performed by the judgement of each physician in charge . at the end of the study ( 12 weeks )", ", the same laboratory tests ( fpg , hba1c , and glycosylated albumin ) were repeated again .", "cgm data were obtained by using the ipro2 ( medtronic ; northridge , ca ) .", "patients were required to use cgm for six consecutive days . over each cgm occasion , at least 288/day cgm glucose values were to be recorded .", "as an index of day - to - day variability , the mean of daily difference ( modd ) was calculated from the absolute difference between paired cgm values during two successive days ( days 2 to 3 and days 4 to 5 ) , and the data were presented as the average of the two values .", "the patient was asked to record 7-point smbg profiles for one day during cgm for before and 2  hr after meals and at bedtime .", "the primary outcome of the study was change in hba1c before and 12 weeks after switching .", "the secondary outcomes based on cgm values were ( 1 ) changes in standard deviation ( sd ) and modd .", "safety variables included the frequencies of severe hypoglycemia , which was defined as low blood glucose level requiring assistance from another person to treat , nocturnal hypoglycemia , and adverse events .", "confirmed hypoglycemia was defined as a glucose value of less than 70  mg / dl by cgm and was reported in percentage (= times < 70  mg / dl / total time of measurement ) .", "hypoglycemic episodes occurring between 0:00 and 5:59 hours were classified as nighttime while daytime episodes occurred between 6:00 and 23:59 .", "data were expressed as mean  sd . the mann - whitney u test was used for analysis of cgm data before and 4 weeks after switching and , for analysis of fpg , hba1c and glycosylated albumin before and 12 weeks after switching were used .", "all statistical analyses were conducted using statview statistical software package , version 5.0 ( sas institute inc . , cary , nc ) .", "two patients withdrew from the study after the first treatment period ; one decided to withdraw during the conduct of the study and the other did not visit the outpatient clinic .", "the mean age and duration of type 1 diabetes mellitus were 54.8  14.5 and 14.6  9.0 years , respectively . fasting", "plasma c - peptide was below the detection limit of the ultrasensitive c - peptide elisa kit in 18 patients ( 81% ) , indicating severely low insulin secretion in most subjects .", "as shown in table 2 , hba1c levels at baseline and 4 and 8 weeks after switching to insulin degludec were 8.5  1.4% , 8.6  1.6% , and 8.7  1.6% , respectively .", "glycosylated albumin levels before and 4 and 8 weeks after switching were 24.9  5.0% , 25.3  5.2% , and 24.7  3.6% , respectively .", "furthermore , fasting blood glucose levels were 203.2  81.2  mg / dl , 165.5  82.1  mg / dl , and 206.5  122.4  mg / dl , respectively . based on these data ,", "it is clear that switching to insulin degludec did not improve glycemic control throughout the study .", "the mean basal insulin and total daily doses at 12 weeks after switching to insulin degludec were significantly reduced compared to the baseline ( 15.2  7.6 versus 11.6  6.9  u , p < 0.01 , and 40.0  17.3 versus 37.9  16.7  u , p < 0.01 , resp . ) whereas the bolus insulin dose did not significantly change after switching . \n", "table 3 summarizes fluctuations in glucose level and the frequency of hypoglycemia recorded by cgm over 4 days before and after switching .", "the averages of blood glucose and standard deviation ( sd ) through the daytime ( 0:0023:59 ) were 184.1  45.8 versus 189.6  52.7  mg / dl and 68.4  14.5 versus 66.5  17.1  mg / dl , respectively .", "furthermore , the modd was 72.1  16.0 versus 74.0  23.0  mg / dl and the frequency of hypoglycemia below 70  mg / dl was 6.1  8.0 versus 6.5  9.7% , respectively .", "table 3 also shows fluctuations in glucose level and the frequency of hypoglycemic episodes recorded by cgm during the nighttime ( 0:005:59 ) .", "the averages of blood glucose before and after switching were 156.5  63.7 versus 174.6  58.5  mg / dl during the nighttime .", "the sd during nighttime did not change significantly ( before : 22.6  10.9 , after : 24.8  10.7  mg / dl ) .", "modd tended to increase during the nighttime both at baseline and after switching , however ; these changes were not significantly different .", "the frequency of hypoglycemic glucose ( below 70  mg / dl ) was 14.4  17.0 versus 11.1  15.0% during the nighttime , before and after switching .", "thus , the frequency of nocturnal hypoglycemia did not change significantly after switching , similar to other parameters , and no severe hypoglycemia was recorded during the study period . \n", "blood glucose level at 2 hours after lunch was significantly low before and after switching ( 207.6  87.7 versus 158.2  90.3  mg / dl , p < 0.01 ) .", "the present study investigated the efficacy and safety of switching from twice - daily basal insulin injections to once - daily insulin degludec injection in japanese patients with type 1 diabetes .", "however , some patients with extremely low insulin secretion capacity often need to use twice - daily basal insulin because the duration of action of insulin detemir is about 16 hours   and that of insulin glargine does not last up to 24 hours [ 4 , 12 ] .", "a few studies from japan have already investigated the outcome of switching from once - daily or twice - daily basal insulin to once - daily insulin degludec in patients with type 1 diabetes [ 1315 ] . specifically , these studies investigated the effects of switching to insulin degludec in type 1 diabetes patients who were being treated with a combination of once- or twice - daily injections of insulin glargine or detemir , though there are no studies that focused on type 1 diabetes patients treated only with twice - daily basal insulin .", "the basal insulin levels were very low in our patients and the level could not be detected in most patients even by using high - sensitivity c - peptide kits with detection limit of < 0.015  ng / ml .", "therefore , the selection of twice - daily basal insulin injections in our study seems reasonable to achieve better glycemic control .", "insulin degludec , an ultra - long - acting basal insulin , became available in japan in 2013 , ahead of other countries , and is known to have longer duration of action ( over 42 hours ) compared with insulin glargine and detemir .", "therefore , it is clinically worthy to investigate the efficacy and safety of switching from twice - daily insulin glargine or detemir to once - daily insulin degludec in type 1 diabetes patients with severely reduced insulin secretion .", "the results showed no significant changes in various parameters of glycemic control , such as fasting plasma glucose , hba1c , and glycosylated albumin , after switching to insulin degludec despite about 20% reduction in basal insulin dose at 12 weeks , indicating that insulin degludec has longer duration of action and a more potent glucose - lowering effect than insulin glargine or insulin detemir .", "the frequency of hypoglycemic episodes recorded by cgm did not increase at 4 weeks after switching to insulin degludec . in our study protocol ,", "the bolus insulin dose was not changed before and after cgm recording because the effect of bolus insulin on glycemic control needed to be minimized . according to a previous report by the begin basal - bolus type 1 trial investigators , the mean doses of basal and premeal bolus insulin", "were significantly decreased by 14% and 10% in the insulin degludec group compared with the insulin glargine group at the end of the trial , leading to similar rate of overall hypoglycemia between insulin glargine and degludec groups .", "therefore , in daily clinical practice , adjustment of the premeal bolus insulin dose also needs be considered when switching to insulin degludec .", "the smbg data in our study showed that postlunch blood glucose level was significantly lower after switching to insulin degludec injected at bedtime , suggesting that the peak action of insulin degludec occurs 14 - 15 hours after injection .", "consistent with this finding , another study showed that the trough blood glucose was recorded at daytime when insulin degludec was injected at bedtime .", "in addition , one review showed that the peak of the glucose - lowering effect of insulin degludec appeared about 12 hours after injection .", "in addition to the longer duration of action of basal insulin , its effects on daily and day - to - day variability of plasma glucose should be noted .", "reported that the use of insulin degludec resulted in lower day - to - day variability in blood glucose compared to insulin glargine in type 1 diabetes patients .", "however , different from this study , our results showed that switching to insulin degludec did not reduce modd , an index of plasma glucose day - to - day variation , which was consistent with a previous study in japan [ 13 , 14 ] .", "heise et al .   examined the glucose fluctuation by the glucose clamp method after very long fasting , which was not different from our method by cgm .", "therefore , the inconsistency might be due to differences between experimental and real - world study .", "our study extended over a short period of time and included a limited sample size .", "in addition , the carry - over effect of hba1c could not be completely excluded because our study was not a randomized controlled trial .", "therefore , a crossover trial or randomized controlled trial of a larger sample size is needed in the future . in conclusion", ", our study demonstrated that glycemic control 12 weeks after switching to once - daily insulin degludec injection with 20% dose reduction was comparable to that in patients treated with twice - daily injection of basal insulin injections and that such switching did not change the frequency of nocturnal hypoglycemia recorded by cgm ."], "abstract_text": ["<S> the aim of this study was to investigate the efficacy of insulin degludec used for basal - bolus insulin regimen after switching from twice - daily basal insulin in japanese patients with type 1 diabetes mellitus . </S>", "<S> the subjects were 22 type 1 diabetes patients treated with basal - bolus insulin regimen with twice - daily basal insulin . </S>", "<S> basal insulin was switched to once - daily injection of insulin degludec with 10% dose reduction . </S>", "<S> hba1c and fasting plasma glucose ( fpg ) were measured before and 12 weeks after switching . </S>", "<S> the frequency of hypoglycemic episodes , standard deviation ( sd ) of blood glucose , and mean of daily difference ( modd ) were evaluated by continuous glucose monitoring ( cgm ) before and 4 weeks after switching . hba1c and fpg before and 12 weeks after switching were comparable ( hba1c 8.5  1.4 versus 8.7  1.6% , p = 0.28 ; fpg 203.2  81.2 versus 206.5  122.4  mg / dl , p = 0.91 ) . </S>", "<S> the frequency of hypoglycemia during nighttime was not significantly different at 4 weeks after switching ( 14.4  17.0 versus 11.1  15.0% , p = 0.45 ) . </S>", "<S> in addition , sd and modd before and 4 weeks after switching were also comparable . in conclusion , </S>", "<S> glycemic control under once - daily insulin degludec injection was almost comparable to that under twice - daily basal insulin injections in japanese type 1 diabetes patients . </S>", "<S> this study was registered with i d : umin000010474 . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Subjects and Methods", "3. Results", "4. Discussion"], "sections": [["intensive insulin therapy using basal - bolus insulin regimen is the standard therapy for patients with type 1 diabetes mellitus . by mimicking the endogenous insulin secretion profile in healthy subjects ,", "it has been shown to improve glycemic control and reduce the risk of long - term complications compared with conventional insulin therapy [ 1 , 2 ] .", "unfortunately , many patients with type 1 diabetes can not achieve the target glycemic control , and insulin therapy leaves room for improvement .", "thus , the efficacy of basal insulin is especially important in this group of patients .", "importantly , in some type 1 diabetes patients with severe loss of endogenous insulin secretion capacity , once - daily injection of basal insulin does not always cover the basal effect of insulin over the 24-hour period [ 35 ] .", "in addition , the intraday and day - to - day variability in insulin agents could sometimes be an obstacle for optimized titration of insulin and a cause of increased frequency of hypoglycemia . given that increased frequency of injection and large fluctuations in blood glucose could be a burden in such patients , any improvement in the efficacy of basal insulin agents should be appreciated .", "insulin degludec is a new ultra - long - acting basal insulin that forms soluble multihexamers at the subcutaneous injection site from which insulin monomers are slowly and continuously absorbed into the circulation , leading to a peakless action profile over 42 hours .", "consistent with this pharmacological action , begin basal - bolus type 1 trial   showed that the rate of nocturnal - confirmed hypoglycemia was 25% lower with insulin degludec than with insulin glargine .", "in addition , it was reported that the day - to - day variability in plasma glucose in type 1 diabetes patients treated with insulin degludec was lower compared to insulin glargine . taking these unique actions of insulin degludec into consideration , switching from twice - daily injections of basal insulin to once - daily injection of insulin degludec", "could provide great benefit to patients with type 1 diabetes . in this study , to evaluate the efficacy of insulin degludec as a basal insulin for basal - bolus regimen for japanese patients with type 1 diabetes mellitus who are treated with twice - daily basal insulin injection therapy , we investigated glycemic control , daily , and day - to - day variability in plasma glucose using continuous glucose monitoring before and after switching to once - daily insulin degludec injection in 22 patients with type 1 diabetes ."], ["we recruited 24 eligible japanese patients ( 8 males and 16 females ) with type 1 diabetes who visited the outpatient clinic of juntendo university hospital between july 2013 and january 2014 .", "patients who satisfied the following conditions were included : ( 1 ) treated with basal - bolus insulin regimen with twice - daily injections by insulin glargine or detemir and ( 2 ) aged more than 20 and less than 80 years . also , patients were excluded if they ( 1 ) had serious liver disease ( ast and/or alt > 100 ", "iu / l ) , ( 2 ) had serious kidney disease ( serum creatinine > 2.0  mg / dl ) , ( 3 ) had untreated severe diabetic retinopathy , ( 4 ) had adrenal or pituitary insufficiency , ( 5 ) had other conditions considered by the attending physician to be contraindicated to inclusion in the study , or ( 6 ) were pregnant or breastfeeding women .", "this trial was conducted in accordance with the declaration of helsinki , and the protocol was approved by the human ethics committee of juntendo university .", "all patients provided a written informed consent prior to trial initiation . in this prospective , single - center , single - arm , open - label , 12-week study , we compared the effects of switching from twice - daily basal insulin to once - daily insulin degludec on glycemic control , daily , and day - to - day variability in plasma glucose .  ", "figure 1 shows the patient enrolment process . at baseline before switching , the following laboratory tests were performed in each patient : fasting plasma glucose ( fpg ) , plasma c - peptide , hba1c , and glycosylated albumin .", "plasma c - peptide assay was performed using ultrasensitive c - peptide elisa kit ( mercodia , uppsala , sweden ) for precise determination of intrinsic basal insulin level .", "then , cgm and 7-point self - measured blood glucose ( smbg ) profiles ( before and 2 hours after meals and bedtime ) were obtained .", "after that , the patient was switched from twice - daily basal insulin to once - daily insulin degludec , which involved 10% reduction in insulin dosage without any change in the rapid acting insulin therapy .", "insulin degludec was administered once - daily at bedtime . at 4 weeks after switching , the same fasting laboratory tests , cgm and 7-point smbg , were repeated .", "after 4 weeks , the basal insulin dose was adjusted for each individual patient based on self - measured fbg levels taken before breakfast .", "the dose of insulin degludec was decreased by 1 unit if fbg was  80  mg / dl over three consecutive days just before the hospital visit .", "then , the increase of the dose of basal insulin or titration of rapid acting insulin was performed by the judgement of each physician in charge . at the end of the study ( 12 weeks )", ", the same laboratory tests ( fpg , hba1c , and glycosylated albumin ) were repeated again .", "cgm data were obtained by using the ipro2 ( medtronic ; northridge , ca ) .", "patients were required to use cgm for six consecutive days . over each cgm occasion , at least 288/day cgm glucose values were to be recorded .", "as an index of day - to - day variability , the mean of daily difference ( modd ) was calculated from the absolute difference between paired cgm values during two successive days ( days 2 to 3 and days 4 to 5 ) , and the data were presented as the average of the two values .", "the patient was asked to record 7-point smbg profiles for one day during cgm for before and 2  hr after meals and at bedtime .", "the primary outcome of the study was change in hba1c before and 12 weeks after switching .", "the secondary outcomes based on cgm values were ( 1 ) changes in standard deviation ( sd ) and modd .", "safety variables included the frequencies of severe hypoglycemia , which was defined as low blood glucose level requiring assistance from another person to treat , nocturnal hypoglycemia , and adverse events .", "confirmed hypoglycemia was defined as a glucose value of less than 70  mg / dl by cgm and was reported in percentage (= times < 70  mg / dl / total time of measurement ) .", "hypoglycemic episodes occurring between 0:00 and 5:59 hours were classified as nighttime while daytime episodes occurred between 6:00 and 23:59 .", "data were expressed as mean  sd . the mann - whitney u test was used for analysis of cgm data before and 4 weeks after switching and , for analysis of fpg , hba1c and glycosylated albumin before and 12 weeks after switching were used .", "all statistical analyses were conducted using statview statistical software package , version 5.0 ( sas institute inc . , cary , nc ) ."], ["two patients withdrew from the study after the first treatment period ; one decided to withdraw during the conduct of the study and the other did not visit the outpatient clinic .", "the mean age and duration of type 1 diabetes mellitus were 54.8  14.5 and 14.6  9.0 years , respectively . fasting", "plasma c - peptide was below the detection limit of the ultrasensitive c - peptide elisa kit in 18 patients ( 81% ) , indicating severely low insulin secretion in most subjects .", "as shown in table 2 , hba1c levels at baseline and 4 and 8 weeks after switching to insulin degludec were 8.5  1.4% , 8.6  1.6% , and 8.7  1.6% , respectively .", "glycosylated albumin levels before and 4 and 8 weeks after switching were 24.9  5.0% , 25.3  5.2% , and 24.7  3.6% , respectively .", "furthermore , fasting blood glucose levels were 203.2  81.2  mg / dl , 165.5  82.1  mg / dl , and 206.5  122.4  mg / dl , respectively . based on these data ,", "it is clear that switching to insulin degludec did not improve glycemic control throughout the study .", "the mean basal insulin and total daily doses at 12 weeks after switching to insulin degludec were significantly reduced compared to the baseline ( 15.2  7.6 versus 11.6  6.9  u , p < 0.01 , and 40.0  17.3 versus 37.9  16.7  u , p < 0.01 , resp . ) whereas the bolus insulin dose did not significantly change after switching . \n", "table 3 summarizes fluctuations in glucose level and the frequency of hypoglycemia recorded by cgm over 4 days before and after switching .", "the averages of blood glucose and standard deviation ( sd ) through the daytime ( 0:0023:59 ) were 184.1  45.8 versus 189.6  52.7  mg / dl and 68.4  14.5 versus 66.5  17.1  mg / dl , respectively .", "furthermore , the modd was 72.1  16.0 versus 74.0  23.0  mg / dl and the frequency of hypoglycemia below 70  mg / dl was 6.1  8.0 versus 6.5  9.7% , respectively .", "table 3 also shows fluctuations in glucose level and the frequency of hypoglycemic episodes recorded by cgm during the nighttime ( 0:005:59 ) .", "the averages of blood glucose before and after switching were 156.5  63.7 versus 174.6  58.5  mg / dl during the nighttime .", "the sd during nighttime did not change significantly ( before : 22.6  10.9 , after : 24.8  10.7  mg / dl ) .", "modd tended to increase during the nighttime both at baseline and after switching , however ; these changes were not significantly different .", "the frequency of hypoglycemic glucose ( below 70  mg / dl ) was 14.4  17.0 versus 11.1  15.0% during the nighttime , before and after switching .", "thus , the frequency of nocturnal hypoglycemia did not change significantly after switching , similar to other parameters , and no severe hypoglycemia was recorded during the study period . \n", "blood glucose level at 2 hours after lunch was significantly low before and after switching ( 207.6  87.7 versus 158.2  90.3  mg / dl , p < 0.01 ) ."], ["the present study investigated the efficacy and safety of switching from twice - daily basal insulin injections to once - daily insulin degludec injection in japanese patients with type 1 diabetes .", "however , some patients with extremely low insulin secretion capacity often need to use twice - daily basal insulin because the duration of action of insulin detemir is about 16 hours   and that of insulin glargine does not last up to 24 hours [ 4 , 12 ] .", "a few studies from japan have already investigated the outcome of switching from once - daily or twice - daily basal insulin to once - daily insulin degludec in patients with type 1 diabetes [ 1315 ] . specifically , these studies investigated the effects of switching to insulin degludec in type 1 diabetes patients who were being treated with a combination of once- or twice - daily injections of insulin glargine or detemir , though there are no studies that focused on type 1 diabetes patients treated only with twice - daily basal insulin .", "the basal insulin levels were very low in our patients and the level could not be detected in most patients even by using high - sensitivity c - peptide kits with detection limit of < 0.015  ng / ml .", "therefore , the selection of twice - daily basal insulin injections in our study seems reasonable to achieve better glycemic control .", "insulin degludec , an ultra - long - acting basal insulin , became available in japan in 2013 , ahead of other countries , and is known to have longer duration of action ( over 42 hours ) compared with insulin glargine and detemir .", "therefore , it is clinically worthy to investigate the efficacy and safety of switching from twice - daily insulin glargine or detemir to once - daily insulin degludec in type 1 diabetes patients with severely reduced insulin secretion .", "the results showed no significant changes in various parameters of glycemic control , such as fasting plasma glucose , hba1c , and glycosylated albumin , after switching to insulin degludec despite about 20% reduction in basal insulin dose at 12 weeks , indicating that insulin degludec has longer duration of action and a more potent glucose - lowering effect than insulin glargine or insulin detemir .", "the frequency of hypoglycemic episodes recorded by cgm did not increase at 4 weeks after switching to insulin degludec . in our study protocol ,", "the bolus insulin dose was not changed before and after cgm recording because the effect of bolus insulin on glycemic control needed to be minimized . according to a previous report by the begin basal - bolus type 1 trial investigators , the mean doses of basal and premeal bolus insulin", "were significantly decreased by 14% and 10% in the insulin degludec group compared with the insulin glargine group at the end of the trial , leading to similar rate of overall hypoglycemia between insulin glargine and degludec groups .", "therefore , in daily clinical practice , adjustment of the premeal bolus insulin dose also needs be considered when switching to insulin degludec .", "the smbg data in our study showed that postlunch blood glucose level was significantly lower after switching to insulin degludec injected at bedtime , suggesting that the peak action of insulin degludec occurs 14 - 15 hours after injection .", "consistent with this finding , another study showed that the trough blood glucose was recorded at daytime when insulin degludec was injected at bedtime .", "in addition , one review showed that the peak of the glucose - lowering effect of insulin degludec appeared about 12 hours after injection .", "in addition to the longer duration of action of basal insulin , its effects on daily and day - to - day variability of plasma glucose should be noted .", "reported that the use of insulin degludec resulted in lower day - to - day variability in blood glucose compared to insulin glargine in type 1 diabetes patients .", "however , different from this study , our results showed that switching to insulin degludec did not reduce modd , an index of plasma glucose day - to - day variation , which was consistent with a previous study in japan [ 13 , 14 ] .", "heise et al .   examined the glucose fluctuation by the glucose clamp method after very long fasting , which was not different from our method by cgm .", "therefore , the inconsistency might be due to differences between experimental and real - world study .", "our study extended over a short period of time and included a limited sample size .", "in addition , the carry - over effect of hba1c could not be completely excluded because our study was not a randomized controlled trial .", "therefore , a crossover trial or randomized controlled trial of a larger sample size is needed in the future . in conclusion", ", our study demonstrated that glycemic control 12 weeks after switching to once - daily insulin degludec injection with 20% dose reduction was comparable to that in patients treated with twice - daily injection of basal insulin injections and that such switching did not change the frequency of nocturnal hypoglycemia recorded by cgm ."]]}
{"article_id": "PMC2323473", "article_text": ["a 33 year old healthy male of afro - caribbean origin presented to the emergency department with acute generalised abdominal pain of three hours duration .", "he had not noticed any recent change in bowel habit , bleeding per rectum or any systemic symptoms .", "it was of note that there was no family history of inflammatory bowel disease or gastrointestinal malignancy .", "he was a non - smoker and did not consume alcohol . on examination he had a heart rate of 110 beats per minute , temperature of 37.2c , stable blood pressure but he was cold and clammy .", "bowel sounds were absent and rectal exam revealed an empty rectum and no palpable masses .", "blood tests revealed a neutrophilia of 25,000 but urea and electrolytes were within normal range .", "he was resuscitated with intravenous fluids and an emergency laparotomy was performed . during the laparotomy , a 0.5 cm perforation was noted in the ileum 25 cm proximal to the ileocaecal valve on the mesenteric border .", "an inflammatory mass measuring 5  3 cm was noted directly adjacent to the perforation in the mesentry .", "30 cm of small bowel was resected with a right hemicolectomy , hence removing the mass and the perforation .", "no obvious cause of sepsis was identified and it was thought that he may have a subphrenic collection . therefore a computed tomography ( ct ) scan was arranged .", "however , he had a severe reaction to the intravenous contrast medium which resulted in hypotension .", "the patient underwent a second laparotomy to drain the subphrenic abscess and no masses were seen during this procedure .", "histological examination of the resected specimen from the first laporotomy revealed mesenteric fibromatosis ( desmoid tumour ) incompletely excised .", "the resected right hemi - colon did not show evidence of dysplasia , malignancy or polyposis .", "the estrogen receptor status was not reported . due to continuing abdominal discomfort on the ward after the second laparotomy , an ultrasound scan was arranged that revealed the following : a rather ill - defined , fairly ovoid , inhomogeneously - hypoechoic mass lesion is identified in the lower abdomen ( infra- umbilical ) para - sagittal plane .", "it measures about 5  3  4.5 cm in maximum dimensions surrounded by excess peritoneal fat and some bowel loops representing a residual of a previously resected abdominal desmoid .", "the pain eventually resolved and after making a good recovery , he was commenced on oral tamoxifen , 20 mg once a day .", "although estrogen receptor status was not reported , the patient was commenced on this therapy as it was assumed that most desmoid tumours have estrogen receptors .", "he was subsequently referred to a specialist in a distant tertiary unit for further evaluation of the residual tumour and for the prospect of further elective surgery .", "a 33 year old healthy male of afro - caribbean origin presented to the emergency department with acute generalised abdominal pain of three hours duration .", "he had not noticed any recent change in bowel habit , bleeding per rectum or any systemic symptoms .", "it was of note that there was no family history of inflammatory bowel disease or gastrointestinal malignancy .", "he was a non - smoker and did not consume alcohol . on examination he had a heart rate of 110 beats per minute , temperature of 37.2c , stable blood pressure but he was cold and clammy .", "bowel sounds were absent and rectal exam revealed an empty rectum and no palpable masses .", "blood tests revealed a neutrophilia of 25,000 but urea and electrolytes were within normal range .", "he was resuscitated with intravenous fluids and an emergency laparotomy was performed . during the laparotomy , a 0.5 cm perforation was noted in the ileum 25 cm proximal to the ileocaecal valve on the mesenteric border .", "an inflammatory mass measuring 5  3 cm was noted directly adjacent to the perforation in the mesentry .", "30 cm of small bowel was resected with a right hemicolectomy , hence removing the mass and the perforation .", "no obvious cause of sepsis was identified and it was thought that he may have a subphrenic collection . therefore a computed tomography ( ct ) scan was arranged .", "however , he had a severe reaction to the intravenous contrast medium which resulted in hypotension .", "the patient underwent a second laparotomy to drain the subphrenic abscess and no masses were seen during this procedure .", "histological examination of the resected specimen from the first laporotomy revealed mesenteric fibromatosis ( desmoid tumour ) incompletely excised .", "the resected right hemi - colon did not show evidence of dysplasia , malignancy or polyposis .", "due to continuing abdominal discomfort on the ward after the second laparotomy , an ultrasound scan was arranged that revealed the following : a rather ill - defined , fairly ovoid , inhomogeneously - hypoechoic mass lesion is identified in the lower abdomen ( infra- umbilical ) para - sagittal plane .", "it measures about 5  3  4.5 cm in maximum dimensions surrounded by excess peritoneal fat and some bowel loops representing a residual of a previously resected abdominal desmoid .", "the pain eventually resolved and after making a good recovery , he was commenced on oral tamoxifen , 20 mg once a day .", "although estrogen receptor status was not reported , the patient was commenced on this therapy as it was assumed that most desmoid tumours have estrogen receptors .", "he was subsequently referred to a specialist in a distant tertiary unit for further evaluation of the residual tumour and for the prospect of further elective surgery .", "desmoid tumours ( fibromatosis ) are benign fibrous neoplasms originating from the musculo - aponeurotic structures throughout the body .", "desmoid tumours can arise from any skeletal muscle but commonly affect the rectus abdominus in post - partum females and in old surgical scars of the abdomen .", "peripheral tumours are smooth , firm and mobile . they are adherent to the surrounding structures", "gardner s syndrome and familial adenomatous polyposis coli ( fap ) should be suspected in patients with such soft tissue growths ( 3 ) .", "an intra - abdominal desmoid , also known as mesenteric fibromatosis , is the most common solid primary neoplasm of the mesentry .", "approximately 80% of intra - abdominal desmoids involve small bowel mesentry , as was the case with our patient .", "involvement of the transverse mesocolon , retroperitoneum , omentum and the ligamentum teres have also been reported ( 1 , 4 ) .", "intra - abdominal desmoids are usually asymptomatic until their growth and infiltration causes compression of the viscera .", "this can lead to intestinal obstruction , ischemic bowel secondary to vascular compression and hydronephrosis due to ureteric compression .", "bowel perforation is extremely rare and my research has yielded one previous case report ( 1 ) .", "other rare manifestations reported in the literature include deep vein thrombosis , pyrexia of unknown origin , gastrointestinal bleeding and intra - abdominal abscess formation ( 5 , 6 ) .", "the subphrenic abscess that our patient developed is very likely to have been a complication of the initial laparotomy .", "histologically , these tumours are composed of collagen that surrounds spindle cells which are poorly circumscribed ( figure 12 ) .", "exact etiology is uncertain but hormonal factors are implicated as there are estrogen eceptors present in some desmoid tumours .", "the adenomatous polyposis coli ( apc ) gene located on chromosome 5 is responsible for familial adenomatous polypsois coli ( fap ) .", "biallelic mutations of the apc gene induces desmoid tumour formation , hence the association between these two disorders ( 8) . in our patient , there was no polyposis in the resected right hemi - colon .", "trauma has also been suggested as another causative factor but there was no such history in our patient ( 1 ) .", "both ct and magnetic resonance imaging ( mri ) are useful in diagnosing and monitoring recurrence .", "they allow accurate staging and also enable one to delineate accurate anatomy prior to embarking upon major resection .", "mri has the advantage of defining the extent of involvement and monitoring post operative recurrence , which can be as high as 70% ( 6 ) .", "these patients should also undergo colonoscopy and examination of the eye to exclude gardner s syndrome .", "colonoscopy may reveal multiple colonic polyps and fundoscopy may demonstrate multiple pigmented lesions affecting the fundus of the eye ( 2 , 3 ) . complete surgical excision may be the only effective way of providing a cure but complete excision is often impossible and therefore adjuvant treatments have been employed with various degrees of success ( 1 , 2 ) .", "other treatment modalities include radiotherapy , if there is recurrence or as primary treatment to avoid radical surgery ( 9 ) .", "anti - estrogens ( e.g. tamoxifen ) , prostaglandin inhibitors and non - steroidal anti - inflammatory drugs ( nsaids ) have also been used .", "cytotoxic chemotherapy may be useful in those with disease recurrence or where surgery is contraindicated ( 10 ) .", "desmoid tumours originate from the musculo - aponeurotic structures of the body and can be peripheral or intra - abdominal .", "patients should be screened for gardner s syndrome and familial adenomatous polyposis coli since these can often present with dermatoid , epidermoid or other benign tumours .", "emergency presentations are not common , but are usually secondary to mass effect on the viscera .", "ct and mri are the imaging modalities of choice , particularly if surgery is contemplated and also in postoperative follow up to detect recurrence .", "surgery is usually the mainstay of treatment but recurrence is common and adjuvant therapy is used .", "some tumours possess estrogen receptors , hence they regress in response to anti - estrogens , such as tamoxifen ."], "abstract_text": ["<S> desmoid tumours are benign tumours originating from the musculoaponeurotic structures of the body . </S>", "<S> they are mainly composed of collagen . </S>", "<S> these tumours commonly occur in post - partum women in whom they originate from the rectus abominus and in old surgical incisions . here </S>", "<S> we present a case report of a young gentleman who presented with an acute surgical abdomen and subsequently underwent a laparotomy and was found to have an inflammatory mass . </S>", "<S> histological analysis of this mass revealed mesenteric fibromatosis ( desmoid tumour ) . </S>", "<S> i wish to present this interesting case because the patient was neither female nor someone who had previously undergone any surgery . </S>", "<S> it is rare for mesenteric fibromatosis to present with intestinal perforation and only one case been reported in the literature thus far ( 1 ) . an important learning point from this case </S>", "<S> is that rare pathology can manifest itself with common signs and symptoms . </S>"], "labels": null, "section_names": ["CASE", "History & examination", "Investigations", "Treatment", "Post-operative course", "DISCUSSION", "CONCLUSION"], "sections": [["a 33 year old healthy male of afro - caribbean origin presented to the emergency department with acute generalised abdominal pain of three hours duration .", "he had not noticed any recent change in bowel habit , bleeding per rectum or any systemic symptoms .", "it was of note that there was no family history of inflammatory bowel disease or gastrointestinal malignancy .", "he was a non - smoker and did not consume alcohol . on examination he had a heart rate of 110 beats per minute , temperature of 37.2c , stable blood pressure but he was cold and clammy .", "bowel sounds were absent and rectal exam revealed an empty rectum and no palpable masses .", "blood tests revealed a neutrophilia of 25,000 but urea and electrolytes were within normal range .", "he was resuscitated with intravenous fluids and an emergency laparotomy was performed . during the laparotomy , a 0.5 cm perforation was noted in the ileum 25 cm proximal to the ileocaecal valve on the mesenteric border .", "an inflammatory mass measuring 5  3 cm was noted directly adjacent to the perforation in the mesentry .", "30 cm of small bowel was resected with a right hemicolectomy , hence removing the mass and the perforation .", "no obvious cause of sepsis was identified and it was thought that he may have a subphrenic collection . therefore a computed tomography ( ct ) scan was arranged .", "however , he had a severe reaction to the intravenous contrast medium which resulted in hypotension .", "the patient underwent a second laparotomy to drain the subphrenic abscess and no masses were seen during this procedure .", "histological examination of the resected specimen from the first laporotomy revealed mesenteric fibromatosis ( desmoid tumour ) incompletely excised .", "the resected right hemi - colon did not show evidence of dysplasia , malignancy or polyposis .", "the estrogen receptor status was not reported . due to continuing abdominal discomfort on the ward after the second laparotomy , an ultrasound scan was arranged that revealed the following : a rather ill - defined , fairly ovoid , inhomogeneously - hypoechoic mass lesion is identified in the lower abdomen ( infra- umbilical ) para - sagittal plane .", "it measures about 5  3  4.5 cm in maximum dimensions surrounded by excess peritoneal fat and some bowel loops representing a residual of a previously resected abdominal desmoid .", "the pain eventually resolved and after making a good recovery , he was commenced on oral tamoxifen , 20 mg once a day .", "although estrogen receptor status was not reported , the patient was commenced on this therapy as it was assumed that most desmoid tumours have estrogen receptors .", "he was subsequently referred to a specialist in a distant tertiary unit for further evaluation of the residual tumour and for the prospect of further elective surgery ."], ["a 33 year old healthy male of afro - caribbean origin presented to the emergency department with acute generalised abdominal pain of three hours duration .", "he had not noticed any recent change in bowel habit , bleeding per rectum or any systemic symptoms .", "it was of note that there was no family history of inflammatory bowel disease or gastrointestinal malignancy .", "he was a non - smoker and did not consume alcohol . on examination he had a heart rate of 110 beats per minute , temperature of 37.2c , stable blood pressure but he was cold and clammy .", "bowel sounds were absent and rectal exam revealed an empty rectum and no palpable masses ."], ["blood tests revealed a neutrophilia of 25,000 but urea and electrolytes were within normal range ."], ["he was resuscitated with intravenous fluids and an emergency laparotomy was performed . during the laparotomy , a 0.5 cm perforation was noted in the ileum 25 cm proximal to the ileocaecal valve on the mesenteric border .", "an inflammatory mass measuring 5  3 cm was noted directly adjacent to the perforation in the mesentry .", "30 cm of small bowel was resected with a right hemicolectomy , hence removing the mass and the perforation ."], ["no obvious cause of sepsis was identified and it was thought that he may have a subphrenic collection . therefore a computed tomography ( ct ) scan was arranged .", "however , he had a severe reaction to the intravenous contrast medium which resulted in hypotension .", "the patient underwent a second laparotomy to drain the subphrenic abscess and no masses were seen during this procedure .", "histological examination of the resected specimen from the first laporotomy revealed mesenteric fibromatosis ( desmoid tumour ) incompletely excised .", "the resected right hemi - colon did not show evidence of dysplasia , malignancy or polyposis .", "due to continuing abdominal discomfort on the ward after the second laparotomy , an ultrasound scan was arranged that revealed the following : a rather ill - defined , fairly ovoid , inhomogeneously - hypoechoic mass lesion is identified in the lower abdomen ( infra- umbilical ) para - sagittal plane .", "it measures about 5  3  4.5 cm in maximum dimensions surrounded by excess peritoneal fat and some bowel loops representing a residual of a previously resected abdominal desmoid .", "the pain eventually resolved and after making a good recovery , he was commenced on oral tamoxifen , 20 mg once a day .", "although estrogen receptor status was not reported , the patient was commenced on this therapy as it was assumed that most desmoid tumours have estrogen receptors .", "he was subsequently referred to a specialist in a distant tertiary unit for further evaluation of the residual tumour and for the prospect of further elective surgery ."], ["desmoid tumours ( fibromatosis ) are benign fibrous neoplasms originating from the musculo - aponeurotic structures throughout the body .", "desmoid tumours can arise from any skeletal muscle but commonly affect the rectus abdominus in post - partum females and in old surgical scars of the abdomen .", "peripheral tumours are smooth , firm and mobile . they are adherent to the surrounding structures", "gardner s syndrome and familial adenomatous polyposis coli ( fap ) should be suspected in patients with such soft tissue growths ( 3 ) .", "an intra - abdominal desmoid , also known as mesenteric fibromatosis , is the most common solid primary neoplasm of the mesentry .", "approximately 80% of intra - abdominal desmoids involve small bowel mesentry , as was the case with our patient .", "involvement of the transverse mesocolon , retroperitoneum , omentum and the ligamentum teres have also been reported ( 1 , 4 ) .", "intra - abdominal desmoids are usually asymptomatic until their growth and infiltration causes compression of the viscera .", "this can lead to intestinal obstruction , ischemic bowel secondary to vascular compression and hydronephrosis due to ureteric compression .", "bowel perforation is extremely rare and my research has yielded one previous case report ( 1 ) .", "other rare manifestations reported in the literature include deep vein thrombosis , pyrexia of unknown origin , gastrointestinal bleeding and intra - abdominal abscess formation ( 5 , 6 ) .", "the subphrenic abscess that our patient developed is very likely to have been a complication of the initial laparotomy .", "histologically , these tumours are composed of collagen that surrounds spindle cells which are poorly circumscribed ( figure 12 ) .", "exact etiology is uncertain but hormonal factors are implicated as there are estrogen eceptors present in some desmoid tumours .", "the adenomatous polyposis coli ( apc ) gene located on chromosome 5 is responsible for familial adenomatous polypsois coli ( fap ) .", "biallelic mutations of the apc gene induces desmoid tumour formation , hence the association between these two disorders ( 8) . in our patient , there was no polyposis in the resected right hemi - colon .", "trauma has also been suggested as another causative factor but there was no such history in our patient ( 1 ) .", "both ct and magnetic resonance imaging ( mri ) are useful in diagnosing and monitoring recurrence .", "they allow accurate staging and also enable one to delineate accurate anatomy prior to embarking upon major resection .", "mri has the advantage of defining the extent of involvement and monitoring post operative recurrence , which can be as high as 70% ( 6 ) .", "these patients should also undergo colonoscopy and examination of the eye to exclude gardner s syndrome .", "colonoscopy may reveal multiple colonic polyps and fundoscopy may demonstrate multiple pigmented lesions affecting the fundus of the eye ( 2 , 3 ) . complete surgical excision may be the only effective way of providing a cure but complete excision is often impossible and therefore adjuvant treatments have been employed with various degrees of success ( 1 , 2 ) .", "other treatment modalities include radiotherapy , if there is recurrence or as primary treatment to avoid radical surgery ( 9 ) .", "anti - estrogens ( e.g. tamoxifen ) , prostaglandin inhibitors and non - steroidal anti - inflammatory drugs ( nsaids ) have also been used .", "cytotoxic chemotherapy may be useful in those with disease recurrence or where surgery is contraindicated ( 10 ) ."], ["desmoid tumours originate from the musculo - aponeurotic structures of the body and can be peripheral or intra - abdominal .", "patients should be screened for gardner s syndrome and familial adenomatous polyposis coli since these can often present with dermatoid , epidermoid or other benign tumours .", "emergency presentations are not common , but are usually secondary to mass effect on the viscera .", "ct and mri are the imaging modalities of choice , particularly if surgery is contemplated and also in postoperative follow up to detect recurrence .", "surgery is usually the mainstay of treatment but recurrence is common and adjuvant therapy is used .", "some tumours possess estrogen receptors , hence they regress in response to anti - estrogens , such as tamoxifen ."]]}
{"article_id": "PMC2698158", "article_text": ["between april 1999 and march 2001 , 32 ( 26 men and 6 women ; age range , 7 - 62 years ; mean age , 43 years ) of 195 patients who had undergone ldlt underwent 42 sessions of conventional arteriography in search of bleeding foci of arterial origin within 6 months of the operation .", "arterial bleeding was suspected clinically if the symptoms or signs were as follows : abnormally increased drainage of fresh blood through the jackson - pratt ( jp ) tube in 26 sessions , hematochezia or melena in 8 sessions , hemothorax or hemoptysis in 5 sessions , hemobilia in 2 sessions , or an acute decrease in the hemoglobin level in one session .", "six patients underwent two or three sessions of arteriography or tae , because of recurrent arterial bleeding .", "however , the bleeding foci were different in each of the sessions conducted for the same patient . in all patients ,", "the arterial bleeding occurred within two months ( mean , 14 days ; range , 1 - 56 days ) after ldlt .", "arteriography was performed within three days ( mean , 1 day ) after the detection of arterial bleeding , depending on the patient 's condition .", "the underlying causes of hepatic failure and ldlt in these 32 patients were hepatitis b - related liver cirrhosis with or without hepatocellular carcinoma ( n=29 ) , secondary biliary cirrhosis due to intrahepatic stones ( n=1 ) , fatal fulminant hepatitis due to wilson 's disease ( n=1 ) , and biliary atresia ( n=1 ) .", "the grafts involved in the ldlt included the right lobe ( n=21 ) , left lobe ( n=8 ) , dual left lobe ( n=2 ) , and left lateral segment ( n=1 ) . in all patients", ", informed consent was obtained from the patient or the patient 's family prior to arteriography .", "once the right or left femoral artery was punctured , a 5-f end - hole catheter was introduced over a 0.035-inch guide wire ( terumo ; radiofocus , tokyo , japan ) . following the superior mesenteric and common hepatic arteriographies , which were performed to evaluate the patency of the portal and hepatic arterial anastomoses and potential bleeding foci , the suspected arteries destined for selective arteriography were selected .", "a provisional identification of the bleeding arteries was made based on the patient 's symptoms or signs , radiological findings , including dynamic ct and rbc scans , and the surgical information obtained during ldlt . in the patients with abnormally increased drainage of fresh blood through the jp tube , selective arteriographies were performed of several specific arteries , for which the arterial bleeding might be stagnant in the jp tube placed area . in those patients with hematochezia or melena , inferior mesenteric or left gastric arteriography was performed following superior mesenteric and common hepatic arteriographies , according to the available clinical information . in those patients with hemothorax or hemoptysis , selective arteriographies of the inferior phrenic artery ,", "intercostal arteries adjacent to the pleural drain tubes , subclavian artery and/or bronchial artery were performed according to the available clinical information .", "aortography for the purpose of gaining an understanding of the arterial anatomy prior to selective arteriography was not routinely performed .", "however , aortography was performed to rule out the possible of there being any missed bleeding foci , if the selective arteriographies of the presumptive arteries did not demonstrate any active bleeding foci . if the bleeding foci were present on the arteriograms , then either tae or surgery was undertaken to control the bleeding , subject to the mutual agreement of the surgeons and interventional radiologists .", "tae was intended to be the primary treatment , however , surgical management was considered as the primary treatment if the bleeding foci were technically too difficult or dangerous to be embolized , such as in the case of a hepatic arterial anastomotic site or hepatic resection margin . for tae", ", the bleeding arteries were superselected using a 3f - microcatheter ( microferret ; cook , bjaerverskov , denmark ) and embolized with 0.018-inch - hilal microcoils ( cook , bloomington , u.s.a . ) or 0.018-inch - tornado microcoils ( cook , bloomington , u.s.a . ) .", "gelatin sponge particles ( gelfoam ; upjohn , kalamazoo , mi ) were also used in the case of several specific arteries ( intercostal , inferior phrenic and epigastric arteries ) prior to embolization using microcoils , to minimize any rebleeding from collateral vessels .", "if the bleeding foci were absent on the selective arteriograms and aortogram , explorative laparotomy or clinical observation was chosen by the surgeons , depending on the patient 's condition . in all patients , a complete retrospective review of the medical and surgical records and radiological imaging were performed .", "the following items were documented retrospectively : the presumptive causes and locations of arterial bleeding , the technical and clinical success rates of tae , and the complications .", "a bleeding focus was defined as an extravasation of the contrast media or a pseudoaneurysm that was a likely cause of bleeding .", "the arterial bleeding was divided into four categories according to the possible related causes : surgical bleeding was defined as an arterial bleeding related to the ldlt itself and included bleeding from the hepatic resection margin , hepatic arterial anastomotic site , incision wounds , and the hepaticojejunostomy site .", "iatrogenic bleeding was defined as bleeding associated with percutaneous invasive procedures , such as transhepatic biliary drainage , central venous access and chest tube insertion .", "spontaneous bleeding was defined as bleeding that developed spontaneously , such as gastrointestinal bleeding remote from the hepaticojejunostomy site .", "non - classifiable bleeding was defined as bleeding that was not able to be classified properly .", "the technical success of tae was defined as the complete disappearance of arterial bleeding following tae . technically impossible or incomplete", "clinical success was defined as an amelioration of the presenting signs or symptoms of arterial bleeding following tae .", "major complications were defined as those necessitating an increased level of care , surgery , prolonged hospital stay , permanent adverse sequelae or death .", "between april 1999 and march 2001 , 32 ( 26 men and 6 women ; age range , 7 - 62 years ; mean age , 43 years ) of 195 patients who had undergone ldlt underwent 42 sessions of conventional arteriography in search of bleeding foci of arterial origin within 6 months of the operation .", "arterial bleeding was suspected clinically if the symptoms or signs were as follows : abnormally increased drainage of fresh blood through the jackson - pratt ( jp ) tube in 26 sessions , hematochezia or melena in 8 sessions , hemothorax or hemoptysis in 5 sessions , hemobilia in 2 sessions , or an acute decrease in the hemoglobin level in one session .", "six patients underwent two or three sessions of arteriography or tae , because of recurrent arterial bleeding .", "however , the bleeding foci were different in each of the sessions conducted for the same patient . in all patients ,", "the arterial bleeding occurred within two months ( mean , 14 days ; range , 1 - 56 days ) after ldlt .", "arteriography was performed within three days ( mean , 1 day ) after the detection of arterial bleeding , depending on the patient 's condition .", "the underlying causes of hepatic failure and ldlt in these 32 patients were hepatitis b - related liver cirrhosis with or without hepatocellular carcinoma ( n=29 ) , secondary biliary cirrhosis due to intrahepatic stones ( n=1 ) , fatal fulminant hepatitis due to wilson 's disease ( n=1 ) , and biliary atresia ( n=1 ) .", "the grafts involved in the ldlt included the right lobe ( n=21 ) , left lobe ( n=8 ) , dual left lobe ( n=2 ) , and left lateral segment ( n=1 ) .", "in all patients , informed consent was obtained from the patient or the patient 's family prior to arteriography .", "once the right or left femoral artery was punctured , a 5-f end - hole catheter was introduced over a 0.035-inch guide wire ( terumo ; radiofocus , tokyo , japan ) . following the superior mesenteric and common hepatic arteriographies , which were performed to evaluate the patency of the portal and hepatic arterial anastomoses and potential bleeding foci , the suspected arteries destined for selective arteriography were selected .", "a provisional identification of the bleeding arteries was made based on the patient 's symptoms or signs , radiological findings , including dynamic ct and rbc scans , and the surgical information obtained during ldlt . in the patients with abnormally increased drainage of fresh blood through the jp tube , selective arteriographies were performed of several specific arteries , for which the arterial bleeding might be stagnant in the jp tube placed area . in those patients with hematochezia or melena , inferior mesenteric or left gastric arteriography was performed following superior mesenteric and common hepatic arteriographies , according to the available clinical information . in those patients with hemothorax or hemoptysis , selective arteriographies of the inferior phrenic artery ,", "intercostal arteries adjacent to the pleural drain tubes , subclavian artery and/or bronchial artery were performed according to the available clinical information .", "aortography for the purpose of gaining an understanding of the arterial anatomy prior to selective arteriography was not routinely performed .", "however , aortography was performed to rule out the possible of there being any missed bleeding foci , if the selective arteriographies of the presumptive arteries did not demonstrate any active bleeding foci . if the bleeding foci were present on the arteriograms , then either tae or surgery was undertaken to control the bleeding , subject to the mutual agreement of the surgeons and interventional radiologists .", "tae was intended to be the primary treatment , however , surgical management was considered as the primary treatment if the bleeding foci were technically too difficult or dangerous to be embolized , such as in the case of a hepatic arterial anastomotic site or hepatic resection margin . for tae", ", the bleeding arteries were superselected using a 3f - microcatheter ( microferret ; cook , bjaerverskov , denmark ) and embolized with 0.018-inch - hilal microcoils ( cook , bloomington , u.s.a . ) or 0.018-inch - tornado microcoils ( cook , bloomington , u.s.a . ) .", "gelatin sponge particles ( gelfoam ; upjohn , kalamazoo , mi ) were also used in the case of several specific arteries ( intercostal , inferior phrenic and epigastric arteries ) prior to embolization using microcoils , to minimize any rebleeding from collateral vessels . if the bleeding foci were absent on the selective arteriograms and aortogram , explorative laparotomy or clinical observation was chosen by the surgeons , depending on the patient 's condition .", "in all patients , a complete retrospective review of the medical and surgical records and radiological imaging were performed .", "the following items were documented retrospectively : the presumptive causes and locations of arterial bleeding , the technical and clinical success rates of tae , and the complications .", "a bleeding focus was defined as an extravasation of the contrast media or a pseudoaneurysm that was a likely cause of bleeding .", "the arterial bleeding was divided into four categories according to the possible related causes : surgical bleeding was defined as an arterial bleeding related to the ldlt itself and included bleeding from the hepatic resection margin , hepatic arterial anastomotic site , incision wounds , and the hepaticojejunostomy site .", "iatrogenic bleeding was defined as bleeding associated with percutaneous invasive procedures , such as transhepatic biliary drainage , central venous access and chest tube insertion .", "spontaneous bleeding was defined as bleeding that developed spontaneously , such as gastrointestinal bleeding remote from the hepaticojejunostomy site .", "non - classifiable bleeding was defined as bleeding that was not able to be classified properly .", "the technical success of tae was defined as the complete disappearance of arterial bleeding following tae . technically impossible or incomplete", "clinical success was defined as an amelioration of the presenting signs or symptoms of arterial bleeding following tae .", "major complications were defined as those necessitating an increased level of care , surgery , prolonged hospital stay , permanent adverse sequelae or death .", "conventional arteriography demonstrated 42 bleeding foci of arterial origin in 30 sessions ( 23 patients ) .", "the number of bleeding foci detected were one in 22 sessions ( 15 patients ) , two in six sessions ( 6 patients ) , and more than three in two sessions ( 2 patients ) .", "the bleeding foci originated most frequently from the hepatic artery ( 14/42 , 33% ) and intercostal artery ( 11/42 , 26% ) .", "based on a complete review of the clinical , interventional and surgical data , the bleeding foci were classified into 4 categories corresponding to the most likely causes of arterial bleeding ( table 2 ) . in this way", ", it was demonstrated that , in addition to surgical bleeding ( 36% ) , iatrogenic bleeding ( 40% ) was also a major cause of arterial bleeding after ldlt .", "tae was technically successful in 33 of the 42 foci of active arterial bleeding ( table 1 ) ( figs . 1 , 2 )", "however , tae failed in the remaining 9 foci of active arterial bleeding , due to technically difficult superselection of the corresponding bleeding artery or the potential high risk associated with tae ( fig .", "3 ) . these bleeding foci consisted of hepatic artery anastomotic sites ( n=2 ) , hepatic resection margins ( n=4 ) and hepaticojejunostomies ( n=3 ) , and they were treated by surgery .", "however , in three sessions , additional immediate surgery had to be conducted following successful tae for the evacuation of hematoma .", "however , there was no evidence of active arterial bleeding in the operation field in any of these cases .", "in 20 of the 21 sessions , the patients showed a clinical improvement in the active arterial bleeding after successful tae .", "one patient did not show any clinical improvement , because of recurrent arterial bleeding of the same focus .", "this patient had been treated by anti - coagulation therapy due to cerebral infarction , resulting in a severe iatrogenic bleeding tendency , and died of multifocal hemorrhage including cerebral hemorrhage several days after tae .", "the overall technical and clinical success rates for the 30 sessions of tae were 21 ( 70% ) and 20 ( 67% ) , respectively .", "these relatively low success rates can be ascribed to the three bleeding foci ( hepatic resection margins , hepatic artery anastomotic sites and hepaticojejunostomy sites ) , in which embolization was either incomplete or impossible . in the case of the three bleeding foci ,", "if these bleeding foci were excluded , the overall technical and clinical success rates were 19 ( 100% ) and 18 ( 95% ) out of 19 sessions .", "six ( 19% ) patients underwent two or three sessions of arteriography , because of recurrent arterial bleeding .", "however , different bleeding foci were treated in each session of arteriography conducted for the same patient . recurrent bleeding at the same foci was present in only one patient , as described above . on the other hand", "these cases were managed either by surgery ( 6 patients ) or close clinical observation ( 5 patients ) with transfusion , depending on the patient 's condition . in three of the former six patients ,", "surgery allowed the discovery of active arterial bleeding at the hepatic artery anastomotic site ( n=1 ) , hepatic resection margin ( n=1 ) , and venous oozing ( n=1 ) at the anastomosis of the hepatic vein and vena cava .", "however , a retrospective review of the selective angiographic images of these patients did not demonstrate the presence of any bleeding focus . in the remaining three patients ,", "surgery did not lead to the discovery of any bleeding focus , and they only underwent hematoma evacuation .", "after either conservative or surgical management , the signs of active bleeding ameliorated in all 11 patients . during the 6 month follow - up period after ldlt , there were no major tae - related complications .", "conventional arteriography demonstrated 42 bleeding foci of arterial origin in 30 sessions ( 23 patients ) .", "the number of bleeding foci detected were one in 22 sessions ( 15 patients ) , two in six sessions ( 6 patients ) , and more than three in two sessions ( 2 patients ) .", "the bleeding foci originated most frequently from the hepatic artery ( 14/42 , 33% ) and intercostal artery ( 11/42 , 26% ) .", "based on a complete review of the clinical , interventional and surgical data , the bleeding foci were classified into 4 categories corresponding to the most likely causes of arterial bleeding ( table 2 ) . in this way", ", it was demonstrated that , in addition to surgical bleeding ( 36% ) , iatrogenic bleeding ( 40% ) was also a major cause of arterial bleeding after ldlt .", "tae was technically successful in 33 of the 42 foci of active arterial bleeding ( table 1 ) ( figs . 1 , 2 )", ". however , tae failed in the remaining 9 foci of active arterial bleeding , due to technically difficult superselection of the corresponding bleeding artery or the potential high risk associated with tae ( fig .", "3 ) . these bleeding foci consisted of hepatic artery anastomotic sites ( n=2 ) , hepatic resection margins ( n=4 ) and hepaticojejunostomies ( n=3 ) , and they were treated by surgery .", "however , in three sessions , additional immediate surgery had to be conducted following successful tae for the evacuation of hematoma .", "however , there was no evidence of active arterial bleeding in the operation field in any of these cases . in 20 of the 21 sessions , the patients showed a clinical improvement in the active arterial bleeding after successful tae .", "one patient did not show any clinical improvement , because of recurrent arterial bleeding of the same focus .", "this patient had been treated by anti - coagulation therapy due to cerebral infarction , resulting in a severe iatrogenic bleeding tendency , and died of multifocal hemorrhage including cerebral hemorrhage several days after tae .", "the overall technical and clinical success rates for the 30 sessions of tae were 21 ( 70% ) and 20 ( 67% ) , respectively .", "these relatively low success rates can be ascribed to the three bleeding foci ( hepatic resection margins , hepatic artery anastomotic sites and hepaticojejunostomy sites ) , in which embolization was either incomplete or impossible . in the case of the three bleeding foci ,", "the technical success rate was only 2 ( 18% ) out of 11 sessions . if these bleeding foci were excluded , the overall technical and clinical success rates were 19 ( 100% ) and 18 ( 95% ) out of 19 sessions .", "six ( 19% ) patients underwent two or three sessions of arteriography , because of recurrent arterial bleeding .", "however , different bleeding foci were treated in each session of arteriography conducted for the same patient .", "recurrent bleeding at the same foci was present in only one patient , as described above .", "on the other hand , arteriography showed no active bleeding focus in 12 sessions ( 11 patients ) .", "these cases were managed either by surgery ( 6 patients ) or close clinical observation ( 5 patients ) with transfusion , depending on the patient 's condition . in three of the former six patients ,", "surgery allowed the discovery of active arterial bleeding at the hepatic artery anastomotic site ( n=1 ) , hepatic resection margin ( n=1 ) , and venous oozing ( n=1 ) at the anastomosis of the hepatic vein and vena cava .", "however , a retrospective review of the selective angiographic images of these patients did not demonstrate the presence of any bleeding focus . in the remaining three patients ,", "surgery did not lead to the discovery of any bleeding focus , and they only underwent hematoma evacuation .", "after either conservative or surgical management , the signs of active bleeding ameliorated in all 11 patients . during the 6 month follow - up period after ldlt , there were no major tae - related complications .", "according to our results , arterial bleeding after ldlt was not confined to surgical causes .", "surgical bleeding was one of the most common causes of bleeding after ldlt , however , iatrogenic bleeding was even more common than surgical bleeding in our retrospective study .", "coagulopathy after ldlt is inevitable for a certain period of time in most liver transplant recipients , until the graft 's function is normalized .", "the risk of iatrogenic bleeding is also high in the early post - ldlt period .", "thus , clinicians should make a particular effort to prevent iatrogenic bleeding during percutaneous procedures after ldlt . some amount of surgical bleeding from the hepatic artery at the hepatic arterial anastomotic site , hepatic resection margin or hepaticojejunostomy site is inevitable , because ldlt generates a liver graft with variable - sized cut surfaces and multiple anastomotic sites .", "in fact , bleeding from the hepatic artery was the most common cause of bleeding in our study . however , performing tae in the case of bleeding from the hepatic artery following ldlt was virtually impossible , because of the difficulty involved in the superselection of the bleeding arteries , due to the existence of fine arterial feeders , hepatic arterial anastomotic stenosis , a tortuous arterial course or multifocal occurrences .", "furthermore , performing tae of the hepatic artery runs the risk of hepatic infarction or failure following inadvertent diffuse proximal embolization ( 16 ) .", "( 17 ) reported that three of nine patients with extrahepatic pseudoaneurysm following liver transplantation underwent effective coil embolization , however all patients subsequently underwent retransplantation due to graft ischemia .", "accordingly , we achieved technical success in only four of the 14 foci involving bleeding from the hepatic arterial branches .", "although tae can be performed in some patients with massive bleeding from the hepatic artery , we consider that surgical management is the treatment of choice for the management of surgically induced bleeding from the hepatic artery anastomotic site , hepatic resection margin or hepaticojejunostomy site .", "the cause of bleeding from the intercostal artery was iatrogenic bleeding in most cases , resulting from either placement of a drain tube or central venous catheterization .", "the cause of bleeding from the inferior phrenic artery was uncertain , however it might have been related to various causes , such as intraoperative retractor injury , chest tube insertion or spontaneous bleeding , and it must be routinely evaluated to rule out active bleeding . knowledge of the clinical symptoms or signs of arterial bleeding provided by the clinicians is very helpful prior to the performance of arteriography . a jp drain was the most common clinical clue to the presence of arterial bleeding after ldlt .", "bleeding can occur from the hepatic , intercostal , inferior phrenic or pancreaticoduodenal arteries or from the jejunal branches of the superior mesenteric artery .", "unusually , bleeding from the deep circumflex iliac and inferior epigastric arteries was manifested as jp bleeding in one case .", "therefore , in our opinion , arteriograms of the inferior phrenic and intercostals arteries , as well as of the superior mesenteric and common hepatic arteries , should be surveyed in cases of jp drain bleeding .", "in addition , those arteries supplying the lower thoracic and abdominal walls should also be considered as possible sources of arterial bleeding . as in the case of general surgical patients , ldlt recipients are also subject to bleeding from stress ulceration , peptic ulcer and angiodysplasia of the colon ( 4 , 6 , 18 ) .", "not uncommonly , this bleeding tends to be resolved only by proper medical treatment ( 4 ) .", "clinical observation may be sufficient whenever the patient 's condition tolerates it . in our study , four patients with hematochezia or melena showed negative outcomes on arteriography , but improved clinically without surgery or tae . in fact , tae was very effective in cases of positive arteriography , and all four patients with arteriographically proven gastrointestinal bleeding improved after only one session of tae . in the conventional arteriogram , a negative outcome does not always guarantee the absence of actual bleeding . in our study ,", "surgery demonstrated active bleeding foci in three of six patients without active bleeding on selective arteriography .", "this discrepancy seems to have been caused by minor or intermittent arterial bleeding or venous oozing .", "in contrast , surgery did not lead to the detection of any bleeding focus in the three remaining patients . in conclusion ,", "technical limitations were encountered when using tae for the management of hepatic arterial bleeding . however , in the other locations , tae seems to be effective and safe for the control of arterial bleeding after ldlt ."], "abstract_text": ["<S> objectiveto analyze the causes of arterial bleeding after living donor liver transplantation ( ldlt ) and to evaluate the efficacy of transcatheter arterial embolization ( tae).materials and methodsforty - two sessions of conventional arteriography were performed in 32 of the 195 patients who underwent ldlt during the past 2 years . </S>", "<S> this was done in search of bleeding foci of arterial origin . </S>", "<S> tae was performed with microcoils or gelatin sponge particles . </S>", "<S> the causes of arterial bleeding , the technical and clinical success rates of tae and the complications were retrospectively evaluated.resultsforty-two bleeding foci of arterial origin were identified on 30 sessions of arteriography in 21 patients . </S>", "<S> the most common cause of bleeding was percutaneous procedures in 40% of the patients ( 17 of the 42 bleeding foci ) followed by surgical procedures in 36% ( 15/42 ) . </S>", "<S> the overall technical and clinical success rates of tae were 21 ( 70% ) and 20 ( 67% ) of the 30 sessions , respectively . </S>", "<S> the overall technical success rate of tae for the treatment of bleeding from the hepatic resection margin , hepatic artery anastomotic site and hepaticojejunostomy was only 18% ( 2/11 ) , whereas for the treatment of bleeding in the other locations the technical and clinical success rates of tae were 100% and 95% , respectively . </S>", "<S> no procedure - related major complications occurred.conclusionin the case of arterial bleeding after ldlt , percutaneous procedure - related hemorrhages were as common as surgery - related hemorrhages . </S>", "<S> there were technical difficulties in using tae for the treatment of hepatic arterial bleeding . </S>", "<S> however , in the other locations , tae seems to be safe and effective for the control of arterial bleeding in ldlt recipients . </S>"], "labels": null, "section_names": ["MATERIALS AND METHODS", "Patient Population", "Conventional Arteriography and Transcatheter Arterial Embolization", "Analysis", "RESULTS", "Causes and Locations of Arterial Bleeding", "Transcatheter Arterial Embolization", "DISCUSSION"], "sections": [["between april 1999 and march 2001 , 32 ( 26 men and 6 women ; age range , 7 - 62 years ; mean age , 43 years ) of 195 patients who had undergone ldlt underwent 42 sessions of conventional arteriography in search of bleeding foci of arterial origin within 6 months of the operation .", "arterial bleeding was suspected clinically if the symptoms or signs were as follows : abnormally increased drainage of fresh blood through the jackson - pratt ( jp ) tube in 26 sessions , hematochezia or melena in 8 sessions , hemothorax or hemoptysis in 5 sessions , hemobilia in 2 sessions , or an acute decrease in the hemoglobin level in one session .", "six patients underwent two or three sessions of arteriography or tae , because of recurrent arterial bleeding .", "however , the bleeding foci were different in each of the sessions conducted for the same patient . in all patients ,", "the arterial bleeding occurred within two months ( mean , 14 days ; range , 1 - 56 days ) after ldlt .", "arteriography was performed within three days ( mean , 1 day ) after the detection of arterial bleeding , depending on the patient 's condition .", "the underlying causes of hepatic failure and ldlt in these 32 patients were hepatitis b - related liver cirrhosis with or without hepatocellular carcinoma ( n=29 ) , secondary biliary cirrhosis due to intrahepatic stones ( n=1 ) , fatal fulminant hepatitis due to wilson 's disease ( n=1 ) , and biliary atresia ( n=1 ) .", "the grafts involved in the ldlt included the right lobe ( n=21 ) , left lobe ( n=8 ) , dual left lobe ( n=2 ) , and left lateral segment ( n=1 ) . in all patients", ", informed consent was obtained from the patient or the patient 's family prior to arteriography .", "once the right or left femoral artery was punctured , a 5-f end - hole catheter was introduced over a 0.035-inch guide wire ( terumo ; radiofocus , tokyo , japan ) . following the superior mesenteric and common hepatic arteriographies , which were performed to evaluate the patency of the portal and hepatic arterial anastomoses and potential bleeding foci , the suspected arteries destined for selective arteriography were selected .", "a provisional identification of the bleeding arteries was made based on the patient 's symptoms or signs , radiological findings , including dynamic ct and rbc scans , and the surgical information obtained during ldlt . in the patients with abnormally increased drainage of fresh blood through the jp tube , selective arteriographies were performed of several specific arteries , for which the arterial bleeding might be stagnant in the jp tube placed area . in those patients with hematochezia or melena , inferior mesenteric or left gastric arteriography was performed following superior mesenteric and common hepatic arteriographies , according to the available clinical information . in those patients with hemothorax or hemoptysis , selective arteriographies of the inferior phrenic artery ,", "intercostal arteries adjacent to the pleural drain tubes , subclavian artery and/or bronchial artery were performed according to the available clinical information .", "aortography for the purpose of gaining an understanding of the arterial anatomy prior to selective arteriography was not routinely performed .", "however , aortography was performed to rule out the possible of there being any missed bleeding foci , if the selective arteriographies of the presumptive arteries did not demonstrate any active bleeding foci . if the bleeding foci were present on the arteriograms , then either tae or surgery was undertaken to control the bleeding , subject to the mutual agreement of the surgeons and interventional radiologists .", "tae was intended to be the primary treatment , however , surgical management was considered as the primary treatment if the bleeding foci were technically too difficult or dangerous to be embolized , such as in the case of a hepatic arterial anastomotic site or hepatic resection margin . for tae", ", the bleeding arteries were superselected using a 3f - microcatheter ( microferret ; cook , bjaerverskov , denmark ) and embolized with 0.018-inch - hilal microcoils ( cook , bloomington , u.s.a . ) or 0.018-inch - tornado microcoils ( cook , bloomington , u.s.a . ) .", "gelatin sponge particles ( gelfoam ; upjohn , kalamazoo , mi ) were also used in the case of several specific arteries ( intercostal , inferior phrenic and epigastric arteries ) prior to embolization using microcoils , to minimize any rebleeding from collateral vessels .", "if the bleeding foci were absent on the selective arteriograms and aortogram , explorative laparotomy or clinical observation was chosen by the surgeons , depending on the patient 's condition . in all patients , a complete retrospective review of the medical and surgical records and radiological imaging were performed .", "the following items were documented retrospectively : the presumptive causes and locations of arterial bleeding , the technical and clinical success rates of tae , and the complications .", "a bleeding focus was defined as an extravasation of the contrast media or a pseudoaneurysm that was a likely cause of bleeding .", "the arterial bleeding was divided into four categories according to the possible related causes : surgical bleeding was defined as an arterial bleeding related to the ldlt itself and included bleeding from the hepatic resection margin , hepatic arterial anastomotic site , incision wounds , and the hepaticojejunostomy site .", "iatrogenic bleeding was defined as bleeding associated with percutaneous invasive procedures , such as transhepatic biliary drainage , central venous access and chest tube insertion .", "spontaneous bleeding was defined as bleeding that developed spontaneously , such as gastrointestinal bleeding remote from the hepaticojejunostomy site .", "non - classifiable bleeding was defined as bleeding that was not able to be classified properly .", "the technical success of tae was defined as the complete disappearance of arterial bleeding following tae . technically impossible or incomplete", "clinical success was defined as an amelioration of the presenting signs or symptoms of arterial bleeding following tae .", "major complications were defined as those necessitating an increased level of care , surgery , prolonged hospital stay , permanent adverse sequelae or death ."], ["between april 1999 and march 2001 , 32 ( 26 men and 6 women ; age range , 7 - 62 years ; mean age , 43 years ) of 195 patients who had undergone ldlt underwent 42 sessions of conventional arteriography in search of bleeding foci of arterial origin within 6 months of the operation .", "arterial bleeding was suspected clinically if the symptoms or signs were as follows : abnormally increased drainage of fresh blood through the jackson - pratt ( jp ) tube in 26 sessions , hematochezia or melena in 8 sessions , hemothorax or hemoptysis in 5 sessions , hemobilia in 2 sessions , or an acute decrease in the hemoglobin level in one session .", "six patients underwent two or three sessions of arteriography or tae , because of recurrent arterial bleeding .", "however , the bleeding foci were different in each of the sessions conducted for the same patient . in all patients ,", "the arterial bleeding occurred within two months ( mean , 14 days ; range , 1 - 56 days ) after ldlt .", "arteriography was performed within three days ( mean , 1 day ) after the detection of arterial bleeding , depending on the patient 's condition .", "the underlying causes of hepatic failure and ldlt in these 32 patients were hepatitis b - related liver cirrhosis with or without hepatocellular carcinoma ( n=29 ) , secondary biliary cirrhosis due to intrahepatic stones ( n=1 ) , fatal fulminant hepatitis due to wilson 's disease ( n=1 ) , and biliary atresia ( n=1 ) .", "the grafts involved in the ldlt included the right lobe ( n=21 ) , left lobe ( n=8 ) , dual left lobe ( n=2 ) , and left lateral segment ( n=1 ) ."], ["in all patients , informed consent was obtained from the patient or the patient 's family prior to arteriography .", "once the right or left femoral artery was punctured , a 5-f end - hole catheter was introduced over a 0.035-inch guide wire ( terumo ; radiofocus , tokyo , japan ) . following the superior mesenteric and common hepatic arteriographies , which were performed to evaluate the patency of the portal and hepatic arterial anastomoses and potential bleeding foci , the suspected arteries destined for selective arteriography were selected .", "a provisional identification of the bleeding arteries was made based on the patient 's symptoms or signs , radiological findings , including dynamic ct and rbc scans , and the surgical information obtained during ldlt . in the patients with abnormally increased drainage of fresh blood through the jp tube , selective arteriographies were performed of several specific arteries , for which the arterial bleeding might be stagnant in the jp tube placed area . in those patients with hematochezia or melena , inferior mesenteric or left gastric arteriography was performed following superior mesenteric and common hepatic arteriographies , according to the available clinical information . in those patients with hemothorax or hemoptysis , selective arteriographies of the inferior phrenic artery ,", "intercostal arteries adjacent to the pleural drain tubes , subclavian artery and/or bronchial artery were performed according to the available clinical information .", "aortography for the purpose of gaining an understanding of the arterial anatomy prior to selective arteriography was not routinely performed .", "however , aortography was performed to rule out the possible of there being any missed bleeding foci , if the selective arteriographies of the presumptive arteries did not demonstrate any active bleeding foci . if the bleeding foci were present on the arteriograms , then either tae or surgery was undertaken to control the bleeding , subject to the mutual agreement of the surgeons and interventional radiologists .", "tae was intended to be the primary treatment , however , surgical management was considered as the primary treatment if the bleeding foci were technically too difficult or dangerous to be embolized , such as in the case of a hepatic arterial anastomotic site or hepatic resection margin . for tae", ", the bleeding arteries were superselected using a 3f - microcatheter ( microferret ; cook , bjaerverskov , denmark ) and embolized with 0.018-inch - hilal microcoils ( cook , bloomington , u.s.a . ) or 0.018-inch - tornado microcoils ( cook , bloomington , u.s.a . ) .", "gelatin sponge particles ( gelfoam ; upjohn , kalamazoo , mi ) were also used in the case of several specific arteries ( intercostal , inferior phrenic and epigastric arteries ) prior to embolization using microcoils , to minimize any rebleeding from collateral vessels . if the bleeding foci were absent on the selective arteriograms and aortogram , explorative laparotomy or clinical observation was chosen by the surgeons , depending on the patient 's condition ."], ["in all patients , a complete retrospective review of the medical and surgical records and radiological imaging were performed .", "the following items were documented retrospectively : the presumptive causes and locations of arterial bleeding , the technical and clinical success rates of tae , and the complications .", "a bleeding focus was defined as an extravasation of the contrast media or a pseudoaneurysm that was a likely cause of bleeding .", "the arterial bleeding was divided into four categories according to the possible related causes : surgical bleeding was defined as an arterial bleeding related to the ldlt itself and included bleeding from the hepatic resection margin , hepatic arterial anastomotic site , incision wounds , and the hepaticojejunostomy site .", "iatrogenic bleeding was defined as bleeding associated with percutaneous invasive procedures , such as transhepatic biliary drainage , central venous access and chest tube insertion .", "spontaneous bleeding was defined as bleeding that developed spontaneously , such as gastrointestinal bleeding remote from the hepaticojejunostomy site .", "non - classifiable bleeding was defined as bleeding that was not able to be classified properly .", "the technical success of tae was defined as the complete disappearance of arterial bleeding following tae . technically impossible or incomplete", "clinical success was defined as an amelioration of the presenting signs or symptoms of arterial bleeding following tae .", "major complications were defined as those necessitating an increased level of care , surgery , prolonged hospital stay , permanent adverse sequelae or death ."], ["conventional arteriography demonstrated 42 bleeding foci of arterial origin in 30 sessions ( 23 patients ) .", "the number of bleeding foci detected were one in 22 sessions ( 15 patients ) , two in six sessions ( 6 patients ) , and more than three in two sessions ( 2 patients ) .", "the bleeding foci originated most frequently from the hepatic artery ( 14/42 , 33% ) and intercostal artery ( 11/42 , 26% ) .", "based on a complete review of the clinical , interventional and surgical data , the bleeding foci were classified into 4 categories corresponding to the most likely causes of arterial bleeding ( table 2 ) . in this way", ", it was demonstrated that , in addition to surgical bleeding ( 36% ) , iatrogenic bleeding ( 40% ) was also a major cause of arterial bleeding after ldlt .", "tae was technically successful in 33 of the 42 foci of active arterial bleeding ( table 1 ) ( figs . 1 , 2 )", "however , tae failed in the remaining 9 foci of active arterial bleeding , due to technically difficult superselection of the corresponding bleeding artery or the potential high risk associated with tae ( fig .", "3 ) . these bleeding foci consisted of hepatic artery anastomotic sites ( n=2 ) , hepatic resection margins ( n=4 ) and hepaticojejunostomies ( n=3 ) , and they were treated by surgery .", "however , in three sessions , additional immediate surgery had to be conducted following successful tae for the evacuation of hematoma .", "however , there was no evidence of active arterial bleeding in the operation field in any of these cases .", "in 20 of the 21 sessions , the patients showed a clinical improvement in the active arterial bleeding after successful tae .", "one patient did not show any clinical improvement , because of recurrent arterial bleeding of the same focus .", "this patient had been treated by anti - coagulation therapy due to cerebral infarction , resulting in a severe iatrogenic bleeding tendency , and died of multifocal hemorrhage including cerebral hemorrhage several days after tae .", "the overall technical and clinical success rates for the 30 sessions of tae were 21 ( 70% ) and 20 ( 67% ) , respectively .", "these relatively low success rates can be ascribed to the three bleeding foci ( hepatic resection margins , hepatic artery anastomotic sites and hepaticojejunostomy sites ) , in which embolization was either incomplete or impossible . in the case of the three bleeding foci ,", "if these bleeding foci were excluded , the overall technical and clinical success rates were 19 ( 100% ) and 18 ( 95% ) out of 19 sessions .", "six ( 19% ) patients underwent two or three sessions of arteriography , because of recurrent arterial bleeding .", "however , different bleeding foci were treated in each session of arteriography conducted for the same patient . recurrent bleeding at the same foci was present in only one patient , as described above . on the other hand", "these cases were managed either by surgery ( 6 patients ) or close clinical observation ( 5 patients ) with transfusion , depending on the patient 's condition . in three of the former six patients ,", "surgery allowed the discovery of active arterial bleeding at the hepatic artery anastomotic site ( n=1 ) , hepatic resection margin ( n=1 ) , and venous oozing ( n=1 ) at the anastomosis of the hepatic vein and vena cava .", "however , a retrospective review of the selective angiographic images of these patients did not demonstrate the presence of any bleeding focus . in the remaining three patients ,", "surgery did not lead to the discovery of any bleeding focus , and they only underwent hematoma evacuation .", "after either conservative or surgical management , the signs of active bleeding ameliorated in all 11 patients . during the 6 month follow - up period after ldlt , there were no major tae - related complications ."], ["conventional arteriography demonstrated 42 bleeding foci of arterial origin in 30 sessions ( 23 patients ) .", "the number of bleeding foci detected were one in 22 sessions ( 15 patients ) , two in six sessions ( 6 patients ) , and more than three in two sessions ( 2 patients ) .", "the bleeding foci originated most frequently from the hepatic artery ( 14/42 , 33% ) and intercostal artery ( 11/42 , 26% ) .", "based on a complete review of the clinical , interventional and surgical data , the bleeding foci were classified into 4 categories corresponding to the most likely causes of arterial bleeding ( table 2 ) . in this way", ", it was demonstrated that , in addition to surgical bleeding ( 36% ) , iatrogenic bleeding ( 40% ) was also a major cause of arterial bleeding after ldlt ."], ["tae was technically successful in 33 of the 42 foci of active arterial bleeding ( table 1 ) ( figs . 1 , 2 )", ". however , tae failed in the remaining 9 foci of active arterial bleeding , due to technically difficult superselection of the corresponding bleeding artery or the potential high risk associated with tae ( fig .", "3 ) . these bleeding foci consisted of hepatic artery anastomotic sites ( n=2 ) , hepatic resection margins ( n=4 ) and hepaticojejunostomies ( n=3 ) , and they were treated by surgery .", "however , in three sessions , additional immediate surgery had to be conducted following successful tae for the evacuation of hematoma .", "however , there was no evidence of active arterial bleeding in the operation field in any of these cases . in 20 of the 21 sessions , the patients showed a clinical improvement in the active arterial bleeding after successful tae .", "one patient did not show any clinical improvement , because of recurrent arterial bleeding of the same focus .", "this patient had been treated by anti - coagulation therapy due to cerebral infarction , resulting in a severe iatrogenic bleeding tendency , and died of multifocal hemorrhage including cerebral hemorrhage several days after tae .", "the overall technical and clinical success rates for the 30 sessions of tae were 21 ( 70% ) and 20 ( 67% ) , respectively .", "these relatively low success rates can be ascribed to the three bleeding foci ( hepatic resection margins , hepatic artery anastomotic sites and hepaticojejunostomy sites ) , in which embolization was either incomplete or impossible . in the case of the three bleeding foci ,", "the technical success rate was only 2 ( 18% ) out of 11 sessions . if these bleeding foci were excluded , the overall technical and clinical success rates were 19 ( 100% ) and 18 ( 95% ) out of 19 sessions .", "six ( 19% ) patients underwent two or three sessions of arteriography , because of recurrent arterial bleeding .", "however , different bleeding foci were treated in each session of arteriography conducted for the same patient .", "recurrent bleeding at the same foci was present in only one patient , as described above .", "on the other hand , arteriography showed no active bleeding focus in 12 sessions ( 11 patients ) .", "these cases were managed either by surgery ( 6 patients ) or close clinical observation ( 5 patients ) with transfusion , depending on the patient 's condition . in three of the former six patients ,", "surgery allowed the discovery of active arterial bleeding at the hepatic artery anastomotic site ( n=1 ) , hepatic resection margin ( n=1 ) , and venous oozing ( n=1 ) at the anastomosis of the hepatic vein and vena cava .", "however , a retrospective review of the selective angiographic images of these patients did not demonstrate the presence of any bleeding focus . in the remaining three patients ,", "surgery did not lead to the discovery of any bleeding focus , and they only underwent hematoma evacuation .", "after either conservative or surgical management , the signs of active bleeding ameliorated in all 11 patients . during the 6 month follow - up period after ldlt , there were no major tae - related complications ."], ["according to our results , arterial bleeding after ldlt was not confined to surgical causes .", "surgical bleeding was one of the most common causes of bleeding after ldlt , however , iatrogenic bleeding was even more common than surgical bleeding in our retrospective study .", "coagulopathy after ldlt is inevitable for a certain period of time in most liver transplant recipients , until the graft 's function is normalized .", "the risk of iatrogenic bleeding is also high in the early post - ldlt period .", "thus , clinicians should make a particular effort to prevent iatrogenic bleeding during percutaneous procedures after ldlt . some amount of surgical bleeding from the hepatic artery at the hepatic arterial anastomotic site , hepatic resection margin or hepaticojejunostomy site is inevitable , because ldlt generates a liver graft with variable - sized cut surfaces and multiple anastomotic sites .", "in fact , bleeding from the hepatic artery was the most common cause of bleeding in our study . however , performing tae in the case of bleeding from the hepatic artery following ldlt was virtually impossible , because of the difficulty involved in the superselection of the bleeding arteries , due to the existence of fine arterial feeders , hepatic arterial anastomotic stenosis , a tortuous arterial course or multifocal occurrences .", "furthermore , performing tae of the hepatic artery runs the risk of hepatic infarction or failure following inadvertent diffuse proximal embolization ( 16 ) .", "( 17 ) reported that three of nine patients with extrahepatic pseudoaneurysm following liver transplantation underwent effective coil embolization , however all patients subsequently underwent retransplantation due to graft ischemia .", "accordingly , we achieved technical success in only four of the 14 foci involving bleeding from the hepatic arterial branches .", "although tae can be performed in some patients with massive bleeding from the hepatic artery , we consider that surgical management is the treatment of choice for the management of surgically induced bleeding from the hepatic artery anastomotic site , hepatic resection margin or hepaticojejunostomy site .", "the cause of bleeding from the intercostal artery was iatrogenic bleeding in most cases , resulting from either placement of a drain tube or central venous catheterization .", "the cause of bleeding from the inferior phrenic artery was uncertain , however it might have been related to various causes , such as intraoperative retractor injury , chest tube insertion or spontaneous bleeding , and it must be routinely evaluated to rule out active bleeding . knowledge of the clinical symptoms or signs of arterial bleeding provided by the clinicians is very helpful prior to the performance of arteriography . a jp drain was the most common clinical clue to the presence of arterial bleeding after ldlt .", "bleeding can occur from the hepatic , intercostal , inferior phrenic or pancreaticoduodenal arteries or from the jejunal branches of the superior mesenteric artery .", "unusually , bleeding from the deep circumflex iliac and inferior epigastric arteries was manifested as jp bleeding in one case .", "therefore , in our opinion , arteriograms of the inferior phrenic and intercostals arteries , as well as of the superior mesenteric and common hepatic arteries , should be surveyed in cases of jp drain bleeding .", "in addition , those arteries supplying the lower thoracic and abdominal walls should also be considered as possible sources of arterial bleeding . as in the case of general surgical patients , ldlt recipients are also subject to bleeding from stress ulceration , peptic ulcer and angiodysplasia of the colon ( 4 , 6 , 18 ) .", "not uncommonly , this bleeding tends to be resolved only by proper medical treatment ( 4 ) .", "clinical observation may be sufficient whenever the patient 's condition tolerates it . in our study , four patients with hematochezia or melena showed negative outcomes on arteriography , but improved clinically without surgery or tae . in fact , tae was very effective in cases of positive arteriography , and all four patients with arteriographically proven gastrointestinal bleeding improved after only one session of tae . in the conventional arteriogram , a negative outcome does not always guarantee the absence of actual bleeding . in our study ,", "surgery demonstrated active bleeding foci in three of six patients without active bleeding on selective arteriography .", "this discrepancy seems to have been caused by minor or intermittent arterial bleeding or venous oozing .", "in contrast , surgery did not lead to the detection of any bleeding focus in the three remaining patients . in conclusion ,", "technical limitations were encountered when using tae for the management of hepatic arterial bleeding . however , in the other locations , tae seems to be effective and safe for the control of arterial bleeding after ldlt ."]]}
{"article_id": "PMC4719173", "article_text": ["fibrodysplasia ossificans progressiva ( fop ) is a rare genetic disoder characterized by bone formation within muscles tendons and ligaments .", "we hereby report a case of fop in a four year male child from a tribal family in orissa .", "4 yr old male child presented with gradual development of stiffness of neck and hard nodules on his body for which his parents had sought all sort of indegenous treatment and manipulations by traditional bone setters .", "patient returned to our hospital at the age of four years with widespread ossification and stiffness of neck , shoulders and back .", "he also had upper tibial osteochondromas and scalp nodules and valgus deformity of bilateral great toes .", "though rare , diagnosis of myositis ossificans progressiva should be considered in a child with heterotopic bone formation and valgus deformities of great toes .", "being a rare condition , treatment guidelines are not clear and this condition need further research .", "fibrodysplasia ossificans progressiva ( fop ) is also known as myositis ossificans progressiva , stone man disease , munchmeyer s disease .", "fop is an extremely rare and most crippling form of heterotopic ossification in humans with an incidence of 1 in 2 millions .", "kitterman et al reported that it takes atleast six physician evaluations to reach the diagnosis and 90% of these individuals will have received some wrong or hazardous treatment before appropriate diagnosis .", "we hereby report a case of fop in a 4 year old boy coming from tribal family in orissa .", "the diagnosis of this patient was delayed due as the parents tried all sorts of indigenous medications and even manipulations by traditional bonesetters whose influence has been deep rooted in minds of the tribal population in this part of the country .", "our patients was a four year old boy who presented with chief complains of stiffness of neck , decreased movements in bilateral shoulders and had bony hard swellings in his back .", ", his parents noticed that he had stiffness of neck and had developed nodules at his scalp , which gradually subsided without any treatment .", "coming from a tribal background , parents consulted many indigenous medicine practitioners and child was subjected to repeated manipulations of the neck by traditional bone setters .", "these swellings hardened over a period of few days and the child developed stiffness of spine .", "he was subjected to further manipulations and gradually developed stiffness of bilateral shoulders and left elbow . on examination ,", "the patient had bony hard swellings in cervico - thoraco - lumbar spine , bilateral axillae and left elbow and left 3rd costochondral junction along with hypoplasia and valgus deformity of great toes ( fig .", "a frontal view of the patient showing ankylosis of bilateral shoulders and left elbow with bony swelling at left 3rd costo chondral junction .", "radiographs revealed hallux valgus and hypoplasia of proximal phalanges of great toes with short widened first metacarpals bilaterally ( fig .", "2b , c ) extensive ossification overlying both side of the neck and in the para vertebral tissues in dorsolumbar area ( fig .", "diagnosis of fop was made in basis of all these radiological and clinical findings radiological findings .", "a- bilateral hallux valgus and hypoplastic proximal phalynx of great toe with short and widened first metatarsal .", "our case once again highlights the deleterious effects of trauma in the form of manipulations and massage that can aggravate the ossification process in case of fop .", "trauma as trivial as intramuscular injections , mandibular block during dental procedures , muscle fatigue , muscle trauma due to bumps , falls and influenza like illness can aggravate the process of heterotopic ossification in these patients . a sporadic mutation in the gene encoding bone morphogenetic protein ( bmp ) activin receptor type ia / activin - like kinase 2 ( acvr1/alk2 ) located on chromosome 2q23 - 24 region has been identified as the main genetic abnormality in fop . recently , additional mutations have been identified in the gs - domain and kinase domain of acvr1 in individuals with a typical forms of fop .", "inheritance is in autosomal dominant manner but less than 10 cases of familial transmission have been reported . in vitro experiments with use of cells derived from patients with fibrodysplasia ossificans progressiva", "show a markedly attenuated response of noggin ( bmp-4 antagonist ) expression to bmp4 stimulation .", "an inadequate bmp - antagonist response following soft - tissue trauma would permit the rapid expansion of a bmp4 morphogenetic gradient in a patient with fibrodysplasia ossificans progressiva and could explain the explosive bone induction seen during flare - ups . a new research by quen et al interpreted that in addition to the above , the mutation also induces chondrogenesis by signaling in a bmp independent and bmp responsive manner .", "this mutation may also sensitize mesenchymal cells to bmp - induced osteoblast differentiation , and stimulates new bone formation .", "individuals with fop are normal at birth except for having valgus deformity of great toes .", "heterotopic ossification is heralded by the rapid appearance of large painful swellings of highly vascular fibro - proliferative tissue involving tendons , ligaments , fascia , and skeletal muscle .", "these preosseous swellings progress along a pathway of endochondral ossification to form mature heterotopic bone .", "typically , the dorsal , axial , cranial , and proximal regions of the body are involved early , and the ventral , appendicular , caudal , and distal regions are involved later .", "several skeletal muscles including the diaphragm , tongue , and extra - ocular muscles are spared from fop . cardiac muscle and smooth muscle", "histological examination of early fop lesions reveals an intense perivascular lymphocytic infiltrate followed by lymphocyte - associated death of skeletal muscle and robust development of fibro proliferative tissue with extensive neovascularity and mast cell infiltration .", "tissues from fop lesions at later stages of maturation exhibit characteristic features of endochondral ossification that support ectopic hematopoiesis .", "definitive genetic testing for fop is now available however is not essential for diagnosis which is purely clinical .", "this condition needs to be differentiated from progressive osseous hyperplasia ( poh ) , which presents with heterotopic ossification .", "poh is distinguished from fop by the absence of congenital skeletal malformations , the absence of predictable regional patterns of heterotopic ossification , the pre- dominance of intramembranous rather than endochondral ossification , and the presence of heterotopic ossification in skin and subcutaneous fat ", "piram et al noted 40% incidence of scalp nodules in patients of fop in their retrospective study on 43 patients .", "deirmengian et al reported 90% incidence of upper tibial osteochondromas in a study on 96 patients .", "our case had this particular finding and cases with combined heterotrophic ossifications with osteochondromas should be considered for this diagnosis . based on these typical and commonly found characteristics , mutlu et", "the great toe malformations along with rapidly developing and waxing / waning soft tissue lesions over head , neck and upper back are characteristics for fop .", "kaplan et al commented that failure to make such association is the commonest cause of misdiagnosis of fop .", "misdiagnosis may lead to inadvertent managements like manipulations , biopsies and surgery . as in our case", ", these strategies just help the disease to flare and make the life of patient much difficult .", "kitterman et al reported incidence of misdiagnosis to be > 90% , with 68% receiving inappropriate treatment which lead to permanent disability in about 50% of cases .", "early diagnosis and high index of suspicion will not only prevent the itrogenic harm but may also slow the disease progress and avoid rapid and early deterioration in patients quality of life . in our case ,", "this lead to rapid progression of disease in our case with early development of deformities .", "short course of steroids are used in during acute flare - ups in large joints and submandibular area .", "other drugs tried are nsaids and cox-2 inhibitors , bisphonates like etidronate and pamindronate , rosiglitazones , mast cell inhibitors , retinoic receptor agonists etc [ 15 , 16 ] . however", "a statement made by julius rosenstirn in 1918 still holds true :  the disease was attacked with all sorts of remedies and alternatives for faulty metabolism ; every one of them with more or less marked success observed solely by its original author but pronounced a complete failure by every other follower . in many cases , the symptoms of the disease disappear often spontaneously , so the therapeutic effect ( of any treatment ) should not be unreservedly endorsed  .", "the rare incidence of this disease and unpredictable nature of flare - ups makes it difficult to formulate an ideal treatment for it .", "kaplan et al reported that bone marrow transplant for replacement of hematopoietic cells was not sufficient to prevent ectopic skeletogenesis in the patient with fibrodysplasia ossificans progressiva but pharmacologic suppression of the apparently normal donor immune system following transplantation in the new host modulated the activity of the fibrodysplasia ossificans progressiva and diminished the expression of skeletal ectopia .", "glaser et al reported bmp4-induced heterotopic ossification can be prevented in vivo either by local delivery of wild - type noggin or after somatic cell gene transfer of a noggin mutein in murine model of fop . recently", ", dorsomorphin has been identified as a powerful orally - available signal transduction inhibitor of bmp signaling and preliminary data suggest that this category of stis may play a powerful role in inhibiting heterotopic ossification in animal models but its safety and efficacy in animal models of fop is still to be determined . although the rate of disease progression is variable , most patients are confined to a wheel- chair by their early twenties and require lifelong assistance with activities of daily living .", "patients with fop usually succumb later in adulthood at mean age of 40 years to cardiopulmonary complications secondary to thoracic insufficiency syndrome and severe restrictive pulmonary disease due to ossification and ankylosis of the joints of thoracic cage .", "thus in conclusion clinical suspicion , early diagnosis and expectant treatment are the main management strategy in cases with fop .", "fop demands further research to treat individuals suffering from this dreaded form of heterotopic ossification .", "valgus deformity of great toes and neck stiffness are important pointers towards diagnosis of fop and this condition needs further research to find an effective treatment modality for it"], "abstract_text": ["<S> introduction : fibrodysplasia ossificans progressiva ( fop ) is a rare genetic disoder characterized by bone formation within muscles tendons and ligaments . it has an incidence of one in two million . </S>", "<S> we hereby report a case of fop in a four year male child from a tribal family in orissa.case report:4 yr old male child presented with gradual development of stiffness of neck and hard nodules on his body for which his parents had sought all sort of indegenous treatment and manipulations by traditional bone setters . </S>", "<S> patient returned to our hospital at the age of four years with widespread ossification and stiffness of neck , shoulders and back . </S>", "<S> he also had upper tibial osteochondromas and scalp nodules and valgus deformity of bilateral great toes . </S>", "<S> a diagnosis of fop was made on clinical and radiological examination.conclusion:though rare , diagnosis of myositis ossificans progressiva should be considered in a child with heterotopic bone formation and valgus deformities of great toes . </S>", "<S> being a rare condition , treatment guidelines are not clear and this condition need further research . </S>"], "labels": null, "section_names": ["Introduction:", "Case Report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["fibrodysplasia ossificans progressiva ( fop ) is a rare genetic disoder characterized by bone formation within muscles tendons and ligaments .", "we hereby report a case of fop in a four year male child from a tribal family in orissa ."], ["4 yr old male child presented with gradual development of stiffness of neck and hard nodules on his body for which his parents had sought all sort of indegenous treatment and manipulations by traditional bone setters .", "patient returned to our hospital at the age of four years with widespread ossification and stiffness of neck , shoulders and back .", "he also had upper tibial osteochondromas and scalp nodules and valgus deformity of bilateral great toes ."], ["though rare , diagnosis of myositis ossificans progressiva should be considered in a child with heterotopic bone formation and valgus deformities of great toes .", "being a rare condition , treatment guidelines are not clear and this condition need further research ."], ["fibrodysplasia ossificans progressiva ( fop ) is also known as myositis ossificans progressiva , stone man disease , munchmeyer s disease .", "fop is an extremely rare and most crippling form of heterotopic ossification in humans with an incidence of 1 in 2 millions .", "kitterman et al reported that it takes atleast six physician evaluations to reach the diagnosis and 90% of these individuals will have received some wrong or hazardous treatment before appropriate diagnosis .", "we hereby report a case of fop in a 4 year old boy coming from tribal family in orissa .", "the diagnosis of this patient was delayed due as the parents tried all sorts of indigenous medications and even manipulations by traditional bonesetters whose influence has been deep rooted in minds of the tribal population in this part of the country ."], ["our patients was a four year old boy who presented with chief complains of stiffness of neck , decreased movements in bilateral shoulders and had bony hard swellings in his back .", ", his parents noticed that he had stiffness of neck and had developed nodules at his scalp , which gradually subsided without any treatment .", "coming from a tribal background , parents consulted many indigenous medicine practitioners and child was subjected to repeated manipulations of the neck by traditional bone setters .", "these swellings hardened over a period of few days and the child developed stiffness of spine .", "he was subjected to further manipulations and gradually developed stiffness of bilateral shoulders and left elbow . on examination ,", "the patient had bony hard swellings in cervico - thoraco - lumbar spine , bilateral axillae and left elbow and left 3rd costochondral junction along with hypoplasia and valgus deformity of great toes ( fig .", "a frontal view of the patient showing ankylosis of bilateral shoulders and left elbow with bony swelling at left 3rd costo chondral junction .", "radiographs revealed hallux valgus and hypoplasia of proximal phalanges of great toes with short widened first metacarpals bilaterally ( fig .", "2b , c ) extensive ossification overlying both side of the neck and in the para vertebral tissues in dorsolumbar area ( fig .", "diagnosis of fop was made in basis of all these radiological and clinical findings radiological findings .", "a- bilateral hallux valgus and hypoplastic proximal phalynx of great toe with short and widened first metatarsal ."], ["our case once again highlights the deleterious effects of trauma in the form of manipulations and massage that can aggravate the ossification process in case of fop .", "trauma as trivial as intramuscular injections , mandibular block during dental procedures , muscle fatigue , muscle trauma due to bumps , falls and influenza like illness can aggravate the process of heterotopic ossification in these patients . a sporadic mutation in the gene encoding bone morphogenetic protein ( bmp ) activin receptor type ia / activin - like kinase 2 ( acvr1/alk2 ) located on chromosome 2q23 - 24 region has been identified as the main genetic abnormality in fop . recently , additional mutations have been identified in the gs - domain and kinase domain of acvr1 in individuals with a typical forms of fop .", "inheritance is in autosomal dominant manner but less than 10 cases of familial transmission have been reported . in vitro experiments with use of cells derived from patients with fibrodysplasia ossificans progressiva", "show a markedly attenuated response of noggin ( bmp-4 antagonist ) expression to bmp4 stimulation .", "an inadequate bmp - antagonist response following soft - tissue trauma would permit the rapid expansion of a bmp4 morphogenetic gradient in a patient with fibrodysplasia ossificans progressiva and could explain the explosive bone induction seen during flare - ups . a new research by quen et al interpreted that in addition to the above , the mutation also induces chondrogenesis by signaling in a bmp independent and bmp responsive manner .", "this mutation may also sensitize mesenchymal cells to bmp - induced osteoblast differentiation , and stimulates new bone formation .", "individuals with fop are normal at birth except for having valgus deformity of great toes .", "heterotopic ossification is heralded by the rapid appearance of large painful swellings of highly vascular fibro - proliferative tissue involving tendons , ligaments , fascia , and skeletal muscle .", "these preosseous swellings progress along a pathway of endochondral ossification to form mature heterotopic bone .", "typically , the dorsal , axial , cranial , and proximal regions of the body are involved early , and the ventral , appendicular , caudal , and distal regions are involved later .", "several skeletal muscles including the diaphragm , tongue , and extra - ocular muscles are spared from fop . cardiac muscle and smooth muscle", "histological examination of early fop lesions reveals an intense perivascular lymphocytic infiltrate followed by lymphocyte - associated death of skeletal muscle and robust development of fibro proliferative tissue with extensive neovascularity and mast cell infiltration .", "tissues from fop lesions at later stages of maturation exhibit characteristic features of endochondral ossification that support ectopic hematopoiesis .", "definitive genetic testing for fop is now available however is not essential for diagnosis which is purely clinical .", "this condition needs to be differentiated from progressive osseous hyperplasia ( poh ) , which presents with heterotopic ossification .", "poh is distinguished from fop by the absence of congenital skeletal malformations , the absence of predictable regional patterns of heterotopic ossification , the pre- dominance of intramembranous rather than endochondral ossification , and the presence of heterotopic ossification in skin and subcutaneous fat ", "piram et al noted 40% incidence of scalp nodules in patients of fop in their retrospective study on 43 patients .", "deirmengian et al reported 90% incidence of upper tibial osteochondromas in a study on 96 patients .", "our case had this particular finding and cases with combined heterotrophic ossifications with osteochondromas should be considered for this diagnosis . based on these typical and commonly found characteristics , mutlu et", "the great toe malformations along with rapidly developing and waxing / waning soft tissue lesions over head , neck and upper back are characteristics for fop .", "kaplan et al commented that failure to make such association is the commonest cause of misdiagnosis of fop .", "misdiagnosis may lead to inadvertent managements like manipulations , biopsies and surgery . as in our case", ", these strategies just help the disease to flare and make the life of patient much difficult .", "kitterman et al reported incidence of misdiagnosis to be > 90% , with 68% receiving inappropriate treatment which lead to permanent disability in about 50% of cases .", "early diagnosis and high index of suspicion will not only prevent the itrogenic harm but may also slow the disease progress and avoid rapid and early deterioration in patients quality of life . in our case ,", "this lead to rapid progression of disease in our case with early development of deformities .", "short course of steroids are used in during acute flare - ups in large joints and submandibular area .", "other drugs tried are nsaids and cox-2 inhibitors , bisphonates like etidronate and pamindronate , rosiglitazones , mast cell inhibitors , retinoic receptor agonists etc [ 15 , 16 ] . however", "a statement made by julius rosenstirn in 1918 still holds true :  the disease was attacked with all sorts of remedies and alternatives for faulty metabolism ; every one of them with more or less marked success observed solely by its original author but pronounced a complete failure by every other follower . in many cases , the symptoms of the disease disappear often spontaneously , so the therapeutic effect ( of any treatment ) should not be unreservedly endorsed  .", "the rare incidence of this disease and unpredictable nature of flare - ups makes it difficult to formulate an ideal treatment for it .", "kaplan et al reported that bone marrow transplant for replacement of hematopoietic cells was not sufficient to prevent ectopic skeletogenesis in the patient with fibrodysplasia ossificans progressiva but pharmacologic suppression of the apparently normal donor immune system following transplantation in the new host modulated the activity of the fibrodysplasia ossificans progressiva and diminished the expression of skeletal ectopia .", "glaser et al reported bmp4-induced heterotopic ossification can be prevented in vivo either by local delivery of wild - type noggin or after somatic cell gene transfer of a noggin mutein in murine model of fop . recently", ", dorsomorphin has been identified as a powerful orally - available signal transduction inhibitor of bmp signaling and preliminary data suggest that this category of stis may play a powerful role in inhibiting heterotopic ossification in animal models but its safety and efficacy in animal models of fop is still to be determined . although the rate of disease progression is variable , most patients are confined to a wheel- chair by their early twenties and require lifelong assistance with activities of daily living .", "patients with fop usually succumb later in adulthood at mean age of 40 years to cardiopulmonary complications secondary to thoracic insufficiency syndrome and severe restrictive pulmonary disease due to ossification and ankylosis of the joints of thoracic cage ."], ["thus in conclusion clinical suspicion , early diagnosis and expectant treatment are the main management strategy in cases with fop .", "fop demands further research to treat individuals suffering from this dreaded form of heterotopic ossification .", "valgus deformity of great toes and neck stiffness are important pointers towards diagnosis of fop and this condition needs further research to find an effective treatment modality for it"]]}
{"article_id": "PMC2652357", "article_text": ["delivery of exogenous nucleic acids such as dna into cells ( transfection ) holds great promise for disease prevention and therapy ( aka : gene therapy).(1 ) genetic material delivered into the cell modifies the function of such cells generally by altering the production of proteins .", "the selection of appropriate dna carriers is a common problem for gene therapy , and better gene delivery agents are still being sought .", "viral vectors ( such as retroviral , lentiviral , adenoviral ) and nonviral vectors ( such as liposomal and polymeric ) have both been used as gene delivery agents in the past .", "although viral vectors are efficient carriers due to their natural ability to penetrate cells , they have also often been shown to provoke undesirable immune responses , thus limiting their usefulness.(2 ) nonviral vectors often prove less immunogenic , but improvement is needed to increase their overall transfection efficiency . to increase such efficiency ,", "effort should be directed at increasing the vector s ability to promote efficient dna condensation , intracellular transport , and sustained gene expression .", "additionally , the cytocompatibility of nonviral vectors must be well understood before they can be further optimized and become a viable option for gene therapy .", "fullerene ( c60 ) derivatives have been extensively studied for a variety of medical applications(8 ) including their use as neuroprotective agents,(9 ) hiv-1 protease inhibitors,(10 ) bone - disorder therapy agents,(11 ) x - ray contrast agents,(12 ) and slow - release agents for drug delivery.(13 ) recently , c60-based reagents have also been examined as dna transfection vectors and tested for the ability to mediate gene transfer . these first generation c60-based transfection vectors have shown promise , but a few have also exhibited high cytotoxicity.(16 ) as many of the c60 derivatives previously described in the literature have only been slightly soluble in aqueous solutions , it has also been suggested that one possible reason for such observed cytotoxicity was due to the use of organic solvents that were necessary to dissolve those first generation c60-based transfection agents .", "thus , the search for new materials and enhanced protocols are key objectives in the continuing development of c60-based transfection vectors , including the design , construction , and testing of new reagents that are water soluble and able to effect enhanced gene delivery without promoting significant cytotoxicity", ". herein we report transfection efficiencies , cytotoxicity profiles , and biophysical structure / activity studies for a new class of water - soluble c60 vectors prepared using the hirschbingel reaction which is one of the simplest , highest - yielding , and most versatile c60 functionalization methods known . as shown in figure 1 ,", "the structurally derivatized c60 vectors reported herein were synthesized to yield either positively charged , negatively charged , or neutral chemical structures when solvated under physiological conditions .", "these vectors , which have been analyzed for their ability to promote dna transfection , offer the opportunity to elucidate the roles that hydrophobicity ( due to the fullerene core ) , hydrophilicity ( due to the water soluble substituents ) , and overall charge state displayed by the c60 derivatives have on dna transfection , gene expression , and cytotoxicity .", "depiction of the structures of the derivatized c60 vectors ; the positively charged amino - c60 compounds ( iiv ) , the neutral serinolamide - c60 compound ( v ) , and the negatively charged c3-c60 compound ( vi ) ( chemical structures depicted at neutral ph in aqueous solution ; counterions in solution not shown ) .", "materials for culturing cells , including dulbecco s modified eagle s medium ( dmem ) , trypsin , and phosphate buffer saline ( pbs ) , were obtained from gibco ( gibco life , grand island , ny ) .", "complete osteogenic medium containing minimum essential medium ( mem ) , 10 mm -glycerophosphate , 50 g / ml ascorbic acid , and 10 vol % fetal bovine serum was obtained from gemini bio - products ( calabasas , ca ) .", "a plasmid dna ( 7.2 kbp ) containing the gene for a red - shifted wavelength variant of the enhanced green fluorescent protein ( egfp ) under the transcriptional control of the cytomegalovirus ( cmv ) immediate - early gene promoter was used as the dna construct from which reporter gene expression was measured in the transfection assays .", "plasmid dna was prepared in bulk from transformed bacterial sources and purified to homogeneity using commercial kits ( qiagen ; germantown , md ) employing anion - exchange chromatography .", "the amino - c60 adducts ( iiv ) , the seri - c60 adduct ( v ) , and the c3-c60 adduct ( vi ) were synthesized and characterized according to previously published procedures .", "stock solutions of individual compounds ivi were prepared by weight and filtered for sterilization using a cellulose acetate membrane filter ( 0.2 m pore diameter ) prior to use in forming transfection mixtures .", "standard solutions were prepared at a concentration 10 g/l for each of these compounds in both tris edta ( te ) solution ( 10 mm tris , 1 mm edta , ph = 7.4 ) , and in serum - free dmem in separate reaction vials .", "nih 3t3 mouse fibroblast cells ( atcc # crl-1658 ) and hek 293 cells ( human embryonic kidney ; atcc # crl-1573 ) were cultured in dmem containing 10% fbs in a 5% co2 atmosphere at 37 c prior to collection and plating in individual assay wells used for transfection studies . for subculture of cells , routinely on reaching 80% confluence ,", "the cells were washed with pbs , detached using trypsin - edta , harvested by centrifugation , resuspended in culture medium and counted in a coulter counter prior to plating in multiwell dishes for subsequent transfection assays .", "marrow stromal cells ( mscs ) were harvested from wistar rats as previously described.(24 ) mscs were cultured for 6 days in the presence of 10 m dexamethasone in complete osteogenic media before use in transfection studies and cultured under identical environmental conditions to 3t3 and hek 293 cells .", "mixtures of individual derivatized c60 vector material and plasmid dna were allowed to form a complex in serum - free dmem , as described below , prior to adding to cells in the transfection assays .", "each c60-vector solution was added dropwise to a solution of plasmid dna to obtain a final volume of 200 l .", "each resulting solution was then vortexed briefly and allowed to stand for 30 min at room temperature to allow complete assembly of the vector / dna complex , which was then used immediately in the transfection experiment .", "the final dna mass of each solution remained constant at 10 g while the concentration of the c60 derivative was varied to obtain a range of c60-reagent - to - dna - base - pair ( bp ) ratios ( defined here as ", "the value of dna mass was fixed at 10 g per well since it showed the most optimum transfection among different dna masses used ( 112 g ) for transfection at r = 16.8 .", "transfections of nih 3t3 cells were carried out with c60/dna complexes in a range of r values ( 0.4242 ) .", "for hek 293 cells and mscs , transfections were performed only at r = 4.2 and r = 16.8 .", "cells were plated at 40,000 cells / cm in 96 well tissue culture plates and incubated overnight to permit cell attachment to the well surface .", "the culture medium was then replaced by the serum - free transfection mixture for various time periods ( 2 h , 8 h , 24 or 48 h ) ( transfection time ) . after exposing the cells for the respective periods of time to the serum - free transfection mixture", ", the cells were washed with pbs and the medium was replaced with complete medium ( containing fbs ) .", "the cells were then further incubated for 8 h , 24 or 48 h ( incubation time ) before gfp fluorescence was measured using a flow cytometer . for comparative purposes", ", control cell populations were also transfected with plasmid dna alone ( no c60 vector ) , or with plasmid dna complexed with an optimal level of one of two commercially available transfection reagents ; such that the dna was complexed with either 25 kda polyethylenimine ( pei ) , or with cytopure transfection reagent , which is reported by the manufacturer to exhibit very low levels of cytotoxicity .", "the dna / pei complexes were assembled using a well - established protocol(25 ) and the cytopure / dna complexes were assembled as per the manufacturer s instructions and optimized to obtain an optimal level of dna transfection / gfp expression within the nih 3t3 cell type .", "below is a brief description of the conditions which gave the optimal level of transfected nih 3t3 cells using 25 kda pei and cytopure . for pei ,", "dna / polymer complexes were prepared in serum - free dmem to achieve a ratio of polymer to dna of 4 .", "the complexes ( 100 l ) were then incubated at 25 c for 1015 min and then added to cell wells that contained 100 l of serum - free dmem .", "for cytopure , 1.1 l of cytopure stock was diluted to 50 l with serum - free dmem .", "the cytopure mixture was then added slowly to 1 g of dna diluted to 50 l with serum - free dmem .", "the transfection mixture was vortexed , left standing for 15 min at room temperature and added to cell wells that contained 100 l of serum - free dmem .", "after 24 h of transfection , the cells were washed and the medium was replaced with the serum - containing medium . for purposes of this study ,", "transfection  as described herein also relates to cell viability for sufficient time to ensure gfp gene expression , which was determined by cell fluorescence levels above a defined background threshold level ( determined using nontransfected cells to set lower detection limit parameter ) with flow cytometry .", "cells were prepared for flow cytometry by trypsinization after being washed with sterile pbs to remove cell debris and any residual gene - delivery agents .", "cells that were transfected successfully expressed gfp protein and were detected at 470/515 nm ( excitation / emission ) by the flow cytometer .", "transfection efficiency has been determined as the percent of cells that express gfp per study sample relative to the total number of cells passing through the flow cytometer per study sample ( 10,000 events counted for each transfection sample).(25 ) the c60/dna complex solutions were prepared as described above .", "c60 vector solutions without dna ( 010 g/l ) were prepared by diluting the c60 stock solution with serum - free dmem .", "the concentration of cultured cells was determined prior to cell plating using a coulter counter and the cells were plated on 96 well clear - bottom plates at a density of 40,000 cells / cm . after incubating overnight to allow cell attachment , the medium in each well was replaced either with 200 l of the c60 vector alone or with c60 vector / dna solutions prepared as described above .", "the cells were exposed to the solutions for either 2 , 8 or 24 h before the cytotoxicity was determined .", "control cell samples were plated and grown under identical conditions as those described for treated cell samples , with the exception that neither c60 compounds nor transfection mixtures were added to the control cells .", "the live / dead viability / cytotoxicity assay ( molecular probes , eugene , or ) was used to determine cytotoxicity levels in cells treated with either c60-vector or c60-vector / dna solutions .", "this assay makes use of 2 different fluorescent dyes , calcein am and ethidium homodimer ( ethd-1 ) , to measure the number of live and dead cells respectively . the lipid permeable dye , calcein am , when taken up by living cells with intact plasma membranes , emits green fluorescence ( excitation / emission : 495/515 nm ) upon being cleaved by esterase enzymes in the cells .", "conversely , the ethd-1 dye emits red fluorescence ( excitation / emission : 528/617 nm ) on binding to nucleic acids released from dead cells , and is excluded by the intact cell membranes of viable cells .", "this assay was preferred over the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay for evaluation of cell viability because c60-vectors compounds i and ii interacted and attached to the mtt - formazan making the formazan crystals insoluble for the next colorimetric assay .", "similar effects have been reported for single - walled carbon nanotubes.(26 ) the reagents were prepared and added to the wells according to the manufacturer s guidelines . at the time points indicated , the media from the wells was aspirated and replaced with the live / dead reagent , after washing the cells with pbs .", "the plates were then incubated in the dark at room temperature for additional 30 min before being measured for fluorescence .", "fluorescence of each sample well was measured by using a fluorescent microplate reader ( tecan safire ) .", "fluorescence of each sample well was normalized to percent of both live ( no c60 compounds or transfection mixtures treatment condition ) and dead ( 70% methanol treated ) control cell groups .", "these normalized fractions are defined as the fraction of live and dead cells , respectively , in each of the c60-treated samples .", "since gfp fluorescence has excitation / emission wavelengths of 470/515 nm , close to that of calcein am , there was a possibility that gfp fluorescence could interfere with the calcein am ( live ) measurement leading to potential errors for those wells containing c60 vector / dna mixtures .", "thus , a baseline fluorescence intensity of each sample well at 495/515 nm was obtained before the addition of the live cell / dead cell dyes .", "this intensity was then subtracted from the final calcein am detection intensity . for the dynamic light scattering ( dls ) experiments ,", "a standard solution with concentration 10 g/l of each of the c60 vectors was prepared in each of these solvents : hplc grade water , 10 mm tris / edta solution , and serum - free dmem .", "these solvents were filtered through a 0.22 m syringe filter ( millpore , millex- gp ) prior to use .", "the size of standard 100 nm polystyrene beads in tris / edta solution and in serum - free dmem was measured to confirm that no discrepancies were detected with the data acquisition system .", "dls measurements were conducted using a wyatt technologies dawn eos instrument equipped with a quasi - elastic light scattering interface module ( wyatt qels unit ) .", "the scattering angle used for detection was 90 at a constant temperature of 25 c .", "data analysis and size distribution computations were performed using the vendor supplied software ( astra v ) and a multi- digital recorder ( wyatt qels ) .", "statistical analysis was performed between groups for the live / dead assay and for flow cytometry studies .", "groups were analyzed by anova with significance defined by a p - value < 0.05 , and pairwise comparison was performed using the tukey test .", "the cytotoxicity studies had four samples per group , and the transfection studies had three samples per group .", "initial screening was performed on each of the six different derivatized c60 samples , compounds ivi ( figure 1 ) .", "the different c60-vector / dna complexes were assembled at various r values ( 0.4242 ) and each analyzed for its ability to affect transfection in nih 3t3 cells under serum - free conditions .", "serum - free conditions were used in these studies to ensure that derivatized nanomaterial / dna complexes were not compromised by serum lipids and/or proteins , and that the dna complexes were imported into the cells based on the individual physical properties of the complexes themselves , without the potential aid of serum proteins acting as carrier .", "the best transfection efficiencies were routinely observed after allowing transfection mixtures to remain on cells for 24 h ( transfection time ) , then removing the transfection mixtures and incubating cells for an additional 24 h ( incubation time ) in the presence of serum for maximal gene expression to be detected . unless otherwise noted , all transfection efficiency data presented below were for cells transfected for 24 h under serum - free conditions and incubated for an additional 24 h in the presence of serum .", "all c60-conjugated transfection mixtures showed the ability to transfect cells with exogenous dna with statistically significant differences relative to unconjugated dna alone ( naked dna ) ( table 1 ) .", " transfection efficiency  as reported in table 1 is defined as the ratio of gfp - positive cells as determined by fluorescence under flow cytometry per total number of cells counted in the flow cytometer .", "no statistical difference was observed between the transfection efficiencies of the different groups at r = 0.42 and r = 4.2 . for r values above 4.2 , compounds iii and iv exhibited a statistical difference in transfection efficiencies compared to the other compounds . both of these compounds exhibited an increased transfection efficiency relative to the other c60 compounds studied , with a maximum transfection efficiency of 31% at r = 16.8 . at larger values of r", "the efficiency dropped off . at r = 16.8 , their transfection efficiencies were comparable to that of 25 kda pei containing and cytopure transfection reagent containing complexes .", "25 kda pei is a widely used and well - studied nonviral vector , while cytopure is a biodegradable polymeric transfection reagent that exhibits very low cytotoxicity and is reported to work on a broad spectrum of cell types , including hard - to - transfect primary cell isolates .", "c60 vectors are listed in order of their measured efficiency at promoting transfection in nih 3t3 cells under serum - free conditions . to assess the transfection ability of c60 vectors in different cell types ,", "studies were also performed on two other cell types : hek 293 cells and mscs .", "hek 293 cells were chosen since they are another standard cell line used routinely for transfection assays , while mscs were chosen due to their eminence as primary cells for tissue engineering .", "based on the initial screening results of each of the various derivatized c60 compounds for transfection - promoting ability in nih 3t3 cells as reported in tables 1 and 2 , transfection studies were also performed on hek 293 cells and mscs using compounds iii and iv at ratios of r = 4.2 and r = 16.8 .", "however , hek 293 cells ' transfection efficiencies were lower than those observed for nih 3t3 cell at similar r values .", "mscs exhibited low transfection efficiencies overall , and no increase in transfection efficiency was observed with increase in r values .", "transfected cells were visualized by fluorescence microscopy for morphological analysis after transfection ( micrographs not shown ) .", "positive transfection of cells with the plasmid coding for green fluorescent protein could be easily discerned by this technique .", "however , variations in transfection efficiency among the different cell types tested here is not surprising , as cell type is a major factor in determining the level of transfection and gene expression from nonviral vectors as noted previously.(27 ) differences in cell characteristics , such as cell mitosis rates , can play an important role in transgene expression , and may explain the differences in the level of gene expression among the various cell lines studied here .", "transfection efficiency values are presented as a quotient of gfp ( + ) cells relative to the total viable cells present in the transfected sample well as measured by flow cytometry .", "figures 2 a , b and c display the fraction of viable nih 3t3 cells as a function of the c60 vector ( no dna present ) concentration after 2 , 8 , or 24 h post - treatment incubation times , respectively .", "incubation with c60 compounds for 2 h resulted in a viable cell fraction greater than 50% for all c60 vector solutions .", "lowest overall cell viability was observed with compound i ( 0.55  0.08 ) at 10 g/l , and no statistically significant increase in cell viability was observed with decreasing compound i concentration . for all other c60 compounds tested", ", there was a general trend of increased cell viability with decreased c60 compound concentration .", "fraction of live nih 3t3 cells after incubation with the different c60 vectors as a function of derivatized fullerene concentration after ( a ) 2 h , ( b ) 8 h , or ( c ) 24 h c60-incubation times respectively , under serum - free conditions .", "error bars represent mean  standard deviation for n = 4 . at the 8 h c60 incubation time , the fraction of live cells was higher than 50% for all c60 solutions tested except those exposed to compound i. for compound i , the cell viability was the lowest ( 0.51  0.04 ) at 10 g/l , but no statistically significant increase in cell viability was observed with decreasing compound i concentration . for all other c60 vectors ,", "there was a general trend of increased cell viability with decreased compound concentration , but at each compound concentration tested , the cell viability was lower in the 8 h time point than in the corresponding sample concentration at the 2 h time point .", "at the 24 h c60 incubation time point , only cells treated with compounds iii and iv exhibited greater than 50% cell viability at all concentrations tested , with an increase in cell viability with decreased c60 compound ( iii and iv ) concentration .", "all other compounds showed a general trend of increased viability with a decrease in compound concentration , with cell viability values lower than 50% at 10 g/l .", "figures 3 a , b and c display the cell viability of nih 3t3 cells cultured in the presence of the various c60-vector / dna mixtures as a function of both c60 compound concentration and r values after 2 , 8 or 24 h post - transfection time , respectively .", "cell viability at the two hour post - transfection time point for compound i / dna and compound ii / dna mixtures increased from 0.19  0.0 at 10 g/l ( r = 42 ) to 0.70  0.15 at 0.1 g/l ( r = 0.42 ) .", "the fraction of live cells was greater than 50% for all other c60 derivative / dna solutions at the 2 h post - transfection time point .", "fraction of live nih 3t3 cells after incubation with the different c60/dna transfection mixtures as a function of c60 compound concentration and varying r value after ( a ) 2 h , ( b ) 8 h , or ( c ) 24 h post - transfection incubation time , under serum - free conditions respectively .", "error bars represent the mean  standard deviation for n = 4 . at the 8 h post - transfection time point ,", "the fraction of live cells for compound i / dna and compound ii / dna was less than 50% at all concentrations and r values tested . for all other samples ,", "the trend was similar to that of the 2 h post - transfection time point , with cell viability greater than 50% and an increase in viability observed with a decrease in transfection mixture concentration .", "at the 24 h time point , cells treated with the compound v / dna mixture exhibited greater than 50% cell viability at all concentrations tested , while compound i / dna , compound ii / dna exhibited less than 50% cell viability .", "cells treated with compounds iii / dna , iv / dna and vi / dna mixtures resulted in cell viabilities less than 50% at 4 g/l ( r = 16.8 ) and 10 g/l ( r = 42 ) , but greater than 50% viability when added at lower concentrations .", "dls and optical microscopy studies were performed to help elucidate the biophysical structure / activity relationships that might exist between the various c60 complexes .", "these studies were carried out to help generate correlations between the different c60 vectors , and the various c60 vector / dna structural attributes resulting in the different transfection efficiencies and cytotoxicity results observed under serum - free conditions .", "table 3 presents the dls measurements on the various c60 vector compounds ivi when dissolved alone in either water ( 4 g/l ) , in a 10 mm tris / edta solution ( 4 g/l ) , in serum - free dmem ( 4 g/l ) , or when dissolved in complex with dna in 10 mm tris / edta ( 4 g/l , r = 16.8 ) or as a complex with dna in serum - free dmem ( 4 g/l , r = 16.8 ) .", "the concentration of 4 g/l was chosen for dls experiments since compounds iii and iv exhibited maximum transfection efficiency at this concentration of c60 .", "the rh values in table 3 provide qualitative information about the transfection mixture aggregate behavior which can readily be seen in figures 4 and 5 .", "the rh parameter obtained from the dls measurements , and shown in table 3 , corresponds to the radius of a monodisperse hypothetical sphere having the same diffusion constant as that of the c60 compound aggregates or the c60/dna mixture aggregates under study .", "pdi values between 0 and 0.05 indicate more monodisperse particles , and values above 0.05 indicate broadened size distributions.(30 ) clearly , the rh values shown in table 3 for the various c60 compounds and c60/dna complexes are average values for quite polydisperse populations of aggregates .", "previous work by our group and others using atomic force microscopy ( afm ) and cryo transmission electron microscopy ( cryo - tem ) have shown that derivatized c60 compounds form aggregates that are irregularly shaped in solution .", "the rh values observed for these c60 compounds and the c60/dna mixtures suggests that these reagents also form such irregularly shaped aggregates when in aqueous solutions , including when dissolved in solutions of physiological ionic strength such as dmem .", "such qualitative information about the aggregate behavior provides valuable information for understanding the transfection and toxicity results , as discussed below .", "representative morphologies of nih 3t3 cells and the c60/dna complexes at r = 0.42 ten minutes after transfection .", "the optical images represent complexes formed with compound ( a ) i , ( b ) ii , ( c ) iii , ( d ) iv , ( e ) v , and ( f ) vi . all images are shown at the same magnification .", "representative optical microscopy images of nih 3t3 cells incubated with c60/dna complexes formed using compounds ( a ) iii and ( b ) vi at r = 0.42 twenty four hours post - transfection .", "all c60 compounds display a broadly distributed aggregate size in each solvent tested ( water , 10 mm tris / edta solution , and serum - free dmem ) .", "the compounds had aggregate sizes between 47 and 650 nm ( pdi = 0.330.51 ) and between 33 and 446 nm ( pdi = 0.290.46 ) in plain water and 10 mm tris / edta solution , respectively .", "in the presence of dna , there is an increase in rh for all formulations . in general", ", the rh values decrease in the order v , vi > i > ii > iii , iv in the presence of dna . presumably , this trend is the result of the influence of the various structures of the c60 derivatives on the condensation behavior of the dna in the transfection mixture . in the case of cationic polymers ,", "the condensation process with dna has been thought to rely predominantly on electrostatic interactions with minor contributions from other interactions such as hydrophobic interactions , hydrogen bonding , and van der waals forces.(5 ) in case of the positively charged c60 derivatives , the hydrophobicity of the c60 core , and electrostatic interactions between the water - soluble functional groups and the dna are the main contributors to the condensation process .", "compound i , with two adducts on the fullerene core , has less positive charge than the hexa - amino c60 adduct of compound ii and the octa - amino adduct of compound iii .", "fewer electrostatic interactions between functionalized c60 vectors and the dna sugarphosphate backbone can lead to a decrease in condensation capacity , resulting in larger c60-vector / dna complexes .", "compound v , although polar , has no net charge at neutral ph ; while compound vi will possess a negative charge , the same as that of dna .", "thus , for compounds v and vi , hydrophobicity is presumably the only interaction contributing to the formation of the dna condensates , and this interaction appears insufficient to fully condense the dna , leading to large c60 vector / dna complex aggregates .", "the relatively large sizes for the c60 vector / dna complexes , and in particularly for those formed from compounds i , ii , v and vi , may be a reason for the poor transfection efficiencies observed with these compounds .", "this may also be a reason for the low transfection efficiency reported for a similar positively charged amino - c60 compound with a monoadduct on the fullerene core.(17 ) however , increased positive charge - state alone is insufficient to solely predict increased transfection capability of amino - derivatized fullerenes .", "variation in transfection protocol and/or cell types can also dramatically affect transfection efficiencies , as positively charged amino - derivatized c60 similar to compound iv have previously been shown to be inefficient at effecting transfection.(17 ) in general , the condensation behavior of nonviral vector / dna complexes is governed by several factors including the molecular weight of the dna condensing agent , the density of the charges on the condensing agent , and the ratio of composition ( r values ) between the condensing vector and the dna.(5 ) in addition , it is now well - documented that the size of the transfection vector / dna complex is one of the most important parameters for efficient dna delivery into cells , with the most efficient uptake observed for complex with sizes < 150200 nm in diameter.(5 ) the rh values shown in table 3 suggest that only dna complexes formed with compounds iii and iv satisfy these criteria .", "on addition of the c60-vector / dna complexes to cells , the complexes formed with compounds i and ii immediately aggregated further to form large clusters , even at the lowest r value tested of 0.42 .", "figure 4af shows the optical microscopy images of all c60-vector / dna complexes at r = 0.42 ten min after their addition to nih 3t3 cells .", "large , irregularly shaped aggregates ( shown as brown ) can clearly be seen for complexes formed with compound i ( figure 4a ) and for complexes formed with compound ii ( figure 4b ) .", "the aggregate concentration is greater and more densely distributed for compound i than for compound ii .", "no such increased aggregation propensity was seen for the other c60 vector / dna complexes ( figure 4cf ) .", "however , time - dependence of aggregation could be observed for compound iii and compound vi .", "figure 5a , b shows the optical microscopy images of compound iii / dna , and compound vi / dna complexes at r = 0.42 at 24 h post - transfection time .", "the images show the presence of large sized aggregates , more densly distributed for compound vi than for compound iii .", "interestingly , addition of compound iii / dna mixtures caused cell morphological changes when added to cultured cells , causing the attached cells to round up from the substratum surface in contrast to the usual planar , extended / elongated shapes the cells assumed in the normal growth state .", "this shape change may be attributed to the depolymerization / disruption of either the actin or tubulin networks of the cytoskeleton by the complexes , and/or to the disruption / inhibition of focal adhesion plaques formed on the cell surface . however , this observation at r = 0.42 does not have any noticeable effects on cell proliferation , as flow cytometry measurements indicate no statistically significant difference in the number of cells for those treated with compound iii / dna complexes relative to the controls cells ( no c60/dna complexes added ) .", "our results indicate that compounds iii and iv , when complexed with dna , bring about the highest levels of transfection when compared to the other derivatized c60 compounds under evaluation in this study . at the optimal ratio of compound iii or iv to dna ( r = 16.8 ) ,", "however , at 24 h post - transfection , there is only approximately 30% viability in cells treated with these two tranfection mixtures .", "this result is in contrast to cell viability of greater than 50% in cells treated with the compounds iii or iv alone at equivalent c60 concentrations ( 4g/l ) as that when in complex with dna at r = 16.8 .", "the temporal increase in aggregate sizes for these c60/dna complexes ( not seen in the free c60 compounds alone ) leads to precipitation onto the cell surface when these mixtures are left in the culture medium for 24 h ( e.g. , see figure 5 ) .", "this effect may explain the higher levels of cell death in the transfection mixture - treated cells .", "the overall trend observed in these studies was that increased cell death was observed with an increase in either c60 concentration alone or for increased r values in c60/dna mixtures , irrespective of the chemical nature of the c60 compound , with compounds iii and iv exhibiting the least toxicity when free in solution , but not necessarily when complexed with dna ( under these conditions , compound v seems to exhibit the least cell toxicity ) .", "also , higher levels of cytotoxicity seem to correlate more with complex aggregate formation induced by the presence of dna in the mixtures , leading to the formation of visible precipitates . such deposition of aggregates onto the cell surface will impair normal functioning of the plasma membrane and thereby contribute to cell death.(31 ) there may also be other slow processes contributing to cell death for the positively charged c60 vectors in the c60/dna solutions at the longer ( 8 and 24 h ) transfection times .", "following cellular entry , the presence of positively charged amino - c60 compounds ( iiv ) ( individuals or as aggregates ) inside the cell or nucleus may lead to disruptions to other cellular functions , such as interfering with histonedna interactions and/or other cellular processes.(7 ) the favorable transfection efficiencies of compounds iii and iv suggest that these compounds have some characteristics in common with other positively charged amino - c60 derivatives previously cited in the literature which are reported to effect transfection in cells .", "these similarities include having a flexible linker situated between the fullerene core and the positively charged amino groups .", "although the results presented here suggest that the structures of compounds iii and iv will result in higher levels of transfection than those previously reported by others , direct comparison of our results with the results from others using different structurally derivatized positively charged amino c60 vectors is not possible , since the structure of the c60 vectors , the cell lines tested , and the methodologies used are not consistent .", "another distinct feature of all the c60 vectors reported here is their water solubility which obviates the need to use organic solvents such as dimethylformamide ( dmf ) in the preparation of the transfection mixture ; the high cytotoxicity observed for some other c60-based transfection agents has been attributed to the toxicity of dmf .", "the comparable transfection efficiencies of compounds iii and iv on nih 3t3 cells to those of commercially available nonviral vectors 25 kda pei and cytopure is encouraging .", "25 kda pei is a widely used nonviral vector and is quite often used as a standard for comparing new nonviral vectors .", "this high cytotoxicity has been attributed more to free pei than to the pei / dna complex . however , in case of compound iii or iv , at the optimal r = 16.8 , their complexes with dna show higher toxicity ( 30% viability ) compared to the free compounds iii or iv alone ( greater than 50% cell viability ) .", "since the cytotoxicity seems to correlate more with complex aggregate formation induced by the presence of dna in the mixtures , future refinement of the c60/dna complex preparation protocol , such as disaggregating these complexes with phosphate buffer and/or nacl , should potentially lead to lower cytotoxicity while maintaining the same level of transfection efficiency , or potentially even increase these transfection efficiency levels .", "furthermore , although not addressed in this study ( for reasons of maintaining uniformity in c60/dna complex formation and cellular entry ) , the presence of serum proteins on enhanced c60/dna complex uptake , and on subsequent cell viability during extended transfection times , would also be worth examining , and could be expected to lower the observed cellular cytotoxicity levels seen in this study .", "the small , but statistically significant , expression of the reporter protein from c60/dna conjugates formed from compounds v and vi was surprising and interesting since these samples do not possess a net positive charge under the physiological conditions used to form the complexes , although there have been limited reports of other nonpositive charged nanoparticles translocating into cells(35 ) and , indeed , also demonstrating gene transfection.(36 ) thus , at least for some derivatized c60 molecules , the hydrophobic nature of the c60 carbon nanostructures may be the common important property in their binding to dna and their subsequent translocation into the interior of the cell .", "there is now a growing body of work that suggests that carbon nanomaterials such as fullerenes , metallofullerenes and carbon nanotubes , due the hydrophobicity of their carbon sheaths , have the ability to efficiently translocate into the interior of cells irrespective of the charge of their water - solubilizing moieties .", "in addition , carbon nanomaterials such as gd@c60 , gd@c82 , gd@c80 and gd@ultrashort single - walled carbon nanotubes ( gd@us - tubes ) have shown potential as high - performance magnetic resonance imaging ( mri ) contrast agents suitable for advanced applications such as tracking and/or delivery of magnetically labeled cells by mri in vivo .", "these reports , along with the favorable c60-based gene transfection results reported here and by others , offer opportunities for development of analogous gd@c60-based vectors to monitor noninvasively the biodistribution and pharmacokinetics of gene therapy vectors in vivo.(49 )", "a new class of water - soluble c60 transfecting agents with positively charged , negatively charged , or neutral chemical functionalities under physiological conditions was prepared using hirschbingel chemistry .", "transfection , cytotoxicity and biophysical structure / activity studies were performed in an effort to elucidate the relationship between the hydrophobicity of the fullerene core , the hydrophilicity of the water - solubilizing groups , and the overall charge state of the c60 vectors in gene delivery / expression .", "however , only two positively charged c60 derivatives , namely , an octa - amino derivatized c60 and a dodeca - amino derivatized c60 vector , showed efficient in vitro transfection .", "increased levels of cellular toxicity were observed for positively charged c60 vectors relative to the negatively charged and neutral vectors .", "structural analyses using dynamic light scattering and optical microscopy confirmed that higher positive charge on c60 compounds is a dominant physical attribute necessary for optimal c60/dna structural features leading to increased transfection efficiency , and that aggregation is the major factor that negatively affected the cytotoxicity profiles of the c60 vector / dna complexes .", "aggregation is presumably also the dominant reason for increased cytotoxicity of certain specific derivatized c60 compounds ( most notable for compound i ) even in the absence of dna , at least when analyzed under the serum - free conditions utilized in this study .", "future studies should be able to capitalize on this information and enable the design of additional c60 derivatives that address these issues specifically and help lower the propensity of these reagents to aggregate in the presence of dna .", "such reagents would be expected to enhance transfection of cells and tissues while simultaneously lowering toxicity levels .", "future studies using these reagents will also more precisely indicate the cellular uptake and endosomal release mechanisms that these compounds inherently possess , thereby lending further insight into potential enhanced chemical design of future generations of these carbon - based nanomaterials specifically for dna delivery into the cell nucleus .", "the successful demonstration of intracellular dna uptake , intracellular transport , and gene expression from dna using c60 vectors suggests the possibility of developing analogous gd@c60-based vectors to serve simultaneously as both therapeutic and diagnostic agents ."], "abstract_text": ["<S> a new class of water - soluble c60 transfecting agents has been prepared using hirschbingel chemistry and assessed for their ability to act as gene - delivery vectors in vitro . in an effort to elucidate the relationship between the hydrophobicity of the fullerene core , the hydrophilicity of the water - solubilizing groups , and the overall charge state of the c60 vectors in gene delivery and expression , several different c60 derivatives were synthesized to yield either positively charged , negatively charged , or neutral chemical functionalities under physiological conditions . </S>", "<S> these fullerene derivatives were then tested for their ability to transfect cells grown in culture with dna carrying the green fluorescent protein ( gfp ) reporter gene . </S>", "<S> statistically significant expression of gfp was observed for all forms of the c60 derivatives when used as dna vectors and compared to the ability of naked dna alone to transfect cells . </S>", "<S> however , efficient in vitro transfection was only achieved with the two positively charged c60 derivatives , namely , an octa - amino derivatized c60 and a dodeca - amino derivatized c60 vector . </S>", "<S> all c60 vectors showed an increase in toxicity in a dose - dependent manner . </S>", "<S> increased levels of cellular toxicity were observed for positively charged c60 vectors relative to the negatively charged and neutral vectors . </S>", "<S> structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized c60 compounds , the c60 vector / dna complexes , their physical attributes ( aggregation , charge ) and their transfection efficiencies . </S>", "<S> recently , similar gd@c60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging ( mri ) . </S>", "<S> thus , the successful demonstration of intracellular dna uptake , intracellular transport , and gene expression from dna using c60 vectors suggests the possibility of developing analogous gd@c60-based vectors to serve simultaneously as both therapeutic and diagnostic agents . </S>"], "labels": null, "section_names": ["Introduction", "Experimental Section", "Results and Discussion", "Conclusion"], "sections": [["delivery of exogenous nucleic acids such as dna into cells ( transfection ) holds great promise for disease prevention and therapy ( aka : gene therapy).(1 ) genetic material delivered into the cell modifies the function of such cells generally by altering the production of proteins .", "the selection of appropriate dna carriers is a common problem for gene therapy , and better gene delivery agents are still being sought .", "viral vectors ( such as retroviral , lentiviral , adenoviral ) and nonviral vectors ( such as liposomal and polymeric ) have both been used as gene delivery agents in the past .", "although viral vectors are efficient carriers due to their natural ability to penetrate cells , they have also often been shown to provoke undesirable immune responses , thus limiting their usefulness.(2 ) nonviral vectors often prove less immunogenic , but improvement is needed to increase their overall transfection efficiency . to increase such efficiency ,", "effort should be directed at increasing the vector s ability to promote efficient dna condensation , intracellular transport , and sustained gene expression .", "additionally , the cytocompatibility of nonviral vectors must be well understood before they can be further optimized and become a viable option for gene therapy .", "fullerene ( c60 ) derivatives have been extensively studied for a variety of medical applications(8 ) including their use as neuroprotective agents,(9 ) hiv-1 protease inhibitors,(10 ) bone - disorder therapy agents,(11 ) x - ray contrast agents,(12 ) and slow - release agents for drug delivery.(13 ) recently , c60-based reagents have also been examined as dna transfection vectors and tested for the ability to mediate gene transfer . these first generation c60-based transfection vectors have shown promise , but a few have also exhibited high cytotoxicity.(16 ) as many of the c60 derivatives previously described in the literature have only been slightly soluble in aqueous solutions , it has also been suggested that one possible reason for such observed cytotoxicity was due to the use of organic solvents that were necessary to dissolve those first generation c60-based transfection agents .", "thus , the search for new materials and enhanced protocols are key objectives in the continuing development of c60-based transfection vectors , including the design , construction , and testing of new reagents that are water soluble and able to effect enhanced gene delivery without promoting significant cytotoxicity", ". herein we report transfection efficiencies , cytotoxicity profiles , and biophysical structure / activity studies for a new class of water - soluble c60 vectors prepared using the hirschbingel reaction which is one of the simplest , highest - yielding , and most versatile c60 functionalization methods known . as shown in figure 1 ,", "the structurally derivatized c60 vectors reported herein were synthesized to yield either positively charged , negatively charged , or neutral chemical structures when solvated under physiological conditions .", "these vectors , which have been analyzed for their ability to promote dna transfection , offer the opportunity to elucidate the roles that hydrophobicity ( due to the fullerene core ) , hydrophilicity ( due to the water soluble substituents ) , and overall charge state displayed by the c60 derivatives have on dna transfection , gene expression , and cytotoxicity .", "depiction of the structures of the derivatized c60 vectors ; the positively charged amino - c60 compounds ( iiv ) , the neutral serinolamide - c60 compound ( v ) , and the negatively charged c3-c60 compound ( vi ) ( chemical structures depicted at neutral ph in aqueous solution ; counterions in solution not shown ) ."], ["materials for culturing cells , including dulbecco s modified eagle s medium ( dmem ) , trypsin , and phosphate buffer saline ( pbs ) , were obtained from gibco ( gibco life , grand island , ny ) .", "complete osteogenic medium containing minimum essential medium ( mem ) , 10 mm -glycerophosphate , 50 g / ml ascorbic acid , and 10 vol % fetal bovine serum was obtained from gemini bio - products ( calabasas , ca ) .", "a plasmid dna ( 7.2 kbp ) containing the gene for a red - shifted wavelength variant of the enhanced green fluorescent protein ( egfp ) under the transcriptional control of the cytomegalovirus ( cmv ) immediate - early gene promoter was used as the dna construct from which reporter gene expression was measured in the transfection assays .", "plasmid dna was prepared in bulk from transformed bacterial sources and purified to homogeneity using commercial kits ( qiagen ; germantown , md ) employing anion - exchange chromatography .", "the amino - c60 adducts ( iiv ) , the seri - c60 adduct ( v ) , and the c3-c60 adduct ( vi ) were synthesized and characterized according to previously published procedures .", "stock solutions of individual compounds ivi were prepared by weight and filtered for sterilization using a cellulose acetate membrane filter ( 0.2 m pore diameter ) prior to use in forming transfection mixtures .", "standard solutions were prepared at a concentration 10 g/l for each of these compounds in both tris edta ( te ) solution ( 10 mm tris , 1 mm edta , ph = 7.4 ) , and in serum - free dmem in separate reaction vials .", "nih 3t3 mouse fibroblast cells ( atcc # crl-1658 ) and hek 293 cells ( human embryonic kidney ; atcc # crl-1573 ) were cultured in dmem containing 10% fbs in a 5% co2 atmosphere at 37 c prior to collection and plating in individual assay wells used for transfection studies . for subculture of cells , routinely on reaching 80% confluence ,", "the cells were washed with pbs , detached using trypsin - edta , harvested by centrifugation , resuspended in culture medium and counted in a coulter counter prior to plating in multiwell dishes for subsequent transfection assays .", "marrow stromal cells ( mscs ) were harvested from wistar rats as previously described.(24 ) mscs were cultured for 6 days in the presence of 10 m dexamethasone in complete osteogenic media before use in transfection studies and cultured under identical environmental conditions to 3t3 and hek 293 cells .", "mixtures of individual derivatized c60 vector material and plasmid dna were allowed to form a complex in serum - free dmem , as described below , prior to adding to cells in the transfection assays .", "each c60-vector solution was added dropwise to a solution of plasmid dna to obtain a final volume of 200 l .", "each resulting solution was then vortexed briefly and allowed to stand for 30 min at room temperature to allow complete assembly of the vector / dna complex , which was then used immediately in the transfection experiment .", "the final dna mass of each solution remained constant at 10 g while the concentration of the c60 derivative was varied to obtain a range of c60-reagent - to - dna - base - pair ( bp ) ratios ( defined here as ", "the value of dna mass was fixed at 10 g per well since it showed the most optimum transfection among different dna masses used ( 112 g ) for transfection at r = 16.8 .", "transfections of nih 3t3 cells were carried out with c60/dna complexes in a range of r values ( 0.4242 ) .", "for hek 293 cells and mscs , transfections were performed only at r = 4.2 and r = 16.8 .", "cells were plated at 40,000 cells / cm in 96 well tissue culture plates and incubated overnight to permit cell attachment to the well surface .", "the culture medium was then replaced by the serum - free transfection mixture for various time periods ( 2 h , 8 h , 24 or 48 h ) ( transfection time ) . after exposing the cells for the respective periods of time to the serum - free transfection mixture", ", the cells were washed with pbs and the medium was replaced with complete medium ( containing fbs ) .", "the cells were then further incubated for 8 h , 24 or 48 h ( incubation time ) before gfp fluorescence was measured using a flow cytometer . for comparative purposes", ", control cell populations were also transfected with plasmid dna alone ( no c60 vector ) , or with plasmid dna complexed with an optimal level of one of two commercially available transfection reagents ; such that the dna was complexed with either 25 kda polyethylenimine ( pei ) , or with cytopure transfection reagent , which is reported by the manufacturer to exhibit very low levels of cytotoxicity .", "the dna / pei complexes were assembled using a well - established protocol(25 ) and the cytopure / dna complexes were assembled as per the manufacturer s instructions and optimized to obtain an optimal level of dna transfection / gfp expression within the nih 3t3 cell type .", "below is a brief description of the conditions which gave the optimal level of transfected nih 3t3 cells using 25 kda pei and cytopure . for pei ,", "dna / polymer complexes were prepared in serum - free dmem to achieve a ratio of polymer to dna of 4 .", "the complexes ( 100 l ) were then incubated at 25 c for 1015 min and then added to cell wells that contained 100 l of serum - free dmem .", "for cytopure , 1.1 l of cytopure stock was diluted to 50 l with serum - free dmem .", "the cytopure mixture was then added slowly to 1 g of dna diluted to 50 l with serum - free dmem .", "the transfection mixture was vortexed , left standing for 15 min at room temperature and added to cell wells that contained 100 l of serum - free dmem .", "after 24 h of transfection , the cells were washed and the medium was replaced with the serum - containing medium . for purposes of this study ,", "transfection  as described herein also relates to cell viability for sufficient time to ensure gfp gene expression , which was determined by cell fluorescence levels above a defined background threshold level ( determined using nontransfected cells to set lower detection limit parameter ) with flow cytometry .", "cells were prepared for flow cytometry by trypsinization after being washed with sterile pbs to remove cell debris and any residual gene - delivery agents .", "cells that were transfected successfully expressed gfp protein and were detected at 470/515 nm ( excitation / emission ) by the flow cytometer .", "transfection efficiency has been determined as the percent of cells that express gfp per study sample relative to the total number of cells passing through the flow cytometer per study sample ( 10,000 events counted for each transfection sample).(25 ) the c60/dna complex solutions were prepared as described above .", "c60 vector solutions without dna ( 010 g/l ) were prepared by diluting the c60 stock solution with serum - free dmem .", "the concentration of cultured cells was determined prior to cell plating using a coulter counter and the cells were plated on 96 well clear - bottom plates at a density of 40,000 cells / cm . after incubating overnight to allow cell attachment , the medium in each well was replaced either with 200 l of the c60 vector alone or with c60 vector / dna solutions prepared as described above .", "the cells were exposed to the solutions for either 2 , 8 or 24 h before the cytotoxicity was determined .", "control cell samples were plated and grown under identical conditions as those described for treated cell samples , with the exception that neither c60 compounds nor transfection mixtures were added to the control cells .", "the live / dead viability / cytotoxicity assay ( molecular probes , eugene , or ) was used to determine cytotoxicity levels in cells treated with either c60-vector or c60-vector / dna solutions .", "this assay makes use of 2 different fluorescent dyes , calcein am and ethidium homodimer ( ethd-1 ) , to measure the number of live and dead cells respectively . the lipid permeable dye , calcein am , when taken up by living cells with intact plasma membranes , emits green fluorescence ( excitation / emission : 495/515 nm ) upon being cleaved by esterase enzymes in the cells .", "conversely , the ethd-1 dye emits red fluorescence ( excitation / emission : 528/617 nm ) on binding to nucleic acids released from dead cells , and is excluded by the intact cell membranes of viable cells .", "this assay was preferred over the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay for evaluation of cell viability because c60-vectors compounds i and ii interacted and attached to the mtt - formazan making the formazan crystals insoluble for the next colorimetric assay .", "similar effects have been reported for single - walled carbon nanotubes.(26 ) the reagents were prepared and added to the wells according to the manufacturer s guidelines . at the time points indicated , the media from the wells was aspirated and replaced with the live / dead reagent , after washing the cells with pbs .", "the plates were then incubated in the dark at room temperature for additional 30 min before being measured for fluorescence .", "fluorescence of each sample well was measured by using a fluorescent microplate reader ( tecan safire ) .", "fluorescence of each sample well was normalized to percent of both live ( no c60 compounds or transfection mixtures treatment condition ) and dead ( 70% methanol treated ) control cell groups .", "these normalized fractions are defined as the fraction of live and dead cells , respectively , in each of the c60-treated samples .", "since gfp fluorescence has excitation / emission wavelengths of 470/515 nm , close to that of calcein am , there was a possibility that gfp fluorescence could interfere with the calcein am ( live ) measurement leading to potential errors for those wells containing c60 vector / dna mixtures .", "thus , a baseline fluorescence intensity of each sample well at 495/515 nm was obtained before the addition of the live cell / dead cell dyes .", "this intensity was then subtracted from the final calcein am detection intensity . for the dynamic light scattering ( dls ) experiments ,", "a standard solution with concentration 10 g/l of each of the c60 vectors was prepared in each of these solvents : hplc grade water , 10 mm tris / edta solution , and serum - free dmem .", "these solvents were filtered through a 0.22 m syringe filter ( millpore , millex- gp ) prior to use .", "the size of standard 100 nm polystyrene beads in tris / edta solution and in serum - free dmem was measured to confirm that no discrepancies were detected with the data acquisition system .", "dls measurements were conducted using a wyatt technologies dawn eos instrument equipped with a quasi - elastic light scattering interface module ( wyatt qels unit ) .", "the scattering angle used for detection was 90 at a constant temperature of 25 c .", "data analysis and size distribution computations were performed using the vendor supplied software ( astra v ) and a multi- digital recorder ( wyatt qels ) .", "statistical analysis was performed between groups for the live / dead assay and for flow cytometry studies .", "groups were analyzed by anova with significance defined by a p - value < 0.05 , and pairwise comparison was performed using the tukey test .", "the cytotoxicity studies had four samples per group , and the transfection studies had three samples per group ."], ["initial screening was performed on each of the six different derivatized c60 samples , compounds ivi ( figure 1 ) .", "the different c60-vector / dna complexes were assembled at various r values ( 0.4242 ) and each analyzed for its ability to affect transfection in nih 3t3 cells under serum - free conditions .", "serum - free conditions were used in these studies to ensure that derivatized nanomaterial / dna complexes were not compromised by serum lipids and/or proteins , and that the dna complexes were imported into the cells based on the individual physical properties of the complexes themselves , without the potential aid of serum proteins acting as carrier .", "the best transfection efficiencies were routinely observed after allowing transfection mixtures to remain on cells for 24 h ( transfection time ) , then removing the transfection mixtures and incubating cells for an additional 24 h ( incubation time ) in the presence of serum for maximal gene expression to be detected . unless otherwise noted , all transfection efficiency data presented below were for cells transfected for 24 h under serum - free conditions and incubated for an additional 24 h in the presence of serum .", "all c60-conjugated transfection mixtures showed the ability to transfect cells with exogenous dna with statistically significant differences relative to unconjugated dna alone ( naked dna ) ( table 1 ) .", " transfection efficiency  as reported in table 1 is defined as the ratio of gfp - positive cells as determined by fluorescence under flow cytometry per total number of cells counted in the flow cytometer .", "no statistical difference was observed between the transfection efficiencies of the different groups at r = 0.42 and r = 4.2 . for r values above 4.2 , compounds iii and iv exhibited a statistical difference in transfection efficiencies compared to the other compounds . both of these compounds exhibited an increased transfection efficiency relative to the other c60 compounds studied , with a maximum transfection efficiency of 31% at r = 16.8 . at larger values of r", "the efficiency dropped off . at r = 16.8 , their transfection efficiencies were comparable to that of 25 kda pei containing and cytopure transfection reagent containing complexes .", "25 kda pei is a widely used and well - studied nonviral vector , while cytopure is a biodegradable polymeric transfection reagent that exhibits very low cytotoxicity and is reported to work on a broad spectrum of cell types , including hard - to - transfect primary cell isolates .", "c60 vectors are listed in order of their measured efficiency at promoting transfection in nih 3t3 cells under serum - free conditions . to assess the transfection ability of c60 vectors in different cell types ,", "studies were also performed on two other cell types : hek 293 cells and mscs .", "hek 293 cells were chosen since they are another standard cell line used routinely for transfection assays , while mscs were chosen due to their eminence as primary cells for tissue engineering .", "based on the initial screening results of each of the various derivatized c60 compounds for transfection - promoting ability in nih 3t3 cells as reported in tables 1 and 2 , transfection studies were also performed on hek 293 cells and mscs using compounds iii and iv at ratios of r = 4.2 and r = 16.8 .", "however , hek 293 cells ' transfection efficiencies were lower than those observed for nih 3t3 cell at similar r values .", "mscs exhibited low transfection efficiencies overall , and no increase in transfection efficiency was observed with increase in r values .", "transfected cells were visualized by fluorescence microscopy for morphological analysis after transfection ( micrographs not shown ) .", "positive transfection of cells with the plasmid coding for green fluorescent protein could be easily discerned by this technique .", "however , variations in transfection efficiency among the different cell types tested here is not surprising , as cell type is a major factor in determining the level of transfection and gene expression from nonviral vectors as noted previously.(27 ) differences in cell characteristics , such as cell mitosis rates , can play an important role in transgene expression , and may explain the differences in the level of gene expression among the various cell lines studied here .", "transfection efficiency values are presented as a quotient of gfp ( + ) cells relative to the total viable cells present in the transfected sample well as measured by flow cytometry .", "figures 2 a , b and c display the fraction of viable nih 3t3 cells as a function of the c60 vector ( no dna present ) concentration after 2 , 8 , or 24 h post - treatment incubation times , respectively .", "incubation with c60 compounds for 2 h resulted in a viable cell fraction greater than 50% for all c60 vector solutions .", "lowest overall cell viability was observed with compound i ( 0.55  0.08 ) at 10 g/l , and no statistically significant increase in cell viability was observed with decreasing compound i concentration . for all other c60 compounds tested", ", there was a general trend of increased cell viability with decreased c60 compound concentration .", "fraction of live nih 3t3 cells after incubation with the different c60 vectors as a function of derivatized fullerene concentration after ( a ) 2 h , ( b ) 8 h , or ( c ) 24 h c60-incubation times respectively , under serum - free conditions .", "error bars represent mean  standard deviation for n = 4 . at the 8 h c60 incubation time , the fraction of live cells was higher than 50% for all c60 solutions tested except those exposed to compound i. for compound i , the cell viability was the lowest ( 0.51  0.04 ) at 10 g/l , but no statistically significant increase in cell viability was observed with decreasing compound i concentration . for all other c60 vectors ,", "there was a general trend of increased cell viability with decreased compound concentration , but at each compound concentration tested , the cell viability was lower in the 8 h time point than in the corresponding sample concentration at the 2 h time point .", "at the 24 h c60 incubation time point , only cells treated with compounds iii and iv exhibited greater than 50% cell viability at all concentrations tested , with an increase in cell viability with decreased c60 compound ( iii and iv ) concentration .", "all other compounds showed a general trend of increased viability with a decrease in compound concentration , with cell viability values lower than 50% at 10 g/l .", "figures 3 a , b and c display the cell viability of nih 3t3 cells cultured in the presence of the various c60-vector / dna mixtures as a function of both c60 compound concentration and r values after 2 , 8 or 24 h post - transfection time , respectively .", "cell viability at the two hour post - transfection time point for compound i / dna and compound ii / dna mixtures increased from 0.19  0.0 at 10 g/l ( r = 42 ) to 0.70  0.15 at 0.1 g/l ( r = 0.42 ) .", "the fraction of live cells was greater than 50% for all other c60 derivative / dna solutions at the 2 h post - transfection time point .", "fraction of live nih 3t3 cells after incubation with the different c60/dna transfection mixtures as a function of c60 compound concentration and varying r value after ( a ) 2 h , ( b ) 8 h , or ( c ) 24 h post - transfection incubation time , under serum - free conditions respectively .", "error bars represent the mean  standard deviation for n = 4 . at the 8 h post - transfection time point ,", "the fraction of live cells for compound i / dna and compound ii / dna was less than 50% at all concentrations and r values tested . for all other samples ,", "the trend was similar to that of the 2 h post - transfection time point , with cell viability greater than 50% and an increase in viability observed with a decrease in transfection mixture concentration .", "at the 24 h time point , cells treated with the compound v / dna mixture exhibited greater than 50% cell viability at all concentrations tested , while compound i / dna , compound ii / dna exhibited less than 50% cell viability .", "cells treated with compounds iii / dna , iv / dna and vi / dna mixtures resulted in cell viabilities less than 50% at 4 g/l ( r = 16.8 ) and 10 g/l ( r = 42 ) , but greater than 50% viability when added at lower concentrations .", "dls and optical microscopy studies were performed to help elucidate the biophysical structure / activity relationships that might exist between the various c60 complexes .", "these studies were carried out to help generate correlations between the different c60 vectors , and the various c60 vector / dna structural attributes resulting in the different transfection efficiencies and cytotoxicity results observed under serum - free conditions .", "table 3 presents the dls measurements on the various c60 vector compounds ivi when dissolved alone in either water ( 4 g/l ) , in a 10 mm tris / edta solution ( 4 g/l ) , in serum - free dmem ( 4 g/l ) , or when dissolved in complex with dna in 10 mm tris / edta ( 4 g/l , r = 16.8 ) or as a complex with dna in serum - free dmem ( 4 g/l , r = 16.8 ) .", "the concentration of 4 g/l was chosen for dls experiments since compounds iii and iv exhibited maximum transfection efficiency at this concentration of c60 .", "the rh values in table 3 provide qualitative information about the transfection mixture aggregate behavior which can readily be seen in figures 4 and 5 .", "the rh parameter obtained from the dls measurements , and shown in table 3 , corresponds to the radius of a monodisperse hypothetical sphere having the same diffusion constant as that of the c60 compound aggregates or the c60/dna mixture aggregates under study .", "pdi values between 0 and 0.05 indicate more monodisperse particles , and values above 0.05 indicate broadened size distributions.(30 ) clearly , the rh values shown in table 3 for the various c60 compounds and c60/dna complexes are average values for quite polydisperse populations of aggregates .", "previous work by our group and others using atomic force microscopy ( afm ) and cryo transmission electron microscopy ( cryo - tem ) have shown that derivatized c60 compounds form aggregates that are irregularly shaped in solution .", "the rh values observed for these c60 compounds and the c60/dna mixtures suggests that these reagents also form such irregularly shaped aggregates when in aqueous solutions , including when dissolved in solutions of physiological ionic strength such as dmem .", "such qualitative information about the aggregate behavior provides valuable information for understanding the transfection and toxicity results , as discussed below .", "representative morphologies of nih 3t3 cells and the c60/dna complexes at r = 0.42 ten minutes after transfection .", "the optical images represent complexes formed with compound ( a ) i , ( b ) ii , ( c ) iii , ( d ) iv , ( e ) v , and ( f ) vi . all images are shown at the same magnification .", "representative optical microscopy images of nih 3t3 cells incubated with c60/dna complexes formed using compounds ( a ) iii and ( b ) vi at r = 0.42 twenty four hours post - transfection .", "all c60 compounds display a broadly distributed aggregate size in each solvent tested ( water , 10 mm tris / edta solution , and serum - free dmem ) .", "the compounds had aggregate sizes between 47 and 650 nm ( pdi = 0.330.51 ) and between 33 and 446 nm ( pdi = 0.290.46 ) in plain water and 10 mm tris / edta solution , respectively .", "in the presence of dna , there is an increase in rh for all formulations . in general", ", the rh values decrease in the order v , vi > i > ii > iii , iv in the presence of dna . presumably , this trend is the result of the influence of the various structures of the c60 derivatives on the condensation behavior of the dna in the transfection mixture . in the case of cationic polymers ,", "the condensation process with dna has been thought to rely predominantly on electrostatic interactions with minor contributions from other interactions such as hydrophobic interactions , hydrogen bonding , and van der waals forces.(5 ) in case of the positively charged c60 derivatives , the hydrophobicity of the c60 core , and electrostatic interactions between the water - soluble functional groups and the dna are the main contributors to the condensation process .", "compound i , with two adducts on the fullerene core , has less positive charge than the hexa - amino c60 adduct of compound ii and the octa - amino adduct of compound iii .", "fewer electrostatic interactions between functionalized c60 vectors and the dna sugarphosphate backbone can lead to a decrease in condensation capacity , resulting in larger c60-vector / dna complexes .", "compound v , although polar , has no net charge at neutral ph ; while compound vi will possess a negative charge , the same as that of dna .", "thus , for compounds v and vi , hydrophobicity is presumably the only interaction contributing to the formation of the dna condensates , and this interaction appears insufficient to fully condense the dna , leading to large c60 vector / dna complex aggregates .", "the relatively large sizes for the c60 vector / dna complexes , and in particularly for those formed from compounds i , ii , v and vi , may be a reason for the poor transfection efficiencies observed with these compounds .", "this may also be a reason for the low transfection efficiency reported for a similar positively charged amino - c60 compound with a monoadduct on the fullerene core.(17 ) however , increased positive charge - state alone is insufficient to solely predict increased transfection capability of amino - derivatized fullerenes .", "variation in transfection protocol and/or cell types can also dramatically affect transfection efficiencies , as positively charged amino - derivatized c60 similar to compound iv have previously been shown to be inefficient at effecting transfection.(17 ) in general , the condensation behavior of nonviral vector / dna complexes is governed by several factors including the molecular weight of the dna condensing agent , the density of the charges on the condensing agent , and the ratio of composition ( r values ) between the condensing vector and the dna.(5 ) in addition , it is now well - documented that the size of the transfection vector / dna complex is one of the most important parameters for efficient dna delivery into cells , with the most efficient uptake observed for complex with sizes < 150200 nm in diameter.(5 ) the rh values shown in table 3 suggest that only dna complexes formed with compounds iii and iv satisfy these criteria .", "on addition of the c60-vector / dna complexes to cells , the complexes formed with compounds i and ii immediately aggregated further to form large clusters , even at the lowest r value tested of 0.42 .", "figure 4af shows the optical microscopy images of all c60-vector / dna complexes at r = 0.42 ten min after their addition to nih 3t3 cells .", "large , irregularly shaped aggregates ( shown as brown ) can clearly be seen for complexes formed with compound i ( figure 4a ) and for complexes formed with compound ii ( figure 4b ) .", "the aggregate concentration is greater and more densely distributed for compound i than for compound ii .", "no such increased aggregation propensity was seen for the other c60 vector / dna complexes ( figure 4cf ) .", "however , time - dependence of aggregation could be observed for compound iii and compound vi .", "figure 5a , b shows the optical microscopy images of compound iii / dna , and compound vi / dna complexes at r = 0.42 at 24 h post - transfection time .", "the images show the presence of large sized aggregates , more densly distributed for compound vi than for compound iii .", "interestingly , addition of compound iii / dna mixtures caused cell morphological changes when added to cultured cells , causing the attached cells to round up from the substratum surface in contrast to the usual planar , extended / elongated shapes the cells assumed in the normal growth state .", "this shape change may be attributed to the depolymerization / disruption of either the actin or tubulin networks of the cytoskeleton by the complexes , and/or to the disruption / inhibition of focal adhesion plaques formed on the cell surface . however , this observation at r = 0.42 does not have any noticeable effects on cell proliferation , as flow cytometry measurements indicate no statistically significant difference in the number of cells for those treated with compound iii / dna complexes relative to the controls cells ( no c60/dna complexes added ) .", "our results indicate that compounds iii and iv , when complexed with dna , bring about the highest levels of transfection when compared to the other derivatized c60 compounds under evaluation in this study . at the optimal ratio of compound iii or iv to dna ( r = 16.8 ) ,", "however , at 24 h post - transfection , there is only approximately 30% viability in cells treated with these two tranfection mixtures .", "this result is in contrast to cell viability of greater than 50% in cells treated with the compounds iii or iv alone at equivalent c60 concentrations ( 4g/l ) as that when in complex with dna at r = 16.8 .", "the temporal increase in aggregate sizes for these c60/dna complexes ( not seen in the free c60 compounds alone ) leads to precipitation onto the cell surface when these mixtures are left in the culture medium for 24 h ( e.g. , see figure 5 ) .", "this effect may explain the higher levels of cell death in the transfection mixture - treated cells .", "the overall trend observed in these studies was that increased cell death was observed with an increase in either c60 concentration alone or for increased r values in c60/dna mixtures , irrespective of the chemical nature of the c60 compound , with compounds iii and iv exhibiting the least toxicity when free in solution , but not necessarily when complexed with dna ( under these conditions , compound v seems to exhibit the least cell toxicity ) .", "also , higher levels of cytotoxicity seem to correlate more with complex aggregate formation induced by the presence of dna in the mixtures , leading to the formation of visible precipitates . such deposition of aggregates onto the cell surface will impair normal functioning of the plasma membrane and thereby contribute to cell death.(31 ) there may also be other slow processes contributing to cell death for the positively charged c60 vectors in the c60/dna solutions at the longer ( 8 and 24 h ) transfection times .", "following cellular entry , the presence of positively charged amino - c60 compounds ( iiv ) ( individuals or as aggregates ) inside the cell or nucleus may lead to disruptions to other cellular functions , such as interfering with histonedna interactions and/or other cellular processes.(7 ) the favorable transfection efficiencies of compounds iii and iv suggest that these compounds have some characteristics in common with other positively charged amino - c60 derivatives previously cited in the literature which are reported to effect transfection in cells .", "these similarities include having a flexible linker situated between the fullerene core and the positively charged amino groups .", "although the results presented here suggest that the structures of compounds iii and iv will result in higher levels of transfection than those previously reported by others , direct comparison of our results with the results from others using different structurally derivatized positively charged amino c60 vectors is not possible , since the structure of the c60 vectors , the cell lines tested , and the methodologies used are not consistent .", "another distinct feature of all the c60 vectors reported here is their water solubility which obviates the need to use organic solvents such as dimethylformamide ( dmf ) in the preparation of the transfection mixture ; the high cytotoxicity observed for some other c60-based transfection agents has been attributed to the toxicity of dmf .", "the comparable transfection efficiencies of compounds iii and iv on nih 3t3 cells to those of commercially available nonviral vectors 25 kda pei and cytopure is encouraging .", "25 kda pei is a widely used nonviral vector and is quite often used as a standard for comparing new nonviral vectors .", "this high cytotoxicity has been attributed more to free pei than to the pei / dna complex . however , in case of compound iii or iv , at the optimal r = 16.8 , their complexes with dna show higher toxicity ( 30% viability ) compared to the free compounds iii or iv alone ( greater than 50% cell viability ) .", "since the cytotoxicity seems to correlate more with complex aggregate formation induced by the presence of dna in the mixtures , future refinement of the c60/dna complex preparation protocol , such as disaggregating these complexes with phosphate buffer and/or nacl , should potentially lead to lower cytotoxicity while maintaining the same level of transfection efficiency , or potentially even increase these transfection efficiency levels .", "furthermore , although not addressed in this study ( for reasons of maintaining uniformity in c60/dna complex formation and cellular entry ) , the presence of serum proteins on enhanced c60/dna complex uptake , and on subsequent cell viability during extended transfection times , would also be worth examining , and could be expected to lower the observed cellular cytotoxicity levels seen in this study .", "the small , but statistically significant , expression of the reporter protein from c60/dna conjugates formed from compounds v and vi was surprising and interesting since these samples do not possess a net positive charge under the physiological conditions used to form the complexes , although there have been limited reports of other nonpositive charged nanoparticles translocating into cells(35 ) and , indeed , also demonstrating gene transfection.(36 ) thus , at least for some derivatized c60 molecules , the hydrophobic nature of the c60 carbon nanostructures may be the common important property in their binding to dna and their subsequent translocation into the interior of the cell .", "there is now a growing body of work that suggests that carbon nanomaterials such as fullerenes , metallofullerenes and carbon nanotubes , due the hydrophobicity of their carbon sheaths , have the ability to efficiently translocate into the interior of cells irrespective of the charge of their water - solubilizing moieties .", "in addition , carbon nanomaterials such as gd@c60 , gd@c82 , gd@c80 and gd@ultrashort single - walled carbon nanotubes ( gd@us - tubes ) have shown potential as high - performance magnetic resonance imaging ( mri ) contrast agents suitable for advanced applications such as tracking and/or delivery of magnetically labeled cells by mri in vivo .", "these reports , along with the favorable c60-based gene transfection results reported here and by others , offer opportunities for development of analogous gd@c60-based vectors to monitor noninvasively the biodistribution and pharmacokinetics of gene therapy vectors in vivo.(49 )"], ["a new class of water - soluble c60 transfecting agents with positively charged , negatively charged , or neutral chemical functionalities under physiological conditions was prepared using hirschbingel chemistry .", "transfection , cytotoxicity and biophysical structure / activity studies were performed in an effort to elucidate the relationship between the hydrophobicity of the fullerene core , the hydrophilicity of the water - solubilizing groups , and the overall charge state of the c60 vectors in gene delivery / expression .", "however , only two positively charged c60 derivatives , namely , an octa - amino derivatized c60 and a dodeca - amino derivatized c60 vector , showed efficient in vitro transfection .", "increased levels of cellular toxicity were observed for positively charged c60 vectors relative to the negatively charged and neutral vectors .", "structural analyses using dynamic light scattering and optical microscopy confirmed that higher positive charge on c60 compounds is a dominant physical attribute necessary for optimal c60/dna structural features leading to increased transfection efficiency , and that aggregation is the major factor that negatively affected the cytotoxicity profiles of the c60 vector / dna complexes .", "aggregation is presumably also the dominant reason for increased cytotoxicity of certain specific derivatized c60 compounds ( most notable for compound i ) even in the absence of dna , at least when analyzed under the serum - free conditions utilized in this study .", "future studies should be able to capitalize on this information and enable the design of additional c60 derivatives that address these issues specifically and help lower the propensity of these reagents to aggregate in the presence of dna .", "such reagents would be expected to enhance transfection of cells and tissues while simultaneously lowering toxicity levels .", "future studies using these reagents will also more precisely indicate the cellular uptake and endosomal release mechanisms that these compounds inherently possess , thereby lending further insight into potential enhanced chemical design of future generations of these carbon - based nanomaterials specifically for dna delivery into the cell nucleus .", "the successful demonstration of intracellular dna uptake , intracellular transport , and gene expression from dna using c60 vectors suggests the possibility of developing analogous gd@c60-based vectors to serve simultaneously as both therapeutic and diagnostic agents ."]]}
{"article_id": "PMC3465928", "article_text": ["previously we reported that deletion of neutral endopeptidase ( nep ) provides protection from obesity- and diabetes - induced neural complications .", "we have also shown that treating obese and streptozotocin - diabetic mice with the vasopeptidase inhibitor ilepatril prevented neural complications including slowing of nerve conduction velocity , thermal hypoalgesia , and decreased intraepidermal nerve fiber density .", "vasopeptidase inhibitors are drugs that simultaneously inhibit nep and angiotensin - converting enzyme ( ace ) activity .", "recent studies have shown increased expression of angiotensin - ii - forming enzymes in adipose tissue and increased activity of the renin - angiotensin system being implicated in the development of insulin resistance and type 2 diabetes .", "nep is found in many tissues including vascular and renal tissue and its activity is increased by fatty acids and glucose in human microvascular cells [ 59 ] . in the peripheral nervous system nep", "is located in schwann cell membranes surrounding dorsal root ganglion cells and nerve fibers [ 10 , 11 ] .", "nep degrades many vaso- and neuroactive peptides including natriuretic peptides , adrenomedullin , bradykinin , and calcitonin - gene - related peptide [ 12 , 13 ] .", "therefore , inhibition of ace and nep activity would be expected to improve vascular and neural function . in this", "regard we have demonstrated that treating type 1 and type 2 diabetic rats as well as a genetic rat model of obesity with ilepatril improves vascular and neural dysfunction [ 1416 ] .", "however , little information is available about the effect of vasopeptidase inhibitors in animal models of diet - induced obesity . in order to further elucidate the effects of vasopeptidase inhibitors in peripheral nerve dysfunction associated with obesity we examined the effect of diet - induced obesity on nerve conduction velocity and thermal response latency in the hindpaw of c57bl/6j mice and mice deficient in nep treated with ilepatril , enalapril , ace inhibitor , or candoxatril , nep inhibitor [ 1 , 2 ] .", "unless stated otherwise all chemicals used in these studies were obtained from sigma chemical co. ( st . louis , mo ) .   c57bl/6jj", "breeding pairs of neutral endopeptidase - deficient ( nep ) mice were provided by drs .", "these mice have been bred and a colony created at the veterans affairs medical center , iowa city , ia .", "deficiency of nep activity was confirmed in nep mice by measuring the specific activity of nep in kidney homogenates using the method described by ayoub and melzig   with modification .", "activity of nep in kidney from c57bl/6j and nep mice was 0.35  0.02 and 0.02  0.02  mm 7-amido-3-methylcoumarin ( amc)/min / mg protein , respectively ( p < 0.001 versus c57bl/6j by unpaired t - test ) .", "this test was performed on all mice used in these studies in order to confirm that nep was functionally  knocked out  in the nep mice .", "mice were housed in a certified animal care facility and food ( harlan teklad , no .", "adequate measures were taken to minimize pain or discomfort and all of the experiments were conducted in accordance with international standards on animal welfare and were compliant with all institutional and national institutes of health guidelines for use of animals ( acurf protocol 1101009 ) .  ", "for the prevention protocol c57bl/6j and nep mice at 12 weeks of age were divided into five groups .", "a second group was fed a high - fat diet containing 24  gm% fat , 24  gm% protein , and 41  gm% carbohydrate ( d12451 ; research diets , new brunswick , nj ) .", "the primary source of the increased fat content in the diet was soybean oil and lard .", "the control and high - fat diet contained 0.4 and 0.3% sodium chloride , respectively .", "the average fat content of the control diet was 4.25  gm% ( harlan teklad , no .", "the third through fifth groups were fed the high - fat diet containing ilepatril ( 500  mg / kg in the diet ) , candoxatril ( 30  mg / kg in the diet ) , or enalapril ( 500  mg / kg in the diet ) .", "we have found that these doses provide maximal inhibition of nep and/or ace activity in vivo [ 2 , 19 ] .", "for the intervention study the same five groups of c57bl/6j and nep mice at 12 weeks of age were fed the control diet ( group 1 ) or high - fat diet ( groups 25 ) for 12 weeks .", "afterwards , the four groups of high - fat - fed mice were fed a high - fat diet with no additions ( group 2 ) or high - fat diet containing ilepatril , candoxatril , or enalapril ( groups 35 ) for 12 weeks .", "after an overnight fast mice were injected with a saline solution containing 2  g / kg glucose , i.p . immediately prior to the glucose injection and for 120 minutes", "afterwards blood samples were taken to measure circulating glucose levels with the use of glucose - dehydrogenase - based reagent strips ( aviva accu - chek , roche , mannheim , germany ) .", "blood samples ( 0.6  l ) were taken from a tail vein that was lanced once .", "thermal nociceptive response in the hindpaw was measured using the hargreaves method with instrumentation provided by iitc life science , woodland hills , ca ( model 390 g ) as previously described . the test was performed in a blind manner .", "thermal nociceptive responses were measured by placing the mouse in the observation chamber on top of the thermal testing apparatus and allowing it to acclimate to the warmed glass surface ( 33c ) and surroundings for a period of 15  min .", "the mobile heat source was maneuvered so that it was under the heal of the hindpaw and then activated , a process that starts a timer and locally warms the glass surface , when the mouse withdrew its paw , the timer , and the heat source was turned off .", "following an initial recording , which was discarded , two measurements were made for each hindpaw , with a rest period of 5  min between each set of measurements .", "the mean of the measurements , reported in seconds , was used as a measure of the thermal nociceptive response latency .", ", abbott laboratories , north chicago , il ) and sensory nerve conduction velocities were determined as previously described [ 1 , 2 ] .", "briefly , sensory nerve conduction velocity was recorded in the digital nerve to the second toe by stimulating with a square - wave pulse of 0.05  ms duration using the smallest intensity current that resulted in a maximal amplitude response ( grass s44 stimulator ; grass medical instruments , quincy , ma ) .", "the maximal sensory nerve conduction velocity was calculated by measuring the latency to the onset / peak of the initial negative deflection and the distance between stimulating and recording electrodes ( measured in millimeters using a vernier caliper ) .", "biopsies of skin of the right hindpaw were fixed , dehydrated , and embedded in paraffin .", "sections ( 7  m ) were collected and immunostained with anti - pgp9.5 antibody ( rabbit anti - human no .", "7863 - 0504 ( this antibody cross - reacts with rat , mouse guinea pig , and other species ) , abd serotic , morpho sys us inc . ,", "raleigh , nc ) overnight followed by treatment with secondary antibody alexa fluor 546 goat anti - rabbit ( invitrogen , eugene , or ) .", "profiles were imaged using a zeiss 710 confocal microscope with a 40x objective and were counted by two individual investigators that were blinded to the sample identity .", "length of the epidermis was determined by drawing a polyline along the contour of the epidermis and recording its length in mm .", "comparisons between the groups for body weight , blood glucose , sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber profiles were conducted using a one - way anova and bonferroni 's test for multiple comparisons ( prism software ; graphpad , san diego , ca ) .", "presented in table 1 are weight and blood glucose changes for c57/bl6j and nep mice used in the prevention study . at 12 weeks of age", "when fed a high - fat diet c57bl/6j and nep mice both gained a similar amount of weight . treating the high - fat diet with ilepatril or enalapril but not candoxatril completely prevented the gain in weight .", "the mass of the epididymal fat pad was significantly increased in high - fat - fed mice and mice fed the high - fat diet treated with candoxatril compared to control mice or mice fed the high - fat diet containing ilepatril or enalapril . when calculating the epididymal fat pad mass as a percent of total body weight epididymal fat pad mass was significantly increased in high - fat - fed c57bl/6j mice and mice fed a high - fat diet containing candoxatril compared to control . treating the high - fat diet with ilepatril and to", "a greater extent enalapril prevented the increase in epididymal fat pad mass when presented as percent of final body weight .", "nonfasting blood glucose levels were not significantly different between c57bl/6j and nep mice fed the control or high - fat diets and not affected by ilepatril , candoxatril , or enalapril treatment . in the intervention study all mice fed the high - fat diet for the initial 12 weeks of", "the experimental design gained a significant amount of weight ( table 2 ) . when transferred to a high - fat diet containing ilepatril or enalapril for an additional 12 weeks c57bl/6j and nep mice lost weight .", "mice remaining on the high diet or high - fat diet containing candoxatril continued to gain weight .", "the epididymal fat mass decreased after c57bl/6j or nep mice , fed a diet containing high fat for 12 weeks , were given a high - fat diet containing enalapril .", "the epididymal fat mass also was decreased after nep mice but not c57bl/6j mice were fed a high - fat diet treated with ilepatril .", "treating high - fat - fed c57bl/6j or nep mice with candoxatril did not reduce epididymal fat mass .", "a similar trend was observed when the epididymal fat mass data was presented as percentage of final body weight .", "nonfasting blood glucose levels were not changed in c57bl/6j or nep mice fed control or high - fat diets with or without ilepatril , candoxatril , or enalapril .   in the prevention study c57bl/6j ( figure 1 ) and nep ( figure 2 ) mice fed the high - fat diet or high - fat diet containing candoxatril had an impaired glucose clearance curve compared to control mice .", "in contrast , the glucose clearance curve was near normal in c57bl/6j and nep mice fed a high - fat diet containing ilepatril or enalapril .", "in the intervention study feeding c57bl/6j ( figure 3 ) or nep ( figure 4 ) mice for 24 weeks a high - fat diet caused an impaired glucose clearance curve . treating high - fat - fed c57bl/6j or nep mice with enalapril for the last 12 weeks of the 24-week period partially corrected glucose utilization . treating nep mice with ilepatril", "treating c57bl/6j mice with ilepatril did not improve glucose utilization nor did treating c57bl/6j or nep with candoxatril .", "data in figure 5 ( left ) demonstrate that sensory nerve conduction velocity was significantly decreased in high - fat - fed c57bl/6j mice compared to control mice and that this was prevented by treating high - fat - fed mice with ilepatril or candoxatril using a prevention protocol .", "data in figure 5 ( center ) demonstrate that thermal nociception was significantly decreased in c57bl/6j mice after 12 weeks of a high - fat diet compared to control mice as indicated by an increase in paw withdrawal latency .", "treatment with ilepatril or candoxatril and to a lesser extent enalapril for the 12-week period prevented thermal hypoalgesia in high - fat - fed c57bl/6j mice .", "data in figure 5 ( right ) demonstrate that the number of intraepidermal nerve fiber profiles was significantly decreased in high - fat - fed c57bl/6j mice and that treatment of these mice with ilepatril or candoxatril but not enalapril for 12 weeks prevented the significant decrease .", "sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber density were not impaired in nep mice fed a high - fat diet ( figure 6 , left , center , and right , resp . ) .  ", "we also determined the effect of treating high - fat - fed c57bl/6j and nep mice using an intervention protocol ( figures 7 and 8 , resp . ) . in this study design", "high - fat - fed mice were untreated for 12 weeks followed by 12 weeks of a high - fat diet with or without treatment .", "feeding c57bl/6j mice a high - fat diet for 24 weeks caused a significant decrease in sensory nerve conduction velocity ( figure 7 , left ) . treating these mice for the last 12 weeks of the 24-week period with ilepatril , candoxatril , or enalapril", "the decrease in thermal nociception in high - fat - fed c57bl/6j mice was reversed by treating mice with ilepatril or candoxatril but not enalapril ( figure 7 , center ) .", "treating high - fat - fed c57bl/6j mice with ilepatril , candoxatril , or enalapril reversed the decrease in intraepidermal nerve fiber profiles ( figure 7 , right ) .", "feeding nep mice a high - fat diet for 24 weeks with or without treatment had no effect on sensory nerve conduction velocity , thermal nociception , or intraepidermal nerve fiber density ( figure 8 , left , center , and right , resp . ) .", "the main findings from these studies were that diet - induced obesity caused significant weight gain and impaired glucose utilization in c57bl/6j and nep mice that was improved when the mice were treated with ilepatril or enalapril using a prevention protocol . using the intervention protocol enalapril but not ilepatril treatment was effective improving glucose utilization by c57bl/6j mice .", "treating c57bl/6j or nep mice with candoxatril did not improve weight gain or glucose utilization .", "these data suggest that inhibition of ace and not nep activity was responsible for improving weight gain and glucose utilization in high - fat - fed mice .", "diet - induced obesity also caused slowing of sensory nerve conduction velocity , thermal hypoalgesia , and decrease in epidermal nerve fiber density in the paw of the hindlimb in c57bl/6j mice but not nep mice .", "the impairment in sensory nerve function endpoints in c57bl/6j mice fed a high - fat diet was prevented / improved when the mice were treated with ilepatril or candoxatril and to a much lesser extent enalapril using either the prevention or intervention protocol .", "these data suggest that increased nep activity / expression but not ace activity contributes to diet - induced obesity - related deficits in sensory nerve function .  ", "previous studies have shown that diet - induced obesity in rodent models can be prevented by ace inhibitors and angiotensin ii receptor blockers [ 2124 ] .", "in addition , diet - induced weight gain and fat mass are reduced , energy expenditure increased , and glucose tolerance improved in mice lacking ace or the angiotensin ii type 1a receptor [ 25 , 26 ] .", "the mechanisms proposed for the improvement in obesity and glucose tolerance with treatment of rodent models with ace inhibitors are increased energy expenditure , liver and adipose tissue metabolic modulation , lower concentration of leptin , improved insulin signaling , and increased glucose and fatty acid utilization by muscle [ 2130 ] . in a study comparing the effects of ramipril , an ace inhibitor , to ilepatril in jcr : la - cp rats , an obese , insulin - resistant , hyperinsulinemic , normoglycemic model , it was found that both compounds reduced the surge of plasma insulin in a meal tolerance test by about 50% but ilepatril was more beneficial in improving vascular reactivity . in our study", "we found that enalapril trended to be more effective than ilepatril preventing / reducing fat pad mass .", "this could be because enalapril at the dosage used was a more effective ace inhibitor than ilepatril in target tissues . in another study using obese zucker rats", "it was found that dual inhibition of ace and nep improved insulin - mediated glucose disposal more effectively than monotherapy and this effect was linked to increased activation of the kinin - nitric oxide pathway . in a similar independent study it was found that omapatrilat , a vasopeptidase inhibitor , induced insulin sensitization and increased myocardial glucose uptake in obese zucker rats and that the effect of omapatrilat was greater than ramipril in part due to stimulation of the b2 receptor .", "later this group reported that treatment of obese zucker rats with a vasopeptidase inhibitor increased muscle glucose uptake independent of insulin signaling . in two of these studies protection of bradykinin from degradation by nep", "interestingly , it has been shown that natriuretic peptides promote muscle mitochondrial biogenesis and fat oxidation as to prevent obesity and glucose intolerance .", "the natriuretic peptides are also degraded by nep . because nep is expressed in skeletal muscle in relatively large amounts and being located on the cell surface , nep is able to hydrolyze peptides in the vicinity of their receptors thereby neutralizing their bioactivity [ 34 , 36 ] .", "however , inhibition of ace may also lead to protection of bradykinin levels by inhibiting kininase - ii - mediated degradation of this nonapeptide .", "reported that ace inhibition by captopril improved glucose transport in insulin - resistant muscle of the obese zucker rat .", "they attributed the improvement to modulation of insulin action by bradykinin mediated through b2 receptors and by an increase in nitric oxide production . since bradykinin and natriuretic peptides may have a role in enhancing insulin action and regulating glucose and fatty acid metabolism by muscle protecting their bioactive function by preventing degradation through inhibition of ace and/or nep may be a therapeutic approach for treatment of obesity and insulin resistance [ 34 , 36 , 38 ] .  ", "we have previously shown that treating diabetic rats with enalapril improved vascular and nerve endpoints but enalapril was less effective in rats fed a high - fat diet [ 19 , 36 , 39 , 40 ] .", "in contrast , we found that treating obese or diabetic rats with ilepatril was more effective than enalapril in improving peripheral nerve dysfunction [ 19 , 36 , 40 ] .", "we have shown that treating diabetic or diet - induced obese rodents with ilepatril improves vasodilation of blood vessels that provide circulation to the sciatic nerve by reducing oxidative stress and preventing degradation of vasoactive peptides by nep including calcitonin gene - related peptide and c - type natriuretic peptide thereby maintaining endoneurial blood flow and preventing ischemia .", "since nep degrades both calcitonin gene - related peptide and substance p , peptides important in pain signaling pathways , it is likely that blocking nep activity or mice deficient in nep would maintain higher levels of both of these neuroactive peptides and perhaps be more sensitive to painful stimuli .", "data from clinical trials clearly suggest that activation of the renin - angiotensin - aldosterone system plays an important role in the pathophysiology of the metabolic syndrome through interaction of a wide range of physiological and molecular mechanisms . more recently in humans activity of nep", "has been shown to correlate with body mass index and measures of insulin resistance with increasing activity found in subjects with multiple cardiovascular risk factors . in these studies we found that feeding mice a high - fat diet causes weight gain , impaired glucose tolerance , and sensory nerve dysfunction . inhibition of ace was effective in reducing weight gain and improving glucose utilization but generally noneffective in improving sensory nerve dysfunction .", "in contrast , inhibition of nep improved sensory nerve dysfunction and had no effect in improving weight gain or glucose utilization . treating high - fat - fed mice with a vasopeptidase inhibitor improved weight gain , glucose utilization , and sensory nerve function .", "thus , we conclude that increased activity of nep is associated with sensory nerve dysfunction in an animal model of diet - induced obesity and that dual inhibition of ace and nep may be more effective than monotherapy in reducing insulin resistance and the sensory nerve complications associated with metabolic syndrome ."], "abstract_text": ["<S> we have demonstrated that treating diet - induced obese ( dio ) mice with the vasopeptidase inhibitor ilepatril improved neural function . </S>", "<S> vasopeptidase inhibitors block angiotensin - converting enzyme ( ace ) and neutral endopeptidase ( nep ) activity . </S>", "<S> we propose that increased activity of ace and nep contributes to pathophysiology of dio . to address this issue c57bl/6j mice or mice deficient in nep </S>", "<S> were fed a high - fat diet and treated with ilepatril , enalapril , ace inhibitor , or candoxatril , nep inhibitor , using both prevention and intervention protocols . </S>", "<S> endpoints included glucose utilization and neural function determination . in the prevention study glucose tolerance </S>", "<S> was impaired in dio c57bl/6j mice and improved with ilepatril or enalapril . </S>", "<S> sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber density were impaired in dio c57bl/6j mice and improved with ilepatril or candoxatril . in the intervention study only enalapril improved glucose tolerance . sensory nerve conduction velocity and intraepidermal nerve fiber density </S>", "<S> were improved by all three treatments , whereas thermal nociception was improved by ilepatril or candoxatril . in nep - deficient mice </S>", "<S> dio impaired glucose utilization and this was improved with enalapril . </S>", "<S> nerve function was not impaired by dio in nep - deficient mice . </S>", "<S> these studies suggest that ace and nep play a role in pathophysiology associated with dio . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["previously we reported that deletion of neutral endopeptidase ( nep ) provides protection from obesity- and diabetes - induced neural complications .", "we have also shown that treating obese and streptozotocin - diabetic mice with the vasopeptidase inhibitor ilepatril prevented neural complications including slowing of nerve conduction velocity , thermal hypoalgesia , and decreased intraepidermal nerve fiber density .", "vasopeptidase inhibitors are drugs that simultaneously inhibit nep and angiotensin - converting enzyme ( ace ) activity .", "recent studies have shown increased expression of angiotensin - ii - forming enzymes in adipose tissue and increased activity of the renin - angiotensin system being implicated in the development of insulin resistance and type 2 diabetes .", "nep is found in many tissues including vascular and renal tissue and its activity is increased by fatty acids and glucose in human microvascular cells [ 59 ] . in the peripheral nervous system nep", "is located in schwann cell membranes surrounding dorsal root ganglion cells and nerve fibers [ 10 , 11 ] .", "nep degrades many vaso- and neuroactive peptides including natriuretic peptides , adrenomedullin , bradykinin , and calcitonin - gene - related peptide [ 12 , 13 ] .", "therefore , inhibition of ace and nep activity would be expected to improve vascular and neural function . in this", "regard we have demonstrated that treating type 1 and type 2 diabetic rats as well as a genetic rat model of obesity with ilepatril improves vascular and neural dysfunction [ 1416 ] .", "however , little information is available about the effect of vasopeptidase inhibitors in animal models of diet - induced obesity . in order to further elucidate the effects of vasopeptidase inhibitors in peripheral nerve dysfunction associated with obesity we examined the effect of diet - induced obesity on nerve conduction velocity and thermal response latency in the hindpaw of c57bl/6j mice and mice deficient in nep treated with ilepatril , enalapril , ace inhibitor , or candoxatril , nep inhibitor [ 1 , 2 ] ."], ["unless stated otherwise all chemicals used in these studies were obtained from sigma chemical co. ( st . louis , mo ) .   c57bl/6jj", "breeding pairs of neutral endopeptidase - deficient ( nep ) mice were provided by drs .", "these mice have been bred and a colony created at the veterans affairs medical center , iowa city , ia .", "deficiency of nep activity was confirmed in nep mice by measuring the specific activity of nep in kidney homogenates using the method described by ayoub and melzig   with modification .", "activity of nep in kidney from c57bl/6j and nep mice was 0.35  0.02 and 0.02  0.02  mm 7-amido-3-methylcoumarin ( amc)/min / mg protein , respectively ( p < 0.001 versus c57bl/6j by unpaired t - test ) .", "this test was performed on all mice used in these studies in order to confirm that nep was functionally  knocked out  in the nep mice .", "mice were housed in a certified animal care facility and food ( harlan teklad , no .", "adequate measures were taken to minimize pain or discomfort and all of the experiments were conducted in accordance with international standards on animal welfare and were compliant with all institutional and national institutes of health guidelines for use of animals ( acurf protocol 1101009 ) .  ", "for the prevention protocol c57bl/6j and nep mice at 12 weeks of age were divided into five groups .", "a second group was fed a high - fat diet containing 24  gm% fat , 24  gm% protein , and 41  gm% carbohydrate ( d12451 ; research diets , new brunswick , nj ) .", "the primary source of the increased fat content in the diet was soybean oil and lard .", "the control and high - fat diet contained 0.4 and 0.3% sodium chloride , respectively .", "the average fat content of the control diet was 4.25  gm% ( harlan teklad , no .", "the third through fifth groups were fed the high - fat diet containing ilepatril ( 500  mg / kg in the diet ) , candoxatril ( 30  mg / kg in the diet ) , or enalapril ( 500  mg / kg in the diet ) .", "we have found that these doses provide maximal inhibition of nep and/or ace activity in vivo [ 2 , 19 ] .", "for the intervention study the same five groups of c57bl/6j and nep mice at 12 weeks of age were fed the control diet ( group 1 ) or high - fat diet ( groups 25 ) for 12 weeks .", "afterwards , the four groups of high - fat - fed mice were fed a high - fat diet with no additions ( group 2 ) or high - fat diet containing ilepatril , candoxatril , or enalapril ( groups 35 ) for 12 weeks .", "after an overnight fast mice were injected with a saline solution containing 2  g / kg glucose , i.p . immediately prior to the glucose injection and for 120 minutes", "afterwards blood samples were taken to measure circulating glucose levels with the use of glucose - dehydrogenase - based reagent strips ( aviva accu - chek , roche , mannheim , germany ) .", "blood samples ( 0.6  l ) were taken from a tail vein that was lanced once .", "thermal nociceptive response in the hindpaw was measured using the hargreaves method with instrumentation provided by iitc life science , woodland hills , ca ( model 390 g ) as previously described . the test was performed in a blind manner .", "thermal nociceptive responses were measured by placing the mouse in the observation chamber on top of the thermal testing apparatus and allowing it to acclimate to the warmed glass surface ( 33c ) and surroundings for a period of 15  min .", "the mobile heat source was maneuvered so that it was under the heal of the hindpaw and then activated , a process that starts a timer and locally warms the glass surface , when the mouse withdrew its paw , the timer , and the heat source was turned off .", "following an initial recording , which was discarded , two measurements were made for each hindpaw , with a rest period of 5  min between each set of measurements .", "the mean of the measurements , reported in seconds , was used as a measure of the thermal nociceptive response latency .", ", abbott laboratories , north chicago , il ) and sensory nerve conduction velocities were determined as previously described [ 1 , 2 ] .", "briefly , sensory nerve conduction velocity was recorded in the digital nerve to the second toe by stimulating with a square - wave pulse of 0.05  ms duration using the smallest intensity current that resulted in a maximal amplitude response ( grass s44 stimulator ; grass medical instruments , quincy , ma ) .", "the maximal sensory nerve conduction velocity was calculated by measuring the latency to the onset / peak of the initial negative deflection and the distance between stimulating and recording electrodes ( measured in millimeters using a vernier caliper ) .", "biopsies of skin of the right hindpaw were fixed , dehydrated , and embedded in paraffin .", "sections ( 7  m ) were collected and immunostained with anti - pgp9.5 antibody ( rabbit anti - human no .", "7863 - 0504 ( this antibody cross - reacts with rat , mouse guinea pig , and other species ) , abd serotic , morpho sys us inc . ,", "raleigh , nc ) overnight followed by treatment with secondary antibody alexa fluor 546 goat anti - rabbit ( invitrogen , eugene , or ) .", "profiles were imaged using a zeiss 710 confocal microscope with a 40x objective and were counted by two individual investigators that were blinded to the sample identity .", "length of the epidermis was determined by drawing a polyline along the contour of the epidermis and recording its length in mm .", "comparisons between the groups for body weight , blood glucose , sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber profiles were conducted using a one - way anova and bonferroni 's test for multiple comparisons ( prism software ; graphpad , san diego , ca ) ."], ["presented in table 1 are weight and blood glucose changes for c57/bl6j and nep mice used in the prevention study . at 12 weeks of age", "when fed a high - fat diet c57bl/6j and nep mice both gained a similar amount of weight . treating the high - fat diet with ilepatril or enalapril but not candoxatril completely prevented the gain in weight .", "the mass of the epididymal fat pad was significantly increased in high - fat - fed mice and mice fed the high - fat diet treated with candoxatril compared to control mice or mice fed the high - fat diet containing ilepatril or enalapril . when calculating the epididymal fat pad mass as a percent of total body weight epididymal fat pad mass was significantly increased in high - fat - fed c57bl/6j mice and mice fed a high - fat diet containing candoxatril compared to control . treating the high - fat diet with ilepatril and to", "a greater extent enalapril prevented the increase in epididymal fat pad mass when presented as percent of final body weight .", "nonfasting blood glucose levels were not significantly different between c57bl/6j and nep mice fed the control or high - fat diets and not affected by ilepatril , candoxatril , or enalapril treatment . in the intervention study all mice fed the high - fat diet for the initial 12 weeks of", "the experimental design gained a significant amount of weight ( table 2 ) . when transferred to a high - fat diet containing ilepatril or enalapril for an additional 12 weeks c57bl/6j and nep mice lost weight .", "mice remaining on the high diet or high - fat diet containing candoxatril continued to gain weight .", "the epididymal fat mass decreased after c57bl/6j or nep mice , fed a diet containing high fat for 12 weeks , were given a high - fat diet containing enalapril .", "the epididymal fat mass also was decreased after nep mice but not c57bl/6j mice were fed a high - fat diet treated with ilepatril .", "treating high - fat - fed c57bl/6j or nep mice with candoxatril did not reduce epididymal fat mass .", "a similar trend was observed when the epididymal fat mass data was presented as percentage of final body weight .", "nonfasting blood glucose levels were not changed in c57bl/6j or nep mice fed control or high - fat diets with or without ilepatril , candoxatril , or enalapril .   in the prevention study c57bl/6j ( figure 1 ) and nep ( figure 2 ) mice fed the high - fat diet or high - fat diet containing candoxatril had an impaired glucose clearance curve compared to control mice .", "in contrast , the glucose clearance curve was near normal in c57bl/6j and nep mice fed a high - fat diet containing ilepatril or enalapril .", "in the intervention study feeding c57bl/6j ( figure 3 ) or nep ( figure 4 ) mice for 24 weeks a high - fat diet caused an impaired glucose clearance curve . treating high - fat - fed c57bl/6j or nep mice with enalapril for the last 12 weeks of the 24-week period partially corrected glucose utilization . treating nep mice with ilepatril", "treating c57bl/6j mice with ilepatril did not improve glucose utilization nor did treating c57bl/6j or nep with candoxatril .", "data in figure 5 ( left ) demonstrate that sensory nerve conduction velocity was significantly decreased in high - fat - fed c57bl/6j mice compared to control mice and that this was prevented by treating high - fat - fed mice with ilepatril or candoxatril using a prevention protocol .", "data in figure 5 ( center ) demonstrate that thermal nociception was significantly decreased in c57bl/6j mice after 12 weeks of a high - fat diet compared to control mice as indicated by an increase in paw withdrawal latency .", "treatment with ilepatril or candoxatril and to a lesser extent enalapril for the 12-week period prevented thermal hypoalgesia in high - fat - fed c57bl/6j mice .", "data in figure 5 ( right ) demonstrate that the number of intraepidermal nerve fiber profiles was significantly decreased in high - fat - fed c57bl/6j mice and that treatment of these mice with ilepatril or candoxatril but not enalapril for 12 weeks prevented the significant decrease .", "sensory nerve conduction velocity , thermal nociception , and intraepidermal nerve fiber density were not impaired in nep mice fed a high - fat diet ( figure 6 , left , center , and right , resp . ) .  ", "we also determined the effect of treating high - fat - fed c57bl/6j and nep mice using an intervention protocol ( figures 7 and 8 , resp . ) . in this study design", "high - fat - fed mice were untreated for 12 weeks followed by 12 weeks of a high - fat diet with or without treatment .", "feeding c57bl/6j mice a high - fat diet for 24 weeks caused a significant decrease in sensory nerve conduction velocity ( figure 7 , left ) . treating these mice for the last 12 weeks of the 24-week period with ilepatril , candoxatril , or enalapril", "the decrease in thermal nociception in high - fat - fed c57bl/6j mice was reversed by treating mice with ilepatril or candoxatril but not enalapril ( figure 7 , center ) .", "treating high - fat - fed c57bl/6j mice with ilepatril , candoxatril , or enalapril reversed the decrease in intraepidermal nerve fiber profiles ( figure 7 , right ) .", "feeding nep mice a high - fat diet for 24 weeks with or without treatment had no effect on sensory nerve conduction velocity , thermal nociception , or intraepidermal nerve fiber density ( figure 8 , left , center , and right , resp . ) ."], ["the main findings from these studies were that diet - induced obesity caused significant weight gain and impaired glucose utilization in c57bl/6j and nep mice that was improved when the mice were treated with ilepatril or enalapril using a prevention protocol . using the intervention protocol enalapril but not ilepatril treatment was effective improving glucose utilization by c57bl/6j mice .", "treating c57bl/6j or nep mice with candoxatril did not improve weight gain or glucose utilization .", "these data suggest that inhibition of ace and not nep activity was responsible for improving weight gain and glucose utilization in high - fat - fed mice .", "diet - induced obesity also caused slowing of sensory nerve conduction velocity , thermal hypoalgesia , and decrease in epidermal nerve fiber density in the paw of the hindlimb in c57bl/6j mice but not nep mice .", "the impairment in sensory nerve function endpoints in c57bl/6j mice fed a high - fat diet was prevented / improved when the mice were treated with ilepatril or candoxatril and to a much lesser extent enalapril using either the prevention or intervention protocol .", "these data suggest that increased nep activity / expression but not ace activity contributes to diet - induced obesity - related deficits in sensory nerve function .  ", "previous studies have shown that diet - induced obesity in rodent models can be prevented by ace inhibitors and angiotensin ii receptor blockers [ 2124 ] .", "in addition , diet - induced weight gain and fat mass are reduced , energy expenditure increased , and glucose tolerance improved in mice lacking ace or the angiotensin ii type 1a receptor [ 25 , 26 ] .", "the mechanisms proposed for the improvement in obesity and glucose tolerance with treatment of rodent models with ace inhibitors are increased energy expenditure , liver and adipose tissue metabolic modulation , lower concentration of leptin , improved insulin signaling , and increased glucose and fatty acid utilization by muscle [ 2130 ] . in a study comparing the effects of ramipril , an ace inhibitor , to ilepatril in jcr : la - cp rats , an obese , insulin - resistant , hyperinsulinemic , normoglycemic model , it was found that both compounds reduced the surge of plasma insulin in a meal tolerance test by about 50% but ilepatril was more beneficial in improving vascular reactivity . in our study", "we found that enalapril trended to be more effective than ilepatril preventing / reducing fat pad mass .", "this could be because enalapril at the dosage used was a more effective ace inhibitor than ilepatril in target tissues . in another study using obese zucker rats", "it was found that dual inhibition of ace and nep improved insulin - mediated glucose disposal more effectively than monotherapy and this effect was linked to increased activation of the kinin - nitric oxide pathway . in a similar independent study it was found that omapatrilat , a vasopeptidase inhibitor , induced insulin sensitization and increased myocardial glucose uptake in obese zucker rats and that the effect of omapatrilat was greater than ramipril in part due to stimulation of the b2 receptor .", "later this group reported that treatment of obese zucker rats with a vasopeptidase inhibitor increased muscle glucose uptake independent of insulin signaling . in two of these studies protection of bradykinin from degradation by nep", "interestingly , it has been shown that natriuretic peptides promote muscle mitochondrial biogenesis and fat oxidation as to prevent obesity and glucose intolerance .", "the natriuretic peptides are also degraded by nep . because nep is expressed in skeletal muscle in relatively large amounts and being located on the cell surface , nep is able to hydrolyze peptides in the vicinity of their receptors thereby neutralizing their bioactivity [ 34 , 36 ] .", "however , inhibition of ace may also lead to protection of bradykinin levels by inhibiting kininase - ii - mediated degradation of this nonapeptide .", "reported that ace inhibition by captopril improved glucose transport in insulin - resistant muscle of the obese zucker rat .", "they attributed the improvement to modulation of insulin action by bradykinin mediated through b2 receptors and by an increase in nitric oxide production . since bradykinin and natriuretic peptides may have a role in enhancing insulin action and regulating glucose and fatty acid metabolism by muscle protecting their bioactive function by preventing degradation through inhibition of ace and/or nep may be a therapeutic approach for treatment of obesity and insulin resistance [ 34 , 36 , 38 ] .  ", "we have previously shown that treating diabetic rats with enalapril improved vascular and nerve endpoints but enalapril was less effective in rats fed a high - fat diet [ 19 , 36 , 39 , 40 ] .", "in contrast , we found that treating obese or diabetic rats with ilepatril was more effective than enalapril in improving peripheral nerve dysfunction [ 19 , 36 , 40 ] .", "we have shown that treating diabetic or diet - induced obese rodents with ilepatril improves vasodilation of blood vessels that provide circulation to the sciatic nerve by reducing oxidative stress and preventing degradation of vasoactive peptides by nep including calcitonin gene - related peptide and c - type natriuretic peptide thereby maintaining endoneurial blood flow and preventing ischemia .", "since nep degrades both calcitonin gene - related peptide and substance p , peptides important in pain signaling pathways , it is likely that blocking nep activity or mice deficient in nep would maintain higher levels of both of these neuroactive peptides and perhaps be more sensitive to painful stimuli ."], ["data from clinical trials clearly suggest that activation of the renin - angiotensin - aldosterone system plays an important role in the pathophysiology of the metabolic syndrome through interaction of a wide range of physiological and molecular mechanisms . more recently in humans activity of nep", "has been shown to correlate with body mass index and measures of insulin resistance with increasing activity found in subjects with multiple cardiovascular risk factors . in these studies we found that feeding mice a high - fat diet causes weight gain , impaired glucose tolerance , and sensory nerve dysfunction . inhibition of ace was effective in reducing weight gain and improving glucose utilization but generally noneffective in improving sensory nerve dysfunction .", "in contrast , inhibition of nep improved sensory nerve dysfunction and had no effect in improving weight gain or glucose utilization . treating high - fat - fed mice with a vasopeptidase inhibitor improved weight gain , glucose utilization , and sensory nerve function .", "thus , we conclude that increased activity of nep is associated with sensory nerve dysfunction in an animal model of diet - induced obesity and that dual inhibition of ace and nep may be more effective than monotherapy in reducing insulin resistance and the sensory nerve complications associated with metabolic syndrome ."]]}
{"article_id": "PMC5181394", "article_text": ["tetracyclines bind to the a - site on the bacterial ribosome , resulting in steric blocking of the aminoacyl - trna binding site , which prevents protein synthesis .", "they are effective against both gram - positive and gram - negative bacteria and , due to the relative lack of major side effects and cheap cost , have been used extensively in the treatment of infections as well as growth promoters in animal husbandry . bacterial resistance to", "tetracycline is often mediated through the acquisition of dna encoding proteins that confer resistance by one of three main mechanisms : atp - dependent efflux , enzymatic inactivation of tetracycline , or ribosomal protection . to date , a total of 60 different classes of tetracycline resistance gene , including oxytetracycline resistance genes , have been reported .", "these include 33 predicted or proven to encode active efflux pumps , 12 encoding ribosomal protection proteins ( rpps ) , 13 encoding inactivating enzymes and 1 reported to confer resistance via an as yet undetermined mechanism , designated tet(u ) ( a full list is periodically updated by roberts ) .", "although it has yet to be assigned a mechanistic class , tet(u ) has been identified in enterococcus and staphylococcus isolates . however", ", a study by caryl et al . reported that when tet(u ) was cloned and expressed in escherichia coli , the transformants were not resistant to tetracycline . to be considered a new class of tetracycline resistance gene", "determinants representing new classes were originally assigned a letter from the english alphabet . however , as all letters are used , they are now assigned an arabic numeral , with new determinants referred to the levy group ( bonnie.marshall@tufts.edu ) in order to obtain a designation prior to publication to avoid duplication and ensure taxonomic consistency .", "rpps are a related group of proteins that , when bound to the ribosome , result in the release of tetracycline from the ribosome , enabling protein synthesis to proceed ( reviewed by thaker et al . ) . of the 12 classes of rpp gene currently reported [ tet(m )", ", ( o ) , ( q ) , ( s ) , ( t ) , ( w ) , ( 32 ) , ( 36 ) , ( 44 ) , b(p ) , otr(a ) and tet ] , tet(m ) is considered the most prevalent due to its association with the broad host range tn916/tn1545 family of conjugative transposons .", "however , a subgroup of rpp genes has been identified that consist of regions of different , already characterized rpp genes that appear to have undergone recombination forming a mosaic gene .", "it must be stressed here that the progenitors of mosaic genes are assumed based purely on the order in which they were discovered and we can not be sure of the directionality of mosaic gene formation .", "in 2003 , stanton and humphery reported two rpp genes in megasphaera elsdenii that encoded predicted proteins showing 89.1% and 91.9% identity to tet(w ) ( accession number aj222769 ) from butyrivibrio fibrisolvens .", "as this was above the < 80% cut - off , they did not qualify as a new resistance class under the nomenclature system . however , further analysis of the amino acid sequence revealed variability in the percentage identity to tet(w ) across its length .", "the large central section in both sequences showed 98.1% identity to tet(w ) , while small sections at the n- and c - terminal ends were found to have a lower amino acid sequence identity to tet(w ) [ between 66.6% and 75.3% ] .", "however , these same n- and c - terminal sections were shown to have between 99.3% and 100% amino acid identity to tet(o ) ( accession number m18896 ) , despite the central section showing identity to tet(w ) . given the evidence , this suggested recombination had occurred , creating a mosaic determinant with a central tet(w ) region flanked by two tet(o ) regions .", "although never before observed between two different rpp classes , recombination resulting in functional genes has previously been reported between different phylotypes of tet(m ) as well as in other antibiotic resistance genes , such as pena and pbp2x , which confer resistance to penicillin .", "furthermore , in vitro experiments have successfully recombined tet(a ) and tet(c ) to create mosaics that confer resistance to tetracycline at levels comparable to the non - mosaic tet(c ) .", "the guideline for determining a new resistance gene class was established prior to the discovery of these mosaic rpp genes and none of the mosaic genes qualified as a new class when analysed as one single continuous sequence .", "it was clear , however , that these mosaic genes were different from their non - mosaic counterparts and that the current classification did not adequately reflect the true evolutionary background of these genes .", "therefore , an expansion of the nomenclature system was suggested whereby the mosaic gene would receive a designation that reflected the structural order and class of the genes they comprised , better reflecting their variable nature .", "for example , the two resistance genes reported in m. elsdenii , which comprised a central tet(w ) region flanked by two tet(o ) regions , were designated tet(o / w / o ) .", "although stanton and humphrey were the first to report mosaic rpp genes , melville et al . had unknowingly reported a mosaic gene 2 years previously .", "this resistance gene , found in clostridium saccharolyticum k10 , encoded a predicted protein that showed 76% amino acid identity to tet(o ) ( accession number y07780 ) .", "as per the original nomenclature guidelines , it was given the new designation tet(32 ) .", "however , subsequent re - examination of the sequence found that only the central section showed < 80% identity to known proteins , while the n- and c - terminal regions flanking the central section shared 100% and 97.7% identity , respectively , to tet(o ) ( accession number m18896 ) .", "the central region was still thought to represent a section of a new tet(32 ) class and therefore the determinant was reclassified tet(o/32/o ) .", "subsequently , the proposed full , non - mosaic sequences of tet(32 ) have been reported in several isolates identified from the human oral cavity , with the tet(o/32/o ) mosaic determinant now showing 89% amino acid identity to these .", "similarly , the previously reported tet(s ) allele ( accession number ay534326 ) on the conjugative transposon tn916s has subsequently been reclassified as a result of in silico analysis .", "the amino acid sequence shows identity to tet(s ) across 595 amino acids ( 1595 inclusive ) , with the final 61 amino acids at the c - terminus end identical to tet(m ) ( accession number u09422 ) , resulting in it being reclassified as tet(s / m ) .", "to date , a total of 30 mosaic genes have been reported in the literature , of which 26 currently have sequences deposited in genbank ( table  1 ) .", "some studies have reported multiple occurrences of known genes ; however , many of these have been characterized by pcr amplification only .", "structurally , these chimeric genes currently comprise either two [ e.g. tet(o / w ) ] , three [ e.g. tet(o / w / o ) ] , four [ e.g. tet(o / w/32/o ) ] or six [ e.g. tet(o / w/32/o / w / o ) ] different regions ( figure  1 ) , with tet(o ) , tet(w ) and tet(32 ) being the predominant rpp genes reported to form mosaic genes , comprising all but two of the reported variants , and tet(m ) and tet(s ) forming the remaining two .", "given the prevalence of tet(m ) in certain samples , and the previous reports of self - recombination , it is surprising that there are so few reports of mosaic genes containing tet(m ) .", "furthermore , alignment of 12 representative rpp gene sequences shows tet(m ) sharing 75% and 70% identity , respectively , to tet(o ) and tet(44 ) , which is higher than the percentage identity observed between the more commonly reported rpp mosaic genes comprising tet(o ) , ( w ) and ( 32 ) ( table  2 ) . however , mosaic genes comprising tet(m ) and any other gene , with the exception of tet(s ) , have yet to be reported .", "it is entirely possible that this may be due to a lack of investigation rather than an absence of recombination followed by fixation of the recombinant allele in the bacterial population .", "alternatively , it is possible that there is little selective pressure for tet(m)-based mosaic genes if the resultant protein is no more efficient than tet(m ) itself and/or there is no indirect selective pressure for mosaicism .", "reported that the protein encoded by the tet(o / w / o ) mosaic genes in m. elsdenii conferred a higher level of resistance to tetracycline than their non - mosaic counterparts , but similar investigations are still to be reported for other rpp genes .", "therefore , the prevalence of certain mosaic gene variants could suggest that they are in some way more beneficial to the host than the non - mosaic genes they comprise . \n", "table  1.a summary of the mosaic tetracycline genes reported to dategeneorganismsource(s)accession numberreference(s)tet(o / w)bifidobacterium thermophilum b0219environmental ( pig slaughterhouse ) sampleam88911832tet(o / w)b .", "faecesam88912232tet(o / w)-2megasphaera elsdenii 25 - 51swine faecesay48512218,27tet(o / w)-1 [ n  =  15]m .", "elsdenii 27 - 51swine faecesay48512627,33tet(o / w / o)-4uncultured bacterial clonepig faecesno accession number21tet(o / w / o)-3 [ n  =  9]uncultured bacterial clonepig", "elsdenii 14 - 14swine caecumay19692013,18,27,33tet(o / w / o)-1 [ n  =  2]m .", "elsdenii 7 - 11swine caecumay19692113,18,27tet(o / w/32/o ) [ n  =  32]uncultured bacterial clonepig faecesef06552321tet(o / w/32/o ) [ n  =  7]streptococcus suis ss1303pig ( brain , lung and spleen ) and human ( csf )", "suisdiseased pig ( blood , brain , heart , joint and lung ) samplesjq74005328tet(o / w/32/o / w / o)lactobacillus johnsonii g41human faecesdq52502332tet(o / w/32/o / w / o)uncultured bacterial clonepig faecesdq67992621tet(o/32/o)s", ". suisdiseased pig ( blood , brain , heart , joint and lung ) samplesjq74005228tet(o/32/o)clostridium saccharolyticum k10human colonaj29523818tet(o/32/o)-2 [ n  =  3]uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)-3uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)-4uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)-5uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)dorea longicatena agr2136rumen microbiomenz_aujs01000017 ( 41  62643  545 bp)direct submission , analysed in this studytet(o/32/o)campylobacter coli 202/04human faecesainh01000038 ( 23614280 bp)direct submission , analysed in this studytet(o/32/o)c .", "coli 317/04human faecesnz_ainj01000054 ( 20944013 bp)direct submission , analysed in this studytet(o/32/o)campylobacter jejuni subspecies jejuni 2008 - 894humanaioq01000025 ( 14  51516  434 bp)direct submission , analysed in this studytet(o/32/o)roseburia intestinalis xb6b4human intestinal tractfp929050 ( 2  873  8142  875  733 bp)direct submission , analysed in this studytet(s / m)streptococcus equinus 1357foodhm36771125tet(s / m)streptococcus intermediushuman isolateay53432623,24tet(w/32/o)b .", "thermophilum b0256pig faecesam71060532the number given in square brackets indicates the instances of that mosaic gene variant reported , if more than one.fourteen of the 15 tet(o / w)-1 variants were only determined by pcr analysis and so could be either tet(o", "/ w)-2.eleven of the 28 tet(o / w / o)-2 variants were only determined by pcr analysis and so could be either tet(o / w / o)-2 or tet(o / w / o)-1.all s. suis isolates , but not the same strain . \n", "table  2.sequence identity matrix showing the percentage nucleotide identity between representatives of all 12 rpp gene classes , in descending order , compared with tet(m)rpp genetet(m)tet(s)tet(o)tet(44)tet(32)tet(w)tet(t)tet(36)tet(q)tetb(p)otr(a)tettet(m)1007875706964574946231111tet(s)10070696762565648111110tet(o)100696965564948151211tet(44)1007164505846151110tet(32)10067554947111210tet(w)10012451551412tet(t)10057561882tet(36)1006491111tet(q)100131212tetb(p)10011otr(a)10063tet100accession numbers of representative genes included in the matrix : tet(m ) , u09422 ; tet(o ) , y07780 ; tetb(p ) , ae001437 ; tet(q ) , x58717 ; tet(s ) , x92946 ; tet(t ) , l42544 ; tet(w ) , aj222769 ; tet(32 ) , dq647324 ; tet(36 ) , aj514254 ; tet(44 ) , fn594949 ; otr(a ) , x53401 ; tet , al939106.shaded boxes represent those genes currently reported to comprise mosaic genes . \n", "the coded bars indicate sequences of high identity to specific rpp genes : vertical line bars for tet(m ) , white bars for tet(o ) , grey bars for tet(s ) , black bars for tet(w ) and checked bars for tet(32 ) .", "indicates those sequences that are incomplete or absent in genbank , with the crossover points taken from the publication", ". indicates sequences that have been analysed in this study due to no specific crossover point(s ) reported .", "a summary of the mosaic tetracycline genes reported to date the number given in square brackets indicates the instances of that mosaic gene variant reported , if more than one .", "fourteen of the 15 tet(o / w)-1 variants were only determined by pcr analysis and so could be either tet(o", "eleven of the 28 tet(o / w / o)-2 variants were only determined by pcr analysis and so could be either tet(o / w / o)-2 or tet(o / w / o)-1 .", "sequence identity matrix showing the percentage nucleotide identity between representatives of all 12 rpp gene classes , in descending order , compared with tet(m ) accession numbers of representative genes included in the matrix : tet(m ) , u09422 ; tet(o ) , y07780 ; tetb(p ) , ae001437 ; tet(q ) , x58717 ; tet(s ) , x92946 ; tet(t ) , l42544 ; tet(w ) , aj222769 ; tet(32 ) , dq647324 ; tet(36 ) , aj514254 ; tet(44 ) , fn594949 ; otr(a ) , x53401 ; tet , al939106 .", "the coded bars indicate sequences of high identity to specific rpp genes : vertical line bars for tet(m ) , white bars for tet(o ) , grey bars for tet(s ) , black bars for tet(w ) and checked bars for tet(32 ) .", "indicates those sequences that are incomplete or absent in genbank , with the crossover points taken from the publication", ". indicates sequences that have been analysed in this study due to no specific crossover point(s ) reported .", "stanton and humphrey describe an assay that distinguished between the non - mosaic genes tet(o ) and tet(w ) and the mosaic tet(o / w / o ) from megasphaera strains , enabling them to detect tet(o / w / o ) variants in six additional m. elsdenii strains .", "patterson et al . investigated the presence of mosaic genes using various specific oligonucleotide sets that either bound within the resistance genes or flanked them .", "amplicons specific to tet(o / w ) , tet(o/32 ) and tet(w/32 ) were detected in faecal samples , with tet(o/32 ) being the most common of these mosaic amplicons ; it was amplified in all 12 pig faecal samples and 6 of 7 human faecal samples tested .", "in contrast , the faecal samples from cows and sheep , as well as human saliva samples , failed to produce any amplicons for these mosaic genes , suggesting they were not present at detectable levels .", "chen et al . also used an oligonucleotide primer set that annealed outside tet(o ) to determine the presence of tetracycline resistance genes in two streptococcus suis isolates .", "although no amplicon was produced using internal , tet(o)-specific primers , the primers binding to flanking dna yielded an amplicon , indicating the presence of mosaic genes [ identified as tet(o/32/o ) and tet(o / w/32/o ) ] .", "this full - length oligonucleotide primer set does aid the identification of mosaic genes ; however , it is only specific for those with regions homologous to tet(o ) flanking sequences . since pcr strategies aimed at identifying resistance genes require knowledge of the sequence of the target , mosaic rpp genes are likely to be largely undetected and under - reported by pcr - based studies .", ", almost all the mosaic genes reported to date have originated from faecal samples , with the majority identified from a porcine origin and less commonly from humans ( table  1 ) .", "the gut houses a complex and diverse bacterial community with potential for widespread horizontal gene transfer , and the mosaic genes found in faecal samples are likely to reflect the pool of non - mosaic genes present within the gut microbiota .", "genes such as tet(w ) and tet(o ) are commonly reported from these types of samples , but the prevalence of tet(32)-containing mosaic genes suggests that tet(32 ) may be more common than initially thought .", "in fact , tet(o/32/o ) was found to be the most common mosaic gene in both the human and pig faecal samples tested and was present in almost as many samples tested as the non - mosaic tet(o ) and tet(w ) genes .", "in contrast , mosaic genes have not yet been reported in faecal samples from bovine and ovine origin or in human saliva . why they are predominantly found in pigs while as yet unreported in other animals", "is not immediately clear , though the extensive use of tetracyclines in the swine industry may have contributed to their selection .", "the advent of high - throughput genomic sequencing has led to an increase in the number of genomes being deposited in sequence databases .", "many contain tetracycline resistance genes that are generically labelled simply as  tetracycline resistance protein or as  tet(m)-like , the designation of which may be a result of automated annotation pipelines . a preliminary search of the ncbi nucleotide database , using tet(o ) ( accession number y07780 ) as the query , found that some of these generically labelled tetracycline resistance genes gave a partial match to tet(o ) .", "further examination indicates that some are as yet uncharacterized and unreported mosaic genes , which have been further defined for this review using the nucleotide sequence to determine the crossover points .", "for example , the tet(m)-like gene ( accession number nz_aujs01000017 , location 41  62643  545 bp ) in the draft genome of dorea longicatena agr2136 from a human faecal sample appears to be a previously unreported variant of tet(o/32/o ) ( figure  1 ) .", "furthermore , the tetracycline resistance genes present in campylobacter jejuni subspecies jejuni 2008 - 894 , campylobacter coli 202/04 , c. coli 317/04 ( accession numbers aioq01000025 , ainh01000038 and nz_ainj01000054 , respectively ) and roseburia intestinalis xb6b4 ( accession number fp929050 ) are also structurally novel variants of tet(o/32/o ) ( figure  1 ) . the three mosaic genes present in the campylobacter spp .", "taking into account these newly defined genes , the total number of mosaic genes reported increases from 30 to 35 ( not including those identified via pcr amplification only ; table  1 ) and suggests that other generically labelled tetracycline resistance genes present in the database [ e.g. those labelled as tet(m)-like ] could be further classified , helping to understand mosaic gene proliferation and diversity .", "our knowledge of the mosaic rpp gene group is steadily increasing since their discovery in 2003 , with the majority derived from tet(o ) , tet(w ) and tet(32 ) and others deriving from tet(m ) and tet(s ) .", "it is clear that these genes are being under - reported both in terms of experimental detection and also within genomic data .", "further work and increased attention on mosaic rpp genes is important if we are to understand the evolutionary selective pressures driving their fixation in bacterial populations and the subsequent effects on resistance and mobile genetic element evolution within their host ."], "abstract_text": ["<S> first reported in 2003 , mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins ( rpps ) . </S>", "<S> they are formed when two or more rpp - encoding genes recombine resulting in a functional chimera . to date , the majority of mosaic genes are derived from sections of three rpp genes , tet(o ) , tet(w ) and tet(32 ) , with others comprising tet(m ) and tet(s ) . in this first review of mosaic genes </S>", "<S> , we report on their structure , diversity and prevalence , and suggest that these genes may be responsible for an under - reported contribution to tetracycline resistance in bacteria . </S>"], "labels": null, "section_names": ["Introduction", "RPPs", "Mosaic RPP genes", "Mosaic gene diversity", "PCR-based analysis", "Draft genome analysis", "Conclusions", "Transparency declarations"], "sections": [["tetracyclines bind to the a - site on the bacterial ribosome , resulting in steric blocking of the aminoacyl - trna binding site , which prevents protein synthesis .", "they are effective against both gram - positive and gram - negative bacteria and , due to the relative lack of major side effects and cheap cost , have been used extensively in the treatment of infections as well as growth promoters in animal husbandry . bacterial resistance to", "tetracycline is often mediated through the acquisition of dna encoding proteins that confer resistance by one of three main mechanisms : atp - dependent efflux , enzymatic inactivation of tetracycline , or ribosomal protection . to date , a total of 60 different classes of tetracycline resistance gene , including oxytetracycline resistance genes , have been reported .", "these include 33 predicted or proven to encode active efflux pumps , 12 encoding ribosomal protection proteins ( rpps ) , 13 encoding inactivating enzymes and 1 reported to confer resistance via an as yet undetermined mechanism , designated tet(u ) ( a full list is periodically updated by roberts ) .", "although it has yet to be assigned a mechanistic class , tet(u ) has been identified in enterococcus and staphylococcus isolates . however", ", a study by caryl et al . reported that when tet(u ) was cloned and expressed in escherichia coli , the transformants were not resistant to tetracycline . to be considered a new class of tetracycline resistance gene", "determinants representing new classes were originally assigned a letter from the english alphabet . however , as all letters are used , they are now assigned an arabic numeral , with new determinants referred to the levy group ( bonnie.marshall@tufts.edu ) in order to obtain a designation prior to publication to avoid duplication and ensure taxonomic consistency ."], ["rpps are a related group of proteins that , when bound to the ribosome , result in the release of tetracycline from the ribosome , enabling protein synthesis to proceed ( reviewed by thaker et al . ) . of the 12 classes of rpp gene currently reported [ tet(m )", ", ( o ) , ( q ) , ( s ) , ( t ) , ( w ) , ( 32 ) , ( 36 ) , ( 44 ) , b(p ) , otr(a ) and tet ] , tet(m ) is considered the most prevalent due to its association with the broad host range tn916/tn1545 family of conjugative transposons .", "however , a subgroup of rpp genes has been identified that consist of regions of different , already characterized rpp genes that appear to have undergone recombination forming a mosaic gene .", "it must be stressed here that the progenitors of mosaic genes are assumed based purely on the order in which they were discovered and we can not be sure of the directionality of mosaic gene formation ."], ["in 2003 , stanton and humphery reported two rpp genes in megasphaera elsdenii that encoded predicted proteins showing 89.1% and 91.9% identity to tet(w ) ( accession number aj222769 ) from butyrivibrio fibrisolvens .", "as this was above the < 80% cut - off , they did not qualify as a new resistance class under the nomenclature system . however , further analysis of the amino acid sequence revealed variability in the percentage identity to tet(w ) across its length .", "the large central section in both sequences showed 98.1% identity to tet(w ) , while small sections at the n- and c - terminal ends were found to have a lower amino acid sequence identity to tet(w ) [ between 66.6% and 75.3% ] .", "however , these same n- and c - terminal sections were shown to have between 99.3% and 100% amino acid identity to tet(o ) ( accession number m18896 ) , despite the central section showing identity to tet(w ) . given the evidence , this suggested recombination had occurred , creating a mosaic determinant with a central tet(w ) region flanked by two tet(o ) regions .", "although never before observed between two different rpp classes , recombination resulting in functional genes has previously been reported between different phylotypes of tet(m ) as well as in other antibiotic resistance genes , such as pena and pbp2x , which confer resistance to penicillin .", "furthermore , in vitro experiments have successfully recombined tet(a ) and tet(c ) to create mosaics that confer resistance to tetracycline at levels comparable to the non - mosaic tet(c ) .", "the guideline for determining a new resistance gene class was established prior to the discovery of these mosaic rpp genes and none of the mosaic genes qualified as a new class when analysed as one single continuous sequence .", "it was clear , however , that these mosaic genes were different from their non - mosaic counterparts and that the current classification did not adequately reflect the true evolutionary background of these genes .", "therefore , an expansion of the nomenclature system was suggested whereby the mosaic gene would receive a designation that reflected the structural order and class of the genes they comprised , better reflecting their variable nature .", "for example , the two resistance genes reported in m. elsdenii , which comprised a central tet(w ) region flanked by two tet(o ) regions , were designated tet(o / w / o ) .", "although stanton and humphrey were the first to report mosaic rpp genes , melville et al . had unknowingly reported a mosaic gene 2 years previously .", "this resistance gene , found in clostridium saccharolyticum k10 , encoded a predicted protein that showed 76% amino acid identity to tet(o ) ( accession number y07780 ) .", "as per the original nomenclature guidelines , it was given the new designation tet(32 ) .", "however , subsequent re - examination of the sequence found that only the central section showed < 80% identity to known proteins , while the n- and c - terminal regions flanking the central section shared 100% and 97.7% identity , respectively , to tet(o ) ( accession number m18896 ) .", "the central region was still thought to represent a section of a new tet(32 ) class and therefore the determinant was reclassified tet(o/32/o ) .", "subsequently , the proposed full , non - mosaic sequences of tet(32 ) have been reported in several isolates identified from the human oral cavity , with the tet(o/32/o ) mosaic determinant now showing 89% amino acid identity to these .", "similarly , the previously reported tet(s ) allele ( accession number ay534326 ) on the conjugative transposon tn916s has subsequently been reclassified as a result of in silico analysis .", "the amino acid sequence shows identity to tet(s ) across 595 amino acids ( 1595 inclusive ) , with the final 61 amino acids at the c - terminus end identical to tet(m ) ( accession number u09422 ) , resulting in it being reclassified as tet(s / m ) ."], ["to date , a total of 30 mosaic genes have been reported in the literature , of which 26 currently have sequences deposited in genbank ( table  1 ) .", "some studies have reported multiple occurrences of known genes ; however , many of these have been characterized by pcr amplification only .", "structurally , these chimeric genes currently comprise either two [ e.g. tet(o / w ) ] , three [ e.g. tet(o / w / o ) ] , four [ e.g. tet(o / w/32/o ) ] or six [ e.g. tet(o / w/32/o / w / o ) ] different regions ( figure  1 ) , with tet(o ) , tet(w ) and tet(32 ) being the predominant rpp genes reported to form mosaic genes , comprising all but two of the reported variants , and tet(m ) and tet(s ) forming the remaining two .", "given the prevalence of tet(m ) in certain samples , and the previous reports of self - recombination , it is surprising that there are so few reports of mosaic genes containing tet(m ) .", "furthermore , alignment of 12 representative rpp gene sequences shows tet(m ) sharing 75% and 70% identity , respectively , to tet(o ) and tet(44 ) , which is higher than the percentage identity observed between the more commonly reported rpp mosaic genes comprising tet(o ) , ( w ) and ( 32 ) ( table  2 ) . however , mosaic genes comprising tet(m ) and any other gene , with the exception of tet(s ) , have yet to be reported .", "it is entirely possible that this may be due to a lack of investigation rather than an absence of recombination followed by fixation of the recombinant allele in the bacterial population .", "alternatively , it is possible that there is little selective pressure for tet(m)-based mosaic genes if the resultant protein is no more efficient than tet(m ) itself and/or there is no indirect selective pressure for mosaicism .", "reported that the protein encoded by the tet(o / w / o ) mosaic genes in m. elsdenii conferred a higher level of resistance to tetracycline than their non - mosaic counterparts , but similar investigations are still to be reported for other rpp genes .", "therefore , the prevalence of certain mosaic gene variants could suggest that they are in some way more beneficial to the host than the non - mosaic genes they comprise . \n", "table  1.a summary of the mosaic tetracycline genes reported to dategeneorganismsource(s)accession numberreference(s)tet(o / w)bifidobacterium thermophilum b0219environmental ( pig slaughterhouse ) sampleam88911832tet(o / w)b .", "faecesam88912232tet(o / w)-2megasphaera elsdenii 25 - 51swine faecesay48512218,27tet(o / w)-1 [ n  =  15]m .", "elsdenii 27 - 51swine faecesay48512627,33tet(o / w / o)-4uncultured bacterial clonepig faecesno accession number21tet(o / w / o)-3 [ n  =  9]uncultured bacterial clonepig", "elsdenii 14 - 14swine caecumay19692013,18,27,33tet(o / w / o)-1 [ n  =  2]m .", "elsdenii 7 - 11swine caecumay19692113,18,27tet(o / w/32/o ) [ n  =  32]uncultured bacterial clonepig faecesef06552321tet(o / w/32/o ) [ n  =  7]streptococcus suis ss1303pig ( brain , lung and spleen ) and human ( csf )", "suisdiseased pig ( blood , brain , heart , joint and lung ) samplesjq74005328tet(o / w/32/o / w / o)lactobacillus johnsonii g41human faecesdq52502332tet(o / w/32/o / w / o)uncultured bacterial clonepig faecesdq67992621tet(o/32/o)s", ". suisdiseased pig ( blood , brain , heart , joint and lung ) samplesjq74005228tet(o/32/o)clostridium saccharolyticum k10human colonaj29523818tet(o/32/o)-2 [ n  =  3]uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)-3uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)-4uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)-5uncultured bacterial clonehuman and animal faecal samplesno accession number21tet(o/32/o)dorea longicatena agr2136rumen microbiomenz_aujs01000017 ( 41  62643  545 bp)direct submission , analysed in this studytet(o/32/o)campylobacter coli 202/04human faecesainh01000038 ( 23614280 bp)direct submission , analysed in this studytet(o/32/o)c .", "coli 317/04human faecesnz_ainj01000054 ( 20944013 bp)direct submission , analysed in this studytet(o/32/o)campylobacter jejuni subspecies jejuni 2008 - 894humanaioq01000025 ( 14  51516  434 bp)direct submission , analysed in this studytet(o/32/o)roseburia intestinalis xb6b4human intestinal tractfp929050 ( 2  873  8142  875  733 bp)direct submission , analysed in this studytet(s / m)streptococcus equinus 1357foodhm36771125tet(s / m)streptococcus intermediushuman isolateay53432623,24tet(w/32/o)b .", "thermophilum b0256pig faecesam71060532the number given in square brackets indicates the instances of that mosaic gene variant reported , if more than one.fourteen of the 15 tet(o / w)-1 variants were only determined by pcr analysis and so could be either tet(o", "/ w)-2.eleven of the 28 tet(o / w / o)-2 variants were only determined by pcr analysis and so could be either tet(o / w / o)-2 or tet(o / w / o)-1.all s. suis isolates , but not the same strain . \n", "table  2.sequence identity matrix showing the percentage nucleotide identity between representatives of all 12 rpp gene classes , in descending order , compared with tet(m)rpp genetet(m)tet(s)tet(o)tet(44)tet(32)tet(w)tet(t)tet(36)tet(q)tetb(p)otr(a)tettet(m)1007875706964574946231111tet(s)10070696762565648111110tet(o)100696965564948151211tet(44)1007164505846151110tet(32)10067554947111210tet(w)10012451551412tet(t)10057561882tet(36)1006491111tet(q)100131212tetb(p)10011otr(a)10063tet100accession numbers of representative genes included in the matrix : tet(m ) , u09422 ; tet(o ) , y07780 ; tetb(p ) , ae001437 ; tet(q ) , x58717 ; tet(s ) , x92946 ; tet(t ) , l42544 ; tet(w ) , aj222769 ; tet(32 ) , dq647324 ; tet(36 ) , aj514254 ; tet(44 ) , fn594949 ; otr(a ) , x53401 ; tet , al939106.shaded boxes represent those genes currently reported to comprise mosaic genes . \n", "the coded bars indicate sequences of high identity to specific rpp genes : vertical line bars for tet(m ) , white bars for tet(o ) , grey bars for tet(s ) , black bars for tet(w ) and checked bars for tet(32 ) .", "indicates those sequences that are incomplete or absent in genbank , with the crossover points taken from the publication", ". indicates sequences that have been analysed in this study due to no specific crossover point(s ) reported .", "a summary of the mosaic tetracycline genes reported to date the number given in square brackets indicates the instances of that mosaic gene variant reported , if more than one .", "fourteen of the 15 tet(o / w)-1 variants were only determined by pcr analysis and so could be either tet(o", "eleven of the 28 tet(o / w / o)-2 variants were only determined by pcr analysis and so could be either tet(o / w / o)-2 or tet(o / w / o)-1 .", "sequence identity matrix showing the percentage nucleotide identity between representatives of all 12 rpp gene classes , in descending order , compared with tet(m ) accession numbers of representative genes included in the matrix : tet(m ) , u09422 ; tet(o ) , y07780 ; tetb(p ) , ae001437 ; tet(q ) , x58717 ; tet(s ) , x92946 ; tet(t ) , l42544 ; tet(w ) , aj222769 ; tet(32 ) , dq647324 ; tet(36 ) , aj514254 ; tet(44 ) , fn594949 ; otr(a ) , x53401 ; tet , al939106 .", "the coded bars indicate sequences of high identity to specific rpp genes : vertical line bars for tet(m ) , white bars for tet(o ) , grey bars for tet(s ) , black bars for tet(w ) and checked bars for tet(32 ) .", "indicates those sequences that are incomplete or absent in genbank , with the crossover points taken from the publication", ". indicates sequences that have been analysed in this study due to no specific crossover point(s ) reported ."], ["stanton and humphrey describe an assay that distinguished between the non - mosaic genes tet(o ) and tet(w ) and the mosaic tet(o / w / o ) from megasphaera strains , enabling them to detect tet(o / w / o ) variants in six additional m. elsdenii strains .", "patterson et al . investigated the presence of mosaic genes using various specific oligonucleotide sets that either bound within the resistance genes or flanked them .", "amplicons specific to tet(o / w ) , tet(o/32 ) and tet(w/32 ) were detected in faecal samples , with tet(o/32 ) being the most common of these mosaic amplicons ; it was amplified in all 12 pig faecal samples and 6 of 7 human faecal samples tested .", "in contrast , the faecal samples from cows and sheep , as well as human saliva samples , failed to produce any amplicons for these mosaic genes , suggesting they were not present at detectable levels .", "chen et al . also used an oligonucleotide primer set that annealed outside tet(o ) to determine the presence of tetracycline resistance genes in two streptococcus suis isolates .", "although no amplicon was produced using internal , tet(o)-specific primers , the primers binding to flanking dna yielded an amplicon , indicating the presence of mosaic genes [ identified as tet(o/32/o ) and tet(o / w/32/o ) ] .", "this full - length oligonucleotide primer set does aid the identification of mosaic genes ; however , it is only specific for those with regions homologous to tet(o ) flanking sequences . since pcr strategies aimed at identifying resistance genes require knowledge of the sequence of the target , mosaic rpp genes are likely to be largely undetected and under - reported by pcr - based studies .", ", almost all the mosaic genes reported to date have originated from faecal samples , with the majority identified from a porcine origin and less commonly from humans ( table  1 ) .", "the gut houses a complex and diverse bacterial community with potential for widespread horizontal gene transfer , and the mosaic genes found in faecal samples are likely to reflect the pool of non - mosaic genes present within the gut microbiota .", "genes such as tet(w ) and tet(o ) are commonly reported from these types of samples , but the prevalence of tet(32)-containing mosaic genes suggests that tet(32 ) may be more common than initially thought .", "in fact , tet(o/32/o ) was found to be the most common mosaic gene in both the human and pig faecal samples tested and was present in almost as many samples tested as the non - mosaic tet(o ) and tet(w ) genes .", "in contrast , mosaic genes have not yet been reported in faecal samples from bovine and ovine origin or in human saliva . why they are predominantly found in pigs while as yet unreported in other animals", "is not immediately clear , though the extensive use of tetracyclines in the swine industry may have contributed to their selection ."], ["the advent of high - throughput genomic sequencing has led to an increase in the number of genomes being deposited in sequence databases .", "many contain tetracycline resistance genes that are generically labelled simply as  tetracycline resistance protein or as  tet(m)-like , the designation of which may be a result of automated annotation pipelines . a preliminary search of the ncbi nucleotide database , using tet(o ) ( accession number y07780 ) as the query , found that some of these generically labelled tetracycline resistance genes gave a partial match to tet(o ) .", "further examination indicates that some are as yet uncharacterized and unreported mosaic genes , which have been further defined for this review using the nucleotide sequence to determine the crossover points .", "for example , the tet(m)-like gene ( accession number nz_aujs01000017 , location 41  62643  545 bp ) in the draft genome of dorea longicatena agr2136 from a human faecal sample appears to be a previously unreported variant of tet(o/32/o ) ( figure  1 ) .", "furthermore , the tetracycline resistance genes present in campylobacter jejuni subspecies jejuni 2008 - 894 , campylobacter coli 202/04 , c. coli 317/04 ( accession numbers aioq01000025 , ainh01000038 and nz_ainj01000054 , respectively ) and roseburia intestinalis xb6b4 ( accession number fp929050 ) are also structurally novel variants of tet(o/32/o ) ( figure  1 ) . the three mosaic genes present in the campylobacter spp .", "taking into account these newly defined genes , the total number of mosaic genes reported increases from 30 to 35 ( not including those identified via pcr amplification only ; table  1 ) and suggests that other generically labelled tetracycline resistance genes present in the database [ e.g. those labelled as tet(m)-like ] could be further classified , helping to understand mosaic gene proliferation and diversity ."], ["our knowledge of the mosaic rpp gene group is steadily increasing since their discovery in 2003 , with the majority derived from tet(o ) , tet(w ) and tet(32 ) and others deriving from tet(m ) and tet(s ) .", "it is clear that these genes are being under - reported both in terms of experimental detection and also within genomic data .", "further work and increased attention on mosaic rpp genes is important if we are to understand the evolutionary selective pressures driving their fixation in bacterial populations and the subsequent effects on resistance and mobile genetic element evolution within their host ."], [""]]}
{"article_id": "PMC4318834", "article_text": ["follicular thyroid cancer ( ftc ) metastasizes most commonly to the lungs and non - cranial bones .", "skull and skin are uncommon sites and usually manifest well after the diagnosis of primary malignancy .", "metastasis to skull and skin as the presenting feature of ftc is infrequently reported in the literature .", "a 65-year - old caucasian woman with a history of thyroid nodule presented with the complaint of rapidly growing skull nodules which had been present for 3 years but were stable previously .", "she denied any history of smoking or head and neck irradiation . on physical examination ,", "she had two non - tender gray cystic lesions  one on her left temporal region and the other on the right parietal region .", "magnetic resonance imaging of the brain demonstrated 7.13.8 cm and 3.74.5 cm fairly homogeneous , enhancing , relatively well - defined masses centered in the posterior and left anterior lateral calvarium with intracranial and extracranial extensions but without any vasogenic edema or mass effect on the brain .", "histopathological studies of the thyroid gland revealed a well - differentiated ftc in the left lobe .", "she did not have any recurrence of the ftc or metastases during the follow - up period and will be receiving radioactive iodine treatment .", "bone and lung are the common sites of metastasis from ftc , but involvement of skull or skin is unusual , particularly as the presenting feature .", "metastases from ftc should be in the differential of patients with new osteolytic hypervascular skull lesions or cutaneous lesions in head and neck area .", "a 65-year - old caucasian woman with a history of thyroid nodule presented to dermatology clinic with the complaint of rapidly growing skull nodules .", "she reported that the nodules had been present for 3 years but had been stable , so she had not sought medical attention .", "she denied any fevers , chills , pain , or redness over the nodules , fatigue , weight loss , recent infections , or history of trauma .", "she admitted to drinking alcohol on social occasions but denied any history of smoking or head and neck irradiation .", "her family history was positive for leukemia in her father and melanoma in her mother .", "she indicated a remote history of thyroid nodule which she reported was benign on biopsy . on physical examination , she had two non - tender gray cystic lesions  one on her left temporal region measuring about 5 cm in diameter and the other on the right parietal region measuring about 7 cm in diameter .", "laboratory tests revealed a normal tsh of 0.52 iu / ml ( reference range 0.35.0 iu / ml ) and t4 of 0.68 ng / dl ( reference range 0.581.64 ng / dl ) .", "a complete metabolic panel including serum creatinine , bun , and hepatic panel was normal .", "biopsy was attempted under local anesthesia which revealed possible extension to the bone with vascular origin and hence the patient was referred to surgery for debulking . during surgery , total excision of the nodules could not be performed as there appeared to be skull erosion underneath .", "an immunohistochemical panel showed positive staining for ck7 , thyroglobulin , and hbme-1 while it was weakly positive for pankeratin , suggesting a thyroid origin of these lesions , that is , metastatic ftc ( fig .", "two large extra - axial enhancing masses with both intra- and extracranial components were seen ( figs . 2 , 3 ) . magnetic resonance imaging ( mri ) of the brain demonstrated 7.13.8 cm and 3.74.5 cm fairly homogeneous , enhancing , relatively well - defined masses centered in the posterior and left anterior lateral calvarium with intracranial and extracranial extensions but without any vasogenic edema or mass effect on the brain ( fig .", "4 ) . positron emission tomography ( pet ) or ct of the skull demonstrated the two skull lesions to be lytic .", "two foci of hypermetabolic activity were also seen in the thyroid gland in the left lobe and thyroid isthmus .", "thyroid ultrasound showed numerous nodules in both lobes , the largest measuring up to 2.5 cm in greatest dimension .", "ct head with and without contrast showing a large , round , relatively smoothly marginated enhancing extra - axial left frontal mass ( 4.4 cm in anteroposterior diameter and 4.1 cm in transverse diameter ) that has eroded and partially destroyed the calvarium and extends into the subcutaneous scalp .", "ct head with and without contrast demonstrating a large , round , relatively smoothly marginated enhancing extra - axial mass ( measuring 6.8 cm in transverse diameter , 5.9 cm in ap diameter ) involving the right and left parietal region , straddling the posterior falx cerebri and superior sagittal sinus .", "mri brain revealing a large 7.13.8 cm fairly homogeneous enhancing relatively well - defined mass centered in the posterior calvarium with intracranial extension into the extra - axial space as well as extracranial extension .", "there is a similar appearing 3.74.5 cm fairly homogeneous relatively well - defined enhancing mass centered in the left anterior lateral calvarium also extending into the extra - axial space as well as extracranially .", "histopathological studies of the thyroid gland revealed a well - differentiated ftc in the left lobe , pathological stage pt2nxm1 , a 1-cm focus of classic papillary thyroid cancer in the right lobe of thyroid , with a background of multinodular goiter ( fig .", "she underwent intravascular embolization of the feeding blood vessels of her metastatic skull lesions from the external carotid system .", "then she underwent resection of the tumor in multiple stages with resection of the posterior and left frontal tumor , and calvaria , followed by cranioplasty .", "although she had other unrelated postoperative complications , she did not have any recurrence of the ftc or metastases during the follow - up period , had a normal thyroglobulin of 4.9 ng / ml ( reference range 2.840.9 ng / ml ) , and will be receiving radioactive iodine treatment .", "it typically presents as a thyroid nodule , either noted by the patient or the physician on routine physical examination or incidentally on routine imaging ( 1 ) .", "vascular invasion is characteristic for follicular carcinoma accounting for more common distant metastasis ( 5 ) .", "these metastases occur in 1015% of patients with ftc , with lung and bone being the commonest sites of involvement .", "bone metastases from ftc tend to be multiple and often involve sternum , ribs , and vertebrae ( 3 ) .", "skull metastases are uncommon with recent decline in incidence because of early detection and treatment of thyroid cancer ; among thyroid carcinomas , these metastases tend to occur more commonly in ftc and have a female preponderance ( 68 ) .", "there were only 12 reported cases of skull metastases out of 473 patients of thyroid cancer in one study , accounting for 2.5% of cases ( 8) .", "likewise cutaneous metastases from dtc are uncommon with less than 30 reported cases of cutaneous metastases in the english literature until 2010 ( 9 ) .", "these cutaneous metastases are more common in head and neck region and can manifest in a variety of histological appearances ( 1012 ) .", "however , in most of these cases metastases occurred long after the diagnosis and institution of treatment for ftc and it is extremely unusual to encounter skull and cutaneous metastases as the presenting feature of ftc ( 3 ) .", "skull metastases from ftc most commonly present as a slow - growing soft , painless usually hemispheric and singular lump in the occipital region ( 6 , 7 , 12 ) .", "unusual presentations include headaches , hemiparesis , exophthalmos , cranial nerve dysfunction , and altered consciousness ( 13 ) .", "these skull lesions are osteolytic on skull x - ray , and ct scans generally show homogeneous lesions with density slightly greater than the brain and a highly vascular appearance on angiographic assessment with blood supply most commonly from external carotid system ( 7 , 14 ) . in patients with scalp lesions , pet / ct can be used to determine the biopsy site ( 5 ) . in patients with established diagnosis of ftc with no documented metastases , evaluation of possible metastatic lesions can be done with ( 131)i single photon emission ct / ct [ ( 131)i - spect / ct ] ( 15 ) .", "( 99m)tc - mibi scan has been reported to be a highly sensitive technique for the detection of dtc metastases that have lost the capability to uptake ( 131)i ; the combined ( 99m)tc - mibi scintigraphy and serum thyroglobulin ( tg ) estimation appear to be an alternative method of radioiodine imaging in cases with dtc and elevated tg ( 16 ) .", "bone metastases uncommonly respond to radioactive iodine therapy and are associated with poor prognosis ( 7 ) .", "surgical approach should be considered as one of the treatments of choice for bone metastasis , if possible , and curative resection of solitary bone metastasis is associated with improved survival ( 7 ) .", "bone defects often require extensive bone resection , bleeding is often profuse during surgical resection , and deaths have been reported from extreme hemorrhage ( 1 , 4 , 13 , 14 ) . because of the life - threatening nature of such hemorrhages , preoperative angiographic assessment of these lesions with prophylactic ligation or embolization of feeding vessels is recommended ( 14 ) . when surgical excision is not possible , internal radiation with i-131", "is recommended ; external radiation is generally reserved for cases where uptake of i-131 by metastatic foci is poor ( 8) .", "thyroid hormone should be administered after complete excision of thyroid gland to suppress endogenous tsh from promoting tumor growth ( 17 ) .", "frequent follow - up examination is recommended and monitoring thyroglobulin measurement can be useful during follow - up ( 13 ) .", "ftc is thought to have the most optimistic prognosis even with metastases to the lymph nodes and lung", ". however , when combined with distant , especially widespread , metastases , the quality of life is compromised and the overall survival rate significantly decreases ( 2 ) . in one case series , the mean time from the diagnosis of thyroid tumor until discovery of skull metastasis was 23.3 years ( 8) .", "prognosis in case of metastasis is generally poor and the 10-year survival with bone metastases from dtc is reported to be 27% ( 7 ) .", "however , mean survival time of 4.5 years was reported with skull metastases in one case series , suggesting even worse prognosis and warranting an aggressive and multidisciplinary approach in this subset of patients ( 5 ) .", "a regular follow - up is crucial in these patients for early detection and management of any residual or recurrent metastatic ftc ( 6 ) .", "bone and lung are the common sites of metastasis from ftc but involvement of skull is unusual , especially as the presenting feature .", "metastases from ftc should be included in the differential of patients with new oteolytic hypervascular skull lesions or cutaneous lesions in head and neck area ( 7 , 14 ) .", "the authors have not received any funding or benefits from industry or elsewhere to conduct this study ."], "abstract_text": ["<S> backgroundfollicular thyroid cancer ( ftc ) metastasizes most commonly to the lungs and non - cranial bones . </S>", "<S> skull and skin are uncommon sites and usually manifest well after the diagnosis of primary malignancy . </S>", "<S> metastasis to skull and skin as the presenting feature of ftc is infrequently reported in the literature.case presentationa 65-year - old caucasian woman with a history of thyroid nodule presented with the complaint of rapidly growing skull nodules which had been present for 3 years but were stable previously . </S>", "<S> she denied any fevers , chills , history of trauma , or weight loss . </S>", "<S> she denied any history of smoking or head and neck irradiation . on physical examination , </S>", "<S> she had two non - tender gray cystic lesions  one on her left temporal region and the other on the right parietal region . </S>", "<S> biopsy was consistent with metastatic ftc . </S>", "<S> magnetic resonance imaging of the brain demonstrated 7.13.8 cm and 3.74.5 cm fairly homogeneous , enhancing , relatively well - defined masses centered in the posterior and left anterior lateral calvarium with intracranial and extracranial extensions but without any vasogenic edema or mass effect on the brain . </S>", "<S> thyroid ultrasound showed numerous nodules in both lobes . </S>", "<S> the patient underwent a total thyroidectomy . </S>", "<S> histopathological studies of the thyroid gland revealed a well - differentiated ftc in the left lobe . </S>", "<S> then she underwent resection of the tumor in multiple stages . </S>", "<S> she did not have any recurrence of the ftc or metastases during the follow - up period and will be receiving radioactive iodine treatment.conclusionbone and lung are the common sites of metastasis from ftc , but involvement of skull or skin is unusual , particularly as the presenting feature . </S>", "<S> metastases from ftc should be in the differential of patients with new osteolytic hypervascular skull lesions or cutaneous lesions in head and neck area . </S>"], "labels": null, "section_names": ["Background", "Case presentation", "Conclusion", "Case presentation", "Discussion", "Conclusion", "Conflict of interest and funding"], "sections": [["follicular thyroid cancer ( ftc ) metastasizes most commonly to the lungs and non - cranial bones .", "skull and skin are uncommon sites and usually manifest well after the diagnosis of primary malignancy .", "metastasis to skull and skin as the presenting feature of ftc is infrequently reported in the literature ."], ["a 65-year - old caucasian woman with a history of thyroid nodule presented with the complaint of rapidly growing skull nodules which had been present for 3 years but were stable previously .", "she denied any history of smoking or head and neck irradiation . on physical examination ,", "she had two non - tender gray cystic lesions  one on her left temporal region and the other on the right parietal region .", "magnetic resonance imaging of the brain demonstrated 7.13.8 cm and 3.74.5 cm fairly homogeneous , enhancing , relatively well - defined masses centered in the posterior and left anterior lateral calvarium with intracranial and extracranial extensions but without any vasogenic edema or mass effect on the brain .", "histopathological studies of the thyroid gland revealed a well - differentiated ftc in the left lobe .", "she did not have any recurrence of the ftc or metastases during the follow - up period and will be receiving radioactive iodine treatment ."], ["bone and lung are the common sites of metastasis from ftc , but involvement of skull or skin is unusual , particularly as the presenting feature .", "metastases from ftc should be in the differential of patients with new osteolytic hypervascular skull lesions or cutaneous lesions in head and neck area ."], ["a 65-year - old caucasian woman with a history of thyroid nodule presented to dermatology clinic with the complaint of rapidly growing skull nodules .", "she reported that the nodules had been present for 3 years but had been stable , so she had not sought medical attention .", "she denied any fevers , chills , pain , or redness over the nodules , fatigue , weight loss , recent infections , or history of trauma .", "she admitted to drinking alcohol on social occasions but denied any history of smoking or head and neck irradiation .", "her family history was positive for leukemia in her father and melanoma in her mother .", "she indicated a remote history of thyroid nodule which she reported was benign on biopsy . on physical examination , she had two non - tender gray cystic lesions  one on her left temporal region measuring about 5 cm in diameter and the other on the right parietal region measuring about 7 cm in diameter .", "laboratory tests revealed a normal tsh of 0.52 iu / ml ( reference range 0.35.0 iu / ml ) and t4 of 0.68 ng / dl ( reference range 0.581.64 ng / dl ) .", "a complete metabolic panel including serum creatinine , bun , and hepatic panel was normal .", "biopsy was attempted under local anesthesia which revealed possible extension to the bone with vascular origin and hence the patient was referred to surgery for debulking . during surgery , total excision of the nodules could not be performed as there appeared to be skull erosion underneath .", "an immunohistochemical panel showed positive staining for ck7 , thyroglobulin , and hbme-1 while it was weakly positive for pankeratin , suggesting a thyroid origin of these lesions , that is , metastatic ftc ( fig .", "two large extra - axial enhancing masses with both intra- and extracranial components were seen ( figs . 2 , 3 ) . magnetic resonance imaging ( mri ) of the brain demonstrated 7.13.8 cm and 3.74.5 cm fairly homogeneous , enhancing , relatively well - defined masses centered in the posterior and left anterior lateral calvarium with intracranial and extracranial extensions but without any vasogenic edema or mass effect on the brain ( fig .", "4 ) . positron emission tomography ( pet ) or ct of the skull demonstrated the two skull lesions to be lytic .", "two foci of hypermetabolic activity were also seen in the thyroid gland in the left lobe and thyroid isthmus .", "thyroid ultrasound showed numerous nodules in both lobes , the largest measuring up to 2.5 cm in greatest dimension .", "ct head with and without contrast showing a large , round , relatively smoothly marginated enhancing extra - axial left frontal mass ( 4.4 cm in anteroposterior diameter and 4.1 cm in transverse diameter ) that has eroded and partially destroyed the calvarium and extends into the subcutaneous scalp .", "ct head with and without contrast demonstrating a large , round , relatively smoothly marginated enhancing extra - axial mass ( measuring 6.8 cm in transverse diameter , 5.9 cm in ap diameter ) involving the right and left parietal region , straddling the posterior falx cerebri and superior sagittal sinus .", "mri brain revealing a large 7.13.8 cm fairly homogeneous enhancing relatively well - defined mass centered in the posterior calvarium with intracranial extension into the extra - axial space as well as extracranial extension .", "there is a similar appearing 3.74.5 cm fairly homogeneous relatively well - defined enhancing mass centered in the left anterior lateral calvarium also extending into the extra - axial space as well as extracranially .", "histopathological studies of the thyroid gland revealed a well - differentiated ftc in the left lobe , pathological stage pt2nxm1 , a 1-cm focus of classic papillary thyroid cancer in the right lobe of thyroid , with a background of multinodular goiter ( fig .", "she underwent intravascular embolization of the feeding blood vessels of her metastatic skull lesions from the external carotid system .", "then she underwent resection of the tumor in multiple stages with resection of the posterior and left frontal tumor , and calvaria , followed by cranioplasty .", "although she had other unrelated postoperative complications , she did not have any recurrence of the ftc or metastases during the follow - up period , had a normal thyroglobulin of 4.9 ng / ml ( reference range 2.840.9 ng / ml ) , and will be receiving radioactive iodine treatment ."], ["it typically presents as a thyroid nodule , either noted by the patient or the physician on routine physical examination or incidentally on routine imaging ( 1 ) .", "vascular invasion is characteristic for follicular carcinoma accounting for more common distant metastasis ( 5 ) .", "these metastases occur in 1015% of patients with ftc , with lung and bone being the commonest sites of involvement .", "bone metastases from ftc tend to be multiple and often involve sternum , ribs , and vertebrae ( 3 ) .", "skull metastases are uncommon with recent decline in incidence because of early detection and treatment of thyroid cancer ; among thyroid carcinomas , these metastases tend to occur more commonly in ftc and have a female preponderance ( 68 ) .", "there were only 12 reported cases of skull metastases out of 473 patients of thyroid cancer in one study , accounting for 2.5% of cases ( 8) .", "likewise cutaneous metastases from dtc are uncommon with less than 30 reported cases of cutaneous metastases in the english literature until 2010 ( 9 ) .", "these cutaneous metastases are more common in head and neck region and can manifest in a variety of histological appearances ( 1012 ) .", "however , in most of these cases metastases occurred long after the diagnosis and institution of treatment for ftc and it is extremely unusual to encounter skull and cutaneous metastases as the presenting feature of ftc ( 3 ) .", "skull metastases from ftc most commonly present as a slow - growing soft , painless usually hemispheric and singular lump in the occipital region ( 6 , 7 , 12 ) .", "unusual presentations include headaches , hemiparesis , exophthalmos , cranial nerve dysfunction , and altered consciousness ( 13 ) .", "these skull lesions are osteolytic on skull x - ray , and ct scans generally show homogeneous lesions with density slightly greater than the brain and a highly vascular appearance on angiographic assessment with blood supply most commonly from external carotid system ( 7 , 14 ) . in patients with scalp lesions , pet / ct can be used to determine the biopsy site ( 5 ) . in patients with established diagnosis of ftc with no documented metastases , evaluation of possible metastatic lesions can be done with ( 131)i single photon emission ct / ct [ ( 131)i - spect / ct ] ( 15 ) .", "( 99m)tc - mibi scan has been reported to be a highly sensitive technique for the detection of dtc metastases that have lost the capability to uptake ( 131)i ; the combined ( 99m)tc - mibi scintigraphy and serum thyroglobulin ( tg ) estimation appear to be an alternative method of radioiodine imaging in cases with dtc and elevated tg ( 16 ) .", "bone metastases uncommonly respond to radioactive iodine therapy and are associated with poor prognosis ( 7 ) .", "surgical approach should be considered as one of the treatments of choice for bone metastasis , if possible , and curative resection of solitary bone metastasis is associated with improved survival ( 7 ) .", "bone defects often require extensive bone resection , bleeding is often profuse during surgical resection , and deaths have been reported from extreme hemorrhage ( 1 , 4 , 13 , 14 ) . because of the life - threatening nature of such hemorrhages , preoperative angiographic assessment of these lesions with prophylactic ligation or embolization of feeding vessels is recommended ( 14 ) . when surgical excision is not possible , internal radiation with i-131", "is recommended ; external radiation is generally reserved for cases where uptake of i-131 by metastatic foci is poor ( 8) .", "thyroid hormone should be administered after complete excision of thyroid gland to suppress endogenous tsh from promoting tumor growth ( 17 ) .", "frequent follow - up examination is recommended and monitoring thyroglobulin measurement can be useful during follow - up ( 13 ) .", "ftc is thought to have the most optimistic prognosis even with metastases to the lymph nodes and lung", ". however , when combined with distant , especially widespread , metastases , the quality of life is compromised and the overall survival rate significantly decreases ( 2 ) . in one case series , the mean time from the diagnosis of thyroid tumor until discovery of skull metastasis was 23.3 years ( 8) .", "prognosis in case of metastasis is generally poor and the 10-year survival with bone metastases from dtc is reported to be 27% ( 7 ) .", "however , mean survival time of 4.5 years was reported with skull metastases in one case series , suggesting even worse prognosis and warranting an aggressive and multidisciplinary approach in this subset of patients ( 5 ) .", "a regular follow - up is crucial in these patients for early detection and management of any residual or recurrent metastatic ftc ( 6 ) ."], ["bone and lung are the common sites of metastasis from ftc but involvement of skull is unusual , especially as the presenting feature .", "metastases from ftc should be included in the differential of patients with new oteolytic hypervascular skull lesions or cutaneous lesions in head and neck area ( 7 , 14 ) ."], ["the authors have not received any funding or benefits from industry or elsewhere to conduct this study ."]]}
{"article_id": "PMC4327199", "article_text": ["although water - bath polymerization is extensively used to process polymethylmethacrylate ( pmma ) , new resins and processing methods are often proposed to obtain better physical and esthetic properties and simplify the technique1,6,8,11,14 .", "one of the advantages of microwave and visible light polymerization methods is the shorter processing time they offer in comparison to water bath .", "in addition , light - polymerized resins can be processed directly in the mouth using a portable light - curing device , which simplifies the clinical procedures9 .", "while denture base acrylic resins polymerized by microwave irradiation and conventional water bath curing systems are composed of pmma6,8 , visible light - polymerized resins are similar to composites , having an organic instead of an inorganic filler content .", "the material is composed of a urethane dimethacrylate matrix plus small amounts of microfine silica .", "the filler consists of acrylic resin beads ( pmma ) of varying sizes that become part of an interpenetrating polymer network .", "light polymerization occurs by exposure to quartz halogen lamps in the shorter blue 400 to 500 nm wavelength spectrum of visible light .", "visible light - polymerized resins were initially proposed for clinical repairs and/or additions in sub - extended removable dentures7 , but due to their easy manipulation and fast polymerization , their are now indicated for many clinical situations , such as transitional and interim dentures , complete and removable partial dentures , provisional splints , denture repairs and additions , orthodontic appliances and record bases5,7,9 .", "although light - polymerized resins have been used in removable partial dentures , it is not clear whether the presence of a metal framework could interfere with their polymerization , by possibly reflecting the light and affecting important physical properties , such as surface roughness and hardness .", "these properties are largely investigated because they indicate polymerization characteristics4,7,11,14 and might influence plaque accumulation later on 2,3,10 .", "thus , the aims of this study were to investigate whether metal interferes with the surface hardness and roughness of a visible light - polymerized acrylic resin and to compare these values to those of water - bath- and microwave - polymerized resins .", "the resins used in this study are listed in table 1 . twelve disc - shaped specimens ,", "30 mm in diameter and 4 mm thick , were fabricated for each resin according to the manufacturers ' directions .", "all specimens were made using a silicone ( optosil , heraeus kulzer , hanau , germany ) matrix ( 30  0.05 mm in diameter and 4  0.05 mm thick ) .", "the matrix was flasked in type iii dental stone ( herodent soli - rock ; vigodent , rio de janeiro , rj , brazil ) using standard metal dental flasks ( uraby ; dlc , so paulo , sr brazil ) or plastic flasks ( onda cryl ; artigo odontolgicos clssico ltd , so paulo , sp , brazil ) for water bath and microwave polymerization , respectively .", "after the gypsum had completely set , the flasks were opened and the matrixes were removed .", "a separating medium ( al - cote , dentsply ltd , rio de janeiro brazil ) was applied to the exposed areas of the mold . before packing the resin ,", "a rectangular ( 28 mm  8 mm  0.5 mm ) insert of co - cr alloy bar ( degussa , hanau , germany ) was centered at the bottom of each mold . as the size of the metal bars was almost the same as that of the mold cavities , once positioned at the bottom of each mold", "microwave acrylic resin specimens were polymerized in a microwave oven ( continental aw-42 , with 2,450 hz frequency and 900w maximum potency ; bosch , manaus , am , brazil ) , according to the manufacturer 's directions ( 3 min at 360 w ; 4 min resting and 3 min at 810 w ) .", "specimens polymerized by hot water bath were processed in an automatic polymerization unit ( termotron p-100 ; termotron piracicaba , sp , brazil ) at 74c for 9 h. once processed , all flasks were allowed to bench cooling for 2h .", "the resin samples were ground with water - cooled 320- , 400- , 600- and 1200-grit silicon carbide papers ( carbimet ; buehler , lake bluff , il , usa ) in a polishing machine ( arotec apl-4 ; so paulo , sp , brazil ) followed by polishing with cloths and 1-m diamond suspension ( metadi diamond suspension ; buehler ) .", "next , they were ultra - sound cleaned ( thornton - inpec eletrnica ltda , vinhedo , sp , brazil ) for 2 min and then immersed in distilled water at 37c for 12 h to release residual monomers6,12,14 .", "light - polymerized resin specimens were prepared using a transparent matrix , consisting of an acrylic resin plate containing a circular hole measuring 30  0.05 mm in diameter and 4  0.05 mm thick .", "first , a co - cr framework was placed at the bottom of the matrix and then the light - polymerized resin was inserted in the matrix .", "this assembly was put into a light box ( edg lux ; edg equipamentos e controles , so paulo , sp , brazil ) containing four 75 w halogen lamps .", "the resin was polymerized for 6 min , according to manufacturer 's instructions . after processing , the same finishing , polishing and cleaning procedures used for heat and microwave polymerized acrylic resins", "surface roughness was determined using a mechanical profilometer with a 2-m radius tip under a measuring force of 0.7 mn and accurate to 0.01 m ( surfcorder se 1700 , kosaka , tokyo , japan ) calibrated at sample length of 0.25 mm , spread of 2.0 mm and speed of 0.5 mms .", "six readings were performed on each specimen and an average ( ra ) was determined .", "these profilometric traces were taken from the edge of specimen , in the middle and at its bottom.12 \n  knoop hardness was assessed using a microhardness tester ( shimadzu hmv-2000 , shimadzu corporation , kyoto , japan ) .", "knoop penetrations were made on the acrylic surface of each sample at distances of 50 , 100 , 200 , 400 and 800 m from the co - cr metal bar ( figure 2 ) .", "knoop hardness means were analyzed by two - way anova with 2 factors : acrylic resins and distances .", "the acrylic resin groups were compared using tukey test and distances were compared by kruskal - wallis test .", "means and standard deviations for acrylic resin surface roughness m ) are presented in table 2 . the visible light - polymerized resin ( triad ) showed the highest means ( p<0.05 ) , but the metal alloy did not interfere with surface roughness .", ". means and standard deviations for knoop hardness ( kg / mm ) of acrylic resins at several distances from the metal are described in table 3 .", "comparisons between acrylic resins showed that the visible light - polymerized resin ( triad ) exhibited significantly higher means ( p<0.05 ) .", "however , no differences in hardness values at several distances from the metal alloy were found .", "mean followed by different letters are statistically different ( p<0.05 ) lowercase letters indicate differences between acrylic resins and uppercase letters indicate differences between distances from metal .", "this technique is considered to be time - saving as it does not require cast inclusion , is non - toxic and biocompatible .", "it is therefore indicated for using in many clinical situations , such as complete and removable partial dentures9 .", "however , little is known about the influence of metal alloys on the polymerization process and on important physical properties , such as hardness and roughness . in this study ,", "a visible light - polymerized resin was compared to microwave- and heat - polymerized acrylic resins , which have similar composition and surface characteristics6,8 .", "smooth surfaces do not readily retain food debris , epithelial cells and bacteria , thus facilitating oral hygiene , reducing the risk of plaque formation and preventing negative effects on periodontal tissues3 .", "therefore , the roughness of intraoral surfaces helps determining their colonization by different microorganisms because retention preferably occurs on rough surfaces , as they provide protection against shear forces2 .", "acrylic resin roughness is dependent on the polishing method used2 . in this study , polishing was done with 360- , 400- , 600- and 1000-grit abrasive papers , resulting in roughness values below 0.2 m , which is considered clinically acceptable for preventing plaque accumulation2 . although the same polishing procedure was carried out for all resins , higher roughness means were found in the light - polymerized resin group ( 0.11  0.02 m ) .", "this result may be explained by the large amounts of inorganic compounds in this resin , which were probably exposed during polishing procedures , causing irregularities on the resin surface .", "the surface roughness means of microwave- ( onda - cryl ) and water - bath - polymerized ( clssico ) resins did not differ statistically ( p>0.05 ) , probably because of the similarity of their composition .", "12 ( 2004 ) , who investigated the influence of denture cleansers on the surface roughness of heat- and microwave - polymerized resins . however , the findings of the present are in contrast with those of ulusoy et al.15 , who reported higher roughness means ( 0.31 m ) .", "it is likely that these differences can be due to the polishing method used in each study because ulusoy et al.15 polished the specimens on a bench lathe using a roller brush with pumice / water paste , followed by a wet polishing wheel and a chalk .", "in contrast , in the present study , the specimens were polished with materials of decreasing granulation,(up to 1-m diamond particles ) .", "the other two resins ( onda cryl and clssico ) , which do not have inorganic compounds , had lower roughness .", "hardness is a property used to predict the wear resistance of a material and its ability to abrade opposing dental structures1 .", "therefore , surface scratching can be determined by its knoop hardness because its weakness explains why the surface is at risk of roughening during professional cleaning or even during habitual oral hygiene procedures10 .", "this test can also be used to verify the efficacy of polymerization . in this study , it was used to assess resin polymerization around a metal alloy .", "the visible light - polymerized resin presented the highest knoop hardness , which is in accordance with khan et al . 7 ( 1987 ) , who compared triad ( dentsply inc . ) hardness with that of a water - bath - polymerized resin .", "this could be explained by the inorganic content of the light - polymerized resin ( silica)9 , which increased the resistance of this resin to the microhardness indenter1 .", "water - bath - and microwave - polymerized resins had similar knoop hardness means , which are in agreement with many authors4,11,12,14 .", "no differences between 50 , 100 , 200 , 400 and 800 m distances from the metal were found in any of the acrylic resins evaluated ( p>0.05 ) .", "the absence of significant differences suggests that light emission and polymerization processes were not affected by the presence of metal .", "these results are in consistent with those of braun et al . 4 ( 1998 ) , who compared water ", "bath- and microwave - polymerized resins with respect to the hardness means at pre - determined distances from a metal alloy . in this study , roughness and knoop", "hardness results indicated that triad has clinically acceptable properties and its polymerization was not affected by the presence of metal .", "however , further studies about the clinical behavior of visible light - polymerized acrylic resins should be conducted .", "within the limitations of this in vitro study , it may be concluded that the presence of a metal framework did not interfere with the roughness and knoop hardness of the tested visible light - polymerized ( triad ) , microwave - polymerized ( onda cryl ) and water - bath - polymerized ( clssico ) acrylic resins , indicating that all materials can be used in removable partial dentures ."], "abstract_text": ["<S> although visible light - polymerized acrylic resins have been used in removable partial dentures , it is not clear whether the presence of a metal framework could interfere with their polymerization , by possibly reflecting the light and affecting important properties , such as roughness and hardness , which would consequently increase biofilm accumulation . </S>", "<S> the aim of this study was to compare the roughness and knoop hardness of a visible light - polymerized acrylic resin and to compare these values to those of water - bath- and microwave - polymerized resins , in the presence of a metal framework . </S>", "<S> thirty - six specimens measuring 30.0  4.0  0.5 mm of a microwave- ( onda cryl ) , a visible light- ( triad ) and a water - bath- polymerized ( clssico ) ( control ) acrylic resins containing a cobalt - chromium metal bar were prepared . </S>", "<S> after processing , specimens were ground with 360 to 1000-grit abrasive papers in a polishing machine , followed by polishing with cloths and 1m diamond particle suspension . roughness was evaluated using a profilometer ( surfcorder se 1700 ) and knoop hardness ( kg / mm2 ) was assayed using a microhardness tester ( shimadzu hmv 2000 ) at distances of 50 , 100 , 200 , 400 and 800 m from the metal bar . </S>", "<S> roughness and knoop hardness means were submitted to two - way anova and compared by tukey and kruskal wallis tests at a 5% significance level statistically significant differences were found ( p<0.05 ) for roughness and knoop hardness , with light - polymerized resin presenting the highest values ( ra = 0.11 m and hardness between 20.2 and 21.4 kg / mm2 ) . </S>", "<S> knoop values at different distances from the metal bar did not differ statistically ( p>0.05 ) . within the limitations of this in vitro study , it was concluded that the presence of metal did not influence roughness and hardness values of any of the tested acrylic resins . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["although water - bath polymerization is extensively used to process polymethylmethacrylate ( pmma ) , new resins and processing methods are often proposed to obtain better physical and esthetic properties and simplify the technique1,6,8,11,14 .", "one of the advantages of microwave and visible light polymerization methods is the shorter processing time they offer in comparison to water bath .", "in addition , light - polymerized resins can be processed directly in the mouth using a portable light - curing device , which simplifies the clinical procedures9 .", "while denture base acrylic resins polymerized by microwave irradiation and conventional water bath curing systems are composed of pmma6,8 , visible light - polymerized resins are similar to composites , having an organic instead of an inorganic filler content .", "the material is composed of a urethane dimethacrylate matrix plus small amounts of microfine silica .", "the filler consists of acrylic resin beads ( pmma ) of varying sizes that become part of an interpenetrating polymer network .", "light polymerization occurs by exposure to quartz halogen lamps in the shorter blue 400 to 500 nm wavelength spectrum of visible light .", "visible light - polymerized resins were initially proposed for clinical repairs and/or additions in sub - extended removable dentures7 , but due to their easy manipulation and fast polymerization , their are now indicated for many clinical situations , such as transitional and interim dentures , complete and removable partial dentures , provisional splints , denture repairs and additions , orthodontic appliances and record bases5,7,9 .", "although light - polymerized resins have been used in removable partial dentures , it is not clear whether the presence of a metal framework could interfere with their polymerization , by possibly reflecting the light and affecting important physical properties , such as surface roughness and hardness .", "these properties are largely investigated because they indicate polymerization characteristics4,7,11,14 and might influence plaque accumulation later on 2,3,10 .", "thus , the aims of this study were to investigate whether metal interferes with the surface hardness and roughness of a visible light - polymerized acrylic resin and to compare these values to those of water - bath- and microwave - polymerized resins ."], ["the resins used in this study are listed in table 1 . twelve disc - shaped specimens ,", "30 mm in diameter and 4 mm thick , were fabricated for each resin according to the manufacturers ' directions .", "all specimens were made using a silicone ( optosil , heraeus kulzer , hanau , germany ) matrix ( 30  0.05 mm in diameter and 4  0.05 mm thick ) .", "the matrix was flasked in type iii dental stone ( herodent soli - rock ; vigodent , rio de janeiro , rj , brazil ) using standard metal dental flasks ( uraby ; dlc , so paulo , sr brazil ) or plastic flasks ( onda cryl ; artigo odontolgicos clssico ltd , so paulo , sp , brazil ) for water bath and microwave polymerization , respectively .", "after the gypsum had completely set , the flasks were opened and the matrixes were removed .", "a separating medium ( al - cote , dentsply ltd , rio de janeiro brazil ) was applied to the exposed areas of the mold . before packing the resin ,", "a rectangular ( 28 mm  8 mm  0.5 mm ) insert of co - cr alloy bar ( degussa , hanau , germany ) was centered at the bottom of each mold . as the size of the metal bars was almost the same as that of the mold cavities , once positioned at the bottom of each mold", "microwave acrylic resin specimens were polymerized in a microwave oven ( continental aw-42 , with 2,450 hz frequency and 900w maximum potency ; bosch , manaus , am , brazil ) , according to the manufacturer 's directions ( 3 min at 360 w ; 4 min resting and 3 min at 810 w ) .", "specimens polymerized by hot water bath were processed in an automatic polymerization unit ( termotron p-100 ; termotron piracicaba , sp , brazil ) at 74c for 9 h. once processed , all flasks were allowed to bench cooling for 2h .", "the resin samples were ground with water - cooled 320- , 400- , 600- and 1200-grit silicon carbide papers ( carbimet ; buehler , lake bluff , il , usa ) in a polishing machine ( arotec apl-4 ; so paulo , sp , brazil ) followed by polishing with cloths and 1-m diamond suspension ( metadi diamond suspension ; buehler ) .", "next , they were ultra - sound cleaned ( thornton - inpec eletrnica ltda , vinhedo , sp , brazil ) for 2 min and then immersed in distilled water at 37c for 12 h to release residual monomers6,12,14 .", "light - polymerized resin specimens were prepared using a transparent matrix , consisting of an acrylic resin plate containing a circular hole measuring 30  0.05 mm in diameter and 4  0.05 mm thick .", "first , a co - cr framework was placed at the bottom of the matrix and then the light - polymerized resin was inserted in the matrix .", "this assembly was put into a light box ( edg lux ; edg equipamentos e controles , so paulo , sp , brazil ) containing four 75 w halogen lamps .", "the resin was polymerized for 6 min , according to manufacturer 's instructions . after processing , the same finishing , polishing and cleaning procedures used for heat and microwave polymerized acrylic resins", "surface roughness was determined using a mechanical profilometer with a 2-m radius tip under a measuring force of 0.7 mn and accurate to 0.01 m ( surfcorder se 1700 , kosaka , tokyo , japan ) calibrated at sample length of 0.25 mm , spread of 2.0 mm and speed of 0.5 mms .", "six readings were performed on each specimen and an average ( ra ) was determined .", "these profilometric traces were taken from the edge of specimen , in the middle and at its bottom.12 \n  knoop hardness was assessed using a microhardness tester ( shimadzu hmv-2000 , shimadzu corporation , kyoto , japan ) .", "knoop penetrations were made on the acrylic surface of each sample at distances of 50 , 100 , 200 , 400 and 800 m from the co - cr metal bar ( figure 2 ) .", "knoop hardness means were analyzed by two - way anova with 2 factors : acrylic resins and distances .", "the acrylic resin groups were compared using tukey test and distances were compared by kruskal - wallis test ."], ["means and standard deviations for acrylic resin surface roughness m ) are presented in table 2 . the visible light - polymerized resin ( triad ) showed the highest means ( p<0.05 ) , but the metal alloy did not interfere with surface roughness .", ". means and standard deviations for knoop hardness ( kg / mm ) of acrylic resins at several distances from the metal are described in table 3 .", "comparisons between acrylic resins showed that the visible light - polymerized resin ( triad ) exhibited significantly higher means ( p<0.05 ) .", "however , no differences in hardness values at several distances from the metal alloy were found .", "mean followed by different letters are statistically different ( p<0.05 ) lowercase letters indicate differences between acrylic resins and uppercase letters indicate differences between distances from metal ."], ["this technique is considered to be time - saving as it does not require cast inclusion , is non - toxic and biocompatible .", "it is therefore indicated for using in many clinical situations , such as complete and removable partial dentures9 .", "however , little is known about the influence of metal alloys on the polymerization process and on important physical properties , such as hardness and roughness . in this study ,", "a visible light - polymerized resin was compared to microwave- and heat - polymerized acrylic resins , which have similar composition and surface characteristics6,8 .", "smooth surfaces do not readily retain food debris , epithelial cells and bacteria , thus facilitating oral hygiene , reducing the risk of plaque formation and preventing negative effects on periodontal tissues3 .", "therefore , the roughness of intraoral surfaces helps determining their colonization by different microorganisms because retention preferably occurs on rough surfaces , as they provide protection against shear forces2 .", "acrylic resin roughness is dependent on the polishing method used2 . in this study , polishing was done with 360- , 400- , 600- and 1000-grit abrasive papers , resulting in roughness values below 0.2 m , which is considered clinically acceptable for preventing plaque accumulation2 . although the same polishing procedure was carried out for all resins , higher roughness means were found in the light - polymerized resin group ( 0.11  0.02 m ) .", "this result may be explained by the large amounts of inorganic compounds in this resin , which were probably exposed during polishing procedures , causing irregularities on the resin surface .", "the surface roughness means of microwave- ( onda - cryl ) and water - bath - polymerized ( clssico ) resins did not differ statistically ( p>0.05 ) , probably because of the similarity of their composition .", "12 ( 2004 ) , who investigated the influence of denture cleansers on the surface roughness of heat- and microwave - polymerized resins . however , the findings of the present are in contrast with those of ulusoy et al.15 , who reported higher roughness means ( 0.31 m ) .", "it is likely that these differences can be due to the polishing method used in each study because ulusoy et al.15 polished the specimens on a bench lathe using a roller brush with pumice / water paste , followed by a wet polishing wheel and a chalk .", "in contrast , in the present study , the specimens were polished with materials of decreasing granulation,(up to 1-m diamond particles ) .", "the other two resins ( onda cryl and clssico ) , which do not have inorganic compounds , had lower roughness .", "hardness is a property used to predict the wear resistance of a material and its ability to abrade opposing dental structures1 .", "therefore , surface scratching can be determined by its knoop hardness because its weakness explains why the surface is at risk of roughening during professional cleaning or even during habitual oral hygiene procedures10 .", "this test can also be used to verify the efficacy of polymerization . in this study , it was used to assess resin polymerization around a metal alloy .", "the visible light - polymerized resin presented the highest knoop hardness , which is in accordance with khan et al . 7 ( 1987 ) , who compared triad ( dentsply inc . ) hardness with that of a water - bath - polymerized resin .", "this could be explained by the inorganic content of the light - polymerized resin ( silica)9 , which increased the resistance of this resin to the microhardness indenter1 .", "water - bath - and microwave - polymerized resins had similar knoop hardness means , which are in agreement with many authors4,11,12,14 .", "no differences between 50 , 100 , 200 , 400 and 800 m distances from the metal were found in any of the acrylic resins evaluated ( p>0.05 ) .", "the absence of significant differences suggests that light emission and polymerization processes were not affected by the presence of metal .", "these results are in consistent with those of braun et al . 4 ( 1998 ) , who compared water ", "bath- and microwave - polymerized resins with respect to the hardness means at pre - determined distances from a metal alloy . in this study , roughness and knoop", "hardness results indicated that triad has clinically acceptable properties and its polymerization was not affected by the presence of metal .", "however , further studies about the clinical behavior of visible light - polymerized acrylic resins should be conducted ."], ["within the limitations of this in vitro study , it may be concluded that the presence of a metal framework did not interfere with the roughness and knoop hardness of the tested visible light - polymerized ( triad ) , microwave - polymerized ( onda cryl ) and water - bath - polymerized ( clssico ) acrylic resins , indicating that all materials can be used in removable partial dentures ."]]}
{"article_id": "PMC3884910", "article_text": ["there is limited data upon skin cancer screening program in western asia region and especially iran .", "the two most common variants are basal cell carcinoma ( bcc ) and squamous cell carcinoma ( scc ) ; regardless of their low mortality rates , these tumors can provoke severe consequence as a result of their treatment . the third most frequent skin cancer type , malignant melanoma ( mm ) , has a more destructive behavior and accordingly a poor prognosis ; malignant melanoma accounts for approximately 75% of all deaths from skin cancer .", "skin cancer screening includes the complete cutaneous examination and 2 - 3 minute visual inspection of the skin .", "there is strong data that whole - body clinical skin examination reduces the incidence of thick melanoma and , as a result , screening would reduce melanoma mortality . in us and", "however , some studies debate on the effective and beneficial method of skin cancer screening as mass screening or high risk group screening and the real impact of skin cancer screening result on the society health . in iran , there are limited studies about prevalence and incidence of skin cancers .", "therefore , this study was planned as a pilot one - week skin cancer screening campaign in tehran , iran to evaluate its profit and failure and design further large - scale screening program more definitely .", "this study was planned as a skin cancer screening campaign in the public health centers of shahid beheshti medical university in a one - week period in tehran , iran .", "we applied for the collaboration of the public health centers in tehran as the location of this screening program .", "the usual services provided in these centers include prenatal and pediatric care , women health , vaccination , health education , environmental or occupational health , etc . ,", "due to distribution of these centers in different areas of tehran , we discussed with the health deputy of the university for the collaboration of these health centers in this screening campaign and finally , thirty one public health centers were selected in different areas of tehran .", "physicians and health staffs of these health centers were volunteered to cooperate in this study .", "due to cultural and religious belief in our country , some female participants might be reluctant to be examined by male physicians . in this regard , we select a male physician and female health staff or vice versa in each center to avoid any limitation in examination of the female participants .", "practical information upon skin cancer risk factors and its early symptoms or signs along with any needed guideline of this screening project provided to them in a two - day course by an expert board certified dermatologist . an illustrated educational pamphlet in the case of the risk factors especially sun exposure , ways of prevention and early detection of skin cancer published and distributed in the health centers for general patient education .", "we invited all people over 40 years old to the mentioned health centers for complete skin examination in a one - week period on october 2008 .", "we emphasized in these pamphlets and brochures that any bizarre or asymmetric skin lesion especially with change in shape , color and size might be important for screening .", "moreover , it has been advocated that any skin erosion or ulcer remained unimproved after one month should be examined by physicians .", "the participants would have undergone the whole - body skin examination by the trained physicians or health staffs .", "any lesion with bizarre or asymmetric shape , uneven color , increasing size , resistant ulcers or erosions considered as suspicious .", "patients with any suspected lesions were referred to the specialized dermatology clinics of shahid beheshti university of medical sciences for further evaluation of their lesions .", "all the participants were instructed about the study , and they signed the informed consent forms .", "a board certified dermatologist performed the whole - body skin examination for the referred patients and further evaluation particularly skin biopsy was done whenever needed .", "the skin examination was done by visual inspection and use of hand lens in the setting of proper lighting .", "the alarming signs including bizarre or asymmetric shape , uneven color , increasing size , resistant ulcers or erosions were considered for decision to perform skin biopsy .", "we could not use dermatoscope because of some limitation in the facilities . in the case of confirmed malignancies", ", the suitable treatment would be provided for the patients in the dermatology ward or referred to the proper department .", "all the data registered and the related questionnaires were completed . after gathering the data , statistical analysis was performed using the statistical software spss 16.0.0 .", "in a one - week period of screening , 1314 patients , 194 males ( 14.8% ) and 1120 females ( 85.2% ) , with mean age of 51.81  10.28 years were attended the allied health centers and underwent the whole - body skin examination . regarding the considerable attendance of female volunteers , most of them had indoor occupation especially being housewives .", "we had the data of the number of melanocytic nevi in 92.9% of the participants .", "25% of them had less than 3 melanocytic lesions and 25% had more than 10 lesions .", "less than one percent of the participant had the positive personal history of skin cancer .", "we have found the personal history of non - skin cancers in 14 ( 1.1% ) of participants . these were ovarian , prostate , breast and gastrointestinal carcinoma .", "physicians found suspected lesions in 182 ( 13.85% ) of participants [ 142 ( 78% ) females and 40 ( 22% ) males ] with possible diagnosis of bcc , scc or malignant melanoma which were located mostly on the head , neck and trunk [ table 1 ] .", "these participants with suspected lesions were referred to the allied dermatology clinics of the university .", "all these patients underwent the whole - body skin examination by a board certified dermatologist and skin biopsy performed in 115 patients for the histopathological examination .", "table 2 shows the final diagnosis of suspected malignant skin lesions referred from the health centers .", "clinical diagnosis of suspected lesions by primary physician final diagnosis of the suspected lesions the diagnosis of skin cancer was finally confirmed by histopathology evaluation in 15 ( 1.14% ) patients out of 1314 volunteers who participated in this skin cancer screening program [ figures 1 and 2 ] .", "these malignancies include 10 ( 0.76% ) cases of bcc , 2 ( 0.15% ) cases of scc and 3 ( 0.23% ) cases of malignant melanoma .", "two patients with bcc had positive personal history of breast and gastrointestinal cancers but no family history of skin and non - skin cancer found in these patients .", "thirty six ( 2.74% ) cases of dysplastic nevus were also diagnosed in this study . in the case of confirmed malignancies , the proper treatment provided for the patients in the dermatology ward or surgery department .", "a morphoeic basal cell carcinoma found on the eyebrow of a middle - age man squamous cell carcinoma on the ear in an elderly man we found no significant difference in the number of common moles , multiple freckling , family history of skin and non - skin cancers between skin - cancer diagnosed patients and other participants . outdoor occupations and personal history of non - skin cancers was significantly higher in skin cancer diagnosed patients ( p < 0.05 , chi - square test ) .", "early diagnosis of skin cancer is very crucial to improve the prognosis , for best treatment results , and increase the quality of life of the patient . in this regard , there are many studies upon skin cancer screening with valuable results upon the importance of early detection of skin malignancies . however , there is no strong evidence to establish whether there is a decrease in mortality due to regular physical examination of the skin . in this study", ", we find 15 cases ( 10 bbc , 2 scc and 2 malignant melanoma ) of skin cancer .", "most of the participants in this campaign were housewives women and this could be due to the working hours of the public health centers that caused less attendance of male participants or those with outdoor jobs . if we could extend the active daily hours of screening to the evening , we could probably detect more skin cancers . regarding the high population of tehran", ", it would better screen more people or extend the campaign period to avoid this selection or estimating bias .", "however , this campaign was planned as pilot skin cancer screening program in tehran and its results and limitation could help us to design the large - scale screening program more definitely .", "there are different methods of skin cancer screening including population - based or targeted screening .", "planning of a population - based or mass skin cancer screening program needed considerable cooperation of different sectors including dermatology or health departments and it need substantial facilities for the project setting , announcement , training of the volunteered health staff , their remunerations and further diagnostic or treatment measure for the participants .", "well - trained physicians should be employed in order to reduce the false positive diagnosis and it may have great financial impact on the health care system . moreover , the current evidence seems to be insufficient to assess the balance of benefits and harms of mass screening for skin cancer .", "therefore , it might be more practical to perform the targeted screening programs for the high - risk groups such as patients with organ transplantation or those with the history of skin cancer or outdoor occupations .", "the media campaign increased the population awareness of skin cancers , promoted effective sun protection and make known the dangers of excessive uv - exposures .", "more educational activities for both patients and physicians regarding the skin cancer risk factors and their early signs and symptoms might be more beneficial and cost - effective to decrease the skin cancer mortality and morbidity . to the best of our knowledge , there was no similar study in iran to perform skin cancer screening and this study provides us useful information upon the profit and failure of a screening program .", "another limitation of this study was lack of comparison between different methods of skin cancer screening such as targeted or mass screening to provide more exact data upon the efficacy and cost - benefit of different methods of skin cancer screening .", "therefore , further controlled study could be beneficial to elucidate the more practical and cost - effective method of skin cancer screening in our country .", "this study gives us many practical clues to plan and perform large - scale skin cancer screening better ."], "abstract_text": ["<S> background : early detection of skin cancers by screening could be very beneficial to decrease their morbidity or mortality . </S>", "<S> there is limited study about skin cancer screening in iran.aim:this essay was planned as a pilot skin cancer screening campaign in tehran , iran to evaluate its profit and failure and further design large - scale screening program more definitely.materials and methods : thirty one public health centers of shahid beheshti medical university were selected in different areas of tehran . </S>", "<S> the project was announced via media and invited all the people above 40 years old to come for the whole - body skin examination in a one - week period . </S>", "<S> patients with any suspected lesions were referred to the dermatology clinics of the university.results:1314 patients , 194 males ( 14.8% ) and 120 females ( 85.2% ) , with mean age of 51.81  10.28 years participated in this screening campaign . </S>", "<S> physicians found suspected lesions in 182 ( 13.85% ) of participants . </S>", "<S> the diagnosis of skin cancer was confirmed in 15 ( 1.14% ) patients . </S>", "<S> these malignancies included 10 ( 0.76% ) cases of basal cell carcinoma , 2 ( 0.15% ) cases of squamous cell carcinoma and 3 ( 0.23% ) cases of malignant melanoma.conclusion:skin cancer screening seems to be valuable to detect skin malignancies in their early course . regarding the considerable amount of facilities needed to perform skin cancer screening program , it might be more beneficial to perform the targeted screening programs for the high - risk groups or emphasis more on public education of skin cancer risk factors and their early signs . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion"], "sections": [["there is limited data upon skin cancer screening program in western asia region and especially iran .", "the two most common variants are basal cell carcinoma ( bcc ) and squamous cell carcinoma ( scc ) ; regardless of their low mortality rates , these tumors can provoke severe consequence as a result of their treatment . the third most frequent skin cancer type , malignant melanoma ( mm ) , has a more destructive behavior and accordingly a poor prognosis ; malignant melanoma accounts for approximately 75% of all deaths from skin cancer .", "skin cancer screening includes the complete cutaneous examination and 2 - 3 minute visual inspection of the skin .", "there is strong data that whole - body clinical skin examination reduces the incidence of thick melanoma and , as a result , screening would reduce melanoma mortality . in us and", "however , some studies debate on the effective and beneficial method of skin cancer screening as mass screening or high risk group screening and the real impact of skin cancer screening result on the society health . in iran , there are limited studies about prevalence and incidence of skin cancers .", "therefore , this study was planned as a pilot one - week skin cancer screening campaign in tehran , iran to evaluate its profit and failure and design further large - scale screening program more definitely ."], ["this study was planned as a skin cancer screening campaign in the public health centers of shahid beheshti medical university in a one - week period in tehran , iran .", "we applied for the collaboration of the public health centers in tehran as the location of this screening program .", "the usual services provided in these centers include prenatal and pediatric care , women health , vaccination , health education , environmental or occupational health , etc . ,", "due to distribution of these centers in different areas of tehran , we discussed with the health deputy of the university for the collaboration of these health centers in this screening campaign and finally , thirty one public health centers were selected in different areas of tehran .", "physicians and health staffs of these health centers were volunteered to cooperate in this study .", "due to cultural and religious belief in our country , some female participants might be reluctant to be examined by male physicians . in this regard , we select a male physician and female health staff or vice versa in each center to avoid any limitation in examination of the female participants .", "practical information upon skin cancer risk factors and its early symptoms or signs along with any needed guideline of this screening project provided to them in a two - day course by an expert board certified dermatologist . an illustrated educational pamphlet in the case of the risk factors especially sun exposure , ways of prevention and early detection of skin cancer published and distributed in the health centers for general patient education .", "we invited all people over 40 years old to the mentioned health centers for complete skin examination in a one - week period on october 2008 .", "we emphasized in these pamphlets and brochures that any bizarre or asymmetric skin lesion especially with change in shape , color and size might be important for screening .", "moreover , it has been advocated that any skin erosion or ulcer remained unimproved after one month should be examined by physicians .", "the participants would have undergone the whole - body skin examination by the trained physicians or health staffs .", "any lesion with bizarre or asymmetric shape , uneven color , increasing size , resistant ulcers or erosions considered as suspicious .", "patients with any suspected lesions were referred to the specialized dermatology clinics of shahid beheshti university of medical sciences for further evaluation of their lesions .", "all the participants were instructed about the study , and they signed the informed consent forms .", "a board certified dermatologist performed the whole - body skin examination for the referred patients and further evaluation particularly skin biopsy was done whenever needed .", "the skin examination was done by visual inspection and use of hand lens in the setting of proper lighting .", "the alarming signs including bizarre or asymmetric shape , uneven color , increasing size , resistant ulcers or erosions were considered for decision to perform skin biopsy .", "we could not use dermatoscope because of some limitation in the facilities . in the case of confirmed malignancies", ", the suitable treatment would be provided for the patients in the dermatology ward or referred to the proper department .", "all the data registered and the related questionnaires were completed . after gathering the data , statistical analysis was performed using the statistical software spss 16.0.0 ."], ["in a one - week period of screening , 1314 patients , 194 males ( 14.8% ) and 1120 females ( 85.2% ) , with mean age of 51.81  10.28 years were attended the allied health centers and underwent the whole - body skin examination . regarding the considerable attendance of female volunteers , most of them had indoor occupation especially being housewives .", "we had the data of the number of melanocytic nevi in 92.9% of the participants .", "25% of them had less than 3 melanocytic lesions and 25% had more than 10 lesions .", "less than one percent of the participant had the positive personal history of skin cancer .", "we have found the personal history of non - skin cancers in 14 ( 1.1% ) of participants . these were ovarian , prostate , breast and gastrointestinal carcinoma .", "physicians found suspected lesions in 182 ( 13.85% ) of participants [ 142 ( 78% ) females and 40 ( 22% ) males ] with possible diagnosis of bcc , scc or malignant melanoma which were located mostly on the head , neck and trunk [ table 1 ] .", "these participants with suspected lesions were referred to the allied dermatology clinics of the university .", "all these patients underwent the whole - body skin examination by a board certified dermatologist and skin biopsy performed in 115 patients for the histopathological examination .", "table 2 shows the final diagnosis of suspected malignant skin lesions referred from the health centers .", "clinical diagnosis of suspected lesions by primary physician final diagnosis of the suspected lesions the diagnosis of skin cancer was finally confirmed by histopathology evaluation in 15 ( 1.14% ) patients out of 1314 volunteers who participated in this skin cancer screening program [ figures 1 and 2 ] .", "these malignancies include 10 ( 0.76% ) cases of bcc , 2 ( 0.15% ) cases of scc and 3 ( 0.23% ) cases of malignant melanoma .", "two patients with bcc had positive personal history of breast and gastrointestinal cancers but no family history of skin and non - skin cancer found in these patients .", "thirty six ( 2.74% ) cases of dysplastic nevus were also diagnosed in this study . in the case of confirmed malignancies , the proper treatment provided for the patients in the dermatology ward or surgery department .", "a morphoeic basal cell carcinoma found on the eyebrow of a middle - age man squamous cell carcinoma on the ear in an elderly man we found no significant difference in the number of common moles , multiple freckling , family history of skin and non - skin cancers between skin - cancer diagnosed patients and other participants . outdoor occupations and personal history of non - skin cancers was significantly higher in skin cancer diagnosed patients ( p < 0.05 , chi - square test ) ."], ["early diagnosis of skin cancer is very crucial to improve the prognosis , for best treatment results , and increase the quality of life of the patient . in this regard , there are many studies upon skin cancer screening with valuable results upon the importance of early detection of skin malignancies . however , there is no strong evidence to establish whether there is a decrease in mortality due to regular physical examination of the skin . in this study", ", we find 15 cases ( 10 bbc , 2 scc and 2 malignant melanoma ) of skin cancer .", "most of the participants in this campaign were housewives women and this could be due to the working hours of the public health centers that caused less attendance of male participants or those with outdoor jobs . if we could extend the active daily hours of screening to the evening , we could probably detect more skin cancers . regarding the high population of tehran", ", it would better screen more people or extend the campaign period to avoid this selection or estimating bias .", "however , this campaign was planned as pilot skin cancer screening program in tehran and its results and limitation could help us to design the large - scale screening program more definitely .", "there are different methods of skin cancer screening including population - based or targeted screening .", "planning of a population - based or mass skin cancer screening program needed considerable cooperation of different sectors including dermatology or health departments and it need substantial facilities for the project setting , announcement , training of the volunteered health staff , their remunerations and further diagnostic or treatment measure for the participants .", "well - trained physicians should be employed in order to reduce the false positive diagnosis and it may have great financial impact on the health care system . moreover , the current evidence seems to be insufficient to assess the balance of benefits and harms of mass screening for skin cancer .", "therefore , it might be more practical to perform the targeted screening programs for the high - risk groups such as patients with organ transplantation or those with the history of skin cancer or outdoor occupations .", "the media campaign increased the population awareness of skin cancers , promoted effective sun protection and make known the dangers of excessive uv - exposures .", "more educational activities for both patients and physicians regarding the skin cancer risk factors and their early signs and symptoms might be more beneficial and cost - effective to decrease the skin cancer mortality and morbidity . to the best of our knowledge , there was no similar study in iran to perform skin cancer screening and this study provides us useful information upon the profit and failure of a screening program .", "another limitation of this study was lack of comparison between different methods of skin cancer screening such as targeted or mass screening to provide more exact data upon the efficacy and cost - benefit of different methods of skin cancer screening .", "therefore , further controlled study could be beneficial to elucidate the more practical and cost - effective method of skin cancer screening in our country .", "this study gives us many practical clues to plan and perform large - scale skin cancer screening better ."]]}
{"article_id": "PMC4446916", "article_text": ["early failure of fistulas due to thrombosis or inadequate maturation is a barrier to increasing the prevalence of fistulas . despite the benefits of avfs as a form of vascular access in end stage renal disease ( esrd ) patients , considerable time", "is required for the fistula to mature and suitably develop into a functional format maturation rate after autogenous avf depends on various factors such as age , body mass index ( bmi ) , venous and arterial diameter , venous distensibility , and arterial elasticity .", "the main cause of avf failure is outflow stenosis as a result of vascular intimal hyperplasia and thrombosis .", "this formation of neointimal hyperplasia and thrombosis may be initiated by activated platelet function , endothelial cell injury and vascular smooth muscle cell proliferation , which may occur due to unphysiologic vascular anastomosis . in view of these postulated mechanisms of avf failure ,", "furthermore , only a few studies have addressed the safety of aspirin use after avf creation .", "van der linden et al . demonstrated that venous potential distensibility was significantly correlated with functional maturation , especially in the group of patients with vein diameter  2 mm .", "as a result of this study , they concluded that venous line distensibility was a better predictive factor than vein size .", "there is no report about the effects of combination therapy after avf creation using clopidogrel in combination with prostacycline analog as a potent vasodilator agent after autogenous avf creation .", "on the basis of these considerations , we performed a study to test the hypothesis that clopidogrel as an irreversible antiplatelet and oral prostacycline analog as an arterial vasodilator and additional antiaggregant drug may increase venous distensibility and arterial elasticity and would prevent primary avf failure in hemodialysis patients with diabetes mellitus .", "we excluded 289 patients [ table 1 ] , and 96 patients met the study criteria for enrollment .", "major exclusion criteria of the patients to detect the quality of the brachial artery ( ba ) and a venous line , doppler usg was used .", "preoperative venous and arterial spectral usg showed that there were no differences when we compared arterial and venous quality and diameter .", "the randomization was stratified according to the medical center with a permuted block scheme , with a block size of four and an equal allocation .", "eligible participants were called to the coordinating center to obtain a randomization protocol , which corresponded to a specific medication . neither the details of the randomization sequence nor the identity of the medication assigned was known to the participant or any personnel at the participating sites .", "the treatment was initiated 710 days prior to the scheduled access surgery and continued postoperatively .", "after randomization , the two groups of subjects are followed in exactly the same way , and the only difference between the care with which they receive drugs , outpatient visits and follow - up calls should be those intrinsic to the treatments being compared . in the first group of patients ,", "710 days prior to the surgery , we started clopidogrel ( 75 mg daily ) and an oral prostacycline analog ( 200 mg orally daily ) .", "patients in the control and study group were operated by two surgeons using the same technique and same suture materials ( 6/0 polypropylene suture ) . to provide the purse or narrowing effect of suture ,", "we noticed that the two surgeons operated on the same number of patients in both the groups .", "clopidogrel and prostacycline were administered orally prior to surgery , and continued during the year .", "data collection was performed at baseline , 4 weeks after fistula creation , and monthly thereafter until ascertainment of fistula suitability .", "study medication was discontinued when there was a sign of fistula thrombosis confirmed by vascular surgeons . in a normally developed fistula", ", there is a continuous thrill from the anastomosis all the way centrally along the outflow vein .", "the thrill gradually decreases in intensity from the anastomosis centrally . in the absence of a stenosis", "we defined the maturation of avf when the mean blood flow from avf was more than 300 ml / min .", "and a minimum arterial end diastolic velocity was 70 cm / s by sonographic examination for both groups .", "venous and arterial line diameters were calculated using sonography . if there was no sign of restriction of blood flow when the patients underwent hemodialysis , we described the hemodialysis access to be good .", "primary patency was defined as the interval from avf creation to abandonment due to thrombosis or low flow , inadequate for sufficient hemodialysis .", "early patency or failure was defined according to a thrill and/or bruit 4 weeks after the surgery .", "the antero - posterior diameter of ba was measured in the transverse dimension on gray scale sonography .", "peak systolic velocity and end - diastolic velocity of the ba were measured in a representative waveform in each patient [ figure 1 ] .", "the resistive index was a measure of the downstream flow resistance and was calculated as the difference between the respective measurements obtained after fist release ( post ) and during fist clenching ( pre ) .", "the primary outcome measure was final avf failure , which required the creation of a new avf .", "first , we investigated the need of re - intervention after avf due to thrombosis or occlusion during the follow - up period .", "suitability was defined as use of the fistula at a dialysis machine blood pump rate of 300 ml / min . or more during 8 of 12 dialysis sessions .", "doppler ultrasonography ( in a gray scale ) shows the brachial artery and its diameter in a patient with insulin - dependent diabetes mellitus and peak velocity the statistical program , spss version 13 ( spss , chicago , il ) was used to analyze the data .", "data are presented as mean  standard error for continuous variables and as percentages for categorical variables .", "the t - test was used when the means of two groups were compared . on the basis of intention - to - treat principles , all other participants were monitored according to the research protocol for whom study medications were continued .", "all hypothesis tests were conducted by using a significance level of 0.05 ( two - sided ) . for evaluation of the relationships between the avf maturation and independent variables ,", "differences between matured and failed groups were tested with the wilcoxon rank - sum test for continuous variables .", "multivariate cox proportional hazards models were used to determine factors associated with reduced avf patency .", "test results were presented as hazard ratios with 95% confidence intervals ( cis ) , and two - sided p < 0.05 was considered statistically significant .", "the statistical program , spss version 13 ( spss , chicago , il ) was used to analyze the data .", "data are presented as mean  standard error for continuous variables and as percentages for categorical variables .", "the t - test was used when the means of two groups were compared . on the basis of intention - to - treat principles , all other participants were monitored according to the research protocol for whom study medications were continued .", "all hypothesis tests were conducted by using a significance level of 0.05 ( two - sided ) . for evaluation of the relationships between the avf maturation and independent variables ,", "differences between matured and failed groups were tested with the wilcoxon rank - sum test for continuous variables .", "multivariate cox proportional hazards models were used to determine factors associated with reduced avf patency .", "test results were presented as hazard ratios with 95% confidence intervals ( cis ) , and two - sided p < 0.05 was considered statistically significant .", "the mean ages were 54.23  2.6 and 55.8  2.8 years in group 1 and 2 respectively ( p > 0.05 ) .", "there were no significant differences in covariables between groups when we compared patient characteristics , blood pressure , blood ldl , calcium , and phosphate levels [ table 2 ] .", "we did not detect any complication during the treatment period such as bleeding in group 1 patients .", "three patients complained from a temporary headache on the 1 day of oral prostacycline analog administration .", "all patients were taking study medication prior to surgery and did not have to be discontinued prematurely .", "four weeks after the surgery , avf maturation failure developed in 14 patients ( 30.4% ) in placebo group and in 4 patients ( 8% ) in clopidogrel and oral prostacycline analog group ( p = 0.001 ) . fistula maturation and survival has been confirmed using physical examinations and by a doppler usg every month for each patient .", "the kaplanmeier survival curve showed avf maturation and survival results in figure 2 [ table 3 ] .", "the mean maturation time of avf was 38  6.5 day and 53  12.8 day in clopidogrel plus oral prostacycline analog and placebo groups , respectively ( p = 0.023 ) .", "patients treated with clopidogrel and oral prostacycline analog had a increased avf survival ( p = 0.001 ) . in the placebo group , early avf failure was detected in two patients due to venous line thrombosis .", "characteristics of patients in the two groups demonstrates the survival rate of arteriovenous fistulas ( avfs ) during the study period in group 1 and group 2 .", "meier estimates of the cumulative incidence of loss of primary avfs patency in both groups .", "the median duration of patency was 5.8 months ( 95% confidence interval ( ci ) : 4.37.1 ) in the group 1 ( study group ) and 4.3 months ( 95% ci : 3.65.4 ) in the group 2 ( placebo group ) clinical outcomes at the end of the study the preoperative mean diameter of ba was 4.3  1.5 mm in clopidogrel plus oral prostacycline analog group and 4.04  1.3 mm in placebo group ( p = 0.94 ) .", "after surgery , usg showed that the mean diameter of ba was 6.3  1.4 mm and 4.74  0.9 mm in the study and placebo group respectively ( p = 0.002 ) .", "the preoperative mean diameter of the vein in the study and placebo groups were 3.1  0.8 and 3.04  0.7 mm ( p = 0.96 ) . in the postoperative period ,", "the corresponding values were 5.4  0.9 and 4.01  0.3 mm respectively ( p = 0.024 ) .", "blood flow from avf was 352  94 ml / min . in placebo group and 604  125 ml / min .", "doppler usg examination of avf showed that a minimum arterial end - diastolic velocity has been detected at 116  14 cm / s in group 1 , 3 months after the surgery .", "however , this value was 72  21 cm / s in the placebo group ( p = 0.036 ) .", "overall , the hazard ratio for primary avf failure was 0.82 ( 95% ci : 0.310.94 ) . during the follow - up period , from 4 weeks to 6 months , tenderness of the extremity ,", "edema , and hematoma occurred in nine patients in study group , and in six in placebo group .", "hemodialysis was successfully performed in 36 patients ( 72% ) in the study group and 22 patients ( 47.8% ) in the placebo group at the end of study ( p = 0.001 ) .", "the hemodialysis period in group 1 was significantly longer and more successful than in the placebo group ( p = 0.001 ) .", "multivariate logistic regression analysis revealed that avf maturation and survival rate was more than in study group ( odds ratio 2.27 ; 95% ci : 1.078.50 ; p = 0.001 ) .", "the survival and maturation time of avf in study group and placebo group has been summarized in figure 3(p = 0.001 ) .", "the flow diagram demonstrates the number of patients who were screened and selected for research [ figure 4 ] . during follow - up", "this graphic demonstrates the cumulative incidence for the percent of primary end points attributable to flow monitoring compared to total end points in each treatment group .", "end points due to flow monitoring in the group 1 ( study group ) and group 2 ( placebo - treated control group ) are compared to total end points demonstrates the comparison of the mean avfs ' flow diagram ( left columns ) and the differences of arterial end - diastolic velocity ( right columns ) between the groups . in the early period , we detected high blood flows and lower arterial velocity in study group when we compared groups no bleeding episode such as intracranial hemorrhage in gastrointestinal tract was recorded during the active treatment period .", "there were no differences between baseline and follow - up hematocrit values or changes in recombinant human erythropoietin doses during the study period for either group .", "in this prospective double - blind research , we presented our experiences of a new combination therapy and its efficacies on avf access after surgery in our esrd population . according to the study , clopidogrel in combination with oral prostacycline analog as an arterial vasodilator", "can be used to provide early avf maturation in esrdp with diabetes . according to previous reports , the size and quality of the arterial and venous access and distensibility after avf creation play important roles .", "therefore , we decided to use our combination therapy to provide more dilated and elastic arterial and venous access after avf creation .", "thus , blood flow from the fistula increased significantly in patients who were given a combination therapy than in patients who were given placebo .", "our study findings support the importance of distensibility and quality of vessel access . as we know that after avf creation , anastomotic or juxta - anastomotic stenosis is the most common cause of a fistula failing to mature .", "the stenosis most likely occurs because of operative trauma . in case of transposed vein fistulas ,", "the loss of vasa vasorum in the mobilized portion of the vein may also contribute to causation of stenosis .", "second , there is the element of neo - intimal hyperplasia . because of a large pressure gradient between arterial and venous circulation , blood accelerates while passing from the artery to the vein causing turbulent flow that is considered responsible for endothelial damage as a result of micro - trauma .", "in addition , unfortunately , some patients have a small arterial and/or venous access diameter .", "therefore , effective hemodialysis can be impossible due to occlusion and/or stenosis of the venous or arterial line .", "it is shown that venous distensibility is a major problem after avf creation . in van der", "'s study , it was demonstrated that venous distensibility was significantly correlated with avf maturation , especially in patients with vein diameter 2 mm .", "thus , if there is no suitable venous distensibility , avf maturation and survival will be a problem . according to this hypothesis", ", we believe that the effective vasodilatation can be required in these particular groups for early avf maturation and longer survival .", "there have been a few small trials evaluating pharmacological agents aimed at reducing the early thrombosis rate .. these trials utilized the antiplatelet agents , aspirin , sulphinpyrazone , and clopidogrel and varied in size from as few as 5 patients up to 250 patients .", "andrassy et al . compared patients given aspirin for 4 weeks with placebo ; the thrombosis rate in the aspirin group was 4% compared with the control group of 24% .", "in another study , kooistra et al . demonstrated that a low - dose aspirin could not prevent thrombovascular accidents in hemodialysis patients during treatment with recombinant human erythropoietin .", "'s study , it was demonstrated that there was a statistical significance when compared to fistula thrombosis rates in clopidogrel groups versus placebo groups ( 12% vs. 19% ) .", "studies using sulphinpyrazone showed variable results , but were underpowered with the largest study enrolling only a small number of patients .", "thus , although there is some data to suggest that antiplatelet agents may increase the primary patency of avf , the limited evidence base and the uncertainty regarding the choice of agent have not supported the widespread use of antiplatelet agents in the prevention of avf thrombosis . however , for the first time , our study showed the effects of clopidogrel in combination with oral prostacycline agent on autogenous avf creation .", "we know that autogenous avf is the preferred hemodialysis access and is associated with lower morbidity and mortality than avgs . however , 2060% of fistulas fail to mature sufficiently to support dialysis .", "thus , the high rate of maturation failure is a major unsolved problem , yet .", "the costs and morbidity problems due to avf maturation failure among hemodialysis patients are still reported worldwide .", "vascular access surgeons are faced with the same challenge all over the world  choosing the best vascular access type for chronic hemodialysis .", "size and quality of the vessels seem to be the important factors known . despite the experiences of avf in esrdp , considerable amount of time", "is required for the fistula to mature and suitably develop into a functional format . a number of authors and dember et al .", "have maintained that outflow venous stenosis and vascular intimal hyperplasia were the main causes of avf failure .", "avf thrombosis resulting from neointimal hyperplasia can be initiated by activated platelet function , endothelial cell injury , vascular smooth muscle cell proliferation , and unphysiologic vascular anastomosis . in view of these postulated mechanisms of avf failure ,", "possible clinical benefit of antiplatelet therapy has been proposed for prevention of avf failure . in particular , aspirin , the most widespread antiplatelet drug , has been evaluated as prophylaxis for avf failure in several studies , mainly among the use of avg . in a study ,", "the authors examined the efficacy of combination therapy of aspirin and clopidogrel for prevention of arteriovenous graft failure , but this study was stopped before completion because the prevalence of hemorrhagic complication was significantly higher in the intervention group . in the study group , only 28% of patients required re - intervention during the 1-year follow - up period .", "however , 52% of placebo group needed re - intervention due to avf failure . during the aspirin therapy ,", "the most important problem is hemorrhage in hemodialysis patients than in the general population because hemodialysis patients are more likely to have platelet dysfunction and they receive intermittent anticoagulant therapy during hemodialysis sessions .", "however , clopidogrel is tolerated by the patient and is not a cause of gastrointestinal bleeding .", "thrombosis or severe occlusion of autogenous avf can be seen within the first several weeks following surgical creation , or inadequate maturation of the vein .", "some of the previous studies have indicated that the frequency of avf failure can be reduced with antiplatelet agents ; but in other studies the authors maintained antiplatelet therapy did not affect avf maturation and survival .", "they also concluded that vessel dilation was not needed for adequate blood flow except at the smaller diameters .", "the successful results of our analysis suggest that to inhibit avf failure , daily administration of 75 mg oral clopidogrel and arterial prostacycline analog administration may be initiated 710 days prior to avf creation . in", "'s trial suggested that the use of antiplatelet was not associated with significant risk reduction in access failure .", "kaufman demonstrated no change in the risk of graft thrombosis with aspirin plus clopidogrel therapy .", "the author reported that in chronic hemodialysis patients there was a trend toward increased or did not change thrombosis rate of avf using aspirin therapy .", "in fiskerstrand et al . 's study , 2 out of 6 patients in the ticlopidine group compared with 5 out of 9 in the placebo group , developed fistulae thromboses in 1 month . in the previously published study by grntoft et al .", ", there were only 2 out of 19 patients who received treatment developed for fistulae thromboses compared to 8 out of 17 on placebo .", "the results of adequacy for avfs developed sufficiently to be used for dialysis in 46.5% of patients .", "allon and robbin have suggested that the higher adequacy rate could be provided in upper arm location and multidisciplinary team approach to vascular access by the surgeons , nurses , and radiologists .", "the arterial diameter , elasticity , and the venous line distensibility are the main factors of fistula maturation . in publications from dac consortium ,", "followed the patients for a long time ( approximately 4 years ) but we could follow the cases for a year only .", "our therapy model may be provided more distensible and large size venous line , and more elastic arterial access .", "our study differences may be related to surgical experiences , patients ' age , smoking history , bmi , patients ' arterial and venous diameters , and distensibility .", "multiple individuals are involved in the management of vascular access , including nephrologists , access surgeons , radiologists , dialysis nurses , and the patient .", "achieving optimal vascular access outcomes requires agreement on a common set of goals by all these individuals , close collaboration , and good communication .", "however , primary avf failure remains a major problem for hemodialysis patients worldwide . according to our study ,", "it is observed that prophylaxis of vascular access thrombosis needs to start earlier in the hemodialysis patients .", "clopidogrel and oral prostacycline analog , which begins 710 days prior to avf creation and continuing during follow - up period seems to be effective and safe , and inhibit primary avf failure with acceptable side effects in hemodialysis patients ."], "abstract_text": ["<S> vascular access is used as a lifeline for hemodialysis in patients with end stage renal disease failure ( esrd ) . </S>", "<S> failure of arteriovenous fistula ( avf ) maturation is still high . </S>", "<S> the purpose of this study was to research the effects of clopidogrel in combination with oral iloprost , a synthetic analog of prostacyclin pgi2 . </S>", "<S> ninety - six diabetic esrd patients were divided into two groups . in the first group ( group 1 , n = 50 ) , clopidogrel ( 75 mg daily dose ) and an oral prostacycline analog ( 200 mg daily dose ) were administered . in the second group ( group 2 , n = 46 ) , placebo was given . </S>", "<S> all patients took study medication 710 days prior to surgery . a doppler ultrasound ( usg ) </S>", "<S> was performed for measurement of arterial and venous diameters , and peak systolic velocity of arterial flow based on subsequent fistula adequacy . </S>", "<S> autogenous avfs were constructed in forearm as distally as possible in all patients . </S>", "<S> both groups were followed - up for a year . in the placebo group , </S>", "<S> early avf thrombosis was detected in two patients ( 4.3% ) . </S>", "<S> avf maturation failure was noted in 14 patients ( 30.4% ) in placebo group and in four patients ( 8% ) in clopidogrel plus oral prostacycline analog group in the early postoperative period ( p = 0.001 ) . </S>", "<S> the mean maturation time was 38  6.5 and 53  12.8 days in study and placebo groups , respectively ( p = 0.023 ) . </S>", "<S> the mean blood flow was 352  94 ml / min in placebo group and 604  125 ml / min in study group ( p = 0.001 ) . </S>", "<S> the arterial end diastolic velocity was 116  14 cm / s in study group and 72  21 cm / s in placebo group ( p = 0.036 ) 1 year after the surgery . </S>", "<S> our data indicated that clopidogrel and oral prostacycline analog combination is effective and safe for the prevention of primary avf failure in hemodialysis patients and decreased acute and chronic thrombotic events . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Statistical analysis", "Results", "Discussion", "Conclusion"], "sections": [["early failure of fistulas due to thrombosis or inadequate maturation is a barrier to increasing the prevalence of fistulas . despite the benefits of avfs as a form of vascular access in end stage renal disease ( esrd ) patients , considerable time", "is required for the fistula to mature and suitably develop into a functional format maturation rate after autogenous avf depends on various factors such as age , body mass index ( bmi ) , venous and arterial diameter , venous distensibility , and arterial elasticity .", "the main cause of avf failure is outflow stenosis as a result of vascular intimal hyperplasia and thrombosis .", "this formation of neointimal hyperplasia and thrombosis may be initiated by activated platelet function , endothelial cell injury and vascular smooth muscle cell proliferation , which may occur due to unphysiologic vascular anastomosis . in view of these postulated mechanisms of avf failure ,", "furthermore , only a few studies have addressed the safety of aspirin use after avf creation .", "van der linden et al . demonstrated that venous potential distensibility was significantly correlated with functional maturation , especially in the group of patients with vein diameter  2 mm .", "as a result of this study , they concluded that venous line distensibility was a better predictive factor than vein size .", "there is no report about the effects of combination therapy after avf creation using clopidogrel in combination with prostacycline analog as a potent vasodilator agent after autogenous avf creation .", "on the basis of these considerations , we performed a study to test the hypothesis that clopidogrel as an irreversible antiplatelet and oral prostacycline analog as an arterial vasodilator and additional antiaggregant drug may increase venous distensibility and arterial elasticity and would prevent primary avf failure in hemodialysis patients with diabetes mellitus ."], ["we excluded 289 patients [ table 1 ] , and 96 patients met the study criteria for enrollment .", "major exclusion criteria of the patients to detect the quality of the brachial artery ( ba ) and a venous line , doppler usg was used .", "preoperative venous and arterial spectral usg showed that there were no differences when we compared arterial and venous quality and diameter .", "the randomization was stratified according to the medical center with a permuted block scheme , with a block size of four and an equal allocation .", "eligible participants were called to the coordinating center to obtain a randomization protocol , which corresponded to a specific medication . neither the details of the randomization sequence nor the identity of the medication assigned was known to the participant or any personnel at the participating sites .", "the treatment was initiated 710 days prior to the scheduled access surgery and continued postoperatively .", "after randomization , the two groups of subjects are followed in exactly the same way , and the only difference between the care with which they receive drugs , outpatient visits and follow - up calls should be those intrinsic to the treatments being compared . in the first group of patients ,", "710 days prior to the surgery , we started clopidogrel ( 75 mg daily ) and an oral prostacycline analog ( 200 mg orally daily ) .", "patients in the control and study group were operated by two surgeons using the same technique and same suture materials ( 6/0 polypropylene suture ) . to provide the purse or narrowing effect of suture ,", "we noticed that the two surgeons operated on the same number of patients in both the groups .", "clopidogrel and prostacycline were administered orally prior to surgery , and continued during the year .", "data collection was performed at baseline , 4 weeks after fistula creation , and monthly thereafter until ascertainment of fistula suitability .", "study medication was discontinued when there was a sign of fistula thrombosis confirmed by vascular surgeons . in a normally developed fistula", ", there is a continuous thrill from the anastomosis all the way centrally along the outflow vein .", "the thrill gradually decreases in intensity from the anastomosis centrally . in the absence of a stenosis", "we defined the maturation of avf when the mean blood flow from avf was more than 300 ml / min .", "and a minimum arterial end diastolic velocity was 70 cm / s by sonographic examination for both groups .", "venous and arterial line diameters were calculated using sonography . if there was no sign of restriction of blood flow when the patients underwent hemodialysis , we described the hemodialysis access to be good .", "primary patency was defined as the interval from avf creation to abandonment due to thrombosis or low flow , inadequate for sufficient hemodialysis .", "early patency or failure was defined according to a thrill and/or bruit 4 weeks after the surgery .", "the antero - posterior diameter of ba was measured in the transverse dimension on gray scale sonography .", "peak systolic velocity and end - diastolic velocity of the ba were measured in a representative waveform in each patient [ figure 1 ] .", "the resistive index was a measure of the downstream flow resistance and was calculated as the difference between the respective measurements obtained after fist release ( post ) and during fist clenching ( pre ) .", "the primary outcome measure was final avf failure , which required the creation of a new avf .", "first , we investigated the need of re - intervention after avf due to thrombosis or occlusion during the follow - up period .", "suitability was defined as use of the fistula at a dialysis machine blood pump rate of 300 ml / min . or more during 8 of 12 dialysis sessions .", "doppler ultrasonography ( in a gray scale ) shows the brachial artery and its diameter in a patient with insulin - dependent diabetes mellitus and peak velocity the statistical program , spss version 13 ( spss , chicago , il ) was used to analyze the data .", "data are presented as mean  standard error for continuous variables and as percentages for categorical variables .", "the t - test was used when the means of two groups were compared . on the basis of intention - to - treat principles , all other participants were monitored according to the research protocol for whom study medications were continued .", "all hypothesis tests were conducted by using a significance level of 0.05 ( two - sided ) . for evaluation of the relationships between the avf maturation and independent variables ,", "differences between matured and failed groups were tested with the wilcoxon rank - sum test for continuous variables .", "multivariate cox proportional hazards models were used to determine factors associated with reduced avf patency .", "test results were presented as hazard ratios with 95% confidence intervals ( cis ) , and two - sided p < 0.05 was considered statistically significant ."], ["the statistical program , spss version 13 ( spss , chicago , il ) was used to analyze the data .", "data are presented as mean  standard error for continuous variables and as percentages for categorical variables .", "the t - test was used when the means of two groups were compared . on the basis of intention - to - treat principles , all other participants were monitored according to the research protocol for whom study medications were continued .", "all hypothesis tests were conducted by using a significance level of 0.05 ( two - sided ) . for evaluation of the relationships between the avf maturation and independent variables ,", "differences between matured and failed groups were tested with the wilcoxon rank - sum test for continuous variables .", "multivariate cox proportional hazards models were used to determine factors associated with reduced avf patency .", "test results were presented as hazard ratios with 95% confidence intervals ( cis ) , and two - sided p < 0.05 was considered statistically significant ."], ["the mean ages were 54.23  2.6 and 55.8  2.8 years in group 1 and 2 respectively ( p > 0.05 ) .", "there were no significant differences in covariables between groups when we compared patient characteristics , blood pressure , blood ldl , calcium , and phosphate levels [ table 2 ] .", "we did not detect any complication during the treatment period such as bleeding in group 1 patients .", "three patients complained from a temporary headache on the 1 day of oral prostacycline analog administration .", "all patients were taking study medication prior to surgery and did not have to be discontinued prematurely .", "four weeks after the surgery , avf maturation failure developed in 14 patients ( 30.4% ) in placebo group and in 4 patients ( 8% ) in clopidogrel and oral prostacycline analog group ( p = 0.001 ) . fistula maturation and survival has been confirmed using physical examinations and by a doppler usg every month for each patient .", "the kaplanmeier survival curve showed avf maturation and survival results in figure 2 [ table 3 ] .", "the mean maturation time of avf was 38  6.5 day and 53  12.8 day in clopidogrel plus oral prostacycline analog and placebo groups , respectively ( p = 0.023 ) .", "patients treated with clopidogrel and oral prostacycline analog had a increased avf survival ( p = 0.001 ) . in the placebo group , early avf failure was detected in two patients due to venous line thrombosis .", "characteristics of patients in the two groups demonstrates the survival rate of arteriovenous fistulas ( avfs ) during the study period in group 1 and group 2 .", "meier estimates of the cumulative incidence of loss of primary avfs patency in both groups .", "the median duration of patency was 5.8 months ( 95% confidence interval ( ci ) : 4.37.1 ) in the group 1 ( study group ) and 4.3 months ( 95% ci : 3.65.4 ) in the group 2 ( placebo group ) clinical outcomes at the end of the study the preoperative mean diameter of ba was 4.3  1.5 mm in clopidogrel plus oral prostacycline analog group and 4.04  1.3 mm in placebo group ( p = 0.94 ) .", "after surgery , usg showed that the mean diameter of ba was 6.3  1.4 mm and 4.74  0.9 mm in the study and placebo group respectively ( p = 0.002 ) .", "the preoperative mean diameter of the vein in the study and placebo groups were 3.1  0.8 and 3.04  0.7 mm ( p = 0.96 ) . in the postoperative period ,", "the corresponding values were 5.4  0.9 and 4.01  0.3 mm respectively ( p = 0.024 ) .", "blood flow from avf was 352  94 ml / min . in placebo group and 604  125 ml / min .", "doppler usg examination of avf showed that a minimum arterial end - diastolic velocity has been detected at 116  14 cm / s in group 1 , 3 months after the surgery .", "however , this value was 72  21 cm / s in the placebo group ( p = 0.036 ) .", "overall , the hazard ratio for primary avf failure was 0.82 ( 95% ci : 0.310.94 ) . during the follow - up period , from 4 weeks to 6 months , tenderness of the extremity ,", "edema , and hematoma occurred in nine patients in study group , and in six in placebo group .", "hemodialysis was successfully performed in 36 patients ( 72% ) in the study group and 22 patients ( 47.8% ) in the placebo group at the end of study ( p = 0.001 ) .", "the hemodialysis period in group 1 was significantly longer and more successful than in the placebo group ( p = 0.001 ) .", "multivariate logistic regression analysis revealed that avf maturation and survival rate was more than in study group ( odds ratio 2.27 ; 95% ci : 1.078.50 ; p = 0.001 ) .", "the survival and maturation time of avf in study group and placebo group has been summarized in figure 3(p = 0.001 ) .", "the flow diagram demonstrates the number of patients who were screened and selected for research [ figure 4 ] . during follow - up", "this graphic demonstrates the cumulative incidence for the percent of primary end points attributable to flow monitoring compared to total end points in each treatment group .", "end points due to flow monitoring in the group 1 ( study group ) and group 2 ( placebo - treated control group ) are compared to total end points demonstrates the comparison of the mean avfs ' flow diagram ( left columns ) and the differences of arterial end - diastolic velocity ( right columns ) between the groups . in the early period , we detected high blood flows and lower arterial velocity in study group when we compared groups no bleeding episode such as intracranial hemorrhage in gastrointestinal tract was recorded during the active treatment period .", "there were no differences between baseline and follow - up hematocrit values or changes in recombinant human erythropoietin doses during the study period for either group ."], ["in this prospective double - blind research , we presented our experiences of a new combination therapy and its efficacies on avf access after surgery in our esrd population . according to the study , clopidogrel in combination with oral prostacycline analog as an arterial vasodilator", "can be used to provide early avf maturation in esrdp with diabetes . according to previous reports , the size and quality of the arterial and venous access and distensibility after avf creation play important roles .", "therefore , we decided to use our combination therapy to provide more dilated and elastic arterial and venous access after avf creation .", "thus , blood flow from the fistula increased significantly in patients who were given a combination therapy than in patients who were given placebo .", "our study findings support the importance of distensibility and quality of vessel access . as we know that after avf creation , anastomotic or juxta - anastomotic stenosis is the most common cause of a fistula failing to mature .", "the stenosis most likely occurs because of operative trauma . in case of transposed vein fistulas ,", "the loss of vasa vasorum in the mobilized portion of the vein may also contribute to causation of stenosis .", "second , there is the element of neo - intimal hyperplasia . because of a large pressure gradient between arterial and venous circulation , blood accelerates while passing from the artery to the vein causing turbulent flow that is considered responsible for endothelial damage as a result of micro - trauma .", "in addition , unfortunately , some patients have a small arterial and/or venous access diameter .", "therefore , effective hemodialysis can be impossible due to occlusion and/or stenosis of the venous or arterial line .", "it is shown that venous distensibility is a major problem after avf creation . in van der", "'s study , it was demonstrated that venous distensibility was significantly correlated with avf maturation , especially in patients with vein diameter 2 mm .", "thus , if there is no suitable venous distensibility , avf maturation and survival will be a problem . according to this hypothesis", ", we believe that the effective vasodilatation can be required in these particular groups for early avf maturation and longer survival .", "there have been a few small trials evaluating pharmacological agents aimed at reducing the early thrombosis rate .. these trials utilized the antiplatelet agents , aspirin , sulphinpyrazone , and clopidogrel and varied in size from as few as 5 patients up to 250 patients .", "andrassy et al . compared patients given aspirin for 4 weeks with placebo ; the thrombosis rate in the aspirin group was 4% compared with the control group of 24% .", "in another study , kooistra et al . demonstrated that a low - dose aspirin could not prevent thrombovascular accidents in hemodialysis patients during treatment with recombinant human erythropoietin .", "'s study , it was demonstrated that there was a statistical significance when compared to fistula thrombosis rates in clopidogrel groups versus placebo groups ( 12% vs. 19% ) .", "studies using sulphinpyrazone showed variable results , but were underpowered with the largest study enrolling only a small number of patients .", "thus , although there is some data to suggest that antiplatelet agents may increase the primary patency of avf , the limited evidence base and the uncertainty regarding the choice of agent have not supported the widespread use of antiplatelet agents in the prevention of avf thrombosis . however , for the first time , our study showed the effects of clopidogrel in combination with oral prostacycline agent on autogenous avf creation .", "we know that autogenous avf is the preferred hemodialysis access and is associated with lower morbidity and mortality than avgs . however , 2060% of fistulas fail to mature sufficiently to support dialysis .", "thus , the high rate of maturation failure is a major unsolved problem , yet .", "the costs and morbidity problems due to avf maturation failure among hemodialysis patients are still reported worldwide .", "vascular access surgeons are faced with the same challenge all over the world  choosing the best vascular access type for chronic hemodialysis .", "size and quality of the vessels seem to be the important factors known . despite the experiences of avf in esrdp , considerable amount of time", "is required for the fistula to mature and suitably develop into a functional format . a number of authors and dember et al .", "have maintained that outflow venous stenosis and vascular intimal hyperplasia were the main causes of avf failure .", "avf thrombosis resulting from neointimal hyperplasia can be initiated by activated platelet function , endothelial cell injury , vascular smooth muscle cell proliferation , and unphysiologic vascular anastomosis . in view of these postulated mechanisms of avf failure ,", "possible clinical benefit of antiplatelet therapy has been proposed for prevention of avf failure . in particular , aspirin , the most widespread antiplatelet drug , has been evaluated as prophylaxis for avf failure in several studies , mainly among the use of avg . in a study ,", "the authors examined the efficacy of combination therapy of aspirin and clopidogrel for prevention of arteriovenous graft failure , but this study was stopped before completion because the prevalence of hemorrhagic complication was significantly higher in the intervention group . in the study group , only 28% of patients required re - intervention during the 1-year follow - up period .", "however , 52% of placebo group needed re - intervention due to avf failure . during the aspirin therapy ,", "the most important problem is hemorrhage in hemodialysis patients than in the general population because hemodialysis patients are more likely to have platelet dysfunction and they receive intermittent anticoagulant therapy during hemodialysis sessions .", "however , clopidogrel is tolerated by the patient and is not a cause of gastrointestinal bleeding .", "thrombosis or severe occlusion of autogenous avf can be seen within the first several weeks following surgical creation , or inadequate maturation of the vein .", "some of the previous studies have indicated that the frequency of avf failure can be reduced with antiplatelet agents ; but in other studies the authors maintained antiplatelet therapy did not affect avf maturation and survival .", "they also concluded that vessel dilation was not needed for adequate blood flow except at the smaller diameters .", "the successful results of our analysis suggest that to inhibit avf failure , daily administration of 75 mg oral clopidogrel and arterial prostacycline analog administration may be initiated 710 days prior to avf creation . in", "'s trial suggested that the use of antiplatelet was not associated with significant risk reduction in access failure .", "kaufman demonstrated no change in the risk of graft thrombosis with aspirin plus clopidogrel therapy .", "the author reported that in chronic hemodialysis patients there was a trend toward increased or did not change thrombosis rate of avf using aspirin therapy .", "in fiskerstrand et al . 's study , 2 out of 6 patients in the ticlopidine group compared with 5 out of 9 in the placebo group , developed fistulae thromboses in 1 month . in the previously published study by grntoft et al .", ", there were only 2 out of 19 patients who received treatment developed for fistulae thromboses compared to 8 out of 17 on placebo .", "the results of adequacy for avfs developed sufficiently to be used for dialysis in 46.5% of patients .", "allon and robbin have suggested that the higher adequacy rate could be provided in upper arm location and multidisciplinary team approach to vascular access by the surgeons , nurses , and radiologists .", "the arterial diameter , elasticity , and the venous line distensibility are the main factors of fistula maturation . in publications from dac consortium ,", "followed the patients for a long time ( approximately 4 years ) but we could follow the cases for a year only .", "our therapy model may be provided more distensible and large size venous line , and more elastic arterial access .", "our study differences may be related to surgical experiences , patients ' age , smoking history , bmi , patients ' arterial and venous diameters , and distensibility ."], ["multiple individuals are involved in the management of vascular access , including nephrologists , access surgeons , radiologists , dialysis nurses , and the patient .", "achieving optimal vascular access outcomes requires agreement on a common set of goals by all these individuals , close collaboration , and good communication .", "however , primary avf failure remains a major problem for hemodialysis patients worldwide . according to our study ,", "it is observed that prophylaxis of vascular access thrombosis needs to start earlier in the hemodialysis patients .", "clopidogrel and oral prostacycline analog , which begins 710 days prior to avf creation and continuing during follow - up period seems to be effective and safe , and inhibit primary avf failure with acceptable side effects in hemodialysis patients ."]]}
{"article_id": "PMC3246515", "article_text": ["tension - free vaginal tape ( tvt ) is now the treatment of choice for female stress urinary incontinence because of its ease of use , minimal invasiveness , and high success rate .", "mesh in the bladder or urethra following midurethral sling has been reported to occur in somewhere between less than 1% to 6% of patients and can result from intraoperative needle penetration or tape erosion [ 2 - 4 ] . even though the trans - obturator tape ( tot ) procedure has less risk of lower urinary tract injury , surgeons should handle with care to prevent this complication .", "bladder injury that is recognized during the operation is not usually related to long - term sequelae . however ,", "when bladder injury is missed or the mesh erodes into the bladder or urethra , patients might suffer from painful urination , recurrent urinary tract infection , irritative or obstructive symptoms , and hematuria .", "since the first case of urethral mesh erosion was reported in 2001 , several case series have provided various techniques to manage this difficult complication [ 7,8 - 10 ] .", "intravesical or intraurethral mesh can be removed by open surgery ( cystorrhapy or urethrolysis and urethroplasty ) or endoscopic surgery .", "the objective of this study was to determine the efficacy and complication associated with transurethral removal ( tur ) of the intravesical or intraurethral mesh following midurethral sling procedures .", "a retrospective chart review was performed for women who had undergone transurethral surgery for bladder or urethral mesh between january 2002 and december 2010 .", "a total of 23 consecutive women were identified , and their demographics , midurethral sling procedures , and removal procedures were reviewed .", "the surgical outcomes and complications were evaluated . to remove the mesh , transurethral resection with an electrode loop ( tur - e )", "was used until march 2010 , when a holmium laser became available in the study center .", "thereafter , transurethral resection with a holmium laser ( tur - h ) was performed .", "the usual surgical techniques of tur - e and tur - h are described below .", "tur - e was performed for 15 women with intravesical mesh and 1 woman with intraurethral mesh . with the patient under spinal anesthesia in the lithotomy position ,", "a urethro - cystoscopic exam was performed with a 25 fr resectoscope and a 0 , 30 , and 70 degree telescope .", "when the stone was attached to the mesh , lithotripsy was performed with a lithoclast .", "the mesh was then resected with an electrode loop until it was not visible or the perivesical fat was reached .", "the resected mesh was removed by foreign body forceps . after confirming that no mesh remained in the bladder or urethra a 16 fr foley catheter was inserted and left in place for a median of 6 days ( range , 3 to 29 days ) .", "the median operation time was 57 minutes ( 40 to 135 minutes ) , and the median hospital stay was 8 days ( 4 to 36 days ) .", "seven patients were treated with the tur - h procedure with a rigid scope : 5 cases of intravesical mesh and 2 cases of intraurethral mesh .", "the surgery was similar to that for tur - e except a holmium laser was used .", "when the laser could not cut deeply enough and strings or a portion of the mesh remained , the remnant was evaporated by the laser .", "the median energy used was 4.3 kj ( range , 0.7 to 14.9 kj ) . after confirming that no mesh remained in the bladder or urethra , a 16 fr foley catheter was inserted and left in place for a median of 1 day ( range , 1 to 2 days ) .", "the median operation time was 125 minutes ( range , 30 to 180 minutes ) and the median hospital stay was 4.5 days ( range , 3 to 10 days ) .", "three women required concomitant transvaginal surgery for remnant mesh after tur - h : 2 with intravesical mesh and 1 with intraurethral mesh .", "a midline vaginal incision was made and submucosal dissection was performed with mezenbaum scissors . after identifying the mesh , it was grasped with kelly forceps and further dissection proceeded toward the exposed side while carefully maintaining traction on the mesh . after exposing the cut edge of the mesh through the vaginal incision , the whole exposed portion of the mesh was resected and removed .", "the wound was closed layer - by - layer with 3 - 0 vicryl sutures .", "a foley catheter was left in place for a median of 14 days ( range , 6 to 21 days ) .", "the median operation time was 215 minutes ( range , 180 to 220 minutes ) and the median hospital stay was 8 days ( range , 6 to 22 days ) .", "tur - e was performed for 15 women with intravesical mesh and 1 woman with intraurethral mesh . with the patient under spinal anesthesia in the lithotomy position ,", "a urethro - cystoscopic exam was performed with a 25 fr resectoscope and a 0 , 30 , and 70 degree telescope .", "when the stone was attached to the mesh , lithotripsy was performed with a lithoclast .", "the mesh was then resected with an electrode loop until it was not visible or the perivesical fat was reached .", "the resected mesh was removed by foreign body forceps . after confirming that no mesh remained in the bladder or urethra a 16 fr foley catheter", "was inserted and left in place for a median of 6 days ( range , 3 to 29 days ) .", "the median operation time was 57 minutes ( 40 to 135 minutes ) , and the median hospital stay was 8 days ( 4 to 36 days ) .", "1 . seven patients were treated with the tur - h procedure with a rigid scope : 5 cases of intravesical mesh and 2 cases of intraurethral mesh . the surgery was similar to that for tur - e except a holmium laser was used .", "when the laser could not cut deeply enough and strings or a portion of the mesh remained , the remnant was evaporated by the laser .", "the median energy used was 4.3 kj ( range , 0.7 to 14.9 kj ) .", "after confirming that no mesh remained in the bladder or urethra , a 16 fr foley catheter was inserted and left in place for a median of 1 day ( range , 1 to 2 days ) .", "the median operation time was 125 minutes ( range , 30 to 180 minutes ) and the median hospital stay was 4.5 days ( range , 3 to 10 days ) .", "three women required concomitant transvaginal surgery for remnant mesh after tur - h : 2 with intravesical mesh and 1 with intraurethral mesh .", "a midline vaginal incision was made and submucosal dissection was performed with mezenbaum scissors . after identifying the mesh , it was grasped with kelly forceps and further dissection proceeded toward the exposed side while carefully maintaining traction on the mesh . after exposing the cut edge of the mesh through the vaginal incision , the whole exposed portion of the mesh was resected and removed .", "the wound was closed layer - by - layer with 3 - 0 vicryl sutures .", "a foley catheter was left in place for a median of 14 days ( range , 6 to 21 days ) .", "the median operation time was 215 minutes ( range , 180 to 220 minutes ) and the median hospital stay was 8 days ( range , 6 to 22 days ) .", "patient demographics and the clinical characteristics of a total of 23 women are described in table 1 according to the removal technique .", "sixteen women had the tur - e procedure and 7 women had the tur - h procedure . at the time of surgery , the median age was 51 years ( range , 40 to 67 years ) .", "the median period between the midurethral sling and mesh removal was 31.3 months ( range , 2.2 to 247.6 months ) , and the median follow - up period was 2.1 months ( range , 0.3 to 35.9 months ) after mesh removal .", "the previous midurethral sling procedures were 11 transobturator slings , 8 retropubic slings , 1 single incision sling , and 3 which were not identified .", "most women presented with multiple symptoms , including dysuria , hematuria , and irritative or obstructive urinary symptoms .", "table 2 summarizes the preoperative findings , surgical outcomes , and complications for all 23 cases .", "overall , 6 women ( 26% ) had a mesh remnant after transurethral surgery : 1 woman ( 6.2% ) after tur - e and 5 women ( 71.4% ) after tur - h .", "the woman who had a mesh remnant after tur - e underwent repeat tur - e for the remnant , and no mesh remained on the follow - up cystoscopic exam .", "of the 5 women who had remnant mesh after tur - h , 3 underwent concomitant transvaginal removal . on the postoperative cystoscopic exam , 2 women had remnant mesh after tur - h .", "one woman had transvaginal removal with no mesh visible on the follow - up cystoscopic exam . in the other woman", "she wanted to delay additional treatment for the remaining mesh because she had none of the preoperative symptoms .", "two cases of vesico - vaginal fistulas occurred in women treated with tur - e .", "one case was found during the tur - e , and the fistula tract was immediately repaired transvaginally .", "a foley catheter was placed for 29 days and no sequelae were observed during 9.5 months of follow - up .", "the woman experienced continuous urine leakage 7 days after the foley catheter was removed . on the vaginal exam ,", "one case of stress incontinence ( stamey grade i ) recurred in a woman who had remaining intraurethral mesh after tur - h .", "in this retrospective study , 26% of women ( 6/23 ) had a mesh remnant after transurethral removal of intravesical or intraurethral mesh .", "mesh exposure in the bladder or urethra is an uncommon but troublesome complication of midurethral slings .", "several authors have described their approaches to this difficult problem , with no consensus on the best approach [ 11 - 16 ] .", "frenkl et al proposed a treatment algorithm after reviewing 22 cases of iatrogenic foreign bodies in the bladder and urethra following pelvic floor surgery .", "they commented on the technical difficulties of endoscopic resection of the mesh due to the encrusted stone material and the difficulty in applying tension to the mesh during resection .", "standard transurethral resection had success rates of 90% to 100% in several case series with a small number of patients [ 7,17 - 19 ] . with the electrode loop", "our series also showed a high success rate ( 93.7% [ 15/16 ] ) after the tur - e procedure .", "on the contrary , the holmium laser completely removed the mesh in only 28% of women ( 2/7 ) .", "doumouchtsis et al reported that 4 of 6 women had recurrent erosion after transurethral resection using a holmium laser .", "the holmium laser allows sharp incision of the mesh at the entrance and exit points . however , complete excision is difficult with a rigid cystoscope when the mesh is located in the bladder neck or urethra . of the 3 successful cases of holmium laser excision reported by giri et al , none were in the bladder neck or urethra , and a flexible instrument was used . in the present study ,", "the mesh was in the bladder neck or urethra in all 5 cases with mesh remaining after tur - h , and we used a rigid scope .", "therefore , we suggest that complete removal of the mesh using tur - h depends on the location of the mesh and the range of motion of the instrument .", "another reason for the low success rate after tur - h might be less experience with the technique using holmium laser .", "two cases of vesico - vaginal fistulas were found during or after the tur - e procedure . as mentioned above", ", one advantage of the electrode loop is that it can resect the mesh down to the submucosal portion .", "because damaged urethral or bladder mucosa is atrophic , the risk of perforation may be increased .", "a laparoscopic trocar was inserted suprapubically into the bladder under cystoscopic control . under traction and fixation by suprapubically", "a disadvantage of this technique is that it requires an additional bladder tract , which can increase the hospital stay and morbidity . to avoid bladder or urethral perforation ,", "nevertheless , transurethral resection using an electrode loop for intraurethral mesh might have a higher possibility of urethral perforation or incomplete resection . also , with a holmium laser and rigid scope", "the median duration from the manifestation of symptoms to diagnosis was more than 7 months .", "the possibility of unrecognized tape perforation or erosion and cystosopic exam should be considered in patients with lower urinary tract symptoms after a midurethral sling procedure .", "stress urinary incontinence recurred in 1 woman who had a mesh remnant after tur - h .", "she wanted to delay further treatment because the grade was minimal and her preoperative symptom was resolved .", "given these results , transurethral resection wolud have a good functional outcome and low risk recurrence of stress incontinence .", "however , there are only limited data on the effectiveness of transurethral management for bladder or urethral mesh", ". our results can provide information on the outcomes and complications of different transurethral removal techniques and can help physicians to choose the best treatment modality .", "transurethral surgery is associated with low morbidity and is familiar to urologists . to remove intravesical or intraurethral mesh after midurethral sling procedures", ", tur - e has a high success rate , although it has a high risk of bladder perforation .", "in contrast , tur - h has less risk of tissue damage , but complete resection seems to depend on the location of the mesh and the range of motion of the instrument .", "the evidence for tur - h is not sufficient to conclude that this is an ineffective method for mesh removal ; however , it should be performed in selected patients .", "transurethral resection has good functional outcomes and a low risk of recurrence of stress incontinence ."], "abstract_text": ["<S> purposeto present outcomes of transurethral removal ( tur ) of intravesical or intraurethral mesh after midurethral slings.materials and methodsthis was a retrospective chart review of 23 consecutive women : 20 with intravesical mesh and 3 with intraurethral mesh.resultsto remove the mesh , transurethral resection with an electrode loop ( tur - e ) was used in 16 women and transurethral resection with a holmium laser ( tur - h ) was used in 7 . </S>", "<S> the median follow - up was 2.1 months . </S>", "<S> twenty - six percent of the women ( 6/23 ) had a mesh remnant : 6.2% ( 1/16 ) of the women treated with tur - e and 71.4% ( 5/7 ) of the women treated with tur - h . </S>", "<S> of the 5 women treated with tur - h , 3 underwent concomitant transvaginal removal . on the follow - up cystoscopic exam </S>", "<S> , a mesh remnant was observed in 3 women ( 1 treated with tur - e and 2 treated with tur - h ) . </S>", "<S> vesico - vaginal fistulas were found in 2 women during and after tur - e , respectively . </S>", "<S> stress urinary incontinence recurred in 1 woman.conclusionstur-e has a high success rate but carries a risk of bladder perforation . </S>", "<S> complete resection using tur - h depends on the location of the mesh and the range of motion of the instrument . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "1. Transurethral resection with an electrode loop", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["tension - free vaginal tape ( tvt ) is now the treatment of choice for female stress urinary incontinence because of its ease of use , minimal invasiveness , and high success rate .", "mesh in the bladder or urethra following midurethral sling has been reported to occur in somewhere between less than 1% to 6% of patients and can result from intraoperative needle penetration or tape erosion [ 2 - 4 ] . even though the trans - obturator tape ( tot ) procedure has less risk of lower urinary tract injury , surgeons should handle with care to prevent this complication .", "bladder injury that is recognized during the operation is not usually related to long - term sequelae . however ,", "when bladder injury is missed or the mesh erodes into the bladder or urethra , patients might suffer from painful urination , recurrent urinary tract infection , irritative or obstructive symptoms , and hematuria .", "since the first case of urethral mesh erosion was reported in 2001 , several case series have provided various techniques to manage this difficult complication [ 7,8 - 10 ] .", "intravesical or intraurethral mesh can be removed by open surgery ( cystorrhapy or urethrolysis and urethroplasty ) or endoscopic surgery .", "the objective of this study was to determine the efficacy and complication associated with transurethral removal ( tur ) of the intravesical or intraurethral mesh following midurethral sling procedures ."], ["a retrospective chart review was performed for women who had undergone transurethral surgery for bladder or urethral mesh between january 2002 and december 2010 .", "a total of 23 consecutive women were identified , and their demographics , midurethral sling procedures , and removal procedures were reviewed .", "the surgical outcomes and complications were evaluated . to remove the mesh , transurethral resection with an electrode loop ( tur - e )", "was used until march 2010 , when a holmium laser became available in the study center .", "thereafter , transurethral resection with a holmium laser ( tur - h ) was performed .", "the usual surgical techniques of tur - e and tur - h are described below .", "tur - e was performed for 15 women with intravesical mesh and 1 woman with intraurethral mesh . with the patient under spinal anesthesia in the lithotomy position ,", "a urethro - cystoscopic exam was performed with a 25 fr resectoscope and a 0 , 30 , and 70 degree telescope .", "when the stone was attached to the mesh , lithotripsy was performed with a lithoclast .", "the mesh was then resected with an electrode loop until it was not visible or the perivesical fat was reached .", "the resected mesh was removed by foreign body forceps . after confirming that no mesh remained in the bladder or urethra a 16 fr foley catheter was inserted and left in place for a median of 6 days ( range , 3 to 29 days ) .", "the median operation time was 57 minutes ( 40 to 135 minutes ) , and the median hospital stay was 8 days ( 4 to 36 days ) .", "seven patients were treated with the tur - h procedure with a rigid scope : 5 cases of intravesical mesh and 2 cases of intraurethral mesh .", "the surgery was similar to that for tur - e except a holmium laser was used .", "when the laser could not cut deeply enough and strings or a portion of the mesh remained , the remnant was evaporated by the laser .", "the median energy used was 4.3 kj ( range , 0.7 to 14.9 kj ) . after confirming that no mesh remained in the bladder or urethra , a 16 fr foley catheter was inserted and left in place for a median of 1 day ( range , 1 to 2 days ) .", "the median operation time was 125 minutes ( range , 30 to 180 minutes ) and the median hospital stay was 4.5 days ( range , 3 to 10 days ) .", "three women required concomitant transvaginal surgery for remnant mesh after tur - h : 2 with intravesical mesh and 1 with intraurethral mesh .", "a midline vaginal incision was made and submucosal dissection was performed with mezenbaum scissors . after identifying the mesh , it was grasped with kelly forceps and further dissection proceeded toward the exposed side while carefully maintaining traction on the mesh . after exposing the cut edge of the mesh through the vaginal incision , the whole exposed portion of the mesh was resected and removed .", "the wound was closed layer - by - layer with 3 - 0 vicryl sutures .", "a foley catheter was left in place for a median of 14 days ( range , 6 to 21 days ) .", "the median operation time was 215 minutes ( range , 180 to 220 minutes ) and the median hospital stay was 8 days ( range , 6 to 22 days ) ."], ["tur - e was performed for 15 women with intravesical mesh and 1 woman with intraurethral mesh . with the patient under spinal anesthesia in the lithotomy position ,", "a urethro - cystoscopic exam was performed with a 25 fr resectoscope and a 0 , 30 , and 70 degree telescope .", "when the stone was attached to the mesh , lithotripsy was performed with a lithoclast .", "the mesh was then resected with an electrode loop until it was not visible or the perivesical fat was reached .", "the resected mesh was removed by foreign body forceps . after confirming that no mesh remained in the bladder or urethra a 16 fr foley catheter", "was inserted and left in place for a median of 6 days ( range , 3 to 29 days ) .", "the median operation time was 57 minutes ( 40 to 135 minutes ) , and the median hospital stay was 8 days ( 4 to 36 days ) .", "1 . seven patients were treated with the tur - h procedure with a rigid scope : 5 cases of intravesical mesh and 2 cases of intraurethral mesh . the surgery was similar to that for tur - e except a holmium laser was used .", "when the laser could not cut deeply enough and strings or a portion of the mesh remained , the remnant was evaporated by the laser .", "the median energy used was 4.3 kj ( range , 0.7 to 14.9 kj ) .", "after confirming that no mesh remained in the bladder or urethra , a 16 fr foley catheter was inserted and left in place for a median of 1 day ( range , 1 to 2 days ) .", "the median operation time was 125 minutes ( range , 30 to 180 minutes ) and the median hospital stay was 4.5 days ( range , 3 to 10 days ) .", "three women required concomitant transvaginal surgery for remnant mesh after tur - h : 2 with intravesical mesh and 1 with intraurethral mesh .", "a midline vaginal incision was made and submucosal dissection was performed with mezenbaum scissors . after identifying the mesh , it was grasped with kelly forceps and further dissection proceeded toward the exposed side while carefully maintaining traction on the mesh . after exposing the cut edge of the mesh through the vaginal incision , the whole exposed portion of the mesh was resected and removed .", "the wound was closed layer - by - layer with 3 - 0 vicryl sutures .", "a foley catheter was left in place for a median of 14 days ( range , 6 to 21 days ) .", "the median operation time was 215 minutes ( range , 180 to 220 minutes ) and the median hospital stay was 8 days ( range , 6 to 22 days ) ."], ["patient demographics and the clinical characteristics of a total of 23 women are described in table 1 according to the removal technique .", "sixteen women had the tur - e procedure and 7 women had the tur - h procedure . at the time of surgery , the median age was 51 years ( range , 40 to 67 years ) .", "the median period between the midurethral sling and mesh removal was 31.3 months ( range , 2.2 to 247.6 months ) , and the median follow - up period was 2.1 months ( range , 0.3 to 35.9 months ) after mesh removal .", "the previous midurethral sling procedures were 11 transobturator slings , 8 retropubic slings , 1 single incision sling , and 3 which were not identified .", "most women presented with multiple symptoms , including dysuria , hematuria , and irritative or obstructive urinary symptoms .", "table 2 summarizes the preoperative findings , surgical outcomes , and complications for all 23 cases .", "overall , 6 women ( 26% ) had a mesh remnant after transurethral surgery : 1 woman ( 6.2% ) after tur - e and 5 women ( 71.4% ) after tur - h .", "the woman who had a mesh remnant after tur - e underwent repeat tur - e for the remnant , and no mesh remained on the follow - up cystoscopic exam .", "of the 5 women who had remnant mesh after tur - h , 3 underwent concomitant transvaginal removal . on the postoperative cystoscopic exam , 2 women had remnant mesh after tur - h .", "one woman had transvaginal removal with no mesh visible on the follow - up cystoscopic exam . in the other woman", "she wanted to delay additional treatment for the remaining mesh because she had none of the preoperative symptoms .", "two cases of vesico - vaginal fistulas occurred in women treated with tur - e .", "one case was found during the tur - e , and the fistula tract was immediately repaired transvaginally .", "a foley catheter was placed for 29 days and no sequelae were observed during 9.5 months of follow - up .", "the woman experienced continuous urine leakage 7 days after the foley catheter was removed . on the vaginal exam ,", "one case of stress incontinence ( stamey grade i ) recurred in a woman who had remaining intraurethral mesh after tur - h ."], ["in this retrospective study , 26% of women ( 6/23 ) had a mesh remnant after transurethral removal of intravesical or intraurethral mesh .", "mesh exposure in the bladder or urethra is an uncommon but troublesome complication of midurethral slings .", "several authors have described their approaches to this difficult problem , with no consensus on the best approach [ 11 - 16 ] .", "frenkl et al proposed a treatment algorithm after reviewing 22 cases of iatrogenic foreign bodies in the bladder and urethra following pelvic floor surgery .", "they commented on the technical difficulties of endoscopic resection of the mesh due to the encrusted stone material and the difficulty in applying tension to the mesh during resection .", "standard transurethral resection had success rates of 90% to 100% in several case series with a small number of patients [ 7,17 - 19 ] . with the electrode loop", "our series also showed a high success rate ( 93.7% [ 15/16 ] ) after the tur - e procedure .", "on the contrary , the holmium laser completely removed the mesh in only 28% of women ( 2/7 ) .", "doumouchtsis et al reported that 4 of 6 women had recurrent erosion after transurethral resection using a holmium laser .", "the holmium laser allows sharp incision of the mesh at the entrance and exit points . however , complete excision is difficult with a rigid cystoscope when the mesh is located in the bladder neck or urethra . of the 3 successful cases of holmium laser excision reported by giri et al , none were in the bladder neck or urethra , and a flexible instrument was used . in the present study ,", "the mesh was in the bladder neck or urethra in all 5 cases with mesh remaining after tur - h , and we used a rigid scope .", "therefore , we suggest that complete removal of the mesh using tur - h depends on the location of the mesh and the range of motion of the instrument .", "another reason for the low success rate after tur - h might be less experience with the technique using holmium laser .", "two cases of vesico - vaginal fistulas were found during or after the tur - e procedure . as mentioned above", ", one advantage of the electrode loop is that it can resect the mesh down to the submucosal portion .", "because damaged urethral or bladder mucosa is atrophic , the risk of perforation may be increased .", "a laparoscopic trocar was inserted suprapubically into the bladder under cystoscopic control . under traction and fixation by suprapubically", "a disadvantage of this technique is that it requires an additional bladder tract , which can increase the hospital stay and morbidity . to avoid bladder or urethral perforation ,", "nevertheless , transurethral resection using an electrode loop for intraurethral mesh might have a higher possibility of urethral perforation or incomplete resection . also , with a holmium laser and rigid scope", "the median duration from the manifestation of symptoms to diagnosis was more than 7 months .", "the possibility of unrecognized tape perforation or erosion and cystosopic exam should be considered in patients with lower urinary tract symptoms after a midurethral sling procedure .", "stress urinary incontinence recurred in 1 woman who had a mesh remnant after tur - h .", "she wanted to delay further treatment because the grade was minimal and her preoperative symptom was resolved .", "given these results , transurethral resection wolud have a good functional outcome and low risk recurrence of stress incontinence .", "however , there are only limited data on the effectiveness of transurethral management for bladder or urethral mesh", ". our results can provide information on the outcomes and complications of different transurethral removal techniques and can help physicians to choose the best treatment modality ."], ["transurethral surgery is associated with low morbidity and is familiar to urologists . to remove intravesical or intraurethral mesh after midurethral sling procedures", ", tur - e has a high success rate , although it has a high risk of bladder perforation .", "in contrast , tur - h has less risk of tissue damage , but complete resection seems to depend on the location of the mesh and the range of motion of the instrument .", "the evidence for tur - h is not sufficient to conclude that this is an ineffective method for mesh removal ; however , it should be performed in selected patients .", "transurethral resection has good functional outcomes and a low risk of recurrence of stress incontinence ."]]}
{"article_id": "PMC3503236", "article_text": ["generalized anxiety disorder ( gad ) is a chronic disorder mainly characterized by pathological worry .", "gad also presents with a variety of somatic and psychological symptoms such as restlessness , muscle tension , sleep disturbance , irritability , difficulty concentrating , and fatigue .", "gad is one of the most prevalent anxiety disorders , with its lifetime prevalence estimated to be of 5.7% in the united states and 2.8% in europe [ 2 , 3 ] .", "patients with gad have a higher likelihood of developing other mood disorders in their lifetime ; one of the most common is major depressive disorder , present in 62% of gad patients .", "since gad affects normal functioning , these patients are usually frequent users of healthcare resources ; in fact , studies aimed to explore gad clinical prevalence have estimated it to be 7.3% in primary care and up to 13% in the psychiatric outpatient setting [ 5 , 6 ] .", "gad is difficult to diagnose by primary care physicians and psychiatrists . in people who suffer gad , commonly occurring comorbid conditions ( e.g. , depression , alcoholism , other anxiety disorders ) often mask the underlying anxiety and result in a missed diagnosis of gad [ 7 , 8 ] .", "other factors complicating the diagnosis of gad are patients tendency to complain about the somatic symptoms , such as sleeplessness and fatigue , rather than the psychic symptoms ( e.g. , nervousness , apprehension , chronic worry ) , and physicians lack of education in the diagnosis of gad .", "diagnostic criteria have been modified in each edition of the diagnostic and statistical manual of mental disorders ( dsm ) [ 1 , 11 , 12 ] .", " duration of excessive worry  criterion and the number of associated symptoms are still questioned today . the duration criterion was increased to 6 month in the dsm - iii - r   to distinguish gad from adjustment disorders or situational stress reactions .", "however , recent studies have suggested that this duration excludes significantly impaired patients from diagnosis .", "dsm - v , which is expected to get released in may 2013 , will revise the wording and organization of gad diagnostic criteria in order to simplify them .  ", "meanwhile , several groups are conducting studies aiming to assess the need for 6 months duration of excessive anxiety and worry criterion [ 13 , 15 - 17 ] .   using data from the us national comorbidity survey replication , ruscio et al . have demonstrated that broadening the gad diagnosis criteria , more than doubles its prevalence .", "this broadening of the criteria consists on a shortening of anxiety duration from  at least 6 months  to  at least 1 month ( but less than 6 months )  , relaxing excessiveness to include excessive and non - excessive worry , and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .", "most of the increase in prevalence comes from reducing the minimum duration to 1 month and , to a lesser extent , from eliminating the excessive worry requirement .", "prevalence is minimally affected by requiring 2 rather than 3 associated symptoms . in a recent study conducted", "both in developed and developing countries , the 6-month duration criterion was not found to influence symptom severity , age of onset , persistence , impairment or comorbidity when compared to shorter criterion durations ( 1 - 2 months and 3 - 5 months ) . to our knowledge , there is no study aimed to evaluate prospectively the evolution of anxiety symptoms in patients diagnosed with broad dsm - iv criteria under medical practice . before considering changing dsm - iv criteria , full consequences of these changes should be explored .", "broadening of gad criteria could lead to earlier diagnosis of gad patients and to diagnosis of otherwise under diagnosed patients .", "earlier diagnosis could translate into a sooner start of treatment and this could lead to an improvement in the course of the illness and the quality of life .", "in fact , gad patients have reported lower perceived quality of life than non - anxious controls and a lower degree of social functioning than patients with arthritis or diabetes . in a recent study by lee et al . , those patients with more than 12 months duration of symptoms were more severely impaired and had a lower rate of recovery than the groups with shorter duration of symptoms , suggesting that an earlier diagnosis could also benefit patient response and recovery .   in an attempt to elucidate the consequences of broadening dsm - iv criteria for generalized anxiety disorder ,", "the current study examined prospectively the evolution of gad symptoms in two groups of diagnosed patients in public and private mental healthcare settings ; one group according to the existing dsm - iv criteria and the other according to broader criteria ( at least 1 month but less than 6 months of excessive or non - excessive worry and 2 associated symptoms listed on dsm - iv for gad diagnosis ) . by using the total ham - a scores , changes in evolution of anxiety symptoms", "were studied in both criteria groups under the basis of routine medical care in a 6 month period .", "finally , self - reported quality of life , disability and other patient - reported - outcomes were studied as a measure of the overall well - being of gad patients .", "this was a multicentre , prospective and observational study carried out in outpatient psychiatric clinics between october 2007 and january 2009 .", "the study was approved by the local ethics committee of the hospital clnico de san carlos ( madrid ) and was conducted according to the helsinki declaration for research in the human being .", "functional and clinical outcome measures were completed in all three visits and included the following instruments : hamilton anxiety rating scale , montgomery - asberg depression rating scale ( baseline and 6 month visit only ) , clinical global impression - severity of illness scale , sleep scale from medical outcomes study ( mos - sleep ) , world health organization disability assessment schedule ii ( baseline and 6 month visit only ) , and the quality of life questionnaire , eq-5d ( all described in detail below ) . at baseline , socio - demographic data ,", "current therapy and psychiatric and medical illnesses information were collected . for the 3 and 6 months visits ,", "psychiatrists participating in the study made a confirmatory diagnosis of gad in 6 consecutive patients , 3 according to dsm - iv criteria and 3 according to broad criteria .", "although patients could have symptoms and receive treatment before entering the study , they had nt been diagnosed before by the psychiatrist .", "eligible patients were men and women over 18 years of age that were diagnosed with gad by a trained psychiatrist at the baseline visit .", "exclusion criteria included previous gad diagnosis , inability or difficulty to understand patient - reported - outcomes questionnaires written in spanish .", "diagnosis was carried out by a trained psychiatrist , familiar with the study procedures , and who worked in an outpatient setting .", "sampling frame included all public and private healthcare settings within the 17 regions of spain .", "first stage consisted on a selection of the outpatient psychiatry clinics ( mental health centres ) in each healthcare region .", "the number of clinics selected in each region was proportional to the region s population , being the probability of choosing each clinic relative to the population in the area covered by the clinic . in the second stage ,", "one psychiatrist per clinic was chosen randomly within those with previous experience in clinical and epidemiological research in psychiatry , and with at least 5 year experience in mental health diseases diagnosis .", "if a selected psychiatrist refused to participate , he or she was replaced by another from the same clinic ( also selected randomly ) .", "participant psychiatrists were asked to invite in a consecutive manner those patients from the daily list of appointments that fulfilled the inclusion criteria .", "psychiatrists were encouraged to maintain the above mentioned proportion of 3 subjects diagnosed following dms - iv criteria and 3 subjects diagnosed following the broad criteria .  ", "sample size was calculated taking into account study s main variable : evolution of anxiety symptoms according to the total ham - a scores for each patient group .", "a sample of 3400 evaluable patients was estimated assuming a 2-tailed 95% confidence interval for the total ham - a scores lower than 0.25 points from the observed mean .", "based on the results of a previous study , a standard deviation lower than 8 was assumed .  ", "gad was diagnosed following the dsm - iv criteria ( dsm - iv criteria group ) in one group .", "the other group ( broad criteria group ) was diagnosed according to broader criteria consisting on shortening of anxiety duration from  at least 6 months  to  at least 1 month ( but less than 6 months )  , including excessive or non - excessive worry and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .", "hamilton anxiety rating scale ( ham - a ) : the validated spanish version of the ham - a scale was used to evaluate the evolution of anxiety symptoms during all 3 visits of the study .", "the ham - a scales includes 14 items , each of them rates from 0 ( absence ) to 4 ( severe ) .", "the total score comes from the addition of all 14 items with a maximum of 56 .", "scores higher than 24 were considered as severe anxiety ; between 16 and 24 , moderate ; between 10 and 15 , mild ; and below 9 , no anxiety . a psychic anxiety sub domain ( addition of items 1 - 6 and 14 ) and a somatic anxiety sub domain ( addition of items 7 - 13 )", "montgomery - asberg depression rating scale ( madrs ) : the validated spanish version of the madrs scale was used to measure the intensity of depression symptoms .", "this observer - rated depression scale consists on 10 items regarding different depressive symptoms , each item being scored from 0 to 6 depending on the severity of symptoms ; the total score results from adding the score of each item , obtaining a minimum of 0 ( no symptoms ) and a maximum of 60 ( very severe ) .", "clinical global impression - severity of illness scale ( cgi - s ) : participant psychiatrists evaluated patient s global severity with the cgi - s scale , which rates illness severity from 0 to 7 , where 0 was considered as  not evaluated ", ", 1 as  no disease  , 2  borderline ill  , 3  mildly ill ", ", 4  moderately ill  , 5  markedly ill  , 6  severely ill  , and 7  extremely ill  .", "sleep scale from medical outcomes study ( mos - sleep ) : mos - sleep scale evaluates the impact or interference with sleep of any disease or treatment .", "the 7 subscales are sleep disturbance ( 4 items ) , snoring ( 1 item ) , awaken short of breath or with headache ( 1 item ) , quantity of sleep ( 1 item ) , optimal sleep ( 1 item ) , sleep adequacy ( 2 items ) , and somnolence ( 3 items ) .", "this scale also has a sleep problems index , rated from 0 ( no interference ) to 100 ( maximum interference ) , and a sleep problems subscale .", "each item is rated independently with more interference scoring higher , except for the sleep adequacy and quantity and optimal sleep subscales [ 21 , 22 ] .", "world health organization disability assessment schedule ii ( who - das ii ) : the who - das ii is a psychometric instrument that measures day to day functioning and disability in the following six activity domains : understanding and communicating , moving and getting around , self care , getting along with people , work activities , and participation in society . the 12-item who - das ii scale", "is rated from 1 ( none or nothing ) to 5 ( extreme or inability to perform a given task ) . the total calculated score ranges from 0 to 100 , with higher scores indicating higher degrees of disability , for the total scale as well as for each subscale .", "eq-5d : this is a standard self - reported quality of life questionnaire developed in several european countries and validated for spain .", "this questionnaire consists of two parts , the first one aims to evaluate patient s health profile in five dimensions ( mobility , self - care , daily living activities , pain , and anxiety / depression ) .", "the patient rates his / her current status as no problems , some / moderate problems , and severe / extreme problems for each dimension .", "results are then converted into 1 of the 243 different eq-5d health state descriptions which are used to calculate a unique index or tariff , between 1 ( as healthy as possible ) and 0 ( usually equivalent to death ) .", "this index is used to calculate the time - trade off tariff which allows calculating quality - adjusted - life - year ( qaly ) gain ( further explanation below ) .", "the second part consists on a visual analogue scale ( vas or thermometer ) in which the patient has to rate his health status between 0 ( equivalent to death ) and 100 ( the best possible healthy status ) .  ", "for statistical analysis , last observation carried forward approach was used for those patients for whom data from the 6 months visit were missing . if the only missing data was that from the 3 months visit , this was estimated proportionally to the change observed at the 6 months visit , given that the missing data at 3 month visit were completely at random .", "patients were considered to respond to treatment when a higher than 50% reduction was observed for the total ham - a scale score at the study final visit when compared to that of the baseline visit .", "descriptive statistics were prepared for the continuous variables in the study , including the assessment of central position and dispersion ( two - tailed 95% confidence interval ) .", "the kolmogorov - smirnov test was applied to check adjustment of data to a gaussian distribution .", "student t tests and chi - square test were used for continuous and categorical variables , respectively , to test homogeneity of baseline data between groups .", "analysis of covariance ( ancova ) or binary logistic regression models were fitted comparing dsm - iv criteria versus broad criteria groups in most variables adjusting by baseline scoring , sex , age , body mass index , educational level , marital status and percentage of patients with comorbid major depression or depressive disorder .", "analysis of variance ( anova ) was applied for quantitative measures and chi square ( mcnemar s test ) for qualitative measures in case of paired comparisons .  ", "the clinical relevance of observed differences in baseline characteristics was ascertained by means of the effect size that was calculated using the difference of means for each group , divided by the combined standard deviation of the variables .", "qalys gain after the 6-months follow - up was calculated from patients responses to the eq-5d as described previously using tariff calculated with this instrument .", "gained qalys were estimated by using the trapezoidal approach , with baseline and 6- month visit values used as reference values .  ", "all statistical tests were two - tailed , and  error of < 0.05 was accepted as statistically significant .", "this was a multicentre , prospective and observational study carried out in outpatient psychiatric clinics between october 2007 and january 2009 .", "the study was approved by the local ethics committee of the hospital clnico de san carlos ( madrid ) and was conducted according to the helsinki declaration for research in the human being .", "functional and clinical outcome measures were completed in all three visits and included the following instruments : hamilton anxiety rating scale , montgomery - asberg depression rating scale ( baseline and 6 month visit only ) , clinical global impression - severity of illness scale , sleep scale from medical outcomes study ( mos - sleep ) , world health organization disability assessment schedule ii ( baseline and 6 month visit only ) , and the quality of life questionnaire , eq-5d ( all described in detail below ) . at baseline , socio - demographic data ,", "current therapy and psychiatric and medical illnesses information were collected . for the 3 and 6 months visits ,", "psychiatrists participating in the study made a confirmatory diagnosis of gad in 6 consecutive patients , 3 according to dsm - iv criteria and 3 according to broad criteria .", "although patients could have symptoms and receive treatment before entering the study , they had nt been diagnosed before by the psychiatrist .", "eligible patients were men and women over 18 years of age that were diagnosed with gad by a trained psychiatrist at the baseline visit .", "exclusion criteria included previous gad diagnosis , inability or difficulty to understand patient - reported - outcomes questionnaires written in spanish .", "diagnosis was carried out by a trained psychiatrist , familiar with the study procedures , and who worked in an outpatient setting .", "sampling frame included all public and private healthcare settings within the 17 regions of spain .", "first stage consisted on a selection of the outpatient psychiatry clinics ( mental health centres ) in each healthcare region .", "the number of clinics selected in each region was proportional to the region s population , being the probability of choosing each clinic relative to the population in the area covered by the clinic . in the second stage ,", "one psychiatrist per clinic was chosen randomly within those with previous experience in clinical and epidemiological research in psychiatry , and with at least 5 year experience in mental health diseases diagnosis .", "if a selected psychiatrist refused to participate , he or she was replaced by another from the same clinic ( also selected randomly ) .", "participant psychiatrists were asked to invite in a consecutive manner those patients from the daily list of appointments that fulfilled the inclusion criteria .", "psychiatrists were encouraged to maintain the above mentioned proportion of 3 subjects diagnosed following dms - iv criteria and 3 subjects diagnosed following the broad criteria .  ", "sample size was calculated taking into account study s main variable : evolution of anxiety symptoms according to the total ham - a scores for each patient group .", "a sample of 3400 evaluable patients was estimated assuming a 2-tailed 95% confidence interval for the total ham - a scores lower than 0.25 points from the observed mean .", "based on the results of a previous study , a standard deviation lower than 8 was assumed .", "gad was diagnosed following the dsm - iv criteria ( dsm - iv criteria group ) in one group .", "the other group ( broad criteria group ) was diagnosed according to broader criteria consisting on shortening of anxiety duration from  at least 6 months  to  at least 1 month ( but less than 6 months )  , including excessive or non - excessive worry and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .", "hamilton anxiety rating scale ( ham - a ) : the validated spanish version of the ham - a scale was used to evaluate the evolution of anxiety symptoms during all 3 visits of the study .", "the ham - a scales includes 14 items , each of them rates from 0 ( absence ) to 4 ( severe ) .", "the total score comes from the addition of all 14 items with a maximum of 56 .", "scores higher than 24 were considered as severe anxiety ; between 16 and 24 , moderate ; between 10 and 15 , mild ; and below 9 , no anxiety . a psychic anxiety sub domain ( addition of items 1 - 6 and 14 ) and a somatic anxiety sub domain ( addition of items 7 - 13 )", "montgomery - asberg depression rating scale ( madrs ) : the validated spanish version of the madrs scale was used to measure the intensity of depression symptoms .", "this observer - rated depression scale consists on 10 items regarding different depressive symptoms , each item being scored from 0 to 6 depending on the severity of symptoms ; the total score results from adding the score of each item , obtaining a minimum of 0 ( no symptoms ) and a maximum of 60 ( very severe ) .", "clinical global impression - severity of illness scale ( cgi - s ) : participant psychiatrists evaluated patient s global severity with the cgi - s scale , which rates illness severity from 0 to 7 , where 0 was considered as  not evaluated ", ", 1 as  no disease  , 2  borderline ill  , 3  mildly ill  , 4  moderately ill  , 5  markedly ill  , 6  severely ill  , and 7  extremely ill  .", "sleep scale from medical outcomes study ( mos - sleep ) : mos - sleep scale evaluates the impact or interference with sleep of any disease or treatment .", "the 7 subscales are sleep disturbance ( 4 items ) , snoring ( 1 item ) , awaken short of breath or with headache ( 1 item ) , quantity of sleep ( 1 item ) , optimal sleep ( 1 item ) , sleep adequacy ( 2 items ) , and somnolence ( 3 items ) .", "this scale also has a sleep problems index , rated from 0 ( no interference ) to 100 ( maximum interference ) , and a sleep problems subscale .", "each item is rated independently with more interference scoring higher , except for the sleep adequacy and quantity and optimal sleep subscales [ 21 , 22 ] .", "world health organization disability assessment schedule ii ( who - das ii ) : the who - das ii is a psychometric instrument that measures day to day functioning and disability in the following six activity domains : understanding and communicating , moving and getting around , self care , getting along with people , work activities , and participation in society .", "the 12-item who - das ii scale is rated from 1 ( none or nothing ) to 5 ( extreme or inability to perform a given task ) . the total calculated score ranges from 0 to 100 , with higher scores indicating higher degrees of disability , for the total scale as well as for each subscale .", "eq-5d : this is a standard self - reported quality of life questionnaire developed in several european countries and validated for spain .", "this questionnaire consists of two parts , the first one aims to evaluate patient s health profile in five dimensions ( mobility , self - care , daily living activities , pain , and anxiety / depression ) .", "the patient rates his / her current status as no problems , some / moderate problems , and severe / extreme problems for each dimension .", "results are then converted into 1 of the 243 different eq-5d health state descriptions which are used to calculate a unique index or tariff , between 1 ( as healthy as possible ) and 0 ( usually equivalent to death ) .", "this index is used to calculate the time - trade off tariff which allows calculating quality - adjusted - life - year ( qaly ) gain ( further explanation below ) .", "the second part consists on a visual analogue scale ( vas or thermometer ) in which the patient has to rate his health status between 0 ( equivalent to death ) and 100 ( the best possible healthy status ) .", "for statistical analysis , last observation carried forward approach was used for those patients for whom data from the 6 months visit were missing .", "if the only missing data was that from the 3 months visit , this was estimated proportionally to the change observed at the 6 months visit , given that the missing data at 3 month visit were completely at random .", "patients were considered to respond to treatment when a higher than 50% reduction was observed for the total ham - a scale score at the study final visit when compared to that of the baseline visit .", "descriptive statistics were prepared for the continuous variables in the study , including the assessment of central position and dispersion ( two - tailed 95% confidence interval ) .", "the kolmogorov - smirnov test was applied to check adjustment of data to a gaussian distribution . for categorical variables", "student t tests and chi - square test were used for continuous and categorical variables , respectively , to test homogeneity of baseline data between groups .", "analysis of covariance ( ancova ) or binary logistic regression models were fitted comparing dsm - iv criteria versus broad criteria groups in most variables adjusting by baseline scoring , sex , age , body mass index , educational level , marital status and percentage of patients with comorbid major depression or depressive disorder .", "analysis of variance ( anova ) was applied for quantitative measures and chi square ( mcnemar s test ) for qualitative measures in case of paired comparisons .  ", "the clinical relevance of observed differences in baseline characteristics was ascertained by means of the effect size that was calculated using the difference of means for each group , divided by the combined standard deviation of the variables .", "qalys gain after the 6-months follow - up was calculated from patients responses to the eq-5d as described previously using tariff calculated with this instrument .", "gained qalys were estimated by using the trapezoidal approach , with baseline and 6- month visit values used as reference values .  ", "all statistical tests were two - tailed , and  error of < 0.05 was accepted as statistically significant .", "a total of 3,549 patients were finally recruited by 618 psychiatrists in spanish psychiatric clinics for this observational study .", "four hundred and fifty three patients ( 12.8% ) were excluded because they did not meet all inclusion criteria .", "remaining patients were diagnosed of gad according to dsm - iv criteria ( 1,815 patients ) or to broad criteria ( 1,281 patients ) as described on the methods section .", "a total of 529 ( 17.0% ) patients drop - out from study , with no significant differences between study groups ( dsm - iv criteria : 313 patients , broad criteria : 216 patients ; p=0.899 ) ( fig .", "however , main reasons for drop - outs were a somewhat different between groups with 52.7% lost to follow - up in dsm - iv group , compared with 67.1% in broad criteria group ( p=0.033 ) , and unknown reasons in 22.0% of withdrawals in dsm - iv group compared with 13.6% in broad criteria group ( p=0.023 ) .", "the mean age was 45.5 years for the dsm - iv group and slightly lower for the broad criteria group ( 42.9 years , p<0.001 vs. dsm - iv group ) .", "regarding gad , all features studied were slightly , but significantly higher in the standard dsm - iv criteria group .", "thus , the mean number of anxiety symptoms was 4.7 for the dsm - iv group compared with 4.3 for the broad criteria group ( p<0.001 ) .", "significant differences were also observed in the percentage of patients presenting with each individual gad symptom . all ham - a scores indicated severe anxiety on both patient groups , being the scores in the dsm - iv group significantly higher ( table 1 ) .", "likewise , statistical differences were observed at baseline in the cgi and madrs scales between the two groups .", "however , the magnitude of the effect was low for all quantitative variables at baseline ( effect size < 0,50 ) so the statistically significant differences observed were of small or negligible clinical relevance .  ", "most patients in both groups ( 87.9% for dsm - iv ; 82.0% for broad criteria ) were following a pharmacological treatment in the six months previous to the baseline visit .", "( 2 ) , benzodiazepines were the most commonly used drugs followed by antidepressants and antiepileptics . at the end of the study", ", there was an increase in the number of patients under pharmacological treatment ( 97% in both groups , p<0.001 versus baseline in all cases ) .", "the use of antiepileptic drugs increased by 3.2 and 3.8 fold in the dsm - iv and broad criteria groups , respectively , by the end - of - trial ( p<0.001 in all cases ) .", "the percentage of antiepileptic drugs users in dsm - iv group at baseline and at 6 months was significantly higher , albeit only slightly ( p=0.002 in both cases , fig .", "both groups showed a significant reduction in the percentage of benzodiazepines users after 6 months of follow - up ( p<0.001 ) , being this reduction even higher in the broad criteria group ( p=0.011 ) .", "also a slight but statistically significant increase was observed in the percentage of anti - depressive users in both groups ( p<0.001 , see fig .", "the percentage of subjects in dsm - iv and broad criteria groups that had at least one psychiatric comorbidity was 51.4% and 43.7% , respectively , ( p<0.001 ) . table 2 describes the most frequent psychiatric comorbidities and medical disorders in both study groups .", "however , neither the mean number of comorbid psychiatric disorders   nor the medical illnesses were different between both criteria groups , except for comorbid major depression and other depressive disorders which were slightly more frequent , but statistically significant in dsm - iv group ( p<0.01 and p=0.05 , respectively , table 2 ) . regarding psychiatric disorders , most of them were present around 5 months before the diagnosis of gad , without significant differences between groups in disease evolution . major depression and panic disorders were the most frequent comorbid psychiatric disorders in both study groups . for medical illnesses ,", "chronic pain was present in more than half of patients in both groups , although a higher percentage was seen in dsm - iv group ; 64.3% versus 52.0% , p<0.001 ( table 2 ) .", "anxiety severity was assessed with ham - a and cgi - s scales at 3 and 6 month visits .", "( 3 ) shows the evolution of ham - a total and sub - scores over study time in the dsm - iv and broad criteria groups .", "all ham - a scores improved after 3 and 6 months in both criteria groups .", "moreover , the percentage of patients without symptoms of anxiety , showing ham - a<9 , increased up to 22.3% and 31.4% at the 3 month visit for the dsm - iv and broad criteria groups ( p<0.001 vs. basal visit in both groups , and p=0.006 between groups ) , and up to 49% and 58% at the 6 month visit ( p<0.001 vs. basal visit in both groups , and p=0.261 between groups ) , indicating similar results for both groups at the end of the study . also , patients responding to anxiety treatment increased when considering the responder criteria ( > 50% reduction of baseline scores ) ; 30.7% and 38.2% for the dms - iv and broad criteria , respectively , at the 3 months visit ( p= 0.004 ) ; increasing up to 59.7% in the dms - iv and 67.7% at the 6 month visit ( p=0.103 ) .", "4 ) , differences between criteria groups were statistically significant at the study time points . regarding each gad symptom , fig .", "( 5 ) shows the reduction in the percentage of patients presenting with each anxiety symptom .", "however , higher significant reductions were observed in dsm - iv group for the following symptoms : fatigue , irritability and sleep disturbance ( p<0.05 in all cases , fig . 5 ) . in this study", ", depression symptoms were evaluated using the madrs . when compared to scores at baseline visit ( table 1 )", ", patients in both criteria groups improved their madrs scores with a mean reduction of 12.1 and 12.5 points for the dms - iv and broad criteria groups , respectively ( p=0.264 dsm - iv vs. broad criteria ) .", "mos - sleep scale scores revealed an overall improvement in sleep patterns over study time in both criteria group .", "as described in table 3 , all sleep domains in the mos - sleep scale changed significantly after 6 months when compared to basal visit scores .", "differences between the dsm - iv and broad criteria groups were observed for the total score ( p= 0.018 ) , sleep disturbance domain ( p=0.012 ) , sleep quantity ( p=0.016 ) , sleep adequacy ( p=0.031 ) , and sleep problems ( p=0.019 ) scores , always favouring the broad criteria group .  ", "health - related quality of life was measured by the eq-5d questionnaire . as seen in table 3 , both the health status ( visual analogue scale ) and the frequency of severe problems in each domain had significantly improved at the 6 month visit in both study groups ( p<0.001 in all cases ) . also , comparisons between both criteria groups in quality - of - life gain by domain were significant in favour of the broad criteria group in health status , daily living activities and mood status .", "mean qalys gain at the 3 month and 6 month visits were 0.034 and 0.103 years , respectively , for the dsm - iv criteria group and 0.034 ( 3 month ) and 0.101 ( 6 month ) for the broad criteria patients , without statistical significance between groups ( table 3 ) . at the 6 month visit , the percentage of patients reporting an improvement in their health status as compared with that of one year", "earlier increased by 13.8% for the dsm - iv criteria group , and 16.6% for the broad criteria group ( differences not significant ) . for measuring the disability degree in both groups of patients ,", ", there was a significant improvement in all 7 domains of the who - das ii scale after the 6 month follow - up in both criteria groups .", "the improvements were not significantly different in the dsm - iv group when compared to the new criteria group except for household activity domain , in which the improvement in the dsm - iv group was significantly better when compared to the broad criteria group .", "nevertheless , a trend toward statistical significance was observed in the total scoring of the disability scale favouring broad criteria group .", "a total of 3,549 patients were finally recruited by 618 psychiatrists in spanish psychiatric clinics for this observational study .", "four hundred and fifty three patients ( 12.8% ) were excluded because they did not meet all inclusion criteria .", "remaining patients were diagnosed of gad according to dsm - iv criteria ( 1,815 patients ) or to broad criteria ( 1,281 patients ) as described on the methods section .", "a total of 529 ( 17.0% ) patients drop - out from study , with no significant differences between study groups ( dsm - iv criteria : 313 patients , broad criteria : 216 patients ; p=0.899 ) ( fig .", "however , main reasons for drop - outs were a somewhat different between groups with 52.7% lost to follow - up in dsm - iv group , compared with 67.1% in broad criteria group ( p=0.033 ) , and unknown reasons in 22.0% of withdrawals in dsm - iv group compared with 13.6% in broad criteria group ( p=0.023 ) .", "the mean age was 45.5 years for the dsm - iv group and slightly lower for the broad criteria group ( 42.9 years , p<0.001 vs. dsm - iv group ) .", "regarding gad , all features studied were slightly , but significantly higher in the standard dsm - iv criteria group .", "thus , the mean number of anxiety symptoms was 4.7 for the dsm - iv group compared with 4.3 for the broad criteria group ( p<0.001 ) .", "significant differences were also observed in the percentage of patients presenting with each individual gad symptom . all ham - a scores indicated severe anxiety on both patient groups , being the scores in the dsm - iv group significantly higher ( table 1 ) .", "likewise , statistical differences were observed at baseline in the cgi and madrs scales between the two groups .", "however , the magnitude of the effect was low for all quantitative variables at baseline ( effect size < 0,50 ) so the statistically significant differences observed were of small or negligible clinical relevance .", "most patients in both groups ( 87.9% for dsm - iv ; 82.0% for broad criteria ) were following a pharmacological treatment in the six months previous to the baseline visit .", "( 2 ) , benzodiazepines were the most commonly used drugs followed by antidepressants and antiepileptics . at the end of the study", ", there was an increase in the number of patients under pharmacological treatment ( 97% in both groups , p<0.001 versus baseline in all cases ) . the use of antiepileptic drugs increased by 3.2 and 3.8 fold in the dsm - iv and broad criteria groups , respectively , by the end - of - trial ( p<0.001 in all cases ) .", "the percentage of antiepileptic drugs users in dsm - iv group at baseline and at 6 months was significantly higher , albeit only slightly ( p=0.002 in both cases , fig .", "both groups showed a significant reduction in the percentage of benzodiazepines users after 6 months of follow - up ( p<0.001 ) , being this reduction even higher in the broad criteria group ( p=0.011 ) . also a slight but", "statistically significant increase was observed in the percentage of anti - depressive users in both groups ( p<0.001 , see fig .", "at the study entry , the percentage of subjects in dsm - iv and broad criteria groups that had at least one psychiatric comorbidity was 51.4% and 43.7% , respectively , ( p<0.001 ) .", "table 2 describes the most frequent psychiatric comorbidities and medical disorders in both study groups .", "however , neither the mean number of comorbid psychiatric disorders   nor the medical illnesses were different between both criteria groups , except for comorbid major depression and other depressive disorders which were slightly more frequent , but statistically significant in dsm - iv group ( p<0.01 and p=0.05 , respectively , table 2 ) . regarding psychiatric disorders , most of them were present around 5 months before the diagnosis of gad , without significant differences between groups in disease evolution .", "major depression and panic disorders were the most frequent comorbid psychiatric disorders in both study groups . for medical illnesses ,", "chronic pain was present in more than half of patients in both groups , although a higher percentage was seen in dsm - iv group ; 64.3% versus 52.0% , p<0.001 ( table 2 ) .", "anxiety severity was assessed with ham - a and cgi - s scales at 3 and 6 month visits . fig .", "( 3 ) shows the evolution of ham - a total and sub - scores over study time in the dsm - iv and broad criteria groups .", "all ham - a scores improved after 3 and 6 months in both criteria groups .", "moreover , the percentage of patients without symptoms of anxiety , showing ham - a<9 , increased up to 22.3% and 31.4% at the 3 month visit for the dsm - iv and broad criteria groups ( p<0.001 vs. basal visit in both groups , and p=0.006 between groups ) , and up to 49% and 58% at the 6 month visit ( p<0.001 vs. basal visit in both groups , and p=0.261 between groups ) , indicating similar results for both groups at the end of the study . also , patients responding to anxiety treatment increased when considering the responder criteria ( > 50% reduction of baseline scores ) ; 30.7% and 38.2% for the dms - iv and broad criteria , respectively , at the 3 months visit ( p= 0.004 ) ; increasing up to 59.7% in the dms - iv and 67.7% at the 6 month visit ( p=0.103 ) .", "4 ) , differences between criteria groups were statistically significant at the study time points . regarding each gad symptom , fig .", "( 5 ) shows the reduction in the percentage of patients presenting with each anxiety symptom .", "however , higher significant reductions were observed in dsm - iv group for the following symptoms : fatigue , irritability and sleep disturbance ( p<0.05 in all cases , fig . 5 ) . in this study ,", "depression symptoms were evaluated using the madrs . when compared to scores at baseline visit ( table 1 )", ", patients in both criteria groups improved their madrs scores with a mean reduction of 12.1 and 12.5 points for the dms - iv and broad criteria groups , respectively ( p=0.264 dsm - iv vs. broad criteria ) .", "mos - sleep scale scores revealed an overall improvement in sleep patterns over study time in both criteria group .", "as described in table 3 , all sleep domains in the mos - sleep scale changed significantly after 6 months when compared to basal visit scores .", "differences between the dsm - iv and broad criteria groups were observed for the total score ( p= 0.018 ) , sleep disturbance domain ( p=0.012 ) , sleep quantity ( p=0.016 ) , sleep adequacy ( p=0.031 ) , and sleep problems ( p=0.019 ) scores , always favouring the broad criteria group .  ", "health - related quality of life was measured by the eq-5d questionnaire . as seen in table 3 , both the health status ( visual analogue scale ) and the frequency of severe problems in each domain had significantly improved at the 6 month visit in both study groups ( p<0.001 in all cases ) . also , comparisons between both criteria groups in quality - of - life gain by domain were significant in favour of the broad criteria group in health status , daily living activities and mood status .", "mean qalys gain at the 3 month and 6 month visits were 0.034 and 0.103 years , respectively , for the dsm - iv criteria group and 0.034 ( 3 month ) and 0.101 ( 6 month ) for the broad criteria patients , without statistical significance between groups ( table 3 ) .", "at the 6 month visit , the percentage of patients reporting an improvement in their health status as compared with that of one year earlier increased by 13.8% for the dsm - iv criteria group , and 16.6% for the broad criteria group ( differences not significant ) . for measuring the disability degree in both groups of patients , the who - das scale was used . as shown in table 4 , there was a significant improvement in all 7 domains of the who - das ii scale after the 6 month follow - up in both criteria groups .", "the improvements were not significantly different in the dsm - iv group when compared to the new criteria group except for household activity domain , in which the improvement in the dsm - iv group was significantly better when compared to the broad criteria group .", "nevertheless , a trend toward statistical significance was observed in the total scoring of the disability scale favouring broad criteria group .", "this real world prospective study in usual medical practice has shown that broadening of gad dsm - iv diagnostic criteria , by reducing the duration of excessive or non - excessive worry to one month and associated symptoms to two , does not significantly affect patient s responsiveness to psychiatrist - prescribed therapy .", "the patient sample of this study seems to be similar to other gad populations presented in previous studies , with a higher prevalence among women and among those aged above 25 years [ 4 , 18 ] .", "the most frequent comorbid psychiatric disorder was major depression ; however , its presence was somewhat smaller than the observed in other studies , if we take into account major depression only .  ", "the main goal when treating a gad patient is to improve the symptoms , hopefully leading in the long - term to their complete remission and to recovery of patient s functionality .", "several guidelines recommend antidepressants , such as the selective serotonin reuptake inhibitors and the serotonin noradrenalin reuptake inhibitors , and antiepileptics as first line treatments , and benzodiazepines as second - line treatment [ 28 , 29 ] .", "although patients in this study received a confirmatory diagnosis of gad , a high percentage of them were already following a therapy recommended for gad at baseline .", "these results suggest that , in our sanitary context , general practitioners and/or family physicians start treatment to control anxiety symptoms , mostly with benzodiazepines , at patients first complaint .", "the general practitioner / family physician usually refers those patients with lower treatment response than expected to the psychiatrist .", "it should be taken into account that refractory patients may be difficult to manage at the primary care level in the spanish health system .", "one of the reasons is the brief period of time devoted by physicians to each patient visit in the spanish primary health care level , as described by duran et al . .", "other studies have explored the consequences of broadening dsm - iv diagnostic criteria for gad [ 3 , 13 , 15 - 16 , 31 ] .", "most of them have considered a reduction in the duration criteria [ 3 , 16 ] , the excessiveness of worry , and the number of associated symptoms . a possible", "learning from all of them is that the socio - demographic characteristics between the populations diagnosed with broad or strict dsm - iv criteria are not substantially different . as expected , the broadening of dsm - iv gad criteria results in an increase in the point - prevalence to more than double [ 15 - 16 ] .", "concluded that the increase was mainly due to the reduction of minimum duration to one month . in regards to gad s severity , ruscio \n et al .   observed a tendency to reduced severity for the broad criteria group , however", "we have observed a similar tendency suggesting that patients diagnosed with broader gad criteria present a slightly less severe form of the disease .  ", "every study that has investigated the possibility of broadening the dsm - iv criteria has done it retrospectively [ 13 , 15 , 16 , 27 , 31 , 32 ] .", "the present study is the first one to compare two groups of gad patients with dsm - iv and broader criteria following a prospective design .", "according to our results , broadening of gad diagnostic criteria does not affect the response to the prescribed therapy , since ham - a scores , total and domains , improved similarly in both groups , and so did the presence of gad symptoms ( fig .", "a significant improvement was perceived by physicians ( cgi - s scores ) at the end of the study .", "the influence of broader gad diagnostic criteria in patient s disability has been studied previously by lee \n et al .", "lee et al .   observed an increase in the sheehan disability scale score when increasing time for the duration criteria although it was not statistically significant .", "the same observation was made by kessler et al .   with data from the ncs .", "parallel to their clinical improvement , patients in our study experienced an improvement in the studied quality of life variables .", "health status improvements in our study were similar to those observed in primary care patients receiving treatment over six months for their gad .", "the biggest improvements were observed in the daily living activities and mood domains of the eq-5d .", "qalys gain observed corresponded to more than one month of perfect health for both groups ; which were similar in magnitude to that observed in other health conditions such as trigeminal neuralgia , successfully treated with pregabalin .", "all other dimensions of functionality included in the who das - ii scale , and sleep scale were substantially ameliorated in both study groups by the end - of - trial ; however , the differences in the magnitude of these improvements for both groups were hardly meaningful from a clinical point of view .  ", "results from this study should be interpreted bearing in mind its observational design with its inherent limitations . since this study", "is based on out - patient psychiatric clinics , patients included in this study might not be representative of the whole gad population , since psychiatrists could deal with more severe and refractory cases .", "also , due to the high degree of expertise required for participant psychiatrists , diagnosis in our study may have been more accurate than in other settings , such as primary care", ". a consequence of broadening gad criteria in other healthcare settings could be the difficulty in distinguishing gad from adjustment disorders or situational stress reactions by other less trained physicians . in this study", "there was a high proportion of patients treated with antiepileptics ; that could be explained by the fact that most subjects included in the study were non responders to recommended first line drug treatment according to the european guidelines on the treatment and management of gad patients .", "finally , as this was a real world prospective study we did not have a control group so further clinical research with randomized controlled studies and newly diagnosed patients needs to be carried out to confirm the consequences of broadening the criteria .", "broadening criteria for gad diagnosis , by reducing excessive and non - excessive anxiety and worry duration to one month and the number of associated symptoms to two , captures a similar population of patients compared to the one identified by applying standard dsm - iv criteria .", "broadening diagnostic criteria is able to identify the core symptoms of gad according to the dsm - iv criteria and could lead to an earlier diagnosis .", "funding for this study was provided by pfizer spain ; pfizer spain participated in the idea and design of the study .", "enrique lvarez , jose l. carrasco and jos m. olivares declare not to have any conflict of interests as a consequence of participating in this study .", "inma vilardaga was employed of the european biometric institute , a consulting agency which was responsible for logistic of study and analysis of data .", "mara perez and vanessa lpez - gmez are employed by pfizer spain , the body funding the study ."], "abstract_text": ["<S> objective : to elucidate the consequences of broadening dsm - iv criteria for generalized anxiety disorder ( gad ) , we examined prospectively the evolution of gad symptoms in two groups of patients ; one group diagnosed according to dsm - iv criteria and the other , according to broader criteria.method:multicentre , prospective and observational study conducted on outpatient psychiatric clinics . </S>", "<S> patients were selected from october 2007 to january 2009 and diagnosed with gad according to dsm - iv criteria ( dsm - iv group ) or broader criteria . </S>", "<S> broader criteria were considered 1-month of excessive or non - excessive worry and only 2 of the associated symptoms listed on dsm - iv for gad diagnosis . </S>", "<S> socio - demographic data , medical history and functional outcome measures were collected three times during a 6-month period.results:3,549 patients were systematically recruited ; 1,815 patients in dsm - iv group ( dg ) and 1,264 in broad group ( bg ) ; 453 patients did not fulfil inclusion criteria and were excluded . </S>", "<S> most patients ( 87.9% in dg , 82.0% in bg ) were currently following pharmacological therapies ( mainly benzodiazepines ) to manage their anxiety symptoms . </S>", "<S> the changes observed during the study were : 49.0% and 58.0% , respectively of patients without anxiety symptoms as per ham - a scale at the 6 month visit ( p=0.261 ) and 59.7% and 67.7% , respectively ( p=0.103 ) of responder rates ( > 50% reduction of baseline scoring).conclusion : broadening of gad criteria does not seem to affect psychiatric care results in subjects with gad , is able to identify the core symptoms of the disease according to the dsm - iv criteria and could lead to an earlier diagnosis . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODOLOGY", "Study Design", "Study Sample", "Diagnostic Assessments", "Clinical and Functional Outcome Measures", "Statistical Analysis", "RESULTS", "Patient Characteristics", "Treatment", "Comorbidity ", "Reduction of Anxiety Symptoms After 6-Month Treatment and Severity of Illness", "Functional Outcome Measures", "DISCUSSION", "CONCLUSION", "CONFLICT OF INTEREST"], "sections": [["generalized anxiety disorder ( gad ) is a chronic disorder mainly characterized by pathological worry .", "gad also presents with a variety of somatic and psychological symptoms such as restlessness , muscle tension , sleep disturbance , irritability , difficulty concentrating , and fatigue .", "gad is one of the most prevalent anxiety disorders , with its lifetime prevalence estimated to be of 5.7% in the united states and 2.8% in europe [ 2 , 3 ] .", "patients with gad have a higher likelihood of developing other mood disorders in their lifetime ; one of the most common is major depressive disorder , present in 62% of gad patients .", "since gad affects normal functioning , these patients are usually frequent users of healthcare resources ; in fact , studies aimed to explore gad clinical prevalence have estimated it to be 7.3% in primary care and up to 13% in the psychiatric outpatient setting [ 5 , 6 ] .", "gad is difficult to diagnose by primary care physicians and psychiatrists . in people who suffer gad , commonly occurring comorbid conditions ( e.g. , depression , alcoholism , other anxiety disorders ) often mask the underlying anxiety and result in a missed diagnosis of gad [ 7 , 8 ] .", "other factors complicating the diagnosis of gad are patients tendency to complain about the somatic symptoms , such as sleeplessness and fatigue , rather than the psychic symptoms ( e.g. , nervousness , apprehension , chronic worry ) , and physicians lack of education in the diagnosis of gad .", "diagnostic criteria have been modified in each edition of the diagnostic and statistical manual of mental disorders ( dsm ) [ 1 , 11 , 12 ] .", " duration of excessive worry  criterion and the number of associated symptoms are still questioned today . the duration criterion was increased to 6 month in the dsm - iii - r   to distinguish gad from adjustment disorders or situational stress reactions .", "however , recent studies have suggested that this duration excludes significantly impaired patients from diagnosis .", "dsm - v , which is expected to get released in may 2013 , will revise the wording and organization of gad diagnostic criteria in order to simplify them .  ", "meanwhile , several groups are conducting studies aiming to assess the need for 6 months duration of excessive anxiety and worry criterion [ 13 , 15 - 17 ] .   using data from the us national comorbidity survey replication , ruscio et al . have demonstrated that broadening the gad diagnosis criteria , more than doubles its prevalence .", "this broadening of the criteria consists on a shortening of anxiety duration from  at least 6 months  to  at least 1 month ( but less than 6 months )  , relaxing excessiveness to include excessive and non - excessive worry , and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .", "most of the increase in prevalence comes from reducing the minimum duration to 1 month and , to a lesser extent , from eliminating the excessive worry requirement .", "prevalence is minimally affected by requiring 2 rather than 3 associated symptoms . in a recent study conducted", "both in developed and developing countries , the 6-month duration criterion was not found to influence symptom severity , age of onset , persistence , impairment or comorbidity when compared to shorter criterion durations ( 1 - 2 months and 3 - 5 months ) . to our knowledge , there is no study aimed to evaluate prospectively the evolution of anxiety symptoms in patients diagnosed with broad dsm - iv criteria under medical practice . before considering changing dsm - iv criteria , full consequences of these changes should be explored .", "broadening of gad criteria could lead to earlier diagnosis of gad patients and to diagnosis of otherwise under diagnosed patients .", "earlier diagnosis could translate into a sooner start of treatment and this could lead to an improvement in the course of the illness and the quality of life .", "in fact , gad patients have reported lower perceived quality of life than non - anxious controls and a lower degree of social functioning than patients with arthritis or diabetes . in a recent study by lee et al . , those patients with more than 12 months duration of symptoms were more severely impaired and had a lower rate of recovery than the groups with shorter duration of symptoms , suggesting that an earlier diagnosis could also benefit patient response and recovery .   in an attempt to elucidate the consequences of broadening dsm - iv criteria for generalized anxiety disorder ,", "the current study examined prospectively the evolution of gad symptoms in two groups of diagnosed patients in public and private mental healthcare settings ; one group according to the existing dsm - iv criteria and the other according to broader criteria ( at least 1 month but less than 6 months of excessive or non - excessive worry and 2 associated symptoms listed on dsm - iv for gad diagnosis ) . by using the total ham - a scores , changes in evolution of anxiety symptoms", "were studied in both criteria groups under the basis of routine medical care in a 6 month period .", "finally , self - reported quality of life , disability and other patient - reported - outcomes were studied as a measure of the overall well - being of gad patients ."], ["this was a multicentre , prospective and observational study carried out in outpatient psychiatric clinics between october 2007 and january 2009 .", "the study was approved by the local ethics committee of the hospital clnico de san carlos ( madrid ) and was conducted according to the helsinki declaration for research in the human being .", "functional and clinical outcome measures were completed in all three visits and included the following instruments : hamilton anxiety rating scale , montgomery - asberg depression rating scale ( baseline and 6 month visit only ) , clinical global impression - severity of illness scale , sleep scale from medical outcomes study ( mos - sleep ) , world health organization disability assessment schedule ii ( baseline and 6 month visit only ) , and the quality of life questionnaire , eq-5d ( all described in detail below ) . at baseline , socio - demographic data ,", "current therapy and psychiatric and medical illnesses information were collected . for the 3 and 6 months visits ,", "psychiatrists participating in the study made a confirmatory diagnosis of gad in 6 consecutive patients , 3 according to dsm - iv criteria and 3 according to broad criteria .", "although patients could have symptoms and receive treatment before entering the study , they had nt been diagnosed before by the psychiatrist .", "eligible patients were men and women over 18 years of age that were diagnosed with gad by a trained psychiatrist at the baseline visit .", "exclusion criteria included previous gad diagnosis , inability or difficulty to understand patient - reported - outcomes questionnaires written in spanish .", "diagnosis was carried out by a trained psychiatrist , familiar with the study procedures , and who worked in an outpatient setting .", "sampling frame included all public and private healthcare settings within the 17 regions of spain .", "first stage consisted on a selection of the outpatient psychiatry clinics ( mental health centres ) in each healthcare region .", "the number of clinics selected in each region was proportional to the region s population , being the probability of choosing each clinic relative to the population in the area covered by the clinic . in the second stage ,", "one psychiatrist per clinic was chosen randomly within those with previous experience in clinical and epidemiological research in psychiatry , and with at least 5 year experience in mental health diseases diagnosis .", "if a selected psychiatrist refused to participate , he or she was replaced by another from the same clinic ( also selected randomly ) .", "participant psychiatrists were asked to invite in a consecutive manner those patients from the daily list of appointments that fulfilled the inclusion criteria .", "psychiatrists were encouraged to maintain the above mentioned proportion of 3 subjects diagnosed following dms - iv criteria and 3 subjects diagnosed following the broad criteria .  ", "sample size was calculated taking into account study s main variable : evolution of anxiety symptoms according to the total ham - a scores for each patient group .", "a sample of 3400 evaluable patients was estimated assuming a 2-tailed 95% confidence interval for the total ham - a scores lower than 0.25 points from the observed mean .", "based on the results of a previous study , a standard deviation lower than 8 was assumed .  ", "gad was diagnosed following the dsm - iv criteria ( dsm - iv criteria group ) in one group .", "the other group ( broad criteria group ) was diagnosed according to broader criteria consisting on shortening of anxiety duration from  at least 6 months  to  at least 1 month ( but less than 6 months )  , including excessive or non - excessive worry and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .", "hamilton anxiety rating scale ( ham - a ) : the validated spanish version of the ham - a scale was used to evaluate the evolution of anxiety symptoms during all 3 visits of the study .", "the ham - a scales includes 14 items , each of them rates from 0 ( absence ) to 4 ( severe ) .", "the total score comes from the addition of all 14 items with a maximum of 56 .", "scores higher than 24 were considered as severe anxiety ; between 16 and 24 , moderate ; between 10 and 15 , mild ; and below 9 , no anxiety . a psychic anxiety sub domain ( addition of items 1 - 6 and 14 ) and a somatic anxiety sub domain ( addition of items 7 - 13 )", "montgomery - asberg depression rating scale ( madrs ) : the validated spanish version of the madrs scale was used to measure the intensity of depression symptoms .", "this observer - rated depression scale consists on 10 items regarding different depressive symptoms , each item being scored from 0 to 6 depending on the severity of symptoms ; the total score results from adding the score of each item , obtaining a minimum of 0 ( no symptoms ) and a maximum of 60 ( very severe ) .", "clinical global impression - severity of illness scale ( cgi - s ) : participant psychiatrists evaluated patient s global severity with the cgi - s scale , which rates illness severity from 0 to 7 , where 0 was considered as  not evaluated ", ", 1 as  no disease  , 2  borderline ill  , 3  mildly ill ", ", 4  moderately ill  , 5  markedly ill  , 6  severely ill  , and 7  extremely ill  .", "sleep scale from medical outcomes study ( mos - sleep ) : mos - sleep scale evaluates the impact or interference with sleep of any disease or treatment .", "the 7 subscales are sleep disturbance ( 4 items ) , snoring ( 1 item ) , awaken short of breath or with headache ( 1 item ) , quantity of sleep ( 1 item ) , optimal sleep ( 1 item ) , sleep adequacy ( 2 items ) , and somnolence ( 3 items ) .", "this scale also has a sleep problems index , rated from 0 ( no interference ) to 100 ( maximum interference ) , and a sleep problems subscale .", "each item is rated independently with more interference scoring higher , except for the sleep adequacy and quantity and optimal sleep subscales [ 21 , 22 ] .", "world health organization disability assessment schedule ii ( who - das ii ) : the who - das ii is a psychometric instrument that measures day to day functioning and disability in the following six activity domains : understanding and communicating , moving and getting around , self care , getting along with people , work activities , and participation in society . the 12-item who - das ii scale", "is rated from 1 ( none or nothing ) to 5 ( extreme or inability to perform a given task ) . the total calculated score ranges from 0 to 100 , with higher scores indicating higher degrees of disability , for the total scale as well as for each subscale .", "eq-5d : this is a standard self - reported quality of life questionnaire developed in several european countries and validated for spain .", "this questionnaire consists of two parts , the first one aims to evaluate patient s health profile in five dimensions ( mobility , self - care , daily living activities , pain , and anxiety / depression ) .", "the patient rates his / her current status as no problems , some / moderate problems , and severe / extreme problems for each dimension .", "results are then converted into 1 of the 243 different eq-5d health state descriptions which are used to calculate a unique index or tariff , between 1 ( as healthy as possible ) and 0 ( usually equivalent to death ) .", "this index is used to calculate the time - trade off tariff which allows calculating quality - adjusted - life - year ( qaly ) gain ( further explanation below ) .", "the second part consists on a visual analogue scale ( vas or thermometer ) in which the patient has to rate his health status between 0 ( equivalent to death ) and 100 ( the best possible healthy status ) .  ", "for statistical analysis , last observation carried forward approach was used for those patients for whom data from the 6 months visit were missing . if the only missing data was that from the 3 months visit , this was estimated proportionally to the change observed at the 6 months visit , given that the missing data at 3 month visit were completely at random .", "patients were considered to respond to treatment when a higher than 50% reduction was observed for the total ham - a scale score at the study final visit when compared to that of the baseline visit .", "descriptive statistics were prepared for the continuous variables in the study , including the assessment of central position and dispersion ( two - tailed 95% confidence interval ) .", "the kolmogorov - smirnov test was applied to check adjustment of data to a gaussian distribution .", "student t tests and chi - square test were used for continuous and categorical variables , respectively , to test homogeneity of baseline data between groups .", "analysis of covariance ( ancova ) or binary logistic regression models were fitted comparing dsm - iv criteria versus broad criteria groups in most variables adjusting by baseline scoring , sex , age , body mass index , educational level , marital status and percentage of patients with comorbid major depression or depressive disorder .", "analysis of variance ( anova ) was applied for quantitative measures and chi square ( mcnemar s test ) for qualitative measures in case of paired comparisons .  ", "the clinical relevance of observed differences in baseline characteristics was ascertained by means of the effect size that was calculated using the difference of means for each group , divided by the combined standard deviation of the variables .", "qalys gain after the 6-months follow - up was calculated from patients responses to the eq-5d as described previously using tariff calculated with this instrument .", "gained qalys were estimated by using the trapezoidal approach , with baseline and 6- month visit values used as reference values .  ", "all statistical tests were two - tailed , and  error of < 0.05 was accepted as statistically significant ."], ["this was a multicentre , prospective and observational study carried out in outpatient psychiatric clinics between october 2007 and january 2009 .", "the study was approved by the local ethics committee of the hospital clnico de san carlos ( madrid ) and was conducted according to the helsinki declaration for research in the human being .", "functional and clinical outcome measures were completed in all three visits and included the following instruments : hamilton anxiety rating scale , montgomery - asberg depression rating scale ( baseline and 6 month visit only ) , clinical global impression - severity of illness scale , sleep scale from medical outcomes study ( mos - sleep ) , world health organization disability assessment schedule ii ( baseline and 6 month visit only ) , and the quality of life questionnaire , eq-5d ( all described in detail below ) . at baseline , socio - demographic data ,", "current therapy and psychiatric and medical illnesses information were collected . for the 3 and 6 months visits ,", "psychiatrists participating in the study made a confirmatory diagnosis of gad in 6 consecutive patients , 3 according to dsm - iv criteria and 3 according to broad criteria .", "although patients could have symptoms and receive treatment before entering the study , they had nt been diagnosed before by the psychiatrist ."], ["eligible patients were men and women over 18 years of age that were diagnosed with gad by a trained psychiatrist at the baseline visit .", "exclusion criteria included previous gad diagnosis , inability or difficulty to understand patient - reported - outcomes questionnaires written in spanish .", "diagnosis was carried out by a trained psychiatrist , familiar with the study procedures , and who worked in an outpatient setting .", "sampling frame included all public and private healthcare settings within the 17 regions of spain .", "first stage consisted on a selection of the outpatient psychiatry clinics ( mental health centres ) in each healthcare region .", "the number of clinics selected in each region was proportional to the region s population , being the probability of choosing each clinic relative to the population in the area covered by the clinic . in the second stage ,", "one psychiatrist per clinic was chosen randomly within those with previous experience in clinical and epidemiological research in psychiatry , and with at least 5 year experience in mental health diseases diagnosis .", "if a selected psychiatrist refused to participate , he or she was replaced by another from the same clinic ( also selected randomly ) .", "participant psychiatrists were asked to invite in a consecutive manner those patients from the daily list of appointments that fulfilled the inclusion criteria .", "psychiatrists were encouraged to maintain the above mentioned proportion of 3 subjects diagnosed following dms - iv criteria and 3 subjects diagnosed following the broad criteria .  ", "sample size was calculated taking into account study s main variable : evolution of anxiety symptoms according to the total ham - a scores for each patient group .", "a sample of 3400 evaluable patients was estimated assuming a 2-tailed 95% confidence interval for the total ham - a scores lower than 0.25 points from the observed mean .", "based on the results of a previous study , a standard deviation lower than 8 was assumed ."], ["gad was diagnosed following the dsm - iv criteria ( dsm - iv criteria group ) in one group .", "the other group ( broad criteria group ) was diagnosed according to broader criteria consisting on shortening of anxiety duration from  at least 6 months  to  at least 1 month ( but less than 6 months )  , including excessive or non - excessive worry and reducing the associated symptoms from a minimum of 3 to a minimum of 2 ."], ["hamilton anxiety rating scale ( ham - a ) : the validated spanish version of the ham - a scale was used to evaluate the evolution of anxiety symptoms during all 3 visits of the study .", "the ham - a scales includes 14 items , each of them rates from 0 ( absence ) to 4 ( severe ) .", "the total score comes from the addition of all 14 items with a maximum of 56 .", "scores higher than 24 were considered as severe anxiety ; between 16 and 24 , moderate ; between 10 and 15 , mild ; and below 9 , no anxiety . a psychic anxiety sub domain ( addition of items 1 - 6 and 14 ) and a somatic anxiety sub domain ( addition of items 7 - 13 )", "montgomery - asberg depression rating scale ( madrs ) : the validated spanish version of the madrs scale was used to measure the intensity of depression symptoms .", "this observer - rated depression scale consists on 10 items regarding different depressive symptoms , each item being scored from 0 to 6 depending on the severity of symptoms ; the total score results from adding the score of each item , obtaining a minimum of 0 ( no symptoms ) and a maximum of 60 ( very severe ) .", "clinical global impression - severity of illness scale ( cgi - s ) : participant psychiatrists evaluated patient s global severity with the cgi - s scale , which rates illness severity from 0 to 7 , where 0 was considered as  not evaluated ", ", 1 as  no disease  , 2  borderline ill  , 3  mildly ill  , 4  moderately ill  , 5  markedly ill  , 6  severely ill  , and 7  extremely ill  .", "sleep scale from medical outcomes study ( mos - sleep ) : mos - sleep scale evaluates the impact or interference with sleep of any disease or treatment .", "the 7 subscales are sleep disturbance ( 4 items ) , snoring ( 1 item ) , awaken short of breath or with headache ( 1 item ) , quantity of sleep ( 1 item ) , optimal sleep ( 1 item ) , sleep adequacy ( 2 items ) , and somnolence ( 3 items ) .", "this scale also has a sleep problems index , rated from 0 ( no interference ) to 100 ( maximum interference ) , and a sleep problems subscale .", "each item is rated independently with more interference scoring higher , except for the sleep adequacy and quantity and optimal sleep subscales [ 21 , 22 ] .", "world health organization disability assessment schedule ii ( who - das ii ) : the who - das ii is a psychometric instrument that measures day to day functioning and disability in the following six activity domains : understanding and communicating , moving and getting around , self care , getting along with people , work activities , and participation in society .", "the 12-item who - das ii scale is rated from 1 ( none or nothing ) to 5 ( extreme or inability to perform a given task ) . the total calculated score ranges from 0 to 100 , with higher scores indicating higher degrees of disability , for the total scale as well as for each subscale .", "eq-5d : this is a standard self - reported quality of life questionnaire developed in several european countries and validated for spain .", "this questionnaire consists of two parts , the first one aims to evaluate patient s health profile in five dimensions ( mobility , self - care , daily living activities , pain , and anxiety / depression ) .", "the patient rates his / her current status as no problems , some / moderate problems , and severe / extreme problems for each dimension .", "results are then converted into 1 of the 243 different eq-5d health state descriptions which are used to calculate a unique index or tariff , between 1 ( as healthy as possible ) and 0 ( usually equivalent to death ) .", "this index is used to calculate the time - trade off tariff which allows calculating quality - adjusted - life - year ( qaly ) gain ( further explanation below ) .", "the second part consists on a visual analogue scale ( vas or thermometer ) in which the patient has to rate his health status between 0 ( equivalent to death ) and 100 ( the best possible healthy status ) ."], ["for statistical analysis , last observation carried forward approach was used for those patients for whom data from the 6 months visit were missing .", "if the only missing data was that from the 3 months visit , this was estimated proportionally to the change observed at the 6 months visit , given that the missing data at 3 month visit were completely at random .", "patients were considered to respond to treatment when a higher than 50% reduction was observed for the total ham - a scale score at the study final visit when compared to that of the baseline visit .", "descriptive statistics were prepared for the continuous variables in the study , including the assessment of central position and dispersion ( two - tailed 95% confidence interval ) .", "the kolmogorov - smirnov test was applied to check adjustment of data to a gaussian distribution . for categorical variables", "student t tests and chi - square test were used for continuous and categorical variables , respectively , to test homogeneity of baseline data between groups .", "analysis of covariance ( ancova ) or binary logistic regression models were fitted comparing dsm - iv criteria versus broad criteria groups in most variables adjusting by baseline scoring , sex , age , body mass index , educational level , marital status and percentage of patients with comorbid major depression or depressive disorder .", "analysis of variance ( anova ) was applied for quantitative measures and chi square ( mcnemar s test ) for qualitative measures in case of paired comparisons .  ", "the clinical relevance of observed differences in baseline characteristics was ascertained by means of the effect size that was calculated using the difference of means for each group , divided by the combined standard deviation of the variables .", "qalys gain after the 6-months follow - up was calculated from patients responses to the eq-5d as described previously using tariff calculated with this instrument .", "gained qalys were estimated by using the trapezoidal approach , with baseline and 6- month visit values used as reference values .  ", "all statistical tests were two - tailed , and  error of < 0.05 was accepted as statistically significant ."], ["a total of 3,549 patients were finally recruited by 618 psychiatrists in spanish psychiatric clinics for this observational study .", "four hundred and fifty three patients ( 12.8% ) were excluded because they did not meet all inclusion criteria .", "remaining patients were diagnosed of gad according to dsm - iv criteria ( 1,815 patients ) or to broad criteria ( 1,281 patients ) as described on the methods section .", "a total of 529 ( 17.0% ) patients drop - out from study , with no significant differences between study groups ( dsm - iv criteria : 313 patients , broad criteria : 216 patients ; p=0.899 ) ( fig .", "however , main reasons for drop - outs were a somewhat different between groups with 52.7% lost to follow - up in dsm - iv group , compared with 67.1% in broad criteria group ( p=0.033 ) , and unknown reasons in 22.0% of withdrawals in dsm - iv group compared with 13.6% in broad criteria group ( p=0.023 ) .", "the mean age was 45.5 years for the dsm - iv group and slightly lower for the broad criteria group ( 42.9 years , p<0.001 vs. dsm - iv group ) .", "regarding gad , all features studied were slightly , but significantly higher in the standard dsm - iv criteria group .", "thus , the mean number of anxiety symptoms was 4.7 for the dsm - iv group compared with 4.3 for the broad criteria group ( p<0.001 ) .", "significant differences were also observed in the percentage of patients presenting with each individual gad symptom . all ham - a scores indicated severe anxiety on both patient groups , being the scores in the dsm - iv group significantly higher ( table 1 ) .", "likewise , statistical differences were observed at baseline in the cgi and madrs scales between the two groups .", "however , the magnitude of the effect was low for all quantitative variables at baseline ( effect size < 0,50 ) so the statistically significant differences observed were of small or negligible clinical relevance .  ", "most patients in both groups ( 87.9% for dsm - iv ; 82.0% for broad criteria ) were following a pharmacological treatment in the six months previous to the baseline visit .", "( 2 ) , benzodiazepines were the most commonly used drugs followed by antidepressants and antiepileptics . at the end of the study", ", there was an increase in the number of patients under pharmacological treatment ( 97% in both groups , p<0.001 versus baseline in all cases ) .", "the use of antiepileptic drugs increased by 3.2 and 3.8 fold in the dsm - iv and broad criteria groups , respectively , by the end - of - trial ( p<0.001 in all cases ) .", "the percentage of antiepileptic drugs users in dsm - iv group at baseline and at 6 months was significantly higher , albeit only slightly ( p=0.002 in both cases , fig .", "both groups showed a significant reduction in the percentage of benzodiazepines users after 6 months of follow - up ( p<0.001 ) , being this reduction even higher in the broad criteria group ( p=0.011 ) .", "also a slight but statistically significant increase was observed in the percentage of anti - depressive users in both groups ( p<0.001 , see fig .", "the percentage of subjects in dsm - iv and broad criteria groups that had at least one psychiatric comorbidity was 51.4% and 43.7% , respectively , ( p<0.001 ) . table 2 describes the most frequent psychiatric comorbidities and medical disorders in both study groups .", "however , neither the mean number of comorbid psychiatric disorders   nor the medical illnesses were different between both criteria groups , except for comorbid major depression and other depressive disorders which were slightly more frequent , but statistically significant in dsm - iv group ( p<0.01 and p=0.05 , respectively , table 2 ) . regarding psychiatric disorders , most of them were present around 5 months before the diagnosis of gad , without significant differences between groups in disease evolution . major depression and panic disorders were the most frequent comorbid psychiatric disorders in both study groups . for medical illnesses ,", "chronic pain was present in more than half of patients in both groups , although a higher percentage was seen in dsm - iv group ; 64.3% versus 52.0% , p<0.001 ( table 2 ) .", "anxiety severity was assessed with ham - a and cgi - s scales at 3 and 6 month visits .", "( 3 ) shows the evolution of ham - a total and sub - scores over study time in the dsm - iv and broad criteria groups .", "all ham - a scores improved after 3 and 6 months in both criteria groups .", "moreover , the percentage of patients without symptoms of anxiety , showing ham - a<9 , increased up to 22.3% and 31.4% at the 3 month visit for the dsm - iv and broad criteria groups ( p<0.001 vs. basal visit in both groups , and p=0.006 between groups ) , and up to 49% and 58% at the 6 month visit ( p<0.001 vs. basal visit in both groups , and p=0.261 between groups ) , indicating similar results for both groups at the end of the study . also , patients responding to anxiety treatment increased when considering the responder criteria ( > 50% reduction of baseline scores ) ; 30.7% and 38.2% for the dms - iv and broad criteria , respectively , at the 3 months visit ( p= 0.004 ) ; increasing up to 59.7% in the dms - iv and 67.7% at the 6 month visit ( p=0.103 ) .", "4 ) , differences between criteria groups were statistically significant at the study time points . regarding each gad symptom , fig .", "( 5 ) shows the reduction in the percentage of patients presenting with each anxiety symptom .", "however , higher significant reductions were observed in dsm - iv group for the following symptoms : fatigue , irritability and sleep disturbance ( p<0.05 in all cases , fig . 5 ) . in this study", ", depression symptoms were evaluated using the madrs . when compared to scores at baseline visit ( table 1 )", ", patients in both criteria groups improved their madrs scores with a mean reduction of 12.1 and 12.5 points for the dms - iv and broad criteria groups , respectively ( p=0.264 dsm - iv vs. broad criteria ) .", "mos - sleep scale scores revealed an overall improvement in sleep patterns over study time in both criteria group .", "as described in table 3 , all sleep domains in the mos - sleep scale changed significantly after 6 months when compared to basal visit scores .", "differences between the dsm - iv and broad criteria groups were observed for the total score ( p= 0.018 ) , sleep disturbance domain ( p=0.012 ) , sleep quantity ( p=0.016 ) , sleep adequacy ( p=0.031 ) , and sleep problems ( p=0.019 ) scores , always favouring the broad criteria group .  ", "health - related quality of life was measured by the eq-5d questionnaire . as seen in table 3 , both the health status ( visual analogue scale ) and the frequency of severe problems in each domain had significantly improved at the 6 month visit in both study groups ( p<0.001 in all cases ) . also , comparisons between both criteria groups in quality - of - life gain by domain were significant in favour of the broad criteria group in health status , daily living activities and mood status .", "mean qalys gain at the 3 month and 6 month visits were 0.034 and 0.103 years , respectively , for the dsm - iv criteria group and 0.034 ( 3 month ) and 0.101 ( 6 month ) for the broad criteria patients , without statistical significance between groups ( table 3 ) . at the 6 month visit , the percentage of patients reporting an improvement in their health status as compared with that of one year", "earlier increased by 13.8% for the dsm - iv criteria group , and 16.6% for the broad criteria group ( differences not significant ) . for measuring the disability degree in both groups of patients ,", ", there was a significant improvement in all 7 domains of the who - das ii scale after the 6 month follow - up in both criteria groups .", "the improvements were not significantly different in the dsm - iv group when compared to the new criteria group except for household activity domain , in which the improvement in the dsm - iv group was significantly better when compared to the broad criteria group .", "nevertheless , a trend toward statistical significance was observed in the total scoring of the disability scale favouring broad criteria group ."], ["a total of 3,549 patients were finally recruited by 618 psychiatrists in spanish psychiatric clinics for this observational study .", "four hundred and fifty three patients ( 12.8% ) were excluded because they did not meet all inclusion criteria .", "remaining patients were diagnosed of gad according to dsm - iv criteria ( 1,815 patients ) or to broad criteria ( 1,281 patients ) as described on the methods section .", "a total of 529 ( 17.0% ) patients drop - out from study , with no significant differences between study groups ( dsm - iv criteria : 313 patients , broad criteria : 216 patients ; p=0.899 ) ( fig .", "however , main reasons for drop - outs were a somewhat different between groups with 52.7% lost to follow - up in dsm - iv group , compared with 67.1% in broad criteria group ( p=0.033 ) , and unknown reasons in 22.0% of withdrawals in dsm - iv group compared with 13.6% in broad criteria group ( p=0.023 ) .", "the mean age was 45.5 years for the dsm - iv group and slightly lower for the broad criteria group ( 42.9 years , p<0.001 vs. dsm - iv group ) .", "regarding gad , all features studied were slightly , but significantly higher in the standard dsm - iv criteria group .", "thus , the mean number of anxiety symptoms was 4.7 for the dsm - iv group compared with 4.3 for the broad criteria group ( p<0.001 ) .", "significant differences were also observed in the percentage of patients presenting with each individual gad symptom . all ham - a scores indicated severe anxiety on both patient groups , being the scores in the dsm - iv group significantly higher ( table 1 ) .", "likewise , statistical differences were observed at baseline in the cgi and madrs scales between the two groups .", "however , the magnitude of the effect was low for all quantitative variables at baseline ( effect size < 0,50 ) so the statistically significant differences observed were of small or negligible clinical relevance ."], ["most patients in both groups ( 87.9% for dsm - iv ; 82.0% for broad criteria ) were following a pharmacological treatment in the six months previous to the baseline visit .", "( 2 ) , benzodiazepines were the most commonly used drugs followed by antidepressants and antiepileptics . at the end of the study", ", there was an increase in the number of patients under pharmacological treatment ( 97% in both groups , p<0.001 versus baseline in all cases ) . the use of antiepileptic drugs increased by 3.2 and 3.8 fold in the dsm - iv and broad criteria groups , respectively , by the end - of - trial ( p<0.001 in all cases ) .", "the percentage of antiepileptic drugs users in dsm - iv group at baseline and at 6 months was significantly higher , albeit only slightly ( p=0.002 in both cases , fig .", "both groups showed a significant reduction in the percentage of benzodiazepines users after 6 months of follow - up ( p<0.001 ) , being this reduction even higher in the broad criteria group ( p=0.011 ) . also a slight but", "statistically significant increase was observed in the percentage of anti - depressive users in both groups ( p<0.001 , see fig ."], ["at the study entry , the percentage of subjects in dsm - iv and broad criteria groups that had at least one psychiatric comorbidity was 51.4% and 43.7% , respectively , ( p<0.001 ) .", "table 2 describes the most frequent psychiatric comorbidities and medical disorders in both study groups .", "however , neither the mean number of comorbid psychiatric disorders   nor the medical illnesses were different between both criteria groups , except for comorbid major depression and other depressive disorders which were slightly more frequent , but statistically significant in dsm - iv group ( p<0.01 and p=0.05 , respectively , table 2 ) . regarding psychiatric disorders , most of them were present around 5 months before the diagnosis of gad , without significant differences between groups in disease evolution .", "major depression and panic disorders were the most frequent comorbid psychiatric disorders in both study groups . for medical illnesses ,", "chronic pain was present in more than half of patients in both groups , although a higher percentage was seen in dsm - iv group ; 64.3% versus 52.0% , p<0.001 ( table 2 ) ."], ["anxiety severity was assessed with ham - a and cgi - s scales at 3 and 6 month visits . fig .", "( 3 ) shows the evolution of ham - a total and sub - scores over study time in the dsm - iv and broad criteria groups .", "all ham - a scores improved after 3 and 6 months in both criteria groups .", "moreover , the percentage of patients without symptoms of anxiety , showing ham - a<9 , increased up to 22.3% and 31.4% at the 3 month visit for the dsm - iv and broad criteria groups ( p<0.001 vs. basal visit in both groups , and p=0.006 between groups ) , and up to 49% and 58% at the 6 month visit ( p<0.001 vs. basal visit in both groups , and p=0.261 between groups ) , indicating similar results for both groups at the end of the study . also , patients responding to anxiety treatment increased when considering the responder criteria ( > 50% reduction of baseline scores ) ; 30.7% and 38.2% for the dms - iv and broad criteria , respectively , at the 3 months visit ( p= 0.004 ) ; increasing up to 59.7% in the dms - iv and 67.7% at the 6 month visit ( p=0.103 ) .", "4 ) , differences between criteria groups were statistically significant at the study time points . regarding each gad symptom , fig .", "( 5 ) shows the reduction in the percentage of patients presenting with each anxiety symptom .", "however , higher significant reductions were observed in dsm - iv group for the following symptoms : fatigue , irritability and sleep disturbance ( p<0.05 in all cases , fig . 5 ) . in this study ,", "depression symptoms were evaluated using the madrs . when compared to scores at baseline visit ( table 1 )", ", patients in both criteria groups improved their madrs scores with a mean reduction of 12.1 and 12.5 points for the dms - iv and broad criteria groups , respectively ( p=0.264 dsm - iv vs. broad criteria ) ."], ["mos - sleep scale scores revealed an overall improvement in sleep patterns over study time in both criteria group .", "as described in table 3 , all sleep domains in the mos - sleep scale changed significantly after 6 months when compared to basal visit scores .", "differences between the dsm - iv and broad criteria groups were observed for the total score ( p= 0.018 ) , sleep disturbance domain ( p=0.012 ) , sleep quantity ( p=0.016 ) , sleep adequacy ( p=0.031 ) , and sleep problems ( p=0.019 ) scores , always favouring the broad criteria group .  ", "health - related quality of life was measured by the eq-5d questionnaire . as seen in table 3 , both the health status ( visual analogue scale ) and the frequency of severe problems in each domain had significantly improved at the 6 month visit in both study groups ( p<0.001 in all cases ) . also , comparisons between both criteria groups in quality - of - life gain by domain were significant in favour of the broad criteria group in health status , daily living activities and mood status .", "mean qalys gain at the 3 month and 6 month visits were 0.034 and 0.103 years , respectively , for the dsm - iv criteria group and 0.034 ( 3 month ) and 0.101 ( 6 month ) for the broad criteria patients , without statistical significance between groups ( table 3 ) .", "at the 6 month visit , the percentage of patients reporting an improvement in their health status as compared with that of one year earlier increased by 13.8% for the dsm - iv criteria group , and 16.6% for the broad criteria group ( differences not significant ) . for measuring the disability degree in both groups of patients , the who - das scale was used . as shown in table 4 , there was a significant improvement in all 7 domains of the who - das ii scale after the 6 month follow - up in both criteria groups .", "the improvements were not significantly different in the dsm - iv group when compared to the new criteria group except for household activity domain , in which the improvement in the dsm - iv group was significantly better when compared to the broad criteria group .", "nevertheless , a trend toward statistical significance was observed in the total scoring of the disability scale favouring broad criteria group ."], ["this real world prospective study in usual medical practice has shown that broadening of gad dsm - iv diagnostic criteria , by reducing the duration of excessive or non - excessive worry to one month and associated symptoms to two , does not significantly affect patient s responsiveness to psychiatrist - prescribed therapy .", "the patient sample of this study seems to be similar to other gad populations presented in previous studies , with a higher prevalence among women and among those aged above 25 years [ 4 , 18 ] .", "the most frequent comorbid psychiatric disorder was major depression ; however , its presence was somewhat smaller than the observed in other studies , if we take into account major depression only .  ", "the main goal when treating a gad patient is to improve the symptoms , hopefully leading in the long - term to their complete remission and to recovery of patient s functionality .", "several guidelines recommend antidepressants , such as the selective serotonin reuptake inhibitors and the serotonin noradrenalin reuptake inhibitors , and antiepileptics as first line treatments , and benzodiazepines as second - line treatment [ 28 , 29 ] .", "although patients in this study received a confirmatory diagnosis of gad , a high percentage of them were already following a therapy recommended for gad at baseline .", "these results suggest that , in our sanitary context , general practitioners and/or family physicians start treatment to control anxiety symptoms , mostly with benzodiazepines , at patients first complaint .", "the general practitioner / family physician usually refers those patients with lower treatment response than expected to the psychiatrist .", "it should be taken into account that refractory patients may be difficult to manage at the primary care level in the spanish health system .", "one of the reasons is the brief period of time devoted by physicians to each patient visit in the spanish primary health care level , as described by duran et al . .", "other studies have explored the consequences of broadening dsm - iv diagnostic criteria for gad [ 3 , 13 , 15 - 16 , 31 ] .", "most of them have considered a reduction in the duration criteria [ 3 , 16 ] , the excessiveness of worry , and the number of associated symptoms . a possible", "learning from all of them is that the socio - demographic characteristics between the populations diagnosed with broad or strict dsm - iv criteria are not substantially different . as expected , the broadening of dsm - iv gad criteria results in an increase in the point - prevalence to more than double [ 15 - 16 ] .", "concluded that the increase was mainly due to the reduction of minimum duration to one month . in regards to gad s severity , ruscio \n et al .   observed a tendency to reduced severity for the broad criteria group , however", "we have observed a similar tendency suggesting that patients diagnosed with broader gad criteria present a slightly less severe form of the disease .  ", "every study that has investigated the possibility of broadening the dsm - iv criteria has done it retrospectively [ 13 , 15 , 16 , 27 , 31 , 32 ] .", "the present study is the first one to compare two groups of gad patients with dsm - iv and broader criteria following a prospective design .", "according to our results , broadening of gad diagnostic criteria does not affect the response to the prescribed therapy , since ham - a scores , total and domains , improved similarly in both groups , and so did the presence of gad symptoms ( fig .", "a significant improvement was perceived by physicians ( cgi - s scores ) at the end of the study .", "the influence of broader gad diagnostic criteria in patient s disability has been studied previously by lee \n et al .", "lee et al .   observed an increase in the sheehan disability scale score when increasing time for the duration criteria although it was not statistically significant .", "the same observation was made by kessler et al .   with data from the ncs .", "parallel to their clinical improvement , patients in our study experienced an improvement in the studied quality of life variables .", "health status improvements in our study were similar to those observed in primary care patients receiving treatment over six months for their gad .", "the biggest improvements were observed in the daily living activities and mood domains of the eq-5d .", "qalys gain observed corresponded to more than one month of perfect health for both groups ; which were similar in magnitude to that observed in other health conditions such as trigeminal neuralgia , successfully treated with pregabalin .", "all other dimensions of functionality included in the who das - ii scale , and sleep scale were substantially ameliorated in both study groups by the end - of - trial ; however , the differences in the magnitude of these improvements for both groups were hardly meaningful from a clinical point of view .  ", "results from this study should be interpreted bearing in mind its observational design with its inherent limitations . since this study", "is based on out - patient psychiatric clinics , patients included in this study might not be representative of the whole gad population , since psychiatrists could deal with more severe and refractory cases .", "also , due to the high degree of expertise required for participant psychiatrists , diagnosis in our study may have been more accurate than in other settings , such as primary care", ". a consequence of broadening gad criteria in other healthcare settings could be the difficulty in distinguishing gad from adjustment disorders or situational stress reactions by other less trained physicians . in this study", "there was a high proportion of patients treated with antiepileptics ; that could be explained by the fact that most subjects included in the study were non responders to recommended first line drug treatment according to the european guidelines on the treatment and management of gad patients .", "finally , as this was a real world prospective study we did not have a control group so further clinical research with randomized controlled studies and newly diagnosed patients needs to be carried out to confirm the consequences of broadening the criteria ."], ["broadening criteria for gad diagnosis , by reducing excessive and non - excessive anxiety and worry duration to one month and the number of associated symptoms to two , captures a similar population of patients compared to the one identified by applying standard dsm - iv criteria .", "broadening diagnostic criteria is able to identify the core symptoms of gad according to the dsm - iv criteria and could lead to an earlier diagnosis ."], ["funding for this study was provided by pfizer spain ; pfizer spain participated in the idea and design of the study .", "enrique lvarez , jose l. carrasco and jos m. olivares declare not to have any conflict of interests as a consequence of participating in this study .", "inma vilardaga was employed of the european biometric institute , a consulting agency which was responsible for logistic of study and analysis of data .", "mara perez and vanessa lpez - gmez are employed by pfizer spain , the body funding the study ."]]}
{"article_id": "PMC2837295", "article_text": ["renal clear cell carcinomas represent about 3% of all visceral cancers and account for 85% of renal cancers in adults .", "the tumours occur most often in older individuals , usually in the sixth and seventh decades of life , and are often diagnosed at incurable stages . in western countries", "the frequency of renal cancer remains relatively high , there being approximately 30,000 new cases and 12,000 deaths per year from the disease .", "the causes of kidney cancer are believed to be environmental ( such as cigarette smoking , asbestos , petroleum products , heavy metals , unopposed oestrogen therapy , hypertension and obesity ) , genetic or a mixture of both .", "to date there are 19 hereditary syndromes described in which renal cell cancer may occur ( table 1 ) .", "genetic syndromes characterised by an increased risk of renal cancer ( familial cancer database - facd , http://facd.uicc.org ) the identification of genetic predispositions to renal cell cancer remains a priority since knowledge about the underlying molecular genetic basis of the disease will allow for a better understanding of the mechanisms giving rise to the disease and , perhaps more importantly , allow for the identification of individuals who are at risk of disease development .", "there are two aspects of these criteria that can be problematic in the clinical setting with respect to the identification of familial renal cell cancer patients .", "the first is the difficulty in fulfilling criteria in countries where large families and extensive pedigrees are impossible to identify , for whatever reason , even though the incidence of hereditary renal cell cancer may be quite high .", "second , the criteria do not take into consideration the existence of family cancer syndromes where renal cell cancer may occur in association with an extra - gastric malignancy . from a clinical perspective", ", there is a necessity to be able to identify hereditary renal cell cancer families with a minimum set of criteria that will provide a high likelihood of ascertainment .", "the aim of this study was to determine whether a minimum set of criteria could be established to identify suspected hereditary renal cell cancer patients when there is restricted information about the familial occurrence of disease .", "a total of 146 clear cell renal carcinoma ( ccrc ) patients comprising 3 groups were enrolled in the study .", "group a ( familial renal cancer ) : comprising 46 patients affected by ccrc from 22 randomly selected families with at least two renal cancers among first or second degree relatives , independent of age at diagnosis of tumours .", "group a1 ( nuclear pedigree ) : comprising 25 patients affected by ccrc from group a. none of the parents of these patients have been diagnosed as affected with renal cell cancer .", "group b : a total of 100 individuals diagnosed with ccrc between the years 1993 and 1997 irrespective of family history were collected from the city of szczecin ( total population 400,000 ) .", "the following inclusion features ( if ) for the identification of suspected hereditary forms of clear cell renal cancer were used and compared against one another for their sensitivity and specificity : if 1 : at least one of the parents of the patient with ccrc was affected by lung cancer if 2 : at least one of the parents of the patient with ccrc was affected by gastric cancer if 3 : ccrc diagnosed at the age of 45 years or younger if 4 : ccrc diagnosed at the age of 50 years or younger if 5 : ccrc diagnosed at the age of 55 years or younger univariate statistical analysis ( chi - squared , odds ratio ( or ) , and sensitivity and specificity of selection were performed using the sas and logit programs .", "univariate statistical analysis ( chi - squared , odds ratio ( or ) , and sensitivity and specificity of selection were performed using the sas and logit programs .", "the comparison of the five ifs was undertaken to identify the most consistent criteria that can be employed in a clinical setting for the identification of suspected hereditary renal cell cancer , based on nuclear pedigree data .", "the first comparison was between group a ( associated with a genetic predisposition to disease ) compared to unselected cases from group b ( table 2 ) .", "the results indicate that all inclusion features are more frequent in group a ( or 1.62 - 4.88 ) .", "the second comparison was performed between group a1 and group b ( table 3 ) .", "the results indicate that there is a very strong correlation between hereditary ( familial ) predisposition to ccrc and occurrence of at least one of the following ifs : if1 , if2 or if5 - or 13.4 ; p < 0.00001 .", "the recognition of features that can be used for the identification of familial predispositions to ccrc in situations where extensive pedigree analysis is unknown or impossible to ascertain but the prevalence of the disease is relatively high in the population will aid in the identification of individuals at increased risk of developing ccrc . by using the criteria", "described herein and the consequent recognition of significant odds ratios for some of the inclusion features to identify ccrc families , we believe that the identification of additional genes associated with this malignancy will be expedited .", "of particular interest are the odds ratio values for the inclusion features if5 between groups a1 and b and if 1 between groups a1 and b , which were relatively high ( 6.21 and 6.09 , respectively ) .", "since these inclusion features are significant we have a relatively high degree of confidence that the reported observations are not biased and are an accurate reflection of the validity of our approach for the identification of hereditary ccrc families .", "indeed , these criteria have been tested in our outpatient clinics to successfully identify hereditary ccrc . therefore if we have families matching if1 or if2 or if5 , we are confident that a diagnosis of familial ccrc can be made . at present", "it seems reasonable to offer the option of ultrasonography examination to all individuals identified by the use of our inclusion features beginning at the age of 5 to 10 years before the youngest ccrc identified within the patient 's family .", "such surveillance should only be an option and not a recommendation because the efficiency of such management procedures has not been rigorously determined to reduce morbidity and/or mortality .", "with respect to surveillance the real value of this will have to be established by studies on large cohorts of individuals from families matching pedigree and clinical criteria of suspected hereditary ccrc with identified constitutional dna variants associated with genetic predispositions .", "so far , the list of genetic changes associated with ccrc is somewhat limited but should be extended in the near future as more knowledge is gained about the genetic factors associated with altered ccrc predisposition . in summary , we advocate the use of our criteria for suspected hereditary clear cell renal cancer identified in this report in order to : a. offer an ultrasound examination option , b. create repositories of nuclear clear cell renal cancer families for future studies on the efficiency of surveillance for individuals with genetic predispositions to renal cancer , c. perform further studies to aid in the identification of genetic factors associated with ccrc ."], "abstract_text": ["<S> renal clear cell carcinomas represent about 3% of all visceral cancers and account for approximately 85% of renal cancers in adults . </S>", "<S> environmental and genetic factors are involved in the development of renal cancer . </S>", "<S> although to date there are 19 hereditary syndromes described in which renal cell cancer may occur , only four syndromes with an unequivocal genetic predisposition to renal cell carcinoma have been identified : vhl syndrome ( mutations in the vhl gene ) , hereditary clear cell carcinoma ( translocations t(3:8 ) , t(2:3 ) ) , hereditary papillary carcinoma ( mutations in the met protooncogene ) and tuberous sclerosis ( mutations in the tsc1 and tsc2 genes ) . </S>", "<S> little is known genetically about the other forms of familial renal cell cancer . since there is a growing awareness about the necessity of early intervention </S>", "<S> , clinical criteria have been developed that aid in the identification of hereditary forms of renal cancer . </S>", "<S> the aim of the current study was to identify minimal inclusion criteria so that nuclear pedigree families can be ascertained for risk assessment and/or kidney tumour screening . </S>", "<S> the results reveal that inclusion features described herein , such as ( a ) renal clear cell cancer diagnosed before 55 years of age , and ( b ) renal clear cell cancer and gastric cancer or lung cancer among first degree relatives , are useful in identifying suspected hereditary clear cell renal cancer patients . </S>"], "labels": null, "section_names": ["Introduction", "Patients and methods", "Statistical analysis", "Results", "Discussion"], "sections": [["renal clear cell carcinomas represent about 3% of all visceral cancers and account for 85% of renal cancers in adults .", "the tumours occur most often in older individuals , usually in the sixth and seventh decades of life , and are often diagnosed at incurable stages . in western countries", "the frequency of renal cancer remains relatively high , there being approximately 30,000 new cases and 12,000 deaths per year from the disease .", "the causes of kidney cancer are believed to be environmental ( such as cigarette smoking , asbestos , petroleum products , heavy metals , unopposed oestrogen therapy , hypertension and obesity ) , genetic or a mixture of both .", "to date there are 19 hereditary syndromes described in which renal cell cancer may occur ( table 1 ) .", "genetic syndromes characterised by an increased risk of renal cancer ( familial cancer database - facd , http://facd.uicc.org ) the identification of genetic predispositions to renal cell cancer remains a priority since knowledge about the underlying molecular genetic basis of the disease will allow for a better understanding of the mechanisms giving rise to the disease and , perhaps more importantly , allow for the identification of individuals who are at risk of disease development .", "there are two aspects of these criteria that can be problematic in the clinical setting with respect to the identification of familial renal cell cancer patients .", "the first is the difficulty in fulfilling criteria in countries where large families and extensive pedigrees are impossible to identify , for whatever reason , even though the incidence of hereditary renal cell cancer may be quite high .", "second , the criteria do not take into consideration the existence of family cancer syndromes where renal cell cancer may occur in association with an extra - gastric malignancy . from a clinical perspective", ", there is a necessity to be able to identify hereditary renal cell cancer families with a minimum set of criteria that will provide a high likelihood of ascertainment .", "the aim of this study was to determine whether a minimum set of criteria could be established to identify suspected hereditary renal cell cancer patients when there is restricted information about the familial occurrence of disease ."], ["a total of 146 clear cell renal carcinoma ( ccrc ) patients comprising 3 groups were enrolled in the study .", "group a ( familial renal cancer ) : comprising 46 patients affected by ccrc from 22 randomly selected families with at least two renal cancers among first or second degree relatives , independent of age at diagnosis of tumours .", "group a1 ( nuclear pedigree ) : comprising 25 patients affected by ccrc from group a. none of the parents of these patients have been diagnosed as affected with renal cell cancer .", "group b : a total of 100 individuals diagnosed with ccrc between the years 1993 and 1997 irrespective of family history were collected from the city of szczecin ( total population 400,000 ) .", "the following inclusion features ( if ) for the identification of suspected hereditary forms of clear cell renal cancer were used and compared against one another for their sensitivity and specificity : if 1 : at least one of the parents of the patient with ccrc was affected by lung cancer if 2 : at least one of the parents of the patient with ccrc was affected by gastric cancer if 3 : ccrc diagnosed at the age of 45 years or younger if 4 : ccrc diagnosed at the age of 50 years or younger if 5 : ccrc diagnosed at the age of 55 years or younger univariate statistical analysis ( chi - squared , odds ratio ( or ) , and sensitivity and specificity of selection were performed using the sas and logit programs ."], ["univariate statistical analysis ( chi - squared , odds ratio ( or ) , and sensitivity and specificity of selection were performed using the sas and logit programs ."], ["the comparison of the five ifs was undertaken to identify the most consistent criteria that can be employed in a clinical setting for the identification of suspected hereditary renal cell cancer , based on nuclear pedigree data .", "the first comparison was between group a ( associated with a genetic predisposition to disease ) compared to unselected cases from group b ( table 2 ) .", "the results indicate that all inclusion features are more frequent in group a ( or 1.62 - 4.88 ) .", "the second comparison was performed between group a1 and group b ( table 3 ) .", "the results indicate that there is a very strong correlation between hereditary ( familial ) predisposition to ccrc and occurrence of at least one of the following ifs : if1 , if2 or if5 - or 13.4 ; p < 0.00001 ."], ["the recognition of features that can be used for the identification of familial predispositions to ccrc in situations where extensive pedigree analysis is unknown or impossible to ascertain but the prevalence of the disease is relatively high in the population will aid in the identification of individuals at increased risk of developing ccrc . by using the criteria", "described herein and the consequent recognition of significant odds ratios for some of the inclusion features to identify ccrc families , we believe that the identification of additional genes associated with this malignancy will be expedited .", "of particular interest are the odds ratio values for the inclusion features if5 between groups a1 and b and if 1 between groups a1 and b , which were relatively high ( 6.21 and 6.09 , respectively ) .", "since these inclusion features are significant we have a relatively high degree of confidence that the reported observations are not biased and are an accurate reflection of the validity of our approach for the identification of hereditary ccrc families .", "indeed , these criteria have been tested in our outpatient clinics to successfully identify hereditary ccrc . therefore if we have families matching if1 or if2 or if5 , we are confident that a diagnosis of familial ccrc can be made . at present", "it seems reasonable to offer the option of ultrasonography examination to all individuals identified by the use of our inclusion features beginning at the age of 5 to 10 years before the youngest ccrc identified within the patient 's family .", "such surveillance should only be an option and not a recommendation because the efficiency of such management procedures has not been rigorously determined to reduce morbidity and/or mortality .", "with respect to surveillance the real value of this will have to be established by studies on large cohorts of individuals from families matching pedigree and clinical criteria of suspected hereditary ccrc with identified constitutional dna variants associated with genetic predispositions .", "so far , the list of genetic changes associated with ccrc is somewhat limited but should be extended in the near future as more knowledge is gained about the genetic factors associated with altered ccrc predisposition . in summary , we advocate the use of our criteria for suspected hereditary clear cell renal cancer identified in this report in order to : a. offer an ultrasound examination option , b. create repositories of nuclear clear cell renal cancer families for future studies on the efficiency of surveillance for individuals with genetic predispositions to renal cancer , c. perform further studies to aid in the identification of genetic factors associated with ccrc ."]]}
{"article_id": "PMC3483330", "article_text": ["intracranial hypotension syndrome ( ihs ) is characterized by a postural headache that is aggravated in the erect or sitting position and relieved in the supine position .", "it is often associated with one or more of the following symptoms : nausea , vomiting , dizziness , diplopia , photophobia , hearing impairment , neck stiffness , and blurred vision6 ) . acquired or secondary intracranial hypotension is less common but has been reported after spinal procedures such as lumbar discectomy and dural puncture for myelogram , spinal anesthesia .", "we report a case of a 54-year - old man who was successfully treated with epidural blood patches for intracranial hypotension due to csf leakage into the lumbosacral area after l4 - 5 discectomy .", "a 54-year - old male admitted to the er complaining of headache \n from 13 days ago .", "it did not occur when lying down but became intensely squeezing around the occipital area within 10 seconds after being seated or standing .", "past medical history was insignificant other than undergoing l4 - 5 laminectomy and discectomy at a local hospital 15 days ago .", "physical examination did not display any neurologic findings such as neck stiffness and laboratory results as well as brain mri findings were not remarkable . under the strong impression of intracranial hypotension", ", he admitted to neurology department and underwent conservative therapy including absolute bed rest ( abr ) and hydration .", "a mr myelogram was performed , showing a pseudomeningocele accompanied by large csf leakage in the lumbosacral area ( fig .", "because resolution of symptoms was unsatisfactory , an epidural blood patch was placed on the sixth admission day . after placing the patient in a prone position", ", a 22 gage touhy needle was inserted through the l5-s1 epidural space through a paraspinal method . after confirming the epidural space by the loss - of - resistance technique , a dye was injected to confirm the epidural space with the c - arm .", "after three days of abr following the procedure , the patient practiced changing positions by sitting .", "five days after the procedure , the patient did not complain of any symptoms ( vas 0 ) .", "2 ) . we decided to perform one more procedure of epidural patch placement because of the residual csf leakage 6 days after the first intervention .", "a total of 10 ml autologous blood was injected , and the patient was discharged the next day . follow up for six months confirmed no recurrence of symptoms ( fig .", "intracranial hypotension syndrome can occur spontaneously . acquired or secondary intracranial hypotension is less common but has been reported after spinal procedures .", "reported that the incidence of incidental durotomy during spine surgery was 3.84% in patients who underwent spinal surgery at a single spine unit5 ) . in one study looking into medicolegal aspects of spine surgery ,", "146 malpractice cases were reviewed and incidental durotomy was second most frequent complication in such cases4 ) .", "the study has shown that potentially serious problems such as pseudomeningocele , csf fistula formation , meningitis and arachnoiditis with subsequent chronic pain are all related to dural tears and csf leakage after spinal surgery .", "good long - term clinical results were noted in all patients with durotomy repaired at identification and are comparable to long - term results of patients undergoing similar surgical procedures but without durotomy . however , cammisa et al.1 ) reported the incidence of clinically significant durotomy occurred during surgery but not indentified at the time was 0.28% , and patients had subsequent surgical repair of dural defects because of failure of conservative therapy . in one study which investigated incidental durotomy during spine surgery and its treatment , in addition to primary repair , drain , bed rest , hydration , antibiotics had been used for treatment by spine surgeon7 ) .", "however to our knowledge , there have been no previous studies which compared the effects of epidural blood patch for the treatment of incidental durotomy with other treatments .", "epidural blood patch has been employed over the last five decades in the treatment of postdural puncture headache ( pdph ) and has proven to be beneficial .", "the mechanism by which an epidural blood patch ( ebp ) relieves pdph is not known .", "the \" plug \" theory for symptom resolution proposes that the blood injected during ebp forms a gelatinous plug , sealing the dural hole and preventing further csf leak into the epidural space . in the absence of continued loss , regeneration of csf restores csf pressure and alleviates the headache3 ) .", "the \" pressure patch \" hypothesis emphasizes the impact of injected blood or other fluid ( crystalloid or colloid ) on the pressure dynamics of the cns .", "the injected fluid increases epidural pressure which , in turn , elevates subarachnoid csf pressure by compressing the dura8 ) .", "the optimal volume of blood that must be injected is controversial but recommended volumes have increased over time .", "crawford 's initial experience using 6 - 15 ml produced a 30% failure rate while later experience with 20 ml produced a 96% success rate , so 20 ml became the recommended volume and has since become commonly cited as a \" target volume \" to enhance patch efficacy2 ) .", "however , the effects of ebp in intracranial hypotension are mostly focused on post - dural puncture during csf tapping or spontaneous intracranial hypotension .", "even patients with rather large amount of csf leakage and patients with severe complications such as intracranial hematomas have been effectively treated with ebp , so we considered epb for postoperative incidental durotomy due to such evidence .", "the effects of ebp in postoperative intracranial hypotension can not be discussed after one successful clinical trial , but additional trials and studies can provide the basis of epb treatment in such cases .", "when incidental durotomy was recognized postoperatively , conservative therapy or surgical correction has been the therapeutic choice .", "however , because there has been no report on indication for ebp according to the degree of dural injury or the size of peudomeningocele , additional research is needed . in our case ,", "the patient underwent l4 - 5 spine surgery at a local orthopedic , but dural injury was not recognized at that time . after admitting to our hospital ,", "there was no significant resolution of symptoms after conservative therapy including the five days of bed rest and hydration .", "epidural blood patch can be used as an effective treatment modality for csf leakage after lumbar spine surgery ."], "abstract_text": ["<S> intracranial hypotension syndrome typically occurs spontaneously or iatrogenically . </S>", "<S> it can be associated with headache , drowsy mentality and intracranial heamorrhage . </S>", "<S> iatrogenic intracranial hypotension can occur due to dural pucture , trauma and spine surgery . </S>", "<S> treatment may include conservative therapy and operation . </S>", "<S> we report a case of a 54-year - old man who was successfully treated with epidural blood patches for intracranial hypotension due to cerebrospinal fluid ( csf ) leakage into the lumbosacral area after spine surgery . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["intracranial hypotension syndrome ( ihs ) is characterized by a postural headache that is aggravated in the erect or sitting position and relieved in the supine position .", "it is often associated with one or more of the following symptoms : nausea , vomiting , dizziness , diplopia , photophobia , hearing impairment , neck stiffness , and blurred vision6 ) . acquired or secondary intracranial hypotension is less common but has been reported after spinal procedures such as lumbar discectomy and dural puncture for myelogram , spinal anesthesia .", "we report a case of a 54-year - old man who was successfully treated with epidural blood patches for intracranial hypotension due to csf leakage into the lumbosacral area after l4 - 5 discectomy ."], ["a 54-year - old male admitted to the er complaining of headache \n from 13 days ago .", "it did not occur when lying down but became intensely squeezing around the occipital area within 10 seconds after being seated or standing .", "past medical history was insignificant other than undergoing l4 - 5 laminectomy and discectomy at a local hospital 15 days ago .", "physical examination did not display any neurologic findings such as neck stiffness and laboratory results as well as brain mri findings were not remarkable . under the strong impression of intracranial hypotension", ", he admitted to neurology department and underwent conservative therapy including absolute bed rest ( abr ) and hydration .", "a mr myelogram was performed , showing a pseudomeningocele accompanied by large csf leakage in the lumbosacral area ( fig .", "because resolution of symptoms was unsatisfactory , an epidural blood patch was placed on the sixth admission day . after placing the patient in a prone position", ", a 22 gage touhy needle was inserted through the l5-s1 epidural space through a paraspinal method . after confirming the epidural space by the loss - of - resistance technique , a dye was injected to confirm the epidural space with the c - arm .", "after three days of abr following the procedure , the patient practiced changing positions by sitting .", "five days after the procedure , the patient did not complain of any symptoms ( vas 0 ) .", "2 ) . we decided to perform one more procedure of epidural patch placement because of the residual csf leakage 6 days after the first intervention .", "a total of 10 ml autologous blood was injected , and the patient was discharged the next day . follow up for six months confirmed no recurrence of symptoms ( fig ."], ["intracranial hypotension syndrome can occur spontaneously . acquired or secondary intracranial hypotension is less common but has been reported after spinal procedures .", "reported that the incidence of incidental durotomy during spine surgery was 3.84% in patients who underwent spinal surgery at a single spine unit5 ) . in one study looking into medicolegal aspects of spine surgery ,", "146 malpractice cases were reviewed and incidental durotomy was second most frequent complication in such cases4 ) .", "the study has shown that potentially serious problems such as pseudomeningocele , csf fistula formation , meningitis and arachnoiditis with subsequent chronic pain are all related to dural tears and csf leakage after spinal surgery .", "good long - term clinical results were noted in all patients with durotomy repaired at identification and are comparable to long - term results of patients undergoing similar surgical procedures but without durotomy . however , cammisa et al.1 ) reported the incidence of clinically significant durotomy occurred during surgery but not indentified at the time was 0.28% , and patients had subsequent surgical repair of dural defects because of failure of conservative therapy . in one study which investigated incidental durotomy during spine surgery and its treatment , in addition to primary repair , drain , bed rest , hydration , antibiotics had been used for treatment by spine surgeon7 ) .", "however to our knowledge , there have been no previous studies which compared the effects of epidural blood patch for the treatment of incidental durotomy with other treatments .", "epidural blood patch has been employed over the last five decades in the treatment of postdural puncture headache ( pdph ) and has proven to be beneficial .", "the mechanism by which an epidural blood patch ( ebp ) relieves pdph is not known .", "the \" plug \" theory for symptom resolution proposes that the blood injected during ebp forms a gelatinous plug , sealing the dural hole and preventing further csf leak into the epidural space . in the absence of continued loss , regeneration of csf restores csf pressure and alleviates the headache3 ) .", "the \" pressure patch \" hypothesis emphasizes the impact of injected blood or other fluid ( crystalloid or colloid ) on the pressure dynamics of the cns .", "the injected fluid increases epidural pressure which , in turn , elevates subarachnoid csf pressure by compressing the dura8 ) .", "the optimal volume of blood that must be injected is controversial but recommended volumes have increased over time .", "crawford 's initial experience using 6 - 15 ml produced a 30% failure rate while later experience with 20 ml produced a 96% success rate , so 20 ml became the recommended volume and has since become commonly cited as a \" target volume \" to enhance patch efficacy2 ) .", "however , the effects of ebp in intracranial hypotension are mostly focused on post - dural puncture during csf tapping or spontaneous intracranial hypotension .", "even patients with rather large amount of csf leakage and patients with severe complications such as intracranial hematomas have been effectively treated with ebp , so we considered epb for postoperative incidental durotomy due to such evidence .", "the effects of ebp in postoperative intracranial hypotension can not be discussed after one successful clinical trial , but additional trials and studies can provide the basis of epb treatment in such cases .", "when incidental durotomy was recognized postoperatively , conservative therapy or surgical correction has been the therapeutic choice .", "however , because there has been no report on indication for ebp according to the degree of dural injury or the size of peudomeningocele , additional research is needed . in our case ,", "the patient underwent l4 - 5 spine surgery at a local orthopedic , but dural injury was not recognized at that time . after admitting to our hospital ,", "there was no significant resolution of symptoms after conservative therapy including the five days of bed rest and hydration ."], ["epidural blood patch can be used as an effective treatment modality for csf leakage after lumbar spine surgery ."]]}
{"article_id": "PMC4004838", "article_text": ["pituitary insufficiency in childhood is often secondary to pituitary or hypothalamic \n space - occupying lesions .", "the differential diagnosis in etiologies of a mass in the sella is \n broad in spectrum , including rathke s cyst , tumor , autoimmune diseases , primary or \n metastatic neoplasms , and granulomatous inflammation . among them , xanthomatous pituitary \n diseases are rare in childhood .", "these conditions include xanthomatous hypophysitis , \n xanthogranulomatous hypophysitis and xanthogranuloma of the sellar region ( 1,2,3,4,5 ) .", "xanthogranuloma of the sellar region is characterized \n by a granulomatous lesion containing cholesterol clefts , macrophages , chronic inflammatory \n infiltrates , and hemosiderin deposit ( 6 ) .", "this \n condition has been traditionally regarded as a hallmark of the adamantinomatous \n craniopharyngioma even in the absence of epithelium ( 6 )", ". however , paulus et al . ( 7 ) suggested that \n xanthogranuloma is likely to constitute a different entity from the classical \n adamantinomatous craniopharyngioma clinically and pathologically .", "after their original \n report , several patients with xanthogranuloma of the sellar region have been described \n ( 8 , 9 ) . \n however , to our knowledge", ", there is no detailed clinical report of xanthogranuloma of the \n sellar region in cases of pediatric age . here ,", "we report the cases of two children with \n xanthogranuloma of the sellar region , presenting central diabetes insipidus ( di ) .", "a 9-yr - old japanese boy was referred to our clinic because of a 6-mo history of polyuria \n and polydipsia .", "his height was 135.3 \n cm ( + 0.7 sd ) and body weight 29.9 kg .", "the growth hormone ( gh ) releasing \n hormone ( ghrh ) and the thyroid stimulating hormone releasing hormone ( trh ) tolerance tests \n showed subnormal responses of gh and tsh , respectively ( table 1table 1 endocrinological findings of cases 1 and 2 ) .", "the corticotropin releasing hormone ( crh ) tolerance test showed normal \n responses of acth and serum cortisol ( table \n 1 ) . magnetic resonance imaging ( mri )", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "the pituitary stalk deviated \n to the frontal direction and the anterior pituitary gland was compressed by the tumor . \n", "there was no calcification of the mass detected by computed tomgraphic ( ct ) scan .", "cyst was suspected preoperatively by the mri findings , however a definite diagnosis could \n not be made .", "the capsule of the tumor \n had tightly adhered to the dura of the sellar floor .", "histological examination of the tumor showed fibrous tissue with cholesterol \n clefts , macrophages , multinucleated giant cells , and lymphocyte infiltration ( fig .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed .", "postoperatively , di remains and has been treated with desmopessin \n acetate ( ddavp ) .", "two months after surgery , gh and tsh responses after stimulation tests \n recovered ( table 1 ) .", "after one - year follow - up , the patient continues to have di , but his basal \n levels of igf-1 , ft3 , ft4 and tsh are within normal ranges .   radiological appearance on magnetic resonance imaging ( mri ) .", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed .", "a 6-yr - old japanese boy was admitted to our hospital for evaluation of polyuria and \n polydipsia of 6-mo duration .", "his \n height was 113.2 cm ( 0.8 sd ) and body weight was 18.8 kg .", "brain mri showed an intrasellar mass , which was high on the t1-weighted ( fig .", "gadolinium \n administration caused no enhancement of the mass . ) and hypointense on the t2-weighted images ( fig . \n", "ghrh , trh , and crh \n tolerance tests showed normal responses one month after surgery ( table 1 ) .", "the patient is currently well only on ddavp \n medication , 12 mo after surgery .", "a 9-yr - old japanese boy was referred to our clinic because of a 6-mo history of polyuria \n and polydipsia .", "his height was 135.3 \n cm ( + 0.7 sd ) and body weight 29.9 kg .", "the growth hormone ( gh ) releasing \n hormone ( ghrh ) and the thyroid stimulating hormone releasing hormone ( trh ) tolerance tests \n showed subnormal responses of gh and tsh , respectively ( table 1table 1 endocrinological findings of cases 1 and 2 ) .", "the corticotropin releasing hormone ( crh ) tolerance test showed normal \n responses of acth and serum cortisol ( table \n 1 ) . magnetic resonance imaging ( mri )", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "the pituitary stalk deviated \n to the frontal direction and the anterior pituitary gland was compressed by the tumor . \n", "there was no calcification of the mass detected by computed tomgraphic ( ct ) scan .", "cyst was suspected preoperatively by the mri findings , however a definite diagnosis could \n not be made .", "the capsule of the tumor \n had tightly adhered to the dura of the sellar floor .", "histological examination of the tumor showed fibrous tissue with cholesterol \n clefts , macrophages , multinucleated giant cells , and lymphocyte infiltration ( fig .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed .", "postoperatively , di remains and has been treated with desmopessin \n acetate ( ddavp ) .", "two months after surgery , gh and tsh responses after stimulation tests \n recovered ( table 1 ) .", "after one - year follow - up , the patient continues to have di , but his basal \n levels of igf-1 , ft3 , ft4 and tsh are within normal ranges .   radiological appearance on magnetic resonance imaging ( mri ) .", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed .", "a 6-yr - old japanese boy was admitted to our hospital for evaluation of polyuria and \n polydipsia of 6-mo duration .", "his \n height was 113.2 cm ( 0.8 sd ) and body weight was 18.8 kg .", "brain mri showed an intrasellar mass , which was high on the t1-weighted ( fig .", ". the mass had a high appearance on the t1-weighted \n images ( a ) .", "gadolinium \n administration caused no enhancement of the mass . ) and hypointense on the t2-weighted images ( fig . \n", "ghrh , trh , and crh \n tolerance tests showed normal responses one month after surgery ( table 1 ) .", "the patient is currently well only on ddavp \n medication , 12 mo after surgery .", "we report the cases of two children with xanthogranuloma in the pituitary region with di . \n", "xanthogranuloma in the sellar region has been traditionally considered to be a variant of \n adamantinomatous craniopharyngioma despite the lack of epithelium ( 6 )", ". however , paulus et al . ( 7 ) \n reviewed histological sections of 110 patients diagnosed with craniopharyngioma and found \n sections from 37 patients were exclusively or predominantly composed of xanthogranulomatous \n tissue . according to their study ,", "pathological findings were characterized by cholesterol \n clefts ( 100% ) , macrophages infiltrates , lympho - plasmacelluar infiltrates ( 100% ) , marked \n hemosiderin deposits ( 97% ) , fibrosis ( 87% ) , and foreign - body giant cells around cholesterol \n clefts ( 86% ) .", "only sections from 3 of these patients had small adamantinomatous components \n ( corresponding to calcifying odontogenic cyst ) , which are histological features of \n adamantinomatous craniopharyngioma . based on these findings , paulus et al . suggested that \n xanthogranuloma of the sellar region is pathologically distinct from adamantinomatous \n craniopharyngioma . in our two patients , histological findings of cholesterol clefts , \n", "macrophages including cholesterol droplets , multinucleated giant cells , lymphocytes , and the \n absence of adamantinomatous components are consistent with those previously reported by \n paulus et al .", "clinically , xanthogranuloma in the sellar region has been reported to cause severe \n endocrinological deficits ( 7 ) .", "( 9 ) also described one adult japanese patient with \n xanthogranuloma in the pituitary showing panhypopituitarism .", "by contrast , our patients did \n not have severe defects of anterior pituitary hormones and showed only central di . in the \n first report", ", xanthogranuloma of the pituitary in children was reported , but the frequency \n in children , and the clinical and endocrinological findings were not mentioned ( 7 ) . to answer whether clinical manifestations of \n xanthogranuloma in children are different from those of adults , more patients with \n xanthogranuloma of pediatric age must be studied . if xanthogranuloma is different form adamantinomatous craniopharyingioma , what is the \n origin of xanthogranuloma in the sellar region ?", "xanthogranulomatous inflammation ( also known \n as cholesterol granuloma ) has been reported in various tissues such as the middle ear , \n mastoid , and choroid plexus .", "also the obstruction of the gall \n flow has been considered to cause inflammation , eventually developing to xanthogranulomatous \n cholecystitis ( 11 ) .", "in addition , it is suggested that \n rathke s cyst and neuroepithelial cyst may undergo xanthogranulomatous changes ( 12 , 13 ) .", "taken \n together , xanthogranuloma of the sellar region is likely to represent a non - specific tissue \n reaction to hemorrhage or degenerative changes originating from heterogeneous etiologies .", "it \n may be plausible that xanthogranulomas of our patients were responses to ruptured cysts , \n however , no confirmation of pre - existing cysts has been identified .", "xanthogranuloma is difficult to differentiate from craniopharyngioma and rathke s cyst by \n mri findings .", "cystic \n and solid lesions may have any pattern of signal intensity on the t1 and t2-weighted images , \n although most often they are hypointense to isointense on the t1-weighted images and \n hyperintense on the t2-weighted images .", "on ct , craniopharyngiomas typically appear as \n moderate - to - large , partially calcified mass .", "rathke s cysts are present as intrasellar \n masses . as with craniopharyngiomas , the signal characteristics are variable on the t1 and \n t2-weighted images due to the variable quality of the cyst fluid . in our two patients , \n", "previously , two \n reports have also described xanthogranuloma as hyperintense on t1-weighted images ( 8 , 9 ) .", "in addition , \n there was no calcification of masses in our patients , as well as in previous reports ( 8 , 9 ) .", "these points \n may be helpful in considering xanthogranuloma of the pituitary region . however , there is no \n reliable way to distinguish xanthogranuloma from craniopharyngioma and rathke s cyst without \n histological examination . in conclusion ,", "currently , whether this condition is a truly distinct entity or merely a different \n manifestation of adamantinomatous craniopharyingioma is unclear . to clarify the natural \n history , pathogenesis and prognosis in childhood , further accumulation of well - characterized \n pediatric cases with xanthogranuloma"], "abstract_text": ["<S>   we report the cases of two japanese children with cystic pituitary enlargement on \n magnetic resonance imaging ( mri ) causing central diabetes insipidus ( di ) . in the first \n patient , </S>", "<S> endocrinological examination demonstrated slightly impaired growth hormone and \n thyroid stimulating hormone secretions , but normal responses of other anterior pituitary \n hormones . </S>", "<S> the second patient had normal basal levels of anterior pituitary hormones . \n </S>", "<S> transsphenoidal resection of the tumors was performed in both patients . </S>", "<S> histological \n analysis of the tumor sections demonstrated granulomatous tissue with cholesterol clefts , \n foamy macrophages , multinucleated giant cells and no epithelial component . </S>", "<S> thus , these \n tumors were pathologically diagnosed as xanthogranuloma of the sellar region , different \n from adamantinomatous craniopharyngioma . </S>", "<S> post - operatively , the two patients continue to \n have di , however other hormone replacement therapy after one year of follow - up has not \n been required . </S>", "<S> currently , it is not clear whether xanthogranuloma is a distinct entity \n from adamantinomatous craniopharyingioma . although , to our knowledge , a clinical report of \n xanthogranuloma of the sellar region has not been reported at pediatric age , it would be \n included in the differential diagnosis of the sellar region . </S>"], "labels": null, "section_names": ["Introduction", "Case Reports", "Case 1", "Case 2", "Discussion"], "sections": [["pituitary insufficiency in childhood is often secondary to pituitary or hypothalamic \n space - occupying lesions .", "the differential diagnosis in etiologies of a mass in the sella is \n broad in spectrum , including rathke s cyst , tumor , autoimmune diseases , primary or \n metastatic neoplasms , and granulomatous inflammation . among them , xanthomatous pituitary \n diseases are rare in childhood .", "these conditions include xanthomatous hypophysitis , \n xanthogranulomatous hypophysitis and xanthogranuloma of the sellar region ( 1,2,3,4,5 ) .", "xanthogranuloma of the sellar region is characterized \n by a granulomatous lesion containing cholesterol clefts , macrophages , chronic inflammatory \n infiltrates , and hemosiderin deposit ( 6 ) .", "this \n condition has been traditionally regarded as a hallmark of the adamantinomatous \n craniopharyngioma even in the absence of epithelium ( 6 )", ". however , paulus et al . ( 7 ) suggested that \n xanthogranuloma is likely to constitute a different entity from the classical \n adamantinomatous craniopharyngioma clinically and pathologically .", "after their original \n report , several patients with xanthogranuloma of the sellar region have been described \n ( 8 , 9 ) . \n however , to our knowledge", ", there is no detailed clinical report of xanthogranuloma of the \n sellar region in cases of pediatric age . here ,", "we report the cases of two children with \n xanthogranuloma of the sellar region , presenting central diabetes insipidus ( di ) ."], ["a 9-yr - old japanese boy was referred to our clinic because of a 6-mo history of polyuria \n and polydipsia .", "his height was 135.3 \n cm ( + 0.7 sd ) and body weight 29.9 kg .", "the growth hormone ( gh ) releasing \n hormone ( ghrh ) and the thyroid stimulating hormone releasing hormone ( trh ) tolerance tests \n showed subnormal responses of gh and tsh , respectively ( table 1table 1 endocrinological findings of cases 1 and 2 ) .", "the corticotropin releasing hormone ( crh ) tolerance test showed normal \n responses of acth and serum cortisol ( table \n 1 ) . magnetic resonance imaging ( mri )", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "the pituitary stalk deviated \n to the frontal direction and the anterior pituitary gland was compressed by the tumor . \n", "there was no calcification of the mass detected by computed tomgraphic ( ct ) scan .", "cyst was suspected preoperatively by the mri findings , however a definite diagnosis could \n not be made .", "the capsule of the tumor \n had tightly adhered to the dura of the sellar floor .", "histological examination of the tumor showed fibrous tissue with cholesterol \n clefts , macrophages , multinucleated giant cells , and lymphocyte infiltration ( fig .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed .", "postoperatively , di remains and has been treated with desmopessin \n acetate ( ddavp ) .", "two months after surgery , gh and tsh responses after stimulation tests \n recovered ( table 1 ) .", "after one - year follow - up , the patient continues to have di , but his basal \n levels of igf-1 , ft3 , ft4 and tsh are within normal ranges .   radiological appearance on magnetic resonance imaging ( mri ) .", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed .", "a 6-yr - old japanese boy was admitted to our hospital for evaluation of polyuria and \n polydipsia of 6-mo duration .", "his \n height was 113.2 cm ( 0.8 sd ) and body weight was 18.8 kg .", "brain mri showed an intrasellar mass , which was high on the t1-weighted ( fig .", "gadolinium \n administration caused no enhancement of the mass . ) and hypointense on the t2-weighted images ( fig . \n", "ghrh , trh , and crh \n tolerance tests showed normal responses one month after surgery ( table 1 ) .", "the patient is currently well only on ddavp \n medication , 12 mo after surgery ."], ["a 9-yr - old japanese boy was referred to our clinic because of a 6-mo history of polyuria \n and polydipsia .", "his height was 135.3 \n cm ( + 0.7 sd ) and body weight 29.9 kg .", "the growth hormone ( gh ) releasing \n hormone ( ghrh ) and the thyroid stimulating hormone releasing hormone ( trh ) tolerance tests \n showed subnormal responses of gh and tsh , respectively ( table 1table 1 endocrinological findings of cases 1 and 2 ) .", "the corticotropin releasing hormone ( crh ) tolerance test showed normal \n responses of acth and serum cortisol ( table \n 1 ) . magnetic resonance imaging ( mri )", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "the pituitary stalk deviated \n to the frontal direction and the anterior pituitary gland was compressed by the tumor . \n", "there was no calcification of the mass detected by computed tomgraphic ( ct ) scan .", "cyst was suspected preoperatively by the mri findings , however a definite diagnosis could \n not be made .", "the capsule of the tumor \n had tightly adhered to the dura of the sellar floor .", "histological examination of the tumor showed fibrous tissue with cholesterol \n clefts , macrophages , multinucleated giant cells , and lymphocyte infiltration ( fig .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed .", "postoperatively , di remains and has been treated with desmopessin \n acetate ( ddavp ) .", "two months after surgery , gh and tsh responses after stimulation tests \n recovered ( table 1 ) .", "after one - year follow - up , the patient continues to have di , but his basal \n levels of igf-1 , ft3 , ft4 and tsh are within normal ranges .   radiological appearance on magnetic resonance imaging ( mri ) .", "the mass was hyperintense on the \n t1-weighted ( a ) and in the t2-weighted images ( b ) .", "( b ) macrophages ( white arrowheads ) \n and multinucleated giant cells ( black arrowheads ) were observed ."], ["a 6-yr - old japanese boy was admitted to our hospital for evaluation of polyuria and \n polydipsia of 6-mo duration .", "his \n height was 113.2 cm ( 0.8 sd ) and body weight was 18.8 kg .", "brain mri showed an intrasellar mass , which was high on the t1-weighted ( fig .", ". the mass had a high appearance on the t1-weighted \n images ( a ) .", "gadolinium \n administration caused no enhancement of the mass . ) and hypointense on the t2-weighted images ( fig . \n", "ghrh , trh , and crh \n tolerance tests showed normal responses one month after surgery ( table 1 ) .", "the patient is currently well only on ddavp \n medication , 12 mo after surgery ."], ["we report the cases of two children with xanthogranuloma in the pituitary region with di . \n", "xanthogranuloma in the sellar region has been traditionally considered to be a variant of \n adamantinomatous craniopharyngioma despite the lack of epithelium ( 6 )", ". however , paulus et al . ( 7 ) \n reviewed histological sections of 110 patients diagnosed with craniopharyngioma and found \n sections from 37 patients were exclusively or predominantly composed of xanthogranulomatous \n tissue . according to their study ,", "pathological findings were characterized by cholesterol \n clefts ( 100% ) , macrophages infiltrates , lympho - plasmacelluar infiltrates ( 100% ) , marked \n hemosiderin deposits ( 97% ) , fibrosis ( 87% ) , and foreign - body giant cells around cholesterol \n clefts ( 86% ) .", "only sections from 3 of these patients had small adamantinomatous components \n ( corresponding to calcifying odontogenic cyst ) , which are histological features of \n adamantinomatous craniopharyngioma . based on these findings , paulus et al . suggested that \n xanthogranuloma of the sellar region is pathologically distinct from adamantinomatous \n craniopharyngioma . in our two patients , histological findings of cholesterol clefts , \n", "macrophages including cholesterol droplets , multinucleated giant cells , lymphocytes , and the \n absence of adamantinomatous components are consistent with those previously reported by \n paulus et al .", "clinically , xanthogranuloma in the sellar region has been reported to cause severe \n endocrinological deficits ( 7 ) .", "( 9 ) also described one adult japanese patient with \n xanthogranuloma in the pituitary showing panhypopituitarism .", "by contrast , our patients did \n not have severe defects of anterior pituitary hormones and showed only central di . in the \n first report", ", xanthogranuloma of the pituitary in children was reported , but the frequency \n in children , and the clinical and endocrinological findings were not mentioned ( 7 ) . to answer whether clinical manifestations of \n xanthogranuloma in children are different from those of adults , more patients with \n xanthogranuloma of pediatric age must be studied . if xanthogranuloma is different form adamantinomatous craniopharyingioma , what is the \n origin of xanthogranuloma in the sellar region ?", "xanthogranulomatous inflammation ( also known \n as cholesterol granuloma ) has been reported in various tissues such as the middle ear , \n mastoid , and choroid plexus .", "also the obstruction of the gall \n flow has been considered to cause inflammation , eventually developing to xanthogranulomatous \n cholecystitis ( 11 ) .", "in addition , it is suggested that \n rathke s cyst and neuroepithelial cyst may undergo xanthogranulomatous changes ( 12 , 13 ) .", "taken \n together , xanthogranuloma of the sellar region is likely to represent a non - specific tissue \n reaction to hemorrhage or degenerative changes originating from heterogeneous etiologies .", "it \n may be plausible that xanthogranulomas of our patients were responses to ruptured cysts , \n however , no confirmation of pre - existing cysts has been identified .", "xanthogranuloma is difficult to differentiate from craniopharyngioma and rathke s cyst by \n mri findings .", "cystic \n and solid lesions may have any pattern of signal intensity on the t1 and t2-weighted images , \n although most often they are hypointense to isointense on the t1-weighted images and \n hyperintense on the t2-weighted images .", "on ct , craniopharyngiomas typically appear as \n moderate - to - large , partially calcified mass .", "rathke s cysts are present as intrasellar \n masses . as with craniopharyngiomas , the signal characteristics are variable on the t1 and \n t2-weighted images due to the variable quality of the cyst fluid . in our two patients , \n", "previously , two \n reports have also described xanthogranuloma as hyperintense on t1-weighted images ( 8 , 9 ) .", "in addition , \n there was no calcification of masses in our patients , as well as in previous reports ( 8 , 9 ) .", "these points \n may be helpful in considering xanthogranuloma of the pituitary region . however , there is no \n reliable way to distinguish xanthogranuloma from craniopharyngioma and rathke s cyst without \n histological examination . in conclusion ,", "currently , whether this condition is a truly distinct entity or merely a different \n manifestation of adamantinomatous craniopharyingioma is unclear . to clarify the natural \n history , pathogenesis and prognosis in childhood , further accumulation of well - characterized \n pediatric cases with xanthogranuloma"]]}
{"article_id": "PMC3030046", "article_text": ["a 52-year - old female patient visited the hospital complaining of pain in both breasts .", "the pain started immediately after breast reduction surgery she underwent 3 years previously at a private plastic surgery hospital , and it had continued despite a subsequent surgery .", "the pattern of the pain included burning , shooting and itching sensations at the operation site as well as unbearable burning sensations when the patient was in the places where the temperature was higher than normal .", "the pain as measured by the visual analogue scale ( vas ) was approximately 70/100 mm .", "in particular , the degree of pain was dependent on the season , and it was unbearable under hot weather , with the patient complaining of pain at vas 80 - 90/100 mm . before coming to the hospital , the patient had taken aceclofenac ( airtal , daewoong pharmaceutical ) 200 mg , pregabalin ( lyrica , pfizer ) 300 mg , acetaminophen 1,300 mg and tramadol hcl 150 mg ( ultracet , janssen korea ) per day , but the pain was not relieved .", "thus , the patient tried pregabalin 600 mg , acetaminophen 1,300 mg and tramadol hcl 150 mg per day , but there was no effect .", "complaining of dizziness due to ultracet , the patient started to take 20 mg of oxycodone hcl ( oxycontin , mundipharma korea ) each day , but she eventually stopped taking it because of dizziness , nausea , and vomiting .", "subsequently , the patient underwent drug treatment with pregabalin 300 mg a day , but she visited the hospital because the pain did not subside .", "there was no specific finding in a simple plane chest x - ray spectrograph , while digital infrared thermographic imaging ( diti ) showed a slight temperature drop in both breasts .", "because the previous treatments were not effective in improving the symptoms and because the patient wanted another method of treatment due to the continued pain , it was decided that a thoracic epidural block would be performed , and informed consent was obtained from the patient .", "the patient was laid on her side and the epidural space was checked using the loss - of - resistance method with a 22-gauge epidural needle ( hakko , japan ) in between the t4 and t5 , applying an aseptic technique in the interlaminar approach .", "0.5% mepivacaine hcl ( emcaine 2% inj. reyon pharm ) 6 ml was then injected .", "the pain was relieved into the vas range of 40 - 50/100 mm after the procedure , and the patient was discharged .", "the vas was decreased to 40 - 50/100 mm after an additional procedure a week later , but the patient complained of continued pain .", "a decision was made to carry out prf to the spinal nerve and its root and . to measure the level and determine the effect", ", informed consent was received from the patient after she was provided with an explanation of the procedure and the side effects that may occur after the procedure . for the diagnosis ,", "selective nerve root block ( snb ) was performed at t3 , t4 and t5 , one level each time in order with an interval of 2 days , by injecting 1% lidocaine 0.5 ml with a 25-gauge quincke needle ( spinocan , bbraun ) .", "prf was carried out at the fourth thoracic spinal nerve and its root after giving an explanation to the patient about the procedure and the side effects that may occur after the procedure and receiving informed consent once again . after supporting the patient 's lower abdomen with a pillow and placing her in a prone position ,", "the site of the procedure was disinfected by a common sterilizing method and a standard monitor was attached .", "the area between t4 and t5 was identified in the anteroposterior view using a c - arm device and the 4 cm point right lateral from the t4 spinous process was chosen as the needle implantation point .", "local anesthesia was carried out with 1% lidocaine , and a 20-gauge 10 cm long rf cannula ( owl sharp curved rf insulated cannula , diros technology inc . ,", "usa ) with a 10 mm - active tip was then introduced into the anesthetized part . by continuously monitoring the position of the cannula with the c - arm", ", it continued to the inferior of end plate of fourth thoracic vertebral body ( fig .", "1 ) , and the tip of the cannula was located at the posterior border of the vertebral space in the lateral view ( fig .", "3 ) , and an rf generator ( rfg-3c plus , radionics , usa ) was then connected and an electric stimulus of 50 hz at 0.1 volt was applied .", "electric stimulus of 2 hz at 0.1 volt was applied , and it was verified that the muscle did not contract up to 0.8 volt .", "one minute after injecting 1% mepivacaine 1 ml , prf was performed two times for 120 seconds at 42. the same procedure was performed on the opposite side after one week . during the procedure , the vital signs of the patient were normal and no side effect took place . after the procedure , the pain was reduced to vas 20 - 30/100 mm .", "the patient currently undergoes oral administration of pregabalin 300 mg / day and nortriptyline ( sensival , ilsung pharm ) 10 mg / day and this was followed up at the hospital for 9 months without particular exacerbation of the pain or any other inconvenience in her daily tasks .", "pain after breast surgery , which is commonly complaint by patients , is known to be related with the injury , ischemia and inflammation of the soft tissue .", "if the pain in the affected part and arm continues for one year after the surgery , it is considered as chronic pain in general .", "the mechanism is not yet accurately known , but the chronic pain may be caused by injuries in various peripheral nerves that arise during the surgery and can develop into neuropathic pain such as stabbing , pricking , burning , shooting and sharp pains .", "wallace et al .   investigated 282 women who underwent different types of breast surgery , and their result showed that 31% of women who underwent mastectomy , 49% of those who underwent mastectomy with reconstruction , 38% of those who underwent cosmetic augmentation and 22% of those who underwent breast reduction complained of pain up to one year after the surgery .", "particularly , as the survival rate of breast cancer patients has increased , the number of patients who complain of chronic pain after breast cancer survey has also increased .", "the patient in this case study was also a chronic neuropathic pain patient whose pain had continued for 3 years following breast reduction surgery .", "she complained of pricking , shooting and burning sensations at the site of the operation .", "although the patient complained of pain immediately after the surgery , she continued to suffer neuropathic pain for 3 years without being given appropriate pain treatment because she was not able to take medicine due to the side effects of the drugs .", "macrae   stated that severe acute pain after the surgery may be a risk factor for chronic pain .", "this can be one possible cause of the chronic pain of the patient in this case report , because she complained severe pain at vas 70 - 80/100 mm immediately after the surgery but the pain was not properly controlled .", "in general , it is thought that the intercostal nerve ( icn ) from t1 to t6 and the nerves that originate from those braches can be damaged by breast surgery . for the patient in this case report , because she complained of the pain at the dermatome of the t4 spinal nerve , neuropathic pain caused by damage at the 4th icn and at the branch was suspected .", "the effect of a nerve block in controlling pain after breast surgery is well known . in particular , it was reported that a paravertebral block ( pvb ) reduced pain , nausea and vomiting after breast surgery .", "in addition , kairaluoma et al .   reported that preoperative pvb reduced chronic pain after breast surgery .", "these results show that a nerve block can be used in controlling pain following breast surgery effectively , preventing it from becoming chronic .", "a nerve block using a local anesthetic is easy to perform in the treatment of pain and is effective in reducing pain .", "however , because the duration of desensibilization may not be sufficient depending on the disease causing the pain and because many repeated procedures may be required , radiofrequency thermocoagulation is sometimes employed to maintain the effect of the nerve block .", "radiofrequency thermocoagulation was first introduced in 1930 's by kirschner , who applied it treating trigeminal neuralgia at the gasserian ganglion .", "recently , the application of radiofrequency thermocoagulation in the case of various diseases has gradually increased as quality of the radiofrequency generator , the associated equipment , and the catheter needle has been improved .", "this treatment is highly advantageous , in that a more accurate lesion can be generated compared to other nerve - destructive procedures . moreover ,", "the mechanism of radiofrequency thermocoagulation is known to change the nerve tissue due to the heat around the electrode , blocking and the inflow of the nerve stimulus . on the other hand ,", "prf was introduced as a new method because another mechanism of radiofrequency - aided treatment arises in that a clinical effect is noted by the heat applied to the nerve and the electromagnetic field that forms around the electrode .", "prf is a radiofrequency - aided treatment in which pulsed radiofrequency stimulus of 20 msec is generated two times every 0.5 second at a temperature of 42 , which is low enough for the nerve tissue to remain undamaged .", "prf is a novel treatment method that has corrected the problems of previous radiofrequency - aided treatments based on heat .", "it does not cause nerve destruction , side effects such as neuritis , or the complications of other types of radiofrequency - aided treatments based on heat .", "it can be performed to treat neuropathic pain even for body parts where side effects take place frequently due to radiofrequency thermocoagulation .", "it can be also performed on parts near bone or scar tissue , where the risk of complications is high when radiofrequency thermocoagulation is performed .", "rohof   applied prf to the suprascapular nerve in the treatment of chronic shoulder pain and reported it to be a novel treatment method that is free from side effects or complications , in contrast to the conventional thermocoagulation .", "moreover , positive effects were reported when prf was applied to peripheral nerves such as the facial nerve , superior laryngeal nerve , suprascapular nerve , intercostals nerve , obturator nerve and the joint - dominating peripheral nerve articular branch , whose main nerve is sensory . in this case report as well , although the pain was controlled by an epidural block with a local anesthetic , repeated procedures were required due to the short duration of the first treatment .", "long - lasting pain reduction was achieved without any specific side effect , as the prf was carried out on the spinal nerve and its root under monitoring by a c - arm device . in this article", ", we report a case in which prf was applied to control the pain of a patient who came to the hospital complaining of chronic severe neuropathic pain at the operation site that arose after breast reduction surgery .", "appropriate pain control after breast surgery can reduce the suffering of the patient due to early pain and greatly affect the quality of life of the patient by preventing the pain from being chronic .", "if an appropriate pain treatment had been given after breast surgery to the patient in this case report , she would have not suffered from chronic pain . in the case of breast surgery , considerable attention should be paid to postoperative pain as well as surgical pain , and pain treatment should be carried out positively .", "it is thought that a nerve block as well as general analgesics can be helpful as a pain control strategy .", "in addition , prf may be helpful in reducing the pain of the patients who are not responsive to a nerve block using analgesics or local anesthetics .", "furthermore , additional studies are needed regarding the reduction of chronic neuropathic pain by prf after breast surgery ."], "abstract_text": ["<S> breast surgery is a common procedure performed in women . </S>", "<S> many women who undergo breast surgery suffer from ill - defined pain syndromes . </S>", "<S> a nerve block is used in the treatment of the acute and chronic pain , but the effectiveness of the treatment has been limited because of its short duration . </S>", "<S> recently , the advent of pulsed radiofrequency lesioning ( prf ) has proved a successful treatment for chronic refractory pain involving the peripheral nerves . </S>", "<S> we experienced a case of a 52-year - old female patient complaining of chronic breast neuropathic pain after breast reduction , which was relieved after prf lesioning of the 4th thoracic spinal nerve and its root . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 52-year - old female patient visited the hospital complaining of pain in both breasts .", "the pain started immediately after breast reduction surgery she underwent 3 years previously at a private plastic surgery hospital , and it had continued despite a subsequent surgery .", "the pattern of the pain included burning , shooting and itching sensations at the operation site as well as unbearable burning sensations when the patient was in the places where the temperature was higher than normal .", "the pain as measured by the visual analogue scale ( vas ) was approximately 70/100 mm .", "in particular , the degree of pain was dependent on the season , and it was unbearable under hot weather , with the patient complaining of pain at vas 80 - 90/100 mm . before coming to the hospital , the patient had taken aceclofenac ( airtal , daewoong pharmaceutical ) 200 mg , pregabalin ( lyrica , pfizer ) 300 mg , acetaminophen 1,300 mg and tramadol hcl 150 mg ( ultracet , janssen korea ) per day , but the pain was not relieved .", "thus , the patient tried pregabalin 600 mg , acetaminophen 1,300 mg and tramadol hcl 150 mg per day , but there was no effect .", "complaining of dizziness due to ultracet , the patient started to take 20 mg of oxycodone hcl ( oxycontin , mundipharma korea ) each day , but she eventually stopped taking it because of dizziness , nausea , and vomiting .", "subsequently , the patient underwent drug treatment with pregabalin 300 mg a day , but she visited the hospital because the pain did not subside .", "there was no specific finding in a simple plane chest x - ray spectrograph , while digital infrared thermographic imaging ( diti ) showed a slight temperature drop in both breasts .", "because the previous treatments were not effective in improving the symptoms and because the patient wanted another method of treatment due to the continued pain , it was decided that a thoracic epidural block would be performed , and informed consent was obtained from the patient .", "the patient was laid on her side and the epidural space was checked using the loss - of - resistance method with a 22-gauge epidural needle ( hakko , japan ) in between the t4 and t5 , applying an aseptic technique in the interlaminar approach .", "0.5% mepivacaine hcl ( emcaine 2% inj. reyon pharm ) 6 ml was then injected .", "the pain was relieved into the vas range of 40 - 50/100 mm after the procedure , and the patient was discharged .", "the vas was decreased to 40 - 50/100 mm after an additional procedure a week later , but the patient complained of continued pain .", "a decision was made to carry out prf to the spinal nerve and its root and . to measure the level and determine the effect", ", informed consent was received from the patient after she was provided with an explanation of the procedure and the side effects that may occur after the procedure . for the diagnosis ,", "selective nerve root block ( snb ) was performed at t3 , t4 and t5 , one level each time in order with an interval of 2 days , by injecting 1% lidocaine 0.5 ml with a 25-gauge quincke needle ( spinocan , bbraun ) .", "prf was carried out at the fourth thoracic spinal nerve and its root after giving an explanation to the patient about the procedure and the side effects that may occur after the procedure and receiving informed consent once again . after supporting the patient 's lower abdomen with a pillow and placing her in a prone position ,", "the site of the procedure was disinfected by a common sterilizing method and a standard monitor was attached .", "the area between t4 and t5 was identified in the anteroposterior view using a c - arm device and the 4 cm point right lateral from the t4 spinous process was chosen as the needle implantation point .", "local anesthesia was carried out with 1% lidocaine , and a 20-gauge 10 cm long rf cannula ( owl sharp curved rf insulated cannula , diros technology inc . ,", "usa ) with a 10 mm - active tip was then introduced into the anesthetized part . by continuously monitoring the position of the cannula with the c - arm", ", it continued to the inferior of end plate of fourth thoracic vertebral body ( fig .", "1 ) , and the tip of the cannula was located at the posterior border of the vertebral space in the lateral view ( fig .", "3 ) , and an rf generator ( rfg-3c plus , radionics , usa ) was then connected and an electric stimulus of 50 hz at 0.1 volt was applied .", "electric stimulus of 2 hz at 0.1 volt was applied , and it was verified that the muscle did not contract up to 0.8 volt .", "one minute after injecting 1% mepivacaine 1 ml , prf was performed two times for 120 seconds at 42. the same procedure was performed on the opposite side after one week . during the procedure , the vital signs of the patient were normal and no side effect took place . after the procedure , the pain was reduced to vas 20 - 30/100 mm .", "the patient currently undergoes oral administration of pregabalin 300 mg / day and nortriptyline ( sensival , ilsung pharm ) 10 mg / day and this was followed up at the hospital for 9 months without particular exacerbation of the pain or any other inconvenience in her daily tasks ."], ["pain after breast surgery , which is commonly complaint by patients , is known to be related with the injury , ischemia and inflammation of the soft tissue .", "if the pain in the affected part and arm continues for one year after the surgery , it is considered as chronic pain in general .", "the mechanism is not yet accurately known , but the chronic pain may be caused by injuries in various peripheral nerves that arise during the surgery and can develop into neuropathic pain such as stabbing , pricking , burning , shooting and sharp pains .", "wallace et al .   investigated 282 women who underwent different types of breast surgery , and their result showed that 31% of women who underwent mastectomy , 49% of those who underwent mastectomy with reconstruction , 38% of those who underwent cosmetic augmentation and 22% of those who underwent breast reduction complained of pain up to one year after the surgery .", "particularly , as the survival rate of breast cancer patients has increased , the number of patients who complain of chronic pain after breast cancer survey has also increased .", "the patient in this case study was also a chronic neuropathic pain patient whose pain had continued for 3 years following breast reduction surgery .", "she complained of pricking , shooting and burning sensations at the site of the operation .", "although the patient complained of pain immediately after the surgery , she continued to suffer neuropathic pain for 3 years without being given appropriate pain treatment because she was not able to take medicine due to the side effects of the drugs .", "macrae   stated that severe acute pain after the surgery may be a risk factor for chronic pain .", "this can be one possible cause of the chronic pain of the patient in this case report , because she complained severe pain at vas 70 - 80/100 mm immediately after the surgery but the pain was not properly controlled .", "in general , it is thought that the intercostal nerve ( icn ) from t1 to t6 and the nerves that originate from those braches can be damaged by breast surgery . for the patient in this case report , because she complained of the pain at the dermatome of the t4 spinal nerve , neuropathic pain caused by damage at the 4th icn and at the branch was suspected .", "the effect of a nerve block in controlling pain after breast surgery is well known . in particular , it was reported that a paravertebral block ( pvb ) reduced pain , nausea and vomiting after breast surgery .", "in addition , kairaluoma et al .   reported that preoperative pvb reduced chronic pain after breast surgery .", "these results show that a nerve block can be used in controlling pain following breast surgery effectively , preventing it from becoming chronic .", "a nerve block using a local anesthetic is easy to perform in the treatment of pain and is effective in reducing pain .", "however , because the duration of desensibilization may not be sufficient depending on the disease causing the pain and because many repeated procedures may be required , radiofrequency thermocoagulation is sometimes employed to maintain the effect of the nerve block .", "radiofrequency thermocoagulation was first introduced in 1930 's by kirschner , who applied it treating trigeminal neuralgia at the gasserian ganglion .", "recently , the application of radiofrequency thermocoagulation in the case of various diseases has gradually increased as quality of the radiofrequency generator , the associated equipment , and the catheter needle has been improved .", "this treatment is highly advantageous , in that a more accurate lesion can be generated compared to other nerve - destructive procedures . moreover ,", "the mechanism of radiofrequency thermocoagulation is known to change the nerve tissue due to the heat around the electrode , blocking and the inflow of the nerve stimulus . on the other hand ,", "prf was introduced as a new method because another mechanism of radiofrequency - aided treatment arises in that a clinical effect is noted by the heat applied to the nerve and the electromagnetic field that forms around the electrode .", "prf is a radiofrequency - aided treatment in which pulsed radiofrequency stimulus of 20 msec is generated two times every 0.5 second at a temperature of 42 , which is low enough for the nerve tissue to remain undamaged .", "prf is a novel treatment method that has corrected the problems of previous radiofrequency - aided treatments based on heat .", "it does not cause nerve destruction , side effects such as neuritis , or the complications of other types of radiofrequency - aided treatments based on heat .", "it can be performed to treat neuropathic pain even for body parts where side effects take place frequently due to radiofrequency thermocoagulation .", "it can be also performed on parts near bone or scar tissue , where the risk of complications is high when radiofrequency thermocoagulation is performed .", "rohof   applied prf to the suprascapular nerve in the treatment of chronic shoulder pain and reported it to be a novel treatment method that is free from side effects or complications , in contrast to the conventional thermocoagulation .", "moreover , positive effects were reported when prf was applied to peripheral nerves such as the facial nerve , superior laryngeal nerve , suprascapular nerve , intercostals nerve , obturator nerve and the joint - dominating peripheral nerve articular branch , whose main nerve is sensory . in this case report as well , although the pain was controlled by an epidural block with a local anesthetic , repeated procedures were required due to the short duration of the first treatment .", "long - lasting pain reduction was achieved without any specific side effect , as the prf was carried out on the spinal nerve and its root under monitoring by a c - arm device . in this article", ", we report a case in which prf was applied to control the pain of a patient who came to the hospital complaining of chronic severe neuropathic pain at the operation site that arose after breast reduction surgery .", "appropriate pain control after breast surgery can reduce the suffering of the patient due to early pain and greatly affect the quality of life of the patient by preventing the pain from being chronic .", "if an appropriate pain treatment had been given after breast surgery to the patient in this case report , she would have not suffered from chronic pain . in the case of breast surgery , considerable attention should be paid to postoperative pain as well as surgical pain , and pain treatment should be carried out positively .", "it is thought that a nerve block as well as general analgesics can be helpful as a pain control strategy .", "in addition , prf may be helpful in reducing the pain of the patients who are not responsive to a nerve block using analgesics or local anesthetics .", "furthermore , additional studies are needed regarding the reduction of chronic neuropathic pain by prf after breast surgery ."]]}
{"article_id": "PMC1160155", "article_text": ["the generation of expressed sequence tags ( ests ) was originally proposed as a strategy for cdna characterization over a decade ago ( 1 ) .", "subsequent improvements in sequencing methods and dramatically reduced unit costs have increased the attractiveness of the est - based research , such that it is now one of the most widely employed methods used for gene discovery and genome characterization .", "consequently , the number of organisms with est sequences deposited in the genbank dbest database is increasing rapidly ( ) . to maximize the value of these ests , ncbi has built unigenes that incorporated est data for a number of species ( ) , and the institute for genomics research ( tigr ) has been working on the gene indices for more than 70 species ( ) . annotating est and cdna sequences", "two tools that have been designed for locating protein - coding regions in cdna and est sequences are orffinder ( ) and estscan ( ) , respectively .", "the estscan server is designed for processing a batch of est sequences for identifying the protein - coding regions with a function for correcting insertions or deletions , but it is only trained for mammals and yeast .", "we tested estscan with our aspergillus niger est sequences and found that the results were not satisfactory .", "for example , using a full - length cdna sequence encoding glucoamylase , a well - characterized enzyme in a.niger , we found that estscan could not identify its correct coding region and had the undesired side effect of inserting nucleotides even when the test sequence was correct .", "we have implemented a web server called orfpredictor for the prediction of protein - coding regions within est - derived sequences .", "the algorithm uses the translation reading frames predicted by using blastx ( 2 ) as a guide for the identification of the coding region in sequences that have a hit ( 3 ) and predicts a coding region ab initio for sequences without a hit .", "a mature eukaryotic mrna molecule , starting from the 5 end , typically consists of a 5 cap , a 5-untranslated region ( 5-utr ) , a protein - coding region [ open reading frame ( orf ) ] and a 3-utr followed by a poly(a ) tail .", "the protein - coding region extends from the start codon aug ( atg in a cdna ) and continues until the reading frame defined by the start codon is terminated by one of three translation stop codons , uga , uaa or uag . most cdna libraries are constructed using oligo(dt ) primers to direct first - strand synthesis by reverse transcriptase .", "essentially , all clones in oligo(dt ) primed cdna libraries will therefore include information for the 3 end of the processed transcript and a poly(a ) region .", "ests are single - pass sequencing reads obtained from either the 5 or 3 end of the cdna insert .", "the high - quality sequence obtained using systems , such as abi 3730xl , is typically 700800 nt per read .", "given that the 3- and 5-utrs are typically much shorter than 500 nt , most ests and the consensus sequences ( contigs ) generated by an est assembler are expected to include some coding sequence useful for predicting gene function . in the annotation and analysis of ests ,", "overlapping est sequences are often assembled into contigs to remove redundancy , reduce the frequency of sequencing errors and extend the length of sequence derived from each mrna species . assuming the ests are sequenced from the 5 end , sequence information from contigs and individual ests fall into 10 categories ( figure 1 ) as follows : \n a full - length sequence that includes the 5-utr with one or more stop codons ( 5 stop codon ) , translation start codon , complete protein - coding region , translation stop codon and the 3-utr .", "the protein - coding orf may have internal atg codons and the 3-utr may possess multiple stop codons .", "the 5-utr may be truncated.a full - length sequence as defined for category ( a ) , except that it does not contain any 5 stop codons.a partial sequence that has a portion of the 5-utr , one or more 5 stop codons , the start codon and a portion of the coding region.a sequence that contains only 5-utr sequence and there is a 5 stop codon.a sequence that contains a 5-utr sequence , the start codon and a portion of the protein - coding orf.a sequence that contains only 5-utr sequence without a 5 stop codon.a sequence that contains a portion of the protein - coding region , but does not contain the start codon or the stop codon.a sequence with the potential protein - coding region truncated at its 5 end , one or more atg codons in the truncated orf , the stop codon and a 3-utr.a sequence that contains a portion of the potential protein - coding region and the 3-utr sequence.a sequence that contains a portion of the 3-utr and a poly(a ) sequence at its 3 end . a full - length sequence that includes the 5-utr with one or more stop codons ( 5 stop codon ) , translation start codon , complete protein - coding region , translation stop codon and the 3-utr .", "the protein - coding orf may have internal atg codons and the 3-utr may possess multiple stop codons .", "the 5-utr may be truncated . a full - length sequence as defined for category ( a ) , except that it does not contain any 5 stop codons . a partial sequence that has a portion of the 5-utr , one or more 5 stop codons , the start codon and a portion of the coding region . a sequence that contains only 5-utr sequence and there is a 5 stop codon . a sequence that contains a 5-utr sequence , the start codon and a portion of the protein - coding orf . a sequence that contains only 5-utr sequence without a 5 stop codon .", "a sequence that contains a portion of the protein - coding region , but does not contain the start codon or the stop codon . a sequence with the potential protein - coding region truncated at its 5 end , one or more atg codons in the truncated orf , the stop codon and a 3-utr . a sequence that contains a portion of the potential protein - coding region and the 3-utr sequence . a sequence that contains a portion of the 3-utr and a poly(a ) sequence at its 3 end . for sequences generated by sequencing cdna inserts from their 3 ends ,", "the categories of their reverse complementary sequences only include ( a ) , ( b ) , ( h ) , ( i ) and ( j ) .", "therefore , it is more challenging to predict the coding region within an est than it is to predict the coding region of a fully sequenced cdna . distinguishing the translation start codon from other atg codons remains a difficult task .", "identifying start codons is further complicated because there is not a universal consensus sequence surrounding eukaryotic start codons , although the conserved consensus sequence , gccrccaugg ( r : purine ; aug : start codon ) is present in mammals ( 4 ) .", "however , blastx using a nucleotide query against a protein database is able to reliably identify protein - coding regions within a dna sequence if sufficient similarity exists between the translated query and an entry in the database ( 3 ) .", "sequencing errors may disrupt the conceptual translation of orfs , blastx could also detect frame shifts if there are insertions / deletions in the coding regions of the query sequences . when significant blastx alignments can be generated our algorithm uses them as a guide to identify the translation reading frames and coding regions . for est - derived sequences without a database match , their frames and coding sequences are predicted based on the presence and the location of intrinsic signals in a sequence that include start codons , 5 or / and 3 stop codons and stretches of poly(a ) ( figure 1 ) .", "our algorithm uses the following rules to locate protein - coding regions and predict the translation reading frame . for cases where blastx identified a significant database match ( e - value lower than a user chosen threshold ) ,", "the frame assignment in the blastx output will be used and rules 19 are applied . if there is a conflict , rules 1 and 2 will override the other rules . for sequences that do not produce a significant blastx alignment rules 310", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx.rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used.rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a).rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j).rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c).rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids).rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i).rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) . although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence.rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .", "however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence.rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx . rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used .", "rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a ) .", "rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j ) .", "rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c ) .", "rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids ) .", "rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i ) .", "rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .", "although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence .", "rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .", "however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence .", "rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .", "the server provides a user interface for copy and paste , or for loading the users ' sequences and blastx outputs .", "these various inputs are summarized as follows : \n a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction.blastx output for all the est - derived query sequences .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "targetidentifier server uses the uniprot / swiss - prot protein database.e - value : the user can also set a threshold e - value for their blastx file .", "if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as ", "no hit. the default threshold is 1  10.strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen .", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , ", "users can select download or use email for receiving their results . a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "e - value : the user can also set a threshold e - value for their blastx file .", "if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as ", "strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen . if the sequences were obtained by sequencing cdnas from their 3 ends , the   strand should be chosen .", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , ", "an identifier for the sequence , the reading frame for the predicted coding region , the location of the beginning and end of the predicted coding region , a flag shown as  fs that locates any translation frame shifts detected in the blastx alignment and the predicted protein sequence .", "the other file contains the query identifiers for those sequences that do not have predicted protein - coding regions ( figure 1j ) .", "most ests encompass only a portion of the mrna sequence . therefore , it is more challenging to predict the coding region within an est than it is to predict the coding region of a fully sequenced cdna . distinguishing the translation start codon from other atg codons", "identifying start codons is further complicated because there is not a universal consensus sequence surrounding eukaryotic start codons , although the conserved consensus sequence , gccrccaugg ( r : purine ; aug : start codon ) is present in mammals ( 4 ) .", "however , blastx using a nucleotide query against a protein database is able to reliably identify protein - coding regions within a dna sequence if sufficient similarity exists between the translated query and an entry in the database ( 3 ) .", "sequencing errors may disrupt the conceptual translation of orfs , blastx could also detect frame shifts if there are insertions / deletions in the coding regions of the query sequences . when significant blastx alignments can be generated our algorithm uses them as a guide to identify the translation reading frames and coding regions . for est - derived sequences without a database match , their frames and coding sequences are predicted based on the presence and the location of intrinsic signals in a sequence that include start codons , 5 or / and 3 stop codons and stretches of poly(a ) ( figure 1 ) .", "our algorithm uses the following rules to locate protein - coding regions and predict the translation reading frame . for cases where blastx identified a significant database match ( e - value lower than a user chosen threshold ) ,", "the frame assignment in the blastx output will be used and rules 19 are applied . if there is a conflict , rules 1 and 2 will override the other rules . for sequences that do not produce a significant blastx alignment rules 310", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx.rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used.rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a).rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j).rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c).rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids).rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i).rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .", "although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence.rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature . however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence.rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx . rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used .", "rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a ) .", "rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j ) . rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c ) .", "rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids ) . rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i ) .", "rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .", "although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence .", "rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .", "however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence .", "rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .", "the server provides a user interface for copy and paste , or for loading the users ' sequences and blastx outputs", "these various inputs are summarized as follows : \n a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction.blastx output for all the est - derived query sequences .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "targetidentifier server uses the uniprot / swiss - prot protein database.e - value : the user can also set a threshold e - value for their blastx file .", "if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as ", "no hit. the default threshold is 1  10.strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen .", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , ", "users can select download or use email for receiving their results . a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "e - value : the user can also set a threshold e - value for their blastx file . if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as  no hit. the default threshold is 1  10 .", "strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen . if the sequences were obtained by sequencing cdnas from their 3 ends , the ", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , ", "it contains the following information for each input sequence . an identifier for the sequence , the reading frame for the predicted coding region , the location of the beginning and end of the predicted coding region , a flag shown as  fs that locates any translation frame shifts detected in the blastx alignment and the predicted protein sequence .", "the other file contains the query identifiers for those sequences that do not have predicted protein - coding regions ( figure 1j ) .", "we evaluated the accuracy of orfpredictor using 2127 arabidopsis cdna sequences that have annotated protein sequences in genbank , and 4289 a.niger and 3065 phanerochaete chrysosporium sequences assembled from ests with phrap ( ) .", "we first compared the predicted arabidopsis protein sequences obtained when blastx alignments were used as a guide with the annotated protein sequences in genbank , and confirmed that our program was able to predict the protein - coding regions with 100% accuracy .", "then , we compared the ab initio predicted protein sequences with the results obtained by using blastx .", "we then examined the prediction accuracy using the a.niger and p.chrysosporium sequences , which had a blastx hit in the ncbi nr database with an e - value 1  10 .", "the ab initio predicted frames were then compared with the frames identified by using blastx .", "we found that the reading frame predicted ab initio was identical with the frames predicted by using blastx for 3943 ( 91.9% ) of the a.niger sequences and 2867 ( 93.5% ) of the p.chrysosporium sequences .", "we implemented a web server , orfpredictor , for predicting protein - coding regions in est - derived sequences .", "orfpredictor uses the reading frame predicted by using blastx when a significant alignment is produced , whereas for sequences that do not return a significant blastx alignment protein - coding regions are predicted ab initio .", "the predicted protein sequences can then be used as the input for additional annotation tools , such as interproscan ( 5 ) , for identifying protein families , domains and functional sites , the conserved domain search service ( 6 ) for the detection of structural and functional domains , and signalp ( 7 ) for locating potential signal peptides .", "categories of information derived from the est sequences . ( a ) a typical full - length cdna sequence including one or more stop codons in the 5-utr , a start codon and a stop codon", ". the coding region may contain multiple atg codons encoding methionine and the 3-utr may harbor additional stop codons .", "( b ) a full - length cdna without a stop codon in the 5-utr . ( c ) a sequence containing a 5-utr with a stop codon and a portion of the coding region .", "( d ) a sequence containing a 5-utr with a stop codon . ( e ) a sequence containing a 5-utr without a 5 stop codon , and a portion of the coding region .", "( f ) a sequence containing a portion of 5-utr without a 5 stop codon . (", "g ) a sequence containing the internal portion of a coding region with or without internal atg codons .", "( h ) a sequence containing a portion of the coding region with an internal atg codon , a 3 stop codon and 3-utr .", "( i ) a sequence containing a portion of the coding region with no internal atg codons , a 3 stop codon and a 3-utr .", "red star : stop codon at 5 end ; green circle : start codon ; blue circle : internal atg codon ; red hexagon : stop codon ; solid line : sequenced portion of the full - length cdna ; and dashed line : unsequenced or truncated portion of the full - length cdna . the orfpredictor server interface for loading data and choosing other parameters ."], "abstract_text": ["<S> orfpredictor is a web server designed for identifying protein - coding regions in expressed sequence tag ( est)-derived sequences . for query sequences with a hit in blastx , </S>", "<S> the program predicts the coding regions based on the translation reading frames identified in blastx alignments , otherwise , it predicts the most probable coding region based on the intrinsic signals of the query sequences . </S>", "<S> the output is the predicted peptide sequences in the fasta format , and a definition line that includes the query i d , the translation reading frame and the nucleotide positions where the coding region begins and ends . </S>", "<S> orfpredictor facilitates the annotation of est - derived sequences , particularly , for large - scale est projects . </S>", "<S> orfpredictor is available at . </S>"], "labels": null, "section_names": ["INTRODUCTION", "OVERVIEW OF THE ALGORITHM AND IMPLEMENTATION", "Algorithm and implementation", "Input", "Output", "EVALUATIONS OF ACCURACY", "SUMMARY", "Figures and Tables"], "sections": [["the generation of expressed sequence tags ( ests ) was originally proposed as a strategy for cdna characterization over a decade ago ( 1 ) .", "subsequent improvements in sequencing methods and dramatically reduced unit costs have increased the attractiveness of the est - based research , such that it is now one of the most widely employed methods used for gene discovery and genome characterization .", "consequently , the number of organisms with est sequences deposited in the genbank dbest database is increasing rapidly ( ) . to maximize the value of these ests , ncbi has built unigenes that incorporated est data for a number of species ( ) , and the institute for genomics research ( tigr ) has been working on the gene indices for more than 70 species ( ) . annotating est and cdna sequences", "two tools that have been designed for locating protein - coding regions in cdna and est sequences are orffinder ( ) and estscan ( ) , respectively .", "the estscan server is designed for processing a batch of est sequences for identifying the protein - coding regions with a function for correcting insertions or deletions , but it is only trained for mammals and yeast .", "we tested estscan with our aspergillus niger est sequences and found that the results were not satisfactory .", "for example , using a full - length cdna sequence encoding glucoamylase , a well - characterized enzyme in a.niger , we found that estscan could not identify its correct coding region and had the undesired side effect of inserting nucleotides even when the test sequence was correct .", "we have implemented a web server called orfpredictor for the prediction of protein - coding regions within est - derived sequences .", "the algorithm uses the translation reading frames predicted by using blastx ( 2 ) as a guide for the identification of the coding region in sequences that have a hit ( 3 ) and predicts a coding region ab initio for sequences without a hit ."], ["a mature eukaryotic mrna molecule , starting from the 5 end , typically consists of a 5 cap , a 5-untranslated region ( 5-utr ) , a protein - coding region [ open reading frame ( orf ) ] and a 3-utr followed by a poly(a ) tail .", "the protein - coding region extends from the start codon aug ( atg in a cdna ) and continues until the reading frame defined by the start codon is terminated by one of three translation stop codons , uga , uaa or uag . most cdna libraries are constructed using oligo(dt ) primers to direct first - strand synthesis by reverse transcriptase .", "essentially , all clones in oligo(dt ) primed cdna libraries will therefore include information for the 3 end of the processed transcript and a poly(a ) region .", "ests are single - pass sequencing reads obtained from either the 5 or 3 end of the cdna insert .", "the high - quality sequence obtained using systems , such as abi 3730xl , is typically 700800 nt per read .", "given that the 3- and 5-utrs are typically much shorter than 500 nt , most ests and the consensus sequences ( contigs ) generated by an est assembler are expected to include some coding sequence useful for predicting gene function . in the annotation and analysis of ests ,", "overlapping est sequences are often assembled into contigs to remove redundancy , reduce the frequency of sequencing errors and extend the length of sequence derived from each mrna species . assuming the ests are sequenced from the 5 end , sequence information from contigs and individual ests fall into 10 categories ( figure 1 ) as follows : \n a full - length sequence that includes the 5-utr with one or more stop codons ( 5 stop codon ) , translation start codon , complete protein - coding region , translation stop codon and the 3-utr .", "the protein - coding orf may have internal atg codons and the 3-utr may possess multiple stop codons .", "the 5-utr may be truncated.a full - length sequence as defined for category ( a ) , except that it does not contain any 5 stop codons.a partial sequence that has a portion of the 5-utr , one or more 5 stop codons , the start codon and a portion of the coding region.a sequence that contains only 5-utr sequence and there is a 5 stop codon.a sequence that contains a 5-utr sequence , the start codon and a portion of the protein - coding orf.a sequence that contains only 5-utr sequence without a 5 stop codon.a sequence that contains a portion of the protein - coding region , but does not contain the start codon or the stop codon.a sequence with the potential protein - coding region truncated at its 5 end , one or more atg codons in the truncated orf , the stop codon and a 3-utr.a sequence that contains a portion of the potential protein - coding region and the 3-utr sequence.a sequence that contains a portion of the 3-utr and a poly(a ) sequence at its 3 end . a full - length sequence that includes the 5-utr with one or more stop codons ( 5 stop codon ) , translation start codon , complete protein - coding region , translation stop codon and the 3-utr .", "the protein - coding orf may have internal atg codons and the 3-utr may possess multiple stop codons .", "the 5-utr may be truncated . a full - length sequence as defined for category ( a ) , except that it does not contain any 5 stop codons . a partial sequence that has a portion of the 5-utr , one or more 5 stop codons , the start codon and a portion of the coding region . a sequence that contains only 5-utr sequence and there is a 5 stop codon . a sequence that contains a 5-utr sequence , the start codon and a portion of the protein - coding orf . a sequence that contains only 5-utr sequence without a 5 stop codon .", "a sequence that contains a portion of the protein - coding region , but does not contain the start codon or the stop codon . a sequence with the potential protein - coding region truncated at its 5 end , one or more atg codons in the truncated orf , the stop codon and a 3-utr . a sequence that contains a portion of the potential protein - coding region and the 3-utr sequence . a sequence that contains a portion of the 3-utr and a poly(a ) sequence at its 3 end . for sequences generated by sequencing cdna inserts from their 3 ends ,", "the categories of their reverse complementary sequences only include ( a ) , ( b ) , ( h ) , ( i ) and ( j ) .", "therefore , it is more challenging to predict the coding region within an est than it is to predict the coding region of a fully sequenced cdna . distinguishing the translation start codon from other atg codons remains a difficult task .", "identifying start codons is further complicated because there is not a universal consensus sequence surrounding eukaryotic start codons , although the conserved consensus sequence , gccrccaugg ( r : purine ; aug : start codon ) is present in mammals ( 4 ) .", "however , blastx using a nucleotide query against a protein database is able to reliably identify protein - coding regions within a dna sequence if sufficient similarity exists between the translated query and an entry in the database ( 3 ) .", "sequencing errors may disrupt the conceptual translation of orfs , blastx could also detect frame shifts if there are insertions / deletions in the coding regions of the query sequences . when significant blastx alignments can be generated our algorithm uses them as a guide to identify the translation reading frames and coding regions . for est - derived sequences without a database match , their frames and coding sequences are predicted based on the presence and the location of intrinsic signals in a sequence that include start codons , 5 or / and 3 stop codons and stretches of poly(a ) ( figure 1 ) .", "our algorithm uses the following rules to locate protein - coding regions and predict the translation reading frame . for cases where blastx identified a significant database match ( e - value lower than a user chosen threshold ) ,", "the frame assignment in the blastx output will be used and rules 19 are applied . if there is a conflict , rules 1 and 2 will override the other rules . for sequences that do not produce a significant blastx alignment rules 310", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx.rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used.rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a).rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j).rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c).rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids).rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i).rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) . although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence.rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .", "however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence.rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx . rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used .", "rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a ) .", "rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j ) .", "rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c ) .", "rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids ) .", "rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i ) .", "rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .", "although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence .", "rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .", "however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence .", "rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .", "the server provides a user interface for copy and paste , or for loading the users ' sequences and blastx outputs .", "these various inputs are summarized as follows : \n a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction.blastx output for all the est - derived query sequences .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "targetidentifier server uses the uniprot / swiss - prot protein database.e - value : the user can also set a threshold e - value for their blastx file .", "if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as ", "no hit. the default threshold is 1  10.strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen .", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , ", "users can select download or use email for receiving their results . a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "e - value : the user can also set a threshold e - value for their blastx file .", "if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as ", "strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen . if the sequences were obtained by sequencing cdnas from their 3 ends , the   strand should be chosen .", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , ", "an identifier for the sequence , the reading frame for the predicted coding region , the location of the beginning and end of the predicted coding region , a flag shown as  fs that locates any translation frame shifts detected in the blastx alignment and the predicted protein sequence .", "the other file contains the query identifiers for those sequences that do not have predicted protein - coding regions ( figure 1j ) ."], ["most ests encompass only a portion of the mrna sequence . therefore , it is more challenging to predict the coding region within an est than it is to predict the coding region of a fully sequenced cdna . distinguishing the translation start codon from other atg codons", "identifying start codons is further complicated because there is not a universal consensus sequence surrounding eukaryotic start codons , although the conserved consensus sequence , gccrccaugg ( r : purine ; aug : start codon ) is present in mammals ( 4 ) .", "however , blastx using a nucleotide query against a protein database is able to reliably identify protein - coding regions within a dna sequence if sufficient similarity exists between the translated query and an entry in the database ( 3 ) .", "sequencing errors may disrupt the conceptual translation of orfs , blastx could also detect frame shifts if there are insertions / deletions in the coding regions of the query sequences . when significant blastx alignments can be generated our algorithm uses them as a guide to identify the translation reading frames and coding regions . for est - derived sequences without a database match , their frames and coding sequences are predicted based on the presence and the location of intrinsic signals in a sequence that include start codons , 5 or / and 3 stop codons and stretches of poly(a ) ( figure 1 ) .", "our algorithm uses the following rules to locate protein - coding regions and predict the translation reading frame . for cases where blastx identified a significant database match ( e - value lower than a user chosen threshold ) ,", "the frame assignment in the blastx output will be used and rules 19 are applied . if there is a conflict , rules 1 and 2 will override the other rules . for sequences that do not produce a significant blastx alignment rules 310", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx.rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used.rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a).rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j).rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c).rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids).rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i).rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .", "although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence.rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature . however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence.rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .", "rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx . rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used .", "rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a ) .", "rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j ) . rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c ) .", "rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids ) . rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i ) .", "rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .", "although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence .", "rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .", "however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence .", "rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region ."], ["the server provides a user interface for copy and paste , or for loading the users ' sequences and blastx outputs", "these various inputs are summarized as follows : \n a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction.blastx output for all the est - derived query sequences .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "targetidentifier server uses the uniprot / swiss - prot protein database.e - value : the user can also set a threshold e - value for their blastx file .", "if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as ", "no hit. the default threshold is 1  10.strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen .", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , ", "users can select download or use email for receiving their results . a sequence file in the fasta format .", "the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction .", "although it is optional , the user is encouraged to provide a pre - run blastx output .", "the user can choose a cut - off e - value when setting up the blastx run .", "if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .", "users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .", "e - value : the user can also set a threshold e - value for their blastx file . if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as  no hit. the default threshold is 1  10 .", "strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the  +  strand should be chosen . if the sequences were obtained by sequencing cdnas from their 3 ends , the ", "if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting , "], ["it contains the following information for each input sequence . an identifier for the sequence , the reading frame for the predicted coding region , the location of the beginning and end of the predicted coding region , a flag shown as  fs that locates any translation frame shifts detected in the blastx alignment and the predicted protein sequence .", "the other file contains the query identifiers for those sequences that do not have predicted protein - coding regions ( figure 1j ) ."], ["we evaluated the accuracy of orfpredictor using 2127 arabidopsis cdna sequences that have annotated protein sequences in genbank , and 4289 a.niger and 3065 phanerochaete chrysosporium sequences assembled from ests with phrap ( ) .", "we first compared the predicted arabidopsis protein sequences obtained when blastx alignments were used as a guide with the annotated protein sequences in genbank , and confirmed that our program was able to predict the protein - coding regions with 100% accuracy .", "then , we compared the ab initio predicted protein sequences with the results obtained by using blastx .", "we then examined the prediction accuracy using the a.niger and p.chrysosporium sequences , which had a blastx hit in the ncbi nr database with an e - value 1  10 .", "the ab initio predicted frames were then compared with the frames identified by using blastx .", "we found that the reading frame predicted ab initio was identical with the frames predicted by using blastx for 3943 ( 91.9% ) of the a.niger sequences and 2867 ( 93.5% ) of the p.chrysosporium sequences ."], ["we implemented a web server , orfpredictor , for predicting protein - coding regions in est - derived sequences .", "orfpredictor uses the reading frame predicted by using blastx when a significant alignment is produced , whereas for sequences that do not return a significant blastx alignment protein - coding regions are predicted ab initio .", "the predicted protein sequences can then be used as the input for additional annotation tools , such as interproscan ( 5 ) , for identifying protein families , domains and functional sites , the conserved domain search service ( 6 ) for the detection of structural and functional domains , and signalp ( 7 ) for locating potential signal peptides ."], ["categories of information derived from the est sequences . ( a ) a typical full - length cdna sequence including one or more stop codons in the 5-utr , a start codon and a stop codon", ". the coding region may contain multiple atg codons encoding methionine and the 3-utr may harbor additional stop codons .", "( b ) a full - length cdna without a stop codon in the 5-utr . ( c ) a sequence containing a 5-utr with a stop codon and a portion of the coding region .", "( d ) a sequence containing a 5-utr with a stop codon . ( e ) a sequence containing a 5-utr without a 5 stop codon , and a portion of the coding region .", "( f ) a sequence containing a portion of 5-utr without a 5 stop codon . (", "g ) a sequence containing the internal portion of a coding region with or without internal atg codons .", "( h ) a sequence containing a portion of the coding region with an internal atg codon , a 3 stop codon and 3-utr .", "( i ) a sequence containing a portion of the coding region with no internal atg codons , a 3 stop codon and a 3-utr .", "red star : stop codon at 5 end ; green circle : start codon ; blue circle : internal atg codon ; red hexagon : stop codon ; solid line : sequenced portion of the full - length cdna ; and dashed line : unsequenced or truncated portion of the full - length cdna . the orfpredictor server interface for loading data and choosing other parameters ."]]}
{"article_id": "PMC1852396", "article_text": ["nature evolved a very limited number of chromophores in the different evolutionary branches of the tree of life because of the similar needs of the most diverse organisms for both the detection of the external world and the interaction with it .", "the study of these chromophores and their distribution inside cellular structures is usually based on extractive procedures , followed by biochemical or spectroscopic assays .", "extractive techniques often include disadvantages : they can modify the nature of the components and they are not successful in isolating the chromophores .", "direct investigation by means of microspectrophotometry of on intact samples has the advantage of preserving the integrity of biological structures or substructures .", "a microspectrophotometer is a simple apparatus consisting of a modified microscope that can measure absorption or emission spectra of very small areas inside a cell 1 .", "a high quality microscope is equipped with a polychromator , i.e. a flat field concave grating , which is connected to the slit - shaped exit pupil of a 19 light - guides probe .", "the dispersion image of the exit pupil produced by the polychromator is focused onto a digital slow scan cooled ccd camera .", "the main feature of this instrumentation is the possibility to measure in vivo absorption or emission spectra at the same time on different sub - cellular compartments using extremely low light intensities .", "this set - up represents a better strategy with respect to traditional instruments , since it eliminates errors typical of microspectrophotometry , such as photobleaching , distributional errors , the schwarzschild - villiger effect , and allows in vivo reliable spectroscopic investigations 2 , 3 .", "the photosynthetic and the photoreceptive compartments are the main location of chromophores inside algal cells . absorption and emission spectra measured in vivo on these compartments can provide very precise and accurate information about the spectral range in which chromophore molecules capture photons in their natural environment and their de - excitation pathways 4 .", "we tested our apparatus by measuring absorption spectra on the eyespot and chloroplast of the unicellular alga dunaliella and emission spectra on the photoreceptor and chloroplast of the unicellular alga euglena gracilis .", "cultures . euglena gracilis strain z ( sammlung von algenkulturen gttingen , 1224 - 5/25 ) cells", "were grown axenically in cramer - myers medium 0.025 m in sodium acetate ( ph 6.8 ) 5 ; cultures of dunaliella spp .", "( sammlung von algenkulturen gttingen , 19 - 5 ) were grown axenically in johnson 's medium 6 .", "both cultures were kept under constant temperature ( 24 c ) and continuous illumination ( 2x10 mol photons m sec ) .", "the hardware platform consists of a zeiss axioplan microscope ( zeiss , oberkoken , de ) , equipped with an epifluorescence system , 100x ( n.a .", "the emission spectra were acquired with a combination of a uv - blue filter set ( 8 nm band pass excitation filter , 365 nm ; chromatic beam splitter , 395 nm ; barrier filter , 397 nm ; irradiance 800 w / cm ) , and a blue - violet filter set ( 8 nm band pass excitation filter , 436 nm ; chromatic beam splitter , 460 nm ; barrier filter , 470 nm ; irradiance 1100 w / cm ) .", "77423 oriel , stratford , connecticut , usa ) connected to a schott kl 1500 probe illuminator system ( schott corporation , mainz , de ) was mounted in the back focal plane of the ocular of the microscope ( figure 1 ) .", "this probe consists of an outer bundle of 24 light - guides and a central bundle of 19 light - guides ( figure 2a ) .", "the outer bundle is used for centering the objects , while the central bundle of the probe is used for acquiring transmitted or emitted light .", "the sub - cellular components on which the spectra have to be measured , are placed in the microscope field and finely adjusted .", "the exit pupil of the probe ( figure 2b ) is connected to a flat field imaging concave grating polychromator ( mod .", "52300070 , jobin yvon , longjumenau , france ) that produces a dispersion image of the probe .", "this image is in turn focused onto a digital slow scan cooled ccd camera ( dta discovery ds260e , pisa , italy ) , and it consists of 19 multi - lines strips showing the distribution of light intensity according to the wavelength .", "the microspectophotometer instrumentation possesses a graphical interface that allows the set - up of both the optics and the frame grabber ( scion corporation , frederick , maryland , usa ) and controls the measurements .", "once the instrumental has been set - up , the operator , on the basis of the light guide positions upon the cell , selects the zones of the dispersion image displayed on the top of the graphical layout ( in the layout r stands for reference , and s for sample ) .", "the resulting spectrum is displayed at the bottom of the graphical layout , ( figure 3 ) .", "absorption measurements are based on the comparison of two radiant fluxes density is and ir .", "is results from the interaction of light with the sample ( it is related to absorption cross section of the molecules and the number of absorbing molecules ) 7 , while ir results from the interaction of light with the reference material . therefore , we can consider the absorbance of a sample ( as ) as derived from the measures as follows : this equation is known as the lambert - beer 's law . for the discussion on the theoretical aspects of image formation and the light transmission in microspectrophotometry see barsanti et al .", "absorption spectra were performed on both the eyespot ( screening device ) and the chloroplast ( photosynthetic apparatus ) of the unicellular alga dunaliella .", "the central bundle of the probe was centered on both the structures in the apical portion of the cell ( is ) , in such a way that some light - guides were located outside the cell ; these guides measure ir .", "measurements of emission microspectroscopy are based on the radiant flux density f. this density is given by : where ia is the amount of light absorbed by the chromophores ; f is their fluorescence quantum yield and z represents the fraction of fluorescence collected by the objective , 9 .", "emission spectra were performed on both the photoreceptor and chloroplast of the photosensitive alga euglena gracilis .", "the outer bundle of the probe was used for centering the photoreceptor and the chloroplast in the apical portion of the cell , while the central bundle of the probe was used for acquiring light emitted by the two structures .", "photographs were taken with an olympus camedia c-30303 digital camera ( olympus , tokyo , japan ) mounted onto the microscope .", "the eyespot is recognizable as a the bright orange spot located on the left side of the cell in the apical portion .", "the size of the eyespot is about 3  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figures 4b ) .", "this spectrum closely resembles the spectrum obtained by batra and tollin 10 on a suspension of eyespot granules , and the other in vivo spectra previously recorded by strother and wolken 11 , by benedetti et al .", "major peaks are due to lutein whose bands are centered at 410 , 479.5 , and 510 nm and -carotene whose bands are centered at 455.5 , 481.5 , and 510.5 nm ( not shown ) .", "it clearly shows that dunaliella belongs to the green lineage of eukaryotic algae since only chlorophylls a and b and carotenoids are present in this spectrum .", "gaussian bands decomposition of this spectrum is easily explained as a combination of chlorophyll a bands centered at ( 410 , 435 , 444 , 585 , 615 , 626 , 634.5 , 663 , 672 , 678 , 683 , 695 nm , chlorophyll b bands centered at 412 , 428.5 , 445 , 452 , 582 , 594 , 607 , 621.5 , 652 nm , and carotenoids lutein and bands centered at 410 , 479.5 , and 510 nm , ( not shown ) , 14 .", "the photoreceptor is recognizable as a the bright green spot located in the apical portion of the cell .", "the size of the photoreceptor is about 2  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figure 6b ) .", "figure 7a shows the emission spectrum of a single photoreceptor under 436 nm excitation light , after 10 seconds of excitation with the 365 nm light .", "gaussian bands decomposition of the emission spectrum reveals 3 bands with different intensity centered at about 500 nm , 525 nm , and 556 nm ( not shown ) .", "the bands obtained by the gaussian decomposition indicate the presence of a mixture of very similar conformers in the photoreceptor , each one capable of photocycling between a non - fluorescent parent species and a fluorescent excited species 15 .", "the presence in the photoreceptor of euglena of a photochromic chromophore , which undergoes light - driven reversible photochromism has been well established by means of digital and fluorescence microscopy 16 .", "the photoreceptor possesses optical bistability , i.e. upon photoexcitation the ground state generates a stable excited state , which can be photochemically driven back to the ground state .", "the 27 kda protein extracted from the photoreceptor shows a similar behavior , the photochromic reaction cycling between two different stable conformers , the parent and the excited conformers 17 .", "figure 7b shows the emission spectrum of the thylakoid compartment of euglena gracilis excited at 463 nm .", "it is the typical emission spectrum of a chloroplast that contains only chlorophyll a and b whose bands are centered at 685 for the photosystem ii and at 725 nm for the photosystem i , 18 .", "set - up of the polychromator - based microspectrophotometer entrance ( a ) and exit ( b ) pupils of the light guide probe layout of the software interface a ) bright field image of dunaliella .", "b ) position of the probe upon dunaliella   absorption spectra of the eyespot ( a ) and a chloroplast ( b ) of dunaliella a ) fluorescent image of euglena gracilis ; b ) position of the probe upon euglena gracilis emission spectra of the photoreceptor ( a ) and a chloroplast ( b ) of euglena gracilis"], "abstract_text": ["<S> a microspectrophotometer is a digital microscope used to measure absorption and fluorescence spectra . in this paper </S>", "<S> we describe a polychromator - based microspectrophotometer that performs in vivo absorption or emission measurements at the same time on different subcellular compartments such as photoreceptive and photosynthetic structures of algal cells . in this system , a flat field imaging concave grating polychromator is connected to the slit - shaped exit pupil of a light - guide probe mounted onto a microscope equipped with an epifluorescence module.the subcellular components , on which the spectra will be measured , are placed in the microscope field and finely adjusted . </S>", "<S> the outer bundle of the probe is used for centering the objects , while the central bundle of the probe , containing 19 light guides , is used for acquiring either transmitted or emitted light ( i.e. fluorescence ) . </S>", "<S> the light transmitted or emitted by the subcellular components is collected by the probe mounted in the back focal plane of the ocular . </S>", "<S> the exit pupil of this probe , connected to a flat field imaging concave grating polychromator , produces a dispersion image that in turn is focused onto a digital slow scan cooled ccd camera . </S>", "<S> absorption and emission spectra of algal subcellular compartments are presented </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "Figures and Tables"], "sections": [["nature evolved a very limited number of chromophores in the different evolutionary branches of the tree of life because of the similar needs of the most diverse organisms for both the detection of the external world and the interaction with it .", "the study of these chromophores and their distribution inside cellular structures is usually based on extractive procedures , followed by biochemical or spectroscopic assays .", "extractive techniques often include disadvantages : they can modify the nature of the components and they are not successful in isolating the chromophores .", "direct investigation by means of microspectrophotometry of on intact samples has the advantage of preserving the integrity of biological structures or substructures .", "a microspectrophotometer is a simple apparatus consisting of a modified microscope that can measure absorption or emission spectra of very small areas inside a cell 1 .", "a high quality microscope is equipped with a polychromator , i.e. a flat field concave grating , which is connected to the slit - shaped exit pupil of a 19 light - guides probe .", "the dispersion image of the exit pupil produced by the polychromator is focused onto a digital slow scan cooled ccd camera .", "the main feature of this instrumentation is the possibility to measure in vivo absorption or emission spectra at the same time on different sub - cellular compartments using extremely low light intensities .", "this set - up represents a better strategy with respect to traditional instruments , since it eliminates errors typical of microspectrophotometry , such as photobleaching , distributional errors , the schwarzschild - villiger effect , and allows in vivo reliable spectroscopic investigations 2 , 3 .", "the photosynthetic and the photoreceptive compartments are the main location of chromophores inside algal cells . absorption and emission spectra measured in vivo on these compartments can provide very precise and accurate information about the spectral range in which chromophore molecules capture photons in their natural environment and their de - excitation pathways 4 .", "we tested our apparatus by measuring absorption spectra on the eyespot and chloroplast of the unicellular alga dunaliella and emission spectra on the photoreceptor and chloroplast of the unicellular alga euglena gracilis ."], ["cultures . euglena gracilis strain z ( sammlung von algenkulturen gttingen , 1224 - 5/25 ) cells", "were grown axenically in cramer - myers medium 0.025 m in sodium acetate ( ph 6.8 ) 5 ; cultures of dunaliella spp .", "( sammlung von algenkulturen gttingen , 19 - 5 ) were grown axenically in johnson 's medium 6 .", "both cultures were kept under constant temperature ( 24 c ) and continuous illumination ( 2x10 mol photons m sec ) .", "the hardware platform consists of a zeiss axioplan microscope ( zeiss , oberkoken , de ) , equipped with an epifluorescence system , 100x ( n.a .", "the emission spectra were acquired with a combination of a uv - blue filter set ( 8 nm band pass excitation filter , 365 nm ; chromatic beam splitter , 395 nm ; barrier filter , 397 nm ; irradiance 800 w / cm ) , and a blue - violet filter set ( 8 nm band pass excitation filter , 436 nm ; chromatic beam splitter , 460 nm ; barrier filter , 470 nm ; irradiance 1100 w / cm ) .", "77423 oriel , stratford , connecticut , usa ) connected to a schott kl 1500 probe illuminator system ( schott corporation , mainz , de ) was mounted in the back focal plane of the ocular of the microscope ( figure 1 ) .", "this probe consists of an outer bundle of 24 light - guides and a central bundle of 19 light - guides ( figure 2a ) .", "the outer bundle is used for centering the objects , while the central bundle of the probe is used for acquiring transmitted or emitted light .", "the sub - cellular components on which the spectra have to be measured , are placed in the microscope field and finely adjusted .", "the exit pupil of the probe ( figure 2b ) is connected to a flat field imaging concave grating polychromator ( mod .", "52300070 , jobin yvon , longjumenau , france ) that produces a dispersion image of the probe .", "this image is in turn focused onto a digital slow scan cooled ccd camera ( dta discovery ds260e , pisa , italy ) , and it consists of 19 multi - lines strips showing the distribution of light intensity according to the wavelength .", "the microspectophotometer instrumentation possesses a graphical interface that allows the set - up of both the optics and the frame grabber ( scion corporation , frederick , maryland , usa ) and controls the measurements .", "once the instrumental has been set - up , the operator , on the basis of the light guide positions upon the cell , selects the zones of the dispersion image displayed on the top of the graphical layout ( in the layout r stands for reference , and s for sample ) .", "the resulting spectrum is displayed at the bottom of the graphical layout , ( figure 3 ) .", "absorption measurements are based on the comparison of two radiant fluxes density is and ir .", "is results from the interaction of light with the sample ( it is related to absorption cross section of the molecules and the number of absorbing molecules ) 7 , while ir results from the interaction of light with the reference material . therefore , we can consider the absorbance of a sample ( as ) as derived from the measures as follows : this equation is known as the lambert - beer 's law . for the discussion on the theoretical aspects of image formation and the light transmission in microspectrophotometry see barsanti et al .", "absorption spectra were performed on both the eyespot ( screening device ) and the chloroplast ( photosynthetic apparatus ) of the unicellular alga dunaliella .", "the central bundle of the probe was centered on both the structures in the apical portion of the cell ( is ) , in such a way that some light - guides were located outside the cell ; these guides measure ir .", "measurements of emission microspectroscopy are based on the radiant flux density f. this density is given by : where ia is the amount of light absorbed by the chromophores ; f is their fluorescence quantum yield and z represents the fraction of fluorescence collected by the objective , 9 .", "emission spectra were performed on both the photoreceptor and chloroplast of the photosensitive alga euglena gracilis .", "the outer bundle of the probe was used for centering the photoreceptor and the chloroplast in the apical portion of the cell , while the central bundle of the probe was used for acquiring light emitted by the two structures .", "photographs were taken with an olympus camedia c-30303 digital camera ( olympus , tokyo , japan ) mounted onto the microscope ."], ["the eyespot is recognizable as a the bright orange spot located on the left side of the cell in the apical portion .", "the size of the eyespot is about 3  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figures 4b ) .", "this spectrum closely resembles the spectrum obtained by batra and tollin 10 on a suspension of eyespot granules , and the other in vivo spectra previously recorded by strother and wolken 11 , by benedetti et al .", "major peaks are due to lutein whose bands are centered at 410 , 479.5 , and 510 nm and -carotene whose bands are centered at 455.5 , 481.5 , and 510.5 nm ( not shown ) .", "it clearly shows that dunaliella belongs to the green lineage of eukaryotic algae since only chlorophylls a and b and carotenoids are present in this spectrum .", "gaussian bands decomposition of this spectrum is easily explained as a combination of chlorophyll a bands centered at ( 410 , 435 , 444 , 585 , 615 , 626 , 634.5 , 663 , 672 , 678 , 683 , 695 nm , chlorophyll b bands centered at 412 , 428.5 , 445 , 452 , 582 , 594 , 607 , 621.5 , 652 nm , and carotenoids lutein and bands centered at 410 , 479.5 , and 510 nm , ( not shown ) , 14 .", "the photoreceptor is recognizable as a the bright green spot located in the apical portion of the cell .", "the size of the photoreceptor is about 2  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figure 6b ) .", "figure 7a shows the emission spectrum of a single photoreceptor under 436 nm excitation light , after 10 seconds of excitation with the 365 nm light .", "gaussian bands decomposition of the emission spectrum reveals 3 bands with different intensity centered at about 500 nm , 525 nm , and 556 nm ( not shown ) .", "the bands obtained by the gaussian decomposition indicate the presence of a mixture of very similar conformers in the photoreceptor , each one capable of photocycling between a non - fluorescent parent species and a fluorescent excited species 15 .", "the presence in the photoreceptor of euglena of a photochromic chromophore , which undergoes light - driven reversible photochromism has been well established by means of digital and fluorescence microscopy 16 .", "the photoreceptor possesses optical bistability , i.e. upon photoexcitation the ground state generates a stable excited state , which can be photochemically driven back to the ground state .", "the 27 kda protein extracted from the photoreceptor shows a similar behavior , the photochromic reaction cycling between two different stable conformers , the parent and the excited conformers 17 .", "figure 7b shows the emission spectrum of the thylakoid compartment of euglena gracilis excited at 463 nm .", "it is the typical emission spectrum of a chloroplast that contains only chlorophyll a and b whose bands are centered at 685 for the photosystem ii and at 725 nm for the photosystem i , 18 ."], ["set - up of the polychromator - based microspectrophotometer entrance ( a ) and exit ( b ) pupils of the light guide probe layout of the software interface a ) bright field image of dunaliella .", "b ) position of the probe upon dunaliella   absorption spectra of the eyespot ( a ) and a chloroplast ( b ) of dunaliella a ) fluorescent image of euglena gracilis ; b ) position of the probe upon euglena gracilis emission spectra of the photoreceptor ( a ) and a chloroplast ( b ) of euglena gracilis"]]}
{"article_id": "PMC4684879", "article_text": ["the rostral ventromedial medulla ( rvm ) is an important relay region that contributes to the descending pain control pathway from the periaqueductal gray ( pag ) to the superficial laminae ( laminae i and ii ) of the spinal cord [ 1 , 2 ] .", "it is well known that the rvm is closely linked to long - lasting activation of descending control circuits that involve descending facilitation , which significantly contributes to the development of persistent pain induced by tissue and nerve injury .", "although many studies have focused on this region , the cellular and molecular mechanisms of descending pain facilitation control remain poorly understood . due to the role of the descending pain facilitation pathway , several types of injuries , such as tissue and nerve injury , often become chronic and persistent , eventually leading to neuropathic pain .", ", significant progress has been made in basic and clinical studies ; however , the currently available therapies for neuropathic pain remain inadequate , and the search continues not only for improved treatments but also for novel targets .", "the mammalian target of rapamycin ( mtor ) , a conserved serine - threonine protein kinase that is inhibited by the effective clinical immunosuppressant rapamycin , regulates several intracellular processes in response to various extracellular signals and thereby modulates mrna translation .", "thus , mtor plays a critical role in the modulation of long - term plasticity and memory processes [ 57 ] .", "activation of the mtor complex with the protein raptor ( mtorc1 ) promotes the phosphorylation of mtor downstream targets , including eukaryotic initiation factor 4e - binding protein ( 4e - bp1/2 ) and s6 kinase ( s6k ) , which can further lead to local protein synthesis .", "it has been reported that deletion of either the 4e - bp1/2 or the s6k gene in mice results in deficits in synaptic plasticity and long - term memory [ 8 , 9 ] . moreover", ", phosphorylated mtor ( p - mtor ) , which is the activated form , is upregulated in the peripheral nervous system as well as at the spinal cord level in several pain models [ 1015 ] .", "inhibition of spinal cord mtor by intrathecal administration has proven to be effective in alleviating the nociceptive behaviors of animals under pain conditions [ 10 , 11 , 16 , 17 ] .", "synaptic plasticity changes in chronic pain conditions can occur not only at the spinal cord level but also at the supraspinal level , including the rvm .", "therefore , considering the important role of the rvm in descending pain facilitation , targeting mtor in the rvm might be a promising way to combat pain . because serotoninergic ( 5-htergic ) neurons are the primary constitutive element in the rvm and can send projections to the superficial spinal dorsal horn ( sdh ) [ 1820 ]", ", we thus hypothesize that 5-htergic spinally projecting neurons in the rvm contain mtor , the activation of which could in turn increase the excitability of the 5-htergic neurons and thus may potently potentiate the descending facilitation pain control pathway and exaggerate neuropathic pain conditions .", "accordingly , we used a spared nerve injury ( sni ) model to evaluate the role of mtor in the rvm in neuropathic pain in rats .", "adult male sprague - dawley ( sd ) rats ( weighing 250290  g ) were used in the present study .", "the ethics committee for animal experiments of the fourth military medical university ( xi'an , china ) approved the animal experiments ( permit number : 10071 ) .", "briefly , rats were anesthetized with pentobarbital ( 45  mg / kg , i.p . ) , and three terminal branches of the sciatic nerve were exposed by direct incision of the skin and a section of the biceps femoris muscle in the left thigh .", "the tibial and common peroneal branches were carefully tight - ligated with 5 - 0 silk sutures and sectioned distal to the ligation , removing 24  mm of the distal nerve stump .", "the surgical procedures for the sham - operated group were identical to those for the sni group , except that the nerves were not lesioned .", "mechanical allodynia , as a behavioral sign of sni - induced neuropathic pain , was assessed by measuring the 50% paw withdrawal threshold ( pwt ) as described previously .", "the 50% pwt in response to a series of ascending von frey filaments ( stoelting , kiel , wi , usa ) was determined by the up - and - down method .", "von frey force was delivered perpendicularly to the plantar surface of the hind paw for 2 to 3 seconds .", "an abrupt withdrawal of the hind paw during stimulation was recorded as a positive response .", "the 50% pwt was calculated using the following formula : 50%  pwt = 10 .", "mechanical allodynia was assessed by measuring the 50% pwt of the ipsilateral hind paw in sni- or sham - operated rats .", "measurements of the paw withdrawal latency ( pwl ) were obtained using a timer that was started by the activation of the heat source and stopped when withdrawal of the paw was detected with a photodetector .", "three measurements of the pwl were taken for each hind paw and were averaged as the result of each test session .", "the ipsilateral hind paw was tested with intervals of more than 5  min between consecutive tests .", "fluoro - gold ( fg ) was used as a retrograde tracer to label the rvm neurons that project to the sdh .", "the procedures for fg injection were essentially the same as in our previous studies [ 20 , 27 ] .", "briefly , after exposing the lumbar cord , 0.1  l of a 4% solution of fg ( fluorochrome , denver , co , usa ) dissolved in 0.9% saline was stereotaxically injected into the left side of the lumbar dorsal horn with a microsyringe attached to a glass micropipette by pressure injection . due to the transportation period of the tracer ,", "after perfusion , the brains and spinal cords of the rats were cut into sections .", "the sections were used to evaluate the fg injection sites in the sdh as well as the distribution patterns of the retrogradely fg - labeled neurons in the rvm under an epifluorescence microscope ( bx-60 ; olympus , tokyo , japan ) using an appropriate filter for fg ( excitation 350395  nm ; emission 430  nm ) .", "after the rats were anesthetized with pentobarbital ( 45  mg / kg , i.p . ) , a 26-gauge stainless steel guide cannula was stereotaxically implanted into a site above the rvm ( 10.52  mm posterior to bregma , 0  mm lateral from the midline , and 10.20  mm beneath the surface of the skull ) .", "intra - rvm microinjections were delivered via a 33-gauge injector needle cannula that was lowered 0.5  mm deeper into the brainstem than the guide cannula . the microinjection apparatus consisted of a hamilton syringe ( 10  l ) connected to an injector ( 33-gauge ) by a thin polyethylene tube and a motorized syringe pump .", "rapamycin ( 250  m/1  l , dissolved in a saline / dmso mix comprising 25% dmso , tocris bioscience , minneapolis , mn , usa ) , the specific inhibitor for mtor , was infused into the rvm at a rate of 0.1  l / min ; an equivalent volume of 25% dmso was used as a vehicle .", "after injection , the microinjection needle was left in place for at least 2  min .", "the injection sites were verified at the end of all of the experiments by nissl staining , and injection sites outside the rvm region were excluded from the study .", "these rats were randomly divided into two groups designed for different purposes , as shown in figure 6(c ) : group 1 : to investigate whether rapamycin could influence the induction stage of sni - induced neuropathic pain , behavioral tests were performed before the first drug or vehicle injection , followed by sni ( pre - sni ) , and 30  min after the second injection on day 1 after sni ( sni - d1 ) ( figure 6(c ) , top ) and group 2 : behavioral tests were performed before sni ( pre - sni ) , 6 days after sni ( sni - d6 ) , and 30  min after drug or vehicle injection on day 7 after sni ( sni - d7 ) for the purpose of demonstrating the effect of rapamycin on the maintenance stage of sni - induced neuropathic pain ( figure 6(c ) , bottom ) .", "the rats were transcardially perfused with 150  ml of 0.01  m phosphate - buffered saline ( pbs , ph 7.4 ) , followed by 500  ml of 4% paraformaldehyde in 0.1  m phosphate buffer ( pb , ph 7.4 ) .", "the brainstems and/or spinal cords were transversely sliced into 25 mm thick coronal sections using a freezing microtome ( cm1950 , leica , heidelberg , germany ) .", "double - immunofluorescence staining for p - mtor / neun , p - mtor / fg , or p - mtor/5-ht was performed .", "the sections were sequentially incubated at room temperature with primary antisera in 0.01  m pbs containing 5% normal donkey serum ( nds ) , 0.3% triton x-100 , 0.05% nan3 , and 0.25% carrageenan ( pbs - nds , ph 7.4 ) for 24  h. then , the sections were incubated with fluorescein - labeled igg ( secondary antisera ) for 6  h. a negative control experiment , in which the primary antisera were omitted , and a peptide competition assay were both carried out .", "after the immunofluorescence histochemical staining , the sections were observed and images were captured using a confocal laser - scanning microscope ( clsm , fv1000 , olympus ) .", "micrographs of 1012 sections per rat , which were 150  m apart within bregma 9.30 to 11.60  mm , were analyzed for p - mtor expression . using imagej software , the rvm area , including the nucleus raphe magnus ( rmg ) and the nucleus reticularis gigantocellularis pars  ,", "the p - mtor - positive cells within the area were counted manually by an observer blinded to the treatment conditions .", "the same counting method was used to evaluate the coexpression of 5-ht / p - mtor as well as that of p - mtor / fg within the rvm .", "cells with visible green cytoplasmic staining represent 5-htergic or fg - labeled cells , while red staining represents p - mtor - positive cells ; thus , cells with 5-ht or fg double - labeling with p - mtor appear yellow .", "the rats were decapitated , and brain slices ( 400  mm ) containing the rvm were cut at 0c with a vibratome ( vt1200s , leica ) in a sucrose cutting solution containing the following ( in mm ) : kcl 2.5 , nah2po4 1.2 , nahco3 26 , sucrose 252 , mgso47h2o 6 , cacl2 0.5 , and glucose 10 , bubbled with 95% o2/5% co2 ( ph 7.4 ) . for the electrophysiology studies ,", "the brain slices were transferred to a submerged recovery chamber with oxygenated artificial cerebrospinal fluid ( acsf ) containing the following ( in mm ) : nacl 124 , kcl 2.5 , mgso47h2o 2 , nah2po4 1 , nahco3 25 , cacl2 2 , and glucose 37 for 2 hours at room temperature before recording .", "for the biochemical experiments , the slices were slowly brought to a final temperature of 30c in acsf gassed with 95% o2/5% co2 and incubated for at least 1 hour before the experiments .", "the brain slices were treated with rapamycin ( 250  m ) and vehicle for 30  min .", "subsequently , the rvm regions were microdissected and snap - frozen over dry ice . following the standard western blot protocol ,", "rats were anesthetized with an overdose of pentobarbital ( 60  mg / kg , i.p . ) , and the rvm regions were carefully dissected and harvested for western blotting . to obtain total protein extracts ,", "the tissues were lysed in 300  l lysis buffer containing 10  mm tris , 150  mm nacl , 1% triton x-100 , 0.5% np-40 , and 1  mm edta at ph 7.4 .", "the samples were adequately mixed at a 100  :  1 ( v / v ) ratio of protease inhibitor cocktail and phosphatase inhibitor cocktail ( roche , tucson , az , usa ) .", "the procedures for the in vitro - infused brain slices were similar to those of the tissue protocols .", "then , 30  g of cell lysis material ( quantitatively measured using the bca protein assay ; thermo scientific , rockford , il , usa ) was resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to pvdf membranes ( immobilon - p , millipore ) . after blocking in nonfat milk for 1  h", ", the membranes were incubated overnight at 4c with the following primary antibodies : rabbit anti - mtor ( 1  :  1000 , cell signaling technology ) ; rabbit anti - p - mtor ( 1  :  1000 , cell signaling technology ) ; rabbit anti - s6k ( 1  :  1000 , cell signaling technology ) ; rabbit anti - p - s6k ( 1  :  1000 , cell signaling technology ) ; and mouse anti--actin ( 1  :  5000 , sigma , st .", "the immunoblots were then reacted with the corresponding horseradish peroxidase- ( hrp- ) conjugated secondary antibodies ( anti - rabbit 1  :  5000 , anti - mouse 1  :  5000 ; amersham pharmacia biotech , piscataway , nj , usa ) .", "all of the reactions were detected by the enhanced chemiluminescence ( ecl ) detection method ( amersham ) and exposure to film .", "target protein levels were normalized against -actin levels and expressed as fold changes relative to the nave control group .", "neurons in the rvm region were targeted for recording using an upright microscope equipped with zeiss ( oberkochen , germany ) infrared - differential interference contrast ( ir - dic ) optics , a 40 water - immersion objective , and a video - imaging camera .", "the patch pipette was filled with intracellular solution containing the following ( in mm ) : k - gluconate 130 , nacl 5 , kcl 15 , egta 0.4 , hepes 10 , mg - atp 4 , and tris - gtp 0.2 , ph 7.257.35 , with an osmotic pressure of 290300  mosm / l . the pipette resistance , as measured in the bath , was typically 4  0.5 m. the voltage was held at 60  mv , and neurons were given at least 3  min to stabilize before data were collected .", "spontaneous discharge and the number of action potentials were used to investigate sni - induced changes in neuronal excitability in the rvm .", "the excitatory postsynaptic currents ( epscs ) of the rvm 5-htergic neurons , which are mediated by ampa receptors , were voltage - clamped and recorded at 60  mv with an axon 700b amplifier ( molecular devices , sunnyvale , ca , usa ) after blocking gabaergic transmission by picrotoxin ( 100  mm , sigma ) , a gabaa receptor antagonist .", "the membrane excitability of the recorded neurons was measured in current - clamp mode by determining the number of action potentials elicited by intracellular injection of 0 , 10 , 20 , 30 , 40 , 50 , and 60  pa depolarizing currents for 400  ms .", "the spike number was determined to estimate the influence of rapamycin on the recorded neurons . in all cases ,", "biocytin ( 0.5% ) was introduced into the intracellular solution to identify the morphological properties of the recorded neurons .", "after recording , the brain slices were immediately fixed in 4% paraformaldehyde in 0.1  m pb for 4  h at room temperature .", "then , sections were rinsed with 3% hydrogen peroxide in 0.01  m pbs for 30  min .", "after thorough washing with pbs , the tissue was incubated with a goat anti-5-ht ( 1  :  500 , immunostar ) antibody in pbs - nds ( ph 7.4 ) for 24  h , followed by incubation with alexa 594 avidin d ( 1  :  1000 , invitrogen ) and alexa 488 donkey anti - goat ( 1  :  500 , invitrogen ) antibodies in pbs for 6  h at room temperature .", "the sections were then observed , and images were captured with a confocal microscope ( olympus ) .", "two - way anova with bonferroni multiple comparisons tests or one - way anova with tukey 's multiple comparisons post hoc tests were used for between - groups comparisons ( e.g. , the western blot data with surgery and drug administration as main effects ) .", "student 's paired t - test was used to analyze the differences between two groups ( e.g. , the difference in the numbers of p - mtor - positive cells between the sni - induced neuropathic pain group and the sham group ) .", "spared nerve injury ( sni ) produced increased nociceptive responses to innocuous mechanical stimulation ( mechanical allodynia ) of the ipsilateral hind paw in rats from as early as post - sni operation day 1 , and this effect was maintained for at least 2 weeks ( figure 1(a ) ) .", "however , sni had no impact on the latency of withdrawal to the radiant heat stimulus ( no thermal hyperalgesia ) ( figure 1(b ) ) .", "in fact , our present data are consistent with a previous report demonstrating that the low mechanical threshold induced by sni could persist even for 9 weeks after surgery , but there was no reported decrease in the hind paw withdrawal latency to the radiant heat stimulus . by using double - immunofluorescence staining", ", we found that the activated form of mtor , p - mtor , was expressed in the rvm , and it was exclusively expressed by neurons based on the observation that p - mtor was almost completely colocalized with neun , a marker for neurons , in rats in both the sham ( figures 2(a)2(c ) ) and post - sni day 7 groups ( figures 2(d)2(f ) ) .", "we observed that the number of p - mtor - positive neurons was significantly increased in the rvm on day 7 after sni compared to the sham group ( figures 2(a ) , 2(d ) , and 2(f ) ) , indicating that activation of mtor in the rvm may contribute to sni - induced neuropathic pain .", "it has been reported that s6k , a main downstream substrate for mtor , is involved in several intracellular processes , including neuronal plasticity and long - term memory .", "therefore , we used western blot analysis to further evaluate the mtor signaling pathway , including mtor and s6k , in the rvm after sni . compared with nave control rats , the expression of both phosphorylated mtor and phosphorylated s6k ( p - mtor and p - s6k ) was not significantly altered in the sham rats ( figures 2(h ) , 2(i ) , and 2(k ) ) . as indicated in figure 2(h ) , p - mtor and p - s6k were significantly elevated in the rvm 3 days after sni , and phosphorylation was maintained for at least 14 days compared to the control group ( figures 2(h ) and 2(i ) ; p - mtor : sni d3 : 1.42  0.25 ; sni d7 : 1.86  0.39 ; sni d14 : 1.49  0.28-fold of naive control , p < 0.05 ; p - s6k : sni d3 : 1.75  0.23 ; sni d7 : 1.96  0.57 ; sni d14 : 1.61  0.28-fold of nave control , p", "< 0.05 , n = 4 ) . the change in p - s6k was similar to that in p - mtor , indicating that the mtor signaling pathway in the rvm was activated by sni 3 days after surgery . compared with the nave control group , p - mtor and p - s6k expression", "was slightly increased , but no significant difference was detected at 1 day after sni ( figures 2(h ) , 2(i ) , and 2(k ) , p - mtor : sni d1 : 1.17  0.36-fold of nave control , p > 0.05 ; p - s6k : sni d1 : 1.14  0.38-fold of nave control , p > 0.05 , n = 4 ) .", "total mtor and s6k were not changed in any of the groups in the present study ( figures 2(h ) , 2(j ) , and 2(l ) ) . in summary , these data suggest that the mtor signaling pathway , including mtor and s6k , was activated in the rvm , which might indicate new protein synthesis and could result in changes in neuroplasticity", ". injections of fg into the lumbar sdh ( figure 3(a ) and 3(b ) ) resulted in many retrogradely labeled spinally projecting neurons in the rvm ( figure 3(d ) ) .", "although fg was injected unilaterally into the ( left ) lumbar sdh , 81.9% ( 203/248 ) of the retrogradely labeled spinally projecting neurons contained p - mtor - immunoreactive ( ir ) staining ( figures 3(c)3(e ) ) , which indicates that more than three - quarters of the spinally projecting neurons in the rvm express p - mtor .", "because 5-htergic neurons have previously been shown to be involved in the descending pain control pathway , particularly those localized within the rvm , we further investigated the relationship between 5-ht and p - mtor .", "our immunofluorescence staining results showed that a majority of the p - mtor - ir neurons contained 5-ht ( figures 4(a)4(f ) ) .", "the number of 5-htergic neurons was slightly increased within the rvm in the sni rats , but no statistical significance was detected compared to the sham control group ( 25.52  5.13 per section in the sni d7 group versus 20.8  4.02 in the sham control group , n = 3 rats / group , p > 0.05 ) .", "interestingly , 5-ht - positive p - mtor - ir neurons were remarkably increased in the rvm 7 days after sni compared to sham rats ( figure 4(g ) ) .", "in contrast , 5-ht - negative p - mtor - ir neurons were not significantly altered 7 days after sni ( figure 4(h ) ) .", "these results indicate that sni - induced neuropathic pain caused a substantial upregulation of p - mtor , which primarily occurred in 5-htergic neurons in the rvm . as reported previously , rapamycin is a specific and effective inhibitor of mtor . to investigate the inhibitory effect of rapamycin in vitro", ", we incubated brain slices containing the rvm with rapamycin ( 250  m ) for 30  min .", "we found that sni significantly increased the expression of both p - mtor and its downstream substrate p - s6k ( figure 5(a ) , p - mtor : sni d7 + vehicle : 1.80  0.48-fold of sham + vehicle , p < 0.05 ; p - s6k : sni d7 + vehicle : 1.77  0.45-fold of sham + vehicle , n", "= 4 , p < 0.05 ) , which is inconsistent with our tissue western blot data . moreover , rapamycin ( 250  m ) significantly reversed the upregulation of p - mtor as well as p - s6k after 30  min of drug infusion ( p - mtor : sni d7 + rapamycin : 1.11  0.27-fold of nave control versus sni d7 + vehicle : 1.80  0.48-fold of nave control , p < 0.05 ; p - s6k : sni 7d + rapamycin : 1.17  0.29-fold of nave control versus sni d7 + vehicle 1.77  0.45-fold of nave control , n = 4 , p < 0.05 ) , indicating that rapamycin could rapidly inhibit the activation of mtor after sni in vitro . to further investigate the neuronal excitability of 5-ht and the effect of rapamycin on 5-htergic neurons in the rvm", "a total of sixty 5-ht - positive neurons ( n = 60 ) from 12 rats were identified by biocytin introduction in combination with 5-ht immunofluorescence staining ( figure 5(b ) ) . moreover ,", "they showed a relatively higher membrane capacitance ( cm ) and a smaller membrane resistance ( rm ) compared to other small neurons in the rvm , indicating that they have a larger membrane surface and smaller electrical resistance .", "we first investigated the frequency and amplitude of spontaneous excitatory postsynaptic currents ( sepscs ) in the rvm neurons .", "most of the 5-htergic neurons recorded in the rvm that were responsive to rapamycin showed increased activity .", "inconsistent with previous reports , the frequency and amplitude of sepscs were significantly increased after sni ( figures 5(c)5(f ) ) , which indicates that the probability of presynaptic transmitter release and the postsynaptic neuronal excitability , respectively , was elevated .", "subsequently , we used rapamycin ( 250  m ) to evaluate whether the enhanced presynaptic and postsynaptic excitability could be reversed .", "we found that the amplitude ( baseline , 45.26  1.89 pa ; rapamycin , 35.83  2.58 pa .", "p < 0.05 , n = 15 , paired t - test ) , but not the frequency ( baseline , 3.41  0.12  hz ; rapamycin , 3.29  0.33 , p > 0.05 , n = 15 , paired t - test ) , of the sepscs was inhibited by rapamycin application in rats with sni but not in the sham control rats ( frequency : baseline , 1.22  0.11  hz ; rapamycin , 1.20  0.19  hz .", "p > 0.05 , n = 15 , paired t - test ; amplitude : baseline , 29.79  1.80 pa ; rapamycin , 28.14  1.28 pa .", "p > 0.05 , n = 15 , paired t - test ) ( figures 5(c)5(f ) ) .", "these results suggest that rapamycin can decrease the postsynaptic excitability of 5-htergic neurons in the rvm after sni .", "we next compared the effects of rapamycin on the action potentials of the 5-htergic neurons and determined that the spike number of the recorded neurons was obviously increased after sni ( figures 5(g ) and 5(h ) ) .", "rapamycin ( 250  m ) did not change the spike number in recorded neurons from the sham group ( f(1,62 ) = 2.25 , p > 0.05 , n = 15 , two - way repeated measures anova ) ( figure 5(g ) ) .", "however , in the presence of rapamycin , the spike number of 5-htergic neurons in the sni group was significantly reduced ( f(1,48 ) = 8.56 , p < 0.05 , n = 15 , two - way repeated measures anova ) ( figure 5(h ) ) .", "these results indicate that rapamycin inhibited the excitability of 5-htergic neurons in the rvm under neuropathic pain conditions .", "we preliminarily investigated nociceptive behaviors in the sni rats mentioned above . in agreement with previous reports", ", significant mechanical allodynia rather than thermal hyperalgesia was observed in the present study ( figure 1 ) .", "based on this observation , we next used mechanical pwt instead of heat pwl to assess the nociceptive behaviors . to determine whether rapamycin could prevent the development of the induction stage of mechanical allodynia , we microinjected rapamycin via a cannula implanted into the rvm ( figures 6(a ) and 6(b ) ) immediately before sni surgery and at day 1 after sni , which was followed by behavioral testing 30  min later ( figure 6(c ) ) .", "after treatment with rapamycin , the pwt in the sni + rapamycin group showed no significant change compared to that in the sni + vehicle control group at day 1 after sni ( figure 6(d ) ) , indicating that rapamycin could not alleviate the neuropathic pain induced by sni in the induction stage .", "subsequently , we investigated whether rapamycin could reverse established neuropathic pain . on day 6 after sni ,", "the rats demonstrated typical increased nociceptive responses to nonnoxious mechanical stimulation ( figure 6(e ) ) . compared with the vehicle control group ,", "the mechanical allodynia was significantly reduced after microinjection of rapamycin into the rvm on day 7 after sni ( figure 6(e ) ) . because our biochemical results showed that the amount of both p - mtor and p - s6k was increased after sni ( figures 2(h ) , 2(i ) , and 2(k ) ) and because rapamycin infusion into the brain slices could effectively reverse the upregulated level of p - mtor and p - s6k ( figure 5(a ) )", ", we concluded that the effect of rapamycin in partially reversing the mechanical allodynia was exerted via inactivation of the mtor signaling pathway , thus decreasing the excitability of 5-htergic spinally projecting neurons in the rvm .", "in the current study , we provide the first demonstration of the following : ( 1 ) mtor is expressed in the rvm region and can be activated in nerve injury - induced neuropathic pain ; ( 2 ) this mtor is largely expressed in 5-htergic neurons , which mainly comprise the descending pain control pathway ; ( 3 ) inhibition of the activated mtor restores the overexcitability of the 5-htergic neurons to normal ; and ( 4 ) inactivation of mtor by intra - rvm rapamycin microinjection alleviates established hyperalgesia ( abolished at the maintenance stage of neuropathic pain ) rather than influencing the beginning priming ( induction stage ) of neuropathic pain .", "these findings suggest that the mtor signaling pathway in the rvm is involved in the maintenance of nerve injury - induced neuropathic pain and that inhibition of mtor in the rvm could effectively alleviate neuropathic pain in sni rats . due to the importance of the descending pain control pathway in mammals ,", "it has been demonstrated that blockade of rvm activity with lidocaine produced conditioned place preference ( cpp ) , which is linked to pain relief , in nerve injury models , indicating that descending pain facilitation pathways modulate injury - induced spontaneous tonic pain .", "wei et al .   have reported that selectively depleting functional 5-ht phenotypes in rvm neurons with shrna interference ( rnai ) of tryptophan hydroxylase-2 ( tph-2 , the rate - limiting enzyme in the synthesis of neuronal 5-ht ) attenuated tissue or nerve injury - induced allodynia and hyperalgesia .", "this finding provides strong evidence that descending 5-ht from the rvm is an important contributor to pain facilitation during the development of persistent pain .", "recently , by taking advantage of optogenetic methods , optogenetic stimulation in tph2-channelrhodopsin 2 ( chr2 ) transgenic mice was shown to decrease both mechanical and thermal pain thresholds .", "however , in contrast , several studies showed the opposite results [ 3235 ] , that 5-ht from the rvm is important for the descending inhibitory pathway .", "these controversial arguments regarding whether rvm 5-ht plays a facilitatory or inhibitory role might be explained by the different subtypes of 5-ht receptors located in the sdh , according to the reports .", "for example , 5-ht3 receptors are reported to mediate descending facilitation and to contribute to pain hypersensitivity , whereas the activation of 5-ht2 receptors can potentiate glycine release in the sdh to inhibit pain transmission . in addition , a previous study also provided evidence that , under conditions of experimental pain , activation of 5-ht7 receptors leads to antinociceptive effects in the spinal cord . in the present study", ", we found that 5-htergic neurons were slightly , although not significantly , increased after nerve injury .", "however , the excitability of these 5-htergic neurons was elevated after sni ( figure 5 ) .", "rapamycin could effectively inhibit 5-ht overexcitability and thus attenuate hyperalgesia ( figure 6 ) , which indicates that 5-ht in the rvm is probably involved in the descending pain facilitation pathway under nerve injury - induced neuropathic pain conditions .", "mtor has been extensively studied in tumors , cardiovascular diseases , and neurodegenerative disorders [ 40 , 41 ] .", "recently , emerging evidence has indicated that mtor plays a role in pain processing , and it is becoming clear that mtor is important in the regulation of nociception , at both the peripheral and spinal cord levels [ 1017 ] . to date , however , no report has investigated mtor at the supraspinal level and its role in nociceptive modulation . here , we provide potent evidence that mtor contributes to neuropathic pain by increasing the neuronal excitability of 5-htergic neurons in the rvm , thus potentiating descending pain facilitation .", "4e - bp1/2 and s6k are involved in the regulation of cell physiology through the modulation of protein synthesis .", "4e - bp1/2 inhibits the interaction of the cap - binding translation initiation factor eif4e with other elongation factors , which is a key regulatory process in translation .", "mtor - mediated phosphorylation of 4e - bp1/2 releases this inhibition , allowing translation initiation to proceed .", "s6k - mediated phosphorylation of s6 promotes the unwinding and initiation of translation of a subgroup of mrnas called 5-terminal oligopyrimidine tract ( top ) mrnas .", "top mrnas encode ribosomal proteins and elongation factors 1a and 2 , which are important in translational control . in the present study", ", we detected that p - mtor and p - s6k levels were significantly elevated in the rvm after sni ( figure 2 ) , which suggests that mtor - mediated protein translation and synthesis are increased .", "in contrast , the number of 5-htergic neurons was slightly increased within the rvm in the sni rats , but no statistical significance was detected compared to the sham control group .", "however , by using whole - cell patch recording , we found that the 5-htergic neurons were overexcited , with significant increases in the amplitude and frequency of sepscs as well as the number of action potentials ( figure 5 ) .", "in addition , rapamycin inhibited only the amplitude and not the frequency of sepscs ( figure 5 ) ; we thus propose that the postsynaptic overexcitability of the 5-htergic neurons , which primarily depends on an increase in glutamate receptors , is mainly due to the activation of mtor .", "it has been reported that mtor signaling can potentiate the insertion of ampa ( -amino-3-hydroxy-5-methyl-4-isoxazole - propionic acid ) receptors into the postsynaptic membrane and lead to long - term potentiation ( ltp ) [ 44 , 45 ] .", "thus , the data collected from our immunofluorescence staining and electrophysiology are consistent with previous reports and suggest that the activation of mtor might lead to an increase in ampa receptors and their insertion into the postsynaptic membrane , resulting in the elevated neuronal excitability of the 5-htergic neurons in the rvm . as an effective immunosuppressant , rapamycin is widely used to prevent transplant rejection .", "chronic treatment of patients with mtor inhibitors is associated with an increased incidence of pain [ 46 , 47 ] , including the possible development of complex regional pain syndrome ( crps ) [ 48 , 49 ] .", "these conflicting results might be due to the following reasons : ( 1 ) the drug concentration of rapamycin or its analogues was not the same as in the other reports in which the inhibition of mtor produces antinociception ; ( 2 ) long - term treatment might lead to feedback activation of other pronociceptive signal proteins or molecules ; and ( 3 ) intrathecal administration of rapamycin ( at the spinal cord level ) might play a very complicated role in pain transmission with unknown mechanisms .", "the present study used intra - rvm , instead of intrathecal , administration of rapamycin ( 250  m , 30  min before the behavioral tests ) , and this treatment remarkably attenuated the nociceptive behaviors induced by sni ( figure 6 ) .", "moreover , intra - rvm rapamycin treatment was effective on day 7 after sni ( the maintenance stage of neuropathic pain ) but not on day 1 after sni ( the induction stage ) .", "the behavioral pharmacological data suggest that inhibition of mtor in the rvm at the late phase ( maintenance stage ) of neuropathic pain might be effective , even though pain has already been well established . by contrast , inhibition of mtor in the rvm had no effect on the development ( induction stage ) of neuropathic pain .", "all of these results are consistent with our biochemical data showing that rvm p - mtor was not greatly enhanced at day 1 but showed a significant increase at 7 days after sni ( figure 2 ) . combining our current results with previous findings , we conclude that the specific inhibition of mtor by rapamycin in the rvm is a promising avenue for the management of neuropathic pain .", "this effect probably occurs via deactivation of 5-htergic spinally projecting neurons in the rvm , which are required for descending pain facilitation .", "due to the lack of a specific mtor activator , reverse experiments involving the activation of mtor in the rvm , which should produce or enhance nociception , are difficult to achieve .", "moreover , optogenetic methods as well as transgenic animals should be further introduced to confirm the role of mtor in the rvm , not only in neuropathic pain but also in inflammatory pain .", "through the deactivation of 5-htergic spinally projecting neurons in the rvm and thus the weakening of descending pain facilitation , specific targeting of the activation of mtor in the rvm is a promising avenue for the management of neuropathic pain ."], "abstract_text": ["<S> the mammalian target of rapamycin ( mtor ) , a serine - threonine protein kinase , integrates extracellular signals , thereby modulating several physiological and pathological processes , including pain . </S>", "<S> previous studies have suggested that rapamycin ( an mtor inhibitor ) can attenuate nociceptive behaviors in many pain models , most likely at the spinal cord level . </S>", "<S> however , the mechanisms of mtor at the supraspinal level , particularly at the level of the rostral ventromedial medulla ( rvm ) , remain unclear . </S>", "<S> thus , the aim of this study was to elucidate the role of mtor in the rvm , a key relay region for the descending pain control pathway , under neuropathic pain conditions . </S>", "<S> phosphorylated mtor was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the rvm after spared nerve injury- ( sni- ) induced neuropathic pain . </S>", "<S> moreover , in sni rat brain slices , rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons . finally , intra - rvm microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain . </S>", "<S> in conclusion , our data provide strong evidence for the role of mtor in the rvm in nerve injury - induced neuropathic pain , indicating a novel mechanism of mtor inhibitor - induced analgesia . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["the rostral ventromedial medulla ( rvm ) is an important relay region that contributes to the descending pain control pathway from the periaqueductal gray ( pag ) to the superficial laminae ( laminae i and ii ) of the spinal cord [ 1 , 2 ] .", "it is well known that the rvm is closely linked to long - lasting activation of descending control circuits that involve descending facilitation , which significantly contributes to the development of persistent pain induced by tissue and nerve injury .", "although many studies have focused on this region , the cellular and molecular mechanisms of descending pain facilitation control remain poorly understood . due to the role of the descending pain facilitation pathway , several types of injuries , such as tissue and nerve injury , often become chronic and persistent , eventually leading to neuropathic pain .", ", significant progress has been made in basic and clinical studies ; however , the currently available therapies for neuropathic pain remain inadequate , and the search continues not only for improved treatments but also for novel targets .", "the mammalian target of rapamycin ( mtor ) , a conserved serine - threonine protein kinase that is inhibited by the effective clinical immunosuppressant rapamycin , regulates several intracellular processes in response to various extracellular signals and thereby modulates mrna translation .", "thus , mtor plays a critical role in the modulation of long - term plasticity and memory processes [ 57 ] .", "activation of the mtor complex with the protein raptor ( mtorc1 ) promotes the phosphorylation of mtor downstream targets , including eukaryotic initiation factor 4e - binding protein ( 4e - bp1/2 ) and s6 kinase ( s6k ) , which can further lead to local protein synthesis .", "it has been reported that deletion of either the 4e - bp1/2 or the s6k gene in mice results in deficits in synaptic plasticity and long - term memory [ 8 , 9 ] . moreover", ", phosphorylated mtor ( p - mtor ) , which is the activated form , is upregulated in the peripheral nervous system as well as at the spinal cord level in several pain models [ 1015 ] .", "inhibition of spinal cord mtor by intrathecal administration has proven to be effective in alleviating the nociceptive behaviors of animals under pain conditions [ 10 , 11 , 16 , 17 ] .", "synaptic plasticity changes in chronic pain conditions can occur not only at the spinal cord level but also at the supraspinal level , including the rvm .", "therefore , considering the important role of the rvm in descending pain facilitation , targeting mtor in the rvm might be a promising way to combat pain . because serotoninergic ( 5-htergic ) neurons are the primary constitutive element in the rvm and can send projections to the superficial spinal dorsal horn ( sdh ) [ 1820 ]", ", we thus hypothesize that 5-htergic spinally projecting neurons in the rvm contain mtor , the activation of which could in turn increase the excitability of the 5-htergic neurons and thus may potently potentiate the descending facilitation pain control pathway and exaggerate neuropathic pain conditions .", "accordingly , we used a spared nerve injury ( sni ) model to evaluate the role of mtor in the rvm in neuropathic pain in rats ."], ["adult male sprague - dawley ( sd ) rats ( weighing 250290  g ) were used in the present study .", "the ethics committee for animal experiments of the fourth military medical university ( xi'an , china ) approved the animal experiments ( permit number : 10071 ) .", "briefly , rats were anesthetized with pentobarbital ( 45  mg / kg , i.p . ) , and three terminal branches of the sciatic nerve were exposed by direct incision of the skin and a section of the biceps femoris muscle in the left thigh .", "the tibial and common peroneal branches were carefully tight - ligated with 5 - 0 silk sutures and sectioned distal to the ligation , removing 24  mm of the distal nerve stump .", "the surgical procedures for the sham - operated group were identical to those for the sni group , except that the nerves were not lesioned .", "mechanical allodynia , as a behavioral sign of sni - induced neuropathic pain , was assessed by measuring the 50% paw withdrawal threshold ( pwt ) as described previously .", "the 50% pwt in response to a series of ascending von frey filaments ( stoelting , kiel , wi , usa ) was determined by the up - and - down method .", "von frey force was delivered perpendicularly to the plantar surface of the hind paw for 2 to 3 seconds .", "an abrupt withdrawal of the hind paw during stimulation was recorded as a positive response .", "the 50% pwt was calculated using the following formula : 50%  pwt = 10 .", "mechanical allodynia was assessed by measuring the 50% pwt of the ipsilateral hind paw in sni- or sham - operated rats .", "measurements of the paw withdrawal latency ( pwl ) were obtained using a timer that was started by the activation of the heat source and stopped when withdrawal of the paw was detected with a photodetector .", "three measurements of the pwl were taken for each hind paw and were averaged as the result of each test session .", "the ipsilateral hind paw was tested with intervals of more than 5  min between consecutive tests .", "fluoro - gold ( fg ) was used as a retrograde tracer to label the rvm neurons that project to the sdh .", "the procedures for fg injection were essentially the same as in our previous studies [ 20 , 27 ] .", "briefly , after exposing the lumbar cord , 0.1  l of a 4% solution of fg ( fluorochrome , denver , co , usa ) dissolved in 0.9% saline was stereotaxically injected into the left side of the lumbar dorsal horn with a microsyringe attached to a glass micropipette by pressure injection . due to the transportation period of the tracer ,", "after perfusion , the brains and spinal cords of the rats were cut into sections .", "the sections were used to evaluate the fg injection sites in the sdh as well as the distribution patterns of the retrogradely fg - labeled neurons in the rvm under an epifluorescence microscope ( bx-60 ; olympus , tokyo , japan ) using an appropriate filter for fg ( excitation 350395  nm ; emission 430  nm ) .", "after the rats were anesthetized with pentobarbital ( 45  mg / kg , i.p . ) , a 26-gauge stainless steel guide cannula was stereotaxically implanted into a site above the rvm ( 10.52  mm posterior to bregma , 0  mm lateral from the midline , and 10.20  mm beneath the surface of the skull ) .", "intra - rvm microinjections were delivered via a 33-gauge injector needle cannula that was lowered 0.5  mm deeper into the brainstem than the guide cannula . the microinjection apparatus consisted of a hamilton syringe ( 10  l ) connected to an injector ( 33-gauge ) by a thin polyethylene tube and a motorized syringe pump .", "rapamycin ( 250  m/1  l , dissolved in a saline / dmso mix comprising 25% dmso , tocris bioscience , minneapolis , mn , usa ) , the specific inhibitor for mtor , was infused into the rvm at a rate of 0.1  l / min ; an equivalent volume of 25% dmso was used as a vehicle .", "after injection , the microinjection needle was left in place for at least 2  min .", "the injection sites were verified at the end of all of the experiments by nissl staining , and injection sites outside the rvm region were excluded from the study .", "these rats were randomly divided into two groups designed for different purposes , as shown in figure 6(c ) : group 1 : to investigate whether rapamycin could influence the induction stage of sni - induced neuropathic pain , behavioral tests were performed before the first drug or vehicle injection , followed by sni ( pre - sni ) , and 30  min after the second injection on day 1 after sni ( sni - d1 ) ( figure 6(c ) , top ) and group 2 : behavioral tests were performed before sni ( pre - sni ) , 6 days after sni ( sni - d6 ) , and 30  min after drug or vehicle injection on day 7 after sni ( sni - d7 ) for the purpose of demonstrating the effect of rapamycin on the maintenance stage of sni - induced neuropathic pain ( figure 6(c ) , bottom ) .", "the rats were transcardially perfused with 150  ml of 0.01  m phosphate - buffered saline ( pbs , ph 7.4 ) , followed by 500  ml of 4% paraformaldehyde in 0.1  m phosphate buffer ( pb , ph 7.4 ) .", "the brainstems and/or spinal cords were transversely sliced into 25 mm thick coronal sections using a freezing microtome ( cm1950 , leica , heidelberg , germany ) .", "double - immunofluorescence staining for p - mtor / neun , p - mtor / fg , or p - mtor/5-ht was performed .", "the sections were sequentially incubated at room temperature with primary antisera in 0.01  m pbs containing 5% normal donkey serum ( nds ) , 0.3% triton x-100 , 0.05% nan3 , and 0.25% carrageenan ( pbs - nds , ph 7.4 ) for 24  h. then , the sections were incubated with fluorescein - labeled igg ( secondary antisera ) for 6  h. a negative control experiment , in which the primary antisera were omitted , and a peptide competition assay were both carried out .", "after the immunofluorescence histochemical staining , the sections were observed and images were captured using a confocal laser - scanning microscope ( clsm , fv1000 , olympus ) .", "micrographs of 1012 sections per rat , which were 150  m apart within bregma 9.30 to 11.60  mm , were analyzed for p - mtor expression . using imagej software , the rvm area , including the nucleus raphe magnus ( rmg ) and the nucleus reticularis gigantocellularis pars  ,", "the p - mtor - positive cells within the area were counted manually by an observer blinded to the treatment conditions .", "the same counting method was used to evaluate the coexpression of 5-ht / p - mtor as well as that of p - mtor / fg within the rvm .", "cells with visible green cytoplasmic staining represent 5-htergic or fg - labeled cells , while red staining represents p - mtor - positive cells ; thus , cells with 5-ht or fg double - labeling with p - mtor appear yellow .", "the rats were decapitated , and brain slices ( 400  mm ) containing the rvm were cut at 0c with a vibratome ( vt1200s , leica ) in a sucrose cutting solution containing the following ( in mm ) : kcl 2.5 , nah2po4 1.2 , nahco3 26 , sucrose 252 , mgso47h2o 6 , cacl2 0.5 , and glucose 10 , bubbled with 95% o2/5% co2 ( ph 7.4 ) . for the electrophysiology studies ,", "the brain slices were transferred to a submerged recovery chamber with oxygenated artificial cerebrospinal fluid ( acsf ) containing the following ( in mm ) : nacl 124 , kcl 2.5 , mgso47h2o 2 , nah2po4 1 , nahco3 25 , cacl2 2 , and glucose 37 for 2 hours at room temperature before recording .", "for the biochemical experiments , the slices were slowly brought to a final temperature of 30c in acsf gassed with 95% o2/5% co2 and incubated for at least 1 hour before the experiments .", "the brain slices were treated with rapamycin ( 250  m ) and vehicle for 30  min .", "subsequently , the rvm regions were microdissected and snap - frozen over dry ice . following the standard western blot protocol ,", "rats were anesthetized with an overdose of pentobarbital ( 60  mg / kg , i.p . ) , and the rvm regions were carefully dissected and harvested for western blotting . to obtain total protein extracts ,", "the tissues were lysed in 300  l lysis buffer containing 10  mm tris , 150  mm nacl , 1% triton x-100 , 0.5% np-40 , and 1  mm edta at ph 7.4 .", "the samples were adequately mixed at a 100  :  1 ( v / v ) ratio of protease inhibitor cocktail and phosphatase inhibitor cocktail ( roche , tucson , az , usa ) .", "the procedures for the in vitro - infused brain slices were similar to those of the tissue protocols .", "then , 30  g of cell lysis material ( quantitatively measured using the bca protein assay ; thermo scientific , rockford , il , usa ) was resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to pvdf membranes ( immobilon - p , millipore ) . after blocking in nonfat milk for 1  h", ", the membranes were incubated overnight at 4c with the following primary antibodies : rabbit anti - mtor ( 1  :  1000 , cell signaling technology ) ; rabbit anti - p - mtor ( 1  :  1000 , cell signaling technology ) ; rabbit anti - s6k ( 1  :  1000 , cell signaling technology ) ; rabbit anti - p - s6k ( 1  :  1000 , cell signaling technology ) ; and mouse anti--actin ( 1  :  5000 , sigma , st .", "the immunoblots were then reacted with the corresponding horseradish peroxidase- ( hrp- ) conjugated secondary antibodies ( anti - rabbit 1  :  5000 , anti - mouse 1  :  5000 ; amersham pharmacia biotech , piscataway , nj , usa ) .", "all of the reactions were detected by the enhanced chemiluminescence ( ecl ) detection method ( amersham ) and exposure to film .", "target protein levels were normalized against -actin levels and expressed as fold changes relative to the nave control group .", "neurons in the rvm region were targeted for recording using an upright microscope equipped with zeiss ( oberkochen , germany ) infrared - differential interference contrast ( ir - dic ) optics , a 40 water - immersion objective , and a video - imaging camera .", "the patch pipette was filled with intracellular solution containing the following ( in mm ) : k - gluconate 130 , nacl 5 , kcl 15 , egta 0.4 , hepes 10 , mg - atp 4 , and tris - gtp 0.2 , ph 7.257.35 , with an osmotic pressure of 290300  mosm / l . the pipette resistance , as measured in the bath , was typically 4  0.5 m. the voltage was held at 60  mv , and neurons were given at least 3  min to stabilize before data were collected .", "spontaneous discharge and the number of action potentials were used to investigate sni - induced changes in neuronal excitability in the rvm .", "the excitatory postsynaptic currents ( epscs ) of the rvm 5-htergic neurons , which are mediated by ampa receptors , were voltage - clamped and recorded at 60  mv with an axon 700b amplifier ( molecular devices , sunnyvale , ca , usa ) after blocking gabaergic transmission by picrotoxin ( 100  mm , sigma ) , a gabaa receptor antagonist .", "the membrane excitability of the recorded neurons was measured in current - clamp mode by determining the number of action potentials elicited by intracellular injection of 0 , 10 , 20 , 30 , 40 , 50 , and 60  pa depolarizing currents for 400  ms .", "the spike number was determined to estimate the influence of rapamycin on the recorded neurons . in all cases ,", "biocytin ( 0.5% ) was introduced into the intracellular solution to identify the morphological properties of the recorded neurons .", "after recording , the brain slices were immediately fixed in 4% paraformaldehyde in 0.1  m pb for 4  h at room temperature .", "then , sections were rinsed with 3% hydrogen peroxide in 0.01  m pbs for 30  min .", "after thorough washing with pbs , the tissue was incubated with a goat anti-5-ht ( 1  :  500 , immunostar ) antibody in pbs - nds ( ph 7.4 ) for 24  h , followed by incubation with alexa 594 avidin d ( 1  :  1000 , invitrogen ) and alexa 488 donkey anti - goat ( 1  :  500 , invitrogen ) antibodies in pbs for 6  h at room temperature .", "the sections were then observed , and images were captured with a confocal microscope ( olympus ) .", "two - way anova with bonferroni multiple comparisons tests or one - way anova with tukey 's multiple comparisons post hoc tests were used for between - groups comparisons ( e.g. , the western blot data with surgery and drug administration as main effects ) .", "student 's paired t - test was used to analyze the differences between two groups ( e.g. , the difference in the numbers of p - mtor - positive cells between the sni - induced neuropathic pain group and the sham group ) ."], ["spared nerve injury ( sni ) produced increased nociceptive responses to innocuous mechanical stimulation ( mechanical allodynia ) of the ipsilateral hind paw in rats from as early as post - sni operation day 1 , and this effect was maintained for at least 2 weeks ( figure 1(a ) ) .", "however , sni had no impact on the latency of withdrawal to the radiant heat stimulus ( no thermal hyperalgesia ) ( figure 1(b ) ) .", "in fact , our present data are consistent with a previous report demonstrating that the low mechanical threshold induced by sni could persist even for 9 weeks after surgery , but there was no reported decrease in the hind paw withdrawal latency to the radiant heat stimulus . by using double - immunofluorescence staining", ", we found that the activated form of mtor , p - mtor , was expressed in the rvm , and it was exclusively expressed by neurons based on the observation that p - mtor was almost completely colocalized with neun , a marker for neurons , in rats in both the sham ( figures 2(a)2(c ) ) and post - sni day 7 groups ( figures 2(d)2(f ) ) .", "we observed that the number of p - mtor - positive neurons was significantly increased in the rvm on day 7 after sni compared to the sham group ( figures 2(a ) , 2(d ) , and 2(f ) ) , indicating that activation of mtor in the rvm may contribute to sni - induced neuropathic pain .", "it has been reported that s6k , a main downstream substrate for mtor , is involved in several intracellular processes , including neuronal plasticity and long - term memory .", "therefore , we used western blot analysis to further evaluate the mtor signaling pathway , including mtor and s6k , in the rvm after sni . compared with nave control rats , the expression of both phosphorylated mtor and phosphorylated s6k ( p - mtor and p - s6k ) was not significantly altered in the sham rats ( figures 2(h ) , 2(i ) , and 2(k ) ) . as indicated in figure 2(h ) , p - mtor and p - s6k were significantly elevated in the rvm 3 days after sni , and phosphorylation was maintained for at least 14 days compared to the control group ( figures 2(h ) and 2(i ) ; p - mtor : sni d3 : 1.42  0.25 ; sni d7 : 1.86  0.39 ; sni d14 : 1.49  0.28-fold of naive control , p < 0.05 ; p - s6k : sni d3 : 1.75  0.23 ; sni d7 : 1.96  0.57 ; sni d14 : 1.61  0.28-fold of nave control , p", "< 0.05 , n = 4 ) . the change in p - s6k was similar to that in p - mtor , indicating that the mtor signaling pathway in the rvm was activated by sni 3 days after surgery . compared with the nave control group , p - mtor and p - s6k expression", "was slightly increased , but no significant difference was detected at 1 day after sni ( figures 2(h ) , 2(i ) , and 2(k ) , p - mtor : sni d1 : 1.17  0.36-fold of nave control , p > 0.05 ; p - s6k : sni d1 : 1.14  0.38-fold of nave control , p > 0.05 , n = 4 ) .", "total mtor and s6k were not changed in any of the groups in the present study ( figures 2(h ) , 2(j ) , and 2(l ) ) . in summary , these data suggest that the mtor signaling pathway , including mtor and s6k , was activated in the rvm , which might indicate new protein synthesis and could result in changes in neuroplasticity", ". injections of fg into the lumbar sdh ( figure 3(a ) and 3(b ) ) resulted in many retrogradely labeled spinally projecting neurons in the rvm ( figure 3(d ) ) .", "although fg was injected unilaterally into the ( left ) lumbar sdh , 81.9% ( 203/248 ) of the retrogradely labeled spinally projecting neurons contained p - mtor - immunoreactive ( ir ) staining ( figures 3(c)3(e ) ) , which indicates that more than three - quarters of the spinally projecting neurons in the rvm express p - mtor .", "because 5-htergic neurons have previously been shown to be involved in the descending pain control pathway , particularly those localized within the rvm , we further investigated the relationship between 5-ht and p - mtor .", "our immunofluorescence staining results showed that a majority of the p - mtor - ir neurons contained 5-ht ( figures 4(a)4(f ) ) .", "the number of 5-htergic neurons was slightly increased within the rvm in the sni rats , but no statistical significance was detected compared to the sham control group ( 25.52  5.13 per section in the sni d7 group versus 20.8  4.02 in the sham control group , n = 3 rats / group , p > 0.05 ) .", "interestingly , 5-ht - positive p - mtor - ir neurons were remarkably increased in the rvm 7 days after sni compared to sham rats ( figure 4(g ) ) .", "in contrast , 5-ht - negative p - mtor - ir neurons were not significantly altered 7 days after sni ( figure 4(h ) ) .", "these results indicate that sni - induced neuropathic pain caused a substantial upregulation of p - mtor , which primarily occurred in 5-htergic neurons in the rvm . as reported previously , rapamycin is a specific and effective inhibitor of mtor . to investigate the inhibitory effect of rapamycin in vitro", ", we incubated brain slices containing the rvm with rapamycin ( 250  m ) for 30  min .", "we found that sni significantly increased the expression of both p - mtor and its downstream substrate p - s6k ( figure 5(a ) , p - mtor : sni d7 + vehicle : 1.80  0.48-fold of sham + vehicle , p < 0.05 ; p - s6k : sni d7 + vehicle : 1.77  0.45-fold of sham + vehicle , n", "= 4 , p < 0.05 ) , which is inconsistent with our tissue western blot data . moreover , rapamycin ( 250  m ) significantly reversed the upregulation of p - mtor as well as p - s6k after 30  min of drug infusion ( p - mtor : sni d7 + rapamycin : 1.11  0.27-fold of nave control versus sni d7 + vehicle : 1.80  0.48-fold of nave control , p < 0.05 ; p - s6k : sni 7d + rapamycin : 1.17  0.29-fold of nave control versus sni d7 + vehicle 1.77  0.45-fold of nave control , n = 4 , p < 0.05 ) , indicating that rapamycin could rapidly inhibit the activation of mtor after sni in vitro . to further investigate the neuronal excitability of 5-ht and the effect of rapamycin on 5-htergic neurons in the rvm", "a total of sixty 5-ht - positive neurons ( n = 60 ) from 12 rats were identified by biocytin introduction in combination with 5-ht immunofluorescence staining ( figure 5(b ) ) . moreover ,", "they showed a relatively higher membrane capacitance ( cm ) and a smaller membrane resistance ( rm ) compared to other small neurons in the rvm , indicating that they have a larger membrane surface and smaller electrical resistance .", "we first investigated the frequency and amplitude of spontaneous excitatory postsynaptic currents ( sepscs ) in the rvm neurons .", "most of the 5-htergic neurons recorded in the rvm that were responsive to rapamycin showed increased activity .", "inconsistent with previous reports , the frequency and amplitude of sepscs were significantly increased after sni ( figures 5(c)5(f ) ) , which indicates that the probability of presynaptic transmitter release and the postsynaptic neuronal excitability , respectively , was elevated .", "subsequently , we used rapamycin ( 250  m ) to evaluate whether the enhanced presynaptic and postsynaptic excitability could be reversed .", "we found that the amplitude ( baseline , 45.26  1.89 pa ; rapamycin , 35.83  2.58 pa .", "p < 0.05 , n = 15 , paired t - test ) , but not the frequency ( baseline , 3.41  0.12  hz ; rapamycin , 3.29  0.33 , p > 0.05 , n = 15 , paired t - test ) , of the sepscs was inhibited by rapamycin application in rats with sni but not in the sham control rats ( frequency : baseline , 1.22  0.11  hz ; rapamycin , 1.20  0.19  hz .", "p > 0.05 , n = 15 , paired t - test ; amplitude : baseline , 29.79  1.80 pa ; rapamycin , 28.14  1.28 pa .", "p > 0.05 , n = 15 , paired t - test ) ( figures 5(c)5(f ) ) .", "these results suggest that rapamycin can decrease the postsynaptic excitability of 5-htergic neurons in the rvm after sni .", "we next compared the effects of rapamycin on the action potentials of the 5-htergic neurons and determined that the spike number of the recorded neurons was obviously increased after sni ( figures 5(g ) and 5(h ) ) .", "rapamycin ( 250  m ) did not change the spike number in recorded neurons from the sham group ( f(1,62 ) = 2.25 , p > 0.05 , n = 15 , two - way repeated measures anova ) ( figure 5(g ) ) .", "however , in the presence of rapamycin , the spike number of 5-htergic neurons in the sni group was significantly reduced ( f(1,48 ) = 8.56 , p < 0.05 , n = 15 , two - way repeated measures anova ) ( figure 5(h ) ) .", "these results indicate that rapamycin inhibited the excitability of 5-htergic neurons in the rvm under neuropathic pain conditions .", "we preliminarily investigated nociceptive behaviors in the sni rats mentioned above . in agreement with previous reports", ", significant mechanical allodynia rather than thermal hyperalgesia was observed in the present study ( figure 1 ) .", "based on this observation , we next used mechanical pwt instead of heat pwl to assess the nociceptive behaviors . to determine whether rapamycin could prevent the development of the induction stage of mechanical allodynia , we microinjected rapamycin via a cannula implanted into the rvm ( figures 6(a ) and 6(b ) ) immediately before sni surgery and at day 1 after sni , which was followed by behavioral testing 30  min later ( figure 6(c ) ) .", "after treatment with rapamycin , the pwt in the sni + rapamycin group showed no significant change compared to that in the sni + vehicle control group at day 1 after sni ( figure 6(d ) ) , indicating that rapamycin could not alleviate the neuropathic pain induced by sni in the induction stage .", "subsequently , we investigated whether rapamycin could reverse established neuropathic pain . on day 6 after sni ,", "the rats demonstrated typical increased nociceptive responses to nonnoxious mechanical stimulation ( figure 6(e ) ) . compared with the vehicle control group ,", "the mechanical allodynia was significantly reduced after microinjection of rapamycin into the rvm on day 7 after sni ( figure 6(e ) ) . because our biochemical results showed that the amount of both p - mtor and p - s6k was increased after sni ( figures 2(h ) , 2(i ) , and 2(k ) ) and because rapamycin infusion into the brain slices could effectively reverse the upregulated level of p - mtor and p - s6k ( figure 5(a ) )", ", we concluded that the effect of rapamycin in partially reversing the mechanical allodynia was exerted via inactivation of the mtor signaling pathway , thus decreasing the excitability of 5-htergic spinally projecting neurons in the rvm ."], ["in the current study , we provide the first demonstration of the following : ( 1 ) mtor is expressed in the rvm region and can be activated in nerve injury - induced neuropathic pain ; ( 2 ) this mtor is largely expressed in 5-htergic neurons , which mainly comprise the descending pain control pathway ; ( 3 ) inhibition of the activated mtor restores the overexcitability of the 5-htergic neurons to normal ; and ( 4 ) inactivation of mtor by intra - rvm rapamycin microinjection alleviates established hyperalgesia ( abolished at the maintenance stage of neuropathic pain ) rather than influencing the beginning priming ( induction stage ) of neuropathic pain .", "these findings suggest that the mtor signaling pathway in the rvm is involved in the maintenance of nerve injury - induced neuropathic pain and that inhibition of mtor in the rvm could effectively alleviate neuropathic pain in sni rats . due to the importance of the descending pain control pathway in mammals ,", "it has been demonstrated that blockade of rvm activity with lidocaine produced conditioned place preference ( cpp ) , which is linked to pain relief , in nerve injury models , indicating that descending pain facilitation pathways modulate injury - induced spontaneous tonic pain .", "wei et al .   have reported that selectively depleting functional 5-ht phenotypes in rvm neurons with shrna interference ( rnai ) of tryptophan hydroxylase-2 ( tph-2 , the rate - limiting enzyme in the synthesis of neuronal 5-ht ) attenuated tissue or nerve injury - induced allodynia and hyperalgesia .", "this finding provides strong evidence that descending 5-ht from the rvm is an important contributor to pain facilitation during the development of persistent pain .", "recently , by taking advantage of optogenetic methods , optogenetic stimulation in tph2-channelrhodopsin 2 ( chr2 ) transgenic mice was shown to decrease both mechanical and thermal pain thresholds .", "however , in contrast , several studies showed the opposite results [ 3235 ] , that 5-ht from the rvm is important for the descending inhibitory pathway .", "these controversial arguments regarding whether rvm 5-ht plays a facilitatory or inhibitory role might be explained by the different subtypes of 5-ht receptors located in the sdh , according to the reports .", "for example , 5-ht3 receptors are reported to mediate descending facilitation and to contribute to pain hypersensitivity , whereas the activation of 5-ht2 receptors can potentiate glycine release in the sdh to inhibit pain transmission . in addition , a previous study also provided evidence that , under conditions of experimental pain , activation of 5-ht7 receptors leads to antinociceptive effects in the spinal cord . in the present study", ", we found that 5-htergic neurons were slightly , although not significantly , increased after nerve injury .", "however , the excitability of these 5-htergic neurons was elevated after sni ( figure 5 ) .", "rapamycin could effectively inhibit 5-ht overexcitability and thus attenuate hyperalgesia ( figure 6 ) , which indicates that 5-ht in the rvm is probably involved in the descending pain facilitation pathway under nerve injury - induced neuropathic pain conditions .", "mtor has been extensively studied in tumors , cardiovascular diseases , and neurodegenerative disorders [ 40 , 41 ] .", "recently , emerging evidence has indicated that mtor plays a role in pain processing , and it is becoming clear that mtor is important in the regulation of nociception , at both the peripheral and spinal cord levels [ 1017 ] . to date , however , no report has investigated mtor at the supraspinal level and its role in nociceptive modulation . here , we provide potent evidence that mtor contributes to neuropathic pain by increasing the neuronal excitability of 5-htergic neurons in the rvm , thus potentiating descending pain facilitation .", "4e - bp1/2 and s6k are involved in the regulation of cell physiology through the modulation of protein synthesis .", "4e - bp1/2 inhibits the interaction of the cap - binding translation initiation factor eif4e with other elongation factors , which is a key regulatory process in translation .", "mtor - mediated phosphorylation of 4e - bp1/2 releases this inhibition , allowing translation initiation to proceed .", "s6k - mediated phosphorylation of s6 promotes the unwinding and initiation of translation of a subgroup of mrnas called 5-terminal oligopyrimidine tract ( top ) mrnas .", "top mrnas encode ribosomal proteins and elongation factors 1a and 2 , which are important in translational control . in the present study", ", we detected that p - mtor and p - s6k levels were significantly elevated in the rvm after sni ( figure 2 ) , which suggests that mtor - mediated protein translation and synthesis are increased .", "in contrast , the number of 5-htergic neurons was slightly increased within the rvm in the sni rats , but no statistical significance was detected compared to the sham control group .", "however , by using whole - cell patch recording , we found that the 5-htergic neurons were overexcited , with significant increases in the amplitude and frequency of sepscs as well as the number of action potentials ( figure 5 ) .", "in addition , rapamycin inhibited only the amplitude and not the frequency of sepscs ( figure 5 ) ; we thus propose that the postsynaptic overexcitability of the 5-htergic neurons , which primarily depends on an increase in glutamate receptors , is mainly due to the activation of mtor .", "it has been reported that mtor signaling can potentiate the insertion of ampa ( -amino-3-hydroxy-5-methyl-4-isoxazole - propionic acid ) receptors into the postsynaptic membrane and lead to long - term potentiation ( ltp ) [ 44 , 45 ] .", "thus , the data collected from our immunofluorescence staining and electrophysiology are consistent with previous reports and suggest that the activation of mtor might lead to an increase in ampa receptors and their insertion into the postsynaptic membrane , resulting in the elevated neuronal excitability of the 5-htergic neurons in the rvm . as an effective immunosuppressant , rapamycin is widely used to prevent transplant rejection .", "chronic treatment of patients with mtor inhibitors is associated with an increased incidence of pain [ 46 , 47 ] , including the possible development of complex regional pain syndrome ( crps ) [ 48 , 49 ] .", "these conflicting results might be due to the following reasons : ( 1 ) the drug concentration of rapamycin or its analogues was not the same as in the other reports in which the inhibition of mtor produces antinociception ; ( 2 ) long - term treatment might lead to feedback activation of other pronociceptive signal proteins or molecules ; and ( 3 ) intrathecal administration of rapamycin ( at the spinal cord level ) might play a very complicated role in pain transmission with unknown mechanisms .", "the present study used intra - rvm , instead of intrathecal , administration of rapamycin ( 250  m , 30  min before the behavioral tests ) , and this treatment remarkably attenuated the nociceptive behaviors induced by sni ( figure 6 ) .", "moreover , intra - rvm rapamycin treatment was effective on day 7 after sni ( the maintenance stage of neuropathic pain ) but not on day 1 after sni ( the induction stage ) .", "the behavioral pharmacological data suggest that inhibition of mtor in the rvm at the late phase ( maintenance stage ) of neuropathic pain might be effective , even though pain has already been well established . by contrast , inhibition of mtor in the rvm had no effect on the development ( induction stage ) of neuropathic pain .", "all of these results are consistent with our biochemical data showing that rvm p - mtor was not greatly enhanced at day 1 but showed a significant increase at 7 days after sni ( figure 2 ) . combining our current results with previous findings , we conclude that the specific inhibition of mtor by rapamycin in the rvm is a promising avenue for the management of neuropathic pain .", "this effect probably occurs via deactivation of 5-htergic spinally projecting neurons in the rvm , which are required for descending pain facilitation .", "due to the lack of a specific mtor activator , reverse experiments involving the activation of mtor in the rvm , which should produce or enhance nociception , are difficult to achieve .", "moreover , optogenetic methods as well as transgenic animals should be further introduced to confirm the role of mtor in the rvm , not only in neuropathic pain but also in inflammatory pain ."], ["through the deactivation of 5-htergic spinally projecting neurons in the rvm and thus the weakening of descending pain facilitation , specific targeting of the activation of mtor in the rvm is a promising avenue for the management of neuropathic pain ."]]}
{"article_id": "PMC3015287", "article_text": ["traditional diagnostic methods and liberal indications for surgery have resulted in negative laparotomy rates ranging from 12 - 40% .", "while negative laparotomy was previously accepted as the inevitable and innocuous result of this policy , recent reports by renz and others have highlighted the 6 - 40% incidence of complications that accompany nontheraputic abdominal exploration . in view of these data , and", "dissatisfied with our own 33% negative laparotomy rate , prospective evaluation of the role of diagnostic laparoscopy in the evaluation of suspected penetrating abdominal injury was undertaken in september 1995 to identify those patients who could safely avoid surgical intervention , with the expectation that the rate of negative trauma celiotomy would markedly decrease . in this report", "we review our experience with 70 consecutive patients who underwent diagnostic laparoscopy , and discuss the benefits and pitfalls in diagnosis we encountered utilizing this technique .", "a prospective study of patients with penetrating injuries to the lower thorax and anterior abdomen admitted to the bellevue hospital trauma and shock unit , a level i trauma center in new york city , was performed between september 1995 through november 1997 .", "patients who were hemodynamically stable without obvious evidence of peritoneal penetration or free intra - abdominal air on upright chest radiograph were entered into the protocol . in light of evidence that co2 pneumoperitoneum may result in an increase in intracranial pressure ,", "patients with known or suspected central nervous system injuries were initially excluded from the protocol . however , the use of diagnostic laparoscopy may be appropriate in head - injured patients when intracranial pressure monitors are in place .", "after initial evaluation in the emergency room , informed consent was obtained and the patient was transferred to the operating room .", "general endotracheal anesthesia was induced , orogastric tube and foley catheter inserted , and open laparoscopy performed with the hasson trocar positioned infraumbilically .", "after insufflation with carbon dioxide gas to 15 mm hg , a 10 mm laparoscope with attached video camera was inserted into the peritoneal cavity , and exploration performed . in patients with thoracoabdominal injuries and suspected diaphragmatic tears , a low pressure pneumoperitoneum of 8 - 10 mm hg was created , the diaphragms visualized , and if no diaphragmatic injuries were present , the pneumoperitoneum was raised to 15 mm hg .", "routine thoracostomy tubes were not placed prophylactically in light of zargut 's reported 1% pneumothorax rate following carbon dioxide insufflation ( 5 of 510 patients ) ; rather , the anesthesia and surgical teams were alerted to the possible development of a tension pneumothorax in patients at risk for diaphragmatic injury . if no peritoneal violation was noted , or an isolated non - bleeding injury to the liver was present beneath the site of peritoneal penetration , the procedure was terminated , and the patient subsequently discharged from the hospital . if peritoneal violation , major organ injury or hematoma were noted , the procedure was converted to open celiotomy . during this period", "we did not utilize the technique of  gasless laparoscopy ,  or perform  minilaparoscopy  in the emergency department with local anesthesia utilizing 3 - 5 mm videoscopes .", "trauma service records were reviewed for the two years which preceded this study ( 1993  1994 ) ; the overall negative exploration rate for all patients with penetrating abdominal trauma during that time period was 33% .", "sixty - four ( 91% ) patients were male and six ( 9% ) female .", "forty - four patients ( 63% ) suffered stab wounds to the anterior abdomen , eleven ( 16% ) stab wounds to the left chest with suspicion of intra - abdominal injury ,   ( 19% ) with abdominal gunshot wounds .", "two additional patients ( 3% ) involved in motor - vehicle accidents were evaluated laparoscopically after indeterminate computed tomo - graphic ( ct ) scans .", "the mean operative time for laparoscopy was 16 minutes ( range : 3  60 ) .", "the mean time for negative laparoscopy was 23 minutes , and 10.8 minutes for laparoscopy with positive findings .", "forty - one ( 59% ) procedures met criteria for positive laparoscopy : thirty - nine were converted to celiotomy and two additional nonbleeding liver injuries were treated nonoperatively .", "twenty - five ( 65% ) of the 39 laparotomies were considered therapeutic by the operating surgeon . among the 14 nontheraputic laparotomies ,", "ten were performed in patients with peritoneal penetration visualized on laparoscopy but found to have no intraperitoneal injury at laparotomy , three revealed only minor nonbleeding liver injuries early in our study that the individual attending surgeon was unwilling to treat nonoperatively , and one was performed for free blood secondary to a minor omental laceration . in patients without associated injuries ,", "the mean length of stay following negative laparoscopy was 1.5 days in 21 patients ( range : 1  4 ; the single 4-day stay was due to social service issues ) , compared to 5.2 days ( range : 3  8) for 12 negative explorations following positive laparoscopy .", "there were no anesthetic complications in the study group , and no tension pneumothoraces or air emboli occurred .", "one small bowel injury resulted from insertion of the hasson trocar , and was repaired primarily in a patient during laparotomy for a thoracoabdominal gunshot wound with liver , adrenal and diaphragmatic injuries .", "no intra - abdominal injuries were missed in the negative laparoscopy group ( table 1 ) .", "however , two of ten patients ( 20% ) who underwent negative laparoscopy for lower left chest stab wounds required urgent return to surgery from the recovery room for missed ongoing hemothoraces .", "one was a 23-year - old female stabbed in the left anterior axillary line at the 7th intercostal space .", "a closed tube thoracostomy was per - formed for a nonbleeding pneumothorax upon initial presentation to the emergency room , and she was transferred to the operating room , where laparoscopy revealed no evidence of peritoneal penetration .", "soon after arrival in the recovery room she drained 750 milliliters of blood from her chest tube and became hypotensive .", "she was returned to the operating room , where thoracotomy revealed an actively bleeding injury to the lingula which was successfully controlled by stapled wedge resection .", "the second patient also required a left thoracotomy to oversew bleeding from the lung parenchyma .", "the mean operative time for laparoscopy was 16 minutes ( range : 3  60 ) .", "the mean time for negative laparoscopy was 23 minutes , and 10.8 minutes for laparoscopy with positive findings .", "forty - one ( 59% ) procedures met criteria for positive laparoscopy : thirty - nine were converted to celiotomy and two additional nonbleeding liver injuries were treated nonoperatively .", "twenty - five ( 65% ) of the 39 laparotomies were considered therapeutic by the operating surgeon . among the 14 nontheraputic laparotomies ,", "ten were performed in patients with peritoneal penetration visualized on laparoscopy but found to have no intraperitoneal injury at laparotomy , three revealed only minor nonbleeding liver injuries early in our study that the individual attending surgeon was unwilling to treat nonoperatively , and one was performed for free blood secondary to a minor omental laceration . in patients without associated injuries , the mean length of stay following negative laparoscopy was 1.5 days in 21 patients ( range : 1  4 ; the single 4-day stay was due to social service issues ) , compared to 5.2 days ( range : 3  8) for 12 negative explorations following positive laparoscopy .", "there were no anesthetic complications in the study group , and no tension pneumothoraces or air emboli occurred .", "one small bowel injury resulted from insertion of the hasson trocar , and was repaired primarily in a patient during laparotomy for a thoracoabdominal gunshot wound with liver , adrenal and diaphragmatic injuries .", "no intra - abdominal injuries were missed in the negative laparoscopy group ( table 1 ) .", "however , two of ten patients ( 20% ) who underwent negative laparoscopy for lower left chest stab wounds required urgent return to surgery from the recovery room for missed ongoing hemothoraces .", "one was a 23-year - old female stabbed in the left anterior axillary line at the 7th intercostal space .", "a closed tube thoracostomy was per - formed for a nonbleeding pneumothorax upon initial presentation to the emergency room , and she was transferred to the operating room , where laparoscopy revealed no evidence of peritoneal penetration .", "soon after arrival in the recovery room she drained 750 milliliters of blood from her chest tube and became hypotensive .", "she was returned to the operating room , where thoracotomy revealed an actively bleeding injury to the lingula which was successfully controlled by stapled wedge resection .", "the second patient also required a left thoracotomy to oversew bleeding from the lung parenchyma .", "the mean operative time for laparoscopy was 16 minutes ( range : 3  60 ) .", "the mean time for negative laparoscopy was 23 minutes , and 10.8 minutes for laparoscopy with positive findings .", "forty - one ( 59% ) procedures met criteria for positive laparoscopy : thirty - nine were converted to celiotomy and two additional nonbleeding liver injuries were treated nonoperatively .", "twenty - five ( 65% ) of the 39 laparotomies were considered therapeutic by the operating surgeon . among the 14 nontheraputic laparotomies ,", "ten were performed in patients with peritoneal penetration visualized on laparoscopy but found to have no intraperitoneal injury at laparotomy , three revealed only minor nonbleeding liver injuries early in our study that the individual attending surgeon was unwilling to treat nonoperatively , and one was performed for free blood secondary to a minor omental laceration .", "in patients without associated injuries , the mean length of stay following negative laparoscopy was 1.5 days in 21 patients ( range : 1  4 ; the single 4-day stay was due to social service issues ) , compared to 5.2 days ( range : 3  8) for 12 negative explorations following positive laparoscopy .", "there were no anesthetic complications in the study group , and no tension pneumothoraces or air emboli occurred .", "one small bowel injury resulted from insertion of the hasson trocar , and was repaired primarily in a patient during laparotomy for a thoracoabdominal gunshot wound with liver , adrenal and diaphragmatic injuries .", "no intra - abdominal injuries were missed in the negative laparoscopy group ( table 1 ) . however , two of ten patients ( 20% ) who underwent negative laparoscopy for lower left chest stab wounds required urgent return to surgery from the recovery room for missed ongoing hemothoraces .", "one was a 23-year - old female stabbed in the left anterior axillary line at the 7th intercostal space .", "a closed tube thoracostomy was per - formed for a nonbleeding pneumothorax upon initial presentation to the emergency room , and she was transferred to the operating room , where laparoscopy revealed no evidence of peritoneal penetration .", "soon after arrival in the recovery room she drained 750 milliliters of blood from her chest tube and became hypotensive .", "she was returned to the operating room , where thoracotomy revealed an actively bleeding injury to the lingula which was successfully controlled by stapled wedge resection .", "the second patient also required a left thoracotomy to oversew bleeding from the lung parenchyma .", "the optimal management of the asymptomatic patient with penetrating thoracoabdominal trauma has yet to be determined . prior to september 1995 ,", "the criteria for mandatory laparotomy at our institution were broad and included all anterior abdominal gunshot injuries , and stab wounds to the anterior abdomen with posterior fascial penetration demonstrated by local wound exploration .", "recent reports have emphasized the complication rate following negative laparotomy for trauma , ranging from a six percent to 20% incidence in renz and feliciano 's series of 81 patients with penetrating abdominal trauma without associated injuries , and 22% in sosa 's recent comparison of mandatory laparotomy and diagnostic laparoscopy for asymptomatic abdominal gunshot wounds .", "leppaniemi et al . from finland reported their 24-year experience of a 40% negative laparotomy and 21% complication rate in 457 patients following mandatory laparotomy for penetrating truncal trauma . while many of the complications following negative laparotomy in these four reports were minor", "sosa et al . prospectively studied 85 patients with asymptomatic abdominal gunshot wounds by diagnostic laparoscopy , and compared them to the preceding four - year period when mandatory laparotomy was performed in a similar group of 817 patients .", "he was able to document a drop in the negative laparotomy rate from 12.4% to 4.7% when routine diagnostic laparoscopy was instituted .", "additionally , there was a significant decline in average length of stay from 5.1 to 1.4 days , with no missed intraperitoneal injuries and only one significant complication ( urinary retention ) following negative laparoscopy .", "the largest experience to date with the use of diagnostic laparoscopy is a retrospective multicenter study of 510 patients with penetrating abdominal trauma . in this series ,", "54.3% of exams revealed either nonpenetration or nonsignificant injuries , and the mean hospital stay was 1.7 days in the absence of associated injuries .", "laparotomy was considered therapeutic in 155 of the 213 patients ( 73% ) who underwent formal abdominal exploration .", "is an additional group of 26 patients who underwent therapeutic laparoscopy , including repair of diaphragmatic and anterior gastric lacerations , and control of hepatic bleeding .", "while it is not clear from this paper whether laparoscopy resulted in a reduction in the rate of negative laparotomy ( which was 25% overall in this combined series ) , it does indicate that laparoscopy can safely and reliably select those patients without intraperitoneal injury who can be spared a negative laparotomy . in view of these reports , and", "dissatisfied with our own negative laparotomy rate , prospective evaluation of diagnostic laparoscopy was undertaken with the expectation that insignificant injuries could be reliably identified , thus reducing both the incidence of negative celiotomy and hospital length of stay . in the 70 consecutive patients who underwent diagnostic laparoscopy ,", "the negative laparotomy rate was 20% , and if the trivial liver injuries explored early in our experience are excluded , the rate drops to 16% .", "this is comparable to the negative laparotomy rates of 11 - 20% following laparoscopy recently reported by other authors .", "overall , the negative exploration rate for all patients with penetrating abdominal trauma treated during this time period at our institution fell from 33% to 20% .", "while a significant decrease in our negative laparotomy rate occurred , it is still not negligible , and this failure to eliminate nontheraputic laparotomy in one - fifth of the cases of penetrating trauma is the  achilles ' heel  of diagnostic laparoscopy as currently practiced . as our ability to laparoscopically explore the peritoneal cavity , reliably examine the large and small bowel , and perform laparoscopic repair of selected injuries improves , the negative laparotomy rate should continue to decline and can be expected to approach five percent . since bellevue hospital charges a fixed comprehensive daily rate , a true cost analysis could not be made , leaving the length of stay as the only valid index of comparison ; during the study period it was 5.2 days following negative laparotomy as compared to 1.5 days after negative laparoscopy . at current charges of $ 1,000 per day , a 3.7 day decrease in length of stay represents a $ 3,700 reduction in cost per patient , which translates into an overall savings of $ 77,700 for the 21 patients who underwent negative laparoscopy and did not require additional hospitalization for associated injuries .", "while primarily used to evaluate penetrating injuries , laparoscopy also proved to be a useful tool in selected blunt trauma patients with benign clinical exams and indeterminate radiologic studies .", "one patient had a minimal amount of free fluid in the pelvis on computed tomography ( ct ) ; laparoscopy revealed turbid fluid , and a perforation of the antimesenteric border of the proximal jejenum was found and repaired at laparotomy .", "the second patient was a pedestrian struck by a taxi on the right flank and hip ; she was hemodynamically stable and complained only of right hip pain upon evaluation in the emergency room .", "there was a question of bowel gas above the left hemidiaphragm on chest roentgenogram , and both ct scan and upper gastrointestinal series were equivocal regarding the presence of a diaphragmatic rupture .", "laparoscopy revealed free blood in the peritoneal cavity , and a splenic laceration and intact left hemidiaphragm were found at exploration .", "another subgroup in whom diagnostic laparoscopy proved efficacious was in the evaluation of the trauma patient infected by the human immunodeficiency virus .", "three hiv+ patients with abdominal stab wounds and equivocal physical exams underwent negative laparoscopy , sparing all three an unnecessary abdominal exploration .", "laparoscopy has been championed by some authors as a routine maneuver in the evaluation of penetrating lower chest injuries to detect clinically and radiologically occult diaphragmatic injuries . in this study ,", "laparoscopy was selectively employed to evaluate those lower chest injuries felt to be at particular risk for intra - abdominal injuries , and one diaphragmatic injury was detected and repaired .", "however , two of the remaining ten patients with thoracoabdominal stab wounds who underwent negative diagnostic laparoscopy manifested ongoing thoracic blood loss that necessitated return to the operating room , reinduction of general anesthesia and thoracotomy for definitive control of pulmonary parenchymal bleeding . clearly , routine diagnostic laparoscopy in these patients resulted in initial evaluation of the wrong body cavity , and lulled us into a false sense of security regarding the extent and severity of injury .", "thus , in patients with penetrating thoracoabdominal trauma and negative diagnostic laparoscopy , we now believe that diagnostic thoracoscopy should be performed before the patient leaves the operating room .", "the applications of thoracoscopy in the trauma patient are expanding ; this technique can be used to detect and repair diaphragmatic injuries , evaluate the pericardium , and treat both ongoing and established hemothoraces .", "this experience confirms that laparoscopy is a safe and accurate tool for identifying peritoneal penetration following penetrating abdominal trauma , and adoption of this technique resulted in significant decreases in both the rate of negative laparotomy , and the hospital length of stay following a negative evaluation .", "one drawback is that the rate of negative laparotomy , while reduced , remains in the range of 1020% in our experience and recent comparable series .", "diagnostic laparoscopy reliably identifies those injuries to the liver that can be safely managed nonoperatively .", "it also proved useful in the evaluation of the hiv+ patient with equivocal findings on physical exam , and of stable patients with indeterminate or ", "while other authors have demonstrated its sensitivity in the detection of diaphragmatic injuries , we urge caution in its use as the primary diagnostic study in penetrating thoracoabdominal trauma based on our experience with missed major intrathoracic injuries , and are currently investigating the complementary role of thoracoscopy in penetrating lower thoracic injuries ."], "abstract_text": ["<S> background : how should the stable patient with penetrating abdominal or lower chest trauma be evaluated ? </S>", "<S> evolving trends have recently included the use of diagnostic laparoscopy . in september 1995 </S>", "<S> we instituted a protocol of diagnostic laparoscopy to identify those patients who could safely avoid surgical intervention.design:prospective case series.materials and methods : hemodynamically stable patients with penetrating injuries to the anterior abdomen and lower chest were prospectively evaluated by diagnostic laparoscopy , performed in the operating room under general anesthesia , and considered negative if no peritoneal violation or an isolated nonbleeding liver injury had occurred . </S>", "<S> if peritoneal violation , major organ injury or hematoma was noted , conversion to open celiotomy was undertaken.results:seventy consecutive patients were evaluated over a two - year period . </S>", "<S> the average length of stay ( los ) following negative laparoscopy was 1.5 days , and for negative celiotomy 5.2 days . </S>", "<S> there were no missed intra - abdominal injuries following 30 negative laparoscopies , and 26 of 40 laparotomies were therapeutic . </S>", "<S> the technique also proved useful in evaluation of selected blunt and hiv+ trauma vic - tims with unclear clinical presentations . </S>", "<S> however , while laparoscopy was accurate in assessing the abdomen following penetrating lower chest injuries , significant thoracic injuries were missed in 2 out of 11 patients who required subsequent return to or for thoracotomy.conclusions:laparoscopy has become a useful and accu - rate diagnostic tool in the evaluation of abdominal trauma . </S>", "<S> nevertheless , laparoscopy still carries a 20% nontheraputic laparotomy rate . </S>", "<S> additionally , significant intrathoracic injuries may be missed when laparoscopy is used as the pri - mary technique to evaluate penetrating lower thoracic trauma . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "None", "Operative Findings:", "Length of Stay:", "Morbidity:", "Missed Injuries:", "DISCUSSION", "CONCLUSIONS"], "sections": [["how should the stable patient with penetrating abdominal or lower chest wall trauma be evaluated ?", "traditional diagnostic methods and liberal indications for surgery have resulted in negative laparotomy rates ranging from 12 - 40% .", "while negative laparotomy was previously accepted as the inevitable and innocuous result of this policy , recent reports by renz and others have highlighted the 6 - 40% incidence of complications that accompany nontheraputic abdominal exploration . in view of these data , and", "dissatisfied with our own 33% negative laparotomy rate , prospective evaluation of the role of diagnostic laparoscopy in the evaluation of suspected penetrating abdominal injury was undertaken in september 1995 to identify those patients who could safely avoid surgical intervention , with the expectation that the rate of negative trauma celiotomy would markedly decrease . in this report", "we review our experience with 70 consecutive patients who underwent diagnostic laparoscopy , and discuss the benefits and pitfalls in diagnosis we encountered utilizing this technique ."], ["a prospective study of patients with penetrating injuries to the lower thorax and anterior abdomen admitted to the bellevue hospital trauma and shock unit , a level i trauma center in new york city , was performed between september 1995 through november 1997 .", "patients who were hemodynamically stable without obvious evidence of peritoneal penetration or free intra - abdominal air on upright chest radiograph were entered into the protocol . in light of evidence that co2 pneumoperitoneum may result in an increase in intracranial pressure ,", "patients with known or suspected central nervous system injuries were initially excluded from the protocol . however , the use of diagnostic laparoscopy may be appropriate in head - injured patients when intracranial pressure monitors are in place .", "after initial evaluation in the emergency room , informed consent was obtained and the patient was transferred to the operating room .", "general endotracheal anesthesia was induced , orogastric tube and foley catheter inserted , and open laparoscopy performed with the hasson trocar positioned infraumbilically .", "after insufflation with carbon dioxide gas to 15 mm hg , a 10 mm laparoscope with attached video camera was inserted into the peritoneal cavity , and exploration performed . in patients with thoracoabdominal injuries and suspected diaphragmatic tears , a low pressure pneumoperitoneum of 8 - 10 mm hg was created , the diaphragms visualized , and if no diaphragmatic injuries were present , the pneumoperitoneum was raised to 15 mm hg .", "routine thoracostomy tubes were not placed prophylactically in light of zargut 's reported 1% pneumothorax rate following carbon dioxide insufflation ( 5 of 510 patients ) ; rather , the anesthesia and surgical teams were alerted to the possible development of a tension pneumothorax in patients at risk for diaphragmatic injury . if no peritoneal violation was noted , or an isolated non - bleeding injury to the liver was present beneath the site of peritoneal penetration , the procedure was terminated , and the patient subsequently discharged from the hospital . if peritoneal violation , major organ injury or hematoma were noted , the procedure was converted to open celiotomy . during this period", "we did not utilize the technique of  gasless laparoscopy ,  or perform  minilaparoscopy  in the emergency department with local anesthesia utilizing 3 - 5 mm videoscopes .", "trauma service records were reviewed for the two years which preceded this study ( 1993  1994 ) ; the overall negative exploration rate for all patients with penetrating abdominal trauma during that time period was 33% ."], ["sixty - four ( 91% ) patients were male and six ( 9% ) female .", "forty - four patients ( 63% ) suffered stab wounds to the anterior abdomen , eleven ( 16% ) stab wounds to the left chest with suspicion of intra - abdominal injury ,   ( 19% ) with abdominal gunshot wounds .", "two additional patients ( 3% ) involved in motor - vehicle accidents were evaluated laparoscopically after indeterminate computed tomo - graphic ( ct ) scans .", "the mean operative time for laparoscopy was 16 minutes ( range : 3  60 ) .", "the mean time for negative laparoscopy was 23 minutes , and 10.8 minutes for laparoscopy with positive findings .", "forty - one ( 59% ) procedures met criteria for positive laparoscopy : thirty - nine were converted to celiotomy and two additional nonbleeding liver injuries were treated nonoperatively .", "twenty - five ( 65% ) of the 39 laparotomies were considered therapeutic by the operating surgeon . among the 14 nontheraputic laparotomies ,", "ten were performed in patients with peritoneal penetration visualized on laparoscopy but found to have no intraperitoneal injury at laparotomy , three revealed only minor nonbleeding liver injuries early in our study that the individual attending surgeon was unwilling to treat nonoperatively , and one was performed for free blood secondary to a minor omental laceration . in patients without associated injuries ,", "the mean length of stay following negative laparoscopy was 1.5 days in 21 patients ( range : 1  4 ; the single 4-day stay was due to social service issues ) , compared to 5.2 days ( range : 3  8) for 12 negative explorations following positive laparoscopy .", "there were no anesthetic complications in the study group , and no tension pneumothoraces or air emboli occurred .", "one small bowel injury resulted from insertion of the hasson trocar , and was repaired primarily in a patient during laparotomy for a thoracoabdominal gunshot wound with liver , adrenal and diaphragmatic injuries .", "no intra - abdominal injuries were missed in the negative laparoscopy group ( table 1 ) .", "however , two of ten patients ( 20% ) who underwent negative laparoscopy for lower left chest stab wounds required urgent return to surgery from the recovery room for missed ongoing hemothoraces .", "one was a 23-year - old female stabbed in the left anterior axillary line at the 7th intercostal space .", "a closed tube thoracostomy was per - formed for a nonbleeding pneumothorax upon initial presentation to the emergency room , and she was transferred to the operating room , where laparoscopy revealed no evidence of peritoneal penetration .", "soon after arrival in the recovery room she drained 750 milliliters of blood from her chest tube and became hypotensive .", "she was returned to the operating room , where thoracotomy revealed an actively bleeding injury to the lingula which was successfully controlled by stapled wedge resection .", "the second patient also required a left thoracotomy to oversew bleeding from the lung parenchyma ."], ["the mean operative time for laparoscopy was 16 minutes ( range : 3  60 ) .", "the mean time for negative laparoscopy was 23 minutes , and 10.8 minutes for laparoscopy with positive findings .", "forty - one ( 59% ) procedures met criteria for positive laparoscopy : thirty - nine were converted to celiotomy and two additional nonbleeding liver injuries were treated nonoperatively .", "twenty - five ( 65% ) of the 39 laparotomies were considered therapeutic by the operating surgeon . among the 14 nontheraputic laparotomies ,", "ten were performed in patients with peritoneal penetration visualized on laparoscopy but found to have no intraperitoneal injury at laparotomy , three revealed only minor nonbleeding liver injuries early in our study that the individual attending surgeon was unwilling to treat nonoperatively , and one was performed for free blood secondary to a minor omental laceration . in patients without associated injuries , the mean length of stay following negative laparoscopy was 1.5 days in 21 patients ( range : 1  4 ; the single 4-day stay was due to social service issues ) , compared to 5.2 days ( range : 3  8) for 12 negative explorations following positive laparoscopy .", "there were no anesthetic complications in the study group , and no tension pneumothoraces or air emboli occurred .", "one small bowel injury resulted from insertion of the hasson trocar , and was repaired primarily in a patient during laparotomy for a thoracoabdominal gunshot wound with liver , adrenal and diaphragmatic injuries .", "no intra - abdominal injuries were missed in the negative laparoscopy group ( table 1 ) .", "however , two of ten patients ( 20% ) who underwent negative laparoscopy for lower left chest stab wounds required urgent return to surgery from the recovery room for missed ongoing hemothoraces .", "one was a 23-year - old female stabbed in the left anterior axillary line at the 7th intercostal space .", "a closed tube thoracostomy was per - formed for a nonbleeding pneumothorax upon initial presentation to the emergency room , and she was transferred to the operating room , where laparoscopy revealed no evidence of peritoneal penetration .", "soon after arrival in the recovery room she drained 750 milliliters of blood from her chest tube and became hypotensive .", "she was returned to the operating room , where thoracotomy revealed an actively bleeding injury to the lingula which was successfully controlled by stapled wedge resection .", "the second patient also required a left thoracotomy to oversew bleeding from the lung parenchyma ."], ["the mean operative time for laparoscopy was 16 minutes ( range : 3  60 ) .", "the mean time for negative laparoscopy was 23 minutes , and 10.8 minutes for laparoscopy with positive findings .", "forty - one ( 59% ) procedures met criteria for positive laparoscopy : thirty - nine were converted to celiotomy and two additional nonbleeding liver injuries were treated nonoperatively .", "twenty - five ( 65% ) of the 39 laparotomies were considered therapeutic by the operating surgeon . among the 14 nontheraputic laparotomies ,", "ten were performed in patients with peritoneal penetration visualized on laparoscopy but found to have no intraperitoneal injury at laparotomy , three revealed only minor nonbleeding liver injuries early in our study that the individual attending surgeon was unwilling to treat nonoperatively , and one was performed for free blood secondary to a minor omental laceration ."], ["in patients without associated injuries , the mean length of stay following negative laparoscopy was 1.5 days in 21 patients ( range : 1  4 ; the single 4-day stay was due to social service issues ) , compared to 5.2 days ( range : 3  8) for 12 negative explorations following positive laparoscopy ."], ["there were no anesthetic complications in the study group , and no tension pneumothoraces or air emboli occurred .", "one small bowel injury resulted from insertion of the hasson trocar , and was repaired primarily in a patient during laparotomy for a thoracoabdominal gunshot wound with liver , adrenal and diaphragmatic injuries ."], ["no intra - abdominal injuries were missed in the negative laparoscopy group ( table 1 ) . however , two of ten patients ( 20% ) who underwent negative laparoscopy for lower left chest stab wounds required urgent return to surgery from the recovery room for missed ongoing hemothoraces .", "one was a 23-year - old female stabbed in the left anterior axillary line at the 7th intercostal space .", "a closed tube thoracostomy was per - formed for a nonbleeding pneumothorax upon initial presentation to the emergency room , and she was transferred to the operating room , where laparoscopy revealed no evidence of peritoneal penetration .", "soon after arrival in the recovery room she drained 750 milliliters of blood from her chest tube and became hypotensive .", "she was returned to the operating room , where thoracotomy revealed an actively bleeding injury to the lingula which was successfully controlled by stapled wedge resection .", "the second patient also required a left thoracotomy to oversew bleeding from the lung parenchyma ."], ["the optimal management of the asymptomatic patient with penetrating thoracoabdominal trauma has yet to be determined . prior to september 1995 ,", "the criteria for mandatory laparotomy at our institution were broad and included all anterior abdominal gunshot injuries , and stab wounds to the anterior abdomen with posterior fascial penetration demonstrated by local wound exploration .", "recent reports have emphasized the complication rate following negative laparotomy for trauma , ranging from a six percent to 20% incidence in renz and feliciano 's series of 81 patients with penetrating abdominal trauma without associated injuries , and 22% in sosa 's recent comparison of mandatory laparotomy and diagnostic laparoscopy for asymptomatic abdominal gunshot wounds .", "leppaniemi et al . from finland reported their 24-year experience of a 40% negative laparotomy and 21% complication rate in 457 patients following mandatory laparotomy for penetrating truncal trauma . while many of the complications following negative laparotomy in these four reports were minor", "sosa et al . prospectively studied 85 patients with asymptomatic abdominal gunshot wounds by diagnostic laparoscopy , and compared them to the preceding four - year period when mandatory laparotomy was performed in a similar group of 817 patients .", "he was able to document a drop in the negative laparotomy rate from 12.4% to 4.7% when routine diagnostic laparoscopy was instituted .", "additionally , there was a significant decline in average length of stay from 5.1 to 1.4 days , with no missed intraperitoneal injuries and only one significant complication ( urinary retention ) following negative laparoscopy .", "the largest experience to date with the use of diagnostic laparoscopy is a retrospective multicenter study of 510 patients with penetrating abdominal trauma . in this series ,", "54.3% of exams revealed either nonpenetration or nonsignificant injuries , and the mean hospital stay was 1.7 days in the absence of associated injuries .", "laparotomy was considered therapeutic in 155 of the 213 patients ( 73% ) who underwent formal abdominal exploration .", "is an additional group of 26 patients who underwent therapeutic laparoscopy , including repair of diaphragmatic and anterior gastric lacerations , and control of hepatic bleeding .", "while it is not clear from this paper whether laparoscopy resulted in a reduction in the rate of negative laparotomy ( which was 25% overall in this combined series ) , it does indicate that laparoscopy can safely and reliably select those patients without intraperitoneal injury who can be spared a negative laparotomy . in view of these reports , and", "dissatisfied with our own negative laparotomy rate , prospective evaluation of diagnostic laparoscopy was undertaken with the expectation that insignificant injuries could be reliably identified , thus reducing both the incidence of negative celiotomy and hospital length of stay . in the 70 consecutive patients who underwent diagnostic laparoscopy ,", "the negative laparotomy rate was 20% , and if the trivial liver injuries explored early in our experience are excluded , the rate drops to 16% .", "this is comparable to the negative laparotomy rates of 11 - 20% following laparoscopy recently reported by other authors .", "overall , the negative exploration rate for all patients with penetrating abdominal trauma treated during this time period at our institution fell from 33% to 20% .", "while a significant decrease in our negative laparotomy rate occurred , it is still not negligible , and this failure to eliminate nontheraputic laparotomy in one - fifth of the cases of penetrating trauma is the  achilles ' heel  of diagnostic laparoscopy as currently practiced . as our ability to laparoscopically explore the peritoneal cavity , reliably examine the large and small bowel , and perform laparoscopic repair of selected injuries improves , the negative laparotomy rate should continue to decline and can be expected to approach five percent . since bellevue hospital charges a fixed comprehensive daily rate , a true cost analysis could not be made , leaving the length of stay as the only valid index of comparison ; during the study period it was 5.2 days following negative laparotomy as compared to 1.5 days after negative laparoscopy . at current charges of $ 1,000 per day , a 3.7 day decrease in length of stay represents a $ 3,700 reduction in cost per patient , which translates into an overall savings of $ 77,700 for the 21 patients who underwent negative laparoscopy and did not require additional hospitalization for associated injuries .", "while primarily used to evaluate penetrating injuries , laparoscopy also proved to be a useful tool in selected blunt trauma patients with benign clinical exams and indeterminate radiologic studies .", "one patient had a minimal amount of free fluid in the pelvis on computed tomography ( ct ) ; laparoscopy revealed turbid fluid , and a perforation of the antimesenteric border of the proximal jejenum was found and repaired at laparotomy .", "the second patient was a pedestrian struck by a taxi on the right flank and hip ; she was hemodynamically stable and complained only of right hip pain upon evaluation in the emergency room .", "there was a question of bowel gas above the left hemidiaphragm on chest roentgenogram , and both ct scan and upper gastrointestinal series were equivocal regarding the presence of a diaphragmatic rupture .", "laparoscopy revealed free blood in the peritoneal cavity , and a splenic laceration and intact left hemidiaphragm were found at exploration .", "another subgroup in whom diagnostic laparoscopy proved efficacious was in the evaluation of the trauma patient infected by the human immunodeficiency virus .", "three hiv+ patients with abdominal stab wounds and equivocal physical exams underwent negative laparoscopy , sparing all three an unnecessary abdominal exploration .", "laparoscopy has been championed by some authors as a routine maneuver in the evaluation of penetrating lower chest injuries to detect clinically and radiologically occult diaphragmatic injuries . in this study ,", "laparoscopy was selectively employed to evaluate those lower chest injuries felt to be at particular risk for intra - abdominal injuries , and one diaphragmatic injury was detected and repaired .", "however , two of the remaining ten patients with thoracoabdominal stab wounds who underwent negative diagnostic laparoscopy manifested ongoing thoracic blood loss that necessitated return to the operating room , reinduction of general anesthesia and thoracotomy for definitive control of pulmonary parenchymal bleeding . clearly , routine diagnostic laparoscopy in these patients resulted in initial evaluation of the wrong body cavity , and lulled us into a false sense of security regarding the extent and severity of injury .", "thus , in patients with penetrating thoracoabdominal trauma and negative diagnostic laparoscopy , we now believe that diagnostic thoracoscopy should be performed before the patient leaves the operating room .", "the applications of thoracoscopy in the trauma patient are expanding ; this technique can be used to detect and repair diaphragmatic injuries , evaluate the pericardium , and treat both ongoing and established hemothoraces ."], ["this experience confirms that laparoscopy is a safe and accurate tool for identifying peritoneal penetration following penetrating abdominal trauma , and adoption of this technique resulted in significant decreases in both the rate of negative laparotomy , and the hospital length of stay following a negative evaluation .", "one drawback is that the rate of negative laparotomy , while reduced , remains in the range of 1020% in our experience and recent comparable series .", "diagnostic laparoscopy reliably identifies those injuries to the liver that can be safely managed nonoperatively .", "it also proved useful in the evaluation of the hiv+ patient with equivocal findings on physical exam , and of stable patients with indeterminate or ", "while other authors have demonstrated its sensitivity in the detection of diaphragmatic injuries , we urge caution in its use as the primary diagnostic study in penetrating thoracoabdominal trauma based on our experience with missed major intrathoracic injuries , and are currently investigating the complementary role of thoracoscopy in penetrating lower thoracic injuries ."]]}
{"article_id": "PMC3103859", "article_text": ["selenium ( se ) has been recognized as an essential nutrient for plant , animal , and human body , but at high concentration it can become toxic . the range between the concentration in which selenium is essential and toxic is very narrow [ 1 , 2 ] .", "this element plays an important role in elderly people as well as in the prevention of many age - associated diseases and in maintenance of normal immune function .", "se is potent antioxidant involved in cellular defense against free radical reactions , and the risk of deficiency seems to increase in proportion to the age .", "evidence is accumulating that most of the degenerative diseases have their origin in deleterious free radical reactions .", "these diseases include atherosclerosis , cancer , inflammatory joint , asthma , diabetes , senile dementia , and degenerative eye disease .", "but high se concentrations in human can cause loosing hair and nails and irritation of skin and eye .", "the required daily amount of se is 20  mcg  day and 4070  mcg day for 46 years old and adult males , respectively .", "the essential role of the se is due to its presence in the active site of some enzymes ( i.e. , glutathione peroxidase and iodotironine-5-deiodinase ) and the catalytic effect of selenium compounds on the reaction of intermediate metabolism and inhibition of the toxic effect of heavy metals .", "it has been established that diets with deficit in se are associated with some human diseases , but diets with se contents higher than 5  mg / kg are toxic and cause important symptoms in humans and animals .", "selenium is present as selenocysteine ( se - cys ) in at least 30 proteins .", "all these factors , together with the fact that the concentration levels are extremely low , call for sensitive and accurate method for the determination of this element .  ", "selenium can be present in 2 , 0 , + 4 , and + 6 oxidation states .", "these forms can not simultaneously be evaluated by direct application of certain analytical techniques such as instrumental neutron activation analysis ( inaa ) [ 6 , 7 ] , as well as hydride generation atomic absorption spectroscopy ( hg - aas ) , inductively coupled plasma atomic emission spectrometry ( icp - aes ) , electrothermal atomic absorption spectrometry ( et - aas ) , or inductively coupled plasma mass spectrometry ( icp - ms ) , after digestion of sample [ 810 ] .", "the most common methods used for determination of various species of selenium were atomic absorption spectrometry ( hydride generation and electrothermal atomization ) [ 1114 ] , molecular [ 15 , 16 ] and atomic fluorescence spectrometry [ 1719 ] , high - performance liquid chromatography ( hplc ) [ 2022 ] , voltammetry [ 23 , 24 ] , atomic emission spectrometry ( aes ) , such as inductively coupled plasma ( icp )   and inductively coupled plasma - mass spectrometry ( icp - ms ) , and spectrophotometry methods [ 2528 ] . in spite of some of the above methods , spectrophotometric methods are popular because of their simplicity and are based on piazselenol complex formation between the reagents and selenium .  ", "an interesting alternative to traditional liquid - liquid extraction is the micelle - mediated extraction , firstly developed by watanabe and tanaka .", "the cloud point is the temperature above which aqueous solutions of nonionic and zwitterionic surfactant become turbid .", "micelles of such well - known nonionic surfactant as triton x-100 or x-114 have a nonpolar core and extended polar layer , where both extractants and extracted complexes can be solubilized . above the cloud point ,", "the solution is separated into two phases , a rich phase containing a high surfactant concentration in a small volume and a poor phase with a surfactant concentration close to the critical micelle concentration ( cmc ) .", "hydrophobic species ( hydrophobic organic compounds or metal ions after reaction with a suitable hydrophobic ligand ) present in sample are able to interact with the micelles , thus being concentrated in the small volume of the surfactant - rich phase .", "the aim of this work was to use a simple , rapid , and sensitive spectrophotometric method ( dithizone as chromogenic reagent ) for determination of trace amounts of selenium ( iv ) ( 5100  ng  ml ) in micellar medium ( triton x-100 ) in cosmetic and pharmaceutical products . to improve", "the detection limit of this method , cloud point extraction ( cpe ) has been used .", "selenium metal ( purity : 99.9% ) was obtained from the riedel de haen ( germany ) . nitric acid", "( > 99.5% ) , sodium hydroxide ( > 97% ) , ascorbic acid , hydrochloric acid ( 36.538% ) , phenol ( > 99.0% ) , methanol ( > 99.8% ) , and sulfuric acid ( 95.097.0% ) were purchased from merck ( germany ) .", "all chemicals were used without any further purification . deionized water from a milli - q system ( millipore , usa ) was used for preparation of all solutions .", "selenium sulphide shampoo ( iran ) , centrum tablet ( usa ) , and selen plus capsule ( germany ) were obtained from market . a shimadzu double - beam uv - vis spectrophotometer ( model 1650 pc , japan ) with 1.0  cm quartz cell", "was used for all spectral measurements . in order to investigate the accuracy of method a perkinelmer ( model optima 7000 dv ) inductively coupled plasma optical emission spectrometer ( icp - oes )", "separation of surfactant phase and aqueous phase was carried out with a hettich centrifuge ( eba 20 , uk ) .", "a microwave system ( microdigest 301 , prolabo , france ) with a maximum irradiation power of 200  w was used .", "ph measurements were made with a metrohm ph meter ( model 744 , switzerland ) .", "the effect of temperature was investigated by a gfl thermostated bath ( model 1003 , germany ) .", "the stock standard solution of selenium ( iv ) ( 1000  g  ml ) was prepared by dissolving 100  mg of selenium metal in hot concentrated nitric acid and diluted to 100  ml with water . from this solution", "serial dilutions were made to obtain different concentration levels of 5 , 15 , 30 , 40 , 55 , 70 , 80 , and 100  ng  ml of selenium ( iv ) .", "the stock solution of dithizone ( 10  mol  l ) was prepared daily by dissolving 12.8  mg of the reagent in 5  ml of naoh solution ( 0.1  m , containing the 50  mg of ascorbic acid for stabilizing the dithizone ) and diluting to 50  ml with water .", "the stock solution was diluted with water to obtain the final dithizone concentration of about 7.5  mol  l. the stock solution of triton x-100 ( 10% ( w / v ) ) was prepared by weighing 10  g of this reagent into 100  ml volumetric flask , dissolving in water by gentle heating , and making up to the mark with water .", "the final concentration of triton x-100 ( 0.2% ) was prepared by suitable dilution of stock solution with the diluent ( solution of 0.5% ( w / v ) of phenol ) .", "an aliquot of 50  ml of a solution containing selenium ( iv ) ( final concentration 5100  ng  ml ) , triton x-100 ( 0.2% ( w / v ) ) , dithizone ( 7.5  mol  l ) , and hcl ( 0.35  m ) was kept for 15  min to complete the color development . then the mixture was placed in a water bath at 40c for 10  min , when 0.5% ( w / v ) of the phenol was used for inducing the cloud point .", "phase separation was accelerated by centrifuging the test tube at 3500  rpm for 15  min .", "the bulk aqueous phases were easily decanted by gently inverting the test tubes , and the surfactant - rich phase ( complex rich ) was made up to 5  ml by adding methanol .", "the same procedure was applied for preparation of blank solution without the selenium and dithizone .", "the absorbance of two methanolic solutions was measured at 424 and 592  nm , against a blank ( prepared in the same way without se and dithizone ) .", "the absorption maxima of the complex ( 424  nm ) and dithizone ( 434  nm ) were overlapping ( figure 1 ) .", "hence , the corrected absorbance , a \n corr , was used to overcome the problem : \n ( 1)acorr = a424(a424a592)la592 , \n where a \n 424 and a \n 592 are the absorbance of the complex measured at 424 and 592  nm , respectively , and ( a \n 424/a \n 592)l is the absorbance ratio of dithizone at 424 and 592  nm . the complex does not show any absorbance at 592  nm .", "under optimum conditions , a calibration graph was used for determination of selenium in cosmetic and pharmaceutical products .", "shampoo ( selenium sulphide , iran ) sample dissolution was prepared using the procedure described by afkhami et al . .", "approximately 1  g of shampoo sample was weighted into a 100  ml beaker . concentrated sulfuric acid ( 1  ml )", "the solution was allowed to cool , and 5  ml of 30% ( w / v ) hydrogen peroxide was added .", "the mixture boiled vigorously to eliminate excess hydrogen peroxide and allowed to cool and then the digest transferred to a 100  ml volumetric flask .", "finally , 2  ml of this solution was taken for determination of selenium ( iv ) as in the recommended procedure .  ", "a centrum tablet ( usa ) containing 0.25  mg se ( as sodium selenate ) was placed in digestion vessel", ". 5  ml of concentrated nitric acid and 2  ml of hydrogen peroxide were added and the mixture subjected to microwave irradiation ( 25% of power , 5  min ) in order to digest the sample .", "then , 10  ml of hydrochloric acid ( 6  m ) was added , and the mixture was irradiated by microwave ( 75% of power , 5  min ) in order to reduce all se to se ( iv ) .", ". 1  ml of treated sample solution was dropwise passed through an eichrom cation exchange resin ( sulfonic acid groups , mesh ranges : 50100  m , 8  cm ) , for removal of interferences , and selenium was determined with the recommended procedure .  ", "a selen plus capsule ( germany ) was dissolved in 10  ml hcl ( 6  m ) , and the mixture was irradiated by microwave ( 75% of power , 5  min ) in order to reduce all se to se ( iv ) .", "the mixture was then diluted to 50  ml with water , and then 1  ml of this mixture was taken for determination of selenium ( iv ) as in the recommended procedure .", "from the plot obtained by job 's method of continuous variation , the se- ( iv ) to- dithizone ( h2dz ) ratio forming the complex was found to be 1 ", ":  4 at ph  <  1 . the complex can , therefore , be represented as se(hdz)4 which is in agreement with the reported composition in the organic phase .", "the conditional stability constant of the 1  :  4 ( se  :  h2dz ) complex was calculated , assuming the following equilibrium in the system :  \n ( 2)h2seo3 + 4  h2dzse(hdz)4 + 3h2o . \n using the plot obtained by job 's method ,", "xmax  ( chdz / ctotal ) was found to be 0.8 , and therefore the ratio of se  :  hdz was calculated 1  :  4 ( figure 1 ) .", "selenium ( iv ) reacts with dithizone ( hdz ) in micellar medium and forms a hydrophobic complex , se(hdz)4 , which is subsequently trapped in the surfactant micelles and separated from the aqueous phase .  ", "figure 2 shows the absorption spectra for the dithizone and selenium - dithizonate complex in surfactant - rich phase against a reagent blank .", "the absorption maximum of the complex was 424  nm , and the absorption maxima of the dithizone were 434 and 592  nm .", "solution ph plays a unique role on metal - chelate function and subsequent extraction .  ", "figure 3 shows the influence of hcl concentration on the corrected absorbance of the selenium dithizonate complex at 424  nm .", "as can be seen , the complex extraction was increased with the increased hcl concentration and then showed a decrease .", "the reason for this behaviour might be because of decreasing the dithizone stability at higher phs . hence , an hcl concentration of 0.35  m was chosen for further studies . the influence of the dithizone ( hdz ) concentration on analytical response is shown in figure 4 .", "this figure shows that the absorbance increased up to a known concentration of dithizone ( 7.5  mol  l ) and then reached a plateau that can be attributed to complete extraction .", "therefore , the concentration of 7.5  mol  l of dithizone was selected as the optimum .  ", "the nonionic surfactant triton x-100 was chosen because of its commercial availability of high purified homogeneous form , low toxicological properties , and cost .", "the cloud point temperature of triton x-100 is 63.7c . to decrease the cloud point temperature of triton x-100 to room temperature ( desirable temperature in cloud point procedure ) ,", "this solution was prepared by the diluent ( solution of 0.5% ( w / v ) of phenol ) .", "the variation of absorbance at  \n max of complex as a function of the concentration of triton x-100 is shown in figure 5 . at lower concentrations of triton x-100", ", the low extraction efficiency of the complex may be due to the inadequacy of the assemblies to entrap the hydrophobic complex quantitatively .", "the concentration of 0.2% ( w / v ) triton x-100 was selected for further studies .", "optimal incubation time and equilibration temperature are necessary to complete the reaction , easy phase separation , and preconcentration as efficient as possible . providing that the complexation reaction has been completed under certain conditions , corrected absorbance data", "show that , for equilibration times of 10 to 30  min , extraction efficiency is almost constant .", "on the other hand , it appears that the phase volume ratio of all nonionic surfactants decreases as the equilibration temperature increases . as can be seen in figure 7 , the corrected absorbance increases very gently up to 40c and starts to decrease afterwards due to decrease in the stability of the dithizone and consequently the extraction efficiency .", "the dependence of corrected absorbance upon centrifugation time was studied within the range of 530  min .", "it was found that a time of 15  min is adequate for separation of two phases .", "calibration graph was obtained by preconcentrating 50  ml of the sample containing 5 , 15 , 30 , 40 , 55 , 70 , 80 , and 100  ng  ml of selenium ( iv ) under the optimal experimental conditions .", "analytical parameters for the determination of the selenium - dithizonate complex are presented in table 1 .", "the relative standard deviation was 2.18% for six replicate measurements of standard selenium solution ( 50  ng  ml ) .  ", "the effect of various cations and anions on the determination of 0.05  g  ml of se(iv ) was studied , and the results are summarized in table 2 .", "the tolerance limit was taken as the concentration of added ion causing less than 5% relative error .", "cations such as ag(i ) , cu(ii ) , hg(ii ) , and fe(iii ) were interfered with the determination of se(iv ) ion .", "these cations can be removed using a cation exchange resin ( sulfonic acid groups , mesh ranges : 50100 , 8  cm ) . for this purpose , 25  ml solution containing 2.5  g of se(iv ) and various amounts of desired cations", "was passed through 5  g cation exchange resin ( 8  cm wet resin ) dropwise and collected in a 50  ml volumetric flask containing optimum amount of dithizone ( 7.5  mol  l ) , hcl ( 0.35  m ) , and triton x-100 ( 0.2% w / v ) .", "after washing the column with water ( three times , with 15  ml ) , 0.5% ( w / v ) phenol was added and the solution was diluted to the mark with water and the amount of se ( iv ) was determined using recommended procedure . to evaluate the analytical applicability ,", "the method was applied to the determination of selenium in two multivitamin tablets ( centrum and selen plus ) and a selenium sulphide shampoo ( for treatment of dandruff ) .", "the results for selenium sulphide shampoo , centrum , and selen plus multivitamins are given in tables 3 , 4 , and 5 , respectively .   in order to evaluate the accuracy of procedure", ", selenium was determined in centrum tablet ( containing 0.25  mg se as sodium selenate ) by icp - oes as reference method . in both methods the results agreed well ( table 6 ) .  ", "the limit of detection and relative standard deviation of the proposed method were compared with methods in the literature ( table 7 ) .", "as observed from the results limit of detection by the proposed method was lower than other methods .", "the simplicity , rapidity , and inexpensiveness of the proposed method in combination with the use of dithizone as a chromogenic reagent were utilized for selenium determination in cosmetic and pharmaceutical samples .", "precision and recovery data clearly indicated the reproducibility and accuracy of the method . in this investigation ,", "spectrophotometric determination of selenium was carried out in micellar medium without the need for an extraction step .", "because of the overlapping of absorption maximum of complex at 424  nm with absorption maximum of dithizone at 434  nm , a \n corr was applied ."], "abstract_text": ["<S> a simple , rapid , and sensitive spectrophotometric method for the determination of trace amounts of selenium ( iv ) was described . in this method </S>", "<S> , all selenium spices reduced to selenium ( iv ) using 6  m  hcl . </S>", "<S> cloud point extraction was applied as a preconcentration method for spectrophotometric determination of selenium ( iv ) in aqueous solution . </S>", "<S> the proposed method is based on the complexation of selenium ( iv ) with dithizone at ph  <  1 in micellar medium ( triton x-100 ) . </S>", "<S> after complexation with dithizone , the analyte was quantitatively extracted to the surfactant - rich phase by centrifugation and diluted to 5 ml with methanol . since the absorption maxima of the complex ( 424  nm ) and dithizone ( 434  nm ) overlap , hence , the corrected absorbance , acorr , was used to overcome the problem . </S>", "<S> with regard to the preconcentration , the tested parameters were the ph of the extraction , the concentration of the surfactant , the concentration of dithizone , and equilibration temperature and time . </S>", "<S> the detection limit is 4.4  ng ml1 ; the relative standard deviation for six replicate measurements is 2.18% for 50  ng ml1 of selenium . </S>", "<S> the procedure was applied successfully to the determination of selenium in two kinds of pharmaceutical samples . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Experimental", "3. Results and Discussion", "4. Conclusion"], "sections": [["selenium ( se ) has been recognized as an essential nutrient for plant , animal , and human body , but at high concentration it can become toxic . the range between the concentration in which selenium is essential and toxic is very narrow [ 1 , 2 ] .", "this element plays an important role in elderly people as well as in the prevention of many age - associated diseases and in maintenance of normal immune function .", "se is potent antioxidant involved in cellular defense against free radical reactions , and the risk of deficiency seems to increase in proportion to the age .", "evidence is accumulating that most of the degenerative diseases have their origin in deleterious free radical reactions .", "these diseases include atherosclerosis , cancer , inflammatory joint , asthma , diabetes , senile dementia , and degenerative eye disease .", "but high se concentrations in human can cause loosing hair and nails and irritation of skin and eye .", "the required daily amount of se is 20  mcg  day and 4070  mcg day for 46 years old and adult males , respectively .", "the essential role of the se is due to its presence in the active site of some enzymes ( i.e. , glutathione peroxidase and iodotironine-5-deiodinase ) and the catalytic effect of selenium compounds on the reaction of intermediate metabolism and inhibition of the toxic effect of heavy metals .", "it has been established that diets with deficit in se are associated with some human diseases , but diets with se contents higher than 5  mg / kg are toxic and cause important symptoms in humans and animals .", "selenium is present as selenocysteine ( se - cys ) in at least 30 proteins .", "all these factors , together with the fact that the concentration levels are extremely low , call for sensitive and accurate method for the determination of this element .  ", "selenium can be present in 2 , 0 , + 4 , and + 6 oxidation states .", "these forms can not simultaneously be evaluated by direct application of certain analytical techniques such as instrumental neutron activation analysis ( inaa ) [ 6 , 7 ] , as well as hydride generation atomic absorption spectroscopy ( hg - aas ) , inductively coupled plasma atomic emission spectrometry ( icp - aes ) , electrothermal atomic absorption spectrometry ( et - aas ) , or inductively coupled plasma mass spectrometry ( icp - ms ) , after digestion of sample [ 810 ] .", "the most common methods used for determination of various species of selenium were atomic absorption spectrometry ( hydride generation and electrothermal atomization ) [ 1114 ] , molecular [ 15 , 16 ] and atomic fluorescence spectrometry [ 1719 ] , high - performance liquid chromatography ( hplc ) [ 2022 ] , voltammetry [ 23 , 24 ] , atomic emission spectrometry ( aes ) , such as inductively coupled plasma ( icp )   and inductively coupled plasma - mass spectrometry ( icp - ms ) , and spectrophotometry methods [ 2528 ] . in spite of some of the above methods , spectrophotometric methods are popular because of their simplicity and are based on piazselenol complex formation between the reagents and selenium .  ", "an interesting alternative to traditional liquid - liquid extraction is the micelle - mediated extraction , firstly developed by watanabe and tanaka .", "the cloud point is the temperature above which aqueous solutions of nonionic and zwitterionic surfactant become turbid .", "micelles of such well - known nonionic surfactant as triton x-100 or x-114 have a nonpolar core and extended polar layer , where both extractants and extracted complexes can be solubilized . above the cloud point ,", "the solution is separated into two phases , a rich phase containing a high surfactant concentration in a small volume and a poor phase with a surfactant concentration close to the critical micelle concentration ( cmc ) .", "hydrophobic species ( hydrophobic organic compounds or metal ions after reaction with a suitable hydrophobic ligand ) present in sample are able to interact with the micelles , thus being concentrated in the small volume of the surfactant - rich phase .", "the aim of this work was to use a simple , rapid , and sensitive spectrophotometric method ( dithizone as chromogenic reagent ) for determination of trace amounts of selenium ( iv ) ( 5100  ng  ml ) in micellar medium ( triton x-100 ) in cosmetic and pharmaceutical products . to improve", "the detection limit of this method , cloud point extraction ( cpe ) has been used ."], ["selenium metal ( purity : 99.9% ) was obtained from the riedel de haen ( germany ) . nitric acid", "( > 99.5% ) , sodium hydroxide ( > 97% ) , ascorbic acid , hydrochloric acid ( 36.538% ) , phenol ( > 99.0% ) , methanol ( > 99.8% ) , and sulfuric acid ( 95.097.0% ) were purchased from merck ( germany ) .", "all chemicals were used without any further purification . deionized water from a milli - q system ( millipore , usa ) was used for preparation of all solutions .", "selenium sulphide shampoo ( iran ) , centrum tablet ( usa ) , and selen plus capsule ( germany ) were obtained from market . a shimadzu double - beam uv - vis spectrophotometer ( model 1650 pc , japan ) with 1.0  cm quartz cell", "was used for all spectral measurements . in order to investigate the accuracy of method a perkinelmer ( model optima 7000 dv ) inductively coupled plasma optical emission spectrometer ( icp - oes )", "separation of surfactant phase and aqueous phase was carried out with a hettich centrifuge ( eba 20 , uk ) .", "a microwave system ( microdigest 301 , prolabo , france ) with a maximum irradiation power of 200  w was used .", "ph measurements were made with a metrohm ph meter ( model 744 , switzerland ) .", "the effect of temperature was investigated by a gfl thermostated bath ( model 1003 , germany ) .", "the stock standard solution of selenium ( iv ) ( 1000  g  ml ) was prepared by dissolving 100  mg of selenium metal in hot concentrated nitric acid and diluted to 100  ml with water . from this solution", "serial dilutions were made to obtain different concentration levels of 5 , 15 , 30 , 40 , 55 , 70 , 80 , and 100  ng  ml of selenium ( iv ) .", "the stock solution of dithizone ( 10  mol  l ) was prepared daily by dissolving 12.8  mg of the reagent in 5  ml of naoh solution ( 0.1  m , containing the 50  mg of ascorbic acid for stabilizing the dithizone ) and diluting to 50  ml with water .", "the stock solution was diluted with water to obtain the final dithizone concentration of about 7.5  mol  l. the stock solution of triton x-100 ( 10% ( w / v ) ) was prepared by weighing 10  g of this reagent into 100  ml volumetric flask , dissolving in water by gentle heating , and making up to the mark with water .", "the final concentration of triton x-100 ( 0.2% ) was prepared by suitable dilution of stock solution with the diluent ( solution of 0.5% ( w / v ) of phenol ) .", "an aliquot of 50  ml of a solution containing selenium ( iv ) ( final concentration 5100  ng  ml ) , triton x-100 ( 0.2% ( w / v ) ) , dithizone ( 7.5  mol  l ) , and hcl ( 0.35  m ) was kept for 15  min to complete the color development . then the mixture was placed in a water bath at 40c for 10  min , when 0.5% ( w / v ) of the phenol was used for inducing the cloud point .", "phase separation was accelerated by centrifuging the test tube at 3500  rpm for 15  min .", "the bulk aqueous phases were easily decanted by gently inverting the test tubes , and the surfactant - rich phase ( complex rich ) was made up to 5  ml by adding methanol .", "the same procedure was applied for preparation of blank solution without the selenium and dithizone .", "the absorbance of two methanolic solutions was measured at 424 and 592  nm , against a blank ( prepared in the same way without se and dithizone ) .", "the absorption maxima of the complex ( 424  nm ) and dithizone ( 434  nm ) were overlapping ( figure 1 ) .", "hence , the corrected absorbance , a \n corr , was used to overcome the problem : \n ( 1)acorr = a424(a424a592)la592 , \n where a \n 424 and a \n 592 are the absorbance of the complex measured at 424 and 592  nm , respectively , and ( a \n 424/a \n 592)l is the absorbance ratio of dithizone at 424 and 592  nm . the complex does not show any absorbance at 592  nm .", "under optimum conditions , a calibration graph was used for determination of selenium in cosmetic and pharmaceutical products .", "shampoo ( selenium sulphide , iran ) sample dissolution was prepared using the procedure described by afkhami et al . .", "approximately 1  g of shampoo sample was weighted into a 100  ml beaker . concentrated sulfuric acid ( 1  ml )", "the solution was allowed to cool , and 5  ml of 30% ( w / v ) hydrogen peroxide was added .", "the mixture boiled vigorously to eliminate excess hydrogen peroxide and allowed to cool and then the digest transferred to a 100  ml volumetric flask .", "finally , 2  ml of this solution was taken for determination of selenium ( iv ) as in the recommended procedure .  ", "a centrum tablet ( usa ) containing 0.25  mg se ( as sodium selenate ) was placed in digestion vessel", ". 5  ml of concentrated nitric acid and 2  ml of hydrogen peroxide were added and the mixture subjected to microwave irradiation ( 25% of power , 5  min ) in order to digest the sample .", "then , 10  ml of hydrochloric acid ( 6  m ) was added , and the mixture was irradiated by microwave ( 75% of power , 5  min ) in order to reduce all se to se ( iv ) .", ". 1  ml of treated sample solution was dropwise passed through an eichrom cation exchange resin ( sulfonic acid groups , mesh ranges : 50100  m , 8  cm ) , for removal of interferences , and selenium was determined with the recommended procedure .  ", "a selen plus capsule ( germany ) was dissolved in 10  ml hcl ( 6  m ) , and the mixture was irradiated by microwave ( 75% of power , 5  min ) in order to reduce all se to se ( iv ) .", "the mixture was then diluted to 50  ml with water , and then 1  ml of this mixture was taken for determination of selenium ( iv ) as in the recommended procedure ."], ["from the plot obtained by job 's method of continuous variation , the se- ( iv ) to- dithizone ( h2dz ) ratio forming the complex was found to be 1 ", ":  4 at ph  <  1 . the complex can , therefore , be represented as se(hdz)4 which is in agreement with the reported composition in the organic phase .", "the conditional stability constant of the 1  :  4 ( se  :  h2dz ) complex was calculated , assuming the following equilibrium in the system :  \n ( 2)h2seo3 + 4  h2dzse(hdz)4 + 3h2o . \n using the plot obtained by job 's method ,", "xmax  ( chdz / ctotal ) was found to be 0.8 , and therefore the ratio of se  :  hdz was calculated 1  :  4 ( figure 1 ) .", "selenium ( iv ) reacts with dithizone ( hdz ) in micellar medium and forms a hydrophobic complex , se(hdz)4 , which is subsequently trapped in the surfactant micelles and separated from the aqueous phase .  ", "figure 2 shows the absorption spectra for the dithizone and selenium - dithizonate complex in surfactant - rich phase against a reagent blank .", "the absorption maximum of the complex was 424  nm , and the absorption maxima of the dithizone were 434 and 592  nm .", "solution ph plays a unique role on metal - chelate function and subsequent extraction .  ", "figure 3 shows the influence of hcl concentration on the corrected absorbance of the selenium dithizonate complex at 424  nm .", "as can be seen , the complex extraction was increased with the increased hcl concentration and then showed a decrease .", "the reason for this behaviour might be because of decreasing the dithizone stability at higher phs . hence , an hcl concentration of 0.35  m was chosen for further studies . the influence of the dithizone ( hdz ) concentration on analytical response is shown in figure 4 .", "this figure shows that the absorbance increased up to a known concentration of dithizone ( 7.5  mol  l ) and then reached a plateau that can be attributed to complete extraction .", "therefore , the concentration of 7.5  mol  l of dithizone was selected as the optimum .  ", "the nonionic surfactant triton x-100 was chosen because of its commercial availability of high purified homogeneous form , low toxicological properties , and cost .", "the cloud point temperature of triton x-100 is 63.7c . to decrease the cloud point temperature of triton x-100 to room temperature ( desirable temperature in cloud point procedure ) ,", "this solution was prepared by the diluent ( solution of 0.5% ( w / v ) of phenol ) .", "the variation of absorbance at  \n max of complex as a function of the concentration of triton x-100 is shown in figure 5 . at lower concentrations of triton x-100", ", the low extraction efficiency of the complex may be due to the inadequacy of the assemblies to entrap the hydrophobic complex quantitatively .", "the concentration of 0.2% ( w / v ) triton x-100 was selected for further studies .", "optimal incubation time and equilibration temperature are necessary to complete the reaction , easy phase separation , and preconcentration as efficient as possible . providing that the complexation reaction has been completed under certain conditions , corrected absorbance data", "show that , for equilibration times of 10 to 30  min , extraction efficiency is almost constant .", "on the other hand , it appears that the phase volume ratio of all nonionic surfactants decreases as the equilibration temperature increases . as can be seen in figure 7 , the corrected absorbance increases very gently up to 40c and starts to decrease afterwards due to decrease in the stability of the dithizone and consequently the extraction efficiency .", "the dependence of corrected absorbance upon centrifugation time was studied within the range of 530  min .", "it was found that a time of 15  min is adequate for separation of two phases .", "calibration graph was obtained by preconcentrating 50  ml of the sample containing 5 , 15 , 30 , 40 , 55 , 70 , 80 , and 100  ng  ml of selenium ( iv ) under the optimal experimental conditions .", "analytical parameters for the determination of the selenium - dithizonate complex are presented in table 1 .", "the relative standard deviation was 2.18% for six replicate measurements of standard selenium solution ( 50  ng  ml ) .  ", "the effect of various cations and anions on the determination of 0.05  g  ml of se(iv ) was studied , and the results are summarized in table 2 .", "the tolerance limit was taken as the concentration of added ion causing less than 5% relative error .", "cations such as ag(i ) , cu(ii ) , hg(ii ) , and fe(iii ) were interfered with the determination of se(iv ) ion .", "these cations can be removed using a cation exchange resin ( sulfonic acid groups , mesh ranges : 50100 , 8  cm ) . for this purpose , 25  ml solution containing 2.5  g of se(iv ) and various amounts of desired cations", "was passed through 5  g cation exchange resin ( 8  cm wet resin ) dropwise and collected in a 50  ml volumetric flask containing optimum amount of dithizone ( 7.5  mol  l ) , hcl ( 0.35  m ) , and triton x-100 ( 0.2% w / v ) .", "after washing the column with water ( three times , with 15  ml ) , 0.5% ( w / v ) phenol was added and the solution was diluted to the mark with water and the amount of se ( iv ) was determined using recommended procedure . to evaluate the analytical applicability ,", "the method was applied to the determination of selenium in two multivitamin tablets ( centrum and selen plus ) and a selenium sulphide shampoo ( for treatment of dandruff ) .", "the results for selenium sulphide shampoo , centrum , and selen plus multivitamins are given in tables 3 , 4 , and 5 , respectively .   in order to evaluate the accuracy of procedure", ", selenium was determined in centrum tablet ( containing 0.25  mg se as sodium selenate ) by icp - oes as reference method . in both methods the results agreed well ( table 6 ) .  ", "the limit of detection and relative standard deviation of the proposed method were compared with methods in the literature ( table 7 ) .", "as observed from the results limit of detection by the proposed method was lower than other methods ."], ["the simplicity , rapidity , and inexpensiveness of the proposed method in combination with the use of dithizone as a chromogenic reagent were utilized for selenium determination in cosmetic and pharmaceutical samples .", "precision and recovery data clearly indicated the reproducibility and accuracy of the method . in this investigation ,", "spectrophotometric determination of selenium was carried out in micellar medium without the need for an extraction step .", "because of the overlapping of absorption maximum of complex at 424  nm with absorption maximum of dithizone at 434  nm , a \n corr was applied ."]]}
{"article_id": "PMC3041200", "article_text": ["data on individuals aged 029 years diagnosed with any type of diabetes ( excluding gestational ) from 1 january 1991 to 31 december 2006 were extracted from clinical notes by a dedicated register manager and entered onto the population - based yorkshire register of diabetes in children and young people for residents of west yorkshire , which has a 97.6% level of completeness ( 1 ) .", "type of diabetes was defined in accordance with world health organization guidelines ( 10 ) as type 1 , type 2 , maturity - onset diabetes of the young ( mody ) , ", "original diagnoses of uncertain / unclassified were reclassified as type 1 for 18% ( n = 32 ) and type 2 for 11% ( n = 20 ) .", "ethnicity was assigned as south asian ( pakistani , indian , bangladeshi ) or non - south asian ( all other ethnicities ) using the south asian names analysis program ( nam pehchan ) and the south asian name and group recognition algorithm ( sangra ) , two independent name recognition programs that are validated and sensitive tools for identifying south asians ( 11,12 ) . where the programs disagreed ( < 1% ) , a local expert examined the names and assigned ethnicity .", "incidence was defined as the number of newly diagnosed cases per calendar year and 100,000 population at risk .", "populations for west yorkshire were provided by the office for national statistics , supplemented by estimated midyear population denominators by age , sex , and ethnic group .", "effects on incidence of age at diagnosis ( 5-year groups ) , sex , ethnicity ( south asian or not ) , and year of diagnosis on all types , type 1 , type 2 and uncertain / unclassified diabetes were analyzed using poisson regression .", "average annual percentage change ( aapc ) and 95% cis were derived using the coefficient for year in the poisson model .", "all analyses were done in stata 11 software ( statacorp lp , college station , tx ) .", "diabetes was diagnosed in 2,889 children and young people , with 83% ( n = 2,410 ) type 1 , 12% ( n = 337 ) type 2 , 0.7% ( n = 19 ) mody , 0.1% ( n = 1 of each )  j-type or other , 0.1% ( n = 4 ) not recorded , and 4%", "the incidence ( 95% ci ) of all diabetes ( aged 029 years ) was 21.0 ( 20.221.7 ) per 100,000 , with no statistically significant differences between south asians and non - south asians .", "the incidence of type 1 diabetes was 17.6 ( 16.918.3 ) , with significantly higher rates in non - south asians ( 18.3 [ 18.319.0 ] ) than in south asians ( 13.1 [ 11.414.8 ] ) .", "type 2 incidence was 2.5 ( 2.22.7 ) , which was significantly lower for non - south asians ( 1.8 [ 1.62.1 ] ) than south asians ( 6.9 [ 5.68.1 ] ) .", "incidence of uncertain / unclassified diabetes was 0.8 ( 0.670.98 ) and was lower for non - south asians .", "aapc was 4.0% ( 95% ci 3.24.8 ) overall , and significantly greater in south asians ( 7.0% [ 4.89.2 ] ) compared with non - south asians ( 3.6% [ 2.74.4 ] ) ( fig . 1 ) and females ( 5.3% [ 4.16.4 ] ) compared with males ( 3.0% [ 1.94.0 ] ) . for type 1 diabetes , aapc was 1.3% ( 0.42.2 ) overall , with a significant increasing trend seen in non - south asians ( 1.4% [ 0.52.3 ] ) compared with no trend in south asians ( 0.0% [ 2.8 to 2.8 ] ) .", "annual incidence increased significantly for children ( aged 014 years ) at 2.9% ( 1.74.0 ) , but was stable for young people aged 1529 years ( 0.6% [ 1.9 to 0.7 ] ) . for type 2 ,", "the incidence in those aged younger than 30 years rose from 0.1 to 4.9 per 100,000 between 1991 and 2006 and aapc was 19.4% ( 16.522.2 ) overall , with incidence increasing at a similar rate for both ethnic groups .", "incidence increased at a faster rate for children aged 014 ( 37.4% [ 21.553.2 ] ) compared with those aged 1529", "( a high - quality color representation of this figure is available in the online issue . )", "the proportion of uncertain / unclassified cases per year rose from 0% in 1991 to 8% in 2006 .", "aapc was 26.4% ( 95% ci 20.732.1 ) overall , with similar increases for both ethnic groups .", "the incidence ( 95% ci ) of all diabetes ( aged 029 years ) was 21.0 ( 20.221.7 ) per 100,000 , with no statistically significant differences between south asians and non - south asians .", "the incidence of type 1 diabetes was 17.6 ( 16.918.3 ) , with significantly higher rates in non - south asians ( 18.3 [ 18.319.0 ] ) than in south asians ( 13.1 [ 11.414.8 ] ) .", "type 2 incidence was 2.5 ( 2.22.7 ) , which was significantly lower for non - south asians ( 1.8 [ 1.62.1 ] ) than south asians ( 6.9 [ 5.68.1 ] ) .", "incidence of uncertain / unclassified diabetes was 0.8 ( 0.670.98 ) and was lower for non - south asians .", "aapc was 4.0% ( 95% ci 3.24.8 ) overall , and significantly greater in south asians ( 7.0% [ 4.89.2 ] ) compared with non - south asians ( 3.6% [ 2.74.4 ] ) ( fig . 1 ) and females ( 5.3% [ 4.16.4 ] ) compared with males ( 3.0% [ 1.94.0 ] ) . for type 1 diabetes , aapc was 1.3% ( 0.42.2 ) overall , with a significant increasing trend seen in non - south asians ( 1.4% [ 0.52.3 ] ) compared with no trend in south asians ( 0.0% [ 2.8 to 2.8 ] ) .", "annual incidence increased significantly for children ( aged 014 years ) at 2.9% ( 1.74.0 ) , but was stable for young people aged 1529 years ( 0.6% [ 1.9 to 0.7 ] ) . for type 2 ,", "the incidence in those aged younger than 30 years rose from 0.1 to 4.9 per 100,000 between 1991 and 2006 and aapc was 19.4% ( 16.522.2 ) overall , with incidence increasing at a similar rate for both ethnic groups .", "incidence increased at a faster rate for children aged 014 ( 37.4% [ 21.553.2 ] ) compared with those aged 1529", "( a high - quality color representation of this figure is available in the online issue . ) the proportion of uncertain / unclassified cases per year rose from 0% in 1991 to 8% in 2006 .", "aapc was 26.4% ( 95% ci 20.732.1 ) overall , with similar increases for both ethnic groups .", "an increasing burden of diabetes was observed in non - south asian and south asian children and young people , with a rising incidence of type 1 diabetes confirmed in children ( 3,4 ) and stable rates for young adults ( 1,2 ) .", "although the proportion of south asians in the diabetes population ( 13% ) was similar to the background population , south asians had a lower incidence of type 1 diabetes but an excess of type 2 , with an apparent shift from diagnosis of type 1 to type 2 from 2000 onwards .", "the previously reported steep rise in incidence of type 1 diabetes in south asian children up to 1999 ( 5 ) was not sustained , possibly as a result of a change in the recognition that type 2 and other forms of diabetes are emerging in younger age groups .", "all south asian children diagnosed with type 2 diabetes between 1991 and 2006 were 10 years or older , and a fall in type 1 in those 15 to 29 years old in the second period is unlikely to be attributable to changes in diagnostic classification .", "there are clinical challenges in the differential diagnosis of type 1 and type 2 diabetes ( 13,14 ) .", "treatment can be based on symptoms with no clear diagnosis , reflected in rising numbers of uncertain / unclassified diagnoses ( two diagnoses in the first 5 years , with 91 thereafter ) .", "we acknowledge that we may have underestimated the incidence of type 2 especially in earlier years , a problem identified in an earlier survey ( 15 ) , but these observations confirm the importance of including all forms of diabetes in our analyses ."], "abstract_text": ["<S> objectiveto investigate incidence trends of all diabetes types in all children and young people and in the south asian subpopulation.research design and methodsannual incidence per 100,000 and time trends ( 19912006 ) were analyzed for 2,889 individuals aged 029 years diagnosed with diabetes while resident in west yorkshire , u.k.resultsdiagnoses comprised type 1 ( 83% ) , type 2 ( 12% ) , maturity - onset diabetes of the young ( 0.7% ) ,  </S>", "<S> j-type / other ( 0.1% ) , and uncertain / unclassified ( 4% ) . </S>", "<S> there was a lower incidence of type 1 and a threefold excess of type 2 in south asians compared with non - south asians . </S>", "<S> type 1 incidence leveled out and type 2 increased after the first south asian case of type 2 was diagnosed in 1999 . </S>", "<S> type 2 and unclassified diabetes incidence rose in all population subgroups.conclusionsthe burden of diabetes increased over time for both ethnic groups , with a significant excess of type 2 diabetes in south asians . </S>", "<S> the rising incidence of type 1 diabetes in south asians attenuated as type 2 diabetes increased after 1999 . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "RESULTS", "Incidence", "Time trends", "CONCLUSIONS"], "sections": [["data on individuals aged 029 years diagnosed with any type of diabetes ( excluding gestational ) from 1 january 1991 to 31 december 2006 were extracted from clinical notes by a dedicated register manager and entered onto the population - based yorkshire register of diabetes in children and young people for residents of west yorkshire , which has a 97.6% level of completeness ( 1 ) .", "type of diabetes was defined in accordance with world health organization guidelines ( 10 ) as type 1 , type 2 , maturity - onset diabetes of the young ( mody ) , ", "original diagnoses of uncertain / unclassified were reclassified as type 1 for 18% ( n = 32 ) and type 2 for 11% ( n = 20 ) .", "ethnicity was assigned as south asian ( pakistani , indian , bangladeshi ) or non - south asian ( all other ethnicities ) using the south asian names analysis program ( nam pehchan ) and the south asian name and group recognition algorithm ( sangra ) , two independent name recognition programs that are validated and sensitive tools for identifying south asians ( 11,12 ) . where the programs disagreed ( < 1% ) , a local expert examined the names and assigned ethnicity .", "incidence was defined as the number of newly diagnosed cases per calendar year and 100,000 population at risk .", "populations for west yorkshire were provided by the office for national statistics , supplemented by estimated midyear population denominators by age , sex , and ethnic group .", "effects on incidence of age at diagnosis ( 5-year groups ) , sex , ethnicity ( south asian or not ) , and year of diagnosis on all types , type 1 , type 2 and uncertain / unclassified diabetes were analyzed using poisson regression .", "average annual percentage change ( aapc ) and 95% cis were derived using the coefficient for year in the poisson model .", "all analyses were done in stata 11 software ( statacorp lp , college station , tx ) ."], ["diabetes was diagnosed in 2,889 children and young people , with 83% ( n = 2,410 ) type 1 , 12% ( n = 337 ) type 2 , 0.7% ( n = 19 ) mody , 0.1% ( n = 1 of each )  j-type or other , 0.1% ( n = 4 ) not recorded , and 4%", "the incidence ( 95% ci ) of all diabetes ( aged 029 years ) was 21.0 ( 20.221.7 ) per 100,000 , with no statistically significant differences between south asians and non - south asians .", "the incidence of type 1 diabetes was 17.6 ( 16.918.3 ) , with significantly higher rates in non - south asians ( 18.3 [ 18.319.0 ] ) than in south asians ( 13.1 [ 11.414.8 ] ) .", "type 2 incidence was 2.5 ( 2.22.7 ) , which was significantly lower for non - south asians ( 1.8 [ 1.62.1 ] ) than south asians ( 6.9 [ 5.68.1 ] ) .", "incidence of uncertain / unclassified diabetes was 0.8 ( 0.670.98 ) and was lower for non - south asians .", "aapc was 4.0% ( 95% ci 3.24.8 ) overall , and significantly greater in south asians ( 7.0% [ 4.89.2 ] ) compared with non - south asians ( 3.6% [ 2.74.4 ] ) ( fig . 1 ) and females ( 5.3% [ 4.16.4 ] ) compared with males ( 3.0% [ 1.94.0 ] ) . for type 1 diabetes , aapc was 1.3% ( 0.42.2 ) overall , with a significant increasing trend seen in non - south asians ( 1.4% [ 0.52.3 ] ) compared with no trend in south asians ( 0.0% [ 2.8 to 2.8 ] ) .", "annual incidence increased significantly for children ( aged 014 years ) at 2.9% ( 1.74.0 ) , but was stable for young people aged 1529 years ( 0.6% [ 1.9 to 0.7 ] ) . for type 2 ,", "the incidence in those aged younger than 30 years rose from 0.1 to 4.9 per 100,000 between 1991 and 2006 and aapc was 19.4% ( 16.522.2 ) overall , with incidence increasing at a similar rate for both ethnic groups .", "incidence increased at a faster rate for children aged 014 ( 37.4% [ 21.553.2 ] ) compared with those aged 1529", "( a high - quality color representation of this figure is available in the online issue . )", "the proportion of uncertain / unclassified cases per year rose from 0% in 1991 to 8% in 2006 .", "aapc was 26.4% ( 95% ci 20.732.1 ) overall , with similar increases for both ethnic groups ."], ["the incidence ( 95% ci ) of all diabetes ( aged 029 years ) was 21.0 ( 20.221.7 ) per 100,000 , with no statistically significant differences between south asians and non - south asians .", "the incidence of type 1 diabetes was 17.6 ( 16.918.3 ) , with significantly higher rates in non - south asians ( 18.3 [ 18.319.0 ] ) than in south asians ( 13.1 [ 11.414.8 ] ) .", "type 2 incidence was 2.5 ( 2.22.7 ) , which was significantly lower for non - south asians ( 1.8 [ 1.62.1 ] ) than south asians ( 6.9 [ 5.68.1 ] ) .", "incidence of uncertain / unclassified diabetes was 0.8 ( 0.670.98 ) and was lower for non - south asians ."], ["aapc was 4.0% ( 95% ci 3.24.8 ) overall , and significantly greater in south asians ( 7.0% [ 4.89.2 ] ) compared with non - south asians ( 3.6% [ 2.74.4 ] ) ( fig . 1 ) and females ( 5.3% [ 4.16.4 ] ) compared with males ( 3.0% [ 1.94.0 ] ) . for type 1 diabetes , aapc was 1.3% ( 0.42.2 ) overall , with a significant increasing trend seen in non - south asians ( 1.4% [ 0.52.3 ] ) compared with no trend in south asians ( 0.0% [ 2.8 to 2.8 ] ) .", "annual incidence increased significantly for children ( aged 014 years ) at 2.9% ( 1.74.0 ) , but was stable for young people aged 1529 years ( 0.6% [ 1.9 to 0.7 ] ) . for type 2 ,", "the incidence in those aged younger than 30 years rose from 0.1 to 4.9 per 100,000 between 1991 and 2006 and aapc was 19.4% ( 16.522.2 ) overall , with incidence increasing at a similar rate for both ethnic groups .", "incidence increased at a faster rate for children aged 014 ( 37.4% [ 21.553.2 ] ) compared with those aged 1529", "( a high - quality color representation of this figure is available in the online issue . ) the proportion of uncertain / unclassified cases per year rose from 0% in 1991 to 8% in 2006 .", "aapc was 26.4% ( 95% ci 20.732.1 ) overall , with similar increases for both ethnic groups ."], ["an increasing burden of diabetes was observed in non - south asian and south asian children and young people , with a rising incidence of type 1 diabetes confirmed in children ( 3,4 ) and stable rates for young adults ( 1,2 ) .", "although the proportion of south asians in the diabetes population ( 13% ) was similar to the background population , south asians had a lower incidence of type 1 diabetes but an excess of type 2 , with an apparent shift from diagnosis of type 1 to type 2 from 2000 onwards .", "the previously reported steep rise in incidence of type 1 diabetes in south asian children up to 1999 ( 5 ) was not sustained , possibly as a result of a change in the recognition that type 2 and other forms of diabetes are emerging in younger age groups .", "all south asian children diagnosed with type 2 diabetes between 1991 and 2006 were 10 years or older , and a fall in type 1 in those 15 to 29 years old in the second period is unlikely to be attributable to changes in diagnostic classification .", "there are clinical challenges in the differential diagnosis of type 1 and type 2 diabetes ( 13,14 ) .", "treatment can be based on symptoms with no clear diagnosis , reflected in rising numbers of uncertain / unclassified diagnoses ( two diagnoses in the first 5 years , with 91 thereafter ) .", "we acknowledge that we may have underestimated the incidence of type 2 especially in earlier years , a problem identified in an earlier survey ( 15 ) , but these observations confirm the importance of including all forms of diabetes in our analyses ."]]}
{"article_id": "PMC5177561", "article_text": ["familial adenomatous polyposis ( fap ) is a rare cause of colorectal cancer and is caused by a wide spectrum of mutations in the apc gene.1 \n 2 fap usually presents with a 100 to 1,000 of precancerous colonic polyps . without colectomy ,", "while pathogenic apc mutations have a complete penetrance in the colon , this is not the case in extracolonic manifestations .", "gardner and richards first described the association of fap with a variety of extracolonic tumors , especially soft tissue lesions , but also osteomas , thyroid cancer , and hepatoblastoma .", "the triad of soft tissue lesions , osteomas , and dental abnormalities in fap patients has therefore been named gardner syndrome.3 \n 4 gardner fibromas ( gf ) have been defined only recently as superficial and poorly circumscribed tumor - like lesions , which consist mainly of thick haphazardly arranged collagen bundles with few interspersed spindle cells of fibroblast type.5 \n 6 \n 7 gf are regarded as a precursor lesion of desmoid tumors which have a more cellular appearance .", "both are associated with fap ( although it is unknown whether this is always the case).8 \n 9 the most common sites are the back , paraspinal region , and chest wall , but gf may occur in any part of the body .", "gf may precede the development of colonic adenomas and therefore lead to an early detection of fap in otherwise asymptomatic patients .", "although it is most frequently observed in the first decade of life , with 78% of the cases being diagnosed before 10 years of age,9 neonatal gf has only been reported in a single case report so far.10 in our report , we relate the cases of two patients with neonatal gf and very different findings .", "a 7-week - old otherwise healthy boy of nonconsangineous parents presented with a subcutaneous tumor on the left parasternal chest wall immediately after birth .", "ultrasound was performed and showed a dense structure lying above rib level measuring 1.4    0.5 cm ( fig .", "analysis of the family history revealed no family members with adenomas , fibromas , or colorectal cancer .", "1b ) . magnetic resonance imaging ( mri ) scans at the age of 5.5 and 9 months revealed a tumor size of 4.5    4.0    1.2 and 6.2    5.6    1.8 cm , respectively .", "furthermore , the tumor had infiltrated the intercostal space and distal sternum at 9 months , eroding the bone , and was lying adjacent to the pericardium ( fig .", "a radical resection was performed with partial resection of the distal sternum , ribs 7 to 9 on the left side and the medial part of the major pectoralis muscle .", "closure of the chest wall was possible with insertion of a 5    4 cm goretexpatch ( w. l. gore & associates , inc .", "mobilization of major and minor pectoralis muscle as well as the abdominal wall muscles allowed full muscular covering and subsequent primary skin closure .", "resection margins were free of tumor except at the inferior end ( cartilaginous part of rib 10 ) .", "regular mri scans every 6 months during follow - up showed no recurrence ( fig .", "( b ) growing tumor size 5 months after first resection : 4.8    3.5    1.4 cm . \n", "h&e stain ( 40 ) shows thick haphazardly arranged collagen bundles with few interspersed spindle cells of fibroblast type .", "( image courtesy of a. kaiser , md , klinikum nrnberg , institute of pathology ) . \n", "mri scans of patient a. ( a ) tumor at the age of 9 months .", "( b ) tumor site 24 months postoperatively without recurrence . mri , magnetic resonance imaging .", "molecular genetic analysis of the apc gene by polymerase chain reaction ( pcr ) and subsequent sanger sequence analysis of all coding exons did not show any causative mutation .", "in addition , multiplex ligation - dependent probe amplification ( mlpa , kit p043 , mrc - holland , the netherlands ) analysis , which allows for the detection of potential deletions and duplications , showed a large heterozygous deletion affecting most of the apc coding region ( exons 415 [ genbank nm_000038.3 ] , equals exons 718 [ genbank nm_000038.5 ] ) up to the most distal part of the gene ( fragment w ) ( fig .", "the deletion was confirmed on a second blood sample and classified as a bona fide pathogenic alteration because of its large size . \n", "deletion / duplication analysis using multiplex ligation - dependent probe amplification showing monoallelic deletion affecting most of the apc coding region , showing that the gene dose of exon 4 onwards is reduced by 50% and proving the heterozygous deletion of a large portion of apc .", "subsequent testing of the parents showed the same apc deletion in the father who had no complaints . after having established the diagnosis he underwent colonoscopy , which led to the diagnosis of cancer of the sigmoid colon .", "anal anastomosis , the histological workup verified a pt3 , pn2 ( 11/52 ) , l1 , g2 adenocarcinoma .", "the paternal grandmother did not show the familial apc deletion , the paternal grandfather was unavailable for investigation .", "the otherwise healthy boy of nonconsangineous parents was diagnosed with a left - sided paravertebral soft tissue tumor measuring 6    3    1 cm at birth . due to", "known fap in the father the soft tissue tumor was assumed to be an extracolonic manifestation of fap . because of tumor progression during the next 4 months ( fig .", "histology confirmed gf consisting of fatty tissue interspersed with thick collagen bundles , many small blood vessels and some dispersed mast cells .", "mri scan 16 weeks after birth tumor size : 8    5 cm .", "he developed osteomas of the skull and intestinal polyps were confirmed by colonoscopy but without the necessity for surgical intervention so far .", "the father of the boy had developed clinical symptoms of fap at the age of 27 and underwent prophylactic restorative proctocolectomy and ileal pouch ", "anal anastomosis at the age of 28 . within 3 years after surgery he developed severe retroperitoneal desmoid disease leading to hydronephrosis necessitating nephroureterectomy .", "after 6 months he underwent a further desmoid tumor resection in the abdominal wall , the attempted resection of a large pelvic desmoid failed due to poor circumscription .", "molecular genetic analysis by pcr and sanger sequence analysis of all coding exons showed a heterozygous frameshift mutation c.4393_4394delag , p.ser1465trpfs*3 in exon 15 , segment h ( equals exon 18 , segment h [ genbank nm_000038.5 ] ) of the apc gene leading to introduction of a stop codon at amino acid position 1467 , thereby causing a truncated nonfunctional or functionally impaired apc protein .", "a 7-week - old otherwise healthy boy of nonconsangineous parents presented with a subcutaneous tumor on the left parasternal chest wall immediately after birth .", "ultrasound was performed and showed a dense structure lying above rib level measuring 1.4    0.5 cm ( fig .", "analysis of the family history revealed no family members with adenomas , fibromas , or colorectal cancer .", "1b ) . magnetic resonance imaging ( mri ) scans at the age of 5.5 and 9 months revealed a tumor size of 4.5    4.0    1.2 and 6.2    5.6    1.8 cm , respectively .", "furthermore , the tumor had infiltrated the intercostal space and distal sternum at 9 months , eroding the bone , and was lying adjacent to the pericardium ( fig .", "a radical resection was performed with partial resection of the distal sternum , ribs 7 to 9 on the left side and the medial part of the major pectoralis muscle .", "closure of the chest wall was possible with insertion of a 5    4 cm goretexpatch ( w. l. gore & associates , inc .", "mobilization of major and minor pectoralis muscle as well as the abdominal wall muscles allowed full muscular covering and subsequent primary skin closure .", "resection margins were free of tumor except at the inferior end ( cartilaginous part of rib 10 ) .", "regular mri scans every 6 months during follow - up showed no recurrence ( fig .", "( b ) growing tumor size 5 months after first resection : 4.8    3.5    1.4 cm . \n", "h&e stain ( 40 ) shows thick haphazardly arranged collagen bundles with few interspersed spindle cells of fibroblast type .", "( image courtesy of a. kaiser , md , klinikum nrnberg , institute of pathology ) . \n", "mri scans of patient a. ( a ) tumor at the age of 9 months .", "( b ) tumor site 24 months postoperatively without recurrence . mri , magnetic resonance imaging .", "molecular genetic analysis of the apc gene by polymerase chain reaction ( pcr ) and subsequent sanger sequence analysis of all coding exons did not show any causative mutation .", "in addition , multiplex ligation - dependent probe amplification ( mlpa , kit p043 , mrc - holland , the netherlands ) analysis , which allows for the detection of potential deletions and duplications , showed a large heterozygous deletion affecting most of the apc coding region ( exons 415 [ genbank nm_000038.3 ] , equals exons 718 [ genbank nm_000038.5 ] ) up to the most distal part of the gene ( fragment w ) ( fig .", "the deletion was confirmed on a second blood sample and classified as a bona fide pathogenic alteration because of its large size . \n", "deletion / duplication analysis using multiplex ligation - dependent probe amplification showing monoallelic deletion affecting most of the apc coding region , showing that the gene dose of exon 4 onwards is reduced by 50% and proving the heterozygous deletion of a large portion of apc .", "subsequent testing of the parents showed the same apc deletion in the father who had no complaints . after having established the diagnosis he underwent colonoscopy , which led to the diagnosis of cancer of the sigmoid colon .", "anal anastomosis , the histological workup verified a pt3 , pn2 ( 11/52 ) , l1 , g2 adenocarcinoma .", "the paternal grandmother did not show the familial apc deletion , the paternal grandfather was unavailable for investigation .", "the otherwise healthy boy of nonconsangineous parents was diagnosed with a left - sided paravertebral soft tissue tumor measuring 6    3    1 cm at birth . due to", "known fap in the father the soft tissue tumor was assumed to be an extracolonic manifestation of fap . because of tumor progression during the next 4 months ( fig .", "histology confirmed gf consisting of fatty tissue interspersed with thick collagen bundles , many small blood vessels and some dispersed mast cells .", "mri scan 16 weeks after birth tumor size : 8    5 cm .", "he developed osteomas of the skull and intestinal polyps were confirmed by colonoscopy but without the necessity for surgical intervention so far .", "the father of the boy had developed clinical symptoms of fap at the age of 27 and underwent prophylactic restorative proctocolectomy and ileal pouch ", "anal anastomosis at the age of 28 . within 3 years after surgery he developed severe retroperitoneal desmoid disease leading to hydronephrosis necessitating nephroureterectomy .", "after 6 months he underwent a further desmoid tumor resection in the abdominal wall , the attempted resection of a large pelvic desmoid failed due to poor circumscription .", "molecular genetic analysis by pcr and sanger sequence analysis of all coding exons showed a heterozygous frameshift mutation c.4393_4394delag , p.ser1465trpfs*3 in exon 15 , segment h ( equals exon 18 , segment h [ genbank nm_000038.5 ] ) of the apc gene leading to introduction of a stop codon at amino acid position 1467 , thereby causing a truncated nonfunctional or functionally impaired apc protein .", "whereas early childhood gf have been reported and the assumption has been made that there is a strong connection to fap,9 to our knowledge there is only one report of a neonatal onset of gf leading to the diagnosis of fap in an otherwise asymptomatic child.10 in this report the patient 's parents were healthy and had negative genetic testing , so that a de novo mutation is most probable . in contrast , in our patient a , testing of the parents revealed the same mutation in the father and the diagnosis of abundant intestinal polyposis and sigmoid cancer in colonoscopy .", "this is to our knowledge the first report of a neonatal gf requiring immediate total proctocolectomy in a parent .", "coffin et al noted that 29% of all patients with gf developed this lesion under the age of 1  year , with an onset at 2 months of age.9 it is possible that in this series some gf may have existed unnoticed since birth .", "although the overall ( and possibly underestimated ) association rate with fap is as high as 69% in this study , no information is available about relevance of gf as a sentinel lesion for fap in neonatal patients .", "viera et al reported two infants with gf which led to previously unrecognized fap in the families.11 in their patients , immunofluorescent staining of -catenin in the tumor , which accumulates in cells with biallelic truncation of apc because of the impairment of apc - mediated degradation of -catenin,4 led to the strong suspicion of underlying fap .", "this may well be a future diagnostic tool in evaluating the role of gf in detection of fap.12 \n  of note , in our patient a the tumor showed a rapid progression which necessitated extensive surgery , while the lesion remained stable in patient b. although the invasive rapid growth is already a sign of a desmoid tumor , it was histologically still regarded as a gf . it may be discussed if site and type of apc mutations may play a role in disease outcome , since patient a carries a large deletion encompassing most of the apc coding region , whereas patient b has an underlying mutation in the desmoid region of the apc gene.4 screening for somatic apc mutations in the tumor indicating loss of heterozygosity might have been instructive .", "the loss of the wildtype allele of apc in the tumor has been shown to be a relatively frequent event in desmoid tumors in adult fap individuals.13 unfortunately , these data are not available in our patients", ". however , it is known that trauma or surgery can be a predisposing factor to desmoid development in fap.9 therefore , the possibility that the first surgery triggered the intensive growth of the gf in patient a can not be excluded .", "if surgery is performed , a total excision of gf with a wide margin should be aimed for , but can be technically demanding or even impossible because of the poor circumscription or anatomic boundaries . in general , prediction of gf behavior", "it has been stated that approximately 5 to 10% appear to resolve spontaneously , 30% undergo cycles of progression and resolution , 50% remain stable after diagnosis , but 10% tend to progress rapidly , growing to massive sizes and infiltrating adjacent tissue.4 it is also known that there is a relationship between frequency of extracolonic manifestations and the position of apc germline mutations in patients with fap.4 \n 14 \n 15 for example , the risk of developing desmoid tumors is increased in patients with an apc mutation 3 to codon 1399,16 and reaches up to 100% in patients with a mutation in codons 1395 to 1493.17 studies correlating the risk of developing gf with a particular genotype are not available .", "nevertheless , surgery is the first - line therapy in fast - growing gf . in older patients with advanced cases with progression into desmoid tumor cytotoxic chemotherapy", "may be discussed , but was not considered promising in our case because of the young age and the gf still being considered a benign entity.18 \n 19 \n  continuous follow - up is mandatory , since early onset of intestinal polyps in children from the age of 4 to 6 years has been reported.20 \n 21 \n 22 in general , routine colonoscopies have been suggested to start at the age of 10 .", "because of the unusual early clinical onset of intestinal symptoms ( bloody stools and  >  75 polyps in colonoscopy ) at the age of 4 years in a previously reported patient with neonatal gf , we recommend early annual routine colonoscopy in patient a starting from the age of 2 years.10 \n  since approximately 25% of fap cases are caused by de novo mutations , the development of a gf in a child must raise suspicion of an underlying fap even if the family history is negative . performing a sound genetic testing for fap including sequencing of the complete coding region of the apc gene and quantitative analysis by , for example , mlpa in every child with gf", "continuous follow - up is mandatory because of unpredictable growth behavior and early onset of intestinal polyps ."], "abstract_text": ["<S> gardner fibromas ( gfs ) have only recently been described as poorly circumscribed tumor - like lesions , which are exceedingly rare in children . </S>", "<S> gfs are associated with apc gene mutations and therefore with familial adenomatous polyposis ( fap ) . </S>", "<S> so far there is only very limited literature on gf in the neonatal period . </S>", "<S> we present two children with gf diagnosed at birth and subsequent fap with very different clinical courses . in one case , the disease led to extensive surgery of the thoracic wall and detection of fap in the father with the need of immediate proctocolectomy . in the other patient ( with a positive family history for fap ) the disease remained stable . </S>", "<S> our cases indicate that the diagnosis of gf in the neonatal period requires the exclusion of fap both in the child as well as the parents . </S>", "<S> since the clinical behavior of gf can not be predicted , continuous monitoring is mandatory . </S>", "<S> depending on tumor site and growth , individual therapeutic options must be thoroughly considered . </S>", "<S> surgical resection , if necessary , has to be inevitably total to prevent recurrence . </S>"], "labels": null, "section_names": ["Introduction", "Patient Information", "Patient A", "Patient B", "Discussion", "New Insights and Importance for the Pediatric Surgeon"], "sections": [["familial adenomatous polyposis ( fap ) is a rare cause of colorectal cancer and is caused by a wide spectrum of mutations in the apc gene.1 \n 2 fap usually presents with a 100 to 1,000 of precancerous colonic polyps . without colectomy ,", "while pathogenic apc mutations have a complete penetrance in the colon , this is not the case in extracolonic manifestations .", "gardner and richards first described the association of fap with a variety of extracolonic tumors , especially soft tissue lesions , but also osteomas , thyroid cancer , and hepatoblastoma .", "the triad of soft tissue lesions , osteomas , and dental abnormalities in fap patients has therefore been named gardner syndrome.3 \n 4 gardner fibromas ( gf ) have been defined only recently as superficial and poorly circumscribed tumor - like lesions , which consist mainly of thick haphazardly arranged collagen bundles with few interspersed spindle cells of fibroblast type.5 \n 6 \n 7 gf are regarded as a precursor lesion of desmoid tumors which have a more cellular appearance .", "both are associated with fap ( although it is unknown whether this is always the case).8 \n 9 the most common sites are the back , paraspinal region , and chest wall , but gf may occur in any part of the body .", "gf may precede the development of colonic adenomas and therefore lead to an early detection of fap in otherwise asymptomatic patients .", "although it is most frequently observed in the first decade of life , with 78% of the cases being diagnosed before 10 years of age,9 neonatal gf has only been reported in a single case report so far.10 in our report , we relate the cases of two patients with neonatal gf and very different findings ."], ["a 7-week - old otherwise healthy boy of nonconsangineous parents presented with a subcutaneous tumor on the left parasternal chest wall immediately after birth .", "ultrasound was performed and showed a dense structure lying above rib level measuring 1.4    0.5 cm ( fig .", "analysis of the family history revealed no family members with adenomas , fibromas , or colorectal cancer .", "1b ) . magnetic resonance imaging ( mri ) scans at the age of 5.5 and 9 months revealed a tumor size of 4.5    4.0    1.2 and 6.2    5.6    1.8 cm , respectively .", "furthermore , the tumor had infiltrated the intercostal space and distal sternum at 9 months , eroding the bone , and was lying adjacent to the pericardium ( fig .", "a radical resection was performed with partial resection of the distal sternum , ribs 7 to 9 on the left side and the medial part of the major pectoralis muscle .", "closure of the chest wall was possible with insertion of a 5    4 cm goretexpatch ( w. l. gore & associates , inc .", "mobilization of major and minor pectoralis muscle as well as the abdominal wall muscles allowed full muscular covering and subsequent primary skin closure .", "resection margins were free of tumor except at the inferior end ( cartilaginous part of rib 10 ) .", "regular mri scans every 6 months during follow - up showed no recurrence ( fig .", "( b ) growing tumor size 5 months after first resection : 4.8    3.5    1.4 cm . \n", "h&e stain ( 40 ) shows thick haphazardly arranged collagen bundles with few interspersed spindle cells of fibroblast type .", "( image courtesy of a. kaiser , md , klinikum nrnberg , institute of pathology ) . \n", "mri scans of patient a. ( a ) tumor at the age of 9 months .", "( b ) tumor site 24 months postoperatively without recurrence . mri , magnetic resonance imaging .", "molecular genetic analysis of the apc gene by polymerase chain reaction ( pcr ) and subsequent sanger sequence analysis of all coding exons did not show any causative mutation .", "in addition , multiplex ligation - dependent probe amplification ( mlpa , kit p043 , mrc - holland , the netherlands ) analysis , which allows for the detection of potential deletions and duplications , showed a large heterozygous deletion affecting most of the apc coding region ( exons 415 [ genbank nm_000038.3 ] , equals exons 718 [ genbank nm_000038.5 ] ) up to the most distal part of the gene ( fragment w ) ( fig .", "the deletion was confirmed on a second blood sample and classified as a bona fide pathogenic alteration because of its large size . \n", "deletion / duplication analysis using multiplex ligation - dependent probe amplification showing monoallelic deletion affecting most of the apc coding region , showing that the gene dose of exon 4 onwards is reduced by 50% and proving the heterozygous deletion of a large portion of apc .", "subsequent testing of the parents showed the same apc deletion in the father who had no complaints . after having established the diagnosis he underwent colonoscopy , which led to the diagnosis of cancer of the sigmoid colon .", "anal anastomosis , the histological workup verified a pt3 , pn2 ( 11/52 ) , l1 , g2 adenocarcinoma .", "the paternal grandmother did not show the familial apc deletion , the paternal grandfather was unavailable for investigation .", "the otherwise healthy boy of nonconsangineous parents was diagnosed with a left - sided paravertebral soft tissue tumor measuring 6    3    1 cm at birth . due to", "known fap in the father the soft tissue tumor was assumed to be an extracolonic manifestation of fap . because of tumor progression during the next 4 months ( fig .", "histology confirmed gf consisting of fatty tissue interspersed with thick collagen bundles , many small blood vessels and some dispersed mast cells .", "mri scan 16 weeks after birth tumor size : 8    5 cm .", "he developed osteomas of the skull and intestinal polyps were confirmed by colonoscopy but without the necessity for surgical intervention so far .", "the father of the boy had developed clinical symptoms of fap at the age of 27 and underwent prophylactic restorative proctocolectomy and ileal pouch ", "anal anastomosis at the age of 28 . within 3 years after surgery he developed severe retroperitoneal desmoid disease leading to hydronephrosis necessitating nephroureterectomy .", "after 6 months he underwent a further desmoid tumor resection in the abdominal wall , the attempted resection of a large pelvic desmoid failed due to poor circumscription .", "molecular genetic analysis by pcr and sanger sequence analysis of all coding exons showed a heterozygous frameshift mutation c.4393_4394delag , p.ser1465trpfs*3 in exon 15 , segment h ( equals exon 18 , segment h [ genbank nm_000038.5 ] ) of the apc gene leading to introduction of a stop codon at amino acid position 1467 , thereby causing a truncated nonfunctional or functionally impaired apc protein ."], ["a 7-week - old otherwise healthy boy of nonconsangineous parents presented with a subcutaneous tumor on the left parasternal chest wall immediately after birth .", "ultrasound was performed and showed a dense structure lying above rib level measuring 1.4    0.5 cm ( fig .", "analysis of the family history revealed no family members with adenomas , fibromas , or colorectal cancer .", "1b ) . magnetic resonance imaging ( mri ) scans at the age of 5.5 and 9 months revealed a tumor size of 4.5    4.0    1.2 and 6.2    5.6    1.8 cm , respectively .", "furthermore , the tumor had infiltrated the intercostal space and distal sternum at 9 months , eroding the bone , and was lying adjacent to the pericardium ( fig .", "a radical resection was performed with partial resection of the distal sternum , ribs 7 to 9 on the left side and the medial part of the major pectoralis muscle .", "closure of the chest wall was possible with insertion of a 5    4 cm goretexpatch ( w. l. gore & associates , inc .", "mobilization of major and minor pectoralis muscle as well as the abdominal wall muscles allowed full muscular covering and subsequent primary skin closure .", "resection margins were free of tumor except at the inferior end ( cartilaginous part of rib 10 ) .", "regular mri scans every 6 months during follow - up showed no recurrence ( fig .", "( b ) growing tumor size 5 months after first resection : 4.8    3.5    1.4 cm . \n", "h&e stain ( 40 ) shows thick haphazardly arranged collagen bundles with few interspersed spindle cells of fibroblast type .", "( image courtesy of a. kaiser , md , klinikum nrnberg , institute of pathology ) . \n", "mri scans of patient a. ( a ) tumor at the age of 9 months .", "( b ) tumor site 24 months postoperatively without recurrence . mri , magnetic resonance imaging .", "molecular genetic analysis of the apc gene by polymerase chain reaction ( pcr ) and subsequent sanger sequence analysis of all coding exons did not show any causative mutation .", "in addition , multiplex ligation - dependent probe amplification ( mlpa , kit p043 , mrc - holland , the netherlands ) analysis , which allows for the detection of potential deletions and duplications , showed a large heterozygous deletion affecting most of the apc coding region ( exons 415 [ genbank nm_000038.3 ] , equals exons 718 [ genbank nm_000038.5 ] ) up to the most distal part of the gene ( fragment w ) ( fig .", "the deletion was confirmed on a second blood sample and classified as a bona fide pathogenic alteration because of its large size . \n", "deletion / duplication analysis using multiplex ligation - dependent probe amplification showing monoallelic deletion affecting most of the apc coding region , showing that the gene dose of exon 4 onwards is reduced by 50% and proving the heterozygous deletion of a large portion of apc .", "subsequent testing of the parents showed the same apc deletion in the father who had no complaints . after having established the diagnosis he underwent colonoscopy , which led to the diagnosis of cancer of the sigmoid colon .", "anal anastomosis , the histological workup verified a pt3 , pn2 ( 11/52 ) , l1 , g2 adenocarcinoma .", "the paternal grandmother did not show the familial apc deletion , the paternal grandfather was unavailable for investigation ."], ["the otherwise healthy boy of nonconsangineous parents was diagnosed with a left - sided paravertebral soft tissue tumor measuring 6    3    1 cm at birth . due to", "known fap in the father the soft tissue tumor was assumed to be an extracolonic manifestation of fap . because of tumor progression during the next 4 months ( fig .", "histology confirmed gf consisting of fatty tissue interspersed with thick collagen bundles , many small blood vessels and some dispersed mast cells .", "mri scan 16 weeks after birth tumor size : 8    5 cm .", "he developed osteomas of the skull and intestinal polyps were confirmed by colonoscopy but without the necessity for surgical intervention so far .", "the father of the boy had developed clinical symptoms of fap at the age of 27 and underwent prophylactic restorative proctocolectomy and ileal pouch ", "anal anastomosis at the age of 28 . within 3 years after surgery he developed severe retroperitoneal desmoid disease leading to hydronephrosis necessitating nephroureterectomy .", "after 6 months he underwent a further desmoid tumor resection in the abdominal wall , the attempted resection of a large pelvic desmoid failed due to poor circumscription .", "molecular genetic analysis by pcr and sanger sequence analysis of all coding exons showed a heterozygous frameshift mutation c.4393_4394delag , p.ser1465trpfs*3 in exon 15 , segment h ( equals exon 18 , segment h [ genbank nm_000038.5 ] ) of the apc gene leading to introduction of a stop codon at amino acid position 1467 , thereby causing a truncated nonfunctional or functionally impaired apc protein ."], ["whereas early childhood gf have been reported and the assumption has been made that there is a strong connection to fap,9 to our knowledge there is only one report of a neonatal onset of gf leading to the diagnosis of fap in an otherwise asymptomatic child.10 in this report the patient 's parents were healthy and had negative genetic testing , so that a de novo mutation is most probable . in contrast , in our patient a , testing of the parents revealed the same mutation in the father and the diagnosis of abundant intestinal polyposis and sigmoid cancer in colonoscopy .", "this is to our knowledge the first report of a neonatal gf requiring immediate total proctocolectomy in a parent .", "coffin et al noted that 29% of all patients with gf developed this lesion under the age of 1  year , with an onset at 2 months of age.9 it is possible that in this series some gf may have existed unnoticed since birth .", "although the overall ( and possibly underestimated ) association rate with fap is as high as 69% in this study , no information is available about relevance of gf as a sentinel lesion for fap in neonatal patients .", "viera et al reported two infants with gf which led to previously unrecognized fap in the families.11 in their patients , immunofluorescent staining of -catenin in the tumor , which accumulates in cells with biallelic truncation of apc because of the impairment of apc - mediated degradation of -catenin,4 led to the strong suspicion of underlying fap .", "this may well be a future diagnostic tool in evaluating the role of gf in detection of fap.12 \n  of note , in our patient a the tumor showed a rapid progression which necessitated extensive surgery , while the lesion remained stable in patient b. although the invasive rapid growth is already a sign of a desmoid tumor , it was histologically still regarded as a gf . it may be discussed if site and type of apc mutations may play a role in disease outcome , since patient a carries a large deletion encompassing most of the apc coding region , whereas patient b has an underlying mutation in the desmoid region of the apc gene.4 screening for somatic apc mutations in the tumor indicating loss of heterozygosity might have been instructive .", "the loss of the wildtype allele of apc in the tumor has been shown to be a relatively frequent event in desmoid tumors in adult fap individuals.13 unfortunately , these data are not available in our patients", ". however , it is known that trauma or surgery can be a predisposing factor to desmoid development in fap.9 therefore , the possibility that the first surgery triggered the intensive growth of the gf in patient a can not be excluded .", "if surgery is performed , a total excision of gf with a wide margin should be aimed for , but can be technically demanding or even impossible because of the poor circumscription or anatomic boundaries . in general , prediction of gf behavior", "it has been stated that approximately 5 to 10% appear to resolve spontaneously , 30% undergo cycles of progression and resolution , 50% remain stable after diagnosis , but 10% tend to progress rapidly , growing to massive sizes and infiltrating adjacent tissue.4 it is also known that there is a relationship between frequency of extracolonic manifestations and the position of apc germline mutations in patients with fap.4 \n 14 \n 15 for example , the risk of developing desmoid tumors is increased in patients with an apc mutation 3 to codon 1399,16 and reaches up to 100% in patients with a mutation in codons 1395 to 1493.17 studies correlating the risk of developing gf with a particular genotype are not available .", "nevertheless , surgery is the first - line therapy in fast - growing gf . in older patients with advanced cases with progression into desmoid tumor cytotoxic chemotherapy", "may be discussed , but was not considered promising in our case because of the young age and the gf still being considered a benign entity.18 \n 19 \n  continuous follow - up is mandatory , since early onset of intestinal polyps in children from the age of 4 to 6 years has been reported.20 \n 21 \n 22 in general , routine colonoscopies have been suggested to start at the age of 10 .", "because of the unusual early clinical onset of intestinal symptoms ( bloody stools and  >  75 polyps in colonoscopy ) at the age of 4 years in a previously reported patient with neonatal gf , we recommend early annual routine colonoscopy in patient a starting from the age of 2 years.10 \n  since approximately 25% of fap cases are caused by de novo mutations , the development of a gf in a child must raise suspicion of an underlying fap even if the family history is negative . performing a sound genetic testing for fap including sequencing of the complete coding region of the apc gene and quantitative analysis by , for example , mlpa in every child with gf"], ["continuous follow - up is mandatory because of unpredictable growth behavior and early onset of intestinal polyps ."]]}
{"article_id": "PMC3841566", "article_text": ["down 's syndrome ( ds ) is one of the common chromosomal disorders and the most common cause of mental retardation .", "it is well - known that patients with ds have an increased prevalence of autoimmune disorders affecting both endocrine and non - endocrine organs .", "although cases of ds associated with diabetes mellitus ( dm ) have been reported in the past , invariably all cases were associated with type 1 dm .", "we present two cases of type 2 dm with ds and believe that this is the first report of type dm with ds .", "a 28-year - old male patient with a body mass index ( bmi ) of 25.1 kg / m and positive family history of dm reported to the out - patient clinic of the department of endocrinology , medwin hospital with the clinical features of polyuria and polydipsia for last 1 year .", "physical examination revealed typical mongolian facies and other features of ds such as short stature ( 145 cm ) , brachycephaly , short neck and pot belly , small mouth with protruding tongue , wide occipital region and characteristic small eyes of ds .", "laboratory data revealed diabetes with fasting plasma glucose of 256 mg / dl , postprandial plasma glucose of 375 mg / dl and glycated hemoglobin ( hba1c ) level of 9.9% .", "c - peptide assay showed fairly well - preserved pancreatic  cell function : fasting values of 1.3 pmol and stimulated values of 2.4 pmol / ml ( normal values in non - diabetic subjects , fasting > 1.5 pmol / ml and stimulated > 4.0 pmol / ml ) .", "glutamic acid decarboxylase ( gad ) antibody testing ( 3.2 iu / ml ) was negative .", "other investigations ( hemogram , renal and liver function parameters , serum electrolytes and lipid profile ) were within the normal limits .", "he was initiated on basal glargine insulin at a dose of 15 units subcutaneously at night along with oral hypoglycemic agent ( glimepiride 2 mg / metformin 1000 mg ) once daily and l - thyroxine 100 g daily .", "later , the doses of insulin were reduced and subsequently withdrawn completely and he is maintained well on oral hypoglycemic agents alone . a 26-year - old female patient with a bmi of 33.4 kg / m with strong family history of dm", "were admitted in the in - patient department of endocrinology , medwin hospital for proper control of hyperglycemia .", "physical examination revealed features of ds ( short stature , obesity , brachycephaly , gynecomastia , protruding tongue , simian crease , pot belly , short neck and acanthosis nigricans , wide occipital region with characteristic small eyes ) .", "the laboratory evaluation revealed fasting and postprandial sugar levels as 175 mg / dl and 240 mg / dl , respectively hba1c of 8.9% .", "his fasting and stimulated c - peptide levels were 1.4 pmol / ml and 2.5 pmol / ml with negative gad - antibody test ( 4.1 iu / ml ) .", "a 28-year - old male patient with a body mass index ( bmi ) of 25.1 kg / m and positive family history of dm reported to the out - patient clinic of the department of endocrinology , medwin hospital with the clinical features of polyuria and polydipsia for last 1 year .", "physical examination revealed typical mongolian facies and other features of ds such as short stature ( 145 cm ) , brachycephaly , short neck and pot belly , small mouth with protruding tongue , wide occipital region and characteristic small eyes of ds .", "laboratory data revealed diabetes with fasting plasma glucose of 256 mg / dl , postprandial plasma glucose of 375 mg / dl and glycated hemoglobin ( hba1c ) level of 9.9% .", "c - peptide assay showed fairly well - preserved pancreatic  cell function : fasting values of 1.3 pmol and stimulated values of 2.4 pmol / ml ( normal values in non - diabetic subjects , fasting > 1.5 pmol / ml and stimulated > 4.0 pmol / ml ) .", "glutamic acid decarboxylase ( gad ) antibody testing ( 3.2 iu / ml ) was negative .", "other investigations ( hemogram , renal and liver function parameters , serum electrolytes and lipid profile ) were within the normal limits .", "he was initiated on basal glargine insulin at a dose of 15 units subcutaneously at night along with oral hypoglycemic agent ( glimepiride 2 mg / metformin 1000 mg ) once daily and l - thyroxine 100 g daily .", "later , the doses of insulin were reduced and subsequently withdrawn completely and he is maintained well on oral hypoglycemic agents alone . a 26-year - old female patient with a bmi of 33.4 kg / m with strong family history of dm", "were admitted in the in - patient department of endocrinology , medwin hospital for proper control of hyperglycemia .", "physical examination revealed features of ds ( short stature , obesity , brachycephaly , gynecomastia , protruding tongue , simian crease , pot belly , short neck and acanthosis nigricans , wide occipital region with characteristic small eyes ) .", "the laboratory evaluation revealed fasting and postprandial sugar levels as 175 mg / dl and 240 mg / dl , respectively hba1c of 8.9% .", "his fasting and stimulated c - peptide levels were 1.4 pmol / ml and 2.5 pmol / ml with negative gad - antibody test ( 4.1 iu / ml ) .", "a 28-year - old male patient with a body mass index ( bmi ) of 25.1 kg / m and positive family history of dm reported to the out - patient clinic of the department of endocrinology , medwin hospital with the clinical features of polyuria and polydipsia for last 1 year .", "physical examination revealed typical mongolian facies and other features of ds such as short stature ( 145 cm ) , brachycephaly , short neck and pot belly , small mouth with protruding tongue , wide occipital region and characteristic small eyes of ds .", "laboratory data revealed diabetes with fasting plasma glucose of 256 mg / dl , postprandial plasma glucose of 375 mg / dl and glycated hemoglobin ( hba1c ) level of 9.9% .", "c - peptide assay showed fairly well - preserved pancreatic  cell function : fasting values of 1.3 pmol and stimulated values of 2.4 pmol / ml ( normal values in non - diabetic subjects , fasting > 1.5 pmol / ml and stimulated > 4.0 pmol / ml ) .", "glutamic acid decarboxylase ( gad ) antibody testing ( 3.2 iu / ml ) was negative .", "other investigations ( hemogram , renal and liver function parameters , serum electrolytes and lipid profile ) were within the normal limits .", "he was initiated on basal glargine insulin at a dose of 15 units subcutaneously at night along with oral hypoglycemic agent ( glimepiride 2 mg / metformin 1000 mg ) once daily and l - thyroxine 100 g daily .", "later , the doses of insulin were reduced and subsequently withdrawn completely and he is maintained well on oral hypoglycemic agents alone .", "a 26-year - old female patient with a bmi of 33.4 kg / m with strong family history of dm were admitted in the in - patient department of endocrinology , medwin hospital for proper control of hyperglycemia .", "physical examination revealed features of ds ( short stature , obesity , brachycephaly , gynecomastia , protruding tongue , simian crease , pot belly , short neck and acanthosis nigricans , wide occipital region with characteristic small eyes ) .", "the laboratory evaluation revealed fasting and postprandial sugar levels as 175 mg / dl and 240 mg / dl , respectively hba1c of 8.9% .", "his fasting and stimulated c - peptide levels were 1.4 pmol / ml and 2.5 pmol / ml with negative gad - antibody test ( 4.1 iu / ml ) .", "previous studies had suggested that type 1 dm is more prevalent in people with ds than in the general population and vice versa . to the best of our knowledge ,", "impaired  cell function and insulin sensitivity are the main factors in the pathogenesis of type 2 dm .", " cell function was fairly preserved in these two patients ( as indicated by c - peptide levels in the blood ) with negative antibody testing for gad - ab .", "this indicated toward the fact that impaired insulin sensitivity might have contributed to a greater extent in the development of type 2 dm in these two patients .", "a variety of genetic syndromes have been described in which dm occurs with increased frequency .", "the etiology of the disturbance in glucose homeostasis in these diverse and unrelated syndromes remain undefined .", "since rare forms a disease often provide insight into the possible mechanisms of the disease and since each candidate gene contributes a small amount of genetic risk to the disease , individuals with ds and type 2 dm may therefore provide the clinical insights into possible mechanisms underlying susceptibility to diabetes .", "further investigations are warranted to prove whether the presence of type diabetes in patients with ds was a chance association or whether there is a genetic basis for this association . however , these two cases point to the fact that one should screen patients with ds for type 2 diabetes ."], "abstract_text": ["<S> down 's syndrome ( ds ) is known to be associated with autoimmune disease including type 1 diabetes . to the best of our knowledge , </S>", "<S> there are no reports of ds with type 2 diabetes mellitus in the literature . </S>", "<S> we hereby report two cases of ds with type 2 diabetes . </S>"], "labels": null, "section_names": ["Introduction", "Case Reports", "None", "Case 1", "Case 2", "Discussion", "Conclusion"], "sections": [["down 's syndrome ( ds ) is one of the common chromosomal disorders and the most common cause of mental retardation .", "it is well - known that patients with ds have an increased prevalence of autoimmune disorders affecting both endocrine and non - endocrine organs .", "although cases of ds associated with diabetes mellitus ( dm ) have been reported in the past , invariably all cases were associated with type 1 dm .", "we present two cases of type 2 dm with ds and believe that this is the first report of type dm with ds ."], ["a 28-year - old male patient with a body mass index ( bmi ) of 25.1 kg / m and positive family history of dm reported to the out - patient clinic of the department of endocrinology , medwin hospital with the clinical features of polyuria and polydipsia for last 1 year .", "physical examination revealed typical mongolian facies and other features of ds such as short stature ( 145 cm ) , brachycephaly , short neck and pot belly , small mouth with protruding tongue , wide occipital region and characteristic small eyes of ds .", "laboratory data revealed diabetes with fasting plasma glucose of 256 mg / dl , postprandial plasma glucose of 375 mg / dl and glycated hemoglobin ( hba1c ) level of 9.9% .", "c - peptide assay showed fairly well - preserved pancreatic  cell function : fasting values of 1.3 pmol and stimulated values of 2.4 pmol / ml ( normal values in non - diabetic subjects , fasting > 1.5 pmol / ml and stimulated > 4.0 pmol / ml ) .", "glutamic acid decarboxylase ( gad ) antibody testing ( 3.2 iu / ml ) was negative .", "other investigations ( hemogram , renal and liver function parameters , serum electrolytes and lipid profile ) were within the normal limits .", "he was initiated on basal glargine insulin at a dose of 15 units subcutaneously at night along with oral hypoglycemic agent ( glimepiride 2 mg / metformin 1000 mg ) once daily and l - thyroxine 100 g daily .", "later , the doses of insulin were reduced and subsequently withdrawn completely and he is maintained well on oral hypoglycemic agents alone . a 26-year - old female patient with a bmi of 33.4 kg / m with strong family history of dm", "were admitted in the in - patient department of endocrinology , medwin hospital for proper control of hyperglycemia .", "physical examination revealed features of ds ( short stature , obesity , brachycephaly , gynecomastia , protruding tongue , simian crease , pot belly , short neck and acanthosis nigricans , wide occipital region with characteristic small eyes ) .", "the laboratory evaluation revealed fasting and postprandial sugar levels as 175 mg / dl and 240 mg / dl , respectively hba1c of 8.9% .", "his fasting and stimulated c - peptide levels were 1.4 pmol / ml and 2.5 pmol / ml with negative gad - antibody test ( 4.1 iu / ml ) ."], ["a 28-year - old male patient with a body mass index ( bmi ) of 25.1 kg / m and positive family history of dm reported to the out - patient clinic of the department of endocrinology , medwin hospital with the clinical features of polyuria and polydipsia for last 1 year .", "physical examination revealed typical mongolian facies and other features of ds such as short stature ( 145 cm ) , brachycephaly , short neck and pot belly , small mouth with protruding tongue , wide occipital region and characteristic small eyes of ds .", "laboratory data revealed diabetes with fasting plasma glucose of 256 mg / dl , postprandial plasma glucose of 375 mg / dl and glycated hemoglobin ( hba1c ) level of 9.9% .", "c - peptide assay showed fairly well - preserved pancreatic  cell function : fasting values of 1.3 pmol and stimulated values of 2.4 pmol / ml ( normal values in non - diabetic subjects , fasting > 1.5 pmol / ml and stimulated > 4.0 pmol / ml ) .", "glutamic acid decarboxylase ( gad ) antibody testing ( 3.2 iu / ml ) was negative .", "other investigations ( hemogram , renal and liver function parameters , serum electrolytes and lipid profile ) were within the normal limits .", "he was initiated on basal glargine insulin at a dose of 15 units subcutaneously at night along with oral hypoglycemic agent ( glimepiride 2 mg / metformin 1000 mg ) once daily and l - thyroxine 100 g daily .", "later , the doses of insulin were reduced and subsequently withdrawn completely and he is maintained well on oral hypoglycemic agents alone . a 26-year - old female patient with a bmi of 33.4 kg / m with strong family history of dm", "were admitted in the in - patient department of endocrinology , medwin hospital for proper control of hyperglycemia .", "physical examination revealed features of ds ( short stature , obesity , brachycephaly , gynecomastia , protruding tongue , simian crease , pot belly , short neck and acanthosis nigricans , wide occipital region with characteristic small eyes ) .", "the laboratory evaluation revealed fasting and postprandial sugar levels as 175 mg / dl and 240 mg / dl , respectively hba1c of 8.9% .", "his fasting and stimulated c - peptide levels were 1.4 pmol / ml and 2.5 pmol / ml with negative gad - antibody test ( 4.1 iu / ml ) ."], ["a 28-year - old male patient with a body mass index ( bmi ) of 25.1 kg / m and positive family history of dm reported to the out - patient clinic of the department of endocrinology , medwin hospital with the clinical features of polyuria and polydipsia for last 1 year .", "physical examination revealed typical mongolian facies and other features of ds such as short stature ( 145 cm ) , brachycephaly , short neck and pot belly , small mouth with protruding tongue , wide occipital region and characteristic small eyes of ds .", "laboratory data revealed diabetes with fasting plasma glucose of 256 mg / dl , postprandial plasma glucose of 375 mg / dl and glycated hemoglobin ( hba1c ) level of 9.9% .", "c - peptide assay showed fairly well - preserved pancreatic  cell function : fasting values of 1.3 pmol and stimulated values of 2.4 pmol / ml ( normal values in non - diabetic subjects , fasting > 1.5 pmol / ml and stimulated > 4.0 pmol / ml ) .", "glutamic acid decarboxylase ( gad ) antibody testing ( 3.2 iu / ml ) was negative .", "other investigations ( hemogram , renal and liver function parameters , serum electrolytes and lipid profile ) were within the normal limits .", "he was initiated on basal glargine insulin at a dose of 15 units subcutaneously at night along with oral hypoglycemic agent ( glimepiride 2 mg / metformin 1000 mg ) once daily and l - thyroxine 100 g daily .", "later , the doses of insulin were reduced and subsequently withdrawn completely and he is maintained well on oral hypoglycemic agents alone ."], ["a 26-year - old female patient with a bmi of 33.4 kg / m with strong family history of dm were admitted in the in - patient department of endocrinology , medwin hospital for proper control of hyperglycemia .", "physical examination revealed features of ds ( short stature , obesity , brachycephaly , gynecomastia , protruding tongue , simian crease , pot belly , short neck and acanthosis nigricans , wide occipital region with characteristic small eyes ) .", "the laboratory evaluation revealed fasting and postprandial sugar levels as 175 mg / dl and 240 mg / dl , respectively hba1c of 8.9% .", "his fasting and stimulated c - peptide levels were 1.4 pmol / ml and 2.5 pmol / ml with negative gad - antibody test ( 4.1 iu / ml ) ."], ["previous studies had suggested that type 1 dm is more prevalent in people with ds than in the general population and vice versa . to the best of our knowledge ,", "impaired  cell function and insulin sensitivity are the main factors in the pathogenesis of type 2 dm .", " cell function was fairly preserved in these two patients ( as indicated by c - peptide levels in the blood ) with negative antibody testing for gad - ab .", "this indicated toward the fact that impaired insulin sensitivity might have contributed to a greater extent in the development of type 2 dm in these two patients .", "a variety of genetic syndromes have been described in which dm occurs with increased frequency .", "the etiology of the disturbance in glucose homeostasis in these diverse and unrelated syndromes remain undefined .", "since rare forms a disease often provide insight into the possible mechanisms of the disease and since each candidate gene contributes a small amount of genetic risk to the disease , individuals with ds and type 2 dm may therefore provide the clinical insights into possible mechanisms underlying susceptibility to diabetes ."], ["further investigations are warranted to prove whether the presence of type diabetes in patients with ds was a chance association or whether there is a genetic basis for this association . however , these two cases point to the fact that one should screen patients with ds for type 2 diabetes ."]]}
{"article_id": "PMC5155712", "article_text": ["recent progress in our understanding of brain development has significantly altered concepts for treating neurodegenerative diseases , including those that affect the retina to cause blindness .", "contrary to previous thought , it is now recognized that neurons are generated in the sub ventricular zone ( svz ) of the lateral ventricle , and the sub - granular zone ( sgz ) in the hippocampus from neural stem cells ( nsc ) of glial origin throughout life ( 1 ) . outside these discrete regions in the mammalian cns , including the retina , active neurogenesis has not been reproducibly demonstrated under normal conditions ( 2 ) .", "however , rare neurogenic changes are observed in the injured adult mammalian retina ; the source of injury - induced neurogenesis is traced to mller glia ( mg ) ( 35 ) .", "recent observations that mammalian mg possess nsc properties and are able to generate retinal neurons in vitro and upon transplantation in vivo posit these cells as the potential target for regenerating diseased or injured photoreceptors and retinal ganglion cells ( 6 ) . while growing evidence confirms that mg possess evolutionarily conserved neurogenic potential , studies from various labs have observed that , unlike their lower vertebrate counterparts , the injury or disease - activated mammalian mg proliferate , but that they convert to neurons rather infrequently ( 4 , 7 , 8) . whether or not the converted neurons are functional , make synaptic connections , and survive for the long term remains unknown .", "the challenge that remains is how to unlock the neurogenic potentials of the mammalian mg in vivo .", "this may essentially involve approaches to reprogram these cells to dedifferentiate and regain their lost ability to generate neurons or trans - differentiate them into neurons .", "to address this challenge , the neurogenic potentials of mg need to be evaluated against the backdrop of two models , with the understanding that examples from lower vertebrates would constitute a framework , but that solutions will be unique to the mammalian retina : ( 1 ) svz / sgz model , where mg have nsc properties and thus the capacity for neuronal differentiation like radial glia - derived nscs in svz and sgz .", "given the observations that the adult nscs are responsive to environmental cues for neuronal differentiation ( 1 ) , mg in this model should be amenable to directed differentiation along neuronal lineage through the manipulation of niche - based signals by recombinant growth factors and/or small molecules .", "( 2 ) extra svz / sgz model , where mg may have neurogenic potential like parenchymal astrocytes but incapable of neuronal differentiation , presumably under the influence of the non - neurogenic niche . for example", ", both mg and astrocytes express nsc regulators such as sox2 ( mg ) and pax6 ( astrocytes ) and when cultured in the presence of mitogens tend to differentiate along neuronal lineage ( 2 , 6 , 9 , 10 ) .", "however , in vivo , although they proliferate and express neural progenitor markers such as nestin in response to injury , they maintain their glial phenotype , with rare expression of neuronal markers ( 2 , 5 , 10 ) .", "given their stable glial identity for supporting neurons despite expressing some of the nsc - specific genes , a trans - differentiation approach for the lineage conversion of mg has emerged as a practical option in this model .", "this notion is supported by recent observations that injury - activated resident astrocytes differentiate into functional neurons upon enforced expression of nsc regulator , sox2 and/or proneural gene , ascl1 ( 11 ) .", "regardless , information gained in deciphering the two models will be useful . in both cases ,", "knowledge of the developmental mechanism for the lineage switch , the reaction of mg to different kinds of injuries , phenotypes of the activated mg in spatial and temporal contexts , transcriptional and epigenetic status , and niche - related signals will be critical .", "these approaches will reveal whether the roadblock to regeneration is cell - intrinsic or cell - extrinsic or both , information that will be essential in formulating strategies for making mg - dependent therapeutic regeneration practical and clinically applicable . toward this effort ,", "the recent success of reprogramming to induce pluripotency in somatic cells and lineage - specific differentiation of pluripotent cells , whether through directed differentiation ( svz / sgz model ) or trans - differentiation ( extra svz / sgz model ) , is owed directly to the knowledge of developmental mechanisms ( 12 ) .", "there is a significant knowledge gap in our understanding of how neurogenesis shifts to the generation of mg in the mammalian retina , information essential to navigate the cellular or molecular roadblocks to neuronal differentiation .", "for example , although we know that notch signaling regulates the generation of mg , how it is incorporated into the gliogenic program and to what extent it is involved in suppressing neuronal differentiation remains rather unknown ( 4 ) .", "given the contextual role of notch signaling ( 4 ) , its complexity due to the oscillatory expression of its effector hes1 ( 13 ) , and its gliogenic interactions with the eye field gene lhx2 ( 14 ) , this information will be essential to formulate niche - based approaches for facilitating neuronal differentiation of mg .", "along this line , it will also be advantageous to know whether genes involved in neurogliogenesis elsewhere in the cns , such as lin28 , a heterochronic gene regulating the developmental timing ( 15 ) and ezh2 , an epigenetic regulator of gene expression ( 16 ) participate in mg differentiation and coordinate the influence of the niche .", "recent observations that the decision of retinal progenitors during late histogenesis to differentiate along glial or neuronal lineage is influenced by lin28 ( 17 ) and that ezh2 plays a role in constraining the generation of mg ( 18 ) posit these genes as potential targets for pivoting mg torwards neuronal differentiation .", "regardless of whether or not mg possesses dormant stem cell properties according to the aforementioned models they must initiate and complete an intertwined program of activation and neuronal conversion for successful regeneration ( 4 ) .", "given that injuries in general lead to proliferation of mg across species , but not to their neuronal differentiation in higher vertebrates , it is likely that the cross - talk between transcriptional networks sub - serving the activation and neuronal differentiation are either not connected to the process , or the network components are in place but are not epigenetically primed for optimal expression in the mammalian mg .", "these probabilities could be examined by a genome - wide screening of prospectively enriched mg in select animal models in quiescent and activated states ( facilitated by lineage reporters and other enrichment protocols such as the side population ( sp ) cell profiling by microarrays , rna seq , and chip seq analyses . a similar approach to characterizing mg in different states of activation and differentiation in controlled conditions in vitro", "will yield corroborative evidence and facilitate the means to test putative mechanism(s ) by manipulating gene expression .", "together , these approaches may provide insight into molecular axes that can be tested against the background of those operational in zebrafish ( 7 , 8 , 19 ) .", "for example , the molecular axis defined by lin28 and ascl1 , which facilitates mg - mediated regeneration in zebrafish , is a valid target for study in mammals .", "a variety of approaches in higher vertebrates have demonstrated important regulatory roles for lin28 in the maintenance of neural progenitors and neuroglial decision ( 20 ) .", "it is likely that lin28 influences different developmental function by contextual recruitment of hmga2 , a gene encoding a dna architecture protein ( 21 , 22 ) and ascl1 ( 23 ) by directly regulating the heterochronic mirna let-7 ( 17 , 21 , 23 ) .", "let-7 targets hmga2 ( 17 , 21 ) and ascl1 ( 23 ) ; therefore , the lin28-let-7-hmga2/ascl1 axis has emerged as an evolutionary conserved axis that could be a candidate for unlocking neurogenic potential of mg .", "for example , overexpression of lin28a ( 24 ) in the enriched mg ( fig .", "2a ) prompted these cells to acquire neuronal morphology and to express immunoreactivities and transcripts corresponding to neuronal genes ( fig .", "more importantly , neuronal differentiation was accompanied by an increase in levels of mir-124 , a proneural mirna ( 25 ) , hmga2 , and ascl1 , and a decrease in those of rest , a global inhibitor of neuronal differentiation ( 26 ) , thus demonstrating the capacity of lin28a in activating the neurogenic program in mg . however , targeting the lin28-let-7-hmga2/ascl1 axis alone may not be sufficient for the functional reprogramming of mg along neuronal lineage , given that its influence may not be able to completely counter the inhibitory resistance of the rest axis .", "rest , by suppressing the expression of proneural mirnas , mir-124 and mir-9 - 9 * , whose targets are proglial genes encoding sox9 and he s family of regulators , may keep mg non - neuronal and therefore non - regenerative ( 25 ) .", "the existence of the rest - mir-124 - 9 - 9 * -sox9/hes1 axis in mg suggested that either inhibiting rest or ectopically expressing mir-124/mir-9 - 9 * or both may direct mg along the neuronal lineage .", "it is likely that the ectopic expression of mir-124/mir-9 - 9 * in itself might be effective , as they directly or indirectly influence the expression and function of rest ( 25 ) .", "for example , over expression of mir-124 - 9 - 9 * ( 27 ) in mg ( fig .", "2a ) facilitated neuronal morphology with accompanied expression and suppression of neuronal- and glial - specific genes , respectively ( fig .", "expression of both ascl1 and lin28a was up regulated and that of rest was down regulated in the perturbed groups , compared to controls .", "however , no significant difference in hmga2 transcript levels was observed , suggesting a threshold requirement of lin28a levels for influencing hmga2 expression . that mir-124 and mir-9 - 9 * could facilitate proneural function of ascl1", "was demonstrated by the improvement of ascl1-mediated reprogramming of mg by ectopically expressed mirnas ( 28 ) .", "together , these observations posit these molecular axes as valid intrinsic targets for studying and unlocking the dormant regenerative potential of mg .", "mg , the guardian of homeostasis in the retina , respond to injuries by proliferating and migrating out of the inner nuclear layer ( 5 ) .", "however , despite proliferation and migration as in zebrafish retina , as mentioned , mammalian mg do not initiate regeneration effectively .", "this raises the possibility that , in addition to internal constraints discussed above , the environment in the mammalian retina might not be conducive for neurogenic conversion of mg . a niche - based approach to unlock neurogenic potential of mg will involve examining the relationship of mg with neighboring retinal cells , microglia , immigrant astrocytes , and endothelial cells in the context of signaling pathways and their capacity to engage the molecular axes involved in reprograming along neuronal lineage .", "for example , the understanding of the role of microglia , one of the first responders to injury , and which may mediate regeneration through inflammatory signals , is still evolving in the retina ( 29 ) .", "studies have demonstrated that notch ( 8 , 30 ) , wnt ( 5 , 6 ) , fgf ( 6 , 8) , insulin and insulin - like growth factor-1 ( 8) , shh ( 31 ) , and cytokines such as tnf ( 8 , 19 ) , leptin , and il-11 ( 32 ) may play important roles in mediating the influence of the niche in reprogramming mg .", "however , the identity of cells delivering the signals and whether or not these signals and their associated pathways act in concert , which would determine niche - based strategies to promote regeneration , remain largely unknown .", "however , studies in zebrafish have begun to shed light on these issues . for example , it has been observed that tnf released by dying photoreceptors in mechanically damaged zebrafish retina may constitute one of the early signals for activating mg for regeneration ( 19 ) .", "also , it was reported that zebrafish mg respond to retinal injury by secreting leptin and il-11 , which help reprogram cells in an autocrine fashion ( 32 ) . in both cases , these cytokines facilitated injury - dependent induction of ascl1 , a key step in reprogramming of mg ( 8) . whether or not signaling - mediated by these factors are similarly involved in the mammalian retina remains to be demonstrated .", "although the identity of cells delivering notch signaling in mg remains speculative , emerging evidence from zebrafish and higher vertebrates posit it as an important niche - based target for mg - dependent regeneration .", "it remains active long after facilitating the differentiation of mg , enabling them to launch the proliferative response when injury takes place ( 4 , 5 ) .", "however , persistence of notch signaling in activated mg , which are poised for neuronal conversion , might be deleterious for regenerative process , given the inhibitory influence of notch signaling on the activation of proneural genes ( 33 ) . the inability to confine the activation of notch signaling in mg to a narrow temporal window following injury", "may be one of the reasons for their anemic neuronal conversion in mammals , compared to that in zebrafish ( 30 ) .", "emerging evidence , once again from the zebrafish model , suggests that these pathways , including notch signaling , may act in concert and mg integrate the diverse niche - based influence of a variety of signaling molecules for a calibrated regenerative response to injury ( 8) .", "therefore , niche - related information is crucial for understanding the temporal and spatial modulation of signaling required for activating mg and shifting them from the state of activation to neuronal differentiation .", "this is fundamental information missing from studies of mg - dependent regeneration , without which the underlying mechanisms remain difficult to grasp and the therapeutic target rather illusory .", "currently , the identity of the activated mg represents cells in the inner nuclear layer of the retina , which have incorporated brdu in response to injury and express mg - specific markers .", "given the transition of mg from a quiescent to a proliferative state and the generation of their progenies in different stages of development , the identity based on brdu incorporation , though important , is of limited value .", "attempts have been made to prospectively enrich activated mg by hoechst dye efflux assays and to characterize them as sp cells , a phenotype shared by the majority of stem cells ( 4 , 6 ) .", "however , since sp cells are also heterogeneous , this functional approach has limitations in the absence of specific cellular markers .", "although several signaling pathways ( e.g. notch signaling ) and intrinsic factors ( e.g. , ascl1 ) have been observed to regulate mg activation in fish , birds , and rodents ( 8) , their status as markers of activated mg in different stages of regeneration remain unspecified .", "progress in understanding the regenerative mechanisms in tissues like blood ( 34 ) , intestine ( 35 ) , and skin ( 36 ) has been facilitated by the identification and characterization of markers of the resident stem cells and their progenies . within the cns ,", "insight into regeneration in the svz has come from the characterization of b1 quiescent and active progenitors and resulting neuroblasts ( 37 ) .", "therefore , to follow mg - mediated regeneration , spatially and temporally , and to identify the stage - specific intrinsic players , a concerted effort is needed to search for reliable and reproducible markers . currently , there is no consensus on reliable and reproducible mammalian models to study the activation and neurogenic potential of mg .", "rodent models representing retinal injuries , ranging from those caused by the exposure to light , neurotoxins , and genetic mutations have been used .", "given the observations that glia respond differently to different types and durations of injuries ( 2 ) , that their levels of activation differ from species to species ( ahmad et al .", ", unpublished observations ) , and that within the same species there are strain differences in responses ( 38 ) , a case may be made for developing injury- and species / strain - specific models for reproducible and unambiguous examination of mg neurogenic potentials .", "these models will be valuable in incorporating transgenic technology for reliable lineage tracing , and more importantly for molecular characterization of activated mg .", "characterization of these cells for gene regulatory network and epigenetic signature is essential to shed light on the status of their neurogenic potential and approaches to unlock it .", "furthermore , cell type specific injury models would test the ability of mg to replace specific neuronal types , which will be helpful in designing approaches for mg - dependent therapeutic regeneration .", "for example , the loss of vision in two of the intractable blinding diseases , age - related macular degeneration ( amd ) and glaucoma , is due to selective loss of photoreceptors and retinal ganglion cells ( rgcs ) , respectively .", "the neurotoxin injury models where exposure of the retina to n - methyl - n - nitrosourea ( mnu ) ablates photoreceptors ( 39 ) and nmda causes the degeneration of rgcs ( 40 ) would reveal whether or not mg could differentiate along specific neuronal types . also , it may shed light on if mg - dependent therapeutic regeneration would be a practical approach to address degenerative changes in amd and/or glaucoma .", "the recent success of reprogramming to induce pluripotency in somatic cells and lineage - specific differentiation of pluripotent cells , whether through directed differentiation ( svz / sgz model ) or trans - differentiation ( extra svz / sgz model ) , is owed directly to the knowledge of developmental mechanisms ( 12 ) .", "there is a significant knowledge gap in our understanding of how neurogenesis shifts to the generation of mg in the mammalian retina , information essential to navigate the cellular or molecular roadblocks to neuronal differentiation .", "for example , although we know that notch signaling regulates the generation of mg , how it is incorporated into the gliogenic program and to what extent it is involved in suppressing neuronal differentiation remains rather unknown ( 4 ) .", "given the contextual role of notch signaling ( 4 ) , its complexity due to the oscillatory expression of its effector hes1 ( 13 ) , and its gliogenic interactions with the eye field gene lhx2 ( 14 ) , this information will be essential to formulate niche - based approaches for facilitating neuronal differentiation of mg . along this line", ", it will also be advantageous to know whether genes involved in neurogliogenesis elsewhere in the cns , such as lin28 , a heterochronic gene regulating the developmental timing ( 15 ) and ezh2 , an epigenetic regulator of gene expression ( 16 ) participate in mg differentiation and coordinate the influence of the niche .", "recent observations that the decision of retinal progenitors during late histogenesis to differentiate along glial or neuronal lineage is influenced by lin28 ( 17 ) and that ezh2 plays a role in constraining the generation of mg ( 18 ) posit these genes as potential targets for pivoting mg torwards neuronal differentiation .", "regardless of whether or not mg possesses dormant stem cell properties according to the aforementioned models they must initiate and complete an intertwined program of activation and neuronal conversion for successful regeneration ( 4 ) . given that injuries in general lead to proliferation of mg across species , but not to their neuronal differentiation in higher vertebrates , it is likely that the cross - talk between transcriptional networks sub - serving the activation and neuronal differentiation are either not connected to the process , or the network components are in place but are not epigenetically primed for optimal expression in the mammalian mg .", "these probabilities could be examined by a genome - wide screening of prospectively enriched mg in select animal models in quiescent and activated states ( facilitated by lineage reporters and other enrichment protocols such as the side population ( sp ) cell profiling by microarrays , rna seq , and chip seq analyses . a similar approach to characterizing mg in different states of activation and differentiation in controlled conditions in vitro", "will yield corroborative evidence and facilitate the means to test putative mechanism(s ) by manipulating gene expression .", "together , these approaches may provide insight into molecular axes that can be tested against the background of those operational in zebrafish ( 7 , 8 , 19 ) .", "for example , the molecular axis defined by lin28 and ascl1 , which facilitates mg - mediated regeneration in zebrafish , is a valid target for study in mammals .", "a variety of approaches in higher vertebrates have demonstrated important regulatory roles for lin28 in the maintenance of neural progenitors and neuroglial decision ( 20 ) .", "it is likely that lin28 influences different developmental function by contextual recruitment of hmga2 , a gene encoding a dna architecture protein ( 21 , 22 ) and ascl1 ( 23 ) by directly regulating the heterochronic mirna let-7 ( 17 , 21 , 23 ) .", "let-7 targets hmga2 ( 17 , 21 ) and ascl1 ( 23 ) ; therefore , the lin28-let-7-hmga2/ascl1 axis has emerged as an evolutionary conserved axis that could be a candidate for unlocking neurogenic potential of mg .", "for example , overexpression of lin28a ( 24 ) in the enriched mg ( fig .", "2a ) prompted these cells to acquire neuronal morphology and to express immunoreactivities and transcripts corresponding to neuronal genes ( fig .", "more importantly , neuronal differentiation was accompanied by an increase in levels of mir-124 , a proneural mirna ( 25 ) , hmga2 , and ascl1 , and a decrease in those of rest , a global inhibitor of neuronal differentiation ( 26 ) , thus demonstrating the capacity of lin28a in activating the neurogenic program in mg . however , targeting the lin28-let-7-hmga2/ascl1 axis alone may not be sufficient for the functional reprogramming of mg along neuronal lineage , given that its influence may not be able to completely counter the inhibitory resistance of the rest axis .", "rest , by suppressing the expression of proneural mirnas , mir-124 and mir-9 - 9 * , whose targets are proglial genes encoding sox9 and he s family of regulators , may keep mg non - neuronal and therefore non - regenerative ( 25 ) .", "the existence of the rest - mir-124 - 9 - 9 * -sox9/hes1 axis in mg suggested that either inhibiting rest or ectopically expressing mir-124/mir-9 - 9 * or both may direct mg along the neuronal lineage .", "it is likely that the ectopic expression of mir-124/mir-9 - 9 * in itself might be effective , as they directly or indirectly influence the expression and function of rest ( 25 ) .", "for example , over expression of mir-124 - 9 - 9 * ( 27 ) in mg ( fig .", "2a ) facilitated neuronal morphology with accompanied expression and suppression of neuronal- and glial - specific genes , respectively ( fig .", "expression of both ascl1 and lin28a was up regulated and that of rest was down regulated in the perturbed groups , compared to controls .", "however , no significant difference in hmga2 transcript levels was observed , suggesting a threshold requirement of lin28a levels for influencing hmga2 expression .", "that mir-124 and mir-9 - 9 * could facilitate proneural function of ascl1 was demonstrated by the improvement of ascl1-mediated reprogramming of mg by ectopically expressed mirnas ( 28 ) .", "together , these observations posit these molecular axes as valid intrinsic targets for studying and unlocking the dormant regenerative potential of mg .", "mg , the guardian of homeostasis in the retina , respond to injuries by proliferating and migrating out of the inner nuclear layer ( 5 ) .", "however , despite proliferation and migration as in zebrafish retina , as mentioned , mammalian mg do not initiate regeneration effectively .", "this raises the possibility that , in addition to internal constraints discussed above , the environment in the mammalian retina might not be conducive for neurogenic conversion of mg . a niche - based approach to unlock neurogenic potential of mg will involve examining the relationship of mg with neighboring retinal cells , microglia , immigrant astrocytes , and endothelial cells in the context of signaling pathways and their capacity to engage the molecular axes involved in reprograming along neuronal lineage .", "for example , the understanding of the role of microglia , one of the first responders to injury , and which may mediate regeneration through inflammatory signals , is still evolving in the retina ( 29 ) .", "studies have demonstrated that notch ( 8 , 30 ) , wnt ( 5 , 6 ) , fgf ( 6 , 8) , insulin and insulin - like growth factor-1 ( 8) , shh ( 31 ) , and cytokines such as tnf ( 8 , 19 ) , leptin , and il-11 ( 32 ) may play important roles in mediating the influence of the niche in reprogramming mg . however , the identity of cells delivering the signals and whether or not these signals and their associated pathways act in concert , which would determine niche - based strategies to promote regeneration , remain largely unknown .", "for example , it has been observed that tnf released by dying photoreceptors in mechanically damaged zebrafish retina may constitute one of the early signals for activating mg for regeneration ( 19 ) .", "also , it was reported that zebrafish mg respond to retinal injury by secreting leptin and il-11 , which help reprogram cells in an autocrine fashion ( 32 ) . in both cases , these cytokines facilitated injury - dependent induction of ascl1 , a key step in reprogramming of mg ( 8) . whether or not signaling - mediated by these factors are similarly involved in the mammalian retina remains to be demonstrated .", "although the identity of cells delivering notch signaling in mg remains speculative , emerging evidence from zebrafish and higher vertebrates posit it as an important niche - based target for mg - dependent regeneration .", "it remains active long after facilitating the differentiation of mg , enabling them to launch the proliferative response when injury takes place ( 4 , 5 ) .", "however , persistence of notch signaling in activated mg , which are poised for neuronal conversion , might be deleterious for regenerative process , given the inhibitory influence of notch signaling on the activation of proneural genes ( 33 ) . the inability to confine", "the activation of notch signaling in mg to a narrow temporal window following injury may be one of the reasons for their anemic neuronal conversion in mammals , compared to that in zebrafish ( 30 ) .", "emerging evidence , once again from the zebrafish model , suggests that these pathways , including notch signaling , may act in concert and mg integrate the diverse niche - based influence of a variety of signaling molecules for a calibrated regenerative response to injury ( 8) .", "therefore , niche - related information is crucial for understanding the temporal and spatial modulation of signaling required for activating mg and shifting them from the state of activation to neuronal differentiation .", "this is fundamental information missing from studies of mg - dependent regeneration , without which the underlying mechanisms remain difficult to grasp and the therapeutic target rather illusory .", "currently , the identity of the activated mg represents cells in the inner nuclear layer of the retina , which have incorporated brdu in response to injury and express mg - specific markers .", "given the transition of mg from a quiescent to a proliferative state and the generation of their progenies in different stages of development , the identity based on brdu incorporation , though important , is of limited value .", "attempts have been made to prospectively enrich activated mg by hoechst dye efflux assays and to characterize them as sp cells , a phenotype shared by the majority of stem cells ( 4 , 6 ) . however , since sp cells are also heterogeneous , this functional approach has limitations in the absence of specific cellular markers .", "although several signaling pathways ( e.g. notch signaling ) and intrinsic factors ( e.g. , ascl1 ) have been observed to regulate mg activation in fish , birds , and rodents ( 8) , their status as markers of activated mg in different stages of regeneration remain unspecified .", "progress in understanding the regenerative mechanisms in tissues like blood ( 34 ) , intestine ( 35 ) , and skin ( 36 ) has been facilitated by the identification and characterization of markers of the resident stem cells and their progenies . within the cns ,", "insight into regeneration in the svz has come from the characterization of b1 quiescent and active progenitors and resulting neuroblasts ( 37 ) .", "therefore , to follow mg - mediated regeneration , spatially and temporally , and to identify the stage - specific intrinsic players , a concerted effort is needed to search for reliable and reproducible markers .", "currently , there is no consensus on reliable and reproducible mammalian models to study the activation and neurogenic potential of mg .", "rodent models representing retinal injuries , ranging from those caused by the exposure to light , neurotoxins , and genetic mutations have been used .", "given the observations that glia respond differently to different types and durations of injuries ( 2 ) , that their levels of activation differ from species to species ( ahmad et al .", ", unpublished observations ) , and that within the same species there are strain differences in responses ( 38 ) , a case may be made for developing injury- and species / strain - specific models for reproducible and unambiguous examination of mg neurogenic potentials .", "these models will be valuable in incorporating transgenic technology for reliable lineage tracing , and more importantly for molecular characterization of activated mg .", "characterization of these cells for gene regulatory network and epigenetic signature is essential to shed light on the status of their neurogenic potential and approaches to unlock it .", "furthermore , cell type specific injury models would test the ability of mg to replace specific neuronal types , which will be helpful in designing approaches for mg - dependent therapeutic regeneration .", "for example , the loss of vision in two of the intractable blinding diseases , age - related macular degeneration ( amd ) and glaucoma , is due to selective loss of photoreceptors and retinal ganglion cells ( rgcs ) , respectively .", "the neurotoxin injury models where exposure of the retina to n - methyl - n - nitrosourea ( mnu ) ablates photoreceptors ( 39 ) and nmda causes the degeneration of rgcs ( 40 ) would reveal whether or not mg could differentiate along specific neuronal types . also , it may shed light on if mg - dependent therapeutic regeneration would be a practical approach to address degenerative changes in amd and/or glaucoma .", "the discipline of mg - based regeneration is a recent one , particularly in mammals . with information on the nature of", "the activated mg and the molecular axes mediating their response to the niche , aided by reproducible animal models and lineage reporters , a pharmacological recruitment of these endogenous progenitors for therapeutic regeneration is a near possibility . in addition , information emerging from these studies will help us understand how the neurogenic potential in the adult mammalian brain is constrained relative to that of lower vertebrates , currently a significant knowledge gap ."], "abstract_text": ["<S> mller glia ( mg ) are the primary support cells in the vertebrate retina , regulating homeostasis in one of the most metabolically active tissues . in lower vertebrates such as fish </S>", "<S> , they respond to injury by proliferating and reprogramming to regenerate retinal neurons . in mammals </S>", "<S> , mg may also react to injury by proliferating , but they fail to initiate regeneration . </S>", "<S> the barriers to regeneration could be intrinsic to mammalian mg or the function of the niche that can not support the mg reprogramming required for lineage conversion or both . </S>", "<S> understanding these mechanisms in light of those being discovered in fish may lead to the formulation of strategies to unlock the neurogenic potential of mg and restore regeneration in the mammalian retina . </S>"], "labels": null, "section_names": ["Introduction", "Neurogenic potentials of MG in mammals", "Mechanisms underlying the neuroglial switch in the mammalian retina", "Molecular axes involved in MG-mediated regeneration", "Influences of the niche and how they are mediated", "Markers of activated MG", "Animal models for MG-mediated therapeutic regeneration", "Conclusions"], "sections": [["recent progress in our understanding of brain development has significantly altered concepts for treating neurodegenerative diseases , including those that affect the retina to cause blindness .", "contrary to previous thought , it is now recognized that neurons are generated in the sub ventricular zone ( svz ) of the lateral ventricle , and the sub - granular zone ( sgz ) in the hippocampus from neural stem cells ( nsc ) of glial origin throughout life ( 1 ) . outside these discrete regions in the mammalian cns , including the retina , active neurogenesis has not been reproducibly demonstrated under normal conditions ( 2 ) .", "however , rare neurogenic changes are observed in the injured adult mammalian retina ; the source of injury - induced neurogenesis is traced to mller glia ( mg ) ( 35 ) .", "recent observations that mammalian mg possess nsc properties and are able to generate retinal neurons in vitro and upon transplantation in vivo posit these cells as the potential target for regenerating diseased or injured photoreceptors and retinal ganglion cells ( 6 ) . while growing evidence confirms that mg possess evolutionarily conserved neurogenic potential , studies from various labs have observed that , unlike their lower vertebrate counterparts , the injury or disease - activated mammalian mg proliferate , but that they convert to neurons rather infrequently ( 4 , 7 , 8) . whether or not the converted neurons are functional , make synaptic connections , and survive for the long term remains unknown .", "the challenge that remains is how to unlock the neurogenic potentials of the mammalian mg in vivo .", "this may essentially involve approaches to reprogram these cells to dedifferentiate and regain their lost ability to generate neurons or trans - differentiate them into neurons ."], ["to address this challenge , the neurogenic potentials of mg need to be evaluated against the backdrop of two models , with the understanding that examples from lower vertebrates would constitute a framework , but that solutions will be unique to the mammalian retina : ( 1 ) svz / sgz model , where mg have nsc properties and thus the capacity for neuronal differentiation like radial glia - derived nscs in svz and sgz .", "given the observations that the adult nscs are responsive to environmental cues for neuronal differentiation ( 1 ) , mg in this model should be amenable to directed differentiation along neuronal lineage through the manipulation of niche - based signals by recombinant growth factors and/or small molecules .", "( 2 ) extra svz / sgz model , where mg may have neurogenic potential like parenchymal astrocytes but incapable of neuronal differentiation , presumably under the influence of the non - neurogenic niche . for example", ", both mg and astrocytes express nsc regulators such as sox2 ( mg ) and pax6 ( astrocytes ) and when cultured in the presence of mitogens tend to differentiate along neuronal lineage ( 2 , 6 , 9 , 10 ) .", "however , in vivo , although they proliferate and express neural progenitor markers such as nestin in response to injury , they maintain their glial phenotype , with rare expression of neuronal markers ( 2 , 5 , 10 ) .", "given their stable glial identity for supporting neurons despite expressing some of the nsc - specific genes , a trans - differentiation approach for the lineage conversion of mg has emerged as a practical option in this model .", "this notion is supported by recent observations that injury - activated resident astrocytes differentiate into functional neurons upon enforced expression of nsc regulator , sox2 and/or proneural gene , ascl1 ( 11 ) .", "regardless , information gained in deciphering the two models will be useful . in both cases ,", "knowledge of the developmental mechanism for the lineage switch , the reaction of mg to different kinds of injuries , phenotypes of the activated mg in spatial and temporal contexts , transcriptional and epigenetic status , and niche - related signals will be critical .", "these approaches will reveal whether the roadblock to regeneration is cell - intrinsic or cell - extrinsic or both , information that will be essential in formulating strategies for making mg - dependent therapeutic regeneration practical and clinically applicable . toward this effort ,", "the recent success of reprogramming to induce pluripotency in somatic cells and lineage - specific differentiation of pluripotent cells , whether through directed differentiation ( svz / sgz model ) or trans - differentiation ( extra svz / sgz model ) , is owed directly to the knowledge of developmental mechanisms ( 12 ) .", "there is a significant knowledge gap in our understanding of how neurogenesis shifts to the generation of mg in the mammalian retina , information essential to navigate the cellular or molecular roadblocks to neuronal differentiation .", "for example , although we know that notch signaling regulates the generation of mg , how it is incorporated into the gliogenic program and to what extent it is involved in suppressing neuronal differentiation remains rather unknown ( 4 ) .", "given the contextual role of notch signaling ( 4 ) , its complexity due to the oscillatory expression of its effector hes1 ( 13 ) , and its gliogenic interactions with the eye field gene lhx2 ( 14 ) , this information will be essential to formulate niche - based approaches for facilitating neuronal differentiation of mg .", "along this line , it will also be advantageous to know whether genes involved in neurogliogenesis elsewhere in the cns , such as lin28 , a heterochronic gene regulating the developmental timing ( 15 ) and ezh2 , an epigenetic regulator of gene expression ( 16 ) participate in mg differentiation and coordinate the influence of the niche .", "recent observations that the decision of retinal progenitors during late histogenesis to differentiate along glial or neuronal lineage is influenced by lin28 ( 17 ) and that ezh2 plays a role in constraining the generation of mg ( 18 ) posit these genes as potential targets for pivoting mg torwards neuronal differentiation .", "regardless of whether or not mg possesses dormant stem cell properties according to the aforementioned models they must initiate and complete an intertwined program of activation and neuronal conversion for successful regeneration ( 4 ) .", "given that injuries in general lead to proliferation of mg across species , but not to their neuronal differentiation in higher vertebrates , it is likely that the cross - talk between transcriptional networks sub - serving the activation and neuronal differentiation are either not connected to the process , or the network components are in place but are not epigenetically primed for optimal expression in the mammalian mg .", "these probabilities could be examined by a genome - wide screening of prospectively enriched mg in select animal models in quiescent and activated states ( facilitated by lineage reporters and other enrichment protocols such as the side population ( sp ) cell profiling by microarrays , rna seq , and chip seq analyses . a similar approach to characterizing mg in different states of activation and differentiation in controlled conditions in vitro", "will yield corroborative evidence and facilitate the means to test putative mechanism(s ) by manipulating gene expression .", "together , these approaches may provide insight into molecular axes that can be tested against the background of those operational in zebrafish ( 7 , 8 , 19 ) .", "for example , the molecular axis defined by lin28 and ascl1 , which facilitates mg - mediated regeneration in zebrafish , is a valid target for study in mammals .", "a variety of approaches in higher vertebrates have demonstrated important regulatory roles for lin28 in the maintenance of neural progenitors and neuroglial decision ( 20 ) .", "it is likely that lin28 influences different developmental function by contextual recruitment of hmga2 , a gene encoding a dna architecture protein ( 21 , 22 ) and ascl1 ( 23 ) by directly regulating the heterochronic mirna let-7 ( 17 , 21 , 23 ) .", "let-7 targets hmga2 ( 17 , 21 ) and ascl1 ( 23 ) ; therefore , the lin28-let-7-hmga2/ascl1 axis has emerged as an evolutionary conserved axis that could be a candidate for unlocking neurogenic potential of mg .", "for example , overexpression of lin28a ( 24 ) in the enriched mg ( fig .", "2a ) prompted these cells to acquire neuronal morphology and to express immunoreactivities and transcripts corresponding to neuronal genes ( fig .", "more importantly , neuronal differentiation was accompanied by an increase in levels of mir-124 , a proneural mirna ( 25 ) , hmga2 , and ascl1 , and a decrease in those of rest , a global inhibitor of neuronal differentiation ( 26 ) , thus demonstrating the capacity of lin28a in activating the neurogenic program in mg . however , targeting the lin28-let-7-hmga2/ascl1 axis alone may not be sufficient for the functional reprogramming of mg along neuronal lineage , given that its influence may not be able to completely counter the inhibitory resistance of the rest axis .", "rest , by suppressing the expression of proneural mirnas , mir-124 and mir-9 - 9 * , whose targets are proglial genes encoding sox9 and he s family of regulators , may keep mg non - neuronal and therefore non - regenerative ( 25 ) .", "the existence of the rest - mir-124 - 9 - 9 * -sox9/hes1 axis in mg suggested that either inhibiting rest or ectopically expressing mir-124/mir-9 - 9 * or both may direct mg along the neuronal lineage .", "it is likely that the ectopic expression of mir-124/mir-9 - 9 * in itself might be effective , as they directly or indirectly influence the expression and function of rest ( 25 ) .", "for example , over expression of mir-124 - 9 - 9 * ( 27 ) in mg ( fig .", "2a ) facilitated neuronal morphology with accompanied expression and suppression of neuronal- and glial - specific genes , respectively ( fig .", "expression of both ascl1 and lin28a was up regulated and that of rest was down regulated in the perturbed groups , compared to controls .", "however , no significant difference in hmga2 transcript levels was observed , suggesting a threshold requirement of lin28a levels for influencing hmga2 expression . that mir-124 and mir-9 - 9 * could facilitate proneural function of ascl1", "was demonstrated by the improvement of ascl1-mediated reprogramming of mg by ectopically expressed mirnas ( 28 ) .", "together , these observations posit these molecular axes as valid intrinsic targets for studying and unlocking the dormant regenerative potential of mg .", "mg , the guardian of homeostasis in the retina , respond to injuries by proliferating and migrating out of the inner nuclear layer ( 5 ) .", "however , despite proliferation and migration as in zebrafish retina , as mentioned , mammalian mg do not initiate regeneration effectively .", "this raises the possibility that , in addition to internal constraints discussed above , the environment in the mammalian retina might not be conducive for neurogenic conversion of mg . a niche - based approach to unlock neurogenic potential of mg will involve examining the relationship of mg with neighboring retinal cells , microglia , immigrant astrocytes , and endothelial cells in the context of signaling pathways and their capacity to engage the molecular axes involved in reprograming along neuronal lineage .", "for example , the understanding of the role of microglia , one of the first responders to injury , and which may mediate regeneration through inflammatory signals , is still evolving in the retina ( 29 ) .", "studies have demonstrated that notch ( 8 , 30 ) , wnt ( 5 , 6 ) , fgf ( 6 , 8) , insulin and insulin - like growth factor-1 ( 8) , shh ( 31 ) , and cytokines such as tnf ( 8 , 19 ) , leptin , and il-11 ( 32 ) may play important roles in mediating the influence of the niche in reprogramming mg .", "however , the identity of cells delivering the signals and whether or not these signals and their associated pathways act in concert , which would determine niche - based strategies to promote regeneration , remain largely unknown .", "however , studies in zebrafish have begun to shed light on these issues . for example , it has been observed that tnf released by dying photoreceptors in mechanically damaged zebrafish retina may constitute one of the early signals for activating mg for regeneration ( 19 ) .", "also , it was reported that zebrafish mg respond to retinal injury by secreting leptin and il-11 , which help reprogram cells in an autocrine fashion ( 32 ) . in both cases , these cytokines facilitated injury - dependent induction of ascl1 , a key step in reprogramming of mg ( 8) . whether or not signaling - mediated by these factors are similarly involved in the mammalian retina remains to be demonstrated .", "although the identity of cells delivering notch signaling in mg remains speculative , emerging evidence from zebrafish and higher vertebrates posit it as an important niche - based target for mg - dependent regeneration .", "it remains active long after facilitating the differentiation of mg , enabling them to launch the proliferative response when injury takes place ( 4 , 5 ) .", "however , persistence of notch signaling in activated mg , which are poised for neuronal conversion , might be deleterious for regenerative process , given the inhibitory influence of notch signaling on the activation of proneural genes ( 33 ) . the inability to confine the activation of notch signaling in mg to a narrow temporal window following injury", "may be one of the reasons for their anemic neuronal conversion in mammals , compared to that in zebrafish ( 30 ) .", "emerging evidence , once again from the zebrafish model , suggests that these pathways , including notch signaling , may act in concert and mg integrate the diverse niche - based influence of a variety of signaling molecules for a calibrated regenerative response to injury ( 8) .", "therefore , niche - related information is crucial for understanding the temporal and spatial modulation of signaling required for activating mg and shifting them from the state of activation to neuronal differentiation .", "this is fundamental information missing from studies of mg - dependent regeneration , without which the underlying mechanisms remain difficult to grasp and the therapeutic target rather illusory .", "currently , the identity of the activated mg represents cells in the inner nuclear layer of the retina , which have incorporated brdu in response to injury and express mg - specific markers .", "given the transition of mg from a quiescent to a proliferative state and the generation of their progenies in different stages of development , the identity based on brdu incorporation , though important , is of limited value .", "attempts have been made to prospectively enrich activated mg by hoechst dye efflux assays and to characterize them as sp cells , a phenotype shared by the majority of stem cells ( 4 , 6 ) .", "however , since sp cells are also heterogeneous , this functional approach has limitations in the absence of specific cellular markers .", "although several signaling pathways ( e.g. notch signaling ) and intrinsic factors ( e.g. , ascl1 ) have been observed to regulate mg activation in fish , birds , and rodents ( 8) , their status as markers of activated mg in different stages of regeneration remain unspecified .", "progress in understanding the regenerative mechanisms in tissues like blood ( 34 ) , intestine ( 35 ) , and skin ( 36 ) has been facilitated by the identification and characterization of markers of the resident stem cells and their progenies . within the cns ,", "insight into regeneration in the svz has come from the characterization of b1 quiescent and active progenitors and resulting neuroblasts ( 37 ) .", "therefore , to follow mg - mediated regeneration , spatially and temporally , and to identify the stage - specific intrinsic players , a concerted effort is needed to search for reliable and reproducible markers . currently , there is no consensus on reliable and reproducible mammalian models to study the activation and neurogenic potential of mg .", "rodent models representing retinal injuries , ranging from those caused by the exposure to light , neurotoxins , and genetic mutations have been used .", "given the observations that glia respond differently to different types and durations of injuries ( 2 ) , that their levels of activation differ from species to species ( ahmad et al .", ", unpublished observations ) , and that within the same species there are strain differences in responses ( 38 ) , a case may be made for developing injury- and species / strain - specific models for reproducible and unambiguous examination of mg neurogenic potentials .", "these models will be valuable in incorporating transgenic technology for reliable lineage tracing , and more importantly for molecular characterization of activated mg .", "characterization of these cells for gene regulatory network and epigenetic signature is essential to shed light on the status of their neurogenic potential and approaches to unlock it .", "furthermore , cell type specific injury models would test the ability of mg to replace specific neuronal types , which will be helpful in designing approaches for mg - dependent therapeutic regeneration .", "for example , the loss of vision in two of the intractable blinding diseases , age - related macular degeneration ( amd ) and glaucoma , is due to selective loss of photoreceptors and retinal ganglion cells ( rgcs ) , respectively .", "the neurotoxin injury models where exposure of the retina to n - methyl - n - nitrosourea ( mnu ) ablates photoreceptors ( 39 ) and nmda causes the degeneration of rgcs ( 40 ) would reveal whether or not mg could differentiate along specific neuronal types . also , it may shed light on if mg - dependent therapeutic regeneration would be a practical approach to address degenerative changes in amd and/or glaucoma ."], ["the recent success of reprogramming to induce pluripotency in somatic cells and lineage - specific differentiation of pluripotent cells , whether through directed differentiation ( svz / sgz model ) or trans - differentiation ( extra svz / sgz model ) , is owed directly to the knowledge of developmental mechanisms ( 12 ) .", "there is a significant knowledge gap in our understanding of how neurogenesis shifts to the generation of mg in the mammalian retina , information essential to navigate the cellular or molecular roadblocks to neuronal differentiation .", "for example , although we know that notch signaling regulates the generation of mg , how it is incorporated into the gliogenic program and to what extent it is involved in suppressing neuronal differentiation remains rather unknown ( 4 ) .", "given the contextual role of notch signaling ( 4 ) , its complexity due to the oscillatory expression of its effector hes1 ( 13 ) , and its gliogenic interactions with the eye field gene lhx2 ( 14 ) , this information will be essential to formulate niche - based approaches for facilitating neuronal differentiation of mg . along this line", ", it will also be advantageous to know whether genes involved in neurogliogenesis elsewhere in the cns , such as lin28 , a heterochronic gene regulating the developmental timing ( 15 ) and ezh2 , an epigenetic regulator of gene expression ( 16 ) participate in mg differentiation and coordinate the influence of the niche .", "recent observations that the decision of retinal progenitors during late histogenesis to differentiate along glial or neuronal lineage is influenced by lin28 ( 17 ) and that ezh2 plays a role in constraining the generation of mg ( 18 ) posit these genes as potential targets for pivoting mg torwards neuronal differentiation ."], ["regardless of whether or not mg possesses dormant stem cell properties according to the aforementioned models they must initiate and complete an intertwined program of activation and neuronal conversion for successful regeneration ( 4 ) . given that injuries in general lead to proliferation of mg across species , but not to their neuronal differentiation in higher vertebrates , it is likely that the cross - talk between transcriptional networks sub - serving the activation and neuronal differentiation are either not connected to the process , or the network components are in place but are not epigenetically primed for optimal expression in the mammalian mg .", "these probabilities could be examined by a genome - wide screening of prospectively enriched mg in select animal models in quiescent and activated states ( facilitated by lineage reporters and other enrichment protocols such as the side population ( sp ) cell profiling by microarrays , rna seq , and chip seq analyses . a similar approach to characterizing mg in different states of activation and differentiation in controlled conditions in vitro", "will yield corroborative evidence and facilitate the means to test putative mechanism(s ) by manipulating gene expression .", "together , these approaches may provide insight into molecular axes that can be tested against the background of those operational in zebrafish ( 7 , 8 , 19 ) .", "for example , the molecular axis defined by lin28 and ascl1 , which facilitates mg - mediated regeneration in zebrafish , is a valid target for study in mammals .", "a variety of approaches in higher vertebrates have demonstrated important regulatory roles for lin28 in the maintenance of neural progenitors and neuroglial decision ( 20 ) .", "it is likely that lin28 influences different developmental function by contextual recruitment of hmga2 , a gene encoding a dna architecture protein ( 21 , 22 ) and ascl1 ( 23 ) by directly regulating the heterochronic mirna let-7 ( 17 , 21 , 23 ) .", "let-7 targets hmga2 ( 17 , 21 ) and ascl1 ( 23 ) ; therefore , the lin28-let-7-hmga2/ascl1 axis has emerged as an evolutionary conserved axis that could be a candidate for unlocking neurogenic potential of mg .", "for example , overexpression of lin28a ( 24 ) in the enriched mg ( fig .", "2a ) prompted these cells to acquire neuronal morphology and to express immunoreactivities and transcripts corresponding to neuronal genes ( fig .", "more importantly , neuronal differentiation was accompanied by an increase in levels of mir-124 , a proneural mirna ( 25 ) , hmga2 , and ascl1 , and a decrease in those of rest , a global inhibitor of neuronal differentiation ( 26 ) , thus demonstrating the capacity of lin28a in activating the neurogenic program in mg . however , targeting the lin28-let-7-hmga2/ascl1 axis alone may not be sufficient for the functional reprogramming of mg along neuronal lineage , given that its influence may not be able to completely counter the inhibitory resistance of the rest axis .", "rest , by suppressing the expression of proneural mirnas , mir-124 and mir-9 - 9 * , whose targets are proglial genes encoding sox9 and he s family of regulators , may keep mg non - neuronal and therefore non - regenerative ( 25 ) .", "the existence of the rest - mir-124 - 9 - 9 * -sox9/hes1 axis in mg suggested that either inhibiting rest or ectopically expressing mir-124/mir-9 - 9 * or both may direct mg along the neuronal lineage .", "it is likely that the ectopic expression of mir-124/mir-9 - 9 * in itself might be effective , as they directly or indirectly influence the expression and function of rest ( 25 ) .", "for example , over expression of mir-124 - 9 - 9 * ( 27 ) in mg ( fig .", "2a ) facilitated neuronal morphology with accompanied expression and suppression of neuronal- and glial - specific genes , respectively ( fig .", "expression of both ascl1 and lin28a was up regulated and that of rest was down regulated in the perturbed groups , compared to controls .", "however , no significant difference in hmga2 transcript levels was observed , suggesting a threshold requirement of lin28a levels for influencing hmga2 expression .", "that mir-124 and mir-9 - 9 * could facilitate proneural function of ascl1 was demonstrated by the improvement of ascl1-mediated reprogramming of mg by ectopically expressed mirnas ( 28 ) .", "together , these observations posit these molecular axes as valid intrinsic targets for studying and unlocking the dormant regenerative potential of mg ."], ["mg , the guardian of homeostasis in the retina , respond to injuries by proliferating and migrating out of the inner nuclear layer ( 5 ) .", "however , despite proliferation and migration as in zebrafish retina , as mentioned , mammalian mg do not initiate regeneration effectively .", "this raises the possibility that , in addition to internal constraints discussed above , the environment in the mammalian retina might not be conducive for neurogenic conversion of mg . a niche - based approach to unlock neurogenic potential of mg will involve examining the relationship of mg with neighboring retinal cells , microglia , immigrant astrocytes , and endothelial cells in the context of signaling pathways and their capacity to engage the molecular axes involved in reprograming along neuronal lineage .", "for example , the understanding of the role of microglia , one of the first responders to injury , and which may mediate regeneration through inflammatory signals , is still evolving in the retina ( 29 ) .", "studies have demonstrated that notch ( 8 , 30 ) , wnt ( 5 , 6 ) , fgf ( 6 , 8) , insulin and insulin - like growth factor-1 ( 8) , shh ( 31 ) , and cytokines such as tnf ( 8 , 19 ) , leptin , and il-11 ( 32 ) may play important roles in mediating the influence of the niche in reprogramming mg . however , the identity of cells delivering the signals and whether or not these signals and their associated pathways act in concert , which would determine niche - based strategies to promote regeneration , remain largely unknown .", "for example , it has been observed that tnf released by dying photoreceptors in mechanically damaged zebrafish retina may constitute one of the early signals for activating mg for regeneration ( 19 ) .", "also , it was reported that zebrafish mg respond to retinal injury by secreting leptin and il-11 , which help reprogram cells in an autocrine fashion ( 32 ) . in both cases , these cytokines facilitated injury - dependent induction of ascl1 , a key step in reprogramming of mg ( 8) . whether or not signaling - mediated by these factors are similarly involved in the mammalian retina remains to be demonstrated .", "although the identity of cells delivering notch signaling in mg remains speculative , emerging evidence from zebrafish and higher vertebrates posit it as an important niche - based target for mg - dependent regeneration .", "it remains active long after facilitating the differentiation of mg , enabling them to launch the proliferative response when injury takes place ( 4 , 5 ) .", "however , persistence of notch signaling in activated mg , which are poised for neuronal conversion , might be deleterious for regenerative process , given the inhibitory influence of notch signaling on the activation of proneural genes ( 33 ) . the inability to confine", "the activation of notch signaling in mg to a narrow temporal window following injury may be one of the reasons for their anemic neuronal conversion in mammals , compared to that in zebrafish ( 30 ) .", "emerging evidence , once again from the zebrafish model , suggests that these pathways , including notch signaling , may act in concert and mg integrate the diverse niche - based influence of a variety of signaling molecules for a calibrated regenerative response to injury ( 8) .", "therefore , niche - related information is crucial for understanding the temporal and spatial modulation of signaling required for activating mg and shifting them from the state of activation to neuronal differentiation ."], ["this is fundamental information missing from studies of mg - dependent regeneration , without which the underlying mechanisms remain difficult to grasp and the therapeutic target rather illusory .", "currently , the identity of the activated mg represents cells in the inner nuclear layer of the retina , which have incorporated brdu in response to injury and express mg - specific markers .", "given the transition of mg from a quiescent to a proliferative state and the generation of their progenies in different stages of development , the identity based on brdu incorporation , though important , is of limited value .", "attempts have been made to prospectively enrich activated mg by hoechst dye efflux assays and to characterize them as sp cells , a phenotype shared by the majority of stem cells ( 4 , 6 ) . however , since sp cells are also heterogeneous , this functional approach has limitations in the absence of specific cellular markers .", "although several signaling pathways ( e.g. notch signaling ) and intrinsic factors ( e.g. , ascl1 ) have been observed to regulate mg activation in fish , birds , and rodents ( 8) , their status as markers of activated mg in different stages of regeneration remain unspecified .", "progress in understanding the regenerative mechanisms in tissues like blood ( 34 ) , intestine ( 35 ) , and skin ( 36 ) has been facilitated by the identification and characterization of markers of the resident stem cells and their progenies . within the cns ,", "insight into regeneration in the svz has come from the characterization of b1 quiescent and active progenitors and resulting neuroblasts ( 37 ) .", "therefore , to follow mg - mediated regeneration , spatially and temporally , and to identify the stage - specific intrinsic players , a concerted effort is needed to search for reliable and reproducible markers ."], ["currently , there is no consensus on reliable and reproducible mammalian models to study the activation and neurogenic potential of mg .", "rodent models representing retinal injuries , ranging from those caused by the exposure to light , neurotoxins , and genetic mutations have been used .", "given the observations that glia respond differently to different types and durations of injuries ( 2 ) , that their levels of activation differ from species to species ( ahmad et al .", ", unpublished observations ) , and that within the same species there are strain differences in responses ( 38 ) , a case may be made for developing injury- and species / strain - specific models for reproducible and unambiguous examination of mg neurogenic potentials .", "these models will be valuable in incorporating transgenic technology for reliable lineage tracing , and more importantly for molecular characterization of activated mg .", "characterization of these cells for gene regulatory network and epigenetic signature is essential to shed light on the status of their neurogenic potential and approaches to unlock it .", "furthermore , cell type specific injury models would test the ability of mg to replace specific neuronal types , which will be helpful in designing approaches for mg - dependent therapeutic regeneration .", "for example , the loss of vision in two of the intractable blinding diseases , age - related macular degeneration ( amd ) and glaucoma , is due to selective loss of photoreceptors and retinal ganglion cells ( rgcs ) , respectively .", "the neurotoxin injury models where exposure of the retina to n - methyl - n - nitrosourea ( mnu ) ablates photoreceptors ( 39 ) and nmda causes the degeneration of rgcs ( 40 ) would reveal whether or not mg could differentiate along specific neuronal types . also , it may shed light on if mg - dependent therapeutic regeneration would be a practical approach to address degenerative changes in amd and/or glaucoma ."], ["the discipline of mg - based regeneration is a recent one , particularly in mammals . with information on the nature of", "the activated mg and the molecular axes mediating their response to the niche , aided by reproducible animal models and lineage reporters , a pharmacological recruitment of these endogenous progenitors for therapeutic regeneration is a near possibility . in addition , information emerging from these studies will help us understand how the neurogenic potential in the adult mammalian brain is constrained relative to that of lower vertebrates , currently a significant knowledge gap ."]]}
{"article_id": "PMC3662402", "article_text": ["the field of utility organometallics is increasingly turning to more complex mixed - metal systems to deliver superior results , whether this be for metal  halogen exchange , alkylation , insertion , or the trapping of reactive intermediates . in theory", ", mixed - metal reagents can offer more flexible , tunable solutions to any problem compared to more traditional monometallic alternatives due to the increased number of components which can be varied .", "furthermore , the different metal centers and ligand sets can exhibit cooperativity ( or synergism ) to produce new reactivities inaccessible to the monometallic components .", "hydrogen abstraction is an area in which the lure of such mixed - metal systems is proving to be irresistible .", "the combination of an alkali metal with a range of different subordinates ( i.e. , metals that would be expected to be less reactive than the alkali metals - zn , mg , mn , cr , fe , cu , cd , al ) have been integrated with a diverse array of ligands ( alkyl , amido , alkoxy , halide ) and have been efficiently deployed in both ethereal and hydrocarbon media to overcome some of the great challenges facing deprotonation chemistry such as the incapability to metalate low brnsted acidity substrates , lack of compatibility with sensitive functionalities , low selectivity and the need to perform reactions under cryogenic conditions .", "recently our own group has started to investigate the use of diamines in a bimetallic context , reporting in a communication the synthesis of the diazaethene zincate complex ( tmeda)li[(ipr)nch = chn(ipr)]zn(tbu ) 1 .", "1,3-diazabutadiene ligands , from which diazaethene ligands are often prepared , are increasingly important within coordination chemistry due to their flexible coordination modes and redox properties .", "the synthesis of this complex from the fully saturated ( ipr)nhch2ch2nh(ipr ) ( diisopropylethylenediamine ) 2 ( scheme 1 ) displayed a new mode of hydrogen activation previously unseen within zincate chemistry .", "we now report a detailed mechanistic overview of this surprising transformation from both experimental and theoretical perspectives .", "as reported previously , the cocomplexation of the monolithiated diamine li[(ipr)nch2ch2nh(ipr ) ] with ( tbu)2zn(tmeda ) at 0 c gives the expected product ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 ( scheme 1 ) . in a fashion similar to that of the two - step mechanism for monoamido zincates originally proposed by uchiyama et al .", ", the amino n  h group of 3 is rapidly deprotonated by a tbu ligand to form the dimetalated diamido ( tmeda)li[(ipr)nch2ch2n(ipr)]zn(tbu ) 4 with the evolution of ibuh .", "although diamido 4 has thus far not crystallized , it has been characterized by multinuclear nmr spectroscopy , and we have succeeded in the crystallographic identification of several analogous structures . using the homologous ( ipr)nhch2ch2ch2nh(ipr ) ( diisopropylpropylenediamine ) , the dimetalated diamido ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) 5 ( scheme 2 and figure 1 ) could be crystallized from the corresponding reaction of li[(ipr)nch2ch2ch2nh(ipr ) ] with ( tbu)2zn(tmeda ) .", "unfortunately , the crystal data are of insufficient quality to allow discussion of the geometric parameters of the structure , but the connectivity is definite .", "zincate 5 contains an ncccnzn six - membered ring in a boat - type conformation .", "the diamido ligand thus chelates the zn center while its two ipr arms are oriented out of the plane of the ring .", "the ( tmeda)li cation is coordinated to the hull of the boat , anchored exclusively to the two secondary amido groups to produce a puckered linznn four - membered ring .", "h nmr spectroscopic studies confirm the fully saturated nature of the propylene bridge with methylene resonances at 3.33 , 2.95 , and 1.93 ppm , while no characteristic diazaethene resonances were present in the 56 ppm region .", "molecular structure of the diamido zincate ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) 5 . hydrogen atoms and disordered tbu / tmeda components have been omitted for clarity .", "on utilizing thf as a donor ligand in the absence of tmeda , in a repeat of the synthesis of 4 , the dimeric thf - solvated product { ( thf)li[(ipr)nch2ch2n(ipr)]zn(tbu)}26 was isolated and subsequently crystallographically characterized ( scheme 2 and figure 2 ) .", "lithium zincate 6 has a centrosymmetric molecular structure consisting of two [ ( ipr)nch2ch2n(ipr)]zn(tbu ) complex anions , juxtaposed , and bridged by two ( thf)li cations to produce an eight - membered ( linznn)2 ring .", "the cyclic conformation of 6 allows for the symmetrical coordination of each amido group to one lithium and one zinc center . both sets of metal centers occupy distorted trigonal planar geometries .", "the ipr groups on the amido ligands lie syn with respect to the znnccn ring , while the nonplanar nature of the diamide ( n1c4c5n2 torsion angle of 50.0(2) ) as well as its n  c [ 1.471(3 )  ] and c  c [ 1.530(3 )  ] bond distances , which are comparable with those found in the ruthenium complex [ ( ipr)nch2ch2n(ipr)]ruh2(co)5 [ n  c , 1.48(1 )  ; c  c , 1.52(1 )  ] , establish that the ch2ch2 backbone remains saturated .", "thermal ellipsoids are shown at the 50% probability level . selected bond lengths [  ] and angles [ deg ] : zn1n1 , 2.019(2 )", "; zn1n2 , 1.986(2 ) ; zn1c9 , 2.019(9 ) ; li1n1 , 2.001(4 ) ; li1n2(a ) , 2.041(4 ) ; li1o1 , 2.004(4 ) ; n1c4 , 1.471(3 ) ; n2c5 , 1.470(3 ) ; c4c5 , 1.530(3 ) ; n1zn1n2 , 91.20(7 ) ; n1zn1c9 , 127.5(2 ) ; n2zn1c9 , 141.2(2 ) ; n1c4c5n2 , 50.0(2 ) ; n1li1n2(a ) , 127.0(2 ) ; n1li1o1 , 117.7(2 ) ; n3li1o1 , 114.9(2 ) . to gain mechanistic insight a detailed nmr spectroscopic study", "was carried out on the reaction between the monometallic compounds li[(ipr)nch2ch2nh(ipr ) ] and ( tbu)2zn(tmeda ) .", "a li nmr spectrum of isolated crystals of the cocomplexation product ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 in c6d6 solution reveals two major resonances at 0.37 ppm and 0.63 ppm , suggesting a mixture of products .", "when this solution is gently warmed and reanalyzed , the resonance at 0.37 ppm is absent , intimating that the product associated with this low - frequency resonance has been fully consumed , and only the resonance at 0.63 ppm remains ( see figure s24 in supporting information ) .", "this is consistent with the disappearing resonance at 0.37 ppm representing the bisalkyl zincate 3 which , via an intramolecular deprotonation and concomitant release of ibuh , forms the diamido complex 4 .", "further support for this interpretation can be found when considering how close the signal assigned to complex 4 ( 0.63 ppm ) comes to that of the structurally analogous propylene derivative 5 ( 0.75 ppm , i.e. a difference of only 0.12 ppm ) .", "when the reaction was repeated in situ at 0 c and an aliquot removed and analyzed by li nmr spectroscopy , two pertinent resonances at 2.39 ppm , and 1.71 ppm , as well as the resonance belonging to the previously identified intermediate 4 , are present ( see figure s24 in supporting information ) .", "li dosy nmr experiments reveal that the intermediate responsible for the resonance at 1.71 ppm has a molecular weight similar to that of 4 ( see figure s25 in supporting information ) , presumably ruling out an aggregation process for the formation of this , as yet , unidentified species . comparison with the li nmr spectra of the diazaethene 1 in c6d6 reveals that the signal at 2.39 ppm is the result of a trace amount of this final product . due to the highly reactive , transient nature of the bis - alkylzincate 3 , its presence is not detected in this in situ nmr study .", "another aliquot of the solution was removed after a 2-day stir at room temperature , but the recorded li nmr spectrum indicated no significant further compositional change had taken place .", "aliquots were then removed after 5 min , 1 h , and 2 h reflux time , and the recorded spectra revealed a gradual transformation from the saturated intermediate 4 to the unsaturated final product 1 .", "after 2 h reflux , decomposition of the reaction mixture is evidenced by the deposition of a black solid .", "any further intermediates along the reaction pathway must be too short lived to be visible on the nmr time scale .", "the next step in the reaction pathway 4  1 has been shown to likely proceed through a hydride intermediate as we successfully trapped a hydride by using the electrophilic ketone ( tbu)2co .", "the hydride produced can then deprotonate the now allylic proton on the ethylene bridge to give the final product .", "the system must then find a way of solubilizing and activating the produced lih , which is normally insoluble in hydrocarbon solvents . a more typical dehydrogenation pathway involving redox active transition metals , such as those reported by brookhart ,", "can be ruled out as there are no redox - active metals present in our alkali metal ", "instead , the reaction could be envisaged to proceed through a ligand - sited oxidation followed by a reduction ( scheme 3 ) .", "this reaction can thus be said to exploit a unique three - way cooperation , where the chemical properties of the two metals and the ethylenediamide ligand combine to allow a type of chemistry otherwise unavailable . in order to explore the possible pathways by which this reaction could proceed we carried out detailed mechanistic investigations using density functional theory ( dft ,", "see the supporting information for full details on the level of theory employed ) in order to explore the potential energy surface ( pes ) associated with the proposed mechanisms .", "we initially inspected the structure and stability of the monomer species 4 at the dft level of theory . in agreement with the three - carbon bridge analogue", "( 5 , figure 1 ) , 4 is found to be a stable minimum on the pes with the diamido ligand coordinated to the zn atom ( n  zn : 2.04  and 1.99  ) to form a five - membered ring ( nccnzn ) in a slightly twisted ( nccn torsion of 31.4 ) envelope conformation with the zn atom below the nccn plane , ( figure 3 ) . the ( tmeda)li cation is coordinated directly above the two n atoms of the five - membered ring by two essentially equivalent li ", "based on the optimized structure of 4 two different mechanisms by which the proposed zinc hydride species could be formed are possible .", "all h atoms , except those involved in the reaction , are omitted for clarity .", "the h atom could be initially abstracted by the li cation to form a zn  h bond below the plane of the ring , or direct abstraction by the zn to form the zn  h bond above the ring plane .", "optimisation of the two hydride intermediate structures revealed that both were stable minima on the pes but were formed in endothermic reactions relative to 4 ( figure 4 ) .", "the intermediate with the h atom above the nccn plane ( inta ) is destabilized by 14.7 kcal / mol ( g ) relative to 4 , while the intermediate with the h atom below the plane ( intb ) is destabilized by 8.6 kcal / mol ( g ) , relative to 4 .", "while both reactions are endothermic , the relative stabilities of the intermediates are not sufficiently different to determine which pathway is more favorable .", "therefore , we characterized the transition states leading to the intermediate structures in order to determine whether there was a kinetic preference toward either species . the activation barrier", "associated with the formation of inta is 23.2 kcal / mol ( g * ) , compared to a barrier of 31.8 kcal / mol ( g * ) for the formation of intb .", "an activation barrier of 31.8 kcal / mol is still accessible under the experimental conditions employed in the reactivity of 4 ; thus , while inta is expected to be preferentially formed as an initial hydride intermediate in the formation of the diazaethene product ( 1 ) , we also considered intb as a potential intermediate from which the second hydrogen abstraction could occur .", "abstraction of the second h atom from inta occurs with a barrier of 17.4 kcal / mol , relative to inta , ( g * ) in an exothermic reaction ( g = 13.2 kcal / mol ) to generate h2 and the final product 1 . however , despite exhaustive sampling , no transition state could be located that corresponded to the formation of h2 and 1 starting from intb .", "therefore , the overall reaction 4  1 + h2 , which is essentially thermoneutral ( g = 1.5 kcal / mol , figure 4 ) , appears to preferentially occur via the hydride inta .", "free energy ( g ) profile for the formation of 1 from 4 via a hydride intermediate .", "the path in red corresponds to the formation of the hydride intermediate above the ring plane ( inta ) , while the path in blue corresponds to the formation of the hydride intermediate below the ring plane ( intb ) .", "the relative stabilities of ts(4-inta ) and ts(4-intb ) ( figure 4 ) are dictated by the different roles that the li and zn centers play in the two different mechanisms . in both transition states , in order for the zn  h bond to be formed , one zn ", "n bond must be broken such that the zn atom is able to accommodate the incoming h and the unsaturated n = c bond can form . within the ts(4-inta )", "n bond breaking is facilitated by the formation of a stabilizing interaction between the n atom and the li .", "this is evidenced by the rotation around the c  c bond [ increase in the dihedral angle from 31.4 in 4 ( figure 3 ) to 56.3 ( figure 5a ) ] , which decreases the li ", "n distance from 2.02  ( figure 3 ) to 1.98  ( figure 5a ) and increases the zn ", "n distance from 2.04  ( figure 3 ) to 3.39  ( figure 5a ) .", "in contrast , the rotation around the c  c bond in ts(4-intb ) is not as great ( 34.1 , figure 5b ) .", "in addition , in ts(4-intb ) the li atom is forced to migrate toward the second n atom in order to facilitate the transfer of the h atom beneath the plane of the ring .", "n distance to 3.60  for the primary n ( figure 5b ) , and consequently , the breaking of the zn ", "n bond length is only slightly increased from 2.04  in 4 ( figure 3 ) to 2.12  in ts(4-intb ) ( figure 5b ) .", "thus , the formation of the hydride intermediate is truly the result of the synergic interplay between the zn ( stabilizing the formation of the zn  h bond ) and li ( stabilizing the breaking of the zn ", "returning to experimental studies , a molecular hydride species [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 has been isolated by using a variation of this mixed - metal approach with the ethylenediamide ligand , demonstrating that this system has the ability to encapsulate lih ( scheme 4 and figure 6 ) .", "dilithium hydridozincate 7 was synthesized while attempting to generate  [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)2  by the n ", "h deprotonation of ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 with a molar equivalent of nbuli(tmeda ) ( scheme 4 ) .", "[ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)2  facilitates the -hydride formation and elimination of ibuh to generate the zinc hydride species 7 .", "dft calculations have confirmed that such a process [ 4 + ( tmeda)lih  7 ] would be exothermic ( g = 28.4 kcal / mol ) .", "molecular hydride 7 can also be produced via the alternative reaction of the usually kinetically challenged base [ ( tmeda)li]2zn(tbu)4 with ( ipr)nhch2ch2nh(ipr ) with an improved yield of 65% relative to 35% .", "molecular structure of the zincate hydride complex [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 with hydrogen atoms ( except for the hydride ion ) omitted for clarity .", "thermal ellipsoids are shown at the 50% probability level . selected bond lengths [  ] and angles [ deg ] : li1h100 , 1.96(2 ) ; li2h100 , 2.07(2 ) ; zn1h100 , 2.14(2 ) ; li1n2 , 1.979(4 ) ; li2n1 , 1.989(4 ) ; zn1n1 , 2.043(2 ) ; zn1n2 , 2.074(2 ) ; zn1c1 , 2.045(3 ) ; n1c9 , 1.481(3 ) ; n2c8 , 1.448(3 ) ; c8c9 , 1.523(4 ) ; n1zn1h100 , 92.8(6 ) ; n1zn1n2 , 89.66(8 ) ; n1zn1c1 , 133.1(1 ) ; n2zn1c1 , 125.19(9 ) ; n2zn1h100 , 92.8(5 ) ; h100zn1c1 , 113.0(6 ) ; n2li1n5 , 121.8(2 ) ; n2li1n6 , 129.6(2 ) ; n2li1h100 , 101.5(6 ) ; n5li1n6 , 85.7(2 ) ; n5li1h100 , 115.9(6 ) ; n6li1h100 , 101.6(6 ) .", "mixed - metal zincate hydrides have repeatedly demonstrated their utility in organic synthesis as reagents for chemoselective , diastereoselective , and catalytic reductions .", "highly soluble , stable , and well - defined zincate hydride species are thus highly desirable . despite this , however , structurally characterized alkali metal zincate hydrides are extremely rare . to date the only examples on the ccdb are the zinc  zinc bonded arzn(-h)(-na)znar ( ar = c6h3 - 2,6-[c6h3-(ipr)2]2 ) , the heterocubanes [ ( tbuoznh)4n(liotbuthf)n ] ( n = 03 ) , and the higher - order zincates na2[(et)2znh]2 and na3[(ipr)3zn(-h)zn(ipr)3 ] .", "although metal  hydride complexes are notoriously insoluble , a reputation perpetuated by common hydride reagents such as lih and cah2 , the hydride carrier 7 is soluble in the nonpolar solvent hexane .", "the structure of zinc hydride 7 bears a partial resemblance to that of the thf solvated dimer 6 , with a [ ( ipr)nch2ch2n(ipr)]zn(tbu ) complex anion having a lithium cation bound to each nitrogen atom . the li ", "n amido bond distances in 7 ( average 1.984  ) are close to those in dimer 6 ( average 2.021  ) . likewise the zn ", "n bond distances in hydride 7 [ 2.074(2 )  and 2.043(2 )  ] are only marginally longer than those in alkylamido 6 ( average 2.002  ) , owing to the increased number of anions bound to the zinc .", "the tbu group has been pushed out of the plane of the amido ligand to allow the zinc center to enter a distorted tetrahedral geometry by binding to the hydride ligand . as in the lithium zincate 6 , the n ", "c [ 1.481(3 )  and 1.448(3 )  ] and c  c [ 1.523(4 )  ] bond lengths in 7 are consistent with an unactivated ethylene bridge .", "the trapped 3-hydride was successfully located and freely refined in the x - ray diffraction study .", "it caps a li2zn triangle asymmetrically with unequal li  h bond distances [ 1.96(2 )  and 2.07(2 )  ] and a longer zn  h bond [ 2.14(2 )  ] .", "the ccdb at the time of writing contains only 45 compounds exhibiting a zn  hydride bond . excluding an anomalously long contact within a zinc borohydride compound ( 2.409  ) , zn  hydride bond lengths range from 1.4092.167  with an average distance of 1.771  .", "the zn  hydride contact in 7 is thus long - range  to the best of our knowledge , the longest zn hydride contact in a non - borohydride complex to date ( the value of 2.167  mentioned above also comes from a borohydride complex ) .", "there are currently 147 compounds in the ccdb containing li hydride contacts with their lengths spanning the wide range 1.6072.802  .", "the average distance ( 2.044  ) is comparable with those found in 7 . to the best of our knowledge", "however , there have been various dilithio 3-hydride compounds reported with other metals such as ga , al , zr , w , sm , ta , and fe .", "variable - concentration multinuclear ( h , c and li ) nmr spectroscopic studies of 7 , as well as exchange spectroscopic ( exsy ) experiments , confirm the presence of a dynamic equilibrium in c6d6 solution . as well as resonances consistent with the solid state structure of 7 ,", "mixed - metal zincate complex 4 can thus be described as a molecular scaffold for the molecularization of the usually insoluble polymeric lih .", "an examination of the li nmr spectrum reveals a doublet with a jli  h coupling constant of 13.3 hz , confirming the retention of the li ", "only a few examples have been detected since the first measurement by bergman of ( cp*)irh2sime3li(pmdeta ) ( cp * = c5me5 , pmdeta = n , n , n,n,n-pentamethyldiethylenetriamine ) in 1985 .", "those detected have coupling constants ranging from 6.414.7 hz , placing our example at the upper end of the literature examples .", "surprisingly , two independent h resonances , at 3.27 and 3.19 ppm , were detected coupling with the doublet in the li nmr spectrum .", "this result can be tentatively assigned to the li resonance for the zincate hydride 7 sharing the same chemical shift as dissociated ( tmeda)lih , in agreement with the presence of a dynamic equilibrium in solution .", "given the equilibrium between 4 and 7 , the formation of a diazaethene compound , analogous to 1 , may also occur using 7 as the starting point .", "the optimized structure of 7 closely matches the experimental x - ray structure , with a mean unsigned error of 0.07  between the measured and calculated bond lengths .", "the largest deviation between the calculated and experimental values occurs for the interactions involving the hydride ion ( e.g. , zn  h : 1.94  calculated vs. 2.14  experimental , see table s1 in supporting information for details ) .", "the deviations in the hydride ion distances may be due to the uncertainty inherent in the crystallographic determination of hydride positions ; however , even with the shorter calculated zn ", "h interaction , at 1.94  the zn  h contact is clearly long - range .", "the introduction of the ( tmeda)lih ligand to 4 to form 7 results in a different mechanism for the formation of the diazaethene compound .", "one of the ethylene c8 hydrogen atoms from 7 ( figure 7 ) is aligned for abstraction by the dilithio hydride to form the intermediate ( intc ) containing a ( lih)2 unit .", "the formation of intc occurs in an endothermic step ( g = 21.7 kcal / mol ) , relative to 7 , with an associated barrier of 30.1 kcal / mol ( g * , figure 7 ) .", "the subsequent abstraction of the h atom from c9 ( figure 7 ) to form h2 and the diazaethene compound 8 , occurs with a barrier ( g * ) of 11.8 kcal / mol , relative to intc , in an exothermic reaction ( g = 16.7 kcal / mol ) .", "overall , the reaction from 7 to form 8 is slightly endothermic ( g = 5.0 kcal / mol , figure 7 ) . from the intermediate structure ( intc )", "the barriers to the forward and reverse reaction ( i.e. , the formation of 8 + h2 and 7 , respectively ) are comparable in energy ( g * = 3.4 kcal / mol ) . therefore , although the equilibrium from intc will be in the direction of 7 , the formation of 8 + h2 from this structure should also occur to some extent .", "free energy ( g ) profile for the formation of 8 from 7 via a dihydride intermediate .", "the possible formation of a zn hydride intermediate , in analogy to the mechanisms explored for the formation of 1 ( via inta ) was also investigated .", "the barrier associated with the formation of this hydride intermediate ( g * = 28.4 kcal / mol ) is accessible under the experimental reaction conditions and the intermediate produced in this reaction is destabilized by 21.1 kcal / mol , relative to 7 .", "however , the barrier to the subsequent second hydrogen abstraction ( i.e. , the formation of 8 + h2 ) is 34.4 kcal / mol , which results in a strong preference for the reverse reaction back to the reactant 7 ( g * = 6.0 kcal / mol ) . as such", ", the mechanism resulting in the formation of dihydride intermediate ( intc , figure 7 ) is expected to be the dominant mechanism for the formation of 8 + h2 , from 7 . despite the comparable energetics for the formation of 8 from 7 ( via intc ) and 1 from 4 ( via inta ) , the mechanisms and the synergic interaction of the two metals are distinctly different .", "the presence of the second li atom in 7 ( relative to 4 ) allows the zn to stabilize the formation of the c = n bond ( the zn ", "n bond increases from 2.07  in 7 to 2.18  in ts(7-intc ) , figure 8) , while the li atom is able to facilitate the c  h bond breaking step .", "finally , the free energy associated with the loss of a ( tmeda)lih ligand from 8 to form 1 is slightly less ( g = 24.9 kcal / mol ) than the equivalent association / dissociation reaction between 4 and 7 . therefore , given the similar overall kinetic barriers associated with the generation of the diazaethene species starting from 4 and 7 , both reaction mechanisms described in figures 5 and 8 , should be competitive and operate in a complementary fashion to generate the observed product 1 .", "the closest analogy to the synthesis of 1 was reported by veith who formed 1,3-diaza-2-silacyclopentene by the double lithiation of ( tbu)nhch2ch2nh(tbu ) ( di - tert - butylethylenediamine ) with nbuli followed by an electrophilic quench with cl2si(me)n(h)tbu .", "it was acknowledged at the time that the process leading to this dehydrogenation was unclear but it was noted that the reaction was highly concentration dependent . for dehydrogenation to occur the lithiation step needed to be carried out in a highly concentrated nbuli hexane solution ( 2 m ) .", "this concentration dependence infers a different mechanism of hydrogen activation from that which results in the synthesis of 1 , possibly involving either larger aggregates or an intermolecular process .", "large aggregates of liotbu have previously been found to activate and retain lih in solution .", "the diamido species was never characterized in veith s study . we have confirmed by nmr spectroscopy that ( ipr)nhch2ch2nh(ipr ) , like its homologue ( tbu)nhch2ch2nh(tbu ) , can be activated when refluxed in a concentrated nbuli solution . when the reaction was carried out at concentrations akin to that used in the synthesis of 1", ", then the dilithiated species could be crystallized as a tmeda solvate ( figure 9 ) .", "this product , [ ( tmeda)li(ipr)nch2ch2n(ipr)li]29 , has a distorted ladder structure in the solid state with the diamido ligands adopting a trans  bent conformation allowing them to bridge between opposite corners of alternate rungs .", "n bond distances of 2.227(2 )  and 1.971(2 )  between the outer rungs and 2.057(2 )  between the inner rungs . maintaining the c2 axis of symmetry , the long and short edges of the ladder alternate as the ladder is ascended .", "both outer lithium centers adopt distorted tetrahedral geometries , while the internal lithium atoms are three - coordinate .", "this trapping of a ladder segment instigated by the lewis base tmeda supports gardiner s assertions .", "selected bond lengths [  ] and angles [ deg ] : li1n1 , 2.227(2 ) ; li1n2 , 1.977(3 ) ; li1n3 , 2.209(3 ) ; li1n4 , 2.199(3 ) ; li2n1 , 2.025(2 ) ; li2n2 , 1.971(2 ) ; li2n1(a ) , 2.057(2 ) ; n1c2 , 1.460(2 ) ; n2c1 , 1.458(2 ) ; c1c2 , 1.521(2 ) ; n1li1n2 , 81.23(9 ) ; n1li1n3 , 125.9(1 ) ; n1li1n4 , 129.1(1 ) ; n2li1n3 , 117.3(1 ) ; n2li1n4 , 125.26(1 ) ; n3li1n4 , 83.34(1 ) ; n1(a)li2n1 , 109.6(1 ) ; n1(a)li2n2 , 151.4(1 ) ; n1li2n2 , 86.7(1 ) .", "the activation of ( ipr)nhch2ch2nh(ipr ) has also been achieved starting from the sodium congener na[(ipr)nch2ch2nh(ipr ) ] and ( tbu)2zn(tmeda ) to generate the sodium diazaethene zincate ( tmeda)na[(ipr)nch = chn(ipr)]zn(tbu ) 10 . although the initial cocomplexation adduct  ( tmeda)na[(ipr)nch2ch2nh(ipr)]zn(tbu)2  with one n ", "h bond still remaining is apparently too short - lived to isolate , when the reaction was carried out at 0 c and the resulting solution stored at 30 c , it was possible to isolate and fully characterize the dimeric species { ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)}211 with no n  h bond but containing the saturated ethylene diamido backbone ( figure 10 ) which must exist further along the reaction pathway that ultimately produces 10 .", "two independent centrosymmetric molecules of dimer 11 as well as a hexane molecule form the asymmetric unit .", "sodium zincate 11 is constructed from a 545 fused ring system in a distorted ladder conformation ; the two znnccn rings lie anti about the strictly planar central znnznn core .", "the n  c [ 1.452(6 )  and 1.463(6 )  ] and c  c [ 1.494(7 )  ] bond distances are indicative of a fully saturated bisamide . contrary to binding in the lithium monomer 5 , the heavier alkali metal binds to only one of the available amido groups , with the core nitrogen atoms ( n1 , n1a ) coordinating exclusively to zinc . despite the variety of amide environments around zinc , the zn ", "n bond distances show little variation ( average 2.11  ) . due to the narrow bite angle of the diamido ligand ( 85.9 )", "the zinc center adopts a distorted tetrahedral geometry . a highly distorted tetrahedral geometry around sodium is also completed by a weak agostic interaction [ 2.977(6 )  ] to a methyl arm of the tertiary butyl ligand on zinc .", "this is moderately longer than a similar interaction found in the bis - alkylamido zincate ( tmeda)na(tmp)zn(tbu)2 ( 2.75  ) .", "545 fused ring systems have been found before in metallate complexes containing saturated nccn diamido ligands .", "examples include the lithium lanthanide complexes [ ( thf)3li[1,2-{n(sime3)}2c6h10]mcl2]2 ( m = nd , yb ) and the dimeric gallium species [ { ( bz)nch2ch2n(bz)}li{(bz)nch2ch2n(bz)}gao]2 ( bz = benzyl , ch2c6h5 ) . molecular structure of one of the independent molecules of the dimeric sodium zincate [ ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)]211 . a weak agostic nac contact is denoted by a broken bond .", "hydrogen atoms , ipr groups , and disordered hexane of crystallization have been omitted for clarity .", "selected bond lengths [  ] and angles [ deg ] : na1n1 , 3.720(4 ) ; na2n2 , 2.326(5 ) ; na1n3 , 2.490(6 ) ; na1n4 , 2.490(5 ) ; na1c4 , 2.977(6 ) ; zn1n1 , 2.105(4 ) ; zn1n2 , 2.103(4 ) ; zn1c1 , 2.072(5 ) ; zn1n1(a ) , 2.122(4 ) ; n1c8 , 1.463(6 ) ; n2c9 , 1.452(6 ) ; c8c9 , 1.494(7 ) ; n4na1n3 , 75.4(2 ) ; n4na1n2 , 121.9(2 ) ; n4na1c4 , 147.7(2 ) ; n3na1n2 , 137.7(2 ) ; n3na1c4 , 91.8(2 ) ; n2na1c4 , 87.4(2 ) ; n2zn1c1 , 117.1(2 ) ; n2zn1n1 , 85.9(2 ) ; n2zn1n1(a ) , 110.5(2 ) ; n1zn1c1 , 127.6(2 ) ; n1zn1n5 , 88.8(2 ) ; n5zn1c1 , 120.0(2 ) .", "as might be expected from polarity considerations , the sodium reagent proves more reactive than the lithium analogue , providing after a 30 min reflux in hexane , the sodium diazaethene 10 in 61% isolated yield .", "although the tmeda solvate 10 could be isolated as a crystalline solid , to obtain crystallographic data of adequate precision , the donor ligand thf had to be used in place of tmeda to produce the analogous thf solvate ( thf)3na[(ipr)nch = chn(ipr)]zn(tbu ) 10a ( figure 11 ) . here , the zinc atom is chelated symmetrically [ zn  n bond distances 1.957(2 )  and 1.971(2 )  ] by the diamide ligand , lying below the plane defined by the nccn core of the diazaethene ligand .", "the methine carbons of the isopropyl groups lie in the plane of the diazaethene ligand while their methyl groups protrude above and below this plane .", "the sodium atom lies above the nccn bridge , though slightly off center , coordinating to both pairs of nitrogen and carbon atoms .", "three thf molecules complete the coordination sphere of the sodium center . comparing the c  n [ 1.398  ( average ) ] and c  c [ 1.353(4 )  ] bond distances of the diazaethene core with those found in the lithium analogue ( tmeda)li[(ipr)nch = chn(ipr)]zn(me ) [ 1.400 ( average ) and 1.349(3 )  ] indicates that the change in alkali metal has little effect on the diazaethene ligand .", "dissolution of 10a in c6d6 to perform an nmr spectroscopic analysis was achieved by the addition of a drop of d8-thf . comparing the h nmr spectra of the thf", "solvate 10a with the tmeda solvate 10 reveals only marginal shift changes of the resonances associated with the diazaethene ligand . comparing the h nmr spectra of the homologues ( tmeda)li[(ipr)nch = chn(ipr)]zn(tbu ) 1 and ( tmeda)na[(ipr)nch = chn(ipr)]zn(tbu ) 10 reveals a 0.24 ppm downfield shift of the signal attributable to the ch = ch backbone of the diazaethene ligand on moving from lithium to sodium .", "interestingly , there are two sets of doublets associated with the methyl groups of the isopropyls of the lithium compound but only one set in the sodium congener .", "this would suggest sterically induced hindered rotation of the isopropyl groups in the lithium compound which is relieved on switching to the larger sodium cation .", "the synthesis of several alkali metal zincate diazaethene complexes has been achieved previously . however , to the best of our knowledge , all previous examples were synthesized by the reduction of unsaturated bis - imino ligands .", "therefore , the synthesis of the sodium zincate 10 , along with the lithium zincates detailed in our recent communication , appear to represent the first such complexes to be synthesized via a double ch activation of a saturated diamido ligand . molecular structure of the diazaethene sodium zincate ( thf)3na[(ipr)nch = chn(ipr)]zn(tbu ) 10a . hydrogen atoms and minor disordered thf components have been omitted for clarity .", "thermal ellipsoids shown at the 50% probability level . selected bond lengths [  ] and angles [ deg ] : na1o1 , 2.350(2 ) ; na1o2 , 2.312(2 ) ; na1o3 , 2.312(2 ) ; na1n1 , 2.587(2 ) ; na1n2 , 2.662(2 ) ; na1c8 , 2.612(2 ) ; na1c9 , 2.649(2 )", "; zn1n1 , 1.957(2 ) ; zn1n2 , 1.971(2 ) ; zn1c1 , 2.009(2 ) ; n1na1n2 , 60.81(6 ) ; n1zn1n2 , 85.14(8 ) ; n1zn1c1 , 139.88(10 ) ; n2zn1c1 , 134.97(10 ) .", "conversions of saturated diamines into unsaturated diazaethenes are usually associated with redox - amenable transition metal catalysts .", "however here we show through a combination of experimental ( nmr spectroscopic , x - ray crystallographic ) and theoretical ( dft ) studies that working co - operatively in a molecular mixed - metal system , an alkali metal ( lithium or sodium ) and zinc can bring about such transformations under challenging redox - inactive conditions ."], "abstract_text": ["<S> the surprising transformation of the saturated diamine ( ipr)nhch2ch2nh(ipr ) to the unsaturated diazaethene [ ( ipr)nch = chn(ipr)]2- via the synergic mixture nbum , ( tbu)2zn and tmeda ( where m = li , na ; tmeda = n , n , n,n-tetramethylethylenediamine ) has been investigated by multinuclear nmr spectroscopic studies and dft calculations . </S>", "<S> several pertinent intermediary and related compounds ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)2 ( 3 ) , ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) ( 5 ) , { ( thf)li[(ipr)nch2ch2n(ipr)]zn(tbu)}2 ( 6 ) , and { ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)}2 ( 11 ) , characterized by single - crystal x - ray diffraction , are discussed in relation to their role in the formation of ( tmeda)m[(ipr)nch = chn(ipr)]zn(tbu ) ( m = li , 1 ; na , 10 ) . in addition , the dilithio zincate molecular hydride [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 has been synthesized from the reaction of ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 with nbuli(tmeda ) and also characterized by both x - ray crystallographic and nmr spectroscopic studies . </S>", "<S> the retention of the li  h bond of 7 in solution was confirmed by 7li1h hsqc experiments . also , the 7li nmr spectrum of 7 in c6d6 solution allowed for the rare observation of a scalar 1jli  h coupling constant of 13.3 hz . </S>", "<S> possible mechanisms for the transformation from diamine to diazaethene , a process involving the formal breakage of four bonds , have been determined computationally using density functional theory . </S>", "<S> the dominant mechanism , starting from ( tmeda)li[(ipr)nch2ch2n(ipr)]zn(tbu ) ( 4 ) , involves the formation of a hydride intermediate and leads directly to the observed diazaethene product . </S>", "<S> in addition the existence of 7 in equilibrium with 4 through the dynamic association and dissociation of a ( tmeda)lih ligand , also provides a secondary mechanism for the formation of the diazaethene . </S>", "<S> the two reaction pathways ( i.e. , starting from 4 or 7 ) are quite distinct and provide excellent examples in which the two distinct metals in the system are able to interact synergically to catalyze this otherwise challenging transformation . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Conclusion"], "sections": [["the field of utility organometallics is increasingly turning to more complex mixed - metal systems to deliver superior results , whether this be for metal  halogen exchange , alkylation , insertion , or the trapping of reactive intermediates . in theory", ", mixed - metal reagents can offer more flexible , tunable solutions to any problem compared to more traditional monometallic alternatives due to the increased number of components which can be varied .", "furthermore , the different metal centers and ligand sets can exhibit cooperativity ( or synergism ) to produce new reactivities inaccessible to the monometallic components .", "hydrogen abstraction is an area in which the lure of such mixed - metal systems is proving to be irresistible .", "the combination of an alkali metal with a range of different subordinates ( i.e. , metals that would be expected to be less reactive than the alkali metals - zn , mg , mn , cr , fe , cu , cd , al ) have been integrated with a diverse array of ligands ( alkyl , amido , alkoxy , halide ) and have been efficiently deployed in both ethereal and hydrocarbon media to overcome some of the great challenges facing deprotonation chemistry such as the incapability to metalate low brnsted acidity substrates , lack of compatibility with sensitive functionalities , low selectivity and the need to perform reactions under cryogenic conditions .", "recently our own group has started to investigate the use of diamines in a bimetallic context , reporting in a communication the synthesis of the diazaethene zincate complex ( tmeda)li[(ipr)nch = chn(ipr)]zn(tbu ) 1 .", "1,3-diazabutadiene ligands , from which diazaethene ligands are often prepared , are increasingly important within coordination chemistry due to their flexible coordination modes and redox properties .", "the synthesis of this complex from the fully saturated ( ipr)nhch2ch2nh(ipr ) ( diisopropylethylenediamine ) 2 ( scheme 1 ) displayed a new mode of hydrogen activation previously unseen within zincate chemistry .", "we now report a detailed mechanistic overview of this surprising transformation from both experimental and theoretical perspectives ."], ["as reported previously , the cocomplexation of the monolithiated diamine li[(ipr)nch2ch2nh(ipr ) ] with ( tbu)2zn(tmeda ) at 0 c gives the expected product ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 ( scheme 1 ) . in a fashion similar to that of the two - step mechanism for monoamido zincates originally proposed by uchiyama et al .", ", the amino n  h group of 3 is rapidly deprotonated by a tbu ligand to form the dimetalated diamido ( tmeda)li[(ipr)nch2ch2n(ipr)]zn(tbu ) 4 with the evolution of ibuh .", "although diamido 4 has thus far not crystallized , it has been characterized by multinuclear nmr spectroscopy , and we have succeeded in the crystallographic identification of several analogous structures . using the homologous ( ipr)nhch2ch2ch2nh(ipr ) ( diisopropylpropylenediamine ) , the dimetalated diamido ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) 5 ( scheme 2 and figure 1 ) could be crystallized from the corresponding reaction of li[(ipr)nch2ch2ch2nh(ipr ) ] with ( tbu)2zn(tmeda ) .", "unfortunately , the crystal data are of insufficient quality to allow discussion of the geometric parameters of the structure , but the connectivity is definite .", "zincate 5 contains an ncccnzn six - membered ring in a boat - type conformation .", "the diamido ligand thus chelates the zn center while its two ipr arms are oriented out of the plane of the ring .", "the ( tmeda)li cation is coordinated to the hull of the boat , anchored exclusively to the two secondary amido groups to produce a puckered linznn four - membered ring .", "h nmr spectroscopic studies confirm the fully saturated nature of the propylene bridge with methylene resonances at 3.33 , 2.95 , and 1.93 ppm , while no characteristic diazaethene resonances were present in the 56 ppm region .", "molecular structure of the diamido zincate ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) 5 . hydrogen atoms and disordered tbu / tmeda components have been omitted for clarity .", "on utilizing thf as a donor ligand in the absence of tmeda , in a repeat of the synthesis of 4 , the dimeric thf - solvated product { ( thf)li[(ipr)nch2ch2n(ipr)]zn(tbu)}26 was isolated and subsequently crystallographically characterized ( scheme 2 and figure 2 ) .", "lithium zincate 6 has a centrosymmetric molecular structure consisting of two [ ( ipr)nch2ch2n(ipr)]zn(tbu ) complex anions , juxtaposed , and bridged by two ( thf)li cations to produce an eight - membered ( linznn)2 ring .", "the cyclic conformation of 6 allows for the symmetrical coordination of each amido group to one lithium and one zinc center . both sets of metal centers occupy distorted trigonal planar geometries .", "the ipr groups on the amido ligands lie syn with respect to the znnccn ring , while the nonplanar nature of the diamide ( n1c4c5n2 torsion angle of 50.0(2) ) as well as its n  c [ 1.471(3 )  ] and c  c [ 1.530(3 )  ] bond distances , which are comparable with those found in the ruthenium complex [ ( ipr)nch2ch2n(ipr)]ruh2(co)5 [ n  c , 1.48(1 )  ; c  c , 1.52(1 )  ] , establish that the ch2ch2 backbone remains saturated .", "thermal ellipsoids are shown at the 50% probability level . selected bond lengths [  ] and angles [ deg ] : zn1n1 , 2.019(2 )", "; zn1n2 , 1.986(2 ) ; zn1c9 , 2.019(9 ) ; li1n1 , 2.001(4 ) ; li1n2(a ) , 2.041(4 ) ; li1o1 , 2.004(4 ) ; n1c4 , 1.471(3 ) ; n2c5 , 1.470(3 ) ; c4c5 , 1.530(3 ) ; n1zn1n2 , 91.20(7 ) ; n1zn1c9 , 127.5(2 ) ; n2zn1c9 , 141.2(2 ) ; n1c4c5n2 , 50.0(2 ) ; n1li1n2(a ) , 127.0(2 ) ; n1li1o1 , 117.7(2 ) ; n3li1o1 , 114.9(2 ) . to gain mechanistic insight a detailed nmr spectroscopic study", "was carried out on the reaction between the monometallic compounds li[(ipr)nch2ch2nh(ipr ) ] and ( tbu)2zn(tmeda ) .", "a li nmr spectrum of isolated crystals of the cocomplexation product ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 in c6d6 solution reveals two major resonances at 0.37 ppm and 0.63 ppm , suggesting a mixture of products .", "when this solution is gently warmed and reanalyzed , the resonance at 0.37 ppm is absent , intimating that the product associated with this low - frequency resonance has been fully consumed , and only the resonance at 0.63 ppm remains ( see figure s24 in supporting information ) .", "this is consistent with the disappearing resonance at 0.37 ppm representing the bisalkyl zincate 3 which , via an intramolecular deprotonation and concomitant release of ibuh , forms the diamido complex 4 .", "further support for this interpretation can be found when considering how close the signal assigned to complex 4 ( 0.63 ppm ) comes to that of the structurally analogous propylene derivative 5 ( 0.75 ppm , i.e. a difference of only 0.12 ppm ) .", "when the reaction was repeated in situ at 0 c and an aliquot removed and analyzed by li nmr spectroscopy , two pertinent resonances at 2.39 ppm , and 1.71 ppm , as well as the resonance belonging to the previously identified intermediate 4 , are present ( see figure s24 in supporting information ) .", "li dosy nmr experiments reveal that the intermediate responsible for the resonance at 1.71 ppm has a molecular weight similar to that of 4 ( see figure s25 in supporting information ) , presumably ruling out an aggregation process for the formation of this , as yet , unidentified species . comparison with the li nmr spectra of the diazaethene 1 in c6d6 reveals that the signal at 2.39 ppm is the result of a trace amount of this final product . due to the highly reactive , transient nature of the bis - alkylzincate 3 , its presence is not detected in this in situ nmr study .", "another aliquot of the solution was removed after a 2-day stir at room temperature , but the recorded li nmr spectrum indicated no significant further compositional change had taken place .", "aliquots were then removed after 5 min , 1 h , and 2 h reflux time , and the recorded spectra revealed a gradual transformation from the saturated intermediate 4 to the unsaturated final product 1 .", "after 2 h reflux , decomposition of the reaction mixture is evidenced by the deposition of a black solid .", "any further intermediates along the reaction pathway must be too short lived to be visible on the nmr time scale .", "the next step in the reaction pathway 4  1 has been shown to likely proceed through a hydride intermediate as we successfully trapped a hydride by using the electrophilic ketone ( tbu)2co .", "the hydride produced can then deprotonate the now allylic proton on the ethylene bridge to give the final product .", "the system must then find a way of solubilizing and activating the produced lih , which is normally insoluble in hydrocarbon solvents . a more typical dehydrogenation pathway involving redox active transition metals , such as those reported by brookhart ,", "can be ruled out as there are no redox - active metals present in our alkali metal ", "instead , the reaction could be envisaged to proceed through a ligand - sited oxidation followed by a reduction ( scheme 3 ) .", "this reaction can thus be said to exploit a unique three - way cooperation , where the chemical properties of the two metals and the ethylenediamide ligand combine to allow a type of chemistry otherwise unavailable . in order to explore the possible pathways by which this reaction could proceed we carried out detailed mechanistic investigations using density functional theory ( dft ,", "see the supporting information for full details on the level of theory employed ) in order to explore the potential energy surface ( pes ) associated with the proposed mechanisms .", "we initially inspected the structure and stability of the monomer species 4 at the dft level of theory . in agreement with the three - carbon bridge analogue", "( 5 , figure 1 ) , 4 is found to be a stable minimum on the pes with the diamido ligand coordinated to the zn atom ( n  zn : 2.04  and 1.99  ) to form a five - membered ring ( nccnzn ) in a slightly twisted ( nccn torsion of 31.4 ) envelope conformation with the zn atom below the nccn plane , ( figure 3 ) . the ( tmeda)li cation is coordinated directly above the two n atoms of the five - membered ring by two essentially equivalent li ", "based on the optimized structure of 4 two different mechanisms by which the proposed zinc hydride species could be formed are possible .", "all h atoms , except those involved in the reaction , are omitted for clarity .", "the h atom could be initially abstracted by the li cation to form a zn  h bond below the plane of the ring , or direct abstraction by the zn to form the zn  h bond above the ring plane .", "optimisation of the two hydride intermediate structures revealed that both were stable minima on the pes but were formed in endothermic reactions relative to 4 ( figure 4 ) .", "the intermediate with the h atom above the nccn plane ( inta ) is destabilized by 14.7 kcal / mol ( g ) relative to 4 , while the intermediate with the h atom below the plane ( intb ) is destabilized by 8.6 kcal / mol ( g ) , relative to 4 .", "while both reactions are endothermic , the relative stabilities of the intermediates are not sufficiently different to determine which pathway is more favorable .", "therefore , we characterized the transition states leading to the intermediate structures in order to determine whether there was a kinetic preference toward either species . the activation barrier", "associated with the formation of inta is 23.2 kcal / mol ( g * ) , compared to a barrier of 31.8 kcal / mol ( g * ) for the formation of intb .", "an activation barrier of 31.8 kcal / mol is still accessible under the experimental conditions employed in the reactivity of 4 ; thus , while inta is expected to be preferentially formed as an initial hydride intermediate in the formation of the diazaethene product ( 1 ) , we also considered intb as a potential intermediate from which the second hydrogen abstraction could occur .", "abstraction of the second h atom from inta occurs with a barrier of 17.4 kcal / mol , relative to inta , ( g * ) in an exothermic reaction ( g = 13.2 kcal / mol ) to generate h2 and the final product 1 . however , despite exhaustive sampling , no transition state could be located that corresponded to the formation of h2 and 1 starting from intb .", "therefore , the overall reaction 4  1 + h2 , which is essentially thermoneutral ( g = 1.5 kcal / mol , figure 4 ) , appears to preferentially occur via the hydride inta .", "free energy ( g ) profile for the formation of 1 from 4 via a hydride intermediate .", "the path in red corresponds to the formation of the hydride intermediate above the ring plane ( inta ) , while the path in blue corresponds to the formation of the hydride intermediate below the ring plane ( intb ) .", "the relative stabilities of ts(4-inta ) and ts(4-intb ) ( figure 4 ) are dictated by the different roles that the li and zn centers play in the two different mechanisms . in both transition states , in order for the zn  h bond to be formed , one zn ", "n bond must be broken such that the zn atom is able to accommodate the incoming h and the unsaturated n = c bond can form . within the ts(4-inta )", "n bond breaking is facilitated by the formation of a stabilizing interaction between the n atom and the li .", "this is evidenced by the rotation around the c  c bond [ increase in the dihedral angle from 31.4 in 4 ( figure 3 ) to 56.3 ( figure 5a ) ] , which decreases the li ", "n distance from 2.02  ( figure 3 ) to 1.98  ( figure 5a ) and increases the zn ", "n distance from 2.04  ( figure 3 ) to 3.39  ( figure 5a ) .", "in contrast , the rotation around the c  c bond in ts(4-intb ) is not as great ( 34.1 , figure 5b ) .", "in addition , in ts(4-intb ) the li atom is forced to migrate toward the second n atom in order to facilitate the transfer of the h atom beneath the plane of the ring .", "n distance to 3.60  for the primary n ( figure 5b ) , and consequently , the breaking of the zn ", "n bond length is only slightly increased from 2.04  in 4 ( figure 3 ) to 2.12  in ts(4-intb ) ( figure 5b ) .", "thus , the formation of the hydride intermediate is truly the result of the synergic interplay between the zn ( stabilizing the formation of the zn  h bond ) and li ( stabilizing the breaking of the zn ", "returning to experimental studies , a molecular hydride species [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 has been isolated by using a variation of this mixed - metal approach with the ethylenediamide ligand , demonstrating that this system has the ability to encapsulate lih ( scheme 4 and figure 6 ) .", "dilithium hydridozincate 7 was synthesized while attempting to generate  [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)2  by the n ", "h deprotonation of ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 with a molar equivalent of nbuli(tmeda ) ( scheme 4 ) .", "[ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)2  facilitates the -hydride formation and elimination of ibuh to generate the zinc hydride species 7 .", "dft calculations have confirmed that such a process [ 4 + ( tmeda)lih  7 ] would be exothermic ( g = 28.4 kcal / mol ) .", "molecular hydride 7 can also be produced via the alternative reaction of the usually kinetically challenged base [ ( tmeda)li]2zn(tbu)4 with ( ipr)nhch2ch2nh(ipr ) with an improved yield of 65% relative to 35% .", "molecular structure of the zincate hydride complex [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 with hydrogen atoms ( except for the hydride ion ) omitted for clarity .", "thermal ellipsoids are shown at the 50% probability level . selected bond lengths [  ] and angles [ deg ] : li1h100 , 1.96(2 ) ; li2h100 , 2.07(2 ) ; zn1h100 , 2.14(2 ) ; li1n2 , 1.979(4 ) ; li2n1 , 1.989(4 ) ; zn1n1 , 2.043(2 ) ; zn1n2 , 2.074(2 ) ; zn1c1 , 2.045(3 ) ; n1c9 , 1.481(3 ) ; n2c8 , 1.448(3 ) ; c8c9 , 1.523(4 ) ; n1zn1h100 , 92.8(6 ) ; n1zn1n2 , 89.66(8 ) ; n1zn1c1 , 133.1(1 ) ; n2zn1c1 , 125.19(9 ) ; n2zn1h100 , 92.8(5 ) ; h100zn1c1 , 113.0(6 ) ; n2li1n5 , 121.8(2 ) ; n2li1n6 , 129.6(2 ) ; n2li1h100 , 101.5(6 ) ; n5li1n6 , 85.7(2 ) ; n5li1h100 , 115.9(6 ) ; n6li1h100 , 101.6(6 ) .", "mixed - metal zincate hydrides have repeatedly demonstrated their utility in organic synthesis as reagents for chemoselective , diastereoselective , and catalytic reductions .", "highly soluble , stable , and well - defined zincate hydride species are thus highly desirable . despite this , however , structurally characterized alkali metal zincate hydrides are extremely rare . to date the only examples on the ccdb are the zinc  zinc bonded arzn(-h)(-na)znar ( ar = c6h3 - 2,6-[c6h3-(ipr)2]2 ) , the heterocubanes [ ( tbuoznh)4n(liotbuthf)n ] ( n = 03 ) , and the higher - order zincates na2[(et)2znh]2 and na3[(ipr)3zn(-h)zn(ipr)3 ] .", "although metal  hydride complexes are notoriously insoluble , a reputation perpetuated by common hydride reagents such as lih and cah2 , the hydride carrier 7 is soluble in the nonpolar solvent hexane .", "the structure of zinc hydride 7 bears a partial resemblance to that of the thf solvated dimer 6 , with a [ ( ipr)nch2ch2n(ipr)]zn(tbu ) complex anion having a lithium cation bound to each nitrogen atom . the li ", "n amido bond distances in 7 ( average 1.984  ) are close to those in dimer 6 ( average 2.021  ) . likewise the zn ", "n bond distances in hydride 7 [ 2.074(2 )  and 2.043(2 )  ] are only marginally longer than those in alkylamido 6 ( average 2.002  ) , owing to the increased number of anions bound to the zinc .", "the tbu group has been pushed out of the plane of the amido ligand to allow the zinc center to enter a distorted tetrahedral geometry by binding to the hydride ligand . as in the lithium zincate 6 , the n ", "c [ 1.481(3 )  and 1.448(3 )  ] and c  c [ 1.523(4 )  ] bond lengths in 7 are consistent with an unactivated ethylene bridge .", "the trapped 3-hydride was successfully located and freely refined in the x - ray diffraction study .", "it caps a li2zn triangle asymmetrically with unequal li  h bond distances [ 1.96(2 )  and 2.07(2 )  ] and a longer zn  h bond [ 2.14(2 )  ] .", "the ccdb at the time of writing contains only 45 compounds exhibiting a zn  hydride bond . excluding an anomalously long contact within a zinc borohydride compound ( 2.409  ) , zn  hydride bond lengths range from 1.4092.167  with an average distance of 1.771  .", "the zn  hydride contact in 7 is thus long - range  to the best of our knowledge , the longest zn hydride contact in a non - borohydride complex to date ( the value of 2.167  mentioned above also comes from a borohydride complex ) .", "there are currently 147 compounds in the ccdb containing li hydride contacts with their lengths spanning the wide range 1.6072.802  .", "the average distance ( 2.044  ) is comparable with those found in 7 . to the best of our knowledge", "however , there have been various dilithio 3-hydride compounds reported with other metals such as ga , al , zr , w , sm , ta , and fe .", "variable - concentration multinuclear ( h , c and li ) nmr spectroscopic studies of 7 , as well as exchange spectroscopic ( exsy ) experiments , confirm the presence of a dynamic equilibrium in c6d6 solution . as well as resonances consistent with the solid state structure of 7 ,", "mixed - metal zincate complex 4 can thus be described as a molecular scaffold for the molecularization of the usually insoluble polymeric lih .", "an examination of the li nmr spectrum reveals a doublet with a jli  h coupling constant of 13.3 hz , confirming the retention of the li ", "only a few examples have been detected since the first measurement by bergman of ( cp*)irh2sime3li(pmdeta ) ( cp * = c5me5 , pmdeta = n , n , n,n,n-pentamethyldiethylenetriamine ) in 1985 .", "those detected have coupling constants ranging from 6.414.7 hz , placing our example at the upper end of the literature examples .", "surprisingly , two independent h resonances , at 3.27 and 3.19 ppm , were detected coupling with the doublet in the li nmr spectrum .", "this result can be tentatively assigned to the li resonance for the zincate hydride 7 sharing the same chemical shift as dissociated ( tmeda)lih , in agreement with the presence of a dynamic equilibrium in solution .", "given the equilibrium between 4 and 7 , the formation of a diazaethene compound , analogous to 1 , may also occur using 7 as the starting point .", "the optimized structure of 7 closely matches the experimental x - ray structure , with a mean unsigned error of 0.07  between the measured and calculated bond lengths .", "the largest deviation between the calculated and experimental values occurs for the interactions involving the hydride ion ( e.g. , zn  h : 1.94  calculated vs. 2.14  experimental , see table s1 in supporting information for details ) .", "the deviations in the hydride ion distances may be due to the uncertainty inherent in the crystallographic determination of hydride positions ; however , even with the shorter calculated zn ", "h interaction , at 1.94  the zn  h contact is clearly long - range .", "the introduction of the ( tmeda)lih ligand to 4 to form 7 results in a different mechanism for the formation of the diazaethene compound .", "one of the ethylene c8 hydrogen atoms from 7 ( figure 7 ) is aligned for abstraction by the dilithio hydride to form the intermediate ( intc ) containing a ( lih)2 unit .", "the formation of intc occurs in an endothermic step ( g = 21.7 kcal / mol ) , relative to 7 , with an associated barrier of 30.1 kcal / mol ( g * , figure 7 ) .", "the subsequent abstraction of the h atom from c9 ( figure 7 ) to form h2 and the diazaethene compound 8 , occurs with a barrier ( g * ) of 11.8 kcal / mol , relative to intc , in an exothermic reaction ( g = 16.7 kcal / mol ) .", "overall , the reaction from 7 to form 8 is slightly endothermic ( g = 5.0 kcal / mol , figure 7 ) . from the intermediate structure ( intc )", "the barriers to the forward and reverse reaction ( i.e. , the formation of 8 + h2 and 7 , respectively ) are comparable in energy ( g * = 3.4 kcal / mol ) . therefore , although the equilibrium from intc will be in the direction of 7 , the formation of 8 + h2 from this structure should also occur to some extent .", "free energy ( g ) profile for the formation of 8 from 7 via a dihydride intermediate .", "the possible formation of a zn hydride intermediate , in analogy to the mechanisms explored for the formation of 1 ( via inta ) was also investigated .", "the barrier associated with the formation of this hydride intermediate ( g * = 28.4 kcal / mol ) is accessible under the experimental reaction conditions and the intermediate produced in this reaction is destabilized by 21.1 kcal / mol , relative to 7 .", "however , the barrier to the subsequent second hydrogen abstraction ( i.e. , the formation of 8 + h2 ) is 34.4 kcal / mol , which results in a strong preference for the reverse reaction back to the reactant 7 ( g * = 6.0 kcal / mol ) . as such", ", the mechanism resulting in the formation of dihydride intermediate ( intc , figure 7 ) is expected to be the dominant mechanism for the formation of 8 + h2 , from 7 . despite the comparable energetics for the formation of 8 from 7 ( via intc ) and 1 from 4 ( via inta ) , the mechanisms and the synergic interaction of the two metals are distinctly different .", "the presence of the second li atom in 7 ( relative to 4 ) allows the zn to stabilize the formation of the c = n bond ( the zn ", "n bond increases from 2.07  in 7 to 2.18  in ts(7-intc ) , figure 8) , while the li atom is able to facilitate the c  h bond breaking step .", "finally , the free energy associated with the loss of a ( tmeda)lih ligand from 8 to form 1 is slightly less ( g = 24.9 kcal / mol ) than the equivalent association / dissociation reaction between 4 and 7 . therefore , given the similar overall kinetic barriers associated with the generation of the diazaethene species starting from 4 and 7 , both reaction mechanisms described in figures 5 and 8 , should be competitive and operate in a complementary fashion to generate the observed product 1 .", "the closest analogy to the synthesis of 1 was reported by veith who formed 1,3-diaza-2-silacyclopentene by the double lithiation of ( tbu)nhch2ch2nh(tbu ) ( di - tert - butylethylenediamine ) with nbuli followed by an electrophilic quench with cl2si(me)n(h)tbu .", "it was acknowledged at the time that the process leading to this dehydrogenation was unclear but it was noted that the reaction was highly concentration dependent . for dehydrogenation to occur the lithiation step needed to be carried out in a highly concentrated nbuli hexane solution ( 2 m ) .", "this concentration dependence infers a different mechanism of hydrogen activation from that which results in the synthesis of 1 , possibly involving either larger aggregates or an intermolecular process .", "large aggregates of liotbu have previously been found to activate and retain lih in solution .", "the diamido species was never characterized in veith s study . we have confirmed by nmr spectroscopy that ( ipr)nhch2ch2nh(ipr ) , like its homologue ( tbu)nhch2ch2nh(tbu ) , can be activated when refluxed in a concentrated nbuli solution . when the reaction was carried out at concentrations akin to that used in the synthesis of 1", ", then the dilithiated species could be crystallized as a tmeda solvate ( figure 9 ) .", "this product , [ ( tmeda)li(ipr)nch2ch2n(ipr)li]29 , has a distorted ladder structure in the solid state with the diamido ligands adopting a trans  bent conformation allowing them to bridge between opposite corners of alternate rungs .", "n bond distances of 2.227(2 )  and 1.971(2 )  between the outer rungs and 2.057(2 )  between the inner rungs . maintaining the c2 axis of symmetry , the long and short edges of the ladder alternate as the ladder is ascended .", "both outer lithium centers adopt distorted tetrahedral geometries , while the internal lithium atoms are three - coordinate .", "this trapping of a ladder segment instigated by the lewis base tmeda supports gardiner s assertions .", "selected bond lengths [  ] and angles [ deg ] : li1n1 , 2.227(2 ) ; li1n2 , 1.977(3 ) ; li1n3 , 2.209(3 ) ; li1n4 , 2.199(3 ) ; li2n1 , 2.025(2 ) ; li2n2 , 1.971(2 ) ; li2n1(a ) , 2.057(2 ) ; n1c2 , 1.460(2 ) ; n2c1 , 1.458(2 ) ; c1c2 , 1.521(2 ) ; n1li1n2 , 81.23(9 ) ; n1li1n3 , 125.9(1 ) ; n1li1n4 , 129.1(1 ) ; n2li1n3 , 117.3(1 ) ; n2li1n4 , 125.26(1 ) ; n3li1n4 , 83.34(1 ) ; n1(a)li2n1 , 109.6(1 ) ; n1(a)li2n2 , 151.4(1 ) ; n1li2n2 , 86.7(1 ) .", "the activation of ( ipr)nhch2ch2nh(ipr ) has also been achieved starting from the sodium congener na[(ipr)nch2ch2nh(ipr ) ] and ( tbu)2zn(tmeda ) to generate the sodium diazaethene zincate ( tmeda)na[(ipr)nch = chn(ipr)]zn(tbu ) 10 . although the initial cocomplexation adduct  ( tmeda)na[(ipr)nch2ch2nh(ipr)]zn(tbu)2  with one n ", "h bond still remaining is apparently too short - lived to isolate , when the reaction was carried out at 0 c and the resulting solution stored at 30 c , it was possible to isolate and fully characterize the dimeric species { ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)}211 with no n  h bond but containing the saturated ethylene diamido backbone ( figure 10 ) which must exist further along the reaction pathway that ultimately produces 10 .", "two independent centrosymmetric molecules of dimer 11 as well as a hexane molecule form the asymmetric unit .", "sodium zincate 11 is constructed from a 545 fused ring system in a distorted ladder conformation ; the two znnccn rings lie anti about the strictly planar central znnznn core .", "the n  c [ 1.452(6 )  and 1.463(6 )  ] and c  c [ 1.494(7 )  ] bond distances are indicative of a fully saturated bisamide . contrary to binding in the lithium monomer 5 , the heavier alkali metal binds to only one of the available amido groups , with the core nitrogen atoms ( n1 , n1a ) coordinating exclusively to zinc . despite the variety of amide environments around zinc , the zn ", "n bond distances show little variation ( average 2.11  ) . due to the narrow bite angle of the diamido ligand ( 85.9 )", "the zinc center adopts a distorted tetrahedral geometry . a highly distorted tetrahedral geometry around sodium is also completed by a weak agostic interaction [ 2.977(6 )  ] to a methyl arm of the tertiary butyl ligand on zinc .", "this is moderately longer than a similar interaction found in the bis - alkylamido zincate ( tmeda)na(tmp)zn(tbu)2 ( 2.75  ) .", "545 fused ring systems have been found before in metallate complexes containing saturated nccn diamido ligands .", "examples include the lithium lanthanide complexes [ ( thf)3li[1,2-{n(sime3)}2c6h10]mcl2]2 ( m = nd , yb ) and the dimeric gallium species [ { ( bz)nch2ch2n(bz)}li{(bz)nch2ch2n(bz)}gao]2 ( bz = benzyl , ch2c6h5 ) . molecular structure of one of the independent molecules of the dimeric sodium zincate [ ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)]211 . a weak agostic nac contact is denoted by a broken bond .", "hydrogen atoms , ipr groups , and disordered hexane of crystallization have been omitted for clarity .", "selected bond lengths [  ] and angles [ deg ] : na1n1 , 3.720(4 ) ; na2n2 , 2.326(5 ) ; na1n3 , 2.490(6 ) ; na1n4 , 2.490(5 ) ; na1c4 , 2.977(6 ) ; zn1n1 , 2.105(4 ) ; zn1n2 , 2.103(4 ) ; zn1c1 , 2.072(5 ) ; zn1n1(a ) , 2.122(4 ) ; n1c8 , 1.463(6 ) ; n2c9 , 1.452(6 ) ; c8c9 , 1.494(7 ) ; n4na1n3 , 75.4(2 ) ; n4na1n2 , 121.9(2 ) ; n4na1c4 , 147.7(2 ) ; n3na1n2 , 137.7(2 ) ; n3na1c4 , 91.8(2 ) ; n2na1c4 , 87.4(2 ) ; n2zn1c1 , 117.1(2 ) ; n2zn1n1 , 85.9(2 ) ; n2zn1n1(a ) , 110.5(2 ) ; n1zn1c1 , 127.6(2 ) ; n1zn1n5 , 88.8(2 ) ; n5zn1c1 , 120.0(2 ) .", "as might be expected from polarity considerations , the sodium reagent proves more reactive than the lithium analogue , providing after a 30 min reflux in hexane , the sodium diazaethene 10 in 61% isolated yield .", "although the tmeda solvate 10 could be isolated as a crystalline solid , to obtain crystallographic data of adequate precision , the donor ligand thf had to be used in place of tmeda to produce the analogous thf solvate ( thf)3na[(ipr)nch = chn(ipr)]zn(tbu ) 10a ( figure 11 ) . here , the zinc atom is chelated symmetrically [ zn  n bond distances 1.957(2 )  and 1.971(2 )  ] by the diamide ligand , lying below the plane defined by the nccn core of the diazaethene ligand .", "the methine carbons of the isopropyl groups lie in the plane of the diazaethene ligand while their methyl groups protrude above and below this plane .", "the sodium atom lies above the nccn bridge , though slightly off center , coordinating to both pairs of nitrogen and carbon atoms .", "three thf molecules complete the coordination sphere of the sodium center . comparing the c  n [ 1.398  ( average ) ] and c  c [ 1.353(4 )  ] bond distances of the diazaethene core with those found in the lithium analogue ( tmeda)li[(ipr)nch = chn(ipr)]zn(me ) [ 1.400 ( average ) and 1.349(3 )  ] indicates that the change in alkali metal has little effect on the diazaethene ligand .", "dissolution of 10a in c6d6 to perform an nmr spectroscopic analysis was achieved by the addition of a drop of d8-thf . comparing the h nmr spectra of the thf", "solvate 10a with the tmeda solvate 10 reveals only marginal shift changes of the resonances associated with the diazaethene ligand . comparing the h nmr spectra of the homologues ( tmeda)li[(ipr)nch = chn(ipr)]zn(tbu ) 1 and ( tmeda)na[(ipr)nch = chn(ipr)]zn(tbu ) 10 reveals a 0.24 ppm downfield shift of the signal attributable to the ch = ch backbone of the diazaethene ligand on moving from lithium to sodium .", "interestingly , there are two sets of doublets associated with the methyl groups of the isopropyls of the lithium compound but only one set in the sodium congener .", "this would suggest sterically induced hindered rotation of the isopropyl groups in the lithium compound which is relieved on switching to the larger sodium cation .", "the synthesis of several alkali metal zincate diazaethene complexes has been achieved previously . however , to the best of our knowledge , all previous examples were synthesized by the reduction of unsaturated bis - imino ligands .", "therefore , the synthesis of the sodium zincate 10 , along with the lithium zincates detailed in our recent communication , appear to represent the first such complexes to be synthesized via a double ch activation of a saturated diamido ligand . molecular structure of the diazaethene sodium zincate ( thf)3na[(ipr)nch = chn(ipr)]zn(tbu ) 10a . hydrogen atoms and minor disordered thf components have been omitted for clarity .", "thermal ellipsoids shown at the 50% probability level . selected bond lengths [  ] and angles [ deg ] : na1o1 , 2.350(2 ) ; na1o2 , 2.312(2 ) ; na1o3 , 2.312(2 ) ; na1n1 , 2.587(2 ) ; na1n2 , 2.662(2 ) ; na1c8 , 2.612(2 ) ; na1c9 , 2.649(2 )", "; zn1n1 , 1.957(2 ) ; zn1n2 , 1.971(2 ) ; zn1c1 , 2.009(2 ) ; n1na1n2 , 60.81(6 ) ; n1zn1n2 , 85.14(8 ) ; n1zn1c1 , 139.88(10 ) ; n2zn1c1 , 134.97(10 ) ."], ["conversions of saturated diamines into unsaturated diazaethenes are usually associated with redox - amenable transition metal catalysts .", "however here we show through a combination of experimental ( nmr spectroscopic , x - ray crystallographic ) and theoretical ( dft ) studies that working co - operatively in a molecular mixed - metal system , an alkali metal ( lithium or sodium ) and zinc can bring about such transformations under challenging redox - inactive conditions ."]]}
{"article_id": "PMC3291213", "article_text": ["we reviewed the literature for reports of e. sakazakii disease in infants . using medical subject heading terms \" e. sakazakii \" or \" enterobacter \" in combination with \" newborn , \" \" infant , \" or \" meningitis ,", "finally , we reviewed e. sakazakii case consultations conducted by the centers for disease control and prevention ( cdc ) from 1998 to 2005 and reviewed results of a french outbreak reported to cdc in 2005 ( 14 ) .", "we defined a case as an infant ( < 12 months of age ) with e. sakazakii cultured from an area of the body that is normally sterile : tissue , blood , cerebrospinal fluid , or urine aspirated through the bladder wall .", "infants with cultures of cerebrospinal fluid or brain abscesses yielding e. sakazakii were considered to have meningitis . because bacteremia is usually an intermediate step during the process of developing meningitis , patients with both bacteremia and meningitis", "bacteremia was defined as e. sakazakii grown from the blood of a case - patient without evidence of meningitis .", "infants with gestational ages < 37 weeks , or those reported to be premature , were considered premature .", "a gestational age of 40 weeks was assigned to 3 ( 27% ) of 11 infants with bacteremia and 4 ( 14% ) of 28 infants with meningitis who were reported as born following term gestation without a specific gestational age mentioned .", "we defined birthweight < 2,500 g as low birthweight ( lbw ) , < 1,500 g as very low birthweight ( vlbw ) , and < 1,000 g as extremely low birthweight ( elbw ) .", "descriptive analysis was performed by using sas software ( sas institute , cary , nc , usa ) .", "forty - six infants met the case definition ( table 1 ) . reported onset years ranged from 1958 to 2005 ; 32 ( 70% ) cases", "cases were reported in 7 countries in north america , europe , and the middle east .", "thirty - three ( 72% ) infants had meningitis , 12 ( 26% ) had bacteremia , and 1 ( 2% ) had a urinary tract infection .", "eight ( 40% ) of 20 infants for whom data were available were delivered by cesarean section .", "twenty - nine ( 69% ) of 42 infants experienced disease onset within a hospital .", "gestational duration was available for 38 infants ; 21 ( 55% ) were born prematurely , and the median gestational age overall was 36 weeks ( range 23.540 weeks ) .", "the median birthweight was 2,063 g ( range 5403,401 ) ; 18 ( 56% ) of 32 infants had lbw ; 9 ( 28% ) of these met the definition for vlbw , and 7 ( 22% ) met the definition for elbw .", "median age at the time of infection onset was 8.5 days ( range 2300 ) .", "* lbw , low birth weight ; vlbw , very low birth weight ; elbw , extremely low birth weight .", "although the proportion of infants who experienced nosocomial disease onset was not significantly different between the meningitis and bacteremia groups , other infant characteristics differed by site of infection ( figure ) .", "the median gestational ages of infants with meningitis and bacteremia were 37 and 27.8 weeks , respectively ( p = 0.02 ) .", "median birthweights were 2,454 g and 850 g , respectively ( p = 0.002 ) .", "however , median age at infection onset was 6 days in the group with meningitis and 35 days in the group with bacteremia ( p < 0.0001 ) .", "thirty ( 94% ) of 32 infants with meningitis , but only 2 ( 18% ) of 11 infants with bacteremia , were < 28 days old when infection was detected .", "one infant ( 8% ) with bacteremia died ; this infant also had necrotizing enterocolitis . the single infant with a urinary tract infection recovered without complication .", "of 19 surviving infants , only those with meningitis suffered adverse outcomes , including brain abscess ( 21% , p = 0.2 ) , developmental delays ( 53% , p = 0.004 ) , motor impairment ( 21% , p = 0.3 ) , and ventricular shunt placement ( 42% , p = 0.01 ) ; 74% experienced at least one of these outcomes .", "box plot with each box indicating range ( vertical lines ) , first and third quartiles ( lower and upper boundaries of box , respectively ) , and median value ( horizontal solid line ) for gestational age in weeks , birth weight in grams , and age of onset in days for infants with bacteremia only or meningitis .", "the dotted lines indicate median values for all cases . * significantly different values (  = 0.05 ) between groups .", "twenty - four ( 92% ) received a powdered formula product , including an infant who received powdered breast milk fortifier but no powdered infant formula ; 1 additional infant received formula of an unspecified type .", "e. sakazakii was cultured from formula associated with 15 ( 68% ) of 22 cases investigated .", "isolates were obtained from prepared formula , opened formula tins , and previously unopened formula tins associated with 2 , 6 , and 7 cases , respectively .", "thirteen ( 87% ) of the 15 formula isolates were indistinguishable from the corresponding clinical strain by biotype or genotype ; multiple formula manufacturers were implicated . in one of the remaining cases ,", "multiple e. sakazakii strains were recovered from powdered formula , but none matched the clinical isolate by pulsed - field gel electrophoresis ( cdc , unpub .", ", 2 e. sakazakii strains were isolated from blood and from rectal swabs ; a third strain , as determined by arbitrarily primed pcr , was recovered from the powdered infant formula ( 9 ) .", "although numerous reports include infants with e. sakazakii isolated from nonsterile sites , such as respiratory secretions or stool , 46 infants identified from the literature and cdc sources met the case definition for this analysis ( 1,9,10,12 ) . of the infants with sterile - site infection", "contrary to previous characterizations of e. sakazakii disease , we found that infants with meningitis and bacteremia alone fell into 2 distinct groups .", "those in whom meningitis developed tended to be of greater gestational age and birthweight than those with bacteremia alone .", "in fact , infants in whom meningitis developed tended to attain near - term gestational age and birthweight .", "in contrast , infants in whom bacteremia alone developed tended to be born very prematurely and have elbw . a second major difference between the group with meningitis and the group with bacteremia was the infants ' chronological ages .", "infants with meningitis were generally < 1 week of age at the onset of infection with e. sakazakii , whereas infants with bacteremia had generally surpassed the neonatal period at the onset of their disease .", "rates of adverse outcome also differed between the 2 groups , although this was not unexpected .", "most infants with e. sakazakii bacteremia fared better than those with meningitis . among those in whom", "meningitis developed , rates of adverse outcome were similar to those reported in the literature : in this case series , 74% of meningitis survivors experienced an adverse neurologic outcome , while other studies cite adverse outcome in 20%78% of neonatal or infant meningitis survivors ( 2123 ) .", "the division of the infant population into 2 distinct groups occurred for unclear reasons . that infants < 1 week of age comprised", "the meningitis group was not surprising ; the disparities in other infant characteristics , however , are not intuitive . since infants in the 2 groups experienced similar rates of nosocomial disease onset , the infants with bacteremia were unlikely to have simply received treatment earlier in the disease course than the infants with meningitis .", "powdered infant formula is a demonstrated source of e. sakazakii infection . a microbiologic survey of powdered infant formulas published in 1988 found e. sakazakii in 20 ( 14% ) of 141 samples tested ( 24 ) .", "however , a survey of 82 powdered infant formula samples in 2003 yielded e. sakazakii in 2 ( 2.4% ) , which suggests that recent rates of powdered formula contamination may be lower ( 25 ) .", "powdered formula has also been implicated both epidemiologically and microbiologically as a vehicle in several cases of e. sakazakii disease in infants ( 1,3,9,10,13 ) .", "although we could not explore feeding exposures fully with the data available in this series , infant feeding practices may relate to the differences we describe in chronological age of infants at onset of meningitis and bacteremia .", "infants with nearly normal gestational ages and birthweights are likely to be tended in normal newborn nurseries during the first 2472 hours of life . in such nurseries , powdered formula is frequently given to babies who are not breast - fed , and it may also be used as a supplement by mothers who have chosen to breast - feed . since infants are at highest risk for meningitis during the first several weeks of life , possibly because of immaturity of the blood - brain barrier , exposures to e. sakazakii in powdered formula or other sources during this time may quickly lead to central nervous system disease ( 26,27 ) .", "conversely , in the intensive care settings where immature and low birthweight infants are tended , babies are not often fed powdered formula in the first few weeks of life .", "they may be given parenteral nutrition initially and may be fed breast milk from their own mothers or from a banked source when they do begin enteral feeds . if breast milk is not available , they are more likely to be given sterile , premixed infant formula than powdered formula , since standard preterm infant formula is only available in this form ( 28 ) .", "powdered breast milk fortifiers are not introduced until premature infants tolerate full - volume feeds , which may not occur for days or weeks after birth .", "thus , infants in intensive care settings may not be exposed to nonsterile formula until they are more mature , which would lead to a greater proportion of e. sakazakii bacteremia than meningitis in this group if powdered formula is a source of infection . in this series", ", we were unable to explore the roles of indwelling enteric tubes , prior gastrointestinal surgery , and antecedent antacid or antimicrobial drug use as risk factors for infection . while the reservoir for e. sakazakii is unknown ,", "e. sakazakii has been isolated from factories used to produce milk powder , chocolate , cereal , potato flour , spices , and pasta ( 29 ) .", "it also has been isolated from household vacuum cleaner bags and from the guts of the stable fly , stomoxys calcitrans , and the mexican fruit fly , anastrpha ludens ( 2931 ) .", "although a human vaginal tract culture yielding e. sakazakii has been reported , vertical transmission is unlikely because nearly half of infants with e. sakazakii disease in this review were delivered by cesarean section , and symptoms developed in only 1 infant earlier than 3 days of age ( 32 ) .", "although cases with more severe outcomes might have been investigated and published more frequently than uncomplicated cases , this possible bias would not likely affect the representativeness of baseline infant characteristics . assigning a gestational age of 40 weeks to term infants without a reported gestational age", "may have falsely elevated the median gestational ages we report , since most term infants are born at < 40 weeks ' gestation ( 33 ) .", "however , a greater proportion of bacteremic infants than meningitic infants received this assignment , and therefore the significance of the differences in gestational age between the groups may be even greater than we report .", "we were unable to explore the effects of concomitant medical problems , treatments , and other environmental factors , and we relied on existing reports of feeding practices and formula testing .", "clearly , additional study is needed to elucidate the lingering questions about e. sakazakii reservoirs , disease risk factors , and disease course .", "other gram - negative organisms , including escherichia coli , enterobacter agglomerans , e. cloacae , klebsiella pneumoniae , k. oxytoca , and citrobacter freundii , can be found in powdered infant formula ( 24,25 ) .", "powdered infant formula also has been associated with outbreaks of illness due to citrobacter and multiple salmonella serotypes ( 13,3438 ) . the degree to", "which e. sakazakii is a marker for a range of neonatal infections possibly related to powdered infant formula remains to be defined .", "certain steps can be taken immediately , however , to prevent or mitigate e. sakazakii disease . in a joint conference on infant formula safety in february , 2004 ,", "the world health organization and food and agriculture organization of the united nations made the following recommendations : 1 ) encourage industry partners to develop a range of affordable sterile formula options ; 2 ) consider setting an industry standard for enterobacteriaecae and e. sakazakii in infant formula ; 3 ) inform infant caregivers of the risks associated with nonsterile , powdered formula ; and 4 ) consider feeding high - risk infants sterile formula if they can not breast - feed ( 39 ) .", "the findings of our case review suggest that all neonates as well as premature infants should be included in this high - risk infant category .", "the american dietetic association has issued guidelines for infant formula preparation , storage , and administration ; these should be followed by infant caregivers in hospitals and private homes ( 40 ) .", "rapid reporting of cases by clinicians could streamline data collection by local health departments and more rapidly resolve remaining questions about this illness .", "manufacturer warning labels on powdered infant formula packages should stress that powdered infant formula is nonsterile and requires proper preparation , handling , and storage , and that sterile , liquid formula alternatives are available", ". these actions , adopted in whole or in part , may decrease the infectious risks associated with powdered formula and prevent this rare but potentially devastating disease ."], "abstract_text": ["<S> enterobacter sakazakii kills 40%80% of infected infants and has been associated with powdered formula . </S>", "<S> we analyzed 46 cases of invasive infant e. sakazakii infection to define risk factors and guide prevention and treatment . </S>", "<S> twelve infants had bacteremia , 33 had meningitis , and 1 had a urinary tract infection . compared with infants with isolated bacteremia , infants with meningitis had greater birthweight ( 2,454 g vs. 850 g , p = 0.002 ) and gestational age ( 37 weeks vs. 27.8 weeks , p = 0.02 ) , and infection developed at a younger age ( 6 days vs. 35 days , p<0.001 ) . among meningitis patients , </S>", "<S> 11 ( 33% ) had seizures , 7 ( 21% ) had brain abscess , and 14 ( 42% ) died . twenty - four ( 92% ) of 26 infants with feeding patterns specified were fed powdered formula . </S>", "<S> formula samples associated with 15 ( 68% ) of 22 cases yielded e. sakazakii ; in 13 cases , clinical and formula strains were indistinguishable . </S>", "<S> further clarification of clinical risk factors and improved powdered formula safety is needed . </S>"], "labels": null, "section_names": ["Methods", "Results", "Discussion"], "sections": [["we reviewed the literature for reports of e. sakazakii disease in infants . using medical subject heading terms \" e. sakazakii \" or \" enterobacter \" in combination with \" newborn , \" \" infant , \" or \" meningitis ,", "finally , we reviewed e. sakazakii case consultations conducted by the centers for disease control and prevention ( cdc ) from 1998 to 2005 and reviewed results of a french outbreak reported to cdc in 2005 ( 14 ) .", "we defined a case as an infant ( < 12 months of age ) with e. sakazakii cultured from an area of the body that is normally sterile : tissue , blood , cerebrospinal fluid , or urine aspirated through the bladder wall .", "infants with cultures of cerebrospinal fluid or brain abscesses yielding e. sakazakii were considered to have meningitis . because bacteremia is usually an intermediate step during the process of developing meningitis , patients with both bacteremia and meningitis", "bacteremia was defined as e. sakazakii grown from the blood of a case - patient without evidence of meningitis .", "infants with gestational ages < 37 weeks , or those reported to be premature , were considered premature .", "a gestational age of 40 weeks was assigned to 3 ( 27% ) of 11 infants with bacteremia and 4 ( 14% ) of 28 infants with meningitis who were reported as born following term gestation without a specific gestational age mentioned .", "we defined birthweight < 2,500 g as low birthweight ( lbw ) , < 1,500 g as very low birthweight ( vlbw ) , and < 1,000 g as extremely low birthweight ( elbw ) .", "descriptive analysis was performed by using sas software ( sas institute , cary , nc , usa ) ."], ["forty - six infants met the case definition ( table 1 ) . reported onset years ranged from 1958 to 2005 ; 32 ( 70% ) cases", "cases were reported in 7 countries in north america , europe , and the middle east .", "thirty - three ( 72% ) infants had meningitis , 12 ( 26% ) had bacteremia , and 1 ( 2% ) had a urinary tract infection .", "eight ( 40% ) of 20 infants for whom data were available were delivered by cesarean section .", "twenty - nine ( 69% ) of 42 infants experienced disease onset within a hospital .", "gestational duration was available for 38 infants ; 21 ( 55% ) were born prematurely , and the median gestational age overall was 36 weeks ( range 23.540 weeks ) .", "the median birthweight was 2,063 g ( range 5403,401 ) ; 18 ( 56% ) of 32 infants had lbw ; 9 ( 28% ) of these met the definition for vlbw , and 7 ( 22% ) met the definition for elbw .", "median age at the time of infection onset was 8.5 days ( range 2300 ) .", "* lbw , low birth weight ; vlbw , very low birth weight ; elbw , extremely low birth weight .", "although the proportion of infants who experienced nosocomial disease onset was not significantly different between the meningitis and bacteremia groups , other infant characteristics differed by site of infection ( figure ) .", "the median gestational ages of infants with meningitis and bacteremia were 37 and 27.8 weeks , respectively ( p = 0.02 ) .", "median birthweights were 2,454 g and 850 g , respectively ( p = 0.002 ) .", "however , median age at infection onset was 6 days in the group with meningitis and 35 days in the group with bacteremia ( p < 0.0001 ) .", "thirty ( 94% ) of 32 infants with meningitis , but only 2 ( 18% ) of 11 infants with bacteremia , were < 28 days old when infection was detected .", "one infant ( 8% ) with bacteremia died ; this infant also had necrotizing enterocolitis . the single infant with a urinary tract infection recovered without complication .", "of 19 surviving infants , only those with meningitis suffered adverse outcomes , including brain abscess ( 21% , p = 0.2 ) , developmental delays ( 53% , p = 0.004 ) , motor impairment ( 21% , p = 0.3 ) , and ventricular shunt placement ( 42% , p = 0.01 ) ; 74% experienced at least one of these outcomes .", "box plot with each box indicating range ( vertical lines ) , first and third quartiles ( lower and upper boundaries of box , respectively ) , and median value ( horizontal solid line ) for gestational age in weeks , birth weight in grams , and age of onset in days for infants with bacteremia only or meningitis .", "the dotted lines indicate median values for all cases . * significantly different values (  = 0.05 ) between groups .", "twenty - four ( 92% ) received a powdered formula product , including an infant who received powdered breast milk fortifier but no powdered infant formula ; 1 additional infant received formula of an unspecified type .", "e. sakazakii was cultured from formula associated with 15 ( 68% ) of 22 cases investigated .", "isolates were obtained from prepared formula , opened formula tins , and previously unopened formula tins associated with 2 , 6 , and 7 cases , respectively .", "thirteen ( 87% ) of the 15 formula isolates were indistinguishable from the corresponding clinical strain by biotype or genotype ; multiple formula manufacturers were implicated . in one of the remaining cases ,", "multiple e. sakazakii strains were recovered from powdered formula , but none matched the clinical isolate by pulsed - field gel electrophoresis ( cdc , unpub .", ", 2 e. sakazakii strains were isolated from blood and from rectal swabs ; a third strain , as determined by arbitrarily primed pcr , was recovered from the powdered infant formula ( 9 ) ."], ["although numerous reports include infants with e. sakazakii isolated from nonsterile sites , such as respiratory secretions or stool , 46 infants identified from the literature and cdc sources met the case definition for this analysis ( 1,9,10,12 ) . of the infants with sterile - site infection", "contrary to previous characterizations of e. sakazakii disease , we found that infants with meningitis and bacteremia alone fell into 2 distinct groups .", "those in whom meningitis developed tended to be of greater gestational age and birthweight than those with bacteremia alone .", "in fact , infants in whom meningitis developed tended to attain near - term gestational age and birthweight .", "in contrast , infants in whom bacteremia alone developed tended to be born very prematurely and have elbw . a second major difference between the group with meningitis and the group with bacteremia was the infants ' chronological ages .", "infants with meningitis were generally < 1 week of age at the onset of infection with e. sakazakii , whereas infants with bacteremia had generally surpassed the neonatal period at the onset of their disease .", "rates of adverse outcome also differed between the 2 groups , although this was not unexpected .", "most infants with e. sakazakii bacteremia fared better than those with meningitis . among those in whom", "meningitis developed , rates of adverse outcome were similar to those reported in the literature : in this case series , 74% of meningitis survivors experienced an adverse neurologic outcome , while other studies cite adverse outcome in 20%78% of neonatal or infant meningitis survivors ( 2123 ) .", "the division of the infant population into 2 distinct groups occurred for unclear reasons . that infants < 1 week of age comprised", "the meningitis group was not surprising ; the disparities in other infant characteristics , however , are not intuitive . since infants in the 2 groups experienced similar rates of nosocomial disease onset , the infants with bacteremia were unlikely to have simply received treatment earlier in the disease course than the infants with meningitis .", "powdered infant formula is a demonstrated source of e. sakazakii infection . a microbiologic survey of powdered infant formulas published in 1988 found e. sakazakii in 20 ( 14% ) of 141 samples tested ( 24 ) .", "however , a survey of 82 powdered infant formula samples in 2003 yielded e. sakazakii in 2 ( 2.4% ) , which suggests that recent rates of powdered formula contamination may be lower ( 25 ) .", "powdered formula has also been implicated both epidemiologically and microbiologically as a vehicle in several cases of e. sakazakii disease in infants ( 1,3,9,10,13 ) .", "although we could not explore feeding exposures fully with the data available in this series , infant feeding practices may relate to the differences we describe in chronological age of infants at onset of meningitis and bacteremia .", "infants with nearly normal gestational ages and birthweights are likely to be tended in normal newborn nurseries during the first 2472 hours of life . in such nurseries , powdered formula is frequently given to babies who are not breast - fed , and it may also be used as a supplement by mothers who have chosen to breast - feed . since infants are at highest risk for meningitis during the first several weeks of life , possibly because of immaturity of the blood - brain barrier , exposures to e. sakazakii in powdered formula or other sources during this time may quickly lead to central nervous system disease ( 26,27 ) .", "conversely , in the intensive care settings where immature and low birthweight infants are tended , babies are not often fed powdered formula in the first few weeks of life .", "they may be given parenteral nutrition initially and may be fed breast milk from their own mothers or from a banked source when they do begin enteral feeds . if breast milk is not available , they are more likely to be given sterile , premixed infant formula than powdered formula , since standard preterm infant formula is only available in this form ( 28 ) .", "powdered breast milk fortifiers are not introduced until premature infants tolerate full - volume feeds , which may not occur for days or weeks after birth .", "thus , infants in intensive care settings may not be exposed to nonsterile formula until they are more mature , which would lead to a greater proportion of e. sakazakii bacteremia than meningitis in this group if powdered formula is a source of infection . in this series", ", we were unable to explore the roles of indwelling enteric tubes , prior gastrointestinal surgery , and antecedent antacid or antimicrobial drug use as risk factors for infection . while the reservoir for e. sakazakii is unknown ,", "e. sakazakii has been isolated from factories used to produce milk powder , chocolate , cereal , potato flour , spices , and pasta ( 29 ) .", "it also has been isolated from household vacuum cleaner bags and from the guts of the stable fly , stomoxys calcitrans , and the mexican fruit fly , anastrpha ludens ( 2931 ) .", "although a human vaginal tract culture yielding e. sakazakii has been reported , vertical transmission is unlikely because nearly half of infants with e. sakazakii disease in this review were delivered by cesarean section , and symptoms developed in only 1 infant earlier than 3 days of age ( 32 ) .", "although cases with more severe outcomes might have been investigated and published more frequently than uncomplicated cases , this possible bias would not likely affect the representativeness of baseline infant characteristics . assigning a gestational age of 40 weeks to term infants without a reported gestational age", "may have falsely elevated the median gestational ages we report , since most term infants are born at < 40 weeks ' gestation ( 33 ) .", "however , a greater proportion of bacteremic infants than meningitic infants received this assignment , and therefore the significance of the differences in gestational age between the groups may be even greater than we report .", "we were unable to explore the effects of concomitant medical problems , treatments , and other environmental factors , and we relied on existing reports of feeding practices and formula testing .", "clearly , additional study is needed to elucidate the lingering questions about e. sakazakii reservoirs , disease risk factors , and disease course .", "other gram - negative organisms , including escherichia coli , enterobacter agglomerans , e. cloacae , klebsiella pneumoniae , k. oxytoca , and citrobacter freundii , can be found in powdered infant formula ( 24,25 ) .", "powdered infant formula also has been associated with outbreaks of illness due to citrobacter and multiple salmonella serotypes ( 13,3438 ) . the degree to", "which e. sakazakii is a marker for a range of neonatal infections possibly related to powdered infant formula remains to be defined .", "certain steps can be taken immediately , however , to prevent or mitigate e. sakazakii disease . in a joint conference on infant formula safety in february , 2004 ,", "the world health organization and food and agriculture organization of the united nations made the following recommendations : 1 ) encourage industry partners to develop a range of affordable sterile formula options ; 2 ) consider setting an industry standard for enterobacteriaecae and e. sakazakii in infant formula ; 3 ) inform infant caregivers of the risks associated with nonsterile , powdered formula ; and 4 ) consider feeding high - risk infants sterile formula if they can not breast - feed ( 39 ) .", "the findings of our case review suggest that all neonates as well as premature infants should be included in this high - risk infant category .", "the american dietetic association has issued guidelines for infant formula preparation , storage , and administration ; these should be followed by infant caregivers in hospitals and private homes ( 40 ) .", "rapid reporting of cases by clinicians could streamline data collection by local health departments and more rapidly resolve remaining questions about this illness .", "manufacturer warning labels on powdered infant formula packages should stress that powdered infant formula is nonsterile and requires proper preparation , handling , and storage , and that sterile , liquid formula alternatives are available", ". these actions , adopted in whole or in part , may decrease the infectious risks associated with powdered formula and prevent this rare but potentially devastating disease ."]]}
{"article_id": "PMC3612189", "article_text": ["sterilization in orthodontics has repeatedly been discussed and emphasized in the dental literature.[16 ] the orthodontic personnel are at a great risk of various contagious diseases and hepatitis b , acquired immuno deficiency syndrome ( aids ) and herpes infections are some of the serious conditions encountered in an orthodontic office .", "therefore , the center for disease control ( cdc ) specifies that all instruments which are not expected to penetrate into soft tissue or bone but will certainly contact oral tissues must be sterilized , too . in cases in which barrier techniques are not used , cross - infection between the orthodontic personnel and patients is possible .", "in addition , since orthodontic clinics have a large number of patients during the day compared to other dental clinics , each clinic requires its own custom - made sterilization processes .", "that 12% of unused orthodontic wires are not sterile and produce bacterial colonies in a culture . therefore , the instructions on the packages generally recommended sterilization before use . at present ,", "orthodontic wires are selected in different stages of the treatment procedure not only based on wire diameter , but also biomechanical and deactivation properties of wires in order to achieve the best treatment results .", "-titanium ( tma ) wires are alloys of titanium and molybdenum , which were introduced in the early 1979 by goldberg and burstone for use in orthodontic treatment .", "these wires are becoming more popular because of their excellent balance of mechanical properties and absence of nickel , resulting in better tolerance by many patients ; some of these mechanical properties include : \n lower elastic modulus in comparison to stainless steelhigh formabilityability of direct weldinggood spring - backlow stiffnesslow force delivery[1116 ] \n  lower elastic modulus in comparison to stainless steel ability of direct welding low force delivery[1116 ] the limitation of -ti wires is their high coefficient of friction", ". however , some manufacturers have used different methods to solve this problem , including incorporation of a titanium nitride and titanium oxide layer on the wire surface through ion implantation , changing their composition , highly polishing of the surface and increasing the surface smoothness.[71724 ] some previous studies have evaluated the effect of sterilization on wires with various alloy combinations ( -ti , niti , ss),[82528 ] with contradictory results in relation to -ti wires . in addition , after the expiration of patent on -ti wires by ormco ( glendora , california , usa ) various manufacturers produce these wires with different compositions and processing methods , resulting in different mechanical properties and different effects as a result of sterilization processes .", "the aim of the present study was to evaluate the effect of sterilization with dry heat and steam on load - deflection characteristics of -ti wires , with different brands .", "in the present study five different types of -titanium wires with a cross - section of 0.43  0.64 mm ( 0.017  0.025 inch ) were evaluated [ table 1 ] .", "the wires were of the straight type . in cases in which only the preformed arch forms were available their straight posterior segments", "a total of 30 wire segments from each type were provided ; each segment measured 30 mm in length .", "-titanium wires tested in the present study group i was assigned as the control group .", "the wire samples in group ii were sterilized by dry heat in an oven ( 12 bahman digital sterilizer , dsl 40 , isfahan , iran ) at 160c for 1 hour .", "the wire samples in group iii were sterilized by steam in an autoclave ( melag euroclav , 23 v - s , berlin , germany ) at 121c and 15 psi for 15 minutes .", "the wire samples were left to gradually cool to temperatures near the ambient temperature in the same sterilizers .", "both process indicators and test strips were used to ensure the sterilization cycles and processes .", "then the samples underwent a three - point load - deflection test in the dry state as described by miura et al .", "the test was carried out using a universal mechanical testing machine ( model 1122 , instron corporation , canton , ma , usa ) using a specially designed setup , consisting of two metallic half - cylindrical structures placed parallel to each other .", "a single maxillary first premolar bracket ( american orthodontics , master series , wis , usa ) , with a 0.022  0.028-inch slot size with zero angulation and zero torque was bonded on each of the half - cylinders [ figure 1 ] , with a distance of 14 mm between the brackets .", "the wire sample to be tested was fixed on the brackets using 0.012-inch elastomeric ligatures ( ortho organizers , carlsbad , california , usa ) .", "the center of each wire was deflected by moving a metallic pole adjusted on the upper head of the testing machine at a crosshead speed of 0.5 mm / min .", "the load was recorded at 0.1-mm intervals from the inactive position up to 1.5 mm of activation and then returning to the zero point to achieve load - deflection characteristics of each wire .", "load - deflection apparatus with the wire in place data was analyzed using repeated measures anova to compare load - deflection properties of the five types of -ti wires and determine the changes in characteristics during loading and unloading separately after sterilization of each wire type .", "scheffe is a post - hoc test , done after anova test , to evaluate the differences between each two groups .", "tables 2 and 3 present means and standard deviations of loading and unloading forces of different types of -ti wires after 1.5 mm of deflection in the three groups under study at 0.1-mm intervals .", "figures 26 show load - deflection curves . means and standard deviations ( sd ) for loading forces at 0.1 mm intervals of deflection for wire samples in groups i - iii means and standard deviations ( sd ) for unloading forces at 0.1 mm intervals of defl ection for wire specimens in groups i - iii load - deflection graph of beta wires in the control group and after sterilization with dry and moist heat load - deflection graph of cna wires in the control group and after sterilization with dry and moist heat load - deflection graph of res wires in the control group and after sterilization with dry and moist heat load - deflection graph of hone wires in the control group and after sterilization with dry and moist heat load - deflection graph of tmal wires in the control group and after sterilization with dry and moist heat repeated measures anova showed that during loading , hone wire had the least force level ( p < 0.05 ) and res wire had the highest force level .", "however , there were no statistically significant differences in force levels between res , cna and beta wires ( p > 0.05 ) .", "no significant differences were observed in force levels between tmal and beta wires ( p > 0.05 ) .", "the force levels of res and cna wires were significantly higher than that of tmal wire ( p < 0.05 ) . during unloading , tmal and hone wires exhibited the lowest force levels ( p < 0.05 ) ; although the force of tmal wire was a little less than that of hone wire", "res wire exhibited the highest force level , which was significantly higher than those of beta , tmal and hone wires ( p < 0.05 ) .", "although res wires exhibited force levels slightly higher than those of cna wires , there were no significant differences between the two wires ( p > 0.05 ) .", "there were no significant differences in force levels between beta and cna wires ( p > 0.05 ) .", "repeated measures anova revealed the following [ figures 26 ] : dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .", "the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .", "the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .", "there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .", "the force levels of various res and cna groups during loading and unloading were as follows : iiii < ii . during loading ,", "the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .", "there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading , the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .", "there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .", "the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .", "there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) .", "according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study .", "repeated measures anova showed that during loading , hone wire had the least force level ( p < 0.05 ) and res wire had the highest force level .", "however , there were no statistically significant differences in force levels between res , cna and beta wires ( p > 0.05 ) .", "no significant differences were observed in force levels between tmal and beta wires ( p > 0.05 ) .", "the force levels of res and cna wires were significantly higher than that of tmal wire ( p < 0.05 ) . during unloading , tmal and hone wires exhibited the lowest force levels ( p < 0.05 ) ; although the force of tmal wire was a little less than that of hone wire", "res wire exhibited the highest force level , which was significantly higher than those of beta , tmal and hone wires ( p < 0.05 ) .", "although res wires exhibited force levels slightly higher than those of cna wires , there were no significant differences between the two wires ( p > 0.05 ) .", "there were no significant differences in force levels between beta and cna wires ( p > 0.05 ) .", "repeated measures anova revealed the following [ figures 26 ] : dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .", "the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .", "the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .", "there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .", "the force levels of various res and cna groups during loading and unloading were as follows :", "the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .", "there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading ,", "the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .", "there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .", "the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .", "there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) . according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study .", "dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .", "the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .", "the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .", "there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .", "the force levels of various res and cna groups during loading and unloading were as follows : iiii < ii .", "during loading , the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .", "there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading ,", "the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .", "there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .", "the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .", "there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) . according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study .", "-ti wires have gained great popularity in orthodontics due to their favorable nature to tooth and supporting tissues .", "multi - stage processing of orthodontic wires by manufacturers has a significant influence on their mechanical properties .", "high autoclave or dry heat sterilizer temperatures , too , might influence the mechanical properties of wires .", "therefore , it is important for the manufacturers to evaluate the effect of sterilization procedures on -ti wires .", "heat sterilization techniques were selected in the present study because they are the most popular type of sterilization technique used in dental offices ; in addition , it is the most typical method recommended until now .", "the load - deflection properties are significant parameters in determining the biologic nature of tooth movement .", "a modified version of the three - point bending test was used in the present study to evaluate load - deflection properties of orthodontic wires .", "this technique provides a simple model by determining the nature of forces applied during orthodontic treatment and closely simulates clinical situations . in the clinic ,", "when a wire undergoes deflection to be placed within a bracket on a malaligned tooth , in fact , it undergoes loading . when a wire is inclined to return to its original shape , unloading takes place , which provides the necessary force to elicit a biologic tissue response , resulting in the alignment of teeth . however , laboratory tests do not necessarily reflect the clinical situations ; rather , they provide a basis for the comparison of various brands of wires .", "of the five brands of wires under study [ tables 2 and 3 ] , the wires manufactured by ormco ( tmal and hone ) exhibited the lowest force during unloading , with tmal forces being higher than those of hone during loading , statically equal to those of beta wires .", "therefore , hone wires , compared to the other types tested , are more easily engaged in the brackets of malaligned teeth due to lower force levels needed to deflect .", "res and cna wires required higher force levels for deflection ; therefore , these wires exhibited greater stiffness in comparison to tmal and hone wires , which might be attributed to differences in composition and processing of the alloys used in the manufacture of each of these orthodontic wires .", "given the curves and steepness of load - deflection graphs of the control wires in the present study [ figures 26 ] , it appears res , beta and cna wires have different behaviors compared to the two products from the ormco company ( tmal and hone ) .", "these results are supported by those of kusy et al . who reported that the same wire manufacturer due to similar composition and surface roughness could have manufactured the -ti new products of res , beta iii and cna .", "the original -ti alloy from the ormco company could have been manufactured with the same characteristics .", "the results of the present study showed that dry heat sterilization significantly increases force levels during loading and unloading of cna , beta and res and loading of hone wires , making them stiffer .", "as indicated by the increased slope of the graphs [ figures 25 ] dry heat sterilization influences the clinical performance of cna , res , beta and hone wires .", "it also results in increased force delivery by cna , res and beta to teeth .", "the differences in the effect of sterilization with dry heat and steam might be attributed to the use of longer times and higher temperatures in the sterilization process by dry heat .", "however , sterilization with dry heat did not result in significant changes in force levels during loading and unloading of tmal wires [ figure 6 ] .", "previous studies on the effect of dry heat sterilization on -ti wires have evaluated only tma wires ( ormco , glendora , california , usa ) with a size of 0.016 inch .", "reported that use of dry heat sterilization with a temperature of 375f ( 191c ) for 10 minutes on tma archform wires ( ormco , glendora , california , usa ) which had already been used clinically did not exert any influence on tensile and load - deflection test results of the wires .", "the results of the present study with tmal wires are consistent with the results of a study by smith et al . with tma wires", "evaluated the effect of dry heat sterilization at 375f for 20 minutes on tma wires ( ormco , glendora , california , usa ) and reported a significant increase in the tensile strength of wires .", "in addition to the type and size of the wires , other factors , which might have resulted in differences between the results of the present study and other studies , mentioned above might be the differences in the sterilization protocols and the type of the mechanical tests applied .", "the results of the present study did not show any effect of steam sterilization ( autoclave ) [ figures 26 ] on load - deflection characteristics of the majority of -ti wires tested ; however , it increased force levels during loading and unloading of beta and unloading of hone wires , which is reflected by an increased slope of loading and unloading of beta and unloading of hone wires [ figures 2 and 5 ]", ". therefore , these wires will deliver greater forces to teeth after sterilization in an autoclave .", "regarding the low force levels of tmal wire , especially during unloading both in the control group and after sterilization , it appears ion implantation process is only related to surface characteristics and a decrease in friction , with no detrimental effects on load - deflection characteristics of the wire even after sterilization .", "the different effects of sterilization on hone and tmal wires might be attributed to differences in the type and concentration of ions or other factors during the manufacturing processes of these wires . it is suggested that clinicians sterilize tmal , res and cna wires in an autoclave before using them without any worries because their favorable load characteristics will not undergo any changes after sterilization , which is consistent with the results of a study carried out by pernier et al .", "on the effect of autoclave sterilization at 134c or 274f for 18 minutes on tma , tmal and res wires .", "they reported no differences in load - displacement curves after sterilization of any wire tested .", "reported no effect of autoclave sterilization ( 250f for 20 minutes ) on tensile strength of tma wires .", "in addition , a study by smith et al . did not show any perceptible clinical differences between new wires and clinically used wires , which are later , sterilized in an autoclave ( 274f for 10 minutes ) .", "\n the results of the present study showed differences in force levels during loading and unloading between the five brands of -ti wires under study after 1.5 mm of deflection ; res , cna and beta wires exhibited different force deflection behaviors in comparison with the two products of ormco company ( tmal and hone).there are different changes in load - deflection characteristics of various -ti wires after sterilization with dry heat and heated steam.the clinicians who want to provide maximum safety for their patients can sterilize res , tmal and cna wires in an autoclave without any worries before placing them in the oral cavity . \n ", "the results of the present study showed differences in force levels during loading and unloading between the five brands of -ti wires under study after 1.5 mm of deflection ; res , cna and beta wires exhibited different force deflection behaviors in comparison with the two products of ormco company ( tmal and hone ) .", "there are different changes in load - deflection characteristics of various -ti wires after sterilization with dry heat and heated steam .", "the clinicians who want to provide maximum safety for their patients can sterilize res , tmal and cna wires in an autoclave without any worries before placing them in the oral cavity .", "however , further in vivo studies are required to substantiate the results of the present study ."], "abstract_text": ["<S> background : sterilization techniques could affect the characteristics of orthodontic wires . </S>", "<S> the aim of the present study was to evaluate the effect of steam and dry heat sterilization techniques on load - deflection behavior of five types of -titanium alloy wires.materials and methods : the samples consisted of 30 straight lengths of five types of -titanium alloy wires : titanium molybdenum alloy ( tma ) low friction ( tmal ) , tma low friction colored ( hone ) , resolve ( res ) , betaforce ( beta ) , and beta cna ( cna ) . </S>", "<S> thirty wire segments were divided into three groups of 10 . </S>", "<S> group 1 was the control group and the group 2 samples were sterilized by dry heat in an oven ( 60 minutes at 160c ) and group 3 by steam in an autoclave ( 15 minutes at 121c ) . then all the wire samples underwent a three - point bending test in a testing machine to evaluate load - deflection properties . </S>", "<S> data was analyzed by repeated measures anova and scheff 's test (  = 0.05).results : the results showed that dry heat sterilization significantly increased force levels during both loading and unloading of cna , beta and res and during loading of hone ( p < 0.05 ) . </S>", "<S> steam sterilization significantly increased force levels during both loading and unloading of beta and during unloading of hone ( p < 0.05 ) , with no effects on the load - deflection characteristics of tmal , cna and res ( p > 0.05).conclusion : it appears dry heat sterilization increases stiffness of res , beta , cna and hone but autoclave sterilization did not have any effect on load - deflection characteristics of most of the -titanium wires tested , indicating that clinicians who want to provide maximum safety for their patients can autoclave tmal , res and cna before applying them . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "Load-deflection characteristics of the five types of -Ti wires", "Effect of sterilization process on loading and unloading characteristics of -Ti wires of various brands", "BETA wires", "RES and CNA wires", "HONE wire", "TMAL wires", "DISCUSSION", "CONCLUSIONS"], "sections": [["sterilization in orthodontics has repeatedly been discussed and emphasized in the dental literature.[16 ] the orthodontic personnel are at a great risk of various contagious diseases and hepatitis b , acquired immuno deficiency syndrome ( aids ) and herpes infections are some of the serious conditions encountered in an orthodontic office .", "therefore , the center for disease control ( cdc ) specifies that all instruments which are not expected to penetrate into soft tissue or bone but will certainly contact oral tissues must be sterilized , too . in cases in which barrier techniques are not used , cross - infection between the orthodontic personnel and patients is possible .", "in addition , since orthodontic clinics have a large number of patients during the day compared to other dental clinics , each clinic requires its own custom - made sterilization processes .", "that 12% of unused orthodontic wires are not sterile and produce bacterial colonies in a culture . therefore , the instructions on the packages generally recommended sterilization before use . at present ,", "orthodontic wires are selected in different stages of the treatment procedure not only based on wire diameter , but also biomechanical and deactivation properties of wires in order to achieve the best treatment results .", "-titanium ( tma ) wires are alloys of titanium and molybdenum , which were introduced in the early 1979 by goldberg and burstone for use in orthodontic treatment .", "these wires are becoming more popular because of their excellent balance of mechanical properties and absence of nickel , resulting in better tolerance by many patients ; some of these mechanical properties include : \n lower elastic modulus in comparison to stainless steelhigh formabilityability of direct weldinggood spring - backlow stiffnesslow force delivery[1116 ] \n  lower elastic modulus in comparison to stainless steel ability of direct welding low force delivery[1116 ] the limitation of -ti wires is their high coefficient of friction", ". however , some manufacturers have used different methods to solve this problem , including incorporation of a titanium nitride and titanium oxide layer on the wire surface through ion implantation , changing their composition , highly polishing of the surface and increasing the surface smoothness.[71724 ] some previous studies have evaluated the effect of sterilization on wires with various alloy combinations ( -ti , niti , ss),[82528 ] with contradictory results in relation to -ti wires . in addition , after the expiration of patent on -ti wires by ormco ( glendora , california , usa ) various manufacturers produce these wires with different compositions and processing methods , resulting in different mechanical properties and different effects as a result of sterilization processes .", "the aim of the present study was to evaluate the effect of sterilization with dry heat and steam on load - deflection characteristics of -ti wires , with different brands ."], ["in the present study five different types of -titanium wires with a cross - section of 0.43  0.64 mm ( 0.017  0.025 inch ) were evaluated [ table 1 ] .", "the wires were of the straight type . in cases in which only the preformed arch forms were available their straight posterior segments", "a total of 30 wire segments from each type were provided ; each segment measured 30 mm in length .", "-titanium wires tested in the present study group i was assigned as the control group .", "the wire samples in group ii were sterilized by dry heat in an oven ( 12 bahman digital sterilizer , dsl 40 , isfahan , iran ) at 160c for 1 hour .", "the wire samples in group iii were sterilized by steam in an autoclave ( melag euroclav , 23 v - s , berlin , germany ) at 121c and 15 psi for 15 minutes .", "the wire samples were left to gradually cool to temperatures near the ambient temperature in the same sterilizers .", "both process indicators and test strips were used to ensure the sterilization cycles and processes .", "then the samples underwent a three - point load - deflection test in the dry state as described by miura et al .", "the test was carried out using a universal mechanical testing machine ( model 1122 , instron corporation , canton , ma , usa ) using a specially designed setup , consisting of two metallic half - cylindrical structures placed parallel to each other .", "a single maxillary first premolar bracket ( american orthodontics , master series , wis , usa ) , with a 0.022  0.028-inch slot size with zero angulation and zero torque was bonded on each of the half - cylinders [ figure 1 ] , with a distance of 14 mm between the brackets .", "the wire sample to be tested was fixed on the brackets using 0.012-inch elastomeric ligatures ( ortho organizers , carlsbad , california , usa ) .", "the center of each wire was deflected by moving a metallic pole adjusted on the upper head of the testing machine at a crosshead speed of 0.5 mm / min .", "the load was recorded at 0.1-mm intervals from the inactive position up to 1.5 mm of activation and then returning to the zero point to achieve load - deflection characteristics of each wire .", "load - deflection apparatus with the wire in place data was analyzed using repeated measures anova to compare load - deflection properties of the five types of -ti wires and determine the changes in characteristics during loading and unloading separately after sterilization of each wire type .", "scheffe is a post - hoc test , done after anova test , to evaluate the differences between each two groups ."], ["tables 2 and 3 present means and standard deviations of loading and unloading forces of different types of -ti wires after 1.5 mm of deflection in the three groups under study at 0.1-mm intervals .", "figures 26 show load - deflection curves . means and standard deviations ( sd ) for loading forces at 0.1 mm intervals of deflection for wire samples in groups i - iii means and standard deviations ( sd ) for unloading forces at 0.1 mm intervals of defl ection for wire specimens in groups i - iii load - deflection graph of beta wires in the control group and after sterilization with dry and moist heat load - deflection graph of cna wires in the control group and after sterilization with dry and moist heat load - deflection graph of res wires in the control group and after sterilization with dry and moist heat load - deflection graph of hone wires in the control group and after sterilization with dry and moist heat load - deflection graph of tmal wires in the control group and after sterilization with dry and moist heat repeated measures anova showed that during loading , hone wire had the least force level ( p < 0.05 ) and res wire had the highest force level .", "however , there were no statistically significant differences in force levels between res , cna and beta wires ( p > 0.05 ) .", "no significant differences were observed in force levels between tmal and beta wires ( p > 0.05 ) .", "the force levels of res and cna wires were significantly higher than that of tmal wire ( p < 0.05 ) . during unloading , tmal and hone wires exhibited the lowest force levels ( p < 0.05 ) ; although the force of tmal wire was a little less than that of hone wire", "res wire exhibited the highest force level , which was significantly higher than those of beta , tmal and hone wires ( p < 0.05 ) .", "although res wires exhibited force levels slightly higher than those of cna wires , there were no significant differences between the two wires ( p > 0.05 ) .", "there were no significant differences in force levels between beta and cna wires ( p > 0.05 ) .", "repeated measures anova revealed the following [ figures 26 ] : dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .", "the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .", "the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .", "there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .", "the force levels of various res and cna groups during loading and unloading were as follows : iiii < ii . during loading ,", "the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .", "there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading , the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .", "there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .", "the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .", "there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) .", "according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study ."], ["repeated measures anova showed that during loading , hone wire had the least force level ( p < 0.05 ) and res wire had the highest force level .", "however , there were no statistically significant differences in force levels between res , cna and beta wires ( p > 0.05 ) .", "no significant differences were observed in force levels between tmal and beta wires ( p > 0.05 ) .", "the force levels of res and cna wires were significantly higher than that of tmal wire ( p < 0.05 ) . during unloading , tmal and hone wires exhibited the lowest force levels ( p < 0.05 ) ; although the force of tmal wire was a little less than that of hone wire", "res wire exhibited the highest force level , which was significantly higher than those of beta , tmal and hone wires ( p < 0.05 ) .", "although res wires exhibited force levels slightly higher than those of cna wires , there were no significant differences between the two wires ( p > 0.05 ) .", "there were no significant differences in force levels between beta and cna wires ( p > 0.05 ) ."], ["repeated measures anova revealed the following [ figures 26 ] : dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .", "the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .", "the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .", "there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .", "the force levels of various res and cna groups during loading and unloading were as follows :", "the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .", "there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading ,", "the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .", "there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .", "the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .", "there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) . according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study ."], ["dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .", "the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii ."], ["the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .", "there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .", "there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .", "the force levels of various res and cna groups during loading and unloading were as follows : iiii < ii ."], ["during loading , the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .", "there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading ,", "the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .", "there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .", "the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii ."], ["there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) . according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study ."], ["-ti wires have gained great popularity in orthodontics due to their favorable nature to tooth and supporting tissues .", "multi - stage processing of orthodontic wires by manufacturers has a significant influence on their mechanical properties .", "high autoclave or dry heat sterilizer temperatures , too , might influence the mechanical properties of wires .", "therefore , it is important for the manufacturers to evaluate the effect of sterilization procedures on -ti wires .", "heat sterilization techniques were selected in the present study because they are the most popular type of sterilization technique used in dental offices ; in addition , it is the most typical method recommended until now .", "the load - deflection properties are significant parameters in determining the biologic nature of tooth movement .", "a modified version of the three - point bending test was used in the present study to evaluate load - deflection properties of orthodontic wires .", "this technique provides a simple model by determining the nature of forces applied during orthodontic treatment and closely simulates clinical situations . in the clinic ,", "when a wire undergoes deflection to be placed within a bracket on a malaligned tooth , in fact , it undergoes loading . when a wire is inclined to return to its original shape , unloading takes place , which provides the necessary force to elicit a biologic tissue response , resulting in the alignment of teeth . however , laboratory tests do not necessarily reflect the clinical situations ; rather , they provide a basis for the comparison of various brands of wires .", "of the five brands of wires under study [ tables 2 and 3 ] , the wires manufactured by ormco ( tmal and hone ) exhibited the lowest force during unloading , with tmal forces being higher than those of hone during loading , statically equal to those of beta wires .", "therefore , hone wires , compared to the other types tested , are more easily engaged in the brackets of malaligned teeth due to lower force levels needed to deflect .", "res and cna wires required higher force levels for deflection ; therefore , these wires exhibited greater stiffness in comparison to tmal and hone wires , which might be attributed to differences in composition and processing of the alloys used in the manufacture of each of these orthodontic wires .", "given the curves and steepness of load - deflection graphs of the control wires in the present study [ figures 26 ] , it appears res , beta and cna wires have different behaviors compared to the two products from the ormco company ( tmal and hone ) .", "these results are supported by those of kusy et al . who reported that the same wire manufacturer due to similar composition and surface roughness could have manufactured the -ti new products of res , beta iii and cna .", "the original -ti alloy from the ormco company could have been manufactured with the same characteristics .", "the results of the present study showed that dry heat sterilization significantly increases force levels during loading and unloading of cna , beta and res and loading of hone wires , making them stiffer .", "as indicated by the increased slope of the graphs [ figures 25 ] dry heat sterilization influences the clinical performance of cna , res , beta and hone wires .", "it also results in increased force delivery by cna , res and beta to teeth .", "the differences in the effect of sterilization with dry heat and steam might be attributed to the use of longer times and higher temperatures in the sterilization process by dry heat .", "however , sterilization with dry heat did not result in significant changes in force levels during loading and unloading of tmal wires [ figure 6 ] .", "previous studies on the effect of dry heat sterilization on -ti wires have evaluated only tma wires ( ormco , glendora , california , usa ) with a size of 0.016 inch .", "reported that use of dry heat sterilization with a temperature of 375f ( 191c ) for 10 minutes on tma archform wires ( ormco , glendora , california , usa ) which had already been used clinically did not exert any influence on tensile and load - deflection test results of the wires .", "the results of the present study with tmal wires are consistent with the results of a study by smith et al . with tma wires", "evaluated the effect of dry heat sterilization at 375f for 20 minutes on tma wires ( ormco , glendora , california , usa ) and reported a significant increase in the tensile strength of wires .", "in addition to the type and size of the wires , other factors , which might have resulted in differences between the results of the present study and other studies , mentioned above might be the differences in the sterilization protocols and the type of the mechanical tests applied .", "the results of the present study did not show any effect of steam sterilization ( autoclave ) [ figures 26 ] on load - deflection characteristics of the majority of -ti wires tested ; however , it increased force levels during loading and unloading of beta and unloading of hone wires , which is reflected by an increased slope of loading and unloading of beta and unloading of hone wires [ figures 2 and 5 ]", ". therefore , these wires will deliver greater forces to teeth after sterilization in an autoclave .", "regarding the low force levels of tmal wire , especially during unloading both in the control group and after sterilization , it appears ion implantation process is only related to surface characteristics and a decrease in friction , with no detrimental effects on load - deflection characteristics of the wire even after sterilization .", "the different effects of sterilization on hone and tmal wires might be attributed to differences in the type and concentration of ions or other factors during the manufacturing processes of these wires . it is suggested that clinicians sterilize tmal , res and cna wires in an autoclave before using them without any worries because their favorable load characteristics will not undergo any changes after sterilization , which is consistent with the results of a study carried out by pernier et al .", "on the effect of autoclave sterilization at 134c or 274f for 18 minutes on tma , tmal and res wires .", "they reported no differences in load - displacement curves after sterilization of any wire tested .", "reported no effect of autoclave sterilization ( 250f for 20 minutes ) on tensile strength of tma wires .", "in addition , a study by smith et al . did not show any perceptible clinical differences between new wires and clinically used wires , which are later , sterilized in an autoclave ( 274f for 10 minutes ) ."], ["\n the results of the present study showed differences in force levels during loading and unloading between the five brands of -ti wires under study after 1.5 mm of deflection ; res , cna and beta wires exhibited different force deflection behaviors in comparison with the two products of ormco company ( tmal and hone).there are different changes in load - deflection characteristics of various -ti wires after sterilization with dry heat and heated steam.the clinicians who want to provide maximum safety for their patients can sterilize res , tmal and cna wires in an autoclave without any worries before placing them in the oral cavity . \n ", "the results of the present study showed differences in force levels during loading and unloading between the five brands of -ti wires under study after 1.5 mm of deflection ; res , cna and beta wires exhibited different force deflection behaviors in comparison with the two products of ormco company ( tmal and hone ) .", "there are different changes in load - deflection characteristics of various -ti wires after sterilization with dry heat and heated steam .", "the clinicians who want to provide maximum safety for their patients can sterilize res , tmal and cna wires in an autoclave without any worries before placing them in the oral cavity .", "however , further in vivo studies are required to substantiate the results of the present study ."]]}
{"article_id": "PMC5033608", "article_text": ["most pituitary adenomas are clinically inactive . in patients with long - standing compression of the optic chiasm , ganglion cells may undergo axonal degeneration .", "spectral domain optical coherence tomography ( sd - oct ) is able to identify retinal nerve fiber layer ( rnfl ) and ganglion cell loss in the retina .", "we present a case in which sd - oct was used to diagnose an asymptomatic pituitary macroadenoma .", "sd - oct identified atrophy of the ganglion cell and nerve layers , with preservation of outer layers bilaterally .", "as macroadenomas enlarge , they can induce uncrossed axon loss , resulting in nasal field defects and reduced visual acuity . in these cases , there is atrophy of the nasal and temporal portions of the optic disc , thus occupying a horizontal band across the disc .", "sd - oct is able to identify rnfl loss in eyes with band atrophy of the optic nerve , which correlates with visual field defects found in perimetry .", "sd - oct is a useful tool to assess the structural and functional damage of ganglion cells . in our case", "the sd - oct demonstrated a symmetrical loss of the rnfl and the ganglion cell layer in both eyes , indicating important optic nerve damage .", "pituitary adenoma is the most common cause of the chiasmal syndrome.1 the tumor is classified based on size as microadenoma , smaller than 10 mm , or as macroadenoma when it exceeds 10 mm in diameter.2 some tumors secrete one or more hormones in excess , so - called secretory pituitary adenomas , but most are clinically inactive.3 classically , the nonsecretory tumors present with vision loss,4 whereas patients with secretory tumors are usually referred to ophthalmologists for evaluation due to hormonal imbalances that affect bodily functions.5 in patients with long - standing compression of the optic chiasm , ganglion cells may undergo axonal degeneration4 .", "optical coherence tomography ( oct ) is able to identify retinal nerve fiber layer ( rnfl ) and ganglion cell loss in retina.6,7 we present a case in which the oct ( 3d oct-2000 spectral domain oct , topcon corporation , tokyo , japan ) was used to diagnose an asymptomatic pituitary macroadenoma .", "the authors adhere to the international standards developed in the 2 conference on research integrity in singapore ( 2010 ) , while performing this study .", "the patient agreed to have their case published , and all information presented is non - identifiable.8", "a 48-year - old , caucasian female presented with progressive vision loss in both eyes for several months .", "her past ocular , medical , and family history was non - contributory with no fatigue , loss of libido , no mood change , and no weight change . on examination , best - corrected visual acuity was hand motion in the right eye and 2/3 in the left eye .", "spectral domain optical coherence tomography ( sd - oct ) demonstrated diffuse atrophy of the ganglion cell and nerve layers , with preservation of outer layers bilaterally ( figure 1 and figure 2 ) .", "magnetic resonance imaging of the brain was performed and a pituitary macroadenoma with suprasellar extension was observed ( figure 3 ) .", "pituitary adenomas are common benign tumors of the pituitary gland that account for 12% of all intracranial tumors.9 people can develop pituitary adenomas at any age .", "some adenomas secrete hormones in excess . of the secretory tumors , the most common are prolactinomas.5 they may be relatively small when detected .", "the most common symptoms include headaches , menstrual changes in females , sexual dysfunction in males , vision problems , and behavioral changes.5 diagnostic imaging may include a high - resolution , t1 weighted , gadolinium enhanced magnetic resonance imaging.10 other secretory tumors may secrete corticotropin , growth hormone , gonadotropins , or thyroid - stimulating hormone.11 adenomas remain confined to the pituitary gland , but when a pituitary adenoma is 10 mm it is called a macroadenoma .", "macroadenomas can compress the rest of the pituitary gland and surrounding structures , such as the crossing retinal ganglion cell axons in the optic chiasm , resulting in bitemporal visual field loss.4 as the tumor enlarges , it has been shown to induce uncrossed axons loss , resulting in nasal field defects and reduced visual acuity . in these cases , there is atrophy of the nasal and temporal portions of the optic disc with relative sparing of the superior and inferior parts where the majority of temporal fibers enter .", "the optic atrophy occupies a horizontal band across the disc , called bowtie or band atrophy.12,13 oct is a noninvasive technique that allows cross - sectional imaging of the retina and quantifies the thickness of the rnfl around the optic nerve head .", "a number of studies have demonstrated that oct is able to identify rnfl loss in eyes with band atrophy of the optic nerve.6,7 the degree of rnfl thickness reduction has been shown to correlate with that of visual field defects using goldmann perimetry4 and automated static perimetry.14,15 this fact gives oct prognostic value regarding the visual outcome , as demonstrated in different studies.16,17 oct is a useful tool to assess the structural and functional damage of ganglion cells , objectively and quantitatively.4 in our case the sd - oct demonstrates a symmetrical loss of the rnfl and the ganglion cell layer in both eyes , indicating important optic nerve damage .", "sd - oct can help in the diagnosis of adenomas in equivocal situations in which there is only subtle optic nerve pallor in the setting of nonspecific visual complaints or visual field defects , or when patients are referred to screen for subclinical compressive optic neuropathies ."], "abstract_text": ["<S> introductionmost pituitary adenomas are clinically inactive . in patients with long - standing compression of the optic chiasm </S>", "<S> , ganglion cells may undergo axonal degeneration . </S>", "<S> spectral domain optical coherence tomography ( sd - oct ) is able to identify retinal nerve fiber layer ( rnfl ) and ganglion cell loss in the retina . </S>", "<S> we present a case in which sd - oct was used to diagnose an asymptomatic pituitary macroadenoma.clinical casea 48-year - old female presented with progressive vision loss in both eyes . </S>", "<S> sd - oct identified atrophy of the ganglion cell and nerve layers , with preservation of outer layers bilaterally . </S>", "<S> magnetic resonance imaging of the brain showed a pituitary macroadenoma . </S>", "<S> the pathological diagnosis was nonfunctioning adenoma.discussionas macroadenomas enlarge , they can induce uncrossed axon loss , resulting in nasal field defects and reduced visual acuity . in these cases , there is atrophy of the nasal and temporal portions of the optic disc , thus occupying a horizontal band across the disc . </S>", "<S> sd - oct is able to identify rnfl loss in eyes with band atrophy of the optic nerve , which correlates with visual field defects found in perimetry . </S>", "<S> sd - oct is a useful tool to assess the structural and functional damage of ganglion cells . in our case </S>", "<S> the sd - oct demonstrated a symmetrical loss of the rnfl and the ganglion cell layer in both eyes , indicating important optic nerve damage . </S>"], "labels": null, "section_names": ["Introduction", "Clinical case", "Discussion", "Introduction", "Clinical case", "Discussion", "Conclusion"], "sections": [["most pituitary adenomas are clinically inactive . in patients with long - standing compression of the optic chiasm , ganglion cells may undergo axonal degeneration .", "spectral domain optical coherence tomography ( sd - oct ) is able to identify retinal nerve fiber layer ( rnfl ) and ganglion cell loss in the retina .", "we present a case in which sd - oct was used to diagnose an asymptomatic pituitary macroadenoma ."], ["sd - oct identified atrophy of the ganglion cell and nerve layers , with preservation of outer layers bilaterally ."], ["as macroadenomas enlarge , they can induce uncrossed axon loss , resulting in nasal field defects and reduced visual acuity . in these cases , there is atrophy of the nasal and temporal portions of the optic disc , thus occupying a horizontal band across the disc .", "sd - oct is able to identify rnfl loss in eyes with band atrophy of the optic nerve , which correlates with visual field defects found in perimetry .", "sd - oct is a useful tool to assess the structural and functional damage of ganglion cells . in our case", "the sd - oct demonstrated a symmetrical loss of the rnfl and the ganglion cell layer in both eyes , indicating important optic nerve damage ."], ["pituitary adenoma is the most common cause of the chiasmal syndrome.1 the tumor is classified based on size as microadenoma , smaller than 10 mm , or as macroadenoma when it exceeds 10 mm in diameter.2 some tumors secrete one or more hormones in excess , so - called secretory pituitary adenomas , but most are clinically inactive.3 classically , the nonsecretory tumors present with vision loss,4 whereas patients with secretory tumors are usually referred to ophthalmologists for evaluation due to hormonal imbalances that affect bodily functions.5 in patients with long - standing compression of the optic chiasm , ganglion cells may undergo axonal degeneration4 .", "optical coherence tomography ( oct ) is able to identify retinal nerve fiber layer ( rnfl ) and ganglion cell loss in retina.6,7 we present a case in which the oct ( 3d oct-2000 spectral domain oct , topcon corporation , tokyo , japan ) was used to diagnose an asymptomatic pituitary macroadenoma .", "the authors adhere to the international standards developed in the 2 conference on research integrity in singapore ( 2010 ) , while performing this study .", "the patient agreed to have their case published , and all information presented is non - identifiable.8"], ["a 48-year - old , caucasian female presented with progressive vision loss in both eyes for several months .", "her past ocular , medical , and family history was non - contributory with no fatigue , loss of libido , no mood change , and no weight change . on examination , best - corrected visual acuity was hand motion in the right eye and 2/3 in the left eye .", "spectral domain optical coherence tomography ( sd - oct ) demonstrated diffuse atrophy of the ganglion cell and nerve layers , with preservation of outer layers bilaterally ( figure 1 and figure 2 ) .", "magnetic resonance imaging of the brain was performed and a pituitary macroadenoma with suprasellar extension was observed ( figure 3 ) ."], ["pituitary adenomas are common benign tumors of the pituitary gland that account for 12% of all intracranial tumors.9 people can develop pituitary adenomas at any age .", "some adenomas secrete hormones in excess . of the secretory tumors , the most common are prolactinomas.5 they may be relatively small when detected .", "the most common symptoms include headaches , menstrual changes in females , sexual dysfunction in males , vision problems , and behavioral changes.5 diagnostic imaging may include a high - resolution , t1 weighted , gadolinium enhanced magnetic resonance imaging.10 other secretory tumors may secrete corticotropin , growth hormone , gonadotropins , or thyroid - stimulating hormone.11 adenomas remain confined to the pituitary gland , but when a pituitary adenoma is 10 mm it is called a macroadenoma .", "macroadenomas can compress the rest of the pituitary gland and surrounding structures , such as the crossing retinal ganglion cell axons in the optic chiasm , resulting in bitemporal visual field loss.4 as the tumor enlarges , it has been shown to induce uncrossed axons loss , resulting in nasal field defects and reduced visual acuity . in these cases , there is atrophy of the nasal and temporal portions of the optic disc with relative sparing of the superior and inferior parts where the majority of temporal fibers enter .", "the optic atrophy occupies a horizontal band across the disc , called bowtie or band atrophy.12,13 oct is a noninvasive technique that allows cross - sectional imaging of the retina and quantifies the thickness of the rnfl around the optic nerve head .", "a number of studies have demonstrated that oct is able to identify rnfl loss in eyes with band atrophy of the optic nerve.6,7 the degree of rnfl thickness reduction has been shown to correlate with that of visual field defects using goldmann perimetry4 and automated static perimetry.14,15 this fact gives oct prognostic value regarding the visual outcome , as demonstrated in different studies.16,17 oct is a useful tool to assess the structural and functional damage of ganglion cells , objectively and quantitatively.4 in our case the sd - oct demonstrates a symmetrical loss of the rnfl and the ganglion cell layer in both eyes , indicating important optic nerve damage ."], ["sd - oct can help in the diagnosis of adenomas in equivocal situations in which there is only subtle optic nerve pallor in the setting of nonspecific visual complaints or visual field defects , or when patients are referred to screen for subclinical compressive optic neuropathies ."]]}
{"article_id": "PMC3015674", "article_text": ["percutaneous nephrolithotomy ( pcnl ) is a widely accepted treatment for urinary calculi > 2 cm or resistant to other modalities .", "complications of this procedure are well reported ; however , to our knowledge no mention has occurred in the literature of a retained nephrostomy tube after pcnl .", "thus , we will present the management of a retained council tip catheter after pcnl .", "a 76-year - old gentleman underwent a pcnl for a 2.2x1.4-cm lower pole calculus via an uneventful upper pole puncture .", "incomplete stone fragmentation was obtained using the ultrasonic lithotripter ; therefore , a 24-french council tip nephrostomy tube was placed , the balloon was inflated with 3 ml of sterile water , and the patient was scheduled for a second look nephroscopy when a holmium laser was available .", "one week later at the second look nephroscopy , attempts to deflate the catheter balloon were unsuccessful .", "after transection of the balloon port valve , attempted passage of a 0.038 movable core bentson guidewire down the balloon port was unsuccessful .", "an antegrade nephrostogram outlined the catheter balloon within the renal pelvis ( figure 1 ) . with the c - arm in a vertical orientation ,", "an 18-gauge chiba needle ( cook , bloomington in ) was advanced through a puncture site directly over the balloon under fluoroscopic guidance .", "the balloon was successfully punctured by the chiba needle with clear efflux through the needle .", "contrast was then instilled through the chiba needle to evaluate the integrity of the balloon and exclude the possibility of retained balloon fragments from a ruptured balloon ( figure 2b ) .", "flexible nephroscopy was performed to evaluate for balloon fragments , which were not identified , and to remove the lower pole stone .", "( a ) chiba needle ( white arrow ) has punctured and drained the catheter balloon .", "( b ) contrast injected via chiba needle has filled the catheter balloon ( dark arrow ) and confirmed its integrity .", "a retained urethral catheter due to balloon malfunction is a well - recognized urologic complication .", "many solutions to this problem have been attempted , including overinflation until rupture , puncture of the balloon via a wire through the balloon port , and puncture of the balloon under ultrasound guidance via a suprapubic or transvaginal route . given this infrequent but known complication of balloon - type cystostomy catheters , it is not surprising that a similar complication occurred in our patient .", "our familiarity with the use of the chiba needle and 3-dimensional fluoroscopy for percutaneous renal access facilitated percutaneous puncture of the catheter balloon .", "if equipment or experience limits the urologist 's ability to perform this technique , collaboration with an interventional radiologist using conventional c - arm or ultrasound for guidance should be considered .", "prevention of a retained catheter due to a balloon - port malfunction requires use of sterile water instead of saline and limiting the time of catheter placement .", "malecot tubes have been utilized for nephrostomy drainage , but these tubes are not without difficulties . tissue bridge formation over flanges of the tube has occurred , requiring endoscopic removal .", "tubeless pcnl would avoid these issues entirely ; however , the tubeless technique is contraindicated in the face of significant postoperative bleeding , urinary extravasation , ureteral obstruction , or anticipated need for a second - look nephroscopy .", "pigtail catheters following pcnl may represent a good alternative for drainage and access , while minimizing the risk of catheter malfunction or retention ."], "abstract_text": ["<S> percutaneous nephrolithotomy is a widely accepted treatment for urinary calculi , but it is not without complications . we present the case of a 76-year - old male with a retained council tip catheter after percutaneous nephrolithotomy . </S>", "<S> fluoroscopic guidance was used to perform percutaneous puncture of the catheter balloon , and the catheter was removed without complication . </S>", "<S> advantages of various nephrostomy tube designs and additional measures to prevent this type of complication are discussed . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["percutaneous nephrolithotomy ( pcnl ) is a widely accepted treatment for urinary calculi > 2 cm or resistant to other modalities .", "complications of this procedure are well reported ; however , to our knowledge no mention has occurred in the literature of a retained nephrostomy tube after pcnl .", "thus , we will present the management of a retained council tip catheter after pcnl ."], ["a 76-year - old gentleman underwent a pcnl for a 2.2x1.4-cm lower pole calculus via an uneventful upper pole puncture .", "incomplete stone fragmentation was obtained using the ultrasonic lithotripter ; therefore , a 24-french council tip nephrostomy tube was placed , the balloon was inflated with 3 ml of sterile water , and the patient was scheduled for a second look nephroscopy when a holmium laser was available .", "one week later at the second look nephroscopy , attempts to deflate the catheter balloon were unsuccessful .", "after transection of the balloon port valve , attempted passage of a 0.038 movable core bentson guidewire down the balloon port was unsuccessful .", "an antegrade nephrostogram outlined the catheter balloon within the renal pelvis ( figure 1 ) . with the c - arm in a vertical orientation ,", "an 18-gauge chiba needle ( cook , bloomington in ) was advanced through a puncture site directly over the balloon under fluoroscopic guidance .", "the balloon was successfully punctured by the chiba needle with clear efflux through the needle .", "contrast was then instilled through the chiba needle to evaluate the integrity of the balloon and exclude the possibility of retained balloon fragments from a ruptured balloon ( figure 2b ) .", "flexible nephroscopy was performed to evaluate for balloon fragments , which were not identified , and to remove the lower pole stone .", "( a ) chiba needle ( white arrow ) has punctured and drained the catheter balloon .", "( b ) contrast injected via chiba needle has filled the catheter balloon ( dark arrow ) and confirmed its integrity ."], ["a retained urethral catheter due to balloon malfunction is a well - recognized urologic complication .", "many solutions to this problem have been attempted , including overinflation until rupture , puncture of the balloon via a wire through the balloon port , and puncture of the balloon under ultrasound guidance via a suprapubic or transvaginal route . given this infrequent but known complication of balloon - type cystostomy catheters , it is not surprising that a similar complication occurred in our patient .", "our familiarity with the use of the chiba needle and 3-dimensional fluoroscopy for percutaneous renal access facilitated percutaneous puncture of the catheter balloon .", "if equipment or experience limits the urologist 's ability to perform this technique , collaboration with an interventional radiologist using conventional c - arm or ultrasound for guidance should be considered .", "prevention of a retained catheter due to a balloon - port malfunction requires use of sterile water instead of saline and limiting the time of catheter placement .", "malecot tubes have been utilized for nephrostomy drainage , but these tubes are not without difficulties . tissue bridge formation over flanges of the tube has occurred , requiring endoscopic removal .", "tubeless pcnl would avoid these issues entirely ; however , the tubeless technique is contraindicated in the face of significant postoperative bleeding , urinary extravasation , ureteral obstruction , or anticipated need for a second - look nephroscopy .", "pigtail catheters following pcnl may represent a good alternative for drainage and access , while minimizing the risk of catheter malfunction or retention ."]]}
{"article_id": "PMC4742442", "article_text": ["radical prostatectomy ( rp ) is a common curative surgical treatment option in localized prostate cancer ( pca ) .", "pelvic lymph node ( ln ) dissection is performed in order to determine the disease stage , prognosis , and the necessary future treatment options [ 1 , 2 ] .", "the fatty tissue overlying the prostate is removed in order to have a better exposure of the dorsal venous complex , the puboprostatic ligaments , and the prostate apex . however ,", "if this fatty tissue is not examined separately by a pathologist , the presence of lns and possible metastasis might be missed and underreported .", "this might ultimately have an impact on the postoperative disease stage , treatment , and oncologic outcomes . in our study", ", we investigated the histopathologic evaluation outcomes of anterior periprostatic fat ( appf ) tissues that were separately sent for pathologic evaluation in order to examine presence of lns and metastasis .", "overall , 129 patients who underwent rarp between january 2012 and october 2014 were included in our study .", "surgical procedures were performed by three robotic surgeons ( afa , aec and mdb ) .", "all of the rarp procedures were performed via a transperitoneal approach and patients who had history of transurethral surgery or radiotherapy were excluded from the study . during the rarp procedures ,", "appf covering prostate , endopelvic fascia , dorsal venous complex , prostatic apex , puboprostatic ligaments , prostate - vesical junction and the bladder neck was dissected en bloc laterally and cephalad .", "if metastatic ln(s ) was /were detected , immunohistochemistry for prostate specific antigen ( psa ) and prostate specific acid phosphatase were performed .", "parameters including serum psa , prostate weight , body mass index ( bmi ) , positive surgical margins ( psm ) and gleason score ( gs ) were included for further evaluation .", "patients were classified according to bmi ( kg / m ) as follows : < 18.5 underweight , 18.524.9 optimal weight , 2529.9 overweight and > 30 obese .", "mean patient age , serum psa , prostate weight and bmi were 62.2 5.5 ( range 4574 ) , 9.3 6.3ng / dl ( range 0.330.3 ) , 60.3 27.2 grams ( range 11.0180 ) and 26.6 1.9 kg / m ( range 20.0 - 30.3 ) , respectively .", "overall , lns in appf tissues were detected in 14 ( 10.6% ) patients with a mean ln yield of 1.1 0.7 lns ( range , 13 ) . among those found ,", "no additional complication or morbidity due to excision of the appf was found in any of the patients . \n ", "patient characteristics postoperative pathologic stages included pt0 ( n = 1 , 0.8% ) , pt2a ( n = 22 , 17.1% ) , pt2b ( n = 15 , 11.6% ) , pt2c ( n = 62 , 48.1% ) , pt3a ( n = 21 , 16.3% ) and pt3b ( n = 8 , 6.2% ) . overall , psm were detected in 32 ( 24.8% ) patients . of the patients with pt2 disease ( n = 99 ) , psm rate was 13.1% ( n = 13 ) .", "of the patients with pt3 disease ( n = 29 ) , psm rate was 65.5% ( n = 19 ) .", "of the patients with pt2a ( n = 22 ) , pt2b ( n = 15 ) , pt2c ( n = 62 ) and pt3a ( n = 21 ) disease , lns in appf tissues were detected in 1 ( 4.6% ) , 1 ( 6.7% ) , 11 ( 17.7% ) and 1 ( 4.8% ) patient in each group , respectively .", "no ln was detected in appf tissue in patients with pt0 ( n = 1 ) and pt3b ( n = 8) disease .", "of the patients with postoperative gs 3 + 3 = 6 ( n = 63 ) , gs 3 + 4 = 7 ( n = 34 ) , gs 4 + 3 = 7 ( n = 14 ) and gs 4 + 4 = 8 ( n = 7 ) diseases , lns in appf tissues were detected in 11 ( 17.5% ) , 1 ( 2.9% ) , 1 ( 7.1% ) and 1 ( 14.3% ) patient , respectively .", "no ln was detected in appf tissue in patients with postoperative gs of 5 + 3 = 8 ( n = 1 ) , 3 + 5 = 8 ( n = 2 ) and 4 + 5 = 9 ( n = 6 ) .", "we did not have any patient with postoperative gs of 5 + 4 = 9 or 5 + 5 = 10 .", "of the patients , lns in appf tissues were detected in 0 ( 0% ) , 5 ( 35.7% ) , 8 ( 57.1% ) and 1 ( 7.1% ) patients of underweight , optimal weight , overweight and obese patients due to bmi , respectively .", "no association was detected between bmi and presence of lns or mean ln count in the appf tissue ( p > 0.05 ) .", "we removed appf tissue as an en bloc excision and sent it as one piece for pathologic evaluation .", "we generally started removing the fat laterally from one side on the endopelvic fascia from , laterally to medially , and then shift to the other side .", "we accumulated both sides in the middle around the superficial dorsal vein on the prostate .", "thereafter , we elevated it above the prostate using maryland bipolar scissors and cauterized and cut its tip over the prostatic apex by applying bipolar energy .", "we then pushed it back up to the junction between the prostate and bladder neck and excised the tissue by applying monopolar and bipolar cautery . following excision of the appf tissue , the area of endopelvic fascia ,", "puboprostatic ligaments , prostatic apex , surface of the prostate and junction between prostate and bladder neck are all cleared ( figures 1 and 2 ) .", "prostate , endopelvic fascia , puboprostatic ligaments , junction between prostate and bladder neck are covered with fat tissue .", "prostate , endopelvic fascia , puboprostatic ligaments , junction between prostate and bladder neck are seen clearly following excision of the fat tissue .", "in our study , we identified lns in appf tissue in 10.9% of the patients . aning et al .  ", "reported 14.7% ( n = 204 ) lns in appf tissue in their series , respectively .", "therefore , our findings are similar to previously published studies . in our study , no metastatic ln was detected in the lymphoid tissue that was obtained from the appf tissue region overlying the prostate .", "however , finley et al .   found ln metastasis in 4 of 204 patients and jeong et al", "due to the published literature , ln metastasis rate in anterior periprostatic lymphoid tissue varied between 1.22.5% [ 58 ] . in our study , no metastatic ln was detected , which might be related to the limited number of patients in our series compared to the other published articles with a larger number of patients .", "in addition , studies that identified ln metastasis in anterior periprostatic lymphoid tissue had a higher volume of patients with high and intermediate risk patients , as compared to our study . in a multi - center study , kim et al", ". included 4261 rrp and rarp patients and there were 40 patients who had metastasis on periprostatic lns .", "of the 40 patients , 31 ( 77.5% ) were in a high - risk group , 6 ( 15% ) of them were in an intermediate group and 3 ( 7.5% ) of them were in a low - risk group according to d'amico risk classifications .", "jeong et al . detected metastatic lns in appf tissue in 3 patients that were all in the high - risk group . in our study , 55 ( 42.6% ) patients were in the high - risk group , 52 ( 40.3% ) patients were in the intermediate - risk group and 22 ( 17.1% ) patients were in the low - risk group .", "we summarized the outcomes of published literature on appf tissue and presence of lns in table 2 . \n ", "outcomes of published literature on periprostatic fat tissue and presence of lymph nodes the european association of urology ( eau )   and national comprehensive cancer network ( nccn )   recommended extended pelvic lymph node dissection ( plnd ) in patients at 10% or greater and 7% or greater calculated risk , respectively , for ln metastases . likewise , the american urological association ( aua ) recommended plnd in patients at higher risk for nodal involvement .", "prognostic significance of ln metastases in prostate cancer   and assessment of rates of lns and ln metastases in periprostatic fat pads in radical prostatectomy were reported before . in our experience , we performed extended plnd in intermediate or high - risk pca patients and in those with at least 5% risk of pelvic ln involvement by pca , according to partin 's tables .", "in addition , we always remove appf tissue and send it separately for histopathologic evaluation in patients with low , intermediate and high - risk in order to identify the presence of lns and possible metastasis that we think could have an impact on identifying the correct stage of the disease .", "we remove appf tissue in low - risk patients because removal of this tissue leads to better exposure of the prostatic apex , puboprostatic ligaments , endopelvic fascia and junction of bladder and prostate .", "kim et al . suggested that removal of appf tissue leads to more accurate staging of the patients . in their series ,", "due to the identification of metastatic lns in the appf tissue , 0.63% of the patients were up - staged pathologically .", "this finding might certainly have an impact on postoperative adjuvant treatment requirement in this patient group .", "the condition of regional lns is one of the most important prognostic indicators of disease free survival and overall survival in pca .", "if a metastatic ln is detected in appf tissue ; patients might require adjuvant hormone therapy or radiotherapy . by dissecting appf tissue anatomically , finley et al .  ", "observed that the anterior fat pad was directly connected to the obturator ln chain at the lateral pelvic wall .", "jeong et al . identified 3 patients with metastatic lns in appf tissue in their series of 258 patients , and 2 of these 3 also had metastasis in pelvic lns .", "harnisch et al . identified 4 patients who had metastasis in appf tissue in their study including 302 patients and 2 of these 4 patients had also pelvic ln metastasis .", "identified 4 patients with metastatic periprostatic lns but only one of them had also pelvic ln metastasis . to assess the need of adjuvant therapy , postoperative serum psa follow - up is important as well as detecting metastatic lns . in their multicenter study including 4261 patients , kim et al .", "reported that 6 of 40 patients with metastasis on periprostatic anterior fatty lymphoid tissue did not require adjuvant therapy at a follow - up of 1.651 months , which might suggest a therapeutic advantage .", "however , kim et al . separately evaluated appf tissue in three pieces as a left packet , middle packet and a right packet . in their results ,", ", increased cost was another issue for pathological evaluation of periprostatic fat tissue . in order to decrease cost , they suggested asking for pathological evaluation of the appf tissue only in intermediate and high - risk patients . in our country , if the surgery is performed in private institutions , pathologic evaluation of the appf tissue increases costs .", "however , in government institutions , it does not increase overall cost because all expenses are covered by the government .", "in addition , because we think that complete removal of appf tissue leads to better exposure of the surgical field to the operating console surgeon , we excise it even in patients with low - risk pca . in our study , no association was detected between bmi and presence of lns or ln count in the appf tissue .", "bmi might have an impact on the amount of periprostatic fat rather than presence of lns .", "not only robotic series but also open retropubic radical prostatectomy ( rrp ) series have evaluated ln existence in appf tissue .", "identified lns in periprostatic region in 19 ( 5.5% ) patients of 356 in their rrp series and 4 ( 1.2% ) of them had metastasis , one of 4 patients had also pelvic ln metastasis . in this study they reported that there is no connection between periprostatic ln meta - stasis and pelvic ln metastasis , but in order to provide exact staging , pathologic examination of periprostatic lymphatic area should be a routine procedure .", "our study has some limitations that include limited number of patients and differences in the experiences of the surgeons that performed the cases . with increasing number of patients and having more high - risk pca patients", "lymph nodes might be present in the appf tissue which should be removed and sent for histopathologic evaluation following rarp .", "identification of metastatic lns in the appf might have an impact on postoperative staging and management of the patients .", "in addition , removal of appf leads to a better exposure of the surgical field ."], "abstract_text": ["<S> introductionwe investigated whether anterior periprostatic fat ( appf ) tissue removed during robotic radical prostatectomy ( rarp ) contains any lymph nodes ( lns).material and methodsappf tissues removed during rarp in 129 patients were evaluated histopathologically . </S>", "<S> correlation with postoperative pathologic stage was made . </S>", "<S> patients with a history of previous prostate or bladder surgery and radiation therapy were excluded.resultsmean patient age , serum prostate specific antigen ( psa ) , prostate weight and body mass index ( bmi ) were 62.2 5.5 ( range 4574 ) , 9.3 6.3 ng / dl ( range 0.26 - 30.3 ) , 60.3 27.2 grams ( range 11.0180 ) and 26.6 1.9 kg / m2 ( range 20.030.3 ) , respectively . </S>", "<S> overall , lns in appf tissues were detected in 14 ( 10.9% ) patients with a mean ln yield of 1.1 0.7 lns ( range , 13 ) . among those found , </S>", "<S> no metastatic ln was detected . </S>", "<S> of the patients with pt2a ( n = 22 ) , pt2b ( n = 15 ) , pt2c ( n = 62 ) and pt3a ( n = 21 ) disease , lns in appf tissues were detected in 1 ( 4.6% ) , 1 ( 6.7% ) , 11 ( 17.7% ) and 1 ( 4.8% ) patient in each group , respectively . among the patients , </S>", "<S> lns in appf tissues were detected in 0 ( 0% ) , 5 ( 35.7% ) , 8 ( 57.1% ) and 1 ( 7.1% ) patients of underweight , optimal weight , overweight and obese patients due to body mass index , respectively.conclusionsin our series , lns were detected in around 10% of the patients . </S>", "<S> therefore , this fat should , not be pushed back during rarp but should be removed and sent for pathologic evaluation . although no metastatic ln was detected in our series , the presence of metastatic lns might have an impact on the oncologic outcomes of the patients and warrants further research . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHOD", "RESULTS", "DISCUSSION", "CONCLUSIONS", "CONFLICTS OF INTEREST"], "sections": [["radical prostatectomy ( rp ) is a common curative surgical treatment option in localized prostate cancer ( pca ) .", "pelvic lymph node ( ln ) dissection is performed in order to determine the disease stage , prognosis , and the necessary future treatment options [ 1 , 2 ] .", "the fatty tissue overlying the prostate is removed in order to have a better exposure of the dorsal venous complex , the puboprostatic ligaments , and the prostate apex . however ,", "if this fatty tissue is not examined separately by a pathologist , the presence of lns and possible metastasis might be missed and underreported .", "this might ultimately have an impact on the postoperative disease stage , treatment , and oncologic outcomes . in our study", ", we investigated the histopathologic evaluation outcomes of anterior periprostatic fat ( appf ) tissues that were separately sent for pathologic evaluation in order to examine presence of lns and metastasis ."], ["overall , 129 patients who underwent rarp between january 2012 and october 2014 were included in our study .", "surgical procedures were performed by three robotic surgeons ( afa , aec and mdb ) .", "all of the rarp procedures were performed via a transperitoneal approach and patients who had history of transurethral surgery or radiotherapy were excluded from the study . during the rarp procedures ,", "appf covering prostate , endopelvic fascia , dorsal venous complex , prostatic apex , puboprostatic ligaments , prostate - vesical junction and the bladder neck was dissected en bloc laterally and cephalad .", "if metastatic ln(s ) was /were detected , immunohistochemistry for prostate specific antigen ( psa ) and prostate specific acid phosphatase were performed .", "parameters including serum psa , prostate weight , body mass index ( bmi ) , positive surgical margins ( psm ) and gleason score ( gs ) were included for further evaluation .", "patients were classified according to bmi ( kg / m ) as follows : < 18.5 underweight , 18.524.9 optimal weight , 2529.9 overweight and > 30 obese ."], ["mean patient age , serum psa , prostate weight and bmi were 62.2 5.5 ( range 4574 ) , 9.3 6.3ng / dl ( range 0.330.3 ) , 60.3 27.2 grams ( range 11.0180 ) and 26.6 1.9 kg / m ( range 20.0 - 30.3 ) , respectively .", "overall , lns in appf tissues were detected in 14 ( 10.6% ) patients with a mean ln yield of 1.1 0.7 lns ( range , 13 ) . among those found ,", "no additional complication or morbidity due to excision of the appf was found in any of the patients . \n ", "patient characteristics postoperative pathologic stages included pt0 ( n = 1 , 0.8% ) , pt2a ( n = 22 , 17.1% ) , pt2b ( n = 15 , 11.6% ) , pt2c ( n = 62 , 48.1% ) , pt3a ( n = 21 , 16.3% ) and pt3b ( n = 8 , 6.2% ) . overall , psm were detected in 32 ( 24.8% ) patients . of the patients with pt2 disease ( n = 99 ) , psm rate was 13.1% ( n = 13 ) .", "of the patients with pt3 disease ( n = 29 ) , psm rate was 65.5% ( n = 19 ) .", "of the patients with pt2a ( n = 22 ) , pt2b ( n = 15 ) , pt2c ( n = 62 ) and pt3a ( n = 21 ) disease , lns in appf tissues were detected in 1 ( 4.6% ) , 1 ( 6.7% ) , 11 ( 17.7% ) and 1 ( 4.8% ) patient in each group , respectively .", "no ln was detected in appf tissue in patients with pt0 ( n = 1 ) and pt3b ( n = 8) disease .", "of the patients with postoperative gs 3 + 3 = 6 ( n = 63 ) , gs 3 + 4 = 7 ( n = 34 ) , gs 4 + 3 = 7 ( n = 14 ) and gs 4 + 4 = 8 ( n = 7 ) diseases , lns in appf tissues were detected in 11 ( 17.5% ) , 1 ( 2.9% ) , 1 ( 7.1% ) and 1 ( 14.3% ) patient , respectively .", "no ln was detected in appf tissue in patients with postoperative gs of 5 + 3 = 8 ( n = 1 ) , 3 + 5 = 8 ( n = 2 ) and 4 + 5 = 9 ( n = 6 ) .", "we did not have any patient with postoperative gs of 5 + 4 = 9 or 5 + 5 = 10 .", "of the patients , lns in appf tissues were detected in 0 ( 0% ) , 5 ( 35.7% ) , 8 ( 57.1% ) and 1 ( 7.1% ) patients of underweight , optimal weight , overweight and obese patients due to bmi , respectively .", "no association was detected between bmi and presence of lns or mean ln count in the appf tissue ( p > 0.05 ) .", "we removed appf tissue as an en bloc excision and sent it as one piece for pathologic evaluation .", "we generally started removing the fat laterally from one side on the endopelvic fascia from , laterally to medially , and then shift to the other side .", "we accumulated both sides in the middle around the superficial dorsal vein on the prostate .", "thereafter , we elevated it above the prostate using maryland bipolar scissors and cauterized and cut its tip over the prostatic apex by applying bipolar energy .", "we then pushed it back up to the junction between the prostate and bladder neck and excised the tissue by applying monopolar and bipolar cautery . following excision of the appf tissue , the area of endopelvic fascia ,", "puboprostatic ligaments , prostatic apex , surface of the prostate and junction between prostate and bladder neck are all cleared ( figures 1 and 2 ) .", "prostate , endopelvic fascia , puboprostatic ligaments , junction between prostate and bladder neck are covered with fat tissue .", "prostate , endopelvic fascia , puboprostatic ligaments , junction between prostate and bladder neck are seen clearly following excision of the fat tissue ."], ["in our study , we identified lns in appf tissue in 10.9% of the patients . aning et al .  ", "reported 14.7% ( n = 204 ) lns in appf tissue in their series , respectively .", "therefore , our findings are similar to previously published studies . in our study , no metastatic ln was detected in the lymphoid tissue that was obtained from the appf tissue region overlying the prostate .", "however , finley et al .   found ln metastasis in 4 of 204 patients and jeong et al", "due to the published literature , ln metastasis rate in anterior periprostatic lymphoid tissue varied between 1.22.5% [ 58 ] . in our study , no metastatic ln was detected , which might be related to the limited number of patients in our series compared to the other published articles with a larger number of patients .", "in addition , studies that identified ln metastasis in anterior periprostatic lymphoid tissue had a higher volume of patients with high and intermediate risk patients , as compared to our study . in a multi - center study , kim et al", ". included 4261 rrp and rarp patients and there were 40 patients who had metastasis on periprostatic lns .", "of the 40 patients , 31 ( 77.5% ) were in a high - risk group , 6 ( 15% ) of them were in an intermediate group and 3 ( 7.5% ) of them were in a low - risk group according to d'amico risk classifications .", "jeong et al . detected metastatic lns in appf tissue in 3 patients that were all in the high - risk group . in our study , 55 ( 42.6% ) patients were in the high - risk group , 52 ( 40.3% ) patients were in the intermediate - risk group and 22 ( 17.1% ) patients were in the low - risk group .", "we summarized the outcomes of published literature on appf tissue and presence of lns in table 2 . \n ", "outcomes of published literature on periprostatic fat tissue and presence of lymph nodes the european association of urology ( eau )   and national comprehensive cancer network ( nccn )   recommended extended pelvic lymph node dissection ( plnd ) in patients at 10% or greater and 7% or greater calculated risk , respectively , for ln metastases . likewise , the american urological association ( aua ) recommended plnd in patients at higher risk for nodal involvement .", "prognostic significance of ln metastases in prostate cancer   and assessment of rates of lns and ln metastases in periprostatic fat pads in radical prostatectomy were reported before . in our experience , we performed extended plnd in intermediate or high - risk pca patients and in those with at least 5% risk of pelvic ln involvement by pca , according to partin 's tables .", "in addition , we always remove appf tissue and send it separately for histopathologic evaluation in patients with low , intermediate and high - risk in order to identify the presence of lns and possible metastasis that we think could have an impact on identifying the correct stage of the disease .", "we remove appf tissue in low - risk patients because removal of this tissue leads to better exposure of the prostatic apex , puboprostatic ligaments , endopelvic fascia and junction of bladder and prostate .", "kim et al . suggested that removal of appf tissue leads to more accurate staging of the patients . in their series ,", "due to the identification of metastatic lns in the appf tissue , 0.63% of the patients were up - staged pathologically .", "this finding might certainly have an impact on postoperative adjuvant treatment requirement in this patient group .", "the condition of regional lns is one of the most important prognostic indicators of disease free survival and overall survival in pca .", "if a metastatic ln is detected in appf tissue ; patients might require adjuvant hormone therapy or radiotherapy . by dissecting appf tissue anatomically , finley et al .  ", "observed that the anterior fat pad was directly connected to the obturator ln chain at the lateral pelvic wall .", "jeong et al . identified 3 patients with metastatic lns in appf tissue in their series of 258 patients , and 2 of these 3 also had metastasis in pelvic lns .", "harnisch et al . identified 4 patients who had metastasis in appf tissue in their study including 302 patients and 2 of these 4 patients had also pelvic ln metastasis .", "identified 4 patients with metastatic periprostatic lns but only one of them had also pelvic ln metastasis . to assess the need of adjuvant therapy , postoperative serum psa follow - up is important as well as detecting metastatic lns . in their multicenter study including 4261 patients , kim et al .", "reported that 6 of 40 patients with metastasis on periprostatic anterior fatty lymphoid tissue did not require adjuvant therapy at a follow - up of 1.651 months , which might suggest a therapeutic advantage .", "however , kim et al . separately evaluated appf tissue in three pieces as a left packet , middle packet and a right packet . in their results ,", ", increased cost was another issue for pathological evaluation of periprostatic fat tissue . in order to decrease cost , they suggested asking for pathological evaluation of the appf tissue only in intermediate and high - risk patients . in our country , if the surgery is performed in private institutions , pathologic evaluation of the appf tissue increases costs .", "however , in government institutions , it does not increase overall cost because all expenses are covered by the government .", "in addition , because we think that complete removal of appf tissue leads to better exposure of the surgical field to the operating console surgeon , we excise it even in patients with low - risk pca . in our study , no association was detected between bmi and presence of lns or ln count in the appf tissue .", "bmi might have an impact on the amount of periprostatic fat rather than presence of lns .", "not only robotic series but also open retropubic radical prostatectomy ( rrp ) series have evaluated ln existence in appf tissue .", "identified lns in periprostatic region in 19 ( 5.5% ) patients of 356 in their rrp series and 4 ( 1.2% ) of them had metastasis , one of 4 patients had also pelvic ln metastasis . in this study they reported that there is no connection between periprostatic ln meta - stasis and pelvic ln metastasis , but in order to provide exact staging , pathologic examination of periprostatic lymphatic area should be a routine procedure .", "our study has some limitations that include limited number of patients and differences in the experiences of the surgeons that performed the cases . with increasing number of patients and having more high - risk pca patients"], ["lymph nodes might be present in the appf tissue which should be removed and sent for histopathologic evaluation following rarp .", "identification of metastatic lns in the appf might have an impact on postoperative staging and management of the patients .", "in addition , removal of appf leads to a better exposure of the surgical field ."], [""]]}
{"article_id": "PMC4976135", "article_text": ["the american marten ( martes americana ) is an arboreal mesocarnivore that ranges from the boreal forests of northern north america into coniferous and mixed coniferous / deciduous forests of the northern and northeastern united states , including the great lakes region ( clark et  al . , 1987 ) .", "the american marten was reintroduced to michigan 's upper peninsula ( up ) and northern lower peninsula ( nlp ) in the mid-20th century after regional extirpation due to habitat loss and over - harvest ( cooley et  al . , 2004 ) .", "over 200 animals were reintroduced to the up over the course of several reintroductions . many fewer animals ( n  =  36 ) were reintroduced in the huron - manistee national forest of the nlp and 49 martens were reintroduced to the pigeon river state forest of the nlp .", "in contrast , the species is considered a regional forester sensitive species and there is no harvest in the nlp ( usda forest service , 2012 ) .", "factors hypothesized to be contributing to the differences in population sustainability between the up and nlp include differences in habitat , genetic diversity , health and others . at the time of the original reintroduction", ", american marten were not examined for parasitic or infectious diseases ( spriggs , unpublished data ) .", "some parasites are of economic or zoonotic importance and may be introduced with animal translocations .", "therefore , reintroduction programs should take into account the presence of parasites which are pathogenic or to which the species of concern is not adapted ( kimber and kollias , 2000 ) .", "a collaborative research effort has begun in order to investigate factors that may be contributing to the difference in sustainability between the up and nlp populations .", "the aim of this parasite survey was to describe the presence and prevalence of parasites in the huron - manistee national forest of the nlp and to determine whether there are differences in presence or prevalence of parasites between the nlp and up .", "should future translocation of animals from the up into the nlp be considered for management of the species , a non - invasive and inexpensive method for screening animals would be desirable . also , american marten are not harvested in the nlp and thus adequate numbers of carcasses are not available for examination .", "while some information exists regarding the prevalence of endoparasitism in american marten in north american , relatively little information is available for the species in michigan ( poole et  al . , 1983 , veine - smith et  al . , 2011 ) .", "this study reviews previous parasite prevalence reports from american marten in north america , presents data from the parasitological examination of live - trapped american marten in michigan , and identifies a possible association between hookworm infection and anaemia in affected american marten .", "american marten ( n  =  49 ) were sampled from the manistee national forest in the nlp ( n  =  31 ) , hiawatha national forest in the up ( n  =  13 ) and ottawa national forest in the up ( n  =  5 ) .", "american marten were trapped and immobilized from 2011 to 2015 as described by desmarchelier et  al .", "nine american marten were recaptured and re - sampled , resulting in a total of 60 faecal samples included in analyses .", "blood was collected from the jugular vein and placed into lithium heparin anticoagulant ( bd microtainer tubes , becton dickinson and company ) .", "we determined haematocrit using microhematocrit capillary tubes ( safecrit ) and the statspin vt centrifuge ( iris ) .", "individuals were identified as anaemic if the haematocrit was < 42% based on a report of normal haematocrit in captive , fed american martens of 47    5% ( nieminen et  al . , 2007 ) .", "other health parameters were collected as part of a complete health assessment ( spriggs , unpublished data ) .", "faecal float and sedimentation examinations were performed at michigan state university 's diagnostic center for population and animal health using standard methods ( zajac and conboy , 2012 ) .", "faecal flotation was performed using sheather 's sugar solution ( specific gravity 1.251.27 ) alone ( n  =  30 ) from may 2011may 2012 or with both sheather 's sugar solution and zinc sulfate solution ( specific gravity 1.18 , n  =  30 ) from june 2012january 2015 and examined by light microscopy .", "fifty - nine of the samples , representing 48 individual animals , were sufficient in quantity for faecal sedimentation procedure .", "ova were identified based on morphologic characteristics including size and in accordance with parasitologic references and previous reports . upon consultation with a wildlife veterinary parasitologist ( gerhold ) , 2 nematode species ( syphacia muris and aspicularis sp . )", "prevalence was calculated as the number of infected hosts divided by the number of hosts examined . because some american marten were sampled more than once , all parasite species found in an individual were considered together for prevalence calculations .", "differences between locations ( up and nlp ) and sexes were examined with a pearson 's  test with p  <  0.05 considered significant .", "significant differences between presence or absence of anaemia and hookworm infection were also examined with a pearson 's  test .", "parasite species richness by host was calculated as the number of parasite species present per host ; species richness by sample was calculated as the number of parasite species present per sample .", "an unidentified capillarid species was not included in the prevalence or host species richness if one or more species of capillaria was identified in other samples from the same host .", "thus , an individual american marten found with unidentified capillarid at the initial exam and aonchotheca sp . at a subsequent exam", "if both samples had unidentified capillaria , then the host was included in the calculation of prevalence of unidentified capillaria .", "differences between sex and location in species richness were examined with a wilcoxon rank sum test , with p  <  0.05 considered significant . the capture and handling protocol", "was approved by the university of tennessee animal care and use committee ( protocol # 2180 ) , and american marten live - trapping and sample collection was an authorized tribal activity under the 2007 inland consent decree between the state of michigan and the little river band of ottawa indians .", "sixty samples from 49 individual american marten ( 28 males , 21 females ) were examined , and results are shown in table  1 .", "of 49 individual american marten examined , 91.8% were positive for 1 or more parasites and 69.4% were infected with 2 or more parasites .", "there was no significant difference in mean species richness by host between sexes or locations ( nlp and up ) .", "trematode egg identification was suspected but not confirmed to be alaria sp . during the early part of the study ( may 2011may 2012 ) when sheather 's sugar solution alone was used for flotation , as alaria species may be distorted due to the osmotic pressure of sugar solution . later in the study", "( june 2012january 2015 ) , trematode ova found on sedimentation were confirmed to be alaria species when the sample was floated with zinc sulfate solution , in which trematode ova will not be distorted .", "once the use of zinc sulfate solution was implemented , no trematode other than alaria species was identified , and the authors concluded that trematode eggs identified in the early samples were most likely alaria species , as suspected . in the up , 3 samples that were positive on sedimentation for trematode ova were suspected to be alaria species and 7 were confirmed via zinc sulfate flotation ; in the nlp , 14 samples positive on sedimentation were suspected to be alaria species and 5 were confirmed via zinc sulfate flotation .", "capillaria eggs were seen in 79% and 78.1% of samples from the up and nlp , respectively .", "capillaria eggs were further identified as eucoleus aerophila , eucoleus boehmi , or aonchotheca putorii based on size and morphologic characteristics . a hookworm egg is shown in fig .  1 .", "there was no significant difference in prevalence of any of the identified parasites between male and female american marten .", "of 9 american marten that were sampled more than once , only one had identical results for each time point .", "the mean haematocrit was 45.6    8.1 ( range 3068 ; n  =  49 ) .", "haematocrit was reported to be 47    5% in the only other report of american marten haematocrit ( nieminen et  al . , 2007 ) . using < 42% as a cut - off", "american marten infected with hookworms were significantly more likely to be anaemic than non - infected american marten ( p  =  0.01 ) with an odds ratio of 8.75 ( 95% confidence interval : 1.456.4 ) .", "parasite species richness per host was similar to that reported by veine - smith et  al .", "we identified more parasite species in the nlp than the up , but this result may be a function of the larger sample size from the nlp , and there was no significant difference in richness between the 2 locations .", "foreyt and langerquist ( 1993 ) found 2 or more parasites in 35% of american marten from eastern washington . in another survey from washington ,", "9 helminth species were found , and 48.4% of hosts had coinfection of 2 or more parasites , with a maximum of 4 ( hoberg et  al . , 1990 ) .", "overall , parasite prevalences were similar between the up and nlp in our samples and we conclude that the up may be suitable source for a translocation of marten to the nlp from the point of view of parasite translocation .", "additional screening of both populations , the use of molecular techniques to definitively speciate parasites , and testing of individual martens marked for translocation are recommended .", "parasites in our study were identified to the genus and/or species level as possible by microscopic examination alone .", "future research could employ the use of molecular tools such as polymerase chain reaction to provide more specific results .", "such techniques were not used in this study because we desired an inexpensive method that could be repeated in any future reintroduction programs that require screening of large numbers of animals .", "parasites identified as a concern for reintroduction of north american river otter included alaria canis , strongyloides lutrae , crenosoma goblei , capillaria and coccidia due to potential for pathogenic effects or high prevalence ( kimber and kollias , 2000 ) .", "river otter reintroduction programs tested and treated parasitized otters , and similar methods could be considered for reintroduction of american marten ( hoover et  al . , 1985 , griess , 1987 , serfass et  al . ,", "of 9 american marten that were sampled more than once , only one had identical results at subsequent sampling , indicating either a change in infection status or inconsistent shedding of ova in the remaining 8 marten .", "alaria species are flukes found in the small intestine of definitive hosts including felids , canids and mustelids .", "alaria mustelae is known to infect mink ( mustela vison ) and short - tailed weasels ( mustela erminea ) , as well as american marten ( veine - smith et  al . ,", "alaria taxidae was identified from 25% ( n  =  6 ) of american marten from the district of mackenzie , northwest territories and was identified in manitoba , canada , at prevalences ranging from 36 to 73% depending on the area ( holmes , 1963 , poole et  al . , 1983 ) .", "a definitive host infected with alaria species in its intestines sheds eggs in its faeces .", "after 2 weeks in wet soil or water , the eggs hatch , producing a miracidium .", "the miracidium invades a freshwater snail , at which point it develops into a cercaria .", "when the tadpole is ingested by an amphibian , reptile , or rodent , the mesocercariae remain in the tissues of the paratenic host .", "the mesocercariae migrate through the stomach , across the diaphragm and into the lungs where the mesocercariae develops into metacercariae .", "the metacercariae are able to travel up the trachea and are then swallowed , at which point they develop into adult trematodes in the small intestines . while infection with alaria species typically does not cause disease in the definitive host , there are reports of the mesocercariae causing neurologic disease due to aberrant migration through the central nervous system and of respiratory illness due to migration through the lungs in domestic dogs ( kimber and kollias , 2000 , kazacos , 2001 )", ". other species of trematodes have caused disease in north american river otters , but the pathogenicity of alaria in american marten is not known ( kimber and kollias , 2000 ) .", "if a pregnant female rodent or carnivore becomes infected with alaria , the mesocercariae can migrate to the mammary glands and can be transmitted to nursing young ( bowman et  al . ,", "not yet weaned , in the nlp , histopathology revealed infection with alaria species in the duodenum , confirming the potential for lactogenic transmission of mesocercariae in this mustelid . in the report by veine - smith et  al .", "( 2011 ) , faeces from the large intestines of 140 american marten carcasses from the up were examined for flukes using a sedimentation technique and found a prevalence of 39% ( n  =  54 ) . the difference in prevalence between that report and ours ( 55.6% , n  =  10 ) may be due to differences in sample size , methodology or a true difference ( veine - smith et  al . , 2011 ) .", "we recommend the use of both faecal sedimentation and zinc sulfate flotation for identification of trematode eggs in faecal samples obtained from live animals .", "euryhelmis squamula has been reported in raccoons ( procyon lotor ) , mink , and american marten in washington and uses amphibian intermediate hosts in this region ( hoberg et  al .", "american marten from washington were documented with 6% prevalence from the southern cascades , confirming that marten are ingesting anuran prey in this area ( hoberg et  al . ,", "e.  squamula has been reported in mink in north america and is a common parasite of the polecat in europe ( ameel , 1938 , miller and harkema , 1964 ) . a related parasite ,", "the mink is the natural host for euparyphium beaveri in michigan , while euparyphium inerme has been reported to infect river otters in the pacific northwest ( miller and harkema , 1964 , hoberg et  al . , 1997 ) .", "hymenolepis nana is a zoonotic cestode of rodents , carnivores and humans found worldwide , but other species of hymenolepis infect galliformes , including potential american marten prey species .", "the parasite may use intermediate hosts or paratenic hosts , including dung beetles , stable flies and fleas ( drew , 2003 , joslin , 2003 , loomis , 2003 , sainsbury , 2003 ) . without knowing the species of hymenolepis found from the american marten in the nlp", ", it is not known whether this may have been a pass - through finding or was truly an infection of the american marten ; the zoonotic potential is not known .", "taenia species are cestodes ( commonly known as tapeworms ) that maintain a completely sylvatic life cycle . the definitive hosts for taenia mustelae and taenia martis americana are primarily mustelids .", "adult parasites live in the small intestine and eggs are passed in the faeces of the host .", "the ingested larvae form cysticercus in skeletal muscle and viscera , and the life cycle is completed when a carnivore consumes the infected intermediate host .", "while the definitive host typically shows no signs of disease , the intermediate host may suffer morbidity or mortality , including liver damage , as a result of infection ( jones and pybus , 2001 ) .", "t.  mustelae has a wide distribution across the northern hemisphere and has been reported in north american mustelids , including american marten , short - tailed weasel ( mustela erminea ) , mink ( neovison vison ) and least weasel ( m.  nivalis ) .", "the cestode has been found in 2 sciurid definitive hosts , marmota broweri and m.  caligata ( jones and pybus , 2001 ) .", "taenia martis americana infects mustelids , including american marten , fisher ( martes pennanti ) and the ringtail ( bassariscus astutus , family : procyonidae ) , as definitive hosts .", "rodents in north america reported with the larval stage of infection include lemmus sibiricus , microtus xanthognathus , mus musculus and ondatra zibethicus ( jones and pybus , 2001 ) .", "two american marten from the southern cascades were co - infected with both t.  mustelae and t.  martis americana ( hoberg et  al .", "adult parasites are found in the small intestine of definitive hosts , which include canids , felids and mustelids ( bowman et  al . ,", "larval or adult parasites can also infect birds , reptiles and other mammals ( wardle and mcleod , 1952 ) .", "eggs , or gravid proglottids , are suspected to be ingested by a first intermediate host , a coprophagic insect or a mite ( chowdhury and aguirre , 2001 , bowman et  al . ,", "the insect is consumed by a second intermediate host , which may include birds , mammals , reptiles and amphibians .", "lastly , the definitive host becomes infected by ingesting the second intermediate host ( bowman et  al .", "definitive hosts may have clinical signs of infection including anorexia , low serum albumin and vomiting ( chowdhury and aguirre , 2001 ) .", "humans can be incidental definitive hosts and become infected by consuming undercooked game ( chowdhury and aguirre , 2001 , fuentes et  al .", "mesocestoides lineatus was identified in the small intestine of 33% ( n  =  14 ) of american marten from eastern washington with significantly higher rates in juveniles than in adults .", "current taxonomy , however , suggests that this species may have actually been m.  variabilis , which occurs in north america , while m.  lineatus is an old world species ( fuentes et  al . , 2003 ) .", "coinfections with m.  lineatus and capillaria putorii occurred in 35% of parasitized american marten , and juveniles had statistically higher rates of coinfection than adults ( foreyt and langerquist , 1993 ) .", "hookworms are zoonotic nematode parasites infecting carnivores ( taylor et  al . , 2007 ) .", "carnivores may become infected via ingestion of larvae or eggs , paratenic hosts , or via percutaneous or lactogenic transmission .", "larvae migrate to the lungs , moult , and are coughed up and swallowed to lay eggs in the small intestine . ingested larvae may bypass pulmonary migration or migrate out of the lungs into the muscle and remain in an infected female mammal until pregnancy occurs , at which point the larvae migrate to the mammary gland leading to lactogenic transmission ( taylor et  al . , 2007 ) .", "hookworm infection in dogs and foxes can result in bloody diarrhoea , anaemia , poor hair coat , poor growth in puppies and respiratory signs ( taylor et  al . , 2007 ) .", "we found that the odds of having anaemia ( haematocrit < 42% as described by nieminen et  al .", ", 2007 ) were 8.75 higher for american marten infected with hookworms than in uninfected american marten in this study . while there was a significant association between presence of hookworm infection and presence of anaemia in this report , other clinical signs related to hookworm infection", "in addition , molecular techniques could be used to determine the species of parasite infecting american marten in michigan . because of the potential for anaemia to affect fitness of an individual american marten or the growth of kits infected via transmammary transmission , treatment of hookworm - infected american marten destined for relocation may be warranted .", "( previously known as capillaria putorii ) infects the gastrointestinal tract of mustelids and other species , while other capillarids infect the respiratory tract , bladder or liver of their respective definitive hosts ( bowman et  al .", "a.  putorii can have a direct or indirect life cycle in which adult parasites shed eggs in the gastrointestinal tract of the mammalian host .", "the eggs are capable of infecting other susceptible hosts directly or using an earthworm as an intermediate host ( segovia et  al . , 2007 , taylor et  al . , 2007 )", "a.  putorii was found in 77.8% ( n  =  14 ) of american marten from the up in the current report , which was significantly higher than the 29.0% ( n  =  9 ) prevalence in the nlp ( p  <  0.05 ) .", "however , during the initial stage of this parasite survey , some capillaria ova were not identified to species , and these are represented as ", "if the 42.0% ( n  =  13 ) prevalence of unidentified capillaria found in the nlp were in fact a.  putorii , then there would not be a significant difference in prevalence of a.  putorii between the up and nlp .", "the prevalence of a.  putorii from the up in this report is higher than the 47% ( n  =  66 ) previously reported from american marten carcasses from the up ( veine - smith et  al . , 2011 ) .", "a.  putorii has also been reported in the stomach of ferret , mink , short - tailed weasel , raccoon , fisher and striped skunk ( mephitis mephitis ) and in the small intestine of bobcats , bears , raccoons , swine , hedgehogs and the domestic cat ( foreyt and langerquist , 1993 , bowman et  al . , 2009 ) .", "in northeastern washington , c.  putorii was found in 86% ( n  =  13 ) of american marten carcasses , mostly in the stomach and less frequently in the large or small intestine of american marten .", "using faecal flotation alone , capillaria species ova were found in 64% ( n  =  21 ) of samples examined from the same population ( foreyt and langerquist , 1993 ) .", "additional future comparisons between carcass and faecal examinations to determine prevalence of capillaria and other parasites is warranted .", "eucoleus aerophilus , previously known as capillaria aerophila , infects the respiratory tract of hosts including american marten and other carnivores , such as fisher , red fox ( vulpes vulpes ) , raccoon , coyote ( canis latrans ) , striped skunk and badger ( taxidea taxus ) ( bowman et  al . , 2009 ) .", "e.  aerophilus rarely infects humans ( laloevi et  al . , 2013 ) .", "the life cycle of e.  aerophilus may be direct or indirect with earthworms serving as intermediate hosts ( bowman et  al .", "aerophilus can lead to respiratory disease and clinical signs include coughing , wheezing , failure to thrive , pneumonia and even death .", "cats and dogs have also been infected but typically do not suffer the same degree of clinical signs as foxes since their infections are not as intense ( bowman et  al . , 2009 ) .", "e.  aerophilus was identified in 4% of the respiratory tracts of american marten from ontario , but it is unknown whether infection resulted in disease or increased risk of being trapped ( seville and addison , 1995 ) .", "eucoleus boehmi has been reported to infect the respiratory tract of foxes and dogs ( bowman et  al . , 2009 ) .", "its ova can be differentiated from the similar e.  aerophilus by its pitted surface ( bowman et  al . , 2009 ) .", "the relative contribution of earthworms to the american marten diet in michigan is not known but may be significant given the high overall prevalence of capillarid parasites seen there .", "physaloptera species are found in the stomach of infected carnivores , including mink , striped skunk , raccoons , dogs , and cats , and eggs are shed intermittently ( chowdhury and aguirre , 2001 , veine - smith et  al . , 2011 ) .", "crickets , beetles or other invertebrates act as intermediate hosts , while rodents and reptiles may be paratenic hosts ( chowdhury and aguirre , 2001 ) .", "while most infections do not cause disease in the host , severe ulcerative gastritis has been reported in the bandicoot , a marsupial ( perameles species ) ( holz , 2003 ) .", "crenosoma species is a lungworm found within the respiratory tract of carnivores and insectivores ( craig and anderson , 1972 ) .", "adult parasites lay eggs in the lungs ; larvae are coughed up , swallowed , and passed in host faeces .", "heavy infection with crenosoma species can cause clinical signs such as coughing , sneezing , nasal discharge and difficulty breathing ( chowdhury and aguirre , 2001 ) .", "the red fox is the typical host for crenosoma vulpis and is sympatric with american marten in both the up and nlp . given the global distribution of c.  vulpis , it is likely that this parasite exists in the nlp red fox population although the parasite was not identified in american marten from the nlp in this study .", "crenosoma petrowi has been reported from free - ranging russian sable , a captive fisher in the united states and a badger from canada ( craig and anderson , 1972 ) .", "a single american marten from ontario was found to be infected with crenosoma petrowi ( < 1% prevalence ) , but the reported prevalence in fisher from the same region was 15% ( seville and addison , 1995 ) .", "olsen ( 1952 ) examined 62 carcasses of martes caurina from colorado and found 18 ( 29% ) to be infected with crenosoma , which he designated crenosoma coloradoensis .", "crenosoma species was found in the lungs of 2% ( n  =  3 ) of american marten from the up of michigan ( veine - smith et  al . , 2011 ) .", "strongyloides martis and s.  lutrae have been reported in river otters ( hoberg et  al . , 1997 ) .", "parasites of this genus are generally species or host - specific and undergo both a direct life cycle and a free - living stage .", "infective larvae or eggs in the soil are consumed by the host ; larvae of some species can also enter the host through the skin ( morris and shima , 2003 ) .", "pathogenicity of strongyloides species in mustelids is not known , but disease could result from migration of the parasite through the lung ( kimber and kollias , 2000 ) .", "dioctophyme renale , commonly known as the giant kidney worm , is one of the largest roundworms infecting wild and domestic species worldwide including wolves , bears , foxes and mink , as well as domestic dogs , cattle , horses and pigs .", "humans have also been reported with d.  renale ( chowdhury and aguirre , 2001 ) .", "the adult worm is typically found in the right kidney because the infective larvae exits the intestinal tract on the right side near the stomach .", "when the parasite dies , the kidney is essentially destroyed , and the host becomes reliant on the remaining left kidney . occasionally , both kidneys are infected or the parasite is found elsewhere in the abdomen .", "an adult female worm lays eggs within the kidney , and the eggs are shed in the urine of the host mammal .", "it takes about 6 months for the egg to become infective , at which point it may be swallowed by the intermediate host , lumbriculus variegatus , an aquatic annelid commonly known as blackworm .", "if the annelid is eaten by an american marten , or other mammalian host , the life cycle is completed when the larvae finds its way to a kidney .", "fish , frogs and crayfish may act as paratenic hosts by consuming the infected annelid ( cheng , 1986 , chowdhury and aguirre , 2001 ) .", "a single american marten from the up was reported with nephritis due to suspected prior infection with d.  renale ( spriggs , unpublished data ) . in an examination of 405 american marten from ontario ,", "d.  renale was found in only 2% of american marten and only from districts with previous reports of infected mink . in 4 of the 5 infected american marten", ", there was evidence only of past infection such as the entire right kidney missing or merely a fibrous capsule remaining , while in 1 american marten the actual parasite was identified ( seville and addison , 1995 ) .", "filaroides martis is a helminth parasite found in the trachea , bronchi and lungs and has been reported to infect mustelids , including mink and american marten , as well as canids ( chowdhury and aguirre , 2001 ) .", "the larvae moults in the stomach mucosa of the definitive host and migrates to the thoracic cavity over the next month .", "larvae increase in size over 100-fold during this timeframe ( ko and anderson , 1972 ) .", "infection with f.  martis has been reported to cause pneumonia in other species , but its effect on american marten is not known ( chowdhury and aguirre , 2001 ) .", "of 405 american marten examined from ontario , 8% had lung or aortic nodules associated with the parasite . because yearlings had a significantly higher prevalence of infection than other ages ,", "the authors suggest that infection could have an effect on survival of yearlings or that american marten are able to recover from infection at older age groups ( seville and addison , 1995 ) .", "f.  martis was found in the lungs of 4% ( n  =  5 ) of american marten carcasses from the up of michigan ( veine - smith et  al . ,", "histopathology revealed lesions consistent with verminous pneumonia in 60% ( n  =  9 ) of american marten carcasses from michigan ( spriggs , unpublished data ) . while these were incidental findings and the causative parasite was not identified", ", it is possible that pneumonia may have resulted in mild respiratory compromise in affected american marten .", "pearsonema plica , previously known as capillaria plica , was identified in the urinary bladder of 6% of american marten from ontario ( seville and addison , 1995 ) .", "p.  plica has been reported in the urinary tract of the domestic cat , dog , raccoon , red fox , coyote , wolf , striped skunk and fisher , as well as american marten ( butterworth and beverley - burton , 1980 ) .", "the definitive host begins to shed eggs of c.  plica in the urine about 8 weeks after consuming an earthworm , the paratenic host ( bowman et  al . , 2009 ) .", "infection usually does not cause disease for the host , but there is a suggestion that p.  plica resulted in poor growth in fox kits ( bowman et  al . ,", "baylisascaris devosi is a nematode reported in both american marten and fisher ( kazacos , 2001 ) .", "eggs shed from the definitive host become infective after 1114 days , at which point small mammals such as rodents and squirrels become infected by ingesting the eggs while foraging .", "the larvae migrate throughout the tissues of these paratenic hosts and typically localize to the muscle of the forelimbs and thorax .", "neural larval migrans , a neurologic disease , is rare or non - existent with b.  devosi ( kazacos , 2001 ) .", "in contrast , the larvae of the related baylisascaris procyonis , or raccoon roundworm , frequently migrate to the central nervous system of non - adapted hosts and cause neural larval migrans .", "humans are susceptible to neural larval migrans caused by baylisascaris species , but b.  devosi is less likely to cause disease in humans than b.  procyonis ( kazacos , 2001 ) .", "adult b.  devosi inhabits the small intestine ; the definitive host does not typically show signs , but intestinal blockage is possible with a severe infection ( chowdhury and aguirre , 2001 ) . in 42 m.  caurina carcasses examined from idaho ,", "one was infected with b.  devosi ( erickson , 1946 , poole et  al . , 1983 ) .", "soboliphyme baturini , commonly known as stomach worm , is a nematode parasite distributed from central siberia across beringia to the pacific northwest of the united states ( koehler et  al . , 2009 ) .", "other mustelids reported with the parasite include ermine ( m.  erminea ) , mink and ferrets ( mustelo putorius furo ) ( levine , 1968 , swartz , 1968 , koehler et  al . , 2009 ) .", "s.  baturini has been reported in other carnivores , including the fox and domestic cat ( levine , 1968 ) .", "mature female worms are found in the stomach or small intestine of the mustelid host , and eggs are passed in the faeces of the host .", "earthworms are the intermediate host , while shrews become paratenic hosts when they ingest the earthworm .", "american marten may be infected by consuming either infected shrews or earthworms ( koehler et  al . , 2009 ) .", "( 2009 ) used genetic molecular data of s.  baturini to shed light on the expansion of the ancestral american marten across beringia into north america , its speciation during isolation in glacial refugia , and re - colonization in alaska and reinfection with s.  baturini .", "clinical manifestation of infection in sable includes anaemia and gastric ulceration ( thomas et  al . , 2008 ) .", "there was a 55% ( n  =  155 ) prevalence of infection with s.  baturini in american marten from prince of wales island in alaska ( table  3 ) .", "there was no correlation between intensity of s.  baturini infection and fat deposits which measured to assess nutritional condition ( thomas et  al .", "american marten carcasses were collected over an 8-year time period from 3 locations in alaska and stomachs were examined for s.  baturini ( table  3 ) .", "none of the american marten in that study had any sign of negative health impact from the parasite infection ( zarnke et  al . , 2004 ) .", "a study conducted in idaho examined 42 m.  caurina carcasses and found one with s.  baturini ( erickson , 1946 ) .", "it is found worldwide except in antarctica and australasia ( chowdhury and aguirre , 2001 ) .", "adult t.  spiralis are found in the small intestine , and the females give birth to larvae which migrate through the body to become encysted in skeletal muscle .", "an american marten may become infected by consuming a rodent or other mammal with the encysted parasite .", "once consumed , the larvae are freed and migrate to the small intestine of the host to complete their life cycle .", "humans can acquire trichinellosis by eating undercooked meat ( taylor et  al . , 2007 ) .", "the first report of t.  spiralis in american marten in north america was from manitoba , canada , where a single yearling was found infected of 139 american marten examined ( poole et  al . , 1983 ) .", "prevalence of trichinella in american marten and other species may vary from year to year ( dick et  al . , 1986 ) .", "american marten from the northern and southern cascades of washington were found to have trichinella encysted in the diaphragm , with a prevalence of 50% and 31% , respectively ( hoberg et  al . , 1990 ) .", "another study examined tongue muscle from 42 american marten from northeastern washington and found only 5% prevalence , which the authors attribute to differences in technique and tissues examined ( foreyt and langerquist , 1993 ) .", "a study conducted in ontario found a 3.4% prevalence ( n  =  68 ) of infection with t.  spiralis in american marten ( dick et  al . , 1986 ) .", "the prevalence in fishers in the study was slightly higher at 4.5% ( n  =  83 ) , and a single mink ( of 12 tested ) was positive .", "the authors suggested that american marten and fishers are key in the sylvatic transmission of trichinella in this part of canada ( dick et  al . , 1986 ) .", "other reports of t.  spiralis in american marten are found in table  3 .", "( 1986 ) in which only mustelids were found with t.  spiralis , this study found a high prevalence in coyote ( 61% , n  =  22 ) and confirmed infection in a variety of other mammals including carnivores and rodents ( schmitt et  al . , 1976 ) .", "dracunculus insignis is a parasite known to infect raccoons , dogs , mink , fishers and skunks in the united states east of the rocky mountains and in ontario , canada ( crichton and beverly - burton , 1973 , cheng , 1986 ) .", "experimental infections in ferrets are used as a model for human dracunculiasis ( eberhard et  al . , 1988 ) .", "dracunculus lutrae infects otters in north america ( crichton and beverly - burton , 1973 ) .", "both species have a similar life cycle to the related the old world parasite didelphis medinensis , commonly called the guinea worm , a zoonotic parasite .", "female d.  lutrae migrate to the tissues under the skin of the animal to give birth to live larvae while secreting a substance that causes a blister to form .", "when the blister ruptures and is exposed to water , the infective larvae is released into the water .", "the free - living larvae can survive for several days until they are consumed by an aquatic copepod , the intermediate host .", "d.  insignis develops through several more stages within the copepod over the next 3 weeks .", "when a definitive host ingests the copepod while drinking water , the larvae are freed in the stomach or small intestine and migrate to abdominal organs and tissues where they continue to develop into adult worms over the next 812 months ( cheng , 1986 ) .", "d.  insignis was found in only a single american marten of 405 examined in ontario despite the parasite being commonly found in raccoons of the same region .", "the authors suggested a higher true prevalence , but detection was low due to the pelt being removed for commercial reasons in most of the examined specimens ( seville and addison , 1995 ) .", "this parasite is of economic concern as it can affect pelt quality in fur - bearer species .", "a single american marten from the nlp was found to be shedding sarcocystis species sporocysts .", "sarcocystis species is a protozoal parasite that has infrequently been reported in carnivores such as domestic cats , dogs , raccoons , cougars , bobcats , mink , striped skunks , sea otters , fishers and pacific harbor seals ( foreyt and langerquist , 1993 , gerhold et  al . , 2005 , larkin et  al . , 2011 ) .", "( 2005 ) reported a case of meningoencephalitis in a fisher caused by sarcocystis neurona in maryland , usa . the virginia opossum ( didelphis virginiana ) is the definitive host for the parasite in north america , but the natural intermediate host for s.  neurona has not been discovered ( gerhold et  al . , 2005 ) .", "sarcocystis species was found in the tongues of 10% ( n  =  4 ) of american marten examined from northeastern washington ( foreyt and langerquist , 1993 ) .", "antibodies to toxoplasma gondii were detected in 10.8% ( n  =  15 ) of american marten in ontario .", "american marten in michigan were found to have a 58% seroprevalence ( n  =  47 ) , and there was no significant difference between the up and nlp ( spriggs , unpublished data ) .", "t.  gondii has been reported to cause mortality in the related black - footed ferret ( mustela nigripes ) , captive - raised american mink ( neovison vison ) and southern sea otters ( enhydra lutris ) ( dubey et  al . ,", "2003 , burns et  al . , 2003 , jones et  al . , 2006", "coccidian parasites have been only rarely reported in american marten , but this is likely because few studies have used faecal flotation and/or histopathology to detect parasites .", "all coccidian parasites are obligate , intracellular parasites and undergo stages of asexual and sexual reproduction in the life cycle , which may be direct or indirect .", "coccidian oocysts from 5 american marten in this report were sporulated to allow identification to the genus level .", "eimeria species were identified in 2 of the 5 sporulated samples from american marten in the current study , and cystoisospora species in the remaining 3 samples .", "it is unknown whether coccidian oocysts seen in faecal samples were pass - through from prey species .", "taxonomy of coccidian parasites has been controversial ; some have considered cystoisospora species to be synonymous with isospora species , while others believe it to be a distinct genus based on molecular and morphological characteristics ( yi - fan et  al . , 2012 ) .", "cystoisospora species were identified in american marten from michigan in this report based on morphological features of the sporulated oocysts .", "( 2012 ) reported cystoisospora species in steppe polecats in china and suggested that previously reported isospora species in various other mustelids , including that of sable , should be reassigned to cystoisospora species .", "faeces from 33 american marten from eastern washington were examined , and 6% were found with coccidian oocysts ( foreyt and langerquist , 1993 ) .", "cryptosporidium and giardia species cysts were detected in the faeces of american marten from the up of michigan , both having a prevalence of 4% ( n  =  5 ) ( veine - smith et  al . , 2011 ) .", "north american marten are infected with a wide variety of endoparasites , but information regarding the pathologic effects of parasitism remains limited .", "american marten infected with hookworms in the current study were found to be at risk for anaemia , and this association warrants further investigation .", "some parasites are infrequently reported in only a single location , while those reported in multiple locations typically have a varying prevalence .", "multiple ecological factors , including habitat , prey availability , sympatric carnivore community , and host adaptation , are likely involved in the variation of prevalence at different geographic locations . a number of parasites known to infect american marten have zoonotic potential . as american marten", "are frequently trapped as a furbearer species , this information could guide prevention of disease transmission to trappers and researchers working with the species .", "parasite infections could cause illness under conditions of stress and presumed immunosuppression associated with reintroduction programs ( kimber and kollias , 2000 ) .", "the majority of previous studies examined american marten carcasses for parasitism , which allows characterization and speciation of the adult parasite and information about intensity of infection .", "however , a non - invasive method of detecting endoparasitism is desired for reintroduction programs and our results provide baseline information about parasites detected by faecal examination from american marten in michigan .", "multiple faecal exams from individuals destined for relocation , including sedimentation and molecular techniques when available , are warranted to identify novel parasites and parasites with pathologic or zoonotic potential and to make appropriate treatment or management decisions ."], "abstract_text": ["<S> the american marten ( martes americana ) was reintroduced to both the upper ( up ) and northern lower peninsula ( nlp ) of michigan during the 20th century . </S>", "<S> this is the first report of endoparasites of american marten from the nlp . </S>", "<S> faeces from live - trapped american marten were examined for the presence of parasitic ova , and blood samples were obtained for haematocrit evaluation . </S>", "<S> the most prevalent parasites were capillaria and alaria species . </S>", "<S> helminth parasites reported in american marten for the first time include eucoleus boehmi , hookworm , and hymenolepis and strongyloides species . </S>", "<S> this is the first report of shedding of sarcocystis species sporocysts in an american marten and identification of 2 coccidian parasites , cystoisospora and eimeria species . </S>", "<S> the pathologic and zoonotic potential of each parasite species is discussed , and previous reports of endoparasites of the american marten in north america are reviewed . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Results", "Discussion", "Conclusion"], "sections": [["the american marten ( martes americana ) is an arboreal mesocarnivore that ranges from the boreal forests of northern north america into coniferous and mixed coniferous / deciduous forests of the northern and northeastern united states , including the great lakes region ( clark et  al . , 1987 ) .", "the american marten was reintroduced to michigan 's upper peninsula ( up ) and northern lower peninsula ( nlp ) in the mid-20th century after regional extirpation due to habitat loss and over - harvest ( cooley et  al . , 2004 ) .", "over 200 animals were reintroduced to the up over the course of several reintroductions . many fewer animals ( n  =  36 ) were reintroduced in the huron - manistee national forest of the nlp and 49 martens were reintroduced to the pigeon river state forest of the nlp .", "in contrast , the species is considered a regional forester sensitive species and there is no harvest in the nlp ( usda forest service , 2012 ) .", "factors hypothesized to be contributing to the differences in population sustainability between the up and nlp include differences in habitat , genetic diversity , health and others . at the time of the original reintroduction", ", american marten were not examined for parasitic or infectious diseases ( spriggs , unpublished data ) .", "some parasites are of economic or zoonotic importance and may be introduced with animal translocations .", "therefore , reintroduction programs should take into account the presence of parasites which are pathogenic or to which the species of concern is not adapted ( kimber and kollias , 2000 ) .", "a collaborative research effort has begun in order to investigate factors that may be contributing to the difference in sustainability between the up and nlp populations .", "the aim of this parasite survey was to describe the presence and prevalence of parasites in the huron - manistee national forest of the nlp and to determine whether there are differences in presence or prevalence of parasites between the nlp and up .", "should future translocation of animals from the up into the nlp be considered for management of the species , a non - invasive and inexpensive method for screening animals would be desirable . also , american marten are not harvested in the nlp and thus adequate numbers of carcasses are not available for examination .", "while some information exists regarding the prevalence of endoparasitism in american marten in north american , relatively little information is available for the species in michigan ( poole et  al . , 1983 , veine - smith et  al . , 2011 ) .", "this study reviews previous parasite prevalence reports from american marten in north america , presents data from the parasitological examination of live - trapped american marten in michigan , and identifies a possible association between hookworm infection and anaemia in affected american marten ."], ["american marten ( n  =  49 ) were sampled from the manistee national forest in the nlp ( n  =  31 ) , hiawatha national forest in the up ( n  =  13 ) and ottawa national forest in the up ( n  =  5 ) .", "american marten were trapped and immobilized from 2011 to 2015 as described by desmarchelier et  al .", "nine american marten were recaptured and re - sampled , resulting in a total of 60 faecal samples included in analyses .", "blood was collected from the jugular vein and placed into lithium heparin anticoagulant ( bd microtainer tubes , becton dickinson and company ) .", "we determined haematocrit using microhematocrit capillary tubes ( safecrit ) and the statspin vt centrifuge ( iris ) .", "individuals were identified as anaemic if the haematocrit was < 42% based on a report of normal haematocrit in captive , fed american martens of 47    5% ( nieminen et  al . , 2007 ) .", "other health parameters were collected as part of a complete health assessment ( spriggs , unpublished data ) .", "faecal float and sedimentation examinations were performed at michigan state university 's diagnostic center for population and animal health using standard methods ( zajac and conboy , 2012 ) .", "faecal flotation was performed using sheather 's sugar solution ( specific gravity 1.251.27 ) alone ( n  =  30 ) from may 2011may 2012 or with both sheather 's sugar solution and zinc sulfate solution ( specific gravity 1.18 , n  =  30 ) from june 2012january 2015 and examined by light microscopy .", "fifty - nine of the samples , representing 48 individual animals , were sufficient in quantity for faecal sedimentation procedure .", "ova were identified based on morphologic characteristics including size and in accordance with parasitologic references and previous reports . upon consultation with a wildlife veterinary parasitologist ( gerhold ) , 2 nematode species ( syphacia muris and aspicularis sp . )", "prevalence was calculated as the number of infected hosts divided by the number of hosts examined . because some american marten were sampled more than once , all parasite species found in an individual were considered together for prevalence calculations .", "differences between locations ( up and nlp ) and sexes were examined with a pearson 's  test with p  <  0.05 considered significant .", "significant differences between presence or absence of anaemia and hookworm infection were also examined with a pearson 's  test .", "parasite species richness by host was calculated as the number of parasite species present per host ; species richness by sample was calculated as the number of parasite species present per sample .", "an unidentified capillarid species was not included in the prevalence or host species richness if one or more species of capillaria was identified in other samples from the same host .", "thus , an individual american marten found with unidentified capillarid at the initial exam and aonchotheca sp . at a subsequent exam", "if both samples had unidentified capillaria , then the host was included in the calculation of prevalence of unidentified capillaria .", "differences between sex and location in species richness were examined with a wilcoxon rank sum test , with p  <  0.05 considered significant . the capture and handling protocol", "was approved by the university of tennessee animal care and use committee ( protocol # 2180 ) , and american marten live - trapping and sample collection was an authorized tribal activity under the 2007 inland consent decree between the state of michigan and the little river band of ottawa indians ."], ["sixty samples from 49 individual american marten ( 28 males , 21 females ) were examined , and results are shown in table  1 .", "of 49 individual american marten examined , 91.8% were positive for 1 or more parasites and 69.4% were infected with 2 or more parasites .", "there was no significant difference in mean species richness by host between sexes or locations ( nlp and up ) .", "trematode egg identification was suspected but not confirmed to be alaria sp . during the early part of the study ( may 2011may 2012 ) when sheather 's sugar solution alone was used for flotation , as alaria species may be distorted due to the osmotic pressure of sugar solution . later in the study", "( june 2012january 2015 ) , trematode ova found on sedimentation were confirmed to be alaria species when the sample was floated with zinc sulfate solution , in which trematode ova will not be distorted .", "once the use of zinc sulfate solution was implemented , no trematode other than alaria species was identified , and the authors concluded that trematode eggs identified in the early samples were most likely alaria species , as suspected . in the up , 3 samples that were positive on sedimentation for trematode ova were suspected to be alaria species and 7 were confirmed via zinc sulfate flotation ; in the nlp , 14 samples positive on sedimentation were suspected to be alaria species and 5 were confirmed via zinc sulfate flotation .", "capillaria eggs were seen in 79% and 78.1% of samples from the up and nlp , respectively .", "capillaria eggs were further identified as eucoleus aerophila , eucoleus boehmi , or aonchotheca putorii based on size and morphologic characteristics . a hookworm egg is shown in fig .  1 .", "there was no significant difference in prevalence of any of the identified parasites between male and female american marten .", "of 9 american marten that were sampled more than once , only one had identical results for each time point .", "the mean haematocrit was 45.6    8.1 ( range 3068 ; n  =  49 ) .", "haematocrit was reported to be 47    5% in the only other report of american marten haematocrit ( nieminen et  al . , 2007 ) . using < 42% as a cut - off", "american marten infected with hookworms were significantly more likely to be anaemic than non - infected american marten ( p  =  0.01 ) with an odds ratio of 8.75 ( 95% confidence interval : 1.456.4 ) ."], ["parasite species richness per host was similar to that reported by veine - smith et  al .", "we identified more parasite species in the nlp than the up , but this result may be a function of the larger sample size from the nlp , and there was no significant difference in richness between the 2 locations .", "foreyt and langerquist ( 1993 ) found 2 or more parasites in 35% of american marten from eastern washington . in another survey from washington ,", "9 helminth species were found , and 48.4% of hosts had coinfection of 2 or more parasites , with a maximum of 4 ( hoberg et  al . , 1990 ) .", "overall , parasite prevalences were similar between the up and nlp in our samples and we conclude that the up may be suitable source for a translocation of marten to the nlp from the point of view of parasite translocation .", "additional screening of both populations , the use of molecular techniques to definitively speciate parasites , and testing of individual martens marked for translocation are recommended .", "parasites in our study were identified to the genus and/or species level as possible by microscopic examination alone .", "future research could employ the use of molecular tools such as polymerase chain reaction to provide more specific results .", "such techniques were not used in this study because we desired an inexpensive method that could be repeated in any future reintroduction programs that require screening of large numbers of animals .", "parasites identified as a concern for reintroduction of north american river otter included alaria canis , strongyloides lutrae , crenosoma goblei , capillaria and coccidia due to potential for pathogenic effects or high prevalence ( kimber and kollias , 2000 ) .", "river otter reintroduction programs tested and treated parasitized otters , and similar methods could be considered for reintroduction of american marten ( hoover et  al . , 1985 , griess , 1987 , serfass et  al . ,", "of 9 american marten that were sampled more than once , only one had identical results at subsequent sampling , indicating either a change in infection status or inconsistent shedding of ova in the remaining 8 marten .", "alaria species are flukes found in the small intestine of definitive hosts including felids , canids and mustelids .", "alaria mustelae is known to infect mink ( mustela vison ) and short - tailed weasels ( mustela erminea ) , as well as american marten ( veine - smith et  al . ,", "alaria taxidae was identified from 25% ( n  =  6 ) of american marten from the district of mackenzie , northwest territories and was identified in manitoba , canada , at prevalences ranging from 36 to 73% depending on the area ( holmes , 1963 , poole et  al . , 1983 ) .", "a definitive host infected with alaria species in its intestines sheds eggs in its faeces .", "after 2 weeks in wet soil or water , the eggs hatch , producing a miracidium .", "the miracidium invades a freshwater snail , at which point it develops into a cercaria .", "when the tadpole is ingested by an amphibian , reptile , or rodent , the mesocercariae remain in the tissues of the paratenic host .", "the mesocercariae migrate through the stomach , across the diaphragm and into the lungs where the mesocercariae develops into metacercariae .", "the metacercariae are able to travel up the trachea and are then swallowed , at which point they develop into adult trematodes in the small intestines . while infection with alaria species typically does not cause disease in the definitive host , there are reports of the mesocercariae causing neurologic disease due to aberrant migration through the central nervous system and of respiratory illness due to migration through the lungs in domestic dogs ( kimber and kollias , 2000 , kazacos , 2001 )", ". other species of trematodes have caused disease in north american river otters , but the pathogenicity of alaria in american marten is not known ( kimber and kollias , 2000 ) .", "if a pregnant female rodent or carnivore becomes infected with alaria , the mesocercariae can migrate to the mammary glands and can be transmitted to nursing young ( bowman et  al . ,", "not yet weaned , in the nlp , histopathology revealed infection with alaria species in the duodenum , confirming the potential for lactogenic transmission of mesocercariae in this mustelid . in the report by veine - smith et  al .", "( 2011 ) , faeces from the large intestines of 140 american marten carcasses from the up were examined for flukes using a sedimentation technique and found a prevalence of 39% ( n  =  54 ) . the difference in prevalence between that report and ours ( 55.6% , n  =  10 ) may be due to differences in sample size , methodology or a true difference ( veine - smith et  al . , 2011 ) .", "we recommend the use of both faecal sedimentation and zinc sulfate flotation for identification of trematode eggs in faecal samples obtained from live animals .", "euryhelmis squamula has been reported in raccoons ( procyon lotor ) , mink , and american marten in washington and uses amphibian intermediate hosts in this region ( hoberg et  al .", "american marten from washington were documented with 6% prevalence from the southern cascades , confirming that marten are ingesting anuran prey in this area ( hoberg et  al . ,", "e.  squamula has been reported in mink in north america and is a common parasite of the polecat in europe ( ameel , 1938 , miller and harkema , 1964 ) . a related parasite ,", "the mink is the natural host for euparyphium beaveri in michigan , while euparyphium inerme has been reported to infect river otters in the pacific northwest ( miller and harkema , 1964 , hoberg et  al . , 1997 ) .", "hymenolepis nana is a zoonotic cestode of rodents , carnivores and humans found worldwide , but other species of hymenolepis infect galliformes , including potential american marten prey species .", "the parasite may use intermediate hosts or paratenic hosts , including dung beetles , stable flies and fleas ( drew , 2003 , joslin , 2003 , loomis , 2003 , sainsbury , 2003 ) . without knowing the species of hymenolepis found from the american marten in the nlp", ", it is not known whether this may have been a pass - through finding or was truly an infection of the american marten ; the zoonotic potential is not known .", "taenia species are cestodes ( commonly known as tapeworms ) that maintain a completely sylvatic life cycle . the definitive hosts for taenia mustelae and taenia martis americana are primarily mustelids .", "adult parasites live in the small intestine and eggs are passed in the faeces of the host .", "the ingested larvae form cysticercus in skeletal muscle and viscera , and the life cycle is completed when a carnivore consumes the infected intermediate host .", "while the definitive host typically shows no signs of disease , the intermediate host may suffer morbidity or mortality , including liver damage , as a result of infection ( jones and pybus , 2001 ) .", "t.  mustelae has a wide distribution across the northern hemisphere and has been reported in north american mustelids , including american marten , short - tailed weasel ( mustela erminea ) , mink ( neovison vison ) and least weasel ( m.  nivalis ) .", "the cestode has been found in 2 sciurid definitive hosts , marmota broweri and m.  caligata ( jones and pybus , 2001 ) .", "taenia martis americana infects mustelids , including american marten , fisher ( martes pennanti ) and the ringtail ( bassariscus astutus , family : procyonidae ) , as definitive hosts .", "rodents in north america reported with the larval stage of infection include lemmus sibiricus , microtus xanthognathus , mus musculus and ondatra zibethicus ( jones and pybus , 2001 ) .", "two american marten from the southern cascades were co - infected with both t.  mustelae and t.  martis americana ( hoberg et  al .", "adult parasites are found in the small intestine of definitive hosts , which include canids , felids and mustelids ( bowman et  al . ,", "larval or adult parasites can also infect birds , reptiles and other mammals ( wardle and mcleod , 1952 ) .", "eggs , or gravid proglottids , are suspected to be ingested by a first intermediate host , a coprophagic insect or a mite ( chowdhury and aguirre , 2001 , bowman et  al . ,", "the insect is consumed by a second intermediate host , which may include birds , mammals , reptiles and amphibians .", "lastly , the definitive host becomes infected by ingesting the second intermediate host ( bowman et  al .", "definitive hosts may have clinical signs of infection including anorexia , low serum albumin and vomiting ( chowdhury and aguirre , 2001 ) .", "humans can be incidental definitive hosts and become infected by consuming undercooked game ( chowdhury and aguirre , 2001 , fuentes et  al .", "mesocestoides lineatus was identified in the small intestine of 33% ( n  =  14 ) of american marten from eastern washington with significantly higher rates in juveniles than in adults .", "current taxonomy , however , suggests that this species may have actually been m.  variabilis , which occurs in north america , while m.  lineatus is an old world species ( fuentes et  al . , 2003 ) .", "coinfections with m.  lineatus and capillaria putorii occurred in 35% of parasitized american marten , and juveniles had statistically higher rates of coinfection than adults ( foreyt and langerquist , 1993 ) .", "hookworms are zoonotic nematode parasites infecting carnivores ( taylor et  al . , 2007 ) .", "carnivores may become infected via ingestion of larvae or eggs , paratenic hosts , or via percutaneous or lactogenic transmission .", "larvae migrate to the lungs , moult , and are coughed up and swallowed to lay eggs in the small intestine . ingested larvae may bypass pulmonary migration or migrate out of the lungs into the muscle and remain in an infected female mammal until pregnancy occurs , at which point the larvae migrate to the mammary gland leading to lactogenic transmission ( taylor et  al . , 2007 ) .", "hookworm infection in dogs and foxes can result in bloody diarrhoea , anaemia , poor hair coat , poor growth in puppies and respiratory signs ( taylor et  al . , 2007 ) .", "we found that the odds of having anaemia ( haematocrit < 42% as described by nieminen et  al .", ", 2007 ) were 8.75 higher for american marten infected with hookworms than in uninfected american marten in this study . while there was a significant association between presence of hookworm infection and presence of anaemia in this report , other clinical signs related to hookworm infection", "in addition , molecular techniques could be used to determine the species of parasite infecting american marten in michigan . because of the potential for anaemia to affect fitness of an individual american marten or the growth of kits infected via transmammary transmission , treatment of hookworm - infected american marten destined for relocation may be warranted .", "( previously known as capillaria putorii ) infects the gastrointestinal tract of mustelids and other species , while other capillarids infect the respiratory tract , bladder or liver of their respective definitive hosts ( bowman et  al .", "a.  putorii can have a direct or indirect life cycle in which adult parasites shed eggs in the gastrointestinal tract of the mammalian host .", "the eggs are capable of infecting other susceptible hosts directly or using an earthworm as an intermediate host ( segovia et  al . , 2007 , taylor et  al . , 2007 )", "a.  putorii was found in 77.8% ( n  =  14 ) of american marten from the up in the current report , which was significantly higher than the 29.0% ( n  =  9 ) prevalence in the nlp ( p  <  0.05 ) .", "however , during the initial stage of this parasite survey , some capillaria ova were not identified to species , and these are represented as ", "if the 42.0% ( n  =  13 ) prevalence of unidentified capillaria found in the nlp were in fact a.  putorii , then there would not be a significant difference in prevalence of a.  putorii between the up and nlp .", "the prevalence of a.  putorii from the up in this report is higher than the 47% ( n  =  66 ) previously reported from american marten carcasses from the up ( veine - smith et  al . , 2011 ) .", "a.  putorii has also been reported in the stomach of ferret , mink , short - tailed weasel , raccoon , fisher and striped skunk ( mephitis mephitis ) and in the small intestine of bobcats , bears , raccoons , swine , hedgehogs and the domestic cat ( foreyt and langerquist , 1993 , bowman et  al . , 2009 ) .", "in northeastern washington , c.  putorii was found in 86% ( n  =  13 ) of american marten carcasses , mostly in the stomach and less frequently in the large or small intestine of american marten .", "using faecal flotation alone , capillaria species ova were found in 64% ( n  =  21 ) of samples examined from the same population ( foreyt and langerquist , 1993 ) .", "additional future comparisons between carcass and faecal examinations to determine prevalence of capillaria and other parasites is warranted .", "eucoleus aerophilus , previously known as capillaria aerophila , infects the respiratory tract of hosts including american marten and other carnivores , such as fisher , red fox ( vulpes vulpes ) , raccoon , coyote ( canis latrans ) , striped skunk and badger ( taxidea taxus ) ( bowman et  al . , 2009 ) .", "e.  aerophilus rarely infects humans ( laloevi et  al . , 2013 ) .", "the life cycle of e.  aerophilus may be direct or indirect with earthworms serving as intermediate hosts ( bowman et  al .", "aerophilus can lead to respiratory disease and clinical signs include coughing , wheezing , failure to thrive , pneumonia and even death .", "cats and dogs have also been infected but typically do not suffer the same degree of clinical signs as foxes since their infections are not as intense ( bowman et  al . , 2009 ) .", "e.  aerophilus was identified in 4% of the respiratory tracts of american marten from ontario , but it is unknown whether infection resulted in disease or increased risk of being trapped ( seville and addison , 1995 ) .", "eucoleus boehmi has been reported to infect the respiratory tract of foxes and dogs ( bowman et  al . , 2009 ) .", "its ova can be differentiated from the similar e.  aerophilus by its pitted surface ( bowman et  al . , 2009 ) .", "the relative contribution of earthworms to the american marten diet in michigan is not known but may be significant given the high overall prevalence of capillarid parasites seen there .", "physaloptera species are found in the stomach of infected carnivores , including mink , striped skunk , raccoons , dogs , and cats , and eggs are shed intermittently ( chowdhury and aguirre , 2001 , veine - smith et  al . , 2011 ) .", "crickets , beetles or other invertebrates act as intermediate hosts , while rodents and reptiles may be paratenic hosts ( chowdhury and aguirre , 2001 ) .", "while most infections do not cause disease in the host , severe ulcerative gastritis has been reported in the bandicoot , a marsupial ( perameles species ) ( holz , 2003 ) .", "crenosoma species is a lungworm found within the respiratory tract of carnivores and insectivores ( craig and anderson , 1972 ) .", "adult parasites lay eggs in the lungs ; larvae are coughed up , swallowed , and passed in host faeces .", "heavy infection with crenosoma species can cause clinical signs such as coughing , sneezing , nasal discharge and difficulty breathing ( chowdhury and aguirre , 2001 ) .", "the red fox is the typical host for crenosoma vulpis and is sympatric with american marten in both the up and nlp . given the global distribution of c.  vulpis , it is likely that this parasite exists in the nlp red fox population although the parasite was not identified in american marten from the nlp in this study .", "crenosoma petrowi has been reported from free - ranging russian sable , a captive fisher in the united states and a badger from canada ( craig and anderson , 1972 ) .", "a single american marten from ontario was found to be infected with crenosoma petrowi ( < 1% prevalence ) , but the reported prevalence in fisher from the same region was 15% ( seville and addison , 1995 ) .", "olsen ( 1952 ) examined 62 carcasses of martes caurina from colorado and found 18 ( 29% ) to be infected with crenosoma , which he designated crenosoma coloradoensis .", "crenosoma species was found in the lungs of 2% ( n  =  3 ) of american marten from the up of michigan ( veine - smith et  al . , 2011 ) .", "strongyloides martis and s.  lutrae have been reported in river otters ( hoberg et  al . , 1997 ) .", "parasites of this genus are generally species or host - specific and undergo both a direct life cycle and a free - living stage .", "infective larvae or eggs in the soil are consumed by the host ; larvae of some species can also enter the host through the skin ( morris and shima , 2003 ) .", "pathogenicity of strongyloides species in mustelids is not known , but disease could result from migration of the parasite through the lung ( kimber and kollias , 2000 ) .", "dioctophyme renale , commonly known as the giant kidney worm , is one of the largest roundworms infecting wild and domestic species worldwide including wolves , bears , foxes and mink , as well as domestic dogs , cattle , horses and pigs .", "humans have also been reported with d.  renale ( chowdhury and aguirre , 2001 ) .", "the adult worm is typically found in the right kidney because the infective larvae exits the intestinal tract on the right side near the stomach .", "when the parasite dies , the kidney is essentially destroyed , and the host becomes reliant on the remaining left kidney . occasionally , both kidneys are infected or the parasite is found elsewhere in the abdomen .", "an adult female worm lays eggs within the kidney , and the eggs are shed in the urine of the host mammal .", "it takes about 6 months for the egg to become infective , at which point it may be swallowed by the intermediate host , lumbriculus variegatus , an aquatic annelid commonly known as blackworm .", "if the annelid is eaten by an american marten , or other mammalian host , the life cycle is completed when the larvae finds its way to a kidney .", "fish , frogs and crayfish may act as paratenic hosts by consuming the infected annelid ( cheng , 1986 , chowdhury and aguirre , 2001 ) .", "a single american marten from the up was reported with nephritis due to suspected prior infection with d.  renale ( spriggs , unpublished data ) . in an examination of 405 american marten from ontario ,", "d.  renale was found in only 2% of american marten and only from districts with previous reports of infected mink . in 4 of the 5 infected american marten", ", there was evidence only of past infection such as the entire right kidney missing or merely a fibrous capsule remaining , while in 1 american marten the actual parasite was identified ( seville and addison , 1995 ) .", "filaroides martis is a helminth parasite found in the trachea , bronchi and lungs and has been reported to infect mustelids , including mink and american marten , as well as canids ( chowdhury and aguirre , 2001 ) .", "the larvae moults in the stomach mucosa of the definitive host and migrates to the thoracic cavity over the next month .", "larvae increase in size over 100-fold during this timeframe ( ko and anderson , 1972 ) .", "infection with f.  martis has been reported to cause pneumonia in other species , but its effect on american marten is not known ( chowdhury and aguirre , 2001 ) .", "of 405 american marten examined from ontario , 8% had lung or aortic nodules associated with the parasite . because yearlings had a significantly higher prevalence of infection than other ages ,", "the authors suggest that infection could have an effect on survival of yearlings or that american marten are able to recover from infection at older age groups ( seville and addison , 1995 ) .", "f.  martis was found in the lungs of 4% ( n  =  5 ) of american marten carcasses from the up of michigan ( veine - smith et  al . ,", "histopathology revealed lesions consistent with verminous pneumonia in 60% ( n  =  9 ) of american marten carcasses from michigan ( spriggs , unpublished data ) . while these were incidental findings and the causative parasite was not identified", ", it is possible that pneumonia may have resulted in mild respiratory compromise in affected american marten .", "pearsonema plica , previously known as capillaria plica , was identified in the urinary bladder of 6% of american marten from ontario ( seville and addison , 1995 ) .", "p.  plica has been reported in the urinary tract of the domestic cat , dog , raccoon , red fox , coyote , wolf , striped skunk and fisher , as well as american marten ( butterworth and beverley - burton , 1980 ) .", "the definitive host begins to shed eggs of c.  plica in the urine about 8 weeks after consuming an earthworm , the paratenic host ( bowman et  al . , 2009 ) .", "infection usually does not cause disease for the host , but there is a suggestion that p.  plica resulted in poor growth in fox kits ( bowman et  al . ,", "baylisascaris devosi is a nematode reported in both american marten and fisher ( kazacos , 2001 ) .", "eggs shed from the definitive host become infective after 1114 days , at which point small mammals such as rodents and squirrels become infected by ingesting the eggs while foraging .", "the larvae migrate throughout the tissues of these paratenic hosts and typically localize to the muscle of the forelimbs and thorax .", "neural larval migrans , a neurologic disease , is rare or non - existent with b.  devosi ( kazacos , 2001 ) .", "in contrast , the larvae of the related baylisascaris procyonis , or raccoon roundworm , frequently migrate to the central nervous system of non - adapted hosts and cause neural larval migrans .", "humans are susceptible to neural larval migrans caused by baylisascaris species , but b.  devosi is less likely to cause disease in humans than b.  procyonis ( kazacos , 2001 ) .", "adult b.  devosi inhabits the small intestine ; the definitive host does not typically show signs , but intestinal blockage is possible with a severe infection ( chowdhury and aguirre , 2001 ) . in 42 m.  caurina carcasses examined from idaho ,", "one was infected with b.  devosi ( erickson , 1946 , poole et  al . , 1983 ) .", "soboliphyme baturini , commonly known as stomach worm , is a nematode parasite distributed from central siberia across beringia to the pacific northwest of the united states ( koehler et  al . , 2009 ) .", "other mustelids reported with the parasite include ermine ( m.  erminea ) , mink and ferrets ( mustelo putorius furo ) ( levine , 1968 , swartz , 1968 , koehler et  al . , 2009 ) .", "s.  baturini has been reported in other carnivores , including the fox and domestic cat ( levine , 1968 ) .", "mature female worms are found in the stomach or small intestine of the mustelid host , and eggs are passed in the faeces of the host .", "earthworms are the intermediate host , while shrews become paratenic hosts when they ingest the earthworm .", "american marten may be infected by consuming either infected shrews or earthworms ( koehler et  al . , 2009 ) .", "( 2009 ) used genetic molecular data of s.  baturini to shed light on the expansion of the ancestral american marten across beringia into north america , its speciation during isolation in glacial refugia , and re - colonization in alaska and reinfection with s.  baturini .", "clinical manifestation of infection in sable includes anaemia and gastric ulceration ( thomas et  al . , 2008 ) .", "there was a 55% ( n  =  155 ) prevalence of infection with s.  baturini in american marten from prince of wales island in alaska ( table  3 ) .", "there was no correlation between intensity of s.  baturini infection and fat deposits which measured to assess nutritional condition ( thomas et  al .", "american marten carcasses were collected over an 8-year time period from 3 locations in alaska and stomachs were examined for s.  baturini ( table  3 ) .", "none of the american marten in that study had any sign of negative health impact from the parasite infection ( zarnke et  al . , 2004 ) .", "a study conducted in idaho examined 42 m.  caurina carcasses and found one with s.  baturini ( erickson , 1946 ) .", "it is found worldwide except in antarctica and australasia ( chowdhury and aguirre , 2001 ) .", "adult t.  spiralis are found in the small intestine , and the females give birth to larvae which migrate through the body to become encysted in skeletal muscle .", "an american marten may become infected by consuming a rodent or other mammal with the encysted parasite .", "once consumed , the larvae are freed and migrate to the small intestine of the host to complete their life cycle .", "humans can acquire trichinellosis by eating undercooked meat ( taylor et  al . , 2007 ) .", "the first report of t.  spiralis in american marten in north america was from manitoba , canada , where a single yearling was found infected of 139 american marten examined ( poole et  al . , 1983 ) .", "prevalence of trichinella in american marten and other species may vary from year to year ( dick et  al . , 1986 ) .", "american marten from the northern and southern cascades of washington were found to have trichinella encysted in the diaphragm , with a prevalence of 50% and 31% , respectively ( hoberg et  al . , 1990 ) .", "another study examined tongue muscle from 42 american marten from northeastern washington and found only 5% prevalence , which the authors attribute to differences in technique and tissues examined ( foreyt and langerquist , 1993 ) .", "a study conducted in ontario found a 3.4% prevalence ( n  =  68 ) of infection with t.  spiralis in american marten ( dick et  al . , 1986 ) .", "the prevalence in fishers in the study was slightly higher at 4.5% ( n  =  83 ) , and a single mink ( of 12 tested ) was positive .", "the authors suggested that american marten and fishers are key in the sylvatic transmission of trichinella in this part of canada ( dick et  al . , 1986 ) .", "other reports of t.  spiralis in american marten are found in table  3 .", "( 1986 ) in which only mustelids were found with t.  spiralis , this study found a high prevalence in coyote ( 61% , n  =  22 ) and confirmed infection in a variety of other mammals including carnivores and rodents ( schmitt et  al . , 1976 ) .", "dracunculus insignis is a parasite known to infect raccoons , dogs , mink , fishers and skunks in the united states east of the rocky mountains and in ontario , canada ( crichton and beverly - burton , 1973 , cheng , 1986 ) .", "experimental infections in ferrets are used as a model for human dracunculiasis ( eberhard et  al . , 1988 ) .", "dracunculus lutrae infects otters in north america ( crichton and beverly - burton , 1973 ) .", "both species have a similar life cycle to the related the old world parasite didelphis medinensis , commonly called the guinea worm , a zoonotic parasite .", "female d.  lutrae migrate to the tissues under the skin of the animal to give birth to live larvae while secreting a substance that causes a blister to form .", "when the blister ruptures and is exposed to water , the infective larvae is released into the water .", "the free - living larvae can survive for several days until they are consumed by an aquatic copepod , the intermediate host .", "d.  insignis develops through several more stages within the copepod over the next 3 weeks .", "when a definitive host ingests the copepod while drinking water , the larvae are freed in the stomach or small intestine and migrate to abdominal organs and tissues where they continue to develop into adult worms over the next 812 months ( cheng , 1986 ) .", "d.  insignis was found in only a single american marten of 405 examined in ontario despite the parasite being commonly found in raccoons of the same region .", "the authors suggested a higher true prevalence , but detection was low due to the pelt being removed for commercial reasons in most of the examined specimens ( seville and addison , 1995 ) .", "this parasite is of economic concern as it can affect pelt quality in fur - bearer species .", "a single american marten from the nlp was found to be shedding sarcocystis species sporocysts .", "sarcocystis species is a protozoal parasite that has infrequently been reported in carnivores such as domestic cats , dogs , raccoons , cougars , bobcats , mink , striped skunks , sea otters , fishers and pacific harbor seals ( foreyt and langerquist , 1993 , gerhold et  al . , 2005 , larkin et  al . , 2011 ) .", "( 2005 ) reported a case of meningoencephalitis in a fisher caused by sarcocystis neurona in maryland , usa . the virginia opossum ( didelphis virginiana ) is the definitive host for the parasite in north america , but the natural intermediate host for s.  neurona has not been discovered ( gerhold et  al . , 2005 ) .", "sarcocystis species was found in the tongues of 10% ( n  =  4 ) of american marten examined from northeastern washington ( foreyt and langerquist , 1993 ) .", "antibodies to toxoplasma gondii were detected in 10.8% ( n  =  15 ) of american marten in ontario .", "american marten in michigan were found to have a 58% seroprevalence ( n  =  47 ) , and there was no significant difference between the up and nlp ( spriggs , unpublished data ) .", "t.  gondii has been reported to cause mortality in the related black - footed ferret ( mustela nigripes ) , captive - raised american mink ( neovison vison ) and southern sea otters ( enhydra lutris ) ( dubey et  al . ,", "2003 , burns et  al . , 2003 , jones et  al . , 2006", "coccidian parasites have been only rarely reported in american marten , but this is likely because few studies have used faecal flotation and/or histopathology to detect parasites .", "all coccidian parasites are obligate , intracellular parasites and undergo stages of asexual and sexual reproduction in the life cycle , which may be direct or indirect .", "coccidian oocysts from 5 american marten in this report were sporulated to allow identification to the genus level .", "eimeria species were identified in 2 of the 5 sporulated samples from american marten in the current study , and cystoisospora species in the remaining 3 samples .", "it is unknown whether coccidian oocysts seen in faecal samples were pass - through from prey species .", "taxonomy of coccidian parasites has been controversial ; some have considered cystoisospora species to be synonymous with isospora species , while others believe it to be a distinct genus based on molecular and morphological characteristics ( yi - fan et  al . , 2012 ) .", "cystoisospora species were identified in american marten from michigan in this report based on morphological features of the sporulated oocysts .", "( 2012 ) reported cystoisospora species in steppe polecats in china and suggested that previously reported isospora species in various other mustelids , including that of sable , should be reassigned to cystoisospora species .", "faeces from 33 american marten from eastern washington were examined , and 6% were found with coccidian oocysts ( foreyt and langerquist , 1993 ) .", "cryptosporidium and giardia species cysts were detected in the faeces of american marten from the up of michigan , both having a prevalence of 4% ( n  =  5 ) ( veine - smith et  al . , 2011 ) ."], ["north american marten are infected with a wide variety of endoparasites , but information regarding the pathologic effects of parasitism remains limited .", "american marten infected with hookworms in the current study were found to be at risk for anaemia , and this association warrants further investigation .", "some parasites are infrequently reported in only a single location , while those reported in multiple locations typically have a varying prevalence .", "multiple ecological factors , including habitat , prey availability , sympatric carnivore community , and host adaptation , are likely involved in the variation of prevalence at different geographic locations . a number of parasites known to infect american marten have zoonotic potential . as american marten", "are frequently trapped as a furbearer species , this information could guide prevention of disease transmission to trappers and researchers working with the species .", "parasite infections could cause illness under conditions of stress and presumed immunosuppression associated with reintroduction programs ( kimber and kollias , 2000 ) .", "the majority of previous studies examined american marten carcasses for parasitism , which allows characterization and speciation of the adult parasite and information about intensity of infection .", "however , a non - invasive method of detecting endoparasitism is desired for reintroduction programs and our results provide baseline information about parasites detected by faecal examination from american marten in michigan .", "multiple faecal exams from individuals destined for relocation , including sedimentation and molecular techniques when available , are warranted to identify novel parasites and parasites with pathologic or zoonotic potential and to make appropriate treatment or management decisions ."]]}
{"article_id": "PMC4968486", "article_text": ["in korea , close to 86.8% of the elderly population ( aged 65 or older ) has chronic \n degenerative disease1 . among these \n degenerative diseases , arthritis has the highest prevalence rate .", "osteoarthritis mainly \n occurs in the hip joints and knee joints , which are weight - bearing joints , and most elderly \n persons aged 65  years or older have this disease1 , \n 2 .", "osteoarthritis induces the growth of \n osteophytes , leading not only to pain but also to the loss of movement and often to a state \n of lethargy , in which the individual avoids walking1 .", "this lethargy hinders the individual from performing functional \n activities , often resulting in muscle weakening and atrophy , including decrease in muscle \n force and motor unit activation of the muscles around the joint affected by \n osteoarthritis3 .", "muscle weakening and \n atrophy further contribute to reduced walking and functional abilities required for daily \n living3 .", "flexion - relaxation phenomenon ( frp ) refers to the appearance of muscular - electrical silence \n on the surface of the erector spine during complete trunk flexion4 , which could be affected by several factors , including \n lumbopelvic postures5 , repetition of \n tasks6 , and muscle fatigue7 .", "chronic osteoarthritis , therefore , often leads to stooped postures \n as a result of the tension experienced when assuming postures in which the erector spine is \n elongated3 , 4 .", "the erector spinae muscle is a posture - maintaining muscle that is \n used in all daily living activities in which trunk forward - tilting and straightening motions \n are performed .", "normal frp of the erector spinae is an important precondition for smoothly \n performing the functional activities of daily living and is an important measurement \n variable in the evaluation of treatment effects at clinics8 .", "there are currently insufficient studies examining the effects of osteoarthritis on the frp \n of the erector spinae and on how the frp of the erector spinae observed in young adults is \n different from that of the elderly in whom osteoarthritis has occurred . therefore , the \n present study compared the frps of the erector spinae occurring during ( 1 ) trunk flexion , \n ( 2 ) complete trunk flexion , and ( 3 ) trunk extension motions in both healthy , young females \n and elderly females with chronic knee osteoarthritis . in addition , data regarding whether \n the frp of the erector spinae can be used as a method of evaluating the degree of pain in \n chronic osteoarthritis in the elderly will be presented .", "if the erector spinae \n flexion - relaxation rates can be used as a pain scale , it may assist healthcare professionals \n to improve elderly patients quality of life .", "this study was a randomized controlled trial , which included healthy adult females \n attending a university and elderly females aged at least 65  years diagnosed with chronic \n osteoarthritis in the leg , among the elderly community residing in a metropolitan area of \n chungcheongnam - do in south korea .", "the selection criteria for elderly subjects were as \n follows : those who had no orthopedic or neurologic disease affecting the leg other than \n chronic osteoarthritis , had not undergone surgical treatment of the leg within the last 6 \n months , had not experienced any fracture in the leg in the last 6 months , had corrected \n visual acuity not lower than 0.8 and had no blurred vision , could understand experimenters \n instructions and cooperate with the experimenters , and had understood the purpose of the \n present study and voluntarily agreed to participate .", "the study was approved by the \n institutional review board of hanyang university , and written informed consent was obtained \n from all patients prior to the experiments .", "the general characteristics of the subjects that \n participated in the present study are shown in table \n 1table 1.general characteristics of study subjects ( n=30)elderly females with chronicosteoarthritis \n ( n=17)healthy adult females ( n=13)age ( years ) , m  sd 75.8  12.0 * 21.1  0.8height ( cm ) , m  sd153.5  6.3159.6  3.7weight(kg ) , m  sd56.5  8.752.9  5.4*p<0.05 . in the present study , surface electromyography ( emg ) ( trigno wireless system , delsys inc . , \n boston , ma , usa ) was used to measure the muscle activity of the healthy adult females and \n elderly females with chronic knee osteoarthritis while they were performing trunk flexion , \n complete trunk flexion , and trunk extension .", "the emg signals measured by the trigno sensor \n and wirelessly transmitted to the trigno base station were analyzed using emgworks 4.0 \n ( delsys inc . , boston , ma , usa ) software .", "all measured muscle activity values were calculated \n as percentages of maximal or submaximal voluntary isometric contraction values .", "the emg \n signal - sampling rate was 1,024  hz , and the signals were band - pass filtered within a range of \n 10500  hz . metronomes were used to enable the experimental subjects of the present study to perform \n trunk flexion , complete trunk flexion , and trunk extension for 5  s each over a total of 15 \n s. the beat was set to 60 bpm , and seconds were counted so that the subjects could hear and \n perform each motion for 5 s. before attaching the emg electrodes , sweat and foreign matter in the experimental muscle \n regions were removed in order to minimize skin resistance .", "the attachment points for \n measuring the thoracic erector spinae were 5  cm to the left and right of the spinous process \n of the t9 , while the attachment points for measuring the lumbar erector spinae were 2  cm to \n the left and right of the spinous process of the l2 .", "the manual muscle force postures for \n measuring of the maximal voluntary isometric contraction ( mvic ) in healthy adults and the \n submaximal voluntary isometric contraction in elderly persons with chronic knee \n osteoarthritis were determined following the method of osullivan et al6 . the values of each subject were measured 3 times for 5  s , \n in succession , over a total of 15 s. measurements during the first and last seconds of each \n measurement period were excluded , and the maximum values during the middle 3  s were used for \n the quantification of the root mean square ( rms ) . to minimize muscle fatigue , 1 ", "the subjects were randomly measured in the \n order of voluntary participation . during the measurement of the flexion - relaxation rates , \n", "three motions were performed : ( a ) trunk flexion , ( b ) complete trunk flexion , and ( c ) trunk \n extension .", "posture 1 was a standing posture , posture 2 was a trunk - bending motion , and \n posture 3 was a maximum trunk - bending motion . in the complete trunk flexion section ,", "the \n subjects were instructed to bend the trunk maximally within the range of bending with no \n pain before measurement .", "preliminary instruction was provided to the subjects by the \n experimenter , who performed the motions in front of the subjects and helped the subjects \n practice the motions at least one time .", "the flexion - relaxation rates were determined using both the values obtained by dividing the \n data for trunk extension by that of complete trunk flexion ( c / b ) and by dividing the data \n for trunk flexion by that of complete trunk flexion ( a / b ) .", "the mean values of the three \n trunk flexion extension motions were used to determine the flexion - relaxation rates .", "the means and standard deviations of \n the general characteristics of the study subjects ( age , gender , height , and weight ) were \n calculated using descriptive statistics . in the present study ,", "the independent variable was \n the presence or absence of chronic knee osteoarthritis , and the dependent variable was the \n frp of the erector spinae .", "independent t - tests were used to examine the erector spine \n flexion - relaxation rates according to whether the presence or absence of chronic knee \n osteoarthritis . to test the statistical significance ,", " was set to 0.05 , and the collected \n data were analyzed using pasw ver .", "the effect of chronic knee osteoarthritis on the frp of the erector spinae was first \n examined .", "the values obtained by dividing the data for trunk extension by that of complete \n trunk flexion were not different for the left and right lumbar erector spinae or right \n thoracic erector spinae ; however , they were significantly different for the left thoracic \n erector spinae ( p<0.05 ) ( table 2table 2.flexion-relaxation phenomenon ( frp ) values obtained by dividing the data for \n trunk extension by those for complete trunk flexion from healthy adults and elderly \n individuals ( units : % ) musclesmean  sdelderly females withchronic osteoarthritishealthy adultfemalesleft thoracic erector spine1.67  0.61 * 1.22  0.45right thoracic erector spine1.87  0.822.31  1.35left", "lumbar erector spine2.35  1.312.08  1.36right lumbar erector spine2.46  1.031.85  1.81*p<0.05 ) .", "the values obtained by dividing the data for trunk flexion by that of \n complete trunk flexion , were not different for the left and right lumbar erector spinae and \n right thoracic erector spinae , but were significantly different for the left thoracic \n erector spinae ( p<0.05 ) ( table 3table 3.flexion-relaxation phenomenon ( frp ) values obtained by dividing the data for \n trunk flexion by those for complete trunk flexion from healthy adults and elderly \n individuals ( units : % ) musclesmean  sdelderly females with chronic osteoarthritishealthy adult femalesleft thoracic erector spine1.47  0.55 * 1.12  0.31right thoracic erector spine1.41  0.491.99  1.43left lumbar erector spine2.00  1.082.58  2.75right lumbar erector spine1.76  0.901.62  1.31*p<0.05 ) .", "in the present study , the frp of the erector spinae in elderly females with chronic knee \n osteoarthritis and healthy young females were measured to determine whether the erector \n spinae flexion - relaxation rates could act as a useful pain evaluation tool for patients with \n knee osteoarthritis . although no significant differences were evident in the \n flexion - relaxation rates of the left and right thoracic erector spinae or right lumbar \n erector spinae , significant differences were shown in the flexion - relaxation rates of the \n left thoracic erector spinae .", "degenerative arthritis develops in most people aged 65 or older , with a higher prevalence \n in females than in males .", "lower - limb osteoarthritis is \n associated with foot postures and affects the dynamic alignment of the lower limbs9 .", "reilly et al.9 reported that patients with knee joint osteoarthritis showed changes \n in the ankle joint postures when compared with healthy individuals , and according to the \n study of hu et al.10 , foot posture and \n stance widths affected the load - sharing mechanism of the lumbar muscle tissues . therefore", ", \n knee osteoarthritis was believed to affect the frp of the lumbar erector spinae .", "the frp can act as a quantitative measure for the evaluation of changes in the \n neuromuscular system functions11 .", "varying \n flexion - relaxation rates among patients are a useful clinical tool that can help in \n diagnosing chronic lower back pain in patients3 .", "this argument is supported by strong evidence that those subjects \n with low back pain do not exhibit the frp during complete trunk flexion12 .", "watson et al.13 demonstrated the validity of measuring the frp of the lumbar \n vertebrae to diagnose lower back issues and reported high specificity and sensitivity . in \n addition , the flexion - relaxation rates are a valuable tool for distinguishing between \n patients with cervical pain and those with no cervical pain , and to indicate changes in the \n neuromuscular control in patients with cervical pain .", "the flexion - relaxation rates of the \n cervical spine were reported to be significantly lower in patients with cervical pain14 .", "shin et \n al.15 advised that the knee angles and \n individuals flexibility levels affected the frp of the lumbar muscles .", "they reported that \n in the case of subjects with high flexibility , knee angles affected the flexion - relaxation \n rates when the trunk angle became 90 , and in the case of those with moderate flexibility , \n the foregoing reactions appeared when trunk angles were 7090. they also reported that in \n the case of those with poor flexibility , knee angles did not affect the flexion - relaxation \n rates .", "according to the study by hashemirad et al.16 , muscle activity pattern of the erector spinae varied with \n flexibility during trunk flexion / extension .", "the flexion - relaxation reactions are affected by \n several factors , including trunk loads , lumbosacral postures , trunk angular speed , number of \n task repetitions , and muscle fatigue6,7,8 , 17 .", "although previous studies were actively \n conducted on the effects of loads , angular speed , muscle fatigue , and postures on the \n flexion - relaxation rates , studies on the effects of changed lumbosacral postures of patients \n with chronic knee osteoarthritis on the frp of the erector spinae have been insufficient . \n", "therefore , the present study intended to examine the differences in the frp of the erector \n spinae between elderly persons with degenerative knee osteoarthritis and healthy adults . \n although not clinically significance , the results showed significant differences only in the \n left thoracic erector spinae , but not in the left and right lumbar erector spinae or right \n thoracic erector spinae .", "first , the study sample was small and was limited to females \n with chronic knee osteoarthritis", "second , \n although the same voice signals were given using a metronome during trunk flexion and \n extension , trunk speeds were different among individual subjects .", "third , the levels of knee \n pain were different among individual patients with chronic knee osteoarthritis , and the \n effects of medication and previous treatment could not be eliminated . to further determine \n the frp of the erector spinae in elderly females with chronic knee osteoarthritis , further \n studies that account for the limitations of the present study may be required ."], "abstract_text": ["<S> [ purpose ] this study investigated the flexion - relaxation phenomenon of the erector spinae \n in elderly women with chronic knee osteoarthritis and determined whether the \n flexion - relaxation phenomenon can be used as a pain evaluation tool in such cases . \n [ subjects and methods ] seventeen elderly females with chronic knee osteoarthritis and 13 \n healthy young females voluntarily participated in this study . </S>", "<S> they performed three \n postural positions in 15  s : trunk flexion , complete trunk flexion , and trunk extension , \n each for 5 s. while these positions were held , muscle activation of the thoracic and \n lumbar erector spinae were measured using surface electromyography . </S>", "<S> the flexion - relaxation \n rate was determined by dividing the values for trunk extension by those of complete trunk \n flexion and by dividing the values for trunk flexion by those of complete trunk flexion . \n </S>", "<S> [ results ] according to our results , the flexion - relaxation phenomenon was different \n between healthy young and elderly females with chronic knee osteoarthritis . </S>", "<S> specifically , \n there was a difference in the left thoracic erector spinae muscle , but not in the left and \n right lumbar erector spinae or right thoracic spinae muscle . </S>", "<S> [ conclusion ] our study \n demonstrated that the erector spinae muscle flexion - relaxation phenomenon can be used as a \n pain evaluation tool in elderly females with chronic knee osteoarthritis . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["in korea , close to 86.8% of the elderly population ( aged 65 or older ) has chronic \n degenerative disease1 . among these \n degenerative diseases , arthritis has the highest prevalence rate .", "osteoarthritis mainly \n occurs in the hip joints and knee joints , which are weight - bearing joints , and most elderly \n persons aged 65  years or older have this disease1 , \n 2 .", "osteoarthritis induces the growth of \n osteophytes , leading not only to pain but also to the loss of movement and often to a state \n of lethargy , in which the individual avoids walking1 .", "this lethargy hinders the individual from performing functional \n activities , often resulting in muscle weakening and atrophy , including decrease in muscle \n force and motor unit activation of the muscles around the joint affected by \n osteoarthritis3 .", "muscle weakening and \n atrophy further contribute to reduced walking and functional abilities required for daily \n living3 .", "flexion - relaxation phenomenon ( frp ) refers to the appearance of muscular - electrical silence \n on the surface of the erector spine during complete trunk flexion4 , which could be affected by several factors , including \n lumbopelvic postures5 , repetition of \n tasks6 , and muscle fatigue7 .", "chronic osteoarthritis , therefore , often leads to stooped postures \n as a result of the tension experienced when assuming postures in which the erector spine is \n elongated3 , 4 .", "the erector spinae muscle is a posture - maintaining muscle that is \n used in all daily living activities in which trunk forward - tilting and straightening motions \n are performed .", "normal frp of the erector spinae is an important precondition for smoothly \n performing the functional activities of daily living and is an important measurement \n variable in the evaluation of treatment effects at clinics8 .", "there are currently insufficient studies examining the effects of osteoarthritis on the frp \n of the erector spinae and on how the frp of the erector spinae observed in young adults is \n different from that of the elderly in whom osteoarthritis has occurred . therefore , the \n present study compared the frps of the erector spinae occurring during ( 1 ) trunk flexion , \n ( 2 ) complete trunk flexion , and ( 3 ) trunk extension motions in both healthy , young females \n and elderly females with chronic knee osteoarthritis . in addition , data regarding whether \n the frp of the erector spinae can be used as a method of evaluating the degree of pain in \n chronic osteoarthritis in the elderly will be presented .", "if the erector spinae \n flexion - relaxation rates can be used as a pain scale , it may assist healthcare professionals \n to improve elderly patients quality of life ."], ["this study was a randomized controlled trial , which included healthy adult females \n attending a university and elderly females aged at least 65  years diagnosed with chronic \n osteoarthritis in the leg , among the elderly community residing in a metropolitan area of \n chungcheongnam - do in south korea .", "the selection criteria for elderly subjects were as \n follows : those who had no orthopedic or neurologic disease affecting the leg other than \n chronic osteoarthritis , had not undergone surgical treatment of the leg within the last 6 \n months , had not experienced any fracture in the leg in the last 6 months , had corrected \n visual acuity not lower than 0.8 and had no blurred vision , could understand experimenters \n instructions and cooperate with the experimenters , and had understood the purpose of the \n present study and voluntarily agreed to participate .", "the study was approved by the \n institutional review board of hanyang university , and written informed consent was obtained \n from all patients prior to the experiments .", "the general characteristics of the subjects that \n participated in the present study are shown in table \n 1table 1.general characteristics of study subjects ( n=30)elderly females with chronicosteoarthritis \n ( n=17)healthy adult females ( n=13)age ( years ) , m  sd 75.8  12.0 * 21.1  0.8height ( cm ) , m  sd153.5  6.3159.6  3.7weight(kg ) , m  sd56.5  8.752.9  5.4*p<0.05 . in the present study , surface electromyography ( emg ) ( trigno wireless system , delsys inc . , \n boston , ma , usa ) was used to measure the muscle activity of the healthy adult females and \n elderly females with chronic knee osteoarthritis while they were performing trunk flexion , \n complete trunk flexion , and trunk extension .", "the emg signals measured by the trigno sensor \n and wirelessly transmitted to the trigno base station were analyzed using emgworks 4.0 \n ( delsys inc . , boston , ma , usa ) software .", "all measured muscle activity values were calculated \n as percentages of maximal or submaximal voluntary isometric contraction values .", "the emg \n signal - sampling rate was 1,024  hz , and the signals were band - pass filtered within a range of \n 10500  hz . metronomes were used to enable the experimental subjects of the present study to perform \n trunk flexion , complete trunk flexion , and trunk extension for 5  s each over a total of 15 \n s. the beat was set to 60 bpm , and seconds were counted so that the subjects could hear and \n perform each motion for 5 s. before attaching the emg electrodes , sweat and foreign matter in the experimental muscle \n regions were removed in order to minimize skin resistance .", "the attachment points for \n measuring the thoracic erector spinae were 5  cm to the left and right of the spinous process \n of the t9 , while the attachment points for measuring the lumbar erector spinae were 2  cm to \n the left and right of the spinous process of the l2 .", "the manual muscle force postures for \n measuring of the maximal voluntary isometric contraction ( mvic ) in healthy adults and the \n submaximal voluntary isometric contraction in elderly persons with chronic knee \n osteoarthritis were determined following the method of osullivan et al6 . the values of each subject were measured 3 times for 5  s , \n in succession , over a total of 15 s. measurements during the first and last seconds of each \n measurement period were excluded , and the maximum values during the middle 3  s were used for \n the quantification of the root mean square ( rms ) . to minimize muscle fatigue , 1 ", "the subjects were randomly measured in the \n order of voluntary participation . during the measurement of the flexion - relaxation rates , \n", "three motions were performed : ( a ) trunk flexion , ( b ) complete trunk flexion , and ( c ) trunk \n extension .", "posture 1 was a standing posture , posture 2 was a trunk - bending motion , and \n posture 3 was a maximum trunk - bending motion . in the complete trunk flexion section ,", "the \n subjects were instructed to bend the trunk maximally within the range of bending with no \n pain before measurement .", "preliminary instruction was provided to the subjects by the \n experimenter , who performed the motions in front of the subjects and helped the subjects \n practice the motions at least one time .", "the flexion - relaxation rates were determined using both the values obtained by dividing the \n data for trunk extension by that of complete trunk flexion ( c / b ) and by dividing the data \n for trunk flexion by that of complete trunk flexion ( a / b ) .", "the mean values of the three \n trunk flexion extension motions were used to determine the flexion - relaxation rates .", "the means and standard deviations of \n the general characteristics of the study subjects ( age , gender , height , and weight ) were \n calculated using descriptive statistics . in the present study ,", "the independent variable was \n the presence or absence of chronic knee osteoarthritis , and the dependent variable was the \n frp of the erector spinae .", "independent t - tests were used to examine the erector spine \n flexion - relaxation rates according to whether the presence or absence of chronic knee \n osteoarthritis . to test the statistical significance ,", " was set to 0.05 , and the collected \n data were analyzed using pasw ver ."], ["the effect of chronic knee osteoarthritis on the frp of the erector spinae was first \n examined .", "the values obtained by dividing the data for trunk extension by that of complete \n trunk flexion were not different for the left and right lumbar erector spinae or right \n thoracic erector spinae ; however , they were significantly different for the left thoracic \n erector spinae ( p<0.05 ) ( table 2table 2.flexion-relaxation phenomenon ( frp ) values obtained by dividing the data for \n trunk extension by those for complete trunk flexion from healthy adults and elderly \n individuals ( units : % ) musclesmean  sdelderly females withchronic osteoarthritishealthy adultfemalesleft thoracic erector spine1.67  0.61 * 1.22  0.45right thoracic erector spine1.87  0.822.31  1.35left", "lumbar erector spine2.35  1.312.08  1.36right lumbar erector spine2.46  1.031.85  1.81*p<0.05 ) .", "the values obtained by dividing the data for trunk flexion by that of \n complete trunk flexion , were not different for the left and right lumbar erector spinae and \n right thoracic erector spinae , but were significantly different for the left thoracic \n erector spinae ( p<0.05 ) ( table 3table 3.flexion-relaxation phenomenon ( frp ) values obtained by dividing the data for \n trunk flexion by those for complete trunk flexion from healthy adults and elderly \n individuals ( units : % ) musclesmean  sdelderly females with chronic osteoarthritishealthy adult femalesleft thoracic erector spine1.47  0.55 * 1.12  0.31right thoracic erector spine1.41  0.491.99  1.43left lumbar erector spine2.00  1.082.58  2.75right lumbar erector spine1.76  0.901.62  1.31*p<0.05 ) ."], ["in the present study , the frp of the erector spinae in elderly females with chronic knee \n osteoarthritis and healthy young females were measured to determine whether the erector \n spinae flexion - relaxation rates could act as a useful pain evaluation tool for patients with \n knee osteoarthritis . although no significant differences were evident in the \n flexion - relaxation rates of the left and right thoracic erector spinae or right lumbar \n erector spinae , significant differences were shown in the flexion - relaxation rates of the \n left thoracic erector spinae .", "degenerative arthritis develops in most people aged 65 or older , with a higher prevalence \n in females than in males .", "lower - limb osteoarthritis is \n associated with foot postures and affects the dynamic alignment of the lower limbs9 .", "reilly et al.9 reported that patients with knee joint osteoarthritis showed changes \n in the ankle joint postures when compared with healthy individuals , and according to the \n study of hu et al.10 , foot posture and \n stance widths affected the load - sharing mechanism of the lumbar muscle tissues . therefore", ", \n knee osteoarthritis was believed to affect the frp of the lumbar erector spinae .", "the frp can act as a quantitative measure for the evaluation of changes in the \n neuromuscular system functions11 .", "varying \n flexion - relaxation rates among patients are a useful clinical tool that can help in \n diagnosing chronic lower back pain in patients3 .", "this argument is supported by strong evidence that those subjects \n with low back pain do not exhibit the frp during complete trunk flexion12 .", "watson et al.13 demonstrated the validity of measuring the frp of the lumbar \n vertebrae to diagnose lower back issues and reported high specificity and sensitivity . in \n addition , the flexion - relaxation rates are a valuable tool for distinguishing between \n patients with cervical pain and those with no cervical pain , and to indicate changes in the \n neuromuscular control in patients with cervical pain .", "the flexion - relaxation rates of the \n cervical spine were reported to be significantly lower in patients with cervical pain14 .", "shin et \n al.15 advised that the knee angles and \n individuals flexibility levels affected the frp of the lumbar muscles .", "they reported that \n in the case of subjects with high flexibility , knee angles affected the flexion - relaxation \n rates when the trunk angle became 90 , and in the case of those with moderate flexibility , \n the foregoing reactions appeared when trunk angles were 7090. they also reported that in \n the case of those with poor flexibility , knee angles did not affect the flexion - relaxation \n rates .", "according to the study by hashemirad et al.16 , muscle activity pattern of the erector spinae varied with \n flexibility during trunk flexion / extension .", "the flexion - relaxation reactions are affected by \n several factors , including trunk loads , lumbosacral postures , trunk angular speed , number of \n task repetitions , and muscle fatigue6,7,8 , 17 .", "although previous studies were actively \n conducted on the effects of loads , angular speed , muscle fatigue , and postures on the \n flexion - relaxation rates , studies on the effects of changed lumbosacral postures of patients \n with chronic knee osteoarthritis on the frp of the erector spinae have been insufficient . \n", "therefore , the present study intended to examine the differences in the frp of the erector \n spinae between elderly persons with degenerative knee osteoarthritis and healthy adults . \n although not clinically significance , the results showed significant differences only in the \n left thoracic erector spinae , but not in the left and right lumbar erector spinae or right \n thoracic erector spinae .", "first , the study sample was small and was limited to females \n with chronic knee osteoarthritis", "second , \n although the same voice signals were given using a metronome during trunk flexion and \n extension , trunk speeds were different among individual subjects .", "third , the levels of knee \n pain were different among individual patients with chronic knee osteoarthritis , and the \n effects of medication and previous treatment could not be eliminated . to further determine \n the frp of the erector spinae in elderly females with chronic knee osteoarthritis , further \n studies that account for the limitations of the present study may be required ."]]}
{"article_id": "PMC4003765", "article_text": ["\n new - onset diabetes after transplantation ( nodat ) represents a common comorbidity after solid organ transplantation .", "nodat after kidney transplantation is associated with inferior outcomes , including higher risk of infections , decreased graft survival , higher rates of cardiovascular disease , and increased overall mortality [ 15 ] .", "risk factors for nodat include use of immunosuppressants , in particular corticosteroids and calcineurin - inhibitors , as well as traditional risk factors for type 2 diabetes including obesity , family history , age , and ethnicity [ 68 ] .", "metformin is the first - line agent of choice for the treatment of type 2 diabetes in the general population .", "though some data suggests metformin is safe in the setting of chronic kidney disease , the use of metformin in kidney transplant patients is still quite controversial due to the concern about lactic acidosis and lack of data regarding metformin use in this patient population [ 911 ] .", "therefore , it is important to identify an alternative first - line oral agent for the treatment of nodat in kidney transplant recipients .", "short - term studies have shown some oral antihyperglycemic agents to be effective in patients with nodat [ 1214 ] .", "however , use of these agents is limited by adverse effects including fluid retention and bone loss with pioglitazone and hypoglycemia and weight gain with insulin secretagogues [ 12 , 15 , 16 ] .", "sitagliptin , an oral dipeptidyl - peptidase-4 ( dpp-4 ) inhibitor , was approved in the united states in 2006 for the treatment of type 2 diabetes .", "sitagliptin has a low incidence of hypoglycemia and does not promote weight gain , and the dosage can be adjusted based on renal function .", "additionally , in a 3-month trial of kidney transplant recipients , sitagliptin use did not alter immunosuppressant levels .", "however , evidence supporting the efficacy and safety of long - term use of sitagliptin in kidney transplant recipients is lacking .", "we hypothesized that in kidney transplant recipients sitagliptin would improve nodat control , as assessed by hemoglobin a1c", "( hba1c ) , with a favorable safety profile including no effect on immunosuppressant levels or graft function .", "in this single center study , we performed a retrospective analysis of kidney transplant recipients in the multidisciplinary transplant clinic at the university of nebraska medical center ( unmc ) between october 2006 ( release date of sitagliptin in the united states ) and december 2012 .", "all subjects in this study received their kidney transplant at unmc . between 2009 and 2013 , 125 to 149 kidney transplants were performed per year at unmc ( http://optn.transplant.hrsa.gov/ ) .", "we have previously reported an incidence of nodat of 18% in our kidney transplant population . the study was approved and monitored by the unmc institutional review board .", "a general query was performed to identify all kidney transplant recipients who had been prescribed sitagliptin during the study dates .", "data for the query and subsequent data collection for the study were obtained from review of the electronic medical records ", "centricity ( ge healthcare ) , ottr ( ottr chronic care solutions ) , and epic ( epic systems corporation ) .", "inclusion criteria for the study included diagnosis of nodat by the 2003 international consensus guidelines criteria ( fasting plasma glucose 126  mg / dl or 7.0 ", "mmol / l after no caloric intake for at least 8 hours or random plasma glucose 200  mg / dl or 11.1  mmol / l with symptoms of diabetes or 2  h plasma glucose in a 75  g oral glucose tolerance test ( ogtt ) 200  mg / dl or 11.1  mmol / l ) and diabetes care received at the unmc diabetes center to allow for accurate record of diabetes medication changes and reporting of side effects . additionally , to specifically understand the efficacy and safety of sitagliptin for the treatment of nodat , patients included in the study either used sitagliptin as the initial agent for nodat or had other diabetes medications stopped prior to or at the time of sitagliptin initiation .", "exclusion criteria included diagnosis of diabetes prior to transplant and death or loss of followup prior to 12 months after initiation of sitagliptin .", "sitagliptin dosing was performed based on the patients ' renal function , as directed by the prescribing information for sitagliptin ( https://www.merck.com/ ) .", "initiation of sitagliptin therapy marked the start of followup for each patient and patients were followed up until discontinuation of sitagliptin or december 2012 , whichever came first .", "the first was a 12-month followup to assess the subacute efficacy and safety of sitagliptin in terms of diabetes control , side effects , immunosuppressant levels , and graft function .", "the second endpoint was at discontinuation of sitagliptin or , for those patients who continued on sitagliptin through the entire study , in december 2012 .", "this longer followup allowed for assessment of long - term efficacy and safety in terms of diabetes control , acute rejection episodes , allograft function , and other side effects .", "the patient medical records were reviewed in detail for a number of different diabetes and transplant - specific outcomes including dates of sitagliptin initiation / discontinuation ; dates of initiation / discontinuation of any other diabetes medications ; patient or physician - reported side effects ; reason for discontinuation of sitagliptin ; acute rejection episodes ; graft loss ; immunosuppressant regimens and dosages ; opportunistic infections ; and death .", "body mass index ( bmi ) was obtained at the time of transplant , at the time of sitagliptin initiation , after 12 months of sitagliptin therapy , and at the end of followup . as part of routine posttransplant and diabetes", "care , patients underwent fasting labs weekly to monthly at the clinical laboratory of the nebraska medical center including serum creatinine , tacrolimus and/or sirolimus trough levels ( as appropriate ) , and fasting glucose levels .", "hba1c was performed every 3 months and fasting lipid panel every 3 to 6 months .", "serum creatinine , fasting serum glucose , hdl cholesterol , and ldl cholesterol measurements were performed using standard calibration protocols and dedicated analyzers and were assayed by colorimetric means . tacrolimus and", "hba1c was performed on a national glycohemoglobin standardization program ( ngsp ) certified high - pressure liquid chromatography ( hplc ) analyzer .", "estimated gfr ( egfr ) was calculated using the modification of diet in renal disease ( mdrd ) equation .", "a total of 65 patients were identified to have received sitagliptin after kidney transplant during the study dates . of these , 37 patients met criteria for nodat ( figure 1 ) . of these 37 patients ,", "a total of 15 were excluded from the study : 9 patients who obtained their diabetes care at outside clinics and clear records of diabetes medication adjustments were not available ; 1 patient who was deceased 8 months after sitagliptin initiation ; and 5 patients because sitagliptin was added to other diabetes medications .", "the remaining twenty - two ( 22 ) patients met inclusion criteria and had at least one year of followup and were subsequently analyzed .", "this cohort of 22 patients does include long - term followup of eight of the fifteen patients analyzed previously at our center in a 3-month prospective study of sitagliptin for the treatment of nodat .", "baseline characteristics of the 22 patients included in the study are shown in table 1 .", "of the 22 patients , nodat was diagnosed in 21 by fasting blood glucose 7.0  mmol / l on two occasions ; one patient was diagnosed with random blood glucose > 11.1  mmol / l with symptoms of diabetes . a majority of patients utilized tacrolimus - based immunosuppression with the addition of either sirolimus or mycophenolate mofetil ( table 1 ) .", "hepatitis c has been reported to be a risk factor for nodat ; however , none of our subjects were hepatitis c positive .", "sitagliptin was the initial diabetes medication for a majority of the patients ( 16/22 ) .", "a total of twenty - two patients were analyzed at the end of 12 months of sitagliptin therapy .", "of these , 19 patients remained on sitagliptin alone as their only diabetes medication , one patient required initiation of an additional oral diabetes medication , and 2 patients had sitagliptin discontinued in favor of other diabetes medications due to hyperglycemia .", "diabetes control , as noted by hba1c , was significantly improved at the end of 6 months and this effect persisted at 12 months ( figure 2(a ) ) .", "significance remained when analyzing just those patients ( n = 19 ) who remained on sitagliptin alone for the entire 12 months ( data not shown ) .", "there was a modest but significant decrease in bmi from the start of sitagliptin to the 12-month followup ( figure 2(b ) ) , though certainly this weight loss may have been due to multiple factors including lifestyle modifications .", "ldl and hdl cholesterol values remained unchanged with sitagliptin therapy ( figures 2(c ) and 2(d ) ) . at the 12-month followup , graft function as noted by serum creatinine and estimated gfr ( egfr ) was no different than at start of sitagliptin ( figures 3(a ) and 3(b ) ) .", "we then reviewed immunosuppressant levels over the 12 months of followup and also meticulously reviewed medical records for immunosuppressant dose changes and , if changes to dose were made , the impetus for doing so .", "tacrolimus and sirolimus levels remained stable throughout the 12-month followup ( figures 3(d ) and 3(e ) ) . in tacrolimus - treated patients ,", "for the remainder of tacrolimus - treated patients only 3 patients required a single protocol dose adjustment and the rest maintained consistent tacrolimus doses .", "similarly , the patient with noncompliance required frequent sirolimus dose adjustments during the follow - up period while 3 patients had single , protocol - related sirolimus dose adjustments . to assess the long - term efficacy and risk for adverse effects of sitagliptin in kidney transplant recipients", ", we followed our cohort from the initiation of sitagliptin until december 2012 or discontinuation of sitagliptin , whichever came first ( mean followup for all 22 patients = 32.5  17.8 months ) . in the cohort , 17/22 patients remained on sitagliptin throughout the study period , for a mean duration of sitagliptin use of 37.9  16.5 months .", "of these 17 patients , 9 remained on sitagliptin alone for the entirety of the study ( 31.8  18.7 months ) .", "diabetes was well controlled in this group , with hba1c maintained below 7% ( 6.5  0.5% ) at the end of followup ( figure 4(a ) ) .", "eight patients continued on sitagliptin for the duration of the study ( 44.9  10.9 months ) but required addition of other diabetes medications to maintain glycemic control ( oral agents , n = 4 ; insulin , n = 4 ) .", "hba1c remained well controlled in this group ( figure 4(b ) ) and two of the four patients on insulin required only basal insulin in addition to sitagliptin . therefore ,", "five patients discontinued sitagliptin in favor of other diabetes medications after 14  4.2 months .", "of these , 4 patients discontinued sitagliptin due to worsening hyperglycemia ( figure 4(c ) ) and need for intensive insulin therapy while one patient was switched from sitagliptin to a sulfonylurea due to cost .", "hemoglobin a1c was significantly lower at baseline in patients who were able to continue on sitagliptin alone for the duration of the study ( n = 9 ) compared to patients who discontinued sitagliptin and were switched to more aggressive diabetes management ( n = 5 ) ( figure 4(d ) ) .", "change in bmi , from sitagliptin initiation to the end of followup , was no different between these groups ( sitagliptin alone : 0.8  2.2 ", "kg / m versus sitagliptin discontinued : 1.1  1.7  kg / m , p = ns ) suggesting the need for intensification of diabetes therapy in the sitagliptin discontinuation group was not necessarily related to weight gain .", "glucagon - like peptide-1 ( glp-1 ) therapies have been implicated to potentially increase the risk of pancreatitis , a matter of significant controversy . in the current study , no episodes of pancreatitis were reported during the follow - up period . additionally , other transplant - specific adverse events were rare in our cohort , including one episode of acute rejection ( 47 months after sitagliptin initiation ) ; two episodes of opportunistic infections ( 13 and 14 months after sitagliptin initiation ) ; no graft loss ; and one death ( due to end - stage liver disease ) .", "our study represents the largest cohort investigating the safety and efficacy of sitagliptin for the treatment of nodat in kidney transplant recipients .", "though our study was retrospective , through meticulous review of the medical records of patients followed up primarily in our transplant endocrine clinic we were able to closely follow medication prescribing changes , laboratory values , and reports of potential side effects and transplant - related comorbid events . in our cohort ,", "sitagliptin was efficacious with a majority of patients meeting a goal hba1c < 7% . specifically , sitagliptin alone was adequate to improve and maintain glycemic control in a majority of patients ( 19/22 ) for 12 months after sitagliptin initiation , consistent with its efficacy in the nontransplant population [ 21 , 22 ] .", "sitagliptin inhibits dpp-4 with several subsequent effects that improve blood glucose control including potentiation of insulin secretion and inhibition of glucagon secretion . in studies utilizing animal models of type 2 diabetes", ", sitagliptin has also been shown to reduce islet inflammation and protect beta cell mass [ 2325 ] .", "beta cells exposed to glp-1 in vitro are resistant to toxicities associated with immunosuppressants , suggesting another potential benefit to dpp-4 inhibitor therapy for the treatment of nodat .", "these beta cell - specific protective effects of sitagliptin may explain why , in our cohort , patients with lower baseline hba1c responded better to sitagliptin monotherapy as these patients may have been earlier into the disease and may have had more beta cell mass that was subsequently protected by sitagliptin .", "overall , these findings suggest that initiating sitagliptin therapy early in the treatment of nodat , when average glucose levels are only modestly elevated , may provide long - term benefits in terms of glycemic control .", "larger cohorts are needed to investigate this point further . in solid organ transplant recipients , drug - drug interactions and adverse effects of medications on graft function", "we set out to better define the safety profile of sitagliptin in kidney transplant recipients in terms of effects on immunosuppressant levels / dosing , graft function , and other side effects .", "renal function and tacrolimus and sirolimus dosing and trough levels remained stable during the initial 12-month follow - up period , indicating sitagliptin does not adversely affect renal function or interfere with immunosuppressant dosing .", "hypoglycemia was not encountered in our cohort , consistent with reports of sitagliptin use in the nontransplant population .", "the use of dpp-4 inhibitors for the treatment of hyperglycemia and nodat in kidney transplant recipients is gaining more interest .", "recently , a 3-month clinical trial of vildagliptin , another oral dpp-4 inhibitor , revealed that this agent improved oral glucose tolerance test outcomes and modestly improved hemoglobin a1c in kidney transplant recipients with impaired glucose tolerance ( igt ) .", "similarly , a small , randomized control trial recently revealed vildagliptin to be a safe and effective therapy for the treatment of nodat in kidney transplant recipients .", "finally , a 24-week retrospective study of linagliptin suggests this agent may be beneficial for treating nodat , as well .", "however , data regarding sitagliptin for the treatment of nodat in kidney transplant recipients is scarce", ". a single pilot study of 15 patients followed up for 3 months revealed no significant effect of sitagliptin on renal function ( as measured by estimated gfr ) or tacrolimus or sirolimus levels .", "additionally , iuppa et al . presented an abstract discussing the use of sitagliptin in solid organ transplant recipients and , although most of the subjects were liver transplant recipients , 18 kidney transplant recipients were reported on with a median followup of all patients ( kidney and liver transplant recipients ) of 178 days .", "these small trials and a case report of a single kidney transplant recipient treated with sitagliptin for 2.5 years encompass the entire literature available on this subject .", "our study provides the most person - years of followup of any trial of sitagliptin for the treatment of nodat in kidney transplant recipients .", "additionally , though retrospective , our study provides the longest followup of any dpp-4 inhibitor used for the treatment of nodat in kidney transplant recipients . limitations of our study include the retrospective nature of our study and the small cohort size .", "however , large , prospective studies regarding glucose - lowering therapy for nodat do not exist currently ( as reviewed ) and our study represents the largest study to date examining the use of sitagliptin in the setting of nodat after kidney transplantation .", "our cohort was comprised mostly of caucasian males and steroids were not part of the maintenance immunosuppressant regimen .", "these factors may limit the ability to generalize our findings . finally , our mean time to diagnosis of nodat ( 56.3  57.7 months after transplant ) was longer than many studies of nodat .", "this likely reflects that the patients in our cohort had slower , less aggressive development of nodat and did not require insulin therapy .", "kidney transplant recipients with viable grafts remain at higher risk of diabetes development , a significant portion of the risk being ascribed to immunosuppressant therapy .", "safe , effective therapies are needed for patients who develop nodat regardless of the timing of the diagnosis , and sitagliptin may be useful for this purpose given the favorable side effect profile and lack of interaction with immunosuppressant medications .", "in conclusion , we have shown that , in a small cohort of kidney transplant recipients who developed nodat , sitagliptin was efficacious as a single agent or in combination with other glucose - lowering medications .", "sitagliptin was also well tolerated and renal function and immunosuppressant levels and dosing were stable during 12 months of therapy .", "sitagliptin may be valuable as a first - line agent in kidney transplant recipients diagnosed with nodat who are candidates for oral therapy ."], "abstract_text": ["<S> new - onset diabetes after transplantation ( nodat ) is a common comorbidity after renal transplantation . </S>", "<S> though metformin is the first - line agent for the treatment of type 2 diabetes , in renal transplant recipients , metformin is frequently avoided due to concerns about renal dysfunction and risk for lactic acidosis . </S>", "<S> therefore , alternative first - line agents for the treatment of nodat in renal transplant recipients are needed . </S>", "<S> sitagliptin , a dipeptidyl - peptidase-4 ( dpp-4 ) inhibitor , has a low incidence of hypoglycemia , is weight neutral , and , in a small study , did not affect immunosuppressant levels . however , long - term sitagliptin use for the treatment of nodat in kidney transplant recipients has not been studied . </S>", "<S> we retrospectively analyzed renal transplant recipients diagnosed with nodat and treated with sitagliptin to assess safety and efficacy . </S>", "<S> twenty - two patients were started on sitagliptin alone . </S>", "<S> after 12 months of followup , 19/22 patients remained on sitagliptin alone with a significant improvement in hemoglobin a1c . </S>", "<S> renal function and immunosuppressant levels remained stable . </S>", "<S> analysis of long - term followup ( 32.5  17.8 months ) revealed that 17/22 patients remained on sitagliptin ( mean hemoglobin a1c </S>", "<S> < 7% ) with 9/17 patients remaining on sitagliptin alone . </S>", "<S> transplant - specific adverse events were rare . </S>", "<S> sitagliptin appears safe and efficacious for the treatment of nodat in kidney transplant recipients . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["\n new - onset diabetes after transplantation ( nodat ) represents a common comorbidity after solid organ transplantation .", "nodat after kidney transplantation is associated with inferior outcomes , including higher risk of infections , decreased graft survival , higher rates of cardiovascular disease , and increased overall mortality [ 15 ] .", "risk factors for nodat include use of immunosuppressants , in particular corticosteroids and calcineurin - inhibitors , as well as traditional risk factors for type 2 diabetes including obesity , family history , age , and ethnicity [ 68 ] .", "metformin is the first - line agent of choice for the treatment of type 2 diabetes in the general population .", "though some data suggests metformin is safe in the setting of chronic kidney disease , the use of metformin in kidney transplant patients is still quite controversial due to the concern about lactic acidosis and lack of data regarding metformin use in this patient population [ 911 ] .", "therefore , it is important to identify an alternative first - line oral agent for the treatment of nodat in kidney transplant recipients .", "short - term studies have shown some oral antihyperglycemic agents to be effective in patients with nodat [ 1214 ] .", "however , use of these agents is limited by adverse effects including fluid retention and bone loss with pioglitazone and hypoglycemia and weight gain with insulin secretagogues [ 12 , 15 , 16 ] .", "sitagliptin , an oral dipeptidyl - peptidase-4 ( dpp-4 ) inhibitor , was approved in the united states in 2006 for the treatment of type 2 diabetes .", "sitagliptin has a low incidence of hypoglycemia and does not promote weight gain , and the dosage can be adjusted based on renal function .", "additionally , in a 3-month trial of kidney transplant recipients , sitagliptin use did not alter immunosuppressant levels .", "however , evidence supporting the efficacy and safety of long - term use of sitagliptin in kidney transplant recipients is lacking .", "we hypothesized that in kidney transplant recipients sitagliptin would improve nodat control , as assessed by hemoglobin a1c", "( hba1c ) , with a favorable safety profile including no effect on immunosuppressant levels or graft function ."], ["in this single center study , we performed a retrospective analysis of kidney transplant recipients in the multidisciplinary transplant clinic at the university of nebraska medical center ( unmc ) between october 2006 ( release date of sitagliptin in the united states ) and december 2012 .", "all subjects in this study received their kidney transplant at unmc . between 2009 and 2013 , 125 to 149 kidney transplants were performed per year at unmc ( http://optn.transplant.hrsa.gov/ ) .", "we have previously reported an incidence of nodat of 18% in our kidney transplant population . the study was approved and monitored by the unmc institutional review board .", "a general query was performed to identify all kidney transplant recipients who had been prescribed sitagliptin during the study dates .", "data for the query and subsequent data collection for the study were obtained from review of the electronic medical records ", "centricity ( ge healthcare ) , ottr ( ottr chronic care solutions ) , and epic ( epic systems corporation ) .", "inclusion criteria for the study included diagnosis of nodat by the 2003 international consensus guidelines criteria ( fasting plasma glucose 126  mg / dl or 7.0 ", "mmol / l after no caloric intake for at least 8 hours or random plasma glucose 200  mg / dl or 11.1  mmol / l with symptoms of diabetes or 2  h plasma glucose in a 75  g oral glucose tolerance test ( ogtt ) 200  mg / dl or 11.1  mmol / l ) and diabetes care received at the unmc diabetes center to allow for accurate record of diabetes medication changes and reporting of side effects . additionally , to specifically understand the efficacy and safety of sitagliptin for the treatment of nodat , patients included in the study either used sitagliptin as the initial agent for nodat or had other diabetes medications stopped prior to or at the time of sitagliptin initiation .", "exclusion criteria included diagnosis of diabetes prior to transplant and death or loss of followup prior to 12 months after initiation of sitagliptin .", "sitagliptin dosing was performed based on the patients ' renal function , as directed by the prescribing information for sitagliptin ( https://www.merck.com/ ) .", "initiation of sitagliptin therapy marked the start of followup for each patient and patients were followed up until discontinuation of sitagliptin or december 2012 , whichever came first .", "the first was a 12-month followup to assess the subacute efficacy and safety of sitagliptin in terms of diabetes control , side effects , immunosuppressant levels , and graft function .", "the second endpoint was at discontinuation of sitagliptin or , for those patients who continued on sitagliptin through the entire study , in december 2012 .", "this longer followup allowed for assessment of long - term efficacy and safety in terms of diabetes control , acute rejection episodes , allograft function , and other side effects .", "the patient medical records were reviewed in detail for a number of different diabetes and transplant - specific outcomes including dates of sitagliptin initiation / discontinuation ; dates of initiation / discontinuation of any other diabetes medications ; patient or physician - reported side effects ; reason for discontinuation of sitagliptin ; acute rejection episodes ; graft loss ; immunosuppressant regimens and dosages ; opportunistic infections ; and death .", "body mass index ( bmi ) was obtained at the time of transplant , at the time of sitagliptin initiation , after 12 months of sitagliptin therapy , and at the end of followup . as part of routine posttransplant and diabetes", "care , patients underwent fasting labs weekly to monthly at the clinical laboratory of the nebraska medical center including serum creatinine , tacrolimus and/or sirolimus trough levels ( as appropriate ) , and fasting glucose levels .", "hba1c was performed every 3 months and fasting lipid panel every 3 to 6 months .", "serum creatinine , fasting serum glucose , hdl cholesterol , and ldl cholesterol measurements were performed using standard calibration protocols and dedicated analyzers and were assayed by colorimetric means . tacrolimus and", "hba1c was performed on a national glycohemoglobin standardization program ( ngsp ) certified high - pressure liquid chromatography ( hplc ) analyzer .", "estimated gfr ( egfr ) was calculated using the modification of diet in renal disease ( mdrd ) equation ."], ["a total of 65 patients were identified to have received sitagliptin after kidney transplant during the study dates . of these , 37 patients met criteria for nodat ( figure 1 ) . of these 37 patients ,", "a total of 15 were excluded from the study : 9 patients who obtained their diabetes care at outside clinics and clear records of diabetes medication adjustments were not available ; 1 patient who was deceased 8 months after sitagliptin initiation ; and 5 patients because sitagliptin was added to other diabetes medications .", "the remaining twenty - two ( 22 ) patients met inclusion criteria and had at least one year of followup and were subsequently analyzed .", "this cohort of 22 patients does include long - term followup of eight of the fifteen patients analyzed previously at our center in a 3-month prospective study of sitagliptin for the treatment of nodat .", "baseline characteristics of the 22 patients included in the study are shown in table 1 .", "of the 22 patients , nodat was diagnosed in 21 by fasting blood glucose 7.0  mmol / l on two occasions ; one patient was diagnosed with random blood glucose > 11.1  mmol / l with symptoms of diabetes . a majority of patients utilized tacrolimus - based immunosuppression with the addition of either sirolimus or mycophenolate mofetil ( table 1 ) .", "hepatitis c has been reported to be a risk factor for nodat ; however , none of our subjects were hepatitis c positive .", "sitagliptin was the initial diabetes medication for a majority of the patients ( 16/22 ) .", "a total of twenty - two patients were analyzed at the end of 12 months of sitagliptin therapy .", "of these , 19 patients remained on sitagliptin alone as their only diabetes medication , one patient required initiation of an additional oral diabetes medication , and 2 patients had sitagliptin discontinued in favor of other diabetes medications due to hyperglycemia .", "diabetes control , as noted by hba1c , was significantly improved at the end of 6 months and this effect persisted at 12 months ( figure 2(a ) ) .", "significance remained when analyzing just those patients ( n = 19 ) who remained on sitagliptin alone for the entire 12 months ( data not shown ) .", "there was a modest but significant decrease in bmi from the start of sitagliptin to the 12-month followup ( figure 2(b ) ) , though certainly this weight loss may have been due to multiple factors including lifestyle modifications .", "ldl and hdl cholesterol values remained unchanged with sitagliptin therapy ( figures 2(c ) and 2(d ) ) . at the 12-month followup , graft function as noted by serum creatinine and estimated gfr ( egfr ) was no different than at start of sitagliptin ( figures 3(a ) and 3(b ) ) .", "we then reviewed immunosuppressant levels over the 12 months of followup and also meticulously reviewed medical records for immunosuppressant dose changes and , if changes to dose were made , the impetus for doing so .", "tacrolimus and sirolimus levels remained stable throughout the 12-month followup ( figures 3(d ) and 3(e ) ) . in tacrolimus - treated patients ,", "for the remainder of tacrolimus - treated patients only 3 patients required a single protocol dose adjustment and the rest maintained consistent tacrolimus doses .", "similarly , the patient with noncompliance required frequent sirolimus dose adjustments during the follow - up period while 3 patients had single , protocol - related sirolimus dose adjustments . to assess the long - term efficacy and risk for adverse effects of sitagliptin in kidney transplant recipients", ", we followed our cohort from the initiation of sitagliptin until december 2012 or discontinuation of sitagliptin , whichever came first ( mean followup for all 22 patients = 32.5  17.8 months ) . in the cohort , 17/22 patients remained on sitagliptin throughout the study period , for a mean duration of sitagliptin use of 37.9  16.5 months .", "of these 17 patients , 9 remained on sitagliptin alone for the entirety of the study ( 31.8  18.7 months ) .", "diabetes was well controlled in this group , with hba1c maintained below 7% ( 6.5  0.5% ) at the end of followup ( figure 4(a ) ) .", "eight patients continued on sitagliptin for the duration of the study ( 44.9  10.9 months ) but required addition of other diabetes medications to maintain glycemic control ( oral agents , n = 4 ; insulin , n = 4 ) .", "hba1c remained well controlled in this group ( figure 4(b ) ) and two of the four patients on insulin required only basal insulin in addition to sitagliptin . therefore ,", "five patients discontinued sitagliptin in favor of other diabetes medications after 14  4.2 months .", "of these , 4 patients discontinued sitagliptin due to worsening hyperglycemia ( figure 4(c ) ) and need for intensive insulin therapy while one patient was switched from sitagliptin to a sulfonylurea due to cost .", "hemoglobin a1c was significantly lower at baseline in patients who were able to continue on sitagliptin alone for the duration of the study ( n = 9 ) compared to patients who discontinued sitagliptin and were switched to more aggressive diabetes management ( n = 5 ) ( figure 4(d ) ) .", "change in bmi , from sitagliptin initiation to the end of followup , was no different between these groups ( sitagliptin alone : 0.8  2.2 ", "kg / m versus sitagliptin discontinued : 1.1  1.7  kg / m , p = ns ) suggesting the need for intensification of diabetes therapy in the sitagliptin discontinuation group was not necessarily related to weight gain .", "glucagon - like peptide-1 ( glp-1 ) therapies have been implicated to potentially increase the risk of pancreatitis , a matter of significant controversy . in the current study , no episodes of pancreatitis were reported during the follow - up period . additionally , other transplant - specific adverse events were rare in our cohort , including one episode of acute rejection ( 47 months after sitagliptin initiation ) ; two episodes of opportunistic infections ( 13 and 14 months after sitagliptin initiation ) ; no graft loss ; and one death ( due to end - stage liver disease ) ."], ["our study represents the largest cohort investigating the safety and efficacy of sitagliptin for the treatment of nodat in kidney transplant recipients .", "though our study was retrospective , through meticulous review of the medical records of patients followed up primarily in our transplant endocrine clinic we were able to closely follow medication prescribing changes , laboratory values , and reports of potential side effects and transplant - related comorbid events . in our cohort ,", "sitagliptin was efficacious with a majority of patients meeting a goal hba1c < 7% . specifically , sitagliptin alone was adequate to improve and maintain glycemic control in a majority of patients ( 19/22 ) for 12 months after sitagliptin initiation , consistent with its efficacy in the nontransplant population [ 21 , 22 ] .", "sitagliptin inhibits dpp-4 with several subsequent effects that improve blood glucose control including potentiation of insulin secretion and inhibition of glucagon secretion . in studies utilizing animal models of type 2 diabetes", ", sitagliptin has also been shown to reduce islet inflammation and protect beta cell mass [ 2325 ] .", "beta cells exposed to glp-1 in vitro are resistant to toxicities associated with immunosuppressants , suggesting another potential benefit to dpp-4 inhibitor therapy for the treatment of nodat .", "these beta cell - specific protective effects of sitagliptin may explain why , in our cohort , patients with lower baseline hba1c responded better to sitagliptin monotherapy as these patients may have been earlier into the disease and may have had more beta cell mass that was subsequently protected by sitagliptin .", "overall , these findings suggest that initiating sitagliptin therapy early in the treatment of nodat , when average glucose levels are only modestly elevated , may provide long - term benefits in terms of glycemic control .", "larger cohorts are needed to investigate this point further . in solid organ transplant recipients , drug - drug interactions and adverse effects of medications on graft function", "we set out to better define the safety profile of sitagliptin in kidney transplant recipients in terms of effects on immunosuppressant levels / dosing , graft function , and other side effects .", "renal function and tacrolimus and sirolimus dosing and trough levels remained stable during the initial 12-month follow - up period , indicating sitagliptin does not adversely affect renal function or interfere with immunosuppressant dosing .", "hypoglycemia was not encountered in our cohort , consistent with reports of sitagliptin use in the nontransplant population .", "the use of dpp-4 inhibitors for the treatment of hyperglycemia and nodat in kidney transplant recipients is gaining more interest .", "recently , a 3-month clinical trial of vildagliptin , another oral dpp-4 inhibitor , revealed that this agent improved oral glucose tolerance test outcomes and modestly improved hemoglobin a1c in kidney transplant recipients with impaired glucose tolerance ( igt ) .", "similarly , a small , randomized control trial recently revealed vildagliptin to be a safe and effective therapy for the treatment of nodat in kidney transplant recipients .", "finally , a 24-week retrospective study of linagliptin suggests this agent may be beneficial for treating nodat , as well .", "however , data regarding sitagliptin for the treatment of nodat in kidney transplant recipients is scarce", ". a single pilot study of 15 patients followed up for 3 months revealed no significant effect of sitagliptin on renal function ( as measured by estimated gfr ) or tacrolimus or sirolimus levels .", "additionally , iuppa et al . presented an abstract discussing the use of sitagliptin in solid organ transplant recipients and , although most of the subjects were liver transplant recipients , 18 kidney transplant recipients were reported on with a median followup of all patients ( kidney and liver transplant recipients ) of 178 days .", "these small trials and a case report of a single kidney transplant recipient treated with sitagliptin for 2.5 years encompass the entire literature available on this subject .", "our study provides the most person - years of followup of any trial of sitagliptin for the treatment of nodat in kidney transplant recipients .", "additionally , though retrospective , our study provides the longest followup of any dpp-4 inhibitor used for the treatment of nodat in kidney transplant recipients . limitations of our study include the retrospective nature of our study and the small cohort size .", "however , large , prospective studies regarding glucose - lowering therapy for nodat do not exist currently ( as reviewed ) and our study represents the largest study to date examining the use of sitagliptin in the setting of nodat after kidney transplantation .", "our cohort was comprised mostly of caucasian males and steroids were not part of the maintenance immunosuppressant regimen .", "these factors may limit the ability to generalize our findings . finally , our mean time to diagnosis of nodat ( 56.3  57.7 months after transplant ) was longer than many studies of nodat .", "this likely reflects that the patients in our cohort had slower , less aggressive development of nodat and did not require insulin therapy .", "kidney transplant recipients with viable grafts remain at higher risk of diabetes development , a significant portion of the risk being ascribed to immunosuppressant therapy .", "safe , effective therapies are needed for patients who develop nodat regardless of the timing of the diagnosis , and sitagliptin may be useful for this purpose given the favorable side effect profile and lack of interaction with immunosuppressant medications ."], ["in conclusion , we have shown that , in a small cohort of kidney transplant recipients who developed nodat , sitagliptin was efficacious as a single agent or in combination with other glucose - lowering medications .", "sitagliptin was also well tolerated and renal function and immunosuppressant levels and dosing were stable during 12 months of therapy .", "sitagliptin may be valuable as a first - line agent in kidney transplant recipients diagnosed with nodat who are candidates for oral therapy ."]]}
{"article_id": "PMC4389205", "article_text": ["substantial improvement in survival of people living with hiv has been observed with the introduction of antiretroviral therapy ( art ) in sub - saharan africa .", "however , the introduction of art has led to new immune - mediated complications from dysfunction of the recovering immune system , termed immune reconstitution inflammatory syndrome ( iris ) . in patients with cryptococcal meningitis ( cm ) , a very common opportunistic infection in sub - saharan africa ,", "iris is a frequent complication that is most often associated with exaggerated inflammatory responses in the central nervous system .", "although cm remains the most common cause of adult meningitis in the overall population in sub - saharan africa , there are only a few recognized , reported cases of gastrointestinal ( gi ) involvement with cryptococcus in the literature .", "an hiv - infected patient on art with a history of recent cm presented with chronic abdominal pain , thickened ileum , and abdominal lymphadenopathy .", "he was initially treated for abdominal tuberculosis ( tb ) but later , he was shown to have a histologically confirmed cryptococcoma in the ileum . the major objective of this case report is to highlight the challenges in diagnosis and management of intra - abdominal cryptococcosis , especially in the setting of recent initiation of art .", "a 37 year old hiv - positive male was initially diagnosed and treated for cm in may of 2009 with amphotericin induction therapy for 2 weeks followed by fluconazole 400  mg / day .", "he had a protracted hospital course lasting 4 weeks because of persistently high intracranial hypertension despite sterilization of his cerebrospinal fluid , and he , required repeated therapeutic lumber punctures .", "he started art ( zidovudine , lamivudine , and efavirenz ) on the 11th june 2009 with a baseline cd4 count of 5  cells/l . at day + 21 of art", ", he reported abdominal pain with hypogastric tenderness that was empirically treated as urinary tract infection with a 5  day course of ciprofloxacin , and he noted some improvement . his cd4 count at the time had risen to 29  cells/l . at day + 35 of art ,", "his general exam was unremarkable , except for a tachypnea of 28 breaths per minute .", "the patient was managed as a possible atypical pneumonia with doxycycline , and his respiratory symptoms improved . however , the abdominal pain persisted intermittently with occasional vomiting . at day + 84 of art and in view of the persistent abdominal pain and an occasional dry cough ,", "another chest radiograph was performed to exclude tb , which was also unremarkable . at this time ,", "his cd4 was only 14  cells/l , and the hiv-1 viral load was 22,000  copies / ml . he reported 100% adherence to his art , and pill counts verified his adherence . at day + 112 of art , the patient still had similar complaints of abdominal pain and occasional vomiting .", "repeat cd4 was 8  cells/l , and the viral load was minimally decreased at 15,475  copies / ml . the patient initially declined an ultrasound - guided biopsy but consented to the procedure at day + 140 of art .", "this second ultrasound revealed a thickened ileum up to 6 mm , with adjacent 1  cm lymph nodes .", "two biopsy specimens from the lymph nodes demonstrated no abnormality at histology . at day + 168 , the patient still had the same complaints with almost unchanged cd4 of 24  cells/l and viral load of 23,755  copies / ml .", "he was presumed to be failing art , possibly due to poor absorptive surface because of the thickened gut wall .", "a decision was made to initiate empiric anti - tb treatment for possible gastrointestinal tb .", "after one week of tb empiric therapy , the patient presented with an acute abdomen , having signs of focal peritonitis in the right iliac fossa .", "an upright abdominal radiograph demonstrated evidence of perforation with free air visible under the right hemidiaphram .", "an emergency laparotomy was performed , revealing fecal matter with purulent fluid , adhesions , pneumoperitonium , and intestinal perforation at 10  cm proximal to the ileocecal junction .", "the confirmatory histopathology report received 2 weeks later revealed a cryptococcoma in the ileal wall as the cause of the perforation with exuberant inflammation demonstrated by the multinucleated giant cell ( figs . 2 and 3 on hematoxylin and eosin stain and figs . 4 and 5 on periodic acid - schiff stain ) .", "the patient 's marked clinical improvement after surgery , the lack of any evidence of cryptococcal infection elsewhere in the body especially in the central nervous system and a well formed granuloma formed the basis for the patient 's continuation with maintenance dose of fluconazole 200  mg / day under observation .", "at this point , his tb medications were stopped and he was switched to second line art with subsequent viral suppression . through december 2014 , he continues to remain in care on second line art without any complaints .", "we have presented a 37 year old hiv positive male , with a history of recent cm who subsequently developed chronic abdominal pain , eventually manifesting as a cryptococcoma of the ileum .", "after treatment for cm and initiating art , he had presented with chronic abdominal pain and low grade fever without diarrhea .", "he subsequently developed an intestinal perforation and presented with an acute surgical abdomen requiring bowel resection .", "we suspected an iris phenomena , in accordance with the patient presentation shortly after initiation of art , recent history of cm , and exuberant inflammation in the granuloma on histology .", "although , the initial immune recovery coupled with falling hiv-1 viral loads is consistent with iris , the subsequent virological failure makes the diagnosis of paradoxical iris less clear . in cryptococcosis ,", "ideally , intra - operative cultures would have been performed which could have helped distinguish iris from cryptococcal relapse , based on culture sterility vs. growth , respectively .", "cryptococcus organisms can be acquired in the gut primarily through hematogenous dissemination   or less commonly through direct inoculation during paracentesis or via a neurosurgical shunt .", "the presentation in these gi cases of cryptococcal infection is usually vague , as seen in our patient , with subacute fevers , constitutional symptoms , asthenia , and anorexia .", "virtually every intra - abdominal organ has been reported to be affected by cryptococcal infection .", "the diagnosis of gi cryptococcosis requires a high index of suspicion , yet as in this case , clinicians may often initially focus on other common etiologies in immunocompromised persons , such as tb .", "although abdominal tb was found to be the most common diagnosis in patients with hiv / aids presenting with chronic abdominal pain and abdominal lymphadenopathy , these studies were conducted predominantly in persons without cryptococcosis . among persons with a known pre - existing opportunistic infection , such as cm , the pre - test probability changes as paradoxical iris enters into the differential diagnosis . in our case ,", "the characteristics of granulomas found in hiv - infected persons varies depending on whether or not they are receiving art . in pulmonary cryptococcomas ,", "persons not receiving art demonstrate yeast proliferation with a histiocytic response but only minor lymphocytic and neutrophilic components .", "conversely , cryptococcal granulomas in persons on art are characterized by the presence of cd4 t cells , greater response of histiocytes , and multinucleated giant - cell formation , as demonstrated in our patient .", "there is a paucity of evidenced - based data for the management of cryptococcomas . in our case ,", "the initial abdominal lymph node biopsy ( 5 weeks prior to the perforation ) did not reveal a diagnosis .", "the question raised is , if we had confirmed the diagnosis of gi cryptococcoma before the perforation , would we have been able to effectively intervene . to answer this question", ", it might be important to know if the cryptococcoma were due to iris or cryptococcal relapse .", "could the patient have benefited from immunosuppressive therapy to treat iris and perhaps , avoid the perforation , or would more enhanced fungal therapy be needed to eradicate the cryptococcus ?", "two case reports have described cryptococcomas due to paradoxical iris ; one in the brain   and the other in the retroperitoneal abdomen . in both cases", ", they simply observed the patients but also emphasized the importance of confirming sterility of contents in the cryptococcoma by culture . in a case report by katchanov et al .", ", a similar presentation of a central nervous system cryptococcoma was initially treated with antifungals exclusively with radiological worsening until steroids were added to direct therapy at paradoxical iris .", "we have previously reported the challenges and dangers of using corticosteroids for cm because they may be contraindicated in cases of fluconazole - resistant cryptococcal relapse .", "surgery , corticosteroids , and interferon - gamma have been tried in iris - like cryptococcomas due cryptococcus gattii \n .", "empiric tb therapy , including rifampin , could have induced the metabolism of fluconazole , possibly lowering plasma levels by ~50% . these lower levels may have removed the fungistatic control of the cryptococcus , precipitating the perforation . however , after the surgery , the patient was observed on secondary prophylaxis doses of fluconazole , and he did well . to treat a cryptococcoma in the setting of recent initiation of art , where iris versus relapse can not be determined due to the absence of culture results , treatment with a combination of enhanced antifungal therapy and anti - inflammatory therapy", "gi cryptococcosis has been described as a rare occurrence , with only a few published case reports .", "we have discussed an hiv - infected patient with profound immunosuppression and recent cm who developed a gi cryptococcoma during initial immune recovery followed by virological failure after initiation of art .", "the cryptococcoma was not identified and he developed perforation of the ileum , requiring surgery . we anticipate that with the roll out of art in sub - sahara africa , we are bound to see rare presentations of some of common conditions such as cm .", "we recommend that health workers have a high index of suspicion for unusual complications of opportunistic infections in the setting of art - associated iris .", "often treatment of iris requires only observation or anti - inflammatory drugs , but the presence of active infection needs to be excluded .", "the authors declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article ."], "abstract_text": ["<S> the introduction of antiretroviral therapy ( art ) may lead to unusual paradoxical and unmasking presentations of opportunistic infections . </S>", "<S> intra - abdominal cryptococcosis is a rare manifestation of cryptococcus . </S>", "<S> we present the case of an hiv - infected patient on art , with a history of cryptococcal meningitis who presented with subacute , worsening abdominal pain during immune recovery . </S>", "<S> this evolved into chronic abdominal pain , with thickened bowel , and abdominal lymphadenopathy , while receiving empiric tuberculosis treatment . at 6-months , he developed intestinal perforation due to a histologically confirmed cryptococcoma . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion", "Conflict of interest"], "sections": [["substantial improvement in survival of people living with hiv has been observed with the introduction of antiretroviral therapy ( art ) in sub - saharan africa .", "however , the introduction of art has led to new immune - mediated complications from dysfunction of the recovering immune system , termed immune reconstitution inflammatory syndrome ( iris ) . in patients with cryptococcal meningitis ( cm ) , a very common opportunistic infection in sub - saharan africa ,", "iris is a frequent complication that is most often associated with exaggerated inflammatory responses in the central nervous system .", "although cm remains the most common cause of adult meningitis in the overall population in sub - saharan africa , there are only a few recognized , reported cases of gastrointestinal ( gi ) involvement with cryptococcus in the literature .", "an hiv - infected patient on art with a history of recent cm presented with chronic abdominal pain , thickened ileum , and abdominal lymphadenopathy .", "he was initially treated for abdominal tuberculosis ( tb ) but later , he was shown to have a histologically confirmed cryptococcoma in the ileum . the major objective of this case report is to highlight the challenges in diagnosis and management of intra - abdominal cryptococcosis , especially in the setting of recent initiation of art ."], ["a 37 year old hiv - positive male was initially diagnosed and treated for cm in may of 2009 with amphotericin induction therapy for 2 weeks followed by fluconazole 400  mg / day .", "he had a protracted hospital course lasting 4 weeks because of persistently high intracranial hypertension despite sterilization of his cerebrospinal fluid , and he , required repeated therapeutic lumber punctures .", "he started art ( zidovudine , lamivudine , and efavirenz ) on the 11th june 2009 with a baseline cd4 count of 5  cells/l . at day + 21 of art", ", he reported abdominal pain with hypogastric tenderness that was empirically treated as urinary tract infection with a 5  day course of ciprofloxacin , and he noted some improvement . his cd4 count at the time had risen to 29  cells/l . at day + 35 of art ,", "his general exam was unremarkable , except for a tachypnea of 28 breaths per minute .", "the patient was managed as a possible atypical pneumonia with doxycycline , and his respiratory symptoms improved . however , the abdominal pain persisted intermittently with occasional vomiting . at day + 84 of art and in view of the persistent abdominal pain and an occasional dry cough ,", "another chest radiograph was performed to exclude tb , which was also unremarkable . at this time ,", "his cd4 was only 14  cells/l , and the hiv-1 viral load was 22,000  copies / ml . he reported 100% adherence to his art , and pill counts verified his adherence . at day + 112 of art , the patient still had similar complaints of abdominal pain and occasional vomiting .", "repeat cd4 was 8  cells/l , and the viral load was minimally decreased at 15,475  copies / ml . the patient initially declined an ultrasound - guided biopsy but consented to the procedure at day + 140 of art .", "this second ultrasound revealed a thickened ileum up to 6 mm , with adjacent 1  cm lymph nodes .", "two biopsy specimens from the lymph nodes demonstrated no abnormality at histology . at day + 168 , the patient still had the same complaints with almost unchanged cd4 of 24  cells/l and viral load of 23,755  copies / ml .", "he was presumed to be failing art , possibly due to poor absorptive surface because of the thickened gut wall .", "a decision was made to initiate empiric anti - tb treatment for possible gastrointestinal tb .", "after one week of tb empiric therapy , the patient presented with an acute abdomen , having signs of focal peritonitis in the right iliac fossa .", "an upright abdominal radiograph demonstrated evidence of perforation with free air visible under the right hemidiaphram .", "an emergency laparotomy was performed , revealing fecal matter with purulent fluid , adhesions , pneumoperitonium , and intestinal perforation at 10  cm proximal to the ileocecal junction .", "the confirmatory histopathology report received 2 weeks later revealed a cryptococcoma in the ileal wall as the cause of the perforation with exuberant inflammation demonstrated by the multinucleated giant cell ( figs . 2 and 3 on hematoxylin and eosin stain and figs . 4 and 5 on periodic acid - schiff stain ) .", "the patient 's marked clinical improvement after surgery , the lack of any evidence of cryptococcal infection elsewhere in the body especially in the central nervous system and a well formed granuloma formed the basis for the patient 's continuation with maintenance dose of fluconazole 200  mg / day under observation .", "at this point , his tb medications were stopped and he was switched to second line art with subsequent viral suppression . through december 2014 , he continues to remain in care on second line art without any complaints ."], ["we have presented a 37 year old hiv positive male , with a history of recent cm who subsequently developed chronic abdominal pain , eventually manifesting as a cryptococcoma of the ileum .", "after treatment for cm and initiating art , he had presented with chronic abdominal pain and low grade fever without diarrhea .", "he subsequently developed an intestinal perforation and presented with an acute surgical abdomen requiring bowel resection .", "we suspected an iris phenomena , in accordance with the patient presentation shortly after initiation of art , recent history of cm , and exuberant inflammation in the granuloma on histology .", "although , the initial immune recovery coupled with falling hiv-1 viral loads is consistent with iris , the subsequent virological failure makes the diagnosis of paradoxical iris less clear . in cryptococcosis ,", "ideally , intra - operative cultures would have been performed which could have helped distinguish iris from cryptococcal relapse , based on culture sterility vs. growth , respectively .", "cryptococcus organisms can be acquired in the gut primarily through hematogenous dissemination   or less commonly through direct inoculation during paracentesis or via a neurosurgical shunt .", "the presentation in these gi cases of cryptococcal infection is usually vague , as seen in our patient , with subacute fevers , constitutional symptoms , asthenia , and anorexia .", "virtually every intra - abdominal organ has been reported to be affected by cryptococcal infection .", "the diagnosis of gi cryptococcosis requires a high index of suspicion , yet as in this case , clinicians may often initially focus on other common etiologies in immunocompromised persons , such as tb .", "although abdominal tb was found to be the most common diagnosis in patients with hiv / aids presenting with chronic abdominal pain and abdominal lymphadenopathy , these studies were conducted predominantly in persons without cryptococcosis . among persons with a known pre - existing opportunistic infection , such as cm , the pre - test probability changes as paradoxical iris enters into the differential diagnosis . in our case ,", "the characteristics of granulomas found in hiv - infected persons varies depending on whether or not they are receiving art . in pulmonary cryptococcomas ,", "persons not receiving art demonstrate yeast proliferation with a histiocytic response but only minor lymphocytic and neutrophilic components .", "conversely , cryptococcal granulomas in persons on art are characterized by the presence of cd4 t cells , greater response of histiocytes , and multinucleated giant - cell formation , as demonstrated in our patient .", "there is a paucity of evidenced - based data for the management of cryptococcomas . in our case ,", "the initial abdominal lymph node biopsy ( 5 weeks prior to the perforation ) did not reveal a diagnosis .", "the question raised is , if we had confirmed the diagnosis of gi cryptococcoma before the perforation , would we have been able to effectively intervene . to answer this question", ", it might be important to know if the cryptococcoma were due to iris or cryptococcal relapse .", "could the patient have benefited from immunosuppressive therapy to treat iris and perhaps , avoid the perforation , or would more enhanced fungal therapy be needed to eradicate the cryptococcus ?", "two case reports have described cryptococcomas due to paradoxical iris ; one in the brain   and the other in the retroperitoneal abdomen . in both cases", ", they simply observed the patients but also emphasized the importance of confirming sterility of contents in the cryptococcoma by culture . in a case report by katchanov et al .", ", a similar presentation of a central nervous system cryptococcoma was initially treated with antifungals exclusively with radiological worsening until steroids were added to direct therapy at paradoxical iris .", "we have previously reported the challenges and dangers of using corticosteroids for cm because they may be contraindicated in cases of fluconazole - resistant cryptococcal relapse .", "surgery , corticosteroids , and interferon - gamma have been tried in iris - like cryptococcomas due cryptococcus gattii \n .", "empiric tb therapy , including rifampin , could have induced the metabolism of fluconazole , possibly lowering plasma levels by ~50% . these lower levels may have removed the fungistatic control of the cryptococcus , precipitating the perforation . however , after the surgery , the patient was observed on secondary prophylaxis doses of fluconazole , and he did well . to treat a cryptococcoma in the setting of recent initiation of art , where iris versus relapse can not be determined due to the absence of culture results , treatment with a combination of enhanced antifungal therapy and anti - inflammatory therapy", "gi cryptococcosis has been described as a rare occurrence , with only a few published case reports .", "we have discussed an hiv - infected patient with profound immunosuppression and recent cm who developed a gi cryptococcoma during initial immune recovery followed by virological failure after initiation of art .", "the cryptococcoma was not identified and he developed perforation of the ileum , requiring surgery . we anticipate that with the roll out of art in sub - sahara africa , we are bound to see rare presentations of some of common conditions such as cm .", "we recommend that health workers have a high index of suspicion for unusual complications of opportunistic infections in the setting of art - associated iris .", "often treatment of iris requires only observation or anti - inflammatory drugs , but the presence of active infection needs to be excluded ."], ["the authors declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article ."]]}
{"article_id": "PMC4559634", "article_text": ["dermatophytosis is a common clinical entity characterized by the infection of keratinized tissues such as skin , hair , and nails .", "these dermatophytes are closely related filamentous fungi and cause the disease by virtue of their unique ability to degrade keratin and invade the skin and its appendages .", "dermatophytic infections are of major importance , as they are widespread and cause discomfort . reactions to dermatophyte infection may range from mild to severe .", "the mildness and severity depend on a variety of factors such as the host reactions to the metabolic products of the fungus , the virulence of infecting species or particular strain , anatomical location of the infection and local environmental factors .", "dermatophytic infections are a common clinical problem encountered in more than 50% of patients attending the dermatology outpatient departments .", "overcrowding , poor hygiene , low standards of living along with high humidity environments are contributing to the increased prevalence of these fungal infections .", "the present study was conducted to know the prevalence , etiology and common clinical presentations of dermatophytosis involving skin and hair .", "a total of 110 samples which included 101 skin samples and 9 hair samples , from clinically suspected to have dermatophytosis were collected .", "a detailed history regarding age , sex , occupation , social status , duration of complaint and others were taken .", "samples were collected after cleaning the affected surface with 70% alcohol . from skin lesions ,", "scales were collected from erythematous growing margins of the lesion with a sterile blunt scalpel , and in case of tinea capitis , hairs were plucked with sterile surgical forceps .", "samples were collected in sterilized whatman filter paper envelope and transported to the microbiological laboratory .", "direct examination of fungal elements from skin scales and hair samples was done by using 10% and 20% koh mounts respectively .", "all the samples were cultured on sabouraud 's dextrose agar ( sda ) with gentamicin and cycloheximide ( sda with actidione ) and dermatophyte test medium ( dtm ) ( hi - media ) .", "fungal growth was identified by colony morphology ; pigment production and microscopic examination by tease mount technique in lactophenol cotton blue .", "urease test and in - vitro hair perforation tests were also performed to differentiate trichophyton rubrum and trichophyton mentagrophytes when there was difficulty in identification by microscopic and macroscopic examination .", "samples were collected after cleaning the affected surface with 70% alcohol . from skin lesions ,", "scales were collected from erythematous growing margins of the lesion with a sterile blunt scalpel , and in case of tinea capitis , hairs were plucked with sterile surgical forceps .", "samples were collected in sterilized whatman filter paper envelope and transported to the microbiological laboratory .", "direct examination of fungal elements from skin scales and hair samples was done by using 10% and 20% koh mounts respectively .", "all the samples were cultured on sabouraud 's dextrose agar ( sda ) with gentamicin and cycloheximide ( sda with actidione ) and dermatophyte test medium ( dtm ) ( hi - media ) .", "fungal growth was identified by colony morphology ; pigment production and microscopic examination by tease mount technique in lactophenol cotton blue .", "urease test and in - vitro hair perforation tests were also performed to differentiate trichophyton rubrum and trichophyton mentagrophytes when there was difficulty in identification by microscopic and macroscopic examination .", "all the samples were cultured on sabouraud 's dextrose agar ( sda ) with gentamicin and cycloheximide ( sda with actidione ) and dermatophyte test medium ( dtm ) ( hi - media ) .", "fungal growth was identified by colony morphology ; pigment production and microscopic examination by tease mount technique in lactophenol cotton blue .", "urease test and in - vitro hair perforation tests were also performed to differentiate trichophyton rubrum and trichophyton mentagrophytes when there was difficulty in identification by microscopic and macroscopic examination .", "males were more predominant 74 ( 67.27% ) compared to females 36 ( 32.73% ) .", "more number of cases were observed between age groups of 2140 years 71 ( 64.54% ) [ table 1 ] .", "clinical presentations of dermatophytosis observed were tinea corporis 44 ( 40% ) , followed by t. corporis with cruris 28 ( 25.45% ) , tinea cruris 10 ( 9.09% ) , tinea capitis 9 ( 8.19% ) , tinea faciei 7 ( 6.36% ) , tinea manuum 6 ( 5.45% ) , tinea pedis 5 ( 4.55% ) and tinea barbae 1 ( 0.91% ) .", "t. corporis and t. corporis with cruris were more common in the age groups of 2140 years .", "age- and sex - wise distribution of cases fungal elements by koh mount were observed in 64 ( 58.18% ) and culture was positive in 62 ( 56.36% ) .", "of 64 ( 58.18% ) koh positive cases 55 ( 85.9% ) yielded growth in culture . among koh negative 46 ( 41.82% ) cases , 7 ( 15.2% ) were culture positive .", "culture positivity was highest in t. corporis ( 38.71% ) and no culture positivity was noted in tinea barbae and tinea pedis .", "dermatophytic species isolated were t. rubrum 58.06% [ figure 1 ] , t. mentagrophytes 22.58% [ figure 2 ] , epidermophyton floccosum 6.45% [ figure 3 ] , trichophyton violaceum 6.54% [ figure 4 ] , trichophyton tonsurans 3.22% [ figure 5 ] and trichophyton schoenleinii 3.22% [ figure 6 ] . \n", "( c ) lacto phenol cotton blue mount showing tear drop shaped microconidia ( 400 ) .", "( d and e ) lacto phenol cotton blue mount showing pencil shaped macroconidia ( 400 ) \n trichophyton mentagrophytes .", "( d ) lacto phenol cotton blue mount showing nodular organ ( 400 ) \n epidermophyton floccosum .", "( c and d ) lacto phenol cotton blue mount showing club shaped macroconidia ( 400 ) .", "( e ) lacto phenol cotton blue mount showing characteristic chlamydospores ( 400 ) \n trichophyton violaceum .", "( a ) growth on sabouraud 's dextrose agar ( obverse with violet color pigmentation ) .", "( b ) lacto phenol cotton blue mount showing hyphae with chains of chlamydospores ( 400 ) trichophyton tonsurans .", "( d ) lacto phenol cotton blue mount showing match stick like microconidia ( 400 ) .", "( e ) lacto phenol cotton blue mount showing macroconidia ( 400 ) \n trichophyton schoenleinii .", "lacto phenol cotton blue mount showing antler hyphae and chlamydospores ( 400 ) t. rubrum was predominantly recovered in each clinical presentation but t. violaceum was the most common isolate in tinea capitis , followed by t. rubrum , t. tonsurans and t. schoenleinii [ table 2 ] .", "dermatophyte species causing different clinical presentations all culture positives were grown both on sda with actidione and dtm on primary isolation .", "on dtm first appearance of growth was within 10 days of inoculation for most of the isolates that is , 56 ( 90.32% ) [ figure 7 ] .", "but , the appearance of growth was only after 10 days for most of the isolates when grown on sda with actidione 57 ( 91.94% ) . species level identification on primary isolation was possible for 42 ( 67.74% ) of culture positives when grown on sda with actidione .", "but , no culture positive was identified up to species level from dtm on primary isolation .", "( f ) trichophyton violaceum \n  among culture positive cases 19.35% were from rural area , and 80.65% were from urban area and 67.74% were from low socio - economic status , 30.65% were from middle socioeconomic status and 1.61% were from high socioeconomic status . in this study ,", "8.06% of patients were diabetics , 6.45% were anemic , 3.23% of patients had a history of atopy and 1.61% were hiv positive .", "use of occlusive or synthetic fabric dressing regularly was there in 40.32% of patients and 32.25% of patients were in contact with soil regularly because of their occupation .", "all culture positives were grown both on sda with actidione and dtm on primary isolation .", "on dtm first appearance of growth was within 10 days of inoculation for most of the isolates that is , 56 ( 90.32% ) [ figure 7 ] .", "but , the appearance of growth was only after 10 days for most of the isolates when grown on sda with actidione 57 ( 91.94% ) . species level identification on primary isolation was possible for 42 ( 67.74% ) of culture positives when grown on sda with actidione .", "but , no culture positive was identified up to species level from dtm on primary isolation .", "( f ) trichophyton violaceum \n  among culture positive cases 19.35% were from rural area , and 80.65% were from urban area and 67.74% were from low socio - economic status , 30.65% were from middle socioeconomic status and 1.61% were from high socioeconomic status . in this study ,", "8.06% of patients were diabetics , 6.45% were anemic , 3.23% of patients had a history of atopy and 1.61% were hiv positive .", "use of occlusive or synthetic fabric dressing regularly was there in 40.32% of patients and 32.25% of patients were in contact with soil regularly because of their occupation .", "in this study , highest incidence of dermatophytosis was observed in the age group of 2140 years and in males .", "this may be due to greater physical activity and increased sweating in this age group favoring the growth of dermatophytes .", "t. corporis , followed by t. corporis with cruris were common clinical presentations of dermatophytosis in the present study which was in correlation with other studies from india .", "t. capitis was more common in children below the age group of 10 years , which was also observed in some studies .", "t. rubrum was the most common dermatophyte to cause all clinical types of dermatophytoses followed by t. mentagrophytes .", "chronic nature of infection and adaptation to human is mainly responsible for predominance of t. rubrum to cause dermatophytosis .", "t. violaceum was predominant species to cause tinea capitis , followed by t. rubrum , t. tonsurans and t. schoenleinii .", "culture positivity was more in koh positive cases 85.9% compared to koh negative cases 15.2% .", "this difference is statistically significant  = 55.29 ; p < 0.001 , this shows that direct microscopy by koh mount is a good screening test in the laboratory diagnosis of dermatophytosis .", "efficacy of sda with actidione and dtm in isolation of dermatophytes is equal in the present study .", "this was in correlation with the study of singh and beena who reported that 96.55% of culture positives were grown on sda and 98.3% of culture positives were grown on dtm . but", "dermatophytes grow earlier on dtm compared to sda with actidione . in the present study even though all 62 culture positive samples yielded growth both on sda with actidione and dtm on primary isolation , appearance of growth was earlier on dtm that is , within 10 days ( 90.32% ) compared to sda with actidione ( 8.06% ) .", "for species level identification of dermatophytes , sda with actidione is preferable compared to dtm . in the present study ,", "species level identification was possible for 67.74% of isolates on primary isolation on sda with actidione .", "but species level identification was not possible with the growth on dtm on primary isolation , as conidial production was low on dtm , which is required for identification . moreover , it was not possible to observe pigment production on dtm . for identification of growth on dtm ,", "dermatophytic infections were more common in people living in urban areas ( 80.65% ) compared to rural areas ( 19.35% ) .", "this may be because the study included patients attending the outpatient department of dermatology of tertiary care teaching hospital , where most of the people were from the urban area .", "high incidence of dermatophytic infections was observed in the low socioeconomic group of people ( 67.74% ) .", "this is because of unhygienic living conditions , overcrowding , illiteracy and poor nutrition among them .", "daily wage workers ( 35.48% ) and students ( 33.87% ) were predominantly affected compared to others .", "this could be because of increased physical activity and hot environmental conditions which results in increased sweating and excessive moisture favoring the growth of dermatophytes .", "this is in correlation with the study of prasad et al . in the present study", "systemic predisposing factors like diabetes ( 8.06% ) , anemia ( 6.45% ) , atopy ( 3.23% ) , and hiv ( 1.61% ) were observed which are in correlation with other authors . local predisposing factors like occlusive or synthetic fabric dressing in 40.32% and contact with soil regularly because of their occupation in 32.25% were noted .", "dtm is a good screening medium in laboratory diagnosis of dermatophytosis compared to sda with actidione ."], "abstract_text": ["<S> introduction : dermatophytic infections are commonly encountered a problem and constitute more than 50% of cases in dermatology outpatient departments . </S>", "<S> diagnosis of these infections requires the proper use of laboratory methods.objectives:this study was conducted to know the etiology of dermatophytosis in patients attending tertiary care level hospital in south india and to compare the efficacy of sabouraud 's dextrose agar ( sda ) with actidione and dermatophyte test medium ( dtm ) in isolating and identifying dermatophytes.materials and methods : a total of 110 samples which included 101 skin samples and 9 hair samples from clinically suspected dermatophytosis were collected . </S>", "<S> direct microscopy by koh and culture on sda with actidione and dtm were done.results:of 110 samples collected , 58.18% were koh positive for fungal filaments and 56.36% were culture positive for dermatophytes . </S>", "<S> more number of cases were observed between age groups of 2140 years . </S>", "<S> males were more affected compared to females . </S>", "<S> tinea corporis was the common clinical presentation observed ( 40% ) . </S>", "<S> trichophyton rubrum ( 58.06% ) was the predominant isolate recovered in all clinical presentations but trichophyton violaceum was the most common isolate in tinea capitis . </S>", "<S> all culture positives were grown on both sda with actidione and dtm . </S>", "<S> appearance of growth was earlier on dtm that is , within 10 days compared to sda with actidione where growth started appearing only after 10 days . </S>", "<S> this is statistically significant p < 0.0001 ( 2 = 71.6 ) . </S>", "<S> species level identification on primary isolation was possible when grown on sda with actidione and it was not possible with the growth on dtm on primary isolation.conclusion:dtm is a good screening medium in laboratory diagnosis of dermatophytosis when compared to sda with actidione . </S>", "<S> but dtm is inferior to sda with actidione in identification of dermatophyte species . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Collection of samples", "Direct microscopic examination", "Isolation by culture", "RESULTS", "Comparison of growth pattern on Sabouraud's dextrose agar with actidione and dermatophyte test medium", "DISCUSSION", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["dermatophytosis is a common clinical entity characterized by the infection of keratinized tissues such as skin , hair , and nails .", "these dermatophytes are closely related filamentous fungi and cause the disease by virtue of their unique ability to degrade keratin and invade the skin and its appendages .", "dermatophytic infections are of major importance , as they are widespread and cause discomfort . reactions to dermatophyte infection may range from mild to severe .", "the mildness and severity depend on a variety of factors such as the host reactions to the metabolic products of the fungus , the virulence of infecting species or particular strain , anatomical location of the infection and local environmental factors .", "dermatophytic infections are a common clinical problem encountered in more than 50% of patients attending the dermatology outpatient departments .", "overcrowding , poor hygiene , low standards of living along with high humidity environments are contributing to the increased prevalence of these fungal infections .", "the present study was conducted to know the prevalence , etiology and common clinical presentations of dermatophytosis involving skin and hair ."], ["a total of 110 samples which included 101 skin samples and 9 hair samples , from clinically suspected to have dermatophytosis were collected .", "a detailed history regarding age , sex , occupation , social status , duration of complaint and others were taken .", "samples were collected after cleaning the affected surface with 70% alcohol . from skin lesions ,", "scales were collected from erythematous growing margins of the lesion with a sterile blunt scalpel , and in case of tinea capitis , hairs were plucked with sterile surgical forceps .", "samples were collected in sterilized whatman filter paper envelope and transported to the microbiological laboratory .", "direct examination of fungal elements from skin scales and hair samples was done by using 10% and 20% koh mounts respectively .", "all the samples were cultured on sabouraud 's dextrose agar ( sda ) with gentamicin and cycloheximide ( sda with actidione ) and dermatophyte test medium ( dtm ) ( hi - media ) .", "fungal growth was identified by colony morphology ; pigment production and microscopic examination by tease mount technique in lactophenol cotton blue .", "urease test and in - vitro hair perforation tests were also performed to differentiate trichophyton rubrum and trichophyton mentagrophytes when there was difficulty in identification by microscopic and macroscopic examination ."], ["samples were collected after cleaning the affected surface with 70% alcohol . from skin lesions ,", "scales were collected from erythematous growing margins of the lesion with a sterile blunt scalpel , and in case of tinea capitis , hairs were plucked with sterile surgical forceps .", "samples were collected in sterilized whatman filter paper envelope and transported to the microbiological laboratory ."], ["direct examination of fungal elements from skin scales and hair samples was done by using 10% and 20% koh mounts respectively .", "all the samples were cultured on sabouraud 's dextrose agar ( sda ) with gentamicin and cycloheximide ( sda with actidione ) and dermatophyte test medium ( dtm ) ( hi - media ) .", "fungal growth was identified by colony morphology ; pigment production and microscopic examination by tease mount technique in lactophenol cotton blue .", "urease test and in - vitro hair perforation tests were also performed to differentiate trichophyton rubrum and trichophyton mentagrophytes when there was difficulty in identification by microscopic and macroscopic examination ."], ["all the samples were cultured on sabouraud 's dextrose agar ( sda ) with gentamicin and cycloheximide ( sda with actidione ) and dermatophyte test medium ( dtm ) ( hi - media ) .", "fungal growth was identified by colony morphology ; pigment production and microscopic examination by tease mount technique in lactophenol cotton blue .", "urease test and in - vitro hair perforation tests were also performed to differentiate trichophyton rubrum and trichophyton mentagrophytes when there was difficulty in identification by microscopic and macroscopic examination ."], ["males were more predominant 74 ( 67.27% ) compared to females 36 ( 32.73% ) .", "more number of cases were observed between age groups of 2140 years 71 ( 64.54% ) [ table 1 ] .", "clinical presentations of dermatophytosis observed were tinea corporis 44 ( 40% ) , followed by t. corporis with cruris 28 ( 25.45% ) , tinea cruris 10 ( 9.09% ) , tinea capitis 9 ( 8.19% ) , tinea faciei 7 ( 6.36% ) , tinea manuum 6 ( 5.45% ) , tinea pedis 5 ( 4.55% ) and tinea barbae 1 ( 0.91% ) .", "t. corporis and t. corporis with cruris were more common in the age groups of 2140 years .", "age- and sex - wise distribution of cases fungal elements by koh mount were observed in 64 ( 58.18% ) and culture was positive in 62 ( 56.36% ) .", "of 64 ( 58.18% ) koh positive cases 55 ( 85.9% ) yielded growth in culture . among koh negative 46 ( 41.82% ) cases , 7 ( 15.2% ) were culture positive .", "culture positivity was highest in t. corporis ( 38.71% ) and no culture positivity was noted in tinea barbae and tinea pedis .", "dermatophytic species isolated were t. rubrum 58.06% [ figure 1 ] , t. mentagrophytes 22.58% [ figure 2 ] , epidermophyton floccosum 6.45% [ figure 3 ] , trichophyton violaceum 6.54% [ figure 4 ] , trichophyton tonsurans 3.22% [ figure 5 ] and trichophyton schoenleinii 3.22% [ figure 6 ] . \n", "( c ) lacto phenol cotton blue mount showing tear drop shaped microconidia ( 400 ) .", "( d and e ) lacto phenol cotton blue mount showing pencil shaped macroconidia ( 400 ) \n trichophyton mentagrophytes .", "( d ) lacto phenol cotton blue mount showing nodular organ ( 400 ) \n epidermophyton floccosum .", "( c and d ) lacto phenol cotton blue mount showing club shaped macroconidia ( 400 ) .", "( e ) lacto phenol cotton blue mount showing characteristic chlamydospores ( 400 ) \n trichophyton violaceum .", "( a ) growth on sabouraud 's dextrose agar ( obverse with violet color pigmentation ) .", "( b ) lacto phenol cotton blue mount showing hyphae with chains of chlamydospores ( 400 ) trichophyton tonsurans .", "( d ) lacto phenol cotton blue mount showing match stick like microconidia ( 400 ) .", "( e ) lacto phenol cotton blue mount showing macroconidia ( 400 ) \n trichophyton schoenleinii .", "lacto phenol cotton blue mount showing antler hyphae and chlamydospores ( 400 ) t. rubrum was predominantly recovered in each clinical presentation but t. violaceum was the most common isolate in tinea capitis , followed by t. rubrum , t. tonsurans and t. schoenleinii [ table 2 ] .", "dermatophyte species causing different clinical presentations all culture positives were grown both on sda with actidione and dtm on primary isolation .", "on dtm first appearance of growth was within 10 days of inoculation for most of the isolates that is , 56 ( 90.32% ) [ figure 7 ] .", "but , the appearance of growth was only after 10 days for most of the isolates when grown on sda with actidione 57 ( 91.94% ) . species level identification on primary isolation was possible for 42 ( 67.74% ) of culture positives when grown on sda with actidione .", "but , no culture positive was identified up to species level from dtm on primary isolation .", "( f ) trichophyton violaceum \n  among culture positive cases 19.35% were from rural area , and 80.65% were from urban area and 67.74% were from low socio - economic status , 30.65% were from middle socioeconomic status and 1.61% were from high socioeconomic status . in this study ,", "8.06% of patients were diabetics , 6.45% were anemic , 3.23% of patients had a history of atopy and 1.61% were hiv positive .", "use of occlusive or synthetic fabric dressing regularly was there in 40.32% of patients and 32.25% of patients were in contact with soil regularly because of their occupation ."], ["all culture positives were grown both on sda with actidione and dtm on primary isolation .", "on dtm first appearance of growth was within 10 days of inoculation for most of the isolates that is , 56 ( 90.32% ) [ figure 7 ] .", "but , the appearance of growth was only after 10 days for most of the isolates when grown on sda with actidione 57 ( 91.94% ) . species level identification on primary isolation was possible for 42 ( 67.74% ) of culture positives when grown on sda with actidione .", "but , no culture positive was identified up to species level from dtm on primary isolation .", "( f ) trichophyton violaceum \n  among culture positive cases 19.35% were from rural area , and 80.65% were from urban area and 67.74% were from low socio - economic status , 30.65% were from middle socioeconomic status and 1.61% were from high socioeconomic status . in this study ,", "8.06% of patients were diabetics , 6.45% were anemic , 3.23% of patients had a history of atopy and 1.61% were hiv positive .", "use of occlusive or synthetic fabric dressing regularly was there in 40.32% of patients and 32.25% of patients were in contact with soil regularly because of their occupation ."], ["in this study , highest incidence of dermatophytosis was observed in the age group of 2140 years and in males .", "this may be due to greater physical activity and increased sweating in this age group favoring the growth of dermatophytes .", "t. corporis , followed by t. corporis with cruris were common clinical presentations of dermatophytosis in the present study which was in correlation with other studies from india .", "t. capitis was more common in children below the age group of 10 years , which was also observed in some studies .", "t. rubrum was the most common dermatophyte to cause all clinical types of dermatophytoses followed by t. mentagrophytes .", "chronic nature of infection and adaptation to human is mainly responsible for predominance of t. rubrum to cause dermatophytosis .", "t. violaceum was predominant species to cause tinea capitis , followed by t. rubrum , t. tonsurans and t. schoenleinii .", "culture positivity was more in koh positive cases 85.9% compared to koh negative cases 15.2% .", "this difference is statistically significant  = 55.29 ; p < 0.001 , this shows that direct microscopy by koh mount is a good screening test in the laboratory diagnosis of dermatophytosis .", "efficacy of sda with actidione and dtm in isolation of dermatophytes is equal in the present study .", "this was in correlation with the study of singh and beena who reported that 96.55% of culture positives were grown on sda and 98.3% of culture positives were grown on dtm . but", "dermatophytes grow earlier on dtm compared to sda with actidione . in the present study even though all 62 culture positive samples yielded growth both on sda with actidione and dtm on primary isolation , appearance of growth was earlier on dtm that is , within 10 days ( 90.32% ) compared to sda with actidione ( 8.06% ) .", "for species level identification of dermatophytes , sda with actidione is preferable compared to dtm . in the present study ,", "species level identification was possible for 67.74% of isolates on primary isolation on sda with actidione .", "but species level identification was not possible with the growth on dtm on primary isolation , as conidial production was low on dtm , which is required for identification . moreover , it was not possible to observe pigment production on dtm . for identification of growth on dtm ,", "dermatophytic infections were more common in people living in urban areas ( 80.65% ) compared to rural areas ( 19.35% ) .", "this may be because the study included patients attending the outpatient department of dermatology of tertiary care teaching hospital , where most of the people were from the urban area .", "high incidence of dermatophytic infections was observed in the low socioeconomic group of people ( 67.74% ) .", "this is because of unhygienic living conditions , overcrowding , illiteracy and poor nutrition among them .", "daily wage workers ( 35.48% ) and students ( 33.87% ) were predominantly affected compared to others .", "this could be because of increased physical activity and hot environmental conditions which results in increased sweating and excessive moisture favoring the growth of dermatophytes .", "this is in correlation with the study of prasad et al . in the present study", "systemic predisposing factors like diabetes ( 8.06% ) , anemia ( 6.45% ) , atopy ( 3.23% ) , and hiv ( 1.61% ) were observed which are in correlation with other authors . local predisposing factors like occlusive or synthetic fabric dressing in 40.32% and contact with soil regularly because of their occupation in 32.25% were noted ."], ["dtm is a good screening medium in laboratory diagnosis of dermatophytosis compared to sda with actidione ."], [""], [""]]}
{"article_id": "PMC4227712", "article_text": ["gemcitabine is a modified \n cytidine analog having two fluorine atoms \n at the 2-position in the deoxyribose sugar moiety ( scheme 1 ) . for nearly 20 years , it has been widely used \n to treat specifically pancreatic cancer .", "it has been proposed that gemcitabine inhibits \n ribonucleotide reductase ( rnr ) activity as well as acting as \n a replication stop , thereby affecting dna synthesis \n and elongation .", "the two highly electronegative \n f - atoms at c2 substantially increase the acidity of h3 \n through the inductive effect .", "it is well established \n that in the absence of oxygen , thiyl radicals \n are able to abstract hydrogen ( h ) atoms to form neutral c - centered \n radicals .", "for example , h - atom abstraction by thiyl radicals has been \n shown to induce isomerization of cis-2,5-dimethyltetrahydrofuran \n to the trans-2,5-dimethyltetrahydrofuran . on this basis , stubbe et al .", ", in their enzymatic and electron \n spin resonance ( esr ) studies with gemcitabine , have proposed that \n inhibition of the rnr activity by gemcitabine should occur via radical \n formation at the c3 site by direct h - atom abstraction to produce \n a c3 via a enzymatic thiyl radical ( reaction 1 ) .", "subsequently , the c3 intermediate \n has been proposed to form 3-keto c2 via hf \n loss .", "this c2 is stabilized by an oxy radical resonance \n contribution ( reaction 2 ) .", "c2 \n and its immediate precursor c3 ( reactions 1 and 2 ) play a key role in \n the rnr inactivation.12 the h - atom abstraction reaction ( reaction 1 ) is not only important in rnr activation but also \n plays a very key \n role toward stable product formation in other biologically damage \n processes in dna , such as oxidative intrastrand cross - link formation and in the formation of sugar radicals that \n are strand break precursors .", "it is noteworthy that pulse radiolysis \n experiments with a 1,4-anhydro-5-deoxy-6-thio - d - ribo - hexofuranitol \n detected the formation of ribosyl - based carbon - centered radical(s ) \n after h - atom abstraction by thiyl radicals .", "these studies are supportive of reactions 1 and 2 but unequivocal , and direct detection \n of c3 and c2 employing esr or pulse \n radiolysis during rnr - catalyzed deoxygenation of the natural substrates or during inactivation by gemcitabine still remains elusive . in this work , we report the formation of c2 from \n a likely c3 in a gemcitabine analog which mimics the \n mechanism proposed above . from the structural formula of gemcitabine \n ( scheme 1 )", ", it is expected that the negative \n inductive effect ( i ) of two highly electronegative f - atoms \n at c2 should increase the acidity of h3. from our \n previous work on nucleoside cation radicals , the gemcitabine cation radical formed upon one - electron oxidation \n is expected to produce c3 after deprotonation of the \n acidic proton h3. in this work , esr spectroscopy has been \n employed to investigate one - electron oxidation of gemcitabine and \n other 2-modified derivatives , for example , 2-deoxy-2-fluoro-2-c - methylcytidine ( mefdc ( psi-6130 ) ; scheme 2 ) and 2-fluoro-2-deoxycytidine ( 2-fdc , scheme 2 ) , in order to test the influence of 2- \n substituent on radical site formation .", "it is noteworthy that mefdc \n is a well - known clinically efficacious inhibitor of hepatitis c virus .", "the esr results clearly identify the c3 formation \n in one - electron oxidized gemcitabine and the production of c2 \n in one - electron oxidized mefdc .", "these calculations show \n that in the case of one - electron oxidized mefdc , the lowest energy \n path is the rapid formation of c2 from c3 \n via f loss .", "this f loss is a \n barrierless reaction between the 2-f - atom and the proximate \n h3o which was formed via deprotonation of h3 \n in the cation radical .", "gemcitabine ( scheme 1 ) and 2-fdc \n ( scheme 2 ) were obtained \n from carbosynth ltd .", "mefdc ( scheme 2 ) was prepared as described or \n purchased from adooq bioscience ( irvine , ca ) .", "lithium chloride ( licl ) ( ultra \n dry , 99.995% ( metals basis ) ) was \n obtained from alfa aesar ( ward hill , ma , usa ) .", "2-deoxycytidine \n ( 2-dc ) was obtained from sigma chemical company ( st louis , \n mo , usa ) .", "deuterium oxide ( d2o ) ( 99.9 atom % d ) was purchased \n from aldrich chemical co. inc .", "( paris , ky , usa ) . cytidine-5,6-d2 ( [ 5,6-d , d]-cyd , 99 atom % d ) was purchased from cdn isotopes ( quebec , \n canada ) .", "homogeneous solutions \n of gemcitabine were prepared by dissolving 210 mg / ml either \n in 7.5 m licl in d2o or in h2o .", "solutions of \n other compounds ( 2-dc , 2-f - dc , mefdc , and [ 5,6-d , d]-cyd ) \n were prepared by dissolving ca .", "k2s2o8 ( 616 \n mg / ml ) was added as an electron scavenger so that only the formation \n of the one - electron oxidized species and its subsequent reactions \n can be followed by employing esr spectroscopy .", "the above - mentioned \n procedure for preparation of solutions is according to our ongoing \n studies on various model systems of dna and rna .", "the ph of gemcitabine in 7.5 m licl / d2o was adjusted to the range of ca .", "the ph of gemcitabine in 7.5 m licl / h2o \n and the ph of other compounds ( 2-dc ,", "2-f - dc , mefdc , \n and [ 5,6-d , d]-cyd ) in 7.5 m licl / d2o was adjusted at ph \n ca . 10 .", "these ph adjustments were performed by adding l amounts \n 1 m naoh as per our previous efforts .", "these solutions have high ionic strength ( 7.5 m licl ) ; therefore , \n the ph meters would not provide accurate ph measurements of these \n solutions . instead , as per our previous works ,", "ph values reported in this work were obtained using ph papers and \n are approximate measurements . as \n per our previous works , these ph - adjusted homogeneous solutions were thoroughly bubbled \n with nitrogen to remove the dissolved oxygen . immediately , these solutions \n were drawn into 4 mm suprasil quartz tubes ( catalog no .", "buena , nj , usa ) and were rapidly cooled in \n liquid nitrogen ( 77 k ) .", "the rapid cooling of these homogeneous liquid \n solutions at 77 k leads to the formation of transparent homogeneous \n glassy solutions .", "these glassy solutions were later used for the irradiation \n and subsequent progressive annealing experiments .", "all glassy samples \n were stored at 77 k in teflon containers in the dark .", "all samples \n were  ( co)-irradiated ( absorbed \n dose = 1.4 kgy ) at 77 k and stored at 77 k in teflon containers in \n dark following our previous efforts .", "a variable - temperature \n assembly was employed which passed liquid nitrogen cooled nitrogen \n gas past a thermister and over the sample as described in our earlier \n studies .", "the glassy samples have been \n annealed anywhere from ( 140170 ) k for 15 min .", "annealing leads \n to one - electron oxidation of the solute by the matrix radical cl2 thus , forming only the cation \n radical of the solute , e.g. , gemcitabine .", "following our earlier studies , immediately after -irradiation of the glassy sample at 77 \n k , the esr spectrum was recorded at 77 k. also , immediately after \n each annealing step , the sample was cooled to 77 k by immersing in \n liquid nitrogen ( 77 k ) , and the esr spectrum was recorded at 77 k \n which maximizes signal height and allows for comparison of signal \n intensities . a varian century series x - band ( 9.3 ghz ) esr spectrometer \n with an e-4531 dual cavity , 9 in .", "magnet , and a 200 mw klystron was \n used , and fremy s salt ( gcenter = 2.0056 , a(n ) = 13.09 g ) was employed for the \n field calibration .", "all esr spectra have been recorded at 77 k and \n at 40 db ( 20  w ) .", "anisotropic simulations of esr spectra \n have been performed using the win - epr and simfonia programs of bruker \n as per our previous works .", "the simulated spectra thus obtained \n were compared to experimental spectra , and esr parameters were adjusted \n for the best fit ( also supporting \n information figure s3 ) .", "gaussview and jmol programs were used to plot the spin densities \n and molecular structures .", "the geometries of all the radicals considered \n in the present study were fully optimized using the b97x functional and 6 - 31g(d ) basis set .", "we note here that b97x \n functional was developed by the group of head  gordon and found \n to be very successful for the calculations of various properties of \n molecules in their different spin states .", "the hyperfine coupling constants ( hfccs ) of the radicals were calculated \n using the same method and basis set , i.e. , b97x/6 - 31g(d ) in \n the gas phase . in order to treat the effect of solvent on hf loss \n from the c3 in gemcitabine and in mefdc , we employ \n the integral equation formalism polarized continuum model ( ief - pcm ) as implemented in gaussian 09 . in \n addition to pcm , for c3 in both systems", "a h3o is placed in the vicinity of the c3-oh bond \n for c3 in gemcitabine and for c3 in \n mefdc and have optimized the structures .", "the electronic energy profile \n of f dissociation from c2 site of c3 \n in mefdc as well as the electronic energy profile of f dissociation for each of the two f - atoms from c2 site of \n c3 in gemcitabine were obtained in the presence of \n a single water molecule at the same level of theory ( supporting information figure s4c , d ) .", "furthermore , employing \n the wb97x/6 - 31++g(d , p ) method along with the ief - pcm model for the \n solvent effect , the pka of the c3-oh \n group for the c3 of gemcitabine and also of the c3-oh \n group for the c3 of 2-dc was calculated .", "in figure 1a , we show the experimentally recorded ( 77 k ) esr spectrum \n ( green ) of one - electron oxidized gemcitabine at ph ( pd ) ca . 7 in a \n homogeneous glassy 7.5 m licl / d2o solution .", "the one - electron \n oxidation of gemcitabine was induced by cl2 attack after annealing at 155 k in the dark .", "912 showed identical spectra after \n one - electron oxidation of gemcitabine by cl2 on annealing at 150155 k. thus , only the spectrum \n obtained from the gemcitabine sample at pd ca .", "10 is presented in \n figure 1b along with the simulated spectrum \n in blue .", "it is evident from figure 1a , b , the \n line shape , total hyperfine splitting , and the center of the simulated \n spectra match with those of the experimentally recorded spectra quite \n well .", "each of the spectra in figure 1a , b show \n two anisotropic -f - atom hyperfine couplings and a -h - atom \n hyperfine coupling .", "the -h - atom hyperfine coupling creates \n the doublet splitting in the line components in figure 1a , b .", "figure 1a is best matched \n with a simulation \n employing the two different anisotropic -f - atom ( nuclear spin \n = 1/2 ) hfcc values of ( 15.0 , 15.0 , 105 ) g and ( 15.0 , 15.0 , 69.0 ) g , \n one -h hfcc as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0080 , 2.0050 , 2.0020 ) along with a mixed ( lorentzian / gaussian \n ( 1:1 ) ) line - width of 14 g. the simulated spectrum in blue is superimposed \n on the experimentally recorded spectrum in figure 1a . on the other hand , the best fit for figure 1b", "is obtained employing the two identical anisotropic -f - atom \n ( nuclear spin = 1/2 ) hfcc as ( 17.0 , 17.0 , 86.0 ) g , one -h hfcc \n as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0060 , 2.0050 , \n 2.0020 ) along with a mixed ( lorentzian / gaussian ( 1:1 ) ) line - width \n of 10 g. following our work on the radicals produced in monomers \n of dna \n and rna , the a ( i.e. , \n the azz ) component of each of the two \n anisotropic -f - atoms ( see table 1 ) as \n well as the a of the -h are directly measured \n from the width of the experimentally recorded spectra with an uncertainty \n of 2 g ( see supporting information figure s3 ) . on the other hand ,", "the theoretically obtained values \n of axx and ayy components of each of the two anisotropic -f - atoms and of the \n -h - atom in table 1 were adjusted to \n fit the experimentally recorded spectra with estimated uncertainty \n of 4 g ( see supporting information figure s3 ) .", "7 show two nonequivalent anisotropic -f - atom hfccs , whereas , \n the one - electron oxidized gemcitabine spectrum at ph ca .", "the -h - atom \n hfcc does not show any observable change in the one - electron oxidized \n gemcitabine spectrum throughout the ph range ca .", "the coupling to two -f - atoms ( c2 ) and one -h - atom \n ( c4 ) is clear evidence for the generation of c3 \n after one electron oxidation of gemcitabine at 150155 k. the \n electron - withdrawing effect of the two electronegative f - atoms at \n c2 increases the acidity of h3 , which leads to deprotonation \n ( see supporting information table t2 ) and \n prevents observation of the initially formed cytosine base -cation \n radical ( c ) in gemcitabine as indicated in reaction 3 .", "therefore , the mechanism of c3 \n formation due to one - electron oxidation of gemcitabine is proposed \n as follows : one - electron oxidation of gemcitabine results in the formation \n of metastable c , which is unstable even at ca . \n", "155 k. the metastable c quickly deprotonates at \n c3 in the sugar moiety producing c3 ( reaction 3 ) via a proton - coupled electron - transfer ( pcet ) mechanism.3 esr \n spectra obtained from matched gemcitabine samples [ concentration \n of gemcitabine in each sample = 2 mg / ml in 7.5 m licl / d2o ] in the presence of the electron scavenger k2s2o8 ( 8 mg / ml in each sample ) .", "each sample has been -irradiated \n ( absorbed dose = 1.4 kgy at 77 k ) , subsequently annealed to 155 k \n for 15 min in the dark at various phs ( a ) ph ca .", "7 ( green ) and ( b ) \n ph ranging ca . 912 ( pink ) . here", "the blue spectra that are superimposed on the experimentally \n recorded spectra are the simulated spectra of c3. \n see text for the details of simulation .", "all esr spectra are recorded \n at 77 k. the three reference markers ( open triangles ) in this figure \n and in the subsequent figures show the position of fremy s \n salt resonance with the central marker at g = 2.0056 .", "the spacing \n separating the markers is 13.09 g. as is evident from the hfcc \n values of the spectra shown in figure 1 and \n also in table 1 , the ph of the solution clearly \n affects the individual -f - atom anisotropic hfcc but not the \n sum of the two -f - atom anisotropic hfcc along with the c4 \n -proton hfcc ( see section above ) . at ph ca .", "912 , the two -f - atom anisotropic hfccs \n are equivalent ; whereas , at ph ca . 7 ,", "the sum of the two -f - atom \n anisotropic hfccs remain the same , but they individually differ .", "the presence of two 2-f - atoms in gemcitabine will lower the \n pka value of both h3 and c3-oh \n hydrogens .", "for example , the oh in 2,2-difluoroethanol has its pka lowered by 3.5 units in comparison with ethanol .", "further , radical formation has also been shown \n to lower the pka of the alcoholic oh group \n ( e.g. , pka ( ch3)2choh = 17.1 , pka ( ch3)2coh = 12.03 ) . based on \n these factors , the pka value of the c3-oh \n group for c3 in gemcitabine is estimated to be in \n the range of 79 .", "employing dft/b97x/6 - 31++g(d , p ) method \n along with the ief - pcm model for the solvent effect , the pka value of the c3-oh group for c3 \n in 2-dc has been calculated as 14.2 .", "the same level of calculation \n for the c3-oh group of c3 in gemcitabine which \n replaces each of the two hydrogens at c2 with fluorine predicts \n a pka of 6.8 ( see supporting information pages s8 , s9 ) .", "therefore , the two 2-f - atoms \n are predicted to lower the pka of the \n c3-oh group in c3 by 7 full units .", "considering \n the sensitivity of the calculations for predicting pka to the small changes in free energy , these results are very reasonable .", "furthermore , the theoretically \n calculated pka value 6.8 of the c3-oh \n group for c3 in gemcitabine is in good agreement with \n its experimentally estimated value ( 79 ) . the spectrum \n in figure 1a ( ph ca .", "7 ) should \n therefore be for c3 in gemcitabine with a c3-oh \n group and the spectrum in figure 1b ( ph ca . \n", "912 ) should be for c3 with the deprotonated \n group , i.e. , c3-o ( reaction 4 ) .", "thus , we attribute the variation of the two -f - atom \n anisotropic hfcc to the deprotonation of the c3-oh group at \n higher phs.4 esr spectral studies \n of one - electron oxidation of gemcitabine in h2o glasses \n ( 7.5 m licl / h2o ) were performed and compared with the results \n found in d2o glasses ( 7.5 m licl / d2o ) .", "no observable difference in spectra other than a small \n line broadening was observed on formation of c3 in \n h2o glasses versus in d2o glasses .", "since c3 \n only has the c3-oh as an exchangeable proton at ph 7 and this \n does not contribute to a significant hyperfine coupling , the esr spectrum \n found on formation of c3 is not altered by a change \n of the solvent from d2o to h2o .", "similar experiments to those performed for gemcitabine \n were carried out for the methyl / fluoro analog mefdc ( scheme 2 ) .", "using mefdc , we investigated whether the formation \n of c3 observed in gemcitabine bearing geminal difluoro unit at c2 ( figure 1 ) is \n affected by the substitution of one of the f - atoms ( i ) with \n a methyl ( me ) group ( + i ) .", "( a ) esr spectrum ( black ) obtained from mefdc [ concentration \n = 2 \n mg / ml in 7.5 m licl / d2o ] in the presence of the electron \n scavenger k2s2o8 ( 8 mg / ml ) , ph ca . \n", "10 , -irradiated to a dose of 1.4 kgy at 77 k and subsequently \n annealed to 155 k for 15 min .", "( c ) \n spectrum ( black ) obtained after subtraction of 60% of spectrum ( b ) \n from spectrum ( a ) .", "for comparison , the c spectrum \n ( blue ) in 2-dc ( supporting information figure s2 and pp s3s5 ) is superimposed .", "( d ) spectrum ( black ) \n assigned to c2 is obtained after subtraction of 50% \n of spectrum ( c ) from spectrum ( a ) .", "the simulated c2 \n spectrum ( for simulation parameters see figure 2 and text ) is superimposed \n on the experimentally isolated spectrum for comparison .", "all the spectra \n are recorded at 77 k. shown in figure 2a is the esr spectrum \n ( black ) \n of a matched sample of mefdc that has been -irradiated ( absorbed \n dose = 1.4 kgy ) , subsequently annealed to 155 k for 15 min in the \n dark , and recorded at 77 k. figure 2b \n was obtained by annealing this \n sample for 15 min to 170 k. comparison of spectrum 2a with spectrum \n 2b shows clearly that a central doublet decreases along with an increase \n of the other line components upon annealing .", "therefore , the central \n doublet ( black ) shown in figure 2c is isolated \n by subtraction of 60% of the spectrum 2b from \n spectrum 2a .", "the doublet due to c spectrum ( blue ) in 2-dc ( see supporting information figure s2 and its discussion ( pp s3s5 ) ) \n is superimposed on it for comparison . from the spectral similarities \n of both doublets , the doublet in black shown in figure 2c", "subtraction \n of 50% c spectrum 2c ( black ) from \n spectrum 2a results in the black spectrum shown in figure 2d .", "this overall quintet spectrum arises from 4 isotropic \n -proton couplings : three methyl -protons ( ca .", "25.5 g due to a -proton \n assigned to the c1-h ( vide infra ) .", "the experimental ( black ) \n spectrum is simulated using the above - mentioned hfcc values along \n with a 10 g line - width and g - value = 2.0033 ( this g - value is typical for c - centered sugar radicals ) . the simulated spectrum ( red ) in figure 2d \n matches the overall line components of the experimental spectrum well . \n", "since the c2 ( reaction 5 ) is \n the only likely radical structure that would explain the large hyperfine \n coupling to a methyl group and the additional -proton hyperfine \n coupling ( assigned to c1 ) , the experimental spectrum in figure 2d has been assigned to c2 ( reaction 5 ) . the spectra 2a and 2b are a composite of c ( black , \n figure 2c ) and c2 ( black , figure 2d ) in different amounts . under the same constant \n gain and constant microwave power and upon gradual and stepwise annealing \n of the sample from 155 k ( spectrum 2a ) to 170 k ( spectrum 2b ) ,", "no \n loss of spectral intensity was observed , and our analyses show an \n additional ( ca .", "20% ) conversion of the c to c2. consideration of the results presented in figures 1 and 2 suggest that for mefdc , c is produced first and on annealing converts to a transient \n c3 , which is not observed .", "in contrast , the line shape , \n line width , and the overall hyperfine splitting of the c3 \n spectrum in gemcitabine do not change upon annealing to ca .", "thus , unlike \n the rapid conversion of c3 to c2 found \n in one - electron oxidized mefdc , a similar conversion of c3 \n to c2 is not observed for one - electron oxidized gemcitabine \n at these low temperatures .", "the lack of observation of the transient \n c3 in one - electron oxidized mefdc and the low temperatures \n employed in these experiments implies a very low activation barrier \n for the conversion of c3 to c2 ; whereas , \n the activation barrier for the conversion of c3 to \n c2 for one - electron oxidized gemcitabine should be \n 4 kcal / mol .", "calculations suggest that the proximity of the \n lost h3 proton as h3o to the 2-f - atom \n quite likely provides the driving force for this rapid unimolecular \n reaction ( see figure 3 ) .", "therefore , we propose \n that c3 in one - electron oxidized mefdc readily converts \n to c2 via a barrierless f loss \n ( see reaction 5 , figure 3 , and supporting information s5 ) . the b97x/6 - 31g(d ) \n", "calculated hfccs of c3 and c2 found \n in gemcitabine and in mefdc along with experimental hfccs ( in gauss ) \n are presented in table 1 .", "it is evident from \n table 1 that experimental and theoretically \n calculated hfccs are in reasonably good agreement .", "estimated errors are of 2 g for \n azz and 4 g for axx and also for ayy .", "calculated in the presence \n of one \n water molecule only isotropic \n hfcc values have \n been considered . to explore the reaction mechanism of c2 \n formation from c3 in one - electron oxidized mefdc and \n gemcitabine , using the dft b97x/6 - 31g(d ) method", ", we considered \n three possible reaction paths : ( i ) hf loss due to deprotonation of \n 3-hydroxyl group , ( ii ) hf loss due to fluorine dissociation , and additionally , ( iii ) hf loss in the presence of a hydronium ion \n ( h3o ) .", "the electronic energy profile \n of hf loss from c3 in mefdc and also from c3 \n in gemcitabine in the presence of a water molecule is shown in figure \n s4 in the supporting information . from supporting information figure s4a ,", "it is evident \n that for c3 of mefdc , the formation of c2 \n via hf loss with deprotonation of 3-oh has a significant barrier \n of ca . 18 kcal / mol .", "for c3 in gemcitabine , the hf \n loss associated with deprotonation of 3-hydroxyl group was \n calculated to be ca .", "we have also considered the dissociation of \n fluorine in c3 \n of mefdc and in c3 of gemcitabine and found that stretching \n the c2-f bond up to 1.8  needs ca .", "this shows that dissociation of fluorine for c3 in \n mefdc occurs at lower energy than dissociation of fluorine for c3 \n in gemcitabine ( supporting information figure \n s4 ) .", "alternatively , we consider the fact that deprotonation \n of h3 \n will form h3o initially in close proximity \n of the c2-c3 bond which may then induce hf loss ( reaction 5 ) . employing the b97x/6 - 31g(d ) method and \n considering the full solvent effect through the polarized continuum \n model ( pcm ) , this mechanism have been modeled by placing a h3o in the vicinity of the c3-oh bond for c3 \n in gemcitabine and for c3 in mefdc and have optimized \n the structures . from our calculations , we have observed that for c3 \n in gemcitabine , a minimum structure exists in the electronic energy \n profile in which the h3o stabilizes the f-atom \n through forming a hydrogen bond ( 1.57  ) ( see figure 3a and supporting information figure s4c ) .", "a second minimum structure was also found in the electronic \n energy profile in which the h3o stabilizes \n the f-atom through forming a hydrogen bond of identical length , \n 1.57  ( see supporting information figure s4d ) .", "a barrier of 5 kcal / mol and the overall reaction energy \n of 7 to 8 kcal / mol were found for hf formation in \n both cases ( see figure 3a and supporting information figures s4c , d ) .", "however , for c3 \n in mefdc , the h3o reacts with the 2-f - atom \n without a barrier and forms hf and c2 ( see figure 3b ) .", "the bond distances of c3-o3 \n and o3-h bonds for c3 in gemcitabine are calculated \n as 1.34  ( primarily c  o single bond character ) and 0.97  , respectively .", "the values of \n the corresponding bond distances of c3-o3 and o3-h \n bonds for c3 in mefdc are obtained as 1.28  \n ( mainly double bond character and 1.0 \n  respectively ) .", "thus , these calculations show that the \n hf loss from c3 \n in gemcitabine has a ca .", "5 kcal / mol barrier ( supporting \n information figure s4c , d ) while for c3 in mefdc , \n the loss of hf is barrierless , and the c2 production \n is exothermic in nature as shown in figure 3 .", "these findings support our experimental observations that in mefdc , \n c3 is too unstable to be observed , and only c2 \n is found .", "in contrast , in gemcitabine only c3 formation \n is observed without any conversion to c2 in the same \n temperature range .", "pcm-b97x/6 - 31g(d ) calculated structures of c3 \n in ( a ) gemcitabine and in ( b ) mefdc .", "as indicated in ( a ) the c3 \n in gemcitabine involves a barrier of 5 kcal / mol ( detailed electronic \n energy profiles are provided in supporting information figure s4c , d ) to hf loss in the presence of h3o . in ( b ) , the c3 in mefdc is unstable in the presence \n of h3o and reacts without a barrier to form \n c2 via hf loss . the animations ( movies ) of the optimization steps for reaction involving h3o for c3 in both gemcitabine ( a ) \n and mefdc ( b ) are provided in the supporting information ( s5 ) .", "our \n work has the following two salient findings : ( i ) one electron \n oxidation leads to cytosine base -cation radical ( c ) in 2-dc and 2-f - dc but to c3 \n in gemcitabine . as expected from the one - electron redox potentials \n of the bases and the backbone , one - electron oxidation of 2-dc and 2-f - dc leads \n to c formation as evidenced by the ca .", "16 g doublet \n that is characteristic of c. however , gemcitabine \n ( scheme 1 ) leads to the formation of c3 \n on one - electron oxidation .", "the two highly electronegative f - atoms \n at 2-position , through their negative inductive effect , lead \n to a substantial increase in the acidity of h3. therefore , \n c in gemcitabine is highly unstable toward the \n loss of h3 as deprotonation at 150155 k. this is evidenced \n by the free energy changes of the cation radical for the loss of h3 \n as deprotonation to the surrounding solvent ( see supporting information table t2 ) ; this deprotonation shifts \n the unpaired spin from the cytosine base of metastable c in gemcitabine to sugar at c3 via a pcet \n process .", "( ii ) c2 formation does not occur in \n gemcitabine \n but does in its analog mefdc .", "it has been proposed in the literature \n that in gemcitabine both c3 and c2 \n ( reactions 1 and 2 ) play \n an important role in the rnr inactivation .", "conversion of \n c3 to c2 takes place via an irreversible \n f loss from c2 during rnr inactivation \n by gemcitabine .", "however , experimental and theoretical results shown \n in this work have clearly demonstrated that in our system ( supercooled \n homogeneous glassy solutions ) , c3 in gemcitabine does \n not convert to c2 on annealing up to 170 k owing to \n theoretically predicted barrier of greater than 5 kcal / mol .", "theoretically , \n dft calculations support the mechanism involving a h3o induced barrierless conversion of c3 to c2 \n in one - electron oxidized mefdc .", "experimentally , c2 \n is observed in one - electron oxidized mefdc upon annealing to ca .", "160170 \n k. thus , our study in one - electron oxidized mefdc provides the first \n evidence of formation of c2 ( via the unstable intermediate \n c3 ( reaction 5 ) ) in a nonenzymatic \n system even at low temperature.5"], "abstract_text": ["<S> gemcitabine \n is a modified cytidine analog having two fluorine atoms \n at the 2-position of the ribose ring . </S>", "<S> it has been proposed \n that gemcitabine inhibits rnr activity by producing a c3 \n intermediate via direct h3-atom abstraction followed by loss \n of hf to yield a c2 with 3-keto moiety . </S>", "<S> direct \n detection of c3 and c2 during rnr \n inactivation by gemcitabine still remains elusive . to test the influence \n of 2- substitution on radical site formation , </S>", "<S> electron spin \n resonance ( esr ) studies are carried out on one - electron oxidized gemcitabine \n and other 2-modified analogs , i.e. , 2-deoxy-2-fluoro-2-c - methylcytidine ( mefdc ) and 2-fluoro-2-deoxycytidine \n ( 2-fdc ) . </S>", "<S> esr line components from two anisotropic -2-f - atom \n hyperfine couplings identify the c3 formation in one - electron \n oxidized gemcitabine , but no further reaction to c2 \n is found . </S>", "<S> one - electron oxidized 2-fdc is unreactive toward \n c3 or c2 formation . in one - electron \n </S>", "<S> oxidized mefdc , esr studies show c2 production presumably \n from a very unstable c3 precursor . </S>", "<S> the experimentally \n observed hyperfine couplings for c2 and c3 \n match well with the theoretically predicted ones . </S>", "<S> c3 \n to c2 conversion in one - electron oxidized gemcitabine \n and mefdc has theoretically been modeled by first considering the \n c3 and h3o+ formation via h3-proton \n deprotonation and the subsequent c2 formation via \n hf loss induced by this proximate h3o+ . </S>", "<S> theoretical \n calculations show that in gemcitabine , c3 to c2 \n conversion in the presence of a proximate h3o+ has a barrier in agreement with the experimentally observed \n lack of c3 to c2 conversion . </S>", "<S> in contrast , \n in mefdc , the loss of hf from c3 in the presence of \n a proximate h3o+ is barrierless resulting in \n c2 formation which agrees with the experimentally \n observed rapid c2 formation . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results and Discussion", "Conclusion"], "sections": [["gemcitabine is a modified \n cytidine analog having two fluorine atoms \n at the 2-position in the deoxyribose sugar moiety ( scheme 1 ) . for nearly 20 years , it has been widely used \n to treat specifically pancreatic cancer .", "it has been proposed that gemcitabine inhibits \n ribonucleotide reductase ( rnr ) activity as well as acting as \n a replication stop , thereby affecting dna synthesis \n and elongation .", "the two highly electronegative \n f - atoms at c2 substantially increase the acidity of h3 \n through the inductive effect .", "it is well established \n that in the absence of oxygen , thiyl radicals \n are able to abstract hydrogen ( h ) atoms to form neutral c - centered \n radicals .", "for example , h - atom abstraction by thiyl radicals has been \n shown to induce isomerization of cis-2,5-dimethyltetrahydrofuran \n to the trans-2,5-dimethyltetrahydrofuran . on this basis , stubbe et al .", ", in their enzymatic and electron \n spin resonance ( esr ) studies with gemcitabine , have proposed that \n inhibition of the rnr activity by gemcitabine should occur via radical \n formation at the c3 site by direct h - atom abstraction to produce \n a c3 via a enzymatic thiyl radical ( reaction 1 ) .", "subsequently , the c3 intermediate \n has been proposed to form 3-keto c2 via hf \n loss .", "this c2 is stabilized by an oxy radical resonance \n contribution ( reaction 2 ) .", "c2 \n and its immediate precursor c3 ( reactions 1 and 2 ) play a key role in \n the rnr inactivation.12 the h - atom abstraction reaction ( reaction 1 ) is not only important in rnr activation but also \n plays a very key \n role toward stable product formation in other biologically damage \n processes in dna , such as oxidative intrastrand cross - link formation and in the formation of sugar radicals that \n are strand break precursors .", "it is noteworthy that pulse radiolysis \n experiments with a 1,4-anhydro-5-deoxy-6-thio - d - ribo - hexofuranitol \n detected the formation of ribosyl - based carbon - centered radical(s ) \n after h - atom abstraction by thiyl radicals .", "these studies are supportive of reactions 1 and 2 but unequivocal , and direct detection \n of c3 and c2 employing esr or pulse \n radiolysis during rnr - catalyzed deoxygenation of the natural substrates or during inactivation by gemcitabine still remains elusive . in this work , we report the formation of c2 from \n a likely c3 in a gemcitabine analog which mimics the \n mechanism proposed above . from the structural formula of gemcitabine \n ( scheme 1 )", ", it is expected that the negative \n inductive effect ( i ) of two highly electronegative f - atoms \n at c2 should increase the acidity of h3. from our \n previous work on nucleoside cation radicals , the gemcitabine cation radical formed upon one - electron oxidation \n is expected to produce c3 after deprotonation of the \n acidic proton h3. in this work , esr spectroscopy has been \n employed to investigate one - electron oxidation of gemcitabine and \n other 2-modified derivatives , for example , 2-deoxy-2-fluoro-2-c - methylcytidine ( mefdc ( psi-6130 ) ; scheme 2 ) and 2-fluoro-2-deoxycytidine ( 2-fdc , scheme 2 ) , in order to test the influence of 2- \n substituent on radical site formation .", "it is noteworthy that mefdc \n is a well - known clinically efficacious inhibitor of hepatitis c virus .", "the esr results clearly identify the c3 formation \n in one - electron oxidized gemcitabine and the production of c2 \n in one - electron oxidized mefdc .", "these calculations show \n that in the case of one - electron oxidized mefdc , the lowest energy \n path is the rapid formation of c2 from c3 \n via f loss .", "this f loss is a \n barrierless reaction between the 2-f - atom and the proximate \n h3o which was formed via deprotonation of h3 \n in the cation radical ."], ["gemcitabine ( scheme 1 ) and 2-fdc \n ( scheme 2 ) were obtained \n from carbosynth ltd .", "mefdc ( scheme 2 ) was prepared as described or \n purchased from adooq bioscience ( irvine , ca ) .", "lithium chloride ( licl ) ( ultra \n dry , 99.995% ( metals basis ) ) was \n obtained from alfa aesar ( ward hill , ma , usa ) .", "2-deoxycytidine \n ( 2-dc ) was obtained from sigma chemical company ( st louis , \n mo , usa ) .", "deuterium oxide ( d2o ) ( 99.9 atom % d ) was purchased \n from aldrich chemical co. inc .", "( paris , ky , usa ) . cytidine-5,6-d2 ( [ 5,6-d , d]-cyd , 99 atom % d ) was purchased from cdn isotopes ( quebec , \n canada ) .", "homogeneous solutions \n of gemcitabine were prepared by dissolving 210 mg / ml either \n in 7.5 m licl in d2o or in h2o .", "solutions of \n other compounds ( 2-dc , 2-f - dc , mefdc , and [ 5,6-d , d]-cyd ) \n were prepared by dissolving ca .", "k2s2o8 ( 616 \n mg / ml ) was added as an electron scavenger so that only the formation \n of the one - electron oxidized species and its subsequent reactions \n can be followed by employing esr spectroscopy .", "the above - mentioned \n procedure for preparation of solutions is according to our ongoing \n studies on various model systems of dna and rna .", "the ph of gemcitabine in 7.5 m licl / d2o was adjusted to the range of ca .", "the ph of gemcitabine in 7.5 m licl / h2o \n and the ph of other compounds ( 2-dc ,", "2-f - dc , mefdc , \n and [ 5,6-d , d]-cyd ) in 7.5 m licl / d2o was adjusted at ph \n ca . 10 .", "these ph adjustments were performed by adding l amounts \n 1 m naoh as per our previous efforts .", "these solutions have high ionic strength ( 7.5 m licl ) ; therefore , \n the ph meters would not provide accurate ph measurements of these \n solutions . instead , as per our previous works ,", "ph values reported in this work were obtained using ph papers and \n are approximate measurements . as \n per our previous works , these ph - adjusted homogeneous solutions were thoroughly bubbled \n with nitrogen to remove the dissolved oxygen . immediately , these solutions \n were drawn into 4 mm suprasil quartz tubes ( catalog no .", "buena , nj , usa ) and were rapidly cooled in \n liquid nitrogen ( 77 k ) .", "the rapid cooling of these homogeneous liquid \n solutions at 77 k leads to the formation of transparent homogeneous \n glassy solutions .", "these glassy solutions were later used for the irradiation \n and subsequent progressive annealing experiments .", "all glassy samples \n were stored at 77 k in teflon containers in the dark .", "all samples \n were  ( co)-irradiated ( absorbed \n dose = 1.4 kgy ) at 77 k and stored at 77 k in teflon containers in \n dark following our previous efforts .", "a variable - temperature \n assembly was employed which passed liquid nitrogen cooled nitrogen \n gas past a thermister and over the sample as described in our earlier \n studies .", "the glassy samples have been \n annealed anywhere from ( 140170 ) k for 15 min .", "annealing leads \n to one - electron oxidation of the solute by the matrix radical cl2 thus , forming only the cation \n radical of the solute , e.g. , gemcitabine .", "following our earlier studies , immediately after -irradiation of the glassy sample at 77 \n k , the esr spectrum was recorded at 77 k. also , immediately after \n each annealing step , the sample was cooled to 77 k by immersing in \n liquid nitrogen ( 77 k ) , and the esr spectrum was recorded at 77 k \n which maximizes signal height and allows for comparison of signal \n intensities . a varian century series x - band ( 9.3 ghz ) esr spectrometer \n with an e-4531 dual cavity , 9 in .", "magnet , and a 200 mw klystron was \n used , and fremy s salt ( gcenter = 2.0056 , a(n ) = 13.09 g ) was employed for the \n field calibration .", "all esr spectra have been recorded at 77 k and \n at 40 db ( 20  w ) .", "anisotropic simulations of esr spectra \n have been performed using the win - epr and simfonia programs of bruker \n as per our previous works .", "the simulated spectra thus obtained \n were compared to experimental spectra , and esr parameters were adjusted \n for the best fit ( also supporting \n information figure s3 ) .", "gaussview and jmol programs were used to plot the spin densities \n and molecular structures .", "the geometries of all the radicals considered \n in the present study were fully optimized using the b97x functional and 6 - 31g(d ) basis set .", "we note here that b97x \n functional was developed by the group of head  gordon and found \n to be very successful for the calculations of various properties of \n molecules in their different spin states .", "the hyperfine coupling constants ( hfccs ) of the radicals were calculated \n using the same method and basis set , i.e. , b97x/6 - 31g(d ) in \n the gas phase . in order to treat the effect of solvent on hf loss \n from the c3 in gemcitabine and in mefdc , we employ \n the integral equation formalism polarized continuum model ( ief - pcm ) as implemented in gaussian 09 . in \n addition to pcm , for c3 in both systems", "a h3o is placed in the vicinity of the c3-oh bond \n for c3 in gemcitabine and for c3 in \n mefdc and have optimized the structures .", "the electronic energy profile \n of f dissociation from c2 site of c3 \n in mefdc as well as the electronic energy profile of f dissociation for each of the two f - atoms from c2 site of \n c3 in gemcitabine were obtained in the presence of \n a single water molecule at the same level of theory ( supporting information figure s4c , d ) .", "furthermore , employing \n the wb97x/6 - 31++g(d , p ) method along with the ief - pcm model for the \n solvent effect , the pka of the c3-oh \n group for the c3 of gemcitabine and also of the c3-oh \n group for the c3 of 2-dc was calculated ."], ["in figure 1a , we show the experimentally recorded ( 77 k ) esr spectrum \n ( green ) of one - electron oxidized gemcitabine at ph ( pd ) ca . 7 in a \n homogeneous glassy 7.5 m licl / d2o solution .", "the one - electron \n oxidation of gemcitabine was induced by cl2 attack after annealing at 155 k in the dark .", "912 showed identical spectra after \n one - electron oxidation of gemcitabine by cl2 on annealing at 150155 k. thus , only the spectrum \n obtained from the gemcitabine sample at pd ca .", "10 is presented in \n figure 1b along with the simulated spectrum \n in blue .", "it is evident from figure 1a , b , the \n line shape , total hyperfine splitting , and the center of the simulated \n spectra match with those of the experimentally recorded spectra quite \n well .", "each of the spectra in figure 1a , b show \n two anisotropic -f - atom hyperfine couplings and a -h - atom \n hyperfine coupling .", "the -h - atom hyperfine coupling creates \n the doublet splitting in the line components in figure 1a , b .", "figure 1a is best matched \n with a simulation \n employing the two different anisotropic -f - atom ( nuclear spin \n = 1/2 ) hfcc values of ( 15.0 , 15.0 , 105 ) g and ( 15.0 , 15.0 , 69.0 ) g , \n one -h hfcc as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0080 , 2.0050 , 2.0020 ) along with a mixed ( lorentzian / gaussian \n ( 1:1 ) ) line - width of 14 g. the simulated spectrum in blue is superimposed \n on the experimentally recorded spectrum in figure 1a . on the other hand , the best fit for figure 1b", "is obtained employing the two identical anisotropic -f - atom \n ( nuclear spin = 1/2 ) hfcc as ( 17.0 , 17.0 , 86.0 ) g , one -h hfcc \n as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0060 , 2.0050 , \n 2.0020 ) along with a mixed ( lorentzian / gaussian ( 1:1 ) ) line - width \n of 10 g. following our work on the radicals produced in monomers \n of dna \n and rna , the a ( i.e. , \n the azz ) component of each of the two \n anisotropic -f - atoms ( see table 1 ) as \n well as the a of the -h are directly measured \n from the width of the experimentally recorded spectra with an uncertainty \n of 2 g ( see supporting information figure s3 ) . on the other hand ,", "the theoretically obtained values \n of axx and ayy components of each of the two anisotropic -f - atoms and of the \n -h - atom in table 1 were adjusted to \n fit the experimentally recorded spectra with estimated uncertainty \n of 4 g ( see supporting information figure s3 ) .", "7 show two nonequivalent anisotropic -f - atom hfccs , whereas , \n the one - electron oxidized gemcitabine spectrum at ph ca .", "the -h - atom \n hfcc does not show any observable change in the one - electron oxidized \n gemcitabine spectrum throughout the ph range ca .", "the coupling to two -f - atoms ( c2 ) and one -h - atom \n ( c4 ) is clear evidence for the generation of c3 \n after one electron oxidation of gemcitabine at 150155 k. the \n electron - withdrawing effect of the two electronegative f - atoms at \n c2 increases the acidity of h3 , which leads to deprotonation \n ( see supporting information table t2 ) and \n prevents observation of the initially formed cytosine base -cation \n radical ( c ) in gemcitabine as indicated in reaction 3 .", "therefore , the mechanism of c3 \n formation due to one - electron oxidation of gemcitabine is proposed \n as follows : one - electron oxidation of gemcitabine results in the formation \n of metastable c , which is unstable even at ca . \n", "155 k. the metastable c quickly deprotonates at \n c3 in the sugar moiety producing c3 ( reaction 3 ) via a proton - coupled electron - transfer ( pcet ) mechanism.3 esr \n spectra obtained from matched gemcitabine samples [ concentration \n of gemcitabine in each sample = 2 mg / ml in 7.5 m licl / d2o ] in the presence of the electron scavenger k2s2o8 ( 8 mg / ml in each sample ) .", "each sample has been -irradiated \n ( absorbed dose = 1.4 kgy at 77 k ) , subsequently annealed to 155 k \n for 15 min in the dark at various phs ( a ) ph ca .", "7 ( green ) and ( b ) \n ph ranging ca . 912 ( pink ) . here", "the blue spectra that are superimposed on the experimentally \n recorded spectra are the simulated spectra of c3. \n see text for the details of simulation .", "all esr spectra are recorded \n at 77 k. the three reference markers ( open triangles ) in this figure \n and in the subsequent figures show the position of fremy s \n salt resonance with the central marker at g = 2.0056 .", "the spacing \n separating the markers is 13.09 g. as is evident from the hfcc \n values of the spectra shown in figure 1 and \n also in table 1 , the ph of the solution clearly \n affects the individual -f - atom anisotropic hfcc but not the \n sum of the two -f - atom anisotropic hfcc along with the c4 \n -proton hfcc ( see section above ) . at ph ca .", "912 , the two -f - atom anisotropic hfccs \n are equivalent ; whereas , at ph ca . 7 ,", "the sum of the two -f - atom \n anisotropic hfccs remain the same , but they individually differ .", "the presence of two 2-f - atoms in gemcitabine will lower the \n pka value of both h3 and c3-oh \n hydrogens .", "for example , the oh in 2,2-difluoroethanol has its pka lowered by 3.5 units in comparison with ethanol .", "further , radical formation has also been shown \n to lower the pka of the alcoholic oh group \n ( e.g. , pka ( ch3)2choh = 17.1 , pka ( ch3)2coh = 12.03 ) . based on \n these factors , the pka value of the c3-oh \n group for c3 in gemcitabine is estimated to be in \n the range of 79 .", "employing dft/b97x/6 - 31++g(d , p ) method \n along with the ief - pcm model for the solvent effect , the pka value of the c3-oh group for c3 \n in 2-dc has been calculated as 14.2 .", "the same level of calculation \n for the c3-oh group of c3 in gemcitabine which \n replaces each of the two hydrogens at c2 with fluorine predicts \n a pka of 6.8 ( see supporting information pages s8 , s9 ) .", "therefore , the two 2-f - atoms \n are predicted to lower the pka of the \n c3-oh group in c3 by 7 full units .", "considering \n the sensitivity of the calculations for predicting pka to the small changes in free energy , these results are very reasonable .", "furthermore , the theoretically \n calculated pka value 6.8 of the c3-oh \n group for c3 in gemcitabine is in good agreement with \n its experimentally estimated value ( 79 ) . the spectrum \n in figure 1a ( ph ca .", "7 ) should \n therefore be for c3 in gemcitabine with a c3-oh \n group and the spectrum in figure 1b ( ph ca . \n", "912 ) should be for c3 with the deprotonated \n group , i.e. , c3-o ( reaction 4 ) .", "thus , we attribute the variation of the two -f - atom \n anisotropic hfcc to the deprotonation of the c3-oh group at \n higher phs.4 esr spectral studies \n of one - electron oxidation of gemcitabine in h2o glasses \n ( 7.5 m licl / h2o ) were performed and compared with the results \n found in d2o glasses ( 7.5 m licl / d2o ) .", "no observable difference in spectra other than a small \n line broadening was observed on formation of c3 in \n h2o glasses versus in d2o glasses .", "since c3 \n only has the c3-oh as an exchangeable proton at ph 7 and this \n does not contribute to a significant hyperfine coupling , the esr spectrum \n found on formation of c3 is not altered by a change \n of the solvent from d2o to h2o .", "similar experiments to those performed for gemcitabine \n were carried out for the methyl / fluoro analog mefdc ( scheme 2 ) .", "using mefdc , we investigated whether the formation \n of c3 observed in gemcitabine bearing geminal difluoro unit at c2 ( figure 1 ) is \n affected by the substitution of one of the f - atoms ( i ) with \n a methyl ( me ) group ( + i ) .", "( a ) esr spectrum ( black ) obtained from mefdc [ concentration \n = 2 \n mg / ml in 7.5 m licl / d2o ] in the presence of the electron \n scavenger k2s2o8 ( 8 mg / ml ) , ph ca . \n", "10 , -irradiated to a dose of 1.4 kgy at 77 k and subsequently \n annealed to 155 k for 15 min .", "( c ) \n spectrum ( black ) obtained after subtraction of 60% of spectrum ( b ) \n from spectrum ( a ) .", "for comparison , the c spectrum \n ( blue ) in 2-dc ( supporting information figure s2 and pp s3s5 ) is superimposed .", "( d ) spectrum ( black ) \n assigned to c2 is obtained after subtraction of 50% \n of spectrum ( c ) from spectrum ( a ) .", "the simulated c2 \n spectrum ( for simulation parameters see figure 2 and text ) is superimposed \n on the experimentally isolated spectrum for comparison .", "all the spectra \n are recorded at 77 k. shown in figure 2a is the esr spectrum \n ( black ) \n of a matched sample of mefdc that has been -irradiated ( absorbed \n dose = 1.4 kgy ) , subsequently annealed to 155 k for 15 min in the \n dark , and recorded at 77 k. figure 2b \n was obtained by annealing this \n sample for 15 min to 170 k. comparison of spectrum 2a with spectrum \n 2b shows clearly that a central doublet decreases along with an increase \n of the other line components upon annealing .", "therefore , the central \n doublet ( black ) shown in figure 2c is isolated \n by subtraction of 60% of the spectrum 2b from \n spectrum 2a .", "the doublet due to c spectrum ( blue ) in 2-dc ( see supporting information figure s2 and its discussion ( pp s3s5 ) ) \n is superimposed on it for comparison . from the spectral similarities \n of both doublets , the doublet in black shown in figure 2c", "subtraction \n of 50% c spectrum 2c ( black ) from \n spectrum 2a results in the black spectrum shown in figure 2d .", "this overall quintet spectrum arises from 4 isotropic \n -proton couplings : three methyl -protons ( ca .", "25.5 g due to a -proton \n assigned to the c1-h ( vide infra ) .", "the experimental ( black ) \n spectrum is simulated using the above - mentioned hfcc values along \n with a 10 g line - width and g - value = 2.0033 ( this g - value is typical for c - centered sugar radicals ) . the simulated spectrum ( red ) in figure 2d \n matches the overall line components of the experimental spectrum well . \n", "since the c2 ( reaction 5 ) is \n the only likely radical structure that would explain the large hyperfine \n coupling to a methyl group and the additional -proton hyperfine \n coupling ( assigned to c1 ) , the experimental spectrum in figure 2d has been assigned to c2 ( reaction 5 ) . the spectra 2a and 2b are a composite of c ( black , \n figure 2c ) and c2 ( black , figure 2d ) in different amounts . under the same constant \n gain and constant microwave power and upon gradual and stepwise annealing \n of the sample from 155 k ( spectrum 2a ) to 170 k ( spectrum 2b ) ,", "no \n loss of spectral intensity was observed , and our analyses show an \n additional ( ca .", "20% ) conversion of the c to c2. consideration of the results presented in figures 1 and 2 suggest that for mefdc , c is produced first and on annealing converts to a transient \n c3 , which is not observed .", "in contrast , the line shape , \n line width , and the overall hyperfine splitting of the c3 \n spectrum in gemcitabine do not change upon annealing to ca .", "thus , unlike \n the rapid conversion of c3 to c2 found \n in one - electron oxidized mefdc , a similar conversion of c3 \n to c2 is not observed for one - electron oxidized gemcitabine \n at these low temperatures .", "the lack of observation of the transient \n c3 in one - electron oxidized mefdc and the low temperatures \n employed in these experiments implies a very low activation barrier \n for the conversion of c3 to c2 ; whereas , \n the activation barrier for the conversion of c3 to \n c2 for one - electron oxidized gemcitabine should be \n 4 kcal / mol .", "calculations suggest that the proximity of the \n lost h3 proton as h3o to the 2-f - atom \n quite likely provides the driving force for this rapid unimolecular \n reaction ( see figure 3 ) .", "therefore , we propose \n that c3 in one - electron oxidized mefdc readily converts \n to c2 via a barrierless f loss \n ( see reaction 5 , figure 3 , and supporting information s5 ) . the b97x/6 - 31g(d ) \n", "calculated hfccs of c3 and c2 found \n in gemcitabine and in mefdc along with experimental hfccs ( in gauss ) \n are presented in table 1 .", "it is evident from \n table 1 that experimental and theoretically \n calculated hfccs are in reasonably good agreement .", "estimated errors are of 2 g for \n azz and 4 g for axx and also for ayy .", "calculated in the presence \n of one \n water molecule only isotropic \n hfcc values have \n been considered . to explore the reaction mechanism of c2 \n formation from c3 in one - electron oxidized mefdc and \n gemcitabine , using the dft b97x/6 - 31g(d ) method", ", we considered \n three possible reaction paths : ( i ) hf loss due to deprotonation of \n 3-hydroxyl group , ( ii ) hf loss due to fluorine dissociation , and additionally , ( iii ) hf loss in the presence of a hydronium ion \n ( h3o ) .", "the electronic energy profile \n of hf loss from c3 in mefdc and also from c3 \n in gemcitabine in the presence of a water molecule is shown in figure \n s4 in the supporting information . from supporting information figure s4a ,", "it is evident \n that for c3 of mefdc , the formation of c2 \n via hf loss with deprotonation of 3-oh has a significant barrier \n of ca . 18 kcal / mol .", "for c3 in gemcitabine , the hf \n loss associated with deprotonation of 3-hydroxyl group was \n calculated to be ca .", "we have also considered the dissociation of \n fluorine in c3 \n of mefdc and in c3 of gemcitabine and found that stretching \n the c2-f bond up to 1.8  needs ca .", "this shows that dissociation of fluorine for c3 in \n mefdc occurs at lower energy than dissociation of fluorine for c3 \n in gemcitabine ( supporting information figure \n s4 ) .", "alternatively , we consider the fact that deprotonation \n of h3 \n will form h3o initially in close proximity \n of the c2-c3 bond which may then induce hf loss ( reaction 5 ) . employing the b97x/6 - 31g(d ) method and \n considering the full solvent effect through the polarized continuum \n model ( pcm ) , this mechanism have been modeled by placing a h3o in the vicinity of the c3-oh bond for c3 \n in gemcitabine and for c3 in mefdc and have optimized \n the structures . from our calculations , we have observed that for c3 \n in gemcitabine , a minimum structure exists in the electronic energy \n profile in which the h3o stabilizes the f-atom \n through forming a hydrogen bond ( 1.57  ) ( see figure 3a and supporting information figure s4c ) .", "a second minimum structure was also found in the electronic \n energy profile in which the h3o stabilizes \n the f-atom through forming a hydrogen bond of identical length , \n 1.57  ( see supporting information figure s4d ) .", "a barrier of 5 kcal / mol and the overall reaction energy \n of 7 to 8 kcal / mol were found for hf formation in \n both cases ( see figure 3a and supporting information figures s4c , d ) .", "however , for c3 \n in mefdc , the h3o reacts with the 2-f - atom \n without a barrier and forms hf and c2 ( see figure 3b ) .", "the bond distances of c3-o3 \n and o3-h bonds for c3 in gemcitabine are calculated \n as 1.34  ( primarily c  o single bond character ) and 0.97  , respectively .", "the values of \n the corresponding bond distances of c3-o3 and o3-h \n bonds for c3 in mefdc are obtained as 1.28  \n ( mainly double bond character and 1.0 \n  respectively ) .", "thus , these calculations show that the \n hf loss from c3 \n in gemcitabine has a ca .", "5 kcal / mol barrier ( supporting \n information figure s4c , d ) while for c3 in mefdc , \n the loss of hf is barrierless , and the c2 production \n is exothermic in nature as shown in figure 3 .", "these findings support our experimental observations that in mefdc , \n c3 is too unstable to be observed , and only c2 \n is found .", "in contrast , in gemcitabine only c3 formation \n is observed without any conversion to c2 in the same \n temperature range .", "pcm-b97x/6 - 31g(d ) calculated structures of c3 \n in ( a ) gemcitabine and in ( b ) mefdc .", "as indicated in ( a ) the c3 \n in gemcitabine involves a barrier of 5 kcal / mol ( detailed electronic \n energy profiles are provided in supporting information figure s4c , d ) to hf loss in the presence of h3o . in ( b ) , the c3 in mefdc is unstable in the presence \n of h3o and reacts without a barrier to form \n c2 via hf loss . the animations ( movies ) of the optimization steps for reaction involving h3o for c3 in both gemcitabine ( a ) \n and mefdc ( b ) are provided in the supporting information ( s5 ) ."], ["our \n work has the following two salient findings : ( i ) one electron \n oxidation leads to cytosine base -cation radical ( c ) in 2-dc and 2-f - dc but to c3 \n in gemcitabine . as expected from the one - electron redox potentials \n of the bases and the backbone , one - electron oxidation of 2-dc and 2-f - dc leads \n to c formation as evidenced by the ca .", "16 g doublet \n that is characteristic of c. however , gemcitabine \n ( scheme 1 ) leads to the formation of c3 \n on one - electron oxidation .", "the two highly electronegative f - atoms \n at 2-position , through their negative inductive effect , lead \n to a substantial increase in the acidity of h3. therefore , \n c in gemcitabine is highly unstable toward the \n loss of h3 as deprotonation at 150155 k. this is evidenced \n by the free energy changes of the cation radical for the loss of h3 \n as deprotonation to the surrounding solvent ( see supporting information table t2 ) ; this deprotonation shifts \n the unpaired spin from the cytosine base of metastable c in gemcitabine to sugar at c3 via a pcet \n process .", "( ii ) c2 formation does not occur in \n gemcitabine \n but does in its analog mefdc .", "it has been proposed in the literature \n that in gemcitabine both c3 and c2 \n ( reactions 1 and 2 ) play \n an important role in the rnr inactivation .", "conversion of \n c3 to c2 takes place via an irreversible \n f loss from c2 during rnr inactivation \n by gemcitabine .", "however , experimental and theoretical results shown \n in this work have clearly demonstrated that in our system ( supercooled \n homogeneous glassy solutions ) , c3 in gemcitabine does \n not convert to c2 on annealing up to 170 k owing to \n theoretically predicted barrier of greater than 5 kcal / mol .", "theoretically , \n dft calculations support the mechanism involving a h3o induced barrierless conversion of c3 to c2 \n in one - electron oxidized mefdc .", "experimentally , c2 \n is observed in one - electron oxidized mefdc upon annealing to ca .", "160170 \n k. thus , our study in one - electron oxidized mefdc provides the first \n evidence of formation of c2 ( via the unstable intermediate \n c3 ( reaction 5 ) ) in a nonenzymatic \n system even at low temperature.5"]]}
{"article_id": "PMC4752095", "article_text": ["it has a significant impact on the patient s quality of life ( 1 ) .", "post - prostatectomy urinary incontinence ( ppi ) is a potential complication of prostate surgery and although it is more frequently seen after radical prostatectomy it can also occur after endoscopic or open surgery for benign prostate hyperplasia ( bph ) ( 2 ) .", "initial management is usually conservative and includes the use of diapers or pads , penile clamps , or various collecting systems ( such as a condom catheter ) .", "mild degrees of ppi in the early postoperative period may be improved by pelvic muscle exercises , physiotherapy , and pharmacotherapy ( 3 ) . however , for most patients who have moderate to severe ppi , conservative methods are not sufficient to return to their normal lives .", "the present study reports our intermediate experience in men who underwent implantation of adjustable perineal male sling using a tissue expander for ppi .", "this prospective study was approved by the local ethics committee and a comprehensive informed consent was obtained from all the patients before the surgery . between september 2007 and may 2013 ,", "a total of 21 men with ppi underwent implantation of adjustable perineal male sling using a tissue expander .", "the underlying etiologies of ppi in patients were : radical prostatectomy for prostate cancer in 13 patients ( open radical retropubic prostatectomy=12 , transperitoneal laparoscopic radical prostatectomy=1 ) , open prostatectomy in 5 patients and transurethral prostate resection for bph in 3 patients .", "patient evaluation consisted of medical history , physical examination , blood and urine laboratory tests .", "all the patients underwent a standard urodynamic study including both cystometry and pressure - flow study to evaluate bladder storage and voiding capabilities , and to exclude overactive bladder .", "also , an urethrocystoscopy was conducted in all the patients to exclude urethral stricture and/or bladder neck contracture .", "the patients who had a history of previous surgery for ppi were not included in the study .", "incontinence in the patients was defined according to the use of incontinence pads over a 24 hours period : mild ( using 1 to 2 pads ) , moderate ( 3 to 5 pads ) and severe ( more than 5 pads ) ( 4 ) .", "the post - operative follow - up was carried out in the second week and every 6 months thereafter consisting of daily pad use , physical examination , maximum urine flow rate ( qmax ) and post - voiding residual urine volume ( pvr ) measurement .", "all patients received prophylactic antibiotics ( 3rd generation cephalosporin ) before the induction of anesthesia . under general or regional anesthesia", "after the insertion of a urethral catheter , a midline perineal incision of 40 to 50 mm was made .", "the critical point is not to remove periurethral fat tissues from urethra . both sides of urethra", "a eurosilicone ( laboratoires eurosilicone , france ) tissue expander device was used with an adjustable male sling in our patients .", "the device contains a silicone balloon expander , a connecting tube and an inflation component ( injection port ) that allows the expander to gradually fill up to a volume of 25 ml saline solution ( figure-1b ) . \n", "figure 1a ) both sides of urethra are dissected ; b ) a balloon expander ; c ) a balloon expander between two layers of the mesh ; d ) the mesh is stapled to the pubic bone using autosuture stat tacktm ; e ) prepared scrotal pouch for placing the inflation component . \n  a standard polypropylene mesh of 100 x 100 mm was used as the sling material .", "first , the mesh was folded and a balloon expander was placed between the two layers of the modification .", "secondly , the balloon was completely filled with the saline solution , and the two layers of the mesh were sutured using 2/0 polypropylene suture to create a pocket between the two layers of the mesh ( figure-1c ) . during this procedure", "third , the mesh was stapled on both sides of the lower border of the pubic bone using an autosuture stat tacktm ( tyco healthcare , uk ) stapler ( figure-1d ) . the original surgical method defined by inci , et al .", "( 5 ) used a polypropylene mesh that was fixed on the pubic bone with non - absorbable sutures . in our case , this method was modified and staples were used to prevent the sutures and the mesh from loosening and moving . after the sling tension was properly adjusted , the sling was placed as tightly as possible in all patients since urinary leakage had been observed in our patients postoperatively .", "the inflation component has two sides ( figure-1e ) . finally , the wound was closed with careful hemostasis .", "after the surgery , all the patients were asked to take an oral antibiotic ( 2th generation cephalosporin ) for 7 days .", "post - operative success was assessed by the number of pads used per 24 hours as follows ; zero to 1 safety pad - dry , 1 to 2 pads - mild , and 3 to 5 pads - moderate . at a minimum of 1 week after surgery", ", patients were questioned regarding their daily pad use . if incontinence persisted 7 to 10 days after surgery , tension over the urethra was increased by saline injection to expand the tissue expander via the injection port using an insulin needle .", "injection was started with 3 cc and increased by 2 cc at each injection . in this way", "the patients were asked to return 2 days after each adjustment to confirm the status of continence .", "the mean values of the parameters were calculated using the statistical package for social sciences version 13.0 for windows ( spss inc . , chicago , il , usa ) .", "the mean values of the parameters were calculated using the statistical package for social sciences version 13.0 for windows ( spss inc . , chicago , il , usa ) .", "the mean age of the patients was 66.27.3 ( 50 - 79 ) years and the mean pad usage was 6.40.6 ( 6 - 8 ) per day .", "the mean volume of the postoperatively inflated balloon was 11.65.7 ( 5 - 25 ) ml .", "after the follow - up period , 16 of the 21 patients ( 76.2% ) were dry ( 11 patients , 0 pads ; 5 patients using safety pads ) , 3 patients ( 14% ) had mild degree ppi and 2 patients ( 9.8% ) had moderate degree ppi . in the last assessment of the patients , qmax and estimated pvr were found to be 15.64.7 ( 10 - 31 ) ml / s and 10 ml , respectively .", "in these two patients , the polypropylene mesh with balloon , connecting tube and inflation component were removed and they did not undergo any additional intervention . local scrotal infection developed around the inflation component in three patients . in these patients ,", "only the inflation component was removed , the connecting tube was clipped and the polypropylene mesh with inflated balloon was kept in place . in these patients , polypropylene mesh with inflated balloon provided suitable pressure on the urethra . in the follow - up period , they were completely dry .", "no complications occurred in relation to mesh erosion , voiding dysfunction , voiding difficulty or mechanical failure .", "six patients reported mild perineal pain in the early postoperative period but this was resolved using non - steroidal anti - inflammatory drugs .", "the rising elderly population and the increasing number of surgical interventions for prostate cancer mean that the incidence of ppi will rise .", "since its introduction in 1973 , the artificial urinary sphincter ( aus ) has been considered the gold standard treatment for stress urinary incontinence after prostatectomy , offering the patient the greatest chance of a cure ( 6 ) .", "the success rates of aus range from 59 to 90% ( 7 - 9 ) .", "although , aus is an effective and durable treatment , many patients are hesitant about implantation or refuse the procedure . most patients could not manipulate the scrotal pump ( 10 ) .", "furthermore , aus is expensive and requires a complex surgical procedure that is associated with significant rates of complications .", "a recent systematic review about aus reported that infection or erosion occurred in 8.5% of cases ( 3.327.8% ) , mechanical failure in 6.2% ( 213.8% ) , and urethral atrophy in 7.9% ( 1.928.6% ) .", "re - operation rate was reported to range from 14.8% to 44.8% ( 11 ) .", "the proact ( uromedica , us ) system was first described in 2005 by hubner et al . ( 12 ) . in theietlr study , there were 117 patients with ppi followed for a mean period of 13 months , after which 67% of the patients were found to be dry .", "( 13 ) reported the results of the proact intervention in 62 men with ppi and found that 71% of the patients wore no pads or used 1 pad per day after 6 months ( following the adjustment ) .", "however , in 19 men the device was removed due to infection and erosion ( n=5 ) , migration ( n=1 ) , and iatrogenic traumatism ( n=2 ) . moreover ,", ", the authors evaluated the results of proact in 114 men with ppi at a mean follow - up time of 58 months and reported an overall dry rate of 50% . in that study , complications included balloon leakage ( 11% ) , migration ( 5% ) and wound erosion ( 4% ) .", "the authors reported that there was a total re - operation rate of 27% , and 12% of the patients underwent aus or a urethral sling procedures due to proact not being effective .", "the argus ( promedon sa , argentina ) sub - urethral sling with an adjustable system is another treatment option in men with ppi that was first described by romano et al .", "the authors reported a cure rate of 73% and the improvement rate was 10% in 48 men with ppi after a mean follow - up of 7.5 months .", "( 16 ) reported mid - term complications after the placement of the argus sling in 29 men with ppi at a mean follow - up of 35 months .", "overall , 24 patients ( 83% ) experienced complications , consisting of acute urinary retention ( 35% ) and removal of the sling ( 35% ) owing to urethral erosion , infection , system dislocation , urinary retention , and persistent pain . furthermore , 27% of the patients complained of significant perineal pain .", "the authors concluded that the argustm sub - urethral sling was associated with serious mechanical and infectious complications , and sparse functional results with negative impact on the patient s quality of life .", "a bone anchored sling ( bas ) compresses the bulbar urethra with a silicone - coated polypropylene mesh by attaching the sling to the inferior pubic ramus with bone screws .", "following initial reports of degradation of organic materials , synthetic mesh ( invance , american medical systems , us ) has become the most commonly utilized material with the bas ( 17 ) .", "( 4 ) reported their results in 46 patients with ppi who undergo bas implantation . in their study , the total cure rate was 37% . furthermore ,", "37% of the patients significantly improved , and the treatment of 26% patients failed after an average of 24 months follow - up .", "they concluded that the male sling procedure is an effective and safe procedure for the management of stress urinary incontinence .", "guimaraes et al . ( 18 ) reported their intermediate results of up to 4 years with the invance sling .", "their cure rate was 65% and the improvement rate was 23% in 54 men with ppi after a mean follow - up of 28 months .", "the authors claimed that the invance sling offers a good intermediate cure and improvement rates with acceptably low rate of complications in patients suffering from ppi .", "another study investigated the use of the invance sling in 40 patients with ppi ( 19 ) .", "the authors observed perineal pain in 73% of the patients , detrusor overactivity in 5% and sling infection in 15% .", "the traditional bas procedure does not provide for the adjustment of the tension of the sling material in the post - operative period and this can result in progressive failure over time . in a study by castle et al .", "social continence was achieved in 67% 50% and 0% of mild , moderate and severe cases , respectively .", "comiter ( 20 ) speculated that the surgeons left the fatty tissue over the bulbospongious intact , and additionally used a piece of porcine dermis between the urethra and the sling a combination of fat necrosis and absorption of the dermis over time likely contributes to diminution of the compression provided by the sling over the bulbar urethra .", "( 21 ) reported a median time to recurrent incontinence as 3 months after bas procedure .", "similarly , cespedes and jacoby reported an early failure ( within 6 months ) in 5% of their patients ( 22 ) . in another study", "( 23 ) , the authors retrospectively reported objective and subjective outcomes in 40 male patients who underwent bas positioning for stress urinary incontinence due to intrinsic sphincter deficiency .", "patients with stress urinary incontinence due to radical retropubic prostatectomy ( n=32 ) , robot assisted laparoscopic prostatectomy ( n=3 ) and transurethral prostate resection ( n=5 ) underwent the bas procedure over a 5 years period .", "previous anti - incontinence procedures , radiotherapy and transurethral procedures due to urethral stricture were performed in 5 , 11 and 5 patients , respectively . at a mean follow - up of 35.2 months , 22 patients ( 55% )", "were cured , the condition of 5 patients ( 12.5% ) improved and the treatment of 13 patients ( 32.5% ) failed .", "the authors concluded that bas is a simple and effective procedure that can produce immediate good results with low morbidity , especially when strictly selected patients are treated .", "an adjustable perineal male sling technique was used for the treatment of ppi as described previously ( 5 ) . in that study", "an adjustable perineal male sling with a 10 ml tissue expander was implanted in 19 consecutive men with severe ppi . according to the results of that study , at a mean follow - up of 17.3 months 15 patients ( 78.9% ) were completely dry and 2 ( 10.5% ) improved significantly using only 1 to 2 pads per day . a total of 11 patients required volume adjustment injections .", "the average number of adjustments was 2 ( range 1 to 3 ) and the average injected volume was 6.3 cc ( range 5 to 10 ) .", "regarding the complications , 1 patient had a superficial wound infection , 1 patient required surgical revision due to infection and 8 patients reported mild to moderate perineal pain .", "the authors concluded that the short term results indicate that this minimally invasive technique seems to be safe and effective , and patient satisfaction appears to be high .", "the same group presented their long term results in the international continence society annual meeting 2013 . in a study by ergen and ozdemir", "the mean follow - up time was 52 months . the completely dry rate decreased from 78.9% to 58% in the long term period .", "the authors concluded that adjustable male sling using a 10 ml tissue expander is an effective method for severe ppi , in the early period , but the effectiveness gradually decreases in the long term .", "the review of our intermediate results at 40.1 months showed that 16 of the 21 patients ( 76.2% ) were completely dry , 3 patients ( 14% ) had mild incontinence and 2 patients ( 9.8% ) had moderate incontinence .", "the success rate of the current study is better than that of the other studies mentioned above ( 5 , 24 ) .", "a possible explanation for our high success rate may be associated with the high capacity of the silicone balloon . in our patients", ", we used a larger size of silicone balloon ( 25 ml ) than the other two studies .", "additionally , we used a stapler which allows the easier attachment of the polypropylene mesh on the lower border of the pubic bone in both sides .", "although previous pelvic radiotherapy history has also been claimed to be a negative predictor of success rate in ppi surgery ( 19 , 23 ) , 2 patients in current study whom had a previous history of pelvic radiotherapy were dry in the intermediate follow - up .", "a new device is a perineal sling with a tissue expander , composed of 2 silicone components , a balloon expander and an injection port , connected by a tube . in turkey", ", this device is relatively cheap compared with aus , proact , argustm ( $ 500 vs. $ 5.000 vs. $ 2330 vs. $ 2400 ) and easy to implant , even in difficult cases .", "although complications occurred in this study , including the removal of the device and the removal of inflation component due to local scrotal infection , our intermediate follow - up study suggests that an adjustable perineal male sling using a tissue expander appears to be an efficient and safe alternative surgical treatment option for patients suffering from ppi .", "the major limitations of our study were the small number of patients and the duration of the follow - up period .", "additional follow - up and larger series of patients are necessary to confirm our results ."], "abstract_text": ["<S> purpose to report our intermediate experience in treating patients with severe incontinence using an adjustable perineal male sling with a tissue expander.materials and methods an adjustable male sling procedure was performed on 21 patients with severe incontinence . </S>", "<S> the underlying etiology of urinary incontinence was radical prostatectomy in 13 patients , open prostatectomy in 5 patients and transurethral prostate resection in 3 patients . </S>", "<S> the difference between the classical and the adjustable sling is that in the latter there is a 25 ml tissue expander between the two layers of polypropylene mesh with an injection port . </S>", "<S> adjustment of the sling was performed with saline via an inflation port , in case of recurrence or persistence of incontinence.results the mean age of the patients was 66.27.3 ( 50 - 79 ) years and mean pad usage was 6.40.6 per day . </S>", "<S> the mean follow - up time was 40.123.2 ( 6 - 74 ) months . </S>", "<S> the balloon was postoperatively inflated on average with 11.65.7 ( 5 - 25 ) ml . </S>", "<S> after the mean 40.1 months of follow - up , 16 of the 21 patients ( 76.2% ) were dry ( 11 patients , 0 pads ; 5 patients using safety pads ) , 3 patients ( 14% ) had mild and 2 ( 9.8% ) had moderate degree post - prostatectomy urinary incontinence ( ppi ) . </S>", "<S> the average maximum urine flow rate of the patients was 15.64.7 ( 10 - 31 ) ml / s . </S>", "<S> no residual urine was found . in 2 patients , all parts of the device </S>", "<S> were removed due to infection and discomfort , and in 3 patients only the inflation component was removed due to local scrotal infection.conclusions our results show that using an adjustable perineal male sling with a tissue expander seems to be an efficient , and safe surgical treatment option in patients with ppi . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "SURGICAL PROCEDURE", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["it has a significant impact on the patient s quality of life ( 1 ) .", "post - prostatectomy urinary incontinence ( ppi ) is a potential complication of prostate surgery and although it is more frequently seen after radical prostatectomy it can also occur after endoscopic or open surgery for benign prostate hyperplasia ( bph ) ( 2 ) .", "initial management is usually conservative and includes the use of diapers or pads , penile clamps , or various collecting systems ( such as a condom catheter ) .", "mild degrees of ppi in the early postoperative period may be improved by pelvic muscle exercises , physiotherapy , and pharmacotherapy ( 3 ) . however , for most patients who have moderate to severe ppi , conservative methods are not sufficient to return to their normal lives .", "the present study reports our intermediate experience in men who underwent implantation of adjustable perineal male sling using a tissue expander for ppi ."], ["this prospective study was approved by the local ethics committee and a comprehensive informed consent was obtained from all the patients before the surgery . between september 2007 and may 2013 ,", "a total of 21 men with ppi underwent implantation of adjustable perineal male sling using a tissue expander .", "the underlying etiologies of ppi in patients were : radical prostatectomy for prostate cancer in 13 patients ( open radical retropubic prostatectomy=12 , transperitoneal laparoscopic radical prostatectomy=1 ) , open prostatectomy in 5 patients and transurethral prostate resection for bph in 3 patients .", "patient evaluation consisted of medical history , physical examination , blood and urine laboratory tests .", "all the patients underwent a standard urodynamic study including both cystometry and pressure - flow study to evaluate bladder storage and voiding capabilities , and to exclude overactive bladder .", "also , an urethrocystoscopy was conducted in all the patients to exclude urethral stricture and/or bladder neck contracture .", "the patients who had a history of previous surgery for ppi were not included in the study .", "incontinence in the patients was defined according to the use of incontinence pads over a 24 hours period : mild ( using 1 to 2 pads ) , moderate ( 3 to 5 pads ) and severe ( more than 5 pads ) ( 4 ) .", "the post - operative follow - up was carried out in the second week and every 6 months thereafter consisting of daily pad use , physical examination , maximum urine flow rate ( qmax ) and post - voiding residual urine volume ( pvr ) measurement ."], ["all patients received prophylactic antibiotics ( 3rd generation cephalosporin ) before the induction of anesthesia . under general or regional anesthesia", "after the insertion of a urethral catheter , a midline perineal incision of 40 to 50 mm was made .", "the critical point is not to remove periurethral fat tissues from urethra . both sides of urethra", "a eurosilicone ( laboratoires eurosilicone , france ) tissue expander device was used with an adjustable male sling in our patients .", "the device contains a silicone balloon expander , a connecting tube and an inflation component ( injection port ) that allows the expander to gradually fill up to a volume of 25 ml saline solution ( figure-1b ) . \n", "figure 1a ) both sides of urethra are dissected ; b ) a balloon expander ; c ) a balloon expander between two layers of the mesh ; d ) the mesh is stapled to the pubic bone using autosuture stat tacktm ; e ) prepared scrotal pouch for placing the inflation component . \n  a standard polypropylene mesh of 100 x 100 mm was used as the sling material .", "first , the mesh was folded and a balloon expander was placed between the two layers of the modification .", "secondly , the balloon was completely filled with the saline solution , and the two layers of the mesh were sutured using 2/0 polypropylene suture to create a pocket between the two layers of the mesh ( figure-1c ) . during this procedure", "third , the mesh was stapled on both sides of the lower border of the pubic bone using an autosuture stat tacktm ( tyco healthcare , uk ) stapler ( figure-1d ) . the original surgical method defined by inci , et al .", "( 5 ) used a polypropylene mesh that was fixed on the pubic bone with non - absorbable sutures . in our case , this method was modified and staples were used to prevent the sutures and the mesh from loosening and moving . after the sling tension was properly adjusted , the sling was placed as tightly as possible in all patients since urinary leakage had been observed in our patients postoperatively .", "the inflation component has two sides ( figure-1e ) . finally , the wound was closed with careful hemostasis .", "after the surgery , all the patients were asked to take an oral antibiotic ( 2th generation cephalosporin ) for 7 days .", "post - operative success was assessed by the number of pads used per 24 hours as follows ; zero to 1 safety pad - dry , 1 to 2 pads - mild , and 3 to 5 pads - moderate . at a minimum of 1 week after surgery", ", patients were questioned regarding their daily pad use . if incontinence persisted 7 to 10 days after surgery , tension over the urethra was increased by saline injection to expand the tissue expander via the injection port using an insulin needle .", "injection was started with 3 cc and increased by 2 cc at each injection . in this way", "the patients were asked to return 2 days after each adjustment to confirm the status of continence .", "the mean values of the parameters were calculated using the statistical package for social sciences version 13.0 for windows ( spss inc . , chicago , il , usa ) ."], ["the mean values of the parameters were calculated using the statistical package for social sciences version 13.0 for windows ( spss inc . , chicago , il , usa ) ."], ["the mean age of the patients was 66.27.3 ( 50 - 79 ) years and the mean pad usage was 6.40.6 ( 6 - 8 ) per day .", "the mean volume of the postoperatively inflated balloon was 11.65.7 ( 5 - 25 ) ml .", "after the follow - up period , 16 of the 21 patients ( 76.2% ) were dry ( 11 patients , 0 pads ; 5 patients using safety pads ) , 3 patients ( 14% ) had mild degree ppi and 2 patients ( 9.8% ) had moderate degree ppi . in the last assessment of the patients , qmax and estimated pvr were found to be 15.64.7 ( 10 - 31 ) ml / s and 10 ml , respectively .", "in these two patients , the polypropylene mesh with balloon , connecting tube and inflation component were removed and they did not undergo any additional intervention . local scrotal infection developed around the inflation component in three patients . in these patients ,", "only the inflation component was removed , the connecting tube was clipped and the polypropylene mesh with inflated balloon was kept in place . in these patients , polypropylene mesh with inflated balloon provided suitable pressure on the urethra . in the follow - up period , they were completely dry .", "no complications occurred in relation to mesh erosion , voiding dysfunction , voiding difficulty or mechanical failure .", "six patients reported mild perineal pain in the early postoperative period but this was resolved using non - steroidal anti - inflammatory drugs ."], ["the rising elderly population and the increasing number of surgical interventions for prostate cancer mean that the incidence of ppi will rise .", "since its introduction in 1973 , the artificial urinary sphincter ( aus ) has been considered the gold standard treatment for stress urinary incontinence after prostatectomy , offering the patient the greatest chance of a cure ( 6 ) .", "the success rates of aus range from 59 to 90% ( 7 - 9 ) .", "although , aus is an effective and durable treatment , many patients are hesitant about implantation or refuse the procedure . most patients could not manipulate the scrotal pump ( 10 ) .", "furthermore , aus is expensive and requires a complex surgical procedure that is associated with significant rates of complications .", "a recent systematic review about aus reported that infection or erosion occurred in 8.5% of cases ( 3.327.8% ) , mechanical failure in 6.2% ( 213.8% ) , and urethral atrophy in 7.9% ( 1.928.6% ) .", "re - operation rate was reported to range from 14.8% to 44.8% ( 11 ) .", "the proact ( uromedica , us ) system was first described in 2005 by hubner et al . ( 12 ) . in theietlr study , there were 117 patients with ppi followed for a mean period of 13 months , after which 67% of the patients were found to be dry .", "( 13 ) reported the results of the proact intervention in 62 men with ppi and found that 71% of the patients wore no pads or used 1 pad per day after 6 months ( following the adjustment ) .", "however , in 19 men the device was removed due to infection and erosion ( n=5 ) , migration ( n=1 ) , and iatrogenic traumatism ( n=2 ) . moreover ,", ", the authors evaluated the results of proact in 114 men with ppi at a mean follow - up time of 58 months and reported an overall dry rate of 50% . in that study , complications included balloon leakage ( 11% ) , migration ( 5% ) and wound erosion ( 4% ) .", "the authors reported that there was a total re - operation rate of 27% , and 12% of the patients underwent aus or a urethral sling procedures due to proact not being effective .", "the argus ( promedon sa , argentina ) sub - urethral sling with an adjustable system is another treatment option in men with ppi that was first described by romano et al .", "the authors reported a cure rate of 73% and the improvement rate was 10% in 48 men with ppi after a mean follow - up of 7.5 months .", "( 16 ) reported mid - term complications after the placement of the argus sling in 29 men with ppi at a mean follow - up of 35 months .", "overall , 24 patients ( 83% ) experienced complications , consisting of acute urinary retention ( 35% ) and removal of the sling ( 35% ) owing to urethral erosion , infection , system dislocation , urinary retention , and persistent pain . furthermore , 27% of the patients complained of significant perineal pain .", "the authors concluded that the argustm sub - urethral sling was associated with serious mechanical and infectious complications , and sparse functional results with negative impact on the patient s quality of life .", "a bone anchored sling ( bas ) compresses the bulbar urethra with a silicone - coated polypropylene mesh by attaching the sling to the inferior pubic ramus with bone screws .", "following initial reports of degradation of organic materials , synthetic mesh ( invance , american medical systems , us ) has become the most commonly utilized material with the bas ( 17 ) .", "( 4 ) reported their results in 46 patients with ppi who undergo bas implantation . in their study , the total cure rate was 37% . furthermore ,", "37% of the patients significantly improved , and the treatment of 26% patients failed after an average of 24 months follow - up .", "they concluded that the male sling procedure is an effective and safe procedure for the management of stress urinary incontinence .", "guimaraes et al . ( 18 ) reported their intermediate results of up to 4 years with the invance sling .", "their cure rate was 65% and the improvement rate was 23% in 54 men with ppi after a mean follow - up of 28 months .", "the authors claimed that the invance sling offers a good intermediate cure and improvement rates with acceptably low rate of complications in patients suffering from ppi .", "another study investigated the use of the invance sling in 40 patients with ppi ( 19 ) .", "the authors observed perineal pain in 73% of the patients , detrusor overactivity in 5% and sling infection in 15% .", "the traditional bas procedure does not provide for the adjustment of the tension of the sling material in the post - operative period and this can result in progressive failure over time . in a study by castle et al .", "social continence was achieved in 67% 50% and 0% of mild , moderate and severe cases , respectively .", "comiter ( 20 ) speculated that the surgeons left the fatty tissue over the bulbospongious intact , and additionally used a piece of porcine dermis between the urethra and the sling a combination of fat necrosis and absorption of the dermis over time likely contributes to diminution of the compression provided by the sling over the bulbar urethra .", "( 21 ) reported a median time to recurrent incontinence as 3 months after bas procedure .", "similarly , cespedes and jacoby reported an early failure ( within 6 months ) in 5% of their patients ( 22 ) . in another study", "( 23 ) , the authors retrospectively reported objective and subjective outcomes in 40 male patients who underwent bas positioning for stress urinary incontinence due to intrinsic sphincter deficiency .", "patients with stress urinary incontinence due to radical retropubic prostatectomy ( n=32 ) , robot assisted laparoscopic prostatectomy ( n=3 ) and transurethral prostate resection ( n=5 ) underwent the bas procedure over a 5 years period .", "previous anti - incontinence procedures , radiotherapy and transurethral procedures due to urethral stricture were performed in 5 , 11 and 5 patients , respectively . at a mean follow - up of 35.2 months , 22 patients ( 55% )", "were cured , the condition of 5 patients ( 12.5% ) improved and the treatment of 13 patients ( 32.5% ) failed .", "the authors concluded that bas is a simple and effective procedure that can produce immediate good results with low morbidity , especially when strictly selected patients are treated .", "an adjustable perineal male sling technique was used for the treatment of ppi as described previously ( 5 ) . in that study", "an adjustable perineal male sling with a 10 ml tissue expander was implanted in 19 consecutive men with severe ppi . according to the results of that study , at a mean follow - up of 17.3 months 15 patients ( 78.9% ) were completely dry and 2 ( 10.5% ) improved significantly using only 1 to 2 pads per day . a total of 11 patients required volume adjustment injections .", "the average number of adjustments was 2 ( range 1 to 3 ) and the average injected volume was 6.3 cc ( range 5 to 10 ) .", "regarding the complications , 1 patient had a superficial wound infection , 1 patient required surgical revision due to infection and 8 patients reported mild to moderate perineal pain .", "the authors concluded that the short term results indicate that this minimally invasive technique seems to be safe and effective , and patient satisfaction appears to be high .", "the same group presented their long term results in the international continence society annual meeting 2013 . in a study by ergen and ozdemir", "the mean follow - up time was 52 months . the completely dry rate decreased from 78.9% to 58% in the long term period .", "the authors concluded that adjustable male sling using a 10 ml tissue expander is an effective method for severe ppi , in the early period , but the effectiveness gradually decreases in the long term .", "the review of our intermediate results at 40.1 months showed that 16 of the 21 patients ( 76.2% ) were completely dry , 3 patients ( 14% ) had mild incontinence and 2 patients ( 9.8% ) had moderate incontinence .", "the success rate of the current study is better than that of the other studies mentioned above ( 5 , 24 ) .", "a possible explanation for our high success rate may be associated with the high capacity of the silicone balloon . in our patients", ", we used a larger size of silicone balloon ( 25 ml ) than the other two studies .", "additionally , we used a stapler which allows the easier attachment of the polypropylene mesh on the lower border of the pubic bone in both sides .", "although previous pelvic radiotherapy history has also been claimed to be a negative predictor of success rate in ppi surgery ( 19 , 23 ) , 2 patients in current study whom had a previous history of pelvic radiotherapy were dry in the intermediate follow - up .", "a new device is a perineal sling with a tissue expander , composed of 2 silicone components , a balloon expander and an injection port , connected by a tube . in turkey", ", this device is relatively cheap compared with aus , proact , argustm ( $ 500 vs. $ 5.000 vs. $ 2330 vs. $ 2400 ) and easy to implant , even in difficult cases ."], ["although complications occurred in this study , including the removal of the device and the removal of inflation component due to local scrotal infection , our intermediate follow - up study suggests that an adjustable perineal male sling using a tissue expander appears to be an efficient and safe alternative surgical treatment option for patients suffering from ppi .", "the major limitations of our study were the small number of patients and the duration of the follow - up period .", "additional follow - up and larger series of patients are necessary to confirm our results ."]]}
{"article_id": "PMC5333001", "article_text": ["fibromyalgia syndrome ( fms ) is a prevalent chronic pain condition among middle - aged females \n and is characterized by chronic widespread pain .", "various comorbid symptoms of fms include \n fatigue , cognitive disturbances , depression , sleep impairments , and weight gain , to name but \n a few .", "it is a challenging disorder , and thus assessment and diagnosis might pose some \n difficulties in various health care settings .", "patients may visit their general practitioners \n repeatedly with numerous symptoms before a diagnosis of fms is made1 , 2 .", "patients are encountered at not only primary but also secondary health care settings and by \n various subspecialities including rheumatologists , pain specialists , physiatrists , and \n psychologists .", "this has led to debate about whether fms is a clinical or epidemiological \n disease3 .", "the 1990 american college of rheumatology ( acr ) criteria included 2 major sections : history \n of widespread pain and pain in 11 of 18 tender points on digital palpation .", "debates over \n reliability , validity , and number of tender points required to make a diagnosis were \n settled .", "many controversies arose over tender points4 , and some authors argued against tender points , claiming that they \n are a measure of general stress3 , 5 , 6 .", "it \n was then suggested that dealing with the symptoms rather than tender points would be more \n realistic and that use of 1990 criteria should be stopped7 .", "the acr 2010 criteria eliminated the controversial tender point examination and included \n the following headings : widespread pain index , including 19 pain locations , and symptom \n severity score for 3 symptoms plus the extent of 41 somatic symptoms in general .", "2010 \n criteria further modified to the 2011 modified criteria ( 2011modcr ) with 6 self - reported \n symptoms instead of 41 somatic symptoms .", "the most recently developed criteria are the \n alternative diagnostic criteria ( acr 2013altcr ) , which include more pain locations than the \n 2011modcr ( 28 instead of 19 ) and 10 symptoms instead of 6 . compared to the 2011modcr ,", "the \n acr 2013altcr have comparable diagnostic sensitivity , better specificity , and a smaller \n number needed to diagnose8 . in addition to the search for best criteria , some other tools were developed for \n epidemiological studies such as the london fibromyalgia study screening questionnaire or \n  survey criteria .", " however , they included only items related to pain and fatigue , neglecting \n other aspects of the condition9 .", "to \n address these problems , some screening procedures have been suggested ; however , they have \n been argued against due to psychometric validation problems and sensitivity and specificity \n issues10 .", "thus , a need for a short , easy - to - use , psychometrically validated screening tool has arisen \n in both clinical and research settings .", "the french rheumatic pain study group decided to \n develop a short , psychometrically sound , self - reported screening tool considering the major \n symptoms and aspects of fms9 .", "the original \n version of the fibromyalgia rapid screening tool ( first ) was demonstrated to have excellent \n discriminative properties , especially divergent validity in terms of psychological aspect , \n which is an important property for an fms screening tool9 . the spanish version of the first", "was also demonstrated to have \n acceptable internal consistency , reliability , and criterion validity10 .", "we aimed to study the reliability and validity of the turkish version of the first by \n correlating this tool with the acr 2013altcr and the hospital anxiety and depression scale \n ( hads ) .", "two hundred and sixty - nine patients ( 257 females and 12 males ) with an average age ( years ) \n of 48.29  12.78 ( range 2279 ) were included in the study .", "the principles of the declaration of helsinki were followed \n and the study protocol was approved by the uludag university ethical committee ( no : \n fr - hyh-19 ) .", "subjects were recruited from outpatient physical medicine and rehabilitation \n clinics from 9 medical faculty hospitals and ministry of health research and training \n hospitals .", "a convenience sample of patients with chronic diffuse pain was included in the study . \n", "literate patients who could read and understand turkish and who were able to complete the \n questionnaire were invited to participate in the study .", "patients with psychiatric disorders ( severe depression , schizophrenia ) or systemic or \n neurological disorders that could adversely affect quality of life or social functioning \n ( such as end - stage heart failure , hemiplegia , spinal cord injury , etc . ) were excluded from \n the study .", "we obtained permission from mapi research trust ( contact information and permission to use : \n mapi research trust , lyon , france .", "e - mail : proinformation@mapi-trust.org-internet : \n www.proqolid.org . ) cross - cultural adaptation was accomplished according to the suggestions \n of the mapi research institute in 3 steps : forward translation , backward translation , and \n patient testing .", "the first was translated into turkish by 2 professional native turkish speakers bilingual \n in turkish and english .", "they discussed on the translated forms with the local project \n manager ( r.c . ) to resolve discrepancies and produced a pooled version , which is the first \n version of the translation ( step 1 ) .", "then , english back - translation was done by the \n professional native english - speaking translator bilingual in english and turkish .", "he \n translated the first version of the questionnaire back to english without access to the \n original questionnaire .", "the local project manager compared the backward version with the \n original during a meeting with the backward translator and prepared the second version ( step \n 2 ) .", "the second version was tested on 5 patients , all of whom were native turkish speakers , \n and comprehension was determined through face - to - face interviews . after this final step , a \n third version of the questionnaire", "patients were asked to complete the questionnaire including the first , 2013altcr , and hads . \n", "they completed the first twice ; the time interval for the 2 first scales was 6 hours .", "the \n first includes 6 items , and a score of 1 is given for the response of  yes  and 0 if the \n response is  no  for each item .", "the total score is calculated as the sum of scores ; the \n cut - off value is designated as 5/69 .", "the examiners were physiatrists and they were blinded to the results of first and hads .", "we \n used the acr 2013altcr8 , which includes \n the pain location inventory ( pli ) including 28 sites and the symptom impact questionnaire \n ( siqr ) consisting of 10 symptom items ( pain ; energy ; stiffness ; sleep ; depression ; memory \n problems ; anxiety ; tenderness to touch ; balance problems ; and sensitivity to loud noises , \n bright colors , odors , and cold ) .", "pli score is between 0 and 28 , and the siqr range is 0100 \n divided by 2 . for a patient to fulfill the acr 2013altcr criteria , the symptoms and pain \n locations should have been persistent for at least 3 months , pli17 and siqr21 .", "one for anxiety and the \n other for depression  and is a questionnaire completed by the patient .", "each item has 4 \n possible answers ( range 03 ) , and the maximum possible score is 21 .", "the validity and \n reliability of the turkish version of the hads was demonstrated previously11 . concurrent validity of anxiety subscale \n with spielberger s trait anxiety inventory , correlation coefficient was 0.7544 and of \n depression subscale with beck depression inventory it was 0.7237 .", "testing the reliability of \n had scale , cronbach alfa coefficient for anxiety subscale was 0.8525 and for depression \n subscale it was 0.778411 .", "we analyzed the data using the statistical package for the social sciences ( spss ) version \n 16 ( chicago , il , usa ) .", "the reliability of the \n hads was tested using cronbach s alpha coefficient . for criterion validity of the first , we \n used the acr 2013altcr for comparison .", "the \n concordance between the acr 2013altcr and the first was evaluated using a mcnemar test .", "the sensitivity , specificity , and positive and negative likelihood ratios of the first were \n calculated according to acr 2013altcr criteria .", "we compared \n the 2 first measurements using wilcoxon signed rank test , and the 2 measurements were \n statistically similar ( p=0.169 ) .", "one hundred fifty - six ( 58% ) patients had score of 5 , which is the cut - off score for the \n first ; 116 of these patients ( 74.4% ) were diagnosed as having fms according to acr 2013altcr \n criteria .", "one hundred thirteen patients ( 42% ) had first scores lower than 5 , and 93 of these \n patients ( 82.3% ) did not meet the 2013altcr criteria .", "the first was not statistically \n different from the acr 2013 altcr in defining patients with fms ( table 1table 1.sensitivity , specificity , and positive and negative likelihood ratios of the \n first to discriminate patients with and without fms according to the 2013 \n altcrvalueconfidence intervalsensitivity83.8276.589.6specificity68.4259.876.2+lr2.652.33.0lr0.240.10.4lr : likelihood ratio ) .", "first total score was correlated with \n subscores of the acr 2013altcr , namely pli ( r=0.619 , p<0.001 ) and siqr scores ( r=0.408 , \n p<0.001 ) .", "similarly , the first and hads were significantly correlated ( r=0.424 , \n p<0.001 ) ( table 2table 2.the coefficient of determinations between first total score and acr 2013 \n subscores ( pli and siqr score ) and hads scores.plisiqrhadsfirst total score0.383 * 0.166 * 0.18*pli0.196 * 0.131*siqr0.194*data expressed as squared spearman s rho correlation coefficients .", "first : \n fibromyalgia rapid screening tool ; hads : hospital anxiety and depression scale ; pli : \n pain location inventory ; siqr : symptom impact questionnaire , * p<0.001 statistically \n significant ) .", "first : \n fibromyalgia rapid screening tool ; hads : hospital anxiety and depression scale ; pli : \n pain location inventory ; siqr : symptom impact questionnaire , * p<0.001 statistically \n significant logistic regression analysis was carried out to find the confounding effect of the hads on \n the first to discriminate the patients with fms .", "the model was able to discriminate patients \n with fibromyalgia and without fibromyalgia ( 2 log likelihood=274.82 ; =95.29 , \n d.f=2 ; p < 0.001 ; nagelkerke r= 0.4 ) .", "the first global score explained 30% of \n the proportion of uncertainty ; each point increase in first global score meant 10 times \n greater odds of experiencing fms .", "in this study , we showed that the turkish version of the first is a reliable \n patient - completed instrument for identifying patients with fms .", "the acr 2013altcr was used \n for comparison , and the first was similar to the acr criteria in terms of defining patients \n with fms .", "the first demonstrated high correlation with acr 2013altcr subscores and hads \n scores . in our study , we examined the consistency of the first over time .", "we used the acr 2013altcr because these criteria were equally efficient with somewhat \n better specificity , and a smaller number was needed to diagnose than in the 2011modcr8 .", "the acr 2013altcr were also marginally more \n efficient in differentiating common chronic pain disorders from fms8 .", "torres et al.10 used acr 1990 criteria , and perrot et al.9 stated that they diagnosed patients on the basis of acr \n criteria , referring to the article by wolfe et al . , who used the symptom intensity scale , \n which is a combination of pain counts in 19 nonarticular regions with a visual analogue \n scale for fatigue12 .", "torres et al . reported the sensitivity of the first as 90.5% and its specificity as 85.7% . \n", "the turkish version demonstrated similar sensitivity and specificity . in this study by \n torres et al .", "positive likelihood ratio was calculated as 1.99 and negative likelihood ratio \n as 0.2 for cut - off point of 5 which is the established score for the original version of \n first . in our study", "positive and negative likelihood ratio values were 2.65 and 0.24 \n respectively , with fairly narrow confidence intervals .", "our study group was similar to torres \n et al.10 ; the majority of the patients in \n the study by torres were female , as in our study group . in the original study of the first \n by perrot et al.9 ,", "first total score was \n not significantly correlated with the beck depression inventory , hads - depression , and \n hads - anxiety scores . in our study", ", we were able to demonstrate that first scores are \n correlated with hads scores ; however , despite the correlation of first with hads scores , the \n first was still able to discriminate between patients with and without fms .", "different from previous studies , we included patients with various chronic pain problems at \n a tertiary care level .", "fitzcharles et al . argued that , if patients in a study have an \n established diagnosis of fms , they may express symptom severity at the extreme end of the \n spectrum , thus increasing construct validity13 .", "similar to our study , bennett et al.8 included a wide range of common pain disorders as the dominating \n principle of their study and claimed to simulate everyday clinical practice .", "both rehabilitation specialists and physical therapists are dealing with various pain \n conditions . among these disorders", "fms is a prevalent condition and can be encountered \n secondarily as in case of rheumatologic disorders .", "first includes 6 items with either yes or \n no answer , thus enables easy and quick scoring .", "it can be used as an adjunct to physical \n examination or evaluation of patients undergoing physical therapy .", ". patients were recruited from tertiary care hospitals however study \n population is not limited to patients referred from primary care clinics .", "an important consideration for the first is ease of use and scoring in the settings \n involved with chronic pain patients .", "it can be completed in less than 3 minutes and seems to be acceptable and relevant for the \n patients10 . since it is a single page \n and evaluation requires simple addition , use of the first may be advantageous in a busy \n clinical setting such as surgical units .", "it was demonstrated that patients with fms were more likely to have surgical \n interventions including back or neck surgery14 .", "surgeons and interventionalists may also benefit from using an \n easy - to - administer patient - completed screening tool to avoid unnecessary procedures ."], "abstract_text": ["<S> [ purpose ] an easy - to - use , psychometrically validated screening tool for fibromyalgia is \n needed . </S>", "<S> this study aims to evaluate the reliability and validity of the turkish version of \n the fibromyalgia rapid screening tool by correlating it with 2013 american college of \n rheumatology alternative diagnostic criteria and the hospital anxiety and depression \n scale . </S>", "<S> [ subjects and methods ] subjects were 269 physical medicine and rehabilitation \n clinic outpatients . </S>", "<S> patients completed a questionnaire including the fibromyalgia rapid \n screening tool ( twice ) , 2013 american college of rheumatology alternative diagnostic \n criteria , and the hospital anxiety and depression scale . </S>", "<S> scale reliability was examined by \n test - retest . </S>", "<S> the 2013 american college of rheumatology alternative diagnostic criteria was \n used for comparison to determine criterion validity . </S>", "<S> the sensitivity , specificity , and \n positive and negative likelihood ratios were calculated according to 2013 american college \n of rheumatology alternative diagnostic criteria . </S>", "<S> logistic regression analysis was \n conducted to find the confounding effect of the hospital anxiety and depression scale on \n fibromyalgia rapid screening tool to distinguish patients with fibromyalgia syndrome . \n </S>", "<S> [ results ] the fibromyalgia rapid screening tool was similar to the 2013 american college \n of rheumatology alternative diagnostic criteria in defining patients with fibromyalgia \n syndrome . </S>", "<S> fibromyalgia rapid screening tool score was correlated with 2013 american \n college of rheumatology alternative diagnostic criteria subscores . </S>", "<S> each point increase in \n fibromyalgia rapid screening tool global score meant 10 times greater odds of experiencing \n fibromyalgia syndrome . </S>", "<S> [ conclusion ] the turkish version of the fibromyalgia rapid \n screening tool is reliable for identifying patients with fibromyalgia . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION", "Conflict of interest"], "sections": [["fibromyalgia syndrome ( fms ) is a prevalent chronic pain condition among middle - aged females \n and is characterized by chronic widespread pain .", "various comorbid symptoms of fms include \n fatigue , cognitive disturbances , depression , sleep impairments , and weight gain , to name but \n a few .", "it is a challenging disorder , and thus assessment and diagnosis might pose some \n difficulties in various health care settings .", "patients may visit their general practitioners \n repeatedly with numerous symptoms before a diagnosis of fms is made1 , 2 .", "patients are encountered at not only primary but also secondary health care settings and by \n various subspecialities including rheumatologists , pain specialists , physiatrists , and \n psychologists .", "this has led to debate about whether fms is a clinical or epidemiological \n disease3 .", "the 1990 american college of rheumatology ( acr ) criteria included 2 major sections : history \n of widespread pain and pain in 11 of 18 tender points on digital palpation .", "debates over \n reliability , validity , and number of tender points required to make a diagnosis were \n settled .", "many controversies arose over tender points4 , and some authors argued against tender points , claiming that they \n are a measure of general stress3 , 5 , 6 .", "it \n was then suggested that dealing with the symptoms rather than tender points would be more \n realistic and that use of 1990 criteria should be stopped7 .", "the acr 2010 criteria eliminated the controversial tender point examination and included \n the following headings : widespread pain index , including 19 pain locations , and symptom \n severity score for 3 symptoms plus the extent of 41 somatic symptoms in general .", "2010 \n criteria further modified to the 2011 modified criteria ( 2011modcr ) with 6 self - reported \n symptoms instead of 41 somatic symptoms .", "the most recently developed criteria are the \n alternative diagnostic criteria ( acr 2013altcr ) , which include more pain locations than the \n 2011modcr ( 28 instead of 19 ) and 10 symptoms instead of 6 . compared to the 2011modcr ,", "the \n acr 2013altcr have comparable diagnostic sensitivity , better specificity , and a smaller \n number needed to diagnose8 . in addition to the search for best criteria , some other tools were developed for \n epidemiological studies such as the london fibromyalgia study screening questionnaire or \n  survey criteria .", " however , they included only items related to pain and fatigue , neglecting \n other aspects of the condition9 .", "to \n address these problems , some screening procedures have been suggested ; however , they have \n been argued against due to psychometric validation problems and sensitivity and specificity \n issues10 .", "thus , a need for a short , easy - to - use , psychometrically validated screening tool has arisen \n in both clinical and research settings .", "the french rheumatic pain study group decided to \n develop a short , psychometrically sound , self - reported screening tool considering the major \n symptoms and aspects of fms9 .", "the original \n version of the fibromyalgia rapid screening tool ( first ) was demonstrated to have excellent \n discriminative properties , especially divergent validity in terms of psychological aspect , \n which is an important property for an fms screening tool9 . the spanish version of the first", "was also demonstrated to have \n acceptable internal consistency , reliability , and criterion validity10 .", "we aimed to study the reliability and validity of the turkish version of the first by \n correlating this tool with the acr 2013altcr and the hospital anxiety and depression scale \n ( hads ) ."], ["two hundred and sixty - nine patients ( 257 females and 12 males ) with an average age ( years ) \n of 48.29  12.78 ( range 2279 ) were included in the study .", "the principles of the declaration of helsinki were followed \n and the study protocol was approved by the uludag university ethical committee ( no : \n fr - hyh-19 ) .", "subjects were recruited from outpatient physical medicine and rehabilitation \n clinics from 9 medical faculty hospitals and ministry of health research and training \n hospitals .", "a convenience sample of patients with chronic diffuse pain was included in the study . \n", "literate patients who could read and understand turkish and who were able to complete the \n questionnaire were invited to participate in the study .", "patients with psychiatric disorders ( severe depression , schizophrenia ) or systemic or \n neurological disorders that could adversely affect quality of life or social functioning \n ( such as end - stage heart failure , hemiplegia , spinal cord injury , etc . ) were excluded from \n the study .", "we obtained permission from mapi research trust ( contact information and permission to use : \n mapi research trust , lyon , france .", "e - mail : proinformation@mapi-trust.org-internet : \n www.proqolid.org . ) cross - cultural adaptation was accomplished according to the suggestions \n of the mapi research institute in 3 steps : forward translation , backward translation , and \n patient testing .", "the first was translated into turkish by 2 professional native turkish speakers bilingual \n in turkish and english .", "they discussed on the translated forms with the local project \n manager ( r.c . ) to resolve discrepancies and produced a pooled version , which is the first \n version of the translation ( step 1 ) .", "then , english back - translation was done by the \n professional native english - speaking translator bilingual in english and turkish .", "he \n translated the first version of the questionnaire back to english without access to the \n original questionnaire .", "the local project manager compared the backward version with the \n original during a meeting with the backward translator and prepared the second version ( step \n 2 ) .", "the second version was tested on 5 patients , all of whom were native turkish speakers , \n and comprehension was determined through face - to - face interviews . after this final step , a \n third version of the questionnaire", "patients were asked to complete the questionnaire including the first , 2013altcr , and hads . \n", "they completed the first twice ; the time interval for the 2 first scales was 6 hours .", "the \n first includes 6 items , and a score of 1 is given for the response of  yes  and 0 if the \n response is  no  for each item .", "the total score is calculated as the sum of scores ; the \n cut - off value is designated as 5/69 .", "the examiners were physiatrists and they were blinded to the results of first and hads .", "we \n used the acr 2013altcr8 , which includes \n the pain location inventory ( pli ) including 28 sites and the symptom impact questionnaire \n ( siqr ) consisting of 10 symptom items ( pain ; energy ; stiffness ; sleep ; depression ; memory \n problems ; anxiety ; tenderness to touch ; balance problems ; and sensitivity to loud noises , \n bright colors , odors , and cold ) .", "pli score is between 0 and 28 , and the siqr range is 0100 \n divided by 2 . for a patient to fulfill the acr 2013altcr criteria , the symptoms and pain \n locations should have been persistent for at least 3 months , pli17 and siqr21 .", "one for anxiety and the \n other for depression  and is a questionnaire completed by the patient .", "each item has 4 \n possible answers ( range 03 ) , and the maximum possible score is 21 .", "the validity and \n reliability of the turkish version of the hads was demonstrated previously11 . concurrent validity of anxiety subscale \n with spielberger s trait anxiety inventory , correlation coefficient was 0.7544 and of \n depression subscale with beck depression inventory it was 0.7237 .", "testing the reliability of \n had scale , cronbach alfa coefficient for anxiety subscale was 0.8525 and for depression \n subscale it was 0.778411 .", "we analyzed the data using the statistical package for the social sciences ( spss ) version \n 16 ( chicago , il , usa ) .", "the reliability of the \n hads was tested using cronbach s alpha coefficient . for criterion validity of the first , we \n used the acr 2013altcr for comparison .", "the \n concordance between the acr 2013altcr and the first was evaluated using a mcnemar test .", "the sensitivity , specificity , and positive and negative likelihood ratios of the first were \n calculated according to acr 2013altcr criteria ."], ["we compared \n the 2 first measurements using wilcoxon signed rank test , and the 2 measurements were \n statistically similar ( p=0.169 ) .", "one hundred fifty - six ( 58% ) patients had score of 5 , which is the cut - off score for the \n first ; 116 of these patients ( 74.4% ) were diagnosed as having fms according to acr 2013altcr \n criteria .", "one hundred thirteen patients ( 42% ) had first scores lower than 5 , and 93 of these \n patients ( 82.3% ) did not meet the 2013altcr criteria .", "the first was not statistically \n different from the acr 2013 altcr in defining patients with fms ( table 1table 1.sensitivity , specificity , and positive and negative likelihood ratios of the \n first to discriminate patients with and without fms according to the 2013 \n altcrvalueconfidence intervalsensitivity83.8276.589.6specificity68.4259.876.2+lr2.652.33.0lr0.240.10.4lr : likelihood ratio ) .", "first total score was correlated with \n subscores of the acr 2013altcr , namely pli ( r=0.619 , p<0.001 ) and siqr scores ( r=0.408 , \n p<0.001 ) .", "similarly , the first and hads were significantly correlated ( r=0.424 , \n p<0.001 ) ( table 2table 2.the coefficient of determinations between first total score and acr 2013 \n subscores ( pli and siqr score ) and hads scores.plisiqrhadsfirst total score0.383 * 0.166 * 0.18*pli0.196 * 0.131*siqr0.194*data expressed as squared spearman s rho correlation coefficients .", "first : \n fibromyalgia rapid screening tool ; hads : hospital anxiety and depression scale ; pli : \n pain location inventory ; siqr : symptom impact questionnaire , * p<0.001 statistically \n significant ) .", "first : \n fibromyalgia rapid screening tool ; hads : hospital anxiety and depression scale ; pli : \n pain location inventory ; siqr : symptom impact questionnaire , * p<0.001 statistically \n significant logistic regression analysis was carried out to find the confounding effect of the hads on \n the first to discriminate the patients with fms .", "the model was able to discriminate patients \n with fibromyalgia and without fibromyalgia ( 2 log likelihood=274.82 ; =95.29 , \n d.f=2 ; p < 0.001 ; nagelkerke r= 0.4 ) .", "the first global score explained 30% of \n the proportion of uncertainty ; each point increase in first global score meant 10 times \n greater odds of experiencing fms ."], ["in this study , we showed that the turkish version of the first is a reliable \n patient - completed instrument for identifying patients with fms .", "the acr 2013altcr was used \n for comparison , and the first was similar to the acr criteria in terms of defining patients \n with fms .", "the first demonstrated high correlation with acr 2013altcr subscores and hads \n scores . in our study , we examined the consistency of the first over time .", "we used the acr 2013altcr because these criteria were equally efficient with somewhat \n better specificity , and a smaller number was needed to diagnose than in the 2011modcr8 .", "the acr 2013altcr were also marginally more \n efficient in differentiating common chronic pain disorders from fms8 .", "torres et al.10 used acr 1990 criteria , and perrot et al.9 stated that they diagnosed patients on the basis of acr \n criteria , referring to the article by wolfe et al . , who used the symptom intensity scale , \n which is a combination of pain counts in 19 nonarticular regions with a visual analogue \n scale for fatigue12 .", "torres et al . reported the sensitivity of the first as 90.5% and its specificity as 85.7% . \n", "the turkish version demonstrated similar sensitivity and specificity . in this study by \n torres et al .", "positive likelihood ratio was calculated as 1.99 and negative likelihood ratio \n as 0.2 for cut - off point of 5 which is the established score for the original version of \n first . in our study", "positive and negative likelihood ratio values were 2.65 and 0.24 \n respectively , with fairly narrow confidence intervals .", "our study group was similar to torres \n et al.10 ; the majority of the patients in \n the study by torres were female , as in our study group . in the original study of the first \n by perrot et al.9 ,", "first total score was \n not significantly correlated with the beck depression inventory , hads - depression , and \n hads - anxiety scores . in our study", ", we were able to demonstrate that first scores are \n correlated with hads scores ; however , despite the correlation of first with hads scores , the \n first was still able to discriminate between patients with and without fms .", "different from previous studies , we included patients with various chronic pain problems at \n a tertiary care level .", "fitzcharles et al . argued that , if patients in a study have an \n established diagnosis of fms , they may express symptom severity at the extreme end of the \n spectrum , thus increasing construct validity13 .", "similar to our study , bennett et al.8 included a wide range of common pain disorders as the dominating \n principle of their study and claimed to simulate everyday clinical practice .", "both rehabilitation specialists and physical therapists are dealing with various pain \n conditions . among these disorders", "fms is a prevalent condition and can be encountered \n secondarily as in case of rheumatologic disorders .", "first includes 6 items with either yes or \n no answer , thus enables easy and quick scoring .", "it can be used as an adjunct to physical \n examination or evaluation of patients undergoing physical therapy .", ". patients were recruited from tertiary care hospitals however study \n population is not limited to patients referred from primary care clinics .", "an important consideration for the first is ease of use and scoring in the settings \n involved with chronic pain patients .", "it can be completed in less than 3 minutes and seems to be acceptable and relevant for the \n patients10 . since it is a single page \n and evaluation requires simple addition , use of the first may be advantageous in a busy \n clinical setting such as surgical units .", "it was demonstrated that patients with fms were more likely to have surgical \n interventions including back or neck surgery14 .", "surgeons and interventionalists may also benefit from using an \n easy - to - administer patient - completed screening tool to avoid unnecessary procedures ."], [""]]}
{"article_id": "PMC3807520", "article_text": ["overweight and obesity are conditions that have been increasing in the united states and these risk factors for diseases are estimated to affect more than one - half of those over the age of 20 . over the last few years", "obesity is a major public health concern because it increases the risk for many chronic conditions , such as cardiovascular diseases , particularly diabetes , hypertension , coronary artery disease , and cancer [ 1,811 ] .", "public health officials have begun to monitor the health status of school - aged children because it is believed that many of them exhibit risk factors that create the potential for developing chronic disease as adults . in 1994 ,", "10.4% among 2 to 5-year - old children , compared to 11.3% and 7.2% respectively for the same age groups in 1988 .", "this increase in overweight children has underscored the challenges faced by young people as they grow into an adult group where over 50% are overweight , and at significant risk for developing cardiovascular and other metabolic disorders [ 1 , 12 ] .", "effective management of risk factors among the youth , such as reducing / eliminating improper dietary practices and incorporating adequate physical activity , must become a universal health priority in order to effectively precipitate any noticeable decline in the adult morbidity and mortality statistics .", "childhood obesity is also associated with several immediate health risk factors , such as orthopedic , neurological , pulmonary , gastroenterological , and endocrine conditions .", "obesity has been linked to negative psychosocial outcomes in children , such as low self - esteem and depression [ 1523 ] , making obesity indirectly linked to academic performance and adverse social outcomes over time [ 2429 ] .", "cardiovascular disease is not only the leading cause of death , but also the single greatest contributor to excess mortality in african - americans .", "unhealthy eating contributes to at least 300,000 preventable deaths each year , and is one of the most identifiable contributors to premature death , second only to tobacco use .", "premature deaths due to cardiovascular disease remain higher for african americans than any other group [ 3137 ] .", "the highest rates of premature coronary heart disease in adults age 3564 occurred in the rural southern region of the united states .", "the rate of cvd for african americans in mississippi was deemed serious enough to compel the national institutes of health ( nih ) to initiate the jackson heart study to focus on identifying risk factors for the development of cvd in african - americans . among the known risk factors for cvd", "according to tom walden of the north american association for the study of obesity , schools can play a key role in preventing the risk behaviors by providing instruction on proper nutrition and physical activity .", "based on this premise , this study was designed to examine the students possible role in changing their own negative practices , by first assessing their perceptions of the obstacles to positive dietary practices and increased physical activity .", "the study also solicited students recommendations for reducing the negative practices that are associated with the rise in obesity and the development of cardiovascular diseases .", "the implementation of the study was intended to grant the students an opportunity to provide their own ideas , suggestions and recommendations for what they feel could be done to improve their quality of life and to prevent premature morbidity and mortality .", "the sample for this study included 300 students from a high school , located in rural , mississippi .", "the high school under investigation in this study educates students in grades 912 , and all students enrolled in the health and physical education courses comprised the sample for this project .", "students were first asked to complete an assent form and to have their parents sign a consent form before they could participate in the survey .", "only students who returned completed assent and consent forms were permitted to participate in the study .", "the data for the study were obtained from the administration of the project health high school survey ( phhss ) , which was a modification of the mississippi youth risk behavior survey ( yrbs ) that was developed by the centers for disease control and prevention ( cdc ) .", "the phhss was designed to measure the prevalence of risk behaviors associated with leading causes of illness and death .", "the survey was modified to include three additional questions that served to fulfill the objectives of this study .", "these questions specifically asked students to respond about their perceptions , and were the following : ", "what do you think is keeping you or other students from eating more nutritious , healthier foods ?", "  what do you think is preventing you or other students from taking part in daily physical activity ?   what suggestions do you have that would help students to start eating better and exercising more ?  these were open - ended questions and the students had the freedom to think and enumerate their perceptions .", "the students were assured that their responses would be confidential and their honest responses could shed some light on a perplexing health dilemma that currently faced public health officials .", "all students enrolled in health and physical education classes , which included students in the 912 grades , were administered the phhss survey during the regular classroom session by the regular health and physical education teacher .", "the students responses on each of these issues were recorded and analyzed to fully understand their perceptions and recommendations .", "this descriptive study sought to ascertain the students perceptions through the use of questions that were presented in the form of a self - administered questionnaire . upon completion of the survey ,", "the statistical package for the social sciences ( spss ) was used to evaluate the student responses .", "the high school students perceptions about current practices and possible solutions were calculated and presented in the tables that follow .", "the students perceptions on each of the areas under investigation were recorded and reported based on the rankings ( highest number of responses received and recorded ) from highest to lowest .", "completed phhss surveys were returned for 126 students which represented 42% of the 300 students enrolled in the health and physical education classes .", "all of the students were between the ages of 14 and 18 , and they were all enrolled in grades 912 .", "the breakdown according to race was as follows : 94.9% were african american ; 2.5% were hispanic ; 1.3% were asian ; and 1.3% were native americans , the participants were 69% female and 31% male .", "students at the high school were asked to respond to the question  what do you think is keeping you or other students from eating better ( more nutritious foods ?  figure 1 provides a graphical image of the students responses about this issue .", "love of sweets , junk food and fatty foods occupied the highest ranked reason selected by 19.8% of the students .", "the students also admitted that most of them ate what they were accustomed to eating .", "they ate what they liked to eat , and the decision about what to eat was made because of the taste of the food without regard for any health consequence or negative health outcomes .", "the number two reason for not eating healthy was that students believed that the food they were served in the school s cafeteria was of poor quality : 15.7% of the students responses supported this view .", "the number three reason given for not eating more nutritious foods was family background , family customs and family heritage : 14.1% of the students acknowledged that they ate the way they did simply because they inherited those patterns from their family .", "in other words , they ate what their parents could afford to buy and prepare for meals .", "many of them asserted that they were simply not accustomed to eating nutritious foods daily .", "these students stated that they buy the types of food they want , and , in some instances , they want the fast foods more than anything else .", "the students placed fast food restaurants as number five among the reasons why they found it difficult to practice positive eating habits .", "ranked number six as a factor that inhibits them from healthy eating habits is the easy access to junk foods and other unhealthy foods : 6.6% of them blamed the easy accessibility of junk foods as a perpetrator of the unhealthy dietary choices and practices .", "another 6.6% of the students cited lack of care and guidance from responsible adults as a factor that prevents them from practicing good dietary habits .", "these students admitted that pressure from videos and corporate advertisements helped to steer them in the wrong direction in reference to the choices they make .", "the negative influences perpetrated by famous actors , sports personalities and performers usually project the wrong messages and oftentimes influence many students to act spontaneously and without proper judgment . as a result ,", "many of them make the decision to eat daily at fast food restaurants , where they often consume an over abundance of unhealthy meals . several students reported that there was no one in their life to motivate them with positive messages or inspiration , and not many people cared enough .", "the number eight ranked reason for students not practicing positive eating habits was the presence of snack machines at school , laden with junk food .", "about 5.0% of the students cited this as a possible obstacle to good eating practices .", "approximately 19.8% of the students did not express an opinion about what is keeping them and other students from eating better ( more nutritious foods ) .", "the students were also asked  what do you think is keeping you or other students from taking part in sufficient exercise ( daily physical activity ) ? ", "the number one reason offered by students for inadequate participation in physical activity is laziness .", "some students insisted that there are many students who believe that they are too  cute  to dress in workout gear , and do not want to sweat during physical activity .", "approximately 38.9% of the students perceived that as the main reason why many students do not participate in activities today .", "the next reason cited by 14.3% of the students was that they preferred to hang out with friends rather than participate in physical activity . since it is not required daily", "many of them would prefer to attend parties , talk on the telephone , ride around in cars or watch television , rather than participate in physical activity . and , besides , as some of them insisted , the school has not been helpful in encouraging them to look at the positive benefits of daily exercising .", "the third ranked reason was that they simply have no desire to take part in physical activity ( see figure 2 ) .", "about 6.3% of the students cited self esteem issues and the feeling of embarrassment as a major contributing factor .", "the number five ranked reason for non - participation was the volume of homework assignments they have .", "these students felt that they just do not have the time to participate in physical activity .", "approximately 3.2% of the students explained that their inadequate participation is due to a lack of adequate mixture of games available at school .", "health problems were cited by 2.4% of the students as obstacles , and 1.6% of them indicated that disagreements with the gym teacher resulted in their non - participation .", "a small number of students believe that being fat or overweight was a contributing factor to their failure to participate in physical activity . there were approximately 19.0% of the students who had no opinion about what was keeping them and other students from taking part in sufficient exercise ( daily physical activity ) .", "the students also responded to the question  what do you think is needed to help students to start eating better and exercising more ?", " the number one suggestion given by students for improving their quality of life was that the school should provide better and more nutritious cafeteria food ( see figure 3 ) .", "this was equaled in number by the need for guidance by school personnel and parents in the area of healthy eating practices .", "they felt that the adults should show more interest and concern , and do more to provide adequate instruction and direction , possibly implementing new programs or activities and increased time in health class to address these important issues .", "they also believed that the parents have a major responsibility to lead the way by providing positive alternatives .", "this was followed by the need for more encouragement and motivation , as expressed by 14.1% of the students .", "about 11.3% of the students felt that the school should increase the volume of physical activity and exercise offered to students daily .", "number five among the students suggestions was the recommendation that the school make exercise mandatory for students .", "the students felt that , as incentives , school officials should use prizes , grades or money to encourage students to participate .", "another 6.3% of the students suggested that school should make exercise and nutrition programs more fun to encourage students to participate .", "some students concluded that increased conversation about the topic would lead to heightened motivation among students to participate in activities that are offered in their health and physical education classes .", "about 4.2% of the students believed that parents and teachers do not care enough to provide them with adequate guidance and opportunity .", "the students felt that the school needs to implement a healthy life program within its curriculum .", "approximately 4.2% of the students indicated that the students themselves should eliminate junk food and fast foods from their daily lunch menus .", "another group of students suggested that more positive advertising of nutritious foods should be encouraged and should be a major part of the plan to improve the students quality of life .", "students felt that they should be constantly apprised of the dangers of negative practices regarding poor eating practices and inadequate physical activity .", "this would facilitate their change to healthier daily practices which could possibly add years to their lives .", "approximately 11.3% of the students said they did not know the answer and could not present any suggestions .", "the major limitation of this study was the fact that only 42% of the total number of students eligible to participate in the study provided surveys that could be analyzed .", ". however , their responses are valuable enough to shed some light on obstacles to good health and possible solutions to improve the quality of life and develop ways of helping them to eliminate negative practices and reduce risk factors .", "the major limitation of this study was the fact that only 42% of the total number of students eligible to participate in the study provided surveys that could be analyzed .", ". however , their responses are valuable enough to shed some light on obstacles to good health and possible solutions to improve the quality of life and develop ways of helping them to eliminate negative practices and reduce risk factors .", "the interpretation of the students responses to these critical health issues identifies certain urgent needs that could and should be addressed by the academic institutions .", "many students understand that there may be negative consequences resulting from the daily choices that they make .", "it is clear that culture and family customs have a great influence on the direction students take in their dietary practices , as well as their involvement in physical activity .", "the convenience and easy accessibility of low nutritious foods is also seen as a contributing factor , as are the negative influences that are presented through advertising of non - nutritious foods .", "it is evident that adequate support and guidance from adults are not available often enough to facilitate the kind of learning and training that could ensure that students are afforded the opportunity to acquire knowledge to help them make positive responsible choices and decisions .", "so children have little desire to take the initiative to make improvements in their daily practices .", "the students suggested that modifying the schools curriculum to address student risk behaviors should become an embedded characteristic of their educational systems .", "the current absence of learning strategies to adequately address and educate students about the pitfalls of negative dietary and physical behaviors is a major shortcoming of the school s education system .", "recommendations for future studies should involve examining the responses of students enrolled in predominantly white institutions with those of the predominantly african american canton public school district . studies could also be designed to compare the responses of students from urban schools and rural schools to determine what their perceptions and recommendations are concerning risk factors and ways of reducing them ."], "abstract_text": ["<S> this study was designed to assess students perceptions of the obstacles to positive dietary practices and increased physical activity and to solicit the students recommendations for addressing and possibly reducing the negative practices that are associated with the rise in obesity and the development of cardiovascular diseases . </S>", "<S> data for the study were obtained from the administration of the 2005 project health high school survey ( phhss ) which measured the students perceptions regarding obstacles to eating more nutritious , healthier foods and obstacles to participating in daily physical activity . </S>", "<S> the reasons for students lack of interest in practicing more life - healthy behaviors are ranked and recorded . </S>", "<S> some of the students indicated that they usually ate what they liked to eat , and the decision about what to eat was made because of the taste of the food without regard for any health consequence or negative health outcomes . </S>", "<S> finding ways to reach these students at their young ages is the key to successfully combating the high prevalence of obesity and the development of other chronic diseases in childhood , as well as in adulthood . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Statistical Analysis", "Results", "Limitations", "Conclusion"], "sections": [["overweight and obesity are conditions that have been increasing in the united states and these risk factors for diseases are estimated to affect more than one - half of those over the age of 20 . over the last few years", "obesity is a major public health concern because it increases the risk for many chronic conditions , such as cardiovascular diseases , particularly diabetes , hypertension , coronary artery disease , and cancer [ 1,811 ] .", "public health officials have begun to monitor the health status of school - aged children because it is believed that many of them exhibit risk factors that create the potential for developing chronic disease as adults . in 1994 ,", "10.4% among 2 to 5-year - old children , compared to 11.3% and 7.2% respectively for the same age groups in 1988 .", "this increase in overweight children has underscored the challenges faced by young people as they grow into an adult group where over 50% are overweight , and at significant risk for developing cardiovascular and other metabolic disorders [ 1 , 12 ] .", "effective management of risk factors among the youth , such as reducing / eliminating improper dietary practices and incorporating adequate physical activity , must become a universal health priority in order to effectively precipitate any noticeable decline in the adult morbidity and mortality statistics .", "childhood obesity is also associated with several immediate health risk factors , such as orthopedic , neurological , pulmonary , gastroenterological , and endocrine conditions .", "obesity has been linked to negative psychosocial outcomes in children , such as low self - esteem and depression [ 1523 ] , making obesity indirectly linked to academic performance and adverse social outcomes over time [ 2429 ] .", "cardiovascular disease is not only the leading cause of death , but also the single greatest contributor to excess mortality in african - americans .", "unhealthy eating contributes to at least 300,000 preventable deaths each year , and is one of the most identifiable contributors to premature death , second only to tobacco use .", "premature deaths due to cardiovascular disease remain higher for african americans than any other group [ 3137 ] .", "the highest rates of premature coronary heart disease in adults age 3564 occurred in the rural southern region of the united states .", "the rate of cvd for african americans in mississippi was deemed serious enough to compel the national institutes of health ( nih ) to initiate the jackson heart study to focus on identifying risk factors for the development of cvd in african - americans . among the known risk factors for cvd", "according to tom walden of the north american association for the study of obesity , schools can play a key role in preventing the risk behaviors by providing instruction on proper nutrition and physical activity .", "based on this premise , this study was designed to examine the students possible role in changing their own negative practices , by first assessing their perceptions of the obstacles to positive dietary practices and increased physical activity .", "the study also solicited students recommendations for reducing the negative practices that are associated with the rise in obesity and the development of cardiovascular diseases .", "the implementation of the study was intended to grant the students an opportunity to provide their own ideas , suggestions and recommendations for what they feel could be done to improve their quality of life and to prevent premature morbidity and mortality ."], ["the sample for this study included 300 students from a high school , located in rural , mississippi .", "the high school under investigation in this study educates students in grades 912 , and all students enrolled in the health and physical education courses comprised the sample for this project .", "students were first asked to complete an assent form and to have their parents sign a consent form before they could participate in the survey .", "only students who returned completed assent and consent forms were permitted to participate in the study .", "the data for the study were obtained from the administration of the project health high school survey ( phhss ) , which was a modification of the mississippi youth risk behavior survey ( yrbs ) that was developed by the centers for disease control and prevention ( cdc ) .", "the phhss was designed to measure the prevalence of risk behaviors associated with leading causes of illness and death .", "the survey was modified to include three additional questions that served to fulfill the objectives of this study .", "these questions specifically asked students to respond about their perceptions , and were the following : ", "what do you think is keeping you or other students from eating more nutritious , healthier foods ?", "  what do you think is preventing you or other students from taking part in daily physical activity ?   what suggestions do you have that would help students to start eating better and exercising more ?  these were open - ended questions and the students had the freedom to think and enumerate their perceptions .", "the students were assured that their responses would be confidential and their honest responses could shed some light on a perplexing health dilemma that currently faced public health officials .", "all students enrolled in health and physical education classes , which included students in the 912 grades , were administered the phhss survey during the regular classroom session by the regular health and physical education teacher .", "the students responses on each of these issues were recorded and analyzed to fully understand their perceptions and recommendations ."], ["this descriptive study sought to ascertain the students perceptions through the use of questions that were presented in the form of a self - administered questionnaire . upon completion of the survey ,", "the statistical package for the social sciences ( spss ) was used to evaluate the student responses .", "the high school students perceptions about current practices and possible solutions were calculated and presented in the tables that follow .", "the students perceptions on each of the areas under investigation were recorded and reported based on the rankings ( highest number of responses received and recorded ) from highest to lowest ."], ["completed phhss surveys were returned for 126 students which represented 42% of the 300 students enrolled in the health and physical education classes .", "all of the students were between the ages of 14 and 18 , and they were all enrolled in grades 912 .", "the breakdown according to race was as follows : 94.9% were african american ; 2.5% were hispanic ; 1.3% were asian ; and 1.3% were native americans , the participants were 69% female and 31% male .", "students at the high school were asked to respond to the question  what do you think is keeping you or other students from eating better ( more nutritious foods ?  figure 1 provides a graphical image of the students responses about this issue .", "love of sweets , junk food and fatty foods occupied the highest ranked reason selected by 19.8% of the students .", "the students also admitted that most of them ate what they were accustomed to eating .", "they ate what they liked to eat , and the decision about what to eat was made because of the taste of the food without regard for any health consequence or negative health outcomes .", "the number two reason for not eating healthy was that students believed that the food they were served in the school s cafeteria was of poor quality : 15.7% of the students responses supported this view .", "the number three reason given for not eating more nutritious foods was family background , family customs and family heritage : 14.1% of the students acknowledged that they ate the way they did simply because they inherited those patterns from their family .", "in other words , they ate what their parents could afford to buy and prepare for meals .", "many of them asserted that they were simply not accustomed to eating nutritious foods daily .", "these students stated that they buy the types of food they want , and , in some instances , they want the fast foods more than anything else .", "the students placed fast food restaurants as number five among the reasons why they found it difficult to practice positive eating habits .", "ranked number six as a factor that inhibits them from healthy eating habits is the easy access to junk foods and other unhealthy foods : 6.6% of them blamed the easy accessibility of junk foods as a perpetrator of the unhealthy dietary choices and practices .", "another 6.6% of the students cited lack of care and guidance from responsible adults as a factor that prevents them from practicing good dietary habits .", "these students admitted that pressure from videos and corporate advertisements helped to steer them in the wrong direction in reference to the choices they make .", "the negative influences perpetrated by famous actors , sports personalities and performers usually project the wrong messages and oftentimes influence many students to act spontaneously and without proper judgment . as a result ,", "many of them make the decision to eat daily at fast food restaurants , where they often consume an over abundance of unhealthy meals . several students reported that there was no one in their life to motivate them with positive messages or inspiration , and not many people cared enough .", "the number eight ranked reason for students not practicing positive eating habits was the presence of snack machines at school , laden with junk food .", "about 5.0% of the students cited this as a possible obstacle to good eating practices .", "approximately 19.8% of the students did not express an opinion about what is keeping them and other students from eating better ( more nutritious foods ) .", "the students were also asked  what do you think is keeping you or other students from taking part in sufficient exercise ( daily physical activity ) ? ", "the number one reason offered by students for inadequate participation in physical activity is laziness .", "some students insisted that there are many students who believe that they are too  cute  to dress in workout gear , and do not want to sweat during physical activity .", "approximately 38.9% of the students perceived that as the main reason why many students do not participate in activities today .", "the next reason cited by 14.3% of the students was that they preferred to hang out with friends rather than participate in physical activity . since it is not required daily", "many of them would prefer to attend parties , talk on the telephone , ride around in cars or watch television , rather than participate in physical activity . and , besides , as some of them insisted , the school has not been helpful in encouraging them to look at the positive benefits of daily exercising .", "the third ranked reason was that they simply have no desire to take part in physical activity ( see figure 2 ) .", "about 6.3% of the students cited self esteem issues and the feeling of embarrassment as a major contributing factor .", "the number five ranked reason for non - participation was the volume of homework assignments they have .", "these students felt that they just do not have the time to participate in physical activity .", "approximately 3.2% of the students explained that their inadequate participation is due to a lack of adequate mixture of games available at school .", "health problems were cited by 2.4% of the students as obstacles , and 1.6% of them indicated that disagreements with the gym teacher resulted in their non - participation .", "a small number of students believe that being fat or overweight was a contributing factor to their failure to participate in physical activity . there were approximately 19.0% of the students who had no opinion about what was keeping them and other students from taking part in sufficient exercise ( daily physical activity ) .", "the students also responded to the question  what do you think is needed to help students to start eating better and exercising more ?", " the number one suggestion given by students for improving their quality of life was that the school should provide better and more nutritious cafeteria food ( see figure 3 ) .", "this was equaled in number by the need for guidance by school personnel and parents in the area of healthy eating practices .", "they felt that the adults should show more interest and concern , and do more to provide adequate instruction and direction , possibly implementing new programs or activities and increased time in health class to address these important issues .", "they also believed that the parents have a major responsibility to lead the way by providing positive alternatives .", "this was followed by the need for more encouragement and motivation , as expressed by 14.1% of the students .", "about 11.3% of the students felt that the school should increase the volume of physical activity and exercise offered to students daily .", "number five among the students suggestions was the recommendation that the school make exercise mandatory for students .", "the students felt that , as incentives , school officials should use prizes , grades or money to encourage students to participate .", "another 6.3% of the students suggested that school should make exercise and nutrition programs more fun to encourage students to participate .", "some students concluded that increased conversation about the topic would lead to heightened motivation among students to participate in activities that are offered in their health and physical education classes .", "about 4.2% of the students believed that parents and teachers do not care enough to provide them with adequate guidance and opportunity .", "the students felt that the school needs to implement a healthy life program within its curriculum .", "approximately 4.2% of the students indicated that the students themselves should eliminate junk food and fast foods from their daily lunch menus .", "another group of students suggested that more positive advertising of nutritious foods should be encouraged and should be a major part of the plan to improve the students quality of life .", "students felt that they should be constantly apprised of the dangers of negative practices regarding poor eating practices and inadequate physical activity .", "this would facilitate their change to healthier daily practices which could possibly add years to their lives .", "approximately 11.3% of the students said they did not know the answer and could not present any suggestions .", "the major limitation of this study was the fact that only 42% of the total number of students eligible to participate in the study provided surveys that could be analyzed .", ". however , their responses are valuable enough to shed some light on obstacles to good health and possible solutions to improve the quality of life and develop ways of helping them to eliminate negative practices and reduce risk factors ."], ["the major limitation of this study was the fact that only 42% of the total number of students eligible to participate in the study provided surveys that could be analyzed .", ". however , their responses are valuable enough to shed some light on obstacles to good health and possible solutions to improve the quality of life and develop ways of helping them to eliminate negative practices and reduce risk factors ."], ["the interpretation of the students responses to these critical health issues identifies certain urgent needs that could and should be addressed by the academic institutions .", "many students understand that there may be negative consequences resulting from the daily choices that they make .", "it is clear that culture and family customs have a great influence on the direction students take in their dietary practices , as well as their involvement in physical activity .", "the convenience and easy accessibility of low nutritious foods is also seen as a contributing factor , as are the negative influences that are presented through advertising of non - nutritious foods .", "it is evident that adequate support and guidance from adults are not available often enough to facilitate the kind of learning and training that could ensure that students are afforded the opportunity to acquire knowledge to help them make positive responsible choices and decisions .", "so children have little desire to take the initiative to make improvements in their daily practices .", "the students suggested that modifying the schools curriculum to address student risk behaviors should become an embedded characteristic of their educational systems .", "the current absence of learning strategies to adequately address and educate students about the pitfalls of negative dietary and physical behaviors is a major shortcoming of the school s education system .", "recommendations for future studies should involve examining the responses of students enrolled in predominantly white institutions with those of the predominantly african american canton public school district . studies could also be designed to compare the responses of students from urban schools and rural schools to determine what their perceptions and recommendations are concerning risk factors and ways of reducing them ."]]}
{"article_id": "PMC3830876", "article_text": ["vascular diseases are among the most common causes of morbidity and mortality , and both number and severity of morbid vascular conditions increase with age . regulations of angiogenesis , coagulation , and inflammation are very important issues in vascular biology , both in normal physiology and pathology .", "it is now well established that disruption of endothelial integrity represents a crucial event in the initiation and development of cardiovascular ( cv ) diseases .", "numerous studies have reported that microparticles ( mps ) play an important role in endothelial dysfunction .", "endothelial dysfunction occurs when a perturbed homeostatic endothelium disrupts vascular competency resulting in reduced vasodilatation and increased proinflammatory and prothrombotic properties of the vascular network .", "recently , mps originating from various cells have been found to be associated with several vascular related diseases .", "moreover , exposed procoagulant phospholipids and specific receptors at the surface of mps act as biomessengers linking inflammation , coagulation , and angiogenesis [ 35 ] .   although mps were first described as  cellular debris  that are believed to have no biological significance , recent studies documented that mps of endothelial and other origins are biological effectors in inflammation , vascular injury , angiogenesis , and thrombosis [ 68 ] .", "mps isolated from granulation tissue are derived from endothelial cells , monocytes , platelets , erythrocytes [ 913 ] , and myofibroblasts .", "they exchange biological signals and information intercellularly and each kind of mp carries the antigens and receptors of the cells they originated . mps may transfer part of their components and content to the selected target cells , thus mediating cell activation , phenotypic modification , and reprogramming of cell function .", "although 70% to 90% of all circulating mps in the peripheral blood of healthy individuals are derived from platelets , marked elevations of all kinds of mps have been observed in many vascular diseases .", "specifically , endothelium - derived microparticles ( emps ) represent a relatively small ( 515% ) but very important subset of all circulating microparticles [ 1618 ] .", "this number may vary in different cardiovascular and inflammatory diseases [ 18 , 19 ] .", "new insights into endothelial dysfunction and alterations in angiogenesis are emerging from studies of vascular microparticles , particularly endothelial microparticles in elderly populations .", "vascular aging with impairment of endothelial cell function leads to altered angiogenesis , a key factor in the etiology of various cardiovascular disorders .", "73% of individuals aged 6079 have a cv disease , including stroke , hypertension , or heart failure , and at > 79 years of age prevalence of these diseases increased to 86% in females and 82% in males ( 2012 nhlbi fact book ) .", "recently published data have shown that these diseases are the leading cause of death for individuals aged > 65   and morbidity increased from 32% for individuals aged 66 to 48% for individuals aged 85 . an important factor which significantly decreases the incidence of coronary heart diseases in postmenopausal women is estrogen [ 2224 ] . in women already having coronary artery disease or ischemic stroke , the therapeutic benefit of estrogen is not clear [ 25 , 26 ] although it has been reported that estrogen induces rapid vasodilation , exerts anti - inflammatory activity , and regulates vascular cell growth , migration , and protection of cardiomyocytes from injury , all of which prevent atherosclerotic deterioration in vessels .", "this review focuses on the role of emps in angiogenesis , coagulation , and inflammation during age - related vascular diseases and the contribution of estrogen to these diseases .", "emps are small vesicles that are released from endothelial cells and can be found circulating in the blood . defined by their small size ( 0.1 to 1.0  m ) , they are a heterogeneous population of vesicles which are shed from plasma membranes in response to cell activation , injury , angiogenesis / neovascularization , and/or apoptosis .", "emps consist of a small amount of cytosol surrounded by a plasma membrane and display negatively charged phospholipids on their surface that can initiate and accelerate coagulation .", "circulating emps have been demonstrated as a marker of preeclampsia [ 29 , 30 ] , acute coronary syndromes , and severe hypertension [ 30 , 32 , 33 ] suggesting their association with pathological processes within the endothelium .", "furthermore , circulating emp levels are also implicated in the progression of atherosclerotic lesions , heart failure , arrhythmias , inflammatory vascular disease , sickle anemia , and endotoxemia .  ", "jimenez et al . demonstrated that the surface antigens of emps are distinctive depending on the type of endothelial cell injury , as in apoptosis versus activation .", "high levels of the surface antigens e - selectin , intracellular adhesion molecule-1 ( icam-1 ) , and vascular cell adhesion molecule-1 ( vcam-1 ) are on emps derived from activated endothelial cells .", "in contrast , the low levels of these antigens are on emps derived from apoptotic endothelial cells .", "platelet cell adhesion molecule ( pecam-1 ) , endoglin , and vascular endothelial - cadherin ( ve - cadherin ) are at low levels on emps derived from activated ecs [ 3740 ] ( figure 1 ) .", "additionally , emps generated from apoptotic endothelial cells have higher levels of phosphatidylserine on their surface and different phospholipid composition and oxidation status compared with emps generated from activated endothelial cells [ 41 , 42 ] .", "these data suggest that there are distinct mechanisms for the formation of emps in apoptotic and activated cells   and several studies suggest that these types of emps have different functions in vascular diseases [ 40 , 44 ] .", "both inflammatory cytokines and coagulation factors participate in the generation of emps ( figure 2 ) .", "recently , it has been shown that p38 mitogen - activated protein kinase ( mapk ) is a critical molecule in the production of proinflammatory emps and increased icam-1 production by endothelial cells , providing a paracrine loop to enhance the endothelial response to inflammation . in vitro studies", "have shown that the proinflammatory agent , tnf , activates endothelial cells and induces release of emps   ( figure 2 ) .", "another potent stimulus for emp formation both in vivo and in vitro is angiotensin ii ( ang ii ) .", "this effect is mediated by ang ii receptor type i that signals through nadph oxidase and rho kinase .", "furthermore , in ang ii - infused apoliprotein e ( apoe / ) hyperlipidemic mice , a model of significant endothelial dysfunction , ang ii has been shown to increase emp formation by a redox - sensitive and blood - pressure - independent process .", "another important factor pai-1 ( plasminogen activator inhibitor type 1 ) plays an important role in the formation of emps .", "it has been shown by brodsky et al . that pai-1 promotes formation of emps with reduced transmembrane asymmetry of phospholipids in a dose dependent manner .", "these findings could possibly link elevated levels of pai-1 with endothelial dysfunction and tendency toward thrombosis [ 4850 ] . increased levels of pai-1", "might serve as an initiator of emp formation followed by increased procoagulant activity and thrombin generation .", "in addition , it is also known that thrombin stimulates pai-1 synthesis   suggesting constant production of these two factors .", "all these data indicate that formation of emps links together inflammation , coagulation , and angiogenesis and causes the impairment of the last two phenomena ( figure 3 ) . among many signaling molecules , t - cadherin ( t - cad ) on the surface of ecs", "might be upregulated and may serve as a characteristic marker of ec activation and stress .", "recently , philippova et al . have demonstrated a mechanism of t - cad - dependent signaling in the vascular endothelium .", "the authors identified that t - cadherin levels in plasma are increased in early atherosclerosis and correlate with endothelial dysfunction , which may lead to increased release of emps from ecs .", "increasing evidence has also accumulated to implicate an impaired coagulation system in many vascular diseases .", "this coagulation imbalance is the net result of activation of coagulation , impaired activity of natural coagulation inhibitors , and suppressed fibrinolysis .", "activation of coagulation proteases , for example , thrombin , is one of the earliest events following tissue injury .", "thrombin modulates tissue repair responses by altering vascular permeability , stimulating endothelial cell , fibroblast , and neutrophil migration , and promoting their spreading and adhesion .", "it activates various cell types and induces secretion of several proimmune , profibrotic , and proinflammatory factors [ 45 , 56 , 57 ] .", "recent studies by sapet et al . have shown that release of emps by endothelial cells in response to thrombin involves a group of genes that regulate angiogenesis and are linked to the cytoskeleton reorganization family . among these genes ,", "rho - kinase rock - ii was transcribed at a high rate and was identified as a target of thrombin in emp generation .", "the involvement of caspase-2 in rock - ii activation , independent of cell death , points out a novel signaling pathway that emphasizes the proteolytic activity of caspase in emp generation in response to cell activation   ( figure 1 ) . however , further studies are needed to determine the molecular mechanisms involved in emp release .", "emp release is initiated when thrombin binds to its receptor , proteolytically activated receptor-1 ( par-1 ) , which induces gene transcription that is mediated by thrombin via trail / apo2l , a cytokine belonging to the tumor necrosis factor alpha ( tnf ) superfamily .", "this mechanism of emp generation depends on the nuclear factor ( nf ) b activation and involves the soluble form of trail , which is secreted by the endothelial cells under thrombin or inflammatory stimulation [ 4 , 59 ] ( figure 2 ) .", "phospholipids expressed on emps bind coagulation factors leading to a prothrombotic state   and an increase in procoagulant activity of tissue factor ( tf ) .", "these phospholipids are exposed on the outer membrane of mp and are considered to be the main initiators of the coagulation cascade .", "additionally , it has been demonstrated that sphingosine 1-phosphate ( s1p ) strongly potentiates thrombin - induced tf expression in ecs suggesting its role in blood coagulation .", "s1p has also been shown to be involved in the process of angiogenesis and inflammation [ 6264 ] .", "another important role of mps is their contribution to the development of platelet- and fibrin - rich thrombi at sites of vascular injury via the recruitment of cells and the accumulation of tf .", "data suggest that emp - mediated coagulation has clinical significance ; for example , an association between the number of circulating mps and the risk of thrombolytic complication has been reported . because emps interact with coagulation proteins and with inflammatory or vascular cells , their role in cardiovascular diseases has been intensively studied .", "it has also been observed by jy et al . that emps carry von willebrand factor ( vwf ) and factor viii that promote platelet aggregates and increase their stability   ( figure 1 ) .", "moreover , the authors postulated that emps released during vascular injury may arrest bleeding by rapid interaction with platelets via membrane - associated vwf multimers and adhesions to stabilized platelet aggregates in the microenvironment .", "sabatier demonstrated that emps also carry tf and bind to monocytes causing further tf expression and resulting in enhanced transmigration of monocytes through endothelial junction [ 66 , 67 ] .  ", "one of the important key genes for aging - associated cardiovascular disorders is plasminogen activator inhibitor-1 ( pai-1 ) , a main inhibitor of fibrinolysis .", "the expression of pai-1 is not only elevated in the elderly but also significantly induced in a variety of pathologies associated with the process of aging .", "increased levels of pai-1 and its procoagulant activity have been recognized as hallmarks of endothelial dysfunction in vascular aging ( figure 4 ) .", "furthermore , elevated levels of pai-1 were found in werner syndrome , a disease characterized by premature aging [ 68 , 69 ] and atherosclerosis , which in advanced stages may lead to myocardial infarction and death .", "recently , it has been shown that emps expressing both activators and inhibitors of coagulation have fibrinolytic properties that counteract their procoagulant activities , which may enable them to contribute to haemostatic balance .", "it has been observed that endothelial and leukocyte microparticles generate fibrinolytic activity , whereas erythrocyte and platelet microparticles do not have this property .", "additionally , different plasminogen activators were identified on leukocyte microparticles , urokinase - type plasminogen activator ( upa ) , and emps where tissue plasminogen activator ( tpa ) has been found .", "the authors provide evidence that microparticles with plasminogen activators are rare in healthy populations but are observed more frequently in pathological conditions   suggesting that plasmin generation on microparticles may be important in the modulation of hemostatic balance . therefore , complex functions of emps have an ambivalent role both in physiological and pathological conditions , either promoting or inhibiting coagulation , inflammation , or angiogenesis .", "inflammatory mediators increase several procoagulant factors , inhibit endogenous anticoagulants , and attenuate the fibrinolytic response", ". however , the interaction between these two systems is bidirectional , as coagulation is also capable of modulating inflammatory activity .", "thrombin mediated inflammatory molecules such as il-8 and il-1ra and il-1 participate in emp release [ 4 , 59 ] ( figure 2 ) and are key factors involved in coagulation , inflammation , and angiogenesis .", "inflammation and coagulation are linked processes in many diseases and emps may amplify the responses by activating the endothelium .", "it has been reported that in addition to activation of d - dimers and c - reactive proteins in coagulation , the inflammatory cytokine il-6 is associated with mortality , declines in all measures of function , and leads to the frailty phenotype in the elderly .", "recently , it has been shown that increased levels of il-6 are present in aged aortas and that aging induces a proinflammatory phenotype in vascular smooth muscle cells ( vsmc ) due in part to increased signaling of toll - like receptor 4 ( tlr4 ) and its signaling adaptor myd 88 .", "these observations support the notion of a high prevalence of proinflammatory conditions in advanced age .", "all of these factors lead to increased generation of emps and impaired coagulation , inflammation , and angiogenesis in cv diseases ( figures 3 and 4 ) .", "the process of angiogenesis is complex and requires endothelial cells ( ecs ) to detach from pericytes and the extracellular matrix ( ecm ) , proliferate , invade the surrounding tissues , migrate , and differentiate to form capillary tubes that connect to newly developed vascular networks leading to vascular stabilization [ 7476 ] .", "defective angiogenesis has been found in many vascular diseases and it has been established that emps play an important role in this process .", "mps can act on angiogenesis directly through ligand / receptor interaction or indirectly by modulating production of soluble factors involved in endothelial cell differentiation , proliferation , migration , and adhesion .", "brodsky et al . have shown that in low concentration , emps did not affect the endothelium . however , increased levels of circulating emps are an important factor in the pathophysiology of cv diseases , directly affecting the endothelium and other circulating cells .", "brodsky et al . have demonstrated that emps directly impair vasorelaxation via diminishing production and/or bioavailability of nitric oxide .", "this result was correlated with increased superoxide levels in aortic rings isolated from rats and cultured endothelial cells treated with microparticles .", "other studies have demonstrated that mps of endothelial origin induce the expression of endothelial cyclooxygenase type 2 , different adhesion molecules , release of cytokines , and impaired release of nitric oxide from vascular endothelial cells .", "a pathological concentration ( 10 ) of emps affects angiogenesis by diminishing the cell proliferation rate and decreasing the total capillary length of human umbilical vein endothelial cells ( huvec ) plated on a matrigel substrate .", "moreover , huvecs or human microvascular endothelial cells ( hmvecs ) treated with emps demonstrated disorganized tube formation in the presence or absence of vegf .", "impairments in the regulation of vascular tone , coagulation , and hemostasis contribute to damage of the vascular system and dysfunctional angiogenesis [ 8183 ] .", "recently , in vitro studies published by burger et al . have proved that a long term culture of mouse aortic ecs leads to a senescent phenotype with increased rock activity and formation of mps ( figure 4 ) .", "furthermore , it has been shown that mps promote premature ec senescence through the stimulation of endothelial cell ros production   ( figure 4 ) .", "brodsky et al . demonstrated a significant increase in the number of circulating emps in obesity - induced diabetes rats as well as premature endothelial cell senescence and vasculopathy .", "this disorder was characterized by impaired vasorelaxation , nitric oxide production , and defective angiogenesis .", "an increased number of circulating emps have been identified in patients with certain diseases , such as hypertension , coronary artery disease , acute coronary syndrome , and stroke . in patients with established endothelial dysfunction , levels of circulating emps", "are inversely correlated with the amplitude of flow - mediated dilation , independent of blood pressure [ 11 , 8587 ] .", "another study published by thomasow et al . has shown elevated levels of cd31 ( pecam-1 ) , which are suggestive of ec apoptosis , in severe and mild chronic obstructive pulmonary disease ( copd ) and emphysema .", "cd31 emps were positively related to emphysema and were inversely associated with pulmonary microvascular blood flow .", "in contrast , cd62e ( e - selectin ) emps indicative of endothelial activation were elevated in severe copd and hyperinflation .", "previous studies have demonstrated an impairment of cell proliferation , migration , tube formation , and sprouting in older individuals ( > 65 years , male or female ) when compared to their younger counterparts ( < 65 years , male or female ) suggesting that these changes contribute to the decrease in effective blood vessel growth and repair mechanisms in the elderly .", "a decrease of proangiogenic factors and loss of circulating endothelial progenitor cells ( epcs ) have also been observed with increased aging ( > 50 years , male ) , which may lead to compromised angiogenesis .", "furthermore , age - related changes in epc number and function may directly correlate with the degree of senescent endothelial impairment .", "furthermore , in older men ( > 60 years ) with myocardial infarction , circulating microparticles selectively impair the nitric oxide transduction pathway in endothelial cells , which contributes to the general vasomotor dysfunction observed after myocardial infarction . additionally , an environmental alteration such as a decrease in ecm proteins particularly fibrillar collagen production and small leucine rich proteoglycans ( slrps )   and changes in the expression of matrix metalloproteinases ( mmps ) have also been observed with increased aging .", "emps are clearly implicated in the impairment of angiogenesis in vascular diseases associated with aging however , the specific pathways through which emps augment this process are unknown .", "moreover , emps may be one of the major factors leading to reduced effectiveness of therapies for treating impaired angiogenesis in humans and should be further explored .", "furthermore , estrogen levels have been connected to thrombin generation , a central molecule in the coagulation cascade in postmenopausal women .", "in particular , estradiol increases migration , proliferation , and formation of capillary - like networks of huvecs   by the classic estrogen receptor ( er ) pathway [ 9698 ] .", "most reports have concentrated on the role of ers in mediating big vessel relaxation and contraction . in a rat model of acute myocardial infarction", "it has been shown that estradiol promotes myocardial angiogenesis by increasing microvascular density through estrogen receptors .", "furthermore , in ovariectomized rats it has been demonstrated that an increased number of genes in the aged heart , including tnf and map kinase - activating death domain protein ( madd ) , play a role in the release of emps [ 100102 ] ( figure 4 ) .", "another in vivo study has shown that ovariectomy in female rats is associated with reduced pai-1 expression , while estrogen replacement counteracts this change promoting emp formation . in vitro studies", "other mechanisms have also been revealed to be involved in the proangiogenic effect of estrogen including increased expression of both vegf and its receptors [ 104 , 105 ] and bfgf , as well as expression of vascular adhesion molecules .", "moreover , estrogen is known to enhance nitric oxide production and release by endothelial cells .", "studies published by reed and edelberg have suggested that a physiological decrease in the concentration of steroid hormones ( e.g. , estrogen and testosterone ) as a result of menopause and  chronological aging  may contribute both directly and indirectly to subsequent deficits in the synthesis and function of the angiogenic growth factor tgf- .   in humans endogenous", "estrogen contributes to the anticoagulant , anti - inflammatory , and antithrombotic properties of the endothelium .", "the numbers of endothelium , platelet , and monocyte - derived microparticles have been found to be elevated in low - estrogen menopausal women .", "the authors implied that increased numbers of procoagulant microparticles provide a resource to study mechanisms for cardiovascular risk development in newly menopausal women . on the other hand , studies demonstrated by rank et al .  ", "had shown that hormone replacement therapy in postmenopausal women increased the concentration of mps derived from platelets .", "the emp levels were unchanged excluding their primary role in the initiation of a thromboembolic event in these women .", "another study has shown that emp concentration was diminished in older patients ( > 80 years , male or female ) but mp procoagulant activity was preserved   in comparison to younger patients .", "the authors suggested that a decreased emp level was associated with age and any effect of gender was ruled out by multivariate analysis .", "the study performed by mateos - caceres   had shown an increased level of circulating emps in elderly ( > 66 years ) male and female patients with acute stroke and that tnf activated emps were the major player in stroke induction .", "furthermore , simak et al . demonstrated that circulating emp phenotypes may be associated with the severity , lesion volume , and outcome of acute ischemic stroke ( ais ) in male patients ( > 78 years ) . on the other hand , studies published by williams et al .", "have shown that in elderly patients of both genders ( > 66 years ) , emp levels were similar in ais and stroke mimic patients . moreover", ", they demonstrated that emps were generated via activation and not by apoptosis / necrosis of endothelial cells .", "this suggested that emps may not be an appropriate marker for ais , given the incapability to distinguish between ais and stroke mimic .", "the tree - city cohort studies have shown that increased thrombin generation is an independent predictor of ais in elderly women suggesting that hypercoagulability may play an important role in the pathogenesis of ais .", "indicated that elevated plasma pai-1 levels are a strong risk factor for stroke at old age in people of both genders ; however , the 4g/5 g polymorphism variant of pai-1 is associated with reduced incidence of stroke .", "it has been shown that the single polymorphism pai-1 4g/5 g genotype is associated with higher central systolic , diastolic , and mean arterial blood pressure in women ( > 70 years ) , while no association was found in men   suggesting gender specific biology of pai-1 in addition to an advanced age specific factor . moreover ,", "elevated levels of circulating mps have been reported in patients ( > 58 years , male or female ) with acute myocardial infarction and coronary artery disease .", "studies published by sinning et al .   have shown that circulating emps , but not mps of other cellular origin , are a strong predictor of cardiovascular mortality and major cardiovascular events in patients ( > 66 years , male or female ) with coronary artery disease and pulmonary hypertension .", "all these data may indicate that estrogen probably does not exert its protective effects on cv diseases through the emp axis .", "however , more analyses are needed in order to confirm if a direct connection occurs between estrogen and emps in age - related vascular diseases ( figure 4 ) .", "endothelial microparticles , as pleiotropic factors , play a role in both physiological and pathological conditions and thus may contribute to regulation of vascular homeostasis .", "emps not only reflect the stage of disease but also play a causative role in the development of various vascular diseases .", "they can modulate coagulation , and their elevated levels have been observed in many conditions associated with inflammation and angiogenesis .", "the prothrombotic properties and proinflammatory effects of microparticles on endothelial cells affect vascular aging and lead to structural changes in the heart and other organs .", "thrombin and pai-1 seem to be key factors involved in emp generation in age - related vascular disease .", "furthermore , in age - related vascular diseases , steroid hormones are among the factors that have been shown to have an influence on vascular homeostasis .", "specifically , estrogen plays a regulatory function on vessel inflammation , injury , and repair .", "lack of estrogen has been suggested to be directly involved in the endothelial cell injury with emp release that is observed in ischemic diseases .", "however , the direct link between emp generation , emp release , and estrogen is understudied and the further investigation of cellular and molecular mechanisms of these correlations is imperative for understanding and providing a basis for new translational investigations .", "furthermore , circulating emps show great promise not only as biomarkers in the diagnostics of vascular diseases but also as a target for the treatment of these disorders , especially in elderly patients ."], "abstract_text": ["<S> endothelial microparticles ( emps ) are complex vesicular structures that originate from plasma membranes of activated or apoptotic endothelial cells . </S>", "<S> emps play a significant role in vascular function by altering the processes of inflammation , coagulation , and angiogenesis , and they are key players in the pathogenesis of several vascular diseases . </S>", "<S> circulating emps are increased in many age - related vascular diseases such as coronary artery disease , peripheral vascular disease , cerebral ischemia , and congestive heart failure . </S>", "<S> their elevation in plasma has been considered as both a biomarker and bioactive effector of vascular damage and a target for vascular diseases . </S>", "<S> this review focuses on the pleiotropic roles of emps and the mechanisms that trigger their formation , particularly the involvement of decreased estrogen levels , thrombin , and pai-1 as major factors that induce emps in age - related vascular diseases . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Endothelial Microparticles and Factors That Stimulate Their Formation and Release", "3. Endothelial Microparticles in Coagulation and Vascular Aging", "4. Endothelial Microparticles in Inflammation", "5. Endothelial Microparticles and Their Effect on Vascular Function in the Elderly", "6. Endothelial Microparticles and Estrogen in Age-Related Vascular Diseases", "7. Conclusions"], "sections": [["vascular diseases are among the most common causes of morbidity and mortality , and both number and severity of morbid vascular conditions increase with age . regulations of angiogenesis , coagulation , and inflammation are very important issues in vascular biology , both in normal physiology and pathology .", "it is now well established that disruption of endothelial integrity represents a crucial event in the initiation and development of cardiovascular ( cv ) diseases .", "numerous studies have reported that microparticles ( mps ) play an important role in endothelial dysfunction .", "endothelial dysfunction occurs when a perturbed homeostatic endothelium disrupts vascular competency resulting in reduced vasodilatation and increased proinflammatory and prothrombotic properties of the vascular network .", "recently , mps originating from various cells have been found to be associated with several vascular related diseases .", "moreover , exposed procoagulant phospholipids and specific receptors at the surface of mps act as biomessengers linking inflammation , coagulation , and angiogenesis [ 35 ] .   although mps were first described as  cellular debris  that are believed to have no biological significance , recent studies documented that mps of endothelial and other origins are biological effectors in inflammation , vascular injury , angiogenesis , and thrombosis [ 68 ] .", "mps isolated from granulation tissue are derived from endothelial cells , monocytes , platelets , erythrocytes [ 913 ] , and myofibroblasts .", "they exchange biological signals and information intercellularly and each kind of mp carries the antigens and receptors of the cells they originated . mps may transfer part of their components and content to the selected target cells , thus mediating cell activation , phenotypic modification , and reprogramming of cell function .", "although 70% to 90% of all circulating mps in the peripheral blood of healthy individuals are derived from platelets , marked elevations of all kinds of mps have been observed in many vascular diseases .", "specifically , endothelium - derived microparticles ( emps ) represent a relatively small ( 515% ) but very important subset of all circulating microparticles [ 1618 ] .", "this number may vary in different cardiovascular and inflammatory diseases [ 18 , 19 ] .", "new insights into endothelial dysfunction and alterations in angiogenesis are emerging from studies of vascular microparticles , particularly endothelial microparticles in elderly populations .", "vascular aging with impairment of endothelial cell function leads to altered angiogenesis , a key factor in the etiology of various cardiovascular disorders .", "73% of individuals aged 6079 have a cv disease , including stroke , hypertension , or heart failure , and at > 79 years of age prevalence of these diseases increased to 86% in females and 82% in males ( 2012 nhlbi fact book ) .", "recently published data have shown that these diseases are the leading cause of death for individuals aged > 65   and morbidity increased from 32% for individuals aged 66 to 48% for individuals aged 85 . an important factor which significantly decreases the incidence of coronary heart diseases in postmenopausal women is estrogen [ 2224 ] . in women already having coronary artery disease or ischemic stroke , the therapeutic benefit of estrogen is not clear [ 25 , 26 ] although it has been reported that estrogen induces rapid vasodilation , exerts anti - inflammatory activity , and regulates vascular cell growth , migration , and protection of cardiomyocytes from injury , all of which prevent atherosclerotic deterioration in vessels .", "this review focuses on the role of emps in angiogenesis , coagulation , and inflammation during age - related vascular diseases and the contribution of estrogen to these diseases ."], ["emps are small vesicles that are released from endothelial cells and can be found circulating in the blood . defined by their small size ( 0.1 to 1.0  m ) , they are a heterogeneous population of vesicles which are shed from plasma membranes in response to cell activation , injury , angiogenesis / neovascularization , and/or apoptosis .", "emps consist of a small amount of cytosol surrounded by a plasma membrane and display negatively charged phospholipids on their surface that can initiate and accelerate coagulation .", "circulating emps have been demonstrated as a marker of preeclampsia [ 29 , 30 ] , acute coronary syndromes , and severe hypertension [ 30 , 32 , 33 ] suggesting their association with pathological processes within the endothelium .", "furthermore , circulating emp levels are also implicated in the progression of atherosclerotic lesions , heart failure , arrhythmias , inflammatory vascular disease , sickle anemia , and endotoxemia .  ", "jimenez et al . demonstrated that the surface antigens of emps are distinctive depending on the type of endothelial cell injury , as in apoptosis versus activation .", "high levels of the surface antigens e - selectin , intracellular adhesion molecule-1 ( icam-1 ) , and vascular cell adhesion molecule-1 ( vcam-1 ) are on emps derived from activated endothelial cells .", "in contrast , the low levels of these antigens are on emps derived from apoptotic endothelial cells .", "platelet cell adhesion molecule ( pecam-1 ) , endoglin , and vascular endothelial - cadherin ( ve - cadherin ) are at low levels on emps derived from activated ecs [ 3740 ] ( figure 1 ) .", "additionally , emps generated from apoptotic endothelial cells have higher levels of phosphatidylserine on their surface and different phospholipid composition and oxidation status compared with emps generated from activated endothelial cells [ 41 , 42 ] .", "these data suggest that there are distinct mechanisms for the formation of emps in apoptotic and activated cells   and several studies suggest that these types of emps have different functions in vascular diseases [ 40 , 44 ] .", "both inflammatory cytokines and coagulation factors participate in the generation of emps ( figure 2 ) .", "recently , it has been shown that p38 mitogen - activated protein kinase ( mapk ) is a critical molecule in the production of proinflammatory emps and increased icam-1 production by endothelial cells , providing a paracrine loop to enhance the endothelial response to inflammation . in vitro studies", "have shown that the proinflammatory agent , tnf , activates endothelial cells and induces release of emps   ( figure 2 ) .", "another potent stimulus for emp formation both in vivo and in vitro is angiotensin ii ( ang ii ) .", "this effect is mediated by ang ii receptor type i that signals through nadph oxidase and rho kinase .", "furthermore , in ang ii - infused apoliprotein e ( apoe / ) hyperlipidemic mice , a model of significant endothelial dysfunction , ang ii has been shown to increase emp formation by a redox - sensitive and blood - pressure - independent process .", "another important factor pai-1 ( plasminogen activator inhibitor type 1 ) plays an important role in the formation of emps .", "it has been shown by brodsky et al . that pai-1 promotes formation of emps with reduced transmembrane asymmetry of phospholipids in a dose dependent manner .", "these findings could possibly link elevated levels of pai-1 with endothelial dysfunction and tendency toward thrombosis [ 4850 ] . increased levels of pai-1", "might serve as an initiator of emp formation followed by increased procoagulant activity and thrombin generation .", "in addition , it is also known that thrombin stimulates pai-1 synthesis   suggesting constant production of these two factors .", "all these data indicate that formation of emps links together inflammation , coagulation , and angiogenesis and causes the impairment of the last two phenomena ( figure 3 ) . among many signaling molecules , t - cadherin ( t - cad ) on the surface of ecs", "might be upregulated and may serve as a characteristic marker of ec activation and stress .", "recently , philippova et al . have demonstrated a mechanism of t - cad - dependent signaling in the vascular endothelium .", "the authors identified that t - cadherin levels in plasma are increased in early atherosclerosis and correlate with endothelial dysfunction , which may lead to increased release of emps from ecs ."], ["increasing evidence has also accumulated to implicate an impaired coagulation system in many vascular diseases .", "this coagulation imbalance is the net result of activation of coagulation , impaired activity of natural coagulation inhibitors , and suppressed fibrinolysis .", "activation of coagulation proteases , for example , thrombin , is one of the earliest events following tissue injury .", "thrombin modulates tissue repair responses by altering vascular permeability , stimulating endothelial cell , fibroblast , and neutrophil migration , and promoting their spreading and adhesion .", "it activates various cell types and induces secretion of several proimmune , profibrotic , and proinflammatory factors [ 45 , 56 , 57 ] .", "recent studies by sapet et al . have shown that release of emps by endothelial cells in response to thrombin involves a group of genes that regulate angiogenesis and are linked to the cytoskeleton reorganization family . among these genes ,", "rho - kinase rock - ii was transcribed at a high rate and was identified as a target of thrombin in emp generation .", "the involvement of caspase-2 in rock - ii activation , independent of cell death , points out a novel signaling pathway that emphasizes the proteolytic activity of caspase in emp generation in response to cell activation   ( figure 1 ) . however , further studies are needed to determine the molecular mechanisms involved in emp release .", "emp release is initiated when thrombin binds to its receptor , proteolytically activated receptor-1 ( par-1 ) , which induces gene transcription that is mediated by thrombin via trail / apo2l , a cytokine belonging to the tumor necrosis factor alpha ( tnf ) superfamily .", "this mechanism of emp generation depends on the nuclear factor ( nf ) b activation and involves the soluble form of trail , which is secreted by the endothelial cells under thrombin or inflammatory stimulation [ 4 , 59 ] ( figure 2 ) .", "phospholipids expressed on emps bind coagulation factors leading to a prothrombotic state   and an increase in procoagulant activity of tissue factor ( tf ) .", "these phospholipids are exposed on the outer membrane of mp and are considered to be the main initiators of the coagulation cascade .", "additionally , it has been demonstrated that sphingosine 1-phosphate ( s1p ) strongly potentiates thrombin - induced tf expression in ecs suggesting its role in blood coagulation .", "s1p has also been shown to be involved in the process of angiogenesis and inflammation [ 6264 ] .", "another important role of mps is their contribution to the development of platelet- and fibrin - rich thrombi at sites of vascular injury via the recruitment of cells and the accumulation of tf .", "data suggest that emp - mediated coagulation has clinical significance ; for example , an association between the number of circulating mps and the risk of thrombolytic complication has been reported . because emps interact with coagulation proteins and with inflammatory or vascular cells , their role in cardiovascular diseases has been intensively studied .", "it has also been observed by jy et al . that emps carry von willebrand factor ( vwf ) and factor viii that promote platelet aggregates and increase their stability   ( figure 1 ) .", "moreover , the authors postulated that emps released during vascular injury may arrest bleeding by rapid interaction with platelets via membrane - associated vwf multimers and adhesions to stabilized platelet aggregates in the microenvironment .", "sabatier demonstrated that emps also carry tf and bind to monocytes causing further tf expression and resulting in enhanced transmigration of monocytes through endothelial junction [ 66 , 67 ] .  ", "one of the important key genes for aging - associated cardiovascular disorders is plasminogen activator inhibitor-1 ( pai-1 ) , a main inhibitor of fibrinolysis .", "the expression of pai-1 is not only elevated in the elderly but also significantly induced in a variety of pathologies associated with the process of aging .", "increased levels of pai-1 and its procoagulant activity have been recognized as hallmarks of endothelial dysfunction in vascular aging ( figure 4 ) .", "furthermore , elevated levels of pai-1 were found in werner syndrome , a disease characterized by premature aging [ 68 , 69 ] and atherosclerosis , which in advanced stages may lead to myocardial infarction and death .", "recently , it has been shown that emps expressing both activators and inhibitors of coagulation have fibrinolytic properties that counteract their procoagulant activities , which may enable them to contribute to haemostatic balance .", "it has been observed that endothelial and leukocyte microparticles generate fibrinolytic activity , whereas erythrocyte and platelet microparticles do not have this property .", "additionally , different plasminogen activators were identified on leukocyte microparticles , urokinase - type plasminogen activator ( upa ) , and emps where tissue plasminogen activator ( tpa ) has been found .", "the authors provide evidence that microparticles with plasminogen activators are rare in healthy populations but are observed more frequently in pathological conditions   suggesting that plasmin generation on microparticles may be important in the modulation of hemostatic balance . therefore , complex functions of emps have an ambivalent role both in physiological and pathological conditions , either promoting or inhibiting coagulation , inflammation , or angiogenesis ."], ["inflammatory mediators increase several procoagulant factors , inhibit endogenous anticoagulants , and attenuate the fibrinolytic response", ". however , the interaction between these two systems is bidirectional , as coagulation is also capable of modulating inflammatory activity .", "thrombin mediated inflammatory molecules such as il-8 and il-1ra and il-1 participate in emp release [ 4 , 59 ] ( figure 2 ) and are key factors involved in coagulation , inflammation , and angiogenesis .", "inflammation and coagulation are linked processes in many diseases and emps may amplify the responses by activating the endothelium .", "it has been reported that in addition to activation of d - dimers and c - reactive proteins in coagulation , the inflammatory cytokine il-6 is associated with mortality , declines in all measures of function , and leads to the frailty phenotype in the elderly .", "recently , it has been shown that increased levels of il-6 are present in aged aortas and that aging induces a proinflammatory phenotype in vascular smooth muscle cells ( vsmc ) due in part to increased signaling of toll - like receptor 4 ( tlr4 ) and its signaling adaptor myd 88 .", "these observations support the notion of a high prevalence of proinflammatory conditions in advanced age .", "all of these factors lead to increased generation of emps and impaired coagulation , inflammation , and angiogenesis in cv diseases ( figures 3 and 4 ) ."], ["the process of angiogenesis is complex and requires endothelial cells ( ecs ) to detach from pericytes and the extracellular matrix ( ecm ) , proliferate , invade the surrounding tissues , migrate , and differentiate to form capillary tubes that connect to newly developed vascular networks leading to vascular stabilization [ 7476 ] .", "defective angiogenesis has been found in many vascular diseases and it has been established that emps play an important role in this process .", "mps can act on angiogenesis directly through ligand / receptor interaction or indirectly by modulating production of soluble factors involved in endothelial cell differentiation , proliferation , migration , and adhesion .", "brodsky et al . have shown that in low concentration , emps did not affect the endothelium . however , increased levels of circulating emps are an important factor in the pathophysiology of cv diseases , directly affecting the endothelium and other circulating cells .", "brodsky et al . have demonstrated that emps directly impair vasorelaxation via diminishing production and/or bioavailability of nitric oxide .", "this result was correlated with increased superoxide levels in aortic rings isolated from rats and cultured endothelial cells treated with microparticles .", "other studies have demonstrated that mps of endothelial origin induce the expression of endothelial cyclooxygenase type 2 , different adhesion molecules , release of cytokines , and impaired release of nitric oxide from vascular endothelial cells .", "a pathological concentration ( 10 ) of emps affects angiogenesis by diminishing the cell proliferation rate and decreasing the total capillary length of human umbilical vein endothelial cells ( huvec ) plated on a matrigel substrate .", "moreover , huvecs or human microvascular endothelial cells ( hmvecs ) treated with emps demonstrated disorganized tube formation in the presence or absence of vegf .", "impairments in the regulation of vascular tone , coagulation , and hemostasis contribute to damage of the vascular system and dysfunctional angiogenesis [ 8183 ] .", "recently , in vitro studies published by burger et al . have proved that a long term culture of mouse aortic ecs leads to a senescent phenotype with increased rock activity and formation of mps ( figure 4 ) .", "furthermore , it has been shown that mps promote premature ec senescence through the stimulation of endothelial cell ros production   ( figure 4 ) .", "brodsky et al . demonstrated a significant increase in the number of circulating emps in obesity - induced diabetes rats as well as premature endothelial cell senescence and vasculopathy .", "this disorder was characterized by impaired vasorelaxation , nitric oxide production , and defective angiogenesis .", "an increased number of circulating emps have been identified in patients with certain diseases , such as hypertension , coronary artery disease , acute coronary syndrome , and stroke . in patients with established endothelial dysfunction , levels of circulating emps", "are inversely correlated with the amplitude of flow - mediated dilation , independent of blood pressure [ 11 , 8587 ] .", "another study published by thomasow et al . has shown elevated levels of cd31 ( pecam-1 ) , which are suggestive of ec apoptosis , in severe and mild chronic obstructive pulmonary disease ( copd ) and emphysema .", "cd31 emps were positively related to emphysema and were inversely associated with pulmonary microvascular blood flow .", "in contrast , cd62e ( e - selectin ) emps indicative of endothelial activation were elevated in severe copd and hyperinflation .", "previous studies have demonstrated an impairment of cell proliferation , migration , tube formation , and sprouting in older individuals ( > 65 years , male or female ) when compared to their younger counterparts ( < 65 years , male or female ) suggesting that these changes contribute to the decrease in effective blood vessel growth and repair mechanisms in the elderly .", "a decrease of proangiogenic factors and loss of circulating endothelial progenitor cells ( epcs ) have also been observed with increased aging ( > 50 years , male ) , which may lead to compromised angiogenesis .", "furthermore , age - related changes in epc number and function may directly correlate with the degree of senescent endothelial impairment .", "furthermore , in older men ( > 60 years ) with myocardial infarction , circulating microparticles selectively impair the nitric oxide transduction pathway in endothelial cells , which contributes to the general vasomotor dysfunction observed after myocardial infarction . additionally , an environmental alteration such as a decrease in ecm proteins particularly fibrillar collagen production and small leucine rich proteoglycans ( slrps )   and changes in the expression of matrix metalloproteinases ( mmps ) have also been observed with increased aging .", "emps are clearly implicated in the impairment of angiogenesis in vascular diseases associated with aging however , the specific pathways through which emps augment this process are unknown .", "moreover , emps may be one of the major factors leading to reduced effectiveness of therapies for treating impaired angiogenesis in humans and should be further explored ."], ["furthermore , estrogen levels have been connected to thrombin generation , a central molecule in the coagulation cascade in postmenopausal women .", "in particular , estradiol increases migration , proliferation , and formation of capillary - like networks of huvecs   by the classic estrogen receptor ( er ) pathway [ 9698 ] .", "most reports have concentrated on the role of ers in mediating big vessel relaxation and contraction . in a rat model of acute myocardial infarction", "it has been shown that estradiol promotes myocardial angiogenesis by increasing microvascular density through estrogen receptors .", "furthermore , in ovariectomized rats it has been demonstrated that an increased number of genes in the aged heart , including tnf and map kinase - activating death domain protein ( madd ) , play a role in the release of emps [ 100102 ] ( figure 4 ) .", "another in vivo study has shown that ovariectomy in female rats is associated with reduced pai-1 expression , while estrogen replacement counteracts this change promoting emp formation . in vitro studies", "other mechanisms have also been revealed to be involved in the proangiogenic effect of estrogen including increased expression of both vegf and its receptors [ 104 , 105 ] and bfgf , as well as expression of vascular adhesion molecules .", "moreover , estrogen is known to enhance nitric oxide production and release by endothelial cells .", "studies published by reed and edelberg have suggested that a physiological decrease in the concentration of steroid hormones ( e.g. , estrogen and testosterone ) as a result of menopause and  chronological aging  may contribute both directly and indirectly to subsequent deficits in the synthesis and function of the angiogenic growth factor tgf- .   in humans endogenous", "estrogen contributes to the anticoagulant , anti - inflammatory , and antithrombotic properties of the endothelium .", "the numbers of endothelium , platelet , and monocyte - derived microparticles have been found to be elevated in low - estrogen menopausal women .", "the authors implied that increased numbers of procoagulant microparticles provide a resource to study mechanisms for cardiovascular risk development in newly menopausal women . on the other hand , studies demonstrated by rank et al .  ", "had shown that hormone replacement therapy in postmenopausal women increased the concentration of mps derived from platelets .", "the emp levels were unchanged excluding their primary role in the initiation of a thromboembolic event in these women .", "another study has shown that emp concentration was diminished in older patients ( > 80 years , male or female ) but mp procoagulant activity was preserved   in comparison to younger patients .", "the authors suggested that a decreased emp level was associated with age and any effect of gender was ruled out by multivariate analysis .", "the study performed by mateos - caceres   had shown an increased level of circulating emps in elderly ( > 66 years ) male and female patients with acute stroke and that tnf activated emps were the major player in stroke induction .", "furthermore , simak et al . demonstrated that circulating emp phenotypes may be associated with the severity , lesion volume , and outcome of acute ischemic stroke ( ais ) in male patients ( > 78 years ) . on the other hand , studies published by williams et al .", "have shown that in elderly patients of both genders ( > 66 years ) , emp levels were similar in ais and stroke mimic patients . moreover", ", they demonstrated that emps were generated via activation and not by apoptosis / necrosis of endothelial cells .", "this suggested that emps may not be an appropriate marker for ais , given the incapability to distinguish between ais and stroke mimic .", "the tree - city cohort studies have shown that increased thrombin generation is an independent predictor of ais in elderly women suggesting that hypercoagulability may play an important role in the pathogenesis of ais .", "indicated that elevated plasma pai-1 levels are a strong risk factor for stroke at old age in people of both genders ; however , the 4g/5 g polymorphism variant of pai-1 is associated with reduced incidence of stroke .", "it has been shown that the single polymorphism pai-1 4g/5 g genotype is associated with higher central systolic , diastolic , and mean arterial blood pressure in women ( > 70 years ) , while no association was found in men   suggesting gender specific biology of pai-1 in addition to an advanced age specific factor . moreover ,", "elevated levels of circulating mps have been reported in patients ( > 58 years , male or female ) with acute myocardial infarction and coronary artery disease .", "studies published by sinning et al .   have shown that circulating emps , but not mps of other cellular origin , are a strong predictor of cardiovascular mortality and major cardiovascular events in patients ( > 66 years , male or female ) with coronary artery disease and pulmonary hypertension .", "all these data may indicate that estrogen probably does not exert its protective effects on cv diseases through the emp axis .", "however , more analyses are needed in order to confirm if a direct connection occurs between estrogen and emps in age - related vascular diseases ( figure 4 ) ."], ["endothelial microparticles , as pleiotropic factors , play a role in both physiological and pathological conditions and thus may contribute to regulation of vascular homeostasis .", "emps not only reflect the stage of disease but also play a causative role in the development of various vascular diseases .", "they can modulate coagulation , and their elevated levels have been observed in many conditions associated with inflammation and angiogenesis .", "the prothrombotic properties and proinflammatory effects of microparticles on endothelial cells affect vascular aging and lead to structural changes in the heart and other organs .", "thrombin and pai-1 seem to be key factors involved in emp generation in age - related vascular disease .", "furthermore , in age - related vascular diseases , steroid hormones are among the factors that have been shown to have an influence on vascular homeostasis .", "specifically , estrogen plays a regulatory function on vessel inflammation , injury , and repair .", "lack of estrogen has been suggested to be directly involved in the endothelial cell injury with emp release that is observed in ischemic diseases .", "however , the direct link between emp generation , emp release , and estrogen is understudied and the further investigation of cellular and molecular mechanisms of these correlations is imperative for understanding and providing a basis for new translational investigations .", "furthermore , circulating emps show great promise not only as biomarkers in the diagnostics of vascular diseases but also as a target for the treatment of these disorders , especially in elderly patients ."]]}
{"article_id": "PMC3429799", "article_text": ["type 2 diabetes is the most common metabolic disorder worldwide and its prevalence is growing at an alarming rate in both developed and developing countries .", "it is characterized by abnormalities in carbohydrate , lipid and lipoprotein metabolism , which lead to hyperglycemia and many complications such hyperlipidemia , hyperinsulinemia , hypertension and atherosclerosis .", "lifestyle changes , weight control , physical activity and healthy nutrition practices are vital for persons with diabetes to maintain quality of life and longevity . also as an alternative approach , medicinal herbs with antihyperglycemic activities are increasingly sought by diabetic patients and health care professionals .", "cinnamon is the bark of cinnamomum zeylanicum and has been used as traditional folk herbs to treat disease thousands of years in asia . both in vitro and in vivo studies", "have shown that cinnamon is an insulin sensitizer.[610 ] in 3t3 l1 adipocyte , cinnamon extract stimulates glucose uptake , glycogen synthesis and activated glycogen synthase . in rats cinnamon enhanced glucose uptake by enhancing insulin stimulated tyrosine phosphorylation of insulin receptor  , insulin receptor substrate 1 , and phosphatidylinositol 3 kinase in a dose dependent fashion .", "several clinical trials[1216 ] have investigated the impact of cinnamon on glucose and plasma lipid concentrations in patients with diabetes but yielded conflicting results .", "khan et al . presented the first data on the effects of cinnamon supplementation in humans .", "they reported a substantial reduction in fasting serum glucose ( 18 - 29% ) and blood lipid profile after 40 d of daily supplementation with one , three , or six grams of whole cinnamon in patients with type 2 diabetes .", "more recently , mang et al . reported a 10.3% reduction in fbg after four months of supplementation with a purified aqueous cinnamon extract .", "however , data from vanschoonbeek et al . shows no effect of cinnamon spice ingestion on whole body insulin sensitivity , oral glucose tolerance or lipid profiles in postmenopausal type 2 diabetic patients .", "the present study investigated the proposed benefit of cinnamon use in patients with type 2 diabetes .", "therefore a randomized , placebo controlled , double blind study was performed in 44 patients with type 2 diabetes . in this study", ", we assessed the effects of eight weeks of three grams per day cinnamon supplementation ( cinnamomum zeylanicum ) on glycemic status , lipid profiles , blood pressure and body composition in type 2 diabetic patients .", "the study was approved by the medical ethical committee of tehran university of medical science ( code p/26/d5/147 ) and was supported by grants from vice chancellor of research ( grant number p/608 ) .", "a total of 44 individuals with type 2 diabetes were selected to participate in this study .", "type 2 diabetes was verified according to the criteria set by the who in 1999 .", "the objectives of study and risks of experimental procedures were explained to the volunteers , after which their written informed consent was obtained .", "inclusion criteria were non - insulin dependent type 2 diabetes , aged between 30 - 65 years , hba1c between 6 - 8% and fbg levels between 126 - 160 mg / dl .", "exclusion criteria were cholesterol > 240 mg / dl , triglyceride > 400 mg / dl , smoking and alcohol consumption , pregnancy and lactation , allergy to cinnamon , liver or renal or thyroid diseases and hemolytic anemia .", "all subjects were stable because medication had not modified over the last month and there were homogeneity regarding their treatments ( metformin : 1 - 1.5 gr / d , gliclazide : 160 - 240 mg / d ) .", "subjects were selected based on inclusion criteria among patients who came to the endocrinology and metabolism center , tehran university of medical sciences and randomly assigned to cinnamon ( n=22 ) or placebo group ( n=22 ) .", "cinnamon and wheat flour were ground finely and put into capsules which could not be distinguished by color , odor , or taste .", "subjects were instructed to ingest two capsules at each main meal ( breakfast , lunch and dinner ) for eight weeks .", "compliance to the supplementation protocol was supervised by a research technician who contacted the subjects once a week .", "each subject was required to return the original bottle of their respective supplement for capsule counts and compliance was monitored by counting the unconsumed capsules each week .", "we chose this dose because it is achievable if we want to use it as a spice in future and there is no side effect for this dose in this target group based on literature.[1316 ] all medication was continued as usual and subjects were advised to maintain their normal diet and continue their habitual physical activity throughout the study . to verify this , subjects completed three day food records ( two weekday and one weekend day ) at baseline and end of study .", "food records were analyzed by nutritionist iv ( n squared computing , san bruno ca ) .", "physical activity levels were measured with international physical activity questionnaire at baseline and end of study .", "two moved and five were unwilling to continue ; so 37 patients completed the study ( 19 patients in the intervention group and 18 in the control group ) .", "weight , height , body composition and blood pressure were measured in the fasting state with minimal clothing and without shoes in the beginning and end of study .", "standing height was measured using a wall mounted stadiometer and body composition was measured with e body 205 ( body composition analyzer ) .", "after 10 - 12 h overnight fasting , blood sample was collected from each subject at the beginning and at the end of eight week trial .", "all blood samples were taken in the morning at approximately the same time of day to minimize diurnal variation and immediately centrifuged at 3000 g for 10 min at 20c .", "fasting plasma glucose and hba1c were analyzed immediately ; aliquots of plasma were stored at 70c until lipid profile and insulin analysis .", "fasting plasma glucose was analyzed by the glucose oxidase method ( pars azmoon kit , iran ) .", "insulin was analyzed by immunoradiometric assay ( irma ) method ( immunotech co. kit , france ) .", "serum concentrations of triglyceride and total cholesterol were analyzed using the glycerol-3-phosphate oxidase - phenol+aminophenazone ( gpo pap ) kit as described in our previous study and the cholesterol oxidase - phenol+aminophenazone ( chod pap ) kit , respectively ( pars azmoon kit , iran ) .", "apo lipoproteins ai and b were analyzed by immunoturbidometry method by using auto analyzer cobas ( pars azmoon kit , iran ) .", "statistical analyses were performed using statistical package for the social sciences ( spss ) software ( version 14 ; spss inc , chicago , usa ) .", "qualitative variables , such as physical activity , were analyzed using chi square test . for comparison variables before and after the intervention within each group , paired t test was used .", "adjustment for differences in baseline covariates and changes in variables during study were performed by analysis of covariance ( ancova ) .", "the study was approved by the medical ethical committee of tehran university of medical science ( code p/26/d5/147 ) and was supported by grants from vice chancellor of research ( grant number p/608 ) .", "a total of 44 individuals with type 2 diabetes were selected to participate in this study .", "type 2 diabetes was verified according to the criteria set by the who in 1999 .", "the objectives of study and risks of experimental procedures were explained to the volunteers , after which their written informed consent was obtained .", "inclusion criteria were non - insulin dependent type 2 diabetes , aged between 30 - 65 years , hba1c between 6 - 8% and fbg levels between 126 - 160 mg / dl .", "exclusion criteria were cholesterol > 240 mg / dl , triglyceride > 400 mg / dl , smoking and alcohol consumption , pregnancy and lactation , allergy to cinnamon , liver or renal or thyroid diseases and hemolytic anemia .", "all subjects were stable because medication had not modified over the last month and there were homogeneity regarding their treatments ( metformin : 1 - 1.5 gr / d , gliclazide : 160 - 240 mg / d ) .", "subjects were selected based on inclusion criteria among patients who came to the endocrinology and metabolism center , tehran university of medical sciences and randomly assigned to cinnamon ( n=22 ) or placebo group ( n=22 ) .", "cinnamon and wheat flour were ground finely and put into capsules which could not be distinguished by color , odor , or taste .", "subjects were instructed to ingest two capsules at each main meal ( breakfast , lunch and dinner ) for eight weeks .", "compliance to the supplementation protocol was supervised by a research technician who contacted the subjects once a week .", "each subject was required to return the original bottle of their respective supplement for capsule counts and compliance was monitored by counting the unconsumed capsules each week .", "we chose this dose because it is achievable if we want to use it as a spice in future and there is no side effect for this dose in this target group based on literature.[1316 ] all medication was continued as usual and subjects were advised to maintain their normal diet and continue their habitual physical activity throughout the study . to verify this , subjects completed three day food records ( two weekday and one weekend day ) at baseline and end of study .", "food records were analyzed by nutritionist iv ( n squared computing , san bruno ca ) .", "physical activity levels were measured with international physical activity questionnaire at baseline and end of study .", "two moved and five were unwilling to continue ; so 37 patients completed the study ( 19 patients in the intervention group and 18 in the control group ) .", "weight , height , body composition and blood pressure were measured in the fasting state with minimal clothing and without shoes in the beginning and end of study .", "standing height was measured using a wall mounted stadiometer and body composition was measured with e body 205 ( body composition analyzer ) .", "after 10 - 12 h overnight fasting , blood sample was collected from each subject at the beginning and at the end of eight week trial .", "all blood samples were taken in the morning at approximately the same time of day to minimize diurnal variation and immediately centrifuged at 3000 g for 10 min at 20c .", "fasting plasma glucose and hba1c were analyzed immediately ; aliquots of plasma were stored at 70c until lipid profile and insulin analysis .", "fasting plasma glucose was analyzed by the glucose oxidase method ( pars azmoon kit , iran ) .", "insulin was analyzed by immunoradiometric assay ( irma ) method ( immunotech co. kit , france ) .", "serum concentrations of triglyceride and total cholesterol were analyzed using the glycerol-3-phosphate oxidase - phenol+aminophenazone ( gpo pap ) kit as described in our previous study and the cholesterol oxidase - phenol+aminophenazone ( chod pap ) kit , respectively ( pars azmoon kit , iran ) .", "apo lipoproteins ai and b were analyzed by immunoturbidometry method by using auto analyzer cobas ( pars azmoon kit , iran ) .", "statistical analyses were performed using statistical package for the social sciences ( spss ) software ( version 14 ; spss inc , chicago , usa ) .", "qualitative variables , such as physical activity , were analyzed using chi square test . for comparison variables before and after the intervention within each group , paired t test was used .", "adjustment for differences in baseline covariates and changes in variables during study were performed by analysis of covariance ( ancova ) .", "the study was approved by the medical ethical committee of tehran university of medical science ( code p/26/d5/147 ) and was supported by grants from vice chancellor of research ( grant number p/608 ) .", "a total of 44 individuals with type 2 diabetes were selected to participate in this study .", "type 2 diabetes was verified according to the criteria set by the who in 1999 .", "the objectives of study and risks of experimental procedures were explained to the volunteers , after which their written informed consent was obtained .", "inclusion criteria were non - insulin dependent type 2 diabetes , aged between 30 - 65 years , hba1c between 6 - 8% and fbg levels between 126 - 160 mg / dl .", "exclusion criteria were cholesterol > 240 mg / dl , triglyceride > 400 mg / dl , smoking and alcohol consumption , pregnancy and lactation , allergy to cinnamon , liver or renal or thyroid diseases and hemolytic anemia .", "all subjects were stable because medication had not modified over the last month and there were homogeneity regarding their treatments ( metformin : 1 - 1.5 gr / d , gliclazide : 160 - 240 mg / d ) .", "subjects were selected based on inclusion criteria among patients who came to the endocrinology and metabolism center , tehran university of medical sciences and randomly assigned to cinnamon ( n=22 ) or placebo group ( n=22 ) .", "cinnamon and wheat flour were ground finely and put into capsules which could not be distinguished by color , odor , or taste .", "subjects were instructed to ingest two capsules at each main meal ( breakfast , lunch and dinner ) for eight weeks .", "compliance to the supplementation protocol was supervised by a research technician who contacted the subjects once a week .", "each subject was required to return the original bottle of their respective supplement for capsule counts and compliance was monitored by counting the unconsumed capsules each week .", "we chose this dose because it is achievable if we want to use it as a spice in future and there is no side effect for this dose in this target group based on literature.[1316 ] all medication was continued as usual and subjects were advised to maintain their normal diet and continue their habitual physical activity throughout the study . to verify this , subjects completed three day food records ( two weekday and one weekend day ) at baseline and end of study .", "food records were analyzed by nutritionist iv ( n squared computing , san bruno ca ) .", "physical activity levels were measured with international physical activity questionnaire at baseline and end of study .", "two moved and five were unwilling to continue ; so 37 patients completed the study ( 19 patients in the intervention group and 18 in the control group ) .", "weight , height , body composition and blood pressure were measured in the fasting state with minimal clothing and without shoes in the beginning and end of study .", "standing height was measured using a wall mounted stadiometer and body composition was measured with e body 205 ( body composition analyzer ) .", "after 10 - 12 h overnight fasting , blood sample was collected from each subject at the beginning and at the end of eight week trial .", "all blood samples were taken in the morning at approximately the same time of day to minimize diurnal variation and immediately centrifuged at 3000 g for 10 min at 20c .", "fasting plasma glucose and hba1c were analyzed immediately ; aliquots of plasma were stored at 70c until lipid profile and insulin analysis .", "fasting plasma glucose was analyzed by the glucose oxidase method ( pars azmoon kit , iran ) .", "insulin was analyzed by immunoradiometric assay ( irma ) method ( immunotech co. kit , france ) .", "serum concentrations of triglyceride and total cholesterol were analyzed using the glycerol-3-phosphate oxidase - phenol+aminophenazone ( gpo pap ) kit as described in our previous study and the cholesterol oxidase - phenol+aminophenazone ( chod pap ) kit , respectively ( pars azmoon kit , iran ) .", "apo lipoproteins ai and b were analyzed by immunoturbidometry method by using auto analyzer cobas ( pars azmoon kit , iran ) .", "statistical analyses were performed using statistical package for the social sciences ( spss ) software ( version 14 ; spss inc , chicago , usa ) .", "qualitative variables , such as physical activity , were analyzed using chi square test . for comparison variables before and after the intervention within each group , paired t test was used .", "adjustment for differences in baseline covariates and changes in variables during study were performed by analysis of covariance ( ancova ) .", "baseline characteristics did not differ between the two groups of the study population [ table 1 ] .", "energy intake , macronutrient and micronutrient composition of the diet did not differ between groups at baseline and did not change in the placebo or cinnamon group during the intervention period .", "physical activity levels did not differ between groups at baseline and did not change during the study .", "table 2 shows the fasting plasma glucose concentrations , hba1c , serum lipids , anthropometric variables and the corresponding differences of these variables for the cinnamon and placebo groups .", "while the fbg , hba1c and triglyceride levels in the cinnamon group was significantly reduced after the intervention compared with baseline ( p=0.005 , p=0.008 , p=0.038 respectively ) , no such effect were observed in the placebo group .", "no significant intragroup differences were observed in insulin or lipid profile ( total cholesterol , hdl cholesterol , ldl cholesterol , apo lipoproteins ai and b ) .", "body weight and bmi in both groups were significantly reduced after the intervention compared with baseline ( p=0.017 , p=0.010 respectively ) .", "fat body mass was significantly reduced in cinnamon group , ( p=0.047 ) but not in placebo group .", "no significant intergroup differences were observed in glycemic parameters , lipid profile , anthropometric variables and blood pressure .", "baseline characteristics of the study population biochemical and anthropometric variables at baseline and after the intervention period", "our findings indicate that consuming three gram per day of cinnamon for eight weeks leads to significant reduction in some biochemical and anthropometric variables compared with baseline ( i.e. , fbg decreased by 9.2% , hba1c decreased by 6.12% , triglyceride decreased by 15.38% , body weight decreased by 1.19% , bmi decreased by 1.54% and fat body mass decreased by 1.36% ) . but these reductions were not significant compare to placebo group .", "khan et al . studied the effects of one , three , and six grams per day of whole cinnamon powder on fbg and serum lipids in 60 people with poorly controlled type 2 diabetes from pakistan .", "after 40 days of supplementation , fbg decreased by 18 - 29% , cholesterol decreased by 12 - 26% , ldl cholesterol decreased by 7 - 27% , and triacylglycerol decreased by 23 - 30% .", "consequently the authors concluded that small amount of cinnamon represent a safe and effective mean to reduce the risk factors associated with type 2 diabetes . in the cinnamon group", ", we observed a significant reduction of plasma glucose ( 9.2% ) after eight weeks of treatment but not in the placebo group .", "the observed reduction in fbg in our study is less than that of khan et al . and similar in magnitude to mang et al . who supplemented 79 diabetic patients with three grams per day cinnamon powder or a placebo for four months", "although no changes in blood lipids or hba1c were noted in their study , researchers did find a 10.3% reduction in fbg .", "also the decrease in plasma glucose correlated significantly with the baseline concentrations indicated that subjects with a higher initial plasma glucose level may show more reduction after cinnamon intake .", "however data from vanschoonbeek et al . showed no effect of cinnamon spice ingestion on whole body insulin sensitivity , oral glucose tolerance or lipid profiles in a postmenopausal type 2 diabetes mellitus ( t2 dm ) group .", "the positive correlation between baseline plasma glucose and the decrease of plasma glucose in mang et al .", "study and the strong decrease of serum glucose in the poorly controlled pakistani diabetics ( mean fasting glucose ( 140 - 400 mg / dl ) concentrations ) , suggest that subjects with poor glycemic control may benefit more from cinnamon intake .", "no significant intergroup and intragroup differences were observed in lipid profile except in triglyceride that decreased significantly in cinnamon group compared with the baseline .", "insulin resistance leads to the overproduction of very low density lipoproteins ( vldls ) and to reduce lipoprotein lipase activity , thereby resulting in dyslipidemia .", "obviously , the decrease of plasma glucose concentrations in our study was not sufficient to induce an improvement in the lipoprotein concentrations . also our subjects lipid levels were not abnormal , while pakistani subjects in khan et al .", "no significant intergroup differences were observed in anthropometric variables . there were no changes regarding the systolic / diastolic blood pressure after the intervention compared with baseline in our study .", "ziegenfuss et al . reported statistically significant decrease in systolic bp ( from 133 to 129 mm hg ) and increases in lean mass ( + 1.1% ) compared with the placebo group .", "additionally , within - group analyses uncovered small , but statistically significant decreases in body fat ( 0.7% ) in the cinnamon group .", "it is possible that the improvements in fbg , as well as plasma antioxidant status are responsible , at least in part , for cinnamon 's beneficial effects on body composition and features of the metabolic syndrome like blood pressure .", "couturier et al . suggested that in animals fed a high fat / high fructose diet to induce insulin resistance , cinnamon alters body composition in association with improved insulin sensitivity .", "the decreases of plasma glucose and insulin concentrations and weight in our study were not sufficient to induce an improvement of the blood pressure .", "regarding the limitations of our study , we can point to the duration of study ( eight weeks ) and dose of the cinnamon ( three grams per day ) .", "there are some studies with longer duration and it would be better if we could have possibility for continuing the intervention which it was impossible because subjects had time limitations to come to the study center ( endocrinology and metabolism center , tehran university of medical sciences ) and compliance was limited if we extend the time .", "there are some studies that use more than three grams per day cinnamon and detect significant effects and it would be better if we could have another group with higher dose of cinnamon for comparing doses that was impossible because of financial limitations .", "in conclusion , our study shows that in type 2 diabetic patients , the intake of three grams per day cinnamon may lead to a moderate effect on glycemic status indicators , but does not improve blood pressure , blood lipid profile or body composition parameters ."], "abstract_text": ["<S> objective : type 2 diabetes is the most common metabolic disorder worldwide . traditional herbs and spices can be used to control blood glucose concentrations . </S>", "<S> the objective of this study was to evaluate the effects of the daily intake of three grams cinnamon over eight weeks on glycemic status , lipid profiles and body composition in type 2 diabetic patients.methods:a double blind , randomized , placebo controlled clinical trial was conducted on 44 patients with type 2 diabetes . </S>", "<S> participants were randomly assigned to take either a three g / day cinnamon supplement ( n=22 ) or a placebo ( n=22 ) for eight weeks . </S>", "<S> weight , height , body fat mass and systolic and diastolic blood pressure were measured at baseline and after intervention . the fasting blood glucose , insulin , hba1c , total cholesterol , ldl c , hdl c , apo lipoprotein a i and b were measured at baseline and endpoint.results:from 44 subjects participated in this study 37 completed the study . </S>", "<S> there were no significant differences in baseline characteristics , dietary intake and physical activity between groups . in the treatment group , the levels of fasting blood glucose , hba1c , triglyceride , weight , bmi and body fat mass decreased significantly compared to baseline , but not in placebo group . </S>", "<S> no significant differences were observed in glycemic status indicators , lipid profile and anthropometric indicators between the groups at the end of intervention.conclusion:these data suggest that cinnamon may have a moderate effect in improving glycemic status indicators . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "None", "Subjects", "Study design and dietary intervention", "Biochemical measurements", "Statistics", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["type 2 diabetes is the most common metabolic disorder worldwide and its prevalence is growing at an alarming rate in both developed and developing countries .", "it is characterized by abnormalities in carbohydrate , lipid and lipoprotein metabolism , which lead to hyperglycemia and many complications such hyperlipidemia , hyperinsulinemia , hypertension and atherosclerosis .", "lifestyle changes , weight control , physical activity and healthy nutrition practices are vital for persons with diabetes to maintain quality of life and longevity . also as an alternative approach , medicinal herbs with antihyperglycemic activities are increasingly sought by diabetic patients and health care professionals .", "cinnamon is the bark of cinnamomum zeylanicum and has been used as traditional folk herbs to treat disease thousands of years in asia . both in vitro and in vivo studies", "have shown that cinnamon is an insulin sensitizer.[610 ] in 3t3 l1 adipocyte , cinnamon extract stimulates glucose uptake , glycogen synthesis and activated glycogen synthase . in rats cinnamon enhanced glucose uptake by enhancing insulin stimulated tyrosine phosphorylation of insulin receptor  , insulin receptor substrate 1 , and phosphatidylinositol 3 kinase in a dose dependent fashion .", "several clinical trials[1216 ] have investigated the impact of cinnamon on glucose and plasma lipid concentrations in patients with diabetes but yielded conflicting results .", "khan et al . presented the first data on the effects of cinnamon supplementation in humans .", "they reported a substantial reduction in fasting serum glucose ( 18 - 29% ) and blood lipid profile after 40 d of daily supplementation with one , three , or six grams of whole cinnamon in patients with type 2 diabetes .", "more recently , mang et al . reported a 10.3% reduction in fbg after four months of supplementation with a purified aqueous cinnamon extract .", "however , data from vanschoonbeek et al . shows no effect of cinnamon spice ingestion on whole body insulin sensitivity , oral glucose tolerance or lipid profiles in postmenopausal type 2 diabetic patients .", "the present study investigated the proposed benefit of cinnamon use in patients with type 2 diabetes .", "therefore a randomized , placebo controlled , double blind study was performed in 44 patients with type 2 diabetes . in this study", ", we assessed the effects of eight weeks of three grams per day cinnamon supplementation ( cinnamomum zeylanicum ) on glycemic status , lipid profiles , blood pressure and body composition in type 2 diabetic patients ."], ["the study was approved by the medical ethical committee of tehran university of medical science ( code p/26/d5/147 ) and was supported by grants from vice chancellor of research ( grant number p/608 ) .", "a total of 44 individuals with type 2 diabetes were selected to participate in this study .", "type 2 diabetes was verified according to the criteria set by the who in 1999 .", "the objectives of study and risks of experimental procedures were explained to the volunteers , after which their written informed consent was obtained .", "inclusion criteria were non - insulin dependent type 2 diabetes , aged between 30 - 65 years , hba1c between 6 - 8% and fbg levels between 126 - 160 mg / dl .", "exclusion criteria were cholesterol > 240 mg / dl , triglyceride > 400 mg / dl , smoking and alcohol consumption , pregnancy and lactation , allergy to cinnamon , liver or renal or thyroid diseases and hemolytic anemia .", "all subjects were stable because medication had not modified over the last month and there were homogeneity regarding their treatments ( metformin : 1 - 1.5 gr / d , gliclazide : 160 - 240 mg / d ) .", "subjects were selected based on inclusion criteria among patients who came to the endocrinology and metabolism center , tehran university of medical sciences and randomly assigned to cinnamon ( n=22 ) or placebo group ( n=22 ) .", "cinnamon and wheat flour were ground finely and put into capsules which could not be distinguished by color , odor , or taste .", "subjects were instructed to ingest two capsules at each main meal ( breakfast , lunch and dinner ) for eight weeks .", "compliance to the supplementation protocol was supervised by a research technician who contacted the subjects once a week .", "each subject was required to return the original bottle of their respective supplement for capsule counts and compliance was monitored by counting the unconsumed capsules each week .", "we chose this dose because it is achievable if we want to use it as a spice in future and there is no side effect for this dose in this target group based on literature.[1316 ] all medication was continued as usual and subjects were advised to maintain their normal diet and continue their habitual physical activity throughout the study . to verify this , subjects completed three day food records ( two weekday and one weekend day ) at baseline and end of study .", "food records were analyzed by nutritionist iv ( n squared computing , san bruno ca ) .", "physical activity levels were measured with international physical activity questionnaire at baseline and end of study .", "two moved and five were unwilling to continue ; so 37 patients completed the study ( 19 patients in the intervention group and 18 in the control group ) .", "weight , height , body composition and blood pressure were measured in the fasting state with minimal clothing and without shoes in the beginning and end of study .", "standing height was measured using a wall mounted stadiometer and body composition was measured with e body 205 ( body composition analyzer ) .", "after 10 - 12 h overnight fasting , blood sample was collected from each subject at the beginning and at the end of eight week trial .", "all blood samples were taken in the morning at approximately the same time of day to minimize diurnal variation and immediately centrifuged at 3000 g for 10 min at 20c .", "fasting plasma glucose and hba1c were analyzed immediately ; aliquots of plasma were stored at 70c until lipid profile and insulin analysis .", "fasting plasma glucose was analyzed by the glucose oxidase method ( pars azmoon kit , iran ) .", "insulin was analyzed by immunoradiometric assay ( irma ) method ( immunotech co. kit , france ) .", "serum concentrations of triglyceride and total cholesterol were analyzed using the glycerol-3-phosphate oxidase - phenol+aminophenazone ( gpo pap ) kit as described in our previous study and the cholesterol oxidase - phenol+aminophenazone ( chod pap ) kit , respectively ( pars azmoon kit , iran ) .", "apo lipoproteins ai and b were analyzed by immunoturbidometry method by using auto analyzer cobas ( pars azmoon kit , iran ) .", "statistical analyses were performed using statistical package for the social sciences ( spss ) software ( version 14 ; spss inc , chicago , usa ) .", "qualitative variables , such as physical activity , were analyzed using chi square test . for comparison variables before and after the intervention within each group , paired t test was used .", "adjustment for differences in baseline covariates and changes in variables during study were performed by analysis of covariance ( ancova ) ."], ["the study was approved by the medical ethical committee of tehran university of medical science ( code p/26/d5/147 ) and was supported by grants from vice chancellor of research ( grant number p/608 ) .", "a total of 44 individuals with type 2 diabetes were selected to participate in this study .", "type 2 diabetes was verified according to the criteria set by the who in 1999 .", "the objectives of study and risks of experimental procedures were explained to the volunteers , after which their written informed consent was obtained .", "inclusion criteria were non - insulin dependent type 2 diabetes , aged between 30 - 65 years , hba1c between 6 - 8% and fbg levels between 126 - 160 mg / dl .", "exclusion criteria were cholesterol > 240 mg / dl , triglyceride > 400 mg / dl , smoking and alcohol consumption , pregnancy and lactation , allergy to cinnamon , liver or renal or thyroid diseases and hemolytic anemia .", "all subjects were stable because medication had not modified over the last month and there were homogeneity regarding their treatments ( metformin : 1 - 1.5 gr / d , gliclazide : 160 - 240 mg / d ) .", "subjects were selected based on inclusion criteria among patients who came to the endocrinology and metabolism center , tehran university of medical sciences and randomly assigned to cinnamon ( n=22 ) or placebo group ( n=22 ) .", "cinnamon and wheat flour were ground finely and put into capsules which could not be distinguished by color , odor , or taste .", "subjects were instructed to ingest two capsules at each main meal ( breakfast , lunch and dinner ) for eight weeks .", "compliance to the supplementation protocol was supervised by a research technician who contacted the subjects once a week .", "each subject was required to return the original bottle of their respective supplement for capsule counts and compliance was monitored by counting the unconsumed capsules each week .", "we chose this dose because it is achievable if we want to use it as a spice in future and there is no side effect for this dose in this target group based on literature.[1316 ] all medication was continued as usual and subjects were advised to maintain their normal diet and continue their habitual physical activity throughout the study . to verify this , subjects completed three day food records ( two weekday and one weekend day ) at baseline and end of study .", "food records were analyzed by nutritionist iv ( n squared computing , san bruno ca ) .", "physical activity levels were measured with international physical activity questionnaire at baseline and end of study .", "two moved and five were unwilling to continue ; so 37 patients completed the study ( 19 patients in the intervention group and 18 in the control group ) .", "weight , height , body composition and blood pressure were measured in the fasting state with minimal clothing and without shoes in the beginning and end of study .", "standing height was measured using a wall mounted stadiometer and body composition was measured with e body 205 ( body composition analyzer ) .", "after 10 - 12 h overnight fasting , blood sample was collected from each subject at the beginning and at the end of eight week trial .", "all blood samples were taken in the morning at approximately the same time of day to minimize diurnal variation and immediately centrifuged at 3000 g for 10 min at 20c .", "fasting plasma glucose and hba1c were analyzed immediately ; aliquots of plasma were stored at 70c until lipid profile and insulin analysis .", "fasting plasma glucose was analyzed by the glucose oxidase method ( pars azmoon kit , iran ) .", "insulin was analyzed by immunoradiometric assay ( irma ) method ( immunotech co. kit , france ) .", "serum concentrations of triglyceride and total cholesterol were analyzed using the glycerol-3-phosphate oxidase - phenol+aminophenazone ( gpo pap ) kit as described in our previous study and the cholesterol oxidase - phenol+aminophenazone ( chod pap ) kit , respectively ( pars azmoon kit , iran ) .", "apo lipoproteins ai and b were analyzed by immunoturbidometry method by using auto analyzer cobas ( pars azmoon kit , iran ) .", "statistical analyses were performed using statistical package for the social sciences ( spss ) software ( version 14 ; spss inc , chicago , usa ) .", "qualitative variables , such as physical activity , were analyzed using chi square test . for comparison variables before and after the intervention within each group , paired t test was used .", "adjustment for differences in baseline covariates and changes in variables during study were performed by analysis of covariance ( ancova ) ."], ["the study was approved by the medical ethical committee of tehran university of medical science ( code p/26/d5/147 ) and was supported by grants from vice chancellor of research ( grant number p/608 ) .", "a total of 44 individuals with type 2 diabetes were selected to participate in this study .", "type 2 diabetes was verified according to the criteria set by the who in 1999 .", "the objectives of study and risks of experimental procedures were explained to the volunteers , after which their written informed consent was obtained .", "inclusion criteria were non - insulin dependent type 2 diabetes , aged between 30 - 65 years , hba1c between 6 - 8% and fbg levels between 126 - 160 mg / dl .", "exclusion criteria were cholesterol > 240 mg / dl , triglyceride > 400 mg / dl , smoking and alcohol consumption , pregnancy and lactation , allergy to cinnamon , liver or renal or thyroid diseases and hemolytic anemia .", "all subjects were stable because medication had not modified over the last month and there were homogeneity regarding their treatments ( metformin : 1 - 1.5 gr / d , gliclazide : 160 - 240 mg / d ) ."], ["subjects were selected based on inclusion criteria among patients who came to the endocrinology and metabolism center , tehran university of medical sciences and randomly assigned to cinnamon ( n=22 ) or placebo group ( n=22 ) .", "cinnamon and wheat flour were ground finely and put into capsules which could not be distinguished by color , odor , or taste .", "subjects were instructed to ingest two capsules at each main meal ( breakfast , lunch and dinner ) for eight weeks .", "compliance to the supplementation protocol was supervised by a research technician who contacted the subjects once a week .", "each subject was required to return the original bottle of their respective supplement for capsule counts and compliance was monitored by counting the unconsumed capsules each week .", "we chose this dose because it is achievable if we want to use it as a spice in future and there is no side effect for this dose in this target group based on literature.[1316 ] all medication was continued as usual and subjects were advised to maintain their normal diet and continue their habitual physical activity throughout the study . to verify this , subjects completed three day food records ( two weekday and one weekend day ) at baseline and end of study .", "food records were analyzed by nutritionist iv ( n squared computing , san bruno ca ) .", "physical activity levels were measured with international physical activity questionnaire at baseline and end of study .", "two moved and five were unwilling to continue ; so 37 patients completed the study ( 19 patients in the intervention group and 18 in the control group ) .", "weight , height , body composition and blood pressure were measured in the fasting state with minimal clothing and without shoes in the beginning and end of study .", "standing height was measured using a wall mounted stadiometer and body composition was measured with e body 205 ( body composition analyzer ) .", "after 10 - 12 h overnight fasting , blood sample was collected from each subject at the beginning and at the end of eight week trial .", "all blood samples were taken in the morning at approximately the same time of day to minimize diurnal variation and immediately centrifuged at 3000 g for 10 min at 20c .", "fasting plasma glucose and hba1c were analyzed immediately ; aliquots of plasma were stored at 70c until lipid profile and insulin analysis ."], ["fasting plasma glucose was analyzed by the glucose oxidase method ( pars azmoon kit , iran ) .", "insulin was analyzed by immunoradiometric assay ( irma ) method ( immunotech co. kit , france ) .", "serum concentrations of triglyceride and total cholesterol were analyzed using the glycerol-3-phosphate oxidase - phenol+aminophenazone ( gpo pap ) kit as described in our previous study and the cholesterol oxidase - phenol+aminophenazone ( chod pap ) kit , respectively ( pars azmoon kit , iran ) .", "apo lipoproteins ai and b were analyzed by immunoturbidometry method by using auto analyzer cobas ( pars azmoon kit , iran ) ."], ["statistical analyses were performed using statistical package for the social sciences ( spss ) software ( version 14 ; spss inc , chicago , usa ) .", "qualitative variables , such as physical activity , were analyzed using chi square test . for comparison variables before and after the intervention within each group , paired t test was used .", "adjustment for differences in baseline covariates and changes in variables during study were performed by analysis of covariance ( ancova ) ."], ["baseline characteristics did not differ between the two groups of the study population [ table 1 ] .", "energy intake , macronutrient and micronutrient composition of the diet did not differ between groups at baseline and did not change in the placebo or cinnamon group during the intervention period .", "physical activity levels did not differ between groups at baseline and did not change during the study .", "table 2 shows the fasting plasma glucose concentrations , hba1c , serum lipids , anthropometric variables and the corresponding differences of these variables for the cinnamon and placebo groups .", "while the fbg , hba1c and triglyceride levels in the cinnamon group was significantly reduced after the intervention compared with baseline ( p=0.005 , p=0.008 , p=0.038 respectively ) , no such effect were observed in the placebo group .", "no significant intragroup differences were observed in insulin or lipid profile ( total cholesterol , hdl cholesterol , ldl cholesterol , apo lipoproteins ai and b ) .", "body weight and bmi in both groups were significantly reduced after the intervention compared with baseline ( p=0.017 , p=0.010 respectively ) .", "fat body mass was significantly reduced in cinnamon group , ( p=0.047 ) but not in placebo group .", "no significant intergroup differences were observed in glycemic parameters , lipid profile , anthropometric variables and blood pressure .", "baseline characteristics of the study population biochemical and anthropometric variables at baseline and after the intervention period"], ["our findings indicate that consuming three gram per day of cinnamon for eight weeks leads to significant reduction in some biochemical and anthropometric variables compared with baseline ( i.e. , fbg decreased by 9.2% , hba1c decreased by 6.12% , triglyceride decreased by 15.38% , body weight decreased by 1.19% , bmi decreased by 1.54% and fat body mass decreased by 1.36% ) . but these reductions were not significant compare to placebo group .", "khan et al . studied the effects of one , three , and six grams per day of whole cinnamon powder on fbg and serum lipids in 60 people with poorly controlled type 2 diabetes from pakistan .", "after 40 days of supplementation , fbg decreased by 18 - 29% , cholesterol decreased by 12 - 26% , ldl cholesterol decreased by 7 - 27% , and triacylglycerol decreased by 23 - 30% .", "consequently the authors concluded that small amount of cinnamon represent a safe and effective mean to reduce the risk factors associated with type 2 diabetes . in the cinnamon group", ", we observed a significant reduction of plasma glucose ( 9.2% ) after eight weeks of treatment but not in the placebo group .", "the observed reduction in fbg in our study is less than that of khan et al . and similar in magnitude to mang et al . who supplemented 79 diabetic patients with three grams per day cinnamon powder or a placebo for four months", "although no changes in blood lipids or hba1c were noted in their study , researchers did find a 10.3% reduction in fbg .", "also the decrease in plasma glucose correlated significantly with the baseline concentrations indicated that subjects with a higher initial plasma glucose level may show more reduction after cinnamon intake .", "however data from vanschoonbeek et al . showed no effect of cinnamon spice ingestion on whole body insulin sensitivity , oral glucose tolerance or lipid profiles in a postmenopausal type 2 diabetes mellitus ( t2 dm ) group .", "the positive correlation between baseline plasma glucose and the decrease of plasma glucose in mang et al .", "study and the strong decrease of serum glucose in the poorly controlled pakistani diabetics ( mean fasting glucose ( 140 - 400 mg / dl ) concentrations ) , suggest that subjects with poor glycemic control may benefit more from cinnamon intake .", "no significant intergroup and intragroup differences were observed in lipid profile except in triglyceride that decreased significantly in cinnamon group compared with the baseline .", "insulin resistance leads to the overproduction of very low density lipoproteins ( vldls ) and to reduce lipoprotein lipase activity , thereby resulting in dyslipidemia .", "obviously , the decrease of plasma glucose concentrations in our study was not sufficient to induce an improvement in the lipoprotein concentrations . also our subjects lipid levels were not abnormal , while pakistani subjects in khan et al .", "no significant intergroup differences were observed in anthropometric variables . there were no changes regarding the systolic / diastolic blood pressure after the intervention compared with baseline in our study .", "ziegenfuss et al . reported statistically significant decrease in systolic bp ( from 133 to 129 mm hg ) and increases in lean mass ( + 1.1% ) compared with the placebo group .", "additionally , within - group analyses uncovered small , but statistically significant decreases in body fat ( 0.7% ) in the cinnamon group .", "it is possible that the improvements in fbg , as well as plasma antioxidant status are responsible , at least in part , for cinnamon 's beneficial effects on body composition and features of the metabolic syndrome like blood pressure .", "couturier et al . suggested that in animals fed a high fat / high fructose diet to induce insulin resistance , cinnamon alters body composition in association with improved insulin sensitivity .", "the decreases of plasma glucose and insulin concentrations and weight in our study were not sufficient to induce an improvement of the blood pressure .", "regarding the limitations of our study , we can point to the duration of study ( eight weeks ) and dose of the cinnamon ( three grams per day ) .", "there are some studies with longer duration and it would be better if we could have possibility for continuing the intervention which it was impossible because subjects had time limitations to come to the study center ( endocrinology and metabolism center , tehran university of medical sciences ) and compliance was limited if we extend the time .", "there are some studies that use more than three grams per day cinnamon and detect significant effects and it would be better if we could have another group with higher dose of cinnamon for comparing doses that was impossible because of financial limitations ."], ["in conclusion , our study shows that in type 2 diabetic patients , the intake of three grams per day cinnamon may lead to a moderate effect on glycemic status indicators , but does not improve blood pressure , blood lipid profile or body composition parameters ."]]}
{"article_id": "PMC3982982", "article_text": [". failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , even death .", "management of an unexpectedly difficult airway consists of laryngeal mask ventilation , gum - elastic bougie and video laryngoscopy - assisted intubation .", "gum - elastic bougie is the easiest and cheapest tool used in case of an unexpected difficult intubation occurring in the operating room .", "a 53-year - old male patient with hypogonadotropic hypogonadism presented as an unexpected difficult intubation after the induction of anesthesia .", "a difficult airway has been described in patients with a variety of endocrine disorders , including pituitary diseases , but not with hypogonadism . there may be an unrevealed relationship between hypogonadism and difficult airway .", "gum - elastic bougie is still the most attainable and effective tool in the operation room in this situation .", "anesthesiologists are responsible for maintaining a patent airway in the anesthetized patient . failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , as even a few minutes of deoxygenation can result in catastrophic outcome like brain damage or even death.1 anesthesiologists are occasionally faced and challenged with this significant problem in practice .", "a difficult airway has been described in patients with pituitary endocrine disorders such as acromegaly and dwarfism ; however , airway problems with hypogonadism have not been well identified in the literature.2", "a 53-year - old married man ( weight : 85 kg , height : 187 cm , body mass index : 24 kg / m ) presented with a history of nasal obstruction for two years .", "he had not had any prior operation under general anesthesia , so he did not have any history of difficult intubation , and he did not have any chronic systemic disease .", "the patient was evaluated for obstructive sleep apnea syndrome ( osas ) with a comprehensive questionnaire on his sleeping habits and medical history ; no complaints or predictors pertaining to osas were identified .", "the patient s preoperative airway assessment was normal , mallampati class was ii , thyromental distance was 7 cm , inter - incisor gap was 5 cm , and head extension was > 35. his physical examination was characterized by lack of secondary sexual characteristics and presence of fine facial wrinkles .", "although , as previously indicated , the patient was married , he had had no children .", "his hormone profile was : testosterone 0.3 ng / ml ( reference range 1.757.81 ) , free testosterone 0.91 ( reference range 4.542.0 ) , prolactin 1.31 ng / ml ( reference range 2.6426.72 ) , luteinizing hormone ( lh ) 0.33 miu / ml ( reference range 1.24103.03 ) .", "no other pathological finding was obtained as the result of magnetic resonance imaging of the pituitary gland .", "he was admitted to the operating theater , and following the induction of anesthesia with a dose of 5 mg / kg intravenous thiopental , bag - mask ventilation was barely sustained .", "fentanyl ( 12 gr / kg ) and , as a muscle relaxant , rocuronium ( 0.6 mg / kg ) were administered . while the patient s head was in the sniffing position , direct laryngoscopy and intubation of the trachea", "were attempted three times with different sizes of macintosh and miller blades by an assistant professor of anesthesiology with 5 years experience . however , unfortunately , the intubation failed .", "the lungs were then ventilated with 100% oxygen via a face mask in order to avoid desaturation .", "glottic visualization was assessed with cook s modification of the cormack  lehane classification ; a grade of 3a ( with direct laryngoscopy , only the epiglottis can be visualized ; the epiglottis can be lifted using an introducer or bougie ) was assigned.3 the patient was subsequently successfully intubated with a gum - elastic bougie .", "after the operation , the patient was extubated successfully without any complication and then examined by otorhinolaryngologists via fexible laryngoscopy .", "the epiglottis was found to be in a slightly lower than normal position ( figure 1 ) .", "the overall incidence of unexpected difficult intubation is estimated to be 1%3% , while failed intubation incidence is 0.05%0.35% in europe.1 difficult endotracheal intubation has been described in patients with pituitary diseases like acromegaly , cushing s disease , lh / follicle - stimulating hormone - secreting adenoma , thyroid - stimulating hormone - secreting adenoma , nonfunctional adenoma and prolactinoma.4 difficult airway with dwarfism has also been described .", "however , difficult airway with a hypogonadotropic patient has not yet been clearly defined . in our case ,", "further , magnetic resonance imaging of the pituitary gland did not reveal any adenoma or mass lesion.2 pulsatile release of follicle stimulating hormone and lh in puberty instigates the development of the male larynx and secondary sexual characteristics .", "the growth of laryngeal cartilages and maturation of the male larynx is also facilitated by androgen .", "high - affinity androgen receptors have been found in the human larynx.5,6 in this case , the patient s laryngeal development was compatible with his age and sex . only the epiglottis was located in a slightly lower position than normal , partially leaning toward the laryngeal passage .", "the patient was infertile and lacked secondary sexual characteristics due to insufficient exposure to androgens .", "the rate of difficult intubation has been found to be higher in osas patients than the normal , healthy population.7 body mass index , mallampati grading , cormack  lehane classification , sternomental distance , and thyromental distance evaluation might be predictive factors for difficult airway in osas patients .", "as such , anesthesiologists must be aware of this fact and be prepared for difficult intubation in osas patients.7 however , the rate of difficult intubation has not been found to be related to the severity of osas.8 no significant relationship between the apnea  hypopnea index and difficult intubation has been found.8 in our patient , no apnea and daytime sleepiness were present in his history . only snoring was present , which might well have been associated with nasal septal deviation .", "his body mass index was 24 kg / m , mallampati class was ii , thyromental distance was 7 cm , inter - incisor gap was 5 cm , and head extension was above 35. these findings did not suggest any evidence of osas or difficult airway .", "this multiparameter test reveals the presence and severity of osas.9 in this case , as there was no clinical index of suspicion for osas and to keep costs down , polysomnography was not required by the otorhinolaryngologists .", "thus , anesthesiologists must take into account difficult airway in endocrinological disorders like hypogonadotropic hypogonadism and be prepared for difficult intubation ."], "abstract_text": ["<S> backgrounda critical aspect of safe general anesthesia is providing adequate ventilation and oxygenation </S>", "<S> . failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , even death . </S>", "<S> anesthesiologists rarely encounter unexpected difficult airway problems in daily routine . </S>", "<S> management of an unexpectedly difficult airway consists of laryngeal mask ventilation , gum - elastic bougie and video laryngoscopy - assisted intubation . </S>", "<S> gum - elastic bougie is the easiest and cheapest tool used in case of an unexpected difficult intubation occurring in the operating room.casea 53-year - old male patient with hypogonadotropic hypogonadism presented as an unexpected difficult intubation after the induction of anesthesia . </S>", "<S> no pathological finding or predictor of difficult intubation was present . </S>", "<S> in addition , bag - mask ventilation was poor and inadequate . </S>", "<S> the patient was finally successfully intubated with a gum - elastic bougie.conclusiona difficult airway has been described in patients with a variety of endocrine disorders , including pituitary diseases , but not with hypogonadism . </S>", "<S> there may be an unrevealed relationship between hypogonadism and difficult airway . </S>", "<S> gum - elastic bougie is still the most attainable and effective tool in the operation room in this situation . </S>"], "labels": null, "section_names": ["Background", "Case", "Conclusion", "Introduction", "Case", "Discussion", "Conclusion"], "sections": [[". failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , even death .", "management of an unexpectedly difficult airway consists of laryngeal mask ventilation , gum - elastic bougie and video laryngoscopy - assisted intubation .", "gum - elastic bougie is the easiest and cheapest tool used in case of an unexpected difficult intubation occurring in the operating room ."], ["a 53-year - old male patient with hypogonadotropic hypogonadism presented as an unexpected difficult intubation after the induction of anesthesia ."], ["a difficult airway has been described in patients with a variety of endocrine disorders , including pituitary diseases , but not with hypogonadism . there may be an unrevealed relationship between hypogonadism and difficult airway .", "gum - elastic bougie is still the most attainable and effective tool in the operation room in this situation ."], ["anesthesiologists are responsible for maintaining a patent airway in the anesthetized patient . failed endotracheal intubation and inadequate ventilation with insufficient oxygenation may lead to serious complications , as even a few minutes of deoxygenation can result in catastrophic outcome like brain damage or even death.1 anesthesiologists are occasionally faced and challenged with this significant problem in practice .", "a difficult airway has been described in patients with pituitary endocrine disorders such as acromegaly and dwarfism ; however , airway problems with hypogonadism have not been well identified in the literature.2"], ["a 53-year - old married man ( weight : 85 kg , height : 187 cm , body mass index : 24 kg / m ) presented with a history of nasal obstruction for two years .", "he had not had any prior operation under general anesthesia , so he did not have any history of difficult intubation , and he did not have any chronic systemic disease .", "the patient was evaluated for obstructive sleep apnea syndrome ( osas ) with a comprehensive questionnaire on his sleeping habits and medical history ; no complaints or predictors pertaining to osas were identified .", "the patient s preoperative airway assessment was normal , mallampati class was ii , thyromental distance was 7 cm , inter - incisor gap was 5 cm , and head extension was > 35. his physical examination was characterized by lack of secondary sexual characteristics and presence of fine facial wrinkles .", "although , as previously indicated , the patient was married , he had had no children .", "his hormone profile was : testosterone 0.3 ng / ml ( reference range 1.757.81 ) , free testosterone 0.91 ( reference range 4.542.0 ) , prolactin 1.31 ng / ml ( reference range 2.6426.72 ) , luteinizing hormone ( lh ) 0.33 miu / ml ( reference range 1.24103.03 ) .", "no other pathological finding was obtained as the result of magnetic resonance imaging of the pituitary gland .", "he was admitted to the operating theater , and following the induction of anesthesia with a dose of 5 mg / kg intravenous thiopental , bag - mask ventilation was barely sustained .", "fentanyl ( 12 gr / kg ) and , as a muscle relaxant , rocuronium ( 0.6 mg / kg ) were administered . while the patient s head was in the sniffing position , direct laryngoscopy and intubation of the trachea", "were attempted three times with different sizes of macintosh and miller blades by an assistant professor of anesthesiology with 5 years experience . however , unfortunately , the intubation failed .", "the lungs were then ventilated with 100% oxygen via a face mask in order to avoid desaturation .", "glottic visualization was assessed with cook s modification of the cormack  lehane classification ; a grade of 3a ( with direct laryngoscopy , only the epiglottis can be visualized ; the epiglottis can be lifted using an introducer or bougie ) was assigned.3 the patient was subsequently successfully intubated with a gum - elastic bougie .", "after the operation , the patient was extubated successfully without any complication and then examined by otorhinolaryngologists via fexible laryngoscopy .", "the epiglottis was found to be in a slightly lower than normal position ( figure 1 ) ."], ["the overall incidence of unexpected difficult intubation is estimated to be 1%3% , while failed intubation incidence is 0.05%0.35% in europe.1 difficult endotracheal intubation has been described in patients with pituitary diseases like acromegaly , cushing s disease , lh / follicle - stimulating hormone - secreting adenoma , thyroid - stimulating hormone - secreting adenoma , nonfunctional adenoma and prolactinoma.4 difficult airway with dwarfism has also been described .", "however , difficult airway with a hypogonadotropic patient has not yet been clearly defined . in our case ,", "further , magnetic resonance imaging of the pituitary gland did not reveal any adenoma or mass lesion.2 pulsatile release of follicle stimulating hormone and lh in puberty instigates the development of the male larynx and secondary sexual characteristics .", "the growth of laryngeal cartilages and maturation of the male larynx is also facilitated by androgen .", "high - affinity androgen receptors have been found in the human larynx.5,6 in this case , the patient s laryngeal development was compatible with his age and sex . only the epiglottis was located in a slightly lower position than normal , partially leaning toward the laryngeal passage .", "the patient was infertile and lacked secondary sexual characteristics due to insufficient exposure to androgens .", "the rate of difficult intubation has been found to be higher in osas patients than the normal , healthy population.7 body mass index , mallampati grading , cormack  lehane classification , sternomental distance , and thyromental distance evaluation might be predictive factors for difficult airway in osas patients .", "as such , anesthesiologists must be aware of this fact and be prepared for difficult intubation in osas patients.7 however , the rate of difficult intubation has not been found to be related to the severity of osas.8 no significant relationship between the apnea  hypopnea index and difficult intubation has been found.8 in our patient , no apnea and daytime sleepiness were present in his history . only snoring was present , which might well have been associated with nasal septal deviation .", "his body mass index was 24 kg / m , mallampati class was ii , thyromental distance was 7 cm , inter - incisor gap was 5 cm , and head extension was above 35. these findings did not suggest any evidence of osas or difficult airway .", "this multiparameter test reveals the presence and severity of osas.9 in this case , as there was no clinical index of suspicion for osas and to keep costs down , polysomnography was not required by the otorhinolaryngologists ."], ["thus , anesthesiologists must take into account difficult airway in endocrinological disorders like hypogonadotropic hypogonadism and be prepared for difficult intubation ."]]}
{"article_id": "PMC3322081", "article_text": ["we reviewed the medical records of patients who had l. monocytogenes isolated from blood and body fluids from sterile sites during 19962008 at the national taiwan university hospital ( ntuh ) , a 2,500-bed hospital in taiwan .", "we evaluated disease severity using modified acute physiology and chronic health evaluation ii scores ( 8) .", "incidence of nontyphiodal salmonella bacteremia ( ntsb ) during 20002008 in ntuh was calculated for trend comparison of the 2 foodborne illnesses .", "only 1 episode was calculated during the same admission for ntsb to avoid the influence of repetitive bacteremia .", "isolates from patients with fetomaternal listeriosis ( i.e. , paired isolates from mother and neonate who had listeriosis ) were considered to be the same and only 1 of them was analyzed .", "all isolates were analyzed for their serotype by pcr as described , and genetic relatedness was evaluated by pulsed - field gel electrophoresis ( pfge ) by using pulsenet standardized protocols and 2 restriction enzymes ( asci and apai ) ( 2,9 ) .", "strains were considered to be of the same cluster if their bands had indistinguishable restriction patterns by both enzymes .", "strains with pfge patterns with > 80% similarity by asci and apai profiles were considered to be closely related .", "a forward stepwise model with a p value of 0.1 was used , and p<0.05 was considered statistically significant in the multivariate cox proportional hazards model . during the study period ,", "listeriosis was diagnosed in 48 patients , and 46 nonduplicated isolates were obtained for further microbiological analysis .", "average annual incidence increased from 0.0287 cases per 1,000 admissions during 19962004 to 0.118 cases per 1,000 admissions during 20052008 ( figure 1 ) .", "the increase in annual incidence of listeriosis was significantly correlated with years ( p = 0.0045 ) .", "the average annual incidences of ntsb were 1.189 and 1.118 per 1,000 admissions during 20002004 and 20052008 , respectively ; it was not significantly correlated with years ( p = 0.50 ) .", "age - specific incidence of listeriosis increased at both extremes of age , but especially among patients > 80 years ( figure 2 ) .", "all of the patients with listeriosis lived in northern taiwan , and no obvious geographic correlation was observed between listeriosis patients in each year .", "incidence ( cases per 1,000 admissions ) of human listeriosis and serotype distribution of all isolates , national taiwan university hospital , taipei , taiwan , 19962008 .", "forty - six isolates were available for analysis , including 2 serotype 4b isolates from fetomaternal transmission in 2005 and 2006 and 1 serotype 4b from a pediatric patient ( 2005 ) .", "patient distribution and incidence of human listeriosis , by age group , national taiwan university hospital , taipei , taiwan , 19962008 .", "all 43 patients had underlying predisposing conditions , and 18 ( 42% ) were > 65 years of age . of the 30 patients with malignancies , 23 ( 77% )", " one female patient had coexisting non - hodgkin lymphoma and colon cancer ; 1 male patient had coexisting non - hodgkin lymphoma , gastric cancer , and bladder cancer . ", "initial modified acute physiology and chronic health evaluation ii score , mean  sd : 18.8  7.3 . among the 46 isolates ,", "serotype 1/2b was identified most frequently ( 46% ) , followed by 1/2a ( 28% ) and 4b ( 26% ) .", "all 46 isolates were susceptible to ampicillin , ertapenem , meropenem , and vancomycin ; 3 isolates were intermediately susceptible to trimethoprim / sulfamethoxazole ; and 4 isolates were nonsusceptible to linezolid ( 10 ) .", "sixteen ( 37% ) patients received cephalosporin alone as initial treatment regimens and empirical treatment was effective for only 14 ( 33% ) .", "the presence of solid - organ malignancies was a significant negative prognostic factor for 14-day mortality in the univariate analysis ( 95% confidence interval [ ci ] 1.16516.694 ; p = 0.029 ) but not in the multivariate analysis .", "the results of the multivariate analysis for 14-day mortality showed that hepatic decompensation at disease onset was a significant negative prognostic factor ( hazard ratio 12.02 , 95% ci 1.84278.470 ; p = 0.009 ) and that the use of effective antimicrobial drugs after culture results were reported was a significant positive prognostic factor ( hazard ratio 0.014 , 95% ci 0.0020.131 ; p<0.001 ) .", "log - rank tests performed to compare the difference in survival between patient groups for the 2 variables also had the same results ( p<0.001 ) .", "we observed an upsurge of listeriosis beginning in 2005 in ntuh . the increase might not be attributable to common - source outbreaks because no clustering was detected .", "the annual incidence of listeriosis has been on the rise in europe since 2000 ( 2,3,11 ) .", "the reason is not clear because the increase could not be attributed to outbreak clusters and no increase in pregnancy - related listeriosis was observed ( 2,3 ) .", "wong et al . found that l. monocytogenes was isolated in > 50% of pork samples and chicken carcasses ( 6 ) .", "semiready foods ( dumplings and meatballs ) and frozen dim sum examined also carried the pathogen ( 34.0% and 4.4% , respectively ) , and > 60% of the isolates were serotype 1 or 4 ( 6 ) . if served undercooked , these foods could be potential transmission sources .", "taiwan currently has no strict regulatory policy regarding listeriosis in the food industry and no disease surveillance system . in france ,", "serotype 4b was the predominant serotype ( 42%56% ) , whereas serotype 1/2b was more common ( 46% ) in our study ( 2 ) .", "none of the isolates were resistant to the tested agents , except for 4 isolates , which were resistant to linezolid .", "the contributions of disease severity and antimicrobial drug treatment are difficult to evaluate in population - based studies ( 12 ) .", "brouwer et al . reported that up to 30% of adults with l. monocytogenes meningitis did not receive initial adequate antimicrobial drug therapy , and they found no association between that variable and outcome ( 13 ) . a high proportion ( 37% ) of patients in our study", "initial disease severity and initial adequate antimicrobial drug therapy was not associated with overall mortality .", "we found that mortality was related to hepatic decompensation and effective antimicrobial drug therapy after culture results were reported .", "therefore , the incidence of and risk factors for human listeriosis in taiwan could not be determined precisely , and potential outbreaks might have been overlooked .", "the retrospective design of our study limited the possibility of identifying the potential vehicles and disease - acquiring behaviors .", "an increase of listeriosis was noted since 2005 in our hospital , and all of the affected patients had predisposing factors that hampered their immunity .", "dietary education and food management information should be provided to high - risk groups in taiwan .", "food monitoring and human disease surveillance systems need to be established in taiwan to control this potentially fatal foodborne disease ."], "abstract_text": ["<S> during 19962008 , a total of 48 patients with listeriosis were identified at a taiwan hospital . </S>", "<S> average annual incidence increased from 0.029 to 0.118 cases per 1,000 admissions before and after january 2005 . </S>", "<S> serotype 1/2b predominated ; serotype 4b emerged since 2004 . </S>", "<S> food monitoring and disease surveillance systems could help control listeriosis in taiwan . </S>"], "labels": null, "section_names": ["The Study", "Conclusions"], "sections": [["we reviewed the medical records of patients who had l. monocytogenes isolated from blood and body fluids from sterile sites during 19962008 at the national taiwan university hospital ( ntuh ) , a 2,500-bed hospital in taiwan .", "we evaluated disease severity using modified acute physiology and chronic health evaluation ii scores ( 8) .", "incidence of nontyphiodal salmonella bacteremia ( ntsb ) during 20002008 in ntuh was calculated for trend comparison of the 2 foodborne illnesses .", "only 1 episode was calculated during the same admission for ntsb to avoid the influence of repetitive bacteremia .", "isolates from patients with fetomaternal listeriosis ( i.e. , paired isolates from mother and neonate who had listeriosis ) were considered to be the same and only 1 of them was analyzed .", "all isolates were analyzed for their serotype by pcr as described , and genetic relatedness was evaluated by pulsed - field gel electrophoresis ( pfge ) by using pulsenet standardized protocols and 2 restriction enzymes ( asci and apai ) ( 2,9 ) .", "strains were considered to be of the same cluster if their bands had indistinguishable restriction patterns by both enzymes .", "strains with pfge patterns with > 80% similarity by asci and apai profiles were considered to be closely related .", "a forward stepwise model with a p value of 0.1 was used , and p<0.05 was considered statistically significant in the multivariate cox proportional hazards model . during the study period ,", "listeriosis was diagnosed in 48 patients , and 46 nonduplicated isolates were obtained for further microbiological analysis .", "average annual incidence increased from 0.0287 cases per 1,000 admissions during 19962004 to 0.118 cases per 1,000 admissions during 20052008 ( figure 1 ) .", "the increase in annual incidence of listeriosis was significantly correlated with years ( p = 0.0045 ) .", "the average annual incidences of ntsb were 1.189 and 1.118 per 1,000 admissions during 20002004 and 20052008 , respectively ; it was not significantly correlated with years ( p = 0.50 ) .", "age - specific incidence of listeriosis increased at both extremes of age , but especially among patients > 80 years ( figure 2 ) .", "all of the patients with listeriosis lived in northern taiwan , and no obvious geographic correlation was observed between listeriosis patients in each year .", "incidence ( cases per 1,000 admissions ) of human listeriosis and serotype distribution of all isolates , national taiwan university hospital , taipei , taiwan , 19962008 .", "forty - six isolates were available for analysis , including 2 serotype 4b isolates from fetomaternal transmission in 2005 and 2006 and 1 serotype 4b from a pediatric patient ( 2005 ) .", "patient distribution and incidence of human listeriosis , by age group , national taiwan university hospital , taipei , taiwan , 19962008 .", "all 43 patients had underlying predisposing conditions , and 18 ( 42% ) were > 65 years of age . of the 30 patients with malignancies , 23 ( 77% )", " one female patient had coexisting non - hodgkin lymphoma and colon cancer ; 1 male patient had coexisting non - hodgkin lymphoma , gastric cancer , and bladder cancer . ", "initial modified acute physiology and chronic health evaluation ii score , mean  sd : 18.8  7.3 . among the 46 isolates ,", "serotype 1/2b was identified most frequently ( 46% ) , followed by 1/2a ( 28% ) and 4b ( 26% ) .", "all 46 isolates were susceptible to ampicillin , ertapenem , meropenem , and vancomycin ; 3 isolates were intermediately susceptible to trimethoprim / sulfamethoxazole ; and 4 isolates were nonsusceptible to linezolid ( 10 ) .", "sixteen ( 37% ) patients received cephalosporin alone as initial treatment regimens and empirical treatment was effective for only 14 ( 33% ) .", "the presence of solid - organ malignancies was a significant negative prognostic factor for 14-day mortality in the univariate analysis ( 95% confidence interval [ ci ] 1.16516.694 ; p = 0.029 ) but not in the multivariate analysis .", "the results of the multivariate analysis for 14-day mortality showed that hepatic decompensation at disease onset was a significant negative prognostic factor ( hazard ratio 12.02 , 95% ci 1.84278.470 ; p = 0.009 ) and that the use of effective antimicrobial drugs after culture results were reported was a significant positive prognostic factor ( hazard ratio 0.014 , 95% ci 0.0020.131 ; p<0.001 ) .", "log - rank tests performed to compare the difference in survival between patient groups for the 2 variables also had the same results ( p<0.001 ) ."], ["we observed an upsurge of listeriosis beginning in 2005 in ntuh . the increase might not be attributable to common - source outbreaks because no clustering was detected .", "the annual incidence of listeriosis has been on the rise in europe since 2000 ( 2,3,11 ) .", "the reason is not clear because the increase could not be attributed to outbreak clusters and no increase in pregnancy - related listeriosis was observed ( 2,3 ) .", "wong et al . found that l. monocytogenes was isolated in > 50% of pork samples and chicken carcasses ( 6 ) .", "semiready foods ( dumplings and meatballs ) and frozen dim sum examined also carried the pathogen ( 34.0% and 4.4% , respectively ) , and > 60% of the isolates were serotype 1 or 4 ( 6 ) . if served undercooked , these foods could be potential transmission sources .", "taiwan currently has no strict regulatory policy regarding listeriosis in the food industry and no disease surveillance system . in france ,", "serotype 4b was the predominant serotype ( 42%56% ) , whereas serotype 1/2b was more common ( 46% ) in our study ( 2 ) .", "none of the isolates were resistant to the tested agents , except for 4 isolates , which were resistant to linezolid .", "the contributions of disease severity and antimicrobial drug treatment are difficult to evaluate in population - based studies ( 12 ) .", "brouwer et al . reported that up to 30% of adults with l. monocytogenes meningitis did not receive initial adequate antimicrobial drug therapy , and they found no association between that variable and outcome ( 13 ) . a high proportion ( 37% ) of patients in our study", "initial disease severity and initial adequate antimicrobial drug therapy was not associated with overall mortality .", "we found that mortality was related to hepatic decompensation and effective antimicrobial drug therapy after culture results were reported .", "therefore , the incidence of and risk factors for human listeriosis in taiwan could not be determined precisely , and potential outbreaks might have been overlooked .", "the retrospective design of our study limited the possibility of identifying the potential vehicles and disease - acquiring behaviors .", "an increase of listeriosis was noted since 2005 in our hospital , and all of the affected patients had predisposing factors that hampered their immunity .", "dietary education and food management information should be provided to high - risk groups in taiwan .", "food monitoring and human disease surveillance systems need to be established in taiwan to control this potentially fatal foodborne disease ."]]}
{"article_id": "PMC5362020", "article_text": ["three cats , siamese or siamese cross , were presented with a chronic thoracic limb weightbearing lameness .", "previous anti - inflammatory administrations were unable to improve lameness consistently in the three cats .", "two of the three cats had undergone onychectomy several years before presentation . a permanent flexion of the proximal interphalangeal joint of one or more digits , associated with a difficult and painful extension of the proximal interphalangeal joint ,", "for all cats , treatment consisted of a tenectomy or tenotomy of the superficial and deep digital flexor tendons in order to release the contracture .", "the three cats responded well to the surgical treatment and became sound around 24 weeks after surgery .", "it should be considered in a differential diagnosis of feline lameness whenever onychectomy has been performed in the past .", "the precise etiology that explains this tendon contracture is unknown , but trauma or breed predisposition could represent potential causes .", "digit lesions are most often associated with fractures , luxations , wounds , shearing injuries or tumors , and , less commonly , osteomyelitis or osteoarthritis .", "various surgical interventions are feasible , and digit amputation or luxation reduction is the most frequently performed treatment .", "feline onychectomy is also a common surgery in north america , despite debate over the ethics of this procedure .", "feline digital flexor tendon pathology , especially tendon contracture , is not well described . to our knowledge ,", "the term contracture refers to an abnormal pathologic process resulting in fibrosis and permanent damage to a muscle , characterized by replacement of the muscle and/or associated tendon with fibrous connective tissue .", "this phenomenon leads to shortening of the tendon or the muscle , and can have functional consequences on the range of motion of adjacent joints .", "most contractures are associated with a previous trauma , weeks to months before the contracture occurs , but repetitive strains , infectious diseases , ischemia , compartment syndrome or neoplasia may also lead to contracture .", "this case series reports three different clinical presentations of digital flexor tendon contracture in three cats , all successfully treated by tenectomy .", "a 4-year - old neutered male siamese cat weighing 4.8 kg was presented to a referral hospital for chronic left forelimb weight - bearing lameness of 1 year s duration .", "the cat showed mild and transient lameness improvement with meloxicam ( 0.05 mg / kg po q24h metacam ; boehringer ingelheim ) , but long - term medication did not show any additional benefits .", "the cat showed very mild lameness , with the left forelimb constantly non - weight bearing at rest .", "a permanent flexion of the proximal interphalangeal joint of the left forelimb second digit was noted .", "extension of the proximal interphalangeal joint was possible but very difficult and palpation of this digit was significantly painful .", "six months after initial presentation , the cat was presented for follow - up , without improvement .", "radio - graphic views of the left forelimb extremity revealed an abnormal positioning of the middle phalanx .", "indeed , the angulation between the middle and proximal phalanges was decreased and reached almost 90 between these two phalanges on the second digit .", "there was no evidence of retention of remnants , osteomyelitis or hyperostosis of the distal phalanx .", "following premedication with hydromorphone ( 0.1 mg / kg iv hydromorphone ; summit ) and acepromazine ( 0.02 mg / kg iv atravent ; boehringer ingelheim ) , general anesthesia was induced with propofol ( 4 mg / kg iv diprivan ; astrazeneca ) and maintained with 2% isoflurane ( forane ; baxter ) in 100% oxygen after orotracheal intubation .", "the cat was placed in dorsal recumbency , and the palmar surface from mid - radius to the extremity of the limb was clipped and aseptically prepared .", "surgery involved a palmar approach to the proximal interphalangeal joint of the second digit , with a midline skin incision extending from the distal aspect of the carpal pad to the proximal aspect of the metacarpal pad .", "following blunt dissection , the deep and superficial digital flexor muscle tendons were visualized and elevated , and a portion of the tendons ( 5 mm ) were transected with a # 15 scalpel blade ( figure 1 ) .", "the surgical wound was copiously lavaged with 0.9% sodium chloride ( 0.9% sodium chloride irrigation ; baxter ) .", "the subcutaneous tissues were closed with a continuous pattern , using a 4 - 0 absorbable monofilament ( poliglecaprone 25 , monocryl ; ethicon ) . the skin was closed with an interrupted pattern , using a 4 - 0 non - absorbable monofilament ( polyamide 6 , ethilon ii ; ethicon ) .", "the patient was discharged from the hospital the day after the surgery with strict rest instructions for 3 weeks and meloxicam ( 0.05 mg / kg q24 h po ) for 2 weeks .", "two weeks after surgery , the owner mentioned that the cat was very active , walking normally at home and was more playful than before surgery . upon", "follow - up examination no lameness was observed , and manipulation of the left proximal interphalangeal joint of the second digit was normal and pain free .", "two years after surgery the owner reported that the cat was totally sound , very active and had returned to its usual activity level .", "intraoperative plantar visualization of the flexor tendons of the third digit before tenectomy ( black arrow ) , exposed with a periosteal elevator ( * ) .", "left is distal , right is proximal , up is medial , down is lateral )", "a 7-month - old neutered female siamese weighing 3 kg was presented for a lameness of the left forelimb of 2 months duration .", "the owner reported that the lameness was most likely due to a trauma that occurred 2 months previously and was permanent since the incident .", "the cat received two different unknown non - steroidal anti - inflammatories before presentation at our hospital and did not show any improvement .", "the lameness was moderate at a walk and severe and non - weight bearing when the cat was running . at rest , a non - weight bearing stance was also obvious on the left forelimb .", "a permanent flexion of the proximal interphalangeal joint was noted on the fourth digit of the left forelimb .", "five months after initial presentation , the cat was presented for the same condition , without any improvement regarding lameness or comfort upon palpation .", "radiographic views of the left forelimb extremity revealed an abnormal positioning of the middle phalanx compared with the proximal phalanx , characterized by a decreased angulation between these two phalanges on the fourth digit .", "following premedication with butorphanol ( 0.1 mg / kg iv torbugesic ; pfizer ) , general anesthesia was induced with propofol ( 4 mg / kg iv ) and maintained with 2% isoflurane in 100% oxygen after orotracheal intubation .", "a tenectomy of the deep and superficial digital flexor muscle tendons was performed as previously described for the first case .", "the surgical preparation and operative technique were similar to those used in case 1 , except that the surgical site was the tendons of the fourth digit .", "the cat was discharged from the hospital the day after surgery with strict rest instructions for 3 weeks and meloxicam ( 0.05 mg / kg q24h orally ) for 2 weeks . at the 2 week re - evaluation ,", "the cat still presented a mild lameness of the left thoracic limb , although the extension of the proximal interphalangeal joint of the fourth digit was improved compared with preoperative examination .", "one month after surgery , the cat was totally sound and manipulation of the digit was not painful .", "at a 1.5 year postoperative follow - up , the owner reported that the cat was not limping anymore , was pain free and had returned to its normal activity .", "a 7-year - old neutered male siamese cross weighing 6 kg presented with right forelimb lameness , which had appeared a month earlier .", "the owner also reported that the cat had undergone onychectomy at 6 months of age .", "the cat received a non - steroidal anti - inflammatory treatment ( 0.05 mg / kg q24h po meloxicam ) for 2 weeks before presentation and did not show any improvement in lameness . upon examination ,", "the cat s right forelimb was non - weight bearing at rest , and the cat was uncomfortable while walking on both forelimbs .", "a permanent flexion of the proximal interphalangeal joint was noted in all four digits of both forelimbs ( figure 2 ) .", "moreover , a 3 mm diameter white firm and painful callus was palpable at the craniodistal aspect , bilaterally , of the third and fourth digital pads .", "radiographic views of the forelimb extremity revealed an abnormal positioning on both limbs between the middle and the proximal phalanx .", "indeed , the angulation between these two phalanges was decreased and reached almost 90 in all digits .", "no bony or articular lesion or osteomyelitis of the proximal and middle phalanges was observed .", "remnants of the distal phalanx were not observed in the radiographs ( figure 3 ) .", "a digital flexor tendon contracture of both forelimb digits was then suspected , and , after discussion , the owner agreed to surgical treatment .", "flexor contracture of all digits can be visualized ( left is distal , right is proximal , up is dorsal , down is plantar ) forelimb oblique extremity radiographic view of the left limb in case 3 .", "an abnormal positioning between the middle and the proximal phalanx is observed in the four digits .", "the angulation between these two phalanges is decreased and reached almost 90 in all digits following premedication with hydromorphone ( 0.1 mg / kg iv ) , general anesthesia was induced with alfaxalone ( 2 mg / kg iv alfaxan ; abbott laboratories ) and maintained with 2% isoflurane in 100% oxygen after orotracheal tube placement .", "a local regional anesthetic bloc was performed bilaterally with bupivacaine ( 0.5 mg / kg sc marcane 0.5% ; hospira healthcare corporation ) .", "the surgical procedure was performed as described in case 1 for digits iii and iv of both forelimbs , and a tenotomy was performed on digits ii and v on both forelimbs ( figure 4 ) .", "the cat was discharged from the hospital the day after the surgery with strict rest instructions for 3 weeks , meloxicam ( 0.05 mg / kg q24h po ) for 6 days and buprenorphine ( 20 g / kg q8h sublingually buprenorphine ; chiron compounding pharmacy ) for 7 days .", "perioperative dorsal view of an operated limb ( ol ) and a preoperative limb ( pl ) in case 3 .", "flexor contracture is well visualized in pl compared with ol ol = on the left ; pl = on the right at the 2 week follow - up , the cat was totally sound and manipulation of the forelimb digits was very comfortable . at rest , digits were in hyperextension ( figure 5 ) .", "histopathologic examination of some parts of the flexor tendons revealed dense and thick collagen bundles associated with a few non - reactive small fibroblasts .", "these observations were considered to be consistent with a chronic degeneration or trauma of this tendon . a definitive diagnosis of flexor tendon contracture was made ( figure 6 ) .", "thoracic limbs of case 3 , 4 weeks after flexor tendon tenectomy and tenotomy photomicrograph of a tendon section from a domestic cat .", "note the presence of increased well - differentiated fibroblasts ( large arrows ) in the tendon accompanied by few small - caliber blood vessels ( arrowhead ) and thin collagen fibers ( thin arrows ) .", "the three cats reported in this study presented with chronic mild forelimb lameness associated with reduction of the normal extension of the proximal interphalangeal joint of one or more digits .", "a contracture of the digital flexor tendon was suspected and confirmed by histopathologic analysis in one case .", "this case series is of particular interest because it describes three different presentations of digital flexor tendon contracture in cats . in case 1 , which involved an onychectomized cat , tendon contracture involved only one digit , which is different from the two cases reported by cooper et al . in case 2 ,", "the cat developed a tendon contracture on a digit , although it had not undergone onychectomy .", "case 3 is more comparable to the cases reported by cooper et al because this case involved tendon contracture of all digits in both forelimbs after onychectomy .", "however , such an important delay , as seen in this case , between onychectomy and the development of digital flexor tendon contracture has not been previously reported .", "we elected to perform only tenotomy on digits ii and v in case 3 because of a satisfactory intraoperative contracture release after tenotomy .", "however , although we thought that tenectomy was not mandatory to relieve contracture in theses digits , it could have been a valuable option for treatment of contracture .", "the surgical outcome was excellent in all cats , as in the two cases reported elsewhere .", "flexor contracture can be painful and disabling and can induce abnormal contact on normally non - contact areas of the digits .", "contracture release by tenectomy or tenotomy seemed to improve the lameness quickly , while long - term anti - inflammatory treatments failed in each of the cases reported here .", "the deep and superficial digital flexor tendons provide digital flexion through contraction of the muscle bellies .", "more precisely , the proximal interphalangeal joint is flexed by the action of the superficial digital flexor and the distal interphalangeal joint is flexed by the deep digital flexor .", "the superficial digital flexor ( flexor digitorum superficialis ) tendon runs on the palmar surface of the deep digital flexor tendon ( flexor digitorum profondus ) .", "it splits distally into four parts , which diverge to the second to fifth metacarpophalangeal joints , with the corresponding terminal tendons of the deep flexor tendon .", "each superficial digital flexor ends on the proximal aspect of the palmar surface of the middle phalanx after being perforated by its respective deep flexor tendon , which ends on the tuberosity of the distal phalanges of digits ii ", "those superficial and deep tendons are bridged by three transverse ligaments , at the metacarpophalangeal joints and at the proximal and middle phalangeal joints , on each of the four main digits . in two of the cases reported here , tendon", "therefore , the cause of the contracture must have been distal to the common tendon . with the third cat", ", the contracture could have been located more proximally than the tendon division , but multiple contractures of the distal portion of each superficial digital flexor tendon were confirmed by histopathologic analysis .", "distal aspect of flexor tendon anatomy on a third thoracic digit ( distal digital annular ligament has been removed ) . * deep flexor tendon ; p1 = proximal phalanx ; p2 = middle phalanx ; p3 = distal phalanx ; ( + ) = superficial flexor tendon a precise etiology is unknown in these cats .", "however , a traumatic episode was reported by the owner of case 2 , and this could have been the initial cause of the flexor tendon damage that led to a chronic problem .", "two cats presented for bilateral and painful flexor tendon contracture following onychectomy have been previously reported .", "both cats underwent onychectomy 6 weeks and 3 months before presentation , respectively , and had all digits of both front paws fixed in flexion .", "tenectomy of each deep digital flexor tendon successfully resolved the condition , and the cats were sound 2 months after surgery .", "the authors hypothesized that the postoperative contracture was the result of inflammation due to a suboptimal surgical technique ( excessive or rough tissue manipulation , use of a dull scalpel blade , improper use of tissue adhesives or poor aseptic technique ) .", "these factors could elicit an abnormal inflammatory response and result in flexor fibrosis , adhesion formation and evolution toward flexor tendon contracture .", "tobias et al reported that the duration of the surgery , which was potentially secondary to a suboptimal surgical technique , was correlated with the occurrence of postoperative lameness .", "this association could also be observed with persistent long - term lameness but the number of cases observed was considered too low to determine such an association .", "the anatomic relation between the deep digital flexor tendon and the middle phalanx could explain the long - term contracture . indeed", ", traumatic resection of the distal phalanx could be the cause of an inflammatory reaction in the area of the distal extremity of the middle phalanx and the deep digital flexor tendon .", "in addition to contracture of the digital flexor , other complications have been associated in 040% of cases of onychectomy in cats , such as pain , hemorrhage , lameness , swelling , non - weightbearing , infection , claw regrowth , middle phalanx protrusion and palmigrade stance .", "lameness is usually an early postoperative complication and is associated with pain , which resolves in 2 days but can persist for up to 2 months .", "the postoperative flexor contracture was previously reported in the first months after onychectomy but long - term postoperative contracture after onychectomy could explain undiagnosed chronic pain in some cats and particularly in cats 1 and 3 .", "thus , digital flexor tendon contracture can not be ruled out because of an increased delay from the onychectomy .", "a trauma was suspected for the second cat , as no previous onychectomy was performed on this patient .", "digital flexor tendon injuries in dogs and cats are uncommon and most often result from laceration by sharp or penetrating foreign objects .", "other conditions of digital flexor tendons are rare , and only a few cases have been reported .", "a report of a 10-month - old male great dane affected by an excessive non - painful exion of the digits of the rightforelimb ( more pronounced on the fourth digit ) has been published .", "this dog was lame for 2 months . restricted extension of the fourth digit was conrmed under general anesthesia , and a modied z - tenotomy was performed .", "surgery provided a very good outcome , as postoperative extension of the affected digit was possible and pain free , and no lameness was noted 7 months after surgery .", "a 5-month - old basset hound was also reported with simple incomplete syndactyly ( fusion of the third and fourth digital pads on a hindlimb ) and secondary contracture of the deep digital flexor tendon of the third and fourth digit . with this dog", ", the deep digital flexor tendon contracture was suspected to be secondary to the primary congenital anomaly and was treated by palmar tenotomy .", "the first two cats reported here were middle - aged indoor siamese cats , and the third case was a siamese cross .", "they are known to be predisposed to many diseases , including cardiovascular , dermatologic , endocrine , gastrointestinal , hematologic , infectious and musculoskeletal conditions , such as congenital myasthenia gravis , mucopolysaccharidosis vi or hip dysplasia .", "however , this hypothesis needs to be confirmed with additional cases and with tendon histopathologic analysis to find a more specific cause to the digital flexor contracture .", "this pathology is a flexor tendon entrapment of the digits that is characterized by locking of the fingers , with or without pain .", "the flexor entrapment is due to hypertrophy , narrowing and relative stenosis of the palmar retinacular sheath ( most often the transverse ligament at the metacarpophalangeal joint ) , which progressively restricts the motion of the flexor tendon .", "the precise etiology is not well understood and repetitive finger movements or local trauma leading to a progressive loss of fingers extension is currently the most plausible hypothesis .", "different treatments , such as medical treatment , joint immobilization , or corticosteroid injections , have been tried to manage the disease but surgical treatment is indicated when conservative management fails to resolve the pain and clinical signs .", "the surgical treatment consists of resection of the metacarpophalangeal transverse ligament , with or without resection of the superficial flexor tendon , and is highly effective with low associated complication and recurrence rates .", "digital flexor contracture can affect an isolated digit or all digits of a limb and is characterized by a chronic lameness and a painful permanent flexion of the affected digit .", "in contrast to medical management , tenectomy of the deep and superficial flexor tendons is associated with a good - to - excellent surgical outcome for this condition .", "etiology of this contracture is unknown but trauma , whether related to an onychectomy or not , seems to predispose a cat to this condition .", "this pathology could therefore be considered as a long - term complication of onychectomy in some cases . however , further cases need to be reported to better understand the pathogenesis of this condition .", "digit evaluation seems to be of paramount importance when investigating cases of chronic lameness in cats ."], "abstract_text": ["<S> case series summarythree cats , siamese or siamese cross , were presented with a chronic thoracic limb weightbearing lameness . </S>", "<S> previous anti - inflammatory administrations were unable to improve lameness consistently in the three cats . </S>", "<S> two of the three cats had undergone onychectomy several years before presentation . </S>", "<S> a permanent flexion of the proximal interphalangeal joint of one or more digits , associated with a difficult and painful extension of the proximal interphalangeal joint , was noticed during orthopedic examination . </S>", "<S> a digital flexor tendon contracture was suspected and confirmed with radiographic examination . </S>", "<S> surgical exploration was then performed . </S>", "<S> for all cats , treatment consisted of a tenectomy or tenotomy of the superficial and deep digital flexor tendons in order to release the contracture . </S>", "<S> the three cats responded well to the surgical treatment and became sound around 24 weeks after surgery.relevance and novel informationdigital flexor tendon contracture is rarely reported as a cause of lameness in cats . </S>", "<S> it should be considered in a differential diagnosis of feline lameness whenever onychectomy has been performed in the past . </S>", "<S> the precise etiology that explains this tendon contracture is unknown , but trauma or breed predisposition could represent potential causes . </S>"], "labels": null, "section_names": ["Case series summary", "Relevance and novel information", "Introduction", "Case 1", "Case 2", "Case 3", "Discussion", "Conclusions"], "sections": [["three cats , siamese or siamese cross , were presented with a chronic thoracic limb weightbearing lameness .", "previous anti - inflammatory administrations were unable to improve lameness consistently in the three cats .", "two of the three cats had undergone onychectomy several years before presentation . a permanent flexion of the proximal interphalangeal joint of one or more digits , associated with a difficult and painful extension of the proximal interphalangeal joint ,", "for all cats , treatment consisted of a tenectomy or tenotomy of the superficial and deep digital flexor tendons in order to release the contracture .", "the three cats responded well to the surgical treatment and became sound around 24 weeks after surgery ."], ["it should be considered in a differential diagnosis of feline lameness whenever onychectomy has been performed in the past .", "the precise etiology that explains this tendon contracture is unknown , but trauma or breed predisposition could represent potential causes ."], ["digit lesions are most often associated with fractures , luxations , wounds , shearing injuries or tumors , and , less commonly , osteomyelitis or osteoarthritis .", "various surgical interventions are feasible , and digit amputation or luxation reduction is the most frequently performed treatment .", "feline onychectomy is also a common surgery in north america , despite debate over the ethics of this procedure .", "feline digital flexor tendon pathology , especially tendon contracture , is not well described . to our knowledge ,", "the term contracture refers to an abnormal pathologic process resulting in fibrosis and permanent damage to a muscle , characterized by replacement of the muscle and/or associated tendon with fibrous connective tissue .", "this phenomenon leads to shortening of the tendon or the muscle , and can have functional consequences on the range of motion of adjacent joints .", "most contractures are associated with a previous trauma , weeks to months before the contracture occurs , but repetitive strains , infectious diseases , ischemia , compartment syndrome or neoplasia may also lead to contracture .", "this case series reports three different clinical presentations of digital flexor tendon contracture in three cats , all successfully treated by tenectomy ."], ["a 4-year - old neutered male siamese cat weighing 4.8 kg was presented to a referral hospital for chronic left forelimb weight - bearing lameness of 1 year s duration .", "the cat showed mild and transient lameness improvement with meloxicam ( 0.05 mg / kg po q24h metacam ; boehringer ingelheim ) , but long - term medication did not show any additional benefits .", "the cat showed very mild lameness , with the left forelimb constantly non - weight bearing at rest .", "a permanent flexion of the proximal interphalangeal joint of the left forelimb second digit was noted .", "extension of the proximal interphalangeal joint was possible but very difficult and palpation of this digit was significantly painful .", "six months after initial presentation , the cat was presented for follow - up , without improvement .", "radio - graphic views of the left forelimb extremity revealed an abnormal positioning of the middle phalanx .", "indeed , the angulation between the middle and proximal phalanges was decreased and reached almost 90 between these two phalanges on the second digit .", "there was no evidence of retention of remnants , osteomyelitis or hyperostosis of the distal phalanx .", "following premedication with hydromorphone ( 0.1 mg / kg iv hydromorphone ; summit ) and acepromazine ( 0.02 mg / kg iv atravent ; boehringer ingelheim ) , general anesthesia was induced with propofol ( 4 mg / kg iv diprivan ; astrazeneca ) and maintained with 2% isoflurane ( forane ; baxter ) in 100% oxygen after orotracheal intubation .", "the cat was placed in dorsal recumbency , and the palmar surface from mid - radius to the extremity of the limb was clipped and aseptically prepared .", "surgery involved a palmar approach to the proximal interphalangeal joint of the second digit , with a midline skin incision extending from the distal aspect of the carpal pad to the proximal aspect of the metacarpal pad .", "following blunt dissection , the deep and superficial digital flexor muscle tendons were visualized and elevated , and a portion of the tendons ( 5 mm ) were transected with a # 15 scalpel blade ( figure 1 ) .", "the surgical wound was copiously lavaged with 0.9% sodium chloride ( 0.9% sodium chloride irrigation ; baxter ) .", "the subcutaneous tissues were closed with a continuous pattern , using a 4 - 0 absorbable monofilament ( poliglecaprone 25 , monocryl ; ethicon ) . the skin was closed with an interrupted pattern , using a 4 - 0 non - absorbable monofilament ( polyamide 6 , ethilon ii ; ethicon ) .", "the patient was discharged from the hospital the day after the surgery with strict rest instructions for 3 weeks and meloxicam ( 0.05 mg / kg q24 h po ) for 2 weeks .", "two weeks after surgery , the owner mentioned that the cat was very active , walking normally at home and was more playful than before surgery . upon", "follow - up examination no lameness was observed , and manipulation of the left proximal interphalangeal joint of the second digit was normal and pain free .", "two years after surgery the owner reported that the cat was totally sound , very active and had returned to its usual activity level .", "intraoperative plantar visualization of the flexor tendons of the third digit before tenectomy ( black arrow ) , exposed with a periosteal elevator ( * ) .", "left is distal , right is proximal , up is medial , down is lateral )"], ["a 7-month - old neutered female siamese weighing 3 kg was presented for a lameness of the left forelimb of 2 months duration .", "the owner reported that the lameness was most likely due to a trauma that occurred 2 months previously and was permanent since the incident .", "the cat received two different unknown non - steroidal anti - inflammatories before presentation at our hospital and did not show any improvement .", "the lameness was moderate at a walk and severe and non - weight bearing when the cat was running . at rest , a non - weight bearing stance was also obvious on the left forelimb .", "a permanent flexion of the proximal interphalangeal joint was noted on the fourth digit of the left forelimb .", "five months after initial presentation , the cat was presented for the same condition , without any improvement regarding lameness or comfort upon palpation .", "radiographic views of the left forelimb extremity revealed an abnormal positioning of the middle phalanx compared with the proximal phalanx , characterized by a decreased angulation between these two phalanges on the fourth digit .", "following premedication with butorphanol ( 0.1 mg / kg iv torbugesic ; pfizer ) , general anesthesia was induced with propofol ( 4 mg / kg iv ) and maintained with 2% isoflurane in 100% oxygen after orotracheal intubation .", "a tenectomy of the deep and superficial digital flexor muscle tendons was performed as previously described for the first case .", "the surgical preparation and operative technique were similar to those used in case 1 , except that the surgical site was the tendons of the fourth digit .", "the cat was discharged from the hospital the day after surgery with strict rest instructions for 3 weeks and meloxicam ( 0.05 mg / kg q24h orally ) for 2 weeks . at the 2 week re - evaluation ,", "the cat still presented a mild lameness of the left thoracic limb , although the extension of the proximal interphalangeal joint of the fourth digit was improved compared with preoperative examination .", "one month after surgery , the cat was totally sound and manipulation of the digit was not painful .", "at a 1.5 year postoperative follow - up , the owner reported that the cat was not limping anymore , was pain free and had returned to its normal activity ."], ["a 7-year - old neutered male siamese cross weighing 6 kg presented with right forelimb lameness , which had appeared a month earlier .", "the owner also reported that the cat had undergone onychectomy at 6 months of age .", "the cat received a non - steroidal anti - inflammatory treatment ( 0.05 mg / kg q24h po meloxicam ) for 2 weeks before presentation and did not show any improvement in lameness . upon examination ,", "the cat s right forelimb was non - weight bearing at rest , and the cat was uncomfortable while walking on both forelimbs .", "a permanent flexion of the proximal interphalangeal joint was noted in all four digits of both forelimbs ( figure 2 ) .", "moreover , a 3 mm diameter white firm and painful callus was palpable at the craniodistal aspect , bilaterally , of the third and fourth digital pads .", "radiographic views of the forelimb extremity revealed an abnormal positioning on both limbs between the middle and the proximal phalanx .", "indeed , the angulation between these two phalanges was decreased and reached almost 90 in all digits .", "no bony or articular lesion or osteomyelitis of the proximal and middle phalanges was observed .", "remnants of the distal phalanx were not observed in the radiographs ( figure 3 ) .", "a digital flexor tendon contracture of both forelimb digits was then suspected , and , after discussion , the owner agreed to surgical treatment .", "flexor contracture of all digits can be visualized ( left is distal , right is proximal , up is dorsal , down is plantar ) forelimb oblique extremity radiographic view of the left limb in case 3 .", "an abnormal positioning between the middle and the proximal phalanx is observed in the four digits .", "the angulation between these two phalanges is decreased and reached almost 90 in all digits following premedication with hydromorphone ( 0.1 mg / kg iv ) , general anesthesia was induced with alfaxalone ( 2 mg / kg iv alfaxan ; abbott laboratories ) and maintained with 2% isoflurane in 100% oxygen after orotracheal tube placement .", "a local regional anesthetic bloc was performed bilaterally with bupivacaine ( 0.5 mg / kg sc marcane 0.5% ; hospira healthcare corporation ) .", "the surgical procedure was performed as described in case 1 for digits iii and iv of both forelimbs , and a tenotomy was performed on digits ii and v on both forelimbs ( figure 4 ) .", "the cat was discharged from the hospital the day after the surgery with strict rest instructions for 3 weeks , meloxicam ( 0.05 mg / kg q24h po ) for 6 days and buprenorphine ( 20 g / kg q8h sublingually buprenorphine ; chiron compounding pharmacy ) for 7 days .", "perioperative dorsal view of an operated limb ( ol ) and a preoperative limb ( pl ) in case 3 .", "flexor contracture is well visualized in pl compared with ol ol = on the left ; pl = on the right at the 2 week follow - up , the cat was totally sound and manipulation of the forelimb digits was very comfortable . at rest , digits were in hyperextension ( figure 5 ) .", "histopathologic examination of some parts of the flexor tendons revealed dense and thick collagen bundles associated with a few non - reactive small fibroblasts .", "these observations were considered to be consistent with a chronic degeneration or trauma of this tendon . a definitive diagnosis of flexor tendon contracture was made ( figure 6 ) .", "thoracic limbs of case 3 , 4 weeks after flexor tendon tenectomy and tenotomy photomicrograph of a tendon section from a domestic cat .", "note the presence of increased well - differentiated fibroblasts ( large arrows ) in the tendon accompanied by few small - caliber blood vessels ( arrowhead ) and thin collagen fibers ( thin arrows ) ."], ["the three cats reported in this study presented with chronic mild forelimb lameness associated with reduction of the normal extension of the proximal interphalangeal joint of one or more digits .", "a contracture of the digital flexor tendon was suspected and confirmed by histopathologic analysis in one case .", "this case series is of particular interest because it describes three different presentations of digital flexor tendon contracture in cats . in case 1 , which involved an onychectomized cat , tendon contracture involved only one digit , which is different from the two cases reported by cooper et al . in case 2 ,", "the cat developed a tendon contracture on a digit , although it had not undergone onychectomy .", "case 3 is more comparable to the cases reported by cooper et al because this case involved tendon contracture of all digits in both forelimbs after onychectomy .", "however , such an important delay , as seen in this case , between onychectomy and the development of digital flexor tendon contracture has not been previously reported .", "we elected to perform only tenotomy on digits ii and v in case 3 because of a satisfactory intraoperative contracture release after tenotomy .", "however , although we thought that tenectomy was not mandatory to relieve contracture in theses digits , it could have been a valuable option for treatment of contracture .", "the surgical outcome was excellent in all cats , as in the two cases reported elsewhere .", "flexor contracture can be painful and disabling and can induce abnormal contact on normally non - contact areas of the digits .", "contracture release by tenectomy or tenotomy seemed to improve the lameness quickly , while long - term anti - inflammatory treatments failed in each of the cases reported here .", "the deep and superficial digital flexor tendons provide digital flexion through contraction of the muscle bellies .", "more precisely , the proximal interphalangeal joint is flexed by the action of the superficial digital flexor and the distal interphalangeal joint is flexed by the deep digital flexor .", "the superficial digital flexor ( flexor digitorum superficialis ) tendon runs on the palmar surface of the deep digital flexor tendon ( flexor digitorum profondus ) .", "it splits distally into four parts , which diverge to the second to fifth metacarpophalangeal joints , with the corresponding terminal tendons of the deep flexor tendon .", "each superficial digital flexor ends on the proximal aspect of the palmar surface of the middle phalanx after being perforated by its respective deep flexor tendon , which ends on the tuberosity of the distal phalanges of digits ii ", "those superficial and deep tendons are bridged by three transverse ligaments , at the metacarpophalangeal joints and at the proximal and middle phalangeal joints , on each of the four main digits . in two of the cases reported here , tendon", "therefore , the cause of the contracture must have been distal to the common tendon . with the third cat", ", the contracture could have been located more proximally than the tendon division , but multiple contractures of the distal portion of each superficial digital flexor tendon were confirmed by histopathologic analysis .", "distal aspect of flexor tendon anatomy on a third thoracic digit ( distal digital annular ligament has been removed ) . * deep flexor tendon ; p1 = proximal phalanx ; p2 = middle phalanx ; p3 = distal phalanx ; ( + ) = superficial flexor tendon a precise etiology is unknown in these cats .", "however , a traumatic episode was reported by the owner of case 2 , and this could have been the initial cause of the flexor tendon damage that led to a chronic problem .", "two cats presented for bilateral and painful flexor tendon contracture following onychectomy have been previously reported .", "both cats underwent onychectomy 6 weeks and 3 months before presentation , respectively , and had all digits of both front paws fixed in flexion .", "tenectomy of each deep digital flexor tendon successfully resolved the condition , and the cats were sound 2 months after surgery .", "the authors hypothesized that the postoperative contracture was the result of inflammation due to a suboptimal surgical technique ( excessive or rough tissue manipulation , use of a dull scalpel blade , improper use of tissue adhesives or poor aseptic technique ) .", "these factors could elicit an abnormal inflammatory response and result in flexor fibrosis , adhesion formation and evolution toward flexor tendon contracture .", "tobias et al reported that the duration of the surgery , which was potentially secondary to a suboptimal surgical technique , was correlated with the occurrence of postoperative lameness .", "this association could also be observed with persistent long - term lameness but the number of cases observed was considered too low to determine such an association .", "the anatomic relation between the deep digital flexor tendon and the middle phalanx could explain the long - term contracture . indeed", ", traumatic resection of the distal phalanx could be the cause of an inflammatory reaction in the area of the distal extremity of the middle phalanx and the deep digital flexor tendon .", "in addition to contracture of the digital flexor , other complications have been associated in 040% of cases of onychectomy in cats , such as pain , hemorrhage , lameness , swelling , non - weightbearing , infection , claw regrowth , middle phalanx protrusion and palmigrade stance .", "lameness is usually an early postoperative complication and is associated with pain , which resolves in 2 days but can persist for up to 2 months .", "the postoperative flexor contracture was previously reported in the first months after onychectomy but long - term postoperative contracture after onychectomy could explain undiagnosed chronic pain in some cats and particularly in cats 1 and 3 .", "thus , digital flexor tendon contracture can not be ruled out because of an increased delay from the onychectomy .", "a trauma was suspected for the second cat , as no previous onychectomy was performed on this patient .", "digital flexor tendon injuries in dogs and cats are uncommon and most often result from laceration by sharp or penetrating foreign objects .", "other conditions of digital flexor tendons are rare , and only a few cases have been reported .", "a report of a 10-month - old male great dane affected by an excessive non - painful exion of the digits of the rightforelimb ( more pronounced on the fourth digit ) has been published .", "this dog was lame for 2 months . restricted extension of the fourth digit was conrmed under general anesthesia , and a modied z - tenotomy was performed .", "surgery provided a very good outcome , as postoperative extension of the affected digit was possible and pain free , and no lameness was noted 7 months after surgery .", "a 5-month - old basset hound was also reported with simple incomplete syndactyly ( fusion of the third and fourth digital pads on a hindlimb ) and secondary contracture of the deep digital flexor tendon of the third and fourth digit . with this dog", ", the deep digital flexor tendon contracture was suspected to be secondary to the primary congenital anomaly and was treated by palmar tenotomy .", "the first two cats reported here were middle - aged indoor siamese cats , and the third case was a siamese cross .", "they are known to be predisposed to many diseases , including cardiovascular , dermatologic , endocrine , gastrointestinal , hematologic , infectious and musculoskeletal conditions , such as congenital myasthenia gravis , mucopolysaccharidosis vi or hip dysplasia .", "however , this hypothesis needs to be confirmed with additional cases and with tendon histopathologic analysis to find a more specific cause to the digital flexor contracture .", "this pathology is a flexor tendon entrapment of the digits that is characterized by locking of the fingers , with or without pain .", "the flexor entrapment is due to hypertrophy , narrowing and relative stenosis of the palmar retinacular sheath ( most often the transverse ligament at the metacarpophalangeal joint ) , which progressively restricts the motion of the flexor tendon .", "the precise etiology is not well understood and repetitive finger movements or local trauma leading to a progressive loss of fingers extension is currently the most plausible hypothesis .", "different treatments , such as medical treatment , joint immobilization , or corticosteroid injections , have been tried to manage the disease but surgical treatment is indicated when conservative management fails to resolve the pain and clinical signs .", "the surgical treatment consists of resection of the metacarpophalangeal transverse ligament , with or without resection of the superficial flexor tendon , and is highly effective with low associated complication and recurrence rates ."], ["digital flexor contracture can affect an isolated digit or all digits of a limb and is characterized by a chronic lameness and a painful permanent flexion of the affected digit .", "in contrast to medical management , tenectomy of the deep and superficial flexor tendons is associated with a good - to - excellent surgical outcome for this condition .", "etiology of this contracture is unknown but trauma , whether related to an onychectomy or not , seems to predispose a cat to this condition .", "this pathology could therefore be considered as a long - term complication of onychectomy in some cases . however , further cases need to be reported to better understand the pathogenesis of this condition .", "digit evaluation seems to be of paramount importance when investigating cases of chronic lameness in cats ."]]}
{"article_id": "PMC3984813", "article_text": ["one of them , adiponectin , also known as acrp30 , adipoq , gbp28 , and apm1 , was discovered by four independent research teams in 1995/1996 , [ 25 ] as the most abundant product of the adipose tissue .", "the hormone is a 244-amino acid protein with 30 kda molecular weight that circulates in the serum in different multimeric forms .", "actions of adiponectin are mediated by two types of receptors : adiponectin receptor 1 ( adipor1 ) and 2 ( adipor2 ) .", "the receptors are highly related and share 67% sequence identity in mice , but they differ in their tissue distribution and the ability to bind various forms of adiponectin .", "adipor1 shows high affinity for the globular form of adiponectin , whereas adipor2 is characterized by intermediate binding affinity for both globular and full - length adiponectin .", "the highest levels of adipor1 expression are observed in skeletal muscles , whereas adipor2 is most highly expressed in the liver [ 7 , 8 ] . due to the extensive distribution of adiponectin receptors in peripheral tissues and organs , adiponectin exerts pleiotropic effects on metabolism , including regulation of homeostasis by fatty acid oxidation , stimulation of glucose uptake and gluconeogenesis inhibition , which leads to intensified thermogenesis and weight loss .", "consequently , adiponectin level correlates negatively with body fat   and positively with insulin sensitivity .", "the existing evidence points to the presence of a common endocrine system comprising several hormones , including ghrelin , leptin , and orexin that controls metabolism and reproductive functions [ 1215 ] .", "adiponectin mrna and protein were found in the ovaries of several species , including humans , rats , and cows [ 1619 ] .", "adiponectin mrna and protein were also detected in the ovaries of prepubertal but not sexually mature gilts .", "the influence of the hormonal status of swine related to the stage of the oestrous cycle on adiponectin expression in corpora lutea , granulosa , and theca interna cells remains unknown .", "studies indicating higher concentrations of adiponectin in female than in male blood suggest that ovarian steroids may regulate adiponectin secretion [ 20 , 21 ] .", "similar conclusions can be drawn from varied plasma levels of adiponectin during the oestrous cycle in pigs . despite the above ,", "the possible influence of adiponectin on the production of steroid hormones by porcine ovarian cells remains unexplored .", "therefore , the aim of this study was to investigate the expression of the adiponectin gene by real - time pcr , to determine the concentrations of adiponectin protein in the porcine ovary ( corpora lutea , granulosa and theca interna cells ) and to compare gene and protein expression levels during different stages of the oestrous cycle in pigs . additionally , we sought to determine the in vitro effect of adiponectin on the secretion of steroid hormones ( progesterone , oestradiol , testosterone , and androstenedione ) by ovarian luteal , granulosa , and theca interna cells of sexually mature swine .", "all experiments were carried out in observance of the ethical standards of the animal ethics committee at the university of warmia and mazury in olsztyn . the experimental material comprised mature gilts ( large white and polish landrace ) from a private breeding farm , aged 7 - 8 months and weighing 130140  kg .", "twenty gilts were divided into four experimental groups as follows : days 2 - 3 , 1012 , 1416 , and 1719 of the oestrous cycle .", "the day of the second oestrus was designated as day 0 of the oestrous cycle .", "the phase of the oestrous cycle was also determined based on ovarian morphology . additionally , to fully confirm correctness of the evaluation of the oestrous cycle phase , the level of progesterone was determined .", "dissected corpora lutea ( cls ) from different stages of the oestrous cycle ( days 2 - 3-corpora haemorrhagica , 1012-mature cls , and 1416-regressing cls ) were either immediately frozen in liquid nitrogen and stored at 80c until rna and protein analysis or , similarly to the ovaries from days 1719 of the cycle , placed in cold pbs buffer and transported to the laboratory where luteal , follicular granulosa , and theca interna cells were isolated .", "granulosa and theca interna cells are precursor cells of large and small luteal cells , respectively .", "dissected corpora lutea from ovaries on days 2 - 3 , 1012 , and 1416 were minced into small fragments and dispersed in f-12 medium containing bovine serum albumin fraction v ( bsa ; 1% ) and antibiotics .", "corpora lutea were enzymatically dissociated in 0.125% trypsin solution ( 46 times ) at 37c , centrifuged ( 300  g , 10  min , 4c ) , and washed three times .", "isolated luteal cells were filtered through nylon mesh ( 40  m in diameter ) and resuspended in fresh f-12 medium .", "the cells were counted using a haemocytometer , and their viability ( ~90% ) was determined by 0.4% trypan blue dye exclusion .", "granulosa and theca interna cells were isolated from large follicles ( diameter > 6  mm ) without signs of atresia .", "granulosa cells were aspirated with a syringe and additionally washed out with a strong stream of media directed to the internal wall of the follicle .", "the theca interna layer was scraped off from granulosa cells and enzymatically dispersed in 0.25% trypsin solution .", "dispersed cells were centrifuged ( 800  g for 10  min ) and washed two and three times to isolate granulosa and theca interna cells , respectively .", "the cells were filtered through nylon mesh ( 40  m in diameter ) and resuspended in eagle 's medium enriched with bsa ( 5% ) and antibiotics .", "the cells were counted using a haemocytometer , and their viability ( ~98% ) was determined by 0.4% trypan blue dye exclusion .", "some of granulosa and theca interna cells ( 5  10 viable cells within each experiment ) were immediately resuspended in trizol reagent ( invitrogen , usa ) and stored at 80c until processing for rna analysis .", "total rna was extracted from luteal tissues from days 2 - 3 , 1012 , and 1416 of the oestrous cycle using the absolutely rna miniprep kit ( stratagene , usa ) . in the case of granulosa and theca cells", "approximately 1  g of rna was reverse - transcribed into cdna in a total volume of 20  l with 0.5  g oligo ( dt)15 primer ( roche , germany ) using the omniscript rt kit ( qiagen , usa ) at 37c for one hour and was terminated by incubation at 93c for 5  min .", "quantitative real - time pcr analysis was performed using a pcr system 7300 ( applied biosystems , usa ) .", "sense and antisense primers ( adiponectin , cyclophilin a ) were chosen according to lord et al .  ", "the chosen primers were as follows : adiponectin forward : 5atgatgtcaccactggcaaattc-3 , reverse : 5-gaccgtgacgtggaaggaga-3 ; cyclophilin a , forward : 5-gcactggtggcaagtccat-3 , reverse : 5-aggacccgtatgcttcagga-3 ; gapdh forward : 5-ccttcattgacctccactacatggt-3 , reverse : 5-ccacaacatacgtagcaccagcatc-3. adiponectin primers ( access no : ay135647 ) were complementary to positions 514536 ( f ) and 565584 ( r ) of pig adiponectin gene sequence ; cyclophilin a primers ( access no : ay266299 ) were complementary to positions 219237 ( f ) and 269299 ( r ) of pig cyclophilin a gene sequence , gapdh primers ( access no : u48832 ) were complementary to positions 6185 ( f ) and 219243 ( r ) of pig gapdh . a constitutively", "expressed genes , cyclophilin a and gapdh , were used as the internal control to verify the quantitative real - time pcr . to ensure that cyclophilin", "a and gapdh were the suitable reference genes for this study , we revealed that there were no statistically significant differences in ct values between the examined ovarian structures throughout all investigated stages of the oestrous cycle .", "both of the housekeeping genes were also indicated as suitable reference genes ( internal controls ) in the independent studies .", "the pcr reaction included 20  ng cdna , 900  nm ( adiponectin forward ) , 300  nm ( adiponectin reverse , cyclophilin a forward and reverse ) and 60  nm ( gapdh forward and reverse ) primers , 12.5  l sybr green pcr master mix ( applied biosystems , usa ) , and rnase free water in a final volume of 25  l .", "quantitative real - time pcr cycling conditions were as follows : initial denaturation and enzyme activation at 95c for 10  min , followed by 40 cycles of denaturation at 95c for 15  s and annealing at 59 for 1  min .", "negative controls were performed in which water was substituted for cdna , or reverse transcription was not performed prior to pcr .", "the specificity of amplification was tested at the end of the pcr by melting - curve analysis .", "calculation of relative expression levels of adiponectin was conducted based on the comparative cycle threshold method ( ct )   and normalized using the geometrical mean of reference genes expression levels : gapdh and cyclophilin a. western blotting analysis was performed as described by smolinska et al . .", "briefly , equal amounts of porcine corpora lutea as well as granulosa and theca cells lysats ( 10  g ) were resolved by sds - page on 12.5% polyacrylamide gel and then transferred to a nitrocellulose membranes ( whatman , usa ) .", "blots were blocked for 5  h at 4c in 1  tbst containing 5% skimmed milk powder and then incubated overnight at 4c with the rabbit polyclonal adiponectin antibodies at the dilution of 1  :  150 ( santa cruz biotechnology , usa ) or rabbit polyclonal actin antibodies ( sigma , usa ) diluted 1  :  200 , which were used as an internal control for equal loading and to quantify porcine adiponectin protein . to identify immunoreactive bands ,", "membranes were incubated for 1.5  h at room temperature with mouse anti - rabbit igg for adiponectin ( sigma , usa ; diluted 1  :  2000 ) , goat anti - rabbit igg for actin ( diluted 1  :  5000 ) conjugated with alkaline phosphatase ( santa cruz biotechnology , usa ) .", "nonspecific foetal calf serum ( mp biomedicals , usa ) was used instead of primary antibodies to produce negative control blots .", "the immunocomplexes were visualized using 4-nitroblue tetrazolium chloride ( nbt ) and 5-bromo-4-chloro-3-indolyl phosphate ( bcip ) , according to the manufacture 's protocol ( promega , usa ) .", "the results of western blotting were quantified by densitometric scanning of immunoblots with gelscan for windows ver . 1.45 software ( kucharczyk , poland ) .", "data were expressed as a ratio of adiponectin protein relative to actin protein in arbitrary optical density units .", "luteal cells ( 250000/1  ml medium ) were resuspended in f-12 medium enriched with foetal calf serum ( fcs ; 20% ) , bsa ( 1% ) and antibiotics and preincubated for 48 hours in a humidified incubator with 95% air and 5% co2 atmosphere .", "the serum - containing medium was discarded , and the cells were washed using serum - free f-12 medium .", "after washing , luteal cells were cultured for 24 hours in f-12 medium with bsa ( 1% ) and antibiotics , with or without treatment agents .", "granulosa and theca interna cells ( 250000/1  ml medium ) were resuspended in eagle 's medium supplemented with 10% fcs , 5% bsa , and antibiotics .", "after 24 hours of preincubation , the medium was discarded , and the cells were washed and cultured for 24 hours in eagle 's medium with 5% bsa and antibiotics , with or without treatment reagents .", "the cultured cells were treated with 1 or 10  g / ml of recombinant human adiponectin ( biovendor , usa ) alone or in combination with insulin ( granulosa and theca interna cells ; 10  ng / ml ; sigma , usa ) , fsh ( granulosa cells ; 10  ng / ml ; nhpp , usa ) and lh ( theca interna cells ; 10  ng / ml ; nhpp , usa ) , and combination of those treatments ( insulin + fsh for granulosa cells and insulin + lh for theca interna cells ) .", "adiponectin doses used in the experiment were close to physiological concentrations of this hormone in the porcine blood .", "those cells are thought to be generally more autonomic than follicular cells which is a reason of luteal cells ' hard response to stimulators [ 3133 ] .", "the alamar blue test revealed that none of the treatments affected the viability of the cultured cells .", "following incubation , the media were harvested , centrifuged ( 800  g for 10  min ) , and the supernatants were collected and stored at 20c until analyses of progesterone ( cultures of luteal , theca interna , and granulosa cells ) , oestradiol ( cultures of theca interna and granulosa cells ) , androstenedione , and testosterone ( cultures of theca interna cells ) . progesterone ( p4 )", "was analysed according to the method described by ciereszko et al . , oestradiol ( e2 ) was determined according to the method of hotchkiss et al .   modified by kotwica , androstenedione ( a4 ) as described by dziadkowiec et al . , and testosterone ( t ) according to kotwica and williams .", "the specificity of the antibodies against a4 , t , and e2 has been reported by ciereszko et al .   and szafranska et al . .", "the sensitivities of the assays for p4 and a4 were 1  pg / ml and for e2 and t were 0.5  pg / ml .", "intra- and interassay coefficients of variation of the p4 , e2 , a4 , and t assays were 0.91% , 0.7% , 0.81% , 0.97% and 8.5% , 10.1% , 14.2% , and 7.8% , respectively .", "data from real - time analysis and western blot were analysed by one - way anova and least significant difference ( lsd ) post hoc test and are reported as the means  sem from five independent observations .", "all data concerning in vitro cell cultures were analysed by anova for repeated measurements and least significant difference ( lsd ) post hoc test and are reported as the means  sem from five ( for all types of cells ) independent observations .", "each experiment was performed on different day using separate pool of luteal or follicular cells .", "the expression of adiponectin mrna was significantly higher in corpora lutea during all investigated days of the luteal phase than in theca interna cells isolated on days 1719 of the cycle ( p < 0.05 ) .", "no significant differences were noted in adiponectin gene expression in granulosa cells compared with theca interna cells and cls ( figure 1(a ) ) . in general ,", "adiponectin protein concentration in the porcine ovary was higher in the luteal phase than in the follicular phase .", "in particular , the adiponectin protein content was the greatest in cls from days 2 - 3 in comparison with cls and follicular cells from the remaining stages of the oestrous cycle ( p < 0.01 ) . within", "the luteal phase adiponectin concentration was the lowest in cls during days 1012 ( p < 0.01 ) .", "there was no significant differences in protein expression between granulosa and theca interna cells ( figure 1(b ) ) .", "treatment of luteal cells from days 1012 of the oestrous cycle with adiponectin at both doses ( 1  g / ml ; 10  g / ml ) resulted in significant ( p < 0.05 ) decrease in progesterone secretion ( figure 2 ) .", "adiponectin at any dose did not affect p4 secretion by luteal cells collected on days 2 - 3 and 1416 ( data not shown ) .", "adiponectin at the higher dose ( 10  g / ml ) in combination with insulin provoked the increase in p4 secretion in comparison with the cells stimulated by insulin alone ( p < 0.05 ) .", "in contrast , there was no effect of adiponectin ( both doses ) on fsh- and fsh + insulin - induced production of p4 in porcine granulosa cells ( figure 3 ) .", "basal secretion of p4 was unaffected independently on adiponectin dose ( data not shown ) .", "adiponectin at the higher dose ( 10  g / ml ) increased basal secretion of e2 ( p < 0.05 ) ( figure 4 ) .", "adiponectin did not change secretion of e2 by granulosa cells cultured with insulin and/or fsh ( data not shown ) .", "adiponectin at both doses reduced basal secretion of t by porcine theca interna cells ( p < 0.05 ) ( figure 5 ) .", "the effect of adiponectin on basal p4 , e2 , and a4 release was negligible ( data not shown ) .", "adiponectin ( 10  g / ml ) decreased lh + insulin - stimulated secretion of p4 by theca interna cells ( p < 0.05 ) .", "there was no effect of adiponectin on lh- or insulin - induced production of p4 by these cells ( figure 6(a ) ) .", "adiponectin did not influence lh- or insulin - stimulated secretion of e2 by theca interna cells .", "however , lh + insulin - induced secretion of e2 by the cells was increased in response to the higher dose ( 10  g / ml ) of adiponectin ( p < 0.05 ) ( figure 6(b ) ) .", "secretion of a4 by theca interna cells was inhibited by combination of adiponectin ( 10  g / ml ) with insulin compared to insulin alone ( p < 0.05 ) .", "adiponectin at the lower dose ( 1  g / ml ) increased lh + insulin - induced a4 secretion by the cells ( p < 0.05 ) ( figure 6(c ) ) .", "induced secretion of t was not changed under adiponectin influence ( data not shown ) .", "this is the first ever study to demonstrate the changes in adiponectin gene and protein expression in the porcine ovary throughout the oestrous cycle .", "our results indicate that adiponectin is more highly expressed in luteal cells derived from ovaries in all examined stages of the luteal phase than in follicular granulosa and theca interna cells .", "adiponectin was also found to affect basal and stimulated secretion of steroid hormones by porcine ovarian cells .", "in addition to porcine ovarian structures , adiponectin gene expression and the presence of the hormone protein were also found in woman theca interna cells   and in theca interna cells , granulosa cells , corpora lutea , and oocytes of rats   and cows [ 18 , 19 ] .", "singh and krishna   localized the adiponectin protein in bat ovarian structures that showed positive immunostaining in theca interna cells , oocytes of growing follicles , and the corpus luteum .", "the expression of adiponectin gene and protein was also determined in porcine granulosa cells of prepubertal gilts .", "the levels of adiponectin expression and also its receptors seem to vary subject to cell type and the stage of follicle and corpora lutea development . in the bovine ovary ,", "the expression of adiponectin and its both receptors was lower in granulosa cells than in theca interna cells and oocytes .", "contrary results were presented by ortega et al .   who observed the highest levels of adiponectin expression in granulosa cells of sheep ovaries .", "throughout follicular development , adiponectin mrna increased in granulosa cells and decreased in theca interna cells of cows . in the corpora lutea ,", "bovine theca interna cells derived from large follicles revealed higher expression of both receptors than cells isolated from medium - sized and small follicles , indicating that follicular growth influences the transcript levels of adiponectin receptors .", "the synthesis of ovarian adiponectin and the expression of adipor1 and adipor2 are probably hormonally controlled , as suggested by an increase in adiponectin concentrations in ovarian follicular fluid of women administrated lh in the in vitro fertilization procedure .", "following pmsg pretreatment , an injection of hcg also significantly increased the expression of adiponectin and adipor1 ( but not adipor2 ) genes in rat ovaries .", "in bovine theca interna cells , lh increased the concentrations of adipor2 mrnas , whereas igf - i suppressed the expression of the above gene .", "the results of the present study seem to confirm the hypothesis that adiponectin production is regulated hormonally .", "higher levels of adiponectin gene and protein expression during the luteal phase and lower expression levels during the follicular phase could point to the stimulatory effect of progesterone and the inhibitory influence of oestradiol on ovarian adiponectin production . in our previous study ,", "adiponectin serum concentrations were higher during the luteal phase than the follicular phase , which suggests that ovarian steroids influence plasma adiponectin levels . in this study", "the results of our in vitro studies and the presence of both adiponectin receptors in porcine ovaries indicate that adiponectin may directly affect ovarian steroidogenesis .", "in fact , progesterone secretion decreased due to adiponectin 's influence on luteal cells in mid - luteal phase ( days 1012 ) .", "greater variations in adiponectin action were observed in porcine follicular cells . induced progesterone production increased in granulosa cells and decreased in theca interna cells .", "basal testosterone output and insulin - induced androstenedione secretion were inhibited , while lh + insulin - stimulated release of androstenedione was enhanced by adiponectin .", "the effect of adiponectin on ovarian steroidogenesis was also suggested in studies of cows , rats , and humans .", "adiponectin was found to inhibit insulin - induced secretion of progesterone and oestradiol by bovine granulosa cells . in a study of theca interna cells of bovine ovaries ,", "lagaly et al .   observed that adiponectin decreased lh + insulin - induced production of progesterone .", "the above authors also noted that adiponectin inhibited the mrna expression of lh receptor in granulosa cells .", "granulosa cells of rats and women treated with igf - i responded to adiponectin by increasing progesterone and oestradiol secretion [ 16 , 17 ] .", "in the cited studies , adiponectin did not influence fsh - induced production of progesterone and oestradiol in human and rat granulosa cells which is consistent with the response of porcine cells noted in this study .", "it seems that adiponectin affects ovarian functions by binding adipor1 and adipor2 , in two ways .", "an experiment where rnai was used to block adipor1 and adipor2 expression in the kgn cell line derived from human granulosa cells contributed to new information about adiponectin 's role in the ovaries .", "the absence of adipor1 in the analysed cells enhanced apoptosis , whereas the elimination of adipor2 reduced fsh- and igf - i - induced the production of progesterone and oestradiol and inhibited mitogen - activated kinase activity , relative to control , in response to adiponectin or fsh treatment .", "the above results suggest that adipor1 is more involved in the survival of granulosa cells , whereas adipor2 regulates steroidogenesis through mapk activation .", "the influence of adiponectin on the ovaries of sexually mature pigs has not been studied to date , but the response of granulosa cells derived from prepubertal gilts to the analysed hormone was described by ledoux et al . .", "the above authors observed higher levels of cyclooxygenase-2 , prostaglandin e synthase , and vegf gene expression in cells primed with adiponectin , an increase in the expression of the star gene and a decrease in the expression of the p450 aromatase gene .", "adiponectin treatment contributed to an increase in progesterone levels and a decrease in androstenedione and oestradiol plasma concentrations in comparison with control bats . in the same study , adiponectin treatment increased p450scc and 3-hsd enzyme levels but decreased aromatase , star and lh - r levels in comparison with controls .", "an in vitro study by caminos et al .   demonstrated that adiponectin significantly inhibited basal and hcg - stimulated testosterone secretion by testicular explants .", "in another study , adiponectin decreased corticosterone secretion by freshly isolated rat adrenocortical cells but did not affect aldosterone production .", "adiponectin treatment enhanced cortisol secretion , which was accompanied by increased expression of steroidogenic pathway genes , including star protein expression , via erk1/2 and ampk - dependent pathways .", "in addition to its direct effects on steroidogenesis in the ovary , adiponectin could also indirectly affect gonadal functions by controlling the secretory activity of the hypothalamus and pituitary .", "a study by wen et al .   demonstrated the inhibition of gnrh release from gt1 - 7 hypothalamic gnrh neurons after the administration of adiponectin . in in vivo study by cheng et al .  ", "adiponectin also lowered gnrh secretion by activating the ampk and inhibiting the erk pathways . in the lower branch of the hpg axis ,", "the release of lh from rat pituitary cells and the murine lt2 cell line was decreased after the administration of adiponectin alone and in combination with gnrh [ 53 , 54 ] . in vivo observations also demonstrated an inhibitory effect of adiponectin on lh secretion : the intravenous administration of the adenovirus expressing the adiponectin gene to male mice decreased plasma lh levels without changes in fsh levels .", "the inhibitory influence of adiponectin on lh secretion could be attributed to a decrease in gnrh receptor gene expression in the pituitary .", "the presence of adiponectin mrna and protein in porcine ovaries observed in this study as well as the presence of adiponectin receptors 1 and 2 in the gonads noted in our previous work could provide evidence for auto / paracrine actions of the analysed hormone .", "the variations in adiponectin gene and protein expression during the oestrous cycle indicate that adiponectin expression is determined by the hormonal status of pigs .", "the effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs . yet for definitive conclusions to be drawn , especially concerning precise localization of adiponectin mrna and protein in different ovarian structures , intracellular mechanisms of adiponectin influence on the gonadal steroidogenesis and its possible effect on other functions of the ovaries , further studies are required to determine the role of adiponectin in ovarian physiology ."], "abstract_text": ["<S> adiponectin is an adipose - secreted hormone that regulates energy homeostasis and is also involved in the control of the reproductive system . </S>", "<S> the goal of the present study was to investigate changes in adiponectin gene and protein expression in porcine ovarian structures during the oestrous cycle and to examine the effects of in vitro administration of adiponectin on basal and gonadotrophin- and/or insulin - induced secretion of ovarian steroid hormones . </S>", "<S> both gene and protein expression of adiponectin were enhanced during the luteal phase of the cycle . </S>", "<S> adiponectin affected basal secretion of progesterone by luteal cells , oestradiol by granulosa cells , and testosterone by theca interna cells . </S>", "<S> the gonadotrophin / insulin - induced release of progesterone from granulosa and theca interna cells and the release of oestradiol and androstenedione from theca cells was also modified by adiponectin . in conclusion , the presence of adiponectin mrna and protein in the porcine ovary coupled with our previous results indicating adiponectin receptors expression suggest that adiponectin may locally affect ovarian functions . </S>", "<S> the changes in adiponectin expression throughout the oestrous cycle seem to be dependent on the hormonal status of pigs related to the stage of the oestrous cycle . </S>", "<S> the effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["one of them , adiponectin , also known as acrp30 , adipoq , gbp28 , and apm1 , was discovered by four independent research teams in 1995/1996 , [ 25 ] as the most abundant product of the adipose tissue .", "the hormone is a 244-amino acid protein with 30 kda molecular weight that circulates in the serum in different multimeric forms .", "actions of adiponectin are mediated by two types of receptors : adiponectin receptor 1 ( adipor1 ) and 2 ( adipor2 ) .", "the receptors are highly related and share 67% sequence identity in mice , but they differ in their tissue distribution and the ability to bind various forms of adiponectin .", "adipor1 shows high affinity for the globular form of adiponectin , whereas adipor2 is characterized by intermediate binding affinity for both globular and full - length adiponectin .", "the highest levels of adipor1 expression are observed in skeletal muscles , whereas adipor2 is most highly expressed in the liver [ 7 , 8 ] . due to the extensive distribution of adiponectin receptors in peripheral tissues and organs , adiponectin exerts pleiotropic effects on metabolism , including regulation of homeostasis by fatty acid oxidation , stimulation of glucose uptake and gluconeogenesis inhibition , which leads to intensified thermogenesis and weight loss .", "consequently , adiponectin level correlates negatively with body fat   and positively with insulin sensitivity .", "the existing evidence points to the presence of a common endocrine system comprising several hormones , including ghrelin , leptin , and orexin that controls metabolism and reproductive functions [ 1215 ] .", "adiponectin mrna and protein were found in the ovaries of several species , including humans , rats , and cows [ 1619 ] .", "adiponectin mrna and protein were also detected in the ovaries of prepubertal but not sexually mature gilts .", "the influence of the hormonal status of swine related to the stage of the oestrous cycle on adiponectin expression in corpora lutea , granulosa , and theca interna cells remains unknown .", "studies indicating higher concentrations of adiponectin in female than in male blood suggest that ovarian steroids may regulate adiponectin secretion [ 20 , 21 ] .", "similar conclusions can be drawn from varied plasma levels of adiponectin during the oestrous cycle in pigs . despite the above ,", "the possible influence of adiponectin on the production of steroid hormones by porcine ovarian cells remains unexplored .", "therefore , the aim of this study was to investigate the expression of the adiponectin gene by real - time pcr , to determine the concentrations of adiponectin protein in the porcine ovary ( corpora lutea , granulosa and theca interna cells ) and to compare gene and protein expression levels during different stages of the oestrous cycle in pigs . additionally , we sought to determine the in vitro effect of adiponectin on the secretion of steroid hormones ( progesterone , oestradiol , testosterone , and androstenedione ) by ovarian luteal , granulosa , and theca interna cells of sexually mature swine ."], ["all experiments were carried out in observance of the ethical standards of the animal ethics committee at the university of warmia and mazury in olsztyn . the experimental material comprised mature gilts ( large white and polish landrace ) from a private breeding farm , aged 7 - 8 months and weighing 130140  kg .", "twenty gilts were divided into four experimental groups as follows : days 2 - 3 , 1012 , 1416 , and 1719 of the oestrous cycle .", "the day of the second oestrus was designated as day 0 of the oestrous cycle .", "the phase of the oestrous cycle was also determined based on ovarian morphology . additionally , to fully confirm correctness of the evaluation of the oestrous cycle phase , the level of progesterone was determined .", "dissected corpora lutea ( cls ) from different stages of the oestrous cycle ( days 2 - 3-corpora haemorrhagica , 1012-mature cls , and 1416-regressing cls ) were either immediately frozen in liquid nitrogen and stored at 80c until rna and protein analysis or , similarly to the ovaries from days 1719 of the cycle , placed in cold pbs buffer and transported to the laboratory where luteal , follicular granulosa , and theca interna cells were isolated .", "granulosa and theca interna cells are precursor cells of large and small luteal cells , respectively .", "dissected corpora lutea from ovaries on days 2 - 3 , 1012 , and 1416 were minced into small fragments and dispersed in f-12 medium containing bovine serum albumin fraction v ( bsa ; 1% ) and antibiotics .", "corpora lutea were enzymatically dissociated in 0.125% trypsin solution ( 46 times ) at 37c , centrifuged ( 300  g , 10  min , 4c ) , and washed three times .", "isolated luteal cells were filtered through nylon mesh ( 40  m in diameter ) and resuspended in fresh f-12 medium .", "the cells were counted using a haemocytometer , and their viability ( ~90% ) was determined by 0.4% trypan blue dye exclusion .", "granulosa and theca interna cells were isolated from large follicles ( diameter > 6  mm ) without signs of atresia .", "granulosa cells were aspirated with a syringe and additionally washed out with a strong stream of media directed to the internal wall of the follicle .", "the theca interna layer was scraped off from granulosa cells and enzymatically dispersed in 0.25% trypsin solution .", "dispersed cells were centrifuged ( 800  g for 10  min ) and washed two and three times to isolate granulosa and theca interna cells , respectively .", "the cells were filtered through nylon mesh ( 40  m in diameter ) and resuspended in eagle 's medium enriched with bsa ( 5% ) and antibiotics .", "the cells were counted using a haemocytometer , and their viability ( ~98% ) was determined by 0.4% trypan blue dye exclusion .", "some of granulosa and theca interna cells ( 5  10 viable cells within each experiment ) were immediately resuspended in trizol reagent ( invitrogen , usa ) and stored at 80c until processing for rna analysis .", "total rna was extracted from luteal tissues from days 2 - 3 , 1012 , and 1416 of the oestrous cycle using the absolutely rna miniprep kit ( stratagene , usa ) . in the case of granulosa and theca cells", "approximately 1  g of rna was reverse - transcribed into cdna in a total volume of 20  l with 0.5  g oligo ( dt)15 primer ( roche , germany ) using the omniscript rt kit ( qiagen , usa ) at 37c for one hour and was terminated by incubation at 93c for 5  min .", "quantitative real - time pcr analysis was performed using a pcr system 7300 ( applied biosystems , usa ) .", "sense and antisense primers ( adiponectin , cyclophilin a ) were chosen according to lord et al .  ", "the chosen primers were as follows : adiponectin forward : 5atgatgtcaccactggcaaattc-3 , reverse : 5-gaccgtgacgtggaaggaga-3 ; cyclophilin a , forward : 5-gcactggtggcaagtccat-3 , reverse : 5-aggacccgtatgcttcagga-3 ; gapdh forward : 5-ccttcattgacctccactacatggt-3 , reverse : 5-ccacaacatacgtagcaccagcatc-3. adiponectin primers ( access no : ay135647 ) were complementary to positions 514536 ( f ) and 565584 ( r ) of pig adiponectin gene sequence ; cyclophilin a primers ( access no : ay266299 ) were complementary to positions 219237 ( f ) and 269299 ( r ) of pig cyclophilin a gene sequence , gapdh primers ( access no : u48832 ) were complementary to positions 6185 ( f ) and 219243 ( r ) of pig gapdh . a constitutively", "expressed genes , cyclophilin a and gapdh , were used as the internal control to verify the quantitative real - time pcr . to ensure that cyclophilin", "a and gapdh were the suitable reference genes for this study , we revealed that there were no statistically significant differences in ct values between the examined ovarian structures throughout all investigated stages of the oestrous cycle .", "both of the housekeeping genes were also indicated as suitable reference genes ( internal controls ) in the independent studies .", "the pcr reaction included 20  ng cdna , 900  nm ( adiponectin forward ) , 300  nm ( adiponectin reverse , cyclophilin a forward and reverse ) and 60  nm ( gapdh forward and reverse ) primers , 12.5  l sybr green pcr master mix ( applied biosystems , usa ) , and rnase free water in a final volume of 25  l .", "quantitative real - time pcr cycling conditions were as follows : initial denaturation and enzyme activation at 95c for 10  min , followed by 40 cycles of denaturation at 95c for 15  s and annealing at 59 for 1  min .", "negative controls were performed in which water was substituted for cdna , or reverse transcription was not performed prior to pcr .", "the specificity of amplification was tested at the end of the pcr by melting - curve analysis .", "calculation of relative expression levels of adiponectin was conducted based on the comparative cycle threshold method ( ct )   and normalized using the geometrical mean of reference genes expression levels : gapdh and cyclophilin a. western blotting analysis was performed as described by smolinska et al . .", "briefly , equal amounts of porcine corpora lutea as well as granulosa and theca cells lysats ( 10  g ) were resolved by sds - page on 12.5% polyacrylamide gel and then transferred to a nitrocellulose membranes ( whatman , usa ) .", "blots were blocked for 5  h at 4c in 1  tbst containing 5% skimmed milk powder and then incubated overnight at 4c with the rabbit polyclonal adiponectin antibodies at the dilution of 1  :  150 ( santa cruz biotechnology , usa ) or rabbit polyclonal actin antibodies ( sigma , usa ) diluted 1  :  200 , which were used as an internal control for equal loading and to quantify porcine adiponectin protein . to identify immunoreactive bands ,", "membranes were incubated for 1.5  h at room temperature with mouse anti - rabbit igg for adiponectin ( sigma , usa ; diluted 1  :  2000 ) , goat anti - rabbit igg for actin ( diluted 1  :  5000 ) conjugated with alkaline phosphatase ( santa cruz biotechnology , usa ) .", "nonspecific foetal calf serum ( mp biomedicals , usa ) was used instead of primary antibodies to produce negative control blots .", "the immunocomplexes were visualized using 4-nitroblue tetrazolium chloride ( nbt ) and 5-bromo-4-chloro-3-indolyl phosphate ( bcip ) , according to the manufacture 's protocol ( promega , usa ) .", "the results of western blotting were quantified by densitometric scanning of immunoblots with gelscan for windows ver . 1.45 software ( kucharczyk , poland ) .", "data were expressed as a ratio of adiponectin protein relative to actin protein in arbitrary optical density units .", "luteal cells ( 250000/1  ml medium ) were resuspended in f-12 medium enriched with foetal calf serum ( fcs ; 20% ) , bsa ( 1% ) and antibiotics and preincubated for 48 hours in a humidified incubator with 95% air and 5% co2 atmosphere .", "the serum - containing medium was discarded , and the cells were washed using serum - free f-12 medium .", "after washing , luteal cells were cultured for 24 hours in f-12 medium with bsa ( 1% ) and antibiotics , with or without treatment agents .", "granulosa and theca interna cells ( 250000/1  ml medium ) were resuspended in eagle 's medium supplemented with 10% fcs , 5% bsa , and antibiotics .", "after 24 hours of preincubation , the medium was discarded , and the cells were washed and cultured for 24 hours in eagle 's medium with 5% bsa and antibiotics , with or without treatment reagents .", "the cultured cells were treated with 1 or 10  g / ml of recombinant human adiponectin ( biovendor , usa ) alone or in combination with insulin ( granulosa and theca interna cells ; 10  ng / ml ; sigma , usa ) , fsh ( granulosa cells ; 10  ng / ml ; nhpp , usa ) and lh ( theca interna cells ; 10  ng / ml ; nhpp , usa ) , and combination of those treatments ( insulin + fsh for granulosa cells and insulin + lh for theca interna cells ) .", "adiponectin doses used in the experiment were close to physiological concentrations of this hormone in the porcine blood .", "those cells are thought to be generally more autonomic than follicular cells which is a reason of luteal cells ' hard response to stimulators [ 3133 ] .", "the alamar blue test revealed that none of the treatments affected the viability of the cultured cells .", "following incubation , the media were harvested , centrifuged ( 800  g for 10  min ) , and the supernatants were collected and stored at 20c until analyses of progesterone ( cultures of luteal , theca interna , and granulosa cells ) , oestradiol ( cultures of theca interna and granulosa cells ) , androstenedione , and testosterone ( cultures of theca interna cells ) . progesterone ( p4 )", "was analysed according to the method described by ciereszko et al . , oestradiol ( e2 ) was determined according to the method of hotchkiss et al .   modified by kotwica , androstenedione ( a4 ) as described by dziadkowiec et al . , and testosterone ( t ) according to kotwica and williams .", "the specificity of the antibodies against a4 , t , and e2 has been reported by ciereszko et al .   and szafranska et al . .", "the sensitivities of the assays for p4 and a4 were 1  pg / ml and for e2 and t were 0.5  pg / ml .", "intra- and interassay coefficients of variation of the p4 , e2 , a4 , and t assays were 0.91% , 0.7% , 0.81% , 0.97% and 8.5% , 10.1% , 14.2% , and 7.8% , respectively .", "data from real - time analysis and western blot were analysed by one - way anova and least significant difference ( lsd ) post hoc test and are reported as the means  sem from five independent observations .", "all data concerning in vitro cell cultures were analysed by anova for repeated measurements and least significant difference ( lsd ) post hoc test and are reported as the means  sem from five ( for all types of cells ) independent observations .", "each experiment was performed on different day using separate pool of luteal or follicular cells ."], ["the expression of adiponectin mrna was significantly higher in corpora lutea during all investigated days of the luteal phase than in theca interna cells isolated on days 1719 of the cycle ( p < 0.05 ) .", "no significant differences were noted in adiponectin gene expression in granulosa cells compared with theca interna cells and cls ( figure 1(a ) ) . in general ,", "adiponectin protein concentration in the porcine ovary was higher in the luteal phase than in the follicular phase .", "in particular , the adiponectin protein content was the greatest in cls from days 2 - 3 in comparison with cls and follicular cells from the remaining stages of the oestrous cycle ( p < 0.01 ) . within", "the luteal phase adiponectin concentration was the lowest in cls during days 1012 ( p < 0.01 ) .", "there was no significant differences in protein expression between granulosa and theca interna cells ( figure 1(b ) ) .", "treatment of luteal cells from days 1012 of the oestrous cycle with adiponectin at both doses ( 1  g / ml ; 10  g / ml ) resulted in significant ( p < 0.05 ) decrease in progesterone secretion ( figure 2 ) .", "adiponectin at any dose did not affect p4 secretion by luteal cells collected on days 2 - 3 and 1416 ( data not shown ) .", "adiponectin at the higher dose ( 10  g / ml ) in combination with insulin provoked the increase in p4 secretion in comparison with the cells stimulated by insulin alone ( p < 0.05 ) .", "in contrast , there was no effect of adiponectin ( both doses ) on fsh- and fsh + insulin - induced production of p4 in porcine granulosa cells ( figure 3 ) .", "basal secretion of p4 was unaffected independently on adiponectin dose ( data not shown ) .", "adiponectin at the higher dose ( 10  g / ml ) increased basal secretion of e2 ( p < 0.05 ) ( figure 4 ) .", "adiponectin did not change secretion of e2 by granulosa cells cultured with insulin and/or fsh ( data not shown ) .", "adiponectin at both doses reduced basal secretion of t by porcine theca interna cells ( p < 0.05 ) ( figure 5 ) .", "the effect of adiponectin on basal p4 , e2 , and a4 release was negligible ( data not shown ) .", "adiponectin ( 10  g / ml ) decreased lh + insulin - stimulated secretion of p4 by theca interna cells ( p < 0.05 ) .", "there was no effect of adiponectin on lh- or insulin - induced production of p4 by these cells ( figure 6(a ) ) .", "adiponectin did not influence lh- or insulin - stimulated secretion of e2 by theca interna cells .", "however , lh + insulin - induced secretion of e2 by the cells was increased in response to the higher dose ( 10  g / ml ) of adiponectin ( p < 0.05 ) ( figure 6(b ) ) .", "secretion of a4 by theca interna cells was inhibited by combination of adiponectin ( 10  g / ml ) with insulin compared to insulin alone ( p < 0.05 ) .", "adiponectin at the lower dose ( 1  g / ml ) increased lh + insulin - induced a4 secretion by the cells ( p < 0.05 ) ( figure 6(c ) ) .", "induced secretion of t was not changed under adiponectin influence ( data not shown ) ."], ["this is the first ever study to demonstrate the changes in adiponectin gene and protein expression in the porcine ovary throughout the oestrous cycle .", "our results indicate that adiponectin is more highly expressed in luteal cells derived from ovaries in all examined stages of the luteal phase than in follicular granulosa and theca interna cells .", "adiponectin was also found to affect basal and stimulated secretion of steroid hormones by porcine ovarian cells .", "in addition to porcine ovarian structures , adiponectin gene expression and the presence of the hormone protein were also found in woman theca interna cells   and in theca interna cells , granulosa cells , corpora lutea , and oocytes of rats   and cows [ 18 , 19 ] .", "singh and krishna   localized the adiponectin protein in bat ovarian structures that showed positive immunostaining in theca interna cells , oocytes of growing follicles , and the corpus luteum .", "the expression of adiponectin gene and protein was also determined in porcine granulosa cells of prepubertal gilts .", "the levels of adiponectin expression and also its receptors seem to vary subject to cell type and the stage of follicle and corpora lutea development . in the bovine ovary ,", "the expression of adiponectin and its both receptors was lower in granulosa cells than in theca interna cells and oocytes .", "contrary results were presented by ortega et al .   who observed the highest levels of adiponectin expression in granulosa cells of sheep ovaries .", "throughout follicular development , adiponectin mrna increased in granulosa cells and decreased in theca interna cells of cows . in the corpora lutea ,", "bovine theca interna cells derived from large follicles revealed higher expression of both receptors than cells isolated from medium - sized and small follicles , indicating that follicular growth influences the transcript levels of adiponectin receptors .", "the synthesis of ovarian adiponectin and the expression of adipor1 and adipor2 are probably hormonally controlled , as suggested by an increase in adiponectin concentrations in ovarian follicular fluid of women administrated lh in the in vitro fertilization procedure .", "following pmsg pretreatment , an injection of hcg also significantly increased the expression of adiponectin and adipor1 ( but not adipor2 ) genes in rat ovaries .", "in bovine theca interna cells , lh increased the concentrations of adipor2 mrnas , whereas igf - i suppressed the expression of the above gene .", "the results of the present study seem to confirm the hypothesis that adiponectin production is regulated hormonally .", "higher levels of adiponectin gene and protein expression during the luteal phase and lower expression levels during the follicular phase could point to the stimulatory effect of progesterone and the inhibitory influence of oestradiol on ovarian adiponectin production . in our previous study ,", "adiponectin serum concentrations were higher during the luteal phase than the follicular phase , which suggests that ovarian steroids influence plasma adiponectin levels . in this study", "the results of our in vitro studies and the presence of both adiponectin receptors in porcine ovaries indicate that adiponectin may directly affect ovarian steroidogenesis .", "in fact , progesterone secretion decreased due to adiponectin 's influence on luteal cells in mid - luteal phase ( days 1012 ) .", "greater variations in adiponectin action were observed in porcine follicular cells . induced progesterone production increased in granulosa cells and decreased in theca interna cells .", "basal testosterone output and insulin - induced androstenedione secretion were inhibited , while lh + insulin - stimulated release of androstenedione was enhanced by adiponectin .", "the effect of adiponectin on ovarian steroidogenesis was also suggested in studies of cows , rats , and humans .", "adiponectin was found to inhibit insulin - induced secretion of progesterone and oestradiol by bovine granulosa cells . in a study of theca interna cells of bovine ovaries ,", "lagaly et al .   observed that adiponectin decreased lh + insulin - induced production of progesterone .", "the above authors also noted that adiponectin inhibited the mrna expression of lh receptor in granulosa cells .", "granulosa cells of rats and women treated with igf - i responded to adiponectin by increasing progesterone and oestradiol secretion [ 16 , 17 ] .", "in the cited studies , adiponectin did not influence fsh - induced production of progesterone and oestradiol in human and rat granulosa cells which is consistent with the response of porcine cells noted in this study .", "it seems that adiponectin affects ovarian functions by binding adipor1 and adipor2 , in two ways .", "an experiment where rnai was used to block adipor1 and adipor2 expression in the kgn cell line derived from human granulosa cells contributed to new information about adiponectin 's role in the ovaries .", "the absence of adipor1 in the analysed cells enhanced apoptosis , whereas the elimination of adipor2 reduced fsh- and igf - i - induced the production of progesterone and oestradiol and inhibited mitogen - activated kinase activity , relative to control , in response to adiponectin or fsh treatment .", "the above results suggest that adipor1 is more involved in the survival of granulosa cells , whereas adipor2 regulates steroidogenesis through mapk activation .", "the influence of adiponectin on the ovaries of sexually mature pigs has not been studied to date , but the response of granulosa cells derived from prepubertal gilts to the analysed hormone was described by ledoux et al . .", "the above authors observed higher levels of cyclooxygenase-2 , prostaglandin e synthase , and vegf gene expression in cells primed with adiponectin , an increase in the expression of the star gene and a decrease in the expression of the p450 aromatase gene .", "adiponectin treatment contributed to an increase in progesterone levels and a decrease in androstenedione and oestradiol plasma concentrations in comparison with control bats . in the same study , adiponectin treatment increased p450scc and 3-hsd enzyme levels but decreased aromatase , star and lh - r levels in comparison with controls .", "an in vitro study by caminos et al .   demonstrated that adiponectin significantly inhibited basal and hcg - stimulated testosterone secretion by testicular explants .", "in another study , adiponectin decreased corticosterone secretion by freshly isolated rat adrenocortical cells but did not affect aldosterone production .", "adiponectin treatment enhanced cortisol secretion , which was accompanied by increased expression of steroidogenic pathway genes , including star protein expression , via erk1/2 and ampk - dependent pathways .", "in addition to its direct effects on steroidogenesis in the ovary , adiponectin could also indirectly affect gonadal functions by controlling the secretory activity of the hypothalamus and pituitary .", "a study by wen et al .   demonstrated the inhibition of gnrh release from gt1 - 7 hypothalamic gnrh neurons after the administration of adiponectin . in in vivo study by cheng et al .  ", "adiponectin also lowered gnrh secretion by activating the ampk and inhibiting the erk pathways . in the lower branch of the hpg axis ,", "the release of lh from rat pituitary cells and the murine lt2 cell line was decreased after the administration of adiponectin alone and in combination with gnrh [ 53 , 54 ] . in vivo observations also demonstrated an inhibitory effect of adiponectin on lh secretion : the intravenous administration of the adenovirus expressing the adiponectin gene to male mice decreased plasma lh levels without changes in fsh levels .", "the inhibitory influence of adiponectin on lh secretion could be attributed to a decrease in gnrh receptor gene expression in the pituitary ."], ["the presence of adiponectin mrna and protein in porcine ovaries observed in this study as well as the presence of adiponectin receptors 1 and 2 in the gonads noted in our previous work could provide evidence for auto / paracrine actions of the analysed hormone .", "the variations in adiponectin gene and protein expression during the oestrous cycle indicate that adiponectin expression is determined by the hormonal status of pigs .", "the effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs . yet for definitive conclusions to be drawn , especially concerning precise localization of adiponectin mrna and protein in different ovarian structures , intracellular mechanisms of adiponectin influence on the gonadal steroidogenesis and its possible effect on other functions of the ovaries , further studies are required to determine the role of adiponectin in ovarian physiology ."]]}
{"article_id": "PMC1458436", "article_text": ["molecular and morphological evidence together support the idea that the anterior end of the amphioxus nerve cord contains regions homologous with vertebrate forebrain and hindbrain 1,2 , though it is generally the ventral portion of these regions that are best represented 3 .", "its most probable position , as defined by gene expression patterns , would be from somewhere forward of the caudal limit of otx expression , which in vertebrates extends through the midbrain , to a point close to the beginning of the zone of hox gene expression .", "in young amphioxus larvae , this corresponds with a region extending from the infundibular cells , which lie at a level roughly midway along somite 1 , through the posterior part of the cerebral vesicle to a level somewhere near or just beyond the middle of somite 2 ( fig .", "this domain begins with an anterior zone of ventral neuropile and commissural fibers followed , near the junction between somites 1 and 2 , by a complex of ventrally - positioned somatic motoneurons and interneurons with caudal projections that initiate and control larval swimming behavior 4 .", "the first members of the visceral motoneuron series lie further back , near the caudal end of somite 2 .", "from there , visceral motoneurons recur at regular intervals along the anterior nerve cord and innervate the body via an extended series of peripheral nerves .", "any attempt to identify an amphioxus homolog of the vertebrate midbrain is hampered by the fact that we currently lack unambiguous criteria for recognizing its presence .", "the quintessential identifying feature in vertebrates is the dorsal optic tectum , but this is absent in amphioxus .", "in fact , except for a pineal homolog , amphioxus appears to lack all of the dorsal structures of the vertebrate brain .", "further , because the amphioxus nerve cord is of uniform dimension along its length , there are no morphological constrictions to separate sub - domains in the anterior cord from one another in the way vertebrate isthmus separates midbrain from hindbrain .", "the isthmus is notable as the site of an important organizing center , the midbrain - hindbrain boundary ( mhb ) , characterized in vertebrates by the expression of a highly conserved set of gene , including fgf8 , engrailed , pax2 , and wnt1 5,6 .", "the comparable site in amphioxus lies somewhere close to the caudal limit of somite 1 , which is where the anterior zone of otx expression abuts that of gbx 7 . however , though engrailed is expressed in small clusters of cells in the embryonic nerve cord 8 , these lie further forward in the cerebral vesicle , at a level near the midpoint of somite 1 .", "in addition , wnt1 is not expressed at all in the anterior cord , nor does the expression of the amphioxus pax2 homolog , amphipax2/5/8 , match the vertebrate pattern . instead ,", "the latter is expressed caudally through much of the nerve cord 9 , and though there is a small anterior zone of later expression , it is too far forward for an mhb marker .", "there is thus no clear molecular evidence for a focal center in amphioxus with the expression profile of an mhb .", "this is perhaps not surprising , considering that the structures organized by the mhb are primarily dorsal ones not present in amphioxus .", "the mhb is , however , also required for normal organization of some ventral brain regions in vertebrates 10 .", "since the ventral midbrain of vertebrates very likely combines vertebrate - specific neuronal cell types along with cell groupings with more primitive organizational features , it would be useful to know whether all of these are mhb - dependent or only the former .", "it may well be that only vertebrate innovations , whether dorsal or ventral , are under the specific control of the mhb .", "this leaves open the question of whether the amphioxus pattern differs from that of vertebrates because it reflects an earlier stage in the evolution of a vertebrate - type mhb 7 , or whether amphioxus has secondarily diverged from a pattern that was once closer to the vertebrate one .", "in addition , the vertebrate midbrain marker dmbx is not expressed in the amphioxus nerve cord 11 .", "the available molecular data thus argues against the presence , in amphioxus , of precise homologs of either the vertebrate midbrain or mhb . among tunicates , the other group of protochordates ,", "it is the ascidians that are best studied , and for these there is again good molecular evidence for homologs of both forebrain and hindbrain in the larval cns 11,12 , corresponding roughly with the sensory vesicle and caudal ganglion respectively .", "in addition , cells in the narrow  neck  region that separates these structures express at least some characteristic mhb genes ( fig .", "data on pax2/5/8 led initially to the proposal that ascidians had an exact counterpart of the vertebrate mhb and that amphioxus , being a later offshoot of the chordate lineage , must have lost this feature secondarily 12 .", "however , the precise spatial arrangement of the expression zones for several key genes in ascidians differs from that in vertebrates , e.g. fgf and dmbx in ascidian larvae are expressed in cells immediately caudal to those expressing pax2/5/8 and en , whereas in vertebrates , dmbx lies forward of the other three genes , whose expression overlaps .", "expression patterns of the same genes in larvaceans , another group of tunicates , is somewhat different yet again 13 , which further complicates the problem of determining the nature of the ancestral pattern .", "the molecular data is thus somewhat inconclusive regarding protochordate homologs of either the vertebrate midbrain or mhb .", "if anatomical and functional considerations are taken into consideration , however , a somewhat stronger case can be made that amphioxus may have an approximate counterpart of the midbrain .", "the key point is that some of the cell types and neural circuits located in the caudal part of the zone of otx expression are similar to those found in the ventral midbrain in vertebrates .", "in amphioxus , as pointed out above , only ventral markers are available for comparison . of these", ", the infundibular cells probably mark the anterior limit of any prospective midbrain - like territory , as their homology with the ventral infundibular region of the vertebrate diencephalon seems to be fairly well accepted . immediately behind this point , in amphioxus", ", there is a zone of ventral neuropile in some ways comparable with the ventral tegmental commissure , which forms part of the early axonal scaffolding in embryonic vertebrate brains .", "this same region in amphioxus also contains the anterior - most motoneurons in the nerve cord along with populations of large interneurons with descending projections , features found in the tegmentum and the reticulospinal plexus of lower vertebrates beginning at midbrain level .", "the ventral midbrain in vertebrates is also where dopaminergic neurons with projections to the forebrain first develop , and these are a key component of the motivational circuitry linking basal brainstem centers with the forebrain .", "dopaminergic neurons are found in the cerebral vesicle in amphioxus and nowhere else in the nerve cord .", "two main populations develop , one in a dorsal position in the anterior cerebral vesicle , the other more ventrally near the junction between somites 1 and 2 14,15 .", "further research is needed on these cells to determine their precise pattern of projections and function , but the more caudal of the two populations is well positioned to be a homolog of the midbrain dopaminergic neurons in vertebrates .", "if the anatomical and functional criteria outlined above are meaningful indicators that amphioxus does indeed have a midbrain homolog , the value of the molecular markers used to date to test this would have to be reconsidered .", "the same could be said of ascidian larvae , but for a different reason , for here there is an alternative explanation for the expression patterns observed .", "the cns of adult ascidians consists of a simple brain - like dorsal ganglion , from which nerves radiate to the body musculature and visceral organs .", "the ganglion is present in only a rudimentary form in the larva , however , and the source of its cells has never been clear . in the few species where this has been investigated in the past", ", the ganglion develops in contact with the neck region of the larval nerve cord near the site where it contacts the neurohypophyseal duct .", "the latter is partly a stomodeal derivative , which raises the question of whether a significant part of the ganglion might be derived from stomodeal ectoderm .", "an analysis of the salp ganglion , compared with the data available on compound ascidians 16,17 , supports the idea that most if not all of the dorsal ganglion is of neural origin and that , in ascidians , it develops as part of the neck region .", "recent data on ciona 18 tends to support this interpretation , as its ganglion develops in contact with the base of an outgrowth that arises from the caudal part of the sensory vesicle very near the neck .", "more importantly , experimental work now in progress on ciona is confirming the neck region as the source of major classes of neurons within the adult ganglion , including motoneurons innervating the visceral organs [ j.f .", "brunet , personal communication ] . evidently then , the cells of the neck region in ascidian larvae serve as a pool of precursors from which most if not all post - metamorphic cns neurons are derived . assuming the genes expressed in the vertebrate mhb include major players in the overall process of neuronal specification and differentiation , one can argue that their expression in the neck region is to be expected , irrespective of any homology between this site and the mhb .", "one would predict the genes would be expressed in combinatorial patterns and in a few cells at most , which is precisely what is observed .", "current thinking on the nature of ancestral chordates favors the view that the separation of adult and larval tissues in ascidians is a secondary specialization , and that the ancestral body plan was a more fully integrated one , as in amphioxus 19,20 .", "this may explain why the expression patterns of some key developmental control genes differ so dramatically between ascidians and amphioxus .", "consider pax2/5/8 , which has an extended zone of expression in amphioxus , but a very restricted one in ascidians ( cf . figs .", "paralleling these , there are differences in innervation patterns of , for example , the visceral organs .", "these are strictly adult structures in ascidians , and the cells responsible for their innervation arise from the neck region , whereas the hox - expressing part of the nerve cord , which innervates the tail , is entirely lost at metamorphosis . in amphioxus , in contrast , visceral motoneurons , along with the rest of the locomotory control system , arise from an extended region of the nerve cord extending well into the hox zone . whether the zone supplying visceral innervation corresponds precisely with that expressing pax2/5/8 has yet to be determined .", "one can nevertheless predict that any gene essential to the development of the visceral innervation will necessarily be expressed over a much greater length of the nerve cord in amphioxus than in ascidians . for at least some of the genes associated with the mhb , therefore", ", the reason their homologs have a very restricted expression zone in ascidian larvae likely has less to do with the presence of a vertebrate - type mhb , and more with the functional necessity of generating a full complement of adult neurons from a single site within the nerve cord .", "what one then wants to know , to determine whether the ascidian pattern and the vertebrate one are more than coincidentally related , is what functional role the genes play in each instance . with regard to pax2/5/8 specifically ,", "a further clue might come from knowing whether its expression in hemichordate embryos is restricted , as in ascidians , or extended , as in amphioxus .", "if the latter , this would be further evidence for the view that the ascidian pattern is indeed the derived one .", "from the above it is clear that there is a certain plasticity in the way expression patterns of genes identified with the mhb have diverged among chordates . in fact , the vertebrates seem to be most conservative : it is their pattern that most closely resembles that of hemichordates 19,20 , the closest available model for an ancestral deuterostome , and there are remarkably similar patterns in protostomes , e.g. drosophila . in hemichordates", "an mhb - like region can nevertheless be recognized , consisting of overlapping zones of en , pax2/5/8 , fgf8 and wnt1 expression near the junction between the collar and trunk in a region where otx and gbx expression overlaps 21 . if , however , as argued above , the specific organizer functions of the vertebrate mhb evolved with vertebrates , why are hemichordate expression patterns so close to vertebrate ones ?", "it could be , that despite the anatomical differences in their nervous systems , homology dictates the position along the body axis at which particular neural functions and cell types are localized .", "this might be due , for example , to their association with conserved structures like the mouth or the first gill slit .", "too little is currently known of hemichordate neuroanatomy to speculate much further , though some essential aspect of ectodermal and/or neural patterning is presumably involved .", "a further problem is to discover why amphioxus and ascidians have altered their mhb - related expression patterns when they are elsewhere so well conserved . in the case of ascidians , there have evidently been major changes in the way key patterning genes , notably those of the hox cluster , are structured and regulated .", "this probably relates to the presence of alternative mechanisms for cell specification in ascidians , which has relaxed some constraints on genome organization 22 .", "it is otherwise somewhat surprising that such comparatively distant relatives as vertebrates and hemichordates are so similar in terms of their mhb - related expression patterns when the main protochordate groups are both so different from vertebrates and from each other .", "there is a second issue integral to any consideration of chordate cns origins that relates to how the main organ systems of the body , including the nervous system , are positioned in relation to the dorsoventral axis .", "comparatively strong evidence now supports the idea that the body of chordates is dorsoventrally inverted relative to that of insects and the other main groups of protostomes .", "a full account of how this might have happened has been difficult to piece together , at least until now , because there was little relevant data on the basal offshoots of the lineage leading to chordates , namely hemichordates and echinoderms .", "their anteroposterior maps are very similar to vertebrates 21 and , while the data on dorsoventral patterning is more ambiguous , it appears that hemichordates are oriented the same way as protostomes , such that the mouth is ventral and the expression domain for homologs of the dorsoventral patterning genes bmp2/4 and bmp7 is dorsal 20 .", "removal of all influence of the latter in hemichordates results in a much expanded mouth , while over - expression suppresses the mouth and generates radialized embryos . in vertebrates , bmp expression and the mouth", "are both ventral , but suppressing bmps has equally dramatic radializing effects 23 . the genes thus appear to be performing essentially similar functions in these two groups , which implies that dorsal in basal deuterostomes has become ventral in vertebrates and , hence , in all chordates .", "inversion must therefore have occurred between the separation of the clade comprising hemichordates plus echinoderms from chordates , possibly in an organism that did not yet have an internalized cns .", "the transition is somewhat easier to imagine , in fact , if the starting point is an animal with circumferential expression zones of all the key patterning genes ( fig .", "it should then be equally easy to concentrate both the expression zones and the structures they pattern either to the dorsal surface or the ventral one ( fig .", "walter garstang 's ideas about chordate origins are fairly typical of hypotheses that predate the renewed interest in dorsoventral inversion .", "he derived neural tube from the dorsal portion of an ancestral system of larval ciliary bands , in that way preserving the dorsoventral orientation of the ancestor ( fig .", "if inversion did , in fact , occur , the brain and nerve cord would have had to come instead from the ventral surface of the ancestral body ( fig .", "2c ) , an alternative explored by claus nielsen in a 1999 analysis 24 . a major premise of garstang 's hypothesis , that the whole of the neural tube should to arise as a single entity , is no longer widely accepted 19 , but it is still useful to consider the consequence of dorsoventrally inverting his hypothetical ancestor . as nielsen points out , so long as the brain arises at the anterior end of an originally ventral neuraxis , it must necessarily form in the postoral region , most likely immediately caudal to the mouth .", "this may seem counterintuitive , but the zone of expression for mhb - type genes , which mark a core brain region in vertebrates , is also postoral in hemichordates .", "the ancestral chordate in this scenario , in its inverted position , would have both its mouth and brain on the dorsal surface of the body . while this may seem peculiar", "what is then needed to generate the body of more advanced chordates like amphioxus and vertebrates is for differential growth of the ectoderm separating the mouth and brain to move the former forward , over the front or side of the snout , to what is now the ventral surface .", "the site of the ancestral apical organ must move as well , and will eventually find itself behind the now - ventral mouth , roughly in the position of the adhesive gland , which is also exactly the path the polar bodies follow from the apical pole of the egg during amphibian development 24 . in consequence ,", "the anterior most part of our brain can not be homologous with the anterior - most parts of protostome brains , because the latter are in part derived from apical structures .", "this is despite the supposed homology between the anterior otx - expressing regions of protostome and deuterostome brains , because in embryos and larvae in both groups , otx can be expressed both pre- and post - orally 25,26 . in summary , though there is good reason to doubt that the chordate neural tube actually derives evolutionarily from larval ciliary bands 19 , nielsen 's point about the site of brain formation being post - oral , though counterintuitive , is largely consistent with current molecular data .", "in addition , the larvae of hemichordates and echinoderms have neurons at this location , and these form comparatively complex ganglion - like assemblages in some instances .", "the function of these simple neural centers is to innervate the larval oral region and pharynx , presumably to control basic feeding behaviors and digestion .", "if these did indeed serve in some way as the core of the evolving chordate brain , then the progressive incorporation of centers for locomotory control into the brain is yet another story , and one that would have unfolded quite independently of the evolutionary process by which a centralized brain and nerve cord arose in the protostome lineage . in this version of events ,", "it is quite unlikely that the internalized neural tube of chordates can be explained by a simple inversion of a protostome - like ancestor with an already - internalized brain and nerve cord , as has sometimes been supposed .", "dorsal views of the larval nerve cords of ( a ) amphioxus and ( b ) ciona , a representative ascidian , showing expression domains for the main cns patterning genes .", "this is somewhat simplified , as the exact extent of the expression domains can vary with developmental stage , and the ascidian hox genes , in particular , are expressed in non - overlapping domains with some gaps .", "two alternatives for explaining the origin of a dorsally positioned brain in chordates , using the hemichordate embryo ( a ) as a starting point ; indicates the mouth ( m ) , anus ( a ) , apical plate ( orange ) , and the ancestral dorsal ( a - dorsal ) and ventral ( a - ventral ) surfaces ; the light blue rectangle is the expression  hot zone  ( hz ) in which homologs of genes characteristic of the vertebrate mhb are expressed , typically in circumferential bands within ectoderm of the collar and anterior trunk .", "older explanations of chordate origins , e.g. garstang 's hypothesis , retain an ancestral dorsoventral orientation ( as in b ) .", "this allows the dorsal condensation of tissue that became the brain ( dark blue ) to form within the hot zone of mhb - like expression , co - opting the latter for neural patterning functions . with inversion ( c , left side ) , the ancestral ventral surface becomes dorsal .", "in contrast with b , any brain - like condensation that forms on the new dorsal surface would necessarily then be post - oral in position , but it could still incorporate the mhb - like hot zone .", "then , to generate the vertebrate condition ( c , right side ) , with mouth and brain on opposing sides of the body , one needs to move the mouth over the front or side of the snout by differential growth of an expansion zone ( ez ) located somewhere forward of the brain ."], "abstract_text": ["<S> the mhb ( midbrain - hindbrain boundary ) is a key organizing center in the vertebrate brain characterized by highly conserved patterns of gene expression . </S>", "<S> the evidence for an mhb homolog in protochordates is equivocal , the \" neck \" region immediately caudal to the sensory vesicle in ascidian larvae being the best accepted candidate . </S>", "<S> it is argued here that similarities in expression patterns between the mhb and the ascidian neck region are more likely due to the latter being the principal source of neurons in the adult brain , and hence where all the genes involved in patterning the latter will necessarily be expressed . </S>", "<S> the contrast with amphioxus is exemplified by pax2/5/8 , expressed in the neck region in ascidian larvae , but more caudally , along much of the nerve cord in amphioxus . </S>", "<S> the zone of expression in each case corresponds with that part of the nerve cord ultimately responsible for innervating the adult body , which suggests the spatially restricted mhb - like expression pattern in ascidians is secondarily reduced from a condition more like that in amphioxus . </S>", "<S> patterns resembling those of the vertebrate mhb are nevertheless found elsewhere among metazoans . </S>", "<S> this suggests that , irrespective of its modern function , the mhb marks the site of an organizing center of considerable antiquity . </S>", "<S> any explanation for how such a center became incorporated into the chordate brain must take account of the dorsoventral inversion chordates have experienced relative to other metazoans . </S>", "<S> especially relevant here is a concept developed by claus nielsen , in which the brain is derived from a neural center located behind the ancestral mouth . while this is somewhat counterintuitive , it accords well with emerging molecular data . </S>"], "labels": null, "section_names": ["1. Midbrains and MHBs in protochordates", "2. The ancestral condition  did it include a precursor of the MHB?", "Figures and Tables"], "sections": [["molecular and morphological evidence together support the idea that the anterior end of the amphioxus nerve cord contains regions homologous with vertebrate forebrain and hindbrain 1,2 , though it is generally the ventral portion of these regions that are best represented 3 .", "its most probable position , as defined by gene expression patterns , would be from somewhere forward of the caudal limit of otx expression , which in vertebrates extends through the midbrain , to a point close to the beginning of the zone of hox gene expression .", "in young amphioxus larvae , this corresponds with a region extending from the infundibular cells , which lie at a level roughly midway along somite 1 , through the posterior part of the cerebral vesicle to a level somewhere near or just beyond the middle of somite 2 ( fig .", "this domain begins with an anterior zone of ventral neuropile and commissural fibers followed , near the junction between somites 1 and 2 , by a complex of ventrally - positioned somatic motoneurons and interneurons with caudal projections that initiate and control larval swimming behavior 4 .", "the first members of the visceral motoneuron series lie further back , near the caudal end of somite 2 .", "from there , visceral motoneurons recur at regular intervals along the anterior nerve cord and innervate the body via an extended series of peripheral nerves .", "any attempt to identify an amphioxus homolog of the vertebrate midbrain is hampered by the fact that we currently lack unambiguous criteria for recognizing its presence .", "the quintessential identifying feature in vertebrates is the dorsal optic tectum , but this is absent in amphioxus .", "in fact , except for a pineal homolog , amphioxus appears to lack all of the dorsal structures of the vertebrate brain .", "further , because the amphioxus nerve cord is of uniform dimension along its length , there are no morphological constrictions to separate sub - domains in the anterior cord from one another in the way vertebrate isthmus separates midbrain from hindbrain .", "the isthmus is notable as the site of an important organizing center , the midbrain - hindbrain boundary ( mhb ) , characterized in vertebrates by the expression of a highly conserved set of gene , including fgf8 , engrailed , pax2 , and wnt1 5,6 .", "the comparable site in amphioxus lies somewhere close to the caudal limit of somite 1 , which is where the anterior zone of otx expression abuts that of gbx 7 . however , though engrailed is expressed in small clusters of cells in the embryonic nerve cord 8 , these lie further forward in the cerebral vesicle , at a level near the midpoint of somite 1 .", "in addition , wnt1 is not expressed at all in the anterior cord , nor does the expression of the amphioxus pax2 homolog , amphipax2/5/8 , match the vertebrate pattern . instead ,", "the latter is expressed caudally through much of the nerve cord 9 , and though there is a small anterior zone of later expression , it is too far forward for an mhb marker .", "there is thus no clear molecular evidence for a focal center in amphioxus with the expression profile of an mhb .", "this is perhaps not surprising , considering that the structures organized by the mhb are primarily dorsal ones not present in amphioxus .", "the mhb is , however , also required for normal organization of some ventral brain regions in vertebrates 10 .", "since the ventral midbrain of vertebrates very likely combines vertebrate - specific neuronal cell types along with cell groupings with more primitive organizational features , it would be useful to know whether all of these are mhb - dependent or only the former .", "it may well be that only vertebrate innovations , whether dorsal or ventral , are under the specific control of the mhb .", "this leaves open the question of whether the amphioxus pattern differs from that of vertebrates because it reflects an earlier stage in the evolution of a vertebrate - type mhb 7 , or whether amphioxus has secondarily diverged from a pattern that was once closer to the vertebrate one .", "in addition , the vertebrate midbrain marker dmbx is not expressed in the amphioxus nerve cord 11 .", "the available molecular data thus argues against the presence , in amphioxus , of precise homologs of either the vertebrate midbrain or mhb . among tunicates , the other group of protochordates ,", "it is the ascidians that are best studied , and for these there is again good molecular evidence for homologs of both forebrain and hindbrain in the larval cns 11,12 , corresponding roughly with the sensory vesicle and caudal ganglion respectively .", "in addition , cells in the narrow  neck  region that separates these structures express at least some characteristic mhb genes ( fig .", "data on pax2/5/8 led initially to the proposal that ascidians had an exact counterpart of the vertebrate mhb and that amphioxus , being a later offshoot of the chordate lineage , must have lost this feature secondarily 12 .", "however , the precise spatial arrangement of the expression zones for several key genes in ascidians differs from that in vertebrates , e.g. fgf and dmbx in ascidian larvae are expressed in cells immediately caudal to those expressing pax2/5/8 and en , whereas in vertebrates , dmbx lies forward of the other three genes , whose expression overlaps .", "expression patterns of the same genes in larvaceans , another group of tunicates , is somewhat different yet again 13 , which further complicates the problem of determining the nature of the ancestral pattern .", "the molecular data is thus somewhat inconclusive regarding protochordate homologs of either the vertebrate midbrain or mhb .", "if anatomical and functional considerations are taken into consideration , however , a somewhat stronger case can be made that amphioxus may have an approximate counterpart of the midbrain .", "the key point is that some of the cell types and neural circuits located in the caudal part of the zone of otx expression are similar to those found in the ventral midbrain in vertebrates .", "in amphioxus , as pointed out above , only ventral markers are available for comparison . of these", ", the infundibular cells probably mark the anterior limit of any prospective midbrain - like territory , as their homology with the ventral infundibular region of the vertebrate diencephalon seems to be fairly well accepted . immediately behind this point , in amphioxus", ", there is a zone of ventral neuropile in some ways comparable with the ventral tegmental commissure , which forms part of the early axonal scaffolding in embryonic vertebrate brains .", "this same region in amphioxus also contains the anterior - most motoneurons in the nerve cord along with populations of large interneurons with descending projections , features found in the tegmentum and the reticulospinal plexus of lower vertebrates beginning at midbrain level .", "the ventral midbrain in vertebrates is also where dopaminergic neurons with projections to the forebrain first develop , and these are a key component of the motivational circuitry linking basal brainstem centers with the forebrain .", "dopaminergic neurons are found in the cerebral vesicle in amphioxus and nowhere else in the nerve cord .", "two main populations develop , one in a dorsal position in the anterior cerebral vesicle , the other more ventrally near the junction between somites 1 and 2 14,15 .", "further research is needed on these cells to determine their precise pattern of projections and function , but the more caudal of the two populations is well positioned to be a homolog of the midbrain dopaminergic neurons in vertebrates .", "if the anatomical and functional criteria outlined above are meaningful indicators that amphioxus does indeed have a midbrain homolog , the value of the molecular markers used to date to test this would have to be reconsidered .", "the same could be said of ascidian larvae , but for a different reason , for here there is an alternative explanation for the expression patterns observed .", "the cns of adult ascidians consists of a simple brain - like dorsal ganglion , from which nerves radiate to the body musculature and visceral organs .", "the ganglion is present in only a rudimentary form in the larva , however , and the source of its cells has never been clear . in the few species where this has been investigated in the past", ", the ganglion develops in contact with the neck region of the larval nerve cord near the site where it contacts the neurohypophyseal duct .", "the latter is partly a stomodeal derivative , which raises the question of whether a significant part of the ganglion might be derived from stomodeal ectoderm .", "an analysis of the salp ganglion , compared with the data available on compound ascidians 16,17 , supports the idea that most if not all of the dorsal ganglion is of neural origin and that , in ascidians , it develops as part of the neck region .", "recent data on ciona 18 tends to support this interpretation , as its ganglion develops in contact with the base of an outgrowth that arises from the caudal part of the sensory vesicle very near the neck .", "more importantly , experimental work now in progress on ciona is confirming the neck region as the source of major classes of neurons within the adult ganglion , including motoneurons innervating the visceral organs [ j.f .", "brunet , personal communication ] . evidently then , the cells of the neck region in ascidian larvae serve as a pool of precursors from which most if not all post - metamorphic cns neurons are derived . assuming the genes expressed in the vertebrate mhb include major players in the overall process of neuronal specification and differentiation , one can argue that their expression in the neck region is to be expected , irrespective of any homology between this site and the mhb .", "one would predict the genes would be expressed in combinatorial patterns and in a few cells at most , which is precisely what is observed .", "current thinking on the nature of ancestral chordates favors the view that the separation of adult and larval tissues in ascidians is a secondary specialization , and that the ancestral body plan was a more fully integrated one , as in amphioxus 19,20 .", "this may explain why the expression patterns of some key developmental control genes differ so dramatically between ascidians and amphioxus .", "consider pax2/5/8 , which has an extended zone of expression in amphioxus , but a very restricted one in ascidians ( cf . figs .", "paralleling these , there are differences in innervation patterns of , for example , the visceral organs .", "these are strictly adult structures in ascidians , and the cells responsible for their innervation arise from the neck region , whereas the hox - expressing part of the nerve cord , which innervates the tail , is entirely lost at metamorphosis . in amphioxus , in contrast , visceral motoneurons , along with the rest of the locomotory control system , arise from an extended region of the nerve cord extending well into the hox zone . whether the zone supplying visceral innervation corresponds precisely with that expressing pax2/5/8 has yet to be determined .", "one can nevertheless predict that any gene essential to the development of the visceral innervation will necessarily be expressed over a much greater length of the nerve cord in amphioxus than in ascidians . for at least some of the genes associated with the mhb , therefore", ", the reason their homologs have a very restricted expression zone in ascidian larvae likely has less to do with the presence of a vertebrate - type mhb , and more with the functional necessity of generating a full complement of adult neurons from a single site within the nerve cord .", "what one then wants to know , to determine whether the ascidian pattern and the vertebrate one are more than coincidentally related , is what functional role the genes play in each instance . with regard to pax2/5/8 specifically ,", "a further clue might come from knowing whether its expression in hemichordate embryos is restricted , as in ascidians , or extended , as in amphioxus .", "if the latter , this would be further evidence for the view that the ascidian pattern is indeed the derived one ."], ["from the above it is clear that there is a certain plasticity in the way expression patterns of genes identified with the mhb have diverged among chordates . in fact , the vertebrates seem to be most conservative : it is their pattern that most closely resembles that of hemichordates 19,20 , the closest available model for an ancestral deuterostome , and there are remarkably similar patterns in protostomes , e.g. drosophila . in hemichordates", "an mhb - like region can nevertheless be recognized , consisting of overlapping zones of en , pax2/5/8 , fgf8 and wnt1 expression near the junction between the collar and trunk in a region where otx and gbx expression overlaps 21 . if , however , as argued above , the specific organizer functions of the vertebrate mhb evolved with vertebrates , why are hemichordate expression patterns so close to vertebrate ones ?", "it could be , that despite the anatomical differences in their nervous systems , homology dictates the position along the body axis at which particular neural functions and cell types are localized .", "this might be due , for example , to their association with conserved structures like the mouth or the first gill slit .", "too little is currently known of hemichordate neuroanatomy to speculate much further , though some essential aspect of ectodermal and/or neural patterning is presumably involved .", "a further problem is to discover why amphioxus and ascidians have altered their mhb - related expression patterns when they are elsewhere so well conserved . in the case of ascidians , there have evidently been major changes in the way key patterning genes , notably those of the hox cluster , are structured and regulated .", "this probably relates to the presence of alternative mechanisms for cell specification in ascidians , which has relaxed some constraints on genome organization 22 .", "it is otherwise somewhat surprising that such comparatively distant relatives as vertebrates and hemichordates are so similar in terms of their mhb - related expression patterns when the main protochordate groups are both so different from vertebrates and from each other .", "there is a second issue integral to any consideration of chordate cns origins that relates to how the main organ systems of the body , including the nervous system , are positioned in relation to the dorsoventral axis .", "comparatively strong evidence now supports the idea that the body of chordates is dorsoventrally inverted relative to that of insects and the other main groups of protostomes .", "a full account of how this might have happened has been difficult to piece together , at least until now , because there was little relevant data on the basal offshoots of the lineage leading to chordates , namely hemichordates and echinoderms .", "their anteroposterior maps are very similar to vertebrates 21 and , while the data on dorsoventral patterning is more ambiguous , it appears that hemichordates are oriented the same way as protostomes , such that the mouth is ventral and the expression domain for homologs of the dorsoventral patterning genes bmp2/4 and bmp7 is dorsal 20 .", "removal of all influence of the latter in hemichordates results in a much expanded mouth , while over - expression suppresses the mouth and generates radialized embryos . in vertebrates , bmp expression and the mouth", "are both ventral , but suppressing bmps has equally dramatic radializing effects 23 . the genes thus appear to be performing essentially similar functions in these two groups , which implies that dorsal in basal deuterostomes has become ventral in vertebrates and , hence , in all chordates .", "inversion must therefore have occurred between the separation of the clade comprising hemichordates plus echinoderms from chordates , possibly in an organism that did not yet have an internalized cns .", "the transition is somewhat easier to imagine , in fact , if the starting point is an animal with circumferential expression zones of all the key patterning genes ( fig .", "it should then be equally easy to concentrate both the expression zones and the structures they pattern either to the dorsal surface or the ventral one ( fig .", "walter garstang 's ideas about chordate origins are fairly typical of hypotheses that predate the renewed interest in dorsoventral inversion .", "he derived neural tube from the dorsal portion of an ancestral system of larval ciliary bands , in that way preserving the dorsoventral orientation of the ancestor ( fig .", "if inversion did , in fact , occur , the brain and nerve cord would have had to come instead from the ventral surface of the ancestral body ( fig .", "2c ) , an alternative explored by claus nielsen in a 1999 analysis 24 . a major premise of garstang 's hypothesis , that the whole of the neural tube should to arise as a single entity , is no longer widely accepted 19 , but it is still useful to consider the consequence of dorsoventrally inverting his hypothetical ancestor . as nielsen points out , so long as the brain arises at the anterior end of an originally ventral neuraxis , it must necessarily form in the postoral region , most likely immediately caudal to the mouth .", "this may seem counterintuitive , but the zone of expression for mhb - type genes , which mark a core brain region in vertebrates , is also postoral in hemichordates .", "the ancestral chordate in this scenario , in its inverted position , would have both its mouth and brain on the dorsal surface of the body . while this may seem peculiar", "what is then needed to generate the body of more advanced chordates like amphioxus and vertebrates is for differential growth of the ectoderm separating the mouth and brain to move the former forward , over the front or side of the snout , to what is now the ventral surface .", "the site of the ancestral apical organ must move as well , and will eventually find itself behind the now - ventral mouth , roughly in the position of the adhesive gland , which is also exactly the path the polar bodies follow from the apical pole of the egg during amphibian development 24 . in consequence ,", "the anterior most part of our brain can not be homologous with the anterior - most parts of protostome brains , because the latter are in part derived from apical structures .", "this is despite the supposed homology between the anterior otx - expressing regions of protostome and deuterostome brains , because in embryos and larvae in both groups , otx can be expressed both pre- and post - orally 25,26 . in summary , though there is good reason to doubt that the chordate neural tube actually derives evolutionarily from larval ciliary bands 19 , nielsen 's point about the site of brain formation being post - oral , though counterintuitive , is largely consistent with current molecular data .", "in addition , the larvae of hemichordates and echinoderms have neurons at this location , and these form comparatively complex ganglion - like assemblages in some instances .", "the function of these simple neural centers is to innervate the larval oral region and pharynx , presumably to control basic feeding behaviors and digestion .", "if these did indeed serve in some way as the core of the evolving chordate brain , then the progressive incorporation of centers for locomotory control into the brain is yet another story , and one that would have unfolded quite independently of the evolutionary process by which a centralized brain and nerve cord arose in the protostome lineage . in this version of events ,", "it is quite unlikely that the internalized neural tube of chordates can be explained by a simple inversion of a protostome - like ancestor with an already - internalized brain and nerve cord , as has sometimes been supposed ."], ["dorsal views of the larval nerve cords of ( a ) amphioxus and ( b ) ciona , a representative ascidian , showing expression domains for the main cns patterning genes .", "this is somewhat simplified , as the exact extent of the expression domains can vary with developmental stage , and the ascidian hox genes , in particular , are expressed in non - overlapping domains with some gaps .", "two alternatives for explaining the origin of a dorsally positioned brain in chordates , using the hemichordate embryo ( a ) as a starting point ; indicates the mouth ( m ) , anus ( a ) , apical plate ( orange ) , and the ancestral dorsal ( a - dorsal ) and ventral ( a - ventral ) surfaces ; the light blue rectangle is the expression  hot zone  ( hz ) in which homologs of genes characteristic of the vertebrate mhb are expressed , typically in circumferential bands within ectoderm of the collar and anterior trunk .", "older explanations of chordate origins , e.g. garstang 's hypothesis , retain an ancestral dorsoventral orientation ( as in b ) .", "this allows the dorsal condensation of tissue that became the brain ( dark blue ) to form within the hot zone of mhb - like expression , co - opting the latter for neural patterning functions . with inversion ( c , left side ) , the ancestral ventral surface becomes dorsal .", "in contrast with b , any brain - like condensation that forms on the new dorsal surface would necessarily then be post - oral in position , but it could still incorporate the mhb - like hot zone .", "then , to generate the vertebrate condition ( c , right side ) , with mouth and brain on opposing sides of the body , one needs to move the mouth over the front or side of the snout by differential growth of an expansion zone ( ez ) located somewhere forward of the brain ."]]}
{"article_id": "PMC3066746", "article_text": ["a seventy five year old libyan man was seen in the urology department of tripoli medical centre , tripoli , libya with six month history of left loin pain .", "the patient noted a mass in the left loin two days before he was assessed in the hospital .", "the urine analysis revealed pus cells , protein , and no sugar , but urine culture yielded no bacterial growth .", "liver function tests showed slightly elevated alkaline phosphatase at 160 iu / l ( 44 to 147 iu / l ) , and lactate dehydrogenase at 152 iu / l ( 105 - 333", "full blood count revealed haemoglobin of 8.3g% , ( 1315g% ) , white cell count of 14.7103/mm3 ( 411103/ mm3 ) , and platelets of 3.4 105/ mm3 ( 1.54.5 105/ mm3 ) .", "the patient 's vomiting subsided with proton pump inhibitors , but the loin pain showed partial response to analgesic .", "abdominal ct - scan showed an irregular mass in the upper pole of left kidney .", "the patient was posted for partial nephrectomy with the provisional diagnosis of renal tuberculosis or renal infarction involving upper pole .", "pathologically , the kidney was noted to have greyish white soft tissue mass , firm in consistency measuring 6x4x2 cms , with adjacent dilated calyces .", "the renal resection margin and external surface appeared free of tumour ( figure 2 ) .", "microscopically , the tumour consisted of pleomorphic squamoid cells arranged in nests and sheets with few foci of epithelial pearl formation and keratinisation . areas of necrosis and frequent mitosis were noted .", "the malignant cells nore were seen to infiltrate the adjacent renal parenchyma as nests of cells and also focally renal capsule ( figures 3 & 4 ) .", "the dilated calysis showed areas of squamous metaplasia and severe dysplasia with focal invasion of basement membrane into renal parenchyma to form the tumour ( figure 5 ) .", "photomicrograph revealing calyseal subepithelial invasion of malignant cells ( left ) and invasion of renal parenchyma adjacent to glomerulus with epithelial pearl formation ( right ) .", "postoperative chest x - ray ( pre - operative chest x - ray was normal ) revealed massive pleural effusion in the left lower lobe ( figure 6 ) .", "however , no histological typing is available as tissue showed only necrotic material with scanty malignant cells .", "he was put on cisplatin and sunitinib on the suspicion of primary lung tumour and showed no response .", "the patient was suspected clinically to have renal tuberculosis and was later found to have renal squamous cell carcinoma with possible lung secondaries .", "the common renal malignancy in adults is of clear cell type , followed by papillary carcinoma and chromophobe cell carcinoma [ 1 , 2 ] .", "primary neoplasms of the renal collecting system are rare , accounting for less than 5% of urothelial tumours in urinary system [ 4 , 5 ] .", "the transitional cell type is the most frequent ( 85%95% ) , followed by squamous cell carcinoma ( 6%15% ) and adenocarcinoma ( 7% ) .", "usually , renal squamous carcinoma is highly aggressive and of a high grade at presentation .", "haematuria , the classical presenting complaint of renal cell carcinoma , is not common in this entity as in this case .", "the incidence of co - existing stone was reported in a wide range of 18% to 100% .", "the present case demonstrates the transition from calyseal urothelium to squamous metaplasia , dysplasia and invasive squamous malignancy .", "there is a well known association between chronic pyelonephritis , renal pelvic stones , phanecetin ingestion and radiotherapy with squamous cell carcinoma .", "the case was clinically suspected as renal tuberculosis because of sterile pyuria and past history of cough with fever .", "the renal malignancy was not considered due to loin pain and absence of haematuria as the renal cell carcinoma classically presents as painless haematuria .", "the tumour can be difficult to diagnose by imaging modalities as the usual features are the presence of calculi and hydronephrotic changes with ureteral obstruction .", "filling defects or obstructive lesions in the renal pelvis by intravenous / retrograde urography or detection of a solid mass by ultrasonography can be the signs of the tumour .", "central renal squamous cell carcinoma presents more with intraluminal components and is usually associated with lymph node metastasis whereas peripheral renal squamous cell carcinoma presents with prominent renal parenchymal thickening and might invade the perirenal fat tissue before lymph node or distant metastasis could be identified .", "the survival of patients with central renal squamous cell carcinoma was reported to be significantly shorter than those with peripheral renal squamous cell carcinoma .", "nativ and colleagues reported that patients with locally invasive renal squamous cell carcinomas had 1 and 2 yearsurvival rates of 33% and 22% , respectively .", "if metastasis develops , adjuvant chemotherapy or irradiation has little effect on the unfavourable prognosis .", "our patient underwent partial nephrectomy because of suspicion of renal tuberculosis and was later found have pulmonary metastasis . in conclusion , this patient presented with lion pain and a mass without haematuria and was found to have renal squamous cell carcinoma with lung metastasis .", "as renal calyseal tumours could present with atypical features , a proper preoperative workup may help in better management though outcome is short lived ."], "abstract_text": ["<S> a seventy five year old gentleman with the clinical diagnosis of renal tuberculosis was found to have renal squamous cell carcinoma . the clinical presentation and management are being discussed . </S>"], "labels": null, "section_names": ["Case presentation", "Diagnosis", "Discussion"], "sections": [["a seventy five year old libyan man was seen in the urology department of tripoli medical centre , tripoli , libya with six month history of left loin pain .", "the patient noted a mass in the left loin two days before he was assessed in the hospital .", "the urine analysis revealed pus cells , protein , and no sugar , but urine culture yielded no bacterial growth .", "liver function tests showed slightly elevated alkaline phosphatase at 160 iu / l ( 44 to 147 iu / l ) , and lactate dehydrogenase at 152 iu / l ( 105 - 333", "full blood count revealed haemoglobin of 8.3g% , ( 1315g% ) , white cell count of 14.7103/mm3 ( 411103/ mm3 ) , and platelets of 3.4 105/ mm3 ( 1.54.5 105/ mm3 ) .", "the patient 's vomiting subsided with proton pump inhibitors , but the loin pain showed partial response to analgesic .", "abdominal ct - scan showed an irregular mass in the upper pole of left kidney .", "the patient was posted for partial nephrectomy with the provisional diagnosis of renal tuberculosis or renal infarction involving upper pole .", "pathologically , the kidney was noted to have greyish white soft tissue mass , firm in consistency measuring 6x4x2 cms , with adjacent dilated calyces .", "the renal resection margin and external surface appeared free of tumour ( figure 2 ) .", "microscopically , the tumour consisted of pleomorphic squamoid cells arranged in nests and sheets with few foci of epithelial pearl formation and keratinisation . areas of necrosis and frequent mitosis were noted .", "the malignant cells nore were seen to infiltrate the adjacent renal parenchyma as nests of cells and also focally renal capsule ( figures 3 & 4 ) .", "the dilated calysis showed areas of squamous metaplasia and severe dysplasia with focal invasion of basement membrane into renal parenchyma to form the tumour ( figure 5 ) .", "photomicrograph revealing calyseal subepithelial invasion of malignant cells ( left ) and invasion of renal parenchyma adjacent to glomerulus with epithelial pearl formation ( right ) .", "postoperative chest x - ray ( pre - operative chest x - ray was normal ) revealed massive pleural effusion in the left lower lobe ( figure 6 ) .", "however , no histological typing is available as tissue showed only necrotic material with scanty malignant cells .", "he was put on cisplatin and sunitinib on the suspicion of primary lung tumour and showed no response ."], ["the patient was suspected clinically to have renal tuberculosis and was later found to have renal squamous cell carcinoma with possible lung secondaries ."], ["the common renal malignancy in adults is of clear cell type , followed by papillary carcinoma and chromophobe cell carcinoma [ 1 , 2 ] .", "primary neoplasms of the renal collecting system are rare , accounting for less than 5% of urothelial tumours in urinary system [ 4 , 5 ] .", "the transitional cell type is the most frequent ( 85%95% ) , followed by squamous cell carcinoma ( 6%15% ) and adenocarcinoma ( 7% ) .", "usually , renal squamous carcinoma is highly aggressive and of a high grade at presentation .", "haematuria , the classical presenting complaint of renal cell carcinoma , is not common in this entity as in this case .", "the incidence of co - existing stone was reported in a wide range of 18% to 100% .", "the present case demonstrates the transition from calyseal urothelium to squamous metaplasia , dysplasia and invasive squamous malignancy .", "there is a well known association between chronic pyelonephritis , renal pelvic stones , phanecetin ingestion and radiotherapy with squamous cell carcinoma .", "the case was clinically suspected as renal tuberculosis because of sterile pyuria and past history of cough with fever .", "the renal malignancy was not considered due to loin pain and absence of haematuria as the renal cell carcinoma classically presents as painless haematuria .", "the tumour can be difficult to diagnose by imaging modalities as the usual features are the presence of calculi and hydronephrotic changes with ureteral obstruction .", "filling defects or obstructive lesions in the renal pelvis by intravenous / retrograde urography or detection of a solid mass by ultrasonography can be the signs of the tumour .", "central renal squamous cell carcinoma presents more with intraluminal components and is usually associated with lymph node metastasis whereas peripheral renal squamous cell carcinoma presents with prominent renal parenchymal thickening and might invade the perirenal fat tissue before lymph node or distant metastasis could be identified .", "the survival of patients with central renal squamous cell carcinoma was reported to be significantly shorter than those with peripheral renal squamous cell carcinoma .", "nativ and colleagues reported that patients with locally invasive renal squamous cell carcinomas had 1 and 2 yearsurvival rates of 33% and 22% , respectively .", "if metastasis develops , adjuvant chemotherapy or irradiation has little effect on the unfavourable prognosis .", "our patient underwent partial nephrectomy because of suspicion of renal tuberculosis and was later found have pulmonary metastasis . in conclusion , this patient presented with lion pain and a mass without haematuria and was found to have renal squamous cell carcinoma with lung metastasis .", "as renal calyseal tumours could present with atypical features , a proper preoperative workup may help in better management though outcome is short lived ."]]}
{"article_id": "PMC3532871", "article_text": ["medicinal plants have been used for centuries in traditional medicine because of their therapeutic value .", "mentha piperita , ( family lamiaceae ) is a species found in iran and many parts of the world which has an economical value for its flavoring , odor , and therapeutic properties in foods and cosmetic industrial products . in addition , the leaves and flowers of m. piperita have medicinal properties [ 2 , 3 ] .", "essential oils are valuable natural products used as raw materials in many fields including perfumes , cosmetics , aromatherapy , phototherapy , spices , and nutrition . peppermint ( m. piperita )", "oil is one of the most popular and widely used essential oils , mostly because of its main components , menthol , and menthone .", "previous studies have shown antiviral , antibacterial [ 6 , 7 ] , antifungal [ 6 , 810 ] , antibiofilm formation [ 1113 ] , radioprotective , antioedema , analgesic , and antioxidant activities   of the eo and methanolic extracts of herbal parts and callus cultures of m. piperita .", "in addition , m. piperita eo has been shown to cause inhibitory effects against radial fungal growth and aflatoxin production by aspergillus species . in the past two decades ,", "azole - resistant candida and aspergillus species are the top pathogens responsible for nosocomial or food - borne infections [ 18 , 19 ] . in addition , the formation of biofilms by candida species have raised concerns due to their increased resistance to antifungal therapy and protects the microbial cells within biofilms from the host immune defenses [ 2023 ] .   an alternative approach to overcome antibiotic resistance might be using natural products and phytochemicals . it has also been shown that some plant extracts efficiently inhibit the biofilm formation of c. albicans .", "moreover , eos especially with known antibacterial effects have the potential to be used in food industry as preservatives and to increase the shelf life of products .", "therefore , determining the antimicrobial properties of eos might help to overcome microorganism resistance to antibiotics and prevent food spoilage .", "the chemical composition of aromatic plants depends largely on the individual genetic variability and different plant parts [ 2527 ] .", "the presence and concentration of certain chemical constituents of eos also fluctuate according to the season , climatic condition , and site of plant growth [ 25 , 26 ] .", "the goal of this study was to investigate the chemical composition and in vitro antifungal and antibiofilm activities of essential oils of the leaves of m. piperita collected in the region of fars from iran .", "at full flowering stage , the aerial parts of the m. piperita were hydrodistillated for 2.5  h , using an all - glass clevenger - type apparatus , according to the method outlined by the british pharmacopoeia .", "the sample oils were dried over anhydrous sodium sulfate and stored in sealed vials at 4c before gas chromatography and gas chromatography - mass spectrometry ( gc - ms ) analysis .  ", "the analysis was carried out on a thermoquest - finnigan trace gc - ms instrument equipped with a db-5 fused silica column ( 60  m  0.25  mm i.d . , film thickness 0.25  mm ) .", "the oven temperature was programmed to increase from 60c to 250c at a rate of 4c / min and finally held for 10  min ; transfer line temperature was 250c .", "helium was used as the carrier gas at a flow rate of 1.1  ml / min with a split ratio equal to 1/50 .", "the quadrupole mass spectrometer was scanned over the 35465  amu with an ionizing voltage of 70  ev and an ionization current of 150  ma .", "gc - flame ionization detector ( fid ) analysis of the oil was conducted using a thermoquest - finnigan instrument equipped with a db-5 fused silica column ( 60  m   0.25  mm i.d . ,", "nitrogen was used as the carrier gas at the constant flow of 1.1  ml / min ; the split ratio was the same as that for gc - ms .", "the oven temperature was raised from 60c to 250c at a rate of 4c / min and held for 10  min .", "the injector and detector ( fid ) temperatures were kept at 250c and 280c , respectively .", "retention indexes ( ris ) were calculated by using retention times of n - alkanes ( c6c24 ) that were injected after the oil at the same temperature and conditions .", "the compounds were identified by comparing their ri with those reported in the literature , and their mass spectrum was compared with those reported in wiley library .", "the antifungal activities of the eo against 25 standard strains of fungi , including c. albicans ( atcc 5982 , 1912 , 562 , 1905 , 1949 , 10261 , and 2730 ) , c. tropicalis ( atcc 750 ) , c. krusei ( atcc 6258 ) , c. glabrata ( atcc 863 , 2192 , 2175 , 6144 , and 90030 ) , c. dubliniensis ( cbs 8501 , atcc 8500 , 7987 , and 7988 ) , c. parapsilosis ( atcc 4344 ) , c. neoformance ( atcc 9011 ) , aspergillus flavus ( atcc 64025 ) , a. fumigatus ( atcc 14110 , cbs 144.89 ) , a. clavatus ( cbs 514.65 ) , and a. oryzae ( cbs 818.72 ) were determined . in addition , the antifungal activities of the eo against 35 clinical isolates of yeasts identified by polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) were also examined [ 29 , 30 ] .", "the antifungal susceptibility of clinical isolates of the tested fungi against fluconazole was examined by microdilution and disk diffusion methods [ 31 , 32 ] .  ", "minimal inhibitory concentrations of the eo against standard and clinical species of the fungi were determined by the broth microdilution method as recommended by the clinical and laboratory standards institute ( clsi ) , with some modifications [ 31 , 32 ] .", "briefly , the rpmi-1640 ( with l - glutamine and phenol red , without bicarbonate ) ( sigma , usa ) was prepared and buffered at ph 7.0 with 0.165  mol 3-(n - morpholino)propane sulfonic acid ( mops ) ( sigma - aldrich , steinheim , germany ) .", "serial dilutions of the eo ( 0.06 to 16 l / ml ) were prepared in 96-well microtiter trays using rpmi-1640 media ( sigma , st .", "double dilutions of fluconazole were also prepared for each of the tested fungi with the final concentration of 0.25 to 128  g / ml . stock inoculums were prepared by suspending three colonies of the examined yeast in 5  ml sterile 0.85% nacl and adjusting the turbidity of the inoculums to 0.5 mcfarland standard at 630  nm wavelength ( this yields stock suspension of 15  10  cells / ml ) . for moulds ( aspergillus spp . ) , conidia were recovered from the 7-day - old cultures grown on potato dextrose agar by a wetting loop with tween 20 .", "the collected conidia were transferred in sterile saline and their turbidity was adjusted to optical density of 0.09 to 0.11 that yields 0.45  10  conidia / ml . working suspension was prepared by making a 1/50 and 1/1000 dilution with rpmi of the stock suspension for moulds and yeasts , respectively . after the addition of 0.1  ml of the inoculums to the wells , the trays were incubated at 30c for 2448  h in a humid atmosphere .", "200  l of the uninoculated medium was included as a sterility control ( blank ) .", "in addition , growth controls ( medium with inoculums and 5% ( v / v ) without the eo or fluconazole ) were also included .", "mics were visually determined and defined as the lowest concentration of the eo that produced no visible growth .", "in addition , minimum fungicidal concentrations ( mfcs ) of all the examined agents were also determined by culturing 10  l from the wells showing no visible growth onto sda plates .", "mfcs were of the lowest concentration that showed either no growth or fewer than 4 colonies , which corresponded to 98% killing activity of the initial inoculums .", "mfcs were determined as the lowest concentration yielding no more than 4 colonies , which corresponds to a mortality of 98% of the fungi in the initial inoculums . according to behnam et al . , c. albicans and c. dubliniensis strains were maintained on sabouraud dextrose agar ( difco ) plates .", "after 48  hrs one loop of the colonies was transferred to 20  ml sabouraud broth in 250  ml erlenmeyer flasks and incubated overnight in an orbital shaker ( 100  rpm ) at 30c under aerobic condition .", "yeast cells were harvested and washed twice in sterile phosphate - buffered saline ( pbs ) ( 0.8% [ w / v ] , nacl ( merck ) ; 0.02% [ w / v ] , kh2po4 ( merck ) ; 0.31% [ w / v ] , na2hpo4 + 12h2o ( merck ) ; 0.02% [ w / v ] , kcl ( panreac ) ; ph 7.4 ) .", "then , they were resuspended in rpmi 1640 supplemented with l - glutamine ( gibco ) and buffered with morpholinopropanesulfonic acid ( mops ) and the cell densities were adjusted to 1.0  10  cells / ml after counting with a hemocytometer", ". serial dilution of the eos ( 0.0158  l / ml ) in rpmi 1640 was prepared in a presterilized , polystyrene , flat - bottom , 96-well microtiter plates ( nunc ) . after the addition of 0.1  ml of the yeast inoculums to the wells ,", "200  l of the uninoculated medium was included as a negative control ( blank ) .", "in addition , rpmi with yeasts but without the eos served as positive controls .  ", "a semiquantitative measure of biofilm formation was assayed using a 2 , 3-bis(2-methoxy-4-nitro-5-sulfo - phenyl)-2h - tetrazolium-5-carbox - anilide ( xtt ) reduction assay .", "xtt ( sigma chemical co. ) was prepared as a saturated solution at a concentration of 0.5  mg / ml in ringer 's lactate .", "this solution was filter - sterilized through a 0.22  m - pore - size filter , divided into aliquots and then stored at 70c . prior to each assay ,", "an aliquot of the xtt stock solution was thawed and treated with menadione sodium bisulfite ( 10  mm prepared in distilled water ; sigma chemical co. ) to obtain a final concentration of 1  m of menadione .", "a 100  l aliquot of xtt menadione was then added to each prewashed wells .", "the plates were then incubated in dark for 2 hours at 37c , and the colorimetric change at 490  nm ( a reflection of the metabolic activity of the biofilm ) was measured with a microtiter plate reader ( titertekplus - ms2 reader , uk ) .", "the qualitative and quantitative compositions of the eo of m. piperita are presented in table 1 .", "gc / ms analyses showed that the main constituent of the eo was menthol ( 53.28% ) followed by menthyl acetate ( 15.1% ) and menthofuran ( 11.18% ) .", "the antifungal activities of m. piperita eo against the tested yeasts are shown in table 2 .", "the eo inhibited the growth of all of the tested yeasts at concentrations of 0.124  l / ml . furthermore , the eo exhibited fungicidal activity ( mfc ) for all of the above - mentioned yeasts at concentrations ranging from 1 to 8  l / ml .", "no significant differences in inhibitory concentrations were found between azole - resistant and -susceptible strains .", "in addition , the eo inhibited the growth and killed the standard strain of cryptococcus neoformance at the concentration of 4  l / ml . all of the aspergillus standard strains were susceptible to m. piperita eo at concentrations of 0.54  l / ml ( table 3 ) .", "as shown in table 4 , the eo completely inhibited the biofilm formation of c. albicans and c. dubliniensis at concentrations of 1  l / ml and 2  l / ml , respectively .  ", "the compositions of the eos might be affected by the developmental stages of the plant . although some authors reported alpha terpinen as the dominant component of m. piperita eo ( 19.7% ) , similar to the previous studies [ 4 , 7 , 33 ] , we identified menthol as one of the main constituents of the eos .", "the higher concentration of menthol in this study as compared to some of those of previous reports [ 4 , 34 ] may reflect variations due to geographical location from which the plants were collected .  ", "in this study , the eos exhibited fungistatic and fungicidal activities against both of the standard and clinical strains of candida species at concentrations ranging from 0.5  l / ml to 8  l / ml , which can be best compared to the previous investigations [ 6 , 8 , 9 , 33 ] .", "one of the encapsulated yeasts , c. neoformance , is a well - known primarily opportunistic pathogen which produces chronic and life - threatening meningitis . according to the findings of this study , the examined oils killed the standard strain of c. neoformance at concentration of 4  l / ml .", "similar to the previous studies [ 10 , 17 ] the eo of m. piperita exhibited a strong anti - aspergillus activity with mic values ranging from 0.5 to 4  l / ml . mfcs of the eo against the tested fungi were almost similar or two times greater than those of their corresponding mics .  ", "since the eos exhibited similar antifungal effect against the tested azole - resistant and azole - susceptible strains , it could be assumed that the mechanism of the action of the eos is different with those of the above - mentioned antifungal drug .", "one of the main characteristics of eos is their hydrophobicity , which enables their incorporation into the cell membrane .", "it has been shown that this phenolic monoterpene has a hydroxyl group around the phenolic ring and exhibits its antimicrobial activity through the disruption of the cytoplasmic membrane [ 35 , 36 ] .", "biofilm formation by candida species is a phenomenon which helps the survival , pathogenesis , and drug resistance .", "the most commonly candida species associated with biofilm formation is c. albicans . in the present study ,", "the formation of biofilm was inhibited completely at a concentration of up to 2  l / ml in a dose - dependent manner , which was comparable to the study of agarwal et al . .", "as the industries tend to reduce the use of chemical preservatives in their products , eo of m. piperita with potential active antimicrobial properties might be considered as a natural source for the maintenance or extension of the shelf life of products .", "in addition , delectable taste of the eo at the concentrations needed for antimicrobial properties was a bonus to its antimicrobial effects . on the other hand , these eos might also be considered for developing products for controlling fungal infections . as these tests have all been done in vitro ,", "the next step may be further investigations in animal models to see if infection can be inhibited by the eo ."], "abstract_text": ["<S> variations in quantity and quality of essential oil ( eo ) from the aerial parts of cultivated mentha piperita were determined . </S>", "<S> the eo of air - dried sample was obtained by a hydrodistillation method and analyzed by a gas chromatography / mass spectrometry ( gc / ms ) . </S>", "<S> the antifungal activity of the eo was investigated by broth microdilution methods as recommended by clinical and laboratory standards institute . </S>", "<S> a biofilm formation inhibition was measured by using an xtt reduction assay . </S>", "<S> menthol ( 53.28% ) was the major compound of the eo followed by menthyl acetate ( 15.1% ) and menthofuran ( 11.18% ) . </S>", "<S> the eo exhibited strong antifungal activities against the examined fungi at concentrations ranging from 0.12 to 8.0  l / ml . in addition , the eo inhibited the biofilm formation of candida albicans and c. dubliniensis at concentrations up to 2  l / ml . considering the wide range of the antifungal activities of the examined eo , it might be potentially used in the management of fungal infections or in the extension of the shelf life of food products . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods and Material ", "3. Results and Discussion", "4. Conclusion "], "sections": [["medicinal plants have been used for centuries in traditional medicine because of their therapeutic value .", "mentha piperita , ( family lamiaceae ) is a species found in iran and many parts of the world which has an economical value for its flavoring , odor , and therapeutic properties in foods and cosmetic industrial products . in addition , the leaves and flowers of m. piperita have medicinal properties [ 2 , 3 ] .", "essential oils are valuable natural products used as raw materials in many fields including perfumes , cosmetics , aromatherapy , phototherapy , spices , and nutrition . peppermint ( m. piperita )", "oil is one of the most popular and widely used essential oils , mostly because of its main components , menthol , and menthone .", "previous studies have shown antiviral , antibacterial [ 6 , 7 ] , antifungal [ 6 , 810 ] , antibiofilm formation [ 1113 ] , radioprotective , antioedema , analgesic , and antioxidant activities   of the eo and methanolic extracts of herbal parts and callus cultures of m. piperita .", "in addition , m. piperita eo has been shown to cause inhibitory effects against radial fungal growth and aflatoxin production by aspergillus species . in the past two decades ,", "azole - resistant candida and aspergillus species are the top pathogens responsible for nosocomial or food - borne infections [ 18 , 19 ] . in addition , the formation of biofilms by candida species have raised concerns due to their increased resistance to antifungal therapy and protects the microbial cells within biofilms from the host immune defenses [ 2023 ] .   an alternative approach to overcome antibiotic resistance might be using natural products and phytochemicals . it has also been shown that some plant extracts efficiently inhibit the biofilm formation of c. albicans .", "moreover , eos especially with known antibacterial effects have the potential to be used in food industry as preservatives and to increase the shelf life of products .", "therefore , determining the antimicrobial properties of eos might help to overcome microorganism resistance to antibiotics and prevent food spoilage .", "the chemical composition of aromatic plants depends largely on the individual genetic variability and different plant parts [ 2527 ] .", "the presence and concentration of certain chemical constituents of eos also fluctuate according to the season , climatic condition , and site of plant growth [ 25 , 26 ] .", "the goal of this study was to investigate the chemical composition and in vitro antifungal and antibiofilm activities of essential oils of the leaves of m. piperita collected in the region of fars from iran ."], ["at full flowering stage , the aerial parts of the m. piperita were hydrodistillated for 2.5  h , using an all - glass clevenger - type apparatus , according to the method outlined by the british pharmacopoeia .", "the sample oils were dried over anhydrous sodium sulfate and stored in sealed vials at 4c before gas chromatography and gas chromatography - mass spectrometry ( gc - ms ) analysis .  ", "the analysis was carried out on a thermoquest - finnigan trace gc - ms instrument equipped with a db-5 fused silica column ( 60  m  0.25  mm i.d . , film thickness 0.25  mm ) .", "the oven temperature was programmed to increase from 60c to 250c at a rate of 4c / min and finally held for 10  min ; transfer line temperature was 250c .", "helium was used as the carrier gas at a flow rate of 1.1  ml / min with a split ratio equal to 1/50 .", "the quadrupole mass spectrometer was scanned over the 35465  amu with an ionizing voltage of 70  ev and an ionization current of 150  ma .", "gc - flame ionization detector ( fid ) analysis of the oil was conducted using a thermoquest - finnigan instrument equipped with a db-5 fused silica column ( 60  m   0.25  mm i.d . ,", "nitrogen was used as the carrier gas at the constant flow of 1.1  ml / min ; the split ratio was the same as that for gc - ms .", "the oven temperature was raised from 60c to 250c at a rate of 4c / min and held for 10  min .", "the injector and detector ( fid ) temperatures were kept at 250c and 280c , respectively .", "retention indexes ( ris ) were calculated by using retention times of n - alkanes ( c6c24 ) that were injected after the oil at the same temperature and conditions .", "the compounds were identified by comparing their ri with those reported in the literature , and their mass spectrum was compared with those reported in wiley library .", "the antifungal activities of the eo against 25 standard strains of fungi , including c. albicans ( atcc 5982 , 1912 , 562 , 1905 , 1949 , 10261 , and 2730 ) , c. tropicalis ( atcc 750 ) , c. krusei ( atcc 6258 ) , c. glabrata ( atcc 863 , 2192 , 2175 , 6144 , and 90030 ) , c. dubliniensis ( cbs 8501 , atcc 8500 , 7987 , and 7988 ) , c. parapsilosis ( atcc 4344 ) , c. neoformance ( atcc 9011 ) , aspergillus flavus ( atcc 64025 ) , a. fumigatus ( atcc 14110 , cbs 144.89 ) , a. clavatus ( cbs 514.65 ) , and a. oryzae ( cbs 818.72 ) were determined . in addition , the antifungal activities of the eo against 35 clinical isolates of yeasts identified by polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) were also examined [ 29 , 30 ] .", "the antifungal susceptibility of clinical isolates of the tested fungi against fluconazole was examined by microdilution and disk diffusion methods [ 31 , 32 ] .  ", "minimal inhibitory concentrations of the eo against standard and clinical species of the fungi were determined by the broth microdilution method as recommended by the clinical and laboratory standards institute ( clsi ) , with some modifications [ 31 , 32 ] .", "briefly , the rpmi-1640 ( with l - glutamine and phenol red , without bicarbonate ) ( sigma , usa ) was prepared and buffered at ph 7.0 with 0.165  mol 3-(n - morpholino)propane sulfonic acid ( mops ) ( sigma - aldrich , steinheim , germany ) .", "serial dilutions of the eo ( 0.06 to 16 l / ml ) were prepared in 96-well microtiter trays using rpmi-1640 media ( sigma , st .", "double dilutions of fluconazole were also prepared for each of the tested fungi with the final concentration of 0.25 to 128  g / ml . stock inoculums were prepared by suspending three colonies of the examined yeast in 5  ml sterile 0.85% nacl and adjusting the turbidity of the inoculums to 0.5 mcfarland standard at 630  nm wavelength ( this yields stock suspension of 15  10  cells / ml ) . for moulds ( aspergillus spp . ) , conidia were recovered from the 7-day - old cultures grown on potato dextrose agar by a wetting loop with tween 20 .", "the collected conidia were transferred in sterile saline and their turbidity was adjusted to optical density of 0.09 to 0.11 that yields 0.45  10  conidia / ml . working suspension was prepared by making a 1/50 and 1/1000 dilution with rpmi of the stock suspension for moulds and yeasts , respectively . after the addition of 0.1  ml of the inoculums to the wells , the trays were incubated at 30c for 2448  h in a humid atmosphere .", "200  l of the uninoculated medium was included as a sterility control ( blank ) .", "in addition , growth controls ( medium with inoculums and 5% ( v / v ) without the eo or fluconazole ) were also included .", "mics were visually determined and defined as the lowest concentration of the eo that produced no visible growth .", "in addition , minimum fungicidal concentrations ( mfcs ) of all the examined agents were also determined by culturing 10  l from the wells showing no visible growth onto sda plates .", "mfcs were of the lowest concentration that showed either no growth or fewer than 4 colonies , which corresponded to 98% killing activity of the initial inoculums .", "mfcs were determined as the lowest concentration yielding no more than 4 colonies , which corresponds to a mortality of 98% of the fungi in the initial inoculums . according to behnam et al . , c. albicans and c. dubliniensis strains were maintained on sabouraud dextrose agar ( difco ) plates .", "after 48  hrs one loop of the colonies was transferred to 20  ml sabouraud broth in 250  ml erlenmeyer flasks and incubated overnight in an orbital shaker ( 100  rpm ) at 30c under aerobic condition .", "yeast cells were harvested and washed twice in sterile phosphate - buffered saline ( pbs ) ( 0.8% [ w / v ] , nacl ( merck ) ; 0.02% [ w / v ] , kh2po4 ( merck ) ; 0.31% [ w / v ] , na2hpo4 + 12h2o ( merck ) ; 0.02% [ w / v ] , kcl ( panreac ) ; ph 7.4 ) .", "then , they were resuspended in rpmi 1640 supplemented with l - glutamine ( gibco ) and buffered with morpholinopropanesulfonic acid ( mops ) and the cell densities were adjusted to 1.0  10  cells / ml after counting with a hemocytometer", ". serial dilution of the eos ( 0.0158  l / ml ) in rpmi 1640 was prepared in a presterilized , polystyrene , flat - bottom , 96-well microtiter plates ( nunc ) . after the addition of 0.1  ml of the yeast inoculums to the wells ,", "200  l of the uninoculated medium was included as a negative control ( blank ) .", "in addition , rpmi with yeasts but without the eos served as positive controls .  ", "a semiquantitative measure of biofilm formation was assayed using a 2 , 3-bis(2-methoxy-4-nitro-5-sulfo - phenyl)-2h - tetrazolium-5-carbox - anilide ( xtt ) reduction assay .", "xtt ( sigma chemical co. ) was prepared as a saturated solution at a concentration of 0.5  mg / ml in ringer 's lactate .", "this solution was filter - sterilized through a 0.22  m - pore - size filter , divided into aliquots and then stored at 70c . prior to each assay ,", "an aliquot of the xtt stock solution was thawed and treated with menadione sodium bisulfite ( 10  mm prepared in distilled water ; sigma chemical co. ) to obtain a final concentration of 1  m of menadione .", "a 100  l aliquot of xtt menadione was then added to each prewashed wells .", "the plates were then incubated in dark for 2 hours at 37c , and the colorimetric change at 490  nm ( a reflection of the metabolic activity of the biofilm ) was measured with a microtiter plate reader ( titertekplus - ms2 reader , uk ) ."], ["the qualitative and quantitative compositions of the eo of m. piperita are presented in table 1 .", "gc / ms analyses showed that the main constituent of the eo was menthol ( 53.28% ) followed by menthyl acetate ( 15.1% ) and menthofuran ( 11.18% ) .", "the antifungal activities of m. piperita eo against the tested yeasts are shown in table 2 .", "the eo inhibited the growth of all of the tested yeasts at concentrations of 0.124  l / ml . furthermore , the eo exhibited fungicidal activity ( mfc ) for all of the above - mentioned yeasts at concentrations ranging from 1 to 8  l / ml .", "no significant differences in inhibitory concentrations were found between azole - resistant and -susceptible strains .", "in addition , the eo inhibited the growth and killed the standard strain of cryptococcus neoformance at the concentration of 4  l / ml . all of the aspergillus standard strains were susceptible to m. piperita eo at concentrations of 0.54  l / ml ( table 3 ) .", "as shown in table 4 , the eo completely inhibited the biofilm formation of c. albicans and c. dubliniensis at concentrations of 1  l / ml and 2  l / ml , respectively .  ", "the compositions of the eos might be affected by the developmental stages of the plant . although some authors reported alpha terpinen as the dominant component of m. piperita eo ( 19.7% ) , similar to the previous studies [ 4 , 7 , 33 ] , we identified menthol as one of the main constituents of the eos .", "the higher concentration of menthol in this study as compared to some of those of previous reports [ 4 , 34 ] may reflect variations due to geographical location from which the plants were collected .  ", "in this study , the eos exhibited fungistatic and fungicidal activities against both of the standard and clinical strains of candida species at concentrations ranging from 0.5  l / ml to 8  l / ml , which can be best compared to the previous investigations [ 6 , 8 , 9 , 33 ] .", "one of the encapsulated yeasts , c. neoformance , is a well - known primarily opportunistic pathogen which produces chronic and life - threatening meningitis . according to the findings of this study , the examined oils killed the standard strain of c. neoformance at concentration of 4  l / ml .", "similar to the previous studies [ 10 , 17 ] the eo of m. piperita exhibited a strong anti - aspergillus activity with mic values ranging from 0.5 to 4  l / ml . mfcs of the eo against the tested fungi were almost similar or two times greater than those of their corresponding mics .  ", "since the eos exhibited similar antifungal effect against the tested azole - resistant and azole - susceptible strains , it could be assumed that the mechanism of the action of the eos is different with those of the above - mentioned antifungal drug .", "one of the main characteristics of eos is their hydrophobicity , which enables their incorporation into the cell membrane .", "it has been shown that this phenolic monoterpene has a hydroxyl group around the phenolic ring and exhibits its antimicrobial activity through the disruption of the cytoplasmic membrane [ 35 , 36 ] .", "biofilm formation by candida species is a phenomenon which helps the survival , pathogenesis , and drug resistance .", "the most commonly candida species associated with biofilm formation is c. albicans . in the present study ,", "the formation of biofilm was inhibited completely at a concentration of up to 2  l / ml in a dose - dependent manner , which was comparable to the study of agarwal et al . ."], ["as the industries tend to reduce the use of chemical preservatives in their products , eo of m. piperita with potential active antimicrobial properties might be considered as a natural source for the maintenance or extension of the shelf life of products .", "in addition , delectable taste of the eo at the concentrations needed for antimicrobial properties was a bonus to its antimicrobial effects . on the other hand , these eos might also be considered for developing products for controlling fungal infections . as these tests have all been done in vitro ,", "the next step may be further investigations in animal models to see if infection can be inhibited by the eo ."]]}
{"article_id": "PMC4346101", "article_text": ["falls of the elderly always lead to serious health issues as the decline of their physical fitness .", "fracture is the most common injury in fall of an elderly and there is also a certain possibility to get coma , brain trauma , and paralysis . at most fall situations , the fall process is the main source of injury because of the high impact . but sometimes the late medical salvage may worsen the situation .", "that means the faster the salvage comes , the less risk the elderly will face .", "mems ( microelectro mechanical systems ) sensors have simplified the design and implementation of sensor system .", "location based service ( lbs ) makes it more convenient to locate the elderly in health monitoring . beside these", "cameras are distributed at limited space to offer pictures or videos of human activities to implement fall detection algorithm .", "external supports such as motion sensors could be used to enhance computer vision based fall detection method , and a data fusion algorithm can operate the validation and correlation among the two subsystems to raise robust performance of fall detection .", "these computer based methods work effectively in indoor environment , but they are hard to realize in outdoor environment as the deployment of cameras is always limited .", "there are several kinds of detection methods which differ in constitution of motion sensors and detection algorithms .", "a single triaxial accelerometer can provide object 's accelerations in three directions which include the influence of gravity .", "the influence of gravity or dynamic acceleration is available by using a low pass filter or a high pass filter .", "some kinds of angular movement information can also be calculated based on the relationship between acceleration components and their vector sum .", "a triaxial magnetometer can detect magnetic strength in three directions , and it can also provide angular motion information in the horizontal plane .", "but the environment magnetic field may disturb the geomagnetic field which reduces the reliability of the magnetometer outputs , for instance , in some steel structure architecture or near some objects with strong electromagnetism . as angular information", "can also be extracted from accelerometer measurements , a state space filter such as the kalman filter is a commonly used technique to combine angular motion information . beside these , sensors such as barometer can also assist pure motion sensors at human gait recognition .", "but , in fact , using more sensors means more power consumption , and it is a challenge to design a proper algorithm to fuse different kinds of sensors .", "a single triaxial accelerometer is quite enough for human fall detection as sufficient information could be extracted from its measurements .", "besides this , the accelerometer coordinate does not have to be fixed if only the magnitude of sum vector is needed , and that is quite convenient for wearable application . in this paper , a fall detection system based on a wearable device is developed .", "the hardware and software realization of the device is mainly based on a single triaxial accelerometer and gps / gsm module .", "the device uses an efficient fall detection algorithm with less resource and power consumption , which means that it is a proper design for outdoor application .", "then it will get the elderly 's geographic position and send fall alarm short message to caregivers .", "so the elderly who has fallen can get timely help to minimize the negative influence .", "information like linear movements ( e.g. , displacement , velocity , and acceleration ) and angular movements ( e.g. , angle , angular velocity , and angular acceleration ) could be obtained directly or indirectly . beside these", ", frequency domain parameters could be extracted from basic sensor measurements by techniques such as fft and wavelet [ 11 , 12 ] .", "for single triaxial accelerometer application , accelerations and derived angular parameters could be used as recognition features . fall detection algorithm design", "according to the recognition feature , fall detection algorithms are classified as threshold based and machine learning based . for threshold", "based method , threshold of recognition feature is set by the designer before application which makes the algorithm have rapid response and less resource consumption .", "but the choice of threshold needs both rigorous schemes and adequate experiments . for machine learning based design", ", the classification of fall and normal activities is available with the assistance of technologies such as support vector machine ( svm ) and neural network [ 14 , 15 ] .", "machine learning assistance may enhance system robustness to some extent , but its algorithm design is always high computing resource consumed which limits its application in wearable device . as the compact wearable device requires low power consumption and a single triaxial accelerometer could provide effective information , threshold based fall detection algorithm will be used in this system . algorithm used in this fall alarm system", "when a real fall happens , collision between human 's body and ground will produce obvious peak value at the sum acceleration a which has magnitude as \n ( 1)a = ax2+ay2+az2 , \n where ax , ay , and az present accelerometer measurements of three axes .", "the system uses the sum acceleration as the first step to distinguish high intensity movements from others .", "but normal motions such as jumping or sitting also produce peak values , which mean that additional detection features are required .", "as human 's motion has low acceleration , it is feasible to get gravity component in each axis by using a low pass filter . if gravity components could be separated before and after human 's fall , then it is possible to calculate the rotation angle of accelerometer coordinate in 3d space , which is also equivalent to the rotation angle of gravity vector relative to fixed coordinate .", "quaternion is an effective tool to describe rotation movement in human 's gait change which also includes falling .", "a quaternion could be described as \n ( 2)q = q0+q1i+q2j+q3k \n which has magnitude as \n ( 3)q = q02+q12+q22+q32 .", "unit quaternion which has magnitude q = 1 can be described as \n ( 4)q = cos2+sin2qxi+sin2qyj+sin2qzk. as shown in figure 3 , rotation angle of q equals . the rotation axis is orthogonal to the rotation plane and its direction is in accordance with right hand screw rule .", "qx , qy , and qz are three components of the unit vector which describes the orientation of the quaternion at the fixed coordinate .", "besides rotation movement , quaternion can also describe a vector in 3d space , such as the gravity vector g which could be described as a quaternion \n ( 5)g=0+gxi+gyj+gzk.gx , gy , and gz are three components of g which has quaternion magnitude as \n ( 6)g = gx2+gy2+gz2=g . a unit quaternion q is used to describe human 's falling movement , which can also be divided into three rotation quaternions q1", ", q2 , and q3 to simplify the calculation as shown in figure 4 . with the help of gravity vector information before and after human 's falling movement as shown in figure 3 , these three separated quaternions are all available . \n", "q \n 1 could be expressed as \n ( 7)q1=cos12+sin12sini+sin12cosj \n which has rotation angle and rotation axis information as \n ( 8)1=arctangbefore , zgbefore , x2+gbefore , y2,sin=gbefore , ygbefore , x2+gbefore , y2,cos=gbefore , xgbefore , x2+gbefore , y2 .", "quaternion q2 can be calculated as \n ( 9)q2=cos22+sin22k \n which has rotation angle as \n ( 10)2=arctan2gafter , ygafter , xarctan2gbefore , ygbefore , x .", "quaternion q3 is \n ( 11)q3=cos32+sin32sini+sin32cosj \n with rotation angle and rotation axis information as \n ( 12)3=arctangafter , zgafter , x2+gafter , y2,sin=gafter , ygafter , x2+gafter , y2,cos=gafter , xgafter , x2+gafter , y2 .", "then the rotation of the fall movement can be expressed by quaternion multiplication as \n ( 13)gafter = qgbeforeq=q3q2q1gbeforeq1q2q3 , \n where q=q0-q1i -- q2j -- q3k- is the conjugate quaternion of q. quaternion algebra is normally implemented based on basic matrix algebra .", "quaternion multiplication used above could be realized by matrix multiplication as \n ( 14)qgbefore = mq0gbefore , xgbefore , ygbefore , z = q0q1q2q3q1q0q3q2q2q3q0q1q3q2q1q00gbefore , xgbefore , ygbefore , z .", "the whole rotation quaternion can also be decomposed as \n ( 15)q = q3q2q1=mq3mq2q1.q1 , q2 , and q3 are all available based on gravity information before and after the falling movement . at last , it is possible to get four elements of quaternion q by the equation above and the rotation angle  could be calculated as \n ( 16)=2arctanq12+q22+q32q0 .", "when an object is falling , magnitude of  will approach 90 which is also a character different from most normal activities .", "the wearable device will be mounted on human 's waist at first to reflect the motion of human body closely , and the device will record gbefore while the elderly is standing still .", "there is no special requirement of the device orientation but only keep stationary during the wear .", "athreshold means the threshold of sum acceleration magnitude and tthreshold means the threshold of oscillation time duration after the break of athreshold . when measurements in three different axes have been acquired , the sum acceleration |a| will be calculated .", "when a real fall happens , sum acceleration will reach peak value of |a|  athreshold . in a real falling", ", the fluctuation of acceleration will stop in time duration tthreshold and then the sum acceleration is |a|  g as the elderly will lie on the ground . then the acceleration a is recorded as gafter .", ", the system will consider it as a fall if the rotation angle ||  90.", "ti 's 16 bits mcu msp430f1611 is used to control the whole system and imply the detection algorithm .", "the measurement range of accelerometer could be set at 2  g , 4  g , 8  g , or 16 ", "g , and the maximal sampling rate is 3.2  khz . as human 's activities are normally at low frequency bands , 100  hz is a proper sampling rate for human fall detection .", "there is an inner digital filter in adxl345 which could weaken noise and reduce the burden of digital signal processing in mcu to some extent .", "the measurements will be sent through iic ( interintegrated circuit ) bus communication between the sensor and the mcu .", "sim908 can offer gps and gsm service on serial port communication with mcu , and it can also work in low power mode .", "each hardware component of the wearable device is working under low voltage and the detection algorithm does not need complex calculation resource , so the power consumption of the whole device is quite low .", "a 1200  mah , 3.7  v polymer lithium battery is quite enough to provide the need of the wearable device for a couple of days .", "hardware structure of the detection device is shown in figure 6 , and the pcb board prototype is shown in figure 7 .", "one of them is the software design in wearable device , and the other is in the caregiver 's handset .", "after initialization of the system , gbefore would be extracted from acceleration measurements by using a low pass filter when the elderly is standing .", "if a fall has been detected , the wearable device will locate the user and send alarm short message to caregivers immediately .", "if the user withdraws the alarm by pressing a button manually , the device will get back to fall detection state and a short message will be sent to inform the caregivers .", "there is a url ( universal resource locator ) which links to a web map in the alarm short message .", "the fall location information will be highlighted on a web map when the caregiver opens this link .", "system test of the fall detection system has been conducted based on the system design described above . the sampling rate of accelerometer is set at 100  hz and the measurement range is 16  g with a maximum precision of 4  mg .", "mcu will read raw measurements from sensor 's inner fifo and apply the detection algorithm .", "the test objects are three different volunteers at the ages of 23 , 42 , and 60 , respectively . based on analysis of these volunteers ' experiment data , athreshold and tthreshold", "are set as 2  g and 2  s , respectively . in order to get g", "when standing and lying after the fall , sum acceleration a which has magnitude between ( 1  0.3 )  g and ( 1 + 0.3 )  g will be considered as gbefore and gafter .", "considering that the tilt of the ground or the lying posture of the elderly may affect the rotation angle , rotation angle  between ( 90  30) and ( 90 + 30) will infer that the elderly has fallen .", "system test contains five kinds of activities of daily living ( i.e. , walking , jumping , squatting , sitting , and resting ) and four kinds of fallings ( i.e. , forward , backward , leftward , and rightward ) .", "each kind of motion has been repeated 20 times on each volunteer , and the detection results of the proposed algorithm and an acceleration threshold based algorithm   are listed in table 1 . the sensitivity and specificity [ 21 , 22 ] of the proposed system can be got from the test data in table 1 .", "test results of acceleration threshold based algorithm show lower sensitivity and specificity at 91.6% and 88.7% , respectively . \n", "figure 9 shows the accelerations in different kinds of fallings which all trigger the fall alarm correctly and the corresponding rotation angles are 91.9 , 100.5 , 104.7 , and 114.1. \n figure 10 shows acceleration during activity of lying down and resting which has rotation angle of 117.0 and similar waveform to falling , but the peak acceleration is much lower .", "they all do not have rotation about 90 ; even some of the peak values are quite high , so the fall alarm has not been triggered .", "an alarm sms ( short message service ) containing a map url has been received by the handset as shown in figure 12 when a fall has been detected . clicking", "the url will open a map in web browser on which the fall location will be displayed accurately as shown in figure 13 .", "this paper developed a fall detection system based on a single triaxial accelerometer based wearable device .", "there is no special requirement of the device 's mounting orientation because the algorithm does not claim the axes of accelerometer to be fixed strictly .", "the system has low power consumed hardware design and highly efficient algorithm which could extend the service time of the wearable device .", "as normal activity of resting also has similar rotation as falling , it may trigger fall alarm when the body hits ground heavily .", "so the choice of athreshold is quite important to distinguish falling from heavily lying activity .", "sufficient sample number collected from subjects with different age and gender will improve the reliability and robustness of the threshold . beside these , technologies such as svm and neural network are considerable to seek out a proper classification method based on the features used in this system ."], "abstract_text": ["<S> fall detection is a major challenge in the public healthcare domain , especially for the elderly as the decline of their physical fitness , and timely and reliable surveillance is necessary to mitigate the negative effects of falls . </S>", "<S> this paper develops a novel fall detection system based on a wearable device . </S>", "<S> the system monitors the movements of human body , recognizes a fall from normal daily activities by an effective quaternion algorithm , and automatically sends request for help to the caregivers with the patient 's location . </S>"], "labels": null, "section_names": ["1. Introduction", "2. System Design", "3. Fall Detection Algorithm", "4. Implementation", "5. System Test", "6. Conclusion"], "sections": [["falls of the elderly always lead to serious health issues as the decline of their physical fitness .", "fracture is the most common injury in fall of an elderly and there is also a certain possibility to get coma , brain trauma , and paralysis . at most fall situations , the fall process is the main source of injury because of the high impact . but sometimes the late medical salvage may worsen the situation .", "that means the faster the salvage comes , the less risk the elderly will face .", "mems ( microelectro mechanical systems ) sensors have simplified the design and implementation of sensor system .", "location based service ( lbs ) makes it more convenient to locate the elderly in health monitoring . beside these", "cameras are distributed at limited space to offer pictures or videos of human activities to implement fall detection algorithm .", "external supports such as motion sensors could be used to enhance computer vision based fall detection method , and a data fusion algorithm can operate the validation and correlation among the two subsystems to raise robust performance of fall detection .", "these computer based methods work effectively in indoor environment , but they are hard to realize in outdoor environment as the deployment of cameras is always limited .", "there are several kinds of detection methods which differ in constitution of motion sensors and detection algorithms .", "a single triaxial accelerometer can provide object 's accelerations in three directions which include the influence of gravity .", "the influence of gravity or dynamic acceleration is available by using a low pass filter or a high pass filter .", "some kinds of angular movement information can also be calculated based on the relationship between acceleration components and their vector sum .", "a triaxial magnetometer can detect magnetic strength in three directions , and it can also provide angular motion information in the horizontal plane .", "but the environment magnetic field may disturb the geomagnetic field which reduces the reliability of the magnetometer outputs , for instance , in some steel structure architecture or near some objects with strong electromagnetism . as angular information", "can also be extracted from accelerometer measurements , a state space filter such as the kalman filter is a commonly used technique to combine angular motion information . beside these , sensors such as barometer can also assist pure motion sensors at human gait recognition .", "but , in fact , using more sensors means more power consumption , and it is a challenge to design a proper algorithm to fuse different kinds of sensors .", "a single triaxial accelerometer is quite enough for human fall detection as sufficient information could be extracted from its measurements .", "besides this , the accelerometer coordinate does not have to be fixed if only the magnitude of sum vector is needed , and that is quite convenient for wearable application . in this paper , a fall detection system based on a wearable device is developed .", "the hardware and software realization of the device is mainly based on a single triaxial accelerometer and gps / gsm module .", "the device uses an efficient fall detection algorithm with less resource and power consumption , which means that it is a proper design for outdoor application ."], ["then it will get the elderly 's geographic position and send fall alarm short message to caregivers .", "so the elderly who has fallen can get timely help to minimize the negative influence ."], ["information like linear movements ( e.g. , displacement , velocity , and acceleration ) and angular movements ( e.g. , angle , angular velocity , and angular acceleration ) could be obtained directly or indirectly . beside these", ", frequency domain parameters could be extracted from basic sensor measurements by techniques such as fft and wavelet [ 11 , 12 ] .", "for single triaxial accelerometer application , accelerations and derived angular parameters could be used as recognition features . fall detection algorithm design", "according to the recognition feature , fall detection algorithms are classified as threshold based and machine learning based . for threshold", "based method , threshold of recognition feature is set by the designer before application which makes the algorithm have rapid response and less resource consumption .", "but the choice of threshold needs both rigorous schemes and adequate experiments . for machine learning based design", ", the classification of fall and normal activities is available with the assistance of technologies such as support vector machine ( svm ) and neural network [ 14 , 15 ] .", "machine learning assistance may enhance system robustness to some extent , but its algorithm design is always high computing resource consumed which limits its application in wearable device . as the compact wearable device requires low power consumption and a single triaxial accelerometer could provide effective information , threshold based fall detection algorithm will be used in this system . algorithm used in this fall alarm system", "when a real fall happens , collision between human 's body and ground will produce obvious peak value at the sum acceleration a which has magnitude as \n ( 1)a = ax2+ay2+az2 , \n where ax , ay , and az present accelerometer measurements of three axes .", "the system uses the sum acceleration as the first step to distinguish high intensity movements from others .", "but normal motions such as jumping or sitting also produce peak values , which mean that additional detection features are required .", "as human 's motion has low acceleration , it is feasible to get gravity component in each axis by using a low pass filter . if gravity components could be separated before and after human 's fall , then it is possible to calculate the rotation angle of accelerometer coordinate in 3d space , which is also equivalent to the rotation angle of gravity vector relative to fixed coordinate .", "quaternion is an effective tool to describe rotation movement in human 's gait change which also includes falling .", "a quaternion could be described as \n ( 2)q = q0+q1i+q2j+q3k \n which has magnitude as \n ( 3)q = q02+q12+q22+q32 .", "unit quaternion which has magnitude q = 1 can be described as \n ( 4)q = cos2+sin2qxi+sin2qyj+sin2qzk. as shown in figure 3 , rotation angle of q equals . the rotation axis is orthogonal to the rotation plane and its direction is in accordance with right hand screw rule .", "qx , qy , and qz are three components of the unit vector which describes the orientation of the quaternion at the fixed coordinate .", "besides rotation movement , quaternion can also describe a vector in 3d space , such as the gravity vector g which could be described as a quaternion \n ( 5)g=0+gxi+gyj+gzk.gx , gy , and gz are three components of g which has quaternion magnitude as \n ( 6)g = gx2+gy2+gz2=g . a unit quaternion q is used to describe human 's falling movement , which can also be divided into three rotation quaternions q1", ", q2 , and q3 to simplify the calculation as shown in figure 4 . with the help of gravity vector information before and after human 's falling movement as shown in figure 3 , these three separated quaternions are all available . \n", "q \n 1 could be expressed as \n ( 7)q1=cos12+sin12sini+sin12cosj \n which has rotation angle and rotation axis information as \n ( 8)1=arctangbefore , zgbefore , x2+gbefore , y2,sin=gbefore , ygbefore , x2+gbefore , y2,cos=gbefore , xgbefore , x2+gbefore , y2 .", "quaternion q2 can be calculated as \n ( 9)q2=cos22+sin22k \n which has rotation angle as \n ( 10)2=arctan2gafter , ygafter , xarctan2gbefore , ygbefore , x .", "quaternion q3 is \n ( 11)q3=cos32+sin32sini+sin32cosj \n with rotation angle and rotation axis information as \n ( 12)3=arctangafter , zgafter , x2+gafter , y2,sin=gafter , ygafter , x2+gafter , y2,cos=gafter , xgafter , x2+gafter , y2 .", "then the rotation of the fall movement can be expressed by quaternion multiplication as \n ( 13)gafter = qgbeforeq=q3q2q1gbeforeq1q2q3 , \n where q=q0-q1i -- q2j -- q3k- is the conjugate quaternion of q. quaternion algebra is normally implemented based on basic matrix algebra .", "quaternion multiplication used above could be realized by matrix multiplication as \n ( 14)qgbefore = mq0gbefore , xgbefore , ygbefore , z = q0q1q2q3q1q0q3q2q2q3q0q1q3q2q1q00gbefore , xgbefore , ygbefore , z .", "the whole rotation quaternion can also be decomposed as \n ( 15)q = q3q2q1=mq3mq2q1.q1 , q2 , and q3 are all available based on gravity information before and after the falling movement . at last , it is possible to get four elements of quaternion q by the equation above and the rotation angle  could be calculated as \n ( 16)=2arctanq12+q22+q32q0 .", "when an object is falling , magnitude of  will approach 90 which is also a character different from most normal activities .", "the wearable device will be mounted on human 's waist at first to reflect the motion of human body closely , and the device will record gbefore while the elderly is standing still .", "there is no special requirement of the device orientation but only keep stationary during the wear .", "athreshold means the threshold of sum acceleration magnitude and tthreshold means the threshold of oscillation time duration after the break of athreshold . when measurements in three different axes have been acquired , the sum acceleration |a| will be calculated .", "when a real fall happens , sum acceleration will reach peak value of |a|  athreshold . in a real falling", ", the fluctuation of acceleration will stop in time duration tthreshold and then the sum acceleration is |a|  g as the elderly will lie on the ground . then the acceleration a is recorded as gafter .", ", the system will consider it as a fall if the rotation angle ||  90."], ["ti 's 16 bits mcu msp430f1611 is used to control the whole system and imply the detection algorithm .", "the measurement range of accelerometer could be set at 2  g , 4  g , 8  g , or 16 ", "g , and the maximal sampling rate is 3.2  khz . as human 's activities are normally at low frequency bands , 100  hz is a proper sampling rate for human fall detection .", "there is an inner digital filter in adxl345 which could weaken noise and reduce the burden of digital signal processing in mcu to some extent .", "the measurements will be sent through iic ( interintegrated circuit ) bus communication between the sensor and the mcu .", "sim908 can offer gps and gsm service on serial port communication with mcu , and it can also work in low power mode .", "each hardware component of the wearable device is working under low voltage and the detection algorithm does not need complex calculation resource , so the power consumption of the whole device is quite low .", "a 1200  mah , 3.7  v polymer lithium battery is quite enough to provide the need of the wearable device for a couple of days .", "hardware structure of the detection device is shown in figure 6 , and the pcb board prototype is shown in figure 7 .", "one of them is the software design in wearable device , and the other is in the caregiver 's handset .", "after initialization of the system , gbefore would be extracted from acceleration measurements by using a low pass filter when the elderly is standing .", "if a fall has been detected , the wearable device will locate the user and send alarm short message to caregivers immediately .", "if the user withdraws the alarm by pressing a button manually , the device will get back to fall detection state and a short message will be sent to inform the caregivers .", "there is a url ( universal resource locator ) which links to a web map in the alarm short message .", "the fall location information will be highlighted on a web map when the caregiver opens this link ."], ["system test of the fall detection system has been conducted based on the system design described above . the sampling rate of accelerometer is set at 100  hz and the measurement range is 16  g with a maximum precision of 4  mg .", "mcu will read raw measurements from sensor 's inner fifo and apply the detection algorithm .", "the test objects are three different volunteers at the ages of 23 , 42 , and 60 , respectively . based on analysis of these volunteers ' experiment data , athreshold and tthreshold", "are set as 2  g and 2  s , respectively . in order to get g", "when standing and lying after the fall , sum acceleration a which has magnitude between ( 1  0.3 )  g and ( 1 + 0.3 )  g will be considered as gbefore and gafter .", "considering that the tilt of the ground or the lying posture of the elderly may affect the rotation angle , rotation angle  between ( 90  30) and ( 90 + 30) will infer that the elderly has fallen .", "system test contains five kinds of activities of daily living ( i.e. , walking , jumping , squatting , sitting , and resting ) and four kinds of fallings ( i.e. , forward , backward , leftward , and rightward ) .", "each kind of motion has been repeated 20 times on each volunteer , and the detection results of the proposed algorithm and an acceleration threshold based algorithm   are listed in table 1 . the sensitivity and specificity [ 21 , 22 ] of the proposed system can be got from the test data in table 1 .", "test results of acceleration threshold based algorithm show lower sensitivity and specificity at 91.6% and 88.7% , respectively . \n", "figure 9 shows the accelerations in different kinds of fallings which all trigger the fall alarm correctly and the corresponding rotation angles are 91.9 , 100.5 , 104.7 , and 114.1. \n figure 10 shows acceleration during activity of lying down and resting which has rotation angle of 117.0 and similar waveform to falling , but the peak acceleration is much lower .", "they all do not have rotation about 90 ; even some of the peak values are quite high , so the fall alarm has not been triggered .", "an alarm sms ( short message service ) containing a map url has been received by the handset as shown in figure 12 when a fall has been detected . clicking", "the url will open a map in web browser on which the fall location will be displayed accurately as shown in figure 13 ."], ["this paper developed a fall detection system based on a single triaxial accelerometer based wearable device .", "there is no special requirement of the device 's mounting orientation because the algorithm does not claim the axes of accelerometer to be fixed strictly .", "the system has low power consumed hardware design and highly efficient algorithm which could extend the service time of the wearable device .", "as normal activity of resting also has similar rotation as falling , it may trigger fall alarm when the body hits ground heavily .", "so the choice of athreshold is quite important to distinguish falling from heavily lying activity .", "sufficient sample number collected from subjects with different age and gender will improve the reliability and robustness of the threshold . beside these , technologies such as svm and neural network are considerable to seek out a proper classification method based on the features used in this system ."]]}
{"article_id": "PMC2667587", "article_text": ["one radiologist retrospectively reviewed the medical records of the patients who received tfesi using the conventional approach at our department during a 1-year period , from june 2003 to may 2004 .", "this involved performing tfesi at the location of the exiting nerve root , regardless of the level of the nerve root compression .", "for example , tfesi for l5 radiculopathy was performed at the neural foramen of l5-s1 even when the l5 nerve root compression was in the paracentral or subarticular region at the level of the l4 - 5 disc .", "after a consensus meeting between three radiologists who were unaware of the clinical results after tfesi , we selected those cases treated by conventional tfesi with supra - adjacent nerve root impingement .", "the inclusion criteria were ( a ) the presence of lumbar radiculopathy , ( b ) nerve root compression in the paracentral or subarticular region at the level of the supra - adjacent intervertebral disc ( for example , at the l4 - 5 disc level for the l5 nerve root ) , ( c ) one level tfesi from l1 to s1 , ( d ) no prior therapeutic tfesi , ( e ) no prior surgery , and ( f ) clear identification of the nerve root compression by using a cross - sectional imaging study ( either computed tomography or magnetic resonance imaging ) .", "thirteen patients who met all the criteria were included and they were referred to as the conventional tfesi group .", "however , since june 2004 , we have used tfesi with the preganglionic approach at the supra - adjacent intervertebral disc ( for example , at the neural foramen of l4 - 5 for a l5 nerve root ) if the nerve root compression was at the level of the supra - adjacent intervertebral disc . using the same inclusion criteria", "as mentioned above , a total of 20 patients were consecutively enrolled and they were referred to as the preganglionic tfesi group .", "ttransforaminal epidural steroid injection using the conventional approach was conducted under biplane fluoroscopic guidance by two radiologists who were very experienced in spinal interventions .", "all the treatments were performed as outpatient procedures , and written informed consents were obtained from all the patients . with a patient lying in the prone position , the tube was rotated obliquely to ensure injection at the neural foramen .", "the goal of positioning was to allow a perpendicular needle tract toward the classic injection site underneath the pedicle , in the so - called \" safe triangle \" ( 5 , 7 ) .", "the safe triangle was defined by the pedicle superiorly , the lateral border of the vertebral body laterally , and the outer margin of the spinal nerve medially . after disinfecting the skin , local anesthetic", "was administered by using a 25-gauge needle . under fluoroscopic guidance , a 12-cm 22-gauge spinal needle", "was then advanced into the safe triangle . at the same time , a lateral view was obtained to verify that the anterior - posterior position of the needle tip was appropriate .", "the needle position was checked by biplane fluoroscopy , and this was followed by the injection of about 1 ml of contrast material ( omnipaque 300 [ iohexol , 300 mg of iodine per milliliter ] ; amersham health , princeton , nj ) .", "the posteroanterior and lateral spot radiographs were obtained to document the distribution of the contrast material .", "bupivacaine hydrochloride ( 0.5 ml , marcaine spinal 0.5% heavy ; astrazeneca , westborough , ma ) and 40 mg ( 1 ml ) of triamcinolon acetonide suspension ( tamcelon ; hanall , seoul , korea ) were slowly injected . in terms of tfesi with using the preganglionic approach ,", "the goal of positioning the needle tip was medial and inferior to that used in the conventional approach . in our department , we target injections just lateral to the pars interarticularis on the oblique view during the preganglionic approach ( fig .", "a fortnight after tfesi , the patients were followed - up at our outpatient department .", "this length of follow - up has been proposed in the literature and relates to the duration of the therapeutic effect of corticosteroid ( 7 ) . to check the effect of tfesi , all the patients", "were recommended not take any drugs or to participate in any physical therapy for their sciatica before this 2-week follow - up .", "the level and cause of nerve root compression on the ct or mr imaging and the level of radiculopathy were documented in the medical charts before performing tfesi .", "after retrospectively reviewing medical records , we classified the cause of radiculopathy as being due to a herniated intervertebral disc ( hivd ) or it was due to spinal stenosis .", "the patients ' demographic variables were also detailed at the initial clinical evaluation . for statistical analysis , we classified the patients ' ages into six age groups ; < 29-years - old , 30 - 39 , 40 - 49 , 50 - 59 , 60 - 69 and > 70 .", "the duration of radiculopathy was also treated as a potential predictive variable , and it was classified as acute or subacute , i.e. , < 6 months or chronic ( > 6 months ) ( 8) .", "the treatment outcome was assessed using a 5-point patient satisfaction scale , i.e. , 0 ( poor ) , 1 ( fair ) , 2 ( good ) , 3 ( very good ) , and 4 ( excellent ) , and by using a vas ( visual assessment scale ) that ranged from 0 to 100 .", "a successful outcome required a patient satisfaction score of 3 ( very good ) or 4 ( excellent ) , and a reduction on the vas of > 50% two weeks after the tfesi .", "the patients with a successful outcome were referred to as having received effective treatment and those patients without a successful outcome were referred to as having received ineffective treatment .", "logistic regression analysis was performed and the factors included were conventional or preganglionic tfesi , the cause of radiculopathy , the patients ' age and gender and the duration of radiculopathy .", "the mann - whitney u test was also used to evaluate for age differences between the two groups ( the conventional and preganglionic groups ) .", "spss , version 10.0 ( spss , chicago , il ) was used throughout the analysis .", "one radiologist retrospectively reviewed the medical records of the patients who received tfesi using the conventional approach at our department during a 1-year period , from june 2003 to may 2004 .", "this involved performing tfesi at the location of the exiting nerve root , regardless of the level of the nerve root compression .", "for example , tfesi for l5 radiculopathy was performed at the neural foramen of l5-s1 even when the l5 nerve root compression was in the paracentral or subarticular region at the level of the l4 - 5 disc .", "after a consensus meeting between three radiologists who were unaware of the clinical results after tfesi , we selected those cases treated by conventional tfesi with supra - adjacent nerve root impingement .", "the inclusion criteria were ( a ) the presence of lumbar radiculopathy , ( b ) nerve root compression in the paracentral or subarticular region at the level of the supra - adjacent intervertebral disc ( for example , at the l4 - 5 disc level for the l5 nerve root ) , ( c ) one level tfesi from l1 to s1 , ( d ) no prior therapeutic tfesi , ( e ) no prior surgery , and ( f ) clear identification of the nerve root compression by using a cross - sectional imaging study ( either computed tomography or magnetic resonance imaging ) .", "thirteen patients who met all the criteria were included and they were referred to as the conventional tfesi group .", "however , since june 2004 , we have used tfesi with the preganglionic approach at the supra - adjacent intervertebral disc ( for example , at the neural foramen of l4 - 5 for a l5 nerve root ) if the nerve root compression was at the level of the supra - adjacent intervertebral disc . using the same inclusion criteria", "as mentioned above , a total of 20 patients were consecutively enrolled and they were referred to as the preganglionic tfesi group .", "ttransforaminal epidural steroid injection using the conventional approach was conducted under biplane fluoroscopic guidance by two radiologists who were very experienced in spinal interventions .", "all the treatments were performed as outpatient procedures , and written informed consents were obtained from all the patients . with a patient lying in the prone position , the tube was rotated obliquely to ensure injection at the neural foramen . the goal of positioning was to allow a perpendicular needle tract toward the classic injection site underneath the pedicle , in the so - called \" safe triangle \" ( 5 , 7 ) .", "the safe triangle was defined by the pedicle superiorly , the lateral border of the vertebral body laterally , and the outer margin of the spinal nerve medially . after disinfecting the skin , local anesthetic", "was administered by using a 25-gauge needle . under fluoroscopic guidance , a 12-cm 22-gauge spinal needle", "was then advanced into the safe triangle . at the same time , a lateral view was obtained to verify that the anterior - posterior position of the needle tip was appropriate .", "the needle position was checked by biplane fluoroscopy , and this was followed by the injection of about 1 ml of contrast material ( omnipaque 300 [ iohexol , 300 mg of iodine per milliliter ] ; amersham health , princeton , nj ) . the posteroanterior and lateral spot radiographs were obtained to document the distribution of the contrast material .", "bupivacaine hydrochloride ( 0.5 ml , marcaine spinal 0.5% heavy ; astrazeneca , westborough , ma ) and 40 mg ( 1 ml ) of triamcinolon acetonide suspension ( tamcelon ; hanall , seoul , korea ) were slowly injected . in terms of tfesi with using the preganglionic approach ,", "the goal of positioning the needle tip was medial and inferior to that used in the conventional approach . in our department , we target injections just lateral to the pars interarticularis on the oblique view during the preganglionic approach ( fig .", "a fortnight after tfesi , the patients were followed - up at our outpatient department .", "this length of follow - up has been proposed in the literature and relates to the duration of the therapeutic effect of corticosteroid ( 7 ) . to check the effect of tfesi ,", "all the patients were recommended not take any drugs or to participate in any physical therapy for their sciatica before this 2-week follow - up .", "the level and cause of nerve root compression on the ct or mr imaging and the level of radiculopathy were documented in the medical charts before performing tfesi .", "after retrospectively reviewing medical records , we classified the cause of radiculopathy as being due to a herniated intervertebral disc ( hivd ) or it was due to spinal stenosis .", "the patients ' demographic variables were also detailed at the initial clinical evaluation . for statistical analysis , we classified the patients ' ages into six age groups ; < 29-years - old , 30 - 39 , 40 - 49 , 50 - 59 , 60 - 69 and > 70 .", "the duration of radiculopathy was also treated as a potential predictive variable , and it was classified as acute or subacute , i.e. , < 6 months or chronic ( > 6 months ) ( 8) . the effectiveness of tfesi was evaluated two weeks after the procedures .", "the treatment outcome was assessed using a 5-point patient satisfaction scale , i.e. , 0 ( poor ) , 1 ( fair ) , 2 ( good ) , 3 ( very good ) , and 4 ( excellent ) , and by using a vas ( visual assessment scale ) that ranged from 0 to 100 . a successful outcome required a patient satisfaction score of 3 ( very good ) or 4 ( excellent ) , and a reduction on the vas of > 50% two weeks after the tfesi .", "the patients with a successful outcome were referred to as having received effective treatment and those patients without a successful outcome were referred to as having received ineffective treatment .", "logistic regression analysis was performed and the factors included were conventional or preganglionic tfesi , the cause of radiculopathy , the patients ' age and gender and the duration of radiculopathy .", "the mann - whitney u test was also used to evaluate for age differences between the two groups ( the conventional and preganglionic groups ) .", "spss , version 10.0 ( spss , chicago , il ) was used throughout the analysis .", "the study subjects were 22 women and 11 men with a mean age of 55.1 years ( age range : 17 - 76 , standard deviation [ sd ] : 14.1 ) .", "the mean age was 53.9 years ( sd : 13.4 ) in the conventional tfesi group and it was was 55.8 years ( sd : 14.8 ) in the preganglionic tfesi group .", "of the 13 patients in the conventional tfesi group , nine showed effective treatment two weeks after tfesi ( 69.2% ) , and 18 of the 20 patients ( 90% ) in the preganglionic tfesi group showed effective treatment , and the difference in the two rates of effective treatment was of borderline significance ( p = 0.056 ; odds ratio 10.483 ) .", "the cause of radiculopathy , the age of the groups , gender and the duration of symptoms were not found to be statistically related to the effectivness of tfesi ( p > 0.05 ) .", "these results are summarized in tables 1 , 2 . in the conventional tfesi group , seven patients ( 53.8% ) had spinal stenosis and six patients ( 46.2% ) had hivd , and in the preganglionic tfesi group , 13 patients ( 65% ) had spinal stenosis and seven patients ( 35% ) had hivd . in the conventional tfesi group ,", "the radiculopathy was acute or subacute in eight patients ( 61.5% ) and chronic in five patients ( 38.5% ) ; in the preganglionic tfesi group , radiculopathy was acute or subacute in six patients ( 30% ) and it was chronic in 14 patients ( 70% ) . in the conventional tfesi group , tfesi at l5-s1 was performed in 10 patients and tfesi at l4 - 5 was performed in three patients ; in the preganglionic tfesi group , tfesi was performed at l5-s1 in five patients , at l4 - 5 in 14 patients and at l3 - 4 in one patient .", "lumbosacral radiculopathy is a common and costly medical disease , and its lifetime prevalence has been estimated to be 40 - 60% ( 9 ) . based on the recent concepts of pain generation in the sciatic condition , it 's believed that concomitant chemical irritation of the nerve root that 's caused by disk material is the decisive factor for the development of severe sciatica , rather than the pain being due to mechanical compression alone ( 10 - 16 ) . according to this concept , the local application of corticosteroids in the area of the compressed", "thus , percutaneous injection based therapies , including tfesi , are being used with increasing frequency to treat the radiculopathy resulting from a herniated disc or degenerative lumbar spinal stenosis ( 17 - 19 ) .", "in contrast to an interlaminar or a caudal epidural steroid injection , tfesi provides a low volume of concentrated medication to a selected nerve root .", "medical journals and texts often incorrectly refer to transforaminal epidural injections as \" selective epidural injections \" , \" selective nerve root blocks \" , or \" nerve root sleeve injections \" .", "however , the international spinal intervention society ( isis ) , has promoted the used of a nomenclature based on precise anatomic descriptors , and it recommends the term \" transforaminal \" ( 5 ) .", "gajraj ( 4 ) mentioned that to be selective , a nerve root block should be performed extraforaminally and distal to the division of the ventral and dorsal rami ; otherwise , the dorsal ramus and its innervated structures would also be anesthetized .", "therefore , they suggested that the therapeutic procedure be referred to as a ' transforaminal epidural steroid injection ' and that the diagnostic procedure should be referred to as a ' selective spinal nerve block ' or a ' selective ventral ramus block . ' in the case of tfesi with using the conventional approach , the spinal needle is positioned within the \" safe triangle \" ( bordering the pedicle , the exiting nerve root and the lateral border of the vertebral body ) , with the bevel placed below the inferior aspect of the pedicle ( 5 , 7 ) . using this conventional approach", ", the injectate typically bathes the epidural space around the dorsal root ganglion and then it descends along the distal aspect of the nerve root . ideally ,", "if there is no stenosis and the needle tip is appropriately positioned , the injectate may spread rostrally to the epidural portion of the preganglionic nerve root as well .", "however , for most cases of lumbosacral radiculopathy that are secondary to stenosis or a herniated disc , the site of impingement usually lies at the level of the supra - adjacent intervertebral disc , which is rostral to the conventional lumbar tfesi bevel position .", "in addition , one can not always guarantee a rostral spread of injectate to the epidural / preganglionic portion of the nerve root .", "thus , performing preganglionic tfesi at the supra - adjacent level may be a more effective method to deliver injectate to a target site .", "for the preganglionic approach , it is important that the injectate distributes itself predominantly in the epidural space at the disc level ( more inferior than that for the conventional approach ) , and that it does not excessively spread along the spinal nerve at the supra - adjacent level , which is not inflamed or compressed .", "lew et al . ( 6 ) inserted the needle into the inferior and anterior neural foramen for performing preganglionic tfesi , and we use a modification of this method .", "the landmark we use for needle insertion is just lateral to the pars interarticularis on the oblique view ; this is medial and inferior to that used for the conventional approach ( fig .", "first , it is not a controlled , randomized prospective study ; thus , it has the limitations of a retrospective study and so a randomized , prospective study is warranted .", "third , other factors that probably influenced the therapeutic effect were not included . in conclusion , preganglionic tfesi was found to have a better therapeutic effect on the radiculopathy caused by compression at the supra - adjacent level than did conventional tfesi , and this result had borderline statistical significance ."], "abstract_text": ["<S> objectivethe present study was undertaken to evaluate the effectiveness of transforaminal epidural steroid injection ( tfesi ) with using a preganglionic approach for treating lumbar radiculopathy when the nerve root compression was located at the level of the supra - adjacent intervertebral disc.materials and methodsthe medical records of the patients who received conventional tfesi at our department from june 2003 to may 2004 were retrospectively reviewed . </S>", "<S> tfesi was performed in a total of 13 cases at the level of the exiting nerve root , in which the nerve root compression was at the level of the supra - adjacent intervertebral disc ( the conventional tfesi group ) . since june 2004 </S>", "<S> , we have performed tfesi with using a preganglionic approach at the level of the supra - adjacent intervertebral disc ( for example , at the neural foramen of l4 - 5 for the l5 nerve root ) if the nerve root compression was at the level of the supra - adjacent intervertebral disc . using the inclusion criteria described above , 20 of these patients </S>", "<S> were also consecutively enrolled in our study ( the preganglionic tfesi group ) . </S>", "<S> the treatment outcome was assessed using a 5-point patient satisfaction scale and by using a vas ( visual assessment scale ) . </S>", "<S> a successful outcome required a patient satisfaction scale score of 3 ( very good ) or 4 ( excellent ) , and a reduction on the vas score of > 50% two weeks after performing tfesi . </S>", "<S> logistic regression analysis was also performed.resultsof the 13 patients in the conventional tfesi group , nine showed satisfactory improvement two weeks after tfesi ( 69.2% ) . </S>", "<S> however , in the preganglionic tfesi group , 18 of the 20 patients ( 90% ) showed satisfactory improvement . </S>", "<S> the difference between the two approaches in terms of tfesi effectiveness was of borderline significance ( p = 0.056 ; odds ratio : 10.483).conclusionwe conclude that preganglionic tfesi has the better therapeutic effect on radiculopathy caused by nerve root compression at the level of the supra - adjacent disc than does conventional tfesi , and the diffence between the two treatments had borderline statistical significance . </S>"], "labels": null, "section_names": ["MATERIALS AND METHODS", "Patients", "Technique", "Review of the Radiologic and Clinical Data", "Statistics", "RESULTS", "DISCUSSION"], "sections": [["one radiologist retrospectively reviewed the medical records of the patients who received tfesi using the conventional approach at our department during a 1-year period , from june 2003 to may 2004 .", "this involved performing tfesi at the location of the exiting nerve root , regardless of the level of the nerve root compression .", "for example , tfesi for l5 radiculopathy was performed at the neural foramen of l5-s1 even when the l5 nerve root compression was in the paracentral or subarticular region at the level of the l4 - 5 disc .", "after a consensus meeting between three radiologists who were unaware of the clinical results after tfesi , we selected those cases treated by conventional tfesi with supra - adjacent nerve root impingement .", "the inclusion criteria were ( a ) the presence of lumbar radiculopathy , ( b ) nerve root compression in the paracentral or subarticular region at the level of the supra - adjacent intervertebral disc ( for example , at the l4 - 5 disc level for the l5 nerve root ) , ( c ) one level tfesi from l1 to s1 , ( d ) no prior therapeutic tfesi , ( e ) no prior surgery , and ( f ) clear identification of the nerve root compression by using a cross - sectional imaging study ( either computed tomography or magnetic resonance imaging ) .", "thirteen patients who met all the criteria were included and they were referred to as the conventional tfesi group .", "however , since june 2004 , we have used tfesi with the preganglionic approach at the supra - adjacent intervertebral disc ( for example , at the neural foramen of l4 - 5 for a l5 nerve root ) if the nerve root compression was at the level of the supra - adjacent intervertebral disc . using the same inclusion criteria", "as mentioned above , a total of 20 patients were consecutively enrolled and they were referred to as the preganglionic tfesi group .", "ttransforaminal epidural steroid injection using the conventional approach was conducted under biplane fluoroscopic guidance by two radiologists who were very experienced in spinal interventions .", "all the treatments were performed as outpatient procedures , and written informed consents were obtained from all the patients . with a patient lying in the prone position , the tube was rotated obliquely to ensure injection at the neural foramen .", "the goal of positioning was to allow a perpendicular needle tract toward the classic injection site underneath the pedicle , in the so - called \" safe triangle \" ( 5 , 7 ) .", "the safe triangle was defined by the pedicle superiorly , the lateral border of the vertebral body laterally , and the outer margin of the spinal nerve medially . after disinfecting the skin , local anesthetic", "was administered by using a 25-gauge needle . under fluoroscopic guidance , a 12-cm 22-gauge spinal needle", "was then advanced into the safe triangle . at the same time , a lateral view was obtained to verify that the anterior - posterior position of the needle tip was appropriate .", "the needle position was checked by biplane fluoroscopy , and this was followed by the injection of about 1 ml of contrast material ( omnipaque 300 [ iohexol , 300 mg of iodine per milliliter ] ; amersham health , princeton , nj ) .", "the posteroanterior and lateral spot radiographs were obtained to document the distribution of the contrast material .", "bupivacaine hydrochloride ( 0.5 ml , marcaine spinal 0.5% heavy ; astrazeneca , westborough , ma ) and 40 mg ( 1 ml ) of triamcinolon acetonide suspension ( tamcelon ; hanall , seoul , korea ) were slowly injected . in terms of tfesi with using the preganglionic approach ,", "the goal of positioning the needle tip was medial and inferior to that used in the conventional approach . in our department , we target injections just lateral to the pars interarticularis on the oblique view during the preganglionic approach ( fig .", "a fortnight after tfesi , the patients were followed - up at our outpatient department .", "this length of follow - up has been proposed in the literature and relates to the duration of the therapeutic effect of corticosteroid ( 7 ) . to check the effect of tfesi , all the patients", "were recommended not take any drugs or to participate in any physical therapy for their sciatica before this 2-week follow - up .", "the level and cause of nerve root compression on the ct or mr imaging and the level of radiculopathy were documented in the medical charts before performing tfesi .", "after retrospectively reviewing medical records , we classified the cause of radiculopathy as being due to a herniated intervertebral disc ( hivd ) or it was due to spinal stenosis .", "the patients ' demographic variables were also detailed at the initial clinical evaluation . for statistical analysis , we classified the patients ' ages into six age groups ; < 29-years - old , 30 - 39 , 40 - 49 , 50 - 59 , 60 - 69 and > 70 .", "the duration of radiculopathy was also treated as a potential predictive variable , and it was classified as acute or subacute , i.e. , < 6 months or chronic ( > 6 months ) ( 8) .", "the treatment outcome was assessed using a 5-point patient satisfaction scale , i.e. , 0 ( poor ) , 1 ( fair ) , 2 ( good ) , 3 ( very good ) , and 4 ( excellent ) , and by using a vas ( visual assessment scale ) that ranged from 0 to 100 .", "a successful outcome required a patient satisfaction score of 3 ( very good ) or 4 ( excellent ) , and a reduction on the vas of > 50% two weeks after the tfesi .", "the patients with a successful outcome were referred to as having received effective treatment and those patients without a successful outcome were referred to as having received ineffective treatment .", "logistic regression analysis was performed and the factors included were conventional or preganglionic tfesi , the cause of radiculopathy , the patients ' age and gender and the duration of radiculopathy .", "the mann - whitney u test was also used to evaluate for age differences between the two groups ( the conventional and preganglionic groups ) .", "spss , version 10.0 ( spss , chicago , il ) was used throughout the analysis ."], ["one radiologist retrospectively reviewed the medical records of the patients who received tfesi using the conventional approach at our department during a 1-year period , from june 2003 to may 2004 .", "this involved performing tfesi at the location of the exiting nerve root , regardless of the level of the nerve root compression .", "for example , tfesi for l5 radiculopathy was performed at the neural foramen of l5-s1 even when the l5 nerve root compression was in the paracentral or subarticular region at the level of the l4 - 5 disc .", "after a consensus meeting between three radiologists who were unaware of the clinical results after tfesi , we selected those cases treated by conventional tfesi with supra - adjacent nerve root impingement .", "the inclusion criteria were ( a ) the presence of lumbar radiculopathy , ( b ) nerve root compression in the paracentral or subarticular region at the level of the supra - adjacent intervertebral disc ( for example , at the l4 - 5 disc level for the l5 nerve root ) , ( c ) one level tfesi from l1 to s1 , ( d ) no prior therapeutic tfesi , ( e ) no prior surgery , and ( f ) clear identification of the nerve root compression by using a cross - sectional imaging study ( either computed tomography or magnetic resonance imaging ) .", "thirteen patients who met all the criteria were included and they were referred to as the conventional tfesi group .", "however , since june 2004 , we have used tfesi with the preganglionic approach at the supra - adjacent intervertebral disc ( for example , at the neural foramen of l4 - 5 for a l5 nerve root ) if the nerve root compression was at the level of the supra - adjacent intervertebral disc . using the same inclusion criteria", "as mentioned above , a total of 20 patients were consecutively enrolled and they were referred to as the preganglionic tfesi group ."], ["ttransforaminal epidural steroid injection using the conventional approach was conducted under biplane fluoroscopic guidance by two radiologists who were very experienced in spinal interventions .", "all the treatments were performed as outpatient procedures , and written informed consents were obtained from all the patients . with a patient lying in the prone position , the tube was rotated obliquely to ensure injection at the neural foramen . the goal of positioning was to allow a perpendicular needle tract toward the classic injection site underneath the pedicle , in the so - called \" safe triangle \" ( 5 , 7 ) .", "the safe triangle was defined by the pedicle superiorly , the lateral border of the vertebral body laterally , and the outer margin of the spinal nerve medially . after disinfecting the skin , local anesthetic", "was administered by using a 25-gauge needle . under fluoroscopic guidance , a 12-cm 22-gauge spinal needle", "was then advanced into the safe triangle . at the same time , a lateral view was obtained to verify that the anterior - posterior position of the needle tip was appropriate .", "the needle position was checked by biplane fluoroscopy , and this was followed by the injection of about 1 ml of contrast material ( omnipaque 300 [ iohexol , 300 mg of iodine per milliliter ] ; amersham health , princeton , nj ) . the posteroanterior and lateral spot radiographs were obtained to document the distribution of the contrast material .", "bupivacaine hydrochloride ( 0.5 ml , marcaine spinal 0.5% heavy ; astrazeneca , westborough , ma ) and 40 mg ( 1 ml ) of triamcinolon acetonide suspension ( tamcelon ; hanall , seoul , korea ) were slowly injected . in terms of tfesi with using the preganglionic approach ,", "the goal of positioning the needle tip was medial and inferior to that used in the conventional approach . in our department , we target injections just lateral to the pars interarticularis on the oblique view during the preganglionic approach ( fig .", "a fortnight after tfesi , the patients were followed - up at our outpatient department .", "this length of follow - up has been proposed in the literature and relates to the duration of the therapeutic effect of corticosteroid ( 7 ) . to check the effect of tfesi ,", "all the patients were recommended not take any drugs or to participate in any physical therapy for their sciatica before this 2-week follow - up ."], ["the level and cause of nerve root compression on the ct or mr imaging and the level of radiculopathy were documented in the medical charts before performing tfesi .", "after retrospectively reviewing medical records , we classified the cause of radiculopathy as being due to a herniated intervertebral disc ( hivd ) or it was due to spinal stenosis .", "the patients ' demographic variables were also detailed at the initial clinical evaluation . for statistical analysis , we classified the patients ' ages into six age groups ; < 29-years - old , 30 - 39 , 40 - 49 , 50 - 59 , 60 - 69 and > 70 .", "the duration of radiculopathy was also treated as a potential predictive variable , and it was classified as acute or subacute , i.e. , < 6 months or chronic ( > 6 months ) ( 8) . the effectiveness of tfesi was evaluated two weeks after the procedures .", "the treatment outcome was assessed using a 5-point patient satisfaction scale , i.e. , 0 ( poor ) , 1 ( fair ) , 2 ( good ) , 3 ( very good ) , and 4 ( excellent ) , and by using a vas ( visual assessment scale ) that ranged from 0 to 100 . a successful outcome required a patient satisfaction score of 3 ( very good ) or 4 ( excellent ) , and a reduction on the vas of > 50% two weeks after the tfesi .", "the patients with a successful outcome were referred to as having received effective treatment and those patients without a successful outcome were referred to as having received ineffective treatment ."], ["logistic regression analysis was performed and the factors included were conventional or preganglionic tfesi , the cause of radiculopathy , the patients ' age and gender and the duration of radiculopathy .", "the mann - whitney u test was also used to evaluate for age differences between the two groups ( the conventional and preganglionic groups ) .", "spss , version 10.0 ( spss , chicago , il ) was used throughout the analysis ."], ["the study subjects were 22 women and 11 men with a mean age of 55.1 years ( age range : 17 - 76 , standard deviation [ sd ] : 14.1 ) .", "the mean age was 53.9 years ( sd : 13.4 ) in the conventional tfesi group and it was was 55.8 years ( sd : 14.8 ) in the preganglionic tfesi group .", "of the 13 patients in the conventional tfesi group , nine showed effective treatment two weeks after tfesi ( 69.2% ) , and 18 of the 20 patients ( 90% ) in the preganglionic tfesi group showed effective treatment , and the difference in the two rates of effective treatment was of borderline significance ( p = 0.056 ; odds ratio 10.483 ) .", "the cause of radiculopathy , the age of the groups , gender and the duration of symptoms were not found to be statistically related to the effectivness of tfesi ( p > 0.05 ) .", "these results are summarized in tables 1 , 2 . in the conventional tfesi group , seven patients ( 53.8% ) had spinal stenosis and six patients ( 46.2% ) had hivd , and in the preganglionic tfesi group , 13 patients ( 65% ) had spinal stenosis and seven patients ( 35% ) had hivd . in the conventional tfesi group ,", "the radiculopathy was acute or subacute in eight patients ( 61.5% ) and chronic in five patients ( 38.5% ) ; in the preganglionic tfesi group , radiculopathy was acute or subacute in six patients ( 30% ) and it was chronic in 14 patients ( 70% ) . in the conventional tfesi group , tfesi at l5-s1 was performed in 10 patients and tfesi at l4 - 5 was performed in three patients ; in the preganglionic tfesi group , tfesi was performed at l5-s1 in five patients , at l4 - 5 in 14 patients and at l3 - 4 in one patient ."], ["lumbosacral radiculopathy is a common and costly medical disease , and its lifetime prevalence has been estimated to be 40 - 60% ( 9 ) . based on the recent concepts of pain generation in the sciatic condition , it 's believed that concomitant chemical irritation of the nerve root that 's caused by disk material is the decisive factor for the development of severe sciatica , rather than the pain being due to mechanical compression alone ( 10 - 16 ) . according to this concept , the local application of corticosteroids in the area of the compressed", "thus , percutaneous injection based therapies , including tfesi , are being used with increasing frequency to treat the radiculopathy resulting from a herniated disc or degenerative lumbar spinal stenosis ( 17 - 19 ) .", "in contrast to an interlaminar or a caudal epidural steroid injection , tfesi provides a low volume of concentrated medication to a selected nerve root .", "medical journals and texts often incorrectly refer to transforaminal epidural injections as \" selective epidural injections \" , \" selective nerve root blocks \" , or \" nerve root sleeve injections \" .", "however , the international spinal intervention society ( isis ) , has promoted the used of a nomenclature based on precise anatomic descriptors , and it recommends the term \" transforaminal \" ( 5 ) .", "gajraj ( 4 ) mentioned that to be selective , a nerve root block should be performed extraforaminally and distal to the division of the ventral and dorsal rami ; otherwise , the dorsal ramus and its innervated structures would also be anesthetized .", "therefore , they suggested that the therapeutic procedure be referred to as a ' transforaminal epidural steroid injection ' and that the diagnostic procedure should be referred to as a ' selective spinal nerve block ' or a ' selective ventral ramus block . ' in the case of tfesi with using the conventional approach , the spinal needle is positioned within the \" safe triangle \" ( bordering the pedicle , the exiting nerve root and the lateral border of the vertebral body ) , with the bevel placed below the inferior aspect of the pedicle ( 5 , 7 ) . using this conventional approach", ", the injectate typically bathes the epidural space around the dorsal root ganglion and then it descends along the distal aspect of the nerve root . ideally ,", "if there is no stenosis and the needle tip is appropriately positioned , the injectate may spread rostrally to the epidural portion of the preganglionic nerve root as well .", "however , for most cases of lumbosacral radiculopathy that are secondary to stenosis or a herniated disc , the site of impingement usually lies at the level of the supra - adjacent intervertebral disc , which is rostral to the conventional lumbar tfesi bevel position .", "in addition , one can not always guarantee a rostral spread of injectate to the epidural / preganglionic portion of the nerve root .", "thus , performing preganglionic tfesi at the supra - adjacent level may be a more effective method to deliver injectate to a target site .", "for the preganglionic approach , it is important that the injectate distributes itself predominantly in the epidural space at the disc level ( more inferior than that for the conventional approach ) , and that it does not excessively spread along the spinal nerve at the supra - adjacent level , which is not inflamed or compressed .", "lew et al . ( 6 ) inserted the needle into the inferior and anterior neural foramen for performing preganglionic tfesi , and we use a modification of this method .", "the landmark we use for needle insertion is just lateral to the pars interarticularis on the oblique view ; this is medial and inferior to that used for the conventional approach ( fig .", "first , it is not a controlled , randomized prospective study ; thus , it has the limitations of a retrospective study and so a randomized , prospective study is warranted .", "third , other factors that probably influenced the therapeutic effect were not included . in conclusion , preganglionic tfesi was found to have a better therapeutic effect on the radiculopathy caused by compression at the supra - adjacent level than did conventional tfesi , and this result had borderline statistical significance ."]]}
{"article_id": "PMC5098230", "article_text": ["one of the key challenges \n in the development of novel materials \n is the ability to tune and control their macroscopic physical and \n chemical properties on a molecular level by external stimuli .", "such a degree of control is challenging but crucial for a wide range \n of applications ranging from catalysis to pharmacological and medical \n applications .", "in photoactive materials light absorbed by chromophores can be converted \n into directed functionality , typically by means of photoinduced isomerization \n of nitrogen or carbon containing double bonds and pericylic reactions .", "indeed , e  z isomerization \n results in large amplitude changes in structure as well as chemical \n and physical properties .", "molecular motors based on overcrowded alkenes are particularly \n attractive due to the synthetically tunable properties , such as thermal \n stability , absorption spectra , etc . , and", "therefore are readily applied \n to a wide range of functional systems . however , although the thermally \n activated step in the unidirectional rotary cycle is well understood \n and is now largely predictable , the photochemical properties and especially \n photochemical quantum yields are less well understood .", "hence both \n from a fundamental perspective and in achieving the goal of complete \n molecular design , gaining insight into the excited - state properties \n of light driven molecular - sized machines is essential .", "the overcrowded alkene - based molecular \n rotary motors are composed \n of a central carbon  carbon double bond ( the axle ) embedded \n in an intrinsically chiral environment .", "the unidirectional rotational \n cycle starts with a photoinduced e  z isomerization around the central c = c bond , generating \n a metastable isomer p * ( figure 1 ) , which relaxes thermally to the stable isomer p , which in \n the case of a symmetric stator unit is identical to i. this isomer \n marks half of the rotation cycle and is the starting point for a second \n photoinduced isomerization step that leads again to a metastable isomer . \n", "thermally activated conformational isomerization of this isomer brings \n the system back to its initial state .", "overall , the rotor part has \n then performed a complete rotation with respect to the stator unit \n of the molecular motor .", "although the rotation includes both \n photochemical and thermal steps \n that involve different parts of the motor , the structural evolution \n is largely governed by the excited - state properties of the central \n c = c group , which , in turn , are dictated by the potential energy \n surfaces of the excited states that are accessed upon electronic excitation . \n despite the large number of theoretical and experimental studies that have focused on characterizing \n this process , there are several key questions outstanding including \n identification of the main structural coordinates and the role of \n various electronic states involved in the excited - state dynamics . \n", "time - resolved studies are essential to elucidate these dynamics but \n have thus far focused on probing the change in the electronic structure \n after photoexcitation through uv / vis absorption and fluorescence studies .", "these techniques offer \n a high time resolution but provide only indirect information as to \n how the structure evolves spatially over time . in the present contribution \n we show that this information can be obtained through a combination \n of picosecond time - resolved vibrational spectroscopy of the electronically \n excited states together with quantum - mechanical calculations .", "the setup for the time - resolved infrared measurements has been \n described in detail previously .", "mid - ir \n probe pulses were generated using an amplified ti : sapphire system \n ( spectra - physics hurricane , 600 j , repetition rate 1 khz ) pumping \n a commercial bbo - based opa ( spectra - physics opa-800c ) , the signal \n and idler output of which were difference - frequency - mixed in aggas2 .", "uv pump pulses ( 400 nm , 1 j ) were generated \n by doubling part of the output of the ti : sapphire laser .", "the diameter \n of the uv pump focus was 180200 m ; that of \n the ir probe focus , 150 m . the sample cell with caf2 windows spaced by 500 m was placed in the ir focus . \n to avoid accumulation of the thermally unstable conformer p * during \n the measurements ,", "the pump  probe delay time was scanned by mechanically \n adjusting the beam path of the uv pump .", "the temporal resolution of \n 200 fs has been determined from the full width at half - maximum of \n the pump probe cross - correlation function .", "the pump pulse was chopped \n at 500 hz , and transient spectra were obtained by subtracting nonpumped \n absorption spectra from the pumped absorption spectra , both of which \n were recorded by spectrally dispersing the probe pulses using an oriel \n ms260i spectrograph and measuring the frequency - dependent ir intensity \n using a 2  32 hgcdte detector array ( infrared associates ) .", "compound \n i was available from earlier studies ( see ref ( 44 ) ) . in all experiments", "we \n investigate a 10 mm solution of compound i in deuterated cyclohexane \n ( aldrich , 99.6 atom% d ) .", "for the investigation of the ground - state \n conformers ( including \n vertical excitation energies and forces acting at the franck ", "condon \n point ) quantum mechanical calculations were performed using the turbomole \n suite of programs .", "geometry optimization \n and normal - mode analysis were performed by ( time - dependent ) density \n functional theory ( ( td)-dft ) using the hybrid functional b3lyp . for the geometry optimization the d3 correction \n method of grimme et al .", "the cc - pvdz or aug - cc - pvdz basis \n sets were employed , respectively .", "post - hartree  fock calculations \n were performed on the basis of spin - component scaled second - order \n mller  plesset perturbation theory and the approximate coupled - cluster singles - and - doubles \n model cc2 .", "all perturbation method calculations were performed using \n the resolution of identity ( ri ) approximation and the efficient ricc2 module implemented \n in the turbomole package together with the standard auxiliary basis \n sets .", "the gaussian09 program package was employed to study the excited - state properties \n in more detail and in particular to optimize the structure of the \n dark - state species .", "geometry optimization and normal - mode analysis \n were performed in the framework of td - dft at the b3lyp / cc - pvdz level .", "the structural evolution from the \n initial state i to isomer p * \n ( figure 1 ) was considered first in terms of the spectral properties \n of the initial state and the first metastable product ( p * ) .", "the steady - state \n uv / vis absorption spectra of the two isomers are distinct , reflecting \n a change in the conjugated  system ( figure 1a ) .", "the ftir spectra of the two isomers ( figure 1c ) show that the \n carbon  carbon stretch and ring vibrations couple to a large \n degree as a consequence of the extended  system ; however , dft \n calculations enable us to assign the bands in the 15501650 \n cm range as being mainly associated with \n modes involving the central c = c double bond ( supporting information ) .", "the metastable isomer p * shows a \n shift of these modes to lower frequencies confirming a decrease in \n bond strength .", "comparison of the optimized geometries of the two isomers \n ( i ( p * ) , cc bond length 1.369 ( 1.377 )  ; cc = cc dihedral \n angle 14.1 ( 29.5 ) ) evidence the corresponding lower  \n bond character as well .", "unidirectional motion of the unidirectional \n motor starting from \n the thermally stable conformer ( i ) via the product of the photoinitiated \n step ( p * ) to the thermally stable conformer ( p ) ( optimized geometries \n on the b3lyp+d / aug - cc - pvdz level ) : ( a ) experimental and calculated \n ( light color and dotted line ) uv  vis spectra of conformer i \n and p * ; ( b ) molecular orbitals of the initial conformer ; ( c ) experimental \n and calculated ir spectra ( b3lyp+d / aug - cc - pvdz ) of the initial state \n ( blue ) and final state ( red ) . in both the measured uv / vis absorption \n and ftir spectra at the photostationary state ( pss ) , i and p * are \n present in a 25:75 ratio .", "time - resolved vibrational spectroscopy was employed to achieve the required temporal resolution as well \n as structural sensitivity to elucidate the structural evolution that \n follows photoexcitation with a short uv - pump pulse ( center wavelength \n 400 nm ) .", "the resulting structural evolution was followed by time - resolved \n differential absorption spectra in cyclohexane - d12 with a time resolution of 200 fs ( figure 2 ) .", "the transient ir spectra show , as expected , \n pronounced differences between the ir absorption spectrum of the molecule \n in the initial and excited states .", "a first point of note is that the \n spectrum does not evolve further after ca .", "30 ps , and the difference \n spectrum corresponds perfectly with the difference spectrum obtained \n upon continuous wave excitation ( figure 1 ) ; i.e. , the metastable isomer p * has formed \n fully within the 30 ps after excitation .", "ftir and time - resolved \n infrared spectra of the molecular motor \n in cyclohexane - d12 : ( a ) ftir spectra of \n the thermal stable conformer ( initial , solvent corrected , top ) , uv - ump / ir - probe \n transient infrared spectra ( uv = 400 nm ) ( middle ) , \n and difference ftir spectra between the two ground - state conformers \n of the molecular motor ; ( b ) kinetics of selected bands and corresponding \n results of the global fit ; ( c ) species associated spectra ( sas ) of \n species 1 and calculated ir spectra ( top ) and sas of the pss ( sp3 ) \n together with ftir spectra ( bottom ) .", "negative bands indicate ground - state \n bleaching , and positive bands , species created after excitation . the time - resolved ir spectra obtained \n within the first 30 ps exhibit \n several remarkable features .", "the first and most striking is a broad \n and unstructured absorption over the entire mid - ir range ( 12003000 \n cm ) .", "this absorption , which decays rapidly , is \n particularly prominent at early time delays ( < 5 ps ) .", "individual \n absorption bands with remarkably high integrated intensities in the \n 14001600 cm range are superimposed on \n the broad spectral feature .", "notably , these bands are significantly \n broader than the bleached bands in the transient spectra and bands \n in the ftir spectra .", "the solvent response and the dependence of the \n uv pump intensity ( supporting information ) confirm that these features are intrinsic to the compound and are \n not due to solvent or two - photon processes .", "the width and intensity \n of the broad excited - state absorption bands \n excludes that they arise from vibrational transitions .", "indeed , the \n absorbance is closer to that expected for electronic transitions , \n albeit these typically occur at much higher energies ( in the uv / vis \n range ) .", "the initial state of the compound \n ( i ) shows an absorption band at 390 nm ( figure 1 ) , which is reproduced well by b3lyp / cc - pvdz \n calculations in the gas phase ( 403 nm ; f = 0.428 ) . \n", "the s1  s0 transition has predominantly \n homo  lumo character ( figure 1 ) .", "in contrast to previous assignments , however , we find that this electronic transition \n is not localized on the stator but involves to a significant extent \n excitation from the bonding  orbital of the central double \n bond to the antibonding  * orbital ( figure 1 ) . as a consequence", ", the double bond character \n of this bond is reduced as well as the barrier to rotation around \n this bond . in line with calculations on ethylene and its derivatives , \n", "we find a dark state ( s2 ) associated with the homo1 \n  lumo excitation at slightly higher excitation energies that \n is not directly accessible from the ground state ( figure 1 , table 1 ) . because the homo1 orbital is localized \n on the aromatic stator unit , s2 has a significantly larger \n dipole moment than s1 ( 4.3 vs 2.2 d at the scs - cc2/cc - pvdz \n level ) .", "it should be emphasized that the description of the lower - lying \n electronic states in the compound studied here is thus entirely different \n from ethylene and its derivatives where , as discussed by salem and \n others , for torsion angles of around 90 the two low - lying electronic \n states have either a biradical or zwitterionic character depending \n on the occupation of the two central 2p - orbitals and require multireference \n methods for a proper description . in the present study , in contrast , \n we investigate the dynamics occurring at small torsional angles ( vide infra ) for which the employed single - reference methods \n not only have been validated .", "in addition \n we employ the results of multireference calculations on smaller motor \n molecules as reported by liu and morokuma to make a further comparison with our experimental results .", "for s0 the optimized \n structure on the b3lyp+d / aug - cc - pvdz level was used to calculate the \n vertical excitation energies . for vertical excitation", "they thus strongly suggest that the broad \n and structureless absorption feature observed in the time - resolved \n ir spectra is associated with the electronic s1  \n s2 transition .", "the presence of two close - lying states also \n explains why the induced absorption bands have such high intensities . \n", "our calculations show that the energy gap between the two states strongly \n depends on structural parameters such as the c = c bond length \n that are involved in the 14001600 cm normal \n modes . for these modes", "one therefore expects that their transition \n moments will be enhanced by vibronic coupling as is indeed observed . \n", "the conclusion that the excited - state dynamics of the molecular rotor \n should be described in a multistate model is further supported by \n recent multireference calculations on smaller rotor molecules that \n also find very small energy differences between the s1 and \n s2 states .", "the excited - state \n dynamics were elucidated further using a global \n analysis of the transient spectra , which shows that the spectra can \n be described well with two exponential decay terms with time constants \n of 1.53  0.03 and 12  1 ps .", "we note that our experiments \n have a time resolution of 150200 fs and therefore do not allow \n observation and confirmation of the ultrafast dynamics reported previously \n in fluorescence - upconversion studies .", "species - associated spectra ( sas ) ( supplementary \n figure 2 ) were derived by using a two - step sequential kinetic \n model .", "the sas of the first species shows an overall background \n signal \n with broad and intense features superimposed on it that indicate vibronic \n coupling .", "furthermore , the increase ( decrease ) of absorbance in the \n 1550 ( 1600 ) cm region indicates a substantial \n change in bond character of the central double bond ( figure 2c ) .", "this spectrum was related \n to the structure of this excited - state species using excited - state \n geometry optimizations .", " \n state without any structural constrains associated with the homo1 \n  lumo excitation leads to a stable minimum that can be reached \n from the franck  condon excited geometry by relatively small \n structural changes ( supplementary figure 4 ) , and an excited - state ir absorption spectrum that is in good agreement \n with the sas of species 1 ( figure 2 ) .", "it is notable that for a proper comparison with \n the experimental spectrum , the theoretically predicted stick spectrum \n needs to be convolved with 20 cm gaussian line \n shapes , suggesting that in the  dark  state a wide range \n of structures is sampled .", "our experiments and calculations thus demonstrate \n that excitation and excited - state decay involve two different electronic \n states . in earlier studies , time - resolved fluorescence and visible \n absorption data were interpreted in terms of a decay from a single , \n isolated electronic state decaying to the ground state after structural \n relaxation in the excited state .", "the present data show that the s1 state actually decays to an intermediate , dark electronic \n state , and from there to the ground state . in the sas of the \n second species ( supplementary \n figure", "2 ) the broad background is absent , and bands have widths \n comparable to those in the s0 state .", "the absence of the \n broad background indicates that this species is no longer in an electronically \n excited state but back in the ( vibrationally hot ) electronic ground \n state .", "indeed , the vibrational activity observed in the 1550 cm region follows closely the ir absorption spectrum \n of p*. in addition , repopulation of the original state ( i ) is observed \n also ( i.e. the quantum yield for isomerization is less than unity ) , and the sas of the second species contains contributions \n from both vibrationally hot ground - state i and metastable p * isomers . \n", "vibrational cooling of these isomers leads to the sas of the final \n species , which is in excellent agreement with the steady - state ftir \n difference spectrum of room - temperature i and p*. the present \n experimental and theoretical data allow a complete \n picture of the mechanism by which the molecular rotary motor undergoes \n isomerization after electronic excitation ( figure 3 ) .", "photon absorption brings the motor to \n a  bright  state from which it decays on an ultrafast \n time scale and within the time resolution of the present experiments \n to a different electronically excited  dark  state . \n in line with previous calculations on similar systems ,", "our calculations \n indicate that this is a barrierless process proceeding via a conical \n intersection .", "previously , it was assumed \n that structural evolution on the homo  lumo potential energy \n surface is responsible for the observed ultrafast decay of the fluorescence , \n and ( following commonly accepted models on photoisomerization of carbon ", "carbon \n double bonds ) that it primarily proceeds along torsional and pyramidalization \n coordinates . the present study , in contrast , leads to the conclusion \n that the fluorescence decay is mediated by the passage through the \n conical intersection of the homo  lumo and homo1  \n lumo potential energy surfaces . inspection of the structural changes \n that occur upon geometry relaxation to the minimum on the homo1 \n  lumo potential energy surface show that the central c = c \n bond length increases from 1.381 to 1.461  and that only relatively \n small changes occur in the torsional and pyramidalization coordinates . \n in combination with the forces that act on the molecule at the franck ", "condon \n excited  bright  state ( supplementary \n figure 3 ) , this suggests that motion along the c = c stretch \n coordinate needs to be taken into account as well for understanding \n the decay of the fluorescence .", "proposed multistate dynamics of the photoinduced \n unidirectional \n rotation of the molecular motor starting from conformer i to conformer \n p * ( projected into one dimension ) : dotted arrow , ultrafast process \n reported in previous work ; fast ( red arrow ) and slow process ( blue \n arrow ) observed in the present time - resolved infrared study . ( a ) calculated \n energy profile along the torsion coordinate showing how the energies \n of the electronic states change as the molecules evolves to conformer \n p*. ( b ) schematic representation of the proposed model .", "hence , it can be concluded that \n internal conversion to the electronic \n ground state takes place from the  dark  state .", "the \n decay rate for this process indicates that it is mediated by conical \n intersections between the two states as has indeed been proposed in \n previous theoretical studies .", "these studies \n concluded , however , as well that to reach these intersections a barrier \n needs to be overcome that results from the steric repulsion of the \n methyl group of the rotor unit with the hydrogen of the stator unit . \n", "previous time - resolved fluorescence measurements reported an ultrafast \n damping mode at 113 cm that was speculated to \n arise from motion along a coordinate involving inversion / pyramidalization \n although no direct support could be found for such an assignment .", "interestingly , our calculations find in the \n  dark  state a mode at 119 cm ( b3lyp / cc - pvdz ; \n not scaled , supporting information ) that \n involves motion of the methyl group with respect to the hydrogen of \n the stator unit .", "one might therefore wonder to what extent this mode \n is related to the oscillatory behavior of the fluorescence .", "our observed \n s2 decay constant of 1.5 ps matches the slow component \n of the overall fluorescence decay ( 0.91.5 ps ) .", "our calculation of the excitation energies of \n the  bright  and  dark  states along the \n 119 cm normal mode did not show a conical intersection \n between the two states , supporting the previous explanation of the \n oscillations observed in the fluorescence measurements by the movement of wave packets and suggesting \n a scenario in which a coherent oscillation in the dark s2 state periodically partly repopulates the fluorescent s1 state .", "however , our experimental methods do not provide the time \n resolution to confirm this .", "the photodynamics \n of a unidirectional molecular motor has been \n studied by time - resolved ir spectroscopy in which both vibrational \n and electronic structures are probed . in combination with quantum \n mechanical calculations , the high structural sensitivity and real - time \n character of the technique allows us to understand fundamental aspects \n of the operation mechanism .", "the results show that conversion of photon \n energy into directed motion proceeds on the potential energy surfaces \n of two different electronic states , which is the more important as \n it implies that the conical intersection between the  dark  \n electronically excited state and the ground state determines the efficiency \n of the motor . to rationally tune", "the performance of photoinitiated \n unidirectional rotation thus involves a multicoupled - state optimization \n of nuclear motion ."], "abstract_text": ["<S> controlling the excited - state properties \n of light driven molecular \n machines is crucial to achieving high efficiency and directed functionality . \n a key challenge in achieving control lies in unravelling the complex \n photodynamics and especially in identifying the role played by dark \n states . </S>", "<S> here we use the structure sensitivity and high time resolution \n of uv - pump / ir - probe spectroscopy to build a detailed and comprehensive \n model of the structural evolution of light driven molecular rotors . \n </S>", "<S> the photodynamics of these chiral overcrowded alkene derivatives are \n determined by two close - lying excited electronic states . </S>", "<S> the potential \n energy landscape of these  bright  and  dark  \n </S>", "<S> states gives rise to a broad excited - state electronic absorption band \n over the entire mid - ir range that is probed for the first time and \n modeled by quantum mechanical calculations . </S>", "<S> the transient ir vibrational \n fingerprints observed in our studies allow for an unambiguous identification \n of the identity of the  dark  electronic excited state \n from which the photon s energy is converted into motion , and \n thereby pave the way for tuning the quantum yield of future molecular \n rotors based on this structural motif . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULT AND DISCUSSION", "SUMMARY AND CONCLUSION"], "sections": [["one of the key challenges \n in the development of novel materials \n is the ability to tune and control their macroscopic physical and \n chemical properties on a molecular level by external stimuli .", "such a degree of control is challenging but crucial for a wide range \n of applications ranging from catalysis to pharmacological and medical \n applications .", "in photoactive materials light absorbed by chromophores can be converted \n into directed functionality , typically by means of photoinduced isomerization \n of nitrogen or carbon containing double bonds and pericylic reactions .", "indeed , e  z isomerization \n results in large amplitude changes in structure as well as chemical \n and physical properties .", "molecular motors based on overcrowded alkenes are particularly \n attractive due to the synthetically tunable properties , such as thermal \n stability , absorption spectra , etc . , and", "therefore are readily applied \n to a wide range of functional systems . however , although the thermally \n activated step in the unidirectional rotary cycle is well understood \n and is now largely predictable , the photochemical properties and especially \n photochemical quantum yields are less well understood .", "hence both \n from a fundamental perspective and in achieving the goal of complete \n molecular design , gaining insight into the excited - state properties \n of light driven molecular - sized machines is essential .", "the overcrowded alkene - based molecular \n rotary motors are composed \n of a central carbon  carbon double bond ( the axle ) embedded \n in an intrinsically chiral environment .", "the unidirectional rotational \n cycle starts with a photoinduced e  z isomerization around the central c = c bond , generating \n a metastable isomer p * ( figure 1 ) , which relaxes thermally to the stable isomer p , which in \n the case of a symmetric stator unit is identical to i. this isomer \n marks half of the rotation cycle and is the starting point for a second \n photoinduced isomerization step that leads again to a metastable isomer . \n", "thermally activated conformational isomerization of this isomer brings \n the system back to its initial state .", "overall , the rotor part has \n then performed a complete rotation with respect to the stator unit \n of the molecular motor .", "although the rotation includes both \n photochemical and thermal steps \n that involve different parts of the motor , the structural evolution \n is largely governed by the excited - state properties of the central \n c = c group , which , in turn , are dictated by the potential energy \n surfaces of the excited states that are accessed upon electronic excitation . \n despite the large number of theoretical and experimental studies that have focused on characterizing \n this process , there are several key questions outstanding including \n identification of the main structural coordinates and the role of \n various electronic states involved in the excited - state dynamics . \n", "time - resolved studies are essential to elucidate these dynamics but \n have thus far focused on probing the change in the electronic structure \n after photoexcitation through uv / vis absorption and fluorescence studies .", "these techniques offer \n a high time resolution but provide only indirect information as to \n how the structure evolves spatially over time . in the present contribution \n we show that this information can be obtained through a combination \n of picosecond time - resolved vibrational spectroscopy of the electronically \n excited states together with quantum - mechanical calculations ."], ["the setup for the time - resolved infrared measurements has been \n described in detail previously .", "mid - ir \n probe pulses were generated using an amplified ti : sapphire system \n ( spectra - physics hurricane , 600 j , repetition rate 1 khz ) pumping \n a commercial bbo - based opa ( spectra - physics opa-800c ) , the signal \n and idler output of which were difference - frequency - mixed in aggas2 .", "uv pump pulses ( 400 nm , 1 j ) were generated \n by doubling part of the output of the ti : sapphire laser .", "the diameter \n of the uv pump focus was 180200 m ; that of \n the ir probe focus , 150 m . the sample cell with caf2 windows spaced by 500 m was placed in the ir focus . \n to avoid accumulation of the thermally unstable conformer p * during \n the measurements ,", "the pump  probe delay time was scanned by mechanically \n adjusting the beam path of the uv pump .", "the temporal resolution of \n 200 fs has been determined from the full width at half - maximum of \n the pump probe cross - correlation function .", "the pump pulse was chopped \n at 500 hz , and transient spectra were obtained by subtracting nonpumped \n absorption spectra from the pumped absorption spectra , both of which \n were recorded by spectrally dispersing the probe pulses using an oriel \n ms260i spectrograph and measuring the frequency - dependent ir intensity \n using a 2  32 hgcdte detector array ( infrared associates ) .", "compound \n i was available from earlier studies ( see ref ( 44 ) ) . in all experiments", "we \n investigate a 10 mm solution of compound i in deuterated cyclohexane \n ( aldrich , 99.6 atom% d ) .", "for the investigation of the ground - state \n conformers ( including \n vertical excitation energies and forces acting at the franck ", "condon \n point ) quantum mechanical calculations were performed using the turbomole \n suite of programs .", "geometry optimization \n and normal - mode analysis were performed by ( time - dependent ) density \n functional theory ( ( td)-dft ) using the hybrid functional b3lyp . for the geometry optimization the d3 correction \n method of grimme et al .", "the cc - pvdz or aug - cc - pvdz basis \n sets were employed , respectively .", "post - hartree  fock calculations \n were performed on the basis of spin - component scaled second - order \n mller  plesset perturbation theory and the approximate coupled - cluster singles - and - doubles \n model cc2 .", "all perturbation method calculations were performed using \n the resolution of identity ( ri ) approximation and the efficient ricc2 module implemented \n in the turbomole package together with the standard auxiliary basis \n sets .", "the gaussian09 program package was employed to study the excited - state properties \n in more detail and in particular to optimize the structure of the \n dark - state species .", "geometry optimization and normal - mode analysis \n were performed in the framework of td - dft at the b3lyp / cc - pvdz level ."], ["the structural evolution from the \n initial state i to isomer p * \n ( figure 1 ) was considered first in terms of the spectral properties \n of the initial state and the first metastable product ( p * ) .", "the steady - state \n uv / vis absorption spectra of the two isomers are distinct , reflecting \n a change in the conjugated  system ( figure 1a ) .", "the ftir spectra of the two isomers ( figure 1c ) show that the \n carbon  carbon stretch and ring vibrations couple to a large \n degree as a consequence of the extended  system ; however , dft \n calculations enable us to assign the bands in the 15501650 \n cm range as being mainly associated with \n modes involving the central c = c double bond ( supporting information ) .", "the metastable isomer p * shows a \n shift of these modes to lower frequencies confirming a decrease in \n bond strength .", "comparison of the optimized geometries of the two isomers \n ( i ( p * ) , cc bond length 1.369 ( 1.377 )  ; cc = cc dihedral \n angle 14.1 ( 29.5 ) ) evidence the corresponding lower  \n bond character as well .", "unidirectional motion of the unidirectional \n motor starting from \n the thermally stable conformer ( i ) via the product of the photoinitiated \n step ( p * ) to the thermally stable conformer ( p ) ( optimized geometries \n on the b3lyp+d / aug - cc - pvdz level ) : ( a ) experimental and calculated \n ( light color and dotted line ) uv  vis spectra of conformer i \n and p * ; ( b ) molecular orbitals of the initial conformer ; ( c ) experimental \n and calculated ir spectra ( b3lyp+d / aug - cc - pvdz ) of the initial state \n ( blue ) and final state ( red ) . in both the measured uv / vis absorption \n and ftir spectra at the photostationary state ( pss ) , i and p * are \n present in a 25:75 ratio .", "time - resolved vibrational spectroscopy was employed to achieve the required temporal resolution as well \n as structural sensitivity to elucidate the structural evolution that \n follows photoexcitation with a short uv - pump pulse ( center wavelength \n 400 nm ) .", "the resulting structural evolution was followed by time - resolved \n differential absorption spectra in cyclohexane - d12 with a time resolution of 200 fs ( figure 2 ) .", "the transient ir spectra show , as expected , \n pronounced differences between the ir absorption spectrum of the molecule \n in the initial and excited states .", "a first point of note is that the \n spectrum does not evolve further after ca .", "30 ps , and the difference \n spectrum corresponds perfectly with the difference spectrum obtained \n upon continuous wave excitation ( figure 1 ) ; i.e. , the metastable isomer p * has formed \n fully within the 30 ps after excitation .", "ftir and time - resolved \n infrared spectra of the molecular motor \n in cyclohexane - d12 : ( a ) ftir spectra of \n the thermal stable conformer ( initial , solvent corrected , top ) , uv - ump / ir - probe \n transient infrared spectra ( uv = 400 nm ) ( middle ) , \n and difference ftir spectra between the two ground - state conformers \n of the molecular motor ; ( b ) kinetics of selected bands and corresponding \n results of the global fit ; ( c ) species associated spectra ( sas ) of \n species 1 and calculated ir spectra ( top ) and sas of the pss ( sp3 ) \n together with ftir spectra ( bottom ) .", "negative bands indicate ground - state \n bleaching , and positive bands , species created after excitation . the time - resolved ir spectra obtained \n within the first 30 ps exhibit \n several remarkable features .", "the first and most striking is a broad \n and unstructured absorption over the entire mid - ir range ( 12003000 \n cm ) .", "this absorption , which decays rapidly , is \n particularly prominent at early time delays ( < 5 ps ) .", "individual \n absorption bands with remarkably high integrated intensities in the \n 14001600 cm range are superimposed on \n the broad spectral feature .", "notably , these bands are significantly \n broader than the bleached bands in the transient spectra and bands \n in the ftir spectra .", "the solvent response and the dependence of the \n uv pump intensity ( supporting information ) confirm that these features are intrinsic to the compound and are \n not due to solvent or two - photon processes .", "the width and intensity \n of the broad excited - state absorption bands \n excludes that they arise from vibrational transitions .", "indeed , the \n absorbance is closer to that expected for electronic transitions , \n albeit these typically occur at much higher energies ( in the uv / vis \n range ) .", "the initial state of the compound \n ( i ) shows an absorption band at 390 nm ( figure 1 ) , which is reproduced well by b3lyp / cc - pvdz \n calculations in the gas phase ( 403 nm ; f = 0.428 ) . \n", "the s1  s0 transition has predominantly \n homo  lumo character ( figure 1 ) .", "in contrast to previous assignments , however , we find that this electronic transition \n is not localized on the stator but involves to a significant extent \n excitation from the bonding  orbital of the central double \n bond to the antibonding  * orbital ( figure 1 ) . as a consequence", ", the double bond character \n of this bond is reduced as well as the barrier to rotation around \n this bond . in line with calculations on ethylene and its derivatives , \n", "we find a dark state ( s2 ) associated with the homo1 \n  lumo excitation at slightly higher excitation energies that \n is not directly accessible from the ground state ( figure 1 , table 1 ) . because the homo1 orbital is localized \n on the aromatic stator unit , s2 has a significantly larger \n dipole moment than s1 ( 4.3 vs 2.2 d at the scs - cc2/cc - pvdz \n level ) .", "it should be emphasized that the description of the lower - lying \n electronic states in the compound studied here is thus entirely different \n from ethylene and its derivatives where , as discussed by salem and \n others , for torsion angles of around 90 the two low - lying electronic \n states have either a biradical or zwitterionic character depending \n on the occupation of the two central 2p - orbitals and require multireference \n methods for a proper description . in the present study , in contrast , \n we investigate the dynamics occurring at small torsional angles ( vide infra ) for which the employed single - reference methods \n not only have been validated .", "in addition \n we employ the results of multireference calculations on smaller motor \n molecules as reported by liu and morokuma to make a further comparison with our experimental results .", "for s0 the optimized \n structure on the b3lyp+d / aug - cc - pvdz level was used to calculate the \n vertical excitation energies . for vertical excitation", "they thus strongly suggest that the broad \n and structureless absorption feature observed in the time - resolved \n ir spectra is associated with the electronic s1  \n s2 transition .", "the presence of two close - lying states also \n explains why the induced absorption bands have such high intensities . \n", "our calculations show that the energy gap between the two states strongly \n depends on structural parameters such as the c = c bond length \n that are involved in the 14001600 cm normal \n modes . for these modes", "one therefore expects that their transition \n moments will be enhanced by vibronic coupling as is indeed observed . \n", "the conclusion that the excited - state dynamics of the molecular rotor \n should be described in a multistate model is further supported by \n recent multireference calculations on smaller rotor molecules that \n also find very small energy differences between the s1 and \n s2 states .", "the excited - state \n dynamics were elucidated further using a global \n analysis of the transient spectra , which shows that the spectra can \n be described well with two exponential decay terms with time constants \n of 1.53  0.03 and 12  1 ps .", "we note that our experiments \n have a time resolution of 150200 fs and therefore do not allow \n observation and confirmation of the ultrafast dynamics reported previously \n in fluorescence - upconversion studies .", "species - associated spectra ( sas ) ( supplementary \n figure 2 ) were derived by using a two - step sequential kinetic \n model .", "the sas of the first species shows an overall background \n signal \n with broad and intense features superimposed on it that indicate vibronic \n coupling .", "furthermore , the increase ( decrease ) of absorbance in the \n 1550 ( 1600 ) cm region indicates a substantial \n change in bond character of the central double bond ( figure 2c ) .", "this spectrum was related \n to the structure of this excited - state species using excited - state \n geometry optimizations .", " \n state without any structural constrains associated with the homo1 \n  lumo excitation leads to a stable minimum that can be reached \n from the franck  condon excited geometry by relatively small \n structural changes ( supplementary figure 4 ) , and an excited - state ir absorption spectrum that is in good agreement \n with the sas of species 1 ( figure 2 ) .", "it is notable that for a proper comparison with \n the experimental spectrum , the theoretically predicted stick spectrum \n needs to be convolved with 20 cm gaussian line \n shapes , suggesting that in the  dark  state a wide range \n of structures is sampled .", "our experiments and calculations thus demonstrate \n that excitation and excited - state decay involve two different electronic \n states . in earlier studies , time - resolved fluorescence and visible \n absorption data were interpreted in terms of a decay from a single , \n isolated electronic state decaying to the ground state after structural \n relaxation in the excited state .", "the present data show that the s1 state actually decays to an intermediate , dark electronic \n state , and from there to the ground state . in the sas of the \n second species ( supplementary \n figure", "2 ) the broad background is absent , and bands have widths \n comparable to those in the s0 state .", "the absence of the \n broad background indicates that this species is no longer in an electronically \n excited state but back in the ( vibrationally hot ) electronic ground \n state .", "indeed , the vibrational activity observed in the 1550 cm region follows closely the ir absorption spectrum \n of p*. in addition , repopulation of the original state ( i ) is observed \n also ( i.e. the quantum yield for isomerization is less than unity ) , and the sas of the second species contains contributions \n from both vibrationally hot ground - state i and metastable p * isomers . \n", "vibrational cooling of these isomers leads to the sas of the final \n species , which is in excellent agreement with the steady - state ftir \n difference spectrum of room - temperature i and p*. the present \n experimental and theoretical data allow a complete \n picture of the mechanism by which the molecular rotary motor undergoes \n isomerization after electronic excitation ( figure 3 ) .", "photon absorption brings the motor to \n a  bright  state from which it decays on an ultrafast \n time scale and within the time resolution of the present experiments \n to a different electronically excited  dark  state . \n in line with previous calculations on similar systems ,", "our calculations \n indicate that this is a barrierless process proceeding via a conical \n intersection .", "previously , it was assumed \n that structural evolution on the homo  lumo potential energy \n surface is responsible for the observed ultrafast decay of the fluorescence , \n and ( following commonly accepted models on photoisomerization of carbon ", "carbon \n double bonds ) that it primarily proceeds along torsional and pyramidalization \n coordinates . the present study , in contrast , leads to the conclusion \n that the fluorescence decay is mediated by the passage through the \n conical intersection of the homo  lumo and homo1  \n lumo potential energy surfaces . inspection of the structural changes \n that occur upon geometry relaxation to the minimum on the homo1 \n  lumo potential energy surface show that the central c = c \n bond length increases from 1.381 to 1.461  and that only relatively \n small changes occur in the torsional and pyramidalization coordinates . \n in combination with the forces that act on the molecule at the franck ", "condon \n excited  bright  state ( supplementary \n figure 3 ) , this suggests that motion along the c = c stretch \n coordinate needs to be taken into account as well for understanding \n the decay of the fluorescence .", "proposed multistate dynamics of the photoinduced \n unidirectional \n rotation of the molecular motor starting from conformer i to conformer \n p * ( projected into one dimension ) : dotted arrow , ultrafast process \n reported in previous work ; fast ( red arrow ) and slow process ( blue \n arrow ) observed in the present time - resolved infrared study . ( a ) calculated \n energy profile along the torsion coordinate showing how the energies \n of the electronic states change as the molecules evolves to conformer \n p*. ( b ) schematic representation of the proposed model .", "hence , it can be concluded that \n internal conversion to the electronic \n ground state takes place from the  dark  state .", "the \n decay rate for this process indicates that it is mediated by conical \n intersections between the two states as has indeed been proposed in \n previous theoretical studies .", "these studies \n concluded , however , as well that to reach these intersections a barrier \n needs to be overcome that results from the steric repulsion of the \n methyl group of the rotor unit with the hydrogen of the stator unit . \n", "previous time - resolved fluorescence measurements reported an ultrafast \n damping mode at 113 cm that was speculated to \n arise from motion along a coordinate involving inversion / pyramidalization \n although no direct support could be found for such an assignment .", "interestingly , our calculations find in the \n  dark  state a mode at 119 cm ( b3lyp / cc - pvdz ; \n not scaled , supporting information ) that \n involves motion of the methyl group with respect to the hydrogen of \n the stator unit .", "one might therefore wonder to what extent this mode \n is related to the oscillatory behavior of the fluorescence .", "our observed \n s2 decay constant of 1.5 ps matches the slow component \n of the overall fluorescence decay ( 0.91.5 ps ) .", "our calculation of the excitation energies of \n the  bright  and  dark  states along the \n 119 cm normal mode did not show a conical intersection \n between the two states , supporting the previous explanation of the \n oscillations observed in the fluorescence measurements by the movement of wave packets and suggesting \n a scenario in which a coherent oscillation in the dark s2 state periodically partly repopulates the fluorescent s1 state .", "however , our experimental methods do not provide the time \n resolution to confirm this ."], ["the photodynamics \n of a unidirectional molecular motor has been \n studied by time - resolved ir spectroscopy in which both vibrational \n and electronic structures are probed . in combination with quantum \n mechanical calculations , the high structural sensitivity and real - time \n character of the technique allows us to understand fundamental aspects \n of the operation mechanism .", "the results show that conversion of photon \n energy into directed motion proceeds on the potential energy surfaces \n of two different electronic states , which is the more important as \n it implies that the conical intersection between the  dark  \n electronically excited state and the ground state determines the efficiency \n of the motor . to rationally tune", "the performance of photoinitiated \n unidirectional rotation thus involves a multicoupled - state optimization \n of nuclear motion ."]]}
{"article_id": "PMC5134784", "article_text": ["the patellofemoral joint is one of the most common sources of knee pain in younger people .", "maltracking and instability of the patellofemoral joint are responsible for the majority of symptoms and are associated with the development and progression of osteoarthritis1 ) .", "the position of the patella relative to the trochlea of the femur is not fixed and changes as the knee moves from extension to flexion .", "differences in the morphology of the patellofemoral joint have been shown to be associated with altered kinematics and patellofemoral instability29 ) .", "a variety of measurements have been described to identify abnormalities of the patellofemoral joint . on a lateral radiograph , or a sagittal magnetic resonance imaging ( mri ) scan , the insall - salvati index10 )", "is measured to assess patella height by comparing the length of the patellar tendon with the length of the patella .", "the caton - deschamps index11 ) can also be used to determine patellar height and is calculated by comparing the length of the patellar articular surface with the distance from the postero - inferior border of the patellar and the anterior edge of the tibial plateau . on the axial view , the bisect offset can be used to assess medial or lateral displacement of the patella .", "patellar tilt is also assessed on the axial view and is normally 05 relative to a line connecting the posterior femoral condyles .", "the position of the knee and its weight bearing status have been shown to influence patellofemoral kinematics and measurement of patellofemoral indices1215 ) .", "mri is commonly used in the investigation of patellofemoral pain and instability and in many cases is the investigation of choice in preference to plain radiography .", "mri provides additional information compared to radiographs , such as the condition of the articular surfaces , integrity of ligamentous stabilising structures ( e.g. the medial patellofemoral ligament ) , and rotational alignment ( tibial tuberosity trochlear groove ) , and facilitates better evaluation of trochlear dysplasia5,1620 ) .", "whilst the patellofemoral indices described above were originally designed for radiographic measurements , they have been applied to mri which has been shown to be an excellent imaging modality for evaluating the patellofemoral joint with good correlation with plain radiographs2124 ) .", "mri is usually taken with the patient in supine position and the knee in extension1,21 ) .", "it has been demonstrated that patellofemoral instability typically manifests during the terminal 30 of knee extension with contraction of the quadriceps femoris muscle14 ) .", "undergoing an mri scan can be an intimidating experience for patients , particularly those prone to claustrophobia25,26 ) .", "such patients are likely to be anxious and tense during the scan and may inadvertently contract their quadriceps .", "this quadriceps contraction may alter the relationship of the patella to the femur , overestimating the degree of instability that may be present .", "the objective of our study was to determine the impact of knee position and quadriceps contraction on patellofemoral indices measured on mri .", "twenty patients undergoing mri scan of the knee were identified from a pool of referrals to the orthopaedic department at our institution .", "patients were selected based on the absence of patellar or patellofemoral symptoms documented in the clinical notes or on the mri request .", "inclusion criteria were 2540 years of age , a body mass index less than 30 , a clinical diagnosis of non - patellar or patellofemoral pathology .", "exclusion criteria were known or suspected patellar or patellofemoral conditions , connective tissue disorders that could cause hyperlaxity , and previous surgery or injury to the knee .", "axial and sagittal mri sequences were performed with the knee fully extended and quadriceps contracted ; knee fully extended and quadriceps relaxed ; knee flexed 30 with quadriceps contracted ; and knee flexed 30 with quadriceps relaxed .", "a triangular wedge was placed under the patient s knee to reproducibly create 30 of flexion .", "when quadriceps contraction was required , subjects were given a rest period between mri sequences to prevent muscle fatigue .", "bisect offset27 ) and patellar tilt angle28 ) were measured on axial mri images using the slice showing the widest view of the patella .", "the insall - salvati index and caton - deschamps index were measured on the sagittal view using the slice showing the widest view of the patella .", "1 ) was determined by drawing a line connecting the posterior femoral condyles and a perpendicular line was then drawn through the deepest point of the trochlea groove to where it intersects the patellar width line . in cases where the trochlea was flattened ,", "the bisect offset is defined as the proportion of the patella lying lateral to the midline as a percentage of the whole patellar width .", "patellar tilt was defined as the angle between a line connecting the posterior femoral condyles and the maximum patellar width ( fig .", "the insall - salvati ratio was determined by comparing the length of the patellar tendon with the length of the patella .", "the caton - deschamps index was defined as the ratio of the distance between the lower edge of the patellar joint surface and the upper edge of the tibial plateau to the length of the patellar articular surface ( fig .", "the student t - test ( unpaired ) was used to compare the difference between these values in the various states of knee flexion and quadriceps contraction .", "all 20 patients were assessed with the knee in extension and flexion , with and without quadriceps contraction .", "the mean values of the patellar tilt , bisect offset , insall - salvati ratio and caton - deschamps index of the patients are detailed in table 1 .", "the mean patella tilt averaged 9 with the knee flexed and quadriceps in any state .", "patella tilt increased but remained within normal limits with the knee extended and quadriceps relaxed .", "however , when the quadriceps was contracted with the knee in extension , the mean patella tilt was 14.6 , the upper limit of the normal range .", "the mean difference in patella tilt from the flexed , relaxed positon to the extended , contracted position was 5.6 ( range , 9.8 to + 25.9 ) , but this did not reach statistical significance ( p=0.06 ) .", "the mean bisect offset angle in the flexed position increased from a normal range of 56% and 57% when the quadriceps were relaxed or contracted , respectively , to an abnormal value of 65% with the knee extended and quadriceps contracted ( p=0.012 ) .", "there were four patients who demonstrated normal patella tilt and bisect offset with the knee flexed and quadriceps relaxed , but abnormally high tilt and bisect offset were observed with the knee extended and quadriceps contracted .", "the mean values of the caton - deschamps index were within the normal range with knee flexed but were abnormally elevated with the knee extended , both with the quadriceps relaxed and contracted .", "the difference between the flexed relaxed position and extended contracted position was 0.05 ( range , 0.15 to + 0.31 ) , which did not reach statistical significance ( p=0.34 ) .", "the mean values of the insall - salvati ratio were normal in all combinations of knee position and quadriceps contraction .", "the change in the insall - salvati ratio from the flexed , relaxed state to the extended , contracted state was 0.07 ( range , 0.17 to + 0.11 ) , which was not statistically significant ( p=0.97 ) .", "mri evaluation of the knee forms an integral part of the investigation of young patients with knee pain .", "the patella begins to engage the trochlea during knee flexion , and patellofemoral instability typically manifests during the terminal 30 of knee extension . in the clinical setting of patellofemoral disorders", ", mri can provide useful information about the bony morphology of the femur and patella as well as the condition of the articular cartilage .", "numerous patellofemoral measurements or indices have been described to quantify abnormalities of the patellofemoral joint .", "many of these measurements were originally described on plain radiographs and their use has been extended to mri .", "they can be of some value when deciding on the most appropriate management for patients with patellofemoral symptoms .", "significant differences in these measurements exist between patients with patellofemoral instability and asymptomatic patients3,6 ) .", "quadriceps contraction and weight bearing have been shown to alter patellar height and patellofemoral kinematics1215,29,30 ) .", "for the majority of patients , mri of the knee is performed with the knee extended .", "patients undergoing mri scanning of the knee may be anxious and as a result involuntarily contract their quadriceps .", "this has the potential to lead to inappropriate management decisions or unnecessary surgery . in our study", ", we aimed to assess the influence of knee position and quadriceps activity on frequently measured patellofemoral indices . in our cohort of patients", ", none of whom had patellofemoral symptoms , an increase in the measured patellofemoral indices was observed with the knee extended and quadriceps contracted .", "the mean values of these indices increased above the normally accepted range in some cases with the knee in this position . in the case of the bisect", "this suggests that the flexed position protects against erroneous or exaggerated patellofemoral measurements caused by inadvertent quadriceps contraction as well as trochlea engagement .", "thus , it is important that potential confounders ( e.g. quadriceps contraction and knee extension ) are minimised in order to achieve more reliable measurements upon which surgical decision - making is based .", "we , therefore , recommend , when investigating patients with suspected patellofemoral pathology , mri of the knee should be performed in 30 of flexion to obtain a more accurate view of the patellofemoral joint and avoid the confounding effects of quadriceps contraction .", "patellofemoral indices measured on mri are affected by the position of the knee and quadriceps muscle activity . with the knee in extension and the quadriceps contracted , as may occur in an anxious patient , there was a tendency towards abnormal patellofemoral measurements in our cohort of asymptomatic patients .", "performing mri of the knee in 30 of flexion helps to reduce the confounding effect of quadriceps contraction and may result in more reliable radiological assessment of the patellofemoral joint ."], "abstract_text": ["<S> purposethe purpose of this study was to investigate the impact of varying knee flexion and quadriceps activity on patellofemoral indices measured on magnetic resonance imaging ( mri).materials and methodsmri of the knee was performed in 20 patients for indications other than patellar or patellofemoral pathology . </S>", "<S> axial and sagittal sequences were performed in full extension of the knee with the quadriceps relaxed , full extension of the knee with the quadriceps contracted , 30 flexion of the knee with the quadriceps relaxed , and 30 flexion with the quadriceps contracted . </S>", "<S> bisect offset , patella tilt angle , insall - salvati ratio and caton - deschamps index were measured.resultswith the knee flexed to 30 and quadriceps relaxed , the mean values of patellar tilt angle , bisect offset , insall - salvati ratio and caton - deschamps index were all within normal limits . with </S>", "<S> the knee extended and quadriceps contracted , the mean patellar tilt angle ( normal value , < 15 ) was 14.6 and the bisect offset ( normal value , < </S>", "<S> 65% ) was 65% , while the caton - deschamps index was 1.34 ( normal range , 0.6 to 1.3 ) . with the knee extended and quadriceps relaxed , </S>", "<S> the mean caton - deschamps index was 1.31.conclusionsmri scanning of the knee in extension with the quadriceps contracted leads to elevated patellofemoral indices . </S>", "<S> mri taken with the knee in 30 of flexion allows more reliable assessment of the patellofemoral joint and minimises the confounding effect of quadriceps contraction . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusions"], "sections": [["the patellofemoral joint is one of the most common sources of knee pain in younger people .", "maltracking and instability of the patellofemoral joint are responsible for the majority of symptoms and are associated with the development and progression of osteoarthritis1 ) .", "the position of the patella relative to the trochlea of the femur is not fixed and changes as the knee moves from extension to flexion .", "differences in the morphology of the patellofemoral joint have been shown to be associated with altered kinematics and patellofemoral instability29 ) .", "a variety of measurements have been described to identify abnormalities of the patellofemoral joint . on a lateral radiograph , or a sagittal magnetic resonance imaging ( mri ) scan , the insall - salvati index10 )", "is measured to assess patella height by comparing the length of the patellar tendon with the length of the patella .", "the caton - deschamps index11 ) can also be used to determine patellar height and is calculated by comparing the length of the patellar articular surface with the distance from the postero - inferior border of the patellar and the anterior edge of the tibial plateau . on the axial view , the bisect offset can be used to assess medial or lateral displacement of the patella .", "patellar tilt is also assessed on the axial view and is normally 05 relative to a line connecting the posterior femoral condyles .", "the position of the knee and its weight bearing status have been shown to influence patellofemoral kinematics and measurement of patellofemoral indices1215 ) .", "mri is commonly used in the investigation of patellofemoral pain and instability and in many cases is the investigation of choice in preference to plain radiography .", "mri provides additional information compared to radiographs , such as the condition of the articular surfaces , integrity of ligamentous stabilising structures ( e.g. the medial patellofemoral ligament ) , and rotational alignment ( tibial tuberosity trochlear groove ) , and facilitates better evaluation of trochlear dysplasia5,1620 ) .", "whilst the patellofemoral indices described above were originally designed for radiographic measurements , they have been applied to mri which has been shown to be an excellent imaging modality for evaluating the patellofemoral joint with good correlation with plain radiographs2124 ) .", "mri is usually taken with the patient in supine position and the knee in extension1,21 ) .", "it has been demonstrated that patellofemoral instability typically manifests during the terminal 30 of knee extension with contraction of the quadriceps femoris muscle14 ) .", "undergoing an mri scan can be an intimidating experience for patients , particularly those prone to claustrophobia25,26 ) .", "such patients are likely to be anxious and tense during the scan and may inadvertently contract their quadriceps .", "this quadriceps contraction may alter the relationship of the patella to the femur , overestimating the degree of instability that may be present .", "the objective of our study was to determine the impact of knee position and quadriceps contraction on patellofemoral indices measured on mri ."], ["twenty patients undergoing mri scan of the knee were identified from a pool of referrals to the orthopaedic department at our institution .", "patients were selected based on the absence of patellar or patellofemoral symptoms documented in the clinical notes or on the mri request .", "inclusion criteria were 2540 years of age , a body mass index less than 30 , a clinical diagnosis of non - patellar or patellofemoral pathology .", "exclusion criteria were known or suspected patellar or patellofemoral conditions , connective tissue disorders that could cause hyperlaxity , and previous surgery or injury to the knee .", "axial and sagittal mri sequences were performed with the knee fully extended and quadriceps contracted ; knee fully extended and quadriceps relaxed ; knee flexed 30 with quadriceps contracted ; and knee flexed 30 with quadriceps relaxed .", "a triangular wedge was placed under the patient s knee to reproducibly create 30 of flexion .", "when quadriceps contraction was required , subjects were given a rest period between mri sequences to prevent muscle fatigue .", "bisect offset27 ) and patellar tilt angle28 ) were measured on axial mri images using the slice showing the widest view of the patella .", "the insall - salvati index and caton - deschamps index were measured on the sagittal view using the slice showing the widest view of the patella .", "1 ) was determined by drawing a line connecting the posterior femoral condyles and a perpendicular line was then drawn through the deepest point of the trochlea groove to where it intersects the patellar width line . in cases where the trochlea was flattened ,", "the bisect offset is defined as the proportion of the patella lying lateral to the midline as a percentage of the whole patellar width .", "patellar tilt was defined as the angle between a line connecting the posterior femoral condyles and the maximum patellar width ( fig .", "the insall - salvati ratio was determined by comparing the length of the patellar tendon with the length of the patella .", "the caton - deschamps index was defined as the ratio of the distance between the lower edge of the patellar joint surface and the upper edge of the tibial plateau to the length of the patellar articular surface ( fig .", "the student t - test ( unpaired ) was used to compare the difference between these values in the various states of knee flexion and quadriceps contraction ."], ["all 20 patients were assessed with the knee in extension and flexion , with and without quadriceps contraction .", "the mean values of the patellar tilt , bisect offset , insall - salvati ratio and caton - deschamps index of the patients are detailed in table 1 .", "the mean patella tilt averaged 9 with the knee flexed and quadriceps in any state .", "patella tilt increased but remained within normal limits with the knee extended and quadriceps relaxed .", "however , when the quadriceps was contracted with the knee in extension , the mean patella tilt was 14.6 , the upper limit of the normal range .", "the mean difference in patella tilt from the flexed , relaxed positon to the extended , contracted position was 5.6 ( range , 9.8 to + 25.9 ) , but this did not reach statistical significance ( p=0.06 ) .", "the mean bisect offset angle in the flexed position increased from a normal range of 56% and 57% when the quadriceps were relaxed or contracted , respectively , to an abnormal value of 65% with the knee extended and quadriceps contracted ( p=0.012 ) .", "there were four patients who demonstrated normal patella tilt and bisect offset with the knee flexed and quadriceps relaxed , but abnormally high tilt and bisect offset were observed with the knee extended and quadriceps contracted .", "the mean values of the caton - deschamps index were within the normal range with knee flexed but were abnormally elevated with the knee extended , both with the quadriceps relaxed and contracted .", "the difference between the flexed relaxed position and extended contracted position was 0.05 ( range , 0.15 to + 0.31 ) , which did not reach statistical significance ( p=0.34 ) .", "the mean values of the insall - salvati ratio were normal in all combinations of knee position and quadriceps contraction .", "the change in the insall - salvati ratio from the flexed , relaxed state to the extended , contracted state was 0.07 ( range , 0.17 to + 0.11 ) , which was not statistically significant ( p=0.97 ) ."], ["mri evaluation of the knee forms an integral part of the investigation of young patients with knee pain .", "the patella begins to engage the trochlea during knee flexion , and patellofemoral instability typically manifests during the terminal 30 of knee extension . in the clinical setting of patellofemoral disorders", ", mri can provide useful information about the bony morphology of the femur and patella as well as the condition of the articular cartilage .", "numerous patellofemoral measurements or indices have been described to quantify abnormalities of the patellofemoral joint .", "many of these measurements were originally described on plain radiographs and their use has been extended to mri .", "they can be of some value when deciding on the most appropriate management for patients with patellofemoral symptoms .", "significant differences in these measurements exist between patients with patellofemoral instability and asymptomatic patients3,6 ) .", "quadriceps contraction and weight bearing have been shown to alter patellar height and patellofemoral kinematics1215,29,30 ) .", "for the majority of patients , mri of the knee is performed with the knee extended .", "patients undergoing mri scanning of the knee may be anxious and as a result involuntarily contract their quadriceps .", "this has the potential to lead to inappropriate management decisions or unnecessary surgery . in our study", ", we aimed to assess the influence of knee position and quadriceps activity on frequently measured patellofemoral indices . in our cohort of patients", ", none of whom had patellofemoral symptoms , an increase in the measured patellofemoral indices was observed with the knee extended and quadriceps contracted .", "the mean values of these indices increased above the normally accepted range in some cases with the knee in this position . in the case of the bisect", "this suggests that the flexed position protects against erroneous or exaggerated patellofemoral measurements caused by inadvertent quadriceps contraction as well as trochlea engagement .", "thus , it is important that potential confounders ( e.g. quadriceps contraction and knee extension ) are minimised in order to achieve more reliable measurements upon which surgical decision - making is based .", "we , therefore , recommend , when investigating patients with suspected patellofemoral pathology , mri of the knee should be performed in 30 of flexion to obtain a more accurate view of the patellofemoral joint and avoid the confounding effects of quadriceps contraction ."], ["patellofemoral indices measured on mri are affected by the position of the knee and quadriceps muscle activity . with the knee in extension and the quadriceps contracted , as may occur in an anxious patient , there was a tendency towards abnormal patellofemoral measurements in our cohort of asymptomatic patients .", "performing mri of the knee in 30 of flexion helps to reduce the confounding effect of quadriceps contraction and may result in more reliable radiological assessment of the patellofemoral joint ."]]}
{"article_id": "PMC4897108", "article_text": ["the knowledge of mechanical materials behaviour is of great importance particularly for some innovative engineering applications , which involve a single material , bimaterials , or multiple materials .", "some bimaterials are obtained by means of a coating process and consist of coupled layers that may or may not contain filler elements , such as a foaming operation , or may be developed as welded substrates . usually , at the end of the process , the material is identified as a new single material .", "the coating procedure developed and studied in this work refers to the hot - dip galvanizing treatment , considered as a better treatment to fight against corrosion .", "the role of a hot - dip galvanizing treatment consists in the deposition of a protective external layer of metallic zinc obtained by immersing the steel in a zinc bath at a temperature of around 460c .", "when the material ( i.e. , steel ) is introduced into the zinc bath and then removed , several changes in the chemical composition and in the mechanical structure can occur .", "these changes produce a new structural arrangement on zinc substrate and are usually revealed by the generation of cracks in the zinc layer .", "the behaviour of materials may change as a result of various events such as fatigue , fracture , wear , fretting fatigue , creep , hydrogen embrittlement , thermal shock processes , or atmospheric attacks .", "an important role in these failure processes is played by any discontinuity in the material that can appear during the manufacturing process or during working period , or as a result of inappropriate use . in our case", "it appears as a consequence of the hot - dip galvanizing treatment applied as described above in association with the action of the mechanical loading .", "recently , krishnan and xu   focused on failure mechanics of adhesive joints ( i.e. , considered as bimaterials ) using a fringe pattern concentrations technique to describe the bimaterials interface .", "as analytical model , they used the fracture mechanics approach , while to compute the stress singularity in the bimaterial layer they took into account the stress intensity factor in mode i. the formula used to express this stress intensity factor is \n ( 1)ki = rekai , \n where k is the stress intensity factor in the case of the bimaterial component : \n ( 2)k = ytaaiei , \n where t = p(3s / w ) and y ,  are the calibrating factors that depend on a / w , b / w , respectively , dundurs ' parameters .", "then a , b , and w represent the length of the possible crack , the thickness , and the length of the specimen .", " is a function of dundurs ' parameters ,  , and is presented as \n ( 3)=12ln11+ , \n where  , dundurs ' parameters material , is expressed by \n ( 4)=112221211122+2121 \n in which 1 and 2 denote the material , while  and  are the shear modulus and poisson 's ratio , respectively .", "the elastic fracture theory is a suitable tool to compute the mechanical behaviour in case of coatings , composites , and welded structures .", "the bimaterial produced by the reactions that occur during the hot - dip galvanized steel process must have the same conditions on the contact surface as the  linear spring - like  model .", "the discontinuity of displacement across the interface is assumed to be linearly proportional to the displacement at the interface of the constituent where the stress source is located .", "the present authors agree that the two materials that form the bimaterial should be considered as a functionally autonomous subsystem with a different young 's modulus and poisson 's coefficients .", "this implies a strain incompatibility between the two solids and the formation of a periodic distribution of tensile and compressive stress in checkerboard patterns under the uniaxial tensile test .", "a robust approach was proposed by yu et al .   in order to formulate analytical solutions through the use of  linear spring - like model .  according to this theory we can define the type of contact between the surfaces by the fraction of the adherent area , applying the following formula : \n ( 5)=aacac , \n where  is used to quantify the extent of bonding at the interface , a is the total area of the interface , and ac is the area covered by paper ( see example on figure 2 from ) . according to the study made by panin et al .", ", the interface of solid materials covers a sinusoidal surface layer due to a rotation of successive constraints in tensile and compression stress in the structure .", "this effect is described by the following equation : \n ( 6)=aysinxlxt2 , \n where ", "t  is the thickness of the coating , x is the distance of crack propagation , y is the stress coefficient .", "this paper addresses two goals : first of all , to experimentally characterize the layer of zinc applied during the hot - dip galvanization process , in order to obtain necessary information about the uniformity of the zinc layer and the number of cracks and their length , and finally to estimate the behaviour of the cracks located in zinc layer when the mechanical loading is imposed .", "secondly , we proposed to corroborate the experimental results with analytical and numerical computations , ascertaining where the crack discontinuities spread while considering the three main possibilities of crack development : arrest at the bimaterial interface , crossing into the second material , in our case steel , and finally creating a deflection between the two materials . in the literature", "k. m. mrz and z. mrz   predicted the behaviour of bi- and multimaterial interfaces according to a simplified approach using the mk - criterion based on the linear elastic fracture mechanics ( lefm ) . in this criterion", "it is assumed that the crack growth follows the direction of minimum distortion energy density at a distance corresponding to a specified value of dilatation energy .  ", "figure 2 shows a specific case in which the crack is considered to propagate perpendicularly to the interlayer and make a bifurcation at the interface .", "the decohesion phase may present as one of the four possible scenarios whereby the crack will propagate as follows : ( i ) the plastically weaker material ( wm ) to the plastically stronger material ( sm ) , wm - sm , ( ii ) the plastically weaker material ( wm ) to the plastically stronger interlayer ( si ) , wm - si , ( iii ) the plastically stronger material ( sm ) to the plastically weaker material ( wm ) , sm - wm , and ( iv ) the plastically stronger material ( sm ) to the plastically weaker interlayer ( wi ) , sm - wi . in simple terms , the criterion for crack initiation and propagation along the interface could be considered , where the maximum stress , max , is expressed as \n ( 7)max=x+y2+x+y22+xy . to solve the problem of deflection at the interface of two materials , hutchinson   proposed the following condition : \n ( 8)gcgc1<gdgp1 , \n where g = gc represents release energy rate of the interface bimaterial .", "studies , related to the problem of cracks running perpendicular to a bimaterial , yields a different expression for the stress intensity factor that is connected to the stress tensor as follows : \n ( 9)ij = kr1fij. to determine the propagation energy of the crack running perpendicular to the interface , also called energy release rate , the following equation studied by madani et al .  ", "can be applied : \n ( 10)gd=(11)/1+(11)/24cosh2k12+k22gp=1222kp2 . \n in ( 8) , ( 10 ) ", "gd and gp are the strain energy release rate for deflection and penetration ; k1 and k2 are the stress intensity factors for the interface crack ; kp is the value of stress intensity factor , for a particular case , when the crack from material 1 penetrates into material 2 ( see figure 2 ) .", "analytical techniques and the green function can be employed to describe the behaviour of bimaterial compounds by calculating numerical solutions of singular integral equations , as was applied by erdogan et al .   and used by pruncu et al .   and azari et al . , which yield the following expression : \n ( 11)ki(a)=211+k1a0g(1)kib=211+k1a0g1 . a more general form of such approach could be structured using an algorithm that describes the numerical procedure for obtaining the different values of the function g(ti ) by the following .", "( 2 ) for k ranging from 1 to n , we need a computing route for xk and f(xk ) .", "( 3 ) for every xk and for i variants from 1 to ( n  1 ) , we have to calculate the first ti that is a weight function of the jacobi polynomials described as follows : \n ( 12)1ng(ti)1tixk+k(xk , ti)=0  withti = cos2i12n ,  i=1,  ,nxk = coskn ,  k=1,  ,n1 .", "( 4 ) the computation then yields a matrix relationship expressed as follows:(13)1t1x1+k(x1,t1)1t2x1+k(x1,t2) .... 1tnx1+k(x1,tn) .............................. 1t1xk+k(xk , t1)1t2xk+k(xk , t2) .... 1tnxk+k(xk , tn)gt1gt2 .... gtn = fx1fx2 .... fxk .", "g  are obtained by multiplying the transposed of the matrix by the vector f(xi ) .", "one has the following : a0 is half - length of the crack ; 1 , 2 are shear modulus for materials 1 and 2 ; c is the distance from the middle of the crack at the interface plane ; r ,  are polar coordinates , k = 3  4 for the plane strain case and k = ( 3)/(1 +  ) for the plane stress case ,  is poisson coefficient , and g is the parameter that expresses the magnitude of the applied load . another analytical technique , which was employed by chen et al .   using complex potential values ,", "was obtained in the following manner : \n ( 14)ki(b)=limr02rx=22k2 + 1a0m=11mmki(a)=22k2 + 1a0m=1m , \n where a0 is half - length of the crack , m = m/b , m = 0,1 , 2,3 ,  ,  b is the distance between the interface and the crack front , x is the distribution of stress in front of the crack , k = 3  4 for the plane strain case and k = ( 3  4)(1 +  ) for the plane stress case , and 2 is shear modulus for the material in which the crack is located .", "because of its multiple properties , steel is one of the most versatile materials used in industrial engineering applications .", "the european standard ( en ) reveals different types of steel employed in fields such as the automotive industry and aeronautical design . in this work we considered two steel alloys : he360dr and s420mc .", "their mechanical characteristics are summarized in table 1 . during the lifetime of service , in contact with the environment", "this metal may develop problems that could be prevented by applying the above - described process of hot - dip galvanization .", "the specimens obtained after hot - dip galvanization were submitted in laboratory to fatigue tensile test in order to detect their behaviour under fatigue conditions conforming to real life and to observe the changes that occurred in the metallic zinc layer .", "the fatigue tests were performed on a servohydraulic testing machine capable of applying axial loads up to 100  kn , using a sinusoidal load wave , with a frequency of 30  hz and a strength ratio r = 0.1 .", "after the fatigue test the specimens were experimentally analysed by optical microscopy and scanning electron microscopy ( sem ) . during observation with the optical microscope ,", "a sample of length 5328  m was considered . to simplify the management of these optical microscope observations , we divided the sample size into 16 units with a length of about 333  m .", "the main purpose of this was to analyse ( i ) the number of cracks per unit of length ; ( ii ) whether the evolution of the number of cracks was constant over all layers applied ; ( iii ) which material was prone to develop longer cracks , and finally if the zinc layer was constant over the whole steel surface .", "the first result was the average number of cracks per unit of length , as summarized in table 2 .", "the letters b and h in table 2 indicate the time of immersion in the zinc bath , b being 3 minutes of immersion and h 7 minutes .", "the numbers 9 , 8 , 7 , and 2 indicate the specimen number analysed . from table 2", "we can observe that the average number of cracks per unit of length is about 6/11 for he360dr and 7/8 for s420mc .", "a first remark proves that the number of cracks increases with the time of immersion .", "the results are plotted in figure 3 and highlight the number of cracks for all 16 units of length .", "figure 3 shows that the number of cracks on the entire length of zinc layer that encloses each unit is overall scattered ; however , on the local case of two / three consecutive units , the number of cracks per unit seems to be almost uniformly distributed . a key element for determination of the efficiency of the coating process", "may be deduced from the evolution of cracks lengths before and after the fatigue tests . according to the observations shown in figure 4", ", there was a significant change of crack length as a consequence of fatigue test .", "a gap of about 25  m in crack length before and after cyclic load was found due to the weakened structure of material .", "this observation was done in a useful sample area ( ua ) and then in the area where the test had less influence on the behaviour of the piece , marked as the nonuseful area ( nua ) .", "the uniformity of the zinc layer was considered as another issue that can stimulate the crack behaviour .", ", the zinc layer deposited over the steel alloy is uniformly distributed , having a value of 5080  m .", "the particularity of the size of substrate of he360dr h8 materials , that is , the thin thickness , may be explained as a consequence of immersion time .", "so , because the time of immersion within the bath of zinc was less than the time imposed by our methodology , the thickness of substrate was lower ; this means that instead of 7 minutes we found that the real time of immersion was about 5 minutes .", "the optical measurements confirmed that the thickness deposited over the steel pieces was uniform and show that the zinc layer is composed of several multilayers .", "the substrates are denominated by gamma (  ) , delta (  ) , zeta (  ) , and zinc eta (  ) , as shown in figure 6 .", "figure 6 proves that most of the cracks are located in substrate  and may occur along the zinc grain boundaries . indeed , from our observations these cracks arise in this  substrate and then spread toward the steel - zinc interface .", "however , the analysis indicates a decrease of the crack magnitude near the interface , which means the crack growth is interrupted before it reaches the interface .", "observations from scanning electron microscopy ( sem ) exhibit even more clearly that , during the hot - dip galvanized process ( hdg ) , the quality of the bonded layers may influence the direction of the cracks , if the cracks touch the steel - zinc interface .", "this troublesome problem produces a sort of  debonding area  which forms a path for surface deflection of the cracks .", "the  debonding area  in steel - zinc adhesive layers is illustrated in figure 7 , created by the propagation of cracks at the interface between the zinc and steel and into the zinc substrates zeta (  ) and eta (  ) . for bimaterials , the interface crack problems could be due to the nonhomogeneity of the materials , which develop in the direction parallel to the crack tip . from these preliminary findings", "we could confirm that during the hdg process the zinc layer is uniformly deposited and the average number of cracks is significant on this layer .", "it seems that during the fatigue process the cracks get longer and spread toward the bonding interface , arresting at the bimaterial interface . in the worst scenario ,", "numerical computational technique was implemented by abaqus software , in order to corroborate the experimental data with analytical and numerical simulations and to highlight the effects of cracks that develop during the hot - dip galvanizing process . in order to implement the experimental data in the numerical model ,", "besides , this value corresponds with the thickness measured under the s420mc h2 materials ( see figure 5 ) , and it allows making the assumption that the crack / cracks imposed in our models will spread only toward the interface steel / zinc . if we consider a smaller thickness , for example , the size of layer measured under he360dr b9 material , and we adopt in the numerical model the critical size of crack detected after the loading test ( see figure 4 ) , it will be obvious that the crack will cross all the coating substrate .", "but this issue is far from our experimental observation , since we had observed that the crack growth is only toward interface steel / zinc .", "experimental data were introduced in our numerical model as the thickness size of zinc layer ( figure 5 ) , while the contour of zinc layer on the entire material surface is considered homogenous and uniform ( figure 5 ) .", "an initial length of crack and the maximum length of crack ( see figure 4 ) were imposed as constraint in the numerical program .", "the principles of linear fracture mechanics ( lfm ) were implemented in the numerical model in order to explain behaviour of crack at the interface steel / zinc that is assuming that all the materials contain a minimum flow that will grow during the mechanical loading .", "so , considering the crack located in zinc substrate ( figure 5 ) that will develop perpendicular to the applied load , it is possible to denote this state by mode i fracture .", "it should be noted that in the numerical model a mechanical loading of 500  mpa corresponding to an average of ultimate tensile strength of galvanized materials ( see table 1 ) was imposed .", "a value of about 30  m was selected as the initial length of the crack / cracks before propagation , in agreement with experimental data ( figure 4 ) .", "however , we know that the crack is bounded by two fronts , a and b. in this model , we assumed that the front of crack b was static and so it could not propagate , whereas the only front of crack a would spread .", "a basic model composed of two materials , m1 and m2 , as shown in figure 8(a ) , which forms the bimaterial body , was implemented .", "m2 represents the steel and m1 the zinc layer , delimited by the following values : h1 = 0.08  mm , h2 = 19.84  mm , c = 0.045  mm , w = 15  mm , 2a0 = 0.030  mm , and  = 0.5  m and submitted to a mechanical load ,  = 500  mpa .", "the applied load in this system will have direct consequence to the crack behaviour and define the presence of stress singularity on both fronts of the crack , tips a and b. the configuration for the crack fronts is shown in figure 8(b ) .", "settlement of the crack propagates performance was evaluated using the stress intensity factor ( sif ) parameter , derived from the singularity 1/r advanced in front of the crack tip .", "since the value of the stress intensity factor ( sif ) is obtained , we proceed to conclude if the crack crosses into the second material , ends at the bonded interface , or deflects between these two bodies .", "( a ) model with one crack in the bimaterial : in this case one single crack of an initial size of 30  m was considered . then the length was increased by 3  m in each simulation up to the maximum of 60  m ( i.e. , close to the experimental analysis shown in figure 4 ) .", "figure 9 shows how the stress distribution increases with the length of crack , assuming that the initial crack had the same stress value at both fronts a and b. two - dimensional finite - element meshes are virtually symmetrical near this bottom ( crack fronts ) and the symmetry showed almost the same value as the stress distribution , for each of the crack fronts , during the simulation .", "the shape of the stress - strain curve as a function of the crack length is illustrated in figure 10 .", "the shape of the curve grows wide with the increasing of crack length in front of crack tip a. the effect of stress distribution in front of crack can be converted into basic parameters that show the impact of crack behaviour , that is , the stress concentration factor ( scf ) . obviously , the stress concentration factor is the ratio of the maximum stress over the nominal stress , denominated kt and expressed as \n ( 15)kt=maxnom. while the front of crack b does change , the main role in this research was played only by the front of crack a located near the interface of the bimaterial .", "thus , figure 11 shows the effect of scf in the front of crack a. these results were corroborated with the theoretical stress concentration factor ( scf ) obtained with the online software .", "the results presented in figure 11 show a good agreement and underline that when the crack reaches the interface , there is a sudden increase in the stress distribution value .", "this sharp rise in values may result in the creation of a surface called a  debonding area ,  as shown in figures 9(c ) and 9(d ) .", "then , by assessing the crack propagation with lfm using ( crack ) stress intensity factors ( sif ) , k , it is possible to establish where the crack stops . to achieve even better accurate results , a comparison between an analytical model reported by erdogan et al .   and", "the difference between the numerical and analytical results is due to the use of a small number of constants in the analytical model .", "the results are shown in figure 12 , demonstrating the trend of the ( crack ) stress intensity factors ( sif ) during applied stress , in front of cracks a and b. from the curves drawn below , it can be seen that the value of the sif increases with the crack length , and the crack propagates up to a length of about 50  m . then stabilization and even a decline of the sif value", "the value of sif will increase sharply only if the flow area denoted as well as ", "since the value of the ( crack ) stress intensity factors ( sif ) is low for the front of both cracks a and b , it seems that the crack can not cross into the second material . \n", "( b ) model with two or more cracks : in the second assumption , a  multiple cracks  model , namely , with 3 cracks , was implemented using almost the correspondent algorithm but specifying 3 initial cracks with an initial size of 30  m for each crack , and then each crack would increase by 3  m , up to the maximum size of 60  m .", "thus , the maximum cracks value is assigned when the cracks reach the steel - zinc interface . in this model with 3 cracks the applied load ( 500  mpa ) was considered as statement from the case of the model with a single crack .", "the motivation to implement a model with three cracks aims to detect whether the situation changed due to the increased ( crack ) stress intensity factor ( sif ) values , as a result of the cumulative energy developed by these 3 cracks .", "related work has been reported by song et al .   who declared that the interface crack will propagate if the principal maximum stress at the crack tip exceeds a critical value .", "thus , confirming our initial supposition , the results presented in figure 14 show an increase for maximum stress value within front of the cracks . at the same time , the surface denominated  debonding area  appears more pronounced .", "the tendency was confirmed from the development of the ( crack ) stress intensity factors ( sif ) , k values , where the results are reported in figure 15 .", "the shapes of the curves in figure 15 show a similar trend obtained by the one in the case with one crack except for the fact that  crack 2  shows the highest influence of propagation , derived from the cumulated extension efforts from another two cracks .", "another thing to note is the gap difference of sif for crack lengths 57  m and 60  m .", "this difference could be explained by the appearance of the previously mentioned  debonding areas ", "( i.e. , imposed length for debonding area considered in this research was a maximum of two initial crack lengths , because after this size the peeling processes occur ) .", "although the difference is evident , it does not exceed the critical value of mode i fracture toughness ; for example , the value cited by ashby   for steel is about 80170  mpa  m. consequently , the crack will not cross into the steel material and in the worst case the crack / cracks will form a large bifurcation at the interface between these two materials .", "this research paper highlights by means of experimental , numerical , and analytical tool the behaviour of a bimaterial zinc / steel interface submitted to mechanical loading . in particular , the case of crack / cracks at the bimaterials interface was considered .", "the main objectives of this paper were to assess the behaviour of the crack / cracks generated at the end of the hot - dip process and to reveal that the numerical computations are in a good agreement with the experimental outcomes .", "optical microscopy and scanning electron microscopy ( sem ) techniques were involved to evaluate the experimental performance of the bimaterials compound .", "postmortem analysis of the fracture surfaces emphasized the uniformity of the zinc layer over the steel surface and provided accurate information related to the average of cracks length ( figure 4 ) .", "in addition , this technique allows quantifying the number of cracks ( figure 3 ) that forms in the zinc layers . in the meantime", ", this assessment provides patterns about the evolution of crack from incipient phase , where the cracks arise , and how far they spread . in numerical computations , simulations of the model considered", "were run in two different situations , namely , when the bimaterial contains just one crack or else 3 cracks , in order to prove the agreement between computation and experimental outcomes .", "besides , to get even accurate results , the analytical model used by erdogan et al .   was also confronted .", "the sif were calculated to obtain value of the stress singularity that characterizes the crack evolution in the bonding area of bimaterial , for both crack tip values , that is , the fronts of cracks a and b. the analytical , numerical , and experimental assessments prove that the crack / cracks that arise during the hot - dip galvanized steel process will propagate during the mechanical fatigue test .", "it was also established that the crack / cracks will stop near the steel - zinc interface at a distance of about 3  m and only in particular cases yield a deflection at the bimaterial interface . to summarize", ", the results may confirm that cracks reduce the life span of the bimaterial but are not responsible for a direct degradation of the steel .", "this method is therefore useful for employment in damage models that include the safety factor condition .", "finally , a nondestructive method should be employed to detect the behaviour of bimaterials for a better calibration ."], "abstract_text": ["<S> the behaviour of materials is governed by the surrounding environment . </S>", "<S> the contact area between the material and the surrounding environment is the likely spot where different forms of degradation , particularly rust , may be generated . </S>", "<S> a rust prevention treatment , like bluing , inhibitors , humidity control , coatings , and galvanization , will be necessary . </S>", "<S> the galvanization process aims to protect the surface of the material by depositing a layer of metallic zinc by either hot - dip galvanizing or electroplating . in the hot - dip galvanizing process </S>", "<S> , a metallic bond between steel and metallic zinc is obtained by immersing the steel in a zinc bath at a temperature of around 460c . </S>", "<S> although the hot - dip galvanizing procedure is recognized to be one of the most effective techniques to combat corrosion , cracks can arise in the intermetallic  layer . </S>", "<S> these cracks can affect the life of the coated material and decrease the lifetime service of the entire structure . </S>", "<S> in the present paper the mechanical response of hot - dip galvanized steel submitted to mechanical loading condition is investigated . </S>", "<S> experimental tests were performed and corroborative numerical and analytical methods were then applied in order to describe both the mechanical behaviour and the processes of crack / cracks propagation in a bimaterial as zinc - coated material . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Objectives", "3. Material and Experimental Procedures", "4. Simulation Procedure", "5. Numerical Results and Discussion", "6. Conclusion"], "sections": [["the knowledge of mechanical materials behaviour is of great importance particularly for some innovative engineering applications , which involve a single material , bimaterials , or multiple materials .", "some bimaterials are obtained by means of a coating process and consist of coupled layers that may or may not contain filler elements , such as a foaming operation , or may be developed as welded substrates . usually , at the end of the process , the material is identified as a new single material .", "the coating procedure developed and studied in this work refers to the hot - dip galvanizing treatment , considered as a better treatment to fight against corrosion .", "the role of a hot - dip galvanizing treatment consists in the deposition of a protective external layer of metallic zinc obtained by immersing the steel in a zinc bath at a temperature of around 460c .", "when the material ( i.e. , steel ) is introduced into the zinc bath and then removed , several changes in the chemical composition and in the mechanical structure can occur .", "these changes produce a new structural arrangement on zinc substrate and are usually revealed by the generation of cracks in the zinc layer .", "the behaviour of materials may change as a result of various events such as fatigue , fracture , wear , fretting fatigue , creep , hydrogen embrittlement , thermal shock processes , or atmospheric attacks .", "an important role in these failure processes is played by any discontinuity in the material that can appear during the manufacturing process or during working period , or as a result of inappropriate use . in our case", "it appears as a consequence of the hot - dip galvanizing treatment applied as described above in association with the action of the mechanical loading .", "recently , krishnan and xu   focused on failure mechanics of adhesive joints ( i.e. , considered as bimaterials ) using a fringe pattern concentrations technique to describe the bimaterials interface .", "as analytical model , they used the fracture mechanics approach , while to compute the stress singularity in the bimaterial layer they took into account the stress intensity factor in mode i. the formula used to express this stress intensity factor is \n ( 1)ki = rekai , \n where k is the stress intensity factor in the case of the bimaterial component : \n ( 2)k = ytaaiei , \n where t = p(3s / w ) and y ,  are the calibrating factors that depend on a / w , b / w , respectively , dundurs ' parameters .", "then a , b , and w represent the length of the possible crack , the thickness , and the length of the specimen .", " is a function of dundurs ' parameters ,  , and is presented as \n ( 3)=12ln11+ , \n where  , dundurs ' parameters material , is expressed by \n ( 4)=112221211122+2121 \n in which 1 and 2 denote the material , while  and  are the shear modulus and poisson 's ratio , respectively .", "the elastic fracture theory is a suitable tool to compute the mechanical behaviour in case of coatings , composites , and welded structures .", "the bimaterial produced by the reactions that occur during the hot - dip galvanized steel process must have the same conditions on the contact surface as the  linear spring - like  model .", "the discontinuity of displacement across the interface is assumed to be linearly proportional to the displacement at the interface of the constituent where the stress source is located .", "the present authors agree that the two materials that form the bimaterial should be considered as a functionally autonomous subsystem with a different young 's modulus and poisson 's coefficients .", "this implies a strain incompatibility between the two solids and the formation of a periodic distribution of tensile and compressive stress in checkerboard patterns under the uniaxial tensile test .", "a robust approach was proposed by yu et al .   in order to formulate analytical solutions through the use of  linear spring - like model .  according to this theory we can define the type of contact between the surfaces by the fraction of the adherent area , applying the following formula : \n ( 5)=aacac , \n where  is used to quantify the extent of bonding at the interface , a is the total area of the interface , and ac is the area covered by paper ( see example on figure 2 from ) . according to the study made by panin et al .", ", the interface of solid materials covers a sinusoidal surface layer due to a rotation of successive constraints in tensile and compression stress in the structure .", "this effect is described by the following equation : \n ( 6)=aysinxlxt2 , \n where ", "t  is the thickness of the coating , x is the distance of crack propagation , y is the stress coefficient ."], ["this paper addresses two goals : first of all , to experimentally characterize the layer of zinc applied during the hot - dip galvanization process , in order to obtain necessary information about the uniformity of the zinc layer and the number of cracks and their length , and finally to estimate the behaviour of the cracks located in zinc layer when the mechanical loading is imposed .", "secondly , we proposed to corroborate the experimental results with analytical and numerical computations , ascertaining where the crack discontinuities spread while considering the three main possibilities of crack development : arrest at the bimaterial interface , crossing into the second material , in our case steel , and finally creating a deflection between the two materials . in the literature", "k. m. mrz and z. mrz   predicted the behaviour of bi- and multimaterial interfaces according to a simplified approach using the mk - criterion based on the linear elastic fracture mechanics ( lefm ) . in this criterion", "it is assumed that the crack growth follows the direction of minimum distortion energy density at a distance corresponding to a specified value of dilatation energy .  ", "figure 2 shows a specific case in which the crack is considered to propagate perpendicularly to the interlayer and make a bifurcation at the interface .", "the decohesion phase may present as one of the four possible scenarios whereby the crack will propagate as follows : ( i ) the plastically weaker material ( wm ) to the plastically stronger material ( sm ) , wm - sm , ( ii ) the plastically weaker material ( wm ) to the plastically stronger interlayer ( si ) , wm - si , ( iii ) the plastically stronger material ( sm ) to the plastically weaker material ( wm ) , sm - wm , and ( iv ) the plastically stronger material ( sm ) to the plastically weaker interlayer ( wi ) , sm - wi . in simple terms , the criterion for crack initiation and propagation along the interface could be considered , where the maximum stress , max , is expressed as \n ( 7)max=x+y2+x+y22+xy . to solve the problem of deflection at the interface of two materials , hutchinson   proposed the following condition : \n ( 8)gcgc1<gdgp1 , \n where g = gc represents release energy rate of the interface bimaterial .", "studies , related to the problem of cracks running perpendicular to a bimaterial , yields a different expression for the stress intensity factor that is connected to the stress tensor as follows : \n ( 9)ij = kr1fij. to determine the propagation energy of the crack running perpendicular to the interface , also called energy release rate , the following equation studied by madani et al .  ", "can be applied : \n ( 10)gd=(11)/1+(11)/24cosh2k12+k22gp=1222kp2 . \n in ( 8) , ( 10 ) ", "gd and gp are the strain energy release rate for deflection and penetration ; k1 and k2 are the stress intensity factors for the interface crack ; kp is the value of stress intensity factor , for a particular case , when the crack from material 1 penetrates into material 2 ( see figure 2 ) .", "analytical techniques and the green function can be employed to describe the behaviour of bimaterial compounds by calculating numerical solutions of singular integral equations , as was applied by erdogan et al .   and used by pruncu et al .   and azari et al . , which yield the following expression : \n ( 11)ki(a)=211+k1a0g(1)kib=211+k1a0g1 . a more general form of such approach could be structured using an algorithm that describes the numerical procedure for obtaining the different values of the function g(ti ) by the following .", "( 2 ) for k ranging from 1 to n , we need a computing route for xk and f(xk ) .", "( 3 ) for every xk and for i variants from 1 to ( n  1 ) , we have to calculate the first ti that is a weight function of the jacobi polynomials described as follows : \n ( 12)1ng(ti)1tixk+k(xk , ti)=0  withti = cos2i12n ,  i=1,  ,nxk = coskn ,  k=1,  ,n1 .", "( 4 ) the computation then yields a matrix relationship expressed as follows:(13)1t1x1+k(x1,t1)1t2x1+k(x1,t2) .... 1tnx1+k(x1,tn) .............................. 1t1xk+k(xk , t1)1t2xk+k(xk , t2) .... 1tnxk+k(xk , tn)gt1gt2 .... gtn = fx1fx2 .... fxk .", "g  are obtained by multiplying the transposed of the matrix by the vector f(xi ) .", "one has the following : a0 is half - length of the crack ; 1 , 2 are shear modulus for materials 1 and 2 ; c is the distance from the middle of the crack at the interface plane ; r ,  are polar coordinates , k = 3  4 for the plane strain case and k = ( 3)/(1 +  ) for the plane stress case ,  is poisson coefficient , and g is the parameter that expresses the magnitude of the applied load . another analytical technique , which was employed by chen et al .   using complex potential values ,", "was obtained in the following manner : \n ( 14)ki(b)=limr02rx=22k2 + 1a0m=11mmki(a)=22k2 + 1a0m=1m , \n where a0 is half - length of the crack , m = m/b , m = 0,1 , 2,3 ,  ,  b is the distance between the interface and the crack front , x is the distribution of stress in front of the crack , k = 3  4 for the plane strain case and k = ( 3  4)(1 +  ) for the plane stress case , and 2 is shear modulus for the material in which the crack is located ."], ["because of its multiple properties , steel is one of the most versatile materials used in industrial engineering applications .", "the european standard ( en ) reveals different types of steel employed in fields such as the automotive industry and aeronautical design . in this work we considered two steel alloys : he360dr and s420mc .", "their mechanical characteristics are summarized in table 1 . during the lifetime of service , in contact with the environment", "this metal may develop problems that could be prevented by applying the above - described process of hot - dip galvanization .", "the specimens obtained after hot - dip galvanization were submitted in laboratory to fatigue tensile test in order to detect their behaviour under fatigue conditions conforming to real life and to observe the changes that occurred in the metallic zinc layer .", "the fatigue tests were performed on a servohydraulic testing machine capable of applying axial loads up to 100  kn , using a sinusoidal load wave , with a frequency of 30  hz and a strength ratio r = 0.1 .", "after the fatigue test the specimens were experimentally analysed by optical microscopy and scanning electron microscopy ( sem ) . during observation with the optical microscope ,", "a sample of length 5328  m was considered . to simplify the management of these optical microscope observations , we divided the sample size into 16 units with a length of about 333  m .", "the main purpose of this was to analyse ( i ) the number of cracks per unit of length ; ( ii ) whether the evolution of the number of cracks was constant over all layers applied ; ( iii ) which material was prone to develop longer cracks , and finally if the zinc layer was constant over the whole steel surface .", "the first result was the average number of cracks per unit of length , as summarized in table 2 .", "the letters b and h in table 2 indicate the time of immersion in the zinc bath , b being 3 minutes of immersion and h 7 minutes .", "the numbers 9 , 8 , 7 , and 2 indicate the specimen number analysed . from table 2", "we can observe that the average number of cracks per unit of length is about 6/11 for he360dr and 7/8 for s420mc .", "a first remark proves that the number of cracks increases with the time of immersion .", "the results are plotted in figure 3 and highlight the number of cracks for all 16 units of length .", "figure 3 shows that the number of cracks on the entire length of zinc layer that encloses each unit is overall scattered ; however , on the local case of two / three consecutive units , the number of cracks per unit seems to be almost uniformly distributed . a key element for determination of the efficiency of the coating process", "may be deduced from the evolution of cracks lengths before and after the fatigue tests . according to the observations shown in figure 4", ", there was a significant change of crack length as a consequence of fatigue test .", "a gap of about 25  m in crack length before and after cyclic load was found due to the weakened structure of material .", "this observation was done in a useful sample area ( ua ) and then in the area where the test had less influence on the behaviour of the piece , marked as the nonuseful area ( nua ) .", "the uniformity of the zinc layer was considered as another issue that can stimulate the crack behaviour .", ", the zinc layer deposited over the steel alloy is uniformly distributed , having a value of 5080  m .", "the particularity of the size of substrate of he360dr h8 materials , that is , the thin thickness , may be explained as a consequence of immersion time .", "so , because the time of immersion within the bath of zinc was less than the time imposed by our methodology , the thickness of substrate was lower ; this means that instead of 7 minutes we found that the real time of immersion was about 5 minutes .", "the optical measurements confirmed that the thickness deposited over the steel pieces was uniform and show that the zinc layer is composed of several multilayers .", "the substrates are denominated by gamma (  ) , delta (  ) , zeta (  ) , and zinc eta (  ) , as shown in figure 6 .", "figure 6 proves that most of the cracks are located in substrate  and may occur along the zinc grain boundaries . indeed , from our observations these cracks arise in this  substrate and then spread toward the steel - zinc interface .", "however , the analysis indicates a decrease of the crack magnitude near the interface , which means the crack growth is interrupted before it reaches the interface .", "observations from scanning electron microscopy ( sem ) exhibit even more clearly that , during the hot - dip galvanized process ( hdg ) , the quality of the bonded layers may influence the direction of the cracks , if the cracks touch the steel - zinc interface .", "this troublesome problem produces a sort of  debonding area  which forms a path for surface deflection of the cracks .", "the  debonding area  in steel - zinc adhesive layers is illustrated in figure 7 , created by the propagation of cracks at the interface between the zinc and steel and into the zinc substrates zeta (  ) and eta (  ) . for bimaterials , the interface crack problems could be due to the nonhomogeneity of the materials , which develop in the direction parallel to the crack tip . from these preliminary findings", "we could confirm that during the hdg process the zinc layer is uniformly deposited and the average number of cracks is significant on this layer .", "it seems that during the fatigue process the cracks get longer and spread toward the bonding interface , arresting at the bimaterial interface . in the worst scenario ,"], ["numerical computational technique was implemented by abaqus software , in order to corroborate the experimental data with analytical and numerical simulations and to highlight the effects of cracks that develop during the hot - dip galvanizing process . in order to implement the experimental data in the numerical model ,", "besides , this value corresponds with the thickness measured under the s420mc h2 materials ( see figure 5 ) , and it allows making the assumption that the crack / cracks imposed in our models will spread only toward the interface steel / zinc . if we consider a smaller thickness , for example , the size of layer measured under he360dr b9 material , and we adopt in the numerical model the critical size of crack detected after the loading test ( see figure 4 ) , it will be obvious that the crack will cross all the coating substrate .", "but this issue is far from our experimental observation , since we had observed that the crack growth is only toward interface steel / zinc .", "experimental data were introduced in our numerical model as the thickness size of zinc layer ( figure 5 ) , while the contour of zinc layer on the entire material surface is considered homogenous and uniform ( figure 5 ) .", "an initial length of crack and the maximum length of crack ( see figure 4 ) were imposed as constraint in the numerical program .", "the principles of linear fracture mechanics ( lfm ) were implemented in the numerical model in order to explain behaviour of crack at the interface steel / zinc that is assuming that all the materials contain a minimum flow that will grow during the mechanical loading .", "so , considering the crack located in zinc substrate ( figure 5 ) that will develop perpendicular to the applied load , it is possible to denote this state by mode i fracture .", "it should be noted that in the numerical model a mechanical loading of 500  mpa corresponding to an average of ultimate tensile strength of galvanized materials ( see table 1 ) was imposed .", "a value of about 30  m was selected as the initial length of the crack / cracks before propagation , in agreement with experimental data ( figure 4 ) .", "however , we know that the crack is bounded by two fronts , a and b. in this model , we assumed that the front of crack b was static and so it could not propagate , whereas the only front of crack a would spread ."], ["a basic model composed of two materials , m1 and m2 , as shown in figure 8(a ) , which forms the bimaterial body , was implemented .", "m2 represents the steel and m1 the zinc layer , delimited by the following values : h1 = 0.08  mm , h2 = 19.84  mm , c = 0.045  mm , w = 15  mm , 2a0 = 0.030  mm , and  = 0.5  m and submitted to a mechanical load ,  = 500  mpa .", "the applied load in this system will have direct consequence to the crack behaviour and define the presence of stress singularity on both fronts of the crack , tips a and b. the configuration for the crack fronts is shown in figure 8(b ) .", "settlement of the crack propagates performance was evaluated using the stress intensity factor ( sif ) parameter , derived from the singularity 1/r advanced in front of the crack tip .", "since the value of the stress intensity factor ( sif ) is obtained , we proceed to conclude if the crack crosses into the second material , ends at the bonded interface , or deflects between these two bodies .", "( a ) model with one crack in the bimaterial : in this case one single crack of an initial size of 30  m was considered . then the length was increased by 3  m in each simulation up to the maximum of 60  m ( i.e. , close to the experimental analysis shown in figure 4 ) .", "figure 9 shows how the stress distribution increases with the length of crack , assuming that the initial crack had the same stress value at both fronts a and b. two - dimensional finite - element meshes are virtually symmetrical near this bottom ( crack fronts ) and the symmetry showed almost the same value as the stress distribution , for each of the crack fronts , during the simulation .", "the shape of the stress - strain curve as a function of the crack length is illustrated in figure 10 .", "the shape of the curve grows wide with the increasing of crack length in front of crack tip a. the effect of stress distribution in front of crack can be converted into basic parameters that show the impact of crack behaviour , that is , the stress concentration factor ( scf ) . obviously , the stress concentration factor is the ratio of the maximum stress over the nominal stress , denominated kt and expressed as \n ( 15)kt=maxnom. while the front of crack b does change , the main role in this research was played only by the front of crack a located near the interface of the bimaterial .", "thus , figure 11 shows the effect of scf in the front of crack a. these results were corroborated with the theoretical stress concentration factor ( scf ) obtained with the online software .", "the results presented in figure 11 show a good agreement and underline that when the crack reaches the interface , there is a sudden increase in the stress distribution value .", "this sharp rise in values may result in the creation of a surface called a  debonding area ,  as shown in figures 9(c ) and 9(d ) .", "then , by assessing the crack propagation with lfm using ( crack ) stress intensity factors ( sif ) , k , it is possible to establish where the crack stops . to achieve even better accurate results , a comparison between an analytical model reported by erdogan et al .   and", "the difference between the numerical and analytical results is due to the use of a small number of constants in the analytical model .", "the results are shown in figure 12 , demonstrating the trend of the ( crack ) stress intensity factors ( sif ) during applied stress , in front of cracks a and b. from the curves drawn below , it can be seen that the value of the sif increases with the crack length , and the crack propagates up to a length of about 50  m . then stabilization and even a decline of the sif value", "the value of sif will increase sharply only if the flow area denoted as well as ", "since the value of the ( crack ) stress intensity factors ( sif ) is low for the front of both cracks a and b , it seems that the crack can not cross into the second material . \n", "( b ) model with two or more cracks : in the second assumption , a  multiple cracks  model , namely , with 3 cracks , was implemented using almost the correspondent algorithm but specifying 3 initial cracks with an initial size of 30  m for each crack , and then each crack would increase by 3  m , up to the maximum size of 60  m .", "thus , the maximum cracks value is assigned when the cracks reach the steel - zinc interface . in this model with 3 cracks the applied load ( 500  mpa ) was considered as statement from the case of the model with a single crack .", "the motivation to implement a model with three cracks aims to detect whether the situation changed due to the increased ( crack ) stress intensity factor ( sif ) values , as a result of the cumulative energy developed by these 3 cracks .", "related work has been reported by song et al .   who declared that the interface crack will propagate if the principal maximum stress at the crack tip exceeds a critical value .", "thus , confirming our initial supposition , the results presented in figure 14 show an increase for maximum stress value within front of the cracks . at the same time , the surface denominated  debonding area  appears more pronounced .", "the tendency was confirmed from the development of the ( crack ) stress intensity factors ( sif ) , k values , where the results are reported in figure 15 .", "the shapes of the curves in figure 15 show a similar trend obtained by the one in the case with one crack except for the fact that  crack 2  shows the highest influence of propagation , derived from the cumulated extension efforts from another two cracks .", "another thing to note is the gap difference of sif for crack lengths 57  m and 60  m .", "this difference could be explained by the appearance of the previously mentioned  debonding areas ", "( i.e. , imposed length for debonding area considered in this research was a maximum of two initial crack lengths , because after this size the peeling processes occur ) .", "although the difference is evident , it does not exceed the critical value of mode i fracture toughness ; for example , the value cited by ashby   for steel is about 80170  mpa  m. consequently , the crack will not cross into the steel material and in the worst case the crack / cracks will form a large bifurcation at the interface between these two materials ."], ["this research paper highlights by means of experimental , numerical , and analytical tool the behaviour of a bimaterial zinc / steel interface submitted to mechanical loading . in particular , the case of crack / cracks at the bimaterials interface was considered .", "the main objectives of this paper were to assess the behaviour of the crack / cracks generated at the end of the hot - dip process and to reveal that the numerical computations are in a good agreement with the experimental outcomes .", "optical microscopy and scanning electron microscopy ( sem ) techniques were involved to evaluate the experimental performance of the bimaterials compound .", "postmortem analysis of the fracture surfaces emphasized the uniformity of the zinc layer over the steel surface and provided accurate information related to the average of cracks length ( figure 4 ) .", "in addition , this technique allows quantifying the number of cracks ( figure 3 ) that forms in the zinc layers . in the meantime", ", this assessment provides patterns about the evolution of crack from incipient phase , where the cracks arise , and how far they spread . in numerical computations , simulations of the model considered", "were run in two different situations , namely , when the bimaterial contains just one crack or else 3 cracks , in order to prove the agreement between computation and experimental outcomes .", "besides , to get even accurate results , the analytical model used by erdogan et al .   was also confronted .", "the sif were calculated to obtain value of the stress singularity that characterizes the crack evolution in the bonding area of bimaterial , for both crack tip values , that is , the fronts of cracks a and b. the analytical , numerical , and experimental assessments prove that the crack / cracks that arise during the hot - dip galvanized steel process will propagate during the mechanical fatigue test .", "it was also established that the crack / cracks will stop near the steel - zinc interface at a distance of about 3  m and only in particular cases yield a deflection at the bimaterial interface . to summarize", ", the results may confirm that cracks reduce the life span of the bimaterial but are not responsible for a direct degradation of the steel .", "this method is therefore useful for employment in damage models that include the safety factor condition .", "finally , a nondestructive method should be employed to detect the behaviour of bimaterials for a better calibration ."]]}
{"article_id": "PMC5363384", "article_text": ["epilepsy is a common and serious chronic neurological disease that affects over 50 million people worldwide .", "anti - epileptic drugs ( aeds ) are effective at controlling seizures in many patients , acting through a range of neuronal targets , including ion channels , neurotransmitter release , and receptor mechanisms.1 , 2 however , a third of patients with epilepsy fail to achieve seizure freedom , and current aeds do not show disease - modifying properties .", "future treatments for epilepsy need to differentiate from currently marketed drugs , for example , by having a novel mechanism(s ) of action or providing disease - modifying effects.1 , 2 acquired epilepsy is thought to result from disturbances to the expression and function of neurotransmitters and their receptors , ion channels , and signaling components , as well as altered metabolism , neuronal and glial microstructure , neuroinflammation , and other mechanisms.3 , 4 , 5 it is likely that disease - modifying treatments will need to influence multiple genes within a given pathway or in various independent pathways .", "potential gene network control points were recently identified and include master regulators of transcription and neuroinflammation , epigenetic factors , and noncoding rnas .", "micrornas ( mirnas ) are a conserved family of endogenous small noncoding rnas that regulate protein levels in cells by post - transcriptional control of mrna stability and translation.9 , 10 this is achieved by sequence - specific binding of the mirna to complementary bases in the mrna target .", "since this generally requires only a 7- to 8-nucleotide match , individual mirnas can potentially target large numbers of mrnas .", "this provides the ability to regulate entire gene networks or multiple biological processes and has been demonstrated in the brain .", "microrna-134 ( mir-134 ) was identified as a brain - enriched , neuronally expressed mirna that controls dendrite spine morphogenesis , via repression of lim domain kinase 1 and other targets.14 , 15 this is potentially important for disorders of hyperexcitability , since dendritic spines are contact points for excitatory communication and there is evidence that reorganization of dendrites is a feature of experimental and human epilepsy.16 , 17 we identified mir-134 among upregulated mirnas profiled in areas of hippocampal damage following status epilepticus induced by intra - amygdala kainic acid ( ka ) in mice .", "expression of mir-134 is also upregulated after neuronal stimulation and seizures in various other in  vitro and in  vivo models,15 , 19 , 20 , 21 , 22 and mir-134 is overexpressed in the resected human neocortex from patients with intractable temporal lobe epilepsy ( tle ) . silencing mir-134 by intracerebroventricular ( i.c.v . )", "injection of locked nucleic acid ( lna ) , cholesterol - tagged antagomirs ( ant-134 ) reduced levels of mir-134 for at least a month and rendered mice refractory to the convulsant effects of both ka and pilocarpine.19 , 22 consistent with known mir-134 targets , lim domain kinase 1 and levels of other mir-134 targets were higher in ant-134-treated mice after status epilepticus and the dendritic spine volume was increased.19 , 22 notably , injection of ant-134 after status epilepticus suppressed the later occurrence of spontaneous recurrent seizures in the intra - amygdala ka model in mice .", "there is significant interest in developing mirna - based therapies.23 , 24 there are also barriers to antisense - based approaches and there has been some disengagement of industry from pursuing such efforts .", "this is likely to be a problem for any new therapy for epilepsy , particularly one that is not based on traditional small molecule chemistry.1 , 2 pre - clinical development of an mir-134-based treatment for epilepsy might be more attractive if additional important questions are answered .", "principally , work to date has only tested ant-134 in mouse models of status epilepticus .", "proof of seizure - suppressive effects of ant-134 in a non - status epilepticus model or in another species would bridge the gap in evidence and facilitate pre - clinical development of ant-134-based treatment for epilepsy.26 , 27 here , we evaluated mir-134 expression in the resected hippocampus from patients with pharmacoresistant tle and we tested ant-134 in three different models .", "we used the pentylenetetrazol ( ptz ) model in mice , a popular method for screening putative anticonvulsive effects that triggers seizures via -amino butyric acid ( gaba ) blockade.26 , 28 second , the perforant pathway stimulation ( pps ) model , a toxin - free model of acquired epilepsy based on nonconvulsive status epilepticus that avoids the confounding influence of exposing the brain to chemoconvulsants , was used in rats .", "finally , we used a high - potassium model of epileptiform activity , using ex  vivo brain slices from rats pre - treated with ant-134 .", "our results establish that targeting mir-134 offers broad anticonvulsant and possibly disease - modifying effects in experimental epilepsy , which may encourage pre - clinical development of ant-134 as a treatment for epilepsy .", "for this , we analyzed levels of mir-134 in the surgically obtained hippocampus from adults with pharmacoresistant tle and we compared findings to autopsy hippocampal samples from people who died of causes unrelated to neurological disease .", "these samples were used previously to analyze expression of other genes , and clinical data for both groups are briefly summarized in tables s1 and s2 .", "there were no between - group differences in patient age , and each group included males and females .", "we detected higher levels ( p  = 0.037 ) of mature mir-134 in the tle specimens compared to autopsy samples ( figure  1a ) .", "this difference is unlikely to be an artifact of post mortem delay , since mir-134 levels did not decrease in simulated autopsy experiments lasting up to 12  hr . we began by establishing dosing parameters in the mouse ptz model .", "generalized tonic - clonic seizures were first reliably elicited at a dose of 60  mg / kg in c57bl/6j mice ( figure  1b ) .", "a dose of 80  mg / kg elicited generalized tonic - clonic seizures in all mice and with a more consistent and shorter latency than with the 60 mg / kg dose ( figure  1b ) .", "a dose of 100  mg / kg also produced rapid - onset tonic - clonic seizures in all mice but was associated with high mortality ( figure  1b and data not shown ) .", "ordinal logistic regression analysis was used to explore the dose dependence of the ptz - induced convulsions .", "ptz dose was significantly associated with racine scores ( figure  1c ) . a dose of 80  mg / kg was selected for further experiments .", "to obtain molecular evidence for ptz - induced seizure activity in the model , we measured expression of activity - regulated genes in the hippocampus .", "transcript levels of fbj osteosarcoma oncogene ( fos ) , a calcium - dependent marker of neuronal activity and activity regulated cytoskeletal - associated protein ( arc ) , another immediate early gene responsive to intense neuronal activity , were strongly increased in the hippocampus 30  min , but not at 4  hr , after ptz - induced seizures ( figures 1d and 1e ) .", "there was no evidence of irreversible neuronal injury in tissue sections from mice after ptz , as assessed by fluoro - jade b ( fjb ) and neun ( rna binding protein , fox-1 homolog [ c.  elegans ] 3 ) staining ( data not shown ) .", "expression of mir-134 is known to increase in various in  vitro and in  vivo seizure models.15 , 19 , 20 , 21 , 22 we therefore investigated whether ptz - induced convulsions alter mir-134 levels in mice .", "taqman mirna assays showed that levels of mature mir-134 were significantly increased in the hippocampus , but not in the cortex , 30  min after ptz - induced generalized tonic - clonic seizures in mice ( figure  1f and data not shown ) .", "injection of antagomirs targeting mir-134 ( ant-134 ) in mice.19 , 22 previous work has established a dose of these antagomirs that reduces hippocampal mir-134 levels by over 90% by 24  hr after injection .", "accordingly , we tested ptz responses 24  hr after injection of ant-134 and compared them to responses in mice that received a scrambled antagomir ( scr ) . in scr mice , delay to onset of first tonic - clonic seizure after ptz injection was similar to that observed before in control mice ( figure  2a , and compare to figure  1b ) .", "thus , the control antagomir did not alter the normal response to ptz in the model .", "the time to first ptz - induced tonic - clonic seizure was significantly longer in mice injected with ant-134 compared to scr - injected animals ( figure  2a ) .", "the severity of racine - scored ptz - induced seizures was also significantly lower in ant-134- compared to scr - injected mice ( figures 2b and 2c ) .", "analysis of electrographically recorded seizure activity determined that electroencephalography ( eeg ) total power was lower in ant-134 mice compared to the control group after ptz ( figure  2d ) .", "we next analyzed brain tissue samples from scr- and ant-134-injected mice for evidence of mirna silencing and differences in expression of activity - regulated genes .", "analysis of the hippocampus obtained after ptz - induced seizures confirmed that mir-134 expression was lower in ant-134 pre - treated mice compared to scr - treated animals ( figure  3a ) .", "ant-134 had no effect on levels of an unrelated mirna ( figure  3b ) .", "next , we assessed expression of arc and c - fos as molecular markers of recent neuronal activity to support the clinically scored behavior and eeg differences in ant-134 mice . quantitative real - time pcr analysis revealed increased c - fos expression in the hippocampus of scr - injected mice after ptz - induced seizures ( figure  3c ) .", "in contrast , c - fos levels were lower in ant-134-injected mice , consistent with reduced seizure severity ( figure  3c ) .", "this difference was also apparent in the neocortex from the same animals ( figure  3c ) .", "expression of arc was increased in the hippocampus of scr - injected mice that received ptz .", "arc levels were not significantly different in ant-134 mice compared to scr - injected animals in the hippocampus and neocortex after ptz - induced seizures ( figure  3d ) .", "we turned next to the pps model in rats , first investigating effects on mir-134 levels .", "taqman mirna assays showed that levels of mature mir-134 were not significantly modified in the hippocampus of rats at two different time points ( figure  4a ) .", "next , we assessed the effect of silencing mir-134 before stimulation on seizure severity during status epilepticus .", "rats were pre - treated with ant-134 or scr ( i.c.v . ) 24  hr before the 8-hr stimulation protocol .", "analysis of electrographically recorded seizure activity determined that scr - injected rats presented a mean (  sem ) increase of 1,304.3%   94.9% in eeg total power relative to baseline .", "this was similar to animals that were stimulated but not receiving the oligonucleotide ( data not shown ) . in rats", "pre - treated with ant-134 , the severity of evoked seizures in the pps model was similar : eeg total power during seizures was a mean (  s.e.m ) of 1,244.6%   82.7% ( percent of baseline ) . despite not seeing an acute anticonvulsant effect of ant-134 in the pps model , we were nevertheless interested in whether silencing mir-134 after status epilepticus in the model would alter the emergence of recurrent spontaneous seizures . a key prior", "finding was that silencing mir-134 after status epilepticus induced by intra - amygdala ka reduced the subsequent occurrence of spontaneous recurrent seizures by over 90% .", "for these studies , rats underwent 8-hr stimulation of the perforant pathway to induce epilepsy and the i.c.v .", "rats were then monitored with video - eeg until a first spontaneous seizure was recorded , typically 1025  days after pps , and they were then followed for a further 8  weeks ( figure  4b ) .", "epilepsy developed in all scr - injected rats over the expected course , with the first spontaneous seizures appearing 14  weeks after status epilepticus ( figures 4b and 4c ) .", "scr - injected rats averaged 20 spontaneous seizures recorded during the 8-week recording time ( figures 4d and 4e ) .", "in contrast , no spontaneous seizures were detected over the 8-week period in six of seven rats injected with ant-134 after status epilepticus ( figures 4d4f ) .", "epilepsy emerged in only a single rat treated with ant-134 ( figures 4d and 4f ) .", "the fisher exact test determined that there was a significant difference in epilepsy development between the groups ( p  = 0.005 ) . in terms of effect size , ant-134 prevented 86% of the risk of epilepsy , with a 95% confidence interval ( ci ) of 12%98% .", "an analysis of the duration of spontaneous seizures ( electrographically defined ) revealed that when spontaneous seizures occurred , they were similar between groups .", "that is , the average duration of a spontaneous seizure in scr - treated rats was 53.7   14.4  s ( n  = 20 seizures , from five rats ) , which was similar to the duration in epileptic rats in the pps model that did not receive an i.c.v .", "injection ( 49.4   15.9 , n  = 20 seizures ) . in the ant-134 rat that developed epilepsy ,", "the average duration of a spontaneous seizure was 52.3   16.2  s ( from n  = 12 seizures ) .", "having observed a disparity between the acute anti - seizure effects of ant-134 pre - treatment in the ptz and pps models , we sought to examine the effect of ant-134 on naive , non - epileptic tissue and whether the effects were due to long range networks or if local circuitry was sufficient to mediate any seizure protection .", "we injected ant-134 in  vivo in rats and subsequently prepared ex  vivo brain slices ( figure  5a ) .", "these slices were then exposed to a 9-mm k seizure challenge , to determine whether ant-134 protects against epilepsy in local circuits of the isolated slice in the absence of epileptogenesis .", "onset of activity was delayed in ant-134-treated slices by 176  s relative to the control ( figures 5b and 5c ) .", "this effect was statistically significant ( mann - whitney u test , p  = 0.002 ,   = 0.025 , power  = 0.87 ) .", "we also compared the power of epileptiform activity and duration of seizure - like events in these data ; these effects were not statistically significant ( power : t test , p  = 0.70 ; duration : mann - whitney u test , p  = 0.067 ) .", "this finding suggests that ant-134 can confer seizure resistance in naive tissue and thus mediate its effect even in the absence of epileptogenesis .", "in addition , preservation of the seizure resistance in isolated slices indicates that at least some of the effects of ant-134 are due to local changes in activity .", "the present study provides important additional evidence of the anticonvulsant and disease - modifying effects of lna - based antagomirs targeting mir-134 in animal models of epilepsy .", "our study shows that ant-134 is an anticonvulsant in an acute chemoconvulsant model ( ptz in mice ) and ant-134 had anti - epileptogenic activity in the perforant pathway stimulation model of epilepsy in rats .", "ant-134 also confers resistance to epileptic activity in naive tissue , reflected as a delay to activity onset following a seizure challenge in ex  vivo brain slices .", "we also report that mir-134 levels are elevated in the hippocampus of patients with pharmacoresistant tle .", "the therapeutic potential of targeting mir-134 in established epilepsy will require additional studies , but our findings may encourage further research or pre - clinical development of this mirna - based treatment for epilepsy . despite the recent introduction of two additional aeds ( perampanel and brivaracetam ) , new drug development in epilepsy", "faces significant challenges.1 , 2 , 32 new targets are required that can treat pharmacoresistant patients or have disease - modifying effects that are not served by current therapies .", "mirnas have emerged as potential targets of the future via their influence on neuronal microstructure , gliosis , neuroinflammation , and ion channels.8 , 33 silencing mir-134 has produced the most convincing anti - seizure effects to date , reducing status epilepticus in pre - treatment paradigms in the intra - amygdala ka and pilocarpine models and exerting potent long - term effects on spontaneous seizure development.19 , 22 this mirna is also an attractive target , since studies show increased levels of mir-134 within the temporal lobe of adult and pediatric patients with intractable epilepsy.19 , 20 we report here that mir-134 is also elevated in the hippocampus of adults with pharmacoresistant tle , although there are potential confounders with comparing resected material from patients to autopsy control samples . the present study has answered key questions that could facilitate pre - clinical development by showing that ant-134 has anticonvulsant effects in non - status epilepticus models and is disease modifying in another species .", "the first important finding was that pre - treatment with ant-134 strongly suppressed seizures in the mouse ptz model .", "effects on both clinical behavior , including delaying the time to first convulsive seizure , and electrographic seizure activity were seen .", "thus , lowering brain levels of mir-134 can exert anti - excitability effects , and this implies that mir-134 has a role in setting excitability thresholds in the brain .", "this is thought to be due to regulation of proteins that control dendritic morphology,14 , 15 although other targets are known .", "ant-134 did not fully prevent seizures in the ptz model ; rather , it delayed and minimized their severity , a finding most closely matching the effect of ant-134 in the pilocarpine model of status epilepticus", ". it may not be possible to fully prevent all seizures using ant-134 in chemoconvulsant models , but further adjustments to antagomir dose or delivery may yield optimal seizure - suppressive effects .", "ptz triggers seizures via gabaergic blockade ; this differs from the mechanism by which status epilepticus is induced by ka and pilocarpine , which work by promoting glutamatergic and cholinergic transmission , respectively .", "this suggests that ant-134 may be effective for epilepsies of various etiologies , which , in patients , can vary considerably between trauma , stroke , infection , genetic , and other causes .", "our studies also show that ptz - induced convulsions increase brain levels of mir-134 , transcription of which is probably mediated by myocyte enhancer factor 2c .", "we did not observe an increase in mir-134 after perforant pathway stimulation in the rat .", "this was surprising , although changes to mir-134 levels are not always detected after chemoconvulsive stimuli , and we can not rule out a technical explanation ( e.g. , the time points selected or a subfield - specific effect that was masked by the use of the whole hippocampus ) .", "our study included an analysis of the short - term effects of ant-134 on neuronal activity - regulated genes in the ptz model .", "expression of genes such as c - fos and arc is a well - established surrogate marker of recent seizure activity , the protein products of which are involved in coordinating synaptic plasticity and adaptation to excessive neuronal stimulation.38 , 39 unexpectedly , only levels of c - fos , and not arc , showed the expected reductions in ant-134 mice after ptz seizures .", "it is known that c - fos induction is calcium dependent , whereas arc expression can be induced via calcium - independent pathways.38 , 39 therefore , ant-134 treatment may have reduced seizure severity sufficient to prevent c - fos induction , such as by minimizing n - methyl - d - aspartate ( nmda ) receptor opening , but enough residual neuronal excitation remained to upregulate arc .", "these findings provide additional evidence of the scale and nature of seizure suppression by ant-134 and suggest molecular markers that can distinguish effects of mirna manipulations on different components of seizure responses . in the present study", ", we observed that pre - treatment of rats with ant-134 did not protect against perforant pathway stimulation - induced status epilepticus .", "while there are examples of anticonvulsants producing effects in some but not all models , these findings suggest that ant-134 may have the most clinical value as an anti - epileptogenic treatment .", "previous work showed that injection of ant-134 after status epilepticus in mice suppressed the later occurrence of spontaneous seizures by 90% , evidence of a disease - modifying effect in the intra - amygdala ka model . here", ", we found nearly identical anti - epileptogenic effects of ant-134 in a different model in rats .", "this is the only demonstration , to our knowledge , of any disease - modifying treatment in the pps model in rats .", "the pps technique induces hippocampal sclerosis and recurrent spontaneous seizures,29 , 40 and it provides a means to trigger epilepsy without the bias or other issues associated with chemoconvulsants .", "ant-134 was injected shortly after triggering status epilepticus in order to model a reasonably realistic clinical scenario , such as a patient being treated shortly after an initial precipitating injury .", "ant-134 had dramatic and potent effects , with the majority of ant-134 rats ( six of seven , 86% ) never developing spontaneous recurrent seizures .", "notably , spontaneous seizures in the study with the intra - amygdala ka model did occur in all ant-134 mice , albeit with dramatically lower seizure frequency .", "this difference may relate to the extent of hippocampal injury or another aspect of the epileptic phenotype , such as the frequency of spontaneous seizures .", "thus , presently we see a possible additional therapeutic action of ant-134 , whereby post - treatment is capable of fully preventing epilepsy at least in some models .", "we can not , of course , exclude that epilepsy might have developed in some of the ant-134 rats at some point in the future . however , we recorded for 8  weeks beyond the standard latent period .", "we further corroborated the anti - seizure effect of ant-134 by using an acute seizure challenge with high potassium in otherwise healthy ( ex  vivo ) tissue .", "inhibitory neurotransmission is retained in this model ( in contrast to ptz ) and there is no hippocampal sclerosis ( unlike pps ) , further suggesting that ant-134 can be therapeutic in multiple epileptic syndromes with varying mechanisms .", "we observed a delay in the onset of epileptiform bursts in brain slices that had been pre - treated with ant-134 .", "this finding suggests that epileptogenesis is not required for ant-134 to mediate its effects on seizure onset .", "we did not see any significant changes in the power or duration of epileptiform activity ; however , these parameters are highly variable in brain slices , which can obscure potential effects . in addition", ", we propose that ant-134 can mediate seizure protection in the hippocampus via effects on local circuitry , because long range connections would likely be severed during the slicing process .", "the anti - epileptogenic actions of ant-134 may be independent of the etiology of the initial precipitating injury , although this will require further testing ( e.g. , in models of trauma or infection - induced epilepsy ) .", "in addition , upregulation of mir-134 is not essential for ant-134 to produce anti - epileptogenic effects , since mir-134 levels were not altered after pps - induced status epilepticus .", "thus , epilepsy can be dramatically suppressed in two different in  vivo models , and one in  vitro , in two different species .", "this provides strong support for and is an argument for further development of ant-134 for epilepsy treatment or prevention .", "antagomirs targeting a liver - expressed mirna have been given to humans and were found to be well tolerated and effective against hepatitis c in clinical trials .", "there are concerns , obviously , that because ant-134 can alter dendritic spines , it could have effects on cognition . studies to date have not identified such problems , with natural exploratory behavior and performance in a hippocampus - dependent task found to be normal in mice a few days after i.c.v .", "injection of ant-134.19 , 22 however , more demanding tests of cognition or learning will need to be performed .", "another factor that argues for general safety is that mice and rats have been recorded for at least 2  months after brain injections of ant-134 .", "nevertheless , pre - clinical development will necessitate an extensive battery of toxicity and safety testing .", "work to date on ant-134 satisfies certain recommendations for testing and evaluating novel therapies for epilepsy , with seizure - suppressive effects demonstrated in five different models and two different species.1 , 2 , 43 , 44 there are , however , additional seizure models that could be used during any future pre - clinical development .", "this includes kindling , a popular model that has been successful in identifying aeds with novel mechanisms of action .", "while this is not an essential step before pre - clinical development , sex differences in mirna inhibitor responses may be worth investigating in future studies and sexual dimorphism in mirna expression has been reported , although not for mir-134.46 , 47 it will also be of value to understand how ant-134 produces its potent seizure - suppressive effects .", "the most likely mechanism is upregulation of one or more proteins due to de - repression of a mir-134 target .", "previous work has shown that mir-134 is uploaded into the rna - induced silencing complex after seizures , which facilitates targeting of mrnas , and in  vitro studies suggest that the neuroprotective effects of ant-134 are due to de - repression of lim domain kinase 1 .", "ant-134 treatment also increases dendritic spine volume and changes spine number in the hippocampus,19 , 22 which could influence excitability and would also be consistent with a mechanism involving protection of mir-134 targets such as lim domain kinase 1 or pumilio rna - binding family member 2", ". altered spine volume and number is consistent with our observation of a robust local effect of suppressing mir-134 on delaying epileptiform activity , although additional effects on long range connections can not be ruled out .", "we must also consider that the acute seizure - suppressive effects and long - term anti - epileptogenesis effects may arise from different mechanisms .", "ultimately , in  vivo studies will be necessary to determine the anti - seizure mechanism of ant-134 ( e.g. , by analyzing gene expression networks , quantifying the proteome , and applying techniques to validate antagomir - mirna targeting such as polysome shift assays ) .", "what are the next steps for translation or pre - clinical development ? an obvious question is whether and how antagomirs could be given to patients .", "their large size precludes their passing an intact blood - brain barrier ( bbb ) .", "recent work has explored methods to circumvent the bbb , including conjugating antagomirs with brain - penetrating peptides or administering antagomirs via intranasal injection.19 , 51 the bbb may also be open after certain epilepsy - precipitating injuries or it can be physically breached , such as through use of ultrasound . another question is whether ant-134 injection into already epileptic animals could alter the frequency or duration of spontaneous seizures or attendant hippocampal pathology .", "success with that approach could provide novel treatment opportunities for the population of drug - resistant patients for whom surgery or other alternatives to aeds are either not possible or not effective currently . in summary ,", "the present study provides important additional validation of the potent seizure - suppressive and possibly disease - modifying effects of ant-134 in two different animal models and one brain slice model .", "the effectiveness of ant-134 and the potency of seizure suppression suggest a target for drug development in epilepsy that could provide additional effects beyond those provided by any currently marketed drug for epilepsy .", "this study was approved by the ethics ( medical research ) committee of beaumont hospital of dublin ( no . 05/18 ) and written informed consent was obtained from all patients .", "the control ( autopsy ) hippocampus ( n  = 12 ) was obtained from 12 individuals .", "patients ( n  = 12 ) were referred for surgical resection of the temporal lobe by an epileptologist ( n.d . ) following neurological assessment , video - eeg recording , and mri / neuroimaging . each patient was determined to have medically intractable tle with a history of recurring seizures ( table s2 ) .", "the hippocampus was obtained and a portion of each specimen was frozen in liquid nitrogen and stored at 70c until use .", "a pathologist ( m.a.f . ) assessed the  hippocampus as part of the pathologic evaluation , addressed the degree of hippocampal sclerosis , and described other pathologic changes .", "all animal experiments were performed in accordance with the european communities council directive ( 2010/63/eu ) .", "procedures in mice were approved by the research ethics committee of the royal college of surgeons in ireland ( rec-842 ) , under license from the ireland health products regulatory authority ( ae19127/001 ) .", "procedures in rats were performed under authorization of the philipps university bioethics committee and were approved by the local regulation authority ( regierungsprsidium gieen ) , or they were performed according to the uk animals ( scientific procedures ) 1986 act ( ppl 70 - 7684 ) and approved by local ethical review ( university college london ) .", "male adult c57bl/6j mice ( 2025 g ) and male sprague - dawley rats ( 325350  g or 200300 g ; all from harlan ) were used in all studies .", "animals were housed in on - site barrier - controlled facilities having a 12-hr/12-hr light / dark cycle with ad libitum access to food and water .", "mice were equipped for eeg recordings under surgical anesthesia ( isoflurane ; 5% induction , 1%2% maintenance ) in a mouse - adapted stereotaxic frame .", "body temperature was maintained within the normal physiological range with a feedback - controlled heat pad ( harvard apparatus ) .", "after a midline scalp incision , partial craniectomies were performed and animals had skull - mounted recording electrodes placed and fixed with dental cement .", "a second cohort of mice had eeg electrodes and a guide cannula implanted to allow i.c.v .", "this cannula was placed on the dura mater with the following coordinates from the bregma : anterior - posterior ( ap ) ,  + 0.3  mm ; lateral ( l ) ,  + 0.9  mm ; and ventral ( v ) , 2.0  mm .", "after recovery from surgery , mice were connected to the lead socket of a swivel commutator , which was connected to a grass twin digital eeg system .", "] ) with different doses of ptz ( 60 , 80 , or 100  mg / kg ) . lorazepam ( 8  mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .", "mice were followed for 30  min or 4  hr after ptz administration for the appearance of seizures by electrographic and behavioral methods .", "after the observation period , mice were deeply anesthetized ( pentobarbital ) and transcardially perfused , and their brains were removed for analysis of gene expression or histological assessment .", "injection of 0.12  nmol/2  l 3-cholesterol - tagged locked nucleic acid oligonucleotide targeting mir-134 ( ant-134 ) or its respective control , a non - targeting scrambled version of the antagomir ( scr ; 0.12  nmol/2  l ) ( exiqon a / s ) , as previously described.19 , 22 twenty - four hours later , mice were connected to an eeg system and a baseline recording was obtained .", "next , mice were injected with ptz ( 80  mg / kg , i.p . ) .", "lorazepam ( 8  mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .", "these mice were followed up for 30  min after ptz administration for the appearance of seizures .", "after this period , mice were deeply anesthetized ( pentobarbital overdose ) and transcardially perfused ( pbs ) for further assessments .", "under isoflurane ( 5% induction , 3% maintenance ) anesthesia , bipolar stainless - steel stimulating electrodes ( nex-200 ; rhodes medical instruments ) were positioned in the angular bundles of the perforant pathway and custom unipolar recording electrodes ( crafted from 796000 ; a - m systems ) were lowered into the dorsal dentate gyrus .", "electrodes and ground screws were connected to miniature wireless transmitters ( ft20 ; data sciences international ) that were implanted subcutaneously on the animal s flank .", "plastic connectors ( gs09plg ; ginder scientific ) joined the electrodes with stimulation / recording equipment .", "the pps protocol utilized a paradigm designed to evoke and maintain hippocampal seizure activity throughout the stimulation , but not convulsive status epilepticus , which consisted of continuous , bilateral 2-hz paired - pulse stimuli , with a 40-ms interpulse interval , plus a 10-s train of 20  hz single - pulse stimuli delivered once per minute , generated by an s88 stimulator ( grass instruments ) .", "all pulses ( 0.1-ms duration ) were delivered at 2024 v , as this voltage reliably evokes granule cell discharging without tissue - damaging hydrolysis.29 , 40 the current associated with these voltages was typically between 15 and 30  a .", "no samples displayed any signs of hydrolysis ( e.g. , holes around the stimulating electrodes ) .", "as described previously , stimulation for 30  min ( on 2 consecutive days ) required only isoflurane to terminate seizures .", "eight - hour stimulation on the third day did not induce convulsive status epilepticus and , therefore , did not require pharmacological termination .", "rats were randomly divided in three cohorts to assess the following : ( 1 ) mir-134 expression after pps ( 24  hr , 4  days , and 14  days ) ; ( 2 ) the effect of ant-134 on pps - induced status epilepticus , in which rats were pre - treated with ant-134 or scr ( 0.36  nmol/6  l , i.c.v . ; exiqon a / s ) 24  hr before the 8-hr stimulation ; and ( 3 ) the effect of ant-134 on epileptogenesis , in which rats were injected with 0.36  nmol/6  l ( i.c.v . ) of ant-134 or scr ( exiqon a / s ) immediately after 8-hr stimulation .", "five rats were excluded from this study due to practical issues ( e.g. , blocked cannula ) . for statistical purposes", ", we pooled samples from 24  hr and 4  days after pps as an  early  time point , while 14-day samples were considered as the  late  time point .", "mouse eeg data were analyzed and quantified using labchart 8 software ( ad instruments ) .", "ptz - induced seizures were defined as high - amplitude ( > 2 baseline ) high - frequency ( > 5  hz ) polyspike discharges lasting > 5 s. for statistical purposes , we assigned a cut - off time of 1,800  s for those animals that did not present seizures during the observation period . from the eeg recordings ,", "we calculated the delay to first seizure , the total power , and the percentage of total power / spectral bands , as previously described.19 , 54 eeg total power was plotted as the percentage of baseline recording ( each animal s eeg power post - seizure compared to its own baseline eeg ) .", "clinical behavior was scored using the following adapted racine scale : score 0 , no seizures observed ; score 1 , rhythmic mouth and facial movement ; score 2 , rhythmic head nodding ( bobbing ) ; score 3 , forelimb clonus ; score 4 , rearing and bilateral forelimb clonus ; and score 5 , rearing and falling ( stay fallen on rear side ; tonic - clonic seizures ) .", "the highest score reached during every 5  min during the 30  min after ptz was recorded .", "for studies in rats , eeg activity during and after pps was amplified and recorded digitally at 2 khz utilizing a powerlab 16/35 and labchart software versions 7 and 8 ( ad instruments ) .", "spontaneous granule cell layer activity was recorded continuously ( 24 hr / day ) and stored digitally and automatically in 3-hr epochs . each day , we analyzed the preceding 24  hr of recordings , as well as all events with amplitudes exceeding 120% of average baseline .", "continuous ( 24 hour/7  day ) video monitoring utilized edimax  7010w infrared cameras .", "video files were captured at 1215  frames / sec and were time - stamped for integration with the electrophysiological data using securityspy surveillance software ( ben software ) and stored digitally . confirmed seizures were scored according to the racine scale .", "rats were anesthetized with isoflurane ( 5% induction,2% maintenance as appropriate ) and mounted in a stereotaxic frame .", "we injected ant-134 ( 0.12  nmol ) in 1  l te buffer ( life technologies ) at coordinates relative to the bregma : ap , 0.92  mm ; medial - lateral ( ml ) ,  + 1.3  mm , and dorsal - ventral ( dv ) , 3.3  mm to target the lateral ventricle . to allow the rats to fully recover from the analgesic drugs and to coincide with the maximal anti - epileptic effect seen in  vivo", "sodium pentobarbital and transcardially perfused with ice - cold sucrose artificial cerebrospinal fluid ( acsf ; in mm : 205 sucrose , 10 glucose , 2.5 kcl , 0.1 cacl2 , 5 mgcl2 , 26 nahco3 , and 1.2 nah2po4.h2o ; osmolarity , 295 mosm ) .", "the brain was quickly dissected into ice - cold sucrose acsf and transferred to a campden 700 smz slicer ( campden instruments ) . slices ( 400  m ) were prepared in the horizontal orientation between the bregma at approximately 7.1 and  5.1  mm . for storage ,", "slices were submerged in oxygenated normal acsf ( in mm : 125 nacl , 10 glucose , 3 kcl , 1 mgcl2 , 2 cacl2 , 26 nahco3 , and 1.25 nah2po4.h2o ; osmolarity , 290 mosm ) and allowed to recover at room temperature for at least 60  min prior to recordings .", "slice recordings were made using a membrane chamber ( scientific systems design ) , a modified submerged chamber , which improves oxygen supply to the tissue and supports greater epileptiform bursts than conventional submerged recording chambers .", "slices were continually perfused with acsf , heated to 32c , at a flow rate of 16  ml / min . for local field potential ( lfp ) recordings ,", "borosilicate glass recording micropipettes ( resistance , 34 m ) were placed into hippocampal ca1b stratum pyramidale and epileptiform activity was induced by raising the perfusing kcl concentration to 9  mm .", "total rna was isolated from brain samples using trizol ( invitrogen ) , as described previously.19 , 54 for mrna qpcr , 1  g total rna was used to generate cdna by reverse transcription using the superscript iii reverse transcriptase enzyme ( invitrogen ) .", "the pcr amplification was performed for 50 cycles on a thermocycler ( applied biosystems ) .", "quantitative real - time pcr was performed using a lightcycler 1.5 ( roche diagnostics ) in combination with quantitect sybr green pcr kit ( qiagen ) as per the manufacturer s protocol with 25  m of primer pairs used .", "specific primers for each gene assayed were purchased from sigma and sequences used were as follows : actin , beta ( actb ) ( forward [ f ] , ggttggccttagggttcagg ; reverse [ r ] , gggtgtgatggtgggaatgg ) ; c - fos ( f , cattcagaccacctcgacaa ; r , ggaattaacctggtgctgga ) ; and arc ( f , agcagcagacctgacatcct ; r , gtgatgccctttccagacat ) .", "data were normalized to actb and relative mrna transcript levels were quantified using the ct method . for mirna ,", "250  ng rna was reverse transcribed using stem - loop specific primers for mmu - mir-134 ( applied biosystems ) and real - time quantitative pcr was carried out on a 7900ht fast real - time system ( applied biosystems ) using taqman mirna assays .", "expression of rnu19 was used for normalization . a relative fold change in expression of mir-134", "was determined using the ct method.19 , 56 specific mirna assays were obtained from thermofisher scientific for mir-134 ( i d 001186 ) , mir-19a ( i d 000395 ) , and rnu19 ( i d 001003 ) .", "data were normalized to rnu19 and relative mirna expression levels were quantified using the ct method .", "statistical analysis was performed using graphpad prism and stata release 14 software , and a p value  < 0.05 was considered significant .", "latencies to tonic - clonic seizures were analyzed by the kruskal - wallis test , followed by the nonparametric dunn multiple comparison test when indicated .", "total time spent in seizures , mean amplitude , frequency , total power of eeg recordings , and mrna levels were analyzed by the student t test or one - way anova followed by the bonferroni test , depending on the experimental design .", "ex  vivo brain slice data were analyzed using the mann - whitney u test .", "three comparisons were made from the same data and  ( significance level ) was consequently reduced to 0.025 for these experiments .", ", d.f.o . , and n.d . provided human samples and data ; c.r.r . , s.s .", "wrote the paper and all co - authors contributed to data interpretation , review , and editing of the final paper .", "the royal college of surgeons in ireland filed for a patent on the inhibition of microrna-134 for the treatment of seizure - related disorders and other neurologic injuries ."], "abstract_text": ["<S> current anti - epileptic drugs ( aeds ) act on a limited set of  neuronal targets , are ineffective in a third of patients with epilepsy , and do not show disease - modifying properties . </S>", "<S> micrornas are small noncoding rnas that regulate levels of proteins by post - transcriptional control of mrna stability and translation . </S>", "<S> microrna-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid ( lna ) , cholesterol - tagged antagomirs targeting microrna-134 ( ant-134 ) reduced evoked and spontaneous seizures in mouse models of status epilepticus . </S>", "<S> translation of these findings would benefit from evidence of efficacy in non - status epilepticus models and validation in another species . here , we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre - treated with ant-134 in the pentylenetetrazol model . </S>", "<S> pre - treatment with ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats , a toxin - free model of acquired epilepsy . </S>", "<S> nevertheless , ant-134 post - treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and ant-134 delayed epileptiform activity in a rat ex  vivo hippocampal slice model . </S>", "<S> the potent anticonvulsant effects of ant-134 in multiple models may encourage pre - clinical development of this approach to epilepsy therapy . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Discussion", "Materials and Methods", "Author Contributions", "Conflicts of Interest"], "sections": [["epilepsy is a common and serious chronic neurological disease that affects over 50 million people worldwide .", "anti - epileptic drugs ( aeds ) are effective at controlling seizures in many patients , acting through a range of neuronal targets , including ion channels , neurotransmitter release , and receptor mechanisms.1 , 2 however , a third of patients with epilepsy fail to achieve seizure freedom , and current aeds do not show disease - modifying properties .", "future treatments for epilepsy need to differentiate from currently marketed drugs , for example , by having a novel mechanism(s ) of action or providing disease - modifying effects.1 , 2 acquired epilepsy is thought to result from disturbances to the expression and function of neurotransmitters and their receptors , ion channels , and signaling components , as well as altered metabolism , neuronal and glial microstructure , neuroinflammation , and other mechanisms.3 , 4 , 5 it is likely that disease - modifying treatments will need to influence multiple genes within a given pathway or in various independent pathways .", "potential gene network control points were recently identified and include master regulators of transcription and neuroinflammation , epigenetic factors , and noncoding rnas .", "micrornas ( mirnas ) are a conserved family of endogenous small noncoding rnas that regulate protein levels in cells by post - transcriptional control of mrna stability and translation.9 , 10 this is achieved by sequence - specific binding of the mirna to complementary bases in the mrna target .", "since this generally requires only a 7- to 8-nucleotide match , individual mirnas can potentially target large numbers of mrnas .", "this provides the ability to regulate entire gene networks or multiple biological processes and has been demonstrated in the brain .", "microrna-134 ( mir-134 ) was identified as a brain - enriched , neuronally expressed mirna that controls dendrite spine morphogenesis , via repression of lim domain kinase 1 and other targets.14 , 15 this is potentially important for disorders of hyperexcitability , since dendritic spines are contact points for excitatory communication and there is evidence that reorganization of dendrites is a feature of experimental and human epilepsy.16 , 17 we identified mir-134 among upregulated mirnas profiled in areas of hippocampal damage following status epilepticus induced by intra - amygdala kainic acid ( ka ) in mice .", "expression of mir-134 is also upregulated after neuronal stimulation and seizures in various other in  vitro and in  vivo models,15 , 19 , 20 , 21 , 22 and mir-134 is overexpressed in the resected human neocortex from patients with intractable temporal lobe epilepsy ( tle ) . silencing mir-134 by intracerebroventricular ( i.c.v . )", "injection of locked nucleic acid ( lna ) , cholesterol - tagged antagomirs ( ant-134 ) reduced levels of mir-134 for at least a month and rendered mice refractory to the convulsant effects of both ka and pilocarpine.19 , 22 consistent with known mir-134 targets , lim domain kinase 1 and levels of other mir-134 targets were higher in ant-134-treated mice after status epilepticus and the dendritic spine volume was increased.19 , 22 notably , injection of ant-134 after status epilepticus suppressed the later occurrence of spontaneous recurrent seizures in the intra - amygdala ka model in mice .", "there is significant interest in developing mirna - based therapies.23 , 24 there are also barriers to antisense - based approaches and there has been some disengagement of industry from pursuing such efforts .", "this is likely to be a problem for any new therapy for epilepsy , particularly one that is not based on traditional small molecule chemistry.1 , 2 pre - clinical development of an mir-134-based treatment for epilepsy might be more attractive if additional important questions are answered .", "principally , work to date has only tested ant-134 in mouse models of status epilepticus .", "proof of seizure - suppressive effects of ant-134 in a non - status epilepticus model or in another species would bridge the gap in evidence and facilitate pre - clinical development of ant-134-based treatment for epilepsy.26 , 27 here , we evaluated mir-134 expression in the resected hippocampus from patients with pharmacoresistant tle and we tested ant-134 in three different models .", "we used the pentylenetetrazol ( ptz ) model in mice , a popular method for screening putative anticonvulsive effects that triggers seizures via -amino butyric acid ( gaba ) blockade.26 , 28 second , the perforant pathway stimulation ( pps ) model , a toxin - free model of acquired epilepsy based on nonconvulsive status epilepticus that avoids the confounding influence of exposing the brain to chemoconvulsants , was used in rats .", "finally , we used a high - potassium model of epileptiform activity , using ex  vivo brain slices from rats pre - treated with ant-134 .", "our results establish that targeting mir-134 offers broad anticonvulsant and possibly disease - modifying effects in experimental epilepsy , which may encourage pre - clinical development of ant-134 as a treatment for epilepsy ."], ["for this , we analyzed levels of mir-134 in the surgically obtained hippocampus from adults with pharmacoresistant tle and we compared findings to autopsy hippocampal samples from people who died of causes unrelated to neurological disease .", "these samples were used previously to analyze expression of other genes , and clinical data for both groups are briefly summarized in tables s1 and s2 .", "there were no between - group differences in patient age , and each group included males and females .", "we detected higher levels ( p  = 0.037 ) of mature mir-134 in the tle specimens compared to autopsy samples ( figure  1a ) .", "this difference is unlikely to be an artifact of post mortem delay , since mir-134 levels did not decrease in simulated autopsy experiments lasting up to 12  hr . we began by establishing dosing parameters in the mouse ptz model .", "generalized tonic - clonic seizures were first reliably elicited at a dose of 60  mg / kg in c57bl/6j mice ( figure  1b ) .", "a dose of 80  mg / kg elicited generalized tonic - clonic seizures in all mice and with a more consistent and shorter latency than with the 60 mg / kg dose ( figure  1b ) .", "a dose of 100  mg / kg also produced rapid - onset tonic - clonic seizures in all mice but was associated with high mortality ( figure  1b and data not shown ) .", "ordinal logistic regression analysis was used to explore the dose dependence of the ptz - induced convulsions .", "ptz dose was significantly associated with racine scores ( figure  1c ) . a dose of 80  mg / kg was selected for further experiments .", "to obtain molecular evidence for ptz - induced seizure activity in the model , we measured expression of activity - regulated genes in the hippocampus .", "transcript levels of fbj osteosarcoma oncogene ( fos ) , a calcium - dependent marker of neuronal activity and activity regulated cytoskeletal - associated protein ( arc ) , another immediate early gene responsive to intense neuronal activity , were strongly increased in the hippocampus 30  min , but not at 4  hr , after ptz - induced seizures ( figures 1d and 1e ) .", "there was no evidence of irreversible neuronal injury in tissue sections from mice after ptz , as assessed by fluoro - jade b ( fjb ) and neun ( rna binding protein , fox-1 homolog [ c.  elegans ] 3 ) staining ( data not shown ) .", "expression of mir-134 is known to increase in various in  vitro and in  vivo seizure models.15 , 19 , 20 , 21 , 22 we therefore investigated whether ptz - induced convulsions alter mir-134 levels in mice .", "taqman mirna assays showed that levels of mature mir-134 were significantly increased in the hippocampus , but not in the cortex , 30  min after ptz - induced generalized tonic - clonic seizures in mice ( figure  1f and data not shown ) .", "injection of antagomirs targeting mir-134 ( ant-134 ) in mice.19 , 22 previous work has established a dose of these antagomirs that reduces hippocampal mir-134 levels by over 90% by 24  hr after injection .", "accordingly , we tested ptz responses 24  hr after injection of ant-134 and compared them to responses in mice that received a scrambled antagomir ( scr ) . in scr mice , delay to onset of first tonic - clonic seizure after ptz injection was similar to that observed before in control mice ( figure  2a , and compare to figure  1b ) .", "thus , the control antagomir did not alter the normal response to ptz in the model .", "the time to first ptz - induced tonic - clonic seizure was significantly longer in mice injected with ant-134 compared to scr - injected animals ( figure  2a ) .", "the severity of racine - scored ptz - induced seizures was also significantly lower in ant-134- compared to scr - injected mice ( figures 2b and 2c ) .", "analysis of electrographically recorded seizure activity determined that electroencephalography ( eeg ) total power was lower in ant-134 mice compared to the control group after ptz ( figure  2d ) .", "we next analyzed brain tissue samples from scr- and ant-134-injected mice for evidence of mirna silencing and differences in expression of activity - regulated genes .", "analysis of the hippocampus obtained after ptz - induced seizures confirmed that mir-134 expression was lower in ant-134 pre - treated mice compared to scr - treated animals ( figure  3a ) .", "ant-134 had no effect on levels of an unrelated mirna ( figure  3b ) .", "next , we assessed expression of arc and c - fos as molecular markers of recent neuronal activity to support the clinically scored behavior and eeg differences in ant-134 mice . quantitative real - time pcr analysis revealed increased c - fos expression in the hippocampus of scr - injected mice after ptz - induced seizures ( figure  3c ) .", "in contrast , c - fos levels were lower in ant-134-injected mice , consistent with reduced seizure severity ( figure  3c ) .", "this difference was also apparent in the neocortex from the same animals ( figure  3c ) .", "expression of arc was increased in the hippocampus of scr - injected mice that received ptz .", "arc levels were not significantly different in ant-134 mice compared to scr - injected animals in the hippocampus and neocortex after ptz - induced seizures ( figure  3d ) .", "we turned next to the pps model in rats , first investigating effects on mir-134 levels .", "taqman mirna assays showed that levels of mature mir-134 were not significantly modified in the hippocampus of rats at two different time points ( figure  4a ) .", "next , we assessed the effect of silencing mir-134 before stimulation on seizure severity during status epilepticus .", "rats were pre - treated with ant-134 or scr ( i.c.v . ) 24  hr before the 8-hr stimulation protocol .", "analysis of electrographically recorded seizure activity determined that scr - injected rats presented a mean (  sem ) increase of 1,304.3%   94.9% in eeg total power relative to baseline .", "this was similar to animals that were stimulated but not receiving the oligonucleotide ( data not shown ) . in rats", "pre - treated with ant-134 , the severity of evoked seizures in the pps model was similar : eeg total power during seizures was a mean (  s.e.m ) of 1,244.6%   82.7% ( percent of baseline ) . despite not seeing an acute anticonvulsant effect of ant-134 in the pps model , we were nevertheless interested in whether silencing mir-134 after status epilepticus in the model would alter the emergence of recurrent spontaneous seizures . a key prior", "finding was that silencing mir-134 after status epilepticus induced by intra - amygdala ka reduced the subsequent occurrence of spontaneous recurrent seizures by over 90% .", "for these studies , rats underwent 8-hr stimulation of the perforant pathway to induce epilepsy and the i.c.v .", "rats were then monitored with video - eeg until a first spontaneous seizure was recorded , typically 1025  days after pps , and they were then followed for a further 8  weeks ( figure  4b ) .", "epilepsy developed in all scr - injected rats over the expected course , with the first spontaneous seizures appearing 14  weeks after status epilepticus ( figures 4b and 4c ) .", "scr - injected rats averaged 20 spontaneous seizures recorded during the 8-week recording time ( figures 4d and 4e ) .", "in contrast , no spontaneous seizures were detected over the 8-week period in six of seven rats injected with ant-134 after status epilepticus ( figures 4d4f ) .", "epilepsy emerged in only a single rat treated with ant-134 ( figures 4d and 4f ) .", "the fisher exact test determined that there was a significant difference in epilepsy development between the groups ( p  = 0.005 ) . in terms of effect size , ant-134 prevented 86% of the risk of epilepsy , with a 95% confidence interval ( ci ) of 12%98% .", "an analysis of the duration of spontaneous seizures ( electrographically defined ) revealed that when spontaneous seizures occurred , they were similar between groups .", "that is , the average duration of a spontaneous seizure in scr - treated rats was 53.7   14.4  s ( n  = 20 seizures , from five rats ) , which was similar to the duration in epileptic rats in the pps model that did not receive an i.c.v .", "injection ( 49.4   15.9 , n  = 20 seizures ) . in the ant-134 rat that developed epilepsy ,", "the average duration of a spontaneous seizure was 52.3   16.2  s ( from n  = 12 seizures ) .", "having observed a disparity between the acute anti - seizure effects of ant-134 pre - treatment in the ptz and pps models , we sought to examine the effect of ant-134 on naive , non - epileptic tissue and whether the effects were due to long range networks or if local circuitry was sufficient to mediate any seizure protection .", "we injected ant-134 in  vivo in rats and subsequently prepared ex  vivo brain slices ( figure  5a ) .", "these slices were then exposed to a 9-mm k seizure challenge , to determine whether ant-134 protects against epilepsy in local circuits of the isolated slice in the absence of epileptogenesis .", "onset of activity was delayed in ant-134-treated slices by 176  s relative to the control ( figures 5b and 5c ) .", "this effect was statistically significant ( mann - whitney u test , p  = 0.002 ,   = 0.025 , power  = 0.87 ) .", "we also compared the power of epileptiform activity and duration of seizure - like events in these data ; these effects were not statistically significant ( power : t test , p  = 0.70 ; duration : mann - whitney u test , p  = 0.067 ) .", "this finding suggests that ant-134 can confer seizure resistance in naive tissue and thus mediate its effect even in the absence of epileptogenesis .", "in addition , preservation of the seizure resistance in isolated slices indicates that at least some of the effects of ant-134 are due to local changes in activity ."], ["the present study provides important additional evidence of the anticonvulsant and disease - modifying effects of lna - based antagomirs targeting mir-134 in animal models of epilepsy .", "our study shows that ant-134 is an anticonvulsant in an acute chemoconvulsant model ( ptz in mice ) and ant-134 had anti - epileptogenic activity in the perforant pathway stimulation model of epilepsy in rats .", "ant-134 also confers resistance to epileptic activity in naive tissue , reflected as a delay to activity onset following a seizure challenge in ex  vivo brain slices .", "we also report that mir-134 levels are elevated in the hippocampus of patients with pharmacoresistant tle .", "the therapeutic potential of targeting mir-134 in established epilepsy will require additional studies , but our findings may encourage further research or pre - clinical development of this mirna - based treatment for epilepsy . despite the recent introduction of two additional aeds ( perampanel and brivaracetam ) , new drug development in epilepsy", "faces significant challenges.1 , 2 , 32 new targets are required that can treat pharmacoresistant patients or have disease - modifying effects that are not served by current therapies .", "mirnas have emerged as potential targets of the future via their influence on neuronal microstructure , gliosis , neuroinflammation , and ion channels.8 , 33 silencing mir-134 has produced the most convincing anti - seizure effects to date , reducing status epilepticus in pre - treatment paradigms in the intra - amygdala ka and pilocarpine models and exerting potent long - term effects on spontaneous seizure development.19 , 22 this mirna is also an attractive target , since studies show increased levels of mir-134 within the temporal lobe of adult and pediatric patients with intractable epilepsy.19 , 20 we report here that mir-134 is also elevated in the hippocampus of adults with pharmacoresistant tle , although there are potential confounders with comparing resected material from patients to autopsy control samples . the present study has answered key questions that could facilitate pre - clinical development by showing that ant-134 has anticonvulsant effects in non - status epilepticus models and is disease modifying in another species .", "the first important finding was that pre - treatment with ant-134 strongly suppressed seizures in the mouse ptz model .", "effects on both clinical behavior , including delaying the time to first convulsive seizure , and electrographic seizure activity were seen .", "thus , lowering brain levels of mir-134 can exert anti - excitability effects , and this implies that mir-134 has a role in setting excitability thresholds in the brain .", "this is thought to be due to regulation of proteins that control dendritic morphology,14 , 15 although other targets are known .", "ant-134 did not fully prevent seizures in the ptz model ; rather , it delayed and minimized their severity , a finding most closely matching the effect of ant-134 in the pilocarpine model of status epilepticus", ". it may not be possible to fully prevent all seizures using ant-134 in chemoconvulsant models , but further adjustments to antagomir dose or delivery may yield optimal seizure - suppressive effects .", "ptz triggers seizures via gabaergic blockade ; this differs from the mechanism by which status epilepticus is induced by ka and pilocarpine , which work by promoting glutamatergic and cholinergic transmission , respectively .", "this suggests that ant-134 may be effective for epilepsies of various etiologies , which , in patients , can vary considerably between trauma , stroke , infection , genetic , and other causes .", "our studies also show that ptz - induced convulsions increase brain levels of mir-134 , transcription of which is probably mediated by myocyte enhancer factor 2c .", "we did not observe an increase in mir-134 after perforant pathway stimulation in the rat .", "this was surprising , although changes to mir-134 levels are not always detected after chemoconvulsive stimuli , and we can not rule out a technical explanation ( e.g. , the time points selected or a subfield - specific effect that was masked by the use of the whole hippocampus ) .", "our study included an analysis of the short - term effects of ant-134 on neuronal activity - regulated genes in the ptz model .", "expression of genes such as c - fos and arc is a well - established surrogate marker of recent seizure activity , the protein products of which are involved in coordinating synaptic plasticity and adaptation to excessive neuronal stimulation.38 , 39 unexpectedly , only levels of c - fos , and not arc , showed the expected reductions in ant-134 mice after ptz seizures .", "it is known that c - fos induction is calcium dependent , whereas arc expression can be induced via calcium - independent pathways.38 , 39 therefore , ant-134 treatment may have reduced seizure severity sufficient to prevent c - fos induction , such as by minimizing n - methyl - d - aspartate ( nmda ) receptor opening , but enough residual neuronal excitation remained to upregulate arc .", "these findings provide additional evidence of the scale and nature of seizure suppression by ant-134 and suggest molecular markers that can distinguish effects of mirna manipulations on different components of seizure responses . in the present study", ", we observed that pre - treatment of rats with ant-134 did not protect against perforant pathway stimulation - induced status epilepticus .", "while there are examples of anticonvulsants producing effects in some but not all models , these findings suggest that ant-134 may have the most clinical value as an anti - epileptogenic treatment .", "previous work showed that injection of ant-134 after status epilepticus in mice suppressed the later occurrence of spontaneous seizures by 90% , evidence of a disease - modifying effect in the intra - amygdala ka model . here", ", we found nearly identical anti - epileptogenic effects of ant-134 in a different model in rats .", "this is the only demonstration , to our knowledge , of any disease - modifying treatment in the pps model in rats .", "the pps technique induces hippocampal sclerosis and recurrent spontaneous seizures,29 , 40 and it provides a means to trigger epilepsy without the bias or other issues associated with chemoconvulsants .", "ant-134 was injected shortly after triggering status epilepticus in order to model a reasonably realistic clinical scenario , such as a patient being treated shortly after an initial precipitating injury .", "ant-134 had dramatic and potent effects , with the majority of ant-134 rats ( six of seven , 86% ) never developing spontaneous recurrent seizures .", "notably , spontaneous seizures in the study with the intra - amygdala ka model did occur in all ant-134 mice , albeit with dramatically lower seizure frequency .", "this difference may relate to the extent of hippocampal injury or another aspect of the epileptic phenotype , such as the frequency of spontaneous seizures .", "thus , presently we see a possible additional therapeutic action of ant-134 , whereby post - treatment is capable of fully preventing epilepsy at least in some models .", "we can not , of course , exclude that epilepsy might have developed in some of the ant-134 rats at some point in the future . however , we recorded for 8  weeks beyond the standard latent period .", "we further corroborated the anti - seizure effect of ant-134 by using an acute seizure challenge with high potassium in otherwise healthy ( ex  vivo ) tissue .", "inhibitory neurotransmission is retained in this model ( in contrast to ptz ) and there is no hippocampal sclerosis ( unlike pps ) , further suggesting that ant-134 can be therapeutic in multiple epileptic syndromes with varying mechanisms .", "we observed a delay in the onset of epileptiform bursts in brain slices that had been pre - treated with ant-134 .", "this finding suggests that epileptogenesis is not required for ant-134 to mediate its effects on seizure onset .", "we did not see any significant changes in the power or duration of epileptiform activity ; however , these parameters are highly variable in brain slices , which can obscure potential effects . in addition", ", we propose that ant-134 can mediate seizure protection in the hippocampus via effects on local circuitry , because long range connections would likely be severed during the slicing process .", "the anti - epileptogenic actions of ant-134 may be independent of the etiology of the initial precipitating injury , although this will require further testing ( e.g. , in models of trauma or infection - induced epilepsy ) .", "in addition , upregulation of mir-134 is not essential for ant-134 to produce anti - epileptogenic effects , since mir-134 levels were not altered after pps - induced status epilepticus .", "thus , epilepsy can be dramatically suppressed in two different in  vivo models , and one in  vitro , in two different species .", "this provides strong support for and is an argument for further development of ant-134 for epilepsy treatment or prevention .", "antagomirs targeting a liver - expressed mirna have been given to humans and were found to be well tolerated and effective against hepatitis c in clinical trials .", "there are concerns , obviously , that because ant-134 can alter dendritic spines , it could have effects on cognition . studies to date have not identified such problems , with natural exploratory behavior and performance in a hippocampus - dependent task found to be normal in mice a few days after i.c.v .", "injection of ant-134.19 , 22 however , more demanding tests of cognition or learning will need to be performed .", "another factor that argues for general safety is that mice and rats have been recorded for at least 2  months after brain injections of ant-134 .", "nevertheless , pre - clinical development will necessitate an extensive battery of toxicity and safety testing .", "work to date on ant-134 satisfies certain recommendations for testing and evaluating novel therapies for epilepsy , with seizure - suppressive effects demonstrated in five different models and two different species.1 , 2 , 43 , 44 there are , however , additional seizure models that could be used during any future pre - clinical development .", "this includes kindling , a popular model that has been successful in identifying aeds with novel mechanisms of action .", "while this is not an essential step before pre - clinical development , sex differences in mirna inhibitor responses may be worth investigating in future studies and sexual dimorphism in mirna expression has been reported , although not for mir-134.46 , 47 it will also be of value to understand how ant-134 produces its potent seizure - suppressive effects .", "the most likely mechanism is upregulation of one or more proteins due to de - repression of a mir-134 target .", "previous work has shown that mir-134 is uploaded into the rna - induced silencing complex after seizures , which facilitates targeting of mrnas , and in  vitro studies suggest that the neuroprotective effects of ant-134 are due to de - repression of lim domain kinase 1 .", "ant-134 treatment also increases dendritic spine volume and changes spine number in the hippocampus,19 , 22 which could influence excitability and would also be consistent with a mechanism involving protection of mir-134 targets such as lim domain kinase 1 or pumilio rna - binding family member 2", ". altered spine volume and number is consistent with our observation of a robust local effect of suppressing mir-134 on delaying epileptiform activity , although additional effects on long range connections can not be ruled out .", "we must also consider that the acute seizure - suppressive effects and long - term anti - epileptogenesis effects may arise from different mechanisms .", "ultimately , in  vivo studies will be necessary to determine the anti - seizure mechanism of ant-134 ( e.g. , by analyzing gene expression networks , quantifying the proteome , and applying techniques to validate antagomir - mirna targeting such as polysome shift assays ) .", "what are the next steps for translation or pre - clinical development ? an obvious question is whether and how antagomirs could be given to patients .", "their large size precludes their passing an intact blood - brain barrier ( bbb ) .", "recent work has explored methods to circumvent the bbb , including conjugating antagomirs with brain - penetrating peptides or administering antagomirs via intranasal injection.19 , 51 the bbb may also be open after certain epilepsy - precipitating injuries or it can be physically breached , such as through use of ultrasound . another question is whether ant-134 injection into already epileptic animals could alter the frequency or duration of spontaneous seizures or attendant hippocampal pathology .", "success with that approach could provide novel treatment opportunities for the population of drug - resistant patients for whom surgery or other alternatives to aeds are either not possible or not effective currently . in summary ,", "the present study provides important additional validation of the potent seizure - suppressive and possibly disease - modifying effects of ant-134 in two different animal models and one brain slice model .", "the effectiveness of ant-134 and the potency of seizure suppression suggest a target for drug development in epilepsy that could provide additional effects beyond those provided by any currently marketed drug for epilepsy ."], ["this study was approved by the ethics ( medical research ) committee of beaumont hospital of dublin ( no . 05/18 ) and written informed consent was obtained from all patients .", "the control ( autopsy ) hippocampus ( n  = 12 ) was obtained from 12 individuals .", "patients ( n  = 12 ) were referred for surgical resection of the temporal lobe by an epileptologist ( n.d . ) following neurological assessment , video - eeg recording , and mri / neuroimaging . each patient was determined to have medically intractable tle with a history of recurring seizures ( table s2 ) .", "the hippocampus was obtained and a portion of each specimen was frozen in liquid nitrogen and stored at 70c until use .", "a pathologist ( m.a.f . ) assessed the  hippocampus as part of the pathologic evaluation , addressed the degree of hippocampal sclerosis , and described other pathologic changes .", "all animal experiments were performed in accordance with the european communities council directive ( 2010/63/eu ) .", "procedures in mice were approved by the research ethics committee of the royal college of surgeons in ireland ( rec-842 ) , under license from the ireland health products regulatory authority ( ae19127/001 ) .", "procedures in rats were performed under authorization of the philipps university bioethics committee and were approved by the local regulation authority ( regierungsprsidium gieen ) , or they were performed according to the uk animals ( scientific procedures ) 1986 act ( ppl 70 - 7684 ) and approved by local ethical review ( university college london ) .", "male adult c57bl/6j mice ( 2025 g ) and male sprague - dawley rats ( 325350  g or 200300 g ; all from harlan ) were used in all studies .", "animals were housed in on - site barrier - controlled facilities having a 12-hr/12-hr light / dark cycle with ad libitum access to food and water .", "mice were equipped for eeg recordings under surgical anesthesia ( isoflurane ; 5% induction , 1%2% maintenance ) in a mouse - adapted stereotaxic frame .", "body temperature was maintained within the normal physiological range with a feedback - controlled heat pad ( harvard apparatus ) .", "after a midline scalp incision , partial craniectomies were performed and animals had skull - mounted recording electrodes placed and fixed with dental cement .", "a second cohort of mice had eeg electrodes and a guide cannula implanted to allow i.c.v .", "this cannula was placed on the dura mater with the following coordinates from the bregma : anterior - posterior ( ap ) ,  + 0.3  mm ; lateral ( l ) ,  + 0.9  mm ; and ventral ( v ) , 2.0  mm .", "after recovery from surgery , mice were connected to the lead socket of a swivel commutator , which was connected to a grass twin digital eeg system .", "] ) with different doses of ptz ( 60 , 80 , or 100  mg / kg ) . lorazepam ( 8  mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .", "mice were followed for 30  min or 4  hr after ptz administration for the appearance of seizures by electrographic and behavioral methods .", "after the observation period , mice were deeply anesthetized ( pentobarbital ) and transcardially perfused , and their brains were removed for analysis of gene expression or histological assessment .", "injection of 0.12  nmol/2  l 3-cholesterol - tagged locked nucleic acid oligonucleotide targeting mir-134 ( ant-134 ) or its respective control , a non - targeting scrambled version of the antagomir ( scr ; 0.12  nmol/2  l ) ( exiqon a / s ) , as previously described.19 , 22 twenty - four hours later , mice were connected to an eeg system and a baseline recording was obtained .", "next , mice were injected with ptz ( 80  mg / kg , i.p . ) .", "lorazepam ( 8  mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .", "these mice were followed up for 30  min after ptz administration for the appearance of seizures .", "after this period , mice were deeply anesthetized ( pentobarbital overdose ) and transcardially perfused ( pbs ) for further assessments .", "under isoflurane ( 5% induction , 3% maintenance ) anesthesia , bipolar stainless - steel stimulating electrodes ( nex-200 ; rhodes medical instruments ) were positioned in the angular bundles of the perforant pathway and custom unipolar recording electrodes ( crafted from 796000 ; a - m systems ) were lowered into the dorsal dentate gyrus .", "electrodes and ground screws were connected to miniature wireless transmitters ( ft20 ; data sciences international ) that were implanted subcutaneously on the animal s flank .", "plastic connectors ( gs09plg ; ginder scientific ) joined the electrodes with stimulation / recording equipment .", "the pps protocol utilized a paradigm designed to evoke and maintain hippocampal seizure activity throughout the stimulation , but not convulsive status epilepticus , which consisted of continuous , bilateral 2-hz paired - pulse stimuli , with a 40-ms interpulse interval , plus a 10-s train of 20  hz single - pulse stimuli delivered once per minute , generated by an s88 stimulator ( grass instruments ) .", "all pulses ( 0.1-ms duration ) were delivered at 2024 v , as this voltage reliably evokes granule cell discharging without tissue - damaging hydrolysis.29 , 40 the current associated with these voltages was typically between 15 and 30  a .", "no samples displayed any signs of hydrolysis ( e.g. , holes around the stimulating electrodes ) .", "as described previously , stimulation for 30  min ( on 2 consecutive days ) required only isoflurane to terminate seizures .", "eight - hour stimulation on the third day did not induce convulsive status epilepticus and , therefore , did not require pharmacological termination .", "rats were randomly divided in three cohorts to assess the following : ( 1 ) mir-134 expression after pps ( 24  hr , 4  days , and 14  days ) ; ( 2 ) the effect of ant-134 on pps - induced status epilepticus , in which rats were pre - treated with ant-134 or scr ( 0.36  nmol/6  l , i.c.v . ; exiqon a / s ) 24  hr before the 8-hr stimulation ; and ( 3 ) the effect of ant-134 on epileptogenesis , in which rats were injected with 0.36  nmol/6  l ( i.c.v . ) of ant-134 or scr ( exiqon a / s ) immediately after 8-hr stimulation .", "five rats were excluded from this study due to practical issues ( e.g. , blocked cannula ) . for statistical purposes", ", we pooled samples from 24  hr and 4  days after pps as an  early  time point , while 14-day samples were considered as the  late  time point .", "mouse eeg data were analyzed and quantified using labchart 8 software ( ad instruments ) .", "ptz - induced seizures were defined as high - amplitude ( > 2 baseline ) high - frequency ( > 5  hz ) polyspike discharges lasting > 5 s. for statistical purposes , we assigned a cut - off time of 1,800  s for those animals that did not present seizures during the observation period . from the eeg recordings ,", "we calculated the delay to first seizure , the total power , and the percentage of total power / spectral bands , as previously described.19 , 54 eeg total power was plotted as the percentage of baseline recording ( each animal s eeg power post - seizure compared to its own baseline eeg ) .", "clinical behavior was scored using the following adapted racine scale : score 0 , no seizures observed ; score 1 , rhythmic mouth and facial movement ; score 2 , rhythmic head nodding ( bobbing ) ; score 3 , forelimb clonus ; score 4 , rearing and bilateral forelimb clonus ; and score 5 , rearing and falling ( stay fallen on rear side ; tonic - clonic seizures ) .", "the highest score reached during every 5  min during the 30  min after ptz was recorded .", "for studies in rats , eeg activity during and after pps was amplified and recorded digitally at 2 khz utilizing a powerlab 16/35 and labchart software versions 7 and 8 ( ad instruments ) .", "spontaneous granule cell layer activity was recorded continuously ( 24 hr / day ) and stored digitally and automatically in 3-hr epochs . each day , we analyzed the preceding 24  hr of recordings , as well as all events with amplitudes exceeding 120% of average baseline .", "continuous ( 24 hour/7  day ) video monitoring utilized edimax  7010w infrared cameras .", "video files were captured at 1215  frames / sec and were time - stamped for integration with the electrophysiological data using securityspy surveillance software ( ben software ) and stored digitally . confirmed seizures were scored according to the racine scale .", "rats were anesthetized with isoflurane ( 5% induction,2% maintenance as appropriate ) and mounted in a stereotaxic frame .", "we injected ant-134 ( 0.12  nmol ) in 1  l te buffer ( life technologies ) at coordinates relative to the bregma : ap , 0.92  mm ; medial - lateral ( ml ) ,  + 1.3  mm , and dorsal - ventral ( dv ) , 3.3  mm to target the lateral ventricle . to allow the rats to fully recover from the analgesic drugs and to coincide with the maximal anti - epileptic effect seen in  vivo", "sodium pentobarbital and transcardially perfused with ice - cold sucrose artificial cerebrospinal fluid ( acsf ; in mm : 205 sucrose , 10 glucose , 2.5 kcl , 0.1 cacl2 , 5 mgcl2 , 26 nahco3 , and 1.2 nah2po4.h2o ; osmolarity , 295 mosm ) .", "the brain was quickly dissected into ice - cold sucrose acsf and transferred to a campden 700 smz slicer ( campden instruments ) . slices ( 400  m ) were prepared in the horizontal orientation between the bregma at approximately 7.1 and  5.1  mm . for storage ,", "slices were submerged in oxygenated normal acsf ( in mm : 125 nacl , 10 glucose , 3 kcl , 1 mgcl2 , 2 cacl2 , 26 nahco3 , and 1.25 nah2po4.h2o ; osmolarity , 290 mosm ) and allowed to recover at room temperature for at least 60  min prior to recordings .", "slice recordings were made using a membrane chamber ( scientific systems design ) , a modified submerged chamber , which improves oxygen supply to the tissue and supports greater epileptiform bursts than conventional submerged recording chambers .", "slices were continually perfused with acsf , heated to 32c , at a flow rate of 16  ml / min . for local field potential ( lfp ) recordings ,", "borosilicate glass recording micropipettes ( resistance , 34 m ) were placed into hippocampal ca1b stratum pyramidale and epileptiform activity was induced by raising the perfusing kcl concentration to 9  mm .", "total rna was isolated from brain samples using trizol ( invitrogen ) , as described previously.19 , 54 for mrna qpcr , 1  g total rna was used to generate cdna by reverse transcription using the superscript iii reverse transcriptase enzyme ( invitrogen ) .", "the pcr amplification was performed for 50 cycles on a thermocycler ( applied biosystems ) .", "quantitative real - time pcr was performed using a lightcycler 1.5 ( roche diagnostics ) in combination with quantitect sybr green pcr kit ( qiagen ) as per the manufacturer s protocol with 25  m of primer pairs used .", "specific primers for each gene assayed were purchased from sigma and sequences used were as follows : actin , beta ( actb ) ( forward [ f ] , ggttggccttagggttcagg ; reverse [ r ] , gggtgtgatggtgggaatgg ) ; c - fos ( f , cattcagaccacctcgacaa ; r , ggaattaacctggtgctgga ) ; and arc ( f , agcagcagacctgacatcct ; r , gtgatgccctttccagacat ) .", "data were normalized to actb and relative mrna transcript levels were quantified using the ct method . for mirna ,", "250  ng rna was reverse transcribed using stem - loop specific primers for mmu - mir-134 ( applied biosystems ) and real - time quantitative pcr was carried out on a 7900ht fast real - time system ( applied biosystems ) using taqman mirna assays .", "expression of rnu19 was used for normalization . a relative fold change in expression of mir-134", "was determined using the ct method.19 , 56 specific mirna assays were obtained from thermofisher scientific for mir-134 ( i d 001186 ) , mir-19a ( i d 000395 ) , and rnu19 ( i d 001003 ) .", "data were normalized to rnu19 and relative mirna expression levels were quantified using the ct method .", "statistical analysis was performed using graphpad prism and stata release 14 software , and a p value  < 0.05 was considered significant .", "latencies to tonic - clonic seizures were analyzed by the kruskal - wallis test , followed by the nonparametric dunn multiple comparison test when indicated .", "total time spent in seizures , mean amplitude , frequency , total power of eeg recordings , and mrna levels were analyzed by the student t test or one - way anova followed by the bonferroni test , depending on the experimental design .", "ex  vivo brain slice data were analyzed using the mann - whitney u test .", "three comparisons were made from the same data and  ( significance level ) was consequently reduced to 0.025 for these experiments ."], [", d.f.o . , and n.d . provided human samples and data ; c.r.r . , s.s .", "wrote the paper and all co - authors contributed to data interpretation , review , and editing of the final paper ."], ["the royal college of surgeons in ireland filed for a patent on the inhibition of microrna-134 for the treatment of seizure - related disorders and other neurologic injuries ."]]}
{"article_id": "PMC3451745", "article_text": ["it is a common discomfort making to the top ten list of complaints in ambulatory medical care , but our understanding of the epidemiology of headache disorders is still incomplete .", "most of the recurrent headache cases are due to benign chronic primary headache disorders , such as tension headache and migraine .", "less frequently , headache could be due to other underlying conditions such as infections , cerebral hemorrhage and brain lesions [ 5 , 6 ] .", "the study of headache epidemiology can address a number of important questions such as variation in the occurrence and severity of headache in the population , and the relationship between headache and other medical disorders .", "in addition , these studies may provide clues to abortive treatments and preventive strategies for headache .", "it is well documented among adults that overuse of headache medication may contribute to the development of chronic headache [ 814 ] .", "epidemiologic studies indicate that chronic headache ( > 15  days per month ) is common in the adult population with a 25% prevalence rate [ 1521 ] and a prevalence of chronic headache associated with medication overuse of about 1% [ 18 , 20 , 21 ] .", "in general , self - medicating for headache is highly prevalent and self - care is likely to increase in the era of health - care reform [ 22 , 23 ] .", ". chronic and inappropriate use of over - the - counter drugs such as analgesics , particularly nonsteroidal anti - inflammatory drugs ( nsaids ) , can lead to overuse syndromes and drug - induced headache [ 25 , 26 ] . in the present study", ", we sought to estimate the association between analgesic use and headache among adults in a large sample of the jordanian population in relation to age , gender and headache frequency .", "in this study , participants ( age range 1885 ) were approached at their work places , classes or homes .", ", researchers asked every participant to nominate two other persons until the desired sample size was obtained .", "the study was ethically approved by the institutional review board ( irb ) at jordan university of science and technology and was carried out in accordance with the principles described in the declaration of helsinki , including all amendments and revisions .", "the study was conducted in various regions of jordan during the period from january 2007 to november 2008 .", "the questionnaire was distributed , in person , by the researchers , and was completed in the presence of the researcher .", "each participant was provided with a full explanation of the study and how to complete the questionnaire .", "participants were informed that the researcher would be available for any required assistance during scoring of the questionnaire .", "for the small group of participants who were illiterate , the questionnaire was administered by the researcher in the form of an interview .", "data were aggregated into groups and only the authors and the investigator were allowed access to the collected data .", "the questionnaire gathered information that included demographic data , frequency and type of headache , and its impact on everyday activities , analgesic use , consultation of a doctor or pharmacist on the use , increase or decrease in the frequency of headache after painkiller usage , increase or decrease in painkiller dose and family history of headache .", "every person who was 18  years or older was allowed to complete the survey .", "retest reliability , 50 subjects were selected randomly ; they answered the questionnaire twice with a 1-week interval . test ", "for each item , correlation coefficients ranged from 0.79 to 0.86 , suggesting that the questionnaire was reliable .", "the data were coded using the statistical package for the social sciences , version 15.0 ( spss inc .", "the data were summarized using frequency tables and means and standard deviation for continuous variables .", "to estimate the prevalence of headache among adults in jordan , a sample of participants ( 4,836 participants ; mean age 27    0.4  years , and male : female ratio of 59:41 ) was allowed to complete a self - conducted screening questionnaire . as shown in table  1 , about half of the participants ( 49.2% ) were university students and 78.3% were jordanian citizens . moreover ,", "70.7% participants were single and 47.4% were smokers.table  1the demographic data of the study samplevariablen ( % ) gender  male2,870 ( 59.4 )  female1,966 ( 40.6)age  1829  years3,572 ( 73.95 )  3039  years506 ( 10.46 )  4049  years402 ( 8.32 )  50  years356 ( 7.37)education  illiterate83 ( 1.7 )  elementary school65 ( 1.3 )  secondary school213 ( 4.4 )  high school610 ( 12.6 )  diploma344 ( 7.1 )  university student2,378 ( 49.2 )  bachelor901 ( 18.6 )  masters181 ( 3.7 )  ph.d.61 ( 1.3)nationality  jordanian3,787 ( 78.3 )  arab ( non - jordanian)841 ( 17.4 )  foreign208 ( 4.3)marital status  single3,422 ( 70.8 )  married1,304 ( 27.0 )  divorced55 ( 1.14 )  other55 ( 1.14)monthly income  low ( < or = 400  jd)257 ( 5.3 )  medium ( > 400  jd to < or = 1,000  jd)3,831 ( 79.2 )  high ( > 1,000  jd)748 ( 15.5)smoking  smoker2,294 ( 47.4 )  non - smoker2,542 ( 52.6)1  jd is equivalent to about 1.4 us dollars the demographic data of the study sample 1  jd is equivalent to about 1.4 us dollars of the 4,836 participants , 82.3% complained of headache at least once per year .", "for both the 1829 and the 3039-year - old groups , the yearly prevalence of headache was found to be the same ( 82.8% ) , while it was 79.9 and 81.7% for the 4049 and older than 50-year - old groups , respectively . among the participants , 17.2% had daily headache attacks ,", "while 25.7% had fewer than daily to weekly headaches , 21.6% had headaches on fewer than weekly up to monthly basis , 17.8% complained of headaches on a fewer than monthly basis and 17.7% of all participants did not experience headache attacks .", "moreover , 38.4% of the headache complainers did not know the exact type of headache they were experiencing , 36.9% complained of tension type headache and only 7.7% were diagnosed with migraine headache ; 51.6% of complainers thought that headache affected their daily activities .", "a large number of participants stated that one or more of their family members complained of headache as well ( table  2).table  2frequency , type , and family history of headachevariablen ( % ) headache frequency  daily832 ( 17.2 )  fewer than daily to weekly1,243 ( 25.7 )  fewer than weekly to monthly1,043 ( 21.6 )  fewer than monthly to 1  year860 ( 17.8 )  no headache858 ( 17.7)type of headache  migraine372 ( 7.7 )  tension1,749 ( 36.1 )  unknown1,857 ( 38.4 )  no headache858 ( 17.7)headache affects daily activities ( if any )  yes2,051 ( 51.6 )  no1,927 ( 48.4)other family members complaining from headaches  father327 ( 6.8 )  mother545 ( 11.3 )  brothers or sisters552 ( 11.4 )  other relatives558 ( 11.5 )  more than one family member860 ( 17.8 )  none1,994 ( 41.1 ) frequency , type , and family history of headache of the migrainers , 37.7% ( n  =  140 ) complained of daily headache , 35.6% ( n  =  132 ) had fewer than daily up to weekly headaches , and 18.1 ( n  =  67 ) and 8.6% ( n  =  32 ) complained of headaches on a fewer than weekly up to monthly basis , and on fewer than monthly basis , respectively .", "additionally , about 75% ( n  =  279 ) of migrainers thought that migraine adversely affected their daily activities .", "concerning tension type headache , 20.3% ( n  =  348 ) complained of daily headache , 34.7% ( n  =  594 ) had fewer than daily up to weekly headaches , and 24.1% ( n  =  413 ) and 20.8 ( n  =  356 ) complained of headaches on fewer than weekly up to monthly basis , and fewer than monthly basis , respectively .", "moreover , about 53% ( n  =  907 ) indicated that tension type headache affected their daily activities .", "the stratified prevalence for both migraine and tension type headache according to age group and gender are shown in table  3.table  3stratified prevalence of migraine and tension - type headache according to age group and genderheadache typetension - type headachemigrainegendermale n ( % ) female n ( % ) male n ( % ) female n ( % ) age category ( years )  1829652 ( 31.6)586 ( 39.0)158 ( 7.7)97 ( 6.5 )  3039120 ( 36.1)73 ( 43.7)34 ( 10.2)11 ( 6.7 )  404972 ( 31.7)68 ( 39.1)17 ( 7.5)18 ( 10.3 )  50 and above92 ( 37.3)49 ( 40.8)28 ( 11.3)8 ( 6.7)total936 ( 32.6)776 ( 39.5)237 ( 8.3)134 ( 6.8 ) stratified prevalence of migraine and tension - type headache according to age group and gender only 17.3% of participants sought medical care for their headaches .", "the percentage of participants using analgesics on a weekly basis was 24.5% , less than monthly but more than weekly was 24.8% , monthly usage was 22.5% , and the percentage of participants who never use a analgesics was 14.6% .", "participants who used analgesics on the advice of a physician were 15.3% , while those who used analgesics on a pharmacist advice compromised 13.6% .", "those who used analgesics based on the experience of a family member or others were 34.8 and 47.4% , respectively ( table  4 ) .", "in addition , 13.1% of participants complained of increased headache severity or frequency on medication use .", "results also revealed that 22.0% of participants had increased their analgesic dose and 78.0% did not change their regimen .", "the most frequently used analgesic among participants was acetaminophen ( 78.00% ) , and to a lesser extent ibuprofen ( 7.49% ) and aspirin ( 5.58% ) ( table  5).table  4approach to medication use among patients with headachevariablen ( % ) seeking medical help for headaches  yes688 ( 17.3 )  no3,290 ( 82.7)advice on using analgesics  physician609 ( 15.3 )  pharmacist541 ( 13.6 )  family1,383 ( 34.8 )  others ( friends , co - workers , neighbors , etc.)1,887 ( 36.3)frequency of analgesics usage  daily606 ( 15.2 )  fewer than daily to weekly974 ( 24.5 )  fewer than weekly to monthly985 ( 24.8 )  fewer than monthly to 1  year829 ( 22.4 )  no analgesics584 ( 14.6)increase in headache frequency after analgesic use  yes520 ( 13.1 )  no3,458 ( 86.9)increase analgesic dose used over time  yes877 ( 22.0 )  no3,101 ( 78.0)some participants have sought the advice of more than one categorytable  5frequency of analgesics use among headache patientsname of the analgesic usedn ( % ) acetaminophen3,103 ( 78.0)ibuprofen298 ( 7.5)aspirin222 ( 5.6)diclofenac sodium64 ( 1.6)naproxen25 ( 0.6)metamizole11 ( 0.3)mefenamic acid10 ( 0.3)ergotamine10 ( 0.3)others137 ( 3.5)note that some patients used more than one medication approach to medication use among patients with headache some participants have sought the advice of more than one category frequency of analgesics use among headache patients note that some patients used more than one medication", "in this study , we report for the first time an overall 1-year period prevalence of headache among adults in jordan . a sample ( 4,836 ) of participants", "were requested to complete a self - conducted screening questionnaire . as much as 82.3% of participants reported that they complained of headache at least once per year .", "this is much higher than the average global prevalence of headache ( 46% ) [ 27 , 28 ] .", "similar percentages were obtained from two studies conducted in other middle eastern countries namely oman   and qatar , and from other developing   and developed countries [ 3234 ] . on the other hand ,", "one study conducted in oman showed a prevalence of headache of about 45% , however , that study was among university medical students and not the general population . in nearby saudi arabia , the prevalence of primary headaches ranged from 8 to 13% [ 30 , 36 , 37 ] , which is much lower than that reported in all other studies from the middle east area .", "the higher prevalence of headache in jordan indicates that it is one of the major health problem .", "tension - type headache was found to be the most prevalent among other types of headaches ( 36.9% compared to 7.7% for migraine ) .", "the prevalence of tension - type headache , in this study , falls within the prevalence range previously reported from studies conducted in the middle east area ( 1139% ) [ 29 , 35 , 38 , 39 ] . additionally , the reported prevalence for tension - type headache , in this study , is lower than its global prevalence ( about 42% ) , which seems to be the case for the whole middle east area .", "studies on migraine headache from other arab countries reported prevalence that ranged from 10 to 12.2% [ 29 , 39 , 40 ] , which is higher than the migraine prevalence in our studied sample .", "as in the case of tension type headache , both the global migraine prevalence ( about 11% ) , and the migraine prevalence in western europe ( about 14% )   is higher than that in jordan . therefore , jordan seems to have lower migraine prevalence than many other parts of the world .", "a recent study from nearby qatar reported a migraine prevalence of about 8.0% , which is very similar to the prevalence reported in the current study .", "the effect of headache on everyday activities was obvious in our study where a significant percentage ( 51.6% ) of participants ascertained that headache adversely affected their daily activities .", "our results showed that 82.7% of participants did not seek medical attention for their headaches . in agreement with other s findings [ 29 , 39 ] , 75.6% of our population used analgesics and acetaminophen was the most popular ( 78.0% ) .", "the popularity of acetaminophen over other analgesics could be related to its availability as an over - the - counter medication , low price , and because it is known to be safe to the gastrointestinal tract .", "previous studies from jordan and surrounding countries reported smoking prevalence of about 2648% , depending on the gender , age and the specific population group studied [ 4144 ] . worldwide", ", the overall prevalence of smoking among adult males and females was estimated to ranges from 21 to 37% in the high - income countries , and 8.9 to 49% in low to middle income countries .", "therefore , the percentage of smokers obtained in this study is comparable to previous studies from the region and to low to middle income countries all over the world . according to data obtained form the jordanian department of statistics , about 38% of jordanian adult population falls into the age range of 1929  year , which is considered a high percentage of young adults . in this study , however , 74% of the studied sample falls within the range of 1829  years , and 47% of them were college students .", "therefore , the studied sample appears to be not population based , which is one limitation of the current study .", "another limitation is the fact that the sensitivity and specificity of the questionnaire was not tested . in summary", ", headache is a major health problem in jordan , where 82.3% of participants experienced troublesome headache attacks at least once per year .", "analgesic overuse without seeking medical advice is regarded as potential for development of chronic headache .", "these results indicate the need for public education to ensure safe practices and to make the use and selling of analgesics more stringent ."], "abstract_text": ["<S> here , we investigated the prevalence of headache among adults in jordan . </S>", "<S> the study was conducted from january 2007 to november 2008 . </S>", "<S> a sample of 4,836 participants were permitted to complete a self - conducted screening questionnaire . as much as 82.3% of participants complained from headache at least once per year . </S>", "<S> 36.1% were tension - type headache and 59% of the participants had other family members who suffered from headache . </S>", "<S> headaches affected everyday activities in 51.6% of the participants ; 82.7% of participants did not seek medical attention for their headaches . among those who used analgesics ( 75.6% ) , acetaminophen was the most common ( 91.43% ) . in conclusion , headache and overuse of analgesics </S>", "<S> were prevalent in a significant part of the society . </S>", "<S> thus , there is a need to educate the public to ensure safe practices and to make the use and selling of analgesics more stringent . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion"], "sections": [["it is a common discomfort making to the top ten list of complaints in ambulatory medical care , but our understanding of the epidemiology of headache disorders is still incomplete .", "most of the recurrent headache cases are due to benign chronic primary headache disorders , such as tension headache and migraine .", "less frequently , headache could be due to other underlying conditions such as infections , cerebral hemorrhage and brain lesions [ 5 , 6 ] .", "the study of headache epidemiology can address a number of important questions such as variation in the occurrence and severity of headache in the population , and the relationship between headache and other medical disorders .", "in addition , these studies may provide clues to abortive treatments and preventive strategies for headache .", "it is well documented among adults that overuse of headache medication may contribute to the development of chronic headache [ 814 ] .", "epidemiologic studies indicate that chronic headache ( > 15  days per month ) is common in the adult population with a 25% prevalence rate [ 1521 ] and a prevalence of chronic headache associated with medication overuse of about 1% [ 18 , 20 , 21 ] .", "in general , self - medicating for headache is highly prevalent and self - care is likely to increase in the era of health - care reform [ 22 , 23 ] .", ". chronic and inappropriate use of over - the - counter drugs such as analgesics , particularly nonsteroidal anti - inflammatory drugs ( nsaids ) , can lead to overuse syndromes and drug - induced headache [ 25 , 26 ] . in the present study", ", we sought to estimate the association between analgesic use and headache among adults in a large sample of the jordanian population in relation to age , gender and headache frequency ."], ["in this study , participants ( age range 1885 ) were approached at their work places , classes or homes .", ", researchers asked every participant to nominate two other persons until the desired sample size was obtained .", "the study was ethically approved by the institutional review board ( irb ) at jordan university of science and technology and was carried out in accordance with the principles described in the declaration of helsinki , including all amendments and revisions .", "the study was conducted in various regions of jordan during the period from january 2007 to november 2008 .", "the questionnaire was distributed , in person , by the researchers , and was completed in the presence of the researcher .", "each participant was provided with a full explanation of the study and how to complete the questionnaire .", "participants were informed that the researcher would be available for any required assistance during scoring of the questionnaire .", "for the small group of participants who were illiterate , the questionnaire was administered by the researcher in the form of an interview .", "data were aggregated into groups and only the authors and the investigator were allowed access to the collected data .", "the questionnaire gathered information that included demographic data , frequency and type of headache , and its impact on everyday activities , analgesic use , consultation of a doctor or pharmacist on the use , increase or decrease in the frequency of headache after painkiller usage , increase or decrease in painkiller dose and family history of headache .", "every person who was 18  years or older was allowed to complete the survey .", "retest reliability , 50 subjects were selected randomly ; they answered the questionnaire twice with a 1-week interval . test ", "for each item , correlation coefficients ranged from 0.79 to 0.86 , suggesting that the questionnaire was reliable .", "the data were coded using the statistical package for the social sciences , version 15.0 ( spss inc .", "the data were summarized using frequency tables and means and standard deviation for continuous variables ."], ["to estimate the prevalence of headache among adults in jordan , a sample of participants ( 4,836 participants ; mean age 27    0.4  years , and male : female ratio of 59:41 ) was allowed to complete a self - conducted screening questionnaire . as shown in table  1 , about half of the participants ( 49.2% ) were university students and 78.3% were jordanian citizens . moreover ,", "70.7% participants were single and 47.4% were smokers.table  1the demographic data of the study samplevariablen ( % ) gender  male2,870 ( 59.4 )  female1,966 ( 40.6)age  1829  years3,572 ( 73.95 )  3039  years506 ( 10.46 )  4049  years402 ( 8.32 )  50  years356 ( 7.37)education  illiterate83 ( 1.7 )  elementary school65 ( 1.3 )  secondary school213 ( 4.4 )  high school610 ( 12.6 )  diploma344 ( 7.1 )  university student2,378 ( 49.2 )  bachelor901 ( 18.6 )  masters181 ( 3.7 )  ph.d.61 ( 1.3)nationality  jordanian3,787 ( 78.3 )  arab ( non - jordanian)841 ( 17.4 )  foreign208 ( 4.3)marital status  single3,422 ( 70.8 )  married1,304 ( 27.0 )  divorced55 ( 1.14 )  other55 ( 1.14)monthly income  low ( < or = 400  jd)257 ( 5.3 )  medium ( > 400  jd to < or = 1,000  jd)3,831 ( 79.2 )  high ( > 1,000  jd)748 ( 15.5)smoking  smoker2,294 ( 47.4 )  non - smoker2,542 ( 52.6)1  jd is equivalent to about 1.4 us dollars the demographic data of the study sample 1  jd is equivalent to about 1.4 us dollars of the 4,836 participants , 82.3% complained of headache at least once per year .", "for both the 1829 and the 3039-year - old groups , the yearly prevalence of headache was found to be the same ( 82.8% ) , while it was 79.9 and 81.7% for the 4049 and older than 50-year - old groups , respectively . among the participants , 17.2% had daily headache attacks ,", "while 25.7% had fewer than daily to weekly headaches , 21.6% had headaches on fewer than weekly up to monthly basis , 17.8% complained of headaches on a fewer than monthly basis and 17.7% of all participants did not experience headache attacks .", "moreover , 38.4% of the headache complainers did not know the exact type of headache they were experiencing , 36.9% complained of tension type headache and only 7.7% were diagnosed with migraine headache ; 51.6% of complainers thought that headache affected their daily activities .", "a large number of participants stated that one or more of their family members complained of headache as well ( table  2).table  2frequency , type , and family history of headachevariablen ( % ) headache frequency  daily832 ( 17.2 )  fewer than daily to weekly1,243 ( 25.7 )  fewer than weekly to monthly1,043 ( 21.6 )  fewer than monthly to 1  year860 ( 17.8 )  no headache858 ( 17.7)type of headache  migraine372 ( 7.7 )  tension1,749 ( 36.1 )  unknown1,857 ( 38.4 )  no headache858 ( 17.7)headache affects daily activities ( if any )  yes2,051 ( 51.6 )  no1,927 ( 48.4)other family members complaining from headaches  father327 ( 6.8 )  mother545 ( 11.3 )  brothers or sisters552 ( 11.4 )  other relatives558 ( 11.5 )  more than one family member860 ( 17.8 )  none1,994 ( 41.1 ) frequency , type , and family history of headache of the migrainers , 37.7% ( n  =  140 ) complained of daily headache , 35.6% ( n  =  132 ) had fewer than daily up to weekly headaches , and 18.1 ( n  =  67 ) and 8.6% ( n  =  32 ) complained of headaches on a fewer than weekly up to monthly basis , and on fewer than monthly basis , respectively .", "additionally , about 75% ( n  =  279 ) of migrainers thought that migraine adversely affected their daily activities .", "concerning tension type headache , 20.3% ( n  =  348 ) complained of daily headache , 34.7% ( n  =  594 ) had fewer than daily up to weekly headaches , and 24.1% ( n  =  413 ) and 20.8 ( n  =  356 ) complained of headaches on fewer than weekly up to monthly basis , and fewer than monthly basis , respectively .", "moreover , about 53% ( n  =  907 ) indicated that tension type headache affected their daily activities .", "the stratified prevalence for both migraine and tension type headache according to age group and gender are shown in table  3.table  3stratified prevalence of migraine and tension - type headache according to age group and genderheadache typetension - type headachemigrainegendermale n ( % ) female n ( % ) male n ( % ) female n ( % ) age category ( years )  1829652 ( 31.6)586 ( 39.0)158 ( 7.7)97 ( 6.5 )  3039120 ( 36.1)73 ( 43.7)34 ( 10.2)11 ( 6.7 )  404972 ( 31.7)68 ( 39.1)17 ( 7.5)18 ( 10.3 )  50 and above92 ( 37.3)49 ( 40.8)28 ( 11.3)8 ( 6.7)total936 ( 32.6)776 ( 39.5)237 ( 8.3)134 ( 6.8 ) stratified prevalence of migraine and tension - type headache according to age group and gender only 17.3% of participants sought medical care for their headaches .", "the percentage of participants using analgesics on a weekly basis was 24.5% , less than monthly but more than weekly was 24.8% , monthly usage was 22.5% , and the percentage of participants who never use a analgesics was 14.6% .", "participants who used analgesics on the advice of a physician were 15.3% , while those who used analgesics on a pharmacist advice compromised 13.6% .", "those who used analgesics based on the experience of a family member or others were 34.8 and 47.4% , respectively ( table  4 ) .", "in addition , 13.1% of participants complained of increased headache severity or frequency on medication use .", "results also revealed that 22.0% of participants had increased their analgesic dose and 78.0% did not change their regimen .", "the most frequently used analgesic among participants was acetaminophen ( 78.00% ) , and to a lesser extent ibuprofen ( 7.49% ) and aspirin ( 5.58% ) ( table  5).table  4approach to medication use among patients with headachevariablen ( % ) seeking medical help for headaches  yes688 ( 17.3 )  no3,290 ( 82.7)advice on using analgesics  physician609 ( 15.3 )  pharmacist541 ( 13.6 )  family1,383 ( 34.8 )  others ( friends , co - workers , neighbors , etc.)1,887 ( 36.3)frequency of analgesics usage  daily606 ( 15.2 )  fewer than daily to weekly974 ( 24.5 )  fewer than weekly to monthly985 ( 24.8 )  fewer than monthly to 1  year829 ( 22.4 )  no analgesics584 ( 14.6)increase in headache frequency after analgesic use  yes520 ( 13.1 )  no3,458 ( 86.9)increase analgesic dose used over time  yes877 ( 22.0 )  no3,101 ( 78.0)some participants have sought the advice of more than one categorytable  5frequency of analgesics use among headache patientsname of the analgesic usedn ( % ) acetaminophen3,103 ( 78.0)ibuprofen298 ( 7.5)aspirin222 ( 5.6)diclofenac sodium64 ( 1.6)naproxen25 ( 0.6)metamizole11 ( 0.3)mefenamic acid10 ( 0.3)ergotamine10 ( 0.3)others137 ( 3.5)note that some patients used more than one medication approach to medication use among patients with headache some participants have sought the advice of more than one category frequency of analgesics use among headache patients note that some patients used more than one medication"], ["in this study , we report for the first time an overall 1-year period prevalence of headache among adults in jordan . a sample ( 4,836 ) of participants", "were requested to complete a self - conducted screening questionnaire . as much as 82.3% of participants reported that they complained of headache at least once per year .", "this is much higher than the average global prevalence of headache ( 46% ) [ 27 , 28 ] .", "similar percentages were obtained from two studies conducted in other middle eastern countries namely oman   and qatar , and from other developing   and developed countries [ 3234 ] . on the other hand ,", "one study conducted in oman showed a prevalence of headache of about 45% , however , that study was among university medical students and not the general population . in nearby saudi arabia , the prevalence of primary headaches ranged from 8 to 13% [ 30 , 36 , 37 ] , which is much lower than that reported in all other studies from the middle east area .", "the higher prevalence of headache in jordan indicates that it is one of the major health problem .", "tension - type headache was found to be the most prevalent among other types of headaches ( 36.9% compared to 7.7% for migraine ) .", "the prevalence of tension - type headache , in this study , falls within the prevalence range previously reported from studies conducted in the middle east area ( 1139% ) [ 29 , 35 , 38 , 39 ] . additionally , the reported prevalence for tension - type headache , in this study , is lower than its global prevalence ( about 42% ) , which seems to be the case for the whole middle east area .", "studies on migraine headache from other arab countries reported prevalence that ranged from 10 to 12.2% [ 29 , 39 , 40 ] , which is higher than the migraine prevalence in our studied sample .", "as in the case of tension type headache , both the global migraine prevalence ( about 11% ) , and the migraine prevalence in western europe ( about 14% )   is higher than that in jordan . therefore , jordan seems to have lower migraine prevalence than many other parts of the world .", "a recent study from nearby qatar reported a migraine prevalence of about 8.0% , which is very similar to the prevalence reported in the current study .", "the effect of headache on everyday activities was obvious in our study where a significant percentage ( 51.6% ) of participants ascertained that headache adversely affected their daily activities .", "our results showed that 82.7% of participants did not seek medical attention for their headaches . in agreement with other s findings [ 29 , 39 ] , 75.6% of our population used analgesics and acetaminophen was the most popular ( 78.0% ) .", "the popularity of acetaminophen over other analgesics could be related to its availability as an over - the - counter medication , low price , and because it is known to be safe to the gastrointestinal tract .", "previous studies from jordan and surrounding countries reported smoking prevalence of about 2648% , depending on the gender , age and the specific population group studied [ 4144 ] . worldwide", ", the overall prevalence of smoking among adult males and females was estimated to ranges from 21 to 37% in the high - income countries , and 8.9 to 49% in low to middle income countries .", "therefore , the percentage of smokers obtained in this study is comparable to previous studies from the region and to low to middle income countries all over the world . according to data obtained form the jordanian department of statistics , about 38% of jordanian adult population falls into the age range of 1929  year , which is considered a high percentage of young adults . in this study , however , 74% of the studied sample falls within the range of 1829  years , and 47% of them were college students .", "therefore , the studied sample appears to be not population based , which is one limitation of the current study .", "another limitation is the fact that the sensitivity and specificity of the questionnaire was not tested . in summary", ", headache is a major health problem in jordan , where 82.3% of participants experienced troublesome headache attacks at least once per year .", "analgesic overuse without seeking medical advice is regarded as potential for development of chronic headache .", "these results indicate the need for public education to ensure safe practices and to make the use and selling of analgesics more stringent ."]]}
{"article_id": "PMC4333850", "article_text": ["a 55-year - old woman visited the emergency clinic of hanyang university seoul hospital with chest pain that had persisted for one month and had become aggravated two days earlier .", "ten years before this admission , the patient had undergone double valve replacement with aortic and mitral mechanical valves due to aortic regurgitation and mitral steno - insufficiency .", "echocardiography revealed normal motion and function of the mitral and aortic valve prostheses , and coronary angiography findings were also normal .", "however , coronary computed tomographic angiography revealed three large lobulated aneurysms with calcified walls bulging from the base of the left ventricle ( fig .", "the leak in the left ventricle was located in the sub - mitral valve prosthesis area .", "based on this finding , we diagnosed the patient with a pseudoaneurysm of the left ventricle , and surgical treatment was planned .", "arterial cannulation via the left femoral artery and venous cannulation via the left femoral vein and left pulmonary artery were performed .", "three communicating aneurysmal sacs were observed in a single plane , identical to that observed in coronary computed tomographic angiography .", "the neck of the first sac was located at the base of the left ventricle . under cardiopulmonary bypass and fibrillation ,", "a defect measuring 15 mm in diameter was identified in the sub - mitral left ventricular wall with calcification but with no infection .", "closure of the defect was performed with a supple peri - guard pericardium patch with apex processing ( synovis , st .", "the walls of the aneurysmal sacs were closed , and the patient was weaned from cardiopulmonary bypass without difficulty .", "the vital signs of the patient remained stable , and she was extubated six hours after the operation .", "follow - up echocardiography was performed on postoperative day eight , and no abnormal findings , including cardiac wall motion , were noted .", "no leakage at the closed aneurysmal neck was observed , and there was no evidence of recurrence of the pseudoaneurysm ( fig .", "she has remained disease - free for six years postoperatively , with regular coumadin anticoagulation therapy due to the presence of the prosthetic aortic and mitral valves .", "a left ventricular ( lv ) pseudoaneurysm , or false aneurysm , is defined as a contained rupture or perforation of the myocardium .", "the rupture of the myocardium occurs rarely in clinical practice ; it is most often associated with myocardial infarction or cardiac surgery , such as mitral valve replacement ( mvr ) , and is usually fatal [ 24 ] . in 1980 , cobb et al .  ", "roberts and morrow   suggested that inadvertent invasion into the lv free wall is possible during excision of the mitral valve if there is poor visualization of the operating field when using the tips of the scissors .", "other causes of complete or incomplete rupture of the lv free wall include an oversized prosthetic valve , excessive extirpation of calcium in the mitral annulus , myocardial erosion caused by the struts of the prosthetic valve , the untethering of the fibrous structures of the left ventricle during resection of mitral leaflets , an increase in lv contractility after aortic cross- clamping , enhanced lv wall stress with the support of inotropic agents , and other mechanical trauma between the free wall and the papillary muscles , such as rubber catheter wedging or metal pump suction during valve replacement .", "the wall of a false aneurysm comprises the fibrous obliteration of the pericardial sac , resulting from adhesion between the parietal and visceral layer of the pericardium .", "while a true aneurysm has nonrestrictive continuity with the lv cavity , a pseudoaneurysm has a defect in the myocardial continuity and a well - defined neck , representing a history of lv wall perforation .", "thus , such pseudoaneurysms are more likely to undergo rapid enlargement and rupture than true aneurysms . therefore , surgical correction , including the resection of the aneurysmal sac and patch repair or primary closure of the aneurysmal neck , is indicated and recommended for pseudoaneurysms . in the present case , we diagnosed a lv pseudoaneurysm using coronary computed tomographic angiography .", "myocardial infarction was excluded as a cause of the lv pseudoaneurysm because coronary angiography revealed normal findings and cardiac markers were in the normal range , although electrocardiography revealed myocardial ischemia in the inferior wall .", "the cause of lv pseudoaneurysm in our patient was a late - term complication of mvr .", "echocardiography revealed normal function of the mitral valve prosthesis ; therefore , mitral reoperation was not indicated . in such patients ,", "if a repeat sternotomy for entering the operative field is considered , it may be difficult to approach the heart due to massive pericardial adhesions , and the possibility of injury or rupture of the pseudoaneurysm during dissection may be higher .", "a direct approach to the operative field via a left lateral thoracotomy can avoid these complications and preserve the previously implanted prosthetic valves .", "further , it has other advantages , such as making it easier to establish cardiopulmonary bypass and obviating the need to cross - clamp the aorta , in contrast to the sternotomy approach . in our operation , a fibrillator was used and the exposure of the operative field was excellent . in the operative field , we found three large lobulated pseudoaneurysms with calcified walls in a line , bulging from the base of the left ventricle .", "the configuration of the pseudoaneurysms suggested a previous episode of lv rupture , which had developed over time . in patients with lv pseudoaneurysms , there is a well - defined approach to surgical correction .", "the neck of the pseudoaneurysm is dissected , and the perforated site can be closed by patch repair or primarily . the mortality rate of this surgery is approximately 10% .", ", we have reported a case of lv pseudoaneurysm that occurred as a late complication after mvr .", "we performed a left lateral thoracotomy via the fifth intercostal space to approach the operative field directly and successfully repaired the sub - mitral lv pseudoaneurysm using a prosthetic patch .", "a few cases of lv pseudoaneurysms have been previously reported ; however , no case reports have previously been presented of korean patients with an lv pseudoaneurysm following mvr who were treated with an operative technique employing a left lateral thoracotomy ."], "abstract_text": ["<S> we present a case of left ventricular pseudoaneurysm , which is a very rare and fatal complication of cardiac procedures such as mitral valve replacement . </S>", "<S> a 55-year - old woman presented to the department of thoracic and cardiovascular surgery at hanyang university seoul hospital with chest pain . </S>", "<S> ten years prior , the patient had undergone double valve replacement due to aortic regurgitation and mitral steno - insufficiency . </S>", "<S> surgical repair was successfully performed using a prosthetic pericardial patch via a left lateral thoracotomy . </S>"], "labels": null, "section_names": ["CASE REPORT", "DISCUSSION"], "sections": [["a 55-year - old woman visited the emergency clinic of hanyang university seoul hospital with chest pain that had persisted for one month and had become aggravated two days earlier .", "ten years before this admission , the patient had undergone double valve replacement with aortic and mitral mechanical valves due to aortic regurgitation and mitral steno - insufficiency .", "echocardiography revealed normal motion and function of the mitral and aortic valve prostheses , and coronary angiography findings were also normal .", "however , coronary computed tomographic angiography revealed three large lobulated aneurysms with calcified walls bulging from the base of the left ventricle ( fig .", "the leak in the left ventricle was located in the sub - mitral valve prosthesis area .", "based on this finding , we diagnosed the patient with a pseudoaneurysm of the left ventricle , and surgical treatment was planned .", "arterial cannulation via the left femoral artery and venous cannulation via the left femoral vein and left pulmonary artery were performed .", "three communicating aneurysmal sacs were observed in a single plane , identical to that observed in coronary computed tomographic angiography .", "the neck of the first sac was located at the base of the left ventricle . under cardiopulmonary bypass and fibrillation ,", "a defect measuring 15 mm in diameter was identified in the sub - mitral left ventricular wall with calcification but with no infection .", "closure of the defect was performed with a supple peri - guard pericardium patch with apex processing ( synovis , st .", "the walls of the aneurysmal sacs were closed , and the patient was weaned from cardiopulmonary bypass without difficulty .", "the vital signs of the patient remained stable , and she was extubated six hours after the operation .", "follow - up echocardiography was performed on postoperative day eight , and no abnormal findings , including cardiac wall motion , were noted .", "no leakage at the closed aneurysmal neck was observed , and there was no evidence of recurrence of the pseudoaneurysm ( fig .", "she has remained disease - free for six years postoperatively , with regular coumadin anticoagulation therapy due to the presence of the prosthetic aortic and mitral valves ."], ["a left ventricular ( lv ) pseudoaneurysm , or false aneurysm , is defined as a contained rupture or perforation of the myocardium .", "the rupture of the myocardium occurs rarely in clinical practice ; it is most often associated with myocardial infarction or cardiac surgery , such as mitral valve replacement ( mvr ) , and is usually fatal [ 24 ] . in 1980 , cobb et al .  ", "roberts and morrow   suggested that inadvertent invasion into the lv free wall is possible during excision of the mitral valve if there is poor visualization of the operating field when using the tips of the scissors .", "other causes of complete or incomplete rupture of the lv free wall include an oversized prosthetic valve , excessive extirpation of calcium in the mitral annulus , myocardial erosion caused by the struts of the prosthetic valve , the untethering of the fibrous structures of the left ventricle during resection of mitral leaflets , an increase in lv contractility after aortic cross- clamping , enhanced lv wall stress with the support of inotropic agents , and other mechanical trauma between the free wall and the papillary muscles , such as rubber catheter wedging or metal pump suction during valve replacement .", "the wall of a false aneurysm comprises the fibrous obliteration of the pericardial sac , resulting from adhesion between the parietal and visceral layer of the pericardium .", "while a true aneurysm has nonrestrictive continuity with the lv cavity , a pseudoaneurysm has a defect in the myocardial continuity and a well - defined neck , representing a history of lv wall perforation .", "thus , such pseudoaneurysms are more likely to undergo rapid enlargement and rupture than true aneurysms . therefore , surgical correction , including the resection of the aneurysmal sac and patch repair or primary closure of the aneurysmal neck , is indicated and recommended for pseudoaneurysms . in the present case , we diagnosed a lv pseudoaneurysm using coronary computed tomographic angiography .", "myocardial infarction was excluded as a cause of the lv pseudoaneurysm because coronary angiography revealed normal findings and cardiac markers were in the normal range , although electrocardiography revealed myocardial ischemia in the inferior wall .", "the cause of lv pseudoaneurysm in our patient was a late - term complication of mvr .", "echocardiography revealed normal function of the mitral valve prosthesis ; therefore , mitral reoperation was not indicated . in such patients ,", "if a repeat sternotomy for entering the operative field is considered , it may be difficult to approach the heart due to massive pericardial adhesions , and the possibility of injury or rupture of the pseudoaneurysm during dissection may be higher .", "a direct approach to the operative field via a left lateral thoracotomy can avoid these complications and preserve the previously implanted prosthetic valves .", "further , it has other advantages , such as making it easier to establish cardiopulmonary bypass and obviating the need to cross - clamp the aorta , in contrast to the sternotomy approach . in our operation , a fibrillator was used and the exposure of the operative field was excellent . in the operative field , we found three large lobulated pseudoaneurysms with calcified walls in a line , bulging from the base of the left ventricle .", "the configuration of the pseudoaneurysms suggested a previous episode of lv rupture , which had developed over time . in patients with lv pseudoaneurysms , there is a well - defined approach to surgical correction .", "the neck of the pseudoaneurysm is dissected , and the perforated site can be closed by patch repair or primarily . the mortality rate of this surgery is approximately 10% .", ", we have reported a case of lv pseudoaneurysm that occurred as a late complication after mvr .", "we performed a left lateral thoracotomy via the fifth intercostal space to approach the operative field directly and successfully repaired the sub - mitral lv pseudoaneurysm using a prosthetic patch .", "a few cases of lv pseudoaneurysms have been previously reported ; however , no case reports have previously been presented of korean patients with an lv pseudoaneurysm following mvr who were treated with an operative technique employing a left lateral thoracotomy ."]]}
{"article_id": "PMC3897633", "article_text": ["posterior urethral valve ( puv ) is the most common cause of congenital bladder outlet obstruction in boys and causes renal failure in 25% to 30% of cases before adolescence .", "puv is associated with considerable morbidity , including urinary tract infection ( uti ) , chronic renal failure , urinary incontinence , and even death [ 1 - 3 ] .", "the diagnosis is made on average in 1 in 1,285 fetal ultrasound screenings . as a result of recent prenatal diagnosis , improvements in respiratory support and resuscitation at birth , and adequate management of end - stage renal disease ,", "the mortality rate in patients with puv has significantly decreased in the past four decades .", "endoscopic ablation of a puv is the current gold standard of therapy , but approximately 10% to 30% of patients require a second procedure to achieve satisfactory valve ablation [ 3,6 - 8 ] . as animal models have proven , even partial outlet obstruction can lead to structural and functional deterioration in the detrusor muscle and bladder if the obstruction is not released soon enough [ 9 - 11 ] , which indicates the need for close follow - up after valve ablation . although studies about prognostic factors for outcome of renal function after puv ablation are available [ 8,12 - 15 ] , to date , the effects of preoperative factors on the rate of residual valves have not been precisely addressed . in this study , therefore , we sought to evaluate any possible relationship between preoperative clinical and imaging findings of patients with puv and remnant leaflets after their valve ablation to identify high - risk patients and achieve purposive follow - up .", "we evaluated 64 patients with clinical evidence of puvs , confirmed by voiding cystourethrography ( vcug ) , who were admitted between 2008 and 2012 at the shiraz university of medical sciences .", "preoperative evaluation included clinical examination , history of uti , serum electrolytes , urinalysis , complete blood count , urine culture , serum creatinine , and radiographic evaluation ( vcug / ultrasonography and dimercaptosuccinic acid [ dmsa ] scan if necessary ) .", "of these patients , 9 did not participate in follow - up sessions and were excluded from our study .", "the median patient age at the time of diagnosis was 10.0 months ( range , 5 days to 120 months ) .", "surgeon preference was to use an 11-fr pediatric resectoscope ( karl storz gmbh & co. kg , tuttlingen , germany ) .", "when the urethra was too small for this instrument , a 3-fr ureteric catheter with a metal stylet was used .", "valves were ablated mainly at the 5 , 7 , and ( in most cases ) 12 o'clock positions .", "the end point of primary ablation was determined by visual assessment of destruction of the valve .", "a foley catheter was left in place and was removed 24 to 48 hours after valve ablation .", "we performed cystoscopy in all patients at a follow - up session , at least 3 months after valve ablation ( range , 3 - 12 months ) .", "patients were divided into two groups on the basis of observation of obstructive residual leaflets in a second cystoscopy to analyze the possible preoperative factors that were related to obstructive remnant leaflets .", "group a had no evidence of obstructive remnant leaflets and in group b a second ablation was done owing to obstructive residual leaflets .", "we evaluated age at surgery , history of uti , level of serum creatinine and blood urea nitrogen , specific gravity of urine , hemoglobin , presence of scarring in the dmsa scan , vesicoureteral reflux ( vur ) and grade of reflux , renal parenchymal thickness , echogenicity of kidney , bladder wall thickness , renal cortical thickness , anteroposterior diameter of the renal pelvis , diameter of the distal ureter , and hydroureteronephrosis and side between groups . for statistical analysis ,", "student t - test and mann - whitney test for quantitative numeric data comparison and chi - square test for categorical variables were adopted by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) .", "follow - up cystoscopy was performed at least 3 months ( range , 3 - 12 months ) after primary valve ablation in 55 boys with a 6.75-f pediatric cystoscope ( richard wolf gmbh , knittlingen , germany ) .", "a total of 37 patients ( 67.3% ) had no significant remnant leaflets ( group a ) , whereas 18 boys ( 32.7% ) required a second ablation as a result of valve remnants .", "the valve remnants were ablated by use of an 11-f pediatric resectoscope ( karl storz gmbh & co. kg ) or a 3-fr ureteric catheter with a metal stylet .", "a pediatric resectoscope was used in 25 boys ( 67.6% ) in group a and in 11 boys ( 61.1% ) in group b , whereas in the other children a 3-fr ureteric catheter was applied . in both groups of patients , no significant statistical relationship was observed between method of ablation ( resectoscope versus urethral catheter method ) and remnant leaflets ( p=0.764 ) .", "the median ages at the time of ablation for groups a and b were 15 and 7 months , respectively ( fig .", "1 ) . the mann - whitney test revealed a significant difference between the groups ( p=0.017 ) .", "other clinical data that we assessed demonstrated no significant differences in children with and without remnant leaflets after valve ablation and are summarized in table 1 .", "radiographic characteristics of patients as shown by ultrasonography , dmsa scan , and vcug were collected retrospectively .", "sixteen patients had a dmsa scan and scarring was detected in all except one boy , with no significant predictive value for valve remnants ( p=1 ) . in vcug , vur and side and grade   of reflux", "significant differences were shown in the presence of vur and grade 4 or 5 reflux ( p<0.05 ) .", "our review of the ultrasound studies showed that the echogenicity of the renal parenchyma separated into normal echogenicity and hyperechogenicity , and echogenicity was higher in 94.4% of the preoperative ultrasounds of patients with residual leaflets ( p=0.000 ) .", "data on renal parenchymal thickness , bladder wall thickness , renal cortical thickness ( in the sagittal plane over a medullary pyramid , perpendicular to the capsule ) , hydroureteronephrosis and side of hydroureteronephrosis , anteroposterior diameter of the renal pelvis , and distal ureteral diameter demonstrated no influence on puv ablation outcome in our patients ( table 2 ) .", "most previous studies have focused on prognostic clinical and imaging factors in relation to the long - term outcome of renal function . to our knowledge , this is the first study to compare preoperative factors regarding the presence of postoperative obstructive leaflets . although endoscopic primary valve ablation is the preferred initial surgical treatment in most patients with puvs , the absence of obstructive residual valve remnants should be confirmed by careful clinical , radiological , and endoscopic evaluation after surgery .", "some investigators suggest vcug to confirm the adequacy of valve ablation , whereas others recommend cystoscopy follow - up in all patients .", "both methods have advantages and disadvantages such as transient dysuria , enuresis , hematuria , and toileting anxiety after vcug   and the need for general anesthesia in cystoscopy and the more invasive nature of cystoscopy than repeated vcugs . whatever the method of follow - up , the necessity of post - ablation evaluation can not be undervalued owing to the high incidence rate of remnant leaflets of 10%-30% in most studies [ 3,6 - 8 ] , even as high as 51.6% in one case series . to decrease the effect of technical components of the initial resection in the presence of residual valves , all surgeries in our study", "smeulders et al .   reported that micturating cystourethrography alone is inexact for excluding residual valve tissue ( positive and negative predictive value of 56% and 50% , respectively ) , so we routinely perform cystoscopy in all of our patients . given that there are no quantitative guidelines for the adequacy of valve ablation , our criterion was no visible obstructive residual valves in follow - up cystoscopy .", "the slightly higher rate of residual valves in our patients ( 32.7% ) than in most cited studies ( 10% to 30% ) [ 3,6 - 8 ] may have been because we applied follow - up cystoscopy in all patients after primary valve ablation , whereas others used follow - up cystoscopy only in cases with abnormal clinical or radiological findings . even though controversy exists about the role of age ( at diagnosis and time of puv surgery ) , it has been mentioned as one of the predictive factors for renal outcome after valve ablation .", "we found no published studies that assessed age as a prognostic factor for remnant leaflets , but in our patients , age ( at the time of surgery ) showed a significant effect on the presence of residual leaflets in follow - up cystoscopy .", "the cause of this difference may be the narrower urethra in younger children , which limits the surgeon 's visibility and complicates the endoscopic maneuver for puv ablation owing to fear of causing stricture . in any event , careful and closer follow - up in younger children seems necessary to achieve better long - term renal outcomes .", "besides age at diagnosis , of the other factors that we analyzed , echogenicity of kidney , presence of reflux , and grade of reflux differed significantly between the two groups ( p<0.05 ) .", "other studies have shown the hyperechogenicity of renal parenchyma as a factor that may help to predict long - time prognosis of renal insufficiency   but did not comment on it as a factor affecting the consequences of valve ablation . in our patients , 17 boys ( 94.4% ) with residual leaflets in follow - up cystoscopy showed increased renal echogenicity in comparison with 6 patients ( 16.2% ) in the other group , with a p - value less than 0.05 . in our patients , 33 children showed evidence of vur ( 16 boys in group a and 17 in group b ) and the incidence of grade 4 and 5 reflux was significantly higher in patients with obstructive remnant leaflets ( table 2 ) . to exclude the tendency in infants to have more echogenic kidneys separate from renal function , we analyzed this factor in three age groups ( under 12 , 12 to 48 , and over 48 months ) , which revealed a significant difference in the first two groups ( p<0.05 ) .", "we suppose that this statistical relationship may be due to longer and thicker valves preoperatively that caused more obstruction initially , which was followed by high grades of vur as well as hyperechoic kidney that persisted after the first ablation .", "the presence of vur and hyperechogenicity of the kidney can affect the long - term prognosis of renal function and should be considered in the management of patients with puv .", "method of ablation , renal parenchymal thickness , bladder wall thickness , renal cortical thickness , hydroureteronephrosis , anteroposterior diameter of the renal pelvis and ureter , serum creatinine , and history of uti demonstrated no relationship with residual leaflets in our patients . considering the lack of studies in this field", ", it seems that more research is inevitable to facilitate proper screening and the detection of patients with residual leaflets .", "the need for confirmation of complete resection of puv is undeniable because valve remnants may result in persistent outflow obstruction or renal failure through time and should be detected quickly to minimize the deterioration of kidney or bladder function .", "the timing of follow - up sessions in most studies is 3 to 6 months after ablation . however , owing to decreases in function and structural deformities in the bladder as a result of partial obstruction , even after as short a period as 2 weeks in animal models [ 9 - 11 ] , earlier follow - up in the first month after ablation , especially in patients at high risk of residual valves , seems more advantageous", ". there may be other operative or preoperative factors , in addition to those we evaluated in our study , with the potential to affect the end result of initial valve ablation and residual leaflets .", "the results of such research can help to provide a better algorithm for management of puvs and achieve purposive follow - up for high - risk patients instead of invasive procedures .", "younger age at surgery time , hyperechogenicity of the renal parenchyma , presence of vur , and grade of reflux before surgery in our patients had a significant relationship with residual valves , but more research is needed to confirm these results .", "more studies may result in enhanced management of patients at high risk of residual valves after puv ablation , because the sooner the obstruction is entirely resolved , the better the long - term outcome ."], "abstract_text": ["<S> purposeto find patients at high risk of obstructive remnant leaflets after valve ablation among boys with posterior urethral valve ( puv ) , we evaluated any possible relationship between preoperative findings in our patients and residual obstructive leaflets after valve ablation.materials and methodswe retrospectively reviewed the medical records of 55 patients with puv that was treated by the same surgeon between 2008 and 2012 . of these , </S>", "<S> 37 patients ( 67.3% ) had no obstructive remnant leaflets ( group a ) and 18 patients ( 32.7% ) had obstructive remnant leaflets ( group b ) in follow - up cystoscopy . </S>", "<S> preoperative clinical and radiological findings were evaluated and compared between the groups.resultsamong all the preoperative data we examined , the analysis revealed that age at the time of surgery ( median age : group a , 15 months ; group b , 7 months ; p=0.017 ) , echogenicity of kidneys ( p<0.05 ) , presence of vesicoureteral reflux ( p<0.05 ) , and grade of reflux ( p<0.05 ) were significantly different between the groups . </S>", "<S> method of valve ablation , anterior - posterior diameters of the renal pelvis , renal cortical thickness , bladder wall thickening , and scarring on the dimercaptosuccinic acid scan showed no significant differences between the two groups.conclusionsin our patients , younger age at surgery time , hyperechogenicity of renal parenchyma , presence of vesicoureteral reflux , and grade 4 or 5 reflux before surgery had a significant relationship with residual valves . </S>", "<S> more studies may result in enhanced management of patients at high risk of residual valves after puv ablation , because the sooner the obstruction is resolved entirely , the better the outcome . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["posterior urethral valve ( puv ) is the most common cause of congenital bladder outlet obstruction in boys and causes renal failure in 25% to 30% of cases before adolescence .", "puv is associated with considerable morbidity , including urinary tract infection ( uti ) , chronic renal failure , urinary incontinence , and even death [ 1 - 3 ] .", "the diagnosis is made on average in 1 in 1,285 fetal ultrasound screenings . as a result of recent prenatal diagnosis , improvements in respiratory support and resuscitation at birth , and adequate management of end - stage renal disease ,", "the mortality rate in patients with puv has significantly decreased in the past four decades .", "endoscopic ablation of a puv is the current gold standard of therapy , but approximately 10% to 30% of patients require a second procedure to achieve satisfactory valve ablation [ 3,6 - 8 ] . as animal models have proven , even partial outlet obstruction can lead to structural and functional deterioration in the detrusor muscle and bladder if the obstruction is not released soon enough [ 9 - 11 ] , which indicates the need for close follow - up after valve ablation . although studies about prognostic factors for outcome of renal function after puv ablation are available [ 8,12 - 15 ] , to date , the effects of preoperative factors on the rate of residual valves have not been precisely addressed . in this study , therefore , we sought to evaluate any possible relationship between preoperative clinical and imaging findings of patients with puv and remnant leaflets after their valve ablation to identify high - risk patients and achieve purposive follow - up ."], ["we evaluated 64 patients with clinical evidence of puvs , confirmed by voiding cystourethrography ( vcug ) , who were admitted between 2008 and 2012 at the shiraz university of medical sciences .", "preoperative evaluation included clinical examination , history of uti , serum electrolytes , urinalysis , complete blood count , urine culture , serum creatinine , and radiographic evaluation ( vcug / ultrasonography and dimercaptosuccinic acid [ dmsa ] scan if necessary ) .", "of these patients , 9 did not participate in follow - up sessions and were excluded from our study .", "the median patient age at the time of diagnosis was 10.0 months ( range , 5 days to 120 months ) .", "surgeon preference was to use an 11-fr pediatric resectoscope ( karl storz gmbh & co. kg , tuttlingen , germany ) .", "when the urethra was too small for this instrument , a 3-fr ureteric catheter with a metal stylet was used .", "valves were ablated mainly at the 5 , 7 , and ( in most cases ) 12 o'clock positions .", "the end point of primary ablation was determined by visual assessment of destruction of the valve .", "a foley catheter was left in place and was removed 24 to 48 hours after valve ablation .", "we performed cystoscopy in all patients at a follow - up session , at least 3 months after valve ablation ( range , 3 - 12 months ) .", "patients were divided into two groups on the basis of observation of obstructive residual leaflets in a second cystoscopy to analyze the possible preoperative factors that were related to obstructive remnant leaflets .", "group a had no evidence of obstructive remnant leaflets and in group b a second ablation was done owing to obstructive residual leaflets .", "we evaluated age at surgery , history of uti , level of serum creatinine and blood urea nitrogen , specific gravity of urine , hemoglobin , presence of scarring in the dmsa scan , vesicoureteral reflux ( vur ) and grade of reflux , renal parenchymal thickness , echogenicity of kidney , bladder wall thickness , renal cortical thickness , anteroposterior diameter of the renal pelvis , diameter of the distal ureter , and hydroureteronephrosis and side between groups . for statistical analysis ,", "student t - test and mann - whitney test for quantitative numeric data comparison and chi - square test for categorical variables were adopted by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) ."], ["follow - up cystoscopy was performed at least 3 months ( range , 3 - 12 months ) after primary valve ablation in 55 boys with a 6.75-f pediatric cystoscope ( richard wolf gmbh , knittlingen , germany ) .", "a total of 37 patients ( 67.3% ) had no significant remnant leaflets ( group a ) , whereas 18 boys ( 32.7% ) required a second ablation as a result of valve remnants .", "the valve remnants were ablated by use of an 11-f pediatric resectoscope ( karl storz gmbh & co. kg ) or a 3-fr ureteric catheter with a metal stylet .", "a pediatric resectoscope was used in 25 boys ( 67.6% ) in group a and in 11 boys ( 61.1% ) in group b , whereas in the other children a 3-fr ureteric catheter was applied . in both groups of patients , no significant statistical relationship was observed between method of ablation ( resectoscope versus urethral catheter method ) and remnant leaflets ( p=0.764 ) .", "the median ages at the time of ablation for groups a and b were 15 and 7 months , respectively ( fig .", "1 ) . the mann - whitney test revealed a significant difference between the groups ( p=0.017 ) .", "other clinical data that we assessed demonstrated no significant differences in children with and without remnant leaflets after valve ablation and are summarized in table 1 .", "radiographic characteristics of patients as shown by ultrasonography , dmsa scan , and vcug were collected retrospectively .", "sixteen patients had a dmsa scan and scarring was detected in all except one boy , with no significant predictive value for valve remnants ( p=1 ) . in vcug , vur and side and grade   of reflux", "significant differences were shown in the presence of vur and grade 4 or 5 reflux ( p<0.05 ) .", "our review of the ultrasound studies showed that the echogenicity of the renal parenchyma separated into normal echogenicity and hyperechogenicity , and echogenicity was higher in 94.4% of the preoperative ultrasounds of patients with residual leaflets ( p=0.000 ) .", "data on renal parenchymal thickness , bladder wall thickness , renal cortical thickness ( in the sagittal plane over a medullary pyramid , perpendicular to the capsule ) , hydroureteronephrosis and side of hydroureteronephrosis , anteroposterior diameter of the renal pelvis , and distal ureteral diameter demonstrated no influence on puv ablation outcome in our patients ( table 2 ) ."], ["most previous studies have focused on prognostic clinical and imaging factors in relation to the long - term outcome of renal function . to our knowledge , this is the first study to compare preoperative factors regarding the presence of postoperative obstructive leaflets . although endoscopic primary valve ablation is the preferred initial surgical treatment in most patients with puvs , the absence of obstructive residual valve remnants should be confirmed by careful clinical , radiological , and endoscopic evaluation after surgery .", "some investigators suggest vcug to confirm the adequacy of valve ablation , whereas others recommend cystoscopy follow - up in all patients .", "both methods have advantages and disadvantages such as transient dysuria , enuresis , hematuria , and toileting anxiety after vcug   and the need for general anesthesia in cystoscopy and the more invasive nature of cystoscopy than repeated vcugs . whatever the method of follow - up , the necessity of post - ablation evaluation can not be undervalued owing to the high incidence rate of remnant leaflets of 10%-30% in most studies [ 3,6 - 8 ] , even as high as 51.6% in one case series . to decrease the effect of technical components of the initial resection in the presence of residual valves , all surgeries in our study", "smeulders et al .   reported that micturating cystourethrography alone is inexact for excluding residual valve tissue ( positive and negative predictive value of 56% and 50% , respectively ) , so we routinely perform cystoscopy in all of our patients . given that there are no quantitative guidelines for the adequacy of valve ablation , our criterion was no visible obstructive residual valves in follow - up cystoscopy .", "the slightly higher rate of residual valves in our patients ( 32.7% ) than in most cited studies ( 10% to 30% ) [ 3,6 - 8 ] may have been because we applied follow - up cystoscopy in all patients after primary valve ablation , whereas others used follow - up cystoscopy only in cases with abnormal clinical or radiological findings . even though controversy exists about the role of age ( at diagnosis and time of puv surgery ) , it has been mentioned as one of the predictive factors for renal outcome after valve ablation .", "we found no published studies that assessed age as a prognostic factor for remnant leaflets , but in our patients , age ( at the time of surgery ) showed a significant effect on the presence of residual leaflets in follow - up cystoscopy .", "the cause of this difference may be the narrower urethra in younger children , which limits the surgeon 's visibility and complicates the endoscopic maneuver for puv ablation owing to fear of causing stricture . in any event , careful and closer follow - up in younger children seems necessary to achieve better long - term renal outcomes .", "besides age at diagnosis , of the other factors that we analyzed , echogenicity of kidney , presence of reflux , and grade of reflux differed significantly between the two groups ( p<0.05 ) .", "other studies have shown the hyperechogenicity of renal parenchyma as a factor that may help to predict long - time prognosis of renal insufficiency   but did not comment on it as a factor affecting the consequences of valve ablation . in our patients , 17 boys ( 94.4% ) with residual leaflets in follow - up cystoscopy showed increased renal echogenicity in comparison with 6 patients ( 16.2% ) in the other group , with a p - value less than 0.05 . in our patients , 33 children showed evidence of vur ( 16 boys in group a and 17 in group b ) and the incidence of grade 4 and 5 reflux was significantly higher in patients with obstructive remnant leaflets ( table 2 ) . to exclude the tendency in infants to have more echogenic kidneys separate from renal function , we analyzed this factor in three age groups ( under 12 , 12 to 48 , and over 48 months ) , which revealed a significant difference in the first two groups ( p<0.05 ) .", "we suppose that this statistical relationship may be due to longer and thicker valves preoperatively that caused more obstruction initially , which was followed by high grades of vur as well as hyperechoic kidney that persisted after the first ablation .", "the presence of vur and hyperechogenicity of the kidney can affect the long - term prognosis of renal function and should be considered in the management of patients with puv .", "method of ablation , renal parenchymal thickness , bladder wall thickness , renal cortical thickness , hydroureteronephrosis , anteroposterior diameter of the renal pelvis and ureter , serum creatinine , and history of uti demonstrated no relationship with residual leaflets in our patients . considering the lack of studies in this field", ", it seems that more research is inevitable to facilitate proper screening and the detection of patients with residual leaflets .", "the need for confirmation of complete resection of puv is undeniable because valve remnants may result in persistent outflow obstruction or renal failure through time and should be detected quickly to minimize the deterioration of kidney or bladder function .", "the timing of follow - up sessions in most studies is 3 to 6 months after ablation . however , owing to decreases in function and structural deformities in the bladder as a result of partial obstruction , even after as short a period as 2 weeks in animal models [ 9 - 11 ] , earlier follow - up in the first month after ablation , especially in patients at high risk of residual valves , seems more advantageous", ". there may be other operative or preoperative factors , in addition to those we evaluated in our study , with the potential to affect the end result of initial valve ablation and residual leaflets .", "the results of such research can help to provide a better algorithm for management of puvs and achieve purposive follow - up for high - risk patients instead of invasive procedures ."], ["younger age at surgery time , hyperechogenicity of the renal parenchyma , presence of vur , and grade of reflux before surgery in our patients had a significant relationship with residual valves , but more research is needed to confirm these results .", "more studies may result in enhanced management of patients at high risk of residual valves after puv ablation , because the sooner the obstruction is entirely resolved , the better the long - term outcome ."]]}
{"article_id": "PMC2905912", "article_text": ["white spot lesions are areas of demineralized enamel that represent an early stage of caries , since they can progress to cavitated lesions if untreated .", "the demineralization process can be arrested or reversed by noninvasive means , including oral hygiene counselling and/or topical fluoride application [ 2 , 3 ] .", "therefore , early detection of white spot lesions ( wsls ) is desirable since early preventive treatment can avert the need for future restorative treatment .  ", "new technologies such as the diagnodent and quantitative light fluorescence ( qlf ) devices have been developed for the detection of early carious lesions .", "these techniques are intended to be adjuncts to clinical decision making and aid in planning preventive treatment . despite the potential of these methods ,", "the results from these tools are affected by confounding factors in the oral environment , thereby compromising the sensitivity and/or specificity of these techniques .", "for example with the diagnodent , stain , calculus , plaque , as well as developmental hypomineralization can produce a fluorescence output that results in false - positive readings . a review of the literature evaluating the diagnodent device found that , compared to visual assessment methods , the sensitivity was consistently higher but the specificity was lower , concluding that the increased risk of false - positives limits its clinical usefulness . for qlf , stain , plaque , fluorosis , or any developmental hypocalcification results in false - positives [ 811 ] .", "composite resins pit and fissure sealants as well as prophy pastes could also lead to false - positive results .", "clearly , a new technology is needed that will not be affected by factors in the oral environment . a new optical approach to detect white spot lesions clinically is jointly being developed at the national research council of canada - institute for biodiagnostics , the university of manitoba and dalhousie university , using a combination of optical coherence tomography ( oct ) and polarized raman spectroscopy ( prs ) .", "oct is a nondestructive technique for high - resolution ( 1020  m ) depth imaging that gives information about the morphology and depth of white spot lesions up to 3  mm into enamel .", "this method is similar to ultrasound technology , but instead of sound waves , light waves are utilized and the imaging is limited to near - surface tissues .", "changes in the refractive indices of structures cause light backscattering , creating an image that is different for sound enamel and demineralized enamel .  ", "prs is a noninvasive spectroscopic method that provides details on the biochemistry and molecular structure of white spot lesions .", "the energy difference between the incoming excitation light and scattered photons is proportional to the vibrational energy of molecules within the sample being studied , known as the raman effect .", "the caries process results in biochemical and structural changes that can be followed by prs , which uses scattered light to determine differences between the mineral matrix of sound enamel and demineralized enamel .", "our previous studies have shown that oct and prs can be used to distinguish sound enamel from white spot lesions ( wsls ) ex vivo [ 14 , 15 , 17 ] . however , the effects of calculus , hypocalcification , and stain on oct and prs have yet to be established . by combining oct and prs ,", "false - positive results from one technology can potentially be eliminated , thereby increasing the sensitivity and specificity of the new method .  ", "calculus consists of an organic matrix with the inorganic components of dicalcium phosphate dehydrate ( dcpd ) , hydroxyapatite ( ha ) , octacalcium phosphate ( ocp ) , and -tricalcium phosphate ( tcp ) .", "raman spectra of human enamel are characterized by a main peak at ~959  cm arising predominantly from the symmetric phosphate groups in carbonated hydroxyapatite , the major mineral component of dental enamel [ 18 , 19 ] .", "clinically , hypocalcification appears visually similar ( chalky white ) to a wsl and must be differentiated from a wsl since hypocalcification is a developmental defect that does not need to be treated . in hypocalcified enamel ,", "the crystals are arranged normally but there are pores due to larger spaces between enamel rods .", "intrinsic stain occurs during tooth development due to alterations in the structure or thickness of enamel or dentin , extrinsic stain accumulates on the acquired pellicle , and internalized stain occurs when extrinsic stain is incorporated into areas of enamel defects after tooth development .   ", "the objective of this study was to determine whether calculus , hypocalcification , and stain are confounding factors affecting wsl detection with optical coherence tomography and polarized raman spectroscopy .", "postextraction , the teeth were rinsed with water and clinically examined by two clinicians independently .", "samples were separated into 5 groups : sound enamel , wsls , calculus , hypocalcification , and stained enamel .", "figure 1 is a diagrammatic representation of sample sizes and allocation criteria for the above groups .", "since patient histories were not available , it can not be determined if hypocalcified enamel was caused by excess ingestion of fluoride ; therefore , this category is referred to as  hypocalcification  .  ", "figure 2 is a diagrammatic summary of data collection and analyses with oct and prs .", "a humphrey 's system 2000 optical coherence tomography scanner ( zeiss humphrey systems , dublin , ca , usa ) operating at 850  nm was used for oct measurements . for all data ,", "the laser was focused to the thinnest line on the tooth surface and the scan length was 2.0  mm .", "three scans were collected across the area of interest with the proximal surface of interest oriented perpendicular to the laser beam .", "three vertical scans were also taken of sound enamel on the same tooth surface for comparison .", "oct images had display resolutions of 500    100 pixels , and transverse resolutions of 1020  m .", "figure 3(a ) displays a photo of a tooth being scanned with the oct system ( laser scan line circled ) with the corresponding 2-dimensional depth image of sound enamel ( figure 3(b ) ) .", "matlab software ( the mathworks , natick , ma , usa ) was used to plot oct images . a labramhr raman microspectrometer ( horiba jobin yvon , edison , nj , usa )", "the laser excitation was 830  nm and a 10x microscope objective was used ( power at the sample was 125  mw ) with an acquisition time of 5  seconds and 6 accumulations . on each surface", ", point measurements were taken with parallel- ( p1 ) and cross- ( p2 ) polarizations at a minimum of three different points .", "raman data were analyzed using matlab software to calculate the depolarization ratios ( i/i ) , where i is the area under the raman band from 9251000  cm for cross - polarization ( p2 ) , and i is the similar area for parallel - polarization ( p1 ) .", "this wavenumber range was used since it centres at 959  cm ( the main peak due to phosphate groups such as those found in apatite ) .", "two - dimensional oct images are shown of sound enamel ( figure 3(b ) ) and a wsl ( figure 4(a ) ) . in the sound enamel image", ", there is intense light backscattering at the tooth - air interface , and no significant signal with depth into the enamel .", "in contrast , the oct image of a wsl shows significant light backscattering beneath the surface with a triangular shape characteristic of a subsurface lesion as observed on histological sections . the two - dimensional oct image displayed as figure 4(b ) shows a deposit of material on the tooth surface that is attributed to calculus .", "the oct image of hypocalcification in figure 4(c ) shows diffuse light back - scattering and a more irregular subsurface pattern of scattering across the entire region scanned compared to sound enamel .", "the light back - scattering of hypocalcified regions is also more irregular than wsls , which have a characteristic triangular shape .", "the oct image of stained enamel in figure 4(d ) demonstrates increased light backscattering when compared to sound enamel , but again there is no triangular - shaped subsurface light back - scattering as observed with wsls .  ", "average raman depolarization ratios (  ) from the various groups are depicted in a box - and - whisker plot ( figure 5 ) . in order to determine whether the mean depolarization ratio values of the various groups were statistically significant from one another ,", "a one - way analysis of variance ( anova ) followed by unequal n hsd post - hoc comparisons ( statistica ) was performed ( table 1 ) .", "it was determined that the mean values were statistically significant in all cases at p < .05 except for three cases .", "sound enamel was not statistically significant from hypocalcified enamel , carious enamel was not statistically significant from stained enamel , and lastly stained enamel was not statistically significant from hypocalcified enamel . in raman spectra acquired from areas of stain ( figure 6 ) , there is a large background sloping fluorescence that is not observed in spectra from areas of sound enamel or wsls without stain where these spectra have a flat background .", "the development of new technologies for wsl assessment requires that these devices undergo clinical validation prior to becoming an accepted clinical method .", "in addition to demonstrating the performance of these methods for providing high sensitivity for wsl detection , high specificity is also desirable . optical coherence tomography and polarized raman spectroscopy are methods that potentially can address the need for a technology with high sensitivity and high specificity . to date there are no studies outside our research group that investigate a combination of oct and prs to detect wsls , and in particular the effects of the calculus , stain , or hypocalcification on the utility of these two technologies .", "oct imaging allows differentiation of sound from demineralized enamel on the basis of the characteristic triangular shape of the back - scattered signal beneath the tooth surface in images of wsls .", "this subsurface scattering pattern is believed to be due to wsls having porous enamel matrices , allowing incident light to travel further into the enamel and causing more scattering to occur , which is detected by the oct system .  ", "when calculus is present , it appears clearly as a deposit in the two - dimensional oct image .", "similar to conventional caries assessment methods , scaling of calculus is recommended before an assessment is made using oct and prs .", "oct can possibly be used to alert the clinician when calculus is still present in the region of interest and that further scaling is necessary .", "oct images of hypocalcification can be differentiated from wsls , since the light back - scattering found with hypocalcification is more irregular than wsls and lacks the characteristic triangular shape . to some extent", ", hypocalcification can be differentiated from sound enamel with oct since hypocalcification has a more irregular pattern of scattering at the surface and subsurface compared to sound enamel .", "although areas of stain are not easily distinguishable from hypocalcified enamel or sound enamel with oct , they can be readily differentiated from wsls because areas of stain lack the subsurface triangular - shaped back - scattering pattern characteristic of wsls .", "further image analysis is required to nonsubjectively distinguish sound enamel from stained and hypocalcified enamel .  ", "statistical analysis revealed that mean raman depolarization ratios were statistically significant in all cases at p < .05 except for three cases : ( a ) sound enamel compared to hypocalcified enamel , ( b ) carious enamel compared to stained enamel , and ( c ) stained enamel from hypocalcified enamel . since hypocalcified enamel can be mistaken for wsls upon visual clinical examination , it is reassuring that the raman depolarization values from hypocalcified enamel are distinct from caries , thereby increasing the specificity of the method .", "based on the analysis focusing on the depolarization ratio of the 959  cm peak , hypocalcified enamel could not be distinguished from sound enamel .", "this observation indicates that , fundamentally , hypocalcified enamel is like healthy sound enamel and not in need of treatment . in order to make this separation ,", "further studies are needed which include examining the peak positions and peak width of the phosphate hydroxyapatite peak , which have been shown to be affected by the mineral crystallinity .", "furthermore , other peaks could be surveyed to look for peaks characteristic of specialized forms of hypocalcification such as fluorosis .  ", "the analyses indicate that stain complicates the use of prs for discriminating stained sound enamel from carious enamel . in reviewing the raman spectra acquired from areas of stain", ", it is observed that stained sound enamel spectra show a large background fluorescence that is not found in spectra from areas of sound enamel or wsls without stain . in these preliminary analyses ,", "a straight - line background in the region of the peak was simply subtracted for calculating the areas under the peak . clearly , this initial approach is not enough as the curved background confounds this calculation .", "further studies are consequently required using various algorithms to robustly fit the fluorescence background for elimination .", "in addition , there are various instrument - based methods proposed to suppress the background fluorescence in raman spectra . with fluorescence - based devices ( diagnodent , qlf ) , stain chromophores from any source", "with prs , the fundamental basis of the method is the phosphate moieties specific to the dominant hydroxyapatite component from the mineral matrix .", "this peak itself is not due to staining and provides information on mineralization states as required for a method to detect demineralization in caries development .", "therefore , with improved methods for fluorescence background subtraction / suppression , it is anticipated that staining will no longer confound prs analyses .", "the statistical analyses also indicated that , based on the raman depolarization ratio , stained sound enamel can not be distinguished from hypocalcified enamel .", "this is also not surprising since both groups are overall noncarious intact enamel with one group containing extrinsic staining .", "the underlying biochemistry of the enamel matrix is largely similar in both cases as revealed by the raman spectra .", "subsequent analyses such as those described above for examining spectra of hypocalcified enamel could provide insights for discriminating these two groups .", "this result suggests that , like regions of demineralization , the apatite in areas of calculus has a disordered crystal structure and orientation as shown by higher depolarization ratio values .", "calculus is easily observed on the oct image , and therefore oct will be the first technology used with the fibre optic probe to screen for wsls and to determine whether prs analysis of the lesion is necessary .", "if calculus is detected on the oct image , the area will be scaled before the prs method is applied to determine the raman depolarization ratio . by combining oct and prs technologies ,", "it is possible to rule out false - positive readings that might occur from using raman depolarization ratios alone .", "furthermore , according to the box- and whisker- plot ( figure 4 ) , setting a depolarization ratio threshold of ~0.18 can help discriminate carious enamel from enamel with a calculus deposit .", "it is important to note that this study was limited to extracted human teeth . in the oral environment , there will be a combination of many possible confounding factors present , and an additive effect may result in the oral cavity that was not observed when testing the possible confounding factors separately .", "these issues will be addressed in our subsequent studies as we transition to using fibre - optic - based devices for in vivo measurements with patient volunteers .   in conclusion , calculus and hypocalcification", "stain does not influence wsl detection with oct . with improved analysis methods , the current limitations with prs analysis in the presence of stain", "the combination of oct and prs technologies can decrease the risk of false - positive reading and increase the potential for the detection of wsls with high sensitivity and specificity .", "this initial study has pointed out limitations that should be taken into consideration when using these methods and highlighted further analyses that need to be undertaken to better understand the effects of calculus , hypocalcification , and stain on oct and prs technologies ."], "abstract_text": ["<S> optical coherence tomography ( oct ) and polarized raman spectroscopy ( prs ) have been shown as useful methods for distinguishing sound enamel from carious lesions ex vivo . however </S>", "<S> , factors in the oral environment such as calculus , hypocalcification , and stain could lead to false - positive results . </S>", "<S> oct and prs were used to investigate extracted human teeth clinically examined for sound enamel , white spot lesion ( wsl ) , calculus , hypocalcification , and stain to determine whether these factors would confound wsl detection with these optical methods . </S>", "<S> results indicate that oct allowed differentiating caries from sound enamel , hypocalcification , and stain , with calculus deposits recognizable on oct images . </S>", "<S> anova and post - hoc unequal n hsd analyses to compare the mean raman depolarization ratios from the various groups showed that the mean values were statistically significant at p < .05 , except for several comparison pairs . with the current prs analysis method , </S>", "<S> the mean depolarization ratios of stained enamel and caries are not significantly different due to the sloping background in the stained enamel spectra . </S>", "<S> overall , calculus and hypocalcification are not confounding factors affecting wsl detection using oct and prs . </S>", "<S> stain does not influence wsl detection with oct . </S>", "<S> improved prs analysis methods are needed to differentiate carious from stained enamel . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods and Materials", "3. Results", "4. Discussion"], "sections": [["white spot lesions are areas of demineralized enamel that represent an early stage of caries , since they can progress to cavitated lesions if untreated .", "the demineralization process can be arrested or reversed by noninvasive means , including oral hygiene counselling and/or topical fluoride application [ 2 , 3 ] .", "therefore , early detection of white spot lesions ( wsls ) is desirable since early preventive treatment can avert the need for future restorative treatment .  ", "new technologies such as the diagnodent and quantitative light fluorescence ( qlf ) devices have been developed for the detection of early carious lesions .", "these techniques are intended to be adjuncts to clinical decision making and aid in planning preventive treatment . despite the potential of these methods ,", "the results from these tools are affected by confounding factors in the oral environment , thereby compromising the sensitivity and/or specificity of these techniques .", "for example with the diagnodent , stain , calculus , plaque , as well as developmental hypomineralization can produce a fluorescence output that results in false - positive readings . a review of the literature evaluating the diagnodent device found that , compared to visual assessment methods , the sensitivity was consistently higher but the specificity was lower , concluding that the increased risk of false - positives limits its clinical usefulness . for qlf , stain , plaque , fluorosis , or any developmental hypocalcification results in false - positives [ 811 ] .", "composite resins pit and fissure sealants as well as prophy pastes could also lead to false - positive results .", "clearly , a new technology is needed that will not be affected by factors in the oral environment . a new optical approach to detect white spot lesions clinically is jointly being developed at the national research council of canada - institute for biodiagnostics , the university of manitoba and dalhousie university , using a combination of optical coherence tomography ( oct ) and polarized raman spectroscopy ( prs ) .", "oct is a nondestructive technique for high - resolution ( 1020  m ) depth imaging that gives information about the morphology and depth of white spot lesions up to 3  mm into enamel .", "this method is similar to ultrasound technology , but instead of sound waves , light waves are utilized and the imaging is limited to near - surface tissues .", "changes in the refractive indices of structures cause light backscattering , creating an image that is different for sound enamel and demineralized enamel .  ", "prs is a noninvasive spectroscopic method that provides details on the biochemistry and molecular structure of white spot lesions .", "the energy difference between the incoming excitation light and scattered photons is proportional to the vibrational energy of molecules within the sample being studied , known as the raman effect .", "the caries process results in biochemical and structural changes that can be followed by prs , which uses scattered light to determine differences between the mineral matrix of sound enamel and demineralized enamel .", "our previous studies have shown that oct and prs can be used to distinguish sound enamel from white spot lesions ( wsls ) ex vivo [ 14 , 15 , 17 ] . however , the effects of calculus , hypocalcification , and stain on oct and prs have yet to be established . by combining oct and prs ,", "false - positive results from one technology can potentially be eliminated , thereby increasing the sensitivity and specificity of the new method .  ", "calculus consists of an organic matrix with the inorganic components of dicalcium phosphate dehydrate ( dcpd ) , hydroxyapatite ( ha ) , octacalcium phosphate ( ocp ) , and -tricalcium phosphate ( tcp ) .", "raman spectra of human enamel are characterized by a main peak at ~959  cm arising predominantly from the symmetric phosphate groups in carbonated hydroxyapatite , the major mineral component of dental enamel [ 18 , 19 ] .", "clinically , hypocalcification appears visually similar ( chalky white ) to a wsl and must be differentiated from a wsl since hypocalcification is a developmental defect that does not need to be treated . in hypocalcified enamel ,", "the crystals are arranged normally but there are pores due to larger spaces between enamel rods .", "intrinsic stain occurs during tooth development due to alterations in the structure or thickness of enamel or dentin , extrinsic stain accumulates on the acquired pellicle , and internalized stain occurs when extrinsic stain is incorporated into areas of enamel defects after tooth development .   ", "the objective of this study was to determine whether calculus , hypocalcification , and stain are confounding factors affecting wsl detection with optical coherence tomography and polarized raman spectroscopy ."], ["postextraction , the teeth were rinsed with water and clinically examined by two clinicians independently .", "samples were separated into 5 groups : sound enamel , wsls , calculus , hypocalcification , and stained enamel .", "figure 1 is a diagrammatic representation of sample sizes and allocation criteria for the above groups .", "since patient histories were not available , it can not be determined if hypocalcified enamel was caused by excess ingestion of fluoride ; therefore , this category is referred to as  hypocalcification  .  ", "figure 2 is a diagrammatic summary of data collection and analyses with oct and prs .", "a humphrey 's system 2000 optical coherence tomography scanner ( zeiss humphrey systems , dublin , ca , usa ) operating at 850  nm was used for oct measurements . for all data ,", "the laser was focused to the thinnest line on the tooth surface and the scan length was 2.0  mm .", "three scans were collected across the area of interest with the proximal surface of interest oriented perpendicular to the laser beam .", "three vertical scans were also taken of sound enamel on the same tooth surface for comparison .", "oct images had display resolutions of 500    100 pixels , and transverse resolutions of 1020  m .", "figure 3(a ) displays a photo of a tooth being scanned with the oct system ( laser scan line circled ) with the corresponding 2-dimensional depth image of sound enamel ( figure 3(b ) ) .", "matlab software ( the mathworks , natick , ma , usa ) was used to plot oct images . a labramhr raman microspectrometer ( horiba jobin yvon , edison , nj , usa )", "the laser excitation was 830  nm and a 10x microscope objective was used ( power at the sample was 125  mw ) with an acquisition time of 5  seconds and 6 accumulations . on each surface", ", point measurements were taken with parallel- ( p1 ) and cross- ( p2 ) polarizations at a minimum of three different points .", "raman data were analyzed using matlab software to calculate the depolarization ratios ( i/i ) , where i is the area under the raman band from 9251000  cm for cross - polarization ( p2 ) , and i is the similar area for parallel - polarization ( p1 ) .", "this wavenumber range was used since it centres at 959  cm ( the main peak due to phosphate groups such as those found in apatite ) ."], ["two - dimensional oct images are shown of sound enamel ( figure 3(b ) ) and a wsl ( figure 4(a ) ) . in the sound enamel image", ", there is intense light backscattering at the tooth - air interface , and no significant signal with depth into the enamel .", "in contrast , the oct image of a wsl shows significant light backscattering beneath the surface with a triangular shape characteristic of a subsurface lesion as observed on histological sections . the two - dimensional oct image displayed as figure 4(b ) shows a deposit of material on the tooth surface that is attributed to calculus .", "the oct image of hypocalcification in figure 4(c ) shows diffuse light back - scattering and a more irregular subsurface pattern of scattering across the entire region scanned compared to sound enamel .", "the light back - scattering of hypocalcified regions is also more irregular than wsls , which have a characteristic triangular shape .", "the oct image of stained enamel in figure 4(d ) demonstrates increased light backscattering when compared to sound enamel , but again there is no triangular - shaped subsurface light back - scattering as observed with wsls .  ", "average raman depolarization ratios (  ) from the various groups are depicted in a box - and - whisker plot ( figure 5 ) . in order to determine whether the mean depolarization ratio values of the various groups were statistically significant from one another ,", "a one - way analysis of variance ( anova ) followed by unequal n hsd post - hoc comparisons ( statistica ) was performed ( table 1 ) .", "it was determined that the mean values were statistically significant in all cases at p < .05 except for three cases .", "sound enamel was not statistically significant from hypocalcified enamel , carious enamel was not statistically significant from stained enamel , and lastly stained enamel was not statistically significant from hypocalcified enamel . in raman spectra acquired from areas of stain ( figure 6 ) , there is a large background sloping fluorescence that is not observed in spectra from areas of sound enamel or wsls without stain where these spectra have a flat background ."], ["the development of new technologies for wsl assessment requires that these devices undergo clinical validation prior to becoming an accepted clinical method .", "in addition to demonstrating the performance of these methods for providing high sensitivity for wsl detection , high specificity is also desirable . optical coherence tomography and polarized raman spectroscopy are methods that potentially can address the need for a technology with high sensitivity and high specificity . to date there are no studies outside our research group that investigate a combination of oct and prs to detect wsls , and in particular the effects of the calculus , stain , or hypocalcification on the utility of these two technologies .", "oct imaging allows differentiation of sound from demineralized enamel on the basis of the characteristic triangular shape of the back - scattered signal beneath the tooth surface in images of wsls .", "this subsurface scattering pattern is believed to be due to wsls having porous enamel matrices , allowing incident light to travel further into the enamel and causing more scattering to occur , which is detected by the oct system .  ", "when calculus is present , it appears clearly as a deposit in the two - dimensional oct image .", "similar to conventional caries assessment methods , scaling of calculus is recommended before an assessment is made using oct and prs .", "oct can possibly be used to alert the clinician when calculus is still present in the region of interest and that further scaling is necessary .", "oct images of hypocalcification can be differentiated from wsls , since the light back - scattering found with hypocalcification is more irregular than wsls and lacks the characteristic triangular shape . to some extent", ", hypocalcification can be differentiated from sound enamel with oct since hypocalcification has a more irregular pattern of scattering at the surface and subsurface compared to sound enamel .", "although areas of stain are not easily distinguishable from hypocalcified enamel or sound enamel with oct , they can be readily differentiated from wsls because areas of stain lack the subsurface triangular - shaped back - scattering pattern characteristic of wsls .", "further image analysis is required to nonsubjectively distinguish sound enamel from stained and hypocalcified enamel .  ", "statistical analysis revealed that mean raman depolarization ratios were statistically significant in all cases at p < .05 except for three cases : ( a ) sound enamel compared to hypocalcified enamel , ( b ) carious enamel compared to stained enamel , and ( c ) stained enamel from hypocalcified enamel . since hypocalcified enamel can be mistaken for wsls upon visual clinical examination , it is reassuring that the raman depolarization values from hypocalcified enamel are distinct from caries , thereby increasing the specificity of the method .", "based on the analysis focusing on the depolarization ratio of the 959  cm peak , hypocalcified enamel could not be distinguished from sound enamel .", "this observation indicates that , fundamentally , hypocalcified enamel is like healthy sound enamel and not in need of treatment . in order to make this separation ,", "further studies are needed which include examining the peak positions and peak width of the phosphate hydroxyapatite peak , which have been shown to be affected by the mineral crystallinity .", "furthermore , other peaks could be surveyed to look for peaks characteristic of specialized forms of hypocalcification such as fluorosis .  ", "the analyses indicate that stain complicates the use of prs for discriminating stained sound enamel from carious enamel . in reviewing the raman spectra acquired from areas of stain", ", it is observed that stained sound enamel spectra show a large background fluorescence that is not found in spectra from areas of sound enamel or wsls without stain . in these preliminary analyses ,", "a straight - line background in the region of the peak was simply subtracted for calculating the areas under the peak . clearly , this initial approach is not enough as the curved background confounds this calculation .", "further studies are consequently required using various algorithms to robustly fit the fluorescence background for elimination .", "in addition , there are various instrument - based methods proposed to suppress the background fluorescence in raman spectra . with fluorescence - based devices ( diagnodent , qlf ) , stain chromophores from any source", "with prs , the fundamental basis of the method is the phosphate moieties specific to the dominant hydroxyapatite component from the mineral matrix .", "this peak itself is not due to staining and provides information on mineralization states as required for a method to detect demineralization in caries development .", "therefore , with improved methods for fluorescence background subtraction / suppression , it is anticipated that staining will no longer confound prs analyses .", "the statistical analyses also indicated that , based on the raman depolarization ratio , stained sound enamel can not be distinguished from hypocalcified enamel .", "this is also not surprising since both groups are overall noncarious intact enamel with one group containing extrinsic staining .", "the underlying biochemistry of the enamel matrix is largely similar in both cases as revealed by the raman spectra .", "subsequent analyses such as those described above for examining spectra of hypocalcified enamel could provide insights for discriminating these two groups .", "this result suggests that , like regions of demineralization , the apatite in areas of calculus has a disordered crystal structure and orientation as shown by higher depolarization ratio values .", "calculus is easily observed on the oct image , and therefore oct will be the first technology used with the fibre optic probe to screen for wsls and to determine whether prs analysis of the lesion is necessary .", "if calculus is detected on the oct image , the area will be scaled before the prs method is applied to determine the raman depolarization ratio . by combining oct and prs technologies ,", "it is possible to rule out false - positive readings that might occur from using raman depolarization ratios alone .", "furthermore , according to the box- and whisker- plot ( figure 4 ) , setting a depolarization ratio threshold of ~0.18 can help discriminate carious enamel from enamel with a calculus deposit .", "it is important to note that this study was limited to extracted human teeth . in the oral environment , there will be a combination of many possible confounding factors present , and an additive effect may result in the oral cavity that was not observed when testing the possible confounding factors separately .", "these issues will be addressed in our subsequent studies as we transition to using fibre - optic - based devices for in vivo measurements with patient volunteers .   in conclusion , calculus and hypocalcification", "stain does not influence wsl detection with oct . with improved analysis methods , the current limitations with prs analysis in the presence of stain", "the combination of oct and prs technologies can decrease the risk of false - positive reading and increase the potential for the detection of wsls with high sensitivity and specificity .", "this initial study has pointed out limitations that should be taken into consideration when using these methods and highlighted further analyses that need to be undertaken to better understand the effects of calculus , hypocalcification , and stain on oct and prs technologies ."]]}
{"article_id": "PMC3830987", "article_text": ["hematoxylin and eosin - stained slides and formalin fixed paraffin embedded blocks of eight cases of hca were retrieved from the archives of the department of pathology , korea university medical center and seoul national university hospital .", "the hematoxylin and eosin slides were reviewed , and immunohistochemical stains were performed on the formalin fixed paraffin embedded blocks as followed .", "hematoxylin and eosin - stained slides of each case were reviewed independently by two authors ( h.k .", "the histological characteristics of the tumors were evaluated with emphasis on steatosis , sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar growth pattern , telangiectasia , and peliosis .", "histological parameters were recorded as negative if less than 10% of the tumor showed the corresponding morphology .", "immunohistochemical stainings was performed using the following antibodies : l - fabp ( 1:50 , rabbit polyclonal ) from abcam ( cambridge , ma , usa ) ; -catenin ( 1:50 , mouse monoclonal ) , saa ( 1:50 , mouse monoclonal ) , and glutamine synthetase ( gs ; 1:200 , mouse monoclonal ) from bd biosciences ( san diego , ca , usa ) ; cd3 ( 1:100 , mouse monoclonal ) , cd20 ( 1:100 , mouse monoclonal ) , p53 ( 1:50 , mouse monoclonal ) and ki-67 ( 1:50 , mouse monoclonal ) from dako ( carpentaria , ca , usa ) ; and glypican3 ( 1:50 , mouse monoclonal ) from cell marque ( rocklin , ca , usa ) .", "cytoplasmic staining of normal hepatic parenchyma served as a positive control for l - fabp immunostaining .", "focal cytoplasmic staining in the perivenular area of normal hepatic parenchyma served as a positive control for gs immunostaining .", "diffuse cytoplasmic granular staining with or without membranous staining was recorded as a positive saa immunostaining result .", "the recent hca classification system proposed by the bordeaux group was used.8 the histologic and immunohistochemical characteristics for each subtype are described below .", "each case was categorized based on combined clinical , histologic , and immunohistochemical parameters . the histologic characteristics of h - hca are marked steatosis and an absence of cytological abnormalities and inflammatory infiltrate.6,7,13 this subtype shows negative staining for l - fabp because hnf1 positively regulates fabp1 in normal liver tissue .", "diffuse or focal cytoplasmic staining was interpreted as a positive result for l - fabp immunostaining .", "the presence of steatosis and negative l - fabp immunostaining were indicators of h - hca .", "the histological characteristics of -hcas are nuclear atypia , an acinar ( pseudoglandular ) growth pattern , and lack of steatosis.6 -catenin and gs immunostaining was used to detect -hcas.12,13 diffuse or focal cytoplasmic gs staining was interpreted as a positive result .", "diffuse or focal nuclear -catenin immunostaining was interpreted as a positive result , whereas cytoplasmic -catenin immunostaining was recorded as aberrant expression .", "characteristic -hca histologic characteristics and positive gs and/or -catenin immunostaining results were indicators of -hca .", "the histologic characteristics of i - hca are presence of inflammatory infiltrate , marked sinusoidal dilatation , and thick - walled arteries.8 steatosis may be present in patients with i - hca but is not as extensive as that in patients with h - hca.8 diffuse or focal cytoplasmic immunostaining for saa was interpreted as a positive result .", "hcas without genetic changes in hnf1 or -catenin and without protein expression of the previously mentioned markers are categorized into this group.8 hcas with complete or near - complete necrosis can also be classified in this group.13", "hematoxylin and eosin - stained slides of each case were reviewed independently by two authors ( h.k .", "the histological characteristics of the tumors were evaluated with emphasis on steatosis , sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar growth pattern , telangiectasia , and peliosis . the percentage of steatotic area was recorded .", "histological parameters were recorded as negative if less than 10% of the tumor showed the corresponding morphology .", "immunohistochemical stainings was performed using the following antibodies : l - fabp ( 1:50 , rabbit polyclonal ) from abcam ( cambridge , ma , usa ) ; -catenin ( 1:50 , mouse monoclonal ) , saa ( 1:50 , mouse monoclonal ) , and glutamine synthetase ( gs ; 1:200 , mouse monoclonal ) from bd biosciences ( san diego , ca , usa ) ; cd3 ( 1:100 , mouse monoclonal ) , cd20 ( 1:100 , mouse monoclonal ) , p53 ( 1:50 , mouse monoclonal ) and ki-67 ( 1:50 , mouse monoclonal ) from dako ( carpentaria , ca , usa ) ; and glypican3 ( 1:50 , mouse monoclonal ) from cell marque ( rocklin , ca , usa ) .", "cytoplasmic staining of normal hepatic parenchyma served as a positive control for l - fabp immunostaining .", "focal cytoplasmic staining in the perivenular area of normal hepatic parenchyma served as a positive control for gs immunostaining .", "diffuse cytoplasmic granular staining with or without membranous staining was recorded as a positive saa immunostaining result .", "the recent hca classification system proposed by the bordeaux group was used.8 the histologic and immunohistochemical characteristics for each subtype are described below .", "the histologic characteristics of h - hca are marked steatosis and an absence of cytological abnormalities and inflammatory infiltrate.6,7,13 this subtype shows negative staining for l - fabp because hnf1 positively regulates fabp1 in normal liver tissue .", "diffuse or focal cytoplasmic staining was interpreted as a positive result for l - fabp immunostaining .", "the presence of steatosis and negative l - fabp immunostaining were indicators of h - hca .", "the histological characteristics of -hcas are nuclear atypia , an acinar ( pseudoglandular ) growth pattern , and lack of steatosis.6 -catenin and gs immunostaining was used to detect -hcas.12,13 diffuse or focal cytoplasmic gs staining was interpreted as a positive result .", "diffuse or focal nuclear -catenin immunostaining was interpreted as a positive result , whereas cytoplasmic -catenin immunostaining was recorded as aberrant expression .", "characteristic -hca histologic characteristics and positive gs and/or -catenin immunostaining results were indicators of -hca .", "the histologic characteristics of i - hca are presence of inflammatory infiltrate , marked sinusoidal dilatation , and thick - walled arteries.8 steatosis may be present in patients with i - hca but is not as extensive as that in patients with h - hca.8 diffuse or focal cytoplasmic immunostaining for saa was interpreted as a positive result .", "hcas without genetic changes in hnf1 or -catenin and without protein expression of the previously mentioned markers are categorized into this group.8 hcas with complete or near - complete necrosis can also be classified in this group.13", "the histologic characteristics of h - hca are marked steatosis and an absence of cytological abnormalities and inflammatory infiltrate.6,7,13 this subtype shows negative staining for l - fabp because hnf1 positively regulates fabp1 in normal liver tissue .", "diffuse or focal cytoplasmic staining was interpreted as a positive result for l - fabp immunostaining .", "the presence of steatosis and negative l - fabp immunostaining were indicators of h - hca .", "the histological characteristics of -hcas are nuclear atypia , an acinar ( pseudoglandular ) growth pattern , and lack of steatosis.6 -catenin and gs immunostaining was used to detect -hcas.12,13 diffuse or focal cytoplasmic gs staining was interpreted as a positive result . diffuse or focal nuclear -catenin immunostaining was interpreted as a positive result , whereas cytoplasmic -catenin immunostaining was recorded as aberrant expression .", "characteristic -hca histologic characteristics and positive gs and/or -catenin immunostaining results were indicators of -hca .", "the histologic characteristics of i - hca are presence of inflammatory infiltrate , marked sinusoidal dilatation , and thick - walled arteries.8 steatosis may be present in patients with i - hca but is not as extensive as that in patients with h - hca.8 diffuse or focal cytoplasmic immunostaining for saa was interpreted as a positive result .", "hcas without genetic changes in hnf1 or -catenin and without protein expression of the previously mentioned markers are categorized into this group.8 hcas with complete or near - complete necrosis can also be classified in this group.13", "there were three cases of h - hca , four cases of i - hca , and one cases of -hca .", "morphologic findings ( table 2 ) and results of immunohistochemical staining ( table 3 ) were analyzed for each case .", "this was confirmed by negativity for glypican 3 and p53 immunostaining and a ki-67 index < 1% in all eight cases . three cases ( a , b , and c ) were classified as h - hca .", "morphologically , cases a and b revealed marked steatosis of 30 - 40% and 20% , respectively , and did not show most of the characteristic histologic features of the other subtypes , such as sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar pattern , thick - walled arteries , or peliosis ( table 2 ) .", "case c did not show steatosis but presented with thick - walled arteries and cytological atypia .", ". there was faint l - fabp staining in the tumoral area , but it was interpreted as negative because the staining intensity was very weak and identical to the non - specific staining of blood vessels .", "cases a ( fig . 1 ) and b were in accordance with the known characters of h - hca , but case c was not fully concordant and shared some characteristics with i - hca . four cases ( d , e , f , and g ) were categorized as i - hca . three were male and one was female .", "although these three cases had a negative history of alcohol consumption , cases d and g had high bmis ( 27.8 kg / m and 25.9 kg / m , respectively ) .", "all three cases had morphological characteristics typical of i - hca , including marked sinusoidal dilation , presence of inflammatory infiltrates , and thick - walled arteries ( table 2 ) .", "in all four cases l - fabp immunostaining was negative and saa immunostaining was positive ( table 3 ) .", "glutamine synthetase immunostaining , should be negative in i - hca , was positive in cases e and f. cases d ( fig .", "morphologic analysis showed lack of steatosis and inflammatory infiltrate , but the presence of an acinar pattern with cytological atypia ( table 2 , fig .", "satining the tumor cells were positive for l - fabp and glutamine synthetase ( fig .", "however , -catenin immunostaining showed an aberrant pattern of diffuse cytoplasmic staining and was interpreted as negative because there was no nuclear staining ( fig .", "this case was classified as -hca because all morphologic and immunohistochemical findings were in agreement with known characteristics of -hca except for the -catenin immunostaining result .", "all eight cases were determined to belong to the above subtypes therefore none were categorized as an unclassified hca .", "three cases ( a , b , and c ) were classified as h - hca .", "morphologically , cases a and b revealed marked steatosis of 30 - 40% and 20% , respectively , and did not show most of the characteristic histologic features of the other subtypes , such as sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar pattern , thick - walled arteries , or peliosis ( table 2 ) .", "case c did not show steatosis but presented with thick - walled arteries and cytological atypia .", ". there was faint l - fabp staining in the tumoral area , but it was interpreted as negative because the staining intensity was very weak and identical to the non - specific staining of blood vessels . based on these findings , these three cases were classified as h - hca .", "cases a ( fig . 1 ) and b were in accordance with the known characters of h - hca , but case c was not fully concordant and shared some characteristics with i - hca .", "four cases ( d , e , f , and g ) were categorized as i - hca . three were male and one was female .", "although these three cases had a negative history of alcohol consumption , cases d and g had high bmis ( 27.8 kg / m and 25.9 kg / m , respectively ) .", "all three cases had morphological characteristics typical of i - hca , including marked sinusoidal dilation , presence of inflammatory infiltrates , and thick - walled arteries ( table 2 ) .", "in all four cases l - fabp immunostaining was negative and saa immunostaining was positive ( table 3 ) .", "glutamine synthetase immunostaining , should be negative in i - hca , was positive in cases e and f. cases d ( fig .", "morphologic analysis showed lack of steatosis and inflammatory infiltrate , but the presence of an acinar pattern with cytological atypia ( table 2 , fig .", "satining the tumor cells were positive for l - fabp and glutamine synthetase ( fig .", "however , -catenin immunostaining showed an aberrant pattern of diffuse cytoplasmic staining and was interpreted as negative because there was no nuclear staining ( fig .", "this case was classified as -hca because all morphologic and immunohistochemical findings were in agreement with known characteristics of -hca except for the -catenin immunostaining result .", "all eight cases were determined to belong to the above subtypes therefore none were categorized as an unclassified hca .", "this is the largest korean hca series review of the past several decades and the first to apply the new subtype classification .", "the incidence of hca in korea is very low and only a few cases of hca have been reported in korea during the past 25 years.19 the recent hca classification was established based on previous studies of genetic and phenotypic characteristics of hca and hepatic nodular hyperplasia.9,10,15,17,18,20 pathologically , the subtype classification enables an accurate diagnosis of this rare entity even with a small biopsy specimen.8,21 l - fabp immunohistochemistry is especially helpful in the identification of h - hca because hnf1 positively regulates fabp1 in normal liver tissue .", "mutation in the hnf1 gene causes steatosis,22 therefore most cases of h - hca also show marked steatosis.7,11 a correlation study between genotype and phenotype of each subtype revealed a strong association between h - hca and the presence of steatosis , the absence of cytological atypia , and absence of inflammatory infiltrate.6 however , one of our l - fabp - negative cases was not consistent with the description in the new classificat ion system because it did not have steatosis but did have cytological atypia .", "although a lack of steatosis in an l - fabp - negative case is unusual , similar l - fabp - negative cases without steatosis can be found in other reports.6,23 according to zucman - rossi et al.,6 fewer than 7% of hnf1-mutated adenomas show no or < 10% steatosis and fewer than 2% present with cytological atypia .", "four cases were categorized as i - hca , and cases showed consistent i - hca findings including positive saa and l - fabp immunostaining in addition to an inflammatory infiltrate .", "atypia in i - hca does not seem to be uncommon , and occurred at a rate of approximately 50% in one previous report.6 the bordeaux group pointed out the presence of cases with overlapping features of i - hca and -hca.13 i - hcas are have been reported to present with a lower than moderate level of steatosis in 94% of cases.6 in our study , two of the four i - hcas presented with 5% steatosis while the remaining three showed none .", "case h was categorized as -hca because of cytological atypia , an acinar growth pattern , and positive l - fabp staininng .", "interpretation of -catenin immunostaining in hca seems to be a common problem ; thus , the bordeaux group reported that they rely more on glutamine synthetase immunostaining as a surrogate marker for detecting -hca than on -catenin immunostaining because of the focal distribution or absence of -catenin positive cells.11 in one japanese report , the authors also recognized a certain case as -hca even if the tumors expressed only gs.24 the discordance between -catenin mutation status and protein expression in hcas is not as striking as that reported in studies of other tumors.25,26 glutamine synthetase , a surrogate marker for -catenin , showed inconsistent results in our series .", "although positive glutamine synthetase staining is supposed to be characteristics of -hcas , four of our seven cases with non--hca subtypes showed a positive result .", "similar findings of inconsistent gs staining results are discussed in a review of hca cases from scotland.27 in addition to these difficulties with immunohistochemistry , -hca can be difficult or impossible to be distinguished from well differentiated hcc because of the cytological atypia and acinar growth pattern.8 if used alone , none of the histological or immunohistochemical parameters included in the subtype classification seem to be able to properly categorize a particular tumor subtype . as summarized in a table created by the bordeaux group , a small number of cases from each subtype share histological features with other subtypes.6 furthermore , immunohistochemistry can give inconsistent results in certain cases .", "however , l - fabp or saa staining alone seemed to be sufficient for discriminating between h - hca and inflammatory hca , respectively .", "the opinion that there is no single histologic or immunohistochemical parameter sufficient for classification of any subtype has been expressed in a previous report.27 in our cases presenting with hnf1-mutated and inflammatory hca , most , but not all , had intratumoral hemorrhage .", "moreover , female predominance is typically observed in hca cases , but 50% of the patients in our series , were male .", "a similar relatively high proportion of males was observed in a japanese study of hca.24 the authors assumed that this was a characteristic of japanese hca cases . when combined with our result", ", it seems likely that the relatively high proportion of males might be the result of genetic and environmental actors associated with an asian population .", "overall , our results both support and contradict other reports on hca . in conclusion ,", "the hca subtype classification based on genetic and phenotypic characteristics provides new insight into the pathological and radiological diagnosis and clinical management of these tumors .", "the diagnostic and clinical implications of the new classification scheme may need to be validated by other studies ."], "abstract_text": ["<S> backgroundhepatocellular adenoma ( hca ) is a rare benign tumor of the liver . </S>", "<S> a subtype classification of hca ( hepatocyte nuclear factor 1 [ hnf1]-mutated , -catenin - mutated hca , inflammatory hca , and unclassified hca ) has recently been established based on a single institutional review of a hca series by the bordeaux group.methodswe used histologic and immunohistochemical parameters to classify and evaluate eight cases from our institution . </S>", "<S> we evaluated the new classification method and analyzed correlations between our results and those of other reports.resultsseven of our eight cases showed histologic and immunohistochemical results consistent with previous reports . </S>", "<S> however , one case showed overlapping histologic features , as previously described by the bordeaux group . </S>", "<S> four cases showed glutamine synthetase immunohistochemical staining inconsistent with their classification , indicating that glutamine synthetase staining may not be diagnostic for -catenin - mutated hca . </S>", "<S> hnf1-mutated hca may be indicated by the absence of liver fatty acid binding protein expression . </S>", "<S> detection of amyloid a may indicate inflammatory hca . </S>", "<S> hca with no mutation in the hnf1 or -catenin genes and no inflammatory protein expression is categorized as unclassified hca.conclusionsalthough the new classification is now generally accepted , validation through follow - up studies is necessary . </S>"], "labels": null, "section_names": ["MATERIALS AND METHODS", "Interpretation of histologic findings", "Immunohistochemistry", "Hepatocellular adenoma subtype classification", "HNF1-mutated hepatocellular adenoma", "-Catenin mutated hepatocellular adenoma", "Inflammatory hepatocellular adenoma", "Unclassified hepatocellular adenoma", "RESULTS", "HNF1-mutated hepatocellular adenoma", "Inflammatory hepatocellular adenoma", "-Catenin-mutated hepatocellular adenoma", "Unclassified HCA", "DISCUSSION"], "sections": [["hematoxylin and eosin - stained slides and formalin fixed paraffin embedded blocks of eight cases of hca were retrieved from the archives of the department of pathology , korea university medical center and seoul national university hospital .", "the hematoxylin and eosin slides were reviewed , and immunohistochemical stains were performed on the formalin fixed paraffin embedded blocks as followed .", "hematoxylin and eosin - stained slides of each case were reviewed independently by two authors ( h.k .", "the histological characteristics of the tumors were evaluated with emphasis on steatosis , sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar growth pattern , telangiectasia , and peliosis .", "histological parameters were recorded as negative if less than 10% of the tumor showed the corresponding morphology .", "immunohistochemical stainings was performed using the following antibodies : l - fabp ( 1:50 , rabbit polyclonal ) from abcam ( cambridge , ma , usa ) ; -catenin ( 1:50 , mouse monoclonal ) , saa ( 1:50 , mouse monoclonal ) , and glutamine synthetase ( gs ; 1:200 , mouse monoclonal ) from bd biosciences ( san diego , ca , usa ) ; cd3 ( 1:100 , mouse monoclonal ) , cd20 ( 1:100 , mouse monoclonal ) , p53 ( 1:50 , mouse monoclonal ) and ki-67 ( 1:50 , mouse monoclonal ) from dako ( carpentaria , ca , usa ) ; and glypican3 ( 1:50 , mouse monoclonal ) from cell marque ( rocklin , ca , usa ) .", "cytoplasmic staining of normal hepatic parenchyma served as a positive control for l - fabp immunostaining .", "focal cytoplasmic staining in the perivenular area of normal hepatic parenchyma served as a positive control for gs immunostaining .", "diffuse cytoplasmic granular staining with or without membranous staining was recorded as a positive saa immunostaining result .", "the recent hca classification system proposed by the bordeaux group was used.8 the histologic and immunohistochemical characteristics for each subtype are described below .", "each case was categorized based on combined clinical , histologic , and immunohistochemical parameters . the histologic characteristics of h - hca are marked steatosis and an absence of cytological abnormalities and inflammatory infiltrate.6,7,13 this subtype shows negative staining for l - fabp because hnf1 positively regulates fabp1 in normal liver tissue .", "diffuse or focal cytoplasmic staining was interpreted as a positive result for l - fabp immunostaining .", "the presence of steatosis and negative l - fabp immunostaining were indicators of h - hca .", "the histological characteristics of -hcas are nuclear atypia , an acinar ( pseudoglandular ) growth pattern , and lack of steatosis.6 -catenin and gs immunostaining was used to detect -hcas.12,13 diffuse or focal cytoplasmic gs staining was interpreted as a positive result .", "diffuse or focal nuclear -catenin immunostaining was interpreted as a positive result , whereas cytoplasmic -catenin immunostaining was recorded as aberrant expression .", "characteristic -hca histologic characteristics and positive gs and/or -catenin immunostaining results were indicators of -hca .", "the histologic characteristics of i - hca are presence of inflammatory infiltrate , marked sinusoidal dilatation , and thick - walled arteries.8 steatosis may be present in patients with i - hca but is not as extensive as that in patients with h - hca.8 diffuse or focal cytoplasmic immunostaining for saa was interpreted as a positive result .", "hcas without genetic changes in hnf1 or -catenin and without protein expression of the previously mentioned markers are categorized into this group.8 hcas with complete or near - complete necrosis can also be classified in this group.13"], ["hematoxylin and eosin - stained slides of each case were reviewed independently by two authors ( h.k .", "the histological characteristics of the tumors were evaluated with emphasis on steatosis , sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar growth pattern , telangiectasia , and peliosis . the percentage of steatotic area was recorded .", "histological parameters were recorded as negative if less than 10% of the tumor showed the corresponding morphology ."], ["immunohistochemical stainings was performed using the following antibodies : l - fabp ( 1:50 , rabbit polyclonal ) from abcam ( cambridge , ma , usa ) ; -catenin ( 1:50 , mouse monoclonal ) , saa ( 1:50 , mouse monoclonal ) , and glutamine synthetase ( gs ; 1:200 , mouse monoclonal ) from bd biosciences ( san diego , ca , usa ) ; cd3 ( 1:100 , mouse monoclonal ) , cd20 ( 1:100 , mouse monoclonal ) , p53 ( 1:50 , mouse monoclonal ) and ki-67 ( 1:50 , mouse monoclonal ) from dako ( carpentaria , ca , usa ) ; and glypican3 ( 1:50 , mouse monoclonal ) from cell marque ( rocklin , ca , usa ) .", "cytoplasmic staining of normal hepatic parenchyma served as a positive control for l - fabp immunostaining .", "focal cytoplasmic staining in the perivenular area of normal hepatic parenchyma served as a positive control for gs immunostaining .", "diffuse cytoplasmic granular staining with or without membranous staining was recorded as a positive saa immunostaining result ."], ["the recent hca classification system proposed by the bordeaux group was used.8 the histologic and immunohistochemical characteristics for each subtype are described below .", "the histologic characteristics of h - hca are marked steatosis and an absence of cytological abnormalities and inflammatory infiltrate.6,7,13 this subtype shows negative staining for l - fabp because hnf1 positively regulates fabp1 in normal liver tissue .", "diffuse or focal cytoplasmic staining was interpreted as a positive result for l - fabp immunostaining .", "the presence of steatosis and negative l - fabp immunostaining were indicators of h - hca .", "the histological characteristics of -hcas are nuclear atypia , an acinar ( pseudoglandular ) growth pattern , and lack of steatosis.6 -catenin and gs immunostaining was used to detect -hcas.12,13 diffuse or focal cytoplasmic gs staining was interpreted as a positive result .", "diffuse or focal nuclear -catenin immunostaining was interpreted as a positive result , whereas cytoplasmic -catenin immunostaining was recorded as aberrant expression .", "characteristic -hca histologic characteristics and positive gs and/or -catenin immunostaining results were indicators of -hca .", "the histologic characteristics of i - hca are presence of inflammatory infiltrate , marked sinusoidal dilatation , and thick - walled arteries.8 steatosis may be present in patients with i - hca but is not as extensive as that in patients with h - hca.8 diffuse or focal cytoplasmic immunostaining for saa was interpreted as a positive result .", "hcas without genetic changes in hnf1 or -catenin and without protein expression of the previously mentioned markers are categorized into this group.8 hcas with complete or near - complete necrosis can also be classified in this group.13"], ["the histologic characteristics of h - hca are marked steatosis and an absence of cytological abnormalities and inflammatory infiltrate.6,7,13 this subtype shows negative staining for l - fabp because hnf1 positively regulates fabp1 in normal liver tissue .", "diffuse or focal cytoplasmic staining was interpreted as a positive result for l - fabp immunostaining .", "the presence of steatosis and negative l - fabp immunostaining were indicators of h - hca ."], ["the histological characteristics of -hcas are nuclear atypia , an acinar ( pseudoglandular ) growth pattern , and lack of steatosis.6 -catenin and gs immunostaining was used to detect -hcas.12,13 diffuse or focal cytoplasmic gs staining was interpreted as a positive result . diffuse or focal nuclear -catenin immunostaining was interpreted as a positive result , whereas cytoplasmic -catenin immunostaining was recorded as aberrant expression .", "characteristic -hca histologic characteristics and positive gs and/or -catenin immunostaining results were indicators of -hca ."], ["the histologic characteristics of i - hca are presence of inflammatory infiltrate , marked sinusoidal dilatation , and thick - walled arteries.8 steatosis may be present in patients with i - hca but is not as extensive as that in patients with h - hca.8 diffuse or focal cytoplasmic immunostaining for saa was interpreted as a positive result ."], ["hcas without genetic changes in hnf1 or -catenin and without protein expression of the previously mentioned markers are categorized into this group.8 hcas with complete or near - complete necrosis can also be classified in this group.13"], ["there were three cases of h - hca , four cases of i - hca , and one cases of -hca .", "morphologic findings ( table 2 ) and results of immunohistochemical staining ( table 3 ) were analyzed for each case .", "this was confirmed by negativity for glypican 3 and p53 immunostaining and a ki-67 index < 1% in all eight cases . three cases ( a , b , and c ) were classified as h - hca .", "morphologically , cases a and b revealed marked steatosis of 30 - 40% and 20% , respectively , and did not show most of the characteristic histologic features of the other subtypes , such as sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar pattern , thick - walled arteries , or peliosis ( table 2 ) .", "case c did not show steatosis but presented with thick - walled arteries and cytological atypia .", ". there was faint l - fabp staining in the tumoral area , but it was interpreted as negative because the staining intensity was very weak and identical to the non - specific staining of blood vessels .", "cases a ( fig . 1 ) and b were in accordance with the known characters of h - hca , but case c was not fully concordant and shared some characteristics with i - hca . four cases ( d , e , f , and g ) were categorized as i - hca . three were male and one was female .", "although these three cases had a negative history of alcohol consumption , cases d and g had high bmis ( 27.8 kg / m and 25.9 kg / m , respectively ) .", "all three cases had morphological characteristics typical of i - hca , including marked sinusoidal dilation , presence of inflammatory infiltrates , and thick - walled arteries ( table 2 ) .", "in all four cases l - fabp immunostaining was negative and saa immunostaining was positive ( table 3 ) .", "glutamine synthetase immunostaining , should be negative in i - hca , was positive in cases e and f. cases d ( fig .", "morphologic analysis showed lack of steatosis and inflammatory infiltrate , but the presence of an acinar pattern with cytological atypia ( table 2 , fig .", "satining the tumor cells were positive for l - fabp and glutamine synthetase ( fig .", "however , -catenin immunostaining showed an aberrant pattern of diffuse cytoplasmic staining and was interpreted as negative because there was no nuclear staining ( fig .", "this case was classified as -hca because all morphologic and immunohistochemical findings were in agreement with known characteristics of -hca except for the -catenin immunostaining result .", "all eight cases were determined to belong to the above subtypes therefore none were categorized as an unclassified hca ."], ["three cases ( a , b , and c ) were classified as h - hca .", "morphologically , cases a and b revealed marked steatosis of 30 - 40% and 20% , respectively , and did not show most of the characteristic histologic features of the other subtypes , such as sinusoidal dilatation , inflammatory infiltrate , cytological atypia , acinar pattern , thick - walled arteries , or peliosis ( table 2 ) .", "case c did not show steatosis but presented with thick - walled arteries and cytological atypia .", ". there was faint l - fabp staining in the tumoral area , but it was interpreted as negative because the staining intensity was very weak and identical to the non - specific staining of blood vessels . based on these findings , these three cases were classified as h - hca .", "cases a ( fig . 1 ) and b were in accordance with the known characters of h - hca , but case c was not fully concordant and shared some characteristics with i - hca ."], ["four cases ( d , e , f , and g ) were categorized as i - hca . three were male and one was female .", "although these three cases had a negative history of alcohol consumption , cases d and g had high bmis ( 27.8 kg / m and 25.9 kg / m , respectively ) .", "all three cases had morphological characteristics typical of i - hca , including marked sinusoidal dilation , presence of inflammatory infiltrates , and thick - walled arteries ( table 2 ) .", "in all four cases l - fabp immunostaining was negative and saa immunostaining was positive ( table 3 ) .", "glutamine synthetase immunostaining , should be negative in i - hca , was positive in cases e and f. cases d ( fig ."], ["morphologic analysis showed lack of steatosis and inflammatory infiltrate , but the presence of an acinar pattern with cytological atypia ( table 2 , fig .", "satining the tumor cells were positive for l - fabp and glutamine synthetase ( fig .", "however , -catenin immunostaining showed an aberrant pattern of diffuse cytoplasmic staining and was interpreted as negative because there was no nuclear staining ( fig .", "this case was classified as -hca because all morphologic and immunohistochemical findings were in agreement with known characteristics of -hca except for the -catenin immunostaining result ."], ["all eight cases were determined to belong to the above subtypes therefore none were categorized as an unclassified hca ."], ["this is the largest korean hca series review of the past several decades and the first to apply the new subtype classification .", "the incidence of hca in korea is very low and only a few cases of hca have been reported in korea during the past 25 years.19 the recent hca classification was established based on previous studies of genetic and phenotypic characteristics of hca and hepatic nodular hyperplasia.9,10,15,17,18,20 pathologically , the subtype classification enables an accurate diagnosis of this rare entity even with a small biopsy specimen.8,21 l - fabp immunohistochemistry is especially helpful in the identification of h - hca because hnf1 positively regulates fabp1 in normal liver tissue .", "mutation in the hnf1 gene causes steatosis,22 therefore most cases of h - hca also show marked steatosis.7,11 a correlation study between genotype and phenotype of each subtype revealed a strong association between h - hca and the presence of steatosis , the absence of cytological atypia , and absence of inflammatory infiltrate.6 however , one of our l - fabp - negative cases was not consistent with the description in the new classificat ion system because it did not have steatosis but did have cytological atypia .", "although a lack of steatosis in an l - fabp - negative case is unusual , similar l - fabp - negative cases without steatosis can be found in other reports.6,23 according to zucman - rossi et al.,6 fewer than 7% of hnf1-mutated adenomas show no or < 10% steatosis and fewer than 2% present with cytological atypia .", "four cases were categorized as i - hca , and cases showed consistent i - hca findings including positive saa and l - fabp immunostaining in addition to an inflammatory infiltrate .", "atypia in i - hca does not seem to be uncommon , and occurred at a rate of approximately 50% in one previous report.6 the bordeaux group pointed out the presence of cases with overlapping features of i - hca and -hca.13 i - hcas are have been reported to present with a lower than moderate level of steatosis in 94% of cases.6 in our study , two of the four i - hcas presented with 5% steatosis while the remaining three showed none .", "case h was categorized as -hca because of cytological atypia , an acinar growth pattern , and positive l - fabp staininng .", "interpretation of -catenin immunostaining in hca seems to be a common problem ; thus , the bordeaux group reported that they rely more on glutamine synthetase immunostaining as a surrogate marker for detecting -hca than on -catenin immunostaining because of the focal distribution or absence of -catenin positive cells.11 in one japanese report , the authors also recognized a certain case as -hca even if the tumors expressed only gs.24 the discordance between -catenin mutation status and protein expression in hcas is not as striking as that reported in studies of other tumors.25,26 glutamine synthetase , a surrogate marker for -catenin , showed inconsistent results in our series .", "although positive glutamine synthetase staining is supposed to be characteristics of -hcas , four of our seven cases with non--hca subtypes showed a positive result .", "similar findings of inconsistent gs staining results are discussed in a review of hca cases from scotland.27 in addition to these difficulties with immunohistochemistry , -hca can be difficult or impossible to be distinguished from well differentiated hcc because of the cytological atypia and acinar growth pattern.8 if used alone , none of the histological or immunohistochemical parameters included in the subtype classification seem to be able to properly categorize a particular tumor subtype . as summarized in a table created by the bordeaux group , a small number of cases from each subtype share histological features with other subtypes.6 furthermore , immunohistochemistry can give inconsistent results in certain cases .", "however , l - fabp or saa staining alone seemed to be sufficient for discriminating between h - hca and inflammatory hca , respectively .", "the opinion that there is no single histologic or immunohistochemical parameter sufficient for classification of any subtype has been expressed in a previous report.27 in our cases presenting with hnf1-mutated and inflammatory hca , most , but not all , had intratumoral hemorrhage .", "moreover , female predominance is typically observed in hca cases , but 50% of the patients in our series , were male .", "a similar relatively high proportion of males was observed in a japanese study of hca.24 the authors assumed that this was a characteristic of japanese hca cases . when combined with our result", ", it seems likely that the relatively high proportion of males might be the result of genetic and environmental actors associated with an asian population .", "overall , our results both support and contradict other reports on hca . in conclusion ,", "the hca subtype classification based on genetic and phenotypic characteristics provides new insight into the pathological and radiological diagnosis and clinical management of these tumors .", "the diagnostic and clinical implications of the new classification scheme may need to be validated by other studies ."]]}
{"article_id": "PMC3361961", "article_text": ["microglia account for approximately 10% of the adult brain cell population and represent the first and main form of immune defense in the central nervous system ( cns ; lawson et al .", "microglia become activated and they can be identified and distinguished from their resting phenotype based on a combination of morphological and immunophenotypic changes ( dheen et al . , 2007 ; ransohoff and perry , 2009 ) .", "microglia initiate immune responses by enhancing the expression of toll - like receptors ( tlr ) and a wide range of pro - inflammatory mediators such as tumor necrosis factor - alpha ( tnf ) , interleukin ( il)-1 and il-6 for the removal of the cns threat ( suzumura et al . , 1996 ;", "microglia may also fulfill a neuroprotective role via the release of neurotrophic factors and promotion of neurogenesis for the restoration of normal physiology ( stadelmann et al . , 2002 ) .", "hence , the acute inflammatory response is generally beneficial , as it tends to minimize injury and promotes tissue repair .", "however , chronic neuroinflammation is closely related to various neurodegenerative disorders such as parkinson s disease ( pd ) , huntington s disease ( hd ) , dementias , and multiple sclerosis ( ms ) , although the consequences of sustained microglial activation in these diseases is unclear .", "activated microglia upregulate expression of the 18-kda translocator protein ( tspo ; chen and guilarte , 2008 ; cosenza - nashat et al .", "tspo are found in abundance throughout the body in peripheral organs ( i.e. , liver and adrenals ) , and hematogenous cells , but are present at very low levels in the normal healthy cns ( banati , 2002 ) .", "functionally , tspo has several biological functions including the control of cholesterol transport and neurosteroid synthesis ( papadopoulos et al . , 2006 ) , and", "may also be involved in the release of pro - inflammatory cytokines during inflammation ( choi et al . , 2002 ; wilms et al . ,", "enhanced tspo expression can be detected in vivo by using positron emission tomography ( pet ) imaging with the selective tspo radioligand c - pk11195 ( benavides et al .", ", 1993 ; banati et al . , 1999 ) , with evidence that increases in c - pk11195 binding potential ( bpnd ) correspond to activation of microglia ( stephenson et al . , 1995 ; conway et al .", "although tspo is also expressed by reactive astrocytes , a c - pk11195 pet study of patients with hippocampal sclerosis , a condition histopathologically characterized by marked astrogliosis , did not yield results that were significantly different to healthy normal controls ( banati et al .", "this is consistent with the view that reactive astrocytes , in vivo , do not significantly contribute to the c - pk11195 signal .", "therefore , in the absence of invading blood borne cells or severe focal leakage of blood - brain barrier , the increased pk11195 binding is likely to indicate the transition of microglia from a resting to an activated state , and is due to an increase in the number , rather than the affinity , of tspo ( banati et al . , 2000 ) .", "hence , the measurement of tspo uptake using pet provides an in vivo tool to monitor progression and severity of neuroinflammation and is a useful indicator of active cns disease .", "this article aims to review the use of pet imaging to promote the understanding of activated microglia in neurodegenerative disease .", "parkinson s disease is the second most common neurodegenerative disorder of the elderly and is associated with the motor symptoms of tremor , bradykinesia , and rigidity .", "it is characterized by the extended loss of dopaminergic neurons in the substantia nigra pars compacta , resulting in a deficiency of dopamine in the striatum ( braak et al . , 2006 ) , and the presence of alpha - synuclein ( -synuclein)-containing lewy bodies .", "pd is the most common of a group of parkinsonian movement disorders that also includes multiple system atrophy ( msa ) , corticobasal degeneration ( cbd ) , and progressive supranuclear palsy ( psp ) .", "the presence of activated microglia close to dopaminergic neurons in post - mortem pd patient brains ( mcgeer et al . , 1988a ; mogi et al . , 1994 ; langston et al . , 1999 ; imamura et al . ,", "2003 ) , and pd animal models ( czlonkowska et al . , 1996 ; kim et al . , 2009 ) suggests a close relationship between neurodegeneration and neuroinflammation in pd .", "numerous investigations have proposed a deleterious role of microglial activation in pd based on the vulnerability of dopaminergic neurons to various microglia - derived pro - inflammatory cytokines ( ferrari et al . , 2006 ; stone et al . , 2009 ; de lella ezcurra et al . , 2010 ) , while -synuclein can directly induce activation of microglia ( zhang et al . , 2005 ) .", "however , it seems that the plasticity of microglia must be considered with regards to their contribution in pd , and their role ; whether beneficial or detrimental , it may depend on the stimuli present and the stage of disease ( li et al .", ", 2007 ; michelucci et al . , 2009 ; sanchez - guajardo et al . ,", "further clues regarding the role of activated microglia has also come from in vivo pet imaging studies ( table 1 ) .", "significant microglial activation , as reflected by an increase in c - pk11195 bpnd was reported in the midbrain and putamen of pd patients when compared to controls , and was found to correlate positively with the motor severity of parkinsonism ( ouchi et al . , 2005 ; bartels et al . , 2010", "these findings suggest that activated microglia has a pathogenic importance in the disease and indicate that the early introduction of a neuroprotective drug to suppress microglial activation could be favorable in pd . additionally , pd patients exhibited significantly increased c - pk11195 bpnd in the basal ganglia , pons , and frontal and temporal cortical regions ( gerhard et al . , 2006a ) .", "in this study , the increased microglial activation remained unchanged for 2  years , while the patients deteriorated clinically during this period .", "hence , it is likely that microglia are activated early in pd , where they remain activated for longer periods and possibly drive progression of the disease ( gerhard et al . , 2006a ) .", "bpnd , binding potential ; cbd , corticobasal degeneration ; msa , multiple system atrophy ; nc , normal control ; pd , parkinson s disease ; psp , progressive supranuclear palsy .", "multiple system atrophy is a sporadic neurodegenerative disorder involving a progressive akinetic - rigid syndrome , autonomic failure , and cerebellar dysfunction .", "it is associated by the appearance of abnormal glial cytoplasmic inclusions ( gci ) containing ( -synuclein aggregates and neuronal loss within the nigrostriatal and olivopontocerebellar regions ( lantos and papp , 1994 ) .", "the presence of activated microglia is also a prominent feature of msa ( schwarz et al . , 1998 ) .", "in an in vivo pet study of msa patients , significant c - pk11195 bpnd was observed in the putamen , pallidum , pons , substantia nigra pars compacta , and dorsolateral prefrontal cortex , reflecting the known distribution of neuropathological changes in msa ( gerhard et al . , 2003 ) . although the role of microglia in msa is inconclusive , microglial activation localization correlated significantly with the locations of gcis in specific neuroanatomical systems affected in msa ( ishizawa et al . , 2004 ) .", "a correlation between extent of microglial activation and dopaminergic neurodegeneration has also been reported ( stefanova et al . , 2007 ) .", "progressive supranuclear palsy is an adult - onset progressive neurodegenerative disease of unknown cause , characterized by pd - like symptoms such as postural instability and bradykinesia .", "the pathological hallmark of the disease is neurofibrillary tangles consisting of hyperphosphorylated tau , accompanied by neuronal loss in the thalamus , basal ganglia , and specific brainstem regions ( hauw et al .", "several early studies including immunohistochemical investigations have confirmed the possible involvement of activated microglia in psp ( kida et al .", ", 1992 ; komori et al . , 1998 ; ishizawa et al . , 2000 ; ishizawa and dickson , 2001 ) .", "c - pk11195 pet have also reported significant levels of activated microglia in brain regions known to be affected by the disease process such as the midbrain , cerebellum , pons , frontal lobe , and basal ganglia ( gerhard et al . , 2006b ) . although these results were unable to support a direct causal contribution to neurodegeneration in psp , they are at least suggestive of a role of microglia in the disease .", "corticobasal degeneration is a neurodegenerative disorder that affects both cortical and basal ganglial regions , with considerable clinical heterogeneity between patients .", "typically , cbd features an asymmetric hypokinetic - rigid syndrome , coupled with alien limb phenomenon and cortical sensory impairment that is unresponsive to dopaminergic therapy ( rebeiz et al . , 1968 ; gibb et al . , 1989", "information on the association of activated microglia in cbd is limited , and mainly coming from immunohistochemical - based assessments ( armstrong et al . , 2000 ; ishizawa and dickson , 2001 ) .", "however , more recent in vivo pet investigations have attempted to quantify microglial activation in cbd patients .", "increased c - pk11195 bpnd was observed in regions such as the caudate nucleus , putamen , substantia nigra pars compacta , pons , and pre- and post central gyrus ( gerhard et al .", "2004 ) that correspond to the expected neuropathological changes seen in cbd ( ishizawa and dickson , 2001 ; dickson et al . , 2002 ) .", "huntington s disease is an autosomal , dominant inherited progressive neurodegenerative disorder associated with motor , cognitive , and psychiatric symptoms .", "it is caused by an abnormal polyglutamine - repeat expansion on the it15 gene that codes huntingtin , and involves the progressive loss of medium spiny dopaminergic receptor - bearing striatal gaba - ergic neurons ( vonsattel and difiglia , 1998 ) .", "although the role of chronic neuroinflammation in the hd pathogenesis is not fully understood , post - mortem assessments have reported high levels of activated microglia close to degenerating neurons ( mcgeer et al .", "upregulated inflammatory cytokines have also been detected in the striatum and plasma , indicative of an inflammatory component in hd ( dalrymple et al . , 2007 ;", "imaging studies using c - pk11195 pet have found increased microglial activation in both premanifest hd gene carriers and manifest hd patients when compared to healthy controls ( table 2 ; pavese et al .", ", 2006 ; tai et al . , 2007 ; politis et al . , 2008 , 2011 ) . in premanifest", "hd patients , significant increases in c - pk11195 bpnd in the striatum and hypothalamus was reported , which correlated inversely with neuronal dysfunction as measured by c - raclopride ; a marker of dopaminergic d2/d3 receptor availability ( tai et al . , 2007 ; politis et al . , 2008 ) .", "interestingly , microglial activation in the striatum , and regions related to cognitive function has been shown to predict the 5-year disease clinical onset in premanifest hd patients ( tai et al .", "these results imply that microglial activation is an early event in the hd disease course , with a possible pathogenic involvement that is associated with a subclinical progression of the disease .", "bpnd , binding potential ; hd , huntington s disease ; nc , normal control . in manifest hd patients ,", "significant c - pk11195 bpnd in the striatum , hypothalamus , and various cortical regions was found , that correlated with greater disease burden and higher motor disability ( pavese et al .", "the cortical microglial activation is likely to indicate the involvement of cortical neurons in hd , a well - recognized phenomenon as the disease progresses .", "collectively , these findings are consistent with the post - mortem studies ( messmer and reynolds , 1998 ; sapp et al . , 2001 ) and suggest a detrimental microglial contribution to the ongoing neuronal degeneration in hd .", "dementias are a group of disorders that are expected to affect more than 100 million people by 2050 raising remarkable financial costs for healthcare ( wimo et al . , 2003 ) .", "ad is the most common cause of dementia and is the most common neurological disorder of the elderly .", "ad is characterized by the presence of amyloid plaques , neurofibrillary tangles , and activated microglia ( for review , see hardy and selkoe , 2002 ) .", "there is a plethora of evidence from post - mortem human ad studies ( mcgeer et al . , 1988a ; venneti et al . , 2009 ) and animal models ( frautschy et al . , 1998 ; stalder et al", ", 1999 ; leung et al . , 2011 ) reporting a high accumulation of activated microglia in close proximity with the amyloid plaques , and upregulated levels of pro - inflammatory cytokines ( akiyama et al . , 2000 ; eikelenboom et al . ,", "positron emission tomography enables a broad range of functional processes to assess the ad brain in vivo ( table 3 ) .", "c - pk11195 has been used to demonstrate increased levels of activated microglia in both ad animal models ( venneti et al . , 2009 ) and ad patients ( cagnin et al . , 2001 ; edison et al . , 2008 ; yokokura et al . , 2011 ) . in ad patients ,", "significant c - pk11195 bpnd was consistently observed in the temporal , parietal , and occipital cortices , regions known to be affected by ad pathology ( cagnin et al . , 2001 ;", "the increased activated microglia also inversely correlated with the patient mini - mental state examination ( mmse ) scores , which is compatible with a role of microglia in neuronal damage ( edison et al . , 2008 ) .", "interestingly , elevated levels of activated microglia were also detected in patients with amnestic mild cognitive impairment ( mci ; okello et al . , 2009a ) , although this was not observed in another study assessing mci patients ( wiley et al .", "mci could represent an early precursor stage of ad , since it was found that mci patients with increased amyloid load were significantly more likely to clinically convert to ad within 3  years ( okello et al . , 2009b ) .", "therefore , microglial activation could be an early event in the ad pathogenesis that begins at the mci stage .", "ad , alzheimer s disease ; bpnd , binding potential ; ftld , frontotemporal lobar degeneration ; mci , mild cognitive impairment ; mmse , mini - mental state examination ; nc , normal controls . despite the evidence suggestive of a pathogenic role of activated microglia in ad", ", it is hypothesized that the accumulation of amyloid plaques is actually due to a failure in microglial clearance mechanisms that would normally remove the protein ( bornemann et al . , 2001 ;", "this indicates a beneficial , rather than detrimental role of microglia in ad . notwithstanding the abundance of activated microglia close to senile plaques , they maybe inefficient in the clearance of amyloid , hence , resulting in aggregate formation ( bolmont et al . , 2008 ) .", "it has been shown that in the presence of pro - inflammatory cytokines , phagocytic functions of microglia are compromised ( koenigsknecht - talboo and landreth , 2005 ) .", "therefore , microglia may confer a dichotomous role in ad , where early microglial activation is possibly neuroprotective involving the removal of amyloid . however , chronic neuroinflammation may downregulate amyloid clearance mechanisms , thus , promoting aggregation and progression of disease .", "frontotemporal lobar degeneration ( ftld ) which includes frontotemporal dementia is the name given to a group of pathologically , clinically , and genetically heterogeneous disorders involving focal atrophy of the frontal and temporal lobes , while unlike ad , with sparing of the parietal and occipital regions ( neary et al . , 1998 ) .", "another important dissimilarity between ad and ftld pathology is the absence of amyloid plaque formation ( paulus et al . , 1993 ;", "rather , the key histopathological features of ftld , depending on subtype , includes tau deposition ( including pick bodies ) and ubiquitin - positive , tau - negative inclusions ( munoz et al . , 2003 ; uchihara et al . , 2003 ) .", "in vivo pet imaging of ftld patients detected enhanced microglial activation in the expected frontotemporal regions ( cagnin et al . ,", "2004 ) . in the same study , significant c - pk11195 bpnd in the bilateral putamen is also consistent with previous neuropathological data showing the involvement of the basal ganglia in ftld ( mirra and hyman , 2002 ) .", "these observations indicate the presence of an active microglial response that reflects progressive neuronal degeneration .", "importantly , the detection of increased microglial activation in affected regions in ftld suggests that microglial responses occur independently of amyloid deposition , and that neuronal loss alone is enough to induce activation ( cagnin et al . , 2004 ) .", "multiple sclerosis is a disease characterized pathologically by inflammatory demyelination and axonal transection , and is the most common cause of non - traumatic disability in young adults ( compston and coles , 2008 ) .", "the involvement of activated microglia has long been proposed in ms ( benveniste , 1997 ) .", "post - mortem investigations have detected activated microglia in the cortical gm of ms patients ( de groot et al .", "2002 ) , while histopathological studies have implicated microglia in lesion pathogenesis ( for review , see lassmann , 2008 ) .", "an observed correlation between neuronal loss and microglial activation was reported in animal experimental ms ( rasmussen et al . , 2007 ) .", "significant levels of activated microglia was also found in ms patients , especially in the progressive forms of disease that are associated with neurodegeneration ( kutzelnigg et al . , 2005 ; magliozzi et al . ,", "2010 ) , and selective ablation of parenchymal microglia was able to prevent demyelination and axonal damage ( heppner et al . , 2005 ) .", "pathological aspects of ms such as neuroinflammation , demyelination , and neurodegeneration may be explored in vivo with pet ( for review , see kiferle et al . , 2011 ) .", "pet with c - pk11195 and other tracers has demonstrated inflammatory processes with microglial involvement in ms ( figure 1 ; table 4 ) . in animal", "experimental ms and human post - mortem brains it has been shown that c - pk11195 uptake corresponds to the distribution pattern of activated microglia ( banati et al . , 2000 ) .", "it has also been demonstrated that there is increased c - pk11195 bpnd in areas of focal pathology identified by t1- and t2-weighted mri ( vowinckel et al . , 1997 ; banati et al . , 2000 ) and in gadolinium - enhanced t1-weighted mri ( debruyne et al . , 2003 ) .", "increased c - pk11195 bpnd was observed in normal - appearing gray and white anatomical structures ( banati et al .", "this is in line with the hypothesis that inflammatory processes initiated by microglia early in ms may constitute the real burden of disease , associated with invisible microglia - mediated damage that occur independently of relapses ( kesselring , 1990 ; confavreux et al . , 2000 ) .", "a positive correlation has been suggested between ligand uptake and disease duration , disability , and brain atrophy ( banati et al . , 2000 ;", "however , recent data from our group found a significant association between high c - pk11195 bpnd in the cortical gray matter and disability in patients with secondary progressive ms , and with higher c - pk11195 bpnd in the secondary progressive group than the relapse - remitting ms group ( politis et al .", "enhanced microglial activation in ms has also been detected using the more recently developed tspo tracers c - vinpocetine and c - pbr28 ( vas et al . , 2008 ; oh et al . , 2011 ) .", "positron emission tomography images showing increased c - pk11195 bpnd in a multiple sclerosis patient ( a ) when compared to a healthy normal control ( b ) . color bar represents intensity of c - pk11195 tracer binding ( bpnd ) .", "bpnd , binding potential ; gm , gray matter ; mri , magnetic resonance image ; ms , multiple sclerosis ; nawm , normal - appearing white matter ; nc , normal control ; pp , primary progressive multiple sclerosis ; rr , relapse - remitting multiple sclerosis ; sp , secondary progressive multiple sclerosis . microglial activation may contribute to the mechanism of axonal injury via the release of soluble factors that may either directly or indirectly cause neuronal dysfunction ( peterson et al .", ", 2001 ; barnett and prineas , 2004 ; dutta and trapp , 2006 ; zipp et al . , 2006 ; dal bianco et al . , 2008 ; lassmann , 2008 ; magliozzi et al . , 2010 ) ,", "however , activated microglia may also exert protective functions with ms through the release of neurotrophic factors ( stadelmann et al .", ", 2002 ; napoli and neumann , 2009 ) , and triggering of remyelination mechanisms ( li et al . , 2005 ; setzu et al . ,", "c - pk11195 was the first tracer to be consistently used for the study of activated microglia and neuroinflammation in vivo .", "however , limitations associated with the application of c - pk11195 include a high level of non - specific binding ( petit - tabou et al . , 1991 ) , and a poor signal to noise ratio , which complicates its quantification ( boutin et al . , 2007 ) .", "this has prompted the search for novel pet tracers ( termed , second generation radioligands ) with improved capacities to quantify tspo expression .", "radioligands such as c - pbr28 , c - daa1106 , f - fedaa1106 , and f - pbr111 have recently been developed to image tspo in vivo ( gulys et al . , 2002 ; ikoma et al . , 2007 ;", ", 2011 ; for a review , see chauveau et al . , 2008 ) .", "published data using the second generation ligands c - daa1106 ( ikoma et al . , 2007 ) and f - fedaa1106 ( fujimura et al . , 2006 ) in humans were promising , with both tracers showing significantly higher cerebral uptake than c - pk11195 .", "furthermore , increased c - daa1106 binding was reported in ad patients ( yasuno et al .", ", 2008 ) that were similar to the previous studies that used c - pk11195 ( cagnin et al . , 2001 ) . the only published study using the second generation radioligand c - pbr28 found areas of focal increases in radiotracer binding in the brain of ms patients ( oh et al . , 2011 )", "interestingly , the increased focal c - pbr28 binding preceded the development of some gadolinium - enhancing lesions . brain parenchymal c - pbr28 binding in ms patients was positively correlated with the duration of the disease , however it was not significantly higher than that of healthy volunteers . interpretation of these results is limited by the lack of characterization of the binding affinity pattern , which might have significantly affected the comparison between subjects .", "it has been recently demonstrated that there are three different affinity patterns for second generation tspo ligands in healthy volunteers as well as patients with ms , which was evident with all the ligands tested ( c - pbr28 ; c - pbr06 ; f - pbr111 ; owen et al . , 2010 ) .", "this presents a methodological problem , as differences in pet signal across subjects can not be safely interpreted as differences in target density , but may reflect differences in the affinity pattern . a possible approach to solve this problem", "is based on the use of peripheral binding affinity , which can be characterized to classify subjects into one of the groups , as differences in affinity status between individuals have been shown to be present on peripheral cells as well ( owen et al .", "interestingly , the difference in binding patterns observed with second generation radioligands was not observed with c - pk11195 .", "also , in vitro autoradiography data using c - pk11195 suggest a receptor density ( bmax ) significantly higher than that found using second generation ligands .", "it could be speculated that c - pk11195 and newer ligands bind to distinct sites within the tspo molecule .", "although , data obtained from first generation studies have been promising and suggested that c - pk11195 could be useful to image acute inflammatory lesions and microglial activation in ms , a conclusive demonstration of the potential of tspo imaging for the application as disease biomarker , indicative of microglial activation in ms , is still lacking .", "furthermore , despite second generation ligands constituting a potential improvement relative to c - pk11195 at least from a methodological point of view , a clear advantage in their clinical application as disease biomarkers has not been demonstrated yet .", "for these reasons , we aim to characterize a second generation tspo pet radioligand in vivo in humans , and to evaluate its application as a disease biomarker in ms . among second generation tspo tracers ,", "f - pbr111 presents different advantages , as there is low difference in its affinity for tspo between high , medium , low affinity binders .", "also , it could be potentially used in clinical applications as it is labeled with fluorine-18 .", "promising preclinical data , and ongoing studies in neurological patients , suggest it could be a good choice amongst second generation tspo ligands to progress into studies in ms patients .", "inflammation coupled with the presence of activated microglia seems to be a common feature of a wide range of cns diseases .", "however , despite a large number of research studies , the exact role of microglia in chronic neurodegenerative diseases remains uncertain . in line with the high plasticity of microglia that allows them to perform numerous cns functions , microglia are likely to play a dichromatic role in disease , depending on signals present in their microenvironment and the duration of activation . while early microglial activation could represents a beneficial response ( i.e. , removal of cns threat , promoting tissue repair and removal of misfolded protein ) , chronic exposure could induces detrimental effects by promoting neuronal death ( i.e. , through the sustained release of neurotoxic factors ) , thus , contributing to progression of disease . pet imaging with the use of tspo radioligands", "provides a valuable tool that allows us to track the progression and severity of neuroinflammation in the living brain , and is a useful indicator of active cns disease .", "therefore , the early detection of microglia using pet could offer opportunities for pharmacological interventions to limit the potential disruptive effects of chronic microglial activation .", "furthermore , with the development of newer tspo tracers , the potential for pet imaging research to promote our understanding of activated microglia in cns disease can only increase .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S> microglia constitute the main immune defense in the central nervous system . in response to neuronal injury , </S>", "<S> microglia become activated , acquire phagocytic properties , and release a wide range of pro - inflammatory mediators that are essential for the annihilation of the neuronal insult . </S>", "<S> although the role of microglial activation in acute neuronal damage is well defined , the pathophysiological processes underlying destructive or protective role to neurons following chronic exposure to microglial activation is still a subject of debate . </S>", "<S> it is likely that chronic exposure induces detrimental effects by promoting neuronal death through the release of neurotoxic factors . </S>", "<S> positron emission tomography ( pet ) imaging with the use of translocator protein ( tspo ) radioligands provides an in vivo tool for tracking the progression and severity of neuroinflammation in neurodegenerative disease . </S>", "<S> tspo expression is correlated to the extent of microglial activation and the measurement of tspo uptake in vivo with pet is a useful indicator of active disease . </S>", "<S> although understanding of the interaction between radioligands and tspo is not completely clear , there is a wide interest in application of tspo imaging in neurodegenerative disease . in this article , we aim to review the applications of in vivo microglia imaging in neurodegenerative disorders such as parkinson s disease , huntington s disease , dementias , and multiple sclerosis . </S>"], "labels": null, "section_names": ["Introduction", "Parkinsons Disease and Related Disorders", "Huntingtons Disease", "Dementia", "Multiple Sclerosis", "New TSPO Ligands", "Conclusion", "Conflict of Interest Statement"], "sections": [["microglia account for approximately 10% of the adult brain cell population and represent the first and main form of immune defense in the central nervous system ( cns ; lawson et al .", "microglia become activated and they can be identified and distinguished from their resting phenotype based on a combination of morphological and immunophenotypic changes ( dheen et al . , 2007 ; ransohoff and perry , 2009 ) .", "microglia initiate immune responses by enhancing the expression of toll - like receptors ( tlr ) and a wide range of pro - inflammatory mediators such as tumor necrosis factor - alpha ( tnf ) , interleukin ( il)-1 and il-6 for the removal of the cns threat ( suzumura et al . , 1996 ;", "microglia may also fulfill a neuroprotective role via the release of neurotrophic factors and promotion of neurogenesis for the restoration of normal physiology ( stadelmann et al . , 2002 ) .", "hence , the acute inflammatory response is generally beneficial , as it tends to minimize injury and promotes tissue repair .", "however , chronic neuroinflammation is closely related to various neurodegenerative disorders such as parkinson s disease ( pd ) , huntington s disease ( hd ) , dementias , and multiple sclerosis ( ms ) , although the consequences of sustained microglial activation in these diseases is unclear .", "activated microglia upregulate expression of the 18-kda translocator protein ( tspo ; chen and guilarte , 2008 ; cosenza - nashat et al .", "tspo are found in abundance throughout the body in peripheral organs ( i.e. , liver and adrenals ) , and hematogenous cells , but are present at very low levels in the normal healthy cns ( banati , 2002 ) .", "functionally , tspo has several biological functions including the control of cholesterol transport and neurosteroid synthesis ( papadopoulos et al . , 2006 ) , and", "may also be involved in the release of pro - inflammatory cytokines during inflammation ( choi et al . , 2002 ; wilms et al . ,", "enhanced tspo expression can be detected in vivo by using positron emission tomography ( pet ) imaging with the selective tspo radioligand c - pk11195 ( benavides et al .", ", 1993 ; banati et al . , 1999 ) , with evidence that increases in c - pk11195 binding potential ( bpnd ) correspond to activation of microglia ( stephenson et al . , 1995 ; conway et al .", "although tspo is also expressed by reactive astrocytes , a c - pk11195 pet study of patients with hippocampal sclerosis , a condition histopathologically characterized by marked astrogliosis , did not yield results that were significantly different to healthy normal controls ( banati et al .", "this is consistent with the view that reactive astrocytes , in vivo , do not significantly contribute to the c - pk11195 signal .", "therefore , in the absence of invading blood borne cells or severe focal leakage of blood - brain barrier , the increased pk11195 binding is likely to indicate the transition of microglia from a resting to an activated state , and is due to an increase in the number , rather than the affinity , of tspo ( banati et al . , 2000 ) .", "hence , the measurement of tspo uptake using pet provides an in vivo tool to monitor progression and severity of neuroinflammation and is a useful indicator of active cns disease .", "this article aims to review the use of pet imaging to promote the understanding of activated microglia in neurodegenerative disease ."], ["parkinson s disease is the second most common neurodegenerative disorder of the elderly and is associated with the motor symptoms of tremor , bradykinesia , and rigidity .", "it is characterized by the extended loss of dopaminergic neurons in the substantia nigra pars compacta , resulting in a deficiency of dopamine in the striatum ( braak et al . , 2006 ) , and the presence of alpha - synuclein ( -synuclein)-containing lewy bodies .", "pd is the most common of a group of parkinsonian movement disorders that also includes multiple system atrophy ( msa ) , corticobasal degeneration ( cbd ) , and progressive supranuclear palsy ( psp ) .", "the presence of activated microglia close to dopaminergic neurons in post - mortem pd patient brains ( mcgeer et al . , 1988a ; mogi et al . , 1994 ; langston et al . , 1999 ; imamura et al . ,", "2003 ) , and pd animal models ( czlonkowska et al . , 1996 ; kim et al . , 2009 ) suggests a close relationship between neurodegeneration and neuroinflammation in pd .", "numerous investigations have proposed a deleterious role of microglial activation in pd based on the vulnerability of dopaminergic neurons to various microglia - derived pro - inflammatory cytokines ( ferrari et al . , 2006 ; stone et al . , 2009 ; de lella ezcurra et al . , 2010 ) , while -synuclein can directly induce activation of microglia ( zhang et al . , 2005 ) .", "however , it seems that the plasticity of microglia must be considered with regards to their contribution in pd , and their role ; whether beneficial or detrimental , it may depend on the stimuli present and the stage of disease ( li et al .", ", 2007 ; michelucci et al . , 2009 ; sanchez - guajardo et al . ,", "further clues regarding the role of activated microglia has also come from in vivo pet imaging studies ( table 1 ) .", "significant microglial activation , as reflected by an increase in c - pk11195 bpnd was reported in the midbrain and putamen of pd patients when compared to controls , and was found to correlate positively with the motor severity of parkinsonism ( ouchi et al . , 2005 ; bartels et al . , 2010", "these findings suggest that activated microglia has a pathogenic importance in the disease and indicate that the early introduction of a neuroprotective drug to suppress microglial activation could be favorable in pd . additionally , pd patients exhibited significantly increased c - pk11195 bpnd in the basal ganglia , pons , and frontal and temporal cortical regions ( gerhard et al . , 2006a ) .", "in this study , the increased microglial activation remained unchanged for 2  years , while the patients deteriorated clinically during this period .", "hence , it is likely that microglia are activated early in pd , where they remain activated for longer periods and possibly drive progression of the disease ( gerhard et al . , 2006a ) .", "bpnd , binding potential ; cbd , corticobasal degeneration ; msa , multiple system atrophy ; nc , normal control ; pd , parkinson s disease ; psp , progressive supranuclear palsy .", "multiple system atrophy is a sporadic neurodegenerative disorder involving a progressive akinetic - rigid syndrome , autonomic failure , and cerebellar dysfunction .", "it is associated by the appearance of abnormal glial cytoplasmic inclusions ( gci ) containing ( -synuclein aggregates and neuronal loss within the nigrostriatal and olivopontocerebellar regions ( lantos and papp , 1994 ) .", "the presence of activated microglia is also a prominent feature of msa ( schwarz et al . , 1998 ) .", "in an in vivo pet study of msa patients , significant c - pk11195 bpnd was observed in the putamen , pallidum , pons , substantia nigra pars compacta , and dorsolateral prefrontal cortex , reflecting the known distribution of neuropathological changes in msa ( gerhard et al . , 2003 ) . although the role of microglia in msa is inconclusive , microglial activation localization correlated significantly with the locations of gcis in specific neuroanatomical systems affected in msa ( ishizawa et al . , 2004 ) .", "a correlation between extent of microglial activation and dopaminergic neurodegeneration has also been reported ( stefanova et al . , 2007 ) .", "progressive supranuclear palsy is an adult - onset progressive neurodegenerative disease of unknown cause , characterized by pd - like symptoms such as postural instability and bradykinesia .", "the pathological hallmark of the disease is neurofibrillary tangles consisting of hyperphosphorylated tau , accompanied by neuronal loss in the thalamus , basal ganglia , and specific brainstem regions ( hauw et al .", "several early studies including immunohistochemical investigations have confirmed the possible involvement of activated microglia in psp ( kida et al .", ", 1992 ; komori et al . , 1998 ; ishizawa et al . , 2000 ; ishizawa and dickson , 2001 ) .", "c - pk11195 pet have also reported significant levels of activated microglia in brain regions known to be affected by the disease process such as the midbrain , cerebellum , pons , frontal lobe , and basal ganglia ( gerhard et al . , 2006b ) . although these results were unable to support a direct causal contribution to neurodegeneration in psp , they are at least suggestive of a role of microglia in the disease .", "corticobasal degeneration is a neurodegenerative disorder that affects both cortical and basal ganglial regions , with considerable clinical heterogeneity between patients .", "typically , cbd features an asymmetric hypokinetic - rigid syndrome , coupled with alien limb phenomenon and cortical sensory impairment that is unresponsive to dopaminergic therapy ( rebeiz et al . , 1968 ; gibb et al . , 1989", "information on the association of activated microglia in cbd is limited , and mainly coming from immunohistochemical - based assessments ( armstrong et al . , 2000 ; ishizawa and dickson , 2001 ) .", "however , more recent in vivo pet investigations have attempted to quantify microglial activation in cbd patients .", "increased c - pk11195 bpnd was observed in regions such as the caudate nucleus , putamen , substantia nigra pars compacta , pons , and pre- and post central gyrus ( gerhard et al .", "2004 ) that correspond to the expected neuropathological changes seen in cbd ( ishizawa and dickson , 2001 ; dickson et al . , 2002 ) ."], ["huntington s disease is an autosomal , dominant inherited progressive neurodegenerative disorder associated with motor , cognitive , and psychiatric symptoms .", "it is caused by an abnormal polyglutamine - repeat expansion on the it15 gene that codes huntingtin , and involves the progressive loss of medium spiny dopaminergic receptor - bearing striatal gaba - ergic neurons ( vonsattel and difiglia , 1998 ) .", "although the role of chronic neuroinflammation in the hd pathogenesis is not fully understood , post - mortem assessments have reported high levels of activated microglia close to degenerating neurons ( mcgeer et al .", "upregulated inflammatory cytokines have also been detected in the striatum and plasma , indicative of an inflammatory component in hd ( dalrymple et al . , 2007 ;", "imaging studies using c - pk11195 pet have found increased microglial activation in both premanifest hd gene carriers and manifest hd patients when compared to healthy controls ( table 2 ; pavese et al .", ", 2006 ; tai et al . , 2007 ; politis et al . , 2008 , 2011 ) . in premanifest", "hd patients , significant increases in c - pk11195 bpnd in the striatum and hypothalamus was reported , which correlated inversely with neuronal dysfunction as measured by c - raclopride ; a marker of dopaminergic d2/d3 receptor availability ( tai et al . , 2007 ; politis et al . , 2008 ) .", "interestingly , microglial activation in the striatum , and regions related to cognitive function has been shown to predict the 5-year disease clinical onset in premanifest hd patients ( tai et al .", "these results imply that microglial activation is an early event in the hd disease course , with a possible pathogenic involvement that is associated with a subclinical progression of the disease .", "bpnd , binding potential ; hd , huntington s disease ; nc , normal control . in manifest hd patients ,", "significant c - pk11195 bpnd in the striatum , hypothalamus , and various cortical regions was found , that correlated with greater disease burden and higher motor disability ( pavese et al .", "the cortical microglial activation is likely to indicate the involvement of cortical neurons in hd , a well - recognized phenomenon as the disease progresses .", "collectively , these findings are consistent with the post - mortem studies ( messmer and reynolds , 1998 ; sapp et al . , 2001 ) and suggest a detrimental microglial contribution to the ongoing neuronal degeneration in hd ."], ["dementias are a group of disorders that are expected to affect more than 100 million people by 2050 raising remarkable financial costs for healthcare ( wimo et al . , 2003 ) .", "ad is the most common cause of dementia and is the most common neurological disorder of the elderly .", "ad is characterized by the presence of amyloid plaques , neurofibrillary tangles , and activated microglia ( for review , see hardy and selkoe , 2002 ) .", "there is a plethora of evidence from post - mortem human ad studies ( mcgeer et al . , 1988a ; venneti et al . , 2009 ) and animal models ( frautschy et al . , 1998 ; stalder et al", ", 1999 ; leung et al . , 2011 ) reporting a high accumulation of activated microglia in close proximity with the amyloid plaques , and upregulated levels of pro - inflammatory cytokines ( akiyama et al . , 2000 ; eikelenboom et al . ,", "positron emission tomography enables a broad range of functional processes to assess the ad brain in vivo ( table 3 ) .", "c - pk11195 has been used to demonstrate increased levels of activated microglia in both ad animal models ( venneti et al . , 2009 ) and ad patients ( cagnin et al . , 2001 ; edison et al . , 2008 ; yokokura et al . , 2011 ) . in ad patients ,", "significant c - pk11195 bpnd was consistently observed in the temporal , parietal , and occipital cortices , regions known to be affected by ad pathology ( cagnin et al . , 2001 ;", "the increased activated microglia also inversely correlated with the patient mini - mental state examination ( mmse ) scores , which is compatible with a role of microglia in neuronal damage ( edison et al . , 2008 ) .", "interestingly , elevated levels of activated microglia were also detected in patients with amnestic mild cognitive impairment ( mci ; okello et al . , 2009a ) , although this was not observed in another study assessing mci patients ( wiley et al .", "mci could represent an early precursor stage of ad , since it was found that mci patients with increased amyloid load were significantly more likely to clinically convert to ad within 3  years ( okello et al . , 2009b ) .", "therefore , microglial activation could be an early event in the ad pathogenesis that begins at the mci stage .", "ad , alzheimer s disease ; bpnd , binding potential ; ftld , frontotemporal lobar degeneration ; mci , mild cognitive impairment ; mmse , mini - mental state examination ; nc , normal controls . despite the evidence suggestive of a pathogenic role of activated microglia in ad", ", it is hypothesized that the accumulation of amyloid plaques is actually due to a failure in microglial clearance mechanisms that would normally remove the protein ( bornemann et al . , 2001 ;", "this indicates a beneficial , rather than detrimental role of microglia in ad . notwithstanding the abundance of activated microglia close to senile plaques , they maybe inefficient in the clearance of amyloid , hence , resulting in aggregate formation ( bolmont et al . , 2008 ) .", "it has been shown that in the presence of pro - inflammatory cytokines , phagocytic functions of microglia are compromised ( koenigsknecht - talboo and landreth , 2005 ) .", "therefore , microglia may confer a dichotomous role in ad , where early microglial activation is possibly neuroprotective involving the removal of amyloid . however , chronic neuroinflammation may downregulate amyloid clearance mechanisms , thus , promoting aggregation and progression of disease .", "frontotemporal lobar degeneration ( ftld ) which includes frontotemporal dementia is the name given to a group of pathologically , clinically , and genetically heterogeneous disorders involving focal atrophy of the frontal and temporal lobes , while unlike ad , with sparing of the parietal and occipital regions ( neary et al . , 1998 ) .", "another important dissimilarity between ad and ftld pathology is the absence of amyloid plaque formation ( paulus et al . , 1993 ;", "rather , the key histopathological features of ftld , depending on subtype , includes tau deposition ( including pick bodies ) and ubiquitin - positive , tau - negative inclusions ( munoz et al . , 2003 ; uchihara et al . , 2003 ) .", "in vivo pet imaging of ftld patients detected enhanced microglial activation in the expected frontotemporal regions ( cagnin et al . ,", "2004 ) . in the same study , significant c - pk11195 bpnd in the bilateral putamen is also consistent with previous neuropathological data showing the involvement of the basal ganglia in ftld ( mirra and hyman , 2002 ) .", "these observations indicate the presence of an active microglial response that reflects progressive neuronal degeneration .", "importantly , the detection of increased microglial activation in affected regions in ftld suggests that microglial responses occur independently of amyloid deposition , and that neuronal loss alone is enough to induce activation ( cagnin et al . , 2004 ) ."], ["multiple sclerosis is a disease characterized pathologically by inflammatory demyelination and axonal transection , and is the most common cause of non - traumatic disability in young adults ( compston and coles , 2008 ) .", "the involvement of activated microglia has long been proposed in ms ( benveniste , 1997 ) .", "post - mortem investigations have detected activated microglia in the cortical gm of ms patients ( de groot et al .", "2002 ) , while histopathological studies have implicated microglia in lesion pathogenesis ( for review , see lassmann , 2008 ) .", "an observed correlation between neuronal loss and microglial activation was reported in animal experimental ms ( rasmussen et al . , 2007 ) .", "significant levels of activated microglia was also found in ms patients , especially in the progressive forms of disease that are associated with neurodegeneration ( kutzelnigg et al . , 2005 ; magliozzi et al . ,", "2010 ) , and selective ablation of parenchymal microglia was able to prevent demyelination and axonal damage ( heppner et al . , 2005 ) .", "pathological aspects of ms such as neuroinflammation , demyelination , and neurodegeneration may be explored in vivo with pet ( for review , see kiferle et al . , 2011 ) .", "pet with c - pk11195 and other tracers has demonstrated inflammatory processes with microglial involvement in ms ( figure 1 ; table 4 ) . in animal", "experimental ms and human post - mortem brains it has been shown that c - pk11195 uptake corresponds to the distribution pattern of activated microglia ( banati et al . , 2000 ) .", "it has also been demonstrated that there is increased c - pk11195 bpnd in areas of focal pathology identified by t1- and t2-weighted mri ( vowinckel et al . , 1997 ; banati et al . , 2000 ) and in gadolinium - enhanced t1-weighted mri ( debruyne et al . , 2003 ) .", "increased c - pk11195 bpnd was observed in normal - appearing gray and white anatomical structures ( banati et al .", "this is in line with the hypothesis that inflammatory processes initiated by microglia early in ms may constitute the real burden of disease , associated with invisible microglia - mediated damage that occur independently of relapses ( kesselring , 1990 ; confavreux et al . , 2000 ) .", "a positive correlation has been suggested between ligand uptake and disease duration , disability , and brain atrophy ( banati et al . , 2000 ;", "however , recent data from our group found a significant association between high c - pk11195 bpnd in the cortical gray matter and disability in patients with secondary progressive ms , and with higher c - pk11195 bpnd in the secondary progressive group than the relapse - remitting ms group ( politis et al .", "enhanced microglial activation in ms has also been detected using the more recently developed tspo tracers c - vinpocetine and c - pbr28 ( vas et al . , 2008 ; oh et al . , 2011 ) .", "positron emission tomography images showing increased c - pk11195 bpnd in a multiple sclerosis patient ( a ) when compared to a healthy normal control ( b ) . color bar represents intensity of c - pk11195 tracer binding ( bpnd ) .", "bpnd , binding potential ; gm , gray matter ; mri , magnetic resonance image ; ms , multiple sclerosis ; nawm , normal - appearing white matter ; nc , normal control ; pp , primary progressive multiple sclerosis ; rr , relapse - remitting multiple sclerosis ; sp , secondary progressive multiple sclerosis . microglial activation may contribute to the mechanism of axonal injury via the release of soluble factors that may either directly or indirectly cause neuronal dysfunction ( peterson et al .", ", 2001 ; barnett and prineas , 2004 ; dutta and trapp , 2006 ; zipp et al . , 2006 ; dal bianco et al . , 2008 ; lassmann , 2008 ; magliozzi et al . , 2010 ) ,", "however , activated microglia may also exert protective functions with ms through the release of neurotrophic factors ( stadelmann et al .", ", 2002 ; napoli and neumann , 2009 ) , and triggering of remyelination mechanisms ( li et al . , 2005 ; setzu et al . ,"], ["c - pk11195 was the first tracer to be consistently used for the study of activated microglia and neuroinflammation in vivo .", "however , limitations associated with the application of c - pk11195 include a high level of non - specific binding ( petit - tabou et al . , 1991 ) , and a poor signal to noise ratio , which complicates its quantification ( boutin et al . , 2007 ) .", "this has prompted the search for novel pet tracers ( termed , second generation radioligands ) with improved capacities to quantify tspo expression .", "radioligands such as c - pbr28 , c - daa1106 , f - fedaa1106 , and f - pbr111 have recently been developed to image tspo in vivo ( gulys et al . , 2002 ; ikoma et al . , 2007 ;", ", 2011 ; for a review , see chauveau et al . , 2008 ) .", "published data using the second generation ligands c - daa1106 ( ikoma et al . , 2007 ) and f - fedaa1106 ( fujimura et al . , 2006 ) in humans were promising , with both tracers showing significantly higher cerebral uptake than c - pk11195 .", "furthermore , increased c - daa1106 binding was reported in ad patients ( yasuno et al .", ", 2008 ) that were similar to the previous studies that used c - pk11195 ( cagnin et al . , 2001 ) . the only published study using the second generation radioligand c - pbr28 found areas of focal increases in radiotracer binding in the brain of ms patients ( oh et al . , 2011 )", "interestingly , the increased focal c - pbr28 binding preceded the development of some gadolinium - enhancing lesions . brain parenchymal c - pbr28 binding in ms patients was positively correlated with the duration of the disease , however it was not significantly higher than that of healthy volunteers . interpretation of these results is limited by the lack of characterization of the binding affinity pattern , which might have significantly affected the comparison between subjects .", "it has been recently demonstrated that there are three different affinity patterns for second generation tspo ligands in healthy volunteers as well as patients with ms , which was evident with all the ligands tested ( c - pbr28 ; c - pbr06 ; f - pbr111 ; owen et al . , 2010 ) .", "this presents a methodological problem , as differences in pet signal across subjects can not be safely interpreted as differences in target density , but may reflect differences in the affinity pattern . a possible approach to solve this problem", "is based on the use of peripheral binding affinity , which can be characterized to classify subjects into one of the groups , as differences in affinity status between individuals have been shown to be present on peripheral cells as well ( owen et al .", "interestingly , the difference in binding patterns observed with second generation radioligands was not observed with c - pk11195 .", "also , in vitro autoradiography data using c - pk11195 suggest a receptor density ( bmax ) significantly higher than that found using second generation ligands .", "it could be speculated that c - pk11195 and newer ligands bind to distinct sites within the tspo molecule .", "although , data obtained from first generation studies have been promising and suggested that c - pk11195 could be useful to image acute inflammatory lesions and microglial activation in ms , a conclusive demonstration of the potential of tspo imaging for the application as disease biomarker , indicative of microglial activation in ms , is still lacking .", "furthermore , despite second generation ligands constituting a potential improvement relative to c - pk11195 at least from a methodological point of view , a clear advantage in their clinical application as disease biomarkers has not been demonstrated yet .", "for these reasons , we aim to characterize a second generation tspo pet radioligand in vivo in humans , and to evaluate its application as a disease biomarker in ms . among second generation tspo tracers ,", "f - pbr111 presents different advantages , as there is low difference in its affinity for tspo between high , medium , low affinity binders .", "also , it could be potentially used in clinical applications as it is labeled with fluorine-18 .", "promising preclinical data , and ongoing studies in neurological patients , suggest it could be a good choice amongst second generation tspo ligands to progress into studies in ms patients ."], ["inflammation coupled with the presence of activated microglia seems to be a common feature of a wide range of cns diseases .", "however , despite a large number of research studies , the exact role of microglia in chronic neurodegenerative diseases remains uncertain . in line with the high plasticity of microglia that allows them to perform numerous cns functions , microglia are likely to play a dichromatic role in disease , depending on signals present in their microenvironment and the duration of activation . while early microglial activation could represents a beneficial response ( i.e. , removal of cns threat , promoting tissue repair and removal of misfolded protein ) , chronic exposure could induces detrimental effects by promoting neuronal death ( i.e. , through the sustained release of neurotoxic factors ) , thus , contributing to progression of disease . pet imaging with the use of tspo radioligands", "provides a valuable tool that allows us to track the progression and severity of neuroinflammation in the living brain , and is a useful indicator of active cns disease .", "therefore , the early detection of microglia using pet could offer opportunities for pharmacological interventions to limit the potential disruptive effects of chronic microglial activation .", "furthermore , with the development of newer tspo tracers , the potential for pet imaging research to promote our understanding of activated microglia in cns disease can only increase ."], ["the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC4983360", "article_text": ["rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disease affecting predominantly joints , synovial membranes , articular cartilages , and subchondral bones .", "disease progression is attributed to increases in reactive oxygen species ( ros ) and oxidative stress ( os ) in the lesion sites .", "proinflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and il-6 , regulate the inflammatory and immune responses and play a pivotal role in the disease .", "overproduction of nitric oxide ( no ) , as a result of induction of inducible nitric oxide synthase ( inos ) due to enhanced production of these cytokines , is associated with persistent inflammation and tissue destruction in experimental arthritis models , including rheumatoid arthritis [ 4 , 5 ] . a number of inflammation stimuli , including tnf- , il-1 , il-6 , or ros , can activate proinflammatory pathways involved in ra pathogenesis , concerning predominantly nuclear factor-b ( nf-b ) , mitogen activated protein kinases ( mapks ) , or janus kinases / signal transducers and activators of transcription ( jak / stat1/3 ) [ 68 ] .", "this results in translocation of relevant downstream transcription factors from the cytoplasm to the nucleus , where they activate messenger rna ( mrna ) expression of target genes , including il-1 , tnf- , inos , and 12/15-lipoxygenase ( lox ) , leading to overproduction of corresponding proteins .", "cytokines released into the synovium reach also the systemic circulation and act in other tissues and organs such as lungs , vascular tissue , liver , and heart . several recent investigations reported damage of vital organs with various degrees of impairment , considered to be secondary complications of ra and a major predictor of mortality in ra patients .", "increasing evidence is pointing to the critical role of the liver in modulating the immune response in autoimmune and chronic inflammatory diseases including ra [ 5 , 11 , 12 ] .", "the hepatic biochemical and immunological alterations are associated with and influenced by changes in the oxidative state of liver cells .", "adjuvant - induced arthritis ( aa ) in rats not only is an experimental model of polyarthritis but also induces pathological changes in a variety of other tissues , including the liver and spleen .", "it is a useful tool to study immunopathologic processes , autoimmune chronic inflammation , and inflammatory cachexia in rodents .", "in addition , at the molecular level , mrna profiling suggests that this model is also similar to human ra , particularly in tissue gene expression and in the activation of regulatory pathways [ 11 , 14 ] .", "numerous studies reported natural polyphenols as potential therapeutic agents of diseases caused by os and inflammation [ 1517 ] .", "n - feruloylserotonin ( n - f-5ht , n - feruloyl-5-hydroxy - tryptamine ) is a conjugated serotonin , a member of the indole hydroxycinnamic acid amides , with serotonin ( 5-ht ) and ferulic acid ( fa ) as representative components of its structure .", "hydroxycinnamic acid amides of serotonin , synthesized by serotonin n - hydroxycinnamoyltransferase , are present in several vegetables   and wild - growing plants whose seeds are used in herbal medicine in eastern countries [ 1820 ] . in cell - based studies , under short - term high - glucose conditions ,", "n - f-5ht exerted an inhibitory effect on overproduction of mitochondrial superoxide by acting as scavenger of superoxide .", "n - f-5ht attenuated the upregulation of mrna and proteins of ros - dependent adhesion ( vascular cell adhesion protein-1 ( vcam-1 ) ) and migration factors ( monocyte chemoattractant protein-1 ( mcp-1 ) ) , crucial in early atherosclerosis lesions in human aortic endothelial cells , and inhibited the activation of transcription factor nf-b .", "furthermore , n - f-5ht showed a protective effect on ros - related neuronal damage by decreasing the activity of proapoptotic caspase-3 .", "n - f-5ht isomers isolated from seeds of leuzea carthamoides were shown to inhibit protein kinase c / ii activation and decrease the oxidative burst of human whole blood and isolated neutrophils in vitro .", "n - f-5ht was also found to have a protective effect against ldl oxidation and atherogenesis in experimental animals and in human studies [ 2426 ] .", "methotrexate ( mtx ) , used as a standard drug in our study , represents the most frequently used pharmacotherapy of ra in clinical practice .", "its administration is , however , limited due to its toxic side effects [ 27 , 28 ] .", "yet application of a combination therapy of mtx with other potential immunomodulators , synthetic drugs   or natural substances [ 3032 ] , might elevate the therapeutic efficacy : decrease the dose of mtx and thus its side effects . in our previous study , we showed that administration of n - f-5ht to mtx - treated arthritic rats lowered the dose of mtx for the required sustained antirheumatic impact . in this study , we focused on the therapeutic impact of n - f-5ht and mtx administered in monotherapy and on details of the inflammatory state in the arthritic rat liver with the aim to elucidate the molecular mechanisms of their effect .", "one of the possible clarifying approaches is to study the mrna expression of key proinflammatory markers ( il-1 , tnf- , and inos ) in the liver of treated and untreated arthritic rats .", "further , it is of particular interest to expand our knowledge on the effect of n - f-5ht and mtx in the aa model , which in turn should allow extrapolations of these results to ra patients .", "to this aim we evaluated also conventional arthritic parameters ( hpv , arthritic score , body weight change , and weight of the liver ) along with changes in plasmatic levels of il-1 and crp and the activity of 12/15-lox in the liver .", "adult male lewis rats weighing 160180  g were obtained from charles river wiga , germany .", "the experimental protocol was approved by the ethics committee of the institute of experimental pharmacology and toxicology and by the slovak state veterinary and food administration in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes and was in line with slovak legislation . to induce a rat model of adjuvant arthritis ( aa ) ,", "rats were intradermally injected with a suspension of heat - inactivated mycobacterium butyricum in incomplete freund 's adjuvant ( difco laboratories , detroit , mi , usa ) .", "the third group comprised adjuvant arthritis rats treated with methotrexate ( methotrexat ebewe sol inj 20  mg/2.0  ml ) in oral dose of 0.4  mg / kg twice a week ( aa - mtx ) . \n", "the fourth group comprised adjuvant arthritis rats treated with n - feruloylserotonin dissolved in suspension of methylcellulose tween 80 at a dose of 3  mg / kg / day orally ( aa - n - f-5ht ) .", "drugs were administered orally by gastric gavage from day 0 ( the day of treatment ) to day 28 of the study .", "blood for plasma preparation was taken by retroorbital puncture on day 14 and by cardiac puncture on day 28 under deep ketamine / xylazine anesthesia .", "after the animals had been sacrificed under deep ketamine / xylazine anesthesia , tissues for liver and spleen homogenate preparation were taken at the end of the experiment ( day 28 ) .", "blood in heparinized tubes for plasma preparation was centrifuged at 3000  rpm for 15 minutes at 4c .", "fraction of four isomers of n - f-5ht ( table 1 ) was isolated from the seeds of leuzea carthamoides ( wild ) dc by solvent extraction .", "this was then followed by column chromatography on silica gel and hplc separations under conditions previously reported [ 35 , 36 ] .", "the hind paw volume ( hpv ) was recorded on days 14 , 21 , and 28 with the use of an electronic water plethysmometer ( ugo basile , comerio , varese , italy ) .", "calculation of the increase in hind paw volume in ml assessed the intensity of the edema .", "the arthritic score was measured as the total score of hpv ( ml , max .", "points 5 ) + diameter of scab in the site of mb application , measured in parallel to the spinal column ( mm , max .", "body weight change ( bwc ; g ) was measured on days 1 , 14 , 21 , and 28 .", "bwc was calculated as the difference of the body mass measured on days 14 , 21 , and 28 to the body weight measured at the beginning of the experiment ( day 1 ) . for the determination of rat crp concentration in plasma ( g / ml ) , the elisa kit from immunology consultant laboratories , inc .", "the reaction of secondary biotin - conjugated anti - rat crp antibody was evaluated by streptavidin - hrp .", "the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 450  nm ( microplate reader , labsystems multiskan rc ) .", "for the determination of il-1 concentration in plasma , the elisa kit from r&d systems quantikine was used .", "rat cytokine present in the samples binds to anti - rat cytokine antibodies absorbed in the microwells .", "the reaction of secondary biotin - conjugated anti - rat cytokine antibody is evaluated by hrp .", "the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 490  nm in comparison with the reference wavelength of 620  nm ( microplate reader mrx ii ) .", "concentration of proteins in liver homogenates was determined by using the bradford method   and expressed in mg / ml of enzyme preparation ( cytosolic fraction from rat lung and liver tissues ) .", "linoleic acid ( 99% , sigma - aldrich , usa ) was used as a substrate prepared in solubilized state as described   in the concentration of 0.2143  100.7143  10  m. the assay of lox was monitored for 60 seconds as an increase in the absorbance at 234  nm , reflecting the formation of hydroperoxylinoleic acid . for the lox activity assay ,", "an uv / vis spectrometer perkin - elmer lambda 35 ( usa ) was used .", "the reaction medium contained a 50  mm tris - hcl buffer ( ph 7.0 ) , 2.5  l of the enzyme , and solubilized linoleic acid .", "total rna was isolated from the rat liver and spleen using rnazol rt ( sigma - aldrich ) and converted into complementary dna ( cdna ) using the primescript rt reagent kit ( takara ) following the protocols of the manufacturers .", "amplification and detection of cdna of reference and target genes were performed on a 7300 real - time pcr system ( applied biosystems ) using hot firepol evagreen qpcr mix plus ( rox ) ( solis biodyne ) .", "relative mrna expressions of il-1 , tnf- , and inos were analyzed using the ct value method .", "the sequences of the primers were designed and checked using primer3 and oligo analyzer 1.0.3 ( table 2 ) .", "mean and sem values were calculated for each parameter in each group ( 810 animals in each experimental group ) .", "statistically significant differences among treated , untreated , and control groups were tested using parametric analysis of variance ( anova ) .", "post hoc tests ( tukey - kramer ( anova ) ) were applied in situations where differences among groups were significant at the level of significance  = 0.05 .", "after post hoc testing , the following significance levels were specified : extremely significant ( p < 0.001 ) , highly significant ( p < 0.01 ) , significant ( p < 0.05 ) , and not significant ( p > 0.05 ) .", "antioxidant properties of polyphenols including n - f-5ht have been reported [ 21 , 40 , 41 ] .", "nevertheless , the n - f-5ht impact on chronic inflammatory and os - inducing arthritis , which could widen the possibilities of the ra therapy , remains to be elucidated . in our previous study in the model of aa , n - f-5ht in the dosage of 15  mg / kg markedly potentiated the therapeutic effect of low - dose ( nontherapeutic dose ) mtx ( 0.3  mg / kg ) on arthritic ( hind paw volume and arthritic score ) and inflammatory parameters ( il-17 , mcp-1 , and crp ) , yet it resulted in insignificant effect in monotherapy . as data about the optimal n - f-5ht dose in the rat model", "are scarce , we decided to study two doses of n - f-5ht : ( i ) when 15  mg / kg exceeded the physiologically acceptable concentration , we used 3  mg / kg , and ( ii ) when 15  mg / kg was too low to reach the maximal effect , we used 30  mg / kg .", "unexpectedly , contrary to the lower dose of n - f-5ht , the higher dose exhibited minor effect on the parameters examined and/or these varied strongly among the animals .", "for this reason , this report shows only the data evaluating the lower dose of n - f-5ht . in this study", ", we used the therapeutic dose of mtx ( 0.4  mg / kg ) with the intention to compare each mechanism of action of mtx and n - f-5ht , both evaluated in monotherapy .", "the significant rise in arthritic parameters , arthritic score , and hpv confirmed the arthritis in our model in rats .", "the arthritic score showed an increase in the untreated arthritic group compared to the control group on all days monitored ( aa versus co , day 14 , p < 0.01 ; day 21 and day 28 , p < 0.001 ; table 3 ) . at the end of the experiment ,", "the arthritic score was almost doubled in the aa group compared to controls . a trend toward reduction", "was observed after administration of n - f-5ht to aa animals on day 28 , but the effect was not statistically significant .", "the treatment with mtx significantly reduced the arthritic score on observation days 21 and 28 , compared to the untreated arthritic group , proving the therapeutic potential of the applied dose of mtx ( aa - mtx versus aa , day 21 , p < 0.05 ; day 28 , p < 0.01 ; table 3 ) .", "similarly , the change in hpv showed an increase in the untreated arthritic group compared to the control group on days 21 and 28 ( aa versus co , day 21 , p < 0.01 ; day 28 , p < 0.05 ; table 3 ) .", "the administration of n - f-5ht induced no modification of hpv of the arthritic animals on any day monitored .", "mtx therapy significantly reduced the observed swelling on days 21 and 28 compared to the untreated arthritic group ( aa - mtx versus aa , day 21 and day 28 , p < 0.001 ; table 3 ) .", "the muscle wasting condition due to high catabolic activity , known as rheumatoid cachexia , occurring in approximately two - thirds of all patients with ra , is mediated by tnf- and il-1 in ra .", "papers published over the past years confirmed that oxidative metabolism was considerably enhanced in the liver of adjuvant - induced arthritis in rats [ 4346 ] .", "rats used in this study revealed signs of cachexia ( table 3 ) . a significant decrease in body weight change ( bwc )", "the bwc of the arthritic rats was 56% on day 14 , 19% on day 21 , and 27% on day 28 ( aa versus co , days 14 , 21 , and 28 , p < 0.001 ; table 3 ) of the bwc of healthy controls .", "n - f-5ht treatment led to a significant increase of bwc on day 28 ( aa - n - f-5ht versus aa , p < 0.05 ; table 3 ) .", "the administration of n - f-5ht in arthritic animals did not change these parameters on any of the days observed .", "the liver weights were significantly lower ( aa - mtx versus aa , p < 0.05 ; table 3 ) only in the group of rats treated with mtx .", "the reduced weight of the liver in mtx - treated rats was assumed to be the result of inhibition of the pathway of de novo dna synthesis by mtx [ 47 , 48 ] . in summary ,", "the statistical significance of 3  mg / kg of n - f-5ht treatment was determined only for bwc . the arthritic score revealed a trend toward the positive effect increasing with time , indicating a late onset of n - f-5ht action ( table 3 ) .", "as expected , significant differences were found in the arthritic score and hpv in the arthritic animals treated with the therapeutic dose of 0.4  mg / kg mtx compared to those treated with the low dose of 0.3  mg / kg mtx .", "il-1 , a prototypic proinflammatory cytokine , is a major mediator of the inflammatory cascade in ra , which is involved in the mechanisms leading to progressive joint destruction . in the model of aa ,", "the early phases of the disease seem to be characterized by a systemic increase of il-1 .", "the plasmatic level of il-1 , a protein of multiorgan origin , was significantly increased in arthritic animals compared to the control group in the early phase of aa , on day 14 ( aa versus co , p < 0.001 ; figure 1(a ) ) , ascertaining the presence of inflammation .", "administration of mtx did not lead to a significant change of plasmatic il-1 concentration ; only a trend toward reduction was observed on day 14 .", "it is noteworthy that n - f-5ht treatment resulted in a significant decrease of il-1 level in plasma ( aa - n - f-5ht versus aa , p < 0.05 ; figure 1(a ) ) .", "this result is interesting , as this molecule was reported to be relevant in driving the transition from the acute phase to the chronic irreversible phase of the disease and it has been suggested that it could be the target of early intervention to stop the course toward the chronic form of the disease .", "the blocking il-1 protects bone and cartilage from progressive destruction in ra and its inhibition could be effective in the treatment of this disease .", "activation of t and b cells , macrophages , and inflammatory mediators tnf- , il-1 , and il-6 aggravates the oxidative damage of the vital organs in rheumatoid arthritis , such as the liver .", "the liver , in turn , influences the systemic inflammation via producing inflammatory cytokines and mediators such as tnf- , il-1 , il-6 , no , crp , and lox .", "il-6 , il-1 , and tnf- promote the synthesis of crp in hepatocytes via stat3 [ 51 , 52 ] and nf-b   pathways .", "the level of the systemic inflammatory parameter crp in plasma , resulting from liver synthesis , was increased significantly in the group of arthritic animals compared with control animals in the chronic phase of the disease on experimental day 28 ( aa versus co , p < 0.001 ; figure 1(b ) ) .", "administration of n - f-5ht and mtx significantly reduced the plasmatic levels of crp on day 28 compared to the untreated group of arthritic animals ( aa - n - f-5ht versus aa , p < 0.05 ; aa - mtx versus aa , p < 0.05 ; figure 1(b ) ) .", "interaction of crp with fc - gamma receptors ( fcr ) fcri and fcriia is known to promote the production of proinflammatory cytokines , resulting in the amplification loop of inflammatory reaction .", "these processes are initiated through the induction of the receptor activator of nuclear factor-b ligand ( rankl ) protein and direct stimulation of osteoclastogenesis , causing a loop between inflammation and bone destruction in ra .", "crp enhances both the proinflammatory response and bone destruction . in the treatment of ra , a lowered crp level thus not only is a significant parameter in terms of disease progression elimination but also has a direct impact on decreasing the degree of bone destruction .", "alterations in the oxidative state lead to the activation of nf-b and nf-b - dependent genes , such as lox .", "the enzyme 5-lox catalyzes the conversion of arachidonic acid to leukotrienes , whose production has been associated with inflammation in arthritis .", "suppression of 5-lox expression ameliorates clinical parameters in ra and aa [ 56 , 57 ] .", "increased levels of nf-b in the lung and liver as well as increased activity of lox in the lung highlight the importance of extra - articular manifestations of aa . in our experiment ,", "liver 12/15 lox activity increased in arthritic animals in comparison to healthy animals ( aa versus co , p < 0.001 ; figure 1(c ) ) .", "the effect of n - f-5ht on the activity of 12/15-lox in liver homogenate was comparable with that of mtx .", "after administration of mtx or n - f-5ht , a significant decrease to control levels was assessed in the liver of the aa group ( aa - n - f-5ht versus aa , p < 0.001 ; aa - mtx versus aa , p < 0.001 ; figure 1(c ) ) .", "thus the anti - inflammatory effect of n - f-5ht in aa was supported by the ability of the molecule to inhibit 12/15-lox activity . similar to this result , recent observations also reported that several other flavonoids may act as lox inhibitors . in aa ,", "the gene expression levels of tnf- and inos produced in the liver were reported to increase [ 60 , 61 ] .", "also , in our study , the levels of tnf- and inos mrna expressions were significantly increased in arthritic animals ( both p < 0.001 , aa versus co ; figures 2(a ) and 2(b ) )", ". it was proposed that these modifications in the liver of arthritic rats not only were a consequence of the metabolic alterations caused by the disease , especially the increased oxidative metabolism , but also depended on increased inflammatory parameters in the liver .", "the same agents that increase oxidative metabolism , tnf- , il-1 , il-6 , and others , are responsible for increasing the activity of inos in several tissues .", "an increase of inos activity as a consequence of elevated inos mrna expression was considered to play a dominant role in the pathogenesis of ra .", "no generation by inos induced in chondrocytes in the initial stage of aa may play a key role in triggering the subsequent events in arthritis . in general , the use of nos inhibitors has been shown to exert beneficial effects in experimentally induced arthritis . however ,", "which types of cells expressing inos are associated with the induction or progression of adjuvant - induced arthritis via no generation remains uncertain .", "mrna expression of inos in rat liver was reduced following mtx ( aa - mtx versus aa , p < 0.001 ; figure 2(a ) ) and n - f-5ht treatment ( aa - n - f-5ht versus aa , p < 0.01 ; figure 2(a ) ) .", "the effect of mtx treatment on tnf- protein and mrna expression differs among studies , depending on the conditions of the given study , concerning gender of patients , type of cell line , duration of treatment , mtx dose , and so forth . in our study in the rat aa model , administration of mtx", "attenuated significantly the mrna expression of tnf- ( aa - mtx versus aa , p < 0.01 ; figure 2(b ) ) . in many patients , however , mtx treatment does not result in lower tnf- plasma concentration .", "when mtx fails to produce an adequate response , newer therapies are used in combination with mtx .", "blocking tnf- with anti - tnf- monoclonal antibodies significantly decreased the signs and symptoms of ra compared to placebo in ra patients with active disease receiving mtx [ 65 , 66 ] .", "thus , the n - f-5ht - driven significant reduction of tnf- mrna expression ( a - n - f-5ht versus aa , p < 0.01 ; figure 2(b ) ) suggests an intriguing effect on ra treatment , calling for deeper investigation .", "increase of mrna expression was observed for il-1 in the liver of arthritic animals ( aa versus co , p < 0.001 ; figure 3(a ) ) as expected .", "administration of mtx did not lead to significant attenuation of il-1 transcription in the liver .", "this is in concert with previous studies of mtx function in different types of cells ( e.g. , human peripheral blood mononuclear cells and murine peritoneal and splenic cells ) [ 67 , 68 ] . on the other hand", ", mtx exhibits another mechanism of il-1 function inhibition , which involves blocking the binding of il-1 to il-1 receptor in the membrane of peripheral blood cells ( monocytes , lymphocytes , and granulocytes ) .", "contrary to mtx , treatment with n - f-5ht led to a substantial inhibition of il-1 gene expression ( aa - n - f-5ht versus aa , p < 0.01 ; figure 3(a ) ) .", "further , we examined il-1 mrna expression in the main immunocompetent organ , in the rat arthritic spleen , which has not been studied previously in terms of the aa model , related to il-1 expression .", "we observed il-1 mrna expression activation comparable to that in the liver ( aa versus co , p < 0.001 ; figure 3(b ) ) .", "interestingly , both mtx and n - f-5ht exhibited a significant and remarkably stronger inhibition of il-1 mrna expression in comparison to that in the liver ( aa - mtx versus aa , p < 0.01 ; aa - n - f-5ht versus aa , p < 0.001 ; figure 3(b ) ) .", "in the spleen of n - f-5ht treated rats , the relative mrna expression decreased even to control level .", "besides other events , mtx treatment leads to suppression of nf-b , a heterodimer consisting of two subunits p65 and p50 , one of the most prominent inflammatory transcription factors activated in ra .", "this was confirmed in our previous work , along with the finding that also n - f-5ht ( 15  mg / kg ) suppressed the activation of nf-b ( p65 ) in the arthritic rat liver [ 21 , 33 ] .", "interestingly , combination therapy ( mtx + n - f-5ht ) potentiated the effect of a single drug , suggesting different mechanisms leading to nf-b inhibition .", "mtx driven reduction of cytokine transcription was attributed to abrogation of ib kinase activation and thereby suppression of ib ( nf-b inhibitor ) phosphorylation and degradation , resulting in retaining the inactive nf-b form in cytoplasm .", "however , the contribution of n - f-5ht to nf-b pathway suppression needs to be further investigated .", "studies of the proposed pathways involved in the transcription of tnf- , il-1 , and inos in ra could help evaluate the mechanism of action of these drugs [ 68 , 71 , 72 ] .", "the gene expression of inos is mostly under the control of synergistically activating nf-b ( il-1 and tnf- stimulated ) and stat1 ( ifn- stimulated ) key proinflammatory signals in the liver .", "in contrast to inos , tnf- does not contain the stat binding element in its promoter region .", "the inhibition of tnf- and inos transcription observed in our study might be mostly attributed to the suppressed nf-b pathway for both mtx and n - f-5ht [ 33 , 64 , 70 ] .", "however , the contribution of ap-1 to tnf- and stat1 for inos can not be excluded .", "mtx - dependent suppression of nf-b was reported [ 33 , 70 , 73 , 74 ] , but in other cases mtx was not found to be effective in the attenuation of arthritic - increased mrna expression of il-1 [ 68 , 75 ] . taking into account our results , where mtx treatment did not lead to inhibition of il-1 mrna expression in the arthritic liver in contrast to the significant n - f-5ht impact , yet treatment of both mtx and n - f-5ht decreased the presumably nf-b - dependent lox activity and inos and tnf- transcription to a similar extent , the involvement of n - f-5ht in another pathway for transcription regulation of this cytokine in the arthritic liver should be considered .", "after analysis of the reported pathways involved in the regulation of il-1 mrna expression , we hypothesized that tnf--driven ap-1 transcription factor activation or jak / stat3 pathway activated via il-6 or ifn- might play a role ( [ 7 , 8 , 71 , 72 , 76 , 77 ] , figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7509653 ) .", "papers reporting involvement of other polyphenols in anti - inflammatory regulation , for example , resveratrol , claim that these compounds exhibit their anti - inflammatory effect through suppression of nf-b and jak / stat signaling pathways [ 78 , 79 ] .", "the enhanced influence of mtx and n - f-5ht on il-1 transcription in the spleen in comparison to the liver may be the consequence of different predominance of inflammatory pathways in this organ , presumably with a stronger nf-b contribution .", "details about the relevance of these pathways and the role of n - f-5ht in the transcription regulation of il-1 , inos , and tnf- in the liver and other organs in ra are to be further elucidated .", "the present study contributed additional evidence about the beneficial effect and mechanism of action of n - f-5ht and of mtx on a systemic inflammatory process in the liver and its association with the pathogenesis of adjuvant arthritis .", "n - f-5ht treatment led to amelioration of inflammatory parameters tested ( plasmatic crp and il-1 protein levels , liver lox activity , and liver and spleen cytokine expression ) .", "however , this did not result in a significant change of hpv , although a trend of improvement of the arthritic score was observed after 28 days .", "a synergistic effect of tnf- and il-1 was shown to influence the balance between protein degradation and protein synthesis causing among others an increase in resting energy expenditure and net efflux of amino acids from muscle to liver .", "the significant increase of bwc in n - f-5ht treated rats , probably sign of the partial improvement of rheumatoid cachexia , might be the result of lowered mrna expression of tnf- and il-1 determined in the arthritic liver . moreover , taking into account the reported association of weight loss with the il-1 production by splenic cells , the n - f-5ht mediated attenuation of increased il-1 mrna expression in the arthritic spleen might contribute to this complex process", ". the contribution of the affected expression of tnf- and il-1 originating from other organs can not be excluded and is to be further elucidated .", "unexpectedly , chronic daily treatment with a high concentration of n - f-5ht ( 30  mg / kg ) exhibited either a minor effect on the parameters examined and/or a strong variation among the animals ( not shown ) and that in contrast to a much lower concentration ( 3  mg / kg ) . since n - f-5ht possesses a serotonin ( 5-hydroxytryptamine , 5-ht ) moiety , the question if there might be some interplay between effects of these two molecules on ra pathogenesis is to be raised . since n - f-5ht inhibited the increase of cytosolic free ca concentration in rat vascular smooth muscle cells induced by serotonin mediated by 5-ht2 receptors , it was hypothesized that at a sufficient concentration n - f-5ht may act as a competitive antagonist , which displaces serotonin from its binding site .", "intake of a high concentration of a 5-ht2 receptor antagonist may lead to a variety of effects : it may influence the receptor density , even enhance the effect of serotonin , or lead to desensitization and with time to receptor resistance ( through inhibitory feedback due to binding - induced enhanced production of serotonin ) .", "interestingly , serotonin is known not only as a neurotransmitter . increasing but contradictory reports associate serotonin with immunoinflammatory pathways in the periphery .", "serotonin , via its 5-ht2a , 5-ht2b , and 5-ht3 receptors , has been implicated to have both proinflammatory and anti - inflammatory roles in a number of studies of rheumatoid arthritis [ 8488 ] .", "the reported effects of 5-ht receptor antagonist on macrophage - like synovial cells encourage the interest to study the effect of n - f-5ht from this point of view . to confirm this hypothesis ,", "a precise characterization of interaction between n - f-5ht and 5-ht receptors is to be done . on comparing the effects of the two drugs , administration of mtx ( 0.4 ", "mg / kg ) or n - f-5ht ( 3  mg / kg ) was found to lead to a decrease of the main plasma marker of systemic inflammation crp , the liver origin protein , and to inhibition of proinflammatory lox in the liver .", "the impact of mtx and n - f-5ht on mrna expression of tnf- , il-1 , and inos in the liver and on the level of crp in plasma was mentioned at the conference .", "mtx and n - f-5ht reduced the arthritis - increased transcription of tnf- and inos in the liver to a comparable extent . we suppose that the inhibition of tnf- and inos transcription might be mostly attributed to the suppressed nf-b pathway for the two drugs [ 21 , 33 , 70 ] .", "as previously reported [ 67 , 68 ] and also proven by our study , mtx was not able to diminish the arthritic - induced il-1 mrna transcription in the liver .", "this handicap might be compensated by coadministration of n - f-5ht , since this drug was shown to lower the level of proinflammatory cytokine il-1 in plasma in the acute phase of aa and to attenuate significantly the elevation of il-1 mrna expression in the arthritic rat liver and spleen in the chronic phase .", "detailed studies are required to confirm the hypothesis that n - f-5ht might function through potentially different mechanisms of inhibition of the inflammatory pathway nf-b and not through mtx , as well as the possibility of an additional pathway influencing il-1 transcription under control of n - f-5ht but not mtx .", "the confirmation would support n - f-5ht as a promising agent for the treatment of ra in combination therapy with mtx .", "the positive effect was shown in our previous study , where n - f-5ht markedly potentiated the therapeutic effect of low - dose mtx .", "as the therapeutic dose of mtx was used in this study and the purpose of combination study is to lower the mtx dose to decrease the side effects of this drug , the effect of combination therapy was not included", ". oral daily intake of n - f-5ht could overcome the inconvenient administration and high costs of biological therapy using il-1 monoclonal antibody , which was shown in clinical trials to be superior to placebo in combination with mtx in reducing signs , symptoms , and radiographic progression in patients with advanced ra [ 91 , 92 ] .", "future studies of n - f-5ht mechanisms of action should shed more light on the immunomodulatory function of this natural polyphenol .", "it is to be expected that n - f-5ht is able to positively affect the activity of other markers of inflammation and oxidative stress not only in the liver and spleen but also in other organs ( lung , brain , etc . ) , a hypothesis to be tested by future work .", "however , to establish the optimal dosing in light of the effects achieved is of primary importance ."], "abstract_text": ["<S> rheumatoid arthritis ( ra ) is a chronic inflammatory disease , leading to progressive destruction of joints and extra - articular tissues , including organs such as liver and spleen . </S>", "<S> the purpose of this study was to compare the effects of a potential immunomodulator , natural polyphenol n - feruloylserotonin ( n - f-5ht ) , with methotrexate ( mtx ) , the standard in ra therapy , in the chronic phase of adjuvant - induced arthritis ( aa ) in male lewis rats . </S>", "<S> the experiment included healthy controls ( co ) , arthritic animals ( aa ) , aa given n - f-5ht ( aa - n - f-5ht ) , and aa given mtx ( aa - mtx ) . </S>", "<S> n - f-5ht did not affect the body weight change and clinical parameters until the 14th experimental day . </S>", "<S> its positive effect was rising during the 28-day experiment , indicating a delayed onset of n - f-5ht action . </S>", "<S> administration of either n - f-5ht or mtx caused reduction of inflammation measured as the level of crp in plasma and the activity of lox in the liver . </S>", "<S> mrna transcription of tnf- and inos in the liver was significantly attenuated in both mtx and n - f-5ht treated groups of arthritic rats . </S>", "<S> interestingly , in contrast to mtx , n - f-5ht significantly lowered the level of il-1 in plasma and il-1 mrna expression in the liver and spleen of arthritic rats . </S>", "<S> this speaks for future investigations of n - f-5ht as an agent in the treatment of ra in combination therapy with mtx . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusions"], "sections": [["rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disease affecting predominantly joints , synovial membranes , articular cartilages , and subchondral bones .", "disease progression is attributed to increases in reactive oxygen species ( ros ) and oxidative stress ( os ) in the lesion sites .", "proinflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and il-6 , regulate the inflammatory and immune responses and play a pivotal role in the disease .", "overproduction of nitric oxide ( no ) , as a result of induction of inducible nitric oxide synthase ( inos ) due to enhanced production of these cytokines , is associated with persistent inflammation and tissue destruction in experimental arthritis models , including rheumatoid arthritis [ 4 , 5 ] . a number of inflammation stimuli , including tnf- , il-1 , il-6 , or ros , can activate proinflammatory pathways involved in ra pathogenesis , concerning predominantly nuclear factor-b ( nf-b ) , mitogen activated protein kinases ( mapks ) , or janus kinases / signal transducers and activators of transcription ( jak / stat1/3 ) [ 68 ] .", "this results in translocation of relevant downstream transcription factors from the cytoplasm to the nucleus , where they activate messenger rna ( mrna ) expression of target genes , including il-1 , tnf- , inos , and 12/15-lipoxygenase ( lox ) , leading to overproduction of corresponding proteins .", "cytokines released into the synovium reach also the systemic circulation and act in other tissues and organs such as lungs , vascular tissue , liver , and heart . several recent investigations reported damage of vital organs with various degrees of impairment , considered to be secondary complications of ra and a major predictor of mortality in ra patients .", "increasing evidence is pointing to the critical role of the liver in modulating the immune response in autoimmune and chronic inflammatory diseases including ra [ 5 , 11 , 12 ] .", "the hepatic biochemical and immunological alterations are associated with and influenced by changes in the oxidative state of liver cells .", "adjuvant - induced arthritis ( aa ) in rats not only is an experimental model of polyarthritis but also induces pathological changes in a variety of other tissues , including the liver and spleen .", "it is a useful tool to study immunopathologic processes , autoimmune chronic inflammation , and inflammatory cachexia in rodents .", "in addition , at the molecular level , mrna profiling suggests that this model is also similar to human ra , particularly in tissue gene expression and in the activation of regulatory pathways [ 11 , 14 ] .", "numerous studies reported natural polyphenols as potential therapeutic agents of diseases caused by os and inflammation [ 1517 ] .", "n - feruloylserotonin ( n - f-5ht , n - feruloyl-5-hydroxy - tryptamine ) is a conjugated serotonin , a member of the indole hydroxycinnamic acid amides , with serotonin ( 5-ht ) and ferulic acid ( fa ) as representative components of its structure .", "hydroxycinnamic acid amides of serotonin , synthesized by serotonin n - hydroxycinnamoyltransferase , are present in several vegetables   and wild - growing plants whose seeds are used in herbal medicine in eastern countries [ 1820 ] . in cell - based studies , under short - term high - glucose conditions ,", "n - f-5ht exerted an inhibitory effect on overproduction of mitochondrial superoxide by acting as scavenger of superoxide .", "n - f-5ht attenuated the upregulation of mrna and proteins of ros - dependent adhesion ( vascular cell adhesion protein-1 ( vcam-1 ) ) and migration factors ( monocyte chemoattractant protein-1 ( mcp-1 ) ) , crucial in early atherosclerosis lesions in human aortic endothelial cells , and inhibited the activation of transcription factor nf-b .", "furthermore , n - f-5ht showed a protective effect on ros - related neuronal damage by decreasing the activity of proapoptotic caspase-3 .", "n - f-5ht isomers isolated from seeds of leuzea carthamoides were shown to inhibit protein kinase c / ii activation and decrease the oxidative burst of human whole blood and isolated neutrophils in vitro .", "n - f-5ht was also found to have a protective effect against ldl oxidation and atherogenesis in experimental animals and in human studies [ 2426 ] .", "methotrexate ( mtx ) , used as a standard drug in our study , represents the most frequently used pharmacotherapy of ra in clinical practice .", "its administration is , however , limited due to its toxic side effects [ 27 , 28 ] .", "yet application of a combination therapy of mtx with other potential immunomodulators , synthetic drugs   or natural substances [ 3032 ] , might elevate the therapeutic efficacy : decrease the dose of mtx and thus its side effects . in our previous study , we showed that administration of n - f-5ht to mtx - treated arthritic rats lowered the dose of mtx for the required sustained antirheumatic impact . in this study , we focused on the therapeutic impact of n - f-5ht and mtx administered in monotherapy and on details of the inflammatory state in the arthritic rat liver with the aim to elucidate the molecular mechanisms of their effect .", "one of the possible clarifying approaches is to study the mrna expression of key proinflammatory markers ( il-1 , tnf- , and inos ) in the liver of treated and untreated arthritic rats .", "further , it is of particular interest to expand our knowledge on the effect of n - f-5ht and mtx in the aa model , which in turn should allow extrapolations of these results to ra patients .", "to this aim we evaluated also conventional arthritic parameters ( hpv , arthritic score , body weight change , and weight of the liver ) along with changes in plasmatic levels of il-1 and crp and the activity of 12/15-lox in the liver ."], ["adult male lewis rats weighing 160180  g were obtained from charles river wiga , germany .", "the experimental protocol was approved by the ethics committee of the institute of experimental pharmacology and toxicology and by the slovak state veterinary and food administration in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes and was in line with slovak legislation . to induce a rat model of adjuvant arthritis ( aa ) ,", "rats were intradermally injected with a suspension of heat - inactivated mycobacterium butyricum in incomplete freund 's adjuvant ( difco laboratories , detroit , mi , usa ) .", "the third group comprised adjuvant arthritis rats treated with methotrexate ( methotrexat ebewe sol inj 20  mg/2.0  ml ) in oral dose of 0.4  mg / kg twice a week ( aa - mtx ) . \n", "the fourth group comprised adjuvant arthritis rats treated with n - feruloylserotonin dissolved in suspension of methylcellulose tween 80 at a dose of 3  mg / kg / day orally ( aa - n - f-5ht ) .", "drugs were administered orally by gastric gavage from day 0 ( the day of treatment ) to day 28 of the study .", "blood for plasma preparation was taken by retroorbital puncture on day 14 and by cardiac puncture on day 28 under deep ketamine / xylazine anesthesia .", "after the animals had been sacrificed under deep ketamine / xylazine anesthesia , tissues for liver and spleen homogenate preparation were taken at the end of the experiment ( day 28 ) .", "blood in heparinized tubes for plasma preparation was centrifuged at 3000  rpm for 15 minutes at 4c .", "fraction of four isomers of n - f-5ht ( table 1 ) was isolated from the seeds of leuzea carthamoides ( wild ) dc by solvent extraction .", "this was then followed by column chromatography on silica gel and hplc separations under conditions previously reported [ 35 , 36 ] .", "the hind paw volume ( hpv ) was recorded on days 14 , 21 , and 28 with the use of an electronic water plethysmometer ( ugo basile , comerio , varese , italy ) .", "calculation of the increase in hind paw volume in ml assessed the intensity of the edema .", "the arthritic score was measured as the total score of hpv ( ml , max .", "points 5 ) + diameter of scab in the site of mb application , measured in parallel to the spinal column ( mm , max .", "body weight change ( bwc ; g ) was measured on days 1 , 14 , 21 , and 28 .", "bwc was calculated as the difference of the body mass measured on days 14 , 21 , and 28 to the body weight measured at the beginning of the experiment ( day 1 ) . for the determination of rat crp concentration in plasma ( g / ml ) , the elisa kit from immunology consultant laboratories , inc .", "the reaction of secondary biotin - conjugated anti - rat crp antibody was evaluated by streptavidin - hrp .", "the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 450  nm ( microplate reader , labsystems multiskan rc ) .", "for the determination of il-1 concentration in plasma , the elisa kit from r&d systems quantikine was used .", "rat cytokine present in the samples binds to anti - rat cytokine antibodies absorbed in the microwells .", "the reaction of secondary biotin - conjugated anti - rat cytokine antibody is evaluated by hrp .", "the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 490  nm in comparison with the reference wavelength of 620  nm ( microplate reader mrx ii ) .", "concentration of proteins in liver homogenates was determined by using the bradford method   and expressed in mg / ml of enzyme preparation ( cytosolic fraction from rat lung and liver tissues ) .", "linoleic acid ( 99% , sigma - aldrich , usa ) was used as a substrate prepared in solubilized state as described   in the concentration of 0.2143  100.7143  10  m. the assay of lox was monitored for 60 seconds as an increase in the absorbance at 234  nm , reflecting the formation of hydroperoxylinoleic acid . for the lox activity assay ,", "an uv / vis spectrometer perkin - elmer lambda 35 ( usa ) was used .", "the reaction medium contained a 50  mm tris - hcl buffer ( ph 7.0 ) , 2.5  l of the enzyme , and solubilized linoleic acid .", "total rna was isolated from the rat liver and spleen using rnazol rt ( sigma - aldrich ) and converted into complementary dna ( cdna ) using the primescript rt reagent kit ( takara ) following the protocols of the manufacturers .", "amplification and detection of cdna of reference and target genes were performed on a 7300 real - time pcr system ( applied biosystems ) using hot firepol evagreen qpcr mix plus ( rox ) ( solis biodyne ) .", "relative mrna expressions of il-1 , tnf- , and inos were analyzed using the ct value method .", "the sequences of the primers were designed and checked using primer3 and oligo analyzer 1.0.3 ( table 2 ) .", "mean and sem values were calculated for each parameter in each group ( 810 animals in each experimental group ) .", "statistically significant differences among treated , untreated , and control groups were tested using parametric analysis of variance ( anova ) .", "post hoc tests ( tukey - kramer ( anova ) ) were applied in situations where differences among groups were significant at the level of significance  = 0.05 .", "after post hoc testing , the following significance levels were specified : extremely significant ( p < 0.001 ) , highly significant ( p < 0.01 ) , significant ( p < 0.05 ) , and not significant ( p > 0.05 ) ."], ["antioxidant properties of polyphenols including n - f-5ht have been reported [ 21 , 40 , 41 ] .", "nevertheless , the n - f-5ht impact on chronic inflammatory and os - inducing arthritis , which could widen the possibilities of the ra therapy , remains to be elucidated . in our previous study in the model of aa , n - f-5ht in the dosage of 15  mg / kg markedly potentiated the therapeutic effect of low - dose ( nontherapeutic dose ) mtx ( 0.3  mg / kg ) on arthritic ( hind paw volume and arthritic score ) and inflammatory parameters ( il-17 , mcp-1 , and crp ) , yet it resulted in insignificant effect in monotherapy . as data about the optimal n - f-5ht dose in the rat model", "are scarce , we decided to study two doses of n - f-5ht : ( i ) when 15  mg / kg exceeded the physiologically acceptable concentration , we used 3  mg / kg , and ( ii ) when 15  mg / kg was too low to reach the maximal effect , we used 30  mg / kg .", "unexpectedly , contrary to the lower dose of n - f-5ht , the higher dose exhibited minor effect on the parameters examined and/or these varied strongly among the animals .", "for this reason , this report shows only the data evaluating the lower dose of n - f-5ht . in this study", ", we used the therapeutic dose of mtx ( 0.4  mg / kg ) with the intention to compare each mechanism of action of mtx and n - f-5ht , both evaluated in monotherapy .", "the significant rise in arthritic parameters , arthritic score , and hpv confirmed the arthritis in our model in rats .", "the arthritic score showed an increase in the untreated arthritic group compared to the control group on all days monitored ( aa versus co , day 14 , p < 0.01 ; day 21 and day 28 , p < 0.001 ; table 3 ) . at the end of the experiment ,", "the arthritic score was almost doubled in the aa group compared to controls . a trend toward reduction", "was observed after administration of n - f-5ht to aa animals on day 28 , but the effect was not statistically significant .", "the treatment with mtx significantly reduced the arthritic score on observation days 21 and 28 , compared to the untreated arthritic group , proving the therapeutic potential of the applied dose of mtx ( aa - mtx versus aa , day 21 , p < 0.05 ; day 28 , p < 0.01 ; table 3 ) .", "similarly , the change in hpv showed an increase in the untreated arthritic group compared to the control group on days 21 and 28 ( aa versus co , day 21 , p < 0.01 ; day 28 , p < 0.05 ; table 3 ) .", "the administration of n - f-5ht induced no modification of hpv of the arthritic animals on any day monitored .", "mtx therapy significantly reduced the observed swelling on days 21 and 28 compared to the untreated arthritic group ( aa - mtx versus aa , day 21 and day 28 , p < 0.001 ; table 3 ) .", "the muscle wasting condition due to high catabolic activity , known as rheumatoid cachexia , occurring in approximately two - thirds of all patients with ra , is mediated by tnf- and il-1 in ra .", "papers published over the past years confirmed that oxidative metabolism was considerably enhanced in the liver of adjuvant - induced arthritis in rats [ 4346 ] .", "rats used in this study revealed signs of cachexia ( table 3 ) . a significant decrease in body weight change ( bwc )", "the bwc of the arthritic rats was 56% on day 14 , 19% on day 21 , and 27% on day 28 ( aa versus co , days 14 , 21 , and 28 , p < 0.001 ; table 3 ) of the bwc of healthy controls .", "n - f-5ht treatment led to a significant increase of bwc on day 28 ( aa - n - f-5ht versus aa , p < 0.05 ; table 3 ) .", "the administration of n - f-5ht in arthritic animals did not change these parameters on any of the days observed .", "the liver weights were significantly lower ( aa - mtx versus aa , p < 0.05 ; table 3 ) only in the group of rats treated with mtx .", "the reduced weight of the liver in mtx - treated rats was assumed to be the result of inhibition of the pathway of de novo dna synthesis by mtx [ 47 , 48 ] . in summary ,", "the statistical significance of 3  mg / kg of n - f-5ht treatment was determined only for bwc . the arthritic score revealed a trend toward the positive effect increasing with time , indicating a late onset of n - f-5ht action ( table 3 ) .", "as expected , significant differences were found in the arthritic score and hpv in the arthritic animals treated with the therapeutic dose of 0.4  mg / kg mtx compared to those treated with the low dose of 0.3  mg / kg mtx .", "il-1 , a prototypic proinflammatory cytokine , is a major mediator of the inflammatory cascade in ra , which is involved in the mechanisms leading to progressive joint destruction . in the model of aa ,", "the early phases of the disease seem to be characterized by a systemic increase of il-1 .", "the plasmatic level of il-1 , a protein of multiorgan origin , was significantly increased in arthritic animals compared to the control group in the early phase of aa , on day 14 ( aa versus co , p < 0.001 ; figure 1(a ) ) , ascertaining the presence of inflammation .", "administration of mtx did not lead to a significant change of plasmatic il-1 concentration ; only a trend toward reduction was observed on day 14 .", "it is noteworthy that n - f-5ht treatment resulted in a significant decrease of il-1 level in plasma ( aa - n - f-5ht versus aa , p < 0.05 ; figure 1(a ) ) .", "this result is interesting , as this molecule was reported to be relevant in driving the transition from the acute phase to the chronic irreversible phase of the disease and it has been suggested that it could be the target of early intervention to stop the course toward the chronic form of the disease .", "the blocking il-1 protects bone and cartilage from progressive destruction in ra and its inhibition could be effective in the treatment of this disease .", "activation of t and b cells , macrophages , and inflammatory mediators tnf- , il-1 , and il-6 aggravates the oxidative damage of the vital organs in rheumatoid arthritis , such as the liver .", "the liver , in turn , influences the systemic inflammation via producing inflammatory cytokines and mediators such as tnf- , il-1 , il-6 , no , crp , and lox .", "il-6 , il-1 , and tnf- promote the synthesis of crp in hepatocytes via stat3 [ 51 , 52 ] and nf-b   pathways .", "the level of the systemic inflammatory parameter crp in plasma , resulting from liver synthesis , was increased significantly in the group of arthritic animals compared with control animals in the chronic phase of the disease on experimental day 28 ( aa versus co , p < 0.001 ; figure 1(b ) ) .", "administration of n - f-5ht and mtx significantly reduced the plasmatic levels of crp on day 28 compared to the untreated group of arthritic animals ( aa - n - f-5ht versus aa , p < 0.05 ; aa - mtx versus aa , p < 0.05 ; figure 1(b ) ) .", "interaction of crp with fc - gamma receptors ( fcr ) fcri and fcriia is known to promote the production of proinflammatory cytokines , resulting in the amplification loop of inflammatory reaction .", "these processes are initiated through the induction of the receptor activator of nuclear factor-b ligand ( rankl ) protein and direct stimulation of osteoclastogenesis , causing a loop between inflammation and bone destruction in ra .", "crp enhances both the proinflammatory response and bone destruction . in the treatment of ra , a lowered crp level thus not only is a significant parameter in terms of disease progression elimination but also has a direct impact on decreasing the degree of bone destruction .", "alterations in the oxidative state lead to the activation of nf-b and nf-b - dependent genes , such as lox .", "the enzyme 5-lox catalyzes the conversion of arachidonic acid to leukotrienes , whose production has been associated with inflammation in arthritis .", "suppression of 5-lox expression ameliorates clinical parameters in ra and aa [ 56 , 57 ] .", "increased levels of nf-b in the lung and liver as well as increased activity of lox in the lung highlight the importance of extra - articular manifestations of aa . in our experiment ,", "liver 12/15 lox activity increased in arthritic animals in comparison to healthy animals ( aa versus co , p < 0.001 ; figure 1(c ) ) .", "the effect of n - f-5ht on the activity of 12/15-lox in liver homogenate was comparable with that of mtx .", "after administration of mtx or n - f-5ht , a significant decrease to control levels was assessed in the liver of the aa group ( aa - n - f-5ht versus aa , p < 0.001 ; aa - mtx versus aa , p < 0.001 ; figure 1(c ) ) .", "thus the anti - inflammatory effect of n - f-5ht in aa was supported by the ability of the molecule to inhibit 12/15-lox activity . similar to this result , recent observations also reported that several other flavonoids may act as lox inhibitors . in aa ,", "the gene expression levels of tnf- and inos produced in the liver were reported to increase [ 60 , 61 ] .", "also , in our study , the levels of tnf- and inos mrna expressions were significantly increased in arthritic animals ( both p < 0.001 , aa versus co ; figures 2(a ) and 2(b ) )", ". it was proposed that these modifications in the liver of arthritic rats not only were a consequence of the metabolic alterations caused by the disease , especially the increased oxidative metabolism , but also depended on increased inflammatory parameters in the liver .", "the same agents that increase oxidative metabolism , tnf- , il-1 , il-6 , and others , are responsible for increasing the activity of inos in several tissues .", "an increase of inos activity as a consequence of elevated inos mrna expression was considered to play a dominant role in the pathogenesis of ra .", "no generation by inos induced in chondrocytes in the initial stage of aa may play a key role in triggering the subsequent events in arthritis . in general , the use of nos inhibitors has been shown to exert beneficial effects in experimentally induced arthritis . however ,", "which types of cells expressing inos are associated with the induction or progression of adjuvant - induced arthritis via no generation remains uncertain .", "mrna expression of inos in rat liver was reduced following mtx ( aa - mtx versus aa , p < 0.001 ; figure 2(a ) ) and n - f-5ht treatment ( aa - n - f-5ht versus aa , p < 0.01 ; figure 2(a ) ) .", "the effect of mtx treatment on tnf- protein and mrna expression differs among studies , depending on the conditions of the given study , concerning gender of patients , type of cell line , duration of treatment , mtx dose , and so forth . in our study in the rat aa model , administration of mtx", "attenuated significantly the mrna expression of tnf- ( aa - mtx versus aa , p < 0.01 ; figure 2(b ) ) . in many patients , however , mtx treatment does not result in lower tnf- plasma concentration .", "when mtx fails to produce an adequate response , newer therapies are used in combination with mtx .", "blocking tnf- with anti - tnf- monoclonal antibodies significantly decreased the signs and symptoms of ra compared to placebo in ra patients with active disease receiving mtx [ 65 , 66 ] .", "thus , the n - f-5ht - driven significant reduction of tnf- mrna expression ( a - n - f-5ht versus aa , p < 0.01 ; figure 2(b ) ) suggests an intriguing effect on ra treatment , calling for deeper investigation .", "increase of mrna expression was observed for il-1 in the liver of arthritic animals ( aa versus co , p < 0.001 ; figure 3(a ) ) as expected .", "administration of mtx did not lead to significant attenuation of il-1 transcription in the liver .", "this is in concert with previous studies of mtx function in different types of cells ( e.g. , human peripheral blood mononuclear cells and murine peritoneal and splenic cells ) [ 67 , 68 ] . on the other hand", ", mtx exhibits another mechanism of il-1 function inhibition , which involves blocking the binding of il-1 to il-1 receptor in the membrane of peripheral blood cells ( monocytes , lymphocytes , and granulocytes ) .", "contrary to mtx , treatment with n - f-5ht led to a substantial inhibition of il-1 gene expression ( aa - n - f-5ht versus aa , p < 0.01 ; figure 3(a ) ) .", "further , we examined il-1 mrna expression in the main immunocompetent organ , in the rat arthritic spleen , which has not been studied previously in terms of the aa model , related to il-1 expression .", "we observed il-1 mrna expression activation comparable to that in the liver ( aa versus co , p < 0.001 ; figure 3(b ) ) .", "interestingly , both mtx and n - f-5ht exhibited a significant and remarkably stronger inhibition of il-1 mrna expression in comparison to that in the liver ( aa - mtx versus aa , p < 0.01 ; aa - n - f-5ht versus aa , p < 0.001 ; figure 3(b ) ) .", "in the spleen of n - f-5ht treated rats , the relative mrna expression decreased even to control level .", "besides other events , mtx treatment leads to suppression of nf-b , a heterodimer consisting of two subunits p65 and p50 , one of the most prominent inflammatory transcription factors activated in ra .", "this was confirmed in our previous work , along with the finding that also n - f-5ht ( 15  mg / kg ) suppressed the activation of nf-b ( p65 ) in the arthritic rat liver [ 21 , 33 ] .", "interestingly , combination therapy ( mtx + n - f-5ht ) potentiated the effect of a single drug , suggesting different mechanisms leading to nf-b inhibition .", "mtx driven reduction of cytokine transcription was attributed to abrogation of ib kinase activation and thereby suppression of ib ( nf-b inhibitor ) phosphorylation and degradation , resulting in retaining the inactive nf-b form in cytoplasm .", "however , the contribution of n - f-5ht to nf-b pathway suppression needs to be further investigated .", "studies of the proposed pathways involved in the transcription of tnf- , il-1 , and inos in ra could help evaluate the mechanism of action of these drugs [ 68 , 71 , 72 ] .", "the gene expression of inos is mostly under the control of synergistically activating nf-b ( il-1 and tnf- stimulated ) and stat1 ( ifn- stimulated ) key proinflammatory signals in the liver .", "in contrast to inos , tnf- does not contain the stat binding element in its promoter region .", "the inhibition of tnf- and inos transcription observed in our study might be mostly attributed to the suppressed nf-b pathway for both mtx and n - f-5ht [ 33 , 64 , 70 ] .", "however , the contribution of ap-1 to tnf- and stat1 for inos can not be excluded .", "mtx - dependent suppression of nf-b was reported [ 33 , 70 , 73 , 74 ] , but in other cases mtx was not found to be effective in the attenuation of arthritic - increased mrna expression of il-1 [ 68 , 75 ] . taking into account our results , where mtx treatment did not lead to inhibition of il-1 mrna expression in the arthritic liver in contrast to the significant n - f-5ht impact , yet treatment of both mtx and n - f-5ht decreased the presumably nf-b - dependent lox activity and inos and tnf- transcription to a similar extent , the involvement of n - f-5ht in another pathway for transcription regulation of this cytokine in the arthritic liver should be considered .", "after analysis of the reported pathways involved in the regulation of il-1 mrna expression , we hypothesized that tnf--driven ap-1 transcription factor activation or jak / stat3 pathway activated via il-6 or ifn- might play a role ( [ 7 , 8 , 71 , 72 , 76 , 77 ] , figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7509653 ) .", "papers reporting involvement of other polyphenols in anti - inflammatory regulation , for example , resveratrol , claim that these compounds exhibit their anti - inflammatory effect through suppression of nf-b and jak / stat signaling pathways [ 78 , 79 ] .", "the enhanced influence of mtx and n - f-5ht on il-1 transcription in the spleen in comparison to the liver may be the consequence of different predominance of inflammatory pathways in this organ , presumably with a stronger nf-b contribution .", "details about the relevance of these pathways and the role of n - f-5ht in the transcription regulation of il-1 , inos , and tnf- in the liver and other organs in ra are to be further elucidated ."], ["the present study contributed additional evidence about the beneficial effect and mechanism of action of n - f-5ht and of mtx on a systemic inflammatory process in the liver and its association with the pathogenesis of adjuvant arthritis .", "n - f-5ht treatment led to amelioration of inflammatory parameters tested ( plasmatic crp and il-1 protein levels , liver lox activity , and liver and spleen cytokine expression ) .", "however , this did not result in a significant change of hpv , although a trend of improvement of the arthritic score was observed after 28 days .", "a synergistic effect of tnf- and il-1 was shown to influence the balance between protein degradation and protein synthesis causing among others an increase in resting energy expenditure and net efflux of amino acids from muscle to liver .", "the significant increase of bwc in n - f-5ht treated rats , probably sign of the partial improvement of rheumatoid cachexia , might be the result of lowered mrna expression of tnf- and il-1 determined in the arthritic liver . moreover , taking into account the reported association of weight loss with the il-1 production by splenic cells , the n - f-5ht mediated attenuation of increased il-1 mrna expression in the arthritic spleen might contribute to this complex process", ". the contribution of the affected expression of tnf- and il-1 originating from other organs can not be excluded and is to be further elucidated .", "unexpectedly , chronic daily treatment with a high concentration of n - f-5ht ( 30  mg / kg ) exhibited either a minor effect on the parameters examined and/or a strong variation among the animals ( not shown ) and that in contrast to a much lower concentration ( 3  mg / kg ) . since n - f-5ht possesses a serotonin ( 5-hydroxytryptamine , 5-ht ) moiety , the question if there might be some interplay between effects of these two molecules on ra pathogenesis is to be raised . since n - f-5ht inhibited the increase of cytosolic free ca concentration in rat vascular smooth muscle cells induced by serotonin mediated by 5-ht2 receptors , it was hypothesized that at a sufficient concentration n - f-5ht may act as a competitive antagonist , which displaces serotonin from its binding site .", "intake of a high concentration of a 5-ht2 receptor antagonist may lead to a variety of effects : it may influence the receptor density , even enhance the effect of serotonin , or lead to desensitization and with time to receptor resistance ( through inhibitory feedback due to binding - induced enhanced production of serotonin ) .", "interestingly , serotonin is known not only as a neurotransmitter . increasing but contradictory reports associate serotonin with immunoinflammatory pathways in the periphery .", "serotonin , via its 5-ht2a , 5-ht2b , and 5-ht3 receptors , has been implicated to have both proinflammatory and anti - inflammatory roles in a number of studies of rheumatoid arthritis [ 8488 ] .", "the reported effects of 5-ht receptor antagonist on macrophage - like synovial cells encourage the interest to study the effect of n - f-5ht from this point of view . to confirm this hypothesis ,", "a precise characterization of interaction between n - f-5ht and 5-ht receptors is to be done . on comparing the effects of the two drugs , administration of mtx ( 0.4 ", "mg / kg ) or n - f-5ht ( 3  mg / kg ) was found to lead to a decrease of the main plasma marker of systemic inflammation crp , the liver origin protein , and to inhibition of proinflammatory lox in the liver .", "the impact of mtx and n - f-5ht on mrna expression of tnf- , il-1 , and inos in the liver and on the level of crp in plasma was mentioned at the conference .", "mtx and n - f-5ht reduced the arthritis - increased transcription of tnf- and inos in the liver to a comparable extent . we suppose that the inhibition of tnf- and inos transcription might be mostly attributed to the suppressed nf-b pathway for the two drugs [ 21 , 33 , 70 ] .", "as previously reported [ 67 , 68 ] and also proven by our study , mtx was not able to diminish the arthritic - induced il-1 mrna transcription in the liver .", "this handicap might be compensated by coadministration of n - f-5ht , since this drug was shown to lower the level of proinflammatory cytokine il-1 in plasma in the acute phase of aa and to attenuate significantly the elevation of il-1 mrna expression in the arthritic rat liver and spleen in the chronic phase .", "detailed studies are required to confirm the hypothesis that n - f-5ht might function through potentially different mechanisms of inhibition of the inflammatory pathway nf-b and not through mtx , as well as the possibility of an additional pathway influencing il-1 transcription under control of n - f-5ht but not mtx .", "the confirmation would support n - f-5ht as a promising agent for the treatment of ra in combination therapy with mtx .", "the positive effect was shown in our previous study , where n - f-5ht markedly potentiated the therapeutic effect of low - dose mtx .", "as the therapeutic dose of mtx was used in this study and the purpose of combination study is to lower the mtx dose to decrease the side effects of this drug , the effect of combination therapy was not included", ". oral daily intake of n - f-5ht could overcome the inconvenient administration and high costs of biological therapy using il-1 monoclonal antibody , which was shown in clinical trials to be superior to placebo in combination with mtx in reducing signs , symptoms , and radiographic progression in patients with advanced ra [ 91 , 92 ] .", "future studies of n - f-5ht mechanisms of action should shed more light on the immunomodulatory function of this natural polyphenol .", "it is to be expected that n - f-5ht is able to positively affect the activity of other markers of inflammation and oxidative stress not only in the liver and spleen but also in other organs ( lung , brain , etc . ) , a hypothesis to be tested by future work .", "however , to establish the optimal dosing in light of the effects achieved is of primary importance ."]]}
{"article_id": "PMC4105607", "article_text": ["although studies on the effects of children s use of computers are still undetermined , some initial studies showed positive and negative effects in this regard .", "children commonly use computers for playing games , completing school assignments , sending / receiving e - mails , and connecting to the internet .", "this may sometimes interfere with other activities such as homework or normal social interchange ( 1 ) .", "studies showed that internet addiction positively correlated with depression , novelty seeking , harm avoidance , and reward dependence . on the other hand , internet addiction negatively correlated with persistence , self - directness , cooperativeness , and self - transcendence .", "in several studies it was demonstrated that significant factors affecting internet addiction were depression , gender , novelty seeking , and self - transcendence ( 2 , 3 ) .", "computer game addiction is excessive or compulsive use of the internet , computer , and video games that may interfere with daily life .", "it is not clear whether internet and video game playing meets the diagnostic criteria of diagnostic and statistical manual of mental disorders , 4 edition ( dsm - iv ) ( 4 ) .", "previous studies about internet addiction have investigated several associated psychological variables such as shyness , loneliness , self - consciousness , anxiety , depression , and interpersonal relations ( 5 ) .", "some studies revealed a significant association between internet addiction and depressive symptoms in adolescents and high school students .", "many investigations suggest the necessity of the evaluation of the potential underlying depression in the treatment of internet - addicted adolescents ( 6 ) . moreover , the level of internet addiction correlated positively with the level of depression and suicidal ideation among high school students . based upon several studies ,", "there were significant positive correlations among internet addiction , depression , and suicidal ideation in adolescents ( 7 ) .", "kim et al . showed that the levels of depression and suicide ideation were highest in the internet - addicts group than other students .", "they advised future studies to investigate the direct relationship between psychological health problems and internet dependency ( 8) . in this study we tried to investigate the prevalence of internet , computer games , and dvd and video addiction among high school students in southern iran , shiraz , and its relation to depression and anxiety", "during the years 2008 and 2009 and in a cross - sectional study 1020 high school students ( males and females ) were selected randomly by area and cluster sampling from different areas of shiraz in southern iran .", "an informed consent was received from each participant and they were assured of the confidentiality of the data .  ", "the students were given a full explanation of the reasons for the implementation of the study and were informed that their responses would be confidential .", "they were asked to express their personal comments and recommendations at the end of the interview .", "special attention was paid to ensure that the students clearly understand the instructions of the interview .", "in addition , they were asked not to say their name or student number in order to encourage them to provide more open and honest answers .", "demographic data ( age , sex , and economic status according to income of the family ) , duration of internet usage , computer games playing , tv and satellite watching , dvd and video cd usage , and the reasons of the usages were asked at the interviews .  ", "the students were interviewed for addiction , generalized anxiety disorder , and major depressive disorder according to the dsm - iv criteria . \n", "mild depression : decreased mood , presence of anxiety symptoms , and increased symptoms in the afternoon , without suicidal idea . \n moderate depression : decreased activity , depressed mood , agitation , decreased energy , and decreased concentration , sense of guilt , hypochondriacs , sleep disturbance , depersonalization symptoms , decreased appetite , and decreased sexual activities .  \n", "dependent : dysfunctional , and withdrawal symptoms ( restlessness , anxiety , decreased concentration , insomnia , irritability , fatigue , and craving ) after discontinuing . \n", "the obtained data were analyzed using descriptive indices and the analytic methods of the chi - squared and pearson regression .", "the data were analyzed with statistical package for social sciences ( spss ) for windows15.0 ( chicago , il .", "277 students ( 27.2% ) were in the first year of high school , 242 ( 23.7% ) were students of the second year , and 501 ( 49.1% ) were in the third year of high school .", "140 students ( 13.7% ) had low economic status ( defined as family monthly income of less than 3000000 rials income ) , 807 ( 79.1% ) had moderate economic status ( 3000000 - 1000000 rials income per month ) , and 62 ( 6.1% ) had high economic status ( more than 10,000,000 rials income per month ) . \n", "table 1 shows the prevalence of depression and anxiety among students according to sex , economic state , and internet , computer , or dvd usage , and history of medical disease .  ", "13 students ( 1.3% ) were computer games abusers , and 634 students ( 85% ) reported playing computer games for amusement .", "215 ( 21.1% ) of student did not use dvd or video cd . 1 student ( 0.1% )", "was dvd or video cd abuser , and 576 students ( 77.7% ) reported using dvd or video cd for amusement . 22 ( 2.2% ) students did not watch tv or satellite ,", "4 students were abusers , and 190 ( 18.6% ) students were dependent to tv or satellite .", "455 ( 53.6% ) students reported watching tv or satellite for film and football , and 306 ( 36.2% ) for amusement .", "94 students ( 9.2% ) had medical problems , and 71 ( 7% ) used some medications regularly .", "prevalence of anxiety and depression were significantly higher in females ( p < 0.05 ) .", "the prevalence of anxiety and depression was significantly lower in students of the third year compared to other grades ( p < 0.05 ) .", "the prevalence of anxiety was significantly higher in students with lower family monthly income status ( p < 0.05 ) .", "the prevalence of depression was significantly higher in students with lower economic status ( p < 0.05 ) .", "the prevalence of depression was significantly higher in students who were internet abusers or dependents ( p < 0.05 ) , but we did not detect a significant relation between anxiety and internet usage .  ", "the prevalence of anxiety was significantly higher in the students who used the internet for chatting , amusement ( such as downloading music , films , and pictures ) , and reading the news compared to the students who used the internet for other reasons ( p < 0.05 ) .", "there was no significant correlation between depression and reason of internet use among the students .", "the prevalence of anxiety and depression were significantly higher in students who were computer games abusers or dependents ( p < 0.05 ) .", "the prevalence of anxiety and depression did not show a relationship with the reason of playing computer games .", "the prevalence of anxiety was significantly higher in students who were dvd or video cd dependents(p < 0.05 ) .", "the prevalence of depression did not relate to the level of dvd or video cd use .", "students who used dvd or video cd for resting had higher levels of anxiety and depression ( p < 0.05 ) .", "the prevalence of depression and anxiety did not relate to the usage of tv or satellite ( p < 0.05 ) .", "students who watched tv or satellite for film , football , and music had lower levels of anxiety ( p < 0.05 ) . students who watched tv or satellite for film and football had lower levels of depression ( p < 0.05 ) .", "the students who used some medications regularly or had medical problems had higher rates of depression and anxiety ( p < 0.05 ) .", "the problem of excessive computer and internet usage is increasing more rapidly and it can occur together with other kinds of addiction .", "the causes of internet addiction do not have only habitual bases , but also demographic and socioeconomic aspects .", "the behaviors related to internet addiction may be a symptom of depressive disorders in adolescents ( 9 - 11 ) .", "prevalence of depression and anxiety among students according to sex , economic state , and internet , computer , or dvd usage , and history of medical disease batthyany et al . reported excessive computer playing and internet usage corresponding to addictive behaviors were found in 12.3% of the adolescents in austria ( 12 ) .", ". showed the rate of internet addiction in the high school students to be about 7% which was more prevalent among males ( 13 ) .", "hur in study on korean adolescents reported significant correlations among internet addiction , depression , and suicidal ideation ( 14 ) .", "ha et al . found that almost one third of subjects in their study diagnosed as internet addicts had a significant level of depressive symptoms that required psychiatric intervention ( 15 ) .", "these users use the internet to such an extent that it interferes with their academic studies and they are very much preoccupied with it .", "it is still very much a matter of debate whether internet addiction is a distinct disorder or a behavioral problem secondary to another disorder .", "suggested that internet communication with people can alleviate depression , at least among socially isolated and moderately depressed populations such as college students ( 11 ) .", "they added that the behaviors related to internet addiction may be a symptom of depressive disorders in adolescents .  ", "educationists and parents should try to understand the root causes of any isolation in the youth .", "it is alarming that excessive internet users are more likely to report having felt sad or depressed most of the days in the past year .", "they opined that the possible co - morbidity of major depressive disorder among internet - dependent adolescents could be explained by the internalizing tendency of the adolescents .", "internalized depressive adolescents can escape from the reality or problems of the real world by losing themselves in the cyber world .", "purposeful activities on the internet , which are focused , for example on academics - related research or discussing a project online , can be completed in relatively shorter periods of time through good planning .", "however , use of the internet for entertainment may not have a time limit and some activities like multi - player games can be highly compulsive .", "parental rules in such situations can set the limit , and thereby reduce excessive use and limit negative outcomes .", "the rate of internet , computer games , and dvd addiction in our study were significantly lower than reports from other parts of the world .", "the prevalence of anxiety was significantly higher in students who did not use the internet .", "these abnormal results may be due to lower economic states among the students who did not use the internet .  ", "the prevalence of anxiety was significantly higher in the students who used internet for chat , amusement , and news .", "similar to other studies , the prevalence of anxiety and depression was significantly higher in students who were computer game abusers or dependents , although it did not have any relation with the reason of playing of the computer games .  ", "for example , ha et al . reported that one third of subjects in their study diagnosed as internet addicts had a significant level of depressive symptoms that required psychiatric intervention ( 15 ) .", "while internet addiction is a new phenomenon , it has already become a subject of numerous studies . on the other hand ,", "it is a very complicated problem and it is difficult to state whether it is more accurate to treat the problem totally as an addiction or not ( 15 - 17 ) . in summary , it can be concluded that the results presented in this study are for the most part in accordance with those of other authors and other studies on this subject .", "some differences could be the result of different conditions , instruments , and differences in the number and structure of the studied group .", "ja conceived and designed the evaluation and helped to draft the manuscript and its revision and re - evaluated the clinical data .", "aa participated in the evaluation and collection of the clinical data , and helped to draft the manuscript .", "agh participated in conceiving and designing the evaluation and helped to draft the manuscript and re - evaluated the clinical data .", "mkh , zkh , and zgh participated in the evaluation and collected the clinical data .", "\n declaration of interest : none . \n citation : ahmadi j , amiri a , ghanizadeh a , khademalhosseini m , khademalhosseini z , gholami z , et al", ". prevalence of addiction to the internet , computer games , dvd and video and its relationship to anxiety and depression in a sample of iranian high school students ."], "abstract_text": ["<S> \n objective : the objective of this study was to assess the prevalence of addiction to the internet , computer games , dvd , and video and its relationship to anxiety and depression in a sample of iranian high school students . \n </S>", "<S> methods : in this cross - sectional study 1020 high school students ( males and females ) were selected randomly from different areas of shiraz city in southern iran . </S>", "<S> they were interviewed according to the diagnostic and statistical manual of mental disorders , 4th ed ( dsm - iv ) criteria . \n </S>", "<S> results : about 50% of the students were females , 277 students ( 27.2% ) were studying in the first year of high school , 242 ( 23.7% ) were in the second year , and others in the third year . </S>", "<S> the prevalence of anxiety was significantly higher in females than in males ( p < 0.05 ) . </S>", "<S> the prevalence of anxiety was lower among students of the third year ( p < 0.05 ) . </S>", "<S> the prevalence of depression was significantly higher in students with lower economic status defined as family monthly income . </S>", "<S> internet dependence was seen only in 5 students . </S>", "<S> the prevalence of anxiety was significantly higher in the students who used internet for chatting , amusement , and reading news ( p < 0.05 ) . </S>", "<S> the prevalence of anxiety was significantly higher in students who were dvd or video cd dependents ( p < 0.05 ) . </S>", "<S> the students who used especial drugs or had especial diseases had higher rates of depression and anxiety ( p < 0.05 ) . \n </S>", "<S> conclusion : internet addiction may cause depression and anxiety in high school students . </S>", "<S> it seems necessary to develop an internet addiction prevention program for adolescents taking into account the psychological factors such as depression and internet use habits . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion", "Authors' contributions", "Footenote"], "sections": [["although studies on the effects of children s use of computers are still undetermined , some initial studies showed positive and negative effects in this regard .", "children commonly use computers for playing games , completing school assignments , sending / receiving e - mails , and connecting to the internet .", "this may sometimes interfere with other activities such as homework or normal social interchange ( 1 ) .", "studies showed that internet addiction positively correlated with depression , novelty seeking , harm avoidance , and reward dependence . on the other hand , internet addiction negatively correlated with persistence , self - directness , cooperativeness , and self - transcendence .", "in several studies it was demonstrated that significant factors affecting internet addiction were depression , gender , novelty seeking , and self - transcendence ( 2 , 3 ) .", "computer game addiction is excessive or compulsive use of the internet , computer , and video games that may interfere with daily life .", "it is not clear whether internet and video game playing meets the diagnostic criteria of diagnostic and statistical manual of mental disorders , 4 edition ( dsm - iv ) ( 4 ) .", "previous studies about internet addiction have investigated several associated psychological variables such as shyness , loneliness , self - consciousness , anxiety , depression , and interpersonal relations ( 5 ) .", "some studies revealed a significant association between internet addiction and depressive symptoms in adolescents and high school students .", "many investigations suggest the necessity of the evaluation of the potential underlying depression in the treatment of internet - addicted adolescents ( 6 ) . moreover , the level of internet addiction correlated positively with the level of depression and suicidal ideation among high school students . based upon several studies ,", "there were significant positive correlations among internet addiction , depression , and suicidal ideation in adolescents ( 7 ) .", "kim et al . showed that the levels of depression and suicide ideation were highest in the internet - addicts group than other students .", "they advised future studies to investigate the direct relationship between psychological health problems and internet dependency ( 8) . in this study we tried to investigate the prevalence of internet , computer games , and dvd and video addiction among high school students in southern iran , shiraz , and its relation to depression and anxiety"], ["during the years 2008 and 2009 and in a cross - sectional study 1020 high school students ( males and females ) were selected randomly by area and cluster sampling from different areas of shiraz in southern iran .", "an informed consent was received from each participant and they were assured of the confidentiality of the data .  ", "the students were given a full explanation of the reasons for the implementation of the study and were informed that their responses would be confidential .", "they were asked to express their personal comments and recommendations at the end of the interview .", "special attention was paid to ensure that the students clearly understand the instructions of the interview .", "in addition , they were asked not to say their name or student number in order to encourage them to provide more open and honest answers .", "demographic data ( age , sex , and economic status according to income of the family ) , duration of internet usage , computer games playing , tv and satellite watching , dvd and video cd usage , and the reasons of the usages were asked at the interviews .  ", "the students were interviewed for addiction , generalized anxiety disorder , and major depressive disorder according to the dsm - iv criteria . \n", "mild depression : decreased mood , presence of anxiety symptoms , and increased symptoms in the afternoon , without suicidal idea . \n moderate depression : decreased activity , depressed mood , agitation , decreased energy , and decreased concentration , sense of guilt , hypochondriacs , sleep disturbance , depersonalization symptoms , decreased appetite , and decreased sexual activities .  \n", "dependent : dysfunctional , and withdrawal symptoms ( restlessness , anxiety , decreased concentration , insomnia , irritability , fatigue , and craving ) after discontinuing . \n", "the obtained data were analyzed using descriptive indices and the analytic methods of the chi - squared and pearson regression .", "the data were analyzed with statistical package for social sciences ( spss ) for windows15.0 ( chicago , il ."], ["277 students ( 27.2% ) were in the first year of high school , 242 ( 23.7% ) were students of the second year , and 501 ( 49.1% ) were in the third year of high school .", "140 students ( 13.7% ) had low economic status ( defined as family monthly income of less than 3000000 rials income ) , 807 ( 79.1% ) had moderate economic status ( 3000000 - 1000000 rials income per month ) , and 62 ( 6.1% ) had high economic status ( more than 10,000,000 rials income per month ) . \n", "table 1 shows the prevalence of depression and anxiety among students according to sex , economic state , and internet , computer , or dvd usage , and history of medical disease .  ", "13 students ( 1.3% ) were computer games abusers , and 634 students ( 85% ) reported playing computer games for amusement .", "215 ( 21.1% ) of student did not use dvd or video cd . 1 student ( 0.1% )", "was dvd or video cd abuser , and 576 students ( 77.7% ) reported using dvd or video cd for amusement . 22 ( 2.2% ) students did not watch tv or satellite ,", "4 students were abusers , and 190 ( 18.6% ) students were dependent to tv or satellite .", "455 ( 53.6% ) students reported watching tv or satellite for film and football , and 306 ( 36.2% ) for amusement .", "94 students ( 9.2% ) had medical problems , and 71 ( 7% ) used some medications regularly .", "prevalence of anxiety and depression were significantly higher in females ( p < 0.05 ) .", "the prevalence of anxiety and depression was significantly lower in students of the third year compared to other grades ( p < 0.05 ) .", "the prevalence of anxiety was significantly higher in students with lower family monthly income status ( p < 0.05 ) .", "the prevalence of depression was significantly higher in students with lower economic status ( p < 0.05 ) .", "the prevalence of depression was significantly higher in students who were internet abusers or dependents ( p < 0.05 ) , but we did not detect a significant relation between anxiety and internet usage .  ", "the prevalence of anxiety was significantly higher in the students who used the internet for chatting , amusement ( such as downloading music , films , and pictures ) , and reading the news compared to the students who used the internet for other reasons ( p < 0.05 ) .", "there was no significant correlation between depression and reason of internet use among the students .", "the prevalence of anxiety and depression were significantly higher in students who were computer games abusers or dependents ( p < 0.05 ) .", "the prevalence of anxiety and depression did not show a relationship with the reason of playing computer games .", "the prevalence of anxiety was significantly higher in students who were dvd or video cd dependents(p < 0.05 ) .", "the prevalence of depression did not relate to the level of dvd or video cd use .", "students who used dvd or video cd for resting had higher levels of anxiety and depression ( p < 0.05 ) .", "the prevalence of depression and anxiety did not relate to the usage of tv or satellite ( p < 0.05 ) .", "students who watched tv or satellite for film , football , and music had lower levels of anxiety ( p < 0.05 ) . students who watched tv or satellite for film and football had lower levels of depression ( p < 0.05 ) .", "the students who used some medications regularly or had medical problems had higher rates of depression and anxiety ( p < 0.05 ) ."], ["the problem of excessive computer and internet usage is increasing more rapidly and it can occur together with other kinds of addiction .", "the causes of internet addiction do not have only habitual bases , but also demographic and socioeconomic aspects .", "the behaviors related to internet addiction may be a symptom of depressive disorders in adolescents ( 9 - 11 ) .", "prevalence of depression and anxiety among students according to sex , economic state , and internet , computer , or dvd usage , and history of medical disease batthyany et al . reported excessive computer playing and internet usage corresponding to addictive behaviors were found in 12.3% of the adolescents in austria ( 12 ) .", ". showed the rate of internet addiction in the high school students to be about 7% which was more prevalent among males ( 13 ) .", "hur in study on korean adolescents reported significant correlations among internet addiction , depression , and suicidal ideation ( 14 ) .", "ha et al . found that almost one third of subjects in their study diagnosed as internet addicts had a significant level of depressive symptoms that required psychiatric intervention ( 15 ) .", "these users use the internet to such an extent that it interferes with their academic studies and they are very much preoccupied with it .", "it is still very much a matter of debate whether internet addiction is a distinct disorder or a behavioral problem secondary to another disorder .", "suggested that internet communication with people can alleviate depression , at least among socially isolated and moderately depressed populations such as college students ( 11 ) .", "they added that the behaviors related to internet addiction may be a symptom of depressive disorders in adolescents .  ", "educationists and parents should try to understand the root causes of any isolation in the youth .", "it is alarming that excessive internet users are more likely to report having felt sad or depressed most of the days in the past year .", "they opined that the possible co - morbidity of major depressive disorder among internet - dependent adolescents could be explained by the internalizing tendency of the adolescents .", "internalized depressive adolescents can escape from the reality or problems of the real world by losing themselves in the cyber world .", "purposeful activities on the internet , which are focused , for example on academics - related research or discussing a project online , can be completed in relatively shorter periods of time through good planning .", "however , use of the internet for entertainment may not have a time limit and some activities like multi - player games can be highly compulsive .", "parental rules in such situations can set the limit , and thereby reduce excessive use and limit negative outcomes .", "the rate of internet , computer games , and dvd addiction in our study were significantly lower than reports from other parts of the world .", "the prevalence of anxiety was significantly higher in students who did not use the internet .", "these abnormal results may be due to lower economic states among the students who did not use the internet .  ", "the prevalence of anxiety was significantly higher in the students who used internet for chat , amusement , and news .", "similar to other studies , the prevalence of anxiety and depression was significantly higher in students who were computer game abusers or dependents , although it did not have any relation with the reason of playing of the computer games .  ", "for example , ha et al . reported that one third of subjects in their study diagnosed as internet addicts had a significant level of depressive symptoms that required psychiatric intervention ( 15 ) ."], ["while internet addiction is a new phenomenon , it has already become a subject of numerous studies . on the other hand ,", "it is a very complicated problem and it is difficult to state whether it is more accurate to treat the problem totally as an addiction or not ( 15 - 17 ) . in summary , it can be concluded that the results presented in this study are for the most part in accordance with those of other authors and other studies on this subject .", "some differences could be the result of different conditions , instruments , and differences in the number and structure of the studied group ."], ["ja conceived and designed the evaluation and helped to draft the manuscript and its revision and re - evaluated the clinical data .", "aa participated in the evaluation and collection of the clinical data , and helped to draft the manuscript .", "agh participated in conceiving and designing the evaluation and helped to draft the manuscript and re - evaluated the clinical data .", "mkh , zkh , and zgh participated in the evaluation and collected the clinical data ."], ["\n declaration of interest : none . \n citation : ahmadi j , amiri a , ghanizadeh a , khademalhosseini m , khademalhosseini z , gholami z , et al", ". prevalence of addiction to the internet , computer games , dvd and video and its relationship to anxiety and depression in a sample of iranian high school students ."]]}
{"article_id": "PMC4381731", "article_text": [", it must develop tolerance to paternal antigens to avoid a lethal immunologic attack against the fetus . on the other hand", ", it must preserve the ability to fight infections from a multitude of commensal and environmental pathogens .", "disruption of this balance can have devastating consequences , including preterm labor and death of the fetus and/or mother .", "the normal maternal immune response to pregnancy is increasingly recognized as a dynamic process , with changes in the maternal pro / anti - inflammatory profile occurring at different stages of gestation [ 13 ] . despite this recognition , a complete , longitudinal immunologic profile of normal", "such analyses are critical for understanding the response to specific infectious and immunologic diseases that show disproportionately negative outcomes during pregnancy , such as influenza   and ulcerative colitis .", "one approach to such immune profiling is through the use of multiplex cytokine arrays , allowing for simultaneous quantification of many proteins with a very small amount of plasma or serum .", "others have used multiplex arrays to study the maternal cytokine milieu , but the studies have been predominantly limited time point cross - sectional [ 68 ] or case - control [ 911 ] in design .", "the results of some longitudinal studies have been published in recent years , although results have been based on relatively few time points ( 5 or fewer ) [ 13 ] . in this study", ", we applied the multiplex array approach to evaluate the changes in 42 cytokines in rich detail during pregnancy .", "the samples are from a cohort of 16 pregnant women , with each subject sampled a median of 18 times . because this represents a significant increase in the number of tests per subject compared to previous studies", ", we also describe a strategy addressing within - subject correlation with repeated measurements using unconditional growth modeling . with this approach", ", we have been able to detail the typical longitudinal variation of serum cytokines throughout pregnancy in this cohort of women .", "each participant had a serum sample collected and cryopreserved biweekly from study enrollment during the first trimester ( average of 9.7 weeks ' gestation for first samples ) until 34 weeks ' gestation , then weekly from 34 weeks until delivery .", "as a result of this repeated sampling , a median of 18 samples per participant were obtained longitudinally throughout pregnancy .", "samples were collected under a protocol approved by the mayo clinic institutional review board ( irb ) that accrued participants from 1987 to 1988 .", "information regarding date of the blood draw , date of delivery , outcome of the delivery , and any major complications that the woman experienced during pregnancy ( hypertensive disorders , preterm labor , and infections ) was available and helped identify appropriate participants to include .", "only women who carried their pregnancies to term and experienced no pregnancy complications were included in this study .", "we only used samples from subjects who had specimens obtained throughout the entire course of pregnancy and whose complete , longitudinally collected specimens appeared intact and nondesiccated .", "most cytokines are stable in storage at 80 degrees c for up to 2 years , but stability beyond that time is not well known . therefore , before proceeding with studies on the samples of interest , we first tested protein levels of a small number of individually stored serum aliquots from other subjects archived on this protocol . since we found detectable levels of cytokines in the pregnant serum samples that were within range of control samples , we proceeded with the entire study involving a cohort of 16 healthy , primigravid women who completed a term pregnancy without any significant complications .", "samples were assayed when freshly thawed to avoid freeze - thaw cycles , which are known to degrade cytokines .", "contemporary plasma samples from 11 nonpregnant control subjects were collected under a separate irb - approved protocol for collection of biospecimens from healthy individuals within the same health system in 2010 and similarly assessed on the same 96-well plates for comparison and as a quality control measure . for contemporary nonpregnant control samples ,", "peripheral venous blood was drawn into heparinized vacutainer tubes that were processed and separated into plasma and peripheral blood mononuclear cells ( pbmcs ) following gradient centrifugation using ficoll - paque ( ge healthcare , uppsala , sweden ) .", "plasma was collected and immediately frozen at 80c in 1  ml aliquots until use .", "protein levels for 42 cytokines , chemokines , and growth factors ( table 1 ) were measured using the milliplex map human cytokine / chemokine kit ( millipore , billerica , ma , usa ) per manufacturer 's instructions . to minimize the potential for interassay variability , each subject had all of their longitudinal specimens analyzed on the same plate .", "protein concentrations were determined using a linear regression standard curve from each plate generated using the high pmt concentrations with sensitivity from 3.2 to 2,000  pg / ml . all samples were tested in duplicate with the mean value of the measurements used for statistical analyses .", "comparison of baseline ( first trimester ) and end of pregnancy levels of cytokines / growth factors was carried out between the 16 primigravid subjects and the 11 nonpregnant control subjects using the kruskal - wallis tests .", "there were extremes of values that fell outside of the limits of the multiplex array .", "for cytokines below the limit of detection ( ld ) , we assigned a value that was half the lowest detectable value for the assay as previously described .", "analytes above the detectable range were assigned a threshold concentration . to obtain a visual representation of overall patterns in the highly dimensional raw data", "as an additional multivariate technique to identify patterns within the data , principle components analysis ( pca ) was also completed on the raw values obtained from the multiplex array . to assess the within - subject correlation , we determined change of cytokines and growth factors from a baseline time point  the values obtained from the participants ' first blood draw in the first trimester  by taking the log of 1 plus fold change from the baseline value : log(1 + x ) , where x = ( cytokine concentration of the sample of interest / cytokine concentration of the baseline sample ) to analyze trends over time .", "first , we graphically assessed the growth trajectory for the 42 cytokines using smoothing splines .", "we then fit unconditional mean models ( umm ) for the 42 cytokines in order to estimate the variance components : the within - subject variance (  ) and the between - subject variance ( 0 ) . estimating the two variance components helps to determine whether there is sufficient variation to warrant further analysis and enabled computations of the intraclass correlation coefficient ,  , which describes the proportion of the total outcome variation that lies between subjects .", "next , we fit unconditional growth models ( ugm ) , which allowed random slope but not random intercept because the starting value of all patients was the same ( baseline value = 0.3 ) due to normalization to each individual 's baseline value . as both gestational age in weeks and trimester", "can be used as the time covariate for growth curve models , and as the trimester can be treated as either a continuous or a categorical variable ( depending on whether we assume linear relationship between trimester and outcome variables ) , six different models were fitted for each cytokines as follows : ( 1 ) model with only the gestational age in weeks as the time covariate , ( 2 ) model with only the trimester ( continuous ) as the time covariate , ( 3 ) model with only the trimester ( categorical ) as the time covariate , where the variance - covariance pattern was assumed to be autoregressive , ( 4 ) model with both gestational age in weeks and continuous trimester as covariates , allowing random slope for gestational time in weeks , ( 5 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slope for trimester , and ( 6 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slopes for both gestational age in weeks and trimester .", "we then compared the six models and the unconditional mean model for each of the cytokines based on logic and statistical fitness , using akaike information criteria ( aic ) .", "because of the exploratory nature of this study , there was no correction for multiple comparisons .", "the multiplex was designed to assess 42 factors , but the limits of detection were exceeded with three factors , and there were 5 cytokines that fell below the limit of detection in > 50% of the samples .", "pdgf - aa , pdgf - ab / bb , and rantes had cytokine levels above detection limits of the assay in 9.9% , 11.2% , and 34.9% of the samples , respectively ( table s1 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/952571 ) .", "il-2 , il-3 , il-4 , il-13 , and tnf had > 50% of the samples below the detection limit ( table s1 ) .", "differences in cytokine profiles between baseline ( first trimester ) pregnancy samples and nonpregnant control samples could be identified by pca ( figure 1 ) .", "specifically , we identified significantly higher levels of gro , tgf , egf , pdgf - aa , and pdgf - ab / bb and significantly lower levels of scd40l , ip-10 , il-6 , il-17 , il-13 , and mcp-1 in the baseline pregnancy samples ( table 1 ) . when all longitudinal samples were included in a pca , no difference", "next , we evaluated whether those differences apparent in early gestation were also present at the end of pregnancy . when comparing the final independent samples obtained at the end of pregnancy to the healthy control samples , the following remained significantly elevated in pregnant women : egf ( 330.6 versus 24.8  pg / ml , p = 0.0013 ) , gro ( 1,153.6 versus 341.6  pg / ml , p < 0.001 ) , sil-2ra ( 17.9  pg / ml versus lld , p = 0.02 ) , and tgf ( 15.8 versus 1.8  pg / ml , p = 0.0006 )", "meanwhile , only eotaxin was lower in women at the end of pregnancy compared to normal controls ( 33.4 versus 330.0  pg / ml , p < 0.001 ) . to begin to assess within - subject variation , we clustered the raw data ( figure 2 ) , where the issue of correlation within subjects becomes visually apparent .", "empirical growth plots demonstrated that the within - subject variation differs among different women ( figures 3 and 4 show a representative example using il-15 , and the remainder of the plots can be viewed in supplementary figures ) .", "for example , patient o displays very large variation in the majority of the cytokines tested , whereas patients b , c , f , and g have comparatively steady levels for most cytokines .", "also , high variability in one cytokine does not imply high variability in other cytokines for the individual women .", "high interclass correlation ( 0.7 ) was observed for scd40l , rantes , il-10 , tnf , il-7 , and gm - csf ( table s2 ) .", "this means that the majority of the variability in these cytokines is attributed to variance between subjects .", "the interclass correlations were comparatively low ( < 0.3 ) for il-3 , il-4 , mcp-3 , and ip-10 , suggesting more variation within subjects for these cytokines .", "the final model for mdc ( table 2 ) was ugm with gw , with a decreasing trajectory .", "the estimated variance for the random slope is 0.000001632 , suggesting that the variability of slope among patients is quite small .", "on the other hand , the average scatter of an individual 's outcome around her own trajectory is larger ( the estimated variance is 0.000937 , p < 0.0001 ) , suggesting that there might be some other important covariates that are not included in the model .", "however , including gestational age in weeks as a covariate does substantially improve the fit of the model .", "when comparing the estimated within - subject variance of the ugm with that from the umm , we found that the linear gestational age in weeks helps to explain 34% of the within - subject variation in mdc .", "the estimated fixed effect suggests that mdc decreases over time ( the estimated slope is 0.00163 , with p  0.0001 , suggesting that the slope is significantly lesser than 0 , figure s2 ) .", "eight other cytokines demonstrated statistical significance , although with increasing trajectories , with ugm and gw as a covariate : il-1 , il-6 , il-8 , il-12p70 , il-13 , il-15 , ip-10 , and flt3-ligand ( table 2 ) .", "six cytokines showed association with trimester as a linear variable : il-1ra , il-3 , il-9 , il-12p40 , ifn2 , and scd40l ( table 3 ) .", "il-1ra showed the greatest percent explanation by inclusion of linear trimester in the model . when comparing the estimated within - subject variance of the umm with that from the ugm", ", we found that the linear trimester helps to explain 27% of the within - subject variation in il-1ra .", "seven other cytokines showed significant associations with trimester as a categorical variable : il-4 , ifn , g - csf , tgf- ,", "tnf- , sil-2ra , and mip-1 ( table 4 ) . the final model for both egf and vegf was umm with intercept only , leading to the conclusion that both egf and vegf do not change over time .", "time - dependent ugm models were identified but did not meet statistical significance for the following cytokines : il-2 , il-5 , pdgf - aa , pdgf - ab , mip-1 , gro , mcp-1 , mcp-3 , rantes , il-17 , il-7 , eotaxin , fgf-2 , il-10 , tnf , il-1ra2 , fracktalkine , and gm - csf . therefore", ", approximately 50% of cytokines measured by multiplex array did not show any association with gestational week or trimester .", "our results suggest that the third trimester of pregnancy is characterized by an increasingly inflammatory ( e.g. , il-1 , il-6 , il-12 , il-15 , ip-10 , and scd40 ligand ) as well as counterregulatory ( e.g. , il-1ra and flt3-ligand ) milieu compared to earlier stages of pregnancy .", "our superimposed growth curves of il-15 ( figure 4 ) and il-1 ( figure s3 ) , the cytokines with the strongest association with gestational week as a linear covariate , suggest that this change begins at approximately week 20 and peaks just after week 30 .", "this is consistent with other reports of systemic immune activation as well as counterregulation in the latter part of pregnancy [ 3 , 1719 ] .", "increasing il-1 and il-15 may predominantly be related to production from placental tissues [ 20 , 21 ] or mononuclear phagocytes [ 22 , 23 ] in late gestation .", "the precise immunologic mechanisms responsible for this shift can not be determined by our study , but if the source of these cytokines is predominantly the innate immune system , one potential stimulus to these cytokines ' secretion is cell - free fetal dna . a recently proposed model links rising circulating levels of fetal dna to maternal innate immune activation via toll - like receptor- ( tlr- ) 9 on neutrophils and macrophages , leading to increasing inflammatory cytokine release , that may result in an immune cascade ultimately leading to parturition .", "a novel finding of this study is the decline in mdc levels throughout pregnancy ( figure 5 ) .", "mdc is chemoattractant for immature dendritic cells   and type 2-biased t cells , and it is possible that decreasing mdc levels may reflect a gradual shift away from a self - amplifying type 2 immune response as pregnancy progresses .", "il-9 is cytokine that has been recognized for decades but only recently had its cell of origin identified : the innate lymphoid cell ( ilc ) .", "the role of il-9 , which has historically been categorized as a th2 cytokine prior to the recognition of ilc , is currently unknown in normal pregnancy .", "because only serum was available for analysis , we can not confirm the cellular source of the changes observed with this cohort .", "nonetheless , increasing transcription levels of several innate immune components , including cd14 , multiple tlr genes , and il-1b , have been observed in peripheral blood leukocytes of women in the third trimester , suggesting that the changes in concentrations of the cytokines identified in our study could plausibly be due to changes in peripheral blood cellular composition .", "further studies will be necessary to determine whether placental tissues , circulating leukocytes , stromal cells , or other sources also contribute to the changes reflected in our results .", "although these results show statistical significance , it is also noteworthy that the slopes of the variance were all fairly small for each of the analytes and that gestational week did not explain any more than 40% of the variation observed in any of the cytokines . pregnancy clearly alters the maternal immune milieu , but the changes from baseline in the setting of normal pregnancy are subtle and more variable in some women than in others .", "what clinical factors contribute to this variation observed between women in the setting of a normal first pregnancy is not entirely known .", "blood draws and resulting data were relatively sparse in the first trimester due to women not returning as frequently for study blood draws , even though they were scheduled per protocol , relative to the second and third trimesters .", "it is also important to note that many analytes were not detectable in samples ; therefore , it is possible that a different method besides multiplex array on serum or plasma , for example , elisa for individual cytokines / growth factors , may be more sensitive to detect change over time .", "previous longitudinal studies of maternal cytokines during pregnancy have shown some concordance with our results .", "investigated the change of maternal plasma cytokines from early to midgestation in a large cohort ( approximately 1,000 patients ) and found that il-12 and ifn levels increased , while il-2 and gm - csf levels decreased as pregnancy progressed .", "our results confirmed increasing il-12 levels , as well as an increase of ifn in the third trimester compared to the first .", "however , kraus et al . published maternal multiplex elisa results from 50 women tested at each trimester as well as postpartum and showed that ifn levels decreased throughout gestation .", "denney et al . also found that basal levels of ifn decreased throughout gestation , as did levels of tnf , il-1 , and il-6 in serum samples of 45 healthy pregnant women collected during each trimester .", "another recent study demonstrated decreasing il-1 levels throughout gestation . while our study did not confirm any time - dependent change of tnf , we instead observed increases in il-1 and il-6 throughout pregnancy , in contrast to these studies .", "clearly , individual heterogeneity exists , and our smaller sample size may account for some of these differences with prior studies .", "although not the original focus of this study , we also identified several differences in cytokine / growth factor milieu when comparing pregnant women to healthy controls .", "notably , the first trimester of pregnancy was characterized by an increase in several growth factors ( gro , egf , tgf , and pdgf ) and a relative decrease in inflammatory markers ( scd40l , il-6 , il-17 , ip-10 , eotaxin , and mcp-1 ) .", "eotaxin , a potent chemoattractant for eosinophils during allergic reactions , has been similarly described to be suppressed during pregnancy by kraus et al . .", "overall , the tolerance induction required for successful pregnancy at the level of the fetomaternal interface may also be observed in a relatively tolerogenic , growth factor - rich maternal systemic environment . the difference in sample source when comparing", "the archived samples from healthy pregnant women ( serum ) and normal controls ( plasma ) may contribute to these findings .", "for example , eotaxin , pdgf , egf , vegf , and soluble cd40 ligand have previously been shown to be significantly different in serum postclotting as compared to plasma samples .", "because of this potential source of variation in comparing our historical to contemporary samples , our results will require validation with similarly processed samples of the same source .", "however , egf levels have been reported to be approximately 65  pg / ml in serum of healthy individuals , well below our median 489.4  pg / ml in our samples obtained from pregnant subjects , suggesting that our results may hold true despite differences in sample source .", "similarly , serum levels of cd40l have been shown to be 830-fold higher in serum as compared to plasma , suggesting again that the very low levels identified in the serum of our cohort of pregnant women may indeed be reflective of the pregnancy state .", "our study is unique in that the longitudinal samples were obtained frequently throughout the course of pregnancy .", "because of this , our study has the potential to more richly describe individual maternal immune variation over time . however , our study is limited by the relatively small sample size , the lack of pre- or postpartum samples , and potentially the age of the specimens . to address these limitations ,", "we have recently conducted a larger longitudinal study of maternal immune changes during pregnancy , with peripheral blood samples obtained monthly throughout gestation as well as 6 weeks postpartum .", "tolerance at the fetomaternal interface is complex and technically challenging to study in humans longitudinally . as a result , many recent studies have sought to understand how pregnancy changes maternal systemic immunity .", "the possibility exists of maternal peripheral blood not completely or accurately reflecting the local changes within decidual tissues .", "an example of such a discrepancy in our study is that of ip-10 , which was low in first - trimester pregnancy compared to healthy controls in this cohort", ". ip-10 is secreted by decidual natural killer cells and is key to trophoblast migration and thus has previously been shown to be elevated during pregnancy [ 31 , 32 ] .", "on the other hand , the decidual t cell compartment at parturition has recently been shown to closely reflect that of peripheral blood , suggesting that for some components of the immune system the maternal peripheral blood is reasonable to study .", "furthermore , sampling maternal peripheral blood is much more feasible to perform longitudinally as compared to more invasive means and may still lend insight into the maternal adaptation to pregnancy .", "successful pregnancy has previously been described as predominantly a type 2 immune response - biased phenomenon [ 3436 ] . in light of the collective evidence", ", it seems that the description of pregnancy in terms of type 2 immune responses may be oversimplified , both in the overall description of key cytokines / growth factors as well as in the temporal dynamics , and it is in need of further refinement ."], "abstract_text": ["<S> several recent studies have shown differences in the maternal immune milieu at different phases of pregnancy , but most studies have been cross - sectional or of relatively few time points . </S>", "<S> levels of 42 cytokines were determined using a multiplex bead - based assay on archived serum from a cohort of pregnant women ( n = 16 ) at median of 18 time points tested , from the first trimester through to parturition , per woman . </S>", "<S> unconditional growth modeling was then used to determine time - dependent changes in levels of these cytokines . </S>", "<S> macrophage - derived chemokine ( mdc , aka ccl22 ) decreases as pregnancy progresses . </S>", "<S> il-1 , il-6 , il-8 , il-12p70 , il-13 , il-15 , ip-10 , and flt3-ligand increase as a function of gestational weeks , and ifn2 , il-1ra , il-3 , il-9 , il-12p40 , and soluble cd40 ligand increase as a function of trimester . as </S>", "<S> pregnancy normally progresses , a maternal shift away from a type 2-biased immune response and toward an inflammatory / counterregulatory response is observed . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [[", it must develop tolerance to paternal antigens to avoid a lethal immunologic attack against the fetus . on the other hand", ", it must preserve the ability to fight infections from a multitude of commensal and environmental pathogens .", "disruption of this balance can have devastating consequences , including preterm labor and death of the fetus and/or mother .", "the normal maternal immune response to pregnancy is increasingly recognized as a dynamic process , with changes in the maternal pro / anti - inflammatory profile occurring at different stages of gestation [ 13 ] . despite this recognition , a complete , longitudinal immunologic profile of normal", "such analyses are critical for understanding the response to specific infectious and immunologic diseases that show disproportionately negative outcomes during pregnancy , such as influenza   and ulcerative colitis .", "one approach to such immune profiling is through the use of multiplex cytokine arrays , allowing for simultaneous quantification of many proteins with a very small amount of plasma or serum .", "others have used multiplex arrays to study the maternal cytokine milieu , but the studies have been predominantly limited time point cross - sectional [ 68 ] or case - control [ 911 ] in design .", "the results of some longitudinal studies have been published in recent years , although results have been based on relatively few time points ( 5 or fewer ) [ 13 ] . in this study", ", we applied the multiplex array approach to evaluate the changes in 42 cytokines in rich detail during pregnancy .", "the samples are from a cohort of 16 pregnant women , with each subject sampled a median of 18 times . because this represents a significant increase in the number of tests per subject compared to previous studies", ", we also describe a strategy addressing within - subject correlation with repeated measurements using unconditional growth modeling . with this approach", ", we have been able to detail the typical longitudinal variation of serum cytokines throughout pregnancy in this cohort of women ."], ["each participant had a serum sample collected and cryopreserved biweekly from study enrollment during the first trimester ( average of 9.7 weeks ' gestation for first samples ) until 34 weeks ' gestation , then weekly from 34 weeks until delivery .", "as a result of this repeated sampling , a median of 18 samples per participant were obtained longitudinally throughout pregnancy .", "samples were collected under a protocol approved by the mayo clinic institutional review board ( irb ) that accrued participants from 1987 to 1988 .", "information regarding date of the blood draw , date of delivery , outcome of the delivery , and any major complications that the woman experienced during pregnancy ( hypertensive disorders , preterm labor , and infections ) was available and helped identify appropriate participants to include .", "only women who carried their pregnancies to term and experienced no pregnancy complications were included in this study .", "we only used samples from subjects who had specimens obtained throughout the entire course of pregnancy and whose complete , longitudinally collected specimens appeared intact and nondesiccated .", "most cytokines are stable in storage at 80 degrees c for up to 2 years , but stability beyond that time is not well known . therefore , before proceeding with studies on the samples of interest , we first tested protein levels of a small number of individually stored serum aliquots from other subjects archived on this protocol . since we found detectable levels of cytokines in the pregnant serum samples that were within range of control samples , we proceeded with the entire study involving a cohort of 16 healthy , primigravid women who completed a term pregnancy without any significant complications .", "samples were assayed when freshly thawed to avoid freeze - thaw cycles , which are known to degrade cytokines .", "contemporary plasma samples from 11 nonpregnant control subjects were collected under a separate irb - approved protocol for collection of biospecimens from healthy individuals within the same health system in 2010 and similarly assessed on the same 96-well plates for comparison and as a quality control measure . for contemporary nonpregnant control samples ,", "peripheral venous blood was drawn into heparinized vacutainer tubes that were processed and separated into plasma and peripheral blood mononuclear cells ( pbmcs ) following gradient centrifugation using ficoll - paque ( ge healthcare , uppsala , sweden ) .", "plasma was collected and immediately frozen at 80c in 1  ml aliquots until use .", "protein levels for 42 cytokines , chemokines , and growth factors ( table 1 ) were measured using the milliplex map human cytokine / chemokine kit ( millipore , billerica , ma , usa ) per manufacturer 's instructions . to minimize the potential for interassay variability , each subject had all of their longitudinal specimens analyzed on the same plate .", "protein concentrations were determined using a linear regression standard curve from each plate generated using the high pmt concentrations with sensitivity from 3.2 to 2,000  pg / ml . all samples were tested in duplicate with the mean value of the measurements used for statistical analyses .", "comparison of baseline ( first trimester ) and end of pregnancy levels of cytokines / growth factors was carried out between the 16 primigravid subjects and the 11 nonpregnant control subjects using the kruskal - wallis tests .", "there were extremes of values that fell outside of the limits of the multiplex array .", "for cytokines below the limit of detection ( ld ) , we assigned a value that was half the lowest detectable value for the assay as previously described .", "analytes above the detectable range were assigned a threshold concentration . to obtain a visual representation of overall patterns in the highly dimensional raw data", "as an additional multivariate technique to identify patterns within the data , principle components analysis ( pca ) was also completed on the raw values obtained from the multiplex array . to assess the within - subject correlation , we determined change of cytokines and growth factors from a baseline time point  the values obtained from the participants ' first blood draw in the first trimester  by taking the log of 1 plus fold change from the baseline value : log(1 + x ) , where x = ( cytokine concentration of the sample of interest / cytokine concentration of the baseline sample ) to analyze trends over time .", "first , we graphically assessed the growth trajectory for the 42 cytokines using smoothing splines .", "we then fit unconditional mean models ( umm ) for the 42 cytokines in order to estimate the variance components : the within - subject variance (  ) and the between - subject variance ( 0 ) . estimating the two variance components helps to determine whether there is sufficient variation to warrant further analysis and enabled computations of the intraclass correlation coefficient ,  , which describes the proportion of the total outcome variation that lies between subjects .", "next , we fit unconditional growth models ( ugm ) , which allowed random slope but not random intercept because the starting value of all patients was the same ( baseline value = 0.3 ) due to normalization to each individual 's baseline value . as both gestational age in weeks and trimester", "can be used as the time covariate for growth curve models , and as the trimester can be treated as either a continuous or a categorical variable ( depending on whether we assume linear relationship between trimester and outcome variables ) , six different models were fitted for each cytokines as follows : ( 1 ) model with only the gestational age in weeks as the time covariate , ( 2 ) model with only the trimester ( continuous ) as the time covariate , ( 3 ) model with only the trimester ( categorical ) as the time covariate , where the variance - covariance pattern was assumed to be autoregressive , ( 4 ) model with both gestational age in weeks and continuous trimester as covariates , allowing random slope for gestational time in weeks , ( 5 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slope for trimester , and ( 6 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slopes for both gestational age in weeks and trimester .", "we then compared the six models and the unconditional mean model for each of the cytokines based on logic and statistical fitness , using akaike information criteria ( aic ) .", "because of the exploratory nature of this study , there was no correction for multiple comparisons ."], ["the multiplex was designed to assess 42 factors , but the limits of detection were exceeded with three factors , and there were 5 cytokines that fell below the limit of detection in > 50% of the samples .", "pdgf - aa , pdgf - ab / bb , and rantes had cytokine levels above detection limits of the assay in 9.9% , 11.2% , and 34.9% of the samples , respectively ( table s1 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/952571 ) .", "il-2 , il-3 , il-4 , il-13 , and tnf had > 50% of the samples below the detection limit ( table s1 ) .", "differences in cytokine profiles between baseline ( first trimester ) pregnancy samples and nonpregnant control samples could be identified by pca ( figure 1 ) .", "specifically , we identified significantly higher levels of gro , tgf , egf , pdgf - aa , and pdgf - ab / bb and significantly lower levels of scd40l , ip-10 , il-6 , il-17 , il-13 , and mcp-1 in the baseline pregnancy samples ( table 1 ) . when all longitudinal samples were included in a pca , no difference", "next , we evaluated whether those differences apparent in early gestation were also present at the end of pregnancy . when comparing the final independent samples obtained at the end of pregnancy to the healthy control samples , the following remained significantly elevated in pregnant women : egf ( 330.6 versus 24.8  pg / ml , p = 0.0013 ) , gro ( 1,153.6 versus 341.6  pg / ml , p < 0.001 ) , sil-2ra ( 17.9  pg / ml versus lld , p = 0.02 ) , and tgf ( 15.8 versus 1.8  pg / ml , p = 0.0006 )", "meanwhile , only eotaxin was lower in women at the end of pregnancy compared to normal controls ( 33.4 versus 330.0  pg / ml , p < 0.001 ) . to begin to assess within - subject variation , we clustered the raw data ( figure 2 ) , where the issue of correlation within subjects becomes visually apparent .", "empirical growth plots demonstrated that the within - subject variation differs among different women ( figures 3 and 4 show a representative example using il-15 , and the remainder of the plots can be viewed in supplementary figures ) .", "for example , patient o displays very large variation in the majority of the cytokines tested , whereas patients b , c , f , and g have comparatively steady levels for most cytokines .", "also , high variability in one cytokine does not imply high variability in other cytokines for the individual women .", "high interclass correlation ( 0.7 ) was observed for scd40l , rantes , il-10 , tnf , il-7 , and gm - csf ( table s2 ) .", "this means that the majority of the variability in these cytokines is attributed to variance between subjects .", "the interclass correlations were comparatively low ( < 0.3 ) for il-3 , il-4 , mcp-3 , and ip-10 , suggesting more variation within subjects for these cytokines .", "the final model for mdc ( table 2 ) was ugm with gw , with a decreasing trajectory .", "the estimated variance for the random slope is 0.000001632 , suggesting that the variability of slope among patients is quite small .", "on the other hand , the average scatter of an individual 's outcome around her own trajectory is larger ( the estimated variance is 0.000937 , p < 0.0001 ) , suggesting that there might be some other important covariates that are not included in the model .", "however , including gestational age in weeks as a covariate does substantially improve the fit of the model .", "when comparing the estimated within - subject variance of the ugm with that from the umm , we found that the linear gestational age in weeks helps to explain 34% of the within - subject variation in mdc .", "the estimated fixed effect suggests that mdc decreases over time ( the estimated slope is 0.00163 , with p  0.0001 , suggesting that the slope is significantly lesser than 0 , figure s2 ) .", "eight other cytokines demonstrated statistical significance , although with increasing trajectories , with ugm and gw as a covariate : il-1 , il-6 , il-8 , il-12p70 , il-13 , il-15 , ip-10 , and flt3-ligand ( table 2 ) .", "six cytokines showed association with trimester as a linear variable : il-1ra , il-3 , il-9 , il-12p40 , ifn2 , and scd40l ( table 3 ) .", "il-1ra showed the greatest percent explanation by inclusion of linear trimester in the model . when comparing the estimated within - subject variance of the umm with that from the ugm", ", we found that the linear trimester helps to explain 27% of the within - subject variation in il-1ra .", "seven other cytokines showed significant associations with trimester as a categorical variable : il-4 , ifn , g - csf , tgf- ,", "tnf- , sil-2ra , and mip-1 ( table 4 ) . the final model for both egf and vegf was umm with intercept only , leading to the conclusion that both egf and vegf do not change over time .", "time - dependent ugm models were identified but did not meet statistical significance for the following cytokines : il-2 , il-5 , pdgf - aa , pdgf - ab , mip-1 , gro , mcp-1 , mcp-3 , rantes , il-17 , il-7 , eotaxin , fgf-2 , il-10 , tnf , il-1ra2 , fracktalkine , and gm - csf . therefore", ", approximately 50% of cytokines measured by multiplex array did not show any association with gestational week or trimester ."], ["our results suggest that the third trimester of pregnancy is characterized by an increasingly inflammatory ( e.g. , il-1 , il-6 , il-12 , il-15 , ip-10 , and scd40 ligand ) as well as counterregulatory ( e.g. , il-1ra and flt3-ligand ) milieu compared to earlier stages of pregnancy .", "our superimposed growth curves of il-15 ( figure 4 ) and il-1 ( figure s3 ) , the cytokines with the strongest association with gestational week as a linear covariate , suggest that this change begins at approximately week 20 and peaks just after week 30 .", "this is consistent with other reports of systemic immune activation as well as counterregulation in the latter part of pregnancy [ 3 , 1719 ] .", "increasing il-1 and il-15 may predominantly be related to production from placental tissues [ 20 , 21 ] or mononuclear phagocytes [ 22 , 23 ] in late gestation .", "the precise immunologic mechanisms responsible for this shift can not be determined by our study , but if the source of these cytokines is predominantly the innate immune system , one potential stimulus to these cytokines ' secretion is cell - free fetal dna . a recently proposed model links rising circulating levels of fetal dna to maternal innate immune activation via toll - like receptor- ( tlr- ) 9 on neutrophils and macrophages , leading to increasing inflammatory cytokine release , that may result in an immune cascade ultimately leading to parturition .", "a novel finding of this study is the decline in mdc levels throughout pregnancy ( figure 5 ) .", "mdc is chemoattractant for immature dendritic cells   and type 2-biased t cells , and it is possible that decreasing mdc levels may reflect a gradual shift away from a self - amplifying type 2 immune response as pregnancy progresses .", "il-9 is cytokine that has been recognized for decades but only recently had its cell of origin identified : the innate lymphoid cell ( ilc ) .", "the role of il-9 , which has historically been categorized as a th2 cytokine prior to the recognition of ilc , is currently unknown in normal pregnancy .", "because only serum was available for analysis , we can not confirm the cellular source of the changes observed with this cohort .", "nonetheless , increasing transcription levels of several innate immune components , including cd14 , multiple tlr genes , and il-1b , have been observed in peripheral blood leukocytes of women in the third trimester , suggesting that the changes in concentrations of the cytokines identified in our study could plausibly be due to changes in peripheral blood cellular composition .", "further studies will be necessary to determine whether placental tissues , circulating leukocytes , stromal cells , or other sources also contribute to the changes reflected in our results .", "although these results show statistical significance , it is also noteworthy that the slopes of the variance were all fairly small for each of the analytes and that gestational week did not explain any more than 40% of the variation observed in any of the cytokines . pregnancy clearly alters the maternal immune milieu , but the changes from baseline in the setting of normal pregnancy are subtle and more variable in some women than in others .", "what clinical factors contribute to this variation observed between women in the setting of a normal first pregnancy is not entirely known .", "blood draws and resulting data were relatively sparse in the first trimester due to women not returning as frequently for study blood draws , even though they were scheduled per protocol , relative to the second and third trimesters .", "it is also important to note that many analytes were not detectable in samples ; therefore , it is possible that a different method besides multiplex array on serum or plasma , for example , elisa for individual cytokines / growth factors , may be more sensitive to detect change over time .", "previous longitudinal studies of maternal cytokines during pregnancy have shown some concordance with our results .", "investigated the change of maternal plasma cytokines from early to midgestation in a large cohort ( approximately 1,000 patients ) and found that il-12 and ifn levels increased , while il-2 and gm - csf levels decreased as pregnancy progressed .", "our results confirmed increasing il-12 levels , as well as an increase of ifn in the third trimester compared to the first .", "however , kraus et al . published maternal multiplex elisa results from 50 women tested at each trimester as well as postpartum and showed that ifn levels decreased throughout gestation .", "denney et al . also found that basal levels of ifn decreased throughout gestation , as did levels of tnf , il-1 , and il-6 in serum samples of 45 healthy pregnant women collected during each trimester .", "another recent study demonstrated decreasing il-1 levels throughout gestation . while our study did not confirm any time - dependent change of tnf , we instead observed increases in il-1 and il-6 throughout pregnancy , in contrast to these studies .", "clearly , individual heterogeneity exists , and our smaller sample size may account for some of these differences with prior studies .", "although not the original focus of this study , we also identified several differences in cytokine / growth factor milieu when comparing pregnant women to healthy controls .", "notably , the first trimester of pregnancy was characterized by an increase in several growth factors ( gro , egf , tgf , and pdgf ) and a relative decrease in inflammatory markers ( scd40l , il-6 , il-17 , ip-10 , eotaxin , and mcp-1 ) .", "eotaxin , a potent chemoattractant for eosinophils during allergic reactions , has been similarly described to be suppressed during pregnancy by kraus et al . .", "overall , the tolerance induction required for successful pregnancy at the level of the fetomaternal interface may also be observed in a relatively tolerogenic , growth factor - rich maternal systemic environment . the difference in sample source when comparing", "the archived samples from healthy pregnant women ( serum ) and normal controls ( plasma ) may contribute to these findings .", "for example , eotaxin , pdgf , egf , vegf , and soluble cd40 ligand have previously been shown to be significantly different in serum postclotting as compared to plasma samples .", "because of this potential source of variation in comparing our historical to contemporary samples , our results will require validation with similarly processed samples of the same source .", "however , egf levels have been reported to be approximately 65  pg / ml in serum of healthy individuals , well below our median 489.4  pg / ml in our samples obtained from pregnant subjects , suggesting that our results may hold true despite differences in sample source .", "similarly , serum levels of cd40l have been shown to be 830-fold higher in serum as compared to plasma , suggesting again that the very low levels identified in the serum of our cohort of pregnant women may indeed be reflective of the pregnancy state .", "our study is unique in that the longitudinal samples were obtained frequently throughout the course of pregnancy .", "because of this , our study has the potential to more richly describe individual maternal immune variation over time . however , our study is limited by the relatively small sample size , the lack of pre- or postpartum samples , and potentially the age of the specimens . to address these limitations ,", "we have recently conducted a larger longitudinal study of maternal immune changes during pregnancy , with peripheral blood samples obtained monthly throughout gestation as well as 6 weeks postpartum .", "tolerance at the fetomaternal interface is complex and technically challenging to study in humans longitudinally . as a result , many recent studies have sought to understand how pregnancy changes maternal systemic immunity .", "the possibility exists of maternal peripheral blood not completely or accurately reflecting the local changes within decidual tissues .", "an example of such a discrepancy in our study is that of ip-10 , which was low in first - trimester pregnancy compared to healthy controls in this cohort", ". ip-10 is secreted by decidual natural killer cells and is key to trophoblast migration and thus has previously been shown to be elevated during pregnancy [ 31 , 32 ] .", "on the other hand , the decidual t cell compartment at parturition has recently been shown to closely reflect that of peripheral blood , suggesting that for some components of the immune system the maternal peripheral blood is reasonable to study .", "furthermore , sampling maternal peripheral blood is much more feasible to perform longitudinally as compared to more invasive means and may still lend insight into the maternal adaptation to pregnancy ."], ["successful pregnancy has previously been described as predominantly a type 2 immune response - biased phenomenon [ 3436 ] . in light of the collective evidence", ", it seems that the description of pregnancy in terms of type 2 immune responses may be oversimplified , both in the overall description of key cytokines / growth factors as well as in the temporal dynamics , and it is in need of further refinement ."]]}
{"article_id": "PMC5069840", "article_text": ["sarcoidosis - related granulomatous reaction of the immune system could be attributed to environmental factors .", "however , data supporting this hypothesis remain controversial \n . clinical features of this disease include the following nonspecific symptoms : cough , dyspnea , erythema nodosum , febrile arthritis , uveitis , and parotitis", "sarcoidosis commonly targets hilar and mediastinal lymph nodes , which are found in more than 90% of the patients \n .", "the diagnosis is established on the basis of compatible clinical and radiological findings and supported by histological evidence in one or more organs of noncaseating epithelioid cell granulomas in the absence of organisms or particles \n .", "the correlation between sarcoidosis and malignancy remains unclear , despite that this topic has been increasingly investigated . in this article", ", we report a case of non - luminal her-2/neu - positive breast cancer in a patient without history of sarcoidosis and initially suspected to have metastatic disease . \n", "a 52-year - old woman was presented to our hospital . she noted a lump in her left breast during self - examination .", "palpation revealed a nodular lump of tight , elastic consistency in the upper inner quadrant of the left breast .", "mammography scan demonstrated a 19 mm  18 mm mass on the border of the inner quadrants on the left breast ( \n\n figure 1 \n ) .", "ultrasound examination revealed enlarged lymph nodes in the left axilla ( 10 mm in diameter ) , left supraclavicular lymph node ( 18 mm  10 mm ) , and multiple right enlarged supraclavicular lymph nodes , with a maximum size of 16 mm  7 mm .", "plain chest x - rays showed no abnormal findings ( \n\n figure 2 \n ) .", "the suspected diagnosis was breast cancer at t \n 1n \n 3 cm \n 0 .", "excisional biopsy of the left supraclavicular lymph node was performed to verify the diagnosis and differentiate the nature of the lesion .", "histological examination of the obtained material revealed no cancer but multiple epithelioid cell granulomas . based on these results , lumpectomy with urgent histology of resection margins", "urgent histodiagnosis revealed clear margins and demonstrated a lump in the breast , which was identified as infiltrative carcinoma .", "routine histological examination revealed infiltrative , moderately differentiated ( g \n 2 ) breast carcinoma with microcalcifications ( \n\n figure 3 \n ) .", "noncaseating epithelioid cell granulomas of sarcoidosis without tumor growth were found in 6 of 15 lymph nodes ( \n\n figure 4 \n ) .", "the molecular type of breast cancer was identified as non - luminal her-2/neu - positive through immunohistochemistry .", "therefore , the post - operative diagnosis of the patient was left breast cancer ( t \n 1n \n 0 m \n 0 ) , with sarcoidosis of left axillary and right supraclavicular lymph nodes . at the time of writing", "this article , the patient had been undergoing radiation therapy and directed to immunologist for sarcoidosis management and follow - up . \n  mammography scan demonstrating a 19 mm  18 mm mass on the border of the inner quadrants of the left breast ( white arrow ) . ( a ) craniocaudal .", "( b ) mediolateral oblique view . plain chest x - ray revealed no abnormal findings .", "infiltrative moderately differentiated ( g \n 2 ) breast carcinoma with microcalcifications ( h&e staining , 200 ) . \n  lymph nodes with noncaseating epithelioid cell granulomas of sarcoidosis without tumor growth ( h&amp;e staining , 200 ) .", "the correlation between sarcoidosis and carcinogenesis remains unproven , although such relation has been described in numerous studies .", "brincker and wilbek \n  first found this link in their study on 2544 sarcoidosis cases ; the incidence rates of lymphomas and lung cancer were 11 and 3 fold higher , respectively , in patients with sarcoidosis than those in the population .", "reported 21 cases of sarcoidosis developing after primary malignancies , including 10 cases after breast cancer .", "blank et al . \n  defined breast cancer , cervical cancer , and b - cell lymphoma as the most common malignancies in patients with sarcoidosis . \n ", "positron emission tomography ( fdg - pet / ct scan ) is one of the most advanced and precise diagnostic tools for such diseases .", "however , the application of this method in the assessment of regional and distant metastasis spread is limited \n . in cases of simultaneous sarcoidosis and malignancies , the functions of fdg - pet /", "identified that the maximal standardized uptake value ( suv ) in patients with maligna- ncies is significantly higher than in patients with benign diseases .", "nevertheless , in patients with granulomatous process , the maximal suv is similar to that in patients with malignant diseases .", "therefore , fdg - pet / ct scan may only be an additional diagnostic tool used to assess the extent of disease spread without differentiation between malignant and granulomatous disorders ; this tool could also create diagnos- tic difficulties and misunderstandings in patients with such simultaneous comorbidities \n 9 , \n 10 .", "this study reports a case of non - luminal her-2/neu - positive breast cancer in a patient presenting non - caseating epithelioid cell granulomas of sarcoidosis after treatment and initially suspected to have metastatic disease .", "a very recent retrospective study described the clinical biological and radiological characteristics of 12 patients presenting sarcoidosis associated with solid tumor ; sarcoidosis frequently affects patients with breast cancer ( 32.3% ) and thus must be considered in the differential diagnosis of the disease \n . in the current study , a review of literature is also presented and included 61 other patients showing similar association between cancer and sarcoidosis . \n ", "this case report emphasizes the importance of differential diagnosis of lymph node involvement in cancer patients .", "diagnosis of sarcoidosis denied that the presence of metastasis could lead to changes in the post - operative management of the patient .", "although several epidemiological studies reported the association between cancer and sarcoidosis , further studies , including case reports , may represent an opportunity to obtain additional biological and clinical information to clarify the mechanisms underlying the association of cancer and sarcoidosis .", "furthermore , the present case report highlights the importance of histological determination in lymph nodes for distinguishing metastasis from sarcoidosis ."], "abstract_text": ["<S> sarcoidosis is a benign systematic granulomatous disorder of unknown etiology and is associated with various malignancies . </S>", "<S> however , granulomatous and metastatic lymph node lesions are difficult to distinguish even when using precise and modern diagnostic methods , such as positron emission tomography . </S>", "<S> thus , histological verification is the only method that can be used to accurately describe the nature of this disease . in this article </S>", "<S> , we report a case of non - luminal her-2/neu - positive breast cancer in a patient without history of sarcoidosis and suspected to have metastatic disease . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion", "Conflict of interest statement"], "sections": [["sarcoidosis - related granulomatous reaction of the immune system could be attributed to environmental factors .", "however , data supporting this hypothesis remain controversial \n . clinical features of this disease include the following nonspecific symptoms : cough , dyspnea , erythema nodosum , febrile arthritis , uveitis , and parotitis", "sarcoidosis commonly targets hilar and mediastinal lymph nodes , which are found in more than 90% of the patients \n .", "the diagnosis is established on the basis of compatible clinical and radiological findings and supported by histological evidence in one or more organs of noncaseating epithelioid cell granulomas in the absence of organisms or particles \n .", "the correlation between sarcoidosis and malignancy remains unclear , despite that this topic has been increasingly investigated . in this article", ", we report a case of non - luminal her-2/neu - positive breast cancer in a patient without history of sarcoidosis and initially suspected to have metastatic disease . \n"], ["a 52-year - old woman was presented to our hospital . she noted a lump in her left breast during self - examination .", "palpation revealed a nodular lump of tight , elastic consistency in the upper inner quadrant of the left breast .", "mammography scan demonstrated a 19 mm  18 mm mass on the border of the inner quadrants on the left breast ( \n\n figure 1 \n ) .", "ultrasound examination revealed enlarged lymph nodes in the left axilla ( 10 mm in diameter ) , left supraclavicular lymph node ( 18 mm  10 mm ) , and multiple right enlarged supraclavicular lymph nodes , with a maximum size of 16 mm  7 mm .", "plain chest x - rays showed no abnormal findings ( \n\n figure 2 \n ) .", "the suspected diagnosis was breast cancer at t \n 1n \n 3 cm \n 0 .", "excisional biopsy of the left supraclavicular lymph node was performed to verify the diagnosis and differentiate the nature of the lesion .", "histological examination of the obtained material revealed no cancer but multiple epithelioid cell granulomas . based on these results , lumpectomy with urgent histology of resection margins", "urgent histodiagnosis revealed clear margins and demonstrated a lump in the breast , which was identified as infiltrative carcinoma .", "routine histological examination revealed infiltrative , moderately differentiated ( g \n 2 ) breast carcinoma with microcalcifications ( \n\n figure 3 \n ) .", "noncaseating epithelioid cell granulomas of sarcoidosis without tumor growth were found in 6 of 15 lymph nodes ( \n\n figure 4 \n ) .", "the molecular type of breast cancer was identified as non - luminal her-2/neu - positive through immunohistochemistry .", "therefore , the post - operative diagnosis of the patient was left breast cancer ( t \n 1n \n 0 m \n 0 ) , with sarcoidosis of left axillary and right supraclavicular lymph nodes . at the time of writing", "this article , the patient had been undergoing radiation therapy and directed to immunologist for sarcoidosis management and follow - up . \n  mammography scan demonstrating a 19 mm  18 mm mass on the border of the inner quadrants of the left breast ( white arrow ) . ( a ) craniocaudal .", "( b ) mediolateral oblique view . plain chest x - ray revealed no abnormal findings .", "infiltrative moderately differentiated ( g \n 2 ) breast carcinoma with microcalcifications ( h&e staining , 200 ) . \n  lymph nodes with noncaseating epithelioid cell granulomas of sarcoidosis without tumor growth ( h&amp;e staining , 200 ) ."], ["the correlation between sarcoidosis and carcinogenesis remains unproven , although such relation has been described in numerous studies .", "brincker and wilbek \n  first found this link in their study on 2544 sarcoidosis cases ; the incidence rates of lymphomas and lung cancer were 11 and 3 fold higher , respectively , in patients with sarcoidosis than those in the population .", "reported 21 cases of sarcoidosis developing after primary malignancies , including 10 cases after breast cancer .", "blank et al . \n  defined breast cancer , cervical cancer , and b - cell lymphoma as the most common malignancies in patients with sarcoidosis . \n ", "positron emission tomography ( fdg - pet / ct scan ) is one of the most advanced and precise diagnostic tools for such diseases .", "however , the application of this method in the assessment of regional and distant metastasis spread is limited \n . in cases of simultaneous sarcoidosis and malignancies , the functions of fdg - pet /", "identified that the maximal standardized uptake value ( suv ) in patients with maligna- ncies is significantly higher than in patients with benign diseases .", "nevertheless , in patients with granulomatous process , the maximal suv is similar to that in patients with malignant diseases .", "therefore , fdg - pet / ct scan may only be an additional diagnostic tool used to assess the extent of disease spread without differentiation between malignant and granulomatous disorders ; this tool could also create diagnos- tic difficulties and misunderstandings in patients with such simultaneous comorbidities \n 9 , \n 10 .", "this study reports a case of non - luminal her-2/neu - positive breast cancer in a patient presenting non - caseating epithelioid cell granulomas of sarcoidosis after treatment and initially suspected to have metastatic disease .", "a very recent retrospective study described the clinical biological and radiological characteristics of 12 patients presenting sarcoidosis associated with solid tumor ; sarcoidosis frequently affects patients with breast cancer ( 32.3% ) and thus must be considered in the differential diagnosis of the disease \n . in the current study , a review of literature is also presented and included 61 other patients showing similar association between cancer and sarcoidosis . \n ", "this case report emphasizes the importance of differential diagnosis of lymph node involvement in cancer patients .", "diagnosis of sarcoidosis denied that the presence of metastasis could lead to changes in the post - operative management of the patient .", "although several epidemiological studies reported the association between cancer and sarcoidosis , further studies , including case reports , may represent an opportunity to obtain additional biological and clinical information to clarify the mechanisms underlying the association of cancer and sarcoidosis .", "furthermore , the present case report highlights the importance of histological determination in lymph nodes for distinguishing metastasis from sarcoidosis ."], [""]]}
{"article_id": "PMC5413303", "article_text": ["this retrospective observational study was approved by the western institutional review board ( olympia , washington , usa ) .", "it complied with the health insurance portability and accountability act of 1996 and followed the tenets of the declaration of helsinki .", "informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study .", "patients with ga were identified from the tertiary practice of retinal specialists ( kbf , js , and ly ) if they exhibited plateau signatures in at least one eye ( study eye ) on oct imaging and if they also had at least 3 years ' follow - up with consecutive eye - tracked spectral - domain ( sd)-oct scans taken at least every 6 months .", "eyes with macular neovascularization , other retinal pathology , or media opacity preventing adequate imaging were excluded .", "medical records and multimodal imaging , including color fundus photography ( cfp ) , red - free photography ( rf ) , fundus autofluorescence ( faf ) , near - infrared reflectance scanning laser ophthalmoscopy ( nir ) , and sd - oct was performed on all the patients .", "cfp and rf were performed with a trc-50ix flood - illuminated fundus camera ( topcon medical systems , oakland , nj , usa ) .", "faf was performed with either the spectralis hra + oct ( heidelberg engineering , heidelberg , germany ) or the topcon trc-50ix fundus camera .", "oct scans taken before the acquisition of eye - tracked spectralis scans were assessed qualitatively but not included in the quantitative analysis .", "the sd - oct protocol used in all eyes comprised 20 horizontal raster line scans over the area of interest , ranging from 19 to 49 b - scans per eye , with each scan spaced 115 to 250 m apart , and automatic real - time averaging set between 4 and 23 . to study the origin and progression of plateaus , the most recent sd - oct with this signature was identified . then", ", serial eye - tracked b - scans of the region of interest were tracked back to baseline . in cases", "in which the scanning protocol varied during the course of follow - up , the b - scan closest to the area of interest was matched manually and extracted for analysis .", "these extracted sd - oct images were then stacked , aligned , and saved as a video file using the fiji distribution (  fiji is just image j , ", "http://fiji.sc ; in the public domain ) for the qualitative analysis of the progression of the lesion ( see supplementary video ) .", "at each time point , the plateau appearance on sd - oct was correlated to the appearance in cfp , nir , and faf .", "three patients had en face oct and oct angiography ( octa ) imaging with 3  3-mm macular cubes ( rtvue xr  avanti  ; optovue , fremont , ca , usa ) .", "the review software ( revue , version 2015.1.0.90 ; optovue , fremont , ca , usa ) displays flow signals superimposed in red on a cross - sectional structural sd - oct image . in a healthy eye ,", "the fourth outer retinal hyperreflective band includes the rpe and brm . in a ped , by definition", "in the course of this separation , either a basal lamina ( bl ) of 0.15-m thickness or a blamd of variable thickness adheres to the rpe and not to the brm .", "therefore , we call the hyperreflective band that anteriorly delimits a ped the rpe - bl complex . the definition of a  plateau  was a distinct oct signature where a mound - like structure with a flattened apex and a wide base was observed within an area of ga after complete loss of the overlying rpe . to determine the volume of the drusenoid ped and ensuing plateau ( fig .", "2 ) , the cavalieri principle of stereology was applied to sd - oct volumetric data acquired at each visit , as follows .", "( 1 ) each b - scan in the volume was scaled to a 1:1 pixel aspect ratio .", "the caliper function within the heidelberg eye explorer software ( version 6.3.4.0 ; heidelberg engineering ) was used to measure the area between the outer boundary of rpe+bl and the inner boundary of brm .", "( 2 ) the volume between two consecutive oct slices ( hereafter called a segment ) was then determined using the following formula : \n \\}\\begin{document}$$d\\left ( { \\left ( { a\\_x + a\\_\\left ( { x + 1 } \\right ) } \\right)/2 } \\right),$$\\end{document}where d is the distance between consecutive slices in m , a is the area between the rpe+bl and brm in m , and x is the oct slice number .", "( 3 ) total ped volume was calculated by summing the volumes of individual segments . the number of segments in the oct volume was ( n  1 ) , where n is the total number of slices that spanned the ped .", "graphical plots of ped volume with respect to time were generated using interval data . \n ", "origin and evolution of a plateau , as revealed by nir ( a ) and oct ( b ) images .", "green arrows on nir images show the levels of corresponding oct cross - sectional images .", "baseline images show several elevations of the rpe with largely hyporeflective interiors . at 3 years after baseline , punctate hyperreflectivity ( red arrowhead )", ", progressive loss of the onl has caused the opl ( yellow arrows ) to approach the surface of the ped .", "also at 4 years , a  mons hyperreflective wedge  ( blue arrowheads ) appears at the ga border . at 5 years", ", the downwardly deflected opl is seen to move progressively toward the edge of the plateau following the advancing border of ga .", "formation of the plateau at 5 years was characterized by the loss of the overlying rpe causing marked thinning of the hyperreflective rpe - basal lamina complex now reduced to just blamd , compared with at 4 years . at 7 years", "the institutional review board at university of alabama at birmingham ( uab ) approved the laboratory study , which complied with the health insurance portability and accountability act and adhered to the tenets of the declaration of helsinki .", "amd eyes were identified through an ex vivo imaging screen of eyes accessioned for research purposes from nondiabetic white donors to the alabama eye bank during the period 1996 to 2012 .", "median death - to - preservation time was 3:49 hours ( range , 0:4011:40 hours ) .", "eyes were preserved by immersion in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer following anterior segment excision .", "after vitreous removal , maculas were photographed in color on a stereomicroscope ( smz - u ; nikon , melville , ny , usa ) . when prepared for histology ( 20112013 ) and uploaded to the project macula web site ( http://projectmacula.cis.uab.edu , in the public domain )", ", eyes underwent additional multimodal ex vivo imaging . from each globe , an 8-mm - diameter full - thickness tissue punch containing the fovea and temporal portion of the optic nerve head was held in a tissue holder mounted on a spectralis , as described . a 30  20 sd - oct volume ( 143 scans , 30-m spacing ,", "automated real - time averaging [ art ] = 25 ) was captured along with red - free and near - infrared reflectance scanning laser ophthalmoscopic images .", "tissue punches were postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular lipid and embedded in epoxy resin ( polybed 812 ; polysciences , warrington , pa , usa ) .", "sub - micrometer - thick ( 0.8 m ) sections at 25- to 30-m intervals were stained with 1% toluidine blue for polychromaticity , scanned with a 40 objective , and reviewed and photodocumented with a 60 oil - immersion objective ( numerical aperture = 1.4 ) and digital camera ( xc10 ; olympus , center valley , pa , usa ) .", "images were adjusted for exposure , contrast , and sharpness ( photoshop cs6 , adobe , san jose , ca , usa ) .", "patients with ga were identified from the tertiary practice of retinal specialists ( kbf , js , and ly ) if they exhibited plateau signatures in at least one eye ( study eye ) on oct imaging and if they also had at least 3 years ' follow - up with consecutive eye - tracked spectral - domain ( sd)-oct scans taken at least every 6 months", ". eyes with macular neovascularization , other retinal pathology , or media opacity preventing adequate imaging were excluded .", "medical records and multimodal imaging , including color fundus photography ( cfp ) , red - free photography ( rf ) , fundus autofluorescence ( faf ) , near - infrared reflectance scanning laser ophthalmoscopy ( nir ) , and sd - oct was performed on all the patients .", "cfp and rf were performed with a trc-50ix flood - illuminated fundus camera ( topcon medical systems , oakland , nj , usa ) .", "faf was performed with either the spectralis hra + oct ( heidelberg engineering , heidelberg , germany ) or the topcon trc-50ix fundus camera .", "oct scans taken before the acquisition of eye - tracked spectralis scans were assessed qualitatively but not included in the quantitative analysis .", "the sd - oct protocol used in all eyes comprised 20 horizontal raster line scans over the area of interest , ranging from 19 to 49 b - scans per eye , with each scan spaced 115 to 250 m apart , and automatic real - time averaging set between 4 and 23 . to study the origin and progression of plateaus , the most recent sd - oct with this signature was identified .", "then , serial eye - tracked b - scans of the region of interest were tracked back to baseline . in cases", "in which the scanning protocol varied during the course of follow - up , the b - scan closest to the area of interest was matched manually and extracted for analysis .", "these extracted sd - oct images were then stacked , aligned , and saved as a video file using the fiji distribution (  fiji is just image j ,  http://fiji.sc ; in the public domain ) for the qualitative analysis of the progression of the lesion ( see supplementary video ) . at each time point , the plateau appearance on sd - oct was correlated to the appearance in cfp , nir , and faf .", "three patients had en face oct and oct angiography ( octa ) imaging with 3  3-mm macular cubes ( rtvue xr  avanti  ; optovue , fremont , ca , usa ) . the review software ( revue , version 2015.1.0.90 ; optovue , fremont , ca , usa ) displays flow signals superimposed in red on a cross - sectional structural sd - oct image", "in a healthy eye , the fourth outer retinal hyperreflective band includes the rpe and brm . in a ped , by definition", ", the rpe is separated from the brm . in the course of this separation , either a basal lamina ( bl ) of 0.15-m thickness or a blamd of variable thickness adheres to the rpe and not to the brm .", "therefore , we call the hyperreflective band that anteriorly delimits a ped the rpe - bl complex . the definition of a  plateau  was a distinct oct signature where a mound - like structure with a flattened apex and a wide base was observed within an area of ga after complete loss of the overlying rpe .", "2 ) , the cavalieri principle of stereology was applied to sd - oct volumetric data acquired at each visit , as follows . ( 1 ) each b - scan in the volume was scaled to a 1:1 pixel aspect ratio . the caliper function within the heidelberg eye explorer software ( version 6.3.4.0 ; heidelberg engineering )", "was used to measure the area between the outer boundary of rpe+bl and the inner boundary of brm .", "( 2 ) the volume between two consecutive oct slices ( hereafter called a segment ) was then determined using the following formula : \n \\}\\begin{document}$$d\\left ( { \\left ( { a\\_x + a\\_\\left ( { x + 1 } \\right ) } \\right)/2 } \\right),$$\\end{document}where d is the distance between consecutive slices in m , a is the area between the rpe+bl and brm in m , and x is the oct slice number . ( 3 ) total ped volume was calculated by summing the volumes of individual segments .", "the number of segments in the oct volume was ( n  1 ) , where n is the total number of slices that spanned the ped .", "graphical plots of ped volume with respect to time were generated using interval data . \n ", "origin and evolution of a plateau , as revealed by nir ( a ) and oct ( b ) images .", "green arrows on nir images show the levels of corresponding oct cross - sectional images .", "baseline images show several elevations of the rpe with largely hyporeflective interiors . at 3 years after baseline , punctate hyperreflectivity ( red arrowhead )", ", progressive loss of the onl has caused the opl ( yellow arrows ) to approach the surface of the ped . also at 4 years , a ", "mons hyperreflective wedge  ( blue arrowheads ) appears at the ga border . at 5 years", ", the downwardly deflected opl is seen to move progressively toward the edge of the plateau following the advancing border of ga .", "formation of the plateau at 5 years was characterized by the loss of the overlying rpe causing marked thinning of the hyperreflective rpe - basal lamina complex now reduced to just blamd , compared with at 4 years . at 7 years , a closed ort ( pink arrowhead ) is present adjacent to the plateau .", "the institutional review board at university of alabama at birmingham ( uab ) approved the laboratory study , which complied with the health insurance portability and accountability act and adhered to the tenets of the declaration of helsinki .", "amd eyes were identified through an ex vivo imaging screen of eyes accessioned for research purposes from nondiabetic white donors to the alabama eye bank during the period 1996 to 2012 .", "median death - to - preservation time was 3:49 hours ( range , 0:4011:40 hours ) .", "eyes were preserved by immersion in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer following anterior segment excision .", "after vitreous removal , maculas were photographed in color on a stereomicroscope ( smz - u ; nikon , melville , ny , usa ) . when prepared for histology ( 20112013 ) and uploaded to the project macula web site ( http://projectmacula.cis.uab.edu , in the public domain )", ", eyes underwent additional multimodal ex vivo imaging . from each globe , an 8-mm - diameter full - thickness tissue punch containing the fovea and temporal portion of the optic nerve head was held in a tissue holder mounted on a spectralis , as described . a 30  20 sd - oct volume ( 143 scans , 30-m spacing ,", "automated real - time averaging [ art ] = 25 ) was captured along with red - free and near - infrared reflectance scanning laser ophthalmoscopic images .", "tissue punches were postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular lipid and embedded in epoxy resin ( polybed 812 ; polysciences , warrington , pa , usa ) .", "sub - micrometer - thick ( 0.8 m ) sections at 25- to 30-m intervals were stained with 1% toluidine blue for polychromaticity , scanned with a 40 objective , and reviewed and photodocumented with a 60 oil - immersion objective ( numerical aperture = 1.4 ) and digital camera ( xc10 ; olympus , center valley , pa , usa ) .", "images were adjusted for exposure , contrast , and sharpness ( photoshop cs6 , adobe , san jose , ca , usa ) .", "plateau signatures were observed in eight eyes of seven patients , of whom six were female .", "mean age was 75 years ( range , 6089 ) and mean follow - up period was 7.7 years ( range , 3.711.6 ) .", "baseline visual acuity ( va ) was logmar 0.42 ( snellen equivalent 20/52 ) ( range , 0.181 ) .", "final va was logmar 1.16 ( snellen equivalent 20/289 ) ( range , 0.182.3 ) . in two eyes ,", "ga spared the fovea throughout the entire course of the follow - up . at baseline", ", the presence of a drusenoid ped with homogeneous hyperreflective contents and an intact overlying rpe was noted in six eyes ( 75% ; figs . 2 , 3 ) .", "one eye ( patient 5 , left eye ) had an extremely large ped measuring 2.24 mm with mostly hyporeflective contents and an overlying vitelliform lesion . in another eye ( patient 4 , left eye ) , the plateau was already present at baseline .", "pigmentary changes noted on cfp were also found on nir , rf , and faf in six eyes , and these corresponded to intraretinal hyperreflective foci on oct ( fig .", "3 ) . overlying the ped surface , the ellipsoid zone and outer nuclear layer ( onl ) were thin , with the opl nearly apposed to the surface of the ped .", "b ) at baseline and follow - up ( a , 4 years ; b , 7 years ) . (", "a ) at baseline , patient 3 has a drusenoid ped with overlying pigment hyperplasia and hyperautofluorescent vitelliform material .", "the oct b - scan shows intraretinal hyperreflective foci ( red arrowhead ) and loss of outer retinal bands over the apex of the ped .", "four years later , a region of hypoautofluorescent ga contains a plateau that can not be appreciated in the cfp , nir , rf , or faf .", "the oct b - scan shows a plateau with a shape similar to the baseline ped .", "the downwardly deflecting opl ( yellow arrow ) has moved laterally as it follows the border of the advancing ga . there was marked attenuation and decreased reflectivity of the rpe - bl band .", "( b ) at baseline , patient 6 has a drusenoid ped with overlying pigment hyperplasia that is located inferior to an area of ga .", "bottom row : seven years later , the ga has markedly enlarged , and the ped is no longer visible on cfp , nir , rf , or faf .", "the oct b - scan shows that following progressive loss of the onl , the opl ( yellow arrow ) appears draped over a plateau with a shape similar to the baseline ped .", "hyporeflective areas ( blue arrowhead ) are visible within the plateau interior . during follow - up ,", "onl thinning resulted in the opl approximating the surface of the ped in all cases in which the ped was present at baseline ( figs . 2 , 3 ) .", "as ga progressed , this point of approximation moved toward the edges of the ped base , following the advancing border of ga ( supplementary video ) . in all eyes , there was also progressive thinning of the hyperreflective rpe - bl complex and diminution of its reflectivity , beginning at the apex of the ped and advancing toward its edges .", "after rpe loss occurred over the entire ped surface , the plateau ( i.e. , a thin hyperreflective inner surface and an interior of heterogeneous and overall reduced reflectivity ) appeared . the plateau surface was highly reflective on both cross - sectional and en face oct ( fig .", "4 , en face oct surface ) and showed the presence of focal defects ( fig .", "4 , blue arrows ) that were usually accompanied by smaller hyperreflective foci seen beneath the hyperreflective border .", "these hyperreflective foci also could be observed within the area of ga as hyperautofluorescent foci on faf and hyperreflective foci on en face oct ( fig .", "4 , green arrows ) . accordingly , on en face oct , plateau contents were seen as honeycomb - like structures with hyporeflective spaces interspersed with hyperreflective foci ( fig .", "4 , green arrows ) increased in abundance within the plateau interior during follow - up in all eyes as seen on cross - sectional and en face oct ( figs . 2 , 3 , 4 ) .", "on both cross - sectional and en face octa , no abnormal flow signals were noted within plateaus ( fig .", "( a ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct ,", "octa , and on oct surface ( an en face oct slab at the level of the rpe - bl atop the plateau ) , the plateau interior is bounded by a distinct hyperreflective band that continues as a thick and highly reflective rpe band .", "this hyperreflective surface shows transient hyporeflective defects ( yellow arrows ) during the evolution of these lesions .", "an en face oct image of the plateau surface allows better appreciation of the focal defects ( yellow arrow ) .", "( b ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct and octa", ", a distinct hyperreflective surface surrounds the plateau interior and focal defects ( yellow arrows ) can be seen in some areas . an en face", "oct taken at the plateau surface allows better appreciation of focal defects in this hyperreflective layer ( en face octa : surface yellow arrow ) .", "deep to these focal defects are hyperreflective features ( faf and en face oct : green arrowheads ) that are also seen on faf as punctate hyperfluorescence within the confluent area of hypoautofluorescence .", "a single plateau was seen in all eyes except for one ( patient 2 ) that had two separate plateaus ( fig .", "patient 6 had one plateau in each eye . in the seven eyes with a baseline ped ,", "the thinning of the hyperreflective band over the ped surface after rpe loss ( at which point we consider the plateau to be formed ) was preceded by a sharp decrease in ped volume ( fig .", "subsequently , the volume of the residual plateau appeared very stable over time ( fig .", "the point at which the plateaus form ( complete loss of rpe from the hyperreflective rpe - bl complex over the ped ) is shown by the dotted line .", "patient 2 has two separate peds that evolve to two separate plateaus ( a , b ) .", "patient 6 has a plateau in both the right eye ( r ) and the left eye ( l ) .", "only patients with an intact hyperreflective rpe - bl complex baseline were included in this analysis .", "when the plateau signature is first observed ( black dotted line ) , a decrease in the volume that stabilizes on further follow - up .", "figure 6 shows histology of a donor eye with ga , for which local relationships and cellular processes in the interior of outer retinal corrugations were particularly clear yet also representative of other ga eyes available for review .", "atrophy spared the fovea in this eye , as seen with ex vivo cfp and 488-nm autofluorescence , and small reflective puncta in the atrophic area were apparent on nir ( figs .", "6e ) , blamd was seen as discontinuous , being present at three separate locations .", "pigmented cells were of the  subducted  phenotype , that is , rpe - originated cells located external to blamd , in contact with brm . for reference , in the henle fiber layer of a normal eye , obliquely oriented mller cell processes are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration ,", "including ort in this ga eye , the trajectories of gliotic mller cell processes were oriented in many directions , as indicated by arrows in figure 6e . in one instance", ", processes seemed to enter an outer retinal corrugation horizontally through an opening in the blamd and sweep the  subducted  cells off brm ( fig .", "6d ) . persistent bl deposit and mller cell processes in ga in a 95-year - old white man .", "cfp ( top row left ) shows a large area of atrophy of rpe extending to the optic nerve head but sparing the fovea .", "green arrowheads approximate the level of the histologic section shown in the top far right and bottom .", "( b ) ex vivo 488-nm autofluorescence shows loss of signal in the atrophic area .", "( c ) ex vivo nir imaging shows small and highly reflective puncta in the atrophic area .", "detailed ( d ) and panoramic ( e ) views of the atrophic area in histology .", "nfl , nerve fiber layer ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; dr , calcified druse .", "( d ) processes from mller cells under a corrugation of persistent blamd in the atrophic area .", "this corrugation overlies  subducted  cells of rpe origin ( teal arrowheads ) , which contain nuclei , spindle - shaped melanosomes , and lipofuscin , and are located external to blamd , on brm .", "mller cell processes appear to enter the corrugation horizontally from the left ( yellow arrow ) and sweep the pigmented cells away .", "( e ) blamd is discontinuous and present at three separate locations ( red arrows ) .", " in the normal henle fiber layer , mller cell processes are obliquely oriented ( outer - center to inner - periphery ) , and they are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , the trajectories of gliotic mller cell processes and remaining photoreceptors are oriented in many directions ( yellow arrows ) .", "plateau signatures were observed in eight eyes of seven patients , of whom six were female .", "mean age was 75 years ( range , 6089 ) and mean follow - up period was 7.7 years ( range , 3.711.6 ) .", "baseline visual acuity ( va ) was logmar 0.42 ( snellen equivalent 20/52 ) ( range , 0.181 ) .", "final va was logmar 1.16 ( snellen equivalent 20/289 ) ( range , 0.182.3 ) . in two eyes ,", "at baseline , the presence of a drusenoid ped with homogeneous hyperreflective contents and an intact overlying rpe was noted in six eyes ( 75% ; figs . 2 , 3 ) .", "one eye ( patient 5 , left eye ) had an extremely large ped measuring 2.24 mm with mostly hyporeflective contents and an overlying vitelliform lesion . in another eye ( patient 4 , left eye ) , the plateau was already present at baseline .", "pigmentary changes noted on cfp were also found on nir , rf , and faf in six eyes , and these corresponded to intraretinal hyperreflective foci on oct ( fig .", "3 ) . overlying the ped surface , the ellipsoid zone and outer nuclear layer ( onl ) were thin , with the opl nearly apposed to the surface of the ped .", "multimodal imaging of two patients ( a , b ) at baseline and follow - up ( a , 4 years ; b , 7 years ) .", "( a ) at baseline , patient 3 has a drusenoid ped with overlying pigment hyperplasia and hyperautofluorescent vitelliform material .", "the oct b - scan shows intraretinal hyperreflective foci ( red arrowhead ) and loss of outer retinal bands over the apex of the ped .", "four years later , a region of hypoautofluorescent ga contains a plateau that can not be appreciated in the cfp , nir , rf , or faf .", "the oct b - scan shows a plateau with a shape similar to the baseline ped .", "the downwardly deflecting opl ( yellow arrow ) has moved laterally as it follows the border of the advancing ga . there was marked attenuation and decreased reflectivity of the rpe - bl band .", "( b ) at baseline , patient 6 has a drusenoid ped with overlying pigment hyperplasia that is located inferior to an area of ga .", "bottom row : seven years later , the ga has markedly enlarged , and the ped is no longer visible on cfp , nir , rf , or faf . the oct b - scan shows that following progressive loss of the onl , the opl ( yellow arrow ) appears draped over a plateau with a shape similar to the baseline ped .", "during follow - up , onl thinning resulted in the opl approximating the surface of the ped in all cases in which the ped was present at baseline ( figs . 2 , 3 ) .", "as ga progressed , this point of approximation moved toward the edges of the ped base , following the advancing border of ga ( supplementary video ) . in all eyes , there was also progressive thinning of the hyperreflective rpe - bl complex and diminution of its reflectivity , beginning at the apex of the ped and advancing toward its edges .", "after rpe loss occurred over the entire ped surface , the plateau ( i.e. , a thin hyperreflective inner surface and an interior of heterogeneous and overall reduced reflectivity ) appeared . the plateau surface was highly reflective on both cross - sectional and en face oct ( fig .", "4 , en face oct surface ) and showed the presence of focal defects ( fig .", "4 , yellow arrows ) . on cross - sectional oct , the inner contents of the plateau consisted of round , hyporeflective areas ( fig .", "4 , blue arrows ) that were usually accompanied by smaller hyperreflective foci seen beneath the hyperreflective border .", "these hyperreflective foci also could be observed within the area of ga as hyperautofluorescent foci on faf and hyperreflective foci on en face oct ( fig .", "4 , green arrows ) . accordingly , on en face oct , plateau contents were seen as honeycomb - like structures with hyporeflective spaces interspersed with hyperreflective foci ( fig .", "4 , green arrows ) increased in abundance within the plateau interior during follow - up in all eyes as seen on cross - sectional and en face oct ( figs . 2 , 3 , 4 ) .", "on both cross - sectional and en face octa , no abnormal flow signals were noted within plateaus ( fig .", "( a ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct ,", "octa , and on oct surface ( an en face oct slab at the level of the rpe - bl atop the plateau ) , the plateau interior is bounded by a distinct hyperreflective band that continues as a thick and highly reflective rpe band .", "this hyperreflective surface shows transient hyporeflective defects ( yellow arrows ) during the evolution of these lesions .", "an en face oct image of the plateau surface allows better appreciation of the focal defects ( yellow arrow ) .", "( b ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct and octa ,", "a distinct hyperreflective surface surrounds the plateau interior and focal defects ( yellow arrows ) can be seen in some areas .", "an en face oct taken at the plateau surface allows better appreciation of focal defects in this hyperreflective layer ( en face octa : surface yellow arrow ) .", "deep to these focal defects are hyperreflective features ( faf and en face oct : green arrowheads ) that are also seen on faf as punctate hyperfluorescence within the confluent area of hypoautofluorescence .", "in seven of eight eyes , the plateau was located in the macula . in one eye ,", "a single plateau was seen in all eyes except for one ( patient 2 ) that had two separate plateaus ( fig .", "patient 6 had one plateau in each eye . in the seven eyes with a baseline ped ,", "the thinning of the hyperreflective band over the ped surface after rpe loss ( at which point we consider the plateau to be formed ) was preceded by a sharp decrease in ped volume ( fig .", "subsequently , the volume of the residual plateau appeared very stable over time ( fig .", "the point at which the plateaus form ( complete loss of rpe from the hyperreflective rpe - bl complex over the ped ) is shown by the dotted line .", "patient 2 has two separate peds that evolve to two separate plateaus ( a , b ) .", "patient 6 has a plateau in both the right eye ( r ) and the left eye ( l ) .", "only patients with an intact hyperreflective rpe - bl complex baseline were included in this analysis . when the plateau signature is first observed ( black dotted line ) , a decrease in the volume that stabilizes on further follow - up .", "figure 6 shows histology of a donor eye with ga , for which local relationships and cellular processes in the interior of outer retinal corrugations were particularly clear yet also representative of other ga eyes available for review .", "atrophy spared the fovea in this eye , as seen with ex vivo cfp and 488-nm autofluorescence , and small reflective puncta in the atrophic area were apparent on nir ( figs .", "6e ) , blamd was seen as discontinuous , being present at three separate locations .", "pigmented cells were of the  subducted  phenotype , that is , rpe - originated cells located external to blamd , in contact with brm . for reference , in the henle fiber layer of a normal eye , obliquely oriented mller cell processes are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , including ort in this ga eye , the trajectories of gliotic mller cell processes were oriented in many directions , as indicated by arrows in figure 6e . in one instance", ", processes seemed to enter an outer retinal corrugation horizontally through an opening in the blamd and sweep the  subducted  cells off brm ( fig .", "6d ) . persistent bl deposit and mller cell processes in ga in a 95-year - old white man .", "( a  c ) ex vivo imaging of postmortem fundus . ( a ) cfp ( top row left ) shows a large area of atrophy of rpe extending to the optic nerve head but sparing the fovea .", "green arrowheads approximate the level of the histologic section shown in the top far right and bottom .", "( b ) ex vivo 488-nm autofluorescence shows loss of signal in the atrophic area .", "( c ) ex vivo nir imaging shows small and highly reflective puncta in the atrophic area .", "detailed ( d ) and panoramic ( e ) views of the atrophic area in histology .", "nfl , nerve fiber layer ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; dr , calcified druse .", "( d ) processes from mller cells under a corrugation of persistent blamd in the atrophic area .", "this corrugation overlies  subducted  cells of rpe origin ( teal arrowheads ) , which contain nuclei , spindle - shaped melanosomes , and lipofuscin , and are located external to blamd , on brm .", "mller cell processes appear to enter the corrugation horizontally from the left ( yellow arrow ) and sweep the pigmented cells away .", "( e ) blamd is discontinuous and present at three separate locations ( red arrows ) .", " in the normal henle fiber layer , mller cell processes are obliquely oriented ( outer - center to inner - periphery ) , and they are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , the trajectories of gliotic mller cell processes and remaining photoreceptors are oriented in many directions ( yellow arrows ) .", "through a retrospective review of amd patients with long - term serial eye - tracked sd - oct , we identified a series of eyes in which drusenoid peds progressed into plateau signatures within areas of ga .", "plateaus were seen on oct to contain heterogeneous reflectivity enclosed by a thin overlying hyperreflective surface . a focal defect in the hyperreflective surface , in addition to intraretinal and sub - rpe hyperreflective dots , was observed during the evolution of ped into plateau signatures .", "opl subsidence was noted to begin at the surface of the ped and progress toward the edge following the areas of expanding atrophy .", "there was a gradual increase in volume due to an increase in hyporeflective contents initially , followed by complete loss of the rpe and a sharp decrease in ped volume , at which time the plateau was considered to have formed .", "the study of drusen life cycles and ped evolution are important to improve our understanding of the pathogenesis of amd . understanding the origins and evolution of various oct findings and correlating these findings to histology enable the in vivo study of microscopic cellular changes occurring in these eyes with progressive ga .", "the development of ga has been previously reported to occur in 19% of eyes with drusenoid peds and to be associated with specific features such as subsidence of the opl and inner nuclear layer and the development of a hyporeflective  wedge - shaped band  within the henle fiber layer described by mons et al .", "the appearance of plateaus should be distinguished from other signatures previously described in ga , such as ort , ghost drusen , hyperreflective crown - like structures , hps , and refractile drusen ( figs . 1 , 2 ) .", "plateau boundaries are difficult to distinguish from the surrounding atrophy using cfp , nir , rf , and faf ( figs . 1 , 3 , 4 , 6 ) .", "previous studies have reported focal areas of hyperautofluorescence within ga , and in our study these correlated with hyperreflective foci seen on en face oct ( fig .", "the presence of many pigmented cells of apparent rpe origin within atrophic areas is now well - documented ( fig .", "6 ) , and such cells could give rise to both autofluorescence and oct hyperreflectivity signals .", "plateaus do not contain the highly refractile material as seen on cfp and nir in eyes with spherules in calcifying drusen .", "1 ) have a similar overlying thin hyperreflective layer consistent with an overlying blamd drape , as previously observed in oct and in histology ( fig .", "our current data support a previous study that suggested that plateaus occur at a low frequency in eyes with ga ( 7.5% ) .", "the plateau is a signature of a residual structure seen on oct once the rpe has progressed into complete atrophy . in our study , after plateau formation as defined by loss of the overlying rpe layer exposing the underlying blamd that then persists , minimal changes in plateau volume were observed on subsequent follow - up , as previously seen . in all eyes of our series , a drusenoid ped was noted to precede the appearance of the plateau . in a prior description ,", "plateaus were thought to evolve in the outer retina and the hyperreflective line that is likely persistent blamd was attributed to the opl .", "in contrast , serial eye - tracked oct imaging in our cases shows that changes occurred in the sub - rpe compartment , defined in atrophic eyes as the space external to persistent blamd . during development of a plateau , its thin hyperreflective surface ,", "having emerged after rpe atrophy , is readily distinguishable from the overlying subsiding opl ( figs . 1 , 2 ) .", "plateaus are one route from drusenoid ped to atrophy , and they can be contrasted with a route via ped growth and collapse , which we recently described . growth and collapse of large ped appear to be on a continuum with similar changes described for smaller drusen", ". why a ped in any one eye proceeds to a plateau or to collapse is not known at present .", "our detailed description of plateaus will facilitate mechanistic thinking and guide the design of future studies with more patients to investigate associated risk factors that may have prognostic value .", "evolution of a plateau entails at least four processes that are discernible at the histologic level and also in sd - oct . in", "approximately chronologic order , the sequence of the evolution is as follows : loss of rpe , persistence of blamd , followed by concurrent clearing of druse contents and occupation of druse space by cellular processes .", "the loss of rpe is the point at which a drusenoid ped becomes a plateau , and it is marked by a thick , roughened , and highly hyperreflective band becoming thinner , smoother , and moderately hyperreflective .", "hyperreflective foci over ped destined for plateaus were noted in our series , suggestive of anterior rpe migration , as also seen over ped destined for collapse .", "the development of ga is hypothesized to proceed via two main pathways of rpe fate , including migration of fully pigmented and nucleated rpe into the retina ( called  sloughed  and  intraretinal  ) and shedding of non - nucleated granule aggregates , possibly apoptotic remnants , into underlying blamd .", "our previous studies correlated intraretinal hyperreflective foci seen on in vivo and ex vivo oct with anteriorly migrating rpe cells .", "thus , we have good reason to suspect that the spherical hyperreflective intraretinal foci directly internal to the plateaus are also migrating rpe cells . the thin , moderately hyperreflective band draped over plateaus", "blamd is a stereotypically structured thick layer of basement membrane material that was initially used by sarks et al . to stage amd eyes and", "a basement membrane is a defining feature of an epithelium , as is the rpe .", "it is crossed by lipoprotein particles en route to brm and choriocapillaris for apparent egress to the systemic circulation .", "when blamd is thick enough to be visible , it is observable as a hyporeflective layer .", "after the rpe atrophies and can no longer cast a shadow , blamd emerges as a moderately hyperreflective line of overall horizontal orientation that is sometimes corrugated .", "outer retinal corrugations were proposed to be contraction folds of persistent blamd created by loss of neovascular tissue or drusen / basal linear deposit previously located in the sub - rpe space . here", "we extend this model to large drusenoid ped , on the grounds that corrugations and plateaus represent a continuum of oct signatures involving persistent blamd .", "further , we propose on the basis of prior pathology literature a possible mechanism for how sub - rpe material may be cleared and the shape of persistent blamd established .", "our clinical series showed that most of the peds that evolved into plateaus were drusenoid , and druse contents ( e.g. , lipids , proteins , and minerals ) appear to be removed , replaced , or both , over time .", "the cells and secreted factors ( e.g. , metalloproteinases , lipases ) responsible for druse removal are not established .", "mller cells are candidates , because previous studies have shown that mller cell processes may protrude into the sub - rpe space , extend over brm , and break up persistent blamd .", "gliosis indicated by intense glial fibrillary acidic protein immunoreactivity is prominent in areas of severe photoreceptor loss and rpe atrophy , and mller cells remain when all photoreceptors have died . in our exemplar histologic case ,", "we hypothesize that plateau and corrugation formation requires extension of mller cell processes through defects in the rpe / blamd complex ( figs . 4 , 6 ) that occur during the life cycle of drusenoid peds .", "we further hypothesize that increasing hyporeflectivity observed within the sub - rpe space during the progression of drusenoid peds represents a change in composition of the sub - rpe material that encourages the entry of these processes .", "the strength of this research is the long follow - up period for our patient cohort , with serial eye - tracked sd - oct imaging for up to 7 years and notably dating back to before the development of ga in all but one eye .", "another strength is the availability of up to nine separate imaging technologies , each specifically highlighting different tissue features in any one case and the availability of one illustrative histology case .", "limitations include the small number of eyes and the absence of direct correlation of in vivo imaging to postmortem histology ; hence , although our hypothesis of mller cells involvement in plateau formation is credible and testable , whether these cellular activities are directly visible in oct is currently unknown .", "our technique of volume measurement may be subject to variations according to oct b - scan density between patients , but for our purpose of studying progression on a per - patient basis , this limitation was acceptable . to minimize inconsistencies", ", all patients in our study had eye - tracked scans at each follow - up visit with similar density scan patterns available for analysis .", "our study adds value to existing literature on ped by suggesting a role of mller cell gliosis , elongation , and elaboration in the pathogenesis of amd , which should be further explored in future studies .", "it is important for clinicians to be aware of plateaus and to distinguish them from oct findings related to neovascular amd , such as vascularized peds or signs of exudation , as octa imaging of three eyes in our study showed no evidence of neovascular flow within plateaus ( fig .", "plateau structures should not be confused with areas of subretinal fluid and treatment is not indicated for this finding .", "finally , the distinctive reflectivity of persistent blamd in outer retinal corrugations , and , now , plateaus , provides strong motivation for renaming the fourth outer retinal hyperreflective band of normal eyes , which is currently called the rpe - brm complex , to the rpe - blam - brm complex ( abbreviated as rbb ) to accommodate the appearance of ped and persistent blamd in eyes with amd ."], "abstract_text": ["<S> purposehistologic details of progression routes to geographic atrophy ( ga ) in amd are becoming available through optical coherence tomography ( oct ) . </S>", "<S> we studied the origins and evolution of an oct signature called plateau in eyes with ga and suggested a histologic correlate.methodsserial eye - tracked oct scans and multimodal imaging were acquired from eight eyes of seven patients with ga and plateau signatures over a mean follow - up of 7.7 years ( range , 3.711.6 ) . </S>", "<S> the histology of unrelated donor eyes with amd was reviewed.resultsdrusenoid pigment epithelial detachment ( ped ) on oct imaging progressed into wide - based mound - like signatures with flattened apices characterized by a hyporeflective yet heterogeneous interior and an overlying hyperreflective exterior , similar to outer retinal corrugations previously ascribed to persistent basal laminar deposit ( blamd ) but larger . </S>", "<S> these new signatures are described as  plateaus . </S>", "<S>  an initial increase of the ped volume and hyporeflectivity of its contents was followed by a decrease in ped volume and thinning of an overlying hyperreflective band attributable to the loss of the overlying rpe leaving persistent blamd . </S>", "<S> both imaging and histology revealed persistent blamd with defects through which gliotic mller cell processes pass.conclusionsplateaus can be traced back to drusenoid peds on oct imaging . </S>", "<S> we hypothesize that during progressive rpe atrophy , mller cell extension through focal defects in the residual persistent blamd may contribute to the heterogeneous internal reflectivity of these entities . </S>", "<S> the role of mller cell activation and extension in the pathogenesis of amd should be explored in future studies . </S>"], "labels": null, "section_names": ["Methods", "Study Cohort", "Imaging Protocol and Analysis", "SD-OCT Terminology", "Quantification of Volume Changes During Progression", "Histology Study", "Results", "Patient Characteristics", "Baseline Features", "Characteristics Seen During Progression of Drusenoid PED into the Plateau", "Changes in Volume of the Drusenoid PED and Plateau Signature With Time", "Illustrative Histologic Case", "Discussion", "Supplementary Material"], "sections": [["this retrospective observational study was approved by the western institutional review board ( olympia , washington , usa ) .", "it complied with the health insurance portability and accountability act of 1996 and followed the tenets of the declaration of helsinki .", "informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study .", "patients with ga were identified from the tertiary practice of retinal specialists ( kbf , js , and ly ) if they exhibited plateau signatures in at least one eye ( study eye ) on oct imaging and if they also had at least 3 years ' follow - up with consecutive eye - tracked spectral - domain ( sd)-oct scans taken at least every 6 months .", "eyes with macular neovascularization , other retinal pathology , or media opacity preventing adequate imaging were excluded .", "medical records and multimodal imaging , including color fundus photography ( cfp ) , red - free photography ( rf ) , fundus autofluorescence ( faf ) , near - infrared reflectance scanning laser ophthalmoscopy ( nir ) , and sd - oct was performed on all the patients .", "cfp and rf were performed with a trc-50ix flood - illuminated fundus camera ( topcon medical systems , oakland , nj , usa ) .", "faf was performed with either the spectralis hra + oct ( heidelberg engineering , heidelberg , germany ) or the topcon trc-50ix fundus camera .", "oct scans taken before the acquisition of eye - tracked spectralis scans were assessed qualitatively but not included in the quantitative analysis .", "the sd - oct protocol used in all eyes comprised 20 horizontal raster line scans over the area of interest , ranging from 19 to 49 b - scans per eye , with each scan spaced 115 to 250 m apart , and automatic real - time averaging set between 4 and 23 . to study the origin and progression of plateaus , the most recent sd - oct with this signature was identified . then", ", serial eye - tracked b - scans of the region of interest were tracked back to baseline . in cases", "in which the scanning protocol varied during the course of follow - up , the b - scan closest to the area of interest was matched manually and extracted for analysis .", "these extracted sd - oct images were then stacked , aligned , and saved as a video file using the fiji distribution (  fiji is just image j , ", "http://fiji.sc ; in the public domain ) for the qualitative analysis of the progression of the lesion ( see supplementary video ) .", "at each time point , the plateau appearance on sd - oct was correlated to the appearance in cfp , nir , and faf .", "three patients had en face oct and oct angiography ( octa ) imaging with 3  3-mm macular cubes ( rtvue xr  avanti  ; optovue , fremont , ca , usa ) .", "the review software ( revue , version 2015.1.0.90 ; optovue , fremont , ca , usa ) displays flow signals superimposed in red on a cross - sectional structural sd - oct image . in a healthy eye ,", "the fourth outer retinal hyperreflective band includes the rpe and brm . in a ped , by definition", "in the course of this separation , either a basal lamina ( bl ) of 0.15-m thickness or a blamd of variable thickness adheres to the rpe and not to the brm .", "therefore , we call the hyperreflective band that anteriorly delimits a ped the rpe - bl complex . the definition of a  plateau  was a distinct oct signature where a mound - like structure with a flattened apex and a wide base was observed within an area of ga after complete loss of the overlying rpe . to determine the volume of the drusenoid ped and ensuing plateau ( fig .", "2 ) , the cavalieri principle of stereology was applied to sd - oct volumetric data acquired at each visit , as follows .", "( 1 ) each b - scan in the volume was scaled to a 1:1 pixel aspect ratio .", "the caliper function within the heidelberg eye explorer software ( version 6.3.4.0 ; heidelberg engineering ) was used to measure the area between the outer boundary of rpe+bl and the inner boundary of brm .", "( 2 ) the volume between two consecutive oct slices ( hereafter called a segment ) was then determined using the following formula : \n \\}\\begin{document}$$d\\left ( { \\left ( { a\\_x + a\\_\\left ( { x + 1 } \\right ) } \\right)/2 } \\right),$$\\end{document}where d is the distance between consecutive slices in m , a is the area between the rpe+bl and brm in m , and x is the oct slice number .", "( 3 ) total ped volume was calculated by summing the volumes of individual segments . the number of segments in the oct volume was ( n  1 ) , where n is the total number of slices that spanned the ped .", "graphical plots of ped volume with respect to time were generated using interval data . \n ", "origin and evolution of a plateau , as revealed by nir ( a ) and oct ( b ) images .", "green arrows on nir images show the levels of corresponding oct cross - sectional images .", "baseline images show several elevations of the rpe with largely hyporeflective interiors . at 3 years after baseline , punctate hyperreflectivity ( red arrowhead )", ", progressive loss of the onl has caused the opl ( yellow arrows ) to approach the surface of the ped .", "also at 4 years , a  mons hyperreflective wedge  ( blue arrowheads ) appears at the ga border . at 5 years", ", the downwardly deflected opl is seen to move progressively toward the edge of the plateau following the advancing border of ga .", "formation of the plateau at 5 years was characterized by the loss of the overlying rpe causing marked thinning of the hyperreflective rpe - basal lamina complex now reduced to just blamd , compared with at 4 years . at 7 years", "the institutional review board at university of alabama at birmingham ( uab ) approved the laboratory study , which complied with the health insurance portability and accountability act and adhered to the tenets of the declaration of helsinki .", "amd eyes were identified through an ex vivo imaging screen of eyes accessioned for research purposes from nondiabetic white donors to the alabama eye bank during the period 1996 to 2012 .", "median death - to - preservation time was 3:49 hours ( range , 0:4011:40 hours ) .", "eyes were preserved by immersion in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer following anterior segment excision .", "after vitreous removal , maculas were photographed in color on a stereomicroscope ( smz - u ; nikon , melville , ny , usa ) . when prepared for histology ( 20112013 ) and uploaded to the project macula web site ( http://projectmacula.cis.uab.edu , in the public domain )", ", eyes underwent additional multimodal ex vivo imaging . from each globe , an 8-mm - diameter full - thickness tissue punch containing the fovea and temporal portion of the optic nerve head was held in a tissue holder mounted on a spectralis , as described . a 30  20 sd - oct volume ( 143 scans , 30-m spacing ,", "automated real - time averaging [ art ] = 25 ) was captured along with red - free and near - infrared reflectance scanning laser ophthalmoscopic images .", "tissue punches were postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular lipid and embedded in epoxy resin ( polybed 812 ; polysciences , warrington , pa , usa ) .", "sub - micrometer - thick ( 0.8 m ) sections at 25- to 30-m intervals were stained with 1% toluidine blue for polychromaticity , scanned with a 40 objective , and reviewed and photodocumented with a 60 oil - immersion objective ( numerical aperture = 1.4 ) and digital camera ( xc10 ; olympus , center valley , pa , usa ) .", "images were adjusted for exposure , contrast , and sharpness ( photoshop cs6 , adobe , san jose , ca , usa ) ."], ["patients with ga were identified from the tertiary practice of retinal specialists ( kbf , js , and ly ) if they exhibited plateau signatures in at least one eye ( study eye ) on oct imaging and if they also had at least 3 years ' follow - up with consecutive eye - tracked spectral - domain ( sd)-oct scans taken at least every 6 months", ". eyes with macular neovascularization , other retinal pathology , or media opacity preventing adequate imaging were excluded ."], ["medical records and multimodal imaging , including color fundus photography ( cfp ) , red - free photography ( rf ) , fundus autofluorescence ( faf ) , near - infrared reflectance scanning laser ophthalmoscopy ( nir ) , and sd - oct was performed on all the patients .", "cfp and rf were performed with a trc-50ix flood - illuminated fundus camera ( topcon medical systems , oakland , nj , usa ) .", "faf was performed with either the spectralis hra + oct ( heidelberg engineering , heidelberg , germany ) or the topcon trc-50ix fundus camera .", "oct scans taken before the acquisition of eye - tracked spectralis scans were assessed qualitatively but not included in the quantitative analysis .", "the sd - oct protocol used in all eyes comprised 20 horizontal raster line scans over the area of interest , ranging from 19 to 49 b - scans per eye , with each scan spaced 115 to 250 m apart , and automatic real - time averaging set between 4 and 23 . to study the origin and progression of plateaus , the most recent sd - oct with this signature was identified .", "then , serial eye - tracked b - scans of the region of interest were tracked back to baseline . in cases", "in which the scanning protocol varied during the course of follow - up , the b - scan closest to the area of interest was matched manually and extracted for analysis .", "these extracted sd - oct images were then stacked , aligned , and saved as a video file using the fiji distribution (  fiji is just image j ,  http://fiji.sc ; in the public domain ) for the qualitative analysis of the progression of the lesion ( see supplementary video ) . at each time point , the plateau appearance on sd - oct was correlated to the appearance in cfp , nir , and faf .", "three patients had en face oct and oct angiography ( octa ) imaging with 3  3-mm macular cubes ( rtvue xr  avanti  ; optovue , fremont , ca , usa ) . the review software ( revue , version 2015.1.0.90 ; optovue , fremont , ca , usa ) displays flow signals superimposed in red on a cross - sectional structural sd - oct image"], ["in a healthy eye , the fourth outer retinal hyperreflective band includes the rpe and brm . in a ped , by definition", ", the rpe is separated from the brm . in the course of this separation , either a basal lamina ( bl ) of 0.15-m thickness or a blamd of variable thickness adheres to the rpe and not to the brm .", "therefore , we call the hyperreflective band that anteriorly delimits a ped the rpe - bl complex . the definition of a  plateau  was a distinct oct signature where a mound - like structure with a flattened apex and a wide base was observed within an area of ga after complete loss of the overlying rpe ."], ["2 ) , the cavalieri principle of stereology was applied to sd - oct volumetric data acquired at each visit , as follows . ( 1 ) each b - scan in the volume was scaled to a 1:1 pixel aspect ratio . the caliper function within the heidelberg eye explorer software ( version 6.3.4.0 ; heidelberg engineering )", "was used to measure the area between the outer boundary of rpe+bl and the inner boundary of brm .", "( 2 ) the volume between two consecutive oct slices ( hereafter called a segment ) was then determined using the following formula : \n \\}\\begin{document}$$d\\left ( { \\left ( { a\\_x + a\\_\\left ( { x + 1 } \\right ) } \\right)/2 } \\right),$$\\end{document}where d is the distance between consecutive slices in m , a is the area between the rpe+bl and brm in m , and x is the oct slice number . ( 3 ) total ped volume was calculated by summing the volumes of individual segments .", "the number of segments in the oct volume was ( n  1 ) , where n is the total number of slices that spanned the ped .", "graphical plots of ped volume with respect to time were generated using interval data . \n ", "origin and evolution of a plateau , as revealed by nir ( a ) and oct ( b ) images .", "green arrows on nir images show the levels of corresponding oct cross - sectional images .", "baseline images show several elevations of the rpe with largely hyporeflective interiors . at 3 years after baseline , punctate hyperreflectivity ( red arrowhead )", ", progressive loss of the onl has caused the opl ( yellow arrows ) to approach the surface of the ped . also at 4 years , a ", "mons hyperreflective wedge  ( blue arrowheads ) appears at the ga border . at 5 years", ", the downwardly deflected opl is seen to move progressively toward the edge of the plateau following the advancing border of ga .", "formation of the plateau at 5 years was characterized by the loss of the overlying rpe causing marked thinning of the hyperreflective rpe - basal lamina complex now reduced to just blamd , compared with at 4 years . at 7 years , a closed ort ( pink arrowhead ) is present adjacent to the plateau ."], ["the institutional review board at university of alabama at birmingham ( uab ) approved the laboratory study , which complied with the health insurance portability and accountability act and adhered to the tenets of the declaration of helsinki .", "amd eyes were identified through an ex vivo imaging screen of eyes accessioned for research purposes from nondiabetic white donors to the alabama eye bank during the period 1996 to 2012 .", "median death - to - preservation time was 3:49 hours ( range , 0:4011:40 hours ) .", "eyes were preserved by immersion in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer following anterior segment excision .", "after vitreous removal , maculas were photographed in color on a stereomicroscope ( smz - u ; nikon , melville , ny , usa ) . when prepared for histology ( 20112013 ) and uploaded to the project macula web site ( http://projectmacula.cis.uab.edu , in the public domain )", ", eyes underwent additional multimodal ex vivo imaging . from each globe , an 8-mm - diameter full - thickness tissue punch containing the fovea and temporal portion of the optic nerve head was held in a tissue holder mounted on a spectralis , as described . a 30  20 sd - oct volume ( 143 scans , 30-m spacing ,", "automated real - time averaging [ art ] = 25 ) was captured along with red - free and near - infrared reflectance scanning laser ophthalmoscopic images .", "tissue punches were postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular lipid and embedded in epoxy resin ( polybed 812 ; polysciences , warrington , pa , usa ) .", "sub - micrometer - thick ( 0.8 m ) sections at 25- to 30-m intervals were stained with 1% toluidine blue for polychromaticity , scanned with a 40 objective , and reviewed and photodocumented with a 60 oil - immersion objective ( numerical aperture = 1.4 ) and digital camera ( xc10 ; olympus , center valley , pa , usa ) .", "images were adjusted for exposure , contrast , and sharpness ( photoshop cs6 , adobe , san jose , ca , usa ) ."], ["plateau signatures were observed in eight eyes of seven patients , of whom six were female .", "mean age was 75 years ( range , 6089 ) and mean follow - up period was 7.7 years ( range , 3.711.6 ) .", "baseline visual acuity ( va ) was logmar 0.42 ( snellen equivalent 20/52 ) ( range , 0.181 ) .", "final va was logmar 1.16 ( snellen equivalent 20/289 ) ( range , 0.182.3 ) . in two eyes ,", "ga spared the fovea throughout the entire course of the follow - up . at baseline", ", the presence of a drusenoid ped with homogeneous hyperreflective contents and an intact overlying rpe was noted in six eyes ( 75% ; figs . 2 , 3 ) .", "one eye ( patient 5 , left eye ) had an extremely large ped measuring 2.24 mm with mostly hyporeflective contents and an overlying vitelliform lesion . in another eye ( patient 4 , left eye ) , the plateau was already present at baseline .", "pigmentary changes noted on cfp were also found on nir , rf , and faf in six eyes , and these corresponded to intraretinal hyperreflective foci on oct ( fig .", "3 ) . overlying the ped surface , the ellipsoid zone and outer nuclear layer ( onl ) were thin , with the opl nearly apposed to the surface of the ped .", "b ) at baseline and follow - up ( a , 4 years ; b , 7 years ) . (", "a ) at baseline , patient 3 has a drusenoid ped with overlying pigment hyperplasia and hyperautofluorescent vitelliform material .", "the oct b - scan shows intraretinal hyperreflective foci ( red arrowhead ) and loss of outer retinal bands over the apex of the ped .", "four years later , a region of hypoautofluorescent ga contains a plateau that can not be appreciated in the cfp , nir , rf , or faf .", "the oct b - scan shows a plateau with a shape similar to the baseline ped .", "the downwardly deflecting opl ( yellow arrow ) has moved laterally as it follows the border of the advancing ga . there was marked attenuation and decreased reflectivity of the rpe - bl band .", "( b ) at baseline , patient 6 has a drusenoid ped with overlying pigment hyperplasia that is located inferior to an area of ga .", "bottom row : seven years later , the ga has markedly enlarged , and the ped is no longer visible on cfp , nir , rf , or faf .", "the oct b - scan shows that following progressive loss of the onl , the opl ( yellow arrow ) appears draped over a plateau with a shape similar to the baseline ped .", "hyporeflective areas ( blue arrowhead ) are visible within the plateau interior . during follow - up ,", "onl thinning resulted in the opl approximating the surface of the ped in all cases in which the ped was present at baseline ( figs . 2 , 3 ) .", "as ga progressed , this point of approximation moved toward the edges of the ped base , following the advancing border of ga ( supplementary video ) . in all eyes , there was also progressive thinning of the hyperreflective rpe - bl complex and diminution of its reflectivity , beginning at the apex of the ped and advancing toward its edges .", "after rpe loss occurred over the entire ped surface , the plateau ( i.e. , a thin hyperreflective inner surface and an interior of heterogeneous and overall reduced reflectivity ) appeared . the plateau surface was highly reflective on both cross - sectional and en face oct ( fig .", "4 , en face oct surface ) and showed the presence of focal defects ( fig .", "4 , blue arrows ) that were usually accompanied by smaller hyperreflective foci seen beneath the hyperreflective border .", "these hyperreflective foci also could be observed within the area of ga as hyperautofluorescent foci on faf and hyperreflective foci on en face oct ( fig .", "4 , green arrows ) . accordingly , on en face oct , plateau contents were seen as honeycomb - like structures with hyporeflective spaces interspersed with hyperreflective foci ( fig .", "4 , green arrows ) increased in abundance within the plateau interior during follow - up in all eyes as seen on cross - sectional and en face oct ( figs . 2 , 3 , 4 ) .", "on both cross - sectional and en face octa , no abnormal flow signals were noted within plateaus ( fig .", "( a ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct ,", "octa , and on oct surface ( an en face oct slab at the level of the rpe - bl atop the plateau ) , the plateau interior is bounded by a distinct hyperreflective band that continues as a thick and highly reflective rpe band .", "this hyperreflective surface shows transient hyporeflective defects ( yellow arrows ) during the evolution of these lesions .", "an en face oct image of the plateau surface allows better appreciation of the focal defects ( yellow arrow ) .", "( b ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct and octa", ", a distinct hyperreflective surface surrounds the plateau interior and focal defects ( yellow arrows ) can be seen in some areas . an en face", "oct taken at the plateau surface allows better appreciation of focal defects in this hyperreflective layer ( en face octa : surface yellow arrow ) .", "deep to these focal defects are hyperreflective features ( faf and en face oct : green arrowheads ) that are also seen on faf as punctate hyperfluorescence within the confluent area of hypoautofluorescence .", "a single plateau was seen in all eyes except for one ( patient 2 ) that had two separate plateaus ( fig .", "patient 6 had one plateau in each eye . in the seven eyes with a baseline ped ,", "the thinning of the hyperreflective band over the ped surface after rpe loss ( at which point we consider the plateau to be formed ) was preceded by a sharp decrease in ped volume ( fig .", "subsequently , the volume of the residual plateau appeared very stable over time ( fig .", "the point at which the plateaus form ( complete loss of rpe from the hyperreflective rpe - bl complex over the ped ) is shown by the dotted line .", "patient 2 has two separate peds that evolve to two separate plateaus ( a , b ) .", "patient 6 has a plateau in both the right eye ( r ) and the left eye ( l ) .", "only patients with an intact hyperreflective rpe - bl complex baseline were included in this analysis .", "when the plateau signature is first observed ( black dotted line ) , a decrease in the volume that stabilizes on further follow - up .", "figure 6 shows histology of a donor eye with ga , for which local relationships and cellular processes in the interior of outer retinal corrugations were particularly clear yet also representative of other ga eyes available for review .", "atrophy spared the fovea in this eye , as seen with ex vivo cfp and 488-nm autofluorescence , and small reflective puncta in the atrophic area were apparent on nir ( figs .", "6e ) , blamd was seen as discontinuous , being present at three separate locations .", "pigmented cells were of the  subducted  phenotype , that is , rpe - originated cells located external to blamd , in contact with brm . for reference , in the henle fiber layer of a normal eye , obliquely oriented mller cell processes are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration ,", "including ort in this ga eye , the trajectories of gliotic mller cell processes were oriented in many directions , as indicated by arrows in figure 6e . in one instance", ", processes seemed to enter an outer retinal corrugation horizontally through an opening in the blamd and sweep the  subducted  cells off brm ( fig .", "6d ) . persistent bl deposit and mller cell processes in ga in a 95-year - old white man .", "cfp ( top row left ) shows a large area of atrophy of rpe extending to the optic nerve head but sparing the fovea .", "green arrowheads approximate the level of the histologic section shown in the top far right and bottom .", "( b ) ex vivo 488-nm autofluorescence shows loss of signal in the atrophic area .", "( c ) ex vivo nir imaging shows small and highly reflective puncta in the atrophic area .", "detailed ( d ) and panoramic ( e ) views of the atrophic area in histology .", "nfl , nerve fiber layer ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; dr , calcified druse .", "( d ) processes from mller cells under a corrugation of persistent blamd in the atrophic area .", "this corrugation overlies  subducted  cells of rpe origin ( teal arrowheads ) , which contain nuclei , spindle - shaped melanosomes , and lipofuscin , and are located external to blamd , on brm .", "mller cell processes appear to enter the corrugation horizontally from the left ( yellow arrow ) and sweep the pigmented cells away .", "( e ) blamd is discontinuous and present at three separate locations ( red arrows ) .", " in the normal henle fiber layer , mller cell processes are obliquely oriented ( outer - center to inner - periphery ) , and they are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , the trajectories of gliotic mller cell processes and remaining photoreceptors are oriented in many directions ( yellow arrows ) ."], ["plateau signatures were observed in eight eyes of seven patients , of whom six were female .", "mean age was 75 years ( range , 6089 ) and mean follow - up period was 7.7 years ( range , 3.711.6 ) .", "baseline visual acuity ( va ) was logmar 0.42 ( snellen equivalent 20/52 ) ( range , 0.181 ) .", "final va was logmar 1.16 ( snellen equivalent 20/289 ) ( range , 0.182.3 ) . in two eyes ,"], ["at baseline , the presence of a drusenoid ped with homogeneous hyperreflective contents and an intact overlying rpe was noted in six eyes ( 75% ; figs . 2 , 3 ) .", "one eye ( patient 5 , left eye ) had an extremely large ped measuring 2.24 mm with mostly hyporeflective contents and an overlying vitelliform lesion . in another eye ( patient 4 , left eye ) , the plateau was already present at baseline .", "pigmentary changes noted on cfp were also found on nir , rf , and faf in six eyes , and these corresponded to intraretinal hyperreflective foci on oct ( fig .", "3 ) . overlying the ped surface , the ellipsoid zone and outer nuclear layer ( onl ) were thin , with the opl nearly apposed to the surface of the ped .", "multimodal imaging of two patients ( a , b ) at baseline and follow - up ( a , 4 years ; b , 7 years ) .", "( a ) at baseline , patient 3 has a drusenoid ped with overlying pigment hyperplasia and hyperautofluorescent vitelliform material .", "the oct b - scan shows intraretinal hyperreflective foci ( red arrowhead ) and loss of outer retinal bands over the apex of the ped .", "four years later , a region of hypoautofluorescent ga contains a plateau that can not be appreciated in the cfp , nir , rf , or faf .", "the oct b - scan shows a plateau with a shape similar to the baseline ped .", "the downwardly deflecting opl ( yellow arrow ) has moved laterally as it follows the border of the advancing ga . there was marked attenuation and decreased reflectivity of the rpe - bl band .", "( b ) at baseline , patient 6 has a drusenoid ped with overlying pigment hyperplasia that is located inferior to an area of ga .", "bottom row : seven years later , the ga has markedly enlarged , and the ped is no longer visible on cfp , nir , rf , or faf . the oct b - scan shows that following progressive loss of the onl , the opl ( yellow arrow ) appears draped over a plateau with a shape similar to the baseline ped ."], ["during follow - up , onl thinning resulted in the opl approximating the surface of the ped in all cases in which the ped was present at baseline ( figs . 2 , 3 ) .", "as ga progressed , this point of approximation moved toward the edges of the ped base , following the advancing border of ga ( supplementary video ) . in all eyes , there was also progressive thinning of the hyperreflective rpe - bl complex and diminution of its reflectivity , beginning at the apex of the ped and advancing toward its edges .", "after rpe loss occurred over the entire ped surface , the plateau ( i.e. , a thin hyperreflective inner surface and an interior of heterogeneous and overall reduced reflectivity ) appeared . the plateau surface was highly reflective on both cross - sectional and en face oct ( fig .", "4 , en face oct surface ) and showed the presence of focal defects ( fig .", "4 , yellow arrows ) . on cross - sectional oct , the inner contents of the plateau consisted of round , hyporeflective areas ( fig .", "4 , blue arrows ) that were usually accompanied by smaller hyperreflective foci seen beneath the hyperreflective border .", "these hyperreflective foci also could be observed within the area of ga as hyperautofluorescent foci on faf and hyperreflective foci on en face oct ( fig .", "4 , green arrows ) . accordingly , on en face oct , plateau contents were seen as honeycomb - like structures with hyporeflective spaces interspersed with hyperreflective foci ( fig .", "4 , green arrows ) increased in abundance within the plateau interior during follow - up in all eyes as seen on cross - sectional and en face oct ( figs . 2 , 3 , 4 ) .", "on both cross - sectional and en face octa , no abnormal flow signals were noted within plateaus ( fig .", "( a ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct ,", "octa , and on oct surface ( an en face oct slab at the level of the rpe - bl atop the plateau ) , the plateau interior is bounded by a distinct hyperreflective band that continues as a thick and highly reflective rpe band .", "this hyperreflective surface shows transient hyporeflective defects ( yellow arrows ) during the evolution of these lesions .", "an en face oct image of the plateau surface allows better appreciation of the focal defects ( yellow arrow ) .", "( b ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct and octa ,", "a distinct hyperreflective surface surrounds the plateau interior and focal defects ( yellow arrows ) can be seen in some areas .", "an en face oct taken at the plateau surface allows better appreciation of focal defects in this hyperreflective layer ( en face octa : surface yellow arrow ) .", "deep to these focal defects are hyperreflective features ( faf and en face oct : green arrowheads ) that are also seen on faf as punctate hyperfluorescence within the confluent area of hypoautofluorescence ."], ["in seven of eight eyes , the plateau was located in the macula . in one eye ,", "a single plateau was seen in all eyes except for one ( patient 2 ) that had two separate plateaus ( fig .", "patient 6 had one plateau in each eye . in the seven eyes with a baseline ped ,", "the thinning of the hyperreflective band over the ped surface after rpe loss ( at which point we consider the plateau to be formed ) was preceded by a sharp decrease in ped volume ( fig .", "subsequently , the volume of the residual plateau appeared very stable over time ( fig .", "the point at which the plateaus form ( complete loss of rpe from the hyperreflective rpe - bl complex over the ped ) is shown by the dotted line .", "patient 2 has two separate peds that evolve to two separate plateaus ( a , b ) .", "patient 6 has a plateau in both the right eye ( r ) and the left eye ( l ) .", "only patients with an intact hyperreflective rpe - bl complex baseline were included in this analysis . when the plateau signature is first observed ( black dotted line ) , a decrease in the volume that stabilizes on further follow - up ."], ["figure 6 shows histology of a donor eye with ga , for which local relationships and cellular processes in the interior of outer retinal corrugations were particularly clear yet also representative of other ga eyes available for review .", "atrophy spared the fovea in this eye , as seen with ex vivo cfp and 488-nm autofluorescence , and small reflective puncta in the atrophic area were apparent on nir ( figs .", "6e ) , blamd was seen as discontinuous , being present at three separate locations .", "pigmented cells were of the  subducted  phenotype , that is , rpe - originated cells located external to blamd , in contact with brm . for reference , in the henle fiber layer of a normal eye , obliquely oriented mller cell processes are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , including ort in this ga eye , the trajectories of gliotic mller cell processes were oriented in many directions , as indicated by arrows in figure 6e . in one instance", ", processes seemed to enter an outer retinal corrugation horizontally through an opening in the blamd and sweep the  subducted  cells off brm ( fig .", "6d ) . persistent bl deposit and mller cell processes in ga in a 95-year - old white man .", "( a  c ) ex vivo imaging of postmortem fundus . ( a ) cfp ( top row left ) shows a large area of atrophy of rpe extending to the optic nerve head but sparing the fovea .", "green arrowheads approximate the level of the histologic section shown in the top far right and bottom .", "( b ) ex vivo 488-nm autofluorescence shows loss of signal in the atrophic area .", "( c ) ex vivo nir imaging shows small and highly reflective puncta in the atrophic area .", "detailed ( d ) and panoramic ( e ) views of the atrophic area in histology .", "nfl , nerve fiber layer ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; dr , calcified druse .", "( d ) processes from mller cells under a corrugation of persistent blamd in the atrophic area .", "this corrugation overlies  subducted  cells of rpe origin ( teal arrowheads ) , which contain nuclei , spindle - shaped melanosomes , and lipofuscin , and are located external to blamd , on brm .", "mller cell processes appear to enter the corrugation horizontally from the left ( yellow arrow ) and sweep the pigmented cells away .", "( e ) blamd is discontinuous and present at three separate locations ( red arrows ) .", " in the normal henle fiber layer , mller cell processes are obliquely oriented ( outer - center to inner - periphery ) , and they are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , the trajectories of gliotic mller cell processes and remaining photoreceptors are oriented in many directions ( yellow arrows ) ."], ["through a retrospective review of amd patients with long - term serial eye - tracked sd - oct , we identified a series of eyes in which drusenoid peds progressed into plateau signatures within areas of ga .", "plateaus were seen on oct to contain heterogeneous reflectivity enclosed by a thin overlying hyperreflective surface . a focal defect in the hyperreflective surface , in addition to intraretinal and sub - rpe hyperreflective dots , was observed during the evolution of ped into plateau signatures .", "opl subsidence was noted to begin at the surface of the ped and progress toward the edge following the areas of expanding atrophy .", "there was a gradual increase in volume due to an increase in hyporeflective contents initially , followed by complete loss of the rpe and a sharp decrease in ped volume , at which time the plateau was considered to have formed .", "the study of drusen life cycles and ped evolution are important to improve our understanding of the pathogenesis of amd . understanding the origins and evolution of various oct findings and correlating these findings to histology enable the in vivo study of microscopic cellular changes occurring in these eyes with progressive ga .", "the development of ga has been previously reported to occur in 19% of eyes with drusenoid peds and to be associated with specific features such as subsidence of the opl and inner nuclear layer and the development of a hyporeflective  wedge - shaped band  within the henle fiber layer described by mons et al .", "the appearance of plateaus should be distinguished from other signatures previously described in ga , such as ort , ghost drusen , hyperreflective crown - like structures , hps , and refractile drusen ( figs . 1 , 2 ) .", "plateau boundaries are difficult to distinguish from the surrounding atrophy using cfp , nir , rf , and faf ( figs . 1 , 3 , 4 , 6 ) .", "previous studies have reported focal areas of hyperautofluorescence within ga , and in our study these correlated with hyperreflective foci seen on en face oct ( fig .", "the presence of many pigmented cells of apparent rpe origin within atrophic areas is now well - documented ( fig .", "6 ) , and such cells could give rise to both autofluorescence and oct hyperreflectivity signals .", "plateaus do not contain the highly refractile material as seen on cfp and nir in eyes with spherules in calcifying drusen .", "1 ) have a similar overlying thin hyperreflective layer consistent with an overlying blamd drape , as previously observed in oct and in histology ( fig .", "our current data support a previous study that suggested that plateaus occur at a low frequency in eyes with ga ( 7.5% ) .", "the plateau is a signature of a residual structure seen on oct once the rpe has progressed into complete atrophy . in our study , after plateau formation as defined by loss of the overlying rpe layer exposing the underlying blamd that then persists , minimal changes in plateau volume were observed on subsequent follow - up , as previously seen . in all eyes of our series , a drusenoid ped was noted to precede the appearance of the plateau . in a prior description ,", "plateaus were thought to evolve in the outer retina and the hyperreflective line that is likely persistent blamd was attributed to the opl .", "in contrast , serial eye - tracked oct imaging in our cases shows that changes occurred in the sub - rpe compartment , defined in atrophic eyes as the space external to persistent blamd . during development of a plateau , its thin hyperreflective surface ,", "having emerged after rpe atrophy , is readily distinguishable from the overlying subsiding opl ( figs . 1 , 2 ) .", "plateaus are one route from drusenoid ped to atrophy , and they can be contrasted with a route via ped growth and collapse , which we recently described . growth and collapse of large ped appear to be on a continuum with similar changes described for smaller drusen", ". why a ped in any one eye proceeds to a plateau or to collapse is not known at present .", "our detailed description of plateaus will facilitate mechanistic thinking and guide the design of future studies with more patients to investigate associated risk factors that may have prognostic value .", "evolution of a plateau entails at least four processes that are discernible at the histologic level and also in sd - oct . in", "approximately chronologic order , the sequence of the evolution is as follows : loss of rpe , persistence of blamd , followed by concurrent clearing of druse contents and occupation of druse space by cellular processes .", "the loss of rpe is the point at which a drusenoid ped becomes a plateau , and it is marked by a thick , roughened , and highly hyperreflective band becoming thinner , smoother , and moderately hyperreflective .", "hyperreflective foci over ped destined for plateaus were noted in our series , suggestive of anterior rpe migration , as also seen over ped destined for collapse .", "the development of ga is hypothesized to proceed via two main pathways of rpe fate , including migration of fully pigmented and nucleated rpe into the retina ( called  sloughed  and  intraretinal  ) and shedding of non - nucleated granule aggregates , possibly apoptotic remnants , into underlying blamd .", "our previous studies correlated intraretinal hyperreflective foci seen on in vivo and ex vivo oct with anteriorly migrating rpe cells .", "thus , we have good reason to suspect that the spherical hyperreflective intraretinal foci directly internal to the plateaus are also migrating rpe cells . the thin , moderately hyperreflective band draped over plateaus", "blamd is a stereotypically structured thick layer of basement membrane material that was initially used by sarks et al . to stage amd eyes and", "a basement membrane is a defining feature of an epithelium , as is the rpe .", "it is crossed by lipoprotein particles en route to brm and choriocapillaris for apparent egress to the systemic circulation .", "when blamd is thick enough to be visible , it is observable as a hyporeflective layer .", "after the rpe atrophies and can no longer cast a shadow , blamd emerges as a moderately hyperreflective line of overall horizontal orientation that is sometimes corrugated .", "outer retinal corrugations were proposed to be contraction folds of persistent blamd created by loss of neovascular tissue or drusen / basal linear deposit previously located in the sub - rpe space . here", "we extend this model to large drusenoid ped , on the grounds that corrugations and plateaus represent a continuum of oct signatures involving persistent blamd .", "further , we propose on the basis of prior pathology literature a possible mechanism for how sub - rpe material may be cleared and the shape of persistent blamd established .", "our clinical series showed that most of the peds that evolved into plateaus were drusenoid , and druse contents ( e.g. , lipids , proteins , and minerals ) appear to be removed , replaced , or both , over time .", "the cells and secreted factors ( e.g. , metalloproteinases , lipases ) responsible for druse removal are not established .", "mller cells are candidates , because previous studies have shown that mller cell processes may protrude into the sub - rpe space , extend over brm , and break up persistent blamd .", "gliosis indicated by intense glial fibrillary acidic protein immunoreactivity is prominent in areas of severe photoreceptor loss and rpe atrophy , and mller cells remain when all photoreceptors have died . in our exemplar histologic case ,", "we hypothesize that plateau and corrugation formation requires extension of mller cell processes through defects in the rpe / blamd complex ( figs . 4 , 6 ) that occur during the life cycle of drusenoid peds .", "we further hypothesize that increasing hyporeflectivity observed within the sub - rpe space during the progression of drusenoid peds represents a change in composition of the sub - rpe material that encourages the entry of these processes .", "the strength of this research is the long follow - up period for our patient cohort , with serial eye - tracked sd - oct imaging for up to 7 years and notably dating back to before the development of ga in all but one eye .", "another strength is the availability of up to nine separate imaging technologies , each specifically highlighting different tissue features in any one case and the availability of one illustrative histology case .", "limitations include the small number of eyes and the absence of direct correlation of in vivo imaging to postmortem histology ; hence , although our hypothesis of mller cells involvement in plateau formation is credible and testable , whether these cellular activities are directly visible in oct is currently unknown .", "our technique of volume measurement may be subject to variations according to oct b - scan density between patients , but for our purpose of studying progression on a per - patient basis , this limitation was acceptable . to minimize inconsistencies", ", all patients in our study had eye - tracked scans at each follow - up visit with similar density scan patterns available for analysis .", "our study adds value to existing literature on ped by suggesting a role of mller cell gliosis , elongation , and elaboration in the pathogenesis of amd , which should be further explored in future studies .", "it is important for clinicians to be aware of plateaus and to distinguish them from oct findings related to neovascular amd , such as vascularized peds or signs of exudation , as octa imaging of three eyes in our study showed no evidence of neovascular flow within plateaus ( fig .", "plateau structures should not be confused with areas of subretinal fluid and treatment is not indicated for this finding .", "finally , the distinctive reflectivity of persistent blamd in outer retinal corrugations , and , now , plateaus , provides strong motivation for renaming the fourth outer retinal hyperreflective band of normal eyes , which is currently called the rpe - brm complex , to the rpe - blam - brm complex ( abbreviated as rbb ) to accommodate the appearance of ped and persistent blamd in eyes with amd ."], [""]]}
{"article_id": "PMC3425033", "article_text": ["endocarditis of the right side of the heart is uncommon by virtue of the low hemodynamic pressure and lack of isolated or significant right - sided valvular deformities .", "the valves fall prey to an infective process in settings of congenital heart disease or endothelial injury .", "pulmonary arterial endarteritis is a rare event , even in patients with congenital heart disease ( chd ) .", "further , a combination of pulmonary endarteritis with a systemic suppuration in chd invites greater attention .", "a 4-year - old male child , born from a non - consanguineous marriage , was admitted with the history of fever and progressive right - sided weakness for 1 week and cyanosis noted since 6 months of age .", "the patient 's mother had noted dyspnea on feeding since 1 year of age . on admission ,", "the patient had dyspnea on exertion ( grade iii / iv , ats grading ) .", "the cardiac apex was at left 5 intercostal space , 3 cm outside the mid - clavicular line . an ejection systolic murmur ( grade iii / vi ) was audible in the pulmonary area . a right - sided complete hemiparesis with extensor plantar was noted .", "an ecg revealed right atrial enlargement , extreme right axis , and poor progression of r wave .", "chest x - ray revealed cardiomegaly with reduced pulmonary vascular markings [ figure 1 ] .", "a full - segmental echocardiography revealed a single ventricle of the double - inlet left ventricle type [ figure 2 ] .", "a rudimentary right ventricle was connected to the main ventricle by a non - restrictive foramen .", "the aorta arising from the rudimentary chamber and pulmonary artery from the main chamber [ figure 3 ] .", "a 7 mm  5 mm fixed structure on the wall of the main pulmonary artery with erratic movement , indicative of a vegetation , suggested pulmonary endarteritis [ figure 4a and b ] .", "a small osteum - secundum atrial septal defect was present , but patent ductus arteriosus was not present .", "ct scan of the brain revealed bilateral multiple cerebral abscesses [ figure 5 ] , with the largest one present superficially in the left parietal region .", "chest x - ray showing cardiomegaly with decreased pulmonary flow echocardiography ( subcostal anatomically corrected view )  showing both mitral and tricuspid valves opening into a single ventricle of lv morphology .", "a rudimentary rv type of ventricle is also visualized echocardiography ( subcostal anatomically corrected view )  showing aorta arising from a rudimentary right ventricle which is connected to the left ventricle through a nonrestrictive foramen ( a ) and ( b ) shows the pulmonary vegetation suggesting pulmonary endarteritis ct scan of the brain showing bilateral cerebral abscesses the child was started on parenteral ceftriaxone ", "80 mg/ kg / day ( plus , gentamicin  3 mg / kg / day for 2 weeks ) for the endarteritis and was continued for 6 weeks for optimal treatment of the cerebral abscess .", "an attempted drainage of the largest cerebral abscess resulted in some improvement of the hemiparesis .", "the patient became afebrile in 2 weeks and was able to walk after 4 weeks of therapy .", "repeat echo before discharge showed substantive decrease in size of the pulmonary artery vegetation and cardiac symptoms .", "the patient has been referred to the cardiac surgery department for corrective surgery for single ventricle .", "the risk of infective endocarditis is considerably increased in children with congenital heart disease , and the disease can occur with almost any lesion , more so with cyanotic congenital heart disease ( cchd ) .", "right - sided infective endocarditis is rare by virtue of the low hemodynamic pressure and lack of isolated or significant right - sided valvar deformities . in adults", ", right - sided infective endocarditis usually follows prolonged intravenous catheterization or intravenous drug - abuse , affecting the tricuspid valve alone or in combination with the pulmonary valve .", "a similar valvar involvement also occurs in infants and in children , but here congenital cardiac anomalies with their accompanying interventions are important pre - disposing factors .", "pulmonary endarteritis as an entity of right - sided endocarditis is rare , even in patients of congenital heart disease .", "most of the reported pulmonary endarteritis cases have been associated with a patent ductus arteriosus ( pda ) .", "there are less than 30 reported cases of pulmonary endarteritis associated with pda in the adult , and overall incidence has dramatically decreased over the last 30 years .", "bilge et al . , in their case report of pulmonary endarteritis in pda , underlines the importance of echocardiography in not only making this rare diagnosis but also as an effective means of following up such a case .", "apart from pda , a single report exists of pulmonary endarteritis occurring after anatomical correction of complete transposition of the great arteries . to the best of our knowledge and available resources , our case report of pulmonary endarteritis in a child of single ventricle is probably the first such so far .", "the onset of pulmonary endarteritis and the formation of vegetation can be explained by several factors , including ", "an obstacle of a valve or bloodstream jet and venturi effect , the formation of a platelet fibrin thrombus , transient bacteremia , and adhesion factor for infection . in the present case", ", an untreated infection in the child could have led to multiple episodes of bacteremia .", "the presence of multiple , bilateral cerebral abscesses is a corroborative evidence to the bacteremia .", "the pulmonary artery in this case arose from the main ventricle ( morphological double - inlet lv ) and was hypoplastic .", "turbulent blood flow distal to the stenotic lesion may increase the risk of developing infective endarteritis in an already hypoplastic pulmonary artery .", "the intima is also damaged in these areas , predisposing to formation of platelet fibrin thrombus , and thus making the region more susceptible to colonization by pathogens .", "congenital heart disease ( chd ) has a reported incidence of 9596/million live births ( 0.95% ) , out of which 14.5% ( 1391/million live births ) have cyanotic congenital heart disease ( cchd ) .", "single ventricle ( 106/million live births ) accounts for 7.6% of cchds and 1.1% of all chds .", "meticulous full - segmental echocardiographic approach is required to diagnose and delineate the specific type of single ventricle .", "single ventricle is a complex cchd and is considered a high risk condition for an infective endocarditis .", "trans - thoracic echocardiography ( tte ) is more sensitive in the pediatric population than in the adult population for detection of vegetation .", "tte is more likely to identify vegetations in children with normal anatomy or isolated valvular pathology than in those with complex cchd .", "the importance of a carefully - performed tte or trans - esophageal echocardiography is , therefore , essential in a complex cchd like single ventricle states .", "cyanotic heart disease accounts for 12.8%-69.4% of all cases of brain abscesses , with the incidence being higher in children .", "the incidence of brain abscess in patients with cyanotic heart disease has been reported to range between 5 and 18.7% . in our case ,", "bacteremia from an unknown source could have bypassed the pulmonary circulation by entering the double - inlet lv ( the dominant ventricle ) and moved through the aorta and seeded in the brain .", "patients with cyanotic heart disease could have low - perfusion areas in the brain due to chronic severe hypoxemia and metabolic acidosis as well as increased viscosity of blood due to secondary polycythemia .", "these low - perfusion areas commonly occur in the junction of gray and white matter , and they are prone to seeding by microorganisms that may be present in the bloodstream . the advent of ct scans and their use in the management of these abscesses has resulted in a fourfold decrease in the mortality rate in patients with brain abscesses , secondary to cyanotic heart disease - from 40%-60% in the pre - ct era to ~10% . the threshold for ct scans in a cchd should be low , and regular ct scans should be obtained to monitor the size of the abscess . in conclusion , this case report aims to highlight the considerable and varied morbidity associated with cyanotic congenital heart diseases ( cchd ) . the presence of a relatively common infectious complication ( cerebral abscesses ) and a rare infectious complication ( pulmonary endarteritis ) in the same child with a cchd ( single ventricle ) validates the need for a heightened clinical awareness and a prompt and meticulous investigational approach for diagnosis and therapy in this group of patients ."], "abstract_text": ["<S> endocarditis of the right side of the heart is otherwise uncommon in children . </S>", "<S> pulmonary endarteritis as a complication of congenital heart disease is even rarer . </S>", "<S> herein , we report the case of pulmonary endarteritis with a 7 mm 5 mm vegetation , involving the main pulmonary artery in a 4-year - old male child , with cyanosis and a 1-week history of fever and rapidly - progressive hemiparesis . a full segmental echocardiography demonstrated a double inlet left ventricle with left - sided subaortic </S>", "<S> hypoplastic right ventricle ( van praagh 's a - iii type  </S>", "<S> single ventricle ) . additionally , ct scan of the brain revealed bilateral cerebral abscesses . to the best of our knowledge , </S>", "<S> the occurrence of pulmonary endarteritis and cerebral abscesses in a case of single ventricle is hitherto unreported . </S>", "<S> this article underlines the importance of heightened clinical awareness and meticulous echocardiography in cases of congenital heart disease so that relatively rare complications may not be missed . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["endocarditis of the right side of the heart is uncommon by virtue of the low hemodynamic pressure and lack of isolated or significant right - sided valvular deformities .", "the valves fall prey to an infective process in settings of congenital heart disease or endothelial injury .", "pulmonary arterial endarteritis is a rare event , even in patients with congenital heart disease ( chd ) .", "further , a combination of pulmonary endarteritis with a systemic suppuration in chd invites greater attention ."], ["a 4-year - old male child , born from a non - consanguineous marriage , was admitted with the history of fever and progressive right - sided weakness for 1 week and cyanosis noted since 6 months of age .", "the patient 's mother had noted dyspnea on feeding since 1 year of age . on admission ,", "the patient had dyspnea on exertion ( grade iii / iv , ats grading ) .", "the cardiac apex was at left 5 intercostal space , 3 cm outside the mid - clavicular line . an ejection systolic murmur ( grade iii / vi ) was audible in the pulmonary area . a right - sided complete hemiparesis with extensor plantar was noted .", "an ecg revealed right atrial enlargement , extreme right axis , and poor progression of r wave .", "chest x - ray revealed cardiomegaly with reduced pulmonary vascular markings [ figure 1 ] .", "a full - segmental echocardiography revealed a single ventricle of the double - inlet left ventricle type [ figure 2 ] .", "a rudimentary right ventricle was connected to the main ventricle by a non - restrictive foramen .", "the aorta arising from the rudimentary chamber and pulmonary artery from the main chamber [ figure 3 ] .", "a 7 mm  5 mm fixed structure on the wall of the main pulmonary artery with erratic movement , indicative of a vegetation , suggested pulmonary endarteritis [ figure 4a and b ] .", "a small osteum - secundum atrial septal defect was present , but patent ductus arteriosus was not present .", "ct scan of the brain revealed bilateral multiple cerebral abscesses [ figure 5 ] , with the largest one present superficially in the left parietal region .", "chest x - ray showing cardiomegaly with decreased pulmonary flow echocardiography ( subcostal anatomically corrected view )  showing both mitral and tricuspid valves opening into a single ventricle of lv morphology .", "a rudimentary rv type of ventricle is also visualized echocardiography ( subcostal anatomically corrected view )  showing aorta arising from a rudimentary right ventricle which is connected to the left ventricle through a nonrestrictive foramen ( a ) and ( b ) shows the pulmonary vegetation suggesting pulmonary endarteritis ct scan of the brain showing bilateral cerebral abscesses the child was started on parenteral ceftriaxone ", "80 mg/ kg / day ( plus , gentamicin  3 mg / kg / day for 2 weeks ) for the endarteritis and was continued for 6 weeks for optimal treatment of the cerebral abscess .", "an attempted drainage of the largest cerebral abscess resulted in some improvement of the hemiparesis .", "the patient became afebrile in 2 weeks and was able to walk after 4 weeks of therapy .", "repeat echo before discharge showed substantive decrease in size of the pulmonary artery vegetation and cardiac symptoms .", "the patient has been referred to the cardiac surgery department for corrective surgery for single ventricle ."], ["the risk of infective endocarditis is considerably increased in children with congenital heart disease , and the disease can occur with almost any lesion , more so with cyanotic congenital heart disease ( cchd ) .", "right - sided infective endocarditis is rare by virtue of the low hemodynamic pressure and lack of isolated or significant right - sided valvar deformities . in adults", ", right - sided infective endocarditis usually follows prolonged intravenous catheterization or intravenous drug - abuse , affecting the tricuspid valve alone or in combination with the pulmonary valve .", "a similar valvar involvement also occurs in infants and in children , but here congenital cardiac anomalies with their accompanying interventions are important pre - disposing factors .", "pulmonary endarteritis as an entity of right - sided endocarditis is rare , even in patients of congenital heart disease .", "most of the reported pulmonary endarteritis cases have been associated with a patent ductus arteriosus ( pda ) .", "there are less than 30 reported cases of pulmonary endarteritis associated with pda in the adult , and overall incidence has dramatically decreased over the last 30 years .", "bilge et al . , in their case report of pulmonary endarteritis in pda , underlines the importance of echocardiography in not only making this rare diagnosis but also as an effective means of following up such a case .", "apart from pda , a single report exists of pulmonary endarteritis occurring after anatomical correction of complete transposition of the great arteries . to the best of our knowledge and available resources , our case report of pulmonary endarteritis in a child of single ventricle is probably the first such so far .", "the onset of pulmonary endarteritis and the formation of vegetation can be explained by several factors , including ", "an obstacle of a valve or bloodstream jet and venturi effect , the formation of a platelet fibrin thrombus , transient bacteremia , and adhesion factor for infection . in the present case", ", an untreated infection in the child could have led to multiple episodes of bacteremia .", "the presence of multiple , bilateral cerebral abscesses is a corroborative evidence to the bacteremia .", "the pulmonary artery in this case arose from the main ventricle ( morphological double - inlet lv ) and was hypoplastic .", "turbulent blood flow distal to the stenotic lesion may increase the risk of developing infective endarteritis in an already hypoplastic pulmonary artery .", "the intima is also damaged in these areas , predisposing to formation of platelet fibrin thrombus , and thus making the region more susceptible to colonization by pathogens .", "congenital heart disease ( chd ) has a reported incidence of 9596/million live births ( 0.95% ) , out of which 14.5% ( 1391/million live births ) have cyanotic congenital heart disease ( cchd ) .", "single ventricle ( 106/million live births ) accounts for 7.6% of cchds and 1.1% of all chds .", "meticulous full - segmental echocardiographic approach is required to diagnose and delineate the specific type of single ventricle .", "single ventricle is a complex cchd and is considered a high risk condition for an infective endocarditis .", "trans - thoracic echocardiography ( tte ) is more sensitive in the pediatric population than in the adult population for detection of vegetation .", "tte is more likely to identify vegetations in children with normal anatomy or isolated valvular pathology than in those with complex cchd .", "the importance of a carefully - performed tte or trans - esophageal echocardiography is , therefore , essential in a complex cchd like single ventricle states .", "cyanotic heart disease accounts for 12.8%-69.4% of all cases of brain abscesses , with the incidence being higher in children .", "the incidence of brain abscess in patients with cyanotic heart disease has been reported to range between 5 and 18.7% . in our case ,", "bacteremia from an unknown source could have bypassed the pulmonary circulation by entering the double - inlet lv ( the dominant ventricle ) and moved through the aorta and seeded in the brain .", "patients with cyanotic heart disease could have low - perfusion areas in the brain due to chronic severe hypoxemia and metabolic acidosis as well as increased viscosity of blood due to secondary polycythemia .", "these low - perfusion areas commonly occur in the junction of gray and white matter , and they are prone to seeding by microorganisms that may be present in the bloodstream . the advent of ct scans and their use in the management of these abscesses has resulted in a fourfold decrease in the mortality rate in patients with brain abscesses , secondary to cyanotic heart disease - from 40%-60% in the pre - ct era to ~10% . the threshold for ct scans in a cchd should be low , and regular ct scans should be obtained to monitor the size of the abscess . in conclusion , this case report aims to highlight the considerable and varied morbidity associated with cyanotic congenital heart diseases ( cchd ) . the presence of a relatively common infectious complication ( cerebral abscesses ) and a rare infectious complication ( pulmonary endarteritis ) in the same child with a cchd ( single ventricle ) validates the need for a heightened clinical awareness and a prompt and meticulous investigational approach for diagnosis and therapy in this group of patients ."]]}
{"article_id": "PMC4071672", "article_text": ["most commonly affects people of african descent . in nigeria , sickle cell anaemia affects about 1 - 3% of the nigerian population .", "individuals who have only one copy of the mutation are said to have sickle cell trait.12 these people are usually healthy but can transmit the disease to their children.3 sickle cell anaemia is characterised by acute episodes of pain ( in the abdomen , chest or joints ) and exhibits a range of severity.45 sickle cell disease is a condition which alters the shape of the red blood cells from round to  sickle  shape , causing them to block small blood vessels and interfere with normal blood flow .", "children affected with sickle cell disease experience chronic episodes of pain and an increased susceptibility to potentially life - threatening conditions , including bacterial infections and organ failure . at the present time , there is no cure for sickle cell anaemia . although there are some altering therapies such as hydroxyurea and hematopoietic cell transplantation for the management of sickle cell disease , but these can have serious side effects and are very risky", "the average life expectancy for people with sickle cell anaemia is less than 50 years .", "the first deoxyribonucleic acid ( dna ) diagnostic procedure for prenatal purposes was reported more than thirty years ago.6 subsequently , it was recognised that the mutation itself affected the cleavage site of a restriction enzyme , ddei , that could recognize the dna sequence of ctnag ( n = a , t , c , or g ) .", "while dna from a normal allele ( ctgag ) would be digested by the enzyme , dna from an affected allele in which a is substituted by t ( ctgtg ) would not.78 the resulting differences between dna fragment sizes can then be recognised by electrophoresis , thus forming the basis for diagnosis . with the advent of polymerase chain reaction ( pcr ) ,", "rapid dna analysis methods have become available , and these techniques are now widely used for prenatal diagnosis.910111213 pre - implantation genetic diagnosis ( pgd ) is a procedure that has emerged against the backdrop of in vitro fertilisation ( ivf ) technology . in nigeria , this process of ivf was first introduced by ashiru et al.14 following their pioneering work , we were able to advance this technique for patients with infertility,1516 and now for patients with advanced maternal age and sickle cell disorders with the use of pgd .", "with pgd , the genetic status of an embryo can be determined before transfer into the uterus after ivf , thus almost completely eliminating the risks of bearing a child with the disease17(pgd is not 100% accurate ) .", "the most common form of pgd involves the extraction of one or two cells from the pre - implantation embryo , often around the 8-cell stage .", "couples at risk for the transmission of genetic diseases can now choose ivf and pgd to avoid the problem of selective abortion that is associated with pre - natal diagnosis during pregnancy .", "the patient was a 29-year - old woman and spouse with heterozygous genotype ( as ) .", "couple already have a 2-year - old child who is affected with sickle cell anaemia .", "prior to ovarian stimulation , hysterosonogram ( hsn ) was done which revealed normal uterine cavity .", "the protocol was also approved by the research and ethics committee of the centre . following 3 weeks of oral contraceptive use , the patient was down - regulated using a gnrhanalog ( leuprolide ) .", "super ovulation was achieved with the use of recombinant follicle stimulating hormone ( fsh ) . when three or more leading follicles measured > 18 mm in diameter , the patient was triggered with human chorionic gonadotropin ( hcg ) 5000 iu .", "the surrounding cumulus and corona cells were then removed and the nuclear maturity of the oocytes was assessed under an inverted microscope . only metaphase ii oocytes were injected with morphologically normal motile spermatozoa .", "further culture of injected oocytes was done in 20 l microdrops of culture medium under lightweight paraffin oil .", "fertilisation was confirmed after 16 - 18 hours by the observation of two distinct pronuclei ( 2pn ) .", "oocytes with 2pn were assessed on day 2 after injection for embryonic development . on day 3 ,", "a single blastomere was biopsied for embryos at 6 - 8 cell stage with less than 50% fragmentation [ figures 1 and 2 ] .", "blastomere showing nucleus fifteen biopsied cells were sent for genetic screening and analysed using pgd / pcr technique [ figure 3 ] .", "results provided after 48 hours showed that 5 ( 33% ) of the biopsied cells were of genotypes unaffected by sickle cell anemia ; of these , three were heterozygous carriers of hb s ( as ) and two were homozygous for hba ( aa ) .", "three unaffected embryos ( two heterozygous normal and one homozygous ) were transferred on day 5 at blastocyst stage .", "endometrial lining was prepared for fet using increasing doses of estradiolvalerate from 2 mg daily up to 8 mg with weekly monitoring of the endometrium .", "two vitrified normal embryos ( one hbas and one hbaa ) were thawed both at hatched blastocyst stage and transferred [ figure 4 ] .", "the advent of ivf as a treatment for infertility has created the opportunity to study the chromosomal constitution of surplus human pre - implantation embryos .", "cultured human pre - implantation embryos have been used to develop methods which allow pgd analyses by pcr on biopsied blastomeres from an embryo.18 beta - thalassaemia and sickle cell anaemia are -globin chain quantitative and structural disorders that lead to anaemia syndromes . until recently", ", the only alternative for couples with a high genetic risk was to undergo prenatal diagnosis followed by termination of an affected pregnancy .", "the pgd of -thalassaemia and sickle cell anaemia is an alternative that avoids therapeutic abortion by diagnosing embryos for -globin defects before implantation into the mother 's womb.19 on a world - wide scale , pgd for -thalassaemia and/or sickle cell anaemia has already been applied on single blastomeres20 and on the first and second polar bodies.21 the molecular strategies used were dna amplification followed by genetic diagnosis by denaturing gradient gel electrophoresis analysis,20 restriction enzyme digestion , the creation of a new restriction enzyme recognition sequence involving the ivs1 nt 110 mutation2122 and the use of fluorescence pcr.22 in the present study , pgd was applied clinically for sickle cell anaemia on a fertile carrier couple with previous experiences of therapeutic abortion for affected foetuses , and a sickle cell disease child . despite the fact that sickle cell anaemia is one of the most common genetic disorders and detailed genetic information is available,23 unaffected pregnancies following pgd for sickle cell anaemia previously have not been reported .", "lack of previous success in this area presumably is due to the length of time and effort required to overcome technical difficulties inherent in these procedures , as well as lack of available research funding.17 our results demonstrate that sickle cell anaemia can be detected in single cells by pcr and restriction enzyme analysis and that unaffected pregnancies can be established by the transfer of embryos of known genetic makeup that have undergone biopsy . in total , one pgd cycle with a frozen embryo transfer cycle were carried out .", "the goal of pgd is to reduce the risk of having children born with genetic diseases from a priori risk of 25% to a value significantly less .", "patients undergoing pgd should be advised that if a pregnancy does occur from this treatment , they should consider undergoing chorionic villi sampling ( cvs ) or amniocentesis to confirm these micro - genomic results . in summary", ", this is the first unaffected pregnancy and delivery after successful pgd for sickle cell anaemia .", "our results demonstrate that pgd for the detection of sickle cell anaemia is a powerful diagnostic tool for carrier couples who desire a healthy child but wish to avoid the difficult decision of whether to abort an affected foetus .", "the procedure , successfully used in this case , may also be applied to other monogenic disorders and further supports the notion that pgd is destined to be an integral aspect of assisted reproductive technology .", "given the current methods and relatively high cost of the procedure , it is unlikely that pgd will totally replace prenatal testing .", "however , it is conceivable that with further refinements , pgd will certainly become an invaluable and powerful diagnostic modality ."], "abstract_text": ["<S> a couple , both carriers of the sickle cell anaemia trait ( genotype hbas ) with an offspring already affected with the genetic disease underwent a pre - implantation genetic diagnosis / polymerase chain reaction screening of biopsied blastomeres . </S>", "<S> dna analysis of single blastomeres was carried out to find out indicated a viable intra - uterine pregnancy with embryos which carried the sickle cell mutation , which resulted in a livebirth ( hbas ) . </S>", "<S> pgd / pcr in combination with ivf appears to be the most suitable treatment plan for patients who are at a higher risk of reproducing offspring affected with inheritable genetic diseases . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["most commonly affects people of african descent . in nigeria , sickle cell anaemia affects about 1 - 3% of the nigerian population .", "individuals who have only one copy of the mutation are said to have sickle cell trait.12 these people are usually healthy but can transmit the disease to their children.3 sickle cell anaemia is characterised by acute episodes of pain ( in the abdomen , chest or joints ) and exhibits a range of severity.45 sickle cell disease is a condition which alters the shape of the red blood cells from round to  sickle  shape , causing them to block small blood vessels and interfere with normal blood flow .", "children affected with sickle cell disease experience chronic episodes of pain and an increased susceptibility to potentially life - threatening conditions , including bacterial infections and organ failure . at the present time , there is no cure for sickle cell anaemia . although there are some altering therapies such as hydroxyurea and hematopoietic cell transplantation for the management of sickle cell disease , but these can have serious side effects and are very risky", "the average life expectancy for people with sickle cell anaemia is less than 50 years .", "the first deoxyribonucleic acid ( dna ) diagnostic procedure for prenatal purposes was reported more than thirty years ago.6 subsequently , it was recognised that the mutation itself affected the cleavage site of a restriction enzyme , ddei , that could recognize the dna sequence of ctnag ( n = a , t , c , or g ) .", "while dna from a normal allele ( ctgag ) would be digested by the enzyme , dna from an affected allele in which a is substituted by t ( ctgtg ) would not.78 the resulting differences between dna fragment sizes can then be recognised by electrophoresis , thus forming the basis for diagnosis . with the advent of polymerase chain reaction ( pcr ) ,", "rapid dna analysis methods have become available , and these techniques are now widely used for prenatal diagnosis.910111213 pre - implantation genetic diagnosis ( pgd ) is a procedure that has emerged against the backdrop of in vitro fertilisation ( ivf ) technology . in nigeria , this process of ivf was first introduced by ashiru et al.14 following their pioneering work , we were able to advance this technique for patients with infertility,1516 and now for patients with advanced maternal age and sickle cell disorders with the use of pgd .", "with pgd , the genetic status of an embryo can be determined before transfer into the uterus after ivf , thus almost completely eliminating the risks of bearing a child with the disease17(pgd is not 100% accurate ) .", "the most common form of pgd involves the extraction of one or two cells from the pre - implantation embryo , often around the 8-cell stage .", "couples at risk for the transmission of genetic diseases can now choose ivf and pgd to avoid the problem of selective abortion that is associated with pre - natal diagnosis during pregnancy ."], ["the patient was a 29-year - old woman and spouse with heterozygous genotype ( as ) .", "couple already have a 2-year - old child who is affected with sickle cell anaemia .", "prior to ovarian stimulation , hysterosonogram ( hsn ) was done which revealed normal uterine cavity .", "the protocol was also approved by the research and ethics committee of the centre . following 3 weeks of oral contraceptive use , the patient was down - regulated using a gnrhanalog ( leuprolide ) .", "super ovulation was achieved with the use of recombinant follicle stimulating hormone ( fsh ) . when three or more leading follicles measured > 18 mm in diameter , the patient was triggered with human chorionic gonadotropin ( hcg ) 5000 iu .", "the surrounding cumulus and corona cells were then removed and the nuclear maturity of the oocytes was assessed under an inverted microscope . only metaphase ii oocytes were injected with morphologically normal motile spermatozoa .", "further culture of injected oocytes was done in 20 l microdrops of culture medium under lightweight paraffin oil .", "fertilisation was confirmed after 16 - 18 hours by the observation of two distinct pronuclei ( 2pn ) .", "oocytes with 2pn were assessed on day 2 after injection for embryonic development . on day 3 ,", "a single blastomere was biopsied for embryos at 6 - 8 cell stage with less than 50% fragmentation [ figures 1 and 2 ] .", "blastomere showing nucleus fifteen biopsied cells were sent for genetic screening and analysed using pgd / pcr technique [ figure 3 ] .", "results provided after 48 hours showed that 5 ( 33% ) of the biopsied cells were of genotypes unaffected by sickle cell anemia ; of these , three were heterozygous carriers of hb s ( as ) and two were homozygous for hba ( aa ) ."], ["three unaffected embryos ( two heterozygous normal and one homozygous ) were transferred on day 5 at blastocyst stage .", "endometrial lining was prepared for fet using increasing doses of estradiolvalerate from 2 mg daily up to 8 mg with weekly monitoring of the endometrium .", "two vitrified normal embryos ( one hbas and one hbaa ) were thawed both at hatched blastocyst stage and transferred [ figure 4 ] ."], ["the advent of ivf as a treatment for infertility has created the opportunity to study the chromosomal constitution of surplus human pre - implantation embryos .", "cultured human pre - implantation embryos have been used to develop methods which allow pgd analyses by pcr on biopsied blastomeres from an embryo.18 beta - thalassaemia and sickle cell anaemia are -globin chain quantitative and structural disorders that lead to anaemia syndromes . until recently", ", the only alternative for couples with a high genetic risk was to undergo prenatal diagnosis followed by termination of an affected pregnancy .", "the pgd of -thalassaemia and sickle cell anaemia is an alternative that avoids therapeutic abortion by diagnosing embryos for -globin defects before implantation into the mother 's womb.19 on a world - wide scale , pgd for -thalassaemia and/or sickle cell anaemia has already been applied on single blastomeres20 and on the first and second polar bodies.21 the molecular strategies used were dna amplification followed by genetic diagnosis by denaturing gradient gel electrophoresis analysis,20 restriction enzyme digestion , the creation of a new restriction enzyme recognition sequence involving the ivs1 nt 110 mutation2122 and the use of fluorescence pcr.22 in the present study , pgd was applied clinically for sickle cell anaemia on a fertile carrier couple with previous experiences of therapeutic abortion for affected foetuses , and a sickle cell disease child . despite the fact that sickle cell anaemia is one of the most common genetic disorders and detailed genetic information is available,23 unaffected pregnancies following pgd for sickle cell anaemia previously have not been reported .", "lack of previous success in this area presumably is due to the length of time and effort required to overcome technical difficulties inherent in these procedures , as well as lack of available research funding.17 our results demonstrate that sickle cell anaemia can be detected in single cells by pcr and restriction enzyme analysis and that unaffected pregnancies can be established by the transfer of embryos of known genetic makeup that have undergone biopsy . in total , one pgd cycle with a frozen embryo transfer cycle were carried out .", "the goal of pgd is to reduce the risk of having children born with genetic diseases from a priori risk of 25% to a value significantly less .", "patients undergoing pgd should be advised that if a pregnancy does occur from this treatment , they should consider undergoing chorionic villi sampling ( cvs ) or amniocentesis to confirm these micro - genomic results . in summary", ", this is the first unaffected pregnancy and delivery after successful pgd for sickle cell anaemia .", "our results demonstrate that pgd for the detection of sickle cell anaemia is a powerful diagnostic tool for carrier couples who desire a healthy child but wish to avoid the difficult decision of whether to abort an affected foetus .", "the procedure , successfully used in this case , may also be applied to other monogenic disorders and further supports the notion that pgd is destined to be an integral aspect of assisted reproductive technology .", "given the current methods and relatively high cost of the procedure , it is unlikely that pgd will totally replace prenatal testing .", "however , it is conceivable that with further refinements , pgd will certainly become an invaluable and powerful diagnostic modality ."]]}
{"article_id": "PMC3480676", "article_text": ["pulse rate , also called heart rate , is the number of heartbeats per unit of time , commonly used as beats per minute ( bpm ) .", "pulse rate is varied in accordance with the body 's need for oxygen changes , such as during exercise or sleep .", "since various health - related phenotypes are closely correlated with pulse rate [ 1 - 3 ] , measuring pulse rate is commonly used for diagnosing and accessing medical conditions .", "pulse rate is known to be positively correlated with triglycerides and negatively correlated with high - density lipoprotein cholesterol .", "metabolic syndrome is associated with poor exercise capacity and poor heart rate recovery in patients who have coronary disease . moreover ,", "high pulse rate is negatively correlated with lifespan in mammals , including humans . despite the importance of pulse rate as a risk factor for various diseases ,", "however , few genes are known to be associated with pulse rate , and the underlying mechanism for pulse rate remains unraveled .", "we previously reported pulse rate - associated loci from the korea association resource ( kare ) genomewide association study ( gwas ) , comprising 8,842 samples in the korean population . in this study", ", we extended the previous study and reexamined the relevance of pulse rate - associated single - nucleotide polymorphisms ( snps ) from an additional gwas of 3,703 korean samples .", "in the discovery stage , the association results for pulse rate were obtained from the kare study .", "subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .", "the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years .", "pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts , the first measurement of the participants , who rested for 5 min ,", "the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,", "genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,", "santa clara , ca , usa ) . from 4,302 genotyped samples , we excluded samples with a low call rate (  95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1  10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals .", "association analyses were performed using plink \n ( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .", "the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .", "the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .", "meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) .", "in the discovery stage , the association results for pulse rate were obtained from the kare study .", "subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .", "the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years .", "pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts ,", "the first measurement of the participants , who rested for 5 min , was used .", "the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,", "genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,", ", we excluded samples with a low call rate (  95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1  10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals .", "association analyses were performed using plink \n ( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .", "the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .", "the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .", "meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) .", "the discovery gwas for pulse rate included 352,225 snps profiled with affymetrix genome - wide human snp array 5.0 in 8,842 individuals from the ansung and ansan cohorts as described previously .", "characteristics of the study , including age , sex , and trait summaries , are presented in table 1 .", "the association analysis identified several genomic locations as potentially associated with pulse rate . unlike the previous gwas", ", we did not apply a threshold with a p < 0.01 in both ansung and ansan cohort in addition to a combined p < 1  10 for the snp selection for the replication study so that we could include more promising snps for their association with pulse rate .", "we observed 61 significant snps by applying only a threshold with a combined p < 1  10 . after excluding snps by cluster plot inspection and removing singletons , 3 independent snps ( that is , with pairwise linkage disequilibrium statistics r < 0.2 and maf  0.05 within a 500-kb window of the genomic region )", "were finally chosen to validate in the independent population - i.e. , the hexa cohort , comprising 3,703 individuals . in the replication study ,", "all 3 snps were confirmed for their association with pulse rate at a p < 0.05 ( table 2 ) . the first pulse rate signal located to 1q32.2a ( rs12731740 , maf = 0.103 , poverall = 2.20  10 ) .", "three genes , such as cd46 , clorf132 , and cd34 , appear near this locus .", "the physiological relevance of the 3 genes in relation to pulse rate is not clear [ 8 , 9 ] .", "thus , fine mapping surrounding this locus by targeted resequencing is highly recommended to discover the causal variants for pulse rate .", "the second pulse rate snp is rs12110693 ( maf = 0.494 , poverall = 1.10  10 ) on 6q22.31c .", "the only gene located near this snp is loc644502 , which has no known function .", "the third locus associated with pulse rate is at 6q22.31c ( rs2892767 , maf = 0.261 , poverall = 2.50  10 ) .", "this locus is near gja1 ( gap junction protein , alpha 1 , 43 kda ) , which is a member of the connexin gene family .", "the protein encoded by gja1 is a component of gap junctions , which are intercellular channels that provide a route for the diffusion of low - molecular - weight materials from cell to cell .", "in this study , we performed a genomewide scan for pulse rate and identified 3 loci accounting for the variation in pulse rate .", "two of 3 loci were previously reported and replicated once again in an independent hexa - shared cohort .", "the other one ( rs2892767 ) near gja1 was previously implicated for its association with pulse rate but was not chosen for the replication study , since the snp did not meet the selection criteria in the discovery stage . in the present study ,", "snp rs2892767 near gja1 was taken forward to the replication study using the independent dataset from the hexa - shared cohort and was nicely confirmed for its association with pulse rate .", "two snps , rs2892767 near gja1 and rs12110693 near loc644502 , are 408 kb apart from each other and are located in a separate ld , indicating that both are independent snps .", "we performed multiple linear regression analyses , conditioning the snps to each other to find a significantly stronger proxy for pulse rate .", "however , no dramatic decrease in statistical significance was observed , and no interaction was found between rs2892767 and rs12110693 ( table 3 ) .", "gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development .", "it is also known that gap junctions , clusters of cell - to - cell channels composed of connexins , mediate the orderly spread of electrical excitation throughout the heart .", "the gja1-encoding protein has been reported to be involved in the development and function of the heart by allowing direct cell - to - cell exchange of molecules , which mediate multiple signaling events .", "cho et al .   previously reported the same locus near gja1 to be responsible for pulse rate without evidence of replication . in the present study", "as indicated from the functional role of gja1 , it is a strong candidate gene for pulse rate .", "thus , further efforts , such as fine mapping or imputation based on large - scale sequencing data near rs2892767 on 6q22.31 , would reveal the causal variant of this locus .", "in addition , functional studies for gja1 would be worthwhile to facilitate a full understanding of its biological role in pulse rate .", "our study was relatively underpowered to discover additional novel loci and demonstrated that large - scale genomewide meta - analysis comprising hundreds of thousands samples is required for understanding the missing heritability of pulse rate ."], "abstract_text": ["<S> pulse rate is known to be related to diverse phenotypes , such as cardiovascular diseases , lifespan , arrhythmia , hypertension , lipids , diabetes , and menopause . </S>", "<S> we have reported two genomewide significant genetic loci responsible for the variation in pulse rate as a part of the korea association resource ( kare ) project , the genomewide association study ( gwas ) that was conducted with 352,228 single nucleoride polymorphisms typed in 8,842 subjects in the korean population . </S>", "<S> gja1 was implied as a functionally causal gene for pulse rate from the kare study , but lacked evidence of replication . </S>", "<S> to re - evaluate the association of a locus near gja1 with pulse rate , we looked up this signal in another gwas conducted in a health examinee - shared cohort of 3,703 samples . </S>", "<S> not only we were able to confirm two pulse rate loci ( 1q32.2a near cd46 and 6q22.13c near locl644502 ) identified in the kare gwas , we also replicated a locus ( 6q22.31c ) near gja1 by the lookup in the health examinee gwas . considering that the gja1-encoded protein is a major component of cardiac gap junctions , </S>", "<S> a functional study might be necessary to validate its genuine molecular biological role in the synchronized contraction of the heart . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Subjects", "The measurement of pulse rate", "Genotyping and quality control", "Statistical analysis", "Results", "Discussion"], "sections": [["pulse rate , also called heart rate , is the number of heartbeats per unit of time , commonly used as beats per minute ( bpm ) .", "pulse rate is varied in accordance with the body 's need for oxygen changes , such as during exercise or sleep .", "since various health - related phenotypes are closely correlated with pulse rate [ 1 - 3 ] , measuring pulse rate is commonly used for diagnosing and accessing medical conditions .", "pulse rate is known to be positively correlated with triglycerides and negatively correlated with high - density lipoprotein cholesterol .", "metabolic syndrome is associated with poor exercise capacity and poor heart rate recovery in patients who have coronary disease . moreover ,", "high pulse rate is negatively correlated with lifespan in mammals , including humans . despite the importance of pulse rate as a risk factor for various diseases ,", "however , few genes are known to be associated with pulse rate , and the underlying mechanism for pulse rate remains unraveled .", "we previously reported pulse rate - associated loci from the korea association resource ( kare ) genomewide association study ( gwas ) , comprising 8,842 samples in the korean population . in this study", ", we extended the previous study and reexamined the relevance of pulse rate - associated single - nucleotide polymorphisms ( snps ) from an additional gwas of 3,703 korean samples ."], ["in the discovery stage , the association results for pulse rate were obtained from the kare study .", "subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .", "the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years .", "pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts , the first measurement of the participants , who rested for 5 min ,", "the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,", "genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,", "santa clara , ca , usa ) . from 4,302 genotyped samples , we excluded samples with a low call rate (  95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1  10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals .", "association analyses were performed using plink \n ( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .", "the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .", "the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .", "meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) ."], ["in the discovery stage , the association results for pulse rate were obtained from the kare study .", "subjects for the replication stage were recruited from the health examinee cohorts , called hexa - shared cohorts .", "the standardized examinations that were applied in this survey included 4,302 participants aged 40 to 70 years ."], ["pulse rate of kare was measured 3 times while the participant was lying down , and the average value was used for the analysis ( before the first measurement , participants rested for 5 min , and 3 measurements were taken at least 30 s apart ) . in hexa - shared cohorts ,", "the first measurement of the participants , who rested for 5 min , was used ."], ["the methods for genotyping and quality control for the kare gwas have been described elsewhere . for the hexa gwas ,", "genomic dna was extracted from 4,302 participants from an urban cohort and genotyped with affymetrix genome - wide human snp array 6.0 ( affymetrix , inc . ,", ", we excluded samples with a low call rate (  95% , n = 443 ) , high heterozygosity ( n = 25 ) , outliers ( n = 26 ) , and gender inconsistencies ( n = 8) , and those obtained from individuals who had developed any kind of cancer ( n = 61 ) were excluded from subsequent analyses , along with related or identical individuals whose computed average pairwise identity - by - state value was higher than that estimated from cryptic first - degree relatives of samples ( n = 33 ) . snp markers with a high missing genotype rate ( > 5% ) , minor allele frequency ( maf ) ( < 0.01 ) , and significant deviation from hardy - weinberg equilibrium ( p < 1  10 ) were excluded , leaving a total of 627,659 markers to be examined in 3,703 individuals ."], ["association analyses were performed using plink \n ( http://pngu.mgh.harvared.edu/~purcell/plink/ ) , sas version 9.2 ( sas institute inc . , cary , nc , usa ) , and r statistics package ( version 2.7.1 ) ( http://r-project.org/ ) .", "the snp association for pulse rate was tested by linear regression analysis with an additive model , adjusting for age , sex , and recruitment area .", "the kare gwas and replication gwas were combined by an inverse - variance meta - analysis method , assuming fixed effects , with cochran 's q test used to assess between - study heterogeneity .", "meta - analysis calculations were performed using metal ( http://www.sph.umich.edu/csg/abecasis/metal ) in the r program ( version 2.7.1 ) ."], ["the discovery gwas for pulse rate included 352,225 snps profiled with affymetrix genome - wide human snp array 5.0 in 8,842 individuals from the ansung and ansan cohorts as described previously .", "characteristics of the study , including age , sex , and trait summaries , are presented in table 1 .", "the association analysis identified several genomic locations as potentially associated with pulse rate . unlike the previous gwas", ", we did not apply a threshold with a p < 0.01 in both ansung and ansan cohort in addition to a combined p < 1  10 for the snp selection for the replication study so that we could include more promising snps for their association with pulse rate .", "we observed 61 significant snps by applying only a threshold with a combined p < 1  10 . after excluding snps by cluster plot inspection and removing singletons , 3 independent snps ( that is , with pairwise linkage disequilibrium statistics r < 0.2 and maf  0.05 within a 500-kb window of the genomic region )", "were finally chosen to validate in the independent population - i.e. , the hexa cohort , comprising 3,703 individuals . in the replication study ,", "all 3 snps were confirmed for their association with pulse rate at a p < 0.05 ( table 2 ) . the first pulse rate signal located to 1q32.2a ( rs12731740 , maf = 0.103 , poverall = 2.20  10 ) .", "three genes , such as cd46 , clorf132 , and cd34 , appear near this locus .", "the physiological relevance of the 3 genes in relation to pulse rate is not clear [ 8 , 9 ] .", "thus , fine mapping surrounding this locus by targeted resequencing is highly recommended to discover the causal variants for pulse rate .", "the second pulse rate snp is rs12110693 ( maf = 0.494 , poverall = 1.10  10 ) on 6q22.31c .", "the only gene located near this snp is loc644502 , which has no known function .", "the third locus associated with pulse rate is at 6q22.31c ( rs2892767 , maf = 0.261 , poverall = 2.50  10 ) .", "this locus is near gja1 ( gap junction protein , alpha 1 , 43 kda ) , which is a member of the connexin gene family .", "the protein encoded by gja1 is a component of gap junctions , which are intercellular channels that provide a route for the diffusion of low - molecular - weight materials from cell to cell ."], ["in this study , we performed a genomewide scan for pulse rate and identified 3 loci accounting for the variation in pulse rate .", "two of 3 loci were previously reported and replicated once again in an independent hexa - shared cohort .", "the other one ( rs2892767 ) near gja1 was previously implicated for its association with pulse rate but was not chosen for the replication study , since the snp did not meet the selection criteria in the discovery stage . in the present study ,", "snp rs2892767 near gja1 was taken forward to the replication study using the independent dataset from the hexa - shared cohort and was nicely confirmed for its association with pulse rate .", "two snps , rs2892767 near gja1 and rs12110693 near loc644502 , are 408 kb apart from each other and are located in a separate ld , indicating that both are independent snps .", "we performed multiple linear regression analyses , conditioning the snps to each other to find a significantly stronger proxy for pulse rate .", "however , no dramatic decrease in statistical significance was observed , and no interaction was found between rs2892767 and rs12110693 ( table 3 ) .", "gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development .", "it is also known that gap junctions , clusters of cell - to - cell channels composed of connexins , mediate the orderly spread of electrical excitation throughout the heart .", "the gja1-encoding protein has been reported to be involved in the development and function of the heart by allowing direct cell - to - cell exchange of molecules , which mediate multiple signaling events .", "cho et al .   previously reported the same locus near gja1 to be responsible for pulse rate without evidence of replication . in the present study", "as indicated from the functional role of gja1 , it is a strong candidate gene for pulse rate .", "thus , further efforts , such as fine mapping or imputation based on large - scale sequencing data near rs2892767 on 6q22.31 , would reveal the causal variant of this locus .", "in addition , functional studies for gja1 would be worthwhile to facilitate a full understanding of its biological role in pulse rate .", "our study was relatively underpowered to discover additional novel loci and demonstrated that large - scale genomewide meta - analysis comprising hundreds of thousands samples is required for understanding the missing heritability of pulse rate ."]]}
{"article_id": "PMC5000837", "article_text": ["this complication usually occurs during abdominal aortic surgery and is an unusual complication following live donor nephrectomy .", "live kidney donors are healthy individuals who willingly undergo major surgery to improve the well - being of another individual .", "therefore , it is of utmost importance to minimize the risks of this procedure and thus maximize donor safety .", "a 39-year - old mediterranean female with a bmi of 26 , underwent an elective , living - related , transperitoneal left - sided totally robot - assisted donor nephrectomy using the da vinci surgical system .", "donor nephrectomy was performed by a transplant surgeon and assisted by a second transplant surgeon , as described elsewhere [ 2 , 3 ] .", "briefly , the procedure was performed with the da vinci surgical system s using three arms with a fenestrated bipolar forceps ( endowrist bipolar cautery instrument ; intuitive surgical , sunnydale , usa ) in arm 1 , a monopolar cautery hook ( endowrist monopolar cautery instrument ; intuitive surgical , sunnydale , usa ) to dissect structures in arm 2 and the camera ( 3d vision system 12 mm 30 endoscope ; intuitive surgical , sunnydale , usa ) in arm 3 .", "after full exposure of the kidney and vascular structures , a pfannenstiel incision was made and an endo - bag ( endocatch ; us surgical , norwalk , usa ) was introduced into the abdomen .", "subsequently , the ureter was clipped and divided with scissors , and the renal artery and the vein were transected using an endoscopic stapler ( endogia ; us surgical , norwalk , usa ) .", "the kidney was then placed in the endobag and extracted through the pfannenstiel incision . before removing all trocars ,", "the renal bed and vascular stumps were inspected using the da vinci , and intra - abdominal hemostasis was performed with the fenestrated bipolar forceps .", "the operation field was found to be dry after which all incisions were closed intracutaneously .", "postoperatively , the patient was placed on dipidolor patient - controlled analgesia combined with paracetamol . during the postoperative recovery", "she was discharged at postoperative day 3 with a normal appetite and no wound problems .", "on postoperative day 11 , she visited the outpatient clinic for a follow - up visit and the nausea was completely gone .", "however , she noted abdominal pain located on the right side near the iliac crest and reported two episodes of hematuria .", "laboratory results showed a decreased c - reactive protein ( crp ) level and normal leukocyte count .", "on postoperative day 19 , the patient noted bright red fluid leakage from the pfannenstiel incision without any further symptoms and contacted the nurse practioner .", "it was decided that she would visit the outpatient clinic if the leakage would worsen or other complaints would emerge . on postoperative day 29 , the patient visited the outpatient clinic for her scheduled 1-month postoperative follow - up visit .", "she still experienced pain in her abdomen which was mildly controlled with anti - analgesia .", "the pain progressed when she laid on her right or left side and , at night , her abdomen was more distended than usual . on physical examination", ", she had a distended abdomen , with tenderness on palpation on the right and the left side , and below the umbilicus , with a shifting dullness on percussion during movement .", "an abdominal ultrasonography showed intraperitoneal fluid in all four quadrants of the abdomen and a diagnostic paracentesis demonstrated yellow fluid with a lipid aspect , suspected of being chylous ascites and confirmed by biochemical analysis showing positive triglycerides of 57.98 mmol / l .", "she was admitted to the hospital for drainage and a medium - chain triglyceride - based diet was started . on day 4", "the medium - chain triglyceride - based diet was continued for a total of 4 weeks after which a second abdominal ultrasonography was done , which showed no fluid in the abdomen .", "in this case report , we describe the first reported complication of chylous ascites after total robot - assisted donor nephrectomy .", "this complication is more common in major oncological surgical procedures , where lymph nodes are often resected .", "therefore , from an oncological point of view these patients often casually accept this complication .", "nevertheless , healthy live kidney donors are different from patients who undergo extensive dissection damaging lymphatic vessels necessitating postoperative treatment . in over three decades", ", > 1500 live donor nephrectomies have been performed at our centre and never before have we encountered a case of postoperative chylous ascites after a donor nephrectomy .", "a review of the literature resulted in a total of 56 reported cases of chylous ascites after donor nephrectomy ( supplement 1 ) . a huge", "contrast has been compared with the thousands of live donor nephrectomies that have been published over the years .", "the occurrence of chylous ascites is low and the expectation is that this is an underestimation .", "there is no clear treatment pathway ; however , a median chain triglyceride diet combined with a therapeutic drain or octreotide seems to be the first step .", "we could not identify any other reported cases of chylous ascites after robot - assisted donor nephrectomy [ 2 , 47 ] . during a robot - assisted donor nephrectomy , it is not necessary to dissect the lymfatic tissues around the aorta and the origo of the real artery .", "larger lymphatics around the renal vessels are either clipped or sealed by modern energy devices . in our case , a fenestrated bipolar forceps and a monopolar cautery hook were used for dissection and sealing .", "however , with monopolar energy , although control the lymphatics for a short time , necrosis can occur later on leading to leakage of large amount of chyle .", "the complication is often diagnosed after a few weeks , causing a delay in treatment .", "fortunately , the complication can be successfully treated with conservative measures within a few weeks as demonstrated in this case ."], "abstract_text": ["<S> we present the first case report of chylous ascites following total robot - assisted donor nephrectomy . </S>", "<S> a 39-year - old female underwent a transperitoneal left - sided total robot - assisted donor nephrectomy . </S>", "<S> the procedure was uneventful and the patient was discharged without any symptoms . at postoperative day 29 , </S>", "<S> the patient presented with abdominal pain , nausea and a distended , painful abdomen with shifting dullness . </S>", "<S> she was diagnosed with chylous ascites by ultrasonography and puncture analysis , and treated with therapeutic drainage and dietary restriction . </S>", "<S> after 4 weeks , she was free of symptoms . </S>", "<S> the occurrence of this complication is rare after donor nephrectomy . </S>", "<S> fortunately , the complication can be successfully treated within a few weeks with minimal discomfort for the patient as demonstrated in this case . </S>", "<S> it is of utmost importance to minimize the risks and limit discomfort for live kidney donors who willingly undergo major surgery to improve the well - being of another individual . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "Conflict of interest statement", "Supplementary material"], "sections": [["this complication usually occurs during abdominal aortic surgery and is an unusual complication following live donor nephrectomy .", "live kidney donors are healthy individuals who willingly undergo major surgery to improve the well - being of another individual .", "therefore , it is of utmost importance to minimize the risks of this procedure and thus maximize donor safety ."], ["a 39-year - old mediterranean female with a bmi of 26 , underwent an elective , living - related , transperitoneal left - sided totally robot - assisted donor nephrectomy using the da vinci surgical system .", "donor nephrectomy was performed by a transplant surgeon and assisted by a second transplant surgeon , as described elsewhere [ 2 , 3 ] .", "briefly , the procedure was performed with the da vinci surgical system s using three arms with a fenestrated bipolar forceps ( endowrist bipolar cautery instrument ; intuitive surgical , sunnydale , usa ) in arm 1 , a monopolar cautery hook ( endowrist monopolar cautery instrument ; intuitive surgical , sunnydale , usa ) to dissect structures in arm 2 and the camera ( 3d vision system 12 mm 30 endoscope ; intuitive surgical , sunnydale , usa ) in arm 3 .", "after full exposure of the kidney and vascular structures , a pfannenstiel incision was made and an endo - bag ( endocatch ; us surgical , norwalk , usa ) was introduced into the abdomen .", "subsequently , the ureter was clipped and divided with scissors , and the renal artery and the vein were transected using an endoscopic stapler ( endogia ; us surgical , norwalk , usa ) .", "the kidney was then placed in the endobag and extracted through the pfannenstiel incision . before removing all trocars ,", "the renal bed and vascular stumps were inspected using the da vinci , and intra - abdominal hemostasis was performed with the fenestrated bipolar forceps .", "the operation field was found to be dry after which all incisions were closed intracutaneously .", "postoperatively , the patient was placed on dipidolor patient - controlled analgesia combined with paracetamol . during the postoperative recovery", "she was discharged at postoperative day 3 with a normal appetite and no wound problems .", "on postoperative day 11 , she visited the outpatient clinic for a follow - up visit and the nausea was completely gone .", "however , she noted abdominal pain located on the right side near the iliac crest and reported two episodes of hematuria .", "laboratory results showed a decreased c - reactive protein ( crp ) level and normal leukocyte count .", "on postoperative day 19 , the patient noted bright red fluid leakage from the pfannenstiel incision without any further symptoms and contacted the nurse practioner .", "it was decided that she would visit the outpatient clinic if the leakage would worsen or other complaints would emerge . on postoperative day 29 , the patient visited the outpatient clinic for her scheduled 1-month postoperative follow - up visit .", "she still experienced pain in her abdomen which was mildly controlled with anti - analgesia .", "the pain progressed when she laid on her right or left side and , at night , her abdomen was more distended than usual . on physical examination", ", she had a distended abdomen , with tenderness on palpation on the right and the left side , and below the umbilicus , with a shifting dullness on percussion during movement .", "an abdominal ultrasonography showed intraperitoneal fluid in all four quadrants of the abdomen and a diagnostic paracentesis demonstrated yellow fluid with a lipid aspect , suspected of being chylous ascites and confirmed by biochemical analysis showing positive triglycerides of 57.98 mmol / l .", "she was admitted to the hospital for drainage and a medium - chain triglyceride - based diet was started . on day 4", "the medium - chain triglyceride - based diet was continued for a total of 4 weeks after which a second abdominal ultrasonography was done , which showed no fluid in the abdomen ."], ["in this case report , we describe the first reported complication of chylous ascites after total robot - assisted donor nephrectomy .", "this complication is more common in major oncological surgical procedures , where lymph nodes are often resected .", "therefore , from an oncological point of view these patients often casually accept this complication .", "nevertheless , healthy live kidney donors are different from patients who undergo extensive dissection damaging lymphatic vessels necessitating postoperative treatment . in over three decades", ", > 1500 live donor nephrectomies have been performed at our centre and never before have we encountered a case of postoperative chylous ascites after a donor nephrectomy .", "a review of the literature resulted in a total of 56 reported cases of chylous ascites after donor nephrectomy ( supplement 1 ) . a huge", "contrast has been compared with the thousands of live donor nephrectomies that have been published over the years .", "the occurrence of chylous ascites is low and the expectation is that this is an underestimation .", "there is no clear treatment pathway ; however , a median chain triglyceride diet combined with a therapeutic drain or octreotide seems to be the first step .", "we could not identify any other reported cases of chylous ascites after robot - assisted donor nephrectomy [ 2 , 47 ] . during a robot - assisted donor nephrectomy , it is not necessary to dissect the lymfatic tissues around the aorta and the origo of the real artery .", "larger lymphatics around the renal vessels are either clipped or sealed by modern energy devices . in our case , a fenestrated bipolar forceps and a monopolar cautery hook were used for dissection and sealing .", "however , with monopolar energy , although control the lymphatics for a short time , necrosis can occur later on leading to leakage of large amount of chyle .", "the complication is often diagnosed after a few weeks , causing a delay in treatment .", "fortunately , the complication can be successfully treated with conservative measures within a few weeks as demonstrated in this case ."], [""], [""]]}
{"article_id": "PMC4430675", "article_text": ["obesity is a worldwide phenomenon , with an incidence increasing in parallel with obesity - associated diseases such as insulin resistance , type 2 diabetes mellitus , and hypertension .", "the combination of obesity and hypertension places patients at a higher risk of hypertensive end - organ damage and vascular events [ 2 , 3 ] .", "blood pressure ( bp ) is subject to diurnal variation , and studies using ambulatory bp monitoring ( abpm ) have demonstrated the clinical significance of disturbances in the diurnal bp profile and have associated nondipping with a progression in end - organ damage [ 4 , 5 ] .", "moreover , a nondipping pattern of bp has been reported to occur in several conditions that are frequently associated with obesity .", "although body mass index ( bmi ) is associated with the severity of ambulatory hypertension and an increase in daytime bp , bmi does not reflect body fat distribution .", "epicardial adipose tissue is related to visceral fat , rather than total adiposity , and shares microcirculation with myocardial tissue and coronary vessels [ 6 , 7 ] . because elevated bp is associated with ectopic fat accumulation in the intrathoracic and epicardial areas , a possibility of association of epicardial adipose tissue with hypertension as well as diurnal bp patterns [ 10 , 11 ] has been found in some recent studies [ 8 , 9 ] .", "epidemiological studies have found that epicardial fat is two to three times more common among females than males and that excessive body weight increases the risk of diabetes mellitus ( dm ) and cardiovascular diseases approximately one - and - a - half to two times higher in women than it does in men .", "furthermore , a higher prevalence of hypertension among overweight adults is found in women than in men , which may indicate different susceptibilities based on gender . given these facts", ", we aimed to investigate the influence of obesity on the association between epicardial fat thickness ( eft ) and circadian bp changes in patients with recently diagnosed essential hypertension ( eh ) , with a gender subgroup analysis .", "this cross - sectional observational single - center cohort study included patients who underwent 24 h abpm and echocardiography between january 2008 and october 2013 .", "demographic characteristics recorded at the first visit included age , sex , height , weight , medication , smoking history , and other diseases .", "blood was drawn for measurement of total serum cholesterol , triglycerides , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) cholesterol , blood glucose , creatinine , uric acid , and high sensitivity c - reactive protein ( hs - crp ) .", "body mass index ( bmi ) was calculated as the ratio of dry weight in kilograms to height squared ( in square meters ) .", "a total of 441 consecutive patients , who underwent office bp measurements , 24-hour abpm , and laboratory measurements for cardiovascular risk factors and echocardiography , were analyzed .", "following the recommendations of the european society of hypertension , a normotensive state was defined as a mean daytime ambulatory systolic and diastolic bp < 135/85  mmhg by abpm , associated with", "true ht was assigned if the average daytime bp was higher than 135/85  mmhg and the average nighttime bp was above 120/75  mmhg .", "obesity was defined as a bmi above 25  kg / m , as per the korean society for the study of obesity .", "the patients with the following diseases were excluded from the study : secondary hypertension , significant liver disease , neurologic disorder , malignant disease , valvular heart disease , heart failure , and history of acute coronary syndrome , myocardial infarction , coronary revascularization procedure , carotid revascularization procedure , ischemic leg ulcer , peripheral revascularization , or amputation .", "because the presence of type 2 dm is known to be associated with increased eft regardless of obesity [ 6 , 7 ] , we also excluded overt type 2 dm from the analysis to avoid its possible confounding impact on the association between eft and circadian bp variability in our hypertensive patients with or without obesity .", "office bp measurements were performed using an automated device ( easy x 800 ( r / l ) , jawon medical , seoul , korea ) .", "measurements were taken after patients had rested for 10  min in a sitting position , with the arm comfortably placed at the heart level .", "each set of two measurements was averaged to give the office systolic and diastolic bp .", "abpm was performed on each patient 's nondominant arm using an automatically oscillometric device ( tonoport v , par medizintechnik , berlin , germany ) on a normal working day .", "all subjects were instructed to rest or sleep between 10:00 pm and 7:00 am ( nighttime ) and to continue their usual activities between 7:00 am and 10:00 pm ( daytime ) .", "the accuracy of the device was checked against the standard auscultatory method to ensure that the difference in bp measurements between methods did not exceed 5  mmhg .", "the device was set to obtain bp readings at 20 min intervals during the day ( 7:00 am10:00 pm ) and at 40 min intervals during the night ( 10:00 pm7:00 am ) .", "each abpm dataset was first automatically scanned to remove artifactual readings according to preselected editing criteria .", "data were edited by omitting all readings of zero , all heart rate readings < 20 or > 200 , diastolic bp readings > 150 and < 40  mmhg , systolic bp readings > 240 and < 70  mmhg , and all readings where the difference between systolic and diastolic bps was less than 10  mmhg .", "the following abpm parameters were evaluated : average ambulatory 24-hour systolic and diastolic bp levels , average ambulatory daytime systolic and diastolic bp levels , average ambulatory nighttime systolic and diastolic bp levels , and mean ambulatory 24-hour , daytime , and nighttime bps .", "additionally , for both sbp and dbp , the magnitude of the nocturnal decline in bp (  nocturnal decline ) was calculated as follows : daytime average bp minus nighttime average ; the percentage change in bp from day to night ( % day  night bp ) was calculated as ( daytime bp  nighttime bp ) 100/daytime bp . with this latter data , a normal dipper pattern was diagnosed when the reduction in the average sbp and dbp during the nighttime was 10% of the average daytime values . the nocturnal bp status was also assessed and expressed as either nocturnal normotension or hypertension , with nocturnal hypertension defined as a nighttime bp of 120/70  mmhg .", "standard two - dimensional ( d ) and strain echocardiography were performed on all subjects while lying in the left lateral decubitus position using a 3.5 mhz transducer ( philips ie33 , philips medical systems , bothell , wa , usa ) , and the echocardiography examiners were blinded to patient information", ". measurements of the thickness of interventricular septum and posterior wall , the diameter of the lv cavity , and the lv mass index ( lvmi ) were performed according to american society of echocardiography criteria .", "pulsed wave doppler of the transmitral lv inflow was performed in an apical 4-chamber view with the sample volume placed at the level of the mitral valve tips , and the following measurements of global lv diastolic function were determined : peak early ( e ) and late ( a ) diastolic mitral flow velocity , and their ratio , e / a ; early ( ea ) diastolic mitral annular velocity ; deceleration time of the e wave ; and lv isovolumetric relaxation time ( ivrt ) .", "echocardiographic assessments of the eft were measured perpendicularly from the free wall of the right ventricle at end - systole in three cardiac cycles according to the method previously described by iacobellis et al .  ", "epicardial fat was described as the echo - free space between the outer wall of the myocardium and the visceral layer of the pericardium .", "the maximum eft was also measured from the point on the free wall of the right ventricle along the midline of the ultrasound beam perpendicular to the aortic annulus as the anatomic landmark ( figure 1(b ) ) and from the apical 4-chamber view focused on the right ventricle ( figure 1(c ) ) , because one of the critical issues in eft measurement is the inconsistency in the measurement location , and mean eft was averaged from the parasternal long axis , parasternal short axis , and apical 4-chamber view images .", "the offline measurement of eft was performed by two cardiologists ( ik shim and ki cho ) who were unaware of the clinical data .", "statistical analyses were performed with the commercially available computer program spss 18.0 for windows ( spss inc .", "values are expressed as mean  standard deviation or as a percentage ( % ) .", "parameter differences among the 3 groups were evaluated using the one - way anova test for normally distributed variables and the kruskal - wallis test for non - normally distributed variables .", "values are expressed as means ( standard deviation ) if numerical variables or as the number of subjects and their percentages ( % ) , if categorical .", "pearson correlations analysis was performed to determine factors that potentially influenced bp and hr variability .", "multivariate logistic regression models for bp variability were built to determine which variables were independently associated with this status .", "a total of 441 patients with eh ( male / female , 236/205 ; mean age , 50.7  13.8 ) and 83 normotensive normal weight patients ( controls , male / female , 42/41 ; mean age , 51.0  13.3 ) were analyzed , and their clinical features and ambulatory blood pressure parameters according to gender are given in table 1 ( male ) and table 2 ( female ) .", "circadian bp profile and bp variability assessed by 24-hour mean bps variation were increased in hypertensive patients , especially in hypertensive obese patients , without gender difference ( all p 's < 0.05 ) . moreover , the proportion of patients with a nondipping pattern was the highest among the hypertensive obese patients .", "although there were no significant differences in hr variability among the groups , hypertensive obese patients had higher daytime and 24-hour mean hr and male hypertensive obese patients also had higher nighttime hr than control patients ( all p 's < 0.05 ) .", "although total cholesterol levels were higher in hypertensive obese patients of both genders than in the controls , male hypertensive obese patients had higher triglycerides and female hypertensive obese patients had lower hdl cholesterol than controls ( all p 's < 0.05 ) .", "although there was no significant difference in systolic function , hypertensive patients showed significantly increased wall thickness and enlarged left atrial diameter , which were more prominent among hypertensive obese patients , without gender difference ( tables 3 and 4 ) .", "interestingly , eft was higher in female than in male patients ( 7.0  2.5 versus 5.9  2.2  mm , p < 0.001 ) . among women , eft was highest in the obese eh group ( 7.5  2.6  mm )", "whereas that of the control was 6.4  2.8  mm and that of the nonobese eh group was 6.7  2.8  mm ; however , eft did not vary significantly among males ( 5.9  1.9 versus 6.0  2.7 versus 5.9  2.4  mm , resp .", ", p = 0.937 ) . when we compared the 24-hour abmp parameters and eft between male and female patients , female patients had significantly higher eft ( 7.0  2.6 versus 5.9  2.2  mm , p < 0.001 ) and a greater daytime bp variation ( 15.1  4.72 versus 13.6  4.45  mmhg , p < 0.001 ) than male subjects", "; males had higher 24-hour mean bp ( 138.8  14.7 versus 137.3  15.3  mmhg , p = 0.043 ) and hr variability ( 15.6  7.45 versus 14.3  6.02 , p = 0.023 ) than female subjects ( table 5 ) .", "variable circadian bp profiles and la size were significantly related to bmi in both females and males ( all p 's < 0.05 , figures 2(a ) and 2(b ) , table 6 ) .", "however , eft was significantly correlated with 24-hour mean bp ( both day and night ) as well as variability ( mainly night ) only in female patients ( figure 2(c ) ) .", "when we investigated the association of quotient of eft / bmi with bp values , eft / bmi showed positive correlation with 24-hour systolic bp ( r = 0.172 ; p = 0.009 ) , 24-hour mean bp ( r = 0.134 ; p = 0.041 ) , day systolic bp ( r = 0.150 ; p = 0.022 ) , day systolic bp variation ( r = 0.133 ; p = 0.043 ) , night systolic bp ( r = 0.217 ; p = 0.001 ) , night systolic bp variation ( r = 0.181 ; p = 0.006 ) , night diastolic bp ( r = 0.132 ; p = 0.047 ) , and night diastolic bp variation ( r = 0.179 ; p = 0.007 ) .", "however , no correlation was shown between eft / bmi and 24-hour abpm parameters in male .", "multivariate logistic regression analysis demonstrated that the 24-hour mean bp variability was associated with sbp ( standardized  coefficient = 0.199 ; p = 0.018 ) and eft ( standardized  coefficient = 0.175 ; p = 0.016 ) in female patients , but not in male ones ( table 7 ) .", "when we performed binary logistic regression analysis to identify the independent determinants of nocturnal nondipping bp pattern , eft was independent contributor to the nondipping pattern only in female ( odds ratio 7.034 , 95% confidence interval 2.258 to 21.909 , p = 0.001 ) ( table 8) .", "the most relevant information obtained from this study is that relationships among eft , obesity , and circadian bp variability are affected by gender in different manners .", "although circadian bp profile and bp variability were increased in hypertensive obese patients without gender differences , female patients had significantly higher eft and greater daytime bp variability than male subjects .", "bp variability was associated with sbp and eft only in female patients ; therefore , eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men .", "bmi is known to be associated with the severity of ambulatory hypertension and increased daytime bp . in the present study", ", we investigated the relationship between the parameters derived from 24-hour abpm ( 24-hour sbp and dbp , and bp variability ) in nondiabetic hypertensive patients with or without obesity .", "in general , bmi is correlated with bp level , and individuals with abdominal obesity have increased bp levels or are at risk for hypertension [ 3 , 5 ] . the mechanism behind obesity - related bp elevation has not been fully established .", "obesity may induce autonomic dysfunction secondary to the elevation of plasma insulin concentration , because hyperinsulinemia has been associated with sympathetic activation [ 18 , 19 ] and marked increases in hr . vice versa , sympathetic hyperactivity is also an independent contributor to the insulin resistance associated with obesity [ 21 , 22 ] .", "thus , it is possible that mean arterial pressure and nighttime hr might remain at daytime levels as a consequence of sustained elevation of plasma insulin concentration .", "our hypertensive obese patients had higher proportions of nondipping patterns and higher daytime and 24-hour mean hr ; moreover , male hypertensive obese patients also had higher nighttime hr than the controls .", "these findings are similar to the report of antic et al . , who showed that the normal dipping pattern of bp and hr is rapidly lost following a switch to a high fat diet , without affecting daytime values .", "moreover , obesity suppresses nighttime parasympathetic activity and increases nighttime hr values . because diurnal variations in mean arterial pressure and hr appear to be autonomically mediated , the loss of nocturnal dipping of mean arterial pressure and hr in our obese patients must represent an underlying change in autonomic function .", "since the nighttime mean bp was altered in obese patients , the presence of hypertension in obese patients might be missed by office bp measurements that were made only during the daytime .", "thus , our results emphasize the importance of continuous 24-hour monitoring of cardiovascular variables , especially in obese patients .", "regarding gender differences , obese females showed a higher tendency towards bp variability than obese males .", "one interesting finding was that females showed a significant relationship between increased eft , obesity , and bp variability , but this was not the case in males .", "still yet , there is no consensus in the literature on the impact of gender on the amount of epicardial fat .", "iacobellis et al . showed bigger size of eft in males than in females , and our result showed the greater values of eft in females .", "recent study by akilli et al .   showed larger eft in females than in males .", "thus , these findings may indicate different gender susceptibilities related to obesity and the regional differences in fat distribution , especially eft distribution .", "several studies have reported gender differences regarding the effects of obesity and change in body weight on bp [ 13 , 26 ] and cardiovascular outcome [ 12 , 27 ] .", "it has been reported that obese women have a higher relative risk of diabetes and heart failure with increasing abdominal circumference than men   and that obesity has an association with diastolic dysfunction and lvmi only in women . from these results", ", women can be considered to be more susceptible to cardiovascular adverse effects related to obesity . in our study , accentuated bp variability in obese hypertensive women may have indicated a role in cardiovascular vulnerability . however , the exact explanation for these gender differences is still unclear .", "one possible mechanism is that such gender differences result from abrupt hormonal and body fat composition changes after menopause .", "thus , obese females have a greater amount of visceral adipose tissue which may lead to insulin resistance .", "insulin resistance and changes in the autonomic nervous system as a result of redistribution of body fat may have an impact on the cardiovascular system in obese women .", "excess fat has traditionally been understood to be found in intra - abdominal organs such as the liver and in subcutaneous tissue .", "recently , however , epicardial adipose tissue has been found to reflect visceral adiposity and has been proposed as a new cardiometabolic risk factor . increased plasma fatty acid levels", "may stimulate the cardiac autonomic nervous system activity through an increase in plasma catecholamine concentrations , which may be related to impaired diurnal bp patterns .", "several reports have found a relationship between bp pattern and eft [ 10 , 31 ] , and echocardiographically assessed elevated eft was independently associated with impaired diurnal blood pressure profiles in hypertensive individuals . also , increased eft has been reported to be associated with diastolic dysfunction and la dilatation due to local or systemic effects in untreated hypertensive patients . in our results , variable circadian bp profiles and la size", "were significantly related tobmi in both males and females , but eft was significantly correlated with la diameter and 24-hour mean bp with variability as well as nocturnal nondipping bp pattern , only in female patients .", "the present study suggests that there would be a sex - dependent regulation of bp associated with epicardial adipose tissue , and a recent study demonstrated a sex - dependent regulation of diet - induced lvh associated with sexual dimorphic expression of adipocytokines in epicardial adipose tissue .", "although our patient sample seems sufficiently large compared to other studies in the literature , in this cross - sectional study we observed predictive factors and outcome variables simultaneously and had no follow - up data .", ", although we suggested that the hyperinsulinemia would be the possible main cause of elevated nighttime hr levels , we did not measure the plasma insulin levels . because both pre- and postmenopausal women were included in the study , we can not assume the impact of estrogen on epicardial fat . finally , all participants were korean , and we thus can not generalize our results to populations of other ethnic groups . in conclusion ,", "the relationship among eft , obesity , and circadian bp variability was affected by gender in different manners .", "eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men ."], "abstract_text": ["<S> this study aimed to investigate the effects of gender on the association between epicardial fat thickness ( eft ) and circadian blood pressure ( bp ) changes in patients with recently diagnosed essential hypertension ( eh ) . </S>", "<S> a total of 441 patients with eh ( male / female : 236/205 , mean age : 50.7  13.8 ) and 83 control patients underwent 24-hour ambulatory bp monitoring and echocardiography . </S>", "<S> obese eh patients had higher circadian bp profile with bp variability , wall thickness , and left ventricular mass than nonobese eh patients and controls ( all p 's < 0.05 ) without gender differences . </S>", "<S> eft was higher in female than in male patients ( 7.0  2.5 versus 5.9  2.2  mm , p < 0.001 ) and higher in the obese female eh group ( 7.5  2.6  mm ) than in the control ( 6.4  2.8  mm ) or nonobese eh group ( 6.7  2.8  mm ) among women , whereas eft did not vary among males ( 5.9  1.9 versus 6.0  2.7 versus 5.9  2.4  mm , p = 0.937 ) . </S>", "<S> multivariate logistic regression analysis demonstrated that the 24-hour mean bp variability was associated with sbp ( p = 0.018 ) and eft ( p = 0.016 ) in female patients , but not in male patients . </S>", "<S> the relationships among circadian bp variability , obesity , and eft were affected by gender in different manners . </S>", "<S> eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["obesity is a worldwide phenomenon , with an incidence increasing in parallel with obesity - associated diseases such as insulin resistance , type 2 diabetes mellitus , and hypertension .", "the combination of obesity and hypertension places patients at a higher risk of hypertensive end - organ damage and vascular events [ 2 , 3 ] .", "blood pressure ( bp ) is subject to diurnal variation , and studies using ambulatory bp monitoring ( abpm ) have demonstrated the clinical significance of disturbances in the diurnal bp profile and have associated nondipping with a progression in end - organ damage [ 4 , 5 ] .", "moreover , a nondipping pattern of bp has been reported to occur in several conditions that are frequently associated with obesity .", "although body mass index ( bmi ) is associated with the severity of ambulatory hypertension and an increase in daytime bp , bmi does not reflect body fat distribution .", "epicardial adipose tissue is related to visceral fat , rather than total adiposity , and shares microcirculation with myocardial tissue and coronary vessels [ 6 , 7 ] . because elevated bp is associated with ectopic fat accumulation in the intrathoracic and epicardial areas , a possibility of association of epicardial adipose tissue with hypertension as well as diurnal bp patterns [ 10 , 11 ] has been found in some recent studies [ 8 , 9 ] .", "epidemiological studies have found that epicardial fat is two to three times more common among females than males and that excessive body weight increases the risk of diabetes mellitus ( dm ) and cardiovascular diseases approximately one - and - a - half to two times higher in women than it does in men .", "furthermore , a higher prevalence of hypertension among overweight adults is found in women than in men , which may indicate different susceptibilities based on gender . given these facts", ", we aimed to investigate the influence of obesity on the association between epicardial fat thickness ( eft ) and circadian bp changes in patients with recently diagnosed essential hypertension ( eh ) , with a gender subgroup analysis ."], ["this cross - sectional observational single - center cohort study included patients who underwent 24 h abpm and echocardiography between january 2008 and october 2013 .", "demographic characteristics recorded at the first visit included age , sex , height , weight , medication , smoking history , and other diseases .", "blood was drawn for measurement of total serum cholesterol , triglycerides , high - density lipoprotein ( hdl ) and low - density lipoprotein ( ldl ) cholesterol , blood glucose , creatinine , uric acid , and high sensitivity c - reactive protein ( hs - crp ) .", "body mass index ( bmi ) was calculated as the ratio of dry weight in kilograms to height squared ( in square meters ) .", "a total of 441 consecutive patients , who underwent office bp measurements , 24-hour abpm , and laboratory measurements for cardiovascular risk factors and echocardiography , were analyzed .", "following the recommendations of the european society of hypertension , a normotensive state was defined as a mean daytime ambulatory systolic and diastolic bp < 135/85  mmhg by abpm , associated with", "true ht was assigned if the average daytime bp was higher than 135/85  mmhg and the average nighttime bp was above 120/75  mmhg .", "obesity was defined as a bmi above 25  kg / m , as per the korean society for the study of obesity .", "the patients with the following diseases were excluded from the study : secondary hypertension , significant liver disease , neurologic disorder , malignant disease , valvular heart disease , heart failure , and history of acute coronary syndrome , myocardial infarction , coronary revascularization procedure , carotid revascularization procedure , ischemic leg ulcer , peripheral revascularization , or amputation .", "because the presence of type 2 dm is known to be associated with increased eft regardless of obesity [ 6 , 7 ] , we also excluded overt type 2 dm from the analysis to avoid its possible confounding impact on the association between eft and circadian bp variability in our hypertensive patients with or without obesity .", "office bp measurements were performed using an automated device ( easy x 800 ( r / l ) , jawon medical , seoul , korea ) .", "measurements were taken after patients had rested for 10  min in a sitting position , with the arm comfortably placed at the heart level .", "each set of two measurements was averaged to give the office systolic and diastolic bp .", "abpm was performed on each patient 's nondominant arm using an automatically oscillometric device ( tonoport v , par medizintechnik , berlin , germany ) on a normal working day .", "all subjects were instructed to rest or sleep between 10:00 pm and 7:00 am ( nighttime ) and to continue their usual activities between 7:00 am and 10:00 pm ( daytime ) .", "the accuracy of the device was checked against the standard auscultatory method to ensure that the difference in bp measurements between methods did not exceed 5  mmhg .", "the device was set to obtain bp readings at 20 min intervals during the day ( 7:00 am10:00 pm ) and at 40 min intervals during the night ( 10:00 pm7:00 am ) .", "each abpm dataset was first automatically scanned to remove artifactual readings according to preselected editing criteria .", "data were edited by omitting all readings of zero , all heart rate readings < 20 or > 200 , diastolic bp readings > 150 and < 40  mmhg , systolic bp readings > 240 and < 70  mmhg , and all readings where the difference between systolic and diastolic bps was less than 10  mmhg .", "the following abpm parameters were evaluated : average ambulatory 24-hour systolic and diastolic bp levels , average ambulatory daytime systolic and diastolic bp levels , average ambulatory nighttime systolic and diastolic bp levels , and mean ambulatory 24-hour , daytime , and nighttime bps .", "additionally , for both sbp and dbp , the magnitude of the nocturnal decline in bp (  nocturnal decline ) was calculated as follows : daytime average bp minus nighttime average ; the percentage change in bp from day to night ( % day  night bp ) was calculated as ( daytime bp  nighttime bp ) 100/daytime bp . with this latter data , a normal dipper pattern was diagnosed when the reduction in the average sbp and dbp during the nighttime was 10% of the average daytime values . the nocturnal bp status was also assessed and expressed as either nocturnal normotension or hypertension , with nocturnal hypertension defined as a nighttime bp of 120/70  mmhg .", "standard two - dimensional ( d ) and strain echocardiography were performed on all subjects while lying in the left lateral decubitus position using a 3.5 mhz transducer ( philips ie33 , philips medical systems , bothell , wa , usa ) , and the echocardiography examiners were blinded to patient information", ". measurements of the thickness of interventricular septum and posterior wall , the diameter of the lv cavity , and the lv mass index ( lvmi ) were performed according to american society of echocardiography criteria .", "pulsed wave doppler of the transmitral lv inflow was performed in an apical 4-chamber view with the sample volume placed at the level of the mitral valve tips , and the following measurements of global lv diastolic function were determined : peak early ( e ) and late ( a ) diastolic mitral flow velocity , and their ratio , e / a ; early ( ea ) diastolic mitral annular velocity ; deceleration time of the e wave ; and lv isovolumetric relaxation time ( ivrt ) .", "echocardiographic assessments of the eft were measured perpendicularly from the free wall of the right ventricle at end - systole in three cardiac cycles according to the method previously described by iacobellis et al .  ", "epicardial fat was described as the echo - free space between the outer wall of the myocardium and the visceral layer of the pericardium .", "the maximum eft was also measured from the point on the free wall of the right ventricle along the midline of the ultrasound beam perpendicular to the aortic annulus as the anatomic landmark ( figure 1(b ) ) and from the apical 4-chamber view focused on the right ventricle ( figure 1(c ) ) , because one of the critical issues in eft measurement is the inconsistency in the measurement location , and mean eft was averaged from the parasternal long axis , parasternal short axis , and apical 4-chamber view images .", "the offline measurement of eft was performed by two cardiologists ( ik shim and ki cho ) who were unaware of the clinical data .", "statistical analyses were performed with the commercially available computer program spss 18.0 for windows ( spss inc .", "values are expressed as mean  standard deviation or as a percentage ( % ) .", "parameter differences among the 3 groups were evaluated using the one - way anova test for normally distributed variables and the kruskal - wallis test for non - normally distributed variables .", "values are expressed as means ( standard deviation ) if numerical variables or as the number of subjects and their percentages ( % ) , if categorical .", "pearson correlations analysis was performed to determine factors that potentially influenced bp and hr variability .", "multivariate logistic regression models for bp variability were built to determine which variables were independently associated with this status ."], ["a total of 441 patients with eh ( male / female , 236/205 ; mean age , 50.7  13.8 ) and 83 normotensive normal weight patients ( controls , male / female , 42/41 ; mean age , 51.0  13.3 ) were analyzed , and their clinical features and ambulatory blood pressure parameters according to gender are given in table 1 ( male ) and table 2 ( female ) .", "circadian bp profile and bp variability assessed by 24-hour mean bps variation were increased in hypertensive patients , especially in hypertensive obese patients , without gender difference ( all p 's < 0.05 ) . moreover , the proportion of patients with a nondipping pattern was the highest among the hypertensive obese patients .", "although there were no significant differences in hr variability among the groups , hypertensive obese patients had higher daytime and 24-hour mean hr and male hypertensive obese patients also had higher nighttime hr than control patients ( all p 's < 0.05 ) .", "although total cholesterol levels were higher in hypertensive obese patients of both genders than in the controls , male hypertensive obese patients had higher triglycerides and female hypertensive obese patients had lower hdl cholesterol than controls ( all p 's < 0.05 ) .", "although there was no significant difference in systolic function , hypertensive patients showed significantly increased wall thickness and enlarged left atrial diameter , which were more prominent among hypertensive obese patients , without gender difference ( tables 3 and 4 ) .", "interestingly , eft was higher in female than in male patients ( 7.0  2.5 versus 5.9  2.2  mm , p < 0.001 ) . among women , eft was highest in the obese eh group ( 7.5  2.6  mm )", "whereas that of the control was 6.4  2.8  mm and that of the nonobese eh group was 6.7  2.8  mm ; however , eft did not vary significantly among males ( 5.9  1.9 versus 6.0  2.7 versus 5.9  2.4  mm , resp .", ", p = 0.937 ) . when we compared the 24-hour abmp parameters and eft between male and female patients , female patients had significantly higher eft ( 7.0  2.6 versus 5.9  2.2  mm , p < 0.001 ) and a greater daytime bp variation ( 15.1  4.72 versus 13.6  4.45  mmhg , p < 0.001 ) than male subjects", "; males had higher 24-hour mean bp ( 138.8  14.7 versus 137.3  15.3  mmhg , p = 0.043 ) and hr variability ( 15.6  7.45 versus 14.3  6.02 , p = 0.023 ) than female subjects ( table 5 ) .", "variable circadian bp profiles and la size were significantly related to bmi in both females and males ( all p 's < 0.05 , figures 2(a ) and 2(b ) , table 6 ) .", "however , eft was significantly correlated with 24-hour mean bp ( both day and night ) as well as variability ( mainly night ) only in female patients ( figure 2(c ) ) .", "when we investigated the association of quotient of eft / bmi with bp values , eft / bmi showed positive correlation with 24-hour systolic bp ( r = 0.172 ; p = 0.009 ) , 24-hour mean bp ( r = 0.134 ; p = 0.041 ) , day systolic bp ( r = 0.150 ; p = 0.022 ) , day systolic bp variation ( r = 0.133 ; p = 0.043 ) , night systolic bp ( r = 0.217 ; p = 0.001 ) , night systolic bp variation ( r = 0.181 ; p = 0.006 ) , night diastolic bp ( r = 0.132 ; p = 0.047 ) , and night diastolic bp variation ( r = 0.179 ; p = 0.007 ) .", "however , no correlation was shown between eft / bmi and 24-hour abpm parameters in male .", "multivariate logistic regression analysis demonstrated that the 24-hour mean bp variability was associated with sbp ( standardized  coefficient = 0.199 ; p = 0.018 ) and eft ( standardized  coefficient = 0.175 ; p = 0.016 ) in female patients , but not in male ones ( table 7 ) .", "when we performed binary logistic regression analysis to identify the independent determinants of nocturnal nondipping bp pattern , eft was independent contributor to the nondipping pattern only in female ( odds ratio 7.034 , 95% confidence interval 2.258 to 21.909 , p = 0.001 ) ( table 8) ."], ["the most relevant information obtained from this study is that relationships among eft , obesity , and circadian bp variability are affected by gender in different manners .", "although circadian bp profile and bp variability were increased in hypertensive obese patients without gender differences , female patients had significantly higher eft and greater daytime bp variability than male subjects .", "bp variability was associated with sbp and eft only in female patients ; therefore , eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men .", "bmi is known to be associated with the severity of ambulatory hypertension and increased daytime bp . in the present study", ", we investigated the relationship between the parameters derived from 24-hour abpm ( 24-hour sbp and dbp , and bp variability ) in nondiabetic hypertensive patients with or without obesity .", "in general , bmi is correlated with bp level , and individuals with abdominal obesity have increased bp levels or are at risk for hypertension [ 3 , 5 ] . the mechanism behind obesity - related bp elevation has not been fully established .", "obesity may induce autonomic dysfunction secondary to the elevation of plasma insulin concentration , because hyperinsulinemia has been associated with sympathetic activation [ 18 , 19 ] and marked increases in hr . vice versa , sympathetic hyperactivity is also an independent contributor to the insulin resistance associated with obesity [ 21 , 22 ] .", "thus , it is possible that mean arterial pressure and nighttime hr might remain at daytime levels as a consequence of sustained elevation of plasma insulin concentration .", "our hypertensive obese patients had higher proportions of nondipping patterns and higher daytime and 24-hour mean hr ; moreover , male hypertensive obese patients also had higher nighttime hr than the controls .", "these findings are similar to the report of antic et al . , who showed that the normal dipping pattern of bp and hr is rapidly lost following a switch to a high fat diet , without affecting daytime values .", "moreover , obesity suppresses nighttime parasympathetic activity and increases nighttime hr values . because diurnal variations in mean arterial pressure and hr appear to be autonomically mediated , the loss of nocturnal dipping of mean arterial pressure and hr in our obese patients must represent an underlying change in autonomic function .", "since the nighttime mean bp was altered in obese patients , the presence of hypertension in obese patients might be missed by office bp measurements that were made only during the daytime .", "thus , our results emphasize the importance of continuous 24-hour monitoring of cardiovascular variables , especially in obese patients .", "regarding gender differences , obese females showed a higher tendency towards bp variability than obese males .", "one interesting finding was that females showed a significant relationship between increased eft , obesity , and bp variability , but this was not the case in males .", "still yet , there is no consensus in the literature on the impact of gender on the amount of epicardial fat .", "iacobellis et al . showed bigger size of eft in males than in females , and our result showed the greater values of eft in females .", "recent study by akilli et al .   showed larger eft in females than in males .", "thus , these findings may indicate different gender susceptibilities related to obesity and the regional differences in fat distribution , especially eft distribution .", "several studies have reported gender differences regarding the effects of obesity and change in body weight on bp [ 13 , 26 ] and cardiovascular outcome [ 12 , 27 ] .", "it has been reported that obese women have a higher relative risk of diabetes and heart failure with increasing abdominal circumference than men   and that obesity has an association with diastolic dysfunction and lvmi only in women . from these results", ", women can be considered to be more susceptible to cardiovascular adverse effects related to obesity . in our study , accentuated bp variability in obese hypertensive women may have indicated a role in cardiovascular vulnerability . however , the exact explanation for these gender differences is still unclear .", "one possible mechanism is that such gender differences result from abrupt hormonal and body fat composition changes after menopause .", "thus , obese females have a greater amount of visceral adipose tissue which may lead to insulin resistance .", "insulin resistance and changes in the autonomic nervous system as a result of redistribution of body fat may have an impact on the cardiovascular system in obese women .", "excess fat has traditionally been understood to be found in intra - abdominal organs such as the liver and in subcutaneous tissue .", "recently , however , epicardial adipose tissue has been found to reflect visceral adiposity and has been proposed as a new cardiometabolic risk factor . increased plasma fatty acid levels", "may stimulate the cardiac autonomic nervous system activity through an increase in plasma catecholamine concentrations , which may be related to impaired diurnal bp patterns .", "several reports have found a relationship between bp pattern and eft [ 10 , 31 ] , and echocardiographically assessed elevated eft was independently associated with impaired diurnal blood pressure profiles in hypertensive individuals . also , increased eft has been reported to be associated with diastolic dysfunction and la dilatation due to local or systemic effects in untreated hypertensive patients . in our results , variable circadian bp profiles and la size", "were significantly related tobmi in both males and females , but eft was significantly correlated with la diameter and 24-hour mean bp with variability as well as nocturnal nondipping bp pattern , only in female patients .", "the present study suggests that there would be a sex - dependent regulation of bp associated with epicardial adipose tissue , and a recent study demonstrated a sex - dependent regulation of diet - induced lvh associated with sexual dimorphic expression of adipocytokines in epicardial adipose tissue .", "although our patient sample seems sufficiently large compared to other studies in the literature , in this cross - sectional study we observed predictive factors and outcome variables simultaneously and had no follow - up data .", ", although we suggested that the hyperinsulinemia would be the possible main cause of elevated nighttime hr levels , we did not measure the plasma insulin levels . because both pre- and postmenopausal women were included in the study , we can not assume the impact of estrogen on epicardial fat . finally , all participants were korean , and we thus can not generalize our results to populations of other ethnic groups . in conclusion ,", "the relationship among eft , obesity , and circadian bp variability was affected by gender in different manners .", "eft may be a more valuable parameter in the evaluation of bp severity and obesity in women than in men ."]]}
{"article_id": "PMC4370038", "article_text": ["during the past 2 decades , laparoscopic surgery has gained worldwide popularity resulting from its well - known characteristics of less postoperative pain , fewer complications , earlier discharge , and better cosmesis .", "smaller skin incisions in laparoscopic surgery were related to fewer unwanted side effects , including less postoperative pain . however , in recent studies comparing conventional and single - incision laparoscopic surgical procedures , the postoperative pain scores were not different .", "keyless abdominal rope - lifting surgery ( kars ) is a novel , gasless ( isobaric ) , single - incision laparoscopic surgical technique used in various gynecologic operations and cholecystectomies . besides being a single - incision laparoscopic technique", ", kars does not require carbon dioxide ( co2 ) use to inflate the abdominal wall and the intra - abdominal pressure is not elevated during the operation .", "in addition , postoperative shoulder pain seems to be more frequently observed after laparoscopic surgical procedures in which the intra - abdominal pressure is elevated to between 12 and 18 mm hg by use of co2 . in this study we aimed to compare the postoperative pain levels for kars and conventional multiport laparoscopy performed for adnexal cysts .", "although kars has been performed in our institute since the first half of 2010 , the study included benign ovarian cysts operated on between november 2011 and june 2013 .", "beginning in november 2011 , intraoperative and postoperative pain management for all operations performed in our unit was standardized ; thus the uniform management approach allowed us to collect and analyze the data from different surgical techniques .", "the study was approved by the local institutional ethical committee of kafkas university school of medicine .", "the included women had cystic masses including simple cysts with or without septation , endometriomas , and benign cystic teratomas .", "most of the participants complained of groin pain , menstrual bleeding disorders , dysmenorrhea , and infertility .", "emergency cases including patients with hemodynamic instability , severe abdominal pain , and inaccurate preoperative diagnoses were excluded .", "malignant cysts were excluded based on the patient 's history , clinical findings , ultrasonography and magnetic resonance imaging , and levels of cancer antigen 125 , alpha fetoprotein , carcinoembryonic antigen , and cancer antigen 199 .", "women who were diagnosed with an adnexal cyst and in whom a laparoscopic operation was indicated were invited to choose one of the operative techniques .", "because kars was performed only in our center , we could not design a randomized trial .", "thus the groups of this cross - sectional prospective clinical study were formed according to the women 's choice as the kars or conventional co2 laparoscopy groups .", "initial visual analog scale ( vas ) scores of 5.7  1.42 and 6.3  1.42 in patients undergoing kars and conventional laparoscopy , respectively , indicated that we needed at least 30 operations in each group to perform a study with a statistical power of 80% at an  level of .05 .", "the demographic and physical characteristics of the participating patients included their age , gravidity , parity , height , weight , and body mass index , as well as the number of abortions , ectopic pregnancies , and offspring .", "oral intake was prohibited starting at 11 pm , and a 135-ml solution containing 19 g of sodium dihydrogen phosphate and 7 g of disodium hydrogen phosphate was administered rectally at 6 am on the operation day .", "we used the operative laparoscopy study group 's classification system for staging intra - abdominal adhesions : 0 , none ; 1 , filmy and avascular ; 2 , dense and vascular ; and 3 , binding and cohesive .", "the abdominal access time in kars patients included the time needed for the construction of the pathway into the abdominal cavity and the placement of the lifting ropes . in conventional laparoscopy patients ,", "the abdominal access time included the time needed for the creation of the co2 pneumoperitoneum at 14 mm hg of pressure and the insertion of the 3 intra - abdominal access ports .", "all included patients were considered class 1 or 2 according to the american society of anesthesiologists classification and received a standard anesthetic management regimen during the operations .", "we used propofol , 1.5 to 2.5 mg / kg , to induce anesthesia and facilitated intubation with rocuronium , 0.4 to 0.6 mg / kg .", "oxygen was supplemented before and after the intubation at rates of 100% and 50% ( mixed with the operating theater 's air ) , respectively .", "we used 2% sevoflurane and 50 g of fentanyl for anesthesia maintenance . to replace the fluid deficit , we used sodium chloride or ringer lactate solution at 10 ml / kg on intravenous insertion at 2 ml / kg per hour .", "unexpected bleeding was managed by use of additional crystalloid solution at 3 ml / kg per hour .", "atropine , 1 to 1.2 mg intravenously , was used to reverse the neuromuscular blockade .", "the postoperative pain management and vas pain score recordings were initiated in the obstetrics and gynecology service .", "all patients , regardless of initial vas pain score , received 50 mg of meperidine intramuscularly at the beginning of the postoperative period and 75 mg of diclofenac sodium during the second postoperative hour .", "vas pain scores were recorded at the beginning and second , fourth , and 24th hours of the postoperative period .", "beginning from the fourth postoperative hour , patients with vas pain scores of > 4 points received an additional dose of meperidine , 25 mg intramuscularly .", "kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,", "the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .", "depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger .", "after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .", "the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage", ", the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .", "the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .", "while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .", "the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .", "the veress needle is unloaded from its cannula , and one tip of a no .", "the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .", "the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .", "it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .", "the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .", "the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip", "then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .", "the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .", "the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .", "there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .", "if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .", "lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .", "if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .", "however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .", "the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .", "3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .", "one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .", "the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .", "co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,", "all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4", "statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .", "the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .", "normally distributed variables of the 2 groups were compared by use of the student t test , and non  normally distributed variables were compared by use of the mann - whitney u test .", "the  test was used to assess the probability of existence of shoulder pain in the 2 groups .", "a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .", "the hosmer - lemeshow test showed that the model fit (  = 13 762 , p = .88 , n = 77 ) .", "kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,", "the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .", "depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .", "the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage", ", the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .", "the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .", "while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .", "the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .", "the veress needle is unloaded from its cannula , and one tip of a no .", "the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .", "the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .", "it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .", "the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .", "the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .", "then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .", "the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .", "the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .", "there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .", "if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .", "lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .", "if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .", "however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .", "the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .", "3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .", "one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .", "the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .", "kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,", "the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .", "depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .", "the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage", ", the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .", "the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .", "while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .", "the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .", "the veress needle is unloaded from its cannula , and one tip of a no .", "the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .", "the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .", "it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .", "the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .", "the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .", "then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .", "the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .", "the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .", "there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .", "if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .", "lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .", "if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .", "however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .", "the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .", "3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .", "one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .", "the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .", "co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,", "all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4 - 0 intracutaneous absorbable sutures .", "statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .", "the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .", "normally distributed variables of the 2 groups were compared by use of the student t test , and non  normally distributed variables were compared by use of the mann - whitney u test .", "the  test was used to assess the probability of existence of shoulder pain in the 2 groups .", "a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .", "the hosmer - lemeshow test showed that the model fit (  = 13 762 , p = .88 , n = 77 ) .", "during a 20-month period , 77 women with benign ovarian cysts were operated on by kars ( n = 32 ) or conventional co2 laparoscopy ( n = 45 ) .", "the demographic data , physical characteristics , and preoperative findings of the participating women were summarized in table 1 .", "the demographic findings , surgical histories , and preoperative hematologic statuses of the groups did not differ significantly ( p > .05 ) .", "demographic data , physical characteristics , and preoperative findings of participating women in both groups data are presented as mean  standard deviation unless otherwise indicated .", "conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) .", "minor bleeding and unintended cyst ruptures were the only observed intraoperative complications . in none of the cases was a blood transfusion or postoperative intensive care admission needed .", "we did not need to convert to conventional laparoscopy or laparotomy in the kars group or to laparotomy or mini - laparotomy in the conventional laparoscopy group .", "simple oral analgesic use after the 24th hour of the postoperative period was adequate for postoperative pain relief in both groups .", "most of the intraoperative and postoperative findings were similar in the 2 groups ( p > .05 ) ; however , the abdominal cavity access and total operative times in the kars group were significantly longer than those in the conventional laparoscopy group ( p < .05 ) .", "intraoperative and postoperative findings according to operative technique data are presented as mean  standard deviation unless otherwise indicated .", "the vas pain scores at the beginning and second , fourth , and 24th hours of the postoperative period were significantly lower in the kars group ( p < .05 ) , although the differences in mean vas scores never exceeded 1 point .", "in addition , the women in the kars group needed less additional analgesia in comparison with those in the conventional laparoscopy group ( p < .05 ) .", "moreover , a higher number and percentage of the patients in the conventional co2 laparoscopy group had shoulder pain ( p < .05 ) .", "postoperative pain scores and additional analgesic requirements according to operative techniques data are presented as mean  standard deviation unless otherwise indicated .", "conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) . the  test analyzing shoulder pain showed that postoperative shoulder pain was significantly less frequent in the kars group (  = 5.304 , p = .021 , n = 77 ) .", "logistic regression for postoperative shoulder pain predicted that shoulder pain was affected only by the operation type and duration ( table 4 ) .", "although the operation duration was longer in the kars group , the frequency of shoulder pain was higher in the conventional laparoscopy group .", "the total operative time correlated positively with the abdominal access and rope - lifting process time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) . in the conventional laparoscopy group ,", "the total operative time correlated positively with the abdominal access time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) .", "in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain .", ", our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .", "postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions .", "although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .", "the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .", "postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .", "pain sensation is subjective and may even differ for the same individual under different conditions .", "it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .", "although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar .", "gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons . however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .", "in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .", "gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .", "single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .", "gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .", "similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .", "however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study", "in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .", "thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .", "recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .", "surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study", "we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .", "in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .", "previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al", ", the postoperative pain scores did not differ between single - incision and multiport laparoscopies .", "however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study", "the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up . in addition", ", there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .", "moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .", "better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .", "although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .", "they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .", "although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .", "the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .", "the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,", "fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .", "in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011", "prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .", "they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .", "although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study , although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .", "our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .", "however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .", "in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum .", "in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain .", "to our knowledge , our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .", "postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions .", "although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .", "the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .", "postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .", "pain sensation is subjective and may even differ for the same individual under different conditions .", "it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .", "although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar .", "gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons .", "however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .", "in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .", "gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .", "single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .", "gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .", "similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .", "however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study", "in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .", "thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .", "recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .", "surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study", "we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .", "in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .", "previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al , the postoperative pain scores did not differ between single - incision and multiport laparoscopies . however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study", "the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up .", "in addition , there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .", "moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .", "better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .", "although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .", "they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .", "although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .", "the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .", "the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,", "fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .", "in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011", "prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .", "they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .", "although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study", ", although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .", "our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .", "however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .", "in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum .", "kars seems to cause less postoperative pain during the management of benign ovarian cysts in comparison with conventional multiport co2 laparoscopy ."], "abstract_text": ["<S> background and objectives : keyless abdominal rope - lifting surgery is a novel , gasless , single - incision laparoscopic surgical technique . in this study we aimed to compare the postoperative pain from keyless abdominal rope - lifting surgery with carbon dioxide laparoscopy performed for benign ovarian cysts.methods:during a 20-month period , 77 women underwent surgery for a benign ovarian cyst . </S>", "<S> keyless abdominal rope - lifting surgery and conventional carbon dioxide laparoscopy techniques were used for the operations in 32 women and 45 women , respectively . </S>", "<S> the 2 operative techniques were compared with regard to demographic characteristics ; preoperative , intraoperative , and postoperative data including early postoperative pain scores ; and frequency of shoulder pain and analgesic requirements.results:data regarding demographic characteristics , preoperative findings , cyst diameters and rupture rates , intra - abdominal adhesions , intraoperative blood loss , and postoperative hospital stay did not differ between groups ( p > .05 ) . </S>", "<S> however , the mean operative and abdominal access times were significantly longer in the keyless abdominal rope - lifting surgery group ( p < .05 ) . </S>", "<S> visual analog scale pain scores at initially and at the second , fourth , and 24th hours of the postoperative period were significantly lower in the keyless abdominal rope - lifting surgery group ( p < .05 ) . </S>", "<S> similarly , keyless abdominal rope - lifting surgery caused significantly less shoulder pain and additional analgesic use ( p < .05).conclusion : keyless abdominal rope - lifting surgery seems to cause less pain in the management of benign ovarian cysts in comparison with conventional carbon dioxide laparoscopy . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Keyless Abdominal Rope-Lifting Surgery", "Abdominal access.", "Rope-lifting process.", "Wound closure.", "CO", "Statistical Analysis", "RESULTS", "DISCUSSION", "Principal Findings", "Strengths of Study", "Limitations of Study", "Comparison With Previous Studies", "CONCLUSION"], "sections": [["during the past 2 decades , laparoscopic surgery has gained worldwide popularity resulting from its well - known characteristics of less postoperative pain , fewer complications , earlier discharge , and better cosmesis .", "smaller skin incisions in laparoscopic surgery were related to fewer unwanted side effects , including less postoperative pain . however , in recent studies comparing conventional and single - incision laparoscopic surgical procedures , the postoperative pain scores were not different .", "keyless abdominal rope - lifting surgery ( kars ) is a novel , gasless ( isobaric ) , single - incision laparoscopic surgical technique used in various gynecologic operations and cholecystectomies . besides being a single - incision laparoscopic technique", ", kars does not require carbon dioxide ( co2 ) use to inflate the abdominal wall and the intra - abdominal pressure is not elevated during the operation .", "in addition , postoperative shoulder pain seems to be more frequently observed after laparoscopic surgical procedures in which the intra - abdominal pressure is elevated to between 12 and 18 mm hg by use of co2 . in this study we aimed to compare the postoperative pain levels for kars and conventional multiport laparoscopy performed for adnexal cysts ."], ["although kars has been performed in our institute since the first half of 2010 , the study included benign ovarian cysts operated on between november 2011 and june 2013 .", "beginning in november 2011 , intraoperative and postoperative pain management for all operations performed in our unit was standardized ; thus the uniform management approach allowed us to collect and analyze the data from different surgical techniques .", "the study was approved by the local institutional ethical committee of kafkas university school of medicine .", "the included women had cystic masses including simple cysts with or without septation , endometriomas , and benign cystic teratomas .", "most of the participants complained of groin pain , menstrual bleeding disorders , dysmenorrhea , and infertility .", "emergency cases including patients with hemodynamic instability , severe abdominal pain , and inaccurate preoperative diagnoses were excluded .", "malignant cysts were excluded based on the patient 's history , clinical findings , ultrasonography and magnetic resonance imaging , and levels of cancer antigen 125 , alpha fetoprotein , carcinoembryonic antigen , and cancer antigen 199 .", "women who were diagnosed with an adnexal cyst and in whom a laparoscopic operation was indicated were invited to choose one of the operative techniques .", "because kars was performed only in our center , we could not design a randomized trial .", "thus the groups of this cross - sectional prospective clinical study were formed according to the women 's choice as the kars or conventional co2 laparoscopy groups .", "initial visual analog scale ( vas ) scores of 5.7  1.42 and 6.3  1.42 in patients undergoing kars and conventional laparoscopy , respectively , indicated that we needed at least 30 operations in each group to perform a study with a statistical power of 80% at an  level of .05 .", "the demographic and physical characteristics of the participating patients included their age , gravidity , parity , height , weight , and body mass index , as well as the number of abortions , ectopic pregnancies , and offspring .", "oral intake was prohibited starting at 11 pm , and a 135-ml solution containing 19 g of sodium dihydrogen phosphate and 7 g of disodium hydrogen phosphate was administered rectally at 6 am on the operation day .", "we used the operative laparoscopy study group 's classification system for staging intra - abdominal adhesions : 0 , none ; 1 , filmy and avascular ; 2 , dense and vascular ; and 3 , binding and cohesive .", "the abdominal access time in kars patients included the time needed for the construction of the pathway into the abdominal cavity and the placement of the lifting ropes . in conventional laparoscopy patients ,", "the abdominal access time included the time needed for the creation of the co2 pneumoperitoneum at 14 mm hg of pressure and the insertion of the 3 intra - abdominal access ports .", "all included patients were considered class 1 or 2 according to the american society of anesthesiologists classification and received a standard anesthetic management regimen during the operations .", "we used propofol , 1.5 to 2.5 mg / kg , to induce anesthesia and facilitated intubation with rocuronium , 0.4 to 0.6 mg / kg .", "oxygen was supplemented before and after the intubation at rates of 100% and 50% ( mixed with the operating theater 's air ) , respectively .", "we used 2% sevoflurane and 50 g of fentanyl for anesthesia maintenance . to replace the fluid deficit , we used sodium chloride or ringer lactate solution at 10 ml / kg on intravenous insertion at 2 ml / kg per hour .", "unexpected bleeding was managed by use of additional crystalloid solution at 3 ml / kg per hour .", "atropine , 1 to 1.2 mg intravenously , was used to reverse the neuromuscular blockade .", "the postoperative pain management and vas pain score recordings were initiated in the obstetrics and gynecology service .", "all patients , regardless of initial vas pain score , received 50 mg of meperidine intramuscularly at the beginning of the postoperative period and 75 mg of diclofenac sodium during the second postoperative hour .", "vas pain scores were recorded at the beginning and second , fourth , and 24th hours of the postoperative period .", "beginning from the fourth postoperative hour , patients with vas pain scores of > 4 points received an additional dose of meperidine , 25 mg intramuscularly .", "kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,", "the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .", "depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger .", "after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .", "the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage", ", the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .", "the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .", "while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .", "the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .", "the veress needle is unloaded from its cannula , and one tip of a no .", "the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .", "the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .", "it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .", "the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .", "the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip", "then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .", "the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .", "the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .", "there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .", "if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .", "lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .", "if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .", "however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .", "the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .", "3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .", "one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .", "the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .", "co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,", "all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4", "statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .", "the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .", "normally distributed variables of the 2 groups were compared by use of the student t test , and non  normally distributed variables were compared by use of the mann - whitney u test .", "the  test was used to assess the probability of existence of shoulder pain in the 2 groups .", "a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .", "the hosmer - lemeshow test showed that the model fit (  = 13 762 , p = .88 , n = 77 ) ."], ["kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,", "the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .", "depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .", "the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage", ", the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .", "the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .", "while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .", "the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .", "the veress needle is unloaded from its cannula , and one tip of a no .", "the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .", "the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .", "it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .", "the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .", "the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .", "then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .", "the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .", "the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .", "there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .", "if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .", "lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .", "if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .", "however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .", "the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .", "3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .", "one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .", "the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery ."], ["kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,", "the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .", "depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .", "the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage", ", the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions ."], ["the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .", "while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .", "the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .", "the veress needle is unloaded from its cannula , and one tip of a no .", "the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .", "the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .", "it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .", "the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .", "the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .", "then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .", "the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .", "the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .", "there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .", "if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .", "lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .", "if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .", "however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed ."], ["the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .", "3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .", "one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .", "the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery ."], ["co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,", "all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4 - 0 intracutaneous absorbable sutures ."], ["statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .", "the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .", "normally distributed variables of the 2 groups were compared by use of the student t test , and non  normally distributed variables were compared by use of the mann - whitney u test .", "the  test was used to assess the probability of existence of shoulder pain in the 2 groups .", "a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .", "the hosmer - lemeshow test showed that the model fit (  = 13 762 , p = .88 , n = 77 ) ."], ["during a 20-month period , 77 women with benign ovarian cysts were operated on by kars ( n = 32 ) or conventional co2 laparoscopy ( n = 45 ) .", "the demographic data , physical characteristics , and preoperative findings of the participating women were summarized in table 1 .", "the demographic findings , surgical histories , and preoperative hematologic statuses of the groups did not differ significantly ( p > .05 ) .", "demographic data , physical characteristics , and preoperative findings of participating women in both groups data are presented as mean  standard deviation unless otherwise indicated .", "conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) .", "minor bleeding and unintended cyst ruptures were the only observed intraoperative complications . in none of the cases was a blood transfusion or postoperative intensive care admission needed .", "we did not need to convert to conventional laparoscopy or laparotomy in the kars group or to laparotomy or mini - laparotomy in the conventional laparoscopy group .", "simple oral analgesic use after the 24th hour of the postoperative period was adequate for postoperative pain relief in both groups .", "most of the intraoperative and postoperative findings were similar in the 2 groups ( p > .05 ) ; however , the abdominal cavity access and total operative times in the kars group were significantly longer than those in the conventional laparoscopy group ( p < .05 ) .", "intraoperative and postoperative findings according to operative technique data are presented as mean  standard deviation unless otherwise indicated .", "the vas pain scores at the beginning and second , fourth , and 24th hours of the postoperative period were significantly lower in the kars group ( p < .05 ) , although the differences in mean vas scores never exceeded 1 point .", "in addition , the women in the kars group needed less additional analgesia in comparison with those in the conventional laparoscopy group ( p < .05 ) .", "moreover , a higher number and percentage of the patients in the conventional co2 laparoscopy group had shoulder pain ( p < .05 ) .", "postoperative pain scores and additional analgesic requirements according to operative techniques data are presented as mean  standard deviation unless otherwise indicated .", "conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) . the  test analyzing shoulder pain showed that postoperative shoulder pain was significantly less frequent in the kars group (  = 5.304 , p = .021 , n = 77 ) .", "logistic regression for postoperative shoulder pain predicted that shoulder pain was affected only by the operation type and duration ( table 4 ) .", "although the operation duration was longer in the kars group , the frequency of shoulder pain was higher in the conventional laparoscopy group .", "the total operative time correlated positively with the abdominal access and rope - lifting process time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) . in the conventional laparoscopy group ,", "the total operative time correlated positively with the abdominal access time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) ."], ["in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain .", ", our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .", "postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions .", "although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .", "the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .", "postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .", "pain sensation is subjective and may even differ for the same individual under different conditions .", "it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .", "although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar .", "gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons . however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .", "in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .", "gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .", "single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .", "gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .", "similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .", "however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study", "in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .", "thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .", "recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .", "surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study", "we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .", "in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .", "previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al", ", the postoperative pain scores did not differ between single - incision and multiport laparoscopies .", "however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study", "the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up . in addition", ", there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .", "moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .", "better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .", "although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .", "they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .", "although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .", "the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .", "the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,", "fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .", "in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011", "prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .", "they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .", "although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study , although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .", "our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .", "however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .", "in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum ."], ["in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain ."], ["to our knowledge , our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .", "postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions ."], ["although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .", "the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .", "postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .", "pain sensation is subjective and may even differ for the same individual under different conditions .", "it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .", "although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar ."], ["gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons .", "however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .", "in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .", "gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .", "single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .", "gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .", "similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .", "however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study", "in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .", "thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .", "recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .", "surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study", "we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .", "in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .", "previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al , the postoperative pain scores did not differ between single - incision and multiport laparoscopies . however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study", "the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up .", "in addition , there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .", "moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .", "better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .", "although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .", "they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .", "although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .", "the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .", "the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,", "fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .", "in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011", "prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .", "they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .", "although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study", ", although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .", "our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .", "however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .", "in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum ."], ["kars seems to cause less postoperative pain during the management of benign ovarian cysts in comparison with conventional multiport co2 laparoscopy ."]]}
{"article_id": "PMC4426942", "article_text": ["pancreatitis , the commonest adverse event following endoscopic retrograde cholangiopancreatography ( ercp ) , has been found to occur at widely varying rates , in 1% to 15.1% of pateints.14 post - ercp pancreatitis ( pep ) has been found at lower rates in recent studies because noninvasive methods , such as magnetic resonance cholangiopancreatography ( mrcp ) and endoscopic ultrasonography ( eus ) , have superseded diagnostic ercp.5 despite improvement of our knowledge , equipment , and methods for ercp , complications are still a significant hazard with this procedure .", "pep is the most common severe complication.1,6,7 numerous studies have looked at risk factors for pep,14,616 with varied results , possibly because of different study designs , different candidate predictor variables , and differences between settings .", "precise identification of risk factors is of great importance for recognition of high - risk cases in which ercp should be avoided if possible or protective modalities should be considered to minimize patients risk of morbidity and mortality .", "we know that inflammatory biomarkers are useful at 2448 hours post procedure in predicting pep , and a few studies have evaluated pre - procedure inflammatory markers , especially c - reactive protein ( crp ) elevation , as predictors of pep.1723 none of the previous studies has focused on pre - ercp erythrocyte sedimentation rate ( esr ) as a potential risk factor for pep . despite the importance of identifying risk factors for pep ,", "the present study examined prospectively the potential patient- and procedure - related risk factors , including esr , for pep in iranian population .", "during a four - year period ( 20082012 ) , 780 patients who underwent ercp in a tertiary care hospital were analyzed in this prospective study .", "the patients were referred to this center from different parts of iran , which enhances the ability to generalize the findings for the iranian population .", "all patients gave their written informed consent to participate in this research before the ercp procedure , and the study protocol was approved by the ethics committee of the research center for gastroenterology and liver diseases , shahid beheshti university of medical sciences .", "indications for ercp were determined by participating endoscopists before the procedure , on the basis of generally accepted diagnostic indications for ercp.5 laboratory evaluations , including those of alkaline phosphatase ( alp ) , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , bilirubin , and serum amylase , were performed on the first day of hospitalization , and the results were used as pre - ercp laboratory parameters .", "patients in acceptable condition were discharged one to two days after ercp ; otherwise , they received longer inpatient care based on the severity of their complications and illness .", "all the patients had a follow - up visit two weeks after discharge from hospital .", "patient- and procedure - related data , including demographics , characteristics , clinical information and technical details , and findings from ercp procedures were recorded prospectively on a detailed data sheet .", "exclusion criteria were a history of biliary sphincterotomy or pre - cut sphincterotomy , pre - procedure active pancreatitis , pregnancy , mental disability , and refusal to participate .", "ercp procedures were performed by a total of eight expert endoscopists , almost always with a trainee performing at least part of the procedure . during the procedures ,", "patients were under conscious sedation using a combination of intravenous lidocaine , midazolam , and propofol .", "successful cannulation was defined as free and deep instrumentation of the biliary tree , and a cannulation attempt was defined as sustained contact with the cannulating device and the papilla for at least five seconds.17 difficult biliary cannulation was defined as the failure of biliary access despite 10 minutes of attempted biliary cannulation or more than five attempted unintentional pancreatic cannulations.18 any complications taking place during or following the procedure were also entered into the records .", "pep was diagnosed according to the generally accepted criteria defined by cotton et al,6 ie , presence of upper abdominal pain 24 hours after an ercp procedure and a serum amylase level more than three times the upper limit of normal .", "results were expressed as the mean  standard deviation ( sd ) for quantitative variables and percentages for categorical variables .", "multiple linear regression analysis was used to develop an optimal model for predicting pep with the presence of confounders .", "the performance of the model was assessed with the hosmer  lemeshow goodness - of - fit test .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . ) for windows .", "between 2008 and 2012 , 780 patients ( male : female ratio 393:387 , mean age 57.5 years ) underwent diagnostic and therapeutic ercp with the primary diagnosis of hepatobiliary disorders .", "of these 780 ercps , 105 procedures ( 13.5% ) were carried out in patients with invasive cancers ( pancreatic ductal adenocarcinoma , biliary carcinoma , metastastic tumor with biliary obstruction , hepatocellular carcinoma , ampullary carcinoma ) because of obstruction and 675 ( 86.5% ) were performed in patients with benign diseases , which included 313 procedures ( 40.1% ) to remove bile duct stones and 362 ( 46.4% ) for benign biliary stricture .", "the patients included 393 men ( 50.4% ) , and 448 ( 57.5% ) were more than 65 years old .", "previous cholecystectomy had been performed in 36.3% of patients and 9.0% had undergone previous ercp . a total of 80 patients had history of biliary stone diagnosed by a similar procedure .", "also , history of confirmed pancreatitis and hepatitis was observed in 27 ( 3.5% ) and 10 ( 1.3% ) patients , respectively ( table 1 ) . regarding laboratory parameters ( table 2 ) ,", "serum amylase was found to be elevated to more than 200 units / l in 102 patients ( 13.7% ) three hours after ercp and more than 800 units / l in 60 patients ( 7.7% ) . in all , 446 ( 70% ) participants underwent wire - guided cannulation and others ( 30% ) underwent sphincterotome biliary cannulation using contrast injection as the conventional method .", "successful biliary cannulation was technically achieved in 82.5% of all patients at ercp , although in 13.0% of them , cannulation failed .", "regarding pathologic changes in papilla , tumoral features and ulcerative changes were found in 3.3% and 0.6% of patients .", "of the 780 patients who underwent diagnostic or therapeutic ercp , pancreatitis developed in 26 patients ( 3.3% ) .", "significant bleeding occurred in two patients ; the only predictor in multivariable analysis was biliary sphincterotomy .", "we diagnosed cholangitis ( using the criteria of transient worsening of clinical states and liver function tests consistent with cholangitis ) in four subjects , all of whom had common bile duct ( cbd ) stones with biliary stent placement .", "table 3 shows the frequency of pep and raised esr according to indication for ercp . in the multivariable risk model for predicting pep ( table 4 ) , significant risk factors with adjusted odds ratios ( ors )", "were age < 65 years ( or = 10.647 , p = 0.023 ) and esr > 30 ( or = 6.414 , p < 0.001 ) .", "female gender , history of recurrent pancreatitis , pre - ercp hyperamylasemia , and difficult or failed cannulation could not predict pep . regarding the cannulation techniques", ", there was no difference in the incidence of pep between guide wire - assisted ercp or conventional contrast - assisted cannulation .", "we have identified two risk factors for the development of pep : age < 65 years and esr > 30 .", "several studies have focused on younger age as an independent predictor of pep.14,617 so in younger individuals , it is important to perform ercp only when the indication is very strong and there is no other noninvasive substitute for ercp .", "another risk factor for the development of pep in our study was increased esr level before ercp ( esr > 30 ) .", "we found that raised esr > 30 mm / hour is a significant factor that independently predicts increased risk of pep .", "although some studies have evaluated the level of post - ercp inflammatory markers , especially post - procedure crp elevation , as predictors of development of pep,1923 it seems that none of the previous studies have focused on pre - ercp esr level as a potential risk factor for pep .", "however , the findings of previous studies suggest that inflammatory biomarkers are helpful at 2448 hours post - procedure , so they are not early predictors .", "although it seems that our study is the first study to evaluate pre - ercp esr level as a risk factor for developing pep , many other studies have evaluated the role of pharmacologic agents in reducing the incidence or severity of pep.2,10,2227 several agents , most of which have an anti - inflammatory effect , have been tested in clinical trials . in terms of attenuating the inflammatory response ,", "the most promising results have been with non - steroidal anti - inflammatory drugs ( nsaids ) , and some standard guidelines recommend the routine use of nsaids to prevent pep.28 most of these studies are experimental clinical trials , but it is possible that the main beneficial effect of these drugs is limiting systemic inflammatory response and lowering the level of esr as a potential risk factor for pep .", "this study could be a guide for future clinical trials on rectal nsaids for the prevention of pep ; the effect of these drugs in prevention of pancreatitis , with attention to the level of esr before ercp , should be evaluated .", "other risk factors such as female gender , younger age , sphincter of oddi dysfunction , cannulation difficulty , prolonged procedure time , and repeated injection to the pancreatic duct were not assessed because ercp procedures were done by an expert and because of the absence of manometry .", "also , other factors such as pre - ercp bacterial infections might increase esr level .", "some studies on both human and animal models , performed to evaluate the potential role of antibiotics in preventing pep , found reduced rates of pep in patients receiving antibiotic prophylaxis prior to ercp when compared to placebo.26 however , most studies did not find any role for infections in pep , except in cases of severe pancreatitis with necrosis .", "the main limitation of this study was the absence of combined evaluation of crp and esr before ercp .", "esr will be raised in almost all patients with any systemic inflammation , so our study is also limited because patients with systemic inflammation were not excluded . to date , many risk factors for pep have been reported from high - volume centers .", "these have included female gender , younger age , sphincter of oddi dysfunction , cannulation difficulty , prolonged procedure time , and repeated injection to the pancreatic duct .", "not all of these factors were analyzed in our study , and our results were not fully consistent with the previous results .", "performing ercp may be safer in the elderly and in patients with low levels of serum esr because they may be less at risk of pep .", "using pre - ercp anti - inflammatory pharmacologic agents like nsaids might be beneficial , by lowering esr level and reducing related risk of pep ."], "abstract_text": ["<S> backgroundpancreatitis remains the most common complication of endoscopic retrograde cholangiopancreatography ( ercp ) , resulting in substantial morbidity and occasional mortality . </S>", "<S> there are notable controversies and conflicting reports about risk factors of post - ercp pancreatitis ( pep).aimto evaluate the potential risk factors for pep at a referral tertiary center , as a sample of the iranian population.materials and methodsbaseline characteristics and clinical as well as paraclinical information of 780 patients undergoing diagnostic and therapeutic ercp at taleghani hospital in tehran between 2008 and 2012 were reviewed . </S>", "<S> data were collected prior to the ercp , at the time of the procedure , and 2472 hours after discharge . </S>", "<S> pep was diagnosed according to consensus criteria.resultsof the 780 patients who underwent diagnostic ercp , pancreatitis developed in 26 patients ( 3.3% ) . in the multivariable risk model , significant risk factors with adjusted odds ratios ( ors ) were age < 65 years ( or = 10.647 , p = 0.023 ) and erythrocyte sedimentation rate ( esr ) > 30 ( or = 6.414 , p < 0.001 ) . </S>", "<S> female gender , history of recurrent pancreatitis , pre - ercp hyperamylasemia , and difficult or failed cannulation could not predict pep . </S>", "<S> there was no significant difference in the rate of pep in wire - guided cannulation versus biliary cannulation using a sphincterotome and contrast injection as the conventional method.conclusionsperforming ercp may be safer in the elderly . </S>", "<S> patients with high esr may be at greater risk of pep , which warrants close observation of these patients for signs of pancreatitis after ercp . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion"], "sections": [["pancreatitis , the commonest adverse event following endoscopic retrograde cholangiopancreatography ( ercp ) , has been found to occur at widely varying rates , in 1% to 15.1% of pateints.14 post - ercp pancreatitis ( pep ) has been found at lower rates in recent studies because noninvasive methods , such as magnetic resonance cholangiopancreatography ( mrcp ) and endoscopic ultrasonography ( eus ) , have superseded diagnostic ercp.5 despite improvement of our knowledge , equipment , and methods for ercp , complications are still a significant hazard with this procedure .", "pep is the most common severe complication.1,6,7 numerous studies have looked at risk factors for pep,14,616 with varied results , possibly because of different study designs , different candidate predictor variables , and differences between settings .", "precise identification of risk factors is of great importance for recognition of high - risk cases in which ercp should be avoided if possible or protective modalities should be considered to minimize patients risk of morbidity and mortality .", "we know that inflammatory biomarkers are useful at 2448 hours post procedure in predicting pep , and a few studies have evaluated pre - procedure inflammatory markers , especially c - reactive protein ( crp ) elevation , as predictors of pep.1723 none of the previous studies has focused on pre - ercp erythrocyte sedimentation rate ( esr ) as a potential risk factor for pep . despite the importance of identifying risk factors for pep ,", "the present study examined prospectively the potential patient- and procedure - related risk factors , including esr , for pep in iranian population ."], ["during a four - year period ( 20082012 ) , 780 patients who underwent ercp in a tertiary care hospital were analyzed in this prospective study .", "the patients were referred to this center from different parts of iran , which enhances the ability to generalize the findings for the iranian population .", "all patients gave their written informed consent to participate in this research before the ercp procedure , and the study protocol was approved by the ethics committee of the research center for gastroenterology and liver diseases , shahid beheshti university of medical sciences .", "indications for ercp were determined by participating endoscopists before the procedure , on the basis of generally accepted diagnostic indications for ercp.5 laboratory evaluations , including those of alkaline phosphatase ( alp ) , alanine transaminase ( alt ) , aspartate aminotransferase ( ast ) , bilirubin , and serum amylase , were performed on the first day of hospitalization , and the results were used as pre - ercp laboratory parameters .", "patients in acceptable condition were discharged one to two days after ercp ; otherwise , they received longer inpatient care based on the severity of their complications and illness .", "all the patients had a follow - up visit two weeks after discharge from hospital .", "patient- and procedure - related data , including demographics , characteristics , clinical information and technical details , and findings from ercp procedures were recorded prospectively on a detailed data sheet .", "exclusion criteria were a history of biliary sphincterotomy or pre - cut sphincterotomy , pre - procedure active pancreatitis , pregnancy , mental disability , and refusal to participate .", "ercp procedures were performed by a total of eight expert endoscopists , almost always with a trainee performing at least part of the procedure . during the procedures ,", "patients were under conscious sedation using a combination of intravenous lidocaine , midazolam , and propofol .", "successful cannulation was defined as free and deep instrumentation of the biliary tree , and a cannulation attempt was defined as sustained contact with the cannulating device and the papilla for at least five seconds.17 difficult biliary cannulation was defined as the failure of biliary access despite 10 minutes of attempted biliary cannulation or more than five attempted unintentional pancreatic cannulations.18 any complications taking place during or following the procedure were also entered into the records .", "pep was diagnosed according to the generally accepted criteria defined by cotton et al,6 ie , presence of upper abdominal pain 24 hours after an ercp procedure and a serum amylase level more than three times the upper limit of normal .", "results were expressed as the mean  standard deviation ( sd ) for quantitative variables and percentages for categorical variables .", "multiple linear regression analysis was used to develop an optimal model for predicting pep with the presence of confounders .", "the performance of the model was assessed with the hosmer  lemeshow goodness - of - fit test .", "all the statistical analyses were performed using spss version 16.0 ( spss inc . ) for windows ."], ["between 2008 and 2012 , 780 patients ( male : female ratio 393:387 , mean age 57.5 years ) underwent diagnostic and therapeutic ercp with the primary diagnosis of hepatobiliary disorders .", "of these 780 ercps , 105 procedures ( 13.5% ) were carried out in patients with invasive cancers ( pancreatic ductal adenocarcinoma , biliary carcinoma , metastastic tumor with biliary obstruction , hepatocellular carcinoma , ampullary carcinoma ) because of obstruction and 675 ( 86.5% ) were performed in patients with benign diseases , which included 313 procedures ( 40.1% ) to remove bile duct stones and 362 ( 46.4% ) for benign biliary stricture .", "the patients included 393 men ( 50.4% ) , and 448 ( 57.5% ) were more than 65 years old .", "previous cholecystectomy had been performed in 36.3% of patients and 9.0% had undergone previous ercp . a total of 80 patients had history of biliary stone diagnosed by a similar procedure .", "also , history of confirmed pancreatitis and hepatitis was observed in 27 ( 3.5% ) and 10 ( 1.3% ) patients , respectively ( table 1 ) . regarding laboratory parameters ( table 2 ) ,", "serum amylase was found to be elevated to more than 200 units / l in 102 patients ( 13.7% ) three hours after ercp and more than 800 units / l in 60 patients ( 7.7% ) . in all , 446 ( 70% ) participants underwent wire - guided cannulation and others ( 30% ) underwent sphincterotome biliary cannulation using contrast injection as the conventional method .", "successful biliary cannulation was technically achieved in 82.5% of all patients at ercp , although in 13.0% of them , cannulation failed .", "regarding pathologic changes in papilla , tumoral features and ulcerative changes were found in 3.3% and 0.6% of patients .", "of the 780 patients who underwent diagnostic or therapeutic ercp , pancreatitis developed in 26 patients ( 3.3% ) .", "significant bleeding occurred in two patients ; the only predictor in multivariable analysis was biliary sphincterotomy .", "we diagnosed cholangitis ( using the criteria of transient worsening of clinical states and liver function tests consistent with cholangitis ) in four subjects , all of whom had common bile duct ( cbd ) stones with biliary stent placement .", "table 3 shows the frequency of pep and raised esr according to indication for ercp . in the multivariable risk model for predicting pep ( table 4 ) , significant risk factors with adjusted odds ratios ( ors )", "were age < 65 years ( or = 10.647 , p = 0.023 ) and esr > 30 ( or = 6.414 , p < 0.001 ) .", "female gender , history of recurrent pancreatitis , pre - ercp hyperamylasemia , and difficult or failed cannulation could not predict pep . regarding the cannulation techniques", ", there was no difference in the incidence of pep between guide wire - assisted ercp or conventional contrast - assisted cannulation ."], ["we have identified two risk factors for the development of pep : age < 65 years and esr > 30 .", "several studies have focused on younger age as an independent predictor of pep.14,617 so in younger individuals , it is important to perform ercp only when the indication is very strong and there is no other noninvasive substitute for ercp .", "another risk factor for the development of pep in our study was increased esr level before ercp ( esr > 30 ) .", "we found that raised esr > 30 mm / hour is a significant factor that independently predicts increased risk of pep .", "although some studies have evaluated the level of post - ercp inflammatory markers , especially post - procedure crp elevation , as predictors of development of pep,1923 it seems that none of the previous studies have focused on pre - ercp esr level as a potential risk factor for pep .", "however , the findings of previous studies suggest that inflammatory biomarkers are helpful at 2448 hours post - procedure , so they are not early predictors .", "although it seems that our study is the first study to evaluate pre - ercp esr level as a risk factor for developing pep , many other studies have evaluated the role of pharmacologic agents in reducing the incidence or severity of pep.2,10,2227 several agents , most of which have an anti - inflammatory effect , have been tested in clinical trials . in terms of attenuating the inflammatory response ,", "the most promising results have been with non - steroidal anti - inflammatory drugs ( nsaids ) , and some standard guidelines recommend the routine use of nsaids to prevent pep.28 most of these studies are experimental clinical trials , but it is possible that the main beneficial effect of these drugs is limiting systemic inflammatory response and lowering the level of esr as a potential risk factor for pep .", "this study could be a guide for future clinical trials on rectal nsaids for the prevention of pep ; the effect of these drugs in prevention of pancreatitis , with attention to the level of esr before ercp , should be evaluated .", "other risk factors such as female gender , younger age , sphincter of oddi dysfunction , cannulation difficulty , prolonged procedure time , and repeated injection to the pancreatic duct were not assessed because ercp procedures were done by an expert and because of the absence of manometry .", "also , other factors such as pre - ercp bacterial infections might increase esr level .", "some studies on both human and animal models , performed to evaluate the potential role of antibiotics in preventing pep , found reduced rates of pep in patients receiving antibiotic prophylaxis prior to ercp when compared to placebo.26 however , most studies did not find any role for infections in pep , except in cases of severe pancreatitis with necrosis .", "the main limitation of this study was the absence of combined evaluation of crp and esr before ercp .", "esr will be raised in almost all patients with any systemic inflammation , so our study is also limited because patients with systemic inflammation were not excluded . to date , many risk factors for pep have been reported from high - volume centers .", "these have included female gender , younger age , sphincter of oddi dysfunction , cannulation difficulty , prolonged procedure time , and repeated injection to the pancreatic duct .", "not all of these factors were analyzed in our study , and our results were not fully consistent with the previous results ."], ["performing ercp may be safer in the elderly and in patients with low levels of serum esr because they may be less at risk of pep .", "using pre - ercp anti - inflammatory pharmacologic agents like nsaids might be beneficial , by lowering esr level and reducing related risk of pep ."]]}
{"article_id": "PMC4229836", "article_text": ["sole aim and the prerequisite for dentistry is an effective pain control during dental procedures . to achieve this goal", "local anesthesia is being used since long . in 1943 , lofgren synthesized the first modern anaesthesia.1 it is lidocaine , which was an amide derivative of diethyl amino acetic acid .", "it is because of the high density and the firm adherence of palatal mucosa to the underlying bone .", "twenty - five years after lidocaine , articaine was first synthesized by muschaneau in 1969 .", "it has a thiophene ring in its molecule instead of usual benzene ring.2,3 this is most commonly used in germany .", "commercially articaine for dental use is available in 4% solution with epinephrine 1:200000 or 1:100000 .", "it also contains maximum 0.6 mg na - sulfite in 1.0 ml and sodium chloride .", "once injected , absorption starts from the site of injection into the vascular compartment.1 the unbound local anesthetic is distributed throughout all the body tissue . due to the presence of thiophene ring", ", it is inactivated in the liver as well as by hydrolyzation in the tissue and blood .", "the aim of the study was to evaluate and compare the efficacy of 4% articaine hydrochloride and 2% lignocaine hydrochloride for the orthodontic extraction .", "the study was carried out on 50 patients at outpatient department of oral and maxillofacial surgery who needed bilateral maxillary premolar extractions for orthodontic purpose .", "patients included in this study were in the age group of 15 - 25 years , both genders and systemically healthy .", "bleeding disorders , hypertensive , diabetic , pregnant , allergic to local anesthetics , reluctant and medically compromised patients were excluded from the study .", "all the patients were explained about visual analog scale ( vas ) before injecting local anesthesia .", "experimental sites ( group 1 ) were injected with 0.5 - 1 ml of 4% articaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule .", "no palatal anesthesia was injected , but the desired anesthetic effect was achieved with the above . on the other hand , control sites ( group 2 ) were injected with 0.8 - 1 ml of 2% lignocaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule .", "when the objective symptoms were checked , it was found that palatal anesthesia was absent hence additional 0.5 ml was injected to obtain a desired result . after assessing the signs and symptoms of obtaining complete anesthesia", "all the parameters , i.e. , drug volume , time of onset , duration of anesthesia and pain rating were recorded for entire patients .", "the mean administered volume of articaine and lignocaine were 0.779  0.1305 and 1.337  0.2369 respectively .", "it should be noted that the articaine volume administered was almost half of the lignocaine ( table 1 ) .", "the mean onset time of lignocaine anesthesia was 1.337  0.2369 , whereas in articaine group the mean time was 1.012  0.2058 min .", "this indicates that onset time of articaine was significantly less than lidocaine ( p < 0.0005 ) ( table 2 ) .", "pain rating showed that there was no significant difference in pain score in articaine palatal and buccal group ( p > 0.8892 ) , whereas a significant difference was noted in lignocaine palatal and buccal group ( tables 3 and 4 ) .", "duration of pain in group 1 was 69.08  18.247 and 55.66  6.414 in group 2 patients .", "duration of anesthesia is articaine group is more than the lignocaine group . in the entire study", ", it goes biotransformation in both liver and plasma and hence gets cleared much quickly .", "recent studies have shown that articaine carries lot of advantages over other anesthetic agents.4 in this study , we observed that the palatal infiltration was required in approximately 98% of cases when lignocaine was used , whereas in articaine group palatal anesthesia was never required .", " thiophene ring  in its molecular structure , which makes it more lipophilic and this accounts for its diffusion properties across all the tissues.5 articaine is metabolized in the liver , tissues and blood and hence it is cleared out very fast from the body .", "this is the only anesthetic agent , which is inactivated from our body in two ways .", "zlkowska et al . has reported that like all other anesthetic agents articaine is safe in epileptic patients.6 this study showed no adverse effects and no complications .", "this study is in accordance with study by malamed et al . suggesting 4% articaine with 1:100000 adrenaline is safe and have a low risk of toxicity.2 statistical analysis in this study showed no significant difference in extraction pain on vas for test and control sites .", "this inference relates to the study done by fan et al.7 oertel et al.8 uckan et al.9 and evans et al.10 when articaine is injected the local concentration of active drug is nearly twice of that obtained with lignocaine .", "oertel et al . in his study showed this by determining the concentration of 4% articaine and 2% lidocaine in alveolar blood using high - performance liquid chromatography.11,12 thiophene derivative articaine blocks ionic channels at lower concentration than benzene derivative lidocaine.13 potocnik et al . in vitro study on rat surap nerve concluded that 2% and 4% of articaine is more effective than 2% and 4 % of lidocaine in depressing compound action potential of the a fibres.14 - 16 this efficacy and safety factors are observed in this study too .", "it is a well - known fact that palatal anesthesia is a very painful experience even though surface anesthesia is used .", "hence , if articaine is used , patients can be relieved from the painful palatal anesthesia without compromising with safety and efficacy .", "certain added advantages like shorter time of onset , longer duration of action and greater diffusion property makes it an ideal anesthetic agent to be used in dentistry ."], "abstract_text": ["<S> background : articaine in an anesthetic agent , which is used less frequently in dentistry . </S>", "<S> it differs from other agents due to the presence of a thiophene ring in its molecular structure . </S>", "<S> few groups of researchers claim that it is superior to lignocaine . </S>", "<S> hence , the purpose of this study was to compare the efficacy of 4% articaine hydrochloride and 2% lignocaine hydrochloride in the orthodontic extraction.materials and methods : the study was carried out in 50 patients who needed the orthodontic extraction in the age group from 15 to 25 years . </S>", "<S> experimental sites were injected with 0.5 - 1 ml of 4% articaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule without palatal anaesthesia . </S>", "<S> control sites were injected with 0.8 - 1 ml of 2% lignocaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule . </S>", "<S> all the parameters , that is volume , duration , time of anesthesia and pain rating were noted and statistically compared.result:when statistically compared mean volume of articaine ( 0.779  0.1305 ) was less than lignocaine ( 1.337  0.2369 ) . </S>", "<S> mean time of onset of articaine was 1.012  0.2058 min , whereas that of was 1.337  0.2369 . </S>", "<S> pain rating showed not much difference , but in the lignocaine group palatal anesthesia was required in all the patients . </S>", "<S> finally , the mean duration of anesthesia in articaine group was 69.08  18.247 , whereas in the lignocaine group was 55.66  6.414.conclusion:articaine has proved its usefulness in all regards . </S>", "<S> literatures have proved its usefulness . like other anesthetic , it is safe and more effective . </S>", "<S> it surpasses the need of additional palatal anesthesia . </S>", "<S> rapid inactivation in liver and plasma reduces the risk of the drug overdose . </S>", "<S> all the above factors make it an ideal anesthetic agent to be used in dentistry . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion"], "sections": [["sole aim and the prerequisite for dentistry is an effective pain control during dental procedures . to achieve this goal", "local anesthesia is being used since long . in 1943 , lofgren synthesized the first modern anaesthesia.1 it is lidocaine , which was an amide derivative of diethyl amino acetic acid .", "it is because of the high density and the firm adherence of palatal mucosa to the underlying bone .", "twenty - five years after lidocaine , articaine was first synthesized by muschaneau in 1969 .", "it has a thiophene ring in its molecule instead of usual benzene ring.2,3 this is most commonly used in germany .", "commercially articaine for dental use is available in 4% solution with epinephrine 1:200000 or 1:100000 .", "it also contains maximum 0.6 mg na - sulfite in 1.0 ml and sodium chloride .", "once injected , absorption starts from the site of injection into the vascular compartment.1 the unbound local anesthetic is distributed throughout all the body tissue . due to the presence of thiophene ring", ", it is inactivated in the liver as well as by hydrolyzation in the tissue and blood .", "the aim of the study was to evaluate and compare the efficacy of 4% articaine hydrochloride and 2% lignocaine hydrochloride for the orthodontic extraction ."], ["the study was carried out on 50 patients at outpatient department of oral and maxillofacial surgery who needed bilateral maxillary premolar extractions for orthodontic purpose .", "patients included in this study were in the age group of 15 - 25 years , both genders and systemically healthy .", "bleeding disorders , hypertensive , diabetic , pregnant , allergic to local anesthetics , reluctant and medically compromised patients were excluded from the study .", "all the patients were explained about visual analog scale ( vas ) before injecting local anesthesia .", "experimental sites ( group 1 ) were injected with 0.5 - 1 ml of 4% articaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule .", "no palatal anesthesia was injected , but the desired anesthetic effect was achieved with the above . on the other hand , control sites ( group 2 ) were injected with 0.8 - 1 ml of 2% lignocaine hcl containing 1:100000 adrenaline , incrementally in the buccal vestibule .", "when the objective symptoms were checked , it was found that palatal anesthesia was absent hence additional 0.5 ml was injected to obtain a desired result . after assessing the signs and symptoms of obtaining complete anesthesia"], ["all the parameters , i.e. , drug volume , time of onset , duration of anesthesia and pain rating were recorded for entire patients .", "the mean administered volume of articaine and lignocaine were 0.779  0.1305 and 1.337  0.2369 respectively .", "it should be noted that the articaine volume administered was almost half of the lignocaine ( table 1 ) .", "the mean onset time of lignocaine anesthesia was 1.337  0.2369 , whereas in articaine group the mean time was 1.012  0.2058 min .", "this indicates that onset time of articaine was significantly less than lidocaine ( p < 0.0005 ) ( table 2 ) .", "pain rating showed that there was no significant difference in pain score in articaine palatal and buccal group ( p > 0.8892 ) , whereas a significant difference was noted in lignocaine palatal and buccal group ( tables 3 and 4 ) .", "duration of pain in group 1 was 69.08  18.247 and 55.66  6.414 in group 2 patients .", "duration of anesthesia is articaine group is more than the lignocaine group . in the entire study"], [", it goes biotransformation in both liver and plasma and hence gets cleared much quickly .", "recent studies have shown that articaine carries lot of advantages over other anesthetic agents.4 in this study , we observed that the palatal infiltration was required in approximately 98% of cases when lignocaine was used , whereas in articaine group palatal anesthesia was never required .", " thiophene ring  in its molecular structure , which makes it more lipophilic and this accounts for its diffusion properties across all the tissues.5 articaine is metabolized in the liver , tissues and blood and hence it is cleared out very fast from the body .", "this is the only anesthetic agent , which is inactivated from our body in two ways .", "zlkowska et al . has reported that like all other anesthetic agents articaine is safe in epileptic patients.6 this study showed no adverse effects and no complications .", "this study is in accordance with study by malamed et al . suggesting 4% articaine with 1:100000 adrenaline is safe and have a low risk of toxicity.2 statistical analysis in this study showed no significant difference in extraction pain on vas for test and control sites .", "this inference relates to the study done by fan et al.7 oertel et al.8 uckan et al.9 and evans et al.10 when articaine is injected the local concentration of active drug is nearly twice of that obtained with lignocaine .", "oertel et al . in his study showed this by determining the concentration of 4% articaine and 2% lidocaine in alveolar blood using high - performance liquid chromatography.11,12 thiophene derivative articaine blocks ionic channels at lower concentration than benzene derivative lidocaine.13 potocnik et al . in vitro study on rat surap nerve concluded that 2% and 4% of articaine is more effective than 2% and 4 % of lidocaine in depressing compound action potential of the a fibres.14 - 16 this efficacy and safety factors are observed in this study too .", "it is a well - known fact that palatal anesthesia is a very painful experience even though surface anesthesia is used .", "hence , if articaine is used , patients can be relieved from the painful palatal anesthesia without compromising with safety and efficacy ."], ["certain added advantages like shorter time of onset , longer duration of action and greater diffusion property makes it an ideal anesthetic agent to be used in dentistry ."]]}
{"article_id": "PMC3337625", "article_text": ["the ras association domain family ( rassf ) consists of 10 members : rassf1 - 10 .", "importantly , all isoforms contain a ras association ( ra ) domain either in their c - terminal ( rassf1 - 6 ) or n - terminal ( rassf7 - 10 ) regions . to date , no known catalytic activity has been described for this family , and the general consensus supposes that rassf proteins function as scaffolds to localize signaling in the cell .", "rassf1 isoform a ( rassf1a ) is the most characterized member of the rassf family .", "the rassf1 gene encodes multiple splice variants , including the two predominant isoforms , rassf1a and c. the rassf1a isoform is the longest variant of the rassf1 gene .", "structurally , rassf1a is a product of exons 1 , 2/ , 3 , 4 , 5 , and 6 , while rassf1c consists of exons 2 , 3 , 4 , 5 , and 6 . both isoforms contain a c - terminal ra domain ; however , rassf1a has an additional c1 domain that is not present in rassf1c .", "this short arm of chromosome 3 is known to exhibit loss of heterozygosity in many tumor models and is thought to harbor tumor suppressor genes . as the literature has shown , rassf1a fits this description .", "the rassf1a promoter contains a cpg island that shows a high frequency of hypermethylation in tumors , thereby silencing rassf1a expression in many human cancers including lung , breast , ovarian , renal , and bladder [ 47 ] .", "rassf1a expression is also lost in numerous cancer cell lines , while rassf1c expression is seemingly unaffected .", "interestingly , recent work suggests that rassf1c may actually promote tumor progression [ 8 , 9 ] , further distinguishing these two splice variants .", "all rassf proteins have an ra domain , which is thought to necessitate their binding to activated , gtp - bound ras proteins . while rassf5 ( nore1 ) is thought to bind ras directly , whether rassf1a is able to associate with ras is less clear .", "it has been shown that rassf1a binds k - ras in vitro , and an interaction between ectopically expressed rassf1a and activated k - ras has been observed in hek293 cells [ 11 , 12 ] .", "however , other work has found that this interaction only occurs in the presence of nore1 , arguing for an indirect association .", "importantly , to our knowledge , there are no reports demonstrating the interaction of endogenous rassf1a and ras proteins .  ", "it has been implicated in the negative regulation of cell cycle progression , cell proliferation , and cell survival .", "rassf1a has been shown to localize to microtubules of proliferating cells , increasing microtubule stability and inhibiting cell division [ 14 , 15 ] .", "this may be mediated through direct binding or though interaction with microtubule - associated proteins such as c19orf5 .", "rassf1a has also been shown to inhibit proliferation by inhibiting the accumulation of cyclin d1 and arresting cell division [ 17 , 18 ] .  ", "rassf1a also promotes apoptosis , which can reportedly occur through multiple mechanisms and is likely cell - type dependent .", "one mechanism that mediates the apoptotic function of rassf1a involves protein interaction with modulator of apoptosis-1 ( moap-1 or map-1 ) .", "moap-1 is normally sequestered in an inactive form in healthy cells . upon death receptor stimulation ,", "rassf1a binds moap-1 , causing its activation and subsequent association with bax , which leads to apoptosis .", "previous work has also demonstrated enhancement of rassf1a / mst - mediated cell death by the scaffold cnk1 .", "rassf1a can also elicit inhibitory effects on growth and survival through engagement of the hippo pathway .", "the hippo signaling pathway is a highly conserved kinase cascade that was originally discovered in drosophila and has been shown to be a critical regulator of cell proliferation , survival , and organ growth .", "three members of this pathway , drassf , salvador and hippo , contain the sarah ( salvador - rassf - hippo ) domain , which is conserved in its mammalian counterparts rassf1 - 6 , ww45 , and mst1/2 , respectively .", "while the drosophila ortholog drassf is known to antagonize hippo activation in the fly , it has been demonstrated that rassf1a promotes phosphorylation and activation of mst 1/2 by inhibiting the phosphatase pp2a in mammalian systems [ 29 , 30 ] .", "the biological relevance of rassf1a - mediated activation of hippo signaling has also been investigated .", "reported a rassf1a - mst2-yap - p73-puma signaling axis that promotes apoptosis in mammalian cells .", "hippo signaling is also important for maintaining intestinal homeostasis and tissue regeneration in response to injury .", "mouse models with conditional disruption of either mst1/2 or sav1 in the intestinal epithelium displayed hyperactivation of yes - associated protein ( yap ) , increased intestinal stem cell ( isc ) proliferation , and increased polyp formation following dextran sodium sulfate ( dss ) treatment [ 32 , 33 ] .", "similarly , loss - of - function mutations of hippo components in the fly midgut caused increased isc proliferation .", "these findings suggest that perhaps hippo signaling serves a more global role in regulating organ integrity , structure , and response to injury , and that perturbation of this pathway can lead to aberrant growth and dysfunction .", "in 2005 , two independent groups generated and published findings regarding the systemic deletion of the rassf1a gene variant in mice [ 35 , 36 ] .", "both described similar phenotypes involving the spontaneous generation of tumors , particularly in aged mice , thus further supporting the notion that rassf1a is a bona fide tumor suppressor [ 35 , 36 ] .", "not surprisingly , nearly all studies involving rassf1a to date are related to cancer biology with few reports related to the cardiovascular field .", "rassf1a is ubiquitously expressed and has been detected in heart tissue [ 3 , 37 , 38 ] .", "initial investigation into the role of rassf1 gene products in a cardiac context came from the neyses laboratory .", "their findings demonstrated that both rassf1a and rassf1c could associate with the sarcolemmal calcium pump , pmca4b , in neonatal rat cardiac myocytes .", "this interaction was shown to mediate the inhibition of erk , and subsequent elk transcription and suggested the possibility that rassf1a could modulate cardiac myocyte growth .  ", "five years later , the same group demonstrated that rassf1a does in fact negatively regulate cardiac hypertrophy in vivo using rassf1a mice .", "although these mice have increased susceptibility to spontaneous tumorigenesis , no apparent cardiovascular phenotype was observed under basal conditions , that is , no differences in heart size , morphology , or function compared to wt .", "however , when rassf1a mice were challenged with pressure overload , they responded with an exaggerated hypertrophic response , evidenced by significantly greater increases in heart weight / body weight and hypertrophic gene expression ( anp , bnp , -mhc ) .", "cardiac myocytes of rassf1a mice were significantly larger , which explains the augmented heart growth .", "chamber dilation of rassf1a mouse hearts was observed by echocardiography , consistent with eccentric hypertrophic remodeling .", "hemodynamic analysis of wt and rassf1a mice showed a rightward shift in pv loops following pressure overload in rassf1a hearts , yet dp / dtmax , dp / dtmin , and fractional shortening were not altered in rassf1a mice compared to wt .   to examine rassf1a function in cardiac myocytes , oceandy et al .", "utilized a neonatal rat cardiac myocyte ( nrcm ) culture and the forced expression of rassf1a through adenoviral gene transfer .", "increased rassf1a expression inhibited phenylephrine-(pe- ) induced cardiac myocyte growth and suppressed raf-1 and erk1/2 activation by pe treatment .", "conversely , both raf-1 and erk1/2 phosphorylation were increased in  rassf1a hearts following pressure overload , suggesting negative regulation of mapk signaling by rassf1a .", "deletion mutants of rassf1a revealed an important function of the n - terminus of rassf1a that disrupts the binding of active ras and raf-1 , thus preventing erk activation and cardiac myocyte growth .   to better understand the function of rassf1a in cardiac myocytes in vivo , we crossed genetically altered mice harboring a floxed rassf1a allele   with mice harboring the cre recombinase transgene driven by the -mhc promoter .", "this strategy disrupted endogenous rassf1a gene expression and ensured cardiac myocyte specificity [ 38 , 40 ] .", "similar to the rassf1a  mice , rassf1a - cre mice had no obvious baseline cardiac phenotype .", "although we also found exaggerated heart growth in the rassf1a mice in response to pressure overload , the rassf1a - cre mice unexpectedly had attenuated hypertrophy , that is , smaller hearts and cardiac myocytes , compared to rassf1a and -mhc - cre controls .", "furthermore , rassf1a - cre mice had significantly less fibrosis and myocyte apoptosis , and better cardiac function following pressure overload .", "this was in stark contrast to the rassf1a mice , which presented significantly more fibrosis and a decline in cardiac function comparable to the levels found in wt mice .  ", "as an alternative approach we also generated two different cardiac - specific transgenic mouse lines : the first expressing wild - type rassf1a and the second expressing a rassf1a sarah domain point mutant ( l308p ) that renders it unable to bind mst1 .", "interestingly , we found that increased rassf1a expression in the heart caused increases in mst1 activation , cardiac myocyte apoptosis , and fibrosis , and led to worsened function following pressure overload .", "conversely , rassf1a l308p tg mice had significant reductions in mst1 activation , apoptosis and fibrosis , while cardiac function was preserved after stress .", "these opposing phenotypes strongly implicate mst1 as a critical effector of rassf1a - mediated myocardial dysfunction .  ", "in cultured nrcms , increased rassf1a expression elicited activation of mst1 and caused mst1-mediated apoptosis .", "however , in primary rat cardiac fibroblasts , rassf1a had a more pronounced effect on inhibition of cell proliferation rather than survival .", "additionally , rassf1a depletion led to an upregulation of nf-b - dependent tnf- expression and secretion in cardiac fibroblasts , while no change in il-1 , il-6 , or tgf-1 was observed . through conditioned medium transfer experiments , we demonstrated that tnf- secretion from fibroblasts promotes cardiac myocyte growth .", "furthermore , treatment of rassf1a mice with a neutralizing antibody against tnf- was able to rescue the augmented heart growth and fibrosis observed following pressure overload .", "these data strongly implicated tnf- as a critical paracrine factor influencing the cardiac myocyte growth response to stress in vivo .", "this work also demonstrated the cell - type specificity of rassf1a signaling in the heart and highlighted a novel signaling pathway downstream of rassf1a / mst1 that mediates a paracrine effect in vivo ( see figure 1 ) .", "this mechanism involving multiple cell types , and paracrine signaling among them is rather unique and contrasts with more established signaling paradigms of cardiac hypertrophy including calcineurin / nfat , hdac / mef2 and mek / erk pathways , which have been elucidated in the cardiac myocyte .  ", "using genetically altered mouse models we showed that increased expression of mst1 , and subsequent activation of the hippo pathway , caused increased apoptosis , dilated cardiomyopathy , and premature death .", "interestingly , expression of mst1 also attenuated cardiac myocyte hypertrophy thereby impairing the heart 's ability to appropriately respond to stress .", "in contrast , expression of a kinase - inactive mst1 mutant ( dn - mst1 ) prevented cell death and protected the heart from insult .", "lats1/2 kinases ( mammalian homologs of warts ) are targets of mst1/2 that can phosphorylate and inactivate yap , thereby inhibiting yap - mediated gene transcription .", "similar to our findings related to mst1 , we demonstrated that transgenic expression of lats2 in the heart led to inhibited growth and worsened function .", "conversely , kinase - inactive lats2 ( dn - lats2 ) transgenic mice had larger hearts both at baseline and following pressure overload and displayed attenuated cardiac myocyte apoptosis in response to stress", ". taken together , these results provide further evidence that activation of hippo signaling , via increased mst1 or lats2 expression , inhibits cardiac myocyte growth and promotes apoptosis in the adult heart", ". furthermore , selective inhibition of hippo signaling in the cardiac myocyte ( dn - mst1 or dn - lats2 tg ) confers protection against insult , similar to what we observed in the cardiac myocyte - specific rassf1a deleted mice .", "however , the hypertrophic response in these two models was opposite , which may result from a hippo - independent pathway(s ) downstream of rassf1a .", "it should be pointed out that studies of adult mouse models using cardiac myocyte - restricted deletion of mst1/2 , lats1/2 or yap have not been published .", "findings from these models should be helpful in further elucidating the role of hippo signaling components in the adult murine heart .", "recent work from the martin laboratory demonstrated the importance of mammalian hippo signaling during cardiac development and cardiac myocyte proliferation .", "conditional deletion of salvador ( sav1 ) in the embryonic heart , driven by nkx2.5-cre expression , caused increased myocyte proliferation and cardiac enlargement and was mediated by hyperactivation of yap and subsequent wnt/-catenin - regulated gene expression . in a similar vein , direct targeting of yap expression in the developing mouse heart further demonstrated its role in governing both myocyte proliferation and heart growth .", "interestingly , both reports described an interaction between yap and wnt signaling , highlighting additional hippo signaling crosstalk in the heart .", "  fueled by the initial reports described herein , investigation into the role of rassf1a in cardiovascular biology has begun to accelerate . yet many questions remain outstanding . among them", ", what are the upstream inputs that regulate rassf1a function ? what is the mechanism responsible for rassf1a cell - type - specific", "what are the molecular constituents of the rassf1a complex ? does rassf1a have additional mst1-independent functions in the heart , as has been demonstrated in tumor cell lines ?", "recent work identified activated k - ras as a promoter of rassf1a signaling in colorectal cancer cells .", "this finding begs the question of whether k - ras or additional ras isoforms regulate rassf1a in other systems and cell types . based on our findings in rassf1a - deleted mice", ", we speculate that the difference in proliferative capacity between cardiac myocytes and fibroblasts may explain the distinct effects of rassf1a signaling in the heart . there may also be differences in the expression or localization of signaling components , thereby modulating their ability to effectively signal in certain cell types .", "exposure to diverse signals and cues in the extracellular milieu may also contribute to varied outcomes downstream rassf1a . as we continue to elucidate the role of rassf1a and hippo signaling in the heart", "our work has shed light on the importance of cell type specificity rassf1a in determining pathological outcomes .", "we also defined a paracrine mechanism functioning downstream of rassf1a in response to cardiac stress .", "it is likely that additional complexities remain to be uncovered and will ultimately influence possible interventions to manipulate rassf1a and treat heart disease .", "rassf1a signaling is diverse and our knowledge regarding rassf1a function is rapidly expanding . given that a bridge from cancer to cardiovascular biology is in place , it is likely that as additional rassf1a mechanisms of action are discovered , its impact on cardiac biology will continue to grow ."], "abstract_text": ["<S> the rassf proteins are a family of polypeptides , each containing a conserved ras association domain , suggesting that these scaffold proteins may be effectors of activated ras or ras - related small gtpases . </S>", "<S> rassf proteins are characterized by their ability to inhibit cell growth and proliferation while promoting cell death . </S>", "<S> rassf1 isoform a is an established tumor suppressor and is frequently silenced in a variety of tumors and human cancer cell lines . </S>", "<S> however , our understanding of its function in terminally differentiated cell types , such as cardiac myocytes , is relatively nascent </S>", "<S> . herein , we review the role of rassf1a in cardiac physiology and disease and highlight signaling pathways that mediate its function . </S>"], "labels": null, "section_names": ["1. Introduction", "2. RASSF1A", "3. Cardiovascular Function of RASSF1A", "4. Conclusion"], "sections": [["the ras association domain family ( rassf ) consists of 10 members : rassf1 - 10 .", "importantly , all isoforms contain a ras association ( ra ) domain either in their c - terminal ( rassf1 - 6 ) or n - terminal ( rassf7 - 10 ) regions . to date , no known catalytic activity has been described for this family , and the general consensus supposes that rassf proteins function as scaffolds to localize signaling in the cell .", "rassf1 isoform a ( rassf1a ) is the most characterized member of the rassf family ."], ["the rassf1 gene encodes multiple splice variants , including the two predominant isoforms , rassf1a and c. the rassf1a isoform is the longest variant of the rassf1 gene .", "structurally , rassf1a is a product of exons 1 , 2/ , 3 , 4 , 5 , and 6 , while rassf1c consists of exons 2 , 3 , 4 , 5 , and 6 . both isoforms contain a c - terminal ra domain ; however , rassf1a has an additional c1 domain that is not present in rassf1c .", "this short arm of chromosome 3 is known to exhibit loss of heterozygosity in many tumor models and is thought to harbor tumor suppressor genes . as the literature has shown , rassf1a fits this description .", "the rassf1a promoter contains a cpg island that shows a high frequency of hypermethylation in tumors , thereby silencing rassf1a expression in many human cancers including lung , breast , ovarian , renal , and bladder [ 47 ] .", "rassf1a expression is also lost in numerous cancer cell lines , while rassf1c expression is seemingly unaffected .", "interestingly , recent work suggests that rassf1c may actually promote tumor progression [ 8 , 9 ] , further distinguishing these two splice variants .", "all rassf proteins have an ra domain , which is thought to necessitate their binding to activated , gtp - bound ras proteins . while rassf5 ( nore1 ) is thought to bind ras directly , whether rassf1a is able to associate with ras is less clear .", "it has been shown that rassf1a binds k - ras in vitro , and an interaction between ectopically expressed rassf1a and activated k - ras has been observed in hek293 cells [ 11 , 12 ] .", "however , other work has found that this interaction only occurs in the presence of nore1 , arguing for an indirect association .", "importantly , to our knowledge , there are no reports demonstrating the interaction of endogenous rassf1a and ras proteins .  ", "it has been implicated in the negative regulation of cell cycle progression , cell proliferation , and cell survival .", "rassf1a has been shown to localize to microtubules of proliferating cells , increasing microtubule stability and inhibiting cell division [ 14 , 15 ] .", "this may be mediated through direct binding or though interaction with microtubule - associated proteins such as c19orf5 .", "rassf1a has also been shown to inhibit proliferation by inhibiting the accumulation of cyclin d1 and arresting cell division [ 17 , 18 ] .  ", "rassf1a also promotes apoptosis , which can reportedly occur through multiple mechanisms and is likely cell - type dependent .", "one mechanism that mediates the apoptotic function of rassf1a involves protein interaction with modulator of apoptosis-1 ( moap-1 or map-1 ) .", "moap-1 is normally sequestered in an inactive form in healthy cells . upon death receptor stimulation ,", "rassf1a binds moap-1 , causing its activation and subsequent association with bax , which leads to apoptosis .", "previous work has also demonstrated enhancement of rassf1a / mst - mediated cell death by the scaffold cnk1 .", "rassf1a can also elicit inhibitory effects on growth and survival through engagement of the hippo pathway .", "the hippo signaling pathway is a highly conserved kinase cascade that was originally discovered in drosophila and has been shown to be a critical regulator of cell proliferation , survival , and organ growth .", "three members of this pathway , drassf , salvador and hippo , contain the sarah ( salvador - rassf - hippo ) domain , which is conserved in its mammalian counterparts rassf1 - 6 , ww45 , and mst1/2 , respectively .", "while the drosophila ortholog drassf is known to antagonize hippo activation in the fly , it has been demonstrated that rassf1a promotes phosphorylation and activation of mst 1/2 by inhibiting the phosphatase pp2a in mammalian systems [ 29 , 30 ] .", "the biological relevance of rassf1a - mediated activation of hippo signaling has also been investigated .", "reported a rassf1a - mst2-yap - p73-puma signaling axis that promotes apoptosis in mammalian cells .", "hippo signaling is also important for maintaining intestinal homeostasis and tissue regeneration in response to injury .", "mouse models with conditional disruption of either mst1/2 or sav1 in the intestinal epithelium displayed hyperactivation of yes - associated protein ( yap ) , increased intestinal stem cell ( isc ) proliferation , and increased polyp formation following dextran sodium sulfate ( dss ) treatment [ 32 , 33 ] .", "similarly , loss - of - function mutations of hippo components in the fly midgut caused increased isc proliferation .", "these findings suggest that perhaps hippo signaling serves a more global role in regulating organ integrity , structure , and response to injury , and that perturbation of this pathway can lead to aberrant growth and dysfunction ."], ["in 2005 , two independent groups generated and published findings regarding the systemic deletion of the rassf1a gene variant in mice [ 35 , 36 ] .", "both described similar phenotypes involving the spontaneous generation of tumors , particularly in aged mice , thus further supporting the notion that rassf1a is a bona fide tumor suppressor [ 35 , 36 ] .", "not surprisingly , nearly all studies involving rassf1a to date are related to cancer biology with few reports related to the cardiovascular field .", "rassf1a is ubiquitously expressed and has been detected in heart tissue [ 3 , 37 , 38 ] .", "initial investigation into the role of rassf1 gene products in a cardiac context came from the neyses laboratory .", "their findings demonstrated that both rassf1a and rassf1c could associate with the sarcolemmal calcium pump , pmca4b , in neonatal rat cardiac myocytes .", "this interaction was shown to mediate the inhibition of erk , and subsequent elk transcription and suggested the possibility that rassf1a could modulate cardiac myocyte growth .  ", "five years later , the same group demonstrated that rassf1a does in fact negatively regulate cardiac hypertrophy in vivo using rassf1a mice .", "although these mice have increased susceptibility to spontaneous tumorigenesis , no apparent cardiovascular phenotype was observed under basal conditions , that is , no differences in heart size , morphology , or function compared to wt .", "however , when rassf1a mice were challenged with pressure overload , they responded with an exaggerated hypertrophic response , evidenced by significantly greater increases in heart weight / body weight and hypertrophic gene expression ( anp , bnp , -mhc ) .", "cardiac myocytes of rassf1a mice were significantly larger , which explains the augmented heart growth .", "chamber dilation of rassf1a mouse hearts was observed by echocardiography , consistent with eccentric hypertrophic remodeling .", "hemodynamic analysis of wt and rassf1a mice showed a rightward shift in pv loops following pressure overload in rassf1a hearts , yet dp / dtmax , dp / dtmin , and fractional shortening were not altered in rassf1a mice compared to wt .   to examine rassf1a function in cardiac myocytes , oceandy et al .", "utilized a neonatal rat cardiac myocyte ( nrcm ) culture and the forced expression of rassf1a through adenoviral gene transfer .", "increased rassf1a expression inhibited phenylephrine-(pe- ) induced cardiac myocyte growth and suppressed raf-1 and erk1/2 activation by pe treatment .", "conversely , both raf-1 and erk1/2 phosphorylation were increased in  rassf1a hearts following pressure overload , suggesting negative regulation of mapk signaling by rassf1a .", "deletion mutants of rassf1a revealed an important function of the n - terminus of rassf1a that disrupts the binding of active ras and raf-1 , thus preventing erk activation and cardiac myocyte growth .   to better understand the function of rassf1a in cardiac myocytes in vivo , we crossed genetically altered mice harboring a floxed rassf1a allele   with mice harboring the cre recombinase transgene driven by the -mhc promoter .", "this strategy disrupted endogenous rassf1a gene expression and ensured cardiac myocyte specificity [ 38 , 40 ] .", "similar to the rassf1a  mice , rassf1a - cre mice had no obvious baseline cardiac phenotype .", "although we also found exaggerated heart growth in the rassf1a mice in response to pressure overload , the rassf1a - cre mice unexpectedly had attenuated hypertrophy , that is , smaller hearts and cardiac myocytes , compared to rassf1a and -mhc - cre controls .", "furthermore , rassf1a - cre mice had significantly less fibrosis and myocyte apoptosis , and better cardiac function following pressure overload .", "this was in stark contrast to the rassf1a mice , which presented significantly more fibrosis and a decline in cardiac function comparable to the levels found in wt mice .  ", "as an alternative approach we also generated two different cardiac - specific transgenic mouse lines : the first expressing wild - type rassf1a and the second expressing a rassf1a sarah domain point mutant ( l308p ) that renders it unable to bind mst1 .", "interestingly , we found that increased rassf1a expression in the heart caused increases in mst1 activation , cardiac myocyte apoptosis , and fibrosis , and led to worsened function following pressure overload .", "conversely , rassf1a l308p tg mice had significant reductions in mst1 activation , apoptosis and fibrosis , while cardiac function was preserved after stress .", "these opposing phenotypes strongly implicate mst1 as a critical effector of rassf1a - mediated myocardial dysfunction .  ", "in cultured nrcms , increased rassf1a expression elicited activation of mst1 and caused mst1-mediated apoptosis .", "however , in primary rat cardiac fibroblasts , rassf1a had a more pronounced effect on inhibition of cell proliferation rather than survival .", "additionally , rassf1a depletion led to an upregulation of nf-b - dependent tnf- expression and secretion in cardiac fibroblasts , while no change in il-1 , il-6 , or tgf-1 was observed . through conditioned medium transfer experiments , we demonstrated that tnf- secretion from fibroblasts promotes cardiac myocyte growth .", "furthermore , treatment of rassf1a mice with a neutralizing antibody against tnf- was able to rescue the augmented heart growth and fibrosis observed following pressure overload .", "these data strongly implicated tnf- as a critical paracrine factor influencing the cardiac myocyte growth response to stress in vivo .", "this work also demonstrated the cell - type specificity of rassf1a signaling in the heart and highlighted a novel signaling pathway downstream of rassf1a / mst1 that mediates a paracrine effect in vivo ( see figure 1 ) .", "this mechanism involving multiple cell types , and paracrine signaling among them is rather unique and contrasts with more established signaling paradigms of cardiac hypertrophy including calcineurin / nfat , hdac / mef2 and mek / erk pathways , which have been elucidated in the cardiac myocyte .  ", "using genetically altered mouse models we showed that increased expression of mst1 , and subsequent activation of the hippo pathway , caused increased apoptosis , dilated cardiomyopathy , and premature death .", "interestingly , expression of mst1 also attenuated cardiac myocyte hypertrophy thereby impairing the heart 's ability to appropriately respond to stress .", "in contrast , expression of a kinase - inactive mst1 mutant ( dn - mst1 ) prevented cell death and protected the heart from insult .", "lats1/2 kinases ( mammalian homologs of warts ) are targets of mst1/2 that can phosphorylate and inactivate yap , thereby inhibiting yap - mediated gene transcription .", "similar to our findings related to mst1 , we demonstrated that transgenic expression of lats2 in the heart led to inhibited growth and worsened function .", "conversely , kinase - inactive lats2 ( dn - lats2 ) transgenic mice had larger hearts both at baseline and following pressure overload and displayed attenuated cardiac myocyte apoptosis in response to stress", ". taken together , these results provide further evidence that activation of hippo signaling , via increased mst1 or lats2 expression , inhibits cardiac myocyte growth and promotes apoptosis in the adult heart", ". furthermore , selective inhibition of hippo signaling in the cardiac myocyte ( dn - mst1 or dn - lats2 tg ) confers protection against insult , similar to what we observed in the cardiac myocyte - specific rassf1a deleted mice .", "however , the hypertrophic response in these two models was opposite , which may result from a hippo - independent pathway(s ) downstream of rassf1a .", "it should be pointed out that studies of adult mouse models using cardiac myocyte - restricted deletion of mst1/2 , lats1/2 or yap have not been published .", "findings from these models should be helpful in further elucidating the role of hippo signaling components in the adult murine heart .", "recent work from the martin laboratory demonstrated the importance of mammalian hippo signaling during cardiac development and cardiac myocyte proliferation .", "conditional deletion of salvador ( sav1 ) in the embryonic heart , driven by nkx2.5-cre expression , caused increased myocyte proliferation and cardiac enlargement and was mediated by hyperactivation of yap and subsequent wnt/-catenin - regulated gene expression . in a similar vein , direct targeting of yap expression in the developing mouse heart further demonstrated its role in governing both myocyte proliferation and heart growth .", "interestingly , both reports described an interaction between yap and wnt signaling , highlighting additional hippo signaling crosstalk in the heart ."], ["  fueled by the initial reports described herein , investigation into the role of rassf1a in cardiovascular biology has begun to accelerate . yet many questions remain outstanding . among them", ", what are the upstream inputs that regulate rassf1a function ? what is the mechanism responsible for rassf1a cell - type - specific", "what are the molecular constituents of the rassf1a complex ? does rassf1a have additional mst1-independent functions in the heart , as has been demonstrated in tumor cell lines ?", "recent work identified activated k - ras as a promoter of rassf1a signaling in colorectal cancer cells .", "this finding begs the question of whether k - ras or additional ras isoforms regulate rassf1a in other systems and cell types . based on our findings in rassf1a - deleted mice", ", we speculate that the difference in proliferative capacity between cardiac myocytes and fibroblasts may explain the distinct effects of rassf1a signaling in the heart . there may also be differences in the expression or localization of signaling components , thereby modulating their ability to effectively signal in certain cell types .", "exposure to diverse signals and cues in the extracellular milieu may also contribute to varied outcomes downstream rassf1a . as we continue to elucidate the role of rassf1a and hippo signaling in the heart", "our work has shed light on the importance of cell type specificity rassf1a in determining pathological outcomes .", "we also defined a paracrine mechanism functioning downstream of rassf1a in response to cardiac stress .", "it is likely that additional complexities remain to be uncovered and will ultimately influence possible interventions to manipulate rassf1a and treat heart disease .", "rassf1a signaling is diverse and our knowledge regarding rassf1a function is rapidly expanding . given that a bridge from cancer to cardiovascular biology is in place , it is likely that as additional rassf1a mechanisms of action are discovered , its impact on cardiac biology will continue to grow ."]]}
{"article_id": "PMC4264083", "article_text": ["it is one of the most commonly used medicines in the treatment of rheumatoid arthritis or psoriatic arthritis .", "we report the case of an adult woman with psoriatic arthritis who developed acute severe hepatitis following long - term treatment with mtx .", "the periodic biological monitoring of liver enzymes : alanine aminotransferase ( alat ) , aspartate aminotransferase ( asat ) , gamma - glutamyl transpeptidase ( gamma - gt ) performed every 6 months did not reveal any abnormalities .", "mtx was stopped in the year 2005 due to remission of the disease . in 2009 ,", "it was reintroduced at a dose of 15 mg / week due to decompensation of the disease .", "monitoring of liver enzymes performed every 6 months showed an elevation < 2 times the normal levels on average of asat and alat since 2010 . assessing the risk - benefit ratio and given the severe decompensation of the disease , mtx was continued . in may 2013 , patient presented with jaundice following the development of asthenia and weight loss .", "laboratory investigation showed the following results : prothrombin time ( 71% ) , asat 580 u / l , alat 620 u / l , serum gamma - gt 146 iu / l , serum alkaline phosphatase 170 iu / l and total serum bilirubin 12 mg / dl .", "antimitochondrial antibodies ( titer > 1/40 ) and antinuclear antibodies ( titer of 1/640 ) were detected in serum .", "routine laboratory investigations for infection with hepatitis a , b , and c viruses and serology for cytomegalovirus and epstein - barr virus were negative .", "methotrexate was stopped , and therapy with oral methylprednisolone ( 80 mg / day ) was initiated .", "a significant improvement in the following parameters was observed a week later : prothrombin time ( 88% ) , asat 270 u / l , alat 340 u / l , s. gamma - gt 122 iu / l , and s. alkaline phosphatase 173 iu / l .", "after 4 months , all laboratory parameters had normalized , and serum was negative for antimitochondrial and antinuclear antibodies .", "patients taking mtx are more likely to discontinue therapy because of adverse drug effects rather than because of lack of efficacy .", "adverse effects of mtx are , usually , mild and self - limiting , but may include serious adverse drug reactions such as hematopoietic suppression , pulmonary , and hepatotoxicity .", "hepatic fibrosis is a usual adverse reaction reported with long - term use of mtx therapy in psoriatic patients .", "causality assessment of the adverse drug event ( ade ) was carried out using who - umc criteria , naranjo 's scale , and roussel uclaf causality assessment method ( rucam ) scale . in this case ,", "patient improved on withdrawal of the drug , and there were no other confounding factors that could have caused this adverse effect .", "hence , the ade was probably caused by mtx ( who - umc criteria : probable ; naranjo 's score : 7 , probable ; and rucam scale : 5 ) .", "in addition , sufficient and continuous exposure to the drug , lack of previous evidence of autoimmune disease and complete resolution of the condition within 4 months of discontinuation of the suspected drug confirmed the diagnostic criteria of drug - induced autoimmunity .", "the first case of autoimmune hepatitis associated with mtx was described in 2011 in a 57-year - old man after 11 years of treatment with mtx .", "a complete normalization of laboratory parameters was obtained 5 months after stopping this drug . to the best of our knowledge ,", "it , usually , occurs at higher doses and also positively correlates with the cumulative dose of drugs .", "other risk factors for the development of elevated liver enzymes and changes in liver biopsy include obesity , higher alcohol intake prior to commencement of mtx and persistent hepatitis b or c infection . in the present case ,", "appearance of autoantibodies and transitory autoimmune disease associated with infliximab in a patient with psoriatic arthritis has also been documented and the preexistent serological signs of autoimmunity are believed to be a risk factor for the development of similar autoimmune reactions .", "this report confirms the need to monitor liver enzymes carefully in patients with psoriatic arthritis using long - term treatment with mtx .", "clinicians must collaborate with the pharmacovigilance center to detect and notify serious adverse effects of mtx .", "further research is needed to identify the exact mechanism and establish , if possible , primary means of prevention of this serious adverse effect ."], "abstract_text": ["<S> methotrexate ( mtx ) is one of the most commonly used medicines in the treatment of psoriatic arthritis . </S>", "<S> the drug can produce steatosis and cirrhosis . </S>", "<S> autoimmune hepatitis is a rare and serious adverse effect . </S>", "<S> we describe the case of a 53-year - old woman who developed autoimmune hepatitis after a long - term use of mtx for psoriatic arthritis . </S>", "<S> hepatitis was completely resolved 4 months after stopping this drug . </S>", "<S> the pathophysiologic mechanisms of a drug - induced autoimmunity are unclear and complex . </S>", "<S> this report confirms the need to monitor liver enzymes carefully in patients using long - term treatment with mtx for psoriasis or rheumatoid arthritis . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["it is one of the most commonly used medicines in the treatment of rheumatoid arthritis or psoriatic arthritis .", "we report the case of an adult woman with psoriatic arthritis who developed acute severe hepatitis following long - term treatment with mtx ."], ["the periodic biological monitoring of liver enzymes : alanine aminotransferase ( alat ) , aspartate aminotransferase ( asat ) , gamma - glutamyl transpeptidase ( gamma - gt ) performed every 6 months did not reveal any abnormalities .", "mtx was stopped in the year 2005 due to remission of the disease . in 2009 ,", "it was reintroduced at a dose of 15 mg / week due to decompensation of the disease .", "monitoring of liver enzymes performed every 6 months showed an elevation < 2 times the normal levels on average of asat and alat since 2010 . assessing the risk - benefit ratio and given the severe decompensation of the disease , mtx was continued . in may 2013 , patient presented with jaundice following the development of asthenia and weight loss .", "laboratory investigation showed the following results : prothrombin time ( 71% ) , asat 580 u / l , alat 620 u / l , serum gamma - gt 146 iu / l , serum alkaline phosphatase 170 iu / l and total serum bilirubin 12 mg / dl .", "antimitochondrial antibodies ( titer > 1/40 ) and antinuclear antibodies ( titer of 1/640 ) were detected in serum .", "routine laboratory investigations for infection with hepatitis a , b , and c viruses and serology for cytomegalovirus and epstein - barr virus were negative .", "methotrexate was stopped , and therapy with oral methylprednisolone ( 80 mg / day ) was initiated .", "a significant improvement in the following parameters was observed a week later : prothrombin time ( 88% ) , asat 270 u / l , alat 340 u / l , s. gamma - gt 122 iu / l , and s. alkaline phosphatase 173 iu / l .", "after 4 months , all laboratory parameters had normalized , and serum was negative for antimitochondrial and antinuclear antibodies ."], ["patients taking mtx are more likely to discontinue therapy because of adverse drug effects rather than because of lack of efficacy .", "adverse effects of mtx are , usually , mild and self - limiting , but may include serious adverse drug reactions such as hematopoietic suppression , pulmonary , and hepatotoxicity .", "hepatic fibrosis is a usual adverse reaction reported with long - term use of mtx therapy in psoriatic patients .", "causality assessment of the adverse drug event ( ade ) was carried out using who - umc criteria , naranjo 's scale , and roussel uclaf causality assessment method ( rucam ) scale . in this case ,", "patient improved on withdrawal of the drug , and there were no other confounding factors that could have caused this adverse effect .", "hence , the ade was probably caused by mtx ( who - umc criteria : probable ; naranjo 's score : 7 , probable ; and rucam scale : 5 ) .", "in addition , sufficient and continuous exposure to the drug , lack of previous evidence of autoimmune disease and complete resolution of the condition within 4 months of discontinuation of the suspected drug confirmed the diagnostic criteria of drug - induced autoimmunity .", "the first case of autoimmune hepatitis associated with mtx was described in 2011 in a 57-year - old man after 11 years of treatment with mtx .", "a complete normalization of laboratory parameters was obtained 5 months after stopping this drug . to the best of our knowledge ,", "it , usually , occurs at higher doses and also positively correlates with the cumulative dose of drugs .", "other risk factors for the development of elevated liver enzymes and changes in liver biopsy include obesity , higher alcohol intake prior to commencement of mtx and persistent hepatitis b or c infection . in the present case ,", "appearance of autoantibodies and transitory autoimmune disease associated with infliximab in a patient with psoriatic arthritis has also been documented and the preexistent serological signs of autoimmunity are believed to be a risk factor for the development of similar autoimmune reactions ."], ["this report confirms the need to monitor liver enzymes carefully in patients with psoriatic arthritis using long - term treatment with mtx .", "clinicians must collaborate with the pharmacovigilance center to detect and notify serious adverse effects of mtx .", "further research is needed to identify the exact mechanism and establish , if possible , primary means of prevention of this serious adverse effect ."]]}
{"article_id": "PMC3015902", "article_text": ["mirizzi syndrome is a rare complication of cholecystolithiasis characterized by jaundice due to compression of the common hepatic duct", ". the diagnosis may not be immediately apparent , and management is controversial with open surgery still recommended by some authors .", "a case is detailed herein of a 67-year - old man who presented with abdominal pain , fever , and jaundice .", "a dilated bile duct was found on ultrasound , but the gallbladder could not be seen .", "the diagnosis of mirizzi syndrome was made at ercp , and a stent was placed through the papilla .", "laparoscopic retrograde ( fundus first ) cholecystectomy was carried out utilizing a laparoscopic liver retractor .", "in this particular case , it was not possible at ercp to get a guidewire and stent past the obstruction . a stent was left through the papilla , below the obstruction and this allowed primary duct closure during surgery .", "acute mirizzi syndrome should be suspected when a patient presents with acute cholecystitis and jaundice with dilated intrahepatic ducts on ultrasound .", "ercp is useful to confirm the diagnosis and allows stenting to alleviate the jaundice and facilitate the subsequent operation .", "laparoscopic ultrasound is useful to locate the impacted stone and to partially replicate the touch of the surgeon 's hand , which is not available in laparoscopic surgery .", "mirizzi syndrome ( ms ) is biliary obstruction from compression of the common hepatic duct ( chd ) by a gallstone impacted in the cystic duct .", "mcsherry classified the syndrome into 2 groups : type i where extrinsic compression of the bile duct occurs and type ii where erosion occurs of the wall of the chd by the stone with formation of a cholecysto - choledochal fistula .", "csendes further subdivided type ii depending on the size of the fistula and the degree of destruction of the wall of the chd .", "the syndrome was initially described in 1948 by pablo luis mirizzi , professor of surgery in cordoba , argentina , and he erroneously postulated that the extrinsic pressure and inflammation induced spasm of the chd .", "precise diagnosis may be difficult initially because the condition may be confused with choledocholithiasis and cholangitis .", "the classical ultrasound findings are of a contracted gallbladder , dilated intrahepatic ducts , and a normal common bile duct ( cbd ) .", "although a rare condition , many controversial aspects still surround the definition and management of ms . to illustrate the true ms and the value of a combination of endoscopic retrograde cholangiopancreatography ( ercp ) and laparoscopic surgery , a case", "a 67-year - old man was admitted to the emergency department with a 3-day history of gradually worsening upper abdominal pain associated initially with vomiting and anorexia . the patient had a history of hypertension and polymyalgia rheumatica .", "he was noted to be jaundiced with a temperature of 38 celsius and right upper quadrant tenderness and guarding .", "his liver function tests were markedly deranged with bilirubin 106 mol / l [ range , 3 to 22 ] , alkaline phosphatase 159 iu / l ( range , 20 to 110 ) , and alanine transaminase 770 iu / l ( range , 5 to 65 ) .", "this showed dilated intrahepatic ducts with the cbd reported as also being dilated and measuring 9 mm in diameter .", "subsequent imaging showed that the ultrasound had actually detected a dilated chd as the cbd was not dilated . at ercp , contrast filled a nondilated cbd up to an obstruction due to a stone ( figure 1 ) .", "attempts were made to pass a guidewire beyond the obstruction up into the chd and intrahepatic ducts but without success .", "the guidewire kept going into the gallbladder , and it became apparent that there was an ms with the obstructing stone in the cystic duct causing compression of the chd ( figure 2 ) .", "after the guidewire had been manipulated for a time , some pus came out of the papilla signifying a gallbladder empyema .", "a 10 french straight plastic stent was placed through the papilla without papillotomy with its proximal tip lying below the obstruction and , as expected , no bile or intrahepatic contrast drained .", "the stone is impacted in the cystic duct , which is anterior and parallel to the common hepatic duct .", "note the narrow common bile duct and the dilatation of the common hepatic and intrahepatic ducts . a computed tomogram ( ct ) scan was done and showed a contracted gallbladder with a calcified stone causing biliary obstruction ( figure 3 ) . at laparoscopic cholecystectomy , an acutely inflamed", "a liver retractor was used to elevate the liver and maintain exposure of the bile duct ( diamond - flex , cardinal health , snowden - dencer mis products , tucker , ga , usa ) ( figure 4 ) .", "it was possible to  feel  the stone via a grasper , and its position was definitely confirmed by laparoscopic ultrasound ( figure 5 ) .", "the duct was incised to release the impacted stone with a single - use cbd blade ( espiner medical ltd .", "the cbd was examined with a 5-mm flexible choledochoscope showing that the duct was clear distally with the stent in place ( figure 7 ) .", "it was not possible to retroflex the choledochoscope to examine the common hepatic or intrahepatic ducts .", "the gallbladder was excised , the opening in the cystic duct sutured with continuous 40 vicryl ( ethicon inc . ,", "somerville , nj , usa ) and a 20 french nonsuction tube drain placed in the subhepatic space near the bile duct .", "the stent was removed at ercp 6 weeks later and a check cholangiogram showed a normal bile duct with no filling of any residual cystic duct and no stricture . during 18 months of follow - up , no delayed sequelae have occurred .", "computed tomograpic scan showing a calcified stone causing bile duct obstruction with the proximal end of the stent just below the stone .", "residual contrast from the ercp is present in the gallbladder ( somatom volume zoom , 4 slice , siemens ag , erlangen , germany ) .", "the gallbladder ( gb ) has been dissected from the liver retrograde , and the liver retractor is in place .", "the arrow points to surgicel ( ethicon , somerville , nj , usa ) in the gallbladder bed of the liver .", "laparoscopic ultrasound image showing the impacted stone ( arrow ) in a thickwalled duct ( aloka inc .", "choledochoscopy using a 5-mm scope showing a clear bile duct with the stent visible ( insert ) .", "ms is a rare cause of jaundice due to extrinsic compression of the chd and is present in approximately 0.35% of cholecystectomies .", "many series report much higher rates ; however , this may represent differing views on what constitutes a case of ms .", "kwon and inui recently reported a prevalence of 1.2% , but only a third of their patients were jaundiced , and despite this the majority still underwent open surgery .", "similarly , planned open operation for mirizzi syndrome is accepted and , in fact , is still advocated by many .", "many surgeons do not view conversion as detrimental and therefore do not persist laparoscopically when cholecystectomy is difficult .", "conversion or an open operation allows the use of proprioception or the touch of the surgeon 's hand and is generally accepted as a way to improve the safety of any operation , especially one in which severe inflammation is present . to replicate this ,", "however , open surgery is associated with significant short- and long - term morbidity , and a difficult operation is not necessarily easier or safer when performed open .", "a degree of tactility is possible via instruments although not by currently available robotic systems , and laparoscopic ultrasound is very useful in stone disease .", "it is generally accepted that an increased risk of bile duct injury exists during surgery for ms , and laparoscopic surgery may increase this risk .", "operative cholangiography is advocated to improve the safety of cholecystectomy , but an accurate transcystic cholangiogram will not be possible in ms . a standard technique in open surgery for the difficult laparoscopic cholecystectomy was the  fundus - first  approach .", "this can be replicated in laparoscopic surgery by the use of a liver retractor and means that exposure does not rely on traction on the fundus of the gallbladder . in ms ,", "the gallbladder is often fibrosed and contracted so that fundic traction gives relatively poor exposure of the hepatobiliary triangle .", "also once the gallbladder is freed from the liver , the obliterated calot 's triangle can be more easily evaluated . the magnified view combined with modern instrumentation", "ercp may provide definitive treatment for ms ; however , the impacted stone can create real difficulties .", "more often , ercp is used to make the diagnosis and insert a stent to alleviate the jaundice and allow planning of an elective operation .", "if ercp is to be used as definitive treatment , sophisticated techniques may be needed for these cases , including the use of a  mother and baby scope  and electrohydraulic or laser lithotripsy . in the case", "described herein , i could have tried to break up the stone with an over the wire mechanical lithotripter , although engaging the stone in the basket may have been difficult .", "alternatively , a mother and baby scope and contact lithotripsy ( laser or electrohydraulic ) could have been tried but would not have been possible in my opinion because the actual cbd was not dilated and may not have taken the 5-mm baby scope .", "because i was planning to remove the gallbladder anyway , i preferred to leave the choledochal sphincter intact to avoid longer term risk of choledocholithiasis from a colonized biliary tract and papillary stenosis . in this case , i could not stent the obstruction from below but a percutaneous transhepatic approach could have been used .", "this would have been relatively straightforward as the hepatic ducts were dilated and might have been a useful strategy if this patient had been unfit for surgery .", "there is purportedly a 5 times higher rate of gallbladder malignancy in mirizzi sydrome compared with that in uncomplicated gallstone disease .", "prasad et al found 5.3% of patients with ms had gallbladder cancer compared with 1% in non - ms cases , and most were diagnosed on histology after cholecystectomy .", "if the patient is fit for surgery , the optimal management of ms should include cholecystectomy .", "the technique described leaves the choledochal sphincter intact and does not require intraoperative antegrade stenting or use of t - tubes . in ms as described herein , it would not have been easy to insert these tubes properly as the opening is into the cystic duct rather than the bile duct proper .", "the preoperative endoscopically placed transpapillary stent temporarily overcame the outflow resistance of the sphincter and allowed safe primary duct closure although periductal drainage was needed .", "in ms , even if an operation is planned , preoperative ercp and stenting should be considered because this approach should simplify and improve the safety of the operation .", "in addition , the fitness of the patient for surgery and the complexity of the pathology are important for planning the therapeutic approach . in my opinion , ms should ideally be treated by a combination of ercp and stenting with laparosocopic ", "stenting overcomes the resistance of the choledochal sphincter , and even if accurate closure of the opening in a friable and inflamed duct is not possible , it should avoid the development of a significant biliary fistula .", "careful planning combined with modern equipment should allow most cases to be managed without t - tubes , destruction of the choledochal sphincter , or open surgery ."], "abstract_text": ["<S> background : mirizzi syndrome is a rare complication of cholecystolithiasis characterized by jaundice due to compression of the common hepatic duct . the diagnosis may not be immediately apparent , and management is controversial with open surgery still recommended by some authors.method:a case is detailed herein of a 67-year - old man who presented with abdominal pain , fever , and jaundice . </S>", "<S> a dilated bile duct was found on ultrasound , but the gallbladder could not be seen . </S>", "<S> the diagnosis of mirizzi syndrome was made at ercp , and a stent was placed through the papilla . </S>", "<S> laparoscopic retrograde ( fundus first ) cholecystectomy was carried out utilizing a laparoscopic liver retractor.results:in this particular case , it was not possible at ercp to get a guidewire and stent past the obstruction . </S>", "<S> a stent was left through the papilla , below the obstruction and this allowed primary duct closure during surgery.conclusion:acute mirizzi syndrome should be suspected when a patient presents with acute cholecystitis and jaundice with dilated intrahepatic ducts on ultrasound . </S>", "<S> ercp is useful to confirm the diagnosis and allows stenting to alleviate the jaundice and facilitate the subsequent operation . </S>", "<S> laparoscopic ultrasound is useful to locate the impacted stone and to partially replicate the touch of the surgeon 's hand , which is not available in laparoscopic surgery . </S>"], "labels": null, "section_names": ["Background:", "Method:", "Results:", "Conclusion:", "INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["mirizzi syndrome is a rare complication of cholecystolithiasis characterized by jaundice due to compression of the common hepatic duct", ". the diagnosis may not be immediately apparent , and management is controversial with open surgery still recommended by some authors ."], ["a case is detailed herein of a 67-year - old man who presented with abdominal pain , fever , and jaundice .", "a dilated bile duct was found on ultrasound , but the gallbladder could not be seen .", "the diagnosis of mirizzi syndrome was made at ercp , and a stent was placed through the papilla .", "laparoscopic retrograde ( fundus first ) cholecystectomy was carried out utilizing a laparoscopic liver retractor ."], ["in this particular case , it was not possible at ercp to get a guidewire and stent past the obstruction . a stent was left through the papilla , below the obstruction and this allowed primary duct closure during surgery ."], ["acute mirizzi syndrome should be suspected when a patient presents with acute cholecystitis and jaundice with dilated intrahepatic ducts on ultrasound .", "ercp is useful to confirm the diagnosis and allows stenting to alleviate the jaundice and facilitate the subsequent operation .", "laparoscopic ultrasound is useful to locate the impacted stone and to partially replicate the touch of the surgeon 's hand , which is not available in laparoscopic surgery ."], ["mirizzi syndrome ( ms ) is biliary obstruction from compression of the common hepatic duct ( chd ) by a gallstone impacted in the cystic duct .", "mcsherry classified the syndrome into 2 groups : type i where extrinsic compression of the bile duct occurs and type ii where erosion occurs of the wall of the chd by the stone with formation of a cholecysto - choledochal fistula .", "csendes further subdivided type ii depending on the size of the fistula and the degree of destruction of the wall of the chd .", "the syndrome was initially described in 1948 by pablo luis mirizzi , professor of surgery in cordoba , argentina , and he erroneously postulated that the extrinsic pressure and inflammation induced spasm of the chd .", "precise diagnosis may be difficult initially because the condition may be confused with choledocholithiasis and cholangitis .", "the classical ultrasound findings are of a contracted gallbladder , dilated intrahepatic ducts , and a normal common bile duct ( cbd ) .", "although a rare condition , many controversial aspects still surround the definition and management of ms . to illustrate the true ms and the value of a combination of endoscopic retrograde cholangiopancreatography ( ercp ) and laparoscopic surgery , a case"], ["a 67-year - old man was admitted to the emergency department with a 3-day history of gradually worsening upper abdominal pain associated initially with vomiting and anorexia . the patient had a history of hypertension and polymyalgia rheumatica .", "he was noted to be jaundiced with a temperature of 38 celsius and right upper quadrant tenderness and guarding .", "his liver function tests were markedly deranged with bilirubin 106 mol / l [ range , 3 to 22 ] , alkaline phosphatase 159 iu / l ( range , 20 to 110 ) , and alanine transaminase 770 iu / l ( range , 5 to 65 ) .", "this showed dilated intrahepatic ducts with the cbd reported as also being dilated and measuring 9 mm in diameter .", "subsequent imaging showed that the ultrasound had actually detected a dilated chd as the cbd was not dilated . at ercp , contrast filled a nondilated cbd up to an obstruction due to a stone ( figure 1 ) .", "attempts were made to pass a guidewire beyond the obstruction up into the chd and intrahepatic ducts but without success .", "the guidewire kept going into the gallbladder , and it became apparent that there was an ms with the obstructing stone in the cystic duct causing compression of the chd ( figure 2 ) .", "after the guidewire had been manipulated for a time , some pus came out of the papilla signifying a gallbladder empyema .", "a 10 french straight plastic stent was placed through the papilla without papillotomy with its proximal tip lying below the obstruction and , as expected , no bile or intrahepatic contrast drained .", "the stone is impacted in the cystic duct , which is anterior and parallel to the common hepatic duct .", "note the narrow common bile duct and the dilatation of the common hepatic and intrahepatic ducts . a computed tomogram ( ct ) scan was done and showed a contracted gallbladder with a calcified stone causing biliary obstruction ( figure 3 ) . at laparoscopic cholecystectomy , an acutely inflamed", "a liver retractor was used to elevate the liver and maintain exposure of the bile duct ( diamond - flex , cardinal health , snowden - dencer mis products , tucker , ga , usa ) ( figure 4 ) .", "it was possible to  feel  the stone via a grasper , and its position was definitely confirmed by laparoscopic ultrasound ( figure 5 ) .", "the duct was incised to release the impacted stone with a single - use cbd blade ( espiner medical ltd .", "the cbd was examined with a 5-mm flexible choledochoscope showing that the duct was clear distally with the stent in place ( figure 7 ) .", "it was not possible to retroflex the choledochoscope to examine the common hepatic or intrahepatic ducts .", "the gallbladder was excised , the opening in the cystic duct sutured with continuous 40 vicryl ( ethicon inc . ,", "somerville , nj , usa ) and a 20 french nonsuction tube drain placed in the subhepatic space near the bile duct .", "the stent was removed at ercp 6 weeks later and a check cholangiogram showed a normal bile duct with no filling of any residual cystic duct and no stricture . during 18 months of follow - up , no delayed sequelae have occurred .", "computed tomograpic scan showing a calcified stone causing bile duct obstruction with the proximal end of the stent just below the stone .", "residual contrast from the ercp is present in the gallbladder ( somatom volume zoom , 4 slice , siemens ag , erlangen , germany ) .", "the gallbladder ( gb ) has been dissected from the liver retrograde , and the liver retractor is in place .", "the arrow points to surgicel ( ethicon , somerville , nj , usa ) in the gallbladder bed of the liver .", "laparoscopic ultrasound image showing the impacted stone ( arrow ) in a thickwalled duct ( aloka inc .", "choledochoscopy using a 5-mm scope showing a clear bile duct with the stent visible ( insert ) ."], ["ms is a rare cause of jaundice due to extrinsic compression of the chd and is present in approximately 0.35% of cholecystectomies .", "many series report much higher rates ; however , this may represent differing views on what constitutes a case of ms .", "kwon and inui recently reported a prevalence of 1.2% , but only a third of their patients were jaundiced , and despite this the majority still underwent open surgery .", "similarly , planned open operation for mirizzi syndrome is accepted and , in fact , is still advocated by many .", "many surgeons do not view conversion as detrimental and therefore do not persist laparoscopically when cholecystectomy is difficult .", "conversion or an open operation allows the use of proprioception or the touch of the surgeon 's hand and is generally accepted as a way to improve the safety of any operation , especially one in which severe inflammation is present . to replicate this ,", "however , open surgery is associated with significant short- and long - term morbidity , and a difficult operation is not necessarily easier or safer when performed open .", "a degree of tactility is possible via instruments although not by currently available robotic systems , and laparoscopic ultrasound is very useful in stone disease .", "it is generally accepted that an increased risk of bile duct injury exists during surgery for ms , and laparoscopic surgery may increase this risk .", "operative cholangiography is advocated to improve the safety of cholecystectomy , but an accurate transcystic cholangiogram will not be possible in ms . a standard technique in open surgery for the difficult laparoscopic cholecystectomy was the  fundus - first  approach .", "this can be replicated in laparoscopic surgery by the use of a liver retractor and means that exposure does not rely on traction on the fundus of the gallbladder . in ms ,", "the gallbladder is often fibrosed and contracted so that fundic traction gives relatively poor exposure of the hepatobiliary triangle .", "also once the gallbladder is freed from the liver , the obliterated calot 's triangle can be more easily evaluated . the magnified view combined with modern instrumentation", "ercp may provide definitive treatment for ms ; however , the impacted stone can create real difficulties .", "more often , ercp is used to make the diagnosis and insert a stent to alleviate the jaundice and allow planning of an elective operation .", "if ercp is to be used as definitive treatment , sophisticated techniques may be needed for these cases , including the use of a  mother and baby scope  and electrohydraulic or laser lithotripsy . in the case", "described herein , i could have tried to break up the stone with an over the wire mechanical lithotripter , although engaging the stone in the basket may have been difficult .", "alternatively , a mother and baby scope and contact lithotripsy ( laser or electrohydraulic ) could have been tried but would not have been possible in my opinion because the actual cbd was not dilated and may not have taken the 5-mm baby scope .", "because i was planning to remove the gallbladder anyway , i preferred to leave the choledochal sphincter intact to avoid longer term risk of choledocholithiasis from a colonized biliary tract and papillary stenosis . in this case , i could not stent the obstruction from below but a percutaneous transhepatic approach could have been used .", "this would have been relatively straightforward as the hepatic ducts were dilated and might have been a useful strategy if this patient had been unfit for surgery .", "there is purportedly a 5 times higher rate of gallbladder malignancy in mirizzi sydrome compared with that in uncomplicated gallstone disease .", "prasad et al found 5.3% of patients with ms had gallbladder cancer compared with 1% in non - ms cases , and most were diagnosed on histology after cholecystectomy .", "if the patient is fit for surgery , the optimal management of ms should include cholecystectomy .", "the technique described leaves the choledochal sphincter intact and does not require intraoperative antegrade stenting or use of t - tubes . in ms as described herein , it would not have been easy to insert these tubes properly as the opening is into the cystic duct rather than the bile duct proper .", "the preoperative endoscopically placed transpapillary stent temporarily overcame the outflow resistance of the sphincter and allowed safe primary duct closure although periductal drainage was needed ."], ["in ms , even if an operation is planned , preoperative ercp and stenting should be considered because this approach should simplify and improve the safety of the operation .", "in addition , the fitness of the patient for surgery and the complexity of the pathology are important for planning the therapeutic approach . in my opinion , ms should ideally be treated by a combination of ercp and stenting with laparosocopic ", "stenting overcomes the resistance of the choledochal sphincter , and even if accurate closure of the opening in a friable and inflamed duct is not possible , it should avoid the development of a significant biliary fistula .", "careful planning combined with modern equipment should allow most cases to be managed without t - tubes , destruction of the choledochal sphincter , or open surgery ."]]}
{"article_id": "PMC3857506", "article_text": ["dirofilaria immitis ( the canine heartworm ) is a filarial nematode naturally hosted by dogs , cats , foxes , muskrat , raccoons , and bears .", "adult worms live in the right heart of these animals and produce a debilitating disease .", "this filarial nematode is transmited by mosquitoes . when an infected mosquito takes blood from a human , the larvae of d. immitis invade and develop for a time in cutaneous tissues , migrate to the right heart ventricle where they eventually die .", "the fragments of worms may reach the pulmonary arteries and lodge in a small caliber vessel to cause infarction or embolism events .", "patients with pulmonary dirofilariasis are usually asymptomatic ; however , occurrence of cough , chest pain , hemoptysis , and wheezing has been described in some patients .", "eosinophilia , commonly relevant in parasite infections , are present only in 15% of patients with pulmonary dirofilariasis . usually ,", "a non - calcified , peripheral coin lesion or nodule is seen in chest x - ray or computed tomography ( ct ) .", "pathologically , spherical subpleural infarct with a central thrombosed artery containing the parasite in stage of degeneration is most commonly seen [ 4 - 6 ] . in the republic of korea ( = korea ) , the first case of human pulmonary dirofilariasis was reported in 2000 . in this report", "a 48-year - old male was referred to seoul national university hospital after a routine check - up at a local hospital due to abnormal findings seen in chest ct .", "he was an ex - smoker ( quit 10 years ago ) and was a social drinker .", "he had a pet dog , but the dog died 3 years ago from unidentified parasite infection .", "complete blood cell count showed a white blood cell count of 6.8610/l ( normal : 4.0 - 10.010/l ) , a hemoglobin concentration of 14.5 g / dl ( normal : 14 - 18 g / dl ) , and a platelet count 26710/l ( normal : 130 - 45010/l ) .", "the differential count showed neutrophils at 36.1% , lymphocytes at 51.2% , and eosinophils at 1.5% .", "the c - reactive protein level was 0.02 mg / dl ( normal : 0.5 mg / dl ) .", "serum sodium , potassium , creatinine , magnesium , calcium , and liver function test were all within normal limits .", "the spirometry was normal ; fvc 4.95l ( 105% of predicted ) , fev1 3.77l ( 107% of predicted ) , and fev1/fvc ( 76% ) .", "the chest ct showed a 1.21.0 cm sized subpleural solitary pulmonary nodule at the right lower lobe .", "although it looked as a benign inflammatory nodule such as cryptococcal infection , malignancy could not be excluded ( fig .", "on positron emission tomography ( pet ) image , the small subpleural nodule at the right lower lobe showed minimal increased fludeoxyglucose ( fdg ) uptake ( standardized uptake value [ suv ] 1.25 ) ( fig .", "at first we considered a percutaneous needle biopsy for diagnosis , but biopsy was considered not technically feasible due to the location of the nodule .", "macroscopic examination of the resected mass revealed a gray , firm , and well - demarcated tumor , measuring 1.21.00.7 cm , with slight pleural indentation and central necrosis , located in the right lower lobe of the lung .", "microscopically , chronic granulomatous inflammation which was composed of coagulation necrosis with rim of fibrous tissues and granulations was recognized . in the center of the necrotic nodules ,", "the parasite was a nematode larva and had prominent internal cuticular ridges and thick cuticle , a well - developed muscle layer , an intestinal tube , and uterine tubules .", "the diameter of the worm was approximately 240 m . based on these morphologic features ,", "after surgical resection , the patient was discharged without any complications and is being followed up in healthy conditions .", "human pulmonary dirofilariasis was first reported in 1887 , when de magalhes documented the discovery of a single male and a single female filarial worm in the left ventricle of a boy from rio de janeiro , brazil . in 1961 , dashiell reported the first case involving the lung .", "usually d. immitis invades the lungs in human cases , but reports of human dirofilariasis involving other organs like the subconjunctiva , liver , and subcutaneous sites are also available .", "another case of dirofilariasis in the liver reported in korea   was actually a misdiagnosis of hepatic capillariasis .", "the chest ct images and pathologic findings of our case were similar to the first korean dirofilariasis case .", "however , our case is the first to show additional pet imaging . in our case ,", "pet - ct was performed for differential diagnosis of malignancy , and the pet - ct showed a small subpleural nodule at the right lower lobe with minimal increased fdg uptake ( suv 1.25 ) . in the life cycle of d. immitis , the dog is the principal host , though other suitable hosts include cats , wolves , coyotes , and foxes .", "are the normal vector - intermediate host , but more than 60 species of mosquitoes in 6 genera are capable of propagating the development of d. immitis . in the usual life cycle ,", "the sexually mature female worm resides in the dog 's right ventricle and sheds thousands of microfilariae into the blood stream .", "when a mosquito bites the dog , it ingests the microfilariae . over the next 10 to 16 days ,", "human are the \" dead - end host \" since larvae can not develop into adults in humans . in our case ,", "human pulmonary dirofilariasis is usually discovered during routine health examinations because it does not cause notable symptoms .", "it is seen as a well - circumscribed , non - calcified , peripheral coin lesion or nodule .", "usually , the pulmonary lesion is a spherical nodule that upon gross examination appears as a well - circumscribed , spherical , grayish - yellow nodule 1 to 3 cm in diameter , surrounded by normal lung parenchyma . on microscopic examinations", ", there is a central core of necrosis surrounded by a granulomatous zone of tissues .", "in our case , the nodule was chronic granulomatous inflammation composed of coagulation necrosis with rim of fibrous tissues and granulations .", "dirofilaria infections can be diagnosed on the basis of worm morphology , such as the thick multilayer cuticle with lateral internal ridges , large lateral cords , and coelomyarian musculature . in cross sections , d. immitis measures 100 - 300", "m in diameter and possesses a multilayered curicle with a thickness of 10 - 25 m .", "the present case showed worm structures such as prominent internal cuticular ridges , no external cuticular ridges , thick cuticle , well - developed muscle layer , and presence of an intestinal tube .", "the diameter of the worm was 240 m , which was compatible with a d. immitis immature female worm ."], "abstract_text": ["<S> dirofilariasis is a rare disease in humans . </S>", "<S> we report here a case of a 48-year - old male who was diagnosed with pulmonary dirofilariasis in korea . on chest radiographs , </S>", "<S> a coin lesion of 1 cm in diameter was shown . </S>", "<S> although it looked like a benign inflammatory nodule , malignancy could not be excluded . </S>", "<S> so , the nodule was resected by video - assisted thoracic surgery . </S>", "<S> pathologically , chronic granulomatous inflammation composed of coagulation necrosis with rim of fibrous tissues and granulations was seen . in the center of the necrotic nodules , </S>", "<S> a degenerating parasitic organism was found . </S>", "<S> the parasite had prominent internal cuticular ridges and thick cuticle , a well - developed muscle layer , an intestinal tube , and uterine tubules . </S>", "<S> the parasite was diagnosed as an immature female worm of dirofilaria immitis . </S>", "<S> this is the second reported case of human pulmonary dirofilariasis in korea . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE RECORD", "DISCUSSION"], "sections": [["dirofilaria immitis ( the canine heartworm ) is a filarial nematode naturally hosted by dogs , cats , foxes , muskrat , raccoons , and bears .", "adult worms live in the right heart of these animals and produce a debilitating disease .", "this filarial nematode is transmited by mosquitoes . when an infected mosquito takes blood from a human , the larvae of d. immitis invade and develop for a time in cutaneous tissues , migrate to the right heart ventricle where they eventually die .", "the fragments of worms may reach the pulmonary arteries and lodge in a small caliber vessel to cause infarction or embolism events .", "patients with pulmonary dirofilariasis are usually asymptomatic ; however , occurrence of cough , chest pain , hemoptysis , and wheezing has been described in some patients .", "eosinophilia , commonly relevant in parasite infections , are present only in 15% of patients with pulmonary dirofilariasis . usually ,", "a non - calcified , peripheral coin lesion or nodule is seen in chest x - ray or computed tomography ( ct ) .", "pathologically , spherical subpleural infarct with a central thrombosed artery containing the parasite in stage of degeneration is most commonly seen [ 4 - 6 ] . in the republic of korea ( = korea ) , the first case of human pulmonary dirofilariasis was reported in 2000 . in this report"], ["a 48-year - old male was referred to seoul national university hospital after a routine check - up at a local hospital due to abnormal findings seen in chest ct .", "he was an ex - smoker ( quit 10 years ago ) and was a social drinker .", "he had a pet dog , but the dog died 3 years ago from unidentified parasite infection .", "complete blood cell count showed a white blood cell count of 6.8610/l ( normal : 4.0 - 10.010/l ) , a hemoglobin concentration of 14.5 g / dl ( normal : 14 - 18 g / dl ) , and a platelet count 26710/l ( normal : 130 - 45010/l ) .", "the differential count showed neutrophils at 36.1% , lymphocytes at 51.2% , and eosinophils at 1.5% .", "the c - reactive protein level was 0.02 mg / dl ( normal : 0.5 mg / dl ) .", "serum sodium , potassium , creatinine , magnesium , calcium , and liver function test were all within normal limits .", "the spirometry was normal ; fvc 4.95l ( 105% of predicted ) , fev1 3.77l ( 107% of predicted ) , and fev1/fvc ( 76% ) .", "the chest ct showed a 1.21.0 cm sized subpleural solitary pulmonary nodule at the right lower lobe .", "although it looked as a benign inflammatory nodule such as cryptococcal infection , malignancy could not be excluded ( fig .", "on positron emission tomography ( pet ) image , the small subpleural nodule at the right lower lobe showed minimal increased fludeoxyglucose ( fdg ) uptake ( standardized uptake value [ suv ] 1.25 ) ( fig .", "at first we considered a percutaneous needle biopsy for diagnosis , but biopsy was considered not technically feasible due to the location of the nodule .", "macroscopic examination of the resected mass revealed a gray , firm , and well - demarcated tumor , measuring 1.21.00.7 cm , with slight pleural indentation and central necrosis , located in the right lower lobe of the lung .", "microscopically , chronic granulomatous inflammation which was composed of coagulation necrosis with rim of fibrous tissues and granulations was recognized . in the center of the necrotic nodules ,", "the parasite was a nematode larva and had prominent internal cuticular ridges and thick cuticle , a well - developed muscle layer , an intestinal tube , and uterine tubules .", "the diameter of the worm was approximately 240 m . based on these morphologic features ,", "after surgical resection , the patient was discharged without any complications and is being followed up in healthy conditions ."], ["human pulmonary dirofilariasis was first reported in 1887 , when de magalhes documented the discovery of a single male and a single female filarial worm in the left ventricle of a boy from rio de janeiro , brazil . in 1961 , dashiell reported the first case involving the lung .", "usually d. immitis invades the lungs in human cases , but reports of human dirofilariasis involving other organs like the subconjunctiva , liver , and subcutaneous sites are also available .", "another case of dirofilariasis in the liver reported in korea   was actually a misdiagnosis of hepatic capillariasis .", "the chest ct images and pathologic findings of our case were similar to the first korean dirofilariasis case .", "however , our case is the first to show additional pet imaging . in our case ,", "pet - ct was performed for differential diagnosis of malignancy , and the pet - ct showed a small subpleural nodule at the right lower lobe with minimal increased fdg uptake ( suv 1.25 ) . in the life cycle of d. immitis , the dog is the principal host , though other suitable hosts include cats , wolves , coyotes , and foxes .", "are the normal vector - intermediate host , but more than 60 species of mosquitoes in 6 genera are capable of propagating the development of d. immitis . in the usual life cycle ,", "the sexually mature female worm resides in the dog 's right ventricle and sheds thousands of microfilariae into the blood stream .", "when a mosquito bites the dog , it ingests the microfilariae . over the next 10 to 16 days ,", "human are the \" dead - end host \" since larvae can not develop into adults in humans . in our case ,", "human pulmonary dirofilariasis is usually discovered during routine health examinations because it does not cause notable symptoms .", "it is seen as a well - circumscribed , non - calcified , peripheral coin lesion or nodule .", "usually , the pulmonary lesion is a spherical nodule that upon gross examination appears as a well - circumscribed , spherical , grayish - yellow nodule 1 to 3 cm in diameter , surrounded by normal lung parenchyma . on microscopic examinations", ", there is a central core of necrosis surrounded by a granulomatous zone of tissues .", "in our case , the nodule was chronic granulomatous inflammation composed of coagulation necrosis with rim of fibrous tissues and granulations .", "dirofilaria infections can be diagnosed on the basis of worm morphology , such as the thick multilayer cuticle with lateral internal ridges , large lateral cords , and coelomyarian musculature . in cross sections , d. immitis measures 100 - 300", "m in diameter and possesses a multilayered curicle with a thickness of 10 - 25 m .", "the present case showed worm structures such as prominent internal cuticular ridges , no external cuticular ridges , thick cuticle , well - developed muscle layer , and presence of an intestinal tube .", "the diameter of the worm was 240 m , which was compatible with a d. immitis immature female worm ."]]}
{"article_id": "PMC4993844", "article_text": ["the maxillary posterior area contains cancellous bones with low bone density and thin cortical bones , the quantity and quality of which are lower than those of the mandibular bone .", "this is due to the small implant - to - bone contact area and the inferior bone quality.123 moreover , the severe bone loss caused by sinus pneumatization or chronic periodontitis , which leads to a reduction in the height of the remaining residual ridge , can further increase the implant failure rate.4 in addition , the occlusal load in the posterior area is 3 - 5 times higher than that in the anterior area.5 to address these problems , various bone graft techniques to augment the insufficient bone volume and thereby ensure the success of maxillary posterior implants have been proposed.6 such surgical methods have been accepted as clinically meaningful procedures .", "however , these procedures have a number of disadvantages , such as the invasiveness of the surgery , high cost , and long treatment duration after bone graft and implant installation.6 in addition , because bone resorption occurs after a bone graft , the increase in vertical height is unpredictable", ". microscopic studies by wallace et al.7 suggested that it was difficult to obtain bones with optimal strength from a bone graft .", "rosen et al.8 asserted that the success rate of the implant placement after the bone graft depended on the residual ridge height .", "this was supported by aka et al.9 who showed that stress applied on the implant was concentrated on the upper part of the implant and that , hence , the stress was not transferred to the grafted bone .", "given the above facts and the disadvantages , success rate of bone grafting in a severely atrophied maxilla is not very promising .", "studies have reported that this method may allow new bone formation without the need for bone graft.1011 however , winter et al.12 stated that the amount of new bone generated by sinus augmentation alone was insufficient and the results were not satisfactory considering the complexity of the technique .", "this approach offers many advantages , which include a shorter treatment time , less cost , fewer complications in patients , and minimal surgical invasiveness . since the introduction of short implant for installation in atrophied residual ridge,13 many previous studies have defined a short implant as measuring 7 mm or less.141516 although short implants have been reported to have a lower survival rate than standard implants due to the small bone - contact area,1415 their survival rates have increased gradually with advancements in surface treatment technologies.16 as a finite element analysis ( fea ) shows that stress transfer at the implant - bone interface is limited to the upper 2 - 3 mm , the long implant is thought to be biomechanically unnecessary.917 therefore , a short implant may be used for achieving primary implant stability in the case of unfavorable bone quantity and may be used as an alternative for surgical approaches such as bone graft or sinus augmentation .", "replacement of a missing molar with a single implant may cause various mechanical failures such as screw loosening , screw fracture , and implant fracture.1819 in a severely atrophied maxillary posterior area , using a single wide implant or splinting multiple implants is a way to overcome the unfavorable crown / root ratio of short implants.202122 two - implant - supported maxillary molars with implants installed in buccal and palatal positions were suggested by balshi and wolfinger.23 the 2 short implants ( 2si , fig .", "1 ) technique can be used to reconstruct a multi - rooted molar by installing 2 short narrow implants without the need for an additional surgical approach .", "the 2si approach is a minimally invasive procedure that solely utilizes residual bones to overcome unfavorable bone quantity and quality in the maxillary posterior area .", "it can yield stable treatment results while minimizing the need for additional procedures and reducing the cost and treatment time .", "a 5-year clinical study demonstrated the clinical success of 2sis.24 the purpose of this study was to compare the stress distribution of various types of 2sis that were installed and restored in severely atrophied maxillary molar sites .", "an internal connection implant with an 11-degree taper interface was used ( gs , osstem , seoul , korea ) . internal connection implants ( length , 7.0 mm ) with narrow platform ( np  3.5 mm ) , regular platform ( rp  4.0 mm ) , and wide platform ( wp  5.0 mm ) were modeled .", "three - dimensional image data of the human maxillary first molar from ct images were transformed into fea meshes and the connecting area between the crown and implant was smoothened .", "a gold crown on the abutment ( rigid , osstem , seoul , korea ) was connected with the implant .", "abutment for each implant diameter was connected , and the occlusal surface area of the definitive prosthesis was constructed to have the same shape . an implant was designed to be installed on the missing maxillary first molar area on the maxilla with a pneumatized sinus , 0.5 mm cortical bone on top and bottom , and 3 mm cancellous bone ( fig .", "2 ) . for the boundary conditions , bottom and mesio - distal parts of the maxillary bone", "the mechanical properties of the bone , gold , and titanium were set as previously described ( table 1).2526 the implant  bone interface was constructed with 100% contact for complete stress transfer to the surrounding bone .", "they were broadly categorized into 3 groups of 2si and 4 groups of single implants .", "the 2si groups were categorized into 1 ) np oblique ( two narrow implants were obliquely installed ) , 2 ) np vertical ( two narrow implants were installed in palatal residual bone due to the resorption of buccal bone ) , and 3 ) np horizontal ( two narrow implants were installed in distal residual bone due to the mesial bone resorption ) .", "single implant installation groups were set to 1 ) rp - cantilever ( a rp implant was installed in stepped bone due to resorption of the mesial bone ) , 2 ) wp - cantilever ( a wp implant was installed in stepped bone due to resorption of the mesial bone ) , 3 ) rp - center ( a rp implant was installed in a flat bone ) , and 4 ) wp - center ( a wp implant was installed in a flat bone ) . meshing and pre - processing were performed using hyper - mesh 10.0 ( altair engineering inc . , troy , mi , usa ) and visual crash for pam version 10.5 ( esi group , paris , france ) .", "the average occlusal force , a 250 n static axial load , was applied to the center of the occlusal surface ( axial loading ) . to simulate an oblique loading condition , a 250 n", "oblique load with a 45 angulation from the implant axis was applied on the center of the buccal cusp ( oblique loading ) .", "the von mises stress was measured in the interface of implant - bone , implant / abutment complex , and at the bone of the peri - implant area in all models . to assess the stress distribution", "the processing and post - processing procedures were performed using pam - medysa v2014 ( esi group , paris , france ) and hyper - view v10.0 ( altair engineering inc . , troy , mi , usa ) .", "analyses of stress distribution in the implant - bone interface of an implant in the buccal side ( np-1 ) showed an evenly distributed stress pattern in the np - horizontal and np - oblique groups , whereas the np - vertical group exhibited a relatively high stress pattern in the top and center areas of the interface ( fig .", "similar stress distribution was observed in the np - horizontal and np - oblique groups , while a different stress pattern was observed in the np - vertical group ( fig .", "stress distribution at the bone of the peri - implant area showed a different pattern to that observed in the interface .", "the np - horizontal and np - vertical groups showed a large stress concentration in stepped areas between different bone levels , while an even stress distribution was observed in the np - oblique group with flat bone level ( fig .", "few differences in stress distribution of the implant / abutment complex were observed in each group and all stress was concentrated on the interface between the abutment and inner surface of implant ( fig .", "4d ) . when oblique loading was applied , similar stress distribution was observed at the bone - implant interface of the buccal ( np-1 ) and palatal ( np-2 ) implants in the np - horizontal and np - oblique groups ( fig .", "although the np - vertical group showed slightly higher stress concentration , the difference was not as evident as with axial loading ( fig .", "an evenly distributed stress pattern at the peri - implant bone was noticed in the np - oblique group compared with the np - vertical or np - horizontal groups ( fig .", "the stress distribution of the implant / abutment complex showed virtually no difference in each group , and the amount and location of stress concentrations were similar to those in axial loading ( fig .", "when axial loading was applied , the wp implant showed lower and evenly distributed stress at the implant - bone interface than the rp implant ( fig .", "6a ) . however , the stress difference was not large between the center and cantilever positions .", "as for the stress on the peri - implant bone , a more even stress distribution was observed in the center position implant than in the cantilever position implant .", "lower stress was observed in the wp implant than in the rp implant ( fig .", "stress patterns at the implant / abutment complex of single implants were similar to that of the 2si groups ( fig .", "when oblique loading was applied , the largest von mises stress was observed in the rp implant .", "the wp implant showed a relatively even stress distribution pattern and had a similar level of von mises stress to that of 2si groups ( fig .", "the stress distribution on the peri - implant bones was similar to that in axial loading but the level of stress was higher .", "stress exerted on the implant / abutment complex was very large ; a high amount of stress was placed on the abutment and implant in both rp and wp implants .", "additionally , larger and wider stress distribution was observed in the implant / abutment contact area on the opposite side of the loading point ( fig .", "the stress distribution at the implant - bone interface was compared by categorizing the bone levels into two ( flat and stepped ) .", "when axial loading was applied , higher von mises stress was observed on the implant - bone interface in the rp implant than in the wp implant or 2si implants .", "the highest stress was concentrated on the region with discontinuity between the different bone levels ( stepped area ) ( fig .", "the 2si groups showed the lowest von mises stress compared with the rp and wp implants .", "the area between the implants showed lower stress distribution than the outer side of the implant in 2si group .", "the 2sis showed the most even stress distribution and rp implant showed the largest stress pattern on the peri - implant bones ( fig .", "when applying oblique loading , the largest stress pattern was noted at the buccal side .", "higher stress was observed in the rp implant than in the wp implant or 2 np implants ( fig .", "stress distribution of the peri - implant bones was relatively small and even in the 2si groups .", "a high level of stress was concentrated in the stepped area of the bone ( fig .", "differences in stress distribution based on implant diameter , number , or position could be observed , but no differences related to types of bone levels were observed ( flat bone vs. stepped bone ) ( fig .", "to identify the biomechanical effectiveness of 2sis , various bone levels and bone morphologies were assumed and implants were positioned on the basis of the bone condition .", "the residual maxillary bone bed was designed with a total length of 4.0 mm and 0.5 mm of cortical bone on the top and bottom for bicortical engagement .", "hence , when load was applied on the implant prosthesis , a large stress concentration was observed in certain regions on the bottom cortical bone located on the maxillary sinus side , unlike the result in another fea study.9 similarly , in clinical situations , stress may be concentrated on the bottom cortical bone as well as on the top portion in the case of severely atrophied bone . in this study ,", "the von mises stress distribution on the bone away from the implant - bone interface was also considered .", "this is because it was difficult to estimate the effect of stress distribution solely based on the stress pattern at the localized implant - bone interface in case of a severely atrophied bone .", "in addition , stress on the implant - abutment complex was analyzed to estimate the risk of fracture in a narrow implant .", "the single rp implant group showed the highest level of stress at the implant - bone interface .", "as the diameter of implant used for restoring the maxillary molar is larger in wp , stress is more evenly distributed and general stress amount ( in mpa ) tends to be lower .", "however , two np implants exhibited more favorable stress distribution than a single wide implant ; this confirms the biomechanical advantage of 2sis . in the 2si groups , the level of stress was not much different between palatal and buccal positioned implants .", "meanwhile , np - vertical , which is a buccal - only or palatal - only positioned condition , exhibited a higher level of stress in the form of a bending moment when axial loading was applied .", "however , under oblique loading , a similar level of stress was exhibited by the three implant positions ( np - oblique , np - horizontal , and np - vertical ) .", "these trends arose because oblique loading induced lopsided loading on the buccal or palatal sides .", "therefore , the bending moment could be generated in a variously positioned implant , whose diameter is smaller than the roots of a natural tooth .", "the stress distributions in the center and cantilever positions of the rp and wp implants did not show a large difference .", "it may be because adjacent teeth were not modeled and both axial and oblique loading were applied on the mesio - distal center .", "these results are consistent with previous studies , which showed that the buccal cantilever was the most unfavorable and the mesio - distal cantilever was unfavorable only in terms of oral hygiene.2728 stress distribution of the peri - implant bone showed similar trends , where von mises stress was lower in implants of 2si groups than in rp or wp implants .", "this may be explained by the contact area between the implant and bone tissue , as placing multiple np implants is an effective way to increase the contact area .", "since installation of a wp implant is difficult in atrophied bones , 2sis can provide better initial osseointegration and its subsequent maintenance with effective stress distribution .", "stress on the implant / abutment complex was lower than that in the implant - bone interface or peri - implant bones .", "the stress was concentrated on the contacting surface of abutment and the inner part of implant when oblique loading was applied on rp or wp implants .", "clinically , fractures are common in the geometric discontinuity between the upper conical part and the lower hex part of the abutment in internal conical connection implants.29 moreover , multiple implants can be advantageous since single molar implants exhibit occasional mechanical failures such as screw loosening , screw fracture , and implant fracture,28 and the oblique loading generated even in physiologic mastication can be detrimental to single maxillary implants . in a previous clinical study on 2sis ,", "none of the np implants using the 2si method was fractured.24 the trend of the presence of higher von mises stress around rp implant than around wp or np implants did not differ by the bone levels .", "prominently , the highest level of stress was exhibited on the stepped area of the bones .", "this can be viewed as a limitation of the fea study ; stress concentration on the discontinuous area of features is unavoidable in the interpretation of the fea . in clinical situations ,", "however , these phenomena are not likely to occur as bone loss occurs continuously . in preliminary modeling ,", "however , real bone images can distort the result when comparing the effect of implant diameter and number .", "therefore , over - interpretation of the importance of stress concentration at the discontinuous region should be avoided .", "as an alternative to bone graft on a severely atrophied maxillary posterior area , single wide implant or splinting of multiple implants can be performed.2123 the results from the this study showed that , multiple implants , even with a short length and narrow diameter , offer a favorable implant - bone interface , peri - implant bone , and implant - abutment complex .", "the use of 2sis , which place np implants in the most dense bone area in the buccal and palatal sides and utilize residual bones per se without surgical difficulties , is a procedure allowing enhanced osseointegration and favorable stress distribution .", "however , 2si could not be used in the case with narrow mesio - distal space .", "the results from the fea study can be the consequence of restricted stress transfer within the 2 - 3 mm of the top of the bone.9 further , as adjacent teeth were not predetermined , the replication was not identical to the actual situation .", "moreover , clinical efficacy can not be validated solely by a biomechanical study , as only a relative comparison is possible . however , this study is important in that it supports the findings of the clinical study that showed favorable clinical success rates of 180 2sis.24", "the stress distributions based on various bone levels and implant installation conditions in a severely atrophied maxillary posterior area were analyzed .", "when axial loading or oblique loading was applied , the highest level of stress was observed on the implant - bone interface and peri - implant bones in a single rp implant and the lowest stress distribution was exhibited in 2si conditions .", "the highest level of stress was concentrated on the implant / abutment - contacting surface of the single wide implant and the lowest level of stress was on the implant / abutment complex of 2sis ."], "abstract_text": ["<S> purposethe aim of this study was to investigate the stress distribution of 2-short implants ( 2sis ) installed in a severely atrophic maxillary molar site.materials and methodsthree different diameters of internal connection implants were modeled : narrow platform ( np ) , regular platform ( rp ) , and wide platform ( wp ) . </S>", "<S> the maxillary first molars were restored with one implant or two short implants . </S>", "<S> three 2si models ( np - oblique , np - vertical , and np - horizontal ) and four single implant models ( rp and wp in a centered or cantilevered position ) were used . </S>", "<S> axial and oblique loadings were applied on the occlusal surface of the crown . </S>", "<S> the von mises stress values were measured at the bone - implant , peri - implant bone , and implant / abutment complex.resultsthe highest stress distribution at the bone - implant interface and the peri - implant bone was noticed in the rp group , and the lowest stress distribution was observed in the 2si groups . </S>", "<S> cantilevered position showed unfavorable stress distribution with axial loading . </S>", "<S> 2si types did not affect the stress distribution in oblique loading . </S>", "<S> the number and installation positions of the implant , rather than the bone level , influenced the stress distribution of 2sis . </S>", "<S> the implant / abutment complex of wp presented the highest stress concentration while that of 2sis showed the lowest stress concentration.conclusion2sis may be useful for achieving stable stress distribution on the surrounding bone and implant - abutment complex in the atrophic posterior maxilla . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["the maxillary posterior area contains cancellous bones with low bone density and thin cortical bones , the quantity and quality of which are lower than those of the mandibular bone .", "this is due to the small implant - to - bone contact area and the inferior bone quality.123 moreover , the severe bone loss caused by sinus pneumatization or chronic periodontitis , which leads to a reduction in the height of the remaining residual ridge , can further increase the implant failure rate.4 in addition , the occlusal load in the posterior area is 3 - 5 times higher than that in the anterior area.5 to address these problems , various bone graft techniques to augment the insufficient bone volume and thereby ensure the success of maxillary posterior implants have been proposed.6 such surgical methods have been accepted as clinically meaningful procedures .", "however , these procedures have a number of disadvantages , such as the invasiveness of the surgery , high cost , and long treatment duration after bone graft and implant installation.6 in addition , because bone resorption occurs after a bone graft , the increase in vertical height is unpredictable", ". microscopic studies by wallace et al.7 suggested that it was difficult to obtain bones with optimal strength from a bone graft .", "rosen et al.8 asserted that the success rate of the implant placement after the bone graft depended on the residual ridge height .", "this was supported by aka et al.9 who showed that stress applied on the implant was concentrated on the upper part of the implant and that , hence , the stress was not transferred to the grafted bone .", "given the above facts and the disadvantages , success rate of bone grafting in a severely atrophied maxilla is not very promising .", "studies have reported that this method may allow new bone formation without the need for bone graft.1011 however , winter et al.12 stated that the amount of new bone generated by sinus augmentation alone was insufficient and the results were not satisfactory considering the complexity of the technique .", "this approach offers many advantages , which include a shorter treatment time , less cost , fewer complications in patients , and minimal surgical invasiveness . since the introduction of short implant for installation in atrophied residual ridge,13 many previous studies have defined a short implant as measuring 7 mm or less.141516 although short implants have been reported to have a lower survival rate than standard implants due to the small bone - contact area,1415 their survival rates have increased gradually with advancements in surface treatment technologies.16 as a finite element analysis ( fea ) shows that stress transfer at the implant - bone interface is limited to the upper 2 - 3 mm , the long implant is thought to be biomechanically unnecessary.917 therefore , a short implant may be used for achieving primary implant stability in the case of unfavorable bone quantity and may be used as an alternative for surgical approaches such as bone graft or sinus augmentation .", "replacement of a missing molar with a single implant may cause various mechanical failures such as screw loosening , screw fracture , and implant fracture.1819 in a severely atrophied maxillary posterior area , using a single wide implant or splinting multiple implants is a way to overcome the unfavorable crown / root ratio of short implants.202122 two - implant - supported maxillary molars with implants installed in buccal and palatal positions were suggested by balshi and wolfinger.23 the 2 short implants ( 2si , fig .", "1 ) technique can be used to reconstruct a multi - rooted molar by installing 2 short narrow implants without the need for an additional surgical approach .", "the 2si approach is a minimally invasive procedure that solely utilizes residual bones to overcome unfavorable bone quantity and quality in the maxillary posterior area .", "it can yield stable treatment results while minimizing the need for additional procedures and reducing the cost and treatment time .", "a 5-year clinical study demonstrated the clinical success of 2sis.24 the purpose of this study was to compare the stress distribution of various types of 2sis that were installed and restored in severely atrophied maxillary molar sites ."], ["an internal connection implant with an 11-degree taper interface was used ( gs , osstem , seoul , korea ) . internal connection implants ( length , 7.0 mm ) with narrow platform ( np  3.5 mm ) , regular platform ( rp  4.0 mm ) , and wide platform ( wp  5.0 mm ) were modeled .", "three - dimensional image data of the human maxillary first molar from ct images were transformed into fea meshes and the connecting area between the crown and implant was smoothened .", "a gold crown on the abutment ( rigid , osstem , seoul , korea ) was connected with the implant .", "abutment for each implant diameter was connected , and the occlusal surface area of the definitive prosthesis was constructed to have the same shape . an implant was designed to be installed on the missing maxillary first molar area on the maxilla with a pneumatized sinus , 0.5 mm cortical bone on top and bottom , and 3 mm cancellous bone ( fig .", "2 ) . for the boundary conditions , bottom and mesio - distal parts of the maxillary bone", "the mechanical properties of the bone , gold , and titanium were set as previously described ( table 1).2526 the implant  bone interface was constructed with 100% contact for complete stress transfer to the surrounding bone .", "they were broadly categorized into 3 groups of 2si and 4 groups of single implants .", "the 2si groups were categorized into 1 ) np oblique ( two narrow implants were obliquely installed ) , 2 ) np vertical ( two narrow implants were installed in palatal residual bone due to the resorption of buccal bone ) , and 3 ) np horizontal ( two narrow implants were installed in distal residual bone due to the mesial bone resorption ) .", "single implant installation groups were set to 1 ) rp - cantilever ( a rp implant was installed in stepped bone due to resorption of the mesial bone ) , 2 ) wp - cantilever ( a wp implant was installed in stepped bone due to resorption of the mesial bone ) , 3 ) rp - center ( a rp implant was installed in a flat bone ) , and 4 ) wp - center ( a wp implant was installed in a flat bone ) . meshing and pre - processing were performed using hyper - mesh 10.0 ( altair engineering inc . , troy , mi , usa ) and visual crash for pam version 10.5 ( esi group , paris , france ) .", "the average occlusal force , a 250 n static axial load , was applied to the center of the occlusal surface ( axial loading ) . to simulate an oblique loading condition , a 250 n", "oblique load with a 45 angulation from the implant axis was applied on the center of the buccal cusp ( oblique loading ) .", "the von mises stress was measured in the interface of implant - bone , implant / abutment complex , and at the bone of the peri - implant area in all models . to assess the stress distribution", "the processing and post - processing procedures were performed using pam - medysa v2014 ( esi group , paris , france ) and hyper - view v10.0 ( altair engineering inc . , troy , mi , usa ) ."], ["analyses of stress distribution in the implant - bone interface of an implant in the buccal side ( np-1 ) showed an evenly distributed stress pattern in the np - horizontal and np - oblique groups , whereas the np - vertical group exhibited a relatively high stress pattern in the top and center areas of the interface ( fig .", "similar stress distribution was observed in the np - horizontal and np - oblique groups , while a different stress pattern was observed in the np - vertical group ( fig .", "stress distribution at the bone of the peri - implant area showed a different pattern to that observed in the interface .", "the np - horizontal and np - vertical groups showed a large stress concentration in stepped areas between different bone levels , while an even stress distribution was observed in the np - oblique group with flat bone level ( fig .", "few differences in stress distribution of the implant / abutment complex were observed in each group and all stress was concentrated on the interface between the abutment and inner surface of implant ( fig .", "4d ) . when oblique loading was applied , similar stress distribution was observed at the bone - implant interface of the buccal ( np-1 ) and palatal ( np-2 ) implants in the np - horizontal and np - oblique groups ( fig .", "although the np - vertical group showed slightly higher stress concentration , the difference was not as evident as with axial loading ( fig .", "an evenly distributed stress pattern at the peri - implant bone was noticed in the np - oblique group compared with the np - vertical or np - horizontal groups ( fig .", "the stress distribution of the implant / abutment complex showed virtually no difference in each group , and the amount and location of stress concentrations were similar to those in axial loading ( fig .", "when axial loading was applied , the wp implant showed lower and evenly distributed stress at the implant - bone interface than the rp implant ( fig .", "6a ) . however , the stress difference was not large between the center and cantilever positions .", "as for the stress on the peri - implant bone , a more even stress distribution was observed in the center position implant than in the cantilever position implant .", "lower stress was observed in the wp implant than in the rp implant ( fig .", "stress patterns at the implant / abutment complex of single implants were similar to that of the 2si groups ( fig .", "when oblique loading was applied , the largest von mises stress was observed in the rp implant .", "the wp implant showed a relatively even stress distribution pattern and had a similar level of von mises stress to that of 2si groups ( fig .", "the stress distribution on the peri - implant bones was similar to that in axial loading but the level of stress was higher .", "stress exerted on the implant / abutment complex was very large ; a high amount of stress was placed on the abutment and implant in both rp and wp implants .", "additionally , larger and wider stress distribution was observed in the implant / abutment contact area on the opposite side of the loading point ( fig .", "the stress distribution at the implant - bone interface was compared by categorizing the bone levels into two ( flat and stepped ) .", "when axial loading was applied , higher von mises stress was observed on the implant - bone interface in the rp implant than in the wp implant or 2si implants .", "the highest stress was concentrated on the region with discontinuity between the different bone levels ( stepped area ) ( fig .", "the 2si groups showed the lowest von mises stress compared with the rp and wp implants .", "the area between the implants showed lower stress distribution than the outer side of the implant in 2si group .", "the 2sis showed the most even stress distribution and rp implant showed the largest stress pattern on the peri - implant bones ( fig .", "when applying oblique loading , the largest stress pattern was noted at the buccal side .", "higher stress was observed in the rp implant than in the wp implant or 2 np implants ( fig .", "stress distribution of the peri - implant bones was relatively small and even in the 2si groups .", "a high level of stress was concentrated in the stepped area of the bone ( fig .", "differences in stress distribution based on implant diameter , number , or position could be observed , but no differences related to types of bone levels were observed ( flat bone vs. stepped bone ) ( fig ."], ["to identify the biomechanical effectiveness of 2sis , various bone levels and bone morphologies were assumed and implants were positioned on the basis of the bone condition .", "the residual maxillary bone bed was designed with a total length of 4.0 mm and 0.5 mm of cortical bone on the top and bottom for bicortical engagement .", "hence , when load was applied on the implant prosthesis , a large stress concentration was observed in certain regions on the bottom cortical bone located on the maxillary sinus side , unlike the result in another fea study.9 similarly , in clinical situations , stress may be concentrated on the bottom cortical bone as well as on the top portion in the case of severely atrophied bone . in this study ,", "the von mises stress distribution on the bone away from the implant - bone interface was also considered .", "this is because it was difficult to estimate the effect of stress distribution solely based on the stress pattern at the localized implant - bone interface in case of a severely atrophied bone .", "in addition , stress on the implant - abutment complex was analyzed to estimate the risk of fracture in a narrow implant .", "the single rp implant group showed the highest level of stress at the implant - bone interface .", "as the diameter of implant used for restoring the maxillary molar is larger in wp , stress is more evenly distributed and general stress amount ( in mpa ) tends to be lower .", "however , two np implants exhibited more favorable stress distribution than a single wide implant ; this confirms the biomechanical advantage of 2sis . in the 2si groups , the level of stress was not much different between palatal and buccal positioned implants .", "meanwhile , np - vertical , which is a buccal - only or palatal - only positioned condition , exhibited a higher level of stress in the form of a bending moment when axial loading was applied .", "however , under oblique loading , a similar level of stress was exhibited by the three implant positions ( np - oblique , np - horizontal , and np - vertical ) .", "these trends arose because oblique loading induced lopsided loading on the buccal or palatal sides .", "therefore , the bending moment could be generated in a variously positioned implant , whose diameter is smaller than the roots of a natural tooth .", "the stress distributions in the center and cantilever positions of the rp and wp implants did not show a large difference .", "it may be because adjacent teeth were not modeled and both axial and oblique loading were applied on the mesio - distal center .", "these results are consistent with previous studies , which showed that the buccal cantilever was the most unfavorable and the mesio - distal cantilever was unfavorable only in terms of oral hygiene.2728 stress distribution of the peri - implant bone showed similar trends , where von mises stress was lower in implants of 2si groups than in rp or wp implants .", "this may be explained by the contact area between the implant and bone tissue , as placing multiple np implants is an effective way to increase the contact area .", "since installation of a wp implant is difficult in atrophied bones , 2sis can provide better initial osseointegration and its subsequent maintenance with effective stress distribution .", "stress on the implant / abutment complex was lower than that in the implant - bone interface or peri - implant bones .", "the stress was concentrated on the contacting surface of abutment and the inner part of implant when oblique loading was applied on rp or wp implants .", "clinically , fractures are common in the geometric discontinuity between the upper conical part and the lower hex part of the abutment in internal conical connection implants.29 moreover , multiple implants can be advantageous since single molar implants exhibit occasional mechanical failures such as screw loosening , screw fracture , and implant fracture,28 and the oblique loading generated even in physiologic mastication can be detrimental to single maxillary implants . in a previous clinical study on 2sis ,", "none of the np implants using the 2si method was fractured.24 the trend of the presence of higher von mises stress around rp implant than around wp or np implants did not differ by the bone levels .", "prominently , the highest level of stress was exhibited on the stepped area of the bones .", "this can be viewed as a limitation of the fea study ; stress concentration on the discontinuous area of features is unavoidable in the interpretation of the fea . in clinical situations ,", "however , these phenomena are not likely to occur as bone loss occurs continuously . in preliminary modeling ,", "however , real bone images can distort the result when comparing the effect of implant diameter and number .", "therefore , over - interpretation of the importance of stress concentration at the discontinuous region should be avoided .", "as an alternative to bone graft on a severely atrophied maxillary posterior area , single wide implant or splinting of multiple implants can be performed.2123 the results from the this study showed that , multiple implants , even with a short length and narrow diameter , offer a favorable implant - bone interface , peri - implant bone , and implant - abutment complex .", "the use of 2sis , which place np implants in the most dense bone area in the buccal and palatal sides and utilize residual bones per se without surgical difficulties , is a procedure allowing enhanced osseointegration and favorable stress distribution .", "however , 2si could not be used in the case with narrow mesio - distal space .", "the results from the fea study can be the consequence of restricted stress transfer within the 2 - 3 mm of the top of the bone.9 further , as adjacent teeth were not predetermined , the replication was not identical to the actual situation .", "moreover , clinical efficacy can not be validated solely by a biomechanical study , as only a relative comparison is possible . however , this study is important in that it supports the findings of the clinical study that showed favorable clinical success rates of 180 2sis.24"], ["the stress distributions based on various bone levels and implant installation conditions in a severely atrophied maxillary posterior area were analyzed .", "when axial loading or oblique loading was applied , the highest level of stress was observed on the implant - bone interface and peri - implant bones in a single rp implant and the lowest stress distribution was exhibited in 2si conditions .", "the highest level of stress was concentrated on the implant / abutment - contacting surface of the single wide implant and the lowest level of stress was on the implant / abutment complex of 2sis ."]]}
{"article_id": "PMC3770877", "article_text": ["hydatid disease ( echionococcosis ; hydatidosis ) is a parasitic disease , endemic in many sheep raising countries ( australia , south america , mediterranean countries , russia , middle east , and central asia ) .", "adult cestodes of echinococcus granulosus inhabit the small intestine of a definitive host ( dogs , foxes , and wolves ) and produce infective eggs . through the feces , either cestode segments or free eggs", "these are ungulates ( sheep , goats , pigs , and horses ) but in some cases accidental ingestion of eggs , usually shed by dogs , could lead to human cystic echinococcosis ( ce ) .", "contaminated food , water , or hand - to - mouth transmission are usual modes of infection in humans [ 2 - 4 ] . so far ,", "once ingested by the intermediate host , in its intestinal tract , parasite eggs transform into the oncosphere .", "oncospheres penetrate the intestinal wall , enters the portal vein , and reaches the liver where they usually are trapped and become the hydatid cyst .", "liver involvement has been described as the most common location of ce , varying from 59% to 78% of all cases [ 1,5 - 8 ] .", "some larvae pass the liver and reach the lungs where they develop into pulmonary hydatidosis .", "lung involvement is present in 15 - 27% of all cases [ 1,5 - 8 ] .", "not so often , larvae may pass these filters and form cysts in the brain , skeletal muscles , bones , kidneys , spleen , or other tissues .", "theoretically , they can occur at any site except the teeth , nails , and hairs .", "epidemiological studies have been conducted in some countries , while in serbia , montenegro , bih , and albania no information is available about the precise incidence in humans .", "the aim of this retrospective study was to investigate the skeletal manifestations of hydatid disease in serbia , it 's demographic distribution , site involvement , complications , and bone tissue comorbidities .", "the study was conducted by reviewing the medical database of institute for pathology ( faculty of medicine in belgrade ) which is a reference institution in serbia for bone pathology .", "materials in the study comprised all the cases with histologically verified bone hydatidosis during the period between 1971 and 2010 in the territory of serbia .", "data obtained from medical records included the age at the moment of diagnosis , sex , localization of hydatid cyst , copresence of hydatid cyst in localizations other than the bone tissue , presence of skeletal complications , bone comorbidities , and diagnosis based on radiographies . in order to standardize the data ,", "the radiological assessment was made on standard radiography , having in mind the length of the study period and diagnostic procedures available for that time .", "changes in the bone structure were classified as tumefaction ( sharply or unshapely demarcated area of altered bone structure ) , cysts ( sharply demarcated lytic bone lesion with geographical borders ) , or fractures ( in cases where fracture line occured ) . in some patients , altered bone structure was able to be classified as cyst or tumefaction preceding the pathological fracture , while in other cases , no alteration of the bone structure was radiologically visible aside of the fracture line .", "the age of the patients at the time of diagnosis was from 7 to 77 years .", "the mean age of all patients was 40.918.8 years . in order to explore the age distribution of ce ,", "we grouped patients into 5 age categories : i ( 1 - 18 years ) , ii ( 19 - 36 years ) , iii ( 37 - 54 years ) , iv ( 55 - 73 years ) , and v ( > 73 years ) . in group", "the mean age of adult patients ( more than 18 years old ) was 44.716.6 years .", "a total of 24 male patients presented 58.5% of all cases , while 17 female patients presented 41.5% .", "the spine was the most commonly involved site with the predominance of the thoracic segment .", "analysis of standard radiographies revealed that fracture was by far the predominant radiological diagnosis , followed by tumefaction and cyst ( table 2 ) .", "pain was the symptom in 41.5% of patients , while some patients demonstrated complications such as paraplegia ( 22.0% ) , pathologic fracture ( 48.8% ) , and scoliosis ( 9.8% ) .", "the pathological fracture was most frequently found in the spine ( 75.0% ) followed by the femur ( 20.0% ) and tibia ( 5.0% ) . however", "in addition to the bone involvement , 2 patients demonstrated extraskeletal hydatid cysts : 1 in the liver and 1 in the lungs .", "bone tissue comorbidities refer to bone tuberculosis in a patient with spinal hydatid disease ( 2.4% ) and to presence of fistula detected in 2 patients ( 4.9% ) , 1 with spinal and 1 with pelvic hydatidosis .", "serbia is recognized as an endemic country for ce , but so far , no epidemiological study has been conducted . since echionococcosis is subjected to an obligatory disease for reporting , the data on the number of reported patients were available through the annual report on infectious diseases on the territory of republic of serbia published by serbian institute for public health , serbia . in the study period , a total of 727 patients with ce", "the frequency was relatively high compared to the reports from other endemic countries , being 0.5 - 4.0% .", "this frequency can be explained at least partially by a larger total number of patients   due to careless reporting of echionococcosis by physicians , which was even noted in the official annual reports from 1978 to 1989 of serbian institute for public health , serbia .", "studies in other endemic countries showed that the prevalence of ce increases with age , resulting that the most of the patients belong to the adult population .", "the mean age at the moment of diagnosis , including all localizations , was from 33 to 52 years [ 13 - 18 ] . in our study ,", "the mean age of the patients with skeletal manifestation of ce was 40.918.8 years , but among the adults the mean age was 44.716.6 .", "higher frequency in aged people could be due to the fact that older people have more chances to be infected with e. granulosus during life , and also , the clinical signs of ce are more probable to appear later in life , as the disease progresses . as it was reported in previous studies [ 15,19 - 21 ] , no significant difference between sex was found . in our study ,", "12.2% of patients were under 18 years , with the mean age being 13.04.2 years .", "it was consistent with the results of a previous report on the frequency of echinococcosis among children in serbia .", "although boys to girls ratio was 1.5:1.0 , no significant difference in the positive rate between sex was found . some other studies reported slight predominance of boys , which could probably be explained by their outdoor playing , closer contact with animals , and low level of personal hygiene , compared to girls .", "symptoms associated with bone ce are not specific and often missing , making the diagnosis difficult and sometimes overlooked .", "our results showed that 19.5% of patients did not report any symptoms and did not develop any complications .", "these asymptomatic patients are not surprising in consideration that hydatid disease is anywhere in the body and often discovered as an incidental finding .", "radiological diagnosis of osseous hydatid disease is also difficult because of non - specific findings .", "radiography usually reveals single or multiple osteolytic lesions and in some cases shows cortical thinning , osteosclerosis ( fig .", "intraosseous foci of hydatid disease predominate in spongious bone tissues and consist of small , separate , and thin - walled cysts .", "while these cysts expand , they destroy the surrounding trabeculae and can reach a considerable size . due to cyst enlargement and consequent cortical thinning", ", pathological fractures may occur . in the case of connective tissue proliferation and bone sclerosis ,", "presentation of ce can be unclear , consisting of mixed radiological signs   ( table 2 ) .", "therefore , preoperative diagnosis of skeletal hydatid disease is difficult , and conclusive diagnosis is often made intraoperatively and by histological verification [ 26 - 28 ] .", "histological examination of the cyst wall usually reveals an outer chitinous ( or fibrous laminar ) and an inner germinal layer , surrounded by either granulation tissue or fibrous capsule ( fig .", "osseous ce is almost invariably related to primary infection and is not the result of extension from neighboring soft tissue lesions . however , some authors report that around 30 - 45% of bone hydatidosis is co - present with the hydatid cysts in other localizations .", "we have reported just in 4.9% of cases the presence of hydatid disease in the liver or lungs ; however , there is no statement in our database about the radiological surveys for the possible diagnoses of hepatic or pulmonary hydatidosis .", "therefore , it is impossible to exclude the presence of secondary cysts without a whole body scan . in patients with bone ce ,", "spine is the most commonly affected site with the frequency of appearance 35 - 50% [ 24,29 , 33 - 36 ] .", "our findings ( table 1 ) are in concordance with the ones reported by other authors , with the exception of the cervical region .", "it is considered to be relatively silent , slowly progressive disease with a latent period of many years .", "early symptoms appear when the small cyst expands and produces significant mechanical compression on surrounding vital structures . at this stage ,", "intermittent pain can be present as a symptom . as the disease progresses and the cyst enlarges , patient starts suffering from persistent backache , gradually leading to severe neurologic deficit and varying degrees of weakness of limbs .", "pain was present in 50% of patients with spinal hydatidosis in our study ( table 3 ) with the frequency of appearance similar to the ones reported by other authors .", "paraplegia is the most serious complication of vertebral hydatidosis appearing in up to 75% of patients with spinal ec .", "this compressive effect of spinal ce was present in 37.5% of patients in our study ( table 3 ) .", "paraplegia is a neurological complication , occurring as a result of invasive intradural and extradural growth of the cyst .", "cysts invade the spinal canal either by direct compression or by ischemic changes in the spinal cord .", "radicular pain usually precedes paraparesis by few weeks , progressing rapidly to paraplegia in the thoracic spine , but slowly in the lumbar spine .", "our results show that , in some patients , paraplegia can be developed as a compressive effect not just of the cyst , but of the spinal pathological fracture that ce can lead to , as well .", "all these data indicate that once formed in the spinal column , hydatid cyst is likely to result in paraplegia during the progression of the disease . in long - term hydatidosis , apart from pain and paraplegia , some cases can result in secondary infections and fistula formation .", ". their presence can resolve the disease in the term of surgical treatment , while bacterial superinfection can lead to sterilization of the parasite , limiting the bone destruction .", "interestingly , in our study , 1 male patient was reported with bone ce and tuberculosis super - infection . despite ce", "shows the spinal tropism causing severe complications , the therapy of spinal ce is still poor .", "they are applied in order to retard the recurrences and control the disease but radical surgery is still the keystone .", "data from the literature reported that the recurrence rate of bone ce , even after several operations , varied from 30% to 100% .", "moreover , the mortality rate of spinal ce is unacceptably high , varying 14 - 58% . when ce is affecting long bones , it is generally the metaphyseal region , extending later to the diaphyses and nearby bones .", "the incidence of long bone involvement in our study ( table 1 ) was similar to the results reported by other authors .", "therapy is surgical resection , performed if lesion is segmental , otherwise , amputation or disarticulation are the only solutions .", "our results ( table 1 ) did not differ from the ones obtained by other authors .", "it 's progression is infiltrating and diffuse and may lead to pain , swelling , and stiffness of the hip joint .", "this disease is hematogenously seeded along the trabeculae and through the medullar canal , leading to extensive bone lesions .", "our results reported that these lesions were mostly complicated with pathological fracture ( 48.8% of all cases ) .", "our results reported primary involvement of the spine ( 75% ) , with the predominance of lumbar over thoracic segment . in patients with bone ce", ", bone tissue destruction develops through 3 mechanisms ; as the consequence of the cystic lesion expansion and its compression on surrounding tissues , ischemic processes through compression and obstruction of nutrient vessels , and cellular processes like osteoclast proliferation around the compressed bone tissue . in the absence of pericyst formation ,", "the wall of the cyst is thin and can not overcome the rigid bone nature .", "therefore , the cyst can not assume it typical , spherical shape , and it enlarges irregularly along the path of least resistance . in patients with skeletal ce ,", "bone destruction lead to its thinning , extension into soft tissues and , as the worse case , can lead to fracture .", "therefore , some authors suggest that all the pathological fractures in endemic countries should be considered as a possible case of ce ."], "abstract_text": ["<S> although serbia is recognized as an endemic country for echinococcosis , no information about precise incidence in humans has been available . </S>", "<S> the aim of this study was to investigate the skeletal manifestations of hydatid disease in serbia . </S>", "<S> this retrospective study was conducted by reviewing the medical database of institute for pathology ( faculty of medicine in belgrade ) , a reference institution for bone pathology in serbia . </S>", "<S> we reported a total of 41 patients with bone cystic echinococcosis ( ce ) during the study period . </S>", "<S> the mean age of 41 patients was 40.918.8 years . in 39% of patients , </S>", "<S> the fracture line was the only visible radiological sign , followed by cyst and tumefaction . </S>", "<S> the spine was the most commonly involved skeletal site ( 55.8% ) , followed by the femur ( 18.6% ) , pelvis ( 13.9% ) , humerus ( 7.0% ) , rib ( 2.3% ) , and tibia ( 2.3% ) . </S>", "<S> pain was the symptom in 41.5% of patients , while some patients demonstrated complications such as paraplegia ( 22.0% ) , pathologic fracture ( 48.8% ) , and scoliosis ( 9.8% ) . </S>", "<S> the pathological fracture most frequently affected the spine ( 75.0% ) followed by the femur ( 20.0% ) and tibia ( 5.0% ) . however </S>", "<S> , 19.5% of patients did n't develop any complication or symptom . in this study </S>", "<S> , we showed that bone ce is not uncommon in serbian population . </S>", "<S> as reported in the literature , therapy of bone ce is controversial and its results are poor . in order to improve the therapy outcome , early diagnosis , </S>", "<S> before symptoms and complications occur , can be contributive . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["hydatid disease ( echionococcosis ; hydatidosis ) is a parasitic disease , endemic in many sheep raising countries ( australia , south america , mediterranean countries , russia , middle east , and central asia ) .", "adult cestodes of echinococcus granulosus inhabit the small intestine of a definitive host ( dogs , foxes , and wolves ) and produce infective eggs . through the feces , either cestode segments or free eggs", "these are ungulates ( sheep , goats , pigs , and horses ) but in some cases accidental ingestion of eggs , usually shed by dogs , could lead to human cystic echinococcosis ( ce ) .", "contaminated food , water , or hand - to - mouth transmission are usual modes of infection in humans [ 2 - 4 ] . so far ,", "once ingested by the intermediate host , in its intestinal tract , parasite eggs transform into the oncosphere .", "oncospheres penetrate the intestinal wall , enters the portal vein , and reaches the liver where they usually are trapped and become the hydatid cyst .", "liver involvement has been described as the most common location of ce , varying from 59% to 78% of all cases [ 1,5 - 8 ] .", "some larvae pass the liver and reach the lungs where they develop into pulmonary hydatidosis .", "lung involvement is present in 15 - 27% of all cases [ 1,5 - 8 ] .", "not so often , larvae may pass these filters and form cysts in the brain , skeletal muscles , bones , kidneys , spleen , or other tissues .", "theoretically , they can occur at any site except the teeth , nails , and hairs .", "epidemiological studies have been conducted in some countries , while in serbia , montenegro , bih , and albania no information is available about the precise incidence in humans .", "the aim of this retrospective study was to investigate the skeletal manifestations of hydatid disease in serbia , it 's demographic distribution , site involvement , complications , and bone tissue comorbidities ."], ["the study was conducted by reviewing the medical database of institute for pathology ( faculty of medicine in belgrade ) which is a reference institution in serbia for bone pathology .", "materials in the study comprised all the cases with histologically verified bone hydatidosis during the period between 1971 and 2010 in the territory of serbia .", "data obtained from medical records included the age at the moment of diagnosis , sex , localization of hydatid cyst , copresence of hydatid cyst in localizations other than the bone tissue , presence of skeletal complications , bone comorbidities , and diagnosis based on radiographies . in order to standardize the data ,", "the radiological assessment was made on standard radiography , having in mind the length of the study period and diagnostic procedures available for that time .", "changes in the bone structure were classified as tumefaction ( sharply or unshapely demarcated area of altered bone structure ) , cysts ( sharply demarcated lytic bone lesion with geographical borders ) , or fractures ( in cases where fracture line occured ) . in some patients , altered bone structure was able to be classified as cyst or tumefaction preceding the pathological fracture , while in other cases , no alteration of the bone structure was radiologically visible aside of the fracture line ."], ["the age of the patients at the time of diagnosis was from 7 to 77 years .", "the mean age of all patients was 40.918.8 years . in order to explore the age distribution of ce ,", "we grouped patients into 5 age categories : i ( 1 - 18 years ) , ii ( 19 - 36 years ) , iii ( 37 - 54 years ) , iv ( 55 - 73 years ) , and v ( > 73 years ) . in group", "the mean age of adult patients ( more than 18 years old ) was 44.716.6 years .", "a total of 24 male patients presented 58.5% of all cases , while 17 female patients presented 41.5% .", "the spine was the most commonly involved site with the predominance of the thoracic segment .", "analysis of standard radiographies revealed that fracture was by far the predominant radiological diagnosis , followed by tumefaction and cyst ( table 2 ) .", "pain was the symptom in 41.5% of patients , while some patients demonstrated complications such as paraplegia ( 22.0% ) , pathologic fracture ( 48.8% ) , and scoliosis ( 9.8% ) .", "the pathological fracture was most frequently found in the spine ( 75.0% ) followed by the femur ( 20.0% ) and tibia ( 5.0% ) . however", "in addition to the bone involvement , 2 patients demonstrated extraskeletal hydatid cysts : 1 in the liver and 1 in the lungs .", "bone tissue comorbidities refer to bone tuberculosis in a patient with spinal hydatid disease ( 2.4% ) and to presence of fistula detected in 2 patients ( 4.9% ) , 1 with spinal and 1 with pelvic hydatidosis ."], ["serbia is recognized as an endemic country for ce , but so far , no epidemiological study has been conducted . since echionococcosis is subjected to an obligatory disease for reporting , the data on the number of reported patients were available through the annual report on infectious diseases on the territory of republic of serbia published by serbian institute for public health , serbia . in the study period , a total of 727 patients with ce", "the frequency was relatively high compared to the reports from other endemic countries , being 0.5 - 4.0% .", "this frequency can be explained at least partially by a larger total number of patients   due to careless reporting of echionococcosis by physicians , which was even noted in the official annual reports from 1978 to 1989 of serbian institute for public health , serbia .", "studies in other endemic countries showed that the prevalence of ce increases with age , resulting that the most of the patients belong to the adult population .", "the mean age at the moment of diagnosis , including all localizations , was from 33 to 52 years [ 13 - 18 ] . in our study ,", "the mean age of the patients with skeletal manifestation of ce was 40.918.8 years , but among the adults the mean age was 44.716.6 .", "higher frequency in aged people could be due to the fact that older people have more chances to be infected with e. granulosus during life , and also , the clinical signs of ce are more probable to appear later in life , as the disease progresses . as it was reported in previous studies [ 15,19 - 21 ] , no significant difference between sex was found . in our study ,", "12.2% of patients were under 18 years , with the mean age being 13.04.2 years .", "it was consistent with the results of a previous report on the frequency of echinococcosis among children in serbia .", "although boys to girls ratio was 1.5:1.0 , no significant difference in the positive rate between sex was found . some other studies reported slight predominance of boys , which could probably be explained by their outdoor playing , closer contact with animals , and low level of personal hygiene , compared to girls .", "symptoms associated with bone ce are not specific and often missing , making the diagnosis difficult and sometimes overlooked .", "our results showed that 19.5% of patients did not report any symptoms and did not develop any complications .", "these asymptomatic patients are not surprising in consideration that hydatid disease is anywhere in the body and often discovered as an incidental finding .", "radiological diagnosis of osseous hydatid disease is also difficult because of non - specific findings .", "radiography usually reveals single or multiple osteolytic lesions and in some cases shows cortical thinning , osteosclerosis ( fig .", "intraosseous foci of hydatid disease predominate in spongious bone tissues and consist of small , separate , and thin - walled cysts .", "while these cysts expand , they destroy the surrounding trabeculae and can reach a considerable size . due to cyst enlargement and consequent cortical thinning", ", pathological fractures may occur . in the case of connective tissue proliferation and bone sclerosis ,", "presentation of ce can be unclear , consisting of mixed radiological signs   ( table 2 ) .", "therefore , preoperative diagnosis of skeletal hydatid disease is difficult , and conclusive diagnosis is often made intraoperatively and by histological verification [ 26 - 28 ] .", "histological examination of the cyst wall usually reveals an outer chitinous ( or fibrous laminar ) and an inner germinal layer , surrounded by either granulation tissue or fibrous capsule ( fig .", "osseous ce is almost invariably related to primary infection and is not the result of extension from neighboring soft tissue lesions . however , some authors report that around 30 - 45% of bone hydatidosis is co - present with the hydatid cysts in other localizations .", "we have reported just in 4.9% of cases the presence of hydatid disease in the liver or lungs ; however , there is no statement in our database about the radiological surveys for the possible diagnoses of hepatic or pulmonary hydatidosis .", "therefore , it is impossible to exclude the presence of secondary cysts without a whole body scan . in patients with bone ce ,", "spine is the most commonly affected site with the frequency of appearance 35 - 50% [ 24,29 , 33 - 36 ] .", "our findings ( table 1 ) are in concordance with the ones reported by other authors , with the exception of the cervical region .", "it is considered to be relatively silent , slowly progressive disease with a latent period of many years .", "early symptoms appear when the small cyst expands and produces significant mechanical compression on surrounding vital structures . at this stage ,", "intermittent pain can be present as a symptom . as the disease progresses and the cyst enlarges , patient starts suffering from persistent backache , gradually leading to severe neurologic deficit and varying degrees of weakness of limbs .", "pain was present in 50% of patients with spinal hydatidosis in our study ( table 3 ) with the frequency of appearance similar to the ones reported by other authors .", "paraplegia is the most serious complication of vertebral hydatidosis appearing in up to 75% of patients with spinal ec .", "this compressive effect of spinal ce was present in 37.5% of patients in our study ( table 3 ) .", "paraplegia is a neurological complication , occurring as a result of invasive intradural and extradural growth of the cyst .", "cysts invade the spinal canal either by direct compression or by ischemic changes in the spinal cord .", "radicular pain usually precedes paraparesis by few weeks , progressing rapidly to paraplegia in the thoracic spine , but slowly in the lumbar spine .", "our results show that , in some patients , paraplegia can be developed as a compressive effect not just of the cyst , but of the spinal pathological fracture that ce can lead to , as well .", "all these data indicate that once formed in the spinal column , hydatid cyst is likely to result in paraplegia during the progression of the disease . in long - term hydatidosis , apart from pain and paraplegia , some cases can result in secondary infections and fistula formation .", ". their presence can resolve the disease in the term of surgical treatment , while bacterial superinfection can lead to sterilization of the parasite , limiting the bone destruction .", "interestingly , in our study , 1 male patient was reported with bone ce and tuberculosis super - infection . despite ce", "shows the spinal tropism causing severe complications , the therapy of spinal ce is still poor .", "they are applied in order to retard the recurrences and control the disease but radical surgery is still the keystone .", "data from the literature reported that the recurrence rate of bone ce , even after several operations , varied from 30% to 100% .", "moreover , the mortality rate of spinal ce is unacceptably high , varying 14 - 58% . when ce is affecting long bones , it is generally the metaphyseal region , extending later to the diaphyses and nearby bones .", "the incidence of long bone involvement in our study ( table 1 ) was similar to the results reported by other authors .", "therapy is surgical resection , performed if lesion is segmental , otherwise , amputation or disarticulation are the only solutions .", "our results ( table 1 ) did not differ from the ones obtained by other authors .", "it 's progression is infiltrating and diffuse and may lead to pain , swelling , and stiffness of the hip joint .", "this disease is hematogenously seeded along the trabeculae and through the medullar canal , leading to extensive bone lesions .", "our results reported that these lesions were mostly complicated with pathological fracture ( 48.8% of all cases ) .", "our results reported primary involvement of the spine ( 75% ) , with the predominance of lumbar over thoracic segment . in patients with bone ce", ", bone tissue destruction develops through 3 mechanisms ; as the consequence of the cystic lesion expansion and its compression on surrounding tissues , ischemic processes through compression and obstruction of nutrient vessels , and cellular processes like osteoclast proliferation around the compressed bone tissue . in the absence of pericyst formation ,", "the wall of the cyst is thin and can not overcome the rigid bone nature .", "therefore , the cyst can not assume it typical , spherical shape , and it enlarges irregularly along the path of least resistance . in patients with skeletal ce ,", "bone destruction lead to its thinning , extension into soft tissues and , as the worse case , can lead to fracture .", "therefore , some authors suggest that all the pathological fractures in endemic countries should be considered as a possible case of ce ."]]}
{"article_id": "PMC3023350", "article_text": ["workers ' health may be protected by his / her own preventive behavior , despite numerous health hazards in the workplace .", "however , worker behavior is constrained by various workplace pressures such as working conditions , including what is considered to be accepted working behavior and overall morale of the working group .", "based on the employment contract , every employer has a certain amount of control / power and the expectation that workers will follow work - related requests .", "this may lead to adverse health effects in workers ; therefore , the government has implemented regulations to protect worker health , such as the occupational safety and health ( osh ) act .", "adverse health effects induced by unexpected incidents may present as occupational injuries , but occupational diseases can appear due to long term continuous exposure to harmful chemicals , dust , noise , etc . to prevent occupational diseases , it is important to manage worker health via health monitoring for early detection of abnormal conditions .", "it is very important for worker health management to monitor the level of worker exposure to hazards in the workplace ; for example , health check - ups including absorption of hazard materials , so called ' biological monitoring ' .", "noise levels in the workplace should also be measured in order to prevent worker 's hearing loss .", "if a worker is exposed to high levels of noise , he / she should be transferred to another work area without noise or provided with proper personal protective equipment ( ppe ) such as earplugs , etc .", "as above , information needed for workers ' health management can be classified into three categories according to 3 questions : 1 ) what is the exact nature of the occupational hazards ?", "3 ) what health problems are induced by these hazards ? these questions should be answered in order to manage workers ' health effectively and efficiently .", "health management for workers is accomplished by two different strategies : 1 ) focusing on reduction of exposure to hazard factors in the workplace based on risk assessment using exposure information ; 2 ) the provision of health care services and compensation for workers with adverse health effects .", "recently , psychosocial factors such as work - related stressors have been included as hazard factors in the workplace , and health promotion activities have become an important component of worker health management before symptoms and signs of occupational diseases manifest . in this article , we reviewed the various kinds of surveillance systems for occupational diseases in korea and resultant data , and suggest reformation of the workers ' health management system .", "the workers ' compensation insurance system is one of the old systems and ensures a worker 's life and his / her family is secure after work - related accident .", "workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .", "now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,", "only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .", "industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .", "occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .", "work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .", "data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act", "the proportion of reported cases is very small ( under 1% of total occupational diseases ) .", "kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases . until now", ", some workers have been excluded from workers ' compensation insurance : \n self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers \n  self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .", "since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular , occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .", "the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .", "the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .", "non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend .", "specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .", "the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .", "there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .", "yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation .", "in particular , brain and heart disease and musculoskeletal disorders have increased since the 1990s and 2000s , respectively .", "recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) .", "another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .", "the first one was done only one time in 1998 for workers in manufacturing sectors .", "the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .", "the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .", "the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .", "every survey has included some questions about worker 's experiences of occupational disease . according to the wcs", ", the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .", "the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) .", "workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .", "now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,", "only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .", "industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .", "occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .", "work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .", "data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act", "the proportion of reported cases is very small ( under 1% of total occupational diseases ) .", "kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases .", "until now , some workers have been excluded from workers ' compensation insurance : \n self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers \n  self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .", "since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular ,", "occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .", "the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .", "the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .", "non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend . specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .", "the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .", "there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .", "yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation . in particular , brain and heart disease and musculoskeletal disorders", "recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) .", "another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .", "the first one was done only one time in 1998 for workers in manufacturing sectors .", "the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .", "the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .", "the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .", "according to the wcs , the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .", "the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) .", "these institutions are examine workers by the request of employers based on the osh act mandatorily .", "for example , health examinations are regulated by government in order to protect worker health through early detection of occupational diseases .", "health examinations for worker health protection are classified into two different types : special health examination and general health examination .", "the special health examinations are comprised of health screening tests for early detection of selective occupational diseases in workers exposed to specific hazard factors such as noise , dust , organic solvent , etc . the general health examination is a mass screening test for finding health problems including occupational diseases of workers without exposure to hazard factors .", "the health examination is focused on early detection of occupational diseases , and its results are used as information for workplace management in order to protect worker health .", "cases diagnosed as ' suspicious cases of occupational disease ' are recommended to be transferred to non - hazardous workplaces . some of them may be compensated based on diagnosis of occupational diseases through the workers ' compensation insurance system", ". detection rate of ' suspicious cases of occupational diseases ' in the special health examination was 0.46% in 2008 , an increase from 2006 .", "the detection rate of ' cases observed for occupational disease ' was 12.85% in 2008 with an increasing trend .", "the health examination is defined as a screening test for occupational diseases , so there is no confirmatory diagnosis in the health examination process . '", "cases to be observed for occupational diseases ' are the result of an examination showing marginal figures for ' suspicious cases for occupational diseases ' . based on results from the health examination ,", "suspicious cases will be recommended to undergo medical treatment , change jobs or tasks for reduction of exposure to hazard workplace , etc .", "cases to be observed are not in need of any immediate intervention ; however , these cases may be also recommended similar treatment , such as wearing personal protective equipment ( ppe ) , changing jobs or tasks , etc . among suspicious cases and cases", "to be observed detected by the special health examination , hearing loss due to noise was the most prevalent ( 3 ) ( table 4 ) .", "detection rate of ' suspicious cases of occupational diseases ' during the general health examination was 0.001% in 2008 , and has been very steady since 2004 . in contrast , the detection rate of ' suspicious cases of general diseases ' has shown fluctuations between 4 and 5% in the last ten years ( 3 ) ( table 5 ) .", "health examinations are very effective in finding asymptomatic chronic diseases , such as pneumoconiosis , hearing loss and some poisonings .", "however , it can barely detect symptomatic diseases with no signs , like asthma , and easily curable diseases like dermatitis because of the characteristics of the periodic screening test .", "therefore , statistics from the health examination include mostly pneumoconiosis and noise - induced hearing loss ( 4 ) ( table 4 ) .", "the working environment measurement institution , regulated by the osh act , is in charge of assessing and , ultimately , reducing the level of worker exposure to hazard factors in the workplace .", "every employer should do working environment measurement in order to identify hazard factors in the workplace and the level of worker exposure to these factors .", "the special health examination is based on working environment measurement , such as selection of the examinee .", "if the level of worker exposure is higher than the occupational exposure level in korea , the employer should immediately act to reduce it .", "the magnitude of occupational diseases and associated fatality rate in korea have gradually increased since 2007 .", "the trend in fatality rate can be reflected by the trend in fatal pneumoconiosis and brain and heart disease , as these two categories of occupational diseases predominate total occupational diseases .", "most pneumoconiosis cases were coal workers ' pneumoconiosis ; however , nowadays most coal mines have closed due to restructuring of the energy production system in korea .", "other dominant fatal occupational diseases , such as brain and heart diseases , will also decrease because general health programs have focused on prevention of hypertension , one of the most important causal factors of brain and heart diseases in workers . in the general population , the morbidity rate of brain and heart diseases has shown a recent downward trend .", "one is the causal factors of occupational diseases for prevention , and the other is affected workers of occupational diseases in order to rehabilitate them through recovery of the labor force .", "occupational diseases are induced by worker exposure to various hazard factors in the workplace , such as physical , chemical , biological and psychosocial factors .", "workers with abnormal conditions can detect acute occupational diseases through self - diagnosis just after exposure to high levels of hazard factors , such as lead , mercury , etc .", "however , it is not so easy to identify abnormal conditions of chronic occupational diseases , such as asbestos - induced diseases .", "furthermore , exposure to low levels of hazard factors may have long - term cumulative effects on worker health ( fig .", "if there is no evidence of work - relatedness , no ill - health can be confirmed as an occupational disease .", "work - relatedness may be classified into two different types : direct and indirect causality .", "first , based on direct causality between work or work environment and ill - health , it is easy to determine work - relatedness , but it is very difficult to evaluate indirect work - relatedness .", "the same condition diagnosed as occupational disease by a physician can also be categorized as non - work - related because a particular hazard factor can not be detected in the working history .", "final determination of work - related illness or death in any individual case requires detailed occupational and non - occupational information ( 5 ) . in the united states", ", there exists occupational health surveillance , such as death certificates , hospital discharge data , physician reports , laboratory reports , workers ' compensation reports , national surveys , employer surveillance programs and occupational health clinics ( 5 ) .", "since 1998 , there were some suggestions for the introduction of a surveillance system for occupational diseases in korea because worker health examinations and work environment measurements had limitations for management of occupational diseases ( 6 , 7 ) .", "the magnitude of occupational diseases in korea is assessed mainly by the workers ' compensation system .", "statistics might exclude unclaimed occupational diseases and those of workers from the public sector and workers who are not covered by compensation insurance ( 4 ) .", "this means that non - compensated cases can be neglected and latent cases can always emerge as compensated cases in the immediate future .", "moreover , statistics based on compensated cases of occupational diseases are not stable because different acceptance criteria for work - relatedness have been applied by the korea labor welfare corporation ( former name of comwel ) and judiciary branch ( 8) . in korea , there are two different kinds of health insurance : national health insurance and workers ' compensation insurance .", "every worker with occupational ill - health can be covered by either of these two .", "if some occupational diseases cases are covered by the national health insurance system , official statistics on occupational diseases can not include these cases . in order to determine the correct number , all occupational diseases covered by the two different insurances", "unfortunately , the national health insurance system has no screening and confirming system for occupational diseases .", "occupational disease compensation will focus on the treatment , compensation , and rehabilitation as secondary or tertiary prevention .", "national surveys of worker health can provide useful information about ill - health workers without treatment or compensation .", "this information can be used to prevent occupational diseases by detection of at - risk workers as a target population for management of occupational diseases .", "the primary prevention of occupational diseases and injuries depends more directly on hazard surveillance , since the identification of hazards enables their reduction prior to the onset of ill - health .", "occupational health surveillance is less well developed than its health counterpart due to scarcity of data sources and , with the exception of the occupational safety and health administration and the national institute for occupational safety and health surveys in the usa , the proprietary and inaccessible nature of hazard data collected by employers ( 5 ) .", "occupational health surveillance entails the systematic monitoring of health events and exposures in working populations in order to prevent and control occupational hazards and their associated diseases and injuries ( 5 ) .", "medical surveillance refers to the ongoing application of medical tests and procedures to individual workers who may be at risk for occupational morbidity to determine whether an occupational disorder may be present ( 5 ) .", "if an individual or population is exposed to a toxin with known effects , and the tests and procedures are highly targeted to detect the likely presence of one or more effects in these persons , then this surveillance activity is more aptly described as medical screening ( 9 ) . in occupational health , there are two kinds of surveillance activities , one is public health and medical surveillance and the other is hazard surveillance .", "the main purpose of occupational health surveillance is to identify the incidence and prevalence of known occupational diseases and injuries ( 5 ) .", "a second broad function is to identify individual cases of occupational disease and injury in order to find and evaluate other individuals from the same workplaces who may be at risk for similar disease and injury .", "another purpose of case identification may be to assure that the affected individual receives appropriate clinical follow - up , an important consideration in view of the scarcity of clinical occupational medicine specialists ( 10 , 11 ) .", "finally , occupational health surveillance is an important means of discovering new associations between occupational agents and accompanying diseases . discovery of rare diseases , patterns of common diseases , or suspicious exposure - disease associations through surveillance activities in the workplace can provide vital leads for a more conclusive scientific evaluation of the problem and possible verification of new occupational diseases ( 5 ) .", "periodic medical screening and surveillance examinations are another frequent service provided by occupational medicine physicians .", "the term screening focuses on the individual ; screening tests are tools for secondary prevention with the goal of early diagnosis and treatment of disease in exposed workers .", "true ' surveillance ' aggregates information about individuals in order to examine patterns within a population . properly applied ,", "surveillance is a tool for primary prevention through the identification and elimination of the cause of disease ( 12 ) .", "screening examinations may be target - organ specific or substance specific , and thus tend to be more focused than preplacement evaluations ( 12 - 14 ) .", "based on considerable information , occupational disease management can be accomplished in order to protect and promote worker health .", "many kinds of information are required to suggest the etiology of poor worker health , such as hazard , exposure level and related information in workplace .", "the present system of surveillance for management of occupational diseases mainly depends upon the workers ' compensation and health examination systems .", "these two systems can not cover all kinds of occupational diseases . in order to focus on primary prevention of occupational diseases , various data sources on hazards and exposure levels in workplaces", "especially , prevention of occupational diseases may be focused on the reduction of worker exposure to the occupational hazards .", "the workers ' compensation insurance system reviews each claim case for evaluating its work - relatedness .", "this means that hazard factors and worker exposure to those hazard factors may be assessed .", "these review data can be some of the most important information for prevention of ill - health by surveillance .", "most of the current surveillance system in korea depends upon the osh act . from the viewpoint of surveillance , the occupational management system in korea is well designed except for the national survey system . in the future , national surveys for detection of occupational hazards and ill - health outcomes of workers should be developed .", "the existing surveillance system of occupational disease can be improved by providing more refined information through statistical analysis of surveillance data .", "recently , data mining has been used in order to estimate the national burden of occupational disease with other international statistics . in korea", ", this technique can be also introduced for development of the management system of occupational diseases ."], "abstract_text": ["<S> the management system of occupational diseases in korea can be assessed from the perspective of a surveillance system . </S>", "<S> workers ' compensation insurance reports are used to produce official statistics on occupational diseases in korea . </S>", "<S> national working conditions surveys are used to monitor the magnitude of work - related symptoms and signs in the labor force . </S>", "<S> a health examination program was introduced to detect occupational diseases through both selective and mass screening programs . </S>", "<S> the working environment measurement institution assesses workers ' exposure to hazards in the workplace . </S>", "<S> government regulates that the employer should do health examinations and working conditions measurement through contracted private agencies and following the occupational safety and health act . </S>", "<S> it is hoped that these institutions may be able to effectively detect and monitor occupational diseases and hazards in the workplace . in view of this , the occupational management system in korea is well designed , except for the national survey system . in the future , national surveys for detection of hazards and ill - health outcomes in workers should be developed . </S>", "<S> the existing surveillance system for occupational disease can be improved by providing more refined information through statistical analysis of surveillance data . </S>"], "labels": null, "section_names": ["INTRODUCTION", "OCCUPATIONAL DISEASES SURVEILLANCE ACTIVITIES IN KOREA", "Workers' compensation insurance report", "National survey report", "HEALTH EXAMINATION SYSTEM", "MONITORING SYSTEM FOR WORKER HAZARD EXPOSURE", "DISCUSSION", "CONCLUSION"], "sections": [["workers ' health may be protected by his / her own preventive behavior , despite numerous health hazards in the workplace .", "however , worker behavior is constrained by various workplace pressures such as working conditions , including what is considered to be accepted working behavior and overall morale of the working group .", "based on the employment contract , every employer has a certain amount of control / power and the expectation that workers will follow work - related requests .", "this may lead to adverse health effects in workers ; therefore , the government has implemented regulations to protect worker health , such as the occupational safety and health ( osh ) act .", "adverse health effects induced by unexpected incidents may present as occupational injuries , but occupational diseases can appear due to long term continuous exposure to harmful chemicals , dust , noise , etc . to prevent occupational diseases , it is important to manage worker health via health monitoring for early detection of abnormal conditions .", "it is very important for worker health management to monitor the level of worker exposure to hazards in the workplace ; for example , health check - ups including absorption of hazard materials , so called ' biological monitoring ' .", "noise levels in the workplace should also be measured in order to prevent worker 's hearing loss .", "if a worker is exposed to high levels of noise , he / she should be transferred to another work area without noise or provided with proper personal protective equipment ( ppe ) such as earplugs , etc .", "as above , information needed for workers ' health management can be classified into three categories according to 3 questions : 1 ) what is the exact nature of the occupational hazards ?", "3 ) what health problems are induced by these hazards ? these questions should be answered in order to manage workers ' health effectively and efficiently .", "health management for workers is accomplished by two different strategies : 1 ) focusing on reduction of exposure to hazard factors in the workplace based on risk assessment using exposure information ; 2 ) the provision of health care services and compensation for workers with adverse health effects .", "recently , psychosocial factors such as work - related stressors have been included as hazard factors in the workplace , and health promotion activities have become an important component of worker health management before symptoms and signs of occupational diseases manifest . in this article , we reviewed the various kinds of surveillance systems for occupational diseases in korea and resultant data , and suggest reformation of the workers ' health management system ."], ["the workers ' compensation insurance system is one of the old systems and ensures a worker 's life and his / her family is secure after work - related accident .", "workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .", "now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,", "only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .", "industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .", "occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .", "work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .", "data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act", "the proportion of reported cases is very small ( under 1% of total occupational diseases ) .", "kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases . until now", ", some workers have been excluded from workers ' compensation insurance : \n self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers \n  self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .", "since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular , occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .", "the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .", "the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .", "non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend .", "specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .", "the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .", "there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .", "yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation .", "in particular , brain and heart disease and musculoskeletal disorders have increased since the 1990s and 2000s , respectively .", "recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) .", "another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .", "the first one was done only one time in 1998 for workers in manufacturing sectors .", "the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .", "the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .", "the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .", "every survey has included some questions about worker 's experiences of occupational disease . according to the wcs", ", the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .", "the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) ."], ["workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .", "now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,", "only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .", "industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .", "occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .", "work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .", "data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act", "the proportion of reported cases is very small ( under 1% of total occupational diseases ) .", "kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases .", "until now , some workers have been excluded from workers ' compensation insurance : \n self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers \n  self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .", "since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular ,", "occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .", "the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .", "the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .", "non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend . specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .", "the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .", "there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .", "yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation . in particular , brain and heart disease and musculoskeletal disorders", "recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) ."], ["another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .", "the first one was done only one time in 1998 for workers in manufacturing sectors .", "the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .", "the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .", "the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .", "according to the wcs , the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .", "the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) ."], ["these institutions are examine workers by the request of employers based on the osh act mandatorily .", "for example , health examinations are regulated by government in order to protect worker health through early detection of occupational diseases .", "health examinations for worker health protection are classified into two different types : special health examination and general health examination .", "the special health examinations are comprised of health screening tests for early detection of selective occupational diseases in workers exposed to specific hazard factors such as noise , dust , organic solvent , etc . the general health examination is a mass screening test for finding health problems including occupational diseases of workers without exposure to hazard factors .", "the health examination is focused on early detection of occupational diseases , and its results are used as information for workplace management in order to protect worker health .", "cases diagnosed as ' suspicious cases of occupational disease ' are recommended to be transferred to non - hazardous workplaces . some of them may be compensated based on diagnosis of occupational diseases through the workers ' compensation insurance system", ". detection rate of ' suspicious cases of occupational diseases ' in the special health examination was 0.46% in 2008 , an increase from 2006 .", "the detection rate of ' cases observed for occupational disease ' was 12.85% in 2008 with an increasing trend .", "the health examination is defined as a screening test for occupational diseases , so there is no confirmatory diagnosis in the health examination process . '", "cases to be observed for occupational diseases ' are the result of an examination showing marginal figures for ' suspicious cases for occupational diseases ' . based on results from the health examination ,", "suspicious cases will be recommended to undergo medical treatment , change jobs or tasks for reduction of exposure to hazard workplace , etc .", "cases to be observed are not in need of any immediate intervention ; however , these cases may be also recommended similar treatment , such as wearing personal protective equipment ( ppe ) , changing jobs or tasks , etc . among suspicious cases and cases", "to be observed detected by the special health examination , hearing loss due to noise was the most prevalent ( 3 ) ( table 4 ) .", "detection rate of ' suspicious cases of occupational diseases ' during the general health examination was 0.001% in 2008 , and has been very steady since 2004 . in contrast , the detection rate of ' suspicious cases of general diseases ' has shown fluctuations between 4 and 5% in the last ten years ( 3 ) ( table 5 ) .", "health examinations are very effective in finding asymptomatic chronic diseases , such as pneumoconiosis , hearing loss and some poisonings .", "however , it can barely detect symptomatic diseases with no signs , like asthma , and easily curable diseases like dermatitis because of the characteristics of the periodic screening test .", "therefore , statistics from the health examination include mostly pneumoconiosis and noise - induced hearing loss ( 4 ) ( table 4 ) ."], ["the working environment measurement institution , regulated by the osh act , is in charge of assessing and , ultimately , reducing the level of worker exposure to hazard factors in the workplace .", "every employer should do working environment measurement in order to identify hazard factors in the workplace and the level of worker exposure to these factors .", "the special health examination is based on working environment measurement , such as selection of the examinee .", "if the level of worker exposure is higher than the occupational exposure level in korea , the employer should immediately act to reduce it ."], ["the magnitude of occupational diseases and associated fatality rate in korea have gradually increased since 2007 .", "the trend in fatality rate can be reflected by the trend in fatal pneumoconiosis and brain and heart disease , as these two categories of occupational diseases predominate total occupational diseases .", "most pneumoconiosis cases were coal workers ' pneumoconiosis ; however , nowadays most coal mines have closed due to restructuring of the energy production system in korea .", "other dominant fatal occupational diseases , such as brain and heart diseases , will also decrease because general health programs have focused on prevention of hypertension , one of the most important causal factors of brain and heart diseases in workers . in the general population , the morbidity rate of brain and heart diseases has shown a recent downward trend .", "one is the causal factors of occupational diseases for prevention , and the other is affected workers of occupational diseases in order to rehabilitate them through recovery of the labor force .", "occupational diseases are induced by worker exposure to various hazard factors in the workplace , such as physical , chemical , biological and psychosocial factors .", "workers with abnormal conditions can detect acute occupational diseases through self - diagnosis just after exposure to high levels of hazard factors , such as lead , mercury , etc .", "however , it is not so easy to identify abnormal conditions of chronic occupational diseases , such as asbestos - induced diseases .", "furthermore , exposure to low levels of hazard factors may have long - term cumulative effects on worker health ( fig .", "if there is no evidence of work - relatedness , no ill - health can be confirmed as an occupational disease .", "work - relatedness may be classified into two different types : direct and indirect causality .", "first , based on direct causality between work or work environment and ill - health , it is easy to determine work - relatedness , but it is very difficult to evaluate indirect work - relatedness .", "the same condition diagnosed as occupational disease by a physician can also be categorized as non - work - related because a particular hazard factor can not be detected in the working history .", "final determination of work - related illness or death in any individual case requires detailed occupational and non - occupational information ( 5 ) . in the united states", ", there exists occupational health surveillance , such as death certificates , hospital discharge data , physician reports , laboratory reports , workers ' compensation reports , national surveys , employer surveillance programs and occupational health clinics ( 5 ) .", "since 1998 , there were some suggestions for the introduction of a surveillance system for occupational diseases in korea because worker health examinations and work environment measurements had limitations for management of occupational diseases ( 6 , 7 ) .", "the magnitude of occupational diseases in korea is assessed mainly by the workers ' compensation system .", "statistics might exclude unclaimed occupational diseases and those of workers from the public sector and workers who are not covered by compensation insurance ( 4 ) .", "this means that non - compensated cases can be neglected and latent cases can always emerge as compensated cases in the immediate future .", "moreover , statistics based on compensated cases of occupational diseases are not stable because different acceptance criteria for work - relatedness have been applied by the korea labor welfare corporation ( former name of comwel ) and judiciary branch ( 8) . in korea , there are two different kinds of health insurance : national health insurance and workers ' compensation insurance .", "every worker with occupational ill - health can be covered by either of these two .", "if some occupational diseases cases are covered by the national health insurance system , official statistics on occupational diseases can not include these cases . in order to determine the correct number , all occupational diseases covered by the two different insurances", "unfortunately , the national health insurance system has no screening and confirming system for occupational diseases .", "occupational disease compensation will focus on the treatment , compensation , and rehabilitation as secondary or tertiary prevention .", "national surveys of worker health can provide useful information about ill - health workers without treatment or compensation .", "this information can be used to prevent occupational diseases by detection of at - risk workers as a target population for management of occupational diseases .", "the primary prevention of occupational diseases and injuries depends more directly on hazard surveillance , since the identification of hazards enables their reduction prior to the onset of ill - health .", "occupational health surveillance is less well developed than its health counterpart due to scarcity of data sources and , with the exception of the occupational safety and health administration and the national institute for occupational safety and health surveys in the usa , the proprietary and inaccessible nature of hazard data collected by employers ( 5 ) .", "occupational health surveillance entails the systematic monitoring of health events and exposures in working populations in order to prevent and control occupational hazards and their associated diseases and injuries ( 5 ) .", "medical surveillance refers to the ongoing application of medical tests and procedures to individual workers who may be at risk for occupational morbidity to determine whether an occupational disorder may be present ( 5 ) .", "if an individual or population is exposed to a toxin with known effects , and the tests and procedures are highly targeted to detect the likely presence of one or more effects in these persons , then this surveillance activity is more aptly described as medical screening ( 9 ) . in occupational health , there are two kinds of surveillance activities , one is public health and medical surveillance and the other is hazard surveillance .", "the main purpose of occupational health surveillance is to identify the incidence and prevalence of known occupational diseases and injuries ( 5 ) .", "a second broad function is to identify individual cases of occupational disease and injury in order to find and evaluate other individuals from the same workplaces who may be at risk for similar disease and injury .", "another purpose of case identification may be to assure that the affected individual receives appropriate clinical follow - up , an important consideration in view of the scarcity of clinical occupational medicine specialists ( 10 , 11 ) .", "finally , occupational health surveillance is an important means of discovering new associations between occupational agents and accompanying diseases . discovery of rare diseases , patterns of common diseases , or suspicious exposure - disease associations through surveillance activities in the workplace can provide vital leads for a more conclusive scientific evaluation of the problem and possible verification of new occupational diseases ( 5 ) .", "periodic medical screening and surveillance examinations are another frequent service provided by occupational medicine physicians .", "the term screening focuses on the individual ; screening tests are tools for secondary prevention with the goal of early diagnosis and treatment of disease in exposed workers .", "true ' surveillance ' aggregates information about individuals in order to examine patterns within a population . properly applied ,", "surveillance is a tool for primary prevention through the identification and elimination of the cause of disease ( 12 ) .", "screening examinations may be target - organ specific or substance specific , and thus tend to be more focused than preplacement evaluations ( 12 - 14 ) ."], ["based on considerable information , occupational disease management can be accomplished in order to protect and promote worker health .", "many kinds of information are required to suggest the etiology of poor worker health , such as hazard , exposure level and related information in workplace .", "the present system of surveillance for management of occupational diseases mainly depends upon the workers ' compensation and health examination systems .", "these two systems can not cover all kinds of occupational diseases . in order to focus on primary prevention of occupational diseases , various data sources on hazards and exposure levels in workplaces", "especially , prevention of occupational diseases may be focused on the reduction of worker exposure to the occupational hazards .", "the workers ' compensation insurance system reviews each claim case for evaluating its work - relatedness .", "this means that hazard factors and worker exposure to those hazard factors may be assessed .", "these review data can be some of the most important information for prevention of ill - health by surveillance .", "most of the current surveillance system in korea depends upon the osh act . from the viewpoint of surveillance , the occupational management system in korea is well designed except for the national survey system . in the future , national surveys for detection of occupational hazards and ill - health outcomes of workers should be developed .", "the existing surveillance system of occupational disease can be improved by providing more refined information through statistical analysis of surveillance data .", "recently , data mining has been used in order to estimate the national burden of occupational disease with other international statistics . in korea", ", this technique can be also introduced for development of the management system of occupational diseases ."]]}
{"article_id": "PMC5042943", "article_text": ["a cross - sectional retrospective study was carried out according to world health organization guidelines over 6  months and included 197 general practitioners ( gps ) and 19,700 prescriptions.this study revealed that the overall index of rational drug prescribing ( irdp ) indicator for gps was below the optimal prescribing practice level.we found disparities in the irdp between gps at different health facilities .", "gps at national insurance fund facilities scored lower than those at state ministry of health facilities or other health facilities such as those owned by private interests , universities , and non - governmental organizations .", "the irrational use of medicines is a major problem worldwide and represents the primary source of medical waste and harm .", "the medicines component of the health services package is more variable and dynamic than other service components , particularly in terms of coverage , quality , and cost . in recent years", "the overuse of medicines is diverse , and drug - prescribing indicators in developing countries are suboptimal , with core indicators varying between countries .", "for instance , antibiotics and injectables are overused and generic medicines are underused in sudan .", "the overuse of medicines not only leads to the development of drug resistance and wastes resources but also increases morbidity and mortality [ 3 , 4 ] .", "thus , the appropriate use of medicines is an essential factor in the provision of quality health services , patient safety , and the rational use of health resources .", "the world health organization ( who ) and the international network of rational use of drugs ( inrud ) have developed widely used core prescribing indicators to assess the prescribing quality of primary healthcare facilities and to enable comparisons between health facilities and prescribers . to our knowledge , no prescriber - based study has been conducted in sudan ; one study of the national health insurance fund ( nhif ) has been conducted in health facilities .", "previous studies have been based on the facilities sampling method ( table  1 ) and have revealed antibiotic overuse [ 8 , 9 ] , underuse of generic medicines , and overprescribing of injectables .", "other studies have revealed a relatively large average number of drugs per prescription and poor adherence to the essential medicines list ( eml ) .", "these previous studies were conducted to investigate prescription patterns ; however , our study investigated the prescribing practices of general practitioners ( gps ) and used the largest sample and a unique study unit .", "we used prescribing indicators as a benchmark among gps at primary healthcare centers ( phccs ) and as a baseline for regular assessment of prescribing quality . in sudan ,", "the objective of this study was to use the who / inrud prescribing indicator to assess the quality of prescribing among gps at different types of phccs .", "we used the assessment regimen created by who / inrud to evaluate prescribing performance according to the adopted standards to monitor the improvement of rationality in prescribing practice . before rational prescribing", "can be promoted , the prescribing practices of those who provide 80  % of the services must be ascertained.table  1previous prescribing quality studies in sudanyeararealevelmean no .", "drugs / prescription% of generic% of antibiotics% of injectable% from eml1991 n. provincephccs1.46363361996 kh statephccs1.9487322981998 kh statehospital and phccs2.1415929992004 kh stateteaching hospitals ( 2)1.9436510.52007 six statesphccs2.344.6662773.52010 kh statephccs243.271.813.792.72010 kh statefour pediatric hospitals249.381.33.52012 five nhif statesphccs2.654.2641499.32012 kh statehospitals and pharmacies2.837.354.338.672.8 \n eml essential medicines list , kh state khartoum state , n. province nile province , nhif national health insurance fund , phccs primary healthcare centers previous prescribing quality studies in sudan \n eml essential medicines list , kh state khartoum state , n. province nile province , nhif national health insurance fund , phccs primary healthcare centers", "the study was conducted in gezira state , which represents 27  % of nhif ; we targeted all gps ( 220 ) providing services for nhif subscribers at the primary healthcare level .", "these gps provide services at three types of health facilities : ( 1 ) those owned by the nhif , ( 2 ) those owned by the state ministry of health ( smoh ) , and ( 3 ) those owned by others ( private interests , universities , and non - governmental organizations [ ngos ] ) .", "insured patients pay 25  % of prescription costs , and pharmaceutical services are provided through a network of pharmacies owned by the nhif , smoh , and others .", "we collected 100 prescriptions for each gp according to who / inrud guidelines for comparisons between prescribers .", "the reference period of retrospective data collection was 6  months , representing the last quarter of 2014 and the first quarter of 2015 .", "prescription collection was based on systematic random sampling , with an interval determined by dividing the gp s total prescriptions by 100 .", "of the 220 gps , 197 ( 90  % ) had valid prescriptions over the 6  months , and the total valid number of observations was 19,700 prescriptions . trained technical professionals collected the data .", "data were reviewed and entered twice in a microsoft excel spreadsheet before being exported to stata-12 for analysis .", "the prescriptions data were transferred to who investigation format , checked again for accuracy , entered into an excel sheet , and individually checked once more for precision and completeness .", "we used the who / inrud standard method for calculating prescribing indicators   ( table  2 ) .", "the unit of analysis used was gp ( 100 prescriptions for each gp).table  2adjusted prescribing standards from who and inrud indicatorsprescribing indicatorsoptimal leveloptimal irdpmean medicines per prescription ( whether or not drugs dispensed)<2 1medicines prescribed by generic name ( % ) 100 1prescriptions with an antibiotic ( % ) 30 1prescriptions with an injectable dosage form ( % ) 10 1medicines prescribed from nhif gp s medicines list ( % ) 1001complementary indicator , average cost of prescription ( sdg)20.31 ( proxy) \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization adjusted prescribing standards from who and inrud indicators \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization to determine prescribing rationality and quality at the gp level , we used the index of rational drug prescribing ( irdp ) developed by yuxin and mingguang .", "the irdp for each gp was calculated by compounding the indices of the five prescribing indicators .", "the index of each prescribing indicator was calculated by dividing the optimal value by the achieved value for each", ". moreover , for each type of health facility , the irdp was applied for comparison .", "finally , we analyzed 19,690 prescriptions from 197 gps  ( 10 invalid prescriptions were excluded ) .", "data at the gp level were clustered to represent the three types of health facilities .", "data were reviewed and entered twice in a microsoft excel spreadsheet before being exported to stata-12 for analysis .", "the prescriptions data were transferred to who investigation format , checked again for accuracy , entered into an excel sheet , and individually checked once more for precision and completeness .", "we used the who / inrud standard method for calculating prescribing indicators   ( table  2 ) .", "the unit of analysis used was gp ( 100 prescriptions for each gp).table  2adjusted prescribing standards from who and inrud indicatorsprescribing indicatorsoptimal leveloptimal irdpmean medicines per prescription ( whether or not drugs dispensed)<2 1medicines prescribed by generic name ( % ) 100 1prescriptions with an antibiotic ( % ) 30 1prescriptions with an injectable dosage form ( % ) 10 1medicines prescribed from nhif gp s medicines list ( % ) 1001complementary indicator , average cost of prescription ( sdg)20.31 ( proxy) \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization adjusted prescribing standards from who and inrud indicators \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization to determine prescribing rationality and quality at the gp level , we used the index of rational drug prescribing ( irdp ) developed by yuxin and mingguang .", "the irdp for each gp was calculated by compounding the indices of the five prescribing indicators .", "the index of each prescribing indicator was calculated by dividing the optimal value by the achieved value for each gp . moreover , for each type of health facility , the irdp was applied for comparison .", "finally , we analyzed 19,690 prescriptions from 197 gps  ( 10 invalid prescriptions were excluded ) .", "data at the gp level were clustered to represent the three types of health facilities .", "the average number of medicines per prescription ( 2.55 : maximum 15 , minimum 1 ) indicated a poly - pharmacy pattern , with prescriptions involving antibiotics ( 54.71  % ) and injectable formulations ( 12.84  % ) .", "a total of 81.19  % of the prescribed medicines were from the eml , and generics accounted for 46.34  % of prescriptions , which is some distance from the optimal required level of 100  % .", "the average cost per prescription was 40.57 sudanese pounds ( sdgs ) ( table  3 ) .", "values for nhif facilities were farther from the prescribing indicator standards than were smoh and other health facilities , except for the percentage of prescriptions containing antibiotics ( nhif 45.91 vs. smoh 57.24 vs. others 56.84  % ) ( table  3).table  3prescribing indicators and health facility typestype of health facility owner or operatorsmohnhifotheroverallnumber of gps1264328197average number of drugs per prescription2.442.972.42.55medicines prescribed by generic name ( % ) 48.9638.4749.2846.34prescriptions containing antibiotics ( % ) 57.2445.9156.8454.71prescriptions containing injectable formulations ( % ) 12.5515.589.9712.84drugs prescribed from gp s medicines list ( % ) 82.9772.6589.2781.19average cost of prescription ( sdgs)36.0759.5431.6940.57irdp3.463.083.753.39 \n gp general practitioner , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , smoh state ministry of health prescribing indicators and health facility types \n gp general practitioner , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , smoh state ministry of health the correlation between gps and irdp was statistically significant at p  <  0.001 .", "the optimal index is 5 ; however , the overall irdp score for gps was 3.39 ( range 2.14.8 ) .", "the prescribing quality indices revealed that prescribing practice was nowhere near the optimal index of 5 , with nhif facilities having a lower index ( 3.08 ) than smoh facilities ( 3.46 ) and health facilities owned by others ( 3.75 ) ( table  3 ) .", "the objectives of this study were to use the who / inrud prescribing indicator to assess the quality of prescribing among gps at different types of phccs .", "we conducted a cross - sectional retrospective study over 6  months that involved 197 gps with valid prescriptions , representing 90  % of the total study population ( 220 ) .", "the study revealed that the mean number of medications per prescription was 2.55    1.32 drugs , which represents poly - pharmacy according to the benchmark we applied of more than two drugs .", "the mean number of drug prescriptions was 2.55 ; this was much closer to the 2.6 found by a study conducted in nhif facilities in 2012 in five other states .", "the mean number of drugs per prescription was higher than most previously reported sudanese studies when we excluded the studies by mustafa   and mahmoud et al . , who reported 2.6 and 2.8 drugs per prescription , respectively ( table  1).when compared with other developing countries , the mean number of drugs per prescription in sudan ( 2.55 ) was less than in mali ( 3.2 ) , yemen ( 3.0 ) , uganda ( 2.9 ) , thailand ( 2.85 ) , and pakistan ( 2.7 ) [ 1923 ] .", "however , gps in sudan prescribed a higher mean number of drugs per prescription than those in india ( 2.4 ) , tanzania ( 2.2 ) , saudi arabia ( 2.08 ) , and malaysia ( 2.0 ) [ 2427 ] .", "collectively , nhif facilities had a higher mean number of medications than other facilities ( table  3 ) . the major implications of poly - pharmacy are additional avoidable costs and an increased probability of adverse drug reactions .", "a limitation of the study was that inter - country comparisons were not adjusted according to the prevalence of chronic diseases in the population .", "the percentage of generic medicines prescribed was 46.34  % , which is considerably lower than the standard of 100  % .", "the calculated percentage represents a severe underuse of generic medicines that was also lower than the middle eastern mediterranean region s average use of generics ( 57.1  % ) .", "this result is consistent with previous studies conducted in different districts in sudan ( table  1 ) . when compared with those of neighboring countries , the percentage of generic drugs per prescription in sudan ( 46.34  % ) was lower than in egypt ( 95.4  % ) , ethiopia ( 99.16  % ) , mali ( 70.4  % ) , uganda ( 91.3  % ) , and yemen ( 67.1  % ) [ 1921 , 29 , 30 ] .", "however , the percentage of generic drugs prescribed was remarkably higher in sudan than in bahrain ( 14.3  % ) and jordan ( 5.1  % ) [ 29 , 30 ] .", "gps in nhif facilities prescribed considerably fewer generics ( 38.47  % ) than those in smoh ( 48.96  % ) and other facilities ( 49.28  % ) .", "the implications of low generic use are primarily the wastage of scarce health resources and a decrease in access to pharmaceuticals because of an affordability barrier .", "the percentage of prescriptions containing antibiotics was 54.71  % , a considerably high result according to the who guideline benchmark of 30  % antibiotic use .", "it was consistent with percentages assessed previously in other states of sudan ( table  1 ) .", "the percentage in the who eastern mediterranean region was 53.6  % , slightly less than found for sudan in the current study .", "the use of antibiotics was lower in nhif facilities ( 45.91  % ) than in smoh ( 57.24  % ) and other facilities ( 56.84  % ) .", "antibiotic overuse can have a devastating impact in terms of the development of multi - drug - resistant bacteria , which can lead to unmanageable infectious diseases .", "the percentage of prescriptions containing injections was 12.84  % , which is considered relatively over the optimal level , although no strict standard exists , as the who benchmark is < 10  % .", "the average prevalence of injection use in the eastern mediterranean region is higher ( 27.1  % ) .", "studies conducted in sudan reveal progress in the rational use of injections ( table  1 ) .", "this reduction is attributable to a new malaria management protocol that focuses strongly on ingestible formulations rather than injection , which was the initial dominant medication formulation .", "the overall percentage of medications prescribed from the eml was 81.19  % , whereas the prescribing quality indicator standard is 100  % .", "eml adherence seems high ; however , in actuality , this figure is misleading because the best achievement in core prescribing indicators was this indicator worldwide . according to the who prescribing database , on average , the middle eastern mediterranean region s percentage of medicines prescribed from the eml was 90.8  % .", "previous studies in sudan have often found this prescribing core indicator to be higher than observed in this study ( table  1 ) .", "interestingly , we found eml adherence was lower in nhif facilities ( 72.65  % ) than in smoh ( 82.97  % ) and other facilities ( 89.27  % ) .", "a total of 197 gps reported an irdp of 2.14.88 , which is relatively low compared with other neighboring countries . for instance , ten health facilities in saudi arabia reported an irdp of 4.375 . in egypt ,", "the same processes were conducted in ten phccs and revealed high irdp rankings , ranging from 3.92 to 4.88 . although the irdp was not high , it was higher than the 3.32 reported in china .", "the index values for nhif facilities ( 3.08 ) were lower than for smoh ( 3.46 ) and other ( 3.75 ) facilities ( table  3 ) .", "the current study revealed that the average prescription cost was 40.57 sdg , almost double the proxy 20.31 sdg .", "a study conducted in five states in 2012 reported an average prescription cost of 20.5 sdg , almost half that observed in the current study .", "the primary cause of the cost increase is attributed to the devaluation of the national currency , particularly over the last 4  years . when comparing average costs with those in other countries , proper economic adjustment of currency values is essential .", "prescriptions from nhif facilities cost more than those from other facilities , with the average prescription costing 59.54 sdg ( nhif ) , 36.07 ( smoh ) , and 31.69 sdg ( other facilities ) .", "these disparities were attributed to the relatively high percentage of patients with chronic diseases in nhif facilities ( 36  % ) , with smoh facilities having fewer such patients ( 18  % ) .", "the authors consider the main cause of the observed suboptimal prescription quality to be low adherence to prescribing guidelines .", "nhif facilities performed worse  than other facilities ,  which could be attributed to the accountability of non - nhif facilities ( all services provided by these facilities are reimbursed according to adherence to regulations ) , while the nhif facilities have not been subjected to that review .", "appropriate medicine use is essential to the provision of quality health services , patient safety , and the rational use of health resources .", "the present study used the largest sample to date and a unique study unit to investigate prescribing rationality among gps at different types of health facilities providing health services for insured patients in gezira state , sudan .", "the study revealed that the overall irdp score for gps was less than optimal and characterized by significant disparities between gps at different health facilities , with nhif facilities receiving a lower index .", "further studies should be conducted to determine the factors causing the considerable discrepancies ( 2.14.88 irdp ) between gps at health facilities owned by the nhif , the smoh , and others ( private interests , universities , and ngos ) .", "the opinions expressed by the authors do not necessarily reflect the opinions of the chulalongkorn university , thailand , or the national health insurance fund , sudan ."], "abstract_text": ["<S> backgroundthe appropriate use of medicines is essential for the provision of quality health services , patient safety , and the rational use of health resources . in sudan , </S>", "<S> general practitioners ( gps ) provide 80  % of insured patients health services . </S>", "<S> pharmaceutical service costs have been increasing since 2010.objectiveswe aimed to use the world health organization ( who ) and international network for the rational use of drugs prescribing indicators to assess prescription quality among gps in different types of primary healthcare centers ( phccs ) within the national health insurance fund ( nhif ) in gezira state , sudan.methodwe followed established who guidelines to conduct a cross - sectional retrospective study . </S>", "<S> the study was carried out over 6  months and involved 197 gps with valid prescriptions , representing 90  % of the total study population of 220 gps . </S>", "<S> we collected a systematic random sample of 100 prescriptions for each gp and used stata 12 to analyze the 19,700 prescriptions.resultsthe mean    standard deviation number of medications was 2.55    1.32 per patient ; 46.32  % of drugs prescribed were generics ; 54.71  % of prescriptions were for antibiotics and 12.84  % were for injectable formulations ; and 81.19  % of prescribed medicines were from the nhif medicines list . </S>", "<S> the overall index of rational drug prescribing ( irdp ) indicator was 3.39 , and the average cost per prescription was 40.57 sudanese pounds ( sdg ) . disregarding prescriptions for antibiotics , </S>", "<S> the prescribing quality of gps in nhif facilities was farther from optimal prescribing practice than those in state ministry of health - owned facilities and facilities owned by private groups , universities , and non - governmental organizations.conclusionthe present study provides strong evidence of irrational prescribing practice among gps , with significant disparities , particularly in terms of antibiotic overuse , generic drug underuse , and adherence to the nhif medicines list . </S>"], "labels": null, "section_names": ["Key Points", "Introduction", "Materials and Methods", "Data Analysis", "Results", "Discussion", "Conclusion", "Conflict of interest", "Funding"], "sections": [["a cross - sectional retrospective study was carried out according to world health organization guidelines over 6  months and included 197 general practitioners ( gps ) and 19,700 prescriptions.this study revealed that the overall index of rational drug prescribing ( irdp ) indicator for gps was below the optimal prescribing practice level.we found disparities in the irdp between gps at different health facilities .", "gps at national insurance fund facilities scored lower than those at state ministry of health facilities or other health facilities such as those owned by private interests , universities , and non - governmental organizations ."], ["the irrational use of medicines is a major problem worldwide and represents the primary source of medical waste and harm .", "the medicines component of the health services package is more variable and dynamic than other service components , particularly in terms of coverage , quality , and cost . in recent years", "the overuse of medicines is diverse , and drug - prescribing indicators in developing countries are suboptimal , with core indicators varying between countries .", "for instance , antibiotics and injectables are overused and generic medicines are underused in sudan .", "the overuse of medicines not only leads to the development of drug resistance and wastes resources but also increases morbidity and mortality [ 3 , 4 ] .", "thus , the appropriate use of medicines is an essential factor in the provision of quality health services , patient safety , and the rational use of health resources .", "the world health organization ( who ) and the international network of rational use of drugs ( inrud ) have developed widely used core prescribing indicators to assess the prescribing quality of primary healthcare facilities and to enable comparisons between health facilities and prescribers . to our knowledge , no prescriber - based study has been conducted in sudan ; one study of the national health insurance fund ( nhif ) has been conducted in health facilities .", "previous studies have been based on the facilities sampling method ( table  1 ) and have revealed antibiotic overuse [ 8 , 9 ] , underuse of generic medicines , and overprescribing of injectables .", "other studies have revealed a relatively large average number of drugs per prescription and poor adherence to the essential medicines list ( eml ) .", "these previous studies were conducted to investigate prescription patterns ; however , our study investigated the prescribing practices of general practitioners ( gps ) and used the largest sample and a unique study unit .", "we used prescribing indicators as a benchmark among gps at primary healthcare centers ( phccs ) and as a baseline for regular assessment of prescribing quality . in sudan ,", "the objective of this study was to use the who / inrud prescribing indicator to assess the quality of prescribing among gps at different types of phccs .", "we used the assessment regimen created by who / inrud to evaluate prescribing performance according to the adopted standards to monitor the improvement of rationality in prescribing practice . before rational prescribing", "can be promoted , the prescribing practices of those who provide 80  % of the services must be ascertained.table  1previous prescribing quality studies in sudanyeararealevelmean no .", "drugs / prescription% of generic% of antibiotics% of injectable% from eml1991 n. provincephccs1.46363361996 kh statephccs1.9487322981998 kh statehospital and phccs2.1415929992004 kh stateteaching hospitals ( 2)1.9436510.52007 six statesphccs2.344.6662773.52010 kh statephccs243.271.813.792.72010 kh statefour pediatric hospitals249.381.33.52012 five nhif statesphccs2.654.2641499.32012 kh statehospitals and pharmacies2.837.354.338.672.8 \n eml essential medicines list , kh state khartoum state , n. province nile province , nhif national health insurance fund , phccs primary healthcare centers previous prescribing quality studies in sudan \n eml essential medicines list , kh state khartoum state , n. province nile province , nhif national health insurance fund , phccs primary healthcare centers"], ["the study was conducted in gezira state , which represents 27  % of nhif ; we targeted all gps ( 220 ) providing services for nhif subscribers at the primary healthcare level .", "these gps provide services at three types of health facilities : ( 1 ) those owned by the nhif , ( 2 ) those owned by the state ministry of health ( smoh ) , and ( 3 ) those owned by others ( private interests , universities , and non - governmental organizations [ ngos ] ) .", "insured patients pay 25  % of prescription costs , and pharmaceutical services are provided through a network of pharmacies owned by the nhif , smoh , and others .", "we collected 100 prescriptions for each gp according to who / inrud guidelines for comparisons between prescribers .", "the reference period of retrospective data collection was 6  months , representing the last quarter of 2014 and the first quarter of 2015 .", "prescription collection was based on systematic random sampling , with an interval determined by dividing the gp s total prescriptions by 100 .", "of the 220 gps , 197 ( 90  % ) had valid prescriptions over the 6  months , and the total valid number of observations was 19,700 prescriptions . trained technical professionals collected the data .", "data were reviewed and entered twice in a microsoft excel spreadsheet before being exported to stata-12 for analysis .", "the prescriptions data were transferred to who investigation format , checked again for accuracy , entered into an excel sheet , and individually checked once more for precision and completeness .", "we used the who / inrud standard method for calculating prescribing indicators   ( table  2 ) .", "the unit of analysis used was gp ( 100 prescriptions for each gp).table  2adjusted prescribing standards from who and inrud indicatorsprescribing indicatorsoptimal leveloptimal irdpmean medicines per prescription ( whether or not drugs dispensed)<2 1medicines prescribed by generic name ( % ) 100 1prescriptions with an antibiotic ( % ) 30 1prescriptions with an injectable dosage form ( % ) 10 1medicines prescribed from nhif gp s medicines list ( % ) 1001complementary indicator , average cost of prescription ( sdg)20.31 ( proxy) \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization adjusted prescribing standards from who and inrud indicators \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization to determine prescribing rationality and quality at the gp level , we used the index of rational drug prescribing ( irdp ) developed by yuxin and mingguang .", "the irdp for each gp was calculated by compounding the indices of the five prescribing indicators .", "the index of each prescribing indicator was calculated by dividing the optimal value by the achieved value for each", ". moreover , for each type of health facility , the irdp was applied for comparison .", "finally , we analyzed 19,690 prescriptions from 197 gps  ( 10 invalid prescriptions were excluded ) .", "data at the gp level were clustered to represent the three types of health facilities ."], ["data were reviewed and entered twice in a microsoft excel spreadsheet before being exported to stata-12 for analysis .", "the prescriptions data were transferred to who investigation format , checked again for accuracy , entered into an excel sheet , and individually checked once more for precision and completeness .", "we used the who / inrud standard method for calculating prescribing indicators   ( table  2 ) .", "the unit of analysis used was gp ( 100 prescriptions for each gp).table  2adjusted prescribing standards from who and inrud indicatorsprescribing indicatorsoptimal leveloptimal irdpmean medicines per prescription ( whether or not drugs dispensed)<2 1medicines prescribed by generic name ( % ) 100 1prescriptions with an antibiotic ( % ) 30 1prescriptions with an injectable dosage form ( % ) 10 1medicines prescribed from nhif gp s medicines list ( % ) 1001complementary indicator , average cost of prescription ( sdg)20.31 ( proxy) \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization adjusted prescribing standards from who and inrud indicators \n inrud international network for the rational use of drugs , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , who world health organization to determine prescribing rationality and quality at the gp level , we used the index of rational drug prescribing ( irdp ) developed by yuxin and mingguang .", "the irdp for each gp was calculated by compounding the indices of the five prescribing indicators .", "the index of each prescribing indicator was calculated by dividing the optimal value by the achieved value for each gp . moreover , for each type of health facility , the irdp was applied for comparison .", "finally , we analyzed 19,690 prescriptions from 197 gps  ( 10 invalid prescriptions were excluded ) .", "data at the gp level were clustered to represent the three types of health facilities ."], ["the average number of medicines per prescription ( 2.55 : maximum 15 , minimum 1 ) indicated a poly - pharmacy pattern , with prescriptions involving antibiotics ( 54.71  % ) and injectable formulations ( 12.84  % ) .", "a total of 81.19  % of the prescribed medicines were from the eml , and generics accounted for 46.34  % of prescriptions , which is some distance from the optimal required level of 100  % .", "the average cost per prescription was 40.57 sudanese pounds ( sdgs ) ( table  3 ) .", "values for nhif facilities were farther from the prescribing indicator standards than were smoh and other health facilities , except for the percentage of prescriptions containing antibiotics ( nhif 45.91 vs. smoh 57.24 vs. others 56.84  % ) ( table  3).table  3prescribing indicators and health facility typestype of health facility owner or operatorsmohnhifotheroverallnumber of gps1264328197average number of drugs per prescription2.442.972.42.55medicines prescribed by generic name ( % ) 48.9638.4749.2846.34prescriptions containing antibiotics ( % ) 57.2445.9156.8454.71prescriptions containing injectable formulations ( % ) 12.5515.589.9712.84drugs prescribed from gp s medicines list ( % ) 82.9772.6589.2781.19average cost of prescription ( sdgs)36.0759.5431.6940.57irdp3.463.083.753.39 \n gp general practitioner , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , smoh state ministry of health prescribing indicators and health facility types \n gp general practitioner , irdp index of rational drug prescribing , nhif national health insurance fund , sdg sudanese pound , smoh state ministry of health the correlation between gps and irdp was statistically significant at p  <  0.001 .", "the optimal index is 5 ; however , the overall irdp score for gps was 3.39 ( range 2.14.8 ) .", "the prescribing quality indices revealed that prescribing practice was nowhere near the optimal index of 5 , with nhif facilities having a lower index ( 3.08 ) than smoh facilities ( 3.46 ) and health facilities owned by others ( 3.75 ) ( table  3 ) ."], ["the objectives of this study were to use the who / inrud prescribing indicator to assess the quality of prescribing among gps at different types of phccs .", "we conducted a cross - sectional retrospective study over 6  months that involved 197 gps with valid prescriptions , representing 90  % of the total study population ( 220 ) .", "the study revealed that the mean number of medications per prescription was 2.55    1.32 drugs , which represents poly - pharmacy according to the benchmark we applied of more than two drugs .", "the mean number of drug prescriptions was 2.55 ; this was much closer to the 2.6 found by a study conducted in nhif facilities in 2012 in five other states .", "the mean number of drugs per prescription was higher than most previously reported sudanese studies when we excluded the studies by mustafa   and mahmoud et al . , who reported 2.6 and 2.8 drugs per prescription , respectively ( table  1).when compared with other developing countries , the mean number of drugs per prescription in sudan ( 2.55 ) was less than in mali ( 3.2 ) , yemen ( 3.0 ) , uganda ( 2.9 ) , thailand ( 2.85 ) , and pakistan ( 2.7 ) [ 1923 ] .", "however , gps in sudan prescribed a higher mean number of drugs per prescription than those in india ( 2.4 ) , tanzania ( 2.2 ) , saudi arabia ( 2.08 ) , and malaysia ( 2.0 ) [ 2427 ] .", "collectively , nhif facilities had a higher mean number of medications than other facilities ( table  3 ) . the major implications of poly - pharmacy are additional avoidable costs and an increased probability of adverse drug reactions .", "a limitation of the study was that inter - country comparisons were not adjusted according to the prevalence of chronic diseases in the population .", "the percentage of generic medicines prescribed was 46.34  % , which is considerably lower than the standard of 100  % .", "the calculated percentage represents a severe underuse of generic medicines that was also lower than the middle eastern mediterranean region s average use of generics ( 57.1  % ) .", "this result is consistent with previous studies conducted in different districts in sudan ( table  1 ) . when compared with those of neighboring countries , the percentage of generic drugs per prescription in sudan ( 46.34  % ) was lower than in egypt ( 95.4  % ) , ethiopia ( 99.16  % ) , mali ( 70.4  % ) , uganda ( 91.3  % ) , and yemen ( 67.1  % ) [ 1921 , 29 , 30 ] .", "however , the percentage of generic drugs prescribed was remarkably higher in sudan than in bahrain ( 14.3  % ) and jordan ( 5.1  % ) [ 29 , 30 ] .", "gps in nhif facilities prescribed considerably fewer generics ( 38.47  % ) than those in smoh ( 48.96  % ) and other facilities ( 49.28  % ) .", "the implications of low generic use are primarily the wastage of scarce health resources and a decrease in access to pharmaceuticals because of an affordability barrier .", "the percentage of prescriptions containing antibiotics was 54.71  % , a considerably high result according to the who guideline benchmark of 30  % antibiotic use .", "it was consistent with percentages assessed previously in other states of sudan ( table  1 ) .", "the percentage in the who eastern mediterranean region was 53.6  % , slightly less than found for sudan in the current study .", "the use of antibiotics was lower in nhif facilities ( 45.91  % ) than in smoh ( 57.24  % ) and other facilities ( 56.84  % ) .", "antibiotic overuse can have a devastating impact in terms of the development of multi - drug - resistant bacteria , which can lead to unmanageable infectious diseases .", "the percentage of prescriptions containing injections was 12.84  % , which is considered relatively over the optimal level , although no strict standard exists , as the who benchmark is < 10  % .", "the average prevalence of injection use in the eastern mediterranean region is higher ( 27.1  % ) .", "studies conducted in sudan reveal progress in the rational use of injections ( table  1 ) .", "this reduction is attributable to a new malaria management protocol that focuses strongly on ingestible formulations rather than injection , which was the initial dominant medication formulation .", "the overall percentage of medications prescribed from the eml was 81.19  % , whereas the prescribing quality indicator standard is 100  % .", "eml adherence seems high ; however , in actuality , this figure is misleading because the best achievement in core prescribing indicators was this indicator worldwide . according to the who prescribing database , on average , the middle eastern mediterranean region s percentage of medicines prescribed from the eml was 90.8  % .", "previous studies in sudan have often found this prescribing core indicator to be higher than observed in this study ( table  1 ) .", "interestingly , we found eml adherence was lower in nhif facilities ( 72.65  % ) than in smoh ( 82.97  % ) and other facilities ( 89.27  % ) .", "a total of 197 gps reported an irdp of 2.14.88 , which is relatively low compared with other neighboring countries . for instance , ten health facilities in saudi arabia reported an irdp of 4.375 . in egypt ,", "the same processes were conducted in ten phccs and revealed high irdp rankings , ranging from 3.92 to 4.88 . although the irdp was not high , it was higher than the 3.32 reported in china .", "the index values for nhif facilities ( 3.08 ) were lower than for smoh ( 3.46 ) and other ( 3.75 ) facilities ( table  3 ) .", "the current study revealed that the average prescription cost was 40.57 sdg , almost double the proxy 20.31 sdg .", "a study conducted in five states in 2012 reported an average prescription cost of 20.5 sdg , almost half that observed in the current study .", "the primary cause of the cost increase is attributed to the devaluation of the national currency , particularly over the last 4  years . when comparing average costs with those in other countries , proper economic adjustment of currency values is essential .", "prescriptions from nhif facilities cost more than those from other facilities , with the average prescription costing 59.54 sdg ( nhif ) , 36.07 ( smoh ) , and 31.69 sdg ( other facilities ) .", "these disparities were attributed to the relatively high percentage of patients with chronic diseases in nhif facilities ( 36  % ) , with smoh facilities having fewer such patients ( 18  % ) .", "the authors consider the main cause of the observed suboptimal prescription quality to be low adherence to prescribing guidelines .", "nhif facilities performed worse  than other facilities ,  which could be attributed to the accountability of non - nhif facilities ( all services provided by these facilities are reimbursed according to adherence to regulations ) , while the nhif facilities have not been subjected to that review ."], ["appropriate medicine use is essential to the provision of quality health services , patient safety , and the rational use of health resources .", "the present study used the largest sample to date and a unique study unit to investigate prescribing rationality among gps at different types of health facilities providing health services for insured patients in gezira state , sudan .", "the study revealed that the overall irdp score for gps was less than optimal and characterized by significant disparities between gps at different health facilities , with nhif facilities receiving a lower index .", "further studies should be conducted to determine the factors causing the considerable discrepancies ( 2.14.88 irdp ) between gps at health facilities owned by the nhif , the smoh , and others ( private interests , universities , and ngos ) ."], ["the opinions expressed by the authors do not necessarily reflect the opinions of the chulalongkorn university , thailand , or the national health insurance fund , sudan ."], [""]]}
{"article_id": "PMC4888757", "article_text": ["blunt traumatic infrarenal aortic injuries are rare , with a few case reports in the literature .", "a more common occurrence is an intimal flap which may form after blunt injury to the aorta , and most of these will resolve with anticoagulation alone [ 1 , 2 ] .", "blunt aortic injury which initially presents as an intimal flap ( grade i ) may progress and evolve into an intramural hematoma ( grade ii ) or a pseudoaneurysm ( grade iii ) .", "aortic intimal flap progressing to dissection or pseudoaneurysm is an occurrence identified in 2% or less of the patients , and the majority of these occur in the thoracic aorta [ 1 , 2 ] .", "this article presents the case of a young woman found to have an intimal flap in the infrarenal abdominal aorta after a car accident , and the injury progressed to a pseudoaneurysm over 3 months .", "a 47-year - old woman with a history of hypertension and smoking was involved in a  high - speed motor vehicle accident and presented to the emergency department with back pain .", "computed tomographic ( ct ) scans were obtained of her abdomen and pelvis . on a single image from the abdominal scan ,", "what appeared to be either an intimal flap or a small amount of contrast extravasation outside of the infrarenal aorta could be seen .", "her care providers had differing opinions as to what was being seen on the image .", "since the abnormality was only seen on a single image , the exact etiology could not be delineated ( fig .", "the patient was observed in the hospital overnight , had minimal complaints the next day , and was discharged on aspirin . \n", "figure 1:initial ct scan shows enhancing vascular abnormality on the right side of the aorta , shown by the black arrow .", "initial ct scan shows enhancing vascular abnormality on the right side of the aorta , shown by the black arrow .", "a follow - up ct scan was obtained 3 months later . at the location of the previous aortic abnormality", ", there was a 3    1  cm pseudoaneurysm that had formed and could now be clearly seen ( figs 2 and 3 ) .", "figure 2:pseudoaneurysm now clearly seen 3 months later . \n figure 3:black arrow pointing to pseudoaneurysm with enhancing flow outside of the aortic lumen .", "operative repair was recommended , but the patient s aorta measured only 18  mm at the aortic bifurcation and was too small for even the smallest endovascular stent graft available at our institution .", "therefore , an open replacement of her infrarenal aorta was performed using a dacron 22  mm graft . in the operating room ,", "after the aorta was clamped and opened , a 4  mm tear in the intima had formed a mature channel into the pseudoaneurysm .", "the patient recovered very well and was discharged from the hospital without complications . at her", "2-year follow - up , she was in good health and had no further problems , but continues to smoke .", "blunt abdominal aortic trauma occurs in only 0.040.1% of all nonpenetrating traumas [ 3 , 4 ] . and", "the vast majority of these ( 92% ) are associated with multiple other injuries [ 3 , 5 ] .", "our patient had no other injuries , and there was no retroperitoneal hematoma or other signs of vascular injury in the area , as is usually seen on ct when the aorta is injured [ 35 ] .", "the lack of other radiographic traumatic findings added to the initial confusion as to whether there was a true injury to the aorta , or if the single image was showing artifact or an enlarged vertebral vessel .", "since the majority of intimal flaps heal with anticoagulation alone ( 55% ) or remain stable ( 40% ) , a conservative plan including daily aspirin and re - imaging in 3 months was chosen . when repeat imaging showed a 3  cm pseudoaneurysm , operative repair was necessary .", "endovascular repair was initially chosen for this case , but after measuring the patient s aorta , femoral vessels and the degree of taper at the bifurcation , there was not an appropriate endovascular prosthesis available in such a small size .", "only one study has compared conventional open repair with endovascular repair for blunt injuries of the abdominal aorta and found similar results in both groups .", "endovascular repair is generally favored since the injuries tend to be focal and discrete , making them quite amenable to a short graft .", "further , the patients usually have other significant injuries that would complicate a long , and potentially morbid , open operation on the aorta .", "although all blunt infrarenal aortic injuries are unusual , most of them consist of intimal flaps or dissections [ 3 , 58 ] .", "a post - traumatic pseudoaneurysm , as this patient had , is even more unusual after blunt trauma .", "one prior publication did document the delayed occurrence of a post - traumatic infrarenal aortic pseudoaneurysm that was diagnosed 3 years after injury .", "endovascular repair for pseudoaneurysms must include post - procedure angiography to document no evidence of a type i or type ii endoleak , which could allow filling of the aneurysm sac and later rupture .", "this case report highlights the need for delayed imaging for even minor intimal flaps or small aortic injuries after trauma , since progression does occur .", "while the vast majority of these injuries improve over time or remain stable , a small percentage will worsen and require intervention ."], "abstract_text": ["<S> blunt traumatic infrarenal aortic injuries are unusual , and the formation of a delayed pseudoaneurysm of the aorta is even more rare . in this report </S>", "<S> , a young woman developed a small intimal flap of the infrarenal aorta after a motor vehicle accident which progressed into a 3  cm pseudoaneurysm after 3 months . </S>", "<S> operative repair was successful and the patient recovered . </S>", "<S> this case illustrates the importance of repeat imaging of small blunt aortic injuries since progression can occur . </S>"], "labels": null, "section_names": ["Introduction", "Case Presentation", "Discussion", "Conflict of Interest Statement"], "sections": [["blunt traumatic infrarenal aortic injuries are rare , with a few case reports in the literature .", "a more common occurrence is an intimal flap which may form after blunt injury to the aorta , and most of these will resolve with anticoagulation alone [ 1 , 2 ] .", "blunt aortic injury which initially presents as an intimal flap ( grade i ) may progress and evolve into an intramural hematoma ( grade ii ) or a pseudoaneurysm ( grade iii ) .", "aortic intimal flap progressing to dissection or pseudoaneurysm is an occurrence identified in 2% or less of the patients , and the majority of these occur in the thoracic aorta [ 1 , 2 ] .", "this article presents the case of a young woman found to have an intimal flap in the infrarenal abdominal aorta after a car accident , and the injury progressed to a pseudoaneurysm over 3 months ."], ["a 47-year - old woman with a history of hypertension and smoking was involved in a  high - speed motor vehicle accident and presented to the emergency department with back pain .", "computed tomographic ( ct ) scans were obtained of her abdomen and pelvis . on a single image from the abdominal scan ,", "what appeared to be either an intimal flap or a small amount of contrast extravasation outside of the infrarenal aorta could be seen .", "her care providers had differing opinions as to what was being seen on the image .", "since the abnormality was only seen on a single image , the exact etiology could not be delineated ( fig .", "the patient was observed in the hospital overnight , had minimal complaints the next day , and was discharged on aspirin . \n", "figure 1:initial ct scan shows enhancing vascular abnormality on the right side of the aorta , shown by the black arrow .", "initial ct scan shows enhancing vascular abnormality on the right side of the aorta , shown by the black arrow .", "a follow - up ct scan was obtained 3 months later . at the location of the previous aortic abnormality", ", there was a 3    1  cm pseudoaneurysm that had formed and could now be clearly seen ( figs 2 and 3 ) .", "figure 2:pseudoaneurysm now clearly seen 3 months later . \n figure 3:black arrow pointing to pseudoaneurysm with enhancing flow outside of the aortic lumen .", "operative repair was recommended , but the patient s aorta measured only 18  mm at the aortic bifurcation and was too small for even the smallest endovascular stent graft available at our institution .", "therefore , an open replacement of her infrarenal aorta was performed using a dacron 22  mm graft . in the operating room ,", "after the aorta was clamped and opened , a 4  mm tear in the intima had formed a mature channel into the pseudoaneurysm .", "the patient recovered very well and was discharged from the hospital without complications . at her", "2-year follow - up , she was in good health and had no further problems , but continues to smoke ."], ["blunt abdominal aortic trauma occurs in only 0.040.1% of all nonpenetrating traumas [ 3 , 4 ] . and", "the vast majority of these ( 92% ) are associated with multiple other injuries [ 3 , 5 ] .", "our patient had no other injuries , and there was no retroperitoneal hematoma or other signs of vascular injury in the area , as is usually seen on ct when the aorta is injured [ 35 ] .", "the lack of other radiographic traumatic findings added to the initial confusion as to whether there was a true injury to the aorta , or if the single image was showing artifact or an enlarged vertebral vessel .", "since the majority of intimal flaps heal with anticoagulation alone ( 55% ) or remain stable ( 40% ) , a conservative plan including daily aspirin and re - imaging in 3 months was chosen . when repeat imaging showed a 3  cm pseudoaneurysm , operative repair was necessary .", "endovascular repair was initially chosen for this case , but after measuring the patient s aorta , femoral vessels and the degree of taper at the bifurcation , there was not an appropriate endovascular prosthesis available in such a small size .", "only one study has compared conventional open repair with endovascular repair for blunt injuries of the abdominal aorta and found similar results in both groups .", "endovascular repair is generally favored since the injuries tend to be focal and discrete , making them quite amenable to a short graft .", "further , the patients usually have other significant injuries that would complicate a long , and potentially morbid , open operation on the aorta .", "although all blunt infrarenal aortic injuries are unusual , most of them consist of intimal flaps or dissections [ 3 , 58 ] .", "a post - traumatic pseudoaneurysm , as this patient had , is even more unusual after blunt trauma .", "one prior publication did document the delayed occurrence of a post - traumatic infrarenal aortic pseudoaneurysm that was diagnosed 3 years after injury .", "endovascular repair for pseudoaneurysms must include post - procedure angiography to document no evidence of a type i or type ii endoleak , which could allow filling of the aneurysm sac and later rupture .", "this case report highlights the need for delayed imaging for even minor intimal flaps or small aortic injuries after trauma , since progression does occur .", "while the vast majority of these injuries improve over time or remain stable , a small percentage will worsen and require intervention ."], [""]]}
{"article_id": "PMC2722381", "article_text": ["strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .", "cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .", "frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .", "ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .", "cell extracts were cleared at 30,000  g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .", "resins were collected at 900  g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .", "bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .", "eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) .", "strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .", "cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .", "frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .", "ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .", "cell extracts were cleared at 30,000  g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .", "resins were collected at 900  g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .", "bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .", "eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) ."], "abstract_text": ["<S> substrates of the proteasome are recognized and unfolded by the regulatory particle ( rp ) , then translocated into the core particle ( cp ) to be degraded1 . </S>", "<S> a hetero - hexameric atpase ring , containing subunits rpt1-rpt6 , is situated within the base subassembly of the rp1 . </S>", "<S> the atpase ring sits atop the cp , with the rpt c - termini inserted into pockets in the cp26 . </S>", "<S> we have identified a novel function of the rpt proteins in proteasome biogenesis through deleting the c - terminal residue from each rpt . </S>", "<S> our results indicate that assembly of the hexameric atpase ring is templated on the cp . </S>", "<S> we have also identified an apparent intermediate in base assembly , bp1 , which contains rpn1 , three rpts , and hsm3 , a chaperone for base assembly . </S>", "<S> the rpt proteins with the strongest assembly phenotypes , rpt4 and rpt6 , were absent from bp1 . </S>", "<S> we propose that rpt4 and rpt6 form a nucleating complex to initiate base assembly , and that this complex is subsequently joined by bp1 to complete the rpt ring . </S>", "<S> our studies show that assembly of the proteasome base is a rapid yet highly orchestrated process . </S>"], "labels": null, "section_names": ["Methods summary", "Purification of BP1", "Pulse-chase analysis of BP1, native PAGE and immunoblot", "Supplementary Material"], "sections": [["strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .", "cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .", "frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .", "ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .", "cell extracts were cleared at 30,000  g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .", "resins were collected at 900  g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .", "bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .", "eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) ."], ["strains carrying proa - tev - rpt1 were grown in 6 l of sd medium to od600 0.8 to 1.2 .", "cells were harvested , washed once with ice - cold water and drop - frozen in liquid nitrogen .", "frozen yeast samples were then ground using an mm301 grinding mill ( restch ) under liquid nitrogen following manufacturer s instructions , or using a mortar and a pestle as previously described24 .", "ground powder was hydrated in proteasome buffer ( 50 mm tris - hcl [ ph7.5 ] , 5 mm mgcl2 , 1 mm edta , and 10% glycerol ) supplemented with 2 mm atp , protease inhibitor tablets ( complete , roche ) , 2 mm pmsf , 1 mm benzamidine , 10 g / ml pepstatin a , and 1 g / ml antipain .", "cell extracts were cleared at 30,000  g for 30 min at 4c , and the supernatants were mixed with rabbit igg resin ( cappel , mp biomedicals ) for 90 min at 4c .", "resins were collected at 900  g for 2 min at 4c and washed with proteasome buffer containing 50 mm nacl three times , followed by a final wash with proteasome buffer alone .", "bp1 was then released from the resin by incubating with actev protease ( invitrogen ) at 2.5 unit / l culture in proteasome buffer containing 2 mm atp and protease inhibitors for 1 hr at 30c .", "eluates were concentrated using ultrafree-0.5 centrifugal filter device with 30 kda nmwl ( millipore ) ."], [""], [""]]}
{"article_id": "PMC4692009", "article_text": ["worldwide , head injury is the single largest cause of death and disability following an injury .", "the burden of head injury is greatest in low- and middle - income countries ( lamic ) , where 85% of the world 's population live .", "the world health organization estimates that almost 90% of deaths due to injuries occur in these settings .", "head injury is the leading cause of disability in people under 40 , severely disabling 150200 people per million annually . in 2005", ", road traffic injuries resulted in the death of an estimated 110,000 persons , 2.5 million hospitalizations , 89 million minor injuries and economic losses to the tune of 3% of the gross domestic product in india .", "the accident rate of 35/1000 vehicles in india is also amongst the highest in the world .", "however , resources have not been diverted adequately by governments toward prevention , management and rehabilitation of head injuries in lamic such as india .", "most patients with severe traumatic brain injuries ( tbi ) in developing countries are discharged to home - based care due to lack of rehabilitative facilities and health insurance .", "those patients who are discharged in an unconscious state are particularly difficult to manage at home and are prone to decubitus ulcers , respiratory infections , inadequate nutrition , and physiotherapy .", "however , data is lacking on the actual load of vegetative patients and their outcomes , especially from developing countries .", "patients , who remain unresponsive to the environment even though their eyes may be open , are considered to be in a vegetative state ( vs ) . a persistent", "vs has been defined as a vs still present 1 month after acute traumatic or nontraumatic brain damage . as by definition", ", vs requires a minimum period of 1 month of remaining unconscious , most patients who remain unconscious are not labeled as vegetative at discharge", ". however , there are no studies assessing conversion to vs , specifically in this group of patients .", "in this retrospective study carried at one trauma center in india , a prospectively maintained neurotrauma registry was queried from may 2010 to february 2013 for patients who had severe traumatic brain injury glasgow coma score ( gcs  8) at admission and had a motor response of m5 or lower on the gcs at discharge .", "unconsciousness was described as patients with a motor score of m5 or lower on the gcs irrespective of the verbal ( v ) or eye ( e ) response .", "demographic and clinical data was analyzed , and outcome assessed at 6 months after injury using a telephonic questionnaire .", "the questions asked were : ( 1 ) is the patient alive ? ( 2 ) if alive , is the patient responding to commands ( conscious ) ? ( 3 ) if the patient is conscious , is he able to perform activities of daily living like feeding himself , going to toilet independently and going to preinjury occupation ? if the family members replied that patient had gone back to preinjury occupation it was considered as glasgow outcome scale ( gos ) 1 , if patient was able to perform activities of daily living , but not able to go back to work it was considered as gos 2 , if patient was conscious but not able to do activities of daily living , it was considered as gos 3 . if a patient remained unconscious , it was gos 4 and if the patient had died it was gos 5 .", "gos 13 was considered as the unfavorable outcome and gos 45 was considered as a good outcome .", "of these 166 ( 10.9% ) were unconscious ( motor response m5 on the gcs or lower ) at discharge from the hospital .", "average hospital stay was 24.31 days ( range : 864 days ) and was 16.71 days for m5 , 20.63 days for m4 , 26.33 days for m3 , 30.59 days for m2 and 16.3 days for m1 patients [ table 1 ] .", "mortality and outcome at 6 months with respect to motor scores at the time of discharge the discharge motor score was m5 in 32 ( 19.3% ) , m4 in 44 ( 26.5% ) , m3 in 59 ( 35.5% ) , m2 in 44 ( 26.5% ) and m1 in 9 ( 5.4% ) .", "54 ( 52.9% ) patients had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months .", "100% of m1 , 90.6% of m2 , 94.3% of m3 , 72.2% of m4 , and 60% of m5 patients had an unfavorable outcome at 6 months .", "only 16 patients ( 15.7% ) had a good outcome ( gos 12 ) at 6 months post injury .", "incidence , as well as the severity of head injuries , the , is rising in developing countries due to rapid industrialization with a lag in legal , healthcare , and safety reforms .", "delhi with a population of around 15 million has the dubious distinction of having the largest number of road traffic accidents of any city in india .", "the enormity of burden can be assessed by the fact that our study had more than 1500 severe head injured patients in <3 years and was conducted at only one hospital !", "one of the reasons for the step - motherly attitude by policy makers toward head injury prevention , management , and rehabilitation , is the lack of hard statistics of the actual burden of disease which head injuries carry . in spite of the improvement in care for head injured patients ,", "there has not been a marked decline in the mortality rates for severe head injuries .", "kagan and baker found that mortality rates were between 26.7% and 41.4% for severe tbi patients in level 1 trauma centers .", "fakhry et al . in their study found 28.8% mortality rate of severe tbi patients .", "it has also been shown that developing ( low and middle income ) countries have a pooled mortality rate of 51% for severe tbi as compared to 30% for high - income countries .", "nevertheless , in - hospital mortality tells only part of the story on the outcome of these patients .", "six months outcome is often poorer due to lack of rehabilitative care and facilities , especially in developing countries .", "most of the patients are discharged to home - based rehabilitation with tracheostomy and oro - gastric tube in situ . in our center ,", "unconscious patients with severe tbi are discharged with tracheostomy tube in situ after ensuring that patient can maintain oxygen saturation on room air and after training relatives regarding care of tracheostomy tube , suctioning , and home - based rehabilitation .", "interestingly , however , there are no studies on the outcome of patients who are discharged from the acute care facility in this unconscious state .", "furthermore , a significant number ( 10.9% in our study ) are discharged in the unconscious state . as all studies are based on admission gcs , it is also often not possible to assess how many patients remained unconscious at the time of discharge and became vegetative at 6 months .", "the outcomes of these patients have also not been studied properly . in our study , patients with m2 motor response had the longest hospital duration with unfavorable outcome occurring in 90% of these patients at 6 months .", "gcs has remained a robust scale for assessment and prognostication in tbi and the motor score ( m response ) continues to have the highest predictive value within the gcs .", "we , therefore , decided to use motor response as criteria to differentiate unconscious and conscious patients .", "it may be sometimes difficult to differentiate unconsciousness from dysphasia as both disease states may show a motor response of 5 . however , left sided contusion in the posterior frontal and/or temporal regions along with the presence of gaze fixation are clues towards dysphasia .", "our study had a reasonable follow - up of 61.4% of the patients at 6 months and showed the poor outcomes in this subgroup of patients .", "54 ( 52.9% ) patients ( who were unconscious at discharge ) had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months with only 16 patients ( 15.7% ) having good outcome ( gos 12 ) at 6 months .", "this study also shows that the majority of the patients ( nearly 85% ) who are discharged in an unconscious state will either die or become vegetative .", "this is the only study of its kind on patients who remain unconscious at discharge following severe tbi and reveals that around 50% will die , and another 30% become vegetative within 6 months of discharge ."], "abstract_text": ["<S> introduction : it is well - known that severe traumatic brain injuries ( tbi ) have a poor outcome . </S>", "<S> however , what is not well - known is the outcome for those who survive but remain unconscious at the time of discharge from the hospital.aims and objectives : to assess the outcome of severe tbi patients who have a motor response of m5 or lower on the glasgow coma score ( gcs ) at discharge from a single centre in india.materials and methods : in this retrospective study carried at one trauma centre in india , a prospectively maintained neurotrauma registry was queried from may 2010 to february 2013 for patients who had severe traumatic brain injury ( gcs  8) at admission and had a motor response of m5 or lower on the gcs at discharge . </S>", "<S> demographic and clinical data were analyzed , and outcome glasgow outcome scale ( gos ) assessed at 6 months using a telephonic questionnaire.observations and results : there were a total of 1525 patients with severe tbi in the study period . </S>", "<S> of these 166 ( 10.9% ) were unconscious ( motor response m5 or lower on the gcs ) at discharge from the hospital . </S>", "<S> 139 were males and 27 females with a mean age of 33.9 years . </S>", "<S> after a mean hospital stay of 24.31 days , the discharge motor score was m5 in 32 ( 19.3% ) , m4 in 44 ( 26.5% ) , m3 in 59 ( 35.5% ) , m2 in 44 ( 26.5% ) , and m1 in 9 ( 5.4% ) . </S>", "<S> telephonic follow - up was available in 102 ( 61.4% ) of the patients . </S>", "<S> 54 ( 52.9% ) patients had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months . </S>", "<S> only 16 patients ( 15.7% ) had a good outcome ( gos 12 ) at 6 months following an injury.conclusions:this is the only study of its kind on patients who remain unconscious at discharge following severe tbi and reveals that around 50% will die and another 30% remains vegetative at 6 months of discharge . </S>", "<S> only a small percentage ( 15% in our study ) will become conscious and partially integrated in the society . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Observations and Results", "Discussion", "Conclusions"], "sections": [["worldwide , head injury is the single largest cause of death and disability following an injury .", "the burden of head injury is greatest in low- and middle - income countries ( lamic ) , where 85% of the world 's population live .", "the world health organization estimates that almost 90% of deaths due to injuries occur in these settings .", "head injury is the leading cause of disability in people under 40 , severely disabling 150200 people per million annually . in 2005", ", road traffic injuries resulted in the death of an estimated 110,000 persons , 2.5 million hospitalizations , 89 million minor injuries and economic losses to the tune of 3% of the gross domestic product in india .", "the accident rate of 35/1000 vehicles in india is also amongst the highest in the world .", "however , resources have not been diverted adequately by governments toward prevention , management and rehabilitation of head injuries in lamic such as india .", "most patients with severe traumatic brain injuries ( tbi ) in developing countries are discharged to home - based care due to lack of rehabilitative facilities and health insurance .", "those patients who are discharged in an unconscious state are particularly difficult to manage at home and are prone to decubitus ulcers , respiratory infections , inadequate nutrition , and physiotherapy .", "however , data is lacking on the actual load of vegetative patients and their outcomes , especially from developing countries .", "patients , who remain unresponsive to the environment even though their eyes may be open , are considered to be in a vegetative state ( vs ) . a persistent", "vs has been defined as a vs still present 1 month after acute traumatic or nontraumatic brain damage . as by definition", ", vs requires a minimum period of 1 month of remaining unconscious , most patients who remain unconscious are not labeled as vegetative at discharge", ". however , there are no studies assessing conversion to vs , specifically in this group of patients ."], ["in this retrospective study carried at one trauma center in india , a prospectively maintained neurotrauma registry was queried from may 2010 to february 2013 for patients who had severe traumatic brain injury glasgow coma score ( gcs  8) at admission and had a motor response of m5 or lower on the gcs at discharge .", "unconsciousness was described as patients with a motor score of m5 or lower on the gcs irrespective of the verbal ( v ) or eye ( e ) response .", "demographic and clinical data was analyzed , and outcome assessed at 6 months after injury using a telephonic questionnaire .", "the questions asked were : ( 1 ) is the patient alive ? ( 2 ) if alive , is the patient responding to commands ( conscious ) ? ( 3 ) if the patient is conscious , is he able to perform activities of daily living like feeding himself , going to toilet independently and going to preinjury occupation ? if the family members replied that patient had gone back to preinjury occupation it was considered as glasgow outcome scale ( gos ) 1 , if patient was able to perform activities of daily living , but not able to go back to work it was considered as gos 2 , if patient was conscious but not able to do activities of daily living , it was considered as gos 3 . if a patient remained unconscious , it was gos 4 and if the patient had died it was gos 5 .", "gos 13 was considered as the unfavorable outcome and gos 45 was considered as a good outcome ."], ["of these 166 ( 10.9% ) were unconscious ( motor response m5 on the gcs or lower ) at discharge from the hospital .", "average hospital stay was 24.31 days ( range : 864 days ) and was 16.71 days for m5 , 20.63 days for m4 , 26.33 days for m3 , 30.59 days for m2 and 16.3 days for m1 patients [ table 1 ] .", "mortality and outcome at 6 months with respect to motor scores at the time of discharge the discharge motor score was m5 in 32 ( 19.3% ) , m4 in 44 ( 26.5% ) , m3 in 59 ( 35.5% ) , m2 in 44 ( 26.5% ) and m1 in 9 ( 5.4% ) .", "54 ( 52.9% ) patients had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months .", "100% of m1 , 90.6% of m2 , 94.3% of m3 , 72.2% of m4 , and 60% of m5 patients had an unfavorable outcome at 6 months .", "only 16 patients ( 15.7% ) had a good outcome ( gos 12 ) at 6 months post injury ."], ["incidence , as well as the severity of head injuries , the , is rising in developing countries due to rapid industrialization with a lag in legal , healthcare , and safety reforms .", "delhi with a population of around 15 million has the dubious distinction of having the largest number of road traffic accidents of any city in india .", "the enormity of burden can be assessed by the fact that our study had more than 1500 severe head injured patients in <3 years and was conducted at only one hospital !", "one of the reasons for the step - motherly attitude by policy makers toward head injury prevention , management , and rehabilitation , is the lack of hard statistics of the actual burden of disease which head injuries carry . in spite of the improvement in care for head injured patients ,", "there has not been a marked decline in the mortality rates for severe head injuries .", "kagan and baker found that mortality rates were between 26.7% and 41.4% for severe tbi patients in level 1 trauma centers .", "fakhry et al . in their study found 28.8% mortality rate of severe tbi patients .", "it has also been shown that developing ( low and middle income ) countries have a pooled mortality rate of 51% for severe tbi as compared to 30% for high - income countries .", "nevertheless , in - hospital mortality tells only part of the story on the outcome of these patients .", "six months outcome is often poorer due to lack of rehabilitative care and facilities , especially in developing countries .", "most of the patients are discharged to home - based rehabilitation with tracheostomy and oro - gastric tube in situ . in our center ,", "unconscious patients with severe tbi are discharged with tracheostomy tube in situ after ensuring that patient can maintain oxygen saturation on room air and after training relatives regarding care of tracheostomy tube , suctioning , and home - based rehabilitation .", "interestingly , however , there are no studies on the outcome of patients who are discharged from the acute care facility in this unconscious state .", "furthermore , a significant number ( 10.9% in our study ) are discharged in the unconscious state . as all studies are based on admission gcs , it is also often not possible to assess how many patients remained unconscious at the time of discharge and became vegetative at 6 months .", "the outcomes of these patients have also not been studied properly . in our study , patients with m2 motor response had the longest hospital duration with unfavorable outcome occurring in 90% of these patients at 6 months .", "gcs has remained a robust scale for assessment and prognostication in tbi and the motor score ( m response ) continues to have the highest predictive value within the gcs .", "we , therefore , decided to use motor response as criteria to differentiate unconscious and conscious patients .", "it may be sometimes difficult to differentiate unconsciousness from dysphasia as both disease states may show a motor response of 5 . however , left sided contusion in the posterior frontal and/or temporal regions along with the presence of gaze fixation are clues towards dysphasia .", "our study had a reasonable follow - up of 61.4% of the patients at 6 months and showed the poor outcomes in this subgroup of patients .", "54 ( 52.9% ) patients ( who were unconscious at discharge ) had died and 32 ( 31.4% ) remained unconscious ( vegetative ) at 6 months with only 16 patients ( 15.7% ) having good outcome ( gos 12 ) at 6 months .", "this study also shows that the majority of the patients ( nearly 85% ) who are discharged in an unconscious state will either die or become vegetative ."], ["this is the only study of its kind on patients who remain unconscious at discharge following severe tbi and reveals that around 50% will die , and another 30% become vegetative within 6 months of discharge ."]]}
{"article_id": "PMC5243954", "article_text": ["their marbling has been increased over many decades to meet domestic consumer preferences . in both countries ,", "highly marbled beef is greatly prized for traditional meat cooking methods such as sukiyaki for japanese and gogigui for korean . because of these demands , the use of heifers and steers instead of bulls , intensive feeding system , and genetic ability of wagyu and hanwoo cattle have resulted in greater fat deposition in these breeds compared to european breeds . as intramuscular fat ( imf ) improves beef quality at least in juiciness and flavor ( hornsterin and wasserman , 1987 ; wheeler et al . , 1994 )", "it is assessed in abattoirs by meat graders in various countries , including usa , australia , japan , and korea . like other kinds of foods , meat has three functions : 1 ) it provides nutrition ; 2 ) it provides deliciousness ; and 3 ) it prevents disease .", "although beef has these three functions , the main food in both japan and korea is boiled rice while beef is a side dish .", "therefore , these two countries have developed the quality of beef rather than its quantity .", "this is quite different from foreign countries where meat is consumed as a main dish . in japanese and korean cuisine , soft and delicious beef with imf and a good red color", "in the past , fat was not given a good image in its role towards human health , although fat is an important energy resource for human .", "recently , fat has been reported to have fewer adverse effects on health than carbohydrates , especially simple carbohydrates .", "in fact , meat has played a crucial role in human evolution of a healthy and well balanced diet ( pereira and vicente , 2013 ) .", "furthermore , meat plays a pivotal role in nutritious diets . high quality marbled beef not only has excellent eating quality , but also contains a lot of beneficial fatty acids ( troy et al . , 2016 ) . in this regard ,", "the current paper reviews the characteristics and health benefit of highly marbled beef from wagyu and hanwoo cattle .", "it has well known that wagyu cattle have high potential of accumulating imf and producing highly marbled beef .", "hanwoo cattle are also known for their high imf for marbled beef similar to wagyu beef .", "highly marbled wagyu loin contains more than 40% of imf , sometimes more than 60% ( horii et al . , 2009 ) , while quality grade 1 hanwoo , the highest quality grade , has approximately 28% of imf in longissimus thoracis muscle ( hwang and joo , 2016 ) .", "wagyu cattle include four types of japanese cattle : the black , brown , short horn , and polled breeds .", "numerous studies have investigated the meat quality , quantity , and muscle physiology of crossbreed wagyu ( japanese black cattle ) in foreign countries ( cafe et al . , 2006 ; cafe et al . , 2009 ; greenwood et al . , 2006 ; greenwood et al . , 2009 ; may et al . , 1993 ) .", "they are identified by different coat color : brown ( major hanwoo ) , black face ( heukwoo ) , black ( jeju heukwoo ) , and tiger color ( chickso ) ( jo et al . , 2012 ) .", "in this review , wagyu and hanwoo are used to describe the japanese black breed and the brown coat color hanwoo , respectively .", "all four types of wagyu cattle have played important roles locally and in the history of mixed farming .", "they also played important roles in the synergies between cattle and crops , especially rice .", "farmers gradually began to replace the role of cattle as draft animals and started to use industrial fertilizers approximately 50 years ago . in recent years , japanese wagyu cattle have been raised more specifically for beef production .", "the famous brand name wagyu not only includes the japanese black cattle produced in japan , but also includes animals or even cross - bred japanese black cattle produced in foreign countries such as australia and the united states .", "similarly , the utilization of hanwoo cattle as an edible meat had been minimal for long time .", "full - scale production of hanwoo as meat - type cattle has started since the 1970s . because hanwoo cattle have maintained stable traits through pure breeding , the current blood lineage is very valuable .", "it is mainly spread in the korean peninsula ( kim and lee , 2000 ) .", "recently , hanwoo beef has been reported to have highly marbled imf similar to wagyu beef .", "especially , hanwoo beef has relatively thin muscle fiber and minimal content of connective tissues ( kim et al . , 1994 ) .", "it has less subcutaneous fat depth with greater ossification scores and marbling scores than those of australian angus ( cho et al . , 2005 ) .", "in 2013 , a total of 2.64 million heads of cattle were fed for beef production in japan .", "approximately 1.71 million heads were japanese black cattle ( maff , 2013 ) , and approximately 873,400 were holstein cattle .", ", the number of farmers producing beef was 613,000 , but 86.5% of these farmers fed less than 50 heads of cattle .", "the mean body weight and carcass weight of beef at slaughter ( 26 - 30 mon of age ) were 725 kg and 470 kg , respectively .", "high performance marbled beef production has caused japanese black cattle to comprise the greatest share of japan s wagyu cattle population ( albrecht et al . , 2011 ;", "recently , the imf percentage of beef from japanese black cattle has an average value of greater than 30% ( albrecht et al . , 2011 ; horii et al . ,", "the total number of slaughter cattle was 1,007,000 , including 883,593 hanwoo cattle , 66,485 holstein cows , and 56,923 holstein heifers and bulls ( kape , 2015 ) .", "the number of cattle farming households was 99,858 , including 89,403 hanwoo farmers ( kape , 2015 ) . during the last decade ,", "the number of households raising hanwoo cattle has drastically decreased from 186,000 households in 2006 to 89,403 households in 2015 ( kape , 2016 ) .", "the average live and carcass weights of hanwoo cattle at slaughter ( 26 - 30 mon of age ) were 719 kg and 430 kg , respectively ( kape , 2015 ) .", "wagyu carcasses are evaluated by accredited graders from the japan meat grading association ( jmga ) in accordance with beef carcass grading standards .", "first established in 1988 , the present grading system assigns both yield grade ( a , b , and c ) and meat quality grade ( 1 , 2 , 3 , 4 , and 5 ) ( jmga , 2014 ) .", "in korea , all cattle carcasses should be evaluated by korean carcass grading system . established in 1992 ,", "the korean carcass grading system presently has three levels of yield grade ( yg ) for meat amount ( a , b , and c ) and five levels of meat quality grade ( qg ) ( 1 , 1 , 1 , 2 , and 3 ) ( kape , 2016 ) . for beef quality grading in japan ,", "all cattle carcasses are graded at the 6 to 7 rib section at least one hour after ribbing .", "the following four items are independently evaluated : beef marbling ; meat color and brightness ; meat firmness and texture ; and fat color , luster , and quality . meat quality grade of the carcass is then assigned according to the lowest grade of these four items .", "korean beef quality grading is also estimated based on several factors , including marbling score , meat color , fat color , firmness and texture of lean meat , and maturity of the exposed loin muscle at the 13 rib interface .", "this means that marbling score is the most dominating determinant in korea because korean consumers have an extraordinary preference for high marbled meats . in 1988 , wagyu marbling levels were assigned by the beef marbling standard ( bms ) using a plastic model made from silicone resin .", "this standard was calculated based on the circumference and area percentage of marbling particles in the rib eye section ( longissimus thoracis ) . in october 2008 , a new marbling standard using carcass photographs replaced the 1988 standard . in march 2014 , an even newer marbling standard", "1 ) . graders now determine the bms number ( 1 to 12 ) by comparing the actual carcass marbling to the standard photograph of marbling . during this process", ", any larger inclusions of fat at the periphery of the rib eye are not considered as marbling according to the japanese grading system .", "the bms of korean carcass grading system has been changed by the addition of marbling number . in 1992 , when the carcass grading system was established for the first time in korea , the bms had only 5 numbers ( 1 to 5 ) with 3 qg ( 1 , 2 , and 3 ) . however , in 1997 , new qg 1 was added with new bms no . 6 and no .", "furthermore , in 2004 , another new qg 1 was added with new bms no . 8 and no .", "hanwoo beef with qg 1 or 1 is considered as a premium class of beef in korea .", "of hanwoo cattle slaughtered in 2015 , 10.0% were qg 1 , 26.4% were qg 1 , and 31.4% were qg 1 ( kape , 2016 ) .", "the plentiful marbling of wagyu and hanwoo beef has attracted attention . in both japan and korea ,", "the value of cattle carcasses is determined by a qg which considers marbling as a decisive determinant . since the liberalization of beef importation", ", marbling has been greatly emphasized to differentiate domestic beef from imported beef ( hirooka , 2014 ; hwang et al . , 2010 ) .", "the high content of imf can improve the texture and juiciness of hanwoo beef and thereby its acceptability ( jung et al . , 2016 ) .", "korean consumers prefer qg 1 or 1 beef because of its high imf content ( kim et al . , 1999 ) .", "2015 ) have demonstrated that an increase in crude fat content ( range 23.8 - 48.6% ) can increase the tenderness , juiciness , and fattiness .", "however , they also reported that an increase in crude fat content can reduce the crude protein content and slightly reduce the content of umami components such as nucleic acid and glutamic acid .", "it is well known that imf content varies depending on feeding time , finishing diet , and breed type . to produce high qg beef ,", "great attention has been paid to more accumulation of imf in wagyu and hanwoo muscle .", "one of good strategy to increase imf content in beef muscle is to extend slaughtering age . although the marbling score is increased and reached a plateau at about 24 mon of age ( choi et al . ,", "2002 ) , the slaughtering age of hanwoo has been extended to increase the bms score ( jo et al . , 2012 ) .", "in korea , the marketing age of hanwoo has been extended to an average of 31 mon with weight of 719 kg to fatten the cattle ( kape , 2015 ) .", "however , average daily gain is decreased due to increased slaughtering weight ( paek et al . , 1993 ) .", "recently , cattle in china are fed for unusually long periods of time before slaughter as wagyu and hanwoo .", "this might have contributed to their high imf and oleic acid contents ( smith , 2016 ; tanaka , 1985 ) .", "it is clear that imf increases with feeding time for grain - fed and pasture - fed cattle .", "however , the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle ( smith et al . , 2009 ) .", "it has been reported that wagyu fed on a high - concentration diet have higher expression of adipogenic transcription factors in the subcutaneous and intramuscular adipocytes than those fed on a high - rough - age diet ( yamada and nakanishi , 2012 ) .", "the imf content and the numbers of preadipocytes and adipocytes are reported to be higher in wagyu than those in angus ( duarte et al . , 2013 ) .", "( 2009 ) have reported that the imf contents in the longissimus muscle of wagyu , german angus , belgain blue , and holstein friesian are 23.3% , 4.4% .", "the wagyu and european cattle breeds did not differ in their mechanisms of postnatal fat accretion .", "however , they differed in their efficiency of accretion of imf ( gotoh et al . , 2009 ) .", "for every 1% increase of imf in the longissimus muscle , the increase amounts of subcutaneous adipose tissue in wagyu , holstein friesian , german angus , and belgain blue were 3.0 , 4.3 , 7.9 , and 10.7 kg , respectively ( gotoh et al . , 2009 ) .", "although imf content is the most dominating determinant of beef quality , the imf content is not the only parameter that decides the quality grade of beef carcass .", "marbling is called  shimo - furi  in japanese and  sang - gang  in korean .", "it literally means  frosting  . in japan , marbling with a fine appearance resembling frost is highly valued , but coarse marbling is not ( motoyama et al . ,", "recently , korea also began to discriminate between fine and coarse marbling in hanwoo beef .", "this marbling quality contributes to the tenderness of beef because imf deposits are found mainly between muscle fiber bundles , resulting in the disorganization of perimysium connective tissue ( nishimura , 2015 ; sasaki et al . , 2012 ) .", "therefore , the sensory of tenderness could be qualitatively affected by histological difference in marbling due to difference in tissue disorganization extent .", "there are several types of fatty acids : 1 ) monounsaturated fatty acids ( mufa ) , 2 ) polyunsaturated fatty acids ( pufa ) , and 3 ) saturated fatty acid ( sfa ) .", "pufa such as linoleic acid , -linolenic acid ( n-3 ) , -linolenic acid ( n-6 ) , arachidonic acid , and so on contain many important compounds such as essential fatty acids .", "the fatty acid that has the highest amount in beef is oleic acid ( c18:1n-9 ) .", "it has been reported that fatty acid compositions are different depending on breeds ( smith et al . , 2006", "the fatty acid compositions in highly marbled wagyu and hanwoo are considerably different from those in other cattle breeds .", "highly marbled wagyu beef has a higher percentage of mufa within fat compared with other breeds ( yang et al . , 1999a ) .", "( 2006 ) have investigated oleic acid concentrations in the subcutaneous adipose tissues of wagyu , hanwoo , australian crossbred , angus ( corn - fed ) , angus ( hay - fed ) , and angus ( weaned ) and found that they are 52.9% , 47.3% , 39.8% , 39.8% , 34.6% , and 32.9% , respectively .", "a higher percentage of mufa will lead to a lower fat - melting point which contributes to the softness of beef fat and favorable beef flavor .", "it may decrease the circulating concentration of ldl cholesterol in consumers ( melton et al .", ", 1982 ; rudel et al . , 1995 ; smith , 1994 ) . therefore , fatty acid compositions of beef have recently become important in the beef industry , especially in highly marbled wagyu and hanwoo cattle", "( 1995 ) have investigated the effect of breed type ( including japanese black ) and sex on fatty acid compositions of subcutaneous and intramuscular lipids in finishing steers and heifers of pure japanese black and holstein as well as crossbred japanese black , holstein , japanese brown , and charolais .", "they have reported that the japanese black is genetically predisposed to producing carcass lipids containing higher concentrations of mufa than holstein , japanese brown , or charolais steers ( zembayashi et al . , 1995 ) .", "1992 ) have also concluded that beef from purebred wagyu cattle raised in japan is rich in mufa .", "( 2011 ) have compared intramuscular fatty acid composition of longissimus muscle in 26-month - old japanese black steers and holstein steers reared and fattened using a standard fattening system ( table 1 ) . in the longissimus muscle of japanese black steers ,", "a higher percentage of unsaturated fatty acid was found than that in holstein steers ( gotoh et al . , 2014 )", "2011 ) have also compared the imf content and fatty acid compositions of 21 major skeletal muscles using the same animals .", "muscles from the japanese black cattle contained a greater proportion of numerous fatty acids , particularly mufa such as c16:1 , c18:1 , and c20:1 compared to fatty acids in holstein cattle .", "in japanese black cattle , the proportion of sfa including c18:0 was much lower compared to that in holstein cattle .", "sfa : saturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .", "2005 ) have investigated the fatty acid compositions of hanwoo and australian angus beef and found a significant difference in fatty acid compositions between these two cattle breeds ( table 2 ) .", "especially , angus beef had significantly higher n-3 pufa while hanwoo beef contained greater n-6 pufa in three different muscles ( cho et al . , 2005 ) .", "the difference in fatty acid composition might be attributed to the influence of different diets , forage , and grain feeding , although fatty acid profile in ruminants is not a direct reflection of the dietary fatty acid composition due to hydrogenation by rumen microorganism ( enser et al . , 1998 ) .", "sfa : saturated fatty acids , usfa : unsaturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .", "f - ratio statistic : * if p<0.05 , * * if p<0.01 , * * * if p<0.001 .", "therefore , it can be easily anticipated that hanwoo beef has a fatty acid profile similar to that of high concentratefed animals ( jo et al . , 2012 ) .", "recently , hwang and joo ( 2016 ) have evaluated the fatty acid profile of ten muscles from high marbled ( qg 1 ) and low marbled ( qg 2 ) hanwoo carcass and found significant differences in fat content and fatty acid composition among 10 muscles and between high and low marbled hanwoo beef . in particular , high marbled hanwoo muscles had significantly higher proportion of mufa due to higher oleic acid ( c18:1 ) proportion , while low marbled hanwoo muscles had higher proportion of sfa due to higher proportion of stearic acid ( c18:0 ) ( hwang and joo , 2016 ) .", "stearoyl - coa desaturase ( scd ) was first identified and reported as one of the genes associated with beef fatty acid composition ( taniguchi et al . ,", "the composition of fatty acids stored in fat depots reflects the earlier action of scd on substrates such as stearic acid and palmitic acid ( kim and ntambi , 1999 ) .", "1999b ) have reported interesting correlations between scd enzyme activity and fatty acid composition in bovine adipose tissue .", "although the adipogenic mechanism is extremely complicated , several genes have been identified and confirmed as either associated with or responsible for the fatty acid composition in wagyu cattle ( gotoh et al . , 2014 ) .", "it is generally accepted that the concentration of oleic acid in beef adipose tissue is dependent on scd expression and activity .", "wagyu cattle are genetically disposed to produce more oleic acid ( smith et al . , 2006 ) .", "very high heritability has been reported for oleic acid in wagyu cattle ( nogi et al . , 2011 ) .", "higher levels of concentrated feed in the later fattening period can lead to higher mufa concentration in the subcutaneous adipose tissues of wagyu steer ( kimura et al . ,", "interest in beef fat and fatty acids has been increasing , especially in highly marbled beef such as wagyu and hanwoo because fatty acids composition in the diet have impact on human health .", "consumption of fat and cholesterol has been reported to be linked to cardiovascular disease , obesity , and cancer ( micha et al . , 2010 ;", "consequently , reduction of total fatty acid intake and replacement of sfa with pufa have been recommended .", "ulbricht and southgate ( 1991 ) have demonstrated that stearic acid has no effect on plasma cholesterol level and that oleic acid can lower serum cholesterol similar to pufa .", "furthermore , pavan and duckett ( 2013 ) have suggested that a higher proportion of oleic acid in beef is desirable because the consumption of high - oleic acid ground beef can increase hdl - cholesterol concentration ( gilmore et al . , 2011 ) . according to smith ( 2016 ) ,", "the amount of fat consumed in a typical portion of beef will not increase risk factors for cardiovascular disease .", "clinical trials have demonstrated that ground beef containing elevated oleic acid can increase the concentration of hdl - cholesterol or at least has no negative effect on the concentration of hdl - cholesterol . in earlier research on oleic acid , the major mufa in beef , grundy et al .", "( 1988 ) have found that it can lower ldl - cholesterol without affecting beneficial hdl - cholesterol .", "( 2014 ) have reported that mufa can normalize or improve lipid metabolism and maintain the balance in cardiac muscle .", "these have implied that mufa have little effect on total cholesterol and that they are heart - healthy dietary fat that can lower ldl - cholesterol and increase hdl - cholesterol ( lahey et al . , 2014 ) .", "this effect is repeatable when natural foods are used to supplement diets with oleic acid . in this", "regard , smith ( 2016 ) have concluded that beef cattle should be raised under production conditions to increase the concentration of oleic acid in their edible tissue , i.e. , by grain feeding over extended periods of time .", "it is obvious that consumer in the world has an overwhelmingly negative attitude toward animal fats , especially saturated fat in meat for the last several decades ( ngapo and dransfield , 2006 ; williams and droulez , 2010 ) .", "according to higgs ( 2000 ) , the per capita decline in beef consumption in the us and other western countries has been attributed in large part to animal fat phobia .", "consumers have been warned to reduce saturated fat in their diet and to avoid meat cuts containing high fat content .", "these health recommendations are obviously in conflict with the health of highly marbled wagyu and hanwoo beef .", "many research studies have shown that the imf of wagyu and hanwoo beef contains a lot of mufa that could prevent arteriosclerosis .", "researches have also demonstrated that high - oleic acid ground beef may reduce risk factors for cardiovascular disease ( adams et al .", "thus , although some consumers in japan and korea consider highly marbled wagyu and hanwoo beef as being unhealthy , there is no scientific evidence to indicate that beef that is high in oleic acid will increase risk factors for diseases ( smith , 2016 ) . consequently , the role of animal fats in the diet should be re - evaluated because scientists around the globe increasingly doubt the validity of the so called  diet - heart hypothesis ", "it is now generally accepted that diets with low fat , high carbohydrate failed to curb obesity ( drewnowski , 2015 ) . on the other hand ,", "more recent functional medicine research studies have suggested that the intake of fat has positive effect on human health ( saito , 2016 ) .", "it is essential to consume fats containing good quality fatty acids while reducing the consumption of food high in simple carbohydrates .", "excessive intake of simple carbohydrates is detrimental to health because they have negative effects on the body ( yu et al . , 2013 ) .", "in this regard , inclusion of high fat foods with superior sensory properties in a balanced diet such as highly marbled wagyu and hanwoo beef is likely to gain wider acceptance as a well - being food in the near future .", "in japan and korea , highly marbled wagyu and hanwoo cattle are greatly prized for traditional meat cooking methods .", "many researches have shown that wagyu and hanwoo cattle have high potential of accumulating imf and producing highly marbled beef .", "the beef quality grading system in both countries is primarily determined by marbling score with bms and additionally adjusted by other carcass traits .", "literature suggests that imf content varies on the basis of feeding time , finishing diet , and breed type .", "great attention has been paid to more accumulation of imf to produce high quality grade beef .", "the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle .", "highly marbled wagyu and hanwoo beef have higher proportions of mufa due to higher concentrations of oleic acid .", "they are heart - healthy dietary fat because they can lower ldl - cholesterol while increasing hdl - cholesterol .", "clinical trials have also indicated that highly marbled beef does not increase ldl - cholesterol and that beef high in oleic acid can consistently increase hdl - cholesterol . finally , literatures have concluded that high - oleic acid beef such as wagyu and hanwoo beef may reduce risk factors for cardiovascular diseases ."], "abstract_text": ["<S> this review addresses the characteristics and health benefit of highly marbled wagyu and hanwoo beef . </S>", "<S> marbling of wagyu and hanwoo beef has been increased in japan and korea to meet domestic consumer preferences . </S>", "<S> wagyu and hanwoo cattle have high potential of accumulating intramuscular fat ( imf ) and producing highly marbled beef . </S>", "<S> the imf content varies depending on the feeding of time , finishing diet , and breed type . </S>", "<S> imf increases when feeding time is increased . </S>", "<S> the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle . </S>", "<S> fatty acid composition are also different depending on breeds . </S>", "<S> highly marbled wagyu and hanwoo beef have higher proportions of monounsaturated fatty acid ( mufa ) due to higher concentrations of oleic acid . </S>", "<S> mufas have little effect on total cholesterol . </S>", "<S> they are heart - healthy dietary fat because they can lower low - density lipoprotein ( ldl)-cholesterol while increasing high - density lipoprotein ( hdl)-cholesterol . </S>", "<S> clinical trials have indicated that highly marbled beef does not increase ldl - cholesterol . </S>", "<S> this review also emphasizes that high oleic acid beef such as wagyu and hanwoo beef might be able to reduce risk factors for cardiovascular disease . </S>"], "labels": null, "section_names": ["Introduction", "Wagyu and Hanwoo Cattle", "Carcass Grading of Wagyu and Hanwoo", "Marbling of Wagyu and Hanwoo Beef", "Fatty Acid Composition of Wagyu and Hanwoo Beef", "Health Implications of Highly Marbled Wagyu and Hanwoo Beef", "Conclusions"], "sections": [["their marbling has been increased over many decades to meet domestic consumer preferences . in both countries ,", "highly marbled beef is greatly prized for traditional meat cooking methods such as sukiyaki for japanese and gogigui for korean . because of these demands , the use of heifers and steers instead of bulls , intensive feeding system , and genetic ability of wagyu and hanwoo cattle have resulted in greater fat deposition in these breeds compared to european breeds . as intramuscular fat ( imf ) improves beef quality at least in juiciness and flavor ( hornsterin and wasserman , 1987 ; wheeler et al . , 1994 )", "it is assessed in abattoirs by meat graders in various countries , including usa , australia , japan , and korea . like other kinds of foods , meat has three functions : 1 ) it provides nutrition ; 2 ) it provides deliciousness ; and 3 ) it prevents disease .", "although beef has these three functions , the main food in both japan and korea is boiled rice while beef is a side dish .", "therefore , these two countries have developed the quality of beef rather than its quantity .", "this is quite different from foreign countries where meat is consumed as a main dish . in japanese and korean cuisine , soft and delicious beef with imf and a good red color", "in the past , fat was not given a good image in its role towards human health , although fat is an important energy resource for human .", "recently , fat has been reported to have fewer adverse effects on health than carbohydrates , especially simple carbohydrates .", "in fact , meat has played a crucial role in human evolution of a healthy and well balanced diet ( pereira and vicente , 2013 ) .", "furthermore , meat plays a pivotal role in nutritious diets . high quality marbled beef not only has excellent eating quality , but also contains a lot of beneficial fatty acids ( troy et al . , 2016 ) . in this regard ,", "the current paper reviews the characteristics and health benefit of highly marbled beef from wagyu and hanwoo cattle ."], ["it has well known that wagyu cattle have high potential of accumulating imf and producing highly marbled beef .", "hanwoo cattle are also known for their high imf for marbled beef similar to wagyu beef .", "highly marbled wagyu loin contains more than 40% of imf , sometimes more than 60% ( horii et al . , 2009 ) , while quality grade 1 hanwoo , the highest quality grade , has approximately 28% of imf in longissimus thoracis muscle ( hwang and joo , 2016 ) .", "wagyu cattle include four types of japanese cattle : the black , brown , short horn , and polled breeds .", "numerous studies have investigated the meat quality , quantity , and muscle physiology of crossbreed wagyu ( japanese black cattle ) in foreign countries ( cafe et al . , 2006 ; cafe et al . , 2009 ; greenwood et al . , 2006 ; greenwood et al . , 2009 ; may et al . , 1993 ) .", "they are identified by different coat color : brown ( major hanwoo ) , black face ( heukwoo ) , black ( jeju heukwoo ) , and tiger color ( chickso ) ( jo et al . , 2012 ) .", "in this review , wagyu and hanwoo are used to describe the japanese black breed and the brown coat color hanwoo , respectively .", "all four types of wagyu cattle have played important roles locally and in the history of mixed farming .", "they also played important roles in the synergies between cattle and crops , especially rice .", "farmers gradually began to replace the role of cattle as draft animals and started to use industrial fertilizers approximately 50 years ago . in recent years , japanese wagyu cattle have been raised more specifically for beef production .", "the famous brand name wagyu not only includes the japanese black cattle produced in japan , but also includes animals or even cross - bred japanese black cattle produced in foreign countries such as australia and the united states .", "similarly , the utilization of hanwoo cattle as an edible meat had been minimal for long time .", "full - scale production of hanwoo as meat - type cattle has started since the 1970s . because hanwoo cattle have maintained stable traits through pure breeding , the current blood lineage is very valuable .", "it is mainly spread in the korean peninsula ( kim and lee , 2000 ) .", "recently , hanwoo beef has been reported to have highly marbled imf similar to wagyu beef .", "especially , hanwoo beef has relatively thin muscle fiber and minimal content of connective tissues ( kim et al . , 1994 ) .", "it has less subcutaneous fat depth with greater ossification scores and marbling scores than those of australian angus ( cho et al . , 2005 ) .", "in 2013 , a total of 2.64 million heads of cattle were fed for beef production in japan .", "approximately 1.71 million heads were japanese black cattle ( maff , 2013 ) , and approximately 873,400 were holstein cattle .", ", the number of farmers producing beef was 613,000 , but 86.5% of these farmers fed less than 50 heads of cattle .", "the mean body weight and carcass weight of beef at slaughter ( 26 - 30 mon of age ) were 725 kg and 470 kg , respectively .", "high performance marbled beef production has caused japanese black cattle to comprise the greatest share of japan s wagyu cattle population ( albrecht et al . , 2011 ;", "recently , the imf percentage of beef from japanese black cattle has an average value of greater than 30% ( albrecht et al . , 2011 ; horii et al . ,", "the total number of slaughter cattle was 1,007,000 , including 883,593 hanwoo cattle , 66,485 holstein cows , and 56,923 holstein heifers and bulls ( kape , 2015 ) .", "the number of cattle farming households was 99,858 , including 89,403 hanwoo farmers ( kape , 2015 ) . during the last decade ,", "the number of households raising hanwoo cattle has drastically decreased from 186,000 households in 2006 to 89,403 households in 2015 ( kape , 2016 ) .", "the average live and carcass weights of hanwoo cattle at slaughter ( 26 - 30 mon of age ) were 719 kg and 430 kg , respectively ( kape , 2015 ) ."], ["wagyu carcasses are evaluated by accredited graders from the japan meat grading association ( jmga ) in accordance with beef carcass grading standards .", "first established in 1988 , the present grading system assigns both yield grade ( a , b , and c ) and meat quality grade ( 1 , 2 , 3 , 4 , and 5 ) ( jmga , 2014 ) .", "in korea , all cattle carcasses should be evaluated by korean carcass grading system . established in 1992 ,", "the korean carcass grading system presently has three levels of yield grade ( yg ) for meat amount ( a , b , and c ) and five levels of meat quality grade ( qg ) ( 1 , 1 , 1 , 2 , and 3 ) ( kape , 2016 ) . for beef quality grading in japan ,", "all cattle carcasses are graded at the 6 to 7 rib section at least one hour after ribbing .", "the following four items are independently evaluated : beef marbling ; meat color and brightness ; meat firmness and texture ; and fat color , luster , and quality . meat quality grade of the carcass is then assigned according to the lowest grade of these four items .", "korean beef quality grading is also estimated based on several factors , including marbling score , meat color , fat color , firmness and texture of lean meat , and maturity of the exposed loin muscle at the 13 rib interface .", "this means that marbling score is the most dominating determinant in korea because korean consumers have an extraordinary preference for high marbled meats . in 1988 , wagyu marbling levels were assigned by the beef marbling standard ( bms ) using a plastic model made from silicone resin .", "this standard was calculated based on the circumference and area percentage of marbling particles in the rib eye section ( longissimus thoracis ) . in october 2008 , a new marbling standard using carcass photographs replaced the 1988 standard . in march 2014 , an even newer marbling standard", "1 ) . graders now determine the bms number ( 1 to 12 ) by comparing the actual carcass marbling to the standard photograph of marbling . during this process", ", any larger inclusions of fat at the periphery of the rib eye are not considered as marbling according to the japanese grading system .", "the bms of korean carcass grading system has been changed by the addition of marbling number . in 1992 , when the carcass grading system was established for the first time in korea , the bms had only 5 numbers ( 1 to 5 ) with 3 qg ( 1 , 2 , and 3 ) . however , in 1997 , new qg 1 was added with new bms no . 6 and no .", "furthermore , in 2004 , another new qg 1 was added with new bms no . 8 and no .", "hanwoo beef with qg 1 or 1 is considered as a premium class of beef in korea .", "of hanwoo cattle slaughtered in 2015 , 10.0% were qg 1 , 26.4% were qg 1 , and 31.4% were qg 1 ( kape , 2016 ) ."], ["the plentiful marbling of wagyu and hanwoo beef has attracted attention . in both japan and korea ,", "the value of cattle carcasses is determined by a qg which considers marbling as a decisive determinant . since the liberalization of beef importation", ", marbling has been greatly emphasized to differentiate domestic beef from imported beef ( hirooka , 2014 ; hwang et al . , 2010 ) .", "the high content of imf can improve the texture and juiciness of hanwoo beef and thereby its acceptability ( jung et al . , 2016 ) .", "korean consumers prefer qg 1 or 1 beef because of its high imf content ( kim et al . , 1999 ) .", "2015 ) have demonstrated that an increase in crude fat content ( range 23.8 - 48.6% ) can increase the tenderness , juiciness , and fattiness .", "however , they also reported that an increase in crude fat content can reduce the crude protein content and slightly reduce the content of umami components such as nucleic acid and glutamic acid .", "it is well known that imf content varies depending on feeding time , finishing diet , and breed type . to produce high qg beef ,", "great attention has been paid to more accumulation of imf in wagyu and hanwoo muscle .", "one of good strategy to increase imf content in beef muscle is to extend slaughtering age . although the marbling score is increased and reached a plateau at about 24 mon of age ( choi et al . ,", "2002 ) , the slaughtering age of hanwoo has been extended to increase the bms score ( jo et al . , 2012 ) .", "in korea , the marketing age of hanwoo has been extended to an average of 31 mon with weight of 719 kg to fatten the cattle ( kape , 2015 ) .", "however , average daily gain is decreased due to increased slaughtering weight ( paek et al . , 1993 ) .", "recently , cattle in china are fed for unusually long periods of time before slaughter as wagyu and hanwoo .", "this might have contributed to their high imf and oleic acid contents ( smith , 2016 ; tanaka , 1985 ) .", "it is clear that imf increases with feeding time for grain - fed and pasture - fed cattle .", "however , the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle ( smith et al . , 2009 ) .", "it has been reported that wagyu fed on a high - concentration diet have higher expression of adipogenic transcription factors in the subcutaneous and intramuscular adipocytes than those fed on a high - rough - age diet ( yamada and nakanishi , 2012 ) .", "the imf content and the numbers of preadipocytes and adipocytes are reported to be higher in wagyu than those in angus ( duarte et al . , 2013 ) .", "( 2009 ) have reported that the imf contents in the longissimus muscle of wagyu , german angus , belgain blue , and holstein friesian are 23.3% , 4.4% .", "the wagyu and european cattle breeds did not differ in their mechanisms of postnatal fat accretion .", "however , they differed in their efficiency of accretion of imf ( gotoh et al . , 2009 ) .", "for every 1% increase of imf in the longissimus muscle , the increase amounts of subcutaneous adipose tissue in wagyu , holstein friesian , german angus , and belgain blue were 3.0 , 4.3 , 7.9 , and 10.7 kg , respectively ( gotoh et al . , 2009 ) .", "although imf content is the most dominating determinant of beef quality , the imf content is not the only parameter that decides the quality grade of beef carcass .", "marbling is called  shimo - furi  in japanese and  sang - gang  in korean .", "it literally means  frosting  . in japan , marbling with a fine appearance resembling frost is highly valued , but coarse marbling is not ( motoyama et al . ,", "recently , korea also began to discriminate between fine and coarse marbling in hanwoo beef .", "this marbling quality contributes to the tenderness of beef because imf deposits are found mainly between muscle fiber bundles , resulting in the disorganization of perimysium connective tissue ( nishimura , 2015 ; sasaki et al . , 2012 ) .", "therefore , the sensory of tenderness could be qualitatively affected by histological difference in marbling due to difference in tissue disorganization extent ."], ["there are several types of fatty acids : 1 ) monounsaturated fatty acids ( mufa ) , 2 ) polyunsaturated fatty acids ( pufa ) , and 3 ) saturated fatty acid ( sfa ) .", "pufa such as linoleic acid , -linolenic acid ( n-3 ) , -linolenic acid ( n-6 ) , arachidonic acid , and so on contain many important compounds such as essential fatty acids .", "the fatty acid that has the highest amount in beef is oleic acid ( c18:1n-9 ) .", "it has been reported that fatty acid compositions are different depending on breeds ( smith et al . , 2006", "the fatty acid compositions in highly marbled wagyu and hanwoo are considerably different from those in other cattle breeds .", "highly marbled wagyu beef has a higher percentage of mufa within fat compared with other breeds ( yang et al . , 1999a ) .", "( 2006 ) have investigated oleic acid concentrations in the subcutaneous adipose tissues of wagyu , hanwoo , australian crossbred , angus ( corn - fed ) , angus ( hay - fed ) , and angus ( weaned ) and found that they are 52.9% , 47.3% , 39.8% , 39.8% , 34.6% , and 32.9% , respectively .", "a higher percentage of mufa will lead to a lower fat - melting point which contributes to the softness of beef fat and favorable beef flavor .", "it may decrease the circulating concentration of ldl cholesterol in consumers ( melton et al .", ", 1982 ; rudel et al . , 1995 ; smith , 1994 ) . therefore , fatty acid compositions of beef have recently become important in the beef industry , especially in highly marbled wagyu and hanwoo cattle", "( 1995 ) have investigated the effect of breed type ( including japanese black ) and sex on fatty acid compositions of subcutaneous and intramuscular lipids in finishing steers and heifers of pure japanese black and holstein as well as crossbred japanese black , holstein , japanese brown , and charolais .", "they have reported that the japanese black is genetically predisposed to producing carcass lipids containing higher concentrations of mufa than holstein , japanese brown , or charolais steers ( zembayashi et al . , 1995 ) .", "1992 ) have also concluded that beef from purebred wagyu cattle raised in japan is rich in mufa .", "( 2011 ) have compared intramuscular fatty acid composition of longissimus muscle in 26-month - old japanese black steers and holstein steers reared and fattened using a standard fattening system ( table 1 ) . in the longissimus muscle of japanese black steers ,", "a higher percentage of unsaturated fatty acid was found than that in holstein steers ( gotoh et al . , 2014 )", "2011 ) have also compared the imf content and fatty acid compositions of 21 major skeletal muscles using the same animals .", "muscles from the japanese black cattle contained a greater proportion of numerous fatty acids , particularly mufa such as c16:1 , c18:1 , and c20:1 compared to fatty acids in holstein cattle .", "in japanese black cattle , the proportion of sfa including c18:0 was much lower compared to that in holstein cattle .", "sfa : saturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .", "2005 ) have investigated the fatty acid compositions of hanwoo and australian angus beef and found a significant difference in fatty acid compositions between these two cattle breeds ( table 2 ) .", "especially , angus beef had significantly higher n-3 pufa while hanwoo beef contained greater n-6 pufa in three different muscles ( cho et al . , 2005 ) .", "the difference in fatty acid composition might be attributed to the influence of different diets , forage , and grain feeding , although fatty acid profile in ruminants is not a direct reflection of the dietary fatty acid composition due to hydrogenation by rumen microorganism ( enser et al . , 1998 ) .", "sfa : saturated fatty acids , usfa : unsaturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .", "f - ratio statistic : * if p<0.05 , * * if p<0.01 , * * * if p<0.001 .", "therefore , it can be easily anticipated that hanwoo beef has a fatty acid profile similar to that of high concentratefed animals ( jo et al . , 2012 ) .", "recently , hwang and joo ( 2016 ) have evaluated the fatty acid profile of ten muscles from high marbled ( qg 1 ) and low marbled ( qg 2 ) hanwoo carcass and found significant differences in fat content and fatty acid composition among 10 muscles and between high and low marbled hanwoo beef . in particular , high marbled hanwoo muscles had significantly higher proportion of mufa due to higher oleic acid ( c18:1 ) proportion , while low marbled hanwoo muscles had higher proportion of sfa due to higher proportion of stearic acid ( c18:0 ) ( hwang and joo , 2016 ) .", "stearoyl - coa desaturase ( scd ) was first identified and reported as one of the genes associated with beef fatty acid composition ( taniguchi et al . ,", "the composition of fatty acids stored in fat depots reflects the earlier action of scd on substrates such as stearic acid and palmitic acid ( kim and ntambi , 1999 ) .", "1999b ) have reported interesting correlations between scd enzyme activity and fatty acid composition in bovine adipose tissue .", "although the adipogenic mechanism is extremely complicated , several genes have been identified and confirmed as either associated with or responsible for the fatty acid composition in wagyu cattle ( gotoh et al . , 2014 ) .", "it is generally accepted that the concentration of oleic acid in beef adipose tissue is dependent on scd expression and activity .", "wagyu cattle are genetically disposed to produce more oleic acid ( smith et al . , 2006 ) .", "very high heritability has been reported for oleic acid in wagyu cattle ( nogi et al . , 2011 ) .", "higher levels of concentrated feed in the later fattening period can lead to higher mufa concentration in the subcutaneous adipose tissues of wagyu steer ( kimura et al . ,"], ["interest in beef fat and fatty acids has been increasing , especially in highly marbled beef such as wagyu and hanwoo because fatty acids composition in the diet have impact on human health .", "consumption of fat and cholesterol has been reported to be linked to cardiovascular disease , obesity , and cancer ( micha et al . , 2010 ;", "consequently , reduction of total fatty acid intake and replacement of sfa with pufa have been recommended .", "ulbricht and southgate ( 1991 ) have demonstrated that stearic acid has no effect on plasma cholesterol level and that oleic acid can lower serum cholesterol similar to pufa .", "furthermore , pavan and duckett ( 2013 ) have suggested that a higher proportion of oleic acid in beef is desirable because the consumption of high - oleic acid ground beef can increase hdl - cholesterol concentration ( gilmore et al . , 2011 ) . according to smith ( 2016 ) ,", "the amount of fat consumed in a typical portion of beef will not increase risk factors for cardiovascular disease .", "clinical trials have demonstrated that ground beef containing elevated oleic acid can increase the concentration of hdl - cholesterol or at least has no negative effect on the concentration of hdl - cholesterol . in earlier research on oleic acid , the major mufa in beef , grundy et al .", "( 1988 ) have found that it can lower ldl - cholesterol without affecting beneficial hdl - cholesterol .", "( 2014 ) have reported that mufa can normalize or improve lipid metabolism and maintain the balance in cardiac muscle .", "these have implied that mufa have little effect on total cholesterol and that they are heart - healthy dietary fat that can lower ldl - cholesterol and increase hdl - cholesterol ( lahey et al . , 2014 ) .", "this effect is repeatable when natural foods are used to supplement diets with oleic acid . in this", "regard , smith ( 2016 ) have concluded that beef cattle should be raised under production conditions to increase the concentration of oleic acid in their edible tissue , i.e. , by grain feeding over extended periods of time .", "it is obvious that consumer in the world has an overwhelmingly negative attitude toward animal fats , especially saturated fat in meat for the last several decades ( ngapo and dransfield , 2006 ; williams and droulez , 2010 ) .", "according to higgs ( 2000 ) , the per capita decline in beef consumption in the us and other western countries has been attributed in large part to animal fat phobia .", "consumers have been warned to reduce saturated fat in their diet and to avoid meat cuts containing high fat content .", "these health recommendations are obviously in conflict with the health of highly marbled wagyu and hanwoo beef .", "many research studies have shown that the imf of wagyu and hanwoo beef contains a lot of mufa that could prevent arteriosclerosis .", "researches have also demonstrated that high - oleic acid ground beef may reduce risk factors for cardiovascular disease ( adams et al .", "thus , although some consumers in japan and korea consider highly marbled wagyu and hanwoo beef as being unhealthy , there is no scientific evidence to indicate that beef that is high in oleic acid will increase risk factors for diseases ( smith , 2016 ) . consequently , the role of animal fats in the diet should be re - evaluated because scientists around the globe increasingly doubt the validity of the so called  diet - heart hypothesis ", "it is now generally accepted that diets with low fat , high carbohydrate failed to curb obesity ( drewnowski , 2015 ) . on the other hand ,", "more recent functional medicine research studies have suggested that the intake of fat has positive effect on human health ( saito , 2016 ) .", "it is essential to consume fats containing good quality fatty acids while reducing the consumption of food high in simple carbohydrates .", "excessive intake of simple carbohydrates is detrimental to health because they have negative effects on the body ( yu et al . , 2013 ) .", "in this regard , inclusion of high fat foods with superior sensory properties in a balanced diet such as highly marbled wagyu and hanwoo beef is likely to gain wider acceptance as a well - being food in the near future ."], ["in japan and korea , highly marbled wagyu and hanwoo cattle are greatly prized for traditional meat cooking methods .", "many researches have shown that wagyu and hanwoo cattle have high potential of accumulating imf and producing highly marbled beef .", "the beef quality grading system in both countries is primarily determined by marbling score with bms and additionally adjusted by other carcass traits .", "literature suggests that imf content varies on the basis of feeding time , finishing diet , and breed type .", "great attention has been paid to more accumulation of imf to produce high quality grade beef .", "the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle .", "highly marbled wagyu and hanwoo beef have higher proportions of mufa due to higher concentrations of oleic acid .", "they are heart - healthy dietary fat because they can lower ldl - cholesterol while increasing hdl - cholesterol .", "clinical trials have also indicated that highly marbled beef does not increase ldl - cholesterol and that beef high in oleic acid can consistently increase hdl - cholesterol . finally , literatures have concluded that high - oleic acid beef such as wagyu and hanwoo beef may reduce risk factors for cardiovascular diseases ."]]}
{"article_id": "PMC3103845", "article_text": ["\n n - acetyl - l - cysteine ( nac ) is an endogenous aminothiol present both in human plasma and in urine .", "n-(2-mercaptopropionyl)glycine ( mpg ) , also known as tiopronin , is a synthetic aminothiol antioxidant .", "nac has been in clinical use for more than 40 years , primarily as a mucolytic agent in a variety of respiratory illness .", "intravenous and oral administration of nac have been extensively used in the management of paracetamol ( acetaminophen ) poisoning .", "mpg is primarily used in the treatment of cystinuria , but studies have shown that mpg can be used as a chelating , cardioprotecting , and radioprotecting agent , as well as an antidote to heavy metal poisoning . a number of electrochemical [ 59 ] , fluorometric [ 1012 ] , chemiluminescence [ 1315 ] , and liquid chromatographic [ 1618 ] methods have been developed for the determination of nac and mpg in biological samples and pharmaceuticals .", "spectrophotometry is the most widely used technique in pharmaceutical analysis because it is simple , economic , and easily available to most quality control laboratories .", "spectrophotometric methods have also been reported for the determination of nac and mpg in pharmaceutical formulations [ 1925 ] .  ", "a coupled redox - complexation reaction has been reported for the spectrophotometric analysis of nac and mpg using 1,10-phenanthroline as the chromogenic reagent . in the present work ,", "we report a simple and cost - effective spectrophotometric method for the reliable analysis of nac and mpg in pharmaceutical formulations .", "the method is also based on the coupled redox - complexation reaction between nac or mpg and fe(iii ) but uses 2,4,6-tripyridyl - s - triazine ( tptz ) as the chromogenic reagent .", "collins et al . have introduced tptz as chromogenic reagent for determination of fe(ii ) .", "the fe(ii ) complex with tptz has a twice higher molar absorptivity coefficient ( 2.2  10  l  mol  cm ) than the fe(ii ) complex with 1,10-phenanthroline ( 1.1  10  l  mol  cm ) .", "all spectrophotometric studies were carried out on an ultraviolet - visible , double - beam spectrophotometer ( uv-1601 shimadzu , kyoto , japan ) , and using 1  cm quartz cells .", "measurements of ph were carried out with a mettler toledo sevenmulti potentiometer ( mettler toledo , schwerzenbach , switzerland ) equipped with a combined glass electrode mettler toledo in lab 413 .", "a thermostated water bath ( mgw lauda , germany ) was used to keep a constant cuvette temperature of 25  0.5c .", "all chemicals were of analytical - reagent grade , and solutions were prepared in milliq deionised water .", "separate 10  mm stock solutions of nac and mpg were prepared by dissolving 163.2  mg ( 1  mmol ) of nac ( merck , darmstadt , germany ) or 163.2  mg ( 1  mmol ) of mpg ( sigma - aldrich , st .", "louis , usa ) in deionised water up to 100.0  ml volume and stored in the dark bottle at 4c .", "dilutions were prepared daily in deionised water .   stock solution of fe(iii ) ( 10  mm ) was prepared by dissolving 270.3  mg ( 1  mmol ) of fecl3  6 h2o ( kemika , zagreb , croatia ) in 50.0  ml deionised water .", "then 0.5  ml of concentrated hcl was added and the volume was adjusted to 100.0  ml with deionised water . a stock solution of 10  mm tptz ( merck , darmstadt , germany )", "was prepared by dissolving 312.3  mg ( 1  mmol ) tptz in 2.0  ml of a 6.0  m hcl , followed by addition of deionised water up to a total volume of 100.0  ml .  ", "m ) was used to cover the ph range 3.24.0 . for solutions of ph 1.0 and 2.0 ,", "two different pharmaceutical formulations of nac were analysed by the present spectrophotometric method , that is , fluimukan 200  mg granules , and fluimukan akut 600  mg dispersible tablets ( lek , ljubljana , slovenia ) .", "an accurately weighed portion of the powder containing about 200  mg of nac was transferred into a 500  ml volumetric flask , and nac was dissolved in and diluted to the nominal volume with deionised water .", "ten tablets of the mpg - containing drug captimer ( mit gesundheit gmbh , germany ) were weighed and pulverised .", "a powder quantity equivalent to 100  mg of mpg was dissolved in 300  ml of deionised water , filtered through filter paper ( blue ribbon , s&s , germany ) , and the filtrate collected in a 500  ml volumetric flask was diluted by deionised water to the nominal volume .", "it is noteworthy that such solutions are not stable and should be analysed within 24 hours .", "these solutions were further diluted quantitatively with water to obtain suitable concentrations for the analysis by the proposed spectrophotometric method .", "acetate buffer ( 20.0  ml , ph 3.6 ) was pipetted into a 25.0  ml calibrated flask . then 1.25  ml of 10.0  mm fe(iii )", ", 1.25  ml of 10.0  mm tptz , and 1.0  ml of nac or mpg solutions were added .", "the flask with reaction solution was filled to the nominal volume with deionised water , mixed well , and kept at room temperature ( about 25c ) for 30  min ( mpg ) or 60  min ( nac ) .", "the absorbance of the produced fe(ii)-tptz complex was measured at  = 593  nm against a blank solution , prepared in the same manner with 1.0  ml water instead of 1.0  ml sample solution .", "nac and mpg concentrations in pharmaceutical preparations were determined by using daily prepared calibration curves .", "the eleven solution of every analyte were prepared for the concentration range from 1.0  m to 100.0  m .", "the proposed method is based on the coupled redox - complexation reaction . in the first ( redox ) step of the reaction ( see ( 1 ) ) , rsh compound ( nac or mpg ) reduces fe(iii ) to fe(ii ) whereas rsh molecules themselves oxidize to thiyl radicals rs which combine to form the disulfide rssr . in the second step of the reaction ( see ( 2 ) ) , in situ formed fe(ii ) is immediately complexed by 2 molecules of tptz to form the deep - blue coloured , highly stable fe(tptz)2 \n  complex which absorbs light at  \n max at 593  nm .", "the net overall reaction can be expressed by reaction ( 3 ) : \n ( 1)fe3++rshfe2++h++rs \n ( 2)fe2++2  tptzfe(tptz)22 + \n ( 3)2  fe3++2  rsh+4  tptz2  fe(tptz)22++rssr+2  h+ \n  krishnamurti and huang have reported that the complexation of tptz is specific for fe(ii ) so that this reaction can be performed in the presence of large amounts of fe(iii ) .", "the results of the present study confirm these previous results for the drugs nac and mpg serving as the reducing agents .   in the literature , we were not able to find the standard reduction potential for nac and mpg .", "however , the calculated formal potential of the fe(iii)/fe(ii ) couple of 0.578  v , equations ( 4 ) and ( 5 ) indicate that its oxidizing power in solution with tptz is more negative than in solution with 1,10-phenanthroline ( 1.197  v ) .", "this means that the proposed method with the tptz is selective for the determination of nac and mpg .", "thiols or other reducing substances with standard ( formal ) potentials higher than 0.6  v would not interfere in the proposed method ( see ( 6 ) ) ;  \n ( 4)e10=efe3+/fe2 + 00.05922log  ( fe2+fe3+[tptz]2)2 , \n ( 5)e10=0.771  v0.0592log  1.2651050.077(3104)2=0.578  v \n ( 6)e20=erssr / rsh00.0592ph . \n  in previous work", ", we found that the initial redox - complexation reaction rate is higher with mpg compared to nac [ 28 , 29 ] . in the coupled redox - complexation reaction with mpg , the steady state value of the absorbance is reached after 30  min while in the reaction with nac the steady state value of the absorbance is reached after 60  min . with both thiols ,", ".   equation ( 6 ) indicates that the potential for the redox system rsh / rssr depends upon the ph value of the reaction mixture .", "the effect of the ph was therefore investigated over the range 1.04.0 using 0.1  m hcl for ph 1 , 0.01  m hcl for ph 2 , and acetate buffer for the ph values 3.2 , 3.5 and 3.6 . in this experiment mpg", "the absorbance at 593  nm increased with increasing ph up to the value of 3.6 . however , precipitation of iron hydroxide occurred at the ph above 3.8 .", "therefore , a buffered reaction medium of ph 3.6 was chosen as a compromise for keeping fe(iii ) in solution and achieving quantitative formation of the fe(tptz)2 \n  complex which is stable in the ph range 3.45.8 .", "the influence of the fe(iii ) concentration on the determination of nac and mpg at the fixed concentration of 40  m each was studied in the concentration range from 20  m to 400  m , allowing a molar ratio fe(iii)/rsh from 0.5 to 10 . in figure 2 , the absorbance measured at 593  nm is plotted versus the molar fe(iii)/rsh ratio for nac and mpg .", "figure 2 shows that the reaction can be forced to completion by increasing the fe(iii)/rsh ratio , for instance by increasing the fe(iii ) concentration .  ", "the influence of the tptz concentration on the analysis of nac and mpg at the fixed concentration of 40  m rsh compound was studied in the range from 20  m to 400  m allowing a tptz / rsh molar ratio of 0.5 to 10 .  ", "figure 3 shows that absorbance increased with increasing tptz / rsh molar ratio , that is , with increasing tptz concentration , to reach its maximal value at a molar excess of five .", "the effect of the reaction temperature on the signal intensity was examined by varying the temperature from 25c to 40c using the thermostated water pump .", "we found that the reaction rate increased by elevating reaction temperature ( see [ 28 , 29 ] ) .", "since the proposed method is an equilibrium method , signal is recorded when the reaction reaches the state of equilibrium .", "the signal intensity in the state of equilibrium is the same for all the examined temperatures .", "the linearity of the method was investigated under the optimized conditions for nac and mpg in the concentration range from 1.0 to 100.0  m .", "straight lines were obtained from linear regression analysis of the absorbance at 593  nm and the drug concentration ( table 1 ) .", "the lowest quantifiable concentration of nac and mpg by this method was 1.0  m each .", "the effect of some possible interfering cations and anions on the analysis of a fixed concentration of 40.0  m for nac and mpg was investigated for the maximum molar ratio of foreign ions .", "the influence of excipients that can commonly accompany nac and mpg in pharmaceutical formulations was also studied .  ", "the tolerance is defined as the foreign - ion / excipient concentration causing an error smaller than 5% for the determination of the analyte of interest .", "the tolerable concentration of kno3 and na2so4 was 40.0  mm ( molar ratio , 1000  :  1 ) .", "the tolerable concentration of glucose , fructose , sucrose , boric acid , and acetic acid was 20.0  mm ( molar ratio , 500  :  1 ) .", "thus , the commonly excipients glucose , fructose , and sucrose do not interfere with the analysis of nac and mpg because they essentially do not react with the oxidizing agents .", "it should be emphasized that the contaminant / analyte concentration ratios studied in the present work are much higher than those normally found in commercial pharmaceutical products .", "the tolerable concentration of some other thiols , that is , d - penicillamine , l - glutathione , and l - cysteine , was 40.0  m ( molar ratio , 1  :  1 ) .", "thiols or other reducing substances with standard ( formal ) potentials higher than 0.6  v would not interfere in the proposed method . in order to evaluate the potential of the proposed method to the analysis of real samples , the method was applied to three pharmaceutical formulations of the drugs nac and mpg .", "the results of these analyses are presented in tables 2 and 3 .   for comparison , the spectrophotometric method reported by raggi et al .  ", "these authors used 1,10-phenantroline as an chromogenic reagent , instead of tptz which is used in the present work .", "as shown in table 2 , there were no significant differences between the values obtained by the reported method   and those obtained by the proposed method ( p > 0.1 , student t - test ) .", "this actually suggests that the proposed method is accurate and precise as the earlier reported method .", "the accuracy of the method was further ascertained through the recovery studies . to the drug solutions of the granules or the tablet powder , the standard solutions of the synthetic nac or mpg", "these results indicate that the proposed method is accurate for the determination of nac and mpg in their commercially available pharmaceutical preparations without any significant interference by common pharmaceutical excipients which do not absorb light in the visible region .  ", "performance characteristic of existing equilibrium spectrophotometric methods [ 19 , 2124 ] and the proposed method are compared in table 4 .", "the proposed method is free from drastic experimental conditions such as heating unlike some of reported methods .", "some other thiol compounds do not interfere in the proposed method at molar ratio 1  :  1 .", "it is also worth mentioning that the proposed method was performed in the visible region (  = 593  nm ) away from the uv - absorbance of the uv - absorbing interfering excipient materials , which might be dissolved from pharmaceutical formulations .", "undoubtedly , hplc is one of the most widely used techniques in routine analysis of pharmaceuticals , but it involves expensive instrumental set which many laboratories in developing and underdeveloped countries can not afford . the proposed equilibrium spectrophotometric method based on the coupled redox - complexation reaction of the thiol drug nac or mpg with fe(iii ) and tptz can be applied in every analytical laboratory as a reliable method for the determination of nac or mpg in pharmaceutical preparations . due to the use of tptz", "the coloured fe(tptz)2 \n  complex is stable in an extended period of time up to 24 hours .", "regular excipients and additives present in the pharmaceutical preparations of nac and mpg do not interfere in this method ."], "abstract_text": ["<S> a simple spectrophotometric method for the determination of \n n - acetyl - l - cysteine ( nac ) and \n n-(2-mercaptopropionyl)glycine ( mpg ) in pharmaceutical preparations was developed , validated , and used . </S>", "<S> the proposed equilibrium method is based on a coupled two - step redox and complexation reaction . in the first step , fe(iii ) </S>", "<S> is reduced to fe(ii ) by nac or mpg . </S>", "<S> subsequently , fe(ii ) is complexed with 2,4,6-tripyridyl - s - triazine ( tptz ) . </S>", "<S> several analytical parameters of the method were optimized for nac and mpg analysis in the concentration range from 1.0  m to 100.0  m . </S>", "<S> regression analysis of the calibration data showed a good correlation coefficient ( 0.9999 ) . </S>", "<S> the detection limit of the method was 0.14  m for nac and 0.13  m for mpg . </S>", "<S> the method was successfully applied to quantify nac and mpg in pharmaceutical preparations . </S>", "<S> no interferences were observed from common pharmaceutical excipients . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Results and Discussion", "4. Conclusion"], "sections": [["\n n - acetyl - l - cysteine ( nac ) is an endogenous aminothiol present both in human plasma and in urine .", "n-(2-mercaptopropionyl)glycine ( mpg ) , also known as tiopronin , is a synthetic aminothiol antioxidant .", "nac has been in clinical use for more than 40 years , primarily as a mucolytic agent in a variety of respiratory illness .", "intravenous and oral administration of nac have been extensively used in the management of paracetamol ( acetaminophen ) poisoning .", "mpg is primarily used in the treatment of cystinuria , but studies have shown that mpg can be used as a chelating , cardioprotecting , and radioprotecting agent , as well as an antidote to heavy metal poisoning . a number of electrochemical [ 59 ] , fluorometric [ 1012 ] , chemiluminescence [ 1315 ] , and liquid chromatographic [ 1618 ] methods have been developed for the determination of nac and mpg in biological samples and pharmaceuticals .", "spectrophotometry is the most widely used technique in pharmaceutical analysis because it is simple , economic , and easily available to most quality control laboratories .", "spectrophotometric methods have also been reported for the determination of nac and mpg in pharmaceutical formulations [ 1925 ] .  ", "a coupled redox - complexation reaction has been reported for the spectrophotometric analysis of nac and mpg using 1,10-phenanthroline as the chromogenic reagent . in the present work ,", "we report a simple and cost - effective spectrophotometric method for the reliable analysis of nac and mpg in pharmaceutical formulations .", "the method is also based on the coupled redox - complexation reaction between nac or mpg and fe(iii ) but uses 2,4,6-tripyridyl - s - triazine ( tptz ) as the chromogenic reagent .", "collins et al . have introduced tptz as chromogenic reagent for determination of fe(ii ) .", "the fe(ii ) complex with tptz has a twice higher molar absorptivity coefficient ( 2.2  10  l  mol  cm ) than the fe(ii ) complex with 1,10-phenanthroline ( 1.1  10  l  mol  cm ) ."], ["all spectrophotometric studies were carried out on an ultraviolet - visible , double - beam spectrophotometer ( uv-1601 shimadzu , kyoto , japan ) , and using 1  cm quartz cells .", "measurements of ph were carried out with a mettler toledo sevenmulti potentiometer ( mettler toledo , schwerzenbach , switzerland ) equipped with a combined glass electrode mettler toledo in lab 413 .", "a thermostated water bath ( mgw lauda , germany ) was used to keep a constant cuvette temperature of 25  0.5c .", "all chemicals were of analytical - reagent grade , and solutions were prepared in milliq deionised water .", "separate 10  mm stock solutions of nac and mpg were prepared by dissolving 163.2  mg ( 1  mmol ) of nac ( merck , darmstadt , germany ) or 163.2  mg ( 1  mmol ) of mpg ( sigma - aldrich , st .", "louis , usa ) in deionised water up to 100.0  ml volume and stored in the dark bottle at 4c .", "dilutions were prepared daily in deionised water .   stock solution of fe(iii ) ( 10  mm ) was prepared by dissolving 270.3  mg ( 1  mmol ) of fecl3  6 h2o ( kemika , zagreb , croatia ) in 50.0  ml deionised water .", "then 0.5  ml of concentrated hcl was added and the volume was adjusted to 100.0  ml with deionised water . a stock solution of 10  mm tptz ( merck , darmstadt , germany )", "was prepared by dissolving 312.3  mg ( 1  mmol ) tptz in 2.0  ml of a 6.0  m hcl , followed by addition of deionised water up to a total volume of 100.0  ml .  ", "m ) was used to cover the ph range 3.24.0 . for solutions of ph 1.0 and 2.0 ,", "two different pharmaceutical formulations of nac were analysed by the present spectrophotometric method , that is , fluimukan 200  mg granules , and fluimukan akut 600  mg dispersible tablets ( lek , ljubljana , slovenia ) .", "an accurately weighed portion of the powder containing about 200  mg of nac was transferred into a 500  ml volumetric flask , and nac was dissolved in and diluted to the nominal volume with deionised water .", "ten tablets of the mpg - containing drug captimer ( mit gesundheit gmbh , germany ) were weighed and pulverised .", "a powder quantity equivalent to 100  mg of mpg was dissolved in 300  ml of deionised water , filtered through filter paper ( blue ribbon , s&s , germany ) , and the filtrate collected in a 500  ml volumetric flask was diluted by deionised water to the nominal volume .", "it is noteworthy that such solutions are not stable and should be analysed within 24 hours .", "these solutions were further diluted quantitatively with water to obtain suitable concentrations for the analysis by the proposed spectrophotometric method .", "acetate buffer ( 20.0  ml , ph 3.6 ) was pipetted into a 25.0  ml calibrated flask . then 1.25  ml of 10.0  mm fe(iii )", ", 1.25  ml of 10.0  mm tptz , and 1.0  ml of nac or mpg solutions were added .", "the flask with reaction solution was filled to the nominal volume with deionised water , mixed well , and kept at room temperature ( about 25c ) for 30  min ( mpg ) or 60  min ( nac ) .", "the absorbance of the produced fe(ii)-tptz complex was measured at  = 593  nm against a blank solution , prepared in the same manner with 1.0  ml water instead of 1.0  ml sample solution .", "nac and mpg concentrations in pharmaceutical preparations were determined by using daily prepared calibration curves .", "the eleven solution of every analyte were prepared for the concentration range from 1.0  m to 100.0  m ."], ["the proposed method is based on the coupled redox - complexation reaction . in the first ( redox ) step of the reaction ( see ( 1 ) ) , rsh compound ( nac or mpg ) reduces fe(iii ) to fe(ii ) whereas rsh molecules themselves oxidize to thiyl radicals rs which combine to form the disulfide rssr . in the second step of the reaction ( see ( 2 ) ) , in situ formed fe(ii ) is immediately complexed by 2 molecules of tptz to form the deep - blue coloured , highly stable fe(tptz)2 \n  complex which absorbs light at  \n max at 593  nm .", "the net overall reaction can be expressed by reaction ( 3 ) : \n ( 1)fe3++rshfe2++h++rs \n ( 2)fe2++2  tptzfe(tptz)22 + \n ( 3)2  fe3++2  rsh+4  tptz2  fe(tptz)22++rssr+2  h+ \n  krishnamurti and huang have reported that the complexation of tptz is specific for fe(ii ) so that this reaction can be performed in the presence of large amounts of fe(iii ) .", "the results of the present study confirm these previous results for the drugs nac and mpg serving as the reducing agents .   in the literature , we were not able to find the standard reduction potential for nac and mpg .", "however , the calculated formal potential of the fe(iii)/fe(ii ) couple of 0.578  v , equations ( 4 ) and ( 5 ) indicate that its oxidizing power in solution with tptz is more negative than in solution with 1,10-phenanthroline ( 1.197  v ) .", "this means that the proposed method with the tptz is selective for the determination of nac and mpg .", "thiols or other reducing substances with standard ( formal ) potentials higher than 0.6  v would not interfere in the proposed method ( see ( 6 ) ) ;  \n ( 4)e10=efe3+/fe2 + 00.05922log  ( fe2+fe3+[tptz]2)2 , \n ( 5)e10=0.771  v0.0592log  1.2651050.077(3104)2=0.578  v \n ( 6)e20=erssr / rsh00.0592ph . \n  in previous work", ", we found that the initial redox - complexation reaction rate is higher with mpg compared to nac [ 28 , 29 ] . in the coupled redox - complexation reaction with mpg , the steady state value of the absorbance is reached after 30  min while in the reaction with nac the steady state value of the absorbance is reached after 60  min . with both thiols ,", ".   equation ( 6 ) indicates that the potential for the redox system rsh / rssr depends upon the ph value of the reaction mixture .", "the effect of the ph was therefore investigated over the range 1.04.0 using 0.1  m hcl for ph 1 , 0.01  m hcl for ph 2 , and acetate buffer for the ph values 3.2 , 3.5 and 3.6 . in this experiment mpg", "the absorbance at 593  nm increased with increasing ph up to the value of 3.6 . however , precipitation of iron hydroxide occurred at the ph above 3.8 .", "therefore , a buffered reaction medium of ph 3.6 was chosen as a compromise for keeping fe(iii ) in solution and achieving quantitative formation of the fe(tptz)2 \n  complex which is stable in the ph range 3.45.8 .", "the influence of the fe(iii ) concentration on the determination of nac and mpg at the fixed concentration of 40  m each was studied in the concentration range from 20  m to 400  m , allowing a molar ratio fe(iii)/rsh from 0.5 to 10 . in figure 2 , the absorbance measured at 593  nm is plotted versus the molar fe(iii)/rsh ratio for nac and mpg .", "figure 2 shows that the reaction can be forced to completion by increasing the fe(iii)/rsh ratio , for instance by increasing the fe(iii ) concentration .  ", "the influence of the tptz concentration on the analysis of nac and mpg at the fixed concentration of 40  m rsh compound was studied in the range from 20  m to 400  m allowing a tptz / rsh molar ratio of 0.5 to 10 .  ", "figure 3 shows that absorbance increased with increasing tptz / rsh molar ratio , that is , with increasing tptz concentration , to reach its maximal value at a molar excess of five .", "the effect of the reaction temperature on the signal intensity was examined by varying the temperature from 25c to 40c using the thermostated water pump .", "we found that the reaction rate increased by elevating reaction temperature ( see [ 28 , 29 ] ) .", "since the proposed method is an equilibrium method , signal is recorded when the reaction reaches the state of equilibrium .", "the signal intensity in the state of equilibrium is the same for all the examined temperatures .", "the linearity of the method was investigated under the optimized conditions for nac and mpg in the concentration range from 1.0 to 100.0  m .", "straight lines were obtained from linear regression analysis of the absorbance at 593  nm and the drug concentration ( table 1 ) .", "the lowest quantifiable concentration of nac and mpg by this method was 1.0  m each .", "the effect of some possible interfering cations and anions on the analysis of a fixed concentration of 40.0  m for nac and mpg was investigated for the maximum molar ratio of foreign ions .", "the influence of excipients that can commonly accompany nac and mpg in pharmaceutical formulations was also studied .  ", "the tolerance is defined as the foreign - ion / excipient concentration causing an error smaller than 5% for the determination of the analyte of interest .", "the tolerable concentration of kno3 and na2so4 was 40.0  mm ( molar ratio , 1000  :  1 ) .", "the tolerable concentration of glucose , fructose , sucrose , boric acid , and acetic acid was 20.0  mm ( molar ratio , 500  :  1 ) .", "thus , the commonly excipients glucose , fructose , and sucrose do not interfere with the analysis of nac and mpg because they essentially do not react with the oxidizing agents .", "it should be emphasized that the contaminant / analyte concentration ratios studied in the present work are much higher than those normally found in commercial pharmaceutical products .", "the tolerable concentration of some other thiols , that is , d - penicillamine , l - glutathione , and l - cysteine , was 40.0  m ( molar ratio , 1  :  1 ) .", "thiols or other reducing substances with standard ( formal ) potentials higher than 0.6  v would not interfere in the proposed method . in order to evaluate the potential of the proposed method to the analysis of real samples , the method was applied to three pharmaceutical formulations of the drugs nac and mpg .", "the results of these analyses are presented in tables 2 and 3 .   for comparison , the spectrophotometric method reported by raggi et al .  ", "these authors used 1,10-phenantroline as an chromogenic reagent , instead of tptz which is used in the present work .", "as shown in table 2 , there were no significant differences between the values obtained by the reported method   and those obtained by the proposed method ( p > 0.1 , student t - test ) .", "this actually suggests that the proposed method is accurate and precise as the earlier reported method .", "the accuracy of the method was further ascertained through the recovery studies . to the drug solutions of the granules or the tablet powder , the standard solutions of the synthetic nac or mpg", "these results indicate that the proposed method is accurate for the determination of nac and mpg in their commercially available pharmaceutical preparations without any significant interference by common pharmaceutical excipients which do not absorb light in the visible region .  ", "performance characteristic of existing equilibrium spectrophotometric methods [ 19 , 2124 ] and the proposed method are compared in table 4 .", "the proposed method is free from drastic experimental conditions such as heating unlike some of reported methods .", "some other thiol compounds do not interfere in the proposed method at molar ratio 1  :  1 .", "it is also worth mentioning that the proposed method was performed in the visible region (  = 593  nm ) away from the uv - absorbance of the uv - absorbing interfering excipient materials , which might be dissolved from pharmaceutical formulations ."], ["undoubtedly , hplc is one of the most widely used techniques in routine analysis of pharmaceuticals , but it involves expensive instrumental set which many laboratories in developing and underdeveloped countries can not afford . the proposed equilibrium spectrophotometric method based on the coupled redox - complexation reaction of the thiol drug nac or mpg with fe(iii ) and tptz can be applied in every analytical laboratory as a reliable method for the determination of nac or mpg in pharmaceutical preparations . due to the use of tptz", "the coloured fe(tptz)2 \n  complex is stable in an extended period of time up to 24 hours .", "regular excipients and additives present in the pharmaceutical preparations of nac and mpg do not interfere in this method ."]]}
{"article_id": "PMC3981438", "article_text": ["vascular malformation is known as one of the major therapeutic problem even to the most experienced clinicians .", "these malformations grow proportionately with age , and may involve multiple anatomical spaces and encase critical neuromuscular structures , associated with nerve damage , massive bleeding , and deformity .", "management of arteriovenous malformations ( avms ) remains challenging because of their unpredictable behavior and high recurrence rate if treated by curettage alone without inadequate ligation of the feeder vessels .", "these lesions are treated by variety of techniques over the years , including surgical excision , irradiation , electrocoagulation , cryotherapy , intravascular magnesium or copper needles , systemic corticosteroids , interferon- , embolization , cryotherapy , lasers and sclerotherapy .", "sclerotherapy is an effective , conservative and low cost technique for the treatment of benign vascular lesions , included vascular malformation .", "the sclerosant agents used are 5% sodium morrhuate , sodium psylliate , quinine urethrone , 5% ethanolamine oleate , 1% polidocanol , sodium tetradecil sulfate , and hypertonic saline .", "we present our experience in treating palatal vascular malformation using direct ethanol sclerotherapy as a cheep therapeutic modality for treatment of deeply seated vascular lesion .", "twenty one - year old saudi male patient presented to the department of oral and maxillofacial surgery clinic at king abulaziz university complaining of swelling on the right side of the face ( figure 1a ) .", "extra - oral clinical examination revealed swelling on the right side of the face , which is soft and compressible .", "intraoral examination showed a bluish swelling at the junction of soft and hard palate that bleed easily on touch .", "computerized tomography ( ct ) and magnetic resonance image ( mri ) studies revealed that it is a vascular malformation that extends to the infratemporal area ( figure 2 ) .", "the patient was advised to have ethanol directly injected into the lesion as surgical excision was hazardous and difficult to achieve .", "patient disagree with that option and disappear for 18 months and returned back with severe gingival inflammation around the whole right maxillary quadrant which appeared covered by heavy calculus as he could not brush his teeth because of bleeding that he described as extensive ( figure 1c ) .", "advised was given to the patient that the only treatment option we can offer him is the ethanol injection .", "the procedure and possible complications were explained to the patient and family and they agreed to that option . under general anesthesia using fibro optic intubations , as the lesion was approaching the soft palate and there was a fear that routine nasal intubations could be hazardous to the airway , contrast medium was injected toward the engorged vascular lesion using a no .", "18 venipuncture catheter ( to determine the volume of the growths before treatment ) , followed by injection of one - third to one - quarter cavity volume of 95% ethyl alcohol into the tumor cavity according to lee and chen ( figure 3 ) .", "the patient was kept intubated for 48 h to guard his airway , as there was edema .", "intravenous dexamethasone ( 0.1 mg / kg ) was given for 3 days postoperatively to control inflammation .", "patient was discharged from the hospital on the fifth post injection day and kept follow up every week for the first three months , then every 2 weeks for another 3 months .", "the lesion remarkably shrinks and the hygienist was able to thoroughly scale his teeth without any bleeding ( figure 4 ) . \n", "figure 1a ) clinical picture of the patient showing facial asymmetry as result of right zygomatic swelling ; b ) intraoral view shows bluish mass extending to the soft palate when the patient presented to the clinic for the first time ; c ) intraoral photograph of the same patient 18 months after first presentation .", "the lesion increased in size , bled easily and appeared darkens with bad oral hygiene and heavy calculus at the right side of the jaw . \n ", "a ) clinical picture of the patient showing facial asymmetry as result of right zygomatic swelling ; b ) intraoral view shows bluish mass extending to the soft palate when the patient presented to the clinic for the first time ; c ) intraoral photograph of the same patient 18 months after first presentation .", "the lesion increased in size , bled easily and appeared darkens with bad oral hygiene and heavy calculus at the right side of the jaw . \n", "figure 2a ) axial and b ) coronal computed tomography scan obtained with contrast material shows dilated vessels that extend into the infratemporal area and multiple phleboliths can be seen .", "c ) and d ) axial t2 weighted magnetic resonance image shows high intensity lesion with flow representing vessels of different sizes . \n ", "a ) axial and b ) coronal computed tomography scan obtained with contrast material shows dilated vessels that extend into the infratemporal area and multiple phleboliths can be seen .", "c ) and d ) axial t2 weighted magnetic resonance image shows high intensity lesion with flow representing vessels of different sizes . \n", "figure 3a ) the catheter loaded with ethanol in place during injection ; b ) contrast medium is injected into the lesion and an x - ray is taken to make sure that the catheter is within the lesion itself ; c ) an angiogram shows ethanol injection within the lesion . \n ", "a ) the catheter loaded with ethanol in place during injection ; b ) contrast medium is injected into the lesion and an x - ray is taken to make sure that the catheter is within the lesion itself ; c ) an angiogram shows ethanol injection within the lesion . \n", "figure 4a ) postoperative picture of the lesion one and b ) three months following injection . \n ", "a ) postoperative picture of the lesion one and b ) three months following injection .", "there were no complications reported in this case except some palatal ulceration which is improving with every visit .", "treatment success was determined clinically by reduction in lesion size , ability of the patient to chew and to clean his teeth without bleeding , patient experience to pain and by patient satisfaction .", "for example : surgical excision can lead to significant loss of motor function , cosmetic problems , nerve damage , or massive bleeding and the complicated anatomy of the face and neck , may result in significant loss of motor function , cosmetic problems , nerve damage , or massive bleeding if surgery is adopted .", "laser therapy presents higher costs to the patient and may result in skin atrophy , transient or permanent hyperpigmentation , slight depression of the skin and scarring .", "intralesional injection of corticosteroids may cause adrenal suppression , slowed weight gain , atrophy , and necrosis .", "the use of sclerotherapy in the management of vascular anomalies has increased and replaced the traditional role of surgical therapy , especially for the venous lesions that are surgically difficult or at inaccessible areas .", "it has the advantage of low cost , no external scaring , and few complications compared to surgical treatment .", "varieties of sclerosing agents have been used for the management of various vascular malformations such as 5% sodium morrhuate , sodium tetradecyl sulphate , ethanolamine oleate , ok432 , bleomycin , ethanol , and hypertonic saline , alone or in various combinations .", "absolute ethanol was adopted as a new scleroagent for this complex form of venous defects to improve overall treatment results with acceptable morbidity and recurrence rates .", "ethanol is used most of often due to its low cost , antiseptic quality , wide availability and easy of use ; however , it requires general anesthesia because the procedure is very painful .", "the aim of treatment is to eradicate the lesion nidus completely , which is the fundamental abnormality as even the smallest residual nidus may expand to form a recurrence . in the presented case it was difficult to achieve complete eradication of the lesion by surgical approach because of the possible hazards that are associated with the affected site .", "it has been suggested that ethanol cause protoplasm precipitation which subsequently causes permanent obliteration of the vessel lumen . also do et al .", "reported that ethanol has shown the ability to induce protein denaturation of the endothelial cells with subsequent vessel wall denudation and interruption , which results in the complete obliteration of the vessel lumen rather than just simple obstruction .", "although various complications were reported to develop during the procedure such as local skin necrosis and fibrosis , haemoglobinuria , anaphylaxis , and some major complications were rarely reported including blood loss , acute pulmonary hypertension with cardio - pulmonary collapse , therefore , lee and chin used rubber bands to compress the patient 's forehead and chin to occlude facial venous return and minimize complications , also hammer et al . recommended to inject the ethanol slowly , over a period of several minutes and not exceeding 1 cc / kg of body weight while monitoring the pulmonary artery pressure to avoid such a catastrophic situation", "also , ethanol should only be used in the head and neck region by individuals and centers with professional expertise and experiences including maxillofacial surgeon , general radiology , general medicine , cardiovascular medicine , physical medicine and rehabilitation , dermatology , psychiatry , nuclear medicine , and social services with maximum efficiency to handle or at least minimize various anticipated complications .", "also , careful and routine monitoring of pulmonary arterial pressure are required by the interventional radiologist , and anesthesiologist .", "vascular malformations were treated with direct percutaneous injection of absolute ethanol , which resulted in remission , and alleviation of their symptoms , with no major complications .", "this technique provides a simple , low cost and reliable alternative treatment for venous malformation in the face and neck .", "absolute ethanol sclerotherapy can deliver excellent results as an independent therapy to the infiltrating type of extra - truncular form of vm .", "complications can occur but are rare . the morbidity involved should be clearly understood and accepted by the patient and family , and the risk of acute and chronic complications , either major or minor ,"], "abstract_text": ["<S> arteriovenous malformation is treated by variety of techniques over the years . </S>", "<S> sclerotherapy is considered an effective and conservative technique for the treatment of benign vascular lesions and replaced the traditional role of surgical therapy , especially for the venous lesions that are surgically difficult or at inaccessible areas . </S>", "<S> absolute ethanol was adopted as a new sclerosant agent for this complex form of venous defects to improve overall treatment results with acceptable morbidity and recurrence rates . </S>", "<S> sclerotherapy has the advantage of no external scaring , low cost , and few complications in comparison to the surgical treatment . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusions"], "sections": [["vascular malformation is known as one of the major therapeutic problem even to the most experienced clinicians .", "these malformations grow proportionately with age , and may involve multiple anatomical spaces and encase critical neuromuscular structures , associated with nerve damage , massive bleeding , and deformity .", "management of arteriovenous malformations ( avms ) remains challenging because of their unpredictable behavior and high recurrence rate if treated by curettage alone without inadequate ligation of the feeder vessels .", "these lesions are treated by variety of techniques over the years , including surgical excision , irradiation , electrocoagulation , cryotherapy , intravascular magnesium or copper needles , systemic corticosteroids , interferon- , embolization , cryotherapy , lasers and sclerotherapy .", "sclerotherapy is an effective , conservative and low cost technique for the treatment of benign vascular lesions , included vascular malformation .", "the sclerosant agents used are 5% sodium morrhuate , sodium psylliate , quinine urethrone , 5% ethanolamine oleate , 1% polidocanol , sodium tetradecil sulfate , and hypertonic saline .", "we present our experience in treating palatal vascular malformation using direct ethanol sclerotherapy as a cheep therapeutic modality for treatment of deeply seated vascular lesion ."], ["twenty one - year old saudi male patient presented to the department of oral and maxillofacial surgery clinic at king abulaziz university complaining of swelling on the right side of the face ( figure 1a ) .", "extra - oral clinical examination revealed swelling on the right side of the face , which is soft and compressible .", "intraoral examination showed a bluish swelling at the junction of soft and hard palate that bleed easily on touch .", "computerized tomography ( ct ) and magnetic resonance image ( mri ) studies revealed that it is a vascular malformation that extends to the infratemporal area ( figure 2 ) .", "the patient was advised to have ethanol directly injected into the lesion as surgical excision was hazardous and difficult to achieve .", "patient disagree with that option and disappear for 18 months and returned back with severe gingival inflammation around the whole right maxillary quadrant which appeared covered by heavy calculus as he could not brush his teeth because of bleeding that he described as extensive ( figure 1c ) .", "advised was given to the patient that the only treatment option we can offer him is the ethanol injection .", "the procedure and possible complications were explained to the patient and family and they agreed to that option . under general anesthesia using fibro optic intubations , as the lesion was approaching the soft palate and there was a fear that routine nasal intubations could be hazardous to the airway , contrast medium was injected toward the engorged vascular lesion using a no .", "18 venipuncture catheter ( to determine the volume of the growths before treatment ) , followed by injection of one - third to one - quarter cavity volume of 95% ethyl alcohol into the tumor cavity according to lee and chen ( figure 3 ) .", "the patient was kept intubated for 48 h to guard his airway , as there was edema .", "intravenous dexamethasone ( 0.1 mg / kg ) was given for 3 days postoperatively to control inflammation .", "patient was discharged from the hospital on the fifth post injection day and kept follow up every week for the first three months , then every 2 weeks for another 3 months .", "the lesion remarkably shrinks and the hygienist was able to thoroughly scale his teeth without any bleeding ( figure 4 ) . \n", "figure 1a ) clinical picture of the patient showing facial asymmetry as result of right zygomatic swelling ; b ) intraoral view shows bluish mass extending to the soft palate when the patient presented to the clinic for the first time ; c ) intraoral photograph of the same patient 18 months after first presentation .", "the lesion increased in size , bled easily and appeared darkens with bad oral hygiene and heavy calculus at the right side of the jaw . \n ", "a ) clinical picture of the patient showing facial asymmetry as result of right zygomatic swelling ; b ) intraoral view shows bluish mass extending to the soft palate when the patient presented to the clinic for the first time ; c ) intraoral photograph of the same patient 18 months after first presentation .", "the lesion increased in size , bled easily and appeared darkens with bad oral hygiene and heavy calculus at the right side of the jaw . \n", "figure 2a ) axial and b ) coronal computed tomography scan obtained with contrast material shows dilated vessels that extend into the infratemporal area and multiple phleboliths can be seen .", "c ) and d ) axial t2 weighted magnetic resonance image shows high intensity lesion with flow representing vessels of different sizes . \n ", "a ) axial and b ) coronal computed tomography scan obtained with contrast material shows dilated vessels that extend into the infratemporal area and multiple phleboliths can be seen .", "c ) and d ) axial t2 weighted magnetic resonance image shows high intensity lesion with flow representing vessels of different sizes . \n", "figure 3a ) the catheter loaded with ethanol in place during injection ; b ) contrast medium is injected into the lesion and an x - ray is taken to make sure that the catheter is within the lesion itself ; c ) an angiogram shows ethanol injection within the lesion . \n ", "a ) the catheter loaded with ethanol in place during injection ; b ) contrast medium is injected into the lesion and an x - ray is taken to make sure that the catheter is within the lesion itself ; c ) an angiogram shows ethanol injection within the lesion . \n", "figure 4a ) postoperative picture of the lesion one and b ) three months following injection . \n ", "a ) postoperative picture of the lesion one and b ) three months following injection .", "there were no complications reported in this case except some palatal ulceration which is improving with every visit .", "treatment success was determined clinically by reduction in lesion size , ability of the patient to chew and to clean his teeth without bleeding , patient experience to pain and by patient satisfaction ."], ["for example : surgical excision can lead to significant loss of motor function , cosmetic problems , nerve damage , or massive bleeding and the complicated anatomy of the face and neck , may result in significant loss of motor function , cosmetic problems , nerve damage , or massive bleeding if surgery is adopted .", "laser therapy presents higher costs to the patient and may result in skin atrophy , transient or permanent hyperpigmentation , slight depression of the skin and scarring .", "intralesional injection of corticosteroids may cause adrenal suppression , slowed weight gain , atrophy , and necrosis .", "the use of sclerotherapy in the management of vascular anomalies has increased and replaced the traditional role of surgical therapy , especially for the venous lesions that are surgically difficult or at inaccessible areas .", "it has the advantage of low cost , no external scaring , and few complications compared to surgical treatment .", "varieties of sclerosing agents have been used for the management of various vascular malformations such as 5% sodium morrhuate , sodium tetradecyl sulphate , ethanolamine oleate , ok432 , bleomycin , ethanol , and hypertonic saline , alone or in various combinations .", "absolute ethanol was adopted as a new scleroagent for this complex form of venous defects to improve overall treatment results with acceptable morbidity and recurrence rates .", "ethanol is used most of often due to its low cost , antiseptic quality , wide availability and easy of use ; however , it requires general anesthesia because the procedure is very painful .", "the aim of treatment is to eradicate the lesion nidus completely , which is the fundamental abnormality as even the smallest residual nidus may expand to form a recurrence . in the presented case it was difficult to achieve complete eradication of the lesion by surgical approach because of the possible hazards that are associated with the affected site .", "it has been suggested that ethanol cause protoplasm precipitation which subsequently causes permanent obliteration of the vessel lumen . also do et al .", "reported that ethanol has shown the ability to induce protein denaturation of the endothelial cells with subsequent vessel wall denudation and interruption , which results in the complete obliteration of the vessel lumen rather than just simple obstruction .", "although various complications were reported to develop during the procedure such as local skin necrosis and fibrosis , haemoglobinuria , anaphylaxis , and some major complications were rarely reported including blood loss , acute pulmonary hypertension with cardio - pulmonary collapse , therefore , lee and chin used rubber bands to compress the patient 's forehead and chin to occlude facial venous return and minimize complications , also hammer et al . recommended to inject the ethanol slowly , over a period of several minutes and not exceeding 1 cc / kg of body weight while monitoring the pulmonary artery pressure to avoid such a catastrophic situation", "also , ethanol should only be used in the head and neck region by individuals and centers with professional expertise and experiences including maxillofacial surgeon , general radiology , general medicine , cardiovascular medicine , physical medicine and rehabilitation , dermatology , psychiatry , nuclear medicine , and social services with maximum efficiency to handle or at least minimize various anticipated complications .", "also , careful and routine monitoring of pulmonary arterial pressure are required by the interventional radiologist , and anesthesiologist ."], ["vascular malformations were treated with direct percutaneous injection of absolute ethanol , which resulted in remission , and alleviation of their symptoms , with no major complications .", "this technique provides a simple , low cost and reliable alternative treatment for venous malformation in the face and neck .", "absolute ethanol sclerotherapy can deliver excellent results as an independent therapy to the infiltrating type of extra - truncular form of vm .", "complications can occur but are rare . the morbidity involved should be clearly understood and accepted by the patient and family , and the risk of acute and chronic complications , either major or minor ,"]]}
{"article_id": "PMC3524437", "article_text": ["in the 1980s , the prevalence of rickets in extremely low birth weight ( elbw ) infants was around 50% and was 17% for fractures ( 1 , 2 ) .", "after introduction of a high mineral containing diet in the late 1980s , the prevalence decreased to 30% or lower ( 3 , 4 ) .", "there is little information , however , beyond 1985 , concerning the incidence of rickets in extreme prematurity and factors that relate to its occurrence .", "in addition to inadequate mineral intake as a major contributing factor ( 5 ) , chronic co - morbidities , like chronic lung disease , short bowel syndrome , and parenteral nutrition associated cholestasis ( pnac ) could also be related factors ( 6 ) .", "medications used in the neonatal intensive care unit ( nicu ) , such as steroids , methylxanthines , and diuretics could theoretically increase the risk of inadequate bone mineralization ( 7 - 9 ) .", "other risk factors , such as immobilization from sepsis , cerebral pathology , muscular disorders and paralysis are associated with bone disease of prematurity ( 10 ) .", "since mineral enriched formula for the preterms began 30 yr ago , it is the standard practice to provide minerals in an attempt to reach in utero mineral accretion rates . in theory , under ideal conditions , the adventage of high mineral intake formulas should be associated with a marked reduction of rickets .", "however , it is often practically difficult to achieve ideal nutrition support in sick elbw infants , the current incidence of rickets in elbw infants is unknown , and the relationship of the severity of overall illness and degree of prematurity in this population is unclear .", "the aim of this study was to characterize the risk factors for development of rickets in elbw infants fed with high mineral containing preterm formula .", "as a retrospective case - control study , medical records of 55 elbw infants admitted into the severance children 's hospital nicu from 2004 to 2008 that survived until 1 month of age were reviewed .", "all infants had their serum alkaline phosphatase ( alp ) , calcium ( ca ) and phosphate ( p ) levels reported weekly and bone screen radiography at 1 month of age . for nutritional care ,", "fortified human milk ( ca 131 mg/100 kcal , p 71 mg/100 kcal ) or a mineral fortified preterm formula ( ca 112 - 141 mg / kcal , p 52 - 63 mg / kcal ) was used . during parenteral nutrition , ca , p and multivitamins with trace minerals were added to parenteral nutrition , appropriately .", "grade 1 was defined as a loss of dense white line , increased lucency of bone shaft , and thinning of cortex ; grade 2 showed irregularity and fraying of a metaphysis with splaying and cupping ; and grade 3 presented evidence of fractures . as co - morbidities , hyaline membrane disease ( hmd ) , patent ductus arteriosus ( pda ) , necrotizing enterocolitis ( nec ; using the modified bell 's staging criteria ) , parenteral nutrition associated cholestasis ( pnac , defined as direct bilirubin [ db ] greater than 2.0 mg / dl ) , bronchopulmonary dysplasia ( bpd ; using nih classification ) , retinopathy of prematurity ( rop , included above stage 2 ) , and intraventricular hemorrhage ( ivh , above grade 3 ) were analyzed .", "severe pnac was defined as elevation of db greater than 4.0 mg / dl for more than 1 month and aspartate aminotransferase ( ast ) and alanine aminotransterase ( alt ) greater than 60", "moderate / severe bpd was defined in infants requiring either 28 days of supplemental oxygen therapy with oxygen , nasal continuous positive airway pressure ( cpap ) or positive pressure ventilation at 36 weeks postmenstrual age or discharge , whichever came first , whose gestational age was younger than 32 weeks , or at 56 days postnatal age or discharge , whichever came first , in infants whose gestational age was older than 32 weeks .", "statistics were calculated with the student t - test and chi - square test for means and frequencies .", "numeric data are presented as mean and standard deviation because the data were normally distributed .", "calculations were performed with spss software version 17.0 ( spss , chicago , il , usa ) .", "the present retrospective review of database in this study was approved by the institutional review board of the yonsei university health system , severance hospital ( approval no . , 4 - 2012 - 0157 ) .", "the present retrospective review of database in this study was approved by the institutional review board of the yonsei university health system , severance hospital ( approval no . , 4 - 2012 - 0157 ) .", "twenty - four elbw infants ( 26.3  1.9 weeks , 792  99 g ) were diagnosed as rickets of prematurity , and 31 infants without rickets ( 27.2  2.3 weeks , 871  93 g ) were the control group .", "rickets of prematurity was diagnosed in 43.9% of elbw infants and fracture in 13% . for the rickets group , 7 infants ( 29% ) showed g1 , 14 infants ( 58% ) g2 and 3 infants ( 13% ) g3 .", "rickets of prematurity was diagnosed at 48.2  16.1 days of life and improved by 85.3  25.3 days of life . in patient characteristics such as mean gestational ages , birth weight , gender , and frequency of intrauterine growth retardation ( iugr )", "the cumulative amounts ( either enteral or parenteral ) of ca and p administration during hospitalization were not different between the groups . for the rickets group in comparison with the control group , mean days on oxygen and ventilator use were significantly longer ( p < 0.05 ) .", "the duration of hospitalization and parenteral nutrition were significantly longer in the rickets group versus the control group ( table 1 ) . in univariate analysis , elbw infants with rickets had a significantly higher occurrence of pda ( p = 0.025 ) , pnac ( p = 0.041 ) , severe pnac ( p = 0.013 ) , bpd ( p = 0.019 ) , and moderate / severe bpd ( p = 0.012 ) ( table 2 ) .", "common neonatal illnesses such as hmd , air leak , nec , rop , ivh and periventricular leucomalacia ( pvl ) were not different between the groups . in multiple regression analysis after adjustment of gestation and birth weight , rickets in elbw infants significantly correlated with severe pnac ( or 18.5 ; 95% ci , 1.1 - 285 ; p = 0.042 ) and moderate / severe bpd ( or 3.2 ; 95% ci , 1.2 - 26.5 ; p = 0.04 ) ( table 3 ) .", "the mean level of peak alp was significantly higher in the rickets group than the control group ( 952.2  413.8 vs 524.7  158.2", "iu / l ) , while the mean level of the lowest ca and p levels were not different between groups ( table 4 ) .", "in the 1980s , fortified mineral formulas were introduced to reduce the rates of rickets and fractures in very low birth weight ( vlbw < 1,500 g ) infants ( 11 ) .", "reports at that time indicated that up to 30% of vlbws had rickets with fractures ( 12 ) . with advances in preterm nutrition , especially with the introduction of mineral fortified formulas , theoretically", "the incidence of rickets of prematurity should have dropped ( 3 , 4 ) . however , for increasing the survival of elbw infants , significant co - morbid conditions could affect actual mineral intake which might mitigate the effect of high mineral fortification ( 13 ) .", "this is the first study to focus on the incidence of rickets in elbw infants after 2 decades of fortified formula use in korea .", "it is also the first to characterize and compare risk factors and neonatal morbidities for rickets of prematurity in elbw infants .", "we found that the incidence of radiologic rickets in elbw infants was extremely high at 44% ( 24/55 ) due to increased survival , and the incidence of significantly increased 18 times and 3 times for severe pnac and moderate to severe bpd , respectively .", "factors commonly thought to relate to rickets of prematurity include mismatch of postnatal intake of minerals compared to intrauterine mineral transfer , low bone loading effects in the nicu , intrauterine growth restriction , chorioamnionitis , steroids , methylxanthines and diuretic usage and necrotizing enterocolitis ( 7 - 10 , 14 - 16 ) .", "inadequate mineral nutrient ( calcium and phosphorous ) intake appears to play a major role in causing rickets of prematurity ( 10 , 17 ) . prolonged parenteral nutrition ( pn ) and delayed enteral nutrition complicate delivery of adequate mineral intake and would be particularly acute in elbw infants ( 10 ) .", "it is standard practice in nicu to provide high mineral preterm formula and human milk fortifier as an attempt to reach in utero mineral accretion rate , including the use of minimal early enteral feeding , which appears to improve bone mass at term ( 3 , 17 - 19 ) .", "however , in elbw infants , co - morbid illnesses often preclude full enteral tolerance and the actual delivery of mineral nutrients ( 13 ) .", "further , when pn is needed , the provision of minerals in pn can be limited by solubility issues compounded by fluid restriction in sick infants ( 3 ) .", "specific chronic co - morbidities , particularly chronic lung disease and short bowel syndrome which is generally associated with prolonged parenteral nutrition and pnac , are potentially serious risk factors for poor bone mineralization ( 6 , 20 , 21 ) .", "thus our findings of markedly higher risk of rickets in elbw infants with pnac ( 18 times ) and bpd ( 3 times ) support these associations", ". the limitations of this study are a small sample size of a single institution and a retrospective design . in conclusion", ", there is a high incidence ( 44% ) of rickets in elbw infants , which shows an 18-fold increase in severe pnac and a 3-fold increase in moderate to severe bpd .", "we suggest that in elbw infants with severe pnac or moderate / severe bpd , aggressive prevention or treatment for rickets and fractures can be instituted earlier in the course of management ."], "abstract_text": ["<S> risk factors for rickets of prematurity have not been re - examined since introduction of high mineral formula , particularly in elbw infants . </S>", "<S> we analyzed the incidence and the risk factors of rickets in extremely low birth weight ( elbw ) infants . </S>", "<S> as a retrospective case - control study from 2004 to 2008 , risk factors were analyzed in 24 patients with rickets versus 31 patients without . </S>", "<S> the frequency of rickets in elbw infants was 24/55 ( 44% ) . </S>", "<S> infants with rickets were diagnosed at 48.2  16.1 days of age , and improved by 85.3  25.3 days . by radiologic evaluation , </S>", "<S> 29% were grade 1 rickets , 58% grade 2 and 13% grade 3 . in univariate analysis , infants with rickets had significantly higher incidence of patent ductus arteriosus , parenteral nutrition associated cholestasis ( pnac ) , severe pnac and moderate / severe bronchopulmonary dysplasia ( bpd ) . </S>", "<S> in multiple regression analysis , after adjustment for gestation and birth weight , rickets significantly correlated with severe pnac and with moderate / severe bpd . </S>", "<S> serum peak alkaline phosphatase levels were significantly elevated in rickets ( p < 0.001 ) . in elbw infants , </S>", "<S> the incidence of rickets of prematurity remains high and the incidence of severe pnac and moderate / severe bpd was significantly increased 18 and 3 times , respectively . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Ethics statement", "RESULTS", "DISCUSSION"], "sections": [["in the 1980s , the prevalence of rickets in extremely low birth weight ( elbw ) infants was around 50% and was 17% for fractures ( 1 , 2 ) .", "after introduction of a high mineral containing diet in the late 1980s , the prevalence decreased to 30% or lower ( 3 , 4 ) .", "there is little information , however , beyond 1985 , concerning the incidence of rickets in extreme prematurity and factors that relate to its occurrence .", "in addition to inadequate mineral intake as a major contributing factor ( 5 ) , chronic co - morbidities , like chronic lung disease , short bowel syndrome , and parenteral nutrition associated cholestasis ( pnac ) could also be related factors ( 6 ) .", "medications used in the neonatal intensive care unit ( nicu ) , such as steroids , methylxanthines , and diuretics could theoretically increase the risk of inadequate bone mineralization ( 7 - 9 ) .", "other risk factors , such as immobilization from sepsis , cerebral pathology , muscular disorders and paralysis are associated with bone disease of prematurity ( 10 ) .", "since mineral enriched formula for the preterms began 30 yr ago , it is the standard practice to provide minerals in an attempt to reach in utero mineral accretion rates . in theory , under ideal conditions , the adventage of high mineral intake formulas should be associated with a marked reduction of rickets .", "however , it is often practically difficult to achieve ideal nutrition support in sick elbw infants , the current incidence of rickets in elbw infants is unknown , and the relationship of the severity of overall illness and degree of prematurity in this population is unclear .", "the aim of this study was to characterize the risk factors for development of rickets in elbw infants fed with high mineral containing preterm formula ."], ["as a retrospective case - control study , medical records of 55 elbw infants admitted into the severance children 's hospital nicu from 2004 to 2008 that survived until 1 month of age were reviewed .", "all infants had their serum alkaline phosphatase ( alp ) , calcium ( ca ) and phosphate ( p ) levels reported weekly and bone screen radiography at 1 month of age . for nutritional care ,", "fortified human milk ( ca 131 mg/100 kcal , p 71 mg/100 kcal ) or a mineral fortified preterm formula ( ca 112 - 141 mg / kcal , p 52 - 63 mg / kcal ) was used . during parenteral nutrition , ca , p and multivitamins with trace minerals were added to parenteral nutrition , appropriately .", "grade 1 was defined as a loss of dense white line , increased lucency of bone shaft , and thinning of cortex ; grade 2 showed irregularity and fraying of a metaphysis with splaying and cupping ; and grade 3 presented evidence of fractures . as co - morbidities , hyaline membrane disease ( hmd ) , patent ductus arteriosus ( pda ) , necrotizing enterocolitis ( nec ; using the modified bell 's staging criteria ) , parenteral nutrition associated cholestasis ( pnac , defined as direct bilirubin [ db ] greater than 2.0 mg / dl ) , bronchopulmonary dysplasia ( bpd ; using nih classification ) , retinopathy of prematurity ( rop , included above stage 2 ) , and intraventricular hemorrhage ( ivh , above grade 3 ) were analyzed .", "severe pnac was defined as elevation of db greater than 4.0 mg / dl for more than 1 month and aspartate aminotransferase ( ast ) and alanine aminotransterase ( alt ) greater than 60", "moderate / severe bpd was defined in infants requiring either 28 days of supplemental oxygen therapy with oxygen , nasal continuous positive airway pressure ( cpap ) or positive pressure ventilation at 36 weeks postmenstrual age or discharge , whichever came first , whose gestational age was younger than 32 weeks , or at 56 days postnatal age or discharge , whichever came first , in infants whose gestational age was older than 32 weeks .", "statistics were calculated with the student t - test and chi - square test for means and frequencies .", "numeric data are presented as mean and standard deviation because the data were normally distributed .", "calculations were performed with spss software version 17.0 ( spss , chicago , il , usa ) .", "the present retrospective review of database in this study was approved by the institutional review board of the yonsei university health system , severance hospital ( approval no . , 4 - 2012 - 0157 ) ."], ["the present retrospective review of database in this study was approved by the institutional review board of the yonsei university health system , severance hospital ( approval no . , 4 - 2012 - 0157 ) ."], ["twenty - four elbw infants ( 26.3  1.9 weeks , 792  99 g ) were diagnosed as rickets of prematurity , and 31 infants without rickets ( 27.2  2.3 weeks , 871  93 g ) were the control group .", "rickets of prematurity was diagnosed in 43.9% of elbw infants and fracture in 13% . for the rickets group , 7 infants ( 29% ) showed g1 , 14 infants ( 58% ) g2 and 3 infants ( 13% ) g3 .", "rickets of prematurity was diagnosed at 48.2  16.1 days of life and improved by 85.3  25.3 days of life . in patient characteristics such as mean gestational ages , birth weight , gender , and frequency of intrauterine growth retardation ( iugr )", "the cumulative amounts ( either enteral or parenteral ) of ca and p administration during hospitalization were not different between the groups . for the rickets group in comparison with the control group , mean days on oxygen and ventilator use were significantly longer ( p < 0.05 ) .", "the duration of hospitalization and parenteral nutrition were significantly longer in the rickets group versus the control group ( table 1 ) . in univariate analysis , elbw infants with rickets had a significantly higher occurrence of pda ( p = 0.025 ) , pnac ( p = 0.041 ) , severe pnac ( p = 0.013 ) , bpd ( p = 0.019 ) , and moderate / severe bpd ( p = 0.012 ) ( table 2 ) .", "common neonatal illnesses such as hmd , air leak , nec , rop , ivh and periventricular leucomalacia ( pvl ) were not different between the groups . in multiple regression analysis after adjustment of gestation and birth weight , rickets in elbw infants significantly correlated with severe pnac ( or 18.5 ; 95% ci , 1.1 - 285 ; p = 0.042 ) and moderate / severe bpd ( or 3.2 ; 95% ci , 1.2 - 26.5 ; p = 0.04 ) ( table 3 ) .", "the mean level of peak alp was significantly higher in the rickets group than the control group ( 952.2  413.8 vs 524.7  158.2", "iu / l ) , while the mean level of the lowest ca and p levels were not different between groups ( table 4 ) ."], ["in the 1980s , fortified mineral formulas were introduced to reduce the rates of rickets and fractures in very low birth weight ( vlbw < 1,500 g ) infants ( 11 ) .", "reports at that time indicated that up to 30% of vlbws had rickets with fractures ( 12 ) . with advances in preterm nutrition , especially with the introduction of mineral fortified formulas , theoretically", "the incidence of rickets of prematurity should have dropped ( 3 , 4 ) . however , for increasing the survival of elbw infants , significant co - morbid conditions could affect actual mineral intake which might mitigate the effect of high mineral fortification ( 13 ) .", "this is the first study to focus on the incidence of rickets in elbw infants after 2 decades of fortified formula use in korea .", "it is also the first to characterize and compare risk factors and neonatal morbidities for rickets of prematurity in elbw infants .", "we found that the incidence of radiologic rickets in elbw infants was extremely high at 44% ( 24/55 ) due to increased survival , and the incidence of significantly increased 18 times and 3 times for severe pnac and moderate to severe bpd , respectively .", "factors commonly thought to relate to rickets of prematurity include mismatch of postnatal intake of minerals compared to intrauterine mineral transfer , low bone loading effects in the nicu , intrauterine growth restriction , chorioamnionitis , steroids , methylxanthines and diuretic usage and necrotizing enterocolitis ( 7 - 10 , 14 - 16 ) .", "inadequate mineral nutrient ( calcium and phosphorous ) intake appears to play a major role in causing rickets of prematurity ( 10 , 17 ) . prolonged parenteral nutrition ( pn ) and delayed enteral nutrition complicate delivery of adequate mineral intake and would be particularly acute in elbw infants ( 10 ) .", "it is standard practice in nicu to provide high mineral preterm formula and human milk fortifier as an attempt to reach in utero mineral accretion rate , including the use of minimal early enteral feeding , which appears to improve bone mass at term ( 3 , 17 - 19 ) .", "however , in elbw infants , co - morbid illnesses often preclude full enteral tolerance and the actual delivery of mineral nutrients ( 13 ) .", "further , when pn is needed , the provision of minerals in pn can be limited by solubility issues compounded by fluid restriction in sick infants ( 3 ) .", "specific chronic co - morbidities , particularly chronic lung disease and short bowel syndrome which is generally associated with prolonged parenteral nutrition and pnac , are potentially serious risk factors for poor bone mineralization ( 6 , 20 , 21 ) .", "thus our findings of markedly higher risk of rickets in elbw infants with pnac ( 18 times ) and bpd ( 3 times ) support these associations", ". the limitations of this study are a small sample size of a single institution and a retrospective design . in conclusion", ", there is a high incidence ( 44% ) of rickets in elbw infants , which shows an 18-fold increase in severe pnac and a 3-fold increase in moderate to severe bpd .", "we suggest that in elbw infants with severe pnac or moderate / severe bpd , aggressive prevention or treatment for rickets and fractures can be instituted earlier in the course of management ."]]}
{"article_id": "PMC3725033", "article_text": ["kyphosis is a progressive curvature of the thoracic spine , which results in rounding or bowing of the back .", "a curved spine accompanied by restricted neck motion and poor lung reserve present challenges for an ophthalmic surgeon , who is used to patients neck and head resting flat on the operating table and the eye in the horizontal position .", "intraoperative difficulties include poor view , inability to maneuver surgical instruments properly and constant threat of elevated vitreous pressure .", "several innovative solutions have been published , including the maximal reverse trendelenburg position and pillows under the head and neck , use of a donut and wedge - shaped head positioner , allowing patients to remain in the upright sitting position while their head is positioned horizontally in a modified waiting room chair , performing surgery while standing [ 5 , 6 ] , using a 4-section orthopedic operating table , or laying the patients on their sides . however", "a 49-year - old obese male with controlled secondary glaucoma needed cataract surgery in the right eye .", "the past ocular history was positive for recurrent uveitis and glaucoma shunt surgery in the right eye .", "cataract density was + 4 , and the best - corrected visual acuity was 20/200 .", "the anterior chamber was shallow and the glaucoma shunt tube was visible at the 11 o'clock position .", "the patient was planned for pupillary dilation using iris retraction hooks , lens capsule staining with trypan blue and phacoemulsification with intraocular implantation under local anesthesia .", "the anesthesiologist was reluctant to use general anesthesia because of the patient 's disability and poor health . in the operating room ,", "the patient could only recline to about 40 from the vertical axis despite maximal reverse trendelenburg position and a number of pillows under his back , neck , head and legs ( fig .", "2 ) . while trying to focus the microscope , it became evident that by grasping the eye at the inferior limbus with a fine forceps and pulling it upwards , it could be moved to a more desirable horizontal position .", "a single 7/0 vicryl ( polyglactin ) corneal retraction suture was placed at the limbus at the 6 o'clock position to achieve the desired view ( fig .", "the assistant surgeon was able to rotate the eye to the desired positions during the whole procedure by relaxing or pulling the suture .", "patients with kyphosis may present several unique challenges to an eye surgeon , the foremost being the inability to lay the head and neck in the ideal horizontal position .", "the resulting poor view makes focusing and manipulation of tissues and instruments difficult . tilting the head end of the operating table down and raising the patient 's legs may compromise venous return from the orbit , causing venous engorgement and elevated posterior vitreous pressure during surgery .", "in addition , the patient 's poor pulmonary reserve itself may further impede orbital / jugular venous return , resulting in increased posterior vitreous pressure . other features of kyphosis that may adversely affect intraocular surgery are tenderness and stiffness of the back and difficulty in breathing .", "several practical solutions have been described by very experienced surgeons but complications may still occur .", "a wedge - shaped pillow with a built - in donut for the positioning of the head has been used by some surgeons .", "livingston and mackool   recommended using more than one of these positioners in severe cases .", "another approach suggested performing cataract surgery while standing , using loupes and making an inferotemporal scleral tunnel [ 5 , 6 ] .", "the surgeons were unable to remove the subincisional cortex because of a shallow anterior chamber secondary to elevated posterior vitreous pressure .", "prasad et al .   recommended using a 4-section orthopedic operating table to enable an extreme reverse trendelenburg position for cataract and glaucoma surgery .", "however , because of a blurred vision , these authors were unable to perform curvilinear capsulorhexis and had to resort to can - opener capsulotomy .", "in short , there is no single solution for the different groups of difficult patients .", "we have found that a single retraction suture at the inferior limbus can rotate the eye to any desired position .", "this may allow both the patient and surgeon to be in a comfortable position without compromising good view .", "it may be tried practically in all patients with kyphosis or other conditions that prevent them from reclining such as torticollis , chronic obstructive pulmonary disease , chronic congestive heart failure or morbid obesity .", "the surgeon can still sit on the preferred temporal side of the patient and perform intraocular surgery of choice . with this maneuver", ", it may be unnecessary to resort to extreme and uncomfortable tilting of the patient and avoid complications such as those described above .", "the authors have no financial or proprietary interest in the materials described in the article ."], "abstract_text": ["<S> we describe a 49-year - old man with advanced kyphosis and dense cataract , who could only recline to about 40 from the vertical axis despite a maximal reverse trendelenburg position and pillows under the head , neck , shoulders and knees . with a single corneal retraction suture at 6 o'clock , the eye could be rotated horizontally , which enabled the surgeon to perform a complex cataract surgery despite prior glaucoma shunt , posterior synechiae , a small pupil and the need to stain the capsule . </S>", "<S> as the eye can be brought into any desired position with a retraction suture , patients with kyphosis or other conditions that prevent them from assuming a supine position can still have safe intraocular procedures . </S>", "<S> this maneuver reduces the need to tilt patients to an uncomfortable position that may cause pain , increased breathing difficulty and elevated posterior vitreous pressure . </S>"], "labels": null, "section_names": ["Introduction", "Case Presentation", "Discussion", "Disclosure Statement"], "sections": [["kyphosis is a progressive curvature of the thoracic spine , which results in rounding or bowing of the back .", "a curved spine accompanied by restricted neck motion and poor lung reserve present challenges for an ophthalmic surgeon , who is used to patients neck and head resting flat on the operating table and the eye in the horizontal position .", "intraoperative difficulties include poor view , inability to maneuver surgical instruments properly and constant threat of elevated vitreous pressure .", "several innovative solutions have been published , including the maximal reverse trendelenburg position and pillows under the head and neck , use of a donut and wedge - shaped head positioner , allowing patients to remain in the upright sitting position while their head is positioned horizontally in a modified waiting room chair , performing surgery while standing [ 5 , 6 ] , using a 4-section orthopedic operating table , or laying the patients on their sides . however"], ["a 49-year - old obese male with controlled secondary glaucoma needed cataract surgery in the right eye .", "the past ocular history was positive for recurrent uveitis and glaucoma shunt surgery in the right eye .", "cataract density was + 4 , and the best - corrected visual acuity was 20/200 .", "the anterior chamber was shallow and the glaucoma shunt tube was visible at the 11 o'clock position .", "the patient was planned for pupillary dilation using iris retraction hooks , lens capsule staining with trypan blue and phacoemulsification with intraocular implantation under local anesthesia .", "the anesthesiologist was reluctant to use general anesthesia because of the patient 's disability and poor health . in the operating room ,", "the patient could only recline to about 40 from the vertical axis despite maximal reverse trendelenburg position and a number of pillows under his back , neck , head and legs ( fig .", "2 ) . while trying to focus the microscope , it became evident that by grasping the eye at the inferior limbus with a fine forceps and pulling it upwards , it could be moved to a more desirable horizontal position .", "a single 7/0 vicryl ( polyglactin ) corneal retraction suture was placed at the limbus at the 6 o'clock position to achieve the desired view ( fig .", "the assistant surgeon was able to rotate the eye to the desired positions during the whole procedure by relaxing or pulling the suture ."], ["patients with kyphosis may present several unique challenges to an eye surgeon , the foremost being the inability to lay the head and neck in the ideal horizontal position .", "the resulting poor view makes focusing and manipulation of tissues and instruments difficult . tilting the head end of the operating table down and raising the patient 's legs may compromise venous return from the orbit , causing venous engorgement and elevated posterior vitreous pressure during surgery .", "in addition , the patient 's poor pulmonary reserve itself may further impede orbital / jugular venous return , resulting in increased posterior vitreous pressure . other features of kyphosis that may adversely affect intraocular surgery are tenderness and stiffness of the back and difficulty in breathing .", "several practical solutions have been described by very experienced surgeons but complications may still occur .", "a wedge - shaped pillow with a built - in donut for the positioning of the head has been used by some surgeons .", "livingston and mackool   recommended using more than one of these positioners in severe cases .", "another approach suggested performing cataract surgery while standing , using loupes and making an inferotemporal scleral tunnel [ 5 , 6 ] .", "the surgeons were unable to remove the subincisional cortex because of a shallow anterior chamber secondary to elevated posterior vitreous pressure .", "prasad et al .   recommended using a 4-section orthopedic operating table to enable an extreme reverse trendelenburg position for cataract and glaucoma surgery .", "however , because of a blurred vision , these authors were unable to perform curvilinear capsulorhexis and had to resort to can - opener capsulotomy .", "in short , there is no single solution for the different groups of difficult patients .", "we have found that a single retraction suture at the inferior limbus can rotate the eye to any desired position .", "this may allow both the patient and surgeon to be in a comfortable position without compromising good view .", "it may be tried practically in all patients with kyphosis or other conditions that prevent them from reclining such as torticollis , chronic obstructive pulmonary disease , chronic congestive heart failure or morbid obesity .", "the surgeon can still sit on the preferred temporal side of the patient and perform intraocular surgery of choice . with this maneuver", ", it may be unnecessary to resort to extreme and uncomfortable tilting of the patient and avoid complications such as those described above ."], ["the authors have no financial or proprietary interest in the materials described in the article ."]]}
{"article_id": "PMC3767350", "article_text": ["using water as solvent in the organic reactions is one of the most important targets to organic chemists because of the easy availability , nontoxicity , and ecofriendly nature of the water [ 17 ] . in this endeavour", ", a number of chemical reactions such as diels alder , hetero diels - alder , 1,3-dipolar cycloaddition , oxidations , reductions , and others are performed successfully in water [ 13 ] .", "also , it is reported that in few cases addition of the water increases the rate and the yield of a reaction and also enhances the enantioselectivity in a chiral synthesis .", "but the main problems associated with water as a solvent is its poor ability to solubilise organic reactants and incapability to create anhydrous condition for moisture sensitive organic compounds and catalysts . to overcome the solubility problem , generally a surfactant is introduced to the reaction mixture .", "the surfactant , due to its hydrophobic and hydrophilic nature , forms micelles with water insoluble organic compounds and promote the desired reactions to occur inside the hydrophobic ambience of the micelle core [ 9 , 10 ] .", "cleavage of the c = n bonds is a very important transformation in organic synthesis as the c = n functionality is widely used to protect both the carbonyl and amines .", "there are a number of methods used for the cleavage of c = n bonds which include acidic reagents [ 1113 ] , oxidizing agents , metallic salts [ 15 , 16 ] , ( phseo)2o , nahso3 , and others .", "most of these methodologies suffer from serious drawbacks like involvement of strong lewis and bronsted acids , use of toxic and costly transition metals ( i.e. , cr , pd , co ) , low temperature , longer reaction time , low yield , and difficulties in isolating the products", ". therefore , development of efficient , mild and environment friendly reagents are always necessary . on the other hand", ", bisindoles are recently emerging as extremely important class of compounds because of their novel antibacterial and anticancer activities [ 1921 ] .", "that is why a number of methodologies have also been postulated for the synthesis of bisindoles [ 2229 ] . in our previous communications", ", we reported that surfactant- ( sds- ) mediated cleavage of c = n bonds could be achieved with acetic anhydride   and surfactant - i2-water can be used for the deprotection of imines to carbonyls .", "we have also shown that bis- and tris ( indolyl)alkanes can be synthesized in presence of bronsted acid in water . in continuation of our research in hydrated media , herein , we wish to disclose the dual catalytic activity of the system i2-sds - h2o which behaves as a lewis acid   for the cleavage of 2,3-diaza-1,3-butadiene , 1-aza-1,3-butadienes , and oximes to produce carbonyls and amines , and the resulting reaction mixture reacts with indoles to produce bis ( indolyl)alkanes in situ at room temperature under neutral conditions .", "for an initial study , molecular iodine was added to a mixture of 1,4-diphenyl-2,3-diaza-1,3-butadiene ( 1a ) ( 1  mmol ) and indole ( 2a ) ( 2  mmol ) in water .", "it was presumed that the activated imine should produce carbonyl compound in the reaction mixture which might be trapped with indole .", "but only a trace amount of 3,3-bis(indolyl)phenylmethane ( 3a ) was found to be formed in the reaction .", "we envisioned that the poor yield of the product may be due to the insolubility of organic substrates in water .", "accordingly , we added a surfactant ( sds ) to the reaction flask . to our delight ,", "the reaction produced isolable amount of 3a as a brown solid but half of the 1,4-diphenyl-2,3-diaza-1,3-butadiene ( 1a ) left unreacted in the reaction mixture .", "so amount of indole ( 2a ) was doubled ( 4  mmol ) and the same reaction condition successfully produced quantitative amount of 3a .", "the product was filtered out and the filtrate was treated with freshly distilled benzaldehyde ( 0.05  mmol ) in ethanol which furnished 1a ( m.p .", "this infers the simultaneous involvement of two c = n bonds of bis - anils , leaving behind hydrazine in the reaction mixture .", "also , no self - reaction of individual starting materials leading to indole dimer   ( 4 ) or pyrrole formation   ( 5 ) were observed under the same reaction conditions ( scheme 1 ) .", "optimization of the reaction conditions was undertaken by employing different catalyst loadings under various surfactant conditions .", "it was found that the best result was obtained by the application of 15  mol% of i2 containing sodium dodecyl sulphate ( sds ) and water at room temperature ( entry 1 , table 1 ) . in absence of the catalyst no formation of 3a", "was observed even after stirring for 24 hours ( entry 5 , table 1 ) . to study the scope and limitations of the reaction ,", "i2-sds - h2o system was applied to the reaction of indole ( 2 ) and 2,3-diaza-1,3-butadiene derivatives ( table 2 , entries a ", "the bisindoles were formed in excellent yields under the reaction condition . it was observed that the reaction was relatively faster when an electron withdrawing substituent , for example , no2 , was present in the phenyl ring of the 2,3-diaza-1,3-butadienes ( table 2 , entry e ) in comparison to the electron donating groups , for example , ome and oh ( table 2 , entries b and f ) .", "identical results were obtained when 2-methylindole ( 2a ) was used in place of indole ( 2 ) ( table 2 , entries g and h ) .", "all the products were characterized by their ir , h nmr , c nmr , and mass spectral data and also by comparison with the literature report ( scheme 2 ) [ 24 , 25 ] . in order to further explore the efficiency of the i2-sds - h2o system the reaction of the oximes 6 and indoles 2 was studied . when oximes ( 1  mmol ) and indole ( 2  mmol ) were allowed to react under the same reaction condition described earlier , bis - indolylalkanes formed ( table 3 ) . the product was filtered off and the filtrate was treated with benzaldehyde in ethanol .", "the resulted product was benzaldoxime , which proved the liberation of hydroxylamine during the reaction .", "it was found that no michael addition product   was formed and only a trace amount of indole dimer 5   could be identified .", "the system was also applied to the reaction of 1-aza-1,3-butadienes 7 with indole ( 2 ) which produced bis ( indolyl)alkanes 8 in very good yield eliminating aryl amine in the reaction mixture ( table 4 ) .", "all the products were well characterized by comparison of their spectral and mass data with that of the reported value [ 24 , 25 ] .", "in conclusion , we have shown the dual catalytic activity of i2-sds - h2o system which deprotects the azadienes , oximes , and azabutadienes and produces bis ( alkyl)indoles in situ when indole is present in the reaction medium .", "the two - step reaction can be carried out without using acid , transition metals , and organic solvents . besides , the reaction condition is mild and can be done in water under neutral condition which contributes to the criteria of green chemistry .", "h nmr spectra were recorded on avance dpx 300  mhz ft - nmr spectrometer .", "chemical shifts are expressed in  units relative to tetramethylsilane ( tms ) signal as internal reference .", "ir spectra were recorded on ft - ir - system-2000 perkin elmer spectrometer on kbr pellets or in chcl3 .", "the solvents for chromatography were distilled before use . in a 50  ml round bottom flask", ", 15  mol% of i2 was first dissolved in water ( 10  ml ) .", "2,3-diaza-1,3-butadiene ( 1  mmol ) and indole ( 4  mmol ) were added and stirred in the presence of sodium dodecyl sulphate ( sds ) ( 0.02  g ) for the stipulated time .", "the product formed was filtered off and washed with water , dried , and recrystallized from ethanol .  ", "identical reaction condition was followed when 1-aza-1,3-butadienes and oximes were used as reactants . in this case , 2  mmol of indoles were used to react with 1  mmol of imines .", "colorless solid ; mp : 150152c ; ftir ( kbr ) :  3418 , 3058 , 1623 , 1611 , 1445 , 1093  cm ; h nmr ( 300  mhz , cdcl3 ) :  5.95 ( s , 1h , ar ", "ch ) , 6.73(s , 2h ) , 7.06 ( t , 2h , j = 6.8  hz ) , 7.187.27 ( m , 3h ) , 7.317.36 ( m , 2h ) , 7.367.42 ( m , 6h ) , 7.98 ( br , s , 2h , nh ) ; c nmr ( 75  mhz , cdcl3 ) : 40.7 , 111.2 , 119.1 , 119.5 , 120.4 , 122.1 , 123.8 , 126.9 , 126.9 , 128.2 , 129.1 , 136.8 , 144.8 ; hrms calcd for c23h18n2 ( m ) : 322.2851 , found 322.2832 ; anal.calcd . : c , 85.70 ; h , 5.59 ; n , 8.69 ; found c , 85.75 ; h , 5.56 ; n , 8.56 .  ", "pink solid ; mp : 76 - 77c ; ftir ( kbr ) :  3415 , 3060 , 1491 , 1465 , 1095  cm ; h nmr ( 300  mhz , cdcl3 ) :  5.91 ( s , 1h , ar  ch ) , 6.76 ( s , 2h ) , 7.08 ( t , 2h , j = 8.3  hz ) , 7.22 ( t , 2h , j = 7.9  hz ) , 7.287.42 ( m , 8h ) , 8.01 ( br , s , 2h , nh ) ; c nmr ( 75  mhz , cdcl3 ) : 39.8 , 111.5 , 122.6 , 123.8 , 127.1,128.4 , 129.8 , 130.1 , 130.9 , 131.0 , 131.6 , 137.2 , 143.5 ; hrms calcd for c23h17n2cl ( m ) : 356.7371 ; found 356.7324 ; anal.calcd .", ": c , 77.52 ; h , 4.77 ; n , 7.86 ; found c , 77.48 ; h , 4.72 ; n , 7.80 .  ", "brown solid ; mp : 323325c ; ftir ( kbr ) :  3420 , 1720 , 1455 , 1258  cm ; h nmr ( 300  mhz , cdcl3 ) :  5.98 ( s , 1h , ar - ch ) , 6.85 ( s , 2h ) , 7.107.55 ( m , 11h ) , 8.05 ( br , s , 2h , nh ) ,", "c nmr ( 75  mhz , cdcl3 ) : 34.8 , 107.0 , 110.0 , 111.5 , 117.8 , 119.9 , 120.0 , 122.5 , 124.8 , 127.1 , 136.8 , 142.2 ; hrms calcd for c21h16n2o2 ( m ) : 312.2621 ; found 312.2611 ; anal.calcd .", "c , 80.84 ; h , 5.12 ; n , 8.97 ; found c , 84.05 ; h , 5.15 ; n , 8.94 .", "the spectral data of the compounds are available in the supplementary material available online at doi : 105402/2012/635835 ."], "abstract_text": ["<S> a novel catalytic system consisting of i2-sds - h2o has been developed which cleaves 2,3-diaza-1,3-butadiene , 1-aza-1,3-butadienes , oximes and in presence of indoles in the medium uses the corresponding aldehyde products to produce bis(indolyl)alkanes in situ . </S>", "<S> this one pot simple and mild dual catalytic system works in water at room temperature under neutral conditions . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Results and Discussion", "3. Conclusions", "4. Experimental"], "sections": [["using water as solvent in the organic reactions is one of the most important targets to organic chemists because of the easy availability , nontoxicity , and ecofriendly nature of the water [ 17 ] . in this endeavour", ", a number of chemical reactions such as diels alder , hetero diels - alder , 1,3-dipolar cycloaddition , oxidations , reductions , and others are performed successfully in water [ 13 ] .", "also , it is reported that in few cases addition of the water increases the rate and the yield of a reaction and also enhances the enantioselectivity in a chiral synthesis .", "but the main problems associated with water as a solvent is its poor ability to solubilise organic reactants and incapability to create anhydrous condition for moisture sensitive organic compounds and catalysts . to overcome the solubility problem , generally a surfactant is introduced to the reaction mixture .", "the surfactant , due to its hydrophobic and hydrophilic nature , forms micelles with water insoluble organic compounds and promote the desired reactions to occur inside the hydrophobic ambience of the micelle core [ 9 , 10 ] .", "cleavage of the c = n bonds is a very important transformation in organic synthesis as the c = n functionality is widely used to protect both the carbonyl and amines .", "there are a number of methods used for the cleavage of c = n bonds which include acidic reagents [ 1113 ] , oxidizing agents , metallic salts [ 15 , 16 ] , ( phseo)2o , nahso3 , and others .", "most of these methodologies suffer from serious drawbacks like involvement of strong lewis and bronsted acids , use of toxic and costly transition metals ( i.e. , cr , pd , co ) , low temperature , longer reaction time , low yield , and difficulties in isolating the products", ". therefore , development of efficient , mild and environment friendly reagents are always necessary . on the other hand", ", bisindoles are recently emerging as extremely important class of compounds because of their novel antibacterial and anticancer activities [ 1921 ] .", "that is why a number of methodologies have also been postulated for the synthesis of bisindoles [ 2229 ] . in our previous communications", ", we reported that surfactant- ( sds- ) mediated cleavage of c = n bonds could be achieved with acetic anhydride   and surfactant - i2-water can be used for the deprotection of imines to carbonyls .", "we have also shown that bis- and tris ( indolyl)alkanes can be synthesized in presence of bronsted acid in water . in continuation of our research in hydrated media , herein , we wish to disclose the dual catalytic activity of the system i2-sds - h2o which behaves as a lewis acid   for the cleavage of 2,3-diaza-1,3-butadiene , 1-aza-1,3-butadienes , and oximes to produce carbonyls and amines , and the resulting reaction mixture reacts with indoles to produce bis ( indolyl)alkanes in situ at room temperature under neutral conditions ."], ["for an initial study , molecular iodine was added to a mixture of 1,4-diphenyl-2,3-diaza-1,3-butadiene ( 1a ) ( 1  mmol ) and indole ( 2a ) ( 2  mmol ) in water .", "it was presumed that the activated imine should produce carbonyl compound in the reaction mixture which might be trapped with indole .", "but only a trace amount of 3,3-bis(indolyl)phenylmethane ( 3a ) was found to be formed in the reaction .", "we envisioned that the poor yield of the product may be due to the insolubility of organic substrates in water .", "accordingly , we added a surfactant ( sds ) to the reaction flask . to our delight ,", "the reaction produced isolable amount of 3a as a brown solid but half of the 1,4-diphenyl-2,3-diaza-1,3-butadiene ( 1a ) left unreacted in the reaction mixture .", "so amount of indole ( 2a ) was doubled ( 4  mmol ) and the same reaction condition successfully produced quantitative amount of 3a .", "the product was filtered out and the filtrate was treated with freshly distilled benzaldehyde ( 0.05  mmol ) in ethanol which furnished 1a ( m.p .", "this infers the simultaneous involvement of two c = n bonds of bis - anils , leaving behind hydrazine in the reaction mixture .", "also , no self - reaction of individual starting materials leading to indole dimer   ( 4 ) or pyrrole formation   ( 5 ) were observed under the same reaction conditions ( scheme 1 ) .", "optimization of the reaction conditions was undertaken by employing different catalyst loadings under various surfactant conditions .", "it was found that the best result was obtained by the application of 15  mol% of i2 containing sodium dodecyl sulphate ( sds ) and water at room temperature ( entry 1 , table 1 ) . in absence of the catalyst no formation of 3a", "was observed even after stirring for 24 hours ( entry 5 , table 1 ) . to study the scope and limitations of the reaction ,", "i2-sds - h2o system was applied to the reaction of indole ( 2 ) and 2,3-diaza-1,3-butadiene derivatives ( table 2 , entries a ", "the bisindoles were formed in excellent yields under the reaction condition . it was observed that the reaction was relatively faster when an electron withdrawing substituent , for example , no2 , was present in the phenyl ring of the 2,3-diaza-1,3-butadienes ( table 2 , entry e ) in comparison to the electron donating groups , for example , ome and oh ( table 2 , entries b and f ) .", "identical results were obtained when 2-methylindole ( 2a ) was used in place of indole ( 2 ) ( table 2 , entries g and h ) .", "all the products were characterized by their ir , h nmr , c nmr , and mass spectral data and also by comparison with the literature report ( scheme 2 ) [ 24 , 25 ] . in order to further explore the efficiency of the i2-sds - h2o system the reaction of the oximes 6 and indoles 2 was studied . when oximes ( 1  mmol ) and indole ( 2  mmol ) were allowed to react under the same reaction condition described earlier , bis - indolylalkanes formed ( table 3 ) . the product was filtered off and the filtrate was treated with benzaldehyde in ethanol .", "the resulted product was benzaldoxime , which proved the liberation of hydroxylamine during the reaction .", "it was found that no michael addition product   was formed and only a trace amount of indole dimer 5   could be identified .", "the system was also applied to the reaction of 1-aza-1,3-butadienes 7 with indole ( 2 ) which produced bis ( indolyl)alkanes 8 in very good yield eliminating aryl amine in the reaction mixture ( table 4 ) .", "all the products were well characterized by comparison of their spectral and mass data with that of the reported value [ 24 , 25 ] ."], ["in conclusion , we have shown the dual catalytic activity of i2-sds - h2o system which deprotects the azadienes , oximes , and azabutadienes and produces bis ( alkyl)indoles in situ when indole is present in the reaction medium .", "the two - step reaction can be carried out without using acid , transition metals , and organic solvents . besides , the reaction condition is mild and can be done in water under neutral condition which contributes to the criteria of green chemistry ."], ["h nmr spectra were recorded on avance dpx 300  mhz ft - nmr spectrometer .", "chemical shifts are expressed in  units relative to tetramethylsilane ( tms ) signal as internal reference .", "ir spectra were recorded on ft - ir - system-2000 perkin elmer spectrometer on kbr pellets or in chcl3 .", "the solvents for chromatography were distilled before use . in a 50  ml round bottom flask", ", 15  mol% of i2 was first dissolved in water ( 10  ml ) .", "2,3-diaza-1,3-butadiene ( 1  mmol ) and indole ( 4  mmol ) were added and stirred in the presence of sodium dodecyl sulphate ( sds ) ( 0.02  g ) for the stipulated time .", "the product formed was filtered off and washed with water , dried , and recrystallized from ethanol .  ", "identical reaction condition was followed when 1-aza-1,3-butadienes and oximes were used as reactants . in this case , 2  mmol of indoles were used to react with 1  mmol of imines .", "colorless solid ; mp : 150152c ; ftir ( kbr ) :  3418 , 3058 , 1623 , 1611 , 1445 , 1093  cm ; h nmr ( 300  mhz , cdcl3 ) :  5.95 ( s , 1h , ar ", "ch ) , 6.73(s , 2h ) , 7.06 ( t , 2h , j = 6.8  hz ) , 7.187.27 ( m , 3h ) , 7.317.36 ( m , 2h ) , 7.367.42 ( m , 6h ) , 7.98 ( br , s , 2h , nh ) ; c nmr ( 75  mhz , cdcl3 ) : 40.7 , 111.2 , 119.1 , 119.5 , 120.4 , 122.1 , 123.8 , 126.9 , 126.9 , 128.2 , 129.1 , 136.8 , 144.8 ; hrms calcd for c23h18n2 ( m ) : 322.2851 , found 322.2832 ; anal.calcd . : c , 85.70 ; h , 5.59 ; n , 8.69 ; found c , 85.75 ; h , 5.56 ; n , 8.56 .  ", "pink solid ; mp : 76 - 77c ; ftir ( kbr ) :  3415 , 3060 , 1491 , 1465 , 1095  cm ; h nmr ( 300  mhz , cdcl3 ) :  5.91 ( s , 1h , ar  ch ) , 6.76 ( s , 2h ) , 7.08 ( t , 2h , j = 8.3  hz ) , 7.22 ( t , 2h , j = 7.9  hz ) , 7.287.42 ( m , 8h ) , 8.01 ( br , s , 2h , nh ) ; c nmr ( 75  mhz , cdcl3 ) : 39.8 , 111.5 , 122.6 , 123.8 , 127.1,128.4 , 129.8 , 130.1 , 130.9 , 131.0 , 131.6 , 137.2 , 143.5 ; hrms calcd for c23h17n2cl ( m ) : 356.7371 ; found 356.7324 ; anal.calcd .", ": c , 77.52 ; h , 4.77 ; n , 7.86 ; found c , 77.48 ; h , 4.72 ; n , 7.80 .  ", "brown solid ; mp : 323325c ; ftir ( kbr ) :  3420 , 1720 , 1455 , 1258  cm ; h nmr ( 300  mhz , cdcl3 ) :  5.98 ( s , 1h , ar - ch ) , 6.85 ( s , 2h ) , 7.107.55 ( m , 11h ) , 8.05 ( br , s , 2h , nh ) ,", "c nmr ( 75  mhz , cdcl3 ) : 34.8 , 107.0 , 110.0 , 111.5 , 117.8 , 119.9 , 120.0 , 122.5 , 124.8 , 127.1 , 136.8 , 142.2 ; hrms calcd for c21h16n2o2 ( m ) : 312.2621 ; found 312.2611 ; anal.calcd .", "c , 80.84 ; h , 5.12 ; n , 8.97 ; found c , 84.05 ; h , 5.15 ; n , 8.94 .", "the spectral data of the compounds are available in the supplementary material available online at doi : 105402/2012/635835 ."]]}
{"article_id": "PMC4266080", "article_text": ["parental drug misuse and its effects on children and families are policy priorities in the uk and elsewhere ( flavin & paltrow , 2010 ; hm government , 2010 ) .", "polydrug use is the norm among drug users in the uk , and benzodiazepine use is common ( bird & robertson , 2011 ; department of health ( england ) , 2007 ; hay , gannon , casey , & mckeganey , 2009 ; jones , mogali , & comer , 2012 ) . increased risks and harm , and a range of adverse effects are associated with long - term benzodiazepine use ( lader , 2011 , 2014 ; obrien , 2005 ) . however , existing research on drug use and parenting has tended to focus on opioids , opioid substitution therapy ( ost ) , or crack cocaine ( radcliffe , 2009 , 2011 ; rhodes et al . , 2010 ) , and in the case of pregnancy , also alcohol , nicotine , marijuana and amphetamines ( behnke & smith , 2013 ) . in this paper ,", "we examine accounts provided by polydrug - dependent parents about their use of , and dependence on , benzodiazepines as compared to ost .", "benzodiazepine use is highly prevalent among drug users worldwide ( jones et al . , 2012 ) .", "opioid - dependent and ost patients use benzodiazepines to prolong and enhance the effects of opiates ( jaffe et al . , 2004 ) , and increased rates of dependence are associated with co - occurring psychosocial problems ( jones et al . , 2012 ) .", "weizman , gelkopf , melamed , adelson , and bleich ( 2003 ) estimated a lifetime prevalence of benzodiazepine dependence in the opiate - dependent population ( in or out of treatment ) ranging from 6194% .", "benzodiazepines were originally marketed in the 1960s for the relief of anxiety , stress and insomnia ( who , 1996 ) and remain one of the most widely used psychoactive drugs ( lader , 2011 ) .", "their use within the general population is common , particularly among females ( obrien , 2005 ) .", "the gendered cultural meanings of diazepam ( valium ) , a well - known benzodiazepine , was cemented in the 1966 rolling stones song  mother s little helper  .", "benzodiazepines are recommended for the short - term treatment of anxiety and insomnia ( baldwin et al . , 2013 ) .", "tolerance and withdrawal effects can develop after only a few weeks of treatment ( lader , 2011 ) .", "adverse effects include sedation , impaired cognitive and psychomotor functioning , and increased likelihood of accidents and injuries ( lader , 2011 ; obrien , 2005 ) .", "withdrawal effects vary in severity and duration , and include anxiety , insomnia , depression , impaired memory , muscle spasms and tension , and rarely , seizures and psychosis ( lader , 2011 ) .", "combined benzodiazepine and opioid use is associated with increased sedation , decreased psychomotor performance ( lintzeris , mitchell , bond , nestor , & strang , 2007 ; lintzeris & nielsen , 2010 ) , poorer psychosocial adjustment and general health , heightened risk - taking behaviour , and increased risk of overdose ( bird & robertson , 2011 ) . however , evidence is limited ( jones et al . , 2012 ) , and", "few studies have focused on the combined effects of opioid and benzodiazepine dependence on pregnancy or parenting ( acmd , 2003 ) , despite evidence to suggest that both drugs are problematic in these contexts . whilst there is evidence that parental opioid use can compromise parenting capacity and pregnancy outcomes ( acmd , 2003 ) , the health and wellbeing of children and families can also be negatively affected by social and structural factors such as poverty , poor housing , unemployment , parental stress , domestic violence and criminal justice involvement ( hepburn , 2004 ; klee , jackson , & lewis , 2002 ) .", "however research indicates that despite this complexity , there is a tendency among service users and service providers to focus on drug use alone ( banwell & bammer , 2006 ; chandler et al . , 2013 ) , and in most cases the primary drug of misuse ( winklbaur et al . , 2008 ) .", "while this focus may relate to concerns regarding the effects of specific drugs per se on parenting capacity and the immediate safety of children , it could also result in a limited view of drug dependence that neglects to consider polydrug use and the impact of socio - economic and structural inequalities . while the use of opioids and ost is stigmatising , especially in the context of pregnancy and parenthood ( holland , forrester , williams , & copello , 2013 ) , use of benzodiazepines in the context of mothering has historically been characterised in a less problematic ( though not entirely positive ) manner ( metzl , 2003 ) .", "we found no published literature which compared benzodiazepine and opioid dependence , from the perspective of parents , or expectant parents .", "therefore the aim of our study was to address this gap in the evidence and to undertake a comparative thematic analysis of the ways in which parents accounted for their use of opioids , ost and benzodiazepines during and after pregnancy .", "this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .", "the truth  , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .", "the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .", "ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .", "there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .", "notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost .", "participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .", "inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .", "all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .", "five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .", "all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .", "eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .", "this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,", "semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services .", "this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .", "the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .", "accounts were compared and contrasted and analytic themes were generated and agreed by the research team .", "participants gave informed consent and were provided with a  20 voucher for each interview completed .", "a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in  normal  daily life , providing a better context in which to undertake parenting .", "positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .", "this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) \n  [ t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period , the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a ", "normal  life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) \n  i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost  supervised   where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .", "such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) \n  sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,", "( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .", "in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .", "most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which ", "carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , ", "mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .", "here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) \n  yes , like that song ,  mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .", "in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .", "this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription : im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) \n  i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews", ", tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) \n  i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but", "i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing .", "accounts of substance use during pregnancy were  for women especially  more similar . in the majority of cases , women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .", "each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) \n  i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .", "those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use . a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .", "as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) \n  i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .", "carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .", "as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets ,  cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) \n  i ve been taking valium , but like the drug worker says , instead of getting it fae the streets ,  cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .", "carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .", "another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .", "these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was  controlled  and unproblematic .", "thus , participants constructed benzodiazepine use and dependence as a  legitimate  means to help them regulate problem emotional states which might otherwise interfere with their ability to live a  normal  life and effectively parent their children . benzodiazepine use and dependence for women", "was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .", "in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .", "one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ?", "where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) \n  i ve got all these people , telling me what s right , and what s wrong , and", "i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .", "all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire  or need  to cease their illicit use of benzodiazepines .", "while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .", "benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .", "one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .", "this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) \n  what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .", "the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .", "this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .", "the truth  , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .", "the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .", "ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .", "there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .", "notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost .", "participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .", "inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .", "all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .", "five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .", "all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .", "eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .", "this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,", "semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services .", "this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .", "the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .", "accounts were compared and contrasted and analytic themes were generated and agreed by the research team .", "participants gave informed consent and were provided with a  20 voucher for each interview completed .", "a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in  normal  daily life , providing a better context in which to undertake parenting .", "positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .", "this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) \n  [ t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,", "the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a ", "normal  life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) \n  i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost  supervised   where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .", "such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) \n  sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot   ", "they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .", "in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .", "most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which ", "carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , ", "mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .", "here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) \n  yes , like that song ,  mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .", "in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .", "this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :", "im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) \n  i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews", ", tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) \n  i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but", "i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were  for women especially  more similar . in the majority of cases", ", women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .", "each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) \n  i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .", "those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use .", "a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in  normal  daily life , providing a better context in which to undertake parenting .", "positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .", "this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) \n  [ t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,", "the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a ", "normal  life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) \n  i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost  supervised   where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .", "such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) \n  sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot   ", "they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .", "in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .", "most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which ", "carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , ", "mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .", "here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) \n  yes , like that song ,  mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .", "in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .", "this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :", "im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) \n  i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews", ", tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) \n  i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but", "i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were  for women especially  more similar . in the majority of cases", ", women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .", "each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) \n  i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .", "those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use .", "a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .", "as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) \n  i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .", "carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .", "as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets ,  cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) \n  i ve been taking valium , but like the drug worker says , instead of getting it fae the streets ,", " cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .", "carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .", "another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .", "these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was", "thus , participants constructed benzodiazepine use and dependence as a  legitimate  means to help them regulate problem emotional states which might otherwise interfere with their ability to live a  normal  life and effectively parent their children . benzodiazepine use and dependence for women", "was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .", "in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .", "one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) \n  i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .", "all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire  or need  to cease their illicit use of benzodiazepines . while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .", "benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .", "one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .", "this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) \n  what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .", "the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .", "benzodiazepine use and dependence for women was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .", "in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .", "one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ?", "where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) \n  i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like", ", because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .", "all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire  or need  to cease their illicit use of benzodiazepines .", "while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .", "benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .", "one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .", "this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) \n  what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .", "the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .", "this longitudinal qualitative study explored accounts of poly - drug use within the context of pregnancy and parenting , highlighting differential understandings and practices surrounding benzodiazepine and opioid use .", "our findings illuminate a number of issues with implications for theory , policy , prevention and education .", "the accounts in our study resonate with others ( klee , 1998 ; radcliffe , 2011 ) in demonstrating the morally difficult nature of being a  drug - using parent  , and the extent to which stigma can play an important role in the day - to - day lives of parents .", "our findings suggest that while drug use , in general , is seen as incompatible with parenting , there are clear indications that some drugs , and types of  addiction  , are more  acceptable  than others .", "crucially , our analysis indicates important differences in the way opioid and benzodiazepine use are understood and narrated within the context of parenthood . unlike benzodiazepine use", ", participants in this study often described ost in negative terms  even when they acknowledged that ost was helpful in treating opioid dependence .", "this dominant negative discourse on ost by drug users themselves , has been described elsewhere ( harris & mcelrath , 2012 ; lloyd , 2013 ; radcliffe & stevens , 2008 ) , and highlights the unique stigma attached to ost for the treatment of opioid dependence ( keane , 2013 ) .", "participants described using benzodiazepines in constructive ways  to enhance social and emotional functioning and engagement in  normal life   appealing to a  self - medicating hypothesis  of addiction to explain and justify continued use ( jones , et al . , 2012 ) .", "these meanings served to legitimise benzodiazepine dependence , contrasting sharply with participants alignment of ost with ", " junkie  identities , as documented elsewhere ( keane , 2013 ; radcliffe & stevens , 2008 ) . in the context of parenthood ,", "parents were more likely to describe a desire to reduce opioid rather than benzodiazepine use , and there was evidence that illicit benzodiazepine use was considered less harmful than opioid use .", "this is noteworthy , because alongside this complacency were occasional notes of concern , particularly in relation to benzodiazepine use by men , and its link with increased aggression and domestic abuse ( reported by some women participants ) . accounts from this study provide a tentative indication of the way gender may shape understandings of benzodiazepine use .", "the long - term use of benzodiazepines by mothers was framed as being therapeutic and unproblematic .", "in contrast , while men reported using benzodiazepine for similar reasons , their accounts indicated that practitioners did not legitimise this and prescriptions were denied .", "these findings sit uneasily with the dominant  recovery  agenda in uk drug policy ( valentine & treloar , 2013 ; wardle , 2012 ) , where abstinence from all drug use is assumed to be both desirable and necessary for  social reintegration  .", "it may be that in practice  recovery  refers to particular categories of drugs , with the hierarchy of acceptability identified among service users in this study being apparently mirrored in prescribing practices and approaches taken to treating addiction by clinicians ( anthierens , habraken , petrovic , & christiaens , 2007 ) .", "thus , while opioid - dependent individuals may express a desire to reduce their opioid prescriptions , this is not necessarily reflected in their orientation towards other drug use , particularly benzodiazepines .", "as such , the claim that opioid - dependent individuals would prefer abstinence - based treatment approaches may result from the unique stigma attached to opioids and may not apply to all drug use , or all drugs of dependence ( lloyd , 2013 ; mckeganey , morris , neale , & robertson , 2004 ; neale , nettleton , & pickering , 2011 ) .", "our findings suggest a need for further preventative measures and targeted education to address benzodiazepine use and dependence within the context of pregnancy and parenting , especially in populations of parents engaged in ost programmes where  stability  on opioids may detract from issues associated with benzodiazepine use and dependence . reviews of benzodiazepine prescribing ( sirdifield et al . , 2013 ) ,", "identify a variety of reasons for inconsistent practice , and conclude that improved education and training , provision of non - pharmacological interventions , and enhanced communication with patients is required .", "our findings support these recommendations , especially the need for enhanced education and training of both clinicians and patients , and extra provision of non - pharmacological interventions to promote the psychological and social functioning of parents on ost .", "this was an in - depth , qualitative exploration of the accounts of nineteen drug - dependent parents on ost in a specific geographical area .", "as such , our findings and conclusions should be read as indicative and suggestive rather than definitive .", "further qualitative exploration in different geographical areas and populations is needed , as it is likely that local or cultural contexts may shape the types of narratives expressed in respect of the range of substances consumed .", "our findings may have limited applicability to opioid dependent parents who are not engaged in ost programmes .", "however , the contrasting accounts depicted here underline the importance of qualitative research in illuminating understandings about the meanings that drugs of addiction have for those who use them ( neale , allen , & coombes , 2005 ) .", "although long - term benzodiazepine use and dependence among opioid - dependent populations has been a topic of concern for some decades ( who , 1996 ) and is associated with increased risks and harm ( lader , 2011 , 2014 ) , research regarding the impact on pregnancy , parenting and family life has been limited ( acmd , 2003 ) .", "our findings suggest that parents frame benzodiazepine use and dependence as largely unproblematic , less stigmatising and more legitimate than ost .", "we suggest these divergent accounts can be understood as drawing on differing cultural meanings attached to ost and benzodiazepine use , which are reflected in policy and practice . as such , prevention ,", "education and policy initiatives should pay greater attention to the kind of narratives and strategies that are employed in addressing benzodiazepine use and dependence within opioid dependent populations , especially in respect of the parenting and child welfare agenda ."], "abstract_text": ["<S> aimsto explore the ways in which opioid - dependent parents accounted for their use of opioids and benzodiazepines during and after pregnancy.methodslongitudinal qualitative interviews [ n  =  45 ] with 19 opioid - dependent adults recruited in scotland , uk , were held during the antenatal and post - natal period . </S>", "<S> interviews focused on parenting and parenting support within the context of problem drug use and were analysed using a narrative informed , thematic analysis . </S>", "<S> findingsthe majority of participants described using benzodiazepines in addition to opioids . </S>", "<S> almost all indicated a desire to stop or reduce opioid use , whereas cessation or reduction of benzodiazepines was rarely prioritised . </S>", "<S> in stark contrast to opioid dependence , benzodiazepine dependence was portrayed as unproblematic , therapeutic and acceptable in the context of family life . whereas opioid dependence was framed as stigmatising , benzodiazepine use and dependence was normalised . </S>", "<S> an exception was benzodiazepine use by men which was occasionally associated with aggression and domestic abuse.conclusionsdrug-dependent parents attach different meanings to opioid and benzodiazepine use and dependence in the context of parenthood . </S>", "<S> divergent meanings , and stigma , may impact on stated commitment to stability or recovery from dependent drug - use . </S>", "<S> attention should be paid to the way in which policy and practice regarding ost and benzodiazepines reflects this divergence . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Study design and setting", "Recruitment, sample and data collection", "Analysis", "Ethics", "Findings", "Benzodiazepine and opioid use for normality and normal life.", "Therapeutic qualities of benzodiazepine and opioid use", "Benzodiazepines: mothers little helper; fathers ruin?", "Discussion", "Limitations", "Conclusion"], "sections": [["parental drug misuse and its effects on children and families are policy priorities in the uk and elsewhere ( flavin & paltrow , 2010 ; hm government , 2010 ) .", "polydrug use is the norm among drug users in the uk , and benzodiazepine use is common ( bird & robertson , 2011 ; department of health ( england ) , 2007 ; hay , gannon , casey , & mckeganey , 2009 ; jones , mogali , & comer , 2012 ) . increased risks and harm , and a range of adverse effects are associated with long - term benzodiazepine use ( lader , 2011 , 2014 ; obrien , 2005 ) . however , existing research on drug use and parenting has tended to focus on opioids , opioid substitution therapy ( ost ) , or crack cocaine ( radcliffe , 2009 , 2011 ; rhodes et al . , 2010 ) , and in the case of pregnancy , also alcohol , nicotine , marijuana and amphetamines ( behnke & smith , 2013 ) . in this paper ,", "we examine accounts provided by polydrug - dependent parents about their use of , and dependence on , benzodiazepines as compared to ost .", "benzodiazepine use is highly prevalent among drug users worldwide ( jones et al . , 2012 ) .", "opioid - dependent and ost patients use benzodiazepines to prolong and enhance the effects of opiates ( jaffe et al . , 2004 ) , and increased rates of dependence are associated with co - occurring psychosocial problems ( jones et al . , 2012 ) .", "weizman , gelkopf , melamed , adelson , and bleich ( 2003 ) estimated a lifetime prevalence of benzodiazepine dependence in the opiate - dependent population ( in or out of treatment ) ranging from 6194% .", "benzodiazepines were originally marketed in the 1960s for the relief of anxiety , stress and insomnia ( who , 1996 ) and remain one of the most widely used psychoactive drugs ( lader , 2011 ) .", "their use within the general population is common , particularly among females ( obrien , 2005 ) .", "the gendered cultural meanings of diazepam ( valium ) , a well - known benzodiazepine , was cemented in the 1966 rolling stones song  mother s little helper  .", "benzodiazepines are recommended for the short - term treatment of anxiety and insomnia ( baldwin et al . , 2013 ) .", "tolerance and withdrawal effects can develop after only a few weeks of treatment ( lader , 2011 ) .", "adverse effects include sedation , impaired cognitive and psychomotor functioning , and increased likelihood of accidents and injuries ( lader , 2011 ; obrien , 2005 ) .", "withdrawal effects vary in severity and duration , and include anxiety , insomnia , depression , impaired memory , muscle spasms and tension , and rarely , seizures and psychosis ( lader , 2011 ) .", "combined benzodiazepine and opioid use is associated with increased sedation , decreased psychomotor performance ( lintzeris , mitchell , bond , nestor , & strang , 2007 ; lintzeris & nielsen , 2010 ) , poorer psychosocial adjustment and general health , heightened risk - taking behaviour , and increased risk of overdose ( bird & robertson , 2011 ) . however , evidence is limited ( jones et al . , 2012 ) , and", "few studies have focused on the combined effects of opioid and benzodiazepine dependence on pregnancy or parenting ( acmd , 2003 ) , despite evidence to suggest that both drugs are problematic in these contexts . whilst there is evidence that parental opioid use can compromise parenting capacity and pregnancy outcomes ( acmd , 2003 ) , the health and wellbeing of children and families can also be negatively affected by social and structural factors such as poverty , poor housing , unemployment , parental stress , domestic violence and criminal justice involvement ( hepburn , 2004 ; klee , jackson , & lewis , 2002 ) .", "however research indicates that despite this complexity , there is a tendency among service users and service providers to focus on drug use alone ( banwell & bammer , 2006 ; chandler et al . , 2013 ) , and in most cases the primary drug of misuse ( winklbaur et al . , 2008 ) .", "while this focus may relate to concerns regarding the effects of specific drugs per se on parenting capacity and the immediate safety of children , it could also result in a limited view of drug dependence that neglects to consider polydrug use and the impact of socio - economic and structural inequalities . while the use of opioids and ost is stigmatising , especially in the context of pregnancy and parenthood ( holland , forrester , williams , & copello , 2013 ) , use of benzodiazepines in the context of mothering has historically been characterised in a less problematic ( though not entirely positive ) manner ( metzl , 2003 ) .", "we found no published literature which compared benzodiazepine and opioid dependence , from the perspective of parents , or expectant parents .", "therefore the aim of our study was to address this gap in the evidence and to undertake a comparative thematic analysis of the ways in which parents accounted for their use of opioids , ost and benzodiazepines during and after pregnancy ."], ["this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .", "the truth  , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .", "the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .", "ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .", "there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .", "notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost .", "participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .", "inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .", "all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .", "five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .", "all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .", "eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .", "this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,", "semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services .", "this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .", "the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .", "accounts were compared and contrasted and analytic themes were generated and agreed by the research team .", "participants gave informed consent and were provided with a  20 voucher for each interview completed .", "a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in  normal  daily life , providing a better context in which to undertake parenting .", "positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .", "this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) \n  [ t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period , the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a ", "normal  life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) \n  i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost  supervised   where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .", "such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) \n  sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,", "( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .", "in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .", "most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which ", "carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , ", "mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .", "here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) \n  yes , like that song ,  mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .", "in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .", "this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription : im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) \n  i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews", ", tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) \n  i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but", "i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing .", "accounts of substance use during pregnancy were  for women especially  more similar . in the majority of cases , women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .", "each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) \n  i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .", "those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use . a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .", "as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) \n  i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .", "carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .", "as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets ,  cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) \n  i ve been taking valium , but like the drug worker says , instead of getting it fae the streets ,  cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .", "carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .", "another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .", "these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was  controlled  and unproblematic .", "thus , participants constructed benzodiazepine use and dependence as a  legitimate  means to help them regulate problem emotional states which might otherwise interfere with their ability to live a  normal  life and effectively parent their children . benzodiazepine use and dependence for women", "was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .", "in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .", "one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ?", "where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) \n  i ve got all these people , telling me what s right , and what s wrong , and", "i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .", "all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire  or need  to cease their illicit use of benzodiazepines .", "while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .", "benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .", "one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .", "this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) \n  what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .", "the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts ."], ["this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .", "the truth  , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .", "the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .", "ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .", "there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .", "notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost ."], ["participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .", "inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .", "all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .", "five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .", "all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .", "eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .", "this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,", "semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services ."], ["this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .", "the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .", "accounts were compared and contrasted and analytic themes were generated and agreed by the research team ."], ["participants gave informed consent and were provided with a  20 voucher for each interview completed ."], ["a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in  normal  daily life , providing a better context in which to undertake parenting .", "positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .", "this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) \n  [ t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,", "the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a ", "normal  life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) \n  i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost  supervised   where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .", "such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) \n  sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot   ", "they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .", "in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .", "most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which ", "carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , ", "mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .", "here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) \n  yes , like that song ,  mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .", "in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .", "this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :", "im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) \n  i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews", ", tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) \n  i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but", "i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were  for women especially  more similar . in the majority of cases", ", women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .", "each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) \n  i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .", "those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use ."], ["a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in  normal  daily life , providing a better context in which to undertake parenting .", "positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .", "this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) \n  [ t]he only way to have a normal life [  ] and keep a normal life to normality , to being able to live again is a methadone prescription .", "( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,", "the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a ", "normal  life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) \n  i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .", "( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost  supervised   where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .", "such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot    they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) \n  sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot   ", "they re standing waiting for their meth and i try and sit in a seat away fae them .", "( melanie , antenatal )   when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [   the locality ] swallowing your methadone in the middle of the shop flair .", "so you get folk looking at you [  ] whether i ve got [ daughter ] wi me or no , so it s just ,  there s the junkie , look at her  .", "( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .", "in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .", "most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which ", "carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , ", "mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .", "here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) \n  yes , like that song ,  mother s little helper  , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .", "( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .", "i said i just take the two at night to help me get to sleep .", "( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .", "in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .", "this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :", "im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) \n  i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [  ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [  ] they help me , to want to get up in time to do stuff and everything .", "( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews", ", tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) \n  i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but", "i mean i dinnae feel like that with my valium [  ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .", "i do need my valium and if they said to me they were going to start cutting me i would panic [  ] but they re not , they re just happy at me being stable just now .", "( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were  for women especially  more similar . in the majority of cases", ", women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .", "each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) \n  i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [  ] and i said it would be a bit much for me doing all of that [  ] but it s no too much for me coming down from the methadone and the valium [  ] . because it s going to be helping the baby and me .", "( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .", "those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use ."], ["a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .", "as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) \n  i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .", "i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .", "i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .", "( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .", "carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .", "as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets ,  cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) \n  i ve been taking valium , but like the drug worker says , instead of getting it fae the streets ,", " cos you do nt know what s in it , she s going to give me a prescription [  ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me", "cos i m a bit , still a bit , like anxious i think that s the word .", "but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .", "( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .", "carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .", "another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .", "these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was", "thus , participants constructed benzodiazepine use and dependence as a  legitimate  means to help them regulate problem emotional states which might otherwise interfere with their ability to live a  normal  life and effectively parent their children . benzodiazepine use and dependence for women", "was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .", "in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .", "one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) \n  i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .", "all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire  or need  to cease their illicit use of benzodiazepines . while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .", "benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .", "one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .", "this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) \n  what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .", "the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts ."], ["benzodiazepine use and dependence for women was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .", "in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .", "one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ?", "where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) \n  i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .", "how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [  ] ? where s their just cause [  ] ? where s the argument , like", ", because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .", "but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .", "all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire  or need  to cease their illicit use of benzodiazepines .", "while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .", "benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .", "one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .", "this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) \n  what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .", "( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .", "the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts ."], ["this longitudinal qualitative study explored accounts of poly - drug use within the context of pregnancy and parenting , highlighting differential understandings and practices surrounding benzodiazepine and opioid use .", "our findings illuminate a number of issues with implications for theory , policy , prevention and education .", "the accounts in our study resonate with others ( klee , 1998 ; radcliffe , 2011 ) in demonstrating the morally difficult nature of being a  drug - using parent  , and the extent to which stigma can play an important role in the day - to - day lives of parents .", "our findings suggest that while drug use , in general , is seen as incompatible with parenting , there are clear indications that some drugs , and types of  addiction  , are more  acceptable  than others .", "crucially , our analysis indicates important differences in the way opioid and benzodiazepine use are understood and narrated within the context of parenthood . unlike benzodiazepine use", ", participants in this study often described ost in negative terms  even when they acknowledged that ost was helpful in treating opioid dependence .", "this dominant negative discourse on ost by drug users themselves , has been described elsewhere ( harris & mcelrath , 2012 ; lloyd , 2013 ; radcliffe & stevens , 2008 ) , and highlights the unique stigma attached to ost for the treatment of opioid dependence ( keane , 2013 ) .", "participants described using benzodiazepines in constructive ways  to enhance social and emotional functioning and engagement in  normal life   appealing to a  self - medicating hypothesis  of addiction to explain and justify continued use ( jones , et al . , 2012 ) .", "these meanings served to legitimise benzodiazepine dependence , contrasting sharply with participants alignment of ost with ", " junkie  identities , as documented elsewhere ( keane , 2013 ; radcliffe & stevens , 2008 ) . in the context of parenthood ,", "parents were more likely to describe a desire to reduce opioid rather than benzodiazepine use , and there was evidence that illicit benzodiazepine use was considered less harmful than opioid use .", "this is noteworthy , because alongside this complacency were occasional notes of concern , particularly in relation to benzodiazepine use by men , and its link with increased aggression and domestic abuse ( reported by some women participants ) . accounts from this study provide a tentative indication of the way gender may shape understandings of benzodiazepine use .", "the long - term use of benzodiazepines by mothers was framed as being therapeutic and unproblematic .", "in contrast , while men reported using benzodiazepine for similar reasons , their accounts indicated that practitioners did not legitimise this and prescriptions were denied .", "these findings sit uneasily with the dominant  recovery  agenda in uk drug policy ( valentine & treloar , 2013 ; wardle , 2012 ) , where abstinence from all drug use is assumed to be both desirable and necessary for  social reintegration  .", "it may be that in practice  recovery  refers to particular categories of drugs , with the hierarchy of acceptability identified among service users in this study being apparently mirrored in prescribing practices and approaches taken to treating addiction by clinicians ( anthierens , habraken , petrovic , & christiaens , 2007 ) .", "thus , while opioid - dependent individuals may express a desire to reduce their opioid prescriptions , this is not necessarily reflected in their orientation towards other drug use , particularly benzodiazepines .", "as such , the claim that opioid - dependent individuals would prefer abstinence - based treatment approaches may result from the unique stigma attached to opioids and may not apply to all drug use , or all drugs of dependence ( lloyd , 2013 ; mckeganey , morris , neale , & robertson , 2004 ; neale , nettleton , & pickering , 2011 ) .", "our findings suggest a need for further preventative measures and targeted education to address benzodiazepine use and dependence within the context of pregnancy and parenting , especially in populations of parents engaged in ost programmes where  stability  on opioids may detract from issues associated with benzodiazepine use and dependence . reviews of benzodiazepine prescribing ( sirdifield et al . , 2013 ) ,", "identify a variety of reasons for inconsistent practice , and conclude that improved education and training , provision of non - pharmacological interventions , and enhanced communication with patients is required .", "our findings support these recommendations , especially the need for enhanced education and training of both clinicians and patients , and extra provision of non - pharmacological interventions to promote the psychological and social functioning of parents on ost ."], ["this was an in - depth , qualitative exploration of the accounts of nineteen drug - dependent parents on ost in a specific geographical area .", "as such , our findings and conclusions should be read as indicative and suggestive rather than definitive .", "further qualitative exploration in different geographical areas and populations is needed , as it is likely that local or cultural contexts may shape the types of narratives expressed in respect of the range of substances consumed .", "our findings may have limited applicability to opioid dependent parents who are not engaged in ost programmes .", "however , the contrasting accounts depicted here underline the importance of qualitative research in illuminating understandings about the meanings that drugs of addiction have for those who use them ( neale , allen , & coombes , 2005 ) ."], ["although long - term benzodiazepine use and dependence among opioid - dependent populations has been a topic of concern for some decades ( who , 1996 ) and is associated with increased risks and harm ( lader , 2011 , 2014 ) , research regarding the impact on pregnancy , parenting and family life has been limited ( acmd , 2003 ) .", "our findings suggest that parents frame benzodiazepine use and dependence as largely unproblematic , less stigmatising and more legitimate than ost .", "we suggest these divergent accounts can be understood as drawing on differing cultural meanings attached to ost and benzodiazepine use , which are reflected in policy and practice . as such , prevention ,", "education and policy initiatives should pay greater attention to the kind of narratives and strategies that are employed in addressing benzodiazepine use and dependence within opioid dependent populations , especially in respect of the parenting and child welfare agenda ."]]}
{"article_id": "PMC5192341", "article_text": ["in industrialized countries , cardiovascular disease ( cvd ) remains the predominant cause of morbidity and mortality .", "it is current scientific opinion that cvd occurs as a part of a series of events that form a continuum .", "the first stage of this continuum corresponds to the presence of risks factors that predispose to tissue injury , such as hypertension , dyslipidemia , and diabetes .", "these risk factors usually activate a complex range of pathogenic pathways leading to the development of atherosclerosis and ischemic heart disease .", "failure to effectively manage any stage of this continuum results in ventricular hypertrophy , heart failure , end - stage heart disease , and cardiovascular death .", "one of the molecular pathways that could be activated in response to these risk factors and be involved in cvd development is that of osteoprotegerin ( opg ) and tnf - related apoptosis - inducing ligand ( trail ) , as reviewed in .", "opg and trail are both members of the tnf superfamily , and they are widely expressed in different tissues , including the heart and the vessels , in health and disease states [ 4 , 5 ] . although initially opg actions seemed to be limited to bone metabolism   and those of trail to host defense against tumors , recent studies have suggested that both molecules might actually be involved in cvd development and progression .", "epidemiological studies have shown that there is an association between circulating opg , trail , and cvd .", "opg was found to be directly correlated [ 5 , 8 , 9 ] , while trail was inversely correlated , with cardiovascular morbidity and mortality [ 10 , 11 ] .", "likewise , opg was significantly associated with the level of c - reactive protein , which is an independent predictor of acute vascular events and adverse outcomes , while trail was negatively correlated with it .", "recently , secchiero and colleagues have shown that patients with coronary artery disease have an increased opg / trail ratio , which further increases in patients developing heart failure .", "consistent with this epidemiological data , experimental studies suggest that opg and trail have opposite actions , as opg has proinflammatory   and proatherogenic effects [ 14 , 15 ] , while trail seems to be anti - inflammatory [ 16 , 17 ] and antiatherogenic [ 1820 ] . based on these premises", ", we hypothesized that dyslipidemia and diabetes , which are two well known risk factors for cvd , could modify the vascular and cardiac expression of opg and trail , leading to an increased opg / trail ratio at a tissue level .", "the aim of this study was to explore the vascular and cardiac changes of opg and trail in experimental models of dyslipidemia and diabetes .", "a total of 12 male c57bl/6j ( cnt ) mice and 24 male apolipoprotein e null ( apoe ) mice on a c57bl/6j background , aged 6 weeks , were studied for 14 weeks . at 6 weeks of age ,", "apoe mice were further randomized to saline ( apoe ) or streptozotocin ( apoe + dm ) , which were delivered intraperitoneally in five consecutive daily doses .", "the daily dose of streptozotocin ( stz ) ( sigma - aldrich ) was 55  mg / kg of body weight .", "after one week , blood glucose was checked and only the mice with blood glucose levels greater than 15  mm were included in the apoe + dm group . during the study period , all the mice were fed with a standard diet .", "the animals were kept in a temperature - controlled room ( 22  1c ) on a 12  h light/12  h dark cycle with free access to food and water and they were fed ad libitum for the length of the study . after 14 weeks , the animals were anesthetized with an intraperitoneal injection of thiobutabarbital sodium ( inactin , sigma - aldrich ) ( 60  mg / kg body weight ) and sacrificed by exsanguination via cardiac puncture .", "all the animal experiments were approved by the animal ethics committee of the university of trieste ( i d 28.0.2008 ) and the italian ministry of health , conforming to the guide for the care and use of laboratory animals . at the end of the study , we measured body weight and systolic blood pressure ( sbp ) .", "in particular , sbp was assessed by a computerized noninvasive tail cuff system in conscious mice .", "glucose , total cholesterol , hdl cholesterol , and triglycerides were measured in the sera by autoanalyzer technique ( hitachi 917 , tokyo , japan ) .", "circulating levels of opg and trail were measured in the sera by elisa ( r&d systems duoset # dy459 and # dy1121 ) . in half of the animals in each group ,", "aortae were collected and placed in 10% neutral - buffered formalin for atherosclerosis quantification , before being processed for immunohistochemical analyses . in the other half ,", "aortae were snap - frozen in liquid nitrogen and stored at 80c for rna extraction and gene expression analyses .", "briefly , aortae were cleaned of excess fat and stained with sudan iv - herxheimer 's solution .", "images were acquired with a dissecting microscope ( olympus bx-50 ) equipped with a high - resolution camera ( sony xc-77ce ) .", "digitized images were then evaluated with an image analysis system ( image - pro plus 6.3 software , media cybernetics ) .", "total plaque area was quantified as the percentage area of aorta stained red . as for the hearts", ", they were divided and half was put in formalin and half was put in liquid nitrogen .", "the part that was fixed in formalin was embedded in paraffin , cut in 4  m thick sections , and stained with hematoxylin and eosin ( h&e ) in order to evaluate cardiomyocyte hypertrophy .", "cardiomyocyte hypertrophy was assessed by measuring the shortest diameter of 200 cells , which were selected when they showed the spindle - shaped transverse section including the elliptical nucleus .", "the shortest transverse diameter was measured 3 times per cell , and the values were averaged , as reported by .", "the other half that was snap - frozen in liquid nitrogen was used either for gene expression analyses or for immunohistochemical stainings . gene expression of opg and trail was determined by real - time quantitative rt - pcr ( reverse transcription - polymerase chain reaction ) in aortic and cardiac tissue . in order to isolate mrna from the aorta and the heart , tissue was homogenized and processed as previously reported .", "then , mrna was treated with the dnase inactivation reagent ( ambion dna - free product # am-1906 ) , and 3  g of treated mrna was subsequently used to synthesize cdna with superscript first strand synthesis system for rt - pcr ( gibco brl ) .", "the gene expression of opg and trail was analyzed by real - time quantitative rt - pcr using the taqman system ( life technologies ) for opg and the sybr green system ( life technologies ) for trail .", "fluorescence for each cycle was quantitatively analyzed by an abi prism 7900ht sequence detection system ( applied biosystems ) .", "gene expression of opg was normalized to 18s mrna , while that of trail was normalized to rps9 .", "results were reported as ratio compared with the level of expression in untreated controls , which were given an arbitrary value of 1 .", "the presence of opg and trail in the aortae was evaluated by immunostainings on 4  m thick paraffin sections . in particular , after antigen retrieval in citrate buffer ( ph6 ) endogenous peroxidase was quenched for 10 minutes using 3% h2o2 in pbs . to localize opg", ", we used a biotinylated goat anti - opg antibody ( r&d systems , 1  :  10 dilution ) followed by a catalyzed signal amplification system ( dako ) . to localize trail", ", we used a monoclonal mouse anti - trail antibody ( r&d systems , 1  :  50 dilution ) , applied overnight at 4c . biotinylated goat anti - mouse immunoglobulins ( vector laboratories , dilution 1  :  200 )", "were then used as secondary antibody , followed by streptavidin - hrp ( dako ) .", "the presence of opg and trail in the hearts was evaluated by immunostaining on 5  m thick frozen sections .", "after neutralization of endogenous peroxidase , sections were incubated overnight with the goat anti - opg antibody ( r&d systems , 1  :  10 dilution ) and the monoclonal mouse anti - trail antibody ( r&d systems , 1  :  50 dilution ) .", "biotinylated immunoglobulins , diluted 1  :  200 , were then applied as secondary antibodies , followed by streptavidin - hrp ( dako ) . in both tissues ,", "after counterstaining with hematoxylin , all the sections were examined by light microscopy ( olympus bx50wi ) and digitized using a high - resolution camera ( q - imaging fast 1394 ) .", "the proportional area of brown staining was measured by an image analysis system ( image - pro plus 6.3 software , media cybernetics ) in order to quantify opg and trail expression in aortae and hearts .", "apoe mice had higher levels of total cholesterol and triglycerides as compared to the cnt mice . in line with the study protocol , the mice in the apoe + dm group were hyperglycemic . moreover , diabetes was associated with a significant reduction of body weight and a significant increase of total cholesterol as compared to nondiabetic apoe mice .", "apoe mice had a significant increase in total plaque area , which increased further after the induction of diabetes ( figures 1(a ) and 1(b ) ) .", "as for cardiac changes , dyslipidemia was associated with a significant increase in cardiomyocyte size ( figures 1(c ) and 1(d ) ) , indicating cardiomyocyte hypertrophy , with no further increase after the induction of diabetes . in the aorta , the induction of diabetes was associated with a significant increase in opg gene expression and in the opg / trail ratio , while dyslipidemia had no effect on opg / trail ratio as compared to controls ( figure 2(a ) ) . by contrast , in the heart , dyslipidemia was the major determinant of opg / trail tissue changes , not only by increasing opg but also by reducing trail gene expression .", "so dyslipidemia increased significantly cardiac opg / trail ratio , with no further differences after the induction of diabetes ( figure 2(b ) ) .", "immunostainings showed that diabetes significantly increased aortic opg expression as compared to the other groups , while trail was unchanged ( figure 3 ) . on the other hand , in the heart , it was dyslipidemia that significantly increased opg protein expression , with no further changes after the induction of diabetes .", "as for cardiac trail , trail protein expression decreased in the apoe and apoe + dm groups ( data not shown ) . the levels of circulating", "circulating opg was 1.91  0.21  ng / ml in the control group , 2.05  0.1  ng / ml in the apoe group , and 3.1  0.19 in the apoe + dm group , such that opg increased significantly in diabetic mice as compared to the other groups ( p < 0.05 , apoe + dm versus cnt and versus apoe group ) .", "as for trail , we were unable to measure its circulating levels , as they were below the detection levels of the elisa we used .", "it has already been argued that opg , which circulates at much higher levels than its ligands ( rankl and trail ) , may be a more stable overall measure of opg / trail activity than trail .", "patients with coronary artery disease have an increased opg / trail ratio , which further increases in the group of patients who develop heart failure . dyslipidemia and diabetes", ", we evaluated the effects of dyslipidemia , alone and in combination with diabetes , on aortic and cardiac opg and trail expression and found that while diabetes is the major determinant of opg / trail tissue changes in the vessels , dyslipidemia is it in the heart .", "this data is in line with previous experimental works showing that diabetes increased opg expression and the opg / trail ratio in the aorta   and that the onset of diabetes was associated with an increase of circulating opg .", "consistent with these observations , in our study , we found that aortic and circulating opg increased significantly only in the group of mice with diabetes .", "taken together , these findings seem to support the hypothesis that the vessels might be the source of increased circulating levels of opg in patients with diabetes and/or cvd .", "it has to be noted that the increase of circulating and tissue opg could be not only a risk marker but also a risk factor for atherosclerosis and cvd development .", "firstly , animal models point to a role for opg in glucose metabolism regulation , as opg injections increased significantly glucose levels [ 12 , 26 ] , which represent one of the risk factors for cvd .", "this effect could be mediated by trail blockade , as trail lowers glucose levels . by offsetting trail effects ,", "opg could also promote body weight gain and dyslipidemia , as well as atherosclerotic plaque development [ 18 , 20 ] .", "secondly , several studies have shown that opg has proinflammatory and profibrotic effects on the vasculature .", "consistent with these actions , our group has shown that opg increases leukocyte adhesion to endothelial cells , that it increases tgf - beta - mediated fibrogenesis and proliferation in vascular smooth muscle cells , and that it increases atherosclerosis extension in diabetic apoe mice .", "by contrast , dyslipidemia alone did not affect opg / trail ratio in the vasculature .", "this data is consistent with a few in vitro studies showing that oxidized ldl did not change opg expression in human coronary artery smooth muscle cells   and lymphocytes .", "nevertheless , in our study , we found that dyslipidemia significantly increased opg / trail ratio in the heart , while diabetes had no additional effect on it .", "it has been shown that dyslipidemia is independently associated with left ventricle ( lv ) hypertrophy [ 5 , 30 , 31 ] .", "this was confirmed by experimental studies on apoe mice , where dyslipidemia was associated with increased lv mass in the absence of hemodynamic stress .", "consistent with previous reports , here we found that dyslipidemia was associated with cardiomyocyte hypertrophy .", "it has been demonstrated that triacylglycerol overload and the increased availability of other lipid intermediates , such as ceramides , diacylglycerol , and oxidized phospholipids , can all activate inflammatory pathways and oxidative stress , leading to cellular apoptosis and myocardial scarring [ 33 , 34 ] .", "it is possible that the increase in cardiac opg / trail ratio that was associated with dyslipidemia represents one of the mediators of lipid - induced cardiac remodeling . as for opg", ", a recent study has demonstrated that opg delivery induced lv hypertrophy , while , in vitro , cardiomyocytes transfected with aav - opg increased dramatically the expression of hypertrophy - related proteins , such as anp , -mhc , and troponin i . in the cardiac fibroblast ,", "by contrast , we showed that trail administration to mice with diabetic cardiomyopathy had cardioprotective effects , as it reduced cardiac fibrosis and apoptosis , which are generally contributing to cardiac remodeling . in conclusion , here we found that both dyslipidemia and diabetes affect opg / trail ratio in the cardiovascular system .", "this could contribute to the changes in circulating opg / trail which are observed in patients with diabetes and cvd .", "most importantly , these changes could mediate / contribute to atherosclerosis development and cardiac remodeling ."], "abstract_text": ["<S> \n background . dyslipidemia and </S>", "<S> diabetes are two of the most well established risk factors for the development of cardiovascular disease ( cvd ) . both of them usually activate a complex range of pathogenic pathways leading to organ damage . here </S>", "<S> we hypothesized that dyslipidemia and diabetes could affect osteoprotegerin ( opg ) and tnf - related apoptosis - inducing ligand ( trail ) expression in the vessels and the heart . </S>", "<S> materials and methods . gene and protein expression of opg , trail , and opg / trail ratio were quantified in the aorta and the hearts of control mice , dyslipidemic mice , and diabetic mice . </S>", "<S> results . </S>", "<S> diabetes significantly increased opg and the opg / trail ratio expression in the aorta , while dyslipidemia was the major determinant of the changes observed in the heart , where it significantly increased opg and reduced trail expression , thus increasing cardiac opg / trail ratio . conclusions . </S>", "<S> this work shows that both dyslipidemia and diabetes affect opg / trail ratio in the cardiovascular system . </S>", "<S> this could contribute to the changes in circulating opg / trail which are observed in patients with diabetes and cvd . </S>", "<S> most importantly , these changes could mediate / contribute to atherosclerosis development and cardiac remodeling . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["in industrialized countries , cardiovascular disease ( cvd ) remains the predominant cause of morbidity and mortality .", "it is current scientific opinion that cvd occurs as a part of a series of events that form a continuum .", "the first stage of this continuum corresponds to the presence of risks factors that predispose to tissue injury , such as hypertension , dyslipidemia , and diabetes .", "these risk factors usually activate a complex range of pathogenic pathways leading to the development of atherosclerosis and ischemic heart disease .", "failure to effectively manage any stage of this continuum results in ventricular hypertrophy , heart failure , end - stage heart disease , and cardiovascular death .", "one of the molecular pathways that could be activated in response to these risk factors and be involved in cvd development is that of osteoprotegerin ( opg ) and tnf - related apoptosis - inducing ligand ( trail ) , as reviewed in .", "opg and trail are both members of the tnf superfamily , and they are widely expressed in different tissues , including the heart and the vessels , in health and disease states [ 4 , 5 ] . although initially opg actions seemed to be limited to bone metabolism   and those of trail to host defense against tumors , recent studies have suggested that both molecules might actually be involved in cvd development and progression .", "epidemiological studies have shown that there is an association between circulating opg , trail , and cvd .", "opg was found to be directly correlated [ 5 , 8 , 9 ] , while trail was inversely correlated , with cardiovascular morbidity and mortality [ 10 , 11 ] .", "likewise , opg was significantly associated with the level of c - reactive protein , which is an independent predictor of acute vascular events and adverse outcomes , while trail was negatively correlated with it .", "recently , secchiero and colleagues have shown that patients with coronary artery disease have an increased opg / trail ratio , which further increases in patients developing heart failure .", "consistent with this epidemiological data , experimental studies suggest that opg and trail have opposite actions , as opg has proinflammatory   and proatherogenic effects [ 14 , 15 ] , while trail seems to be anti - inflammatory [ 16 , 17 ] and antiatherogenic [ 1820 ] . based on these premises", ", we hypothesized that dyslipidemia and diabetes , which are two well known risk factors for cvd , could modify the vascular and cardiac expression of opg and trail , leading to an increased opg / trail ratio at a tissue level .", "the aim of this study was to explore the vascular and cardiac changes of opg and trail in experimental models of dyslipidemia and diabetes ."], ["a total of 12 male c57bl/6j ( cnt ) mice and 24 male apolipoprotein e null ( apoe ) mice on a c57bl/6j background , aged 6 weeks , were studied for 14 weeks . at 6 weeks of age ,", "apoe mice were further randomized to saline ( apoe ) or streptozotocin ( apoe + dm ) , which were delivered intraperitoneally in five consecutive daily doses .", "the daily dose of streptozotocin ( stz ) ( sigma - aldrich ) was 55  mg / kg of body weight .", "after one week , blood glucose was checked and only the mice with blood glucose levels greater than 15  mm were included in the apoe + dm group . during the study period , all the mice were fed with a standard diet .", "the animals were kept in a temperature - controlled room ( 22  1c ) on a 12  h light/12  h dark cycle with free access to food and water and they were fed ad libitum for the length of the study . after 14 weeks , the animals were anesthetized with an intraperitoneal injection of thiobutabarbital sodium ( inactin , sigma - aldrich ) ( 60  mg / kg body weight ) and sacrificed by exsanguination via cardiac puncture .", "all the animal experiments were approved by the animal ethics committee of the university of trieste ( i d 28.0.2008 ) and the italian ministry of health , conforming to the guide for the care and use of laboratory animals . at the end of the study , we measured body weight and systolic blood pressure ( sbp ) .", "in particular , sbp was assessed by a computerized noninvasive tail cuff system in conscious mice .", "glucose , total cholesterol , hdl cholesterol , and triglycerides were measured in the sera by autoanalyzer technique ( hitachi 917 , tokyo , japan ) .", "circulating levels of opg and trail were measured in the sera by elisa ( r&d systems duoset # dy459 and # dy1121 ) . in half of the animals in each group ,", "aortae were collected and placed in 10% neutral - buffered formalin for atherosclerosis quantification , before being processed for immunohistochemical analyses . in the other half ,", "aortae were snap - frozen in liquid nitrogen and stored at 80c for rna extraction and gene expression analyses .", "briefly , aortae were cleaned of excess fat and stained with sudan iv - herxheimer 's solution .", "images were acquired with a dissecting microscope ( olympus bx-50 ) equipped with a high - resolution camera ( sony xc-77ce ) .", "digitized images were then evaluated with an image analysis system ( image - pro plus 6.3 software , media cybernetics ) .", "total plaque area was quantified as the percentage area of aorta stained red . as for the hearts", ", they were divided and half was put in formalin and half was put in liquid nitrogen .", "the part that was fixed in formalin was embedded in paraffin , cut in 4  m thick sections , and stained with hematoxylin and eosin ( h&e ) in order to evaluate cardiomyocyte hypertrophy .", "cardiomyocyte hypertrophy was assessed by measuring the shortest diameter of 200 cells , which were selected when they showed the spindle - shaped transverse section including the elliptical nucleus .", "the shortest transverse diameter was measured 3 times per cell , and the values were averaged , as reported by .", "the other half that was snap - frozen in liquid nitrogen was used either for gene expression analyses or for immunohistochemical stainings . gene expression of opg and trail was determined by real - time quantitative rt - pcr ( reverse transcription - polymerase chain reaction ) in aortic and cardiac tissue . in order to isolate mrna from the aorta and the heart , tissue was homogenized and processed as previously reported .", "then , mrna was treated with the dnase inactivation reagent ( ambion dna - free product # am-1906 ) , and 3  g of treated mrna was subsequently used to synthesize cdna with superscript first strand synthesis system for rt - pcr ( gibco brl ) .", "the gene expression of opg and trail was analyzed by real - time quantitative rt - pcr using the taqman system ( life technologies ) for opg and the sybr green system ( life technologies ) for trail .", "fluorescence for each cycle was quantitatively analyzed by an abi prism 7900ht sequence detection system ( applied biosystems ) .", "gene expression of opg was normalized to 18s mrna , while that of trail was normalized to rps9 .", "results were reported as ratio compared with the level of expression in untreated controls , which were given an arbitrary value of 1 .", "the presence of opg and trail in the aortae was evaluated by immunostainings on 4  m thick paraffin sections . in particular , after antigen retrieval in citrate buffer ( ph6 ) endogenous peroxidase was quenched for 10 minutes using 3% h2o2 in pbs . to localize opg", ", we used a biotinylated goat anti - opg antibody ( r&d systems , 1  :  10 dilution ) followed by a catalyzed signal amplification system ( dako ) . to localize trail", ", we used a monoclonal mouse anti - trail antibody ( r&d systems , 1  :  50 dilution ) , applied overnight at 4c . biotinylated goat anti - mouse immunoglobulins ( vector laboratories , dilution 1  :  200 )", "were then used as secondary antibody , followed by streptavidin - hrp ( dako ) .", "the presence of opg and trail in the hearts was evaluated by immunostaining on 5  m thick frozen sections .", "after neutralization of endogenous peroxidase , sections were incubated overnight with the goat anti - opg antibody ( r&d systems , 1  :  10 dilution ) and the monoclonal mouse anti - trail antibody ( r&d systems , 1  :  50 dilution ) .", "biotinylated immunoglobulins , diluted 1  :  200 , were then applied as secondary antibodies , followed by streptavidin - hrp ( dako ) . in both tissues ,", "after counterstaining with hematoxylin , all the sections were examined by light microscopy ( olympus bx50wi ) and digitized using a high - resolution camera ( q - imaging fast 1394 ) .", "the proportional area of brown staining was measured by an image analysis system ( image - pro plus 6.3 software , media cybernetics ) in order to quantify opg and trail expression in aortae and hearts ."], ["apoe mice had higher levels of total cholesterol and triglycerides as compared to the cnt mice . in line with the study protocol , the mice in the apoe + dm group were hyperglycemic . moreover , diabetes was associated with a significant reduction of body weight and a significant increase of total cholesterol as compared to nondiabetic apoe mice .", "apoe mice had a significant increase in total plaque area , which increased further after the induction of diabetes ( figures 1(a ) and 1(b ) ) .", "as for cardiac changes , dyslipidemia was associated with a significant increase in cardiomyocyte size ( figures 1(c ) and 1(d ) ) , indicating cardiomyocyte hypertrophy , with no further increase after the induction of diabetes . in the aorta , the induction of diabetes was associated with a significant increase in opg gene expression and in the opg / trail ratio , while dyslipidemia had no effect on opg / trail ratio as compared to controls ( figure 2(a ) ) . by contrast , in the heart , dyslipidemia was the major determinant of opg / trail tissue changes , not only by increasing opg but also by reducing trail gene expression .", "so dyslipidemia increased significantly cardiac opg / trail ratio , with no further differences after the induction of diabetes ( figure 2(b ) ) .", "immunostainings showed that diabetes significantly increased aortic opg expression as compared to the other groups , while trail was unchanged ( figure 3 ) . on the other hand , in the heart , it was dyslipidemia that significantly increased opg protein expression , with no further changes after the induction of diabetes .", "as for cardiac trail , trail protein expression decreased in the apoe and apoe + dm groups ( data not shown ) . the levels of circulating", "circulating opg was 1.91  0.21  ng / ml in the control group , 2.05  0.1  ng / ml in the apoe group , and 3.1  0.19 in the apoe + dm group , such that opg increased significantly in diabetic mice as compared to the other groups ( p < 0.05 , apoe + dm versus cnt and versus apoe group ) .", "as for trail , we were unable to measure its circulating levels , as they were below the detection levels of the elisa we used .", "it has already been argued that opg , which circulates at much higher levels than its ligands ( rankl and trail ) , may be a more stable overall measure of opg / trail activity than trail ."], ["patients with coronary artery disease have an increased opg / trail ratio , which further increases in the group of patients who develop heart failure . dyslipidemia and diabetes", ", we evaluated the effects of dyslipidemia , alone and in combination with diabetes , on aortic and cardiac opg and trail expression and found that while diabetes is the major determinant of opg / trail tissue changes in the vessels , dyslipidemia is it in the heart .", "this data is in line with previous experimental works showing that diabetes increased opg expression and the opg / trail ratio in the aorta   and that the onset of diabetes was associated with an increase of circulating opg .", "consistent with these observations , in our study , we found that aortic and circulating opg increased significantly only in the group of mice with diabetes .", "taken together , these findings seem to support the hypothesis that the vessels might be the source of increased circulating levels of opg in patients with diabetes and/or cvd .", "it has to be noted that the increase of circulating and tissue opg could be not only a risk marker but also a risk factor for atherosclerosis and cvd development .", "firstly , animal models point to a role for opg in glucose metabolism regulation , as opg injections increased significantly glucose levels [ 12 , 26 ] , which represent one of the risk factors for cvd .", "this effect could be mediated by trail blockade , as trail lowers glucose levels . by offsetting trail effects ,", "opg could also promote body weight gain and dyslipidemia , as well as atherosclerotic plaque development [ 18 , 20 ] .", "secondly , several studies have shown that opg has proinflammatory and profibrotic effects on the vasculature .", "consistent with these actions , our group has shown that opg increases leukocyte adhesion to endothelial cells , that it increases tgf - beta - mediated fibrogenesis and proliferation in vascular smooth muscle cells , and that it increases atherosclerosis extension in diabetic apoe mice .", "by contrast , dyslipidemia alone did not affect opg / trail ratio in the vasculature .", "this data is consistent with a few in vitro studies showing that oxidized ldl did not change opg expression in human coronary artery smooth muscle cells   and lymphocytes .", "nevertheless , in our study , we found that dyslipidemia significantly increased opg / trail ratio in the heart , while diabetes had no additional effect on it .", "it has been shown that dyslipidemia is independently associated with left ventricle ( lv ) hypertrophy [ 5 , 30 , 31 ] .", "this was confirmed by experimental studies on apoe mice , where dyslipidemia was associated with increased lv mass in the absence of hemodynamic stress .", "consistent with previous reports , here we found that dyslipidemia was associated with cardiomyocyte hypertrophy .", "it has been demonstrated that triacylglycerol overload and the increased availability of other lipid intermediates , such as ceramides , diacylglycerol , and oxidized phospholipids , can all activate inflammatory pathways and oxidative stress , leading to cellular apoptosis and myocardial scarring [ 33 , 34 ] .", "it is possible that the increase in cardiac opg / trail ratio that was associated with dyslipidemia represents one of the mediators of lipid - induced cardiac remodeling . as for opg", ", a recent study has demonstrated that opg delivery induced lv hypertrophy , while , in vitro , cardiomyocytes transfected with aav - opg increased dramatically the expression of hypertrophy - related proteins , such as anp , -mhc , and troponin i . in the cardiac fibroblast ,", "by contrast , we showed that trail administration to mice with diabetic cardiomyopathy had cardioprotective effects , as it reduced cardiac fibrosis and apoptosis , which are generally contributing to cardiac remodeling . in conclusion , here we found that both dyslipidemia and diabetes affect opg / trail ratio in the cardiovascular system .", "this could contribute to the changes in circulating opg / trail which are observed in patients with diabetes and cvd .", "most importantly , these changes could mediate / contribute to atherosclerosis development and cardiac remodeling ."]]}
{"article_id": "PMC4251536", "article_text": ["compounds 14 , 7 , 8 , and 11 were \n synthesized following our previously reported synthetic routes .", "saturated derivatives 5 and 6 , unique to this work , were synthesized via the hydrogenation \n of diastereomers 1 and 2 , respectively . \n palmyrolide alcohol ( 9 ) and acid ( 10 )", "were \n prepared exploiting the known acid - catalyzed ring opening of 1 , followed by treating the resultant \n aldehyde with either nabh4 ( for 9 ) or naclo2 ( for 10 ) .", "alcohol 12 could be accessed \n in a similar manner from 14-epi - palmyrolide ( 2 ) . in order to detect an interaction of ( )-palmyrolide \n a and \n its analogues with voltage - gated sodium channels ( vgscs )", ", we examined \n the ability of these compounds to antagonize veratridine - stimulated \n na influx in murine primary cerebrocortical neurons .", "we \n previously demonstrated that veratridine is a partial agonist at neurotoxin \n site 2 on the vgsc  subunit .", "murine \n cerebrocortical neurons were loaded with the na - binding \n benzofuran isophthalate ( sbfi ) , and the ability of palmyrolide analogues \n to block veratridine - induced elevation of neuronal [ na]i was determined .", "sodium influx was monitored with sbfi ( 340/380 ) responses to veratridine \n in the absence and presence of ( )-palmyrolide a and its analogues . \n", "the compounds shown are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide a ( 5 ) ; ( c ) ( + ) -palmyrolide \n a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide \n a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) \n [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) .", "data shown are from a representative \n experiment performed in 2 or 3 replicates for each concentration . \n in total 47 experiments", "nonlinear regression analysis of concentration  response \n data for active analogues of ( )-palmyrolide a. a three - parameter \n logistic fit of the palmyrolide a analogue inhibition of the response \n to veratridine is shown for each active compound .", "the compounds shown \n are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide \n a ( 5 ) ; ( c ) ( + ) -palmyrolide a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) . data points \n shown represent the mean  sem of 25 experiments performed \n with 210 replicates each ( a  f ) .", "values represent mean  sem \n of 25 experiments each performed with 210 replicates . naturally occurring ( )-palmyrolide \n", "a is a neuroactive macrolide \n that was previously shown to block veratridine - induced sodium influx \n in neuro-2a cells ( ic50 = 5.2 m ) and to display significant inhibition of ca oscillation in murine cerebrocortical neurons , with an ic50 value of 3.70 m ( 2.295.98 m , 95% ci ) .", "we have confirmed and extended these original \n findings by demonstrating that synthetic ( )-palmyrolide a \n ( 1 , figures 2a and 3a ) and its enantiomer ( 3 , figures 2c and 3c ) produced concentration - dependent \n antagonism of veratridine - induced increase in neuronal [ na]i .", "the concentration  response curves for these \n compounds were best fit by a three - parameter logistic equation .", "this \n analysis yielded an ic50 value ( 95% confidence interval ) \n of 2.1 m ( 1.23.1 , 95% ci ) , which was in good agreement \n with the ic50 value reported earlier for natural ( )-palmyrolide \n a. we found that ( )-palmyrolide \n a ( 1 ) and macrolide analogues 5 and 3 were approximately 39-fold more potent than the \n other analogues tested ( figures 2a ", "the observation that saturated \n congener 5 showed a comparable ic50 to synthetic \n ( )-palmyrolide a suggests that the enamide double bond is \n not essential for activity .", "it is moreover noteworthy that acyclic \n analogues of ( )-palmyrolide a with the natural absolute configuration \n were inactive as blockers of veratridine - stimulated na influx .", "these data indicate that the three - dimensional molecular \n shape of ( )-palmyrolide a was crucial for the interaction \n with vgscs . in order to support these observations and to better \n", "understand \n how structure relates to biological function , an exhaustive nmr and \n computational analysis was next conducted on 1 and three \n diastereomers ( i.e. , 2 , 7 , and 8) .", "saturated derivatives 5 and 6 exist \n as a mixture of rotational isomers and precluded precise nmr assignments \n from being made .", "chemical shift assignments of ( )-palmyrolide \n a ( 1 ) and synthetic diastereomers 2 , 7 , and 8 were made using dqf - cosy , hsqc , hmbc , \n and h2bc spectra . \n", "the chemical shifts of these four diastereomeric compounds follow \n the same general trends but differ slightly , indicating that the conformations \n of the macrolide ring are different . for example , in each diastereomer \n a different methylene group exhibited degenerate chemical shifts for \n its two protons .", "stereospecific assignments were made for most of \n the prochiral atoms that possessed nondegenerate shifts .", "the noesy \n spectra provided 74 nonredundant distance restraints for 1 , 83 for 2 , 84 for 7 , and 77 for 8 .", "h coupling in the h nmr spectra \n and from the heteronuclear couplings measured by the hetloc experiments \n ( see supporting information for all restraints ) . \n applying the distance and torsion restraints during simulated annealing \n calculations produced fairly well - defined ensembles of structures . \n", "table 2 summarizes the inputs \n and results of the structure calculations . to test the specificity \n of the restraints and their ability to discriminate between the four \n diastereomers ,", "restrained simulated annealing calculations were performed \n for all 16 combinations of starting structure and restraint set . if \n the restraints correctly discriminated between conformations , then \n the correct combination should have the least restraint violations \n and the lowest energy .", "for all four structures we found the best restraint \n set for each starting structure was the correct one , namely , the restraints \n derived from nmr spectra of that compound ( table 3 ) .", "this confirms that the restraint sets were able to correctly \n discriminate between the diastereomers .", "the four ensembles generally \n have the same overall conformation , \n with the major differences being due to the orientations of the substitutents . \n", "all four diastereomers have a cisoid configuration \n of the n - methyl group ( n - ch3 ) and the \n adjacent carbonyl despite the starting structures having a transoid configuration .", "examining the trajectories reveals \n that the configuration converts early in the simulated annealing as \n the restraints take effect .", "the driving force in these simulations \n appears to be noes between h-18 and the h-2 methylene group . to quantify differences between the ensembles , we calculated root - mean - square \n deviations ( rmsd s ) between the representative structures of \n each ensemble ( table 4 ) .", "the rmsd s \n were calculated using the differences in the positions of the carbon , \n nitrogen , and oxygen atoms of the macrolide ring after aligning the \n structures to minimize these differences .", "representative structures \n were selected as the structure having the smallest rmsd among all \n other structures in its ensemble .", "the rmsd s between all four \n representative structures were similar ( table 4 ) , with 2 having the lowest values overall , suggesting \n that this ensemble is the most  central  in comparison \n to the other diastereomers .", "visual inspection of the ensembles \n shows that the macrolide ring \n is relatively flat , but the orientation of the tert - butyl and methyl groups alters the global shape ( figure 4 ) . in 1", "the tert - butyl \n and methyl groups lie close to the plane of the ring , but in 7 the tert - butyl group projects almost perpendicular \n to the plane of the ring , and in 8 the two methyl groups \n project in opposite directions above and below the ring . examining \n the surface electrostatic potential of the representative structures \n ( figure 5 ) we find that 8 is the \n most similar to the active compound 1 . however , the c-14 \n methyl group in 1 projects to the side , whereas in 8 it is situated above the macrolide ring . taken together , \n these differences between diastereomers may account , at least in part , \n for the observed differences in biological activity .", "in addition , \n due to the highly flexible nature of the macrolide , as evidenced through \n computational modeling , the minimized ligand ensembles reported here \n may not necessarily reflect the structure these molecules adopt when \n bound to their molecular target .", "further consideration and investigations \n into structure  activity relationships are ongoing and will \n be reported in due course .", "number of diastereotopic hydrogens \n for which stereospecific assignments were obtained , and total number \n of protons with nondegenerate chemical shifts .", "the three numbers reported for each \n combination of starting structure and restraint set are number of \n selected structures , average amber energy , and average violation energy . \n", "the italic cells indicate the best set of restraints for each starting \n structure , with respect to amber energy and violation energy , and \n also correspond to the correct pairing .", "the final row is the sum of the \n three rmsd s above , a measure of the total difference from \n the other structures .", "the four structures in the top row are shown in \n a similar orientation to figure 4 . in the lower \n row", "the structures have been rotated 180 about the horizontal \n axis to show the rear face .", "the electrostatic potential ranges from \n red ( 2 kcal / mole ) through white ( 0 ) to blue ( + 2 kcal / mole ) .", "these \n findings suggest that both synthetic ( )-palmyrolide \n a and its enantiomer function with equal potency as vgsc antagonists \n to block veratridine - induced sodium influx . with regard to identification \n of the pharmacophore of this natural product", ", it appears that the \n intact macrolide is required since acyclic versions were inactive \n as inhibitors of vgscs .", "moreover , analogues lacking the enamide double \n bond were of high potency in this assay , and thus this functionality \n appears unnecessary for blocking vgscs . finally ,", "using detailed nmr \n and computational approaches with the natural product and three diastereomeric \n analogues , it was found that the c-5c-7 anti / c-7c14 syn arrangement of stereocenters \n produces a unique combination of three - dimensional shape and electrostatic \n potential that is responsible for the potent biological activity of \n the natural product . in an effort to understand", "the similar biological \n activity found for the natural stereoisomer and its enantiomer , continued \n investigations in this area will focus on uncovering the specific \n molecular target and associated binding site , which may also assist \n in future analogue development of novel sodium channel blocking analgesics \n derived from palmyrolide a.", "unless otherwise noted , \n reactions were performed in flame - dried glassware under an atmosphere \n of dry nitrogen .", "reaction solvents ( ch2cl2 , \n thf , and et2o ) were purified before use in a solvent purification \n system under a flow of dry nitrogen .", "all other solvents and reagents \n were purchased from commercial suppliers and used as received , unless \n otherwise specified .", "thin - layer chromatography ( tlc ) was performed \n using plates precoated with silica gel 60  f-254 ( 250 m ) \n and visualized by uv light , kmno4 , or anisaldehyde stains , \n followed by heating .", "ir samples were prepared by evaporation \n from chcl3 or ch2cl2 on nacl plates \n and run on a perkinelmer spectrum one ft - ir spectrometer . for the \n synthetic studies ,", "h and c nmr spectra were \n recorded at 300 and 75 mhz ( oxford magnet with a varian 300 console ) , \n at 400 and 100 mhz ( oxford magnet with a varian unity 400 console ) , \n and at 600 and 150 mhz ( magnex magnet with a bruker avance", "iii 600 \n console ) , respectively , and are reported relative to residual solvent \n peak ( h 7.26 and c 77.0 for h and c in cdcl3 ) .", "high - resolution \n mass spectra were obtained using positive electrospray ionization \n on a bruker 12 t apex - qe fticr - ms with an apollo ii ion source at \n the cosmic laboratory facility at old dominion university , va . to a solution of ( s , e)-6-iodo-2-methylhex-5-enoic \n acid ( 0.096 g , 0.37 mmol ) in thf ( 3.7 ml )", "was added diisopropylethylamine \n ( 0.100 ml , 0.60 mmol ) followed by the yamaguchi reagent ( 0.071 ml , \n 0.45 mmol ) .", "the mixture was allowed to stir at room temperature for \n 3 h before being concentrated in vacuo .", "the resultant \n mixed anhydride was dissolved in toluene ( 7.4 ml ) and then added , \n via cannula , to a flask containing ( 5r,7r)-7-hydroxy-5,8,8-trimethylnonanamide ( 0.051 g , 0.23 mmol ) and dmap \n ( 0.046 g , 0.37 mmol ) . after stirring at room temperature ( rt ) for \n 12 h", ", the reaction mixture was diluted with ch2cl2 , washed with a saturated aqueous solution of nahco3 , \n dried over mgso4 , and concentrated .", "purification via flash \n column chromatography ( 1:1 hexanes / etoac ) afforded 0.088 g ( 81% ) of \n the coupled product as a colorless oil : [", "]d22.5 + 13.3 ( c 1.98 , \n chcl3 ) ; ir ( neat , thin film )  3429 , 3351 , 3203 , \n 2962 , 2934 , 2868 , 1725 , 1665 , 1607 , 1461 , 1380 , 1366 , 1259 , 1181 , \n 1119 , 1067 , 957 , 935 cm ; h nmr ( 300 \n mhz , cdcl3 )  6.49 ( dt , j = 7.2 , \n 14.4 hz , 1 h ) , 6.02 ( d , j = 14.4 hz , 1h ) , 5.88 ( bs , \n 1 h ) , 5.46 ( bs , 1 h ) , 4.79 ( dd , j = 4.5 , 6.3 hz , \n 1 h ) , 2.45 ( sextet , j = 6.9 hz , 1 h ) , 2.232.15 \n ( m , 2 h ) , 2.122.05 ( m , 2 h ) , 1.861.69 ( m , 2 h ) , 1.611.25 \n ( m , 6 h ) , 1.17 ( d , j = 7.2 hz , 3 h ) , 1.110.99 \n ( m , 1 h ) , 0.910.87 ( overlapping doublet / singlet , 12 h ) ; c nmr ( 75 mhz , cdcl3 )  176.5 , 175.8 , 145.6 , \n 79.0 , 75.5 , 39.3 , 37.7 , 35.7 , 34.79 , 34.77 , 33.9 , 32.3 , 29.2 , 26.1 , \n 22.8 , 21.1 , 17.5 ; hresims m / z 474.1471 \n [ m + na ]", "( calcd for c19h34ino3na , 474.1476 ) . to the coupled product ( 0.088 g , 0.19 \n mmol ) in thf ( 19.0 ml )", "was added cui ( 0.005 g , 0.03 mmol ) and cs2co3 ( 0.100 g , 0.30 mmol ) .", "the mixture was first \n degassed using nitrogen ( 10 min ) before n , n-dimethylethylenediamine ( 0.050 ml , 0.46 mmol ) \n was added .", "the rubber septum was quickly replaced with a glass stopper , \n and the reaction mixture heated to 60 c for 19 h. after cooling \n to rt , the reaction mixture was diluted with etoac , filtered through \n a short plug of silica , and concentrated .", "the crude product was purified \n via flash column chromatography ( 8:2  7:3  1:1 hexanes / etoac ) \n to yield the n - h enamide intermediate ( 0.022 g ) , \n which was immediately dissolved in thf ( 0.7 ml ) and cooled to 0 c . \n to this", "was added nah ( 0.014 g , 0.34 mmol , 60% in mineral oil ) in \n a single portion .", "the reaction mixture was allowed to stir for 20 \n min before mei ( 0.100 ml ) was added dropwise , followed by removal \n of the ice water bath . after being allowed to stir at rt for 20 min , \n", "the reaction mixture was again cooled to 0 c before being quenched \n with h2o , extracted using etoac , dried over mgso4 , and concentrated .", "the crude product was purified via flash column \n chromatography ( 9:1 hexanes / etoac ) to yield 14-epi - palmyrolide a ( 0.016 g , 25% over two steps ) as a colorless oil : \n [ ]d24.5 11.4 ( c 0.58 , chcl3 ) ; ir ( neat , \n thin film )  2959 , 2927 , 2873 , 1728 , 1675 , 1646 , 1466 , 1413 , \n 1384 , 1366 , 1333 , 1298 , 1240 , 1205 , 1193 , 1171,1121 , 933 cm ; h nmr ( 400 mhz , cdcl3 )  6.63 ( d , j = 13.6 hz , 1 h ) , 4.92 ( ddd , j = 5.2 , \n 8.8 , 13.6 hz , 1 h ) , 4.85 ( dd , j = 2.0 , 9.6 , 1 h ) , \n 3.05 ( s , 3 h ) , 2.602.42 ( m , 3 h ) , 2.32 ( dt , j = 7.2 , 13.6 hz , 1 h ) , 2.242.17 ( m , 1 h ) , 2.031.95 \n ( m , 1 h ) ,", "1.841.74 ( m , 1 h ) , 1.681.54 ( m , 2 h ) , 1.491.30 \n ( m , 4 h ) , 1.26 ( d , j = 7.2 hz , 3 h ) , 1.030.93 \n ( m , 1 h ) , 0.89 ( d , j = 6.4 hz , 3 h ) , 0.86 ( s , 9 h ) ; c nmr ( 100 mhz , cdcl3 )  175.4 , 172.8 , 129.8 , \n 110.2 , 76.5 , 37.2 , 36.6 , 35.4 , 34.3 , 33.6 , 31.0 , 29.8 , 29.5 , 27.3 , \n 25.9 , 24.9 , 20.0 , 19.2 ; hresims m / z 360.2504 [ m + na ] ( calcd for c20h35no3na , 360.2509 ) .", "to a solution \n of ( )-palmyrolidea ( 0.008 g , 0.022 mmol ) in anhydrous meoh \n ( 1 ml ) was added 10% palladium on carbon ( 0.005 g ) .", "the atmosphere \n in the flask was replaced with hydrogen , and the reaction mixture \n was allowed to stir at rt for 18 h before being filtered through a \n short plug of silica .", "after rinsing several times with ether , the \n filtrate was concentrated and purified via column chromatography ( 8:2 \n hexanes / etoac ) to afford compound 5 ( 0.006 g , 77% ) as \n a mixture of rotational isomers ( 2:1 ratio ) : [ ]d23 + 35.6 ( c 0.48 , chcl3 ) ; ir ( neat , thin film )  \n 2954 , 2931 , 2871 , 1720 , 1651 , 1269 , 1250 , 1073 cm ; h nmr ( 300 mhz , cdcl3 , mixture of rotational \n isomers with mostly overlapping peaks , making the assignment of major \n and minor components difficult )  ( major isomer ) 4.82 ( d , j = 10.0 hz , 1 h ) , 3.30 ( t , j = 5.0 hz , \n 1 h ) , 2.98 ( s , 3 h ) , 2.532.65 ( m , 2 h ) , 2.212.33 ( m , \n 2 h ) , 1.211.73 ( m , 14 h ) , 1.18 ( d , j = 6.9 \n hz , 3 h ) , 0.94 ( d , j = 5.9 hz , 3 h ) , 0.86 ( s , 9 h ) , \n ( minor isomer , partial ) 4.92 ( d , j = 10.8 hz , 0.6 \n h ) , 2.89 ( s , 1.5 h ) , 0.91 ( d , j = 6.1 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , mixture of rotational isomers ) \n  175.5 , 174.8 , 173.5 , 77.8 , 77.2 , 48.9 , 46.3 , 40.4 , 40.0 , 38.3 , \n 37.2 , 35.2 , 35.0 , 33.6 , 33.2 , 32.98 , 32.96 , 31.2 , 39.7 , 27.9 , 27.6 , \n 27.0 , 26.0 , 25.9 , 25.4 , 23.5 , 23.2 , 22.2 , 20.4 , 20.1 , 18.0 , 16.5 ; \n hresims m / z 362.2660 [ m + na ] ( calcd for c20h37no3na , \n 362.2665 ) .", "the crude product was purified via column \n chromatography ( 8:2 hexanes / etoac ) to afford compound 6 ( 0.006 g , 96% ) as a mixture of rotational isomers ( 2:1 ratio ) : \n [ ]d23 + 42.1 ( c 0.5 , chcl3 ) ; ir ( neat , thin \n film )  2956 , 2930 , 2872 , 1727 , 1647 , 1459 , 1192 , 1156 cm ; h nmr ( 300 mhz , cdcl3 , mixture \n of rotational isomers with mostly overlapping peaks , making the assignment \n of major and minor components difficult )  ( major isomer ) 4.86 \n ( d , j = 10.5 hz , 1 h ) , 3.513.42 ( m , 1 h ) , \n 3.233.14 ( m , 1 hz ) , 2.88 ( s , 3 h ) , 2.312.48 ( m , 2 \n h ) , 2.182.31 ( m , 2 h ) , 1.971.79 ( m , 2 h ) , 1.231.66 \n ( m , 15 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.92 ( d , j = 5.8 hz , 3 h ) , 0.86 ( s , 11 h ) , ( minor isomer , partial ) \n 4.83 ( d , j = 11.6 hz , 0.6 h ) , 2.91 ( s , 1 h ) , 1.15 \n ( d , j = 7.2 hz , 1.5 h ) , 0.96 ( d , j = 5.7 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , \n mixture of rotational isomers )  176.8 , 176.6 , 173.3 , 173.0 , \n 77.2 , 49.1 , 46.0 , 40.6 , 40.1 , 38.0 , 37.3 , 35.2 , 34.0 , 33.9 , 33.2 , \n 33.1 , 33.0 , 31.3 , 29.7 , 28.9 , 27.2 , 25.9 , 24.8 , 23.9 , 22.8 , 19.9 , \n 19.8 , 19.2 , 19.1 ; hresims m / z 362.2659 \n [ m + na ]", "( calcd for c20h37no3na , 362.2665 ) . to a solution \n of 1 ( 0.007 g , 0.021 mmol ) in chcl3 ( 2 ml ) \n", "was added a 6 n aqueous hcl solution dropwise until tlc analysis showed \n complete consumption of the starting material .", "the reaction mixture \n was then quenched with a saturated aqueous solution of nahco3 , followed by extraction using etoac .", "the \n resultant crude aldehyde was dissolved in anhydrous meoh ( 1 ml ) and \n treated with nabh4 ( 0.015 g , 0.39 mmol ) .", "the reaction mixture \n was allowed to stir at rt for 1 h before being quenched with h2o , extracted using etoac , dried over mgso4 , filtered , \n and concentrated .", "the crude alcohol was purified by column chromatography \n ( 100% etoac ) to afford alcohol 9 ( 0.006 g , 73% over two \n steps ) as a colorless oil : [ ]d24.3 + 26.8 ( c 0.20 , chcl3 ) ; ir ( neat , thin film )  3296 , 2956 , 1728 , 1651 , 1562 , \n 1462 , 1366 , 1164 cm ; h nmr ( 300 mhz , \n cdcl3 )  6.10 ( bs , 1 h ) , 4.76 ( dd , j = 2.2 , 9.5 hz , 1 h ) , 3.63 ( t , j = 6.4 hz , 2 h ) , \n 2.79 ( d , j = 4.8 hz , 3 h ) , 2.392.51 ( m , 1 \n h ) , 2.002.23 ( m , 2 h ) , 1.91 ( bs , 1 h ) , 1.631.80 ( m , \n 2 h ) , 1.221.60 ( m , 10 h ) , 1.16 ( d , j = 7.0 \n hz , 3 h ) , 0.87 ( d , overlapped , 3 h ) , 0.86 ( s , 9 h ) ; c \n nmr ( 75 mhz , cdcl3 )  176.8 , 174.0 , 78.6 , 62.5 , 40.2 , \n 37.6 , 36.2 , 34.5 , 33.3 , 32.5 , 28.8 , 26.2 , 25.9 , 25.9 , 23.5 , 23.0 , \n 20.8 , 17.4 ; hresims m / z 380.2764 \n [ m + na ] ( calcd for c20h39no4na , 380.2771 ) . approximately 0.3 \n ml of an oxidant solution prepared via the mixing of naclo2 ( 0.060 g ) and nahpo4 ( 0.040 g ) in h2o ( 1 ml ) \n was added to a solution of palmyrolide aldehyde ( prepared as above , \n 0.006 g , 0.016 mmol ) in tert - butanol ( 0.25 ml ) and \n 2-methyl-2-butene ( 0.1 ml ) . the reaction mixture was allowed to stir \n for 30 min before being quenched with water , extracted with etoac , \n dried over mgso4 , filtered , and concentrated .", "the crude \n acid was purified by column chromatography ( 100% etoac ) to afford \n acid 10 ( 0.002 g , 25% ) as a colorless oil : [ ]d23 + 10.8 ( c 0.10 , chcl3 ) ; ir ( neat , thin film )  \n 3306 , 2960 , 1727 , 1633 , 1164 cm ; h nmr ( 300 mhz , cdcl3 )  6.04 ( bs , 1 h ) , 4.78 ( dd , j = 10.2 , 1.9 hz , 1 h ) , 2.82 ( d , j = 4.8 \n hz , 3 h ) , 2.48 ( q , j = 6.9 hz , 1 h ) , 2.312.41 \n ( m , 2 h ) , 2.022.23 ( m , 3 h ) , 1.601.82 ( m , 4 h ) , 1.211.59 \n ( m , 6 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.921.05 \n ( m , 1 h ) , 0.88 ( d , overlapped , 3 h ) , 0.87 ( s , 9 h ) ; c \n nmr ( 75 mhz , cdcl3 )  176.4 , 78.5 , 40.1 , 37.6 , 36.2 , \n 34.6 , 34.4 , 33.0 , 29.7 , 28.7 , 26.3 , 26.0 , 23.0 , 22.7 , 20.7 , 17.2 ; \n hresims m / z 394.2558 [ m + na ] ( calcd for c20h37no5na , \n 394.2563 ) .", "the crude product was purified via column chromatography \n ( 100% etoac ) to afford compound 12 ( 0.004 g , 94% ) : [ ]d25.5 + 30.8 ( c 0.24 , chcl3 ) ; ir ( neat , thin film )  \n 3304 , 2950 , 1729 , 1649 , 1560 , 1462 , 1366 , 1163 , 1074 cm ; h nmr ( 300 mhz , cdcl3 )  6.25 ( bs , \n 1 h ) , 4.76 ( dd , j = 9.9 , 1.6 hz , 1 h ) , 3.553.70 \n ( m , 2 h ) , 2.77 ( d , j = 4.8 hz , 3h ) , 2.372.50 \n ( m , 1 h ) , 2.27 ( bs , 1 h ) , 2.002.20 ( m , 2 h ) , 1.631.83 \n ( m , 2 h ) , 1.211.60 ( m , 10 h ) , 1.16 ( d , j = \n 7.0 hz , 3 h ) , 0.921.04 ( m , 1 h ) , 0.86 ( d , overlapped , 3 h ) , \n 0.85 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )  \n 177.0 , 174.1 , 78.4 , 62.2 , 40.0 , 37.5 , 36.2 , 34.6 , 34.5 , 33.1 , 32.4 , \n 28.8 , 26.1 , 25.9 , 23.6 , 23.1 , 20.7 , 17.7 ; hresims m / z 380.2764 [ m + na ] ( calcd for c20h39no4na , 380.2771 ) .", "primary cultures of \n cerebrocortical neurons were obtained from embryonic day 17 swiss - webster \n mice as described elsewhere .", "briefly , \n pregnant mice were euthanized by co2 asphyxiation , and \n embryos were removed under sterile conditions .", "cerebral cortices were \n collected , stripped of meninges , minced by trituration with a pasteur \n pipet , and treated with trypsin for 25 min at 37 c .", "the cells \n were then dissociated by two successive trituration and sedimentation \n steps in soybean trypsin inhibitor and dnase - containing isolation \n buffer , centrifuged , and resuspended in eagle s minimal essential \n medium with earle s salt ( mem ) supplemented with 1 mm l - glutamine , 10% fetal bovine serum , 10% horse serum , 100 iu / ml penicillin , \n and 0.10 mg / ml streptomycin ( ph 7.4 ) .", "cells were plated onto poly - l - lysine - coated 96-well ( 9 mm ) , clear - bottomed , black - well culture \n plates ( midsci ) at a density of 1.5  10 cells / well . \n", "cells were then incubated at 37 c in a 5% co2 and \n 95% humidity atmosphere .", "the culture media was changed every other \n day , starting from day 5 in vitro using a serum - free \n growth medium containing neurobasal medium supplemented with b-27 , \n 100 iu / ml penicillin , 0.10 mg / ml streptomycin , and 0.2 mm l - glutamine .", "all animal use protocols were \n approved by the institutional animal care and use committee ( iacuc ) \n of creighton university .", "[ na]i measurement and full in situ calibration of sodium - binding benzofuran isophthalate fluorescence \n ratio were performed as described previously .", "cells were grown in 96-well plates and washed four times with locke s \n buffer ( 8.6 mm hepes , 5.6 mm kcl , 154 mm nacl , 5.6 mm glucose , 1.0 \n mm mgcl2 , 2.3 mm cacl2 , 0.1 mm glycine , ph 7.4 ) \n using an automated microplate washer ( biotek instruments ) . after measuring \n the background fluorescence of each well , cells were incubated for \n 1 h at 37 c with dye - loading buffer ( 100 l / well ) containing \n 10 m sbfi - am ( invitrogen ) and 0.02% pluronic f-127 ( invitrogen ) . \n", "cells were washed five times with locke s buffer , leaving a \n final volume of 100 l in each well .", "the plate was then transferred \n back to the incubator for 15 min to allow the cells to equilibrate \n after washing and placed in a flexstation ii ( molecular devices ) chamber \n to detect na - bound sbfi emission at 505 nm ( cells were \n excited at 340 and 380 nm ) .", "fluorescence readings were taken once \n every 5 s for 60 s to establish the baseline , and then 50 l \n of different concentrations of ( )-palmyrolide a and/or veratridine \n was added to each well from the compound plate at a rate of 26 l / s , \n yielding a final volume of 200 l / well . after correcting for \n background fluorescence , sbfi fluorescence ratios ( 340/380 ) and concentration ", "each palmyrolide a analogue was dissolved in \n dmso at a stock concentration of 50 mm and stored at 20 c \n until used . on the day of an experiment ,", "this dilution in locke s \n buffer resulted in palmyrolide a analogue solutions that were 4 times \n the desired final concentration .", "( )-palmyrolide \n a ( 1 ) and analogues ( 2 , 7 , \n and 8) were dissolved in cdcl3 at a concentration \n of 2.5 mg in 50 l and transferred to a 1.7 mm nmr tube . \n", "the noesy mixing time was 500 ms , the hmbc delay was set to 6.25 ms \n ( 8 hz ) , and the hetloc used a dispi-2 spinlock of 60 ms duration . \n", "chemical shift assignments were based on analysis of the dqf - cosy , \n hsqc , and hmbc 2d experiments using ucsf sparky .", "topspin ( bruker biospin ) was used to analyze coupling constants \n in the h nmr and hetloc spectra .", "noesy peak heights were \n converted to upper distance restraints of 3 , 4 , 5 , or 6  using \n in - house perl scripts . where restraints involved a group of atoms , \n pseudoatom corrections were applied by the amber restraint generation \n tools .", "force field parameters were estimated using \n the gaff force field and the antechamber \n program in the amber 14 package .", "molecular \n dynamics simulations were nonperiodic , vacuum simulations with a distance - dependent \n dielectric term .", "using sander in the amber 14 package each starting \n structure was energy minimized , then subjected to 20 ps of simulated \n annealing with the nmr - derived restraints in which the temperature \n was raised from 10 k to 600 k then cooled to near 0 k. the simulated \n annealing was run 20 times for each structure with a different random \n number seed each time .", "each collection of 20 structures was sorted \n by restraint violation energy , and outliers were discarded . to align \n structures ,", "the selected ensemble was superimposed on c17 , \n o12 , c1318 , and n19 using cpptraj . to stereospecifically assign pairs of diastereotopic hydrogens , \n pairs of noes that involved the diastereotopic atoms and had differential \n intensities", "were identified , then compared with the distances between \n these atoms in the ensemble of selected structures .", "if the ensemble \n mean of one distance was more than one standard deviation from the \n alternative , then the diastereotopic hydrogens were stereospecifically \n assigned ."], "abstract_text": ["<S> a small library of synthetic ( )-palmyrolide \n a diastereomers , \n analogues , and acyclic precursors have been examined with respect \n to their interaction with voltage - gated sodium channels ( vgscs ) . toward \n this goal , </S>", "<S> the ability of ( )-palmyrolide a and analogues to \n antagonize veratridine - stimulated na+ influx in primary \n cultures of mouse cerebrocortical neurons was assessed . </S>", "<S> we found that \n synthetic ( )-palmyrolide a and its enantiomer functioned as \n vgsc antagonists to block veratridine - induced sodium influx . a detailed \n nmr and computational analysis of four diastereomers revealed that \n none had the same combination of shape and electrostatic potential \n as exhibited by natural ( )-palmyrolide a. these data indicate \n that the relative configuration about the tert - butyl \n and methyl substituents appears to be a prerequisite for biological \n function . additional testing revealed that the enamide double bond \n was not necessary for blocking veratridine - induced sodium influx , \n whereas the acyclic analogues and other macrolide diastereomers tested \n were inactive as inhibitors of vgscs , suggesting that the intact macrolide \n was required . </S>"], "labels": null, "section_names": ["Results and Discussion", "Conclusion", "Experimental Section"], "sections": [["compounds 14 , 7 , 8 , and 11 were \n synthesized following our previously reported synthetic routes .", "saturated derivatives 5 and 6 , unique to this work , were synthesized via the hydrogenation \n of diastereomers 1 and 2 , respectively . \n palmyrolide alcohol ( 9 ) and acid ( 10 )", "were \n prepared exploiting the known acid - catalyzed ring opening of 1 , followed by treating the resultant \n aldehyde with either nabh4 ( for 9 ) or naclo2 ( for 10 ) .", "alcohol 12 could be accessed \n in a similar manner from 14-epi - palmyrolide ( 2 ) . in order to detect an interaction of ( )-palmyrolide \n a and \n its analogues with voltage - gated sodium channels ( vgscs )", ", we examined \n the ability of these compounds to antagonize veratridine - stimulated \n na influx in murine primary cerebrocortical neurons .", "we \n previously demonstrated that veratridine is a partial agonist at neurotoxin \n site 2 on the vgsc  subunit .", "murine \n cerebrocortical neurons were loaded with the na - binding \n benzofuran isophthalate ( sbfi ) , and the ability of palmyrolide analogues \n to block veratridine - induced elevation of neuronal [ na]i was determined .", "sodium influx was monitored with sbfi ( 340/380 ) responses to veratridine \n in the absence and presence of ( )-palmyrolide a and its analogues . \n", "the compounds shown are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide a ( 5 ) ; ( c ) ( + ) -palmyrolide \n a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide \n a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) \n [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) .", "data shown are from a representative \n experiment performed in 2 or 3 replicates for each concentration . \n in total 47 experiments", "nonlinear regression analysis of concentration  response \n data for active analogues of ( )-palmyrolide a. a three - parameter \n logistic fit of the palmyrolide a analogue inhibition of the response \n to veratridine is shown for each active compound .", "the compounds shown \n are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide \n a ( 5 ) ; ( c ) ( + ) -palmyrolide a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) . data points \n shown represent the mean  sem of 25 experiments performed \n with 210 replicates each ( a  f ) .", "values represent mean  sem \n of 25 experiments each performed with 210 replicates . naturally occurring ( )-palmyrolide \n", "a is a neuroactive macrolide \n that was previously shown to block veratridine - induced sodium influx \n in neuro-2a cells ( ic50 = 5.2 m ) and to display significant inhibition of ca oscillation in murine cerebrocortical neurons , with an ic50 value of 3.70 m ( 2.295.98 m , 95% ci ) .", "we have confirmed and extended these original \n findings by demonstrating that synthetic ( )-palmyrolide a \n ( 1 , figures 2a and 3a ) and its enantiomer ( 3 , figures 2c and 3c ) produced concentration - dependent \n antagonism of veratridine - induced increase in neuronal [ na]i .", "the concentration  response curves for these \n compounds were best fit by a three - parameter logistic equation .", "this \n analysis yielded an ic50 value ( 95% confidence interval ) \n of 2.1 m ( 1.23.1 , 95% ci ) , which was in good agreement \n with the ic50 value reported earlier for natural ( )-palmyrolide \n a. we found that ( )-palmyrolide \n a ( 1 ) and macrolide analogues 5 and 3 were approximately 39-fold more potent than the \n other analogues tested ( figures 2a ", "the observation that saturated \n congener 5 showed a comparable ic50 to synthetic \n ( )-palmyrolide a suggests that the enamide double bond is \n not essential for activity .", "it is moreover noteworthy that acyclic \n analogues of ( )-palmyrolide a with the natural absolute configuration \n were inactive as blockers of veratridine - stimulated na influx .", "these data indicate that the three - dimensional molecular \n shape of ( )-palmyrolide a was crucial for the interaction \n with vgscs . in order to support these observations and to better \n", "understand \n how structure relates to biological function , an exhaustive nmr and \n computational analysis was next conducted on 1 and three \n diastereomers ( i.e. , 2 , 7 , and 8) .", "saturated derivatives 5 and 6 exist \n as a mixture of rotational isomers and precluded precise nmr assignments \n from being made .", "chemical shift assignments of ( )-palmyrolide \n a ( 1 ) and synthetic diastereomers 2 , 7 , and 8 were made using dqf - cosy , hsqc , hmbc , \n and h2bc spectra . \n", "the chemical shifts of these four diastereomeric compounds follow \n the same general trends but differ slightly , indicating that the conformations \n of the macrolide ring are different . for example , in each diastereomer \n a different methylene group exhibited degenerate chemical shifts for \n its two protons .", "stereospecific assignments were made for most of \n the prochiral atoms that possessed nondegenerate shifts .", "the noesy \n spectra provided 74 nonredundant distance restraints for 1 , 83 for 2 , 84 for 7 , and 77 for 8 .", "h coupling in the h nmr spectra \n and from the heteronuclear couplings measured by the hetloc experiments \n ( see supporting information for all restraints ) . \n applying the distance and torsion restraints during simulated annealing \n calculations produced fairly well - defined ensembles of structures . \n", "table 2 summarizes the inputs \n and results of the structure calculations . to test the specificity \n of the restraints and their ability to discriminate between the four \n diastereomers ,", "restrained simulated annealing calculations were performed \n for all 16 combinations of starting structure and restraint set . if \n the restraints correctly discriminated between conformations , then \n the correct combination should have the least restraint violations \n and the lowest energy .", "for all four structures we found the best restraint \n set for each starting structure was the correct one , namely , the restraints \n derived from nmr spectra of that compound ( table 3 ) .", "this confirms that the restraint sets were able to correctly \n discriminate between the diastereomers .", "the four ensembles generally \n have the same overall conformation , \n with the major differences being due to the orientations of the substitutents . \n", "all four diastereomers have a cisoid configuration \n of the n - methyl group ( n - ch3 ) and the \n adjacent carbonyl despite the starting structures having a transoid configuration .", "examining the trajectories reveals \n that the configuration converts early in the simulated annealing as \n the restraints take effect .", "the driving force in these simulations \n appears to be noes between h-18 and the h-2 methylene group . to quantify differences between the ensembles , we calculated root - mean - square \n deviations ( rmsd s ) between the representative structures of \n each ensemble ( table 4 ) .", "the rmsd s \n were calculated using the differences in the positions of the carbon , \n nitrogen , and oxygen atoms of the macrolide ring after aligning the \n structures to minimize these differences .", "representative structures \n were selected as the structure having the smallest rmsd among all \n other structures in its ensemble .", "the rmsd s between all four \n representative structures were similar ( table 4 ) , with 2 having the lowest values overall , suggesting \n that this ensemble is the most  central  in comparison \n to the other diastereomers .", "visual inspection of the ensembles \n shows that the macrolide ring \n is relatively flat , but the orientation of the tert - butyl and methyl groups alters the global shape ( figure 4 ) . in 1", "the tert - butyl \n and methyl groups lie close to the plane of the ring , but in 7 the tert - butyl group projects almost perpendicular \n to the plane of the ring , and in 8 the two methyl groups \n project in opposite directions above and below the ring . examining \n the surface electrostatic potential of the representative structures \n ( figure 5 ) we find that 8 is the \n most similar to the active compound 1 . however , the c-14 \n methyl group in 1 projects to the side , whereas in 8 it is situated above the macrolide ring . taken together , \n these differences between diastereomers may account , at least in part , \n for the observed differences in biological activity .", "in addition , \n due to the highly flexible nature of the macrolide , as evidenced through \n computational modeling , the minimized ligand ensembles reported here \n may not necessarily reflect the structure these molecules adopt when \n bound to their molecular target .", "further consideration and investigations \n into structure  activity relationships are ongoing and will \n be reported in due course .", "number of diastereotopic hydrogens \n for which stereospecific assignments were obtained , and total number \n of protons with nondegenerate chemical shifts .", "the three numbers reported for each \n combination of starting structure and restraint set are number of \n selected structures , average amber energy , and average violation energy . \n", "the italic cells indicate the best set of restraints for each starting \n structure , with respect to amber energy and violation energy , and \n also correspond to the correct pairing .", "the final row is the sum of the \n three rmsd s above , a measure of the total difference from \n the other structures .", "the four structures in the top row are shown in \n a similar orientation to figure 4 . in the lower \n row", "the structures have been rotated 180 about the horizontal \n axis to show the rear face .", "the electrostatic potential ranges from \n red ( 2 kcal / mole ) through white ( 0 ) to blue ( + 2 kcal / mole ) ."], ["these \n findings suggest that both synthetic ( )-palmyrolide \n a and its enantiomer function with equal potency as vgsc antagonists \n to block veratridine - induced sodium influx . with regard to identification \n of the pharmacophore of this natural product", ", it appears that the \n intact macrolide is required since acyclic versions were inactive \n as inhibitors of vgscs .", "moreover , analogues lacking the enamide double \n bond were of high potency in this assay , and thus this functionality \n appears unnecessary for blocking vgscs . finally ,", "using detailed nmr \n and computational approaches with the natural product and three diastereomeric \n analogues , it was found that the c-5c-7 anti / c-7c14 syn arrangement of stereocenters \n produces a unique combination of three - dimensional shape and electrostatic \n potential that is responsible for the potent biological activity of \n the natural product . in an effort to understand", "the similar biological \n activity found for the natural stereoisomer and its enantiomer , continued \n investigations in this area will focus on uncovering the specific \n molecular target and associated binding site , which may also assist \n in future analogue development of novel sodium channel blocking analgesics \n derived from palmyrolide a."], ["unless otherwise noted , \n reactions were performed in flame - dried glassware under an atmosphere \n of dry nitrogen .", "reaction solvents ( ch2cl2 , \n thf , and et2o ) were purified before use in a solvent purification \n system under a flow of dry nitrogen .", "all other solvents and reagents \n were purchased from commercial suppliers and used as received , unless \n otherwise specified .", "thin - layer chromatography ( tlc ) was performed \n using plates precoated with silica gel 60  f-254 ( 250 m ) \n and visualized by uv light , kmno4 , or anisaldehyde stains , \n followed by heating .", "ir samples were prepared by evaporation \n from chcl3 or ch2cl2 on nacl plates \n and run on a perkinelmer spectrum one ft - ir spectrometer . for the \n synthetic studies ,", "h and c nmr spectra were \n recorded at 300 and 75 mhz ( oxford magnet with a varian 300 console ) , \n at 400 and 100 mhz ( oxford magnet with a varian unity 400 console ) , \n and at 600 and 150 mhz ( magnex magnet with a bruker avance", "iii 600 \n console ) , respectively , and are reported relative to residual solvent \n peak ( h 7.26 and c 77.0 for h and c in cdcl3 ) .", "high - resolution \n mass spectra were obtained using positive electrospray ionization \n on a bruker 12 t apex - qe fticr - ms with an apollo ii ion source at \n the cosmic laboratory facility at old dominion university , va . to a solution of ( s , e)-6-iodo-2-methylhex-5-enoic \n acid ( 0.096 g , 0.37 mmol ) in thf ( 3.7 ml )", "was added diisopropylethylamine \n ( 0.100 ml , 0.60 mmol ) followed by the yamaguchi reagent ( 0.071 ml , \n 0.45 mmol ) .", "the mixture was allowed to stir at room temperature for \n 3 h before being concentrated in vacuo .", "the resultant \n mixed anhydride was dissolved in toluene ( 7.4 ml ) and then added , \n via cannula , to a flask containing ( 5r,7r)-7-hydroxy-5,8,8-trimethylnonanamide ( 0.051 g , 0.23 mmol ) and dmap \n ( 0.046 g , 0.37 mmol ) . after stirring at room temperature ( rt ) for \n 12 h", ", the reaction mixture was diluted with ch2cl2 , washed with a saturated aqueous solution of nahco3 , \n dried over mgso4 , and concentrated .", "purification via flash \n column chromatography ( 1:1 hexanes / etoac ) afforded 0.088 g ( 81% ) of \n the coupled product as a colorless oil : [", "]d22.5 + 13.3 ( c 1.98 , \n chcl3 ) ; ir ( neat , thin film )  3429 , 3351 , 3203 , \n 2962 , 2934 , 2868 , 1725 , 1665 , 1607 , 1461 , 1380 , 1366 , 1259 , 1181 , \n 1119 , 1067 , 957 , 935 cm ; h nmr ( 300 \n mhz , cdcl3 )  6.49 ( dt , j = 7.2 , \n 14.4 hz , 1 h ) , 6.02 ( d , j = 14.4 hz , 1h ) , 5.88 ( bs , \n 1 h ) , 5.46 ( bs , 1 h ) , 4.79 ( dd , j = 4.5 , 6.3 hz , \n 1 h ) , 2.45 ( sextet , j = 6.9 hz , 1 h ) , 2.232.15 \n ( m , 2 h ) , 2.122.05 ( m , 2 h ) , 1.861.69 ( m , 2 h ) , 1.611.25 \n ( m , 6 h ) , 1.17 ( d , j = 7.2 hz , 3 h ) , 1.110.99 \n ( m , 1 h ) , 0.910.87 ( overlapping doublet / singlet , 12 h ) ; c nmr ( 75 mhz , cdcl3 )  176.5 , 175.8 , 145.6 , \n 79.0 , 75.5 , 39.3 , 37.7 , 35.7 , 34.79 , 34.77 , 33.9 , 32.3 , 29.2 , 26.1 , \n 22.8 , 21.1 , 17.5 ; hresims m / z 474.1471 \n [ m + na ]", "( calcd for c19h34ino3na , 474.1476 ) . to the coupled product ( 0.088 g , 0.19 \n mmol ) in thf ( 19.0 ml )", "was added cui ( 0.005 g , 0.03 mmol ) and cs2co3 ( 0.100 g , 0.30 mmol ) .", "the mixture was first \n degassed using nitrogen ( 10 min ) before n , n-dimethylethylenediamine ( 0.050 ml , 0.46 mmol ) \n was added .", "the rubber septum was quickly replaced with a glass stopper , \n and the reaction mixture heated to 60 c for 19 h. after cooling \n to rt , the reaction mixture was diluted with etoac , filtered through \n a short plug of silica , and concentrated .", "the crude product was purified \n via flash column chromatography ( 8:2  7:3  1:1 hexanes / etoac ) \n to yield the n - h enamide intermediate ( 0.022 g ) , \n which was immediately dissolved in thf ( 0.7 ml ) and cooled to 0 c . \n to this", "was added nah ( 0.014 g , 0.34 mmol , 60% in mineral oil ) in \n a single portion .", "the reaction mixture was allowed to stir for 20 \n min before mei ( 0.100 ml ) was added dropwise , followed by removal \n of the ice water bath . after being allowed to stir at rt for 20 min , \n", "the reaction mixture was again cooled to 0 c before being quenched \n with h2o , extracted using etoac , dried over mgso4 , and concentrated .", "the crude product was purified via flash column \n chromatography ( 9:1 hexanes / etoac ) to yield 14-epi - palmyrolide a ( 0.016 g , 25% over two steps ) as a colorless oil : \n [ ]d24.5 11.4 ( c 0.58 , chcl3 ) ; ir ( neat , \n thin film )  2959 , 2927 , 2873 , 1728 , 1675 , 1646 , 1466 , 1413 , \n 1384 , 1366 , 1333 , 1298 , 1240 , 1205 , 1193 , 1171,1121 , 933 cm ; h nmr ( 400 mhz , cdcl3 )  6.63 ( d , j = 13.6 hz , 1 h ) , 4.92 ( ddd , j = 5.2 , \n 8.8 , 13.6 hz , 1 h ) , 4.85 ( dd , j = 2.0 , 9.6 , 1 h ) , \n 3.05 ( s , 3 h ) , 2.602.42 ( m , 3 h ) , 2.32 ( dt , j = 7.2 , 13.6 hz , 1 h ) , 2.242.17 ( m , 1 h ) , 2.031.95 \n ( m , 1 h ) ,", "1.841.74 ( m , 1 h ) , 1.681.54 ( m , 2 h ) , 1.491.30 \n ( m , 4 h ) , 1.26 ( d , j = 7.2 hz , 3 h ) , 1.030.93 \n ( m , 1 h ) , 0.89 ( d , j = 6.4 hz , 3 h ) , 0.86 ( s , 9 h ) ; c nmr ( 100 mhz , cdcl3 )  175.4 , 172.8 , 129.8 , \n 110.2 , 76.5 , 37.2 , 36.6 , 35.4 , 34.3 , 33.6 , 31.0 , 29.8 , 29.5 , 27.3 , \n 25.9 , 24.9 , 20.0 , 19.2 ; hresims m / z 360.2504 [ m + na ] ( calcd for c20h35no3na , 360.2509 ) .", "to a solution \n of ( )-palmyrolidea ( 0.008 g , 0.022 mmol ) in anhydrous meoh \n ( 1 ml ) was added 10% palladium on carbon ( 0.005 g ) .", "the atmosphere \n in the flask was replaced with hydrogen , and the reaction mixture \n was allowed to stir at rt for 18 h before being filtered through a \n short plug of silica .", "after rinsing several times with ether , the \n filtrate was concentrated and purified via column chromatography ( 8:2 \n hexanes / etoac ) to afford compound 5 ( 0.006 g , 77% ) as \n a mixture of rotational isomers ( 2:1 ratio ) : [ ]d23 + 35.6 ( c 0.48 , chcl3 ) ; ir ( neat , thin film )  \n 2954 , 2931 , 2871 , 1720 , 1651 , 1269 , 1250 , 1073 cm ; h nmr ( 300 mhz , cdcl3 , mixture of rotational \n isomers with mostly overlapping peaks , making the assignment of major \n and minor components difficult )  ( major isomer ) 4.82 ( d , j = 10.0 hz , 1 h ) , 3.30 ( t , j = 5.0 hz , \n 1 h ) , 2.98 ( s , 3 h ) , 2.532.65 ( m , 2 h ) , 2.212.33 ( m , \n 2 h ) , 1.211.73 ( m , 14 h ) , 1.18 ( d , j = 6.9 \n hz , 3 h ) , 0.94 ( d , j = 5.9 hz , 3 h ) , 0.86 ( s , 9 h ) , \n ( minor isomer , partial ) 4.92 ( d , j = 10.8 hz , 0.6 \n h ) , 2.89 ( s , 1.5 h ) , 0.91 ( d , j = 6.1 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , mixture of rotational isomers ) \n  175.5 , 174.8 , 173.5 , 77.8 , 77.2 , 48.9 , 46.3 , 40.4 , 40.0 , 38.3 , \n 37.2 , 35.2 , 35.0 , 33.6 , 33.2 , 32.98 , 32.96 , 31.2 , 39.7 , 27.9 , 27.6 , \n 27.0 , 26.0 , 25.9 , 25.4 , 23.5 , 23.2 , 22.2 , 20.4 , 20.1 , 18.0 , 16.5 ; \n hresims m / z 362.2660 [ m + na ] ( calcd for c20h37no3na , \n 362.2665 ) .", "the crude product was purified via column \n chromatography ( 8:2 hexanes / etoac ) to afford compound 6 ( 0.006 g , 96% ) as a mixture of rotational isomers ( 2:1 ratio ) : \n [ ]d23 + 42.1 ( c 0.5 , chcl3 ) ; ir ( neat , thin \n film )  2956 , 2930 , 2872 , 1727 , 1647 , 1459 , 1192 , 1156 cm ; h nmr ( 300 mhz , cdcl3 , mixture \n of rotational isomers with mostly overlapping peaks , making the assignment \n of major and minor components difficult )  ( major isomer ) 4.86 \n ( d , j = 10.5 hz , 1 h ) , 3.513.42 ( m , 1 h ) , \n 3.233.14 ( m , 1 hz ) , 2.88 ( s , 3 h ) , 2.312.48 ( m , 2 \n h ) , 2.182.31 ( m , 2 h ) , 1.971.79 ( m , 2 h ) , 1.231.66 \n ( m , 15 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.92 ( d , j = 5.8 hz , 3 h ) , 0.86 ( s , 11 h ) , ( minor isomer , partial ) \n 4.83 ( d , j = 11.6 hz , 0.6 h ) , 2.91 ( s , 1 h ) , 1.15 \n ( d , j = 7.2 hz , 1.5 h ) , 0.96 ( d , j = 5.7 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , \n mixture of rotational isomers )  176.8 , 176.6 , 173.3 , 173.0 , \n 77.2 , 49.1 , 46.0 , 40.6 , 40.1 , 38.0 , 37.3 , 35.2 , 34.0 , 33.9 , 33.2 , \n 33.1 , 33.0 , 31.3 , 29.7 , 28.9 , 27.2 , 25.9 , 24.8 , 23.9 , 22.8 , 19.9 , \n 19.8 , 19.2 , 19.1 ; hresims m / z 362.2659 \n [ m + na ]", "( calcd for c20h37no3na , 362.2665 ) . to a solution \n of 1 ( 0.007 g , 0.021 mmol ) in chcl3 ( 2 ml ) \n", "was added a 6 n aqueous hcl solution dropwise until tlc analysis showed \n complete consumption of the starting material .", "the reaction mixture \n was then quenched with a saturated aqueous solution of nahco3 , followed by extraction using etoac .", "the \n resultant crude aldehyde was dissolved in anhydrous meoh ( 1 ml ) and \n treated with nabh4 ( 0.015 g , 0.39 mmol ) .", "the reaction mixture \n was allowed to stir at rt for 1 h before being quenched with h2o , extracted using etoac , dried over mgso4 , filtered , \n and concentrated .", "the crude alcohol was purified by column chromatography \n ( 100% etoac ) to afford alcohol 9 ( 0.006 g , 73% over two \n steps ) as a colorless oil : [ ]d24.3 + 26.8 ( c 0.20 , chcl3 ) ; ir ( neat , thin film )  3296 , 2956 , 1728 , 1651 , 1562 , \n 1462 , 1366 , 1164 cm ; h nmr ( 300 mhz , \n cdcl3 )  6.10 ( bs , 1 h ) , 4.76 ( dd , j = 2.2 , 9.5 hz , 1 h ) , 3.63 ( t , j = 6.4 hz , 2 h ) , \n 2.79 ( d , j = 4.8 hz , 3 h ) , 2.392.51 ( m , 1 \n h ) , 2.002.23 ( m , 2 h ) , 1.91 ( bs , 1 h ) , 1.631.80 ( m , \n 2 h ) , 1.221.60 ( m , 10 h ) , 1.16 ( d , j = 7.0 \n hz , 3 h ) , 0.87 ( d , overlapped , 3 h ) , 0.86 ( s , 9 h ) ; c \n nmr ( 75 mhz , cdcl3 )  176.8 , 174.0 , 78.6 , 62.5 , 40.2 , \n 37.6 , 36.2 , 34.5 , 33.3 , 32.5 , 28.8 , 26.2 , 25.9 , 25.9 , 23.5 , 23.0 , \n 20.8 , 17.4 ; hresims m / z 380.2764 \n [ m + na ] ( calcd for c20h39no4na , 380.2771 ) . approximately 0.3 \n ml of an oxidant solution prepared via the mixing of naclo2 ( 0.060 g ) and nahpo4 ( 0.040 g ) in h2o ( 1 ml ) \n was added to a solution of palmyrolide aldehyde ( prepared as above , \n 0.006 g , 0.016 mmol ) in tert - butanol ( 0.25 ml ) and \n 2-methyl-2-butene ( 0.1 ml ) . the reaction mixture was allowed to stir \n for 30 min before being quenched with water , extracted with etoac , \n dried over mgso4 , filtered , and concentrated .", "the crude \n acid was purified by column chromatography ( 100% etoac ) to afford \n acid 10 ( 0.002 g , 25% ) as a colorless oil : [ ]d23 + 10.8 ( c 0.10 , chcl3 ) ; ir ( neat , thin film )  \n 3306 , 2960 , 1727 , 1633 , 1164 cm ; h nmr ( 300 mhz , cdcl3 )  6.04 ( bs , 1 h ) , 4.78 ( dd , j = 10.2 , 1.9 hz , 1 h ) , 2.82 ( d , j = 4.8 \n hz , 3 h ) , 2.48 ( q , j = 6.9 hz , 1 h ) , 2.312.41 \n ( m , 2 h ) , 2.022.23 ( m , 3 h ) , 1.601.82 ( m , 4 h ) , 1.211.59 \n ( m , 6 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.921.05 \n ( m , 1 h ) , 0.88 ( d , overlapped , 3 h ) , 0.87 ( s , 9 h ) ; c \n nmr ( 75 mhz , cdcl3 )  176.4 , 78.5 , 40.1 , 37.6 , 36.2 , \n 34.6 , 34.4 , 33.0 , 29.7 , 28.7 , 26.3 , 26.0 , 23.0 , 22.7 , 20.7 , 17.2 ; \n hresims m / z 394.2558 [ m + na ] ( calcd for c20h37no5na , \n 394.2563 ) .", "the crude product was purified via column chromatography \n ( 100% etoac ) to afford compound 12 ( 0.004 g , 94% ) : [ ]d25.5 + 30.8 ( c 0.24 , chcl3 ) ; ir ( neat , thin film )  \n 3304 , 2950 , 1729 , 1649 , 1560 , 1462 , 1366 , 1163 , 1074 cm ; h nmr ( 300 mhz , cdcl3 )  6.25 ( bs , \n 1 h ) , 4.76 ( dd , j = 9.9 , 1.6 hz , 1 h ) , 3.553.70 \n ( m , 2 h ) , 2.77 ( d , j = 4.8 hz , 3h ) , 2.372.50 \n ( m , 1 h ) , 2.27 ( bs , 1 h ) , 2.002.20 ( m , 2 h ) , 1.631.83 \n ( m , 2 h ) , 1.211.60 ( m , 10 h ) , 1.16 ( d , j = \n 7.0 hz , 3 h ) , 0.921.04 ( m , 1 h ) , 0.86 ( d , overlapped , 3 h ) , \n 0.85 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )  \n 177.0 , 174.1 , 78.4 , 62.2 , 40.0 , 37.5 , 36.2 , 34.6 , 34.5 , 33.1 , 32.4 , \n 28.8 , 26.1 , 25.9 , 23.6 , 23.1 , 20.7 , 17.7 ; hresims m / z 380.2764 [ m + na ] ( calcd for c20h39no4na , 380.2771 ) .", "primary cultures of \n cerebrocortical neurons were obtained from embryonic day 17 swiss - webster \n mice as described elsewhere .", "briefly , \n pregnant mice were euthanized by co2 asphyxiation , and \n embryos were removed under sterile conditions .", "cerebral cortices were \n collected , stripped of meninges , minced by trituration with a pasteur \n pipet , and treated with trypsin for 25 min at 37 c .", "the cells \n were then dissociated by two successive trituration and sedimentation \n steps in soybean trypsin inhibitor and dnase - containing isolation \n buffer , centrifuged , and resuspended in eagle s minimal essential \n medium with earle s salt ( mem ) supplemented with 1 mm l - glutamine , 10% fetal bovine serum , 10% horse serum , 100 iu / ml penicillin , \n and 0.10 mg / ml streptomycin ( ph 7.4 ) .", "cells were plated onto poly - l - lysine - coated 96-well ( 9 mm ) , clear - bottomed , black - well culture \n plates ( midsci ) at a density of 1.5  10 cells / well . \n", "cells were then incubated at 37 c in a 5% co2 and \n 95% humidity atmosphere .", "the culture media was changed every other \n day , starting from day 5 in vitro using a serum - free \n growth medium containing neurobasal medium supplemented with b-27 , \n 100 iu / ml penicillin , 0.10 mg / ml streptomycin , and 0.2 mm l - glutamine .", "all animal use protocols were \n approved by the institutional animal care and use committee ( iacuc ) \n of creighton university .", "[ na]i measurement and full in situ calibration of sodium - binding benzofuran isophthalate fluorescence \n ratio were performed as described previously .", "cells were grown in 96-well plates and washed four times with locke s \n buffer ( 8.6 mm hepes , 5.6 mm kcl , 154 mm nacl , 5.6 mm glucose , 1.0 \n mm mgcl2 , 2.3 mm cacl2 , 0.1 mm glycine , ph 7.4 ) \n using an automated microplate washer ( biotek instruments ) . after measuring \n the background fluorescence of each well , cells were incubated for \n 1 h at 37 c with dye - loading buffer ( 100 l / well ) containing \n 10 m sbfi - am ( invitrogen ) and 0.02% pluronic f-127 ( invitrogen ) . \n", "cells were washed five times with locke s buffer , leaving a \n final volume of 100 l in each well .", "the plate was then transferred \n back to the incubator for 15 min to allow the cells to equilibrate \n after washing and placed in a flexstation ii ( molecular devices ) chamber \n to detect na - bound sbfi emission at 505 nm ( cells were \n excited at 340 and 380 nm ) .", "fluorescence readings were taken once \n every 5 s for 60 s to establish the baseline , and then 50 l \n of different concentrations of ( )-palmyrolide a and/or veratridine \n was added to each well from the compound plate at a rate of 26 l / s , \n yielding a final volume of 200 l / well . after correcting for \n background fluorescence , sbfi fluorescence ratios ( 340/380 ) and concentration ", "each palmyrolide a analogue was dissolved in \n dmso at a stock concentration of 50 mm and stored at 20 c \n until used . on the day of an experiment ,", "this dilution in locke s \n buffer resulted in palmyrolide a analogue solutions that were 4 times \n the desired final concentration .", "( )-palmyrolide \n a ( 1 ) and analogues ( 2 , 7 , \n and 8) were dissolved in cdcl3 at a concentration \n of 2.5 mg in 50 l and transferred to a 1.7 mm nmr tube . \n", "the noesy mixing time was 500 ms , the hmbc delay was set to 6.25 ms \n ( 8 hz ) , and the hetloc used a dispi-2 spinlock of 60 ms duration . \n", "chemical shift assignments were based on analysis of the dqf - cosy , \n hsqc , and hmbc 2d experiments using ucsf sparky .", "topspin ( bruker biospin ) was used to analyze coupling constants \n in the h nmr and hetloc spectra .", "noesy peak heights were \n converted to upper distance restraints of 3 , 4 , 5 , or 6  using \n in - house perl scripts . where restraints involved a group of atoms , \n pseudoatom corrections were applied by the amber restraint generation \n tools .", "force field parameters were estimated using \n the gaff force field and the antechamber \n program in the amber 14 package .", "molecular \n dynamics simulations were nonperiodic , vacuum simulations with a distance - dependent \n dielectric term .", "using sander in the amber 14 package each starting \n structure was energy minimized , then subjected to 20 ps of simulated \n annealing with the nmr - derived restraints in which the temperature \n was raised from 10 k to 600 k then cooled to near 0 k. the simulated \n annealing was run 20 times for each structure with a different random \n number seed each time .", "each collection of 20 structures was sorted \n by restraint violation energy , and outliers were discarded . to align \n structures ,", "the selected ensemble was superimposed on c17 , \n o12 , c1318 , and n19 using cpptraj . to stereospecifically assign pairs of diastereotopic hydrogens , \n pairs of noes that involved the diastereotopic atoms and had differential \n intensities", "were identified , then compared with the distances between \n these atoms in the ensemble of selected structures .", "if the ensemble \n mean of one distance was more than one standard deviation from the \n alternative , then the diastereotopic hydrogens were stereospecifically \n assigned ."]]}
{"article_id": "PMC2955242", "article_text": ["the online version of this article ( doi:10.1007/s00414 - 009 - 0352 - 9 ) contains supplementary material , which is available to authorized users .", "microbes can negatively interfere with the postmortem assessment of alcohol abuse and in this way pose problems for forensic investigators .", "however , microbial forensics is often chiefly associated with the detection of highly pathogenic microbes to which humans are deliberately exposed in cases of biological terrorism [ 2 , 3 ] . however , human fingertip microflora left behind on touched objects at crime scenes may potentially contain forensically relevant information that may be useful for human host inferences accessible via microbial dna fingerprinting of physical fingerprints .", "for example , if endogenous microbial skin species / strains with a geographically restricted distribution could be retrieved from touched objects via microbial dna analysis , the geographic origin of the human host individual could be determined indirectly .", "information about the geographic region of origin can be relevant in suspect - less forensic cases where the evidence dna sample does not match either a suspect s dna profile or any in a criminal dna database .", "in such cases , geographic information derived from crime scene samples is expected to reduce the potential pool of suspects by allowing police investigations to concentrate on specific groups of people , i.e. , those from a restricted geographic region .", "numerous human genetic markers have been suggested for inferring human genetic ancestry mostly to the continental level [ 47 ] , and a recent study indicated that inferring the subregion of origin of an unknown european may be feasible from autosomal genetic data .", "however , direct ancestry inference based on human genetic markers is currently far from perfect , initiating the question whether microbial dna may be used to supplement human dna markers in reliable ancestry reconstruction of unknown persons .", "recently , it has been shown that the gastric pathogenic bacteria helicobacter pylori has intimately coevolved with its human host [ 9 , 10 ] .", "although this example may be of limited direct relevance for forensics , because samples containing h. pylori are usually not found at crime scenes ( with the exception of bodies in cases of missing persons ) , it shows that in principle human geographic signatures are inferable from microbial genomes .", "the human skin is a complex microbial ecosystem consisting of multiple niches , which can differ drastically from each other .", "interactions between skin microbes and the human host , as well as between the microbial occupants , are still poorly understood .", "the current knowledge on skin microbiota primarily derives from cultivation - based studies [ 12 , 13 ] , although molecular fingerprinting techniques have been employed more recently [ 14 , 15 ] .", "if a comparable relationship exists between humans and their skin microbiota , as has been observed for h. pylori , new methods for human geographic origin determination could be developed based on dna analysis of fingertip microflora , with interesting new applications to molecular analyses of physical fingerprints left at crime scenes .", "volunteers and microbiological procedures five dutch european subjects from rotterdam and three volunteers from dhaka , bangladesh , placed their right index finger on a colombia iii blood agar plate ( biomerieux , marcy - letoile , france ) .", "fingerprint sampling for dutch volunteers was performed in rotterdam whereas that of bangladeshi volunteers was performed in dhaka . after washing this finger with hospital soap and air drying , another fingerprint , from the same finger ,", "was placed next to the first . whereas the first fingerprint was expected to provide a mixture of transient and resident microbiota", ", the second one was assumed to provide resident microbiota only as soap removes the upper dead - skin layers including their transient microflora .", "after overnight incubation at 37c , colonies with different morphologies were isolated , with 20 per fingerprint ( two each ) from the five dutch individuals ( n  =  200 strains ) .", "three weeks later , this procedure was repeated for assessment of bacterial persistence in the dutch volunteers .", "agar plates from bangladeshi volunteers were shipped by courier to rotterdam after overnight incubation at 37c for further analyses . given the diversity in genotypes encountered in the time - wise resampling experiments using dutch volunteers ( see below ) , this procedure was not repeated for the volunteers from bangladesh .", "gram staining and the determination of catalase activity were applied for the identification of staphylococci using commercially available test kits ( biomrieux , marcy - letoile , france ) .", "staphylococcus epidermidis sensu stricto was identified among the isolates by a species - specific polymerase chain reaction assay as described elsewhere .", "a coagulase test with slidex staph plus ( biomrieux , marcy - letoile , france ) was performed to detect staphylococcus aureus isolates .", "were classified with the i d 32 staph api system ( biomrieux , marcy - letoile , france ) .", "genotyping bacterial isolates in order to generate dna fingerprints for the most relevant bacterial isolates , all staphylococcal isolates were subjected to pulsed - field gel electrophoresis ( pfge ) .", "pfge can be used to electrophoretically separate dna restriction fragments ranging in length between 50 and 1,000  kb .", "after lysostaphin and proteinase treatment and smai macrorestriction , pfge was performed in a 1% incert agarose ( fmc ) gel in 0.5 tbe at 14c , using a constant electric field of 6  v cm with pulse ramping from 0.1 to 30  s at a 60/60 angle for 18  h. pfge patterns were analyzed for levels of similarity with bionumerics 4.5 ( applied - maths , sint martens latem , belgium ) .", "developing forensic dna markers for human host inferences from physical fingerprints relies on the examination of the transient exogenous microflora left behind together with the physical fingerprint .", "however , any human host inference from fingertip microflora using physical fingerprints would require that the transient microflora is representative of the resident endogenous microflora of the same individual and is not simply reflecting microbial species / strains picked up from the environment . to test this prerequisite , we investigated and compared by means of dna fingerprinting techniques the transient superficial skin microbiota and the resident endogenous microflora of human fingertips within several individuals .", "in addition , we collected data at two time points to investigate time - wise microflora persistency within individuals . to this end , we used samples from five dutch volunteers and gram staining as well as catalase activity testing revealed that all bacterial skin isolates that were selected were gram - positive cocci .", "most isolates were staphylococcus spp . , a small number of colonies represented micrococci ( ~5% of total isolates ) , and the vast majority ( 97% ) of staphylococci were coagulase negative .", "healthy skin is known to harbor gram - positive pleomorphic bacilli , including corynebacterium , brevibacteria , and propionibacteria sp . , but these were not detected here .", "pfge is the most discriminatory typing method that is currently available for typing of staphylococci .", "transient microflora ( tm ) was collected from physical fingerprints before washing with soap and endogenous microflora ( rm ) after soap washing ( see  materials and methods ", "the tm showed higher numbers of pfge types than those seen in the rm in three of the five dutch subjects ( table  1 ) .", "this can be explained by nonendogenous species picked up from the environment and being transferred .", "significant variability between the subjects was seen for both the tm and rm , as has been previously reported for other skin areas [ 10 , 13 ] .", "notably , the rm types encountered after washing were very similar intraindividually but very different interindividually .", "this suggests that certain types of bacteria are able to efficiently and persistently colonize the skin of a given individual . within the rm , all dutch subjects had one or two dominant types , mostly staphylococcus warneri species .", "these types were also present within the tm of three of the five subjects , while s. epidermidis was present at various time points in all of the subjects tm . only one subject ( subject 1 ) showed the major rm type as the major tm type . in several subjects , the dominant rm type did not appear at all in the tm , although there was considerable overlap in nondominant species between the tm and rm in many subjects . in general , pfge types within the rm and within the tm for the same subject appeared consistent over time .", "although the degree of variability changed in the 3-week period for every subject , in some subjects , it changed more drastically than in others . at time point 1 , overlapping pfge types were seen in four of the five subjects in the tm and rm .", "however , by time point 2 , only one subject ( subject 4 ) showed any overlap .", "in one subject ( subject 4 ) , the major rm type did not occur in the tm of the same sampling time point 2 but was present in considerable frequency in the tm of the earlier time point 1 .", "thus , although we see some resemblance between rm and tm as well as some time - wise consistency , the pattern detected is far from perfect . by viewing our results only qualitatively ( ignoring type frequencies ) ,", "rm signals were detectable from the tm analyses in only 50% of the dutch experiments ( combining individuals and time points ) , which appears discouraging as basis for developing molecular markers for forensic applications to infer individual host information . \n", "table  1staphylococcus species and pfge types from dutch fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) staphylococcus species and pfge types from dutch fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) we chose geographic inference as a test example for the type of human host information that would be interesting to retrieve from physical fingerprints by means of microbial dna analysis for forensic applications .", "a prerequisite for developing microbial skin markers for inferring human geographic origins would be that the endogenous fingertip microflora of humans from different parts of the world shows differences in their genetic diversity and that retrievable microbial genotypes cluster according to geographic origin of the host individuals . to test this , we obtained fingerprint microflora from native inhabitants of bangladesh collected in dhaka , bangladesh , and compared their microbiological profiles with those of the dutch volunteers .", "fingertip microflora of three bangladeshi volunteers were isolated and processed in the same manner as for the dutch volunteers , although only at one time point ( three individuals , two fingerprints each and 20 bacterial isolates per fingerprint , hence 3    2    20  =  120 strains ; table  2 ) . correspondence between rm and tm", "was observed in two of the three subjects for one pfge type , although the persistent pfge type found differed between the subjects .", "notably , subject 3 from bangladesh showed an unexpectedly higher variety of staphylococci in the rm compared to tm . comparing pfge types between dutch and bangladeshi subjects revealed some differences , e.g. , bangladeshi skin microbiota contained less s. warneri and much less s. epidermidis types compared with the dutch ones .", "were found among the tm in bangladeshi ( 45% ) than among the dutch ( 7% ) .", "furthermore , none of the pfge types from the bangladesh fingerprints were seen in the dutch collection , indicating extensive geographic heterogeneity within the staphylococcus species . however , when each pfge type obtained during this study was subjected to dice - based cluster analysis for visualizing geographic structure in the skin staphylococci ( see supplementary material fig .  1 ) , the bangladeshi pfge patterns efficiently mixed with the dutch ones . hence , information about geographic origin of the dutch and bangladeshi donors was not obvious among genotype data of the staphylococci inhabiting the human fingertip skin .", "although in general a limited number of five and three persons tested would not allow for cluster analysis , the full absence of clusters as identified in the present study already shows that individual geographic inferences for forensics purposes is not possible using the approach employed here . \n", "table  2staphylococcus species and pfge types from bangladeshi fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) staphylococcus species and pfge types from bangladeshi fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) to conclude , we see only limited geographic differentiation between microbial dna fingerprints from dutch europeans and bangladeshi south asians , indicating that geographic inferences of human hosts from fingertip microbial dna analysis is not feasible .", "furthermore , our results from dna profiling of transient and resident fingertip microflora show that human fingertip microflora is too dynamic and thus does not fulfill the criteria required for forensic marker developments to infer any human host information from physical fingerprints ."], "abstract_text": ["<S> human fingertip microflora is transferred to touched objects and may provide forensically relevant information on individual hosts , such as on geographic origins , if endogenous microbial skin species / strains would be retrievable from physical fingerprints and would carry geographically restricted dna diversity . </S>", "<S> we tested the suitability of physical fingerprints for revealing human host information , with geographic inference as example , via microbial dna fingerprinting . </S>", "<S> we showed that the transient exogenous fingertip microflora is frequently different from the resident endogenous bacteria of the same individuals . in only 54% of the experiments , the dna analysis of the transient fingertip microflora allowed the detection of defined , but often not the major , elements of the resident microflora . </S>", "<S> although we found microbial persistency in certain individuals , time - wise variation of transient and resident microflora within individuals was also observed when resampling fingerprints after 3  weeks . while microbial species differed considerably in their frequency spectrum between fingerprint samples from volunteers in europe and southern asia </S>", "<S> , there was no clear geographic distinction between staphylococcus strains in a cluster analysis , although bacterial genotypes did not overlap between both continental regions . </S>", "<S> our results , though limited in quantity , clearly demonstrate that the dynamic fingerprint microflora challenges human host inferences for forensic purposes including geographic ones . </S>", "<S> overall , our results suggest that human fingerprint microflora is too dynamic to allow for forensic marker developments for retrieving human information.electronic supplementary materialthe online version of this article ( doi:10.1007/s00414 - 009 - 0352 - 9 ) contains supplementary material , which is available to authorized users . </S>"], "labels": null, "section_names": ["Electronic supplementary material", "Introduction", "Material and methods", "Results and discussion", "Electronic supplementary material"], "sections": [["the online version of this article ( doi:10.1007/s00414 - 009 - 0352 - 9 ) contains supplementary material , which is available to authorized users ."], ["microbes can negatively interfere with the postmortem assessment of alcohol abuse and in this way pose problems for forensic investigators .", "however , microbial forensics is often chiefly associated with the detection of highly pathogenic microbes to which humans are deliberately exposed in cases of biological terrorism [ 2 , 3 ] . however , human fingertip microflora left behind on touched objects at crime scenes may potentially contain forensically relevant information that may be useful for human host inferences accessible via microbial dna fingerprinting of physical fingerprints .", "for example , if endogenous microbial skin species / strains with a geographically restricted distribution could be retrieved from touched objects via microbial dna analysis , the geographic origin of the human host individual could be determined indirectly .", "information about the geographic region of origin can be relevant in suspect - less forensic cases where the evidence dna sample does not match either a suspect s dna profile or any in a criminal dna database .", "in such cases , geographic information derived from crime scene samples is expected to reduce the potential pool of suspects by allowing police investigations to concentrate on specific groups of people , i.e. , those from a restricted geographic region .", "numerous human genetic markers have been suggested for inferring human genetic ancestry mostly to the continental level [ 47 ] , and a recent study indicated that inferring the subregion of origin of an unknown european may be feasible from autosomal genetic data .", "however , direct ancestry inference based on human genetic markers is currently far from perfect , initiating the question whether microbial dna may be used to supplement human dna markers in reliable ancestry reconstruction of unknown persons .", "recently , it has been shown that the gastric pathogenic bacteria helicobacter pylori has intimately coevolved with its human host [ 9 , 10 ] .", "although this example may be of limited direct relevance for forensics , because samples containing h. pylori are usually not found at crime scenes ( with the exception of bodies in cases of missing persons ) , it shows that in principle human geographic signatures are inferable from microbial genomes .", "the human skin is a complex microbial ecosystem consisting of multiple niches , which can differ drastically from each other .", "interactions between skin microbes and the human host , as well as between the microbial occupants , are still poorly understood .", "the current knowledge on skin microbiota primarily derives from cultivation - based studies [ 12 , 13 ] , although molecular fingerprinting techniques have been employed more recently [ 14 , 15 ] .", "if a comparable relationship exists between humans and their skin microbiota , as has been observed for h. pylori , new methods for human geographic origin determination could be developed based on dna analysis of fingertip microflora , with interesting new applications to molecular analyses of physical fingerprints left at crime scenes ."], ["volunteers and microbiological procedures five dutch european subjects from rotterdam and three volunteers from dhaka , bangladesh , placed their right index finger on a colombia iii blood agar plate ( biomerieux , marcy - letoile , france ) .", "fingerprint sampling for dutch volunteers was performed in rotterdam whereas that of bangladeshi volunteers was performed in dhaka . after washing this finger with hospital soap and air drying , another fingerprint , from the same finger ,", "was placed next to the first . whereas the first fingerprint was expected to provide a mixture of transient and resident microbiota", ", the second one was assumed to provide resident microbiota only as soap removes the upper dead - skin layers including their transient microflora .", "after overnight incubation at 37c , colonies with different morphologies were isolated , with 20 per fingerprint ( two each ) from the five dutch individuals ( n  =  200 strains ) .", "three weeks later , this procedure was repeated for assessment of bacterial persistence in the dutch volunteers .", "agar plates from bangladeshi volunteers were shipped by courier to rotterdam after overnight incubation at 37c for further analyses . given the diversity in genotypes encountered in the time - wise resampling experiments using dutch volunteers ( see below ) , this procedure was not repeated for the volunteers from bangladesh .", "gram staining and the determination of catalase activity were applied for the identification of staphylococci using commercially available test kits ( biomrieux , marcy - letoile , france ) .", "staphylococcus epidermidis sensu stricto was identified among the isolates by a species - specific polymerase chain reaction assay as described elsewhere .", "a coagulase test with slidex staph plus ( biomrieux , marcy - letoile , france ) was performed to detect staphylococcus aureus isolates .", "were classified with the i d 32 staph api system ( biomrieux , marcy - letoile , france ) .", "genotyping bacterial isolates in order to generate dna fingerprints for the most relevant bacterial isolates , all staphylococcal isolates were subjected to pulsed - field gel electrophoresis ( pfge ) .", "pfge can be used to electrophoretically separate dna restriction fragments ranging in length between 50 and 1,000  kb .", "after lysostaphin and proteinase treatment and smai macrorestriction , pfge was performed in a 1% incert agarose ( fmc ) gel in 0.5 tbe at 14c , using a constant electric field of 6  v cm with pulse ramping from 0.1 to 30  s at a 60/60 angle for 18  h. pfge patterns were analyzed for levels of similarity with bionumerics 4.5 ( applied - maths , sint martens latem , belgium ) ."], ["developing forensic dna markers for human host inferences from physical fingerprints relies on the examination of the transient exogenous microflora left behind together with the physical fingerprint .", "however , any human host inference from fingertip microflora using physical fingerprints would require that the transient microflora is representative of the resident endogenous microflora of the same individual and is not simply reflecting microbial species / strains picked up from the environment . to test this prerequisite , we investigated and compared by means of dna fingerprinting techniques the transient superficial skin microbiota and the resident endogenous microflora of human fingertips within several individuals .", "in addition , we collected data at two time points to investigate time - wise microflora persistency within individuals . to this end , we used samples from five dutch volunteers and gram staining as well as catalase activity testing revealed that all bacterial skin isolates that were selected were gram - positive cocci .", "most isolates were staphylococcus spp . , a small number of colonies represented micrococci ( ~5% of total isolates ) , and the vast majority ( 97% ) of staphylococci were coagulase negative .", "healthy skin is known to harbor gram - positive pleomorphic bacilli , including corynebacterium , brevibacteria , and propionibacteria sp . , but these were not detected here .", "pfge is the most discriminatory typing method that is currently available for typing of staphylococci .", "transient microflora ( tm ) was collected from physical fingerprints before washing with soap and endogenous microflora ( rm ) after soap washing ( see  materials and methods ", "the tm showed higher numbers of pfge types than those seen in the rm in three of the five dutch subjects ( table  1 ) .", "this can be explained by nonendogenous species picked up from the environment and being transferred .", "significant variability between the subjects was seen for both the tm and rm , as has been previously reported for other skin areas [ 10 , 13 ] .", "notably , the rm types encountered after washing were very similar intraindividually but very different interindividually .", "this suggests that certain types of bacteria are able to efficiently and persistently colonize the skin of a given individual . within the rm , all dutch subjects had one or two dominant types , mostly staphylococcus warneri species .", "these types were also present within the tm of three of the five subjects , while s. epidermidis was present at various time points in all of the subjects tm . only one subject ( subject 1 ) showed the major rm type as the major tm type . in several subjects , the dominant rm type did not appear at all in the tm , although there was considerable overlap in nondominant species between the tm and rm in many subjects . in general , pfge types within the rm and within the tm for the same subject appeared consistent over time .", "although the degree of variability changed in the 3-week period for every subject , in some subjects , it changed more drastically than in others . at time point 1 , overlapping pfge types were seen in four of the five subjects in the tm and rm .", "however , by time point 2 , only one subject ( subject 4 ) showed any overlap .", "in one subject ( subject 4 ) , the major rm type did not occur in the tm of the same sampling time point 2 but was present in considerable frequency in the tm of the earlier time point 1 .", "thus , although we see some resemblance between rm and tm as well as some time - wise consistency , the pattern detected is far from perfect . by viewing our results only qualitatively ( ignoring type frequencies ) ,", "rm signals were detectable from the tm analyses in only 50% of the dutch experiments ( combining individuals and time points ) , which appears discouraging as basis for developing molecular markers for forensic applications to infer individual host information . \n", "table  1staphylococcus species and pfge types from dutch fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) staphylococcus species and pfge types from dutch fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) we chose geographic inference as a test example for the type of human host information that would be interesting to retrieve from physical fingerprints by means of microbial dna analysis for forensic applications .", "a prerequisite for developing microbial skin markers for inferring human geographic origins would be that the endogenous fingertip microflora of humans from different parts of the world shows differences in their genetic diversity and that retrievable microbial genotypes cluster according to geographic origin of the host individuals . to test this , we obtained fingerprint microflora from native inhabitants of bangladesh collected in dhaka , bangladesh , and compared their microbiological profiles with those of the dutch volunteers .", "fingertip microflora of three bangladeshi volunteers were isolated and processed in the same manner as for the dutch volunteers , although only at one time point ( three individuals , two fingerprints each and 20 bacterial isolates per fingerprint , hence 3    2    20  =  120 strains ; table  2 ) . correspondence between rm and tm", "was observed in two of the three subjects for one pfge type , although the persistent pfge type found differed between the subjects .", "notably , subject 3 from bangladesh showed an unexpectedly higher variety of staphylococci in the rm compared to tm . comparing pfge types between dutch and bangladeshi subjects revealed some differences , e.g. , bangladeshi skin microbiota contained less s. warneri and much less s. epidermidis types compared with the dutch ones .", "were found among the tm in bangladeshi ( 45% ) than among the dutch ( 7% ) .", "furthermore , none of the pfge types from the bangladesh fingerprints were seen in the dutch collection , indicating extensive geographic heterogeneity within the staphylococcus species . however , when each pfge type obtained during this study was subjected to dice - based cluster analysis for visualizing geographic structure in the skin staphylococci ( see supplementary material fig .  1 ) , the bangladeshi pfge patterns efficiently mixed with the dutch ones . hence , information about geographic origin of the dutch and bangladeshi donors was not obvious among genotype data of the staphylococci inhabiting the human fingertip skin .", "although in general a limited number of five and three persons tested would not allow for cluster analysis , the full absence of clusters as identified in the present study already shows that individual geographic inferences for forensics purposes is not possible using the approach employed here . \n", "table  2staphylococcus species and pfge types from bangladeshi fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) staphylococcus species and pfge types from bangladeshi fingerprints before washing with soap representing elements of the transient ( tm ) microflora and after washing representing resident fingertip microflora ( rm ) to conclude , we see only limited geographic differentiation between microbial dna fingerprints from dutch europeans and bangladeshi south asians , indicating that geographic inferences of human hosts from fingertip microbial dna analysis is not feasible .", "furthermore , our results from dna profiling of transient and resident fingertip microflora show that human fingertip microflora is too dynamic and thus does not fulfill the criteria required for forensic marker developments to infer any human host information from physical fingerprints ."], [""]]}
{"article_id": "PMC3706931", "article_text": ["assisted / supported mechanical ventilation is designed to interact with patient muscle activity and ' share ' the work of breathing . if properly done , assisted /", ", the ventilatory 's flow and pressure delivery must synchronize with patient effort during all three phases of breath delivery : breath initiation , flow delivery , and breath termination ( cycling ) .", "dys - synchronous interactions can overload ventilatory muscles ( ' imposed ' loads ) , compromise alveolar ventilation , overdistend alveolar units , disrupt sleep patterns , and cause patient discomfort , prompting additional sedation .", "carlucci and colleagues   have recently studied patient - ventilator interactions in 69 acutely ill patients receiving non - invasive ventilation ( niv ) for a variety of reasons .", "they grouped these patients according to obstructive or restrictive pathophysiologies and carefully measured respiratory system mechanics ( including esophageal pressure - a surrogate for pleural pressure ) .", "the patients were then initiated on niv according to a standard protocol and were assessed for triggering dys - synchronies defined as missed triggers , delayed triggers , or double triggers .", "importantly , they found that these dys - synchronies were common ( 58% of patients experienced them with missed triggers being the most frequent ) .", "trigger dys - synchronies were associated with niv intolerance and their prevalence was similar among the different patho - physiologic patterns .", "this study is important as it underscores that patient - ventilator trigger dys - synchrony is common   and can be a major barrier to niv effectiveness .", "this study also underscores the fact that the causes of trigger dys - synchrony are complex and affect all types of patients requiring assisted / supported mechanical ventilation [ 3 - 5 ] . like invasive mechanical ventilation , niv trigger dys - synchrony can stem from insensitive / poorly responsive breath triggering mechanisms . however , other factors also involved likely include ventilatory muscle loading patterns and their effects on the ventilatory controller , ventilatory muscle function , lung volumes at end - expiration ( that is , intrinsic positive end - expiratory pressure ( peep ) ) , gas exchange effectiveness , and cortical inputs to the patient 's ventilatory control center such as pain [ 3 - 5 ] . in addition , the triggering process with niv can be further hampered by the mask system with its inherent leaks and claustrophobia exaggerating dyspnea and neural drive .", "importantly , this study did not address issues of flow synchrony or cycle synchrony , two other forms of patient ventilator interactions likely to impact niv tolerance but not well studied in the current literature .", "flow synchrony is defined as the ability of the ventilator to provide flow in accordance with patient demand as manifest by the ventilatory muscle contraction pattern .", "if flow is synchronous with that contraction pattern , the inspiratory muscle pressure time profile conceptually should resemble a near normal pattern .", "instead it means providing flow to ' re - shape ' the inspiratory muscle 's pressure - time or pressure - volume profile to a more physiologic shape . in general", ", the variable flow features of pressure targeted breaths tend to flow synchronize easier with patient effort than clinician set flow - volume targeted breaths .", "an assisted / supported mechanical breath termination shorter than the neural inspiratory time ( machine ti < neural ti ) can lead to muscle activity beyond the machine 's flow delivery phase , which can lead to high muscle loading , excessive tidal volumes and/or triggering of a second breath .", "in contrast , when mechanical breath cycling terminates after the inspiratory effort has ended ( machine ti > neural ti ) , dyspnea and expiratory muscle recruitment may occur in an effort to terminate the breath .", "importantly , it is worth noting that dys - synchronous interactions often result in anxiety and dyspnea , which can stimulate overall ventilatory drive .", "thus , improving synchrony in one area ( for example , triggering ) can help facilitate achieving synchrony in other areas ( for example , flow demand ) . in summary ,", "patient - ventilator interactions are complex and are important to recognize and manage during both invasive and non - invasive mechanical ventilation .", "carlucci and colleagues have demonstrated how common the trigger dys - synchronies are during niv and that understanding them requires assessment not only of respiratory system mechanics , but also the complex interactions of neural drive , gas exchange , muscle function , and muscle loading .", "much research is needed in this area as dys - synchronies of all types can lead to overloaded muscles and dyspneic patients requiring sedation - two factors that clearly can lead to prolonged mechanical ventilation days and poor outcomes ."], "abstract_text": ["<S> patient - ventilator trigger dys - synchronies are common with the use of assisted forms of mechanical ventilatory support , including non - invasive mechanical ventilatory support ( niv ) . </S>", "<S> future system designs need to address this in order to improve the effectiveness of niv . </S>"], "labels": null, "section_names": ["None", "Abbreviations", "Competing interests"], "sections": [["assisted / supported mechanical ventilation is designed to interact with patient muscle activity and ' share ' the work of breathing . if properly done , assisted /", ", the ventilatory 's flow and pressure delivery must synchronize with patient effort during all three phases of breath delivery : breath initiation , flow delivery , and breath termination ( cycling ) .", "dys - synchronous interactions can overload ventilatory muscles ( ' imposed ' loads ) , compromise alveolar ventilation , overdistend alveolar units , disrupt sleep patterns , and cause patient discomfort , prompting additional sedation .", "carlucci and colleagues   have recently studied patient - ventilator interactions in 69 acutely ill patients receiving non - invasive ventilation ( niv ) for a variety of reasons .", "they grouped these patients according to obstructive or restrictive pathophysiologies and carefully measured respiratory system mechanics ( including esophageal pressure - a surrogate for pleural pressure ) .", "the patients were then initiated on niv according to a standard protocol and were assessed for triggering dys - synchronies defined as missed triggers , delayed triggers , or double triggers .", "importantly , they found that these dys - synchronies were common ( 58% of patients experienced them with missed triggers being the most frequent ) .", "trigger dys - synchronies were associated with niv intolerance and their prevalence was similar among the different patho - physiologic patterns .", "this study is important as it underscores that patient - ventilator trigger dys - synchrony is common   and can be a major barrier to niv effectiveness .", "this study also underscores the fact that the causes of trigger dys - synchrony are complex and affect all types of patients requiring assisted / supported mechanical ventilation [ 3 - 5 ] . like invasive mechanical ventilation , niv trigger dys - synchrony can stem from insensitive / poorly responsive breath triggering mechanisms . however , other factors also involved likely include ventilatory muscle loading patterns and their effects on the ventilatory controller , ventilatory muscle function , lung volumes at end - expiration ( that is , intrinsic positive end - expiratory pressure ( peep ) ) , gas exchange effectiveness , and cortical inputs to the patient 's ventilatory control center such as pain [ 3 - 5 ] . in addition , the triggering process with niv can be further hampered by the mask system with its inherent leaks and claustrophobia exaggerating dyspnea and neural drive .", "importantly , this study did not address issues of flow synchrony or cycle synchrony , two other forms of patient ventilator interactions likely to impact niv tolerance but not well studied in the current literature .", "flow synchrony is defined as the ability of the ventilator to provide flow in accordance with patient demand as manifest by the ventilatory muscle contraction pattern .", "if flow is synchronous with that contraction pattern , the inspiratory muscle pressure time profile conceptually should resemble a near normal pattern .", "instead it means providing flow to ' re - shape ' the inspiratory muscle 's pressure - time or pressure - volume profile to a more physiologic shape . in general", ", the variable flow features of pressure targeted breaths tend to flow synchronize easier with patient effort than clinician set flow - volume targeted breaths .", "an assisted / supported mechanical breath termination shorter than the neural inspiratory time ( machine ti < neural ti ) can lead to muscle activity beyond the machine 's flow delivery phase , which can lead to high muscle loading , excessive tidal volumes and/or triggering of a second breath .", "in contrast , when mechanical breath cycling terminates after the inspiratory effort has ended ( machine ti > neural ti ) , dyspnea and expiratory muscle recruitment may occur in an effort to terminate the breath .", "importantly , it is worth noting that dys - synchronous interactions often result in anxiety and dyspnea , which can stimulate overall ventilatory drive .", "thus , improving synchrony in one area ( for example , triggering ) can help facilitate achieving synchrony in other areas ( for example , flow demand ) . in summary ,", "patient - ventilator interactions are complex and are important to recognize and manage during both invasive and non - invasive mechanical ventilation .", "carlucci and colleagues have demonstrated how common the trigger dys - synchronies are during niv and that understanding them requires assessment not only of respiratory system mechanics , but also the complex interactions of neural drive , gas exchange , muscle function , and muscle loading .", "much research is needed in this area as dys - synchronies of all types can lead to overloaded muscles and dyspneic patients requiring sedation - two factors that clearly can lead to prolonged mechanical ventilation days and poor outcomes ."], [""], [""]]}
{"article_id": "PMC3649235", "article_text": ["postanginal septicaemia in the early 20 century ; originally associated with a mortality reaching 90% it involves a classic triad of oropahryngeal sepsis , internal jugular vein ( ijv ) thrombophlebitis and metastatic abscess formation ( 1 ) . ", "clinical findings can therefore vary considerably from neck pain and swelling associated with thrombophlebitis to pleuritic chest pain , haemoptysis , dyspnoea or arthralgia as a result of septic thromboemboli .", "we report a case of predominantly external jugular vein ( ejv ) involvement in an adolescent male with negative blood culture results .", "a twelve year old boy presented to the children s assessment unit with a 24 hour history of unilateral face and neck swelling on a background of seven days progressive sore throat and dysphagia .", "minor systemic upset was also noted including headache , vomiting and non specific abdominal pain .", "a provisional diagnosis of tonsillitis had been made five days earlier by the local gp and a course of erythromycin and penicillinv commenced .", "examination revealed tender swellings over the left temporal area , angle of the mandible and upper third of the sternocleidomastoid extending into the submandibular region .", "examination of the oral cavity was complicated by trismus , however no obvious tonsillar enlargement , erythema or exudate was recorded .", "initial blood results showed an elevated c - reactive protein of 155mg / l and white cell count of 16.6(10 * 9l ) .", "initial imaging of the neck comprised an ultrasound scan ; this demonstrated normal right sided vasculature but abnormal left ejv and retromandibular veins .", "these veins appeared dilated with increased wall vascularity and showed no recordable signal on doppler imaging , an appearance in keeping with an inflammatory thrombophlebitis . to note", "an mri brain with venography ( mrv ) sequences provided greater anatomical delineation ( figure 1 ) . despite a coincidental finding of a smaller calibre left venous sinus ; thrombus was not seen to extend intracranially .", "lastly an echocardiogram was sought confirming a normal heart with no septal defects , vegetations or thrombus .", "mrv brain images demonstrate bilateral sagittal , straight , transverse and sigmoid sinuses with no evidence of thrombus extension . both right and left ijv as well as right ejv are clearly patent . the left ejv could not be demonstrated .", "treatment was started promptly on clinical suspicion of ls and consisted of intravenous ( iv ) metronidazole , benzyl penicillin and dexamethasone ; anticoagulation was not given .", "the patient remained in hospital on iv antibiotics for a total of 14 days , the noted swellings slowly decreased in size , blood results normalized and the trismus resolved .", "he remained afebrile throughout , developed no subsequent complications and was discharged home to complete seven weeks of oral antibiotics .", "despite ijv thrombophlebitis forming a main diagnostic criterion for ls , we report a case in which the primary vascular involvement is that of the ejv , with a corresponding normal ijv .", "studies have previously described ls with thrombosis of the carotid artery , intracranial venous sinus and abdominopelvic vessels ( 1 ) ; only three have ever described involvement of the ejv .", "this unusual finding helps to highlight the variable nature of this condition and the diagnostic confusion it may herald . to note there are reported cases of ls in which there was no demonstrable vascular involvement .", "numerous theories have tried to explain the spread of organisms from oropharynx to the surrounding vein ; haematogenous via the tonsillar vein , secondary to lymphangitis or via direct spread through deep neck spaces .", "( 3 ) the incidence of ls within developed countries is now thought to lie around one per million per year , significantly reduced from the pre antibiotic era , however a resurgence over the last decade has recently been proposed .", "mortality has been recorded at 6.9% however morbidity and complications remain high as the early diagnosis can often be missed and treatment delayed .", "( 3 ) although classically described as following an oropharyngeal infection ls has been reported developing from other primary infective sites including the middle ear , sinuses , mastoid and teeth ( 1 ) .", "the development of sepsis in ls is often delayed , presenting three to seven days from signs of the primary infection , which may have all but resolved ( 4 ) , making accurate history taking and a high index of clinical suspicion ever more important .", "ls is classically but not exclusively associated with an obligate oropharyngeal gram negative bacillus ; fusobacterium necrophorum .", "other causative pathogens described in the literature include streptococcus , staphylococci and bacteroides species ( 5 ) .", "it has however been reported that in almost 13% of cases no pathogen is isolated on culture ( 6 ) .", "ls can also be associated with thrombophillia ( thought to be endotoxin induced ) ( 5 ) .", "the role of anticoagulation however remains controversial with no conclusive literature evidence ; the decision should therefore be made on clinical grounds ; assessing severity of symptoms , poor treatment response and degree of thrombus extension .", "a recent history of a sore throat in conjunction with sepsis and or a suggestive and often declining clinical picture should always prompt early antibiotic therapy whilst a diagnosis of ls is sought ; hopefully preventing the development of late embolic complications .", "differential diagnoses to consider include infectious mononucleosis , parapharyngeal abscess , lymphoma , pneumonia ( including atypical forms ) and other embolic sources ( e.g. infective endocarditis ) ."], "abstract_text": ["<S> oropharyngeal infections are routinely encountered within general practice and accident and emergency departments . </S>", "<S> most settle with simple analgesia and antibiotics ; occasionally such patients may develop lemierre s syndrome ( ls ) a rare and potentially fatal sequela that can be easily overlooked . </S>", "<S> we aim to highlight the main symptoms , pathology , investigations and management . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["postanginal septicaemia in the early 20 century ; originally associated with a mortality reaching 90% it involves a classic triad of oropahryngeal sepsis , internal jugular vein ( ijv ) thrombophlebitis and metastatic abscess formation ( 1 ) . ", "clinical findings can therefore vary considerably from neck pain and swelling associated with thrombophlebitis to pleuritic chest pain , haemoptysis , dyspnoea or arthralgia as a result of septic thromboemboli .", "we report a case of predominantly external jugular vein ( ejv ) involvement in an adolescent male with negative blood culture results ."], ["a twelve year old boy presented to the children s assessment unit with a 24 hour history of unilateral face and neck swelling on a background of seven days progressive sore throat and dysphagia .", "minor systemic upset was also noted including headache , vomiting and non specific abdominal pain .", "a provisional diagnosis of tonsillitis had been made five days earlier by the local gp and a course of erythromycin and penicillinv commenced .", "examination revealed tender swellings over the left temporal area , angle of the mandible and upper third of the sternocleidomastoid extending into the submandibular region .", "examination of the oral cavity was complicated by trismus , however no obvious tonsillar enlargement , erythema or exudate was recorded .", "initial blood results showed an elevated c - reactive protein of 155mg / l and white cell count of 16.6(10 * 9l ) .", "initial imaging of the neck comprised an ultrasound scan ; this demonstrated normal right sided vasculature but abnormal left ejv and retromandibular veins .", "these veins appeared dilated with increased wall vascularity and showed no recordable signal on doppler imaging , an appearance in keeping with an inflammatory thrombophlebitis . to note", "an mri brain with venography ( mrv ) sequences provided greater anatomical delineation ( figure 1 ) . despite a coincidental finding of a smaller calibre left venous sinus ; thrombus was not seen to extend intracranially .", "lastly an echocardiogram was sought confirming a normal heart with no septal defects , vegetations or thrombus .", "mrv brain images demonstrate bilateral sagittal , straight , transverse and sigmoid sinuses with no evidence of thrombus extension . both right and left ijv as well as right ejv are clearly patent . the left ejv could not be demonstrated .", "treatment was started promptly on clinical suspicion of ls and consisted of intravenous ( iv ) metronidazole , benzyl penicillin and dexamethasone ; anticoagulation was not given .", "the patient remained in hospital on iv antibiotics for a total of 14 days , the noted swellings slowly decreased in size , blood results normalized and the trismus resolved .", "he remained afebrile throughout , developed no subsequent complications and was discharged home to complete seven weeks of oral antibiotics ."], ["despite ijv thrombophlebitis forming a main diagnostic criterion for ls , we report a case in which the primary vascular involvement is that of the ejv , with a corresponding normal ijv .", "studies have previously described ls with thrombosis of the carotid artery , intracranial venous sinus and abdominopelvic vessels ( 1 ) ; only three have ever described involvement of the ejv .", "this unusual finding helps to highlight the variable nature of this condition and the diagnostic confusion it may herald . to note there are reported cases of ls in which there was no demonstrable vascular involvement .", "numerous theories have tried to explain the spread of organisms from oropharynx to the surrounding vein ; haematogenous via the tonsillar vein , secondary to lymphangitis or via direct spread through deep neck spaces .", "( 3 ) the incidence of ls within developed countries is now thought to lie around one per million per year , significantly reduced from the pre antibiotic era , however a resurgence over the last decade has recently been proposed .", "mortality has been recorded at 6.9% however morbidity and complications remain high as the early diagnosis can often be missed and treatment delayed .", "( 3 ) although classically described as following an oropharyngeal infection ls has been reported developing from other primary infective sites including the middle ear , sinuses , mastoid and teeth ( 1 ) .", "the development of sepsis in ls is often delayed , presenting three to seven days from signs of the primary infection , which may have all but resolved ( 4 ) , making accurate history taking and a high index of clinical suspicion ever more important .", "ls is classically but not exclusively associated with an obligate oropharyngeal gram negative bacillus ; fusobacterium necrophorum .", "other causative pathogens described in the literature include streptococcus , staphylococci and bacteroides species ( 5 ) .", "it has however been reported that in almost 13% of cases no pathogen is isolated on culture ( 6 ) .", "ls can also be associated with thrombophillia ( thought to be endotoxin induced ) ( 5 ) .", "the role of anticoagulation however remains controversial with no conclusive literature evidence ; the decision should therefore be made on clinical grounds ; assessing severity of symptoms , poor treatment response and degree of thrombus extension .", "a recent history of a sore throat in conjunction with sepsis and or a suggestive and often declining clinical picture should always prompt early antibiotic therapy whilst a diagnosis of ls is sought ; hopefully preventing the development of late embolic complications .", "differential diagnoses to consider include infectious mononucleosis , parapharyngeal abscess , lymphoma , pneumonia ( including atypical forms ) and other embolic sources ( e.g. infective endocarditis ) ."]]}
{"article_id": "PMC4640059", "article_text": ["helicobacter pylori is a gram - negative , spiral and microaerophilic microorganism that was first isolated from the stomach of patients afflicted with peptic ulcer ( 1 , 2 ) .", "this bacterium can cause gastritis and peptic ulcer in the short - term and carcinoma of the stomach in the long - term ( 3 , 4 ) .", "polymerase chain reaction ( pcr ) is the desired method of proving the presence of h. pylori in tissues due to its high sensitivity and specificity ( 5 , 6 ) .", "it was previously thought that the human stomach was the sole source of h. pylori ; however , it was later found that they are present in human dental plaques , mouth , saliva , tonsils , adenoids , middle ear , nasal polyps and sinus mucosa ( 7 ) .", "it is believed that gastroesophageal reflux is involved in many cases of upper respiratory tract disorders including pharyngoesophageal disorders , croup , oropharyngeal dysphagia , rhinosinusitis , otalgia and different forms of otitis media ( 8 , 9 ) .", "samples from tonsil and adenoid tissues showed that they might be sources of h. pylori ( 10 , 11 ) .", "researchers have proved that there is a connection between chronic tubotympanal disorders and gastroesophageal reflux , but the mechanism is unknown ( 12 , 13 ) .", "it is suspected that gastroesophageal reflux is involved in the pathogenesis of chronic otitis media .", "evidence showed that stomach pepsin is present in the middle ear cavity of patients afflicted with chronic otitis media , as compared to the control group , supports this suspicion ( 14 , 15 ) .", "gastroesophageal reflux results in the appearance of otolaryngology symptoms in children and anti - reflux treatments reduce frequency of infection in the middle ear ( 16 ) .", "many studies have been conducted on otitis media with effusion and on its correlation with h. pylori ( 15 , 17 - 19 ) .", "however , the correlation between chronic suppurative otitis media and its various aspects , such as tympanosclerosis in which deposition of acellular hyaline and calcified deposits accumulate in the lamina propria of the middle ear has not been sufficiently studied ( 20 , 21 ) .", "so , we decided to use the pcr technique to determine the role of h. pylori in tympanosclerosis .", "this case - control and cross - sectional study was conducted on patients who were diagnosed with chronic infection of the middle ear and were candidates to undergo surgery in the imam khomeini or apadana hospitals of ahvaz in 2013 .", "patients who had operations for chronic infections of the middle ear and those who had ventilation tube inserted into their ears in the past were excluded from the study .", "the middle ear mucosa of the patients was examined during the surgical operations and based on this clinical evaluation , they were placed in one of the four types of normal , edema and hypertrophy , hyalinized and sclerotic and polyploid with or without cholesteatoma and this classification was recorded in the questionnaire . during the operations", ", samples were taken of the middle ear mucosa of the patients . in patients with sclerotic plaques ,", "the samples were kept under sterilized conditions at 20 degrees centigrade , and were eventually sent to the laboratory of jundishapur university of medical sciences , ahvaz , iran . in the laboratory , dna was extracted using the roche high - pure template pcr kit ( roche , germany ) according to the manufacture protocol .", "primers used to amplify the typing region were va1-f ( a3436 ) 5-atggaaatacaacaaacacac-313 and va1-r ( c1226 ) 5-ctgcttgaatgcgccaaac-313 for amplified 286-bp band and vag - f 5-caatctgtcaatcaagcgag-14 and vag - r 5-ctagcgtcaaaataattccaagg-314 for amplified 630-bp .", "the extracted dna was amplified using 5 l dna template , 0.3 l taq polymerase , 1 l dntp , 0.25 l each forward and reveres primers , 2 l mgcl2 and 5 l 10x pcr buffer , all mixed in a micro - tube and the ultimate volume reach to 50 l using nuclease - free distilled water .", "after activation of reaction in 95c for 5 minutes , amplification performed for 35 cycles .", "cycle program for first pair primers was as : 94c for 60 , 52c for 60 and 72c for 90 seconds . a final extension 72c for 5 minutes was the ultimate step of cycles .", "for the second pair primers , cycle program was as : 94c for 30 seconds , 56c for 60 seconds and 72c for 90 seconds .", "the chi - squared and the independent - samples t - test were also used to analyze the data . the selected level of significance was p < 0.05 .", "fifty - six patients participated in this study , of whom 29 ( 51.8% ) were men and 27 ( 48.2% ) women .", "the biopsy samples of the middle ear mucosa of 31 ( 55.4% ) patients were h. pylori - positive . of the 29 male patients , 15 cases were ( 51.7% ) h. pylori - positive , while of the 27 female patients 16 cases ( 59.3% ) were h. pylori - positive .", "this difference was not significant in pearson s chi - squared test ( p = 0.571 ) .", "the average age of h. pylori - positive patients was 30.3 years old ( standard deviation = 7.6 ) , while the average age of h. pylori - negative patients was 33.4 years ( standard deviation = 9.7 ) .", "this difference was not significant in the independent samples t - test ( p = 0.571 ) .", "therefore , there was no significant correlation between the presence of h. pylori and the age and gender of the patients .", "the most common pathological type of middle ear mucosa was hyalinized and sclerotic type in 19 cases ( 33.9% ) .", "the polyploid type with or without cholesteatoma type was reported in 15 ( 26% ) cases and the edematous and proliferative type with 12 ( 21% ) cases .", "examination of the middle ear mucosa of the 19 patients of the hyalinized and sclerotic type revealed that 16 ( 84.2% ) were h. pylori - positive ( figure 1 ) and 3 ( 15.8% ) were h. pylori - negative ; while of the 37 patients whose middle ear mucosa was not sclerotic,15 cases ( 40.7% ) were h. pylori - positive ( figure 2 ) 22 ( 59.3% ) h. pylori - negative .", "this was a significant difference ( p = 0.002 ) , which means there was a correlation between the presence of h. pylori and tympanosclerosis of the middle ear ( table 1 ) .", "as for other types of middle ear mucosa ( normal , polypoid with or without cholesteatoma and edema with hypertrophy ) , there was no significant correlation between them and the presence of h. pylori .", "l = ladder ( 100 bp ) , p = positive control , lanes are numbered in the following order from 1 to 2 : case h. pylori - positive ( 286 bp ) , case h. pylori - negative .", "l = ladder ( 100 bp ) , p = positive control , lanes are numbered in the following order from 1 to 3 : case h. pylori - positive ( 630 bp ) , negative control , case h. pylori - negative .", "results showed that 26 ( 46.4% ) of 56 patients had dry ears for at least three months before surgery .", "of the 30 patients who did not have dry ears , 13 cases ( 34.3% ) were h. pylori - negative and 17(56.7% ) were h. pylori - positive .", "this was not a significant difference in pearson s chi - squared test ( p = 0.832 ) .", "40 ( 45.2% ) of the 31 patients with perforations of more than 50% were h. pylori - negative and 17 ( 54.8% ) were h. pylori - positive .", "h. pylori was not significant in pearson s chi - squared test ( p = 0.931 ) .", "therefore , there was no correlation between the presence of h. pylori and the size of the perforation or dryness of the middle ear .", "zollner ( 22 ) was the first to use the term tympanosclerosis in 1956 to describe a common complication resulting from middle ear infections .", "tympanosclerosis is an abnormal condition in the middle ear cavity in which lime particles accumulate in the tympanic membrane , tympanic cavity and ossicular chain ( 23 , 24 ) .", "it is a long - term complication of infections of the middle ear ( 4 , 25 - 27 ) . in available reports ,", "the prevalence of tympanosclerosis varies from 14.1% in bhaya s report ( 28 ) to 35.64% in wu s study(21 ) . despite the extensive available information on the clinical , histologic and pathologic aspects of tympanosclerosis ,", "tympanosclerosis is observed as irreversible changes in the temporal bone ( 13 , 24 ) .", "it is thought that gastroesophageal reflux may be involved in the pathogenesis of otitis media and evidence indicating the presence of stomach pepsin in the middle ear cavity of patients with otitis media , as compared to the control group , confirms it ( 14 , 29 ) .", "gastroesophageal reflux results in the appearance of otolaryngology symptoms in children and anti - reflux treatments make frequent middle ear infections go away ( 16 ) .", "many studies have been conducted on otitis media with effusion and on its correlation with h. pylori ( 15 , 17 , 18 ) ; however , the correlation between chronic suppurative otitis media and its various aspects such as tympanosclerosis has not been sufficiently investigated . in tympanosclerosis ,", "the process of deposition of acellular hyaline and calcium deposits in the lamina propria of the middle ear takes place , following which it is even possible for fixation of ossicles to occur leading to conductive hearing loss ( 20 ) . the exact etiology and pathogenesis of tympanosclerosis", "are not completely known ( 9 ) . in some studies , tympanosclerosis has been observed also as a complication of frequent otitis ( 21 , 29 ) . in a recent research", ", it has been suggested that h. pylori is the pathologic factor in tympanosclerosis ( 21 ) . in our research ,", "designed to study the correlation between h. pylori and tympanosclerosis , we found that h. pylori has a prevalence of 84.2% among patients with suppurative infection of the middle ear , while its prevalence was 40.5% in patients without tympanosclerosis .", "this difference ( p = 0.002 ) indicates that h. pylori plays a role in the pathogenesis of tympanosclerosis .", "( 26 ) showed that there are similar risk factors , such as increased homocysteine levels , between coronary artery atherosclerosis and tympanosclerosis .", "although tympanosclerosis and atherosclerosis are two separate pathologies , they have similar histological and pathophysiologic elements and the same genetic reason has been proposed for both ( 2 ) .", "( 30 ) studied evidence concerning the development of inflammation caused by h. pylori in 38 patients with atherosclerotic plaques who had undergone endarterectomy and found that in most of these patients h. pylori was correlated to the inflammation , which indicated the correlation between h. pylori and atherosclerosis .", "( 31 ) show that contact with h. pylori increases the risk of cardiovascular diseases even in the absence of other risk factors .", "our findings suggest that h. pylori probably plays a role in the development of tympanosclerosis .", "( 26 ) carried out a case - control study and found that 100% of the patients with tympanosclerosis were h. pylori - positive while only 26.9% of the patients not suffering from tympanosclerosis were h. pylori - positive ( p < 0.01 ) ( 21 ) . results of our study , while confirming those of iriz et al .", "( 26 ) used the campylobacter - like organism ( clo ) test in h. pylori identification , while we employed the pcr method . since the pcr method is more accurate than the clo test , our results are probably more acceptable than those iriz et al .", "( 26 ) , both show that there is a correlation between the presence of h. pylori and tympanosclerosis , and suggest the presence of h. pylori as the etiology for this disease ; however , neither study can consider h. pylori as the definite pathogenesis for tympanosclerosis . to prove this correlation", ", a study should be designed to evaluate the correlation between tympanosclerosis and h. pylori by considering those pathological changes found in the examination of the middle ear mucosa that attribute the presence of inflammatory symptoms to the activity of h. pylori in the middle ear .", "this study offers a better understanding of the etiological and pathogenic aspects of h. pylori in tympanosclerosis that can be used in designing new treatment methods for this disease ."], "abstract_text": ["<S> background : tympanosclerosis is a condition caused by calcification of tissues in the middle ear mucosa that sometimes results hearing loss . </S>", "<S> helicobacter pylori is one of the pathological and etiologic factors in the development of tympanosclerosis.objectives:the purpose of this study was to show the role of h. pylori in the different aspects of chronic suppurative otitis media using the polymerase chain reaction ( pcr ) technique.patients and methods : this case - control and cross - sectional study was performed on all patients with chronic otitis media , candidates for surgical operations , in 2013 . </S>", "<S> they were allocated into the case group with tympanosclerosis and the control group without tympanosclerosis . during the surgical operation </S>", "<S> , biopsy was done from middle ear and the samples were studied to see if they contained h. pylori using the pcr method.results:from a total of 19 patients with tympanosclerosis , 16 cases ( 84.2% ) were h. pylori positive , while in the control group 15 ( 45.4% ) cases out of the 37 cases were h. pylori positive , which showed a significant difference ( p = 0.002 ) . </S>", "<S> age and gender of the patients , ear dryness and perforation size were not correlated with the presence or absence of h. pylori.conclusions:there is a significant correlation between tympanosclerosis and h. pylori ( p = 0.002 ) . </S>", "<S> this correlation can single out h. pylori as a pathological factor in the development of tympanosclerosis ; however , further studies are needed to prove this correlation . </S>"], "labels": null, "section_names": ["1. Background", "2. Objectives", "3. Patients and Methods", "4. Results", "5. Discussion"], "sections": [["helicobacter pylori is a gram - negative , spiral and microaerophilic microorganism that was first isolated from the stomach of patients afflicted with peptic ulcer ( 1 , 2 ) .", "this bacterium can cause gastritis and peptic ulcer in the short - term and carcinoma of the stomach in the long - term ( 3 , 4 ) .", "polymerase chain reaction ( pcr ) is the desired method of proving the presence of h. pylori in tissues due to its high sensitivity and specificity ( 5 , 6 ) .", "it was previously thought that the human stomach was the sole source of h. pylori ; however , it was later found that they are present in human dental plaques , mouth , saliva , tonsils , adenoids , middle ear , nasal polyps and sinus mucosa ( 7 ) .", "it is believed that gastroesophageal reflux is involved in many cases of upper respiratory tract disorders including pharyngoesophageal disorders , croup , oropharyngeal dysphagia , rhinosinusitis , otalgia and different forms of otitis media ( 8 , 9 ) .", "samples from tonsil and adenoid tissues showed that they might be sources of h. pylori ( 10 , 11 ) .", "researchers have proved that there is a connection between chronic tubotympanal disorders and gastroesophageal reflux , but the mechanism is unknown ( 12 , 13 ) .", "it is suspected that gastroesophageal reflux is involved in the pathogenesis of chronic otitis media .", "evidence showed that stomach pepsin is present in the middle ear cavity of patients afflicted with chronic otitis media , as compared to the control group , supports this suspicion ( 14 , 15 ) .", "gastroesophageal reflux results in the appearance of otolaryngology symptoms in children and anti - reflux treatments reduce frequency of infection in the middle ear ( 16 ) .", "many studies have been conducted on otitis media with effusion and on its correlation with h. pylori ( 15 , 17 - 19 ) .", "however , the correlation between chronic suppurative otitis media and its various aspects , such as tympanosclerosis in which deposition of acellular hyaline and calcified deposits accumulate in the lamina propria of the middle ear has not been sufficiently studied ( 20 , 21 ) ."], ["so , we decided to use the pcr technique to determine the role of h. pylori in tympanosclerosis ."], ["this case - control and cross - sectional study was conducted on patients who were diagnosed with chronic infection of the middle ear and were candidates to undergo surgery in the imam khomeini or apadana hospitals of ahvaz in 2013 .", "patients who had operations for chronic infections of the middle ear and those who had ventilation tube inserted into their ears in the past were excluded from the study .", "the middle ear mucosa of the patients was examined during the surgical operations and based on this clinical evaluation , they were placed in one of the four types of normal , edema and hypertrophy , hyalinized and sclerotic and polyploid with or without cholesteatoma and this classification was recorded in the questionnaire . during the operations", ", samples were taken of the middle ear mucosa of the patients . in patients with sclerotic plaques ,", "the samples were kept under sterilized conditions at 20 degrees centigrade , and were eventually sent to the laboratory of jundishapur university of medical sciences , ahvaz , iran . in the laboratory , dna was extracted using the roche high - pure template pcr kit ( roche , germany ) according to the manufacture protocol .", "primers used to amplify the typing region were va1-f ( a3436 ) 5-atggaaatacaacaaacacac-313 and va1-r ( c1226 ) 5-ctgcttgaatgcgccaaac-313 for amplified 286-bp band and vag - f 5-caatctgtcaatcaagcgag-14 and vag - r 5-ctagcgtcaaaataattccaagg-314 for amplified 630-bp .", "the extracted dna was amplified using 5 l dna template , 0.3 l taq polymerase , 1 l dntp , 0.25 l each forward and reveres primers , 2 l mgcl2 and 5 l 10x pcr buffer , all mixed in a micro - tube and the ultimate volume reach to 50 l using nuclease - free distilled water .", "after activation of reaction in 95c for 5 minutes , amplification performed for 35 cycles .", "cycle program for first pair primers was as : 94c for 60 , 52c for 60 and 72c for 90 seconds . a final extension 72c for 5 minutes was the ultimate step of cycles .", "for the second pair primers , cycle program was as : 94c for 30 seconds , 56c for 60 seconds and 72c for 90 seconds .", "the chi - squared and the independent - samples t - test were also used to analyze the data . the selected level of significance was p < 0.05 ."], ["fifty - six patients participated in this study , of whom 29 ( 51.8% ) were men and 27 ( 48.2% ) women .", "the biopsy samples of the middle ear mucosa of 31 ( 55.4% ) patients were h. pylori - positive . of the 29 male patients , 15 cases were ( 51.7% ) h. pylori - positive , while of the 27 female patients 16 cases ( 59.3% ) were h. pylori - positive .", "this difference was not significant in pearson s chi - squared test ( p = 0.571 ) .", "the average age of h. pylori - positive patients was 30.3 years old ( standard deviation = 7.6 ) , while the average age of h. pylori - negative patients was 33.4 years ( standard deviation = 9.7 ) .", "this difference was not significant in the independent samples t - test ( p = 0.571 ) .", "therefore , there was no significant correlation between the presence of h. pylori and the age and gender of the patients .", "the most common pathological type of middle ear mucosa was hyalinized and sclerotic type in 19 cases ( 33.9% ) .", "the polyploid type with or without cholesteatoma type was reported in 15 ( 26% ) cases and the edematous and proliferative type with 12 ( 21% ) cases .", "examination of the middle ear mucosa of the 19 patients of the hyalinized and sclerotic type revealed that 16 ( 84.2% ) were h. pylori - positive ( figure 1 ) and 3 ( 15.8% ) were h. pylori - negative ; while of the 37 patients whose middle ear mucosa was not sclerotic,15 cases ( 40.7% ) were h. pylori - positive ( figure 2 ) 22 ( 59.3% ) h. pylori - negative .", "this was a significant difference ( p = 0.002 ) , which means there was a correlation between the presence of h. pylori and tympanosclerosis of the middle ear ( table 1 ) .", "as for other types of middle ear mucosa ( normal , polypoid with or without cholesteatoma and edema with hypertrophy ) , there was no significant correlation between them and the presence of h. pylori .", "l = ladder ( 100 bp ) , p = positive control , lanes are numbered in the following order from 1 to 2 : case h. pylori - positive ( 286 bp ) , case h. pylori - negative .", "l = ladder ( 100 bp ) , p = positive control , lanes are numbered in the following order from 1 to 3 : case h. pylori - positive ( 630 bp ) , negative control , case h. pylori - negative .", "results showed that 26 ( 46.4% ) of 56 patients had dry ears for at least three months before surgery .", "of the 30 patients who did not have dry ears , 13 cases ( 34.3% ) were h. pylori - negative and 17(56.7% ) were h. pylori - positive .", "this was not a significant difference in pearson s chi - squared test ( p = 0.832 ) .", "40 ( 45.2% ) of the 31 patients with perforations of more than 50% were h. pylori - negative and 17 ( 54.8% ) were h. pylori - positive .", "h. pylori was not significant in pearson s chi - squared test ( p = 0.931 ) .", "therefore , there was no correlation between the presence of h. pylori and the size of the perforation or dryness of the middle ear ."], ["zollner ( 22 ) was the first to use the term tympanosclerosis in 1956 to describe a common complication resulting from middle ear infections .", "tympanosclerosis is an abnormal condition in the middle ear cavity in which lime particles accumulate in the tympanic membrane , tympanic cavity and ossicular chain ( 23 , 24 ) .", "it is a long - term complication of infections of the middle ear ( 4 , 25 - 27 ) . in available reports ,", "the prevalence of tympanosclerosis varies from 14.1% in bhaya s report ( 28 ) to 35.64% in wu s study(21 ) . despite the extensive available information on the clinical , histologic and pathologic aspects of tympanosclerosis ,", "tympanosclerosis is observed as irreversible changes in the temporal bone ( 13 , 24 ) .", "it is thought that gastroesophageal reflux may be involved in the pathogenesis of otitis media and evidence indicating the presence of stomach pepsin in the middle ear cavity of patients with otitis media , as compared to the control group , confirms it ( 14 , 29 ) .", "gastroesophageal reflux results in the appearance of otolaryngology symptoms in children and anti - reflux treatments make frequent middle ear infections go away ( 16 ) .", "many studies have been conducted on otitis media with effusion and on its correlation with h. pylori ( 15 , 17 , 18 ) ; however , the correlation between chronic suppurative otitis media and its various aspects such as tympanosclerosis has not been sufficiently investigated . in tympanosclerosis ,", "the process of deposition of acellular hyaline and calcium deposits in the lamina propria of the middle ear takes place , following which it is even possible for fixation of ossicles to occur leading to conductive hearing loss ( 20 ) . the exact etiology and pathogenesis of tympanosclerosis", "are not completely known ( 9 ) . in some studies , tympanosclerosis has been observed also as a complication of frequent otitis ( 21 , 29 ) . in a recent research", ", it has been suggested that h. pylori is the pathologic factor in tympanosclerosis ( 21 ) . in our research ,", "designed to study the correlation between h. pylori and tympanosclerosis , we found that h. pylori has a prevalence of 84.2% among patients with suppurative infection of the middle ear , while its prevalence was 40.5% in patients without tympanosclerosis .", "this difference ( p = 0.002 ) indicates that h. pylori plays a role in the pathogenesis of tympanosclerosis .", "( 26 ) showed that there are similar risk factors , such as increased homocysteine levels , between coronary artery atherosclerosis and tympanosclerosis .", "although tympanosclerosis and atherosclerosis are two separate pathologies , they have similar histological and pathophysiologic elements and the same genetic reason has been proposed for both ( 2 ) .", "( 30 ) studied evidence concerning the development of inflammation caused by h. pylori in 38 patients with atherosclerotic plaques who had undergone endarterectomy and found that in most of these patients h. pylori was correlated to the inflammation , which indicated the correlation between h. pylori and atherosclerosis .", "( 31 ) show that contact with h. pylori increases the risk of cardiovascular diseases even in the absence of other risk factors .", "our findings suggest that h. pylori probably plays a role in the development of tympanosclerosis .", "( 26 ) carried out a case - control study and found that 100% of the patients with tympanosclerosis were h. pylori - positive while only 26.9% of the patients not suffering from tympanosclerosis were h. pylori - positive ( p < 0.01 ) ( 21 ) . results of our study , while confirming those of iriz et al .", "( 26 ) used the campylobacter - like organism ( clo ) test in h. pylori identification , while we employed the pcr method . since the pcr method is more accurate than the clo test , our results are probably more acceptable than those iriz et al .", "( 26 ) , both show that there is a correlation between the presence of h. pylori and tympanosclerosis , and suggest the presence of h. pylori as the etiology for this disease ; however , neither study can consider h. pylori as the definite pathogenesis for tympanosclerosis . to prove this correlation", ", a study should be designed to evaluate the correlation between tympanosclerosis and h. pylori by considering those pathological changes found in the examination of the middle ear mucosa that attribute the presence of inflammatory symptoms to the activity of h. pylori in the middle ear .", "this study offers a better understanding of the etiological and pathogenic aspects of h. pylori in tympanosclerosis that can be used in designing new treatment methods for this disease ."]]}
{"article_id": "PMC3202097", "article_text": ["trigeminal neuralgia ( tn ) is a disorder of cranial nerve ( cn ) v that results in severe episodes of shock - like or lancinating pain in one or more of its three divisions ( v1v3 ) .", "idiopathic tn and cases due to vascular compression of cn v are categorized as classical tn .", "patients diagnosed with symptomatic tn experience trigeminal - related facial pain secondary to a brain tumor , skull deformity , or multiple sclerosis ( ms ) .", "evidence suggests that the majority of cases of tn are the consequence of focal compression of the entry zone of the root of the trigeminal nerve , while only 2% of cases are observed in patients diagnosed with ms .", "other than excruciating facial pain , there are no other direct medical symptoms associated with tn , and the condition does not decrease life expectancy .", "however , many patients with tn struggle with accomplishing tasks that affect quality of life , which is how this disorder elicits a negative impact on the social and mental wellness of the patients who suffer from this illness .   following the diagnosis of tn", "however , many patients experience only limited relief from medication or are unable to endure the side effects of the prescribed drugs , and in turn seek neurosurgical intervention .", "currently , surgical approaches include microvascular decompression ( mvd ) or a number of techniques that target the trigeminal ganglion or root which involve the destruction or blockage of portions of those anatomical structures [ 1 , 2 ] .", "although the neurosurgical modalities are preferred in many clinical situations and have proven to be effective in achieving initial pain control , they are known to come with a variety of complications , and facial pain recurrence is likely .  ", "stereotactic radiosurgery ( srs ) has proven to be an effective management approach for patients with medically   and surgically   refractory tn as a primary and repeat treatment modality .", "the use of radiosurgery in the treatment of tn dates back to sweden in the 1950 's , where professor lars leksell performed radiogangliotomies directed at the gasserian ganglion .", "since the time of leksell , advancements in radiosurgery and imaging technologies has led to the increasing popularity of srs as a treatment option for patients with tn .", "one form of srs that can be delivered to a patient is through a machine called the gamma knife ( gk ) .", "the gk device is a cobalt-60-based machine , with 201 separate 4 to 18  mm collimator openings , that emits multiple gamma rays that converge on a target specified by computer planning . for specific medication intolerable patient subsets , gamma knife radiosurgery ( gkrs )", "can be used as an initial management approach , or as a secondary management approach following radiosurgery or one or more of the various surgical modalities . as the evidence examining the role of gkrs in the management of patients with tn is increasing ,", "it is of utmost importance for physicians to understand the criteria associated with gkrs , so that the optimal course of treatment for their patients can be prescribed .   an evidence - based review on the evaluation and treatment of tn by gronseth et al .   found level c evidence indicating that gasserian ganglion percutaneous techniques , gkrs , and mvd may be considered for facial pain management for medically refractory patients .", "however , questions remain regarding optimal treatment modalities in specific patient subsets . for this reason ,", "the goal of this paper is to provide a modern review of the literature thoroughly analyzing the efficacy of gkrs in the treatment of patients with tn , as well as evaluating the treatment planning and methods associated with this evolving modality .", "to identify contemporary studies assessing the clinical outcomes of patients treated with gkrs for tn , a pubmed search from 2006 to april 2011 was performed .", "keywords for search included  gamma knife or gamma knife radiosurgery or stereotactic radiosurgery trigeminal neuralgia or tic douloureux .", " studies analyzed in this review included retrospective cohort studies and prospective cohort studies with 5 evaluated patients .", "studies published only in abstract form and studies published in a language other than english were excluded from our analysis . due to our broad search strategy and the vast amount of world literature ,", "we reviewed a total of 19 studies [ 4 , 5 , 925 ] analyzing the efficacy of patients with tn who were treated once with gkrs ( table 1 ) .", "thirteen of the 19 evaluated studies [ 4 , 5 , 919 ] utilized the barrow neurological institute ( bni ) pain intensity scale   as a measurement of response to treatment ( see section 3 ) .", "of these 13 studies , only two [ 9 , 13 ] analyzed patients treated with gkrs as an initial management approach . with a median followup of 31 months , sheehan et al .  ", "classified 87% of patients in bni class i - iiib , while chen et al .  ", "classified 91% of patients in bni class i - iiib ( median followup = 15 months ) .", "chen et al .   also reported that five of the 44 patients ( 11% ) treated with gkrs developed hypoesthesia following the procedure .  ", "the other 11 bni pain intensity scale studies we reviewed included patients where previous surgical procedures were performed in a fraction of patients [ 4 , 5 , 1012 , 1519 ] or all patients . of the 10 studies where previous surgical procedures were performed in a fraction of patients , nine reported outcomes in terms of categorizing patients in bni class i - iiib [ 4 , 5 , 1012 , 1518 ] .", "classified 58.6% of patients in bni class i - iiib ( median followup = 48 months ) , kondziolka et al .  ", "classified 71% of patients in bni class i - iiib at three years , dhople et al .  ", "classified 72% of patients in bni class i - iiib ( median followup = 29 months ) , han et al", ".   classified 76.7% of patients in bni class i - iiib ( mean followup = 58 months ) , dhople et al .  ", "classified 81% of patients in bni class i - iiib ( median followup = 5.6 years ) , matsuda et al .  ", "classified 82% of patients in bni class i - iiib ( median followup = 37 months ) , little et al .  ", "classified 83% of patients in bni class i - iiib ( median followup = 6.3 years ) , dellaretti et al .  ", "classified 89.5% of patients in bni class i - iiib ( mean followup = 20.3 months ) , and park and hwang   classified 94% of patients in bni class i - iiib with a minimum followup of 3 years .", "reported clinical outcomes with respect to bni class i , which contained only 5.7% of patients .", "the study that evaluated gkrs where previous surgical procedures were performed in 100% of patients classified 85% of patients in bni class i - iiib , with a median followup of 36 months .", "we also reviewed two studies that used the excellent - good - fair - poor ( egfp ) categorical scale to assess patient outcomes [ 20 , 21 ] ( see section 3 ) .", "treated 30 patients with tn with gkrs at iran gamma knife center between 2006 and 2007 .", "the authors reported that 40% of patients had an excellent outcome , 10% of patients had a good outcome , 33% of patients had a fair outcome , and 17% of patients had a poor outcome following the procedure .", "after surgery , 15 patients ( 54% ) reported an excellent outcome , one patient ( 4% ) reported a good outcome , two patients ( 7% ) reported a fair outcome , and 10 patients ( 36% ) reported a poor outcome .", "the complications from mvd included facial numbness in six patients ( 21% ) , dysesthesias in three patients ( 11% ) , and delayed facial palsy in one patient ( 4% ) .   ", "four of the 19 studies we evaluated [ 2225 ] used other methodologies in determining the effectiveness of gkrs . in a prospective controlled trial ,", "rgis et al .   analyzed 100 patients with tn treated with gkrs and reported that 83 patients ( 83% ) were completely pain free , 58 of which ( 58% ) discontinued all medication following the procedure ( minimum followup = 12 months ) .", "ten patients ( 10% ) experienced radiation - induced complications , which included facial paresthesia or hypesthesia .", "performed a study investigating the short - term efficacy of gkrs in 67 patients with medically refractory tn .", "overall , 77.6% of patients witnessed some degree of pain relief , with 32.6% of those patients becoming completely pain free .", "of the 67 patients , 10 ( 14.9% ) experienced complications from the procedure , which included hypoesthesia and paresthesia .", "sixty eight patients ( 42.5% ) underwent prior invasive treatments . in clinical analysis , it was found that 61% of patients were pain free without medication , 29% of patients were pain free with medication , and 10% of patients did not respond to gkrs .", "thirty eight patients ( 49.4% ) exhibited some level of pain improvement following gk treatment , with 23 of those patients ( 29.9% ) reporting a pain - free outcome .", "twelve patients ( 15.6% ) experienced complications , which were reported to be mild facial sensory changes and mild facial nerve dysfunction .", "as gkrs has proven to be an effective initial treatment for tn , numerous reports have been published analyzing patients treated on multiple occasions ( > 1 ) with gkrs .", "we reviewed six studies evaluating patients treated more than once with gkrs [ 2732 ] ( table 2 ) .", "of these six articles , two [ 29 , 32 ] utilized the bni pain intensity scale .", "sixteen patients ( 76.2% ) exhibited compelling improvements and were placed in bni class i - ii .", "huang et al .   analyzed 65 medically refractory patients with tn who were treated with gkrs as a second treatment modality .", "of these 65 patients , 30 ( 46% ) had undergone gkrs as an initial management approach .", "the authors placed 22 patients ( 34% ) in bni class i , 11 patients ( 17% ) in bni class ii , four patients ( 6% ) in bni class iiia , and five patients ( 8% ) in bni class iiib .", "overall , with a median followup of 64 months , 65% of patients reported successful results in terms of pain control rates .   a total of three of the six reviewed studies evaluated patients using the egfp categorical scale [ 28 , 30 , 31 ] .", "analyzed 37 patients treated a second time with gkrs for recurrent tn and reported that 17 patients ( 46% ) achieved excellent pain relief , nine patients ( 24% ) achieved good pain relief , five patients ( 14% ) achieved fair pain relief , and six patients ( 16% ) achieved poor pain relief .", "however , the authors concluded that 57% of patients experienced some form of trigeminal dysfunction following repeat radiosurgery .", ", huang et al .   treated 28 patients with repeat gkrs and reported that 12 patients ( 43% ) exhibited excellent pain relief , five patients ( 18% ) exhibited good pain relief , and two patients ( 7% ) exhibited fair pain relief .", "in addition , the authors found a statistically significant ( p = 0.047 ) correlation between cumulative radiation doses > 115  gy and facial numbness . in a separate study , huang et al .  ", "specifically , a total of eight patients underwent mvd a mean of 7.6 months following repeat gkrs .", "of the eight patients , seven ( 87.5% ) were completely pain free at a mean of 21 months following neurosurgery .", "this data supports the use of mvd if multiple gk procedures are deemed ineffective .  ", "kimball et al .   treated 53 patients with repeat gkrs and analyzed the patients not lost during followup using the marseille scale , which categorizes patients into one of five classes , with a higher class statistically indicating a worse prognosis for the patient . with a mean", "followup of 42 months , 20 patients ( 43.5% ) were categorized in marseille class i - ii , six patients ( 13% ) were categorized in marseille class iii - iv , and 20 patients ( 43.5% ) were categorized in marseille class v. the authors also reported a statistically significant ( p = 0.047 ) correlation between facial numbness and superior long - term pain relief .", "a total of 22 patients ( 48% ) experienced trigeminal dysfunction of any kind , while 21 patients ( 46% ) experienced numbness in the face .", "since gkrs can be performed as both initial and salvage treatment options for patients who suffer from tn , its efficacy has been compared in patients who undergo one versus multiple radiosurgery procedures .", "we reviewed eight studies to further examine this matter [ 3 , 3339 ] ( table 3 ) .", "four of the eight studies utilized the bni pain intensity scale   to evaluate patient outcomes [ 3 , 3335 ] .", ", it was reported that 75% , 60% , and 58% of patients with idiopathic tn had bni scores of i  iiib at 1 , 3 , and 5 years , respectively .", "the 1- , 3- , and 5-year - bni scores of i  iiib in patients with ms - related tn were 56% , 30% , and 20% , respectively .", "the authors concluded that repeat gkrs exhibited similar success rates when compared to the initial procedure .", "similar to verheul et al . , park et al .   did not find differences in terms of time to initial response , time to pain recurrence , and overall pain relief when comparing patients who underwent one versus two gk treatments .", "however , it was observed that patients who received two gk treatments were more likely to have facial sensory changes when compared to patients treated a single time with radiosurgery .", "little et al .   performed a study where 79 patients with typical tn were treated with gkrs as a salvage procedure .", "approximately five years following salvage gkrs , the authors reported that 50% of patients experienced pain relief and 20% of those patients were completely pain free .", "in addition , a statistically significant ( p = 0.029 ) correlation between gkrs failure and prior mvd was found .", "treated 37 patients ( 78% had failed prior surgery ) with ms - related tn with gkrs .", "nine patients ( 24% ) underwent gkrs as their first procedure . the reported 1 , 3 , and 5 year bni scores of i  iiib were 82.6% , 73.9% , and 54% , respectively .   the other four studies we reviewed utilized the egfp categorical scale as a measurement of response to treatment [ 3639 ] .", "two of the evaluated studies [ 36 , 37 ] were conducted by fountas et al . and analyzed patients treated with gkrs for idiopathic tn based on whether or not they had undergone previous surgical or radiosurgical procedures for facial pain control .", "one of the studies evaluated 106 patients ( 19 previous radiosurgery procedures ) and concluded that the treatment group without a previous history of surgical or radiosurgical procedures exhibited superior clinical outcomes , with 1-year and 2-year complete pain relief rates of 82.5% and 78% , respectively .", "the 1-year and 2-year complete pain relief rate in the patient group with a history of surgical or radiosurgical procedures was 69.4% and 63.5% , respectively .", "as expected , similar results were found in the other study by fountas et al .", "; however , no prior radiosurgical procedures were performed in the patient group with a history of prior procedures .", "huang et al .   conducted a study where 89 patients with idiopathic tn were treated with gkrs as an initial management approach , 20 of which underwent a subsequent gkrs procedure for facial pain recurrence . following the initial radiosurgical procedure ,", "50 patients ( 56% ) had an excellent response , 12 patients ( 13.5% ) had a good response , and 7 patients ( 7.9% ) had a fair response .", "following the second radiosurgical procedure , 11 patients ( 55% ) had an excellent response and one patient ( 5% ) had a good response . in a separate study , huang et al .  ", "assessed 21 patients with benign tumor - related tn who were treated with gkrs as an initial or repeat procedure .", "following the initial gk procedure to the tumor , 12 patients ( 57% ) had an excellent response and 1 patient ( 5% ) had a good response .", "a total of eight patients were treated with a subsequent gkrs procedure targeted at the ipsilateral trigeminal root or ganglion due to facial pain recurrence . following the second radiosurgical procedure , the authors reported four patients ( 50% ) with an excellent response .", "we identified six studies comparing patients treated with gkrs with patients treated with one of the various surgical modalities [ 2 , 4044 ] ( table 4 ) .", "the authors of this review acknowledge the importance of percutaneous techniques in the management of tn ; however , our modern literature search predominantly yielded comparison studies analyzing the efficacy of mvd when compared to gkrs .", "specifically , four of the six studies [ 2 , 4042 ] analyzed patients treated with gkrs against patients treated with mvd .", "specifically , 36 were treated with mvd ( 45% ) , while 44 were treated with gkrs ( 55% ) .", "the mvd treatment arm statistically differed from the gkrs treatment arm with respect to age ( median of 54 versus 74 years ) , preoperative symptom duration ( median of 2.6 versus 7.5 years ) , and the presence of major comorbidities ( 2.8 versus 58.3% ) .", "the authors reported that patients treated with mvd exhibited superior levels of initial ( 100% ) , 2 year ( 88% ) , and 5 year ( 80% ) actuarial pain - free rates when compared to the patients treated with gkrs ( 78 , 50 , and 33% , resp . ) , with a p value of 0.0002 .", "in addition to increased levels of patient satisfaction , as reported by required patient surveys , the mvd treatment group also had a decreased level of permanent mild ( 5.6% ) and severe sensory loss ( 0% ) when compared to the gkrs treatment group ( 6.8% and 2.3% , resp . ) .", "two patients ( 5.6% ) in the mvd group experienced permanent mild paresthesias or numbness , one ( 2.8% ) patient experienced a cerebrospinal fluid leak from the wound , and one patient ( 2.8% ) experienced hearing loss and diplopia .", "three patients ( 6.8% ) in the gkrs group experienced permanent mild paresthesias or numbness , one patient ( 2.3% ) experienced a more permanent sensory numbness , and one patient ( 2.3% ) experienced a transient headache and nausea following the gk procedure .  ", "all patients were diagnosed with typical tn and did not undergo previous gk or mvd procedures . it was reported that patients treated with mvd exhibited superior levels of complete pain relief at 12 ( 68% ) and 18 months ( 68% ) when compared to the gkrs group , who 's complete pain relief rate was 58% at 12 months and 24% at 18 months ( p = 0.089 ) .", "the treatment arms did not statistically differ in terms of 90% pain relief at 12 and 18 months .", "this study could be criticized due to the large difference in the number of patients constituting the two treatment arms .  ", "oh et al .   evaluated a total of 45 elderly patients ( > 65 years of age ) diagnosed with idiopathic tn who were treated with either mvd ( 27 patients ) or gkrs ( 18 patients ) .", "it was reported that three mvd patients ( 11% ) and three gk patients ( 17% ) underwent previous percutaneous procedures .", "the mean followup period was reported to be 35.9 months for the mvd group and 33.1 months for the gkrs group . according to the bni pain intensity scale ,", "the mvd group had a superior prognosis , with 17 patients ( 63% ) classified in bni class i - ii compared with the 10 patients ( 56% ) in the gkrs group classified in bni class i - ii .", "the observed complications following mvd included constant headache in 11 patients ( 40.7% ) , facial paresthesia in five patients ( 18.5% ) , paresthesia of the tongue in two patients ( 7.4% ) , infection at the site of incision in one patient ( 3.7% ) , an acute subdural hemorrhage in one patient ( 3.7% ) , temporary hearing loss in one patient ( 3.7% ) , and otitis media with cerebrospinal leakage in one patient ( 3.7% ) . two patients ( 11% ) in the gkrs group experienced paresthesia .  ", "compared the clinical outcomes of 19 patients treated with mvd with 15 patients treated with gkrs .", "nine gk patients ( 60% ) and four mvd patients ( 21% ) underwent previous surgical procedures . the treatment", "arms statistically differed ( p = 0.0005 ) with respect to mean patient age , with the mean age of the gkrs group exceeding the mvd group by 13 years ( 74 versus 61 years ) .", "in addition , patient satisfaction was graded on a scale of 1 ( unsatisfied ) to 10 ( completely satisfied ) .", "it was reported that the mean tn complexity grade was statistically different ( p < 0.001 ) between the treatment arms ( gk = 5.8 ; mvd = 3 ) .", "the average response following the procedure was determined to be 3.4 for the mvd group and 2.4 for the gkrs group ( p = 0.017 ) . also , it was found that the satisfaction score for the mvd group was superior to the gkrs group ( 8.7 versus 6.4 ) , with a p value of 0.02 .", "the authors reported a statistically significant correlation between tn complexity grade and clinical response ( p < 0.001 ) , as well as tn complexity grade and patient satisfaction ( p < 0.001 ) .   to date", ", no randomized trials have been conducted analyzing the outcomes of patients with tn who are treated with mvd compared to gkrs . in a large review on tn management ,", "zakrzewska and linskey   found evidence that mvd is an effective treatment for long - term facial pain relief but comes with an increased risk of ipsilateral hearing loss .", "in addition , the authors concluded that single - dose srs is an effective treatment for long - term facial pain relief but puts patients at risk for facial numbness or facial paresthesias .", "investigation into this matter in the form of a randomized controlled trial would provide the best evidence in terms of facial pain relief and procedure - related complications .  ", "in addition , we reviewed two studies comparing patients treated with gkrs with patients treated with posterior fossa exploration ( pfe ) [ 43 , 44 ] , both of which were conducted by pollock and colleagues at the mayo clinic college of medicine .", "one of the studies   was a specific prospective comparison of 91 patients treated with pfe and 49 patients treated with gkrs for idiopathic tn as an initial management approach .", "the treatment arms statistically differed in terms of age ( gkrs = 67.1 years ; pfe = 58.2 years ) , with a p value < 0.001 .", "it was reported that patients treated with pfe were more likely to be pain free and off medications at 1 year ( 84% ) and 4 years ( 77% ) when compared to the gkrs group ( 66 and 56% , resp . )", "retreatment for recurrent facial pain was performed in 15% of the patients in the pfe treatment arm and 35% of patients in the gkrs treatment arm ( p = 0.009 ) .", "also , it was found that nonbothersome facial numbness occurred more frequently in the gkrs group ( p = 0.04 ) .", "an additional study from the mayo clinic evaluated patients with recurrent tn who underwent 3 or more surgical procedures .", "the authors reported that patients treated with pfe exhibited superior levels of complete pain relief at 3 years of followup when compared to patients treated with gkrs , balloon compression , and glycerol rhizotomy ( p < 0.01 ) and underwent additional surgery for recurrent facial pain less often when compared to patients treated with the other modalities ( p = 0.02 ) .", "clinical 006futcomes did not differ between patients treated with gkrs and patients treated with the percutaneous techniques .", "srs can be performed by a variety of tools , which include gkrs , cyberknife technology , and linear accelerator ( linac)-based treatment .", "our analysis yielded one study whose primary endpoint was to devise a method using cyberknife treatment planning that would mimic the dosimetric characteristics of the gk treatment plan in five patients undergoing radiosurgery for tn .", "both the isodose lines and critical structures were identified using the gkrs treatment plan and were transferred to the cyberknife treatment planning system .", "it was reported that the average length of the trigeminal nerve receiving a dose of 60  gy was 4.5  mm for the gk , 4.5  mm for the nonisocentric cyberknife , and 4.4  mm for the isocentric cyberknife .", "the authors found it more difficult to minimize the dose to critical structures when using cyberknife technology .", "also , the dose falloff of gkrs was found to be steeper when compared to cyberknife technology due to , what the authors hypothesized , the large number of gamma rays produced which converge on the focal point with precision .  ", "as previously mentioned , the gk machine 's primary functional unit is cobalt-60 , which is used to emit photon energy through 201 separate 4 to 18  mm collimator openings that converge on a target specified by a treatment planning system .", "balamucki et al .   performed a study examining if the half life of cobalt ( 5.26 years ) relates to the outcomes for patients being treated for tn with gkrs .", "the authors collected data on 239 gkrs procedures performed at their institution between 1999 and 2004 .", "patient surveys were used to measure responses to radiosurgery . with the followup time ranging from one to six months , it was reported that 80% of patients experienced some degree of pain relief and that 56% of those patients were pain free .", "the authors concluded that clinical outcomes remained consistent during the first half life of cobalt-60 .", "an area of controversy in the treatment of patients with tn is defining the optimal maximum radiosurgery dose that can be delivered to specific patient subsets .", "we analyzed five studies whose primary endpoint was to assess gkrs - dosing efficacy [ 4852 ] .", "kim et al .   utilized the bni pain intensity scale   to assess 66 tn patients treated with a gk maximum dose of 80  gy and 44 tn patients treated with a gk dose of 85  gy . although the two groups did not statistically differ in terms of facial rain relief and procedure - related complications , the authors did report that patients treated with a gk dose of 85  gy experienced a more rapid response to treatment when compared to the patients treated with a gk dose of 80  gy .", "arai et al .   analyzed 165 patients with tn treated with a gkrs dose of 80  gy .", "specifically , the authors divided the patients into two groups , which differed in the radiation dose rate received ( low - dose rate = 1.212.05  gy / min ; high - dose rate = 2.063.74  gy / min ) . using the bni pain intensity scale   as a clinical evaluation method", ", it was reported that the low - dose - rate group and the high - dose - rate group did not statistically differ in terms of initial pain relief , maintenance of pain relief , and clinical complications .  ", "patients in group one were treated with a gk dose < 90  gy with no beam channel plugging , patients in group two were treated with a gk dose equal to 90  gy with no beam channel plugging , and patients in group three were treated with a gk dose equal to 90  gy with beam channel plugging . although the trend did not reach full statistical significance ( p = 0.054 ) , patients in group three exhibited the highest level of pain relief , while patients in group one exhibited the lowest level of pain relief .", "the authors also observed that the three groups statistically differed ( p < 0.0001 ) in terms of trigeminal nerve dysfunction , with patients in group three experiencing the highest rate of mild and bothersome complications and patients in group one experiencing the lowest rate of mild and bothersome complications .", "similar to the results of massager et al . , morbidini - gaffney et al .   reported positive outcomes in patients treated with gk doses > 85  gy .", "the authors also found that patients treated with two isocenters were more likely to have superior bni pain intensity scale   scores during their course of followup when compared to patients treated with a single isocenter .  ", "the median initial dose was 80  gy , and the median retreatment dose was 45  gy . although the authors did not report any predictors in terms of facial pain control and patient morbidity , they did compare the results of their study with seven published retreatment articles and found that successful levels of pain control ( > 50% ) were significantly correlated with cumulative gkrs doses > 130  gy , as well as new trigeminal nerve dysfunction ( > 20% ) . in addition to dose selection efficacy in select patient cohorts , the radiosurgical target of cn v is another subject matter that requires further clinical investigation .", "we reviewed three studies [ 5355 ] analyzing specific gkrs targeting methods in the treatment of tn and one study   that examined the accuracy of gkrs to its image - guided target .", "compared patients treated with gkrs targeted at the dorsal root entry zone ( 59 patients ) with patients whose radiosurgical target was the retrogasserian zone of the trigeminal nerve ( 41 patients ) . with a median followup of 30 months , the dorsal root entry target group exhibited statistically superior levels of initial complete pain remission ( p = 0.003 ) and experienced less complications than the retrogasserian zone group ( p = 0.009 ) .", "chen et al .   also reported positive results with the dorsal root entry zone - targeting approach , with a success rate of 82.8% and a complication rate of 15% .", "park et al .   compared the dorsal root entry zone and retrogasserian zone - targeting methods in the treatment of 39 patients with medically refractory tn .", "the authors reported that the two treatment arms did not statistically differ in treatment success ( bni class i - iiib ) with respect to the bni pain intensity scale .", "however , patients treated with the retrogasserian zone - targeting method experienced a substantially shorter time of response to gkrs than patients treated with the dorsal root entry zone - targeting method ( p = 0.044 ) .", "although the two groups did not statistically differ with regard to treatment - related morbidities , it was found that the patients whose targeting approach was the dorsal root entry zone experienced a greater amount of bothersome complications than the retrogasserian zone group .  ", "massager et al .   analyzed the targeting accuracy of gkrs in 65 patients treated for tn whose six month followup mri showed focal contrast enhancement of the trigeminal nerve .", "the authors found that the median deviation of the coordinates between the intended radiosurgical target and the center of contrast enhancement was 0.91  mm in euclidean space .", "the median radiation dose fitting into the contrast enhancement region was determined to be 77  8.7  gy .", "this small deviation from the gkrs target explains the high accuracy and precise nature of the machine .", "the two most common methods of measuring patient outcomes from gkrs in the management of tn are the barrow neurological institute pain intensity scale   ( table 5 ) and the excellent - good - fair - poor ( egfp ) categorical scale ( table 6 ) .", "the bni pain intensity scale divides patients into one of five classes , with a higher class indicating a worse prognosis for the patient .", "patients in bni class iiia do not experience trigeminal pain but require the use of medication .", "patients in bni class iiib experience some trigeminal pain that can be satisfactorily managed with medication . patients in bni class iv experience some trigeminal pain that is not satisfactorily managed with medication . patients in bni class", " excellent  outcomes are defined as complete pain relief without the need of medication . ", "good  outcomes are defined as complete pain relief with the need of medication . ", "poor  outcomes are defined as a < 50% pain relief rate or treatment failure .", "for patients with medically refractory forms of tn , gkrs has proven to be an effective initial and repeat treatment option .", "cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs .", "however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs .", "although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive .", "further evidence in the form of a phase iii - randomized trial is needed to confirm the clinical outcomes of patients treated with either modality .", "questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter ."], "abstract_text": ["<S> since its introduction by leksell , gamma knife radiosurgery ( gkrs ) has become increasingly popular as a management approach for patients diagnosed with trigeminal neuralgia ( tn ) . </S>", "<S> for this reason , we performed a modern review of the literature analyzing the efficacy of gkrs in the treatment of patients who suffer from tn . for patients with medically refractory forms of the condition , </S>", "<S> gkrs has proven to be an effective initial and repeat treatment option . </S>", "<S> cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs . </S>", "<S> however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs . </S>", "<S> although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive . </S>", "<S> questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Review of Gamma Knife Radiosurgery for Trigeminal Neuralgia", "3. Treatment Planning and Methods", "4. Conclusions"], "sections": [["trigeminal neuralgia ( tn ) is a disorder of cranial nerve ( cn ) v that results in severe episodes of shock - like or lancinating pain in one or more of its three divisions ( v1v3 ) .", "idiopathic tn and cases due to vascular compression of cn v are categorized as classical tn .", "patients diagnosed with symptomatic tn experience trigeminal - related facial pain secondary to a brain tumor , skull deformity , or multiple sclerosis ( ms ) .", "evidence suggests that the majority of cases of tn are the consequence of focal compression of the entry zone of the root of the trigeminal nerve , while only 2% of cases are observed in patients diagnosed with ms .", "other than excruciating facial pain , there are no other direct medical symptoms associated with tn , and the condition does not decrease life expectancy .", "however , many patients with tn struggle with accomplishing tasks that affect quality of life , which is how this disorder elicits a negative impact on the social and mental wellness of the patients who suffer from this illness .   following the diagnosis of tn", "however , many patients experience only limited relief from medication or are unable to endure the side effects of the prescribed drugs , and in turn seek neurosurgical intervention .", "currently , surgical approaches include microvascular decompression ( mvd ) or a number of techniques that target the trigeminal ganglion or root which involve the destruction or blockage of portions of those anatomical structures [ 1 , 2 ] .", "although the neurosurgical modalities are preferred in many clinical situations and have proven to be effective in achieving initial pain control , they are known to come with a variety of complications , and facial pain recurrence is likely .  ", "stereotactic radiosurgery ( srs ) has proven to be an effective management approach for patients with medically   and surgically   refractory tn as a primary and repeat treatment modality .", "the use of radiosurgery in the treatment of tn dates back to sweden in the 1950 's , where professor lars leksell performed radiogangliotomies directed at the gasserian ganglion .", "since the time of leksell , advancements in radiosurgery and imaging technologies has led to the increasing popularity of srs as a treatment option for patients with tn .", "one form of srs that can be delivered to a patient is through a machine called the gamma knife ( gk ) .", "the gk device is a cobalt-60-based machine , with 201 separate 4 to 18  mm collimator openings , that emits multiple gamma rays that converge on a target specified by computer planning . for specific medication intolerable patient subsets , gamma knife radiosurgery ( gkrs )", "can be used as an initial management approach , or as a secondary management approach following radiosurgery or one or more of the various surgical modalities . as the evidence examining the role of gkrs in the management of patients with tn is increasing ,", "it is of utmost importance for physicians to understand the criteria associated with gkrs , so that the optimal course of treatment for their patients can be prescribed .   an evidence - based review on the evaluation and treatment of tn by gronseth et al .   found level c evidence indicating that gasserian ganglion percutaneous techniques , gkrs , and mvd may be considered for facial pain management for medically refractory patients .", "however , questions remain regarding optimal treatment modalities in specific patient subsets . for this reason ,", "the goal of this paper is to provide a modern review of the literature thoroughly analyzing the efficacy of gkrs in the treatment of patients with tn , as well as evaluating the treatment planning and methods associated with this evolving modality ."], ["to identify contemporary studies assessing the clinical outcomes of patients treated with gkrs for tn , a pubmed search from 2006 to april 2011 was performed .", "keywords for search included  gamma knife or gamma knife radiosurgery or stereotactic radiosurgery trigeminal neuralgia or tic douloureux .", " studies analyzed in this review included retrospective cohort studies and prospective cohort studies with 5 evaluated patients .", "studies published only in abstract form and studies published in a language other than english were excluded from our analysis . due to our broad search strategy and the vast amount of world literature ,", "we reviewed a total of 19 studies [ 4 , 5 , 925 ] analyzing the efficacy of patients with tn who were treated once with gkrs ( table 1 ) .", "thirteen of the 19 evaluated studies [ 4 , 5 , 919 ] utilized the barrow neurological institute ( bni ) pain intensity scale   as a measurement of response to treatment ( see section 3 ) .", "of these 13 studies , only two [ 9 , 13 ] analyzed patients treated with gkrs as an initial management approach . with a median followup of 31 months , sheehan et al .  ", "classified 87% of patients in bni class i - iiib , while chen et al .  ", "classified 91% of patients in bni class i - iiib ( median followup = 15 months ) .", "chen et al .   also reported that five of the 44 patients ( 11% ) treated with gkrs developed hypoesthesia following the procedure .  ", "the other 11 bni pain intensity scale studies we reviewed included patients where previous surgical procedures were performed in a fraction of patients [ 4 , 5 , 1012 , 1519 ] or all patients . of the 10 studies where previous surgical procedures were performed in a fraction of patients , nine reported outcomes in terms of categorizing patients in bni class i - iiib [ 4 , 5 , 1012 , 1518 ] .", "classified 58.6% of patients in bni class i - iiib ( median followup = 48 months ) , kondziolka et al .  ", "classified 71% of patients in bni class i - iiib at three years , dhople et al .  ", "classified 72% of patients in bni class i - iiib ( median followup = 29 months ) , han et al", ".   classified 76.7% of patients in bni class i - iiib ( mean followup = 58 months ) , dhople et al .  ", "classified 81% of patients in bni class i - iiib ( median followup = 5.6 years ) , matsuda et al .  ", "classified 82% of patients in bni class i - iiib ( median followup = 37 months ) , little et al .  ", "classified 83% of patients in bni class i - iiib ( median followup = 6.3 years ) , dellaretti et al .  ", "classified 89.5% of patients in bni class i - iiib ( mean followup = 20.3 months ) , and park and hwang   classified 94% of patients in bni class i - iiib with a minimum followup of 3 years .", "reported clinical outcomes with respect to bni class i , which contained only 5.7% of patients .", "the study that evaluated gkrs where previous surgical procedures were performed in 100% of patients classified 85% of patients in bni class i - iiib , with a median followup of 36 months .", "we also reviewed two studies that used the excellent - good - fair - poor ( egfp ) categorical scale to assess patient outcomes [ 20 , 21 ] ( see section 3 ) .", "treated 30 patients with tn with gkrs at iran gamma knife center between 2006 and 2007 .", "the authors reported that 40% of patients had an excellent outcome , 10% of patients had a good outcome , 33% of patients had a fair outcome , and 17% of patients had a poor outcome following the procedure .", "after surgery , 15 patients ( 54% ) reported an excellent outcome , one patient ( 4% ) reported a good outcome , two patients ( 7% ) reported a fair outcome , and 10 patients ( 36% ) reported a poor outcome .", "the complications from mvd included facial numbness in six patients ( 21% ) , dysesthesias in three patients ( 11% ) , and delayed facial palsy in one patient ( 4% ) .   ", "four of the 19 studies we evaluated [ 2225 ] used other methodologies in determining the effectiveness of gkrs . in a prospective controlled trial ,", "rgis et al .   analyzed 100 patients with tn treated with gkrs and reported that 83 patients ( 83% ) were completely pain free , 58 of which ( 58% ) discontinued all medication following the procedure ( minimum followup = 12 months ) .", "ten patients ( 10% ) experienced radiation - induced complications , which included facial paresthesia or hypesthesia .", "performed a study investigating the short - term efficacy of gkrs in 67 patients with medically refractory tn .", "overall , 77.6% of patients witnessed some degree of pain relief , with 32.6% of those patients becoming completely pain free .", "of the 67 patients , 10 ( 14.9% ) experienced complications from the procedure , which included hypoesthesia and paresthesia .", "sixty eight patients ( 42.5% ) underwent prior invasive treatments . in clinical analysis , it was found that 61% of patients were pain free without medication , 29% of patients were pain free with medication , and 10% of patients did not respond to gkrs .", "thirty eight patients ( 49.4% ) exhibited some level of pain improvement following gk treatment , with 23 of those patients ( 29.9% ) reporting a pain - free outcome .", "twelve patients ( 15.6% ) experienced complications , which were reported to be mild facial sensory changes and mild facial nerve dysfunction .", "as gkrs has proven to be an effective initial treatment for tn , numerous reports have been published analyzing patients treated on multiple occasions ( > 1 ) with gkrs .", "we reviewed six studies evaluating patients treated more than once with gkrs [ 2732 ] ( table 2 ) .", "of these six articles , two [ 29 , 32 ] utilized the bni pain intensity scale .", "sixteen patients ( 76.2% ) exhibited compelling improvements and were placed in bni class i - ii .", "huang et al .   analyzed 65 medically refractory patients with tn who were treated with gkrs as a second treatment modality .", "of these 65 patients , 30 ( 46% ) had undergone gkrs as an initial management approach .", "the authors placed 22 patients ( 34% ) in bni class i , 11 patients ( 17% ) in bni class ii , four patients ( 6% ) in bni class iiia , and five patients ( 8% ) in bni class iiib .", "overall , with a median followup of 64 months , 65% of patients reported successful results in terms of pain control rates .   a total of three of the six reviewed studies evaluated patients using the egfp categorical scale [ 28 , 30 , 31 ] .", "analyzed 37 patients treated a second time with gkrs for recurrent tn and reported that 17 patients ( 46% ) achieved excellent pain relief , nine patients ( 24% ) achieved good pain relief , five patients ( 14% ) achieved fair pain relief , and six patients ( 16% ) achieved poor pain relief .", "however , the authors concluded that 57% of patients experienced some form of trigeminal dysfunction following repeat radiosurgery .", ", huang et al .   treated 28 patients with repeat gkrs and reported that 12 patients ( 43% ) exhibited excellent pain relief , five patients ( 18% ) exhibited good pain relief , and two patients ( 7% ) exhibited fair pain relief .", "in addition , the authors found a statistically significant ( p = 0.047 ) correlation between cumulative radiation doses > 115  gy and facial numbness . in a separate study , huang et al .  ", "specifically , a total of eight patients underwent mvd a mean of 7.6 months following repeat gkrs .", "of the eight patients , seven ( 87.5% ) were completely pain free at a mean of 21 months following neurosurgery .", "this data supports the use of mvd if multiple gk procedures are deemed ineffective .  ", "kimball et al .   treated 53 patients with repeat gkrs and analyzed the patients not lost during followup using the marseille scale , which categorizes patients into one of five classes , with a higher class statistically indicating a worse prognosis for the patient . with a mean", "followup of 42 months , 20 patients ( 43.5% ) were categorized in marseille class i - ii , six patients ( 13% ) were categorized in marseille class iii - iv , and 20 patients ( 43.5% ) were categorized in marseille class v. the authors also reported a statistically significant ( p = 0.047 ) correlation between facial numbness and superior long - term pain relief .", "a total of 22 patients ( 48% ) experienced trigeminal dysfunction of any kind , while 21 patients ( 46% ) experienced numbness in the face .", "since gkrs can be performed as both initial and salvage treatment options for patients who suffer from tn , its efficacy has been compared in patients who undergo one versus multiple radiosurgery procedures .", "we reviewed eight studies to further examine this matter [ 3 , 3339 ] ( table 3 ) .", "four of the eight studies utilized the bni pain intensity scale   to evaluate patient outcomes [ 3 , 3335 ] .", ", it was reported that 75% , 60% , and 58% of patients with idiopathic tn had bni scores of i  iiib at 1 , 3 , and 5 years , respectively .", "the 1- , 3- , and 5-year - bni scores of i  iiib in patients with ms - related tn were 56% , 30% , and 20% , respectively .", "the authors concluded that repeat gkrs exhibited similar success rates when compared to the initial procedure .", "similar to verheul et al . , park et al .   did not find differences in terms of time to initial response , time to pain recurrence , and overall pain relief when comparing patients who underwent one versus two gk treatments .", "however , it was observed that patients who received two gk treatments were more likely to have facial sensory changes when compared to patients treated a single time with radiosurgery .", "little et al .   performed a study where 79 patients with typical tn were treated with gkrs as a salvage procedure .", "approximately five years following salvage gkrs , the authors reported that 50% of patients experienced pain relief and 20% of those patients were completely pain free .", "in addition , a statistically significant ( p = 0.029 ) correlation between gkrs failure and prior mvd was found .", "treated 37 patients ( 78% had failed prior surgery ) with ms - related tn with gkrs .", "nine patients ( 24% ) underwent gkrs as their first procedure . the reported 1 , 3 , and 5 year bni scores of i  iiib were 82.6% , 73.9% , and 54% , respectively .   the other four studies we reviewed utilized the egfp categorical scale as a measurement of response to treatment [ 3639 ] .", "two of the evaluated studies [ 36 , 37 ] were conducted by fountas et al . and analyzed patients treated with gkrs for idiopathic tn based on whether or not they had undergone previous surgical or radiosurgical procedures for facial pain control .", "one of the studies evaluated 106 patients ( 19 previous radiosurgery procedures ) and concluded that the treatment group without a previous history of surgical or radiosurgical procedures exhibited superior clinical outcomes , with 1-year and 2-year complete pain relief rates of 82.5% and 78% , respectively .", "the 1-year and 2-year complete pain relief rate in the patient group with a history of surgical or radiosurgical procedures was 69.4% and 63.5% , respectively .", "as expected , similar results were found in the other study by fountas et al .", "; however , no prior radiosurgical procedures were performed in the patient group with a history of prior procedures .", "huang et al .   conducted a study where 89 patients with idiopathic tn were treated with gkrs as an initial management approach , 20 of which underwent a subsequent gkrs procedure for facial pain recurrence . following the initial radiosurgical procedure ,", "50 patients ( 56% ) had an excellent response , 12 patients ( 13.5% ) had a good response , and 7 patients ( 7.9% ) had a fair response .", "following the second radiosurgical procedure , 11 patients ( 55% ) had an excellent response and one patient ( 5% ) had a good response . in a separate study , huang et al .  ", "assessed 21 patients with benign tumor - related tn who were treated with gkrs as an initial or repeat procedure .", "following the initial gk procedure to the tumor , 12 patients ( 57% ) had an excellent response and 1 patient ( 5% ) had a good response .", "a total of eight patients were treated with a subsequent gkrs procedure targeted at the ipsilateral trigeminal root or ganglion due to facial pain recurrence . following the second radiosurgical procedure , the authors reported four patients ( 50% ) with an excellent response .", "we identified six studies comparing patients treated with gkrs with patients treated with one of the various surgical modalities [ 2 , 4044 ] ( table 4 ) .", "the authors of this review acknowledge the importance of percutaneous techniques in the management of tn ; however , our modern literature search predominantly yielded comparison studies analyzing the efficacy of mvd when compared to gkrs .", "specifically , four of the six studies [ 2 , 4042 ] analyzed patients treated with gkrs against patients treated with mvd .", "specifically , 36 were treated with mvd ( 45% ) , while 44 were treated with gkrs ( 55% ) .", "the mvd treatment arm statistically differed from the gkrs treatment arm with respect to age ( median of 54 versus 74 years ) , preoperative symptom duration ( median of 2.6 versus 7.5 years ) , and the presence of major comorbidities ( 2.8 versus 58.3% ) .", "the authors reported that patients treated with mvd exhibited superior levels of initial ( 100% ) , 2 year ( 88% ) , and 5 year ( 80% ) actuarial pain - free rates when compared to the patients treated with gkrs ( 78 , 50 , and 33% , resp . ) , with a p value of 0.0002 .", "in addition to increased levels of patient satisfaction , as reported by required patient surveys , the mvd treatment group also had a decreased level of permanent mild ( 5.6% ) and severe sensory loss ( 0% ) when compared to the gkrs treatment group ( 6.8% and 2.3% , resp . ) .", "two patients ( 5.6% ) in the mvd group experienced permanent mild paresthesias or numbness , one ( 2.8% ) patient experienced a cerebrospinal fluid leak from the wound , and one patient ( 2.8% ) experienced hearing loss and diplopia .", "three patients ( 6.8% ) in the gkrs group experienced permanent mild paresthesias or numbness , one patient ( 2.3% ) experienced a more permanent sensory numbness , and one patient ( 2.3% ) experienced a transient headache and nausea following the gk procedure .  ", "all patients were diagnosed with typical tn and did not undergo previous gk or mvd procedures . it was reported that patients treated with mvd exhibited superior levels of complete pain relief at 12 ( 68% ) and 18 months ( 68% ) when compared to the gkrs group , who 's complete pain relief rate was 58% at 12 months and 24% at 18 months ( p = 0.089 ) .", "the treatment arms did not statistically differ in terms of 90% pain relief at 12 and 18 months .", "this study could be criticized due to the large difference in the number of patients constituting the two treatment arms .  ", "oh et al .   evaluated a total of 45 elderly patients ( > 65 years of age ) diagnosed with idiopathic tn who were treated with either mvd ( 27 patients ) or gkrs ( 18 patients ) .", "it was reported that three mvd patients ( 11% ) and three gk patients ( 17% ) underwent previous percutaneous procedures .", "the mean followup period was reported to be 35.9 months for the mvd group and 33.1 months for the gkrs group . according to the bni pain intensity scale ,", "the mvd group had a superior prognosis , with 17 patients ( 63% ) classified in bni class i - ii compared with the 10 patients ( 56% ) in the gkrs group classified in bni class i - ii .", "the observed complications following mvd included constant headache in 11 patients ( 40.7% ) , facial paresthesia in five patients ( 18.5% ) , paresthesia of the tongue in two patients ( 7.4% ) , infection at the site of incision in one patient ( 3.7% ) , an acute subdural hemorrhage in one patient ( 3.7% ) , temporary hearing loss in one patient ( 3.7% ) , and otitis media with cerebrospinal leakage in one patient ( 3.7% ) . two patients ( 11% ) in the gkrs group experienced paresthesia .  ", "compared the clinical outcomes of 19 patients treated with mvd with 15 patients treated with gkrs .", "nine gk patients ( 60% ) and four mvd patients ( 21% ) underwent previous surgical procedures . the treatment", "arms statistically differed ( p = 0.0005 ) with respect to mean patient age , with the mean age of the gkrs group exceeding the mvd group by 13 years ( 74 versus 61 years ) .", "in addition , patient satisfaction was graded on a scale of 1 ( unsatisfied ) to 10 ( completely satisfied ) .", "it was reported that the mean tn complexity grade was statistically different ( p < 0.001 ) between the treatment arms ( gk = 5.8 ; mvd = 3 ) .", "the average response following the procedure was determined to be 3.4 for the mvd group and 2.4 for the gkrs group ( p = 0.017 ) . also , it was found that the satisfaction score for the mvd group was superior to the gkrs group ( 8.7 versus 6.4 ) , with a p value of 0.02 .", "the authors reported a statistically significant correlation between tn complexity grade and clinical response ( p < 0.001 ) , as well as tn complexity grade and patient satisfaction ( p < 0.001 ) .   to date", ", no randomized trials have been conducted analyzing the outcomes of patients with tn who are treated with mvd compared to gkrs . in a large review on tn management ,", "zakrzewska and linskey   found evidence that mvd is an effective treatment for long - term facial pain relief but comes with an increased risk of ipsilateral hearing loss .", "in addition , the authors concluded that single - dose srs is an effective treatment for long - term facial pain relief but puts patients at risk for facial numbness or facial paresthesias .", "investigation into this matter in the form of a randomized controlled trial would provide the best evidence in terms of facial pain relief and procedure - related complications .  ", "in addition , we reviewed two studies comparing patients treated with gkrs with patients treated with posterior fossa exploration ( pfe ) [ 43 , 44 ] , both of which were conducted by pollock and colleagues at the mayo clinic college of medicine .", "one of the studies   was a specific prospective comparison of 91 patients treated with pfe and 49 patients treated with gkrs for idiopathic tn as an initial management approach .", "the treatment arms statistically differed in terms of age ( gkrs = 67.1 years ; pfe = 58.2 years ) , with a p value < 0.001 .", "it was reported that patients treated with pfe were more likely to be pain free and off medications at 1 year ( 84% ) and 4 years ( 77% ) when compared to the gkrs group ( 66 and 56% , resp . )", "retreatment for recurrent facial pain was performed in 15% of the patients in the pfe treatment arm and 35% of patients in the gkrs treatment arm ( p = 0.009 ) .", "also , it was found that nonbothersome facial numbness occurred more frequently in the gkrs group ( p = 0.04 ) .", "an additional study from the mayo clinic evaluated patients with recurrent tn who underwent 3 or more surgical procedures .", "the authors reported that patients treated with pfe exhibited superior levels of complete pain relief at 3 years of followup when compared to patients treated with gkrs , balloon compression , and glycerol rhizotomy ( p < 0.01 ) and underwent additional surgery for recurrent facial pain less often when compared to patients treated with the other modalities ( p = 0.02 ) .", "clinical 006futcomes did not differ between patients treated with gkrs and patients treated with the percutaneous techniques ."], ["srs can be performed by a variety of tools , which include gkrs , cyberknife technology , and linear accelerator ( linac)-based treatment .", "our analysis yielded one study whose primary endpoint was to devise a method using cyberknife treatment planning that would mimic the dosimetric characteristics of the gk treatment plan in five patients undergoing radiosurgery for tn .", "both the isodose lines and critical structures were identified using the gkrs treatment plan and were transferred to the cyberknife treatment planning system .", "it was reported that the average length of the trigeminal nerve receiving a dose of 60  gy was 4.5  mm for the gk , 4.5  mm for the nonisocentric cyberknife , and 4.4  mm for the isocentric cyberknife .", "the authors found it more difficult to minimize the dose to critical structures when using cyberknife technology .", "also , the dose falloff of gkrs was found to be steeper when compared to cyberknife technology due to , what the authors hypothesized , the large number of gamma rays produced which converge on the focal point with precision .  ", "as previously mentioned , the gk machine 's primary functional unit is cobalt-60 , which is used to emit photon energy through 201 separate 4 to 18  mm collimator openings that converge on a target specified by a treatment planning system .", "balamucki et al .   performed a study examining if the half life of cobalt ( 5.26 years ) relates to the outcomes for patients being treated for tn with gkrs .", "the authors collected data on 239 gkrs procedures performed at their institution between 1999 and 2004 .", "patient surveys were used to measure responses to radiosurgery . with the followup time ranging from one to six months , it was reported that 80% of patients experienced some degree of pain relief and that 56% of those patients were pain free .", "the authors concluded that clinical outcomes remained consistent during the first half life of cobalt-60 .", "an area of controversy in the treatment of patients with tn is defining the optimal maximum radiosurgery dose that can be delivered to specific patient subsets .", "we analyzed five studies whose primary endpoint was to assess gkrs - dosing efficacy [ 4852 ] .", "kim et al .   utilized the bni pain intensity scale   to assess 66 tn patients treated with a gk maximum dose of 80  gy and 44 tn patients treated with a gk dose of 85  gy . although the two groups did not statistically differ in terms of facial rain relief and procedure - related complications , the authors did report that patients treated with a gk dose of 85  gy experienced a more rapid response to treatment when compared to the patients treated with a gk dose of 80  gy .", "arai et al .   analyzed 165 patients with tn treated with a gkrs dose of 80  gy .", "specifically , the authors divided the patients into two groups , which differed in the radiation dose rate received ( low - dose rate = 1.212.05  gy / min ; high - dose rate = 2.063.74  gy / min ) . using the bni pain intensity scale   as a clinical evaluation method", ", it was reported that the low - dose - rate group and the high - dose - rate group did not statistically differ in terms of initial pain relief , maintenance of pain relief , and clinical complications .  ", "patients in group one were treated with a gk dose < 90  gy with no beam channel plugging , patients in group two were treated with a gk dose equal to 90  gy with no beam channel plugging , and patients in group three were treated with a gk dose equal to 90  gy with beam channel plugging . although the trend did not reach full statistical significance ( p = 0.054 ) , patients in group three exhibited the highest level of pain relief , while patients in group one exhibited the lowest level of pain relief .", "the authors also observed that the three groups statistically differed ( p < 0.0001 ) in terms of trigeminal nerve dysfunction , with patients in group three experiencing the highest rate of mild and bothersome complications and patients in group one experiencing the lowest rate of mild and bothersome complications .", "similar to the results of massager et al . , morbidini - gaffney et al .   reported positive outcomes in patients treated with gk doses > 85  gy .", "the authors also found that patients treated with two isocenters were more likely to have superior bni pain intensity scale   scores during their course of followup when compared to patients treated with a single isocenter .  ", "the median initial dose was 80  gy , and the median retreatment dose was 45  gy . although the authors did not report any predictors in terms of facial pain control and patient morbidity , they did compare the results of their study with seven published retreatment articles and found that successful levels of pain control ( > 50% ) were significantly correlated with cumulative gkrs doses > 130  gy , as well as new trigeminal nerve dysfunction ( > 20% ) . in addition to dose selection efficacy in select patient cohorts , the radiosurgical target of cn v is another subject matter that requires further clinical investigation .", "we reviewed three studies [ 5355 ] analyzing specific gkrs targeting methods in the treatment of tn and one study   that examined the accuracy of gkrs to its image - guided target .", "compared patients treated with gkrs targeted at the dorsal root entry zone ( 59 patients ) with patients whose radiosurgical target was the retrogasserian zone of the trigeminal nerve ( 41 patients ) . with a median followup of 30 months , the dorsal root entry target group exhibited statistically superior levels of initial complete pain remission ( p = 0.003 ) and experienced less complications than the retrogasserian zone group ( p = 0.009 ) .", "chen et al .   also reported positive results with the dorsal root entry zone - targeting approach , with a success rate of 82.8% and a complication rate of 15% .", "park et al .   compared the dorsal root entry zone and retrogasserian zone - targeting methods in the treatment of 39 patients with medically refractory tn .", "the authors reported that the two treatment arms did not statistically differ in treatment success ( bni class i - iiib ) with respect to the bni pain intensity scale .", "however , patients treated with the retrogasserian zone - targeting method experienced a substantially shorter time of response to gkrs than patients treated with the dorsal root entry zone - targeting method ( p = 0.044 ) .", "although the two groups did not statistically differ with regard to treatment - related morbidities , it was found that the patients whose targeting approach was the dorsal root entry zone experienced a greater amount of bothersome complications than the retrogasserian zone group .  ", "massager et al .   analyzed the targeting accuracy of gkrs in 65 patients treated for tn whose six month followup mri showed focal contrast enhancement of the trigeminal nerve .", "the authors found that the median deviation of the coordinates between the intended radiosurgical target and the center of contrast enhancement was 0.91  mm in euclidean space .", "the median radiation dose fitting into the contrast enhancement region was determined to be 77  8.7  gy .", "this small deviation from the gkrs target explains the high accuracy and precise nature of the machine .", "the two most common methods of measuring patient outcomes from gkrs in the management of tn are the barrow neurological institute pain intensity scale   ( table 5 ) and the excellent - good - fair - poor ( egfp ) categorical scale ( table 6 ) .", "the bni pain intensity scale divides patients into one of five classes , with a higher class indicating a worse prognosis for the patient .", "patients in bni class iiia do not experience trigeminal pain but require the use of medication .", "patients in bni class iiib experience some trigeminal pain that can be satisfactorily managed with medication . patients in bni class iv experience some trigeminal pain that is not satisfactorily managed with medication . patients in bni class", " excellent  outcomes are defined as complete pain relief without the need of medication . ", "good  outcomes are defined as complete pain relief with the need of medication . ", "poor  outcomes are defined as a < 50% pain relief rate or treatment failure ."], ["for patients with medically refractory forms of tn , gkrs has proven to be an effective initial and repeat treatment option .", "cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs .", "however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs .", "although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive .", "further evidence in the form of a phase iii - randomized trial is needed to confirm the clinical outcomes of patients treated with either modality .", "questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter ."]]}
{"article_id": "PMC3427810", "article_text": ["a 55-year - old caucasian female was undergoing elective coronary artery bypass surgery following a recent myocardial infarction .", "intravenous midazolam was given to the patient in the holding area , and the right radial artery was cannulated . in the operating room , intraoperative monitors , included five - lead electrocardiography , pulse oximetry , a radial artery catheter , capnography ( etco2 ) , and bladder temperature .", "the patient was intubated without any difficulty after routine intravenous induction with etomidate , sufentanil , and rocuronium .", "the right internal jugular vein was cannulated with the patient in the trendelenburg position , and a pulmonary artery catheter was advanced to the wedge position without difficulty .", "additional monitors included a pulmonary artery catheter with venous saturation and continuous cardiac output monitoring as well as central venous pressure .", "an adult multiplane 6.0 mhz transesophageal echocardiography ( tee ) probe ( acuson , siemens , washington , dc , usa ) was placed .", "systematic tee images were obtained according to the american society of echocardiography - society of cardiovascular anesthesiologists guidelines .", "as the cardiac surgeon harvested the left internal mammary artery , the first assistant harvested the saphenous vein from the left leg via an endoscopic approach ( stryker endoscopy , san jose , ca , usa ) using co2 insufflation at 4 l / min and a pressure of 14 mmhg .", "approximately 20 minutes after endoscopic vein harvesting started , we noticed the patient 's end - tidal co2 ( etco2 ) had increased from 36 to 44 mmhg followed by increasing pulmonary artery pressure from 42/20 to 60/39 mmhg within 1 minute .", "subsequently , systemic blood pressure dropped from 101/60 mmhg to 65/30 mmhg while o2 saturation remained at 100% .", "the cardiac index dropped approximately 15% from 2.2 to 1.9 l / min / m , but heart rate and central venous pressure were only slightly elevated .", "the inspiratory oxygen concentration fraction was adjusted from 50% to 100% , and vasopressors were administered to support the pressure .", "mid - esophageal four - chamber and mid - esophageal aortic valve long - axis views were examined , but no new mitral or aortic regurgitation was noted .", "however , the mid - esophageal right ventricular inflow - outflow view revealed a massive snowflake appearance suggestive of gas bubbles from the right atrium to the right ventricle ( fig .", "however , mild dilatation of the right ventricle was noted . at that time , all intravenous fluids through the central line were stopped , and all intravenous connections were checked to make sure no intravenous fluids were running . despite these maneuvers , the snowflake appearance suggestive of gas bubbles was still present .", "we concluded that the gas bubbles were not caused from turbulence due to intravenous fluids or from a disconnected intravenous line , so a co2 embolism was suspected .", "after this observation , we kept the patient in the trendelenburg position and increased the ventilation rate to expire more co2 . a nitroglycerine drip was started to lower pulmonary artery pressure , and phenylephrine boluses were administered to maintain systemic blood pressure within an acceptable range .", "further examination of tee in the mid - esophageal bicaval view revealed that the gas bubbles were originating from the inferior vena cava ( fig .", ", we concluded that the source of the gas embolism was most likely from co2 insufflation .", "the surgical team was informed of these findings and they reassured us that the proximal saphenous vein was completely ligated and confirmed that most branches were adequately cauterized .", "the surgical team requested 5 additional minutes to complete vein harvesting based on patient stability .", "our hemodynamic assessment revealed that the patient was stable , and the tee examination revealed a significant reduction in air bubbles , so the decision was made to proceed with the endoscopic vein harvesting .", "over that course , the patient remained on 100% fio2 , and a higher ventilation rate .", "the nitroglycerin drip was discontinued and intermittent small epinephrine boluses were administered for inotropic support , and phenylephrine or ephedrine were administered to maintain systemic blood pressure > 90/50 mmhg .", "the patient 's hemodynamic status improved shortly after vein harvesting was completed , and co2 insufflation was discontinued .", "we noted the patient 's etco2 had returned back to 35 mmhg , pulmonary artery pressure had decreased steadily back to a baseline level of 40/20 mmhg , and continued down to 30/18 mmhg , whereas systemic blood pressure stabilized at 100/60 mmhg without vasopressor support .", "a tee examination in the mid - esophageal right ventricular inflow - outflow view and mid - esophageal bicaval view revealed no further evidence of gas embolism .", "coronary artery bypass grafting was completed without further events , and the patient recovered without any adverse neurological sequelae .", "our case not only confirms the vital role of tee in the quick and accurate diagnosis of a co2 embolism during endoscopic vein harvesting but also allowed us to focus on the mid - esophageal bicaval view , a view not commonly used to identify a co2 embolism .", "further review of the mid - esophageal right ventricular inflow - outflow view revealed that gas was entering the right side of the heart , but we could not determine whether the bubbles originated from the superior vena cava or the inferior vena cava . using the mid - esophageal bicaval view , we were able to confirm that the gas embolism originated from the inferior vena cava , and that the lower extremity endoscopic site was the most likely suspect .", "the era of minimally invasive surgery started two decades ago and has extended its influence into cardiac surgery .", "the first endoscopic vein harvesting was described by lumsden et al .   in 1994 .", "the endoscopic technique is associated with a shorter time ( 51.07 vs. 75.94 min ) , better leg wound healing , and a lower incidence of wound infections ( 12.0% vs. 8.8% ) compared to those of conventional open vein harvesting .", "other advantages of endoscopic vein harvesting compared to conventional open vein harvesting include a smaller incision , less post - operative pain , a shorter hospital stay , and better cosmetic appearance .", "the endoscopic vein harvesting procedure requires co2 insufflation at a flow of 4 l / min and pressure of approximately 14 mmhg to create a potential subcutaneous space .", "in contrast , there have been reports of co2 embolism , pneumothorax , subcutaneous emphysema , and hypercarbia .", "the reported incidence rate of co2 embolism ranges from 1 - 200 cases in every one million laparoscopic surgeries .", "the incidence of a co2 embolism during endoscopic saphenous vein harvesting with co2 insufflation procedures decreases as the severity of clinical symptoms and the volume of embolized gas increases .", "the incidence of venous co2 embolism of any size is 17.1% , whereas the incidence of massive gas emboli is only 0.5% .", "the incidence of a co2 embolism is even lower when an endoscopic approach is used to harvest arterial conduits . in our hospital", ", the endoscopic vein harvesting technique has been used in approximately 1,500 cases since 2005 , with no reported incidence of a co2 embolism .", "manifestations of a co2 embolism include hypotension , reduced cardiac output , and decreased mixed venous oxygen saturation , along with elevated pulmonary artery and central venous pressures .", "however , this sign may not be as dependable as was the case in our patient , as we observed an initial rise in etco2 instead of a reduction .", "a drop in etco2 is an indication of increased dead space and insufficient pulmonary perfusion .", "this condition does not generally occur unless the trapped gas in the pulmonary vascular system or the right ventricular outflow prevents blood from flowing into the lung , a condition that is generally observed with a massive embolism . whenever co2 is used for endoscopic insufflation , co2", "can directly enter the injured saphenous vein or its branches , resulting in elevated etco2 .", "furthermore , a smaller amount of co2 embolized to the right heart could be released acutely , resulting in elevated etco2 . as was seen in our case ,", "treatment for a co2 embolism includes immediate discontinuation of co2 insufflation , placing the patient in the trendelenburg position , increasing fio2 to 100% , and aspirating the central line .", "the latter intervention is less successful with a pulmonary artery catheter and may divert the attention of the anesthesia team from other rescue efforts . furthermore , inotropic agents should be started to support cardiac output to push the co2 embolism into the lungs .", "cardiac massage and even urgent cardio - pulmonary bypass or extracorporeal membrane oxygenation have also been described if the patient does not respond to initial resuscitation efforts . in our case", ", we choose to allow continued co2 insufflation to complete saphenous vein harvesting based on the patients stabile hemodynamics and on the observation that a reduction in gas entry to the right heart occurred shortly after detecting the co2 embolism .", "this finding was interesting , as the surgical team did not reduce insufflation flow or pressure .", "we believe that the source of co2 absorption was eliminated at this point , as the surgical team cauterized the open vascular structures .", "in summary , the use of endoscopic vein harvesting for the saphenous vein has become increasingly more common and awareness and prevention of the potential for a co2 embolism is important . as seen in our case , tee played a vital role in detection , and we believe that the mid - esophageal bicaval view should be used as the standard view during endoscopic vein harvesting .", "it has been suggested that the surgical team should pay special attention when cauterizing or ligating all vein branches to reduce the potential for co2 entry into the venous system to prevent further co2 embolisms .", "although we allowed endoscopic vein harvesting to continue after confirmation of the co2 embolism with direct inspection of the saphenous - femoral vein junction and meticulous closure of the tears were confirmed , further investigation into the safety of this practice is warranted ."], "abstract_text": ["<S> a carbon dioxide ( co2 ) embolism during endoscopic vein harvesting is a rare but potentially fatal complication . </S>", "<S> early and accurate diagnosis is crucial for limiting the extent of the embolism and stabilizing the resulting cardiovascular compromise . </S>", "<S> we report a case of co2 embolization during endoscopic vein harvesting . </S>", "<S> transesophageal echocardiography was instrumental in the diagnosis and management of this patient by further improving the decision making process , which resulted in the best outcome . </S>", "<S> mid - esophageal bicaval view is the best view to determine whether a co2 embolism is coming from the upper or lower extremities . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 55-year - old caucasian female was undergoing elective coronary artery bypass surgery following a recent myocardial infarction .", "intravenous midazolam was given to the patient in the holding area , and the right radial artery was cannulated . in the operating room , intraoperative monitors , included five - lead electrocardiography , pulse oximetry , a radial artery catheter , capnography ( etco2 ) , and bladder temperature .", "the patient was intubated without any difficulty after routine intravenous induction with etomidate , sufentanil , and rocuronium .", "the right internal jugular vein was cannulated with the patient in the trendelenburg position , and a pulmonary artery catheter was advanced to the wedge position without difficulty .", "additional monitors included a pulmonary artery catheter with venous saturation and continuous cardiac output monitoring as well as central venous pressure .", "an adult multiplane 6.0 mhz transesophageal echocardiography ( tee ) probe ( acuson , siemens , washington , dc , usa ) was placed .", "systematic tee images were obtained according to the american society of echocardiography - society of cardiovascular anesthesiologists guidelines .", "as the cardiac surgeon harvested the left internal mammary artery , the first assistant harvested the saphenous vein from the left leg via an endoscopic approach ( stryker endoscopy , san jose , ca , usa ) using co2 insufflation at 4 l / min and a pressure of 14 mmhg .", "approximately 20 minutes after endoscopic vein harvesting started , we noticed the patient 's end - tidal co2 ( etco2 ) had increased from 36 to 44 mmhg followed by increasing pulmonary artery pressure from 42/20 to 60/39 mmhg within 1 minute .", "subsequently , systemic blood pressure dropped from 101/60 mmhg to 65/30 mmhg while o2 saturation remained at 100% .", "the cardiac index dropped approximately 15% from 2.2 to 1.9 l / min / m , but heart rate and central venous pressure were only slightly elevated .", "the inspiratory oxygen concentration fraction was adjusted from 50% to 100% , and vasopressors were administered to support the pressure .", "mid - esophageal four - chamber and mid - esophageal aortic valve long - axis views were examined , but no new mitral or aortic regurgitation was noted .", "however , the mid - esophageal right ventricular inflow - outflow view revealed a massive snowflake appearance suggestive of gas bubbles from the right atrium to the right ventricle ( fig .", "however , mild dilatation of the right ventricle was noted . at that time , all intravenous fluids through the central line were stopped , and all intravenous connections were checked to make sure no intravenous fluids were running . despite these maneuvers , the snowflake appearance suggestive of gas bubbles was still present .", "we concluded that the gas bubbles were not caused from turbulence due to intravenous fluids or from a disconnected intravenous line , so a co2 embolism was suspected .", "after this observation , we kept the patient in the trendelenburg position and increased the ventilation rate to expire more co2 . a nitroglycerine drip was started to lower pulmonary artery pressure , and phenylephrine boluses were administered to maintain systemic blood pressure within an acceptable range .", "further examination of tee in the mid - esophageal bicaval view revealed that the gas bubbles were originating from the inferior vena cava ( fig .", ", we concluded that the source of the gas embolism was most likely from co2 insufflation .", "the surgical team was informed of these findings and they reassured us that the proximal saphenous vein was completely ligated and confirmed that most branches were adequately cauterized .", "the surgical team requested 5 additional minutes to complete vein harvesting based on patient stability .", "our hemodynamic assessment revealed that the patient was stable , and the tee examination revealed a significant reduction in air bubbles , so the decision was made to proceed with the endoscopic vein harvesting .", "over that course , the patient remained on 100% fio2 , and a higher ventilation rate .", "the nitroglycerin drip was discontinued and intermittent small epinephrine boluses were administered for inotropic support , and phenylephrine or ephedrine were administered to maintain systemic blood pressure > 90/50 mmhg .", "the patient 's hemodynamic status improved shortly after vein harvesting was completed , and co2 insufflation was discontinued .", "we noted the patient 's etco2 had returned back to 35 mmhg , pulmonary artery pressure had decreased steadily back to a baseline level of 40/20 mmhg , and continued down to 30/18 mmhg , whereas systemic blood pressure stabilized at 100/60 mmhg without vasopressor support .", "a tee examination in the mid - esophageal right ventricular inflow - outflow view and mid - esophageal bicaval view revealed no further evidence of gas embolism .", "coronary artery bypass grafting was completed without further events , and the patient recovered without any adverse neurological sequelae ."], ["our case not only confirms the vital role of tee in the quick and accurate diagnosis of a co2 embolism during endoscopic vein harvesting but also allowed us to focus on the mid - esophageal bicaval view , a view not commonly used to identify a co2 embolism .", "further review of the mid - esophageal right ventricular inflow - outflow view revealed that gas was entering the right side of the heart , but we could not determine whether the bubbles originated from the superior vena cava or the inferior vena cava . using the mid - esophageal bicaval view , we were able to confirm that the gas embolism originated from the inferior vena cava , and that the lower extremity endoscopic site was the most likely suspect .", "the era of minimally invasive surgery started two decades ago and has extended its influence into cardiac surgery .", "the first endoscopic vein harvesting was described by lumsden et al .   in 1994 .", "the endoscopic technique is associated with a shorter time ( 51.07 vs. 75.94 min ) , better leg wound healing , and a lower incidence of wound infections ( 12.0% vs. 8.8% ) compared to those of conventional open vein harvesting .", "other advantages of endoscopic vein harvesting compared to conventional open vein harvesting include a smaller incision , less post - operative pain , a shorter hospital stay , and better cosmetic appearance .", "the endoscopic vein harvesting procedure requires co2 insufflation at a flow of 4 l / min and pressure of approximately 14 mmhg to create a potential subcutaneous space .", "in contrast , there have been reports of co2 embolism , pneumothorax , subcutaneous emphysema , and hypercarbia .", "the reported incidence rate of co2 embolism ranges from 1 - 200 cases in every one million laparoscopic surgeries .", "the incidence of a co2 embolism during endoscopic saphenous vein harvesting with co2 insufflation procedures decreases as the severity of clinical symptoms and the volume of embolized gas increases .", "the incidence of venous co2 embolism of any size is 17.1% , whereas the incidence of massive gas emboli is only 0.5% .", "the incidence of a co2 embolism is even lower when an endoscopic approach is used to harvest arterial conduits . in our hospital", ", the endoscopic vein harvesting technique has been used in approximately 1,500 cases since 2005 , with no reported incidence of a co2 embolism .", "manifestations of a co2 embolism include hypotension , reduced cardiac output , and decreased mixed venous oxygen saturation , along with elevated pulmonary artery and central venous pressures .", "however , this sign may not be as dependable as was the case in our patient , as we observed an initial rise in etco2 instead of a reduction .", "a drop in etco2 is an indication of increased dead space and insufficient pulmonary perfusion .", "this condition does not generally occur unless the trapped gas in the pulmonary vascular system or the right ventricular outflow prevents blood from flowing into the lung , a condition that is generally observed with a massive embolism . whenever co2 is used for endoscopic insufflation , co2", "can directly enter the injured saphenous vein or its branches , resulting in elevated etco2 .", "furthermore , a smaller amount of co2 embolized to the right heart could be released acutely , resulting in elevated etco2 . as was seen in our case ,", "treatment for a co2 embolism includes immediate discontinuation of co2 insufflation , placing the patient in the trendelenburg position , increasing fio2 to 100% , and aspirating the central line .", "the latter intervention is less successful with a pulmonary artery catheter and may divert the attention of the anesthesia team from other rescue efforts . furthermore , inotropic agents should be started to support cardiac output to push the co2 embolism into the lungs .", "cardiac massage and even urgent cardio - pulmonary bypass or extracorporeal membrane oxygenation have also been described if the patient does not respond to initial resuscitation efforts . in our case", ", we choose to allow continued co2 insufflation to complete saphenous vein harvesting based on the patients stabile hemodynamics and on the observation that a reduction in gas entry to the right heart occurred shortly after detecting the co2 embolism .", "this finding was interesting , as the surgical team did not reduce insufflation flow or pressure .", "we believe that the source of co2 absorption was eliminated at this point , as the surgical team cauterized the open vascular structures .", "in summary , the use of endoscopic vein harvesting for the saphenous vein has become increasingly more common and awareness and prevention of the potential for a co2 embolism is important . as seen in our case , tee played a vital role in detection , and we believe that the mid - esophageal bicaval view should be used as the standard view during endoscopic vein harvesting .", "it has been suggested that the surgical team should pay special attention when cauterizing or ligating all vein branches to reduce the potential for co2 entry into the venous system to prevent further co2 embolisms .", "although we allowed endoscopic vein harvesting to continue after confirmation of the co2 embolism with direct inspection of the saphenous - femoral vein junction and meticulous closure of the tears were confirmed , further investigation into the safety of this practice is warranted ."]]}
{"article_id": "PMC2980652", "article_text": ["drywood termites cause significant damage to wood in structures in the united states , with two species , incisitermes minor ( hagen ) and cryptotermes brevis ( walker ) , being responsible for the majority of damage ( su and scheffrahn , 1990 ; grace , 2009 ) .", "the economic cost of control and repair of damage is second only to that for subterranean termites ( su and scheffrahn , 1990 ) .", "remedial control of drywood termites in the united states relies primarily on either ( 1 ) fumigation of the entire structure with a toxic gas or heated air , or ( 2 ) localized chemical or physical treatments designed to eradicate small , localized colonies ( lewis and haverty , 1996 ; lewis , 2008 ; lewis and rust , 2009 ) .", "fumigation treatments are likely to kill all of the termites in a structure , while localized treatments can destroy all termites in a colony , provided the treatment is delivered to the entire gallery system .", "the size , number , and dispersion of drywood termite colonies in a structure can vary greatly , depending on the age of the infestation and the success of preventative or remedial treatments .", "drywood termite colonies also are considered single piece nesters , i.e. , they nest within their food source and do not forage from the nesting site to a food source ( abe , 1987 ) .", "in fact , large , dispersed colonies ( or even small colonies ) can live in a single piece of wood or in multiple pieces of wood that are joined together ( grace et al . ,", "drywood termites are cryptic , seldom leaving obvious or visible external signs of their presence in wood . a commonly used sign for determining the presence of drywood termites is the occurrence of fecal pellets , ejected through a  kick - out hole  in the external surface of wood from the internal galleries ( ebeling , 1975 ) .", "these pellets often are found as conical piles or are scattered on horizontal surfaces below infested wood .", "these hexagonally sided pellets are diagnostic for drywood termites , and can be used to distinguish damage from that by other wood - destroying insects ( ebeling , 1975 ; moore , 1992 ) .", "grace and yamamoto ( 2009 ) demonstrated the relationship between the cellulose and lignin content of the food utilized by small groups of c. brevis and incisitermes immigrans ( light ) , and the quantity of fecal pellets produced over time .", "they also discussed the use of the size and number of fecal pellets for estimating both size and age of drywood termite colonies .", "drywood termites excrete feces in the form of hard , even - shaped fecal pellets .", "these fecal pellets contain the same mixture of hydrocarbons as the insects that produced them , albeit in slightly different proportions ( haverty et al .", ", 2005 ) . because cuticular hydrocarbons are species specific in termites ( page et al . , 2002 )", "rather than simply signaling the general presence of termites or providing a diagnosis of the species of termite inhabiting the wood , we postulated that these pellets could be chemically characterized so as to determine the status of a colony as active ( alive ) or inactive ( dead ) . here , we report quantification of the hydrocarbons in pellets of i. minor aged for up to 1  year after they were produced .", "we document the changes in proportions of selected hydrocarbons as an indication of the length of time since the pellets were excreted .", "collection of termites and preparation of termite containment unit termites , i. minor , were removed from one naturally infested board ( 98.9    13.3    271.8  cm ) collected on 19 july 2006 from lakeview , california , and stored at the university of california richmond field station .", "the board was cut across the grain into pieces , 58  cm thick , and stored at room temperature .", "a wood chisel was used to separate pieces of wood into smaller pieces , 0.51.0  cm thick .", "all remaining live termites , including soldiers and primary reproductives ( alates ) , were placed in a termite containment unit ( tcu ) .", "termites thus collected were likely from a large mixed colony ( booth et al . , 2010 ) or from multiple colonies .", "we were not able to assign the various galleries in the wood to any particular colony.fifteen birch tongue depressors were bundled together , and three equally spaced holes drilled across the length of the bundle using a 2.8-mm bit .", "bamboo skewers , 1013  cm in length , were inserted into each of the three holes in the bundle of tongue depressors .", "spaces , 36  mm , were left between each tongue depressor allowing termites access to all tongue depressors .", "two sets of skewered tongue depressors were placed into a clean plastic container ( 17.5    12.5    6  cm ) , one on top of the other , such that the tongue depressors on the top layer fit into the gaps created by the tongue depressors on the bottom layer .", "tongue depressors were lightly misted with water , and then 6,662 drywood termites ( a mixture of pseudergates or workers , alates , and soldiers ) were placed in the tcu .", "tcus , with termites , were maintained in a dark cabinet in the laboratory under ambient conditions prior to collection of fecal pellets .", "a single colony or a mixture of two or more colonies , representing a single location within california , was prepared in this manner . pellet collection and aging process", "after all the tcus had acclimated to laboratory conditions over several weeks , new holding chambers were prepared .", "all wood debris , pellets , dead termites , and damaged tongue depressors were discarded .", "the termites in the new holding chambers were maintained in the laboratory under ambient conditions for 2  weeks . at the end of this period", ", all pellets were removed , and sub - samples collected and readied for the aging study .", "concurrently , three samples of 20 workers , three samples of 20 alates , and one sample of 20 soldiers were also collected for hydrocarbon analysis .", "live termites were placed in a 20-ml scintillation vial and frozen until extraction.fecal pellets were separated from debris by sequentially sifting them through successively smaller sieves ( haverty et al . , 2005 ) .", "thus , all substances such as hand lotion , lip balm , parafilm , and waxed paper were kept away from the work area .", "gloves were worn when handling pellets , and implements that came in contact with pellets were wiped with a paper towel dampened with absolute ethanol to remove any oils or waxes .", "pellets were separated from fine debris by removing individual pellets with forceps or with an aspirator , until a sample weighing approximately 200  mg was obtained.in developing this protocol we made one estimate and one critical assumption .", "the estimate was that at least 1,000 pellets were needed for each hydrocarbon analysis ( based on preliminary analyses of pellets collected from drywood termites maintained in our laboratory ) .", "( 1997 ) using c. brevis and i. snyderi ( light ) from southern florida . in that study , 40 i. snyderi ( pseudergates or workers ) produced 573 pellets over 4  weeks , slightly less than 150 pellets / wk or about 3.5 pellets / individual / wk . in a study with small groups of c. brevis and i. immigrans , grace and yamamoto ( 2009 ) found that these species produced 4.9 to 7.0 pellets / individual / wk . for our study , we assumed that i. minor would produce pellets at roughly the same rate and , therefore , we needed about 3,000 to 5,000 individuals in a tcu for this study .", "we prepared one tcu to collect the requisite quantity of fecal pellets.four sub - sampling intervals , each replicated three times , were used : 0 , 30 , 90 , and 365  days from the initial collection date .", "sub - samples were stored in clean , 20-ml scintillation vials , sealed with 1.5    1.5  mm mesh screen .", "vials were stored in a dark cabinet at the university of california richmond field station at ambient temperature.voucher samples of i. minor pseudergates or workers and soldiers from this study ( fresh , not dried ) were preserved in 85% ethanol and deposited in the essig museum , university of california at berkeley ( haverty et al . ,", "extraction procedure and characterization of hydrocarbons hydrocarbons from workers , alates , soldiers , and fecal pellets of i. minor were extracted , characterized , and quantified as previously reported ( haverty et al . , 2005 ) .", "frozen termite samples were thawed and dried at 70c for approximately 1  h before extraction . each sub - sample of fecal pellets or termites", "was placed in a 20-ml scintillation vial , and immersed in 10  ml of n - hexane for 10  min . after extraction", ", hydrocarbons were separated from other compounds through 4  cm of activated sigma silica gel ( 70230 mesh ) in pasteur pipette mini - columns .", "the resulting hydrocarbon fractions were evaporated to dryness under a stream of nitrogen and redissolved in 60 l of n - hexane for gas chromatography - mass spectrometry ( gc - ms ) analysis .", "a 3-l aliquot was injected into the gc-ms.gc-ms analyses were performed on an agilent 6890 gas chromatograph interfaced with an agilent 5973 mass selective detector , using agilent chemstation data analysis software ( g1701ca version c.00.00 ) .", "the gc - ms was equipped with an hp-1ms , fused silica capillary column ( 30  m    0.25  mm i.d , 0.25  m film thickness ) , and was operated in split mode ( split ratio of 30:1 ) , using helium as carrier gas .", "the column oven was programmed from 200320c at 3c  min , with a final hold of 11  min .", "electron impact ( ei ) mass spectra were obtained at 70  ev.all chemicals of interest were identified by their retention times and mass spectra .", "mass spectra of methyl - branched alkanes were interpreted as described by blomquist et al .", "olefins were identified by their mass spectra , although double bond positions were not determined ( haverty et al . , 2005).in the text , tables , and figures , we use shorthand nomenclature to identify individual hydrocarbons or mixtures of hydrocarbons .", "this shorthand uses a descriptor for the location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) .", "thus , pentacosane is represented as n - c25 , 11-methylnonacosane as 11-mec29 , 13,17-dimethylhentriacontane as 13,17-dimec31 , and heptatriacontatriene as c37:3 .", "hydrocarbons are presented in the tables for each caste and worker fecal pellets in the order of elution from our gc - ms system .", "statistical analysis gc - ms peak ( some peaks contained more than one compound or a mixture of positional isomers ) areas were converted to percentages of total hydrocarbon fraction , enabling mean ( sd ) relative amounts of each hydrocarbon peak for workers and alates , and overall mean ( sd ) of each hydrocarbon peak for the fecal pellets , to be calculated.the percentage of each hydrocarbon peak for pellets of each aging period was transformed to the log of the percentage ( dependent variable y ) and regressed against the days of aging ( independent variable x ) .", "hydrocarbons with slopes different from 0 (   =  0.05 ) were separated into two groups : those with a significant positive slope and those with a significant negative slope . for each aging period ( 0 , 30 , 90 , and 365  day ) , an index of age ( iage ) was created by subtracting the sum of the percentages of the hydrocarbons with a negative slope over time from the sum of the percentages of the hydrocarbons with a positive slope over time [ iage  =   ( peakpositive ) minus  ( peaknegative ) , for each aging period ] .", "the iage for each aging period then was regressed against the aging period ( r development core team , 2004 ) .", "the cuticular hydrocarbons for i. minor pseudergates and fecal pellets were characterized previously ( haverty et al . , 2000 , 2005 ) .", "the composition of the hydrocarbon mixture for this species and the hydrocarbons from their fecal pellets in the present study are displayed in fig . ", "1total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colontable  1relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbonspseudergates n  =  3alates n  =  3soldiers n  =  1fecal pellets n  =  122-mec220.06 ( 0.02)0.08 ( 0.02)0.090.11 ( 0.02)n - c231.30 ( 0.06)1.34 ( 0.05)1.481.35 ( 0.18)2-mec231.45 ( 0.40)1.72 ( 0.35)2.112.13 ( 0.28)3-mec231.31 ( 0.38)1.38 ( 0.30)1.712.06 ( 0.29)n - c240.46 ( 0.01)0.43 ( 0.02)0.480.32 ( 0.05)2-mec240.13 ( 0.03)0.19 ( 0.03)0.190.15 ( 0.02)3-mec240.08 ( 0.02)0.10 ( 0.02)0.120.11 ( 0.02)n - c2516.35 ( 0.16)11.75 ( 0.40)12.648.65 ( 1.05)2-mec250.18 ( 0.01)0.27 ( 0.01)0.240.14 ( 0.02)3-mec250.23 ( 0.04)0.36 ( 0.04)0.340.26 ( 0.03)n - c262.25 ( 0.15)1.17 ( 0.07)1.510.87 ( 0.12)2-mec260.14 ( 0.01)0.13 ( 0.01)0.140.07 ( 0.01)n - c2714.04 ( 0.55)6.81 ( 0.31)10.055.91 ( 0.65)2-mec270.13 ( 0.01)0.09 ( 0.01)0.130.06 ( 0.01)3-mec270.20 ( 0.01)0.19 ( 0.01)0.250.12 ( 0.01)n - c280.43 ( 0.05)0.17 ( 0.01)0.350.15 ( 0.03)n - c293.38 ( 0.33)1.27 ( 0.07)2.971.38 ( 0.18)11-mec290.06 ( 0.01)0.15 ( 0.01)0.100.09 ( 0.01)9,13-dimec290.70 ( 0.02)0.82 ( 0.01)0.910.63 ( 0.06)9,13,17-trimec290.11 ( 0.02)0.27 ( 0.00)0.160.22 ( 0.03)n - c300.04 ( 0.00)0.07 ( 0.00)0.100.0013-mec300.01 ( 0.02)0.08 ( 0.00)0.040.0010,14-dimec300.64 ( 0.06)1.45 ( 0.02)0.880.90 ( 0.03)10,14,18-trimec300.18 ( 0.02)0.43 ( 0.01)0.240.28 ( 0.02)n - c310.14 ( 0.01)0.10 ( 0.01)0.200.09 ( 0.01)15- ; 13- ; 11- ; 9-mec310.36 ( 0.03)0.82 ( 0.01)0.420.49 ( 0.02)13,17- ; 11,15-dimec317.45 ( 0.68)13.74 ( 0.52)8.5510.67 ( 1.80)9,13,17-trimec310.57 ( 0.05)1.19 ( 0.02)0.720.89 ( 0.06)7,11,15-trimec310.54 ( 0.04)0.99 ( 0.01)0.720.81 ( 0.09)14- ; 12-mec320.17 ( 0.01)0.33 ( 0.02)0.190.27 ( 0.03)12,16-dimec322.01 ( 0.14)3.57 ( 0.05)2.222.97 ( 0.28)10,14,18- ; 8,12,16-trimec320.46 ( 0.03)0.80 ( 0.02)0.610.82 ( 0.06)15- ; 13-mec330.93 ( 0.05)1.57 ( 0.03)0.921.30 ( 0.08)13,17- ; 11,15- ; 9,13-dimec334.63 ( 0.31)6.87 ( 0.11)5.117.41 ( 1.03)9,13,17-trimec331.30 ( 0.08)2.02 ( 0.02)1.863.47 ( 0.33)7,13,17-trimec330.21 ( 0.02)0.32 ( 0.01)0.320.47 ( 0.07)c35:31.01 ( 0.03)2.93 ( 0.17)1.641.76 ( 0.17)c35:22.31 ( 0.06)4.46 ( 0.14)2.713.55 ( 0.15)12,16-dimec34 ; c35:10.68 ( 0.02)1.16 ( 0.03)0.851.40 ( 0.10)10,14,18- ; 8,12,16-trimec340.28 ( 0.00)0.50 ( 0.04)0.450.89 ( 0.14)15- ; 13- ; 11-mec350.56 ( 0.02)0.85 ( 0.05)0.671.11 ( 0.09)13,17-dimec351.27 ( 0.09)1.96 ( 0.02)1.833.95 ( 0.46)9,13,17-trimec350.14 ( 0.01)0.28 ( 0.03)0.270.93 ( 0.14)c37:31.74 ( 0.06)3.33 ( 0.16)2.723.28 ( 0.34)c37:21.67 ( 0.06)2.55 ( 0.08)2.182.90 ( 0.10)12,16-dimec360.17 ( 0.01)0.34 ( 0.02)0.280.71 ( 0.08)c37:10.78 ( 0.02)0.76 ( 0.03)0.810.83 ( 0.06)10,14,18-trimec36 ; c37:10.44 ( 0.03)0.35 ( 0.05)0.430.58 ( 0.10)13 ; 11-mec370.71 ( 0.01)0.60 ( 0.04)0.770.89 ( 0.14)13,17- ; 11,15-dimec370.87 ( 0.05)1.27 ( 0.01)1.252.13 ( 0.22)9,13,17-trimec370.09 ( 0.01)0.13 ( 0.01)0.130.30 ( 0.06)c39:30.09 ( 0.02)0.18 ( 0.02)0.290.49 ( 0.16)c39:3 ; 12-mec380.49 ( 0.04)0.56 ( 0.03)0.761.03 ( 0.18)14,18 ; 12,16-dimec380.02 ( 0.03)0.14 ( 0.01)0.140.24 ( 0.04)c39:1 ; 10,14,18-trimec382.73 ( 0.19)1.73 ( 0.04)2.531.74 ( 0.23)13- ; 11-mec391.75 ( 0.09)1.03 ( 0.03)1.701.26 ( 0.20)13,17- ; 11,15-dimec390.90 ( 0.01)1.04 ( 0.14)1.361.44 ( 0.08)9,13,17-trimec390.41 ( 0.03)0.17 ( 0.09)0.110.20 ( 0.03)12- ; 10-mec400.38 ( 0.05)0.20 ( 0.01)0.350.26 ( 0.07)12,16-dimec400.27 ( 0.00)0.27 ( 0.02)0.340.32 ( 0.05)c41:13.31 ( 0.27)1.70 ( 0.04)2.791.58 ( 0.25)13- ; 11-mec412.78 ( 0.20)1.45 ( 0.06)2.591.74 ( 0.31)13,17- ; 11,15-dimec413.34 ( 0.13)2.90 ( 0.15)4.003.33 ( 0.19)9,13,17-trimec410.79 ( 0.02)0.30 ( 0.02)0.330.42 ( 0.19)12- ; 10-mec420.30 ( 0.03)0.15 ( 0.01)0.250.26 ( 0.27)12,16-dimec420.48 ( 0.04)0.37 ( 0.02)0.520.47 ( 0.12)c43:12.72 ( 0.28)1.25 ( 0.06)2.271.31 ( 0.26)15- ; 13-mec431.00 ( 0.14)0.46 ( 0.05)0.870.61 ( 0.16)13,17-dimec431.99 ( 0.16)1.18 ( 0.16)1.931.68 ( 0.28)14,18-dimec440.12 ( 0.02)0.07 ( 0.01)0.120.13 ( 0.06)c45:10.42 ( 0.09)0.16 ( 0.02)0.330.21 ( 0.07)13-mec450.000.000.000.04 ( 0.03)13,17-dimec450.37 ( 0.08)0.18 ( 0.02)0.340.43 ( 0.13)mean percent ( sd ) of total hydrocarbon composition", ". compounds are listed in elution orderacronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y)these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .", "( 2005)an isomeric mixture or two or more compounds co - eluted in this peak total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) mean percent ( sd ) of total hydrocarbon composition .", "compounds are listed in elution order acronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .", "( 2005 ) an isomeric mixture or two or more compounds co - eluted in this peak hydrocarbons from termites", "n - alkanes and dimethylalkanes were the predominant classes of hydrocarbons in all castes and in fecal pellets ( table  2 ) , as previously reported by haverty et al .", "n - alkanes comprised from 23.12% to 38.39% , unsaturated components from 15.21% to 19.03% , terminally branched monomethylalkanes from 3.90% to 5.32% , internally branched monomethylalkanes from 7.70% to 9.01% , and dimethylalkanes from 25.25% to 36.18% , of the total hydrocarbon content .", "there was also a homologous series of trimethylalkanes , representing 8.26% to 9.48% of the total hydrocarbon content . \n", "table  2relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbon classpseudergatesalatessoldiersfecal pelletsnormal alkanes38.3923.1229.7718.71olefins18.3821.1120.3120.66terminally branched methylalkanes3.904.495.325.19internally branched methylalkanes9.017.708.888.33dimethylalkanes25.2436.1829.7937.40trimethylalkanes5.097.405.929.71percent of total hydrocarbon compositionall peaks with co - eluting olefins and saturated compounds are summarized as olefinsthe hydrocarbons of i. minor characterized in this study included all of the compounds reported by haverty et al .", "( 2005 ) , as well as 18 additional hydrocarbons ( table  1 ) .", "these additional hydrocarbons were not abundant , never representing more than 0.85% of the total hydrocarbon ( as for the mixture of 12,16-dimec34 and c35:1 in soldiers ) content , and usually much less .", "the detection of these additional hydrocarbons in this study are likely due to the greater concentration of the extracted hydrocarbons used or , possibly , colony to colony variation .", "relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) percent of total hydrocarbon composition all peaks with co - eluting olefins and saturated compounds are summarized as olefins hydrocarbons from termite fecal pellets in general , the hydrocarbons from whole - body extracts of i. minor were represented in extracts of fecal pellets . as with whole - body extracts of i. minor , dimethylalkanes were the predominant class of hydrocarbon , followed by olefins and normal alkanes ( table  2 ) .", "1 ) were found in termites and not in fecal pellets , but these were present in small quantities , less than 0.1% of the total hydrocarbon content .", "the lack of these two hydrocarbons in fecal pellets may be a function simply of the concentration of the extracts .", "changes in hydrocarbons of fecal pellets over time we identified 73 hydrocarbon peaks in fecal pellets of i. minor ( table  1 ) . of these peaks", "( data from non - significant slopes not reported ) , 19 of 73 ( 26% ) had a significant change , either positive ( five ) or negative ( 14 ) , over time ( fig .  2 ) .", "this is a much higher number of statistically significant slopes ( different from 0 ) , than would be expected by chance alone ( 5% of 73 peaks is < 4 peaks ) . \n", "2changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant (   =  0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colonthe index , iage , for each of the three replications ( a , b , and c at 0 , 30 , 90 , and 365  day ) was plotted against the associated number of days ( fig . ", "the fitted line , with a 95% confidence interval for the means to show the variability of the replicates , had a high adjusted r (= 0.888 ) value , suggesting that it might be possible to predict fecal age by the index . \n", "3fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .", "values for 30  day are jittered a small amount so that all three data points can be seen changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant (   =  0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .", "values for 30  day are jittered a small amount so that all three data points can be seen we do not know the basis by which peaks increase or decrease over the 1-year period .", "in general , those that increased in relative abundance tended to have higher initial relative abundances ; i.e. , mono- , di- , or tri - methylalkanes with carbon chains of 31 , 32 , or 33 .", "those that decreased were represented in five of the six classes of hydrocarbons , ranging from c26 to c45 .", "the composition of the hydrocarbon mixture of insects is genetically controlled ( toolson and kuper - simbrn , 1989 ; kaib et al . , 1991 ; page et al . , 1991 ; coyne et al . , 1994 ) .", "this composition can be slightly affected by diet and environmental conditions ( hadley , 1977 ; espelie et al . , 1994 ; chapman et al . , 1995", "( 1996 ) demonstrated significant seasonal variation in quantities of some hydrocarbons of coptotermes formosanus shiraki from hawaii ; these differences were small and associated with the production of alates .", "however , the qualitative mix of hydrocarbons has been shown to be stable over decades among species of cone beetles in the genus conophthorus ( page et al . , 1990 ) .", "the motivation for our study was to devise a method for the evaluation of the success or failure of drywood termite treatments , including fumigation and local application methods .", "with the recent classification of sulfuryl fluoride ( the only fumigant active ingredient registered in california ) as a greenhouse gas ( mhle et al . , 2009 ) and", "the anticipated increase in local treatments , we felt that there would be considerable interest by the industry , regulatory agencies , and consumers for a simple and accurate means to determine whether or not a targeted colony / infestation is still in a structure .", "our method , based on changes in hydrocarbon chemistry over time , shows promise to this end .", "future research includes : ( 1 ) validating these findings for additional species of drywood termites and geographic populations of the same species , ( 2 ) validating these findings at different times of the year , and ( 3 ) exploring seasonality in pellet production and hydrocarbon quality in pellets ."], "abstract_text": ["<S> hydrocarbon mixtures extracted from fecal pellets of drywood termites are species - specific and can be characterized to identify the termites responsible for damage , even when termites are no longer present or are unable to be recovered easily . in structures infested by drywood termites , it is common to find fecal pellets , but difficult to sample termites from the wood . when fecal pellets appear after remedial treatment of a structure , it is difficult to determine whether this indicates that termites in the structure are still alive and active or not . </S>", "<S> we examined the hydrocarbon composition of workers , alates , and soldiers of incisitermes minor ( hagen ) ( family kalotermitidae ) and of fecal pellets of workers . </S>", "<S> hydrocarbons were qualitatively similar among castes and pellets . </S>", "<S> fecal pellets that were aged for periods of 0 , 30 , 90 , and 365  days after collection were qualitatively similar across all time periods , however , the relative quantities of certain individual hydrocarbons changed over time , with 19 of the 73 hydrocarbon peaks relatively increasing or decreasing . </S>", "<S> when the sums of the positive and negative slopes of these 19 hydrocarbons were indexed , they produced a highly significant linear correlation ( r2  =  0.89 ) . </S>", "<S> consequently , the quantitative differences of these hydrocarbons peaks can be used to determine the age of worker fecal pellets , and thus help determine whether the colony that produced them is alive or dead . </S>"], "labels": null, "section_names": ["Introduction", "Methods and Materials", "Results and Discussion"], "sections": [["drywood termites cause significant damage to wood in structures in the united states , with two species , incisitermes minor ( hagen ) and cryptotermes brevis ( walker ) , being responsible for the majority of damage ( su and scheffrahn , 1990 ; grace , 2009 ) .", "the economic cost of control and repair of damage is second only to that for subterranean termites ( su and scheffrahn , 1990 ) .", "remedial control of drywood termites in the united states relies primarily on either ( 1 ) fumigation of the entire structure with a toxic gas or heated air , or ( 2 ) localized chemical or physical treatments designed to eradicate small , localized colonies ( lewis and haverty , 1996 ; lewis , 2008 ; lewis and rust , 2009 ) .", "fumigation treatments are likely to kill all of the termites in a structure , while localized treatments can destroy all termites in a colony , provided the treatment is delivered to the entire gallery system .", "the size , number , and dispersion of drywood termite colonies in a structure can vary greatly , depending on the age of the infestation and the success of preventative or remedial treatments .", "drywood termite colonies also are considered single piece nesters , i.e. , they nest within their food source and do not forage from the nesting site to a food source ( abe , 1987 ) .", "in fact , large , dispersed colonies ( or even small colonies ) can live in a single piece of wood or in multiple pieces of wood that are joined together ( grace et al . ,", "drywood termites are cryptic , seldom leaving obvious or visible external signs of their presence in wood . a commonly used sign for determining the presence of drywood termites is the occurrence of fecal pellets , ejected through a  kick - out hole  in the external surface of wood from the internal galleries ( ebeling , 1975 ) .", "these pellets often are found as conical piles or are scattered on horizontal surfaces below infested wood .", "these hexagonally sided pellets are diagnostic for drywood termites , and can be used to distinguish damage from that by other wood - destroying insects ( ebeling , 1975 ; moore , 1992 ) .", "grace and yamamoto ( 2009 ) demonstrated the relationship between the cellulose and lignin content of the food utilized by small groups of c. brevis and incisitermes immigrans ( light ) , and the quantity of fecal pellets produced over time .", "they also discussed the use of the size and number of fecal pellets for estimating both size and age of drywood termite colonies .", "drywood termites excrete feces in the form of hard , even - shaped fecal pellets .", "these fecal pellets contain the same mixture of hydrocarbons as the insects that produced them , albeit in slightly different proportions ( haverty et al .", ", 2005 ) . because cuticular hydrocarbons are species specific in termites ( page et al . , 2002 )", "rather than simply signaling the general presence of termites or providing a diagnosis of the species of termite inhabiting the wood , we postulated that these pellets could be chemically characterized so as to determine the status of a colony as active ( alive ) or inactive ( dead ) . here , we report quantification of the hydrocarbons in pellets of i. minor aged for up to 1  year after they were produced .", "we document the changes in proportions of selected hydrocarbons as an indication of the length of time since the pellets were excreted ."], ["collection of termites and preparation of termite containment unit termites , i. minor , were removed from one naturally infested board ( 98.9    13.3    271.8  cm ) collected on 19 july 2006 from lakeview , california , and stored at the university of california richmond field station .", "the board was cut across the grain into pieces , 58  cm thick , and stored at room temperature .", "a wood chisel was used to separate pieces of wood into smaller pieces , 0.51.0  cm thick .", "all remaining live termites , including soldiers and primary reproductives ( alates ) , were placed in a termite containment unit ( tcu ) .", "termites thus collected were likely from a large mixed colony ( booth et al . , 2010 ) or from multiple colonies .", "we were not able to assign the various galleries in the wood to any particular colony.fifteen birch tongue depressors were bundled together , and three equally spaced holes drilled across the length of the bundle using a 2.8-mm bit .", "bamboo skewers , 1013  cm in length , were inserted into each of the three holes in the bundle of tongue depressors .", "spaces , 36  mm , were left between each tongue depressor allowing termites access to all tongue depressors .", "two sets of skewered tongue depressors were placed into a clean plastic container ( 17.5    12.5    6  cm ) , one on top of the other , such that the tongue depressors on the top layer fit into the gaps created by the tongue depressors on the bottom layer .", "tongue depressors were lightly misted with water , and then 6,662 drywood termites ( a mixture of pseudergates or workers , alates , and soldiers ) were placed in the tcu .", "tcus , with termites , were maintained in a dark cabinet in the laboratory under ambient conditions prior to collection of fecal pellets .", "a single colony or a mixture of two or more colonies , representing a single location within california , was prepared in this manner . pellet collection and aging process", "after all the tcus had acclimated to laboratory conditions over several weeks , new holding chambers were prepared .", "all wood debris , pellets , dead termites , and damaged tongue depressors were discarded .", "the termites in the new holding chambers were maintained in the laboratory under ambient conditions for 2  weeks . at the end of this period", ", all pellets were removed , and sub - samples collected and readied for the aging study .", "concurrently , three samples of 20 workers , three samples of 20 alates , and one sample of 20 soldiers were also collected for hydrocarbon analysis .", "live termites were placed in a 20-ml scintillation vial and frozen until extraction.fecal pellets were separated from debris by sequentially sifting them through successively smaller sieves ( haverty et al . , 2005 ) .", "thus , all substances such as hand lotion , lip balm , parafilm , and waxed paper were kept away from the work area .", "gloves were worn when handling pellets , and implements that came in contact with pellets were wiped with a paper towel dampened with absolute ethanol to remove any oils or waxes .", "pellets were separated from fine debris by removing individual pellets with forceps or with an aspirator , until a sample weighing approximately 200  mg was obtained.in developing this protocol we made one estimate and one critical assumption .", "the estimate was that at least 1,000 pellets were needed for each hydrocarbon analysis ( based on preliminary analyses of pellets collected from drywood termites maintained in our laboratory ) .", "( 1997 ) using c. brevis and i. snyderi ( light ) from southern florida . in that study , 40 i. snyderi ( pseudergates or workers ) produced 573 pellets over 4  weeks , slightly less than 150 pellets / wk or about 3.5 pellets / individual / wk . in a study with small groups of c. brevis and i. immigrans , grace and yamamoto ( 2009 ) found that these species produced 4.9 to 7.0 pellets / individual / wk . for our study , we assumed that i. minor would produce pellets at roughly the same rate and , therefore , we needed about 3,000 to 5,000 individuals in a tcu for this study .", "we prepared one tcu to collect the requisite quantity of fecal pellets.four sub - sampling intervals , each replicated three times , were used : 0 , 30 , 90 , and 365  days from the initial collection date .", "sub - samples were stored in clean , 20-ml scintillation vials , sealed with 1.5    1.5  mm mesh screen .", "vials were stored in a dark cabinet at the university of california richmond field station at ambient temperature.voucher samples of i. minor pseudergates or workers and soldiers from this study ( fresh , not dried ) were preserved in 85% ethanol and deposited in the essig museum , university of california at berkeley ( haverty et al . ,", "extraction procedure and characterization of hydrocarbons hydrocarbons from workers , alates , soldiers , and fecal pellets of i. minor were extracted , characterized , and quantified as previously reported ( haverty et al . , 2005 ) .", "frozen termite samples were thawed and dried at 70c for approximately 1  h before extraction . each sub - sample of fecal pellets or termites", "was placed in a 20-ml scintillation vial , and immersed in 10  ml of n - hexane for 10  min . after extraction", ", hydrocarbons were separated from other compounds through 4  cm of activated sigma silica gel ( 70230 mesh ) in pasteur pipette mini - columns .", "the resulting hydrocarbon fractions were evaporated to dryness under a stream of nitrogen and redissolved in 60 l of n - hexane for gas chromatography - mass spectrometry ( gc - ms ) analysis .", "a 3-l aliquot was injected into the gc-ms.gc-ms analyses were performed on an agilent 6890 gas chromatograph interfaced with an agilent 5973 mass selective detector , using agilent chemstation data analysis software ( g1701ca version c.00.00 ) .", "the gc - ms was equipped with an hp-1ms , fused silica capillary column ( 30  m    0.25  mm i.d , 0.25  m film thickness ) , and was operated in split mode ( split ratio of 30:1 ) , using helium as carrier gas .", "the column oven was programmed from 200320c at 3c  min , with a final hold of 11  min .", "electron impact ( ei ) mass spectra were obtained at 70  ev.all chemicals of interest were identified by their retention times and mass spectra .", "mass spectra of methyl - branched alkanes were interpreted as described by blomquist et al .", "olefins were identified by their mass spectra , although double bond positions were not determined ( haverty et al . , 2005).in the text , tables , and figures , we use shorthand nomenclature to identify individual hydrocarbons or mixtures of hydrocarbons .", "this shorthand uses a descriptor for the location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) .", "thus , pentacosane is represented as n - c25 , 11-methylnonacosane as 11-mec29 , 13,17-dimethylhentriacontane as 13,17-dimec31 , and heptatriacontatriene as c37:3 .", "hydrocarbons are presented in the tables for each caste and worker fecal pellets in the order of elution from our gc - ms system .", "statistical analysis gc - ms peak ( some peaks contained more than one compound or a mixture of positional isomers ) areas were converted to percentages of total hydrocarbon fraction , enabling mean ( sd ) relative amounts of each hydrocarbon peak for workers and alates , and overall mean ( sd ) of each hydrocarbon peak for the fecal pellets , to be calculated.the percentage of each hydrocarbon peak for pellets of each aging period was transformed to the log of the percentage ( dependent variable y ) and regressed against the days of aging ( independent variable x ) .", "hydrocarbons with slopes different from 0 (   =  0.05 ) were separated into two groups : those with a significant positive slope and those with a significant negative slope . for each aging period ( 0 , 30 , 90 , and 365  day ) , an index of age ( iage ) was created by subtracting the sum of the percentages of the hydrocarbons with a negative slope over time from the sum of the percentages of the hydrocarbons with a positive slope over time [ iage  =   ( peakpositive ) minus  ( peaknegative ) , for each aging period ] .", "the iage for each aging period then was regressed against the aging period ( r development core team , 2004 ) ."], ["the cuticular hydrocarbons for i. minor pseudergates and fecal pellets were characterized previously ( haverty et al . , 2000 , 2005 ) .", "the composition of the hydrocarbon mixture for this species and the hydrocarbons from their fecal pellets in the present study are displayed in fig . ", "1total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colontable  1relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbonspseudergates n  =  3alates n  =  3soldiers n  =  1fecal pellets n  =  122-mec220.06 ( 0.02)0.08 ( 0.02)0.090.11 ( 0.02)n - c231.30 ( 0.06)1.34 ( 0.05)1.481.35 ( 0.18)2-mec231.45 ( 0.40)1.72 ( 0.35)2.112.13 ( 0.28)3-mec231.31 ( 0.38)1.38 ( 0.30)1.712.06 ( 0.29)n - c240.46 ( 0.01)0.43 ( 0.02)0.480.32 ( 0.05)2-mec240.13 ( 0.03)0.19 ( 0.03)0.190.15 ( 0.02)3-mec240.08 ( 0.02)0.10 ( 0.02)0.120.11 ( 0.02)n - c2516.35 ( 0.16)11.75 ( 0.40)12.648.65 ( 1.05)2-mec250.18 ( 0.01)0.27 ( 0.01)0.240.14 ( 0.02)3-mec250.23 ( 0.04)0.36 ( 0.04)0.340.26 ( 0.03)n - c262.25 ( 0.15)1.17 ( 0.07)1.510.87 ( 0.12)2-mec260.14 ( 0.01)0.13 ( 0.01)0.140.07 ( 0.01)n - c2714.04 ( 0.55)6.81 ( 0.31)10.055.91 ( 0.65)2-mec270.13 ( 0.01)0.09 ( 0.01)0.130.06 ( 0.01)3-mec270.20 ( 0.01)0.19 ( 0.01)0.250.12 ( 0.01)n - c280.43 ( 0.05)0.17 ( 0.01)0.350.15 ( 0.03)n - c293.38 ( 0.33)1.27 ( 0.07)2.971.38 ( 0.18)11-mec290.06 ( 0.01)0.15 ( 0.01)0.100.09 ( 0.01)9,13-dimec290.70 ( 0.02)0.82 ( 0.01)0.910.63 ( 0.06)9,13,17-trimec290.11 ( 0.02)0.27 ( 0.00)0.160.22 ( 0.03)n - c300.04 ( 0.00)0.07 ( 0.00)0.100.0013-mec300.01 ( 0.02)0.08 ( 0.00)0.040.0010,14-dimec300.64 ( 0.06)1.45 ( 0.02)0.880.90 ( 0.03)10,14,18-trimec300.18 ( 0.02)0.43 ( 0.01)0.240.28 ( 0.02)n - c310.14 ( 0.01)0.10 ( 0.01)0.200.09 ( 0.01)15- ; 13- ; 11- ; 9-mec310.36 ( 0.03)0.82 ( 0.01)0.420.49 ( 0.02)13,17- ; 11,15-dimec317.45 ( 0.68)13.74 ( 0.52)8.5510.67 ( 1.80)9,13,17-trimec310.57 ( 0.05)1.19 ( 0.02)0.720.89 ( 0.06)7,11,15-trimec310.54 ( 0.04)0.99 ( 0.01)0.720.81 ( 0.09)14- ; 12-mec320.17 ( 0.01)0.33 ( 0.02)0.190.27 ( 0.03)12,16-dimec322.01 ( 0.14)3.57 ( 0.05)2.222.97 ( 0.28)10,14,18- ; 8,12,16-trimec320.46 ( 0.03)0.80 ( 0.02)0.610.82 ( 0.06)15- ; 13-mec330.93 ( 0.05)1.57 ( 0.03)0.921.30 ( 0.08)13,17- ; 11,15- ; 9,13-dimec334.63 ( 0.31)6.87 ( 0.11)5.117.41 ( 1.03)9,13,17-trimec331.30 ( 0.08)2.02 ( 0.02)1.863.47 ( 0.33)7,13,17-trimec330.21 ( 0.02)0.32 ( 0.01)0.320.47 ( 0.07)c35:31.01 ( 0.03)2.93 ( 0.17)1.641.76 ( 0.17)c35:22.31 ( 0.06)4.46 ( 0.14)2.713.55 ( 0.15)12,16-dimec34 ; c35:10.68 ( 0.02)1.16 ( 0.03)0.851.40 ( 0.10)10,14,18- ; 8,12,16-trimec340.28 ( 0.00)0.50 ( 0.04)0.450.89 ( 0.14)15- ; 13- ; 11-mec350.56 ( 0.02)0.85 ( 0.05)0.671.11 ( 0.09)13,17-dimec351.27 ( 0.09)1.96 ( 0.02)1.833.95 ( 0.46)9,13,17-trimec350.14 ( 0.01)0.28 ( 0.03)0.270.93 ( 0.14)c37:31.74 ( 0.06)3.33 ( 0.16)2.723.28 ( 0.34)c37:21.67 ( 0.06)2.55 ( 0.08)2.182.90 ( 0.10)12,16-dimec360.17 ( 0.01)0.34 ( 0.02)0.280.71 ( 0.08)c37:10.78 ( 0.02)0.76 ( 0.03)0.810.83 ( 0.06)10,14,18-trimec36 ; c37:10.44 ( 0.03)0.35 ( 0.05)0.430.58 ( 0.10)13 ; 11-mec370.71 ( 0.01)0.60 ( 0.04)0.770.89 ( 0.14)13,17- ; 11,15-dimec370.87 ( 0.05)1.27 ( 0.01)1.252.13 ( 0.22)9,13,17-trimec370.09 ( 0.01)0.13 ( 0.01)0.130.30 ( 0.06)c39:30.09 ( 0.02)0.18 ( 0.02)0.290.49 ( 0.16)c39:3 ; 12-mec380.49 ( 0.04)0.56 ( 0.03)0.761.03 ( 0.18)14,18 ; 12,16-dimec380.02 ( 0.03)0.14 ( 0.01)0.140.24 ( 0.04)c39:1 ; 10,14,18-trimec382.73 ( 0.19)1.73 ( 0.04)2.531.74 ( 0.23)13- ; 11-mec391.75 ( 0.09)1.03 ( 0.03)1.701.26 ( 0.20)13,17- ; 11,15-dimec390.90 ( 0.01)1.04 ( 0.14)1.361.44 ( 0.08)9,13,17-trimec390.41 ( 0.03)0.17 ( 0.09)0.110.20 ( 0.03)12- ; 10-mec400.38 ( 0.05)0.20 ( 0.01)0.350.26 ( 0.07)12,16-dimec400.27 ( 0.00)0.27 ( 0.02)0.340.32 ( 0.05)c41:13.31 ( 0.27)1.70 ( 0.04)2.791.58 ( 0.25)13- ; 11-mec412.78 ( 0.20)1.45 ( 0.06)2.591.74 ( 0.31)13,17- ; 11,15-dimec413.34 ( 0.13)2.90 ( 0.15)4.003.33 ( 0.19)9,13,17-trimec410.79 ( 0.02)0.30 ( 0.02)0.330.42 ( 0.19)12- ; 10-mec420.30 ( 0.03)0.15 ( 0.01)0.250.26 ( 0.27)12,16-dimec420.48 ( 0.04)0.37 ( 0.02)0.520.47 ( 0.12)c43:12.72 ( 0.28)1.25 ( 0.06)2.271.31 ( 0.26)15- ; 13-mec431.00 ( 0.14)0.46 ( 0.05)0.870.61 ( 0.16)13,17-dimec431.99 ( 0.16)1.18 ( 0.16)1.931.68 ( 0.28)14,18-dimec440.12 ( 0.02)0.07 ( 0.01)0.120.13 ( 0.06)c45:10.42 ( 0.09)0.16 ( 0.02)0.330.21 ( 0.07)13-mec450.000.000.000.04 ( 0.03)13,17-dimec450.37 ( 0.08)0.18 ( 0.02)0.340.43 ( 0.13)mean percent ( sd ) of total hydrocarbon composition", ". compounds are listed in elution orderacronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y)these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .", "( 2005)an isomeric mixture or two or more compounds co - eluted in this peak total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) mean percent ( sd ) of total hydrocarbon composition .", "compounds are listed in elution order acronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .", "( 2005 ) an isomeric mixture or two or more compounds co - eluted in this peak hydrocarbons from termites", "n - alkanes and dimethylalkanes were the predominant classes of hydrocarbons in all castes and in fecal pellets ( table  2 ) , as previously reported by haverty et al .", "n - alkanes comprised from 23.12% to 38.39% , unsaturated components from 15.21% to 19.03% , terminally branched monomethylalkanes from 3.90% to 5.32% , internally branched monomethylalkanes from 7.70% to 9.01% , and dimethylalkanes from 25.25% to 36.18% , of the total hydrocarbon content .", "there was also a homologous series of trimethylalkanes , representing 8.26% to 9.48% of the total hydrocarbon content . \n", "table  2relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbon classpseudergatesalatessoldiersfecal pelletsnormal alkanes38.3923.1229.7718.71olefins18.3821.1120.3120.66terminally branched methylalkanes3.904.495.325.19internally branched methylalkanes9.017.708.888.33dimethylalkanes25.2436.1829.7937.40trimethylalkanes5.097.405.929.71percent of total hydrocarbon compositionall peaks with co - eluting olefins and saturated compounds are summarized as olefinsthe hydrocarbons of i. minor characterized in this study included all of the compounds reported by haverty et al .", "( 2005 ) , as well as 18 additional hydrocarbons ( table  1 ) .", "these additional hydrocarbons were not abundant , never representing more than 0.85% of the total hydrocarbon ( as for the mixture of 12,16-dimec34 and c35:1 in soldiers ) content , and usually much less .", "the detection of these additional hydrocarbons in this study are likely due to the greater concentration of the extracted hydrocarbons used or , possibly , colony to colony variation .", "relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) percent of total hydrocarbon composition all peaks with co - eluting olefins and saturated compounds are summarized as olefins hydrocarbons from termite fecal pellets in general , the hydrocarbons from whole - body extracts of i. minor were represented in extracts of fecal pellets . as with whole - body extracts of i. minor , dimethylalkanes were the predominant class of hydrocarbon , followed by olefins and normal alkanes ( table  2 ) .", "1 ) were found in termites and not in fecal pellets , but these were present in small quantities , less than 0.1% of the total hydrocarbon content .", "the lack of these two hydrocarbons in fecal pellets may be a function simply of the concentration of the extracts .", "changes in hydrocarbons of fecal pellets over time we identified 73 hydrocarbon peaks in fecal pellets of i. minor ( table  1 ) . of these peaks", "( data from non - significant slopes not reported ) , 19 of 73 ( 26% ) had a significant change , either positive ( five ) or negative ( 14 ) , over time ( fig .  2 ) .", "this is a much higher number of statistically significant slopes ( different from 0 ) , than would be expected by chance alone ( 5% of 73 peaks is < 4 peaks ) . \n", "2changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant (   =  0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colonthe index , iage , for each of the three replications ( a , b , and c at 0 , 30 , 90 , and 365  day ) was plotted against the associated number of days ( fig . ", "the fitted line , with a 95% confidence interval for the means to show the variability of the replicates , had a high adjusted r (= 0.888 ) value , suggesting that it might be possible to predict fecal age by the index . \n", "3fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .", "values for 30  day are jittered a small amount so that all three data points can be seen changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant (   =  0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .", "acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .", "values for 30  day are jittered a small amount so that all three data points can be seen we do not know the basis by which peaks increase or decrease over the 1-year period .", "in general , those that increased in relative abundance tended to have higher initial relative abundances ; i.e. , mono- , di- , or tri - methylalkanes with carbon chains of 31 , 32 , or 33 .", "those that decreased were represented in five of the six classes of hydrocarbons , ranging from c26 to c45 .", "the composition of the hydrocarbon mixture of insects is genetically controlled ( toolson and kuper - simbrn , 1989 ; kaib et al . , 1991 ; page et al . , 1991 ; coyne et al . , 1994 ) .", "this composition can be slightly affected by diet and environmental conditions ( hadley , 1977 ; espelie et al . , 1994 ; chapman et al . , 1995", "( 1996 ) demonstrated significant seasonal variation in quantities of some hydrocarbons of coptotermes formosanus shiraki from hawaii ; these differences were small and associated with the production of alates .", "however , the qualitative mix of hydrocarbons has been shown to be stable over decades among species of cone beetles in the genus conophthorus ( page et al . , 1990 ) .", "the motivation for our study was to devise a method for the evaluation of the success or failure of drywood termite treatments , including fumigation and local application methods .", "with the recent classification of sulfuryl fluoride ( the only fumigant active ingredient registered in california ) as a greenhouse gas ( mhle et al . , 2009 ) and", "the anticipated increase in local treatments , we felt that there would be considerable interest by the industry , regulatory agencies , and consumers for a simple and accurate means to determine whether or not a targeted colony / infestation is still in a structure .", "our method , based on changes in hydrocarbon chemistry over time , shows promise to this end .", "future research includes : ( 1 ) validating these findings for additional species of drywood termites and geographic populations of the same species , ( 2 ) validating these findings at different times of the year , and ( 3 ) exploring seasonality in pellet production and hydrocarbon quality in pellets ."]]}
{"article_id": "PMC4065164", "article_text": ["there \n is a need for computer methods that can calculate the aqueous solvation \n free energies of solute molecules accurately and efficiently .", "explicit - solvent models provide a physically \n accurate and atomistically detailed model of solvent , but they can \n be computationally expensive .", "boltzmann \n ( pb ) , generalized born ( gb ) , and \n weighted surface area ( wsa ) approaches , are computationally efficient because they treat \n water as a continuum .", "however , they are sometimes inaccurate , smearing \n out the particulate nature of water molecules , and they may \n have limited transferability to situations for which they have not \n been optimized .", "solvation modeling has \n often been improved by combining the advantages of implicit- and explicit - solvent \n models .", "one such approach is the semi - explicit assembly ( sea ) water model . in the sea approach ,", "water s solvation behavior is precomputed \n in explicit - solvent simulations of water around model spheres that \n are then combined together as building blocks to represent any given \n solute structure at run time .", "the solvation free energy gsolv for a target solute molecule is calculated \n from a sum over solvating waters .", "sea has been shown to predict efficiently \n the air - to - water transfer free energies ( ghyd ) of small - molecule solutes in both prospective and \n retrospective studies with reasonable accuracy .", "even \n so , the sea model does not give accurate solvation free energies of \n ions or solutes having high charge density .", "here , we describe \n field - sea , a considerable improvement over sea , which gives accurate \n free energies of transfer of ions and charged solutes , at no additional \n computational cost and with no degradation of the predictions for \n nonpolar and uncharged polar solutes . in overview , we studied full \n md simulations of ionic and neutral solutes in tip3p water , described \n below , and found that the results could be captured by using a solvation \n free - energy field surface ( in field - sea ) , instead of using precomputed \n waters ( as in sea ) .", "moreover , \n we found that a weakly charged solute atom that is adjacent to a strongly \n charged solute atom retains some of the restrictions of its solvating \n water molecules that its neighboring charge has ; see figure 1 .", "field - sea captures this effect using an adaptive \n boundary method . a solvent  water molecule around a solute molecule . \n", "on the left , each solute atom ( weakly charged ) has a predefined radius , \n irrespective of its neighboring solute atoms , leading to the locus \n of water centers shown by the black dashed curve . on the right ,", "one \n solute atom is strongly charged , leading to two consequences : its \n own solvating water molecule is pulled in tightly , and neighboring \n solute atoms have tighter water interactions too .", "such effects may not be captured in simplified solvation \n models that treat atoms as having fixed radii , independent of neighboring \n atoms .", "below , we describe the field - sea approach to computing solvation \n free energies . the original sea model is described elsewhere , and our related explicit - solvent and linearized poisson ", "boltzmann \n equation modeling ( lpbe ) are described \n in the supporting information ( si ) . in both the original sea and the new \n field - sea described here ,", "first , in a precomputation step , various \n model spheres are solvated in an explicit - solvent model , such as tip3p . \n", "this provides a database of component free energies that are used \n in the second step . in the second step , at the run time ,", "any given \n solute molecule of interest is assembled from an appropriate concatenation \n of these spheres .", "the solute s solvation free energy is computed \n by summing the component sphere free energies . on the one hand , this \n approach provides the speed advantages of simple additivity - based \n models as we only need to perform the first step once for a given \n solvent model . on the other hand , this procedure is more accurate \n than additivity - based approaches because ( 1 ) sea sums are regional , \n not local , and ( 2 ) the database encodes the microscopic response observed \n in explicit - solvent simulations .", "the new field - sea instead captures \n the solvation free energy using a continuous solvation field . to do this ,", "the charging free energies for a series of lennard - jones \n ( lj ) spheres solvated in tip3p water were calculated with thermodynamics \n integration ( ti ) in the set of precomputations ( see explicit solvent \n free energy calculations in the si ) .", "we \n construct a free - energy contour ( see si figure s1 ) as a combination of the electric field at the first solvation \n shell boundary ( e = q / rw , where e is the signed \n magnitude of the electric field , q is the sphere s \n charge , and rw is the distance from the \n sphere center to the first peak of the water oxygen s radial \n distribution function , rdf , around the given sphere ) , the curvature c = 1/rw at this boundary , and \n the charging free energy of the spheres . within each charge step ( q ) , all of the data of free energy , electric field , and \n curvature were fitted to a formula , which can be taken as an expansion \n of the born model1 equation 1 will \n be used to calculate the free energy associated with any surface spot \n on an arbitrary solute  solvent boundary ( esub ) .", "this free energy could also be calculated from interpolation \n between data points on the free - energy contour .", "in addition \n to a charging free - energy contour , we also need a contour for estimating \n the explicit - solvent - accessible surface of a given solute molecule . \n to generate this", ", we calculated the rdf of water oxygen around each \n charged sphere and picked the first peak in the rdf , denoted as the \n boundary - sphere distance rw .", "all of the rw values from these spheres and their charge \n and lj parameters ( lj and lj ) \n were used to build an rw contour . to generate a solvent - accessible \n surface for a given solute", ", an rw for \n each atom is first calculated from interpolation with its partial \n charge , lj , and lj on the rw contour .", "a lee  richards surface is then constructed from the nonoverlapping \n sections of these rw spheres centered \n on their associated atom sites .", "the fixed rw boundary is dependent upon only the individual parameters \n of the surface atoms .", "for solute molecules with multiple partial charge \n sites ( especially molecular ions , where strong electric fields are \n involved ) , a more physical representation of the explicit - solvent - accessible \n surface would be one that responds to the whole solute s electric \n field .", "in other words , the surface  atom distance , rw , is determined not only by the surface atom s \n partial charge , but also by neighboring atoms charges .", "taking \n into account such collective electrostatic effects , we adjust the \n fixed rw boundary in an adaptive manner , \n described by the following three - step procedure : ( 1)we cull \n all surface segments within 1.4  of any solute atom . as we are \n starting with a lee  richards solvent - accessible surface , the \n minimum rw possible in the surface construction \n is half of a water molecule diameter ( corresponding to a solute atom \n with a 0  radius ) .", "this partial - culling process simply removes \n potential numerical instabilities from surface sites overlapping solute \n atom centers while providing some adjustable starting surface sites \n that penetrate within the fixed rw boundary .", "we calculate \n the electric field at a given dot a about atom a2where n is the number \n of solute atoms , ria represents a vector from atom i to surface \n dot a , and ria is its length ; signa = 1 when raai=1n  ( qi / ria3)ria  0 , and signa = 1 when raai=1n  ( qi / ria3)ria < 0 , and raa is the vector from atom a to dot a. from the electric field , we \n calculate the corresponding charge by3where raa is the length of vector raa . we interpolate with this new charge and \n atom a s lj parameters to get a new rw , rw , new .", "in addition , \n we assume that rw can not expand ( if rw , new > rw , original , we let rw , new = rw , original ) . this nonexpansion assumption is supported by solute ", "solvent \n boundary plots of model diatomic solutes ( see si figure s2 ) . finally , the rw , new s of all atom s potential surface dots are averaged \n to yield an rw for this atom , used in \n the later culling process . ( 3 ) we cull the buried dots adaptively . \n", "because each dot  atom distance ( e.g. , daa ) is adjusted independently in the above \n procedure , the shell of potential surface dots about an atom will \n no longer be spherical .", "thus , culling buried dots is no longer a simple \n process of eliminating dots within a neighboring atom s rw , and an adaptive culling process is needed . \n to adaptively cull ", "buried  surface dots , when we check \n whether a given dot a is buried by a neighboring \n atom b , we first determine a corresponding charge \n at dot a from the electric field at this dot .", "this \n is used along with atom b s lj parameters \n to get a new rw , rw , badp , following the above rw adjustment procedure .", "atom \n distance , dab , with rw , badp , to determine if the \n dot is buried in atom b. if dab is less than rw , badp , it is removed from the set of potential surface \n dots .", "we call the new dot surface resulting from the above culling \n procedure the adaptive boundary .", "our term field - sea refers both to \n the field and to the adaptive boundary .", "the adaptive nature of the \n boundary only pertains to multiatom solutes ; the adaptive and fixed rw boundaries are identical for single - atom solutes , \n like monatomic ions . while our adaptation procedure could , in principle , \n be applied iteratively , we found no further improvement after a single \n calculation .", "the charging \n free energy can be calculated for any given field - sea surface via4where ndot is the total number of surface - exposed dots , mi is the total number of dots \n ( exposed + occluded ) for corresponding atom i ( each \n surface dot , j , of atom i , only \n corresponds to 1/mi of \n this atom sphere s total surface area or total solvation free \n energy ) , and esub , j is \n the subenergy associated with exposed surface dot j , calculated from the free - energy contour . here ,", "esub , j is calculated from eq 1 using the signed magnitude of the electric field , ej , and the curvature at dot j , 1/rij .", "ej is defined as negative when ejrij < 0 , where ej is the electric \n field ( vector ) at dot j , rij is the vector from atom i to its surface dot j , and rij is this vector s length .", "now , \n we take the total solvation free energy to be the sum of polar and \n nonpolar components5 assuming the polar component of the free energy of transfer \n ( gpol ) can be uniquely described \n by both the surface electric field and geometry of the solute , this \n molecular gpol can be accurately \n calculated from the simple summation of the surface dots energies \n process described above .", "we consider the gsolv of monatomic ions as an initial test . for molecular \n solutes ,", "to account for the role of solute conformation in the solvation \n free energy , we average gsolv results \n from calculations on 50 conformations ( though 10 conformations are \n usually enough ; see the si ) . these solute \n conformations were generated from 5 ns tip3p water md simulations \n with a 100 ps snapshot interval .", "we developed field - sea because of the errors that we \n observed in simpler methods in solvating ions ; see figure 2 .", "the explicit - solvent curve shows the hydration \n asymmetry discussed by others , where positively charged solutes are less favored than negatively \n charged solutes of similar size .", "gsolv as the function of a model lj sphere (  \n = 0.22 nm ,  = 0.06538 kj / mol ) charge for tip3p , lpbe , and field - sea . \n for comparison at infinite - dilution conditions ,", "an ewald correction \n is applied to the tip3p and field - sea results . here , we tested field - sea on the solvation free energies \n of monatomic ions using ion parameters developed by aqvist or joung and cheatham ( table s2 , si ) .", "these ions have different \n sizes and charges ; therefore , they have different charge densities . \n", "table 1 and figure s3 ( si ) show the results of field - sea calculations , compared with \n lpbe and tip3p .", "lpbe results using the lj surface do not capture quantitatively \n the gsolv of ions with high charge \n densities .", "in contrast , field - sea reproduces the tip3p gsolv values regardless of the specific lj parameters \n and charges .", "these results indicate that field - sea is accurately reproducing \n explicit - solvent free energies of solvation of monatomic ions , provided \n that their charge and lj parameters are near or encompassed within \n the range of the rw and free - energy contours .", "a common approximation in simplified \n solvation models is to suppose that atoms have fixed ", "atom \n types  , where any particular solute atom has a given value \n of charge and radius , independent of its atomic neighbors . however , \n our tip3p explicit - solvent simulations described below show that this \n approximation is a source of error . how the solvation surface is constructed \n", "our explicit - solvent simulations show that the solvation shell is \n not just a simple union of the spherical surfaces of all of the atoms \n making up the solute molecule .", "imagine a diatomic solute with partially \n charged atom a covalently connected to partially \n charged atom b. our tip3p simulations show that when \n atom a attracts water molecules , it also shrinks \n the solvation shell of waters around atom b. in this \n way , the solvation surface around the diatomic molecule a  b can be more complex than the simple union \n of independent spherical solvation shells around atoms a and b. to address such situations , we developed \n an adaptive method that gives a more explicit - like solvation boundary . to test our adaptive boundary method , we made up 22 fictitious \n diatomic solutes ( see si table s4 ) , computed \n their solvation free energies , and compared to their solvation in \n tip3p .", "we constructed these solutes by taking pairs of ordinary simulation \n atom types , placing them at fixed covalent bond distance apart , and \n giving each atom a charge and radius that we could vary systematically \n over the series . because of the fictitious charges that we give them , \n these are not molecules observed in nature", "however , they are physically \n plausible molecules that provide us with a systematic series for learning \n about how explicit - solvent models handle solvated charges .", "figure 3 shows the computed free energies of these diatomic \n solutes from field - sea when using the fixed rw and adaptive boundaries in comparison to explicit - solvent \n tip3p simulations .", "lpbe and original sea results are shown in the si instead of here for the sake of simplicity . \n in summary", ", we find that when the tip3p gsolv is weak , for example , when the charge density of solute \n atoms is low , the fixed rw boundary works \n fairly well .", "when the gsolv grows \n to 100 kcal / mol and larger ( more negative ) , it becomes increasingly \n important to use an adaptive boundary .", "these strongly solvated model \n molecules often have large , unbalanced partial charges on the solute \n atoms , and these situations lead to exaggerated distortions of the \n explicit solvation shell .", "field - sea gsolv for model diatomic solutes ( triangles : fixed rw ; circles : adaptive boundary ) compared to tip3p simulations . \n", "figure 4 shows how a neighboring \n solute atom can affect the solvation shell about another atom .", "the \n fixed rw and adaptive boundaries are identical \n in figure 3a as both carbon atoms are weakly \n charged and there is only minor collective electrostatic perturbation \n on the boundary . however ,", "when the solutes are more highly charged \n and large charging energies are involved , as in figure 3b , the weakly charged carbon atom s solvation shell \n will be distorted by its neighboring highly charged oxygen atom . in \n this case ,", "water molecules penetrate more deeply into the carbon s \n solvent - accessible surface to better solvate the oxygen charge ( the \n right part of the white curve in the dark blue region ) .", "also , water \n molecules pack more tightly around the carbon atom and reduce its \n apparent rw ( the left part of the white \n curve in the blue and light blue regions ) .", "both of these effects are \n captured well by adaptive field - sea boundaries , leading to field - sea \n charging energies that are in excellent agreement with explicit simulation \n results .", "oxygen density around ( a ) weakly charged \n and ( b ) strongly charged diatomic solutes .", "the blue contours indicate \n water density greater than the bulk value , with the darker blue regions \n indicating the enhanced water probability density .", "the black line \n shows the nonadaptive fixed rw boundary . \n for the weakly charged diatomic , the adaptive and nonadaptive boundaries \n coincide .", "the adaptive and nonadaptive boundaries differ . in order to establish that the accuracy of field - sea is not degraded \n relative to sea on nonionic solutes , we applied field - sea with both \n fixed rw and adaptive boundaries to a \n standard set of 504 neutral small molecules .", "this set contains an alchemically diverse \n range of functional groups , for which solvation free energies are \n available from experiments and tip3p simulations .", "figure 5 shows 504 neutral solutes solvation free \n energy from tip3p simulation , from the lpbe ( white diamonds ) and field - sea \n ( white circles ) ( see si figure s4 for nonadaptive \n fixed rw field - sea results ) .", "field - sea \n shows a rmse ( root - mean - square error ) of 1kt with a negligible mse ( mean - signed error ) , comparable to the original \n sea and considerably better than the \n lpbe , which bears a systematically negative error .", "while the fixed rw boundary field - sea results are comparable \n to the lpbe ( table 1 ) , it overestimates the \n free energy when weakly charged c atoms are neighbored by highly charged \n o or n atoms , as in the cases of alcohols , amines , ethers , and esters \n ( si table s5 ) .", "these errors arise because \n the fixed rw boundary can not capture water \n molecule penetration into the c atom s solvent - accessible surface \n ( see figure 4b ) , situations the adaptive boundary \n handles directly . therefore , while these are simply neutral solutes , \n collective solute interactions still play a clear role in their overall \n hydration .", "figure s5 ( si ) compares \n the total solvation free energy from field - sea with experimental results . \n", "the mse / rmse of field - sea to experimental solvation free energy ( 0.67/1.45 \n kcal / mol , table s6 ( si ) ) is comparable \n to that of the much more computationally expensive tip3p water model \n ( 0.66/1.22 kcal / mol ) .", "these results indicate \n that field - sea can accurately compute solvation free energies over \n the full range from charge - dense ions to neutral polar and nonpolar \n molecular solutes . here , we test field - sea on an expanded set of molecular \n ions and biomolecules ( e.g. , acetate , butylammonium , etc . ; see si table s7 for the complete list and results ) .", "these are ions that are larger and more complex than the simple \n atomic and diatomic ions described above and should better test the \n need for adaptive boundary considerations .", "figure 5 compares solvation energies from lpbe ( orange diamond ) and \n field - sea ( orange circle ; see si figure \n s6 for field - sea results with a fixed rw boundary ) with tip3p results for 35 molecular ions .", "the errors for \n both lpbe and field - sea with a fixed rw boundary are around 10 kcal / mol ( table 1 ) . \n", "while this might be regarded as acceptable in light of the nearly \n 100 kcal / mol span of free energies covered in the tip3p simulations , \n using an adaptive boundary with field - sea has half of this rmse .", "field - sea \n also performs well in multivalent molecular ion solvation ( si figure s7 ) and is as accurate as explicit - solvent \n calculations compared to experimental values for ionic solute solvation \n ( table s6 and figure s8 , si ) .", "md simulations \n of gsolv for 504 neutral solutes \n ( white ) , 35 molecular ions ( orange ) , and 22 capped amino acid dipeptides \n ( cyan ) in tip3p water , compared to ( a ) lpbe and ( b ) field - sea .", "we also tested our methods on 22 capped amino \n acids ( n - acetyl - x - methylamide , x = glu , arg , leu , \n etc . ;", "si table s8 ) , which are widely used \n biological models in both theoretical studies and experiments .", "these are useful precursor \n structures for the foundation of hydrophobicity scales , used in estimating \n the solvation of larger biomolecular structures . here", ", we investigate the solvation \n free energies of a full series of capped amino acids with field - sea . \n as the size of solute increases , computing the total solvation", "show , lpbe \n ( cyan diamond ) and field - sea ( cyan circle ) both yield solvation free \n energies that agree well with tip3p calculations .", "again , the adaptive \n boundary helps field - sea considerably , cutting the rmse to half of \n that seen from the lpbe or fixed rw cases . \n", "these results indicate that the accuracy of field - sea does not degrade \n as the solute size further increases .", "the solvation boundaries \n in field - sea are made from a set of surface dots .", "above , we used 80 dots / atom , \n the same as was used in previous sea studies .", "what is the minimum number of grid dots that we need to properly \n represent the first - shell boundary ? to investigate this , we performed \n an analysis of accuracy versus relative computational time as a function \n of the granularity of the boundary .", "this analysis indicates that the \n rmse for field - sea results on the 504 neutral molecule set is essentially \n uncompromised even down to a granularity of 5 dots / atom , without increasing \n the rmse above 1 kcal / mol ( figure s9 and s10 ( si ) , the granularity does not affect the accuracy for charged \n solutes either ) . at 5 dots / atom ,", "field - sea is roughly 5-fold faster \n than dipolar sea at 80 dots / atom , the minimum surface granularity \n recommended for this method .", "these results \n indicate that while field - sea is sensitive to the physicality of the \n solvation boundary , it is less sensitive to the granularity of its \n depiction .", "we have \n described field - sea , a method for computing solvation free energies \n of solutes in water .", "it improves upon an earlier method called sea \n ( semi - explicit assembly ) .", "sea captured the physics of solvation by \n presimulating toy spheres in explicit water , collecting a database \n of structural and energetic properties of those waters and then assembling \n at run time the solvation physics as sums over appropriate toy spheres \n to properly represent a given solute . in field - sea , this procedure \n differs in using a solvation free - energy field , rather than explicit \n waters .", "furthermore , field - sea uses an adaptive boundary , allowing \n solvating waters to approach a solute atom to different degrees depending \n on neighboring atoms .", "relative to sea , field - sea captures the solvation \n free energies of ions and charged solutes accurately , is faster to \n compute , and has no degradation of performance on nonpolar and polar \n solutes .", "both sea and field - sea offer advantages over implicit - solvent \n modeling in that they entail no adjustable solute atom radii parameters . \n", "sea and field - sea are built upon a corresponding force field and explicit - solvation \n model . here", "one of the \n key observations that arises from our md simulations of charged solutes \n in tip3p water , which is captured by field - sea , is that atoms that \n are adjacent to charged atoms in solutes acquire partial characteristics \n of those charged atoms .", "for example , a weakly charged atom s \n solvation shell is shrunk by its neighboring big charges .", "an implication \n of this for implicit - solvent modeling is that atomic radii should \n not be treated as fixed for solvation in water ; an atom s radius \n in implicit - solvent modeling can depend on the nature of its neighboring \n atom ."], "abstract_text": ["<S> previous \n work describes a computational solvation model called semi - explicit \n assembly ( sea ) . </S>", "<S> the sea water model computes the free energies of \n solvation of nonpolar and polar solutes in water with good efficiency \n and accuracy . </S>", "<S> however , sea gives systematic errors in the solvation \n free energies of ions and charged solutes . </S>", "<S> here , we describe field - sea , \n an improved treatment that gives accurate solvation free energies \n of charged solutes , including monatomic and polyatomic ions and model \n dipeptides , as well as nonpolar and polar molecules . field - sea is \n computationally inexpensive for a given solute because explicit - solvent \n model simulations are relegated to a precomputation step and because \n it represents solvating waters in terms of a solute s free - energy \n field . </S>", "<S> in essence , field - sea approximates the physics of explicit - model \n simulations within a computationally efficient framework . </S>", "<S> a key finding \n is that an atom s solvation shell inherits characteristics \n of a neighboring atom , especially strongly charged neighbors . </S>", "<S> field - sea \n may be useful where there is a need for solvation free - energy computations \n that are faster than explicit - solvent simulations and more accurate \n than traditional implicit - solvent simulations for a wide range of \n solutes . </S>"], "labels": null, "section_names": ["Introduction", "Theory and Methods", "Results and Discussion", "Conclusions"], "sections": [["there \n is a need for computer methods that can calculate the aqueous solvation \n free energies of solute molecules accurately and efficiently .", "explicit - solvent models provide a physically \n accurate and atomistically detailed model of solvent , but they can \n be computationally expensive .", "boltzmann \n ( pb ) , generalized born ( gb ) , and \n weighted surface area ( wsa ) approaches , are computationally efficient because they treat \n water as a continuum .", "however , they are sometimes inaccurate , smearing \n out the particulate nature of water molecules , and they may \n have limited transferability to situations for which they have not \n been optimized .", "solvation modeling has \n often been improved by combining the advantages of implicit- and explicit - solvent \n models .", "one such approach is the semi - explicit assembly ( sea ) water model . in the sea approach ,", "water s solvation behavior is precomputed \n in explicit - solvent simulations of water around model spheres that \n are then combined together as building blocks to represent any given \n solute structure at run time .", "the solvation free energy gsolv for a target solute molecule is calculated \n from a sum over solvating waters .", "sea has been shown to predict efficiently \n the air - to - water transfer free energies ( ghyd ) of small - molecule solutes in both prospective and \n retrospective studies with reasonable accuracy .", "even \n so , the sea model does not give accurate solvation free energies of \n ions or solutes having high charge density .", "here , we describe \n field - sea , a considerable improvement over sea , which gives accurate \n free energies of transfer of ions and charged solutes , at no additional \n computational cost and with no degradation of the predictions for \n nonpolar and uncharged polar solutes . in overview , we studied full \n md simulations of ionic and neutral solutes in tip3p water , described \n below , and found that the results could be captured by using a solvation \n free - energy field surface ( in field - sea ) , instead of using precomputed \n waters ( as in sea ) .", "moreover , \n we found that a weakly charged solute atom that is adjacent to a strongly \n charged solute atom retains some of the restrictions of its solvating \n water molecules that its neighboring charge has ; see figure 1 .", "field - sea captures this effect using an adaptive \n boundary method . a solvent  water molecule around a solute molecule . \n", "on the left , each solute atom ( weakly charged ) has a predefined radius , \n irrespective of its neighboring solute atoms , leading to the locus \n of water centers shown by the black dashed curve . on the right ,", "one \n solute atom is strongly charged , leading to two consequences : its \n own solvating water molecule is pulled in tightly , and neighboring \n solute atoms have tighter water interactions too .", "such effects may not be captured in simplified solvation \n models that treat atoms as having fixed radii , independent of neighboring \n atoms ."], ["below , we describe the field - sea approach to computing solvation \n free energies . the original sea model is described elsewhere , and our related explicit - solvent and linearized poisson ", "boltzmann \n equation modeling ( lpbe ) are described \n in the supporting information ( si ) . in both the original sea and the new \n field - sea described here ,", "first , in a precomputation step , various \n model spheres are solvated in an explicit - solvent model , such as tip3p . \n", "this provides a database of component free energies that are used \n in the second step . in the second step , at the run time ,", "any given \n solute molecule of interest is assembled from an appropriate concatenation \n of these spheres .", "the solute s solvation free energy is computed \n by summing the component sphere free energies . on the one hand , this \n approach provides the speed advantages of simple additivity - based \n models as we only need to perform the first step once for a given \n solvent model . on the other hand , this procedure is more accurate \n than additivity - based approaches because ( 1 ) sea sums are regional , \n not local , and ( 2 ) the database encodes the microscopic response observed \n in explicit - solvent simulations .", "the new field - sea instead captures \n the solvation free energy using a continuous solvation field . to do this ,", "the charging free energies for a series of lennard - jones \n ( lj ) spheres solvated in tip3p water were calculated with thermodynamics \n integration ( ti ) in the set of precomputations ( see explicit solvent \n free energy calculations in the si ) .", "we \n construct a free - energy contour ( see si figure s1 ) as a combination of the electric field at the first solvation \n shell boundary ( e = q / rw , where e is the signed \n magnitude of the electric field , q is the sphere s \n charge , and rw is the distance from the \n sphere center to the first peak of the water oxygen s radial \n distribution function , rdf , around the given sphere ) , the curvature c = 1/rw at this boundary , and \n the charging free energy of the spheres . within each charge step ( q ) , all of the data of free energy , electric field , and \n curvature were fitted to a formula , which can be taken as an expansion \n of the born model1 equation 1 will \n be used to calculate the free energy associated with any surface spot \n on an arbitrary solute  solvent boundary ( esub ) .", "this free energy could also be calculated from interpolation \n between data points on the free - energy contour .", "in addition \n to a charging free - energy contour , we also need a contour for estimating \n the explicit - solvent - accessible surface of a given solute molecule . \n to generate this", ", we calculated the rdf of water oxygen around each \n charged sphere and picked the first peak in the rdf , denoted as the \n boundary - sphere distance rw .", "all of the rw values from these spheres and their charge \n and lj parameters ( lj and lj ) \n were used to build an rw contour . to generate a solvent - accessible \n surface for a given solute", ", an rw for \n each atom is first calculated from interpolation with its partial \n charge , lj , and lj on the rw contour .", "a lee  richards surface is then constructed from the nonoverlapping \n sections of these rw spheres centered \n on their associated atom sites .", "the fixed rw boundary is dependent upon only the individual parameters \n of the surface atoms .", "for solute molecules with multiple partial charge \n sites ( especially molecular ions , where strong electric fields are \n involved ) , a more physical representation of the explicit - solvent - accessible \n surface would be one that responds to the whole solute s electric \n field .", "in other words , the surface  atom distance , rw , is determined not only by the surface atom s \n partial charge , but also by neighboring atoms charges .", "taking \n into account such collective electrostatic effects , we adjust the \n fixed rw boundary in an adaptive manner , \n described by the following three - step procedure : ( 1)we cull \n all surface segments within 1.4  of any solute atom . as we are \n starting with a lee  richards solvent - accessible surface , the \n minimum rw possible in the surface construction \n is half of a water molecule diameter ( corresponding to a solute atom \n with a 0  radius ) .", "this partial - culling process simply removes \n potential numerical instabilities from surface sites overlapping solute \n atom centers while providing some adjustable starting surface sites \n that penetrate within the fixed rw boundary .", "we calculate \n the electric field at a given dot a about atom a2where n is the number \n of solute atoms , ria represents a vector from atom i to surface \n dot a , and ria is its length ; signa = 1 when raai=1n  ( qi / ria3)ria  0 , and signa = 1 when raai=1n  ( qi / ria3)ria < 0 , and raa is the vector from atom a to dot a. from the electric field , we \n calculate the corresponding charge by3where raa is the length of vector raa . we interpolate with this new charge and \n atom a s lj parameters to get a new rw , rw , new .", "in addition , \n we assume that rw can not expand ( if rw , new > rw , original , we let rw , new = rw , original ) . this nonexpansion assumption is supported by solute ", "solvent \n boundary plots of model diatomic solutes ( see si figure s2 ) . finally , the rw , new s of all atom s potential surface dots are averaged \n to yield an rw for this atom , used in \n the later culling process . ( 3 ) we cull the buried dots adaptively . \n", "because each dot  atom distance ( e.g. , daa ) is adjusted independently in the above \n procedure , the shell of potential surface dots about an atom will \n no longer be spherical .", "thus , culling buried dots is no longer a simple \n process of eliminating dots within a neighboring atom s rw , and an adaptive culling process is needed . \n to adaptively cull ", "buried  surface dots , when we check \n whether a given dot a is buried by a neighboring \n atom b , we first determine a corresponding charge \n at dot a from the electric field at this dot .", "this \n is used along with atom b s lj parameters \n to get a new rw , rw , badp , following the above rw adjustment procedure .", "atom \n distance , dab , with rw , badp , to determine if the \n dot is buried in atom b. if dab is less than rw , badp , it is removed from the set of potential surface \n dots .", "we call the new dot surface resulting from the above culling \n procedure the adaptive boundary .", "our term field - sea refers both to \n the field and to the adaptive boundary .", "the adaptive nature of the \n boundary only pertains to multiatom solutes ; the adaptive and fixed rw boundaries are identical for single - atom solutes , \n like monatomic ions . while our adaptation procedure could , in principle , \n be applied iteratively , we found no further improvement after a single \n calculation .", "the charging \n free energy can be calculated for any given field - sea surface via4where ndot is the total number of surface - exposed dots , mi is the total number of dots \n ( exposed + occluded ) for corresponding atom i ( each \n surface dot , j , of atom i , only \n corresponds to 1/mi of \n this atom sphere s total surface area or total solvation free \n energy ) , and esub , j is \n the subenergy associated with exposed surface dot j , calculated from the free - energy contour . here ,", "esub , j is calculated from eq 1 using the signed magnitude of the electric field , ej , and the curvature at dot j , 1/rij .", "ej is defined as negative when ejrij < 0 , where ej is the electric \n field ( vector ) at dot j , rij is the vector from atom i to its surface dot j , and rij is this vector s length .", "now , \n we take the total solvation free energy to be the sum of polar and \n nonpolar components5 assuming the polar component of the free energy of transfer \n ( gpol ) can be uniquely described \n by both the surface electric field and geometry of the solute , this \n molecular gpol can be accurately \n calculated from the simple summation of the surface dots energies \n process described above .", "we consider the gsolv of monatomic ions as an initial test . for molecular \n solutes ,", "to account for the role of solute conformation in the solvation \n free energy , we average gsolv results \n from calculations on 50 conformations ( though 10 conformations are \n usually enough ; see the si ) . these solute \n conformations were generated from 5 ns tip3p water md simulations \n with a 100 ps snapshot interval ."], ["we developed field - sea because of the errors that we \n observed in simpler methods in solvating ions ; see figure 2 .", "the explicit - solvent curve shows the hydration \n asymmetry discussed by others , where positively charged solutes are less favored than negatively \n charged solutes of similar size .", "gsolv as the function of a model lj sphere (  \n = 0.22 nm ,  = 0.06538 kj / mol ) charge for tip3p , lpbe , and field - sea . \n for comparison at infinite - dilution conditions ,", "an ewald correction \n is applied to the tip3p and field - sea results . here , we tested field - sea on the solvation free energies \n of monatomic ions using ion parameters developed by aqvist or joung and cheatham ( table s2 , si ) .", "these ions have different \n sizes and charges ; therefore , they have different charge densities . \n", "table 1 and figure s3 ( si ) show the results of field - sea calculations , compared with \n lpbe and tip3p .", "lpbe results using the lj surface do not capture quantitatively \n the gsolv of ions with high charge \n densities .", "in contrast , field - sea reproduces the tip3p gsolv values regardless of the specific lj parameters \n and charges .", "these results indicate that field - sea is accurately reproducing \n explicit - solvent free energies of solvation of monatomic ions , provided \n that their charge and lj parameters are near or encompassed within \n the range of the rw and free - energy contours .", "a common approximation in simplified \n solvation models is to suppose that atoms have fixed ", "atom \n types  , where any particular solute atom has a given value \n of charge and radius , independent of its atomic neighbors . however , \n our tip3p explicit - solvent simulations described below show that this \n approximation is a source of error . how the solvation surface is constructed \n", "our explicit - solvent simulations show that the solvation shell is \n not just a simple union of the spherical surfaces of all of the atoms \n making up the solute molecule .", "imagine a diatomic solute with partially \n charged atom a covalently connected to partially \n charged atom b. our tip3p simulations show that when \n atom a attracts water molecules , it also shrinks \n the solvation shell of waters around atom b. in this \n way , the solvation surface around the diatomic molecule a  b can be more complex than the simple union \n of independent spherical solvation shells around atoms a and b. to address such situations , we developed \n an adaptive method that gives a more explicit - like solvation boundary . to test our adaptive boundary method , we made up 22 fictitious \n diatomic solutes ( see si table s4 ) , computed \n their solvation free energies , and compared to their solvation in \n tip3p .", "we constructed these solutes by taking pairs of ordinary simulation \n atom types , placing them at fixed covalent bond distance apart , and \n giving each atom a charge and radius that we could vary systematically \n over the series . because of the fictitious charges that we give them , \n these are not molecules observed in nature", "however , they are physically \n plausible molecules that provide us with a systematic series for learning \n about how explicit - solvent models handle solvated charges .", "figure 3 shows the computed free energies of these diatomic \n solutes from field - sea when using the fixed rw and adaptive boundaries in comparison to explicit - solvent \n tip3p simulations .", "lpbe and original sea results are shown in the si instead of here for the sake of simplicity . \n in summary", ", we find that when the tip3p gsolv is weak , for example , when the charge density of solute \n atoms is low , the fixed rw boundary works \n fairly well .", "when the gsolv grows \n to 100 kcal / mol and larger ( more negative ) , it becomes increasingly \n important to use an adaptive boundary .", "these strongly solvated model \n molecules often have large , unbalanced partial charges on the solute \n atoms , and these situations lead to exaggerated distortions of the \n explicit solvation shell .", "field - sea gsolv for model diatomic solutes ( triangles : fixed rw ; circles : adaptive boundary ) compared to tip3p simulations . \n", "figure 4 shows how a neighboring \n solute atom can affect the solvation shell about another atom .", "the \n fixed rw and adaptive boundaries are identical \n in figure 3a as both carbon atoms are weakly \n charged and there is only minor collective electrostatic perturbation \n on the boundary . however ,", "when the solutes are more highly charged \n and large charging energies are involved , as in figure 3b , the weakly charged carbon atom s solvation shell \n will be distorted by its neighboring highly charged oxygen atom . in \n this case ,", "water molecules penetrate more deeply into the carbon s \n solvent - accessible surface to better solvate the oxygen charge ( the \n right part of the white curve in the dark blue region ) .", "also , water \n molecules pack more tightly around the carbon atom and reduce its \n apparent rw ( the left part of the white \n curve in the blue and light blue regions ) .", "both of these effects are \n captured well by adaptive field - sea boundaries , leading to field - sea \n charging energies that are in excellent agreement with explicit simulation \n results .", "oxygen density around ( a ) weakly charged \n and ( b ) strongly charged diatomic solutes .", "the blue contours indicate \n water density greater than the bulk value , with the darker blue regions \n indicating the enhanced water probability density .", "the black line \n shows the nonadaptive fixed rw boundary . \n for the weakly charged diatomic , the adaptive and nonadaptive boundaries \n coincide .", "the adaptive and nonadaptive boundaries differ . in order to establish that the accuracy of field - sea is not degraded \n relative to sea on nonionic solutes , we applied field - sea with both \n fixed rw and adaptive boundaries to a \n standard set of 504 neutral small molecules .", "this set contains an alchemically diverse \n range of functional groups , for which solvation free energies are \n available from experiments and tip3p simulations .", "figure 5 shows 504 neutral solutes solvation free \n energy from tip3p simulation , from the lpbe ( white diamonds ) and field - sea \n ( white circles ) ( see si figure s4 for nonadaptive \n fixed rw field - sea results ) .", "field - sea \n shows a rmse ( root - mean - square error ) of 1kt with a negligible mse ( mean - signed error ) , comparable to the original \n sea and considerably better than the \n lpbe , which bears a systematically negative error .", "while the fixed rw boundary field - sea results are comparable \n to the lpbe ( table 1 ) , it overestimates the \n free energy when weakly charged c atoms are neighbored by highly charged \n o or n atoms , as in the cases of alcohols , amines , ethers , and esters \n ( si table s5 ) .", "these errors arise because \n the fixed rw boundary can not capture water \n molecule penetration into the c atom s solvent - accessible surface \n ( see figure 4b ) , situations the adaptive boundary \n handles directly . therefore , while these are simply neutral solutes , \n collective solute interactions still play a clear role in their overall \n hydration .", "figure s5 ( si ) compares \n the total solvation free energy from field - sea with experimental results . \n", "the mse / rmse of field - sea to experimental solvation free energy ( 0.67/1.45 \n kcal / mol , table s6 ( si ) ) is comparable \n to that of the much more computationally expensive tip3p water model \n ( 0.66/1.22 kcal / mol ) .", "these results indicate \n that field - sea can accurately compute solvation free energies over \n the full range from charge - dense ions to neutral polar and nonpolar \n molecular solutes . here , we test field - sea on an expanded set of molecular \n ions and biomolecules ( e.g. , acetate , butylammonium , etc . ; see si table s7 for the complete list and results ) .", "these are ions that are larger and more complex than the simple \n atomic and diatomic ions described above and should better test the \n need for adaptive boundary considerations .", "figure 5 compares solvation energies from lpbe ( orange diamond ) and \n field - sea ( orange circle ; see si figure \n s6 for field - sea results with a fixed rw boundary ) with tip3p results for 35 molecular ions .", "the errors for \n both lpbe and field - sea with a fixed rw boundary are around 10 kcal / mol ( table 1 ) . \n", "while this might be regarded as acceptable in light of the nearly \n 100 kcal / mol span of free energies covered in the tip3p simulations , \n using an adaptive boundary with field - sea has half of this rmse .", "field - sea \n also performs well in multivalent molecular ion solvation ( si figure s7 ) and is as accurate as explicit - solvent \n calculations compared to experimental values for ionic solute solvation \n ( table s6 and figure s8 , si ) .", "md simulations \n of gsolv for 504 neutral solutes \n ( white ) , 35 molecular ions ( orange ) , and 22 capped amino acid dipeptides \n ( cyan ) in tip3p water , compared to ( a ) lpbe and ( b ) field - sea .", "we also tested our methods on 22 capped amino \n acids ( n - acetyl - x - methylamide , x = glu , arg , leu , \n etc . ;", "si table s8 ) , which are widely used \n biological models in both theoretical studies and experiments .", "these are useful precursor \n structures for the foundation of hydrophobicity scales , used in estimating \n the solvation of larger biomolecular structures . here", ", we investigate the solvation \n free energies of a full series of capped amino acids with field - sea . \n as the size of solute increases , computing the total solvation", "show , lpbe \n ( cyan diamond ) and field - sea ( cyan circle ) both yield solvation free \n energies that agree well with tip3p calculations .", "again , the adaptive \n boundary helps field - sea considerably , cutting the rmse to half of \n that seen from the lpbe or fixed rw cases . \n", "these results indicate that the accuracy of field - sea does not degrade \n as the solute size further increases .", "the solvation boundaries \n in field - sea are made from a set of surface dots .", "above , we used 80 dots / atom , \n the same as was used in previous sea studies .", "what is the minimum number of grid dots that we need to properly \n represent the first - shell boundary ? to investigate this , we performed \n an analysis of accuracy versus relative computational time as a function \n of the granularity of the boundary .", "this analysis indicates that the \n rmse for field - sea results on the 504 neutral molecule set is essentially \n uncompromised even down to a granularity of 5 dots / atom , without increasing \n the rmse above 1 kcal / mol ( figure s9 and s10 ( si ) , the granularity does not affect the accuracy for charged \n solutes either ) . at 5 dots / atom ,", "field - sea is roughly 5-fold faster \n than dipolar sea at 80 dots / atom , the minimum surface granularity \n recommended for this method .", "these results \n indicate that while field - sea is sensitive to the physicality of the \n solvation boundary , it is less sensitive to the granularity of its \n depiction ."], ["we have \n described field - sea , a method for computing solvation free energies \n of solutes in water .", "it improves upon an earlier method called sea \n ( semi - explicit assembly ) .", "sea captured the physics of solvation by \n presimulating toy spheres in explicit water , collecting a database \n of structural and energetic properties of those waters and then assembling \n at run time the solvation physics as sums over appropriate toy spheres \n to properly represent a given solute . in field - sea , this procedure \n differs in using a solvation free - energy field , rather than explicit \n waters .", "furthermore , field - sea uses an adaptive boundary , allowing \n solvating waters to approach a solute atom to different degrees depending \n on neighboring atoms .", "relative to sea , field - sea captures the solvation \n free energies of ions and charged solutes accurately , is faster to \n compute , and has no degradation of performance on nonpolar and polar \n solutes .", "both sea and field - sea offer advantages over implicit - solvent \n modeling in that they entail no adjustable solute atom radii parameters . \n", "sea and field - sea are built upon a corresponding force field and explicit - solvation \n model . here", "one of the \n key observations that arises from our md simulations of charged solutes \n in tip3p water , which is captured by field - sea , is that atoms that \n are adjacent to charged atoms in solutes acquire partial characteristics \n of those charged atoms .", "for example , a weakly charged atom s \n solvation shell is shrunk by its neighboring big charges .", "an implication \n of this for implicit - solvent modeling is that atomic radii should \n not be treated as fixed for solvation in water ; an atom s radius \n in implicit - solvent modeling can depend on the nature of its neighboring \n atom ."]]}
{"article_id": "PMC4401064", "article_text": ["hydatidosisis term used to refer to infection with the parasite larva in humans and echinococcosis restricted to infection with the adult stage in carnivorous animals ( 1 ) .", "recently , the genetics , structure and function of the human cytosolic gst enzyme with emphasis on their roles in the cellular metabolism has been defined ( 2 ) .", "gst activity was detected in most mammalian tissues , especially in the liver which plays a key role in detoxification .", "there are different classes of gst isozymes that diverge in their specificity to xenobiotic or endogenous substrates ( 3 ) .", "gst enzymes involved in the cellular detoxification of a broad range of chemical substrates ( 4 ) . apart from reaction from their endogenous metabolism , gsts of helminth parasite may protect against exogenous xenobiotics as a result of immune effectors mechanisms from the host ( 5 ) .", "significantly higher activity has been found in intestinal cestodes and digeneas , compared with parasitic nematodes ( 6 ) .", "gsts activity assay has been demonstrated in the cytosol of protoscolices from sheep hydatid cysts ( 7 ) .", "diagnosis of hydatid disease is done by a combination of clinical signs , imaging techniques , cyst fluid examination , serological tests and molecular techniques ( 9 ) .", "the prevalence of liver hydatid cyst was reported 4.7% in people of peru country using recombinant antigen , epc1 glutathione s - transferase [ repc1-gst ] , in western blot technique ( 10 ) .", "however , the hydatid cyst diagnosis technique is under developing due to specificity and sensitivity problems . in the present study , gst enzyme activity of hydatid cyst protoscolices ( parasite ) ,", "healthy and cystic liver tissues were compared and mentioned gst enzyme importance for diagnostic biomarker in hydatid cyst disease .", "ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) .", "parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) .", "sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 .", "then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) . the protein concentration in the extract solutions of protoscolices and sheep liver tissues", "were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) .", "gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates .", "protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice .", "cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette .", "to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) .", "sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively .", "molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands", "was calculated , standard curve was prepared in excel software and finally proteins mw were determined .", "independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues .", "ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) .", "parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) .", "sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 . then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) .", "the protein concentration in the extract solutions of protoscolices and sheep liver tissues were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) .", "gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates .", "protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice .", "cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette . to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) .", "sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively .", "molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands", "was calculated , standard curve was prepared in excel software and finally proteins mw were determined .", "independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues .", "the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 .", "protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) .", "significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) .", "statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) .", "extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig .", "1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples .", "similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse .", "the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page .", "lanes 1 - 3 are healthy liver samples . lanes 4 - 6 are cystic liver samples .", "lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no .", "64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org )", "the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 .", "protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) .", "significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) .", "statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) .", "extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig .", "1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples .", "similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse .", "the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page .", "lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no .", "64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org )", "function of hepatocytesmay be disturbed in the presence of infections ( 15 ) . in our study", "the reduction of protein synthesis as a result of hydatid cyst causes to decrease protein concentration .", "gsts can make up to 10% of cytosolic protein in some mammalian organs ( 16 ) .", "hepatic cells contain high levels of gst enzyme which has been found to be an indicator of hepatocyte injury in transplantation , toxicity and infections ( 17 ) .", "the hydatid cyst infection stimulates oxidative stress and toxin production in hepatic cells . from the point of biochemical defense view gst enzyme", "be able to neutralization of these toxins , therefore we expect to increase activity level of this enzyme .", "increase of liver protein and gst in infected mice indicates the occurrence of oxidative stress in hepatocytes due to infection ( 18 ) .", "serum gsta is a more sensitive marker than transaminases ( alanine aminotransferase , asparatate aminotransferase and alkaline transferase ) for monitoring and as an early analyst of hepatic damage ( 19 ) .", "therefore gst activity difference between healthy and infected host liver tissue could be concerned for hydatid cyst diagnosis . however , other infections cause to hepatocyte damage , thus gst elevation must be evaluated with other clinical and paraclinical parameters of hydatid cyst disease .", "mammalian cytosolic gsts are dimeric , with both subunits being from the same class of gsts , although not necessarily identical .", "gst enzyme molecular weight of parasite is reported 24 - 27 kda ( 13 ) . in this", "research protein bands with 24kda and 25kda were found in parasite and liver tissues respectively .", "gst activity in cystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other parameters of hydatidosis ."], "abstract_text": ["<S> abstractbackgroundthe aim of this study was to detect the glutathione s - transferase(gst ) enzyme activity of healthy / cystic liver as a diagnostic biomarker for hydatidosis . in order to compare with liver tissue , </S>", "<S> the level of the gsts enzyme activity of parasite was also determined.methodsparasites were collected from sheep liver tissue with hydatid cysts at a local abattoir and washed with pbs buffer . </S>", "<S> collected parasites and liver tissues were sonicated or homogenized respectively . </S>", "<S> extract solution samples were centrifuged and stored at - 20c . </S>", "<S> gst enzyme activities were measured in the extract of parasite and liver tissue samples ( healthy and infected livers ) . </S>", "<S> protein amounts and protein bands were detected using bradford and sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) methods respectively . to determine significant difference between two groups , two - sample t - test was performed.resultsgst specific activities of healthy / infected livers and parasites were estimated 304 , 1297 and 146 u / ml / mg respectively . </S>", "<S> significant higher gst specific activities in cystic liver than healthy liver was observed ( p < 0.05 ) . </S>", "<S> t - test analysis showed gst activity of parasite was lower than healthy liver tissue . </S>", "<S> sds - page showed gst protein bands with 24 kda in parasite samples and25 kda in liver tissues.conclusiongst activity incystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other hydatid disease parameters . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Preparation of protoscolices (parasite) extracts solution", "Preparation of Liver extracts solution", "Protein assay in the solutions", "GSTs activity assay in the solutions", "SDS-PAGE analysis of samples", "Statistical analysis", "Results", "Protein concentration, enzyme activities and statistical analysis results", "SDS-PAGE analysis results", "Discussion", "Conclusion"], "sections": [["hydatidosisis term used to refer to infection with the parasite larva in humans and echinococcosis restricted to infection with the adult stage in carnivorous animals ( 1 ) .", "recently , the genetics , structure and function of the human cytosolic gst enzyme with emphasis on their roles in the cellular metabolism has been defined ( 2 ) .", "gst activity was detected in most mammalian tissues , especially in the liver which plays a key role in detoxification .", "there are different classes of gst isozymes that diverge in their specificity to xenobiotic or endogenous substrates ( 3 ) .", "gst enzymes involved in the cellular detoxification of a broad range of chemical substrates ( 4 ) . apart from reaction from their endogenous metabolism , gsts of helminth parasite may protect against exogenous xenobiotics as a result of immune effectors mechanisms from the host ( 5 ) .", "significantly higher activity has been found in intestinal cestodes and digeneas , compared with parasitic nematodes ( 6 ) .", "gsts activity assay has been demonstrated in the cytosol of protoscolices from sheep hydatid cysts ( 7 ) .", "diagnosis of hydatid disease is done by a combination of clinical signs , imaging techniques , cyst fluid examination , serological tests and molecular techniques ( 9 ) .", "the prevalence of liver hydatid cyst was reported 4.7% in people of peru country using recombinant antigen , epc1 glutathione s - transferase [ repc1-gst ] , in western blot technique ( 10 ) .", "however , the hydatid cyst diagnosis technique is under developing due to specificity and sensitivity problems . in the present study , gst enzyme activity of hydatid cyst protoscolices ( parasite ) ,", "healthy and cystic liver tissues were compared and mentioned gst enzyme importance for diagnostic biomarker in hydatid cyst disease ."], ["ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) .", "parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) .", "sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 .", "then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) . the protein concentration in the extract solutions of protoscolices and sheep liver tissues", "were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) .", "gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates .", "protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice .", "cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette .", "to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) .", "sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively .", "molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands", "was calculated , standard curve was prepared in excel software and finally proteins mw were determined .", "independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues ."], ["ten samples of parasites were obtained from 10 liver infected with hydatid cysts of sheep slaughtered at a localabattoir ( karaj , iran ) .", "parasites samples were washed 3 times with pbs buffer , ph 7.2 , freeze - thawed 3 - 6 times in liquid nitrogen and and water bath 37c respectively and sonicated in a 150w ultrasonic disintegrator,10 sec on and 5 sec off on ice until no intact psc were visible microscopically ( approximately 15 min ) . then resulted suspension was centrifuged ( 10000 g for 30 min at 4c ) and supernatant was stored at -20c ( 11 ) ."], ["sheep livers ( 10 health liver samples and 10 infected samples ) were obtained at a local abattoir and washed 3 times with pbs buffer ph 7.2 . then they were homogenized with 3 volumes of homogenizing buffer , pbs ph 6.5 , in a glass homogenizer , so the suspension were centrifuged ( 10000 g for 30 minat 4c ) and supernatant stored at -20c ( 12 ) ."], ["the protein concentration in the extract solutions of protoscolices and sheep liver tissues were estimated by the method of bradford using bovine serum albumin as the standard ( 12 ) ."], ["gsts activity was assayed spectro photometrically at 25c with reduced glutathione ( gsh ) and 1-chloro-2 , 4-dinitrobenzene ( cdnb ) as substrates .", "protosolices and liver extract samples were removed from -20c freezer and allowed to thaw on ice .", "cdnb 100 mm from 4c and gsh 100 mm from -20c freezer were removed and allowed to thaw at room temperature , when thawed , incubated at 30c in water bath . for each assay was prepared one ml of assay cocktail ( 980lpbs ph 6.5 , 10l of 100 mm cdnb and 10 l of 100 mm gsh ) , then removed 100 l of cocktail and its remaining placed 900 l of it into 1.5 ml cuvette . to zero spectrophotometer , was used 1 ml of distilled water and to the blank cuveet added 100l pbs to 900 l of cocktail and measured absorbance at 340 nm , every1 minute , for 3 min . to the test cuvette was added 100 l of sample to 900 l cocktail , mixed and measured absorbance at 340 nm as above ( 13 ) ."], ["sds - page and coomassie blue staining were used to separate and stain the protein components of samples respectively .", "molecular weights of sample proteins were compared with respect to the protein marker ( 12 ) . to detect the molecular weight , rf(ratio factor ) of ladder bands", "was calculated , standard curve was prepared in excel software and finally proteins mw were determined ."], ["independent t - test was performed to compare the mean values of protein concentration and enzyme activity between healthy and cystic liver tissues or parasite and liver tissues ."], ["the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 .", "protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) .", "significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) .", "statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) .", "extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig .", "1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples .", "similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse .", "the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page .", "lanes 1 - 3 are healthy liver samples . lanes 4 - 6 are cystic liver samples .", "lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no .", "64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org )"], ["the mean values of protein concentrations and enzyme activities for parasite , healthy and infected liver samples are presented in table 1 .", "protein concentration of healthy liver was higher than infected liver ( p < 0.05 ) .", "significant higher gst specific activities in cystic liver samples was observed as compared with healthy liver ( p < 0.05 ) .", "statistical t - test showed gst enzyme activity of parasite was lower than healthy liver tissues ( p < 0.05 ) ."], ["extract samples of protoscolices , healthy and cystic liver tissues were analyzed by sds - page electrophoresis and the results are shown in fig .", "1 . sds - page gel shows gst protein bands with 24kda in parasite and 25kda in healthy liver samples .", "similar cross protein bands is observing in parasite and liver sample but has been not recorded by databse .", "the mean values of protein amounts and gsts activity for parasite , healthy and cystic liver samples molecular weight of proteins ( kda ) from the extracts of parasite and liver tissues in sds - page .", "lanes 7 - 9 are parasite samples ( protein marker , peqgold i , lot - no .", "64072 ) identified proteins of parasite according to molecular weight by using protein database(http://web.expasy.org ) identified proteins of sheep liver tissue according to molecular weight by using protein database(http://web.expasy.org )"], ["function of hepatocytesmay be disturbed in the presence of infections ( 15 ) . in our study", "the reduction of protein synthesis as a result of hydatid cyst causes to decrease protein concentration .", "gsts can make up to 10% of cytosolic protein in some mammalian organs ( 16 ) .", "hepatic cells contain high levels of gst enzyme which has been found to be an indicator of hepatocyte injury in transplantation , toxicity and infections ( 17 ) .", "the hydatid cyst infection stimulates oxidative stress and toxin production in hepatic cells . from the point of biochemical defense view gst enzyme", "be able to neutralization of these toxins , therefore we expect to increase activity level of this enzyme .", "increase of liver protein and gst in infected mice indicates the occurrence of oxidative stress in hepatocytes due to infection ( 18 ) .", "serum gsta is a more sensitive marker than transaminases ( alanine aminotransferase , asparatate aminotransferase and alkaline transferase ) for monitoring and as an early analyst of hepatic damage ( 19 ) .", "therefore gst activity difference between healthy and infected host liver tissue could be concerned for hydatid cyst diagnosis . however , other infections cause to hepatocyte damage , thus gst elevation must be evaluated with other clinical and paraclinical parameters of hydatid cyst disease .", "mammalian cytosolic gsts are dimeric , with both subunits being from the same class of gsts , although not necessarily identical .", "gst enzyme molecular weight of parasite is reported 24 - 27 kda ( 13 ) . in this", "research protein bands with 24kda and 25kda were found in parasite and liver tissues respectively ."], ["gst activity in cystic liver tissue could be concerned as a biomarker for hydatid cyst diagnosis with other parameters of hydatidosis ."]]}
{"article_id": "PMC328088", "article_text": ["the risk of stroke and myocardial infarction is considerably increased in subjects with diabetes . already at the time of diagnosis of type 2 diabetes , many patients have manifest cardiovascular disease ( cvd ) .", "this could be due to a long presymtomatic period with increased glucose level as evident by the increased cvd risk already present with impaired glucose tolerance ( igt ) .", "thus , interest has focused on factors , not only glucose levels , in the prediabetic state that would increase the atherothrombotic process . a common , possibly genetic , antecedent of both type 2 diabetes and cvd has been proposed as the \" common soil \" hypotheses .", "recently , markers of inflammation , such as highly sensitive c - reactive protein , have been found to predict type 2 diabetes in long - time follow up of population samples , although adjusting for measurements of obesity attenuates the relationship [ 8 - 12 ] .", "such markers are weakly related to variables reflecting endothelial function such as adhesion molecules and von willebrant factor . in a large population - based study , also increased plasma levels of plasminogen activator inhibitor-1 ( pai-1 ) were strongly related to the development of diabetes independent from insulin resistance and obesity .", "the possibility of elevated pai-1 being a very early risk marker of the insulin resistance syndrome and diabetes was raised . furthermore , haemostatic variables related to endothelial function , such as von willebrant factor and factor viii , also predicted diabetes , especially in women . data are lacking regarding other aspects of the fibrinolytic system and the development of diabetes , such as the activities of pai-1 and tissue plasminogen activator ( tpa ) or the mass concentration of the endothelial - derived tpa ( \" tpa antigen \" ) , factors related to cvd [ 15 - 19 ] thus , there are two questions related to fibrinolytic variables and the development of diabetes .", "firstly , do subjects with normal glucose tolerance , who later convert to diabetes , have disturbances in the fibrinolytic system and endothelial function that could explain their increased risk of cvd even before diabetes ensues ?", "if so , do these changes predict future diabetes , independent of the influence of the insulin resistance syndrome , thereby pointing to other pathways to the diabetic state such as endothelial dysfunction ?", "we studied fibrinolytic activity , as measured by tpa and pai-1 activity , and a marker of endothelial dysfunction , as measured by tpa antigen , in a population sample with normal glucose tolerance at baseline and analysed data according to conversion to diabetes or not over nine years of follow - up .", "this study was performed within the framework of the northern sweden monica study . in 1990", "a total of 2,000 randomly selected subjects aged 25 to 64 years were invited . in all , 1,583 persons participated ( 79.2 % ) .", "a 75-gram oral glucose tolerance test ( ogtt ) was performed after an overnight fast in a randomly selected subset of subjects without known diabetes .", "venous plasma glucose samples were analysed by the hexokinase method ( boehringer mannheim automated analysis for bm / hitachi system 717 ) .", "glucose tolerance was classified according to who criteria from 1999 : normal glucose tolerance ( ngt )  fasting glucose < 7 mmol / l and post load glucose < 7.8 mmol / l , impaired glucose tolerance ( igt )  fasting glucose < 7 mmol / l and post load glucose 7.811.0 mmol / l , and diabetes  fasting glucose 7.0", "weight was measured with a balance scale to the nearest 0.2 kg , and height was measured to the nearest centimetre .", "bmi was calculated as total body weight ( kg ) / height ( m ) .", "waist circumference was measured midway between the lower rib margin and the iliac crest , which in most occasions was identical with the level of the umbilicus .", "all measurements were done in a standing position while breathing normally , and participants were asked to wear light underwear and remove shoes . for the measurement of waist circumference , the recording was done after a gentle breath - out .", "tpa and pai-1 activities were determined by chromogenic assays , spectrolyse / fibrin and spectrolyse / pl kit ( biopool ab , ume , sweden ) , respectively .", "tpa antigen was measured by an enzyme - linked immunosorbent assay method ( tintelize tpa , biopool ab ) .", "serum insulin was determined by radio immunoassay with a double - antibody solid - phase technique ( phadaseph insulin ria , pharmacia diagnostics ab , uppsala , sweden ) in 1999 , all participants , alive and still living in the area , were recalled for a follow - up examination . the same questionnaire and anthropometrical measurements as in the baseline survey were used .", "altogether 1,148 ( 72.5 % ) subjects turned up for re - examination . subjects who previously had performed an ogtt with normal or impaired glucose tolerance , and answered no to the question \"", "participants , who denied having diabetes in the baseline survey and answered affirmative to the question \" do you have diabetes mellitus ? \" at follow up , were defined as incident cases of known diabetes mellitus according tho the who criteria from 1999 . in november 2002 , a questionnaire exploring details related to the diagnosis of diabetes , and to the type and duration of current hypoglycaemic treatment was sent to them .", "case records were scrutinised to confirm diabetes diagnosis and time for start of insulin therapy .", "subjects that started their insulin treatment within two years of diagnosis were classified as type 1 .", "however , for subjects fulfilling these criteria but with a age of 40 or above at onset , current bmi had to be < 27 to be classified as type 1 .", "the research ethics committee of ume university and the national computer data inspection board have approved the northern sweden monica study .", "means or medians are given for those who remained non - diabetic and those who converted to diabetes . because the distributions for serum triglycerides , insulin and the fibrinolytic variables are highly skewed , we used logarithmically transformed values ( i.e. geometric means ) .", "the risk of incident diabetes was compared across quartiles of the fibrinolytic variables , calculated from the total sample .", "logistic regression was used for calculation of odds ratios and 95% confidence intervals ( ci ) for the development of diabetes comparing the quartile with highest risk with the other three pooled quartiles .", "stepwise adjustment for age and sex , waist , serum insulin , triglycerides and diastolic blood pressure was used .", "five hundred and fifty - one subjects who initially had normal glucose tolerance returned for re - examination . a new ogtt was done in 477 of those with normal glucose tolerance and without clinically diagnosed diabetes .", "thus , there was no repeated ogtt in 13 % , mostly due to logistic reasons such as not being able to turn up in the morning after an over night fast . among persons with initially normal glucose tolerance , in all 15 subjects ( 2.7 % ) had type 2 diabetes diagnosed , either clinically ( n = 4 ) or by ogtt ( n = 11 ) after a 9-year follow - up or 4,959 person years .", "fifty - one subjects ( 10,7 % ) worsened from normal glucose tolerance to impaired glucose tolerance .", "converters from ngt to diabetes were 7 years older and had higher bmi and waist circumference .", "diastolic blood pressure did not differ but both fasting triglycerides , insulin , fasting and post load plasma glucose were higher in subjects with subsequent diabetes .", "plasma levels of tpa antigen and pai-1 activity were higher and tpa activity was lower .", "baseline characteristics of subjects with normal glucose tolerance according to diabetes or not during 9 years of follow - up data are means ( sd ) , median ( interquartile range ) and [ geometric means ] .", "the risk of developing diabetes increased with increasing quartiles of tpa antigen and pai-1 activity and decreased in a similar way for tpa activity ( figure 1 ) . in the highest quartile of tpa antigen ( above 8.7", "g / l ) nearly 9 % had incident diabetes compared to none in the lowest quartile .", "incidence of type 2 diabetes over nine years in subjects with normal glucose tolerance according to baseline quartiles of fibrinolytic variables .", "the number of cases in each quartile for tpa activity was 7 , 4 , 3 and 1 .", "corresponding numbers for tpa antigen was 0 , 1 , 2 and 12 and for pai-1 activity 2 , 1 , 3 and 9 , respectively .", "p values for x(linear by linear association ) were 0.026 for tpa activity , < 0,001 for tpa antigen and 0,007 for pai-1 activity .", "logistic regression was performed with an initial model adjusting for age and sex . in a second model", ", waist circumference was added and in a third model , also diastolic blood pressure , serum insulin and triglycerides were added .", "quartiles 13 of tpa antigen and pai-1 activity were contrasted with the fourth quartile and , for tpa activity , the first quartiles with quartile 24 ( table 2 ) . logistic regression analysis for fibrinolytic variables at baseline with incident type 2 diabetes as the dependent variable .", "the lowest quartile of tpa activity was associated with a 3-fold increase in the risk of diabetes , which was attenuated after adjustment for waist circumference .", "after further adjustment for diastolic blood pressure , fasting insulin and triglycerides , no prediction from low tpa activity on diabetes development was seen .", "subjects in the highest quartile of tpa antigen experienced more than a ten - fold increase in risk , although the confidence intervals were wide due to few cases .", "taking waist circumference into account , the odds ratio diminished to 6.7 and diminished further to 5.5 when adjusting for blood pressure , triglycerides and insulin .", "this lead to somewhat higher age- and gender adjusted odds ratio ( 7.4 ; ci 1.830 , p value 0.005 ) . exchanging diastolic with systolic blood pressure did not change the findings .", "furter adjustment for leisure time physical activity reduced the odds ratio from 5.5 to 5.1 ( p value 0.03 ) .", "high pai-1 activity increased the risk of diabetes four - fold , although the result was attenuated to an insignificant or of 2.0 when all factors were adjusted for simultaneously .", "the risk of developing diabetes or igt during follow up ( n = 66 ) increased stepwise across quartiles of tpa antigen , from 7.8% to 19.8% ( p = 0,003 , test for linear trend ) . when adjusted for age and sex , the odds ratio for the highest vs. the three lower quartiles of tpa antigen was 1.9 ( ci 1.011.6 , p = 0.04 ) but diminished further when also waist circumference was adjusted for , or = 1.3 ( ci 0.636.5 , p = 0.5 ) .", "subjects with normal glucose tolerance , who subsequently develop type 2 diabetes over the ensuing nine years , are already at baseline characterised by impaired fibrinolysis and endothelial dysfunction .", "low tpa activity and high pai-1 activity is mainly explained by the presence of abdominal obesity but also by other metabolic disturbances characteristic of the insulin resistance syndrome , such as high serum levels of triglycerides and insulin .", "a novel finding , in this context , is that a high plasma level of the endothelial - derived tpa is only slightly explained by such factors .", "thus , the development of type 2 diabetes is preceded by many years of perturbations in fibrinolytic and endothelial function , which increases the risk of atherothrombotic disease long before overt diabetes is present . pa1 - 1 is partly synthesised in fat cells and its activity is strongly related to abdominal obesity as mirrored by high waist circumference or whr .", "high circulating levels of pai-1 inhibit tpa released from the vessel walls and lead to low levels of free tpa , i.e. low tpa activity and impaired capability of thrombolysis .", "the only report of an association between fibrinolysis and the development of diabetes is a recent study where pai-1 antigen was an independent predictor of diabetes in american - mexicans that were followed up to 5 years .", "there is no known pathway whereby impaired fibrinolysis would increase insulin resistance or decrease insulin release and our finding that high pai-1 and low tpa activity precedes diabetes probably mirrors the strong relationship with intraabdominal fat and circulating levels of free fatty acids .", "a recent experimental study show an almost three fold increase in the level of pai-1 antigen with an infusion of triacylglycerol in healthy subjects without any impact on tpa .", "this effect was noted with constant levels of insulin and glucose and also increased levels of soluble vascular cell adhesion molecules-1 was found .", "this support our findings that adjustment for baseline triglyceride levels , which were increased in converters , did not significantly change the relationships noted for tpa antigen but did decrease those for pai-1 .", "it is thus possible that also tpa concentration is an inflammatory marker although we found low correlations between tpa antigen and fibrinogen   and highly sensitive crp ( own unpublished data ) .", "recently , it was described in type 2 diabetes patients , that increased levels of tpa antigen , among other endothelial markers , predicted the development of urinary albumin excretion , a strong marker for cvd risk .", "these findings should be related to recent reports where inflammatory markers predict the development of diabetes in longitudinal studies . in most cases ,", "pro - inflammatory substances derived from abdominal fat tissue may be the common background for these findings .", "we therefore find it plausible that the relation between high tpa and diabetes is a marker of some early disturbance in the vascular wall that operates independent of obesity and inflammation .", "already borderline hypertension is associated with high concentrations of tpa antigen , even after adjustment for abdominal obesity , insulin and triglycerides .", "thus , tpa mass may mirror an endothelial dysfunction that is not causally related to the insulin resistance syndrome but acts by a parallel pathogenetic mechanism leading to atherothrombosis [ 30 - 32 ] .", "insulin resistance , on the other hand , is associated with endothelial dysfunction and impaired vasodilatation due to defects in endothelium - derived nitric oxide . taken together , these findings put focus on the role of the vessel walls in the pathogenesis of type 2 diabetes .", "the relevance of impaired fibrinolysis , i.e. low activity of tissue plasminogen activator ( tpa ) or high activity of its inhibitor ( pai-1 ) , as independent risk factors for cvd is unclear , as these variables seem to mirror most components of the insulin resistance syndrome . in some studies , decreased fibrinolysis , as measured by high pai-1 activity , has been associated with cardiovascular events .", "high levels of tpa antigen independently predict cardiovascular events both in a healthy population   and in patients with prevalent coronary disease .", "the major shortcoming of our study is the small number of incident cases of diabetes leading to wide confidence intervals . on the other hand , the odds ratios for tpa antigen are high and consistent and based on a truly representative population sample where basal normal glucose tolerance was well defined , as were the incident cases of diabetes .", "thus , the risk of bias due to selection or case ascertainment should be low .", "admittedly , the lack of a renewed ogtt in 13 % of the subjects could contribute to some misclassifiaction and dilute the results somewhat .", "the early prediabetic state , with normal glucose tolerance , is characterised by both impaired fibrinolysis and endothelial dysfunction years before glucose levels increase .", "changes in endothelial function are independent from the insulin resistance syndrome and point towards a distinctly different pathway both for cardiovascular disease and for the development of diabetes . to answer a classical question from 1990    yes", ", the clock for coronary heart disease seems to start ticking before the onset of clinical diabetes !", "bmi = body mass index ci = confidence interval crp = c - reactive protein cvd = cardiovascular disease igt = impaired glucose tolerance monica = monitoring of trends and determinants of cardiovascular disease ngt = normal glucose tolerance ogtt = oral glucose tolerance test pai-1 = plasminogen activator inhibitor-1 tpa = tissue plasminogen activator", "me drafted the research on fibrinolysis within the monica project , performed the statistical analysis and drafted the manuscript .", "bs and bl participated in the design of northern sweden monica study and participated in its design and coordination .", "supported by grants from norrbotten and vsterbotten counties , by the joint committee of northern sweden health care region , the swedish public health institute and the swedish medical research council ."], "abstract_text": ["<S> backgroundimpaired fibrinolysis is found in impaired glucose tolerance and type 2 diabetes , associated with components of the metabolic syndrome . </S>", "<S> there are no data concerning fibrinolysis in subjects with normal glucose tolerance that convert to diabetes.methodswe studied the activities of tissue plasminogen activator ( tpa ) and plasminogen activator inhibitor-1 ( pai-1 ) and the levels of tpa antigen ( a marker of endothelial dysfunction ) in 551 subjects with normal glucose tolerance in 1990 in relation to incident diabetes during nine years of follow-up.resultssubjects with diabetes at follow - up ( n = 15 ) had significantly lower baseline tpa activity and higher pai-1 activity and tpa antigen than non - converters . </S>", "<S> the risk of diabetes increased linearly across quartiles of pai - activity ( p = 0.007 ) and tpa antigen ( p < 0.001 ) and decreased across quartiles of tpa activity ( p = 0.026 ) . </S>", "<S> the risk of diabetes with low tpa activity or high pai-1 activity persisted after adjustment for age and sex but diminished to a non - significant level after further adjustments . </S>", "<S> the odds ratio of diabetes for high tpa antigen was 10.4 ( 95% confidence interval 2.740 ) adjusted for age and sex . </S>", "<S> after further adjustment for diastolic blood pressure , waist circumference , insulin , triglycerides , fasting and post load glucose the odds ratio was 6.5 ( 1.333 , p = 0.024).conclusionsimpaired fibrinolysis and endothelial dysfunction are evident in subjects with normal glucose tolerance who later develop diabetes . </S>", "<S> high tpa antigen is predictive of future diabetes independent from the metabolic syndrome . </S>"], "labels": null, "section_names": ["Background", "Methods", "Statistics", "Results", "Discussion", "Conclusions", "Abbreviations", "Competing interests", "Authors' contributions", "Acknowledgements"], "sections": [["the risk of stroke and myocardial infarction is considerably increased in subjects with diabetes . already at the time of diagnosis of type 2 diabetes , many patients have manifest cardiovascular disease ( cvd ) .", "this could be due to a long presymtomatic period with increased glucose level as evident by the increased cvd risk already present with impaired glucose tolerance ( igt ) .", "thus , interest has focused on factors , not only glucose levels , in the prediabetic state that would increase the atherothrombotic process . a common , possibly genetic , antecedent of both type 2 diabetes and cvd has been proposed as the \" common soil \" hypotheses .", "recently , markers of inflammation , such as highly sensitive c - reactive protein , have been found to predict type 2 diabetes in long - time follow up of population samples , although adjusting for measurements of obesity attenuates the relationship [ 8 - 12 ] .", "such markers are weakly related to variables reflecting endothelial function such as adhesion molecules and von willebrant factor . in a large population - based study , also increased plasma levels of plasminogen activator inhibitor-1 ( pai-1 ) were strongly related to the development of diabetes independent from insulin resistance and obesity .", "the possibility of elevated pai-1 being a very early risk marker of the insulin resistance syndrome and diabetes was raised . furthermore , haemostatic variables related to endothelial function , such as von willebrant factor and factor viii , also predicted diabetes , especially in women . data are lacking regarding other aspects of the fibrinolytic system and the development of diabetes , such as the activities of pai-1 and tissue plasminogen activator ( tpa ) or the mass concentration of the endothelial - derived tpa ( \" tpa antigen \" ) , factors related to cvd [ 15 - 19 ] thus , there are two questions related to fibrinolytic variables and the development of diabetes .", "firstly , do subjects with normal glucose tolerance , who later convert to diabetes , have disturbances in the fibrinolytic system and endothelial function that could explain their increased risk of cvd even before diabetes ensues ?", "if so , do these changes predict future diabetes , independent of the influence of the insulin resistance syndrome , thereby pointing to other pathways to the diabetic state such as endothelial dysfunction ?", "we studied fibrinolytic activity , as measured by tpa and pai-1 activity , and a marker of endothelial dysfunction , as measured by tpa antigen , in a population sample with normal glucose tolerance at baseline and analysed data according to conversion to diabetes or not over nine years of follow - up ."], ["this study was performed within the framework of the northern sweden monica study . in 1990", "a total of 2,000 randomly selected subjects aged 25 to 64 years were invited . in all , 1,583 persons participated ( 79.2 % ) .", "a 75-gram oral glucose tolerance test ( ogtt ) was performed after an overnight fast in a randomly selected subset of subjects without known diabetes .", "venous plasma glucose samples were analysed by the hexokinase method ( boehringer mannheim automated analysis for bm / hitachi system 717 ) .", "glucose tolerance was classified according to who criteria from 1999 : normal glucose tolerance ( ngt )  fasting glucose < 7 mmol / l and post load glucose < 7.8 mmol / l , impaired glucose tolerance ( igt )  fasting glucose < 7 mmol / l and post load glucose 7.811.0 mmol / l , and diabetes  fasting glucose 7.0", "weight was measured with a balance scale to the nearest 0.2 kg , and height was measured to the nearest centimetre .", "bmi was calculated as total body weight ( kg ) / height ( m ) .", "waist circumference was measured midway between the lower rib margin and the iliac crest , which in most occasions was identical with the level of the umbilicus .", "all measurements were done in a standing position while breathing normally , and participants were asked to wear light underwear and remove shoes . for the measurement of waist circumference , the recording was done after a gentle breath - out .", "tpa and pai-1 activities were determined by chromogenic assays , spectrolyse / fibrin and spectrolyse / pl kit ( biopool ab , ume , sweden ) , respectively .", "tpa antigen was measured by an enzyme - linked immunosorbent assay method ( tintelize tpa , biopool ab ) .", "serum insulin was determined by radio immunoassay with a double - antibody solid - phase technique ( phadaseph insulin ria , pharmacia diagnostics ab , uppsala , sweden ) in 1999 , all participants , alive and still living in the area , were recalled for a follow - up examination . the same questionnaire and anthropometrical measurements as in the baseline survey were used .", "altogether 1,148 ( 72.5 % ) subjects turned up for re - examination . subjects who previously had performed an ogtt with normal or impaired glucose tolerance , and answered no to the question \"", "participants , who denied having diabetes in the baseline survey and answered affirmative to the question \" do you have diabetes mellitus ? \" at follow up , were defined as incident cases of known diabetes mellitus according tho the who criteria from 1999 . in november 2002 , a questionnaire exploring details related to the diagnosis of diabetes , and to the type and duration of current hypoglycaemic treatment was sent to them .", "case records were scrutinised to confirm diabetes diagnosis and time for start of insulin therapy .", "subjects that started their insulin treatment within two years of diagnosis were classified as type 1 .", "however , for subjects fulfilling these criteria but with a age of 40 or above at onset , current bmi had to be < 27 to be classified as type 1 .", "the research ethics committee of ume university and the national computer data inspection board have approved the northern sweden monica study ."], ["means or medians are given for those who remained non - diabetic and those who converted to diabetes . because the distributions for serum triglycerides , insulin and the fibrinolytic variables are highly skewed , we used logarithmically transformed values ( i.e. geometric means ) .", "the risk of incident diabetes was compared across quartiles of the fibrinolytic variables , calculated from the total sample .", "logistic regression was used for calculation of odds ratios and 95% confidence intervals ( ci ) for the development of diabetes comparing the quartile with highest risk with the other three pooled quartiles .", "stepwise adjustment for age and sex , waist , serum insulin , triglycerides and diastolic blood pressure was used ."], ["five hundred and fifty - one subjects who initially had normal glucose tolerance returned for re - examination . a new ogtt was done in 477 of those with normal glucose tolerance and without clinically diagnosed diabetes .", "thus , there was no repeated ogtt in 13 % , mostly due to logistic reasons such as not being able to turn up in the morning after an over night fast . among persons with initially normal glucose tolerance , in all 15 subjects ( 2.7 % ) had type 2 diabetes diagnosed , either clinically ( n = 4 ) or by ogtt ( n = 11 ) after a 9-year follow - up or 4,959 person years .", "fifty - one subjects ( 10,7 % ) worsened from normal glucose tolerance to impaired glucose tolerance .", "converters from ngt to diabetes were 7 years older and had higher bmi and waist circumference .", "diastolic blood pressure did not differ but both fasting triglycerides , insulin , fasting and post load plasma glucose were higher in subjects with subsequent diabetes .", "plasma levels of tpa antigen and pai-1 activity were higher and tpa activity was lower .", "baseline characteristics of subjects with normal glucose tolerance according to diabetes or not during 9 years of follow - up data are means ( sd ) , median ( interquartile range ) and [ geometric means ] .", "the risk of developing diabetes increased with increasing quartiles of tpa antigen and pai-1 activity and decreased in a similar way for tpa activity ( figure 1 ) . in the highest quartile of tpa antigen ( above 8.7", "g / l ) nearly 9 % had incident diabetes compared to none in the lowest quartile .", "incidence of type 2 diabetes over nine years in subjects with normal glucose tolerance according to baseline quartiles of fibrinolytic variables .", "the number of cases in each quartile for tpa activity was 7 , 4 , 3 and 1 .", "corresponding numbers for tpa antigen was 0 , 1 , 2 and 12 and for pai-1 activity 2 , 1 , 3 and 9 , respectively .", "p values for x(linear by linear association ) were 0.026 for tpa activity , < 0,001 for tpa antigen and 0,007 for pai-1 activity .", "logistic regression was performed with an initial model adjusting for age and sex . in a second model", ", waist circumference was added and in a third model , also diastolic blood pressure , serum insulin and triglycerides were added .", "quartiles 13 of tpa antigen and pai-1 activity were contrasted with the fourth quartile and , for tpa activity , the first quartiles with quartile 24 ( table 2 ) . logistic regression analysis for fibrinolytic variables at baseline with incident type 2 diabetes as the dependent variable .", "the lowest quartile of tpa activity was associated with a 3-fold increase in the risk of diabetes , which was attenuated after adjustment for waist circumference .", "after further adjustment for diastolic blood pressure , fasting insulin and triglycerides , no prediction from low tpa activity on diabetes development was seen .", "subjects in the highest quartile of tpa antigen experienced more than a ten - fold increase in risk , although the confidence intervals were wide due to few cases .", "taking waist circumference into account , the odds ratio diminished to 6.7 and diminished further to 5.5 when adjusting for blood pressure , triglycerides and insulin .", "this lead to somewhat higher age- and gender adjusted odds ratio ( 7.4 ; ci 1.830 , p value 0.005 ) . exchanging diastolic with systolic blood pressure did not change the findings .", "furter adjustment for leisure time physical activity reduced the odds ratio from 5.5 to 5.1 ( p value 0.03 ) .", "high pai-1 activity increased the risk of diabetes four - fold , although the result was attenuated to an insignificant or of 2.0 when all factors were adjusted for simultaneously .", "the risk of developing diabetes or igt during follow up ( n = 66 ) increased stepwise across quartiles of tpa antigen , from 7.8% to 19.8% ( p = 0,003 , test for linear trend ) . when adjusted for age and sex , the odds ratio for the highest vs. the three lower quartiles of tpa antigen was 1.9 ( ci 1.011.6 , p = 0.04 ) but diminished further when also waist circumference was adjusted for , or = 1.3 ( ci 0.636.5 , p = 0.5 ) ."], ["subjects with normal glucose tolerance , who subsequently develop type 2 diabetes over the ensuing nine years , are already at baseline characterised by impaired fibrinolysis and endothelial dysfunction .", "low tpa activity and high pai-1 activity is mainly explained by the presence of abdominal obesity but also by other metabolic disturbances characteristic of the insulin resistance syndrome , such as high serum levels of triglycerides and insulin .", "a novel finding , in this context , is that a high plasma level of the endothelial - derived tpa is only slightly explained by such factors .", "thus , the development of type 2 diabetes is preceded by many years of perturbations in fibrinolytic and endothelial function , which increases the risk of atherothrombotic disease long before overt diabetes is present . pa1 - 1 is partly synthesised in fat cells and its activity is strongly related to abdominal obesity as mirrored by high waist circumference or whr .", "high circulating levels of pai-1 inhibit tpa released from the vessel walls and lead to low levels of free tpa , i.e. low tpa activity and impaired capability of thrombolysis .", "the only report of an association between fibrinolysis and the development of diabetes is a recent study where pai-1 antigen was an independent predictor of diabetes in american - mexicans that were followed up to 5 years .", "there is no known pathway whereby impaired fibrinolysis would increase insulin resistance or decrease insulin release and our finding that high pai-1 and low tpa activity precedes diabetes probably mirrors the strong relationship with intraabdominal fat and circulating levels of free fatty acids .", "a recent experimental study show an almost three fold increase in the level of pai-1 antigen with an infusion of triacylglycerol in healthy subjects without any impact on tpa .", "this effect was noted with constant levels of insulin and glucose and also increased levels of soluble vascular cell adhesion molecules-1 was found .", "this support our findings that adjustment for baseline triglyceride levels , which were increased in converters , did not significantly change the relationships noted for tpa antigen but did decrease those for pai-1 .", "it is thus possible that also tpa concentration is an inflammatory marker although we found low correlations between tpa antigen and fibrinogen   and highly sensitive crp ( own unpublished data ) .", "recently , it was described in type 2 diabetes patients , that increased levels of tpa antigen , among other endothelial markers , predicted the development of urinary albumin excretion , a strong marker for cvd risk .", "these findings should be related to recent reports where inflammatory markers predict the development of diabetes in longitudinal studies . in most cases ,", "pro - inflammatory substances derived from abdominal fat tissue may be the common background for these findings .", "we therefore find it plausible that the relation between high tpa and diabetes is a marker of some early disturbance in the vascular wall that operates independent of obesity and inflammation .", "already borderline hypertension is associated with high concentrations of tpa antigen , even after adjustment for abdominal obesity , insulin and triglycerides .", "thus , tpa mass may mirror an endothelial dysfunction that is not causally related to the insulin resistance syndrome but acts by a parallel pathogenetic mechanism leading to atherothrombosis [ 30 - 32 ] .", "insulin resistance , on the other hand , is associated with endothelial dysfunction and impaired vasodilatation due to defects in endothelium - derived nitric oxide . taken together , these findings put focus on the role of the vessel walls in the pathogenesis of type 2 diabetes .", "the relevance of impaired fibrinolysis , i.e. low activity of tissue plasminogen activator ( tpa ) or high activity of its inhibitor ( pai-1 ) , as independent risk factors for cvd is unclear , as these variables seem to mirror most components of the insulin resistance syndrome . in some studies , decreased fibrinolysis , as measured by high pai-1 activity , has been associated with cardiovascular events .", "high levels of tpa antigen independently predict cardiovascular events both in a healthy population   and in patients with prevalent coronary disease .", "the major shortcoming of our study is the small number of incident cases of diabetes leading to wide confidence intervals . on the other hand , the odds ratios for tpa antigen are high and consistent and based on a truly representative population sample where basal normal glucose tolerance was well defined , as were the incident cases of diabetes .", "thus , the risk of bias due to selection or case ascertainment should be low .", "admittedly , the lack of a renewed ogtt in 13 % of the subjects could contribute to some misclassifiaction and dilute the results somewhat ."], ["the early prediabetic state , with normal glucose tolerance , is characterised by both impaired fibrinolysis and endothelial dysfunction years before glucose levels increase .", "changes in endothelial function are independent from the insulin resistance syndrome and point towards a distinctly different pathway both for cardiovascular disease and for the development of diabetes . to answer a classical question from 1990    yes", ", the clock for coronary heart disease seems to start ticking before the onset of clinical diabetes !"], ["bmi = body mass index ci = confidence interval crp = c - reactive protein cvd = cardiovascular disease igt = impaired glucose tolerance monica = monitoring of trends and determinants of cardiovascular disease ngt = normal glucose tolerance ogtt = oral glucose tolerance test pai-1 = plasminogen activator inhibitor-1 tpa = tissue plasminogen activator"], [""], ["me drafted the research on fibrinolysis within the monica project , performed the statistical analysis and drafted the manuscript .", "bs and bl participated in the design of northern sweden monica study and participated in its design and coordination ."], ["supported by grants from norrbotten and vsterbotten counties , by the joint committee of northern sweden health care region , the swedish public health institute and the swedish medical research council ."]]}
{"article_id": "PMC4296960", "article_text": ["selective serotonin reuptake inhibitors ( ssris ) , serotonin and norepinephrine reuptake inhibitors , and noradrenergic and specific serotonergic antidepressants are categorized as first - line treatments in many countries , including japan.1,2 thus , health care providers have the opportunity to choose an antidepressant from a variety of first - line treatment options . to select the most appropriate treatment for each patient , it is important for clinicians to be aware of the differences in efficacy between comparable antidepressants at the doses approved in their countries .", "one long - standing approach to comparing efficacy has been meta - analysis of several randomized controlled trials that compare widely used treatments.310 however , results have been inconsistent , perhaps partly due to the use of different methodologies .", "recent meta - analyses have used mixed treatment comparisons in which both direct and indirect comparisons were used.11,12 duloxetine , a second - generation norepinephrine reuptake inhibitor that has demonstrated efficacy , safety , and tolerability in patients with mdd,1316 has been the subject of several recent mixed treatment comparisons . a recent analysis by cipriani et al12 compared the effects of 12 new - generation antidepressants in adults with mdd , and gartlehner et al11 conducted an analysis of 234 studies that included placebo as a comparator . however , these results are not applicable to clinical practice in japan , because they included data from patients taking duloxetine up to 120 mg / day , which is twice the maximum dosage ( 60 mg / day ) approved in japan .", "another drawback was the inclusion of patients who took antidepressants not approved or not even available for use in japan .", "therefore , it is uncertain whether these results can be applied to daily clinical practice in countries ( eg , japan ) where the approved dosage for duloxetine is 4060 mg / day and ssris are limited to four compounds , ie , paroxetine , sertraline , escitalopram , and fluvoxamine . to the best of our knowledge , there are no meta - analyses that compare the efficacy of duloxetine 60 mg / day with these ssris .", "the primary purpose of the current study was to provide clinicians with efficacy results that would allow a comparison of duloxetine 60 mg / day with four approved ssris in japan .", "the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .", "we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .", "for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,", "a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,", "patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .", "common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery  sberg depression rating scale score 22 .", "the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .", "the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .", "the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .", "the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .", "the study included 8 weeks of active treatment followed by a one - week dose - tapering period .", "the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .", "this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram .", "the primary endpoint was mean change from baseline in hamd17 total score at week 8 .", "secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .", "response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .", "our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .", "this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .", "continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .", "effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .", "overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .", "the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .", "relative risk is presented with 95% confidence intervals ( ci ) and p - values .", "the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .", "absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .", "safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .", "reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .", "safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test .", "the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .", "we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .", "for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,", "a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,", "patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .", "common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery  sberg depression rating scale score 22 .", "the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .", "the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .", "the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .", "the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .", "the study included 8 weeks of active treatment followed by a one - week dose - tapering period .", "the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .", "this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram .", "the primary endpoint was mean change from baseline in hamd17 total score at week 8 .", "secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .", "response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .", "our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .", "this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .", "continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .", "effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .", "overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .", "the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .", "relative risk is presented with 95% confidence intervals ( ci ) and p - values .", "the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .", "absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .", "safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .", "reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .", "safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test .", "at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .", "the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .", "completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .", "adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .", "discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) .", "a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population", ", no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .", "duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .", "for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .", "these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) .", "the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .", "no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .", "statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) .", "the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .", "no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) .", "the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients ,", "nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth .", "at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .", "the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .", "completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .", "adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .", "discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) .", "a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population", ", no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .", "duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .", "for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .", "these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) .", "the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .", "no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .", "statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) .", "the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .", "no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) .", "the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients , nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth .", "our results show that duloxetine and ssri treatment within their respective approved dosage in japan demonstrated comparable efficacy with regard to overall depressive symptoms", ". however , certain hamd17 subscale scores responded differently to duloxetine compared with ssris , and this may clarify the difference in clinical attributes among these antidepressants .", "first , when assessing overall efficacy using the mean changes in hamd17 total scores from baseline , both duloxetine and ssri demonstrated greater efficacy than placebo , but did not indicate any significant difference between each other for either the total population ( hamd17 total score 15 ) or the more severe population ( hamd17 total score 19 ) . on the one hand , this is consistent with a prior report by thase et al,4 where all randomized patients across any severity were analyzed and no significant difference in efficacy between duloxetine and ssri was found .", "however , on the other hand , this is inconsistent with prior reports that duloxetine 120 mg / day showed greater efficacy than ssris in patients with at least moderately severe depression.4,16 given this and the finding of a modest dose response trend for duloxetine efficacy by mallinckrodt et al16 this inconsistency may be explained by the upper limit on the dosage of duloxetine ( 60 mg / day ) in this study .", "second , when assessing energy and interest using retardation subscale scores , duloxetine showed greater efficacy regardless of baseline depression severity ( hamd17 total score 15 or 19 ) .", "this is consistent with previous reports by mallinckrodt et al16 and martinez et al.24 furthermore , katz et al25 reported that desipramine ( a norepinephrine reuptake inhibitor ) demonstrated better efficacy than paroxetine ( an ssri ) on psychomotor retardation symptoms .", "these results may be explained by the effect of duloxetine on norepinephrine , which has been shown to be associated with arousal and activity.26 the current results show some inconsistency with a report by cipriani et al in which some ssris were significantly more efficacious than duloxetine .", "this is partly due to the different methodologies used in that study and in our current study . in cipriani", "et al,12 approximately one third of the comparisons were direct comparisons from randomized controlled trials and many of the pairwise comparisons were based on only one study .", "degree of heterogeneity and statistical incoherence ( estimates based on the direct comparisons are not contained in the 95% ci for indirect comparisons ) in the study network were detected .", "study conclusions based on direct comparisons were much more conservative than those based on indirect ones . on the other hand ,", "the strengths of this database analysis are that it analyzed individual patient data , used the same analysis method and endpoint across studies , and included similar patients .", "the adopted database analysis design allowed for a homogeneous patient population , with almost the same inclusion criteria and the exact same endpoint . although our analysis resulted in a comparatively smaller number of studies and patients , it achieved high internal validity and reproducibility .", "in addition , direct comparisons of head - to - head data enabled us to avoid the uncertainty of how much the two different evidence levels are weighted when pooling direct and indirect comparisons .", "the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated .", "using this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .", "similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .", "another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .", "furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .", "further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .", "in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .", "lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed .", "the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated . using", "this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .", "similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .", "another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .", "furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .", "further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .", "in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .", "lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed .", "comparison between duloxetine and ssri demonstrated comparable efficacy within the japan - approved dose range in patients with mdd .", "results of the hamd17 subscale analysis indicated that duloxetine might be superior to ssris in improving loss of energy and interest ."], "abstract_text": ["<S> backgroundapproved doses of antidepressants in japan are usually lower than those in the usa and european union , but to date meta - analyses comparing antidepressants have all used the higher doses approved in the usa and european union and often have used indirect comparisons . </S>", "<S> the purpose of this study was to conduct an integrated database analysis of patient level data to compare the effects of duloxetine with those of selective serotonin reuptake inhibitors ( ssris ) at the doses approved in japan.methodspooled data were analyzed from four randomized , double - blind , placebo - controlled studies that compared duloxetine at the dose range approved in japan ( 4060 mg / day ) with other ssris ( paroxetine 20 mg / day or escitalopram 10 mg / day ) and placebo in patients with major depressive disorder . in total , 1,694 patients were included in the analysis ( duloxetine , n=688 ; selective serotonin reuptake inhibitors , n=690 ; placebo , n=316 ) . </S>", "<S> the primary outcome measure was the mean change from baseline at week 8 in 17-item hamilton rating scale for depression ( hamd17 ) total and subscale scores.resultsduloxetine and both selective serotonin reuptake inhibitors were superior to placebo in hamd17 total score at week 8 in both the all - randomized group and the more severe subgroup ( hamd17 total scores 19 ) . </S>", "<S> duloxetine was superior to ssris in improving the hamd17 retardation subscale score ( least squares mean difference [ 95% confidence interval ] ) : all - randomized group , 0.33 [ 0.60 , 0.07 ] , p=0.015 ; severe subgroup , 0.45 [ 0.83 , 0.07 ] , p=0.020).conclusionwithin the dose range approved in japan for patients with major depressive disorder , duloxetine and selective serotonin reuptake inhibitors demonstrated comparable overall efficacy , with a possible advantage for duloxetine in improving loss of energy and interest . to the best of our knowledge , this analysis is unique not only in evaluating dosages specific to japan , but also in using individual patient data and the same endpoint across studies to allow for strictly direct head - to - head data comparisons as opposed to pooling direct and indirect comparisons . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Study selection and data collection", "Statistical analysis", "Results", "Patient demographics and disposition", "Efficacy comparison using HAMD", "Response", "Remission", "Safety", "Discussion", "Limitations", "Conclusion"], "sections": [["selective serotonin reuptake inhibitors ( ssris ) , serotonin and norepinephrine reuptake inhibitors , and noradrenergic and specific serotonergic antidepressants are categorized as first - line treatments in many countries , including japan.1,2 thus , health care providers have the opportunity to choose an antidepressant from a variety of first - line treatment options . to select the most appropriate treatment for each patient , it is important for clinicians to be aware of the differences in efficacy between comparable antidepressants at the doses approved in their countries .", "one long - standing approach to comparing efficacy has been meta - analysis of several randomized controlled trials that compare widely used treatments.310 however , results have been inconsistent , perhaps partly due to the use of different methodologies .", "recent meta - analyses have used mixed treatment comparisons in which both direct and indirect comparisons were used.11,12 duloxetine , a second - generation norepinephrine reuptake inhibitor that has demonstrated efficacy , safety , and tolerability in patients with mdd,1316 has been the subject of several recent mixed treatment comparisons . a recent analysis by cipriani et al12 compared the effects of 12 new - generation antidepressants in adults with mdd , and gartlehner et al11 conducted an analysis of 234 studies that included placebo as a comparator . however , these results are not applicable to clinical practice in japan , because they included data from patients taking duloxetine up to 120 mg / day , which is twice the maximum dosage ( 60 mg / day ) approved in japan .", "another drawback was the inclusion of patients who took antidepressants not approved or not even available for use in japan .", "therefore , it is uncertain whether these results can be applied to daily clinical practice in countries ( eg , japan ) where the approved dosage for duloxetine is 4060 mg / day and ssris are limited to four compounds , ie , paroxetine , sertraline , escitalopram , and fluvoxamine . to the best of our knowledge , there are no meta - analyses that compare the efficacy of duloxetine 60 mg / day with these ssris .", "the primary purpose of the current study was to provide clinicians with efficacy results that would allow a comparison of duloxetine 60 mg / day with four approved ssris in japan ."], ["the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .", "we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .", "for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,", "a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,", "patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .", "common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery  sberg depression rating scale score 22 .", "the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .", "the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .", "the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .", "the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .", "the study included 8 weeks of active treatment followed by a one - week dose - tapering period .", "the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .", "this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram .", "the primary endpoint was mean change from baseline in hamd17 total score at week 8 .", "secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .", "response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .", "our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .", "this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .", "continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .", "effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .", "overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .", "the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .", "relative risk is presented with 95% confidence intervals ( ci ) and p - values .", "the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .", "absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .", "safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .", "reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .", "safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test ."], ["the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .", "we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .", "for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,", "a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,", "patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .", "common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery  sberg depression rating scale score 22 .", "the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .", "the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .", "the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .", "the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .", "the study included 8 weeks of active treatment followed by a one - week dose - tapering period .", "the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .", "this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram ."], ["the primary endpoint was mean change from baseline in hamd17 total score at week 8 .", "secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .", "response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .", "our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .", "this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .", "continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .", "effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .", "overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .", "the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .", "relative risk is presented with 95% confidence intervals ( ci ) and p - values .", "the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .", "absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .", "safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .", "reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .", "safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test ."], ["at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .", "the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .", "completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .", "adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .", "discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) .", "a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population", ", no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .", "duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .", "for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .", "these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) .", "the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .", "no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .", "statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) .", "the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .", "no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) .", "the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients ,", "nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth ."], ["at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .", "the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .", "completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .", "adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .", "discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) ."], ["a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population", ", no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .", "duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .", "for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .", "these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) ."], ["the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .", "no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .", "statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) ."], ["the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .", "no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) ."], ["the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients , nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth ."], ["our results show that duloxetine and ssri treatment within their respective approved dosage in japan demonstrated comparable efficacy with regard to overall depressive symptoms", ". however , certain hamd17 subscale scores responded differently to duloxetine compared with ssris , and this may clarify the difference in clinical attributes among these antidepressants .", "first , when assessing overall efficacy using the mean changes in hamd17 total scores from baseline , both duloxetine and ssri demonstrated greater efficacy than placebo , but did not indicate any significant difference between each other for either the total population ( hamd17 total score 15 ) or the more severe population ( hamd17 total score 19 ) . on the one hand , this is consistent with a prior report by thase et al,4 where all randomized patients across any severity were analyzed and no significant difference in efficacy between duloxetine and ssri was found .", "however , on the other hand , this is inconsistent with prior reports that duloxetine 120 mg / day showed greater efficacy than ssris in patients with at least moderately severe depression.4,16 given this and the finding of a modest dose response trend for duloxetine efficacy by mallinckrodt et al16 this inconsistency may be explained by the upper limit on the dosage of duloxetine ( 60 mg / day ) in this study .", "second , when assessing energy and interest using retardation subscale scores , duloxetine showed greater efficacy regardless of baseline depression severity ( hamd17 total score 15 or 19 ) .", "this is consistent with previous reports by mallinckrodt et al16 and martinez et al.24 furthermore , katz et al25 reported that desipramine ( a norepinephrine reuptake inhibitor ) demonstrated better efficacy than paroxetine ( an ssri ) on psychomotor retardation symptoms .", "these results may be explained by the effect of duloxetine on norepinephrine , which has been shown to be associated with arousal and activity.26 the current results show some inconsistency with a report by cipriani et al in which some ssris were significantly more efficacious than duloxetine .", "this is partly due to the different methodologies used in that study and in our current study . in cipriani", "et al,12 approximately one third of the comparisons were direct comparisons from randomized controlled trials and many of the pairwise comparisons were based on only one study .", "degree of heterogeneity and statistical incoherence ( estimates based on the direct comparisons are not contained in the 95% ci for indirect comparisons ) in the study network were detected .", "study conclusions based on direct comparisons were much more conservative than those based on indirect ones . on the other hand ,", "the strengths of this database analysis are that it analyzed individual patient data , used the same analysis method and endpoint across studies , and included similar patients .", "the adopted database analysis design allowed for a homogeneous patient population , with almost the same inclusion criteria and the exact same endpoint . although our analysis resulted in a comparatively smaller number of studies and patients , it achieved high internal validity and reproducibility .", "in addition , direct comparisons of head - to - head data enabled us to avoid the uncertainty of how much the two different evidence levels are weighted when pooling direct and indirect comparisons .", "the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated .", "using this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .", "similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .", "another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .", "furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .", "further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .", "in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .", "lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed ."], ["the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated . using", "this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .", "similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .", "another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .", "furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .", "further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .", "in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .", "lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed ."], ["comparison between duloxetine and ssri demonstrated comparable efficacy within the japan - approved dose range in patients with mdd .", "results of the hamd17 subscale analysis indicated that duloxetine might be superior to ssris in improving loss of energy and interest ."]]}
{"article_id": "PMC3525911", "article_text": ["we all have a very different understanding of how we define a  need for help  .", "this is most clearly felt when encountering individuals who , despite outside temperatures below zero , prefer sleeping rough in the street over the warm place in an overnight homeless shelter that a social worker or other care worker has offered them .", "if they additionally show no response to medical diagnoses such as open ulcers , flu infections or severe breathing difficulties , we can safely say that this person s behaviour deviates fundamentally from the expectations and understanding that health professionals would normally associate with  need for action  .", "being exposed to people who reject such support services , which seem well - motivated and necessary from a medical point of view , is only one of several fruitful challenges that are deliberately provoked as part of the new teaching concept in the institute for the history of medicine at giessen university .", "the objectives are as follows :   to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ; to get students to reflect upon central concepts such as  bedarf / bedrfnis  ( needs in  objective  vs.  subjective  terms ) that are normative in character and have a structuring and guiding effect on medical care services ; to advance and refine students  soft skills  , including the ability to put themselves in the position of other people , in particular those using medical services ; to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals . to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ;   to get students to reflect upon central concepts such as  bedarf / bedrfnis  ( needs in  objective  vs.  subjective  terms ) that are normative in character and have a structuring and guiding effect on medical care services ;   to advance and refine students  soft skills  , including the ability to put themselves in the position of other people , in particular those using medical services ;   to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and   to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals .", "these teaching objectives are implemented and pursued , among others , in the interdisciplinary field querschnittsbereich 2 / q 2  history , ethics and theory of medicine  ,  ", "that is part of the approbationsordnung fr rzte/(ao : regulations for licensing doctors ) ( ao , 2002 ) . as a matter of fact , some of the objectives", "are also presented and discussed in some medical school - curricula in medical sociology and psychology , as well as in community medicine / public health , however , in these contexts , the primary emphasis here is on the methodological and conceptual repertoire from their related disciplines , sociology and psychology . -", "similar teaching concepts as those presented in q 2 can also be found at an international level , where they tend to be associated with the concept of  medical humanities  ( \" kulturwissenschaften in der medizin  ) , .", "medical faculties vary in their interpretation and weighting of these teaching concepts ; however , in the context of research and debate in medical education , the main focus is on the relationship between the three approaches ( history , ethics , theory : additive or integrative ; weighting of individual components ) , which are specified explicitly in the ao and which intend to introduce a reflective perspective on current medicine , .", "this paper sets out to discuss the possibility of supplementing the above - described methods with methodological approaches and concepts from another discipline based in cultural studies , namely medical anthropology ( or medical ethnology ) , and to apply these in the context of a specifically designed form of experience to which students are exposed . outside of germany", ", medical anthropology is an established discipline in university departments , degree courses and associations .", "the seminar  medical care on the fringes of society : participant observation and change in perspectives  has been designed with these approaches and experiences in mind .", "it has been offered as a seminar option in q 2 at the medical school of giessen university since the 2010 - 11 winter semester .", "the new teaching concept was developed on the basis of the following findings and considerations : in the standard degree courses required by the regulations for licensing doctors medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result ,", "the focus is on  problems  as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .", "the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness (  patients  from a doctor s point of view ) will often be new to this environment .", "this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .", "the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting.as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .", "however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .", "the priorities for action that result from the individual  illness  of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years", ", the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances  according to their own subjective criteria , .", "however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training .", "the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the  risks  of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .", "the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair ", "bedarf/bedrfnis  , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term  bedarf  presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term ", "bedrfnis  is far more closely linked to the subjective perception of an individual or a particular predefined group.empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and  problems  as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary", ", these  problems  tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .", "if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .", "moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .", "such behaviour is frequently perceived as  exotic  and therefore attributed to the  culture  or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation", "may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .", "another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .", "stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups . in the standard degree courses required by the regulations for licensing doctors", "medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result , the focus is on  problems  as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .", "the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness (  patients  from a doctor s point of view ) will often be new to this environment .", "this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .", "the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting . as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .", "however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .", "the priorities for action that result from the individual  illness  of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years", ", the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances  according to their own subjective criteria , .", "however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training . the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the  risks  of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .", "the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair ", "bedarf/bedrfnis  , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term  bedarf  presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term ", "bedrfnis  is far more closely linked to the subjective perception of an individual or a particular predefined group .", "empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and  problems  as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary ,", "these  problems  tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .", "if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .", "moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .", "such behaviour is frequently perceived as  exotic  and therefore attributed to the  culture  or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation", "may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .", "another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .", "stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups .", "the three findings outlined above provided the basis for a teaching concept that incorporates the educational potential provided by concepts and procedures used in medical anthropology .", "moreover , it draws on the specific experience that students gathered working with people from socially marginalised groups , or more precisely : without a permanent address .", "the aim was to systematically draw students attention to the following aspects and dimensions :   the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught;the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed;getting to know and  immersing  themselves in the living environments , health problems and specific needs of people living on  the fringe  of society following the model of  cultural immersion  approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services .", "the aim is to  look closely  at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory;exposing the problems of the concept  need for help  in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds ,  objective  ) needs to be meaningfully and systematically complemented by including the patient s perspective (  subjective  , in the context of their biography and life situation , partly ambivalence / rejection of support services ) .", "the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught ; the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed ; getting to know and  immersing  themselves in the living environments , health problems and specific needs of people living on  the fringe  of society following the model of  cultural immersion  approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services . the aim is to ", "look closely  at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory ; exposing the problems of the concept  need for help  in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds ,  objective  ) needs to be meaningfully and systematically complemented by including the patient s perspective (  subjective  , in the context of their biography and life situation , partly ambivalence / rejection of support services ) . in achieving these objectives , as briefly outlined above , in addition to the medical body of knowledge , concepts and methods from ( medical ) anthropology were introduced as central components of the seminar .", "this essentially involves the method of field research including participant observation , : central to this method is that those at the focus of medical attention will be visited in their everyday living settings .", "an additional effort is made to find out about their physical and social environment and their daily routines in order to comprehend the importance of these factors in relation to the patients perceptions of their body and state of mind , and in relation their help - seeking behaviour .", "in addition to visiting the living environment of those affected , systematic monitoring and  as far as possible  participating in their everyday lives , interviewing informers from the group in focus , together with the systematic documentation of experiences and reflections , form essential elements of this method , .", "this will enable students to get a better grasp , for example , of the behaviour displayed by the strangers they encounter ( which at first glance may often seem obscure and inadequate ) within the logic and conditionality of the particular contextual setting .", "students will also be introduced to the central concept and methodological approach of counter - transference ( as modified by the ethnologist and psychoanalyst george devereux ) : according to this concept  contrary to a widely shared assumption  researchers and other professionals working with people too respond emotionally to these persons and the conditions or behaviours observed in connection with them .", "such emotional responses range from sympathy to various feelings of ambivalence to fear or aggression , which can impact upon professional interaction as well as prolong or shorten it , or even lead to selective perception and specific interpretation of observed behaviours and even persons . following devereux , researchers or doctors", "are asked to systematically observe and reflect upon their own subjectivity and employ their findings , as it were , as an examination tool in its own right that will  in addition to other methods  yield a more holistic and rounded image of the other .", "encounters with homeless people can  as a result of their often radically different way of living and occasionally extreme physical neglect etc .  trigger particularly striking responses from those providing help ; a situation that serves well as an introduction to systematic self - reflection , among other things , on behavioural patterns that are likely to result from such emotional responses .", "in addition to concepts and methods based in anthropology / ethnology and ethno - psychoanalysis , the seminar also teaches the basic principles about the general social , economic and legal conditions affecting the life of people without a permanent address .", "cooperation with relevant institutions plays a major role in this : our main partner is frankfurter verein fr soziale heimsttten e.v . ( fv ) ,", "in the summer semester we additionally have elisabeth strassenambulanz ( esa ) ( backed by : caritasverband frankfurt / m . ) on board .", "the fv is an institution that has close ties with the city and offers an extensive range of services to different groups of homeless in frankfurt / main . during the winter months fv social workers drive the klte - bus to those spots where the homeless", "are known to congregate and , if required , supply them with tea , blankets and sleeping pads .", "the social workers are the first points of contact for the homeless and , if required , will arrange placements offering follow - on support services , including transport organisation .", "the esa provides services aimed at improving the health of women and men in housing difficulties , bearing in mind the unusual living conditions of these people . that a profound knowledge of the particular needs of homeless people as well as of institutional and legal possibilities in dealing with homeless people plays a central role in designing and implementing meaningful teaching concepts", "however , the focus there was on teaching concepts that concentrate mainly on providing medical treatment for socially marginalised groups , and less on the meta - level of systematic self - reflection and the ability to take on a different perspective , which is the focal point of the project presented here .", "the seminar is structured into a preparatory theoretical - methodological module , a practical module , namely the field work ( in the winter semester with the klte - bus , in the summer semester visiting social work in the context of the esa ) , and a follow - up module .", "the seminar groups consist of around 10 students ; this figure is based upon the limited number of individuals when accompanying the klte - bus and in the context of the esa ( one student per appointment ) .", "the first part of the preparatory sessions ( 2 hours ) takes place in the classrooms at giessen university . here", "students are informed about the legal , social and medical situation of people  on the fringe of society  ; previous experience and expectations from students are likewise discussed .", "in addition , students are familiarised with the experience of patients rejecting their offer of support , which can often be disconcerting ; this includes introducing them to the different perceptions people have of  illness  , and to the concept of  counter - transference  .", "the structure of the responsible social institutions , particularly those based in frankfurt ( fv , esa ; among others ) is presented and practical information on the planned field trip given .", "people on the fringe of society  :  the field work starts now .  the second part of the preparatory session ( 2 hours ) takes place on site in the rooms of the fv in frankfurt  a preliminary stage of the field trip : the students leave their familiar environment in order to gain new and unfamiliar impressions . in the rooms of the overnight shelter , amidst the homeless persons staying there , fv social workers provide in - depth information interspersed with specialist and personal contributions .", "they also shed light on the medical and social situation of the individuals in the shelter and discuss expectations and particularities as regards the organisation of this part of the field work with the students .", "the  field work  proper does not take place in the group setting , but students go out invidudually : one student at a time accompanies the fv or esa social workers on their on - site visits to homeless people .", "a 6-hour period is scheduled for this hands - on experience ; in the winter months it includes a trip on the klte - bus between 8 p.m. and 2 a.m. ; in summer the field trip lasts just as long but takes place during the day or in the evening .", "students accompany the social workers on their visits on foot or by bike or in the car ; some students get to attend call - outs on the esa s medical bus .   during the feed - back sessions ( 2x2 hours )", "students relate the different impressions they have had , contextualise them and explain how they felt , thus creating a wide spectrum of different impressions , evaluations and responses , which provide ample opportunities to reflect upon ways of action and potential changes in perception when dealing with patients .", "possible essay topics ( the seminar is assessed by essay ) begin to take shape ; these are discussed in - depth and in relation to how individual students approach their work , as well as formal specifications . in the essay students", "are asked to present their own personal observations and experiences during the field trip ( e.g. , feeling insecure in the unfamiliar environment , personal responses to coming face to face with the living environment , the appearance and behaviour of homeless people ) ; in a second step the aim is to reflect upon these observations and their significance for medical thinking and acting .", "given the limited number of participants ( ten students per semester ) and the relative short period for which the seminar has been available ( since the 2010 - 11 winter semester ; only three seminar groups have fully completed the class ) we are thus far only able to qualitatively comment on the course ; the total number of participants is still too low to allow for any meaningful quantitative - statistical analysis .   to date", ", 31 students overall have participated in the seminar : 16 women and 15 men .", "the anonymous written evaluation contained three open questions (  positive aspects include   ;  negative aspects include   ;  what do you take out of this experience for your further studies ?  ) and nine questions to be answered by way of a graded scale ( school grades a to f ) . among these ,", "three questions were of a general nature (  please assess how much you have learned  ;  relevance for training as a medical doctor  ;  overall school grade  ) , six related to specific aspects of the seminar , such as design and structure of the teaching concept , preparation of the lecturer , availability and comprehensibility of relevant information / literature or learning environment .   the overall feedback on the structure of the course , on the preparation of the lecturer ( b.m . ) and on the learning environment was between positive and very positive ( grade b or a in analogy to the school grading system ) .", "the questions  was the seminar interesting ?  and  the seminar encouraged reflection  received the top grade (  a  ) .", "somewhat in contrast  likewise an unanimous perception  the relevance for hands - on practical application (   relevant for my training as a doctor  ) was recognised but felt to be less positive ( grades b and c ) . in answering the open questions , the  on - site  experience with social workers and ( in a different response ) the", " individual tuition  as a result of this was perceived to be particularly worthwhile .", "another positive comment was that attention was drawn to how a person s concrete living environment affected their health .", "finally ,  the opportunity to  look outside the box  was felt to be a  profound experience  that would shape the students further course of studies .", "the essays too contained statements that reflected the intensity of the seminar and the potential inherent in the anthropological approach complemented by field work and participant observation .", "the following statement has been taken from the discussion section of one of the essays : ", "never before did i look at frankfurt s bahnhofsviertel in this way [  ] .", "another participant in the seminar felt that the field research method  opened up an exotic world [ for him ] .", "the human being that crosses our daily horizon as a penniless and neglected figure on the periphery suddenly gets to be at the heart of our attention .", " both in the evaluation and occasionally also in the essays the wish was expressed to actually extend the practical part of the seminar .   arrangements for receiving written feedback from seminar participants at predefined intervals ( one , two and five years ) have been made , and interviews about the long - term effects of learning and reflection processes are planned with focus groups .", "the preliminary experiences we gained with the teaching concept  providing medical care on the fringe of society : participant observation and change in perspective  illustrate that the spectrum of methodological approaches in q 2 ( ao ) can indeed be complemented by concepts from medical anthropology , in combination with exposing students to people in extreme social situations .", "the objective of getting students to reflect systematically upon historical , ethical - normative and theoretical foundations in medicine can therefore be extended and intensified in particular as regards the following three aspects :   making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) , the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent .   making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) ,   the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and   the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent .   at a more general level students", "will learn how to put themselves in the position of different protagonists in a range of medical settings , and practice the skill of reflecting critically upon putative conceptual / theoretical and normative - ethical assumptions in medicine .", "the teaching project therefore ties in with international models that likewise seek to integrate these teaching objectives in their medical training .", "the method of field research ( or inspired by this in the context of medical training that of  cultural immersion  ) has proven to be very helpful for understanding the perceptions and needs of socially or ethically marginalised groups , and in principle all other groups in health care , , , , [ . in relation to this in the context of the new teaching concept the attention on  social determinants of health  and how they impact on the pathogenesis and course of health conditions", "yield synergies with other teaching concepts , for example in medical sociology , community medicine / public health or general medicine , , which focus on similar matters .", "collaborations with community medicine practitioners working for example at technical colleges / fachhochschulen or other institutions outside universities could likewise bear relevance .", ", it will be necessary to know and familiarise oneself with the central ( medical ) anthropological concepts and methods .", "further , opportunities will have to be created that will enable cooperation with such institutions that are willing to set up the organisational framework and make available their expertise to enable the direct contact of students with people on the fringe of society in their everyday living environments .", "the new curricular option described here could only be realized through the co - operation and engagement of many members of staff at the frankfurter verein fr soziale heimsttten e.v . as well as the elisabeth - straen - ambulanz frankfurt / m .", "( esa ) . we want to express special thanks to heiko ewald and christine heinrichs , together with the staff of the  kltebus-project , the ostpark - project , the beratungszentrum fr aufsuchende sozialarbeit , as well as dr ."], "abstract_text": ["<S> this paper examines the new teaching concept  providing medical care on the fringe of society : participant observation and change in perspectives  in the context of the interdisciplinary field of querschnittsbereich 2/q 2 ( the transdisciplinary section under ao , the german regulations for licensed physicians ) that explores the history , theory and ethics of medicine . </S>", "<S> the disciplinary approach usually adopted in q 2 is supplemented with concepts from medical anthropology ; in addition students will be exposed to people in extreme social situations . the aim is to make students aware of and invite them to reflect upon : the importance of participant observation in the specific on - site setting of medical thinking and acting ; the importance of the subjectivity of all those involved in doctor / patient interaction ; and the fact that key medical terms ( such as the  need  as seen by the physician vs. the need as seen by the patient ) are essentially context - dependent in their interpretation . at a more </S>", "<S> general level students will learn how to put themselves in the position of different protagonists in a range of medical settings , and practice the skill of reflecting critically upon putative conceptual / theoretical and normative - ethical assumptions in medicine . </S>"], "labels": null, "section_names": ["Introduction", "Project description", "Implementation", "Evaluation", "Discussion", "Acknowledgement", "Competing interests"], "sections": [["we all have a very different understanding of how we define a  need for help  .", "this is most clearly felt when encountering individuals who , despite outside temperatures below zero , prefer sleeping rough in the street over the warm place in an overnight homeless shelter that a social worker or other care worker has offered them .", "if they additionally show no response to medical diagnoses such as open ulcers , flu infections or severe breathing difficulties , we can safely say that this person s behaviour deviates fundamentally from the expectations and understanding that health professionals would normally associate with  need for action  .", "being exposed to people who reject such support services , which seem well - motivated and necessary from a medical point of view , is only one of several fruitful challenges that are deliberately provoked as part of the new teaching concept in the institute for the history of medicine at giessen university .", "the objectives are as follows :   to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ; to get students to reflect upon central concepts such as  bedarf / bedrfnis  ( needs in  objective  vs.  subjective  terms ) that are normative in character and have a structuring and guiding effect on medical care services ; to advance and refine students  soft skills  , including the ability to put themselves in the position of other people , in particular those using medical services ; to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals . to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ;   to get students to reflect upon central concepts such as  bedarf / bedrfnis  ( needs in  objective  vs.  subjective  terms ) that are normative in character and have a structuring and guiding effect on medical care services ;   to advance and refine students  soft skills  , including the ability to put themselves in the position of other people , in particular those using medical services ;   to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and   to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals .", "these teaching objectives are implemented and pursued , among others , in the interdisciplinary field querschnittsbereich 2 / q 2  history , ethics and theory of medicine  ,  ", "that is part of the approbationsordnung fr rzte/(ao : regulations for licensing doctors ) ( ao , 2002 ) . as a matter of fact , some of the objectives", "are also presented and discussed in some medical school - curricula in medical sociology and psychology , as well as in community medicine / public health , however , in these contexts , the primary emphasis here is on the methodological and conceptual repertoire from their related disciplines , sociology and psychology . -", "similar teaching concepts as those presented in q 2 can also be found at an international level , where they tend to be associated with the concept of  medical humanities  ( \" kulturwissenschaften in der medizin  ) , .", "medical faculties vary in their interpretation and weighting of these teaching concepts ; however , in the context of research and debate in medical education , the main focus is on the relationship between the three approaches ( history , ethics , theory : additive or integrative ; weighting of individual components ) , which are specified explicitly in the ao and which intend to introduce a reflective perspective on current medicine , .", "this paper sets out to discuss the possibility of supplementing the above - described methods with methodological approaches and concepts from another discipline based in cultural studies , namely medical anthropology ( or medical ethnology ) , and to apply these in the context of a specifically designed form of experience to which students are exposed . outside of germany", ", medical anthropology is an established discipline in university departments , degree courses and associations .", "the seminar  medical care on the fringes of society : participant observation and change in perspectives  has been designed with these approaches and experiences in mind .", "it has been offered as a seminar option in q 2 at the medical school of giessen university since the 2010 - 11 winter semester ."], ["the new teaching concept was developed on the basis of the following findings and considerations : in the standard degree courses required by the regulations for licensing doctors medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result ,", "the focus is on  problems  as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .", "the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness (  patients  from a doctor s point of view ) will often be new to this environment .", "this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .", "the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting.as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .", "however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .", "the priorities for action that result from the individual  illness  of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years", ", the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances  according to their own subjective criteria , .", "however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training .", "the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the  risks  of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .", "the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair ", "bedarf/bedrfnis  , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term  bedarf  presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term ", "bedrfnis  is far more closely linked to the subjective perception of an individual or a particular predefined group.empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and  problems  as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary", ", these  problems  tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .", "if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .", "moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .", "such behaviour is frequently perceived as  exotic  and therefore attributed to the  culture  or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation", "may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .", "another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .", "stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups . in the standard degree courses required by the regulations for licensing doctors", "medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result , the focus is on  problems  as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .", "the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness (  patients  from a doctor s point of view ) will often be new to this environment .", "this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .", "the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting . as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .", "however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .", "the priorities for action that result from the individual  illness  of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years", ", the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances  according to their own subjective criteria , .", "however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training . the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the  risks  of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .", "the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair ", "bedarf/bedrfnis  , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term  bedarf  presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term ", "bedrfnis  is far more closely linked to the subjective perception of an individual or a particular predefined group .", "empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and  problems  as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary ,", "these  problems  tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .", "if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .", "moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .", "such behaviour is frequently perceived as  exotic  and therefore attributed to the  culture  or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation", "may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .", "another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .", "stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups .", "the three findings outlined above provided the basis for a teaching concept that incorporates the educational potential provided by concepts and procedures used in medical anthropology .", "moreover , it draws on the specific experience that students gathered working with people from socially marginalised groups , or more precisely : without a permanent address .", "the aim was to systematically draw students attention to the following aspects and dimensions :   the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught;the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed;getting to know and  immersing  themselves in the living environments , health problems and specific needs of people living on  the fringe  of society following the model of  cultural immersion  approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services .", "the aim is to  look closely  at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory;exposing the problems of the concept  need for help  in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds ,  objective  ) needs to be meaningfully and systematically complemented by including the patient s perspective (  subjective  , in the context of their biography and life situation , partly ambivalence / rejection of support services ) .", "the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught ; the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed ; getting to know and  immersing  themselves in the living environments , health problems and specific needs of people living on  the fringe  of society following the model of  cultural immersion  approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services . the aim is to ", "look closely  at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory ; exposing the problems of the concept  need for help  in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds ,  objective  ) needs to be meaningfully and systematically complemented by including the patient s perspective (  subjective  , in the context of their biography and life situation , partly ambivalence / rejection of support services ) . in achieving these objectives , as briefly outlined above , in addition to the medical body of knowledge , concepts and methods from ( medical ) anthropology were introduced as central components of the seminar .", "this essentially involves the method of field research including participant observation , : central to this method is that those at the focus of medical attention will be visited in their everyday living settings .", "an additional effort is made to find out about their physical and social environment and their daily routines in order to comprehend the importance of these factors in relation to the patients perceptions of their body and state of mind , and in relation their help - seeking behaviour .", "in addition to visiting the living environment of those affected , systematic monitoring and  as far as possible  participating in their everyday lives , interviewing informers from the group in focus , together with the systematic documentation of experiences and reflections , form essential elements of this method , .", "this will enable students to get a better grasp , for example , of the behaviour displayed by the strangers they encounter ( which at first glance may often seem obscure and inadequate ) within the logic and conditionality of the particular contextual setting .", "students will also be introduced to the central concept and methodological approach of counter - transference ( as modified by the ethnologist and psychoanalyst george devereux ) : according to this concept  contrary to a widely shared assumption  researchers and other professionals working with people too respond emotionally to these persons and the conditions or behaviours observed in connection with them .", "such emotional responses range from sympathy to various feelings of ambivalence to fear or aggression , which can impact upon professional interaction as well as prolong or shorten it , or even lead to selective perception and specific interpretation of observed behaviours and even persons . following devereux , researchers or doctors", "are asked to systematically observe and reflect upon their own subjectivity and employ their findings , as it were , as an examination tool in its own right that will  in addition to other methods  yield a more holistic and rounded image of the other .", "encounters with homeless people can  as a result of their often radically different way of living and occasionally extreme physical neglect etc .  trigger particularly striking responses from those providing help ; a situation that serves well as an introduction to systematic self - reflection , among other things , on behavioural patterns that are likely to result from such emotional responses .", "in addition to concepts and methods based in anthropology / ethnology and ethno - psychoanalysis , the seminar also teaches the basic principles about the general social , economic and legal conditions affecting the life of people without a permanent address .", "cooperation with relevant institutions plays a major role in this : our main partner is frankfurter verein fr soziale heimsttten e.v . ( fv ) ,", "in the summer semester we additionally have elisabeth strassenambulanz ( esa ) ( backed by : caritasverband frankfurt / m . ) on board .", "the fv is an institution that has close ties with the city and offers an extensive range of services to different groups of homeless in frankfurt / main . during the winter months fv social workers drive the klte - bus to those spots where the homeless", "are known to congregate and , if required , supply them with tea , blankets and sleeping pads .", "the social workers are the first points of contact for the homeless and , if required , will arrange placements offering follow - on support services , including transport organisation .", "the esa provides services aimed at improving the health of women and men in housing difficulties , bearing in mind the unusual living conditions of these people . that a profound knowledge of the particular needs of homeless people as well as of institutional and legal possibilities in dealing with homeless people plays a central role in designing and implementing meaningful teaching concepts", "however , the focus there was on teaching concepts that concentrate mainly on providing medical treatment for socially marginalised groups , and less on the meta - level of systematic self - reflection and the ability to take on a different perspective , which is the focal point of the project presented here ."], ["the seminar is structured into a preparatory theoretical - methodological module , a practical module , namely the field work ( in the winter semester with the klte - bus , in the summer semester visiting social work in the context of the esa ) , and a follow - up module .", "the seminar groups consist of around 10 students ; this figure is based upon the limited number of individuals when accompanying the klte - bus and in the context of the esa ( one student per appointment ) .", "the first part of the preparatory sessions ( 2 hours ) takes place in the classrooms at giessen university . here", "students are informed about the legal , social and medical situation of people  on the fringe of society  ; previous experience and expectations from students are likewise discussed .", "in addition , students are familiarised with the experience of patients rejecting their offer of support , which can often be disconcerting ; this includes introducing them to the different perceptions people have of  illness  , and to the concept of  counter - transference  .", "the structure of the responsible social institutions , particularly those based in frankfurt ( fv , esa ; among others ) is presented and practical information on the planned field trip given .", "people on the fringe of society  :  the field work starts now .  the second part of the preparatory session ( 2 hours ) takes place on site in the rooms of the fv in frankfurt  a preliminary stage of the field trip : the students leave their familiar environment in order to gain new and unfamiliar impressions . in the rooms of the overnight shelter , amidst the homeless persons staying there , fv social workers provide in - depth information interspersed with specialist and personal contributions .", "they also shed light on the medical and social situation of the individuals in the shelter and discuss expectations and particularities as regards the organisation of this part of the field work with the students .", "the  field work  proper does not take place in the group setting , but students go out invidudually : one student at a time accompanies the fv or esa social workers on their on - site visits to homeless people .", "a 6-hour period is scheduled for this hands - on experience ; in the winter months it includes a trip on the klte - bus between 8 p.m. and 2 a.m. ; in summer the field trip lasts just as long but takes place during the day or in the evening .", "students accompany the social workers on their visits on foot or by bike or in the car ; some students get to attend call - outs on the esa s medical bus .   during the feed - back sessions ( 2x2 hours )", "students relate the different impressions they have had , contextualise them and explain how they felt , thus creating a wide spectrum of different impressions , evaluations and responses , which provide ample opportunities to reflect upon ways of action and potential changes in perception when dealing with patients .", "possible essay topics ( the seminar is assessed by essay ) begin to take shape ; these are discussed in - depth and in relation to how individual students approach their work , as well as formal specifications . in the essay students", "are asked to present their own personal observations and experiences during the field trip ( e.g. , feeling insecure in the unfamiliar environment , personal responses to coming face to face with the living environment , the appearance and behaviour of homeless people ) ; in a second step the aim is to reflect upon these observations and their significance for medical thinking and acting ."], ["given the limited number of participants ( ten students per semester ) and the relative short period for which the seminar has been available ( since the 2010 - 11 winter semester ; only three seminar groups have fully completed the class ) we are thus far only able to qualitatively comment on the course ; the total number of participants is still too low to allow for any meaningful quantitative - statistical analysis .   to date", ", 31 students overall have participated in the seminar : 16 women and 15 men .", "the anonymous written evaluation contained three open questions (  positive aspects include   ;  negative aspects include   ;  what do you take out of this experience for your further studies ?  ) and nine questions to be answered by way of a graded scale ( school grades a to f ) . among these ,", "three questions were of a general nature (  please assess how much you have learned  ;  relevance for training as a medical doctor  ;  overall school grade  ) , six related to specific aspects of the seminar , such as design and structure of the teaching concept , preparation of the lecturer , availability and comprehensibility of relevant information / literature or learning environment .   the overall feedback on the structure of the course , on the preparation of the lecturer ( b.m . ) and on the learning environment was between positive and very positive ( grade b or a in analogy to the school grading system ) .", "the questions  was the seminar interesting ?  and  the seminar encouraged reflection  received the top grade (  a  ) .", "somewhat in contrast  likewise an unanimous perception  the relevance for hands - on practical application (   relevant for my training as a doctor  ) was recognised but felt to be less positive ( grades b and c ) . in answering the open questions , the  on - site  experience with social workers and ( in a different response ) the", " individual tuition  as a result of this was perceived to be particularly worthwhile .", "another positive comment was that attention was drawn to how a person s concrete living environment affected their health .", "finally ,  the opportunity to  look outside the box  was felt to be a  profound experience  that would shape the students further course of studies .", "the essays too contained statements that reflected the intensity of the seminar and the potential inherent in the anthropological approach complemented by field work and participant observation .", "the following statement has been taken from the discussion section of one of the essays : ", "never before did i look at frankfurt s bahnhofsviertel in this way [  ] .", "another participant in the seminar felt that the field research method  opened up an exotic world [ for him ] .", "the human being that crosses our daily horizon as a penniless and neglected figure on the periphery suddenly gets to be at the heart of our attention .", " both in the evaluation and occasionally also in the essays the wish was expressed to actually extend the practical part of the seminar .   arrangements for receiving written feedback from seminar participants at predefined intervals ( one , two and five years ) have been made , and interviews about the long - term effects of learning and reflection processes are planned with focus groups ."], ["the preliminary experiences we gained with the teaching concept  providing medical care on the fringe of society : participant observation and change in perspective  illustrate that the spectrum of methodological approaches in q 2 ( ao ) can indeed be complemented by concepts from medical anthropology , in combination with exposing students to people in extreme social situations .", "the objective of getting students to reflect systematically upon historical , ethical - normative and theoretical foundations in medicine can therefore be extended and intensified in particular as regards the following three aspects :   making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) , the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent .   making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) ,   the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and   the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent .   at a more general level students", "will learn how to put themselves in the position of different protagonists in a range of medical settings , and practice the skill of reflecting critically upon putative conceptual / theoretical and normative - ethical assumptions in medicine .", "the teaching project therefore ties in with international models that likewise seek to integrate these teaching objectives in their medical training .", "the method of field research ( or inspired by this in the context of medical training that of  cultural immersion  ) has proven to be very helpful for understanding the perceptions and needs of socially or ethically marginalised groups , and in principle all other groups in health care , , , , [ . in relation to this in the context of the new teaching concept the attention on  social determinants of health  and how they impact on the pathogenesis and course of health conditions", "yield synergies with other teaching concepts , for example in medical sociology , community medicine / public health or general medicine , , which focus on similar matters .", "collaborations with community medicine practitioners working for example at technical colleges / fachhochschulen or other institutions outside universities could likewise bear relevance .", ", it will be necessary to know and familiarise oneself with the central ( medical ) anthropological concepts and methods .", "further , opportunities will have to be created that will enable cooperation with such institutions that are willing to set up the organisational framework and make available their expertise to enable the direct contact of students with people on the fringe of society in their everyday living environments ."], ["the new curricular option described here could only be realized through the co - operation and engagement of many members of staff at the frankfurter verein fr soziale heimsttten e.v . as well as the elisabeth - straen - ambulanz frankfurt / m .", "( esa ) . we want to express special thanks to heiko ewald and christine heinrichs , together with the staff of the  kltebus-project , the ostpark - project , the beratungszentrum fr aufsuchende sozialarbeit , as well as dr ."], [""]]}
{"article_id": "PMC3712104", "article_text": ["foodborne liver and intestinal flukes are important groups of parasites from the public health point of view [ 1 - 4 ] .", "lao peoples democratic republic ( lao pdr ) is located in the middle of the indochina peninsula , and the mekong river runs through the whole length of the country from north to south . until the 1990s , the liver fluke opisthorchis viverrini and soil - transmitted nematodes , including ascaris , trichuris , and hookworms , were the major helminths prevalent among the laotian people [ 5 - 8 ] . in 1991 ,", "haplorchis taichui adult flukes were recovered for the first time from 5 laotian students studying in czechoslovakia . since then , other intestinal flukes , including haplorchis pumilio , haplorchis yokogawai , prosthodendrium molenkampi , phaneropsolus bonnei , centrocestus formosanus , echinostoma revolutum , artyfechinostomum malayanum , echinochasmus japonicus , and euparyphium sp .", "have been recovered from infected residents of vientiane municipality , phongsaly , saravane , khammouane , and savannakhet province [ 10 - 16 ] .", "the predominating species and the the intensity of infection with each fluke species varied by locality .", "for example , o. viverrini infection was predominant over h. taichui in vientiane municipality , whereas h. taichui infection was dominant over o. viverrini in saravane province . on the other hand , in phongsaly province , located in the northernmost area of lao pdr , high prevalences of h. taichui and h. yokogawai were detected with no detection of o. viverrini .", "after completion of the international collaboration project   between the korea association of health promotion and ministry of public health ( moph ) , lao pdr on the control of intestinal helminthiases in lao pdr from 2000 - 2004 , a new 5-year project plan was started in 2007 to control foodborne trematode infections by the korea foundation for international healthcare and moph , lao pdr . in this new project ,", "the primary goal was to control opisthorchiasis in endemic areas and to reduce the incidence of cholangiocarcinoma in the mekong river basin .", "for the successful implementation of this project , a desirable pre - requisite should be the elucidation of the actual status of infection with o. viverrini in each target village .", "the recovery of eggs only in the feces is not sufficient for this purpose , because the eggs of o. viverrini and those of minute intestinal flukes are practically indistinguishable in routine fecal examinations .", "the present study was performed to determine the actual status of infection with foodborne trematodes by recovery of adult flukes from riparian people of saravane and champasak province , lao pdr .", "saravane province ( capital city , saravane ) is located about 500 km south from vientiane , and champasak province ( capital city , pakse ) is located nearby and just south of saravane province ( fig .", "1 ) . small riverside villages , each having 200 - 300 residents , located near the mainstream or tributaries of the mekong river were selected for this study , which included 12 villages ( in 6 districts ) in both saravane and champasak province .", "most villagers were agricultural workers , and some had caught freshwater fish from small streams and ponds and consumed them raw or improperly cooked . a total of 1,460 fecal samples ( 1 sample per person ) were collected from the villagers ( 717 men and 743 women , 12 - 82 years - of - age ) from december 2009 to june 2010 .", "samples were transported to the malaria stations in saravane and champasak province within 2 - 3 days of collection and were stored at 4 until examined .", "trematode eggs 23 - 32 m in size were collectively recorded as small trematode eggs ( ste ) .", "fecal examinations and anthelmintic treatments were officially approved by the ministry of public health , laos , under the terms of the korea - laos agreement on foodborne trematodiases control in laos ( 2007 - 2011 ) .", "thirty people who were positive for ste were selected for adult worm recovery at the malaria station . after obtaining informed consent ,", "they were treated with a single oral dose of 40 mg / kg praziquantel ( distocide@ ; shinpoong pharm . ,", "worms were counted and some were fixed with 10% formalin under cover slip pressure , acetocarmine - stained , and morphologically identified using a light microscope .", "fecal examination results were analyzed using the student 's t - test and the chi - square test .", "the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively ( table 1 ) .", "there were some mixed infection cases involving different kinds of nematodes , trematodes , and cestodes .", "the most highly prevalent was ste , which included eggs of o. viverrini , heterophyids , and lecithodendriids , with a prevalence of 69.9% ( 46.5 - 87.2% by district ) and 46.3% ( 7.5 - 73.9% by district ) in saravane and champasak province , respectively ( table 1 ) .", "the district showing the highest ste prevalence was toumlane ( 87.2% ) in saravane province and pathumphon ( 73.9% ) in champasak province .", "paksong , an urban area of champasak province , displayed the lowest ste prevalence ( 7.5% ) among the 12 districts surveyed .", "other parasite eggs detected in saravane and champasak province included hookworms ( 20.1% and 22.1% , respectively ) , ascaris lumbricoides ( 5.7% and 4.0% , respectively ) , trichuris trichiura ( 0.8% and 5.6% , respectively ) , taenia spp .", "the egg positive rates were not significantly ( p>0.01 ) different between men and women ( data not shown ) .", "the adult worm collection was completed in 30 people ( 15 men and 15 women ; 20 - 65 years ; 19 in saravane and 11 in champasak ) ( table 2 ) .", "a total of 543,852 helminth specimens were recovered ( average of 18,128 per person ; range 6 - 129,238 worms per person ) .", "intestinal flukes were recovered in all 30 cases ( 100.0% ) , and o. viverrini was recovered in 17 cases ( 56.7% ) .", "the total number of h. taichui specimens recovered in saravane and champasak province were 409,738 ( n=19 ; average 21,562 per person ) and 132,865 ( n=11 ; average 12,078 per person ) , respectively , which was markedly higher than the other helminth parasite species ( table 2 ) .", "the next most frequent species was h. pumilio , with 796 ( n=19 ; average 41.9 per person ) and 247 ( n=11 ; average 22.5 per person ) in saravane and champasak province , respectively ( table 2 ) , followed by o. viverrini , with 179 ( n=19 ; average 9.4 per person ) and 17 ( n=11 ; average 1.5 per person ) in saravane and champasak province , respectively ( table 2 ) .", "small numbers of specimens were collected for taenia saginata , c. formosanus , p. molenkampi , and e. japonicus from a small number of people ( table 2 ) .", "two haplorchis spp . comprised 99.96% ( 99.95% and 99.98% in saravane and champasak province , respectively ) of the total helminth specimens recovered , whereas o. viverrini comprised only 0.04% ( 0.04% and 0.01% in saravane and champasak province , respectively ) .", "the highest worm load of h. taichui collected from a single person was 129,159 in champasak province , followed by 97,791 and 71,647 in saravane province .", "the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively ( table 1 ) .", "there were some mixed infection cases involving different kinds of nematodes , trematodes , and cestodes .", "the most highly prevalent was ste , which included eggs of o. viverrini , heterophyids , and lecithodendriids , with a prevalence of 69.9% ( 46.5 - 87.2% by district ) and 46.3% ( 7.5 - 73.9% by district ) in saravane and champasak province , respectively ( table 1 ) .", "the district showing the highest ste prevalence was toumlane ( 87.2% ) in saravane province and pathumphon ( 73.9% ) in champasak province .", "paksong , an urban area of champasak province , displayed the lowest ste prevalence ( 7.5% ) among the 12 districts surveyed .", "other parasite eggs detected in saravane and champasak province included hookworms ( 20.1% and 22.1% , respectively ) , ascaris lumbricoides ( 5.7% and 4.0% , respectively ) , trichuris trichiura ( 0.8% and 5.6% , respectively ) , taenia spp .", "the egg positive rates were not significantly ( p>0.01 ) different between men and women ( data not shown ) .", "the adult worm collection was completed in 30 people ( 15 men and 15 women ; 20 - 65 years ; 19 in saravane and 11 in champasak ) ( table 2 ) .", "a total of 543,852 helminth specimens were recovered ( average of 18,128 per person ; range 6 - 129,238 worms per person ) .", "intestinal flukes were recovered in all 30 cases ( 100.0% ) , and o. viverrini was recovered in 17 cases ( 56.7% ) .", "the total number of h. taichui specimens recovered in saravane and champasak province were 409,738 ( n=19 ; average 21,562 per person ) and 132,865 ( n=11 ; average 12,078 per person ) , respectively , which was markedly higher than the other helminth parasite species ( table 2 ) .", "the next most frequent species was h. pumilio , with 796 ( n=19 ; average 41.9 per person ) and 247 ( n=11 ; average 22.5 per person ) in saravane and champasak province , respectively ( table 2 ) , followed by o. viverrini , with 179 ( n=19 ; average 9.4 per person ) and 17 ( n=11 ; average 1.5 per person ) in saravane and champasak province , respectively ( table 2 ) .", "small numbers of specimens were collected for taenia saginata , c. formosanus , p. molenkampi , and e. japonicus from a small number of people ( table 2 ) .", "two haplorchis spp . comprised 99.96% ( 99.95% and 99.98% in saravane and champasak province , respectively ) of the total helminth specimens recovered , whereas o. viverrini comprised only 0.04% ( 0.04% and 0.01% in saravane and champasak province , respectively ) .", "the highest worm load of h. taichui collected from a single person was 129,159 in champasak province , followed by 97,791 and 71,647 in saravane province .", "an extremely high predominance of intestinal flukes , in particular , h. taichui , was evident in saravane and champasak province . the liver fluke , o. viverrini , constituted only a very small portion of the helminths infected among the riparian people in the surveyed localities .", "therefore , we could confirm that the real o. viverrini heavy endemic areas in lao pdr include vientiane municipality and savannakhet province , based on the previous results from our research group .", "dominate in khammouane , saravane , and champasak provinces with only a low - grade endemicity of opisthorchiasis , based on our previous observations   and those of the present study .", "phongsaly province   and luang prabang and xiengkhouang provinces are also haplorchis - dominant areas without o. viverrini endemicity ( to be published ) .", "the individual worm load of h. taichui was extremely high in this study , particularly in saravane province .", "it was 67 times higher than in khammouane province ( 324 vs. 21,565 ) , 209 times that of savannakhet province ( 103 vs. 21,565 ) , and 799 times that of vientiane municipality ( 27 vs. 21,565 ) .", "indeed , it was even 3-times higher than that of our previous study in saravane province .", "this may have been due to the fact that different districts were surveyed in our earlier and present studies .", "the present report of intestinal flukes , including h. taichui and h. pumilio , from people in champasak province is novel .", "however , recovery of o. viverrini was previously reported in 3 people ( 5 , 10 , and 395 specimens ) residing near pakse , the capital of champasak province .", "therefore , in the present study , it was surprising to see that the worm load of h. taichui and h. pumilio was markedly higher than that of o. viverrini in champasak province .", "one patient , a 35-year - old man , expelled as many as 129,159 specimens of h. taichui , and thus he represents the most heavily infected case with h. taichui ever recorded in the literature .", "the hyperendemicity of h. taichui infection among riparian people in saravane province was closely correlated with the report of metacercarial prevalence in fish in saravane province .", "h. taichui metacercariae were detected in various freshwater fish species from saravane that included hampala dispar , cyclocheilichthys enoplos , mystacoleucus greenwayi , and puntioplites proctozysron with the average metacercarial density per fish of 1,532 ( range 16 - 6,050 ) , 346 ( 14 - 986 ) , 337 ( 8 - 1,625 ) , and 175 ( 2 - 863 ) , respectively .", "however , the metacercariae of o. viverrini were also detected heavily in h. dispar with the average number per fish of 453 .", "no or very few o. viverrini metacercariae were detected in other species of fish examined in saravane province .", "it is strongly suggested that h. dispar is an important source of infection both for h. taichui and o. viverrini in this area .", "the present study indicated that the sole use of fecal examinations to detect helminth eggs does not fully allow the determination of the actual prevalence and intensity of liver and intestinal flukes in lao pdr .", "for example , 2 recent studies performed in champasak province reported the results of fecal examinations on the residents [ 18 - 20 ] ; the ste were interpreted only as o. viverrini eggs , and the possibility for them to be eggs of intestinal flukes , in particular , haplorchis spp .", ", was overlooked . the adult worm recovery after chemotherapy and purgation , like our studies", "[ 10,11,14 - 16 ] and others , is helpful to determine the actual and relative predominance of liver and intestinal flukes in each area , although it is a tedious work and needs experts who have much experience and skill . it would be preferable to have a proper technique to distinguish the species of ste in fecal specimens .", "several diagnostic techniques have been reported to be useful for morphologic discrimination of ste in fecal samples [ 21 - 23 ] .", "iodine staining was helpful for discriminating lecithodendriid eggs ( p. molenkampi and p. bonnei ) from those of o. viverrini .", "potassium permanganate temporary staining , using 1% ( w / v ) solution for 1 min , was also applied to discriminate the eggs of o. viverrini , h. taichui , and p. bonnei .", "molecular techniques have been applied to detect o. viverrini dna in fecal samples [ 24 - 26 ] . in khon", "kaen , thailand , a 98% specificity and a 100% sensitivity of the pcr technique were obtained in cases of moderate to severe infections ( > 1,000 epg ) but only a 68% sensitivity was shown in light infections ( < 200 epg ) . in lao pdr ,", "the pcr sensitivity was lower ; only around 50% in cases even with high epg counts > 1,000 .", "cetyltrimethyl - ammoniumbromide was used to remove such inhibitors , which resulted in an enhanced sensitivity of pcr technique to detect fecal o. viverrini dna , from 44.8% to 79.3% .", "it was stated that the primers used for pcr did not cross - react with haplorchis spp . .", "in this respect , the possibility of mixed infections with o. viverrini with minute intestinal flukes , including haplorchis spp .", ", was raised as a reason for low sensitivity of pcr technique to detect o. viverrini dna .", "subsequently , pcr assays to discriminate o. viverrini from those of h. taichui and h. pumilio were developed using the internal transcribed spacer ( its ) regions , its1 and its2 .", "the pcr amplicons of o. viverrini , h. taichui , and h. pumilio were 800 , 930 , and 1,250 bp , respectively , for its1 , which successfully differentiated the 3 species , whereas those for its2 were 380 , 530 , and 380 , respectively , which could not discriminate o. viverrini and h. pumilio .", "the sensitivity of its1 pcr using fecal samples , however , was relatively low ( 76.2% ) .", "it may be argued that not all of the o. viverrini worms were expelled from the subjected cases by a 40 mg / kg single dose treatment with praziquantel .", "however , in our previous studies in vientiane municipality and savannakhet province , the same protocol as in the present study was applied , and 3 - 315 o. viverrini specimens in vientiane and 1 - 1,350 o. viverrini specimens in savannakhet were recovered individually in the diarrheic stools of the subjects within 5 - 6 hr ( 4 - 5 successive stools ) after praziquantel administration .", "these observations indicate that if a person is infected with o. viverrini , a majority , if not all , of the worms are expelled by this procedure . a previous study in thailand supports this hypothesis ; in 9 patients treated with 40 mg / kg praziquantel in a single dose followed by purging and whose diarrheal feces were collected at least 24 hr after purgation , 95% of all worms were expelled within the first 4 hr . a study performed in khon", "kaen , thailand , a hyperendemic area of opisthorchiasis , reported that the worm load of o. viverrini among 181 human autopsy cases averaged 182.4 and ranged from 1 - 2,946 per individual . in our previous studies in savannakhet and vientiane , the average worm load measured by worm recovery after praziquantel treatment and purging was 115.4 ( n=29 ; 0 - 1,350 per individual ) and 57.8 ( n=18 ; 3 - 315 per individual ) , respectively", "therefore , it is suggested that the procedure we used is a reliable method for recovery of both liver and intestinal flukes from humans ."], "abstract_text": ["<S> in this study , we found that haplorchis taichui , a heterophyid intestinal fluke , is highly prevalent , with heavy worm loads , among riparian people in saravane and champasak province , lao pdr . </S>", "<S> fecal specimens were collected from 1,460 people ( 717 men and 743 women ) in 12 riparian ( mekong river ) districts and were examined by the kato - katz fecal smear technique . </S>", "<S> the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively . </S>", "<S> the positive rate for small trematode eggs ( ste ) , which included h. taichui and other heterophyids , opisthorchis viverrini , and lecithodendriids , was 69.9% and 46.3% in saravane and champasak province , respectively . to obtain adult flukes , </S>", "<S> 30 ste - positive people were treated with 40 mg / kg praziquantel and then purged . </S>", "<S> whole diarrheic stools were collected 4 - 5 times for each person and searched for fluke specimens using a stereomicroscope . </S>", "<S> mixed infections with various species of trematodes ( h. taichui , haplorchis pumilio , o. viverrini , prosthodendrium molenkampi , centrocestus formosanus , and echinochasmus japonicus ) and a species of cestode ( taenia saginata ) were found . </S>", "<S> however , the worm load was exceptionally high for h. taichui compared with other trematode species , with an average of 21,565 and 12,079 specimens per infected person in saravane and champasak province , respectively , followed by h. pumilio ( 41.9 and 22.5 , respectively ) and o. viverrini ( 9.4 and 1.5 , respectively ) . </S>", "<S> these results show that diverse species of intestinal and liver flukes are prevalent among riparian people in saravane and champasak province , lao pdr , with h. taichui being the exceptionally dominant species . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "Fecal examination", "Worm collection", "DISCUSSION"], "sections": [["foodborne liver and intestinal flukes are important groups of parasites from the public health point of view [ 1 - 4 ] .", "lao peoples democratic republic ( lao pdr ) is located in the middle of the indochina peninsula , and the mekong river runs through the whole length of the country from north to south . until the 1990s , the liver fluke opisthorchis viverrini and soil - transmitted nematodes , including ascaris , trichuris , and hookworms , were the major helminths prevalent among the laotian people [ 5 - 8 ] . in 1991 ,", "haplorchis taichui adult flukes were recovered for the first time from 5 laotian students studying in czechoslovakia . since then , other intestinal flukes , including haplorchis pumilio , haplorchis yokogawai , prosthodendrium molenkampi , phaneropsolus bonnei , centrocestus formosanus , echinostoma revolutum , artyfechinostomum malayanum , echinochasmus japonicus , and euparyphium sp .", "have been recovered from infected residents of vientiane municipality , phongsaly , saravane , khammouane , and savannakhet province [ 10 - 16 ] .", "the predominating species and the the intensity of infection with each fluke species varied by locality .", "for example , o. viverrini infection was predominant over h. taichui in vientiane municipality , whereas h. taichui infection was dominant over o. viverrini in saravane province . on the other hand , in phongsaly province , located in the northernmost area of lao pdr , high prevalences of h. taichui and h. yokogawai were detected with no detection of o. viverrini .", "after completion of the international collaboration project   between the korea association of health promotion and ministry of public health ( moph ) , lao pdr on the control of intestinal helminthiases in lao pdr from 2000 - 2004 , a new 5-year project plan was started in 2007 to control foodborne trematode infections by the korea foundation for international healthcare and moph , lao pdr . in this new project ,", "the primary goal was to control opisthorchiasis in endemic areas and to reduce the incidence of cholangiocarcinoma in the mekong river basin .", "for the successful implementation of this project , a desirable pre - requisite should be the elucidation of the actual status of infection with o. viverrini in each target village .", "the recovery of eggs only in the feces is not sufficient for this purpose , because the eggs of o. viverrini and those of minute intestinal flukes are practically indistinguishable in routine fecal examinations .", "the present study was performed to determine the actual status of infection with foodborne trematodes by recovery of adult flukes from riparian people of saravane and champasak province , lao pdr ."], ["saravane province ( capital city , saravane ) is located about 500 km south from vientiane , and champasak province ( capital city , pakse ) is located nearby and just south of saravane province ( fig .", "1 ) . small riverside villages , each having 200 - 300 residents , located near the mainstream or tributaries of the mekong river were selected for this study , which included 12 villages ( in 6 districts ) in both saravane and champasak province .", "most villagers were agricultural workers , and some had caught freshwater fish from small streams and ponds and consumed them raw or improperly cooked . a total of 1,460 fecal samples ( 1 sample per person ) were collected from the villagers ( 717 men and 743 women , 12 - 82 years - of - age ) from december 2009 to june 2010 .", "samples were transported to the malaria stations in saravane and champasak province within 2 - 3 days of collection and were stored at 4 until examined .", "trematode eggs 23 - 32 m in size were collectively recorded as small trematode eggs ( ste ) .", "fecal examinations and anthelmintic treatments were officially approved by the ministry of public health , laos , under the terms of the korea - laos agreement on foodborne trematodiases control in laos ( 2007 - 2011 ) .", "thirty people who were positive for ste were selected for adult worm recovery at the malaria station . after obtaining informed consent ,", "they were treated with a single oral dose of 40 mg / kg praziquantel ( distocide@ ; shinpoong pharm . ,", "worms were counted and some were fixed with 10% formalin under cover slip pressure , acetocarmine - stained , and morphologically identified using a light microscope .", "fecal examination results were analyzed using the student 's t - test and the chi - square test ."], ["the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively ( table 1 ) .", "there were some mixed infection cases involving different kinds of nematodes , trematodes , and cestodes .", "the most highly prevalent was ste , which included eggs of o. viverrini , heterophyids , and lecithodendriids , with a prevalence of 69.9% ( 46.5 - 87.2% by district ) and 46.3% ( 7.5 - 73.9% by district ) in saravane and champasak province , respectively ( table 1 ) .", "the district showing the highest ste prevalence was toumlane ( 87.2% ) in saravane province and pathumphon ( 73.9% ) in champasak province .", "paksong , an urban area of champasak province , displayed the lowest ste prevalence ( 7.5% ) among the 12 districts surveyed .", "other parasite eggs detected in saravane and champasak province included hookworms ( 20.1% and 22.1% , respectively ) , ascaris lumbricoides ( 5.7% and 4.0% , respectively ) , trichuris trichiura ( 0.8% and 5.6% , respectively ) , taenia spp .", "the egg positive rates were not significantly ( p>0.01 ) different between men and women ( data not shown ) .", "the adult worm collection was completed in 30 people ( 15 men and 15 women ; 20 - 65 years ; 19 in saravane and 11 in champasak ) ( table 2 ) .", "a total of 543,852 helminth specimens were recovered ( average of 18,128 per person ; range 6 - 129,238 worms per person ) .", "intestinal flukes were recovered in all 30 cases ( 100.0% ) , and o. viverrini was recovered in 17 cases ( 56.7% ) .", "the total number of h. taichui specimens recovered in saravane and champasak province were 409,738 ( n=19 ; average 21,562 per person ) and 132,865 ( n=11 ; average 12,078 per person ) , respectively , which was markedly higher than the other helminth parasite species ( table 2 ) .", "the next most frequent species was h. pumilio , with 796 ( n=19 ; average 41.9 per person ) and 247 ( n=11 ; average 22.5 per person ) in saravane and champasak province , respectively ( table 2 ) , followed by o. viverrini , with 179 ( n=19 ; average 9.4 per person ) and 17 ( n=11 ; average 1.5 per person ) in saravane and champasak province , respectively ( table 2 ) .", "small numbers of specimens were collected for taenia saginata , c. formosanus , p. molenkampi , and e. japonicus from a small number of people ( table 2 ) .", "two haplorchis spp . comprised 99.96% ( 99.95% and 99.98% in saravane and champasak province , respectively ) of the total helminth specimens recovered , whereas o. viverrini comprised only 0.04% ( 0.04% and 0.01% in saravane and champasak province , respectively ) .", "the highest worm load of h. taichui collected from a single person was 129,159 in champasak province , followed by 97,791 and 71,647 in saravane province ."], ["the overall helminth egg positive rate was 78.8% and 66.4% in saravane and champasak province , respectively ( table 1 ) .", "there were some mixed infection cases involving different kinds of nematodes , trematodes , and cestodes .", "the most highly prevalent was ste , which included eggs of o. viverrini , heterophyids , and lecithodendriids , with a prevalence of 69.9% ( 46.5 - 87.2% by district ) and 46.3% ( 7.5 - 73.9% by district ) in saravane and champasak province , respectively ( table 1 ) .", "the district showing the highest ste prevalence was toumlane ( 87.2% ) in saravane province and pathumphon ( 73.9% ) in champasak province .", "paksong , an urban area of champasak province , displayed the lowest ste prevalence ( 7.5% ) among the 12 districts surveyed .", "other parasite eggs detected in saravane and champasak province included hookworms ( 20.1% and 22.1% , respectively ) , ascaris lumbricoides ( 5.7% and 4.0% , respectively ) , trichuris trichiura ( 0.8% and 5.6% , respectively ) , taenia spp .", "the egg positive rates were not significantly ( p>0.01 ) different between men and women ( data not shown ) ."], ["the adult worm collection was completed in 30 people ( 15 men and 15 women ; 20 - 65 years ; 19 in saravane and 11 in champasak ) ( table 2 ) .", "a total of 543,852 helminth specimens were recovered ( average of 18,128 per person ; range 6 - 129,238 worms per person ) .", "intestinal flukes were recovered in all 30 cases ( 100.0% ) , and o. viverrini was recovered in 17 cases ( 56.7% ) .", "the total number of h. taichui specimens recovered in saravane and champasak province were 409,738 ( n=19 ; average 21,562 per person ) and 132,865 ( n=11 ; average 12,078 per person ) , respectively , which was markedly higher than the other helminth parasite species ( table 2 ) .", "the next most frequent species was h. pumilio , with 796 ( n=19 ; average 41.9 per person ) and 247 ( n=11 ; average 22.5 per person ) in saravane and champasak province , respectively ( table 2 ) , followed by o. viverrini , with 179 ( n=19 ; average 9.4 per person ) and 17 ( n=11 ; average 1.5 per person ) in saravane and champasak province , respectively ( table 2 ) .", "small numbers of specimens were collected for taenia saginata , c. formosanus , p. molenkampi , and e. japonicus from a small number of people ( table 2 ) .", "two haplorchis spp . comprised 99.96% ( 99.95% and 99.98% in saravane and champasak province , respectively ) of the total helminth specimens recovered , whereas o. viverrini comprised only 0.04% ( 0.04% and 0.01% in saravane and champasak province , respectively ) .", "the highest worm load of h. taichui collected from a single person was 129,159 in champasak province , followed by 97,791 and 71,647 in saravane province ."], ["an extremely high predominance of intestinal flukes , in particular , h. taichui , was evident in saravane and champasak province . the liver fluke , o. viverrini , constituted only a very small portion of the helminths infected among the riparian people in the surveyed localities .", "therefore , we could confirm that the real o. viverrini heavy endemic areas in lao pdr include vientiane municipality and savannakhet province , based on the previous results from our research group .", "dominate in khammouane , saravane , and champasak provinces with only a low - grade endemicity of opisthorchiasis , based on our previous observations   and those of the present study .", "phongsaly province   and luang prabang and xiengkhouang provinces are also haplorchis - dominant areas without o. viverrini endemicity ( to be published ) .", "the individual worm load of h. taichui was extremely high in this study , particularly in saravane province .", "it was 67 times higher than in khammouane province ( 324 vs. 21,565 ) , 209 times that of savannakhet province ( 103 vs. 21,565 ) , and 799 times that of vientiane municipality ( 27 vs. 21,565 ) .", "indeed , it was even 3-times higher than that of our previous study in saravane province .", "this may have been due to the fact that different districts were surveyed in our earlier and present studies .", "the present report of intestinal flukes , including h. taichui and h. pumilio , from people in champasak province is novel .", "however , recovery of o. viverrini was previously reported in 3 people ( 5 , 10 , and 395 specimens ) residing near pakse , the capital of champasak province .", "therefore , in the present study , it was surprising to see that the worm load of h. taichui and h. pumilio was markedly higher than that of o. viverrini in champasak province .", "one patient , a 35-year - old man , expelled as many as 129,159 specimens of h. taichui , and thus he represents the most heavily infected case with h. taichui ever recorded in the literature .", "the hyperendemicity of h. taichui infection among riparian people in saravane province was closely correlated with the report of metacercarial prevalence in fish in saravane province .", "h. taichui metacercariae were detected in various freshwater fish species from saravane that included hampala dispar , cyclocheilichthys enoplos , mystacoleucus greenwayi , and puntioplites proctozysron with the average metacercarial density per fish of 1,532 ( range 16 - 6,050 ) , 346 ( 14 - 986 ) , 337 ( 8 - 1,625 ) , and 175 ( 2 - 863 ) , respectively .", "however , the metacercariae of o. viverrini were also detected heavily in h. dispar with the average number per fish of 453 .", "no or very few o. viverrini metacercariae were detected in other species of fish examined in saravane province .", "it is strongly suggested that h. dispar is an important source of infection both for h. taichui and o. viverrini in this area .", "the present study indicated that the sole use of fecal examinations to detect helminth eggs does not fully allow the determination of the actual prevalence and intensity of liver and intestinal flukes in lao pdr .", "for example , 2 recent studies performed in champasak province reported the results of fecal examinations on the residents [ 18 - 20 ] ; the ste were interpreted only as o. viverrini eggs , and the possibility for them to be eggs of intestinal flukes , in particular , haplorchis spp .", ", was overlooked . the adult worm recovery after chemotherapy and purgation , like our studies", "[ 10,11,14 - 16 ] and others , is helpful to determine the actual and relative predominance of liver and intestinal flukes in each area , although it is a tedious work and needs experts who have much experience and skill . it would be preferable to have a proper technique to distinguish the species of ste in fecal specimens .", "several diagnostic techniques have been reported to be useful for morphologic discrimination of ste in fecal samples [ 21 - 23 ] .", "iodine staining was helpful for discriminating lecithodendriid eggs ( p. molenkampi and p. bonnei ) from those of o. viverrini .", "potassium permanganate temporary staining , using 1% ( w / v ) solution for 1 min , was also applied to discriminate the eggs of o. viverrini , h. taichui , and p. bonnei .", "molecular techniques have been applied to detect o. viverrini dna in fecal samples [ 24 - 26 ] . in khon", "kaen , thailand , a 98% specificity and a 100% sensitivity of the pcr technique were obtained in cases of moderate to severe infections ( > 1,000 epg ) but only a 68% sensitivity was shown in light infections ( < 200 epg ) . in lao pdr ,", "the pcr sensitivity was lower ; only around 50% in cases even with high epg counts > 1,000 .", "cetyltrimethyl - ammoniumbromide was used to remove such inhibitors , which resulted in an enhanced sensitivity of pcr technique to detect fecal o. viverrini dna , from 44.8% to 79.3% .", "it was stated that the primers used for pcr did not cross - react with haplorchis spp . .", "in this respect , the possibility of mixed infections with o. viverrini with minute intestinal flukes , including haplorchis spp .", ", was raised as a reason for low sensitivity of pcr technique to detect o. viverrini dna .", "subsequently , pcr assays to discriminate o. viverrini from those of h. taichui and h. pumilio were developed using the internal transcribed spacer ( its ) regions , its1 and its2 .", "the pcr amplicons of o. viverrini , h. taichui , and h. pumilio were 800 , 930 , and 1,250 bp , respectively , for its1 , which successfully differentiated the 3 species , whereas those for its2 were 380 , 530 , and 380 , respectively , which could not discriminate o. viverrini and h. pumilio .", "the sensitivity of its1 pcr using fecal samples , however , was relatively low ( 76.2% ) .", "it may be argued that not all of the o. viverrini worms were expelled from the subjected cases by a 40 mg / kg single dose treatment with praziquantel .", "however , in our previous studies in vientiane municipality and savannakhet province , the same protocol as in the present study was applied , and 3 - 315 o. viverrini specimens in vientiane and 1 - 1,350 o. viverrini specimens in savannakhet were recovered individually in the diarrheic stools of the subjects within 5 - 6 hr ( 4 - 5 successive stools ) after praziquantel administration .", "these observations indicate that if a person is infected with o. viverrini , a majority , if not all , of the worms are expelled by this procedure . a previous study in thailand supports this hypothesis ; in 9 patients treated with 40 mg / kg praziquantel in a single dose followed by purging and whose diarrheal feces were collected at least 24 hr after purgation , 95% of all worms were expelled within the first 4 hr . a study performed in khon", "kaen , thailand , a hyperendemic area of opisthorchiasis , reported that the worm load of o. viverrini among 181 human autopsy cases averaged 182.4 and ranged from 1 - 2,946 per individual . in our previous studies in savannakhet and vientiane , the average worm load measured by worm recovery after praziquantel treatment and purging was 115.4 ( n=29 ; 0 - 1,350 per individual ) and 57.8 ( n=18 ; 3 - 315 per individual ) , respectively", "therefore , it is suggested that the procedure we used is a reliable method for recovery of both liver and intestinal flukes from humans ."]]}
{"article_id": "PMC4435025", "article_text": ["for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information  a misdiagnosis , an incomplete medication list , the wrong demographic data ", "thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality . building on a framework for data quality ,", "as the source of patient - centered data , consumers have important roles to play in reviewing and confirming their own health information and should be engaged partners in efforts to understand and improve the quality of electronic health data .", "for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information  a misdiagnosis , an incomplete medication list , the wrong demographic data ", "thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality .", "consumers are not only the foundation of the data quality framework kahn et al . , but have important roles to play in reviewing and confirming their own health information along the chain of data stewardship . recognizing patients and families as sources of valuable information and powerful forces for change in identifying and preventing errors will also help to foster a culture of safety and build a sense of partnership between providers and patients in the mutual pursuit of better health and care . building on kahn et al.s figure following the chain of data stewardship ,", "i have overlaid opportunities for patient engagement at each step of the process , underscoring the critical role of individuals as the original data stewards ( see figure 1 ) .", "the number of people with online access to their electronic health information is growing by leaps and bounds . before the health information technology for economic and clinical health ( hitech ) act , patients had a right to receive a copy of their health records within 3090 days under the hipaa privacy rule .", "now , thanks to meaningful use , patients have the ability to view online , download , and transmit their own records within 36 hours after a hospital discharge , or within four business days after a doctor receives the health data.1 health information is increasingly available at the click of a button with blue button .", "blue button is an easy , secure method for individuals to download their medical records in digital form from a secure website offered by doctors , insurers , pharmacies or other health - related services.2 implementation of these policy changes is underway , and there are undoubtedly individuals who still struggle to access their own health information ( electronically or otherwise ) , and work is necessary to improve the experience and utility of online access for patients .", "nonetheless , online access to clinical information is an opportunity to partner with patients and families to assess and enhance the quality of health care data .", "as patients and families get easier access to their medical records and health data , possibly for the first time , it is inevitable that they will have feedback  corrections , additions , and observations to share .", "in fact , the absence of health information produced , shared , and reviewed by patients themselves contributes to poor data quality .", "patients and their caregivers can provide information critical to high - quality research and care delivery , including family health history , treatment side effects , changes in health status or functional status , and even treatment outcomes.3 too often , records are missing key data about health outcomes  did the patient get better after the recommended course of treatment ? corrections and amendments are another important type of pghd .", "patients are often the first to identify errors in their own records . increased access by individuals to their own health information", "these could include updated medication lists , nontolerated medications , other allergies , and more .", "these and other patient generated health data complement clinical information generated by care teams to provide a comprehensive view of an individual s health , thus enhancing data quality , as well as enabling more meaningful conclusions to be drawn from secondary uses of this data . with greater online access to their health information and new tools to electronically communicate and share information with providers , patients and families can help improve the accuracy and reliability of data stored in medical records .", "i recently heard a speaker at the office of the national coordinator ( onc ) s consumer summit affectionately refer to the benefits of this new capability as an  army of free fact checkers . ", "a 2014 nationwide survey found that patients with online access to their health information overwhelmingly use this capability : 86 percent log on at least once a year , and more than half ( 55 percent ) log on three or more times per year .", "survey data show that online access has a positive impact on patients knowledge of their health , ability to communicate with doctors , and ability to correct information ( for 63 percent of patients ) .", "more frequent online access has an even greater impact on ability to correct information ( 71 percent for frequent users , compared with 48 percent for less frequent users).4", "as the original generators and stewards of their own health data , individual patients and consumers are the foundation in the data stewardship chain outlined in kahn et al.s data quality framework .", "consumers also want to continue to be involved , considered , and consulted in all aspects of data capture , assessment , and use .", "figure 1 takes kahn s figure and illustrates the potential data contributions of individual patients and consumers , as well as opportunities to support critical data management activities at multiple points along the chain of data stewardship . in particular , there are opportunities for consumer and patient involvement in improving the quality and integrity of data stored in electronic health records ( ehrs ) for clinical use ( data originator box ) . for example , providers could share their computer screen with patients during clinical encounters , allowing patients to see their medication lists or medical records .", "this gesture not only engages patients in the data entry , but also offers an opportunity for patients to contribute corrections or new information to their record in person . the growing ability for patients to view , download , and", "patients and their families can view their health records and supply missing data or correct inaccurate data via patient portals or secure messaging with their providers .", "individuals can review and verify data further down the stewardship chain via a portal offered by a health information exchange ( hie ) .", "hies collect data from multiple institutions , and some portals allow the patient to see the collective longitudinal record and to offer corrections or amendments to the whole .", "finally , at the end of the data stewardship chain , consumers could review and offer feedback on the relevance or usefulness of the analysis and visualizations being created by clinical researchers and other investigators .", "of course , questions remain about how best to collect and ingest information from patients into the electronic medical record .", "questions , for example , such as how to authenticate data coming from patients prior to use ( how to confirm when the data were created , whether they were modified in some way , what instrument was used to collect them , etc . ) .", "policies and procedures are still necessary to ensure that providers can receive , review , record , and respond to pghd shared by patients and families.5 \n a comprehensive data quality reporting framework for assessing and describing data quality may help to validate pghd and incorporate them into the medical record , and use them to inform clinical care or health care policy . we must also more effectively engage patients and families in an online health environment and educate them about the impact of data quality on their health and health care .", "underlying changes to clinical culture are also necessary : moving toward viewing patients as active partners in their care , rather than as passive recipients.6 great strides are being made in patient - centered outcomes research , as exemplified by the patient centered outcomes research institute ( pcori ) , which funds research and supports patients and other stakeholders to meaningfully partner with researchers throughout the research continuum . looking forward , we must also broaden our focus beyond current data types and quality issues to consider relevant health data outside traditional clinical settings , such as social determinants of health ( building on the institute of medicine s recommendations),7 and their place and impact in the data quality framework .", "consumers have much to gain from , and contribute to , activities to assess and improve data quality.8 efforts to adopt and implement a comprehensive data quality framework should preserve and reinforce the role of individuals as the original data stewards , generating and sharing critical information to inform better research , safer clinical care , and  ultimately  improved health outcomes ."], "abstract_text": ["<S> introduction : for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information  a misdiagnosis , an incomplete medication list , the wrong demographic data  care safety , quality , and outcomes suffer  and patients and families suffer.opportunity for change : thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality . </S>", "<S> building on a framework for data quality , there are numerous opportunities for patients to be part of  </S>", "<S> feedback loops to understand and improve data quality are presented.foundation for the future : as the source of patient - centered data , consumers have important roles to play in reviewing and confirming their own health information and should be engaged partners in efforts to understand and improve the quality of electronic health data . </S>"], "labels": null, "section_names": ["Introduction:", "Opportunity for Change:", "Foundation for the Future:", "Introduction", "The Role of Online Access", "Patient Generated Health Data (PGHD)", "Data Quality Framework"], "sections": [["for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information  a misdiagnosis , an incomplete medication list , the wrong demographic data "], ["thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality . building on a framework for data quality ,"], ["as the source of patient - centered data , consumers have important roles to play in reviewing and confirming their own health information and should be engaged partners in efforts to understand and improve the quality of electronic health data ."], ["for patients and families , the issue of data quality is personal . when treatment decisions or research conclusions are based on incomplete or inaccurate information  a misdiagnosis , an incomplete medication list , the wrong demographic data ", "thanks in part to the power of health information technology , individuals have new opportunities to be part of the solution when it comes to assessing and improving data quality .", "consumers are not only the foundation of the data quality framework kahn et al . , but have important roles to play in reviewing and confirming their own health information along the chain of data stewardship . recognizing patients and families as sources of valuable information and powerful forces for change in identifying and preventing errors will also help to foster a culture of safety and build a sense of partnership between providers and patients in the mutual pursuit of better health and care . building on kahn et al.s figure following the chain of data stewardship ,", "i have overlaid opportunities for patient engagement at each step of the process , underscoring the critical role of individuals as the original data stewards ( see figure 1 ) ."], ["the number of people with online access to their electronic health information is growing by leaps and bounds . before the health information technology for economic and clinical health ( hitech ) act , patients had a right to receive a copy of their health records within 3090 days under the hipaa privacy rule .", "now , thanks to meaningful use , patients have the ability to view online , download , and transmit their own records within 36 hours after a hospital discharge , or within four business days after a doctor receives the health data.1 health information is increasingly available at the click of a button with blue button .", "blue button is an easy , secure method for individuals to download their medical records in digital form from a secure website offered by doctors , insurers , pharmacies or other health - related services.2 implementation of these policy changes is underway , and there are undoubtedly individuals who still struggle to access their own health information ( electronically or otherwise ) , and work is necessary to improve the experience and utility of online access for patients .", "nonetheless , online access to clinical information is an opportunity to partner with patients and families to assess and enhance the quality of health care data ."], ["as patients and families get easier access to their medical records and health data , possibly for the first time , it is inevitable that they will have feedback  corrections , additions , and observations to share .", "in fact , the absence of health information produced , shared , and reviewed by patients themselves contributes to poor data quality .", "patients and their caregivers can provide information critical to high - quality research and care delivery , including family health history , treatment side effects , changes in health status or functional status , and even treatment outcomes.3 too often , records are missing key data about health outcomes  did the patient get better after the recommended course of treatment ? corrections and amendments are another important type of pghd .", "patients are often the first to identify errors in their own records . increased access by individuals to their own health information", "these could include updated medication lists , nontolerated medications , other allergies , and more .", "these and other patient generated health data complement clinical information generated by care teams to provide a comprehensive view of an individual s health , thus enhancing data quality , as well as enabling more meaningful conclusions to be drawn from secondary uses of this data . with greater online access to their health information and new tools to electronically communicate and share information with providers , patients and families can help improve the accuracy and reliability of data stored in medical records .", "i recently heard a speaker at the office of the national coordinator ( onc ) s consumer summit affectionately refer to the benefits of this new capability as an  army of free fact checkers . ", "a 2014 nationwide survey found that patients with online access to their health information overwhelmingly use this capability : 86 percent log on at least once a year , and more than half ( 55 percent ) log on three or more times per year .", "survey data show that online access has a positive impact on patients knowledge of their health , ability to communicate with doctors , and ability to correct information ( for 63 percent of patients ) .", "more frequent online access has an even greater impact on ability to correct information ( 71 percent for frequent users , compared with 48 percent for less frequent users).4"], ["as the original generators and stewards of their own health data , individual patients and consumers are the foundation in the data stewardship chain outlined in kahn et al.s data quality framework .", "consumers also want to continue to be involved , considered , and consulted in all aspects of data capture , assessment , and use .", "figure 1 takes kahn s figure and illustrates the potential data contributions of individual patients and consumers , as well as opportunities to support critical data management activities at multiple points along the chain of data stewardship . in particular , there are opportunities for consumer and patient involvement in improving the quality and integrity of data stored in electronic health records ( ehrs ) for clinical use ( data originator box ) . for example , providers could share their computer screen with patients during clinical encounters , allowing patients to see their medication lists or medical records .", "this gesture not only engages patients in the data entry , but also offers an opportunity for patients to contribute corrections or new information to their record in person . the growing ability for patients to view , download , and", "patients and their families can view their health records and supply missing data or correct inaccurate data via patient portals or secure messaging with their providers .", "individuals can review and verify data further down the stewardship chain via a portal offered by a health information exchange ( hie ) .", "hies collect data from multiple institutions , and some portals allow the patient to see the collective longitudinal record and to offer corrections or amendments to the whole .", "finally , at the end of the data stewardship chain , consumers could review and offer feedback on the relevance or usefulness of the analysis and visualizations being created by clinical researchers and other investigators .", "of course , questions remain about how best to collect and ingest information from patients into the electronic medical record .", "questions , for example , such as how to authenticate data coming from patients prior to use ( how to confirm when the data were created , whether they were modified in some way , what instrument was used to collect them , etc . ) .", "policies and procedures are still necessary to ensure that providers can receive , review , record , and respond to pghd shared by patients and families.5 \n a comprehensive data quality reporting framework for assessing and describing data quality may help to validate pghd and incorporate them into the medical record , and use them to inform clinical care or health care policy . we must also more effectively engage patients and families in an online health environment and educate them about the impact of data quality on their health and health care .", "underlying changes to clinical culture are also necessary : moving toward viewing patients as active partners in their care , rather than as passive recipients.6 great strides are being made in patient - centered outcomes research , as exemplified by the patient centered outcomes research institute ( pcori ) , which funds research and supports patients and other stakeholders to meaningfully partner with researchers throughout the research continuum . looking forward , we must also broaden our focus beyond current data types and quality issues to consider relevant health data outside traditional clinical settings , such as social determinants of health ( building on the institute of medicine s recommendations),7 and their place and impact in the data quality framework .", "consumers have much to gain from , and contribute to , activities to assess and improve data quality.8 efforts to adopt and implement a comprehensive data quality framework should preserve and reinforce the role of individuals as the original data stewards , generating and sharing critical information to inform better research , safer clinical care , and  ultimately  improved health outcomes ."]]}
{"article_id": "PMC3325014", "article_text": ["adverse drug reactions ( adrs ) , including interactions , in older people are a common cause of admission to hospital1 and are also a common cause of disease in hospitalized patients .", "frail elderly patients appear to be particularly at risk of adrs . in some cases , this is because insufficient account is taken of the effect of age and frailty on the pharmacokinetic and pharmacodynamic of the drug , especially in relation to hepatic2 and renal elimination.3 although many adrs are recognized during the drug development process and before licensing , this is not always the case , especially when they are uncommon or rare.3 moreover , patients with severe cognitive impairment may not be able to refer symptoms related to an adr , making the recognition of this clinical condition a medical problem.4 rhabdomyolysis is a clinical and laboratory syndrome that is caused by various etiologies , involving the skeletal muscle.5 rhabdomyolysis is characterized by the leaking of myoglobin and other intracellular proteins and electrolytes into the circulation , and may be associated with a wide variety of diseases , injuries , medications , and toxins.5 it is important to recognize drug - induced rhabdomyolysis because clinical effects are usually reversible.4 rhabdomyolysis related to macrolide - statin interaction has previously been described.68 clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins like simvastatin and atorvastain.69 to date , rhabdomyolysis related to clarithromycin has been reported in only one previous case report.10 we report the case of a 90-year - old caucasian male , admitted to the university hospital of pisa for dyspnea , who experienced rhabdomyolysis related to clarithromycin treatment .", "a 90-year - old caucasian male was admitted to the university hospital of pisa for dyspnea .", "he had experienced dyspnea for 2 weeks , and showed signs of pneumonia after a chest x - ray was performed .", "the patient was affected by alzheimer s disease , in advanced phase , and did not take any home - medication as referred by his general physician . at admission in the emergency room ,", "clinical workup revealed mental confusion ( the relatives referred progressive cognitive impairment for several months ) , sinus tachycardia ( 115 beats per minute ) , basilar wheezes , and rhonchi .", "body temperature was 36.6c with normal systemic blood pressure ( 140/80 mmhg ) , diuresis , and blood oxygen saturation ( so2% 96.2% ) .", "routine laboratory exams , including complete blood cell count , thyroid hormones , liver ( aspartate aminotransferase and alanine aminotransferase ) and muscle ( creatine kinase [ ck ] ) enzymes , as well as the coagulation pattern , resulted within normal limits .", "serum brain natriuretic peptide was measured and a cardiac markers curve ( including myoglobin ) was also started , ruling out cardiogenic dyspnea .", "blood potassium and creatinine concentrations were 4.96 meq / l ( reference range 3.55 meq / l ) and 122.8 mol / l ( reference range 4590", "a diagnosis of acquired pneumonia was made and treatment with ceftriaxone ( 1 g four times daily , intravenous ) , hydration ( crystalloid 1.5 l / day ) , aerosol with salbutamol and oxitropium , o2 inhalation ( 3 l / minute ) , and dalteparin ( 2500 ui , subcutaneous ) was started .", "after the geriatric ward admission , clarithromycin ( 500 mg two times daily , intravenous ) was also started .", "on the second day of hospitalization , for continuing sinus tachycardia , in the absence of any evident causes ( fever , hypotension , arterial - venous fistula , thyrotoxicosis , suspected drug treatments , etc ) beta - blocker treatment ( bisoprolol 1.25 mg , daily ) was added and a cardiac markers curve was restarted ( figure 1 ) .", "the second point of the curve , almost 6 hours after the third clarithromycin dose , revealed a huge myoglobin increase ( 1144 ng / ml ; 8.5 times the upper normal range [ 28138 ng / ml ] ) without electrocardiogram modifications .", "u / l ; 6.5 times the upper normal range [ 38180 u / l ] ) along with a mild creatinine increase ( 152.9 mol / l ) , in the absence of liver enzyme alterations ( aspartate aminotransferase , alanine aminotransferase ) .", "urine color concomitantly changed toward dark brown and hemogasanalysis revealed a slight respiratory insufficiency type 1 with normal ph .", "a diagnosis of rhabdomyolysis was made and clarithromycin was stopped , while the concomitant therapy with ceftriaxone , bisoprolol , and aerosol with salbutamol and oxitropium was continued .", "the patient was immediately treated with intravenous crystalloid hypotonic solution ( 80 ml / hour ) and corticosteroid ( 40 mg methylprednisolone , intravenous ) .", "serum ck and myoglobin levels decreased within a few days and a chest x - ray showed significant improvement .", "the patient was discharged home on the sixth hospital day ( figure 1 ) with the diagnosis of iatrogenic rhabdomyolysis and acquired pneumonia of unknown etiology .", "indeed , serum and urinary antigen and antibody exams excluded the presence of legionella pneumophila , streptococcus pneumoniae , and mycoplasma pneumoniae while virus infection assessments were not performed .", "clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins as simvastatin and atorvastatin.3,69 rhabdomyolysis related to macrolide and statins interaction has previously been described ; however , rhabdomyolysis related to clarithromycin has been reported in only one previous case report.8,10 in our case , there was an evident temporal sequence between clarithromycin exposure - withdrawal and the onset - recovery from rhabdomyolysis biochemical signs , while other potential causes of muscle damage were reasonably excluded .", "accordingly , the naranjo probability scale confirmed a probable relationship ( score 6/9).13 a general physician and a nurse followed the patient at home , thus we are confident the home care was carried out in a correct manner , ruling out mistakes and accidental drug intake .", "moreover , an interaction with other concomitant medications was improbable considering the rapid resolution after clarithromycin withdrawal and the elimination pathways of the concomitant drugs ( both bisoprolol and ceftriaxone have hepatic and renal elimination routes).11,12 in addition , bisoprolol was administered at the minimal dose ( 1.25 mg ) and both bisoprolol and ceftriaxone , as single therapy , were never related to an adverse drug reaction like rhabdomyolysis .", "we ruled out infection as the possible cause of muscle damage considering the sequence of the events .", "in fact , the patient was admitted to the hospital for dyspnea developed at home since 2 weeks , while serum ck suddenly increased 2 days after hospital admission .", "if rhabdomyolysis had been caused by the infection we would have expected that serum ck increased earlier , thus being already evident at hospital admission . moreover ,", "the diagnosis of rhabdomyolysis , unless there is a high index of suspicion , can be missed since muscular pain , swelling , and tenderness may not be prominent or even absent .", "the definitive diagnosis should be made by laboratory tests including serum ck and urine myoglobin.5 in our case , the patient had advanced cognitive impairment making him unable to easily refer any symptom , and the diagnosis was made by the huge elevation of biochemical markers ( 6.5 times the upper normal value for ck , 8.5 times for myoglobin ) .", "overall , these findings allowed us to make the diagnosis of rhabdomyolysis with sufficient warranty .", "although clarithromycin associated rhabdomyolysis is mainly due to pharmacokinetic interactions , a direct muscle toxicity might be postuled.5,10"], "abstract_text": ["<S> rhabdomyolysis is a clinical and laboratory syndrome that is caused by various etiologies , involving the skeletal muscle . </S>", "<S> clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins . </S>", "<S> rhabdomyolysis related to macrolide  statin interaction has previously been described . to date , rhabdomyolysis induced by clarithromycin has been described in only one previous report . </S>", "<S> we describe the case of a 90-year - old caucasian male , admitted to the university hospital of pisa for dyspnea , who developed rhabdomyolysis associated with clarithromycin administration . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["adverse drug reactions ( adrs ) , including interactions , in older people are a common cause of admission to hospital1 and are also a common cause of disease in hospitalized patients .", "frail elderly patients appear to be particularly at risk of adrs . in some cases , this is because insufficient account is taken of the effect of age and frailty on the pharmacokinetic and pharmacodynamic of the drug , especially in relation to hepatic2 and renal elimination.3 although many adrs are recognized during the drug development process and before licensing , this is not always the case , especially when they are uncommon or rare.3 moreover , patients with severe cognitive impairment may not be able to refer symptoms related to an adr , making the recognition of this clinical condition a medical problem.4 rhabdomyolysis is a clinical and laboratory syndrome that is caused by various etiologies , involving the skeletal muscle.5 rhabdomyolysis is characterized by the leaking of myoglobin and other intracellular proteins and electrolytes into the circulation , and may be associated with a wide variety of diseases , injuries , medications , and toxins.5 it is important to recognize drug - induced rhabdomyolysis because clinical effects are usually reversible.4 rhabdomyolysis related to macrolide - statin interaction has previously been described.68 clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins like simvastatin and atorvastain.69 to date , rhabdomyolysis related to clarithromycin has been reported in only one previous case report.10 we report the case of a 90-year - old caucasian male , admitted to the university hospital of pisa for dyspnea , who experienced rhabdomyolysis related to clarithromycin treatment ."], ["a 90-year - old caucasian male was admitted to the university hospital of pisa for dyspnea .", "he had experienced dyspnea for 2 weeks , and showed signs of pneumonia after a chest x - ray was performed .", "the patient was affected by alzheimer s disease , in advanced phase , and did not take any home - medication as referred by his general physician . at admission in the emergency room ,", "clinical workup revealed mental confusion ( the relatives referred progressive cognitive impairment for several months ) , sinus tachycardia ( 115 beats per minute ) , basilar wheezes , and rhonchi .", "body temperature was 36.6c with normal systemic blood pressure ( 140/80 mmhg ) , diuresis , and blood oxygen saturation ( so2% 96.2% ) .", "routine laboratory exams , including complete blood cell count , thyroid hormones , liver ( aspartate aminotransferase and alanine aminotransferase ) and muscle ( creatine kinase [ ck ] ) enzymes , as well as the coagulation pattern , resulted within normal limits .", "serum brain natriuretic peptide was measured and a cardiac markers curve ( including myoglobin ) was also started , ruling out cardiogenic dyspnea .", "blood potassium and creatinine concentrations were 4.96 meq / l ( reference range 3.55 meq / l ) and 122.8 mol / l ( reference range 4590", "a diagnosis of acquired pneumonia was made and treatment with ceftriaxone ( 1 g four times daily , intravenous ) , hydration ( crystalloid 1.5 l / day ) , aerosol with salbutamol and oxitropium , o2 inhalation ( 3 l / minute ) , and dalteparin ( 2500 ui , subcutaneous ) was started .", "after the geriatric ward admission , clarithromycin ( 500 mg two times daily , intravenous ) was also started .", "on the second day of hospitalization , for continuing sinus tachycardia , in the absence of any evident causes ( fever , hypotension , arterial - venous fistula , thyrotoxicosis , suspected drug treatments , etc ) beta - blocker treatment ( bisoprolol 1.25 mg , daily ) was added and a cardiac markers curve was restarted ( figure 1 ) .", "the second point of the curve , almost 6 hours after the third clarithromycin dose , revealed a huge myoglobin increase ( 1144 ng / ml ; 8.5 times the upper normal range [ 28138 ng / ml ] ) without electrocardiogram modifications .", "u / l ; 6.5 times the upper normal range [ 38180 u / l ] ) along with a mild creatinine increase ( 152.9 mol / l ) , in the absence of liver enzyme alterations ( aspartate aminotransferase , alanine aminotransferase ) .", "urine color concomitantly changed toward dark brown and hemogasanalysis revealed a slight respiratory insufficiency type 1 with normal ph .", "a diagnosis of rhabdomyolysis was made and clarithromycin was stopped , while the concomitant therapy with ceftriaxone , bisoprolol , and aerosol with salbutamol and oxitropium was continued .", "the patient was immediately treated with intravenous crystalloid hypotonic solution ( 80 ml / hour ) and corticosteroid ( 40 mg methylprednisolone , intravenous ) .", "serum ck and myoglobin levels decreased within a few days and a chest x - ray showed significant improvement .", "the patient was discharged home on the sixth hospital day ( figure 1 ) with the diagnosis of iatrogenic rhabdomyolysis and acquired pneumonia of unknown etiology .", "indeed , serum and urinary antigen and antibody exams excluded the presence of legionella pneumophila , streptococcus pneumoniae , and mycoplasma pneumoniae while virus infection assessments were not performed ."], ["clarithromycin , like other macrolides , is an inhibitor of cyp450 3a4 , the major enzyme responsible for the metabolism of several drugs , in particular some statins as simvastatin and atorvastatin.3,69 rhabdomyolysis related to macrolide and statins interaction has previously been described ; however , rhabdomyolysis related to clarithromycin has been reported in only one previous case report.8,10 in our case , there was an evident temporal sequence between clarithromycin exposure - withdrawal and the onset - recovery from rhabdomyolysis biochemical signs , while other potential causes of muscle damage were reasonably excluded .", "accordingly , the naranjo probability scale confirmed a probable relationship ( score 6/9).13 a general physician and a nurse followed the patient at home , thus we are confident the home care was carried out in a correct manner , ruling out mistakes and accidental drug intake .", "moreover , an interaction with other concomitant medications was improbable considering the rapid resolution after clarithromycin withdrawal and the elimination pathways of the concomitant drugs ( both bisoprolol and ceftriaxone have hepatic and renal elimination routes).11,12 in addition , bisoprolol was administered at the minimal dose ( 1.25 mg ) and both bisoprolol and ceftriaxone , as single therapy , were never related to an adverse drug reaction like rhabdomyolysis .", "we ruled out infection as the possible cause of muscle damage considering the sequence of the events .", "in fact , the patient was admitted to the hospital for dyspnea developed at home since 2 weeks , while serum ck suddenly increased 2 days after hospital admission .", "if rhabdomyolysis had been caused by the infection we would have expected that serum ck increased earlier , thus being already evident at hospital admission . moreover ,", "the diagnosis of rhabdomyolysis , unless there is a high index of suspicion , can be missed since muscular pain , swelling , and tenderness may not be prominent or even absent .", "the definitive diagnosis should be made by laboratory tests including serum ck and urine myoglobin.5 in our case , the patient had advanced cognitive impairment making him unable to easily refer any symptom , and the diagnosis was made by the huge elevation of biochemical markers ( 6.5 times the upper normal value for ck , 8.5 times for myoglobin ) .", "overall , these findings allowed us to make the diagnosis of rhabdomyolysis with sufficient warranty .", "although clarithromycin associated rhabdomyolysis is mainly due to pharmacokinetic interactions , a direct muscle toxicity might be postuled.5,10"]]}
{"article_id": "PMC3466899", "article_text": ["supravalvar mitral ring is a circumferential ridge or membrane arising from the left atrial wall overlying the mitral valve and frequently attached to the mitral valve.1 variable in thickness and extent , the ring ranges from a thin membrane to a thick discrete fibrous ridge.2 the membranous variety may be difficult to detect because the membrane often adheres to the anterior mitral valve leaflet while remaining just proximal to the posterior mitral leaflet .", "adhesion to the valve may impair the opening of the leaflets , and this impairment may be the main mechanism of mitral valve inflow obstruction in some patients .", "in other patients , the ring may be large enough to protrude into the mitral valve inflow and cause obstruction .", "the supramitral ring may initially be incomplete and eccentric , allowing for an unobstructed flow through the mitral valve .", "however , turbulence can cause a progressive increase in the supravalvar membrane or ridge thickness , worsening mitral inflow obstruction.3 the same mechanism is responsible for the acquired variety of supravalvar mitral stenosis , which occurs after mitral annuloplasty for the repair of mitral regurgitation .", "we herein present a case of a supraventricular ring in a 17-year - old male , who presented with syncope .", "about 15 years ago , he had undergone a cardiac operation for ventricular septal defect ( vsd ) closure and mitral valve repair . in chest x - ray", "both lungs were clear , and the electrocardiogram result showed normal sinus rhythm , had undergone right bundle branch block , and right ventricular hypertrophy .", "transthoracic echocardiography , transesophageal echocardiography , and finally cardiac catheterization revealed ejection fraction of 5055% , pulmonary hypertension with pulmonary artery pressure ( pap ) of 80/40 mm hg , pulmonary capillary wedge pressure of 23 mm hg , gradient in the mitral valve plane of 40 mm hg , no residual vsd , no patent ductus arteriosus ( pda ) , bicuspid aortic valve , no aortic insufficiency , no aortic stenosis , no sub - aortic web , normal coronary arteries , mild mitral regurgitation , moderate to severe mitral stenosis , supra mitral valve ring , normal left ventricle size , moderate right ventricle enlargement , and moderate systolic dysfunction .", "there was a free floating high redundant obstructive membrane in the left atrium that was attached to the mid portion of the anterior mitral valve leaflet and also in the medial and base of the posterior mitral valve leaflet and large papillary muscles in the left ventricle ( figure 1 ) .", "it also extended just adjacent to the lower limb of the left atrium appendage mouth with a small orifice ( 6 mm ) in the lateral side and also multiple small fenestrations ( by 2 dimensional echocardiography ) , resulting in a severely restricted diastolic flow ( mean peak gradient = 9.3 mm hg ) .", "this web was not found in mri cine turbo - flash images acquired on a 1.5 t scanner ( magnetom vision - siemens ) in the four - chamber view ( 3a ) and the transverse plane , short - axis view ( figure 2 ) . the patient s ascending aorta , descending aorta , and aortic arch were tortuous with moderate narrowing of the descending aorta distal to the subclavian artery with a systolic gradient of about 30 mm hg , which was suggestive of insignificant coarctation of the aorta .", "after heparinization , extracorporeal circulation was established between the venae cava and the ascending aorta . following a median sternotomy ,", "there was a long supravalvar muscular band , which was resected ( figure 3 ) .", "supravalvar mitral ring rarely occurs as an isolated defect ; other congenital heart defects coexist in 90% of patients .", "the mitral valve itself is often abnormal and stenotic at the valvar or subvalvar level ; fusion of leaflets , a small valve orifice , and abnormal papillary muscles are common abnormalities .", "shone s complex is a combination of four congenital heart defects : supravalvar mitral ring , parachute mitral valve , subvalvar aortic stenosis , and aortic coarctation .", "other common associated lesions in patients with supravalvar mitral ring are ventricular septal defect ( vsd ) , pda , atrioventricular ( av ) canal defect , and tetralogy of fallout.4 uncommonly associated defects include atrial septal defect , cor triatriatum , left superior vena cava , unroofed coronary sinus , partial anomalous pulmonary venous drainage , pulmonary venous obstruction , double - orifice mitral valve , and wolff - parkinson - white syndrome.5 lesions such as transposition of the great arteries , atrioventricular discordance , and double - outlet right ventricle are occasionally complicated by a supravalvar left atrioventricular valvular ring .", "obstruction to the mitral inflow results from the reduced area of the mitral valve orifice .", "when clinically significant , a diastolic pressure difference occurs between the left atrium and the left ventricle . left atrial and pulmonary venous pressures increase , leading to exudation of fluid into the pulmonary interstitial , which increases lung stiffness .", "breathlessness and tachypnea are secondary to the interstitial edema and diminished pulmonary compliance.6 in severe cases , frank pulmonary edema can occur.7 an associated atrial septal defect may decompress the left atrium , reducing or masking the severity of the mitral valve obstruction .", "associated lesions such as vsd or pda , which increase the left ventricle output , exacerbate the manifestations of mitral inflow obstruction.8 conversely , a supravalvar mitral ring may be difficult to detect in conditions with a diminished pulmonary blood flow such as tetralogy of fallot.9 persistently elevated pulmonary venous hypertension leads to pulmonary arterial hypertension , rise in pulmonary vascular resistance , and eventual failure of the right ventricle and tricuspid regurgitation .", "it also deserves of note that predilection for race , age , and sex has not been found yet .", "treatment is the surgical resection of the web , but balloon dilatation is reported to have been successfully performed under fluoroscopic and transesophageal echocardiographic guidance by some authors.10", "preoperative identification of a supravalvular mitral ring is the target for obtaining good surgical results . for the identification of this congenital heart disease ,"], "abstract_text": ["<S> supravalvar mitral ring is a rare congenital heart defect of surgical importance . </S>", "<S> the condition is characterized by an abnormal ridge of the connective tissue on the atrial side of the mitral valve . </S>", "<S> it often substantially obstructs the mitral valve inflow . </S>", "<S> we herein introduce a case of a supravalvar mitral ring in a 17-year - old male , who was admitted to our hospital with cardiac syncope . </S>", "<S> he had undergone a cardiac operation for ventricular septal defect ( vsd ) closure and mitral valve repair 15 years before . </S>", "<S> transthoracic echocardiography , transesophageal echocardiography , and finally cardiac catheterization revealed a neglected supravalvular mitral ring . </S>", "<S> the ring was resected in a second operation , and the patient was discharged from the hospital symptom free . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["supravalvar mitral ring is a circumferential ridge or membrane arising from the left atrial wall overlying the mitral valve and frequently attached to the mitral valve.1 variable in thickness and extent , the ring ranges from a thin membrane to a thick discrete fibrous ridge.2 the membranous variety may be difficult to detect because the membrane often adheres to the anterior mitral valve leaflet while remaining just proximal to the posterior mitral leaflet .", "adhesion to the valve may impair the opening of the leaflets , and this impairment may be the main mechanism of mitral valve inflow obstruction in some patients .", "in other patients , the ring may be large enough to protrude into the mitral valve inflow and cause obstruction .", "the supramitral ring may initially be incomplete and eccentric , allowing for an unobstructed flow through the mitral valve .", "however , turbulence can cause a progressive increase in the supravalvar membrane or ridge thickness , worsening mitral inflow obstruction.3 the same mechanism is responsible for the acquired variety of supravalvar mitral stenosis , which occurs after mitral annuloplasty for the repair of mitral regurgitation .", "we herein present a case of a supraventricular ring in a 17-year - old male , who presented with syncope ."], ["about 15 years ago , he had undergone a cardiac operation for ventricular septal defect ( vsd ) closure and mitral valve repair . in chest x - ray", "both lungs were clear , and the electrocardiogram result showed normal sinus rhythm , had undergone right bundle branch block , and right ventricular hypertrophy .", "transthoracic echocardiography , transesophageal echocardiography , and finally cardiac catheterization revealed ejection fraction of 5055% , pulmonary hypertension with pulmonary artery pressure ( pap ) of 80/40 mm hg , pulmonary capillary wedge pressure of 23 mm hg , gradient in the mitral valve plane of 40 mm hg , no residual vsd , no patent ductus arteriosus ( pda ) , bicuspid aortic valve , no aortic insufficiency , no aortic stenosis , no sub - aortic web , normal coronary arteries , mild mitral regurgitation , moderate to severe mitral stenosis , supra mitral valve ring , normal left ventricle size , moderate right ventricle enlargement , and moderate systolic dysfunction .", "there was a free floating high redundant obstructive membrane in the left atrium that was attached to the mid portion of the anterior mitral valve leaflet and also in the medial and base of the posterior mitral valve leaflet and large papillary muscles in the left ventricle ( figure 1 ) .", "it also extended just adjacent to the lower limb of the left atrium appendage mouth with a small orifice ( 6 mm ) in the lateral side and also multiple small fenestrations ( by 2 dimensional echocardiography ) , resulting in a severely restricted diastolic flow ( mean peak gradient = 9.3 mm hg ) .", "this web was not found in mri cine turbo - flash images acquired on a 1.5 t scanner ( magnetom vision - siemens ) in the four - chamber view ( 3a ) and the transverse plane , short - axis view ( figure 2 ) . the patient s ascending aorta , descending aorta , and aortic arch were tortuous with moderate narrowing of the descending aorta distal to the subclavian artery with a systolic gradient of about 30 mm hg , which was suggestive of insignificant coarctation of the aorta .", "after heparinization , extracorporeal circulation was established between the venae cava and the ascending aorta . following a median sternotomy ,", "there was a long supravalvar muscular band , which was resected ( figure 3 ) ."], ["supravalvar mitral ring rarely occurs as an isolated defect ; other congenital heart defects coexist in 90% of patients .", "the mitral valve itself is often abnormal and stenotic at the valvar or subvalvar level ; fusion of leaflets , a small valve orifice , and abnormal papillary muscles are common abnormalities .", "shone s complex is a combination of four congenital heart defects : supravalvar mitral ring , parachute mitral valve , subvalvar aortic stenosis , and aortic coarctation .", "other common associated lesions in patients with supravalvar mitral ring are ventricular septal defect ( vsd ) , pda , atrioventricular ( av ) canal defect , and tetralogy of fallout.4 uncommonly associated defects include atrial septal defect , cor triatriatum , left superior vena cava , unroofed coronary sinus , partial anomalous pulmonary venous drainage , pulmonary venous obstruction , double - orifice mitral valve , and wolff - parkinson - white syndrome.5 lesions such as transposition of the great arteries , atrioventricular discordance , and double - outlet right ventricle are occasionally complicated by a supravalvar left atrioventricular valvular ring .", "obstruction to the mitral inflow results from the reduced area of the mitral valve orifice .", "when clinically significant , a diastolic pressure difference occurs between the left atrium and the left ventricle . left atrial and pulmonary venous pressures increase , leading to exudation of fluid into the pulmonary interstitial , which increases lung stiffness .", "breathlessness and tachypnea are secondary to the interstitial edema and diminished pulmonary compliance.6 in severe cases , frank pulmonary edema can occur.7 an associated atrial septal defect may decompress the left atrium , reducing or masking the severity of the mitral valve obstruction .", "associated lesions such as vsd or pda , which increase the left ventricle output , exacerbate the manifestations of mitral inflow obstruction.8 conversely , a supravalvar mitral ring may be difficult to detect in conditions with a diminished pulmonary blood flow such as tetralogy of fallot.9 persistently elevated pulmonary venous hypertension leads to pulmonary arterial hypertension , rise in pulmonary vascular resistance , and eventual failure of the right ventricle and tricuspid regurgitation .", "it also deserves of note that predilection for race , age , and sex has not been found yet .", "treatment is the surgical resection of the web , but balloon dilatation is reported to have been successfully performed under fluoroscopic and transesophageal echocardiographic guidance by some authors.10"], ["preoperative identification of a supravalvular mitral ring is the target for obtaining good surgical results . for the identification of this congenital heart disease ,"]]}
{"article_id": "PMC4263394", "article_text": ["in the recent millennium , the constant trend of change in the demands of the community as well as transforming the trend of knowledge production has highlighted the necessity for researchers to adopt a more comprehensive approach .", "increasingly , many academic disciplines are utilizing qualitative research ( qr ) as the qualitative method investigating the why and how of the process of a developed concept ( 1 , 2 ) .", "qualitative research is sometimes defined as interpretive research , and as interpretations can be incorrect or biased , the findings may be controversial ( 3 ) . however , qualitative research is not only useful as the first stage of quantitative research , but can also play a key role in  validating it or in providing a different viewpoint on the same social phenomena ( 4 ) .", "qualitative studies tend to use methods that result in text production rather than numerical outputs .", "given that the researcher is considered to be the research instrument , and the plan of inquiry needs to be developed and altered as the study progresses , a qualitative researcher can not depend upon traditional approaches to address certain concerns such as bias and credibility .", "therefore , learning from a series of mistakes is often considered an integral part of qualitative research ( 5 , 6 ) . in this study ,", "a literature review was carried out in international electronic databases including pubmed , web of sciences , cumulative index to nursing and allied health literature ( cinahl ) , scopus , ebsco , embase and science direct without any time limitation , using the search terms  qualitative research  , ", "these keywords were also searched on national electronic databases including scientific information database ( sid ) , iran medex and medical articles library ( medlib ) using the same strategy .", "authors of the present article endeavor to shine a light on the ethical issues affecting researchers and propose strategies to face the ethical challenges of qualitative studies , so as to provide applicable and trustworthy outcomes", ". this could be the basis for the formulation of specific ethical guidelines in this regard .", "up to the 1970s , qualitative research was solely employed by anthropologists and sociologists . during the 1970s and 1980s ,", "however , it was favored by various disciplines and experts of different branches of science and humanity such as health care , psychology , nursing , management , political science , education , and communication studies ( 2 , 7 ) .", "qualitative research has been conducted in the field of nursing in order to identify , describe and explain related concepts , experiences and phenomena and to develop the nursing knowledge .", ", qualitative research has been performed to achieve the concepts of patient care and other main perceptions in the nursing profession .", "qualitative studies provide nurses with sensitivity to the lived experiences of individuals from different nursing care aspects ( 4 , 8) .", "in the case of nurses who perform qualitative research , ethical issues are raised when the nurse - patient relationship in the research area leads to some degree of therapeutic communication for the participants ( 9 ) .", "thus , nurse researchers must be aware of the impact of the questioning on the participants , and in order to decrease such harmful effects on human subjects , the  reflexive approach  is recommended ( 10 ) . in qualitative studies researchers", "are often required to clarify their role in the research process ( 11 ) . in the qr procedure", "the researcher is involved in all stages of the study from defining a concept to design , interview , transcription , analysis , verification and reporting the concepts and themes .", "therefore , whenever instruments are involved in qualitative research , a human being will be an integral part of the process ( 12 ) .", "it is argued that humans have increasingly become the  instrument of choice  for naturalistic research due to certain characteristics : they are highly responsive to environmental stimuli , have the ability to interact with the situation , pull together different pieces of information at multiple levels simultaneously , and perceive situations holistically ; moreover , they are able to process findings the instant they become available , can present immediate feedback , and feel unusual responses .", "nevertheless , researchers need to improve the abilities that make them appropriate human instruments and consequently , their interpersonal skills are of major importance in natural settings and study processes ( table 1 ) ( 13 , 14 ) .", "the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .", "ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .", "in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term ", "confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .", "the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .", "when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .", "it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .", "informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .", "the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .", "clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .", "informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .", "many people consider it necessary to participate in research that their peers , community and/or society may benefit from .", "therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) .", "the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .", "therefore , researchers can interpret people s experiences because they are involved in human activities .", "the principle of  no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects .", "obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .", "there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .", "researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .", "it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .", "researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .", "furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .", "therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .", "overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 ) . in qualitative research ,", "data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .", "the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .", "the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .", "data collection needs to be as overt as possible , and findings should be recorded .", "although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research , the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods , ethnography has singular characteristics .", "when a researcher aims to study the culture of certain people , living amongst them is inevitable .", "this method of collecting data is a subject of debate from an ethical point of view .", "participants should always be aware of the information that has been obtained and is being recorded , and consent to it .", "sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .", "the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .", "for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .", "ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .", "researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35  37 ) . in order to gain explicit data ,", "these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .", "researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .", "the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .", "interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .", "it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40  42 ) .", "the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .", "ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .", "in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term ", "confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .", "the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .", "when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .", "it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .", "informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .", "the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .", "clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .", "informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .", "many people consider it necessary to participate in research that their peers , community and/or society may benefit from .", "therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) .", "the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .", "therefore , researchers can interpret people s experiences because they are involved in human activities .", "the principle of  no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects . obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .", "there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .", "researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .", "it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .", "researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .", "furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .", "therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .", "overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 )", "in qualitative research , data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .", "the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .", "the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .", "data collection needs to be as overt as possible , and findings should be recorded . although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research", ", the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods ,", "ethnography has singular characteristics . when a researcher aims to study the culture of certain people , living amongst them is inevitable .", "this method of collecting data is a subject of debate from an ethical point of view .", "participants should always be aware of the information that has been obtained and is being recorded , and consent to it .", "sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .", "the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .", "for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .", "ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .", "researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35  37 ) . in order to gain explicit data , ethnographers need to know the role of instrument details .", "these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .", "researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .", "the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .", "interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .", "it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40  42 ) .", "it is argued that qualitative research that deals with sensitive topics in depth can pose emotional and other risks to both participants and researchers .", "clear protocols for dealing with distress should be in place so that both parties involved in research can use them if necessary .", "it is not usually easy to predict what topics are likely to lead to distress , and researchers should therefore receive sufficient training in predicting traumatic situations .", "preventive measures for researchers who carry out sensitive qualitative studies should include official arrangements for a peer support program consisting of a list of researchers who are involved , or a constellation of researcher support activities aiming at improving psychological fitness in the form of a professional confidence building module .", "other such measures include offering adequate supervision to provide opportunities for self - development and self - care , and facilitating the process of self - reflection and self - monitoring .", "strategies for emotional distancing need to be considered and adopted if the research topic or participants have the potential to be emotionally challenging .", "an appropriate planning should be in place before the commencement of the fieldwork , and it must be perfectly clear how the study should be conducted and what level of relationship development is necessary .", "measures must also be taken so that levels of self - disclosure , objective displays of emotion during the interviews , and strategies to end the relationships are well defined and communicated .", "one of the most prominent tasks of qualitative researchers is to minimize the flaws in observation and endeavor to gain truthful knowledge .", "therefore , it is necessary for researchers to continuously update their investigation skills in terms of methodology and find novel techniques to better carry out studies in the field of health and sociology . as explained before ,", "qualitative research is carried out in natural settings , which requires researchers to work in close collaboration with other members of the team and under direct supervision to discuss and resolve issues as they arise .", "therefore , development of practical strategies and communicating them to researchers can be of great benefit and assist them in conducting more perceptive qualitative studies .", "it is noteworthy that such research should be directed towards making a difference in people s lives , improving care delivery in different settings and at all levels , and providing a framework for health sciences without any ethical disturbances . as a result of the extensive body of research in the field of medical sciences ,", "research ethics committees are formed to provide independent advice to participants , researchers , funders / sponsors and healthcare organizations on the extent to which research proposals comply with universally endorsed ethical standards . in the history of social and medical science , there have been a few research studies that seriously injured people , and many more in which their welfare was not sufficiently protected .", "to return to the matter of privacy , the researcher should not rely solely on the informant to identify possible intrusion , but needs to work at anticipating it in advance .", "confidentiality does not necessarily preclude intrusion , as anonymity by itself is not enough to protect a person s privacy or prevent disclosure of personal issues .", "investigators should refrain from soliciting private information that is not closely related to the research question .", "considering the aforementioned challenges , it is recommended to conduct further research in order to provide meticulous and explicit ethical protocols , guidelines and codes with respect to qualitative studies ."], "abstract_text": ["<S> considering the nature of qualitative studies , the interaction between researchers and participants can be ethically challenging for the former , as they are personally involved in different stages of the study . </S>", "<S> therefore , formulation of specific ethical guidelines in this respect seems to be essential . </S>", "<S> the present paper aimed to discuss the necessity to develop explicit guidelines for conducting qualitative studies with regard to the researchers role . for this purpose , </S>", "<S> a literature review was carried out in domestic and international databases by related keywords.health care providers who carry out qualitative research have an immense responsibility . </S>", "<S> as there is no statistical analysis in qualitative studies , the researcher has to both evaluate what he or she observes and to interpret it . </S>", "<S> providing researchers with the necessary skills and applying stringent supervision can lead to better extraction of reliable information from qualitative studies . </S>", "<S> this article presents a debate in order to illustrate how researchers could cover the ethical challenges of qualitative studies and provide applicable and trustworthy outcomes.researchers face ethical challenges in all stages of the study , from designing to reporting . </S>", "<S> these include anonymity , confidentiality , informed consent , researchers potential impact on the participants and vice versa . </S>", "<S> it seems of paramount importance that health care providers , educators and clinicians be well informed of all the different aspects of their roles when acting as qualitative researchers . </S>", "<S> hence , these adroit roles need to be well defined , and the use of practical guidelines and protocols in all stages of qualitative studies should be encouraged . </S>"], "labels": null, "section_names": ["Introduction", "An overview on qualitative research in health care", "Role of researchers in qualitative studies", "Ethical challenges in qualitative studies:", "The researcher-participant relationship", "Research design", "Data gathering and data analysis", "Conclusion"], "sections": [["in the recent millennium , the constant trend of change in the demands of the community as well as transforming the trend of knowledge production has highlighted the necessity for researchers to adopt a more comprehensive approach .", "increasingly , many academic disciplines are utilizing qualitative research ( qr ) as the qualitative method investigating the why and how of the process of a developed concept ( 1 , 2 ) .", "qualitative research is sometimes defined as interpretive research , and as interpretations can be incorrect or biased , the findings may be controversial ( 3 ) . however , qualitative research is not only useful as the first stage of quantitative research , but can also play a key role in  validating it or in providing a different viewpoint on the same social phenomena ( 4 ) .", "qualitative studies tend to use methods that result in text production rather than numerical outputs .", "given that the researcher is considered to be the research instrument , and the plan of inquiry needs to be developed and altered as the study progresses , a qualitative researcher can not depend upon traditional approaches to address certain concerns such as bias and credibility .", "therefore , learning from a series of mistakes is often considered an integral part of qualitative research ( 5 , 6 ) . in this study ,", "a literature review was carried out in international electronic databases including pubmed , web of sciences , cumulative index to nursing and allied health literature ( cinahl ) , scopus , ebsco , embase and science direct without any time limitation , using the search terms  qualitative research  , ", "these keywords were also searched on national electronic databases including scientific information database ( sid ) , iran medex and medical articles library ( medlib ) using the same strategy .", "authors of the present article endeavor to shine a light on the ethical issues affecting researchers and propose strategies to face the ethical challenges of qualitative studies , so as to provide applicable and trustworthy outcomes", ". this could be the basis for the formulation of specific ethical guidelines in this regard ."], ["up to the 1970s , qualitative research was solely employed by anthropologists and sociologists . during the 1970s and 1980s ,", "however , it was favored by various disciplines and experts of different branches of science and humanity such as health care , psychology , nursing , management , political science , education , and communication studies ( 2 , 7 ) .", "qualitative research has been conducted in the field of nursing in order to identify , describe and explain related concepts , experiences and phenomena and to develop the nursing knowledge .", ", qualitative research has been performed to achieve the concepts of patient care and other main perceptions in the nursing profession .", "qualitative studies provide nurses with sensitivity to the lived experiences of individuals from different nursing care aspects ( 4 , 8) ."], ["in the case of nurses who perform qualitative research , ethical issues are raised when the nurse - patient relationship in the research area leads to some degree of therapeutic communication for the participants ( 9 ) .", "thus , nurse researchers must be aware of the impact of the questioning on the participants , and in order to decrease such harmful effects on human subjects , the  reflexive approach  is recommended ( 10 ) . in qualitative studies researchers", "are often required to clarify their role in the research process ( 11 ) . in the qr procedure", "the researcher is involved in all stages of the study from defining a concept to design , interview , transcription , analysis , verification and reporting the concepts and themes .", "therefore , whenever instruments are involved in qualitative research , a human being will be an integral part of the process ( 12 ) .", "it is argued that humans have increasingly become the  instrument of choice  for naturalistic research due to certain characteristics : they are highly responsive to environmental stimuli , have the ability to interact with the situation , pull together different pieces of information at multiple levels simultaneously , and perceive situations holistically ; moreover , they are able to process findings the instant they become available , can present immediate feedback , and feel unusual responses .", "nevertheless , researchers need to improve the abilities that make them appropriate human instruments and consequently , their interpersonal skills are of major importance in natural settings and study processes ( table 1 ) ( 13 , 14 ) ."], ["the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .", "ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .", "in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term ", "confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .", "the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .", "when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .", "it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .", "informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .", "the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .", "clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .", "informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .", "many people consider it necessary to participate in research that their peers , community and/or society may benefit from .", "therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) .", "the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .", "therefore , researchers can interpret people s experiences because they are involved in human activities .", "the principle of  no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects .", "obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .", "there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .", "researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .", "it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .", "researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .", "furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .", "therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .", "overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 ) . in qualitative research ,", "data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .", "the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .", "the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .", "data collection needs to be as overt as possible , and findings should be recorded .", "although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research , the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods , ethnography has singular characteristics .", "when a researcher aims to study the culture of certain people , living amongst them is inevitable .", "this method of collecting data is a subject of debate from an ethical point of view .", "participants should always be aware of the information that has been obtained and is being recorded , and consent to it .", "sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .", "the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .", "for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .", "ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .", "researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35  37 ) . in order to gain explicit data ,", "these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .", "researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .", "the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .", "interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .", "it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40  42 ) ."], ["the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .", "ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .", "in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term ", "confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .", "the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .", "when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .", "it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .", "informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .", "the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .", "clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .", "informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .", "many people consider it necessary to participate in research that their peers , community and/or society may benefit from .", "therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) ."], ["the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .", "therefore , researchers can interpret people s experiences because they are involved in human activities .", "the principle of  no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects . obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .", "there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .", "researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .", "it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .", "researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .", "furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .", "therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .", "overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 )"], ["in qualitative research , data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .", "the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .", "the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .", "data collection needs to be as overt as possible , and findings should be recorded . although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research", ", the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods ,", "ethnography has singular characteristics . when a researcher aims to study the culture of certain people , living amongst them is inevitable .", "this method of collecting data is a subject of debate from an ethical point of view .", "participants should always be aware of the information that has been obtained and is being recorded , and consent to it .", "sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .", "the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .", "for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .", "ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .", "researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35  37 ) . in order to gain explicit data , ethnographers need to know the role of instrument details .", "these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .", "researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .", "the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .", "interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .", "it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40  42 ) ."], ["it is argued that qualitative research that deals with sensitive topics in depth can pose emotional and other risks to both participants and researchers .", "clear protocols for dealing with distress should be in place so that both parties involved in research can use them if necessary .", "it is not usually easy to predict what topics are likely to lead to distress , and researchers should therefore receive sufficient training in predicting traumatic situations .", "preventive measures for researchers who carry out sensitive qualitative studies should include official arrangements for a peer support program consisting of a list of researchers who are involved , or a constellation of researcher support activities aiming at improving psychological fitness in the form of a professional confidence building module .", "other such measures include offering adequate supervision to provide opportunities for self - development and self - care , and facilitating the process of self - reflection and self - monitoring .", "strategies for emotional distancing need to be considered and adopted if the research topic or participants have the potential to be emotionally challenging .", "an appropriate planning should be in place before the commencement of the fieldwork , and it must be perfectly clear how the study should be conducted and what level of relationship development is necessary .", "measures must also be taken so that levels of self - disclosure , objective displays of emotion during the interviews , and strategies to end the relationships are well defined and communicated .", "one of the most prominent tasks of qualitative researchers is to minimize the flaws in observation and endeavor to gain truthful knowledge .", "therefore , it is necessary for researchers to continuously update their investigation skills in terms of methodology and find novel techniques to better carry out studies in the field of health and sociology . as explained before ,", "qualitative research is carried out in natural settings , which requires researchers to work in close collaboration with other members of the team and under direct supervision to discuss and resolve issues as they arise .", "therefore , development of practical strategies and communicating them to researchers can be of great benefit and assist them in conducting more perceptive qualitative studies .", "it is noteworthy that such research should be directed towards making a difference in people s lives , improving care delivery in different settings and at all levels , and providing a framework for health sciences without any ethical disturbances . as a result of the extensive body of research in the field of medical sciences ,", "research ethics committees are formed to provide independent advice to participants , researchers , funders / sponsors and healthcare organizations on the extent to which research proposals comply with universally endorsed ethical standards . in the history of social and medical science , there have been a few research studies that seriously injured people , and many more in which their welfare was not sufficiently protected .", "to return to the matter of privacy , the researcher should not rely solely on the informant to identify possible intrusion , but needs to work at anticipating it in advance .", "confidentiality does not necessarily preclude intrusion , as anonymity by itself is not enough to protect a person s privacy or prevent disclosure of personal issues .", "investigators should refrain from soliciting private information that is not closely related to the research question .", "considering the aforementioned challenges , it is recommended to conduct further research in order to provide meticulous and explicit ethical protocols , guidelines and codes with respect to qualitative studies ."]]}
{"article_id": "PMC5055980", "article_text": ["since the discovery of penicillin in 1928 by a scottish scientist alexander fleming followed by the release of many other earlier drugs onto the market to treat infection , the development of drug resistance in various sectors including aquaculture has been reported [ 13 ] .", "the misuse and abuse of the antimicrobial drugs are among the important factors that have contributed to the rise of resistant microbes around the world .", "antibiotics , which have saved millions of lives and were also known as miracle drug in the past , are no longer the ultimate way for the treatment of infections because bacteria have continued to develop multiple resistance towards many different types or classes of the drugs .", "antimicrobial agents have been widely used in fish farming for either therapeutic , prophylactic , or other purposes .", "the antibiotics are normally used to increase growth as well as feed efficiency in the animals . however , some of the antibiotics have been frequently used in both veterinary and human medicine such as sulfonamides , chloramphenicol , tetracycline , nitrofurans , oxytetracycline , neomycin , erythromycin , streptomycin , prefuran , and enrofloxacin .", "the evolution of bacteria towards antibiotic resistance has been accelerated distinctly by selective pressure due to inappropriate and overuse of the antibiotics [ 3 , 9 ] . in the efforts to cope with this problem", ", scientists have accelerated the search for alternative antimicrobial agents by screening many potential sources including medicinal plants [ 10 , 11 ] and microbes [ 12 , 13 ] .", "aquaculture is an important sector in the agriculture industry and is rapidly growing to meet the world 's demands for protein source .", "this sector is challenged with the diverse type of diseases and bacterial infections ; and antibiotics are an excellent tool to circumvent the problem [ 14 , 15 ] . the presence of bacteria with multiple antibiotic resistance found in food products has become a threat to public health as there is potential that the carried or acquired genes are transferred to other bacteria of clinical significance [ 1618 ] . some antibiotics which are commonly used in food - producing animals are also used in human medicine , reducing the antibiotic 's efficiency when treating infections and increasing the morbidity and mortality associated with diseases .", "the resistance limits the choice of antibiotics for the disease treatment [ 4 , 16 , 19 , 20 ] .", "the use of antibiotics needs to be monitored from time to time to evaluate the emergence and spread of bacterial resistance towards antimicrobial agents [ 5 , 21 ] .", "there is limited data on the antibiotic resistance of bacteria in fish and other cultured organisms sampled directly from fish farms as well as the aquaculture environment .", "sampling was carried out at aquaculture farms located at selected districts in sarawak , malaysian borneo , including kuching , bintulu , limbang , miri , and sampadi ( lundu ) .", "three types of samples were collected which were the sediment , water , and cultured species .", "cultured species refers to fish or shrimp . in kuching , bintulu , and miri ,", "the cultured organisms collected were the fish while in limbang and sampadi ( lundu ) , the cultured organisms collected were shrimps .", "the isolation , designation , and identification of the isolates were carried out as reported by kathleen and coworkers earlier .", "ninety - four bacterial isolates from 17 different genera   were assessed for their susceptibility to different antibiotics utilizing the disk diffusion method according to method described by clinical and laboratory standards institutes ( clsi )   on mueller - hinton agar ( mha ) .", "the bacterial groups and the antibiotics tested are listed in table 1 . briefly , fresh bacterial culture with 0.5 mcfarland turbidity was swabbed onto the mha surface using sterile cotton buds .", "commercial antimicrobial discs ( oxoid , uk ) were evenly embedded onto the inoculated agar incubated at 37c for 18 to 24 hours .", "escherichia coli strain from american type culture collection ( atcc ) 25922 was used as control .", "the diameter of complete inhibition zone formed around the antibiotic discs was measured to the nearest whole millimeter using standardized ruler .", "the results obtained were analyzed as resistant or susceptible according to standard interpretative table by clsi   and bonnet .", "multiple antibiotic resistance ( mar ) index was then determined for each isolate by dividing the number of antibiotics to which an isolate is resistant with the total number of antibiotics tested .", "the mar index is an indicator to identify the risk contamination that is potentially hazardous to human .", "calculated value of more than 0.2 indicates that the isolates were isolated from high risk sources .", "in this study , commonly used antibiotics in both veterinary and human medicine were selected for the antibiotic susceptibility testing .", "the antibiotic selection also depends on the bacterial genera because different bacterial genera require different classes of antibiotics for optimal antibacterial activity .", "some of the antibiotics were not tested in this study because some bacterial genera are naturally resistant to certain classes of antibiotics ; hence the antibiotics were excluded from the analysis .", "the antibiotic profile of the bacterial isolates from the aquaculture fish and shrimps and their environment is shown in table 1 .", "the bacterial isolates showing the top five highest percentages of resistant were towards streptomycin ( 85% , n = 20 ) , followed by ampicillin ( 56.8% , n = 74 ) , penicillin ( 47.1% , n = 51 ) , erythromycin ( 43.1% , n = 51 ) , and cephalotin ( 42.3% , n = 71 ) . the bacterial isolates showing the top five highest percentages of susceptible were towards gentamicin ( 1.1% , n = 90 ) , followed by tobramycin ( 2.2% , n = 90 ) , chloramphenicol ( 4.0% , n = 75 ) , norfloxacin ( 5% , n = 80 ) , and amikacin ( 5.6% , n = 90 ) . the other bacterial isolates and their percentage of resistant are shown in table 1 . in this study , the antibiotic resistant patterns for all isolates were also determined to monitor the spread of antibiotic resistance .", "sixty - one different antibiotic resistance patterns were observed among the isolates through this study ( data not shown ) .", "the resistance patterns were highly variable ; 20.2% ( n = 94 ) isolates have no resistance towards any antibiotics tested , 16% ( n = 94 ) isolates were resistant to only one antibiotic , and 63.8% ( n = 94 ) isolates were resistant to multiple antibiotics .", ". isolated from water in limbang aquaculture farm was resistant towards 12 out of 19 antibiotics tested , which was the highest amount of antibiotic resistance observed in this study .", "most isolates ( 53.2% , n = 94 ) possess distinctive pattern . however , there are also patterns ( 11 patterns ) shared by 2 or more bacteria isolates .", "the pattern shared by most isolates ( 19 isolates , n = 94 ) is 0% resistance ( mar index equal to 0 ) .", "there is at least an isolate with 0% resistance towards all antibiotics tested in all sampling locations except for bintulu .", "there is difference in the resistance pattern for bacteria isolated from water , sediment , and the cultured organisms .", "bacteria that were isolated from the same pond and same source of origin ( sediment , water , or cultured organisms ) possess different antibiotic patterns .", "the antibiotic resistant patterns of the bacterial genera are more influenced by the location from where the isolates were isolated rather than their genus .", "this analysis has been used to group the different sources from where the bacteria were recovered using the frequency of antibiotics resistance .", "isolates with mar < 0.2 were determined as isolates recovered from low risk sources of contamination while isolates with mar > 0.2 were from high risk sources . in this study ,", "analysis on overall isolates regardless of sampling location revealed that 63.1% ( n = 94 ) isolates belong to group mar < 0.2 while 36.9% ( n = 94 ) isolates belong to group mar > 0.2 .", "bintulu ( btl ) has the highest percentage ( 73.3% , n = 16 ) of bacteria isolated from high antibiotic - contaminated sources while sampadi ( spd ) has the lowest percentage ( 11.7% , n = 17 ) .", "assessment of the antibiotic resistance among aquaculture bacteria against antimicrobial agents is important for update on the bacterial antibiotic resistance patterns .", "it is part of a surveillance system aiming at monitoring emerging antibiotic resistant bacteria and their widespread .", "isolation of antibiotic resistant bacteria from aquaculture products and aquaculture environment indicates the health risk associated with the aquaculture .", "there had been reports on detection of antibiotic resistance genes in bacteria isolated from aquaculture products that can be transferred to human microbiota .", "this matter is becoming critical if the resistance genes are transferrable to human pathogens . providing effective treatment towards this infection becomes a problem to the medical practitioners as the choice of antibiotics is limited .", "thus , any source of antibiotic resistant bacteria must be carefully monitored . in antibiotic resistance analysis", ", the history of antibiotic application in particular area is reflected by the percentage of bacterial resistance to antibiotics .", "the frequency of antibiotics usage is related to the level of resistance among bacteria [ 2 , 29 ] . in this present study ,", "similarly , lim and kasing   and hatha et al .   in their respective studies observed that almost none of the bacteria tested were resistant to gentamicin and chloramphenicol .", "low frequency of antibiotic resistant bacteria may indicate the less activity associated with the contamination of antibiotics in the area .", "the use of chloramphenicol in aquaculture has been banned in certain countries including malaysia , korea , and japan since 1983 .", "this is because of the adverse effect of chloramphenicol in humans , even at very low dosage , which can cause other side effects like severe or fatal blood problems .", "the problems associated with blood are like anemia and  grey syndrome ,  a syndrome of cyanosis and cardiovascular collapse , which occurs particularly in newborn babies .", "this poses risks to the workers handling the products containing this antibiotic [ 5 , 31 ] .", "banning of antibiotics has aided in reducing the number of antibiotic resistant bacteria in an environment .", "reported the significant reduction in the frequency of vancomycin - resistant enterococci from broiler after the banning of vancomycin in denmark in 1995 .", "high percentage of streptomycin , ampicillin , and penicillin g resistance was observed in this study .", "similarly , high ampicillin and streptomycin resistance were also observed by zhang et al .   in their study on antibiotic resistance detection in e. coli strains isolated from two different aquaculture systems in south china .", "also recorded high resistance of ampicillin . in another study , identified isolates from mangrove soil in malaysia were 100% resistant towards ampicillin and penicillin while 77.8% of the isolates were resistant towards streptomycin .", "a study carried out by akinbowale et al .   recorded 54.8% ampicillin resistance of aquaculture bacteria in australia .", "in contrast with the results in this study , low resistance of streptomycin ( 21.2% ) and ampicillin ( 6.1% ) was observed in a study on 33 marine bacteria isolates by you et al . .", "in this study , farmers in the aquaculture farms where the sampling was carried out stated that there was no history of utilization of antibiotics in their farms . despite the absence of antibiotics as medicines or in feeds ,", "high resistance was observed to commonly used antibiotics such as streptomycin , ampicillin , and penicillin .", "high resistance of ampicillin and streptomycin in this study and other researches was not surprising as these antibiotics were among the first antibiotics introduced since the discovery of penicillin .", "although antibiotic usage in the studied farms has been stopped decades ago , antibiotic contamination is still possible as there may still be residues of antibiotics left in the environment .", "bacteria isolated from the sediment of the aquaculture pond may have acquired antibiotic resistance characteristics through unconsumed foods and the cultured organism 's faeces that contain the remaining antibiotics [ 3941 ] .", "the unconsumed foods and faeces will be deposited in the sediment and the composition of sediment microbiota will be altered due to selective pressure .", "fish feeds were a possible reservoir for antibiotic resistant genes in the farm sediments . in this present study ,", "bintulu ( btl ) recorded the highest percentage ( 73.3% , n = 16 ) of bacteria isolated from high antibiotic - contaminated sources ( mar > 0.2 ) while sampadi ( spd ) aquaculture farm recorded the least number ( 11.7% , n = 17 ) of mar index > 0.2 isolates .", "the aquaculture farm is located at an area that has many agriculture activities ( e.g. , pig farming , duck farming , and dragon fruit cultivation ) surrounding it .", "there is the possibility that antibiotics from the animal feeds or medications were absorbed into the sediment causing bacterial selection in the nearby environment .", "multiple antibiotic resistant bacteria might have travelled through water from these agriculture activities to the aquaculture ponds .", "buschmann et al .   in their study suggested that antibiotic resistant bacteria in mariculture farm may be transported by water current which flows from surrounding farms that utilize antibiotics excessively .", "antibiotic resistance pattern may vary depending on the geographical locations and selective pressure [ 43 , 44 ] and these patterns change rapidly from time to time .", "the different patterns exhibited by different strains or species suggest how complex is the understanding of the antibiotics resistance in the study area . in this study ,", "the resistance patterns were highly variable ; 20.2% ( n = 94 ) isolates have no resistance towards any antibiotics tested , 16% ( n = 94 ) isolates were resistant to only one antibiotic , and 63.8% ( n = 94 ) isolates were resistant to multiple antibiotics .", "awareness on antibiotic resistance threat should be instilled in the community regardless of age as precaution and prevention step against dissemination of antibiotic resistant bacteria .", "many surveillance programs had also been introduced to monitor the emergence and spread of antibiotic resistant bacteria .", "it has also been suggested by son et al .   that plasmid screening should be considered as an additional procedure in the monitoring programs to trace antibiotic resistance dissemination .", "alternatives to treatment using antibiotics like probiotics , vaccines , and antimicrobials from plants should be also considered .", "however , most of the alternatives could not really effectively replace antibiotics , so they act as additional preventive measures rather than alternatives .", "the mar indexing has revealed that 63.1% of the isolates came from low antibiotic usage area .", "although antibiotic resistance in aquaculture in the malaysian borneo is still in its infancy , the need for continuous monitoring of the antibiotic resistance patterns should not be overlooked and the community should be educated on the awareness of antibiotic resistance and its implication on human health and environment ."], "abstract_text": ["<S> the administration of antimicrobials in aquaculture provides a selective pressure creating a reservoir of multiple resistant bacteria in the cultured fish and shrimps as well as the aquaculture environment . </S>", "<S> the objective of this study was to determine the extent of antibiotic resistance in aquaculture products and aquaculture 's surrounding environment in sarawak , malaysian borneo . </S>", "<S> ninety - four identified bacterial isolates constituted of 17 genera were isolated from sediment , water , and cultured organisms ( fish and shrimp ) in selected aquaculture farms . </S>", "<S> these isolates were tested for their antibiotic resistance against 22 antibiotics from several groups using the disk diffusion method . </S>", "<S> the results show that the highest resistance was observed towards streptomycin ( 85% , n = 20 ) , while the lowest resistance was towards gentamicin ( 1.1% , n = 90 ) . </S>", "<S> the multiple antibiotic resistant ( mar ) index of the isolates tested ranged between 0 and 0.63 . </S>", "<S> it was suggested that isolates with mar index > 0.2 were recovered from sources with high risk of antibiotic resistant contamination . </S>", "<S> this study revealed low level of antibiotic resistance in the aquaculture bacterial isolates except for streptomycin and ampicillin ( > 50% resistance , n = 94 ) which have been used in the aquaculture industry for several decades . </S>", "<S> antibiotic resistant patterns should be continuously monitored to predict the emergence and widespread of mar . </S>", "<S> effective action is needed to keep the new resistance from further developing and spreading . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["since the discovery of penicillin in 1928 by a scottish scientist alexander fleming followed by the release of many other earlier drugs onto the market to treat infection , the development of drug resistance in various sectors including aquaculture has been reported [ 13 ] .", "the misuse and abuse of the antimicrobial drugs are among the important factors that have contributed to the rise of resistant microbes around the world .", "antibiotics , which have saved millions of lives and were also known as miracle drug in the past , are no longer the ultimate way for the treatment of infections because bacteria have continued to develop multiple resistance towards many different types or classes of the drugs .", "antimicrobial agents have been widely used in fish farming for either therapeutic , prophylactic , or other purposes .", "the antibiotics are normally used to increase growth as well as feed efficiency in the animals . however , some of the antibiotics have been frequently used in both veterinary and human medicine such as sulfonamides , chloramphenicol , tetracycline , nitrofurans , oxytetracycline , neomycin , erythromycin , streptomycin , prefuran , and enrofloxacin .", "the evolution of bacteria towards antibiotic resistance has been accelerated distinctly by selective pressure due to inappropriate and overuse of the antibiotics [ 3 , 9 ] . in the efforts to cope with this problem", ", scientists have accelerated the search for alternative antimicrobial agents by screening many potential sources including medicinal plants [ 10 , 11 ] and microbes [ 12 , 13 ] .", "aquaculture is an important sector in the agriculture industry and is rapidly growing to meet the world 's demands for protein source .", "this sector is challenged with the diverse type of diseases and bacterial infections ; and antibiotics are an excellent tool to circumvent the problem [ 14 , 15 ] . the presence of bacteria with multiple antibiotic resistance found in food products has become a threat to public health as there is potential that the carried or acquired genes are transferred to other bacteria of clinical significance [ 1618 ] . some antibiotics which are commonly used in food - producing animals are also used in human medicine , reducing the antibiotic 's efficiency when treating infections and increasing the morbidity and mortality associated with diseases .", "the resistance limits the choice of antibiotics for the disease treatment [ 4 , 16 , 19 , 20 ] .", "the use of antibiotics needs to be monitored from time to time to evaluate the emergence and spread of bacterial resistance towards antimicrobial agents [ 5 , 21 ] .", "there is limited data on the antibiotic resistance of bacteria in fish and other cultured organisms sampled directly from fish farms as well as the aquaculture environment ."], ["sampling was carried out at aquaculture farms located at selected districts in sarawak , malaysian borneo , including kuching , bintulu , limbang , miri , and sampadi ( lundu ) .", "three types of samples were collected which were the sediment , water , and cultured species .", "cultured species refers to fish or shrimp . in kuching , bintulu , and miri ,", "the cultured organisms collected were the fish while in limbang and sampadi ( lundu ) , the cultured organisms collected were shrimps .", "the isolation , designation , and identification of the isolates were carried out as reported by kathleen and coworkers earlier .", "ninety - four bacterial isolates from 17 different genera   were assessed for their susceptibility to different antibiotics utilizing the disk diffusion method according to method described by clinical and laboratory standards institutes ( clsi )   on mueller - hinton agar ( mha ) .", "the bacterial groups and the antibiotics tested are listed in table 1 . briefly , fresh bacterial culture with 0.5 mcfarland turbidity was swabbed onto the mha surface using sterile cotton buds .", "commercial antimicrobial discs ( oxoid , uk ) were evenly embedded onto the inoculated agar incubated at 37c for 18 to 24 hours .", "escherichia coli strain from american type culture collection ( atcc ) 25922 was used as control .", "the diameter of complete inhibition zone formed around the antibiotic discs was measured to the nearest whole millimeter using standardized ruler .", "the results obtained were analyzed as resistant or susceptible according to standard interpretative table by clsi   and bonnet .", "multiple antibiotic resistance ( mar ) index was then determined for each isolate by dividing the number of antibiotics to which an isolate is resistant with the total number of antibiotics tested .", "the mar index is an indicator to identify the risk contamination that is potentially hazardous to human .", "calculated value of more than 0.2 indicates that the isolates were isolated from high risk sources ."], ["in this study , commonly used antibiotics in both veterinary and human medicine were selected for the antibiotic susceptibility testing .", "the antibiotic selection also depends on the bacterial genera because different bacterial genera require different classes of antibiotics for optimal antibacterial activity .", "some of the antibiotics were not tested in this study because some bacterial genera are naturally resistant to certain classes of antibiotics ; hence the antibiotics were excluded from the analysis .", "the antibiotic profile of the bacterial isolates from the aquaculture fish and shrimps and their environment is shown in table 1 .", "the bacterial isolates showing the top five highest percentages of resistant were towards streptomycin ( 85% , n = 20 ) , followed by ampicillin ( 56.8% , n = 74 ) , penicillin ( 47.1% , n = 51 ) , erythromycin ( 43.1% , n = 51 ) , and cephalotin ( 42.3% , n = 71 ) . the bacterial isolates showing the top five highest percentages of susceptible were towards gentamicin ( 1.1% , n = 90 ) , followed by tobramycin ( 2.2% , n = 90 ) , chloramphenicol ( 4.0% , n = 75 ) , norfloxacin ( 5% , n = 80 ) , and amikacin ( 5.6% , n = 90 ) . the other bacterial isolates and their percentage of resistant are shown in table 1 . in this study , the antibiotic resistant patterns for all isolates were also determined to monitor the spread of antibiotic resistance .", "sixty - one different antibiotic resistance patterns were observed among the isolates through this study ( data not shown ) .", "the resistance patterns were highly variable ; 20.2% ( n = 94 ) isolates have no resistance towards any antibiotics tested , 16% ( n = 94 ) isolates were resistant to only one antibiotic , and 63.8% ( n = 94 ) isolates were resistant to multiple antibiotics .", ". isolated from water in limbang aquaculture farm was resistant towards 12 out of 19 antibiotics tested , which was the highest amount of antibiotic resistance observed in this study .", "most isolates ( 53.2% , n = 94 ) possess distinctive pattern . however , there are also patterns ( 11 patterns ) shared by 2 or more bacteria isolates .", "the pattern shared by most isolates ( 19 isolates , n = 94 ) is 0% resistance ( mar index equal to 0 ) .", "there is at least an isolate with 0% resistance towards all antibiotics tested in all sampling locations except for bintulu .", "there is difference in the resistance pattern for bacteria isolated from water , sediment , and the cultured organisms .", "bacteria that were isolated from the same pond and same source of origin ( sediment , water , or cultured organisms ) possess different antibiotic patterns .", "the antibiotic resistant patterns of the bacterial genera are more influenced by the location from where the isolates were isolated rather than their genus .", "this analysis has been used to group the different sources from where the bacteria were recovered using the frequency of antibiotics resistance .", "isolates with mar < 0.2 were determined as isolates recovered from low risk sources of contamination while isolates with mar > 0.2 were from high risk sources . in this study ,", "analysis on overall isolates regardless of sampling location revealed that 63.1% ( n = 94 ) isolates belong to group mar < 0.2 while 36.9% ( n = 94 ) isolates belong to group mar > 0.2 .", "bintulu ( btl ) has the highest percentage ( 73.3% , n = 16 ) of bacteria isolated from high antibiotic - contaminated sources while sampadi ( spd ) has the lowest percentage ( 11.7% , n = 17 ) ."], ["assessment of the antibiotic resistance among aquaculture bacteria against antimicrobial agents is important for update on the bacterial antibiotic resistance patterns .", "it is part of a surveillance system aiming at monitoring emerging antibiotic resistant bacteria and their widespread .", "isolation of antibiotic resistant bacteria from aquaculture products and aquaculture environment indicates the health risk associated with the aquaculture .", "there had been reports on detection of antibiotic resistance genes in bacteria isolated from aquaculture products that can be transferred to human microbiota .", "this matter is becoming critical if the resistance genes are transferrable to human pathogens . providing effective treatment towards this infection becomes a problem to the medical practitioners as the choice of antibiotics is limited .", "thus , any source of antibiotic resistant bacteria must be carefully monitored . in antibiotic resistance analysis", ", the history of antibiotic application in particular area is reflected by the percentage of bacterial resistance to antibiotics .", "the frequency of antibiotics usage is related to the level of resistance among bacteria [ 2 , 29 ] . in this present study ,", "similarly , lim and kasing   and hatha et al .   in their respective studies observed that almost none of the bacteria tested were resistant to gentamicin and chloramphenicol .", "low frequency of antibiotic resistant bacteria may indicate the less activity associated with the contamination of antibiotics in the area .", "the use of chloramphenicol in aquaculture has been banned in certain countries including malaysia , korea , and japan since 1983 .", "this is because of the adverse effect of chloramphenicol in humans , even at very low dosage , which can cause other side effects like severe or fatal blood problems .", "the problems associated with blood are like anemia and  grey syndrome ,  a syndrome of cyanosis and cardiovascular collapse , which occurs particularly in newborn babies .", "this poses risks to the workers handling the products containing this antibiotic [ 5 , 31 ] .", "banning of antibiotics has aided in reducing the number of antibiotic resistant bacteria in an environment .", "reported the significant reduction in the frequency of vancomycin - resistant enterococci from broiler after the banning of vancomycin in denmark in 1995 .", "high percentage of streptomycin , ampicillin , and penicillin g resistance was observed in this study .", "similarly , high ampicillin and streptomycin resistance were also observed by zhang et al .   in their study on antibiotic resistance detection in e. coli strains isolated from two different aquaculture systems in south china .", "also recorded high resistance of ampicillin . in another study , identified isolates from mangrove soil in malaysia were 100% resistant towards ampicillin and penicillin while 77.8% of the isolates were resistant towards streptomycin .", "a study carried out by akinbowale et al .   recorded 54.8% ampicillin resistance of aquaculture bacteria in australia .", "in contrast with the results in this study , low resistance of streptomycin ( 21.2% ) and ampicillin ( 6.1% ) was observed in a study on 33 marine bacteria isolates by you et al . .", "in this study , farmers in the aquaculture farms where the sampling was carried out stated that there was no history of utilization of antibiotics in their farms . despite the absence of antibiotics as medicines or in feeds ,", "high resistance was observed to commonly used antibiotics such as streptomycin , ampicillin , and penicillin .", "high resistance of ampicillin and streptomycin in this study and other researches was not surprising as these antibiotics were among the first antibiotics introduced since the discovery of penicillin .", "although antibiotic usage in the studied farms has been stopped decades ago , antibiotic contamination is still possible as there may still be residues of antibiotics left in the environment .", "bacteria isolated from the sediment of the aquaculture pond may have acquired antibiotic resistance characteristics through unconsumed foods and the cultured organism 's faeces that contain the remaining antibiotics [ 3941 ] .", "the unconsumed foods and faeces will be deposited in the sediment and the composition of sediment microbiota will be altered due to selective pressure .", "fish feeds were a possible reservoir for antibiotic resistant genes in the farm sediments . in this present study ,", "bintulu ( btl ) recorded the highest percentage ( 73.3% , n = 16 ) of bacteria isolated from high antibiotic - contaminated sources ( mar > 0.2 ) while sampadi ( spd ) aquaculture farm recorded the least number ( 11.7% , n = 17 ) of mar index > 0.2 isolates .", "the aquaculture farm is located at an area that has many agriculture activities ( e.g. , pig farming , duck farming , and dragon fruit cultivation ) surrounding it .", "there is the possibility that antibiotics from the animal feeds or medications were absorbed into the sediment causing bacterial selection in the nearby environment .", "multiple antibiotic resistant bacteria might have travelled through water from these agriculture activities to the aquaculture ponds .", "buschmann et al .   in their study suggested that antibiotic resistant bacteria in mariculture farm may be transported by water current which flows from surrounding farms that utilize antibiotics excessively .", "antibiotic resistance pattern may vary depending on the geographical locations and selective pressure [ 43 , 44 ] and these patterns change rapidly from time to time .", "the different patterns exhibited by different strains or species suggest how complex is the understanding of the antibiotics resistance in the study area . in this study ,", "the resistance patterns were highly variable ; 20.2% ( n = 94 ) isolates have no resistance towards any antibiotics tested , 16% ( n = 94 ) isolates were resistant to only one antibiotic , and 63.8% ( n = 94 ) isolates were resistant to multiple antibiotics .", "awareness on antibiotic resistance threat should be instilled in the community regardless of age as precaution and prevention step against dissemination of antibiotic resistant bacteria .", "many surveillance programs had also been introduced to monitor the emergence and spread of antibiotic resistant bacteria .", "it has also been suggested by son et al .   that plasmid screening should be considered as an additional procedure in the monitoring programs to trace antibiotic resistance dissemination .", "alternatives to treatment using antibiotics like probiotics , vaccines , and antimicrobials from plants should be also considered .", "however , most of the alternatives could not really effectively replace antibiotics , so they act as additional preventive measures rather than alternatives ."], ["the mar indexing has revealed that 63.1% of the isolates came from low antibiotic usage area .", "although antibiotic resistance in aquaculture in the malaysian borneo is still in its infancy , the need for continuous monitoring of the antibiotic resistance patterns should not be overlooked and the community should be educated on the awareness of antibiotic resistance and its implication on human health and environment ."]]}
{"article_id": "PMC4357682", "article_text": ["with an incidence rate of 0.92% , maternal puerperal lower extremity nerve injuries are rare .", "lateral femoral cutaneous neuropathy ( meralgia paraesthetica ) is the most common , followed by femoral neuropathy .", "nerve injuries are more likely to occur in nulliparae , in cases of prolonged stages of labour and assisted vaginal deliveries .", "the presented case involved a 32-year - old woman with bilateral heel numbness due to bilateral neuropathy of the medial calcaneal nerve due to external compression during vaginal delivery .", "a 32-year - old woman presented to our neurology outpatient clinic with tingling and numbness in both heels .", "she continuously complained about these sensations ever since she had given vaginal birth to her first child 3 months earlier in a hospital .", "spinal analgesia ( lumbar level , bupivacaine / sufentanyl bolus , followed by a continuous administration of 8 ml / h ) was used during labour .", "the first stage of labour was prolonged , and because of fetal compromise during the expulsion phase , a vacuum delivery system ( palmpump kiwi ) , together with episiotomy , was used for fetal extraction .", "after delivery , the epidural analgesia was continued during the closure of the episiotomy with sutures .", "the patient did not use any other medication and had no history of neurological disorders or risk factors for neuropathy ( diabetes , hypothyroidism or hereditary liability to nerve pressure palsy ) . on examination , she had hypoaesthesia in the area of both heels .", "the strength of both the leg and foot muscles was normal , and so were the deep tendon reflexes .", ", a near - nerve needle conduction study of the medial calcaneal nerve was not performed .", "a diagnosis of bilateral neuropathy of the medial calcaneal nerve , most likely due to longstanding pressure on both heels with epidural analgesia as a predisposing factor , was made .", "three months after presentation ( 6 months after delivery ) , her numbness had diminished , but had not completely disappeared .", "bilateral neuropathy of the medial calcaneal nerve due to external compression during delivery using epidural analgesia is very rare .", "only 1 patient with numb heels has been reported in a large study , describing 2,615 women who received epidural anaesthesia during delivery .", "however , no details about delivery or analgesia in that patient were mentioned . in epidural analgesia during delivery , a motor block is unwanted , and thus voluntary muscle movements are possible , which makes longstanding external compression of nerves uncommon . in our patient ,", "prolonged labour with spinal analgesia and a continuation of analgesia during episiotomy probably masked signs of external compression on both heels .", "the medial calcaneal nerve arises from the tibial nerve at the medial side of the ankle , perforates the laciniate ligament , travels downwards , passing the bony projection below on the inner side of the ankle , and supplies the skin over the medial aspect of the heel .", "patients with risk factors , such as diabetes , are at a greater risk of developing neuropathy during epidural anaesthesia .", "however , also without any risk factors , the clinician should be aware of the absence of the normal reaction to longstanding pressure on a nerve during epidural analgesia .", "preventative measures like soft gel pads or intermittent posture changes could possibly prevent neuropathy of the medial calcaneal nerve in long - lasting epidural analgesia .", "bilateral medial calcaneal nerve neuropathy as a result of external compression is a rare complication of epidural obstetric analgesia ."], "abstract_text": ["<S> we describe the case of a 32-year - old woman who developed bilateral heel numbness after obstetric epidural analgesia . </S>", "<S> we diagnosed her with bilateral neuropathy of the medial calcaneal nerve , most likely due to longstanding pressure on both heels . </S>", "<S> risk factors for the development of this neuropathy were prolonged labour with spinal analgesia and a continuation of analgesia during episiotomy . </S>", "<S> padded footrests decrease pressure and can possibly prevent this neuropathy . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion", "Disclosure Statement"], "sections": [["with an incidence rate of 0.92% , maternal puerperal lower extremity nerve injuries are rare .", "lateral femoral cutaneous neuropathy ( meralgia paraesthetica ) is the most common , followed by femoral neuropathy .", "nerve injuries are more likely to occur in nulliparae , in cases of prolonged stages of labour and assisted vaginal deliveries .", "the presented case involved a 32-year - old woman with bilateral heel numbness due to bilateral neuropathy of the medial calcaneal nerve due to external compression during vaginal delivery ."], ["a 32-year - old woman presented to our neurology outpatient clinic with tingling and numbness in both heels .", "she continuously complained about these sensations ever since she had given vaginal birth to her first child 3 months earlier in a hospital .", "spinal analgesia ( lumbar level , bupivacaine / sufentanyl bolus , followed by a continuous administration of 8 ml / h ) was used during labour .", "the first stage of labour was prolonged , and because of fetal compromise during the expulsion phase , a vacuum delivery system ( palmpump kiwi ) , together with episiotomy , was used for fetal extraction .", "after delivery , the epidural analgesia was continued during the closure of the episiotomy with sutures .", "the patient did not use any other medication and had no history of neurological disorders or risk factors for neuropathy ( diabetes , hypothyroidism or hereditary liability to nerve pressure palsy ) . on examination , she had hypoaesthesia in the area of both heels .", "the strength of both the leg and foot muscles was normal , and so were the deep tendon reflexes .", ", a near - nerve needle conduction study of the medial calcaneal nerve was not performed .", "a diagnosis of bilateral neuropathy of the medial calcaneal nerve , most likely due to longstanding pressure on both heels with epidural analgesia as a predisposing factor , was made .", "three months after presentation ( 6 months after delivery ) , her numbness had diminished , but had not completely disappeared ."], ["bilateral neuropathy of the medial calcaneal nerve due to external compression during delivery using epidural analgesia is very rare .", "only 1 patient with numb heels has been reported in a large study , describing 2,615 women who received epidural anaesthesia during delivery .", "however , no details about delivery or analgesia in that patient were mentioned . in epidural analgesia during delivery , a motor block is unwanted , and thus voluntary muscle movements are possible , which makes longstanding external compression of nerves uncommon . in our patient ,", "prolonged labour with spinal analgesia and a continuation of analgesia during episiotomy probably masked signs of external compression on both heels .", "the medial calcaneal nerve arises from the tibial nerve at the medial side of the ankle , perforates the laciniate ligament , travels downwards , passing the bony projection below on the inner side of the ankle , and supplies the skin over the medial aspect of the heel .", "patients with risk factors , such as diabetes , are at a greater risk of developing neuropathy during epidural anaesthesia .", "however , also without any risk factors , the clinician should be aware of the absence of the normal reaction to longstanding pressure on a nerve during epidural analgesia .", "preventative measures like soft gel pads or intermittent posture changes could possibly prevent neuropathy of the medial calcaneal nerve in long - lasting epidural analgesia ."], ["bilateral medial calcaneal nerve neuropathy as a result of external compression is a rare complication of epidural obstetric analgesia ."], [""]]}
{"article_id": "PMC3953429", "article_text": ["haemotropic mycoplasmas ( haemoplasmas ) are bacterial organisms without cell walls that attach to and grow on the surface of red blood cells .", "three feline haemoplasma species are described : mycoplasma haemofelis ( mhf ) ,  candidatus mycoplasma haemominutum  ( cmhm ) , and  candidatus mycoplasma turicensis  ( cmt ) .", "these feline haemoplasmas are the causative agents of infectious anemia in cats and in several mammalian species .", "there is also potential interspecies transmission of some of these agents as recorded from cats to immunocompromised dogs .", "their zoonotic potential has recently been substantiated by the molecular identification of a feline haemoplasma isolate ( mhf ) in an hiv - positive immunocompromised human patient .", "the three feline haemoplasma species have different pathogenicities , mhf often resulting in haemolysis and severe anaemia in contrast to cmhm and cmt which are less pathogenic .", "although several studies worldwide have reported on the epidemiology of feline hemoplasmosis in sick or healthy client - owned pet cats with prevalences ranging from 7.2% to 45.8% [ 519 ] and few studies have focused on stray cat ( with prevalences from 11.5% to 60% [ 2024 ] ) , there have been no studies investigating stray cats in northern italy .", "information on regional prevalence of haemoplasmas could be used to limit the spread of diseases in feline populations and for predicting the likelihood of infection in cats presented with anemia .", "the vector potential of ctenocephalides felis has been demonstrated in experimental mhf infections , and stray cats may play a role in multiplying the organism in fleas that then infest pet cats and dogs and human beings .", "direct transmission , by aggressive interaction of cats , or interspecies transmission might play a role in the epidemiology of these organisms .", "in addition , haemoplasmas can be directly transmitted through intravenous infusion of infected blood [ 4 , 26 ] and have been shown to survive for up to 1 week in stored blood products .", "as administration of fresh or stored whole blood becoming more common in feline medicine , the knowledge of the regional prevalence of blood transmitted pathogens in owned and stray cats that share the same environment and parasites is increasingly important .", "this would provide useful information for evaluating the risks of transmission of blood - borne infections from potential blood donors and in the development of optimal screening protocols in blood donors .", "the aim of this study was to evaluate , using a conventional pcr assay , the prevalence of mhf and cmhm infections in stray cats from colonies in milan and to identify possible risk factors for these infections .", "during a 2-year collection period ( january 2008 to january 2010 ) , blood samples were taken from 260 stray cats from urban colonies in milan ( northern italy ) , under a trap - neuter - release ( tnr ) program approved by the local authority of the city council .", "age ( estimated based on dentition , animals < 6 months of age were considered juvenile , whereas all others were considered adult ) , gender ( male or female ) , origin ( provenance area of colonies ) , and body condition score ( bcs 46 , indicating normal weight , 13 underweight ) were recorded together with data obtained from physical examination of the cats ( healthy or unhealthy ) .", "unhealthy cats were defined as cats with one or more of the following clinical abnormalities : lymph node enlargement , pale mucous membranes , stomatitis , or signs of ocular and respiratory infections . the seasonal analysis based on the date of sample collection", "was grouped as follows : winter ( january , february , and march ) , spring ( april , may , and june ) , summer ( july , august , and september ) , autumn ( october , november , and december ) .", "the cats were not systematically examined for the presence of ticks or fleas and so rates of ectoparasitism were not recorded .", "blood samples were collected aseptically from the jugular vein while cats were anaesthetized for neutering and placed in edta - treated tubes and in serum separator tubes . within 24  h of sample collection , a complete blood count ( cbc )", "was performed on whole blood using an advia 120 system ( siemens healthcare diagnostics , milan , italy ) .", "cats were categorized in terms of presence or absence of anaemia ( hct < 24% ) , leukopenia ( wbcs count < 10,570/l ) , leukocytosis ( wbc > 14,390/l ) , and thrombocytopenia ( plt < 200,670/l ) .", "following separation , serum samples were tested for antibodies to fiv ( relative to gp40 and p24 fiv antigens ) and for felv p27 antigen with a commercial enzyme - linked immunosorbent assay ( elisa ) kit ( snap felv / fiv combo plus test ; idexx laboratories , hoofddorp , the netherlands ) .", "toxoplasma gondii sera igg antibodies were detected using indirect fluorescent antibody tests ( ifat ) performed with a commercial kit ( fuller - laboratories , fullerton , ca , usa ) .", "titres 1  :  64 were considered seroreactive and , therefore , indicative of t. gondii exposure .", "the results of these serological tests have been already published   and were reanalyzed with the present results .", "conventional pcr was performed on blood samples to amplify dna associated with haemoplasmas ( mhf and cmhm ) .", "the reaction mixture included 2  l of template dna , 0.25  mm dntps , 0.4  mm of each primer , 1x reaction buffer , and 2.5  u tap dna polymerase ( gotaq dna polymerase , promega , madison , wi , usa ) .", "primers were used that target part of the 16s rrna gene , producing a 170 base pair ( bp ) product from mhf and 193  bp amplicon from mchm ( forward primer , 5-acg aaa gtc tga tgg agc aat a-3 and reverse primer 5-acg ccc aat aaa tcc gra taa t-3 ) [ 6 , 31 ] .", "pcr products were resolved using 2% agarose gels and fragment size was estimated using a dna molecular weight marker ( 50 bp dna ladder ; promega madison , wi , usa ) .", "control reactions were done in the absence of template dna to rule out contaminations during pcr .", "association between the three groups of haemoplasma status ( haemoplasma positive , mhf positive only , and cmhm positive only ) and categorical variables ( age , gender , colony of origin , bcs , season of sampling , health status , presence or absence of selected clinical and cbc abnormalities , felv / fiv status , and t. gondii test results ) were analysed by univariate analysis using the chi - squared test ( cell frequencies of > 5 ) or fisher 's exact test ( cell frequencies of 5 ) .", "any parameters statistically linked to positive pcr results were used in a logistic regression model to test for independent risk factors associated with infection .", "descriptive statistics ( including minimum , maximum , mean , median , and standard deviation ( sd ) ) were obtained for the continuous variables rbcs count , hct , hb , wbc count , and plt count values .", "distribution of the data for normality was assessed with kolmogorov smirnov test , and a t - test or a mann - whitney u test , respectively , was used to test the differences between the feline haemoplasma positive and negative cats depending on whether data was normally distributed or not .", "associations were considered statistically significant when p < 0.05 . both the p value and odds ratio ( or ) with 95% confidence interval ( ci ) are reported .", "haemoplasmic dna was detected in 33.1% ( 86/260 , 95% ci 26.540.9% ) of blood samples . of the positive samples , 28 cats ( 10.8% , 95% ci 7.215.6% )", "were infected with mhf and 58 cats ( 22.3% , 95% ci 16.928.8% ) were infected with cmhm .", "characteristics of the population and association between risk factors and haemoplasma pcr status ( negative or positive ) are reported in table 1 and between mhf alone and cmhm alone positive results in table 2 .", "none of the risk factors were associated with the pcr results for haemoplasmas ( tables 1 and 2 ) , with the exception of negative associations at univariate and multivariate analysis between winter season of sampling and haemoplasma positive status ( p = 0.01 , or = 0.29 , 95% ci = 0.140.61 , p = 0.001 ) and cmhm positive status ( p = 0.01 , or = 0.29 , 95% ci = 0.120.70 , p = 0.01 ) .", "no significant associations were found with cbc abnormalities and haemoplasma pcr - positive results ( figure 1 ) or in the number of anaemic and nonanaemic cats using a t - test .", "we present the first study investigating the prevalence of haemoplasmas in urban stray colony cats from the city of milan in northern italy .", "the prevalence of haemoplasma infection in our sample showed a similar pattern to that reported worldwide in client - owned [ 57 , 915 , 1719 ] and stray cats [ 1923 ] .", "cmhm infection is reported to be the most common infection , ranging from 8% in arizona   to 41.6% in portugal ; mhf infection was less common ranging from 0.5% in switzerland   to 12.8% in portugal   and dual infection was absent or rare ( no more than 5.4% as found in korea ) .", "the prevalence of haemoplasmas in stray cats in our study ( 33.1% ) was higher than that reported in other studies on stray cats performed worldwide ( table 4 ) .", "differences in prevalence among countries can be explained by geographical variation , such as climate , vector distribution , and the cat population surveyed .", "moreover , direct comparisons of prevalence results are of limited value because of the characteristics of sample of cat populations investigated ( number of cats included in the study , healthy versus sick cats ) , inclusion criteria , diagnostic techniques ( molecular tools versus microscopical detection ) , different statistical methods , stage of infection ( acute versus chronic ) , or a combination of all them , resulting in differences between studies .", "stray cats in this study had higher rates of infections than those reported for pet cats in northern italy , in which 18.9% of pcr tested cats were reported to be haemoplasma positive .", "a high incidence of haemoplasmas in stray cats is not surprising as outdoor access is a recognized risk factor for infection .", "for example , in a study on haemoplasma in client - owned cats from barcelona ( spain ) , outdoor access was found to be a risk factor for infection ( or = 3.8 ) .", "additionally , in a study involving feline blood donors from the usa , the prevalence of haemoplasmas was 19.7% in domestic cats with outdoor access and only 3.6% in domestic cats not allowed outdoors .", "free - ranging cats may have more exposure to bloodsucking arthropods and exhibit a higher fighting activity than owned indoor cats ; thus they might experience a higher infection risk for haemoplasmas . in our study", "there was a negative statistical association between sampling in the winter season and haemoplasma pcr - positive status and cmhm positive status .", "this may be due to reduced outdoor activity of fleas in winter seasons , although this hypothesis is purely speculative .", "there were no statistically significant differences or any relationship between the presence of haemoplasma infection and anaemia status or hct levels between positive and negative haemoplasma group .", "this finding was somewhat surprising and was in agreement with studies undertaken in client - owned cats in switzerland   and in italy , which also found no association between haemoplasma infection and anaemia or hct variations .", "in addition , other studies have failed to demonstrate a significant difference in prevalence rates of haemoplasma between healthy and anaemic cats [ 10 , 32 ] .", "these results could be explained by the higher prevalence of cmhm ( a less pathogenic species than mhf ) in this and previous studies or by the fact that the stage of infection is not known in our cats with a positive pcr result , since chronically infected cats that recover from acute illness may be asymptomatic .", "another explanation for the lack of association between infection status and the presence of anaemia in the present study could be that the hct was known only for 150 of the 260 cats tested .", "epidemiological data on haemoplasma are particularly important in areas where feline blood donor programmes are active , as in milan where a donor programme has been running since 2010 at university veterinary transfusion unit ( rev , reparto di medicina emotrasfusionale veterinaria ) .", "feline haemoplasmas can be directly transmitted by intravenous infusion of fresh edta - anticoagulated blood , heparinized blood , and by infusion of blood stored in cpda-1 solution for 1  h ( mhf ) and 1 week ( cmhm ) .", "cats do not reliably eliminate the organism following infection   and most infections with cmhm are chronic and asymptomatic .", "a significant number of asymptomatic cats are positive for haemoplasma infection   and may play a role , along with infected cats , in the maintenance of haemoplasma infection within a population .", "all these characteristics of feline haemotropic mycoplasma infection need to be considered when choosing potential blood donors . the limitations of this study include the lack of information on  candidatus mycoplasma turicensis  in our study population .", "lastly , statistical analysis was limited in some groups because of the sample size and so some conclusions or associations may be affected by type i errors ; that is , no difference between haemoplasma positive and negative groups was observed due to insufficient sample size ; for example , no association was recorded between anaemia and a positive haemoplasma result because of the low number of mhf positive cats ( 28/260 ) .", "regardless of these limitations , we believe that this study provides new and useful information on feline haemoplasma infections in stray cats in italy .", "from this study it can be concluded that feline haemotropic mycoplasma mhf and cmhm are common in the stray cat population of milan .", "indeed , pet cats with outdoor access in this region should be regularly monitored and treated for ectoparasites to minimize health risks .", "importantly , feline blood donors in this area should undergo pcr screening for these infections before donations and preferably donors should be drawn from exclusively indoor cats that receive regular flea control ."], "abstract_text": ["<S> this study investigated the prevalence of feline haemoplasma infections in a number of stray cat colonies in milan , northern italy . </S>", "<S> blood samples from 260 stray cats were evaluated , with conventional pcr , for the presence of dna associated with mycoplasma haemofelis ( mhf ) and  candidatus mycoplasma haemominutum  ( cmhm ) . </S>", "<S> odd ratios ( or ) were calculated to identify risk factors for haemoplasma infections . </S>", "<S> pcr was positive in 86 out of 260 subjects ( 33.1% ) , with a prevalence of 10.8% ( 28/260 cats ) for mhf and 22.3% ( 58/260 cats ) for cmhm . </S>", "<S> no coinfections were registered . </S>", "<S> there were significant associations between infections and season of sampling , that is , a negative association between winter sampling and a haemoplasma positive status ( or = 0.29 , p = 0.001 ) , or cmhm positive status ( or = 0.29 , p = 0.01 ) . </S>", "<S> haemoplasma infections are common in stray cats in milan . </S>", "<S> thus , domestic cats with outdoor access should be routinely monitored and treated for ectoparasites to minimize risks of disease acquisition . moreover , as these infections are transmitted via blood , feline blood donors from this area should be screened by pcr and preferably be drawn from a population of indoor cats regularly treated for fleas . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["haemotropic mycoplasmas ( haemoplasmas ) are bacterial organisms without cell walls that attach to and grow on the surface of red blood cells .", "three feline haemoplasma species are described : mycoplasma haemofelis ( mhf ) ,  candidatus mycoplasma haemominutum  ( cmhm ) , and  candidatus mycoplasma turicensis  ( cmt ) .", "these feline haemoplasmas are the causative agents of infectious anemia in cats and in several mammalian species .", "there is also potential interspecies transmission of some of these agents as recorded from cats to immunocompromised dogs .", "their zoonotic potential has recently been substantiated by the molecular identification of a feline haemoplasma isolate ( mhf ) in an hiv - positive immunocompromised human patient .", "the three feline haemoplasma species have different pathogenicities , mhf often resulting in haemolysis and severe anaemia in contrast to cmhm and cmt which are less pathogenic .", "although several studies worldwide have reported on the epidemiology of feline hemoplasmosis in sick or healthy client - owned pet cats with prevalences ranging from 7.2% to 45.8% [ 519 ] and few studies have focused on stray cat ( with prevalences from 11.5% to 60% [ 2024 ] ) , there have been no studies investigating stray cats in northern italy .", "information on regional prevalence of haemoplasmas could be used to limit the spread of diseases in feline populations and for predicting the likelihood of infection in cats presented with anemia .", "the vector potential of ctenocephalides felis has been demonstrated in experimental mhf infections , and stray cats may play a role in multiplying the organism in fleas that then infest pet cats and dogs and human beings .", "direct transmission , by aggressive interaction of cats , or interspecies transmission might play a role in the epidemiology of these organisms .", "in addition , haemoplasmas can be directly transmitted through intravenous infusion of infected blood [ 4 , 26 ] and have been shown to survive for up to 1 week in stored blood products .", "as administration of fresh or stored whole blood becoming more common in feline medicine , the knowledge of the regional prevalence of blood transmitted pathogens in owned and stray cats that share the same environment and parasites is increasingly important .", "this would provide useful information for evaluating the risks of transmission of blood - borne infections from potential blood donors and in the development of optimal screening protocols in blood donors .", "the aim of this study was to evaluate , using a conventional pcr assay , the prevalence of mhf and cmhm infections in stray cats from colonies in milan and to identify possible risk factors for these infections ."], ["during a 2-year collection period ( january 2008 to january 2010 ) , blood samples were taken from 260 stray cats from urban colonies in milan ( northern italy ) , under a trap - neuter - release ( tnr ) program approved by the local authority of the city council .", "age ( estimated based on dentition , animals < 6 months of age were considered juvenile , whereas all others were considered adult ) , gender ( male or female ) , origin ( provenance area of colonies ) , and body condition score ( bcs 46 , indicating normal weight , 13 underweight ) were recorded together with data obtained from physical examination of the cats ( healthy or unhealthy ) .", "unhealthy cats were defined as cats with one or more of the following clinical abnormalities : lymph node enlargement , pale mucous membranes , stomatitis , or signs of ocular and respiratory infections . the seasonal analysis based on the date of sample collection", "was grouped as follows : winter ( january , february , and march ) , spring ( april , may , and june ) , summer ( july , august , and september ) , autumn ( october , november , and december ) .", "the cats were not systematically examined for the presence of ticks or fleas and so rates of ectoparasitism were not recorded .", "blood samples were collected aseptically from the jugular vein while cats were anaesthetized for neutering and placed in edta - treated tubes and in serum separator tubes . within 24  h of sample collection , a complete blood count ( cbc )", "was performed on whole blood using an advia 120 system ( siemens healthcare diagnostics , milan , italy ) .", "cats were categorized in terms of presence or absence of anaemia ( hct < 24% ) , leukopenia ( wbcs count < 10,570/l ) , leukocytosis ( wbc > 14,390/l ) , and thrombocytopenia ( plt < 200,670/l ) .", "following separation , serum samples were tested for antibodies to fiv ( relative to gp40 and p24 fiv antigens ) and for felv p27 antigen with a commercial enzyme - linked immunosorbent assay ( elisa ) kit ( snap felv / fiv combo plus test ; idexx laboratories , hoofddorp , the netherlands ) .", "toxoplasma gondii sera igg antibodies were detected using indirect fluorescent antibody tests ( ifat ) performed with a commercial kit ( fuller - laboratories , fullerton , ca , usa ) .", "titres 1  :  64 were considered seroreactive and , therefore , indicative of t. gondii exposure .", "the results of these serological tests have been already published   and were reanalyzed with the present results .", "conventional pcr was performed on blood samples to amplify dna associated with haemoplasmas ( mhf and cmhm ) .", "the reaction mixture included 2  l of template dna , 0.25  mm dntps , 0.4  mm of each primer , 1x reaction buffer , and 2.5  u tap dna polymerase ( gotaq dna polymerase , promega , madison , wi , usa ) .", "primers were used that target part of the 16s rrna gene , producing a 170 base pair ( bp ) product from mhf and 193  bp amplicon from mchm ( forward primer , 5-acg aaa gtc tga tgg agc aat a-3 and reverse primer 5-acg ccc aat aaa tcc gra taa t-3 ) [ 6 , 31 ] .", "pcr products were resolved using 2% agarose gels and fragment size was estimated using a dna molecular weight marker ( 50 bp dna ladder ; promega madison , wi , usa ) .", "control reactions were done in the absence of template dna to rule out contaminations during pcr .", "association between the three groups of haemoplasma status ( haemoplasma positive , mhf positive only , and cmhm positive only ) and categorical variables ( age , gender , colony of origin , bcs , season of sampling , health status , presence or absence of selected clinical and cbc abnormalities , felv / fiv status , and t. gondii test results ) were analysed by univariate analysis using the chi - squared test ( cell frequencies of > 5 ) or fisher 's exact test ( cell frequencies of 5 ) .", "any parameters statistically linked to positive pcr results were used in a logistic regression model to test for independent risk factors associated with infection .", "descriptive statistics ( including minimum , maximum , mean , median , and standard deviation ( sd ) ) were obtained for the continuous variables rbcs count , hct , hb , wbc count , and plt count values .", "distribution of the data for normality was assessed with kolmogorov smirnov test , and a t - test or a mann - whitney u test , respectively , was used to test the differences between the feline haemoplasma positive and negative cats depending on whether data was normally distributed or not .", "associations were considered statistically significant when p < 0.05 . both the p value and odds ratio ( or ) with 95% confidence interval ( ci ) are reported ."], ["haemoplasmic dna was detected in 33.1% ( 86/260 , 95% ci 26.540.9% ) of blood samples . of the positive samples , 28 cats ( 10.8% , 95% ci 7.215.6% )", "were infected with mhf and 58 cats ( 22.3% , 95% ci 16.928.8% ) were infected with cmhm .", "characteristics of the population and association between risk factors and haemoplasma pcr status ( negative or positive ) are reported in table 1 and between mhf alone and cmhm alone positive results in table 2 .", "none of the risk factors were associated with the pcr results for haemoplasmas ( tables 1 and 2 ) , with the exception of negative associations at univariate and multivariate analysis between winter season of sampling and haemoplasma positive status ( p = 0.01 , or = 0.29 , 95% ci = 0.140.61 , p = 0.001 ) and cmhm positive status ( p = 0.01 , or = 0.29 , 95% ci = 0.120.70 , p = 0.01 ) .", "no significant associations were found with cbc abnormalities and haemoplasma pcr - positive results ( figure 1 ) or in the number of anaemic and nonanaemic cats using a t - test ."], ["we present the first study investigating the prevalence of haemoplasmas in urban stray colony cats from the city of milan in northern italy .", "the prevalence of haemoplasma infection in our sample showed a similar pattern to that reported worldwide in client - owned [ 57 , 915 , 1719 ] and stray cats [ 1923 ] .", "cmhm infection is reported to be the most common infection , ranging from 8% in arizona   to 41.6% in portugal ; mhf infection was less common ranging from 0.5% in switzerland   to 12.8% in portugal   and dual infection was absent or rare ( no more than 5.4% as found in korea ) .", "the prevalence of haemoplasmas in stray cats in our study ( 33.1% ) was higher than that reported in other studies on stray cats performed worldwide ( table 4 ) .", "differences in prevalence among countries can be explained by geographical variation , such as climate , vector distribution , and the cat population surveyed .", "moreover , direct comparisons of prevalence results are of limited value because of the characteristics of sample of cat populations investigated ( number of cats included in the study , healthy versus sick cats ) , inclusion criteria , diagnostic techniques ( molecular tools versus microscopical detection ) , different statistical methods , stage of infection ( acute versus chronic ) , or a combination of all them , resulting in differences between studies .", "stray cats in this study had higher rates of infections than those reported for pet cats in northern italy , in which 18.9% of pcr tested cats were reported to be haemoplasma positive .", "a high incidence of haemoplasmas in stray cats is not surprising as outdoor access is a recognized risk factor for infection .", "for example , in a study on haemoplasma in client - owned cats from barcelona ( spain ) , outdoor access was found to be a risk factor for infection ( or = 3.8 ) .", "additionally , in a study involving feline blood donors from the usa , the prevalence of haemoplasmas was 19.7% in domestic cats with outdoor access and only 3.6% in domestic cats not allowed outdoors .", "free - ranging cats may have more exposure to bloodsucking arthropods and exhibit a higher fighting activity than owned indoor cats ; thus they might experience a higher infection risk for haemoplasmas . in our study", "there was a negative statistical association between sampling in the winter season and haemoplasma pcr - positive status and cmhm positive status .", "this may be due to reduced outdoor activity of fleas in winter seasons , although this hypothesis is purely speculative .", "there were no statistically significant differences or any relationship between the presence of haemoplasma infection and anaemia status or hct levels between positive and negative haemoplasma group .", "this finding was somewhat surprising and was in agreement with studies undertaken in client - owned cats in switzerland   and in italy , which also found no association between haemoplasma infection and anaemia or hct variations .", "in addition , other studies have failed to demonstrate a significant difference in prevalence rates of haemoplasma between healthy and anaemic cats [ 10 , 32 ] .", "these results could be explained by the higher prevalence of cmhm ( a less pathogenic species than mhf ) in this and previous studies or by the fact that the stage of infection is not known in our cats with a positive pcr result , since chronically infected cats that recover from acute illness may be asymptomatic .", "another explanation for the lack of association between infection status and the presence of anaemia in the present study could be that the hct was known only for 150 of the 260 cats tested .", "epidemiological data on haemoplasma are particularly important in areas where feline blood donor programmes are active , as in milan where a donor programme has been running since 2010 at university veterinary transfusion unit ( rev , reparto di medicina emotrasfusionale veterinaria ) .", "feline haemoplasmas can be directly transmitted by intravenous infusion of fresh edta - anticoagulated blood , heparinized blood , and by infusion of blood stored in cpda-1 solution for 1  h ( mhf ) and 1 week ( cmhm ) .", "cats do not reliably eliminate the organism following infection   and most infections with cmhm are chronic and asymptomatic .", "a significant number of asymptomatic cats are positive for haemoplasma infection   and may play a role , along with infected cats , in the maintenance of haemoplasma infection within a population .", "all these characteristics of feline haemotropic mycoplasma infection need to be considered when choosing potential blood donors . the limitations of this study include the lack of information on  candidatus mycoplasma turicensis  in our study population .", "lastly , statistical analysis was limited in some groups because of the sample size and so some conclusions or associations may be affected by type i errors ; that is , no difference between haemoplasma positive and negative groups was observed due to insufficient sample size ; for example , no association was recorded between anaemia and a positive haemoplasma result because of the low number of mhf positive cats ( 28/260 ) .", "regardless of these limitations , we believe that this study provides new and useful information on feline haemoplasma infections in stray cats in italy ."], ["from this study it can be concluded that feline haemotropic mycoplasma mhf and cmhm are common in the stray cat population of milan .", "indeed , pet cats with outdoor access in this region should be regularly monitored and treated for ectoparasites to minimize health risks .", "importantly , feline blood donors in this area should undergo pcr screening for these infections before donations and preferably donors should be drawn from exclusively indoor cats that receive regular flea control ."]]}
{"article_id": "PMC3395946", "article_text": ["in a case - control study , 140 patients including two equal groups of traumatic due to traffic injuries and non - traumatic patients were evaluated . using the persian version of the conners adult adhd rating scale ( caars ) self - report ( screening version )", "the study was performed in two university hospitals in tabriz , iran during a 13- month period - august 2008 to september 2009 .", "the sample size was calculated using sas software version 9.1 based on proportion comparison tests to ensure 80% statistical power and type 1 error less than 0.05 .", "the required data for sample size calculation were obtained from a previous study with conservative rounding of figures to come up with possible dissimilarities ( 11 ) .", "inclusion criteria were as follows : physical trauma , motor vehicle traumas ( motorcycle and car ) , being the driver / rider when injured , age range of 18 to 65 years , and ability to complete the conner 's questionnaire .", "the exclusion criteria were as follows : brain trauma resulting in decreased consciousness , non - driver / rider victims of motor vehicle accidents , psychiatric comorbidity , factors affecting the attention and concentration such as drug and alcohol use while driving , and illiteracy .", "patients with physical trauma ( case group ) and those patients admitted due to non - trauma reasons without previous history of trauma hospitalization were recruited during the study period .", "adhd diagnosis was performed using caars , and the psychiatric comorbidities were assessed according to dsm - iv - tr using scid , and those with a psychiatric comorbidity were excluded .", "all of the patients were informed about the design and purpose of the study , and a written consent form was obtained from each volunteer patient .", "the assessed factors in both groups included age , sex , educational level , job , accident history , daily driving amount , weekly driving amount , adhd index , a subscale ( attention deficit symptoms ) , b subscale ( hyperactivity - impulsivity symptoms ) , and c subscale ( total adhd symptoms ) .", "in addition , type of vehicle , light intensity at accident time , and the location of the accident were recorded in the case group .", "the conners adult adhd rating scale ( caars ) conner 's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . among its good characteristics", "is having multiple indices including attention deficit index ( subscale a ) , hyperactivity - impulsivity index ( subscale b ) , adhd symptoms total index ( subscale c ) , and adhd index ( subscale d ) .", "this questionnaire had been translated to persian by icss ( institute for cognitive science studies ) in tehran . in this study ,", "the overall internal consistency of the conner 's adult adhd rating scale was estimated as cronbaches  of 0.83 . in case", "the diagnosis of adhd was for children , a rating scale for diagnosing adhd in children designed by delavar & et al can be used ( 13).structured clinical interview for dsm - iv ( scid ) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria .", "the reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories ( kappa > 0.6 ) .", "comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution .", "in addition , the chi - square and fisher exact tests were used for categorical variables . in all cases , the p values less than 0.05 were considered statistically significant .", "the conners adult adhd rating scale ( caars ) conner 's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . among its good characteristics", "is having multiple indices including attention deficit index ( subscale a ) , hyperactivity - impulsivity index ( subscale b ) , adhd symptoms total index ( subscale c ) , and adhd index ( subscale d ) .", "this questionnaire had been translated to persian by icss ( institute for cognitive science studies ) in tehran . in this study ,", "the overall internal consistency of the conner 's adult adhd rating scale was estimated as cronbaches  of 0.83 . in case", "the diagnosis of adhd was for children , a rating scale for diagnosing adhd in children designed by delavar & et al can be used ( 13).structured clinical interview for dsm - iv ( scid ) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria .", "the reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories ( kappa > 0.6 ) .", "comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution .", "in addition , the chi - square and fisher exact tests were used for categorical variables . in all cases , the p values less than 0.05 were considered statistically significant .", "no significant statistical difference was found between case and control groups on age , gender , educational level and occupation . with respect to type of vehicle , 25 cases had a car accident ( 35.7% ) and 45 had a motorcycle accident ( 64.3% ) .", "the light condition was bright in fifty four ( 77.1% ) , semi - bright in eight ( 11.4% ) and dark in eight cases ( 11.4% ) .", "accident location in the case group was within the town in 30 cases ( 42.9% ) , side road in 20 ( 28.6% ) , main road in 13 ( 18.6% ) , and highway in 7 cases ( 10% ) .", "accordingly , the percent of subjects with accident history was significantly higher in case group ( p= 0.24 ) . furthermore , the mean driving hours in case group was significantly more compared to controls .", "no significant difference was observed between the two groups with respect to other factors . \n ", "comparison of measured variables in two groups * \n  the data are shown as mean standard deviation and frequency ( percent ) . in self - report questionnaire of conner 's adult adhd rating scale ,", "subscales ' scores higher than 70 were considered positive.adhd index was positive in 3 ( 4.3% ) subjects in both case and control groups .", "adhd total symptoms were positive in 7 ( 10% ) and 10 ( 14.3% ) cases in both case and control groups respectively p= 0.438 , or= 1.5 ( 0.5- 4.2 ) , ci : 95% . in a subscale", ", inattentive symptoms were observed in 5 ( 7.1% ) and 6 ( 8.6% ) participants in case and control groups respectively p= 0.753 , or=1.2 ( 0.4- 4.2 ) , ci : 95% . in b subscale ( hyperactive - impulsive symptoms were observed in 7 ( 10% ) and 6 ( 8.6% ) participants in case and control groups respectively pv= 0.771 , or= 0.8 ( 0.3- 2.7 ) , ci : 95% .", "in this study , the frequency of adult adhd was compared between traumatic adult patients ( due to traffic injuries ) and a control group . accordingly , adhd index was positive 4.3% in both case and control groups , adhd symptoms total were positive 10% , 14.3% in case and control groups respectively .", "a subscale ( inattentive symptoms ) was 7.1% , 8.6% in case and control groups respectively ; and b subscale ( hyperactive / impulsive symptoms ) was 10% , 8.6% in case and control groups respectively .", "hechtman et al ( 1984 ) first described the higher rate of traffic injuries in children with adhd ( 15 ) .", "the next studies performed by barkley et al . , and jerome et al . , accredited the initial report ( 1619 ) .", "the association of adhd and traffic injuries is so important that driving center of canada was listed the uncontrolled adhd as an item for giving certificate to the drivers .", "the higher frequency of each type of accident in adhd patients was compared to healthy subjects ( odds ratio 1.7 ) and the car accidents are a subgroup of these events ( 20 ) . despite of numerous studies about the association of adhd and car accidents ,", "ludolph et al . , reported twofold higher rate of car accidents in these patients compared with the normal population ( 21 ) . in barkley", "studies and cox et al ( 2000 , 2006 ) , this ratio has been different from two to six ( 16 , 2224 ) .", "sobanski et al . , evaluated 27 patients with adult adhd and 27 age and sex matched subjects as a control group .", "the frequency of car accidents in first group was 2.6 fold higher than the control group patients , the other related factors in this field were also ( 25 ) . besides the higher rate of car accidents in adhd higher . among these", ", we can point to the higher frequency of losing driving certificate , driving after alcohol and substance use , exceeding the speed limit , and other abnormal traffic behaviors , car accidents trauma , high rate of traffic punishments and rate of suspension of driving certificate and higher rate of being responsible for the accidents ( 2630 ) .", "besides the epidemiological studies , there are numerous clinical trials showing the strong association between adhd and car accidents .", "cox et al . , in two different studies showed that the frequency of car accidents in adults with adhd receiving methylphenidate was lower than the group without treatment ( 23 , 31 ) .", "barkley et al . , in a similar study demonstrated that treatment of adult adhd may result in decreased rate of car accidents and related injuries ( 32 ) .", "in a study on selegiline in comparison with methylphenidate in adhd children and adolescents showed that selegiline is also effective and well tolerated for adhd ( 33 ) .", ", in a study on modafinil as a treatment for adhd in children and adolescents found that this drug significantly improves symptoms of adhd and is well tolerated ( 34 ) . on the other hand , it is shown that high risk behaviors of adhd patients during driving would persist despite controlling other factors .", "in other words , high risk behaviors during driving are an independent factor associated with adhd ( 25 , 30 ) .", "different mechanisms are suggested including attention deficit , inability to control impulsivity , early fatigue , anger and aggressiveness ( 25 , 3537 ) . according to the mentioned studies", ", it may be concluded that there is a significant association between adult adhd and car accidents .", "it should be mentioned that in all studies , the patients with adhd and controls were either under investigation for driving - models or their driving situation while we compared the frequency of adhd in subjects with and without car accidents .", "the current study is the first report from tabriz- iran on the association of adhd to traffic injuries .", "the lack of difference between two groups regarding frequency of adhd may be due to the fact that the two groups were matched for job and educational level in this study .", "it was previously reported that adult adhd is one of the main causes of educational and job problems ( 30 ) .", "hence , matching of two groups for these variables may be a kind of selection bias . also driving skill level", "unfortunately , we could not evaluate this condition in our sample . according to available reports ,", "the low driving skill level and lack of sufficient information about driving rules ( especially in young person ) is one of the main causes for car accidents in our country ( 38 , 39 ) . in other words ,", "high frequency of car accidents in our society is mainly due to lack of sufficient training of young drivers and lack of sufficient respect to the driving rules .", "hence , it is very difficult to determine the role of adhd in this field .", "high frequency of motorcycle accidents in this study ( 64.3% ) is a congruent factor because of higher rule destruction in this group .", "difference in driving duration is another major factor and the mean daily driving hours were significantly higher in patients with car accidents .", "on the other hand , it has previously been shown that the mortality or major injuries and severity of injury in adhd patients experiencing car accidents is more than healthy subjects ( 26 , 27 ) . despite the mentioned restrictions ,", "accordingly , it seems that most car accidents involving driver injury are not related to adhd .", "this study showed that the odds of adult adhd was not significantly higher in drivers with traffic injuries compared with the matched controls . therefore ,", "larger scale studies stratified for different types of traffic accidents are recommended to further investigate such an association", "most of the traffic injuries cause death but these case were not included in our study ."], "abstract_text": ["<S> objectivenowadays , it is well known that the attention - deficit / hyperactivity disorder ( adhd ) is not confined to children and adolescents . </S>", "<S> recent data showed that a considerable portion of the general adult population may be affected by adhd . on the other hand , </S>", "<S> the impact of adhd on driving performance , a major area of adult life , has gained enthusiasm . </S>", "<S> more recent studies revealed an association between adult adhd and undesirable driving problems . </S>", "<S> this study was performed to determine the association between presence of adult adhd and traffic injuries.methodsin this case - control study , in a 13 month period , 140 subjects ( 70 drivers / riders injured in traffic accidents , and 70 age- and sex - matched non traumatic controls ) were selected to participate in the study and were placed in two groups . </S>", "<S> subjects with psychiatric comorbidities were excluded . </S>", "<S> the conners adult adhd rating scale ( caars ) self - report ( screening version ) was used for screening adult adhd in both groups . </S>", "<S> finally , the occurrence of this condition was compared between the case and control groups.resultssixty- nine males and one female were recruited in each group with a mean age of 29.319.32 ( 18 - 61 ) years in the case and 29.039.07 ( range : 18 - 60 ) years in the control groups ( p > 0.05 ) . </S>", "<S> previous history of traffic accident was significantly higher in the case group ( 15.7% vs. 4.3% , p=0.024 ) . </S>", "<S> the mean driving time in a day was also significantly higher in the case group . in the case group , </S>", "<S> the scores of caars ( the adhd index , adhd symptoms total , inattentive subscale and hyperactive / impulsive subscale ) were positive ( higher than 70 ) in 4.3 , 10 , 7.1 and 10 percent of patients respectively . among the controls , </S>", "<S> the corresponding proportions were 4.3 , 14.3 , 8.6 and 8.6 percent respectively . </S>", "<S> the two groups were comparable for the mentioned rates.conclusionthe results of this study revealed no significant association between adhd and traffic injuries in tabriz- iran . </S>"], "labels": null, "section_names": ["Materials and Method", "Measurement", "Statistical analysis", "Results", "Discussion", "Conclusion", "Limitation"], "sections": [["in a case - control study , 140 patients including two equal groups of traumatic due to traffic injuries and non - traumatic patients were evaluated . using the persian version of the conners adult adhd rating scale ( caars ) self - report ( screening version )", "the study was performed in two university hospitals in tabriz , iran during a 13- month period - august 2008 to september 2009 .", "the sample size was calculated using sas software version 9.1 based on proportion comparison tests to ensure 80% statistical power and type 1 error less than 0.05 .", "the required data for sample size calculation were obtained from a previous study with conservative rounding of figures to come up with possible dissimilarities ( 11 ) .", "inclusion criteria were as follows : physical trauma , motor vehicle traumas ( motorcycle and car ) , being the driver / rider when injured , age range of 18 to 65 years , and ability to complete the conner 's questionnaire .", "the exclusion criteria were as follows : brain trauma resulting in decreased consciousness , non - driver / rider victims of motor vehicle accidents , psychiatric comorbidity , factors affecting the attention and concentration such as drug and alcohol use while driving , and illiteracy .", "patients with physical trauma ( case group ) and those patients admitted due to non - trauma reasons without previous history of trauma hospitalization were recruited during the study period .", "adhd diagnosis was performed using caars , and the psychiatric comorbidities were assessed according to dsm - iv - tr using scid , and those with a psychiatric comorbidity were excluded .", "all of the patients were informed about the design and purpose of the study , and a written consent form was obtained from each volunteer patient .", "the assessed factors in both groups included age , sex , educational level , job , accident history , daily driving amount , weekly driving amount , adhd index , a subscale ( attention deficit symptoms ) , b subscale ( hyperactivity - impulsivity symptoms ) , and c subscale ( total adhd symptoms ) .", "in addition , type of vehicle , light intensity at accident time , and the location of the accident were recorded in the case group .", "the conners adult adhd rating scale ( caars ) conner 's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . among its good characteristics", "is having multiple indices including attention deficit index ( subscale a ) , hyperactivity - impulsivity index ( subscale b ) , adhd symptoms total index ( subscale c ) , and adhd index ( subscale d ) .", "this questionnaire had been translated to persian by icss ( institute for cognitive science studies ) in tehran . in this study ,", "the overall internal consistency of the conner 's adult adhd rating scale was estimated as cronbaches  of 0.83 . in case", "the diagnosis of adhd was for children , a rating scale for diagnosing adhd in children designed by delavar & et al can be used ( 13).structured clinical interview for dsm - iv ( scid ) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria .", "the reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories ( kappa > 0.6 ) .", "comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution .", "in addition , the chi - square and fisher exact tests were used for categorical variables . in all cases , the p values less than 0.05 were considered statistically significant ."], ["the conners adult adhd rating scale ( caars ) conner 's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . among its good characteristics", "is having multiple indices including attention deficit index ( subscale a ) , hyperactivity - impulsivity index ( subscale b ) , adhd symptoms total index ( subscale c ) , and adhd index ( subscale d ) .", "this questionnaire had been translated to persian by icss ( institute for cognitive science studies ) in tehran . in this study ,", "the overall internal consistency of the conner 's adult adhd rating scale was estimated as cronbaches  of 0.83 . in case", "the diagnosis of adhd was for children , a rating scale for diagnosing adhd in children designed by delavar & et al can be used ( 13).structured clinical interview for dsm - iv ( scid ) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria .", "the reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories ( kappa > 0.6 ) ."], ["comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution .", "in addition , the chi - square and fisher exact tests were used for categorical variables . in all cases , the p values less than 0.05 were considered statistically significant ."], ["no significant statistical difference was found between case and control groups on age , gender , educational level and occupation . with respect to type of vehicle , 25 cases had a car accident ( 35.7% ) and 45 had a motorcycle accident ( 64.3% ) .", "the light condition was bright in fifty four ( 77.1% ) , semi - bright in eight ( 11.4% ) and dark in eight cases ( 11.4% ) .", "accident location in the case group was within the town in 30 cases ( 42.9% ) , side road in 20 ( 28.6% ) , main road in 13 ( 18.6% ) , and highway in 7 cases ( 10% ) .", "accordingly , the percent of subjects with accident history was significantly higher in case group ( p= 0.24 ) . furthermore , the mean driving hours in case group was significantly more compared to controls .", "no significant difference was observed between the two groups with respect to other factors . \n ", "comparison of measured variables in two groups * \n  the data are shown as mean standard deviation and frequency ( percent ) . in self - report questionnaire of conner 's adult adhd rating scale ,", "subscales ' scores higher than 70 were considered positive.adhd index was positive in 3 ( 4.3% ) subjects in both case and control groups .", "adhd total symptoms were positive in 7 ( 10% ) and 10 ( 14.3% ) cases in both case and control groups respectively p= 0.438 , or= 1.5 ( 0.5- 4.2 ) , ci : 95% . in a subscale", ", inattentive symptoms were observed in 5 ( 7.1% ) and 6 ( 8.6% ) participants in case and control groups respectively p= 0.753 , or=1.2 ( 0.4- 4.2 ) , ci : 95% . in b subscale ( hyperactive - impulsive symptoms were observed in 7 ( 10% ) and 6 ( 8.6% ) participants in case and control groups respectively pv= 0.771 , or= 0.8 ( 0.3- 2.7 ) , ci : 95% ."], ["in this study , the frequency of adult adhd was compared between traumatic adult patients ( due to traffic injuries ) and a control group . accordingly , adhd index was positive 4.3% in both case and control groups , adhd symptoms total were positive 10% , 14.3% in case and control groups respectively .", "a subscale ( inattentive symptoms ) was 7.1% , 8.6% in case and control groups respectively ; and b subscale ( hyperactive / impulsive symptoms ) was 10% , 8.6% in case and control groups respectively .", "hechtman et al ( 1984 ) first described the higher rate of traffic injuries in children with adhd ( 15 ) .", "the next studies performed by barkley et al . , and jerome et al . , accredited the initial report ( 1619 ) .", "the association of adhd and traffic injuries is so important that driving center of canada was listed the uncontrolled adhd as an item for giving certificate to the drivers .", "the higher frequency of each type of accident in adhd patients was compared to healthy subjects ( odds ratio 1.7 ) and the car accidents are a subgroup of these events ( 20 ) . despite of numerous studies about the association of adhd and car accidents ,", "ludolph et al . , reported twofold higher rate of car accidents in these patients compared with the normal population ( 21 ) . in barkley", "studies and cox et al ( 2000 , 2006 ) , this ratio has been different from two to six ( 16 , 2224 ) .", "sobanski et al . , evaluated 27 patients with adult adhd and 27 age and sex matched subjects as a control group .", "the frequency of car accidents in first group was 2.6 fold higher than the control group patients , the other related factors in this field were also ( 25 ) . besides the higher rate of car accidents in adhd higher . among these", ", we can point to the higher frequency of losing driving certificate , driving after alcohol and substance use , exceeding the speed limit , and other abnormal traffic behaviors , car accidents trauma , high rate of traffic punishments and rate of suspension of driving certificate and higher rate of being responsible for the accidents ( 2630 ) .", "besides the epidemiological studies , there are numerous clinical trials showing the strong association between adhd and car accidents .", "cox et al . , in two different studies showed that the frequency of car accidents in adults with adhd receiving methylphenidate was lower than the group without treatment ( 23 , 31 ) .", "barkley et al . , in a similar study demonstrated that treatment of adult adhd may result in decreased rate of car accidents and related injuries ( 32 ) .", "in a study on selegiline in comparison with methylphenidate in adhd children and adolescents showed that selegiline is also effective and well tolerated for adhd ( 33 ) .", ", in a study on modafinil as a treatment for adhd in children and adolescents found that this drug significantly improves symptoms of adhd and is well tolerated ( 34 ) . on the other hand , it is shown that high risk behaviors of adhd patients during driving would persist despite controlling other factors .", "in other words , high risk behaviors during driving are an independent factor associated with adhd ( 25 , 30 ) .", "different mechanisms are suggested including attention deficit , inability to control impulsivity , early fatigue , anger and aggressiveness ( 25 , 3537 ) . according to the mentioned studies", ", it may be concluded that there is a significant association between adult adhd and car accidents .", "it should be mentioned that in all studies , the patients with adhd and controls were either under investigation for driving - models or their driving situation while we compared the frequency of adhd in subjects with and without car accidents .", "the current study is the first report from tabriz- iran on the association of adhd to traffic injuries .", "the lack of difference between two groups regarding frequency of adhd may be due to the fact that the two groups were matched for job and educational level in this study .", "it was previously reported that adult adhd is one of the main causes of educational and job problems ( 30 ) .", "hence , matching of two groups for these variables may be a kind of selection bias . also driving skill level", "unfortunately , we could not evaluate this condition in our sample . according to available reports ,", "the low driving skill level and lack of sufficient information about driving rules ( especially in young person ) is one of the main causes for car accidents in our country ( 38 , 39 ) . in other words ,", "high frequency of car accidents in our society is mainly due to lack of sufficient training of young drivers and lack of sufficient respect to the driving rules .", "hence , it is very difficult to determine the role of adhd in this field .", "high frequency of motorcycle accidents in this study ( 64.3% ) is a congruent factor because of higher rule destruction in this group .", "difference in driving duration is another major factor and the mean daily driving hours were significantly higher in patients with car accidents .", "on the other hand , it has previously been shown that the mortality or major injuries and severity of injury in adhd patients experiencing car accidents is more than healthy subjects ( 26 , 27 ) . despite the mentioned restrictions ,", "accordingly , it seems that most car accidents involving driver injury are not related to adhd ."], ["this study showed that the odds of adult adhd was not significantly higher in drivers with traffic injuries compared with the matched controls . therefore ,", "larger scale studies stratified for different types of traffic accidents are recommended to further investigate such an association"], ["most of the traffic injuries cause death but these case were not included in our study ."]]}
{"article_id": "PMC4589633", "article_text": ["crh is a 41-amino - acid peptide hormone synthesized in the paraventricular nucleus of the hypothalamus .", "its function in the central nervous system is to stimulate the hypothalamic - pituitary - adrenal ( hpa ) axis as part of the stress response .", "crh is also produced in peripheral tissues including the placenta of humans and hominine primates [ 13 ] .", "placental crh secretion results in an exponential rise of crh concentration in maternal plasma during the third trimester of gestation .", "the rate of the increase is related to gestational length , since the rise of crh level is accelerated in pregnancies ending with preterm birth , while the increase is retarded in pregnancies that continue after term .", "because of this relationship , it is believed that the regulation of placental crh production is linked to the mechanism that determines the length of pregnancy and triggers labour and delivery .", "the mechanism regulating the exponential increase in placental crh expression remains unclear although positive feedback by glucocorticoids and increasing numbers of syncytial cell nuclei are suggested explanations [ 5 , 6 ] .", "spontaneous and agonist - induced syncytium formation by cytotrophoblasts is associated with crh expression with camp being a strong stimulant of both syncytial differentiation and crh gene activity .", "molecular studies using crh promoter - reporter constructs indicated that transcription factor complexes bound to a consensus cyclic - amp response element ( cre ) at 224  bp upstream of the major transcription initiation site mediated the camp - stimulation , although a nonconsensus second promoter site was also implicated in the cyclic nucleotide response [ 811 ] .", "it has been suggested that these molecular interactions are involved in the gestational age dependent control of crh gene activity .", "epigenetic chromatin modifications define cell - specific gene expression potential and alter gene expression patterns during cell differentiation and development .", "methylation of cytosines at cpg dinucleotides in dna is a well - characterized epigenetic chromatin modification generally associated with closed chromatin structure and gene repression .", "furthermore , methylation of cpg sites within particular transcription factor binding sequences may modify transcription factor binding affinity and alter regulatory changes in gene expression [ 1417 ] .", "the human crh proximal promoter contains 9  cpg dinucleotides with one located within the methylation sensitive cre sequence .", "in addition , the promoter is within 1000  bp distance from an intragenic cpg island , which corresponds to a  cpg island shore ", "region , the methylation of which is related to tissue specific gene expression . therefore , in the present investigation we have explored the possibility that methylation of the promoter contributes to the control of crh expression in trophoblast cells .", "we used the bewo choriocarcinoma cell line in the experiments , which is a well - characterized trophoblast model exhibiting dynamic dna methylation as well as ability to syncytialise and increase crh expression when stimulated with camp [ 1921 ] .", "the bewo human choriocarcinoma - derived cell line was obtained from the american type culture collection ( manassas , va , usa ) ( atcc # ccl-98 ) .", "the culture medium was dmem / f12 ( with hepes and l - glutamine , without phenol red ) supplemented with 10%  ( v / v ) fetal bovine serum ( fbs ) and 1x antibiotic - antimycotic ( gibco / life technologies , mulgrave , vic , australia ) .", "cells were cultured at 37c in a humidified atmosphere of 5% co2 in air . at approximately 80% confluence cells", "were detached with trypsin / edta solution ( gibco ) , washed , counted , and transferred into six - well plates at a density of 0.8  10  cells / well .", "cultures reaching 50% confluence were incubated with fresh medium with or without 8-br - camp ( 8-bromoadenosine-3,5-cyclic monophosphate , 2.5  10  mol  l ) and/or 5-aza - dc ( 5-aza-2-deoxycytidine , 5", " 10mol  l ) ( sigma - aldrich , sydney , nsw , australia ) followed by harvesting for rna and dna isolation .", "drug concentrations were optimised in previous studies and showed no toxic effects in bewo cells at the concentrations and exposure times employed [ 19 , 22 , 23 ] .", "rna was extracted from cells using the rneasy mini kit ( qiagen , chadstone centre , vic , australia ) according to the manufacturer 's protocol .", "rna was eluted from the rneasy spin columns with 30  l of rnase - free water and quantified using a nanodrop 1000 spectrophotometer ( thermo fisher scientific australia , scoresby , vic , australia ) .", "contaminating dna was removed by dnase treatment using the turbo dna - free kit ( ambion / life technologies ) following the  routine  protocol .", "the total reaction volume was 20  l including 2  l of 10x dnase buffer , 1  l of dnase , and up to 2  g of rna .", "rna integrity in all samples was assessed by agarose gel electrophoresis . prior to reverse transcription , the rna samples were spiked with 5  10 copies of alien rna transcript ( supplied with the alien qrt - pcr inhibitor alert kit , stratagene / integrated sciences , chatswood , nsw , australia ) per microgram rna .", "the alien rna transcript served as a reference rna of equal abundance in all samples and pcr runs .", "rna was reverse transcribed using the superscript iii first - strand synthesis system for rt - pcr ( invitrogen ) with random hexamer primers .", "real - time pcr was performed using an applied biosystems 7500 real - time pcr system with reagents supplied by the manufacturer ( applied biosystems / life technologies ) .", "the amplification reaction contained template cdna from 20  ng of reverse - transcribed rna , 6  10  moles  l forward and 3  10  moles  l reverse primer , sybr green master mix , and milliq water to a total volume of 25  l .", "the crh cdna primers were designed and optimised by sehringer et al .   and are listed in table 1 .", "primer sequences for amplifying alien cdna are proprietary and were used according to the manufacturer 's instructions ( stratagen / integrated sciences ) .", "the temperature sequence was 50c for 2  min , 95c for 10  min , 40 cycles of 95c for 15  s , and 60c for 1  min , followed by melting curve analysis .", "no - template control and no - reverse transcriptase controls for all samples were included to detect residual genomic dna .", "expression levels of the crh mrna were determined relative to alien rna according to the ct method .", "cells grown in six - well plates were collected in 750  l pbs ( phosphate - buffered saline ; 137  10mol  l nacl , 2.7  10  mol  l kcl , 8  10mol  l na2hpo4 , and 2  10  mol  l kh2po4 , ph 7.4 ) using cell scrapers and centrifuged for 5  min at 300  g .", "cell pellets were resuspended in 200  l of pbs and processed for dna isolation as per the manufacturer 's instructions .", "genomic dna was eluted from the mini spin columns with 200  l milliq water , quantified using the nanodrop 1000 spectrophotometer , and stored at 4c .", "up to 300  ng of genomic dna was bisulfite - converted and purified using the methylseqr bisulfite conversion kit ( applied biosystems ) .", "the crh proximal promoter regions were pcr - amplified using the toptaq master mix kit ( qiagen ) and two sets of nested primers designed with the methyl primer express software v.1.0 ( applied biosystems ) .", "pcr reactions contained purified bisulfite - converted dna template , 25  l of 2x toptaq master mix , 4  10  mol  l of each primer , and milliq water up to 50  l final volume .", "the conditions for the first pcr amplification included an initial step at 94c for 3  min , followed by 30 cycles of 94c for 30  s , 54c for 30  s , and 72c for 1  min and a final extension step at 72c for 10  min .", "one microliter of a 20-fold diluted aliquot of the first pcr reaction was used as template for the second pcr amplification using the nested primer set .", "pcr conditions were 94c 30  min , 30 cycles of 94c for 30  s , 50c for 30  s , and 65c for 1  min and an extension step at 65c for 10  min . following amplification , 20  l of the pcr reaction mixture", "was separated by agarose gel electrophoresis and the amplification product was visualised with ethidium bromide .", "the gel slice containing the amplified dna fragment was excised , extracted , and purified using the wizard sv gel and pcr clean - up system ( promega , auburn , vic , australia ) .", "the dna was collected in 50  l milliq water by the centrifugation of the sv minicolumn .", "the bisulfite - converted and pcr - amplified dna was ligated into the pgem - t easy vector using reagents provided by the manufacturer ( promega ) . the 10  l reaction mixtures contained ligation buffer , 3 weiss units of t4 dna ligase , 50  ng of pgem - t easy vector , and pcr product at 3  :  1 insert : vector molar ratio .", "a positive control using the control dna provided and a negative control ( no pcr product ) were also included .", "the ligation mixture was used subsequently to transform jm109 competent cells ( promega ) according to the manufacturer 's protocol .", "fifty l of transformed cell suspension was spread onto duplicate lb / ampicillin / iptg / x - gal plates and incubated at 37c overnight .", "the white streak colonies were picked the next day and cultured in 5  ml luria broth at 37c overnight .", "plasmids were isolated from the minicultures using the genelute plasmid miniprep kit ( sigma - aldrich , castle hill , nsw , australia ) .", "the presence of inserts was verified by digesting an aliquot from each plasmid preparation with ecor i ( promega ) followed by agarose gel electrophoresis .", "plasmids containing the expected size inserts ( 300  bp ) were sequenced from both directions by the australian genome research facility ( agrf , brisbane , qld , australia ) .", "the sequencing primers were designed by promega and produced by invitrogen / life technologies ( forward : 5-tatttaggtgacactatag-3 , reverse : 5-tatttaggtgacactatag-3 ) .", "quality control was automatically performed and any sequence with an unacceptably low conversion rate or high number of sequencing errors was excluded .", "ten randomly selected clones , representing individual gene copies , were processed for methylation frequency analyses from each dna sample using the chi - square test and fisher 's exact test as appropriate .", "significant one - sided tests are reported in cases when the two - sided tests showed borderline significance . the stata ( college station , tx , usa ) software package was used for the statistical calculations .", "as shown in figure 1 , abundance was not significantly different between 0  h and 24  h and between 24  h and 28  h of incubation", ". between 48  h and 72  h , a 2.9-fold increase ( p = 0.029 ) was observed , which was coincident with the reported spontaneous syncytialisation of bewo cells . in the presence of 8-br - camp", "( 2.5  10  mol  l ) , a powerful stimulant of syncytial differentiation , significant increases of crh mrna level were detected between 0  h and 24  h ( p < 0.0001 ) , 24  h and 48  h ( p = 0.03 ) , and 48  h and 72  h ( p = 0.049 , one - sided t - test ) with a maximum at 72  h , which was 23.2-fold higher than the 0  h level ( figure 1 ) . in cultures treated with the dna methyltransferase inhibitor 5-aza - dc ( 5  10mol  l ) a slight , but significant , increase of crh mrna abundance", "was observed between 0  h and 24  h ( 1.43-fold , p = 0.028 , one - sided t - test ) , but there was no further change between 24  h and 48  h and between 48  h and 72  h. combined treatment with 8-br - camp and 5-aza - dc resulted in robust increases of crh mrna levels between 0  h and 24  h ( p < 0.0001 ) , 24  h and 48  h ( p = 0.0026 ) , and 48  h and 72  h ( p = 0.039 ) reaching a 86.4-fold rise at 72  h compared to 0  h ( figure 1 ) .", "8-br - camp increased crh mrna abundance relative to vehicle at all time points ( 24  h , p = 0.0042 ; 48  h , p = 0.0066 ; 72  h , p = 0.0004 ; figure 1 ) , which confirmed previous findings of the stimulatory effects of 8-br - camp on crh gene expression in bewo cells .", "5-aza - dc treatment had no effect at 24  h and 48  h and reduced crh mrna abundance relative to vehicle at 72  h , effectively blocking the increase seen between 48  h and 72  h in vehicle - treated cells ( p = 0.0021 , figure 1 ) . combined treatment with the cyclic nucleotide and the dna methyltransferase inhibitor upregulated crh mrna expression at all time points beyond the level reached in response to 8-br - camp alone ( 24  h , p < 0.0001 ; 48  h , p = 0.0002 ; 72  h , p = 0.0029 ; figure 1 ) .", "the significant effects of 5-aza - dc on crh mrna expression suggested that dna methylation was involved in the control of crh gene activity . to explore this further", ", we have determined the effects of 8-br - camp and 5-aza - dc on the methylation of the 9  cpg sites present in the crh proximal promoter .", "bisulfite sequencing revealed partial methylation ( 38% of the 9  cpg sites combined ) at 0  h , before treatments commenced ( figure 2 ) , which increased spontaneously to 57% ( p = 0.001 ) by 72  h of culture .", "treatment with 8-br - camp ( 250  m ) resulted in a similar increase of methylation ( to 61% ) , not significantly different from the vehicle - treated control .", "treatment with 5-aza - dc for 72  h reduced promoter methylation to 23% , which was significantly less than the control ( p = 0.001 ) . combined treatment with 5-aza - dc and 8-br - camp increased the level of methylation compared to 5-aza - dc alone ( to 33% , p = 0.011 ) but did not reach the methylation level observed in cells treated with 8-br - camp only ( p = 0.001 , figure 2 ) .", "clonal bisulfite sequencing determines cytosine methylation with single base resolution in individual alleles ( gene copies ) .", "the technique enabled us to determine the particular cpg sites that undergo methylation changes under the treatment conditions that influence methylation levels overall , as presented in figure 2 .", "the scheme in figure 3 shows the positions of the 9 methylatable cpg dinucleotides in the human crh proximal promoter .", "the two major transcription initiation sites and the two sequence regions implicated in the camp - response are also indicated with cpg2 residing within the cre [ 811 , 29 ] .", "the heatmap in figure 4 illustrates the methylation levels of the 9 cpgs under the treatment conditions employed .", "methylation levels were significantly different among the cpg sites ranging from 10% to 70% at 0  h and from 13% to 80% at 72  h of culture ( p < 0.001 , figure 4 ) indicating site - specific differential methylation .", "the methylation level of cpgs 1 , 2 , 3 , and 7 , but not of cpgs 4 , 5 , 6 , 8 , and 9 increased significantly during the 72  h culture period demonstrating that methylation was dynamic at these sites .  ", "figure 5 shows the methylation of each cpg in the cloned copies of the crh proximal promoter .", "the scattered distribution of methylated and unmethylated cpg sites suggests that the partial methylation observed was allele independent both at 0  h and at 72  h of culture .", "cells treated with 8-br - camp and 5-aza - dc had similar scattered distribution of methylated cpgs in individual alleles ( not shown ) .", "in cells treated with 8-br - camp for 72  h , the cpg sites remained differentially methylated ( from 23% to 73% , p = 0.004 ) , and the methylation levels of individual cpgs were not significantly different from the corresponding sites in the vehicle - treated control ( figure 4 ) .", "treatment with the dna methyltransferase inhibitor 5-aza - dc decreased methylation at all cpg sites compared to vehicle , except for cpg 4 , where methylation was relatively low . furthermore , 5-aza - dc abolished the differences between the methylation levels of the individual cpgs .", "the methyltransferase inhibitor eliminated the methylation differences among individual cpgs in the presence of 8-br - camp as well ( figure 4 ) .", "cotreatment with 8-br - camp prevented , however , the demethylating action of 5-aza - dc at cpgs 1 , and 8 , but not at cpgs 2 , 3 , 5 , 7 , and 9 ( camp versus camp + aza in figure 4 ) .", "finally , no individual cpg site exhibited a statistically significant methylation difference in 8-br - camp + 5-aza - dc - treated cells compared to treatment with the methyltransferase inhibitor alone ( aza versus camp + aza in figure 4 ) despite the small , but significant , overall increase in methylation ( aza 72  h versus camp + aza 72  h , in figure 2 ) .", "the aim of this study was to explore the involvement of promoter methylation in crh gene regulation in human trophoblast cells .", "placental crh expression is predictive of gestational length and is influenced by pregnancy disorders [ 4 , 30 ] .", "dna methylation is a developmentally regulated epigenetic modification influenced by environmental inputs [ 31 , 32 ] , which can potentially control crh gene expression during pregnancy and in response to pathogenic factors . in our experiments we used the choriocarcinoma - derived bewo cell line , which is a well - characterized trophoblast model exhibiting increased crh gene expression during spontaneous and camp - induced syncytial differentiation similar to normal trophoblasts .", "our dna sequencing data show that the crh proximal promoter sequence is identical in bewo cells and in normal trophoblasts including all reported transcription factor response elements and the methylation sensitive cpg sites ( figure 3 ) .", "we have shown by clonal bisulfite sequencing that the cpgs within the crh proximal promoter are partially methylated with significant differences among the individual sites .", "the size of the analysed sequence ( 261  bp ) and the methylation level correspond to an  intermediate methylation region ,  which is a genomic feature implicated in tissue specific epigenetic gene regulation .", "this is similar to normal trophoblasts , which also exhibit allele independent partial and differential methylation in the crh promoter region .", "dna methylation increases in bewo cells during culture and the dna methyltransferase inhibitor 5-aza - dc changes cell phenotype and gene expression levels [ 21 , 22 , 35 ] .", "our results show that the crh promoter follows this general trend , which is different from primary trophoblasts , where crh promoter methylation does not change in culture and remains unaltered by 5-aza - dc and 8-br - camp under the conditions where bewo cells show methylation changes . for this reason , bewo cells", "are uniquely suited to explore the relationship between crh promoter methylation level and gene activity .", "methylation of cpg island promoters is associated generally with the repression of gene activity [ 12 , 14 , 21 ] .", "the crh promoter is located in a cpg island shore region relative to a crh intragenic cpg island ( chr8:67,089,250 - 67,089,962 in the grch37/hg19 assembly , ucsc genome browser ) .", "our results showed , however , that neither the spontaneous nor the 8-br - camp - evoked increase of crh gene activity was associated with the demethylation of the crh promoter ( figures 1 and 2 ) .", "treatment with 5-aza - dc decreased crh promoter methylation and abolished the cpg site - specific methylation differences as expected ( figures 2 and 4 ) , but it also blocked the spontaneous increase of crh gene expression in culture ( figure 1 ) .", "8-br - camp - stimulated crh expression strongly in the presence of 5-aza - dc ( figure 1 ) , but the cyclic nucleotide actually increased methylation in 5-aza - dc - treated cells ( aza 72  h versus camp + aza 72  h in figure 2 ) .", "thus , crh expression was directly , and not reciprocally , related to promoter methylation under these conditions", ". this relationship may be unexpected in view of the well - documented global association between gene repression and promoter methylation , but genome wide trends do not necessarily predict the behavior of individual genes .", "in fact , the methylation level of the cpg - poor class of promoters was found to be uncorrelated with gene activity .", "the crh promoter falls into the cpg - poor class according to established criteria , with partial methylation in both the hypothalamus and the trophoblast [ 34 , 37 , 38 ] ( figure 2 ) .", "methylation reduces crh gene expression in the hypothalamus as expected ; in trophoblastic bewo cells , however , promoter methylation appears to have the opposite effect as detailed before .", "there is evidence to suggest that this cell - specific regulation may result from the methylation - dependent change of the functional properties of the camp - response element ( cre ) in the crh promoter ( figure 3 ) .", "the cre is critical in regulating the activity of transfected crh promoter - reporter constructs in trophoblast cells [ 911 ] .", "it contains a cpg dinucleotide that , when methylated , reduces the affinity of cre to its cognate transcription factor , creb , and renders it unresponsive to camp - stimulation [ 15 , 39 ] . at the same time , the methylated cre has increased affinity to bind the transcription factor c / ebp - alpha , which often activates tissue specific genes during differentiation .", "the cpg within the cre was 30% methylated under basal ( 0  h incubation ) conditions ( cpg2 in figure 4 ) .", "culturing for 72  h increased cpg2 methylation to 60% concomitantly with enhanced gene expression , while treatment with 5-aza - dc reduced cpg2 methylation to 23% and diminished crh gene activity ( figures 1 and 4 ) .", "considering that c / ebp - alpha is expressed in bewo cells , it is reasonable to conjecture that methylation - evoked changes in the transcription factor binding specificity of the cre may have contributed to the enhanced crh expression observed in association with increased promoter methylation .", "the cpg2 in the cre was partially methylated under all experimental conditions , which suggests that the unmethylated portion could have mediated stimulation by 8-br - camp using the canonical , creb - dependent pathway .", "moreover , the crh proximal promoter contains a second , noncanonical camp - response element ( figure 3 ) , which contributes to the regulation of the gene specifically in the trophoblast [ 9 , 11 ] .", "methylation of the cre may thus function to influence the relative contribution of the two camp - response elements , their associated transcription factors , and the coupled signaling pathways to the overall activity of the crh gene under basal and camp - stimulated conditions .", "cotreatment with 5-aza - dc strongly augmented the stimulation of crh mrna expression by 8-br - camp , although the dna methyltransferase inhibitor alone had no stimulatory effect under the same culture conditions ( figure 1 ) .", "cotreatment with 5-aza - dc , however , decreased cpg methylation in the cre ( cpg2 , figure 4 ) to 43.3% from 70% measured after 8-br - camp treatment ( p = 0.034 , fisher 's exact test , one - sided ) .", "the increased proportion of unmethylated cres in the cell population could explain , at least partially , the augmented response to 8-br - camp - stimulation .", "moreover , 5-aza - dc decreases dna methylation globally increasing or repressing the activity of numerous genes [ 4143 ] .", "this suggests the possibility that 5-aza - dc may potentially influence crh expression indirectly through intervening gene products generating synergy between 8-br - camp and 5-aza - dc .", "although gene activation occurs in 5-aza - dc - treated bewo cells [ 22 , 23 , 35 ] , it remains to be established whether transcription factors or other gene products controlled by dna methylation regulate crh expression in trophoblasts .", "we have utilized the dynamic changes of dna methylation in the bewo cell line to explore the relationship between this epigenetic chromatin modification and crh gene expression in a trophoblastic cell type .", "clonal bisulfite sequencing revealed the cpg site - specific and allele independent partial methylation of the crh proximal promoter and classified it as an intermediately methylated region of the genome .", "the data suggest that promoter methylation determines the contribution of the cre , its various associated transcription factors , and a trophoblast specific alternative camp - response element to crh gene regulation .", "furthermore , our results are consistent with the possibility that dna methylation controls crh expression indirectly , but any intervening factor that may regulate crh expression by a dna methylation - dependent mechanism remains to be determined ."], "abstract_text": ["<S> corticotropin releasing hormone ( crh ) production by the human placenta increases exponentially as pregnancy advances , and the rate of increase predicts gestational length . </S>", "<S> crh gene expression is regulated by camp in trophoblasts through a cyclic amp - response element ( cre ) , which changes its transcription factor binding properties upon methylation . </S>", "<S> here we determined whether methylation of the crh proximal promoter controls basal and camp - stimulated crh expression in bewo cells , a well - characterized trophoblastic cell line . </S>", "<S> we treated the cells with 8-br - camp and the dna methyltransferase inhibitor 5-aza-2 deoxycytidine ( 5-aza - dc ) and determined the effects on crh mrna level and promoter methylation . </S>", "<S> clonal bisulfite sequencing showed partial and allele independent methylation of cpgs in the crh promoter . </S>", "<S> crh mrna expression and the methylation of a subset of cpgs ( including cpg2 in the cre ) increased spontaneously during culture . </S>", "<S> 8-br - camp stimulated crh expression without affecting the increase in methylation . </S>", "<S> 5-aza - dc decreased methylation and augmented 8-br - camp - stimulated crh expression , but it blocked the spontaneous increase of crh mrna level . </S>", "<S> we conclude that the crh promoter is a dynamically and intermediately methylated genomic region in bewo cells . </S>", "<S> promoter methylation did not inhibit crh gene expression under the conditions employed ; rather it determined the contribution of alternative camp - independent pathways and camp - independent mechanisms to crh expression control . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["crh is a 41-amino - acid peptide hormone synthesized in the paraventricular nucleus of the hypothalamus .", "its function in the central nervous system is to stimulate the hypothalamic - pituitary - adrenal ( hpa ) axis as part of the stress response .", "crh is also produced in peripheral tissues including the placenta of humans and hominine primates [ 13 ] .", "placental crh secretion results in an exponential rise of crh concentration in maternal plasma during the third trimester of gestation .", "the rate of the increase is related to gestational length , since the rise of crh level is accelerated in pregnancies ending with preterm birth , while the increase is retarded in pregnancies that continue after term .", "because of this relationship , it is believed that the regulation of placental crh production is linked to the mechanism that determines the length of pregnancy and triggers labour and delivery .", "the mechanism regulating the exponential increase in placental crh expression remains unclear although positive feedback by glucocorticoids and increasing numbers of syncytial cell nuclei are suggested explanations [ 5 , 6 ] .", "spontaneous and agonist - induced syncytium formation by cytotrophoblasts is associated with crh expression with camp being a strong stimulant of both syncytial differentiation and crh gene activity .", "molecular studies using crh promoter - reporter constructs indicated that transcription factor complexes bound to a consensus cyclic - amp response element ( cre ) at 224  bp upstream of the major transcription initiation site mediated the camp - stimulation , although a nonconsensus second promoter site was also implicated in the cyclic nucleotide response [ 811 ] .", "it has been suggested that these molecular interactions are involved in the gestational age dependent control of crh gene activity .", "epigenetic chromatin modifications define cell - specific gene expression potential and alter gene expression patterns during cell differentiation and development .", "methylation of cytosines at cpg dinucleotides in dna is a well - characterized epigenetic chromatin modification generally associated with closed chromatin structure and gene repression .", "furthermore , methylation of cpg sites within particular transcription factor binding sequences may modify transcription factor binding affinity and alter regulatory changes in gene expression [ 1417 ] .", "the human crh proximal promoter contains 9  cpg dinucleotides with one located within the methylation sensitive cre sequence .", "in addition , the promoter is within 1000  bp distance from an intragenic cpg island , which corresponds to a  cpg island shore ", "region , the methylation of which is related to tissue specific gene expression . therefore , in the present investigation we have explored the possibility that methylation of the promoter contributes to the control of crh expression in trophoblast cells .", "we used the bewo choriocarcinoma cell line in the experiments , which is a well - characterized trophoblast model exhibiting dynamic dna methylation as well as ability to syncytialise and increase crh expression when stimulated with camp [ 1921 ] ."], ["the bewo human choriocarcinoma - derived cell line was obtained from the american type culture collection ( manassas , va , usa ) ( atcc # ccl-98 ) .", "the culture medium was dmem / f12 ( with hepes and l - glutamine , without phenol red ) supplemented with 10%  ( v / v ) fetal bovine serum ( fbs ) and 1x antibiotic - antimycotic ( gibco / life technologies , mulgrave , vic , australia ) .", "cells were cultured at 37c in a humidified atmosphere of 5% co2 in air . at approximately 80% confluence cells", "were detached with trypsin / edta solution ( gibco ) , washed , counted , and transferred into six - well plates at a density of 0.8  10  cells / well .", "cultures reaching 50% confluence were incubated with fresh medium with or without 8-br - camp ( 8-bromoadenosine-3,5-cyclic monophosphate , 2.5  10  mol  l ) and/or 5-aza - dc ( 5-aza-2-deoxycytidine , 5", " 10mol  l ) ( sigma - aldrich , sydney , nsw , australia ) followed by harvesting for rna and dna isolation .", "drug concentrations were optimised in previous studies and showed no toxic effects in bewo cells at the concentrations and exposure times employed [ 19 , 22 , 23 ] .", "rna was extracted from cells using the rneasy mini kit ( qiagen , chadstone centre , vic , australia ) according to the manufacturer 's protocol .", "rna was eluted from the rneasy spin columns with 30  l of rnase - free water and quantified using a nanodrop 1000 spectrophotometer ( thermo fisher scientific australia , scoresby , vic , australia ) .", "contaminating dna was removed by dnase treatment using the turbo dna - free kit ( ambion / life technologies ) following the  routine  protocol .", "the total reaction volume was 20  l including 2  l of 10x dnase buffer , 1  l of dnase , and up to 2  g of rna .", "rna integrity in all samples was assessed by agarose gel electrophoresis . prior to reverse transcription , the rna samples were spiked with 5  10 copies of alien rna transcript ( supplied with the alien qrt - pcr inhibitor alert kit , stratagene / integrated sciences , chatswood , nsw , australia ) per microgram rna .", "the alien rna transcript served as a reference rna of equal abundance in all samples and pcr runs .", "rna was reverse transcribed using the superscript iii first - strand synthesis system for rt - pcr ( invitrogen ) with random hexamer primers .", "real - time pcr was performed using an applied biosystems 7500 real - time pcr system with reagents supplied by the manufacturer ( applied biosystems / life technologies ) .", "the amplification reaction contained template cdna from 20  ng of reverse - transcribed rna , 6  10  moles  l forward and 3  10  moles  l reverse primer , sybr green master mix , and milliq water to a total volume of 25  l .", "the crh cdna primers were designed and optimised by sehringer et al .   and are listed in table 1 .", "primer sequences for amplifying alien cdna are proprietary and were used according to the manufacturer 's instructions ( stratagen / integrated sciences ) .", "the temperature sequence was 50c for 2  min , 95c for 10  min , 40 cycles of 95c for 15  s , and 60c for 1  min , followed by melting curve analysis .", "no - template control and no - reverse transcriptase controls for all samples were included to detect residual genomic dna .", "expression levels of the crh mrna were determined relative to alien rna according to the ct method .", "cells grown in six - well plates were collected in 750  l pbs ( phosphate - buffered saline ; 137  10mol  l nacl , 2.7  10  mol  l kcl , 8  10mol  l na2hpo4 , and 2  10  mol  l kh2po4 , ph 7.4 ) using cell scrapers and centrifuged for 5  min at 300  g .", "cell pellets were resuspended in 200  l of pbs and processed for dna isolation as per the manufacturer 's instructions .", "genomic dna was eluted from the mini spin columns with 200  l milliq water , quantified using the nanodrop 1000 spectrophotometer , and stored at 4c .", "up to 300  ng of genomic dna was bisulfite - converted and purified using the methylseqr bisulfite conversion kit ( applied biosystems ) .", "the crh proximal promoter regions were pcr - amplified using the toptaq master mix kit ( qiagen ) and two sets of nested primers designed with the methyl primer express software v.1.0 ( applied biosystems ) .", "pcr reactions contained purified bisulfite - converted dna template , 25  l of 2x toptaq master mix , 4  10  mol  l of each primer , and milliq water up to 50  l final volume .", "the conditions for the first pcr amplification included an initial step at 94c for 3  min , followed by 30 cycles of 94c for 30  s , 54c for 30  s , and 72c for 1  min and a final extension step at 72c for 10  min .", "one microliter of a 20-fold diluted aliquot of the first pcr reaction was used as template for the second pcr amplification using the nested primer set .", "pcr conditions were 94c 30  min , 30 cycles of 94c for 30  s , 50c for 30  s , and 65c for 1  min and an extension step at 65c for 10  min . following amplification , 20  l of the pcr reaction mixture", "was separated by agarose gel electrophoresis and the amplification product was visualised with ethidium bromide .", "the gel slice containing the amplified dna fragment was excised , extracted , and purified using the wizard sv gel and pcr clean - up system ( promega , auburn , vic , australia ) .", "the dna was collected in 50  l milliq water by the centrifugation of the sv minicolumn .", "the bisulfite - converted and pcr - amplified dna was ligated into the pgem - t easy vector using reagents provided by the manufacturer ( promega ) . the 10  l reaction mixtures contained ligation buffer , 3 weiss units of t4 dna ligase , 50  ng of pgem - t easy vector , and pcr product at 3  :  1 insert : vector molar ratio .", "a positive control using the control dna provided and a negative control ( no pcr product ) were also included .", "the ligation mixture was used subsequently to transform jm109 competent cells ( promega ) according to the manufacturer 's protocol .", "fifty l of transformed cell suspension was spread onto duplicate lb / ampicillin / iptg / x - gal plates and incubated at 37c overnight .", "the white streak colonies were picked the next day and cultured in 5  ml luria broth at 37c overnight .", "plasmids were isolated from the minicultures using the genelute plasmid miniprep kit ( sigma - aldrich , castle hill , nsw , australia ) .", "the presence of inserts was verified by digesting an aliquot from each plasmid preparation with ecor i ( promega ) followed by agarose gel electrophoresis .", "plasmids containing the expected size inserts ( 300  bp ) were sequenced from both directions by the australian genome research facility ( agrf , brisbane , qld , australia ) .", "the sequencing primers were designed by promega and produced by invitrogen / life technologies ( forward : 5-tatttaggtgacactatag-3 , reverse : 5-tatttaggtgacactatag-3 ) .", "quality control was automatically performed and any sequence with an unacceptably low conversion rate or high number of sequencing errors was excluded .", "ten randomly selected clones , representing individual gene copies , were processed for methylation frequency analyses from each dna sample using the chi - square test and fisher 's exact test as appropriate .", "significant one - sided tests are reported in cases when the two - sided tests showed borderline significance . the stata ( college station , tx , usa ) software package was used for the statistical calculations ."], ["as shown in figure 1 , abundance was not significantly different between 0  h and 24  h and between 24  h and 28  h of incubation", ". between 48  h and 72  h , a 2.9-fold increase ( p = 0.029 ) was observed , which was coincident with the reported spontaneous syncytialisation of bewo cells . in the presence of 8-br - camp", "( 2.5  10  mol  l ) , a powerful stimulant of syncytial differentiation , significant increases of crh mrna level were detected between 0  h and 24  h ( p < 0.0001 ) , 24  h and 48  h ( p = 0.03 ) , and 48  h and 72  h ( p = 0.049 , one - sided t - test ) with a maximum at 72  h , which was 23.2-fold higher than the 0  h level ( figure 1 ) . in cultures treated with the dna methyltransferase inhibitor 5-aza - dc ( 5  10mol  l ) a slight , but significant , increase of crh mrna abundance", "was observed between 0  h and 24  h ( 1.43-fold , p = 0.028 , one - sided t - test ) , but there was no further change between 24  h and 48  h and between 48  h and 72  h. combined treatment with 8-br - camp and 5-aza - dc resulted in robust increases of crh mrna levels between 0  h and 24  h ( p < 0.0001 ) , 24  h and 48  h ( p = 0.0026 ) , and 48  h and 72  h ( p = 0.039 ) reaching a 86.4-fold rise at 72  h compared to 0  h ( figure 1 ) .", "8-br - camp increased crh mrna abundance relative to vehicle at all time points ( 24  h , p = 0.0042 ; 48  h , p = 0.0066 ; 72  h , p = 0.0004 ; figure 1 ) , which confirmed previous findings of the stimulatory effects of 8-br - camp on crh gene expression in bewo cells .", "5-aza - dc treatment had no effect at 24  h and 48  h and reduced crh mrna abundance relative to vehicle at 72  h , effectively blocking the increase seen between 48  h and 72  h in vehicle - treated cells ( p = 0.0021 , figure 1 ) . combined treatment with the cyclic nucleotide and the dna methyltransferase inhibitor upregulated crh mrna expression at all time points beyond the level reached in response to 8-br - camp alone ( 24  h , p < 0.0001 ; 48  h , p = 0.0002 ; 72  h , p = 0.0029 ; figure 1 ) .", "the significant effects of 5-aza - dc on crh mrna expression suggested that dna methylation was involved in the control of crh gene activity . to explore this further", ", we have determined the effects of 8-br - camp and 5-aza - dc on the methylation of the 9  cpg sites present in the crh proximal promoter .", "bisulfite sequencing revealed partial methylation ( 38% of the 9  cpg sites combined ) at 0  h , before treatments commenced ( figure 2 ) , which increased spontaneously to 57% ( p = 0.001 ) by 72  h of culture .", "treatment with 8-br - camp ( 250  m ) resulted in a similar increase of methylation ( to 61% ) , not significantly different from the vehicle - treated control .", "treatment with 5-aza - dc for 72  h reduced promoter methylation to 23% , which was significantly less than the control ( p = 0.001 ) . combined treatment with 5-aza - dc and 8-br - camp increased the level of methylation compared to 5-aza - dc alone ( to 33% , p = 0.011 ) but did not reach the methylation level observed in cells treated with 8-br - camp only ( p = 0.001 , figure 2 ) .", "clonal bisulfite sequencing determines cytosine methylation with single base resolution in individual alleles ( gene copies ) .", "the technique enabled us to determine the particular cpg sites that undergo methylation changes under the treatment conditions that influence methylation levels overall , as presented in figure 2 .", "the scheme in figure 3 shows the positions of the 9 methylatable cpg dinucleotides in the human crh proximal promoter .", "the two major transcription initiation sites and the two sequence regions implicated in the camp - response are also indicated with cpg2 residing within the cre [ 811 , 29 ] .", "the heatmap in figure 4 illustrates the methylation levels of the 9 cpgs under the treatment conditions employed .", "methylation levels were significantly different among the cpg sites ranging from 10% to 70% at 0  h and from 13% to 80% at 72  h of culture ( p < 0.001 , figure 4 ) indicating site - specific differential methylation .", "the methylation level of cpgs 1 , 2 , 3 , and 7 , but not of cpgs 4 , 5 , 6 , 8 , and 9 increased significantly during the 72  h culture period demonstrating that methylation was dynamic at these sites .  ", "figure 5 shows the methylation of each cpg in the cloned copies of the crh proximal promoter .", "the scattered distribution of methylated and unmethylated cpg sites suggests that the partial methylation observed was allele independent both at 0  h and at 72  h of culture .", "cells treated with 8-br - camp and 5-aza - dc had similar scattered distribution of methylated cpgs in individual alleles ( not shown ) .", "in cells treated with 8-br - camp for 72  h , the cpg sites remained differentially methylated ( from 23% to 73% , p = 0.004 ) , and the methylation levels of individual cpgs were not significantly different from the corresponding sites in the vehicle - treated control ( figure 4 ) .", "treatment with the dna methyltransferase inhibitor 5-aza - dc decreased methylation at all cpg sites compared to vehicle , except for cpg 4 , where methylation was relatively low . furthermore , 5-aza - dc abolished the differences between the methylation levels of the individual cpgs .", "the methyltransferase inhibitor eliminated the methylation differences among individual cpgs in the presence of 8-br - camp as well ( figure 4 ) .", "cotreatment with 8-br - camp prevented , however , the demethylating action of 5-aza - dc at cpgs 1 , and 8 , but not at cpgs 2 , 3 , 5 , 7 , and 9 ( camp versus camp + aza in figure 4 ) .", "finally , no individual cpg site exhibited a statistically significant methylation difference in 8-br - camp + 5-aza - dc - treated cells compared to treatment with the methyltransferase inhibitor alone ( aza versus camp + aza in figure 4 ) despite the small , but significant , overall increase in methylation ( aza 72  h versus camp + aza 72  h , in figure 2 ) ."], ["the aim of this study was to explore the involvement of promoter methylation in crh gene regulation in human trophoblast cells .", "placental crh expression is predictive of gestational length and is influenced by pregnancy disorders [ 4 , 30 ] .", "dna methylation is a developmentally regulated epigenetic modification influenced by environmental inputs [ 31 , 32 ] , which can potentially control crh gene expression during pregnancy and in response to pathogenic factors . in our experiments we used the choriocarcinoma - derived bewo cell line , which is a well - characterized trophoblast model exhibiting increased crh gene expression during spontaneous and camp - induced syncytial differentiation similar to normal trophoblasts .", "our dna sequencing data show that the crh proximal promoter sequence is identical in bewo cells and in normal trophoblasts including all reported transcription factor response elements and the methylation sensitive cpg sites ( figure 3 ) .", "we have shown by clonal bisulfite sequencing that the cpgs within the crh proximal promoter are partially methylated with significant differences among the individual sites .", "the size of the analysed sequence ( 261  bp ) and the methylation level correspond to an  intermediate methylation region ,  which is a genomic feature implicated in tissue specific epigenetic gene regulation .", "this is similar to normal trophoblasts , which also exhibit allele independent partial and differential methylation in the crh promoter region .", "dna methylation increases in bewo cells during culture and the dna methyltransferase inhibitor 5-aza - dc changes cell phenotype and gene expression levels [ 21 , 22 , 35 ] .", "our results show that the crh promoter follows this general trend , which is different from primary trophoblasts , where crh promoter methylation does not change in culture and remains unaltered by 5-aza - dc and 8-br - camp under the conditions where bewo cells show methylation changes . for this reason , bewo cells", "are uniquely suited to explore the relationship between crh promoter methylation level and gene activity .", "methylation of cpg island promoters is associated generally with the repression of gene activity [ 12 , 14 , 21 ] .", "the crh promoter is located in a cpg island shore region relative to a crh intragenic cpg island ( chr8:67,089,250 - 67,089,962 in the grch37/hg19 assembly , ucsc genome browser ) .", "our results showed , however , that neither the spontaneous nor the 8-br - camp - evoked increase of crh gene activity was associated with the demethylation of the crh promoter ( figures 1 and 2 ) .", "treatment with 5-aza - dc decreased crh promoter methylation and abolished the cpg site - specific methylation differences as expected ( figures 2 and 4 ) , but it also blocked the spontaneous increase of crh gene expression in culture ( figure 1 ) .", "8-br - camp - stimulated crh expression strongly in the presence of 5-aza - dc ( figure 1 ) , but the cyclic nucleotide actually increased methylation in 5-aza - dc - treated cells ( aza 72  h versus camp + aza 72  h in figure 2 ) .", "thus , crh expression was directly , and not reciprocally , related to promoter methylation under these conditions", ". this relationship may be unexpected in view of the well - documented global association between gene repression and promoter methylation , but genome wide trends do not necessarily predict the behavior of individual genes .", "in fact , the methylation level of the cpg - poor class of promoters was found to be uncorrelated with gene activity .", "the crh promoter falls into the cpg - poor class according to established criteria , with partial methylation in both the hypothalamus and the trophoblast [ 34 , 37 , 38 ] ( figure 2 ) .", "methylation reduces crh gene expression in the hypothalamus as expected ; in trophoblastic bewo cells , however , promoter methylation appears to have the opposite effect as detailed before .", "there is evidence to suggest that this cell - specific regulation may result from the methylation - dependent change of the functional properties of the camp - response element ( cre ) in the crh promoter ( figure 3 ) .", "the cre is critical in regulating the activity of transfected crh promoter - reporter constructs in trophoblast cells [ 911 ] .", "it contains a cpg dinucleotide that , when methylated , reduces the affinity of cre to its cognate transcription factor , creb , and renders it unresponsive to camp - stimulation [ 15 , 39 ] . at the same time , the methylated cre has increased affinity to bind the transcription factor c / ebp - alpha , which often activates tissue specific genes during differentiation .", "the cpg within the cre was 30% methylated under basal ( 0  h incubation ) conditions ( cpg2 in figure 4 ) .", "culturing for 72  h increased cpg2 methylation to 60% concomitantly with enhanced gene expression , while treatment with 5-aza - dc reduced cpg2 methylation to 23% and diminished crh gene activity ( figures 1 and 4 ) .", "considering that c / ebp - alpha is expressed in bewo cells , it is reasonable to conjecture that methylation - evoked changes in the transcription factor binding specificity of the cre may have contributed to the enhanced crh expression observed in association with increased promoter methylation .", "the cpg2 in the cre was partially methylated under all experimental conditions , which suggests that the unmethylated portion could have mediated stimulation by 8-br - camp using the canonical , creb - dependent pathway .", "moreover , the crh proximal promoter contains a second , noncanonical camp - response element ( figure 3 ) , which contributes to the regulation of the gene specifically in the trophoblast [ 9 , 11 ] .", "methylation of the cre may thus function to influence the relative contribution of the two camp - response elements , their associated transcription factors , and the coupled signaling pathways to the overall activity of the crh gene under basal and camp - stimulated conditions .", "cotreatment with 5-aza - dc strongly augmented the stimulation of crh mrna expression by 8-br - camp , although the dna methyltransferase inhibitor alone had no stimulatory effect under the same culture conditions ( figure 1 ) .", "cotreatment with 5-aza - dc , however , decreased cpg methylation in the cre ( cpg2 , figure 4 ) to 43.3% from 70% measured after 8-br - camp treatment ( p = 0.034 , fisher 's exact test , one - sided ) .", "the increased proportion of unmethylated cres in the cell population could explain , at least partially , the augmented response to 8-br - camp - stimulation .", "moreover , 5-aza - dc decreases dna methylation globally increasing or repressing the activity of numerous genes [ 4143 ] .", "this suggests the possibility that 5-aza - dc may potentially influence crh expression indirectly through intervening gene products generating synergy between 8-br - camp and 5-aza - dc .", "although gene activation occurs in 5-aza - dc - treated bewo cells [ 22 , 23 , 35 ] , it remains to be established whether transcription factors or other gene products controlled by dna methylation regulate crh expression in trophoblasts ."], ["we have utilized the dynamic changes of dna methylation in the bewo cell line to explore the relationship between this epigenetic chromatin modification and crh gene expression in a trophoblastic cell type .", "clonal bisulfite sequencing revealed the cpg site - specific and allele independent partial methylation of the crh proximal promoter and classified it as an intermediately methylated region of the genome .", "the data suggest that promoter methylation determines the contribution of the cre , its various associated transcription factors , and a trophoblast specific alternative camp - response element to crh gene regulation .", "furthermore , our results are consistent with the possibility that dna methylation controls crh expression indirectly , but any intervening factor that may regulate crh expression by a dna methylation - dependent mechanism remains to be determined ."]]}
{"article_id": "PMC3360846", "article_text": ["the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig . ", "1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .", "1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig . ", "1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .", "these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .", "1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .", "the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .", "the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .", "lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .", "the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .", "however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .", "lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case", "gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .", "lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .", "also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .", "unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .", "one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .", "2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation", ". bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .", "the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .  ", "ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .", "they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .", "rajeswaran et al .   solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,", "there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .", "their crystal structures as well as the structure of the native ribosome   are available through the protein data bank ( pdb ) .", "these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .", "two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .", "theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .", "although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,", "no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .", "the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .", "these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .", "the methods applied in the calculations are described in the first section of the paper .", "we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds .", "four structures of clindamycin are available in the protein data bank   ( as of may 2011 ) .", "three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .", "the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome  clindamycin complexes of deinococcus radiodurans   ( pdb code 1jzx ) and haloarcula marismortui   ( pdb code 1yjn ) .", "therefore , based on the known x - ray structures of clindamycin ( fig . ", "2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .", ", we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig . ", "2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .", "the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .", "the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .", "no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called  exp - opt ,  were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .", "to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .", "therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,", "the solvent is described by a dielectric constant , which was set to 80 in our work .", "the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .", "the positions of these point charges were obtained from the x - ray structures of the ribosome ", "the coordinates of residues within 10  of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .", "next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .", "the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .", "nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin  spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .", "shielding constants were calculated using the b3lyp / aug - pcs-1   method and the giao routine [ 2630 ] .", "the chemical shifts of the i - th nuclei were calculated as \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\delta ( i ) = \\sigma_i(x ) - \\sigma_i(c ) , $ $ \\end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .", "spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .", "the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .", "the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .", "data were analyzed using gabedit , and the visualizations were carried out with vmd   and xdrawchem .", "the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 . the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol )", "are presented in fig .  5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,", "the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .", "to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .", "in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig .  6 ) . the numerical values for the electron density ( bcp ) and", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions", ". however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure", ", we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .", "as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and", "its chemical shift is lower for the b conformer than for the a conformer of clindamycin .", "the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer", ". however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum", "the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method", "in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 .", "the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol ) are presented in fig . ", "5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize", ", nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig . ", "the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table  4 .", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9  h39c152.3700.01460.0131cp7c17cl28 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .", "bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .", "however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as", "chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group ,", "the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer .", "however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer . this value is large enough to be measured experimentally.fig .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum", "we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .", "internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,", "we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin  spin coupling constants to the conformation of clindamycin .", "the two most stable conformers of clindamycin exhibit c = oh  o intramolecular hydrogen bonds . according to nbo and aim analyses ,", "the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water ."], "abstract_text": ["<S> lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens . </S>", "<S> this group of drugs inhibits the activity of the bacterial ribosome by binding to the 23s rna of the large ribosomal subunit and blocking protein synthesis . </S>", "<S> currently , three x - ray structures of the ribosome in complex with clindamycin are available in the protein data bank , which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin . in this work , we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23s rna . </S>", "<S> we studied three lincosamide antibiotics : clindamycin , lincomycin , and pirlimycin at the b3lyp level with the 6 - 31 g * * basis set . </S>", "<S> the focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties . </S>", "<S> for both functional conformers , we applied natural bond orbital ( nbo ) analysis and the atoms in molecules ( aim ) theory , and calculated the nmr parameters . </S>", "<S> based on the results obtained , we were able to show that the structure with the intramolecular hydrogen bond c = o  h  o is the most stable conformer of clindamycin . </S>", "<S> the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , was found to be the dominant factor influencing the high stability of this conformer.figuremolecular graph of more stable conformer of clindamycin . </S>"], "labels": null, "section_names": ["Introduction", "Computational details", "Results and discussion", "Conformational analysis", "Natural bond analysis", "Atoms in Molecules analysis", "NMR chemical shifts", "Conclusions"], "sections": [["the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig . ", "1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .", "1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig . ", "1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .", "these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .", "1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .", "the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .", "the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .", "lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .", "the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .", "however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .", "lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case", "gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .", "lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .", "also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .", "unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .", "one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .", "2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation", ". bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .", "the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .  ", "ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .", "they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .", "rajeswaran et al .   solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,", "there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .", "their crystal structures as well as the structure of the native ribosome   are available through the protein data bank ( pdb ) .", "these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .", "two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .", "theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .", "although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,", "no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .", "the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .", "these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .", "the methods applied in the calculations are described in the first section of the paper .", "we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds ."], ["four structures of clindamycin are available in the protein data bank   ( as of may 2011 ) .", "three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .", "the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome  clindamycin complexes of deinococcus radiodurans   ( pdb code 1jzx ) and haloarcula marismortui   ( pdb code 1yjn ) .", "therefore , based on the known x - ray structures of clindamycin ( fig . ", "2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .", ", we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig . ", "2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .", "the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .", "the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .", "no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called  exp - opt ,  were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .", "to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .", "therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,", "the solvent is described by a dielectric constant , which was set to 80 in our work .", "the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .", "the positions of these point charges were obtained from the x - ray structures of the ribosome ", "the coordinates of residues within 10  of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .", "next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .", "the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .", "nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin  spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .", "shielding constants were calculated using the b3lyp / aug - pcs-1   method and the giao routine [ 2630 ] .", "the chemical shifts of the i - th nuclei were calculated as \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\delta ( i ) = \\sigma_i(x ) - \\sigma_i(c ) , $ $ \\end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .", "spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .", "the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .", "the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .", "data were analyzed using gabedit , and the visualizations were carried out with vmd   and xdrawchem ."], ["the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 . the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol )", "are presented in fig .  5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,", "the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .", "to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .", "in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig .  6 ) . the numerical values for the electron density ( bcp ) and", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions", ". however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure", ", we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .", "as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and", "its chemical shift is lower for the b conformer than for the a conformer of clindamycin .", "the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer", ". however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum"], ["the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method"], ["in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 .", "the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol ) are presented in fig . ", "5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize", ", nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer ."], ["the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig . ", "the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table  4 .", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9  h39c152.3700.01460.0131cp7c17cl28 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .", "bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .", "however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule ."], ["the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as", "chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group ,", "the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer .", "however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer . this value is large enough to be measured experimentally.fig .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum"], ["we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .", "internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,", "we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin  spin coupling constants to the conformation of clindamycin .", "the two most stable conformers of clindamycin exhibit c = oh  o intramolecular hydrogen bonds . according to nbo and aim analyses ,", "the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water ."]]}
{"article_id": "PMC5178859", "article_text": ["the rising prevalence of alzheimer s disease and related dementias ( henceforth referred to as dementia ) is emerging as a leading global health system challenge .", "effective early diagnosis and management models are required to mitigate its impact on patients , caregivers , and health - care systems . enhancing primary care capacity", "however , dementia care predominantly resides with geriatric specialists , who are in short supply in canada and elsewhere , delaying access to care . to enhance the care of persons with dementia ,", "many of these have been established as primary care - based memory clinics ( pcmcs ) .", "initial evaluations suggest that pcmcs can provide timelier assessment , lead to a high degree of satisfaction among referring physicians , patients , and caregivers , and streamline access to specialists . in order to retain the fidelity of such programs and consistency with initial training and practice guidelines , and", "practice drift and ensure ongoing high quality of care , quality assurance frameworks are needed .", "quality indicators ( qis ) , based on best practices , define achievable benchmarks and a quality assurance framework facilitates practice improvement through targeted , educational quality improvement mechanisms . to our knowledge", ", there is no quality assurance framework specific to dementia care in primary care - based settings .", "this paper describes the results of a consensus approach to identify qis and quality improvement mechanisms in an ontario - wide network of interprofessional pcmcs , and identify potential barriers and facilitators to the implementation of a quality assurance framework .", "a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .", "the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .", "we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .", "respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important .", "links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .", "after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .", "qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .", "data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round .", "student s t - test was used to identify significant differences between pcmc clinicians and specialists .", "spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .", "two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round .", "a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .", "the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .", "we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .", "respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important .", "links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .", "after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .", "qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .", "data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round .", "student s t - test was used to identify significant differences between pcmc clinicians and specialists .", "spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .", "two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round .", "two survey rounds were conducted between april and june 2014 . in the first round , 842 surveys were distributed with a response rate of 21.3% .", "the majority of respondents were physicians , and nurses and other health professionals equally represented the remainder of respondents ( table 2 ) .", "respondents had an average of 14.6  10.1 years of clinical practice with older adults . among pcmc respondents ,", "31% were from urban centres , 42% rural settings , and 25% from mixed urban / rural populations .", "in contrast , 73% of specialists worked in urban settings , and 24% served mixed populations .", "only 11 neurologists responded to the first survey , and their specialty was not included in round 2 . in round 2 ,", "respondents had an average of 17.42  10.08 years of clinical practice with older adults and practice settings were similar to round 1 .", "a third delphi round was not conducted due to the substantial drop in response rate between rounds 1 and 2 .", "table 3 presents the results of the consensus process and table 4 presents the final list of quality indicators .", "quality improvement mechanisms characterized by specialist integration , including case discussions , shared care , observerships , and mentorships , ranked highly ( table 5 ) .", "other preferred quality improvement mechanisms included standardized electronic charting forms , self - directed learning activities , and interactive programs .", "survey respondents recommended that between 1030% of patients seen in a pcmc also be reviewed by a specialist .", "descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process . almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .", "most recognized a formal quality assurance framework and individual qis as essential .  great indicators and very necessary  [ pcmc clinician , round 1 ] . ", "our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family  [ pcmc clinician , delphi round 2 ] .", " all are relevant quality indicators for a pcmc  [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .", "others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work . ", "is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ?  [ pcmc clinician , round 1 ] .", " this is the canadian consensus guideline that every doc should know  [ specialist , round 1 ; emphasis added by authors ] .", "a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .", "respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .", "information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources  [ pcmc clinician , round 2 ] .", "this lack of clarity was considered most problematic with regard to patient follow - up .", " patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out .  [ specialist , round 1 ] .", "similar concerns were raised regarding the management of comorbidities .   [ my ] assumption is that management of comorbidities is the primary care physician s domain .", "recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician  [ pcmc clinician , round 2 ] .", "it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment .  referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral  [ pcmc clinician , round 1 ] .", " my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source  [ specialist , round 1 ] .", "respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .", "one of the main obstacles was the perceived burden of documentation required to implement qis . ", "[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search  [ pcmc clinician , round 2 ] . integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .", "making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it ", "a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .", " i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist  [ specialist , round 1 ] .", "access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .  having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics  [ pcmc clinician , round 2 ] .", " the [ social work ] and [ occupational therapy ] members of the team do an excellent job  the [ alzheimer s society ] representative has been excellent as well when given an opportunity  [ pcmc clinician , round 2 ] .", "an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease . ", "palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced", ". family physician team will do this in following up patient  [ pcmc clinician , round 1 ] .", "lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .", "they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .", " this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team  [ pcmc clinician , round 1 ] .", "barriers to the implementation of this framework included role confusion among stakeholders and limited resources .", "descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process .", "almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .", "most recognized a formal quality assurance framework and individual qis as essential .  great indicators and very necessary  [ pcmc clinician , round 1 ] . ", "our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family  [ pcmc clinician , delphi round 2 ] .", " all are relevant quality indicators for a pcmc  [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .", "others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work . ", "is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ?  [ pcmc clinician , round 1 ] .", " this is the canadian consensus guideline that every doc should know  [ specialist , round 1 ; emphasis added by authors ] .", "a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .", "respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement . ", "information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources  [ pcmc clinician , round 2 ] .", "this lack of clarity was considered most problematic with regard to patient follow - up .", " patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out .  [ specialist , round 1 ] .", "similar concerns were raised regarding the management of comorbidities .   [ my ] assumption is that management of comorbidities is the primary care physician s domain .", "recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician  [ pcmc clinician , round 2 ] .", "it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment .  referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral  [ pcmc clinician , round 1 ] . ", "my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source  [ specialist , round 1 ] .", "respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .", "one of the main obstacles was the perceived burden of documentation required to implement qis . ", "[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search  [ pcmc clinician , round 2 ] .", "integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .", "making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it  [ pcmc clinician , round 2 ] . a second issue identified in relation to the implementation of the quality assurance framework", " i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist ", "access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .  having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics  [ pcmc clinician , round 2 ] .", " the [ social work ] and [ occupational therapy ] members of the team do an excellent job  the [ alzheimer s society ] representative has been excellent as well when given an opportunity  [ pcmc clinician , round 2 ] .", "an important finding is the relative lack of importance ascribed to end - of - life planning .", "while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease . ", "palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .", "family physician team will do this in following up patient  [ pcmc clinician , round 1 ] .", "lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .", "they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach . ", "this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team  [ pcmc clinician , round 1 ] .", "barriers to the implementation of this framework included role confusion among stakeholders and limited resources .", "almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .", "most recognized a formal quality assurance framework and individual qis as essential .  great indicators and very necessary  [ pcmc clinician , round 1 ] . ", "our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family  [ pcmc clinician , delphi round 2 ] .", " all are relevant quality indicators for a pcmc  [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .", "others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .", " is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ?  [ pcmc clinician , round 1 ] .", " this is the canadian consensus guideline that every doc should know  [ specialist , round 1 ; emphasis added by authors ] .", "a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .", "respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement . ", "information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources  [ pcmc clinician , round 2 ] .", "this lack of clarity was considered most problematic with regard to patient follow - up .", " patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out .  [ specialist , round 1 ] .", "similar concerns were raised regarding the management of comorbidities .   [ my ] assumption is that management of comorbidities is the primary care physician s domain .", "recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician  [ pcmc clinician , round 2 ] .", "it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment . ", "referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral  [ pcmc clinician , round 1 ] . ", "my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source  [ specialist , round 1 ] .", "respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .", "one of the main obstacles was the perceived burden of documentation required to implement qis . ", "[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search  [ pcmc clinician , round 2 ] .", "integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .", "making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it ", "a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .", " i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist  [ specialist , round 1 ] .", "access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .", " having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics  [ pcmc clinician , round 2 ] .", " the [ social work ] and [ occupational therapy ] members of the team do an excellent job  the [ alzheimer s society ] representative has been excellent as well when given an opportunity  [ pcmc clinician , round 2 ] .", "an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance", ", many expressed that such planning discussions were not appropriate for patients with less advanced disease .  palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .", "family physician team will do this in following up patient  [ pcmc clinician , round 1 ] .", "lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .", "they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach . ", "this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team  [ pcmc clinician , round 1 ] .", "barriers to the implementation of this framework included role confusion among stakeholders and limited resources .", "this study used a consultative process among primary and specialty providers to identify a core group of qis and preferred quality improvement mechanisms for dementia care .", "quality assurance can be an effective method to ensure fidelity to best practices and maintain a high standard of care quality .", "selected qis address care processes , assessment and reassessment , medication review and management , investigations , non - pharmacological management , pharmacological management , and managing concomitant conditions .", "selected quality improvement mechanisms emphasize a desire for multimodal education and closer collaboration with specialists . assessing care quality and publically reporting the findings are increasingly commonplace .", "however , quality assurance still mainly focuses on specific care sectors or episodes , rather than on overall care for conditions that require coordination and collaboration across multiple sectors . in that context", "critical considerations include educating clinicians about the role of quality assurance , improving role clarity among providers involved in dementia care , expanding access to allied health professionals , and creating standardized electronic medical record templates to simplify qi documentation .", "quality assurance must not create additional burden on clinicians often working with limited resources and time , a burden that could also impact the quality of the clinician - patient interaction .", "respondents proposed that 1030% of patients seen in pcmcs be reviewed with a specialist , preferably in a shared care approach .", "closer integration of urban specialists with rural pcmcs represents a tremendous opportunity to extend quality dementia care , particularly to rural patients .", ", it will be necessary to identify barriers ( e.g. , allocation of funding ) and facilitators ( e.g. , resources for coordination , electronic medical records , telemedicine ) .", "another important finding was the relatively low rankings of qis related to end - of - life planning with dementia patients .", "in addition to potential discomfort among clinicians to address such issues , this finding may also stem from the understandable desire to maintain hope and engagement early in the course of the illness , though undue delays may leave patients and caregivers less able to meaningfully participate in such discussions . the most important identified barrier to the implementation of the quality assurance framework", "is the need for greater clarity on responsibilities of referring family physicians , pcmc clinicians , and specialists , particularly with respect to follow - up , physical examination , and care of complex comorbidities .", "quality assurance often targets relatively simple conditions , such as hypertension , or restricts its scope to specific aspects , processes or locations of care .", "in contrast , dementia , like all major chronic conditions , follows a course of progressive decline punctuated by increasingly frequent health complications ( related to dementia itself , as well as to exacerbations of concurrent comorbidities ) , multiple care transitions , progressive caregiver stress and health service utilization , and ultimately death .", "optimal dementia care thus requires a systems approach of integration and coordination . addressing greater role clarity among all dementia stakeholders , a task that with proper resources could be coordinated from within pcmcs , requires immediate attention , in order to ensure a stable clinical infrastructure that is able to safely and effectively address the needs of these patients , wherever they may be and whenever they arise . until such integration is achieved", "first , only two delphi rounds were conducted because the response rate fell markedly after the first .", "however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .", "a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .", "as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .", "the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .", "fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .", "fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .", "finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked .", "first , only two delphi rounds were conducted because the response rate fell markedly after the first . however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .", "a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .", "as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .", "the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .", "fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .", "fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .", "finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked .", "while this study has identified qis and quality improvement mechanisms to assess care quality for dementia , findings underscore the importance of system integration for the provision of quality dementia care , with specifically defined and mutually understood roles among stakeholders and , where necessary , the reallocation of existing resources to support this approach to care .", "an approach whereby clear roles are negotiated among dementia stakeholders can provide sufficient flexibility to meet regional needs ( especially in rural areas where access to specialists is more limited ) , foster more effective collaboration and accountability , and thus facilitate the delivery and measurement of care quality for dementia . within that context , proper field - testing , validation , and evaluation of selected qis and quality improvement mechanisms", "this work has significant implications on the organization of care for aging patients with complex conditions .", "primary and specialist providers share the responsibility of providing and supporting integrated dementia quality care . as such", ", the care of persons with cognitive impairment would be enhanced by the development of a practical and realistically feasible quality assurance framework , under whose umbrella both pcmc and specialist services are integrated , and which ensures high fidelity to intended design and best practices , and thus maintains a high level of care quality ."], "abstract_text": ["<S> backgroundprimary care - based memory clinics ( pcmcs ) have been established in several jurisdictions to improve the care for persons with alzheimer s disease and related dementias . </S>", "<S> we sought to identify key quality indicators ( qis ) , quality improvement mechanisms , and potential barriers and facilitators to the establishment of a quality assurance framework for pcmcs.methodswe employed a delphi approach to obtain consensus from pcmc clinicians and specialist physicians on qis and quality improvement mechanisms . </S>", "<S> thirty - eight candidate qis and 19 potential quality improvement mechanisms were presented to participants in two rounds of electronic delphi surveys . </S>", "<S> written comments were collected and descriptively analyzed.resultsthe response rate for the first and second rounds were 21.3% ( n = 179 ) and 12.8% ( n = 88 ) , respectively . </S>", "<S> the majority of respondents were physicians . </S>", "<S> fourteen qis remained after the consensus process . </S>", "<S> ten quality improvement mechanisms were selected with those characterized by specialist integration , such as case discussions and mentorships , being ranked highly . </S>", "<S> written comments revealed three major themes related to potential barriers and facilitators to quality assurance : 1 ) perceived importance , 2 ) collaboration and role clarity , and 3 ) implementation process.conclusionwe successfully utilized a consultative process among primary and specialty providers to identify core qis and quality improvement mechanisms for pcmcs . identified quality improvement mechanisms highlight desire for multi - modal education . system integration and closer integration between pcmcs and specialists were emphasized as essential for the provision of high - quality dementia care in community settings . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Protocol and Process", "Data Collection", "Data Analysis", "RESULTS", "Respondent Comments", "1. Need for and Relevance of Quality Assurance for Dementia Care", "2. Collaboration and Role Clarity", "3. Process of QA Framework Implementation in PCMCs", "DISCUSSION", "Limitations of the Study", "CONCLUSIONS"], "sections": [["the rising prevalence of alzheimer s disease and related dementias ( henceforth referred to as dementia ) is emerging as a leading global health system challenge .", "effective early diagnosis and management models are required to mitigate its impact on patients , caregivers , and health - care systems . enhancing primary care capacity", "however , dementia care predominantly resides with geriatric specialists , who are in short supply in canada and elsewhere , delaying access to care . to enhance the care of persons with dementia ,", "many of these have been established as primary care - based memory clinics ( pcmcs ) .", "initial evaluations suggest that pcmcs can provide timelier assessment , lead to a high degree of satisfaction among referring physicians , patients , and caregivers , and streamline access to specialists . in order to retain the fidelity of such programs and consistency with initial training and practice guidelines , and", "practice drift and ensure ongoing high quality of care , quality assurance frameworks are needed .", "quality indicators ( qis ) , based on best practices , define achievable benchmarks and a quality assurance framework facilitates practice improvement through targeted , educational quality improvement mechanisms . to our knowledge", ", there is no quality assurance framework specific to dementia care in primary care - based settings .", "this paper describes the results of a consensus approach to identify qis and quality improvement mechanisms in an ontario - wide network of interprofessional pcmcs , and identify potential barriers and facilitators to the implementation of a quality assurance framework ."], ["a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .", "the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .", "we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .", "respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important .", "links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .", "after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .", "qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .", "data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round .", "student s t - test was used to identify significant differences between pcmc clinicians and specialists .", "spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .", "two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round ."], ["a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .", "the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .", "we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .", "respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important ."], ["links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .", "after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .", "qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .", "data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round ."], ["student s t - test was used to identify significant differences between pcmc clinicians and specialists .", "spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .", "two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round ."], ["two survey rounds were conducted between april and june 2014 . in the first round , 842 surveys were distributed with a response rate of 21.3% .", "the majority of respondents were physicians , and nurses and other health professionals equally represented the remainder of respondents ( table 2 ) .", "respondents had an average of 14.6  10.1 years of clinical practice with older adults . among pcmc respondents ,", "31% were from urban centres , 42% rural settings , and 25% from mixed urban / rural populations .", "in contrast , 73% of specialists worked in urban settings , and 24% served mixed populations .", "only 11 neurologists responded to the first survey , and their specialty was not included in round 2 . in round 2 ,", "respondents had an average of 17.42  10.08 years of clinical practice with older adults and practice settings were similar to round 1 .", "a third delphi round was not conducted due to the substantial drop in response rate between rounds 1 and 2 .", "table 3 presents the results of the consensus process and table 4 presents the final list of quality indicators .", "quality improvement mechanisms characterized by specialist integration , including case discussions , shared care , observerships , and mentorships , ranked highly ( table 5 ) .", "other preferred quality improvement mechanisms included standardized electronic charting forms , self - directed learning activities , and interactive programs .", "survey respondents recommended that between 1030% of patients seen in a pcmc also be reviewed by a specialist .", "descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process . almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .", "most recognized a formal quality assurance framework and individual qis as essential .  great indicators and very necessary  [ pcmc clinician , round 1 ] . ", "our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family  [ pcmc clinician , delphi round 2 ] .", " all are relevant quality indicators for a pcmc  [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .", "others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work . ", "is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ?  [ pcmc clinician , round 1 ] .", " this is the canadian consensus guideline that every doc should know  [ specialist , round 1 ; emphasis added by authors ] .", "a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .", "respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .", "information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources  [ pcmc clinician , round 2 ] .", "this lack of clarity was considered most problematic with regard to patient follow - up .", " patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out .  [ specialist , round 1 ] .", "similar concerns were raised regarding the management of comorbidities .   [ my ] assumption is that management of comorbidities is the primary care physician s domain .", "recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician  [ pcmc clinician , round 2 ] .", "it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment .  referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral  [ pcmc clinician , round 1 ] .", " my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source  [ specialist , round 1 ] .", "respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .", "one of the main obstacles was the perceived burden of documentation required to implement qis . ", "[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search  [ pcmc clinician , round 2 ] . integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .", "making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it ", "a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .", " i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist  [ specialist , round 1 ] .", "access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .  having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics  [ pcmc clinician , round 2 ] .", " the [ social work ] and [ occupational therapy ] members of the team do an excellent job  the [ alzheimer s society ] representative has been excellent as well when given an opportunity  [ pcmc clinician , round 2 ] .", "an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease . ", "palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced", ". family physician team will do this in following up patient  [ pcmc clinician , round 1 ] .", "lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .", "they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .", " this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team  [ pcmc clinician , round 1 ] .", "barriers to the implementation of this framework included role confusion among stakeholders and limited resources ."], ["descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process .", "almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .", "most recognized a formal quality assurance framework and individual qis as essential .  great indicators and very necessary  [ pcmc clinician , round 1 ] . ", "our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family  [ pcmc clinician , delphi round 2 ] .", " all are relevant quality indicators for a pcmc  [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .", "others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work . ", "is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ?  [ pcmc clinician , round 1 ] .", " this is the canadian consensus guideline that every doc should know  [ specialist , round 1 ; emphasis added by authors ] .", "a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .", "respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement . ", "information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources  [ pcmc clinician , round 2 ] .", "this lack of clarity was considered most problematic with regard to patient follow - up .", " patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out .  [ specialist , round 1 ] .", "similar concerns were raised regarding the management of comorbidities .   [ my ] assumption is that management of comorbidities is the primary care physician s domain .", "recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician  [ pcmc clinician , round 2 ] .", "it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment .  referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral  [ pcmc clinician , round 1 ] . ", "my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source  [ specialist , round 1 ] .", "respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .", "one of the main obstacles was the perceived burden of documentation required to implement qis . ", "[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search  [ pcmc clinician , round 2 ] .", "integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .", "making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it  [ pcmc clinician , round 2 ] . a second issue identified in relation to the implementation of the quality assurance framework", " i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist ", "access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .  having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics  [ pcmc clinician , round 2 ] .", " the [ social work ] and [ occupational therapy ] members of the team do an excellent job  the [ alzheimer s society ] representative has been excellent as well when given an opportunity  [ pcmc clinician , round 2 ] .", "an important finding is the relative lack of importance ascribed to end - of - life planning .", "while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease . ", "palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .", "family physician team will do this in following up patient  [ pcmc clinician , round 1 ] .", "lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .", "they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach . ", "this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team  [ pcmc clinician , round 1 ] .", "barriers to the implementation of this framework included role confusion among stakeholders and limited resources ."], ["almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .", "most recognized a formal quality assurance framework and individual qis as essential .  great indicators and very necessary  [ pcmc clinician , round 1 ] . ", "our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family  [ pcmc clinician , delphi round 2 ] .", " all are relevant quality indicators for a pcmc  [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .", "others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .", " is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ?  [ pcmc clinician , round 1 ] .", " this is the canadian consensus guideline that every doc should know  [ specialist , round 1 ; emphasis added by authors ] ."], ["a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .", "respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement . ", "information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources  [ pcmc clinician , round 2 ] .", "this lack of clarity was considered most problematic with regard to patient follow - up .", " patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out .  [ specialist , round 1 ] .", "similar concerns were raised regarding the management of comorbidities .   [ my ] assumption is that management of comorbidities is the primary care physician s domain .", "recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician  [ pcmc clinician , round 2 ] .", "it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment . ", "referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral  [ pcmc clinician , round 1 ] . ", "my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source  [ specialist , round 1 ] ."], ["respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .", "one of the main obstacles was the perceived burden of documentation required to implement qis . ", "[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search  [ pcmc clinician , round 2 ] .", "integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .", "making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it ", "a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .", " i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist  [ specialist , round 1 ] .", "access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .", " having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics  [ pcmc clinician , round 2 ] .", " the [ social work ] and [ occupational therapy ] members of the team do an excellent job  the [ alzheimer s society ] representative has been excellent as well when given an opportunity  [ pcmc clinician , round 2 ] .", "an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance", ", many expressed that such planning discussions were not appropriate for patients with less advanced disease .  palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .", "family physician team will do this in following up patient  [ pcmc clinician , round 1 ] .", "lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .", "they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach . ", "this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team  [ pcmc clinician , round 1 ] .", "barriers to the implementation of this framework included role confusion among stakeholders and limited resources ."], ["this study used a consultative process among primary and specialty providers to identify a core group of qis and preferred quality improvement mechanisms for dementia care .", "quality assurance can be an effective method to ensure fidelity to best practices and maintain a high standard of care quality .", "selected qis address care processes , assessment and reassessment , medication review and management , investigations , non - pharmacological management , pharmacological management , and managing concomitant conditions .", "selected quality improvement mechanisms emphasize a desire for multimodal education and closer collaboration with specialists . assessing care quality and publically reporting the findings are increasingly commonplace .", "however , quality assurance still mainly focuses on specific care sectors or episodes , rather than on overall care for conditions that require coordination and collaboration across multiple sectors . in that context", "critical considerations include educating clinicians about the role of quality assurance , improving role clarity among providers involved in dementia care , expanding access to allied health professionals , and creating standardized electronic medical record templates to simplify qi documentation .", "quality assurance must not create additional burden on clinicians often working with limited resources and time , a burden that could also impact the quality of the clinician - patient interaction .", "respondents proposed that 1030% of patients seen in pcmcs be reviewed with a specialist , preferably in a shared care approach .", "closer integration of urban specialists with rural pcmcs represents a tremendous opportunity to extend quality dementia care , particularly to rural patients .", ", it will be necessary to identify barriers ( e.g. , allocation of funding ) and facilitators ( e.g. , resources for coordination , electronic medical records , telemedicine ) .", "another important finding was the relatively low rankings of qis related to end - of - life planning with dementia patients .", "in addition to potential discomfort among clinicians to address such issues , this finding may also stem from the understandable desire to maintain hope and engagement early in the course of the illness , though undue delays may leave patients and caregivers less able to meaningfully participate in such discussions . the most important identified barrier to the implementation of the quality assurance framework", "is the need for greater clarity on responsibilities of referring family physicians , pcmc clinicians , and specialists , particularly with respect to follow - up , physical examination , and care of complex comorbidities .", "quality assurance often targets relatively simple conditions , such as hypertension , or restricts its scope to specific aspects , processes or locations of care .", "in contrast , dementia , like all major chronic conditions , follows a course of progressive decline punctuated by increasingly frequent health complications ( related to dementia itself , as well as to exacerbations of concurrent comorbidities ) , multiple care transitions , progressive caregiver stress and health service utilization , and ultimately death .", "optimal dementia care thus requires a systems approach of integration and coordination . addressing greater role clarity among all dementia stakeholders , a task that with proper resources could be coordinated from within pcmcs , requires immediate attention , in order to ensure a stable clinical infrastructure that is able to safely and effectively address the needs of these patients , wherever they may be and whenever they arise . until such integration is achieved", "first , only two delphi rounds were conducted because the response rate fell markedly after the first .", "however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .", "a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .", "as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .", "the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .", "fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .", "fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .", "finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked ."], ["first , only two delphi rounds were conducted because the response rate fell markedly after the first . however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .", "a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .", "as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .", "the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .", "fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .", "fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .", "finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked ."], ["while this study has identified qis and quality improvement mechanisms to assess care quality for dementia , findings underscore the importance of system integration for the provision of quality dementia care , with specifically defined and mutually understood roles among stakeholders and , where necessary , the reallocation of existing resources to support this approach to care .", "an approach whereby clear roles are negotiated among dementia stakeholders can provide sufficient flexibility to meet regional needs ( especially in rural areas where access to specialists is more limited ) , foster more effective collaboration and accountability , and thus facilitate the delivery and measurement of care quality for dementia . within that context , proper field - testing , validation , and evaluation of selected qis and quality improvement mechanisms", "this work has significant implications on the organization of care for aging patients with complex conditions .", "primary and specialist providers share the responsibility of providing and supporting integrated dementia quality care . as such", ", the care of persons with cognitive impairment would be enhanced by the development of a practical and realistically feasible quality assurance framework , under whose umbrella both pcmc and specialist services are integrated , and which ensures high fidelity to intended design and best practices , and thus maintains a high level of care quality ."]]}
{"article_id": "PMC4421382", "article_text": ["symptomatic supravesical obstructive uropathy in a patient with dual functioning kidneys is classically characterized by bilateral hydroureters / hydronephrosis and an empty urinary bladder .", "this obstruction may be secondary to metastatic abdomino - pelvic and retroperitoneal malignancies , ureteric calculi and retroperitoneal fibrosis [ 25 ]", ". the evidence for obstruction may be partially or totally absent on ultrasound or computerized tomography , in one or both kidneys [ 68 ] .", "our recent experience with a uraemic 56-year - old caucasian showing only unilateral moderate right - sided hydronephrosis is presented .", "a 56-year - old caucasian male patient with a past medical history that included hypertension and coronary artery disease and a recent serum creatinine of 88.4 mol / l , was diagnosed 10 days earlier with urinary tract infection ( uti ) .", "two days before presentation to us , he developed worsening anorexia , nausea with vomiting and oliguria .", "his father had prostate cancer and died at age 75 years from colon cancer ; a sister had breast cancer and a maternal uncle had throat cancer . physical examination revealed a blood pressure of 165/89 mmhg , pulse rate of 56/min and respiratory rate of 16/min .", "he was not dehydrated nor orthostatic . except for trace ankle oedema and bilateral costo - vertebral angle punch tenderness ,", "mol / l , co2 14 mmol / l and potassium 5.5 mmol / l .", "urinalysis showed 510 wbc per high - power field ( phf ) and 25 rbc phf .", "alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) and total creatine kinase ( ck ) levels were normal .", "a post - void urinary bladder scan had demonstrated only 60 ml of residual urine .", "a renal ultrasound showed unilateral moderate right - sided hydronephrosis with suspected mass effect on the inferior urinary bladder .", "the left kidney appeared normal , measuring 16.2 cm  8.1 cm , with preserved cortical thickness .", "the next day , with increasing vomiting , serum creatinine 894.6 mol / l , phosphorus 2.6 mmol / l , potassium 6.2 mmol / l and oliguria ( table 1 ) , he started daily haemodialysis .", "a non - contrast computerized tomography examination , on day 2 , again showed unilateral right - sided hydronephrosis , bilateral nephric stranding and urinary bladder wall thickening suspicious for transitional cell cancer ( figure 1 ) .", "cystoscopy , on day 3 , revealed a sessile urinary bladder tumour , which was resected .", "the right ureter was successfully cannulated and a right ureteric stent was placed with prompt urine drainage .", "the next day , with a strong push from nephrology , the patient consented to a percutaneous left nephrostomy procedure despite the normal appearing left kidney .", "a percutaneous left nephrostogram revealed a previously unrecognized mild hydronephrosis / hydroureter with obstruction at the ureterovesical junction .", "the pathology report revealed high - grade urothelial carcinoma , grade 3 of 3 , with invasion of the muscularis propria .", "a patient with dual functioning kidneys presenting with uraemic symptoms and suspected to have obstructive uropathy must be presumed to necessarily have bilateral renal obstruction [ 610 ] .", "this is without prejudice to the findings on conventional renal imaging with ultrasound or computerized tomography [ 3,610 ] .", "there are false negative tests with these imaging modalities , the so - called syndrome of  non - dilated obstructive uropathy or non - dilated obstructive nephropathy [ 610 ] .", "clinical conditions associated with the absence of hydronephrosis on ultrasound and computerized tomography despite obstructed kidney(s ) include acute early obstruction , the presence of retroperitoneal fibrosis or infiltrative metastatic abdomino - pelvic cancers , dehydration or septic shock and severe oliguria [ 210 ] .", "our patient was not dehydrated and was not hypotensive but was severely oliguric ( table 1 ) .", "we note that we did not rule out the presence of retroperitoneal fibrosis in our patient .", "the classic picture of bilateral hydronephrosis with hydroureters and an empty urinary bladder , in symptomatic uraemia following supravesical obstruction , in patients with dual functioning kidneys , is well acknowledged .", "however , the presentation of new - onset symptomatic uraemia concurrent with only unilateral hydronephrosis / hydroureter on conventional imaging ( ultrasound , computerized tomography ) should raise the plausibility of non - apparent obstruction of the contra lateral kidney .", "in such instances , the more sensitive albeit invasive percutaneous nephrostogram of the apparently normal appearing kidney is therapeutic and will lead to greater renal salvage [ 68 ] .", "we would like to remind practicing providers that symptomatic uraemia presenting in the setting of suspected obstructive uropathy must be assumed to imply bilateral renal obstruction , regardless of the results / interpretations of any form of conventional renal imaging . therefore , necessarily , every attempt to decompress both kidneys must be the rule .", "this approach would result in early and improved renal salvage . left undiagnosed and therefore untreated", ", this potentially reversible cause of renal failure can lead to irreversible renal failure if bilateral , , or to significant residual loss of renal function if missed on one side only .", "we note that we were not able to carry out any split renal functional testing after recovery as the patient 's primary attention at this point was to find out treatment options for his cancer ."], "abstract_text": ["<S> bilateral hydronephrosis is classic for supravesical obstructive uropathy causing uraemia with dual functioning kidneys . recently , a patient presented with uraemia and metastatic urinary bladder carcinoma but only unilateral right - sided hydronephrosis . </S>", "<S> a right ureteral stent was placed retrograde and no further intervention was planned since the left kidney appeared normal , and since the left ureteric orifice was not visualized . </S>", "<S> we insisted on a left percutaneous nephrostomy which was successful with prompt urine return . </S>", "<S> a left nephrostogram revealed unrecognized hydroureter / hydronephrosis . </S>", "<S> following haemodialysis , kidney function normalized at 3 weeks . for symptomatic </S>", "<S> uraemia from obstruction , an antegrade and/or a retrograde decompression must be attempted bilaterally to improve renal salvage . </S>"], "labels": null, "section_names": ["Background", "Case report", "Discussion"], "sections": [["symptomatic supravesical obstructive uropathy in a patient with dual functioning kidneys is classically characterized by bilateral hydroureters / hydronephrosis and an empty urinary bladder .", "this obstruction may be secondary to metastatic abdomino - pelvic and retroperitoneal malignancies , ureteric calculi and retroperitoneal fibrosis [ 25 ]", ". the evidence for obstruction may be partially or totally absent on ultrasound or computerized tomography , in one or both kidneys [ 68 ] .", "our recent experience with a uraemic 56-year - old caucasian showing only unilateral moderate right - sided hydronephrosis is presented ."], ["a 56-year - old caucasian male patient with a past medical history that included hypertension and coronary artery disease and a recent serum creatinine of 88.4 mol / l , was diagnosed 10 days earlier with urinary tract infection ( uti ) .", "two days before presentation to us , he developed worsening anorexia , nausea with vomiting and oliguria .", "his father had prostate cancer and died at age 75 years from colon cancer ; a sister had breast cancer and a maternal uncle had throat cancer . physical examination revealed a blood pressure of 165/89 mmhg , pulse rate of 56/min and respiratory rate of 16/min .", "he was not dehydrated nor orthostatic . except for trace ankle oedema and bilateral costo - vertebral angle punch tenderness ,", "mol / l , co2 14 mmol / l and potassium 5.5 mmol / l .", "urinalysis showed 510 wbc per high - power field ( phf ) and 25 rbc phf .", "alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) and total creatine kinase ( ck ) levels were normal .", "a post - void urinary bladder scan had demonstrated only 60 ml of residual urine .", "a renal ultrasound showed unilateral moderate right - sided hydronephrosis with suspected mass effect on the inferior urinary bladder .", "the left kidney appeared normal , measuring 16.2 cm  8.1 cm , with preserved cortical thickness .", "the next day , with increasing vomiting , serum creatinine 894.6 mol / l , phosphorus 2.6 mmol / l , potassium 6.2 mmol / l and oliguria ( table 1 ) , he started daily haemodialysis .", "a non - contrast computerized tomography examination , on day 2 , again showed unilateral right - sided hydronephrosis , bilateral nephric stranding and urinary bladder wall thickening suspicious for transitional cell cancer ( figure 1 ) .", "cystoscopy , on day 3 , revealed a sessile urinary bladder tumour , which was resected .", "the right ureter was successfully cannulated and a right ureteric stent was placed with prompt urine drainage .", "the next day , with a strong push from nephrology , the patient consented to a percutaneous left nephrostomy procedure despite the normal appearing left kidney .", "a percutaneous left nephrostogram revealed a previously unrecognized mild hydronephrosis / hydroureter with obstruction at the ureterovesical junction .", "the pathology report revealed high - grade urothelial carcinoma , grade 3 of 3 , with invasion of the muscularis propria ."], ["a patient with dual functioning kidneys presenting with uraemic symptoms and suspected to have obstructive uropathy must be presumed to necessarily have bilateral renal obstruction [ 610 ] .", "this is without prejudice to the findings on conventional renal imaging with ultrasound or computerized tomography [ 3,610 ] .", "there are false negative tests with these imaging modalities , the so - called syndrome of  non - dilated obstructive uropathy or non - dilated obstructive nephropathy [ 610 ] .", "clinical conditions associated with the absence of hydronephrosis on ultrasound and computerized tomography despite obstructed kidney(s ) include acute early obstruction , the presence of retroperitoneal fibrosis or infiltrative metastatic abdomino - pelvic cancers , dehydration or septic shock and severe oliguria [ 210 ] .", "our patient was not dehydrated and was not hypotensive but was severely oliguric ( table 1 ) .", "we note that we did not rule out the presence of retroperitoneal fibrosis in our patient .", "the classic picture of bilateral hydronephrosis with hydroureters and an empty urinary bladder , in symptomatic uraemia following supravesical obstruction , in patients with dual functioning kidneys , is well acknowledged .", "however , the presentation of new - onset symptomatic uraemia concurrent with only unilateral hydronephrosis / hydroureter on conventional imaging ( ultrasound , computerized tomography ) should raise the plausibility of non - apparent obstruction of the contra lateral kidney .", "in such instances , the more sensitive albeit invasive percutaneous nephrostogram of the apparently normal appearing kidney is therapeutic and will lead to greater renal salvage [ 68 ] .", "we would like to remind practicing providers that symptomatic uraemia presenting in the setting of suspected obstructive uropathy must be assumed to imply bilateral renal obstruction , regardless of the results / interpretations of any form of conventional renal imaging . therefore , necessarily , every attempt to decompress both kidneys must be the rule .", "this approach would result in early and improved renal salvage . left undiagnosed and therefore untreated", ", this potentially reversible cause of renal failure can lead to irreversible renal failure if bilateral , , or to significant residual loss of renal function if missed on one side only .", "we note that we were not able to carry out any split renal functional testing after recovery as the patient 's primary attention at this point was to find out treatment options for his cancer ."]]}
{"article_id": "PMC2842971", "article_text": ["in 1985 , smith pioneered phage display technology , an in vitro methodology and system for presenting , selecting and evolving proteins and peptides displayed on the surface of phage virion . since then , phage display has developed rapidly and become an increasingly popular tool for both basic research such as the exploration of protein - protein interaction networks and sites [ 24 ] , and applied research such as the development of new diagnostics , therapeutics , and vaccines [ 510 ] .", "usually , the protein used to screen the phage display library is termed as target and the genuine partner binding to the target is called template .", "peptide mimicking the binding site on the template and binding to the target is defined as mimotope , which was first introduced by geysen et al . .", "one type of the most frequently used targets is monoclonal antibody . in this situation ,", "the template is the corresponding antigen inducing the antibody , and the mimotope is a mimic of the genuine epitope .", ". goes  a mimotope is defined as a molecule able to bind to the antigen combining site of an antibody molecule , not necessarily identical with the epitope inducing the antibody , but an acceptable mimic of the essential features of the epitope .", " mimotopes and the corresponding epitope are considered to have similar physicochemical properties and spatial organization .", "the mimicry between mimotopes and genuine epitope makes mimotopes reasonable solutions to epitope mapping , network inferring , and new diagnostics , therapeutics , and vaccines developing .   powered by phage display technology , mimotopes can be acquired in a relatively cheap , efficient and convenient way , that is , screening phage - displayed random peptides libraries with a given target .", "however , not all phages selected out are target - specific , because the target itself is only one component of the screening system . from time to time , phages reacting with contaminants in the target sample or other components of the screening system such as the solid phase ( e.g. , plastic plates ) and the capturing molecule ( e.g. , streptavidin , secondary antibody ) rather than binding to the actual target are recovered with those target - specific binders ( displaying mimotopes ) during the rounds of panning .", "peptides displayed on these phages are called target - unrelated peptides ( tup ) , a term coined recently by menendez and scott in a review .  ", "the results from phage display technology might be a mixture of target - unrelated peptides and mimotopes , and it can be difficult to discriminate tup from mimotopes since the binding assays used to confirm the affinity of peptides for the target often employ the same components as the initial panning experiment .", "therefore , target - unrelated peptides might be taken into study as mimotopes if the researchers are not careful enough .", "obviously , target - unrelated peptides are not appropriate candidates for the development of new diagnostics , therapeutics , and vaccines . for mimotope - based epitope mapping ,", "if tup is included in the mapping , the input data is improper and the result might be misleading .", "there are now quite a few programs for mimotope based epitope mapping , none of them , however , has a procedure to scan , report and exclude target - unrelated peptides [ 1523 ] . in this study , we describe a web server named sarotup , which is an acronym for  scanner and reporter of target - unrelated peptides  .", "sarotup was coded with perl as a cgi program and can be freely accessed and used to scan peptides acquired from phage display technology .", "it is capable of finding , reporting , and precluding possible target - unrelated peptides , which is very helpful for the development of mimotope - based diagnostics , therapeutics , and vaccines .", "the power and efficiency of sarotup was also demonstrated by preliminary tests in the present study .", "recently , menendez and scott reviewed a collection of target - unrelated peptides recovered in the screening of phage - displayed random peptide libraries with antibodies .", "they divided their collection into several categories according to the component of the screening system to which target - unrelated peptides bind .", "very recently , brammer et al . reported a completely new type of target - unrelated peptides . in the review of menendez and scott", ", target - unrelated selection is due to the binding to contaminants or components other than target ; however , in the report of brammer et al .", ", target - unrelated selection is due to a coincident point mutation in the phage library [ 12 , 13 ] .", "we compiled a set of 23 tup motifs from the above two references [ 12 , 13 ] , including 12 motifs specific for the capturing agents , 5 motifs specific for the constant region of antibody , 3 motifs specific for the screening solid phase , 2 motifs specific for the contaminants in the target sample , and 1 motif for a mutation in phage library ( table 1 ) .", "the sarotup was implemented as a free online service , powered by apache and perl .", "three pages are designed and integrated into a tabbed web interface with cascading style sheets codes .", "the core program of sarotup was sar.pl , a cgi script coded with perl . in this script , the 23 tup motifs were converted to regular expressions , which were then used to match each input peptide sequence .", "we constructed two - test data sets from [ 12 , 13 , 1523 , 25 , 26 ] .", "the first data set contains 8 cases ; 6 of them are sourced from test cases used in extant programs for mimotope - based epitope mapping [ 1523 ] ; the left 2 are cases studies published recently [ 25 , 26 ] .", "as shown in table 2 , the target of each case in the first data set is monoclonal antibody and the structure of corresponding antigen - antibody complex has been resolved , which is used to derive its structural epitope as the golden standard for evaluation . for each case", "if target - unrelated peptides were found , a new panel of peptides excluding tup was produced .", "the old and the new panel of peptides were then used to predict epitope using mapitope or pepsurf [ 15 , 21 , 22 ] . finally , the results were compared to show if sarotup could improve the performance of mimotope - based epitope mapping .   the second data set is composed of 100 peptides in raw sequence format .", "the first group has 77 sequences compiled from the first data set without any known tup motifs ; the second group has 23 sequences sourced from [ 12 , 13 ] with various tup motifs . the mixture of the two groups of sequences made the second data set , which was then used as the sample input and can be used to evaluate the efficiency of sarotup .", "as a free online service , the web interface of sarotup has successfully been implemented as a tabbed web page .", "the left tab is the default page , providing a brief introduction to this web service .", "the users can either paste a set of peptide sequences in the text box or upload a sequence file to the sarotup server for scanning .", "as shown in figure 1 , a panel of peptides in raw sequence format taken from the b12 test case was pasted in the text box . besides the raw sequences", "however , only the standard iupac one - letter amino acid codes are accepted at present .", "the lower section of the form has a series of options ( figure 2 ) .", "it includes three drop lists for the screening target , screened library , and screening solid phase , respectively .", "it also has two groups of check boxes for the capturing reagents and contaminants in the target sample or screening system . by default", "however , the users can customize their scan according to their experiment at this section .", "after the users submit their request , the scanning results of sarotup will be displayed on the middle tabbed page .", "if any target - unrelated peptides are found , they will be reported in a table . at the same time , a new panel of peptides excluding target - unrelated peptides is produced and can be downloaded from the hyperlink created by the sarotup server ( figure 3 ) .", "the file of the new panel of peptides will be stored on the server for a month and then automatically deleted .", "we have tested sarotup on the internet explorer ( version 6.0 ) , mozilla firefox ( version 3.5.2 ) , and google chrome ( version 3.0 ) .", "although sarotup looks a little bit different among different browsers , it works normally on all browsers tested . as shown in table 2 , the first test data set has 11 panels of peptides acquired from phage display libraries screened with 8 targets . in", "the 11 panels of peptides sarotup scanned , there were target - unrelated peptides in 3 panels from cetuximab , 80r , and b12 test case , respectively ( table 3 ) .", "this result suggested it was not rare that target - unrelated peptides sneaked into biopanning results and then were taken as mimotopes in study .", "in all , 7 target - unrelated peptides were found ; 4 of them were due to binding to plastic ; the left 3 were due to binding to the fc fragment ( table 3 ) .", "for the above 3 cases , the genuine epitopes recognized by cetuximab , 80r , and b12 monoclonal antibodies are compiled according to the ced records   and pdbsum entries .", "mapitope or pepsurf [ 15 , 21 , 22 ] were used to perform mimotope - based epitope prediction with or without sarotup procedure . for mapitope and pepsurf algorithm ,", "; the stop codon modification was set to  none  ; and all other options were in default .", "the cluster with best score was taken as the predicted epitope . in the cetuximab case ,", "pepsurf was used because there are only four or three peptides in the panel , statistically too few for mapitope . in the case of 80r and b12 ,", "mapitope was used because many peptides in the two cases exceeding the length limit of pepsurf , that is , 14 amino acids .", "if a predicted residue is identical with a residue in the true epitope , it is underlined ( table 4 ) .   as shown in table 4 ,", "the number of true positives improved from zero to four in the cetuximab case with sarotup procedure . when it came to the b12 case", "sarotup did not improve the number of true positives in the 80r case when the parameters are same to the cetuximab and b12 cases .", "however , when the distance parameter was adjusted from default ( i.e. , 9   ) to 10   , sarotup did increase the number of true positive residues from eight to eleven .", "these results indicate : ( 1 ) epitope prediction based on mimotope will be interfered if target - unrelated peptides are taken as mimotopes ; ( 2 ) sarotup can improve the performance of mimotope based epitope mapping through cleaning the input data .", "the second data set has 100 peptides , varying from 6 to 22 residues long .", "suppose that matching each pattern to each peptide manually costs 10 seconds , then it would take a researcher more than 6 hours ( 23,000 seconds ) to look through the second data set for target - unrelated peptides , even if he is as prompt during the whole period .", "besides , a table of target - unrelated peptides and a new panel of peptides excluding tup was produced at the same time by sarotup .", "it is true that some target - unrelated peptides can be identified through control and binding competition experiments .", "however , using sarotup first will certainly save a lot of labor , money , and time for researchers in this area .", "although the target of all tests described previously were monoclonal antibodies , sarotup can be customized and used in scanning the results from phage display technology using other targets such as enzymes and receptors .", "for the same reason , we can also expect that sarotup will extend its use to other similar in vitro evolution techniques , such as ribosome display [ 2931 ] , yeast display , and bacterial display [ 3335 ] .  ", "furthermore , sarotup will not only benefit the mimotope - based epitope mapping , but also the development of new diagnostics , therapeutics , and vaccines .", "target - unrelated peptides are not appropriate candidates for mimotope based diagnostics , therapeutics , and vaccines , since they are mimics to components or contaminants of the screening system rather than target .", "therefore , it is reasonable to find and exclude possible target - unrelated peptides from the candidate list of new diagnostics , therapeutics , and vaccines .", "screened a phage - displayed random peptides library with the cetuximab and got four different peptides , that is , qfdlstrrlk , qynlssralk , vwqrwqksyv , and mwdrfsrwyk .", "as described previously , we scanned the four  mimotopes  with sarotup and the result suggested that the peptide vwqrwqksyv might be a tup .", "indeed , the dot blot analysis of riemer et al . showed that qynlssralk bound the cetuximab with high affinity but vwqrwqksyv was less reactive with the cetuximab . trying to develop a mimotope vaccine , riemer et al .", "after immunization mice with these constructs , they found that either the cetuximab or the antibodies induced by the qynlssralk vaccine construct inhibited the growth of a431 cancer cells significantly .", "the inhibition of the antibodies induced by the vwqrwqksyv vaccine construct however , was not statistically significant when compared with the inhibition caused by the isotype control antibody .", "sarotup must be used with caution since it is a tool only based on pattern matching at present .", "as these tups are not embedded in sarotup at present , it is possible that a true tup can not be detected by sarotup . to reduce this kind of false negatives", "besides the motif - based search , the database - based search can find out the known tup without known motifs .", "it is also possible that a sarotup predicted target unrelated peptide is actually target - specific . to decrease this kind of false positives", ", the users should customize the scan according to their experiment at the section of advance options .", "for example , the user should select  antibody without fc fragment  as the target if fab was used in biopanning ; this will prevent sarotup from reporting peptides bearing the fc - binding motifs as tup .", "as described above , sarotup in future will also provide an exact match tool based on database search . in this way", ", a match might mean that different research groups have isolated the same peptide with a variety of targets .", "thus , the false positive rate of sarotup can be decreased further when its new feature become available . at last , we must point out that the controlled experiment is still the gold standard to distinguish tups from the specific mimotopes .", "sarotup , a web application for scanning , reporting and excluding target - unrelated peptides has been coded with perl .", "it is also useful in the development of diagnostics , therapeutics , and vaccines . to our knowledge"], "abstract_text": ["<S> as epitope mimics , mimotopes have been widely utilized in the study of epitope prediction and the development of new diagnostics , therapeutics , and vaccines . screening the random peptide libraries constructed with phage display or any other surface display technologies </S>", "<S> provides an efficient and convenient approach to acquire mimotopes . however , target - unrelated peptides creep into mimotopes from time to time through binding to contaminants or other components of the screening system . in this study , we present sarotup , a free web tool for scanning , reporting and excluding possible target - unrelated peptides from real mimotopes . </S>", "<S> preliminary tests show that sarotup is efficient and capable of improving the accuracy of mimotope - based epitope mapping . </S>", "<S> it is also helpful for the development of mimotope - based diagnostics , therapeutics , and vaccines . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusions"], "sections": [["in 1985 , smith pioneered phage display technology , an in vitro methodology and system for presenting , selecting and evolving proteins and peptides displayed on the surface of phage virion . since then , phage display has developed rapidly and become an increasingly popular tool for both basic research such as the exploration of protein - protein interaction networks and sites [ 24 ] , and applied research such as the development of new diagnostics , therapeutics , and vaccines [ 510 ] .", "usually , the protein used to screen the phage display library is termed as target and the genuine partner binding to the target is called template .", "peptide mimicking the binding site on the template and binding to the target is defined as mimotope , which was first introduced by geysen et al . .", "one type of the most frequently used targets is monoclonal antibody . in this situation ,", "the template is the corresponding antigen inducing the antibody , and the mimotope is a mimic of the genuine epitope .", ". goes  a mimotope is defined as a molecule able to bind to the antigen combining site of an antibody molecule , not necessarily identical with the epitope inducing the antibody , but an acceptable mimic of the essential features of the epitope .", " mimotopes and the corresponding epitope are considered to have similar physicochemical properties and spatial organization .", "the mimicry between mimotopes and genuine epitope makes mimotopes reasonable solutions to epitope mapping , network inferring , and new diagnostics , therapeutics , and vaccines developing .   powered by phage display technology , mimotopes can be acquired in a relatively cheap , efficient and convenient way , that is , screening phage - displayed random peptides libraries with a given target .", "however , not all phages selected out are target - specific , because the target itself is only one component of the screening system . from time to time , phages reacting with contaminants in the target sample or other components of the screening system such as the solid phase ( e.g. , plastic plates ) and the capturing molecule ( e.g. , streptavidin , secondary antibody ) rather than binding to the actual target are recovered with those target - specific binders ( displaying mimotopes ) during the rounds of panning .", "peptides displayed on these phages are called target - unrelated peptides ( tup ) , a term coined recently by menendez and scott in a review .  ", "the results from phage display technology might be a mixture of target - unrelated peptides and mimotopes , and it can be difficult to discriminate tup from mimotopes since the binding assays used to confirm the affinity of peptides for the target often employ the same components as the initial panning experiment .", "therefore , target - unrelated peptides might be taken into study as mimotopes if the researchers are not careful enough .", "obviously , target - unrelated peptides are not appropriate candidates for the development of new diagnostics , therapeutics , and vaccines . for mimotope - based epitope mapping ,", "if tup is included in the mapping , the input data is improper and the result might be misleading .", "there are now quite a few programs for mimotope based epitope mapping , none of them , however , has a procedure to scan , report and exclude target - unrelated peptides [ 1523 ] . in this study , we describe a web server named sarotup , which is an acronym for  scanner and reporter of target - unrelated peptides  .", "sarotup was coded with perl as a cgi program and can be freely accessed and used to scan peptides acquired from phage display technology .", "it is capable of finding , reporting , and precluding possible target - unrelated peptides , which is very helpful for the development of mimotope - based diagnostics , therapeutics , and vaccines .", "the power and efficiency of sarotup was also demonstrated by preliminary tests in the present study ."], ["recently , menendez and scott reviewed a collection of target - unrelated peptides recovered in the screening of phage - displayed random peptide libraries with antibodies .", "they divided their collection into several categories according to the component of the screening system to which target - unrelated peptides bind .", "very recently , brammer et al . reported a completely new type of target - unrelated peptides . in the review of menendez and scott", ", target - unrelated selection is due to the binding to contaminants or components other than target ; however , in the report of brammer et al .", ", target - unrelated selection is due to a coincident point mutation in the phage library [ 12 , 13 ] .", "we compiled a set of 23 tup motifs from the above two references [ 12 , 13 ] , including 12 motifs specific for the capturing agents , 5 motifs specific for the constant region of antibody , 3 motifs specific for the screening solid phase , 2 motifs specific for the contaminants in the target sample , and 1 motif for a mutation in phage library ( table 1 ) .", "the sarotup was implemented as a free online service , powered by apache and perl .", "three pages are designed and integrated into a tabbed web interface with cascading style sheets codes .", "the core program of sarotup was sar.pl , a cgi script coded with perl . in this script , the 23 tup motifs were converted to regular expressions , which were then used to match each input peptide sequence .", "we constructed two - test data sets from [ 12 , 13 , 1523 , 25 , 26 ] .", "the first data set contains 8 cases ; 6 of them are sourced from test cases used in extant programs for mimotope - based epitope mapping [ 1523 ] ; the left 2 are cases studies published recently [ 25 , 26 ] .", "as shown in table 2 , the target of each case in the first data set is monoclonal antibody and the structure of corresponding antigen - antibody complex has been resolved , which is used to derive its structural epitope as the golden standard for evaluation . for each case", "if target - unrelated peptides were found , a new panel of peptides excluding tup was produced .", "the old and the new panel of peptides were then used to predict epitope using mapitope or pepsurf [ 15 , 21 , 22 ] . finally , the results were compared to show if sarotup could improve the performance of mimotope - based epitope mapping .   the second data set is composed of 100 peptides in raw sequence format .", "the first group has 77 sequences compiled from the first data set without any known tup motifs ; the second group has 23 sequences sourced from [ 12 , 13 ] with various tup motifs . the mixture of the two groups of sequences made the second data set , which was then used as the sample input and can be used to evaluate the efficiency of sarotup ."], ["as a free online service , the web interface of sarotup has successfully been implemented as a tabbed web page .", "the left tab is the default page , providing a brief introduction to this web service .", "the users can either paste a set of peptide sequences in the text box or upload a sequence file to the sarotup server for scanning .", "as shown in figure 1 , a panel of peptides in raw sequence format taken from the b12 test case was pasted in the text box . besides the raw sequences", "however , only the standard iupac one - letter amino acid codes are accepted at present .", "the lower section of the form has a series of options ( figure 2 ) .", "it includes three drop lists for the screening target , screened library , and screening solid phase , respectively .", "it also has two groups of check boxes for the capturing reagents and contaminants in the target sample or screening system . by default", "however , the users can customize their scan according to their experiment at this section .", "after the users submit their request , the scanning results of sarotup will be displayed on the middle tabbed page .", "if any target - unrelated peptides are found , they will be reported in a table . at the same time , a new panel of peptides excluding target - unrelated peptides is produced and can be downloaded from the hyperlink created by the sarotup server ( figure 3 ) .", "the file of the new panel of peptides will be stored on the server for a month and then automatically deleted .", "we have tested sarotup on the internet explorer ( version 6.0 ) , mozilla firefox ( version 3.5.2 ) , and google chrome ( version 3.0 ) .", "although sarotup looks a little bit different among different browsers , it works normally on all browsers tested . as shown in table 2 , the first test data set has 11 panels of peptides acquired from phage display libraries screened with 8 targets . in", "the 11 panels of peptides sarotup scanned , there were target - unrelated peptides in 3 panels from cetuximab , 80r , and b12 test case , respectively ( table 3 ) .", "this result suggested it was not rare that target - unrelated peptides sneaked into biopanning results and then were taken as mimotopes in study .", "in all , 7 target - unrelated peptides were found ; 4 of them were due to binding to plastic ; the left 3 were due to binding to the fc fragment ( table 3 ) .", "for the above 3 cases , the genuine epitopes recognized by cetuximab , 80r , and b12 monoclonal antibodies are compiled according to the ced records   and pdbsum entries .", "mapitope or pepsurf [ 15 , 21 , 22 ] were used to perform mimotope - based epitope prediction with or without sarotup procedure . for mapitope and pepsurf algorithm ,", "; the stop codon modification was set to  none  ; and all other options were in default .", "the cluster with best score was taken as the predicted epitope . in the cetuximab case ,", "pepsurf was used because there are only four or three peptides in the panel , statistically too few for mapitope . in the case of 80r and b12 ,", "mapitope was used because many peptides in the two cases exceeding the length limit of pepsurf , that is , 14 amino acids .", "if a predicted residue is identical with a residue in the true epitope , it is underlined ( table 4 ) .   as shown in table 4 ,", "the number of true positives improved from zero to four in the cetuximab case with sarotup procedure . when it came to the b12 case", "sarotup did not improve the number of true positives in the 80r case when the parameters are same to the cetuximab and b12 cases .", "however , when the distance parameter was adjusted from default ( i.e. , 9   ) to 10   , sarotup did increase the number of true positive residues from eight to eleven .", "these results indicate : ( 1 ) epitope prediction based on mimotope will be interfered if target - unrelated peptides are taken as mimotopes ; ( 2 ) sarotup can improve the performance of mimotope based epitope mapping through cleaning the input data .", "the second data set has 100 peptides , varying from 6 to 22 residues long .", "suppose that matching each pattern to each peptide manually costs 10 seconds , then it would take a researcher more than 6 hours ( 23,000 seconds ) to look through the second data set for target - unrelated peptides , even if he is as prompt during the whole period .", "besides , a table of target - unrelated peptides and a new panel of peptides excluding tup was produced at the same time by sarotup .", "it is true that some target - unrelated peptides can be identified through control and binding competition experiments .", "however , using sarotup first will certainly save a lot of labor , money , and time for researchers in this area .", "although the target of all tests described previously were monoclonal antibodies , sarotup can be customized and used in scanning the results from phage display technology using other targets such as enzymes and receptors .", "for the same reason , we can also expect that sarotup will extend its use to other similar in vitro evolution techniques , such as ribosome display [ 2931 ] , yeast display , and bacterial display [ 3335 ] .  ", "furthermore , sarotup will not only benefit the mimotope - based epitope mapping , but also the development of new diagnostics , therapeutics , and vaccines .", "target - unrelated peptides are not appropriate candidates for mimotope based diagnostics , therapeutics , and vaccines , since they are mimics to components or contaminants of the screening system rather than target .", "therefore , it is reasonable to find and exclude possible target - unrelated peptides from the candidate list of new diagnostics , therapeutics , and vaccines .", "screened a phage - displayed random peptides library with the cetuximab and got four different peptides , that is , qfdlstrrlk , qynlssralk , vwqrwqksyv , and mwdrfsrwyk .", "as described previously , we scanned the four  mimotopes  with sarotup and the result suggested that the peptide vwqrwqksyv might be a tup .", "indeed , the dot blot analysis of riemer et al . showed that qynlssralk bound the cetuximab with high affinity but vwqrwqksyv was less reactive with the cetuximab . trying to develop a mimotope vaccine , riemer et al .", "after immunization mice with these constructs , they found that either the cetuximab or the antibodies induced by the qynlssralk vaccine construct inhibited the growth of a431 cancer cells significantly .", "the inhibition of the antibodies induced by the vwqrwqksyv vaccine construct however , was not statistically significant when compared with the inhibition caused by the isotype control antibody .", "sarotup must be used with caution since it is a tool only based on pattern matching at present .", "as these tups are not embedded in sarotup at present , it is possible that a true tup can not be detected by sarotup . to reduce this kind of false negatives", "besides the motif - based search , the database - based search can find out the known tup without known motifs .", "it is also possible that a sarotup predicted target unrelated peptide is actually target - specific . to decrease this kind of false positives", ", the users should customize the scan according to their experiment at the section of advance options .", "for example , the user should select  antibody without fc fragment  as the target if fab was used in biopanning ; this will prevent sarotup from reporting peptides bearing the fc - binding motifs as tup .", "as described above , sarotup in future will also provide an exact match tool based on database search . in this way", ", a match might mean that different research groups have isolated the same peptide with a variety of targets .", "thus , the false positive rate of sarotup can be decreased further when its new feature become available . at last , we must point out that the controlled experiment is still the gold standard to distinguish tups from the specific mimotopes ."], ["sarotup , a web application for scanning , reporting and excluding target - unrelated peptides has been coded with perl .", "it is also useful in the development of diagnostics , therapeutics , and vaccines . to our knowledge"]]}
{"article_id": "PMC2908801", "article_text": ["reversible focal lesions in the splenium of the corpus callosum ( scc ) have been found in patients with various conditions including seizures , antiepileptic drug toxicity or withdrawal , viral encephalitis , hypoglycemia , wernicke 's encephalopathy , marchiafava - bignami disease , sympathomimetic - induced kaleidoscopic visual illusion syndrome , hemolytic uremic syndrome , altitude brain injury , and acute axonal injury .", "analysis of the current literature revealed that the most frequent cause of reversible focal lesions of the scc is influenza virus - associated encephalitis / encephalopathy ( iaee ) .", "the abnormality has been reported in association with various viruses ( e.g. , epstein - barr virus , rotavirus , influenza virus ) ( 1 - 3 ) .", "the brain lesions described in patients with iaee include restricted diffusion involving the cerebral cortex and subcortical white matter in various locations , symmetric lesions in the brainstem , basal ganglia , thalamus , and cerebellar white matter with or without brain edema , and mild brain atrophy .", "transient restricted diffusion of the scc in patients with iaee has also been described ( 4 ) . here ,", "we report an adult who presented with non - specific dizziness and lesions of the scc on magnetic resonance imaging ( mri ) who was diagnosed with hemorrhagic fever with renal failure syndrome ( hfrs ) with serologically proven hantaan virus infection .", "a previously healthy 53-yr - old man presented with a 6 - 7-day history of fever up to 40 and dizziness .", "the initial diffusion - weighted images revealed high signal intensity in the corpus callosum with a low signal on the apparent diffusion coefficient ( adc ) map ( fig .", "1 ) . the next day , he developed thrombocytopenia ( 4010/l ) and was transferred to our emergency room for further evaluation .", "he stated that he had been tying rice straw into sheaves several days earlier , but not using any drugs , like metronidazole , antiepileptic drug or alcohol , and had not been exposed to toxins or experienced weight loss . on neurologic examination , he was slightly confused , and scored 24/30 on the mini mental status exam ( mmse ) .", "the cranial nerve , motor , and sensory examinations , deep tendon reflexes , and cerebellar function tests were normal .", "his blood tests showed marked thrombocytopenia ( 2110/l ) and azotemia ( bun / cr 23 mg / dl/1.5 mg / dl ) .", "he was started on cefotaxime and clindamycin for a suspected infection and transfused with fresh frozen plasma , but his fever ( > 38.0 ) was sustained .", "two days after admission , he was alert and his fever had subsided , but the oliguria , azotemia ( bun / cr 49 mg / dl/3.0 mg / dl ) , and thrombocytopenia ( 610/l ) had worsened ( figs . 2 , 3 ) .", "four days later , the thrombocytopenia had recovered , although the azotemia ( bun / cr 82 mg / dl/7.1 mg / dl ) had not improved .", "he was diagnosed with hfrs based on the detection of hantaan virus antibody in his serum .", "six days later , he was alert , and his electroencephalograph ( eeg ) was normal . his platelet count had stabilized ( 31110/l ) , and a lumbar puncture was performed .", "the cerebrospinal fluid ( csf ) analysis revealed no cells , a nor mal protein level of 26.0 mg / l , a normal glucose level of 80 mg / dl , and no organisms on gram staining .", "the mri findings had resolved completely in follow - up studies conducted on day 14 ( fig .", "hemorrhagic fever with renal failure syndrome occurs mainly in europe and asia and is characterized by a fever and renal failure associated with hemorrhagic manifestations .", "hfrs is caused by airborne contact with secretions from rodent hosts infected with viruses belonging to the genus hantavirus in the family bunyaviridae .", "the clinical features consist of a triad of fever , hemorrhage , and renal insufficiency ( 5 ) .", "other common symptoms during the initial phase of the illness include , myalgia , abdominal and back pain , and diarrhea . in most cases , mild neurological symptoms such as headache , vertigo , and nausea", "the pathogenesis is largely unknown , but findings from several studies have suggested that immune mechanisms play an important role .", "after the infection , marked cytokine production , kallikrein - kinin activation , complement pathway activation , and increased levels of circulating immune complexes occur .", "damage to the vascular endothelium , capillary dilatation , and leakage are clinically significant features of the disease .", "myelin sheaths in the scc have a relatively high water component , and the scc is more susceptible to cytotoxic edema than are other brain areas ( 2 ) .", "reversible brain lesions have been attributed to the transient development of intramyelinic edema due to the separation of myelin layers , which is a possible mechanism for the transiently decreased diffusion of the scc lesion ( 2 , 11 ) .", "the influx of inflammatory cells and molecules , possibly combined with related cytotoxic edema , might have decreased the adc ( 2 , 12 , 14 ) .", "the most common causes of reversible focal lesions of the scc are viral encephalitis , antiepileptic drug toxicity / withdrawal , and hypoglycemic encephalopathy .", "they are not specific to iaee and have been reported secondary to various infectious agents , including rotavirus , measles , herpesvirus , salmonella organisms , mumps , varicella - zoster virus , adenovirus , o157 escherichia coli - associated hemolytic - uremic syndrome , legionnaires ' disease , and unknown pathogens ( 4 , 9 , 14 ) .", "the similar clinical and imaging features suggest a common mechanism induced by different pathological events leading to the same results ( 1 ) .", "the most likely causes of these transient lesions of the scc have been explained as a rapidly resolving intramyelinic infiltrate or the influx of inflammatory cells and macromolecules , combined with related cytotoxic edema , which is very similar to the pathogenesis of hfrs ( 2 , 7 , 9 , 10 , 13 ) .", "this argues for a non - specific cause related to vasogenic edema , probably secondary to the inflammatory response ( 2 , 7 ) our patient was diagnosed with hfrs , and his mri showed scc lesions .", "we report a reversible scc lesion associated with hfrs . to our knowledge , this is the first case of virus - associated encephalitis / encephalopathy in which the pathogen was a hantaan virus .", "although it is not certain whether the neurologic symptoms in this patient might have been caused by fever , or scc lesions , or both , we suggest that hfrs patients with neurologic symptoms like dizziness and mental slowing should be considered to have structural brain lesions and to require brain imaging studies ."], "abstract_text": ["<S> this is the first case of virus - associated encephalitis / encephalopathy in which the pathogen was hantaan virus . </S>", "<S> a 53-yr - old man presented fever , renal failure and a hemorrhagic tendency and he was diagnosed with hemorrhagic fever with renal failure syndrome ( hfrs ) . in the course of his illness , mild neurologic symptoms such as dizziness and confusion developed and magnetic resonance images revealed a reversible lesion in the splenium of the corpus callosum . </S>", "<S> this case suggests that hfrs patients with neurologic symptoms like dizziness and mental slowing should be considered to have structural brain lesions and to require brain imaging studies . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["reversible focal lesions in the splenium of the corpus callosum ( scc ) have been found in patients with various conditions including seizures , antiepileptic drug toxicity or withdrawal , viral encephalitis , hypoglycemia , wernicke 's encephalopathy , marchiafava - bignami disease , sympathomimetic - induced kaleidoscopic visual illusion syndrome , hemolytic uremic syndrome , altitude brain injury , and acute axonal injury .", "analysis of the current literature revealed that the most frequent cause of reversible focal lesions of the scc is influenza virus - associated encephalitis / encephalopathy ( iaee ) .", "the abnormality has been reported in association with various viruses ( e.g. , epstein - barr virus , rotavirus , influenza virus ) ( 1 - 3 ) .", "the brain lesions described in patients with iaee include restricted diffusion involving the cerebral cortex and subcortical white matter in various locations , symmetric lesions in the brainstem , basal ganglia , thalamus , and cerebellar white matter with or without brain edema , and mild brain atrophy .", "transient restricted diffusion of the scc in patients with iaee has also been described ( 4 ) . here ,", "we report an adult who presented with non - specific dizziness and lesions of the scc on magnetic resonance imaging ( mri ) who was diagnosed with hemorrhagic fever with renal failure syndrome ( hfrs ) with serologically proven hantaan virus infection ."], ["a previously healthy 53-yr - old man presented with a 6 - 7-day history of fever up to 40 and dizziness .", "the initial diffusion - weighted images revealed high signal intensity in the corpus callosum with a low signal on the apparent diffusion coefficient ( adc ) map ( fig .", "1 ) . the next day , he developed thrombocytopenia ( 4010/l ) and was transferred to our emergency room for further evaluation .", "he stated that he had been tying rice straw into sheaves several days earlier , but not using any drugs , like metronidazole , antiepileptic drug or alcohol , and had not been exposed to toxins or experienced weight loss . on neurologic examination , he was slightly confused , and scored 24/30 on the mini mental status exam ( mmse ) .", "the cranial nerve , motor , and sensory examinations , deep tendon reflexes , and cerebellar function tests were normal .", "his blood tests showed marked thrombocytopenia ( 2110/l ) and azotemia ( bun / cr 23 mg / dl/1.5 mg / dl ) .", "he was started on cefotaxime and clindamycin for a suspected infection and transfused with fresh frozen plasma , but his fever ( > 38.0 ) was sustained .", "two days after admission , he was alert and his fever had subsided , but the oliguria , azotemia ( bun / cr 49 mg / dl/3.0 mg / dl ) , and thrombocytopenia ( 610/l ) had worsened ( figs . 2 , 3 ) .", "four days later , the thrombocytopenia had recovered , although the azotemia ( bun / cr 82 mg / dl/7.1 mg / dl ) had not improved .", "he was diagnosed with hfrs based on the detection of hantaan virus antibody in his serum .", "six days later , he was alert , and his electroencephalograph ( eeg ) was normal . his platelet count had stabilized ( 31110/l ) , and a lumbar puncture was performed .", "the cerebrospinal fluid ( csf ) analysis revealed no cells , a nor mal protein level of 26.0 mg / l , a normal glucose level of 80 mg / dl , and no organisms on gram staining .", "the mri findings had resolved completely in follow - up studies conducted on day 14 ( fig ."], ["hemorrhagic fever with renal failure syndrome occurs mainly in europe and asia and is characterized by a fever and renal failure associated with hemorrhagic manifestations .", "hfrs is caused by airborne contact with secretions from rodent hosts infected with viruses belonging to the genus hantavirus in the family bunyaviridae .", "the clinical features consist of a triad of fever , hemorrhage , and renal insufficiency ( 5 ) .", "other common symptoms during the initial phase of the illness include , myalgia , abdominal and back pain , and diarrhea . in most cases , mild neurological symptoms such as headache , vertigo , and nausea", "the pathogenesis is largely unknown , but findings from several studies have suggested that immune mechanisms play an important role .", "after the infection , marked cytokine production , kallikrein - kinin activation , complement pathway activation , and increased levels of circulating immune complexes occur .", "damage to the vascular endothelium , capillary dilatation , and leakage are clinically significant features of the disease .", "myelin sheaths in the scc have a relatively high water component , and the scc is more susceptible to cytotoxic edema than are other brain areas ( 2 ) .", "reversible brain lesions have been attributed to the transient development of intramyelinic edema due to the separation of myelin layers , which is a possible mechanism for the transiently decreased diffusion of the scc lesion ( 2 , 11 ) .", "the influx of inflammatory cells and molecules , possibly combined with related cytotoxic edema , might have decreased the adc ( 2 , 12 , 14 ) .", "the most common causes of reversible focal lesions of the scc are viral encephalitis , antiepileptic drug toxicity / withdrawal , and hypoglycemic encephalopathy .", "they are not specific to iaee and have been reported secondary to various infectious agents , including rotavirus , measles , herpesvirus , salmonella organisms , mumps , varicella - zoster virus , adenovirus , o157 escherichia coli - associated hemolytic - uremic syndrome , legionnaires ' disease , and unknown pathogens ( 4 , 9 , 14 ) .", "the similar clinical and imaging features suggest a common mechanism induced by different pathological events leading to the same results ( 1 ) .", "the most likely causes of these transient lesions of the scc have been explained as a rapidly resolving intramyelinic infiltrate or the influx of inflammatory cells and macromolecules , combined with related cytotoxic edema , which is very similar to the pathogenesis of hfrs ( 2 , 7 , 9 , 10 , 13 ) .", "this argues for a non - specific cause related to vasogenic edema , probably secondary to the inflammatory response ( 2 , 7 ) our patient was diagnosed with hfrs , and his mri showed scc lesions .", "we report a reversible scc lesion associated with hfrs . to our knowledge , this is the first case of virus - associated encephalitis / encephalopathy in which the pathogen was a hantaan virus .", "although it is not certain whether the neurologic symptoms in this patient might have been caused by fever , or scc lesions , or both , we suggest that hfrs patients with neurologic symptoms like dizziness and mental slowing should be considered to have structural brain lesions and to require brain imaging studies ."]]}
{"article_id": "PMC5136738", "article_text": ["as is the most common cause of left ventricular outflow obstruction in children and adults . it is most common type of valvular heart disease in europe and north america , occurring in 27% of the population over 65 years of age .", "medical therapy alone is not effective for the long - term management of aortic valve disease , thus valve replacement remains the standard of care in patients with an acceptable risk profile .", "we present a case of critical as with significant symptom manifested as chest pain that was managed successfully without surgical avr or tavr .", "we will also briefly review incidence , etiology , grading of as and current guidelines for avr .", "a 91-year - old gentleman with history of hypertension , dyslipidemia and as presented to our office on 7/2015 with complaints of new onset sub sternal burning pain of 6 weeks duration .", "prior to 6 weeks patient could walk 2 blocks on level ground without shortness of breath or chest pain .", "blood pressure was 140/90 mm hg and pulse was regular at 60 beats per minute .", "cardiovascular exam revealed a grade 4/6 ejection systolic murmur best heard in the right second intercostal region radiating bilaterally to the neck .", "ecg revealed left ventricular hypertrophy ( lvh ) with non specific st - t wave changes in inferior leads ( figure 1 ) .", "echocardiography ( echo ) done in may of 2014 had revealed critical as with aortic valve area of 0.6 - 0.7 centimeter square and ejection fraction of 65 percent . the aortic valve ( av ) was heavily calcified .", "aortic valve peak velocity ( av vmax ) 4.66 m / s ( figure 2 ) .", "the patient was directly referred for a cardiac catheterization to evaluate his coronary anatomy prior to avr .", "hemodynamic measurement revealed left ventricle ( lv ) pressure 240/0 with left ventricular end diastolic pressure ( lvedp ) of 10 .", "pulmonary artery wedge pressure ( pawp ) , mean of 24 , right atrium ( ra ) mean of 8 , right ventricle ( rv ) 60/10 with an right ventricular end diastolic pressure ( rvedp ) of 14 , pulmonary artery ( pa ) 60/17 .", "the aortic valve area was calculated at 0.6 centimeter square , which was unchanged from echo done may 1 , 2014 .", "the left ventriculogram in the right anterior oblique ( rao ) view revealed normal lv systolic motion and ejection fraction of 55 - 60 percent . left main coronary artery revealed a common ostium - giving rise to the left anterior descending ( lad ) and the circumflex .", "right coronary artery ( rca ) revealed a 99 % ostial narrowing ( figure 3 ) .", "lad revealed a 60 % narrowing in its proximal segment and a 90% ostial diagonal narrowing ( figure 4 ) .", "after a detailed discussion involving the patient , his son , interventional cardiologist and the cardio thoracic surgeon , patient requested that only pci of the rca be considered , as the chest pain was of recent duration with no change in aortic valve finding .", "thus , a successful pci was done in the proximal rca with a bare metal stent ( figure 6 ) .", "patient did extremely well after pci of the rca and the patient has remained asymptomatic to date .", "thus , this new onset chest pain in patient with critical as was due to concomitant coronary artery lesion and was amenable to stenting and thus spared this elderly patient from the aortic valve replacement which would have been a high risk surgery for him .", "as is the most common valvular heart condition in the developed world , affecting 3% of people between ages 75 and 85 and 4% of people over age 85 .", "congenitally unicuspid , bicuspid , tricuspid , or even quadricuspid valves may be the cause of as . in adults who develop symptoms from congenital as", "the main causes of acquired as include degenerative calcification and , less commonly , rheumatic heart disease .", "other , infrequent causes of as include obstructive vegetations , homozygous type ii hypercholesterolemia , paget disease , fabry disease , ochronosis , and irradiation . based upon a variety of hemodynamic and natural history data", ", clinicians generally grade the severity of stenosis as mild , moderate , severe , or critical .", "grading of as are as follows : i ) mild : valve area exceeds 1.5 cm ; transvalvular velocity 2.0 to 2.9 m / s ; mean gradient < 20 mmhg ; ii ) moderate : valve area of 1.0 to 1.5 cm ; transvalvular velocity 3.0 to 3.9 m / s ; mean gradient 20 to 39 mmhg ; iii ) severe : valve area is less than 1.0 cm ; transvalvular velocity 4 m / s ; mean gradient 40 mmhg .", "the term critical stenosis was defined based upon theoretical considerations showing that the aortic valve area must be reduced to one - fourth of its natural size before significant changes in circulation occur . as a result , since the triangular orifice area of the normal ( adult ) aortic valve is approximately 3.0 cm , an area exceeding 0.75 cm would not be defined as critical .", "avr and tavr remain the only treatment proven to reduce the rates of mortality and morbidity in this condition . under current guidelines , the onset of symptoms of exertional angina , syncope and dyspnea in a patient who has severe as is a class i indication for surgery .", "the annual rate of sudden death in patients with this condition is estimated at 1% to 3% but the surgical mortality rate in avr has been as high as 6% . with improvements in surgical techniques and prostheses , mortality rates have been reduced to 2.42% making a case for earlier intervention .", "tavr has become widely available , but further investigation into its use in this patient cohort is warranted . while assessing the cases of asymptomatic as we have both traditional as well as novel markers at our disposal now . left ventricular ejection fraction ( lvef ) < 50 percent , peak aortic jet velocity > 4.0 m / s , valve area < 1 cm and mean pressure gradient > 40 mm hg are the traditional markers to denote severe as in asymptomatic patients . while , indexed left atrial size > 12.2 cm / m , lvh with wall thickness > 15 mm , global left ventricular longitudinal strain < 15.9 , bnp ( b - natriuretic peptide ) level > 130 pg / ml and increase in mean pressure gradient of > 20 mm hg during exercise testing are the novel markers of asymptomatic severe as .", "bnp level does not appear to be significantly associated with the degree of as severity but does reflect heart failure status .", "the american college of cardiology and american heart association ( acc / aha ) have issued the following recommendations for avr , based on the severity of stenosis and on whether the patient has symptoms : i ) severe stenosis , with symptoms : class i recommendation ( surgery should be done ) . without surgery ,", "these patients have a very poor prognosis , with an overall mortality rate of 75% at 3 years ; ii ) severe stenosis , no symptoms , in patients undergoing cardiac surgery for another indication ( example coronary artery bypass grafting , ascending aortic surgery , or surgery on other valves ) : class i recommendation for concomitant aortic valve replacement ; iii ) moderate stenosis , no symptoms , in patients undergoing cardiac surgery for another indication : class iia recommendation ( i.e. , aortic valve replacement is reasonable ) ; iv ) very severe stenosis ( aortic peak velocity > 5.0 m / s or mean pressure gradient 60 mm hg ) , no symptoms , and low risk of death during surgery : class iia recommendation ; v ) severe stenosis , no symptoms , and an increase in transaortic velocity of 0.3 m / s or more per year on serial testing or in patients considered to be at high risk for rapid disease progression , such as elderly patients with severe calcification : class iib recommendation ( surgery can be considered ) .", "on revisiting the above case description we realize that the patient did have critical as but was asymptomatic .", "his chest pain was only due to concomitant coronary artery disease but was not due to as per se .", "age and comorbidity of the patient posed a high risk for coronary artery bypass graft ( cabg ) with avr .", "besides , the patient s symptoms were of new onset and subsequent cardiac catheterization revealing critical rca stenosis and the patient s preference for treating the cause of his recent symptoms , encouraged us to think otherwise . the decision to perform pci alone with the belief that this chest pain and cad would be amenable to the minimal risk procedure paid dividends .", "in addition , patient received a bare metal stent with the option of undergoing surgical avr if symptoms were not relieved .", "awareness amongst physicians about the fact that all critical aortic stenosis with chest pain may not require aortic valve replacement is important .", "this can lead to less invasive treatment tailored to the need of the patient especially in those with advanced age , significant comorbidities and an extremely high risk for cabg with avr and can also result in decreased cost of care .", "pharmacological nuclear stress testing may be another modality that can be used to differentiate etiology of the symptoms ."], "abstract_text": ["<S> aortic valve replacement ( avr ) remains the cornerstone of treatment for symptomatic critical aortic stenosis ( as ) . </S>", "<S> it is a class i indication that symptomatic patients with critical as undergo either surgical or transcatheter aortic valve replacement ( tavr ) . </S>", "<S> we present a patient with critical as and new angina that was managed successfully with percutaneous coronary intervention ( pci ) of the right coronary artery . </S>", "<S> physicians should consider that not all patients with critical as and angina necessarily require avr . </S>", "<S> concomitant pathology leading to the symptoms should be carefully ruled out . </S>", "<S> this leads to a less invasive , cost effective care plan especially in patients with advanced age and comorbidities for which any type of surgical valvular intervention may pose high risk . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusions"], "sections": [["as is the most common cause of left ventricular outflow obstruction in children and adults . it is most common type of valvular heart disease in europe and north america , occurring in 27% of the population over 65 years of age .", "medical therapy alone is not effective for the long - term management of aortic valve disease , thus valve replacement remains the standard of care in patients with an acceptable risk profile .", "we present a case of critical as with significant symptom manifested as chest pain that was managed successfully without surgical avr or tavr .", "we will also briefly review incidence , etiology , grading of as and current guidelines for avr ."], ["a 91-year - old gentleman with history of hypertension , dyslipidemia and as presented to our office on 7/2015 with complaints of new onset sub sternal burning pain of 6 weeks duration .", "prior to 6 weeks patient could walk 2 blocks on level ground without shortness of breath or chest pain .", "blood pressure was 140/90 mm hg and pulse was regular at 60 beats per minute .", "cardiovascular exam revealed a grade 4/6 ejection systolic murmur best heard in the right second intercostal region radiating bilaterally to the neck .", "ecg revealed left ventricular hypertrophy ( lvh ) with non specific st - t wave changes in inferior leads ( figure 1 ) .", "echocardiography ( echo ) done in may of 2014 had revealed critical as with aortic valve area of 0.6 - 0.7 centimeter square and ejection fraction of 65 percent . the aortic valve ( av ) was heavily calcified .", "aortic valve peak velocity ( av vmax ) 4.66 m / s ( figure 2 ) .", "the patient was directly referred for a cardiac catheterization to evaluate his coronary anatomy prior to avr .", "hemodynamic measurement revealed left ventricle ( lv ) pressure 240/0 with left ventricular end diastolic pressure ( lvedp ) of 10 .", "pulmonary artery wedge pressure ( pawp ) , mean of 24 , right atrium ( ra ) mean of 8 , right ventricle ( rv ) 60/10 with an right ventricular end diastolic pressure ( rvedp ) of 14 , pulmonary artery ( pa ) 60/17 .", "the aortic valve area was calculated at 0.6 centimeter square , which was unchanged from echo done may 1 , 2014 .", "the left ventriculogram in the right anterior oblique ( rao ) view revealed normal lv systolic motion and ejection fraction of 55 - 60 percent . left main coronary artery revealed a common ostium - giving rise to the left anterior descending ( lad ) and the circumflex .", "right coronary artery ( rca ) revealed a 99 % ostial narrowing ( figure 3 ) .", "lad revealed a 60 % narrowing in its proximal segment and a 90% ostial diagonal narrowing ( figure 4 ) .", "after a detailed discussion involving the patient , his son , interventional cardiologist and the cardio thoracic surgeon , patient requested that only pci of the rca be considered , as the chest pain was of recent duration with no change in aortic valve finding .", "thus , a successful pci was done in the proximal rca with a bare metal stent ( figure 6 ) .", "patient did extremely well after pci of the rca and the patient has remained asymptomatic to date .", "thus , this new onset chest pain in patient with critical as was due to concomitant coronary artery lesion and was amenable to stenting and thus spared this elderly patient from the aortic valve replacement which would have been a high risk surgery for him ."], ["as is the most common valvular heart condition in the developed world , affecting 3% of people between ages 75 and 85 and 4% of people over age 85 .", "congenitally unicuspid , bicuspid , tricuspid , or even quadricuspid valves may be the cause of as . in adults who develop symptoms from congenital as", "the main causes of acquired as include degenerative calcification and , less commonly , rheumatic heart disease .", "other , infrequent causes of as include obstructive vegetations , homozygous type ii hypercholesterolemia , paget disease , fabry disease , ochronosis , and irradiation . based upon a variety of hemodynamic and natural history data", ", clinicians generally grade the severity of stenosis as mild , moderate , severe , or critical .", "grading of as are as follows : i ) mild : valve area exceeds 1.5 cm ; transvalvular velocity 2.0 to 2.9 m / s ; mean gradient < 20 mmhg ; ii ) moderate : valve area of 1.0 to 1.5 cm ; transvalvular velocity 3.0 to 3.9 m / s ; mean gradient 20 to 39 mmhg ; iii ) severe : valve area is less than 1.0 cm ; transvalvular velocity 4 m / s ; mean gradient 40 mmhg .", "the term critical stenosis was defined based upon theoretical considerations showing that the aortic valve area must be reduced to one - fourth of its natural size before significant changes in circulation occur . as a result , since the triangular orifice area of the normal ( adult ) aortic valve is approximately 3.0 cm , an area exceeding 0.75 cm would not be defined as critical .", "avr and tavr remain the only treatment proven to reduce the rates of mortality and morbidity in this condition . under current guidelines , the onset of symptoms of exertional angina , syncope and dyspnea in a patient who has severe as is a class i indication for surgery .", "the annual rate of sudden death in patients with this condition is estimated at 1% to 3% but the surgical mortality rate in avr has been as high as 6% . with improvements in surgical techniques and prostheses , mortality rates have been reduced to 2.42% making a case for earlier intervention .", "tavr has become widely available , but further investigation into its use in this patient cohort is warranted . while assessing the cases of asymptomatic as we have both traditional as well as novel markers at our disposal now . left ventricular ejection fraction ( lvef ) < 50 percent , peak aortic jet velocity > 4.0 m / s , valve area < 1 cm and mean pressure gradient > 40 mm hg are the traditional markers to denote severe as in asymptomatic patients . while , indexed left atrial size > 12.2 cm / m , lvh with wall thickness > 15 mm , global left ventricular longitudinal strain < 15.9 , bnp ( b - natriuretic peptide ) level > 130 pg / ml and increase in mean pressure gradient of > 20 mm hg during exercise testing are the novel markers of asymptomatic severe as .", "bnp level does not appear to be significantly associated with the degree of as severity but does reflect heart failure status .", "the american college of cardiology and american heart association ( acc / aha ) have issued the following recommendations for avr , based on the severity of stenosis and on whether the patient has symptoms : i ) severe stenosis , with symptoms : class i recommendation ( surgery should be done ) . without surgery ,", "these patients have a very poor prognosis , with an overall mortality rate of 75% at 3 years ; ii ) severe stenosis , no symptoms , in patients undergoing cardiac surgery for another indication ( example coronary artery bypass grafting , ascending aortic surgery , or surgery on other valves ) : class i recommendation for concomitant aortic valve replacement ; iii ) moderate stenosis , no symptoms , in patients undergoing cardiac surgery for another indication : class iia recommendation ( i.e. , aortic valve replacement is reasonable ) ; iv ) very severe stenosis ( aortic peak velocity > 5.0 m / s or mean pressure gradient 60 mm hg ) , no symptoms , and low risk of death during surgery : class iia recommendation ; v ) severe stenosis , no symptoms , and an increase in transaortic velocity of 0.3 m / s or more per year on serial testing or in patients considered to be at high risk for rapid disease progression , such as elderly patients with severe calcification : class iib recommendation ( surgery can be considered ) .", "on revisiting the above case description we realize that the patient did have critical as but was asymptomatic .", "his chest pain was only due to concomitant coronary artery disease but was not due to as per se .", "age and comorbidity of the patient posed a high risk for coronary artery bypass graft ( cabg ) with avr .", "besides , the patient s symptoms were of new onset and subsequent cardiac catheterization revealing critical rca stenosis and the patient s preference for treating the cause of his recent symptoms , encouraged us to think otherwise . the decision to perform pci alone with the belief that this chest pain and cad would be amenable to the minimal risk procedure paid dividends .", "in addition , patient received a bare metal stent with the option of undergoing surgical avr if symptoms were not relieved ."], ["awareness amongst physicians about the fact that all critical aortic stenosis with chest pain may not require aortic valve replacement is important .", "this can lead to less invasive treatment tailored to the need of the patient especially in those with advanced age , significant comorbidities and an extremely high risk for cabg with avr and can also result in decreased cost of care .", "pharmacological nuclear stress testing may be another modality that can be used to differentiate etiology of the symptoms ."]]}
{"article_id": "PMC3146985", "article_text": ["the various blood cell lineages in mammals arise from a multipotent haematopoietic stem cell via particular differentiation pathways .", "one of these pathways , erythropoiesis , leads to the production of red blood cells ( rbcs ) , which transport oxygen and carbon dioxide around the body by means of the intracellular metalloprotein haemoglobin ( hb ) .", "hb is a tetramer , consisting of two -globin and two -globin subunits . in mammals ,", "these globin chains are encoded by two gene loci : the -globin locus and the -globin locus . in humans ,", "the -globin locus consists of the embryonic - and adult -globin genes , and the -globin locus comprises the embryonic - , foetal - and - , and adult - and -globin genes     [ 1 , 2 ] .", "the globin genes expressed from these loci differ from embryonic to adult erythropoiesis in order to meet varying oxygen demands and facilitate placental transfer of oxygen from mother to embryo .  ", "there are a number of severe diseases caused by the disruption of adult globin genes , including thalassaemias and certain types of anaemia . according to the world health organisation ,", "approximately 5% of the world 's population carry genes involved in hb disorders , and as such , they present an enormous health burden .", "thalassaemia is caused by a reduction or abolition of the expression of one or more globin genes , resulting in an imbalance of - and -globin chains in red blood cells and consequent anaemia     [ 1 , 4 ] .", "sickle cell anaemia is another prevalent haemoglobinopathy and is caused by a mutation in the adult -globin gene which generates a single glutamic acid to valine amino acid substitution .", "this mutation leads to the polymerisation of globins in venous circulation     [ 5 , 6 ] , which can trigger a rigid and sickled cell phenotype     [ 7 , 8 ] and results in a number of acute conditions such as vaso - occlusion , splenic sequestration , and haemolytic anaemia .  ", "there are currently a number of treatments available for patients suffering from thalassaemia and sickle cell anaemia .", "the most common is packed red blood cell transfusion , but this is associated with a number of problems , such as sufficiency of supply , bacterial and viral infection , biochemical and biomechanical changes during storage ( red blood cell storage lesions ) , and the risk of immune rejection from the patient     [ 10 , 11 ] .", "furthermore , blood transfusions are ongoing throughout a patient 's life and often lead to a potentially fatal buildup of iron and associated reduction in organ activity .  ", "residual production of foetal -globin persists naturally throughout life , and levels vary between individuals     [ 12 , 13 ] .", "this persistent expression allows two -globin chains to combine with two adult -globin chains to form what is known as foetal hb ( hbf ) . as only the adult -globin gene", "is mutated in sickle cell anaemia , affected infants are protected from severe symptoms until they reach several months of age , due to the large amount of hbf still in circulation at birth .", "furthermore , patients who have inherited alleles associated with increased levels of hbf , known as hereditary persistence of foetal hb ( hpfh ) , are protected into adulthood . similarly , a more asymptomatic disease phenotype has also been shown in patients with -thalassaemia who exhibit higher levels of hbf     .", "together , these observations indicate that increased foetal -globin is able to compensate in part for the loss of adult -globin function and thus ameliorates the symptoms of certain adult haemoglobinopathies .", "accordingly ,   a number of drug treatments for -thalassaemia and sickle cell disease , for instance , 5-azacitidine , hydroxyurea , and butyrate , all act by nonspecifically reactivating foetal -globin gene expression by various mechanisms .", "there is evidence that long - term administration of these drugs has chronic side effects , consistent with their lack of specificity     [ 8 , 17 ] .   as the existing methods of treatment for these haemoglobinopathies remain inadequate ,", "alternative forms of therapy are currently being sought , and stem cell therapies should be considered .", "this paper will discuss progress in utilising novel cellular reprogramming techniques to treat rbc diseases .", "stem cells , both embryonic and adult , have the ability to differentiate into various cell types , making them a potentially attractive treatment option . embryonic stem cells ( escs ) and adult stem cells ( ascs ) each have their own strengths and disadvantages in these strategies .", "escs are more easily grown in culture and are pluripotent , meaning that they are able to differentiate into any cell of the body .", "the practicality of widespread esc use for therapeutic purposes , however , has been questioned due to issues of supply and ethical and legal considerations . moreover", "ascs , on the other hand , overcome some of these problems as they can be harvested from each individual patient .", "they are not abundant and are difficult to obtain , often being harboured in internal organs such as the gut and bone marrow .", "they have proven difficult to culture in vitro , and furthermore , they are widely believed to be multipotent rather than pluripotent and are thus only able to differentiate into certain cell types .", "cellular reprogramming potentially overcomes these issues and may offer new treatment methods for a range of diseases , including those of rbcs . takahashi and yamanaka      were the first to show that it is possible to take differentiated somatic cells and transform them into cells with pluripotent potential .", "they began by identifying a pool of 24 transcription factors which are important in maintaining stem cell traits and used retroviral transduction to express these factors in murine embryonic and adult fibroblasts .", "they found that these cells then displayed characteristics and properties comparable to those of pluripotent escs .", "they were able to further refine the required transcription factors by setting up numerous combinations to determine which were essential to this process and identified four factors , oct4 , sox2 , klf4 , and c - myc , needed to transform a somatic cell to an induced pluripotent stem cell ( ipsc ) .", "takahashi et al .      then applied these four factors to human cells and transformed neonatal and adult human fibroblasts into human ipscs ( hipscs ) .", "this was a valuable subsequent step as it showed that this process can be reproduced with human cells and could thus potentially be utilised for stem cell - based therapies of human disease .", "several other transcription factor combinations have since been shown to be sufficient to reprogram somatic cells     [ 2224 ] and one particular combination , involving oct4 , sox2 , nanog , and lin28 , is particularly efficient and is now widely utilised .  ", "for example , a number of the transcription factors used to generate these cells , including c - myc , klf4 , and lin28 , are oncogenes , and their misexpression can lead to cancer     [ 2628 ] . in order for ipscs to be differentiated into a cell type of choice ,", "the retrovirally transmitted genes need to be switched off or removed to reduce the possibility of their inducing tumours [ 22 , 29 ] .", "another potential issue is that the transplantation of any cells that have not been fully differentiated from the ipsc state could lead to the formation of cancerous teratomas .", "any cells remaining which are still in a pluripotent state could multiply and , without the appropriate growth controls , would have the potential to result in tumours in transplant patients . to potentially avoid the issue of tumour formation and uncontrolled proliferation of ipscs , an alternative methodology known as transdifferentiation is also being considered as a potential treatment strategy .  ", "transdifferentiation is achieved by introducing various exogenous factors into a differentiated cell , such as a fibroblast , to directly convert it into another type of differentiated cell , thereby bypassing the pluripotent state ( figure 1(b ) ) . as early as 1990 , choi et al .   were able to convert various cells types , including dermal fibroblasts and chondroblasts , into mononucleated , striated myoblasts that were indistinguishable from normal myoblasts in vivo", "this was achieved through the expression of the myogenic regulatory factor myod , a transcription factor known to be involved in the determination of muscle cells . shortly after this", ", another group showed that it was possible to turn myeloid 416b cells into mast cells through the forced expression of gata-2 and gata-3 , two transcription factors which play important roles in haematopoiesis .", "investigated the effects of ectopically expressing the gene eyeless ( ey ) , an orthologue of the mammalian pax6 gene , in drosophila and found that eye structures formed in places such as the wings and legs .  ", "since takahashi and yamanaka 's pioneering studies in cellular reprogramming , the field of transdifferentiation has advanced considerably .", "zhou et al .   investigated the effects of expressing ngn3 , pdx1 , and mafa , transcription factors involved in -cell differentiation , on exocrine cells of the adult pancreas .", "they found that the coexpression of these factors was able to convert the exocrine cells into -cells .", "the induced -cells were identical to endogenous -cells in morphology and showed similar expression of genes associated with -cell function .", "these cells can also rescue the phenotype of hyperglycaemia , as they are able to secrete insulin and remodel surrounding vasculature . in other work ,", "utilised the neural - specific transcription factors , ascl1 , brn2 , and myt1l , to rapidly convert both murine embryonic fibroblasts and adult fibroblasts directly into functional neurons .", "these induced neuronal cells express neuron - specific proteins , generate action potentials , and form functional synapses .", "a further advance has shown that it is also possible to transdifferentiate fibroblasts into functional neural progenitor cells by transient induction of oct4 , sox2 , klf4 , and c - myc in cells cultured in a defined neural reprogramming medium . this process bypassed the generation of ipscs and gave rise to multipotent progenitors with the capacity to expand and differentiate into a number of neural lineages .", "all of these experiments indicate that it is possible to direct a differentiated cell to another cell fate through the application of extragenic factors .", "advances in cellular reprogramming have raised the attractive possibility that this technology could be utilised to generate a limitless source of immune - matched , pathogen - free erythrocytes for transfusion .", "they found that hipscs are capable of generating haematopoietic cells with phenotypic and morphological characteristics similar to those derived from hescs ; however , these hipsc - derived cells exhibited a dramatically reduced capacity ( by greater than 1000-fold ) to generate erythroid cells .   a subsequent study by lapillone et al .", ", however , showed that it was indeed possible to produce significant numbers of mature erythroid cells from hipscs in vitro .", "this group employed the methods outlined by thomson 's group   using oct4 , sox2 , nanog , and lin28 to convert fibroblasts to   hipscs .", "they then cultured these cells in medium containing the cytokines scf , tpo , flt3 ligand , rhu bmp4 , rhu vegf - a165 , il-3 , il-6 , and erythropoietin ( epo ) .", "they analysed the expression profiles of these cells over 20 days of culture and found that pluripotent stem cell markers decreased whilst the erythroid markers cd36 , cd235a , and cd71 increased .", "cells at day 20 were found to have a high erythroid potential and were plated in sequential cocktails of cytokines comprising scf , il-3 , and/or epo .", "erythroid maturation was achieved after another 25 days and was confirmed by morphological examination and by flow cytometric analysis of erythroid markers ( cd235a , cd71 , and cd36 ) .", "furthermore , these erythroid cells were able to enucleate , albeit with reduced capacity compared to hesc - derived erythroid cells , and were found to express functional hb ( predominantly hbf ) .", "these hipsc - derived erythroid cells were compared to those differentiated from hescs , and no significant differences were detected in terms of erythroid commitment , expression of erythroid markers , and type and functionality of hb .  ", "revealed that while functional rbcs could be generated from hipscs , when compared to hescs , hipscs were shown to have reduced ( approximately 8-fold ) amplification potential in producing mature erythroid cells .", "a recent study has set out to determine whether this reduced efficiency is an intrinsic property of hipscs , or whether it can be increased by altered culture conditions .", "this study showed not only that it is possible to generate rbcs from hipscs with similar efficiency to hescs , but also that these cells can be differentiated from hipscs generated using episomal vectors , obviating the need for potentially deleterious retroviral transfection .", "this group cultured hipscs derived from human fibroblasts with the op9 bone marrow stroma line to induce hematopoietic differentiation and followed this with selective expansion of erythroid cells in serum - free media with cytokines supporting rbc differentiation .", "the erythroid cultures established in this study consisted of a pure population of cd235a+ cd45 leukocyte - free rbcs which had robust expansion capabilities , as well as a long lifespan of up to 90 days .", "these hipsc - derived cells can enucleate and were shown to express foetal - and embryonic -globin demonstrating successful reprogramming of the -globin locus .", "the results from this study show that it is possible to produce significant numbers of erythroid cells from fibroblast - derived hipscs , and that thus cellular reprogramming could contribute to the treatment of haemoglobinopathies . furthermore , since these rbcs are generated from transgene - free hipscs , they circumvent problems associated with genomic integration and undesired reactivation of reprogramming factors .   another recent study has shown that it is possible to use transdifferentiation to produce haematopoietic cell lineages , including mature erythrocytes . in this work , szabo et al .", "transduced human dermal fibroblasts with oct4 alone and showed that these cells then express the panhaematopoietic marker cd45 but lack the pluripotency marker tra-1 - 60 .", "they then cultured these cells in a haematopoietic cytokine cocktail containing scf , g - csf , flt3lg , il-3 , il-6 , and bmp-4   supplemented with epo and observed an emergence of cells expressing adult -globin protein and the red blood cell marker glycophorin - a .", "in addition , the authors noted that these erythroid cells express adult rather than embryonic globins , unlike cells derived from human pluripotent stem cells     [ 39 , 40 ] , perhaps suggesting that they have indeed been transdifferentiated from adult fibroblasts , bypassing the hipsc stage .", "as the techniques used in this study avoid the pluripotent state as well as have a high yield , expansion capacity , and clinical feasibility , this strategy could provide a reasonable basis for autologous cell replacement therapies .  ", "these studies demonstrate that it is possible to differentiate hipscs into mature erythrocytes in vitro for transfusions . in order to utilise these methods to treat rbc diseases successfully", ", fibroblasts could potentially be taken from either a healthy immunomatched individual , or a universal donor , and be converted to erythrocytes via the hipsc state .", "a study has already developed hipscs from an individual with a bombay phenotype of the abo blood group system , where the abh antigen is not expressed on rbcs , and blood can be donated to anyone     .", "this group has further demonstrated that it is possible to differentiate haematopoietic lineages from these cells although they have not explicitly shown mature erythrocyte differentiation .", "this possible treatment method circumvents many of the problems associated with current transfusions , such as questionable quality , lack of supply , and immune - rejection .", "however , issues still remain , such as cost and the risk of iron overloading .", "in order to cure , rather than treat , rbc diseases , healthy progenitor cells must be transplanted into patients and subsequently repopulate the haematopoietic system .", "a promising study has already shown that it is possible to use reprogrammed cells to treat sickle cell anaemia in mice .", "hanna et al .      took cells from the tail tips of a humanized sickle cell anaemic mouse model , in which the mouse -globin genes have been replaced with human -globin , and the mouse -globin genes have been replaced with human  and  ( sickle ) globin genes , and through adaptation of the protocol developed by takahashi and yamanaka , who reprogrammed these cells into ipscs .", "once this was accomplished , they were able to correct the mutant -globin gene by replacing it with a copy of the wildtype human -globin gene through homologous recombination . following this ,", "the ipscs were differentiated into haematopoietic progenitors through the ectopic expression of hoxb4 , and by growing in haematopoietic cytokines on an op9 bone marrow stroma cell line .", "this group then carried out extensive testing to determine whether the treated mice displayed any further signs of the disease .", "firstly , functional correction was evaluated by electrophoresis for the human -globin proteins  and  , and they found a significant increase in - and a decrease in -globin .  ", "blood counts were also performed 12 weeks after transplant , which showed that treated mice had increased rbcs , hb , and packed cell volume as well as reduced reticulocytes , a common indicator of sickle cell disease and severity .", "lastly , they examined symptomatic indicators of sickle cell anaemia such as urine concentration , body weight , and breathing rate and found that all three of these parameters were ameliorated in the treated mice .", "this study thus provides an important proof of principle that cellular reprogramming can be employed to correct erythroid disorders , albeit in this case , in conjunction with gene therapy .", "normal erythropoiesis is dependent upon the correct expression of globin genes . where globin genes are incorrectly expressed , or are mutated , anaemia or thalassemia results .", "current therapy for these disorders involves packed red blood cell transfusions , which are limited by supply , risk of infection , expense , and patient rejection .", "drug - based therapies involve the nonspecific reactivation of foetal globins and have long - term side effects . in seeking alternative strategies", ", recent advances have shown that cellular reprogramming can now generate large quantities of red blood cells in culture , potentially for use in transfusions .", "furthermore , these strategies have been successfully combined with gene therapy to treat a sickle cell anaemia mouse model , suggesting that cellular reprogramming will provide a realistic future alternative to conventional treatment of haemoglobinopathies ."], "abstract_text": ["<S> haemoglobinopathies such as thalassaemia and sickle cell disease present a major health burden . </S>", "<S> currently , the main forms of treatment for these diseases are packed red blood cell transfusions and the administration of drugs which act to nonspecifically reactivate the production of foetal haemoglobin . </S>", "<S> these treatments are ongoing throughout the life of the patient and are associated with a number of risks , such as limitations in available blood for transfusion , infections , iron overload , immune rejection , and side effects associated with the drug treatments . </S>", "<S> the field of cellular reprogramming has advanced significantly in the last few years and has recently culminated in the successful production of erythrocytes in culture . </S>", "<S> this paper will discuss cellular reprogramming and its potential relevance to the treatment of haemoglobinopathies . </S>"], "labels": null, "section_names": ["1. Introduction: Globin Gene Regulation and Haemoglobinopathies", "2. Cellular Reprogramming", "3. Cellular Reprogramming as a Potential Treatment for Anaemia", "4. Cellular Reprogramming as a Potential Cure for Anaemia", "5. Conclusions"], "sections": [["the various blood cell lineages in mammals arise from a multipotent haematopoietic stem cell via particular differentiation pathways .", "one of these pathways , erythropoiesis , leads to the production of red blood cells ( rbcs ) , which transport oxygen and carbon dioxide around the body by means of the intracellular metalloprotein haemoglobin ( hb ) .", "hb is a tetramer , consisting of two -globin and two -globin subunits . in mammals ,", "these globin chains are encoded by two gene loci : the -globin locus and the -globin locus . in humans ,", "the -globin locus consists of the embryonic - and adult -globin genes , and the -globin locus comprises the embryonic - , foetal - and - , and adult - and -globin genes     [ 1 , 2 ] .", "the globin genes expressed from these loci differ from embryonic to adult erythropoiesis in order to meet varying oxygen demands and facilitate placental transfer of oxygen from mother to embryo .  ", "there are a number of severe diseases caused by the disruption of adult globin genes , including thalassaemias and certain types of anaemia . according to the world health organisation ,", "approximately 5% of the world 's population carry genes involved in hb disorders , and as such , they present an enormous health burden .", "thalassaemia is caused by a reduction or abolition of the expression of one or more globin genes , resulting in an imbalance of - and -globin chains in red blood cells and consequent anaemia     [ 1 , 4 ] .", "sickle cell anaemia is another prevalent haemoglobinopathy and is caused by a mutation in the adult -globin gene which generates a single glutamic acid to valine amino acid substitution .", "this mutation leads to the polymerisation of globins in venous circulation     [ 5 , 6 ] , which can trigger a rigid and sickled cell phenotype     [ 7 , 8 ] and results in a number of acute conditions such as vaso - occlusion , splenic sequestration , and haemolytic anaemia .  ", "there are currently a number of treatments available for patients suffering from thalassaemia and sickle cell anaemia .", "the most common is packed red blood cell transfusion , but this is associated with a number of problems , such as sufficiency of supply , bacterial and viral infection , biochemical and biomechanical changes during storage ( red blood cell storage lesions ) , and the risk of immune rejection from the patient     [ 10 , 11 ] .", "furthermore , blood transfusions are ongoing throughout a patient 's life and often lead to a potentially fatal buildup of iron and associated reduction in organ activity .  ", "residual production of foetal -globin persists naturally throughout life , and levels vary between individuals     [ 12 , 13 ] .", "this persistent expression allows two -globin chains to combine with two adult -globin chains to form what is known as foetal hb ( hbf ) . as only the adult -globin gene", "is mutated in sickle cell anaemia , affected infants are protected from severe symptoms until they reach several months of age , due to the large amount of hbf still in circulation at birth .", "furthermore , patients who have inherited alleles associated with increased levels of hbf , known as hereditary persistence of foetal hb ( hpfh ) , are protected into adulthood . similarly , a more asymptomatic disease phenotype has also been shown in patients with -thalassaemia who exhibit higher levels of hbf     .", "together , these observations indicate that increased foetal -globin is able to compensate in part for the loss of adult -globin function and thus ameliorates the symptoms of certain adult haemoglobinopathies .", "accordingly ,   a number of drug treatments for -thalassaemia and sickle cell disease , for instance , 5-azacitidine , hydroxyurea , and butyrate , all act by nonspecifically reactivating foetal -globin gene expression by various mechanisms .", "there is evidence that long - term administration of these drugs has chronic side effects , consistent with their lack of specificity     [ 8 , 17 ] .   as the existing methods of treatment for these haemoglobinopathies remain inadequate ,", "alternative forms of therapy are currently being sought , and stem cell therapies should be considered .", "this paper will discuss progress in utilising novel cellular reprogramming techniques to treat rbc diseases ."], ["stem cells , both embryonic and adult , have the ability to differentiate into various cell types , making them a potentially attractive treatment option . embryonic stem cells ( escs ) and adult stem cells ( ascs ) each have their own strengths and disadvantages in these strategies .", "escs are more easily grown in culture and are pluripotent , meaning that they are able to differentiate into any cell of the body .", "the practicality of widespread esc use for therapeutic purposes , however , has been questioned due to issues of supply and ethical and legal considerations . moreover", "ascs , on the other hand , overcome some of these problems as they can be harvested from each individual patient .", "they are not abundant and are difficult to obtain , often being harboured in internal organs such as the gut and bone marrow .", "they have proven difficult to culture in vitro , and furthermore , they are widely believed to be multipotent rather than pluripotent and are thus only able to differentiate into certain cell types .", "cellular reprogramming potentially overcomes these issues and may offer new treatment methods for a range of diseases , including those of rbcs . takahashi and yamanaka      were the first to show that it is possible to take differentiated somatic cells and transform them into cells with pluripotent potential .", "they began by identifying a pool of 24 transcription factors which are important in maintaining stem cell traits and used retroviral transduction to express these factors in murine embryonic and adult fibroblasts .", "they found that these cells then displayed characteristics and properties comparable to those of pluripotent escs .", "they were able to further refine the required transcription factors by setting up numerous combinations to determine which were essential to this process and identified four factors , oct4 , sox2 , klf4 , and c - myc , needed to transform a somatic cell to an induced pluripotent stem cell ( ipsc ) .", "takahashi et al .      then applied these four factors to human cells and transformed neonatal and adult human fibroblasts into human ipscs ( hipscs ) .", "this was a valuable subsequent step as it showed that this process can be reproduced with human cells and could thus potentially be utilised for stem cell - based therapies of human disease .", "several other transcription factor combinations have since been shown to be sufficient to reprogram somatic cells     [ 2224 ] and one particular combination , involving oct4 , sox2 , nanog , and lin28 , is particularly efficient and is now widely utilised .  ", "for example , a number of the transcription factors used to generate these cells , including c - myc , klf4 , and lin28 , are oncogenes , and their misexpression can lead to cancer     [ 2628 ] . in order for ipscs to be differentiated into a cell type of choice ,", "the retrovirally transmitted genes need to be switched off or removed to reduce the possibility of their inducing tumours [ 22 , 29 ] .", "another potential issue is that the transplantation of any cells that have not been fully differentiated from the ipsc state could lead to the formation of cancerous teratomas .", "any cells remaining which are still in a pluripotent state could multiply and , without the appropriate growth controls , would have the potential to result in tumours in transplant patients . to potentially avoid the issue of tumour formation and uncontrolled proliferation of ipscs , an alternative methodology known as transdifferentiation is also being considered as a potential treatment strategy .  ", "transdifferentiation is achieved by introducing various exogenous factors into a differentiated cell , such as a fibroblast , to directly convert it into another type of differentiated cell , thereby bypassing the pluripotent state ( figure 1(b ) ) . as early as 1990 , choi et al .   were able to convert various cells types , including dermal fibroblasts and chondroblasts , into mononucleated , striated myoblasts that were indistinguishable from normal myoblasts in vivo", "this was achieved through the expression of the myogenic regulatory factor myod , a transcription factor known to be involved in the determination of muscle cells . shortly after this", ", another group showed that it was possible to turn myeloid 416b cells into mast cells through the forced expression of gata-2 and gata-3 , two transcription factors which play important roles in haematopoiesis .", "investigated the effects of ectopically expressing the gene eyeless ( ey ) , an orthologue of the mammalian pax6 gene , in drosophila and found that eye structures formed in places such as the wings and legs .  ", "since takahashi and yamanaka 's pioneering studies in cellular reprogramming , the field of transdifferentiation has advanced considerably .", "zhou et al .   investigated the effects of expressing ngn3 , pdx1 , and mafa , transcription factors involved in -cell differentiation , on exocrine cells of the adult pancreas .", "they found that the coexpression of these factors was able to convert the exocrine cells into -cells .", "the induced -cells were identical to endogenous -cells in morphology and showed similar expression of genes associated with -cell function .", "these cells can also rescue the phenotype of hyperglycaemia , as they are able to secrete insulin and remodel surrounding vasculature . in other work ,", "utilised the neural - specific transcription factors , ascl1 , brn2 , and myt1l , to rapidly convert both murine embryonic fibroblasts and adult fibroblasts directly into functional neurons .", "these induced neuronal cells express neuron - specific proteins , generate action potentials , and form functional synapses .", "a further advance has shown that it is also possible to transdifferentiate fibroblasts into functional neural progenitor cells by transient induction of oct4 , sox2 , klf4 , and c - myc in cells cultured in a defined neural reprogramming medium . this process bypassed the generation of ipscs and gave rise to multipotent progenitors with the capacity to expand and differentiate into a number of neural lineages .", "all of these experiments indicate that it is possible to direct a differentiated cell to another cell fate through the application of extragenic factors ."], ["advances in cellular reprogramming have raised the attractive possibility that this technology could be utilised to generate a limitless source of immune - matched , pathogen - free erythrocytes for transfusion .", "they found that hipscs are capable of generating haematopoietic cells with phenotypic and morphological characteristics similar to those derived from hescs ; however , these hipsc - derived cells exhibited a dramatically reduced capacity ( by greater than 1000-fold ) to generate erythroid cells .   a subsequent study by lapillone et al .", ", however , showed that it was indeed possible to produce significant numbers of mature erythroid cells from hipscs in vitro .", "this group employed the methods outlined by thomson 's group   using oct4 , sox2 , nanog , and lin28 to convert fibroblasts to   hipscs .", "they then cultured these cells in medium containing the cytokines scf , tpo , flt3 ligand , rhu bmp4 , rhu vegf - a165 , il-3 , il-6 , and erythropoietin ( epo ) .", "they analysed the expression profiles of these cells over 20 days of culture and found that pluripotent stem cell markers decreased whilst the erythroid markers cd36 , cd235a , and cd71 increased .", "cells at day 20 were found to have a high erythroid potential and were plated in sequential cocktails of cytokines comprising scf , il-3 , and/or epo .", "erythroid maturation was achieved after another 25 days and was confirmed by morphological examination and by flow cytometric analysis of erythroid markers ( cd235a , cd71 , and cd36 ) .", "furthermore , these erythroid cells were able to enucleate , albeit with reduced capacity compared to hesc - derived erythroid cells , and were found to express functional hb ( predominantly hbf ) .", "these hipsc - derived erythroid cells were compared to those differentiated from hescs , and no significant differences were detected in terms of erythroid commitment , expression of erythroid markers , and type and functionality of hb .  ", "revealed that while functional rbcs could be generated from hipscs , when compared to hescs , hipscs were shown to have reduced ( approximately 8-fold ) amplification potential in producing mature erythroid cells .", "a recent study has set out to determine whether this reduced efficiency is an intrinsic property of hipscs , or whether it can be increased by altered culture conditions .", "this study showed not only that it is possible to generate rbcs from hipscs with similar efficiency to hescs , but also that these cells can be differentiated from hipscs generated using episomal vectors , obviating the need for potentially deleterious retroviral transfection .", "this group cultured hipscs derived from human fibroblasts with the op9 bone marrow stroma line to induce hematopoietic differentiation and followed this with selective expansion of erythroid cells in serum - free media with cytokines supporting rbc differentiation .", "the erythroid cultures established in this study consisted of a pure population of cd235a+ cd45 leukocyte - free rbcs which had robust expansion capabilities , as well as a long lifespan of up to 90 days .", "these hipsc - derived cells can enucleate and were shown to express foetal - and embryonic -globin demonstrating successful reprogramming of the -globin locus .", "the results from this study show that it is possible to produce significant numbers of erythroid cells from fibroblast - derived hipscs , and that thus cellular reprogramming could contribute to the treatment of haemoglobinopathies . furthermore , since these rbcs are generated from transgene - free hipscs , they circumvent problems associated with genomic integration and undesired reactivation of reprogramming factors .   another recent study has shown that it is possible to use transdifferentiation to produce haematopoietic cell lineages , including mature erythrocytes . in this work , szabo et al .", "transduced human dermal fibroblasts with oct4 alone and showed that these cells then express the panhaematopoietic marker cd45 but lack the pluripotency marker tra-1 - 60 .", "they then cultured these cells in a haematopoietic cytokine cocktail containing scf , g - csf , flt3lg , il-3 , il-6 , and bmp-4   supplemented with epo and observed an emergence of cells expressing adult -globin protein and the red blood cell marker glycophorin - a .", "in addition , the authors noted that these erythroid cells express adult rather than embryonic globins , unlike cells derived from human pluripotent stem cells     [ 39 , 40 ] , perhaps suggesting that they have indeed been transdifferentiated from adult fibroblasts , bypassing the hipsc stage .", "as the techniques used in this study avoid the pluripotent state as well as have a high yield , expansion capacity , and clinical feasibility , this strategy could provide a reasonable basis for autologous cell replacement therapies .  ", "these studies demonstrate that it is possible to differentiate hipscs into mature erythrocytes in vitro for transfusions . in order to utilise these methods to treat rbc diseases successfully", ", fibroblasts could potentially be taken from either a healthy immunomatched individual , or a universal donor , and be converted to erythrocytes via the hipsc state .", "a study has already developed hipscs from an individual with a bombay phenotype of the abo blood group system , where the abh antigen is not expressed on rbcs , and blood can be donated to anyone     .", "this group has further demonstrated that it is possible to differentiate haematopoietic lineages from these cells although they have not explicitly shown mature erythrocyte differentiation .", "this possible treatment method circumvents many of the problems associated with current transfusions , such as questionable quality , lack of supply , and immune - rejection .", "however , issues still remain , such as cost and the risk of iron overloading ."], ["in order to cure , rather than treat , rbc diseases , healthy progenitor cells must be transplanted into patients and subsequently repopulate the haematopoietic system .", "a promising study has already shown that it is possible to use reprogrammed cells to treat sickle cell anaemia in mice .", "hanna et al .      took cells from the tail tips of a humanized sickle cell anaemic mouse model , in which the mouse -globin genes have been replaced with human -globin , and the mouse -globin genes have been replaced with human  and  ( sickle ) globin genes , and through adaptation of the protocol developed by takahashi and yamanaka , who reprogrammed these cells into ipscs .", "once this was accomplished , they were able to correct the mutant -globin gene by replacing it with a copy of the wildtype human -globin gene through homologous recombination . following this ,", "the ipscs were differentiated into haematopoietic progenitors through the ectopic expression of hoxb4 , and by growing in haematopoietic cytokines on an op9 bone marrow stroma cell line .", "this group then carried out extensive testing to determine whether the treated mice displayed any further signs of the disease .", "firstly , functional correction was evaluated by electrophoresis for the human -globin proteins  and  , and they found a significant increase in - and a decrease in -globin .  ", "blood counts were also performed 12 weeks after transplant , which showed that treated mice had increased rbcs , hb , and packed cell volume as well as reduced reticulocytes , a common indicator of sickle cell disease and severity .", "lastly , they examined symptomatic indicators of sickle cell anaemia such as urine concentration , body weight , and breathing rate and found that all three of these parameters were ameliorated in the treated mice .", "this study thus provides an important proof of principle that cellular reprogramming can be employed to correct erythroid disorders , albeit in this case , in conjunction with gene therapy ."], ["normal erythropoiesis is dependent upon the correct expression of globin genes . where globin genes are incorrectly expressed , or are mutated , anaemia or thalassemia results .", "current therapy for these disorders involves packed red blood cell transfusions , which are limited by supply , risk of infection , expense , and patient rejection .", "drug - based therapies involve the nonspecific reactivation of foetal globins and have long - term side effects . in seeking alternative strategies", ", recent advances have shown that cellular reprogramming can now generate large quantities of red blood cells in culture , potentially for use in transfusions .", "furthermore , these strategies have been successfully combined with gene therapy to treat a sickle cell anaemia mouse model , suggesting that cellular reprogramming will provide a realistic future alternative to conventional treatment of haemoglobinopathies ."]]}
{"article_id": "PMC3888851", "article_text": ["he had a left ventricular ejection fraction of 42% and regional wall motion abnormalities compatible with multi - vessel territories .", "coronary angiography findings revealed total occlusion at the proximal left anterior descending artery ( lad ) , 50 - 75% stenosis at the mid to distal left circumflex artery , and 90% stenosis at the mid right coronary artery ( rca ) .", "cardiac surgery was performed through a median sternotomy , and the heart was displaced using a deep pericardial retraction suture , gauze swab , and tissue stabilizer . after confirming the lack of atheroma burden in the ascending aorta by epiaortic ultrasound examination , the left radial artery was connected to the ascending aorta . after completing the lad grafting using the left internal thoracic artery ,", "the patient was in the trendelenburg position , and norepinephrine was infused at a dose of 0.15 g / kg / min . after stabilizer application , there was a temporary deterioration in the patient 's hemodynamic status , which was stabilized without the aid of incremental doses of norepinephrine dosage .", "after opening the anastomotic site at the om branch , the surgeon informed us the patient was bleeding from the arteriotomy site despite placement of a proximal snare .", "the co2 blower was more aggressively used to aid grafting at a flow rate of 3 - 5 l / min .", "bleeding was replaced by colloid solution , and all hemodynamic variables were stable : systemic blood pressure 121/58 mmhg , heart rate 59 beats / min , pulmonary arterial pressure 24/11 mmhg , cardiac index 2.6 l / min / m and mixed venous oxygen saturation 82% . approximately 5 minutes after the commencement of grafting , the surgeon decided to insert an intracoronary shunt for better visualization .", "suddenly , the patient 's systemic blood pressure decreased to 64/56 mmhg despite increasing the norepinephrine dose from 0.05 g / kg / min to 0.30 g / kg / min .", "the pulmonary arterial pressure increased to 34/20 mmhg , and the mixed venous oxygen saturation decreased to 66% .", "vasopressin 0.6 iu was quickly administered intravenously , and the transesophageal echocardiography ( tee ) revealed a massive gas floating at the orifice of the rca ( fig .", "although the patient remained in normal sinus rhythm , complete heart block occurred within minutes , and ventricular pacing was commenced . over the next several minutes , ventricular fibrillation developed ; despite aggressive treatment , including defibrillation , epinephrine , and direct manual cardiac massage , adequate circulation could not be restored , and cardiac massage was continued . upon preparing for emergent conversion to cardiopulmonary bypass ( cpb ) , epinephrine", "300 g was injected directly to the aortic root by the surgeon using a 26-gauge needle .", "subsequently , manual cardiac massage was continued for several cycles , and spontaneous recovery of unspecified cardiac rhythm was observed .", "then the patient 's systemic mean arterial pressure gradually rose from 38 to 103 mmhg , and recovery of normal sinus rhythm and contractile performance followed immediately .", "the remaining grafts were performed successfully without conversion to cpb , and no sign of bleeding was observed at the puncture site in the aorta .", "the patient had an unremarkable postoperative course without neurologic deficits and was discharged 10 days after the surgery .", "despite using a proximal coronary snare , this method of hemostasis can be incomplete in the presence of severe atheroma or calcification , causing bleeding through the arteriotomy site .", "cardiac displacement invariably causes hemodynamic deterioration , and when the diastolic systemic blood pressure decreases below the pressure generated by the co2 gas blower , gas may migrate in a retrograde fashion to the aortic root . even in the presence of a proximal snare", ", the proximal coronary lumen may not be completely obstructed due to the presence of calcified atheroma , as evidenced by continued bleeding from the arteriotomy site after application of the snare . as the rca ostium is in the highest position of the sinus of valsalva", ", gaseous materials would be prone to migrate into the rca , causing embolic infarction .", "a minor gas embolism may pass unnoticed or be overcome with supportive measures to aid emboli clearance .", "however , in the case of a massive embolism with the possible introduction of room air due to venturi effect , hemodynamic collapse can occur and may require aggressive treatment , such as cardiopulmonary resuscitation ( cpr ) with aspiration of the air by direct puncture of the aortic root .", "it may even require emergent conversion to cpb , which is known to be associated with increased morbidity and mortality .", "therefore , when bleeding from the arteriotomy site persists despite the use of a proximal snare or intracoronary shunt , anesthesiologists should be aware of the possibility of a gas embolism and advise the surgical team to stop or limit the use of the co2 blower .", "when a gas embolism results in hemodynamic deterioration , supportive measures should be immediately undertaken to increase the coronary perfusion pressure and facilitate emboli clearance .", "gas embolism should also be confirmed with tee , and a pacemaker should be at hand given that complete heart block due to atrioventricular nodal ischemia may occur in the case of an embolism involving the rca .", "finally , in the case of failure of supportive measures such as drug administration to the venous side , direct aortic injection of the drugs can be attemped before initiating cpb . in coronary artery bypass graft surgery", ", direct cardiac massage may not be aggressively and effectively performed due to the fear of disrupting the previously grafted conduits , limiting the drug delivery from the venous side to the systemic circulation .", "prior to the 1960s , intracardiac injection of medications was used blindly or directly , but this approach was abandoned due to the devastating complication of lacerating the epicardial coronary arteries and cardiac tamponade .", "however , direct injection to the aortic root following a sternotomy appears to be feasible and may be life - saving , as severe right ventricular dysfunction may limit systemic drug delivery injected through the venous side despite the performance of direct cardiac massage . in the current case , intra - aortic injection of epinephrine may have resulted in a more effective increase in the systemic vascular resistance , thereby increasing the coronary perfusion pressure and thus facilitating emboli clearance . in conclusion , in the rare case of a gas embolism occurring during opcab occurs , the potential for catastrophic results necessitates immediate and appropriate managements including supportive measures , needle aspiration of gas , and cpr with direct cardiac massage . in refractory cases , while considering emergent conversion to cpb , direct injection of drugs to the aortic root can be attempted before initiating cpb .", "this may be a simple , relatively safe , and life - saving approach for the management of a gas embolism during opcab because a sternotomy has already been conducted ."], "abstract_text": ["<S> although compressed gas ( co2 ) blowers have been used safely to aid accurate grafting during off - pump coronary bypass surgery , hemodynamic collapse due to gas embolism into the right coronary artery may occur . </S>", "<S> supportive measures to facilitate gas clearance by increasing the coronary perfusion pressure have been reported to be successful in restoring hemodynamic stability . however , right ventricular dysfunction and atrioventricular nodal ischemia may hinder effective systemic delivery of the vasoactive medications , even when performing resuscitative measures such as direct cardiac massage . </S>", "<S> we herein report a case of cardiac arrest that was caused by a right coronary gas embolism and that could not be restored by cardiac resuscitation . </S>", "<S> when supportive measures fail , direct aortic injection of epinephrine to increase the coronary perfusion pressure can be attempted before initiating cardiopulmonary bypass , and this approach may be life - saving in situations that limit systemic drug delivery from the venous side despite the performance of direct cardiac massage . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["he had a left ventricular ejection fraction of 42% and regional wall motion abnormalities compatible with multi - vessel territories .", "coronary angiography findings revealed total occlusion at the proximal left anterior descending artery ( lad ) , 50 - 75% stenosis at the mid to distal left circumflex artery , and 90% stenosis at the mid right coronary artery ( rca ) .", "cardiac surgery was performed through a median sternotomy , and the heart was displaced using a deep pericardial retraction suture , gauze swab , and tissue stabilizer . after confirming the lack of atheroma burden in the ascending aorta by epiaortic ultrasound examination , the left radial artery was connected to the ascending aorta . after completing the lad grafting using the left internal thoracic artery ,", "the patient was in the trendelenburg position , and norepinephrine was infused at a dose of 0.15 g / kg / min . after stabilizer application , there was a temporary deterioration in the patient 's hemodynamic status , which was stabilized without the aid of incremental doses of norepinephrine dosage .", "after opening the anastomotic site at the om branch , the surgeon informed us the patient was bleeding from the arteriotomy site despite placement of a proximal snare .", "the co2 blower was more aggressively used to aid grafting at a flow rate of 3 - 5 l / min .", "bleeding was replaced by colloid solution , and all hemodynamic variables were stable : systemic blood pressure 121/58 mmhg , heart rate 59 beats / min , pulmonary arterial pressure 24/11 mmhg , cardiac index 2.6 l / min / m and mixed venous oxygen saturation 82% . approximately 5 minutes after the commencement of grafting , the surgeon decided to insert an intracoronary shunt for better visualization .", "suddenly , the patient 's systemic blood pressure decreased to 64/56 mmhg despite increasing the norepinephrine dose from 0.05 g / kg / min to 0.30 g / kg / min .", "the pulmonary arterial pressure increased to 34/20 mmhg , and the mixed venous oxygen saturation decreased to 66% .", "vasopressin 0.6 iu was quickly administered intravenously , and the transesophageal echocardiography ( tee ) revealed a massive gas floating at the orifice of the rca ( fig .", "although the patient remained in normal sinus rhythm , complete heart block occurred within minutes , and ventricular pacing was commenced . over the next several minutes , ventricular fibrillation developed ; despite aggressive treatment , including defibrillation , epinephrine , and direct manual cardiac massage , adequate circulation could not be restored , and cardiac massage was continued . upon preparing for emergent conversion to cardiopulmonary bypass ( cpb ) , epinephrine", "300 g was injected directly to the aortic root by the surgeon using a 26-gauge needle .", "subsequently , manual cardiac massage was continued for several cycles , and spontaneous recovery of unspecified cardiac rhythm was observed .", "then the patient 's systemic mean arterial pressure gradually rose from 38 to 103 mmhg , and recovery of normal sinus rhythm and contractile performance followed immediately .", "the remaining grafts were performed successfully without conversion to cpb , and no sign of bleeding was observed at the puncture site in the aorta .", "the patient had an unremarkable postoperative course without neurologic deficits and was discharged 10 days after the surgery ."], ["despite using a proximal coronary snare , this method of hemostasis can be incomplete in the presence of severe atheroma or calcification , causing bleeding through the arteriotomy site .", "cardiac displacement invariably causes hemodynamic deterioration , and when the diastolic systemic blood pressure decreases below the pressure generated by the co2 gas blower , gas may migrate in a retrograde fashion to the aortic root . even in the presence of a proximal snare", ", the proximal coronary lumen may not be completely obstructed due to the presence of calcified atheroma , as evidenced by continued bleeding from the arteriotomy site after application of the snare . as the rca ostium is in the highest position of the sinus of valsalva", ", gaseous materials would be prone to migrate into the rca , causing embolic infarction .", "a minor gas embolism may pass unnoticed or be overcome with supportive measures to aid emboli clearance .", "however , in the case of a massive embolism with the possible introduction of room air due to venturi effect , hemodynamic collapse can occur and may require aggressive treatment , such as cardiopulmonary resuscitation ( cpr ) with aspiration of the air by direct puncture of the aortic root .", "it may even require emergent conversion to cpb , which is known to be associated with increased morbidity and mortality .", "therefore , when bleeding from the arteriotomy site persists despite the use of a proximal snare or intracoronary shunt , anesthesiologists should be aware of the possibility of a gas embolism and advise the surgical team to stop or limit the use of the co2 blower .", "when a gas embolism results in hemodynamic deterioration , supportive measures should be immediately undertaken to increase the coronary perfusion pressure and facilitate emboli clearance .", "gas embolism should also be confirmed with tee , and a pacemaker should be at hand given that complete heart block due to atrioventricular nodal ischemia may occur in the case of an embolism involving the rca .", "finally , in the case of failure of supportive measures such as drug administration to the venous side , direct aortic injection of the drugs can be attemped before initiating cpb . in coronary artery bypass graft surgery", ", direct cardiac massage may not be aggressively and effectively performed due to the fear of disrupting the previously grafted conduits , limiting the drug delivery from the venous side to the systemic circulation .", "prior to the 1960s , intracardiac injection of medications was used blindly or directly , but this approach was abandoned due to the devastating complication of lacerating the epicardial coronary arteries and cardiac tamponade .", "however , direct injection to the aortic root following a sternotomy appears to be feasible and may be life - saving , as severe right ventricular dysfunction may limit systemic drug delivery injected through the venous side despite the performance of direct cardiac massage . in the current case , intra - aortic injection of epinephrine may have resulted in a more effective increase in the systemic vascular resistance , thereby increasing the coronary perfusion pressure and thus facilitating emboli clearance . in conclusion , in the rare case of a gas embolism occurring during opcab occurs , the potential for catastrophic results necessitates immediate and appropriate managements including supportive measures , needle aspiration of gas , and cpr with direct cardiac massage . in refractory cases , while considering emergent conversion to cpb , direct injection of drugs to the aortic root can be attempted before initiating cpb .", "this may be a simple , relatively safe , and life - saving approach for the management of a gas embolism during opcab because a sternotomy has already been conducted ."]]}
{"article_id": "PMC3663610", "article_text": ["multiple sclerosis ( ms ) is a chronic progressive inflammatory and neurodegenerative disease of human central nervous system ( cns ) which usually affects young adults , with a reported median age of onset of approximately 30 years.1 in approximately 85% of patients with ms , the disease is initially recognized by a relapsing - remitting course which may eventually lead to a progressive deterioration of neurological status along with significant loss of neurological function.2 neuropathologically , ms presents with various patterns , however , inflammatory demyelination and oligodendrocyte loss and neuronal and axonal degeneration3 constitute salient features of microscopic examination of ms lesions .", "currently , the cause and cure for ms remain elusive and in many cases ms ends with significant neurological disabilities . while massive activation of the inflammatory arm of the immune system against putative cns antigen(s ) such as myelin basis protein plays a major role in the pathogenesis of the demyelinating process of ms , the neurodegenerative process prevails in the long run and causes significant cognitive and functional deterioration in affected patients .", "electrophysiologic examination of demyelinated lesions indicates conduction block as the predominant feature of these lesions .", "the pathophysiologic basis of such conduction block in ms rests on segmental demyelination with loss of whole internodes of myelin . in normal individuals , blocking the voltage - gated potassium channels does not have any significant impact on conduction of the action potential since these channels are enclosed by layers of myelin sheath .", "however , loss of myelin during pathogenesis of ms , exposes the potassium channels and interferes with production and conduction of the action potential .", "in fact , the impulse conduction fails specifically at the site of demyelination , while the normal segments of the axons on both sides continue their impulse conduction normally .", "certain features which determine the impulse conduction block in demyelinated axon consist of the size of demyelinated area , extent of myelin loss along the internode , and the time elapsed since demyelination happened .", "the duration of demyelination is an important factor since myelin loss sets a number of adaptive axonal responses in motion .", "for example , development of a revised group of ion channels along the demyelinated membrane may result in restoration of impulse conduction .", "the size of the demyelinated lesion is another significant factor since axons repair centripetally inside the lesion and extensive lengths of demyelination are repaired slowly which in turn reflects the fact that the chance of functional recovery correlated inversely with the lesion size .", "it appears that myelin loss involving the paranodal area is stronger factor in blocking conduction than a comparable loss of distributed along the internode .", "apart from the segmental demyelination which is associated with conduction block , histopathologic examination of ms tissue has shown that some node are inappropriately wide in ms lesions due to paranodal demyelination or retraction of the paranodes.4 another salient feature of demyelinated axons is abnormal reorganization of the ion channels . the speedy conduction velocity and uninterrupted transmission of electric signals in mammalian myelinated axons rest on the appropriate spatial distribution ofion - selective channels . na channels are gathered at nodes of ranvier in concentrations which are ~25 times that of internodal regions.5 these clusters are essential for efflux of ion currents at nodes and allow fast saltatory conduction .", "on the contrary , other studies have demonstrated that k currents appear only after loosening of the myelin sheath from the axonal membrane.6 k channels are located only in paranodal and internodal areas and normally they do not contribute to generation of the action potential.7 scientific studies which have focused on action potential propagation in normal and demyelinated axons have revealed a reorganization of axolemmal ion channels , which in turn leads to conduction impairments . loss or decrease of sodium channels from the nodes of ranvier8 causes the nodes to be inexcitable and leads to conduction block .", "sherratt et al9 demonstrated that in demyelinated regions sodium channel concentrations are reduced , while voltage - sensitive potassium channels ( which are not active on normal myelinated axons ) become detectable .", "this exposure of k channels at juxtaparanodal region leads to efflux through fast k channels and causes axonal conduction block by preventing sufficient depolarization and initiation of the action potential at the node of ranvier.10 demyelination also exposes slow k channels , further interrupting normal hyperpolarization and blunting normal repetitive impulse release from the presynaptic area terminals.11 in 1981 , bostock et al12 demonstrated that potassium channel blockers dendrotoxin and 4-aminopyridine improved conduction impairments in experimentally demyelinated axons and laid the scientific rationale for clinical studies of potassium channel blockers in ms patients .", "fampridine ( or 4-aminopyridine [ 4-ap ] ) is a broad - spectrum blocker of voltage - sensitive potassium channels , which has been demonstrated to improve conduction and propagation of the action potential along the demyelinated axons .", "in addition , fampridine increases the duration of the pre - synaptic action potential and amplitude which in turn leads to increased transmitter release.13 apart from being a voltage - activated potassium channel blocker , 4-aminopyridine potentiates synaptic and neuromuscular transmission by affecting the beta subunit of voltage - activated calcium channels.14 this novel mode of action of 4-aminopyridine which occurs independent from its blocking effect on the potassium channels , provides another rationale for its positive effect on the neuromuscular function in patients with spinal cord injury , myasthenia gravis and ms .", "hawthorne , ny , usa ) is one of the three isomeric amines of pyridine with chemical formula h2nc5h4n , which acts a selective potassium - channel blocker .", "dalfampridine , which is a sustained - release oral form of fampridine , is rapidly and completely absorbed from the gastrointestinal tract , however , its absolute bioavailability has not been determined .", "compared to an aqueous oral solution , the extended release tablet shows a relative bioavailability of 96% , with a delayed absorption pattern which provides a less rapid rise to a lower maximum plasma concentration . in healthy volunteers who took a single 10 mg dose of dalfampridine", ", peak serum concentrations were reached 3 to 4 hours following oral administration and it was almost completely and rapidly eliminated by urinary excretion .", "the elimination half - life of dalfampridine is 6.4 hours in healthy individuals . as a lipid - soluble agent", ", dalfampridine passes through the blood brain barrier and blocks potassium channels in both the central and peripheral nervous systems.15 daldampridine binds reversibly to the cytoplasmic side of potassium channels , blocking the ion conductance pathway which in turn prolongs the action potentials in unmyelinated nerve fibers and enhances neurotransmitter release at synaptic endings.16 dalfampridine has been studied extensively in symptomatic treatment of ms patients with walking impairment .", "impairment of ambulation is a major neurological deficit in ms patients and significantly interferes with their quality of life .", "it is hypothesized that dalfampridine improves clinical symptoms of ms by restoring conduction in demyelinated axons through voltage - dependent potassium channel blockade.15 in addition , dalfampridine has been used for treatment of lambert - eaton myasthenic syndrome,17 where it prolongs neurotransmitter release at the neuromuscular junction .", "studies involving animal models have demonstrated that can reverse tetrodotoxin toxicity.18 pharmacokinetic assessmentsof both immediate - release and sustained release formulations of fampridine have been done in normal individuals as well as those patients with ms and spinal cord damage .", "uges et al19 performed a pharmacokinetic study involving 9 healthy individuals ( 7 men and 2 women ) to assess immediate - release fampridine in intravenous , entric - coated , and uncoated oral formulations .", "the bioavailability of the enteric coated tablets was calculated from the area under the serum concentration curve 95%  29% ( mean  sd ) , which was not much different from that calculated from urinary excretion 98%  8% . there was no evidence that metabolism of fampridine involved glucoronidation , sulfonation , or n - acetylation .", "investigators reported that 89.6%  7.5% of the medication was excreted in the urine unchanged after 24 hours of the intravenous formulation and 86.1%  2.7% with the entric coated formulation .", "one dose - ranging pharmacokinetic study included 12 ms patients who received increasing doses of sustained - release fampridine from 7.5 to 25 mg every 12 hours.20 the investigators reported a mean time to peak concentration of 5 hours and mean serum half - life of 5.2 hours .", "vollmer et al21 studied the pharmacokinetics of dalfampridine in clinical trials of ms patients and found that multiple dosing of this medication was associated with its accumulation .", "in addition , the investigators noted that the steady - state pharmacokinetic profile of fampridine sustained - release 20 mg bid administered for 2 weeks appeared to support the administration of twice - daily dosing in this population .", "dalfampridine ( ampyra ) is available as 10 mg extended release tablets with relative bioavailability of 96% , peak of plasma concentration of 34 hours and half life of 5.26.5 hours .", "the major goals of treating ms patients consist of prevention of exacerbations , limiting disability progression , and improving cognitive decline . the available treatments for ms fall under two categories : disease modifying agents such as beta - interferons and glatiramer acetate and medications for symptomatic treatment such as baclofen for treatment of spasticity and dalfampridine which is used to restore the impulse conduction along the demyelinated axons and improve muscle strength and walking ability . in january 2010", ", dalfampridine was approved by the fda for symptomatic treatment of ms patients with specific indication for improving walking . in the past", "4-aminopyridine has been used as an experimental agent which presumably enhances nerve conduction of demyelinated axons through its effects on potassium channels for treatment of fatigue in ms .", "sheean et al22 reported that use of fampridine in ms patients was associated with improvements in fatigue as well motor and visual symptoms .", "in addition , dalfampridine has been assessed and studied in other cohorts of ms patients in the context of various clinical trials to determine its efficacy in improving ms patients ambulation . in this manuscript only some of the more significant clinical trials have been cited and discussed .", "using quantitative measures of motor function , schwid et al23 performed one of the earlier clinical trials of sustained - release fampridine - sr in ms patients .", "this was a randomized , double - blind , placebo - controlled , crossover clinical trial included 10 patients with clinically definite ms , limb weakness and stable motor deficits ( edss 6.07.5 ) .", "study subjects were randomized to be treated with sustain - release fampridine 17.5 mg orally twice daily for a period of one week or placebo for a period of one week .", "study outcomes included time to walk 8 meters , time to climb four stairs , maximum voluntary isometric contraction measured quiantitatively , manual muscle testing , grip strength , edss , and the patient s global impression .", "of these outcomes , only timed gait progressed on sustain - release fampridine compared with placebo in 9 of the study participants .", "seven participants preferred the medication over placebo based on the findings from the patient s global impression .", "goodman et al24 reported the results of a four - center randomized , double - blind placebo - controlled study of controlled release aminopyridine in 31 ms patients .", "the study participants were treated with increasing doses of 20 to 80 mg of aminopyridine daily in divided doses .", "a total of 25 participants received the active medication and 11 participants were treated with placebo .", "outcome measures of this clinical trial consisted of timed ambulation , manual testing of leg strength , paced auditory serial addition task , 9-hole peg test , and a fatigue diary .", "the results of this clinical trial indicated a statistically significant and dose - related enhancement of timed ambulation in ap - treated patients compared to those who received placebo .", "a significant enhancement of leg strength was also observed in approximately 11% of ap - treated participants compared with a 4% worsening of placebo - treated participants , p = 0.01 .", "the reported side effects consisted of dizziness , insomnia , paresthesia , nausea , headache , tremor , pain and anxiety . on daily doses of 60 and 70 mg", "a phase 2 , multi - center , randomized , double - blind , placebo - controlled , parallel - group , dose - comparison ( 10 , 15 , or 20 mg twice daily orally ) clinical trial of sustained - release fampridine in ms patients demonstrated significantly more consistent responders as determined by improvement in ambulation as compared to the placebo - group : 36.7% versus 8.5%.25 fampridine was generally well tolerated and serious and severe adverse effects were more common in those patients who received the highest dose . a multi - center randomized , double - blinded , placebo - controlled phase 2 dose - raning clinical trial was conducted by goodman et al which assessed the concept that whether the dose of fampridine - sr could be safely elevated to 80 mg / daily in patients with ms.26 a cohort of 36 ms patients were randomized in a 2 to 1 ratio to be treated with an increasing dose of fampridine - sr for a period of 8 weeks . the dosage of fampridine - sr was escalated from 10 to 40 mg twice daily by increments of 5 mg twice daily on a weekly schedule .", "a group of evaluations were done once a week which included ms functional composite ( msfc ) , fatigue questionnaires , and lower extremity manual muscle testing .", "the objectives of this clinical trial included assessment of the safety of higher doses of fampridine - sr as well as the efficacy and dose - response to this drug by ms patients .", "five study participants were terminated from fampridine - sr due to adverse events at doses larger than 25 mg and these events consisted of seizures in two individuals at doses of 30 and 35 mg twice daily .", "no subjects in the placebo arm of the study dropped out due to the adverse events . in the group which was treated with fampridine - sr", "compared to the group which was treated with placebo showed improvement in the lower extremity muscle strength ( prospective analysis ) and walking speed ( post - hoc analysis ) .", "the investigators concluded that future clinical trials should restrict the dose of fampridine - sr to 20 mg twice daily and walking speed and lower extremity muscle strength tests are potential efficacy measurements .", "another phase 3 multicenter clinical trial of dafampridine which was controlled and double - blind included 301 ms patients of different types ( 27% with relapsing - remitting ms and 73% with progressive ms ) . during the 14 weeks duration of this trial", ", participants were assigned in a ratio of 3 to 1 to be treated with dalfampridine 10 mg ( n = 229 ) or placebo ( n = 72 ) orally twice daily for a period of 14 weeks.27 the primary aim of this study included steady progress on timed 25-foot walk to define responses with proportion of timed walk responders in each arm .", "the investigators reported a higher proportion of timed walk responders in the fampridine - treated arm ( 78/224 or 35% ) compared to those who were treated with placebo ( 6/72 or 8% ) .", "based on the results of this study , treatment of ms patients with fampridine was associated with gait improvement with a decrease in patient s gait impairment .", "a more recent executed phase 3 multi - center double - blind clinical trial of extended - release dalfampridine in patients with definite ms of any clinical course assessed the efficacy , safety , and pharmacodynamics of extended release oral dalfampridine in these patients.28 the length of this study was 9 weeks and the participants were randomized to be treated with dalfampridine ( 10 mg twice daily ; n = 120 ) or placebo ( n = 119 ) for a period of 9 weeks .", "the primary aim of this study consisted of constant progress on the timed 25-foot walk , with percentage of time walk responders ( twrs ) in each treatment arm .", "the last on - treatment assessment collected information from 8 to 12 hours postdose , to assess and determine the persistence of the impact of the medication .", "the results of this clinical trial indicated that the quantity of twrs was higher in the dalfampridine - treated group ( 51/119 or 42.9% ) compared to the placebo - treated group ( 11/118 or 9.3% .", "the average enhancement of walking speed among the dalfampridine - treated twrs during the 8-week efficacy assessment period was 24.7% compared to baseline ; the mean enhancement during the finalon - treatment visit was 25.7% , reflecting continuation of the impact of medication over the interdosing phase .", "the authors concluded that this clinical trial generated class 1 evidence that treatment of patients with definite ms with extended - release dalfampridinetablets caused meaningful enhancement of walking ability in and the effect of the medication continued persisted between doses .", "based on the animal studies , dalfampridine demonstrates properties of a powerful convulsant stemming from its broad - spectrum suppressing activity on the potassium channels in the peripheral and central nervous systems.11 based on a number of clinical trials of 4-ap side effects consist of dizziness , insomnia , paresthesia , nausea of mild to moderate intensity , tremor , pain , anxiety and seizures.2934 dalfampridine is neither a substrate for nor an inhibitor of the p - glycoprotein transporter , therefore its pharmacokinetics is not affected by the medications which suppress this transporter .", "it does not interact with the medications which are substrates for the p - glycoprotein transporter .", "dalfampridine is contraindicated in patients with history of epilepsy or history of severe renal insufficiency.35 the reason that dalfampridine use is not recommended in epileptic patients is that epilepsy is characterized by abnormal neuronal depolarization with excessive inhibition on potassium channels , hence a further reduction of potassium channel activity by dalfampridine is not advised .", "pathogenesis of ms involves loss of oligodendrocyte / myelin complex which turn results in re - organization of axolemmal ion channels which leads to conduction abnormalities including conduction delay or block .", "pharmacological agents which block potassium channels have been investigated in the context of clinical trials with positive impact on impulse conduction in experimentally - induced demyelination as well as in patients with ms .", "dalfampridine ( which is marketed as ampyra ) , is an extended release form of fampridine ( 4-aminopyridine ) , which has been recently approved by the us fda for symptomatic treatment of ms patients .", "while this new oral blocker of voltage - gated potassium channels does not have any impact on the underlying pathology of ms , it has been demonstrated to improve fatigue and walking ability in these patients ."], "abstract_text": ["<S> multiple sclerosis ( ms ) is a progressive immune - mediated neurodegenerative disease of human central nervous system ( cns ) , which causes irreversible disability in young adults . </S>", "<S> the cause and cure for ms remain unknown . </S>", "<S> pathophysiology of ms includes two arms : inflammatory demyelination and neurodegeneration . </S>", "<S> the inflammatory demyelination of ms which is mainly promoted by a massive activation of the immune system against putative cns antigen(s ) leads to loss of oligodendrocyte / myelin complex which slows down or halts impulse conduction in denuded axons . </S>", "<S> practically , loss of myelin significantly reduces signal conduction along the demyelinated axons through alterations in the distribution of axonal ion channels . </S>", "<S> dalfampridine ( 4-aminopyridine or 4-ap ) is an oral potassium channel blocker , which was recently approved by fda for symptomatic treatment of ms . </S>", "<S> dalfampridine , which acts at the central and peripheral nervous systems , enhances conduction in demyelinated axons and improves walking ability of ms patients . </S>", "<S> a number of clinical trials have evaluated the safety and efficacy of fampridine in ms patients with the degree of gait improvement as the main outcome . </S>", "<S> the objective of this manuscript is to provide an overview of the pharmacology , pharmacokinetics , clinical trials , side effects and interactions of dalfampridine used in treatment of ms patients . </S>"], "labels": null, "section_names": ["Introduction", "Dalfampridine: Introduction and Mechanism of Action", "Pharmacokinetics", "Dosage, Formulation, and Administration", "Clinical trials of Dalfampridine in Multiple Sclerosis", "Side Effects, Interactions, and Contraindications", "Conclusion"], "sections": [["multiple sclerosis ( ms ) is a chronic progressive inflammatory and neurodegenerative disease of human central nervous system ( cns ) which usually affects young adults , with a reported median age of onset of approximately 30 years.1 in approximately 85% of patients with ms , the disease is initially recognized by a relapsing - remitting course which may eventually lead to a progressive deterioration of neurological status along with significant loss of neurological function.2 neuropathologically , ms presents with various patterns , however , inflammatory demyelination and oligodendrocyte loss and neuronal and axonal degeneration3 constitute salient features of microscopic examination of ms lesions .", "currently , the cause and cure for ms remain elusive and in many cases ms ends with significant neurological disabilities . while massive activation of the inflammatory arm of the immune system against putative cns antigen(s ) such as myelin basis protein plays a major role in the pathogenesis of the demyelinating process of ms , the neurodegenerative process prevails in the long run and causes significant cognitive and functional deterioration in affected patients .", "electrophysiologic examination of demyelinated lesions indicates conduction block as the predominant feature of these lesions .", "the pathophysiologic basis of such conduction block in ms rests on segmental demyelination with loss of whole internodes of myelin . in normal individuals , blocking the voltage - gated potassium channels does not have any significant impact on conduction of the action potential since these channels are enclosed by layers of myelin sheath .", "however , loss of myelin during pathogenesis of ms , exposes the potassium channels and interferes with production and conduction of the action potential .", "in fact , the impulse conduction fails specifically at the site of demyelination , while the normal segments of the axons on both sides continue their impulse conduction normally .", "certain features which determine the impulse conduction block in demyelinated axon consist of the size of demyelinated area , extent of myelin loss along the internode , and the time elapsed since demyelination happened .", "the duration of demyelination is an important factor since myelin loss sets a number of adaptive axonal responses in motion .", "for example , development of a revised group of ion channels along the demyelinated membrane may result in restoration of impulse conduction .", "the size of the demyelinated lesion is another significant factor since axons repair centripetally inside the lesion and extensive lengths of demyelination are repaired slowly which in turn reflects the fact that the chance of functional recovery correlated inversely with the lesion size .", "it appears that myelin loss involving the paranodal area is stronger factor in blocking conduction than a comparable loss of distributed along the internode .", "apart from the segmental demyelination which is associated with conduction block , histopathologic examination of ms tissue has shown that some node are inappropriately wide in ms lesions due to paranodal demyelination or retraction of the paranodes.4 another salient feature of demyelinated axons is abnormal reorganization of the ion channels . the speedy conduction velocity and uninterrupted transmission of electric signals in mammalian myelinated axons rest on the appropriate spatial distribution ofion - selective channels . na channels are gathered at nodes of ranvier in concentrations which are ~25 times that of internodal regions.5 these clusters are essential for efflux of ion currents at nodes and allow fast saltatory conduction .", "on the contrary , other studies have demonstrated that k currents appear only after loosening of the myelin sheath from the axonal membrane.6 k channels are located only in paranodal and internodal areas and normally they do not contribute to generation of the action potential.7 scientific studies which have focused on action potential propagation in normal and demyelinated axons have revealed a reorganization of axolemmal ion channels , which in turn leads to conduction impairments . loss or decrease of sodium channels from the nodes of ranvier8 causes the nodes to be inexcitable and leads to conduction block .", "sherratt et al9 demonstrated that in demyelinated regions sodium channel concentrations are reduced , while voltage - sensitive potassium channels ( which are not active on normal myelinated axons ) become detectable .", "this exposure of k channels at juxtaparanodal region leads to efflux through fast k channels and causes axonal conduction block by preventing sufficient depolarization and initiation of the action potential at the node of ranvier.10 demyelination also exposes slow k channels , further interrupting normal hyperpolarization and blunting normal repetitive impulse release from the presynaptic area terminals.11 in 1981 , bostock et al12 demonstrated that potassium channel blockers dendrotoxin and 4-aminopyridine improved conduction impairments in experimentally demyelinated axons and laid the scientific rationale for clinical studies of potassium channel blockers in ms patients ."], ["fampridine ( or 4-aminopyridine [ 4-ap ] ) is a broad - spectrum blocker of voltage - sensitive potassium channels , which has been demonstrated to improve conduction and propagation of the action potential along the demyelinated axons .", "in addition , fampridine increases the duration of the pre - synaptic action potential and amplitude which in turn leads to increased transmitter release.13 apart from being a voltage - activated potassium channel blocker , 4-aminopyridine potentiates synaptic and neuromuscular transmission by affecting the beta subunit of voltage - activated calcium channels.14 this novel mode of action of 4-aminopyridine which occurs independent from its blocking effect on the potassium channels , provides another rationale for its positive effect on the neuromuscular function in patients with spinal cord injury , myasthenia gravis and ms ."], ["hawthorne , ny , usa ) is one of the three isomeric amines of pyridine with chemical formula h2nc5h4n , which acts a selective potassium - channel blocker .", "dalfampridine , which is a sustained - release oral form of fampridine , is rapidly and completely absorbed from the gastrointestinal tract , however , its absolute bioavailability has not been determined .", "compared to an aqueous oral solution , the extended release tablet shows a relative bioavailability of 96% , with a delayed absorption pattern which provides a less rapid rise to a lower maximum plasma concentration . in healthy volunteers who took a single 10 mg dose of dalfampridine", ", peak serum concentrations were reached 3 to 4 hours following oral administration and it was almost completely and rapidly eliminated by urinary excretion .", "the elimination half - life of dalfampridine is 6.4 hours in healthy individuals . as a lipid - soluble agent", ", dalfampridine passes through the blood brain barrier and blocks potassium channels in both the central and peripheral nervous systems.15 daldampridine binds reversibly to the cytoplasmic side of potassium channels , blocking the ion conductance pathway which in turn prolongs the action potentials in unmyelinated nerve fibers and enhances neurotransmitter release at synaptic endings.16 dalfampridine has been studied extensively in symptomatic treatment of ms patients with walking impairment .", "impairment of ambulation is a major neurological deficit in ms patients and significantly interferes with their quality of life .", "it is hypothesized that dalfampridine improves clinical symptoms of ms by restoring conduction in demyelinated axons through voltage - dependent potassium channel blockade.15 in addition , dalfampridine has been used for treatment of lambert - eaton myasthenic syndrome,17 where it prolongs neurotransmitter release at the neuromuscular junction .", "studies involving animal models have demonstrated that can reverse tetrodotoxin toxicity.18 pharmacokinetic assessmentsof both immediate - release and sustained release formulations of fampridine have been done in normal individuals as well as those patients with ms and spinal cord damage .", "uges et al19 performed a pharmacokinetic study involving 9 healthy individuals ( 7 men and 2 women ) to assess immediate - release fampridine in intravenous , entric - coated , and uncoated oral formulations .", "the bioavailability of the enteric coated tablets was calculated from the area under the serum concentration curve 95%  29% ( mean  sd ) , which was not much different from that calculated from urinary excretion 98%  8% . there was no evidence that metabolism of fampridine involved glucoronidation , sulfonation , or n - acetylation .", "investigators reported that 89.6%  7.5% of the medication was excreted in the urine unchanged after 24 hours of the intravenous formulation and 86.1%  2.7% with the entric coated formulation .", "one dose - ranging pharmacokinetic study included 12 ms patients who received increasing doses of sustained - release fampridine from 7.5 to 25 mg every 12 hours.20 the investigators reported a mean time to peak concentration of 5 hours and mean serum half - life of 5.2 hours .", "vollmer et al21 studied the pharmacokinetics of dalfampridine in clinical trials of ms patients and found that multiple dosing of this medication was associated with its accumulation .", "in addition , the investigators noted that the steady - state pharmacokinetic profile of fampridine sustained - release 20 mg bid administered for 2 weeks appeared to support the administration of twice - daily dosing in this population ."], ["dalfampridine ( ampyra ) is available as 10 mg extended release tablets with relative bioavailability of 96% , peak of plasma concentration of 34 hours and half life of 5.26.5 hours ."], ["the major goals of treating ms patients consist of prevention of exacerbations , limiting disability progression , and improving cognitive decline . the available treatments for ms fall under two categories : disease modifying agents such as beta - interferons and glatiramer acetate and medications for symptomatic treatment such as baclofen for treatment of spasticity and dalfampridine which is used to restore the impulse conduction along the demyelinated axons and improve muscle strength and walking ability . in january 2010", ", dalfampridine was approved by the fda for symptomatic treatment of ms patients with specific indication for improving walking . in the past", "4-aminopyridine has been used as an experimental agent which presumably enhances nerve conduction of demyelinated axons through its effects on potassium channels for treatment of fatigue in ms .", "sheean et al22 reported that use of fampridine in ms patients was associated with improvements in fatigue as well motor and visual symptoms .", "in addition , dalfampridine has been assessed and studied in other cohorts of ms patients in the context of various clinical trials to determine its efficacy in improving ms patients ambulation . in this manuscript only some of the more significant clinical trials have been cited and discussed .", "using quantitative measures of motor function , schwid et al23 performed one of the earlier clinical trials of sustained - release fampridine - sr in ms patients .", "this was a randomized , double - blind , placebo - controlled , crossover clinical trial included 10 patients with clinically definite ms , limb weakness and stable motor deficits ( edss 6.07.5 ) .", "study subjects were randomized to be treated with sustain - release fampridine 17.5 mg orally twice daily for a period of one week or placebo for a period of one week .", "study outcomes included time to walk 8 meters , time to climb four stairs , maximum voluntary isometric contraction measured quiantitatively , manual muscle testing , grip strength , edss , and the patient s global impression .", "of these outcomes , only timed gait progressed on sustain - release fampridine compared with placebo in 9 of the study participants .", "seven participants preferred the medication over placebo based on the findings from the patient s global impression .", "goodman et al24 reported the results of a four - center randomized , double - blind placebo - controlled study of controlled release aminopyridine in 31 ms patients .", "the study participants were treated with increasing doses of 20 to 80 mg of aminopyridine daily in divided doses .", "a total of 25 participants received the active medication and 11 participants were treated with placebo .", "outcome measures of this clinical trial consisted of timed ambulation , manual testing of leg strength , paced auditory serial addition task , 9-hole peg test , and a fatigue diary .", "the results of this clinical trial indicated a statistically significant and dose - related enhancement of timed ambulation in ap - treated patients compared to those who received placebo .", "a significant enhancement of leg strength was also observed in approximately 11% of ap - treated participants compared with a 4% worsening of placebo - treated participants , p = 0.01 .", "the reported side effects consisted of dizziness , insomnia , paresthesia , nausea , headache , tremor , pain and anxiety . on daily doses of 60 and 70 mg", "a phase 2 , multi - center , randomized , double - blind , placebo - controlled , parallel - group , dose - comparison ( 10 , 15 , or 20 mg twice daily orally ) clinical trial of sustained - release fampridine in ms patients demonstrated significantly more consistent responders as determined by improvement in ambulation as compared to the placebo - group : 36.7% versus 8.5%.25 fampridine was generally well tolerated and serious and severe adverse effects were more common in those patients who received the highest dose . a multi - center randomized , double - blinded , placebo - controlled phase 2 dose - raning clinical trial was conducted by goodman et al which assessed the concept that whether the dose of fampridine - sr could be safely elevated to 80 mg / daily in patients with ms.26 a cohort of 36 ms patients were randomized in a 2 to 1 ratio to be treated with an increasing dose of fampridine - sr for a period of 8 weeks . the dosage of fampridine - sr was escalated from 10 to 40 mg twice daily by increments of 5 mg twice daily on a weekly schedule .", "a group of evaluations were done once a week which included ms functional composite ( msfc ) , fatigue questionnaires , and lower extremity manual muscle testing .", "the objectives of this clinical trial included assessment of the safety of higher doses of fampridine - sr as well as the efficacy and dose - response to this drug by ms patients .", "five study participants were terminated from fampridine - sr due to adverse events at doses larger than 25 mg and these events consisted of seizures in two individuals at doses of 30 and 35 mg twice daily .", "no subjects in the placebo arm of the study dropped out due to the adverse events . in the group which was treated with fampridine - sr", "compared to the group which was treated with placebo showed improvement in the lower extremity muscle strength ( prospective analysis ) and walking speed ( post - hoc analysis ) .", "the investigators concluded that future clinical trials should restrict the dose of fampridine - sr to 20 mg twice daily and walking speed and lower extremity muscle strength tests are potential efficacy measurements .", "another phase 3 multicenter clinical trial of dafampridine which was controlled and double - blind included 301 ms patients of different types ( 27% with relapsing - remitting ms and 73% with progressive ms ) . during the 14 weeks duration of this trial", ", participants were assigned in a ratio of 3 to 1 to be treated with dalfampridine 10 mg ( n = 229 ) or placebo ( n = 72 ) orally twice daily for a period of 14 weeks.27 the primary aim of this study included steady progress on timed 25-foot walk to define responses with proportion of timed walk responders in each arm .", "the investigators reported a higher proportion of timed walk responders in the fampridine - treated arm ( 78/224 or 35% ) compared to those who were treated with placebo ( 6/72 or 8% ) .", "based on the results of this study , treatment of ms patients with fampridine was associated with gait improvement with a decrease in patient s gait impairment .", "a more recent executed phase 3 multi - center double - blind clinical trial of extended - release dalfampridine in patients with definite ms of any clinical course assessed the efficacy , safety , and pharmacodynamics of extended release oral dalfampridine in these patients.28 the length of this study was 9 weeks and the participants were randomized to be treated with dalfampridine ( 10 mg twice daily ; n = 120 ) or placebo ( n = 119 ) for a period of 9 weeks .", "the primary aim of this study consisted of constant progress on the timed 25-foot walk , with percentage of time walk responders ( twrs ) in each treatment arm .", "the last on - treatment assessment collected information from 8 to 12 hours postdose , to assess and determine the persistence of the impact of the medication .", "the results of this clinical trial indicated that the quantity of twrs was higher in the dalfampridine - treated group ( 51/119 or 42.9% ) compared to the placebo - treated group ( 11/118 or 9.3% .", "the average enhancement of walking speed among the dalfampridine - treated twrs during the 8-week efficacy assessment period was 24.7% compared to baseline ; the mean enhancement during the finalon - treatment visit was 25.7% , reflecting continuation of the impact of medication over the interdosing phase .", "the authors concluded that this clinical trial generated class 1 evidence that treatment of patients with definite ms with extended - release dalfampridinetablets caused meaningful enhancement of walking ability in and the effect of the medication continued persisted between doses ."], ["based on the animal studies , dalfampridine demonstrates properties of a powerful convulsant stemming from its broad - spectrum suppressing activity on the potassium channels in the peripheral and central nervous systems.11 based on a number of clinical trials of 4-ap side effects consist of dizziness , insomnia , paresthesia , nausea of mild to moderate intensity , tremor , pain , anxiety and seizures.2934 dalfampridine is neither a substrate for nor an inhibitor of the p - glycoprotein transporter , therefore its pharmacokinetics is not affected by the medications which suppress this transporter .", "it does not interact with the medications which are substrates for the p - glycoprotein transporter .", "dalfampridine is contraindicated in patients with history of epilepsy or history of severe renal insufficiency.35 the reason that dalfampridine use is not recommended in epileptic patients is that epilepsy is characterized by abnormal neuronal depolarization with excessive inhibition on potassium channels , hence a further reduction of potassium channel activity by dalfampridine is not advised ."], ["pathogenesis of ms involves loss of oligodendrocyte / myelin complex which turn results in re - organization of axolemmal ion channels which leads to conduction abnormalities including conduction delay or block .", "pharmacological agents which block potassium channels have been investigated in the context of clinical trials with positive impact on impulse conduction in experimentally - induced demyelination as well as in patients with ms .", "dalfampridine ( which is marketed as ampyra ) , is an extended release form of fampridine ( 4-aminopyridine ) , which has been recently approved by the us fda for symptomatic treatment of ms patients .", "while this new oral blocker of voltage - gated potassium channels does not have any impact on the underlying pathology of ms , it has been demonstrated to improve fatigue and walking ability in these patients ."]]}
{"article_id": "PMC4847162", "article_text": ["several imaging modalities including pa , occlusal and panoramic radiography , cone beam computed tomography ( cbct ) and ct are used before and after implant treatment as well as in the maintenance sessions . after successful implant placement ,", "periapical radiography is usually a suitable imaging modality for long - term follow - ups .", "radiographically , a thin radiolucent margin around the implant indicates implant mobility and is an important sign of failed osseointegration [ 13 ] .", "the popularity of digital radiography in dentistry is mainly due to the adjustability of digital images by using image inversion , embossed tools , brightness , contrast and magnification enhancement filters [ 49 ] . by applying image inversion to digital images ,", "inverted digital images are used for various purposes in dentistry such as the measurement of bone loss due to periodontal disease and localization of the mandibular canal and mental foramen [ 10,1117 ] .", "several previous studies have compared the diagnostic accuracy of unprocessed digital images ( psp and charge - coupled device or ccd ) , film - based radiographs and inverted images for detection of peri - implant bone defects and simulated periodontal lesions using image tool and adobe photoshop software programs [ 1214 ] .", "this study aimed to compare the diagnostic value of unprocessed ( psp ) and inverted digital images for detection of peri - implant defects using scanora software .", "soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . in between imaging stages , the rib segments were stored at 1c temperature to minimize moisture loss .", "osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another .", "a total of 30 implant placement sites including 10 control sites , 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created .", "the sicace  screw implants ( sicace , basel switzerland ) ( 3.4mm/14.5 mm ; cylindrical parallel walled ) were placed in the marked areas .", "osteotomy sites were prepared in two steps . in step 1 , holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length ( sic extension drill 3.10 mm , short ) . in step 2 of osteotomy ,", "the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill ( sic extension drill , basel , switzerland)(4.25 mm , short ) to create 0.425 mm peri - implant space .", "the coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill ( astra tech drill , stockholm , sweden ) ( 4.85 , 819 mm ) to create 0.725 mm peri - implant space . to stabilize fixtures in bone , drilling", "was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap .", "all fixtures measuring 3.414.5 mm ( the sicace screw implants 3.4mm/14.5 mm incl . ) were placed into the osteotomy sites at the level of the superior border of the rib ( bone level ) and cover screws were inserted ( figure 1 ) . to simulate soft tissue , an acrylic block ( polymethyl methacrylate ) with 1 cm thickness", "periapical radiographs were obtained from the bone segments at the same day the fixtures were placed .", "all radiographs were obtained using digora optime imaging system ( soredex corporation , helsinki , finland ) , psp size 2 sensor ( 40.0mm30.0 mm ) and minray dental x ray system ( soredex , tuusula , finland ) with exposure settings of 60 kv and 7ma for 0.16s . digital intraoral sensor holder ( kerr dental europe , bioggio , switzerland )", "the x ray tube ( 2 ) the simulated soft tissue ( 3 ) bone segment with implant fixtures placed .", "after exposure of sensors , images were stored in dicom files on a computer using a scanora lite software ( palodex , tuusula , finland ) with standard resolution . by applying image inversion , 60 digital images including 30 unprocessed and 30 inverted images were obtained .", "four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale : \n definite defectprobable defectnot sureprobably no defectdefinitely no defect \n  examiners viewed the images on an 18.5 samsung monitor ( samsung syncmaster e1945nx , 1360 768 ) under adequate lighting .", "the examiners were only allowed to adjust the brightness and contrast of digital images ( figure 3 ) .", "study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers .", "the sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared .", "independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities .", "soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . in between imaging stages , the rib segments were stored at 1c temperature to minimize moisture loss .", "osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another .", "a total of 30 implant placement sites including 10 control sites , 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created .", "the sicace  screw implants ( sicace , basel switzerland ) ( 3.4mm/14.5 mm ; cylindrical parallel walled ) were placed in the marked areas .", "osteotomy sites were prepared in two steps . in step 1 , holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length ( sic extension drill 3.10 mm , short ) . in step 2 of osteotomy ,", "the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill ( sic extension drill , basel , switzerland)(4.25 mm , short ) to create 0.425 mm peri - implant space .", "the coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill ( astra tech drill , stockholm , sweden ) ( 4.85 , 819 mm ) to create 0.725 mm peri - implant space .", "to stabilize fixtures in bone , drilling was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap . all fixtures measuring 3.414.5 mm ( the sicace screw implants 3.4mm/14.5 mm incl . )", "were placed into the osteotomy sites at the level of the superior border of the rib ( bone level ) and cover screws were inserted ( figure 1 ) . to simulate soft tissue , an acrylic block ( polymethyl methacrylate ) with 1 cm thickness", "periapical radiographs were obtained from the bone segments at the same day the fixtures were placed .", "all radiographs were obtained using digora optime imaging system ( soredex corporation , helsinki , finland ) , psp size 2 sensor ( 40.0mm30.0 mm ) and minray dental x ray system ( soredex , tuusula , finland ) with exposure settings of 60 kv and 7ma for 0.16s . digital intraoral sensor holder ( kerr dental europe , bioggio , switzerland )", "the x ray tube ( 2 ) the simulated soft tissue ( 3 ) bone segment with implant fixtures placed .", "after exposure of sensors , images were stored in dicom files on a computer using a scanora lite software ( palodex , tuusula , finland ) with standard resolution . by applying image inversion , 60 digital images including 30 unprocessed and 30 inverted images were obtained .", "four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale : \n definite defectprobable defectnot sureprobably no defectdefinitely no defect \n  examiners viewed the images on an 18.5 samsung monitor ( samsung syncmaster e1945nx , 1360 768 ) under adequate lighting .", "the examiners were only allowed to adjust the brightness and contrast of digital images ( figure 3 ) .", "in this study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers .", "the sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared .", "independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities .", "a total of 60 digital pa radiographs including 30 unprocessed and 30 inverted radiographs were obtained of 30 implants with the same diameter and length : 10 implants with no defects , 10 with small ( 0.425 mm ) diameter peri - implant defects in the coronal 8 mm and 10 with large ( 0.725 mm ) diameter peri - implant defects in the coronal 8 mm .", "based on the results , unprocessed images had a higher diagnostic value than inverted images . according to the t - test , in the equality of the means , no significant difference was detected in the sensitivity of unprocessed and inverted images for detection of peri - implant defects ( absolute positive predictive value ) and for detection of a probable lesion ( complete positive predictive value)(p>0.05 ) .", "considering the equality of variances , the t - test showed that unprocessed images had higher absolute negative predictive value ( p=0.049 ) and complete negative predictive value ( p=0.017 ) than inverted images ( table 1 ) .", "the mean  standard deviation of absolute and complete negative and positive predictive values of unprocessed and inverted images the mean  standard deviation of sensitivity and specificity of unprocessed and inverted images for detection of small and large defects significantly different from zero ( p<0.05 ) considering the equality of variances , the t - test showed that unprocessed images had a higher absolute sensitivity for detection of small defects than inverted images ( p=0.48 ) .", "however , the mean complete sensitivity of the two modalities for detection of small defects despite the non - equality of variances ( p=0.001 ) and equality of the means , was not significantly different ( p=0.81 ) ( table 2 ) . considering the equality of variances ( p=0.055 ) , the t - test failed to find a significant difference in the absolute sensitivity for detection of large defects between the unprocessed and inverted images ( p=1.0 ) .", "however , despite the equality of variances ( p=0.003 ) , the t - test found a statistically significant difference in complete sensitivity for detection of large defects and unprocessed images had a higher mean value in this respect ( p=0.019 ) ( table 2 ) . in the non - equality of the means ,", "the mean absolute specificity of unprocessed and inverted images was significantly different based on the t - test ( p<0.05 ) and unprocessed images had a higher diagnostic value .", "however , in the equality of the means , the complete specificity of unprocessed and inverted images was not significantly different ( p>0.05 ) ( table 2 ) .", "periapical radiography is conventionally used as the standard follow up radiography after implant placement to assess the peri - implant tissue status .", "the digital imaging techniques and different enhancement filters have enabled more accurate diagnoses in dentistry .", "this study showed that unprocessed images had a higher diagnostic value than inverted images for detection of small and large peri - implant defects .", "this finding may be due to the unfamiliarity of the inverted images to the eyes of the observers .", "our obtained results were in accord with those of kavadella et al ,   de molon et al ,   and jorgenson et al ,   despite the differences in the type of receptors ( film and digital sensor ) , type of defects and method of creating the defects .", "however , our results were in contrast to those of scaf et al , in 2007 .", "compared unprocessed and inverted digitized images for detection of bone loss due to periodontal disease and found no significant difference in the diagnostic value of unprocessed and inverted images .", "scaf et al , in their study used pa radiographs available in the records of patients with periodontal disease .", "the difference between our results and those of scaf et al . may be attributed to the different methodology and the software programs used .", "kavadella et al ,   in their in - vitro study compared film - based conventional radiography with unprocessed and inverted digital radiography with ccd sensors for detection of peri - implant lesions .", "our results had some differences with those of kavadella et al . in our study ,", "the sensitivity and specificity of unprocessed images were higher than those of inverted digital images .", "the complete sensitivity for detection of small defects was 0.90 for unprocessed and 0.62 for inverted images .", "the complete sensitivity for large defects was 1 for unprocessed and 0.85 for inverted images .", ". however , kavadella et al .   showed high specificity and low sensitivity values .", "such differences may be due to the methodology of studies i.e. method of defect formation , using fresh bone in our study and use of magnification enhancement ( x2 ) by the observers in the study by kavadella et al .", "however , our results regarding the lower accuracy of inverted digital images compared to unprocessed images were similar to those of kavadella et al .", "molon et al ,   in their study compared the diagnostic accuracy of unprocessed and converted ( cmos sensor ) images with film - based conventional radiographs for detection of bone loss due to simulated periodontal disease .", "the results showed that inverted digital images had lower accuracy than film - based radiographs . despite the difference between the type of sensors used in our study and the study by molon et al ,", "similar results were obtained indicating the higher diagnostic accuracy of unprocessed digital images and film - based radiographs than inverted images .", "jorgenson et al ,   in their comparative study evaluated the diagnostic accuracy of conventional f - speed film radiographs , unprocessed digital radiographs and inverted digital images ( psp , digora ) in patients with vertical bone defects .", "the digital sensor used in our study was similar to that used by jorgenson et al , and our results were in accord with their findings .", "based on the results , we found that unprocessed digital images had higher diagnostic accuracy than inverted images for detection of small and large peri - implant defects ."], "abstract_text": ["<S> objectives : this study aimed to compare the diagnostic accuracy of inverted and unprocessed digitized periapical radiographs for detection of peri - implant defects.materials and methods : a total of 30 osteotomy sites were prepared in three groups of control , study group 1 with 0.425 mm defects and study group 2 with 0.725 mm defects using the sic and astra tech drill systems with 4.25 mm and 4.85 mm diameters . </S>", "<S> small and large defects were randomly created in the coronal 8 mm of 20 implant sites ; implants ( 3.4 mm diameter , 14.5 mm length ) were then placed . </S>", "<S> thirty periapical ( pa ) radiographs were obtained using digora imaging system ( soredex corporation , helsinki , finland ) , size 2 photostimulable storage phosphor ( psp ) plate sensors ( 40.0mm30.0 mm ) and scanora software . </S>", "<S> unprocessed images were inverted using scanora software by applying image inversion and a total of 60 images were obtained and randomly evaluated by four oral and maxillofacial radiologists . </S>", "<S> data were analyzed using the t-test.results:significant differences were observed in absolute and complete sensitivity and specificity of the two imaging modalities for detection of small and large defects ( p<0.05 ) . </S>", "<S> unprocessed digital images had a higher mean in terms of absolute sensitivity for detection of small defects , complete sensitivity for detection of large peri - implant defects and definite rule out of defects compared with inverted images.conclusion:unprocessed digital images have a higher diagnostic value for detection of small and large peri - implant defects and also for definite rule out of defects compared with inverted images . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Preparation of bone segments:", "Preparation of osteotomy sites and implant placement:", "Radiographic procedure:", "Radiographic evaluation:", "Data analysis:", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["several imaging modalities including pa , occlusal and panoramic radiography , cone beam computed tomography ( cbct ) and ct are used before and after implant treatment as well as in the maintenance sessions . after successful implant placement ,", "periapical radiography is usually a suitable imaging modality for long - term follow - ups .", "radiographically , a thin radiolucent margin around the implant indicates implant mobility and is an important sign of failed osseointegration [ 13 ] .", "the popularity of digital radiography in dentistry is mainly due to the adjustability of digital images by using image inversion , embossed tools , brightness , contrast and magnification enhancement filters [ 49 ] . by applying image inversion to digital images ,", "inverted digital images are used for various purposes in dentistry such as the measurement of bone loss due to periodontal disease and localization of the mandibular canal and mental foramen [ 10,1117 ] .", "several previous studies have compared the diagnostic accuracy of unprocessed digital images ( psp and charge - coupled device or ccd ) , film - based radiographs and inverted images for detection of peri - implant bone defects and simulated periodontal lesions using image tool and adobe photoshop software programs [ 1214 ] .", "this study aimed to compare the diagnostic value of unprocessed ( psp ) and inverted digital images for detection of peri - implant defects using scanora software ."], ["soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . in between imaging stages , the rib segments were stored at 1c temperature to minimize moisture loss .", "osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another .", "a total of 30 implant placement sites including 10 control sites , 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created .", "the sicace  screw implants ( sicace , basel switzerland ) ( 3.4mm/14.5 mm ; cylindrical parallel walled ) were placed in the marked areas .", "osteotomy sites were prepared in two steps . in step 1 , holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length ( sic extension drill 3.10 mm , short ) . in step 2 of osteotomy ,", "the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill ( sic extension drill , basel , switzerland)(4.25 mm , short ) to create 0.425 mm peri - implant space .", "the coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill ( astra tech drill , stockholm , sweden ) ( 4.85 , 819 mm ) to create 0.725 mm peri - implant space . to stabilize fixtures in bone , drilling", "was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap .", "all fixtures measuring 3.414.5 mm ( the sicace screw implants 3.4mm/14.5 mm incl . ) were placed into the osteotomy sites at the level of the superior border of the rib ( bone level ) and cover screws were inserted ( figure 1 ) . to simulate soft tissue , an acrylic block ( polymethyl methacrylate ) with 1 cm thickness", "periapical radiographs were obtained from the bone segments at the same day the fixtures were placed .", "all radiographs were obtained using digora optime imaging system ( soredex corporation , helsinki , finland ) , psp size 2 sensor ( 40.0mm30.0 mm ) and minray dental x ray system ( soredex , tuusula , finland ) with exposure settings of 60 kv and 7ma for 0.16s . digital intraoral sensor holder ( kerr dental europe , bioggio , switzerland )", "the x ray tube ( 2 ) the simulated soft tissue ( 3 ) bone segment with implant fixtures placed .", "after exposure of sensors , images were stored in dicom files on a computer using a scanora lite software ( palodex , tuusula , finland ) with standard resolution . by applying image inversion , 60 digital images including 30 unprocessed and 30 inverted images were obtained .", "four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale : \n definite defectprobable defectnot sureprobably no defectdefinitely no defect \n  examiners viewed the images on an 18.5 samsung monitor ( samsung syncmaster e1945nx , 1360 768 ) under adequate lighting .", "the examiners were only allowed to adjust the brightness and contrast of digital images ( figure 3 ) .", "study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers .", "the sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared .", "independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities ."], ["soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . in between imaging stages , the rib segments were stored at 1c temperature to minimize moisture loss ."], ["osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another .", "a total of 30 implant placement sites including 10 control sites , 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created .", "the sicace  screw implants ( sicace , basel switzerland ) ( 3.4mm/14.5 mm ; cylindrical parallel walled ) were placed in the marked areas .", "osteotomy sites were prepared in two steps . in step 1 , holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length ( sic extension drill 3.10 mm , short ) . in step 2 of osteotomy ,", "the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill ( sic extension drill , basel , switzerland)(4.25 mm , short ) to create 0.425 mm peri - implant space .", "the coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill ( astra tech drill , stockholm , sweden ) ( 4.85 , 819 mm ) to create 0.725 mm peri - implant space .", "to stabilize fixtures in bone , drilling was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap . all fixtures measuring 3.414.5 mm ( the sicace screw implants 3.4mm/14.5 mm incl . )", "were placed into the osteotomy sites at the level of the superior border of the rib ( bone level ) and cover screws were inserted ( figure 1 ) . to simulate soft tissue , an acrylic block ( polymethyl methacrylate ) with 1 cm thickness", "periapical radiographs were obtained from the bone segments at the same day the fixtures were placed ."], ["all radiographs were obtained using digora optime imaging system ( soredex corporation , helsinki , finland ) , psp size 2 sensor ( 40.0mm30.0 mm ) and minray dental x ray system ( soredex , tuusula , finland ) with exposure settings of 60 kv and 7ma for 0.16s . digital intraoral sensor holder ( kerr dental europe , bioggio , switzerland )", "the x ray tube ( 2 ) the simulated soft tissue ( 3 ) bone segment with implant fixtures placed ."], ["after exposure of sensors , images were stored in dicom files on a computer using a scanora lite software ( palodex , tuusula , finland ) with standard resolution . by applying image inversion , 60 digital images including 30 unprocessed and 30 inverted images were obtained .", "four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale : \n definite defectprobable defectnot sureprobably no defectdefinitely no defect \n  examiners viewed the images on an 18.5 samsung monitor ( samsung syncmaster e1945nx , 1360 768 ) under adequate lighting .", "the examiners were only allowed to adjust the brightness and contrast of digital images ( figure 3 ) ."], ["in this study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers .", "the sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared .", "independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities ."], ["a total of 60 digital pa radiographs including 30 unprocessed and 30 inverted radiographs were obtained of 30 implants with the same diameter and length : 10 implants with no defects , 10 with small ( 0.425 mm ) diameter peri - implant defects in the coronal 8 mm and 10 with large ( 0.725 mm ) diameter peri - implant defects in the coronal 8 mm .", "based on the results , unprocessed images had a higher diagnostic value than inverted images . according to the t - test , in the equality of the means , no significant difference was detected in the sensitivity of unprocessed and inverted images for detection of peri - implant defects ( absolute positive predictive value ) and for detection of a probable lesion ( complete positive predictive value)(p>0.05 ) .", "considering the equality of variances , the t - test showed that unprocessed images had higher absolute negative predictive value ( p=0.049 ) and complete negative predictive value ( p=0.017 ) than inverted images ( table 1 ) .", "the mean  standard deviation of absolute and complete negative and positive predictive values of unprocessed and inverted images the mean  standard deviation of sensitivity and specificity of unprocessed and inverted images for detection of small and large defects significantly different from zero ( p<0.05 ) considering the equality of variances , the t - test showed that unprocessed images had a higher absolute sensitivity for detection of small defects than inverted images ( p=0.48 ) .", "however , the mean complete sensitivity of the two modalities for detection of small defects despite the non - equality of variances ( p=0.001 ) and equality of the means , was not significantly different ( p=0.81 ) ( table 2 ) . considering the equality of variances ( p=0.055 ) , the t - test failed to find a significant difference in the absolute sensitivity for detection of large defects between the unprocessed and inverted images ( p=1.0 ) .", "however , despite the equality of variances ( p=0.003 ) , the t - test found a statistically significant difference in complete sensitivity for detection of large defects and unprocessed images had a higher mean value in this respect ( p=0.019 ) ( table 2 ) . in the non - equality of the means ,", "the mean absolute specificity of unprocessed and inverted images was significantly different based on the t - test ( p<0.05 ) and unprocessed images had a higher diagnostic value .", "however , in the equality of the means , the complete specificity of unprocessed and inverted images was not significantly different ( p>0.05 ) ( table 2 ) ."], ["periapical radiography is conventionally used as the standard follow up radiography after implant placement to assess the peri - implant tissue status .", "the digital imaging techniques and different enhancement filters have enabled more accurate diagnoses in dentistry .", "this study showed that unprocessed images had a higher diagnostic value than inverted images for detection of small and large peri - implant defects .", "this finding may be due to the unfamiliarity of the inverted images to the eyes of the observers .", "our obtained results were in accord with those of kavadella et al ,   de molon et al ,   and jorgenson et al ,   despite the differences in the type of receptors ( film and digital sensor ) , type of defects and method of creating the defects .", "however , our results were in contrast to those of scaf et al , in 2007 .", "compared unprocessed and inverted digitized images for detection of bone loss due to periodontal disease and found no significant difference in the diagnostic value of unprocessed and inverted images .", "scaf et al , in their study used pa radiographs available in the records of patients with periodontal disease .", "the difference between our results and those of scaf et al . may be attributed to the different methodology and the software programs used .", "kavadella et al ,   in their in - vitro study compared film - based conventional radiography with unprocessed and inverted digital radiography with ccd sensors for detection of peri - implant lesions .", "our results had some differences with those of kavadella et al . in our study ,", "the sensitivity and specificity of unprocessed images were higher than those of inverted digital images .", "the complete sensitivity for detection of small defects was 0.90 for unprocessed and 0.62 for inverted images .", "the complete sensitivity for large defects was 1 for unprocessed and 0.85 for inverted images .", ". however , kavadella et al .   showed high specificity and low sensitivity values .", "such differences may be due to the methodology of studies i.e. method of defect formation , using fresh bone in our study and use of magnification enhancement ( x2 ) by the observers in the study by kavadella et al .", "however , our results regarding the lower accuracy of inverted digital images compared to unprocessed images were similar to those of kavadella et al .", "molon et al ,   in their study compared the diagnostic accuracy of unprocessed and converted ( cmos sensor ) images with film - based conventional radiographs for detection of bone loss due to simulated periodontal disease .", "the results showed that inverted digital images had lower accuracy than film - based radiographs . despite the difference between the type of sensors used in our study and the study by molon et al ,", "similar results were obtained indicating the higher diagnostic accuracy of unprocessed digital images and film - based radiographs than inverted images .", "jorgenson et al ,   in their comparative study evaluated the diagnostic accuracy of conventional f - speed film radiographs , unprocessed digital radiographs and inverted digital images ( psp , digora ) in patients with vertical bone defects .", "the digital sensor used in our study was similar to that used by jorgenson et al , and our results were in accord with their findings ."], ["based on the results , we found that unprocessed digital images had higher diagnostic accuracy than inverted images for detection of small and large peri - implant defects ."]]}
{"article_id": "PMC4339898", "article_text": ["upper gastrointestinal bleeding ( ugib ) is a major cause of mortality and morbidity accounting for about 56.5/100,000 hospitalization with a mortality rate of 8.2% .", "aorto - esophageal fistula ( aef ) , an abnormal anatomical communication between the aorta and esophagus is a rare cause of ugib .", "as the thoracic aneurysm grows in size , the increasing tension on its wall will erode the aorta and rupture into the surrounding esophagus leading to uncontrolled bleeding .", "as ulcer related or variceal bleeding constitutes for most of the ugib bleeding , even in elderly who are at risk of aef and the inability of the esophagogastroduodenoscopy ( egd ) to detect most cases , aef is often not diagnosed timely .", "all these factors make the timely diagnosis of aef challenging , and at times missed altogether leading to catastrophic consequences .", "we present a case of aef which was initially treated as variceal bleeding and later promptly diagnosed with computed tomography ( ct ) .", "a 70-year - old male patient with hypertension with regular ethanol use , presented to a local hospital with complaints of hematemesis and melena from the past 1-day .", "he was hemodynamically stable on presentation with initial hemoglobin level of 7.2 g / dl .", "he underwent timely egd , which showed grade 1 mid and distal esophageal varices without any active bleeding .", "subsequently , his condition deteriorated with the development of hypotension and transferred to our medical center for further management .", "weiss tear without active bleeding along with the presence of large blood clots in the gastric fundus .", "as the source of bleeding was not clear despite two egd and clinical evidence of gi bleeding , he underwent emergent colonoscopy which once again failed to detect any source lower gi bleeding .", "however , later in the day , he developed massive hematemesis and hematochezia leading to persistent hemorrhagic shock .", "emergent intubation was done for airway protection , massive transfusion protocol was initiated , and a ct angiogram was performed .", "it revealed type b aortic dissection with rupture and the active extravasation of blood into the esophagus , confirming the diagnosis of aef [ figure 1 ] .", "he underwent emergency percutaneous thoracic endovascular aortic repair with aortic stent placement [ figure 2 ] .", "his shock resolved hemoglobin stabilized and did not have any more episodes of bleeding thereafter .", "repeat egd showed a 3-cm opening in the midesophagus with ragged and irregular borders , not seen earlier egds .", "his hospital course was complicated by the infection of the endograft which was replaced and fortified with an omental flap and being is presently being , followed by for reconstruction surgeries .", "( a ) communication and extravasation between aortia and esophagus ( blue arrow head ) in the arterial phase .", "( b ) accumulation of blood in lower esophagus in venous phase ( yellow arrow head ) .", "( c ) significant accumulation of blood in the stomach in the kidney delay images ( red arrow head ) .", "( d ) sagittal reconstruction images shows commination ( blue arrow ) and extravasation ( yellow arrow ) of blood between aorta and esophagus endovascular graft in the thoracic aorta", "aorto - esophageal fistula is an abnormal anatomical communication between the aorta and esophagus . in 1818", ", dubrueil described this condition for the 1 time in a french soldier who ingested a beef rib , leading to massive hematemesis .", "almost a century later , chiari described the triad of midthoracic pain , herald hemorrhage , and fatal hematemesis seen in these patients .", "the exact incidence of this condition is not known , as the vast majority of these patients die before a definitive diagnosis can be made . in the past , infections such as tuberculosis and syphilis , leading to aortitis were the common cause of this condition . in this antibiotics era , thoracic aortic aneurysm constitutes for more than half of these cases .", "other causes described in the literature are cancer , acid reflux disease , trauma , caustic ingestion , and esophageal biopsies .", "aefs are classified as primary if they are caused by the spontaneous erosion of the aortic wall into the esophagus and as secondary if they occur as a complication following aortic or esophageal surgeries .", "owing to the increasing number of interventions of the aorta , secondary aef is 10 times more common than primary .", "the herald bleeding described by chiari is only seen in half of these patients . once the bleeding starts , rapid extravasation from aorta will cause a sudden drop in blood pressure .", ", the blood pressure will raise again dislodging the fresh and loose clot around the aef leading to terminal bleeding .", "commonly used resuscitation fluids including pseudo random binary sequence lack adequate clotting factors add to the compromise of clot integrity . unlike in other structures ,", "the lax mediastinum around the aorta and esophagus prevent autotamponade , to prevent persistent extravasation of blood .", "these factors can contribute to torrential bleeding , the final hematemesis as described by chiari .", "the risk of aortic aneurysm increases with age , with an incidence of 12.5% in elderly americans .", "the sensitivity of egd to identify the source of bleeding in other causes of ugib is 90% , but for aef it is < 10% .", "it may show pulsatile mass , bluish discoloration , or simply blood clotting in the esophagus in one - third of cases .", "a ct arteriogram is diagnostic in most cases , which show active extravasation of blood from the aorta to esophagus .", "once diagnosed , aef should be treated with emergent aortic stenting to control the bleeding .", "owing to contamination from the gi tract , one - quarter of stents become infected . apart from broad - spectrum antibiotics and antifungals ,", "once the infection is controlled , the complex aortic and esophageal reconstruction is done in multiples stages .", "aef , once considered to be uniformly fatal , is now a treatable condition with timely intervention .", "aorto - esophageal fistula is a rare , but life - threatening condition presenting as ugib .", "a high level of suspicion is needed , as routine tests performed for ugib typically only detect one - third of cases . avoiding over - resuscitation can be helpful in preventing subsequent hemorrhages ."], "abstract_text": ["<S> aorto - esophageal fistula is a rare cause of upper gastrointestinal bleeding . </S>", "<S> thoracic aneurysm , the most common cause of this condition , will slowly increase over time and can erode the wall of the aorta creating a fistula and leading to torrential bleeding . </S>", "<S> high clinical suspicion is required for timely diagnosis as common investigations routinely done for gastrointestinal ( gi ) bleeding , including esophagogastroduodenoscopy , fails to detect most cases . </S>", "<S> the classical triad of midthoracic pain , herald bleeding and fatal hematemesis described in this condition is seen in only one - third of cases . </S>", "<S> physician should be wary of this condition , especially in elderly patients with uncontrolled gi bleeding and who are also at risk of thoracic aneurysm . </S>", "<S> computed tomography angiogram detects most cases and emergent endovascular repair with stents controls the initial bleeding . </S>", "<S> later , both the aorta and the esophagus are repaired and reconstructed in staged procedures . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["upper gastrointestinal bleeding ( ugib ) is a major cause of mortality and morbidity accounting for about 56.5/100,000 hospitalization with a mortality rate of 8.2% .", "aorto - esophageal fistula ( aef ) , an abnormal anatomical communication between the aorta and esophagus is a rare cause of ugib .", "as the thoracic aneurysm grows in size , the increasing tension on its wall will erode the aorta and rupture into the surrounding esophagus leading to uncontrolled bleeding .", "as ulcer related or variceal bleeding constitutes for most of the ugib bleeding , even in elderly who are at risk of aef and the inability of the esophagogastroduodenoscopy ( egd ) to detect most cases , aef is often not diagnosed timely .", "all these factors make the timely diagnosis of aef challenging , and at times missed altogether leading to catastrophic consequences .", "we present a case of aef which was initially treated as variceal bleeding and later promptly diagnosed with computed tomography ( ct ) ."], ["a 70-year - old male patient with hypertension with regular ethanol use , presented to a local hospital with complaints of hematemesis and melena from the past 1-day .", "he was hemodynamically stable on presentation with initial hemoglobin level of 7.2 g / dl .", "he underwent timely egd , which showed grade 1 mid and distal esophageal varices without any active bleeding .", "subsequently , his condition deteriorated with the development of hypotension and transferred to our medical center for further management .", "weiss tear without active bleeding along with the presence of large blood clots in the gastric fundus .", "as the source of bleeding was not clear despite two egd and clinical evidence of gi bleeding , he underwent emergent colonoscopy which once again failed to detect any source lower gi bleeding .", "however , later in the day , he developed massive hematemesis and hematochezia leading to persistent hemorrhagic shock .", "emergent intubation was done for airway protection , massive transfusion protocol was initiated , and a ct angiogram was performed .", "it revealed type b aortic dissection with rupture and the active extravasation of blood into the esophagus , confirming the diagnosis of aef [ figure 1 ] .", "he underwent emergency percutaneous thoracic endovascular aortic repair with aortic stent placement [ figure 2 ] .", "his shock resolved hemoglobin stabilized and did not have any more episodes of bleeding thereafter .", "repeat egd showed a 3-cm opening in the midesophagus with ragged and irregular borders , not seen earlier egds .", "his hospital course was complicated by the infection of the endograft which was replaced and fortified with an omental flap and being is presently being , followed by for reconstruction surgeries .", "( a ) communication and extravasation between aortia and esophagus ( blue arrow head ) in the arterial phase .", "( b ) accumulation of blood in lower esophagus in venous phase ( yellow arrow head ) .", "( c ) significant accumulation of blood in the stomach in the kidney delay images ( red arrow head ) .", "( d ) sagittal reconstruction images shows commination ( blue arrow ) and extravasation ( yellow arrow ) of blood between aorta and esophagus endovascular graft in the thoracic aorta"], ["aorto - esophageal fistula is an abnormal anatomical communication between the aorta and esophagus . in 1818", ", dubrueil described this condition for the 1 time in a french soldier who ingested a beef rib , leading to massive hematemesis .", "almost a century later , chiari described the triad of midthoracic pain , herald hemorrhage , and fatal hematemesis seen in these patients .", "the exact incidence of this condition is not known , as the vast majority of these patients die before a definitive diagnosis can be made . in the past , infections such as tuberculosis and syphilis , leading to aortitis were the common cause of this condition . in this antibiotics era , thoracic aortic aneurysm constitutes for more than half of these cases .", "other causes described in the literature are cancer , acid reflux disease , trauma , caustic ingestion , and esophageal biopsies .", "aefs are classified as primary if they are caused by the spontaneous erosion of the aortic wall into the esophagus and as secondary if they occur as a complication following aortic or esophageal surgeries .", "owing to the increasing number of interventions of the aorta , secondary aef is 10 times more common than primary .", "the herald bleeding described by chiari is only seen in half of these patients . once the bleeding starts , rapid extravasation from aorta will cause a sudden drop in blood pressure .", ", the blood pressure will raise again dislodging the fresh and loose clot around the aef leading to terminal bleeding .", "commonly used resuscitation fluids including pseudo random binary sequence lack adequate clotting factors add to the compromise of clot integrity . unlike in other structures ,", "the lax mediastinum around the aorta and esophagus prevent autotamponade , to prevent persistent extravasation of blood .", "these factors can contribute to torrential bleeding , the final hematemesis as described by chiari .", "the risk of aortic aneurysm increases with age , with an incidence of 12.5% in elderly americans .", "the sensitivity of egd to identify the source of bleeding in other causes of ugib is 90% , but for aef it is < 10% .", "it may show pulsatile mass , bluish discoloration , or simply blood clotting in the esophagus in one - third of cases .", "a ct arteriogram is diagnostic in most cases , which show active extravasation of blood from the aorta to esophagus .", "once diagnosed , aef should be treated with emergent aortic stenting to control the bleeding .", "owing to contamination from the gi tract , one - quarter of stents become infected . apart from broad - spectrum antibiotics and antifungals ,", "once the infection is controlled , the complex aortic and esophageal reconstruction is done in multiples stages .", "aef , once considered to be uniformly fatal , is now a treatable condition with timely intervention ."], ["aorto - esophageal fistula is a rare , but life - threatening condition presenting as ugib .", "a high level of suspicion is needed , as routine tests performed for ugib typically only detect one - third of cases . avoiding over - resuscitation can be helpful in preventing subsequent hemorrhages ."]]}
{"article_id": "PMC2894203", "article_text": ["the discovery of the silicon pn junction is a landmark in the development of modern microelectronics .", "silicon and other inorganic semiconductors are the predominant materials in electronic manufacturing today ; however , the development of organic - based electronic components has been a focus of intensive research and development .", "organic electronics have become an active research area in recent years because they are an alternative to the traditional semiconductor technology and challenges in design on a smaller scale from microscale to nanoscale .", "moreover , organic materials have vast potential for integration in low - cost microelectronic devices .", "organic - based semiconductors , for example polymeric gels , may have superior properties due to their flexibility against to regulate some physical properties of the materials even after the production , e.g. , by changing the volume of the gel .", "circuits and displays based on organic electronics may also be flexible , low - weight , and environment friendly .", "due to great diversity and functionality of the polymers , they may also be produced for specific requirements , like biocompatible semiconductors .", "last , some water - tolerant organic devices may be biocompatible , that is , they may be used in circuits that directly reside on or are implanted inside animal and human tissue and could perform various sensing , interfacing , and controlling functions for drug delivery , prosthetics , and neural - electronic integration .", "insulating polymers have been made conductive so far by using various methods such as ion implantation [ 1 - 4 ] , press contacting , and photochemical doping .", "the impressive properties of these materials that have been taken advantage are the ability to manipulate electronic properties by changing their molecular structure .", "differently current rectifying in semiconductor diodes via diffusion of ionic charges across a pn junction sets up a built in potential ; there is unidirectional current is observed across a membrane where a built in potential arises from diffusion of anions and cations in organic junctions .", "we considered that the principle of the electrical conductivity in polymeric gels can be changed by doping static ( inactive ) ions having counter ions as charge carriers and these gels can be used to form pn junctions that work similar to inorganic semiconductors .", "these semiconductor polymeric gels can be doped with both negative ( n - type ) and positive ( p - type ) counter ions as charge carriers .", "we have shown that the pn junction formed by combining p - type and n - type gels together in much closer contact rectifies the current .", "p - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide ( aam ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) in varying amount , 10 , 10 , 10 mol / l [ 10 - 12 ] .", "the ratios of aam to bis and aam to aps were kept fixed for all samples ; aam / bis = 31 and aam / aps = 43 in bidistilled water .", "the samples were deoxygenated by bubbling nitrogen during 10 min , and the gelation was performed in the 1  1 cm square - shaped holders ( 4 cm of volume ) at 60c in a heat bath .", "after gelation processes were completed , gels were washed with pure water during 15 days with changing their water every day .", "thus , pollutions and unreacted chemicals washed out from the gel , and only pyranines , which were bound to the polymer strands chemically [ 10 - 12 ] , were stayed in the gel .", "after the gels dried at 40c furnace , they were cut into thin slices with ~1 mm thickness for electrical measurements .", "n - type gels were synthesized , similar to p - type , as free radical cross - linking co - polymerization of n - isopropylacrylamide ( nipa ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride ( maptac ) in varying amount , 3  10 , 3  10 , 3  10 mol / l [ 13 - 15 ] .", "the ratios of nipa to bis and nipa to aibn were kept fixed for all samples ; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide ( dmso ) . these gels were prepared for electrical measurements in similar way with p - type gels .", "aam , nipa , and doping concentrations of the samples were summarized in table 1 .", "properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties , samples p1 , p5 , n1 , and n5 were synthesized without doping agents for comparison with the other samples .", "the gels , prepared in the slice shapes of area a and thickness d , were swollen to a certain swelling ratio , and then placed between platinum electrodes , and sealed from the air against drying .", "the current densities per unit mass , j / m , of the gel were measured against the time , the applied voltage and the swelling ratio of the gels .", "p - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide ( aam ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) in varying amount , 10 , 10 , 10 mol / l [ 10 - 12 ] .", "the ratios of aam to bis and aam to aps were kept fixed for all samples ; aam / bis = 31 and aam / aps = 43 in bidistilled water .", "the samples were deoxygenated by bubbling nitrogen during 10 min , and the gelation was performed in the 1  1 cm square - shaped holders ( 4 cm of volume ) at 60c in a heat bath .", "after gelation processes were completed , gels were washed with pure water during 15 days with changing their water every day .", "thus , pollutions and unreacted chemicals washed out from the gel , and only pyranines , which were bound to the polymer strands chemically [ 10 - 12 ] , were stayed in the gel .", "after the gels dried at 40c furnace , they were cut into thin slices with ~1 mm thickness for electrical measurements .", "n - type gels were synthesized , similar to p - type , as free radical cross - linking co - polymerization of n - isopropylacrylamide ( nipa ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride ( maptac ) in varying amount , 3  10 , 3  10 , 3  10 mol / l [ 13 - 15 ] .", "the ratios of nipa to bis and nipa to aibn were kept fixed for all samples ; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide ( dmso ) . these gels were prepared for electrical measurements in similar way with p - type gels .", "aam , nipa , and doping concentrations of the samples were summarized in table 1 .", "properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties , samples p1 , p5 , n1 , and n5 were synthesized without doping agents for comparison with the other samples .", "the gels , prepared in the slice shapes of area a and thickness d , were swollen to a certain swelling ratio , and then placed between platinum electrodes , and sealed from the air against drying .", "the current densities per unit mass , j / m , of the gel were measured against the time , the applied voltage and the swelling ratio of the gels .", "the pyranine binds to the polymer chains , over its oh group , chemically during the polymerization as it is discussed in detail in references [ 10 - 12 ] , and they form stable charged sites doped with positive counter ions . thus , the polyacrylamide gel is doped with pyranine having   ions as side groups and na as counter ions , so - called p - type semiconducting gel .", "similarly , nipa gel was doped with maptac , having cl as counter ions , so - called n - type semiconducting gel .", "the maptac binds to the polymer chains chemically during the polymerization , thus they form stable charged sites doped with negative counter ions as represented in scheme 1together with the p - type gel .", "this doping process was also discussed in detail in references , . when these gels are in the collapsed state , no current is observed .", "when the gels start to swell , the current starts to flow through the gels .", "schematic representation of the p - type ( a ) and n - type ( b ) gels doped with pyranine and maptac the currents decrease with time for a constant applied voltage as shown in fig .", "as the charge carriers ( the ions in water phase of the gel ) were gathered on the electrodes , the density of the charge carriers decrease in time , and thus the current . as seen , the initial values of the currents measured for the doped gels are considerably bigger than that of the  neat  gel ( the gel that was not doped with ions ) at a certain swelling ratio , v / v0 = 1.28 and under constant applied voltage , u = 5 v. furthermore , the initial amplitude of the currents increases with increasing doping concentration .", "this clearly shows that the net charge in the gel increases with increasing doping concentration .", "aj / mgel vs time plots for the doped ( p2 , p3 , p4 ) and neat ( p1 ) gels under constant applied voltage , 5 v , and swelling ratio , v / v0 ~ 1,28 .", "bj / mgel versus time plots upon short - cut of the circuit for samples p4 , p3 , p2 , p1 .", "the vertical bar represents the standard error for each plot examples for the short - cut experiments -the source of the voltage was removed and the current was measured by means of an ammeter without changing its polarity- were also indicated in ( fig .", "1b ) where all the current takes first a negative value , as it is expected , and then goes to zero with time .", "figure 1 clearly indicates that the free charges leading the current are accumulated on the surfaces of the gel on which the voltage is applied ( similar to charging a parallel plate capacitor ) and the gels have ionic charge carriers .", "figure 2a shows typical current - time plots of the slab gels for increasing pyranine content .", "the gels were swollen in pure water up to certain swelling degree ( v / v0 = 1,3 ) , under a fixed applied voltage , 1 v. as seen , the currents for all samples decrease with time ; however , the initial values of them increase with increasing doping agent ( pyranine content ) .", "aj / mgel versus time plots of the samples p6 , p7 , and p8 , b the ratios of j / mgel for the doped and neat gels ( p8/p5 ) as function of the applied voltage for varying swelling ratio , v / v0 figure 2b shows the ratio of the normalized current densities ( j / mgel ) for doped ( p8 ) to neat ( p5 ) gels as function of the applied voltage for varying swelling ratio .", "the currents were measured 5 s later just after applying voltage . as seen , the doping effect on the current decreases when the swelling ratio is increased .", "this may be due to the vast increase in the free ions coming from the water which causes screening .", "this picture may indicate that doping effect ( p - type behavior ) can be fulfilled up to a certain swelling degree of the above - mentioned gel which semiconducting behavior will be lost .", "the instantaneous currents  the currents that were measured 5 s later just after changing the voltage- as function of the volume of the gels for varying voltages were also measured , and the results were indicated in fig .", "as seen from this figure , distinct peaks appear as the solvent uptake , v / v0 , increases .", "the peaks almost disappear for the gels doped with pyranine due to increasing homogeneity of the gels in the presence of the ions .", "the results coincide with the recent literature where three dominant peaks , each corresponding to a different swelling stage in the conductivity , were observed .", "figure 3 indicates that the volume of the gel is one of the main parameters determining the current .", "variation in j / mgel against v / v0 for ( a ) the neat gel , p1 , and for ( b ) the doped gel , p2 these peaks were related to the distribution of dense polymer regions ; they are defined as the  blobs  appearing in a microstructure of the given paam gel having at least some average sizes .", "this special behavior in these gels may result specific features related to the electrical features .", "the heterogeneity of the gel may decrease essentially in case when it is prepared with the charged groups .", "the internal electric field due to the charged sites could disturb the tendency between the polymer units ( affinity of the aam momoners to the cross - linker molecules ) because the charge excess has a tendency to form denser polymer regions especially in mesoscale regions .", "the formation of the rigid network in the charged gel makes it more homogenous in comparison with the neutral gel .", "this could cause the decreasing amplitude of the peaks , and overall decrease in the conductivity as it is confirmed by fig .", "3 ( see also fig . 2 of reference ) . therefore , in the charged gel ,", "the penetration of neutral water molecules through the system of large channels located in the vicinity of small blobs is facilitated .", "figure 4 represents the normalized current density per unit mass as function of the voltage for the gels ( sample p3 ) swollen with water ( fig .", "dmso is not a polar molecule ; therefore , the ions like h or oh are absent in it . as seen from this figure , the current density decreases fairly for the gels swollen with dmso since no contribution to the current occurs via h and oh ions .", "the current occurs mainly due to doped ions ( the counter ions of the doping agent ) .", "this observation clearly indicates that the possible negative effects of the water upon the pnjunctions that may be formed by means of our gels can be eliminated .", "j / mgel versus applied voltage , u , for the doped gel ( p3 ) swollen in ( a ) pure water , b and in dmso all the above - mentioned measurements were repeated for n - type gels , and the similar results were reproduced . here the results are not given to avoid the repetition . in fig .", "5 , representative examples of i - u(current  voltage ) character of polymeric diode are given for the neat and the doped gels .", "figure 5a shows the current  voltage character that was made by means of close connection of p-(sample p4 ) and n - type ( sample n4 ) slab gels- putting one of the gel on the top of the other for each gel .", "the swelling ratio of each gel was kept the same , v / v0 = 1,2 . when the neat gels are used for both the p - and n - type sides ,", "figure 5a clearly shows that the current of the interface between the cationic and anionic gels , which is passing through only one direction , originates directly from the doping effect of the gels .", "a i versus u plot for pn junction formed with p - and n - type gels ( p4-n4 ) , b i versus u plot for the junction formed with neat gels ( p1-n1 )", "in this study , we have shown that p- and n - type doped hydrogels can be synthesized and be used to built pn junction .", "the synthesis of these gels and making junctions are more simple , inexpensive , and scalable than traditional pn junctions .", "the current  voltage characteristic of the polymeric pn junction can be changed by changing doping concentration , swelling ratio etc that will be discussed in our feature works .", "the percentage of the standard deviations changes around 20% as can be seen from fig .", "the reasons for this high standard deviations can be explained by the following arguments : ( 1 ) the electrical futures of these gels depend strictly on the swelling ratio of the gels as can be seen from fig . 3 and from the detailed discussion given in a recent work   and ( 2 ) on the internal morphology ( nonergodicity or random distribution of the heterogeneous regions in the gels ) .", "this strict volume dependence and the randomness of the internal distributions of the heterogeneous regions   set a barrier for comparison of the samples from the point of the current amplitudes due to the difficulties to bring the gels to exactly the same swelling ratios and to repeat the synthesis with exactly the same internal morphology .", "therefore , the stability and reversibility problems need detailed experiments and probably more controllable polymer synthesis .", "this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited ."], "abstract_text": ["<S> in this study , we synthesized the new kinds of semiconducting polymeric gels having negative ( n - type ) and positive ( p - type ) counter ions as charge carriers . </S>", "<S> the polyacrylamide gel was doped with pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) , havingions as side groups and na+ as counter ions , so - called p - type semiconducting gel . </S>", "<S> the doping process was performed during the polymerization where the pyranine binds to the polymer strands over oh group chemically via radical addition . in a similar way , </S>", "<S> n - isopropylacrylamide ( nipa ) gel was doped with methacrylamidopropyltrimethyl ammonium chloride ( maptac ) , having cl as counter ions , so - called n - type semiconducting gel . here </S>", "<S> maptac was embedded by copolymerization within the polymer network ( nipa ) . </S>", "<S> these semiconducting gels can show different electrical properties by changing the concentration of the doping agents , swelling ratio etc . </S>", "<S> we have shown that the pn junction , formed by combining p - type and n - type gels together in close contact , rectifies the current similar to the conventional si and ge diodes . </S>"], "labels": null, "section_names": ["Introduction", "Experiment", "Preparation of ", "Preparation of ", "Electrical Measurements", "Results and Discussion", "Conclusion", "Open Access"], "sections": [["the discovery of the silicon pn junction is a landmark in the development of modern microelectronics .", "silicon and other inorganic semiconductors are the predominant materials in electronic manufacturing today ; however , the development of organic - based electronic components has been a focus of intensive research and development .", "organic electronics have become an active research area in recent years because they are an alternative to the traditional semiconductor technology and challenges in design on a smaller scale from microscale to nanoscale .", "moreover , organic materials have vast potential for integration in low - cost microelectronic devices .", "organic - based semiconductors , for example polymeric gels , may have superior properties due to their flexibility against to regulate some physical properties of the materials even after the production , e.g. , by changing the volume of the gel .", "circuits and displays based on organic electronics may also be flexible , low - weight , and environment friendly .", "due to great diversity and functionality of the polymers , they may also be produced for specific requirements , like biocompatible semiconductors .", "last , some water - tolerant organic devices may be biocompatible , that is , they may be used in circuits that directly reside on or are implanted inside animal and human tissue and could perform various sensing , interfacing , and controlling functions for drug delivery , prosthetics , and neural - electronic integration .", "insulating polymers have been made conductive so far by using various methods such as ion implantation [ 1 - 4 ] , press contacting , and photochemical doping .", "the impressive properties of these materials that have been taken advantage are the ability to manipulate electronic properties by changing their molecular structure .", "differently current rectifying in semiconductor diodes via diffusion of ionic charges across a pn junction sets up a built in potential ; there is unidirectional current is observed across a membrane where a built in potential arises from diffusion of anions and cations in organic junctions .", "we considered that the principle of the electrical conductivity in polymeric gels can be changed by doping static ( inactive ) ions having counter ions as charge carriers and these gels can be used to form pn junctions that work similar to inorganic semiconductors .", "these semiconductor polymeric gels can be doped with both negative ( n - type ) and positive ( p - type ) counter ions as charge carriers .", "we have shown that the pn junction formed by combining p - type and n - type gels together in much closer contact rectifies the current ."], ["p - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide ( aam ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) in varying amount , 10 , 10 , 10 mol / l [ 10 - 12 ] .", "the ratios of aam to bis and aam to aps were kept fixed for all samples ; aam / bis = 31 and aam / aps = 43 in bidistilled water .", "the samples were deoxygenated by bubbling nitrogen during 10 min , and the gelation was performed in the 1  1 cm square - shaped holders ( 4 cm of volume ) at 60c in a heat bath .", "after gelation processes were completed , gels were washed with pure water during 15 days with changing their water every day .", "thus , pollutions and unreacted chemicals washed out from the gel , and only pyranines , which were bound to the polymer strands chemically [ 10 - 12 ] , were stayed in the gel .", "after the gels dried at 40c furnace , they were cut into thin slices with ~1 mm thickness for electrical measurements .", "n - type gels were synthesized , similar to p - type , as free radical cross - linking co - polymerization of n - isopropylacrylamide ( nipa ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride ( maptac ) in varying amount , 3  10 , 3  10 , 3  10 mol / l [ 13 - 15 ] .", "the ratios of nipa to bis and nipa to aibn were kept fixed for all samples ; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide ( dmso ) . these gels were prepared for electrical measurements in similar way with p - type gels .", "aam , nipa , and doping concentrations of the samples were summarized in table 1 .", "properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties , samples p1 , p5 , n1 , and n5 were synthesized without doping agents for comparison with the other samples .", "the gels , prepared in the slice shapes of area a and thickness d , were swollen to a certain swelling ratio , and then placed between platinum electrodes , and sealed from the air against drying .", "the current densities per unit mass , j / m , of the gel were measured against the time , the applied voltage and the swelling ratio of the gels ."], ["p - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide ( aam ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of pyranine ( 8-hydroxypyrene-1,3,6-trisulfonic acid , trisodium salt ) in varying amount , 10 , 10 , 10 mol / l [ 10 - 12 ] .", "the ratios of aam to bis and aam to aps were kept fixed for all samples ; aam / bis = 31 and aam / aps = 43 in bidistilled water .", "the samples were deoxygenated by bubbling nitrogen during 10 min , and the gelation was performed in the 1  1 cm square - shaped holders ( 4 cm of volume ) at 60c in a heat bath .", "after gelation processes were completed , gels were washed with pure water during 15 days with changing their water every day .", "thus , pollutions and unreacted chemicals washed out from the gel , and only pyranines , which were bound to the polymer strands chemically [ 10 - 12 ] , were stayed in the gel .", "after the gels dried at 40c furnace , they were cut into thin slices with ~1 mm thickness for electrical measurements ."], ["n - type gels were synthesized , similar to p - type , as free radical cross - linking co - polymerization of n - isopropylacrylamide ( nipa ) and n , n-methylene bisacrylamide ( bis ) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride ( maptac ) in varying amount , 3  10 , 3  10 , 3  10 mol / l [ 13 - 15 ] .", "the ratios of nipa to bis and nipa to aibn were kept fixed for all samples ; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide ( dmso ) . these gels were prepared for electrical measurements in similar way with p - type gels .", "aam , nipa , and doping concentrations of the samples were summarized in table 1 .", "properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties , samples p1 , p5 , n1 , and n5 were synthesized without doping agents for comparison with the other samples ."], ["the gels , prepared in the slice shapes of area a and thickness d , were swollen to a certain swelling ratio , and then placed between platinum electrodes , and sealed from the air against drying .", "the current densities per unit mass , j / m , of the gel were measured against the time , the applied voltage and the swelling ratio of the gels ."], ["the pyranine binds to the polymer chains , over its oh group , chemically during the polymerization as it is discussed in detail in references [ 10 - 12 ] , and they form stable charged sites doped with positive counter ions . thus , the polyacrylamide gel is doped with pyranine having   ions as side groups and na as counter ions , so - called p - type semiconducting gel .", "similarly , nipa gel was doped with maptac , having cl as counter ions , so - called n - type semiconducting gel .", "the maptac binds to the polymer chains chemically during the polymerization , thus they form stable charged sites doped with negative counter ions as represented in scheme 1together with the p - type gel .", "this doping process was also discussed in detail in references , . when these gels are in the collapsed state , no current is observed .", "when the gels start to swell , the current starts to flow through the gels .", "schematic representation of the p - type ( a ) and n - type ( b ) gels doped with pyranine and maptac the currents decrease with time for a constant applied voltage as shown in fig .", "as the charge carriers ( the ions in water phase of the gel ) were gathered on the electrodes , the density of the charge carriers decrease in time , and thus the current . as seen , the initial values of the currents measured for the doped gels are considerably bigger than that of the  neat  gel ( the gel that was not doped with ions ) at a certain swelling ratio , v / v0 = 1.28 and under constant applied voltage , u = 5 v. furthermore , the initial amplitude of the currents increases with increasing doping concentration .", "this clearly shows that the net charge in the gel increases with increasing doping concentration .", "aj / mgel vs time plots for the doped ( p2 , p3 , p4 ) and neat ( p1 ) gels under constant applied voltage , 5 v , and swelling ratio , v / v0 ~ 1,28 .", "bj / mgel versus time plots upon short - cut of the circuit for samples p4 , p3 , p2 , p1 .", "the vertical bar represents the standard error for each plot examples for the short - cut experiments -the source of the voltage was removed and the current was measured by means of an ammeter without changing its polarity- were also indicated in ( fig .", "1b ) where all the current takes first a negative value , as it is expected , and then goes to zero with time .", "figure 1 clearly indicates that the free charges leading the current are accumulated on the surfaces of the gel on which the voltage is applied ( similar to charging a parallel plate capacitor ) and the gels have ionic charge carriers .", "figure 2a shows typical current - time plots of the slab gels for increasing pyranine content .", "the gels were swollen in pure water up to certain swelling degree ( v / v0 = 1,3 ) , under a fixed applied voltage , 1 v. as seen , the currents for all samples decrease with time ; however , the initial values of them increase with increasing doping agent ( pyranine content ) .", "aj / mgel versus time plots of the samples p6 , p7 , and p8 , b the ratios of j / mgel for the doped and neat gels ( p8/p5 ) as function of the applied voltage for varying swelling ratio , v / v0 figure 2b shows the ratio of the normalized current densities ( j / mgel ) for doped ( p8 ) to neat ( p5 ) gels as function of the applied voltage for varying swelling ratio .", "the currents were measured 5 s later just after applying voltage . as seen , the doping effect on the current decreases when the swelling ratio is increased .", "this may be due to the vast increase in the free ions coming from the water which causes screening .", "this picture may indicate that doping effect ( p - type behavior ) can be fulfilled up to a certain swelling degree of the above - mentioned gel which semiconducting behavior will be lost .", "the instantaneous currents  the currents that were measured 5 s later just after changing the voltage- as function of the volume of the gels for varying voltages were also measured , and the results were indicated in fig .", "as seen from this figure , distinct peaks appear as the solvent uptake , v / v0 , increases .", "the peaks almost disappear for the gels doped with pyranine due to increasing homogeneity of the gels in the presence of the ions .", "the results coincide with the recent literature where three dominant peaks , each corresponding to a different swelling stage in the conductivity , were observed .", "figure 3 indicates that the volume of the gel is one of the main parameters determining the current .", "variation in j / mgel against v / v0 for ( a ) the neat gel , p1 , and for ( b ) the doped gel , p2 these peaks were related to the distribution of dense polymer regions ; they are defined as the  blobs  appearing in a microstructure of the given paam gel having at least some average sizes .", "this special behavior in these gels may result specific features related to the electrical features .", "the heterogeneity of the gel may decrease essentially in case when it is prepared with the charged groups .", "the internal electric field due to the charged sites could disturb the tendency between the polymer units ( affinity of the aam momoners to the cross - linker molecules ) because the charge excess has a tendency to form denser polymer regions especially in mesoscale regions .", "the formation of the rigid network in the charged gel makes it more homogenous in comparison with the neutral gel .", "this could cause the decreasing amplitude of the peaks , and overall decrease in the conductivity as it is confirmed by fig .", "3 ( see also fig . 2 of reference ) . therefore , in the charged gel ,", "the penetration of neutral water molecules through the system of large channels located in the vicinity of small blobs is facilitated .", "figure 4 represents the normalized current density per unit mass as function of the voltage for the gels ( sample p3 ) swollen with water ( fig .", "dmso is not a polar molecule ; therefore , the ions like h or oh are absent in it . as seen from this figure , the current density decreases fairly for the gels swollen with dmso since no contribution to the current occurs via h and oh ions .", "the current occurs mainly due to doped ions ( the counter ions of the doping agent ) .", "this observation clearly indicates that the possible negative effects of the water upon the pnjunctions that may be formed by means of our gels can be eliminated .", "j / mgel versus applied voltage , u , for the doped gel ( p3 ) swollen in ( a ) pure water , b and in dmso all the above - mentioned measurements were repeated for n - type gels , and the similar results were reproduced . here the results are not given to avoid the repetition . in fig .", "5 , representative examples of i - u(current  voltage ) character of polymeric diode are given for the neat and the doped gels .", "figure 5a shows the current  voltage character that was made by means of close connection of p-(sample p4 ) and n - type ( sample n4 ) slab gels- putting one of the gel on the top of the other for each gel .", "the swelling ratio of each gel was kept the same , v / v0 = 1,2 . when the neat gels are used for both the p - and n - type sides ,", "figure 5a clearly shows that the current of the interface between the cationic and anionic gels , which is passing through only one direction , originates directly from the doping effect of the gels .", "a i versus u plot for pn junction formed with p - and n - type gels ( p4-n4 ) , b i versus u plot for the junction formed with neat gels ( p1-n1 )"], ["in this study , we have shown that p- and n - type doped hydrogels can be synthesized and be used to built pn junction .", "the synthesis of these gels and making junctions are more simple , inexpensive , and scalable than traditional pn junctions .", "the current  voltage characteristic of the polymeric pn junction can be changed by changing doping concentration , swelling ratio etc that will be discussed in our feature works .", "the percentage of the standard deviations changes around 20% as can be seen from fig .", "the reasons for this high standard deviations can be explained by the following arguments : ( 1 ) the electrical futures of these gels depend strictly on the swelling ratio of the gels as can be seen from fig . 3 and from the detailed discussion given in a recent work   and ( 2 ) on the internal morphology ( nonergodicity or random distribution of the heterogeneous regions in the gels ) .", "this strict volume dependence and the randomness of the internal distributions of the heterogeneous regions   set a barrier for comparison of the samples from the point of the current amplitudes due to the difficulties to bring the gels to exactly the same swelling ratios and to repeat the synthesis with exactly the same internal morphology .", "therefore , the stability and reversibility problems need detailed experiments and probably more controllable polymer synthesis ."], ["this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited ."]]}
{"article_id": "PMC5295487", "article_text": ["in june 2014 , a 61-year - old female was admitted to keimyung university dongsan medical center because of a severe dry cough that had lasted for over 20 days . according to her medical history ,", "she had undergone a hysterectomy in 2005 , and the pathologic diagnosis was leiomyoma of the uterus . in 2006 ,", "chest computed tomography ( ct ) findings demonstrated multiple nodules in both lungs ( fig .", "microscopic examination revealed a proliferating spindle cell tumor showing relatively bland nuclei , no pleomorphism , and no mitoses .", "in addition , immunohistochemical staining analysis was positive for smooth muscle actin , desmin , vimentin , estrogen receptor ( er ) , and progesterone receptor ( pr ) , and negative for cd34 , pan - ck , and s100 proteins . the result of the pathologic study was benign leiomyoma , and the nodules were diagnosed as pulmonary benign metastasizing leiomyoma ( pbml ) .", ", the patient did not return to our outpatient clinic for follow - up . upon the patient s return ,", "a posteroanterior chest x - ray showed a very large mass on the right cardiac border ( fig .", "chest ct demonstrated a 101-mm mass on the right lower lobe that compressed the posterobasal segmental bronchus ( fig .", "a ct - guided needle biopsy of the huge mass revealed it to be leiomyoma .", "after right thoracotomy , a very large mass , 100 mm in size , originating from the visceral pleura of the right lower lobe was observed ( fig .", "the mass was in contact with the parietal and diaphragmatic pleura , but no invasion was noted . near the mass , multiple fine ,", "scattered , whitish nodules measuring less than 1 cm were found on the diaphragmatic , parietal , and visceral pleura .", "we performed an excision of the mass in the right lower lobe and excised fine nodules from the parietal and diaphragmatic pleura for biopsy .", "intrapulmonary nodules found in the middle lobe and upper lobe were not excised , because they had not increased in size .", "the necrotic lesion showed severe cellular atypia , frequent mitoses ( more than 15 per 10 high power fields [ hpfs ] ) , and continuity of the leiomyoma and leiomyosarcoma ( fig . 2c ) .", "immunohistochemical staining revealed that the tumor was positive for smooth muscle actin ( sma ) , er , and pr , but negative for cd34 , pan - ck , s100 , and hmb45 ( fig .", "the nodular lesions taken from the parietal and diaphragmatic pleura showed no malignant cells . the patient was discharged 8 days after the operation without complications .", "large heterogeneous hypermetabolic masses were found from the left para - aortic area to the left pelvic cavity ( fig .", "3 ) , and we diagnosed them as metastatic leiomyosarcoma . additionally , increased fluorodeoxyglucose ( fdg ) uptake was observed in the peribronchial lymph nodes , the mediastinum , and the supraclavicular lymph nodes .", "two months later , en - bloc resection of the retroperitoneal masses with left nephrectomy was performed , and the pathologic diagnosis was metastatic leiomyosarcoma .", "we decided to continue observing fdg uptake in the mediastinal and peribronchial lymph nodes rather than prescribing chemotherapy , because metastasis of leiomyosarcoma to the lymph nodes is rare . a chest ct scan done 1 year after surgery demonstrated no changes in the pulmonary nodules and no recurrence of the mass .", "benign metastasizing leiomyoma ( bml ) results from metastasis of a uterine leiomyoma ; thus , bml patients have a history of uterine leiomyoma", ". the condition may occur at any age , but occurs most often in women from 30 to 74 years old .", "bml can occur in a number of different areas of the body , including but not limited to the lungs , para - aortic lymph nodes , abdominal lymph nodes , heart , breast , liver , and esophagus .", "the most common site of occurrence is in the lungs , in which case it is known as pbml .", "the pathogenesis of pbml is unknown , but the most common theory is hematogenous spreading of leiomyoma during uterine surgery .", "immunohistochemical staining of affected tissue is positive for desmin , sma , pr , and er . most cases of pbml do not require treatment , but pbml that metastasizes or increases in size does need to be treated , as it can cause respiratory failure , chest pain , or hemoptysis .", "surgical excision is the treatment of choice , but this is only possible in cases of resectable pbml .", "in some cases of pbml that a repositive for er or pr , reducing hormone release via hysterectomy , bilateral adnexectomy , and/or hormone therapy may decrease the tumor s size .", "the incidence of the sarcomatous transformation of benign uterine leiomyomas is only 0.1%0.8% , and is most often seen in women in their 50s .", "pbml can also undergo malignant transformation ; excluding our case , just 1 case of the malignant transformation of pbml has been reported .", "radiologically , it is difficult to differentiate between normal pbml and pbml that has become malignant .", "on fdg pet - ct , heterogeneous fdg uptake of the tumor may reflect necrosis within the leiomyosarcoma . in our case ,", "pathologically , the current criteria used to differentiate a malignant smooth muscle tumor from a benign mass are the presence of necrosis , a high mitotic index ( > 5 mitoses seen in 50 hpfs ) , nuclear atypia , cellularity , and an ill - defined tumor border .", "however , in primary pulmonary leiomyosarcoma ( ppl ) , progress is slow , and metastasis of ppl occurs late in the disease process .", "if the condition is diagnosed early , wide excision with negative margins ( r0 ) is the standard treatment .", "the 5-year survival rate in ppl patients who undergo complete resection is over 50% , and recurrence is rare .", "lymph node resections are generally not necessary because ppl rarely shows lymph node involvement . for advanced lesions , depending on the size and grade of the tumor , radiation therapy , chemotherapy , or both may be indicated as an adjuvant therapy . however , the median survival of patients with an unresectable sarcoma or metastatic disease is about 12 months .", "overall , early diagnosis and complete mass resection are vital in cases of the malignant transformation of pbml .", "however , preventive resection or diagnostic excisional biopsy is not recommended for all pbml patients , since the occurrence of a malignant change is extremely rare", "if there is a very large , newly developed mass that is increasing in size rapidly , or if a malignant mass is found on pet - ct , diagnostic mass resection should be considered ."], "abstract_text": ["<S> pulmonary benign metastasizing leiomyoma ( pbml ) is defined as metastasis of a leiomyoma to lung tissue . </S>", "<S> it was first reported in 1937 . </S>", "<S> p bml is known as a benign disease , but can undergo malignant transformation . only 1 case of the malignant transformation of pbml to leiomyosarcoma has been reported previously . in this report , we present a case of malignant transformation of pbml . </S>"], "labels": null, "section_names": ["Case report", "Discussion"], "sections": [["in june 2014 , a 61-year - old female was admitted to keimyung university dongsan medical center because of a severe dry cough that had lasted for over 20 days . according to her medical history ,", "she had undergone a hysterectomy in 2005 , and the pathologic diagnosis was leiomyoma of the uterus . in 2006 ,", "chest computed tomography ( ct ) findings demonstrated multiple nodules in both lungs ( fig .", "microscopic examination revealed a proliferating spindle cell tumor showing relatively bland nuclei , no pleomorphism , and no mitoses .", "in addition , immunohistochemical staining analysis was positive for smooth muscle actin , desmin , vimentin , estrogen receptor ( er ) , and progesterone receptor ( pr ) , and negative for cd34 , pan - ck , and s100 proteins . the result of the pathologic study was benign leiomyoma , and the nodules were diagnosed as pulmonary benign metastasizing leiomyoma ( pbml ) .", ", the patient did not return to our outpatient clinic for follow - up . upon the patient s return ,", "a posteroanterior chest x - ray showed a very large mass on the right cardiac border ( fig .", "chest ct demonstrated a 101-mm mass on the right lower lobe that compressed the posterobasal segmental bronchus ( fig .", "a ct - guided needle biopsy of the huge mass revealed it to be leiomyoma .", "after right thoracotomy , a very large mass , 100 mm in size , originating from the visceral pleura of the right lower lobe was observed ( fig .", "the mass was in contact with the parietal and diaphragmatic pleura , but no invasion was noted . near the mass , multiple fine ,", "scattered , whitish nodules measuring less than 1 cm were found on the diaphragmatic , parietal , and visceral pleura .", "we performed an excision of the mass in the right lower lobe and excised fine nodules from the parietal and diaphragmatic pleura for biopsy .", "intrapulmonary nodules found in the middle lobe and upper lobe were not excised , because they had not increased in size .", "the necrotic lesion showed severe cellular atypia , frequent mitoses ( more than 15 per 10 high power fields [ hpfs ] ) , and continuity of the leiomyoma and leiomyosarcoma ( fig . 2c ) .", "immunohistochemical staining revealed that the tumor was positive for smooth muscle actin ( sma ) , er , and pr , but negative for cd34 , pan - ck , s100 , and hmb45 ( fig .", "the nodular lesions taken from the parietal and diaphragmatic pleura showed no malignant cells . the patient was discharged 8 days after the operation without complications .", "large heterogeneous hypermetabolic masses were found from the left para - aortic area to the left pelvic cavity ( fig .", "3 ) , and we diagnosed them as metastatic leiomyosarcoma . additionally , increased fluorodeoxyglucose ( fdg ) uptake was observed in the peribronchial lymph nodes , the mediastinum , and the supraclavicular lymph nodes .", "two months later , en - bloc resection of the retroperitoneal masses with left nephrectomy was performed , and the pathologic diagnosis was metastatic leiomyosarcoma .", "we decided to continue observing fdg uptake in the mediastinal and peribronchial lymph nodes rather than prescribing chemotherapy , because metastasis of leiomyosarcoma to the lymph nodes is rare . a chest ct scan done 1 year after surgery demonstrated no changes in the pulmonary nodules and no recurrence of the mass ."], ["benign metastasizing leiomyoma ( bml ) results from metastasis of a uterine leiomyoma ; thus , bml patients have a history of uterine leiomyoma", ". the condition may occur at any age , but occurs most often in women from 30 to 74 years old .", "bml can occur in a number of different areas of the body , including but not limited to the lungs , para - aortic lymph nodes , abdominal lymph nodes , heart , breast , liver , and esophagus .", "the most common site of occurrence is in the lungs , in which case it is known as pbml .", "the pathogenesis of pbml is unknown , but the most common theory is hematogenous spreading of leiomyoma during uterine surgery .", "immunohistochemical staining of affected tissue is positive for desmin , sma , pr , and er . most cases of pbml do not require treatment , but pbml that metastasizes or increases in size does need to be treated , as it can cause respiratory failure , chest pain , or hemoptysis .", "surgical excision is the treatment of choice , but this is only possible in cases of resectable pbml .", "in some cases of pbml that a repositive for er or pr , reducing hormone release via hysterectomy , bilateral adnexectomy , and/or hormone therapy may decrease the tumor s size .", "the incidence of the sarcomatous transformation of benign uterine leiomyomas is only 0.1%0.8% , and is most often seen in women in their 50s .", "pbml can also undergo malignant transformation ; excluding our case , just 1 case of the malignant transformation of pbml has been reported .", "radiologically , it is difficult to differentiate between normal pbml and pbml that has become malignant .", "on fdg pet - ct , heterogeneous fdg uptake of the tumor may reflect necrosis within the leiomyosarcoma . in our case ,", "pathologically , the current criteria used to differentiate a malignant smooth muscle tumor from a benign mass are the presence of necrosis , a high mitotic index ( > 5 mitoses seen in 50 hpfs ) , nuclear atypia , cellularity , and an ill - defined tumor border .", "however , in primary pulmonary leiomyosarcoma ( ppl ) , progress is slow , and metastasis of ppl occurs late in the disease process .", "if the condition is diagnosed early , wide excision with negative margins ( r0 ) is the standard treatment .", "the 5-year survival rate in ppl patients who undergo complete resection is over 50% , and recurrence is rare .", "lymph node resections are generally not necessary because ppl rarely shows lymph node involvement . for advanced lesions , depending on the size and grade of the tumor , radiation therapy , chemotherapy , or both may be indicated as an adjuvant therapy . however , the median survival of patients with an unresectable sarcoma or metastatic disease is about 12 months .", "overall , early diagnosis and complete mass resection are vital in cases of the malignant transformation of pbml .", "however , preventive resection or diagnostic excisional biopsy is not recommended for all pbml patients , since the occurrence of a malignant change is extremely rare", "if there is a very large , newly developed mass that is increasing in size rapidly , or if a malignant mass is found on pet - ct , diagnostic mass resection should be considered ."]]}
{"article_id": "PMC4993276", "article_text": ["copd is a common , usually progressive disease characterized by chronic inflammation of the airways and persistent airflow limitation .", "its prevalence is increasing worldwide , and it is expected to be the third largest global cause of mortality by the year 2030.1 in japan , a large epidemiological study ( the nippon copd epidemiological study ) reported that the prevalence of airflow limitation was 10.6% , and at least 8.6% of subjects were sought to have copd.2 several studies have shown that the prevalence of potentially undiagnosed airflow limitation in both western35 and asian6 countries is ~ 3%15% .", "many patients with copd continue to be underdiagnosed and untreated.2,7 since copd is a preventable and treatable disease , the importance of early detection has been emphasized.8 the use of simple copd screening questionnaires to detect persistent airflow limitation may help in the early diagnosis of copd .", "these tools reliably detect airflow limitation in the general population and may facilitate the early , accurate diagnosis of copd in general practice settings.9,10 some copd diagnostic questionnaires have already been reported.1114 the copd population screener ( copd - ps ) , which was developed by a clinician working group in the united states , is a five - item , self - administered questionnaire that was validated for screening individuals in the general population who are at high risk of copd .", "it is composed of three copd - related items ( breathlessness , productive cough , and activity limitation ) and one question , each regarding smoking history and age.15 in a previous population - based study , we verified the validity of the japanese version of the copd - ps questionnaire for the identification of individuals at increased risk of airflow limitation.16 this instrument is easy to score and would be suitable for large - scale screening of possible airflow obstruction ( ao ) .", "another copd screening tool , the international primary care airway group ( ipag ) questionnaire,11 is in use worldwide .", "it consists of eight items and takes more time to administer than the copd - ps questionnaire .", "the purpose of this study was to compare these two questionnaires in the general japanese population , as no previous research has done so , as well as to assess the ability of both instruments to discriminate between subjects with and without persistent ao .", "this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .", "the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .", "the population of the town is ~8,000 and has been stable for over 50 years .", "according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17 this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 ,", "registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry . of the 2,643 subjects who were enrolled between june 2012 and october 2012 ,", "307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .", "the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .", "the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .", "each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .", "the results were assessed by two pulmonary physicians , who visually inspected the flow  volume curve and excluded subjects without at least two satisfactory tests .", "the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .", "the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 . the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1", "< 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study .", "the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .", "it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .", "these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire , a cutoff point of 4", "has been found to be useful for copd screening.16 the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .", "each question is scored individually , with a summed total score ranging from 0 to 38 .", "a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan . however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study .", "baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal ", "spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,", "another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .", "likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .", "receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .", "the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .", "we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .", "the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .", "in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .", "we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .", "all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 .", "this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .", "the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .", "the population of the town is ~8,000 and has been stable for over 50 years .", "according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17", "this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 , registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry .", "of the 2,643 subjects who were enrolled between june 2012 and october 2012 , 307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .", "the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .", "the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .", "each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .", "the results were assessed by two pulmonary physicians , who visually inspected the flow  volume curve and excluded subjects without at least two satisfactory tests .", "the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .", "the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 .", "the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1 < 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study .", "the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .", "it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .", "these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire ,", "the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .", "each question is scored individually , with a summed total score ranging from 0 to 38 .", "a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan .", "however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study .", "baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal ", "spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,", "another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .", "likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .", "receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .", "the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .", "we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .", "the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .", "in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .", "we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .", "all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 .", "the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .", "the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,", "almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .", "the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and", "for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .", "the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) . the previously identified cutoff point of 4 was used for the copd - ps questionnaire .", "the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% .", "in this population - based study that included never - smokers , a cutoff point of 20 on the ipag questionnaire would be adequate for screening for ao ( table 3 ) .", "the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .", "if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .", "if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .", "roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .", "the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .", "there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,", "no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .", "however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) .", "reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,", "classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao", ", classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .", "thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .", "the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .", "the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao .", "the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .", "the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,", "almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .", "the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and", "for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .", "the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) .", "the previously identified cutoff point of 4 was used for the copd - ps questionnaire .", "the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% . in this population - based study that included never - smokers , a cutoff point of 20 on", "the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .", "if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .", "if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .", "roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .", "the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .", "there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,", "no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .", "however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) . reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,", "classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao , classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .", "thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .", "the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .", "the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao .", "in the present population - based study , the japanese version of the copd - ps questionnaire was compared with the ipag questionnaire in a general japanese population at the age of 40 years or older .", "the ipag questionnaire had superior likelihood ratios of both positive and negative tests compared with those of the copd - ps questionnaire .", "however , in comparison with two questionnaires , no significant differences were founded in the auc values obtained from the roc curves discriminating between subjects with and without ao and in the sensitivity , specificity , or positive and negative predictive values .", "the two questionnaires were correlated with each other , fev1/fvc , and for both of them , the ors were significantly greater than 1.0 .", "these findings suggest that the copd - ps and the ipag questionnaires are useful screening tools for detecting persistent ao . in addition , the subjects with a 4 point on the copd - ps questionnaire and those with a 20 point on the ipag questionnaire are at increased risk for ao .", "overall , the categorical and continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire were 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) , 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) , and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively .", "reclassifications showed the ipag questionnaire to be superior to the copd - ps questionnaire ; however , there was no significant difference between the two questionnaires .", "a cutoff point of 17 on the ipag questionnaire is used in general health checkup settings and general practices in japan.24 in this study , we found that a cutoff point of 20 was superior .", "the reason for this discrepancy is not clear , although it may be due at least in part to the backgrounds of the study subjects .", "the present study was population based and included never - smokers . in contrast , in a previous study that enrolled subjects in general health checkup settings , a cutoff point of 17 resulted in a higher sensitivity of 86.0% and a smaller auc of 0.66 ( lower than 0.70 ) than a cutoff point of 20.24 the present study was implemented in the town of hisayama , in which the age and occupational distributions of the population have been almost identical to those of japan as a whole , and thus , we recommend a cutoff point of 20 for the general japanese population .", "there were only marginal differences between the two questionnaires in terms of ability to discriminate between subjects with and without ao .", "moreover , the copd - ps questionnaire consists of fewer items than the ipag questionnaire and requires less time to complete .", "it takes ~5 minutes to fill out copd - ps questionnaire , however , the completion of the ipag questionnaire takes ~510 minutes . in this regard ,", "the japanese version of the copd - ps questionnaire should be an adequate measure for large - scale screening for possible ao ."], "abstract_text": ["<S> backgroundthe incidence of chronic obstructive pulmonary disease ( copd ) is increasing worldwide . in japan and other countries , </S>", "<S> epidemiological studies have found that many patients with copd are underdiagnosed and untreated , and thus , early detection and treatment of copd has been emphasized . </S>", "<S> screening questionnaires may have utility in the initial detection of copd.objectivethis study aimed to validate and compare the copd population screener ( copd - ps ) and the international primary care airway group ( ipag ) questionnaires in a general japanese population.patients and methodseligible subjects 40 years of age and older living in the town of hisayama were solicited to participate in a health checkup in 2012 . </S>", "<S> all subjects 4079 years of age without physician - diagnosed asthma or lung resection were recruited , and 2,336 subjects who fully completed both questionnaires and who had valid spirometry measurements were analyzed . </S>", "<S> persistent airflow obstruction ( ao ) was defined by a postbronchodilator forced expiratory volume in 1 second / forced vital capacity < 0.70 . </S>", "<S> receiver operating characteristic curves , net reclassification improvement , and integrated discrimination improvement were used to examine the ability of the copd - ps and ipag questionnaires to discriminate between subjects with and without ao.resultsthe overall area under the receiver operating characteristic curve for the copd - ps questionnaire was 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) and for the ipag was 0.775 ( 95% ci , 0.7350.816 ) , with no significant difference ( p=0.09 ) . </S>", "<S> the net reclassification improvement and integrated discrimination improvement were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively.conclusionthe five - item copd - ps questionnaire was comparable to the eight - item ipag for discriminating between subjects with and without ao . the copd - ps is a simple and useful screening questionnaire for persistent ao . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Study population", "Study design", "COPD-PS", "IPAG questionnaire", "Statistical analysis", "Results", "Participant characteristics", "Discriminating subjects with and without AO", "Discussion", "Conclusion"], "sections": [["copd is a common , usually progressive disease characterized by chronic inflammation of the airways and persistent airflow limitation .", "its prevalence is increasing worldwide , and it is expected to be the third largest global cause of mortality by the year 2030.1 in japan , a large epidemiological study ( the nippon copd epidemiological study ) reported that the prevalence of airflow limitation was 10.6% , and at least 8.6% of subjects were sought to have copd.2 several studies have shown that the prevalence of potentially undiagnosed airflow limitation in both western35 and asian6 countries is ~ 3%15% .", "many patients with copd continue to be underdiagnosed and untreated.2,7 since copd is a preventable and treatable disease , the importance of early detection has been emphasized.8 the use of simple copd screening questionnaires to detect persistent airflow limitation may help in the early diagnosis of copd .", "these tools reliably detect airflow limitation in the general population and may facilitate the early , accurate diagnosis of copd in general practice settings.9,10 some copd diagnostic questionnaires have already been reported.1114 the copd population screener ( copd - ps ) , which was developed by a clinician working group in the united states , is a five - item , self - administered questionnaire that was validated for screening individuals in the general population who are at high risk of copd .", "it is composed of three copd - related items ( breathlessness , productive cough , and activity limitation ) and one question , each regarding smoking history and age.15 in a previous population - based study , we verified the validity of the japanese version of the copd - ps questionnaire for the identification of individuals at increased risk of airflow limitation.16 this instrument is easy to score and would be suitable for large - scale screening of possible airflow obstruction ( ao ) .", "another copd screening tool , the international primary care airway group ( ipag ) questionnaire,11 is in use worldwide .", "it consists of eight items and takes more time to administer than the copd - ps questionnaire .", "the purpose of this study was to compare these two questionnaires in the general japanese population , as no previous research has done so , as well as to assess the ability of both instruments to discriminate between subjects with and without persistent ao ."], ["this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .", "the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .", "the population of the town is ~8,000 and has been stable for over 50 years .", "according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17 this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 ,", "registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry . of the 2,643 subjects who were enrolled between june 2012 and october 2012 ,", "307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .", "the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .", "the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .", "each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .", "the results were assessed by two pulmonary physicians , who visually inspected the flow  volume curve and excluded subjects without at least two satisfactory tests .", "the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .", "the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 . the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1", "< 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study .", "the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .", "it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .", "these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire , a cutoff point of 4", "has been found to be useful for copd screening.16 the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .", "each question is scored individually , with a summed total score ranging from 0 to 38 .", "a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan . however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study .", "baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal ", "spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,", "another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .", "likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .", "receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .", "the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .", "we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .", "the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .", "in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .", "we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .", "all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 ."], ["this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .", "the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .", "the population of the town is ~8,000 and has been stable for over 50 years .", "according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17"], ["this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 , registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry .", "of the 2,643 subjects who were enrolled between june 2012 and october 2012 , 307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .", "the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .", "the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .", "each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .", "the results were assessed by two pulmonary physicians , who visually inspected the flow  volume curve and excluded subjects without at least two satisfactory tests .", "the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .", "the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 .", "the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1 < 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study ."], ["the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .", "it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .", "these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire ,"], ["the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .", "each question is scored individually , with a summed total score ranging from 0 to 38 .", "a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan .", "however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study ."], ["baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal ", "spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,", "another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .", "likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .", "receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .", "the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .", "we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .", "the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .", "in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .", "we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .", "all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 ."], ["the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .", "the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,", "almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .", "the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and", "for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .", "the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) . the previously identified cutoff point of 4 was used for the copd - ps questionnaire .", "the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% .", "in this population - based study that included never - smokers , a cutoff point of 20 on the ipag questionnaire would be adequate for screening for ao ( table 3 ) .", "the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .", "if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .", "if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .", "roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .", "the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .", "there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,", "no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .", "however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) .", "reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,", "classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao", ", classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .", "thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .", "the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .", "the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao ."], ["the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .", "the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,", "almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .", "the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and", "for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .", "the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) ."], ["the previously identified cutoff point of 4 was used for the copd - ps questionnaire .", "the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% . in this population - based study that included never - smokers , a cutoff point of 20 on", "the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .", "if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .", "if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .", "roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .", "the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .", "there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,", "no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .", "however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) . reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,", "classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao , classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .", "thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .", "the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .", "the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao ."], ["in the present population - based study , the japanese version of the copd - ps questionnaire was compared with the ipag questionnaire in a general japanese population at the age of 40 years or older .", "the ipag questionnaire had superior likelihood ratios of both positive and negative tests compared with those of the copd - ps questionnaire .", "however , in comparison with two questionnaires , no significant differences were founded in the auc values obtained from the roc curves discriminating between subjects with and without ao and in the sensitivity , specificity , or positive and negative predictive values .", "the two questionnaires were correlated with each other , fev1/fvc , and for both of them , the ors were significantly greater than 1.0 .", "these findings suggest that the copd - ps and the ipag questionnaires are useful screening tools for detecting persistent ao . in addition , the subjects with a 4 point on the copd - ps questionnaire and those with a 20 point on the ipag questionnaire are at increased risk for ao .", "overall , the categorical and continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire were 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) , 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) , and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively .", "reclassifications showed the ipag questionnaire to be superior to the copd - ps questionnaire ; however , there was no significant difference between the two questionnaires .", "a cutoff point of 17 on the ipag questionnaire is used in general health checkup settings and general practices in japan.24 in this study , we found that a cutoff point of 20 was superior .", "the reason for this discrepancy is not clear , although it may be due at least in part to the backgrounds of the study subjects .", "the present study was population based and included never - smokers . in contrast , in a previous study that enrolled subjects in general health checkup settings , a cutoff point of 17 resulted in a higher sensitivity of 86.0% and a smaller auc of 0.66 ( lower than 0.70 ) than a cutoff point of 20.24 the present study was implemented in the town of hisayama , in which the age and occupational distributions of the population have been almost identical to those of japan as a whole , and thus , we recommend a cutoff point of 20 for the general japanese population ."], ["there were only marginal differences between the two questionnaires in terms of ability to discriminate between subjects with and without ao .", "moreover , the copd - ps questionnaire consists of fewer items than the ipag questionnaire and requires less time to complete .", "it takes ~5 minutes to fill out copd - ps questionnaire , however , the completion of the ipag questionnaire takes ~510 minutes . in this regard ,", "the japanese version of the copd - ps questionnaire should be an adequate measure for large - scale screening for possible ao ."]]}
{"article_id": "PMC2715896", "article_text": ["the human cytomegalovirus ( cmv ) or human herpesvirus 5 is one of the major causes of congenital infections .", "its clinical manifestations range from asymptomatic forms ( 90% of cases ) to severe fetal damage and , in rare cases , death due to abortion . furthermore", ", 10%15% of the children who are asymptomatic at birth may develop late sequelae , especially hearing defects , after a period of months or even years .", "latency following a primary infection ( first contact with the virus ) may be punctuated by periodic reactivations that give rise to recurrent infections , and in utero   transmission may occur during either primary or recurrent infections .", "actually recurrent infections may be due to reinfection with a new strain or to reactivation , but it is likely that most recurrent infections are due to reinfection .", "the risk of congenital infection is much higher during primary infection [ 25 ] , when the rate of transmission from mother to fetus is 30%40% [ 1 , 6 ] , as against 0.15%2.2% during reactivations or reinfections [ 1 , 69 ] when , furthermore , most of the newborns are asymptomatic .", "symptomatic cases are due more to reinfection than reactivation [ 2 , 10 ] .      ", "it has been reported that the risk of fetal damage is greater if the primary infection occurs during the first trimester of pregnancy [ 1113 ] .", "the prevalence of congenital infection ranges from 0.2% to 2.5% in different populations [ 1420 ] , in which the risk factors include particular races or ethnic groups , a low socioeconomic status , premature birth , and admission to an intensive care unit [ 6 , 17 ] .", "furthermore , the prevalence of congenital infection varies with the prevalence of the infection in the population .     ", "the seroprevalence of cmv among women of childbearing age ranges from 35% to 95% in different countries   [ 12 , 2124 ] and , as well as increasing with age , may also depend on sexual activity and occupation , particularly occupations involving close contacts with children in a community setting . in the case of parents ,", "contact with the urine or saliva of their children is a major source of infection [ 2527 ] .      ", "the incidence of primary infection among pregnant women ranges from 0.5% and 4% [ 28 , 29 ] ; the rate of seroconversion during pregnancy ranges from 0.4% to 2% [ 12 , 13 , 30 , 31 ] and depends on the seroprevalence of the infection in the population , being 3.7% among women belonging to populations with a low seroprevalence ( 55% ) and 1.0%1.6% among those belonging to populations with a high seroprevalence ( 85% ) .", "the risk of acquiring infection during pregnancy is 0.71.38  100 pregnancies [ 23 , 29 ] among seronegative women , and 0.20.8  100 pregnancies among women as a whole .    ", "as far as prevention is concerned , in addition to health education campaigns , the serological screening of pregnant women has been proposed . however , there is no consensus in the scientific community concerning the implementation of screening , and it is not recommended by any public health system because of its cost / benefit ratio , although many doctors in israel , belgium , and france do test their pregnant patients .", "furthermore , although the current public health legislation in italy ( law decree 245 of 10 september 1998 ) does not include free cmv antibody screening during pregnancy , it is prescribed by many general practitioners .     ", "the aim of this study was to assess the incidence and risk of acquiring cmv infection in pregnant women in an urban area in northern italy in the period 20052007 .", "during the three years 20052007 , the microbiology unit of hospital of legnano , received samples for the detection of cmv antibodies from 2817 pregnant women ( mean age 32 years , range 1546 ; 2522 ( 89.5% ) were italian and 295 ( 10.5% ) of foreign origin ) .      ", "forty - eight women ( 1.7% ) were 20 or less than 20 years of age , 928 ( 32.9% ) women were aged between 21 and 30 , 1750 ( 62.1% ) aged between 31 and 40 , and 91 ( 3.2% ) between 41 and 50 .", "2318 women ( 82.3% ) underwent their first screening in the first trimester ( group a ) , 316 ( 11.2% ) in the second trimester ( group b ) , and 183 ( 6.5% ) in the third ( group c ) .", "the requests were made by the general practitioners as part of the routine screening required during pregnancy .      ", "all of the samples were analyzed for the presence of anti - cmv igg and igm antibodies by means of an enzyme - linked immunosorbent assay ( elisa ) ( eti - cytok - g - plus , eti - cytok - m reverse plus , diasorin , saluggia , italy ) .", "the cutoff value used to determine igg was 0.4 iu / ml , whereas the samples were considered igm - positive when their absorbence was equal to , or greater than the control cutoff value .     ", "the igm - positive samples were confirmed using an enzyme - linked fluorescent assay ( elfa ) ( vidas cmv igm , biomrieux , lyon , france ) and were considered positive when their index was   0.90 , borderline when their index was between 0.70 and 0.90 , and negative when their index was   > 0.70 . as igm anti", "- cmv antibodies may test positive for more than 12 months and may be produced during reactivation or reinfection , the samples that were igm - positive at elisa were also tested for igg avidity ( liaison cmv igg avidity , diasorin saluggia , italy ) , which was considered low if the index was   < 0.2 , moderate if it was between 0.2 and 0.3 , and high if it was   0.3 .", "low igg avidity levels strongly suggest an infection contracted less than three months before , whereas a high avidity tends to exclude this .the elisa igm - positive samples were also tested   for the presence of rheumatoid factor ( arthri - slidex , biomrieux , lyon , france ) . in the case of positivity for igm ,", "the patients ' general practitioners were contacted and advised to evaluate the case and refer patients to a reference center .    ", "at the first screening , the elisas showed that 1925 women ( 68.3% ; 95% ci : 66.6%70.0% ) were positive for anti - cmv igg ( positive or negative for igm ) and 26 ( 0.9% ; 95% ci : 0.55%1.25% ) were positive for igm antibodies ( 25 in the first trimester , and one in the second trimester for whom no previous data were available as she did not undergo screening during the first trimester ) .", "table 1 shows the results of igm and igg elisa by trimester of first screening ( groups a ", "elfa of the 26 elisa igm - positive samples showed that 17 ( 65.4% ) were positive or borderline , and nine were negative ( 34.6% ) , including the sample that was igm - positive at elisa screening in the second trimester .", "none of the differences in the prevalence of igg and igm between contiguous age classes was statistically significant .", "of the 892 women who were anti - cmv antibody negative at first screening , 687 ( 77.0% ) were in the first trimester of pregnancy ( group a ) , 131 ( 14.7% ) in the second trimester ( group b ) , and 74 ( 8.3% ) in the third ( group c ) .     ", "three hundred and seventy - four of the women of group a ( 54.4% ) were also screened in the second trimester , and 258 ( 37.6% ) were also screened in the third ( table 2 ) .", "of these , two became positive for igm ( confirmed by elfa ) and igg , one in the second trimester ( 0.3% ) , and one in the third ( 0.4% ) , for a mean seroconversion rate of 0.32% .      ", "of the 131 women of group b , 64 ( 48.9% ) were also screened in the third trimester ; there were no cases of seroconversion ( table 2 ) .       of the 1899 women who were anti -", "cmv igg antibody positive ( and negative for igm ) at first screening , 1606 ( 84.6% ) were in the first trimester of pregnancy ( group a ) , 184 ( 9.7% ) in the second trimester ( group b ) , and 109 ( 5.7% ) in the third ( group c ) .", "one hundred and fifteen of the women of group a ( 7.2% ) were also screened in the second trimester , and 66 ( 4.1% ) were also screened in the third .", "of the 184 women of group b , 20 ( 10.9% ) were also screened in the third trimester ( table 2 ) .", "nineteen of the 28 igm - positive samples at elisa ( 67.9% ) were confirmed as being igm - positive by means of elfa , and 13 ( 46.4% ) showed low or moderate igg avidity .", "the 19 elfa - confirmed cases ( 17 first screened in the first trimester , and the two seroconversions ) included six ( 31.6% ) with a high degree of igg avidity , five ( 26.3% ) with moderate avidity , and eight ( 42.1% ) with a low degree of avidity ( table 3 ) .  \n", "table 4 shows the results of the confirmatory igm elfa and igg avidity tests by trimester of pregnancy .", "in particular , of the five cases showing moderate avidity , one was recorded in our files as having been seronegative for both igg and igm four and a half months before ( i.e. , about two months before conception ) , and another was the case of seroconversion in the third trimester after being seronegative in the first and second . in the remaining three cases ,", "the only data available were those of the initial positive sample , but the general practitioner of one of these women , who was contacted after the detection of igm positivity , reported symptoms compatible with ongoing cmv infection .", "no symptoms were reported by the general practitioners in any of the other two cases nor additional information was available .", "all of the 13 cases with low or moderate igg avidity were therefore considered as having primary infection : 11 ( 84.6% ) occurring in the first trimester , one ( 7.7% ) in the second , and one ( 7.7% ) in the third ( both seroconversions ) .      ", "unfortunately there were no data regarding the transmission of infection to the fetus because the igm - positive women were all referred for further investigations to reference centers throughout the area .", "the cumulative incidence of cmv infection ( new cases observed during pregnancy ) calculated on the basis of the 13 cases with low or moderate igg avidity was 1.4% ( 95% ci : 0.971.83 ) , and the risk of infection during pregnancy , calculated on the basis of all of the women ( seronegative and seropositive ) , was 0.5  100 pregnancies ( 95% ci : 0.240.76 ) . the differences in incidence and risk by age was not statistically significant . to take into account the loss to follow - up within the groups and different admission to the study of women during different trimester ,", "there were also calculated the density incidence as cases / pregnant woman - trimester and the correlated risk for all women ( seronegative and seropositive ) :   they were , respectively , 0.8% ( 95% ci : 0.471.13 ) and 0.4% ( 95% ci : 0.170.63 ) .", "the overall prevalence of anti - cmv igg antibodies in our pregnant women was 68.3% ( 95% ci : 66.670.0 ) , without any significant differences between age classes .     ", "as first pregnancies in italy generally occur later than they did in the past , the majority of women have already recovered from primary infection by the time they reach childbearing age and almost certainly by the time of their first pregnancy . moreover , in this study 95% of the women had an age between 21 and 40 years while age classes of 20 or less than 20 and over 40 years were under - represented ; so this could be a further cause of the lack of difference in seroprevalence .     ", "on the basis of the results of the igg avidity test , the cumulative incidence of cmv infection was 1.4% ( 95% ci : 0.971.83% ) , the density incidence was 0.8% ( 95% ci : 0.471.13 ) , and the risk of infection was 0.5% ( 95% ci : 0.240.76% ) without any significant differences by age .      ", "seroconversion or clinical data indicating acute infection were available for three of the five cases with moderate avidity in this study , thus moderate avidity was considered as a potential marker of acute infection .", "moderate and low igg avidity were considered together , and both were included in the calculation of incidence .", "however , the incidence may be an underestimate because only about half of the seronegative women underwent further screening in the second trimester and about one - third in the third , and so some cases of seroconversion may have been missed .      ", "for the same reason , the proportion of primary infections ( 84.6% ) occurring in the first trimester may be overestimated ; however , assuming the same rate of seroconversion among the seronegative women who did not undergo further screening , the majority of primary infections occurred in the first trimester .       the fact that 84.6% of the primary infections occurred in the first trimester may have been due to different behaviors before the pregnancy was recognized , whereas greater care during pregnancy may lead to less exposure .", "the fact that there were no differences related to the age of the women indicates the same type of behavior at different ages .", "it is therefore important to start screening in the first trimester of pregnancy , when there is a greater risk of infection and in order to have initial findings to compare with subsequent follow - up . in the absence of baseline data ,", "the presence of igg without igm in women undergoing their first screening in the third trimester raises doubts as it may be the result of a previous infection occurring at any time in life before the pregnancy ; however , although this is statistically the most probable situation , the possibility of an infection occurring in the first trimester with the subsequent loss of igm can not be excluded .      ", "finally some limitations of the study must be taken into account as no outcome data for newborns , substantial loss to follow - up , and limited testing of igg positive women for reinfections or reactivations .", "however , for the latter two cases , as there are no official recommendations , the follow - up was performed at the discretion of the general practitioner with compliance of pregnant woman who , above all , must pay for cmv antibody screening .       in conclusion , although screening is not recommended by any public health system ( including italy 's ) because of its cost / benefit ratio , it is actually adopted by many general practitioners in our area . such screening provides an opportunity to identify seronegative women who can be counselled about using appropriate hygienic measures to prevent infection , especially in relation to their behavior with children , who are a major source of infection .", "furthermore , the screening identified primary infections in pregnant women who could be referred to reference centers to check for prenatal infection .", "amniocentesis , funicolocentesis , ultrasonography , and magnetic resonance imaging can all be used to detect infection and allow the planning of appropriate interventions ( e.g. , antiviral therapy , termination of pregnancy ) .        although some authors consider that screening is not justified on the grounds of its economic cost , the imperfect nature of congenital infection prognostic criteria , the risk of spontaneous abortions induced by invasive tests such as amniocentesis , and the few data concerning effective treatments during pregnancy , it is unthinkable to deny pregnant women appropriate information concerning the health of their unborn child as this raises a number of ethical and legal questions .", "the incidence and risk of cmv infection in pregnancy found in our area , therefore , support the use of serological screening , certainly in the first trimester when the risk of infection is higher and , in the case of seronegative women , possibly also one screening in the second trimester and one in the third ."], "abstract_text": ["<S> the fetal consequences of cmv infection make it one of the most serious infections contracted during pregnancy , but the scientific community is divided over the proposed implementation of preventive screening for anti - cmv antibodies . </S>", "<S> the aim of this study was to assess the incidence and risk of infection during pregnancy in 2817 women who underwent anti - cmv igg and igm antibody screening during the period 20052007 . </S>", "<S> the prevalence of anti - cmv igg antibodies was 68.3% ( 95% ci : 66.670.0 ) ; the seroconversion rate in the 892 seronegative women was 0.32% ; the results of igg avidity testing revealed an cumulative incidence of 1.4% ( 95% ci : 0.971.83 ) , density incidence of 0.8% ( as cases / pregnant woman - trimester ) ( 95% ci : 0.471.13 ) , and a risk of infection of 0.5% ( 95% ci : 0.240.76 ) . the screening identified 13 cases of primary infection ( 84.6% of which occurred in the first trimester of pregnancy ) . </S>", "<S> the possibility to identify these cases and consequently to plan appropriate interventions , supports the use of screening during pregnancy , especially in the first trimester when the risk of infection is greater . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["the human cytomegalovirus ( cmv ) or human herpesvirus 5 is one of the major causes of congenital infections .", "its clinical manifestations range from asymptomatic forms ( 90% of cases ) to severe fetal damage and , in rare cases , death due to abortion . furthermore", ", 10%15% of the children who are asymptomatic at birth may develop late sequelae , especially hearing defects , after a period of months or even years .", "latency following a primary infection ( first contact with the virus ) may be punctuated by periodic reactivations that give rise to recurrent infections , and in utero   transmission may occur during either primary or recurrent infections .", "actually recurrent infections may be due to reinfection with a new strain or to reactivation , but it is likely that most recurrent infections are due to reinfection .", "the risk of congenital infection is much higher during primary infection [ 25 ] , when the rate of transmission from mother to fetus is 30%40% [ 1 , 6 ] , as against 0.15%2.2% during reactivations or reinfections [ 1 , 69 ] when , furthermore , most of the newborns are asymptomatic .", "symptomatic cases are due more to reinfection than reactivation [ 2 , 10 ] .      ", "it has been reported that the risk of fetal damage is greater if the primary infection occurs during the first trimester of pregnancy [ 1113 ] .", "the prevalence of congenital infection ranges from 0.2% to 2.5% in different populations [ 1420 ] , in which the risk factors include particular races or ethnic groups , a low socioeconomic status , premature birth , and admission to an intensive care unit [ 6 , 17 ] .", "furthermore , the prevalence of congenital infection varies with the prevalence of the infection in the population .     ", "the seroprevalence of cmv among women of childbearing age ranges from 35% to 95% in different countries   [ 12 , 2124 ] and , as well as increasing with age , may also depend on sexual activity and occupation , particularly occupations involving close contacts with children in a community setting . in the case of parents ,", "contact with the urine or saliva of their children is a major source of infection [ 2527 ] .      ", "the incidence of primary infection among pregnant women ranges from 0.5% and 4% [ 28 , 29 ] ; the rate of seroconversion during pregnancy ranges from 0.4% to 2% [ 12 , 13 , 30 , 31 ] and depends on the seroprevalence of the infection in the population , being 3.7% among women belonging to populations with a low seroprevalence ( 55% ) and 1.0%1.6% among those belonging to populations with a high seroprevalence ( 85% ) .", "the risk of acquiring infection during pregnancy is 0.71.38  100 pregnancies [ 23 , 29 ] among seronegative women , and 0.20.8  100 pregnancies among women as a whole .    ", "as far as prevention is concerned , in addition to health education campaigns , the serological screening of pregnant women has been proposed . however , there is no consensus in the scientific community concerning the implementation of screening , and it is not recommended by any public health system because of its cost / benefit ratio , although many doctors in israel , belgium , and france do test their pregnant patients .", "furthermore , although the current public health legislation in italy ( law decree 245 of 10 september 1998 ) does not include free cmv antibody screening during pregnancy , it is prescribed by many general practitioners .     ", "the aim of this study was to assess the incidence and risk of acquiring cmv infection in pregnant women in an urban area in northern italy in the period 20052007 ."], ["during the three years 20052007 , the microbiology unit of hospital of legnano , received samples for the detection of cmv antibodies from 2817 pregnant women ( mean age 32 years , range 1546 ; 2522 ( 89.5% ) were italian and 295 ( 10.5% ) of foreign origin ) .      ", "forty - eight women ( 1.7% ) were 20 or less than 20 years of age , 928 ( 32.9% ) women were aged between 21 and 30 , 1750 ( 62.1% ) aged between 31 and 40 , and 91 ( 3.2% ) between 41 and 50 .", "2318 women ( 82.3% ) underwent their first screening in the first trimester ( group a ) , 316 ( 11.2% ) in the second trimester ( group b ) , and 183 ( 6.5% ) in the third ( group c ) .", "the requests were made by the general practitioners as part of the routine screening required during pregnancy .      ", "all of the samples were analyzed for the presence of anti - cmv igg and igm antibodies by means of an enzyme - linked immunosorbent assay ( elisa ) ( eti - cytok - g - plus , eti - cytok - m reverse plus , diasorin , saluggia , italy ) .", "the cutoff value used to determine igg was 0.4 iu / ml , whereas the samples were considered igm - positive when their absorbence was equal to , or greater than the control cutoff value .     ", "the igm - positive samples were confirmed using an enzyme - linked fluorescent assay ( elfa ) ( vidas cmv igm , biomrieux , lyon , france ) and were considered positive when their index was   0.90 , borderline when their index was between 0.70 and 0.90 , and negative when their index was   > 0.70 . as igm anti", "- cmv antibodies may test positive for more than 12 months and may be produced during reactivation or reinfection , the samples that were igm - positive at elisa were also tested for igg avidity ( liaison cmv igg avidity , diasorin saluggia , italy ) , which was considered low if the index was   < 0.2 , moderate if it was between 0.2 and 0.3 , and high if it was   0.3 .", "low igg avidity levels strongly suggest an infection contracted less than three months before , whereas a high avidity tends to exclude this .the elisa igm - positive samples were also tested   for the presence of rheumatoid factor ( arthri - slidex , biomrieux , lyon , france ) . in the case of positivity for igm ,", "the patients ' general practitioners were contacted and advised to evaluate the case and refer patients to a reference center .    "], ["at the first screening , the elisas showed that 1925 women ( 68.3% ; 95% ci : 66.6%70.0% ) were positive for anti - cmv igg ( positive or negative for igm ) and 26 ( 0.9% ; 95% ci : 0.55%1.25% ) were positive for igm antibodies ( 25 in the first trimester , and one in the second trimester for whom no previous data were available as she did not undergo screening during the first trimester ) .", "table 1 shows the results of igm and igg elisa by trimester of first screening ( groups a ", "elfa of the 26 elisa igm - positive samples showed that 17 ( 65.4% ) were positive or borderline , and nine were negative ( 34.6% ) , including the sample that was igm - positive at elisa screening in the second trimester .", "none of the differences in the prevalence of igg and igm between contiguous age classes was statistically significant .", "of the 892 women who were anti - cmv antibody negative at first screening , 687 ( 77.0% ) were in the first trimester of pregnancy ( group a ) , 131 ( 14.7% ) in the second trimester ( group b ) , and 74 ( 8.3% ) in the third ( group c ) .     ", "three hundred and seventy - four of the women of group a ( 54.4% ) were also screened in the second trimester , and 258 ( 37.6% ) were also screened in the third ( table 2 ) .", "of these , two became positive for igm ( confirmed by elfa ) and igg , one in the second trimester ( 0.3% ) , and one in the third ( 0.4% ) , for a mean seroconversion rate of 0.32% .      ", "of the 131 women of group b , 64 ( 48.9% ) were also screened in the third trimester ; there were no cases of seroconversion ( table 2 ) .       of the 1899 women who were anti -", "cmv igg antibody positive ( and negative for igm ) at first screening , 1606 ( 84.6% ) were in the first trimester of pregnancy ( group a ) , 184 ( 9.7% ) in the second trimester ( group b ) , and 109 ( 5.7% ) in the third ( group c ) .", "one hundred and fifteen of the women of group a ( 7.2% ) were also screened in the second trimester , and 66 ( 4.1% ) were also screened in the third .", "of the 184 women of group b , 20 ( 10.9% ) were also screened in the third trimester ( table 2 ) .", "nineteen of the 28 igm - positive samples at elisa ( 67.9% ) were confirmed as being igm - positive by means of elfa , and 13 ( 46.4% ) showed low or moderate igg avidity .", "the 19 elfa - confirmed cases ( 17 first screened in the first trimester , and the two seroconversions ) included six ( 31.6% ) with a high degree of igg avidity , five ( 26.3% ) with moderate avidity , and eight ( 42.1% ) with a low degree of avidity ( table 3 ) .  \n", "table 4 shows the results of the confirmatory igm elfa and igg avidity tests by trimester of pregnancy .", "in particular , of the five cases showing moderate avidity , one was recorded in our files as having been seronegative for both igg and igm four and a half months before ( i.e. , about two months before conception ) , and another was the case of seroconversion in the third trimester after being seronegative in the first and second . in the remaining three cases ,", "the only data available were those of the initial positive sample , but the general practitioner of one of these women , who was contacted after the detection of igm positivity , reported symptoms compatible with ongoing cmv infection .", "no symptoms were reported by the general practitioners in any of the other two cases nor additional information was available .", "all of the 13 cases with low or moderate igg avidity were therefore considered as having primary infection : 11 ( 84.6% ) occurring in the first trimester , one ( 7.7% ) in the second , and one ( 7.7% ) in the third ( both seroconversions ) .      ", "unfortunately there were no data regarding the transmission of infection to the fetus because the igm - positive women were all referred for further investigations to reference centers throughout the area .", "the cumulative incidence of cmv infection ( new cases observed during pregnancy ) calculated on the basis of the 13 cases with low or moderate igg avidity was 1.4% ( 95% ci : 0.971.83 ) , and the risk of infection during pregnancy , calculated on the basis of all of the women ( seronegative and seropositive ) , was 0.5  100 pregnancies ( 95% ci : 0.240.76 ) . the differences in incidence and risk by age was not statistically significant . to take into account the loss to follow - up within the groups and different admission to the study of women during different trimester ,", "there were also calculated the density incidence as cases / pregnant woman - trimester and the correlated risk for all women ( seronegative and seropositive ) :   they were , respectively , 0.8% ( 95% ci : 0.471.13 ) and 0.4% ( 95% ci : 0.170.63 ) ."], ["the overall prevalence of anti - cmv igg antibodies in our pregnant women was 68.3% ( 95% ci : 66.670.0 ) , without any significant differences between age classes .     ", "as first pregnancies in italy generally occur later than they did in the past , the majority of women have already recovered from primary infection by the time they reach childbearing age and almost certainly by the time of their first pregnancy . moreover , in this study 95% of the women had an age between 21 and 40 years while age classes of 20 or less than 20 and over 40 years were under - represented ; so this could be a further cause of the lack of difference in seroprevalence .     ", "on the basis of the results of the igg avidity test , the cumulative incidence of cmv infection was 1.4% ( 95% ci : 0.971.83% ) , the density incidence was 0.8% ( 95% ci : 0.471.13 ) , and the risk of infection was 0.5% ( 95% ci : 0.240.76% ) without any significant differences by age .      ", "seroconversion or clinical data indicating acute infection were available for three of the five cases with moderate avidity in this study , thus moderate avidity was considered as a potential marker of acute infection .", "moderate and low igg avidity were considered together , and both were included in the calculation of incidence .", "however , the incidence may be an underestimate because only about half of the seronegative women underwent further screening in the second trimester and about one - third in the third , and so some cases of seroconversion may have been missed .      ", "for the same reason , the proportion of primary infections ( 84.6% ) occurring in the first trimester may be overestimated ; however , assuming the same rate of seroconversion among the seronegative women who did not undergo further screening , the majority of primary infections occurred in the first trimester .       the fact that 84.6% of the primary infections occurred in the first trimester may have been due to different behaviors before the pregnancy was recognized , whereas greater care during pregnancy may lead to less exposure .", "the fact that there were no differences related to the age of the women indicates the same type of behavior at different ages .", "it is therefore important to start screening in the first trimester of pregnancy , when there is a greater risk of infection and in order to have initial findings to compare with subsequent follow - up . in the absence of baseline data ,", "the presence of igg without igm in women undergoing their first screening in the third trimester raises doubts as it may be the result of a previous infection occurring at any time in life before the pregnancy ; however , although this is statistically the most probable situation , the possibility of an infection occurring in the first trimester with the subsequent loss of igm can not be excluded .      ", "finally some limitations of the study must be taken into account as no outcome data for newborns , substantial loss to follow - up , and limited testing of igg positive women for reinfections or reactivations .", "however , for the latter two cases , as there are no official recommendations , the follow - up was performed at the discretion of the general practitioner with compliance of pregnant woman who , above all , must pay for cmv antibody screening .       in conclusion , although screening is not recommended by any public health system ( including italy 's ) because of its cost / benefit ratio , it is actually adopted by many general practitioners in our area . such screening provides an opportunity to identify seronegative women who can be counselled about using appropriate hygienic measures to prevent infection , especially in relation to their behavior with children , who are a major source of infection .", "furthermore , the screening identified primary infections in pregnant women who could be referred to reference centers to check for prenatal infection .", "amniocentesis , funicolocentesis , ultrasonography , and magnetic resonance imaging can all be used to detect infection and allow the planning of appropriate interventions ( e.g. , antiviral therapy , termination of pregnancy ) .        although some authors consider that screening is not justified on the grounds of its economic cost , the imperfect nature of congenital infection prognostic criteria , the risk of spontaneous abortions induced by invasive tests such as amniocentesis , and the few data concerning effective treatments during pregnancy , it is unthinkable to deny pregnant women appropriate information concerning the health of their unborn child as this raises a number of ethical and legal questions .", "the incidence and risk of cmv infection in pregnancy found in our area , therefore , support the use of serological screening , certainly in the first trimester when the risk of infection is higher and , in the case of seronegative women , possibly also one screening in the second trimester and one in the third ."]]}
{"article_id": "PMC4626912", "article_text": ["bladder cancer is the second most commonly diagnosed genitourinary malignancy in the usa , with an estimated 74,000 newly diagnosed cases and 16,000 deaths in 2015 .", "the incidence of bladder cancer rises with age , peaking between age 50 and 70 years , and is three times more common in men than in women .", "commonly accepted risk factors for bladder cancer include cigarette smoking , occupational exposure to aniline dyes , benzidene compounds , analgesic abuse ( phenacetin ) and chronic irritation , such as indwelling catheters . of all newly diagnosed cases of bladder cancer , about 70% present as non - muscle invasive bladder cancer ( nmibc ) ,", "it comprises a heterogeneous group of patients and includes pathological stage ta ( confinement to the epithelium or mucosa ) , t1 ( invasion of the subepithelial connective tissue or lamina propria ) and cis ( tis : flat , high - grade , non - papillary carcinomas confined to the urothelium ) . of all newly diagnosed nmibc , 70% present as stage ta , 20% as t1 and 10% as cis .", "approximately 50 - 70% of nmibc will recur and roughly 1020% will progress to muscle ( i.e. , muscularis propria ) invasive disease . in patients with low - grade ta disease ,", "patients with high - grade ta tumors had a progression - free survival of 61% and a disease - specific survival of 74% , whereas patients with t1 disease had a progression - free survival of 44% and a disease - specific survival of 62% , providing support to the view that grouping all patients with nmibc as one disease is misleading as one patient 's prognosis can be quite different from that of another patient .", "when considering a patient 's prognosis with nmibc , it is necessary to consider not only clinical and pathological factors but also take into account the potential effect of the intravesical treatment received and molecular alterations present in the tumors .", "table 1 summarizes the available predictive models to predict recurrence and progression in patients with nmibc .", "the most important risk factor for progression is grade , not stage , because patients with high - grade tumors progress with similar frequency regardless of whether they were invasive ( t1 ) or non - invasive ( ta ) .", "millan - rodriguez et al . evaluated a cohort of 1529 primary nmibc patients treated with transurethral resection ( tur ) and random bladder biopsy and identified prognostic factors for recurrence , progression and disease - specific mortality ( median follow - up : 40 months ) .", "multivariate analysis demonstrated that the main prognostic factors of recurrence were multiplicity , tumor size > 3 cm , presence of cis and treatment with bacillus calmette  guerin ( bcg ) . the prognostic factors for progression were grade 3 disease , multiplicity , tumor size > 3 cm , cis and treatment with bcg .", "furthermore , the prognostic factors for mortality were presence of grade 3 disease and cis .", "prediction of disease recurrence and progression in patients with non - muscle invasive bladder cancer in another study , millan - rodriguez et al .", "risk groups were classified as : \n low risk ( grade 1 stage ta disease and a single grade 1 stage t1 tumor)intermediate risk ( multiple grade 1 stage t1 tumors , grade 2 stage ta disease and a single grade 2 stage t1 tumor)high risk ( multiple grade 2 stage t1 tumors , grade 3 stages ta and t1 disease and any stage disease associated with cis ) . \n  low risk ( grade 1 stage ta disease and a single grade 1 stage t1 tumor ) intermediate risk ( multiple grade 1 stage t1 tumors , grade 2 stage ta disease and a single grade 2 stage t1 tumor ) high risk ( multiple grade 2 stage t1 tumors , grade 3 stages ta and t1 disease and any stage disease associated with cis ) .", "the rates of recurrence , progression and mortality were 37% , 0% and 0% in the low - risk group , 45% , 1.8% and 0.73% in the intermediate - risk group and 54% , 15% and 9.5% in the high - risk group .", "the relative risks of recurrence , progression and mortality in the low - risk versus intermediate - risk and high - risk groups were 1.37 , 2.84 and 1 , and 1.87 , 24.76 and 14.69 , respectively .", "the european organization for research and treatment of cancer ( eortc ) developed risk tables to predict recurrence and progression in patients with nmibc using clinical and pathologic information from 2596 patients enrolled in seven clinical trials that utilized prophylactic treatments after tur .", "the median follow - up was 3.9 years and 78% of patients had received intravesical treatment , mostly with chemotherapy . in their study cohort , 80% of patients had a maximum tumor diameter < 3 cm and 56% of patients had pta tumors .", "the eortc risk tables [ tables 2 and 3 ] for recurrence and progression were based on the scoring system derived from the following six clinical and pathological factors : \n number of tumorstumor sizet categorytumor grade ( who 1973)prior recurrence ratepresence of concurrent cis . \n ", "weights used to calculate eortc risk tables recurrence and progression scores probability of recurrence and progression according to the eortc risk tables total score based on these eortc risk tables ( electronic calculator is available at http://www.eortc.be/tools/bladdercalculator/ ) , the probability of recurrence varied from 15% to 61% at 1 year and from 31% to 78% at 5 years . the probability of progression varied from 0.2% to 17% at 1 year and from 0.8% to 45% at 5 years . for their recurrence and progression models , harrell 's concordance indices at 1 year were 0.66 and 0.74 , respectively .", "of note , only 6.6% ( 171 of 2596 ) patients received bcg for six induction instillations .", "in addition , patients with high - grade disease did not have a second tur or receive maintenance bcg therapy .", "bcg is currently the most effective intravesical therapy for nmibc , especially in intermediate- and high - risk tumors . a meta - analysis of 24 randomized trials ( n = 4863 ) showed that bcg significantly reduces the risk of progression to muscle - invasive disease after tur in patients with nmibc who receive maintenance bcg .", "the patient 's response to bcg at 3 or 6 months is an important prognostic factor to predict subsequent tumor recurrence and progression .", "because the eortc risk tables were generated using nmibc patient 's clinical and pathological information , where majority of the patients did not receive bcg induction and/or bcg maintenance therapy , the eortc risk tables tend to overestimate patient 's risk for recurrence and progression after bcg therapy . the spanish club urolgicoespaol de tratamientooncolgico ( cueto ) group developed a scoring model using information from 1062 patients treated with intravesical adjuvant bcg therapy to predict risk of recurrence and progression .", "patients had bcg induction as weekly instillations for 6 weeks and six additional instillations repeated at 2-week intervals as a maintenance therapy . by the end of the study ,", "4.2% of patients received fewer than six instillations , 22.5% of patients received six to nine instillations and 73.3% of patients received more than 10 instillations .", "the risk tables for recurrence and progression were based on the scoring systems derived from seven clinical and pathological factors : \n agegendernumber of tumorsrecurrent tumort categorytumor grade ( who 1973)presence of concurrent cis . \n  tumor grade ( who 1973 ) presence of concurrent cis . based on the cueto risk tables , the probability of recurrence varied from 8.24% to 41.79% at 1 year to 20.98% to 67.61% at 5 years .", "the probability of progression varied from 1.17% to 13.97% at 1 year to 3.76% to 33.57% at 5 years . for their recurrence and progression models , harrell 's concordance indices at 1 year were 0.636 and 0.687 , respectively . using the cueto risk tables ,", "the calculated risk of recurrence were lower than that obtained by the eortc risk tables .", "the lower risks in the cueto tables is likely attributable to using bcg , which is a more effective intravesical therapy .", "although the eortc and cueto risk tables are the two best - established predictive multivariate models for recurrence and progression risk calculation in patients with nmibc , limitations include retrospective analysis , use of the 1987 tnm classification and use of the who 1973 grading system .", "in addition , repeat tur for high - grade cancer and immediate instillation of chemotherapy , such as mitomycin , was not routinely performed after tur ; hence , both models are prone to overestimate the risk of recurrence and progression in patients treated with current standard of care .", "a restaging tur is routinely performed these days in patients with high - grade nmibc , especially for high - grade t1 , as the rate of residual tumor is high and 30% of tumors are upstaged when muscle is absent in the first obtained specimen .", "photodynamic diagnosis with blue light and narrow - band imaging have been shown to reduce residual tumor rates by roughly 20% and to improve recurrence - free survival of nmibc patients compared with white light cystoscopy .", "a meta - analysis of seven randomized trials ( n = 1476 ) confirmed the effectiveness of single immediate intravesical instillation of chemotherapy in treated patients compared with tur alone , reporting a 39% decrease in the odds of recurrence .", "the efficacy of intravesical mitomycin c can be optimized by administering a dose of 40 mg at a concentration of 2 mg / ml in water , complete bladder emptying just before dose administration , fluid restriction and oral bicarbonate to alkalinize the urine .", "this approach improved the recurrence - free rate at 5 years from 24.6% to 41% and increased the interval to tumor recurrence from 11.9 to 29 months . using a multi - institutional cohort of 4689 patients with nmibc , xylinas et al .", "showed that both the eortc and cueto risk tables exhibit a poor discrimination for disease recurrence and progression .", "these models overestimated the risk of disease recurrence and progression in high - risk patients .", "additional factors not included in the eortc or the cueto models could be added to new prognostic models to enhance their usefulness .", "bladder neck involvement , prostatic urethra involvement , lymphovascular invasion and depth of lamina propria invasion , i.e. , whether the tumor is superficial to , into or beyond the muscularis mucosae ( t1a , t1b , t1c ) , have been identified as risk factors for progression in patients with nmibc . in the prognostic factor meta - analysis of 33 studies of high - grade t1 bladder cancer patients (", "n = 8880 ) , the highest impact risk factor for progression and cancer - specific survival was depth of invasion ( t1b / c ) into the lamina propria .", "in this meta - analysis , several other previously proposed factors , i.e. lymphovascular invasion , associated cis , non - use of bcg , tumor size > 3 cm , gender , multiple tumors and older age , also predicted progression .", "nmibc patients with progression to muscle - invasive disease tend to have worse prognosis compared with patients with primary muscle - invasive disease , and it underscores the need to significantly improve risk stratification and earlier definitive treatment for high - risk nmibc . risk groupings and risk tables provide average predictions , which may or may not apply to the patient interested in knowing individualized risk .", "nomograms have been proposed as a method that avoids the arbitrary division of patients into risk groups .", "developed nomograms to estimate the risk of disease recurrence and progression in patients with nmibc using a large international cohort .", "nomograms based on age , gender , urine cytology and dichotomized nmp22 had accuracy of 0.811 for recurrence of any bladder cancer ; 0.772 for recurrence of grade 3 ta or t1 or cis and 0.774 for recurrence of stage t2 bladder cancer after bootstrap validation . however , this nomogram had several limitations , including failure to incorporate established predictors , such as pathologic grade and stage , number and pattern of previous recurrences , time since the original diagnosis and prior use of intravesical therapy .", "furthermore , the performance of the nomograms varied significantly among the institutions , emphasizing the need for external validation prior to its clinical use . artificial neural networks ( ann ) are algorithms that can be trained to identify complex patterns between input variables and outcomes in the data sets and then apply these patterns to new cases .", "they have a theoretical advantage over conventional statistics as they are not limited by predefined mathematical relationships between input variables and outcomes ; thus , they are able to model complex non - linear parameters .", "qureshi et al . used ann to predict bladder cancer recurrence and progression within 6 months of diagnosis in a small cohort of about 100 patients with nmibc .", "the accuracy of ann in predicting stage progression and recurrence was 80% and 75% , respectively .", "however , there was no significant difference between the ann and the clinicians predictions of progression and recurrence in the patients with nmibc .", "on restricting the validation subset to patients with t1g3 tumors in relation to stage progression , ann accuracy was better than predictions of clinicians .", "several investigators have compared ann and other machine learning techniques with standard statistical approaches to predict outcomes and did not find improvement in predictive accuracy with ann and other machine learning techniques over standard statistical approaches .", "most of the currently available predictive tools assume that nmibc is pure urothelial carcinoma and does not consider the impact of variant histology on prognosis prediction .", "variant histology such as micropapillary , sarcomatoid and plasmacytoid has been shown to be associated with poor prognosis , and early cystectomy is generally advocated in such cases .", "small cell carcinoma of the bladder is considered a systemic disease and chemotherapy followed by tailored local therapy is recommended for patients with non - muscle invasive small cell carcinoma of the bladder .", "however , spaliviero et al . reported not significantly worse outcomes in conservatively managed t1 micropapillary bladder cancer patients compared with patients treated with early radical cystectomy . given the conflicting findings on the impact of variant histology in smaller studies , the association of variant histology with prognosis deserves further evaluation in larger studies .", "for molecular biomarkers to be of clinical use , they should be able to increase the predictive accuracy beyond the standard clinical and pathological parameter models .", "several investigators have attempted to use molecular biomarkers as prognostic factors to predict outcomes in patients with nmibc .", "however , molecular biomarkers have shown mixed results so far and are not sufficiently validated to be used in clinical practice at this time .", "it is becoming clear that superficial low - grade cancers and invasive or high - grade cancers harbor distinctive genetic defects : the low - grade , non - invasive papillary tumors are characterized by activating mutations in the h - ras gene and fibroblast growth factor receptor 3 ( fgfr3 ) gene and the high - grade invasive tumors are characterized by structural and functional defects in the p53 and retinoblastoma protein ( rb ) tumor - suppressor pathways .", "the deletion of both arms of chromosome 9 occurs frequently in bladder cancer during the earliest stages of tumorigenesis .", "however , these chromosomal aberrations do not seem to distinguish between the two tumor development pathways .", "tumor invasion and progression in the bladder seems to be a multifactorial process , promoted by micro - environmental changes that include the up - regulation of n - cadherin , the down - regulation of e - cadherin , the overexpression of matrix metalloproteinases 2 and 9 , an imbalance between angiogenic and anti - angiogenic factors and increased synthesis of prostaglandin . in patients with t1 bladder cancer , nuclear overexpression of p53 protein", "has been reported to have a higher probability of disease progression . in a meta - analysis , p53 was a predictor for recurrence , progression and mortality in patients with bladder cancer .", "however , investigators of this meta - analysis had concerns of overestimating findings because of publication and reporting bias , and suggested that current evidence is not sufficient to conclude whether changes in p53 act as markers of outcome in patients with bladder cancer .", "hernandez et al . , in a prospective cohort of 772 patients with nmibc , showed that fgfr3 mutations are prevalent in low - grade ta and that these mutations are independent predictors of recurrence in patients with low - grade ta tumors .", "van rhijn et al . showed that molecular grading , the grading system based on fgfr3 and mib-1 ( ki-67 ) status , is an independent predictor for recurrence , progression and disease - specific survival .", "van rhijn et al . validated the utility of molecular grading as a prognostic factor to predict outcomes in patients with nmibc .", "the addition of molecular grade to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7% .", "evaluated nmp22 for detecting recurrence and progression in patients with nmibc in a large multi - institutional international cohort .", "there was no clearly defined nmp22 cut - off that could indicate higher risk disease , but there was a continuum of risk for recurrence and progression .", "the international consensus panel on cytology and bladder tumor markers evaluated the prognostic utility of molecular markers for bladder cancer .", "molecular markers were classified into six groups , i.e. microsatellite - associated markers ( e.g. fish , loh ) , proto - oncogenes / oncogenes ( e.g. her-2/neu , h - ras , bcl-2 , mdm-2 , fgfr-3 , c - myc ) tumor suppressor genes ( e.g. , p53 , rb ) , cell cycle regulators ( e.g. , p21 , p27 , ki-67 , cyclin - d1 , cyclin - e ) , angiogenesis - related factors ( vegf , cox-2 , tsp-1 ) and extracellular matrix adhesion molecules ( e.g. e - cadherin , mmps , timps , cd44 , u - pa ) . the panel concluded that although certain biomarkers , such as ki-67 and p53 , appear to be promising in predicting recurrence and progression in patients with bladder cancer , the data are still heterogeneous .", "the panel recommended that identifying definitive criteria for test positivity , a clearly defined patient population , standardization of techniques used to evaluate markers and clearly specified endpoints and statistical methods will help to bring accurate independent prognostic indicators into the clinical management of patients with bladder cancer .", "nmibc comprises of a heterogeneous group of patients and includes pathological stage ta , t1 and cis .", "patients with low - grade ta disease have very low risk of progression while patients with t1 disease with concurrent cis have a much higher risk of progression , approaching 50% .", "the eortc and cueto risk tables are the two best - established predictive models for recurrence and progression risk calculation in patients with nmibc . however", ", both the eortc and the cueto risk tables exhibit a poor discrimination for prognostic outcomes and overestimate the risk of disease recurrence and progression in high - risk patients in external validation .", "additional prognostic factors such as depth of lamina propria invasion should be added to new prognostic models to enhance their usefulness .", "molecular biomarkers such as ki-67 , fgfr3 and p53 appear to be promising in predicting recurrence and progression , but need further validation prior to using them in clinical practice .", "future research should focus to enhance the predictive accuracy of the risk assessment tools by incorporating additional prognostic factors such as depth of lamina propria invasion and molecular biomarkers after rigorous validation in multi - institutional cohorts ."], "abstract_text": ["<S> introduction : non - muscle invasive bladder cancer ( nmibc ) comprises about 70% of all newly diagnosed bladder cancer , and includes tumors with stage ta , t1 and carcinoma in situ ( cis . ) since , nmibc patients with progression to muscle - invasive disease tend to have worse prognosis than with patients with primary muscle - invasive disease , there is a need to significantly improve risk stratification and earlier definitive treatment for high - risk nmibc.materials and methods : a detailed medline search was performed to identify all publications on the topic of prognostic factors and risk predictions for superficial bladder cancer / nmibc . </S>", "<S> the manuscripts were reviewed to identify variables that could predict recurrence and progression.results:the most important prognostic factor for progression is grade of tumor . </S>", "<S> t category , tumor size , number of tumors , concurrent cis , intravesical therapy , response to bacillus calmette  </S>", "<S> guerin at 3- or 6-month follow - up , prior recurrence rate , age , gender , lymphovascular invasion and depth of lamina propria invasion are other important clinical and pathological parameters to predict recurrence and progression in patients with nmibc . </S>", "<S> the european organization for research and treatment of cancer ( eortc ) and the spanish club urolgicoespaol de tratamiento oncolgico ( cueto ) risk tables are the two best - established predictive models for recurrence and progression risk calculation , although they tend to overestimate risk and have poor discrimination for prognostic outcomes in external validation . </S>", "<S> molecular biomarkers such as ki-67 , fgfr3 and p53 appear to be promising in predicting recurrence and progression but need further validation prior to using them in clinical practice.conclusion:eortc and cueto risk tables are the two best - established models to predict recurrence and progression in patients with nmibc though they tend to overestimate risk and have poor discrimination for prognostic outcomes in external validation . </S>", "<S> future research should focus on enhancing the predictive accuracy of risk assessment tools by incorporating additional prognostic factors such as depth of lamina propria invasion and molecular biomarkers after rigorous validation in multi - institutional cohorts . </S>"], "labels": null, "section_names": ["INTRODUCTION", "RISK STRATIFICATION BASED ON CLINICAL AND PATHOLOGICAL PARAMETERS", "RISK STRATIFICATION BASED ON MOLECULAR BIOMARKERS", "CONCLUSIONS", "None", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["bladder cancer is the second most commonly diagnosed genitourinary malignancy in the usa , with an estimated 74,000 newly diagnosed cases and 16,000 deaths in 2015 .", "the incidence of bladder cancer rises with age , peaking between age 50 and 70 years , and is three times more common in men than in women .", "commonly accepted risk factors for bladder cancer include cigarette smoking , occupational exposure to aniline dyes , benzidene compounds , analgesic abuse ( phenacetin ) and chronic irritation , such as indwelling catheters . of all newly diagnosed cases of bladder cancer , about 70% present as non - muscle invasive bladder cancer ( nmibc ) ,", "it comprises a heterogeneous group of patients and includes pathological stage ta ( confinement to the epithelium or mucosa ) , t1 ( invasion of the subepithelial connective tissue or lamina propria ) and cis ( tis : flat , high - grade , non - papillary carcinomas confined to the urothelium ) . of all newly diagnosed nmibc , 70% present as stage ta , 20% as t1 and 10% as cis .", "approximately 50 - 70% of nmibc will recur and roughly 1020% will progress to muscle ( i.e. , muscularis propria ) invasive disease . in patients with low - grade ta disease ,", "patients with high - grade ta tumors had a progression - free survival of 61% and a disease - specific survival of 74% , whereas patients with t1 disease had a progression - free survival of 44% and a disease - specific survival of 62% , providing support to the view that grouping all patients with nmibc as one disease is misleading as one patient 's prognosis can be quite different from that of another patient .", "when considering a patient 's prognosis with nmibc , it is necessary to consider not only clinical and pathological factors but also take into account the potential effect of the intravesical treatment received and molecular alterations present in the tumors ."], ["table 1 summarizes the available predictive models to predict recurrence and progression in patients with nmibc .", "the most important risk factor for progression is grade , not stage , because patients with high - grade tumors progress with similar frequency regardless of whether they were invasive ( t1 ) or non - invasive ( ta ) .", "millan - rodriguez et al . evaluated a cohort of 1529 primary nmibc patients treated with transurethral resection ( tur ) and random bladder biopsy and identified prognostic factors for recurrence , progression and disease - specific mortality ( median follow - up : 40 months ) .", "multivariate analysis demonstrated that the main prognostic factors of recurrence were multiplicity , tumor size > 3 cm , presence of cis and treatment with bacillus calmette  guerin ( bcg ) . the prognostic factors for progression were grade 3 disease , multiplicity , tumor size > 3 cm , cis and treatment with bcg .", "furthermore , the prognostic factors for mortality were presence of grade 3 disease and cis .", "prediction of disease recurrence and progression in patients with non - muscle invasive bladder cancer in another study , millan - rodriguez et al .", "risk groups were classified as : \n low risk ( grade 1 stage ta disease and a single grade 1 stage t1 tumor)intermediate risk ( multiple grade 1 stage t1 tumors , grade 2 stage ta disease and a single grade 2 stage t1 tumor)high risk ( multiple grade 2 stage t1 tumors , grade 3 stages ta and t1 disease and any stage disease associated with cis ) . \n  low risk ( grade 1 stage ta disease and a single grade 1 stage t1 tumor ) intermediate risk ( multiple grade 1 stage t1 tumors , grade 2 stage ta disease and a single grade 2 stage t1 tumor ) high risk ( multiple grade 2 stage t1 tumors , grade 3 stages ta and t1 disease and any stage disease associated with cis ) .", "the rates of recurrence , progression and mortality were 37% , 0% and 0% in the low - risk group , 45% , 1.8% and 0.73% in the intermediate - risk group and 54% , 15% and 9.5% in the high - risk group .", "the relative risks of recurrence , progression and mortality in the low - risk versus intermediate - risk and high - risk groups were 1.37 , 2.84 and 1 , and 1.87 , 24.76 and 14.69 , respectively .", "the european organization for research and treatment of cancer ( eortc ) developed risk tables to predict recurrence and progression in patients with nmibc using clinical and pathologic information from 2596 patients enrolled in seven clinical trials that utilized prophylactic treatments after tur .", "the median follow - up was 3.9 years and 78% of patients had received intravesical treatment , mostly with chemotherapy . in their study cohort , 80% of patients had a maximum tumor diameter < 3 cm and 56% of patients had pta tumors .", "the eortc risk tables [ tables 2 and 3 ] for recurrence and progression were based on the scoring system derived from the following six clinical and pathological factors : \n number of tumorstumor sizet categorytumor grade ( who 1973)prior recurrence ratepresence of concurrent cis . \n ", "weights used to calculate eortc risk tables recurrence and progression scores probability of recurrence and progression according to the eortc risk tables total score based on these eortc risk tables ( electronic calculator is available at http://www.eortc.be/tools/bladdercalculator/ ) , the probability of recurrence varied from 15% to 61% at 1 year and from 31% to 78% at 5 years . the probability of progression varied from 0.2% to 17% at 1 year and from 0.8% to 45% at 5 years . for their recurrence and progression models , harrell 's concordance indices at 1 year were 0.66 and 0.74 , respectively .", "of note , only 6.6% ( 171 of 2596 ) patients received bcg for six induction instillations .", "in addition , patients with high - grade disease did not have a second tur or receive maintenance bcg therapy .", "bcg is currently the most effective intravesical therapy for nmibc , especially in intermediate- and high - risk tumors . a meta - analysis of 24 randomized trials ( n = 4863 ) showed that bcg significantly reduces the risk of progression to muscle - invasive disease after tur in patients with nmibc who receive maintenance bcg .", "the patient 's response to bcg at 3 or 6 months is an important prognostic factor to predict subsequent tumor recurrence and progression .", "because the eortc risk tables were generated using nmibc patient 's clinical and pathological information , where majority of the patients did not receive bcg induction and/or bcg maintenance therapy , the eortc risk tables tend to overestimate patient 's risk for recurrence and progression after bcg therapy . the spanish club urolgicoespaol de tratamientooncolgico ( cueto ) group developed a scoring model using information from 1062 patients treated with intravesical adjuvant bcg therapy to predict risk of recurrence and progression .", "patients had bcg induction as weekly instillations for 6 weeks and six additional instillations repeated at 2-week intervals as a maintenance therapy . by the end of the study ,", "4.2% of patients received fewer than six instillations , 22.5% of patients received six to nine instillations and 73.3% of patients received more than 10 instillations .", "the risk tables for recurrence and progression were based on the scoring systems derived from seven clinical and pathological factors : \n agegendernumber of tumorsrecurrent tumort categorytumor grade ( who 1973)presence of concurrent cis . \n  tumor grade ( who 1973 ) presence of concurrent cis . based on the cueto risk tables , the probability of recurrence varied from 8.24% to 41.79% at 1 year to 20.98% to 67.61% at 5 years .", "the probability of progression varied from 1.17% to 13.97% at 1 year to 3.76% to 33.57% at 5 years . for their recurrence and progression models , harrell 's concordance indices at 1 year were 0.636 and 0.687 , respectively . using the cueto risk tables ,", "the calculated risk of recurrence were lower than that obtained by the eortc risk tables .", "the lower risks in the cueto tables is likely attributable to using bcg , which is a more effective intravesical therapy .", "although the eortc and cueto risk tables are the two best - established predictive multivariate models for recurrence and progression risk calculation in patients with nmibc , limitations include retrospective analysis , use of the 1987 tnm classification and use of the who 1973 grading system .", "in addition , repeat tur for high - grade cancer and immediate instillation of chemotherapy , such as mitomycin , was not routinely performed after tur ; hence , both models are prone to overestimate the risk of recurrence and progression in patients treated with current standard of care .", "a restaging tur is routinely performed these days in patients with high - grade nmibc , especially for high - grade t1 , as the rate of residual tumor is high and 30% of tumors are upstaged when muscle is absent in the first obtained specimen .", "photodynamic diagnosis with blue light and narrow - band imaging have been shown to reduce residual tumor rates by roughly 20% and to improve recurrence - free survival of nmibc patients compared with white light cystoscopy .", "a meta - analysis of seven randomized trials ( n = 1476 ) confirmed the effectiveness of single immediate intravesical instillation of chemotherapy in treated patients compared with tur alone , reporting a 39% decrease in the odds of recurrence .", "the efficacy of intravesical mitomycin c can be optimized by administering a dose of 40 mg at a concentration of 2 mg / ml in water , complete bladder emptying just before dose administration , fluid restriction and oral bicarbonate to alkalinize the urine .", "this approach improved the recurrence - free rate at 5 years from 24.6% to 41% and increased the interval to tumor recurrence from 11.9 to 29 months . using a multi - institutional cohort of 4689 patients with nmibc , xylinas et al .", "showed that both the eortc and cueto risk tables exhibit a poor discrimination for disease recurrence and progression .", "these models overestimated the risk of disease recurrence and progression in high - risk patients .", "additional factors not included in the eortc or the cueto models could be added to new prognostic models to enhance their usefulness .", "bladder neck involvement , prostatic urethra involvement , lymphovascular invasion and depth of lamina propria invasion , i.e. , whether the tumor is superficial to , into or beyond the muscularis mucosae ( t1a , t1b , t1c ) , have been identified as risk factors for progression in patients with nmibc . in the prognostic factor meta - analysis of 33 studies of high - grade t1 bladder cancer patients (", "n = 8880 ) , the highest impact risk factor for progression and cancer - specific survival was depth of invasion ( t1b / c ) into the lamina propria .", "in this meta - analysis , several other previously proposed factors , i.e. lymphovascular invasion , associated cis , non - use of bcg , tumor size > 3 cm , gender , multiple tumors and older age , also predicted progression .", "nmibc patients with progression to muscle - invasive disease tend to have worse prognosis compared with patients with primary muscle - invasive disease , and it underscores the need to significantly improve risk stratification and earlier definitive treatment for high - risk nmibc . risk groupings and risk tables provide average predictions , which may or may not apply to the patient interested in knowing individualized risk .", "nomograms have been proposed as a method that avoids the arbitrary division of patients into risk groups .", "developed nomograms to estimate the risk of disease recurrence and progression in patients with nmibc using a large international cohort .", "nomograms based on age , gender , urine cytology and dichotomized nmp22 had accuracy of 0.811 for recurrence of any bladder cancer ; 0.772 for recurrence of grade 3 ta or t1 or cis and 0.774 for recurrence of stage t2 bladder cancer after bootstrap validation . however , this nomogram had several limitations , including failure to incorporate established predictors , such as pathologic grade and stage , number and pattern of previous recurrences , time since the original diagnosis and prior use of intravesical therapy .", "furthermore , the performance of the nomograms varied significantly among the institutions , emphasizing the need for external validation prior to its clinical use . artificial neural networks ( ann ) are algorithms that can be trained to identify complex patterns between input variables and outcomes in the data sets and then apply these patterns to new cases .", "they have a theoretical advantage over conventional statistics as they are not limited by predefined mathematical relationships between input variables and outcomes ; thus , they are able to model complex non - linear parameters .", "qureshi et al . used ann to predict bladder cancer recurrence and progression within 6 months of diagnosis in a small cohort of about 100 patients with nmibc .", "the accuracy of ann in predicting stage progression and recurrence was 80% and 75% , respectively .", "however , there was no significant difference between the ann and the clinicians predictions of progression and recurrence in the patients with nmibc .", "on restricting the validation subset to patients with t1g3 tumors in relation to stage progression , ann accuracy was better than predictions of clinicians .", "several investigators have compared ann and other machine learning techniques with standard statistical approaches to predict outcomes and did not find improvement in predictive accuracy with ann and other machine learning techniques over standard statistical approaches .", "most of the currently available predictive tools assume that nmibc is pure urothelial carcinoma and does not consider the impact of variant histology on prognosis prediction .", "variant histology such as micropapillary , sarcomatoid and plasmacytoid has been shown to be associated with poor prognosis , and early cystectomy is generally advocated in such cases .", "small cell carcinoma of the bladder is considered a systemic disease and chemotherapy followed by tailored local therapy is recommended for patients with non - muscle invasive small cell carcinoma of the bladder .", "however , spaliviero et al . reported not significantly worse outcomes in conservatively managed t1 micropapillary bladder cancer patients compared with patients treated with early radical cystectomy . given the conflicting findings on the impact of variant histology in smaller studies , the association of variant histology with prognosis deserves further evaluation in larger studies ."], ["for molecular biomarkers to be of clinical use , they should be able to increase the predictive accuracy beyond the standard clinical and pathological parameter models .", "several investigators have attempted to use molecular biomarkers as prognostic factors to predict outcomes in patients with nmibc .", "however , molecular biomarkers have shown mixed results so far and are not sufficiently validated to be used in clinical practice at this time .", "it is becoming clear that superficial low - grade cancers and invasive or high - grade cancers harbor distinctive genetic defects : the low - grade , non - invasive papillary tumors are characterized by activating mutations in the h - ras gene and fibroblast growth factor receptor 3 ( fgfr3 ) gene and the high - grade invasive tumors are characterized by structural and functional defects in the p53 and retinoblastoma protein ( rb ) tumor - suppressor pathways .", "the deletion of both arms of chromosome 9 occurs frequently in bladder cancer during the earliest stages of tumorigenesis .", "however , these chromosomal aberrations do not seem to distinguish between the two tumor development pathways .", "tumor invasion and progression in the bladder seems to be a multifactorial process , promoted by micro - environmental changes that include the up - regulation of n - cadherin , the down - regulation of e - cadherin , the overexpression of matrix metalloproteinases 2 and 9 , an imbalance between angiogenic and anti - angiogenic factors and increased synthesis of prostaglandin . in patients with t1 bladder cancer , nuclear overexpression of p53 protein", "has been reported to have a higher probability of disease progression . in a meta - analysis , p53 was a predictor for recurrence , progression and mortality in patients with bladder cancer .", "however , investigators of this meta - analysis had concerns of overestimating findings because of publication and reporting bias , and suggested that current evidence is not sufficient to conclude whether changes in p53 act as markers of outcome in patients with bladder cancer .", "hernandez et al . , in a prospective cohort of 772 patients with nmibc , showed that fgfr3 mutations are prevalent in low - grade ta and that these mutations are independent predictors of recurrence in patients with low - grade ta tumors .", "van rhijn et al . showed that molecular grading , the grading system based on fgfr3 and mib-1 ( ki-67 ) status , is an independent predictor for recurrence , progression and disease - specific survival .", "van rhijn et al . validated the utility of molecular grading as a prognostic factor to predict outcomes in patients with nmibc .", "the addition of molecular grade to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7% .", "evaluated nmp22 for detecting recurrence and progression in patients with nmibc in a large multi - institutional international cohort .", "there was no clearly defined nmp22 cut - off that could indicate higher risk disease , but there was a continuum of risk for recurrence and progression .", "the international consensus panel on cytology and bladder tumor markers evaluated the prognostic utility of molecular markers for bladder cancer .", "molecular markers were classified into six groups , i.e. microsatellite - associated markers ( e.g. fish , loh ) , proto - oncogenes / oncogenes ( e.g. her-2/neu , h - ras , bcl-2 , mdm-2 , fgfr-3 , c - myc ) tumor suppressor genes ( e.g. , p53 , rb ) , cell cycle regulators ( e.g. , p21 , p27 , ki-67 , cyclin - d1 , cyclin - e ) , angiogenesis - related factors ( vegf , cox-2 , tsp-1 ) and extracellular matrix adhesion molecules ( e.g. e - cadherin , mmps , timps , cd44 , u - pa ) . the panel concluded that although certain biomarkers , such as ki-67 and p53 , appear to be promising in predicting recurrence and progression in patients with bladder cancer , the data are still heterogeneous .", "the panel recommended that identifying definitive criteria for test positivity , a clearly defined patient population , standardization of techniques used to evaluate markers and clearly specified endpoints and statistical methods will help to bring accurate independent prognostic indicators into the clinical management of patients with bladder cancer ."], ["nmibc comprises of a heterogeneous group of patients and includes pathological stage ta , t1 and cis .", "patients with low - grade ta disease have very low risk of progression while patients with t1 disease with concurrent cis have a much higher risk of progression , approaching 50% .", "the eortc and cueto risk tables are the two best - established predictive models for recurrence and progression risk calculation in patients with nmibc . however", ", both the eortc and the cueto risk tables exhibit a poor discrimination for prognostic outcomes and overestimate the risk of disease recurrence and progression in high - risk patients in external validation .", "additional prognostic factors such as depth of lamina propria invasion should be added to new prognostic models to enhance their usefulness .", "molecular biomarkers such as ki-67 , fgfr3 and p53 appear to be promising in predicting recurrence and progression , but need further validation prior to using them in clinical practice .", "future research should focus to enhance the predictive accuracy of the risk assessment tools by incorporating additional prognostic factors such as depth of lamina propria invasion and molecular biomarkers after rigorous validation in multi - institutional cohorts ."], [""], [""], [""]]}
{"article_id": "PMC3834650", "article_text": ["personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies in order to achieve the best medical outcomes for individual patients , thus improving public health . despite the variety of clinical , morphological , and molecular parameters", "used to classify human malignancies , patients receiving the same diagnosis can have markedly different clinical courses and treatment responses . since there is no simple way to determine who will have an adverse reaction ,", "the current system of  one - size - fits - all-  diagnoses is simply not good enough .  ", "an increasing number of studies have demonstrated sex differences in drug reactions to the same drug treatment .", "migeon   implied that males and females responded differently to drug treatments and that sex plays a key role in cancer .", "in addition , females are historically less studied subjects due to the complication of estrous cycle , and therefore such studies would further benefit women 's health and promote public health .", "recent advancements in biotechnology have accelerated the search for molecular biomarkers useful in the diagnosis and treatment of disease .", "molecular biomarkers of disease risk and status are critical to an accurate treatment by identifying patients most likely to benefit from particular drugs or experience adverse reactions . because medicine is always practiced on individuals rather than populations , the goal is to change the assignment of therapies from a population - based approach to an individualized approach .", "gene - expression data can be used to identify patients with a good disease prognosis , thereby preventing some patients from unnecessary therapies and toxicity .", "for example , gene - expression profiling was used to predict clinical outcomes in pediatric patients with acute myeloid leukemia and to find genes whose aberrant expression leads to a poor prognosis .", "thus , accurate classification of prognosis of patients leads to efficient cancer treatment and prolonged survival of patients .", "classification algorithms are needed for biomedical decision making in clinical assignment of patients to treatment therapies based on individual risk factors and disease characteristics . since many of those genes are not relevant , feature selection is a commonly addressed problem in classification [ 3 , 4 ] .", "the goal of gene selection is to identify a set of genes which plays an important role in the classification .", "a common approach is to select a fixed number of the highest ranked genes based on t - test - like statistics , some discrimination measures [ 6 , 7 ] , or classification algorithms including support vector machines ( svm ) [ 8 , 9 ] and random forest ( rf ) [ 10 , 11 ] .  ", "development of a biomarker classifier involves two distinct components : ( 1 ) a procedure for building a classifier and ( 2 ) validation / evaluation procedure for estimating the error rate of biomarker .", "the most important consideration is the validation / evaluation of a biomarker classifier to assess whether it can accurately and unbiasedly predict new samples based on a set of selected features .", "two methods are commonly used to develop and assess performance of a classifier : the split - sample procedure and cross - validation procedure . in the split - sample procedure , the sample dataset", "is randomly split into two subsets : a training set for model building and a test set for performance assessment .", "that is , the training dataset is used for building the biomarker classifiers and the test set is used for evaluating the classifier .", "however , samples are often insufficient to split into two sample sets of approximately equal size for model building and model testing , and a cross - validation ( cv ) procedure is commonly used for performance assessment [ 12 , 13 ]", ". a cv can be regarded as a generalization of the split - sample method .", "it involves repeatedly splitting the data into a training set containing most of the samples and applying the prediction rule to the test set of the remaining samples to estimate the prediction accuracy rate .", "the prediction accuracy is the average accuracy of the numerous training - test partitions .   for model building ,", "given that differences in the biology of lung cancer and other diseases exist between men and women [ 14 , 15 ] , investigations to identify genomic biomarkers for clinical assignment of therapies on an individual patient basis are crucial . in this paper , the ratio of between - group to within - group sums of squares ( bw ratio , ) gene selection algorithm is used to obtain a feasible set of influential genes via variable importance ranking procedure   in the training phases of 20 trials of 10-fold cv within leave - one - out cross - validation ( loocv ) procedure as illustrated below .", "the genes with largest bw ratios are ranked high as significant genes .   in the development of biomarker classifier", ", the predictive accuracy of the classifier must be evaluated on a separate set of data . to derive an unbiased accuracy estimate , a nested cross - validation procedure , 20 trials of 10-fold cv within loocv ,", "is used in this paper . in other words , in each loocv , 90% of the", "wherefore genes are selected only using learning data sets of size ( n    1 ) each , and in the evaluation / validation step a never - touched and left - out single observation in loocv is used to assess the predictive performance of selected genes . in this way , each test case is never used for gene selection . to avoid a bias due to partitions of data ,", "if the performance is assessed using the very same data that are used for developing the classifier , this obviously leads to a biased down estimate of classification error . in other words , if one applies a method to the original data with 20 trials of 10-fold cv only as a way of building the classifier and selects a set of genes and then on that very same data calculates the classification error , it clearly leads to a biased upward estimate of classification accuracy .", "three publicly available data sets of interest in this paper are pediatric acute myeloid leukemia ( aml ) , b - cell chronic lymphocytic leukemia ( b - cll ) , and primary cutaneous melanoma .", "the data sets are downloaded from the brb - arraytools data archive for human cancer gene expression located at the website http://linus.nci.nih.gov/~brb/dataarchive_new.html .", "sex differences in disease rates or in rates of adverse reactions to treatment are common , which we intend to exploit to obtain sex - specific biomarkers from gene - expression data .", "we hypothesize that genomic biomarkers developed from the sex - specific application of classification algorithms will further improve class prediction accuracy .  ", "the summary of our algorithm to find sex - specific predictive / prognostic genomic biomarkers for efficacy or toxicity in individualized treatment of patients for serious diseases is as follows . in each loocv trial , firstly , the data is partitioned into a test data set with one observation and the remaining data as the learning data set .", "the learning data set is further separated into male and female patients ' learning data sets .", "thus , this process will be applied n times , where n represents the total number of patients .", "secondly , within each trial of loocv , twenty trials of 10-fold cv are conducted for each set of male and female patients in the learning data set . at each 10-fold cv step ,", "two sets of top - ranked genes , one for male patients ( smi ) and one for female patients ( sfi ) , are obtained via the bw ratio to differentiate types of patients such as diseased versus nondiseased .", "this can be done by applying variable importance ranking approach in order to extract most influential genes and by combining a measure of importance in each gene .", "a score taking the average of the measure in cross - validation ( cv ) is prioritized in the list of genes according to variable importance .", "thirdly , the mutually exclusive sets of male - specific genes and female - specific genes are obtained .", "fourthly , within each loocv trial , after the sets of potential sex - specific genes are determined in the second and the third steps , they are fitted to a model with diagonal linear discriminant analysis ( dlda ; ) using each learning data set for male and female patients .", "finally , the performance is measured with the test data with one observation in each loocv trial .", "this method is implemented in r. the r code is available upon request . within each trial of loocv , each set of top - ranked genes for male and female patients is obtained in the second and the third steps as follows .", "first , in order to build genomic biomarkers , 20 trials of 10-fold cv are applied to each learning data set for males and females , where 90% were randomly selected without replacement as a set for each trial of cv .", "next , for each trial of 10-fold cv , the bw ratio was applied to this 90 percent learning set and the top 25 ranked genes were selected in each process with the target endpoint of a dataset .", "the bw ratio for a gene j is defined as \n ( 1)bwj=iki(yi = k)(xkjxj)2iki(yi = k)(xijxkj)2 , \n where x-j=ixij / n indicates the average value of a gene j across all the training samples , x - kj=i(yi = k)xij / nk indicates the average value of a gene j for a class k , and i indicates an observation . here", "this criterion has been shown to be reliable for variable selection from high - dimensional data sets [ 6 , 12 ] .  ", "next , to avoid selection bias from a pattern of selection of learning samples , we repeat the entire process 20 times by shuffling samples at every 10-fold cv . in order to obtain the variable importance ranking , 200 sets of top 25 ranked genes were combined so that the maximum possible rank score of a gene would be 200 .", "a set of genes that has been selected at least once ( rank score > 0 ) was obtained separately for males and females .", "these most influential genes used in the classification process are identified in order to extract a feasible set of sex - specific genes . for both male and female learning data sets ,", "the final product of the 20 trials of 10-fold cv described in each loocv is the sets of genes selected for male and female patients in the learning data set smj = { g1 ,  , gxmj } and sfj = { g1 ,  , gxfj } , j = 1 , 2 ,  , n , respectively .", "the sets smj and sfj contain prognostic / predictive genes for male and female patients , respectively", ". there would be n sets of selected genes . within each trial of loocv , genes that are commonly identified between male - specific and female - specific genes", "finally , the test sample with one observation is tested using these sex - specific genes in each loocv . at the end of the entire loocv", ", each selected gene has a combined variable importance score that is less than or equal to n.   to verify sex - specific biomarkers , we consider the following four different cases : we classify the outcome of ( 1 ) male patients with a set of male - specific genes smj,(2 ) female patients with a set of male - specific genes smj,(3 ) male patients with a set of female - specific genes sfj , and ( 4 ) female patients with a set of female - specific genes sfj .", "we anticipate that data with a set of male - specific genes have higher predictable power to predict male patients than the data with female - specific genes .", "similarly , data with female - specific genes have higher predictable power to predict female patients than the data with male - specific genes . upon completion of loocv", ", the performance of sex - specific biomarkers is obtained . in order to validly evaluate the performance of a gene set selected by the proposed method ,", "cv utilizes resampling without replacement of the entire data set to repeatedly develop classifiers on a training set and to evaluate these classifiers on a separate test set and then averages the results over the resamples .", "in this section , we apply the proposed algorithm to the following genomic data sets to find the most meaningful sex - specific predictive / prognostic genomic biomarkers for improving individualized treatment of patients and for evaluating the biomarkers from the proposed algorithm . current chemotherapy enables a high percentage of pediatric patients with aml to enter complete remission ( cr ) , but a large number of them experience relapse ( r ) with resistant disease . because of the wide heterogeneity of aml , predicting a patient 's risk for treatment failure or relapse at the time of diagnosis is critical for the optimal treatment .   this gene - expression data set consists of 54 aml pediatric patients ( < 15 years old ) with an oligonucleotide microarray containing 12,566 probe sets and it is also available at ftp://ftp.ncbi.nih.gov/pub/geo/data/soft/gds/gds1059.soft.gz .", "patients with cr for more than 3 years are classified as having a good prognosis , while patients experienced relapse within 1 year of the first cr are considered as having a poor prognosis . in this data set , there are 28 patients with cr and 25 patients with r. since a five - month male patient experienced induction failure ( no cr achievement ) within 3 months of the start of treatment is considered neither cr nor r , we exclude this subject . therefore , there are 32 male patients and 21 female patients in this data set .   with the prognostic endpoint ( r / cr ) ,", "the average accuracy of 66% ( sd 3.0% ) for pediatric patient classification was obtained when no gene selection was introduced . when a set of 200 genes was selected in a learning phase of each cv ,", "the average accuracy of 68.0% ( sd 3.0% ) was achieved . when a set of 20 genes was selected in a learning phase of each cv ,", "the average accuracy of 71.0% ( sd 5.0% ) was obtained . since it appeared to be no substantial difference between accuracy and the number of genes selected , a feasible set of 25 genes in the learning phase of each cv", "was collectively ranked by the bw ratio to find sex - specific biomarkers . at the end of loocv trials , a set of male - specific genes that were selected at least 75% of the time in the entire loocv were 1882_g_at ( mecom : mds1 and evi1 complex locus ) , 37902_at ( cryz : crystallin , zeta ( quinone reductase ) ) , 38789_at ( tkt : transketolase ( wernicke - korsakoff syndrome ) ) , 39105_at ( vasp : vasodilator - stimulated phosphoprotein ) , 40844_at ( ctr9 : ctr9 , paf1/rna polymerase ii complex component , homolog ( s. cerevisiae ) ) , 36981_at ( srp9 : signal recognition particle 9  kda ) , 36338_at ( luzp1 : leucine zipper protein 1 ) , 31870_at ( cd37 : cd37 antigen ) , 1624_at ( rap1gds1 : rap1 , gtp - gdp dissociation stimulator 1 ) , and 39142_at ( nudt21 : cleavage and polyadenylation specific factor 5 , 25  kda ) .", "these ten top - ranked male - specific genes were selected as potential male - specific genomic biomarkers to classify male patients into r / cr . among them", "38789_at ( tkt : transketolase ) and 36338_at ( est ) were also included in the thirty - five genes associated with prognosis of pediatric aml identified by yagi et al . .", "in order to select a feasible set of sex - specific biomarkers a cut - off criterion of 75 percent is used . since", "every dataset may have a different sample size , the number of genes selected is given by the percentage , which is a rank score of the selected genes greater than 75% of the sample size in our case .", "for example , if a sample size is 100 , then genes that have rank scores greater than 75 have been selected .", "similarly , nine top - ranked genes for classifying female patients into r / cr were 40601_at ( tm2d1 : tm2 domain containing 1 ) , 36330_at ( ccbl1 : cysteine conjugate beta- lyase ; cytoplasmic ( glutamine transaminase k , kynurenine aminotransferase ) ) , 40586_at ( eef1e1 : eukaryotic translation elongation factor 1 epsilon 1 ) , 36648_at ( crsp9 : cofactor required for sp1 transcriptional activation , subunit 9 , 33  kda ) , 32351_at ( gpr20 : g protein - coupled receptor 20 ) , 1718_at ( arpc2 : actin - related protein 2/3 complex , subunit 2 , 34  kda ) , 38622_at ( mtg1 : mitochondrial gtpase 1 homolog ( s. cerevisiae ) ) , 36496_at ( impa2 : inositol(myo)-1(or 4)-monophosphatase 2 ) , and 38337_at ( znf193 : zinc finger protein 193 ) .", "these nine top - ranked genes which were selected at least 75% of the time in the entire loocv were considered as potential female - specific genomic biomarkers to classify female patients into r / cr .", "data with male - specific genes showed higher prediction accuracy ( 71.9% ) to classify male patients than the accuracy ( 43.8% ) to classify male patients from data with female - specific genes .", "similarly , data with female - specific genes showed higher prediction accuracy ( 76.2% ) to classify female patients than the accuracy ( 61.9% ) to classify female patients from data with male - specific genes .", "as shown in table 1 , sensitivity and specificity were also higher in using sex - specific genes for each sex .", "genomic aberrations and mutational status of the immunoglobulin variable heavy chain ( vh ) gene have been shown to be among the most important predictors for outcome in patients with b - cll .", "b - cll is the most common leukemia in the western world , and due to its clinical heterogeneity ( wide range of life expectancy ) and correlations to genomic aberrations , it is important to predict a patient 's vh mutation status at the time of diagnosis for optimal treatment .", "in addition , the study presented by haslinger et al .   suggested that the genomic signature for vh mutational status might be sex related .  ", "the gene - expression data consists of 100 b - cll patients with an oligonucleotide microarray containing around 12,000 probe sets , and it is available at http://linus.nci.nih.gov/~brb/dataarchive_new.html .", "patients are classified as either vh - mutated or unmutated ( m / nm ) .", "there are 62 males ( 33  m and 29  nm ) and 38 females ( 18  m and 20  nm ) , with a total of 51 mutated and 49 unmutated patients .   in step 1 , for each data set with male only and female only patients using the target endpoint ( i.e. , vh - mutated ( m ) versus unmutated ( nm ) ) , we separately selected and ranked 25 potential prognostic genes for males and for females in every cv trial and separately combined ranks of these genes for males and females during the learning phase of 20 trials of 10-fold cv within each loocv trial . in every loocv trial , we prioritized and combined the final top - ranked 200 genes from the male patients result ( sm ) and 200 genes from the female patients result ( sf ) as explained in section 3.1 . at the end of the entire loocv trials , a set of potential sex - specific genes", "are obtained for each sex by prioritizing and combining n sets of top - ranked 200 genes .  ", "after deletion of overlapped genes in both males and females , eleven potential male - specific genes were obtained to classify male patients into m / nm . they were 41209_at ( lpl : lipoprotein lipase ) , 41755_at ( cobll1 : cobl - like 1 ) , 39878_at ( pcdh9 : protocadherin 9 ) , 38211_at ( zbtb20 : zinc finger and btb domain containing 20 ) , 39488_at ( pcdh9 : protocadherin 9 ) , 36886_f_at ( kir2dl3 : killer cell immunoglobulin - like receptor , two domains , long cytoplasmic tail , 3 ) , 32140_at ( sorl1 : sortilin - related receptor , l(dlr class ) a repeats - containing ) , 33535_at ( p2rx1 : purinergic receptor p2x , ligand - gated ion channel , 1 ) , 39967_at ( ldoc1 : leucine zipper , downregulated in cancer 1 ) , 32842_at ( bcl7a : b - cell cll / lymphoma 7a ) , and 36899_at ( satb1 : special at - rich sequence binding protein 1 ( binds to nuclear matrix / scaffold - associating dna 's ) ) . for female patients ,", "only five genes were selected at least 75% of the time in the entire loocv trial as potential female - specific genomic biomarkers to classify female patients into m / nm . they were 33745_at ( phkg2 : phosphorylase kinase , gamma 2 ( testis ) ) , 38152_at ( loh11cr2a : loss of heterozygosity , 11 , chromosomal region 2 , gene", "a ) , 34142_at ( pde8a : phosphodiesterase 8a ) , 39593_at ( fgl2 : fibrinogen - like 2 ) , and 217_at ( klk2 : kallikrein 2 , prostatic ) .   to verify the sex - specific genomic biomarkers , we considered the performance of four different cases in the loocv trials as described in section 2 .", "data with male - specific genomic biomarkers showed higher accuracy ( 67.7% ) to classify male patients into m / nm than the accuracy to classify male patients into m / nm from data with female - specific genomic biomarkers ( 40.3% ) .", "however , female data with female - specific genes showed prediction accuracy similar to random guess close to 50% .", "therefore , there is insufficient evidence to support that the selected female - specific genes were female - specific genes ( see table 2 ) with 38 female patients in this data set .  ", "although cutaneous melanoma represents a small subset , it is the most life - threatening neoplasm of the skin , and its incidence and mortality have been increasing worldwide .", "the key underlying molecular events have not been clearly elucidated , which may explain why no target has been developed and why almost no clinical benefits from new therapies have been clearly demonstrated in patients with melanoma since the late 1970s .", "additionally , gene - expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors .", "this gene - expression data set was collected from 83 patients corresponding to the training data set and 17 patients corresponding to the validation data set .", "the probes are from tumor tissue and from reference tissue that are differentially labeled by the incorporation of cyanine 3 ( cy3 ) and cyanine 5 ( cy5 ) , respectively .", "set , the endpoint was patient prognosis and survival along with tumor stages , defined as follows . in stage", "i , cure rates are excellent with surgical removal , since they are the least likely to spread . in stage ii , melanomas can be cured , but the success rate lags behind that of stage i because a small number of cancer cells may have spread to distant sites . in stage iii , since the tumor has started to metastasize ( the spreading of a disease from one organ or part to another nonadjacent organ or part ) , the survival rate for these stages is lower than the earlier ones .", "stage iv is associated with metastasis beyond the regional lymph nodes to distant sites in the body , such as the lung , liver , or brain , or to distant areas of the skin .", "based on the tumor size , descriptions , and number of lymph nodes the stages are categorized in two classes .", "a class of high survival and small tumor size ( hs / st ) is defined and composed of stages 1 and 2 .", "the second is defined as low survival and non - small tumor size ( ls / nst ) , which is composed of stages 3 and 4 .  ", "tables 3 and 4 show the distribution of the primary cutaneous melanoma data based on sex , the defined clinical endpoint hs / st and ls / nst for the training data and validation data . among 83 patients in the training dataset ,", "there are 27 males ( 12 hs / st and 15 ls / nst ) and 56 females ( 30 hs / st and 26 ls / nst ) .", "there are 42 cases of hs / st and 41 cases of ls / nst in total .", "after preprocessing the data the final gene count is 4641 genes . among 17 patients in the validation data", "set , there are 8 males ( 1 hs / st and 7 ls / nst ) and 9 females ( 1 hs / st and 8 ls / nst )", ". there are 2 cases of hs / st and 15 cases of ls / nst in total .  ", "since the validation set was separately provided , the sex - specific genes were selected via 20 trials of 10-fold cv in the learning set .", "the following ten male - specific genes were selected at least 75% of the time during 20 trials of 10-fold cv : a_23_p128263 ( prb1 : proline - rich protein bstni subfamily 1 ) , a_23_p83838 ( ca8 : carbonic anhydrase viii ) , a_24_p212990 ( mgc70863 : similar to rpl23ap7 protein ) , a_23_p333650 ( rad9b : rad9 homolog b ( s. cerevisiae ) ) , a_32_p125251 ( n / a ) , a_23_p108835 ( ypel5 : yippee - like 5 ( drosophila ) ) , a_32_p150856 ( loc407835 : mitogen - activated protein kinase kinase 2 pseudogene ) , a_23_p11936 ( ubxn11 : ubx domain protein 11 ) , a_24_p169976 ( n / a ) , and a_32_p3998 ( znf600 : zinc finger protein 600 ) . for female patients ,", "the following eight female - specific genes were selected from 20 trials of 10-fold cv : a_23_p69497 ( clec3b : c - type lectin domain family 3 , member b ) , a_24_p118884 ( n / a ) , a_23_p152420 ( kiaa0182 ) , a_24_p265177 ( phc3 : polyhomeotic - like 3 ( drosophila ) ) , a_23_p251421 ( cdca7 : cell division cycle associated 7 ) , a_23_p211738 ( ubp1 : upstream binding protein 1 ( lbp-1a ) ) , a_23_p47377 ( hsd17b12 : hydroxysteroid ( 17-beta ) dehydrogenase 12 ) , and a_32_p113646 ( cdna flj45341 fis , clone brhip3009672 ) . using a given validation set ,", "the sex - specific genes were verified . as shown in the confusion matrix in table 5 , there was no difference in the performance from female data with female genes compared with the performance of female data with male genes .", "however , there was only one misclassification for male patients with male genes as shown in table 6 , while there were four misclassifications for male patients with female genes as shown in table 7 .", "therefore , we conclude that there exists evidence of male - specific genes in a classification of primary cutaneous melanoma .", "large inter individual differences in benefit from chemotherapy highlight the need to develop predictive genomic biomarkers for selecting the right treatment for the right patient .", "inappropriate chemotherapy can result in the selection of more resistant and aggressive tumor cells . to date", ", no reliable genomic biomarkers have been developed to provide the physician with prechemotherapy information to accurately predict the efficacy of a specific therapy .", "we proposed a procedure to find sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm using variable importance ranking via combination of 20 trials of 10-fold cv and loocv .", "the proposed procedure was applied to data sets obtained from the brb arraytools data human cancer archive .", "however , the issue arose out of there being not enough samples , and the data was unbalanced ( i.e. , more males than females or more positives ( disease patients ) than negatives ( nondisease patients ) ) . while patient 's sex information was not specified for many of the publicly available data sets adding to that the difficulty of searching for data , we found the following three genomic data sets that had sex information : ( 1 ) pediatric patients with aml , ( 2 ) b - cell chronic lymphocytic leukemia ( b - cll ) , and ( 3 ) primary cutaneous melanoma . in one application , pediatric patients with aml were classified by the algorithms as having either a good or poor prognosis , in terms of the likelihood of induction failure or relapse within one year of the first complete remission , based on gene - expression profiles .", "if this were brought into clinical application , a patient with a confidently predicted good prognosis might want to elect out of adjuvant chemotherapy and its associated debilitating side effects . with current rule - based decisions ,", "the overall average accuracy of this data set with a variable selection from pooled patients ( males and females ) was about 71.0% .", "however , using male - specific genes found by the proposed procedure , the accuracy was improved to about 72% as we found in the model validation studies . similarly , using female - specific genes found by the proposed procedure ,", "the average accuracy was improved to about 76% ( see table 1 ) .    in the b - cell chronic lymphocytic leukemia ( b - cll )", "dataset we found male - specific prognostic genomic biomarkers associated with b - cell chronic lymphocytic leukemia and its average classification accuracy was improved to about 68% .", "there was no substantial evidence to find female - specific prognostic genomic biomarkers in this data set .", "similarly , male - specific predictive genomic biomarkers associated with a classification of primary cutaneous melanoma were found with the classification accuracy of about 88% .  ", "the scope of our paper was to find sex - specific genomic biomarkers , if any , imbedded in the data instead of finding genomic biomarkers from the data .", "if commonly identified genes were kept in the proposed procedure , our procedure was not sex - specific genomic biomarker classifier involving two populations ( males and females ) any more but rather it became genomic biomarker classifier involving one combined population .", "in fact , even though commonly identified genes were kept , it did not necessarily improve the classification accuracy .", "for a counterexample , for the pediatric aml data of yagi et al . , the accuracy for female patients with female - specific genes by keeping the commonly identified genes was 62% ; however , the accuracy without commonly identified genes was 76% .", "for the male patients with male - specific genes by keeping the commonly identified genes the accuracy was 59% , but the accuracy without the commonly identified genes was 72% .", "for the related note , the accuracy of this data can be found anywhere between 59% and 66% with gene preprocessing and between 58% and 71% with gene selection .", "it is not an easy task to find sex - specific genes , let alone verifying and proving that they are indeed sexspecific .", "we have presented a procedure for finding sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm .", "the procedure is shown to have good  sensitivity  and  specificity  in the sense that the sex - specific genes obtained can improve prediction accuracy in classification of individual patient 's prognosis .", "the proposed procedure to discover predictive and prognostic sex - specific genomic biomarkers for individualized treatment of diseases can play a critical role in developing safer and more effective therapies that replace one - size - fits - all drugs with treatments that focus on specific patient needs ."], "abstract_text": ["<S> numerous studies have demonstrated sex differences in drug reactions to the same drug treatment , steering away from the traditional view of one - size - fits - all medicine . </S>", "<S> a premise of this study is that the sex of a patient influences difference in disease characteristics and risk factors . in this study , we intend to exploit and to obtain better sex - specific biomarkers from gene - expression data . </S>", "<S> we propose a procedure to isolate a set of important genes as sex - specific genomic biomarkers , which may enable more effective patient treatment . </S>", "<S> a set of sex - specific genes is obtained by a variable importance ranking using a combination of cross - validation methods . </S>", "<S> the proposed procedure is applied to three gene - expression datasets . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Method for Identifying Sex-Specific Predictive/Prognostic Genomic Biomarkers", "3. Results: Identification and Evaluation of Predictive/Prognostic Genomic Biomarkers", "4. Discussion and Conclusions"], "sections": [["personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies in order to achieve the best medical outcomes for individual patients , thus improving public health . despite the variety of clinical , morphological , and molecular parameters", "used to classify human malignancies , patients receiving the same diagnosis can have markedly different clinical courses and treatment responses . since there is no simple way to determine who will have an adverse reaction ,", "the current system of  one - size - fits - all-  diagnoses is simply not good enough .  ", "an increasing number of studies have demonstrated sex differences in drug reactions to the same drug treatment .", "migeon   implied that males and females responded differently to drug treatments and that sex plays a key role in cancer .", "in addition , females are historically less studied subjects due to the complication of estrous cycle , and therefore such studies would further benefit women 's health and promote public health .", "recent advancements in biotechnology have accelerated the search for molecular biomarkers useful in the diagnosis and treatment of disease .", "molecular biomarkers of disease risk and status are critical to an accurate treatment by identifying patients most likely to benefit from particular drugs or experience adverse reactions . because medicine is always practiced on individuals rather than populations , the goal is to change the assignment of therapies from a population - based approach to an individualized approach .", "gene - expression data can be used to identify patients with a good disease prognosis , thereby preventing some patients from unnecessary therapies and toxicity .", "for example , gene - expression profiling was used to predict clinical outcomes in pediatric patients with acute myeloid leukemia and to find genes whose aberrant expression leads to a poor prognosis .", "thus , accurate classification of prognosis of patients leads to efficient cancer treatment and prolonged survival of patients .", "classification algorithms are needed for biomedical decision making in clinical assignment of patients to treatment therapies based on individual risk factors and disease characteristics . since many of those genes are not relevant , feature selection is a commonly addressed problem in classification [ 3 , 4 ] .", "the goal of gene selection is to identify a set of genes which plays an important role in the classification .", "a common approach is to select a fixed number of the highest ranked genes based on t - test - like statistics , some discrimination measures [ 6 , 7 ] , or classification algorithms including support vector machines ( svm ) [ 8 , 9 ] and random forest ( rf ) [ 10 , 11 ] .  ", "development of a biomarker classifier involves two distinct components : ( 1 ) a procedure for building a classifier and ( 2 ) validation / evaluation procedure for estimating the error rate of biomarker .", "the most important consideration is the validation / evaluation of a biomarker classifier to assess whether it can accurately and unbiasedly predict new samples based on a set of selected features .", "two methods are commonly used to develop and assess performance of a classifier : the split - sample procedure and cross - validation procedure . in the split - sample procedure , the sample dataset", "is randomly split into two subsets : a training set for model building and a test set for performance assessment .", "that is , the training dataset is used for building the biomarker classifiers and the test set is used for evaluating the classifier .", "however , samples are often insufficient to split into two sample sets of approximately equal size for model building and model testing , and a cross - validation ( cv ) procedure is commonly used for performance assessment [ 12 , 13 ]", ". a cv can be regarded as a generalization of the split - sample method .", "it involves repeatedly splitting the data into a training set containing most of the samples and applying the prediction rule to the test set of the remaining samples to estimate the prediction accuracy rate .", "the prediction accuracy is the average accuracy of the numerous training - test partitions .   for model building ,", "given that differences in the biology of lung cancer and other diseases exist between men and women [ 14 , 15 ] , investigations to identify genomic biomarkers for clinical assignment of therapies on an individual patient basis are crucial . in this paper , the ratio of between - group to within - group sums of squares ( bw ratio , ) gene selection algorithm is used to obtain a feasible set of influential genes via variable importance ranking procedure   in the training phases of 20 trials of 10-fold cv within leave - one - out cross - validation ( loocv ) procedure as illustrated below .", "the genes with largest bw ratios are ranked high as significant genes .   in the development of biomarker classifier", ", the predictive accuracy of the classifier must be evaluated on a separate set of data . to derive an unbiased accuracy estimate , a nested cross - validation procedure , 20 trials of 10-fold cv within loocv ,", "is used in this paper . in other words , in each loocv , 90% of the", "wherefore genes are selected only using learning data sets of size ( n    1 ) each , and in the evaluation / validation step a never - touched and left - out single observation in loocv is used to assess the predictive performance of selected genes . in this way , each test case is never used for gene selection . to avoid a bias due to partitions of data ,", "if the performance is assessed using the very same data that are used for developing the classifier , this obviously leads to a biased down estimate of classification error . in other words , if one applies a method to the original data with 20 trials of 10-fold cv only as a way of building the classifier and selects a set of genes and then on that very same data calculates the classification error , it clearly leads to a biased upward estimate of classification accuracy .", "three publicly available data sets of interest in this paper are pediatric acute myeloid leukemia ( aml ) , b - cell chronic lymphocytic leukemia ( b - cll ) , and primary cutaneous melanoma .", "the data sets are downloaded from the brb - arraytools data archive for human cancer gene expression located at the website http://linus.nci.nih.gov/~brb/dataarchive_new.html ."], ["sex differences in disease rates or in rates of adverse reactions to treatment are common , which we intend to exploit to obtain sex - specific biomarkers from gene - expression data .", "we hypothesize that genomic biomarkers developed from the sex - specific application of classification algorithms will further improve class prediction accuracy .  ", "the summary of our algorithm to find sex - specific predictive / prognostic genomic biomarkers for efficacy or toxicity in individualized treatment of patients for serious diseases is as follows . in each loocv trial , firstly , the data is partitioned into a test data set with one observation and the remaining data as the learning data set .", "the learning data set is further separated into male and female patients ' learning data sets .", "thus , this process will be applied n times , where n represents the total number of patients .", "secondly , within each trial of loocv , twenty trials of 10-fold cv are conducted for each set of male and female patients in the learning data set . at each 10-fold cv step ,", "two sets of top - ranked genes , one for male patients ( smi ) and one for female patients ( sfi ) , are obtained via the bw ratio to differentiate types of patients such as diseased versus nondiseased .", "this can be done by applying variable importance ranking approach in order to extract most influential genes and by combining a measure of importance in each gene .", "a score taking the average of the measure in cross - validation ( cv ) is prioritized in the list of genes according to variable importance .", "thirdly , the mutually exclusive sets of male - specific genes and female - specific genes are obtained .", "fourthly , within each loocv trial , after the sets of potential sex - specific genes are determined in the second and the third steps , they are fitted to a model with diagonal linear discriminant analysis ( dlda ; ) using each learning data set for male and female patients .", "finally , the performance is measured with the test data with one observation in each loocv trial .", "this method is implemented in r. the r code is available upon request . within each trial of loocv , each set of top - ranked genes for male and female patients is obtained in the second and the third steps as follows .", "first , in order to build genomic biomarkers , 20 trials of 10-fold cv are applied to each learning data set for males and females , where 90% were randomly selected without replacement as a set for each trial of cv .", "next , for each trial of 10-fold cv , the bw ratio was applied to this 90 percent learning set and the top 25 ranked genes were selected in each process with the target endpoint of a dataset .", "the bw ratio for a gene j is defined as \n ( 1)bwj=iki(yi = k)(xkjxj)2iki(yi = k)(xijxkj)2 , \n where x-j=ixij / n indicates the average value of a gene j across all the training samples , x - kj=i(yi = k)xij / nk indicates the average value of a gene j for a class k , and i indicates an observation . here", "this criterion has been shown to be reliable for variable selection from high - dimensional data sets [ 6 , 12 ] .  ", "next , to avoid selection bias from a pattern of selection of learning samples , we repeat the entire process 20 times by shuffling samples at every 10-fold cv . in order to obtain the variable importance ranking , 200 sets of top 25 ranked genes were combined so that the maximum possible rank score of a gene would be 200 .", "a set of genes that has been selected at least once ( rank score > 0 ) was obtained separately for males and females .", "these most influential genes used in the classification process are identified in order to extract a feasible set of sex - specific genes . for both male and female learning data sets ,", "the final product of the 20 trials of 10-fold cv described in each loocv is the sets of genes selected for male and female patients in the learning data set smj = { g1 ,  , gxmj } and sfj = { g1 ,  , gxfj } , j = 1 , 2 ,  , n , respectively .", "the sets smj and sfj contain prognostic / predictive genes for male and female patients , respectively", ". there would be n sets of selected genes . within each trial of loocv , genes that are commonly identified between male - specific and female - specific genes", "finally , the test sample with one observation is tested using these sex - specific genes in each loocv . at the end of the entire loocv", ", each selected gene has a combined variable importance score that is less than or equal to n.   to verify sex - specific biomarkers , we consider the following four different cases : we classify the outcome of ( 1 ) male patients with a set of male - specific genes smj,(2 ) female patients with a set of male - specific genes smj,(3 ) male patients with a set of female - specific genes sfj , and ( 4 ) female patients with a set of female - specific genes sfj .", "we anticipate that data with a set of male - specific genes have higher predictable power to predict male patients than the data with female - specific genes .", "similarly , data with female - specific genes have higher predictable power to predict female patients than the data with male - specific genes . upon completion of loocv", ", the performance of sex - specific biomarkers is obtained . in order to validly evaluate the performance of a gene set selected by the proposed method ,", "cv utilizes resampling without replacement of the entire data set to repeatedly develop classifiers on a training set and to evaluate these classifiers on a separate test set and then averages the results over the resamples ."], ["in this section , we apply the proposed algorithm to the following genomic data sets to find the most meaningful sex - specific predictive / prognostic genomic biomarkers for improving individualized treatment of patients and for evaluating the biomarkers from the proposed algorithm . current chemotherapy enables a high percentage of pediatric patients with aml to enter complete remission ( cr ) , but a large number of them experience relapse ( r ) with resistant disease . because of the wide heterogeneity of aml , predicting a patient 's risk for treatment failure or relapse at the time of diagnosis is critical for the optimal treatment .   this gene - expression data set consists of 54 aml pediatric patients ( < 15 years old ) with an oligonucleotide microarray containing 12,566 probe sets and it is also available at ftp://ftp.ncbi.nih.gov/pub/geo/data/soft/gds/gds1059.soft.gz .", "patients with cr for more than 3 years are classified as having a good prognosis , while patients experienced relapse within 1 year of the first cr are considered as having a poor prognosis . in this data set , there are 28 patients with cr and 25 patients with r. since a five - month male patient experienced induction failure ( no cr achievement ) within 3 months of the start of treatment is considered neither cr nor r , we exclude this subject . therefore , there are 32 male patients and 21 female patients in this data set .   with the prognostic endpoint ( r / cr ) ,", "the average accuracy of 66% ( sd 3.0% ) for pediatric patient classification was obtained when no gene selection was introduced . when a set of 200 genes was selected in a learning phase of each cv ,", "the average accuracy of 68.0% ( sd 3.0% ) was achieved . when a set of 20 genes was selected in a learning phase of each cv ,", "the average accuracy of 71.0% ( sd 5.0% ) was obtained . since it appeared to be no substantial difference between accuracy and the number of genes selected , a feasible set of 25 genes in the learning phase of each cv", "was collectively ranked by the bw ratio to find sex - specific biomarkers . at the end of loocv trials , a set of male - specific genes that were selected at least 75% of the time in the entire loocv were 1882_g_at ( mecom : mds1 and evi1 complex locus ) , 37902_at ( cryz : crystallin , zeta ( quinone reductase ) ) , 38789_at ( tkt : transketolase ( wernicke - korsakoff syndrome ) ) , 39105_at ( vasp : vasodilator - stimulated phosphoprotein ) , 40844_at ( ctr9 : ctr9 , paf1/rna polymerase ii complex component , homolog ( s. cerevisiae ) ) , 36981_at ( srp9 : signal recognition particle 9  kda ) , 36338_at ( luzp1 : leucine zipper protein 1 ) , 31870_at ( cd37 : cd37 antigen ) , 1624_at ( rap1gds1 : rap1 , gtp - gdp dissociation stimulator 1 ) , and 39142_at ( nudt21 : cleavage and polyadenylation specific factor 5 , 25  kda ) .", "these ten top - ranked male - specific genes were selected as potential male - specific genomic biomarkers to classify male patients into r / cr . among them", "38789_at ( tkt : transketolase ) and 36338_at ( est ) were also included in the thirty - five genes associated with prognosis of pediatric aml identified by yagi et al . .", "in order to select a feasible set of sex - specific biomarkers a cut - off criterion of 75 percent is used . since", "every dataset may have a different sample size , the number of genes selected is given by the percentage , which is a rank score of the selected genes greater than 75% of the sample size in our case .", "for example , if a sample size is 100 , then genes that have rank scores greater than 75 have been selected .", "similarly , nine top - ranked genes for classifying female patients into r / cr were 40601_at ( tm2d1 : tm2 domain containing 1 ) , 36330_at ( ccbl1 : cysteine conjugate beta- lyase ; cytoplasmic ( glutamine transaminase k , kynurenine aminotransferase ) ) , 40586_at ( eef1e1 : eukaryotic translation elongation factor 1 epsilon 1 ) , 36648_at ( crsp9 : cofactor required for sp1 transcriptional activation , subunit 9 , 33  kda ) , 32351_at ( gpr20 : g protein - coupled receptor 20 ) , 1718_at ( arpc2 : actin - related protein 2/3 complex , subunit 2 , 34  kda ) , 38622_at ( mtg1 : mitochondrial gtpase 1 homolog ( s. cerevisiae ) ) , 36496_at ( impa2 : inositol(myo)-1(or 4)-monophosphatase 2 ) , and 38337_at ( znf193 : zinc finger protein 193 ) .", "these nine top - ranked genes which were selected at least 75% of the time in the entire loocv were considered as potential female - specific genomic biomarkers to classify female patients into r / cr .", "data with male - specific genes showed higher prediction accuracy ( 71.9% ) to classify male patients than the accuracy ( 43.8% ) to classify male patients from data with female - specific genes .", "similarly , data with female - specific genes showed higher prediction accuracy ( 76.2% ) to classify female patients than the accuracy ( 61.9% ) to classify female patients from data with male - specific genes .", "as shown in table 1 , sensitivity and specificity were also higher in using sex - specific genes for each sex .", "genomic aberrations and mutational status of the immunoglobulin variable heavy chain ( vh ) gene have been shown to be among the most important predictors for outcome in patients with b - cll .", "b - cll is the most common leukemia in the western world , and due to its clinical heterogeneity ( wide range of life expectancy ) and correlations to genomic aberrations , it is important to predict a patient 's vh mutation status at the time of diagnosis for optimal treatment .", "in addition , the study presented by haslinger et al .   suggested that the genomic signature for vh mutational status might be sex related .  ", "the gene - expression data consists of 100 b - cll patients with an oligonucleotide microarray containing around 12,000 probe sets , and it is available at http://linus.nci.nih.gov/~brb/dataarchive_new.html .", "patients are classified as either vh - mutated or unmutated ( m / nm ) .", "there are 62 males ( 33  m and 29  nm ) and 38 females ( 18  m and 20  nm ) , with a total of 51 mutated and 49 unmutated patients .   in step 1 , for each data set with male only and female only patients using the target endpoint ( i.e. , vh - mutated ( m ) versus unmutated ( nm ) ) , we separately selected and ranked 25 potential prognostic genes for males and for females in every cv trial and separately combined ranks of these genes for males and females during the learning phase of 20 trials of 10-fold cv within each loocv trial . in every loocv trial , we prioritized and combined the final top - ranked 200 genes from the male patients result ( sm ) and 200 genes from the female patients result ( sf ) as explained in section 3.1 . at the end of the entire loocv trials , a set of potential sex - specific genes", "are obtained for each sex by prioritizing and combining n sets of top - ranked 200 genes .  ", "after deletion of overlapped genes in both males and females , eleven potential male - specific genes were obtained to classify male patients into m / nm . they were 41209_at ( lpl : lipoprotein lipase ) , 41755_at ( cobll1 : cobl - like 1 ) , 39878_at ( pcdh9 : protocadherin 9 ) , 38211_at ( zbtb20 : zinc finger and btb domain containing 20 ) , 39488_at ( pcdh9 : protocadherin 9 ) , 36886_f_at ( kir2dl3 : killer cell immunoglobulin - like receptor , two domains , long cytoplasmic tail , 3 ) , 32140_at ( sorl1 : sortilin - related receptor , l(dlr class ) a repeats - containing ) , 33535_at ( p2rx1 : purinergic receptor p2x , ligand - gated ion channel , 1 ) , 39967_at ( ldoc1 : leucine zipper , downregulated in cancer 1 ) , 32842_at ( bcl7a : b - cell cll / lymphoma 7a ) , and 36899_at ( satb1 : special at - rich sequence binding protein 1 ( binds to nuclear matrix / scaffold - associating dna 's ) ) . for female patients ,", "only five genes were selected at least 75% of the time in the entire loocv trial as potential female - specific genomic biomarkers to classify female patients into m / nm . they were 33745_at ( phkg2 : phosphorylase kinase , gamma 2 ( testis ) ) , 38152_at ( loh11cr2a : loss of heterozygosity , 11 , chromosomal region 2 , gene", "a ) , 34142_at ( pde8a : phosphodiesterase 8a ) , 39593_at ( fgl2 : fibrinogen - like 2 ) , and 217_at ( klk2 : kallikrein 2 , prostatic ) .   to verify the sex - specific genomic biomarkers , we considered the performance of four different cases in the loocv trials as described in section 2 .", "data with male - specific genomic biomarkers showed higher accuracy ( 67.7% ) to classify male patients into m / nm than the accuracy to classify male patients into m / nm from data with female - specific genomic biomarkers ( 40.3% ) .", "however , female data with female - specific genes showed prediction accuracy similar to random guess close to 50% .", "therefore , there is insufficient evidence to support that the selected female - specific genes were female - specific genes ( see table 2 ) with 38 female patients in this data set .  ", "although cutaneous melanoma represents a small subset , it is the most life - threatening neoplasm of the skin , and its incidence and mortality have been increasing worldwide .", "the key underlying molecular events have not been clearly elucidated , which may explain why no target has been developed and why almost no clinical benefits from new therapies have been clearly demonstrated in patients with melanoma since the late 1970s .", "additionally , gene - expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors .", "this gene - expression data set was collected from 83 patients corresponding to the training data set and 17 patients corresponding to the validation data set .", "the probes are from tumor tissue and from reference tissue that are differentially labeled by the incorporation of cyanine 3 ( cy3 ) and cyanine 5 ( cy5 ) , respectively .", "set , the endpoint was patient prognosis and survival along with tumor stages , defined as follows . in stage", "i , cure rates are excellent with surgical removal , since they are the least likely to spread . in stage ii , melanomas can be cured , but the success rate lags behind that of stage i because a small number of cancer cells may have spread to distant sites . in stage iii , since the tumor has started to metastasize ( the spreading of a disease from one organ or part to another nonadjacent organ or part ) , the survival rate for these stages is lower than the earlier ones .", "stage iv is associated with metastasis beyond the regional lymph nodes to distant sites in the body , such as the lung , liver , or brain , or to distant areas of the skin .", "based on the tumor size , descriptions , and number of lymph nodes the stages are categorized in two classes .", "a class of high survival and small tumor size ( hs / st ) is defined and composed of stages 1 and 2 .", "the second is defined as low survival and non - small tumor size ( ls / nst ) , which is composed of stages 3 and 4 .  ", "tables 3 and 4 show the distribution of the primary cutaneous melanoma data based on sex , the defined clinical endpoint hs / st and ls / nst for the training data and validation data . among 83 patients in the training dataset ,", "there are 27 males ( 12 hs / st and 15 ls / nst ) and 56 females ( 30 hs / st and 26 ls / nst ) .", "there are 42 cases of hs / st and 41 cases of ls / nst in total .", "after preprocessing the data the final gene count is 4641 genes . among 17 patients in the validation data", "set , there are 8 males ( 1 hs / st and 7 ls / nst ) and 9 females ( 1 hs / st and 8 ls / nst )", ". there are 2 cases of hs / st and 15 cases of ls / nst in total .  ", "since the validation set was separately provided , the sex - specific genes were selected via 20 trials of 10-fold cv in the learning set .", "the following ten male - specific genes were selected at least 75% of the time during 20 trials of 10-fold cv : a_23_p128263 ( prb1 : proline - rich protein bstni subfamily 1 ) , a_23_p83838 ( ca8 : carbonic anhydrase viii ) , a_24_p212990 ( mgc70863 : similar to rpl23ap7 protein ) , a_23_p333650 ( rad9b : rad9 homolog b ( s. cerevisiae ) ) , a_32_p125251 ( n / a ) , a_23_p108835 ( ypel5 : yippee - like 5 ( drosophila ) ) , a_32_p150856 ( loc407835 : mitogen - activated protein kinase kinase 2 pseudogene ) , a_23_p11936 ( ubxn11 : ubx domain protein 11 ) , a_24_p169976 ( n / a ) , and a_32_p3998 ( znf600 : zinc finger protein 600 ) . for female patients ,", "the following eight female - specific genes were selected from 20 trials of 10-fold cv : a_23_p69497 ( clec3b : c - type lectin domain family 3 , member b ) , a_24_p118884 ( n / a ) , a_23_p152420 ( kiaa0182 ) , a_24_p265177 ( phc3 : polyhomeotic - like 3 ( drosophila ) ) , a_23_p251421 ( cdca7 : cell division cycle associated 7 ) , a_23_p211738 ( ubp1 : upstream binding protein 1 ( lbp-1a ) ) , a_23_p47377 ( hsd17b12 : hydroxysteroid ( 17-beta ) dehydrogenase 12 ) , and a_32_p113646 ( cdna flj45341 fis , clone brhip3009672 ) . using a given validation set ,", "the sex - specific genes were verified . as shown in the confusion matrix in table 5 , there was no difference in the performance from female data with female genes compared with the performance of female data with male genes .", "however , there was only one misclassification for male patients with male genes as shown in table 6 , while there were four misclassifications for male patients with female genes as shown in table 7 .", "therefore , we conclude that there exists evidence of male - specific genes in a classification of primary cutaneous melanoma ."], ["large inter individual differences in benefit from chemotherapy highlight the need to develop predictive genomic biomarkers for selecting the right treatment for the right patient .", "inappropriate chemotherapy can result in the selection of more resistant and aggressive tumor cells . to date", ", no reliable genomic biomarkers have been developed to provide the physician with prechemotherapy information to accurately predict the efficacy of a specific therapy .", "we proposed a procedure to find sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm using variable importance ranking via combination of 20 trials of 10-fold cv and loocv .", "the proposed procedure was applied to data sets obtained from the brb arraytools data human cancer archive .", "however , the issue arose out of there being not enough samples , and the data was unbalanced ( i.e. , more males than females or more positives ( disease patients ) than negatives ( nondisease patients ) ) . while patient 's sex information was not specified for many of the publicly available data sets adding to that the difficulty of searching for data , we found the following three genomic data sets that had sex information : ( 1 ) pediatric patients with aml , ( 2 ) b - cell chronic lymphocytic leukemia ( b - cll ) , and ( 3 ) primary cutaneous melanoma . in one application , pediatric patients with aml were classified by the algorithms as having either a good or poor prognosis , in terms of the likelihood of induction failure or relapse within one year of the first complete remission , based on gene - expression profiles .", "if this were brought into clinical application , a patient with a confidently predicted good prognosis might want to elect out of adjuvant chemotherapy and its associated debilitating side effects . with current rule - based decisions ,", "the overall average accuracy of this data set with a variable selection from pooled patients ( males and females ) was about 71.0% .", "however , using male - specific genes found by the proposed procedure , the accuracy was improved to about 72% as we found in the model validation studies . similarly , using female - specific genes found by the proposed procedure ,", "the average accuracy was improved to about 76% ( see table 1 ) .    in the b - cell chronic lymphocytic leukemia ( b - cll )", "dataset we found male - specific prognostic genomic biomarkers associated with b - cell chronic lymphocytic leukemia and its average classification accuracy was improved to about 68% .", "there was no substantial evidence to find female - specific prognostic genomic biomarkers in this data set .", "similarly , male - specific predictive genomic biomarkers associated with a classification of primary cutaneous melanoma were found with the classification accuracy of about 88% .  ", "the scope of our paper was to find sex - specific genomic biomarkers , if any , imbedded in the data instead of finding genomic biomarkers from the data .", "if commonly identified genes were kept in the proposed procedure , our procedure was not sex - specific genomic biomarker classifier involving two populations ( males and females ) any more but rather it became genomic biomarker classifier involving one combined population .", "in fact , even though commonly identified genes were kept , it did not necessarily improve the classification accuracy .", "for a counterexample , for the pediatric aml data of yagi et al . , the accuracy for female patients with female - specific genes by keeping the commonly identified genes was 62% ; however , the accuracy without commonly identified genes was 76% .", "for the male patients with male - specific genes by keeping the commonly identified genes the accuracy was 59% , but the accuracy without the commonly identified genes was 72% .", "for the related note , the accuracy of this data can be found anywhere between 59% and 66% with gene preprocessing and between 58% and 71% with gene selection .", "it is not an easy task to find sex - specific genes , let alone verifying and proving that they are indeed sexspecific .", "we have presented a procedure for finding sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm .", "the procedure is shown to have good  sensitivity  and  specificity  in the sense that the sex - specific genes obtained can improve prediction accuracy in classification of individual patient 's prognosis .", "the proposed procedure to discover predictive and prognostic sex - specific genomic biomarkers for individualized treatment of diseases can play a critical role in developing safer and more effective therapies that replace one - size - fits - all drugs with treatments that focus on specific patient needs ."]]}
{"article_id": "PMC2693659", "article_text": ["in the last decade , preimplantation genetic diagnosis ( pgd ) has become an important alternative to prenatal diagnosis for couples at high risk of transmitting inherited disorders to their children .", "fluorescence in situ hybridization ( fish ) has been offered for gender selection in x - linked diseases ( 1 , 2 ) , chromosomal translocations ( 3 - 5 ) , aneuploidy , and recurrent implantation failure ( 6 , 7 ) .", "procedures using polymerase chain reaction ( pcr ) has been applied for single gene defects including x - linked diseases ( 8 - 11 ) .", "ornithine transcarbamylase ( otc ; ec 2.1.3.3 ) is placed in the mitochondrial matrix where it contributes to the detoxification of nitrogenous wastes through the urea cycle , and catalyzes the synthesis of citruline from carbamyl phosphate and ornithine .", "the otc gene is located on the short arm of the x chromosome within band xp21.1 ( 12 ) .", "it spans 73 kb , has an open reading frame of 1,062 nucleotides , and contains 10 exons and 9 introns ( 13 , 14 ) .", "otc deficiency ( mim-#311250 ) is an x - linked and co - dominant metabolic disorder with partial penetrance in females ( 15 ) .", "the phenotype of otc deficiency is extremely heterogeneous , ranging from asymptomatic adult hemizygous males to acute neonatal hyperammonemic coma in the first week of affected male babies .", "approximately 80% of heterozygous females are asymptomatic and remaining 20% show clinical severity similar to males with partial deficiencies ( 16 , 17 ) .", "there are more than 341 mutations known to cause otc deficiency , all of which are specific for the individual families .", "mutations have been found in all exons and introns with a relative paucity of mutations in the sequence encoding the leader peptide ( exon 1 and a part of exon 2 ) and in exon 7 ( 17 , 18 ) . to our knowledge , only two cases have been reported where specific pgd for otc deficiency were carried out using multiplex or duplex - nested pcr assay and healthy babies devoid of the otc mutation were born ( 19 , 20 ) . in this study", ", we describe the successful pregnancy and birth after pgd for otc deficiency with simultaneous analyses of fish and duplex - nested pcr in korea .", "we have applied a duplex - nested pcr for the amplification of both the causative mutation and the y chromosome specific sry gene , and fish for aneuploidy screening of chromosome x , y and 18 .", "a couple was referred to our center after therapeutic termination of the second pregnancy following prenatal diagnosis for otc deficiency . in the first pregnancy ,", "a male baby was born but died at 10 days after birth due to liver dysfunction accompanying hyperammonemia .", "molecular genetic analysis of the couple 's otc gene was performed by pcr and direct sequencing at seoul asan hospital and revealed a single base substitution ( r320x ) in the exon 9 of the female partner . in the second pregnancy , the fetus", "was identified to be an affected boy by pcr and direct sequencing on a chorionic villus sample , and the pregnancy was therapeutically terminated . in order to avoid a pregnancy with an affected baby , the couple was counseled for pgd in cheil general hospital . in order to determine the efficiency of single cell pcr and allele drop - out ( ado ) rate , primers used for the detection of the causative mutation and y chromosome specific sry gene were tested using single lymphocytes ( table 1 ) .", "ovarian stimulation , in vitro fertilization , and blastomere biopsy were done as previously described ( 4 , 5 ) .", "after the blastomere biopsy procedure , the embryos were washed several times and transferred to the g2.2 medium ( vitrolife sweden ab , kungsbacka , sweden ) .", "each blastomere was washed twice through two drops of g2.2 medium and transferred into sterile 0.2 ml pcr tubes containing 5 l of alkaline lysis buffer .", "the nested - duplex pcr analysis was performed by previously described protocol ( 6 ) .", "mutation analysis by restriction fragment length polynorphism ( rflp ) with bcli restriction enzyme was carried out only for positively amplified pcr products of blastomeres .", "the fish analysis for chromosome x , y and 18 was done as previously described and the manufacture 's protocol ( 4 , 5 ) .", "the embryos with the normal genotype were selected and transferred on day 4 after fertilization .", "all 10 exons and exon - intron boundaries of the otc gene were screened by pcr followed by direct dna sequencing analysis .", "the heterozygous otc gene with r320x nonsense mutation ( cgatga ) and wild type was detected in the exon 9 of the female partner by rflp ( fig . 1 ) and direct sequencing ( data not shown ) .", "this r320x nonsense mutation was associated with acute neonatal hyperammonemia in their first pregnancy and reduced enzyme activity by 10 - 15% ( 21 ) .", "the efficiency and accuracy of the duplex - nested pcr analysis were tested on single lymphocytes from the heterozygous female partner and normal male partner .", "pcr on single lymphocytes resulted in 90.6% ( 58/64 ) amplification rate , whereas none of the negative controls showed a positive band .", "after ovarian hyperstimulation , a total of 18 oocytes were retrieved and 17 oocytes were injected using intracytoplasmic sperm injection in order to prevent dna contamination from inseminated sperm . among a total of 11 embryos ,", "two blastomeres per embryo from 9 embryos were biopsied and simultaneously analyzed by duplex - nested pcr and fish , and one blastomere per embryo from 2 embryos by only duplex - nested pcr . as a result of duplex - nested pcr and rflp analysis with the bcli restriction enzyme ,", "ten showed successful amplification for the exon 9 of the otc gene , and the amplification rate was 90.9% , almost same as that of the single lymphocyte test .", "there were two unaffected males , five affected males , two unaffected females , and one heterozygous female ( fig . 2 and table 2 ) .", "on the other hand , out of nine blastomeres analyzed by fish , only six blastomeres were identified as chromosomally normal embryos ( fig .", "however , among total four unaffected embryos ( embryo 1 , 2 , 3 , and 8) diagnosed by pcr , only two embryos ( embryo 3 and 8) were identified as chromosomally normal by fish .", "the other two embryos ( embryo 1 and 2 ) were shown to have trisomy 18 and monosomy 18 , respectively . only one male embryo ( embryo 8) was transferred , and another female unaffected embryo ( embryo 3 ) was cryopreserved at blastocyst stage on day 5 after fertilization .", "this pgd cycle resulted in a singleton pregnancy and the genotype of the fetus was confirmed to be a normal male by amniocentesis at 16 weeks of gestation .", "the genotype of the baby for the exon 9 of otc gene and level of ammonia were confirmed as normal .", "most pgd cycles for x - linked disorders such as duchenne muscular dystrophy and fragile x syndrome involved the selection of female embryos which were diagnosed as normal or heterozygous . these gender selections were supported by couples at risk of x - linked recessive conditions .", "ado for affected allele could lead to yielding an affected heterozygous baby , even if the female embryo was diagnosed as normal by pgd for otc deficiency .", "gender selection with fish for y chromosome - specific loci is more reliable because amplification failure could occasionally occur in single cell pcr .", "thus , our protocol with pcr and fish in this study provides higher accuracy for the selection of genetically and chromosomally normal embryos in the pgd for x - linked co - dominant defects .", "we applied duplex - nested pcr and fish analysis in two blastomeres from each embryo , which were more than 6-cell stage .", "biopsy of more than a quarter of blastomeres from each embryo could interfere with developmental potential of embryos .", "thus , single blastomere biopsy was applied to less than 6-cell stage embryos in this study .", "sequential pcr and fish analysis in one blastomere ( cell recycling ) was reported and could be used to detect the aneuploidy , but the rate of ado after pcr seems to be higher using this method ( 22 , 23 ) .", "a duplex - nested pcr analysis was developed for the simultaneous diagnosis of a r320x mutation and a gender selection , and fish analysis was used to detect aneuploidy for chromosome x , y and 18 . as a result of duplex nested pcr and fish analysis ,", "concordant results have been obtained from two blastomeres at the sex locus , and the amplification efficiency was 100% ( 9/9 except two blastomeres , that were not analyzed by fish analysis ) for sry .", "and the amplification rate was 90.9% ( 10/11 ) for exon 9 of the otc gene .", "several strategies have been developed to decrease amplification failure and ado in single cell analysis of pgd .", "these included the development of sequential first and second polar body analysis ( 24 ) , improvement of the pcr condition by increasing the denaturation temperature and time ( 19 ) , using a more powerful lysis method ( 25 - 28 ) and the use of fluorescent pcr for mutations or linked markers and multiplex pcr ( 29 - 32 ) .", "as mentioned above , we have also endeavored to optimize single cell pcr conditions , which included longer initial denaturation time , higher denaturation temperature , and the selection of taq polymerase , the concentration of mgcl2 , and lysis methods .", "the incidence of ado was 5 - 15% in many pgd laboratories ( 27 , 28 ) . at a preliminary experiment on single lymphocytes in this study ,", "the ado rate for exon 9 of the otc gene was about 13.0% when using duplex - nested pcr .", "we could not absolutely exclude the possibility of ado in the diagnosis of unaffected female embryo owing to the relatively higher ado .", "thus , fish was simultaneously applied for the aneuploidy screening and sex selection to prevent the pregnancy of heterozygous female . in this study", ", we report the successful pgd and delivery of a healthy boy in a couple at risk of transmitting the otc deficiency .", "this was carried out by the simultaneous analysis of the duplex - nested pcr for a r320x mutation in the otc gene underlying the otc deficiency and y chromosome - specific loci , and fish for aneuploidy screening of chromosome x , y and 18 .", "this strategy could provide higher accuracy for the selection of genetically and chromosomally normal embryos in pgd for single gene defects ."], "abstract_text": ["<S> ornithine transcarbamylase ( otc ) deficiency is an x - linked co - dominant disorder . </S>", "<S> a couple , with a previous history of a neonatal death and a therapeutical termination due to otc deficiency , was referred to our center for preimplantation genetic diagnosis ( pgd ) . </S>", "<S> the female partner has a nonsense mutation in the exon 9 of the otc gene ( r320x ) . </S>", "<S> we carried out nested polymerase chain reaction ( pcr ) for r320x mutation and fluorescence in situ hybridization ( fish ) for aneuploidy screening . among a total of 11 embryos , two blastomeres per embryo from 9 embryos were biopsied and analyzed by duplex - nested pcr and fish , and one blastomere per embryo from 2 embryos by only duplex - nested pcr . as a result of pcr and restriction fragment length polymorphism analysis , four embryos were diagnosed as unaffected embryos having the normal otc gene . among these embryos , only one embryo was confirmed as euploidy for chromosome x , y and 18 by fish analysis . a single normal embryo was transferred to the mother , yielding an unaffected pregnancy and birth of a healthy boy . </S>", "<S> based on our results , pcr for mutation loci and fish for aneuploidy screening with two blastomeres from an embryo could provide higher accuracy for the selection of genetically and chromosomally normal embryos in the pgd for single gene defects . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["in the last decade , preimplantation genetic diagnosis ( pgd ) has become an important alternative to prenatal diagnosis for couples at high risk of transmitting inherited disorders to their children .", "fluorescence in situ hybridization ( fish ) has been offered for gender selection in x - linked diseases ( 1 , 2 ) , chromosomal translocations ( 3 - 5 ) , aneuploidy , and recurrent implantation failure ( 6 , 7 ) .", "procedures using polymerase chain reaction ( pcr ) has been applied for single gene defects including x - linked diseases ( 8 - 11 ) .", "ornithine transcarbamylase ( otc ; ec 2.1.3.3 ) is placed in the mitochondrial matrix where it contributes to the detoxification of nitrogenous wastes through the urea cycle , and catalyzes the synthesis of citruline from carbamyl phosphate and ornithine .", "the otc gene is located on the short arm of the x chromosome within band xp21.1 ( 12 ) .", "it spans 73 kb , has an open reading frame of 1,062 nucleotides , and contains 10 exons and 9 introns ( 13 , 14 ) .", "otc deficiency ( mim-#311250 ) is an x - linked and co - dominant metabolic disorder with partial penetrance in females ( 15 ) .", "the phenotype of otc deficiency is extremely heterogeneous , ranging from asymptomatic adult hemizygous males to acute neonatal hyperammonemic coma in the first week of affected male babies .", "approximately 80% of heterozygous females are asymptomatic and remaining 20% show clinical severity similar to males with partial deficiencies ( 16 , 17 ) .", "there are more than 341 mutations known to cause otc deficiency , all of which are specific for the individual families .", "mutations have been found in all exons and introns with a relative paucity of mutations in the sequence encoding the leader peptide ( exon 1 and a part of exon 2 ) and in exon 7 ( 17 , 18 ) . to our knowledge , only two cases have been reported where specific pgd for otc deficiency were carried out using multiplex or duplex - nested pcr assay and healthy babies devoid of the otc mutation were born ( 19 , 20 ) . in this study", ", we describe the successful pregnancy and birth after pgd for otc deficiency with simultaneous analyses of fish and duplex - nested pcr in korea .", "we have applied a duplex - nested pcr for the amplification of both the causative mutation and the y chromosome specific sry gene , and fish for aneuploidy screening of chromosome x , y and 18 ."], ["a couple was referred to our center after therapeutic termination of the second pregnancy following prenatal diagnosis for otc deficiency . in the first pregnancy ,", "a male baby was born but died at 10 days after birth due to liver dysfunction accompanying hyperammonemia .", "molecular genetic analysis of the couple 's otc gene was performed by pcr and direct sequencing at seoul asan hospital and revealed a single base substitution ( r320x ) in the exon 9 of the female partner . in the second pregnancy , the fetus", "was identified to be an affected boy by pcr and direct sequencing on a chorionic villus sample , and the pregnancy was therapeutically terminated . in order to avoid a pregnancy with an affected baby , the couple was counseled for pgd in cheil general hospital . in order to determine the efficiency of single cell pcr and allele drop - out ( ado ) rate , primers used for the detection of the causative mutation and y chromosome specific sry gene were tested using single lymphocytes ( table 1 ) .", "ovarian stimulation , in vitro fertilization , and blastomere biopsy were done as previously described ( 4 , 5 ) .", "after the blastomere biopsy procedure , the embryos were washed several times and transferred to the g2.2 medium ( vitrolife sweden ab , kungsbacka , sweden ) .", "each blastomere was washed twice through two drops of g2.2 medium and transferred into sterile 0.2 ml pcr tubes containing 5 l of alkaline lysis buffer .", "the nested - duplex pcr analysis was performed by previously described protocol ( 6 ) .", "mutation analysis by restriction fragment length polynorphism ( rflp ) with bcli restriction enzyme was carried out only for positively amplified pcr products of blastomeres .", "the fish analysis for chromosome x , y and 18 was done as previously described and the manufacture 's protocol ( 4 , 5 ) .", "the embryos with the normal genotype were selected and transferred on day 4 after fertilization .", "all 10 exons and exon - intron boundaries of the otc gene were screened by pcr followed by direct dna sequencing analysis .", "the heterozygous otc gene with r320x nonsense mutation ( cgatga ) and wild type was detected in the exon 9 of the female partner by rflp ( fig . 1 ) and direct sequencing ( data not shown ) .", "this r320x nonsense mutation was associated with acute neonatal hyperammonemia in their first pregnancy and reduced enzyme activity by 10 - 15% ( 21 ) .", "the efficiency and accuracy of the duplex - nested pcr analysis were tested on single lymphocytes from the heterozygous female partner and normal male partner .", "pcr on single lymphocytes resulted in 90.6% ( 58/64 ) amplification rate , whereas none of the negative controls showed a positive band .", "after ovarian hyperstimulation , a total of 18 oocytes were retrieved and 17 oocytes were injected using intracytoplasmic sperm injection in order to prevent dna contamination from inseminated sperm . among a total of 11 embryos ,", "two blastomeres per embryo from 9 embryos were biopsied and simultaneously analyzed by duplex - nested pcr and fish , and one blastomere per embryo from 2 embryos by only duplex - nested pcr . as a result of duplex - nested pcr and rflp analysis with the bcli restriction enzyme ,", "ten showed successful amplification for the exon 9 of the otc gene , and the amplification rate was 90.9% , almost same as that of the single lymphocyte test .", "there were two unaffected males , five affected males , two unaffected females , and one heterozygous female ( fig . 2 and table 2 ) .", "on the other hand , out of nine blastomeres analyzed by fish , only six blastomeres were identified as chromosomally normal embryos ( fig .", "however , among total four unaffected embryos ( embryo 1 , 2 , 3 , and 8) diagnosed by pcr , only two embryos ( embryo 3 and 8) were identified as chromosomally normal by fish .", "the other two embryos ( embryo 1 and 2 ) were shown to have trisomy 18 and monosomy 18 , respectively . only one male embryo ( embryo 8) was transferred , and another female unaffected embryo ( embryo 3 ) was cryopreserved at blastocyst stage on day 5 after fertilization .", "this pgd cycle resulted in a singleton pregnancy and the genotype of the fetus was confirmed to be a normal male by amniocentesis at 16 weeks of gestation .", "the genotype of the baby for the exon 9 of otc gene and level of ammonia were confirmed as normal ."], ["most pgd cycles for x - linked disorders such as duchenne muscular dystrophy and fragile x syndrome involved the selection of female embryos which were diagnosed as normal or heterozygous . these gender selections were supported by couples at risk of x - linked recessive conditions .", "ado for affected allele could lead to yielding an affected heterozygous baby , even if the female embryo was diagnosed as normal by pgd for otc deficiency .", "gender selection with fish for y chromosome - specific loci is more reliable because amplification failure could occasionally occur in single cell pcr .", "thus , our protocol with pcr and fish in this study provides higher accuracy for the selection of genetically and chromosomally normal embryos in the pgd for x - linked co - dominant defects .", "we applied duplex - nested pcr and fish analysis in two blastomeres from each embryo , which were more than 6-cell stage .", "biopsy of more than a quarter of blastomeres from each embryo could interfere with developmental potential of embryos .", "thus , single blastomere biopsy was applied to less than 6-cell stage embryos in this study .", "sequential pcr and fish analysis in one blastomere ( cell recycling ) was reported and could be used to detect the aneuploidy , but the rate of ado after pcr seems to be higher using this method ( 22 , 23 ) .", "a duplex - nested pcr analysis was developed for the simultaneous diagnosis of a r320x mutation and a gender selection , and fish analysis was used to detect aneuploidy for chromosome x , y and 18 . as a result of duplex nested pcr and fish analysis ,", "concordant results have been obtained from two blastomeres at the sex locus , and the amplification efficiency was 100% ( 9/9 except two blastomeres , that were not analyzed by fish analysis ) for sry .", "and the amplification rate was 90.9% ( 10/11 ) for exon 9 of the otc gene .", "several strategies have been developed to decrease amplification failure and ado in single cell analysis of pgd .", "these included the development of sequential first and second polar body analysis ( 24 ) , improvement of the pcr condition by increasing the denaturation temperature and time ( 19 ) , using a more powerful lysis method ( 25 - 28 ) and the use of fluorescent pcr for mutations or linked markers and multiplex pcr ( 29 - 32 ) .", "as mentioned above , we have also endeavored to optimize single cell pcr conditions , which included longer initial denaturation time , higher denaturation temperature , and the selection of taq polymerase , the concentration of mgcl2 , and lysis methods .", "the incidence of ado was 5 - 15% in many pgd laboratories ( 27 , 28 ) . at a preliminary experiment on single lymphocytes in this study ,", "the ado rate for exon 9 of the otc gene was about 13.0% when using duplex - nested pcr .", "we could not absolutely exclude the possibility of ado in the diagnosis of unaffected female embryo owing to the relatively higher ado .", "thus , fish was simultaneously applied for the aneuploidy screening and sex selection to prevent the pregnancy of heterozygous female . in this study", ", we report the successful pgd and delivery of a healthy boy in a couple at risk of transmitting the otc deficiency .", "this was carried out by the simultaneous analysis of the duplex - nested pcr for a r320x mutation in the otc gene underlying the otc deficiency and y chromosome - specific loci , and fish for aneuploidy screening of chromosome x , y and 18 .", "this strategy could provide higher accuracy for the selection of genetically and chromosomally normal embryos in pgd for single gene defects ."]]}
{"article_id": "PMC4540876", "article_text": ["musculoskeletal stiffness and limited range of motion ( rom ) can restrict functional joint \n movement , particularly during ankle supination following neurological diseases1 .", "ankle dorsiflexion rom restrictions impair \n dynamic balance and gait , which may contribute to secondary injuries ; reduced ankle rom and \n tight calf muscles can cause poor gait , inefficient energy use , impaired balance , and an \n increased risk of falls2 .", "musculoskeletal \n transformations manifesting as abnormal joint stiffness and limited rom result in numerous \n restrictions in the function and joint movements of patients3 .", "limited passive ankle dorsiflexion rom during knee extension can \n alter foot positioning and result in compensatory foot movements , leading to an abnormal \n gait , which can result in ankle sprains and lower extremity overuse injuries4 .", "studies have reported that balance - training \n programs that alter the somatosensory input , such as the use of an unstable surface to \n induce equalized weight distribution , can be helpful in hemiplegic patients5 .", "kim et al . used an ankle board with a slope \n in their center to achieve efficient ankle stretching and increased dorsiflexion6 .", "they suggested that stretching exercises \n using their device resulted in a greater increase in foot pressure compared to that with \n stretching exercises using existing inclined boards during ankle dorsiflexion \n stretching6 .", "therefore , we investigated \n the effects of an unstable inclined board on the active and passive ankle rom in patients \n with ankle stiffness .", "the study included 10 female patients ( mean age , 22.8  5.3  years ; height , 158.9  3.0  cm ; \n weight , 61.2  4.7  kg ) with ankle stiffness caused by diseases or medical conditions .", "the \n patients were selected based on ankle stiffness scores obtained using a symptom checker \n tool .", "the patients were assigned randomly to two groups ( both n = 5 ) and their height and \n weight were measured .", "the patients were not cognitively impaired and were capable of \n independent walking for 20  m without difficulty .", "patients were excluded if they had a \n history of or current neurological conditions . before participating ,", "the purpose and methods \n of the study were explained to the patients , and all provided informed consent , according to \n the principles of the declaration of helsinki .", "active and passive ankle dorsiflexion were measured using a standard 31.75  4.45  cm clear \n plastic goniometer , with one increment , a 360 scale , and a bubble level at each end .", "group \n 1 performed the ankle dorsiflexion stretching exercise using a wooden inclined board , and \n group 2 performed ankle dorsiflexion stretching exercises using an air - cushioned inclined \n board that provided an unstable surface .", "rubber sheets were attached to the inclined boards to prevent falls due to \n sliding .", "the patients performed ankle dorsiflexion stretching exercises for 5  min , with a \n 10-min rest between each set , five times a day for 1 week .", "the active and passive ankle \n dorsiflexion angles were measured bilaterally using the goniometer before and after the \n study .", "independent and paired t - tests were used to assess within- and between - group differences in \n ankle dorsiflexion , with spss for windows ( spss , chicago , il , usa ) .", "the amount of active and passive dorsiflexion did not differ between the two groups before \n the exercises , with active dorsiflexion angles of 18.2  6.4 and 19.3  4.2 and passive \n dorsiflexion angles of 23.4  5.5 and 22.6  5.7 in groups 1 and 2 , respectively ( both p \n > 0.05 ) .", "the stretching exercises significantly ( p < 0.05 ) increased the active and \n passive ankle dorsiflexion angles in both groups compared to those at baseline .", "the active \n dorsiflexion angle was significantly increased ( p < 0.05 ) in group 2 compared to that in \n group 1 after the exercises .", "the active dorsiflexion angles before and after the exercises \n were 18.2  6.4 and 22.1  5.0 in group 1 and 19.3  4.2 and 28.1  6.9 in group 2 , \n respectively .", "the passive dorsiflexion angles did not differ significantly between the \n groups after the exercises ( p>0.05 ) and were 23.4  5.5 and 32.2  5.3 in group 1 and \n 22.6  5.7 and 35.1  6.0 in group 2 before and after exercising , respectively .", "the \n air - cushioned inclined board provided an unstable surface for performing ankle dorsiflexion \n stretching exercises .", "the results showed that ankle stretching exercises using the unstable \n inclined board significantly increased the active dorsiflexion angle compared to that with \n exercises using a wooden inclined board .", "we believe that the use of the air - cushioned \n inclined board provides somatosensory input and subsequently stimulates active ankle \n dorsiflexion by promoting automatic postural control .", "sensory proprioception of the ankle \n and sensory compression of the plantar are important in the control of sway7 .", "an increase in the number of somatosensory \n inputs affects the muscles , and the use of an unstable support surface result in many \n different somatosensory inputs that limit compensatory action7 .", "performing cognitive tasks in a standing posture reduces internal \n focus and induces external focus , which promotes automatic postural control8 .", "stretching improve the flexibility of \n tendons , and changes in the mechanical condition of tendons depend on the type of stretching \n performed9 .", ", the passive dorsiflexion angles did not differ significantly between the two \n groups after ankle stretching exercises .", "however , stretching exercises significantly \n increased the active and passive ankle dorsiflexion angles in both groups compared to those \n at baseline .", "these results suggest that passive ankle exercises using a wooden inclined \n board only stretch muscle and tissue , while exercises using an unstable inclined board \n stretch muscle and tissue , and stimulate activation of the ankle dorsiflexor .", "active ankle \n dorsiflexion was more effectively improved with ankle stretching exercises using an unstable \n inclined board than with exercises using a wooden inclined board ."], "abstract_text": ["<S> [ purpose ] the present study assessed the effects of using an unstable inclined board on \n the active and passive ankle range of motion in patients with ankle stiffness . [ subjects ] \n the study included 10 young female patients with ankle stiffness . </S>", "<S> [ methods ] the patients \n were divided into the following two groups : a group that performed ankle dorsiflexion \n stretching exercises using a wooden inclined board and a group that performed stretching \n exercises using an air - cushioned inclined board ( unstable inclined board ) . </S>", "<S> active and \n passive ankle dorsiflexion angles were measured bilaterally using a goniometer . </S>", "<S> [ results ] \n both inclined boards significantly increased active and passive ankle dorsiflexion . </S>", "<S> after \n performing ankle stretching exercises , active dorsiflexion significantly increased the \n unstable inclined board compared to that using the wooden inclined board . </S>", "<S> however , the \n passive dorsiflexion angles did not differ significantly between the two groups after \n ankle stretching exercises . </S>", "<S> [ conclusion ] the use of an unstable inclined board might \n stimulate activation of the ankle dorsiflexors in addition to stretching muscle or tissue . \n active ankle dorsiflexion was more effectively improved with stretching exercises using an \n unstable inclined board than with exercises using a wooden inclined board . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["musculoskeletal stiffness and limited range of motion ( rom ) can restrict functional joint \n movement , particularly during ankle supination following neurological diseases1 .", "ankle dorsiflexion rom restrictions impair \n dynamic balance and gait , which may contribute to secondary injuries ; reduced ankle rom and \n tight calf muscles can cause poor gait , inefficient energy use , impaired balance , and an \n increased risk of falls2 .", "musculoskeletal \n transformations manifesting as abnormal joint stiffness and limited rom result in numerous \n restrictions in the function and joint movements of patients3 .", "limited passive ankle dorsiflexion rom during knee extension can \n alter foot positioning and result in compensatory foot movements , leading to an abnormal \n gait , which can result in ankle sprains and lower extremity overuse injuries4 .", "studies have reported that balance - training \n programs that alter the somatosensory input , such as the use of an unstable surface to \n induce equalized weight distribution , can be helpful in hemiplegic patients5 .", "kim et al . used an ankle board with a slope \n in their center to achieve efficient ankle stretching and increased dorsiflexion6 .", "they suggested that stretching exercises \n using their device resulted in a greater increase in foot pressure compared to that with \n stretching exercises using existing inclined boards during ankle dorsiflexion \n stretching6 .", "therefore , we investigated \n the effects of an unstable inclined board on the active and passive ankle rom in patients \n with ankle stiffness ."], ["the study included 10 female patients ( mean age , 22.8  5.3  years ; height , 158.9  3.0  cm ; \n weight , 61.2  4.7  kg ) with ankle stiffness caused by diseases or medical conditions .", "the \n patients were selected based on ankle stiffness scores obtained using a symptom checker \n tool .", "the patients were assigned randomly to two groups ( both n = 5 ) and their height and \n weight were measured .", "the patients were not cognitively impaired and were capable of \n independent walking for 20  m without difficulty .", "patients were excluded if they had a \n history of or current neurological conditions . before participating ,", "the purpose and methods \n of the study were explained to the patients , and all provided informed consent , according to \n the principles of the declaration of helsinki .", "active and passive ankle dorsiflexion were measured using a standard 31.75  4.45  cm clear \n plastic goniometer , with one increment , a 360 scale , and a bubble level at each end .", "group \n 1 performed the ankle dorsiflexion stretching exercise using a wooden inclined board , and \n group 2 performed ankle dorsiflexion stretching exercises using an air - cushioned inclined \n board that provided an unstable surface .", "rubber sheets were attached to the inclined boards to prevent falls due to \n sliding .", "the patients performed ankle dorsiflexion stretching exercises for 5  min , with a \n 10-min rest between each set , five times a day for 1 week .", "the active and passive ankle \n dorsiflexion angles were measured bilaterally using the goniometer before and after the \n study .", "independent and paired t - tests were used to assess within- and between - group differences in \n ankle dorsiflexion , with spss for windows ( spss , chicago , il , usa ) ."], ["the amount of active and passive dorsiflexion did not differ between the two groups before \n the exercises , with active dorsiflexion angles of 18.2  6.4 and 19.3  4.2 and passive \n dorsiflexion angles of 23.4  5.5 and 22.6  5.7 in groups 1 and 2 , respectively ( both p \n > 0.05 ) .", "the stretching exercises significantly ( p < 0.05 ) increased the active and \n passive ankle dorsiflexion angles in both groups compared to those at baseline .", "the active \n dorsiflexion angle was significantly increased ( p < 0.05 ) in group 2 compared to that in \n group 1 after the exercises .", "the active dorsiflexion angles before and after the exercises \n were 18.2  6.4 and 22.1  5.0 in group 1 and 19.3  4.2 and 28.1  6.9 in group 2 , \n respectively .", "the passive dorsiflexion angles did not differ significantly between the \n groups after the exercises ( p>0.05 ) and were 23.4  5.5 and 32.2  5.3 in group 1 and \n 22.6  5.7 and 35.1  6.0 in group 2 before and after exercising , respectively ."], ["the \n air - cushioned inclined board provided an unstable surface for performing ankle dorsiflexion \n stretching exercises .", "the results showed that ankle stretching exercises using the unstable \n inclined board significantly increased the active dorsiflexion angle compared to that with \n exercises using a wooden inclined board .", "we believe that the use of the air - cushioned \n inclined board provides somatosensory input and subsequently stimulates active ankle \n dorsiflexion by promoting automatic postural control .", "sensory proprioception of the ankle \n and sensory compression of the plantar are important in the control of sway7 .", "an increase in the number of somatosensory \n inputs affects the muscles , and the use of an unstable support surface result in many \n different somatosensory inputs that limit compensatory action7 .", "performing cognitive tasks in a standing posture reduces internal \n focus and induces external focus , which promotes automatic postural control8 .", "stretching improve the flexibility of \n tendons , and changes in the mechanical condition of tendons depend on the type of stretching \n performed9 .", ", the passive dorsiflexion angles did not differ significantly between the two \n groups after ankle stretching exercises .", "however , stretching exercises significantly \n increased the active and passive ankle dorsiflexion angles in both groups compared to those \n at baseline .", "these results suggest that passive ankle exercises using a wooden inclined \n board only stretch muscle and tissue , while exercises using an unstable inclined board \n stretch muscle and tissue , and stimulate activation of the ankle dorsiflexor .", "active ankle \n dorsiflexion was more effectively improved with ankle stretching exercises using an unstable \n inclined board than with exercises using a wooden inclined board ."]]}
{"article_id": "PMC4495481", "article_text": ["renal tubular involvement in sjgren 's syndrome ( ss ) usually manifests with fanconi syndrome , distal ( type 1 ) renal tubular acidosis ( rta ) , nephrogenic diabetes insipidus , and hypokalemia .", ", we describe a case a young female with ss who presented with features of gitelman syndrome and hypokalemic paralysis .", "a 29-year - old female presented with weakness of both upper and lower limb weakness since 15 days .", "she had a history of dry eyes and dry mouth for the past 6 months . on examination ,", "laboratory evaluation showed hemoglobin - 9.5 g / dl , platelet 2.95 lakh / mm .", "blood urea nitrogen was 7 mg / dl , serum creatinine was 0.65 mg / dl , serum sodium was 134 meq / l , serum potassium was 1.81 meq / l and serum chloride was 84.3", "urine chloride ( spot ) was 36.7 meq / l , urine creatinine ( spot ) 23.5 mg / dl , and urine calcium ( spot ) 1.23 mg / dl .", "this patient satisfied the american european consensus criteria for diagnosis of primary sjgren 's syndrome ( ss ) .", "ten days after admission , she was discharged on spironolactone and prednisolone . on follow - up", "this patient presented with muscle weakness and her laboratory reports showed hypokalemia and metabolic alkalosis with normal blood pressure .", "her urinary chloride levels were above 20 meq / l , which ruled out extra - renal causes of metabolic alkalosis .", "the presence of hypomagnesemia and absence of hypercalciuria ( urinary calcium creatinine ratio < 0.2 ) ruled out bartter syndrome .", "hypokalemic alkalosis , high urinary chloride , hypomagnesemia , absence of hypercalciuria in background of no history of diuretic use favored the diagnosis of gitelman syndrome .", "tubular involvement in ss is usually distal tubular dysfunction , type i ( distal ) renal tubular acidosis and nephrogenic diabetes insipidus .", "proximal tubular abnormalities are less frequent , and rarely fanconi 's syndrome has been reported in patients with ss .", "acquired gitelman syndrome is relatively rare . to the best of our knowledge , only five cases of acquired gitelman syndrome have been reported in english literature so far . among these five cases ,", "four had ss , one was a case of chronic sialoadenitis . acquired gitelman syndrome secondary to ss presenting with hypokalemic weakness is very rare .", "only two cases have been reported so far . though gitelman syndrome is an inherited disorder ,", "given the paucity of reports , we believe that ss presenting as acquired gitelman syndrome may be relatively rare . acquired gitelman syndrome should be considered in differential diagnosis of renal involvement in patients with ss ."], "abstract_text": ["<S> we report a case of a young female patient who presented with weakness of upper and lower limbs . on evaluation , she had hypokalemia , hypomagnesemia , metabolic alkalosis and hypocalciuria . </S>", "<S> anti - ro ( ssa ) antibody was positive . </S>", "<S> she had an acquired gitelman syndrome due to primary sjgren 's syndrome ( ss ) . </S>", "<S> ss presenting with features of gitelman syndrome is very rare . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["renal tubular involvement in sjgren 's syndrome ( ss ) usually manifests with fanconi syndrome , distal ( type 1 ) renal tubular acidosis ( rta ) , nephrogenic diabetes insipidus , and hypokalemia .", ", we describe a case a young female with ss who presented with features of gitelman syndrome and hypokalemic paralysis ."], ["a 29-year - old female presented with weakness of both upper and lower limb weakness since 15 days .", "she had a history of dry eyes and dry mouth for the past 6 months . on examination ,", "laboratory evaluation showed hemoglobin - 9.5 g / dl , platelet 2.95 lakh / mm .", "blood urea nitrogen was 7 mg / dl , serum creatinine was 0.65 mg / dl , serum sodium was 134 meq / l , serum potassium was 1.81 meq / l and serum chloride was 84.3", "urine chloride ( spot ) was 36.7 meq / l , urine creatinine ( spot ) 23.5 mg / dl , and urine calcium ( spot ) 1.23 mg / dl .", "this patient satisfied the american european consensus criteria for diagnosis of primary sjgren 's syndrome ( ss ) .", "ten days after admission , she was discharged on spironolactone and prednisolone . on follow - up"], ["this patient presented with muscle weakness and her laboratory reports showed hypokalemia and metabolic alkalosis with normal blood pressure .", "her urinary chloride levels were above 20 meq / l , which ruled out extra - renal causes of metabolic alkalosis .", "the presence of hypomagnesemia and absence of hypercalciuria ( urinary calcium creatinine ratio < 0.2 ) ruled out bartter syndrome .", "hypokalemic alkalosis , high urinary chloride , hypomagnesemia , absence of hypercalciuria in background of no history of diuretic use favored the diagnosis of gitelman syndrome .", "tubular involvement in ss is usually distal tubular dysfunction , type i ( distal ) renal tubular acidosis and nephrogenic diabetes insipidus .", "proximal tubular abnormalities are less frequent , and rarely fanconi 's syndrome has been reported in patients with ss .", "acquired gitelman syndrome is relatively rare . to the best of our knowledge , only five cases of acquired gitelman syndrome have been reported in english literature so far . among these five cases ,", "four had ss , one was a case of chronic sialoadenitis . acquired gitelman syndrome secondary to ss presenting with hypokalemic weakness is very rare .", "only two cases have been reported so far . though gitelman syndrome is an inherited disorder ,", "given the paucity of reports , we believe that ss presenting as acquired gitelman syndrome may be relatively rare . acquired gitelman syndrome should be considered in differential diagnosis of renal involvement in patients with ss ."]]}
{"article_id": "PMC3564424", "article_text": ["inadequate organization has been identified as an important cause of inappropriate courses of treatment for patients in the health care system [ 1 , 2 ] .", "a response to this challenge has been to focus on integrated health care and co - ordination between the actors involved in the process [ 37 ] .", "an illustrative example of this can be found when looking at frail elderly patients on their discharge from hospital .", "studies show that readmission can be diminished by increased collaboration between hospital , district nurses and general practitioners [ 8 , 9 ] .", "follow - up home visits by the district nurse and general practitioner to frail elderly patients after discharge have shown promising results [ 10 , 11 ] .", "the aim of follow - up home visits to elderly and frail persons is to enable them to remain in their own home , avoid readmission to hospital , admission to nursing homes or other forms of sheltered housing and to improve the functional ability and general wellbeing of the elderly .", "a follow - up home visit comprises a home visit by a general practitioner ( gp ) and district nurse within about one week after discharge from hospital .", "the visit is expected to last approximately one hour , a relatively long period compared to other types of doctor - patient contact .", "this visit can be supplemented with two subsequent home visits or visits to the gp if this is considered necessary .", "the latest study from denmark has shown that the readmission rate in the intervention group was 40% while it was 53% in the control group .", "at the same time , studies have shown that there are indications that patients prefer primary care follow - up to specialist care .", "these results are overall convincing and , as a result , on - going work is being done by health authorities to test and further develop follow - up home visits as an initiative , including a programme in copenhagen carried out from 20082011 .", "the copenhagen programme was conducted as a randomized controlled trial which implies strict procedures for the implementation process .", "a total of approximately 300 patients from bispebjerg hospital ( a major hospital in copenhagen ) were included in the intervention and control group .", "approximately 100 follow - up home visits to patients in the intervention group have been made . in the remaining approximately 200 cases in the intervention group it has not been possible to carry out a follow - up home visit , first of all because gps could not meet the requirements . in this feasibility study gps", "were not carefully selected for the programme as they were in an earlier study , and , as a result , more gps chose not to take part in the programme .", "the effect of the copenhagen programme will be reported in 2012 by the danish institute of health services research .", "this paper will focus on the implementation of follow - up home visits using a qualitative methodological approach .", "we know some of the general dynamics in the area   but we do not know more precisely what the practical limitations of the home visitor 's programmes are and how these limitations could make a difference to the outcomes assessed [ 14 , 15 ] . why do so few gps take part in the follow - up home visit programme ?", "why is the documented effect of such visits not motivation enough for gps to encourage them to do so ?", "addressing such questions could help us understand how to design and implement these visits in order to fully exploit the potential benefits of the intervention .", "moreover , such results could generate useful hypotheses which could be addressed in future trials attempting to examine the effects of home visiting programmes .", "the aim of this study is to explore one particular aspect of follow - up home visits : the motivation and rationale of local care providers to invest time and effort in follow - up home visits .", "what is the main motivation of gps , district nurses and hospital staff to take part in a cross - sectoral activity like follow - up home visits ?", "the case study is placed in a danish setting . in order to understand the organizational dynamics involved a short description of the setting", "is needed : the danish health care system is mainly a public system based on general taxation , and characterized by universal access to health care services .", "gps have a central position in the system since they have a kind of  gatekeeper  function , they are usually the first professional confronted with the patient 's problems and they see it as their responsibility to guide patients through the system .", "gps are self - employed , general practices are small and typically privately owned ; only a minority of gps are organized in health centres .", "home care organizations ( district nurses ) have occupied an ever more central role since a major administrative reform in 2007 gave the local level the main responsibility for preventive health care .", "co - ordination between organizational units in primary and secondary health care is determined by agreements between regional and municipality authorities , which include procedures for such cases as when frail and elderly people are discharged from hospital .", "in general , the danish system is considered quite integrated , in particular because the climate of trust in denmark makes co - operation possible [ 7 , 16 ] . as a result ,", "follow - up home visits and other cross - sector interventions , such as  follow - home arrangements  and  case managers  have been quite regularly implemented on an experimental basis as a possible alternative to hospital - based measures .", "the interventions have shown promising results but studies in the area also point to problems in connection with implementing the interventions [ 7 , 8 , 10 , 12 , 17 , 18 ] .", "empirical data were gathered to develop hypotheses and theoretical concepts . at the same time , however , a rough theoretical framework was used as an underlying foundation in the study to ensure that a relevant focus and approach were applied .", "resource dependency theory tells us that network formation can largely be explained by an organization 's need to reduce costs or gain resources and power .", "organizations only work together when administrative and economic structures are designed which makes co - operation a profitable pursuit .", "organizations thus typically follow their own agenda rather than involve themselves in cross - sectoral activities . in the health sector some support can be found for this basic claim .", "studies have shown that network formation in general is limited and poor co - ordination predominant in the area [ 13 , 21 ] .", "resource dependency theory also tells us , however , that organizations need to reduce uncertainty . to reduce uncertainty , organisations tend to co - operate with organisations they trust and depend on to achieve common goals [ 19 , 22 ] .", "hence , in order to understand co - operation you also need to look at the question of trust and the quality of inter - organizational networks , as done in inter - organizational network theory .", "this perspective has shown us that we need to have a comprehensive view of the factors which make health sector organisations work together .", "cross - sectoral programs and inter - organizational activities are not based purely on partners ' own needs and resources [ 23 , 24 ] .", "motivation plays a key role in inter - organizational theory [ 22 , 25 ] .", "working together is a challenging task ; it is typically regarded as uncertain whether it will bring benefits equivalent to the resources invested .", "motivation and commitment to the process are therefore needed among the actors involved in order to keep things moving and overcome the uncertainties inherent in the process . what motivates organizations to work together ?", "the literature points to two aspects in particular : the object needs to be seen as highly relevant and benefits should be obvious for all involved [ 22 , 25 ] .", "this paper is based on an evaluation report made by one of the authors for health and social care , city of copenhagen .", "the interviews were structured around the following questions : \n to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant?how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ? are the benefits obvious ? \n  to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant ? how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ?", "two focus groups and seven individual interviews were conducted in march  may 2010 , giving a total of 23 respondents .", "the focus group approach was chosen because the interaction between some of the key respondents ( hospital staff , district nurses ) was considered to be of importance , since a key goal in the study was to understand the shared views on collaboration among these key actors in the programme .", "one focus group interview was conducted with seven employees at bispebjerg hospital who were involved in the follow - up home visit programme .", "recruitment criteria were designed in order to include a suitable spread of respondents , i.e. to ensure that members of all staff groups directly involved in the project were represented .", "we ensured that the project co - ordinator at the hospital responsible for the selection of patients for the programme was represented in the focus group . at the same time we also ensured that a doctor and nurses from the most relevant wards were represented .", "another focus group interview was conducted with nine district nurses who had been involved in the project .", "nurses from different districts in copenhagen were selected for the focus group in order to ensure that different ways of organising follow - up home visits were represented .", "gps were not as motivated to take part in the research project as district nurses and hospital staff , so a less time - consuming method than focus groups was considered appropriate . as it turned out , in - depth interviews proved to be highly relevant for gps because gps had divergent and less firm views about follow - up home visits than other groups in the study and the methodological design allowed these views to be fully expressed .", "five individual interviews were held with gps involved in the project . both gps with a positive attitude towards follow - up home visits and gps with a less positive attitude were recruited to ensure that a wide range of attitudes was included in the study .", "these recruitment criteria were based on data registered by the city of copenhagen showing which gps had refused to participate in follow - up home visits and which had agreed . at the same time we ensured that the gps represented covered different districts in copenhagen .", "two individual interviews were held with employees from referrals in the city of copenhagen home nursing services .", "there is a purchaser - provider split in the city of copenhagen , and the referrals purchase while the district nurses provides .", "the two referrals came from different districts and had different positions in the organization , ensuring that the most important experiences from this organization were covered .", "the focus group interviews took place in a neutral meeting room arranged by the project manager , and the in - depth interviews took place in the gps ' offices .", "the focus group interviews lasted between 90 and 120 minutes , and the in - depth interviews between 25 and 60 minutes . both were recorded with a digital voice recorder and transcribed verbatim .", "the interview transcripts were analyzed under the two headings which form the basic empirical questions in the study : relevance of collaboration and benefits of collaboration .", "quotes from the interviews were grouped under each heading in order to find the quotes which were most illustrative of the views of gps , district nurses and hospital staff . to ensure reliability ,", "results were presented and discussed in a steering group with representatives from the main professional groups involved in the programme ( gps , district nurses and hospital staff ) .", "a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .", "gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .", "the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .", "the gps generally see the follow - up home visit as a relevant type of contact .", "a gp said :  it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .", ", it can give the patient extra resources when you visit the patient on the patient 's home ground  .", "the district nurses also generally see the follow - up home visits as a relevant type of contact .", "the gps find that the medication review is an especially important element in follow - up home visits .", "a gp said :  we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes  .", "at the same time the gps also stress that follow - up home visits are not only about reviewing medication :  the purpose of a follow - up home visit is first of all about making a patient review .", "it is not only the medicine we should look at , but the patient  in this sense follow - up home visits are very relevant  .", "the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient :  the guide creates a good structure for the home visit .", "the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are  .", "the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital", "the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector :  the guide is quite voluminous .", "we have already made a lot of the tests at the hospital so there is no need to repeat these at home .", "you should make sure that the gp has read the discharge summary before the follow - up home visit ", "the target group of the programme is elderly and frail patients just out of from hospital .", "a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital", "a nurse at the hospital expressed it this way :  it is typically this type of patient that we see again and again : deteriorating health and a small social network  .", "the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .", "other patients would have benefited from a follow - up home visit but they were not included in the programme  gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .", "this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients :  it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .", "a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc . ", "a gp said :  the hospital has contact with the patient at the time of discharge .", "in the present situation there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .", "it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems :  if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .", "but it is often difficult to find out who is responsible and it is difficult to sort out the problem  ( gp ) .", "the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur :  we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .", "a fax from the hospital to the home nursing services is not enough  ( doctor , hospital ) . in the city of copenhagen", "the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .", "it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having :  follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility  .", "the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .", "today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do  .", "the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .", "these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .", "such information is essential since follow - up home visits are new to many gps .", "the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low : ", "i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases", "i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit ", "the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .", "a gp said :  you get to talk  that is the biggest gain , to get the dialogue .", "we work in two parallel organizational systems . by not having the dialogue we as gps miss some observations .", "the gps want , however , more information about the efficacy of follow - up home visits .", "gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health :  if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .", "information could be better  ( gp ) . at the hospital the staff involved in the programme", "a nurse at the hospital said :  follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .", "it is really nice to know that there is this form of support when patients get home  .", "communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .", "doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote :  at meetings we have often asked for the ", "good story. it would make it much more meaningful for us if we were told that mrs .", "that is the most annoying  that we are not told what the results of follow - up home visits are ", "a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .", "gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .", "the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .", "the gps generally see the follow - up home visit as a relevant type of contact .", "a gp said :  it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .", ", it can give the patient extra resources when you visit the patient on the patient 's home ground  .", "the district nurses also generally see the follow - up home visits as a relevant type of contact .", "the gps find that the medication review is an especially important element in follow - up home visits .", "a gp said :  we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes  .", "at the same time the gps also stress that follow - up home visits are not only about reviewing medication :  the purpose of a follow - up home visit is first of all about making a patient review .", "it is not only the medicine we should look at , but the patient  in this sense follow - up home visits are very relevant  .", "the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient :  the guide creates a good structure for the home visit .", "the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are  .", "the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital", "the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector :  the guide is quite voluminous .", "we have already made a lot of the tests at the hospital so there is no need to repeat these at home .", "you should make sure that the gp has read the discharge summary before the follow - up home visit ", "the target group of the programme is elderly and frail patients just out of from hospital .", "a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital", "a nurse at the hospital expressed it this way :  it is typically this type of patient that we see again and again : deteriorating health and a small social network  .", "the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .", "other patients would have benefited from a follow - up home visit but they were not included in the programme ", "gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .", "this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients :  it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .", "a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc . ", "a gp said :  the hospital has contact with the patient at the time of discharge .", "there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .", "it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems :  if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .", "but it is often difficult to find out who is responsible and it is difficult to sort out the problem  ( gp ) .", "the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur :  we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .", "a fax from the hospital to the home nursing services is not enough  ( doctor , hospital ) . in the city of copenhagen", "the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .", "it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having :  follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility  .", "the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .", "today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do  .", "the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .", "these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .", "such information is essential since follow - up home visits are new to many gps .", "the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low : ", "i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases", "i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit ", "the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .", "a gp said :  you get to talk  that is the biggest gain , to get the dialogue .", "the gps want , however , more information about the efficacy of follow - up home visits . gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health :  if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .", "the staff involved in the programme is very positive towards follow - up home visits .", "a nurse at the hospital said :  follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .", "it is really nice to know that there is this form of support when patients get home  .", "communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .", "doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote :  at meetings we have often asked for the ", "good story. it would make it much more meaningful for us if we were told that mrs .", "that is the most annoying  that we are not told what the results of follow - up home visits are ", "the analysis illustrates that it is a big challenge to co - ordinate activities across organizational boundaries and motivate actors to participate in cross - sectoral health programs .", "resources clearly matter ( fees for gps etc . ) but the paper also points to other key factors which need to be taken into consideration when trying to motivate partners to engage actively in a cross - sectoral programme like a follow - up home visit programme . in the following these key factors will be discussed under three sub - headings : the focus of collaboration , a partnership of equals , and the will to co - operate .", "the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .", "the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result , gps and", "district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .", "collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .", "the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result ,", "communication between the gp and the district nurse about a patient 's medicine is typically improved in the treatment following the visit .", "this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .", "the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .", "studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .", "thus , the results indicate that the object at the centre of these efforts  the focus of collaboration  needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .", "inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .", "providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts .", "they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .", "the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .", "this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .", "gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .", "moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .", "this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .", "hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .", "the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .", "the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .", "this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .", "it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .", "if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .", "there needs to be a shared responsibility  a partnership of equals  to engage all partners in a cross - sectoral activity like follow - up home visits .", "research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .", "research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .", "for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .", "they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating .", "the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .", "gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .", "danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .", "the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .", "hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .", "as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .", "district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .", "basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular", "there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .", "follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .", "the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated .", "the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .", "the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result", ", gps and district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .", "collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .", "the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result , communication between the gp and the district nurse about a patient 's medicine", "this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .", "the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .", "studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .", "thus , the results indicate that the object at the centre of these efforts  the focus of collaboration  needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .", "inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .", "providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts .", "they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .", "the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .", "this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .", "gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .", "moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .", "this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .", "hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .", "the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .", "the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .", "this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .", "it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .", "if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .", "there needs to be a shared responsibility  a partnership of equals  to engage all partners in a cross - sectoral activity like follow - up home visits .", "research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .", "research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .", "for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .", "they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating .", "the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .", "gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .", "danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .", "the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .", "hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .", "as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .", "district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .", "basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .", "follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .", "the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated .", "the findings support a basic claim in inter - organizational theory that other factors than resources matter if we want to understand what drives efforts at collaboration .", "this analysis shows that the focus of collaboration needs to be clearly defined and agreed upon , there needs to be a high degree of equality between the professionals involved , and there has to be a will to co - operate based on a shared understanding of values and learning potentials .", "one lesson for current policy is that motivational factors need to be addressed in future collaborative programs in order to fully exploit the potential health benefits .", "the follow - up home visit programme in copenhagen suffered from a lack of motivation among key actors ( especially gps ) to participate and , as a result , readmissions to hospital can be expected to be higher than anticipated prior to the programme .", "susanna bihari axelsson , senior lecturer , associate professor nordic school of public health , box 12133 , nya varvet building 25 , se-402 42 gteborg sweden janne seemann , assistant professor , phd , department of sociology and social work , aalborg university , denmark j.d.h .", "van wijngaarden , dr , assistant professor , institute of health policy and management , erasmus university rotterdam , p.o ."], "abstract_text": ["<S> objectivesthe aim of follow - up visits by the general practitioner and district nurse ( within a week after discharge from hospital ) is to reduce hospital readmissions and improve the overall wellbeing of the patient . </S>", "<S> there is strong evidence that these programmes are effective , but are difficult to implement because of a number of organizational obstacles , including co - ordination between the organizations involved in the process . in this paper </S>", "<S> we look at the factors that affect motivation to participate in a cross - sectoral programme in copenhagen , denmark , implementing follow - up home visits to elderly persons.theory and methodsthe analysis is based on inter - organizational network theory in an attempt to explain the role of motivation in network formation between organizational systems . </S>", "<S> the empirical findings are based on focus groups and in - depth interviews with hospital staff , general practitioners , and district nurses.resultscare providers are motivated to collaborate by a number of factors . </S>", "<S> the focus of collaboration needs to be clearly defined and agreed upon , there needs to be a high degree of equality between the professionals involved , and there has to be a will to co - operate based on a shared understanding of values and learning potentials.conclusionsthe study concludes that we need to focus on specific care fields and actors to reduce complexity in the area and more fully understand what motivates care providers to participate in cross - sectoral activities , such as a follow - up home visit programme . </S>", "<S> one lesson for current policy is that motivational factors need to be addressed in future collaborative programmes in order to fully exploit the potential health benefits . </S>"], "labels": null, "section_names": ["Introduction", "Theoretical framework", "Methodology", "Results", "Relevance of collaboration", "Benefits from collaboration", "Discussion", "The focus of collaboration", "A partnership of equals", "The will to co-operate", "Conclusions", "Reviewers"], "sections": [["inadequate organization has been identified as an important cause of inappropriate courses of treatment for patients in the health care system [ 1 , 2 ] .", "a response to this challenge has been to focus on integrated health care and co - ordination between the actors involved in the process [ 37 ] .", "an illustrative example of this can be found when looking at frail elderly patients on their discharge from hospital .", "studies show that readmission can be diminished by increased collaboration between hospital , district nurses and general practitioners [ 8 , 9 ] .", "follow - up home visits by the district nurse and general practitioner to frail elderly patients after discharge have shown promising results [ 10 , 11 ] .", "the aim of follow - up home visits to elderly and frail persons is to enable them to remain in their own home , avoid readmission to hospital , admission to nursing homes or other forms of sheltered housing and to improve the functional ability and general wellbeing of the elderly .", "a follow - up home visit comprises a home visit by a general practitioner ( gp ) and district nurse within about one week after discharge from hospital .", "the visit is expected to last approximately one hour , a relatively long period compared to other types of doctor - patient contact .", "this visit can be supplemented with two subsequent home visits or visits to the gp if this is considered necessary .", "the latest study from denmark has shown that the readmission rate in the intervention group was 40% while it was 53% in the control group .", "at the same time , studies have shown that there are indications that patients prefer primary care follow - up to specialist care .", "these results are overall convincing and , as a result , on - going work is being done by health authorities to test and further develop follow - up home visits as an initiative , including a programme in copenhagen carried out from 20082011 .", "the copenhagen programme was conducted as a randomized controlled trial which implies strict procedures for the implementation process .", "a total of approximately 300 patients from bispebjerg hospital ( a major hospital in copenhagen ) were included in the intervention and control group .", "approximately 100 follow - up home visits to patients in the intervention group have been made . in the remaining approximately 200 cases in the intervention group it has not been possible to carry out a follow - up home visit , first of all because gps could not meet the requirements . in this feasibility study gps", "were not carefully selected for the programme as they were in an earlier study , and , as a result , more gps chose not to take part in the programme .", "the effect of the copenhagen programme will be reported in 2012 by the danish institute of health services research .", "this paper will focus on the implementation of follow - up home visits using a qualitative methodological approach .", "we know some of the general dynamics in the area   but we do not know more precisely what the practical limitations of the home visitor 's programmes are and how these limitations could make a difference to the outcomes assessed [ 14 , 15 ] . why do so few gps take part in the follow - up home visit programme ?", "why is the documented effect of such visits not motivation enough for gps to encourage them to do so ?", "addressing such questions could help us understand how to design and implement these visits in order to fully exploit the potential benefits of the intervention .", "moreover , such results could generate useful hypotheses which could be addressed in future trials attempting to examine the effects of home visiting programmes .", "the aim of this study is to explore one particular aspect of follow - up home visits : the motivation and rationale of local care providers to invest time and effort in follow - up home visits .", "what is the main motivation of gps , district nurses and hospital staff to take part in a cross - sectoral activity like follow - up home visits ?", "the case study is placed in a danish setting . in order to understand the organizational dynamics involved a short description of the setting", "is needed : the danish health care system is mainly a public system based on general taxation , and characterized by universal access to health care services .", "gps have a central position in the system since they have a kind of  gatekeeper  function , they are usually the first professional confronted with the patient 's problems and they see it as their responsibility to guide patients through the system .", "gps are self - employed , general practices are small and typically privately owned ; only a minority of gps are organized in health centres .", "home care organizations ( district nurses ) have occupied an ever more central role since a major administrative reform in 2007 gave the local level the main responsibility for preventive health care .", "co - ordination between organizational units in primary and secondary health care is determined by agreements between regional and municipality authorities , which include procedures for such cases as when frail and elderly people are discharged from hospital .", "in general , the danish system is considered quite integrated , in particular because the climate of trust in denmark makes co - operation possible [ 7 , 16 ] . as a result ,", "follow - up home visits and other cross - sector interventions , such as  follow - home arrangements  and  case managers  have been quite regularly implemented on an experimental basis as a possible alternative to hospital - based measures .", "the interventions have shown promising results but studies in the area also point to problems in connection with implementing the interventions [ 7 , 8 , 10 , 12 , 17 , 18 ] ."], ["empirical data were gathered to develop hypotheses and theoretical concepts . at the same time , however , a rough theoretical framework was used as an underlying foundation in the study to ensure that a relevant focus and approach were applied .", "resource dependency theory tells us that network formation can largely be explained by an organization 's need to reduce costs or gain resources and power .", "organizations only work together when administrative and economic structures are designed which makes co - operation a profitable pursuit .", "organizations thus typically follow their own agenda rather than involve themselves in cross - sectoral activities . in the health sector some support can be found for this basic claim .", "studies have shown that network formation in general is limited and poor co - ordination predominant in the area [ 13 , 21 ] .", "resource dependency theory also tells us , however , that organizations need to reduce uncertainty . to reduce uncertainty , organisations tend to co - operate with organisations they trust and depend on to achieve common goals [ 19 , 22 ] .", "hence , in order to understand co - operation you also need to look at the question of trust and the quality of inter - organizational networks , as done in inter - organizational network theory .", "this perspective has shown us that we need to have a comprehensive view of the factors which make health sector organisations work together .", "cross - sectoral programs and inter - organizational activities are not based purely on partners ' own needs and resources [ 23 , 24 ] .", "motivation plays a key role in inter - organizational theory [ 22 , 25 ] .", "working together is a challenging task ; it is typically regarded as uncertain whether it will bring benefits equivalent to the resources invested .", "motivation and commitment to the process are therefore needed among the actors involved in order to keep things moving and overcome the uncertainties inherent in the process . what motivates organizations to work together ?", "the literature points to two aspects in particular : the object needs to be seen as highly relevant and benefits should be obvious for all involved [ 22 , 25 ] ."], ["this paper is based on an evaluation report made by one of the authors for health and social care , city of copenhagen .", "the interviews were structured around the following questions : \n to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant?how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ? are the benefits obvious ? \n  to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant ? how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ?", "two focus groups and seven individual interviews were conducted in march  may 2010 , giving a total of 23 respondents .", "the focus group approach was chosen because the interaction between some of the key respondents ( hospital staff , district nurses ) was considered to be of importance , since a key goal in the study was to understand the shared views on collaboration among these key actors in the programme .", "one focus group interview was conducted with seven employees at bispebjerg hospital who were involved in the follow - up home visit programme .", "recruitment criteria were designed in order to include a suitable spread of respondents , i.e. to ensure that members of all staff groups directly involved in the project were represented .", "we ensured that the project co - ordinator at the hospital responsible for the selection of patients for the programme was represented in the focus group . at the same time we also ensured that a doctor and nurses from the most relevant wards were represented .", "another focus group interview was conducted with nine district nurses who had been involved in the project .", "nurses from different districts in copenhagen were selected for the focus group in order to ensure that different ways of organising follow - up home visits were represented .", "gps were not as motivated to take part in the research project as district nurses and hospital staff , so a less time - consuming method than focus groups was considered appropriate . as it turned out , in - depth interviews proved to be highly relevant for gps because gps had divergent and less firm views about follow - up home visits than other groups in the study and the methodological design allowed these views to be fully expressed .", "five individual interviews were held with gps involved in the project . both gps with a positive attitude towards follow - up home visits and gps with a less positive attitude were recruited to ensure that a wide range of attitudes was included in the study .", "these recruitment criteria were based on data registered by the city of copenhagen showing which gps had refused to participate in follow - up home visits and which had agreed . at the same time we ensured that the gps represented covered different districts in copenhagen .", "two individual interviews were held with employees from referrals in the city of copenhagen home nursing services .", "there is a purchaser - provider split in the city of copenhagen , and the referrals purchase while the district nurses provides .", "the two referrals came from different districts and had different positions in the organization , ensuring that the most important experiences from this organization were covered .", "the focus group interviews took place in a neutral meeting room arranged by the project manager , and the in - depth interviews took place in the gps ' offices .", "the focus group interviews lasted between 90 and 120 minutes , and the in - depth interviews between 25 and 60 minutes . both were recorded with a digital voice recorder and transcribed verbatim .", "the interview transcripts were analyzed under the two headings which form the basic empirical questions in the study : relevance of collaboration and benefits of collaboration .", "quotes from the interviews were grouped under each heading in order to find the quotes which were most illustrative of the views of gps , district nurses and hospital staff . to ensure reliability ,", "results were presented and discussed in a steering group with representatives from the main professional groups involved in the programme ( gps , district nurses and hospital staff ) ."], ["a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .", "gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .", "the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .", "the gps generally see the follow - up home visit as a relevant type of contact .", "a gp said :  it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .", ", it can give the patient extra resources when you visit the patient on the patient 's home ground  .", "the district nurses also generally see the follow - up home visits as a relevant type of contact .", "the gps find that the medication review is an especially important element in follow - up home visits .", "a gp said :  we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes  .", "at the same time the gps also stress that follow - up home visits are not only about reviewing medication :  the purpose of a follow - up home visit is first of all about making a patient review .", "it is not only the medicine we should look at , but the patient  in this sense follow - up home visits are very relevant  .", "the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient :  the guide creates a good structure for the home visit .", "the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are  .", "the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital", "the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector :  the guide is quite voluminous .", "we have already made a lot of the tests at the hospital so there is no need to repeat these at home .", "you should make sure that the gp has read the discharge summary before the follow - up home visit ", "the target group of the programme is elderly and frail patients just out of from hospital .", "a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital", "a nurse at the hospital expressed it this way :  it is typically this type of patient that we see again and again : deteriorating health and a small social network  .", "the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .", "other patients would have benefited from a follow - up home visit but they were not included in the programme  gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .", "this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients :  it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .", "a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc . ", "a gp said :  the hospital has contact with the patient at the time of discharge .", "in the present situation there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .", "it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems :  if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .", "but it is often difficult to find out who is responsible and it is difficult to sort out the problem  ( gp ) .", "the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur :  we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .", "a fax from the hospital to the home nursing services is not enough  ( doctor , hospital ) . in the city of copenhagen", "the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .", "it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having :  follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility  .", "the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .", "today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do  .", "the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .", "these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .", "such information is essential since follow - up home visits are new to many gps .", "the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low : ", "i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases", "i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit ", "the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .", "a gp said :  you get to talk  that is the biggest gain , to get the dialogue .", "we work in two parallel organizational systems . by not having the dialogue we as gps miss some observations .", "the gps want , however , more information about the efficacy of follow - up home visits .", "gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health :  if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .", "information could be better  ( gp ) . at the hospital the staff involved in the programme", "a nurse at the hospital said :  follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .", "it is really nice to know that there is this form of support when patients get home  .", "communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .", "doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote :  at meetings we have often asked for the ", "good story. it would make it much more meaningful for us if we were told that mrs .", "that is the most annoying  that we are not told what the results of follow - up home visits are "], ["a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .", "gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .", "the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .", "the gps generally see the follow - up home visit as a relevant type of contact .", "a gp said :  it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .", ", it can give the patient extra resources when you visit the patient on the patient 's home ground  .", "the district nurses also generally see the follow - up home visits as a relevant type of contact .", "the gps find that the medication review is an especially important element in follow - up home visits .", "a gp said :  we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes  .", "at the same time the gps also stress that follow - up home visits are not only about reviewing medication :  the purpose of a follow - up home visit is first of all about making a patient review .", "it is not only the medicine we should look at , but the patient  in this sense follow - up home visits are very relevant  .", "the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient :  the guide creates a good structure for the home visit .", "the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are  .", "the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital", "the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector :  the guide is quite voluminous .", "we have already made a lot of the tests at the hospital so there is no need to repeat these at home .", "you should make sure that the gp has read the discharge summary before the follow - up home visit ", "the target group of the programme is elderly and frail patients just out of from hospital .", "a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital", "a nurse at the hospital expressed it this way :  it is typically this type of patient that we see again and again : deteriorating health and a small social network  .", "the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .", "other patients would have benefited from a follow - up home visit but they were not included in the programme "], ["gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .", "this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients :  it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .", "a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc . ", "a gp said :  the hospital has contact with the patient at the time of discharge .", "there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .", "it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems :  if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .", "but it is often difficult to find out who is responsible and it is difficult to sort out the problem  ( gp ) .", "the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur :  we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .", "a fax from the hospital to the home nursing services is not enough  ( doctor , hospital ) . in the city of copenhagen", "the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .", "it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having :  follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility  .", "the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .", "today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do  .", "the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .", "these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .", "such information is essential since follow - up home visits are new to many gps .", "the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low : ", "i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases", "i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit ", "the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .", "a gp said :  you get to talk  that is the biggest gain , to get the dialogue .", "the gps want , however , more information about the efficacy of follow - up home visits . gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health :  if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .", "the staff involved in the programme is very positive towards follow - up home visits .", "a nurse at the hospital said :  follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .", "it is really nice to know that there is this form of support when patients get home  .", "communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .", "doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote :  at meetings we have often asked for the ", "good story. it would make it much more meaningful for us if we were told that mrs .", "that is the most annoying  that we are not told what the results of follow - up home visits are "], ["the analysis illustrates that it is a big challenge to co - ordinate activities across organizational boundaries and motivate actors to participate in cross - sectoral health programs .", "resources clearly matter ( fees for gps etc . ) but the paper also points to other key factors which need to be taken into consideration when trying to motivate partners to engage actively in a cross - sectoral programme like a follow - up home visit programme . in the following these key factors will be discussed under three sub - headings : the focus of collaboration , a partnership of equals , and the will to co - operate .", "the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .", "the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result , gps and", "district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .", "collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .", "the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result ,", "communication between the gp and the district nurse about a patient 's medicine is typically improved in the treatment following the visit .", "this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .", "the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .", "studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .", "thus , the results indicate that the object at the centre of these efforts  the focus of collaboration  needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .", "inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .", "providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts .", "they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .", "the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .", "this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .", "gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .", "moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .", "this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .", "hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .", "the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .", "the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .", "this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .", "it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .", "if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .", "there needs to be a shared responsibility  a partnership of equals  to engage all partners in a cross - sectoral activity like follow - up home visits .", "research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .", "research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .", "for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .", "they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating .", "the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .", "gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .", "danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .", "the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .", "hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .", "as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .", "district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .", "basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular", "there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .", "follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .", "the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated ."], ["the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .", "the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result", ", gps and district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .", "collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .", "the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result , communication between the gp and the district nurse about a patient 's medicine", "this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .", "the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .", "studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .", "thus , the results indicate that the object at the centre of these efforts  the focus of collaboration  needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .", "inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .", "providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts ."], ["they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .", "the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .", "this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .", "gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .", "moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .", "this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .", "hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .", "the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .", "the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .", "this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .", "it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .", "if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .", "there needs to be a shared responsibility  a partnership of equals  to engage all partners in a cross - sectoral activity like follow - up home visits .", "research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .", "research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .", "for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .", "they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating ."], ["the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .", "gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .", "danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .", "the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .", "hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .", "as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .", "district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .", "basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .", "follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .", "the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated ."], ["the findings support a basic claim in inter - organizational theory that other factors than resources matter if we want to understand what drives efforts at collaboration .", "this analysis shows that the focus of collaboration needs to be clearly defined and agreed upon , there needs to be a high degree of equality between the professionals involved , and there has to be a will to co - operate based on a shared understanding of values and learning potentials .", "one lesson for current policy is that motivational factors need to be addressed in future collaborative programs in order to fully exploit the potential health benefits .", "the follow - up home visit programme in copenhagen suffered from a lack of motivation among key actors ( especially gps ) to participate and , as a result , readmissions to hospital can be expected to be higher than anticipated prior to the programme ."], ["susanna bihari axelsson , senior lecturer , associate professor nordic school of public health , box 12133 , nya varvet building 25 , se-402 42 gteborg sweden janne seemann , assistant professor , phd , department of sociology and social work , aalborg university , denmark j.d.h .", "van wijngaarden , dr , assistant professor , institute of health policy and management , erasmus university rotterdam , p.o ."]]}
{"article_id": "PMC3443483", "article_text": ["alzheimer s disease ( ad ) is a progressive neurodegenerative disorder starting with mild memory loss and manifesting in severe cognitive decline .", "neuropathologically , the disease is characterized by an extensive deposition of amyloid  ( a ) peptides in extracellular amyloid plaques and by intracellular neurofibrillary tangles ( nfts ) of hyper - phosphorylated tau protein .", "sporadic late - onset ad is the most common form of dementia in the elderly and is strongly associated with age . in rare cases", "( < 5% ) , ad is inherited and results in an early disease onset .", "one genetic risk factor for late - onset ad that has been reported throughout many studies is apolipoprotein e ( apoe ) which physiologically functions as a ligand in receptor - mediated endocytosis of lipoprotein particles [ 2 , 3 ] .", "several single nucleotide polymorphisms in apoe lead to alterations in the coding sequence and result in three common isoforms called apoe2 , apoe3 and apoe4 with the e4 allele being an ad risk factor and e2 being protective [ 46 ] .", "strong evidence suggests that apoe influences ad via its effect on a metabolism ; however , the details of this process have to be fully elucidated .", "positional cloning studies of familial ad ( fad ) cases have identified mutations in three genes , amyloid precursor protein ( app ) , presenilin 1 and 2 ( ps1 , ps2 ) , which are tightly linked to the generation of a peptides .", "proteolytic processing of amyloid precursor protein ( app ) by bace ( -site app cleaving enzyme ) is followed by cleavage by -secretase containing presenilins and results in the release of a peptides of different length . a40 and a42 ( 40 or 42 amino acids , respectively )", "are the most prominent a peptides and since the c - terminus has important implication for toxicity , the longer a42 peptide exhibits stronger neurotoxic properties .", "the fad - linked mutations cause an elevated production of amyloidogenic a , which is the basis for the amyloid hypothesis of ad , stating that a peptides are the key - players in neuronal dysfunction and subsequent neurodegeneration in ad .", "recently , it has been suggested that the soluble oligomeric forms of a42 are the potentially harmful species and that these oligomers affect various important cellular mechanisms , resulting in decreased functionality and survival of neuronal cells .", "however , a peptides are not exclusively generated under pathological conditions as they are part of the normal cellular metabolism and a significant load of amyloid plaques is also observed in brains of healthy aged individuals [ 10 , 11 ] .", "this challenges the amyloid hypothesis of ad and up to date the nature of the disease - relevant alteration in aged neurons remains unclear .", "although alois alzheimer already described protein deposits in the brain of demented patients , the question why distinct proteins that are potentially causative for the disease accumulate in discrete brain regions in the course of aging remains unanswered .", "today , we know that the progressive deposition of misfolded and aggregation - prone proteins in defined regions of the nervous system is not unique to ad , but a well - described characteristic of several neurodegenerative disorders , including parkinson s disease ( pd ) , huntington s disease ( hd ) and amyotrophic lateral sclerosis ( als ) . recently , it has been proposed that the accumulation of disease - relevant proteins in aggregates might actually even be considered as a protective mechanism to dispose these proteins and remove them from the cellular metabolism [ 13 , 14 ] . generally , within the crowded cellular environment", ", proteins are always at risk for denaturation . changing genetic and environmental factors , as during pathology or aging , challenge the integrity of the cellular protein homeostasis ( proteostasis ) . therefore , the stability and metabolism of the proteome needs to be controlled carefully and is carried out by a complex network of several hundred evolutionarily conserved proteins .", "this network , including molecular chaperones , the ubiquitin  proteasome system ( ups ) and the autophagy system is stringently regulated and indispensable for the maintenance of proteostasis .", "all main players of this network are ubiquitously expressed and consequently also present in neuronal tissue .", "molecular chaperones , most be prominently represented by the heat shock protein 70 ( hsp70 ) family and their regulators , are responsible for correct folding and refolding of proteins as well as for the transport of misfolded proteins to the protein - degradation systems .", "the chaperone network is controlled by the activity of transcription factors , e.g. heat shock transcription factor 1 ( hsf1 ) , which rapidly adjusts the cellular chaperone capacity during stress conditions .", "the ups is the major protein - degradation machinery of the cell and controls the metabolism of cytosolic proteins and the degradation of misfolded proteins .", "proteins that are destined for proteasomal degradation become specifically tagged with the protein ubiquitin in a well - organized enzymatic cascade .", "interestingly , also a ubiquitin - independent way of proteasomal degradation has been described recently that mostly involves small and misfolded proteins . as an additional mechanism for protein degradation", "the major autophagic systems described so far are macroautophagy and chaperone - mediated autophagy ( cma ) .", "macroautophagy is a complex process that involves the formation of a membrane structure , the autophagosome , which engulfs bulk cellular material ( proteins and organelles ) and subsequently fuses with the lysosome .", "cma involves the constitutively expressed heat shock cognate 70 ( hsc70 ) that targets client proteins with a specific consensus motif directly to the lysosome .", "in fact , hsp70 is also an important factor to assure selectivity of macroautophagy of degradation - prone proteins in a distinct pathway , mediated via the co - chaperone bcl2-associated athanogene 3 ( bag3 ) . aging and disease challenge the proteostasis network and the accumulation of instable proteins results in wide - spread protein aggregation [ 21 , 22 ] .", "our discussion presented here attempts to integrate two major factors that are associated with neurodegeneration , protein homeostasis and aging .", "most importantly , we focus on the role of specific pathways which are needed to restore and maintain proteostasis during aging and their interplay with a special focus on impaired proteostasis in ad , which may add to a novel view and understanding of ad pathogenesis .", "since recently many excellent reviews on various aspects providing in depth discussions have been presented , we are concentrating here on the integration of these different aspects .", "therefore , we frequently refer to review work with the recommendation of further reading rather than trying to fully cover the discussed issues by their original publications .", "cells permanently encounter the problem to maintain the integrity and functionality of the proteome . within the crowded cellular environment ,", "the correct conformation of proteins must be controlled and misfolded and irreversibly damaged proteins must be efficiently refolded or removed .", "central players of the protein homeostasis system are molecular chaperones that sense misfolded proteins and , when refolding fails , direct them to the protein - degradation pathways .", "molecular chaperones are specified as proteins that interact with and participate in folding or refolding of non - native proteins .", "therefore , chaperones help unfolded proteins to achieve their functional conformation without being present in the final structure [ 15 , 2327 ] .", "they exert a multitude of activities , including de novo folding , refolding of denatured proteins , transport to subcellular compartments , oligomeric assembly and disposal by proteolytic degradation [ 28 , 29 ] .", "different classes of chaperones work together to form co - operative networks and are often termed heat shock proteins ( hsps ) , because they are sensitively up - regulated under stress conditions in which the amount of misfolded proteins is increased .", "chaperones , which are involved in protein folding and refolding , such as hsp70 , hsp90 and chaperonin ( hsp60 ) , promote folding activity through atp / cofactor - regulated binding and release cycles [ 15 , 31 ] .", "they recognize short hydrophobic amino acid stretches of misfolded proteins and can co - operate with atp - independent chaperones , e.g. small hsps ( shsps ) , to prevent protein aggregation .", "hsp70 proteins are central players in proteostasis control and increasing hsp70 levels prevent protein aggregation in various disease models that are based on the expression of aggregation - prone proteins [ 32 , 33 ] .", "the activity of hsp70 is atp dependent and is controlled by chaperones of the hsp40 family as well as nucleotide exchange factors ( nefs ) [ 34 , 35 ] .", "after atp hydrolysis , a nef binds to the atpase domain of hsp70 and catalyzes the nucleotide exchange , which results in substrate release .", "thus , chaperone binding to the hydrophobic region of misfolded proteins transiently blocks aggregation and the subsequent atp - dependent release allows controlled folding of the client protein .", "hsp40/dnajs , hsc70-interacting protein ( hip ) , bcl2-associated athanogene 1 ( bag1 ) and bag3 are prominent co - chaperones that control the atp - dependent activity of hsp70 .", "dnaj proteins generally enhance the atpase activity of hsp70 , but their action can lead to different fates of their bound substrates .", "dnajb1 is an example for a dnaj protein that supports substrate folding , whereas dnajb2-bound clients are degraded by the proteasome .", "the co - chaperone hip stabilizes the adp - bound state of hsp70 and co - operates with hsp70 in protein folding .", "other hsp70-binding cofactors are the ubiquitin ligases carboxy terminus of hsc70-interacting protein ( chip ) and parkin , which provide a link between hsp70 , co - chaperones and the ups , resulting in client ubiquitination and degradation by the proteasome [ 34 , 38 , 39 ] .", "recently , it has been shown that bag3 interacts with hsp70 and directs the chaperone and its substrates into the autophagic pathway , providing an association of hsp70 , co - chaperones and autophagy .", "an extensive description of the hsp70 machinery , the exact structure - function relationship between hsp70 and its various cofactors , has been excellently reviewed elsewhere .", "the functional variability of the chaperone system that is exerted by a multitude of single proteins highlights the complexity of this proteostasis network .", "however , limited knowledge exists on the exact composition and capacity of the chaperone system within a cell or a certain tissue .", "a general view assumes that total levels of cellular chaperones exceed the actual requirements and that a sufficient amount of chaperones is free of clients .", "thus , the network has excess capacity to initially deal with sudden additional chaperone requirements , as exhibited during stress conditions .", "an opposite view proposes that the capacity of the chaperone network is always closely titrated to the actual demand of chaperone activity and that the system is otherwise rapidly adapted to conditions of increased requirements .", "the analysis of the chaperone capacity of neuronal and muscular tissue of caenorhabditis elegans ( c. elegans ) demonstrates that these tissues indeed display different folding activities and that neurons are particularly sensitive to protein denaturation during heat stress . however , the adaptation of the chaperone network during aging is critical as it is well acknowledged that protein aggregation and disruption of proteostasis are characteristic for aged cells .", "the periodical application of mild heat stress decreases mortality in drosophila melanogaster and c. elegans , which is mediated by hsp70 activity and the overexpression of hsp70 in c. elegans increases the life span of the nematode [ 4345 ] .", "several reports have analyzed chaperone protein or mrna levels in aged cells and found increased or basal amounts , whereas the stress - mediated induction of chaperone expression is impaired .", "the transcription of chaperone genes in response to stress conditions is controlled by the transcription factor hsf1 , which shows an impaired dna - binding potential in aged cells .", "a reduced activity of hsf1 in c. elegans results in a shortened life span and , conversely , the enhanced expression of the transcription factor increases the life span .", "activity is also essential for the extended life span of the extremely long - lived daf-2/insulin / igf-1 receptor mutants of c. elegans [ 47 , 48 ] .", "thus , hsf1 and chaperone activity can promote longevity , demonstrating a clear association of chaperones , proteostasis and aging .", "some of the factors mentioned so far are involved in linking chaperone functions with cellular protein - degradation pathways , the ups and autophagy , for the removal of misfolded proteins . besides protein aggregation ,", "one factor that induces ubiquitination is protein damage caused by free radical oxygen species ( ros ) and oxidative stress .", "most likely , irreversible oxidation may activate chaperones and the ups to induce protein repair of misfolded proteins and lead to ubiquitination and protein degradation . during aging ,", "in particular , the mitochondrial respiratory chain is strongly linked to the production of ros and as one consequence may cause protein dysfunction , apoptosis , necrosis , aging and disease [ 49 , 50 ] .", "protein oxidation leads to a change in protein conformation and function and chaperones may sense such changes and in turn activate the ups as a quality control system .", "depending on the degree of oxidation , irreversible oxidation and loss of protein function may lead to degradation and/or accumulation of damaged proteins and to the formation of so - called aggresomes , which display a high autophagic activity [ 51 , 52 ] .", "the ups is a complex enzymatic pathway that starts with the ligation of ubiquitin , a 76-amino - acid - long and highly conserved protein , to other cellular proteins and thus labels them for degradation .", "initially the c - terminal end of ubiquitin is activated by atp - dependent phosphorylation and formation of a thiol ester via an activating enzyme , e1 .", "it is then transferred to a thiol group of an ubiquitin - carrier protein , e2 .", "the e3 ligase directs ubiquitin from e2 to an -amino group of the target protein [ 53 , 54 ] .", "the c - terminus of an additional ubiquitin protein can be ligated onto one of the seven lysine residues within the attached ubiquitin molecule . for degradation via the proteasome", "ubiquitin linkages between either the c - terminus and lysine residues k48 or k63 are the major recognition signals for proteasomal degradation .", "ubiquitin chains also occur among other lysine residues , whereas ubiquitin extension via k6 is associated with dna repair , k11 with endoplasmatic reticulum - associated protein degradation and cell cycle regulation , k27 with ubiquitin fusion degradation , k29 with lysosomal degradation and k33 with kinase modification .", "monoubiquitination can modify the activity of the protein transport machinery and when attached to transmembrane proteins can serve as a sorting signal to direct their movement between different cellular compartments [ 5659 ] . the polyubiquitinated proteins destined for degradation", "are processed by the multienzymatic proteasome complex : first they become deubiquitinated and then degraded by the 26s proteasome , a system that is composed of various proteasome subunits .", "the eukaryotic 20s proteasome core complex consists of four heptameric rings comprising two classes of seven non - identical but homologous subunits .", "the outer rings contain alpha - type subunits with gating function for substrate entrance and product release , while the beta - subunits exhibit peptide hydrolyzing activity .", "the 19s regulatory complex binds to the 20s catalytic core in a flexible manner and consists of six atpases , forming a ring at the entrance of the core and exerting chaperone - like activity .", "the ups is the major degradation system in the cell for the degradation of short - lived , misfolded and defective proteins .", "an accumulation of polyubiquitinated proteins is reported for numerous neurological disorders , which suggests that ups dysfunction plays a prominent role in the pathogenesis of neurodegenerative diseases and moreover in aging . since proteasomal", "activity decreases during aging over all , the degradation demand of the cell increases , which results in an age - associated induction of the autophagy pathway that delivers substrates for degradation ultimately to lysosomes [ 20 , 63 ] .", "autophagy is negatively regulated via the evolutionarily conserved enzymatic mammalian target of rapamycin complex 1 ( mtorc1 ) . in turn , if mtor activity is inhibited by rapamycin , the autophagy pathway is switched on .", "rapamycin exerts its stimulatory effect on autophagy by preventing mtor phosphorylation at ser-2448 and thereby subsequently blocks mtor signaling .", "so far , many different autophagic systems have been described including macroautophagy and cma that form the main pathways .", "macroautophagy is a multi - step process by which cytosolic material is sequestered into a double - layered membrane structure , the autophagosome , and is delivered to the lysosome for degradation .", "it is a highly orchestrated process and involves autophagy - related ( atg ) proteins .", "more than 30 genes have been identified in genetic analyses of yeast mutants which have defects in autophagic function .", "after autophagy stimulation through mtor inhibition , the formation of phagophores ( autophagosome precursors / pre - autophagosomal structures ) is initiated in a not yet completely understood mechanism .", "a subset of atg proteins is required for autophagosome formation : first , the atg9 system including atg9 , the atg1 kinase complex ( atg1 and atg13 ) , atg2 and atg18 ; second , the phosphatidylinositol 3-oh kinase ( pi(3)k ) complex consisting of vacuolar protein sorting ( vps)34 , vps15 , beclin 1/atg6 and atg14 and third , the ubiquitin - like protein ( ubl ) system composed of two ubl proteins ( lc3/atg8 and atg12 ) , an e1 enzyme ( atg7 ) , two analogues of e2 ubiquitin - conjugating enzymes ( atg10 and atg3 ) , an lc3/atg8-modifying protease ( atg4 ) , the protein target of atg12 attachment ( atg5 ) and atg16 .", "beclin 1/atg6 regulates autophagic trafficking and membrane trafficking in a variety of physiological and pathological processes .", "microtubule - associated protein 1 light chain 3 ( lc3/atg8 ) is commonly used as a marker for autophagic activity . with its e1-like activity , atg7 converts cytosolic lc3-i to the membrane - associated lc3-ii , which then is conjugated to phosphatidylethanolamine .", "lc3-ii binds the inner and outer membrane of the forming autophagosome and provides a physical link between the autophagosome and lc3 interacting proteins , such as sequestosome 1 ( p62/sqstm1 ) or neighbor of brca1 gene 1 ( nbr1 ) .", "these so - called autophagy receptors can simultaneously bind to ubiquitinated proteins and lc3 , employing their lc3-interacting motif and ubiquitin - associated domain .", "the identification of autophagy receptors , such as p62/sqstm1 and nbr1 has provided a molecular link between ubiquitination and autophagy .", "thus , p62/sqstm1 and nbr1 are possibly also important bridging factors between ups and autophagy . through self - oligomerization , which is stimulated by ubiquitin binding , p62/sqstm1 sequesters ubiquitinated substrates in form of inclusion bodies .", "these inclusions are then specifically engulfed by the autophagosome membrane by recruiting lc3 [ 69 , 70 ] . while macroautophagy is considered as a rather unspecific robust degradation process , cma is a highly selective lysosomal pathway that removes a distinct subset of proteins containing a pentapeptide lysosome - targeting motif .", "these substrates can on the one hand directly be translocated into the lysosome after docking to the lysosomal receptor lysosomal - associated membrane protein 2a or on the other hand unfolded by a chaperone complex containing hsc70 and the co - chaperones bag1 , hip , hsp70hsp90 organizing protein ( hop ) and hsp40/dnajb1 .", "these co - chaperones provide a direct link to the ups and the folding and refolding activity of molecular chaperones .", "in the course of aging , a variety of proteins tend to aggregate and impair the ups directly .", "therefore , these proteins , most of which are ubiquitinated , can not be handled by the proteasome and have to be degraded by different protein clearance pathways .", "recently , it has been shown that the cellular protein quality control ( pqc ) of polyubiquitinated proteins by proteasomal and autophagic systems is regulated by the hsp70 co - chaperones bag1 and bag3 , respectively .", "proteasome activity decreases in an age - dependent manner in a cell model of replicative senescent human fibroblasts and is associated with an increased autophagic activity in aged cells .", "this correlates with decreased levels of bag1 and increased levels of bag3 in aged cells , an age - related bag1/bag3 switch that serves as an induction control of the macroautophagic pathway ( bag3-mediated selective macroautophagy ) and may be considered as a backup of the ups in pqc ( [ 40 , 72 ] ) .", "therefore , the expression shift from bag1 to bag3 during aging , but also upon acute stress ( e.g. proteasome inhibition , oxidative stress ) , can be considered as a physiologically important adaptive response [ 20 , 40 ] .", "the bag3-mediated selective pathways have been shown to be involved in a variety of disease causing processes . in a model for hd ,", "bag3 in concert with hspb8 facilitates the disposal of polyq43-huntingtin by stimulating macroautophagy and this complex is also involved in z - disc maintenance in flies , mice and men [ 73 , 74 ] . additionally , the transport of target proteins to the aggresome , a compartment with high autophagic activity , has been shown to be mediated by bag3 .", "this aggresome - targeting pathway involving bag3 and hsp70 is distinct from other before - described mechanisms as it does not depend on substrate ubiquitination . taken together the molecular chaperone machinery , the ups and the autophagy system are involved in pqc and maintaining proteostasis within aging and disease to prevent protein misfolding and aggregation .", "a summary of the main routes for protein degradation is shown in fig .  1 .", "as aggregated proteins are found in ad patients and aging is a prevailing risk factor for ad it is important to further characterize how these pathways are regulated and how misregulation possibly contributes to the pathology of ad.fig .", "1the ubiquitin  proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria ) the ubiquitin  proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria )", "ad is characterized by the accumulation of intracellular a ( oligomers ) , extracellular high molecular weight deposits of a peptides ( fibrilliar forms ) , and the intracellular aggregation of hyper - phosphorylated tau . indeed ,", "modification of the cellular proteostasis and the metabolism of a and tau have been proven to cause neuronal dysfunction and cell death .", "the malfunctioning proteostasis control results in the accumulation of misfolded and aggregated proteins , which is a general hallmark of aging and neurodegeneration , as observed in pd , hd , als and ad . aging in particular is characterized by decreasing proteostasis capacity and increasing protein damage which are in combination a major challenge for the cell due to the inability to maintain metastable proteins in folded states . to counteract a cascade of protein destabilization caused by metastable proteins which escaped the pqc", "the proteostasis network including molecular chaperones , the ups and autophagic pathways must manage the increasing burden of protein misfolding to maintain proteome stability .", "since aging is the major risk factor of ad [ 7880 ] , the age - dependent loss of proteostasis might be an important contributor to the pathology of ad .", "an overview of proteins that affect proteostasis in ad is shown in table  1.table 1proteins affecting proteostasis in alzheimer s diseaseproteinfunctionreferencea40/42decreases proteasome activity , modulates autophagy through mtor signaling[99102 , 104 , 143146]tauinhibitory effect of tau aggregates on proteasome activityhsf1induces app gene during stress[81 , 82]grp78 ( er isoform of hsp70)modulates app maturation and reduces a40/42 secretion[84 , 85]hsps ( hsp22 , 27 , 70 , 90)small hsps ( hsp22 , hsp27 ) bind to fibrillar amyloid plaques and inhibit their fibrillarisation ; hsp70 , hsp90 inhibit early stages of amyloid aggregation[88 , 89]chipe3 enzyme for phosphorylated tau[93 , 117]bag1regulates proteasomal degradation of tau together with hsp70ps1increases production of a42 , essential for lysosomal proteolysis and autophagy by enabling the acidification of lysosomes required for protease activation[163 , 165]ubb+1potently inhibits the degradation of polyubiquitinated substrates and therefore induces neuronal cell death[120 , 121]uch - l1dub needed for proteasomal degradation of client proteins , oxidized and down - regulated in ad[124 , 125]ubqln1ubqln1 activity is necessary to regulate the production of app and app fragments[127 , 128]mtorinhibitory and/or activating modulation of mtor through a42 but not a40[143146]beclin1beclin1 is down - regulated in ad brains and consequently increases app levels and its metabolites[161 , 162]a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum proteins affecting proteostasis in alzheimer s disease a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .", "interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .", "as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .", "there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .", "the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .", "in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .", "the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .", "moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .", "hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,", "furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1  day and decreased the amount after 3  days in vitro .", "these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .", "besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .", "post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .", "these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .", "hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .", "hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .", "protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .", "vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models", ", it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .", "tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .", "collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . the ubiquitin ", "proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .", "a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .", "a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .", "it still has to be elucidated which form of a is now affecting proteasome function .", "there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell", "free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .", "there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .", "consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .", "so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons", ", it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain", ", a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism", "thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .", "this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .", "the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .", "furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .", "it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .", "in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .", "in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .", "these data show that different aggregation states of tau modify its turnover via the proteasome .", "it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,", "the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .", "in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .", "moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .", "taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .", "ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .", "overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .", "oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .", "it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .", "uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .", "interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .", "several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .", "for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .", "recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .", "in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .", "taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .", "this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .", "ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .", "interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration .", "in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .", "these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .", "these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .", "a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .", "neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .", "furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .", "genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients .", "however , enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .", "the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .", "attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .", "interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself . the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and", "hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .", "furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .", "in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .", "similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .", "interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .", "the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .", "these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .", "interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .", "later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .", "this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .", "the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .", "thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner .", "consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .", "the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .", "the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .", "this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .", "interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .", "as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .", "hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .", "another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .", "beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .", "in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .", "moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .", "however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .", "recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .", "ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,", "the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .", "this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .", "-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .", "in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .", "the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease .", "taken together , all these findings suggest an important role for autophagy in the pathogenesis of ad .", "the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .", "interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .", "as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .", "there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .", "the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .", "in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .", "the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .", "moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .", "hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,", "furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1  day and decreased the amount after 3  days in vitro .", "these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .", "besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .", "post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .", "these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .", "hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .", "hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .", "protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .", "vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models", ", it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .", "tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .", "collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation .", "the ubiquitin  proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .", "a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .", "a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .", "it still has to be elucidated which form of a is now affecting proteasome function .", "there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell", "free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .", "there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .", "consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .", "so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons", ", it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain ,", "a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism", "thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .", "this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .", "the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .", "furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .", "it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .", "in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .", "in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .", "these data show that different aggregation states of tau modify its turnover via the proteasome . particularly intriguing", "it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,", "the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .", "in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .", "moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .", "taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .", "ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .", "overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .", "oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .", "it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .", "uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .", "interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .", "several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .", "for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .", "recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .", "in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .", "taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .", "this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .", "ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .", "interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration .", "in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .", "these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .", "these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .", "a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .", "neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .", "furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .", "genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients . however ,", "enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .", "the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .", "attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .", "interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself .", "the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and", "hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .", "furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .", "in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .", "similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .", "interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .", "the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .", "these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .", "interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .", "later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .", "this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .", "the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .", "thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner . consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .", "the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .", "the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .", "this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .", "interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .", "as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .", "hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .", "another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .", "beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .", "in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .", "moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .", "however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .", "recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .", "ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,", "the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .", "this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .", "-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .", "in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .", "the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease . taken together", ", all these findings suggest an important role for autophagy in the pathogenesis of ad .", "there is quite some evidence that in ad , the proteostasis network is impaired . based on pathological analysis , ad mouse models , in vitro and cellular investigations a molecular link between chaperones , the ubiquitin  proteasome system , autophagy pathways and pathogenetic mechanisms of ad can be suggested .", "deregulation and changes of chaperones and proteasome activity might have serious implications for aging as well as for age - associated diseases .", "autophagy pathways are key mechanisms and of vital importance for cellular function and , especially , for cell survival under adverse conditions .", "consequently , an effect of proteostasis control on neurodegeneration or , vice versa , of neurodegeneration on proteostasis is reasonable . as with other pathomechanisms that are investigated in the search for the cause of ad , it is still open whether an impairment of proteostasis is an upstream or downstream event during ad onset and progression .", "experimental approaches employing mouse models clearly demonstrate that stabilization or induction of proteostasis can be neuroprotective . whether this can be translated into the human condition and , most importantly ,", "whether supporting proteome integrity can be a real target for pharmacological intervention for the prevention and treatment of ad is currently still open .", "also , one has to consider that , e.g. the stimulation of general autophagy may in the long term lead to the stimulation of proliferation of non - neuronal cells , eventually resulting in tumor formation . indeed , a permanent autophagy induction is known as one cellular mechanism that promotes the escape of cells from proliferation control . as beneficial", "it may be to support autophagy in post - mitotic neurons that are confronted with disturbed proteostasis and an impairment of proteasome function , this stimulation should target specific autophagy pathways , such as selective chaperone - mediated macroautophagy involved in the degradation of disease - associated protein aggregation .", "although there are obviously still many questions to be answered to understand the role of proteostasis in ad ( and also in other age - associated neurodegenerative disorders ) , it is a big step forward in ad research to consider a possible role of pathogenetic pathways that are not directly linked to the usual suspects a and tau", ". a better understanding of how the proteostasis network is regulated might help to identify targets which lead to prevention of the deleterious loss of neuronal cells and tissue in ad ."], "abstract_text": ["<S> alzheimer s disease ( ad ) is one key medical challenge of the aging society and despite a great amount of effort and a huge collection of acquired data on molecular mechanisms that are associated with the onset and progression of this devastating disorder , no causal therapy is in sight . </S>", "<S> the two main hypotheses of ad , the amyloid cascade hypothesis and the tau hypothesis , are still in the focus of ad research . with aging </S>", "<S> as the accepted main risk factor of the most important non familial and late onset sporadic forms of ad , it is now mandatory to discuss more intensively aspects of cellular aging and aging biochemistry and its impact on neurodegeneration . </S>", "<S> since aging is accompanied by changes in cellular protein homeostasis and an increasing demand for protein degradation , aspects of protein folding , misfolding , refolding and , importantly , protein degradation need to be linked to ad pathogenesis . </S>", "<S> this is the purpose of this short review . </S>"], "labels": null, "section_names": ["Introduction", "Maintenance of Proteostasis via Molecular Chaperones: the First Line of Defense Against Protein Misfolding", "Molecular Chaperones Get in Touch with the Protein Degradation Machineries", "Proteostasis and AD", "Chaperones and AD", "UbiquitinProteasome System and AD", "Autophagy and AD", "Outlook"], "sections": [["alzheimer s disease ( ad ) is a progressive neurodegenerative disorder starting with mild memory loss and manifesting in severe cognitive decline .", "neuropathologically , the disease is characterized by an extensive deposition of amyloid  ( a ) peptides in extracellular amyloid plaques and by intracellular neurofibrillary tangles ( nfts ) of hyper - phosphorylated tau protein .", "sporadic late - onset ad is the most common form of dementia in the elderly and is strongly associated with age . in rare cases", "( < 5% ) , ad is inherited and results in an early disease onset .", "one genetic risk factor for late - onset ad that has been reported throughout many studies is apolipoprotein e ( apoe ) which physiologically functions as a ligand in receptor - mediated endocytosis of lipoprotein particles [ 2 , 3 ] .", "several single nucleotide polymorphisms in apoe lead to alterations in the coding sequence and result in three common isoforms called apoe2 , apoe3 and apoe4 with the e4 allele being an ad risk factor and e2 being protective [ 46 ] .", "strong evidence suggests that apoe influences ad via its effect on a metabolism ; however , the details of this process have to be fully elucidated .", "positional cloning studies of familial ad ( fad ) cases have identified mutations in three genes , amyloid precursor protein ( app ) , presenilin 1 and 2 ( ps1 , ps2 ) , which are tightly linked to the generation of a peptides .", "proteolytic processing of amyloid precursor protein ( app ) by bace ( -site app cleaving enzyme ) is followed by cleavage by -secretase containing presenilins and results in the release of a peptides of different length . a40 and a42 ( 40 or 42 amino acids , respectively )", "are the most prominent a peptides and since the c - terminus has important implication for toxicity , the longer a42 peptide exhibits stronger neurotoxic properties .", "the fad - linked mutations cause an elevated production of amyloidogenic a , which is the basis for the amyloid hypothesis of ad , stating that a peptides are the key - players in neuronal dysfunction and subsequent neurodegeneration in ad .", "recently , it has been suggested that the soluble oligomeric forms of a42 are the potentially harmful species and that these oligomers affect various important cellular mechanisms , resulting in decreased functionality and survival of neuronal cells .", "however , a peptides are not exclusively generated under pathological conditions as they are part of the normal cellular metabolism and a significant load of amyloid plaques is also observed in brains of healthy aged individuals [ 10 , 11 ] .", "this challenges the amyloid hypothesis of ad and up to date the nature of the disease - relevant alteration in aged neurons remains unclear .", "although alois alzheimer already described protein deposits in the brain of demented patients , the question why distinct proteins that are potentially causative for the disease accumulate in discrete brain regions in the course of aging remains unanswered .", "today , we know that the progressive deposition of misfolded and aggregation - prone proteins in defined regions of the nervous system is not unique to ad , but a well - described characteristic of several neurodegenerative disorders , including parkinson s disease ( pd ) , huntington s disease ( hd ) and amyotrophic lateral sclerosis ( als ) . recently , it has been proposed that the accumulation of disease - relevant proteins in aggregates might actually even be considered as a protective mechanism to dispose these proteins and remove them from the cellular metabolism [ 13 , 14 ] . generally , within the crowded cellular environment", ", proteins are always at risk for denaturation . changing genetic and environmental factors , as during pathology or aging , challenge the integrity of the cellular protein homeostasis ( proteostasis ) . therefore , the stability and metabolism of the proteome needs to be controlled carefully and is carried out by a complex network of several hundred evolutionarily conserved proteins .", "this network , including molecular chaperones , the ubiquitin  proteasome system ( ups ) and the autophagy system is stringently regulated and indispensable for the maintenance of proteostasis .", "all main players of this network are ubiquitously expressed and consequently also present in neuronal tissue .", "molecular chaperones , most be prominently represented by the heat shock protein 70 ( hsp70 ) family and their regulators , are responsible for correct folding and refolding of proteins as well as for the transport of misfolded proteins to the protein - degradation systems .", "the chaperone network is controlled by the activity of transcription factors , e.g. heat shock transcription factor 1 ( hsf1 ) , which rapidly adjusts the cellular chaperone capacity during stress conditions .", "the ups is the major protein - degradation machinery of the cell and controls the metabolism of cytosolic proteins and the degradation of misfolded proteins .", "proteins that are destined for proteasomal degradation become specifically tagged with the protein ubiquitin in a well - organized enzymatic cascade .", "interestingly , also a ubiquitin - independent way of proteasomal degradation has been described recently that mostly involves small and misfolded proteins . as an additional mechanism for protein degradation", "the major autophagic systems described so far are macroautophagy and chaperone - mediated autophagy ( cma ) .", "macroautophagy is a complex process that involves the formation of a membrane structure , the autophagosome , which engulfs bulk cellular material ( proteins and organelles ) and subsequently fuses with the lysosome .", "cma involves the constitutively expressed heat shock cognate 70 ( hsc70 ) that targets client proteins with a specific consensus motif directly to the lysosome .", "in fact , hsp70 is also an important factor to assure selectivity of macroautophagy of degradation - prone proteins in a distinct pathway , mediated via the co - chaperone bcl2-associated athanogene 3 ( bag3 ) . aging and disease challenge the proteostasis network and the accumulation of instable proteins results in wide - spread protein aggregation [ 21 , 22 ] .", "our discussion presented here attempts to integrate two major factors that are associated with neurodegeneration , protein homeostasis and aging .", "most importantly , we focus on the role of specific pathways which are needed to restore and maintain proteostasis during aging and their interplay with a special focus on impaired proteostasis in ad , which may add to a novel view and understanding of ad pathogenesis .", "since recently many excellent reviews on various aspects providing in depth discussions have been presented , we are concentrating here on the integration of these different aspects .", "therefore , we frequently refer to review work with the recommendation of further reading rather than trying to fully cover the discussed issues by their original publications ."], ["cells permanently encounter the problem to maintain the integrity and functionality of the proteome . within the crowded cellular environment ,", "the correct conformation of proteins must be controlled and misfolded and irreversibly damaged proteins must be efficiently refolded or removed .", "central players of the protein homeostasis system are molecular chaperones that sense misfolded proteins and , when refolding fails , direct them to the protein - degradation pathways .", "molecular chaperones are specified as proteins that interact with and participate in folding or refolding of non - native proteins .", "therefore , chaperones help unfolded proteins to achieve their functional conformation without being present in the final structure [ 15 , 2327 ] .", "they exert a multitude of activities , including de novo folding , refolding of denatured proteins , transport to subcellular compartments , oligomeric assembly and disposal by proteolytic degradation [ 28 , 29 ] .", "different classes of chaperones work together to form co - operative networks and are often termed heat shock proteins ( hsps ) , because they are sensitively up - regulated under stress conditions in which the amount of misfolded proteins is increased .", "chaperones , which are involved in protein folding and refolding , such as hsp70 , hsp90 and chaperonin ( hsp60 ) , promote folding activity through atp / cofactor - regulated binding and release cycles [ 15 , 31 ] .", "they recognize short hydrophobic amino acid stretches of misfolded proteins and can co - operate with atp - independent chaperones , e.g. small hsps ( shsps ) , to prevent protein aggregation .", "hsp70 proteins are central players in proteostasis control and increasing hsp70 levels prevent protein aggregation in various disease models that are based on the expression of aggregation - prone proteins [ 32 , 33 ] .", "the activity of hsp70 is atp dependent and is controlled by chaperones of the hsp40 family as well as nucleotide exchange factors ( nefs ) [ 34 , 35 ] .", "after atp hydrolysis , a nef binds to the atpase domain of hsp70 and catalyzes the nucleotide exchange , which results in substrate release .", "thus , chaperone binding to the hydrophobic region of misfolded proteins transiently blocks aggregation and the subsequent atp - dependent release allows controlled folding of the client protein .", "hsp40/dnajs , hsc70-interacting protein ( hip ) , bcl2-associated athanogene 1 ( bag1 ) and bag3 are prominent co - chaperones that control the atp - dependent activity of hsp70 .", "dnaj proteins generally enhance the atpase activity of hsp70 , but their action can lead to different fates of their bound substrates .", "dnajb1 is an example for a dnaj protein that supports substrate folding , whereas dnajb2-bound clients are degraded by the proteasome .", "the co - chaperone hip stabilizes the adp - bound state of hsp70 and co - operates with hsp70 in protein folding .", "other hsp70-binding cofactors are the ubiquitin ligases carboxy terminus of hsc70-interacting protein ( chip ) and parkin , which provide a link between hsp70 , co - chaperones and the ups , resulting in client ubiquitination and degradation by the proteasome [ 34 , 38 , 39 ] .", "recently , it has been shown that bag3 interacts with hsp70 and directs the chaperone and its substrates into the autophagic pathway , providing an association of hsp70 , co - chaperones and autophagy .", "an extensive description of the hsp70 machinery , the exact structure - function relationship between hsp70 and its various cofactors , has been excellently reviewed elsewhere .", "the functional variability of the chaperone system that is exerted by a multitude of single proteins highlights the complexity of this proteostasis network .", "however , limited knowledge exists on the exact composition and capacity of the chaperone system within a cell or a certain tissue .", "a general view assumes that total levels of cellular chaperones exceed the actual requirements and that a sufficient amount of chaperones is free of clients .", "thus , the network has excess capacity to initially deal with sudden additional chaperone requirements , as exhibited during stress conditions .", "an opposite view proposes that the capacity of the chaperone network is always closely titrated to the actual demand of chaperone activity and that the system is otherwise rapidly adapted to conditions of increased requirements .", "the analysis of the chaperone capacity of neuronal and muscular tissue of caenorhabditis elegans ( c. elegans ) demonstrates that these tissues indeed display different folding activities and that neurons are particularly sensitive to protein denaturation during heat stress . however , the adaptation of the chaperone network during aging is critical as it is well acknowledged that protein aggregation and disruption of proteostasis are characteristic for aged cells .", "the periodical application of mild heat stress decreases mortality in drosophila melanogaster and c. elegans , which is mediated by hsp70 activity and the overexpression of hsp70 in c. elegans increases the life span of the nematode [ 4345 ] .", "several reports have analyzed chaperone protein or mrna levels in aged cells and found increased or basal amounts , whereas the stress - mediated induction of chaperone expression is impaired .", "the transcription of chaperone genes in response to stress conditions is controlled by the transcription factor hsf1 , which shows an impaired dna - binding potential in aged cells .", "a reduced activity of hsf1 in c. elegans results in a shortened life span and , conversely , the enhanced expression of the transcription factor increases the life span .", "activity is also essential for the extended life span of the extremely long - lived daf-2/insulin / igf-1 receptor mutants of c. elegans [ 47 , 48 ] .", "thus , hsf1 and chaperone activity can promote longevity , demonstrating a clear association of chaperones , proteostasis and aging ."], ["some of the factors mentioned so far are involved in linking chaperone functions with cellular protein - degradation pathways , the ups and autophagy , for the removal of misfolded proteins . besides protein aggregation ,", "one factor that induces ubiquitination is protein damage caused by free radical oxygen species ( ros ) and oxidative stress .", "most likely , irreversible oxidation may activate chaperones and the ups to induce protein repair of misfolded proteins and lead to ubiquitination and protein degradation . during aging ,", "in particular , the mitochondrial respiratory chain is strongly linked to the production of ros and as one consequence may cause protein dysfunction , apoptosis , necrosis , aging and disease [ 49 , 50 ] .", "protein oxidation leads to a change in protein conformation and function and chaperones may sense such changes and in turn activate the ups as a quality control system .", "depending on the degree of oxidation , irreversible oxidation and loss of protein function may lead to degradation and/or accumulation of damaged proteins and to the formation of so - called aggresomes , which display a high autophagic activity [ 51 , 52 ] .", "the ups is a complex enzymatic pathway that starts with the ligation of ubiquitin , a 76-amino - acid - long and highly conserved protein , to other cellular proteins and thus labels them for degradation .", "initially the c - terminal end of ubiquitin is activated by atp - dependent phosphorylation and formation of a thiol ester via an activating enzyme , e1 .", "it is then transferred to a thiol group of an ubiquitin - carrier protein , e2 .", "the e3 ligase directs ubiquitin from e2 to an -amino group of the target protein [ 53 , 54 ] .", "the c - terminus of an additional ubiquitin protein can be ligated onto one of the seven lysine residues within the attached ubiquitin molecule . for degradation via the proteasome", "ubiquitin linkages between either the c - terminus and lysine residues k48 or k63 are the major recognition signals for proteasomal degradation .", "ubiquitin chains also occur among other lysine residues , whereas ubiquitin extension via k6 is associated with dna repair , k11 with endoplasmatic reticulum - associated protein degradation and cell cycle regulation , k27 with ubiquitin fusion degradation , k29 with lysosomal degradation and k33 with kinase modification .", "monoubiquitination can modify the activity of the protein transport machinery and when attached to transmembrane proteins can serve as a sorting signal to direct their movement between different cellular compartments [ 5659 ] . the polyubiquitinated proteins destined for degradation", "are processed by the multienzymatic proteasome complex : first they become deubiquitinated and then degraded by the 26s proteasome , a system that is composed of various proteasome subunits .", "the eukaryotic 20s proteasome core complex consists of four heptameric rings comprising two classes of seven non - identical but homologous subunits .", "the outer rings contain alpha - type subunits with gating function for substrate entrance and product release , while the beta - subunits exhibit peptide hydrolyzing activity .", "the 19s regulatory complex binds to the 20s catalytic core in a flexible manner and consists of six atpases , forming a ring at the entrance of the core and exerting chaperone - like activity .", "the ups is the major degradation system in the cell for the degradation of short - lived , misfolded and defective proteins .", "an accumulation of polyubiquitinated proteins is reported for numerous neurological disorders , which suggests that ups dysfunction plays a prominent role in the pathogenesis of neurodegenerative diseases and moreover in aging . since proteasomal", "activity decreases during aging over all , the degradation demand of the cell increases , which results in an age - associated induction of the autophagy pathway that delivers substrates for degradation ultimately to lysosomes [ 20 , 63 ] .", "autophagy is negatively regulated via the evolutionarily conserved enzymatic mammalian target of rapamycin complex 1 ( mtorc1 ) . in turn , if mtor activity is inhibited by rapamycin , the autophagy pathway is switched on .", "rapamycin exerts its stimulatory effect on autophagy by preventing mtor phosphorylation at ser-2448 and thereby subsequently blocks mtor signaling .", "so far , many different autophagic systems have been described including macroautophagy and cma that form the main pathways .", "macroautophagy is a multi - step process by which cytosolic material is sequestered into a double - layered membrane structure , the autophagosome , and is delivered to the lysosome for degradation .", "it is a highly orchestrated process and involves autophagy - related ( atg ) proteins .", "more than 30 genes have been identified in genetic analyses of yeast mutants which have defects in autophagic function .", "after autophagy stimulation through mtor inhibition , the formation of phagophores ( autophagosome precursors / pre - autophagosomal structures ) is initiated in a not yet completely understood mechanism .", "a subset of atg proteins is required for autophagosome formation : first , the atg9 system including atg9 , the atg1 kinase complex ( atg1 and atg13 ) , atg2 and atg18 ; second , the phosphatidylinositol 3-oh kinase ( pi(3)k ) complex consisting of vacuolar protein sorting ( vps)34 , vps15 , beclin 1/atg6 and atg14 and third , the ubiquitin - like protein ( ubl ) system composed of two ubl proteins ( lc3/atg8 and atg12 ) , an e1 enzyme ( atg7 ) , two analogues of e2 ubiquitin - conjugating enzymes ( atg10 and atg3 ) , an lc3/atg8-modifying protease ( atg4 ) , the protein target of atg12 attachment ( atg5 ) and atg16 .", "beclin 1/atg6 regulates autophagic trafficking and membrane trafficking in a variety of physiological and pathological processes .", "microtubule - associated protein 1 light chain 3 ( lc3/atg8 ) is commonly used as a marker for autophagic activity . with its e1-like activity , atg7 converts cytosolic lc3-i to the membrane - associated lc3-ii , which then is conjugated to phosphatidylethanolamine .", "lc3-ii binds the inner and outer membrane of the forming autophagosome and provides a physical link between the autophagosome and lc3 interacting proteins , such as sequestosome 1 ( p62/sqstm1 ) or neighbor of brca1 gene 1 ( nbr1 ) .", "these so - called autophagy receptors can simultaneously bind to ubiquitinated proteins and lc3 , employing their lc3-interacting motif and ubiquitin - associated domain .", "the identification of autophagy receptors , such as p62/sqstm1 and nbr1 has provided a molecular link between ubiquitination and autophagy .", "thus , p62/sqstm1 and nbr1 are possibly also important bridging factors between ups and autophagy . through self - oligomerization , which is stimulated by ubiquitin binding , p62/sqstm1 sequesters ubiquitinated substrates in form of inclusion bodies .", "these inclusions are then specifically engulfed by the autophagosome membrane by recruiting lc3 [ 69 , 70 ] . while macroautophagy is considered as a rather unspecific robust degradation process , cma is a highly selective lysosomal pathway that removes a distinct subset of proteins containing a pentapeptide lysosome - targeting motif .", "these substrates can on the one hand directly be translocated into the lysosome after docking to the lysosomal receptor lysosomal - associated membrane protein 2a or on the other hand unfolded by a chaperone complex containing hsc70 and the co - chaperones bag1 , hip , hsp70hsp90 organizing protein ( hop ) and hsp40/dnajb1 .", "these co - chaperones provide a direct link to the ups and the folding and refolding activity of molecular chaperones .", "in the course of aging , a variety of proteins tend to aggregate and impair the ups directly .", "therefore , these proteins , most of which are ubiquitinated , can not be handled by the proteasome and have to be degraded by different protein clearance pathways .", "recently , it has been shown that the cellular protein quality control ( pqc ) of polyubiquitinated proteins by proteasomal and autophagic systems is regulated by the hsp70 co - chaperones bag1 and bag3 , respectively .", "proteasome activity decreases in an age - dependent manner in a cell model of replicative senescent human fibroblasts and is associated with an increased autophagic activity in aged cells .", "this correlates with decreased levels of bag1 and increased levels of bag3 in aged cells , an age - related bag1/bag3 switch that serves as an induction control of the macroautophagic pathway ( bag3-mediated selective macroautophagy ) and may be considered as a backup of the ups in pqc ( [ 40 , 72 ] ) .", "therefore , the expression shift from bag1 to bag3 during aging , but also upon acute stress ( e.g. proteasome inhibition , oxidative stress ) , can be considered as a physiologically important adaptive response [ 20 , 40 ] .", "the bag3-mediated selective pathways have been shown to be involved in a variety of disease causing processes . in a model for hd ,", "bag3 in concert with hspb8 facilitates the disposal of polyq43-huntingtin by stimulating macroautophagy and this complex is also involved in z - disc maintenance in flies , mice and men [ 73 , 74 ] . additionally , the transport of target proteins to the aggresome , a compartment with high autophagic activity , has been shown to be mediated by bag3 .", "this aggresome - targeting pathway involving bag3 and hsp70 is distinct from other before - described mechanisms as it does not depend on substrate ubiquitination . taken together the molecular chaperone machinery , the ups and the autophagy system are involved in pqc and maintaining proteostasis within aging and disease to prevent protein misfolding and aggregation .", "a summary of the main routes for protein degradation is shown in fig .  1 .", "as aggregated proteins are found in ad patients and aging is a prevailing risk factor for ad it is important to further characterize how these pathways are regulated and how misregulation possibly contributes to the pathology of ad.fig .", "1the ubiquitin  proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria ) the ubiquitin  proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria )"], ["ad is characterized by the accumulation of intracellular a ( oligomers ) , extracellular high molecular weight deposits of a peptides ( fibrilliar forms ) , and the intracellular aggregation of hyper - phosphorylated tau . indeed ,", "modification of the cellular proteostasis and the metabolism of a and tau have been proven to cause neuronal dysfunction and cell death .", "the malfunctioning proteostasis control results in the accumulation of misfolded and aggregated proteins , which is a general hallmark of aging and neurodegeneration , as observed in pd , hd , als and ad . aging in particular is characterized by decreasing proteostasis capacity and increasing protein damage which are in combination a major challenge for the cell due to the inability to maintain metastable proteins in folded states . to counteract a cascade of protein destabilization caused by metastable proteins which escaped the pqc", "the proteostasis network including molecular chaperones , the ups and autophagic pathways must manage the increasing burden of protein misfolding to maintain proteome stability .", "since aging is the major risk factor of ad [ 7880 ] , the age - dependent loss of proteostasis might be an important contributor to the pathology of ad .", "an overview of proteins that affect proteostasis in ad is shown in table  1.table 1proteins affecting proteostasis in alzheimer s diseaseproteinfunctionreferencea40/42decreases proteasome activity , modulates autophagy through mtor signaling[99102 , 104 , 143146]tauinhibitory effect of tau aggregates on proteasome activityhsf1induces app gene during stress[81 , 82]grp78 ( er isoform of hsp70)modulates app maturation and reduces a40/42 secretion[84 , 85]hsps ( hsp22 , 27 , 70 , 90)small hsps ( hsp22 , hsp27 ) bind to fibrillar amyloid plaques and inhibit their fibrillarisation ; hsp70 , hsp90 inhibit early stages of amyloid aggregation[88 , 89]chipe3 enzyme for phosphorylated tau[93 , 117]bag1regulates proteasomal degradation of tau together with hsp70ps1increases production of a42 , essential for lysosomal proteolysis and autophagy by enabling the acidification of lysosomes required for protease activation[163 , 165]ubb+1potently inhibits the degradation of polyubiquitinated substrates and therefore induces neuronal cell death[120 , 121]uch - l1dub needed for proteasomal degradation of client proteins , oxidized and down - regulated in ad[124 , 125]ubqln1ubqln1 activity is necessary to regulate the production of app and app fragments[127 , 128]mtorinhibitory and/or activating modulation of mtor through a42 but not a40[143146]beclin1beclin1 is down - regulated in ad brains and consequently increases app levels and its metabolites[161 , 162]a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum proteins affecting proteostasis in alzheimer s disease a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .", "interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .", "as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .", "there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .", "the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .", "in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .", "the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .", "moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .", "hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,", "furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1  day and decreased the amount after 3  days in vitro .", "these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .", "besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .", "post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .", "these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .", "hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .", "hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .", "protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .", "vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models", ", it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .", "tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .", "collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . the ubiquitin ", "proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .", "a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .", "a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .", "it still has to be elucidated which form of a is now affecting proteasome function .", "there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell", "free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .", "there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .", "consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .", "so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons", ", it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain", ", a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism", "thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .", "this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .", "the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .", "furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .", "it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .", "in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .", "in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .", "these data show that different aggregation states of tau modify its turnover via the proteasome .", "it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,", "the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .", "in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .", "moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .", "taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .", "ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .", "overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .", "oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .", "it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .", "uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .", "interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .", "several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .", "for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .", "recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .", "in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .", "taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .", "this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .", "ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .", "interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration .", "in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .", "these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .", "these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .", "a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .", "neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .", "furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .", "genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients .", "however , enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .", "the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .", "attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .", "interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself . the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and", "hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .", "furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .", "in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .", "similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .", "interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .", "the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .", "these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .", "interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .", "later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .", "this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .", "the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .", "thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner .", "consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .", "the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .", "the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .", "this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .", "interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .", "as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .", "hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .", "another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .", "beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .", "in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .", "moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .", "however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .", "recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .", "ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,", "the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .", "this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .", "-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .", "in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .", "the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease .", "taken together , all these findings suggest an important role for autophagy in the pathogenesis of ad ."], ["the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .", "interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .", "as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .", "there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .", "the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .", "in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .", "the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .", "moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .", "hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,", "furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1  day and decreased the amount after 3  days in vitro .", "these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .", "besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .", "post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .", "these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .", "hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .", "hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .", "protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .", "vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models", ", it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .", "tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .", "collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation ."], ["the ubiquitin  proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .", "a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .", "a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .", "it still has to be elucidated which form of a is now affecting proteasome function .", "there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell", "free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .", "there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .", "consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .", "so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons", ", it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain ,", "a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism", "thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .", "this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .", "the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .", "furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .", "it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .", "in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .", "in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .", "these data show that different aggregation states of tau modify its turnover via the proteasome . particularly intriguing", "it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,", "the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .", "in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .", "moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .", "taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .", "ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .", "overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .", "oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .", "it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .", "uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .", "interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .", "several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .", "for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .", "recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .", "in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .", "taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .", "this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .", "ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .", "interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration ."], ["in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .", "these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .", "these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .", "a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .", "neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .", "furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .", "genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients . however ,", "enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .", "the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .", "attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .", "interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself .", "the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and", "hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .", "furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .", "in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .", "similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .", "interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .", "the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .", "these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .", "interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .", "later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .", "this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .", "the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .", "thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner . consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .", "the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .", "the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .", "this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .", "interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .", "as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .", "hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .", "another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .", "beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .", "in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .", "moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .", "however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .", "recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .", "ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,", "the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .", "this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .", "-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .", "in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .", "the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease . taken together", ", all these findings suggest an important role for autophagy in the pathogenesis of ad ."], ["there is quite some evidence that in ad , the proteostasis network is impaired . based on pathological analysis , ad mouse models , in vitro and cellular investigations a molecular link between chaperones , the ubiquitin  proteasome system , autophagy pathways and pathogenetic mechanisms of ad can be suggested .", "deregulation and changes of chaperones and proteasome activity might have serious implications for aging as well as for age - associated diseases .", "autophagy pathways are key mechanisms and of vital importance for cellular function and , especially , for cell survival under adverse conditions .", "consequently , an effect of proteostasis control on neurodegeneration or , vice versa , of neurodegeneration on proteostasis is reasonable . as with other pathomechanisms that are investigated in the search for the cause of ad , it is still open whether an impairment of proteostasis is an upstream or downstream event during ad onset and progression .", "experimental approaches employing mouse models clearly demonstrate that stabilization or induction of proteostasis can be neuroprotective . whether this can be translated into the human condition and , most importantly ,", "whether supporting proteome integrity can be a real target for pharmacological intervention for the prevention and treatment of ad is currently still open .", "also , one has to consider that , e.g. the stimulation of general autophagy may in the long term lead to the stimulation of proliferation of non - neuronal cells , eventually resulting in tumor formation . indeed , a permanent autophagy induction is known as one cellular mechanism that promotes the escape of cells from proliferation control . as beneficial", "it may be to support autophagy in post - mitotic neurons that are confronted with disturbed proteostasis and an impairment of proteasome function , this stimulation should target specific autophagy pathways , such as selective chaperone - mediated macroautophagy involved in the degradation of disease - associated protein aggregation .", "although there are obviously still many questions to be answered to understand the role of proteostasis in ad ( and also in other age - associated neurodegenerative disorders ) , it is a big step forward in ad research to consider a possible role of pathogenetic pathways that are not directly linked to the usual suspects a and tau", ". a better understanding of how the proteostasis network is regulated might help to identify targets which lead to prevention of the deleterious loss of neuronal cells and tissue in ad ."]]}
{"article_id": "PMC5293457", "article_text": ["the union rate and hardware - related complications remain a major concern in these patients , especially following multilevel spinal fusion surgeries .", "developing an adjunct therapy that essentially supplements the mainstay surgical management is of great importance to these patients .", "multitudinous research to gain insight into use of teriparatide ( 134 recombinant human parathyroid hormone ) for enhancing bone formation in osteoporosis is readily available in the literature .", "its action as a direct anabolic agent in bone tissue , consistently increases bone mineral density and thus improves skeletal architecture . since its availability in market", ", indications are unravelling and is being attempted in many conditions including fracture healing , dental stability , hypoparathyroidism , and hypocalcaemia .", "another evolving field of interest is the role of teriparatide for enhancing bone graft union and osseo - integration of implants .", "it is said to accelerate bone graft union and reduce the incidence of pedicle screw loosening after lumbar spinal fusion surgeries .", "we intended to study this effect and analyze if teriparatide can be an effective adjunct therapy to achieve solid posterolateral fusion mass and reduce the incidence of subsequent screw loosening after multilevel instrumented lumbar fusion surgeries in elderly patients .", "an open - labeled , nonplacebo controlled , retrospective , single center study is formulated by shortlisting 62 elderly patients ( male 9 ; female  =  53 ) between the age group of 65 to 78 years ( mean age    standard deviation [ sd ]  =  70.1    3.7 ) , who underwent instrumented lumbar fusion for various degenerative pathologies over a period of time .", "all of them had received multilevel lumbar fixation (  3 levels or 4 vertebral segments ) with pedicle screw constructs and postero - lateral intertransverse fusion with a combination of local bone graft and allograft .", "the prime indication for surgery was chronic unresolving low back pain , with or without neurological symptoms , associated with any one of the following conditions : a)multisegmental spinal instability ( spondylolisthesis  grade 1),b)multilevel degenerative spondylosis with or without segmental instability , andc)degenerative scoliosis with or without segmental instability .", "multisegmental spinal instability ( spondylolisthesis  grade 1 ) , multilevel degenerative spondylosis with or without segmental instability , and degenerative scoliosis with or without segmental instability .", "we excluded : a)those patients operated with oligo - level spinal fusions (  2 levels or 3 vertebral segments).b)those with 1 or more osteoporotic vertebral compression fractures among the fused levels.c)those with previous surgery in the same level.d)those in whom infection or tumor was an indication for surgery.e)those with 1 or more potential data missing .", "those patients operated with oligo - level spinal fusions (  2 levels or 3 vertebral segments ) .", "those with 1 or more osteoporotic vertebral compression fractures among the fused levels . those with previous surgery in the same level . those in whom infection or tumor was an indication for surgery . those with 1 or more potential data missing .", "selection was irrespective of individual bone mineral density ( bmd ) status , as bmd scoring was not done as a part of routine preoperative evaluation in patients undergoing multilevel spinal fusions .", "our sample included elderly patients  65 years who are expected to have detoriating osteogenesis potential .", "associated medical comorbidities included diabetes mellitus ( dm ) and bronchial asthma ( ba ) in 25 and 8 patients , respectively .", "a mixture of fresh frozen allograft and local bone chips were used as graft . local bone consisted of spinous process , lamina , and facets from the decompression site .", "occasionally , additional interbody cages were used at insufficient discs especially at the caudal end of the construct to increase stability . following surgery", ", patients were given an option of postoperative teriparatide use and opting to it was solely based on their own decision . in total , 30 patients who accepted for long - term daily use of teriparatide were in the experimental group and remaining 32 patients were in the control group .", "teriparatide ( self - administered ; 20 mcg sc injection ) was started for those patients in the experimental group from the first postoperative day , once daily using delivery device .", "the control group received similar standardized and accepted treatment protocols as those in the experimental group except for the use of postoperative teriparatide .", "all patients were instructed to take calcium and vitamin d supplements every day following surgery .", "outcome analysis was done using radiological data which included antero - posterior ( ap ) , conventional lateral view , and dynamic stress lateral view radiographs taken at 12 months follow - up ( figs . 1 and 2 ) .", "( c ) mri images showing severe degeneration of disc levels and canal stenosis from l1l5 .", "( d ) 12 months follow - up ap view x - ray which was considered as solid fusion ( lenke type 1 ) .", "ap = antero - posterior , mri = magnetic resonance imaging . radiological outcome illustration of a random case from the control group . ( a )", "( b ) preoperative dynamic stress ( extension and flexion ) lateral view radiographs showing degeneration from l3s1 segments .", "( c ) mri images showing severe degeneration of disc levels and canal stenosis from l3s1 .", "( d ) 12 months follow - up ap view x - ray showing bilateral signs of pseudo - arthrosis interpreted as nonunion ( lenke type 4 ) .", "they divide postero lateral fusion mass into 4 grades based on the appearance in an ap radiograph .", "grade a represents definitive fusion with bilateral thick bony masses , grade b represents probable fusion with unilateral thick bony mass and thin bony mass on other side , grade c represents no probable fusion with thin bony mass on the one side and pseudo - arthrosis on the other side , grade d represents no fusion with thin bony mass on both the sides showing obvious pseudo - arthrosis or resorption of graft bilaterally .", "we interpreted grade 1 and grade 2 of lenke 's criteria as solid fusion which is explained as unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebra showing continuous trabeculation in an ap radiograph .", "although most of the patients had long fixations , segmental cobb 's angle change involving the upper and lower ends of the construct as seen in dynamic ( flexion and extension ) stress lateral views will also be considered as nonunion .", "screw loosening was determined when there was occurrence of radiolucency ( halo sign ) around the pedicle screws in either ap or lateral view radiographs .", "change in the screw position noticed in dynamic stress lateral view radiographs will additionally be considered as screw loosening , which may occur at the cephalic and caudal ends of the construct .", "results were tabulated and statistical analyses were carried out using graph pad prism 5 ( graphpad software inc . , san diego , ca ) .", "analyses were done using unpaired t test for continuous variables and fisher 's exact test for categorical variables .", "probability values of less than 0.05 were considered statistically significant . written informed consent was obtained from all patients including acceptance of daily usage of teriparatide delivery device from those in the experimental group .", "this study was approved by the institutional review board of chang gung memorial hospital with irb registry no - 201600800b0 and was performed in accordance with the ethical standards stated in the most recent version of the 1964 declaration of helsinki .", "sixty - two elderly patients ( age  =  6578 years ; mean  =  70.11 years ; male  =  9 , female  =  53 ) are divided into 2 groups based on teriparatide administration following surgery .", "group 1 or the experimental group ( n  =  30 ; mean age    sd  =  69.8    3.8 years ) was those in whom teriparatide was administered and group 2 ( n  =  32 ; mean age    sd  =  70.4    3.6 years ) was the control group .", "the presence of concomitant medical comorbidities in group 1 ( dm  =  14 ; ba  =  4 ) and group 2 ( dm  =  11 ; ba  =  4 ) was matched .", "the demographic variation in characteristics of the groups is statistically insignificant ( table 1 ) .", "surgical parameters of the experimental group ( fusion levels  =  137 ; pedicle screws  =  269 ) and the control group ( fusion levels  =  144 ; pedicle screws  =  283 ) had no statistically significant variance .", "duration of teriparatide administration averaged 7.4    2.4 months in the experimental group .", "follow - up data were available for a mean duration of 25.4 months for those in the experimental group and 27 months for those in the control group . even though follow - up was done for a longer duration , analyzing outcome further after 1 year may not be related to teriparatide use .", "considering the duration of administration of teriparatide , radiological outcome analysis was decided to be done at 1 year follow - up .", "none of our patients missed the 1st year follow - up and required x - rays were available in our database for all the patients .", "radiological outcome analysis was done using ap , lateral , and dynamic stress lateral view radiographs taken at 12 months follow - up .", "two or more observers concurred stating that there was unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebras showing continuous trabeculation confirming solid fusion in 20 patients ( 66.7% ) of the teriparatide group and 16 patients ( 50% ) in the control group .", "nonunion was noted in 10 patients ( 33.3% ) of teriparatide group and 16 patients ( 50% ) in the control group . the 16.7% advantage procured with teriparatide use was not statistically significant ( p  =  0.20 ) ( table 2 ) .", "screw loosening was calculated comparing the number of screws showing signs of loosening to total number of screws in each group .", "the incidence of screw loosening was 13.4% in the teriparatide group and 24.4% in the control group .", "this difference was statistically significant with p  =  0.001 ( table 2 ) .", "elderly patients with chronic unresolving symptoms due to degenerative spine disease are prone to have poor surgical outcomes .", "they also predispose for more hardware - related complications , especially following long spinal fusions . considering the possibility of poor outcomes , treating these conditions", "can not be ignored but rather a potential solution needs to be developed which effectively supplements the mainstay surgical management .", "it is said to play a reasonable role in treatment of osteoporosis . apart from being used in osteoporotic individuals , insights into its action on many other conditions are unraveling .", "animal studies yielded promising results for teriparatide usage to enhance postero - lateral fusion mass in rat models . in human studies relating to teriparatide", ", most authors studied its effect only in osteoporotic individuals . in our sample , patients were between 65 and 78 years of age and are more susceptible to have poor bmd .", "patients had comorbidities such as dm and bronchial asthma that have negative influence over bmd .", "dm is said to reduce bmd at a higher rate in elderly women , even if the baseline bmd was high .", "along with postero - lateral fusion , additional interbody cages were used especially at very insufficient discs often seen in the caudal end of the construct .", "this step also allows the graft to access the pluripotent stem cells of the bone marrow .", "since a large amount of graft is required for long fusions , there usually is a lack of host bone .", "static and dynamic radiographs are used for radiological assessment of spinal fusion by many authors , but computerized tomography ( ct ) is considered more accurate .", "most of the radiographic criteria developed to assess fusion mass consider movement in dynamic radiographs as a main component for assessment .", "they lack reliability as the absence of motion does not confirm fusion , especially in the case of long instrumented levels . based on ap radiographs ,", "we considered grade a ( definitive fusion ; bilateral thick bony mass ) and grade b ( probable fusion ; unilateral thick bony mass ) described by lenke et al as solid fusion .", "fusion masses that did not favorably appear to have definitive or probable fusion were considered as nonunion .", "we also considered change in segmental cobb 's angle , visualized in dynamic stress lateral view radiographs as an additional criterion to interpret as nonunion .", "formation of a radiolucent zone ( halo sign ) around the pedicle screws seen in conventional ap / lateral view radiographs and change in screw position seen in dynamic stress lateral view radiographs were considered definitive indicators for screw loosening .", "teriparatide is said to have potential to increase the quality of lumbar spine bone marrow and pedicle cortex and thus reduces the incidence of pedicle screw loosening .", "it is even believed to increase the insertional torque of pedicle screws when administered from at least 1 month prior to surgery .", "long - term teriparatide use can be associated with significant improvement in bone microarchitecture and trabeculation .", "it is also said that ; more than 6 months of daily injection facilitates effective bone union following postero - lateral fusion . considering this ,", "we administered teriparatide for a mean duration of 7.4 months and none of the patients had any drug induced complications .", "most of the similar studies in literature included only short segment postero - lateral fusions .", "our study is foremost to discuss the effect of teriparatide on multilevel posterolateral fusions in elderly patients .", "according to our preliminary results , the percentage of patients achieving solid fusion following teriparatide use was found to be more than that of the control group .", "besides that , use of teriparatide effectively reduced the incidence of subsequent screw loosening which proved to be statistically significant .", "our study evaluates the radiological outcome following postoperative teriparatide administration in patients undergoing multilevel instrumented lumbar fusion surgery .", "even though percentage of solid fusion was more among patients who used teriparatide , the difference was not statistically significant . however , teriparatide was more significantly influential in reducing the incidence of subsequent pedicle screw loosening .", "being a retrospective study , there are few methodological shortcomings that may have influenced our outcome .", "the lack of preoperative bmd data , variation in surgical indications , and differential treatment durations of teriparatide administration may have biased the sample .", "use of ct as an evaluation tool to assess bone graft union could have been more empirical .", "considering the selected sample size , the study may be underpowered to draw potential conclusions ."], "abstract_text": ["<S> abstractelderly patients with chronic nonresolving symptoms due to degenerative spine pathologies are prone to have poor surgical outcomes and hardware - related complications , especially following multilevel instrumented lumbar fusion surgeries . </S>", "<S> with intention of analyzing if teriparatide can be an effective adjunct therapy to surgical management , radiological outcomes are studied . </S>", "<S> sixty - two elderly patients were divided into 2 similar groups . </S>", "<S> group 1 ( n  =  30 ; mean age  =  69.83 years ; fusion levels  =  137 ; screws  =  269 ) had taken teriparatide ( 20 mcg sc injection , once daily ) for a duration of 7.4    2.4 months following surgery and group 2 ( n  =  32 ; mean age  =  70.38 years ; fusion levels  =  144 ; screws  =  283 ) did not take teriparatide . </S>", "<S> radiological evaluation was done to determine the state of postero lateral fusion mass and to investigate the incidence of pedicle screw loosening at 1-year follow - up . </S>", "<S> unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebras showing continuous trabeculation suggestive of solid fusion was obtained in 66.7% patients in the teriparatide group and 50% patients in the control group ( p  =  0.20 ) . </S>", "<S> 13.4% of the total no . of screws showed signs of loosening in the teriparatide group , compared to 24.4% in the control group ( p  =  0.001 ) . </S>", "<S> percentage of patients achieving solid fusion following teriparatide use was found to be more than that of the control group . </S>", "<S> this difference may have clinical importance but was not statistically significant . </S>", "<S> however , teriparatide use was more significantly influential in reducing the incidence of subsequent pedicle screw loosening . </S>"], "labels": null, "section_names": ["Introduction", "Patients and methods", "Results", "Discussion", "Conclusion", "Limitations"], "sections": [["the union rate and hardware - related complications remain a major concern in these patients , especially following multilevel spinal fusion surgeries .", "developing an adjunct therapy that essentially supplements the mainstay surgical management is of great importance to these patients .", "multitudinous research to gain insight into use of teriparatide ( 134 recombinant human parathyroid hormone ) for enhancing bone formation in osteoporosis is readily available in the literature .", "its action as a direct anabolic agent in bone tissue , consistently increases bone mineral density and thus improves skeletal architecture . since its availability in market", ", indications are unravelling and is being attempted in many conditions including fracture healing , dental stability , hypoparathyroidism , and hypocalcaemia .", "another evolving field of interest is the role of teriparatide for enhancing bone graft union and osseo - integration of implants .", "it is said to accelerate bone graft union and reduce the incidence of pedicle screw loosening after lumbar spinal fusion surgeries .", "we intended to study this effect and analyze if teriparatide can be an effective adjunct therapy to achieve solid posterolateral fusion mass and reduce the incidence of subsequent screw loosening after multilevel instrumented lumbar fusion surgeries in elderly patients ."], ["an open - labeled , nonplacebo controlled , retrospective , single center study is formulated by shortlisting 62 elderly patients ( male 9 ; female  =  53 ) between the age group of 65 to 78 years ( mean age    standard deviation [ sd ]  =  70.1    3.7 ) , who underwent instrumented lumbar fusion for various degenerative pathologies over a period of time .", "all of them had received multilevel lumbar fixation (  3 levels or 4 vertebral segments ) with pedicle screw constructs and postero - lateral intertransverse fusion with a combination of local bone graft and allograft .", "the prime indication for surgery was chronic unresolving low back pain , with or without neurological symptoms , associated with any one of the following conditions : a)multisegmental spinal instability ( spondylolisthesis  grade 1),b)multilevel degenerative spondylosis with or without segmental instability , andc)degenerative scoliosis with or without segmental instability .", "multisegmental spinal instability ( spondylolisthesis  grade 1 ) , multilevel degenerative spondylosis with or without segmental instability , and degenerative scoliosis with or without segmental instability .", "we excluded : a)those patients operated with oligo - level spinal fusions (  2 levels or 3 vertebral segments).b)those with 1 or more osteoporotic vertebral compression fractures among the fused levels.c)those with previous surgery in the same level.d)those in whom infection or tumor was an indication for surgery.e)those with 1 or more potential data missing .", "those patients operated with oligo - level spinal fusions (  2 levels or 3 vertebral segments ) .", "those with 1 or more osteoporotic vertebral compression fractures among the fused levels . those with previous surgery in the same level . those in whom infection or tumor was an indication for surgery . those with 1 or more potential data missing .", "selection was irrespective of individual bone mineral density ( bmd ) status , as bmd scoring was not done as a part of routine preoperative evaluation in patients undergoing multilevel spinal fusions .", "our sample included elderly patients  65 years who are expected to have detoriating osteogenesis potential .", "associated medical comorbidities included diabetes mellitus ( dm ) and bronchial asthma ( ba ) in 25 and 8 patients , respectively .", "a mixture of fresh frozen allograft and local bone chips were used as graft . local bone consisted of spinous process , lamina , and facets from the decompression site .", "occasionally , additional interbody cages were used at insufficient discs especially at the caudal end of the construct to increase stability . following surgery", ", patients were given an option of postoperative teriparatide use and opting to it was solely based on their own decision . in total , 30 patients who accepted for long - term daily use of teriparatide were in the experimental group and remaining 32 patients were in the control group .", "teriparatide ( self - administered ; 20 mcg sc injection ) was started for those patients in the experimental group from the first postoperative day , once daily using delivery device .", "the control group received similar standardized and accepted treatment protocols as those in the experimental group except for the use of postoperative teriparatide .", "all patients were instructed to take calcium and vitamin d supplements every day following surgery .", "outcome analysis was done using radiological data which included antero - posterior ( ap ) , conventional lateral view , and dynamic stress lateral view radiographs taken at 12 months follow - up ( figs . 1 and 2 ) .", "( c ) mri images showing severe degeneration of disc levels and canal stenosis from l1l5 .", "( d ) 12 months follow - up ap view x - ray which was considered as solid fusion ( lenke type 1 ) .", "ap = antero - posterior , mri = magnetic resonance imaging . radiological outcome illustration of a random case from the control group . ( a )", "( b ) preoperative dynamic stress ( extension and flexion ) lateral view radiographs showing degeneration from l3s1 segments .", "( c ) mri images showing severe degeneration of disc levels and canal stenosis from l3s1 .", "( d ) 12 months follow - up ap view x - ray showing bilateral signs of pseudo - arthrosis interpreted as nonunion ( lenke type 4 ) .", "they divide postero lateral fusion mass into 4 grades based on the appearance in an ap radiograph .", "grade a represents definitive fusion with bilateral thick bony masses , grade b represents probable fusion with unilateral thick bony mass and thin bony mass on other side , grade c represents no probable fusion with thin bony mass on the one side and pseudo - arthrosis on the other side , grade d represents no fusion with thin bony mass on both the sides showing obvious pseudo - arthrosis or resorption of graft bilaterally .", "we interpreted grade 1 and grade 2 of lenke 's criteria as solid fusion which is explained as unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebra showing continuous trabeculation in an ap radiograph .", "although most of the patients had long fixations , segmental cobb 's angle change involving the upper and lower ends of the construct as seen in dynamic ( flexion and extension ) stress lateral views will also be considered as nonunion .", "screw loosening was determined when there was occurrence of radiolucency ( halo sign ) around the pedicle screws in either ap or lateral view radiographs .", "change in the screw position noticed in dynamic stress lateral view radiographs will additionally be considered as screw loosening , which may occur at the cephalic and caudal ends of the construct .", "results were tabulated and statistical analyses were carried out using graph pad prism 5 ( graphpad software inc . , san diego , ca ) .", "analyses were done using unpaired t test for continuous variables and fisher 's exact test for categorical variables .", "probability values of less than 0.05 were considered statistically significant . written informed consent was obtained from all patients including acceptance of daily usage of teriparatide delivery device from those in the experimental group .", "this study was approved by the institutional review board of chang gung memorial hospital with irb registry no - 201600800b0 and was performed in accordance with the ethical standards stated in the most recent version of the 1964 declaration of helsinki ."], ["sixty - two elderly patients ( age  =  6578 years ; mean  =  70.11 years ; male  =  9 , female  =  53 ) are divided into 2 groups based on teriparatide administration following surgery .", "group 1 or the experimental group ( n  =  30 ; mean age    sd  =  69.8    3.8 years ) was those in whom teriparatide was administered and group 2 ( n  =  32 ; mean age    sd  =  70.4    3.6 years ) was the control group .", "the presence of concomitant medical comorbidities in group 1 ( dm  =  14 ; ba  =  4 ) and group 2 ( dm  =  11 ; ba  =  4 ) was matched .", "the demographic variation in characteristics of the groups is statistically insignificant ( table 1 ) .", "surgical parameters of the experimental group ( fusion levels  =  137 ; pedicle screws  =  269 ) and the control group ( fusion levels  =  144 ; pedicle screws  =  283 ) had no statistically significant variance .", "duration of teriparatide administration averaged 7.4    2.4 months in the experimental group .", "follow - up data were available for a mean duration of 25.4 months for those in the experimental group and 27 months for those in the control group . even though follow - up was done for a longer duration , analyzing outcome further after 1 year may not be related to teriparatide use .", "considering the duration of administration of teriparatide , radiological outcome analysis was decided to be done at 1 year follow - up .", "none of our patients missed the 1st year follow - up and required x - rays were available in our database for all the patients .", "radiological outcome analysis was done using ap , lateral , and dynamic stress lateral view radiographs taken at 12 months follow - up .", "two or more observers concurred stating that there was unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebras showing continuous trabeculation confirming solid fusion in 20 patients ( 66.7% ) of the teriparatide group and 16 patients ( 50% ) in the control group .", "nonunion was noted in 10 patients ( 33.3% ) of teriparatide group and 16 patients ( 50% ) in the control group . the 16.7% advantage procured with teriparatide use was not statistically significant ( p  =  0.20 ) ( table 2 ) .", "screw loosening was calculated comparing the number of screws showing signs of loosening to total number of screws in each group .", "the incidence of screw loosening was 13.4% in the teriparatide group and 24.4% in the control group .", "this difference was statistically significant with p  =  0.001 ( table 2 ) ."], ["elderly patients with chronic unresolving symptoms due to degenerative spine disease are prone to have poor surgical outcomes .", "they also predispose for more hardware - related complications , especially following long spinal fusions . considering the possibility of poor outcomes , treating these conditions", "can not be ignored but rather a potential solution needs to be developed which effectively supplements the mainstay surgical management .", "it is said to play a reasonable role in treatment of osteoporosis . apart from being used in osteoporotic individuals , insights into its action on many other conditions are unraveling .", "animal studies yielded promising results for teriparatide usage to enhance postero - lateral fusion mass in rat models . in human studies relating to teriparatide", ", most authors studied its effect only in osteoporotic individuals . in our sample , patients were between 65 and 78 years of age and are more susceptible to have poor bmd .", "patients had comorbidities such as dm and bronchial asthma that have negative influence over bmd .", "dm is said to reduce bmd at a higher rate in elderly women , even if the baseline bmd was high .", "along with postero - lateral fusion , additional interbody cages were used especially at very insufficient discs often seen in the caudal end of the construct .", "this step also allows the graft to access the pluripotent stem cells of the bone marrow .", "since a large amount of graft is required for long fusions , there usually is a lack of host bone .", "static and dynamic radiographs are used for radiological assessment of spinal fusion by many authors , but computerized tomography ( ct ) is considered more accurate .", "most of the radiographic criteria developed to assess fusion mass consider movement in dynamic radiographs as a main component for assessment .", "they lack reliability as the absence of motion does not confirm fusion , especially in the case of long instrumented levels . based on ap radiographs ,", "we considered grade a ( definitive fusion ; bilateral thick bony mass ) and grade b ( probable fusion ; unilateral thick bony mass ) described by lenke et al as solid fusion .", "fusion masses that did not favorably appear to have definitive or probable fusion were considered as nonunion .", "we also considered change in segmental cobb 's angle , visualized in dynamic stress lateral view radiographs as an additional criterion to interpret as nonunion .", "formation of a radiolucent zone ( halo sign ) around the pedicle screws seen in conventional ap / lateral view radiographs and change in screw position seen in dynamic stress lateral view radiographs were considered definitive indicators for screw loosening .", "teriparatide is said to have potential to increase the quality of lumbar spine bone marrow and pedicle cortex and thus reduces the incidence of pedicle screw loosening .", "it is even believed to increase the insertional torque of pedicle screws when administered from at least 1 month prior to surgery .", "long - term teriparatide use can be associated with significant improvement in bone microarchitecture and trabeculation .", "it is also said that ; more than 6 months of daily injection facilitates effective bone union following postero - lateral fusion . considering this ,", "we administered teriparatide for a mean duration of 7.4 months and none of the patients had any drug induced complications .", "most of the similar studies in literature included only short segment postero - lateral fusions .", "our study is foremost to discuss the effect of teriparatide on multilevel posterolateral fusions in elderly patients .", "according to our preliminary results , the percentage of patients achieving solid fusion following teriparatide use was found to be more than that of the control group .", "besides that , use of teriparatide effectively reduced the incidence of subsequent screw loosening which proved to be statistically significant ."], ["our study evaluates the radiological outcome following postoperative teriparatide administration in patients undergoing multilevel instrumented lumbar fusion surgery .", "even though percentage of solid fusion was more among patients who used teriparatide , the difference was not statistically significant . however , teriparatide was more significantly influential in reducing the incidence of subsequent pedicle screw loosening ."], ["being a retrospective study , there are few methodological shortcomings that may have influenced our outcome .", "the lack of preoperative bmd data , variation in surgical indications , and differential treatment durations of teriparatide administration may have biased the sample .", "use of ct as an evaluation tool to assess bone graft union could have been more empirical .", "considering the selected sample size , the study may be underpowered to draw potential conclusions ."]]}
{"article_id": "PMC3095978", "article_text": ["according to the world health organization 's recent update , diabetes , hypertension , and obesity are one of the top five continuing risk factors for cardiovascular deaths in the world .", "obesity is increasing substantially and is one of the major contributors of disease prevalence due to its pathophysiological link to other cardiovascular risks such as hypertension and diabetes .", "it is estimated that , in 2010 , 6.4% of adults would have diabetes mellitus affecting 285 million in the world and it will increase to 7.7% by 2030 , affecting 439 million adults .", "of special note is that there will be a 67% increase in the prevalence of diabetes in developing countries from 2010 to 2030 .", "metabolic syndrome ( ms ) is a constellation of overweight / obesity , hypertension , and disturbances of lipid and carbohydrate metabolism .", "the definition of ms was debated for a long time to produce a standardized clinical criterion .", "the world health organisation describes ms as the presence of type 2 diabetes or impaired glucose tolerance with any two of the following characteristics : obesity , high levels of triglycerides , low levels of high - density lipoprotein , and hypertension .", "the international diabetes federation ( idf ) takes central obesity as a prerequisite for the diagnosis of ms with the association of any two of the other factors , that is , high blood pressure , abnormal blood glucose , high levels of triglycerides , and low levels of high - density lipoprotein .", "also , the idf has derived specific reference values for central obesity for different ethnicities .", "the national cholesterol education programme ( ncep ) expert panel on detection , evaluation , and treatment of high blood cholesterol in adults ( adult treatment panel , or atp , iii ) , the national heart , lung and blood institute , and the american heart association   have released a report on the criteria for diagnosing and managing ms .", "the panel describes ms as the presence of any three of the following : abdominal obesity , dislipidemia ( high levels of triglycerides , low hdl ) , increased blood pressure , and elevated fasting glucose .", "for the purpose of this paper , the atp iii and idf 's definitions are used and compared .", "each component of ms is a known risk factor for the development of type 2 diabetes , atherosclerosis , and coronary artery disease ( cad ) .", "people with ms are 310 times more likely to develop cardiovascular disease commensurate with a high risk of morbidity and mortality [ 5 , 6 ] .", "central obesity , one of the components of ms , predicts the occurrence of diabetes and overall cardiovascular risk .", "the ncep atp iii   states that ms is equal to cigarette smoking as a contributing factor for premature cardiovascular disease .", "the prevalence of metabolic syndrome is increasing all over the world with different regions having individual clusters of epidemic risk factors [ 6 , 8 ] , and in particular there is evidence of a high prevalence of ms and diabetes in south asians .", "substantial increase in the prevalence of type 2 diabetes in asia in recent years has raised serious concerns about cardiovascular consequences for these populations [ 5 , 10 ] . however , in developing countries , many of these subclinical conditions are not diagnosed until the onset of complications such as myocardial infarction or stroke .", "it is essential to initiate early detection of these chronic diseases in underdeveloped countries in asia , such as nepal , so that preventative action can minimize the consequences .", "this study aims to establish the prevalence of hypertension , diabetes , obesity , and metabolic syndrome in the participants of a major health screening programme in nepal .", "this study also aims to establish the relationship between the components of ms and lifestyle of the participants .", "nepal is one of the poorest countries of the world at the 136th position of human development index .", "the subjects were the participants of the  programme for detection and management of chronic kidney disease , hypertension , diabetes and cardiovascular disease ,  a community - based screening programme in eastern nepal . in this community - based programme", "a series of community awareness programmes were conducted in a specific locality with the help of local leaders , medical students , and community volunteers .", "various screening centres such as permanent centers ( in health clinics , community centers , etc . ) and temporary screening centers ( in schools , clubs , houses of worship , and private homes ) were used to screen the population .", "each center used a group of five to seven people as community volunteers and consisted of a local leader ( priest , administrator , school teachers , and local political leaders ) , a laboratory technician , and nurse .", "medical students ( approximately 100 in number ) and nursing students ( around 25 ) assisted the community volunteers .   prior to screening ,", "the community volunteers went from door to door to record the number of family members residing permanently and to inform the members of the family , about the need of the project .", "all people of 20 years were invited to come to a predefined place in very close vicinity to their house .", "pregnant or menstruating women at the time of analysis , people with a fever or acute illness , and those who had recently engaged in heavy exercise were excluded .", "the research team also collected general information on the participants ' demographic data , diet , smoking , alcohol consumption , and physical activity . the data recorded included family and medical history for kidney disease , high blood pressure , diabetes , cardiovascular disease and any current medication or treatment .", "blood pressure was measured by the auscultatory method with a random zero mercury sphygmomanometer and standard cuff ( 12  34  cm ) .", "the blood pressure measurement was taken in the seated position , quietly in a chair with feet on the floor and an arm support at the heart level .  ", "hypertension was defined according to the guidelines of the seventh report of the joint national committee on prevention , detection , evaluation and treatment of high blood pressure , that is , systolic blood pressure  140  mm  hg or diastolic blood pressure  90  mm  hg and/or concomitant use of antihypertensive medications .", "body weight and height were assessed with all subjects standing without shoes and heavy outer garments to the nearest 0.1  kg and 1  cm , respectively .", "waist circumference was measured over light clothing at a level midway between the lower rib margin and the iliac crest in centimetres rounded up to nearest 0.5  cm .", "abdominal obesity is defined as an abdominal circumference > 102  cm ( 40 in ) in males and > 88  cm ( 35 in ) in females for ncep criteria and > 90  cm in males and > 80  cm in females for idf criteria for south asians .  ", "plasma glucose concentration was determined by the glucose oxidase - peroxidase method ( vitalab selectra-2 , merck , germany ) .", "the diagnosis of diabetes was defined by either casual plasma glucose  200  mgdl associated with symptoms of diabetes and on fasting samples  plasma glucose  126  mgdl .", "individuals with self reported , prior physician - diagnosis of diabetes were classified as having previously diagnosed diabetes .  ", "serum lipids that include total cholesterol , high - density lipoprotein ( hdl ) , low - density lipoprotein ( ldl ) , and triglycerides ( tg ) were also measured ( vitalab selectra-2 , merck , germany ) .", "the results from any person having a history of hypertension or found to have hypertension were verified by qualified doctors .", "the biochemical tests were completed in semiautomatic analysers ( microlab 300 , vital scientific , the netherelands ) .", "epidata refers to a group of applications used in combination for creating documented data structures and analysis of quantitative data . in this study ,", "the idf and ncep atp iii 's criteria for metabolic syndrome were used to calculate and compare the frequency of metabolic syndrome .", "the relationships between the prevalence of cardiovascular risk factors , demographic details , lifestyle , and physiological test results were analysed using the spearman correlation test .", "further , the differences in the categorical variables were examined using chi - squared test .", "odds ratios ( ors ) and their 95% confidence interval were calculated using binary logistic regression ( for gender and age ) and multinomial logistic regression ( for life style factors ) .", "in total , 14,425 people , aged 20100 ( mean age 41.4  15.1 ) , were included in the study . among them ,", "the percentage of education level is illustrated in accordance to the number of years in education ( 15 years ", "the participants were divided into four categories according to their work : labourer / farm , office , house , and none / unknown .", "participants ' physical activities were defined according to the time spent every day on physical activity as > 60  min , 3060  min , < 30  min / day , and none .", "abdominal obesity was observed in 11.5% ( n = 1607/14002 ) of the participants as per ncep criteria ( mean waist circumference : male107.38  6.19  cm , female94.84  5.84  cm ) and in 34.7% ( n = 5006/14418 ) of the participants as per idf criteria . according to the revised bmi , 10.6% ( n = 1534/14423 ) were underweight ( bmi < 18.5 ) , 28.2% ( n = 4065/14423 ) were overweight ( bmi = 2224.9 ) , and 32.5% ( n = 4689/14423 ) were obese ( bmi > 25 ) .  ", "diabetic prevalence was 6.3% ( 889/14008 ) of which 4.8% ( n = 673/14008 ) were under treatment .", "a figure of 12.3% ( n = 1718/14009 ) had a family history of diabetes .", "hypertension was observed in 33.9% ( n = 4894/14422 ) of the participants ( mean systolic 138.72  18.03  mm  hg and mean diastolic 93.09  8.45  mm  hg ) .", "only 12.9% ( 1812/14009 ) were previously diagnosed , and 8.5% were receiving treatment for hypertension .", "a history of coronary artery disease was present in 1.6% ( n = 218/14007 ) , and 1% ( n = 142 ) were under treatment for ischemic heart disease or stroke . \n", "table 2 shows the goodness of fit for the prevalence of obesity , hypertension , and diabetes .", "prevalence of hypertension showed no difference from these data , and obesity showed only a small difference .", "the percentages of the participants who had abnormal lipid profile that includes total serum cholesterol , serum ldl cholesterol , serum hdl cholesterol , serum triglycerides are listed in table 3 . there were 2191 sets of data eligible to meet the criteria for metabolic syndrome .", "ms was observed in 22.5% ( n = 494/2191 ) of the participants according to the idf criteria and 20.7% ( 454/2191 ) according to the ncep criteria .", "the percentages of individual ms risk factors among the total participants and the participants with ms are illustrated in figures 1 and 2 .", "generally , among the total participants and the specific participants with ms , the presence of abnormal lipids was higher than the other factors defining ms . however , the presence of abdominal obesity was higher among ms participants using idf criteria ( figure 2 ) .  \n", "the females had a higher prevalence of ms than males . according to the ncep criteria , the age groups 4160 and 6180 had a higher prevalence of ms than the lower age group .", "according to idf criteria , the age groups 4160 and 2040 had a higher prevalence of ms .", "the participants who worked at home had a high incidence of ms according to both the criteria used .", "the sedentary group had a higher incidence of ms than the participants who were physically active .", "the univariate correlations between cardiac risk factors are shown in table 5 , and the chi - squared independence of them in the metabolic syndrome prevalence is listed in table 6 .  ", "the prevalence of ms ( ncep scores ) had a significant positive relationship with education levels and physical activity .", "there were significant positive correlations between physical activity and the three individual ms components : high glucose ( r = 0.03 , p < .01 ) , high bp ( r = 0.04 , p < .01 ) , and low hdl ( r = 0.23 , p < .01 ) .", "there was no correlation between physical activity and the other two ms components : high triglyceride ( r = 0.003 , p > .05 ) and abdominal obesity ( r = 0.003 , p >", "the ncep scores had a positive correlation between the family history of diabetes ( r = 0.83 , p < .01 ) and hypertension ( r = 0.115 , p < .01 ) .", "although a number of these correlations show high levels of significance , the common variance is extremely low , suggesting that the sample size is having a major impact on the significance . as a result of this", "table 7 lists the chi - squared independence , odds ratios , and confidence intervals in the association between age , gender , and specific lifestyle factors in metabolic syndrome prevalence .", "gender , age , education level , and physical activity show a positive association with the prevalence of metabolic syndrome .", "it is important to observe the prevalence of diabetes , hypertension , and obesity individually and also the combination of risk factors as metabolic syndrome to predict the risk of cardiovascular disease .", "any association between lifestyle factors and these risk factors would provide the opportunity to encourage a change in lifestyle to promote lower levels of subsequent cvd .  ", "the large number of poorly educated people and the large number of school dropouts could be linked to the disease prevalence .", "the prevalence of hypertension and metabolic syndrome in poorly educated people was large when compared with the educated participants .", "though the results are not generalized , the relationship between education levels and the prevalence of hypertension agrees with earlier studies [ 19 , 20 ] .", "these found that education levels significantly influence the knowledge of hypertension and the awareness of cardiovascular risk .", "this suggests that there is a need to improve the awareness of health and use education to prevent or reduce the risk of ms and cardiovascular risks in these groups .", "the office workers had a lower prevalence of ms ( ncep scores ) than the other groups .", "a considerable number of office workers ( 64% ) undertook regular physical activity of more than 30  min / day .", "the home workers education levels and physical activities were comparatively lower than the other work groups .", "these findings clearly show that education and physical activity have an influence on the prevalence of ms .", "most of the females were home workers ( 75.5% ) , and their education was comparatively lower than the males .", "the amount of physical activity involved in home workers is unknown , but the results suggest it is less than that undertaken by other workers .", "asian populations continue to modernize , and levels of physical activity are declining as ( i ) home and work place jobs become more automated and sedentary and ( ii ) transportation is more readily available .", "the prevalence of ms among the participants who had no physical activity was surprisingly no different than others .", "this may be due to a higher than average number of missing values in these data ( 2191/14425 complete data to meet the criteria for ms ) or to other unknown socioeconomic factors .", "controversially , there was a high prevalence of ms among people who regularly ate fruit and vegetables .", ".   found that a higher intake of macronutrients such as fruits and vegetables is associated with general obesity .", "however , it is not clear how the vegetables and fruits were eaten , for example , overcooked , processed , and so forth .", "the exact quantity of the dietary intake was not recorded as it was not the primary area of focus of the study . in these populations ,", "these tend to report a low intake of mono - unsaturated fats ( mufas ) , n-3 polyunsaturated fats ( pufa ) , and transfatty acids ( mostly related to widespread use of vanaspati , a hydrogenated oil ) .", "the healthy traditional plant - based diets are being replaced by cheaper calorie dense high - fat foods .", "these changes are resulting in a rapid increase in the prevalence of obesity throughout asia and the subsequent development of ms .", "ness and powles also found in their review   that many studies were reporting the null or negative effects of fruit and vegetable intake on the prevalence of cardiovascular diseases .", "however , the correlations found in those studies were generally low , as seen in our study .", "further , they suggest that a food - based analysis would complement the nutrient - based analysis to clarify these issues . in nepal ,", "the regular diet in addition to fruits and vegetables , that is , such as rice , which is high in carbohydrates , and the methods of cooking may be dietary causes of metabolic syndrome .  ", "the age groups 4060 had a large prevalence of ms in this study . also , it is important to note that this middle - aged group had a high incidence of overweight or general obesity and abdominal obesity .", "the other age groups had a lower prevalence of ms than the 4060 years old , yet it was still relatively high .", "inadequate maternal nutrition in pregnancy , low birth weight , and childhood obesity may be important factors for the development of metabolic syndrome and diabetes . specifically in children and young individuals , a high intake of n-6 pufa is correlated with hyperinsulinaemia . in adults ,", "unger described metabolic syndrome as  a failure of the system of intracellular lipid homeostasis which prevents lipotoxicity in organs of overnourished individuals  . in this study", "in addition to low levels of hdl , the hdl particles are small , dense , and dysfunctional in south asians .", "hypertriglyceridaemia is a direct reflection of an insulin resistance condition , and it is interrelated to the low hdl concentrations in developing endothelial dysfunction .   in nepal ,", "a high number of the participants had abdominal obesity and were overweight / obese , according to their bmi .", "the bmi is a simple useful measure for overall abnormal weight , yet not a standard measure for obesity .", "bmi can not differentiate between whether the condition was due to unusual muscular development or the accumulation or distribution of fat in the body [ 26 , 27 ] . despite", "the low prevalence of general body obesity compared to western countries , metabolic syndrome is growing into a significant public health problem in asia .", "this may be mainly due to the large number of people with central obesity , a feature which was also observed in this study", ". the higher prevalence of ms in females is also more likely to be due to a higher incidence of abdominal obesity .", "abdominal obesity is an important factor because metabolic syndrome and increased abdominal fat are related to a reduction of adiponectin , an adopicyte - derived hormone with antiatherogenic and anti - inflammatory properties .", "the abdominal adipose tissue results in release of free fatty acids directly in the portal veins and altered lipid levels in the blood .", "further abdominal adiposity increases insulin secretion , and it would be exaggerated by decreased hepatic clearance leading to hyperinsulinemia .", "thus abdominal obesity is an important indicator of cardiovascular disease due to its link to dyslipidemia , hyperinsulinemia , hypertension , and impaired fibrinolytic capacity .", "state that if the ncep 's criteria were applied to the asian population , it might underestimate the prevalence of metabolic syndrome and the risk of cardiovascular disease .", "idf 's specific reference values for abdominal obesity make a substantial difference to the prevalence of ms between the two criteria . the idf 's cut - off points for south asians ' waist circumference", "are lower than the ncep 's general cut - off points ( 90  cm versus 102  cm in men and 80  cm versus 88  cm in women ) .", "another study on chinese population also found a large increase in the prevalence of metabolic syndrome using idf criteria compared with ncep criteria .", "however , in our study both definitions demonstrated a higher prevalence of metabolic syndrome ( 20.722.5% ) in nepal when compared with the studies done in other southeast asian countries such as thailand ( 1218% using ncep definition ) and india ( 18.3% using idf definition ) .", "these findings suggest the need for specific attention to control the disease prevalence in nepal .", "although data were prospectively collected , they may not be generalizable outside of eastern nepal .", "the results did not show substantiate relationship between smoking histories , diet , family history of cardiovascular , and metabolic syndrome .", "abdominal obesity , with the revised reference values , is an important risk due to its physiological relationship to the other ms risk factors .", "the ms prevalence may be due to lack of awareness and unhealthy lifestyles , so health education and more preventive measures should decrease the prevalence of ms and cardiac risks in nepal ."], "abstract_text": ["<S> \n background . this study was carried out to establish the prevalence of cardiovascular risks such as hypertension , obesity , and diabetes in eastern nepal . </S>", "<S> this study also establishes the prevalence of metabolic syndrome ( ms ) and its relationships to these cardiovascular risk factors and lifestyle . methods . </S>", "<S> 14,425 subjects aged 20100 ( mean 41.4  15.1 ) were screened with a physical examination and blood tests . </S>", "<S> both the international diabetic federation ( idf ) and national cholesterol education programme 's ( ncep ) definitions for ms were used and compared . </S>", "<S> results . </S>", "<S> 34% of the participants had hypertension , and 6.3% were diabetic . </S>", "<S> 28% were overweight , and 32% were obese . </S>", "<S> 22.5% of the participants had metabolic syndrome based on idf criteria and 20.7% according to the ncep definition . </S>", "<S> prevalence was higher in the less educated , people working at home , and females . </S>", "<S> there was no significant correlation between the participants ' lifestyle factors and the prevalence of ms . </S>", "<S> conclusion </S>", "<S> . the high incidence of dyslipidemia and abdominal obesity could be the major contributors to ms in nepal . </S>", "<S> education also appears to be related to the prevalence of ms . </S>", "<S> this study confirms the need to initiate appropriate treatment options for a condition which is highly prevalent in eastern nepal . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["according to the world health organization 's recent update , diabetes , hypertension , and obesity are one of the top five continuing risk factors for cardiovascular deaths in the world .", "obesity is increasing substantially and is one of the major contributors of disease prevalence due to its pathophysiological link to other cardiovascular risks such as hypertension and diabetes .", "it is estimated that , in 2010 , 6.4% of adults would have diabetes mellitus affecting 285 million in the world and it will increase to 7.7% by 2030 , affecting 439 million adults .", "of special note is that there will be a 67% increase in the prevalence of diabetes in developing countries from 2010 to 2030 .", "metabolic syndrome ( ms ) is a constellation of overweight / obesity , hypertension , and disturbances of lipid and carbohydrate metabolism .", "the definition of ms was debated for a long time to produce a standardized clinical criterion .", "the world health organisation describes ms as the presence of type 2 diabetes or impaired glucose tolerance with any two of the following characteristics : obesity , high levels of triglycerides , low levels of high - density lipoprotein , and hypertension .", "the international diabetes federation ( idf ) takes central obesity as a prerequisite for the diagnosis of ms with the association of any two of the other factors , that is , high blood pressure , abnormal blood glucose , high levels of triglycerides , and low levels of high - density lipoprotein .", "also , the idf has derived specific reference values for central obesity for different ethnicities .", "the national cholesterol education programme ( ncep ) expert panel on detection , evaluation , and treatment of high blood cholesterol in adults ( adult treatment panel , or atp , iii ) , the national heart , lung and blood institute , and the american heart association   have released a report on the criteria for diagnosing and managing ms .", "the panel describes ms as the presence of any three of the following : abdominal obesity , dislipidemia ( high levels of triglycerides , low hdl ) , increased blood pressure , and elevated fasting glucose .", "for the purpose of this paper , the atp iii and idf 's definitions are used and compared .", "each component of ms is a known risk factor for the development of type 2 diabetes , atherosclerosis , and coronary artery disease ( cad ) .", "people with ms are 310 times more likely to develop cardiovascular disease commensurate with a high risk of morbidity and mortality [ 5 , 6 ] .", "central obesity , one of the components of ms , predicts the occurrence of diabetes and overall cardiovascular risk .", "the ncep atp iii   states that ms is equal to cigarette smoking as a contributing factor for premature cardiovascular disease .", "the prevalence of metabolic syndrome is increasing all over the world with different regions having individual clusters of epidemic risk factors [ 6 , 8 ] , and in particular there is evidence of a high prevalence of ms and diabetes in south asians .", "substantial increase in the prevalence of type 2 diabetes in asia in recent years has raised serious concerns about cardiovascular consequences for these populations [ 5 , 10 ] . however , in developing countries , many of these subclinical conditions are not diagnosed until the onset of complications such as myocardial infarction or stroke .", "it is essential to initiate early detection of these chronic diseases in underdeveloped countries in asia , such as nepal , so that preventative action can minimize the consequences .", "this study aims to establish the prevalence of hypertension , diabetes , obesity , and metabolic syndrome in the participants of a major health screening programme in nepal .", "this study also aims to establish the relationship between the components of ms and lifestyle of the participants ."], ["nepal is one of the poorest countries of the world at the 136th position of human development index .", "the subjects were the participants of the  programme for detection and management of chronic kidney disease , hypertension , diabetes and cardiovascular disease ,  a community - based screening programme in eastern nepal . in this community - based programme", "a series of community awareness programmes were conducted in a specific locality with the help of local leaders , medical students , and community volunteers .", "various screening centres such as permanent centers ( in health clinics , community centers , etc . ) and temporary screening centers ( in schools , clubs , houses of worship , and private homes ) were used to screen the population .", "each center used a group of five to seven people as community volunteers and consisted of a local leader ( priest , administrator , school teachers , and local political leaders ) , a laboratory technician , and nurse .", "medical students ( approximately 100 in number ) and nursing students ( around 25 ) assisted the community volunteers .   prior to screening ,", "the community volunteers went from door to door to record the number of family members residing permanently and to inform the members of the family , about the need of the project .", "all people of 20 years were invited to come to a predefined place in very close vicinity to their house .", "pregnant or menstruating women at the time of analysis , people with a fever or acute illness , and those who had recently engaged in heavy exercise were excluded .", "the research team also collected general information on the participants ' demographic data , diet , smoking , alcohol consumption , and physical activity . the data recorded included family and medical history for kidney disease , high blood pressure , diabetes , cardiovascular disease and any current medication or treatment .", "blood pressure was measured by the auscultatory method with a random zero mercury sphygmomanometer and standard cuff ( 12  34  cm ) .", "the blood pressure measurement was taken in the seated position , quietly in a chair with feet on the floor and an arm support at the heart level .  ", "hypertension was defined according to the guidelines of the seventh report of the joint national committee on prevention , detection , evaluation and treatment of high blood pressure , that is , systolic blood pressure  140  mm  hg or diastolic blood pressure  90  mm  hg and/or concomitant use of antihypertensive medications .", "body weight and height were assessed with all subjects standing without shoes and heavy outer garments to the nearest 0.1  kg and 1  cm , respectively .", "waist circumference was measured over light clothing at a level midway between the lower rib margin and the iliac crest in centimetres rounded up to nearest 0.5  cm .", "abdominal obesity is defined as an abdominal circumference > 102  cm ( 40 in ) in males and > 88  cm ( 35 in ) in females for ncep criteria and > 90  cm in males and > 80  cm in females for idf criteria for south asians .  ", "plasma glucose concentration was determined by the glucose oxidase - peroxidase method ( vitalab selectra-2 , merck , germany ) .", "the diagnosis of diabetes was defined by either casual plasma glucose  200  mgdl associated with symptoms of diabetes and on fasting samples  plasma glucose  126  mgdl .", "individuals with self reported , prior physician - diagnosis of diabetes were classified as having previously diagnosed diabetes .  ", "serum lipids that include total cholesterol , high - density lipoprotein ( hdl ) , low - density lipoprotein ( ldl ) , and triglycerides ( tg ) were also measured ( vitalab selectra-2 , merck , germany ) .", "the results from any person having a history of hypertension or found to have hypertension were verified by qualified doctors .", "the biochemical tests were completed in semiautomatic analysers ( microlab 300 , vital scientific , the netherelands ) .", "epidata refers to a group of applications used in combination for creating documented data structures and analysis of quantitative data . in this study ,", "the idf and ncep atp iii 's criteria for metabolic syndrome were used to calculate and compare the frequency of metabolic syndrome .", "the relationships between the prevalence of cardiovascular risk factors , demographic details , lifestyle , and physiological test results were analysed using the spearman correlation test .", "further , the differences in the categorical variables were examined using chi - squared test .", "odds ratios ( ors ) and their 95% confidence interval were calculated using binary logistic regression ( for gender and age ) and multinomial logistic regression ( for life style factors ) ."], ["in total , 14,425 people , aged 20100 ( mean age 41.4  15.1 ) , were included in the study . among them ,", "the percentage of education level is illustrated in accordance to the number of years in education ( 15 years ", "the participants were divided into four categories according to their work : labourer / farm , office , house , and none / unknown .", "participants ' physical activities were defined according to the time spent every day on physical activity as > 60  min , 3060  min , < 30  min / day , and none .", "abdominal obesity was observed in 11.5% ( n = 1607/14002 ) of the participants as per ncep criteria ( mean waist circumference : male107.38  6.19  cm , female94.84  5.84  cm ) and in 34.7% ( n = 5006/14418 ) of the participants as per idf criteria . according to the revised bmi , 10.6% ( n = 1534/14423 ) were underweight ( bmi < 18.5 ) , 28.2% ( n = 4065/14423 ) were overweight ( bmi = 2224.9 ) , and 32.5% ( n = 4689/14423 ) were obese ( bmi > 25 ) .  ", "diabetic prevalence was 6.3% ( 889/14008 ) of which 4.8% ( n = 673/14008 ) were under treatment .", "a figure of 12.3% ( n = 1718/14009 ) had a family history of diabetes .", "hypertension was observed in 33.9% ( n = 4894/14422 ) of the participants ( mean systolic 138.72  18.03  mm  hg and mean diastolic 93.09  8.45  mm  hg ) .", "only 12.9% ( 1812/14009 ) were previously diagnosed , and 8.5% were receiving treatment for hypertension .", "a history of coronary artery disease was present in 1.6% ( n = 218/14007 ) , and 1% ( n = 142 ) were under treatment for ischemic heart disease or stroke . \n", "table 2 shows the goodness of fit for the prevalence of obesity , hypertension , and diabetes .", "prevalence of hypertension showed no difference from these data , and obesity showed only a small difference .", "the percentages of the participants who had abnormal lipid profile that includes total serum cholesterol , serum ldl cholesterol , serum hdl cholesterol , serum triglycerides are listed in table 3 . there were 2191 sets of data eligible to meet the criteria for metabolic syndrome .", "ms was observed in 22.5% ( n = 494/2191 ) of the participants according to the idf criteria and 20.7% ( 454/2191 ) according to the ncep criteria .", "the percentages of individual ms risk factors among the total participants and the participants with ms are illustrated in figures 1 and 2 .", "generally , among the total participants and the specific participants with ms , the presence of abnormal lipids was higher than the other factors defining ms . however , the presence of abdominal obesity was higher among ms participants using idf criteria ( figure 2 ) .  \n", "the females had a higher prevalence of ms than males . according to the ncep criteria , the age groups 4160 and 6180 had a higher prevalence of ms than the lower age group .", "according to idf criteria , the age groups 4160 and 2040 had a higher prevalence of ms .", "the participants who worked at home had a high incidence of ms according to both the criteria used .", "the sedentary group had a higher incidence of ms than the participants who were physically active .", "the univariate correlations between cardiac risk factors are shown in table 5 , and the chi - squared independence of them in the metabolic syndrome prevalence is listed in table 6 .  ", "the prevalence of ms ( ncep scores ) had a significant positive relationship with education levels and physical activity .", "there were significant positive correlations between physical activity and the three individual ms components : high glucose ( r = 0.03 , p < .01 ) , high bp ( r = 0.04 , p < .01 ) , and low hdl ( r = 0.23 , p < .01 ) .", "there was no correlation between physical activity and the other two ms components : high triglyceride ( r = 0.003 , p > .05 ) and abdominal obesity ( r = 0.003 , p >", "the ncep scores had a positive correlation between the family history of diabetes ( r = 0.83 , p < .01 ) and hypertension ( r = 0.115 , p < .01 ) .", "although a number of these correlations show high levels of significance , the common variance is extremely low , suggesting that the sample size is having a major impact on the significance . as a result of this", "table 7 lists the chi - squared independence , odds ratios , and confidence intervals in the association between age , gender , and specific lifestyle factors in metabolic syndrome prevalence .", "gender , age , education level , and physical activity show a positive association with the prevalence of metabolic syndrome ."], ["it is important to observe the prevalence of diabetes , hypertension , and obesity individually and also the combination of risk factors as metabolic syndrome to predict the risk of cardiovascular disease .", "any association between lifestyle factors and these risk factors would provide the opportunity to encourage a change in lifestyle to promote lower levels of subsequent cvd .  ", "the large number of poorly educated people and the large number of school dropouts could be linked to the disease prevalence .", "the prevalence of hypertension and metabolic syndrome in poorly educated people was large when compared with the educated participants .", "though the results are not generalized , the relationship between education levels and the prevalence of hypertension agrees with earlier studies [ 19 , 20 ] .", "these found that education levels significantly influence the knowledge of hypertension and the awareness of cardiovascular risk .", "this suggests that there is a need to improve the awareness of health and use education to prevent or reduce the risk of ms and cardiovascular risks in these groups .", "the office workers had a lower prevalence of ms ( ncep scores ) than the other groups .", "a considerable number of office workers ( 64% ) undertook regular physical activity of more than 30  min / day .", "the home workers education levels and physical activities were comparatively lower than the other work groups .", "these findings clearly show that education and physical activity have an influence on the prevalence of ms .", "most of the females were home workers ( 75.5% ) , and their education was comparatively lower than the males .", "the amount of physical activity involved in home workers is unknown , but the results suggest it is less than that undertaken by other workers .", "asian populations continue to modernize , and levels of physical activity are declining as ( i ) home and work place jobs become more automated and sedentary and ( ii ) transportation is more readily available .", "the prevalence of ms among the participants who had no physical activity was surprisingly no different than others .", "this may be due to a higher than average number of missing values in these data ( 2191/14425 complete data to meet the criteria for ms ) or to other unknown socioeconomic factors .", "controversially , there was a high prevalence of ms among people who regularly ate fruit and vegetables .", ".   found that a higher intake of macronutrients such as fruits and vegetables is associated with general obesity .", "however , it is not clear how the vegetables and fruits were eaten , for example , overcooked , processed , and so forth .", "the exact quantity of the dietary intake was not recorded as it was not the primary area of focus of the study . in these populations ,", "these tend to report a low intake of mono - unsaturated fats ( mufas ) , n-3 polyunsaturated fats ( pufa ) , and transfatty acids ( mostly related to widespread use of vanaspati , a hydrogenated oil ) .", "the healthy traditional plant - based diets are being replaced by cheaper calorie dense high - fat foods .", "these changes are resulting in a rapid increase in the prevalence of obesity throughout asia and the subsequent development of ms .", "ness and powles also found in their review   that many studies were reporting the null or negative effects of fruit and vegetable intake on the prevalence of cardiovascular diseases .", "however , the correlations found in those studies were generally low , as seen in our study .", "further , they suggest that a food - based analysis would complement the nutrient - based analysis to clarify these issues . in nepal ,", "the regular diet in addition to fruits and vegetables , that is , such as rice , which is high in carbohydrates , and the methods of cooking may be dietary causes of metabolic syndrome .  ", "the age groups 4060 had a large prevalence of ms in this study . also , it is important to note that this middle - aged group had a high incidence of overweight or general obesity and abdominal obesity .", "the other age groups had a lower prevalence of ms than the 4060 years old , yet it was still relatively high .", "inadequate maternal nutrition in pregnancy , low birth weight , and childhood obesity may be important factors for the development of metabolic syndrome and diabetes . specifically in children and young individuals , a high intake of n-6 pufa is correlated with hyperinsulinaemia . in adults ,", "unger described metabolic syndrome as  a failure of the system of intracellular lipid homeostasis which prevents lipotoxicity in organs of overnourished individuals  . in this study", "in addition to low levels of hdl , the hdl particles are small , dense , and dysfunctional in south asians .", "hypertriglyceridaemia is a direct reflection of an insulin resistance condition , and it is interrelated to the low hdl concentrations in developing endothelial dysfunction .   in nepal ,", "a high number of the participants had abdominal obesity and were overweight / obese , according to their bmi .", "the bmi is a simple useful measure for overall abnormal weight , yet not a standard measure for obesity .", "bmi can not differentiate between whether the condition was due to unusual muscular development or the accumulation or distribution of fat in the body [ 26 , 27 ] . despite", "the low prevalence of general body obesity compared to western countries , metabolic syndrome is growing into a significant public health problem in asia .", "this may be mainly due to the large number of people with central obesity , a feature which was also observed in this study", ". the higher prevalence of ms in females is also more likely to be due to a higher incidence of abdominal obesity .", "abdominal obesity is an important factor because metabolic syndrome and increased abdominal fat are related to a reduction of adiponectin , an adopicyte - derived hormone with antiatherogenic and anti - inflammatory properties .", "the abdominal adipose tissue results in release of free fatty acids directly in the portal veins and altered lipid levels in the blood .", "further abdominal adiposity increases insulin secretion , and it would be exaggerated by decreased hepatic clearance leading to hyperinsulinemia .", "thus abdominal obesity is an important indicator of cardiovascular disease due to its link to dyslipidemia , hyperinsulinemia , hypertension , and impaired fibrinolytic capacity .", "state that if the ncep 's criteria were applied to the asian population , it might underestimate the prevalence of metabolic syndrome and the risk of cardiovascular disease .", "idf 's specific reference values for abdominal obesity make a substantial difference to the prevalence of ms between the two criteria . the idf 's cut - off points for south asians ' waist circumference", "are lower than the ncep 's general cut - off points ( 90  cm versus 102  cm in men and 80  cm versus 88  cm in women ) .", "another study on chinese population also found a large increase in the prevalence of metabolic syndrome using idf criteria compared with ncep criteria .", "however , in our study both definitions demonstrated a higher prevalence of metabolic syndrome ( 20.722.5% ) in nepal when compared with the studies done in other southeast asian countries such as thailand ( 1218% using ncep definition ) and india ( 18.3% using idf definition ) .", "these findings suggest the need for specific attention to control the disease prevalence in nepal .", "although data were prospectively collected , they may not be generalizable outside of eastern nepal .", "the results did not show substantiate relationship between smoking histories , diet , family history of cardiovascular , and metabolic syndrome ."], ["abdominal obesity , with the revised reference values , is an important risk due to its physiological relationship to the other ms risk factors .", "the ms prevalence may be due to lack of awareness and unhealthy lifestyles , so health education and more preventive measures should decrease the prevalence of ms and cardiac risks in nepal ."]]}
{"article_id": "PMC5360995", "article_text": ["balance is very important in daily activities of the elderly and greatly affects their \n quality of life . due to age - related changes , balance ability deteriorates .", "osteoarthritis is \n a degenerative disease that causes severe pain in elderly patients with low pain thresholds . \n", "research has shown that it results in a high disability score , low quality of life , and \n limited range of motion , in addition to weak muscle strength , poor balance ability , and high \n risk of falls1,2,3,4,5 .", "as osteoarthritis causes \n severe pain and functional disorders , in general , the clinical treatment is focused on pain \n management and amelioration of related disorders .", "the treatments include muscle strength \n training , electrical stimulation , manual therapy , aquatic therapy , balance therapy , and \n pharmacotherapy6 .", "as balance ability and falls are closely related in the elderly , exercise therapies need to \n focus on the maintenance of such ability7 . \n", "horseback riding exercise is one such therapy . in research conducted by arajo et al .", "8 where elderly individuals performed \n horseback riding exercise for eight weeks , the researchers reported that the exercise \n significantly improved the participants balance and muscle strength .", "unlike other \n interventions , horseback riding exercise can arouse the interest of patients and motivate \n them while improving their balance , bone density , muscle strength , and mental state9,10,11 . according to some studies ,", "horseback \n riding exercise increased the static muscular contraction of the rider against the movement \n of the horse and improved the muscle strength in the quadriceps femoris and knee flexors of \n young riders12 , 13 .", "most people do not indulge in horse riding because of where they live ( i.e. , urban rather \n than rural areas ) or the costs and risks involved in horse riding .", "hence , simulator \n horseback riding exercise equipment using virtual reality has been developed for use at home \n and in small spaces .", "previous studies reported that horseback riding exercise significantly \n increased oxygen intake , minute ventilation , met , calorie consumption , and muscle14 , 15 .", "although various therapeutic approaches have made with horseback \n riding exercise in the previous studies there is a lack of research on the effects of \n horseback riding exercise equipment on the balance and gait abilities of elderly patients \n with osteoarthritis .", "experimental procedures were performed in accordance with the protocols established by the \n institution of animal care and use committee ( iacuc ) at the daegu university . include criteria was aged 65  years or over ( table \n 1table 1.general characteristics of the subjectsvariablessubjects ( n=15)gender ( m / f)6/9age ( years)76.5  7.7height ( cm)160.6  9.7weight ( kg)54.7  9.8mean  sd ) , and with a history of none falls in previous year , lower pain for perform \n experiment , and visual or auditory damage , nervous system or of the vestibular organ \n problems , who ca nt understand the contents of the experiment were excluded . after providing \n information study all subjects received a participation agreement . in this study , the subjects , while providing conventional physical therapy , 8 weeks three \n times a week the movement of the horse riding equipment , was carried out for 30 minutes . \n before performing horse riding simulation exercise", ", subjects were educated about exercise \n program through a research assistant s demonstration . in this study , used the slimrider \n ( shinhwa , mx-0004se , korea ) , which is a horse riding simulator . while performing a horse \n riding equipment exercise , subjects were effort to maintain the correct posture .", "exercise \n intensity was increased gradually according to the state of the subjects while performing \n the exercise , the research assistant were always observed to prevent falling . in order to evaluate the balance of the elderly , it was using the frt ( functional reach \n test ) and sbbsf ( short form berg balance scale ) .", "the spss for windows ( version 18.0 ) was used for data analysis of the study . in order to \n compare the differences between before and after , it was analyzed using the paired t - test . \n", "in this study , sfbbs scores and range of frt increased significantly after horse riding \n simulator exercise ( table 2table 2.comparison of measurement values at pre - test and post - testvariablesprepostsfbbs ( score)18.6  5.822.3  4.4**frt ( cm)19.7  9.526.3  7.5*mean  sd .", "osteoarthritis causes severe pain in elderly patients who have a low threshold for pain and \n high disability scores , thereby resulting in a low quality of life , increased risk of falls , \n and poor balance ability1 , 2 , 6 . in the present \n study", ", the static , dynamic , and functional balance abilities of osteoarthritis patients who \n undertook horseback riding exercises on exercise equipment were measured using a frt and \n sfbbs .", "the findings are in \n accordance with those of study that stroke patients performed horseback riding exercise for \n 35  min five times a week for eight weeks and arajo8 in which elderly patients performed horseback riding exercise for \n eight weeks .", "they are also in accordance with the results of han jun - young16 in a study of stroke patients who \n performed horseback riding exercise for 12 weeks that an eight - week - long horseback riding \n exercise significantly improved the bbs scores of osteoarthritis patients . in general \n elderly , the average bbs score is 20.60  4.0817 . in the present study ,", "after eight weeks of the exercise , the average score \n was 22.3  4.4 , higher than the average .", "the results suggest that horseback riding exercise \n equipment can improve dynamic and functional balance abilities .", "furthermore , according to the frt results , the static balance ability of the participants \n improved .", "these results are in line with the research findings of yasuhiro et al.18 and kim sung - gil11 where the frt scores improved significantly after \n 12-week - long and 8-week - long horseback riding exercise , respectively .", "an increase in the frt \n score is considered an improvement in static balance ability .", "based on the findings of the \n present study , horseback riding exercise equipment seems to improve the static balance \n ability of the elderly ."], "abstract_text": ["<S> [ purpose ] the aim of this study was to examine the effects of horse - riding simulator \n exercise on balance in elderly with knee osteoarthritis . [ subjects and methods ] fifty \n elderly patients with knee osteoarthritis were recruited , a horse - riding simulator group \n performed exercise three times a week for eight weeks . and each exercise was performed for \n 30 minutes . </S>", "<S> [ results ] the horse - riding simulator group showed significant differences \n after the intervention in short form berg balance scale , functional reaching test . \n </S>", "<S> [ conclusion ] the results of this study indicate that horse - riding simulator exercise is \n effective on knee osteoarthritis . </S>", "<S> therefore , horse - riding simulator exercise can be used \n balance training for knee osteoarthritis . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["balance is very important in daily activities of the elderly and greatly affects their \n quality of life . due to age - related changes , balance ability deteriorates .", "osteoarthritis is \n a degenerative disease that causes severe pain in elderly patients with low pain thresholds . \n", "research has shown that it results in a high disability score , low quality of life , and \n limited range of motion , in addition to weak muscle strength , poor balance ability , and high \n risk of falls1,2,3,4,5 .", "as osteoarthritis causes \n severe pain and functional disorders , in general , the clinical treatment is focused on pain \n management and amelioration of related disorders .", "the treatments include muscle strength \n training , electrical stimulation , manual therapy , aquatic therapy , balance therapy , and \n pharmacotherapy6 .", "as balance ability and falls are closely related in the elderly , exercise therapies need to \n focus on the maintenance of such ability7 . \n", "horseback riding exercise is one such therapy . in research conducted by arajo et al .", "8 where elderly individuals performed \n horseback riding exercise for eight weeks , the researchers reported that the exercise \n significantly improved the participants balance and muscle strength .", "unlike other \n interventions , horseback riding exercise can arouse the interest of patients and motivate \n them while improving their balance , bone density , muscle strength , and mental state9,10,11 . according to some studies ,", "horseback \n riding exercise increased the static muscular contraction of the rider against the movement \n of the horse and improved the muscle strength in the quadriceps femoris and knee flexors of \n young riders12 , 13 .", "most people do not indulge in horse riding because of where they live ( i.e. , urban rather \n than rural areas ) or the costs and risks involved in horse riding .", "hence , simulator \n horseback riding exercise equipment using virtual reality has been developed for use at home \n and in small spaces .", "previous studies reported that horseback riding exercise significantly \n increased oxygen intake , minute ventilation , met , calorie consumption , and muscle14 , 15 .", "although various therapeutic approaches have made with horseback \n riding exercise in the previous studies there is a lack of research on the effects of \n horseback riding exercise equipment on the balance and gait abilities of elderly patients \n with osteoarthritis ."], ["experimental procedures were performed in accordance with the protocols established by the \n institution of animal care and use committee ( iacuc ) at the daegu university . include criteria was aged 65  years or over ( table \n 1table 1.general characteristics of the subjectsvariablessubjects ( n=15)gender ( m / f)6/9age ( years)76.5  7.7height ( cm)160.6  9.7weight ( kg)54.7  9.8mean  sd ) , and with a history of none falls in previous year , lower pain for perform \n experiment , and visual or auditory damage , nervous system or of the vestibular organ \n problems , who ca nt understand the contents of the experiment were excluded . after providing \n information study all subjects received a participation agreement . in this study , the subjects , while providing conventional physical therapy , 8 weeks three \n times a week the movement of the horse riding equipment , was carried out for 30 minutes . \n before performing horse riding simulation exercise", ", subjects were educated about exercise \n program through a research assistant s demonstration . in this study , used the slimrider \n ( shinhwa , mx-0004se , korea ) , which is a horse riding simulator . while performing a horse \n riding equipment exercise , subjects were effort to maintain the correct posture .", "exercise \n intensity was increased gradually according to the state of the subjects while performing \n the exercise , the research assistant were always observed to prevent falling . in order to evaluate the balance of the elderly , it was using the frt ( functional reach \n test ) and sbbsf ( short form berg balance scale ) .", "the spss for windows ( version 18.0 ) was used for data analysis of the study . in order to \n compare the differences between before and after , it was analyzed using the paired t - test . \n"], ["in this study , sfbbs scores and range of frt increased significantly after horse riding \n simulator exercise ( table 2table 2.comparison of measurement values at pre - test and post - testvariablesprepostsfbbs ( score)18.6  5.822.3  4.4**frt ( cm)19.7  9.526.3  7.5*mean  sd ."], ["osteoarthritis causes severe pain in elderly patients who have a low threshold for pain and \n high disability scores , thereby resulting in a low quality of life , increased risk of falls , \n and poor balance ability1 , 2 , 6 . in the present \n study", ", the static , dynamic , and functional balance abilities of osteoarthritis patients who \n undertook horseback riding exercises on exercise equipment were measured using a frt and \n sfbbs .", "the findings are in \n accordance with those of study that stroke patients performed horseback riding exercise for \n 35  min five times a week for eight weeks and arajo8 in which elderly patients performed horseback riding exercise for \n eight weeks .", "they are also in accordance with the results of han jun - young16 in a study of stroke patients who \n performed horseback riding exercise for 12 weeks that an eight - week - long horseback riding \n exercise significantly improved the bbs scores of osteoarthritis patients . in general \n elderly , the average bbs score is 20.60  4.0817 . in the present study ,", "after eight weeks of the exercise , the average score \n was 22.3  4.4 , higher than the average .", "the results suggest that horseback riding exercise \n equipment can improve dynamic and functional balance abilities .", "furthermore , according to the frt results , the static balance ability of the participants \n improved .", "these results are in line with the research findings of yasuhiro et al.18 and kim sung - gil11 where the frt scores improved significantly after \n 12-week - long and 8-week - long horseback riding exercise , respectively .", "an increase in the frt \n score is considered an improvement in static balance ability .", "based on the findings of the \n present study , horseback riding exercise equipment seems to improve the static balance \n ability of the elderly ."]]}
{"article_id": "PMC4993422", "article_text": [", medulloblastomas can be classified in 4 different molecular subtypes : wnt , shh , group 3 and group 4 .", "each of these molecular subtypes has specific underlying molecular features , gene expression , demographic characteristics and prognosis . sonic hedgehog ( shh ) medulloblastomas", "are characterized by activation of the hedgehog ( hh ) signaling pathway and are often driven by mutations in hh pathway components . here", "we focus on shh medulloblastoma and discuss how cell senescence shapes the natural history and molecular evolution of this childhood cancer .", "for many years , it was thought that tp53 mutations were infrequent in medulloblastomas and that p53 signaling was dispensable for medulloblastoma tumor suppression .", "however , recent studies reported that tp53 mutations are frequent in human wnt ( with a rate of 16% ) and shh primary medulloblastomas ( with a rate of 13% to 21% , depending on the study ) and are indicators of poor prognosis exclusively in shh medulloblastoma .", "moreover , tp53 mutations have recently been identified as a key event in the pathogenesis of medulloblastoma recurrence .", "although the specific roles of p53 in medulloblastoma are still largely unknown , the presence of recurrent tp53 mutations suggests that p53 signaling plays an important role in driving medulloblastoma tumorigenesis .", "human genomic studies also established that tp53 mutations co - exist with mutations or amplifications of hh signaling components such as shh , smo , sufu , gli2 and mycn .", "these studies , however , do not establish the sequential order of genetic events that lead to medulloblastoma formation and how these mutations correlate with presumptive histopathological stages of medulloblastoma .", "nevertheless , patients with li - fraumeni syndrome , caused by germ - line mutations in tp53 , offer an opportunity to study how medulloblastomas arise at the genetic level .", "li - fraumeni patients are cancer - prone and sometimes develop medulloblastoma ; therefore , at least for this subset of patients , tp53 mutations are the first genetic event leading to medulloblastoma formation ( fig . ", "notably , most li - fraumeni medulloblastomas belong to the shh subgroup , indicating that tp53 mutations specifically predispose to shh medulloblastoma . because cerebellum granule cell precursors ( gcps ) are the cells of origin of shh medulloblastoma", "moreover , these shh medulloblastomas seem to be the result of chromothripsis , a massive genome rearrangement caused by chromosomal shattering likely occurring in a single event . these chromothriptic events", "likely lead to mutations in components of hh signaling , such as gli2 , boc and mycn amplifications .", "since hh signaling is the most important mitogenic pathway for gcps , it is therefore expected that acquisition of hh pathway mutations efficiently causes shh medulloblastoma in li - fraumeni patients . \n", "( a ) in li - fraumeni patients with germ - line tp53 mutations , cerebellum gcps experience chromosomal instability .", "chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn .", "( b ) in ptch1 mice , loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia .", "spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . proliferation and senescence levels during medulloblastoma formation are indicated . \n ", "( a ) in li - fraumeni patients with germ - line tp53 mutations , cerebellum gcps experience chromosomal instability .", "chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn .", "( b ) in ptch1 mice , loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia .", "spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . proliferation and senescence levels during medulloblastoma formation are indicated .", "although p53 knockout mice or mouse models of li - fraumeni syndrome do not develop medulloblastoma , elegant studies have demonstrated that the inactivation of p53 together with other dna repair factors such as as xrcc4 , ligase iv , xrcc2 and brca2 leads to shh medulloblastoma .", "interestingly , those medulloblastomas also harbored spontaneous mutations in hh signaling components such as ptch1 , mycn and gli2 .", "this result not only highlights how important it is to maintain genomic stability to prevent shh medulloblastoma formation , but it also indicates that hh signaling activation seems to be necessary for medulloblastoma formation even when p53 and dna repair mechanisms are absent . in summary ,", "germ - line p53 mutations in both mouse and human cause genomic instability and lead to mutations in hh signaling components , conducing to medulloblastoma formation ( fig . ", "this is an interesting paradigm showing that hh signaling mutations happen subsequent to tp53 mutations in li - fraumeni syndrome .", "half of the shh medulloblastomas with tp53 mutations have a germ - line ( li - fraumeni ) origin and are potentially explained by the mechanism described above .", "however , it is not known how the other shh medulloblastoma cases ( including the ones with somatic tp53 mutations and the ones without tp53 mutations ) develop and the temporal order in which they acquire their mutations .", "another unresolved , yet related , question is whether advanced medulloblastomas arise in a step - wise manner from subclinical precancerous lesions . for many epithelial cancers ,", "the availability of preneoplastic lesions allowed the establishment of tumor progression models with sequential histopathological stages and the molecular changes that characterize them .", "however , the problem for understanding brain tumor development lies in the inability to detect and obtain precancerous lesions ; therefore , genome sequencing of advanced brain tumors only offers a snapshot of the mutations present in advanced tumors but does not show the order in which mutations are acquired during tumor progression . to address this question , we used ptch1 heterozygous mice , a well - established model of shh medulloblastoma ( fig .  2 ) .", "ptch1 mice develop preneoplastic lesions with high frequency , but only a fraction of those animals develop advanced medulloblastoma .", "therefore , we hypothesized that an unidentified tumor suppressive mechanism might restrain the progression of medulloblastoma preneoplasia into advanced tumors .", "we found that apoptosis levels are the same between preneoplastic lesions and advanced medulloblastoma , eliminating apoptosis as an essential tumor suppressor in this model of shh medulloblastoma .", "surprisingly , when we looked at cell senescence , we found that while preneoplastic lesions display high numbers of p21 and p16 positive cells ( which are effectors and markers of cell senescence and cell cycle arrest ) , advanced medulloblastomas have very low levels of senescence . these high levels of senescence", "are paralleled by lower levels of proliferation and correlate with loss of heterozygosity ( loh ) of the ptch1 wild - type allele in preneoplastic lesions ( fig . ", "1b ) , suggesting that high levels of hh signaling may contribute to cell senescence .", "using laser capture microdissection , we found that one - third of all advanced medulloblastomas acquired p53 mutations that were not present in preneoplastic lesions , supporting the notion that p53 mutations allow senescence evasion and medulloblastoma progression ( fig . ", "moreover , we found that engineered p53 mutations prevent cellular senescence and accelerate medulloblastoma formation , showing that p53 mutations lead to senescence evasion . further supporting the idea that senescence evasion is necessary for medulloblastoma progression , advanced tumors without p53 mutations display low expression of p16 due to promoter methylation . in summary", ", we found that , contrary to li - fraumeni syndrome ( where tp53 germ - line mutations lead to hh signaling mutations ) , hh signaling hyperactivity leads to cell senescence in preneoplastic lesions and creates selection pressure for the inactivation of p53 or p16 , which allows the progression from preneoplasia to advanced medulloblastoma ( fig . ", "figure 2.medulloblastoma formation in ptch1 mice . during postnatal development , granule cell precursors ( gcps ) of the cerebellum ,", "the cells of origin of shh medulloblastoma , are located in the external granule - cell layer ( egl ) .", "after their proliferation , most gcps differentiate , populate the internal granule - cell layer ( igl ) , and disappear from the egl after the second postnatal week in the mouse .", "loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . while most preneoplastic lesions disappear , some of them progress to advanced medulloblastoma .", "medulloblastoma formation in ptch1 mice . during postnatal development , granule cell precursors ( gcps ) of the cerebellum ,", "the cells of origin of shh medulloblastoma , are located in the external granule - cell layer ( egl ) .", "after their proliferation , most gcps differentiate , populate the internal granule - cell layer ( igl ) , and disappear from the egl after the second postnatal week in the mouse .", "loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . while most preneoplastic lesions disappear , some of them progress to advanced medulloblastoma .", "importantly , the finding of spontaneous p53 mutations is not limited to the ptch1 model , as we also found spontaneous p53 mutations in tumors from olig1-gnas mice , another model of shh medulloblastoma .", "1b ) , this may highlight some possible interactions between p53 and hh signaling in the brain .", "shh , a protein secreted by purkinje cells of the cerebellum , is the most potent mitogen for gcps .", "in contrast to these proliferative effects of shh , p53 activity supresses proliferation and self - renewal of adult neural stem cells of the subventricular zone , and this effect might be mediated by p21 .", "this may explain why p53 activity is downregulated during neurogenesis to allow cell proliferation and brain formation .", "interestingly , gli activity has been shown to downregulate p53 protein levels in cell lines .", "specifically , it has been proposed that high levels of hh signaling in cell lines caused by expression of constitutively active smo mutants or overexpression of gli1 and gli2 leads to an mdm2-dependent degradation of p53 .", "consistently , low levels of mdm2 in vivo increase p53 levels , lead to cerebellar hypoplasia and reduce medulloblastoma development in ptch1 mice , showing that p53 signaling negatively controls cerebellum growth and implying that p53 signaling is important for medulloblastoma tumor suppression .", "similarly , the proto - oncogene ppm1d , a negative regulator of p53 , is overexpressed in medulloblastomas and increases medulloblastoma formation in mice when overexpressed together with shh .", "together , these findings provide strong evidence that hh signaling leads to a functional inactivation of p53 signaling that allows gcp proliferation and , in some instances , medulloblastoma formation .", "however , the presence of somatic p53 mutations in ptch1 medulloblastoma demonstrates that the ability of shh signaling to functionally suppress p53 signaling is not always sufficient to inactivate p53 activity in a tumorigenic context . for many years", ", it has been known that p53 deletion accelerates medulloblastoma formation and increases medulloblastoma incidence in ptch1 mice ; however , the mechanism responsible for this was never investigated .", "the fact that p53 mutations , which are acquired spontaneously during medulloblastoma formation , lead to senescence evasion provides an explanation for the presence of somatic tp53 mutations in human shh medulloblastoma .", "recently , we also found that the ligand shh induces dna damage in gcps , an effect that requires the presence of the hh receptor boc and cyclind1 .", "these results support the hypothesis that gcps are sensitive to replicative stress and that hedgehog signaling likely causes replication stress .", "it has been demonstrated that oncogene activation leads to oncogene - induced cell senescence ( ois ) , a tumor - suppressive mechanism that prevents transformation of premalignant lesions into tumors .", "moreover , oncogene - induced dna damage seems to be required for ois . in light of this", ", the fact that we found cell senescence and ptch1 loh in medulloblastoma preneoplastic lesions suggests that high levels of hedgehog signaling in preneoplasia likely cause high levels of oncogenic stress and this leads to cell senescence .", "therefore , we propose that high levels of hedgehog signaling shape the molecular evolution of medulloblastoma by leading to ois and creating selection pressure to inactivate p53 in order to evade ois .", "in addition to oncogenes , loss of tumor suppressor genes has been shown to cause ois .", "for example , in the context of prostate cancer , pten inactivation causes senescence and leads to the acquisition of p53 mutations . \n", "neurofibromin ( nf1 ) loss also leads to ras - mediated induction of cell senescence . since we observed a strong association between ptch1 loss and cell senescence in medulloblastoma preneoplasia , ptch1 seems to be a new tumor suppressor whose absence may be capable of causing ois .", "however , the molecular mechanism leading to tp53 mutations has never been investigated in wnt medulloblastoma .", "interesting work has shown that , at least in cell lines , overexpression of the downstream wnt effector  - catenin leads to p53 stabilization and activation .", "wnt activation in colorectal cancer has been associated with decreased levels of proliferation and accumulation of p53 protein in early tumor stages , suggesting that wnt signaling may lead to cell senescence in precancerous lesions and this could be the cause of the acquisition of tp53 mutations at late stages of colorectal cancer .", "we thus speculate that wnt activation in the brainstem may also lead to changes compatible with cell senescence and this may be an explanation for the presence of tp53 mutations in advanced wnt medulloblastomas", ". this is supported by the fact that expression of an active  - catenin mutant in the lower rhombic lip of mice leads to the formation of hyperplastic lesions that only progress to advanced medulloblastomas when p53 is also deleted .", "it has been shown that low - grade astrocytoma lesions display a dna damage response and that loss of components of the atm - chk2-p53 pathway accelerates astrocytoma development .", "although cell senescence was not interrogated in those reports , another study showed that pilocytic astrocytomas ( pa ) , the most benign type of astrocytomas , display mapk activation and ois ; interestingly , pa are indolent tumors that display long periods of growth arrest , rarely become high - grade astrocytomas , and do not display tp53 mutations .", "this is in agreement with the idea that pa are long - term senescent astrocytic lesions that do not progress .", "\n eglexternal granule - cell layergcpsgranule cell precursorshhhedgehogiglinternal granule - cell layeroisoncogene - induced senescenceshhsonic hedgehog external granule - cell layer granule cell precursors internal granule - cell layer oncogene - induced senescence", "work done in the charron lab was supported by grants from the canadian institutes of health research ( cihr ) , the fonds de recherche du qubec - sant ( frqs ) , and the canada foundation for innovation ( cfi ) .", "sms is recipient of the mcgill james o. and maria meadows fellowship and ircm challenge - gupards et gazelles gourmands scholarship ."], "abstract_text": ["<S> abstractthe mechanisms leading to brain tumor formation are poorly understood . using ptch1+/ mice as a medulloblastoma model </S>", "<S> , sequential mutations were found to shape tumor evolution . </S>", "<S> initially , medulloblastoma preneoplastic lesions display loss of heterozygosity of the ptch1 wild - type allele , an event associated with cell senescence in preneoplasia . </S>", "<S> subsequently , p53 mutations lead to senescence evasion and progression from preneoplasia to medulloblastoma . </S>", "<S> these findings are consistent with a model where high levels of hedgehog signaling caused by the loss of the tumor suppressor ptch1 lead to oncogene - induced senescence and drive p53 mutations . </S>", "<S> thus , cell senescence is an important characteristic of a subset of shh medulloblastoma and might explain the acquisition of somatic tp53 mutations in human medulloblastoma . </S>", "<S> this mode of medulloblastoma formation contrasts with the one characterizing li - fraumeni patients with medulloblastoma , where tp53 germ - line mutations cause chromothriptic genomic instability and lead to mutations in hedgehog signaling genes , which drive medulloblastoma growth . </S>", "<S> here we discuss in detail these 2 alternative mechanisms leading to medulloblastoma tumorigenesis . </S>"], "labels": null, "section_names": ["Introduction", "None", "Hh and p53 signaling in medulloblastoma", "Cell senescence as a driver of ", "Abbreviations", "Disclosure of potential conflicts of interest", "Funding"], "sections": [[", medulloblastomas can be classified in 4 different molecular subtypes : wnt , shh , group 3 and group 4 .", "each of these molecular subtypes has specific underlying molecular features , gene expression , demographic characteristics and prognosis . sonic hedgehog ( shh ) medulloblastomas", "are characterized by activation of the hedgehog ( hh ) signaling pathway and are often driven by mutations in hh pathway components . here", "we focus on shh medulloblastoma and discuss how cell senescence shapes the natural history and molecular evolution of this childhood cancer ."], ["for many years , it was thought that tp53 mutations were infrequent in medulloblastomas and that p53 signaling was dispensable for medulloblastoma tumor suppression .", "however , recent studies reported that tp53 mutations are frequent in human wnt ( with a rate of 16% ) and shh primary medulloblastomas ( with a rate of 13% to 21% , depending on the study ) and are indicators of poor prognosis exclusively in shh medulloblastoma .", "moreover , tp53 mutations have recently been identified as a key event in the pathogenesis of medulloblastoma recurrence .", "although the specific roles of p53 in medulloblastoma are still largely unknown , the presence of recurrent tp53 mutations suggests that p53 signaling plays an important role in driving medulloblastoma tumorigenesis .", "human genomic studies also established that tp53 mutations co - exist with mutations or amplifications of hh signaling components such as shh , smo , sufu , gli2 and mycn .", "these studies , however , do not establish the sequential order of genetic events that lead to medulloblastoma formation and how these mutations correlate with presumptive histopathological stages of medulloblastoma .", "nevertheless , patients with li - fraumeni syndrome , caused by germ - line mutations in tp53 , offer an opportunity to study how medulloblastomas arise at the genetic level .", "li - fraumeni patients are cancer - prone and sometimes develop medulloblastoma ; therefore , at least for this subset of patients , tp53 mutations are the first genetic event leading to medulloblastoma formation ( fig . ", "notably , most li - fraumeni medulloblastomas belong to the shh subgroup , indicating that tp53 mutations specifically predispose to shh medulloblastoma . because cerebellum granule cell precursors ( gcps ) are the cells of origin of shh medulloblastoma", "moreover , these shh medulloblastomas seem to be the result of chromothripsis , a massive genome rearrangement caused by chromosomal shattering likely occurring in a single event . these chromothriptic events", "likely lead to mutations in components of hh signaling , such as gli2 , boc and mycn amplifications .", "since hh signaling is the most important mitogenic pathway for gcps , it is therefore expected that acquisition of hh pathway mutations efficiently causes shh medulloblastoma in li - fraumeni patients . \n", "( a ) in li - fraumeni patients with germ - line tp53 mutations , cerebellum gcps experience chromosomal instability .", "chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn .", "( b ) in ptch1 mice , loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia .", "spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . proliferation and senescence levels during medulloblastoma formation are indicated . \n ", "( a ) in li - fraumeni patients with germ - line tp53 mutations , cerebellum gcps experience chromosomal instability .", "chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn .", "( b ) in ptch1 mice , loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia .", "spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . proliferation and senescence levels during medulloblastoma formation are indicated .", "although p53 knockout mice or mouse models of li - fraumeni syndrome do not develop medulloblastoma , elegant studies have demonstrated that the inactivation of p53 together with other dna repair factors such as as xrcc4 , ligase iv , xrcc2 and brca2 leads to shh medulloblastoma .", "interestingly , those medulloblastomas also harbored spontaneous mutations in hh signaling components such as ptch1 , mycn and gli2 .", "this result not only highlights how important it is to maintain genomic stability to prevent shh medulloblastoma formation , but it also indicates that hh signaling activation seems to be necessary for medulloblastoma formation even when p53 and dna repair mechanisms are absent . in summary ,", "germ - line p53 mutations in both mouse and human cause genomic instability and lead to mutations in hh signaling components , conducing to medulloblastoma formation ( fig . ", "this is an interesting paradigm showing that hh signaling mutations happen subsequent to tp53 mutations in li - fraumeni syndrome .", "half of the shh medulloblastomas with tp53 mutations have a germ - line ( li - fraumeni ) origin and are potentially explained by the mechanism described above .", "however , it is not known how the other shh medulloblastoma cases ( including the ones with somatic tp53 mutations and the ones without tp53 mutations ) develop and the temporal order in which they acquire their mutations .", "another unresolved , yet related , question is whether advanced medulloblastomas arise in a step - wise manner from subclinical precancerous lesions . for many epithelial cancers ,", "the availability of preneoplastic lesions allowed the establishment of tumor progression models with sequential histopathological stages and the molecular changes that characterize them .", "however , the problem for understanding brain tumor development lies in the inability to detect and obtain precancerous lesions ; therefore , genome sequencing of advanced brain tumors only offers a snapshot of the mutations present in advanced tumors but does not show the order in which mutations are acquired during tumor progression . to address this question , we used ptch1 heterozygous mice , a well - established model of shh medulloblastoma ( fig .  2 ) .", "ptch1 mice develop preneoplastic lesions with high frequency , but only a fraction of those animals develop advanced medulloblastoma .", "therefore , we hypothesized that an unidentified tumor suppressive mechanism might restrain the progression of medulloblastoma preneoplasia into advanced tumors .", "we found that apoptosis levels are the same between preneoplastic lesions and advanced medulloblastoma , eliminating apoptosis as an essential tumor suppressor in this model of shh medulloblastoma .", "surprisingly , when we looked at cell senescence , we found that while preneoplastic lesions display high numbers of p21 and p16 positive cells ( which are effectors and markers of cell senescence and cell cycle arrest ) , advanced medulloblastomas have very low levels of senescence . these high levels of senescence", "are paralleled by lower levels of proliferation and correlate with loss of heterozygosity ( loh ) of the ptch1 wild - type allele in preneoplastic lesions ( fig . ", "1b ) , suggesting that high levels of hh signaling may contribute to cell senescence .", "using laser capture microdissection , we found that one - third of all advanced medulloblastomas acquired p53 mutations that were not present in preneoplastic lesions , supporting the notion that p53 mutations allow senescence evasion and medulloblastoma progression ( fig . ", "moreover , we found that engineered p53 mutations prevent cellular senescence and accelerate medulloblastoma formation , showing that p53 mutations lead to senescence evasion . further supporting the idea that senescence evasion is necessary for medulloblastoma progression , advanced tumors without p53 mutations display low expression of p16 due to promoter methylation . in summary", ", we found that , contrary to li - fraumeni syndrome ( where tp53 germ - line mutations lead to hh signaling mutations ) , hh signaling hyperactivity leads to cell senescence in preneoplastic lesions and creates selection pressure for the inactivation of p53 or p16 , which allows the progression from preneoplasia to advanced medulloblastoma ( fig . ", "figure 2.medulloblastoma formation in ptch1 mice . during postnatal development , granule cell precursors ( gcps ) of the cerebellum ,", "the cells of origin of shh medulloblastoma , are located in the external granule - cell layer ( egl ) .", "after their proliferation , most gcps differentiate , populate the internal granule - cell layer ( igl ) , and disappear from the egl after the second postnatal week in the mouse .", "loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . while most preneoplastic lesions disappear , some of them progress to advanced medulloblastoma .", "medulloblastoma formation in ptch1 mice . during postnatal development , granule cell precursors ( gcps ) of the cerebellum ,", "the cells of origin of shh medulloblastoma , are located in the external granule - cell layer ( egl ) .", "after their proliferation , most gcps differentiate , populate the internal granule - cell layer ( igl ) , and disappear from the egl after the second postnatal week in the mouse .", "loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . while most preneoplastic lesions disappear , some of them progress to advanced medulloblastoma .", "importantly , the finding of spontaneous p53 mutations is not limited to the ptch1 model , as we also found spontaneous p53 mutations in tumors from olig1-gnas mice , another model of shh medulloblastoma ."], ["1b ) , this may highlight some possible interactions between p53 and hh signaling in the brain .", "shh , a protein secreted by purkinje cells of the cerebellum , is the most potent mitogen for gcps .", "in contrast to these proliferative effects of shh , p53 activity supresses proliferation and self - renewal of adult neural stem cells of the subventricular zone , and this effect might be mediated by p21 .", "this may explain why p53 activity is downregulated during neurogenesis to allow cell proliferation and brain formation .", "interestingly , gli activity has been shown to downregulate p53 protein levels in cell lines .", "specifically , it has been proposed that high levels of hh signaling in cell lines caused by expression of constitutively active smo mutants or overexpression of gli1 and gli2 leads to an mdm2-dependent degradation of p53 .", "consistently , low levels of mdm2 in vivo increase p53 levels , lead to cerebellar hypoplasia and reduce medulloblastoma development in ptch1 mice , showing that p53 signaling negatively controls cerebellum growth and implying that p53 signaling is important for medulloblastoma tumor suppression .", "similarly , the proto - oncogene ppm1d , a negative regulator of p53 , is overexpressed in medulloblastomas and increases medulloblastoma formation in mice when overexpressed together with shh .", "together , these findings provide strong evidence that hh signaling leads to a functional inactivation of p53 signaling that allows gcp proliferation and , in some instances , medulloblastoma formation .", "however , the presence of somatic p53 mutations in ptch1 medulloblastoma demonstrates that the ability of shh signaling to functionally suppress p53 signaling is not always sufficient to inactivate p53 activity in a tumorigenic context . for many years", ", it has been known that p53 deletion accelerates medulloblastoma formation and increases medulloblastoma incidence in ptch1 mice ; however , the mechanism responsible for this was never investigated .", "the fact that p53 mutations , which are acquired spontaneously during medulloblastoma formation , lead to senescence evasion provides an explanation for the presence of somatic tp53 mutations in human shh medulloblastoma ."], ["recently , we also found that the ligand shh induces dna damage in gcps , an effect that requires the presence of the hh receptor boc and cyclind1 .", "these results support the hypothesis that gcps are sensitive to replicative stress and that hedgehog signaling likely causes replication stress .", "it has been demonstrated that oncogene activation leads to oncogene - induced cell senescence ( ois ) , a tumor - suppressive mechanism that prevents transformation of premalignant lesions into tumors .", "moreover , oncogene - induced dna damage seems to be required for ois . in light of this", ", the fact that we found cell senescence and ptch1 loh in medulloblastoma preneoplastic lesions suggests that high levels of hedgehog signaling in preneoplasia likely cause high levels of oncogenic stress and this leads to cell senescence .", "therefore , we propose that high levels of hedgehog signaling shape the molecular evolution of medulloblastoma by leading to ois and creating selection pressure to inactivate p53 in order to evade ois .", "in addition to oncogenes , loss of tumor suppressor genes has been shown to cause ois .", "for example , in the context of prostate cancer , pten inactivation causes senescence and leads to the acquisition of p53 mutations . \n", "neurofibromin ( nf1 ) loss also leads to ras - mediated induction of cell senescence . since we observed a strong association between ptch1 loss and cell senescence in medulloblastoma preneoplasia , ptch1 seems to be a new tumor suppressor whose absence may be capable of causing ois .", "however , the molecular mechanism leading to tp53 mutations has never been investigated in wnt medulloblastoma .", "interesting work has shown that , at least in cell lines , overexpression of the downstream wnt effector  - catenin leads to p53 stabilization and activation .", "wnt activation in colorectal cancer has been associated with decreased levels of proliferation and accumulation of p53 protein in early tumor stages , suggesting that wnt signaling may lead to cell senescence in precancerous lesions and this could be the cause of the acquisition of tp53 mutations at late stages of colorectal cancer .", "we thus speculate that wnt activation in the brainstem may also lead to changes compatible with cell senescence and this may be an explanation for the presence of tp53 mutations in advanced wnt medulloblastomas", ". this is supported by the fact that expression of an active  - catenin mutant in the lower rhombic lip of mice leads to the formation of hyperplastic lesions that only progress to advanced medulloblastomas when p53 is also deleted .", "it has been shown that low - grade astrocytoma lesions display a dna damage response and that loss of components of the atm - chk2-p53 pathway accelerates astrocytoma development .", "although cell senescence was not interrogated in those reports , another study showed that pilocytic astrocytomas ( pa ) , the most benign type of astrocytomas , display mapk activation and ois ; interestingly , pa are indolent tumors that display long periods of growth arrest , rarely become high - grade astrocytomas , and do not display tp53 mutations .", "this is in agreement with the idea that pa are long - term senescent astrocytic lesions that do not progress ."], ["\n eglexternal granule - cell layergcpsgranule cell precursorshhhedgehogiglinternal granule - cell layeroisoncogene - induced senescenceshhsonic hedgehog external granule - cell layer granule cell precursors internal granule - cell layer oncogene - induced senescence"], [""], ["work done in the charron lab was supported by grants from the canadian institutes of health research ( cihr ) , the fonds de recherche du qubec - sant ( frqs ) , and the canada foundation for innovation ( cfi ) .", "sms is recipient of the mcgill james o. and maria meadows fellowship and ircm challenge - gupards et gazelles gourmands scholarship ."]]}
{"article_id": "PMC2884340", "article_text": ["a 33-year - old woman , ( gravida 3 , para 2 ) , at 34 weeks of gestation , presented with episodes of lightheadedness , and dyspnea .", "she was asymptomatic until 18 weeks of gestation , when she started having episodes of palpitations associated with presyncope .", "initially , the episodes were infrequent ( less than once per day ) but a gradual increase in frequency and severity was noted by the patient .", "one month prior to presentation , she noted episodes of paroxysmal nocturnal dyspnea , but no dyspnea on exertion , chest pain , diaphoresis , pedal edema , orthopnea , or neurological symptoms .", "she had no significant past medical history and had not had complications relating to this pregnancy .", "all prior pregnancies had progressed to full term without similar symptoms or complications , and resulted in spontaneous vaginal deliveries of healthy newborns .", "physical examination revealed an afebrile patient with a regular pulse of 80 beats per minute , blood pressure 110/70 mmhg , and an oxygen saturation of 100% on room air .", "the examination was normal except for mild non - pitting edema and fine bibasilar crackles .", "laboratory investigations revealed normal complete blood count , electrolytes , renal function , cardiac markers and tsh .", "cardiac monitoring showed multiple episodes of monomorphic , non - sustained ventricular tachycardia ( vt ) with left bundle branch morphology , consistent with vt originating from the right ventricular outflow tract ( fig .", "urgent two - dimensional echocardiogram showed normal left ventricular size and wall thickness with no segmental wall motion abnormalities and normal systolic and diastolic function with a left ventricular ejection fraction of 57% .", "a 12-lead electrocardiogram showed sinus rhythm with average rate of 80 beats / min with a normal axis , pr - interval , and qrs duration with no evidence of chamber enlargement or infarction .", "the clinical working diagnosis was pregnancy - related vt of right ventricular outflow tract origin .", "a 24 year woman ( gravida 4 , para 2 ) presented to her local emergency department at 24 weeks gestation because of shortness of breath , cough , and chest tightness developing over the previous day .", "she had a history of mild asthma but had not had asthma - related symptoms for several years and had never had an asthma exacerbation requiring inpatient or emergency department care . past medical history and family history", "there had been no similar symptoms or medical complications in her previous three pregnancies , one of which had ended with spontaneous abortion and the other two with term delivery of healthy children .", "she smoked one - half to one pack of cigarettes per day but did not drink alcohol or use recreational drugs .", "physical examination revealed an afebrile patient with a regular pulse of 78 beats per minute , blood pressure 100/60 mmhg , and an oxygen saturation of 96% on room air . on respiratory exam", "the rest of the examination was normal except for mild non - pitting peripheral edema .", "laboratory investigations revealed normal complete blood count , electrolytes , renal function , cardiac markers , and tsh .", "the patient was diagnosed with an asthma exacerbation and treated with nebulized salbutamol 2.5 mg , which relieved her symptoms . shortly thereafter , she developed runs of asymptomatic nonsustained vt with a left bundle branch block pattern ( fig .", "nebulized salbutamol 5 mg was once again administered , whereupon the patient developed sustained runs of vt of similar morphology .", "intravenous lidocaine and magnesium sulfate were administered , leading to resolution of the ventricular arrhythmia .", "no further salbutamol was administered , and two further days of electrocardiographic monitoring showed only occasional pvcs .", "twelve lead electrocardiogram showed sinus tachycardia at 102 beats per minute with occasional premature ventricular beats , normal corrected qt interval and no evidence of ischemia or chamber enlargement .", "echocardiography showed normal left ventricular size with normal systolic function with an ejection fraction of 57% , and no valvular abnormalities .", "given the absence of structural heart disease and risk factors for coronary disease or arrhythmia , the clinical diagnosis was catecholamine - sensitive pregnancy - related vt of right ventricular outflow tract origin .", "ventricular tachycardias ( vt ) may be seen in pregnancy and can manifest as a new onset arrhythmia or be exacerbated by pregnancy and can cause concern for the well - being of both the mother and the fetus .", "a study of 11 pregnant women who experienced a new onset of vt during pregnancy showed that onset was distributed equally over the three trimesters and disappeared completely during the postpartum period.1 the characteristics and underlying mechanisms of new - onset vt during pregnancy have not been adequately investigated .", "although the majority of vts that occur during the pregnancy are benign , in women with structural heart disease , arrhythmias ( especially those of ventricular origin ) is one of the five independent predictors of having an adverse outcome during pregnancy.2,3 peripartum cardiomyopathy ( ppcm ) needs to be considered in women presenting with vt in the last month of pregnancy.4,5 the criteria for the diagnosis of ppcm include : development of congestive heart failure secondary to left ventricular systolic dysfunction in the last month of pregnancy or within 5 months after delivery , absence of preexisting cardiac dysfunction , and absence of identifiable cause of heart failure and lv systolic dysfunction demonstrated by classic echocardiographic criteria.6 hence in women presenting with vt in the last month of pregnancy , an urgent echocardiographic study can assist in establishing a diagnosis of ppcm .", "women with certain types of congenital heart disease ( chd ) either repaired or not , are particularly vulnerable to vt . for example", ", patients with previous surgeries for correction of tetralogy of fallot may experience vt arising from the right ventricular outflow tract as a result of right ventricular volume overload due to decades of severe pulmonary regurgitation as a consequence of patch augmentation of the right ventricular outflow tract at the time of original corrective surgery.7 a study of 28 female patients with repaired chd demonstrated women with chd had a significantly higher incidence of tachyarrhythmia during pregnancy and in the postpartum period compared to controls.8 congenital long qt syndrome ( lqts ) is a hereditary disorder of cardiac ion channels causing abnormal electrical activation of the heart.9 women with congenital lqts may experience life - threatening polymorphic vt ; however recent reports indicates that their incidence appears to be significantly increased during the postpartum period.10,11 patients with congenital lqts are particularly vulnerable to adverse arrhythmic effects of electrolyte depletion ( further prolongation of qt interval ) a possible complication in some pregnant women with hyperemesis .", "cautious use of mediations that may further prolong the qt interval is necessary in these individuals to avoid precipitation of polymorphic vt .", "in addition to cardiac diseases , several systemic non - cardiac disorders , including severe electrolyte derangements , thyroid function abnormalities , pulmonary embolism , anemia , and drug overdose may present with ventricular arrhythmias .", "there are few reports of new onset vt during pregnancy in absence of structural heart disease.12,13 increased sympathetic activity , as well as physiological changes associated with normal pregnancy such as increased heart rate , decreased peripheral resistance , increased stroke volume and psychological stresses are thought to be the most common precipitants of vt in pregnant women with a structurally normal hearts.14,15 the majority of outflow tract vt in structurally normal hearts originate from the right ventricular outflow tract ( rvot ) just below the pulmonary valve .", "this is also the most common focus of initiation of pregnancy - related idiopathic vt and usually does not deteriorate to an unstable rhythm .", "it will often manifest as ectopic beats , bigeminal rhythms or short runs of non - sustained vt .", "norepinephrine concentration in the myocardial synaptic cleft has been suggested as a potential mechanism for this arrhythmia.16 only 10% of vt originates from the left ventricular outflow tract ( lvot).17 to make a diagnosis , a 12-lead ecg should be recorded ideally during the arrhythmia , although this can be difficult .", "a typical resting ecg of a pregnant woman may show an increased heart rate with decreased pr , qrs and qt intervals , but there is usually no significant change in the amplitudes of p and t - waves and qrs - complex.15 premature atrial and ventricular beats as well as q wave and inverted t waves in the inferior leads can also be seen during pregnancy.18 on ecg , rvot vt appears as wide qrs complex tachycardia with left bundle brunch block ( lbbb ) morphology and an inferior axis.17,19 two phenotypic forms of rvot vt can occur , non - sustained monomorphic vt and paroxysmal sustained vt.20 in contrast , lvot vt morphology depends on the site of origin with either lbbb and early precordial transition in leads v12 , or rbbb pattern in v1 with broad monophasic r in the precordial leads .", "in addition , the ecg in patients with vt originating in the lvot will show r : s amplitude ratio of 30% or more or an r : qrs duration ratio of 50% in leads v12.21 a routine 24 to 48 hour - holter monitor is helpful in capturing frequently occurring paroxysmal arrhythmias . to elicit symptoms ,", "an exercise ecg can be reasonably carried out during pregnancy in absence of obstetrical contraindications for exercise .", "no fetal adverse events are reported for adenosine , except for one case of transient fetal bradycardia.22 the first step in acute management of vt in pregnancy to determine the hemodynamic stability of the pregnant woman .", "if the woman is unstable or there is evidence of significant fetal compromise which is thought to be related to the vt , dc cardioversion with 50100 j should be given immediately .", "dc shocks can be repeated at higher levels of energy ( 100360 j ) if indicated . the risk for the fetus is minimal for all stages of pregnancy , because the amount of current reaching the fetus is small.23 in stable vt , an accurate diagnosis of the type of arrhythmia should be made with a twelve - lead ecg prior to any intervention .", "invasive electrophysiological studies are rarely required during pregnancy , as the arrhythmias can be effectively managed pharmacologically .", "conservative medical treatment of vt arising during pregnancy is indicated in any patient with sustained vt as long as the patient is hemodynamically stable.14 if possible drug therapy should be avoided during the first trimester and drugs with the longest records of safety should be used as the first - line therapies.24 the literature safety data has examined digoxin , adenosine , flecainide , procainamide , propranolol , propafenone , quinidine , sotalol and verapamil , however ; the experience is limited to single or small case series .", "in stable patients with sustained vt acute therapy can be initiated with either intravenous procainamide , which is safe , well - tolerated and is not associated with teratogenicity .", "alternatively lidocaine can also be used to treat stable vt.14 idiopathic vt originating from the rvot and with a lbbb morphology responds well to beta - blockers , while idiopathic lvot vt with rbbb morphology generally responds well to verapamil .", "given the sensitivity of majority of vts in pregnancy to catecholamines , a cardioselective beta - blocker , in particular metoprolol , in the absence of contraindications is considered first line therapy.12", "in our cases , further investigations did not reveal a secondary cause of the patients arrhythmias .", "based on the ecgs ( qrs morphology of a left bundle branch block pattern with an inferior axis , and clinical findings ) both patients were diagnosed with idiopathic vt originating from the rvot .", "the lower dose for the second patient was chosen because of suspected asthma exacerbation ; however the dose was slowly increased to 50 mg po every 12 hours .", "both patients tolerated treatment well and had no further documented episodes of vt on long - term cardiac monitoring .", "patient 1 was scheduled for elective c - section at a high risk obstetric center and was discharged home . on the follow - up", "patient 2 had a spontaneous vaginal delivery at 36 weeks and continued to be on metoprolol after discharge .", "pregnant patients may present with ventricular tachycardia during any trimester careful and timely clinical , electrocardiographic , as well as echocardiographic assessment help identify those individuals with structural cardiac abnormalities such as congenital heart disease or peripartum cardiomyopathy , as well as those with long qt syndrome who will need specific management beyond therapy for the arrhythmia . in those stable patients with structurally normal hearts ,", "identification of the location of origin of tachycardia will help in choice of appropriate medical therapy ."], "abstract_text": ["<S> ventricular tachycardia although not common , can occasionally complicate pregnancy . </S>", "<S> its presence may indicate an underlying cardiac structural abnormality , or undiagnosed congenital arrhythmic disease . </S>", "<S> however , some pregnant patients with ventricular tachycardia have structurally normal hearts . </S>", "<S> two cases of ventricular tachycardia in pregnant patients with structurally normal hearts are presented and an approach to diagnosis and management of such patients are discussed . </S>"], "labels": null, "section_names": ["Case 1", "Case 2", "Discussion", "Return to the Cases", "Conclusion"], "sections": [["a 33-year - old woman , ( gravida 3 , para 2 ) , at 34 weeks of gestation , presented with episodes of lightheadedness , and dyspnea .", "she was asymptomatic until 18 weeks of gestation , when she started having episodes of palpitations associated with presyncope .", "initially , the episodes were infrequent ( less than once per day ) but a gradual increase in frequency and severity was noted by the patient .", "one month prior to presentation , she noted episodes of paroxysmal nocturnal dyspnea , but no dyspnea on exertion , chest pain , diaphoresis , pedal edema , orthopnea , or neurological symptoms .", "she had no significant past medical history and had not had complications relating to this pregnancy .", "all prior pregnancies had progressed to full term without similar symptoms or complications , and resulted in spontaneous vaginal deliveries of healthy newborns .", "physical examination revealed an afebrile patient with a regular pulse of 80 beats per minute , blood pressure 110/70 mmhg , and an oxygen saturation of 100% on room air .", "the examination was normal except for mild non - pitting edema and fine bibasilar crackles .", "laboratory investigations revealed normal complete blood count , electrolytes , renal function , cardiac markers and tsh .", "cardiac monitoring showed multiple episodes of monomorphic , non - sustained ventricular tachycardia ( vt ) with left bundle branch morphology , consistent with vt originating from the right ventricular outflow tract ( fig .", "urgent two - dimensional echocardiogram showed normal left ventricular size and wall thickness with no segmental wall motion abnormalities and normal systolic and diastolic function with a left ventricular ejection fraction of 57% .", "a 12-lead electrocardiogram showed sinus rhythm with average rate of 80 beats / min with a normal axis , pr - interval , and qrs duration with no evidence of chamber enlargement or infarction .", "the clinical working diagnosis was pregnancy - related vt of right ventricular outflow tract origin ."], ["a 24 year woman ( gravida 4 , para 2 ) presented to her local emergency department at 24 weeks gestation because of shortness of breath , cough , and chest tightness developing over the previous day .", "she had a history of mild asthma but had not had asthma - related symptoms for several years and had never had an asthma exacerbation requiring inpatient or emergency department care . past medical history and family history", "there had been no similar symptoms or medical complications in her previous three pregnancies , one of which had ended with spontaneous abortion and the other two with term delivery of healthy children .", "she smoked one - half to one pack of cigarettes per day but did not drink alcohol or use recreational drugs .", "physical examination revealed an afebrile patient with a regular pulse of 78 beats per minute , blood pressure 100/60 mmhg , and an oxygen saturation of 96% on room air . on respiratory exam", "the rest of the examination was normal except for mild non - pitting peripheral edema .", "laboratory investigations revealed normal complete blood count , electrolytes , renal function , cardiac markers , and tsh .", "the patient was diagnosed with an asthma exacerbation and treated with nebulized salbutamol 2.5 mg , which relieved her symptoms . shortly thereafter , she developed runs of asymptomatic nonsustained vt with a left bundle branch block pattern ( fig .", "nebulized salbutamol 5 mg was once again administered , whereupon the patient developed sustained runs of vt of similar morphology .", "intravenous lidocaine and magnesium sulfate were administered , leading to resolution of the ventricular arrhythmia .", "no further salbutamol was administered , and two further days of electrocardiographic monitoring showed only occasional pvcs .", "twelve lead electrocardiogram showed sinus tachycardia at 102 beats per minute with occasional premature ventricular beats , normal corrected qt interval and no evidence of ischemia or chamber enlargement .", "echocardiography showed normal left ventricular size with normal systolic function with an ejection fraction of 57% , and no valvular abnormalities .", "given the absence of structural heart disease and risk factors for coronary disease or arrhythmia , the clinical diagnosis was catecholamine - sensitive pregnancy - related vt of right ventricular outflow tract origin ."], ["ventricular tachycardias ( vt ) may be seen in pregnancy and can manifest as a new onset arrhythmia or be exacerbated by pregnancy and can cause concern for the well - being of both the mother and the fetus .", "a study of 11 pregnant women who experienced a new onset of vt during pregnancy showed that onset was distributed equally over the three trimesters and disappeared completely during the postpartum period.1 the characteristics and underlying mechanisms of new - onset vt during pregnancy have not been adequately investigated .", "although the majority of vts that occur during the pregnancy are benign , in women with structural heart disease , arrhythmias ( especially those of ventricular origin ) is one of the five independent predictors of having an adverse outcome during pregnancy.2,3 peripartum cardiomyopathy ( ppcm ) needs to be considered in women presenting with vt in the last month of pregnancy.4,5 the criteria for the diagnosis of ppcm include : development of congestive heart failure secondary to left ventricular systolic dysfunction in the last month of pregnancy or within 5 months after delivery , absence of preexisting cardiac dysfunction , and absence of identifiable cause of heart failure and lv systolic dysfunction demonstrated by classic echocardiographic criteria.6 hence in women presenting with vt in the last month of pregnancy , an urgent echocardiographic study can assist in establishing a diagnosis of ppcm .", "women with certain types of congenital heart disease ( chd ) either repaired or not , are particularly vulnerable to vt . for example", ", patients with previous surgeries for correction of tetralogy of fallot may experience vt arising from the right ventricular outflow tract as a result of right ventricular volume overload due to decades of severe pulmonary regurgitation as a consequence of patch augmentation of the right ventricular outflow tract at the time of original corrective surgery.7 a study of 28 female patients with repaired chd demonstrated women with chd had a significantly higher incidence of tachyarrhythmia during pregnancy and in the postpartum period compared to controls.8 congenital long qt syndrome ( lqts ) is a hereditary disorder of cardiac ion channels causing abnormal electrical activation of the heart.9 women with congenital lqts may experience life - threatening polymorphic vt ; however recent reports indicates that their incidence appears to be significantly increased during the postpartum period.10,11 patients with congenital lqts are particularly vulnerable to adverse arrhythmic effects of electrolyte depletion ( further prolongation of qt interval ) a possible complication in some pregnant women with hyperemesis .", "cautious use of mediations that may further prolong the qt interval is necessary in these individuals to avoid precipitation of polymorphic vt .", "in addition to cardiac diseases , several systemic non - cardiac disorders , including severe electrolyte derangements , thyroid function abnormalities , pulmonary embolism , anemia , and drug overdose may present with ventricular arrhythmias .", "there are few reports of new onset vt during pregnancy in absence of structural heart disease.12,13 increased sympathetic activity , as well as physiological changes associated with normal pregnancy such as increased heart rate , decreased peripheral resistance , increased stroke volume and psychological stresses are thought to be the most common precipitants of vt in pregnant women with a structurally normal hearts.14,15 the majority of outflow tract vt in structurally normal hearts originate from the right ventricular outflow tract ( rvot ) just below the pulmonary valve .", "this is also the most common focus of initiation of pregnancy - related idiopathic vt and usually does not deteriorate to an unstable rhythm .", "it will often manifest as ectopic beats , bigeminal rhythms or short runs of non - sustained vt .", "norepinephrine concentration in the myocardial synaptic cleft has been suggested as a potential mechanism for this arrhythmia.16 only 10% of vt originates from the left ventricular outflow tract ( lvot).17 to make a diagnosis , a 12-lead ecg should be recorded ideally during the arrhythmia , although this can be difficult .", "a typical resting ecg of a pregnant woman may show an increased heart rate with decreased pr , qrs and qt intervals , but there is usually no significant change in the amplitudes of p and t - waves and qrs - complex.15 premature atrial and ventricular beats as well as q wave and inverted t waves in the inferior leads can also be seen during pregnancy.18 on ecg , rvot vt appears as wide qrs complex tachycardia with left bundle brunch block ( lbbb ) morphology and an inferior axis.17,19 two phenotypic forms of rvot vt can occur , non - sustained monomorphic vt and paroxysmal sustained vt.20 in contrast , lvot vt morphology depends on the site of origin with either lbbb and early precordial transition in leads v12 , or rbbb pattern in v1 with broad monophasic r in the precordial leads .", "in addition , the ecg in patients with vt originating in the lvot will show r : s amplitude ratio of 30% or more or an r : qrs duration ratio of 50% in leads v12.21 a routine 24 to 48 hour - holter monitor is helpful in capturing frequently occurring paroxysmal arrhythmias . to elicit symptoms ,", "an exercise ecg can be reasonably carried out during pregnancy in absence of obstetrical contraindications for exercise .", "no fetal adverse events are reported for adenosine , except for one case of transient fetal bradycardia.22 the first step in acute management of vt in pregnancy to determine the hemodynamic stability of the pregnant woman .", "if the woman is unstable or there is evidence of significant fetal compromise which is thought to be related to the vt , dc cardioversion with 50100 j should be given immediately .", "dc shocks can be repeated at higher levels of energy ( 100360 j ) if indicated . the risk for the fetus is minimal for all stages of pregnancy , because the amount of current reaching the fetus is small.23 in stable vt , an accurate diagnosis of the type of arrhythmia should be made with a twelve - lead ecg prior to any intervention .", "invasive electrophysiological studies are rarely required during pregnancy , as the arrhythmias can be effectively managed pharmacologically .", "conservative medical treatment of vt arising during pregnancy is indicated in any patient with sustained vt as long as the patient is hemodynamically stable.14 if possible drug therapy should be avoided during the first trimester and drugs with the longest records of safety should be used as the first - line therapies.24 the literature safety data has examined digoxin , adenosine , flecainide , procainamide , propranolol , propafenone , quinidine , sotalol and verapamil , however ; the experience is limited to single or small case series .", "in stable patients with sustained vt acute therapy can be initiated with either intravenous procainamide , which is safe , well - tolerated and is not associated with teratogenicity .", "alternatively lidocaine can also be used to treat stable vt.14 idiopathic vt originating from the rvot and with a lbbb morphology responds well to beta - blockers , while idiopathic lvot vt with rbbb morphology generally responds well to verapamil .", "given the sensitivity of majority of vts in pregnancy to catecholamines , a cardioselective beta - blocker , in particular metoprolol , in the absence of contraindications is considered first line therapy.12"], ["in our cases , further investigations did not reveal a secondary cause of the patients arrhythmias .", "based on the ecgs ( qrs morphology of a left bundle branch block pattern with an inferior axis , and clinical findings ) both patients were diagnosed with idiopathic vt originating from the rvot .", "the lower dose for the second patient was chosen because of suspected asthma exacerbation ; however the dose was slowly increased to 50 mg po every 12 hours .", "both patients tolerated treatment well and had no further documented episodes of vt on long - term cardiac monitoring .", "patient 1 was scheduled for elective c - section at a high risk obstetric center and was discharged home . on the follow - up", "patient 2 had a spontaneous vaginal delivery at 36 weeks and continued to be on metoprolol after discharge ."], ["pregnant patients may present with ventricular tachycardia during any trimester careful and timely clinical , electrocardiographic , as well as echocardiographic assessment help identify those individuals with structural cardiac abnormalities such as congenital heart disease or peripartum cardiomyopathy , as well as those with long qt syndrome who will need specific management beyond therapy for the arrhythmia . in those stable patients with structurally normal hearts ,", "identification of the location of origin of tachycardia will help in choice of appropriate medical therapy ."]]}
{"article_id": "PMC3468919", "article_text": ["common sources include backyard burning , incineration of plastics , and chlorine bleaching of pulp in paper mills .", "documented health effects include acute and chronic effects , including chloracne , various types of cancers , reproductive diseases , circulatory and respiratory diseases , and diabetes .", "the traditional approach to environmental remediation includes a host of physical and chemical methods , depending on the characteristics of the polluted site and the extent of contamination present .", "bioremediation , which is the use of biogenic materials and organisms for environmental cleanup , has also been proposed , including phytoremediation using plants   and microbial degradation using primarily bacteria and fungi .", "bioremediation is an attractive strategy , as it can destroy the pollutant rather than transferring it from one environmental compartment to another .", "common bioremediation strategies include the addition of nutrients , degradative microorganisms , or both . \n", "sphingomonas wittichii rw1 is a microorganism of great interest to the bioremediation community for its ability to biotransform a large number of toxic polychlorinated dioxins and to utilize both nonchlorinated dibenzo - p - dioxin and nonchlorinated dibenzofuran as a growth substrate and sole source of carbon and energy .", "one of the major challenges in bioaugmentation strategies relying on the addition of nonnative microbes is to ensure their viability and degradative activity toward the target compounds .", "monitoring bioremediation is critical to ensure the efficacy of the process and the reduction of contaminant mass to acceptable levels .", "traditionally , the most important characteristics investigated for microorganisms used in bioremediation were their ability to transform the substrate , the rate of substrate removal , and the resulting metabolites [ 69 ] . to optimize the bacterial degradation of pollutants , it is important to understand how these organisms function during growth on recalcitrant substrates and which factors influence their degradative abilities .", "this includes analyzing not only the degradative pathways [ 1012 ] , but also the peripheral processes and mechanisms that are involved in taxis ( i.e. , directed motion in a chemical gradient ) , uptake , and transport during exposures to specific substrates .", "analysis of dna and rna   can shed light on an organism 's metabolic potential ; however , these measurements poorly correlate to actual protein expression profiles .", "therefore , global analyses of protein expression profiles may be a more informative tool for understanding the physiological mechanisms of biodegradation .", "in addition to identifying important degradative enzymes in a variety of important microbes [ 1517 ] , proteomic studies have opened the door to a better understanding of system - wide changes in response to differing substrates .", "the imperative to perform proteomic analyses is particularly true for s. wittichii rw1 because the enzymes in the dioxin degradation pathway are encoded on different loci throughout the genome , certain elements in the pathway are located on a plasmid , and there may be alternative pathways at work .", "the present study builds on previous work   and utilized difference gel eletrophoresis ( dige ) coupled with mass spectrometry ( ms ) to exploit recently gathered rw1 genome data . when used together , these tools yield information on the response of cells of s. wittichii rw1 to dioxin exposure and the bacterium 's degradative activity toward this recalcitrant compound .", "the aim of this study was to investigate system - wide changes in protein expression during growth on dibenzofuran , a nontoxic surrogate for dibenzo - p - dioxin , as compared to nonselective growth media .", "acetate was selected as the nonselective alternate substrate , as growth on this compound was observed to influence expression of select proteins , including the dioxin dioxygenase .", "thus , any changes measured in response to cells grown on dibenzofuran should represent cell - wide effects related specifically to the growth substrate and not to unanticipated extraneous effects .", "cultures of s. wittichii strain rw1 ( 100  ml to 1.0  l ) were grown to mid log phase at 30c in m9 phosphate - buffered minimal medium ( ph 7.05 ) supplemented with either dibenzofuran crystals or 50  mm acetate as growth substrates .", "cells were grown overnight on dibenzofuran and acetate as sole carbon sources to an optical density of 0.40.6 absorbance units ( 560  nm ) . following biomass processing ,", "protein levels in the samples were on the order of 75200  g / ml .", "protein concentrations were normalized prior to analysis by concentration and resuspension in dige sample preparation buffer .", "culture purity was confirmed by the streak plate method using luria bertani medium supplemented with 1.5% agar .", "harvested biomass was washed , spun again , and the resultant pellet suspended in a small volume of 100  mm ammonium bicarbonate ( ph ~7.0 ) . this microbial suspension", "was then sonicated under cooling with ice , using a microtip sonicator ( fisher scientific , pittsburgh , pa , usa ) in a sequence of three 10-second bursts delivered in thirty - second intervals .", "the sonicated cells were then immediately centrifuged at 10,000  xg for 10 minutes at 4c .", "briefly , 8 parts of 10% tca in acetone ( 20c ) were added per volume of supernatant and , following mixing on a vortex , the resultant dilution was incubated at 20c overnight .", "following centrifugation ( 10,000  xg , 10 minutes , 4c ) , harvested biomass was washed in cold acetone for 10 minutes at 20c .", "following a subsequent centrifugation , the pellet was resuspended in sample preparation buffer ( 7  m urea , 2  m thiourea , 2% chaps , 0.2% dtt , 0.02% bromophenol blue ) and stored at 20c until analyzed .", "protein concentrations were measured using the bicinchoninic acid assay ( pierce , rockford , il , usa ) following dilution to reduce the concentration of interfering agents .", "twenty - five g of crude cell lysates of rw1 biological replicates grown on dibenzofuran ( n = 3 ) and acetate ( n = 3 ) were labeled using cy dyes ( ge healthcare ) as described elsewhere .", "briefly , samples were adjusted to 1  g/l using sample preparation buffer and the ph checked .", "subsequently , 0.25  l of 1  pmol/l  cy dyes were added to samples for 30 minutes in the dark on ice . to stop the labeling reaction ,", "0.5  l of 10  mm lysine was added to the samples , which were mixed and incubated on ice for 10 minutes prior to storage at 20c until analysis .  ", "unless stated otherwise , all procedures were carried out in the dark or minimal light to protect the integrity of the fluorescent dyes .", "samples were randomized to reduce the effect of dye bias and in - gel variations .", "a global pool consisting of fractions of each sample was labeled as outlined above using cy 2 and added to each sample as an internal standard .", "one cy-3- and one cy-5-labeled sample were added to each gel , as defined by the experimental randomizing procedures . to each sample", ", an additional 175  g of unlabeled sample were added ; the volume was increased to a total of 450  l using sample preparation buffer .", "the reducing agent dtt ( dithiothreitol ; 1.3  mg per tube ) and ipg ( immobilized ph gradient ) buffer ( 0.5% ) were added , and the samples incubated and mixed in the dark at room temperature for approximately 1  h. samples were then applied to 24  cm ph 47 ipg strips ( ge healthcare ) and focused for 60  kvh using the following protocol : 12  h rehydration at 30  v ; 1  h step and hold at 500  v ; 7 hour gradient to 1,000  v ; step and hold at 1,000  v for 1 hour ; gradient to 8,000  v for 3  h ; step and hold at 8,000  v until 60  kvh .", "strips were then reduced and equilibrated using 10  mg / ml dtt ( 15  min ) followed by 25  mg / ml iodoacetamide ( 15  min ) .", "the ipg strips were overlaid on 24  26  cm 816% gradient tris - hcl ph 8.8 precast gels ( nextgen sciences , ann arbor ,", "approximately 1  ml of agarose was applied to fix the gels and a cy-2-labeled molecular weight marker was applied adjacent to the acidic side of the strip .", "the gels were then run 1 - 2  w per gel overnight ( ~2224 hours ) at 20c until the marker dye ran off the gel .", "gels were then imaged with a typhoon 9400 scanner and processed using decyder v6.5 ( ge healthcare ) bva batch processor tool .", "images were uploaded to decyder ( version 6.5 ) and spurious image objects ( water spots , streaks , and mismatches ) were identified and excluded from further analysis .", "following allocation of changed proteins , individual spots were manually inspected and excluded from analysis if they fell outside acceptable parameters for peak height , area , and slope .  ", "pick lists were generated by selecting proteins whose expression was statistically changed in the two growth conditions ( p < 0.05 ) following digital image analysis using decyder , and the corresponding spots were automatically picked using an ettan spot picker ( ge healthcare ) with ettan spot pick software v.1.1", ". spots were delivered in 100  mm ammonium bicarbonate to a 96-well plate and digested using established protocols .", "briefly , gel pieces were sequentially dried using three exchanges of 100% acetonitrile followed by a 10-minute speedvac ( savant ) drying .", "gel pieces were rehydrated in 40  l of 10  ng/l trypsin in 100  mm nh4hco3 on ice for 45 minutes .", "the supernatant was removed and replaced with 100  mm nh4hco3 and digested at 37c overnight .", "peptides were then extracted using 50% acetonitrile/0.1% tfa ( trifluoroacetic acid ) for 30 minutes at 37c .", "the peptides were microextracted using omix c18 tips ( varian , palo alto , ca , usa ) following the manufacturer 's instructions and then deposited on a stainless - steel target plate in a matrix consisting of 10  mg / ml 2,5-dihydroxybenzoic acid", ".   mass spectra were acquired using a voyager de - str matrix - assisted laser desorption / ionization time - of - flight ms ( applied biosystems , foster city , ca , usa ) in positive reflector mode with delayed extraction using the following parameters : laser energy , 1400 arbitrary units ; mass range , 5005,000  da ; 120  nsec delay , 100 laser shots per spectrum .", "external calibration was conducted using a standard peptide mixture ( bradykinin , insulin b chain , p14r , and acth ) , and internal calibration was carried out using trypsin autolysis peaks .", "data were processed in data explorer v1.1 ( applied biosystems , foster city , ca , usa ) using noise reduction ( 2 standard deviations ) and peak deisotoping .", "peak masses were searched using the mascot online search engine ( http://www.matrixscience.com/ ) with the following settings : database , ncbi entire database ( 5.6 million entries ) ; no missed cleavages ; monoisotopic peaks ; no fixed modifications ; variable modification of methionine oxidation ; error tolerance of 150  ppm .", "database and literature searches were used to further characterize and classify the proteins identified by maldi - tof ms . where ambiguous names were encountered , blastp searches   were used to identify homologous proteins from orthologous species .", "image analysis of 24  cm 2d - dige gels loaded with protein of s. wittichii rw1 cells grown on either dibenzofuran or acetate revealed 937 unique spots .", "differential in - gel analysis of individual gels determined gel - specific parameters for selection criteria and allowed visual examination of changes between growth on the two substrates ( figure 1 ) . of the 937 identified spots ,", "595 were matched between all the gels used to statistically compare the quantitative abundance of proteins .", "statistical analysis compared triplicate biological observations for each condition , normalized to the internal pooled standard ( figure 2 ) .  ", "crude cell lysates from s. wittichii rw1 grown on dibenzofuran showed that , of all proteins observed , 22 proteins were modulated in response to changes in culture conditions .", "these candidate biomarkers of metabolic activity and phenotype were observed in at least 6 of 9 dige images and were modulated as follows : 16 showed an apparent increase and 6 an apparent decrease ( figure 2 ) .", "these proteins , along with 22 proteins selected due to their high abundance in both growth conditions , were further analyzed and identified using mass spectrometry ( figure 3 ) .", "a mascot search of the entire ncbi database using mass spectral data generated by peptide mass fingerprinting identified 23 of the 44 proteins ( 52% ) .", "all protein identifications corresponded to the genome of s. wittichii strain rw1 . among the 16 proteins upregulated during growth on dibenzofuran ,", "7 were successfully identified ( table 1 ) . among the 6 proteins downregulated during growth on dibenzofuran ,", "an additional 13 proteins were identified whose expression level remained unchanged regardless of culture conditions ( table 3 ) .  ", "of the 7 identified proteins increased during growth on dibenzofuran , 2 were directly related to the dibenzofuran degradation pathway ( figure 4 ) ; the others were involved in downstream metabolic processes ( catechol 1,2-dioxygenase , adenosylhomocysteinase ) , cell growth ( elongation factor ts ) , and cell protection ( cold shock dna - binding domain protein , alkyl hydroperoxide reductase ) .", "the three identified proteins whose expression was decreased ( fumarylacetoacetate hydrolase , tonb - dependent receptor , and acyl - coa dehydrogenase ) are involved in biosynthesis , catabolism , and transport .", "the unchanged proteins represented basic cell functions , although biosynthesis , catabolism , and transport proteins dominated the identities .", "the alpha subunit of the dioxin dioxygenase , the first step in the dioxin degradation pathway , was also identified as unchanged .", "dige and 2d electrophoresis are an accepted strategy for mining microbial proteomes for biomarkers related to a number of processes [ 2830 ] .", "the complete protein content of s. wittichii rw1 consists of approximately 5,000 putative proteins from the bacterial chromosome and two megaplasmids . using simple extraction and purification techniques followed by dige", ", over 500 protein spots were resolved on a large ( 24  cm ) 2-dimensional gel and matched between the three biological replicates , representing approximately 10% of the entire protein content .", "of these 500 proteins , 22 were found to be regulated in response to growth condition changes .  ", "of the genes they identified as being changed in their experiments , only two overlapped with the proteins identified in our study : swit_3144 ( tonb - dependent receptor , downregulated ) and swit_3376 ( chaperonin groel , upregulated ) .", "both of these gene levels responded to short - term perturbation with peg8000 but not sodium chloride in the transcriptomic study and were unchanged in our study . in our study", ", we also identified a number of proteins that are related to dioxin / dibenzofuran degradation ( e.g. , dioxin dioxygenase , meta - cleavage product hydrolase , and 2,3-dihydroxybiphenyl 1,2-dioxygenase ) .", "other proteins were identified that showed increases in abundance but whose role was not directly related to the dibenzofuran degradation pathway . the increase in the presence of antioxidants such as alkyl", "hydroperoxide reductase suggests that there is an increasing stress upon the bacterial cell during growth on dibenzofuran , perhaps due to a change in catabolism resulting in an increase in endogenous peroxide generation .", "increases in a cold - shock dna - binding protein may be further evidence of an increased cellular stress .", "however , proteins of the cold shock family and related ones are also known to have transport and protein processing roles .   among the proteins in the dioxin degradation pathway , the most prominent on the gel was the meta - cleavage product hydrolase .", "this identification was produced from two adjacent spots , likely representing an artifact due to the protein 's extremely high expression or a reflection of the presence of multiple isoforms or a modified enzyme .", "the one identified in the present study is the product of the swit_3055 locus , a gene also known as dxnb2 .", "the glyoxalase / bleomycin resistance protein / dioxygenase identified in this study is also annotated as a 2,3-dihydroxybiphenyl-1,2-dioxygenase located on the chromosome at the swit_3046 locus .", "again , there are multiple isoforms of this enzyme found both on the chromosome and the megaplasmids .", "the kegg dioxin degradation pathway identifies swit_4182 as the dihydroxybiphenyl dioxygenase involved in biphenyl metabolism and swit_4902 as the trihydroxybiphenyl dioxygenase in both dioxin and dibenzofuran metabolism .", "the increased expression in response to dibenzofuran suggests that the swit_3046 dioxygenase plays a more important role in dibenzofuran degradation in vivo .", "the high degree of redundancy in the dioxin and dibenzofuran degradation pathways , that is , the presence of multiple ring - hydroxylating alpha and beta subunits , glyoxalase / bleomycin resistance protein / dioxygenases , and meta - cleavage product hydrolases , remains to be explained .", "one possibility is that the various isoforms have different affinities for chlorinated metabolites that would result from chlorinated dioxins and furans .", "further experiments are needed to fully distinguish the roles of these enzymes in s. wittichii rw1 degradation pathways .", "although not directly implicated in dioxin degradation , the fumarylacetoacetate hydrolase is also of interest because the gene encoding this protein ( swit_5089 ) flanks the ferredoxin fdx1 ( swit_5088 ) that has been identified as part of the electron supply chain supporting dioxin dioxygenase activity . of the multiple isoforms of this enzyme ,", "the electron supply chain also contains two isofunctional reductases . neither the ferredoxin itself nor the reductases could be identified . in previous studies ,", "the reductase was present as a much smaller fraction of the soluble cell proteome than either the ferredoxin or the dioxin dioxygenase , so gel - based methods may not be sensitive enough to detect this protein .", "if transcription of the ferredoxin is linked to the other genes at that locus , as is predicted , the decreased expression in response to dibenzofuran suggests that another ferredoxin is more important in the dioxin degradation pathway in vivo .", "the detection of the dioxin dioxygenase alpha subunit and related enzymes in both acetate- and dibenzofuran - grown cells is potentially of importance for the field of bioremediation because it suggests an avenue of biostimulation .", "when utilizing s. wittichii rw1 as a bioremediation agent , it may be possible to induce the expression of the dioxin degradation pathway using acetate .", "induction of the dioxin degradation pathway has not been observed when s. wittichii rw1 is grown on glucose or rich medium , and growth in a complex environmental medium ( landfill leachate ) was correlated with a decrease in copy number of the gene encoding the dioxin dioxygenase alpha subunit .", "previous studies using s. wittichii rw1 to transform chlorinated dioxins in soil or fly ash have observed a progressive decrease in degradative activity   or viable cells [ 38 , 39 ] , respectively .", "the addition of acetate may generate sufficient relevant protein biomass to catalyze the successful degradation of dioxin and dioxin - like compounds in environments bioaugmented with s. wittichii rw1 .", "proteomic technology has emerged in microbiology more rapidly than in other fields for several reasons .", "the relatively small genomes code for relatively limited proteomes featuring no or very limited posttranslational modifications compared to higher organisms .", "furthermore , microbes are easily controlled and manipulated in the laboratory , both during growth and gene expression .", "these factors will continue to drive biomarker discovery in microbial proteomes , including phenotypic biomarkers informing on the degradative activity of biomass produced for bioaugmentation of contaminated environments .", "furthermore , the field of bioremediation can benefit from methods suitable for monitoring microbial biomarkers in field samples to inform on progress in site bioremediation .", "this study highlights a number of proteins that were changed in response to dibenzofuran exposure , opens the door to a greater understanding of how s. wittichii rw1 performs and regulates the degradation of dioxins , and suggests ways to enhance the biodegradation of dioxins ."], "abstract_text": ["<S> \n sphingomonas wittichii rw1 is a bacterium of interest due to its ability to degrade polychlorinated dioxins , which represent priority pollutants in the usa and worldwide . although its genome has been fully sequenced , many questions exist regarding changes in protein expression of s. wittichii rw1 in response to dioxin metabolism . </S>", "<S> we used difference gel electrophoresis ( dige ) and matrix - assisted laser desorption / ionization mass spectrometry ( maldi - ms ) to identify proteomic changes induced by growth on dibenzofuran , a surrogate for dioxin , as compared to acetate . </S>", "<S> approximately 10% of the entire putative proteome of rw1 could be observed . </S>", "<S> several components of the dioxin and dibenzofuran degradation pathway were shown to be upregulated , thereby highlighting the utility of using proteomic analyses for studying bioremediation agents . </S>", "<S> this is the first global protein analysis of a microorganism capable of utilizing the carbon backbone of both polychlorinated dioxins and dibenzofurans as the sole source for carbon and energy . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["common sources include backyard burning , incineration of plastics , and chlorine bleaching of pulp in paper mills .", "documented health effects include acute and chronic effects , including chloracne , various types of cancers , reproductive diseases , circulatory and respiratory diseases , and diabetes .", "the traditional approach to environmental remediation includes a host of physical and chemical methods , depending on the characteristics of the polluted site and the extent of contamination present .", "bioremediation , which is the use of biogenic materials and organisms for environmental cleanup , has also been proposed , including phytoremediation using plants   and microbial degradation using primarily bacteria and fungi .", "bioremediation is an attractive strategy , as it can destroy the pollutant rather than transferring it from one environmental compartment to another .", "common bioremediation strategies include the addition of nutrients , degradative microorganisms , or both . \n", "sphingomonas wittichii rw1 is a microorganism of great interest to the bioremediation community for its ability to biotransform a large number of toxic polychlorinated dioxins and to utilize both nonchlorinated dibenzo - p - dioxin and nonchlorinated dibenzofuran as a growth substrate and sole source of carbon and energy .", "one of the major challenges in bioaugmentation strategies relying on the addition of nonnative microbes is to ensure their viability and degradative activity toward the target compounds .", "monitoring bioremediation is critical to ensure the efficacy of the process and the reduction of contaminant mass to acceptable levels .", "traditionally , the most important characteristics investigated for microorganisms used in bioremediation were their ability to transform the substrate , the rate of substrate removal , and the resulting metabolites [ 69 ] . to optimize the bacterial degradation of pollutants , it is important to understand how these organisms function during growth on recalcitrant substrates and which factors influence their degradative abilities .", "this includes analyzing not only the degradative pathways [ 1012 ] , but also the peripheral processes and mechanisms that are involved in taxis ( i.e. , directed motion in a chemical gradient ) , uptake , and transport during exposures to specific substrates .", "analysis of dna and rna   can shed light on an organism 's metabolic potential ; however , these measurements poorly correlate to actual protein expression profiles .", "therefore , global analyses of protein expression profiles may be a more informative tool for understanding the physiological mechanisms of biodegradation .", "in addition to identifying important degradative enzymes in a variety of important microbes [ 1517 ] , proteomic studies have opened the door to a better understanding of system - wide changes in response to differing substrates .", "the imperative to perform proteomic analyses is particularly true for s. wittichii rw1 because the enzymes in the dioxin degradation pathway are encoded on different loci throughout the genome , certain elements in the pathway are located on a plasmid , and there may be alternative pathways at work .", "the present study builds on previous work   and utilized difference gel eletrophoresis ( dige ) coupled with mass spectrometry ( ms ) to exploit recently gathered rw1 genome data . when used together , these tools yield information on the response of cells of s. wittichii rw1 to dioxin exposure and the bacterium 's degradative activity toward this recalcitrant compound .", "the aim of this study was to investigate system - wide changes in protein expression during growth on dibenzofuran , a nontoxic surrogate for dibenzo - p - dioxin , as compared to nonselective growth media .", "acetate was selected as the nonselective alternate substrate , as growth on this compound was observed to influence expression of select proteins , including the dioxin dioxygenase .", "thus , any changes measured in response to cells grown on dibenzofuran should represent cell - wide effects related specifically to the growth substrate and not to unanticipated extraneous effects ."], ["cultures of s. wittichii strain rw1 ( 100  ml to 1.0  l ) were grown to mid log phase at 30c in m9 phosphate - buffered minimal medium ( ph 7.05 ) supplemented with either dibenzofuran crystals or 50  mm acetate as growth substrates .", "cells were grown overnight on dibenzofuran and acetate as sole carbon sources to an optical density of 0.40.6 absorbance units ( 560  nm ) . following biomass processing ,", "protein levels in the samples were on the order of 75200  g / ml .", "protein concentrations were normalized prior to analysis by concentration and resuspension in dige sample preparation buffer .", "culture purity was confirmed by the streak plate method using luria bertani medium supplemented with 1.5% agar .", "harvested biomass was washed , spun again , and the resultant pellet suspended in a small volume of 100  mm ammonium bicarbonate ( ph ~7.0 ) . this microbial suspension", "was then sonicated under cooling with ice , using a microtip sonicator ( fisher scientific , pittsburgh , pa , usa ) in a sequence of three 10-second bursts delivered in thirty - second intervals .", "the sonicated cells were then immediately centrifuged at 10,000  xg for 10 minutes at 4c .", "briefly , 8 parts of 10% tca in acetone ( 20c ) were added per volume of supernatant and , following mixing on a vortex , the resultant dilution was incubated at 20c overnight .", "following centrifugation ( 10,000  xg , 10 minutes , 4c ) , harvested biomass was washed in cold acetone for 10 minutes at 20c .", "following a subsequent centrifugation , the pellet was resuspended in sample preparation buffer ( 7  m urea , 2  m thiourea , 2% chaps , 0.2% dtt , 0.02% bromophenol blue ) and stored at 20c until analyzed .", "protein concentrations were measured using the bicinchoninic acid assay ( pierce , rockford , il , usa ) following dilution to reduce the concentration of interfering agents .", "twenty - five g of crude cell lysates of rw1 biological replicates grown on dibenzofuran ( n = 3 ) and acetate ( n = 3 ) were labeled using cy dyes ( ge healthcare ) as described elsewhere .", "briefly , samples were adjusted to 1  g/l using sample preparation buffer and the ph checked .", "subsequently , 0.25  l of 1  pmol/l  cy dyes were added to samples for 30 minutes in the dark on ice . to stop the labeling reaction ,", "0.5  l of 10  mm lysine was added to the samples , which were mixed and incubated on ice for 10 minutes prior to storage at 20c until analysis .  ", "unless stated otherwise , all procedures were carried out in the dark or minimal light to protect the integrity of the fluorescent dyes .", "samples were randomized to reduce the effect of dye bias and in - gel variations .", "a global pool consisting of fractions of each sample was labeled as outlined above using cy 2 and added to each sample as an internal standard .", "one cy-3- and one cy-5-labeled sample were added to each gel , as defined by the experimental randomizing procedures . to each sample", ", an additional 175  g of unlabeled sample were added ; the volume was increased to a total of 450  l using sample preparation buffer .", "the reducing agent dtt ( dithiothreitol ; 1.3  mg per tube ) and ipg ( immobilized ph gradient ) buffer ( 0.5% ) were added , and the samples incubated and mixed in the dark at room temperature for approximately 1  h. samples were then applied to 24  cm ph 47 ipg strips ( ge healthcare ) and focused for 60  kvh using the following protocol : 12  h rehydration at 30  v ; 1  h step and hold at 500  v ; 7 hour gradient to 1,000  v ; step and hold at 1,000  v for 1 hour ; gradient to 8,000  v for 3  h ; step and hold at 8,000  v until 60  kvh .", "strips were then reduced and equilibrated using 10  mg / ml dtt ( 15  min ) followed by 25  mg / ml iodoacetamide ( 15  min ) .", "the ipg strips were overlaid on 24  26  cm 816% gradient tris - hcl ph 8.8 precast gels ( nextgen sciences , ann arbor ,", "approximately 1  ml of agarose was applied to fix the gels and a cy-2-labeled molecular weight marker was applied adjacent to the acidic side of the strip .", "the gels were then run 1 - 2  w per gel overnight ( ~2224 hours ) at 20c until the marker dye ran off the gel .", "gels were then imaged with a typhoon 9400 scanner and processed using decyder v6.5 ( ge healthcare ) bva batch processor tool .", "images were uploaded to decyder ( version 6.5 ) and spurious image objects ( water spots , streaks , and mismatches ) were identified and excluded from further analysis .", "following allocation of changed proteins , individual spots were manually inspected and excluded from analysis if they fell outside acceptable parameters for peak height , area , and slope .  ", "pick lists were generated by selecting proteins whose expression was statistically changed in the two growth conditions ( p < 0.05 ) following digital image analysis using decyder , and the corresponding spots were automatically picked using an ettan spot picker ( ge healthcare ) with ettan spot pick software v.1.1", ". spots were delivered in 100  mm ammonium bicarbonate to a 96-well plate and digested using established protocols .", "briefly , gel pieces were sequentially dried using three exchanges of 100% acetonitrile followed by a 10-minute speedvac ( savant ) drying .", "gel pieces were rehydrated in 40  l of 10  ng/l trypsin in 100  mm nh4hco3 on ice for 45 minutes .", "the supernatant was removed and replaced with 100  mm nh4hco3 and digested at 37c overnight .", "peptides were then extracted using 50% acetonitrile/0.1% tfa ( trifluoroacetic acid ) for 30 minutes at 37c .", "the peptides were microextracted using omix c18 tips ( varian , palo alto , ca , usa ) following the manufacturer 's instructions and then deposited on a stainless - steel target plate in a matrix consisting of 10  mg / ml 2,5-dihydroxybenzoic acid", ".   mass spectra were acquired using a voyager de - str matrix - assisted laser desorption / ionization time - of - flight ms ( applied biosystems , foster city , ca , usa ) in positive reflector mode with delayed extraction using the following parameters : laser energy , 1400 arbitrary units ; mass range , 5005,000  da ; 120  nsec delay , 100 laser shots per spectrum .", "external calibration was conducted using a standard peptide mixture ( bradykinin , insulin b chain , p14r , and acth ) , and internal calibration was carried out using trypsin autolysis peaks .", "data were processed in data explorer v1.1 ( applied biosystems , foster city , ca , usa ) using noise reduction ( 2 standard deviations ) and peak deisotoping .", "peak masses were searched using the mascot online search engine ( http://www.matrixscience.com/ ) with the following settings : database , ncbi entire database ( 5.6 million entries ) ; no missed cleavages ; monoisotopic peaks ; no fixed modifications ; variable modification of methionine oxidation ; error tolerance of 150  ppm .", "database and literature searches were used to further characterize and classify the proteins identified by maldi - tof ms . where ambiguous names were encountered , blastp searches   were used to identify homologous proteins from orthologous species ."], ["image analysis of 24  cm 2d - dige gels loaded with protein of s. wittichii rw1 cells grown on either dibenzofuran or acetate revealed 937 unique spots .", "differential in - gel analysis of individual gels determined gel - specific parameters for selection criteria and allowed visual examination of changes between growth on the two substrates ( figure 1 ) . of the 937 identified spots ,", "595 were matched between all the gels used to statistically compare the quantitative abundance of proteins .", "statistical analysis compared triplicate biological observations for each condition , normalized to the internal pooled standard ( figure 2 ) .  ", "crude cell lysates from s. wittichii rw1 grown on dibenzofuran showed that , of all proteins observed , 22 proteins were modulated in response to changes in culture conditions .", "these candidate biomarkers of metabolic activity and phenotype were observed in at least 6 of 9 dige images and were modulated as follows : 16 showed an apparent increase and 6 an apparent decrease ( figure 2 ) .", "these proteins , along with 22 proteins selected due to their high abundance in both growth conditions , were further analyzed and identified using mass spectrometry ( figure 3 ) .", "a mascot search of the entire ncbi database using mass spectral data generated by peptide mass fingerprinting identified 23 of the 44 proteins ( 52% ) .", "all protein identifications corresponded to the genome of s. wittichii strain rw1 . among the 16 proteins upregulated during growth on dibenzofuran ,", "7 were successfully identified ( table 1 ) . among the 6 proteins downregulated during growth on dibenzofuran ,", "an additional 13 proteins were identified whose expression level remained unchanged regardless of culture conditions ( table 3 ) .  ", "of the 7 identified proteins increased during growth on dibenzofuran , 2 were directly related to the dibenzofuran degradation pathway ( figure 4 ) ; the others were involved in downstream metabolic processes ( catechol 1,2-dioxygenase , adenosylhomocysteinase ) , cell growth ( elongation factor ts ) , and cell protection ( cold shock dna - binding domain protein , alkyl hydroperoxide reductase ) .", "the three identified proteins whose expression was decreased ( fumarylacetoacetate hydrolase , tonb - dependent receptor , and acyl - coa dehydrogenase ) are involved in biosynthesis , catabolism , and transport .", "the unchanged proteins represented basic cell functions , although biosynthesis , catabolism , and transport proteins dominated the identities .", "the alpha subunit of the dioxin dioxygenase , the first step in the dioxin degradation pathway , was also identified as unchanged ."], ["dige and 2d electrophoresis are an accepted strategy for mining microbial proteomes for biomarkers related to a number of processes [ 2830 ] .", "the complete protein content of s. wittichii rw1 consists of approximately 5,000 putative proteins from the bacterial chromosome and two megaplasmids . using simple extraction and purification techniques followed by dige", ", over 500 protein spots were resolved on a large ( 24  cm ) 2-dimensional gel and matched between the three biological replicates , representing approximately 10% of the entire protein content .", "of these 500 proteins , 22 were found to be regulated in response to growth condition changes .  ", "of the genes they identified as being changed in their experiments , only two overlapped with the proteins identified in our study : swit_3144 ( tonb - dependent receptor , downregulated ) and swit_3376 ( chaperonin groel , upregulated ) .", "both of these gene levels responded to short - term perturbation with peg8000 but not sodium chloride in the transcriptomic study and were unchanged in our study . in our study", ", we also identified a number of proteins that are related to dioxin / dibenzofuran degradation ( e.g. , dioxin dioxygenase , meta - cleavage product hydrolase , and 2,3-dihydroxybiphenyl 1,2-dioxygenase ) .", "other proteins were identified that showed increases in abundance but whose role was not directly related to the dibenzofuran degradation pathway . the increase in the presence of antioxidants such as alkyl", "hydroperoxide reductase suggests that there is an increasing stress upon the bacterial cell during growth on dibenzofuran , perhaps due to a change in catabolism resulting in an increase in endogenous peroxide generation .", "increases in a cold - shock dna - binding protein may be further evidence of an increased cellular stress .", "however , proteins of the cold shock family and related ones are also known to have transport and protein processing roles .   among the proteins in the dioxin degradation pathway , the most prominent on the gel was the meta - cleavage product hydrolase .", "this identification was produced from two adjacent spots , likely representing an artifact due to the protein 's extremely high expression or a reflection of the presence of multiple isoforms or a modified enzyme .", "the one identified in the present study is the product of the swit_3055 locus , a gene also known as dxnb2 .", "the glyoxalase / bleomycin resistance protein / dioxygenase identified in this study is also annotated as a 2,3-dihydroxybiphenyl-1,2-dioxygenase located on the chromosome at the swit_3046 locus .", "again , there are multiple isoforms of this enzyme found both on the chromosome and the megaplasmids .", "the kegg dioxin degradation pathway identifies swit_4182 as the dihydroxybiphenyl dioxygenase involved in biphenyl metabolism and swit_4902 as the trihydroxybiphenyl dioxygenase in both dioxin and dibenzofuran metabolism .", "the increased expression in response to dibenzofuran suggests that the swit_3046 dioxygenase plays a more important role in dibenzofuran degradation in vivo .", "the high degree of redundancy in the dioxin and dibenzofuran degradation pathways , that is , the presence of multiple ring - hydroxylating alpha and beta subunits , glyoxalase / bleomycin resistance protein / dioxygenases , and meta - cleavage product hydrolases , remains to be explained .", "one possibility is that the various isoforms have different affinities for chlorinated metabolites that would result from chlorinated dioxins and furans .", "further experiments are needed to fully distinguish the roles of these enzymes in s. wittichii rw1 degradation pathways .", "although not directly implicated in dioxin degradation , the fumarylacetoacetate hydrolase is also of interest because the gene encoding this protein ( swit_5089 ) flanks the ferredoxin fdx1 ( swit_5088 ) that has been identified as part of the electron supply chain supporting dioxin dioxygenase activity . of the multiple isoforms of this enzyme ,", "the electron supply chain also contains two isofunctional reductases . neither the ferredoxin itself nor the reductases could be identified . in previous studies ,", "the reductase was present as a much smaller fraction of the soluble cell proteome than either the ferredoxin or the dioxin dioxygenase , so gel - based methods may not be sensitive enough to detect this protein .", "if transcription of the ferredoxin is linked to the other genes at that locus , as is predicted , the decreased expression in response to dibenzofuran suggests that another ferredoxin is more important in the dioxin degradation pathway in vivo .", "the detection of the dioxin dioxygenase alpha subunit and related enzymes in both acetate- and dibenzofuran - grown cells is potentially of importance for the field of bioremediation because it suggests an avenue of biostimulation .", "when utilizing s. wittichii rw1 as a bioremediation agent , it may be possible to induce the expression of the dioxin degradation pathway using acetate .", "induction of the dioxin degradation pathway has not been observed when s. wittichii rw1 is grown on glucose or rich medium , and growth in a complex environmental medium ( landfill leachate ) was correlated with a decrease in copy number of the gene encoding the dioxin dioxygenase alpha subunit .", "previous studies using s. wittichii rw1 to transform chlorinated dioxins in soil or fly ash have observed a progressive decrease in degradative activity   or viable cells [ 38 , 39 ] , respectively .", "the addition of acetate may generate sufficient relevant protein biomass to catalyze the successful degradation of dioxin and dioxin - like compounds in environments bioaugmented with s. wittichii rw1 .", "proteomic technology has emerged in microbiology more rapidly than in other fields for several reasons .", "the relatively small genomes code for relatively limited proteomes featuring no or very limited posttranslational modifications compared to higher organisms .", "furthermore , microbes are easily controlled and manipulated in the laboratory , both during growth and gene expression .", "these factors will continue to drive biomarker discovery in microbial proteomes , including phenotypic biomarkers informing on the degradative activity of biomass produced for bioaugmentation of contaminated environments .", "furthermore , the field of bioremediation can benefit from methods suitable for monitoring microbial biomarkers in field samples to inform on progress in site bioremediation .", "this study highlights a number of proteins that were changed in response to dibenzofuran exposure , opens the door to a greater understanding of how s. wittichii rw1 performs and regulates the degradation of dioxins , and suggests ways to enhance the biodegradation of dioxins ."]]}
{"article_id": "PMC4235203", "article_text": ["among dermatological disorders , erythematous dermatitis , or eczema , and psoriasis are the most common.1 dermatitis is defined as a superficial skin inflammation , characterized by poorly delimited erythema , and usually itchy crusts and scales.2 conventional measures to prevent eczema include avoidance of irritants and potential allergens , as well as continued hydration of the skin with thick emollients .", "other therapies including phototherapy , antimicrobials , antihistamines , and systemic immunosuppressives are also therapeutic options.1 psoriasis is an autoimmune , chronic , noncontagious , recurrent , inflammatory skin disorder . in western populations ,", "its prevalence is estimated to be as high as 2.8%.3 the typical skin lesions of psoriatic patients are sharply demarcated erythematous plaques covered with silvery or opalescent scales.4 the inflammatory response is supported by the infiltration of polymorphic neutrophils in the skin .", "neutrophils are triggered by the chemokines and lymphokines released by keratinocytes and t - lymphocytes , respectively , and , in turn , trigger the activity of lymphocytes and keratinocytes , thus generating a vicious cycle limited to acute lesions but linked in any case to the extended inflammation.5 treatments for psoriasis include immunosuppressive drugs such as methotrexate , cyclosporine , and fumaric acid esters.6 however , advances in mechanistic understanding of signaling pathways involved in the pathogenesis of psoriasis have led to the testing of biological therapies .", "these include immune suppressive drugs ( eg , alefacept ) and anticytokine therapies ( antitumor necrosis factor [ tnf ] therapies [ adalimumab , etanercept , infliximab , ustekinumab]).7 topical application of anti - inflammatory molecules is a feasible alternative to systemic approaches to treat psoriatic and eczematous symptoms by directly acting on inflammatory processes and generating a marked soothing response in skin.8 a recent study on costs of psoriasis medications has demonstrated that biological treatments represent 67% of total medication costs , and only 5% of patients purchase them .", "in contrast , topical medications are the most commonly purchased treatments and account for the 18% of total costs.9 furthermore , since almost 10% of patients require at least three topical therapy switches over a 1-year period to gain health benefit , the identification and formulation of novel anti - inflammatory compounds that could be used in topical application are a medical need for psoriasis treatment and a possible approach to reduce costs.9 boswellia serrata gum resin extracts ( bses ) ( or olibanum ) are used in traditional ayurvedic medicine to treat inflammatory diseases .", "the prevalent component of lipophilic fraction of olibanum is represented by the pentacyclic triterpenes boswellic acids ( bas ) , several forms of which have been characterized by analytical techniques , including - and -configured bas ( figure 1 ) .", "another structural variety is given by the presence of a carbonyl group and an acetyl group in 11-keto bas ( kba ) and 3-o - acetyl - bas ( akba ) , respectively.10 animal studies and pilot clinical trials support the efficacy of bses for the treatment of a variety of inflammatory diseases such as inflammatory bowel disease , rheumatoid arthritis , osteoarthritis , and asthma.1114 in 2002 , the european medicines agency classified bse as an  orphan drug  for the treatment of peritumoral brain edema.15 the main anti - inflammatory properties of bas are attributed to suppression of leukotriene formation via inhibition of 5-lipoxygenase ( 5-lo ) by kba and akba.15 the mechanistic model has to be fully elucidated , but there is some evidence of an indirect action of bas leading to an irreversible inhibition of 5-lo by a ca2 + -mediated pathway.16 bas also inhibit the human leukocyte elastase , released in inflammation- and hypersensitivity - based human disorders , thus further supporting ba s antiphlogistic properties.17 in activated human monocytes , akba conveys inhibition of nuclear factor ( nf)-kb and subsequent downregulation of tnf- expression , via its direct inhibition of i kappa b kinase ( ikk).18 noteworthy is that when the ikk inhibition by bas has been investigated in the cd18 hypomorphic ( cd18[hypo ] ) mouse model of psoriasis , the suppressive effects on nf - kb signaling have been confirmed , thus suggesting that targeting nf - kb with bas may be an effective strategy for the treatment of psoriasis.19 since both psoriasis and erythematous dermatitis present a prevalent inflammatory component and bas have a documented inhibitory activity in some pathways with a major role in inflammation , especially in nf - kb pathway , we developed a formulation containing bas with potential efficacy in patients with psoriasis and erythematous dermatitis . in this double - blind study ,", "we compare , for the first time , a novel ba formulation ( containing bosexil , inci : lecithin , bse ) with a placebo formulation in the treatment of psoriasis or eczema .", "a third arm of the trial received a formulation of vaccinium myrtillus seed oil , which was previously demonstrated as an efficient local treatment for psoriatic lesions.20", "this is a randomized , placebo - controlled , double - blind study performed at the department of dermatology at the university of varese ( italy ) in collaboration with velleja research and the local health agency 1 ( asl1 ) in the piacenza ( italy ) area .", "we separately considered in the study patients who were affected either by psoriasis or by erythematous dermatitis . the diagnosis of erythematous atopic eczema or psoriasis was made by the physician in charge of the department of dermatology involved in the trial .", "patients had to be free from any topical or systemic anti - inflammatory treatment for at least 3 months .", "patients were randomly assigned , in a 1:1:1 ratio and according to a computer - generated numerical table , to one of the following groups : bosexil , v. myrtillus seed oil , or placebo .", "bosexil was formulated according to the phytosome delivery system that is aimed at improving the bioavailability and skin affinity of bioactive molecules.21 all preparations could not be distinguished without a chemical analysis .", "patients were asked to apply the cream twice a day to the affected areas in a sufficient quantity to cover the whole skin area involved , for a total period of 30 days .", "other concomitant pharmacological treatments were not interrupted or modified , except for those , dermatological or oral , finalized to treat psoriasis .", "clinical assessments were carried out at baseline ( t0 ) and at the end of the trial ( t30 ) , and included the evaluation of scales and erythema in the case of psoriasis and the evaluation of itch and erythema in the case of eczema .", "lesions and clinical manifestations were classified as absent ( score 0 ) , moderate ( score 1 ) , severe ( score 2 ) , or very severe ( score 3 ) .", "efficacy was statistically assessed through a procedure based on the individual s  permanence  or  migration state  ( primary end point ) depending on the entity of the score assigned to a symptom at t30 in comparison with the corresponding baseline score ( t0 ) .", "thus , the  change of condition  generated the following categories of patients : in remission : when individuals with scores > 0 at t0 have score = 0 at t30 ; improved : when individuals have lower scores at t30 than at t0 ; unchanged : when individuals have the same scores at t0 and t30 ; worsened : when individuals have higher scores at t30 than at t0 .", "we validated this statistical procedure in a previous study on the efficacy of v. myrtillus seed oil.20 as an internal control , we compared the effects of bosexil and v. myrtillus with the same placebo . the psoriasis area severity index ( pasi ) and the eczema area and severity index ( easi )", "the comparison between groups was performed using pearson s chi - square test ; responses were evaluated with a markov - like procedure .", "the comparison between groups was performed using pearson s chi - square test ; responses were evaluated with a markov - like procedure .", "each group consisted of ten patients affected from psoriasis and ten patients with erythematous dermatitis ( except the bosexil group , in which patients with dermatitis numbered nine ) . at t0 ,", "all groups were similar in terms of severity of the disease . however , three patients in the placebo group , one in the v. myrtillus group , and two in the bosexil group suspended the treatment before t30 and were included with a score of 3 in the statistical analysis . no significant difference in terms of age or sex", "the results from the  change of condition  ( primary end point ) analysis between placebo ( group a ) and two formulations ( bosexil and v. myrtillus seed oil ) are summarized in tables 5 and 6 . in patients with psoriasis ( table 5 ) ,", "signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .", "in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .", "treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,", "p=0.0281 ) without any case of worsening . treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) . in patients with erythematous eczema ( table 6 ) , the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening . v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and 77.8% ( p=0.0068 ) of patients , respectively .", "the pasi and easi scores observed in the three groups are reported in tables 7 and 8 , respectively .", "figure 2 shows pictures of some psoriasis patients  one treated with bosexil , one treated with v. myrtillus seed oil , and the other assigned to placebo .", "the results from the  change of condition  ( primary end point ) analysis between placebo ( group a ) and two formulations ( bosexil and v. myrtillus seed oil ) are summarized in tables 5 and 6 . in patients with psoriasis ( table 5 ) ,", "signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .", "in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .", "treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,", "treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) . in patients with erythematous eczema ( table 6 ) ,", "the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening . v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and", "in patients with psoriasis ( table 5 ) , signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .", "in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .", "treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,", "p=0.0281 ) without any case of worsening . treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) .", "in patients with erythematous eczema ( table 6 ) , the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening .", "v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and 77.8% ( p=0.0068 ) of patients , respectively .", "the pasi and easi scores observed in the three groups are reported in tables 7 and 8 , respectively .", "figure 2 shows pictures of some psoriasis patients  one treated with bosexil , one treated with v. myrtillus seed oil , and the other assigned to placebo .", "this study aimed to compare , for the first time to our knowledge , the efficacy of topical preparation of bosexil and v. myrtillus seed oil vs a placebo in individuals with a diagnosis of psoriasis or erythematous dermatitis .", "indeed , these formulations are particularly rich in polyunsatured fatty acids ( v. myrtillus seed oil ) or bas ( bosexil ) that exert anti - inflammatory effects , thus may provide benefits in skin inflammatory diseases .", "bas in bosexil formulation were more active in the treatment of psoriatic scales than erythema , and were similarly efficient to treat itch and erythema in patients affected by eczema .", "this formulation was designed according to the phytosome delivery system21 in order to improve the bioavailability and skin affinity of bas .", "consistently with previous results,20 this study confirmed the efficacy of v. myrtillus seed oil preparation in the treatment of major symptoms in both psoriasis and eczema , such as erythema and scales and erythema and itch , respectively .", "noteworthy is that v. myrtillus seed oil was associated with three remissions in the treatment of erythema in psoriatic patients .", "bilberry fruits contain small seeds rich in alpha - linolenic acid and linoleic acid , which are considered essential nutrients ( the human body , in fact , lacks the necessary enzymes to introduce an unsaturation beyond position omega-9 ) .", "linoleic acid and alpha - linolenic acid are involved in the skin barrier function.22 one issue in the management of chronic diseases such as psoriasis is the adherence to treatment .", "few data are available in literature , but the most frequently mentioned reasons for nonadherence to topical treatment are low efficacy , time consumption , and poor cosmetic characteristics of topical agents.23 this study has tested a twice - daily administration which determined an effective improvement of disease . however , the study lasted a short period of time and included an overall limited number of patients ; therefore , long - term , larger studies should be recommended to assess the adherence to this protocol in real life .", "moreover , earlier assessments ( eg , at day 15 ) could have been performed to collect information on the early efficacy of both bosexil and v. myrtillus seed oil .", "last , other end points ( eg , patient - related severity score or the dermatology life quality index ) have not been evaluated in this study .", "in addition to the anti - inflammatory / barrier - protecting effects of polyunsatured fatty acids or bas , a cosmeceutical formulation of these creams may provide the adjunctive benefits that can restore and protect the skin barrier function , increase the remission times between flare - ups , and enhance the pharmaceutical effects of active compounds.24", "in conclusion , bas contained in bosexil and formulated according to the phytosome delivery system21 may be eligible components for the formulation of preparation with anti - inflammatory properties for the treatment of psoriatic and eczematous symptoms .", "we speculate that this formulation could also be applied to other inflammatory skin disorders , including contact , atopic , seborrheic , nummular , chronic palmoplantar , and generalized exfoliative or chronic lichen simplex dermatitis ."], "abstract_text": ["<S> backgroundboswellic acids ( bas ) show anti - inflammatory properties in a variety of inflammatory diseases , including rheumatoid arthritis , osteoarthritis , and asthma . </S>", "<S> a topical administration route is currently used to deliver active compounds in psoriatic and eczematous patients . in this double - blind study </S>", "<S> we compare a novel ba formulation ( containing bosexil , inci [ international nomenclature of cosmetic ingredients ] : lecithin , boswellia serrata resin extract ) with a placebo formulation . </S>", "<S> a third arm of the trial received a formulation of vaccinium myrtillus seed oil , previously demonstrated as an effective local treatment for psoriatic lesions.methodspatients with psoriasis or erythematous eczema were randomly assigned , in a 1:1:1 ratio , to bosexil , v. myrtillus seed oil , or placebo . in order to evaluate the effects of treatment , the changes of scales and erythema from diagnosis to the end of treatment </S>", "<S> were scored in psoriatic patients , while changes in itch and erythema were analyzed for erythematous eczema patients . </S>", "<S> psoriasis area severity index and eczema area and severity index scores were also calculated.resultsin patients with psoriasis , scales and erythema improved both with bosexil and the v. myrtillus seed oil treatment in comparison with placebo . in particular , the treatment with bosexil formulation improved scales ( 70% of cases ) and erythema ( 50% of cases ) without any case of worsening . in patients with eczema , the administration of placebo did not result in any improvement in 90% of cases , and in the remaining 10% worsened both itch and erythema . </S>", "<S> bosexil formulation improved both itch ( 60% of cases ) and erythema ( 60% of cases ) without any case of worsening . </S>", "<S> v. myrtillus seed oil improved itch and erythema in 66.7% and 77.8% of patients , respectively.conclusiona topical formulation of bosexil may be promising for the treatment of psoriasis and erythematous eczema . </S>", "<S> long - term studies are recommended to evaluate the adherence to this topical treatment and its clinical benefits in real life . </S>"], "labels": null, "section_names": ["Introduction", "Patients and methods", "Statistical analysis", "Results", "Change of condition", "Psoriasis", "Erythematous eczema", "PASI and EASI", "Discussion", "Conclusion"], "sections": [["among dermatological disorders , erythematous dermatitis , or eczema , and psoriasis are the most common.1 dermatitis is defined as a superficial skin inflammation , characterized by poorly delimited erythema , and usually itchy crusts and scales.2 conventional measures to prevent eczema include avoidance of irritants and potential allergens , as well as continued hydration of the skin with thick emollients .", "other therapies including phototherapy , antimicrobials , antihistamines , and systemic immunosuppressives are also therapeutic options.1 psoriasis is an autoimmune , chronic , noncontagious , recurrent , inflammatory skin disorder . in western populations ,", "its prevalence is estimated to be as high as 2.8%.3 the typical skin lesions of psoriatic patients are sharply demarcated erythematous plaques covered with silvery or opalescent scales.4 the inflammatory response is supported by the infiltration of polymorphic neutrophils in the skin .", "neutrophils are triggered by the chemokines and lymphokines released by keratinocytes and t - lymphocytes , respectively , and , in turn , trigger the activity of lymphocytes and keratinocytes , thus generating a vicious cycle limited to acute lesions but linked in any case to the extended inflammation.5 treatments for psoriasis include immunosuppressive drugs such as methotrexate , cyclosporine , and fumaric acid esters.6 however , advances in mechanistic understanding of signaling pathways involved in the pathogenesis of psoriasis have led to the testing of biological therapies .", "these include immune suppressive drugs ( eg , alefacept ) and anticytokine therapies ( antitumor necrosis factor [ tnf ] therapies [ adalimumab , etanercept , infliximab , ustekinumab]).7 topical application of anti - inflammatory molecules is a feasible alternative to systemic approaches to treat psoriatic and eczematous symptoms by directly acting on inflammatory processes and generating a marked soothing response in skin.8 a recent study on costs of psoriasis medications has demonstrated that biological treatments represent 67% of total medication costs , and only 5% of patients purchase them .", "in contrast , topical medications are the most commonly purchased treatments and account for the 18% of total costs.9 furthermore , since almost 10% of patients require at least three topical therapy switches over a 1-year period to gain health benefit , the identification and formulation of novel anti - inflammatory compounds that could be used in topical application are a medical need for psoriasis treatment and a possible approach to reduce costs.9 boswellia serrata gum resin extracts ( bses ) ( or olibanum ) are used in traditional ayurvedic medicine to treat inflammatory diseases .", "the prevalent component of lipophilic fraction of olibanum is represented by the pentacyclic triterpenes boswellic acids ( bas ) , several forms of which have been characterized by analytical techniques , including - and -configured bas ( figure 1 ) .", "another structural variety is given by the presence of a carbonyl group and an acetyl group in 11-keto bas ( kba ) and 3-o - acetyl - bas ( akba ) , respectively.10 animal studies and pilot clinical trials support the efficacy of bses for the treatment of a variety of inflammatory diseases such as inflammatory bowel disease , rheumatoid arthritis , osteoarthritis , and asthma.1114 in 2002 , the european medicines agency classified bse as an  orphan drug  for the treatment of peritumoral brain edema.15 the main anti - inflammatory properties of bas are attributed to suppression of leukotriene formation via inhibition of 5-lipoxygenase ( 5-lo ) by kba and akba.15 the mechanistic model has to be fully elucidated , but there is some evidence of an indirect action of bas leading to an irreversible inhibition of 5-lo by a ca2 + -mediated pathway.16 bas also inhibit the human leukocyte elastase , released in inflammation- and hypersensitivity - based human disorders , thus further supporting ba s antiphlogistic properties.17 in activated human monocytes , akba conveys inhibition of nuclear factor ( nf)-kb and subsequent downregulation of tnf- expression , via its direct inhibition of i kappa b kinase ( ikk).18 noteworthy is that when the ikk inhibition by bas has been investigated in the cd18 hypomorphic ( cd18[hypo ] ) mouse model of psoriasis , the suppressive effects on nf - kb signaling have been confirmed , thus suggesting that targeting nf - kb with bas may be an effective strategy for the treatment of psoriasis.19 since both psoriasis and erythematous dermatitis present a prevalent inflammatory component and bas have a documented inhibitory activity in some pathways with a major role in inflammation , especially in nf - kb pathway , we developed a formulation containing bas with potential efficacy in patients with psoriasis and erythematous dermatitis . in this double - blind study ,", "we compare , for the first time , a novel ba formulation ( containing bosexil , inci : lecithin , bse ) with a placebo formulation in the treatment of psoriasis or eczema .", "a third arm of the trial received a formulation of vaccinium myrtillus seed oil , which was previously demonstrated as an efficient local treatment for psoriatic lesions.20"], ["this is a randomized , placebo - controlled , double - blind study performed at the department of dermatology at the university of varese ( italy ) in collaboration with velleja research and the local health agency 1 ( asl1 ) in the piacenza ( italy ) area .", "we separately considered in the study patients who were affected either by psoriasis or by erythematous dermatitis . the diagnosis of erythematous atopic eczema or psoriasis was made by the physician in charge of the department of dermatology involved in the trial .", "patients had to be free from any topical or systemic anti - inflammatory treatment for at least 3 months .", "patients were randomly assigned , in a 1:1:1 ratio and according to a computer - generated numerical table , to one of the following groups : bosexil , v. myrtillus seed oil , or placebo .", "bosexil was formulated according to the phytosome delivery system that is aimed at improving the bioavailability and skin affinity of bioactive molecules.21 all preparations could not be distinguished without a chemical analysis .", "patients were asked to apply the cream twice a day to the affected areas in a sufficient quantity to cover the whole skin area involved , for a total period of 30 days .", "other concomitant pharmacological treatments were not interrupted or modified , except for those , dermatological or oral , finalized to treat psoriasis .", "clinical assessments were carried out at baseline ( t0 ) and at the end of the trial ( t30 ) , and included the evaluation of scales and erythema in the case of psoriasis and the evaluation of itch and erythema in the case of eczema .", "lesions and clinical manifestations were classified as absent ( score 0 ) , moderate ( score 1 ) , severe ( score 2 ) , or very severe ( score 3 ) .", "efficacy was statistically assessed through a procedure based on the individual s  permanence  or  migration state  ( primary end point ) depending on the entity of the score assigned to a symptom at t30 in comparison with the corresponding baseline score ( t0 ) .", "thus , the  change of condition  generated the following categories of patients : in remission : when individuals with scores > 0 at t0 have score = 0 at t30 ; improved : when individuals have lower scores at t30 than at t0 ; unchanged : when individuals have the same scores at t0 and t30 ; worsened : when individuals have higher scores at t30 than at t0 .", "we validated this statistical procedure in a previous study on the efficacy of v. myrtillus seed oil.20 as an internal control , we compared the effects of bosexil and v. myrtillus with the same placebo . the psoriasis area severity index ( pasi ) and the eczema area and severity index ( easi )", "the comparison between groups was performed using pearson s chi - square test ; responses were evaluated with a markov - like procedure ."], ["the comparison between groups was performed using pearson s chi - square test ; responses were evaluated with a markov - like procedure ."], ["each group consisted of ten patients affected from psoriasis and ten patients with erythematous dermatitis ( except the bosexil group , in which patients with dermatitis numbered nine ) . at t0 ,", "all groups were similar in terms of severity of the disease . however , three patients in the placebo group , one in the v. myrtillus group , and two in the bosexil group suspended the treatment before t30 and were included with a score of 3 in the statistical analysis . no significant difference in terms of age or sex", "the results from the  change of condition  ( primary end point ) analysis between placebo ( group a ) and two formulations ( bosexil and v. myrtillus seed oil ) are summarized in tables 5 and 6 . in patients with psoriasis ( table 5 ) ,", "signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .", "in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .", "treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,", "p=0.0281 ) without any case of worsening . treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) . in patients with erythematous eczema ( table 6 ) , the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening . v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and 77.8% ( p=0.0068 ) of patients , respectively .", "the pasi and easi scores observed in the three groups are reported in tables 7 and 8 , respectively .", "figure 2 shows pictures of some psoriasis patients  one treated with bosexil , one treated with v. myrtillus seed oil , and the other assigned to placebo ."], ["the results from the  change of condition  ( primary end point ) analysis between placebo ( group a ) and two formulations ( bosexil and v. myrtillus seed oil ) are summarized in tables 5 and 6 . in patients with psoriasis ( table 5 ) ,", "signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .", "in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .", "treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,", "treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) . in patients with erythematous eczema ( table 6 ) ,", "the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening . v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and"], ["in patients with psoriasis ( table 5 ) , signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo .", "in particular , patients who received placebo had unchanged scales and erythema , and in 10% of cases worsened .", "treatment with bosexil formulation improved scales ( 70% of cases , p=0.0001 ) and erythema ( 60% of cases ,", "p=0.0281 ) without any case of worsening . treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases , but 30% of patients were in remission at the end of therapy , therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% ( p=0.253 ) of cases . in terms of scales , v. myrtillus seed oil improved the symptomatology in 80% of patients , and 20% was unchanged ( p=0.0003 ) ."], ["in patients with erythematous eczema ( table 6 ) , the bosexil - containing formulation improved both itch ( 60% of cases , p=0.0115 ) and erythema ( 60% of cases , p=0.0115 ) , without any case of worsening .", "v. myrtillus seed oil treatment improved itch and erythema in 66.7% ( p=0.0020 ) and 77.8% ( p=0.0068 ) of patients , respectively ."], ["the pasi and easi scores observed in the three groups are reported in tables 7 and 8 , respectively .", "figure 2 shows pictures of some psoriasis patients  one treated with bosexil , one treated with v. myrtillus seed oil , and the other assigned to placebo ."], ["this study aimed to compare , for the first time to our knowledge , the efficacy of topical preparation of bosexil and v. myrtillus seed oil vs a placebo in individuals with a diagnosis of psoriasis or erythematous dermatitis .", "indeed , these formulations are particularly rich in polyunsatured fatty acids ( v. myrtillus seed oil ) or bas ( bosexil ) that exert anti - inflammatory effects , thus may provide benefits in skin inflammatory diseases .", "bas in bosexil formulation were more active in the treatment of psoriatic scales than erythema , and were similarly efficient to treat itch and erythema in patients affected by eczema .", "this formulation was designed according to the phytosome delivery system21 in order to improve the bioavailability and skin affinity of bas .", "consistently with previous results,20 this study confirmed the efficacy of v. myrtillus seed oil preparation in the treatment of major symptoms in both psoriasis and eczema , such as erythema and scales and erythema and itch , respectively .", "noteworthy is that v. myrtillus seed oil was associated with three remissions in the treatment of erythema in psoriatic patients .", "bilberry fruits contain small seeds rich in alpha - linolenic acid and linoleic acid , which are considered essential nutrients ( the human body , in fact , lacks the necessary enzymes to introduce an unsaturation beyond position omega-9 ) .", "linoleic acid and alpha - linolenic acid are involved in the skin barrier function.22 one issue in the management of chronic diseases such as psoriasis is the adherence to treatment .", "few data are available in literature , but the most frequently mentioned reasons for nonadherence to topical treatment are low efficacy , time consumption , and poor cosmetic characteristics of topical agents.23 this study has tested a twice - daily administration which determined an effective improvement of disease . however , the study lasted a short period of time and included an overall limited number of patients ; therefore , long - term , larger studies should be recommended to assess the adherence to this protocol in real life .", "moreover , earlier assessments ( eg , at day 15 ) could have been performed to collect information on the early efficacy of both bosexil and v. myrtillus seed oil .", "last , other end points ( eg , patient - related severity score or the dermatology life quality index ) have not been evaluated in this study .", "in addition to the anti - inflammatory / barrier - protecting effects of polyunsatured fatty acids or bas , a cosmeceutical formulation of these creams may provide the adjunctive benefits that can restore and protect the skin barrier function , increase the remission times between flare - ups , and enhance the pharmaceutical effects of active compounds.24"], ["in conclusion , bas contained in bosexil and formulated according to the phytosome delivery system21 may be eligible components for the formulation of preparation with anti - inflammatory properties for the treatment of psoriatic and eczematous symptoms .", "we speculate that this formulation could also be applied to other inflammatory skin disorders , including contact , atopic , seborrheic , nummular , chronic palmoplantar , and generalized exfoliative or chronic lichen simplex dermatitis ."]]}
{"article_id": "PMC4591152", "article_text": ["cell lines , cultures and transfections : a madin - darby bovine kidney cell \n line ( mdbk ) ( hsrrb , osaka , japan ) was cultured in dulbecco s modified eagle s medium ( dmem ) \n with 10% fetal calf serum ( fcs ) . a human cervical carcinoma cell line ( hela )", "( riken cell \n bank , tsukuba , japan ) was cultured as described in previous studies [ 10 , 14 ] .", "a cattle xlf gene \n ( nm_001075393.1 ) with an artificial ecori site at the 5 end and bamhi site at the 3 end \n was synthesized .", "the fragment was confirmed by sequencing and ligated to the ecori and bamhi \n sites of the peyfp - c1 vector to give the in - frame fusion gene .", "peyfp - cattle xlf , \n peyfp - cattle xlf ( 162299 ) , peyfp - cattle xlf ( 162287 ) or peyfp - c1 was transient transfected \n in cells using fugene hd ( promega , madison , wi , u.s.a . )", "the cells were cultured for 2 days and then monitored under an fv300 confocal \n laser scanning microscope ( olympus , tokyo , japan ) as previously described [ 9 , 11 , 12 ] .", "immunoblotting : the extraction of total lysates and western blot analysis \n were conducted based on the previous methods [ 11 , \n 13 ] .", "the membranes \n were blocked in blocking one ( nacalai tesque , kyoto , japan ) for 30 min .", "the following \n antibodies were used : a rabbit anti - xlf polyclonal antibody ( a300 - 730a ) ( bethyl \n laboratories , montgomery , tx , u.s.a . ) , a rabbit anti - gfp polyclonal antibody ( fl ) ( santa \n cruz biotechnology , santa cruz , ca , u.s.a . ) , a mouse anti - ku70 monoclonal antibody ( n3h10 ) \n ( neomarkers , fremont , ca , u.s.a . ) or a mouse anti--actin monoclonal antibody ( sigma , st . \n", "three antibodies ( i.e. , anti - xlf antibody , anti - ku70 antibody and \n anti - gfp antibody ) were diluted in signal enhancer hikari ( nacalai tesque ) , respectively . \n", "the binding to each protein was visualized using a select western blotting detection system \n ( ge healthcare bio - sci .", "piscataway , nj , u.s.a . ) , in accordance with the manufacturer s \n instructions .", "immunofluorescence staining : immunofluorescence staining was conducted as \n previously described [ 9 , 11 ] .", "briefly , the fixed cells were blocked for 10 min using a blocking \n solution and then incubated for 30 min at room temperature with a mouse anti-h2ax \n monoclonal antibody ( jbw301 ) ( upstate biotechnology inc . , charlottesville , va , u.s.a . ) or a \n rabbit anti - xlf polyclonal antibody ( x4754 ) ( sigma ) . after washing with pbs , detection of \n each protein", "was performed using alexa fluor 568-conjugated secondary antibodies ( molecular \n probes , eugene , or , u.s.a . ) .", "local dna damage induction using laser and cell imaging : local dna damage \n induction using laser and cell imaging was conducted as described previously [ 9 , 11,12,13 ] .", "briefly , a \n 530% power scan ( for 1 sec ) from a 405 nm laser was used to induce local dsbs .", "images of \n living cells or fixed cells expressing eyfp - tagged cattle proteins or eyfp alone were \n obtained using an fv300 confocal scanning laser microscopy system ( olympus ) .", "expression and localization of cattle xlf in cattle cells : we examined the \n expression and subcellular localization of xlf in cattle cells .", "first , we examined the \n expression of xlf and ku70 in the cattle cell line mdbk and the human cell line hela by \n western blot analysis using the anti - xlf antibody and anti - ku70 antibody .", "( a ) total cell lysates from each \n cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed \n by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an \n anti--actin antibody .", "( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells \n during the cell cycle .", "the images shown \n are a representative example for interphase cells or mitotic phase cells ( c ) .", "these results demonstrate that the core nhej factors , xlf \n and ku70 , are expressed in cattle cells .", "total cell lysates from each \n cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed \n by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an \n anti--actin antibody .", "( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells \n during the cell cycle .", "the images shown \n are a representative example for interphase cells or mitotic phase cells ( c ) . to elucidate the localization of xlf in cattle cells", ", we studied the distribution of xlf by \n confocal laser microscopy ( fig . 1b and 1c ) . \n", "indirect immunofluorescence staining using the anti - xlf antibody showed that fluorescence \n was detected in the nucleoplasm of mdbk cells during the interphase . on the other hand ,", "the \n fluorescence was detected throughout the cytoplasm of mdbk cells during the mitotic phase , \n but not in the condensed chromosomes of the mitotic cells .", "these observations indicate that \n the localization of cattle xlf changes dynamically during the cell cycle .", "to clarify the \n localization of xlf in living cattle cells during the interphase , we examined the expression \n and localization of eyfp - cattle xlf in mdbk cells .", "the expression vector peyfp - c1 containing cattle xlf \n ( peyfp - cattle xlf ) was transfected into mdbk cells ( fig .", "( a ) schematics of eyfp - cattle \n xlf chimeric protein and control protein ( eyfp ) .", "( b ) extracts from cattle ( mdbk ) cells \n transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western \n blotting using the anti - xlf , anti - gfp or anti--actin antibody .", "living mdbk cells transiently expressing \n eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .", "eyfp images for \n the same cells are shown alone ( left panel ) or merged ( right panel ) with differential \n interference contrast images ( dic ) ( center panel ) . ) .", "2b , a signal of \n eyfp - cattle xlf was detected in the transfectants by western blot analysis using the \n anti - xlf antibody and anti - gfp antibody . by confocal laser microscopy", ", we clarified that \n eyfp - cattle xlf was localized in the nuclei of living interphase cells in eyfp - cattle xlf \n transfectants ( fig . 2c ) .", "expectedly , in eyfp \n transfectants , we confirmed that eyfp was distributed throughout the cell excluding the \n nucleolus ( fig . 2c ) .", "( a ) schematics of eyfp - cattle \n xlf chimeric protein and control protein ( eyfp ) .", "( b ) extracts from cattle ( mdbk ) cells \n transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western \n blotting using the anti - xlf , anti - gfp or anti--actin antibody .", "living mdbk cells transiently expressing \n eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .", "eyfp images for \n the same cells are shown alone ( left panel ) or merged ( right panel ) with differential \n interference contrast images ( dic ) ( center panel ) .", "eyfp - cattle xlf accumulates quickly at dsbs induced by laser \n microirradiation : we examined whether eyfp - cattle xlf accumulates quickly at the \n 405 nm laser - induced dsb sites ( fig . 3afig .", "( a ) \n the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser \n irradiation were examined .", "( b ) imaging of living eyfp - cattle xlf - transfected mdbk \n cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left \n panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) . \n", "( c ) immunostaining of microirradiated \n eyfp - cattle xlf - transfected cells with anti-h2ax antibody .", "the cells were fixed and \n stained with the anti- h2ax antibody at 5 min postirradiation . left panel , \n eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .", "( d ) \n time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after \n irradiation .", "upper panel , eyfp - cattle xlf ; lower panel , differential interference \n contrast images ( dic ) . ) . as shown in fig .", "3b , we observed that \n eyfp - cattle xlf accumulated at the microirradiated sites in the living cattle cells .", "next , \n we investigated whether cattle xlf accumulated at 405 nm laser - induced dsb sites by \n immunostaining with an antibody that detects h2ax . as shown in fig .", "3c , eyfp - cattle xlf colocalized with the dsb marker h2ax at \n microirradiated sites in mdbk cells .", "next , we carried out time - lapse imaging of eyfp - cattle \n xlf - transfected mdbk cells .", "3d , \n we observed eyfp - cattle xlf accumulation at the microirradiated sites 5 sec after \n irradiation . in eyfp - cattle xlf - transfected cells ,", "these results reveal that after irradiation , \n eyfp - cattle xlf quickly accumulates and formes foci at laser - induced dsbs in living \n cells .", "( a ) \n the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser \n irradiation were examined .", "( b ) imaging of living eyfp - cattle xlf - transfected mdbk \n cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left \n panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) . \n", "( c ) immunostaining of microirradiated \n eyfp - cattle xlf - transfected cells with anti-h2ax antibody .", "the cells were fixed and \n stained with the anti- h2ax antibody at 5 min postirradiation . left panel , \n eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .", "( d ) \n time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after \n irradiation .", "upper panel , eyfp - cattle xlf ; lower panel , differential interference \n contrast images ( dic ) .", "the c - terminal region ( ctr ) of cattle xlf is essential for the nuclear localization \n and recruitment of xlf to dsbs in cattle cells : to determine the region essential \n for nuclear localization of cattle xlf , we investigated the localization of cattle xlf and \n its mutant .", "firstly , the peyfp - cattle xlf and its mutants were transfected into mdbk cells . \n as shown in fig .", "4.the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of \n cattle xlf to dsbs in vivo .", "( a ) extracts from cattle ( mdbk ) cells \n transiently expressing the indicated cattle xlf deletions were prepared and subjected \n to western blotting using the anti - gfp or anti--actin antibody .", "( b , c ) identification \n of essential domain of cattle xlf for nuclear localization and for accumulation at \n dsbs .", "the localization \n and accumulation of the chimeric proteins at laser - induced dsbs were investigated via \n live cell imaging .", "the results are summarized on the right : cellular localization ( n , \n nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated \n sites ; - , not accumulated at microirradiated sites ) .", "( d ) immunostaining of microirradiated eyfp - cattle xlf \n ( 162287)-transfected cells with anti-h2ax antibody .", "the cells were fixed and stained \n with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf \n ( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .", "( e ) identity between \n the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino \n acids 288299 ) .", "( f ) the alignment of the primary sequence among homologous xlf \n proteins . for comparison , the basic ( red ) or non - basic residues ( black )", ", a signal of each eyfp - cattle xlf mutant was detected in the extracts of each \n transfectant by western blot analysis using the anti - gfp antibody . by confocal laser \n microscopy", ", we confirmed that eyfp - cattle xlf was localized in the nuclei of living \n interphase cells .", "we observed that n - terminal deletion mutant eyfp - cattle xlf ( 162299 ) \n localized predominantly in the nuclei , whereas eyfp - cattle xlf ( 162287 ) as well as eyfp was \n distributed throughout the cell excluding the nucleolus in mdbk cells ( figs . 2c , 4b and 4c ) .", "these results indicate that 12 c - terminal amino \n acids ( amino acids 288299 ) of cattle xlf are vital for the nuclear localization of xlf in \n cattle cells ( fig .", "which region \n of cattle xlf is essential for its accumulation at dsbs in vivo , we tested \n whether the xlf mutant proteins could be recruited to dsbs induced by microirradiation .", "we \n observed that a n - terminal deletion mutant eyfp - cattle xlf ( 162299 ) as well as the wild \n type eyfp - cattle xlf , accumulated at the dsbs sites in the living mdbk cells ( fig . 3c and \n data not shown ) . on the other hand , the mutant protein eyfp - cattle xlf ( 162287 ) failed to \n accumulate at the dsbs sites , which was detected by the dsb marker h2ax ( fig . 4c and 4d ) , indicating that deletion of the \n c - terminal end 12 amino acids abolished the recruitment of cattle xlf to dsbs .", "the cattle \n and human xlf proteins are 81.9% identical at the amino acid level , whereas the c - terminal \n domain ( amino acids 220299 ) of cattle xlf retains only 56.4% identity to human ( fig .", "interestingly , we confirmed that the basic \n amino acids in the ctr of xlf are evolutionarily highly conserved among humans and domestic \n animal species , e.g. , cattle , goats , horses and avian , but not in yeast ( fig .", "4f and data not shown ) , which strongly suggests \n the biological significance of the xlf ctr in domestic animals .", "the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of \n cattle xlf to dsbs in vivo . ( a )", "extracts from cattle ( mdbk ) cells \n transiently expressing the indicated cattle xlf deletions were prepared and subjected \n to western blotting using the anti - gfp or anti--actin antibody .", "( b , c ) identification \n of essential domain of cattle xlf for nuclear localization and for accumulation at \n dsbs .", "the localization \n and accumulation of the chimeric proteins at laser - induced dsbs were investigated via \n live cell imaging .", "the results are summarized on the right : cellular localization ( n , \n nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated \n sites ; - , not accumulated at microirradiated sites ) .", "( d ) immunostaining of microirradiated eyfp - cattle xlf \n ( 162287)-transfected cells with anti-h2ax antibody .", "the cells were fixed and stained \n with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf \n ( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .", "( e ) identity between \n the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino \n acids 288299 ) .", "( f ) the alignment of the primary sequence among homologous xlf \n proteins . for comparison , the basic ( red ) or non - basic residues ( black )", "to develop next - generation chemotherapeutics for cancer and other disease is important to \n clarify the molecular mechanisms of c - nhej .", "human xlf is the most recently identified core \n nhej factor , and it appears to play essential roles in c - nhej .", "expectedly , xlf - deficient cells derived from human patients and from \n knockout mice show ionizing radiation sensitivity [ 3 , \n 15 ] . in addition", ", sirna - mediated downregulation of \n xlf in human cell lines leads to radiosensitivity and impaired nhej . .", "homologues of the xlf gene were predicted in several eukaryotic \n organisms . on the other hand , the expression , \n function and regulation mechanism of xlf", "have not been elucidated in animal species other \n than mice and humans [ 1 , 3 , 11 , 16 , 21 ] .", "domestic cattle are \n important domestic animals as livestock and draft animals in not only japan , but also many \n countries .", "recently , cattle have been an ideal animal model for assessing chronic radiation \n exposure [ 5 , 20 ] . however , the molecular mechanism of c - nhej is still unknown in cattle cells . in \n this study", ", we examined the expression and subcellular localization of cattle xlf and its \n mutants in cattle cell line mdbk .", "we found that xlf as well as other core nhej protein ku70 \n is expressed in cattle cells , and the localization of cattle xlf changes dynamically during \n the cell cycle .", "in addition , xlf might play a vital role in the repair of dsb immediately \n after microirradiation of cattle cells .", "moreover , our data showed that the ctr of cattle xlf \n is vital for the nuclear localization of xlf and for the accumulation of xlf at dsbs \n in vivo .", "these findings suggest that the mechanisms regulating of the \n localization and recruitment to dsbs play a key role in the function of cattle xlf .", "cattle xlf ( nm_001075393.1 ) as well as goat xlf ( xp_005676612.1 ) and sheep xlf \n ( xp_004004987.1 ) is a 299-amino acid protein .", "the cattle and goat xlf genes are 94.6% \n identical at the amino acid level .", "in addition , the cattle and sheep xlf genes are 94.3% \n identical at the amino acid level . on the other hand ,", "human xlf is a 299-amino acid protein , which contains an n - terminal head \n domain ( amino acids 1141 ) , a coiled - coil central domain ( amino acids 142230 ) and a non \n structured c - terminal domain ( amino acids 231299 ) [ 1 , \n 2 , 17 ] . \n", "comparison with other eukaryotic homologues shows a high degree of sequence similarity \n within the 220 n - terminal amino acids , while the c - terminal domain ( amino acids 225299 ) is \n less conserved [ 1 , 2 , 17 ] . on the basis of experimental \n findings", ", there are some reports concerning the role of the c - terminal domain of xlf in \n humans , but not in animals including cattle .", "yano et al . reported that a \n 10-amino - acid deletion at the c - terminal end completely abolishes the ku - xlf interaction and \n the accumulation of xlf at dsbs . on the other \n hand , malivert et al .", "have reported that the c - terminal end ( amino acids \n 231299 ) of human xlf is dispensable for dna repair in vivo . in this study", ", our data showed that a 12-amino - acid \n deletion at the c - terminal end abolishes the accumulation of cattle xlf at dsbs . altogether", ", \n we conclude that the xlf ctr is important for the accumulation of xlf at dsbs in both human \n and cattle cells , although the role of the c - terminal region of xlf remains controversial in \n human cells .", "it was demonstrated that there is a general absence of conservation in the 75 c - terminal \n amino acids among humans and other species , although the extreme c terminus of xlf contains \n a small conserved basic cluster , which was proposed as a putative nls ( krkk ) [ 1 , 2 ] .", "our data \n revealed experimentally that the ctr of cattle xlf is critical for the nuclear localization \n of xlf and recruitment to dsbs , whereas the n - terminal domain is not essential .", "in addition , \n basic amino acids in the ctr of cattle xlf are evolutionarily conserved among ctr of \n domestic animals , which suggests the common biological significance of the xlf ctr in \n domestic animals .", "we consider that there is only one nls ( kvkrkklr ) in cattle xlf , and the \n nls is a classical monopartite nls having a single cluster of basic amino acid residues .", "we \n speculate that the c - terminus of 75 amino acids is important for a specific function in each \n species , whereas the xlf ctr is critical for the regulation of common functions in domestic \n animals .", ", we showed that xlf is expressed in cattle cells and the localization of \n cattle xlf changes dynamically during the cell cycle .", "in addition , our data showed that the \n localization and recruitment of cattle xlf to dsb sites at an early stage following \n irradiation are dependent on the ctr .", "these basic informations might be useful to develop \n the molecular - targeting therapeutic drug taking xlf as a target molecule for human and \n domestic animals .", "further studies to elucidate the mechanisms regulating cattle xlf at dsbs \n will lead to a better understanding of the physiological function of xlf not only in cattle \n cells , but also in cells of human and other domestic animals .", "inherited mutations of core \n c - nhej factors ( e.g. , dna - pkcs , dna ligase iv and xlf ) , have been discovered in humans \n . on the other hand , inherited", "mutations of the \n dna - pkcs , which cause scid , have been identified in not only humans , but also domestic \n animals , i.e. , mice , horses or dogs .", "therefore , \n further comparative studies might provide available information for the development of new \n clinical medicines and new chemoradiotherapies for humans and domestic animals including \n cattle ."], "abstract_text": ["<S> clinically , many chemotherapeutics and ionizing radiation ( ir ) have been \n applied for the treatment of various types of human and animal malignancies . </S>", "<S> these \n treatments kill tumor cells by causing dna double - strand breaks ( dsbs ) . </S>", "<S> core factors of \n classical nonhomologous dna - end joining ( c - nhej ) play a vital role in dsb repair . </S>", "<S> thus , it \n is indispensable to clarify the mechanisms of c - nhej in order to develop next - generation \n chemotherapeutics for cancer . the xrcc4-like factor ( xlf ; also called cernunnos or nhej1 ) \n </S>", "<S> is the lastly identified core nhej factor . </S>", "<S> the localization of core nhej factors might \n play a critical role in regulating nhej activity . </S>", "<S> the localization and function of xlf \n have not been elucidated in animal species other than mice and humans . </S>", "<S> domestic cattle \n ( bos taurus ) are the most common and vital domestic animals in many \n countries . here </S>", "<S> , we show that the localization of cattle xlf changes dynamically during \n the cell cycle . </S>", "<S> furthermore , eyfp - cattle xlf accumulates quickly at microirradiated sites \n and colocalizes with the dsb marker h2ax . </S>", "<S> moreover , nuclear localization and accumulation \n of cattle xlf at dsb sites are dependent on 12 amino acids ( 288299 ) of the c - terminal \n region of xlf ( xlf ctr ) . </S>", "<S> furthermore , basic amino acids on the xlf ctr are highly \n conserved among domestic animals including cattle , goat and horses , suggesting that the \n ctr is essential for the function of xlf in domestic animals . </S>", "<S> these findings might be \n useful to develop the molecular - targeting therapeutic drug taking xlf as a target molecule \n for human and domestic animals . </S>"], "labels": null, "section_names": ["MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["cell lines , cultures and transfections : a madin - darby bovine kidney cell \n line ( mdbk ) ( hsrrb , osaka , japan ) was cultured in dulbecco s modified eagle s medium ( dmem ) \n with 10% fetal calf serum ( fcs ) . a human cervical carcinoma cell line ( hela )", "( riken cell \n bank , tsukuba , japan ) was cultured as described in previous studies [ 10 , 14 ] .", "a cattle xlf gene \n ( nm_001075393.1 ) with an artificial ecori site at the 5 end and bamhi site at the 3 end \n was synthesized .", "the fragment was confirmed by sequencing and ligated to the ecori and bamhi \n sites of the peyfp - c1 vector to give the in - frame fusion gene .", "peyfp - cattle xlf , \n peyfp - cattle xlf ( 162299 ) , peyfp - cattle xlf ( 162287 ) or peyfp - c1 was transient transfected \n in cells using fugene hd ( promega , madison , wi , u.s.a . )", "the cells were cultured for 2 days and then monitored under an fv300 confocal \n laser scanning microscope ( olympus , tokyo , japan ) as previously described [ 9 , 11 , 12 ] .", "immunoblotting : the extraction of total lysates and western blot analysis \n were conducted based on the previous methods [ 11 , \n 13 ] .", "the membranes \n were blocked in blocking one ( nacalai tesque , kyoto , japan ) for 30 min .", "the following \n antibodies were used : a rabbit anti - xlf polyclonal antibody ( a300 - 730a ) ( bethyl \n laboratories , montgomery , tx , u.s.a . ) , a rabbit anti - gfp polyclonal antibody ( fl ) ( santa \n cruz biotechnology , santa cruz , ca , u.s.a . ) , a mouse anti - ku70 monoclonal antibody ( n3h10 ) \n ( neomarkers , fremont , ca , u.s.a . ) or a mouse anti--actin monoclonal antibody ( sigma , st . \n", "three antibodies ( i.e. , anti - xlf antibody , anti - ku70 antibody and \n anti - gfp antibody ) were diluted in signal enhancer hikari ( nacalai tesque ) , respectively . \n", "the binding to each protein was visualized using a select western blotting detection system \n ( ge healthcare bio - sci .", "piscataway , nj , u.s.a . ) , in accordance with the manufacturer s \n instructions .", "immunofluorescence staining : immunofluorescence staining was conducted as \n previously described [ 9 , 11 ] .", "briefly , the fixed cells were blocked for 10 min using a blocking \n solution and then incubated for 30 min at room temperature with a mouse anti-h2ax \n monoclonal antibody ( jbw301 ) ( upstate biotechnology inc . , charlottesville , va , u.s.a . ) or a \n rabbit anti - xlf polyclonal antibody ( x4754 ) ( sigma ) . after washing with pbs , detection of \n each protein", "was performed using alexa fluor 568-conjugated secondary antibodies ( molecular \n probes , eugene , or , u.s.a . ) .", "local dna damage induction using laser and cell imaging : local dna damage \n induction using laser and cell imaging was conducted as described previously [ 9 , 11,12,13 ] .", "briefly , a \n 530% power scan ( for 1 sec ) from a 405 nm laser was used to induce local dsbs .", "images of \n living cells or fixed cells expressing eyfp - tagged cattle proteins or eyfp alone were \n obtained using an fv300 confocal scanning laser microscopy system ( olympus ) ."], ["expression and localization of cattle xlf in cattle cells : we examined the \n expression and subcellular localization of xlf in cattle cells .", "first , we examined the \n expression of xlf and ku70 in the cattle cell line mdbk and the human cell line hela by \n western blot analysis using the anti - xlf antibody and anti - ku70 antibody .", "( a ) total cell lysates from each \n cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed \n by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an \n anti--actin antibody .", "( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells \n during the cell cycle .", "the images shown \n are a representative example for interphase cells or mitotic phase cells ( c ) .", "these results demonstrate that the core nhej factors , xlf \n and ku70 , are expressed in cattle cells .", "total cell lysates from each \n cell line ( mdbk , 50 g ; hela , 10 g ) were analyzed \n by western blotting using an anti - xlf antibody , an anti - ku70 antibody or an \n anti--actin antibody .", "( b , c ) subcellular localization of xlf in cattle ( mdbk ) cells \n during the cell cycle .", "the images shown \n are a representative example for interphase cells or mitotic phase cells ( c ) . to elucidate the localization of xlf in cattle cells", ", we studied the distribution of xlf by \n confocal laser microscopy ( fig . 1b and 1c ) . \n", "indirect immunofluorescence staining using the anti - xlf antibody showed that fluorescence \n was detected in the nucleoplasm of mdbk cells during the interphase . on the other hand ,", "the \n fluorescence was detected throughout the cytoplasm of mdbk cells during the mitotic phase , \n but not in the condensed chromosomes of the mitotic cells .", "these observations indicate that \n the localization of cattle xlf changes dynamically during the cell cycle .", "to clarify the \n localization of xlf in living cattle cells during the interphase , we examined the expression \n and localization of eyfp - cattle xlf in mdbk cells .", "the expression vector peyfp - c1 containing cattle xlf \n ( peyfp - cattle xlf ) was transfected into mdbk cells ( fig .", "( a ) schematics of eyfp - cattle \n xlf chimeric protein and control protein ( eyfp ) .", "( b ) extracts from cattle ( mdbk ) cells \n transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western \n blotting using the anti - xlf , anti - gfp or anti--actin antibody .", "living mdbk cells transiently expressing \n eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .", "eyfp images for \n the same cells are shown alone ( left panel ) or merged ( right panel ) with differential \n interference contrast images ( dic ) ( center panel ) . ) .", "2b , a signal of \n eyfp - cattle xlf was detected in the transfectants by western blot analysis using the \n anti - xlf antibody and anti - gfp antibody . by confocal laser microscopy", ", we clarified that \n eyfp - cattle xlf was localized in the nuclei of living interphase cells in eyfp - cattle xlf \n transfectants ( fig . 2c ) .", "expectedly , in eyfp \n transfectants , we confirmed that eyfp was distributed throughout the cell excluding the \n nucleolus ( fig . 2c ) .", "( a ) schematics of eyfp - cattle \n xlf chimeric protein and control protein ( eyfp ) .", "( b ) extracts from cattle ( mdbk ) cells \n transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western \n blotting using the anti - xlf , anti - gfp or anti--actin antibody .", "living mdbk cells transiently expressing \n eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy .", "eyfp images for \n the same cells are shown alone ( left panel ) or merged ( right panel ) with differential \n interference contrast images ( dic ) ( center panel ) .", "eyfp - cattle xlf accumulates quickly at dsbs induced by laser \n microirradiation : we examined whether eyfp - cattle xlf accumulates quickly at the \n 405 nm laser - induced dsb sites ( fig . 3afig .", "( a ) \n the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser \n irradiation were examined .", "( b ) imaging of living eyfp - cattle xlf - transfected mdbk \n cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left \n panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) . \n", "( c ) immunostaining of microirradiated \n eyfp - cattle xlf - transfected cells with anti-h2ax antibody .", "the cells were fixed and \n stained with the anti- h2ax antibody at 5 min postirradiation . left panel , \n eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .", "( d ) \n time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after \n irradiation .", "upper panel , eyfp - cattle xlf ; lower panel , differential interference \n contrast images ( dic ) . ) . as shown in fig .", "3b , we observed that \n eyfp - cattle xlf accumulated at the microirradiated sites in the living cattle cells .", "next , \n we investigated whether cattle xlf accumulated at 405 nm laser - induced dsb sites by \n immunostaining with an antibody that detects h2ax . as shown in fig .", "3c , eyfp - cattle xlf colocalized with the dsb marker h2ax at \n microirradiated sites in mdbk cells .", "next , we carried out time - lapse imaging of eyfp - cattle \n xlf - transfected mdbk cells .", "3d , \n we observed eyfp - cattle xlf accumulation at the microirradiated sites 5 sec after \n irradiation . in eyfp - cattle xlf - transfected cells ,", "these results reveal that after irradiation , \n eyfp - cattle xlf quickly accumulates and formes foci at laser - induced dsbs in living \n cells .", "( a ) \n the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser \n irradiation were examined .", "( b ) imaging of living eyfp - cattle xlf - transfected mdbk \n cells before ( upper panel ) and at 1 min after ( lower panel ) microirradiation . left \n panel , eyfp - cattle xlf ; right panel , differential interference contrast images ( dic ) . \n", "( c ) immunostaining of microirradiated \n eyfp - cattle xlf - transfected cells with anti-h2ax antibody .", "the cells were fixed and \n stained with the anti- h2ax antibody at 5 min postirradiation . left panel , \n eyfp - cattle xlf ; center panel , h2ax image ; right panel , merged image .", "( d ) \n time - dependent eyfp - cattle xlf accumulation in living cells ( 5 - 120 sec ) after \n irradiation .", "upper panel , eyfp - cattle xlf ; lower panel , differential interference \n contrast images ( dic ) .", "the c - terminal region ( ctr ) of cattle xlf is essential for the nuclear localization \n and recruitment of xlf to dsbs in cattle cells : to determine the region essential \n for nuclear localization of cattle xlf , we investigated the localization of cattle xlf and \n its mutant .", "firstly , the peyfp - cattle xlf and its mutants were transfected into mdbk cells . \n as shown in fig .", "4.the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of \n cattle xlf to dsbs in vivo .", "( a ) extracts from cattle ( mdbk ) cells \n transiently expressing the indicated cattle xlf deletions were prepared and subjected \n to western blotting using the anti - gfp or anti--actin antibody .", "( b , c ) identification \n of essential domain of cattle xlf for nuclear localization and for accumulation at \n dsbs .", "the localization \n and accumulation of the chimeric proteins at laser - induced dsbs were investigated via \n live cell imaging .", "the results are summarized on the right : cellular localization ( n , \n nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated \n sites ; - , not accumulated at microirradiated sites ) .", "( d ) immunostaining of microirradiated eyfp - cattle xlf \n ( 162287)-transfected cells with anti-h2ax antibody .", "the cells were fixed and stained \n with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf \n ( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .", "( e ) identity between \n the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino \n acids 288299 ) .", "( f ) the alignment of the primary sequence among homologous xlf \n proteins . for comparison , the basic ( red ) or non - basic residues ( black )", ", a signal of each eyfp - cattle xlf mutant was detected in the extracts of each \n transfectant by western blot analysis using the anti - gfp antibody . by confocal laser \n microscopy", ", we confirmed that eyfp - cattle xlf was localized in the nuclei of living \n interphase cells .", "we observed that n - terminal deletion mutant eyfp - cattle xlf ( 162299 ) \n localized predominantly in the nuclei , whereas eyfp - cattle xlf ( 162287 ) as well as eyfp was \n distributed throughout the cell excluding the nucleolus in mdbk cells ( figs . 2c , 4b and 4c ) .", "these results indicate that 12 c - terminal amino \n acids ( amino acids 288299 ) of cattle xlf are vital for the nuclear localization of xlf in \n cattle cells ( fig .", "which region \n of cattle xlf is essential for its accumulation at dsbs in vivo , we tested \n whether the xlf mutant proteins could be recruited to dsbs induced by microirradiation .", "we \n observed that a n - terminal deletion mutant eyfp - cattle xlf ( 162299 ) as well as the wild \n type eyfp - cattle xlf , accumulated at the dsbs sites in the living mdbk cells ( fig . 3c and \n data not shown ) . on the other hand , the mutant protein eyfp - cattle xlf ( 162287 ) failed to \n accumulate at the dsbs sites , which was detected by the dsb marker h2ax ( fig . 4c and 4d ) , indicating that deletion of the \n c - terminal end 12 amino acids abolished the recruitment of cattle xlf to dsbs .", "the cattle \n and human xlf proteins are 81.9% identical at the amino acid level , whereas the c - terminal \n domain ( amino acids 220299 ) of cattle xlf retains only 56.4% identity to human ( fig .", "interestingly , we confirmed that the basic \n amino acids in the ctr of xlf are evolutionarily highly conserved among humans and domestic \n animal species , e.g. , cattle , goats , horses and avian , but not in yeast ( fig .", "4f and data not shown ) , which strongly suggests \n the biological significance of the xlf ctr in domestic animals .", "the c - terminal region ( ctr ) is vital for the nuclear localization and recruitment of \n cattle xlf to dsbs in vivo . ( a )", "extracts from cattle ( mdbk ) cells \n transiently expressing the indicated cattle xlf deletions were prepared and subjected \n to western blotting using the anti - gfp or anti--actin antibody .", "( b , c ) identification \n of essential domain of cattle xlf for nuclear localization and for accumulation at \n dsbs .", "the localization \n and accumulation of the chimeric proteins at laser - induced dsbs were investigated via \n live cell imaging .", "the results are summarized on the right : cellular localization ( n , \n nucleus ; c , cytoplasm ) and formation of focus ( + , accumulated at microirradiated \n sites ; - , not accumulated at microirradiated sites ) .", "( d ) immunostaining of microirradiated eyfp - cattle xlf \n ( 162287)-transfected cells with anti-h2ax antibody .", "the cells were fixed and stained \n with the anti-h2ax antibody at 5 min postirradiation . left panel , eyfp - cattle xlf \n ( 162 - 287 ) ; center panel , h2ax image ; right panel , merged image .", "( e ) identity between \n the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf ( amino \n acids 288299 ) .", "( f ) the alignment of the primary sequence among homologous xlf \n proteins . for comparison , the basic ( red ) or non - basic residues ( black )"], ["to develop next - generation chemotherapeutics for cancer and other disease is important to \n clarify the molecular mechanisms of c - nhej .", "human xlf is the most recently identified core \n nhej factor , and it appears to play essential roles in c - nhej .", "expectedly , xlf - deficient cells derived from human patients and from \n knockout mice show ionizing radiation sensitivity [ 3 , \n 15 ] . in addition", ", sirna - mediated downregulation of \n xlf in human cell lines leads to radiosensitivity and impaired nhej . .", "homologues of the xlf gene were predicted in several eukaryotic \n organisms . on the other hand , the expression , \n function and regulation mechanism of xlf", "have not been elucidated in animal species other \n than mice and humans [ 1 , 3 , 11 , 16 , 21 ] .", "domestic cattle are \n important domestic animals as livestock and draft animals in not only japan , but also many \n countries .", "recently , cattle have been an ideal animal model for assessing chronic radiation \n exposure [ 5 , 20 ] . however , the molecular mechanism of c - nhej is still unknown in cattle cells . in \n this study", ", we examined the expression and subcellular localization of cattle xlf and its \n mutants in cattle cell line mdbk .", "we found that xlf as well as other core nhej protein ku70 \n is expressed in cattle cells , and the localization of cattle xlf changes dynamically during \n the cell cycle .", "in addition , xlf might play a vital role in the repair of dsb immediately \n after microirradiation of cattle cells .", "moreover , our data showed that the ctr of cattle xlf \n is vital for the nuclear localization of xlf and for the accumulation of xlf at dsbs \n in vivo .", "these findings suggest that the mechanisms regulating of the \n localization and recruitment to dsbs play a key role in the function of cattle xlf .", "cattle xlf ( nm_001075393.1 ) as well as goat xlf ( xp_005676612.1 ) and sheep xlf \n ( xp_004004987.1 ) is a 299-amino acid protein .", "the cattle and goat xlf genes are 94.6% \n identical at the amino acid level .", "in addition , the cattle and sheep xlf genes are 94.3% \n identical at the amino acid level . on the other hand ,", "human xlf is a 299-amino acid protein , which contains an n - terminal head \n domain ( amino acids 1141 ) , a coiled - coil central domain ( amino acids 142230 ) and a non \n structured c - terminal domain ( amino acids 231299 ) [ 1 , \n 2 , 17 ] . \n", "comparison with other eukaryotic homologues shows a high degree of sequence similarity \n within the 220 n - terminal amino acids , while the c - terminal domain ( amino acids 225299 ) is \n less conserved [ 1 , 2 , 17 ] . on the basis of experimental \n findings", ", there are some reports concerning the role of the c - terminal domain of xlf in \n humans , but not in animals including cattle .", "yano et al . reported that a \n 10-amino - acid deletion at the c - terminal end completely abolishes the ku - xlf interaction and \n the accumulation of xlf at dsbs . on the other \n hand , malivert et al .", "have reported that the c - terminal end ( amino acids \n 231299 ) of human xlf is dispensable for dna repair in vivo . in this study", ", our data showed that a 12-amino - acid \n deletion at the c - terminal end abolishes the accumulation of cattle xlf at dsbs . altogether", ", \n we conclude that the xlf ctr is important for the accumulation of xlf at dsbs in both human \n and cattle cells , although the role of the c - terminal region of xlf remains controversial in \n human cells .", "it was demonstrated that there is a general absence of conservation in the 75 c - terminal \n amino acids among humans and other species , although the extreme c terminus of xlf contains \n a small conserved basic cluster , which was proposed as a putative nls ( krkk ) [ 1 , 2 ] .", "our data \n revealed experimentally that the ctr of cattle xlf is critical for the nuclear localization \n of xlf and recruitment to dsbs , whereas the n - terminal domain is not essential .", "in addition , \n basic amino acids in the ctr of cattle xlf are evolutionarily conserved among ctr of \n domestic animals , which suggests the common biological significance of the xlf ctr in \n domestic animals .", "we consider that there is only one nls ( kvkrkklr ) in cattle xlf , and the \n nls is a classical monopartite nls having a single cluster of basic amino acid residues .", "we \n speculate that the c - terminus of 75 amino acids is important for a specific function in each \n species , whereas the xlf ctr is critical for the regulation of common functions in domestic \n animals .", ", we showed that xlf is expressed in cattle cells and the localization of \n cattle xlf changes dynamically during the cell cycle .", "in addition , our data showed that the \n localization and recruitment of cattle xlf to dsb sites at an early stage following \n irradiation are dependent on the ctr .", "these basic informations might be useful to develop \n the molecular - targeting therapeutic drug taking xlf as a target molecule for human and \n domestic animals .", "further studies to elucidate the mechanisms regulating cattle xlf at dsbs \n will lead to a better understanding of the physiological function of xlf not only in cattle \n cells , but also in cells of human and other domestic animals .", "inherited mutations of core \n c - nhej factors ( e.g. , dna - pkcs , dna ligase iv and xlf ) , have been discovered in humans \n . on the other hand , inherited", "mutations of the \n dna - pkcs , which cause scid , have been identified in not only humans , but also domestic \n animals , i.e. , mice , horses or dogs .", "therefore , \n further comparative studies might provide available information for the development of new \n clinical medicines and new chemoradiotherapies for humans and domestic animals including \n cattle ."]]}
{"article_id": "PMC3065795", "article_text": ["folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis .", "our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells .", "this modification can be used for successful targeting of cancer cells expressing the folate receptor .", "many conventional cancer chemotherapies are ineffective because of an inability to reach the tumor site in effective concentrations.1 there is little doubt that nanoparticles offer new opportunities in many fields.2 nanotechnology is expected to revolutionize medicine .", "nanostructures can play a major role in medicine , especially in cancer diagnosis and therapy.3 magnetic nanoparticles have been investigated for various biomedical applications , nanoparticles , and prospected in diagnostic research for magnetic resonance eg , fe3o4 imaging and application of nanotechnologies in medicine.4 magnetic nanoparticles could enhance therapeutic effects and reduce side effects of drugs when used in combination with conventional cancer treatment.5 the combination of fe3o4 magnetic nanoparticles with different chemotherapeutics may provide new strategies in the treatment of specific cancer cells.6 moreover , fe3o4 nanoparticles are the only magnetic nanomaterials approved for clinical use by the us food and drug administration , and the preparation method is relatively simple.7 we aimed to determine whether the anticancer effects of methacrylamido - folic acid ( ma - fol ) would have improved anticancer activity if incorporated into magnetic nanoparticles .", "we demonstrated that magnetic fe3o4 nanoparticles coupled with folic acid can inhibit tumor proliferation and induce apoptosis of cancer cells in a dose- and time - dependent manner .", "the folate receptor is significantly overexpressed on the surface of human cancer cells.8,9 folate receptor - mediated drug delivery is based on conjugation with folic acid , which is internalized by folate receptor - mediated endocytosis .", "folic acid has been immobilized on superparamagnetic particles,10 polymer nanoparticles,11 and incorporated into dendrimer - based therapeutic nanodevices12 for selective targeting of tumor cells .", "folate receptors exhibit limited expression on healthy cells , but are often present in large numbers on cancer cells.13 folic acid receptors are overexpressed by epithelial cancers in the ovary , mammary gland , colon , lung , prostate , nose , throat , and brain,14 so represent an important target for tumor - specific delivery of anticancer drugs .", "apoptosis , or programmed cell death , is an active process characterized by cytoplasmic shrinkage , chromatin condensation , nuclear fragmentation , and activation of caspases.13 in addition , phosphatidylserine is exposed on the external surface of the cell in the early phase of apoptosis , and this exposure precedes membrane damage and dna fragmentation.15 on the other hand , necrosis is passive , and is characterized by cell swelling , rupture of the plasma membrane , and cell lysis , with leakage of cytoplasmic components , such as lactate dehydrogenase.13 in the present study , folic acid was coupled on the surface of fe3o4 for selective binding to cancer cells and immobilized on the surfaces of magnetic nanoparticles , to disperse particles and improve their cell internalization and target cancer cells , respectively .", "further , the apoptotic effects of ma - fol - modified fe3o4 nanoparticles were determined in a 5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and in a nih/3t3 control cell line ( normal mouse embryonic fibroblasts ) by flow cytometry and transmission electron microscopy ( tem ) .", "nanoparticles are generally internalized into cells via fluid - phase endocytosis,16,17 receptor - mediated endocytosis , or phagocytosis .", "one strategy to realize efficient and specific cellular uptake of nanoparticles is to modify the nanoparticle surface with a ligand that is efficiently taken up by target cells via receptor - mediated endocytosis.18 the objective of this research was to assess the potential effects of fe3o4 magnetic nanoparticles modified with ma - fol on 5rp7 cancer cells and nih/3t3 cells .", "ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .", "a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .", "completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .", "reaction mixture was extracted with ethyl acetate ( 3  20 ml ) to remove 1h - benzotriazole .", "water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .", "h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 )  was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .", "c nmr ( 125 mhz , dmso - d6 )  : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .", "superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .", "the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .", "these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) .", "5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .", "cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .", ", the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .", "the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .", "cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem ,", "ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .", "the cells were cultured and then reseeded with the nano - particle - dispersed culture media . after 24 and 48 hours of incubation at 37c", ", the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,", "the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density .", "annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .", "it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells . a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .", "cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1  10 cells / ml , after which 100 l of solution ( 1  10 cells ) was transferred to a 5 ml culture tube .", "annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria .", "uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .", "cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,", "the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .", "they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm .", "ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .", "a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .", "completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .", "reaction mixture was extracted with ethyl acetate ( 3  20 ml ) to remove 1h - benzotriazole .", "water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .", "h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 )  was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .", "c nmr ( 125 mhz , dmso - d6 )  : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .", "superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .", "the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .", "these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) .", "5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .", "cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .", ", the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .", "the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .", "cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem", ", ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .", "the cells were cultured and then reseeded with the nano - particle - dispersed culture media .", "after 24 and 48 hours of incubation at 37c , the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,", "the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density .", "annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .", "it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells", ". a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .", "cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1  10 cells / ml , after which 100 l of solution ( 1  10 cells ) was transferred to a 5 ml culture tube .", "annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria .", "uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .", "cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,", "the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .", "they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm .", "a logical method to promote internalization of nanoparticles is to modify their surface with a ligand such as folic acid which can be efficiently taken up by cells through receptor - mediated endocytosis.23 folic acid binds to the folate receptors at cell surfaces with very high affinity and is internalized by receptor - mediated endocytosis.24,26 more importantly , folic acid is stable , poorly immunogenic , and has the ability to target cancer cells preferentially because the folate receptor is frequently overexpressed on the surface of cancer cells .", "flow cytometry is a process whereby physical or biochemical parameters of single biological cells or particles are measured as the cells move through a fluidic channel.27 the annexin v binding assay provides a very specific , rapid , and reliable technique to detect apoptosis by flow cytometry or by fluorescence microscopy.28,29 fitc is a very useful fluorescent moiety that can be used to label essentially any protein .", "fitc - conjugated annexin v is used to detect apoptotic cells in a diverse range of cell types and in response to many different proapoptotic stimuli.28,29 iron nanoparticles were successfully modified with ma - fol as described in the methods section .", "ma - fol was used to target preferentially cancer cells with folate receptors expressed on their surfaces , and to facilitate the nanoparticles to transit across the cell membrane .", "annexin v - fitc allows detection of cell surface changes that occur early during apoptosis , because annexin v binds to phosphatidylserine which becomes exposed on the outer surface of the plasma membranes by flipping from the inner side of the membrane.22,30,31 exposure of phosphatidylserine during apoptosis precedes nuclear changes .", "this change in membrane surface is important for macrophages to recognize cells undergoing apoptosis.30,32 annexin v , an anticoagulant protein , binds to phosphatidylserine with high affinity .", "thus , by staining cells with annexin v - fitc and additionally with dna - specific fluorochrome , eg , propidium iodide , it is possible to identify live cells , early apoptotic cells , as well as late apoptotic and necrotic cells , by flow cytometry.22,33 subsequently , annexin v - fitc binds to cells expressing phosphatidylserine , an early marker of apoptosis on the cell surface .", "target cells are gated upon as propidium iodide - negative and quantified with respect to their annexin v positivity .", "the shift from annexin v to annexin v is a discrete event , such that all target cells fall within discernible populations with respect to annexin v. dead or dying cells are then stained with annexin v - fitc.34 annexin v has been used to detect apoptotic cells in a wide variety of cell types .", "externalization of phosphatidylserine has been demonstrated in plasma membrane permeability changes , as measured by propidium iodide uptake and annexin v binding , and precedes the morphological features of apoptosis as assessed by flow cytometric analysis of cell shrinkage.29,34 the sensitivity and early kinetics of annexin v binding make it an ideal marker for cell death in a flow cytometric assay.34 analysis of annexin v / propidium iodide - stained cells by flow cytometry allows quantitation of the fraction of cells that are annexin v - negative and propidium iodide - negative ( double negative ) , annexin v - positive and propidium iodide - negative ( single positive ) , or annexin v - positive and propidium iodide - positive ( double positive).28 a number of detectors are aimed at the point where the stream passes through the light beam , ie , one in line with the light beam ( forward scatter ) and several perpendicular to it ( side scatter ) and one or more fluorescent detectors . forward", "scatter correlates with the cell volume , and side scatter depends on the inner complexity of the particle ( ie , shape of the nucleus , amount and type of cytoplasmic granules , or membrane roughness).35 data analysis was performed with diva software ( bd facs diva software 6.0 ; bd corporation , bedford , ma ) .", "apoptosis was quantitatively confirmed by analyzing the percentage of early apoptotic cells using annexin v - fitc / propidium iodide double staining .", "a marker of early apoptosis , measured by annexin - v , is phosphatidylserine , which is released as a result of redistribution of the plasma membrane of the cells.22 three main populations of cells were distributed in dot - plots for viable cells q3 ( annexin v - negative / propidium iodide - negative ) , early apoptotic cells q4 ( annexin v - positive / propidium iodide - negative ) , and late apoptotic and necrotic cells q2", "the cytotoxicity of folic acid - modified nanoparticles was determined by exposing normal cells and cancer cells to various concentrations in dulbecco s modified eagle s medium for 24 and 48 hours .", "the results and respective percentages of cells in apoptotic regions for both nih/3t3 and 5rp7 cells are given in figures 1 and 2 .", "the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol - modified nanoparticles was 4.5 g / ml or below in cancer cells .", "as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .", "these results confirm that the cells are saturated with ma - fol - modified nanoparticles at a concentration of 4.5 g / ml . up to this concentration", "figure 2 shows cell death due to the ma - fol - modified magnetic nanoparticles at 4.5 g / ml for 24 hours in cancer cells .", "when cells were exposed to a nanoparticle concentration of 4.5 g / ml for 24 hours , cell survival decreased to 83% . when incubation time was extended to 48 hours", "as shown in figure 2 , folic acid - modified nanoparticles caused worse damage to cancer cells than to normal cells .", "the population of annexin v - positive live cells increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours ( figure 2 ) .", "when cancer cells were incubated with modified magnetic ma - fol nanoparticles for 48 hours ( 9 g / ml ) , the population of annexin v - positive live cells was found to be 7.3% ( figures 2a and 2b ) .", "cells in the late stages of apoptosis are located in the bottom right quadrant of the dot - plot as double positive annexin v - fitc- and propidium iodide - binding cells because , at this stage , the cell membranes are damaged and propidium iodide has penetrated into the cell . in the later stages of apoptosis , propidium iodide enters the cell , leading to a double positive population.28 cells undergoing necrotic cell death also stain as double positive for annexin v and propidium iodide , as discussed earlier .", "flow cytometry by this double staining method enables clear detection of three populations of cells ( viable , apoptotic , and necrotic ) .", "thus , the early apoptotic cells bind only to annexin - v fitc , and late apoptotic cells to both annexin v - fitc and propidium iodide , and viable cells do not take up any of the dye . the annexin v - fitc", "/ propidium iodide population was considered to reflect normal healthy cells , whereas annexin v - fitc - positive / propidium iodide - negative cells were taken to show early apoptosis .", "annexin v - fitc -positive / propidium iodide - negative cells were in late apoptosis or necrosis.35 our flow cytometric analysis has shown that48 hours of treatment with ma - fol - modified magnetic nanoparticles caused apoptosis in a 5rp7 cell line in a concentration - dependent manner .", "ma - fol - modified magnetic nanoparticles at a dose of 4.5 g / ml showed 12.9% cells in the apoptotic zone in the case of nih/3t3 cells , and the same concentration produced apoptosis in 4.7% of cells in dot - plot analysis .", "late apoptosis and necrosis increased steadily following the increase at a concentration of 4.5 g / ml . flow cytometric determination of cell cycle phase distribution has further confirmed that folic acid - modified magnetic nanoparticles cause cell cycle deformation in a cancer cell line .", "the results of this study confirm that cancer cells have more folate receptors , and that a 4.5 g / ml concentration of folic acid - modified magnetic nanoparticles causes apoptosis in 5rp7 cells .", "the uptake of folic acid - modified nanoparticles by cancer cells was also much higher than that of normal cells .", "this indicates that folic acid modification not only facilitates the nanoparticles to target specific cells , but also increases the yield of cell internalization .", "the interaction between folic acid and folate receptors expressed on the surface of cancer cells might have contributed to the improvement of nanoparticle uptake , based on receptor - mediated endocytosis.37 the schemes for the modification of nanoparticles display tem images ( figure 3 ) of ma - fol - modified with fe3o4 nanoparticles for this purpose , firstly , magnetic nanoparticles were imaged .", "figure 4 shows the tem images of nih/3t3 cells not treated with ma - fol - modified magnetic nanoparticles .", "figure 5 shows the uptake of folic acid - modified nanoparticles into normal cells at 24 hours and 48 hours .", "the viability of normal cells after each incubation time with the nanoparticles was close to that of control cells , and was in the 95%96% range in flow cytometric analysis .", "the uptake of magnetic nanoparticles by cancerous rat 5rp7 fibroblasts is shown in figure 7 .", "after 48 hours of culture in media containing folic acid - modified nanoparticles , the morphology and viability of normal fibroblast cells containing the modified nanoparticles were close to that of the control cells , suggesting biocompatibility of the nanoparticles .", "the tem and flow cytometry results showed that folic acid - modified nanoparticles were internalized into both normal cells and cancer cells .", "the immobilization of folic acid on the nanoparticles was demonstrated by increasing the amount of nanoparticle uptake into cancer cells in comparison with normal cells .", "this suggests that the modification of magnetic nanoparticles with ma - fol could be used to resist nonspecific uptake and thus avoid their recognition by macrophage cells , and simultaneously facilitate nanoparticle uptake to specific cancer cells for cancer therapy . in flow cytometry results , the population of annexin v - positive live cells in the cancer cell line increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours at a concentration of 4.5 g / ml .", "the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol modified nanoparticles is 4.5 g / ml or below in cancer cells .", "as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .", "these results confirm that cells saturated with folic acid - modified nanoparticles are detected in the 4.5 g / ml concentration .", "up to this concentration , magnetic nanoparticles were aggregated and had difficulty penetrating the cells . in tem analysis ,", "the concentration of 4.5 g / ml was used to compare the cancer cells and normal cells at 24 and 48 hours . in normal cells , organelles and cell structures", "were not affected by the presence of ma - fol - modified nanoparticles inside the cells ( figure 5 ) . however , cancer cells treated with the 4.5 g / ml concentration of ma - fol - modified nanoparticles underwent apoptosis .", "a representative picture of intracellular nanoconjugate uptake by nih/3t3 cells during a 24-hour and 48-hour treatment is shown in figures 5 and 6 .", "the amount of intracellular uptake of the nanoparticles can be seen in normal cells , whereas in the 5rp7 cell line the amount of the nanoconjugate was increased and cell modifications were damaged , figure 7 clearly illustrates the internalization of ma - fol - modified nanoparticles and their localization near the nucleus and a great number of apoptotic particles .", "this picture shows clearly that nanoconjugate uptake was maximum , with 5rp7 cells having maximum receptor expression , and hence the most intense color of the pellet , whereas the uptake gradually decreased with reduction in folate receptor expression and , accordingly , the intensity of the color of the pellet in the case of nih/3t3 .", "the picture confirms our hypothesis about the targeting efficacy of nanoconjugates containing folic acid , ie , that a positive correlation exists between maximum folate receptor expression and maximum uptake of nanoconjugates .", "the 5rp7 cells with 90% receptor expression showed maximum uptake , and the nih/3t3 cells showed the least .", "this is further substantiated by tem and flow cytometry analysis , confirming that folic acid - modified magnetic nano - particles can be used as a targeting agent .", "ma - fol - modified magnetic nanoparticles represent a potential novel delivery system for compounds with anticancer activity because the folate receptor is frequently overexpressed in cancer cells .", "moreover , it may increase our ability to target drugs to tumor cells , protect the drug from in vivo degradation , and reduce drug toxicity .", "this modification containing folic acid can be used for successful targeting of tumor cells expressing the folate receptor .", "future studies will focus on determining the stability and targeting efficacy of this modification in vivo .", "this study has important implications in cancer cell imaging , tumor ablation , and drug delivery because of its targeting efficacy .", "furthermore , it needs to be investigated whether nanoparticles could cause long - term changes in systemic activity .", "however , the effect and usage of such combinations need to be investigated further in in vivo experiments ."], "abstract_text": ["<S> background : magnetic nanoparticles show great promise for use as tools in a wide variety of biomedical applications . </S>", "<S> the purpose of this study was to investigate the potential effects of methacrylamido - folic acid ( ma - fol)-modified magnetic nanoparticles on 5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( normal mouse embryonic fibroblasts).methods : the cytotoxicity and viability of 5rp7 and nih/3t3 cells were detected . </S>", "<S> the percentage of cells undergoing apoptosis was analyzed by flow cytometry using annexin v - fluorescein isothiocyanate staining . </S>", "<S> nanoparticle internalization into 5rp7 and nih/3t3 cells was visualized by transmission electron microscopy.conclusion:in this study , folic acid coupled to the surface of iron oxide for selective binding to cancer cells and immobilized the surfaces of magnetic nanoparticles </S>", "<S> . this complex improves cell internalization and targeting of cancer cells . </S>", "<S> we detected increased apoptosis using flow cytometry and transmission electron microscopy.results:folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis . </S>", "<S> our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells . </S>", "<S> this modification can be used for successful targeting of cancer cells expressing the folate receptor . </S>"], "labels": null, "section_names": ["Results:", "Introduction", "Materials and methods", "Synthesis and characterization of magnetic nanoparticles with Ma-Fol", "Cell culture", "Flow cytometry", "Transmission electron microscopic analysis", "Results and discussion", "Conclusion"], "sections": [["folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis .", "our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells .", "this modification can be used for successful targeting of cancer cells expressing the folate receptor ."], ["many conventional cancer chemotherapies are ineffective because of an inability to reach the tumor site in effective concentrations.1 there is little doubt that nanoparticles offer new opportunities in many fields.2 nanotechnology is expected to revolutionize medicine .", "nanostructures can play a major role in medicine , especially in cancer diagnosis and therapy.3 magnetic nanoparticles have been investigated for various biomedical applications , nanoparticles , and prospected in diagnostic research for magnetic resonance eg , fe3o4 imaging and application of nanotechnologies in medicine.4 magnetic nanoparticles could enhance therapeutic effects and reduce side effects of drugs when used in combination with conventional cancer treatment.5 the combination of fe3o4 magnetic nanoparticles with different chemotherapeutics may provide new strategies in the treatment of specific cancer cells.6 moreover , fe3o4 nanoparticles are the only magnetic nanomaterials approved for clinical use by the us food and drug administration , and the preparation method is relatively simple.7 we aimed to determine whether the anticancer effects of methacrylamido - folic acid ( ma - fol ) would have improved anticancer activity if incorporated into magnetic nanoparticles .", "we demonstrated that magnetic fe3o4 nanoparticles coupled with folic acid can inhibit tumor proliferation and induce apoptosis of cancer cells in a dose- and time - dependent manner .", "the folate receptor is significantly overexpressed on the surface of human cancer cells.8,9 folate receptor - mediated drug delivery is based on conjugation with folic acid , which is internalized by folate receptor - mediated endocytosis .", "folic acid has been immobilized on superparamagnetic particles,10 polymer nanoparticles,11 and incorporated into dendrimer - based therapeutic nanodevices12 for selective targeting of tumor cells .", "folate receptors exhibit limited expression on healthy cells , but are often present in large numbers on cancer cells.13 folic acid receptors are overexpressed by epithelial cancers in the ovary , mammary gland , colon , lung , prostate , nose , throat , and brain,14 so represent an important target for tumor - specific delivery of anticancer drugs .", "apoptosis , or programmed cell death , is an active process characterized by cytoplasmic shrinkage , chromatin condensation , nuclear fragmentation , and activation of caspases.13 in addition , phosphatidylserine is exposed on the external surface of the cell in the early phase of apoptosis , and this exposure precedes membrane damage and dna fragmentation.15 on the other hand , necrosis is passive , and is characterized by cell swelling , rupture of the plasma membrane , and cell lysis , with leakage of cytoplasmic components , such as lactate dehydrogenase.13 in the present study , folic acid was coupled on the surface of fe3o4 for selective binding to cancer cells and immobilized on the surfaces of magnetic nanoparticles , to disperse particles and improve their cell internalization and target cancer cells , respectively .", "further , the apoptotic effects of ma - fol - modified fe3o4 nanoparticles were determined in a 5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and in a nih/3t3 control cell line ( normal mouse embryonic fibroblasts ) by flow cytometry and transmission electron microscopy ( tem ) .", "nanoparticles are generally internalized into cells via fluid - phase endocytosis,16,17 receptor - mediated endocytosis , or phagocytosis .", "one strategy to realize efficient and specific cellular uptake of nanoparticles is to modify the nanoparticle surface with a ligand that is efficiently taken up by target cells via receptor - mediated endocytosis.18 the objective of this research was to assess the potential effects of fe3o4 magnetic nanoparticles modified with ma - fol on 5rp7 cancer cells and nih/3t3 cells ."], ["ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .", "a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .", "completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .", "reaction mixture was extracted with ethyl acetate ( 3  20 ml ) to remove 1h - benzotriazole .", "water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .", "h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 )  was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .", "c nmr ( 125 mhz , dmso - d6 )  : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .", "superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .", "the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .", "these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) .", "5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .", "cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .", ", the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .", "the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .", "cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem ,", "ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .", "the cells were cultured and then reseeded with the nano - particle - dispersed culture media . after 24 and 48 hours of incubation at 37c", ", the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,", "the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density .", "annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .", "it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells . a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .", "cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1  10 cells / ml , after which 100 l of solution ( 1  10 cells ) was transferred to a 5 ml culture tube .", "annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria .", "uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .", "cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,", "the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .", "they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm ."], ["ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .", "a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .", "completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .", "reaction mixture was extracted with ethyl acetate ( 3  20 ml ) to remove 1h - benzotriazole .", "water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .", "h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 )  was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .", "c nmr ( 125 mhz , dmso - d6 )  : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .", "superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .", "the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .", "these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) ."], ["5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .", "cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .", ", the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .", "the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .", "cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem", ", ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .", "the cells were cultured and then reseeded with the nano - particle - dispersed culture media .", "after 24 and 48 hours of incubation at 37c , the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,", "the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density ."], ["annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .", "it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells", ". a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .", "cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1  10 cells / ml , after which 100 l of solution ( 1  10 cells ) was transferred to a 5 ml culture tube .", "annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria ."], ["uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .", "cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,", "the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .", "they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm ."], ["a logical method to promote internalization of nanoparticles is to modify their surface with a ligand such as folic acid which can be efficiently taken up by cells through receptor - mediated endocytosis.23 folic acid binds to the folate receptors at cell surfaces with very high affinity and is internalized by receptor - mediated endocytosis.24,26 more importantly , folic acid is stable , poorly immunogenic , and has the ability to target cancer cells preferentially because the folate receptor is frequently overexpressed on the surface of cancer cells .", "flow cytometry is a process whereby physical or biochemical parameters of single biological cells or particles are measured as the cells move through a fluidic channel.27 the annexin v binding assay provides a very specific , rapid , and reliable technique to detect apoptosis by flow cytometry or by fluorescence microscopy.28,29 fitc is a very useful fluorescent moiety that can be used to label essentially any protein .", "fitc - conjugated annexin v is used to detect apoptotic cells in a diverse range of cell types and in response to many different proapoptotic stimuli.28,29 iron nanoparticles were successfully modified with ma - fol as described in the methods section .", "ma - fol was used to target preferentially cancer cells with folate receptors expressed on their surfaces , and to facilitate the nanoparticles to transit across the cell membrane .", "annexin v - fitc allows detection of cell surface changes that occur early during apoptosis , because annexin v binds to phosphatidylserine which becomes exposed on the outer surface of the plasma membranes by flipping from the inner side of the membrane.22,30,31 exposure of phosphatidylserine during apoptosis precedes nuclear changes .", "this change in membrane surface is important for macrophages to recognize cells undergoing apoptosis.30,32 annexin v , an anticoagulant protein , binds to phosphatidylserine with high affinity .", "thus , by staining cells with annexin v - fitc and additionally with dna - specific fluorochrome , eg , propidium iodide , it is possible to identify live cells , early apoptotic cells , as well as late apoptotic and necrotic cells , by flow cytometry.22,33 subsequently , annexin v - fitc binds to cells expressing phosphatidylserine , an early marker of apoptosis on the cell surface .", "target cells are gated upon as propidium iodide - negative and quantified with respect to their annexin v positivity .", "the shift from annexin v to annexin v is a discrete event , such that all target cells fall within discernible populations with respect to annexin v. dead or dying cells are then stained with annexin v - fitc.34 annexin v has been used to detect apoptotic cells in a wide variety of cell types .", "externalization of phosphatidylserine has been demonstrated in plasma membrane permeability changes , as measured by propidium iodide uptake and annexin v binding , and precedes the morphological features of apoptosis as assessed by flow cytometric analysis of cell shrinkage.29,34 the sensitivity and early kinetics of annexin v binding make it an ideal marker for cell death in a flow cytometric assay.34 analysis of annexin v / propidium iodide - stained cells by flow cytometry allows quantitation of the fraction of cells that are annexin v - negative and propidium iodide - negative ( double negative ) , annexin v - positive and propidium iodide - negative ( single positive ) , or annexin v - positive and propidium iodide - positive ( double positive).28 a number of detectors are aimed at the point where the stream passes through the light beam , ie , one in line with the light beam ( forward scatter ) and several perpendicular to it ( side scatter ) and one or more fluorescent detectors . forward", "scatter correlates with the cell volume , and side scatter depends on the inner complexity of the particle ( ie , shape of the nucleus , amount and type of cytoplasmic granules , or membrane roughness).35 data analysis was performed with diva software ( bd facs diva software 6.0 ; bd corporation , bedford , ma ) .", "apoptosis was quantitatively confirmed by analyzing the percentage of early apoptotic cells using annexin v - fitc / propidium iodide double staining .", "a marker of early apoptosis , measured by annexin - v , is phosphatidylserine , which is released as a result of redistribution of the plasma membrane of the cells.22 three main populations of cells were distributed in dot - plots for viable cells q3 ( annexin v - negative / propidium iodide - negative ) , early apoptotic cells q4 ( annexin v - positive / propidium iodide - negative ) , and late apoptotic and necrotic cells q2", "the cytotoxicity of folic acid - modified nanoparticles was determined by exposing normal cells and cancer cells to various concentrations in dulbecco s modified eagle s medium for 24 and 48 hours .", "the results and respective percentages of cells in apoptotic regions for both nih/3t3 and 5rp7 cells are given in figures 1 and 2 .", "the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol - modified nanoparticles was 4.5 g / ml or below in cancer cells .", "as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .", "these results confirm that the cells are saturated with ma - fol - modified nanoparticles at a concentration of 4.5 g / ml . up to this concentration", "figure 2 shows cell death due to the ma - fol - modified magnetic nanoparticles at 4.5 g / ml for 24 hours in cancer cells .", "when cells were exposed to a nanoparticle concentration of 4.5 g / ml for 24 hours , cell survival decreased to 83% . when incubation time was extended to 48 hours", "as shown in figure 2 , folic acid - modified nanoparticles caused worse damage to cancer cells than to normal cells .", "the population of annexin v - positive live cells increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours ( figure 2 ) .", "when cancer cells were incubated with modified magnetic ma - fol nanoparticles for 48 hours ( 9 g / ml ) , the population of annexin v - positive live cells was found to be 7.3% ( figures 2a and 2b ) .", "cells in the late stages of apoptosis are located in the bottom right quadrant of the dot - plot as double positive annexin v - fitc- and propidium iodide - binding cells because , at this stage , the cell membranes are damaged and propidium iodide has penetrated into the cell . in the later stages of apoptosis , propidium iodide enters the cell , leading to a double positive population.28 cells undergoing necrotic cell death also stain as double positive for annexin v and propidium iodide , as discussed earlier .", "flow cytometry by this double staining method enables clear detection of three populations of cells ( viable , apoptotic , and necrotic ) .", "thus , the early apoptotic cells bind only to annexin - v fitc , and late apoptotic cells to both annexin v - fitc and propidium iodide , and viable cells do not take up any of the dye . the annexin v - fitc", "/ propidium iodide population was considered to reflect normal healthy cells , whereas annexin v - fitc - positive / propidium iodide - negative cells were taken to show early apoptosis .", "annexin v - fitc -positive / propidium iodide - negative cells were in late apoptosis or necrosis.35 our flow cytometric analysis has shown that48 hours of treatment with ma - fol - modified magnetic nanoparticles caused apoptosis in a 5rp7 cell line in a concentration - dependent manner .", "ma - fol - modified magnetic nanoparticles at a dose of 4.5 g / ml showed 12.9% cells in the apoptotic zone in the case of nih/3t3 cells , and the same concentration produced apoptosis in 4.7% of cells in dot - plot analysis .", "late apoptosis and necrosis increased steadily following the increase at a concentration of 4.5 g / ml . flow cytometric determination of cell cycle phase distribution has further confirmed that folic acid - modified magnetic nanoparticles cause cell cycle deformation in a cancer cell line .", "the results of this study confirm that cancer cells have more folate receptors , and that a 4.5 g / ml concentration of folic acid - modified magnetic nanoparticles causes apoptosis in 5rp7 cells .", "the uptake of folic acid - modified nanoparticles by cancer cells was also much higher than that of normal cells .", "this indicates that folic acid modification not only facilitates the nanoparticles to target specific cells , but also increases the yield of cell internalization .", "the interaction between folic acid and folate receptors expressed on the surface of cancer cells might have contributed to the improvement of nanoparticle uptake , based on receptor - mediated endocytosis.37 the schemes for the modification of nanoparticles display tem images ( figure 3 ) of ma - fol - modified with fe3o4 nanoparticles for this purpose , firstly , magnetic nanoparticles were imaged .", "figure 4 shows the tem images of nih/3t3 cells not treated with ma - fol - modified magnetic nanoparticles .", "figure 5 shows the uptake of folic acid - modified nanoparticles into normal cells at 24 hours and 48 hours .", "the viability of normal cells after each incubation time with the nanoparticles was close to that of control cells , and was in the 95%96% range in flow cytometric analysis .", "the uptake of magnetic nanoparticles by cancerous rat 5rp7 fibroblasts is shown in figure 7 .", "after 48 hours of culture in media containing folic acid - modified nanoparticles , the morphology and viability of normal fibroblast cells containing the modified nanoparticles were close to that of the control cells , suggesting biocompatibility of the nanoparticles .", "the tem and flow cytometry results showed that folic acid - modified nanoparticles were internalized into both normal cells and cancer cells .", "the immobilization of folic acid on the nanoparticles was demonstrated by increasing the amount of nanoparticle uptake into cancer cells in comparison with normal cells .", "this suggests that the modification of magnetic nanoparticles with ma - fol could be used to resist nonspecific uptake and thus avoid their recognition by macrophage cells , and simultaneously facilitate nanoparticle uptake to specific cancer cells for cancer therapy . in flow cytometry results , the population of annexin v - positive live cells in the cancer cell line increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours at a concentration of 4.5 g / ml .", "the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol modified nanoparticles is 4.5 g / ml or below in cancer cells .", "as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .", "these results confirm that cells saturated with folic acid - modified nanoparticles are detected in the 4.5 g / ml concentration .", "up to this concentration , magnetic nanoparticles were aggregated and had difficulty penetrating the cells . in tem analysis ,", "the concentration of 4.5 g / ml was used to compare the cancer cells and normal cells at 24 and 48 hours . in normal cells , organelles and cell structures", "were not affected by the presence of ma - fol - modified nanoparticles inside the cells ( figure 5 ) . however , cancer cells treated with the 4.5 g / ml concentration of ma - fol - modified nanoparticles underwent apoptosis .", "a representative picture of intracellular nanoconjugate uptake by nih/3t3 cells during a 24-hour and 48-hour treatment is shown in figures 5 and 6 .", "the amount of intracellular uptake of the nanoparticles can be seen in normal cells , whereas in the 5rp7 cell line the amount of the nanoconjugate was increased and cell modifications were damaged , figure 7 clearly illustrates the internalization of ma - fol - modified nanoparticles and their localization near the nucleus and a great number of apoptotic particles .", "this picture shows clearly that nanoconjugate uptake was maximum , with 5rp7 cells having maximum receptor expression , and hence the most intense color of the pellet , whereas the uptake gradually decreased with reduction in folate receptor expression and , accordingly , the intensity of the color of the pellet in the case of nih/3t3 .", "the picture confirms our hypothesis about the targeting efficacy of nanoconjugates containing folic acid , ie , that a positive correlation exists between maximum folate receptor expression and maximum uptake of nanoconjugates .", "the 5rp7 cells with 90% receptor expression showed maximum uptake , and the nih/3t3 cells showed the least .", "this is further substantiated by tem and flow cytometry analysis , confirming that folic acid - modified magnetic nano - particles can be used as a targeting agent .", "ma - fol - modified magnetic nanoparticles represent a potential novel delivery system for compounds with anticancer activity because the folate receptor is frequently overexpressed in cancer cells .", "moreover , it may increase our ability to target drugs to tumor cells , protect the drug from in vivo degradation , and reduce drug toxicity ."], ["this modification containing folic acid can be used for successful targeting of tumor cells expressing the folate receptor .", "future studies will focus on determining the stability and targeting efficacy of this modification in vivo .", "this study has important implications in cancer cell imaging , tumor ablation , and drug delivery because of its targeting efficacy .", "furthermore , it needs to be investigated whether nanoparticles could cause long - term changes in systemic activity .", "however , the effect and usage of such combinations need to be investigated further in in vivo experiments ."]]}
{"article_id": "PMC4355506", "article_text": ["this is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax .", "data on age , weight , height , and smoking habits are collected routinely when students join the institute , and then the sport in which the student is injured in the low back is registered in the institute health service files .", "only students injured in the first or second year were taken into account , because the following years involve only athletics and gymnastics .", "body mass index ( bmi ; kg / m ) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . in this study", "we used the standard bmi with categories of underweight ( bmi of < 18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obesity ( bmi of 30 ) .", "smoking was categorized as non - smoker , < 20 cigarettes per day , and 20 cigarettes per day .", "bmi , smoking habits , and history of any pain or injuries were taken for all the students in the beginning of the first grade .", "on the basis of the collected data , participants were dichotomized into those with or without lbp , while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . in 1 week , all students practice for a total of 16.5 h , spending 1.5 h in each of the following sports : soccer , handball , basketball , volleyball , judo , weightlifting , swimming , athletics , and gymnastics .", "lbp was defined as pain or discomfort in the low - back region , from the lower rib curvature to the lower part of the seat region .", "the question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past .", "furthermore , every registered injury causing lbp was recorded with its severity , but the most important detail for us was the sport in which the student was injured .", "students who were injured more than one time were taken into account as subjects with lbp only for their first injury .", "the scientific council of the university of sfax in tunisia gave written permission for the study protocol .", "statistical analyses were done using the spss statistical package , version 11.5 ( spss , inc . ,", "chicago , il , usa ) . the chi - square test was used to test associations between lbp and other categorical variables ( sex , obesity , and smoking habit ) , and to compare the prevalence of lbp between males and female . statistical significance", "this is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax .", "data on age , weight , height , and smoking habits are collected routinely when students join the institute , and then the sport in which the student is injured in the low back is registered in the institute health service files .", "only students injured in the first or second year were taken into account , because the following years involve only athletics and gymnastics .", "body mass index ( bmi ; kg / m ) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . in this study", "we used the standard bmi with categories of underweight ( bmi of < 18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obesity ( bmi of 30 ) .", "smoking was categorized as non - smoker , < 20 cigarettes per day , and 20 cigarettes per day .", "bmi , smoking habits , and history of any pain or injuries were taken for all the students in the beginning of the first grade .", "on the basis of the collected data , participants were dichotomized into those with or without lbp , while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . in 1 week , all students practice for a total of 16.5 h , spending 1.5 h in each of the following sports : soccer , handball , basketball , volleyball , judo , weightlifting , swimming , athletics , and gymnastics .", "lbp was defined as pain or discomfort in the low - back region , from the lower rib curvature to the lower part of the seat region .", "the question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past .", "furthermore , every registered injury causing lbp was recorded with its severity , but the most important detail for us was the sport in which the student was injured .", "students who were injured more than one time were taken into account as subjects with lbp only for their first injury .", "the scientific council of the university of sfax in tunisia gave written permission for the study protocol .", "statistical analyses were done using the spss statistical package , version 11.5 ( spss , inc . ,", "chicago , il , usa ) . the chi - square test was used to test associations between lbp and other categorical variables ( sex , obesity , and smoking habit ) , and to compare the prevalence of lbp between males and female . statistical significance", "table 2 shows that the prevalence of lbp was significantly higher ( p<0.001 ) among females ( 17.6% ) than among males ( 12.5% ) .", "lbp occurred in 17.1% of those classified by bmi as lean , 14.8% of those in the normal range , 14.3% among those who were overweight , and 12.7% of those who were obese .", "in addition , 13.9% of students smoking < 20 cigarettes and 13.3% of students smoking 20 cigarettes had lbp .", "prevalence of lower back pain ( lbp ) by gender , obesity and smoking lbp : low back pain ; standard body mass index ( bmi ) categories : underweight ( bmi of<18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obese ( bmi of 30 ) ; smoking : cigarettes per day ; no smoking : not currently smoking . in the analysis of risk factors for lbp in sports training , we found that in both sexes combined , lbp was positively associated with fatigue ( caused by the long time spent in training ) , gymnastics , judo , handball , and volleyball .", "the increased prevalence of lbp is less in basketball and athletics and even lower in soccer , weightlifting , and swimming ( fig .", "percent prevalence of lower back pain related to fatigue and type of sports in males and females combined . \n", "figure 2 shows that there is no significant difference ( p>0.05 ) in the prevalence of lbp among males and females separately if they are compared in each practiced sport at the institute .", "however , if we analyze the prevalence of lbp in terms of sex in each sport , we find that the prevalence of lbp was significantly higher among males than females in handball ( p<0.05 ) and significantly lower in athletics ( p<0.05 )", ". percent prevalence of lower back pain related to fatigue and type of sports in males and females .", "its incidence and prevalence are so high that it should be studied as an epidemic and social disorder ( 35 ) .", "the first salient feature that emerges from this study is that lbp is widespread among students in high schools for athletes ( 14.8% ) .", "the students were injured mainly in gymnastics , judo , handball , and volleyball , in addition to lbp caused by fatigue due to the long time spent in training .", "these findings are generally in accordance with the literature . in a study focused on identifying the types of physical activity associated with increased occurrence of lbp in schoolchildren , skoffer and foldspang ( 6 ) noted that lbp was increased by the number of hours jogging , playing handball , and doing gymnastics .", "in addition , sato et al . ( 33 ) found that the odds ratio for the risk of experiencing lbp was significantly higher in most sports items compared with the no - sports group .", "the most frequently implicated sports were volleyball , athletics , judo , gymnastics , golf , and rugby , where the odds ratio exceeded 2 .", "kirialanis et al . reported that injuries among gymnasts are anatomically widely distributed ( 36 ) .", "as in our study , much research ( 6 , 33 , 37 ) noted that gymnastics was associated with an increased prevalence of lbp .", "judo is a grappling style martial art with emphasis on dynamic throws and submissions , which can lead to substantial injuries due to the high velocity of the maneuvers ( 38 ) .", "we registered a high prevalence of lbp in judo , as noted in the study of sato et al .", "handball , which also includes repeated sudden and often rather violent movements , has been shown to be associated with lbp in our population and in other studies ( 6 , 33 ) .", "we observed a high prevalence of lbp associated with the fatigue caused by the long time spent in physical activity ( 16.5 h per week ) .", ". however , this point of view is attractive in terms of prevention as physical activity is amenable to behavioral intervention , and physical training programs at school and leisure activities may be adjusted to aim at strengthening the back ( 6 ) .", "moreover , both physical inactivity and intensive sports activity have been associated with lbp in some studies ( 28 , 32 , 3941 ) .", "our results show that a greater amount of physical activity increased the risk of lbp mainly in gymnastics , judo , handball , and volleyball .", "the time spent in athletics and basketball was associated with lbp to a lesser extent .", "moreover , it appears that soccer , weightlifting , and swimming are correlated with relatively low prevalence of lbp .", "skoffer and foldspang ( 6 ) reported that of several sports activities , only swimming was associated with a decreased prevalence of lbp .", "swimming activates the muscles of the trunk , for example , the erector muscles of the spine , so swimming might be a means to prevent lbp . however , our results on soccer are at odds with those of bejia et al . ( 42 ) , who reported that soccer was positively associated with chronic lbp in tunisian schoolchildren and adolescents . similarly , our findings on weightlifting contrast with those of calhoon and fry ( 43 ) , who reported that lbp was one of the most frequent injuries in this sport .", "the relatively low prevalence of lbp in our study in soccer and weightlifting may be due to the system of this sports and physical education institute which is focused mainly on knowing how to teach these sports ( gestural technique and pedagogy ) and not on researching performance .", "our study shows that the percentage of females ( 17.6% ) with lbp was significantly higher than that of males ( 12.5% ) , as frequently reported in the literature ( 1217 ) . however , the prevalence rates of lbp do not differ significantly between males and females ( p>0.05 ) if they are compared in each practiced sport separately .", "there was no significant difference in lbp by bmi ( p>0.05 ) , in males and in females combined as reported in other studies ( 44 , 45 ) .", "in addition , many other studies have reported that active smoking is a risk factor for back pain ( 47 , 48 ) .", "however , we did not find a significant relationship between smoking and lbp ( p>0.05 ) .", "( 49 ) was that parental smoking seemed to increase the risk of spinal pain for both genders .", "we believe that students should perform exercises to strengthen the muscles of the arms and back , and increase coordination of the upper extremities and the lumbar region with regular activities that have been proven to be effective for the prevention of lbp .", "swimming or gymnastics are the nearest sports to reach this aim as they seem to reduce lbp significantly ( 50 ) .", "this study was a survey based on data from the institute health service files of 8 years and therefore had some limitations . to closely evaluate the relationship between lbp and sports activities ,", "more information on the students is needed , including basic information such as their physical activity outside the institute . in this study , the diagnostic of lbp was performed by the doctor of the institute health service and mostly without medical imaging , which could have shown the severity of lbp .", "imaging is used to show the severity of the injury rather than the severity of the pain associated with it .", "lbp was strongly associated with the amount of time reserved to practice the different sports planned in this sports and physical education institute .", "the sports identified as posing the greatest risk for lbp were gymnastics , judo , handball , and volleyball , followed by basketball and athletics .", "imed ajili ) and the administration of the sports and physical education institute of sfax ( tunisia ) for their assistance ."], "abstract_text": ["<S> introductionfor obvious reasons , athletes are at greater risk of sustaining a lumber ( lower ) spine injury due to physical activity . to our knowledge , no previous studies have examined the prevalence of low back pain ( lbp ) in a tunisian sports and physical education institute.aimto assess the prevalence of lbp in different sports among students studying in a sports and physical education institute in tunisia , to determine the causes of the injuries , and to propose solutions.methodsa total of 3,379 boys and 2,579 girls were studied . </S>", "<S> a retrospective cross - sectional survey was conducted on a group of students aged 18.524.5 years at the higher institute of sport and physical education of sfax to estimate the prevalence of lbp and its relation to the type of sports . </S>", "<S> data on age , weight , height , smoking , and the sport in which the student was injured in the low back were collected from the institute health service records from 2005 until 2013.resultslbp was reported by 879 of the 5,958 study participants ( 14.8% ) . </S>", "<S> the prevalence of lbp was significantly higher ( p<0.001 ) in females ( 17.6% ) than in males ( 12.5% ) . </S>", "<S> lbp prevalence did not differ by body mass index or smoking habit ( p>0.05 ) . </S>", "<S> the sports associated with the higher rates of lbp were gymnastics , judo , handball , and volleyball , followed by basketball and athletics.conclusionlbp is frequent among undergraduate students of a sports and physical education institute in tunisia . </S>", "<S> it is strongly associated with fatigue after the long periods of training in different sports . </S>", "<S> gymnastics , judo , handball , and volleyball were identified as high - risk sports for causing lbp . </S>"], "labels": null, "section_names": ["Methods", "Participants", "Statistical analysis", "Results", "Discussion", "Conclusion", "Acknowledgements", "Conflict of interest and funding"], "sections": [["this is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax .", "data on age , weight , height , and smoking habits are collected routinely when students join the institute , and then the sport in which the student is injured in the low back is registered in the institute health service files .", "only students injured in the first or second year were taken into account , because the following years involve only athletics and gymnastics .", "body mass index ( bmi ; kg / m ) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . in this study", "we used the standard bmi with categories of underweight ( bmi of < 18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obesity ( bmi of 30 ) .", "smoking was categorized as non - smoker , < 20 cigarettes per day , and 20 cigarettes per day .", "bmi , smoking habits , and history of any pain or injuries were taken for all the students in the beginning of the first grade .", "on the basis of the collected data , participants were dichotomized into those with or without lbp , while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . in 1 week , all students practice for a total of 16.5 h , spending 1.5 h in each of the following sports : soccer , handball , basketball , volleyball , judo , weightlifting , swimming , athletics , and gymnastics .", "lbp was defined as pain or discomfort in the low - back region , from the lower rib curvature to the lower part of the seat region .", "the question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past .", "furthermore , every registered injury causing lbp was recorded with its severity , but the most important detail for us was the sport in which the student was injured .", "students who were injured more than one time were taken into account as subjects with lbp only for their first injury .", "the scientific council of the university of sfax in tunisia gave written permission for the study protocol .", "statistical analyses were done using the spss statistical package , version 11.5 ( spss , inc . ,", "chicago , il , usa ) . the chi - square test was used to test associations between lbp and other categorical variables ( sex , obesity , and smoking habit ) , and to compare the prevalence of lbp between males and female . statistical significance"], ["this is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax .", "data on age , weight , height , and smoking habits are collected routinely when students join the institute , and then the sport in which the student is injured in the low back is registered in the institute health service files .", "only students injured in the first or second year were taken into account , because the following years involve only athletics and gymnastics .", "body mass index ( bmi ; kg / m ) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . in this study", "we used the standard bmi with categories of underweight ( bmi of < 18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obesity ( bmi of 30 ) .", "smoking was categorized as non - smoker , < 20 cigarettes per day , and 20 cigarettes per day .", "bmi , smoking habits , and history of any pain or injuries were taken for all the students in the beginning of the first grade .", "on the basis of the collected data , participants were dichotomized into those with or without lbp , while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . in 1 week , all students practice for a total of 16.5 h , spending 1.5 h in each of the following sports : soccer , handball , basketball , volleyball , judo , weightlifting , swimming , athletics , and gymnastics .", "lbp was defined as pain or discomfort in the low - back region , from the lower rib curvature to the lower part of the seat region .", "the question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past .", "furthermore , every registered injury causing lbp was recorded with its severity , but the most important detail for us was the sport in which the student was injured .", "students who were injured more than one time were taken into account as subjects with lbp only for their first injury .", "the scientific council of the university of sfax in tunisia gave written permission for the study protocol ."], ["statistical analyses were done using the spss statistical package , version 11.5 ( spss , inc . ,", "chicago , il , usa ) . the chi - square test was used to test associations between lbp and other categorical variables ( sex , obesity , and smoking habit ) , and to compare the prevalence of lbp between males and female . statistical significance"], ["table 2 shows that the prevalence of lbp was significantly higher ( p<0.001 ) among females ( 17.6% ) than among males ( 12.5% ) .", "lbp occurred in 17.1% of those classified by bmi as lean , 14.8% of those in the normal range , 14.3% among those who were overweight , and 12.7% of those who were obese .", "in addition , 13.9% of students smoking < 20 cigarettes and 13.3% of students smoking 20 cigarettes had lbp .", "prevalence of lower back pain ( lbp ) by gender , obesity and smoking lbp : low back pain ; standard body mass index ( bmi ) categories : underweight ( bmi of<18.5 ) , normal weight ( bmi of 18.5<25 ) , overweight ( bmi of 25<30 ) , and obese ( bmi of 30 ) ; smoking : cigarettes per day ; no smoking : not currently smoking . in the analysis of risk factors for lbp in sports training , we found that in both sexes combined , lbp was positively associated with fatigue ( caused by the long time spent in training ) , gymnastics , judo , handball , and volleyball .", "the increased prevalence of lbp is less in basketball and athletics and even lower in soccer , weightlifting , and swimming ( fig .", "percent prevalence of lower back pain related to fatigue and type of sports in males and females combined . \n", "figure 2 shows that there is no significant difference ( p>0.05 ) in the prevalence of lbp among males and females separately if they are compared in each practiced sport at the institute .", "however , if we analyze the prevalence of lbp in terms of sex in each sport , we find that the prevalence of lbp was significantly higher among males than females in handball ( p<0.05 ) and significantly lower in athletics ( p<0.05 )", ". percent prevalence of lower back pain related to fatigue and type of sports in males and females ."], ["its incidence and prevalence are so high that it should be studied as an epidemic and social disorder ( 35 ) .", "the first salient feature that emerges from this study is that lbp is widespread among students in high schools for athletes ( 14.8% ) .", "the students were injured mainly in gymnastics , judo , handball , and volleyball , in addition to lbp caused by fatigue due to the long time spent in training .", "these findings are generally in accordance with the literature . in a study focused on identifying the types of physical activity associated with increased occurrence of lbp in schoolchildren , skoffer and foldspang ( 6 ) noted that lbp was increased by the number of hours jogging , playing handball , and doing gymnastics .", "in addition , sato et al . ( 33 ) found that the odds ratio for the risk of experiencing lbp was significantly higher in most sports items compared with the no - sports group .", "the most frequently implicated sports were volleyball , athletics , judo , gymnastics , golf , and rugby , where the odds ratio exceeded 2 .", "kirialanis et al . reported that injuries among gymnasts are anatomically widely distributed ( 36 ) .", "as in our study , much research ( 6 , 33 , 37 ) noted that gymnastics was associated with an increased prevalence of lbp .", "judo is a grappling style martial art with emphasis on dynamic throws and submissions , which can lead to substantial injuries due to the high velocity of the maneuvers ( 38 ) .", "we registered a high prevalence of lbp in judo , as noted in the study of sato et al .", "handball , which also includes repeated sudden and often rather violent movements , has been shown to be associated with lbp in our population and in other studies ( 6 , 33 ) .", "we observed a high prevalence of lbp associated with the fatigue caused by the long time spent in physical activity ( 16.5 h per week ) .", ". however , this point of view is attractive in terms of prevention as physical activity is amenable to behavioral intervention , and physical training programs at school and leisure activities may be adjusted to aim at strengthening the back ( 6 ) .", "moreover , both physical inactivity and intensive sports activity have been associated with lbp in some studies ( 28 , 32 , 3941 ) .", "our results show that a greater amount of physical activity increased the risk of lbp mainly in gymnastics , judo , handball , and volleyball .", "the time spent in athletics and basketball was associated with lbp to a lesser extent .", "moreover , it appears that soccer , weightlifting , and swimming are correlated with relatively low prevalence of lbp .", "skoffer and foldspang ( 6 ) reported that of several sports activities , only swimming was associated with a decreased prevalence of lbp .", "swimming activates the muscles of the trunk , for example , the erector muscles of the spine , so swimming might be a means to prevent lbp . however , our results on soccer are at odds with those of bejia et al . ( 42 ) , who reported that soccer was positively associated with chronic lbp in tunisian schoolchildren and adolescents . similarly , our findings on weightlifting contrast with those of calhoon and fry ( 43 ) , who reported that lbp was one of the most frequent injuries in this sport .", "the relatively low prevalence of lbp in our study in soccer and weightlifting may be due to the system of this sports and physical education institute which is focused mainly on knowing how to teach these sports ( gestural technique and pedagogy ) and not on researching performance .", "our study shows that the percentage of females ( 17.6% ) with lbp was significantly higher than that of males ( 12.5% ) , as frequently reported in the literature ( 1217 ) . however , the prevalence rates of lbp do not differ significantly between males and females ( p>0.05 ) if they are compared in each practiced sport separately .", "there was no significant difference in lbp by bmi ( p>0.05 ) , in males and in females combined as reported in other studies ( 44 , 45 ) .", "in addition , many other studies have reported that active smoking is a risk factor for back pain ( 47 , 48 ) .", "however , we did not find a significant relationship between smoking and lbp ( p>0.05 ) .", "( 49 ) was that parental smoking seemed to increase the risk of spinal pain for both genders .", "we believe that students should perform exercises to strengthen the muscles of the arms and back , and increase coordination of the upper extremities and the lumbar region with regular activities that have been proven to be effective for the prevention of lbp .", "swimming or gymnastics are the nearest sports to reach this aim as they seem to reduce lbp significantly ( 50 ) .", "this study was a survey based on data from the institute health service files of 8 years and therefore had some limitations . to closely evaluate the relationship between lbp and sports activities ,", "more information on the students is needed , including basic information such as their physical activity outside the institute . in this study , the diagnostic of lbp was performed by the doctor of the institute health service and mostly without medical imaging , which could have shown the severity of lbp .", "imaging is used to show the severity of the injury rather than the severity of the pain associated with it ."], ["lbp was strongly associated with the amount of time reserved to practice the different sports planned in this sports and physical education institute .", "the sports identified as posing the greatest risk for lbp were gymnastics , judo , handball , and volleyball , followed by basketball and athletics ."], ["imed ajili ) and the administration of the sports and physical education institute of sfax ( tunisia ) for their assistance ."], [""]]}
{"article_id": "PMC5074748", "article_text": ["in modern society , health is a n important component , which requires sustained development ( 1 ) . in recent decades", ", a person s health has become one of the most important human rights among social groups throughout the world ( 2 ) .", "the meaning of health has been extended beyond borders , and different definitions have emerged , owing to diverse geographic regions and cultural views in societies ( 3 ) .", "the definition of health may vary by cultural background , and may influence health practices and compliance to medical treatment ( 4 ) .", "individuals perceive health on the basis of their experiences of ill health ( 5 ) .", "so , understanding health as a subjective experience can enable health care providers to manage the care giving procedure based on their clients needs .", "furthermore , one of the most important aspects with regard to health perception , manifests when patients suffer from a chronic disease , through which their perception of health is affected ( 5 , 6 ) .", "diabetes is incurable , and has the third highest mortality rate on the planet ( 8) . the probability of the incidence and prevalence of this disease has increased recently ( 9 ) . in 2004 , it was estimated that by 2030 , this number will have grown to 366 million ( 10 ) and in 2010 , the number of people afflicted by diabetes was estimated to increase up to 439 million by 2030 ( 8) .", "current estimates indicate that the number of diabetic patients in iran is around 3.5 million out of a total population of just over 68 million ( 11 ) .", "previous studies have demonstrated that chronic illnesses such as diabetes can influence a person s perception of health ( 12 ) .", "in addition , the attitudes and beliefs regarding health undoubtedly play an important role in promoting healthy behaviour ( 13 ) .", "therefore , the understanding of diabetic patients perceptions can help the development of precautionary programmes for diabetics ( 14 ) .", "although the concept of health is gaining more importance and it has constantly been improved and changed by the passage of time ( 15 , 16 ) , few studies are reported to have focused on health and illness , particularly with regard to diabetic patients belonging to different ethnic groups ( 17 ) .", "health is a relative concept ; nonetheless , several criteria have been proposed for exploring it through various ethnic and cultural groups ( 2 , 18 ) .", "prior studies have shown lack of awareness about the concept of health among iranian kurdish diabetic patients .", "kurds are an ethnic group living in the middle east , particularly in iran , who are estimated to be the third largest ethnic group , constituting 9 percent of the total population ( 19 ) .", "consequently , investigating the perception of health - related meaning among this ethnic group can increase the knowledge about the nature of health and its effects .", "therefore , this study explored the meaning of health as perceived by iranian kurdish diabetic patients in order to develop an appropriate multidisciplinary outlook toward this ethnicity of patients .", "considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .", "content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) .", "the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .", "a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .", "the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .", "the sample size was determined only when the interviewing stage reached saturation ( 22 ) .", "the sample was restricted to 20 because no new data emerged after the twentieth interview .", "therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon . in - depth , face - to - face , semi - structured interviews were used for data collection .", "the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,", "an interview guideline was defined to direct the interviews toward the aims of the study .", "the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .", "examples of questions asked during the interview were as follows : when do you feel healthy ?", "further , the patients were asked to describe all the factors that could affect their health .", "twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .", "each interview spanned 4090 minutes ( average duration : 60 minutes ) . each interview was transcribed and then used as a guide for the other interviews .", "two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .", "the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .", "the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers . through these inquiries ,", "the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers .", "this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .", "this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .", "all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .", "the participants were assured that they could leave the study at any time even after they had signed the consent form .", "they were assured that their care would not be affected if they chose not to participate in the study .", "they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .", "all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions . in this study ,", "the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .", "this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .", "the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .", "this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .", "researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,", "one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) .", "considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .", "content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) .", "the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .", "a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .", "the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .", "the sample size was determined only when the interviewing stage reached saturation ( 22 ) .", "the sample was restricted to 20 because no new data emerged after the twentieth interview .", "therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon .", "in - depth , face - to - face , semi - structured interviews were used for data collection .", "the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,", "an interview guideline was defined to direct the interviews toward the aims of the study .", "the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .", "examples of questions asked during the interview were as follows : when do you feel healthy ?", "further , the patients were asked to describe all the factors that could affect their health .", "twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .", "two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .", "the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .", "the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers .", "through these inquiries , the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers .", "this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .", "this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .", "all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .", "the participants were assured that they could leave the study at any time even after they had signed the consent form .", "they were assured that their care would not be affected if they chose not to participate in the study .", "they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .", "all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions .", "in this study , the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .", "this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .", "the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .", "this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .", "researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,", "one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) .", "twenty diabetic patients participated in this study ; the participants included seven men with an age range from 53 to 80 years ( average age : 68 years ) and thirteen women with an age range from 32 to 55 ( average age : 40 years ) .", "meanwhile , the average number of years which had lapsed since diagnosis of diabetes was seven years with a range of 517 years . based on the thoughts and feelings of the participants , the following three main themes emerged : the syndrome of the healthy body and the happy heart life without compulsory limitations the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .", "this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .", "it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .", "the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .", "similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .", "one of the participants in the study said ,  a person with a healthy and contented heart is well - behaved and kind to others  ( p1 ) .", "the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .", "another participant said ,  health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy  ", "the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises . ", "health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .", "health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety  ( p 11 ) .", "another participant said ,  health means that we do not have any problems or complaints of illness  ", "( p6 ) , and ,  health brings calmness , and it is like praying to god without having to think about stressful matters  (p2 ) , and ,  when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families .", "so , the complaints gradually go away   ( p 18 ) all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .", "one of the women ( 43 years old ) said ,  when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak  (", "sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me ", "when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines  , this freedom is very fantastic ", ", spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .", "participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .", "most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old ) said ,  some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and", ", a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace  ", "( p 18 ) , and ,  healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet  (p 11 ) another participant said ,  when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy  (p 6 ) .", "the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .", "this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .", "it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .", "the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .", "similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .", "one of the participants in the study said ,  a person with a healthy and contented heart is well - behaved and kind to others  ( p1 ) .", "the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .", "another participant said ,  health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy  ", "the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises . ", "health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .", "health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety  ( p 11 ) .", "another participant said ,  health means that we do not have any problems or complaints of illness  ", "( p6 ) , and ,  health brings calmness , and it is like praying to god without having to think about stressful matters  (p2 ) , and ,  when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families .", "all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .", "one of the women ( 43 years old ) said ,  when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak  ( p 13 ) .", "sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me ", "for some participants , the limitations had led to the feeling that they were different from others ", "when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines  , this freedom is very fantastic ", "in general , spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .", "participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .", "most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old )", "said ,  some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and , a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace  ", "( p 18 ) , and ,  healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet  (p 11 ) another participant said ,  when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy  (p 6 ) .", "as the results for this group of diabetic patients indicated , health is composed of the syndrome of the healthy body and the happy heart , life without compulsory limitations , and exalted spirituality .", "many studies have shown that health perception is a reflection on , not only health but also health as a culture .", "for all patients , health perception and points of view are the important factors that affect health behaviour and determine any related behaviour ( 25 , 26 ) . an individual may be ill and yet may feel healthy ; therefore , many people can compare their health status during suffering from illness with that in the past ( 22 , 27 ) .", "the themes that emerged in our study showed these instances briefly . in this study , participants talked about the effects of diabetes on physical and psychological health , which indicated that our results were similar to those of other studies ( 28 , 29 ) .", "the most important theme of this study was the syndrome of the healthy body and the happy heart .", "the patients always used local terminology for health description , and these terminologies have extended meanings . in this context , health is considered as a feeling that can affect all components of the human body , including their physical , psychological , social , emotional , and spiritual facets .", "this view about health is mainly accepted about diabetes because diabetes affects all aspects of a patient s life ( 11 , 28 , 29 ) . in this study , participants admitted that , not only physical health but also vivacity , satisfaction , and calmness of mind , good relationship with family and good economic status are all considered as aspects of health . hence , health care professionals should strongly consider the issue that health implies lack of having any problems , complaints , illnesses , etc .", "furthermore , physical illness and its adverse effects are important topics in the health experiences of diabetic patients ; however , when these patients were healthy , they said that without peace of mind one could not feel healthy .", "moreover , in a study about health in spanish immigrants , ailinger and causey found that some concepts of health are related to the patients psychological point of view and peace of mind , calmness in family , absence of complaints , and so on ( 30 ) .", "assessed the effectiveness of self - efficacy and family inter - relationships among young turkish patients afflicted with diabetes type i ( 29 ) .", "their study showed that calmness of mind and some other related factors such as relationship among family members , conflicts or compatibility in the family , and emotional situations can affect self - efficacy , and accordingly , these factors affect their overall health .", "hence , considering the important effect of calmness of mind on diabetic patients health perception and points of view , we , as health care professionals , ought to be obliged to associate a disturbed mind with bad health .", "overall , by using these implications as the bases of patients culture , we can improve health programmes for all patients .", "one of the most plausible themes in our interviews was health as life without compulsory limitations .", "miklaucich studied the limitations of life as meaningful health experiences in patients with coronary angina ( 31 ) .", "abazari et al . also demonstrated that diabetic patients have dietary restrictions as well as limitations with regards to bearing children ( 18 ) .", "thomson & gifford indicated that trying to keep a balance was the preferred meaning of health for diabetic participants ( 32 ) .", "it is more important for the professional health care teams to help diabetic patients feel that they are not different from others and in this regard , consultation with dieticians can help to obviate some part of this limitation of life .", "studies also show that having a regular activity program and regular follow- up consultation with other health care professionals can help diabetic patients to expel limitations ( 33 , 34 ) . in this article", ", all participants talked about the importance of the spiritual dimension of health and achieving exalted spirituality .", "some other studies showed that spirituality is one of the significant factors that can affect health ; hence , spirituality plays a very important role in gaining adaptation to a lifestyle , with regards to chronic diseases , and helps patients to manage their health - related problems ( 35 ) .", "this theme was plausible in traditional societies ; hence , nursing professionals must pay more attention to this influential factor .", "patients can adopt a spiritual approach by becoming a member of a religious group , praying , and meditating .", "exalted spirituality , in fact , helps patients to cope with the disease , and feel better about their lives ( 36 , 37 ) .", "he showed that praying helps patients to not only connect with the higher power , but also to relate to each other .", "their act of praying is a response to their anxieties about themselves or their families .", "walton showed that women with chronic diseases , used prayer to calm themselves and connect with others to support each other and cope with diseases ( 39 ) .", "therefore , health care professionals should be aware of the effects of spirituality on patients having a chronic disease .", "the results of our study primarily indicated that the factors that affect health perception include complaining about the illness , side effects and outcomes of the disease , and the extent of the complaints that make the patient feel sick .", "patients with chronic diseases encountered some social and emotional problems due to changes in their lifestyle ( 36 ) .", "diabetes affects patients quality of life , and especially their perception and attitude toward health ( 12 ) .", "it fully depends on how patients and health professionals define health ( 40 , 41 ) . due to chronic and acute complaints of diabetic patients ,", "we need to create a programme aiming to reduce these adverse effects and promote self - care for diabetic patients . especially , a program must be developed based on paying attention to body and mind simultaneously , trying to remove the limitation in life of patients by considering the extent of the limitations and limitations that exist for the patients which compel them to believe that the disease has been brought about just for them .", "this program should not affect or limit the beliefs and spiritual activity of patients because a person s previous experiences influence the perception of present time , which also impact on their future .", "besides , having been brought up in a different society and within a different ethnic group , influences a person s health - related behavior .", "so , health care providers must manage care situations by enhancing health experience to enable patients lives with the least limitations , and giving them an opportunity to preserve exalted spirituality .", "studying health perception among patients with chronic diseases is an important factor that facilitates the development of appropriate programmes , to improve health levels among such patients . in this regard ,", "the professional health - care provider team ought to consider health perception among patients , and then they apply this knowledge for setting up suitable health improvement programs .", "nurses need to pay more attention to diabetic patients limitations and facilitate a programme to improve their health in conjunction with other health care teams .", "although nursing and other health care professionals have the responsibility to help those patients achieve peace of mind , the patients families should also play an active role in the health care programme and treatment process ."], "abstract_text": ["<S> introductionhealth is an exclusive and subjective phenomenon , and one of the most important situations with regard to perception of health , arises when patients suffer from a chronic disease.this study was conducted within the qualitative research framework and aimed to explore the meanings of health as perceived by a group of iranian diabetic patients.methodsa descriptive qualitative analysis design was used . </S>", "<S> data were collected through semi - structured interviews with 20 participants among diabetic patients , who were admitted to the diabetes care centre of tohid hospital of the kurdistan university of medical sciences , sanandaj , iran during a ten - month period in 2014 . </S>", "<S> interviews were transcribed and analysed through conventional content analysis.resultsbased on the findings of the study , three major health - related themes emerged : 1 ) the syndrome of the healthy body and the happy heart ( physical well - being vivacity , satisfaction , and calmness of the mind ) , 2 ) life without compulsory limitations ( lack of dietary limitations , no activity limitations , lack of social limitations ) , and 3 ) exalted spirituality ( satisfying self and others , trusting god , remembering god).conclusionhealth care providers should consider the meaning of health in special groups , chiefly in patients with chronic diseases . </S>", "<S> it facilitates the development of appropriate programmes to improve desirable health levels among diabetic patients . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "2.1. Design", "2.2. Participants", "2.3. Data collection", "2.4. Rigor", "2.5. Ethical Considerations", "2.6. Data Analysis", "3. Results", "3.1. The syndrome of the healthy body and the happy heart", "3.2. Life without compulsory limitations", "3.3. Exalted spirituality", "4. Discussion", "5. Conclusions"], "sections": [["in modern society , health is a n important component , which requires sustained development ( 1 ) . in recent decades", ", a person s health has become one of the most important human rights among social groups throughout the world ( 2 ) .", "the meaning of health has been extended beyond borders , and different definitions have emerged , owing to diverse geographic regions and cultural views in societies ( 3 ) .", "the definition of health may vary by cultural background , and may influence health practices and compliance to medical treatment ( 4 ) .", "individuals perceive health on the basis of their experiences of ill health ( 5 ) .", "so , understanding health as a subjective experience can enable health care providers to manage the care giving procedure based on their clients needs .", "furthermore , one of the most important aspects with regard to health perception , manifests when patients suffer from a chronic disease , through which their perception of health is affected ( 5 , 6 ) .", "diabetes is incurable , and has the third highest mortality rate on the planet ( 8) . the probability of the incidence and prevalence of this disease has increased recently ( 9 ) . in 2004 , it was estimated that by 2030 , this number will have grown to 366 million ( 10 ) and in 2010 , the number of people afflicted by diabetes was estimated to increase up to 439 million by 2030 ( 8) .", "current estimates indicate that the number of diabetic patients in iran is around 3.5 million out of a total population of just over 68 million ( 11 ) .", "previous studies have demonstrated that chronic illnesses such as diabetes can influence a person s perception of health ( 12 ) .", "in addition , the attitudes and beliefs regarding health undoubtedly play an important role in promoting healthy behaviour ( 13 ) .", "therefore , the understanding of diabetic patients perceptions can help the development of precautionary programmes for diabetics ( 14 ) .", "although the concept of health is gaining more importance and it has constantly been improved and changed by the passage of time ( 15 , 16 ) , few studies are reported to have focused on health and illness , particularly with regard to diabetic patients belonging to different ethnic groups ( 17 ) .", "health is a relative concept ; nonetheless , several criteria have been proposed for exploring it through various ethnic and cultural groups ( 2 , 18 ) .", "prior studies have shown lack of awareness about the concept of health among iranian kurdish diabetic patients .", "kurds are an ethnic group living in the middle east , particularly in iran , who are estimated to be the third largest ethnic group , constituting 9 percent of the total population ( 19 ) .", "consequently , investigating the perception of health - related meaning among this ethnic group can increase the knowledge about the nature of health and its effects .", "therefore , this study explored the meaning of health as perceived by iranian kurdish diabetic patients in order to develop an appropriate multidisciplinary outlook toward this ethnicity of patients ."], ["considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .", "content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) .", "the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .", "a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .", "the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .", "the sample size was determined only when the interviewing stage reached saturation ( 22 ) .", "the sample was restricted to 20 because no new data emerged after the twentieth interview .", "therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon . in - depth , face - to - face , semi - structured interviews were used for data collection .", "the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,", "an interview guideline was defined to direct the interviews toward the aims of the study .", "the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .", "examples of questions asked during the interview were as follows : when do you feel healthy ?", "further , the patients were asked to describe all the factors that could affect their health .", "twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .", "each interview spanned 4090 minutes ( average duration : 60 minutes ) . each interview was transcribed and then used as a guide for the other interviews .", "two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .", "the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .", "the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers . through these inquiries ,", "the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers .", "this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .", "this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .", "all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .", "the participants were assured that they could leave the study at any time even after they had signed the consent form .", "they were assured that their care would not be affected if they chose not to participate in the study .", "they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .", "all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions . in this study ,", "the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .", "this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .", "the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .", "this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .", "researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,", "one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) ."], ["considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .", "content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) ."], ["the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .", "a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .", "the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .", "the sample size was determined only when the interviewing stage reached saturation ( 22 ) .", "the sample was restricted to 20 because no new data emerged after the twentieth interview .", "therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon ."], ["in - depth , face - to - face , semi - structured interviews were used for data collection .", "the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,", "an interview guideline was defined to direct the interviews toward the aims of the study .", "the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .", "examples of questions asked during the interview were as follows : when do you feel healthy ?", "further , the patients were asked to describe all the factors that could affect their health .", "twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .", "two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .", "the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .", "the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers ."], ["through these inquiries , the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers ."], ["this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .", "this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .", "all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .", "the participants were assured that they could leave the study at any time even after they had signed the consent form .", "they were assured that their care would not be affected if they chose not to participate in the study .", "they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .", "all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions ."], ["in this study , the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .", "this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .", "the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .", "this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .", "researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,", "one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) ."], ["twenty diabetic patients participated in this study ; the participants included seven men with an age range from 53 to 80 years ( average age : 68 years ) and thirteen women with an age range from 32 to 55 ( average age : 40 years ) .", "meanwhile , the average number of years which had lapsed since diagnosis of diabetes was seven years with a range of 517 years . based on the thoughts and feelings of the participants , the following three main themes emerged : the syndrome of the healthy body and the happy heart life without compulsory limitations the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .", "this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .", "it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .", "the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .", "similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .", "one of the participants in the study said ,  a person with a healthy and contented heart is well - behaved and kind to others  ( p1 ) .", "the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .", "another participant said ,  health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy  ", "the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises . ", "health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .", "health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety  ( p 11 ) .", "another participant said ,  health means that we do not have any problems or complaints of illness  ", "( p6 ) , and ,  health brings calmness , and it is like praying to god without having to think about stressful matters  (p2 ) , and ,  when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families .", "so , the complaints gradually go away   ( p 18 ) all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .", "one of the women ( 43 years old ) said ,  when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak  (", "sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me ", "when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines  , this freedom is very fantastic ", ", spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .", "participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .", "most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old ) said ,  some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and", ", a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace  ", "( p 18 ) , and ,  healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet  (p 11 ) another participant said ,  when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy  (p 6 ) ."], ["the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .", "this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .", "it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .", "the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .", "similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .", "one of the participants in the study said ,  a person with a healthy and contented heart is well - behaved and kind to others  ( p1 ) .", "the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .", "another participant said ,  health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy  ", "the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises . ", "health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .", "health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety  ( p 11 ) .", "another participant said ,  health means that we do not have any problems or complaints of illness  ", "( p6 ) , and ,  health brings calmness , and it is like praying to god without having to think about stressful matters  (p2 ) , and ,  when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families ."], ["all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .", "one of the women ( 43 years old ) said ,  when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak  ( p 13 ) .", "sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me ", "for some participants , the limitations had led to the feeling that they were different from others ", "when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines  , this freedom is very fantastic "], ["in general , spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .", "participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .", "most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old )", "said ,  some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and , a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace  ", "( p 18 ) , and ,  healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet  (p 11 ) another participant said ,  when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy  (p 6 ) ."], ["as the results for this group of diabetic patients indicated , health is composed of the syndrome of the healthy body and the happy heart , life without compulsory limitations , and exalted spirituality .", "many studies have shown that health perception is a reflection on , not only health but also health as a culture .", "for all patients , health perception and points of view are the important factors that affect health behaviour and determine any related behaviour ( 25 , 26 ) . an individual may be ill and yet may feel healthy ; therefore , many people can compare their health status during suffering from illness with that in the past ( 22 , 27 ) .", "the themes that emerged in our study showed these instances briefly . in this study , participants talked about the effects of diabetes on physical and psychological health , which indicated that our results were similar to those of other studies ( 28 , 29 ) .", "the most important theme of this study was the syndrome of the healthy body and the happy heart .", "the patients always used local terminology for health description , and these terminologies have extended meanings . in this context , health is considered as a feeling that can affect all components of the human body , including their physical , psychological , social , emotional , and spiritual facets .", "this view about health is mainly accepted about diabetes because diabetes affects all aspects of a patient s life ( 11 , 28 , 29 ) . in this study , participants admitted that , not only physical health but also vivacity , satisfaction , and calmness of mind , good relationship with family and good economic status are all considered as aspects of health . hence , health care professionals should strongly consider the issue that health implies lack of having any problems , complaints , illnesses , etc .", "furthermore , physical illness and its adverse effects are important topics in the health experiences of diabetic patients ; however , when these patients were healthy , they said that without peace of mind one could not feel healthy .", "moreover , in a study about health in spanish immigrants , ailinger and causey found that some concepts of health are related to the patients psychological point of view and peace of mind , calmness in family , absence of complaints , and so on ( 30 ) .", "assessed the effectiveness of self - efficacy and family inter - relationships among young turkish patients afflicted with diabetes type i ( 29 ) .", "their study showed that calmness of mind and some other related factors such as relationship among family members , conflicts or compatibility in the family , and emotional situations can affect self - efficacy , and accordingly , these factors affect their overall health .", "hence , considering the important effect of calmness of mind on diabetic patients health perception and points of view , we , as health care professionals , ought to be obliged to associate a disturbed mind with bad health .", "overall , by using these implications as the bases of patients culture , we can improve health programmes for all patients .", "one of the most plausible themes in our interviews was health as life without compulsory limitations .", "miklaucich studied the limitations of life as meaningful health experiences in patients with coronary angina ( 31 ) .", "abazari et al . also demonstrated that diabetic patients have dietary restrictions as well as limitations with regards to bearing children ( 18 ) .", "thomson & gifford indicated that trying to keep a balance was the preferred meaning of health for diabetic participants ( 32 ) .", "it is more important for the professional health care teams to help diabetic patients feel that they are not different from others and in this regard , consultation with dieticians can help to obviate some part of this limitation of life .", "studies also show that having a regular activity program and regular follow- up consultation with other health care professionals can help diabetic patients to expel limitations ( 33 , 34 ) . in this article", ", all participants talked about the importance of the spiritual dimension of health and achieving exalted spirituality .", "some other studies showed that spirituality is one of the significant factors that can affect health ; hence , spirituality plays a very important role in gaining adaptation to a lifestyle , with regards to chronic diseases , and helps patients to manage their health - related problems ( 35 ) .", "this theme was plausible in traditional societies ; hence , nursing professionals must pay more attention to this influential factor .", "patients can adopt a spiritual approach by becoming a member of a religious group , praying , and meditating .", "exalted spirituality , in fact , helps patients to cope with the disease , and feel better about their lives ( 36 , 37 ) .", "he showed that praying helps patients to not only connect with the higher power , but also to relate to each other .", "their act of praying is a response to their anxieties about themselves or their families .", "walton showed that women with chronic diseases , used prayer to calm themselves and connect with others to support each other and cope with diseases ( 39 ) .", "therefore , health care professionals should be aware of the effects of spirituality on patients having a chronic disease .", "the results of our study primarily indicated that the factors that affect health perception include complaining about the illness , side effects and outcomes of the disease , and the extent of the complaints that make the patient feel sick .", "patients with chronic diseases encountered some social and emotional problems due to changes in their lifestyle ( 36 ) .", "diabetes affects patients quality of life , and especially their perception and attitude toward health ( 12 ) .", "it fully depends on how patients and health professionals define health ( 40 , 41 ) . due to chronic and acute complaints of diabetic patients ,", "we need to create a programme aiming to reduce these adverse effects and promote self - care for diabetic patients . especially , a program must be developed based on paying attention to body and mind simultaneously , trying to remove the limitation in life of patients by considering the extent of the limitations and limitations that exist for the patients which compel them to believe that the disease has been brought about just for them .", "this program should not affect or limit the beliefs and spiritual activity of patients because a person s previous experiences influence the perception of present time , which also impact on their future .", "besides , having been brought up in a different society and within a different ethnic group , influences a person s health - related behavior .", "so , health care providers must manage care situations by enhancing health experience to enable patients lives with the least limitations , and giving them an opportunity to preserve exalted spirituality ."], ["studying health perception among patients with chronic diseases is an important factor that facilitates the development of appropriate programmes , to improve health levels among such patients . in this regard ,", "the professional health - care provider team ought to consider health perception among patients , and then they apply this knowledge for setting up suitable health improvement programs .", "nurses need to pay more attention to diabetic patients limitations and facilitate a programme to improve their health in conjunction with other health care teams .", "although nursing and other health care professionals have the responsibility to help those patients achieve peace of mind , the patients families should also play an active role in the health care programme and treatment process ."]]}
{"article_id": "PMC4955308", "article_text": ["however , proper amount of daily water intake in a healthy individual is controversial . there is a prevalent thought that we all have to drink eight glasses of water daily", ", we try to clarify this topic from the perspective of avicenna and traditional persian medicine ( tpm ) .", "the most important traditional persian medical encyclopedia , al - qanun fil - tibb ( canon of medicine ) , was reviewed .", "furthermore , medline , embase , scopus , iranmedex , and science iranian database ( sid ) focusing on the keywords  traditional medicine  ,  water intake  , ", "fluid , and  complementary and alternative medicine  were reviewed to find relevant information .", "avicenna believed that the demand for daily water is not the same in different individuals .", "the determinant variables mentioned in his book , the canon of medicine , comprises mizaj ( temperament ) , health status , age , sex , season , place , habits , occupation , etc .", "he believed that water in extra amount quenches the hararat - e - ghariezi , which is the basal internal heat to convey normal homeostasis and metabolism in the body .", "several factors determine the actual need of any person to drink water . consequently , recommending a specific amount of daily water intake for all is illogical .", "moreover , important recommendations of tpm sages on an appropriate amount of water intake should be considered to prevent associated disorders ."], "abstract_text": ["<S> background : fluid intake is an essential factor in having a normal homeostasis . </S>", "<S> however , proper amount of daily water intake in a healthy individual is controversial . </S>", "<S> there is a prevalent thought that we all have to drink eight glasses of water daily , however , it lacks enough supportive evidence at least for healthy individuals . here , we try to clarify this topic from the perspective of avicenna and traditional persian medicine ( tpm).methods : the most important traditional persian medical encyclopedia , al - qanun fil - tibb ( canon of medicine ) , was reviewed . furthermore , medline , embase , scopus , iranmedex , and science iranian database ( sid ) focusing on the keywords  traditional medicine  ,  water intake  ,  </S>", "<S> fluid , and  complementary and alternative medicine  were reviewed to find relevant information.results:avicenna believed that the demand for daily water is not the same in different individuals . </S>", "<S> the determinant variables mentioned in his book , the canon of medicine , comprises mizaj ( temperament ) , health status , age , sex , season , place , habits , occupation , etc . </S>", "<S> he believed that water in extra amount quenches the hararat - e - ghariezi , which is the basal internal heat to convey normal homeostasis and metabolism in the body.conclusion:several factors determine the actual need of any person to drink water . </S>", "<S> consequently , recommending a specific amount of daily water intake for all is illogical . moreover , important recommendations of tpm sages on an appropriate amount of water intake should be considered to prevent associated disorders . </S>"], "labels": null, "section_names": ["Background:", "Methods:", "Results:", "Conclusion:"], "sections": [["however , proper amount of daily water intake in a healthy individual is controversial . there is a prevalent thought that we all have to drink eight glasses of water daily", ", we try to clarify this topic from the perspective of avicenna and traditional persian medicine ( tpm ) ."], ["the most important traditional persian medical encyclopedia , al - qanun fil - tibb ( canon of medicine ) , was reviewed .", "furthermore , medline , embase , scopus , iranmedex , and science iranian database ( sid ) focusing on the keywords  traditional medicine  ,  water intake  , ", "fluid , and  complementary and alternative medicine  were reviewed to find relevant information ."], ["avicenna believed that the demand for daily water is not the same in different individuals .", "the determinant variables mentioned in his book , the canon of medicine , comprises mizaj ( temperament ) , health status , age , sex , season , place , habits , occupation , etc .", "he believed that water in extra amount quenches the hararat - e - ghariezi , which is the basal internal heat to convey normal homeostasis and metabolism in the body ."], ["several factors determine the actual need of any person to drink water . consequently , recommending a specific amount of daily water intake for all is illogical .", "moreover , important recommendations of tpm sages on an appropriate amount of water intake should be considered to prevent associated disorders ."]]}
{"article_id": "PMC4982520", "article_text": ["a 34year - old male presented for general examination on day 49 of a 50-day self - imposed fast , seeking approval to continue his fasting behavior . during this period ,", "the patient reported only consuming water , tea , coffee ( with no milk or sugar ) , and a daily multivitamin tablet ( centrum performance ; pfizer ) .", "patient history prior to fasting behavior revealed a family history of obesity and the presence of pathologies related to body mass .", "personal history involved an oscillating mass between 85 and 134 kg over a 10-year period . at the start of fasting ,", "the patient reported body mass as 96.8 kg , and 29.2% body fat , as recorded by a personal bioelectrical impedance device .", "history following the onset of fasting behavior was unremarkable , but included severe abdominal cramps between days 7 and 21 of fasting , development and frequent occurrence of vasovagal syncope on transition from sitting to standing , and the near - cessation of fecal movement .", "self - reported physical activity and mobility was reduced , but not prevented ; this participant was not bedridden .", "substantial water and coffee intake was noted ( 812 cups of coffee daily and 23 bottles of water daily ) . as expected , body mass loss during fasting was severe and noticeable ( fig .", "resting metabolic rate ( 30 minutes by indirect calorimetry ) was 1626 kcal day . during the 30-minute rest period", "resting ecg appeared morphologically normal , patient was bradycardic ( 52 bpm ) and notably hypertensive ( 150/78 mmhg ) .", "body composition was measured by whole - body air displacement plethysmography ( bod pod ) as 19.8% body fat , while height was 180.9 cm and mass was 75.4 kg , with a bmi of 23.5 kg m , all recorded on calibrated laboratory devices .", "sum of four skinfolds1 was 30.0 mm , approximately half of that of a reference population of similarly aged males ( 62  25 mm , aged 3039 ) .", "it was visually noted that subcutaneous fat in the abdominal region was still present , despite severe losses in the peripheral limbs , protruding ribs , and lack of prominent chest and shoulder musculature .", "2 ) . during a six - minute walk test , this individual covered 342 m , which was below expected performance for a healthy male adult.2 a mild scoliosis was observed while walking ; however , it is unclear if this is related to fasting .", "venous blood was taken for serum markers of metabolism , liver , and renal function .", "noteworthy findings include plasma glucose of 3.1 mmol l and alkaline phosphatase of 36 u l. total bilirubin was 19 mol l , direct bilirubin was 7 mol l. uric acid was 620 mol l. urine was straw colored , lacked sediment , and was noticeably sweet in aroma", ". rapid dipstick analysis ( multistix 2161 ; siemens ) was negative for blood glucose , contained trace proteins , and indicated urine ph of below 5 .", "one month following initial presentation , the patient returned for follow - up examination during which body composition , resting metabolic rate , urine , and venous blood were recorded .", "resumption of feeding initially involved liquids such as vege table soups , with the resumption of solid foods three days following breaking of fast .", "days 35 involved one solid meal per day , with 23 solid meals during days 57 , and the resumption of three solid food meals per day for 7 days following breaking of fast . in the following 23 days", ", diet was reported to be of caffeine and alcohol free , low carbohydrate ( a piece of bread every 13 days ) , and high servings of fruit and vegetables .", "self - estimation of diet by volume was 20% animal protein ( chicken and fish ) and 80% fruit or vegetables ; validated , quantifiable measures of intake and caloric content are not available .", "heart rate remained bradycardic at rest ( 48 bpm ) , and blood pressure was notably reduced ( 132/78 ) .", "metabolic rate was reduced ( 1413 kcal day ) , and body composition was unchanged ( 75.0 kg ; bmi , 23.4 kg m ; 16.1% fat mass ) .", "blood biochemistry returned to within normal range , with the exception of direct bilirubin ( table 1 ) .", "it is noteworthy , therefore , in this case study that moderate physical function appears maintained at day 49 , as assessed by general observation and results of the six - minute walk test .", "frommel et al.3 suggested that fasting in healthy lean individuals was well tolerated until 18% of body mass was lost ; the patient reported here lost 20.7% of body mass without substantial loss of function .", "one prior report of long - term fasting in obese individuals for 3040 days demonstrated body mass losses between 10.6% and 20.5% , but does not report on function of individuals.4 it seems self - apparent and supported by this limited evidence , such that the starting mass is a key variable in survival of extreme fasting , and not the amount of loss .", "this patient started fasting while obese and ended his fast when anthropometric variables were within the targeted ranges for the general population .", "indeed , among the few ( nonpublished ) examples of complete fasting and mortality,5 patients were frail between 30 and 50 days , and death was noted to occur between days 43 and 70 , which this case study has reached without obvious or significant frailty occurring .", "current world medical association guidelines prioritize autonomy , recommending nonintervention in fasting to the point of harm and/or death , if the patient makes a written informed statement of intent.5 lack of research into this field precludes evidence - based decision - making or advice . in the limited light of this case study and historic data of fasting in obesity,4", "it is tempting to suggest that guidelines to physicians for advice to the fasting individual reflect body mass , and specifically mass of adiposity at start of fasting behavior .", "survival in starvation is ultimately governed by physics , the calories available to maintain metabolic function .", "availability of adipose tissue for metabolic usage may thus delay the catastrophic degradation of muscular protein , increasing survival time . that said , small animal models reveal organ - specific effects of starvation , with loss of liver and gut size and function noted after only four to six days.6 functional damage to digestive organs can not be ruled out , and indeed , the elevated direct bilirubin is suggestive of reduced liver filtration . of concern ,", "direct bilirubin remained elevated 30 days post breaking of fast . elevated uric acid is likely due to reduced kidney filtration , as has been reported in caloric restriction previously.7 further , hypertension was an unexpected finding .", "this finding is counter to one of the few published examinations of cardiology in severe starvation , where bradycardia and hypotension ( ~20 mmhg below fed state ) were noted in juvenile pigs following 29 days of complete starvation.8 the porcine heart contains several structural differences relative to the human , most related to bipedal vs. quadrupedal stance.9 as frequent syncope was also noted in that our case study , it is plausible that dysregulated autonomic regulation of blood pressure underlies the observed hypertension .", "blood pressure was measured in a supine position , increasing venous return in the human relative to standing position .", "simple frank  starling mechanics combined with a failure of autonomic regulation may thus explain elevated blood pressure .", "thus , it is tempting to suggest that differences observed may be species dependent ; however , this is highly speculative on a single case study .", "further , long - lasting cardiovascular deficits were noted in the above porcine model on refeeding , as well as chronically following experimental conclusion.8 as death resulting from starvation often occurs via cardiovascular complications , this elevated blood pressure during fast is key to note ; this should be carefully monitered in fasting individuals", ". caloric restriction and weight loss in overweight or obese individuals is often associated with subsequent regain of weight.10,11 having access to this case study 30 days following resumption of feeding was of interest due to a chance to examine any occurrence of such a rebound effect .", "it is of interest to note that this was not seen here ; however , future gains in weight can not be ruled out . here , we report the maintenance of physical function of an individual after 49 days of complete fast .", "this report does not indicate support for such behavioral choices , and the projections of metabolic energy provision by starting adiposity assume no other perturbations to homeostasis , such as impaired immune response , liver and renal function , or cardiovascular events ."], "abstract_text": ["<S> a 34-year - old obese male ( 96.8 kg ; bmi , 30.2 kg m1 ) volitionally undertook a 50-day fast with the stated goal of losing body mass . during this time , only tea , coffee , water , and a daily multivitamin were consumed . </S>", "<S> severe and linear loss of body mass is recorded during these 50 days ( final 75.4 kg ; bmi , 23.5 kg m1 ) . a surprising resilience to effects of fasting on activity levels and physical function is noted . </S>", "<S> plasma samples are suggestive of early impairment of liver function , and perturbations to cardiovascular dynamics are also noted . </S>", "<S> one month following resumption of feeding behavior , body weight was maintained ( 75.0 kg ; bmi , 23.4 kg m1 ) . </S>", "<S> evidence - based decision - making with the fasting or hunger striking patient is limited by a lack of evidence . </S>", "<S> this case report suggests that total body mass , not mass lost , may be a key observation in clinical decision - making during fasting and starvation . </S>"], "labels": null, "section_names": ["Introduction", "One Month Past Breaking of Fast", "Discussion"], "sections": [["a 34year - old male presented for general examination on day 49 of a 50-day self - imposed fast , seeking approval to continue his fasting behavior . during this period ,", "the patient reported only consuming water , tea , coffee ( with no milk or sugar ) , and a daily multivitamin tablet ( centrum performance ; pfizer ) .", "patient history prior to fasting behavior revealed a family history of obesity and the presence of pathologies related to body mass .", "personal history involved an oscillating mass between 85 and 134 kg over a 10-year period . at the start of fasting ,", "the patient reported body mass as 96.8 kg , and 29.2% body fat , as recorded by a personal bioelectrical impedance device .", "history following the onset of fasting behavior was unremarkable , but included severe abdominal cramps between days 7 and 21 of fasting , development and frequent occurrence of vasovagal syncope on transition from sitting to standing , and the near - cessation of fecal movement .", "self - reported physical activity and mobility was reduced , but not prevented ; this participant was not bedridden .", "substantial water and coffee intake was noted ( 812 cups of coffee daily and 23 bottles of water daily ) . as expected , body mass loss during fasting was severe and noticeable ( fig .", "resting metabolic rate ( 30 minutes by indirect calorimetry ) was 1626 kcal day . during the 30-minute rest period", "resting ecg appeared morphologically normal , patient was bradycardic ( 52 bpm ) and notably hypertensive ( 150/78 mmhg ) .", "body composition was measured by whole - body air displacement plethysmography ( bod pod ) as 19.8% body fat , while height was 180.9 cm and mass was 75.4 kg , with a bmi of 23.5 kg m , all recorded on calibrated laboratory devices .", "sum of four skinfolds1 was 30.0 mm , approximately half of that of a reference population of similarly aged males ( 62  25 mm , aged 3039 ) .", "it was visually noted that subcutaneous fat in the abdominal region was still present , despite severe losses in the peripheral limbs , protruding ribs , and lack of prominent chest and shoulder musculature .", "2 ) . during a six - minute walk test , this individual covered 342 m , which was below expected performance for a healthy male adult.2 a mild scoliosis was observed while walking ; however , it is unclear if this is related to fasting .", "venous blood was taken for serum markers of metabolism , liver , and renal function .", "noteworthy findings include plasma glucose of 3.1 mmol l and alkaline phosphatase of 36 u l. total bilirubin was 19 mol l , direct bilirubin was 7 mol l. uric acid was 620 mol l. urine was straw colored , lacked sediment , and was noticeably sweet in aroma", ". rapid dipstick analysis ( multistix 2161 ; siemens ) was negative for blood glucose , contained trace proteins , and indicated urine ph of below 5 ."], ["one month following initial presentation , the patient returned for follow - up examination during which body composition , resting metabolic rate , urine , and venous blood were recorded .", "resumption of feeding initially involved liquids such as vege table soups , with the resumption of solid foods three days following breaking of fast .", "days 35 involved one solid meal per day , with 23 solid meals during days 57 , and the resumption of three solid food meals per day for 7 days following breaking of fast . in the following 23 days", ", diet was reported to be of caffeine and alcohol free , low carbohydrate ( a piece of bread every 13 days ) , and high servings of fruit and vegetables .", "self - estimation of diet by volume was 20% animal protein ( chicken and fish ) and 80% fruit or vegetables ; validated , quantifiable measures of intake and caloric content are not available .", "heart rate remained bradycardic at rest ( 48 bpm ) , and blood pressure was notably reduced ( 132/78 ) .", "metabolic rate was reduced ( 1413 kcal day ) , and body composition was unchanged ( 75.0 kg ; bmi , 23.4 kg m ; 16.1% fat mass ) .", "blood biochemistry returned to within normal range , with the exception of direct bilirubin ( table 1 ) ."], ["it is noteworthy , therefore , in this case study that moderate physical function appears maintained at day 49 , as assessed by general observation and results of the six - minute walk test .", "frommel et al.3 suggested that fasting in healthy lean individuals was well tolerated until 18% of body mass was lost ; the patient reported here lost 20.7% of body mass without substantial loss of function .", "one prior report of long - term fasting in obese individuals for 3040 days demonstrated body mass losses between 10.6% and 20.5% , but does not report on function of individuals.4 it seems self - apparent and supported by this limited evidence , such that the starting mass is a key variable in survival of extreme fasting , and not the amount of loss .", "this patient started fasting while obese and ended his fast when anthropometric variables were within the targeted ranges for the general population .", "indeed , among the few ( nonpublished ) examples of complete fasting and mortality,5 patients were frail between 30 and 50 days , and death was noted to occur between days 43 and 70 , which this case study has reached without obvious or significant frailty occurring .", "current world medical association guidelines prioritize autonomy , recommending nonintervention in fasting to the point of harm and/or death , if the patient makes a written informed statement of intent.5 lack of research into this field precludes evidence - based decision - making or advice . in the limited light of this case study and historic data of fasting in obesity,4", "it is tempting to suggest that guidelines to physicians for advice to the fasting individual reflect body mass , and specifically mass of adiposity at start of fasting behavior .", "survival in starvation is ultimately governed by physics , the calories available to maintain metabolic function .", "availability of adipose tissue for metabolic usage may thus delay the catastrophic degradation of muscular protein , increasing survival time . that said , small animal models reveal organ - specific effects of starvation , with loss of liver and gut size and function noted after only four to six days.6 functional damage to digestive organs can not be ruled out , and indeed , the elevated direct bilirubin is suggestive of reduced liver filtration . of concern ,", "direct bilirubin remained elevated 30 days post breaking of fast . elevated uric acid is likely due to reduced kidney filtration , as has been reported in caloric restriction previously.7 further , hypertension was an unexpected finding .", "this finding is counter to one of the few published examinations of cardiology in severe starvation , where bradycardia and hypotension ( ~20 mmhg below fed state ) were noted in juvenile pigs following 29 days of complete starvation.8 the porcine heart contains several structural differences relative to the human , most related to bipedal vs. quadrupedal stance.9 as frequent syncope was also noted in that our case study , it is plausible that dysregulated autonomic regulation of blood pressure underlies the observed hypertension .", "blood pressure was measured in a supine position , increasing venous return in the human relative to standing position .", "simple frank  starling mechanics combined with a failure of autonomic regulation may thus explain elevated blood pressure .", "thus , it is tempting to suggest that differences observed may be species dependent ; however , this is highly speculative on a single case study .", "further , long - lasting cardiovascular deficits were noted in the above porcine model on refeeding , as well as chronically following experimental conclusion.8 as death resulting from starvation often occurs via cardiovascular complications , this elevated blood pressure during fast is key to note ; this should be carefully monitered in fasting individuals", ". caloric restriction and weight loss in overweight or obese individuals is often associated with subsequent regain of weight.10,11 having access to this case study 30 days following resumption of feeding was of interest due to a chance to examine any occurrence of such a rebound effect .", "it is of interest to note that this was not seen here ; however , future gains in weight can not be ruled out . here , we report the maintenance of physical function of an individual after 49 days of complete fast .", "this report does not indicate support for such behavioral choices , and the projections of metabolic energy provision by starting adiposity assume no other perturbations to homeostasis , such as impaired immune response , liver and renal function , or cardiovascular events ."]]}
{"article_id": "PMC3162299", "article_text": ["tangential tractions on the vitreomacular interface are usually responsible for the development of idiopathic macular holes .", "the prevalence is 3.3/1000 and it shows a strong female predominance.1 in the past , it was known as a rare , untreatable disorder leading to central loss of vision . in 1991 ,", "kelly and wendel2 reported that idiopathic macular holes could be closed by vitrectomy and gas tamponade . with a better understanding of the pathogenesis of macular hole development and innovations in macular surgery ,", "one of the important developments in the surgical approach is peeling of the internal limiting membrane ( ilm ) .", "some authors have argued that ilm peeling has beneficial effects on the surgical outcome,3,4 whereas others have stated that this approach does not affect surgical success,5,6 and may lead to complications by increasing the surgery duration.7,8 the ilm is formed by mller cell extensions and functions as a kind of basal membrane between retina and vitreus , acting as a surface for glial cell proliferation .", "vitreoretinal interface changes due to cellular proliferation lead to ilm distortion and eventually to the development of epiretinal membrane ( erm ) , macular hole , and recurring macular edema.9 with the removal of the ilm , successful results may be obtained in surgical treatment of such vitreoretinal diseases .", "the most difficult aspect of surgery is due to the tight attachment of the ilm to the retina and its extreme thinness and transparent nature . to increase the visibility of the ilm", "indocyanine green ( icg ) and trypan blue have been used by many researchers in peeling of the erm and ilm .", "there are different opinions on the amount and concentration of dye required , and exposure duration in the vitreous space . in many studies , because of intraretinal accumulation of icg and its toxic effects on retinal pigment epithelium , visual field defects after surgery and possible optical nerve atrophy have been considered.8,10,11 recently , brilliant blue ( bb ) has been used in preclinical studies as it has minimal toxic effect and provides effective membrane staining.9,12 in this study , the anatomical and visual outcomes of pars plana vitrectomy ( ppv ) and ilm peeling with bb in the treatment of idiopathic macular holes has been examined .", "fifty eyes of 48 patients who had 23 g ppv and ilm peeling as a treatment for macular holes between july 2007 and december 2009 were retrospectively evaluated in the authors clinic .", "all patients underwent measurement of best - corrected visual acuity and intraocular pressure , anterior segment and fundus examinations before surgery .", "the possible merits and risks of the treatment were explained to the patients , and informed consent was obtained from all patients in accordance with the helsinki declaration .", "inclusion criteria were : diagnosis using noncontact - lens biomicroscopy and optical coherence tomography ( oct ) of stages 2 , 3 , and 4 idiopathic macular hole according to the gass classification .", "exclusion criteria were : trauma history ; previous macular surgery , rhegmatogenous retinal detachment together with macular hole ; myopia higher than 10d ; macular hole for more than 2 years ; and previous retinal vessel disease .", "macular examination was performed before surgery and 1 , 3 , and 6 months after surgery using spectral oct ( optovue inc , fremont , ca ) .", "all patients had a 23 g transconjunctival three port pars plana vitrectomy . in four eyes having both macular hole and erm", ", both ilm and erm peeling were performed . to visualize the ilm , 0.3 ml", ", 16% c3f8 gas was injected into 28 eyes ( 56% ) and 18% c2f6 gas into 22 eyes ( 44% ) .", "follow - up time points included 1-day , 1-week , 1-month , 3-month , and 6-month postsurgical evaluations and thereafter visits at 3-month intervals .", "best - corrected visual acuity , intraocular pressure , and complications were recorded for each visit .", "anatomical and functional outcomes of macular hole patients who underwent 23 g ppv and ilm peeling with bb were evaluated .", "cases in different macular hole stages were compared in terms of anatomical and functional results .", "all operations were performed under local anesthesia . following the introduction of a 23 g infusion cannula at the inferotemporal site ,", "imaging systems used were the volk miniquad xl ( volk optical ltd , mentor , oh ) during vitrectomy and volk central retina ( volk optical ltd ) during ilm peeling . after core vitrectomy , triamcinolone acetonide ( kenacort - a ; bristol myers squibb , new york , ny ) was used to remove the posterior hyaloid .", "brilliant blue 0.3 ml ( fluoron gmbh , ludwigsfeld , germany ) was injected to the posterior pole via a 23 g dualbore cannula .", "after 60 seconds , peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper ( synergetics inc , fort collins , co ) and 23 g eckardt end - gripping forceps ( dorc , zuidland , the netherlands ) for an area of at least 3 disc diameters .", "after liquid  air exchange , air  gas exchange was performed ( 16% c3f8 or 18% c2f6 ) . none of the patients required sutures .", "all operations were performed under local anesthesia . following the introduction of a 23 g infusion cannula at the inferotemporal site ,", "imaging systems used were the volk miniquad xl ( volk optical ltd , mentor , oh ) during vitrectomy and volk central retina ( volk optical ltd ) during ilm peeling . after core vitrectomy , triamcinolone acetonide ( kenacort - a ; bristol myers squibb , new york , ny ) was used to remove the posterior hyaloid .", "brilliant blue 0.3 ml ( fluoron gmbh , ludwigsfeld , germany ) was injected to the posterior pole via a 23 g dualbore cannula .", "after 60 seconds , peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper ( synergetics inc , fort collins , co ) and 23 g eckardt end - gripping forceps ( dorc , zuidland , the netherlands ) for an area of at least 3 disc diameters .", "after liquid  air exchange , air  gas exchange was performed ( 16% c3f8 or 18% c2f6 ) .", "the patient group consisted of 33 ( 66% ) female and 17 ( 34% ) male subjects ( mean age  sd ; 63.34  9.6 ) . of these cases ,", "preoperative oct examination revealed that , according to gass classification , 17 eyes ( 34% ) had stage 2 macular holes , 24 eyes ( 48% ) had stage 3 , and 9 eyes ( 18% ) had stage 4 .", "anatomic success was assessed by the closure of the hole on oct imaging and the withdrawal of subretinal fluid .", "the hole was closed in 46 ( 92% ) of the 50 eyes after surgery but not closed in 4 ( 8% ) eyes .", "when postoperative oct controls revealed that subretinal fluid had disappeared , there was no need for a second surgery .", "hole persistence was observed in only one patient 2 months after the surgery and it was closed by reoperation .", "figure 3 shows pre- and postoperative third - month spectral oct imaging of a patient with a stage 4 macular hole .", "the mean best - corrected visual acuity of all patients before surgery was 0.71  0.25 logmar .", "postoperative mean best - corrected visual acuity ( and comparison with preoperative values ) was obtained as : 0.48  0.26 at 3 months ( p < 0.05 ) , 0.52  0.27 at 6 months ( p < 0.05 ) , 0.42  0.30 at 9 months ( p < 0.05 ) , and 0.41  0.31 at 12 months ( p < 0.05 ) . when assessed together , the final visits ( at 12 months ) demonstrated that visual acuity had increased in 41 eyes ( 82% ) , whereas , in 6 eyes ( 12% ) , visual acuity remained unchanged . because of recurrent macular holes , three cases showed a decrease in visual acuity .", "two of these three cases had stage 4 macular holes , and one had a stage 3 macular hole .", "best - corrected visual acuity results of patients before and after surgery are given in figure 1 and table 2 . when evaluated according to the stage of macular hole , in stage 2 patients , all measurements of visual acuity after surgery showed that it was significantly higher when compared with preoperative measurements ( p < 0.05 ) .", "increase in visual acuity between 6 and 12 months after surgery was statistically significant due to cataract surgery ( p < 0.05 ) . in stage 3", "patients , preoperative visual acuity compared with postoperative 3-month scores showed an increase , but this was not statistically significant ( p = 0.056 ) . during the postoperative period , visual acuity reduced between months 3 and 6 due to cataract development . in patients who underwent phacoemulsification after 6 months ,", "therefore , comparisons of postoperative 912-month values showed significant increases ( p < 0.05 ) .", "in contrast , in stage 4 patients , visual acuity scores of all time points after surgery failed to reach statistically significant levels with respect to preoperative values .", "therefore , the first month visual acuity measurements were excluded from analysis . in 22 phakic patients ,", "the mean preoperative visual acuity of stage 2 macular hole patients was higher than that of stage 3 or 4 patients .", "consequently , the most prominent increase in visual acuity was observed in the stage 2 group after surgery .", "there was no significant difference between stage 2 and 3 macular hole patients in terms of anatomical and visual outcomes , but the functional successes of these two groups were significantly higher than those of stage 4 patients .", "iatrogenic retinal tears developed in four patients during surgery and were successfully treated intraoperatively . in one patient , a nasal retinal tear developed near the optic nerve while removing the posterior hyaloid .", "one of the phakic cases had preoperative lens opacity and vitrectomy and phacoemulsification was performed together with intracapsular lens implantation .", "since the first report of kelly and wendel2 on the treatment of macular holes by pars plana vitrectomy and gas tamponade , anatomical and functional success rates have increased with developments in vitreoretinal surgery .", "previous studies reported success rates over 90% after one session of surgery13,14 and anatomical success rates as high as 100% in stage 2 or the initial phase of stage 3 macular holes.15 in histopathological studies,16,17 various surgical approaches have been proposed based on the idea that collagen - containing myofibroblasts and actin - containing cells in the structure of ilm and erm may cause contraction and lead to hole formation or widening of an existing hole .", "many studies1821 have reported that ilm peeling increases anatomical and functional success in the treatment of macular hole , and during the late phases it reduces reformation of the hole .", "kwok et al18 compared anatomical closure of stage 3 or 4 holes in 40 patients with or without ilm peeling .", "success rate was 89% in the ilm peeling group whereas it was 59% in the group without ilm peeling .", "the differences in anatomical closure success may be due to stage differences of macular holes , inclusion of traumatic cases , high myopic eyes , and recurrent cases .", "although there is no consensus on the effects of ilm peeling on anatomical and functional success , publications stating that ilm peeling increases hole closure are in the majority.22,23 there are several opinions regarding which patients require ilm peeling .", "previous studies have stated that ilm peeling should be considered especially in patients with a hole wider than 400 microns and that is chronic ( 6 months or longer ) , traumatic , or recurring.2125", "ilm peeling was reported to increase anatomical success and prevent reopening of the hole by decreasing erm development.25,26 besides reports that suggest that ilm peeling increases anatomical success but not functional success,27 there are also reports that suggest functional outcomes are better in ilm peeling groups.18 in this study , ilm peeling was applied to all patients who underwent macular hole surgery .", "anatomic closure was obtained in 46 ( 92% ) of the 50 eyes , but the hole was not closed in four patients .", "visual outcomes were significantly better in stage 2 or 3 patients than in stage 4 patients ( p < 0.05 ) .", "the authors consider that ilm peeling improved anatomical and functional success rates in stages 2 and 3 patients whereas it had no effect on visual outcomes in stage 4 patients due to photoreceptor loss .", "cataract development is the most prevalent complication of macular hole surgery in phakic eyes . in this study group , cataract development", "was observed in 23 ( 74% ) of 31 patients due to an endotamponade application . besides publications that report", " early cataract surgery following macular hole surgery may increase the risk of re - opening of the hole,28 there are also studies that report no such risk.29,30 therefore , in patients developing cataract , phacoemulsification surgery was performed at least 6 months after macular hole surgery to prevent a recurrence . as the thin and semitransparent structure of the ilm complicates visualization during surgery , in patients with ilm peeling , there may be small asymptomatic paracentral scotomas , irregularity in nerve fiber layers , and retinal microhemorrhages due to iatrogenic retinal trauma.3136 chromovitrectomy has been developed as a method to improve ilm visibility , shorten the duration of surgery , and reduce iatrogenic retinal trauma.36 many dyes , such as indocyanine green ( icg ) , infracyanine green ( ifcg ) , trypan blue ( tb ) , bb , and triamcinolone acetonide ( ta ) , are used to dye the ilm .", "since icg was first introduced for ilm peeling in macular hole surgery , several potential side effects have been noted.37,38 visual field defects , retinal pigment epithelium , and ganglion cell defects are among the most reported side effects.26,32,33 dose - dependent biochemical damage to the retinal pigment epithelium and ganglion cells , photo - oxidative cell damage due to the phototoxic properties of icg , and retinal pigment epithelium damage due to hypoosmotic solution are considered responsible for icg - mediated ocular toxicity.3941 because of the potential toxic side effects of icg in chromovitrectomy , other vital dyes with minimal toxicity have been tried.32,33,38,42 ifcg , unlike icg , does not contain iodine , and it is widely thought that its toxicity to the retinal pigment epithelium is less than that of icg.31 triamcinolone acetonide was reported as both toxic4345 and nontoxic46 to retinal pigment epithelium in preclinical studies . in the authors opinion , there is an insufficient number of randomized clinical trials to make a conclusion .", "however , in some studies of ta - assisted ilm peeling , it was found similar to icg.43,47 another vital dye , tb , is recommended for erm staining because of its high affinity for proliferation - dense intraocular tissues.48 several preclinical studies with high - concentration tb reported toxic effects on tissue culture of retinal pigment epithelium.49,50 however , many other researchers claim that if it is used in lower concentrations , there will be no toxic effects.51,52 in this study , bb , a relatively new type of vital dye , was used to stain the ilm .", "enaida et al9 studied icg on rats and found that low - dose icg administration resulted in no retinal cell damage but high dose icg led to morphological damage of retinal cells . in a rat study by hisatomi et al and enaida et al,53,54 low dose intravitreal bb administration caused no effects on retinal cells , but in higher doses electron microscopy revealed cyst formation in the inner layers of the retina .", "the authors of this present study think that biological adaptation to bb is better than to icg . the use of bb is easier than icg and tb because of its granular structure and easy dissolution in intraocular irrigation .", "when compared with bb , higher concentrations of icg are required to dye the ilm.12 in addition , bb is not a fluorescent dye , so its phototoxicity is lower than icg .", "the authors of this study have observed intraretinal accumulation in postoperative fundus fluorescein angiography of patients for whom icg was used .", "ilm peeling in macular hole surgery provides beneficial effects on anatomical outcome , independent of disease stage . in terms of visual outcome"], "abstract_text": ["<S> purpose : the evaluation of anatomic and visual outcomes in macular hole cases treated with internal limiting membrane ( ilm ) peeling , brilliant blue ( bb ) , and 23-gauge pars plana vitrectomy ( ppv).materials and methods : fifty eyes of 48 patients who presented between july 2007 and december 2009 with the diagnosis of stage 2 , 3 , or 4 macular holes according to gass classification who had undergone ppv and ilm peeling were included in this study . </S>", "<S> pre- and postoperative macular examinations were assessed with spectral - domain optical coherence tomography . </S>", "<S> 23 g sutureless ppv and ilm peeling with bb was performed on all patients.results:the mean age of patients was 63.34  9.6 years . </S>", "<S> stage 2 macular hole was determined in 17 eyes ( 34% ) , stage 3 in 24 eyes ( 48% ) , and stage 4 in 9 eyes ( 18% ) . </S>", "<S> the mean follow - up time was 13.6  1.09 months . </S>", "<S> anatomic closure was detected in 46/50 eyes ( 92% ) , whereas , in four cases , macular hole persisted and a second operation was not required due to subretinal fluid drainage . at follow - up after 2 months , persistant macular hole was detected in one case and it was closed with reoperation . at 12 months , </S>", "<S> an increase in visual acuity in 41 eyes was observed , while it remained at the same level in six eyes . in three eyes visual acuity </S>", "<S> decreased . </S>", "<S> there was a postoperative statistically significant increase in visual acuity in stage 2 and 3 cases ( p < 0.05 ) , however , no increase in visual acuity in stage 4 cases was observed.conclusion:ppv and ilm peeling in stage 2 , 3 , and 4 macular hole cases provide successful anatomic outcomes , however , in delayed cases , due to photoreceptor loss , it has no effect on functional recovery . </S>", "<S> bb , used for clarity of ilm , may be beneficial due to its low retinal toxicity . </S>"], "labels": null, "section_names": ["Introduction", "Methods and materials", "Surgical technique", "Results", "Discussion", "Conclusion"], "sections": [["tangential tractions on the vitreomacular interface are usually responsible for the development of idiopathic macular holes .", "the prevalence is 3.3/1000 and it shows a strong female predominance.1 in the past , it was known as a rare , untreatable disorder leading to central loss of vision . in 1991 ,", "kelly and wendel2 reported that idiopathic macular holes could be closed by vitrectomy and gas tamponade . with a better understanding of the pathogenesis of macular hole development and innovations in macular surgery ,", "one of the important developments in the surgical approach is peeling of the internal limiting membrane ( ilm ) .", "some authors have argued that ilm peeling has beneficial effects on the surgical outcome,3,4 whereas others have stated that this approach does not affect surgical success,5,6 and may lead to complications by increasing the surgery duration.7,8 the ilm is formed by mller cell extensions and functions as a kind of basal membrane between retina and vitreus , acting as a surface for glial cell proliferation .", "vitreoretinal interface changes due to cellular proliferation lead to ilm distortion and eventually to the development of epiretinal membrane ( erm ) , macular hole , and recurring macular edema.9 with the removal of the ilm , successful results may be obtained in surgical treatment of such vitreoretinal diseases .", "the most difficult aspect of surgery is due to the tight attachment of the ilm to the retina and its extreme thinness and transparent nature . to increase the visibility of the ilm", "indocyanine green ( icg ) and trypan blue have been used by many researchers in peeling of the erm and ilm .", "there are different opinions on the amount and concentration of dye required , and exposure duration in the vitreous space . in many studies , because of intraretinal accumulation of icg and its toxic effects on retinal pigment epithelium , visual field defects after surgery and possible optical nerve atrophy have been considered.8,10,11 recently , brilliant blue ( bb ) has been used in preclinical studies as it has minimal toxic effect and provides effective membrane staining.9,12 in this study , the anatomical and visual outcomes of pars plana vitrectomy ( ppv ) and ilm peeling with bb in the treatment of idiopathic macular holes has been examined ."], ["fifty eyes of 48 patients who had 23 g ppv and ilm peeling as a treatment for macular holes between july 2007 and december 2009 were retrospectively evaluated in the authors clinic .", "all patients underwent measurement of best - corrected visual acuity and intraocular pressure , anterior segment and fundus examinations before surgery .", "the possible merits and risks of the treatment were explained to the patients , and informed consent was obtained from all patients in accordance with the helsinki declaration .", "inclusion criteria were : diagnosis using noncontact - lens biomicroscopy and optical coherence tomography ( oct ) of stages 2 , 3 , and 4 idiopathic macular hole according to the gass classification .", "exclusion criteria were : trauma history ; previous macular surgery , rhegmatogenous retinal detachment together with macular hole ; myopia higher than 10d ; macular hole for more than 2 years ; and previous retinal vessel disease .", "macular examination was performed before surgery and 1 , 3 , and 6 months after surgery using spectral oct ( optovue inc , fremont , ca ) .", "all patients had a 23 g transconjunctival three port pars plana vitrectomy . in four eyes having both macular hole and erm", ", both ilm and erm peeling were performed . to visualize the ilm , 0.3 ml", ", 16% c3f8 gas was injected into 28 eyes ( 56% ) and 18% c2f6 gas into 22 eyes ( 44% ) .", "follow - up time points included 1-day , 1-week , 1-month , 3-month , and 6-month postsurgical evaluations and thereafter visits at 3-month intervals .", "best - corrected visual acuity , intraocular pressure , and complications were recorded for each visit .", "anatomical and functional outcomes of macular hole patients who underwent 23 g ppv and ilm peeling with bb were evaluated .", "cases in different macular hole stages were compared in terms of anatomical and functional results .", "all operations were performed under local anesthesia . following the introduction of a 23 g infusion cannula at the inferotemporal site ,", "imaging systems used were the volk miniquad xl ( volk optical ltd , mentor , oh ) during vitrectomy and volk central retina ( volk optical ltd ) during ilm peeling . after core vitrectomy , triamcinolone acetonide ( kenacort - a ; bristol myers squibb , new york , ny ) was used to remove the posterior hyaloid .", "brilliant blue 0.3 ml ( fluoron gmbh , ludwigsfeld , germany ) was injected to the posterior pole via a 23 g dualbore cannula .", "after 60 seconds , peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper ( synergetics inc , fort collins , co ) and 23 g eckardt end - gripping forceps ( dorc , zuidland , the netherlands ) for an area of at least 3 disc diameters .", "after liquid  air exchange , air  gas exchange was performed ( 16% c3f8 or 18% c2f6 ) . none of the patients required sutures ."], ["all operations were performed under local anesthesia . following the introduction of a 23 g infusion cannula at the inferotemporal site ,", "imaging systems used were the volk miniquad xl ( volk optical ltd , mentor , oh ) during vitrectomy and volk central retina ( volk optical ltd ) during ilm peeling . after core vitrectomy , triamcinolone acetonide ( kenacort - a ; bristol myers squibb , new york , ny ) was used to remove the posterior hyaloid .", "brilliant blue 0.3 ml ( fluoron gmbh , ludwigsfeld , germany ) was injected to the posterior pole via a 23 g dualbore cannula .", "after 60 seconds , peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper ( synergetics inc , fort collins , co ) and 23 g eckardt end - gripping forceps ( dorc , zuidland , the netherlands ) for an area of at least 3 disc diameters .", "after liquid  air exchange , air  gas exchange was performed ( 16% c3f8 or 18% c2f6 ) ."], ["the patient group consisted of 33 ( 66% ) female and 17 ( 34% ) male subjects ( mean age  sd ; 63.34  9.6 ) . of these cases ,", "preoperative oct examination revealed that , according to gass classification , 17 eyes ( 34% ) had stage 2 macular holes , 24 eyes ( 48% ) had stage 3 , and 9 eyes ( 18% ) had stage 4 .", "anatomic success was assessed by the closure of the hole on oct imaging and the withdrawal of subretinal fluid .", "the hole was closed in 46 ( 92% ) of the 50 eyes after surgery but not closed in 4 ( 8% ) eyes .", "when postoperative oct controls revealed that subretinal fluid had disappeared , there was no need for a second surgery .", "hole persistence was observed in only one patient 2 months after the surgery and it was closed by reoperation .", "figure 3 shows pre- and postoperative third - month spectral oct imaging of a patient with a stage 4 macular hole .", "the mean best - corrected visual acuity of all patients before surgery was 0.71  0.25 logmar .", "postoperative mean best - corrected visual acuity ( and comparison with preoperative values ) was obtained as : 0.48  0.26 at 3 months ( p < 0.05 ) , 0.52  0.27 at 6 months ( p < 0.05 ) , 0.42  0.30 at 9 months ( p < 0.05 ) , and 0.41  0.31 at 12 months ( p < 0.05 ) . when assessed together , the final visits ( at 12 months ) demonstrated that visual acuity had increased in 41 eyes ( 82% ) , whereas , in 6 eyes ( 12% ) , visual acuity remained unchanged . because of recurrent macular holes , three cases showed a decrease in visual acuity .", "two of these three cases had stage 4 macular holes , and one had a stage 3 macular hole .", "best - corrected visual acuity results of patients before and after surgery are given in figure 1 and table 2 . when evaluated according to the stage of macular hole , in stage 2 patients , all measurements of visual acuity after surgery showed that it was significantly higher when compared with preoperative measurements ( p < 0.05 ) .", "increase in visual acuity between 6 and 12 months after surgery was statistically significant due to cataract surgery ( p < 0.05 ) . in stage 3", "patients , preoperative visual acuity compared with postoperative 3-month scores showed an increase , but this was not statistically significant ( p = 0.056 ) . during the postoperative period , visual acuity reduced between months 3 and 6 due to cataract development . in patients who underwent phacoemulsification after 6 months ,", "therefore , comparisons of postoperative 912-month values showed significant increases ( p < 0.05 ) .", "in contrast , in stage 4 patients , visual acuity scores of all time points after surgery failed to reach statistically significant levels with respect to preoperative values .", "therefore , the first month visual acuity measurements were excluded from analysis . in 22 phakic patients ,", "the mean preoperative visual acuity of stage 2 macular hole patients was higher than that of stage 3 or 4 patients .", "consequently , the most prominent increase in visual acuity was observed in the stage 2 group after surgery .", "there was no significant difference between stage 2 and 3 macular hole patients in terms of anatomical and visual outcomes , but the functional successes of these two groups were significantly higher than those of stage 4 patients .", "iatrogenic retinal tears developed in four patients during surgery and were successfully treated intraoperatively . in one patient , a nasal retinal tear developed near the optic nerve while removing the posterior hyaloid .", "one of the phakic cases had preoperative lens opacity and vitrectomy and phacoemulsification was performed together with intracapsular lens implantation ."], ["since the first report of kelly and wendel2 on the treatment of macular holes by pars plana vitrectomy and gas tamponade , anatomical and functional success rates have increased with developments in vitreoretinal surgery .", "previous studies reported success rates over 90% after one session of surgery13,14 and anatomical success rates as high as 100% in stage 2 or the initial phase of stage 3 macular holes.15 in histopathological studies,16,17 various surgical approaches have been proposed based on the idea that collagen - containing myofibroblasts and actin - containing cells in the structure of ilm and erm may cause contraction and lead to hole formation or widening of an existing hole .", "many studies1821 have reported that ilm peeling increases anatomical and functional success in the treatment of macular hole , and during the late phases it reduces reformation of the hole .", "kwok et al18 compared anatomical closure of stage 3 or 4 holes in 40 patients with or without ilm peeling .", "success rate was 89% in the ilm peeling group whereas it was 59% in the group without ilm peeling .", "the differences in anatomical closure success may be due to stage differences of macular holes , inclusion of traumatic cases , high myopic eyes , and recurrent cases .", "although there is no consensus on the effects of ilm peeling on anatomical and functional success , publications stating that ilm peeling increases hole closure are in the majority.22,23 there are several opinions regarding which patients require ilm peeling .", "previous studies have stated that ilm peeling should be considered especially in patients with a hole wider than 400 microns and that is chronic ( 6 months or longer ) , traumatic , or recurring.2125", "ilm peeling was reported to increase anatomical success and prevent reopening of the hole by decreasing erm development.25,26 besides reports that suggest that ilm peeling increases anatomical success but not functional success,27 there are also reports that suggest functional outcomes are better in ilm peeling groups.18 in this study , ilm peeling was applied to all patients who underwent macular hole surgery .", "anatomic closure was obtained in 46 ( 92% ) of the 50 eyes , but the hole was not closed in four patients .", "visual outcomes were significantly better in stage 2 or 3 patients than in stage 4 patients ( p < 0.05 ) .", "the authors consider that ilm peeling improved anatomical and functional success rates in stages 2 and 3 patients whereas it had no effect on visual outcomes in stage 4 patients due to photoreceptor loss .", "cataract development is the most prevalent complication of macular hole surgery in phakic eyes . in this study group , cataract development", "was observed in 23 ( 74% ) of 31 patients due to an endotamponade application . besides publications that report", " early cataract surgery following macular hole surgery may increase the risk of re - opening of the hole,28 there are also studies that report no such risk.29,30 therefore , in patients developing cataract , phacoemulsification surgery was performed at least 6 months after macular hole surgery to prevent a recurrence . as the thin and semitransparent structure of the ilm complicates visualization during surgery , in patients with ilm peeling , there may be small asymptomatic paracentral scotomas , irregularity in nerve fiber layers , and retinal microhemorrhages due to iatrogenic retinal trauma.3136 chromovitrectomy has been developed as a method to improve ilm visibility , shorten the duration of surgery , and reduce iatrogenic retinal trauma.36 many dyes , such as indocyanine green ( icg ) , infracyanine green ( ifcg ) , trypan blue ( tb ) , bb , and triamcinolone acetonide ( ta ) , are used to dye the ilm .", "since icg was first introduced for ilm peeling in macular hole surgery , several potential side effects have been noted.37,38 visual field defects , retinal pigment epithelium , and ganglion cell defects are among the most reported side effects.26,32,33 dose - dependent biochemical damage to the retinal pigment epithelium and ganglion cells , photo - oxidative cell damage due to the phototoxic properties of icg , and retinal pigment epithelium damage due to hypoosmotic solution are considered responsible for icg - mediated ocular toxicity.3941 because of the potential toxic side effects of icg in chromovitrectomy , other vital dyes with minimal toxicity have been tried.32,33,38,42 ifcg , unlike icg , does not contain iodine , and it is widely thought that its toxicity to the retinal pigment epithelium is less than that of icg.31 triamcinolone acetonide was reported as both toxic4345 and nontoxic46 to retinal pigment epithelium in preclinical studies . in the authors opinion , there is an insufficient number of randomized clinical trials to make a conclusion .", "however , in some studies of ta - assisted ilm peeling , it was found similar to icg.43,47 another vital dye , tb , is recommended for erm staining because of its high affinity for proliferation - dense intraocular tissues.48 several preclinical studies with high - concentration tb reported toxic effects on tissue culture of retinal pigment epithelium.49,50 however , many other researchers claim that if it is used in lower concentrations , there will be no toxic effects.51,52 in this study , bb , a relatively new type of vital dye , was used to stain the ilm .", "enaida et al9 studied icg on rats and found that low - dose icg administration resulted in no retinal cell damage but high dose icg led to morphological damage of retinal cells . in a rat study by hisatomi et al and enaida et al,53,54 low dose intravitreal bb administration caused no effects on retinal cells , but in higher doses electron microscopy revealed cyst formation in the inner layers of the retina .", "the authors of this present study think that biological adaptation to bb is better than to icg . the use of bb is easier than icg and tb because of its granular structure and easy dissolution in intraocular irrigation .", "when compared with bb , higher concentrations of icg are required to dye the ilm.12 in addition , bb is not a fluorescent dye , so its phototoxicity is lower than icg .", "the authors of this study have observed intraretinal accumulation in postoperative fundus fluorescein angiography of patients for whom icg was used ."], ["ilm peeling in macular hole surgery provides beneficial effects on anatomical outcome , independent of disease stage . in terms of visual outcome"]]}
{"article_id": "PMC3046818", "article_text": ["the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .", "these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute  casa sollievo della sofferenza  from 2001 to 2008 and monitored until the end of 2009 .", "ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .", "they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .", "exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress", ", only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .", "two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .", "this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .", "exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .", "blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .", "of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .", "the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .", "information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events", "was obtained by telephone interview , from their primary care physicians , or from death certificates .", "deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .", "a total of 339 white patients with type 2 diabetes who had survived an mi were studied .", "they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,", " casa sollievo della sofferenza  in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .", "most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .", "another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .", "all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .", "a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .", "a random blood sample was obtained from the cross - sectional study participants recruited in boston .", "bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .", "presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .", "current and former smokers were considered as one group and compared with those who never smoked .", "all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration .", "genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) .", "patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .", "comparisons between genotype groups were performed by pearson  or mann - whitney u tests for continuous or categoric variables , respectively .", "because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies ,", "a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event", "was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,", "the time variable was defined as the time between the enrollment date and the date of the last available clinical data .", "the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,", "the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as  regression coefficients .", "pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for  study sample  ) after excluding genotype - by - sample interactions .", "genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .", "the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) .", "all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) .", "the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .", "these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute  casa sollievo della sofferenza  from 2001 to 2008 and monitored until the end of 2009 .", "ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .", "they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .", "exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress", ", only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .", "two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .", "this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .", "exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .", "blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .", "of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .", "the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .", "information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events", "was obtained by telephone interview , from their primary care physicians , or from death certificates .", "deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .", "a total of 339 white patients with type 2 diabetes who had survived an mi were studied .", "they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,", " casa sollievo della sofferenza  in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .", "most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .", "another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .", "the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .", "these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute  casa sollievo della sofferenza  from 2001 to 2008 and monitored until the end of 2009 .", "ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .", "they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .", "exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress", ", only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .", "two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .", "this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .", "exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .", "blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .", "of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .", "the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .", "information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events", "was obtained by telephone interview , from their primary care physicians , or from death certificates .", "deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .", "a total of 339 white patients with type 2 diabetes who had survived an mi were studied .", "they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,", " casa sollievo della sofferenza  in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .", "most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .", "another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .", "all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .", "a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .", "a random blood sample was obtained from the cross - sectional study participants recruited in boston .", "bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .", "presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .", "current and former smokers were considered as one group and compared with those who never smoked .", "all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration . written informed consent was obtained from each study participant .", "genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) .", "patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .", "comparisons between genotype groups were performed by pearson  or mann - whitney u tests for continuous or categoric variables , respectively .", "deviations from hardy - weinberg equilibrium were investigated by exact  test . because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies , a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event", "was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,", "the time variable was defined as the time between the enrollment date and the date of the last available clinical data .", "the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,", "the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as  regression coefficients .", "pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for  study sample  ) after excluding genotype - by - sample interactions .", "genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .", "the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) . a value of p < 0.05 was considered significant .", "all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) .", "we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs  individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .", "no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .", "clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) .", "the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .", "the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .", "no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .", "incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .", "the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .", "1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,", "the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .", "hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .", "bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .", "the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .", "kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .", "the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) .", "given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .", "an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) . no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) .", "because approximately 85% of these two cohorts consisted of nondiabetic individuals , we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .", "indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .", "thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .", "2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) .", "the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .", "by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .", "survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .", "curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,", "the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .", "a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) . to seek replication of the gene - by - obesity interaction observed in patients with diabetes", ", we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .", "one sample was from the gargano area in italy , the other was from boston .", "salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for  study sample .", " to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi  and diabetes  because all study participants were diabetic )", "64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .", "in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .", "clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .", "virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .", "in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers (  = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .", "3a ) , but not among kk individuals (  = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .", "3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .", "data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston .", "we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs  individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .", "no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .", "clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) .", "the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .", "the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .", "no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .", "incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .", "the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .", "1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,", "the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .", "age at study entry ( hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .", "bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .", "the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .", "kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .", "the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) .", "given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .", "an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) .", "no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) . because approximately 85% of these two cohorts consisted of nondiabetic individuals", ", we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .", "indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .", "thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .", "2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) . the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .", "by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .", "survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .", "curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,", "the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .", "a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) .", "to seek replication of the gene - by - obesity interaction observed in patients with diabetes , we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .", "one sample was from the gargano area in italy , the other was from boston .", "salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for  study sample .", " to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi  and diabetes  because all study participants were diabetic ) were included as covariates . among obese subjects ,", "64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .", "in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .", "clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .", "virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .", "in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers (  = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .", "3a ) , but not among kk individuals (  = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .", "3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .", "data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston .", "our results indicate that the enpp1 k121q polymorphism predicts acceleration of major cardiovascular events in very high - risk patients .", "the increased risk conferred by the q121 variant is independent of that of age , sex , bmi , diabetes , and cigarette smoking .", "our findings are in agreement with a previous cross - sectional study of 445 mi survivors from central europe ( 29 ) .", "by contrast , case - control genome - wide association studies reported that a single nucleotide polymorphism ( snp , rs7767502 ) , which is in perfect linkage disequilibrium with the enpp1 k121q polymorphism , was not associated with cad ( 610 ) .", "several differences between our study and the genome - wide association studies , such as the prospective versus cross - sectional designs , the different end points under investigation , and the different baseline cardiovascular risk , with only the patients enrolled in our study being very high - risk as per selection criteria , might be responsible for this apparent discordance .", "an additional important result of our study is that the effect of the q121 variant was modulated by obesity in diabetic patients among whom the risk of incident events was five times higher in q121 than in kk genotype carriers .", "although not the aim of our study , one can infer that obese individuals ( fig .", "2a ) as a whole tend to have a lower risk of future cardiovascular events than nonobese patients ( fig . 2b ; adjusted hr 0.68 [ 95% ci 0.4111.124 ] , p = 0.13 ) .", "this paradoxic protective effect of obesity resembles that observed in patients with cad ( 41 ) , esrd ( 42 ) , heart failure ( 43 ) , and older age ( 44 ) , all conditions heavily over - represented in our samples . in this context , the q121 variant seems to eliminate the paradoxic protective effect of obesity .", "an important finding was that the q121 variant - by - obesity interaction observed in the prospective study was replicated in a cross - sectional study on age at mi in diabetic patients .", "information on the k121q genotypes tended to improve risk prediction in these patients when the improvement was measured by the idi , the approach that is currently favored to evaluate predictive ability increase conferred by a new marker when added to a well - performing model ( 39 ) .", "thus , pending further validation in larger studies , one can hypothesize clinical implementation of the q121 variant as a marker of early cardiovascular events among obese diabetic patients .", "given the increasing incidence worldwide of both obesity and diabetes ( 24 ) and the poor ability to stratify cardiovascular risk among diabetic patients , a large sector of society would be likely to benefit in the future from the availability of such a test .", "the synergistic effect of the genetic marker and obesity in the modulation of cardiovascular risk resembles results repeatedly reported in the risk modulation of insulin resistance and related traits ( 2527,30,33,4549 ) .", "placed in a broader perspective , this is an excellent example of genetic heterogeneity ( i.e. , different genetic effects being at play in different population subgroups ) and clearly illustrates how accounting for such heterogeneity may be critical to dissect the genetic architecture of multifactorial diseases .", "understanding the mechanisms through which the q121 variant is associated with cvd is beyond the scope of this study .", "however , one can speculate that the q121 variant exacerbates cardiovascular risk by inducing systemic insulin resistance ( 22,2527 ) and proatherogenic phenotypes ( 24,29,30,33 )", ". it may also act by way of a direct detrimental effect on insulin - dependent endothelial function , as suggested by the observation that human endothelial cells carrying the q121 variant show impaired insulin receptor signaling and , most importantly , reduced release of nitric oxide ( 24 ) , a potent vasodilator whose deficiency is an established early step in the pathway development of atherosclerosis ( 50 ) .", "one can hypothesize that the interaction between the q121 variant and obesity is sustained by the different sites of action on the insulin - signaling pathway .", "although enpp1 acts at the insulin receptor level ( 21,23 ) , obesity acts by different mechanisms , mostly at a postreceptor level ( 51 ) .", "it is , therefore , possible that postreceptor insulin - signaling abnormalities are necessary for the q121 variant to be fully effective in inducing insulin resistance and , eventually , related clinical outcomes .", "the three cohorts of very high - risk individuals that we studied were quite different from each other : one comprised only patients with type 2 diabetes and cad , another included patients with a previous mi who did not have frank type 2 diabetes , and the third included only patients with esrds . despite such apparent phenotypic heterogeneity , the effect of the q121 variant was not heterogeneous across the three studies .", "not only did this allow us to analyze the three cohorts together , increasing statistical power , but it also suggests that our findings may be generalizable to all high - risk patients , irrespective of their background clinical characteristics .", "whether the predictive role of the q121 variant extends to situations characterized by a more moderate cardiovascular risk remains to be determined .", "we acknowledge that , mainly because of the relatively small size of our samples , the significance level of our findings is still compatible with a false - positive result .", "however , this seems unlikely given that the association between q121 was not heterogeneous across the three cohorts and , importantly , was further confirmed in cross - sectional studies as far as the interaction with obesity in diabetic patients is concerned .", "we also acknowledge that due to the relatively small sample size of the studies that we analyzed , we can not exclude that the gene - by - obesity interaction that we observed among diabetic patients also occurs among nondiabetic individuals , as is the case for the modulation of insulin resistance ( 2527 ) .", "therefore , our findings need further replication in larger samples before they can be considered as established . finally , because this study was entirely performed in individuals of european ancestry , we do not know whether our findings can be extended to populations of different race . in conclusion ,", "pending confirmation in further larger studies , the q121 variant has the potential to become a clinical tool for identifying those very high - risk patients who are especially prone to major cardiovascular events and need , therefore , to be targeted with specific and even more aggressive preventive strategies ."], "abstract_text": ["<S> objectiveinsulin resistance ( ir ) and cardiovascular disease may share a common genetic background . </S>", "<S> we investigated the role of ir - associated enpp1 k121q polymorphism ( rs1044498 ) on cardiovascular disease in high - risk individuals.research design and methodsa prospective study ( average follow - up , 37 months ) was conducted for major cardiovascular events ( myocardial infarction [ mi ] , stroke , cardiovascular death ) from the gargano heart study ( ghs ; n = 330 with type 2 diabetes and coronary artery disease ) , the tor vergata atherosclerosis study ( tvas ; n = 141 who had mi ) , and the cardiovascular risk extended evaluation in dialysis ( creed ) database ( n = 266 with end - stage renal disease ) </S>", "<S> . age at mi was investigated in cross - sectional studies of 339 type 2 diabetic patients ( n = 169 from italy , n = 170 from the u.s.).resultsincidence of cardiovascular events per 100 person -- years was 4.2 in ghs , 10.8 in tvas , and 11.7 in creed . hazard ratios ( hrs ) for kq+qq versus individuals carrying the k121/k121 genotype ( kk ) individuals were 1.47 ( 95% ci 0.802.70 ) in ghs , 2.31 ( 95% ci 1.224.34 ) in tvas , and 1.36 ( 95% ci 0.882.10 ) in creed , and 1.56 ( 95% ci 1.152.12 ) in the three cohorts combined . in the 395 diabetic patients , the q121 variant predicted cardiovascular events among obese but not among nonobese individuals </S>", "<S> ( hr 5.94 vs. 0.62 , p = 0.003 for interaction ) . </S>", "<S> a similar synergism was observed in cross - sectional studies , with age at mi being 3 years younger in q121 carriers than in kk homozygotes among obese but not among nonobese patients ( p = 0.035 for interaction).conclusionsthe enpp1 k121q polymorphism is an independent predictor of major cardiovascular events in high - risk individuals . in type 2 diabetes , this effect is exacerbated by obesity . </S>", "<S> future larger studies are needed to confirm our finding . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Study participants", "Prospective studies.", "Cross-sectional study of age at MI.", "Data collection.", "Genotyping.", "Statistical analysis.", "RESULTS", "Characteristics of cohort members at baseline.", "None", "Interaction between Q121 variant and obesity in predicting major cardiovascular events.", "Interaction between Q121 variant and obesity on age at MI in cross-sectional studies.", "DISCUSSION"], "sections": [["the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .", "these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute  casa sollievo della sofferenza  from 2001 to 2008 and monitored until the end of 2009 .", "ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .", "they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .", "exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress", ", only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .", "two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .", "this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .", "exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .", "blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .", "of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .", "the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .", "information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events", "was obtained by telephone interview , from their primary care physicians , or from death certificates .", "deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .", "a total of 339 white patients with type 2 diabetes who had survived an mi were studied .", "they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,", " casa sollievo della sofferenza  in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .", "most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .", "another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .", "all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .", "a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .", "a random blood sample was obtained from the cross - sectional study participants recruited in boston .", "bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .", "presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .", "current and former smokers were considered as one group and compared with those who never smoked .", "all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration .", "genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) .", "patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .", "comparisons between genotype groups were performed by pearson  or mann - whitney u tests for continuous or categoric variables , respectively .", "because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies ,", "a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event", "was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,", "the time variable was defined as the time between the enrollment date and the date of the last available clinical data .", "the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,", "the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as  regression coefficients .", "pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for  study sample  ) after excluding genotype - by - sample interactions .", "genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .", "the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) .", "all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) ."], ["the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .", "these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute  casa sollievo della sofferenza  from 2001 to 2008 and monitored until the end of 2009 .", "ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .", "they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .", "exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress", ", only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .", "two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .", "this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .", "exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .", "blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .", "of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .", "the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .", "information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events", "was obtained by telephone interview , from their primary care physicians , or from death certificates .", "deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .", "a total of 339 white patients with type 2 diabetes who had survived an mi were studied .", "they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,", " casa sollievo della sofferenza  in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .", "most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .", "another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) ."], ["the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .", "these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute  casa sollievo della sofferenza  from 2001 to 2008 and monitored until the end of 2009 .", "ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .", "they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .", "exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress", ", only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .", "two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .", "this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .", "exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .", "blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .", "of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .", "the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .", "information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events", "was obtained by telephone interview , from their primary care physicians , or from death certificates .", "deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) ."], ["a total of 339 white patients with type 2 diabetes who had survived an mi were studied .", "they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,", " casa sollievo della sofferenza  in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .", "most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .", "another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) ."], ["all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .", "a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .", "a random blood sample was obtained from the cross - sectional study participants recruited in boston .", "bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .", "presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .", "current and former smokers were considered as one group and compared with those who never smoked .", "all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration . written informed consent was obtained from each study participant ."], ["genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) ."], ["patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .", "comparisons between genotype groups were performed by pearson  or mann - whitney u tests for continuous or categoric variables , respectively .", "deviations from hardy - weinberg equilibrium were investigated by exact  test . because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies , a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event", "was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,", "the time variable was defined as the time between the enrollment date and the date of the last available clinical data .", "the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,", "the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as  regression coefficients .", "pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for  study sample  ) after excluding genotype - by - sample interactions .", "genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .", "the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) . a value of p < 0.05 was considered significant .", "all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) ."], ["we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs  individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .", "no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .", "clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) .", "the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .", "the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .", "no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .", "incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .", "the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .", "1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,", "the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .", "hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .", "bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .", "the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .", "kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .", "the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) .", "given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .", "an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) . no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) .", "because approximately 85% of these two cohorts consisted of nondiabetic individuals , we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .", "indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .", "thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .", "2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) .", "the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .", "by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .", "survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .", "curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,", "the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .", "a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) . to seek replication of the gene - by - obesity interaction observed in patients with diabetes", ", we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .", "one sample was from the gargano area in italy , the other was from boston .", "salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for  study sample .", " to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi  and diabetes  because all study participants were diabetic )", "64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .", "in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .", "clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .", "virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .", "in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers (  = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .", "3a ) , but not among kk individuals (  = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .", "3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .", "data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston ."], ["we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs  individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .", "no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .", "clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) ."], ["the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .", "the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .", "no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .", "incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .", "the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .", "1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,", "the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .", "age at study entry ( hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .", "bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .", "the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .", "kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .", "the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) ."], ["given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .", "an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) .", "no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) . because approximately 85% of these two cohorts consisted of nondiabetic individuals", ", we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .", "indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .", "thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .", "2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) . the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .", "by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .", "survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .", "curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,", "the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .", "a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) ."], ["to seek replication of the gene - by - obesity interaction observed in patients with diabetes , we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .", "one sample was from the gargano area in italy , the other was from boston .", "salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for  study sample .", " to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi  and diabetes  because all study participants were diabetic ) were included as covariates . among obese subjects ,", "64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .", "in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .", "clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .", "virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .", "in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers (  = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .", "3a ) , but not among kk individuals (  = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .", "3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .", "data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston ."], ["our results indicate that the enpp1 k121q polymorphism predicts acceleration of major cardiovascular events in very high - risk patients .", "the increased risk conferred by the q121 variant is independent of that of age , sex , bmi , diabetes , and cigarette smoking .", "our findings are in agreement with a previous cross - sectional study of 445 mi survivors from central europe ( 29 ) .", "by contrast , case - control genome - wide association studies reported that a single nucleotide polymorphism ( snp , rs7767502 ) , which is in perfect linkage disequilibrium with the enpp1 k121q polymorphism , was not associated with cad ( 610 ) .", "several differences between our study and the genome - wide association studies , such as the prospective versus cross - sectional designs , the different end points under investigation , and the different baseline cardiovascular risk , with only the patients enrolled in our study being very high - risk as per selection criteria , might be responsible for this apparent discordance .", "an additional important result of our study is that the effect of the q121 variant was modulated by obesity in diabetic patients among whom the risk of incident events was five times higher in q121 than in kk genotype carriers .", "although not the aim of our study , one can infer that obese individuals ( fig .", "2a ) as a whole tend to have a lower risk of future cardiovascular events than nonobese patients ( fig . 2b ; adjusted hr 0.68 [ 95% ci 0.4111.124 ] , p = 0.13 ) .", "this paradoxic protective effect of obesity resembles that observed in patients with cad ( 41 ) , esrd ( 42 ) , heart failure ( 43 ) , and older age ( 44 ) , all conditions heavily over - represented in our samples . in this context , the q121 variant seems to eliminate the paradoxic protective effect of obesity .", "an important finding was that the q121 variant - by - obesity interaction observed in the prospective study was replicated in a cross - sectional study on age at mi in diabetic patients .", "information on the k121q genotypes tended to improve risk prediction in these patients when the improvement was measured by the idi , the approach that is currently favored to evaluate predictive ability increase conferred by a new marker when added to a well - performing model ( 39 ) .", "thus , pending further validation in larger studies , one can hypothesize clinical implementation of the q121 variant as a marker of early cardiovascular events among obese diabetic patients .", "given the increasing incidence worldwide of both obesity and diabetes ( 24 ) and the poor ability to stratify cardiovascular risk among diabetic patients , a large sector of society would be likely to benefit in the future from the availability of such a test .", "the synergistic effect of the genetic marker and obesity in the modulation of cardiovascular risk resembles results repeatedly reported in the risk modulation of insulin resistance and related traits ( 2527,30,33,4549 ) .", "placed in a broader perspective , this is an excellent example of genetic heterogeneity ( i.e. , different genetic effects being at play in different population subgroups ) and clearly illustrates how accounting for such heterogeneity may be critical to dissect the genetic architecture of multifactorial diseases .", "understanding the mechanisms through which the q121 variant is associated with cvd is beyond the scope of this study .", "however , one can speculate that the q121 variant exacerbates cardiovascular risk by inducing systemic insulin resistance ( 22,2527 ) and proatherogenic phenotypes ( 24,29,30,33 )", ". it may also act by way of a direct detrimental effect on insulin - dependent endothelial function , as suggested by the observation that human endothelial cells carrying the q121 variant show impaired insulin receptor signaling and , most importantly , reduced release of nitric oxide ( 24 ) , a potent vasodilator whose deficiency is an established early step in the pathway development of atherosclerosis ( 50 ) .", "one can hypothesize that the interaction between the q121 variant and obesity is sustained by the different sites of action on the insulin - signaling pathway .", "although enpp1 acts at the insulin receptor level ( 21,23 ) , obesity acts by different mechanisms , mostly at a postreceptor level ( 51 ) .", "it is , therefore , possible that postreceptor insulin - signaling abnormalities are necessary for the q121 variant to be fully effective in inducing insulin resistance and , eventually , related clinical outcomes .", "the three cohorts of very high - risk individuals that we studied were quite different from each other : one comprised only patients with type 2 diabetes and cad , another included patients with a previous mi who did not have frank type 2 diabetes , and the third included only patients with esrds . despite such apparent phenotypic heterogeneity , the effect of the q121 variant was not heterogeneous across the three studies .", "not only did this allow us to analyze the three cohorts together , increasing statistical power , but it also suggests that our findings may be generalizable to all high - risk patients , irrespective of their background clinical characteristics .", "whether the predictive role of the q121 variant extends to situations characterized by a more moderate cardiovascular risk remains to be determined .", "we acknowledge that , mainly because of the relatively small size of our samples , the significance level of our findings is still compatible with a false - positive result .", "however , this seems unlikely given that the association between q121 was not heterogeneous across the three cohorts and , importantly , was further confirmed in cross - sectional studies as far as the interaction with obesity in diabetic patients is concerned .", "we also acknowledge that due to the relatively small sample size of the studies that we analyzed , we can not exclude that the gene - by - obesity interaction that we observed among diabetic patients also occurs among nondiabetic individuals , as is the case for the modulation of insulin resistance ( 2527 ) .", "therefore , our findings need further replication in larger samples before they can be considered as established . finally , because this study was entirely performed in individuals of european ancestry , we do not know whether our findings can be extended to populations of different race . in conclusion ,", "pending confirmation in further larger studies , the q121 variant has the potential to become a clinical tool for identifying those very high - risk patients who are especially prone to major cardiovascular events and need , therefore , to be targeted with specific and even more aggressive preventive strategies ."]]}
{"article_id": "PMC2988444", "article_text": ["in the past two decades , molecular biology research has revealed the intimate mechanisms of epidemiologically significant diseases , such as cancer , infections and immunological disorders . as the next step beyond seeking the mechanisms involved ,", "scientists are now increasingly making it possible to regulate human biological reactions . in recent years", ", there have been breakthroughs in genetic engineering related to the inventory and methods necessary to physically construct and assemble biomolecular parts , such as synthetic rna - based regulatory systems .", "synthetic biology relies on the engineering of biological systems that perform human - defined functions and on the synthesis of complex , biologically based systems that show functions that do not exist in nature . despite the possible advantages for clinical applications ,", "more work remains to be done to elucidate the principles of biological design , and to overcome the scientific and technical challenges in designing and building more effective systems that are harmless to humans and therefore useful for clinical applications .", "a recent study by chen et al .   has produced a significant advance in solving such issues and therefore potentially bridging the gap between the bench and the bedside for synthetic rna - based regulatory systems .", "the authors   developed a modular device composed of a sensor ( an aptamer ) and a gene - regulatory component ( a hammerhead ribozyme ) and tested its ability to affect the expression of cytokines important for the function of t - lymphocytes in mouse and human systems .", "first , it represents the logical continuation of years of experimental work performed by the same group , coming from a team that understands the way a synthetic rna - based regulatory system works and its immediate practical applications .", "in fact , in a previous study , also published in proceedings of the national academy of sciences of the united states of america , the authors were the first to develop and set up universal rna - based regulatory platforms , called ribozyme switches , by using engineering design principles . in the present report", ", the authors expanded the advantages of such biomodular platforms to a broader range of applications .", "they were able to do so by the reliable de novo construction of modular , portable and scalable control systems that can achieve flexible regulatory properties , such as up- and down - regulation of target expression levels and tuning of regulatory responses to fit application - specific performance requirements .", "second , the authors   applied the synthetic rna regulatory device to a significant medical issue , the use of adoptive cell transfer ( act ) .", "the act strategy uses t - cell - based cytotoxic responses to attack malignant cells ( or any other types of abnormal cells ) that escape the body 's natural surveillance by using t cells that have a natural or genetically engineered reactivity to a patient 's cancer cells . for this purpose ,", "t cells have first to be naturally or genetically engineered to react against a tumor - specific antigen , then expanded and made more effective in vitro , and finally adoptively transferred into a cancer patient . however , the clinical efficacy of act , so far , has been limited .", "there are many reasons for this , and insufficient persistence and reactivation of infused t cells are among the main ones .", "conventional strategies for enhancing the persistence of transferred t cells include ablation of all white blood cells ( myeloablative methods ) , such as total body irradiation and administration of toxic levels of interleukin ( il)-2 .", "however , myeloablation is associated with considerable morbidity , caused by decreased immune response and increased risk of infection .", "report on a synthetic rna regulatory system , which marks a new era in adoptive t - cell therapy because of the increase in the amount and survival of infused t cells found with this system .", "their system for the control of mammalian t - cell proliferation is based on a platform of assembled rna devices formed by a modular sensor ( aptamer ) and a gene - regulatory ( hammerhead ribozyme ) component .", "this device converts a small - molecule input to an increased gene expression output , in this particular case cytokine production . in more detail , the authors   fused a theophylline ribozyme switch to the 3 ' untranslated region of a tri - functional transgene ( cd19-tk - t2a - il15 ) encoding il-15 ( potent survival / proliferative cytokine of t cells ) , mutant hsv-1 thymidine kinase ( acting as a reporter and as a suicide protein in the presence of ganciclovir ) and cd19 ( a marker for fluorescence - activated cell sorting and immunomagnetic selection ) . using this system ,", "they could strictly measure ( by monitoring the expression of cd19 ) and control ( by modulating the levels of the input molecule ) the biological response ( cell proliferation / viability ) .", "in addition to all the in vitro evidence , the authors   demonstrated that this system worked in vivo and effectively modulated the t - cell growth rate in mice in response to theophylline administration .", "the growth rate was increased to 32% in the presence of theophylline over a 14 day study in mice .", "they further investigated its possible clinical application by transducing primary human central memory t cells with this system . in vitro results showed that the population of live central memory t cells increased by 24% and that apoptotic cell population was decreased by 54% in the theophylline - responsive system .", "finally , the presented gene regulatory system   showed significant advantages over available gene regulatory techniques ( synthetic inducible promoters ) ; in particular , it provides a wide range of flexibility for clinical settings .", "firstly , the ribozyme switches can be easily programmed to respond to different drug molecules .", "secondly , the system can be stringently controlled and finely tuned by adding additional drug - responsive ribozyme switches ( up to four ) , therefore achieving lower basal gene expression levels .", "thirdly , this system shows tight drug - mediated regulation of growth over an extended time period . taking all these features into consideration , it is feasible that this new synthetic rna - based regulatory system could have straightforward clinical utility .", "it is likely that combining this new modular device framework with upcoming advances in synthetic biology will strongly support the tailoring of rna - based regulatory systems to diverse applications in various clinical and laboratory environments . yet applying these rna - based regulatory systems in clinical practice", "in addition , there are many other factors that limit the use of adoptive t - cell therapy for cancer . for example", ", the failure of adoptive immunotherapy against cancers lies in the absence of tumor - specific sources of t cells .", "if such obstacles are not overcome , efficacy of these systems will be significantly limited in clinical practice .", "also , recent data support the combined roles of protein - coding genes and non - coding rnas , such as micrornas , in the pathogenesis of frequent diseases ( such as cancer , immune and cardiac disorders ) .", "one question for the future is whether such devices can be adapted for the regulation of the functions of non - coding rnas and micrornas .", "the published research is good news , but it would be better to hold our cheers until the clinical trials are successfully completed , which we hope will be in the near future .", "gac received his md and phd at carol davila university of medicine in bucharest , romania . after working on cytogenetics as an undergraduate student with dragos stefanescu in bucharest , he completed training in cancer genomics in massimo negrini 's laboratory at the university of ferrara , italy . in 2000", "he became a postdoctoral fellow at the kimmel cancer center in philadelphia , in carlo croce 's laboratory .", "since july 2007 he has been an associate professor in experimental therapeutics at the md anderson cancer center and studies the roles of micrornas and other non - coding rnas in cancer initiation and progression , as well as the mechanisms of cancer predisposition , and explores new rna therapeutic options for cancer patients .", "skl graduated from yonsei university medical school , seoul , south korea with an md and phd and is an assistant professor in the department of gastroenterology , severance hospital , seoul .", "the focus of his scientific research is understanding the roles of non - coding rnas in gastrointestinal cancers . in march 2009", ", he began working in gac 's laboratory studying the roles of non - coding rnas , including micrornas in the initiation and development of gastrointestinal cancers , as well as the identification of new non - coding rna biomarkers .", "gac is supported as a fellow at the university of texas md anderson research trust , as a fellow of the university of texas system regents research scholar and by the cll global research foundation .", "work in gac 's laboratory is supported in part by nih , by dod , by developmental research awards in breast cancer , ovarian cancer and leukemia spores , and by a 2009 seena magowitz pancreatic cancer action network aacr pilot grant ."], "abstract_text": ["<S> synthetic rna - based regulatory systems are used to program higher - level biological functions that could be exploited , among many applications , for in vivo diagnostic and therapeutic applications . chen and colleagues have recently reported a significant technological advance by producing an rna modular device based on a hammerhead ribozyme and successfully tested its ability to control the proliferation of mammalian t lymphocytes . </S>", "<S> like all exciting research , this work raises a lot of significant questions . </S>", "<S> how quickly will such knowledge be translated into clinical practice ? how efficient will this system be in human clinical trials involving adaptive t - cell therapy ? </S>", "<S> we discuss the possible advantages of using such new technologies for specific therapeutic applications . </S>"], "labels": null, "section_names": ["Commentary", "Abbreviations", "Competing interests", "Authors' contributions", "Author information", "Acknowledgements"], "sections": [["in the past two decades , molecular biology research has revealed the intimate mechanisms of epidemiologically significant diseases , such as cancer , infections and immunological disorders . as the next step beyond seeking the mechanisms involved ,", "scientists are now increasingly making it possible to regulate human biological reactions . in recent years", ", there have been breakthroughs in genetic engineering related to the inventory and methods necessary to physically construct and assemble biomolecular parts , such as synthetic rna - based regulatory systems .", "synthetic biology relies on the engineering of biological systems that perform human - defined functions and on the synthesis of complex , biologically based systems that show functions that do not exist in nature . despite the possible advantages for clinical applications ,", "more work remains to be done to elucidate the principles of biological design , and to overcome the scientific and technical challenges in designing and building more effective systems that are harmless to humans and therefore useful for clinical applications .", "a recent study by chen et al .   has produced a significant advance in solving such issues and therefore potentially bridging the gap between the bench and the bedside for synthetic rna - based regulatory systems .", "the authors   developed a modular device composed of a sensor ( an aptamer ) and a gene - regulatory component ( a hammerhead ribozyme ) and tested its ability to affect the expression of cytokines important for the function of t - lymphocytes in mouse and human systems .", "first , it represents the logical continuation of years of experimental work performed by the same group , coming from a team that understands the way a synthetic rna - based regulatory system works and its immediate practical applications .", "in fact , in a previous study , also published in proceedings of the national academy of sciences of the united states of america , the authors were the first to develop and set up universal rna - based regulatory platforms , called ribozyme switches , by using engineering design principles . in the present report", ", the authors expanded the advantages of such biomodular platforms to a broader range of applications .", "they were able to do so by the reliable de novo construction of modular , portable and scalable control systems that can achieve flexible regulatory properties , such as up- and down - regulation of target expression levels and tuning of regulatory responses to fit application - specific performance requirements .", "second , the authors   applied the synthetic rna regulatory device to a significant medical issue , the use of adoptive cell transfer ( act ) .", "the act strategy uses t - cell - based cytotoxic responses to attack malignant cells ( or any other types of abnormal cells ) that escape the body 's natural surveillance by using t cells that have a natural or genetically engineered reactivity to a patient 's cancer cells . for this purpose ,", "t cells have first to be naturally or genetically engineered to react against a tumor - specific antigen , then expanded and made more effective in vitro , and finally adoptively transferred into a cancer patient . however , the clinical efficacy of act , so far , has been limited .", "there are many reasons for this , and insufficient persistence and reactivation of infused t cells are among the main ones .", "conventional strategies for enhancing the persistence of transferred t cells include ablation of all white blood cells ( myeloablative methods ) , such as total body irradiation and administration of toxic levels of interleukin ( il)-2 .", "however , myeloablation is associated with considerable morbidity , caused by decreased immune response and increased risk of infection .", "report on a synthetic rna regulatory system , which marks a new era in adoptive t - cell therapy because of the increase in the amount and survival of infused t cells found with this system .", "their system for the control of mammalian t - cell proliferation is based on a platform of assembled rna devices formed by a modular sensor ( aptamer ) and a gene - regulatory ( hammerhead ribozyme ) component .", "this device converts a small - molecule input to an increased gene expression output , in this particular case cytokine production . in more detail , the authors   fused a theophylline ribozyme switch to the 3 ' untranslated region of a tri - functional transgene ( cd19-tk - t2a - il15 ) encoding il-15 ( potent survival / proliferative cytokine of t cells ) , mutant hsv-1 thymidine kinase ( acting as a reporter and as a suicide protein in the presence of ganciclovir ) and cd19 ( a marker for fluorescence - activated cell sorting and immunomagnetic selection ) . using this system ,", "they could strictly measure ( by monitoring the expression of cd19 ) and control ( by modulating the levels of the input molecule ) the biological response ( cell proliferation / viability ) .", "in addition to all the in vitro evidence , the authors   demonstrated that this system worked in vivo and effectively modulated the t - cell growth rate in mice in response to theophylline administration .", "the growth rate was increased to 32% in the presence of theophylline over a 14 day study in mice .", "they further investigated its possible clinical application by transducing primary human central memory t cells with this system . in vitro results showed that the population of live central memory t cells increased by 24% and that apoptotic cell population was decreased by 54% in the theophylline - responsive system .", "finally , the presented gene regulatory system   showed significant advantages over available gene regulatory techniques ( synthetic inducible promoters ) ; in particular , it provides a wide range of flexibility for clinical settings .", "firstly , the ribozyme switches can be easily programmed to respond to different drug molecules .", "secondly , the system can be stringently controlled and finely tuned by adding additional drug - responsive ribozyme switches ( up to four ) , therefore achieving lower basal gene expression levels .", "thirdly , this system shows tight drug - mediated regulation of growth over an extended time period . taking all these features into consideration , it is feasible that this new synthetic rna - based regulatory system could have straightforward clinical utility .", "it is likely that combining this new modular device framework with upcoming advances in synthetic biology will strongly support the tailoring of rna - based regulatory systems to diverse applications in various clinical and laboratory environments . yet applying these rna - based regulatory systems in clinical practice", "in addition , there are many other factors that limit the use of adoptive t - cell therapy for cancer . for example", ", the failure of adoptive immunotherapy against cancers lies in the absence of tumor - specific sources of t cells .", "if such obstacles are not overcome , efficacy of these systems will be significantly limited in clinical practice .", "also , recent data support the combined roles of protein - coding genes and non - coding rnas , such as micrornas , in the pathogenesis of frequent diseases ( such as cancer , immune and cardiac disorders ) .", "one question for the future is whether such devices can be adapted for the regulation of the functions of non - coding rnas and micrornas .", "the published research is good news , but it would be better to hold our cheers until the clinical trials are successfully completed , which we hope will be in the near future ."], [""], [""], [""], ["gac received his md and phd at carol davila university of medicine in bucharest , romania . after working on cytogenetics as an undergraduate student with dragos stefanescu in bucharest , he completed training in cancer genomics in massimo negrini 's laboratory at the university of ferrara , italy . in 2000", "he became a postdoctoral fellow at the kimmel cancer center in philadelphia , in carlo croce 's laboratory .", "since july 2007 he has been an associate professor in experimental therapeutics at the md anderson cancer center and studies the roles of micrornas and other non - coding rnas in cancer initiation and progression , as well as the mechanisms of cancer predisposition , and explores new rna therapeutic options for cancer patients .", "skl graduated from yonsei university medical school , seoul , south korea with an md and phd and is an assistant professor in the department of gastroenterology , severance hospital , seoul .", "the focus of his scientific research is understanding the roles of non - coding rnas in gastrointestinal cancers . in march 2009", ", he began working in gac 's laboratory studying the roles of non - coding rnas , including micrornas in the initiation and development of gastrointestinal cancers , as well as the identification of new non - coding rna biomarkers ."], ["gac is supported as a fellow at the university of texas md anderson research trust , as a fellow of the university of texas system regents research scholar and by the cll global research foundation .", "work in gac 's laboratory is supported in part by nih , by dod , by developmental research awards in breast cancer , ovarian cancer and leukemia spores , and by a 2009 seena magowitz pancreatic cancer action network aacr pilot grant ."]]}
{"article_id": "PMC2733967", "article_text": ["the wilms ' tumor suppressor gene ( wt1 ) , located at chromosome 11p13 , has 10 exons , spans -50 kb , and encodes a zinc - finger - transcription factor presumed to regulate the expression of numerous target genes through dna binding ( 1 ) .", "wt1 was initially identified as a gene inactivated in wilms ' tumor , although the estimated percentage of wilms ' tumors ( wts ) with wt1 mutations is only 10 - 15% .", "investigations of wt1 have revealed the normal physiologic functions of this gene in embryogenesis , gonadogenesis and nephrogenesis ( 1 ) .", "wt1 has been shown to play a role in kidney induction ( 2 ) and during later steps of nephrogenesis ( 3 ) , and there are suggestions that wt1 may play an important role in the maintenance of normal podocyte function ( 4 ) . besides wilms ' tumor , a number of human diseases have been shown to be associated with mutations of the wt1 gene ( 5 ) .", "genital abnormalities are noted in three of these disorders , wagr syndrome ( wilms ' tumor , aniridia , genitourinary malformation , and mental retardation ) , denys - drash syndrome ( dds ) , and frasier syndrome ( fs ) , and the analysis of wt1 knockout mice also suggested a fundamental role of wt1 in gonad development ( 6 ) .", "wilhelm and englert reported that the wt1 regulates early gonad development by activation of steroidogenesis factor 1 , sf1 , which encodes an orphan nuclear receptor that regulates the expression of several genes involved in steroidogenesis and gonadal development ( 7 ) .", "dds traditionally encompasses patients with the triad of congenital nephrotic syndrome leading to end - stage renal failure ( esrf ) , xy pseudohermaphroditism and wilms ' tumor ( 8 , 9 ) .", "subsequent reports described patients with incomplete forms of this syndrome ( 10 - 12 ) , and the definition of dds has now been widened to include xy individuals with mild to severe genital anomalies as well as xx individuals displaying nephropathy and wt ( 13 ) .", "the nephropathies in dds are characterized by the histological finding of diffuse mesangial sclerosis ( dms ) , a finding also described in some cases of congenital nephrotic syndrome ( isolated diffuse mesangial sclerosis , idms ) .", "analysis of genotype - phenotype correlation in idms suggests that at least some idms patients have a variant form of dds due to wt1 mutations ( 14 ) .", "attempts to correlate the phenotype of dds and different wt1 mutations did not provide clear - cut results ( 15 ) , and no correlation could be established between any particular mutation and the occurrence of wilms ' tumor .", "however , analysis of a large number of patients might provide clues to the role of each mutation , and might help in understanding the normal physiology of nephrogenesis and the prediction for tumorigenesis . in this report", ", we present two cases of idms with different wt1 gene mutations that had previously been reported in association with dds .", "she was born at 37 weeks ' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema , ascites and normal female external genitalia .", "laboratory findings showed ; hemoglobin , 8.2 g / dl ; cholesterol , 147 mg / dl ; total serum protein , 3.5 g / dl ; serum albumin , 2.1 g / dl ; serum creatinine , 1.7 mg / dl ; bun , 27 mg / dl ; total calcium , 5.6 mg / dl ; phosphorus , 11.7 mg / dl ; sodium , 116 meq / l ; potassium , 6.0 meq / l ; chloride 102 meq / l ; tco2 5.7 meq / l .", "a renal biopsy was performed at the age of 111 days , and 30 glomeruli were examined .", "a wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells .", "analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution , converting arg to his ( fig .", "the patient is now 13 months old , and is well and on peritoneal dialysis , expecting renal transplantation .", "she was born at 38 weeks ' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . at a local clinic , hypocalcemia , hyperkalemia and azotemia", "laboratory findings showed : hemoglobin , 9.8 g / dl ; cholesterol , 120 mg / dl ; total serum protein , 3.7 g / dl ; serum albumin , 1.5 g / dl ; bun , 75 mg / dl ; serum creatinine , 4.1 mg / dl ; total calcium , 5.9 mg / dl ; phosphorus , 12.7 mg / dl ; sodium , 136 meq / l ; potassium , 5.5 meq / l ; chloride , 113 meq / l ; tco2 7.8 meq / l . albuminuria and", "a renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis ( fig .", "analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution , converting asp to tyr , and a heterozygous 395 ser ( tcc ) > ser ( tca ) polymorphism ( fig .", "peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . sustained hypertension ( 120/80 mmhg ) responded to an angiotensin - converting enzyme inhibitor .", "she was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization , and the patient died at the age of six months due to multiple serious infections and failure to thrive .", "she was born at 37 weeks ' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema , ascites and normal female external genitalia .", "laboratory findings showed ; hemoglobin , 8.2 g / dl ; cholesterol , 147 mg / dl ; total serum protein , 3.5 g / dl ; serum albumin , 2.1 g / dl ; serum creatinine , 1.7 mg / dl ; bun , 27 mg / dl ; total calcium , 5.6 mg / dl ; phosphorus , 11.7 mg / dl ; sodium , 116 meq / l ; potassium , 6.0 meq / l ; chloride 102 meq / l ; tco2 5.7 meq / l .", "a renal biopsy was performed at the age of 111 days , and 30 glomeruli were examined .", "a wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells .", "analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution , converting arg to his ( fig .", "the patient is now 13 months old , and is well and on peritoneal dialysis , expecting renal transplantation .", "she was born at 38 weeks ' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . at a local clinic , hypocalcemia , hyperkalemia and azotemia", "were detected and she was referred to us . on admission , generalized edema was noted .", "laboratory findings showed : hemoglobin , 9.8 g / dl ; cholesterol , 120 mg / dl ; total serum protein , 3.7 g / dl ; serum albumin , 1.5 g / dl ; bun , 75 mg / dl ; serum creatinine , 4.1 mg / dl ; total calcium , 5.9 mg / dl ; phosphorus , 12.7 mg / dl ; sodium , 136 meq / l ; potassium , 5.5 meq / l ; chloride , 113 meq / l ; tco2 7.8 meq / l . albuminuria and hematuria were detected .", "a renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis ( fig .", "analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution , converting asp to tyr , and a heterozygous 395 ser ( tcc ) > ser ( tca ) polymorphism ( fig .", "peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . sustained hypertension ( 120/80 mmhg ) responded to an angiotensin - converting enzyme inhibitor .", "she was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization , and the patient died at the age of six months due to multiple serious infections and failure to thrive .", "the mutation present in the second patient was previously reported in a dds patient with early onset esrf resulting from dms , ambiguous genitalia , and a 46 xy karyotype .", "autopsy showed a streak gonad and nephroblastomatosis , but no wts were revealed ( 16 ) .", "as in our second patient , this boy reached esrf within a few weeks . considering that esrf in dms", "is reached within a few months or years ( 17 ) , early onset esrf in patients is noted as a feature of this mutation .", "the mutation in the first patient was also previously reported in a dds patient ( 46 xy , female genitalia , streak gonad , dms , gonadoblastoma , without wt ) ( 18 ) .", "it is well known that wt1 has many roles during gonadogenesis , and since wt1 is expressed in the same cell lineage as sry , the y - located testis determinant , wt1 may be acting upstream to sry during development of the genital ridge ; perhaps controlling sry expression , or interacting directly with sry during sex determination , or functioning immediately downstream of sry ( 19 ) .", "wt1-null mutant mice of both sexes fail to develop kidneys and gonads , indicating that wt1 acts upstream of the sex - determining decision ( 6 ) .", "frasier syndrome ( fs ) is another distinctive disease associated with wt1 mutation , characterized by male pseudohermaphroditism and nephropathy with late onset esrf , and a frequent association with gonadoblastoma ( 20 ) .", "molecular analysis of a familial case of fs has been described , which provided a clue to wt1 's effects on gonadogenesis .", "two sisters in their late teens showed signs of renal disease and each sister has a donor splice - site mutation that was predicted to result in an imbalance of the kts+/kts- isoforms .", "one sister was 46 xy , with complete gonadal dysgenesis with normal female phenotype , while the other was 46 xx , with apparently normal ovarian development and function .", "this suggests that either wt1 has a male - specific function in sex determination or that testis formation is much more sensitive to dosage effects than ovarian formation ( 21 ) .", "our two cases are female with karyotype 46 xx , which might be the reason for the absence of gonadal abnormalities , and we suspect that if a patient is born with karyotype 46 xy , there may be a possibility of dds development . although male patients have been described ( 14 , 22 ) , idms most often occurs in females ; however , the percentage of patients with wt1 mutation - positive idms , the sex ratio and the associated risk of wilms ' tumor are still unknown .", "a recent study of seven japanese idms patients reported a low rate of wt1 mutation ( 2/7 ) and a low risk of wilms ' tumor ( 0/7 ) ( 23 ) .", "the mutations observed in these japanese patients were different from those observed in the previous reports of dds , and in the removed kidneys , nephrogenic nests were not found . therefore , it was concluded that in idms with wt1 mutations , the risk of wilms ' tumor might not be very high .", "our report is limited to two patients , but both mutations were already associated with dds , and the early onset esrf in patients with the 396 asp ( gac ) > tyr ( tac ) mutation is distinctively similar .", "this supports the concept that , at least in part , idms is a variant form of dds , and we suggest that idms patients require the same attention as dds patients because of the possible association with wilms ' tumor and/or gonadoblastoma .", "hu et al . ( 24 ) recently reported two boys with incomplete dds with wt1 mutations who underwent prophylactic bilateral nephrectomy , and the removed kidneys showed nephroblastomatosis , which has malignant potential .", "the authors suggested that missense mutations in exons 8 and 9 of wt1 can be regarded as risk factors for development of wilms ' tumor and proposed prophylactic bilateral nephrectomy and early renal transplantation .", "it is still debatable whether prophylactic nephrectomies should be carried out in all children with dms before renal transplantation , and schumacher et al .", "( 15 ) suggested that regular renal imaging might be necessary to evaluate the need for bilateral nephrectomy .", "considering the clinical courses of the patients in the previous reports , our patients also might have had the possibility of malignant transformation , but we chose regular renal and gonadal imaging rather than prophylactic nephrectomy , and three - monthly ultrasonogram until death did not show evidence of malignancy in the second patient .", "the first patient is well on peritoneal dialysis with no sign of malignancy on three - monthly screening and we are considering of nephrectomy at the time of transplantation .", "the reason for the heterogeneity of the clinical features of patients with wt1 mutations is not yet clear , and we suggest that a classification based on genetic diagnosis rather than syndromatic diagnosis might be more applicable for the prediction of outcome , and routine genetic analysis to detect wt1 mutation should be applied to all idms patients .", "if wt1 mutation is documented , karyotyping and serial abdominal ultrasonographies are warranted for the evaluation of tumor development in the gonad and kidney , and the analysis of genotype - phenotype correlations should be continued ."], "abstract_text": ["<S> here we report two cases of isolated diffuse mesangial sclerosis ( idms ) with early onset end - stage renal failure . </S>", "<S> these female patients did not show abnormalities of the gonads or external genitalia . </S>", "<S> direct sequencing of wt1 pcr products from genomic dna identified wt1 mutations in exons 8 ( 366 arg > his ) and 9 ( 396 asp > tyr ) . </S>", "<S> these mutations have been reported previously in association with denys - drash syndrome ( dds ) with early onset renal failure . </S>", "<S> therefore we suggest that , at least in part , idms is a variant of dds and that investigations for the wt1 mutations should be performed in idms patients . in cases with identified wt1 mutations , the same attention to tumor development </S>", "<S> should be required as in dds patients , and karyotyping and serial abdominal ultrasonograms to evaluate the gonads and kidney are warranted . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "Case 1", "Case 2", "DISCUSSION"], "sections": [["the wilms ' tumor suppressor gene ( wt1 ) , located at chromosome 11p13 , has 10 exons , spans -50 kb , and encodes a zinc - finger - transcription factor presumed to regulate the expression of numerous target genes through dna binding ( 1 ) .", "wt1 was initially identified as a gene inactivated in wilms ' tumor , although the estimated percentage of wilms ' tumors ( wts ) with wt1 mutations is only 10 - 15% .", "investigations of wt1 have revealed the normal physiologic functions of this gene in embryogenesis , gonadogenesis and nephrogenesis ( 1 ) .", "wt1 has been shown to play a role in kidney induction ( 2 ) and during later steps of nephrogenesis ( 3 ) , and there are suggestions that wt1 may play an important role in the maintenance of normal podocyte function ( 4 ) . besides wilms ' tumor , a number of human diseases have been shown to be associated with mutations of the wt1 gene ( 5 ) .", "genital abnormalities are noted in three of these disorders , wagr syndrome ( wilms ' tumor , aniridia , genitourinary malformation , and mental retardation ) , denys - drash syndrome ( dds ) , and frasier syndrome ( fs ) , and the analysis of wt1 knockout mice also suggested a fundamental role of wt1 in gonad development ( 6 ) .", "wilhelm and englert reported that the wt1 regulates early gonad development by activation of steroidogenesis factor 1 , sf1 , which encodes an orphan nuclear receptor that regulates the expression of several genes involved in steroidogenesis and gonadal development ( 7 ) .", "dds traditionally encompasses patients with the triad of congenital nephrotic syndrome leading to end - stage renal failure ( esrf ) , xy pseudohermaphroditism and wilms ' tumor ( 8 , 9 ) .", "subsequent reports described patients with incomplete forms of this syndrome ( 10 - 12 ) , and the definition of dds has now been widened to include xy individuals with mild to severe genital anomalies as well as xx individuals displaying nephropathy and wt ( 13 ) .", "the nephropathies in dds are characterized by the histological finding of diffuse mesangial sclerosis ( dms ) , a finding also described in some cases of congenital nephrotic syndrome ( isolated diffuse mesangial sclerosis , idms ) .", "analysis of genotype - phenotype correlation in idms suggests that at least some idms patients have a variant form of dds due to wt1 mutations ( 14 ) .", "attempts to correlate the phenotype of dds and different wt1 mutations did not provide clear - cut results ( 15 ) , and no correlation could be established between any particular mutation and the occurrence of wilms ' tumor .", "however , analysis of a large number of patients might provide clues to the role of each mutation , and might help in understanding the normal physiology of nephrogenesis and the prediction for tumorigenesis . in this report", ", we present two cases of idms with different wt1 gene mutations that had previously been reported in association with dds ."], ["she was born at 37 weeks ' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema , ascites and normal female external genitalia .", "laboratory findings showed ; hemoglobin , 8.2 g / dl ; cholesterol , 147 mg / dl ; total serum protein , 3.5 g / dl ; serum albumin , 2.1 g / dl ; serum creatinine , 1.7 mg / dl ; bun , 27 mg / dl ; total calcium , 5.6 mg / dl ; phosphorus , 11.7 mg / dl ; sodium , 116 meq / l ; potassium , 6.0 meq / l ; chloride 102 meq / l ; tco2 5.7 meq / l .", "a renal biopsy was performed at the age of 111 days , and 30 glomeruli were examined .", "a wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells .", "analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution , converting arg to his ( fig .", "the patient is now 13 months old , and is well and on peritoneal dialysis , expecting renal transplantation .", "she was born at 38 weeks ' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . at a local clinic , hypocalcemia , hyperkalemia and azotemia", "laboratory findings showed : hemoglobin , 9.8 g / dl ; cholesterol , 120 mg / dl ; total serum protein , 3.7 g / dl ; serum albumin , 1.5 g / dl ; bun , 75 mg / dl ; serum creatinine , 4.1 mg / dl ; total calcium , 5.9 mg / dl ; phosphorus , 12.7 mg / dl ; sodium , 136 meq / l ; potassium , 5.5 meq / l ; chloride , 113 meq / l ; tco2 7.8 meq / l . albuminuria and", "a renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis ( fig .", "analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution , converting asp to tyr , and a heterozygous 395 ser ( tcc ) > ser ( tca ) polymorphism ( fig .", "peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . sustained hypertension ( 120/80 mmhg ) responded to an angiotensin - converting enzyme inhibitor .", "she was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization , and the patient died at the age of six months due to multiple serious infections and failure to thrive ."], ["she was born at 37 weeks ' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema , ascites and normal female external genitalia .", "laboratory findings showed ; hemoglobin , 8.2 g / dl ; cholesterol , 147 mg / dl ; total serum protein , 3.5 g / dl ; serum albumin , 2.1 g / dl ; serum creatinine , 1.7 mg / dl ; bun , 27 mg / dl ; total calcium , 5.6 mg / dl ; phosphorus , 11.7 mg / dl ; sodium , 116 meq / l ; potassium , 6.0 meq / l ; chloride 102 meq / l ; tco2 5.7 meq / l .", "a renal biopsy was performed at the age of 111 days , and 30 glomeruli were examined .", "a wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells .", "analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution , converting arg to his ( fig .", "the patient is now 13 months old , and is well and on peritoneal dialysis , expecting renal transplantation ."], ["she was born at 38 weeks ' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . at a local clinic , hypocalcemia , hyperkalemia and azotemia", "were detected and she was referred to us . on admission , generalized edema was noted .", "laboratory findings showed : hemoglobin , 9.8 g / dl ; cholesterol , 120 mg / dl ; total serum protein , 3.7 g / dl ; serum albumin , 1.5 g / dl ; bun , 75 mg / dl ; serum creatinine , 4.1 mg / dl ; total calcium , 5.9 mg / dl ; phosphorus , 12.7 mg / dl ; sodium , 136 meq / l ; potassium , 5.5 meq / l ; chloride , 113 meq / l ; tco2 7.8 meq / l . albuminuria and hematuria were detected .", "a renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis ( fig .", "analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution , converting asp to tyr , and a heterozygous 395 ser ( tcc ) > ser ( tca ) polymorphism ( fig .", "peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . sustained hypertension ( 120/80 mmhg ) responded to an angiotensin - converting enzyme inhibitor .", "she was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization , and the patient died at the age of six months due to multiple serious infections and failure to thrive ."], ["the mutation present in the second patient was previously reported in a dds patient with early onset esrf resulting from dms , ambiguous genitalia , and a 46 xy karyotype .", "autopsy showed a streak gonad and nephroblastomatosis , but no wts were revealed ( 16 ) .", "as in our second patient , this boy reached esrf within a few weeks . considering that esrf in dms", "is reached within a few months or years ( 17 ) , early onset esrf in patients is noted as a feature of this mutation .", "the mutation in the first patient was also previously reported in a dds patient ( 46 xy , female genitalia , streak gonad , dms , gonadoblastoma , without wt ) ( 18 ) .", "it is well known that wt1 has many roles during gonadogenesis , and since wt1 is expressed in the same cell lineage as sry , the y - located testis determinant , wt1 may be acting upstream to sry during development of the genital ridge ; perhaps controlling sry expression , or interacting directly with sry during sex determination , or functioning immediately downstream of sry ( 19 ) .", "wt1-null mutant mice of both sexes fail to develop kidneys and gonads , indicating that wt1 acts upstream of the sex - determining decision ( 6 ) .", "frasier syndrome ( fs ) is another distinctive disease associated with wt1 mutation , characterized by male pseudohermaphroditism and nephropathy with late onset esrf , and a frequent association with gonadoblastoma ( 20 ) .", "molecular analysis of a familial case of fs has been described , which provided a clue to wt1 's effects on gonadogenesis .", "two sisters in their late teens showed signs of renal disease and each sister has a donor splice - site mutation that was predicted to result in an imbalance of the kts+/kts- isoforms .", "one sister was 46 xy , with complete gonadal dysgenesis with normal female phenotype , while the other was 46 xx , with apparently normal ovarian development and function .", "this suggests that either wt1 has a male - specific function in sex determination or that testis formation is much more sensitive to dosage effects than ovarian formation ( 21 ) .", "our two cases are female with karyotype 46 xx , which might be the reason for the absence of gonadal abnormalities , and we suspect that if a patient is born with karyotype 46 xy , there may be a possibility of dds development . although male patients have been described ( 14 , 22 ) , idms most often occurs in females ; however , the percentage of patients with wt1 mutation - positive idms , the sex ratio and the associated risk of wilms ' tumor are still unknown .", "a recent study of seven japanese idms patients reported a low rate of wt1 mutation ( 2/7 ) and a low risk of wilms ' tumor ( 0/7 ) ( 23 ) .", "the mutations observed in these japanese patients were different from those observed in the previous reports of dds , and in the removed kidneys , nephrogenic nests were not found . therefore , it was concluded that in idms with wt1 mutations , the risk of wilms ' tumor might not be very high .", "our report is limited to two patients , but both mutations were already associated with dds , and the early onset esrf in patients with the 396 asp ( gac ) > tyr ( tac ) mutation is distinctively similar .", "this supports the concept that , at least in part , idms is a variant form of dds , and we suggest that idms patients require the same attention as dds patients because of the possible association with wilms ' tumor and/or gonadoblastoma .", "hu et al . ( 24 ) recently reported two boys with incomplete dds with wt1 mutations who underwent prophylactic bilateral nephrectomy , and the removed kidneys showed nephroblastomatosis , which has malignant potential .", "the authors suggested that missense mutations in exons 8 and 9 of wt1 can be regarded as risk factors for development of wilms ' tumor and proposed prophylactic bilateral nephrectomy and early renal transplantation .", "it is still debatable whether prophylactic nephrectomies should be carried out in all children with dms before renal transplantation , and schumacher et al .", "( 15 ) suggested that regular renal imaging might be necessary to evaluate the need for bilateral nephrectomy .", "considering the clinical courses of the patients in the previous reports , our patients also might have had the possibility of malignant transformation , but we chose regular renal and gonadal imaging rather than prophylactic nephrectomy , and three - monthly ultrasonogram until death did not show evidence of malignancy in the second patient .", "the first patient is well on peritoneal dialysis with no sign of malignancy on three - monthly screening and we are considering of nephrectomy at the time of transplantation .", "the reason for the heterogeneity of the clinical features of patients with wt1 mutations is not yet clear , and we suggest that a classification based on genetic diagnosis rather than syndromatic diagnosis might be more applicable for the prediction of outcome , and routine genetic analysis to detect wt1 mutation should be applied to all idms patients .", "if wt1 mutation is documented , karyotyping and serial abdominal ultrasonographies are warranted for the evaluation of tumor development in the gonad and kidney , and the analysis of genotype - phenotype correlations should be continued ."]]}
{"article_id": "PMC5008198", "article_text": ["titanium dioxide ( tio2 ) is a component of many sunscreens , soaps , shampoos , toothpastes , cosmetics , paper products , plastics , ink , paint , and building materials 1 in both its bulk form and its nanoform .", "it is also used in human food as a colorant and inactive ingredient , where it can also be present in both forms 1 , 2 . from 1916 to 2011 , an estimated total of 165 050 000 metric tonnes of tio2 pigment were produced worldwide ( bulk form and nanoform combined ) , with a current annual estimated production of more than 6 million tonnes / yr 2 .", "reviews of nanotio2 toxicology are available across various evolutionary groups of species 3 , 4 , 5 , 6 , 7 , 8 , often summarizing half maximal effective concentration ( ec50 ) , half maximal inhibitory concentration ( ic50 ) , and median lethal concentration ( lc50 ) values .", "nanotio2 is also photoactive and produces reactive oxygen species ( ros ) on illumination 9 .", "reactive oxygen species can be detrimental to many organisms , causing oxidative damage , cell injury , and ultimately death 10 .", "recently , it has been argued that photoactivation of nanotio2 under natural levels of sunlight is sufficient to affect the output of lc50 and ec50 values in standard toxicology tests 11 , 12 .", "the majority of studies investigating nanotio2 toxicity did not take photoactivation into account and performed tests either in dark conditions or under indoor commercial artificial lighting that did not simulate natural solar irradiation .", "the aim of present study was to derive a phototoxicity ratio between the results of the nanotio2 experiments conducted in the absence of sunlight and conducted in the presence of solar or simulated solar radiation ( ssr ) . to achieve this aim , we searched the literature for studies that included nanotio2 experiments both with and without irradiance under the same experimental setup and otherwise identical conditions .", "therefore , the phototoxicity ratio can be used to correct endpoints of the toxicity tests with nanotio2 that were performed in absence of natural sunlight or ssr .", "such corrections also may be important for regulators and risk assessors when reviewing previously published data .", "for example , one of the current challenges for conducting risk assessment of nanoparticles such as tio2 is lack of consistent toxicity data because of the varieties of materials and test conditions .", "regulatory thresholds for nanotio2 do not exist currently ; however , regulation of nanoparticles discharge and monitoring in aquatic environment is anticipated in the future .", "it is expected that regulators will use the phototoxicity ratio when deriving thresholds for nanotio2 because the majority of the published literature reported toxicity endpoints for nanotio2 in the absence of natural sunlight or ssr .", "of course , the phototoxicity ratio value calculated in present study is not absolutely precise and correct for all environmental conditions and species ; it does , however , considerably reduce the possible error of data endpoints obtained in the absence of natural sunlight or ssr .", "it also mitigates uncertainties in the risk assessment process by taking into account the photoactivation and phototoxicity of nanotio2 .", "a comprehensive literature review was conducted to collect available toxicity endpoints for nanotio2 . the literature search ( september 2014 ) was performed within 4 databases  web of science , scopus , google scholar , and the university of british columbia library database  using the following keywords in various combinations : titanium dioxide , tio2 , nanoparticles , phototoxicity , photoactivation , ec50 , lc50 , ic50 , and lowestobservedeffect concentration ( loec ) .", "abstracts of numerous hits were read , and downloaded papers were checked for useful information . only papers that reported results under the same environmental conditions for 2 different nanotio2 exposure groups ( with and without ssr ) in the form of ec50 , lc50 , ic50 , loec , or a ratio were selected .", "thus , all included data were based on dose  response curves , ensuring the highest possible quality .", "the phototoxicity ratio ( pr ) was calculated in the form of a ratio : \n pr = tio2  lc50 ,  ec50 ,  ic50 ,  loec  without  sunlight  or  ssrtio2  lc50 ,  ec50 ,  ic50 ,  loec  with  sunlight  or  ssra phototoxicity ratio greater than 1 means nanotio2 is phototoxic . in a few isolated cases ,", "results were not given in the form of a number ; but it was possible to derive a number based on figures provided . because the papers reported only irradiance ( power of electromagnetic radiation per unit area ) intensity and not actual insolation ( total amount of solar radiation or ssr energy received on a given surface area during a given time ) , insolation value was calculated when possible .", "insolation was calculated based on the irradiance ( w / m ) , actual duration of irradiance ( h ) , and total duration of the toxicity test . in cases", "in which irradiance intensity was reported in units other than w / m , the data were converted for consistency . for the studies in which data existed only in the form of full spectrum insolation", ", it was important to at least approximate the levels of ultraviolet a ( uva ) and ultraviolet b ( uvb ) used in the studies . at sea level ,", "uva spectra is accountable for 5.7% of the total sunlight , whereas uvb is accountable for 0.3% of the total sunlight 14 .", "thus , uva and uvb approximations were performed on the studies reporting full spectrum with factors of 0.057 and 0.003 for uva and uvb , respectively .", "such conversions allowed us to determine whether uva and uvb levels used in the studies were of environmental relevance by comparing the data with published averaged uva and uvb levels over europe .", "because the focus of the present study is environmental relevance , in all cases , data points were excluded from evaluation if testing conditions did not represent environmentally relevant exposure conditions , such as in vitro toxicity tests with cells .", "nanotio2 toxicity was reported as greater than the highest exposure concentration with no negative toxic effects . in those cases ,", "greater than  value was from the control tio2 group that was not exposed to ssr or sunlight .", "this might have led to a slight underestimation of the phototoxicity ratio value , which can be seen as a conservative approach . in all cases ,", "the crystal structure of the nanotio2 particles , their primary particle size , and their hydrodynamic diameter were reported , and the data are presented in table 1 .", "therefore , collected data are a mixture of both anatase and rutile crystal forms as well as various particle sizes . ecosystems generally contain a mixture of all sizes and types of crystal structures of anthropogenically introduced nanoparticles with which decision makers have to cope simultaneously ; thus , the aim of the phototoxicity ratio is to provide a distinct value within a muddle .", "the coating of nanotio2 was not taken into account when evaluating phototoxicity of nanotio2 , since all of the collected studies have investigated exclusively bare nanotio2 .", "review of nanotitanium dioxide ( tio2 ) phototoxicity to various species anatase ( a ) or rutile ( r ) .", "derived data based on the presented data . calculated as 0.45  m distance ; 30% shadow angle ; and 250  w light source . not in total darkness .", "ec50  =  median effective concentration ; ic50  =  median inhibitory concentration ; lc50  =  median lethal concentration ; loec  =  lowestobservedeffect concentration ; uva  =  ultraviolet a ; uvb  =  ultraviolet b ; pr  =  phototoxicity ratio ; noec  =  noobservedeffect concentration ; na = data are not available .", "data were checked for normality with the kolmogorovsmirnov test and were found not to be of normal distribution .", "spearman rank correlation was performed between the phototoxicity ratio value and time duration of the reported toxicity test , time duration of irradiance , irradiance intensity , insolation , and the organism taxa to determine whether any of these variables drove the output value . in the case of organism taxa , for the purpose of analysis ,", "a code of 5 different digits was assigned to bacteria , algae , invertebrates , fish , and amphibians .", "kruskalwallis analysis of variance with a post hoc multiple comparison and/or mannwhitney u test were also performed where applicable .", "validation of phototoxicity ratios in correction of toxicity tests endpoint values was performed on data obtained in absence of sunlight or ssr . ", "phototoxicity data summarized in table 1 ( obtained in the presence of sunlight or ssr ) , served as a control group .", "results were log 10 transformed and then statistically compared with either log 10 ( data ) , log 10 ( data / median phototoxicity ratio ) , or log 10 ( data/75% phototoxicity ratio quartile ) .", "these 3 groups of results originated from the same set of analyzed studies but were obtained in the absence of sunlight or ssr .", "the literature search resulted in 25 usable references , from which 62 pairs of data were generated for calculation of a phototoxicity ratio ( table 1 ) . in total , experiments were performed on 20 different species , ranging from bacteria to amphibians .", "applied total irradiance was between 0.46  w / m and 231  w / m ( mean , 35.63  w / m ; median , 17  w / m ) , and effective total insolation was between 0.013  wh / m and 200  wh / m ( mean , 17.44  wh / m ; median , 2.83  wh / m ) .", "the recalculated and approximated insolation mean and median data are , respectively , 5.64  w / m and 1.7  w / m for uva , and 0.243  w / m and 0.015  w / m for uvb .", "the majority of the studies have used the same nanotio2 products ( p25 degussa ) , resulting in a fairly similar size span of primary particle diameter .", "phototoxicity ratio minimum and maximum values were 0.84 and 16 778 , respectively , and mean and median values were 407.5 and 3.7 .", "the discrepancy between the mean and median was caused primarily by the data associated with the cladocera taxon .", "when the data were analyzed for susceptibility of bacteria , algae , invertebrates , fish , and amphibians to phototoxicity , the invertebrates were significantly different compared with other groups .", "nanotio2 was significantly more toxic to invertebrates after exposure to light compared with other groups , resulting in a greater phototoxicity ratio ( kruskalwallis followed by post hoc multiple comparison ) .", "on the other hand , the spearman rank correlation test was not statistically significant for phylogeny and phototoxicity ratio ( decreased or increased phototoxicity of nanotio2 from species on the lower organism stadium , such as bacteria , toward more complex organisms , such as amphibians ) .", "also , there was no correlation between phototoxicity ratio and irradiation intensity , duration of irradiation , or received insolation .", "indeed , when exclusive cladocera data were analyzed against all other taxa ( figure 1 ) , the statistical difference was highly significant ( mannwhitney u test , p  <  0.01 ) . because of the clear need for data segregation , separate descriptive statistics were performed for cladocera and noncladocera phototoxicity ratio values ( table 2 ) . on average ,", "nanotio2 was 20 times more toxic to noncladocera and 1867 times more toxic to cladocera ( median values , 3.3 and 24.7 , respectively ) after illumination .", "descriptive statistics of phototoxicity ratio ( pr ) values significant statistical difference was observed between  true ", "phototoxicity data and data obtained in the absence of sunlight or ssr ( figure 2 ) .", "once the data were corrected by dividing data obtained in the absence of sunlight or ssr with a median phototoxicity ratio or a 75% quartile phototoxicity ratio value , there was no longer statistical difference compared with the data obtained in the presence of sunlight or ssr ( figure 2 ) .", "however , we do not claim that values for the median phototoxicity ratio and the 75% phototoxicity ratio quartile are definite , because they will change over time as more data points become available from future studies .", "comparison of uncorrected data ( top ) , corrected data with median phototoxicity ratio ( middle ) , and corrected data with 75% phototoxicity ratio quartile ( bottom ) obtained in the absence of sunlight or simulated solar radiation ( ssr ) with true data obtained in the presence of sunlight or ssr .", "cladocera and noncladocera data were corrected separately with the group corresponding median or 75% quartile values .", "the fact that the cladocera taxon was more sensitive to nanotio2 phototoxicity can not be explained by the intensity of irradiation or received insolation during testing , because such correlation was not statistically significant ( spearman rank correlation test ) . in cladocerarelated experiments ,", "the original publications from which data were derived provided no evidence that cladocera were exposed to any specific grade , type , or size of nanotio2 particles to which other taxa were not exposed .", "ultraviolet sensitivity of the taxon has to be ruled out as well , because appropriate exposure controls to uv were included and no increase in toxicity was detected . although uv is toxic and lethal to cladocera at higher exposure doses , numerous protection mechanisms prevent hazardous occurrences at lower doses 15 .", "both uv and nanotio2 toxicity are based on ros , and oxidative stress was indicated in cladocera exposed to either uv 15 , 16 or nanotio2 \n 17 .", "however , this does not necessarily mean that uv and nanotio2 have the same toxicity mechanism .", "whereas generation of ros and consequently oxidative stress following exposure to uv radiation requires endogenous photosensitizer molecules , generation of ros by nanotio2 under uv radiation is a direct photochemical process , and the substantial ros production can readily damage or kill cells or organisms such as cladocera .", "why cladocera are more sensitive to irradiated nanotio2 remains unclear , and more targeted research is needed .", "however , one possible explanation for the high sensitivity of cladocera to nanotio2 phototoxicity is that photoinduced ros on the surface of cladocera carapace may interfere with the respiratory gas exchange .", "in fact , surface attachment of nanotio2 to cladocera carapace has been observed in previous studies 18 , 19 , and the inner wall of the carapace is a major site of respiratory gas exchange for cladocera 20 .", "sunlight is composed of visible , uv , and infrared spectra . some of the analyzed studies reported irradiance values exclusively within the uv spectrum , whereas others reported values for the full spectrum .", "thus , insolation data also were presented based on reported irradiance spectrum . when the data were segregated into what appeared to be insolation values for the full spectrum , the mean insolation was 25.9  wh / m , and the median was 5  wh / m . the studies that supposedly only reported values for the uv spectrum had a mean insolation of 8.72  wh / m , with a median of 2.83  wh / m . for the purpose of comparison ,", "a solar constant ( irradiance of the sun when positioned at 1 astronomical unit compared with earth at zenith ) measured at the outer surface of earth 's atmosphere is approximately 1360  w / m \n 21 .", "a significant amount of the solar constant is lost by the time sunlight reaches a location on the earth 's surface , depending on atmosphere , latitude , and time of day .", "for example , average insolation of the visible spectrum during a decade of measurements over europe is between 5  wh / m and 302  wh / m in winter and between 285  wh / m and 430  wh / m in summer 22 .", "therefore , both the mean and median ( 25.9  wh / m and 5  wh / m , respectively ) insolation used in the studies reporting only values for full spectrum are much less than the insolation values over europe .", "the most likely culprits for tio2 phototoxicity are uva and uvb spectrum because those photons would have enough quantum energy ( uva , 3.103.94  ev per photon ; uvb , 3.944.43  ev per photon ) 23 versus energy of visible light photon ( 1.63.4  ev ) to overcome the band gap .", "when uva and uvb level approximations were performed on the studies reporting full spectrum and combined with studies directly reporting uva and uvb , mean and median values were 5.64  w / m and 1.7  w / m , respectively , for uva and 0.243  w / m and 0.015  w / m , respectively , for uvb .", "the actual uv spectrum insolation over europe is , on average , 0.7  wh / m to 37.7  wh / m in winter and 34  wh / m to 64.2  wh / m in summer for uva ; for uvb , the average is 0.001  wh / m to 1.08  wh / m in winter and 0.77  wh / m to 2.05  wh / m in summer 22 .", "the mean and the median values for uva and uvb used in toxicity studies are well within the range of uva and uvb values over europe 22 .", "therefore , the current experimental setups represent realistic and natural conditions , and the obtained results should not be doubted .", "thus , levels used in experimental setups are credible for the purpose of risk assessment , since they do not exceed natural conditions .", "it is important to note , however , that approximation to the uva and uvb values were based on the assumption that all of the irradiation lamps spectra used in the phototoxicity studies fully corresponded to sunlight spectrum .", "although the technology has advanced significantly over the years , there is no absolute guarantee that all of the studies had the proper irradiation lamps .", "furthermore , a significant amount of irradiation at sea level altitude is lost because of reflection and adsorption in the water column , according to the beerlambert law \n iz = i0ekzwhere z is depth , e is natural logorithm , k is attenuation coefficient , and i \n 0 is the energy of the sunlight at the surface of the water .", "although attenuation in pure water might not affect energy of uv light , reflectance of the water surface may , thus reducing the actual uv energy to which aquatic organisms are exposed . however", ", an opposite effect may occur in shallow waters because of strong scattering of light , thus multiplying the uv exposure levels 25 .", "therefore , although uv insolation levels currently used in nanotio2 phototoxicity studies are credible at the water surface level , it is still not clear whether they are credible for risk assessment below the water surface .", "the fact that different studies used different exposure time and different irradiances only suggest that current scientific community does not really have a standardized toxicity test to check for the phototoxicity effects of nanoparticles .", "therefore , we strongly recommend that universal agreement on irradiation time and irradiance in a standard nanomaterial phototoxicity test is necessary", ". a validation test of phototoxicity ratio correction ( figure 2 ) showed that after correction with a median phototoxicity ratio value the corrected data are no longer statistically different from the real data obtained in the presence of sunlight or ssr .", "data correction for the 75% quartile of the phototoxicity ratio was still not significantly different from the real data ( p  =  0.052 ) but in general generated much lower endpoints ( higher toxicity ) , as expected . however , the value of 75% quartile application is that , compared with the median phototoxicity ratio , it can more successfully prevent false toxicity underestimation .", "the use of phototoxicity ratio does not mean that the newly corrected data are the true representation of endpoints from toxicity tests , but rather that they are likely as close as possible .", "the true correction of data can be achieved only by defining a function through regression analysis . however , because many variables such as particle size , hydrodynamic diameter , crystal structure , illumination time , irradiance , insolation , species , and organic matter content in test media  will likely influence the phototoxicity of nanotio2 ( even if their effect is not statistically significant , they will contribute certain percentage of variability ) , generating such a function will be difficult .", "therefore , the use of a phototoxicity ratio is an oversimplified method that can provide an approximate correction with lots of versatility .", "one recent study 13 deployed a similar methodology to the present phototoxicity ratio approach to determine which is more toxic in the environment , nanosized or dissolved metals .", "toxicity ratio was calculated between median lethal dose , lc50 , ec50 , and ic50 values of dissolved and nanoparticulated metals to provide corrections for threshold values in existing regulatory standards .", "therefore , the ratio metric approach  whether toxicity ratio , phototoxicity ratio , or nanoratio  is an inexpensive , straightforward method that mitigates uncertainties for the purpose of risk assessment and management , assuming enough literature is available . in conclusion", ", the present study found that nanotio2 is phototoxic to aquatic species , because the phototoxicity ratio values were substantially greater than 1 for the majority of analyzed studies .", "existing literature on the subject is likely credible for the purpose of risk assessment because the insolation levels used in experimental setups did not exceed uv levels under natural conditions at the water surface .", "a significant difference was observed between the phototoxicity ratios of 2 analyzed groups : aquatic species belonging to order cladocera , and all other aquatic species .", "the order cladocera is very sensitive and prone to nanotio2 phototoxicity , at least in laboratorybased toxicity tests .", "a median phototoxicity ratio value and a 75% quartile were chosen as the most practical approach for correcting nanotio2 toxicity endpoints obtained in the absence of sunlight or ssr . using a median phototoxicity ratio value in correction is a more conservative approach , whereas using the 75% quartile lowers the chance of underestimating toxicity and may be favored by risk assessors when analyzing previously published data .", "the values for the phototoxicity ratio are not definite and may change as more data become available in the future .", "all of the data used for calculation and statistical analysis are presented in table 1 of the present study , with corresponding references ."], "abstract_text": ["<S> abstracttitanium dioxide nanoparticles are photoactive and produce reactive oxygen species under natural sunlight . </S>", "<S> reactive oxygen species can be detrimental to many organisms , causing oxidative damage , cell injury , and death . </S>", "<S> most studies investigating tio2 nanoparticle toxicity did not consider photoactivation and performed tests either in dark conditions or under artificial lighting that did not simulate natural irradiation . </S>", "<S> the present study summarizes the literature and derives a phototoxicity ratio between the results of nanotitanium dioxide ( nanotio2 ) experiments conducted in the absence of sunlight and those conducted under solar or simulated solar radiation ( ssr ) for aquatic species . </S>", "<S> therefore , the phototoxicity ratio can be used to correct endpoints of the toxicity tests with nanotio2 that were performed in absence of sunlight . </S>", "<S> such corrections also may be important for regulators and risk assessors when reviewing previously published data . </S>", "<S> a significant difference was observed between the phototoxicity ratios of 2 distinct groups : aquatic species belonging to order cladocera , and all other aquatic species . </S>", "<S> order cladocera appeared very sensitive and prone to nanotio2 phototoxicity . on average </S>", "<S> nanotio2 was 20 times more toxic to noncladocera and 1867 times more toxic to cladocera ( median values 3.3 and 24.7 , respectively ) after illumination . </S>", "<S> both median value and 75% quartile of the phototoxicity ratio are chosen as the most practical values for the correction of endpoints of nanotio2 toxicity tests that were performed in dark conditions , or in the absence of sunlight . </S>", "<S> environ toxicol chem 2015;34:10701077 . </S>", "<S>  2015 the author </S>", "<S> . published by setac . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION", "Data Availability"], "sections": [["titanium dioxide ( tio2 ) is a component of many sunscreens , soaps , shampoos , toothpastes , cosmetics , paper products , plastics , ink , paint , and building materials 1 in both its bulk form and its nanoform .", "it is also used in human food as a colorant and inactive ingredient , where it can also be present in both forms 1 , 2 . from 1916 to 2011 , an estimated total of 165 050 000 metric tonnes of tio2 pigment were produced worldwide ( bulk form and nanoform combined ) , with a current annual estimated production of more than 6 million tonnes / yr 2 .", "reviews of nanotio2 toxicology are available across various evolutionary groups of species 3 , 4 , 5 , 6 , 7 , 8 , often summarizing half maximal effective concentration ( ec50 ) , half maximal inhibitory concentration ( ic50 ) , and median lethal concentration ( lc50 ) values .", "nanotio2 is also photoactive and produces reactive oxygen species ( ros ) on illumination 9 .", "reactive oxygen species can be detrimental to many organisms , causing oxidative damage , cell injury , and ultimately death 10 .", "recently , it has been argued that photoactivation of nanotio2 under natural levels of sunlight is sufficient to affect the output of lc50 and ec50 values in standard toxicology tests 11 , 12 .", "the majority of studies investigating nanotio2 toxicity did not take photoactivation into account and performed tests either in dark conditions or under indoor commercial artificial lighting that did not simulate natural solar irradiation .", "the aim of present study was to derive a phototoxicity ratio between the results of the nanotio2 experiments conducted in the absence of sunlight and conducted in the presence of solar or simulated solar radiation ( ssr ) . to achieve this aim , we searched the literature for studies that included nanotio2 experiments both with and without irradiance under the same experimental setup and otherwise identical conditions .", "therefore , the phototoxicity ratio can be used to correct endpoints of the toxicity tests with nanotio2 that were performed in absence of natural sunlight or ssr .", "such corrections also may be important for regulators and risk assessors when reviewing previously published data .", "for example , one of the current challenges for conducting risk assessment of nanoparticles such as tio2 is lack of consistent toxicity data because of the varieties of materials and test conditions .", "regulatory thresholds for nanotio2 do not exist currently ; however , regulation of nanoparticles discharge and monitoring in aquatic environment is anticipated in the future .", "it is expected that regulators will use the phototoxicity ratio when deriving thresholds for nanotio2 because the majority of the published literature reported toxicity endpoints for nanotio2 in the absence of natural sunlight or ssr .", "of course , the phototoxicity ratio value calculated in present study is not absolutely precise and correct for all environmental conditions and species ; it does , however , considerably reduce the possible error of data endpoints obtained in the absence of natural sunlight or ssr .", "it also mitigates uncertainties in the risk assessment process by taking into account the photoactivation and phototoxicity of nanotio2 ."], ["a comprehensive literature review was conducted to collect available toxicity endpoints for nanotio2 . the literature search ( september 2014 ) was performed within 4 databases  web of science , scopus , google scholar , and the university of british columbia library database  using the following keywords in various combinations : titanium dioxide , tio2 , nanoparticles , phototoxicity , photoactivation , ec50 , lc50 , ic50 , and lowestobservedeffect concentration ( loec ) .", "abstracts of numerous hits were read , and downloaded papers were checked for useful information . only papers that reported results under the same environmental conditions for 2 different nanotio2 exposure groups ( with and without ssr ) in the form of ec50 , lc50 , ic50 , loec , or a ratio were selected .", "thus , all included data were based on dose  response curves , ensuring the highest possible quality .", "the phototoxicity ratio ( pr ) was calculated in the form of a ratio : \n pr = tio2  lc50 ,  ec50 ,  ic50 ,  loec  without  sunlight  or  ssrtio2  lc50 ,  ec50 ,  ic50 ,  loec  with  sunlight  or  ssra phototoxicity ratio greater than 1 means nanotio2 is phototoxic . in a few isolated cases ,", "results were not given in the form of a number ; but it was possible to derive a number based on figures provided . because the papers reported only irradiance ( power of electromagnetic radiation per unit area ) intensity and not actual insolation ( total amount of solar radiation or ssr energy received on a given surface area during a given time ) , insolation value was calculated when possible .", "insolation was calculated based on the irradiance ( w / m ) , actual duration of irradiance ( h ) , and total duration of the toxicity test . in cases", "in which irradiance intensity was reported in units other than w / m , the data were converted for consistency . for the studies in which data existed only in the form of full spectrum insolation", ", it was important to at least approximate the levels of ultraviolet a ( uva ) and ultraviolet b ( uvb ) used in the studies . at sea level ,", "uva spectra is accountable for 5.7% of the total sunlight , whereas uvb is accountable for 0.3% of the total sunlight 14 .", "thus , uva and uvb approximations were performed on the studies reporting full spectrum with factors of 0.057 and 0.003 for uva and uvb , respectively .", "such conversions allowed us to determine whether uva and uvb levels used in the studies were of environmental relevance by comparing the data with published averaged uva and uvb levels over europe .", "because the focus of the present study is environmental relevance , in all cases , data points were excluded from evaluation if testing conditions did not represent environmentally relevant exposure conditions , such as in vitro toxicity tests with cells .", "nanotio2 toxicity was reported as greater than the highest exposure concentration with no negative toxic effects . in those cases ,", "greater than  value was from the control tio2 group that was not exposed to ssr or sunlight .", "this might have led to a slight underestimation of the phototoxicity ratio value , which can be seen as a conservative approach . in all cases ,", "the crystal structure of the nanotio2 particles , their primary particle size , and their hydrodynamic diameter were reported , and the data are presented in table 1 .", "therefore , collected data are a mixture of both anatase and rutile crystal forms as well as various particle sizes . ecosystems generally contain a mixture of all sizes and types of crystal structures of anthropogenically introduced nanoparticles with which decision makers have to cope simultaneously ; thus , the aim of the phototoxicity ratio is to provide a distinct value within a muddle .", "the coating of nanotio2 was not taken into account when evaluating phototoxicity of nanotio2 , since all of the collected studies have investigated exclusively bare nanotio2 .", "review of nanotitanium dioxide ( tio2 ) phototoxicity to various species anatase ( a ) or rutile ( r ) .", "derived data based on the presented data . calculated as 0.45  m distance ; 30% shadow angle ; and 250  w light source . not in total darkness .", "ec50  =  median effective concentration ; ic50  =  median inhibitory concentration ; lc50  =  median lethal concentration ; loec  =  lowestobservedeffect concentration ; uva  =  ultraviolet a ; uvb  =  ultraviolet b ; pr  =  phototoxicity ratio ; noec  =  noobservedeffect concentration ; na = data are not available .", "data were checked for normality with the kolmogorovsmirnov test and were found not to be of normal distribution .", "spearman rank correlation was performed between the phototoxicity ratio value and time duration of the reported toxicity test , time duration of irradiance , irradiance intensity , insolation , and the organism taxa to determine whether any of these variables drove the output value . in the case of organism taxa , for the purpose of analysis ,", "a code of 5 different digits was assigned to bacteria , algae , invertebrates , fish , and amphibians .", "kruskalwallis analysis of variance with a post hoc multiple comparison and/or mannwhitney u test were also performed where applicable .", "validation of phototoxicity ratios in correction of toxicity tests endpoint values was performed on data obtained in absence of sunlight or ssr . ", "phototoxicity data summarized in table 1 ( obtained in the presence of sunlight or ssr ) , served as a control group .", "results were log 10 transformed and then statistically compared with either log 10 ( data ) , log 10 ( data / median phototoxicity ratio ) , or log 10 ( data/75% phototoxicity ratio quartile ) .", "these 3 groups of results originated from the same set of analyzed studies but were obtained in the absence of sunlight or ssr ."], ["the literature search resulted in 25 usable references , from which 62 pairs of data were generated for calculation of a phototoxicity ratio ( table 1 ) . in total , experiments were performed on 20 different species , ranging from bacteria to amphibians .", "applied total irradiance was between 0.46  w / m and 231  w / m ( mean , 35.63  w / m ; median , 17  w / m ) , and effective total insolation was between 0.013  wh / m and 200  wh / m ( mean , 17.44  wh / m ; median , 2.83  wh / m ) .", "the recalculated and approximated insolation mean and median data are , respectively , 5.64  w / m and 1.7  w / m for uva , and 0.243  w / m and 0.015  w / m for uvb .", "the majority of the studies have used the same nanotio2 products ( p25 degussa ) , resulting in a fairly similar size span of primary particle diameter .", "phototoxicity ratio minimum and maximum values were 0.84 and 16 778 , respectively , and mean and median values were 407.5 and 3.7 .", "the discrepancy between the mean and median was caused primarily by the data associated with the cladocera taxon .", "when the data were analyzed for susceptibility of bacteria , algae , invertebrates , fish , and amphibians to phototoxicity , the invertebrates were significantly different compared with other groups .", "nanotio2 was significantly more toxic to invertebrates after exposure to light compared with other groups , resulting in a greater phototoxicity ratio ( kruskalwallis followed by post hoc multiple comparison ) .", "on the other hand , the spearman rank correlation test was not statistically significant for phylogeny and phototoxicity ratio ( decreased or increased phototoxicity of nanotio2 from species on the lower organism stadium , such as bacteria , toward more complex organisms , such as amphibians ) .", "also , there was no correlation between phototoxicity ratio and irradiation intensity , duration of irradiation , or received insolation .", "indeed , when exclusive cladocera data were analyzed against all other taxa ( figure 1 ) , the statistical difference was highly significant ( mannwhitney u test , p  <  0.01 ) . because of the clear need for data segregation , separate descriptive statistics were performed for cladocera and noncladocera phototoxicity ratio values ( table 2 ) . on average ,", "nanotio2 was 20 times more toxic to noncladocera and 1867 times more toxic to cladocera ( median values , 3.3 and 24.7 , respectively ) after illumination .", "descriptive statistics of phototoxicity ratio ( pr ) values significant statistical difference was observed between  true ", "phototoxicity data and data obtained in the absence of sunlight or ssr ( figure 2 ) .", "once the data were corrected by dividing data obtained in the absence of sunlight or ssr with a median phototoxicity ratio or a 75% quartile phototoxicity ratio value , there was no longer statistical difference compared with the data obtained in the presence of sunlight or ssr ( figure 2 ) .", "however , we do not claim that values for the median phototoxicity ratio and the 75% phototoxicity ratio quartile are definite , because they will change over time as more data points become available from future studies .", "comparison of uncorrected data ( top ) , corrected data with median phototoxicity ratio ( middle ) , and corrected data with 75% phototoxicity ratio quartile ( bottom ) obtained in the absence of sunlight or simulated solar radiation ( ssr ) with true data obtained in the presence of sunlight or ssr .", "cladocera and noncladocera data were corrected separately with the group corresponding median or 75% quartile values ."], ["the fact that the cladocera taxon was more sensitive to nanotio2 phototoxicity can not be explained by the intensity of irradiation or received insolation during testing , because such correlation was not statistically significant ( spearman rank correlation test ) . in cladocerarelated experiments ,", "the original publications from which data were derived provided no evidence that cladocera were exposed to any specific grade , type , or size of nanotio2 particles to which other taxa were not exposed .", "ultraviolet sensitivity of the taxon has to be ruled out as well , because appropriate exposure controls to uv were included and no increase in toxicity was detected . although uv is toxic and lethal to cladocera at higher exposure doses , numerous protection mechanisms prevent hazardous occurrences at lower doses 15 .", "both uv and nanotio2 toxicity are based on ros , and oxidative stress was indicated in cladocera exposed to either uv 15 , 16 or nanotio2 \n 17 .", "however , this does not necessarily mean that uv and nanotio2 have the same toxicity mechanism .", "whereas generation of ros and consequently oxidative stress following exposure to uv radiation requires endogenous photosensitizer molecules , generation of ros by nanotio2 under uv radiation is a direct photochemical process , and the substantial ros production can readily damage or kill cells or organisms such as cladocera .", "why cladocera are more sensitive to irradiated nanotio2 remains unclear , and more targeted research is needed .", "however , one possible explanation for the high sensitivity of cladocera to nanotio2 phototoxicity is that photoinduced ros on the surface of cladocera carapace may interfere with the respiratory gas exchange .", "in fact , surface attachment of nanotio2 to cladocera carapace has been observed in previous studies 18 , 19 , and the inner wall of the carapace is a major site of respiratory gas exchange for cladocera 20 .", "sunlight is composed of visible , uv , and infrared spectra . some of the analyzed studies reported irradiance values exclusively within the uv spectrum , whereas others reported values for the full spectrum .", "thus , insolation data also were presented based on reported irradiance spectrum . when the data were segregated into what appeared to be insolation values for the full spectrum , the mean insolation was 25.9  wh / m , and the median was 5  wh / m . the studies that supposedly only reported values for the uv spectrum had a mean insolation of 8.72  wh / m , with a median of 2.83  wh / m . for the purpose of comparison ,", "a solar constant ( irradiance of the sun when positioned at 1 astronomical unit compared with earth at zenith ) measured at the outer surface of earth 's atmosphere is approximately 1360  w / m \n 21 .", "a significant amount of the solar constant is lost by the time sunlight reaches a location on the earth 's surface , depending on atmosphere , latitude , and time of day .", "for example , average insolation of the visible spectrum during a decade of measurements over europe is between 5  wh / m and 302  wh / m in winter and between 285  wh / m and 430  wh / m in summer 22 .", "therefore , both the mean and median ( 25.9  wh / m and 5  wh / m , respectively ) insolation used in the studies reporting only values for full spectrum are much less than the insolation values over europe .", "the most likely culprits for tio2 phototoxicity are uva and uvb spectrum because those photons would have enough quantum energy ( uva , 3.103.94  ev per photon ; uvb , 3.944.43  ev per photon ) 23 versus energy of visible light photon ( 1.63.4  ev ) to overcome the band gap .", "when uva and uvb level approximations were performed on the studies reporting full spectrum and combined with studies directly reporting uva and uvb , mean and median values were 5.64  w / m and 1.7  w / m , respectively , for uva and 0.243  w / m and 0.015  w / m , respectively , for uvb .", "the actual uv spectrum insolation over europe is , on average , 0.7  wh / m to 37.7  wh / m in winter and 34  wh / m to 64.2  wh / m in summer for uva ; for uvb , the average is 0.001  wh / m to 1.08  wh / m in winter and 0.77  wh / m to 2.05  wh / m in summer 22 .", "the mean and the median values for uva and uvb used in toxicity studies are well within the range of uva and uvb values over europe 22 .", "therefore , the current experimental setups represent realistic and natural conditions , and the obtained results should not be doubted .", "thus , levels used in experimental setups are credible for the purpose of risk assessment , since they do not exceed natural conditions .", "it is important to note , however , that approximation to the uva and uvb values were based on the assumption that all of the irradiation lamps spectra used in the phototoxicity studies fully corresponded to sunlight spectrum .", "although the technology has advanced significantly over the years , there is no absolute guarantee that all of the studies had the proper irradiation lamps .", "furthermore , a significant amount of irradiation at sea level altitude is lost because of reflection and adsorption in the water column , according to the beerlambert law \n iz = i0ekzwhere z is depth , e is natural logorithm , k is attenuation coefficient , and i \n 0 is the energy of the sunlight at the surface of the water .", "although attenuation in pure water might not affect energy of uv light , reflectance of the water surface may , thus reducing the actual uv energy to which aquatic organisms are exposed . however", ", an opposite effect may occur in shallow waters because of strong scattering of light , thus multiplying the uv exposure levels 25 .", "therefore , although uv insolation levels currently used in nanotio2 phototoxicity studies are credible at the water surface level , it is still not clear whether they are credible for risk assessment below the water surface .", "the fact that different studies used different exposure time and different irradiances only suggest that current scientific community does not really have a standardized toxicity test to check for the phototoxicity effects of nanoparticles .", "therefore , we strongly recommend that universal agreement on irradiation time and irradiance in a standard nanomaterial phototoxicity test is necessary", ". a validation test of phototoxicity ratio correction ( figure 2 ) showed that after correction with a median phototoxicity ratio value the corrected data are no longer statistically different from the real data obtained in the presence of sunlight or ssr .", "data correction for the 75% quartile of the phototoxicity ratio was still not significantly different from the real data ( p  =  0.052 ) but in general generated much lower endpoints ( higher toxicity ) , as expected . however , the value of 75% quartile application is that , compared with the median phototoxicity ratio , it can more successfully prevent false toxicity underestimation .", "the use of phototoxicity ratio does not mean that the newly corrected data are the true representation of endpoints from toxicity tests , but rather that they are likely as close as possible .", "the true correction of data can be achieved only by defining a function through regression analysis . however , because many variables such as particle size , hydrodynamic diameter , crystal structure , illumination time , irradiance , insolation , species , and organic matter content in test media  will likely influence the phototoxicity of nanotio2 ( even if their effect is not statistically significant , they will contribute certain percentage of variability ) , generating such a function will be difficult .", "therefore , the use of a phototoxicity ratio is an oversimplified method that can provide an approximate correction with lots of versatility .", "one recent study 13 deployed a similar methodology to the present phototoxicity ratio approach to determine which is more toxic in the environment , nanosized or dissolved metals .", "toxicity ratio was calculated between median lethal dose , lc50 , ec50 , and ic50 values of dissolved and nanoparticulated metals to provide corrections for threshold values in existing regulatory standards .", "therefore , the ratio metric approach  whether toxicity ratio , phototoxicity ratio , or nanoratio  is an inexpensive , straightforward method that mitigates uncertainties for the purpose of risk assessment and management , assuming enough literature is available . in conclusion", ", the present study found that nanotio2 is phototoxic to aquatic species , because the phototoxicity ratio values were substantially greater than 1 for the majority of analyzed studies .", "existing literature on the subject is likely credible for the purpose of risk assessment because the insolation levels used in experimental setups did not exceed uv levels under natural conditions at the water surface .", "a significant difference was observed between the phototoxicity ratios of 2 analyzed groups : aquatic species belonging to order cladocera , and all other aquatic species .", "the order cladocera is very sensitive and prone to nanotio2 phototoxicity , at least in laboratorybased toxicity tests .", "a median phototoxicity ratio value and a 75% quartile were chosen as the most practical approach for correcting nanotio2 toxicity endpoints obtained in the absence of sunlight or ssr . using a median phototoxicity ratio value in correction is a more conservative approach , whereas using the 75% quartile lowers the chance of underestimating toxicity and may be favored by risk assessors when analyzing previously published data .", "the values for the phototoxicity ratio are not definite and may change as more data become available in the future ."], ["all of the data used for calculation and statistical analysis are presented in table 1 of the present study , with corresponding references ."]]}
{"article_id": "PMC2600383", "article_text": ["we report 2 patients infected with panton - valentine leukocidin ( pvl)positive mrsa t034 . each patient had a medical history typical of that reported for community - acquired mrsa of other lineages , which in most cases are pvl positive ( 8) . the first patient , a previously healthy 36-year - old male physiotherapist , sought medical care in march 2006 for a small abscess in his axilla . culture of the abscess grew mrsa .", "presence of meca gene was confirmed by pcr ( 9 ) . during the next 2 months ,", "his youngest child , adopted from china , had been found to be mrsa positive ( throat , perineum , and a small wound ) a month earlier during routine screening for adopted children . during subsequent screening of the family , the older sister , adopted from south korea ,", "both parents were negative for mrsa at that time , which suggests that the father was newly infected when his abscess developed and that he had not acquired the strain abroad .", "also , spa typing indicated that the children carried different strains from that of the father and from each other ( t286 , t1434 ) ( 10 ) .", "subsequent screening of family members for mrsa on several occasions found only the father to be repeatedly positive .", "the second patient , a 43-year - old male clerk , also previously healthy , sought medical attention during the summer of 2007 for a mrsa - infected elbow wound .", "follow - up examination determined that he carried mrsa also in the perineum and in a chronic external otitis eczema .", "the patients lived in geographically distinct areas in the western part of sweden and had no connection to each other . no animal contact", "( e.g. , pets , farming ) was reported by the 2 patients , their family members , or other close contacts . both patient strains carried pvl , confirmed by identification of the luks - lukf genes ( 11 ) , and were resistant to digestion with restriction endonuclease smai when typing by pfge was attempted .", "they produced -hemolysin according to phenotypic detection methods that used rabbit blood agar with hot  cold analysis , which further indicated their animal origin ( 12 ) .", "their drug - susceptibility profiles differed ; 1 was resistant to doxycycline and the other was resistant to ciprofloxacin , erythromycin , and clindamycin .", "these strains carry pvl , a toxin partly responsible for the increased virulence of several of the mrsa clones in the community ( 8) . despite several recent publications concerning st398 mrsa ,", "few have reported pvl in this lineage , which is believed to be of animal origin ( 2,4,6,13 ) .", "most previous reports have described asymptomatic carriage in persons exposed to occupational hazards ( e.g. , veterinary personal and pig farmers ) ( 1,2,7 ) . however , severe clinical infections have been described ( 46 ) . in our patients these strains caused repeated infections that needed medical attention , even hospitalization .", "since neither patient had even a remote connection to animals and we found no common source of infection , these strains may already be more common in our region than we had thought .", "these case reports suggest that strains of this lineage may impose a threat in the community , even to patients with no obvious animal contact ."], "abstract_text": ["<S> panton - valentine leukocidin ( pvl)positive methicillin - resistant staphylococcus aureus ( mrsa ) , sequence type 398 is believed to be of animal origin . </S>", "<S> we report 2 cases of infection due to pvl  positive mrsa , spa type t034 , in patients in sweden who had had no animal contact . </S>"], "labels": null, "section_names": ["The Cases", "Conclusions"], "sections": [["we report 2 patients infected with panton - valentine leukocidin ( pvl)positive mrsa t034 . each patient had a medical history typical of that reported for community - acquired mrsa of other lineages , which in most cases are pvl positive ( 8) . the first patient , a previously healthy 36-year - old male physiotherapist , sought medical care in march 2006 for a small abscess in his axilla . culture of the abscess grew mrsa .", "presence of meca gene was confirmed by pcr ( 9 ) . during the next 2 months ,", "his youngest child , adopted from china , had been found to be mrsa positive ( throat , perineum , and a small wound ) a month earlier during routine screening for adopted children . during subsequent screening of the family , the older sister , adopted from south korea ,", "both parents were negative for mrsa at that time , which suggests that the father was newly infected when his abscess developed and that he had not acquired the strain abroad .", "also , spa typing indicated that the children carried different strains from that of the father and from each other ( t286 , t1434 ) ( 10 ) .", "subsequent screening of family members for mrsa on several occasions found only the father to be repeatedly positive .", "the second patient , a 43-year - old male clerk , also previously healthy , sought medical attention during the summer of 2007 for a mrsa - infected elbow wound .", "follow - up examination determined that he carried mrsa also in the perineum and in a chronic external otitis eczema .", "the patients lived in geographically distinct areas in the western part of sweden and had no connection to each other . no animal contact", "( e.g. , pets , farming ) was reported by the 2 patients , their family members , or other close contacts . both patient strains carried pvl , confirmed by identification of the luks - lukf genes ( 11 ) , and were resistant to digestion with restriction endonuclease smai when typing by pfge was attempted .", "they produced -hemolysin according to phenotypic detection methods that used rabbit blood agar with hot  cold analysis , which further indicated their animal origin ( 12 ) .", "their drug - susceptibility profiles differed ; 1 was resistant to doxycycline and the other was resistant to ciprofloxacin , erythromycin , and clindamycin ."], ["these strains carry pvl , a toxin partly responsible for the increased virulence of several of the mrsa clones in the community ( 8) . despite several recent publications concerning st398 mrsa ,", "few have reported pvl in this lineage , which is believed to be of animal origin ( 2,4,6,13 ) .", "most previous reports have described asymptomatic carriage in persons exposed to occupational hazards ( e.g. , veterinary personal and pig farmers ) ( 1,2,7 ) . however , severe clinical infections have been described ( 46 ) . in our patients these strains caused repeated infections that needed medical attention , even hospitalization .", "since neither patient had even a remote connection to animals and we found no common source of infection , these strains may already be more common in our region than we had thought .", "these case reports suggest that strains of this lineage may impose a threat in the community , even to patients with no obvious animal contact ."]]}
{"article_id": "PMC5083936", "article_text": ["the selective melatonin receptor agonism effect of ramelteon is useful for insomnia.1 ) here we wanted to present a refractory chronic migraine case , who had significant improvements in migraine after using ramelteon .", "the precipitating factors were the stress , increase in physical activities and sudden cold weather .", "the moderate or severe pain intensity was aggravated by or causing avoidance of increased physical activity .", "the residual symptom between the migraine episodes were the head tense feeling , but not achieveing headache severity .", "the computed tomography revealed no significant findings of other causes for headache , such as tumor or hemorrhage .", "she ever received many kinds of medications , such as aspirin , nonsteroidal anti - inflammatory drugs , triptans , ergots , anticonvulsants and glucocorticoids .", "however , the severity of migraine still remained moderate ( migraine disability assessment test [ midas ] scores , 19 ) and the associated symptoms were moderate headache , dizziness , nausea , and sensitivity to sound .", "the migraine also exacerbated her insomnia problems , with fragmented sleep and inadequate sleep duration as 12 hours ( insomnia severity index [ isi ] score , 23 ) .", "she did nt have any comorbid mental disorder except insomnia . due to the fear of abuse potential related to hypnotics", "after 2 weeks treatment of ramelteon 8 mg / day , her sleep duration prolonged to 45 hours with less fragmentation .", "in addition , her migraine severity started to decline ( midas scores , 11 ) .", "after 6 months of ramelteon 8 mg / day treatment , the migraine severity continued to relieve ( midas scores , 6 ) with stable sleeping quality ( isi score , 14 ) .", "in this case , we found significant improvements in migraine severity and insomnia after the use of ramelteon . since the patient did nt have insomnia before the first onset of migraine .", "therefore we could speculate that the ramelteon had significant effects in migraine in such mild prolongation of sleep duration .", "the melatonin might relieve the headache via the following possible mechanisms , such as anti - inflammatory effect , free radical scavenging , reduction of pro - inflammatory cytokine , membrane stabilization , nitric oxide synthase activity and dopamine release inhibition , gaba and opioid analgesia potentitation , glutamate neurotoxicity protection , neurovascular regulation,2 ) cytoprotection and antiallodynic action.3 ) the pineal gland , the primary source of serotonin and melatonin , might also play a significant role in the analgesic effect.4 ) however , the clinical trial of melatonin administration showed no significant improvements in migraine severity,5 ) which suggested the modulation of melatonin receptor might be a better option for the analgesic effects .", "the modulation of serotonin system by the ramelteon would also relieve the pain sensation.2 ) in addition , the chemical structure of ramelteon was similar with that of indomethacin , which is a kind of non - steroidal anti - inflammatory medicine.2 ) in the study of animal model , the antinociceptive and antiallodynic actions of melatonin have been observed in different kinds of pathways , such as intrathecally or intracerebroventricular routes .", "the use of ramelteon , a kind of mt1 and mt2 agonist , could also relieve the pain even lack of free radical scavenger effects.3 ) the state - of - art treatment of refractory chronic migraine also includes the ramelteon due to selective m1 receptor agonism.6 ) from the successful experience in this case , we can consider the use of ramelteon for refractory chronic migraine with insomnia ."], "abstract_text": ["<S> the selective melatonin receptor agonism effect of ramelteon is useful for insomnia . </S>", "<S> here we wanted to present a refractory chronic migraine case , who had significant improvements in migraine after using ramelteon . </S>", "<S> the possible mechanism for the ramelteon in the migraine relief might be related to melatonin effects . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE", "DISCUSSION"], "sections": [["the selective melatonin receptor agonism effect of ramelteon is useful for insomnia.1 ) here we wanted to present a refractory chronic migraine case , who had significant improvements in migraine after using ramelteon ."], ["the precipitating factors were the stress , increase in physical activities and sudden cold weather .", "the moderate or severe pain intensity was aggravated by or causing avoidance of increased physical activity .", "the residual symptom between the migraine episodes were the head tense feeling , but not achieveing headache severity .", "the computed tomography revealed no significant findings of other causes for headache , such as tumor or hemorrhage .", "she ever received many kinds of medications , such as aspirin , nonsteroidal anti - inflammatory drugs , triptans , ergots , anticonvulsants and glucocorticoids .", "however , the severity of migraine still remained moderate ( migraine disability assessment test [ midas ] scores , 19 ) and the associated symptoms were moderate headache , dizziness , nausea , and sensitivity to sound .", "the migraine also exacerbated her insomnia problems , with fragmented sleep and inadequate sleep duration as 12 hours ( insomnia severity index [ isi ] score , 23 ) .", "she did nt have any comorbid mental disorder except insomnia . due to the fear of abuse potential related to hypnotics", "after 2 weeks treatment of ramelteon 8 mg / day , her sleep duration prolonged to 45 hours with less fragmentation .", "in addition , her migraine severity started to decline ( midas scores , 11 ) .", "after 6 months of ramelteon 8 mg / day treatment , the migraine severity continued to relieve ( midas scores , 6 ) with stable sleeping quality ( isi score , 14 ) ."], ["in this case , we found significant improvements in migraine severity and insomnia after the use of ramelteon . since the patient did nt have insomnia before the first onset of migraine .", "therefore we could speculate that the ramelteon had significant effects in migraine in such mild prolongation of sleep duration .", "the melatonin might relieve the headache via the following possible mechanisms , such as anti - inflammatory effect , free radical scavenging , reduction of pro - inflammatory cytokine , membrane stabilization , nitric oxide synthase activity and dopamine release inhibition , gaba and opioid analgesia potentitation , glutamate neurotoxicity protection , neurovascular regulation,2 ) cytoprotection and antiallodynic action.3 ) the pineal gland , the primary source of serotonin and melatonin , might also play a significant role in the analgesic effect.4 ) however , the clinical trial of melatonin administration showed no significant improvements in migraine severity,5 ) which suggested the modulation of melatonin receptor might be a better option for the analgesic effects .", "the modulation of serotonin system by the ramelteon would also relieve the pain sensation.2 ) in addition , the chemical structure of ramelteon was similar with that of indomethacin , which is a kind of non - steroidal anti - inflammatory medicine.2 ) in the study of animal model , the antinociceptive and antiallodynic actions of melatonin have been observed in different kinds of pathways , such as intrathecally or intracerebroventricular routes .", "the use of ramelteon , a kind of mt1 and mt2 agonist , could also relieve the pain even lack of free radical scavenger effects.3 ) the state - of - art treatment of refractory chronic migraine also includes the ramelteon due to selective m1 receptor agonism.6 ) from the successful experience in this case , we can consider the use of ramelteon for refractory chronic migraine with insomnia ."]]}
{"article_id": "PMC3697295", "article_text": ["it was previously suggested that atherothrombotic infarction ( ati ) and lacunar infarction ( li ) , as two different subtypes of ischemic stroke , might also differ in the set of the relevant risk factors , with ati being more associated to the atherogenic risk factors in contrast to li .", ".   however , decreased insulin sensitivity ( is ) , that is , insulin resistance , was observed both in ati and li , which was frequently accompanied with compensatory hyperinsulinemia in t2d patients as well as in nondiabetics [ 2 , 3 ] .", "simultaneously , it has been shown that impaired balance between products of oxidative stress and the level of antioxidant enzyme activities might be the important mechanism underlying the occurrence of ischemic stroke . moreover , it was elucidated that the brain has only moderate content of glutathione - dependent enzymes , for example , glutathione peroxidase ( gsh - px ) , glutathione reductase ( gr ) , and superoxide dismutase ( sod ) , together with the fact that the intact antioxidant defense could provide first line of protection from initiation and exacerbation of ischemic cerebral injury .  ", "in addition , changes in enzymatic antioxidative defense mechanisms in patients with stroke are still controversial .", "previous results implied that the majority of antioxidant enzyme activity was significantly reduced in acute ischemic stroke , possibly as a consequence of increased oxidative stress   while the recent finding suggested increased levels of glutathione dependent enzymes as an adaptive mechanisms during acute cerebral ischemia . finally , due to novel facts", ", oxidative stress can be an important component for astrocytic cell death following metabolic stress .   until now , experimental studies provided evidence of an association between ischemic stroke and increased oxidative stress [ 8 , 9 ] , but data in humans are still heterogeneous and limited .", "therefore , our study was aimed to determine is levels and three different types of antioxidant enzyme activities gsh - px , gr , and sod , in t2d with ati and li .", "in this study we included a total of 93 patients with t2d , ascribed to the following groups : t2d patients with ati ( group a , n = 30 ) , and t2d with li ( group b , n = 30 ) , and t2d without ischemic stroke ( group c , n = 33 ) .", "simultaneously , we involved a total of 93 nondiabetics , matched with the t2d patients regarding gender and age , and also comprising the following groups : nondiabetics with ati ( group d , n = 30 ) , nondiabetics with li ( group e , n = 30 ) , and nondiabetics without stroke ( group f , n = 33 ) .   t2d was diagnosed in accordance with the criteria of the world health organization .", "diagnosis of ati and li was done by a neurologist due to clinical features and brain imaging methods such as cranial computerized scan and/or magnetic resonance imaging in two consecutive examinations , during the first 7 days from the appearance of ischemic stroke .", "the patients with ati or li were included in the study provided that they had not shown signs of cardioembolic cerebral infarction , or coronary heart disease based on a history of myocardial infarction with definite elevation of serum cardiac enzymes or coronary angiography .", "t2d patients were treated with insulin therapy , and/or ingestion of antioxidant supplements and drugs , which might affect free radical and antioxidant activity potential ; likewise patients who had other endocrine disease or autoimmune diseases , renal or hepatic failure , current infections , neoplasms , polycythemia , or rheumatic diseases were also excluded as well as the patients with history of trauma or operation within the last 3 months .", "no patient had uncontrolled hypertension , severe alcohol consumption , acute infection , or an inflammatory disease during the last 4 weeks .", "all the patients , with or without ati and li , showed the similar level of their physical activity .", "in addition , they were required not to smoke at least 12  hr before the tests were performed .", "the patients were fully informed about the study and gave the inform consent to participate .  ", "the study was conducted at the clinic for endocrinology , diabetes and metabolic diseases and at the clinic for neurology , clinical centre of serbia , faculty of medicine , university of belgrade , and was approved by the institutional ethics committee .", "the interview , physical examination , metabolic test , and evaluation of antioxidant enzyme activities were performed in all the patients included in the study , for each patient within the same day .", "hypertension was diagnosed according to world health organisation criteria ( systolic / diastolic blood pressure 140/90  mm  hg ) or by the use of antihypertensive agents .", "the metabolic tests were implemented at least after 6 months from the occurrence of the ischemic stroke .   ", "the evaluation of insulin sensitivity was done by frequently sampled intravenous glucose tolerance test ( fsigt ) with minimal model analysis .", "briefly , before testing , each patient was required to be at a 12  hr fasting state . during the fsigt ,", "0.3  g / kg body weight of glucose was injected and the blood samples for plasma glucose ( pg ) and plasma insulin ( pi ) determination were taken immediately before and 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 14 , 16 , 20 , 23 , 24 , 25 , 27 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 160 , and 180 minutes after intravenous glucose stimulation .", "insulin was injected as a continuous infusion 4  mu / kg / min between minutes 20 and 25 in order to avoid the effect of the potentially blunted insulin response .", "the insulin sensitivity index ( si ) was calculated from the results of pg and pi levels by computerized minimal model analysis , using the minmod program ( kindly provided by dr .", "determination of the antioxidant enzymes sod , gsh - px , gr was conducted using the commercial assays ( produced by randox laboratories ltd . , uk ) , based on spectrophotometer determination methods as described previously .", "pg was determined by glucose oxidase method using a beckman glucose analyser ( beckman instruments , fullerton , ca ) .", "pi was tested by radioimmunoassay ( inep , zemun , rs , double antibody kits ) .", "total cholesterol , hdl cholesterol , and triglyceride concentrations were determined with the chromatography method using commercial kits ( produced by boehringer mannheim ) .", "the continuous variables within each subtype of ischemic stroke were analyzed with analysis of variance ( anova ) with a post hoc bonferroni test .", "all analyses were performed with the spss statistical package ( version 16.0 for windows ) .", "the clinical characteristics and biochemistry parameters of t2d patients and nondiabetics with ati or li as different subtypes of ischemic stroke are shown in table 1 .", "the age , gender , duration of diabetes , and duration from the onset of ischemic stroke were similar in t2d patients and nondiabetics with different subtypes of ischemic stroke , together with hba1c levels , implying satisfactory metabolic control before metabolic investigation was done . however , ldl - c level was significantly higher in t2d patients with ati compared to the t2d patients with li and t2d patients without stroke and also in nondiabetics with ati compared to nondiabetics with li and healthy controls .", "there is no difference in prevalence of hypertension in patients with t2d and ati or li and t2d without ischemic stroke , while it was significantly higher in nondiabetics with ati or li than in healthy controls .", "we found that si levels were significantly lower in t2d patients with ati ( group a ) and li ( group b ) compared to t2d patients without ischemic stroke ( group c ) ( 1.14  0.58 and 1.00  0.26 versus 3.14  0.62  min / mu / l  10 , resp .", "also , the results showed significantly lower si levels in nondiabetics with ati ( group d ) and li ( group e ) compared to healthy controls ( group f ) ( 3.38  0.77 and 3.03  0.72 versus 6.03  1.69  min / mu / l  10 , resp .", "simultaneously , pi levels were higher in t2d patients , ati ( group a ) , and li ( group b ) than in t2d patients without ischemic stroke ( group c ) ( 20.94  4.31 and 20.07  0.88 versus 16.06  0.91 ", "mu / l , respectively , p < 0.001 ) and in nondiabetics with ati ( group d ) or li ( group e ) in comparison to healthy controls ( group f ) ( 15.57  1.86 and 15.59  1.26 versus 7.54  2.03 ", "mu / l , respectively , p < 0.001 ) ( figure 2 ) .  ", "when we evaluated antioxidant enzyme activities in t2d patients with ati ( group a ) and li ( group b ) and without ischemic stroke ( group c ) and in nondiabetics with ati ( group d ) and li ( group e ) and healthy controls ( group d ) , we detected the levels of the gsh - px and gr activity being significantly lower in group a and b versus c and in group d and e versus f ( gshpx : a : 21.96  3.56 versus b : 22.51  1.23 versus c : 25.12  1.67  u / ghb ,  p < 0.001 ; d : 24.75  3.02 versus e : 25.57  1.92 versus f : 28.56  3.91  u / ghb ,  p < 0.001 ; gr : a : 44.37  3.58 versus b : 43.50  2.39 versus c : 48.58  3.67  u / ghb , p < 0.001 ; d : 24.75  3.02 versus e : 25.57  1.92 versus f : 28.56  3.91  u / ghb , resp . , p < 0.001 ) ( figure 3 ) , while the sod levels did not differ between the investigated groups ( a : 769.57  72.36 versus b : 768.97  34.50 versus c : 789.18  60.28 , d : 795.23  48.28 versus e : 797.80  69.21 versus f : 813.88  45.80  mu / mghb , resp .", "this analysis identified that decreased insulin sensitivity si and the decreases of gr were related to both ati and li in t2d patients .", "simultaneously , this model identified decreased insulin sensitivity si and the level of gr and gsh - px in nondiabetics with ati , but predominantly decreased insulin sensitivity si in nondiabetics with li ( table 2 ) .", "in this study , we directly measured the is level , together with three different types of antioxidant enzyme activities , gsh - px , sod , and gr , in t2d patients and nondiabetic individuals with two different subtypes of ischemic stroke in type 2 diabetics .", "our results have shown decreased is level in t2d patients with two different subtypes of ischemic stroke , ati and li , compared to t2d without stroke , while we could not demonstrate the difference between the subtypes , and the same pattern of is changes was found in the nondiabetics .   to our knowledge", ", there are scarce data regarding the changes in is in t2d with ischemic stroke subtypes .", "previous study also suggested that insulin resistance measured using different methodology , the short insulin tolerance test , is independently associated with markers of atherosclerosis detected on carotid arteries in t2d patients .", "simultaneously , an association has been documented between insulin resistance and other markers at the large vessels , such as the pulsatility index on cerebral arteries . on the other hand", ", it has been shown that insulin resistance , evaluated by the homeostasis model assessment ( homa ) index , was higher in diabetic in contrast to nondiabetic patients with li being a small vessel disease .", "additionally , it has been suggested that diabetes , hypertension , and metabolic syndrome which share insulin resistance as a common mechanism , contribute to li occurrence [ 1820 ] .", "however , detailed mechanisms of the possible link between the presence of ischemic stroke subtypes and insulin resistance remain to be clarified .", "in addition , when we measured the level of insulinemia in these patients , the significantly higher level of insulinemia was detected in both groups of t2d patients , with ati and li .", "the increases in insulin levels might be primarily consequence of the simultaneous presence of insulin resistance in the relevant groups .", "however , the increases in insulin might contribute to the appearance of the ischemic stroke subtypes .", "insulin , as a growth factor , might interfere with the beneficial effects of nitric oxide ( no ) on the vasculature .", "moreover , insulin infusion during euglycemic insulin clamp was able to suppress endothelium - dependent vasodilation in large arteries , which is reported to be based on increased availability of endothelin-1 , leading to downregulation of nad(p)h oxidase and superoxide anion production , and endothelial dysfunction is proposed to play an important role in the pathogenesis of cerebral small vessel disease [ 23 , 24 ] .  ", "also , our results demonstrated lower values of antioxidant enzymes in t2d patients with ati or li than in type 2 diabetics without ischemic stroke .", "the patients with ati and li did not differ regarding the level of antioxidant enzymes .", "these findings could be explained by the fact that in diabetes there is already reduced capacity of antioxidant protection , which is further significantly disturbed in the acute phase of ischemic stroke [ 4 , 5 ] . in our data , in patients with ati and li , we found the decreases in glutathione - dependent enzymes activity in contrast to other types of antioxidant enzyme activity , for example , sod .", "these results imply a prolonged and severe depression of gluthatione dependent antioxidative defense mechanisms irrespective of stroke subtypes .  ", "since it has been documented that free radicals are extremely difficult to measure directly , antioxidant enzymes have been proposed to represent indirect markers of oxidative stress .  ", "numerous , but mostly experimental , studies provided evidence of an association between ischemic stroke and decreased antioxidant enzyme activity , although this possible association in humans has been less investigated [ 9 , 25 ] . moreover ,", "the analyses of treatment with agents in stroke showing an ability to prevent further depression of antioxidant protection and scavenging reactive free radicals were reported to fail to restore gsh - px and gr activities [ 26 , 27 ] .", "generally , a recently study that aimed to assess total antioxidant capacity and oxidative stress in diabetic and nondiabetic acute stroke patients with 2 different stroke subtypes , large and small vessel disease strokes , concluded that oxidative stress and counterbalancing antioxidant capacity are more pronounced in diabetic acute stroke patients than in nondiabetics .", "the study provided different data in comparison to the previous investigations showing decreased gsh - px activity in both diabetic and nondiabetic patients with coronary heart disease when compared to controls [ 17 , 29 , 30 ] .  ", "however , the results from our study have shown the diminished activity of both gsh - px and gr in t2d patients with ati and li , which is consistent with previous reports of decreased gsh - px levels patients with stroke [ 6 , 8 , 31 , 32 ] .  ", "the levels of sod are found to exhibit great variations in the previous study in patients with the stroke [ 6 , 8 , 26 , 29 , 3137 ] .", "our results could not detect the differences in the sod levels in different groups of patients , and thus they are in line with data showing no changes in respect to sod level in both diabetics and nondiabetics irrespective of different subtypes of ischemic stroke [ 33 , 34 , 37 ] . the tentative explanation for the inconsistent findings regarding sod might reflect the predominant role of intracellular versus extracellular fraction of sod in free radical scavenging . in our study , the detected antioxidant enzyme activities were not affected by other important factors potentially influencing the enzyme levels , for example , by hyperglycemia , aging , duration of diabetes , and presence of macrovascular complications , due to the fact that in all groups of t2d patients had similar levels of metabolic and satisfactory metabolic control and that patients were matched in respect of age , duration of diabetes , and the prevalence of macrovascular complications .  ", "the multiple regression analysis applied to our data has demonstrated that decreased is together with the decreases in gr are related to both ati and li in t2d patients . in nondiabetics , the decreases in si levels and the diminished gr", "the analysis reveals the potential difference in the mechanisms underlying the onset of the two subtypes of the stroke in t2d patients compared to nondiabetics .", "the results imply that higher levels of insulin resistance combined with lower levels of gr , detected in t2d patients compared to nondiabetics , were underlying the onset of li together ati in t2d in contrast to the findings in nondiabetics . in this context ,", "our results are consistent with the findings that li represents the most frequent ischemic stroke subtype in t2d .", "taken together , our data imply that insulin resistance exerts its pathogenic influence on the level of gluthatione dependent antioxidant enzymes , especially in t2d .", "in conclusion , our results suggest that the presence of different subtypes of ischemic stroke is associated with insulin resistance and diminished antioxidant enzyme activity in both subtypes of ischemic stroke in t2d .", "the results also imply that atherogenic influence of decreased is in the different subtypes of stroke in t2d might be exerted through a significantly reduced glutathione dependent antioxidant enzyme activity , while the mechanisms relating the effects of insulin resistance and decreased antioxidant enzyme activity remain to be clarified ."], "abstract_text": ["<S> we analyzed ( a ) insulin sensitivity ( is ) and ( b ) glutathione peroxidase ( gsh - px ) , glutathione reductase ( gr ) , and superoxide dismutase ( sod ) antioxidant enzyme activity in type 2 diabetic ( t2d ) patients with atherothrombotic infarction ( ati ) ( group a ) , lacunar infarction ( li ) ( b ) , or without stroke ( c ) and in nondiabetics with ati ( d ) , li ( e ) , or without stroke ( f ) . </S>", "<S> ati and li were confirmed by brain imaging is levels were determined by \n minimal model ( si index ) , and the enzyme activity by spectrophotometry . in t2d patients , </S>", "<S> si was lower in a and b versus \n c ( 1.14  0.58 , 1.00  0.26 versus 3.14  0.62  min1/mu / l  104 , p < 0.001 ) and in nondiabetics in d and e versus f ( 3.38  0.77 , 3.03  0.72 versus 6.03  1.69  min1/mu / l  104 , p < 0.001 ) . </S>", "<S> also , gsh - px and gr activities were lower in a and b versus c ( gsh - px : 21.96  3.56 , 22.51  1.23 versus 25.12  1.67 ; gr : 44.37  3.58 , 43.50  2.39 versus 48.58  3.67  u / ghb ; p < 0.001 ) and in d and e versus f ( gsh - px : 24.75  3.02 , 25.57  1.92 versus 28.56  3.91 ; gr : 48.27  6.81 , 49.17  6.24 versus 53.67  3.96  u / ghb ; p < 0.001 ) </S>", "<S> . decreases in si and gr were significantly related to both ati and li in t2d . </S>", "<S> our results showed that decreased is and impaired antioxidant enzymes activity influence ischemic stroke subtypes in t2d . </S>", "<S> the influence of insulin resistance might be exerted on the level of glutathione - dependent antioxidant enzymes . </S>"], "labels": null, "section_names": ["1. Introduction ", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["it was previously suggested that atherothrombotic infarction ( ati ) and lacunar infarction ( li ) , as two different subtypes of ischemic stroke , might also differ in the set of the relevant risk factors , with ati being more associated to the atherogenic risk factors in contrast to li .", ".   however , decreased insulin sensitivity ( is ) , that is , insulin resistance , was observed both in ati and li , which was frequently accompanied with compensatory hyperinsulinemia in t2d patients as well as in nondiabetics [ 2 , 3 ] .", "simultaneously , it has been shown that impaired balance between products of oxidative stress and the level of antioxidant enzyme activities might be the important mechanism underlying the occurrence of ischemic stroke . moreover , it was elucidated that the brain has only moderate content of glutathione - dependent enzymes , for example , glutathione peroxidase ( gsh - px ) , glutathione reductase ( gr ) , and superoxide dismutase ( sod ) , together with the fact that the intact antioxidant defense could provide first line of protection from initiation and exacerbation of ischemic cerebral injury .  ", "in addition , changes in enzymatic antioxidative defense mechanisms in patients with stroke are still controversial .", "previous results implied that the majority of antioxidant enzyme activity was significantly reduced in acute ischemic stroke , possibly as a consequence of increased oxidative stress   while the recent finding suggested increased levels of glutathione dependent enzymes as an adaptive mechanisms during acute cerebral ischemia . finally , due to novel facts", ", oxidative stress can be an important component for astrocytic cell death following metabolic stress .   until now , experimental studies provided evidence of an association between ischemic stroke and increased oxidative stress [ 8 , 9 ] , but data in humans are still heterogeneous and limited .", "therefore , our study was aimed to determine is levels and three different types of antioxidant enzyme activities gsh - px , gr , and sod , in t2d with ati and li ."], ["in this study we included a total of 93 patients with t2d , ascribed to the following groups : t2d patients with ati ( group a , n = 30 ) , and t2d with li ( group b , n = 30 ) , and t2d without ischemic stroke ( group c , n = 33 ) .", "simultaneously , we involved a total of 93 nondiabetics , matched with the t2d patients regarding gender and age , and also comprising the following groups : nondiabetics with ati ( group d , n = 30 ) , nondiabetics with li ( group e , n = 30 ) , and nondiabetics without stroke ( group f , n = 33 ) .   t2d was diagnosed in accordance with the criteria of the world health organization .", "diagnosis of ati and li was done by a neurologist due to clinical features and brain imaging methods such as cranial computerized scan and/or magnetic resonance imaging in two consecutive examinations , during the first 7 days from the appearance of ischemic stroke .", "the patients with ati or li were included in the study provided that they had not shown signs of cardioembolic cerebral infarction , or coronary heart disease based on a history of myocardial infarction with definite elevation of serum cardiac enzymes or coronary angiography .", "t2d patients were treated with insulin therapy , and/or ingestion of antioxidant supplements and drugs , which might affect free radical and antioxidant activity potential ; likewise patients who had other endocrine disease or autoimmune diseases , renal or hepatic failure , current infections , neoplasms , polycythemia , or rheumatic diseases were also excluded as well as the patients with history of trauma or operation within the last 3 months .", "no patient had uncontrolled hypertension , severe alcohol consumption , acute infection , or an inflammatory disease during the last 4 weeks .", "all the patients , with or without ati and li , showed the similar level of their physical activity .", "in addition , they were required not to smoke at least 12  hr before the tests were performed .", "the patients were fully informed about the study and gave the inform consent to participate .  ", "the study was conducted at the clinic for endocrinology , diabetes and metabolic diseases and at the clinic for neurology , clinical centre of serbia , faculty of medicine , university of belgrade , and was approved by the institutional ethics committee .", "the interview , physical examination , metabolic test , and evaluation of antioxidant enzyme activities were performed in all the patients included in the study , for each patient within the same day .", "hypertension was diagnosed according to world health organisation criteria ( systolic / diastolic blood pressure 140/90  mm  hg ) or by the use of antihypertensive agents .", "the metabolic tests were implemented at least after 6 months from the occurrence of the ischemic stroke .   ", "the evaluation of insulin sensitivity was done by frequently sampled intravenous glucose tolerance test ( fsigt ) with minimal model analysis .", "briefly , before testing , each patient was required to be at a 12  hr fasting state . during the fsigt ,", "0.3  g / kg body weight of glucose was injected and the blood samples for plasma glucose ( pg ) and plasma insulin ( pi ) determination were taken immediately before and 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 12 , 14 , 16 , 20 , 23 , 24 , 25 , 27 , 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 120 , 160 , and 180 minutes after intravenous glucose stimulation .", "insulin was injected as a continuous infusion 4  mu / kg / min between minutes 20 and 25 in order to avoid the effect of the potentially blunted insulin response .", "the insulin sensitivity index ( si ) was calculated from the results of pg and pi levels by computerized minimal model analysis , using the minmod program ( kindly provided by dr .", "determination of the antioxidant enzymes sod , gsh - px , gr was conducted using the commercial assays ( produced by randox laboratories ltd . , uk ) , based on spectrophotometer determination methods as described previously .", "pg was determined by glucose oxidase method using a beckman glucose analyser ( beckman instruments , fullerton , ca ) .", "pi was tested by radioimmunoassay ( inep , zemun , rs , double antibody kits ) .", "total cholesterol , hdl cholesterol , and triglyceride concentrations were determined with the chromatography method using commercial kits ( produced by boehringer mannheim ) .", "the continuous variables within each subtype of ischemic stroke were analyzed with analysis of variance ( anova ) with a post hoc bonferroni test .", "all analyses were performed with the spss statistical package ( version 16.0 for windows ) ."], ["the clinical characteristics and biochemistry parameters of t2d patients and nondiabetics with ati or li as different subtypes of ischemic stroke are shown in table 1 .", "the age , gender , duration of diabetes , and duration from the onset of ischemic stroke were similar in t2d patients and nondiabetics with different subtypes of ischemic stroke , together with hba1c levels , implying satisfactory metabolic control before metabolic investigation was done . however , ldl - c level was significantly higher in t2d patients with ati compared to the t2d patients with li and t2d patients without stroke and also in nondiabetics with ati compared to nondiabetics with li and healthy controls .", "there is no difference in prevalence of hypertension in patients with t2d and ati or li and t2d without ischemic stroke , while it was significantly higher in nondiabetics with ati or li than in healthy controls .", "we found that si levels were significantly lower in t2d patients with ati ( group a ) and li ( group b ) compared to t2d patients without ischemic stroke ( group c ) ( 1.14  0.58 and 1.00  0.26 versus 3.14  0.62  min / mu / l  10 , resp .", "also , the results showed significantly lower si levels in nondiabetics with ati ( group d ) and li ( group e ) compared to healthy controls ( group f ) ( 3.38  0.77 and 3.03  0.72 versus 6.03  1.69  min / mu / l  10 , resp .", "simultaneously , pi levels were higher in t2d patients , ati ( group a ) , and li ( group b ) than in t2d patients without ischemic stroke ( group c ) ( 20.94  4.31 and 20.07  0.88 versus 16.06  0.91 ", "mu / l , respectively , p < 0.001 ) and in nondiabetics with ati ( group d ) or li ( group e ) in comparison to healthy controls ( group f ) ( 15.57  1.86 and 15.59  1.26 versus 7.54  2.03 ", "mu / l , respectively , p < 0.001 ) ( figure 2 ) .  ", "when we evaluated antioxidant enzyme activities in t2d patients with ati ( group a ) and li ( group b ) and without ischemic stroke ( group c ) and in nondiabetics with ati ( group d ) and li ( group e ) and healthy controls ( group d ) , we detected the levels of the gsh - px and gr activity being significantly lower in group a and b versus c and in group d and e versus f ( gshpx : a : 21.96  3.56 versus b : 22.51  1.23 versus c : 25.12  1.67  u / ghb ,  p < 0.001 ; d : 24.75  3.02 versus e : 25.57  1.92 versus f : 28.56  3.91  u / ghb ,  p < 0.001 ; gr : a : 44.37  3.58 versus b : 43.50  2.39 versus c : 48.58  3.67  u / ghb , p < 0.001 ; d : 24.75  3.02 versus e : 25.57  1.92 versus f : 28.56  3.91  u / ghb , resp . , p < 0.001 ) ( figure 3 ) , while the sod levels did not differ between the investigated groups ( a : 769.57  72.36 versus b : 768.97  34.50 versus c : 789.18  60.28 , d : 795.23  48.28 versus e : 797.80  69.21 versus f : 813.88  45.80  mu / mghb , resp .", "this analysis identified that decreased insulin sensitivity si and the decreases of gr were related to both ati and li in t2d patients .", "simultaneously , this model identified decreased insulin sensitivity si and the level of gr and gsh - px in nondiabetics with ati , but predominantly decreased insulin sensitivity si in nondiabetics with li ( table 2 ) ."], ["in this study , we directly measured the is level , together with three different types of antioxidant enzyme activities , gsh - px , sod , and gr , in t2d patients and nondiabetic individuals with two different subtypes of ischemic stroke in type 2 diabetics .", "our results have shown decreased is level in t2d patients with two different subtypes of ischemic stroke , ati and li , compared to t2d without stroke , while we could not demonstrate the difference between the subtypes , and the same pattern of is changes was found in the nondiabetics .   to our knowledge", ", there are scarce data regarding the changes in is in t2d with ischemic stroke subtypes .", "previous study also suggested that insulin resistance measured using different methodology , the short insulin tolerance test , is independently associated with markers of atherosclerosis detected on carotid arteries in t2d patients .", "simultaneously , an association has been documented between insulin resistance and other markers at the large vessels , such as the pulsatility index on cerebral arteries . on the other hand", ", it has been shown that insulin resistance , evaluated by the homeostasis model assessment ( homa ) index , was higher in diabetic in contrast to nondiabetic patients with li being a small vessel disease .", "additionally , it has been suggested that diabetes , hypertension , and metabolic syndrome which share insulin resistance as a common mechanism , contribute to li occurrence [ 1820 ] .", "however , detailed mechanisms of the possible link between the presence of ischemic stroke subtypes and insulin resistance remain to be clarified .", "in addition , when we measured the level of insulinemia in these patients , the significantly higher level of insulinemia was detected in both groups of t2d patients , with ati and li .", "the increases in insulin levels might be primarily consequence of the simultaneous presence of insulin resistance in the relevant groups .", "however , the increases in insulin might contribute to the appearance of the ischemic stroke subtypes .", "insulin , as a growth factor , might interfere with the beneficial effects of nitric oxide ( no ) on the vasculature .", "moreover , insulin infusion during euglycemic insulin clamp was able to suppress endothelium - dependent vasodilation in large arteries , which is reported to be based on increased availability of endothelin-1 , leading to downregulation of nad(p)h oxidase and superoxide anion production , and endothelial dysfunction is proposed to play an important role in the pathogenesis of cerebral small vessel disease [ 23 , 24 ] .  ", "also , our results demonstrated lower values of antioxidant enzymes in t2d patients with ati or li than in type 2 diabetics without ischemic stroke .", "the patients with ati and li did not differ regarding the level of antioxidant enzymes .", "these findings could be explained by the fact that in diabetes there is already reduced capacity of antioxidant protection , which is further significantly disturbed in the acute phase of ischemic stroke [ 4 , 5 ] . in our data , in patients with ati and li , we found the decreases in glutathione - dependent enzymes activity in contrast to other types of antioxidant enzyme activity , for example , sod .", "these results imply a prolonged and severe depression of gluthatione dependent antioxidative defense mechanisms irrespective of stroke subtypes .  ", "since it has been documented that free radicals are extremely difficult to measure directly , antioxidant enzymes have been proposed to represent indirect markers of oxidative stress .  ", "numerous , but mostly experimental , studies provided evidence of an association between ischemic stroke and decreased antioxidant enzyme activity , although this possible association in humans has been less investigated [ 9 , 25 ] . moreover ,", "the analyses of treatment with agents in stroke showing an ability to prevent further depression of antioxidant protection and scavenging reactive free radicals were reported to fail to restore gsh - px and gr activities [ 26 , 27 ] .", "generally , a recently study that aimed to assess total antioxidant capacity and oxidative stress in diabetic and nondiabetic acute stroke patients with 2 different stroke subtypes , large and small vessel disease strokes , concluded that oxidative stress and counterbalancing antioxidant capacity are more pronounced in diabetic acute stroke patients than in nondiabetics .", "the study provided different data in comparison to the previous investigations showing decreased gsh - px activity in both diabetic and nondiabetic patients with coronary heart disease when compared to controls [ 17 , 29 , 30 ] .  ", "however , the results from our study have shown the diminished activity of both gsh - px and gr in t2d patients with ati and li , which is consistent with previous reports of decreased gsh - px levels patients with stroke [ 6 , 8 , 31 , 32 ] .  ", "the levels of sod are found to exhibit great variations in the previous study in patients with the stroke [ 6 , 8 , 26 , 29 , 3137 ] .", "our results could not detect the differences in the sod levels in different groups of patients , and thus they are in line with data showing no changes in respect to sod level in both diabetics and nondiabetics irrespective of different subtypes of ischemic stroke [ 33 , 34 , 37 ] . the tentative explanation for the inconsistent findings regarding sod might reflect the predominant role of intracellular versus extracellular fraction of sod in free radical scavenging . in our study , the detected antioxidant enzyme activities were not affected by other important factors potentially influencing the enzyme levels , for example , by hyperglycemia , aging , duration of diabetes , and presence of macrovascular complications , due to the fact that in all groups of t2d patients had similar levels of metabolic and satisfactory metabolic control and that patients were matched in respect of age , duration of diabetes , and the prevalence of macrovascular complications .  ", "the multiple regression analysis applied to our data has demonstrated that decreased is together with the decreases in gr are related to both ati and li in t2d patients . in nondiabetics , the decreases in si levels and the diminished gr", "the analysis reveals the potential difference in the mechanisms underlying the onset of the two subtypes of the stroke in t2d patients compared to nondiabetics .", "the results imply that higher levels of insulin resistance combined with lower levels of gr , detected in t2d patients compared to nondiabetics , were underlying the onset of li together ati in t2d in contrast to the findings in nondiabetics . in this context ,", "our results are consistent with the findings that li represents the most frequent ischemic stroke subtype in t2d .", "taken together , our data imply that insulin resistance exerts its pathogenic influence on the level of gluthatione dependent antioxidant enzymes , especially in t2d ."], ["in conclusion , our results suggest that the presence of different subtypes of ischemic stroke is associated with insulin resistance and diminished antioxidant enzyme activity in both subtypes of ischemic stroke in t2d .", "the results also imply that atherogenic influence of decreased is in the different subtypes of stroke in t2d might be exerted through a significantly reduced glutathione dependent antioxidant enzyme activity , while the mechanisms relating the effects of insulin resistance and decreased antioxidant enzyme activity remain to be clarified ."]]}
{"article_id": "PMC3132637", "article_text": ["the formation of spores is a survival mechanism of microorganisms when exposed to unfavorable environmental conditions ( e.g. , heavy metal stress , nutrient limitations ) leading to a  dormant  or  resting", " growth state [ 1 , 2 ] . a variety of bacteria identified in diverse habitats including soil is able to form endospores .", "these physiological groups include aerobic heterotrophs ( e.g. , bacillus , paenibacillus , brevibacillus , geobacillus , thermoactinomyces , and sporolactobacillus ) , anaerobes ( clostridium , anaerobacter , and   desulfotomaculum ) , microaerophiles ( sporolactobacillus ) , halophiles ( sporohalobacter ) , and phototrophs ( heliobacterium , heliophilum ) [ 3 , 4 ] . bacterial spores are characterized by a series of unique chemical features which can facilitate their identification in natural environments . besides the high content of minerals ( particularly calcium ) , spores contain high amounts of dipicolinic acid , dpa .", "dpa is uniquely found in bacterial spores in amounts of up to 25% of the spore dry weight and depends on the bacterial species [ 6 , 7 ] . in solution ,", "a complex is formed in the presence of terbium which shows a very strong and distinctive fluorescence spectrum .", "originally , dpa was used to detect very low concentrations of terbium ( iii ) . on this basis ,", "methods for the detection of bacterial endospores have been developed [ 1013 ] : by the addition of terbium , the dpa content was determined .", "however , terbium - dpa fluorescence might be interfered by a series of compounds , especially when dpa has to be determined in complex samples such as sediments or soils .", "it has been reported that the presence of phosphorus compounds ( especially ortho - phosphate ) reduced terbium fluorescence by as much as 98% .", "the addition of aluminium compounds ( especially aluminium chloride , alcl3 ) , however , ameliorated the interference caused by the quenching substances . from a series of organic compounds (", "benzoate , tryptophan , tyrosine , phenylalanine , glucose , malate , riboflavin , nad , and tryptone ) only the latter two ( especially tryptone ) reduced fluorescence significantly .", "carbohydrates ( e.g. , starch , dextrine ) were reported not to interfere with the terbium fluorescence .", "inorganic compounds such as calcium carbonate , sodium chloride , potassium chloride , ammonium sulphate , ammonium nitrate , and sodium nitrate did not lead to a reduction of the fluorescence , but only dipotassium phosphate did .", "the aim of this study was to adopt the fluorescence - based method to determine the spore content in soils sampled from various locations .", "in particular , we were interested in the differentiation between different types of soil such as grasslands ( pasture , meadow ) , allotment gardens , and forests , as well as fluvial sediments , the relationship of soil parameters ( carbon - to - nitrogen ratio ) on the occurrence of bacterial spores , and the distribution of spores in relation to sampling depth .", "different bacillus species ( b. megaterium , b. subtilis ) were cultivated in liquid medium containing ( in g / l ) : glucose ( 3.6 ) , ammonium chloride ( 2.5 ) , magnesium sulfate ( 0.2 ) , calcium chloride ( 0.07 ) , iron sulfate ( 0.01 ) , edta ( 0.01 ) , potassium dihydrogen phosphate ( 0.6 ) , dipotassium hydrogen phosphate ( 0.9 ) , and yeast extract ( 1.0 ) .", "erlenmeyer flasks ( 250  ml ) containing 100  ml of growth medium were inoculated and incubated for 10 to 15 days ( 150  rpm , 30c ) . to initiate and stimulate sporulation ,", "bacteria were subsequently transferred to a sporulation medium ( identical composition , but without glucose and less ammonium chloride [ only 1  g / l ] ) .", "after additional 30 days of incubation  until vegetative cells were not present anymore after inspection by microscopy  spores were harvested by centrifugation , immediately frozen in liquid nitrogen followed by lyophilization .", "samples from different locations were collected using a stainless steel soil corer ( 15  mm in diameter ) , which was sterilized before each sampling .", "cores with a maximum length of 25  cm were obtained , cut in sections of 5  cm , transferred to sterile screw cap falcon tubes ( 20  ml ) , and stored on dry ice .", "after return to the laboratory , samples were immediately lyophylized or stored at 80c until further processing .", "sampling sites were located in the surroundings of zurich ( switzerland ) : grassland soil , meadow ( municipalities of mnnedorf ; uerikon ; stfa ; and dbendorf ) , allotment garden ( university of zurich , irchel campus ) , pasture ( university of zurich , irchel campus ; municipality of wdenswil ) , forest soil ( municipality of stfa ) , and aquatic sediments ( river glatt in dbendorf ) . lyophilized aliquots of approximately 1  g were transferred to an eppendorf micro test tube ( 2  ml ) and ground ( by adding a 6  mm glass bead ) in tissuelyser ( retsch , haan , germany ) for 5  1 min .", "elemental composition ( carbon , hydrogen , nitrogen ) of soil was performed with a chn-932 elemental analyzer ( leco corp . , st .", "composition ( in % of dry soil ) varied between 2.2 and 15.4 , 0.2 and 1.4 , and 0.2 and 2.0 for total carbon , total hydrogen , and total nitrogen , respectively .", "phosphate in aqueous soil extracts ( 250  mg soil in 5  ml sodium acetate buffer ; 0.2 m , ph 5 ) was determined using commercially available kits ( lck 348 and 349 ; hach lange ag , hegnau , switzerland ) .", "10  mg of dry spore powder was resuspended in 10  ml sodium acetate buffer ( 0.2  m , ph 5 ) .", "soil samples were thawed and 50  mg were suspended in 0.9  ml sodium acetate buffer and 0.1  ml aluminium chloride ( alcl3 , 0.5  m ) .", "samples were microwaved ( berghof microwave digester mws-1 , with built - in in situ infrared temperature control ) in teflon tfm screw cap digestion vessels .", "temperature and power were set to 140c and approximately 680 w ( 80% ) , respectively .", "alternatively , dpa was released from spores by autoclaving the samples in screw cap glass test tubes for 15 minutes at 121c .", "the identical dpa extraction protocol was applied for soil samples . however , microscopy was not possible due to the presence of mineral particles interfering the observation .", "after cooling for 30 minutes , 100  l of the spore suspensions were mixed with 100  l terbium chloride solution ( tbcl3 , 30  m ) in white 96-well microtiter plates ( in 8 replicates ) .", "fluorescence was immediately measured using a plate reader ( spectramax m2 , bucher biotec , basel , switzerland ) with the following settings : time - resolved fluorescence ( delay 50  s , interval 1200  s ) at an excitation wavelength of 272  nm , emission wavelength of 545  nm , and 10 endpoint readings per sample at 30c .", "the number of spores in the soil samples was determined using standard addition method with spores of b. subtilis .", "microwave treatment of spore suspensions and soil samples led to a fast release of dpa ( figure 1 ) . within two minutes , maximum release was obtained .", "highest spore numbers up to 4   10 spores per gram dry soil were found in agriculturally used land ( meadow , pasture ) , less in forest soil .", "the interference of different compounds present in soil ( e.g. , phosphate ) might lead to quenching of the fluorescence signal .", "this drawback has been overcome by the addition of aluminium chloride as already shown for the determination of bacterial spores in aquatic sediments .", "concentration of ortho - phosphate in soil extracts ( 22.5  m ) was reduced by the addition of aluminium chloride to concentrations below the detection limit ( < 1.2  m ) .", "concomitantly , a de - colorization of the extract was observed suggesting the removal of humic acids which have also the potential to form complexes with terbium and quench the fluorescence signal .", "the method based on terbium fluorescence for the detection of total numbers of bacterial endospores in soils is fast and easy .", "a transect ( approximately 100  m in length ) through a grass field with different land use management ( unused meadow , allotment garden , and pasture ) gave spore numbers in the range of 5 to 9  10 spores per gram of dry soil ( figure 3 ) .", "spore counts were not related to the type of land use : in allotment garden soil , counts were not significantly different from soil samples taken from a pasture ( p = 0.423 ; t - test ) . regarding the different sampling sites ,", "our results show that grassland soils ( meadow , allotment garden , and pasture ) contains much more bacterial spores than forest soils and fluvial sediments .", "spore content was related to the carbon - to - nitrogen ratio ( figure 4 ) . at c / n ratios > 20 only low spore counts ( 0.5  10 spores per gram of dry soil ) were detected as compared to c / n ratios < 20 .", "it has been demonstrated in pure cultures of bacillus thuringiensis in a stirred bioreactor that low carbon - to - nitrogen ratios of 4  :  1 resulted in high spore counts .", "in contrast however , spore formation in streptomyces coelicolor was stimulated under nitrogen - limiting conditions .", "in particular , c / n ratios between 50 and 100 promoted sporulation , whereas c / n rations < 40 did not allow spore formation .", "our results showed that in soils with extremely high c / n ratios , spore content was low .", "the importance of c / n ratio was stressed by gao and coworkers regarding the sporulation of fungi , although fungal spores do not contain dpa . a carbon - to - nitrogen ( c / n ) ratio of 20 stimulated spore formation by fungi such as penicillium camembertii .", "the fungus colletotrichum coccodes produced highest spore counts at a c / n ratio of 5 to 10 , whereas at a ratio of 40 , spore formation was significantly lower .", "similarly , in plectosporium tabacinum optimal spore formation was found when c / n rations were between 5 and 10 .", "the distribution of spores in marine sediments ( determined as dpa ) showed only a low correlation with the content of total organic carbon and varied with the sediment type .", "highest numbers have been found in organic - rich black sediments , lowest number in sandy sediments .", "it was hypothesized from anthrax outbreaks , that the high numbers of bacillus spores might be related to soils rich in organic matter that is , to a high c / n ratio .", "these soil environmental conditions are suggested to support the presence and viability of b. anthracis spores .", "depth distribution of spores from an area currently used as allotment garden ( cultivation of flowers and vegetables ) showed highest numbers in a horizon of 5 to 10  cm ( figure 5 ) .", "the two methods evaluated ( microwaving , autoclaving ) for the mobilization of dpa from bacterial spores gave similar results .", "however , microwaving was less time - consuming , whereas autoclaving allowed faster throughput of samples .", "in summary , microwave treatment of soil samples followed by the measurement of fluorescence after addition of terbium proved to be a fast and easy method to assess the content of bacterial spores .", "our study might provide a basis for the detection of  hot spots  of endospores in soil ."], "abstract_text": ["<S> spore formation is a survival mechanism of microorganisms when facing unfavorable environmental conditions resulting in  dormant  states . </S>", "<S> we investigated the occurrence of bacterial endospores in soils from various locations including grasslands ( pasture , meadow ) , allotment gardens , and forests , as well as fluvial sediments . </S>", "<S> bacterial spores are characterized by their high content of dipicolinic acid ( dpa ) . in the presence of terbium , dpa forms a complex showing a distinctive photoluminescence spectrum . </S>", "<S> dpa was released from soil by microwaving or autoclaving . </S>", "<S> the addition of aluminium chloride reduced signal quenching by interfering compounds such as phosphate . </S>", "<S> the highest spore content ( up to 109 spores per gram of dry soil ) was found in grassland soils . </S>", "<S> spore content is related to soil type , to soil depth , and to soil carbon - to - nitrogen ratio . </S>", "<S> our study might provide a basis for the detection of  hot spots  of bacterial spores in soil . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusions"], "sections": [["the formation of spores is a survival mechanism of microorganisms when exposed to unfavorable environmental conditions ( e.g. , heavy metal stress , nutrient limitations ) leading to a  dormant  or  resting", " growth state [ 1 , 2 ] . a variety of bacteria identified in diverse habitats including soil is able to form endospores .", "these physiological groups include aerobic heterotrophs ( e.g. , bacillus , paenibacillus , brevibacillus , geobacillus , thermoactinomyces , and sporolactobacillus ) , anaerobes ( clostridium , anaerobacter , and   desulfotomaculum ) , microaerophiles ( sporolactobacillus ) , halophiles ( sporohalobacter ) , and phototrophs ( heliobacterium , heliophilum ) [ 3 , 4 ] . bacterial spores are characterized by a series of unique chemical features which can facilitate their identification in natural environments . besides the high content of minerals ( particularly calcium ) , spores contain high amounts of dipicolinic acid , dpa .", "dpa is uniquely found in bacterial spores in amounts of up to 25% of the spore dry weight and depends on the bacterial species [ 6 , 7 ] . in solution ,", "a complex is formed in the presence of terbium which shows a very strong and distinctive fluorescence spectrum .", "originally , dpa was used to detect very low concentrations of terbium ( iii ) . on this basis ,", "methods for the detection of bacterial endospores have been developed [ 1013 ] : by the addition of terbium , the dpa content was determined .", "however , terbium - dpa fluorescence might be interfered by a series of compounds , especially when dpa has to be determined in complex samples such as sediments or soils .", "it has been reported that the presence of phosphorus compounds ( especially ortho - phosphate ) reduced terbium fluorescence by as much as 98% .", "the addition of aluminium compounds ( especially aluminium chloride , alcl3 ) , however , ameliorated the interference caused by the quenching substances . from a series of organic compounds (", "benzoate , tryptophan , tyrosine , phenylalanine , glucose , malate , riboflavin , nad , and tryptone ) only the latter two ( especially tryptone ) reduced fluorescence significantly .", "carbohydrates ( e.g. , starch , dextrine ) were reported not to interfere with the terbium fluorescence .", "inorganic compounds such as calcium carbonate , sodium chloride , potassium chloride , ammonium sulphate , ammonium nitrate , and sodium nitrate did not lead to a reduction of the fluorescence , but only dipotassium phosphate did .", "the aim of this study was to adopt the fluorescence - based method to determine the spore content in soils sampled from various locations .", "in particular , we were interested in the differentiation between different types of soil such as grasslands ( pasture , meadow ) , allotment gardens , and forests , as well as fluvial sediments , the relationship of soil parameters ( carbon - to - nitrogen ratio ) on the occurrence of bacterial spores , and the distribution of spores in relation to sampling depth ."], ["different bacillus species ( b. megaterium , b. subtilis ) were cultivated in liquid medium containing ( in g / l ) : glucose ( 3.6 ) , ammonium chloride ( 2.5 ) , magnesium sulfate ( 0.2 ) , calcium chloride ( 0.07 ) , iron sulfate ( 0.01 ) , edta ( 0.01 ) , potassium dihydrogen phosphate ( 0.6 ) , dipotassium hydrogen phosphate ( 0.9 ) , and yeast extract ( 1.0 ) .", "erlenmeyer flasks ( 250  ml ) containing 100  ml of growth medium were inoculated and incubated for 10 to 15 days ( 150  rpm , 30c ) . to initiate and stimulate sporulation ,", "bacteria were subsequently transferred to a sporulation medium ( identical composition , but without glucose and less ammonium chloride [ only 1  g / l ] ) .", "after additional 30 days of incubation  until vegetative cells were not present anymore after inspection by microscopy  spores were harvested by centrifugation , immediately frozen in liquid nitrogen followed by lyophilization .", "samples from different locations were collected using a stainless steel soil corer ( 15  mm in diameter ) , which was sterilized before each sampling .", "cores with a maximum length of 25  cm were obtained , cut in sections of 5  cm , transferred to sterile screw cap falcon tubes ( 20  ml ) , and stored on dry ice .", "after return to the laboratory , samples were immediately lyophylized or stored at 80c until further processing .", "sampling sites were located in the surroundings of zurich ( switzerland ) : grassland soil , meadow ( municipalities of mnnedorf ; uerikon ; stfa ; and dbendorf ) , allotment garden ( university of zurich , irchel campus ) , pasture ( university of zurich , irchel campus ; municipality of wdenswil ) , forest soil ( municipality of stfa ) , and aquatic sediments ( river glatt in dbendorf ) . lyophilized aliquots of approximately 1  g were transferred to an eppendorf micro test tube ( 2  ml ) and ground ( by adding a 6  mm glass bead ) in tissuelyser ( retsch , haan , germany ) for 5  1 min .", "elemental composition ( carbon , hydrogen , nitrogen ) of soil was performed with a chn-932 elemental analyzer ( leco corp . , st .", "composition ( in % of dry soil ) varied between 2.2 and 15.4 , 0.2 and 1.4 , and 0.2 and 2.0 for total carbon , total hydrogen , and total nitrogen , respectively .", "phosphate in aqueous soil extracts ( 250  mg soil in 5  ml sodium acetate buffer ; 0.2 m , ph 5 ) was determined using commercially available kits ( lck 348 and 349 ; hach lange ag , hegnau , switzerland ) .", "10  mg of dry spore powder was resuspended in 10  ml sodium acetate buffer ( 0.2  m , ph 5 ) .", "soil samples were thawed and 50  mg were suspended in 0.9  ml sodium acetate buffer and 0.1  ml aluminium chloride ( alcl3 , 0.5  m ) .", "samples were microwaved ( berghof microwave digester mws-1 , with built - in in situ infrared temperature control ) in teflon tfm screw cap digestion vessels .", "temperature and power were set to 140c and approximately 680 w ( 80% ) , respectively .", "alternatively , dpa was released from spores by autoclaving the samples in screw cap glass test tubes for 15 minutes at 121c .", "the identical dpa extraction protocol was applied for soil samples . however , microscopy was not possible due to the presence of mineral particles interfering the observation .", "after cooling for 30 minutes , 100  l of the spore suspensions were mixed with 100  l terbium chloride solution ( tbcl3 , 30  m ) in white 96-well microtiter plates ( in 8 replicates ) .", "fluorescence was immediately measured using a plate reader ( spectramax m2 , bucher biotec , basel , switzerland ) with the following settings : time - resolved fluorescence ( delay 50  s , interval 1200  s ) at an excitation wavelength of 272  nm , emission wavelength of 545  nm , and 10 endpoint readings per sample at 30c .", "the number of spores in the soil samples was determined using standard addition method with spores of b. subtilis ."], ["microwave treatment of spore suspensions and soil samples led to a fast release of dpa ( figure 1 ) . within two minutes , maximum release was obtained .", "highest spore numbers up to 4   10 spores per gram dry soil were found in agriculturally used land ( meadow , pasture ) , less in forest soil .", "the interference of different compounds present in soil ( e.g. , phosphate ) might lead to quenching of the fluorescence signal .", "this drawback has been overcome by the addition of aluminium chloride as already shown for the determination of bacterial spores in aquatic sediments .", "concentration of ortho - phosphate in soil extracts ( 22.5  m ) was reduced by the addition of aluminium chloride to concentrations below the detection limit ( < 1.2  m ) .", "concomitantly , a de - colorization of the extract was observed suggesting the removal of humic acids which have also the potential to form complexes with terbium and quench the fluorescence signal .", "the method based on terbium fluorescence for the detection of total numbers of bacterial endospores in soils is fast and easy .", "a transect ( approximately 100  m in length ) through a grass field with different land use management ( unused meadow , allotment garden , and pasture ) gave spore numbers in the range of 5 to 9  10 spores per gram of dry soil ( figure 3 ) .", "spore counts were not related to the type of land use : in allotment garden soil , counts were not significantly different from soil samples taken from a pasture ( p = 0.423 ; t - test ) . regarding the different sampling sites ,", "our results show that grassland soils ( meadow , allotment garden , and pasture ) contains much more bacterial spores than forest soils and fluvial sediments .", "spore content was related to the carbon - to - nitrogen ratio ( figure 4 ) . at c / n ratios > 20 only low spore counts ( 0.5  10 spores per gram of dry soil ) were detected as compared to c / n ratios < 20 .", "it has been demonstrated in pure cultures of bacillus thuringiensis in a stirred bioreactor that low carbon - to - nitrogen ratios of 4  :  1 resulted in high spore counts .", "in contrast however , spore formation in streptomyces coelicolor was stimulated under nitrogen - limiting conditions .", "in particular , c / n ratios between 50 and 100 promoted sporulation , whereas c / n rations < 40 did not allow spore formation .", "our results showed that in soils with extremely high c / n ratios , spore content was low .", "the importance of c / n ratio was stressed by gao and coworkers regarding the sporulation of fungi , although fungal spores do not contain dpa . a carbon - to - nitrogen ( c / n ) ratio of 20 stimulated spore formation by fungi such as penicillium camembertii .", "the fungus colletotrichum coccodes produced highest spore counts at a c / n ratio of 5 to 10 , whereas at a ratio of 40 , spore formation was significantly lower .", "similarly , in plectosporium tabacinum optimal spore formation was found when c / n rations were between 5 and 10 .", "the distribution of spores in marine sediments ( determined as dpa ) showed only a low correlation with the content of total organic carbon and varied with the sediment type .", "highest numbers have been found in organic - rich black sediments , lowest number in sandy sediments .", "it was hypothesized from anthrax outbreaks , that the high numbers of bacillus spores might be related to soils rich in organic matter that is , to a high c / n ratio .", "these soil environmental conditions are suggested to support the presence and viability of b. anthracis spores .", "depth distribution of spores from an area currently used as allotment garden ( cultivation of flowers and vegetables ) showed highest numbers in a horizon of 5 to 10  cm ( figure 5 ) .", "the two methods evaluated ( microwaving , autoclaving ) for the mobilization of dpa from bacterial spores gave similar results .", "however , microwaving was less time - consuming , whereas autoclaving allowed faster throughput of samples ."], ["in summary , microwave treatment of soil samples followed by the measurement of fluorescence after addition of terbium proved to be a fast and easy method to assess the content of bacterial spores .", "our study might provide a basis for the detection of  hot spots  of endospores in soil ."]]}
{"article_id": "PMC5146794", "article_text": ["alzheimer 's disease ( ad ) is a common neurodegenerative disease in the central nervous system .", "the pathological features of ad include senile plaques formed by extracellular amyloid  ( a ) aggregation , neurofibrillary tangles formed by abnormal accumulation of hyperphosphorylated tau protein , synaptic impairments , and neuronal loss in the cerebral cortex and hippocampus .", "a aggregation in the brain is central to the pathological changes of ad and leads to a series of pathological events , which further promote a aggregation , resulting in cascade amplification .", "the endoplasmic reticulum ( er ) , a dynamic membranous organelle , is involved in protein synthesis , posttranslational modification , folding , calcium storage , lipid metabolism , and steroid hormone synthesis .", "specific stress conditions such as hyperglycemia , hyperlipidemia , hypoxia , chemical toxicants , and genetic mutations can induce the accumulation of unfolded or misfolded proteins in the er and changes in er functions , resulting in endoplasmic reticulum stress ( ers ) . in turn , ers stimulates the unfolded protein response ( upr ) and restores cellular homeostasis while sustained ers leads to cell apoptosis .", "many studies have shown that ers is involved in the development and progression of neurodegenerative diseases such as ad , parkinson 's disease , huntington 's disease , and amyotrophic lateral sclerosis although the underlying mechanisms still remain blurred .", "the activation of the upr pathway requires the activation of three sensor proteins : inositol - requiring enzyme 1 ( ire-1 ) , activating transcription factor 6 ( atf6 ) , and protein kinase rna - like er kinase .", "these sensors serve to promote the expression of chaperone protein , glucose - regulated protein ( grp ) 78 , to repair misfolded or unfolded proteins , to accelerate er - associated protein degradation ( erad ) , and to aggravate the phosphorylation of eukaryotic translation initiation factor 2a .", "sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of the transcriptional activator ccaat / enhancer - binding protein homologous protein ( chop ) and caspase-12 activation .", "activated ire-1 interacts with tumor necrosis factor receptor - associated factor 2 ( traf2 ) and apoptosis signal - regulating kinase 1 ( ask1 ) via the cytosolic enzyme domain to form ire-1-traf2-ask1 complexes , which in turn stimulate the c - jun amino - terminal kinase ( jnk ) pathway to promote cell apoptosis , thus preventing the damaging impact of misfolded and secreted proteins on tissues and organisms .", "genetic mutations in ad lead to a overexpression in the brain and subsequent neurotoxicity , which leads to pathogenesis .", "the five familial alzheimer 's disease ( 5fad ) mice were established by overexpressing the five familial - inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida ] + v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter .", "this mouse model is characterized by many pathological features similar to ad , including amyloid plaque deposition , gliosis , neuronal degeneration , neuronal loss , and cognitive deficits at the age of 45 months .", "more importantly , this mouse model has been documented to show the earliest signs of intracellular a aggregation in neurons in 1.5-month - old mice , which indicates these mice as a suitable candidate to investigate the early events of ad . in this study", ", we investigated the time - ordered changes of pro - apoptotic and anti - apoptotic factors to determine the role of the upr signaling pathway in cognitive decline of 5fad mice", ". the outcome may clarify the role of ers in the pathological progression of ad .", "the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .", "marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .", "experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .", "they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study", "were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health ", "the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) .", "the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .", "the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded .", "mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) .", "then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .", "brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%", "paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .", "immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .", "the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .", "biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .", "the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .", "the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis , 6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .", "the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .", "the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected .", " cells  that were lightly stained or had irregular shapes were excluded from quantification .", "brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .", "the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .", "alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) .", "triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .", "louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .", "the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .", "supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .", "subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .", "horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis .", "quantitative data were expressed as the mean  standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .", "escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova .", "the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .", "marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .", "experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .", "they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study", "were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health ", "the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) .", "morris water maze tests were performed as described previously . the swimming behavior of mice was evaluated four times a day for 5 days .", "the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .", "the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded .", "mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) . then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .", "brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%", "paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .", "immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .", "the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .", "biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .", "the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .", "the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis ,", "6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .", "the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .", "the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected .  cells  that were lightly stained or had irregular shapes were excluded from quantification .", "brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .", "the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .", "alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) .", "isolated mouse cortical tissues were added into tissue lysates ( 1% triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .", "louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .", "the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .", "supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .", "subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .", "horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis .", "quantitative data were expressed as the mean  standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .", "escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova .", "to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .", "we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,", "when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .", "immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .", "a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .", "furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .", "levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .", "cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .", "the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .", "the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .", "escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *", "p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .", "( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .", "( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .", "( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .", "scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid  ; v : the fifth layer of the frontal cortex .", "grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,", "grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,", "grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .", "interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .", "grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,", "grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .", "furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile , the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .", "these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .", "significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice .", "( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) . ( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .", "( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .", "( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .", "syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,", "however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] .", "although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,", "western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .", "of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p", "the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .", "punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .", "furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .", "meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .", "these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .", "the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,", "caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .", "this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p < 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] .", "significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .", "( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .", "( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <", "0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .", "( c ) western blots analysis showed the expression of p - jnk / jnk .", "( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .", "( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis . however , it remains unknown if jnk signaling is activated to trigger chop .", "the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .", "these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together", ", these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice .", "to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .", "we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,", "when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .", "immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .", "a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .", "furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .", "levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .", "cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .", "the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .", "the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .", "escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *", "p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .", "( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .", "( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .", "( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .", "scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid  ; v : the fifth layer of the frontal cortex .", "grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,", "grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,", "grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .", "interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .", "grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,", "grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .", "furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile ,", "the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .", "these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .", "significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice . ( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) .", "( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .", "( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .", "( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .", "syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,", "however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] .", "although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,", "western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .", "of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p < 0.05 ) [ figure 3a ] .", "the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .", "punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .", "furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .", "meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .", "these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .", "the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,", "caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .", "this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p", "< 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] . significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .", "( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .", "( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <", "0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .", "( c ) western blots analysis showed the expression of p - jnk / jnk .", "( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .", "( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis .", "the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .", "these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together", ", these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice .", "in 5fad mice , we observed that neuronal loss and cleaved caspase-3 increase at the age of 12 months , cognitive deficit at the age of 7 months and 12 months .", "interestingly , at the age of 2-month - old 5fad mice , the related factors involved in the ers - associated upr pathway , including chop , cleaved caspase-12 , grp 78 , and syvn1 , were significantly increased compared with those in age - matched wt mice .", "these findings suggest that 2-month - old 5fad mice exhibit enhanced ers - associated upr pathway , consistent with intracellular a aggregation in neurons .", "oakley et al . reported , in 5fad brain , accumulated intraneuronal a42 starting at 1.5 months of age , the presence of cerebral amyloid plaques and gliosis at 2 months of age , decreased synaptic markers including synaptophysin and postsynaptic density 95 ( psd95 ) accompanied by cognition deficit at 4 months of age , and pyramidal neuron loss in cortical layer 5 at 9 months of age .", "consistently , in our study , the deposit of intra- and extra - neuronal a increased with age in 5fad brain ; neuronal loss in cortical layer 5 was observed at 12 months of age ; however , before significant neuron loss , impaired memory of 5fad mice at 7 months of age was monitored in the morris water maze .", "our previous study has also shown that cognitive impairment present in 5fad mice was accompanied by structural degradation of synapses and decreased expression of synaptophysin and psd95 in the brain at the age of 67 months . under physiological conditions , approximately one - third of proteins in the er are assembled abnormally and do not form mature proteins .", "aberrant proteins are bound by chaperone proteins , such as grp 78 , to be repaired or transported into the er - associated ubiquitin - proteasome system for degradation via the erad pathway , which serves as an accurate quality control system .", "erad promotes the interaction between ubiquitin and proteins waiting for degradation via e1 , e2 , and e3 .", "an impaired proteasome system leads to accumulation of aberrant proteins and increased risk of cell death .", "sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of chop and caspase-12 activation .", "ers characterized by increased ers - specific apoptosis is obvious in postmortem examinations of brain tissues of ad patients . in vitro experiments have also confirmed that a induces the cellular ers response directly , indicating that abnormal aggregation of a is responsible for ers induction .", "other studies have shown that astrocytes and macrophages also induce ers . however , in our study , the co - immunostaining of grp 78 and chop with astrocytic or neuronal markers showed that ers in cortical tissues occurred mainly in neurons .", "therefore , the ers - associated proteins examined here represent neuronal ers and reveal the impact of the upr on neuronal functions under a-induced stress conditions . in this study , we selected mice at 2 , 7 , and 12 months of age to investigate the changes of ers - associated proteins in 5fad mice and wt mice .", "our results showed that the expression of grp 78 and syvn1 changed without statistical significance but displayed an early - increase and subsequent - decline tendency from 2 to 12 months in wt mice .", "interestingly , at the age of 2 months , the expression of grp 78 and syvn1 in 5fad brains was significantly higher than that in wt mice ; however , at the age of 12 months , grp 78 level in 5fad brains significantly declined when compared with that of 2-month - old 5fad mice .", "meanwhile , the expression of chop and cleaved caspase-12 increased continuously with age , either in 5fad mice or in wt mice . of note , the level of chop and cleaved caspase-12 in 5fad mice was obviously higher than that in wt mice .", "altogether , we speculate that a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via pire1a - xbp1s and atf6a pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) .", "interestingly , no significant differences in pjnk / jnk levels were found between 5fad mice and wt mice at different periods , which indicates that the pjnk / jnk pathway is not involved in a-induced ers . under sustained stress conditions caused by the a-associated toxic effects ,", "the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .", "the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death [ figure 4 ] .", "schematic drawing shows that activated upr pathways of ers induced by intraneuronal -amyloid in 5fad mice .", "a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via p - ire-1-xbp1s and atf6 pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) . under sustained stress conditions caused by the a-associated toxic effects ,", "the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .", "the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death .", "upr : unfolded protein response ; ers : endoplasmic reticulum stress ; a : amyloid  ; 5fad : transgenic mice with five familiar alzheimer 's disease ; p - perk : phosphorylated protein kinase rna - like er kinase ; p - eif2 : phosphorylated eukaryotic translation initiation factor 2 ; atf4 : activating transcription factor 4 ; chop : ccaat / enhancer - binding protein homologous protein ; p - ire-1 : phosphorylated inositol - requiring enzyme 1 ; xbp1s : spliced x box - binding protein 1 ; atf6 : activating transcription factor 6 ; grp 78 : glucose - regulated protein 78 ; erad : endoplasmic reticulum - associated protein degradation ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; traf2 : tumor necrosis factor - receptor - associated factor 2 ; ask1 : apoptosis signal - regulating kinase 1 ; jnk : c - jun amino - terminal kinase . in conclusion ,", "current data reveal that intracellular a aggregation induces obvious ers in neurons at the early stage of 5fad brains .", "imbalanced regulation of grp 78 or syvn1 and chop or cleaved caspase-12 may be involved in the resistance to the restoration of er homeostasis in a-secreting neurons . if the development of ers can be delayed or reduced appropriately or the protective functions of the upr can be enhanced exogenously , the capacity of the er to tolerate abnormal proteins can be increased to delay cell damage and disease progression .", "targeting at ers to control the pathophysiological changes at the early stage of ad may be a better strategy for the prevention and treatment of ad .", "this work was supported by grants from the national natural science foundation of china ( no .", "xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs .", "this work was supported by grants from the national natural science foundation of china ( no .", "xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs ."], "abstract_text": ["<S> background : amyloid  ( a ) deposits and the endoplasmic reticulum stress ( ers ) are both well established in the development and progression of alzheimer 's disease ( ad ) . </S>", "<S> however , the mechanism and role of a-induced ers in ad - associated pathological progression remain to be elucidated.methods:the five familial ad ( 5fad ) mice and wild - type ( wt ) mice aged 2 , 7 , and 12 months were used in the present study . </S>", "<S> morris water maze test was used to evaluate their cognitive performance . </S>", "<S> immunofluorescence and western blot analyses were used to examine the dynamic changes of pro - apoptotic ( ccaat / enhancer - binding protein homologous protein [ chop ] and cleaved caspase-12 ) and anti - apoptotic factors ( chaperone glucose - regulated protein [ grp ] 78 and endoplasmic reticulum - associated protein degradation - associated ubiquitin ligase synovial apoptosis inhibitor 1 [ syvn1 ] ) in the ers - associated unfolded protein response ( upr ) pathway.results:compared with age - matched wt mice , 5fad mice showed higher cleaved caspase-3 , lower neuron - positive staining at the age of 12 months , but earlier cognitive deficit at the age of 7 months ( all p < 0.05 ) . </S>", "<S> interestingly , for 2-month - old 5fad mice , the related proteins involved in the ers - associated upr pathway , including chop , cleaved caspase-12 , grp 78 , and syvn1 , were significantly increased when compared with those in age - matched wt mice ( all p < 0.05 ) . </S>", "<S> moreover , ers occurred mainly in neurons , not in astrocytes.conclusions:these findings suggest that compared with those of age - matched wt mice , ers - associated pro - apoptotic and anti - apoptotic proteins are upregulated in 2-month - old 5fad mice , consistent with intracellular a aggregation in neurons . </S>"], "labels": null, "section_names": ["I", "M", "Animal feeding and grouping", "Behavior analysis", "Immunohistochemistry", "Immunocytochemistry", "Western blots", "Statistical analysis", "R", "Increased amyloid  induces the declined cognition in 7-month-old five familial Alzheimer's disease mice and the loss of neurons in the frontal cortex of the 12-month-old ones", "Anti-apoptotic factors, glucose-regulated protein 78 and SYVN1, are significantly upregulated in 2-month-old five familial Alzheimer's disease mice", "Pro-apoptotic factors, CHOP and cleaved caspase-12, are significantly increased in five familial Alzheimer's disease mice", "D", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["alzheimer 's disease ( ad ) is a common neurodegenerative disease in the central nervous system .", "the pathological features of ad include senile plaques formed by extracellular amyloid  ( a ) aggregation , neurofibrillary tangles formed by abnormal accumulation of hyperphosphorylated tau protein , synaptic impairments , and neuronal loss in the cerebral cortex and hippocampus .", "a aggregation in the brain is central to the pathological changes of ad and leads to a series of pathological events , which further promote a aggregation , resulting in cascade amplification .", "the endoplasmic reticulum ( er ) , a dynamic membranous organelle , is involved in protein synthesis , posttranslational modification , folding , calcium storage , lipid metabolism , and steroid hormone synthesis .", "specific stress conditions such as hyperglycemia , hyperlipidemia , hypoxia , chemical toxicants , and genetic mutations can induce the accumulation of unfolded or misfolded proteins in the er and changes in er functions , resulting in endoplasmic reticulum stress ( ers ) . in turn , ers stimulates the unfolded protein response ( upr ) and restores cellular homeostasis while sustained ers leads to cell apoptosis .", "many studies have shown that ers is involved in the development and progression of neurodegenerative diseases such as ad , parkinson 's disease , huntington 's disease , and amyotrophic lateral sclerosis although the underlying mechanisms still remain blurred .", "the activation of the upr pathway requires the activation of three sensor proteins : inositol - requiring enzyme 1 ( ire-1 ) , activating transcription factor 6 ( atf6 ) , and protein kinase rna - like er kinase .", "these sensors serve to promote the expression of chaperone protein , glucose - regulated protein ( grp ) 78 , to repair misfolded or unfolded proteins , to accelerate er - associated protein degradation ( erad ) , and to aggravate the phosphorylation of eukaryotic translation initiation factor 2a .", "sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of the transcriptional activator ccaat / enhancer - binding protein homologous protein ( chop ) and caspase-12 activation .", "activated ire-1 interacts with tumor necrosis factor receptor - associated factor 2 ( traf2 ) and apoptosis signal - regulating kinase 1 ( ask1 ) via the cytosolic enzyme domain to form ire-1-traf2-ask1 complexes , which in turn stimulate the c - jun amino - terminal kinase ( jnk ) pathway to promote cell apoptosis , thus preventing the damaging impact of misfolded and secreted proteins on tissues and organisms .", "genetic mutations in ad lead to a overexpression in the brain and subsequent neurotoxicity , which leads to pathogenesis .", "the five familial alzheimer 's disease ( 5fad ) mice were established by overexpressing the five familial - inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida ] + v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter .", "this mouse model is characterized by many pathological features similar to ad , including amyloid plaque deposition , gliosis , neuronal degeneration , neuronal loss , and cognitive deficits at the age of 45 months .", "more importantly , this mouse model has been documented to show the earliest signs of intracellular a aggregation in neurons in 1.5-month - old mice , which indicates these mice as a suitable candidate to investigate the early events of ad . in this study", ", we investigated the time - ordered changes of pro - apoptotic and anti - apoptotic factors to determine the role of the upr signaling pathway in cognitive decline of 5fad mice", ". the outcome may clarify the role of ers in the pathological progression of ad ."], ["the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .", "marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .", "experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .", "they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study", "were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health ", "the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) .", "the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .", "the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded .", "mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) .", "then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .", "brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%", "paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .", "immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .", "the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .", "biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .", "the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .", "the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis , 6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .", "the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .", "the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected .", " cells  that were lightly stained or had irregular shapes were excluded from quantification .", "brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .", "the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .", "alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) .", "triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .", "louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .", "the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .", "supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .", "subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .", "horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis .", "quantitative data were expressed as the mean  standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .", "escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova ."], ["the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .", "marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .", "experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .", "they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study", "were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health ", "the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) ."], ["morris water maze tests were performed as described previously . the swimming behavior of mice was evaluated four times a day for 5 days .", "the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .", "the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded ."], ["mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) . then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .", "brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%", "paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .", "immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .", "the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .", "biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .", "the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .", "the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis ,", "6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .", "the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .", "the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected .  cells  that were lightly stained or had irregular shapes were excluded from quantification ."], ["brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .", "the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .", "alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) ."], ["isolated mouse cortical tissues were added into tissue lysates ( 1% triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .", "louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .", "the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .", "supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .", "subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .", "horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis ."], ["quantitative data were expressed as the mean  standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .", "escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova ."], ["to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .", "we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,", "when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .", "immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .", "a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .", "furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .", "levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .", "cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .", "the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .", "the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .", "escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *", "p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .", "( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .", "( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .", "( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .", "scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid  ; v : the fifth layer of the frontal cortex .", "grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,", "grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,", "grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .", "interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .", "grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,", "grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .", "furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile , the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .", "these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .", "significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice .", "( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) . ( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .", "( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .", "( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .", "syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,", "however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] .", "although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,", "western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .", "of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p", "the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .", "punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .", "furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .", "meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .", "these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .", "the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,", "caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .", "this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p < 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] .", "significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .", "( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .", "( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <", "0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .", "( c ) western blots analysis showed the expression of p - jnk / jnk .", "( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .", "( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis . however , it remains unknown if jnk signaling is activated to trigger chop .", "the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .", "these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together", ", these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice ."], ["to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .", "we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,", "when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .", "immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .", "a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .", "furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .", "levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .", "cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .", "the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .", "the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .", "escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *", "p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .", "( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .", "( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .", "( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .", "scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid  ; v : the fifth layer of the frontal cortex ."], ["grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,", "grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,", "grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .", "interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .", "grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,", "grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .", "furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile ,", "the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .", "these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .", "significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice . ( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) .", "( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .", "( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .", "( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .", "syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,", "however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] ."], ["although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,", "western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .", "of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p < 0.05 ) [ figure 3a ] .", "the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .", "punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .", "furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .", "meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .", "these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .", "the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,", "caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .", "this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p", "< 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] . significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .", "( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .", "( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <", "0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .", "( c ) western blots analysis showed the expression of p - jnk / jnk .", "( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .", "( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .", "the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .", "5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid  ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis .", "the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .", "these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together", ", these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice ."], ["in 5fad mice , we observed that neuronal loss and cleaved caspase-3 increase at the age of 12 months , cognitive deficit at the age of 7 months and 12 months .", "interestingly , at the age of 2-month - old 5fad mice , the related factors involved in the ers - associated upr pathway , including chop , cleaved caspase-12 , grp 78 , and syvn1 , were significantly increased compared with those in age - matched wt mice .", "these findings suggest that 2-month - old 5fad mice exhibit enhanced ers - associated upr pathway , consistent with intracellular a aggregation in neurons .", "oakley et al . reported , in 5fad brain , accumulated intraneuronal a42 starting at 1.5 months of age , the presence of cerebral amyloid plaques and gliosis at 2 months of age , decreased synaptic markers including synaptophysin and postsynaptic density 95 ( psd95 ) accompanied by cognition deficit at 4 months of age , and pyramidal neuron loss in cortical layer 5 at 9 months of age .", "consistently , in our study , the deposit of intra- and extra - neuronal a increased with age in 5fad brain ; neuronal loss in cortical layer 5 was observed at 12 months of age ; however , before significant neuron loss , impaired memory of 5fad mice at 7 months of age was monitored in the morris water maze .", "our previous study has also shown that cognitive impairment present in 5fad mice was accompanied by structural degradation of synapses and decreased expression of synaptophysin and psd95 in the brain at the age of 67 months . under physiological conditions , approximately one - third of proteins in the er are assembled abnormally and do not form mature proteins .", "aberrant proteins are bound by chaperone proteins , such as grp 78 , to be repaired or transported into the er - associated ubiquitin - proteasome system for degradation via the erad pathway , which serves as an accurate quality control system .", "erad promotes the interaction between ubiquitin and proteins waiting for degradation via e1 , e2 , and e3 .", "an impaired proteasome system leads to accumulation of aberrant proteins and increased risk of cell death .", "sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of chop and caspase-12 activation .", "ers characterized by increased ers - specific apoptosis is obvious in postmortem examinations of brain tissues of ad patients . in vitro experiments have also confirmed that a induces the cellular ers response directly , indicating that abnormal aggregation of a is responsible for ers induction .", "other studies have shown that astrocytes and macrophages also induce ers . however , in our study , the co - immunostaining of grp 78 and chop with astrocytic or neuronal markers showed that ers in cortical tissues occurred mainly in neurons .", "therefore , the ers - associated proteins examined here represent neuronal ers and reveal the impact of the upr on neuronal functions under a-induced stress conditions . in this study , we selected mice at 2 , 7 , and 12 months of age to investigate the changes of ers - associated proteins in 5fad mice and wt mice .", "our results showed that the expression of grp 78 and syvn1 changed without statistical significance but displayed an early - increase and subsequent - decline tendency from 2 to 12 months in wt mice .", "interestingly , at the age of 2 months , the expression of grp 78 and syvn1 in 5fad brains was significantly higher than that in wt mice ; however , at the age of 12 months , grp 78 level in 5fad brains significantly declined when compared with that of 2-month - old 5fad mice .", "meanwhile , the expression of chop and cleaved caspase-12 increased continuously with age , either in 5fad mice or in wt mice . of note , the level of chop and cleaved caspase-12 in 5fad mice was obviously higher than that in wt mice .", "altogether , we speculate that a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via pire1a - xbp1s and atf6a pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) .", "interestingly , no significant differences in pjnk / jnk levels were found between 5fad mice and wt mice at different periods , which indicates that the pjnk / jnk pathway is not involved in a-induced ers . under sustained stress conditions caused by the a-associated toxic effects ,", "the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .", "the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death [ figure 4 ] .", "schematic drawing shows that activated upr pathways of ers induced by intraneuronal -amyloid in 5fad mice .", "a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via p - ire-1-xbp1s and atf6 pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) . under sustained stress conditions caused by the a-associated toxic effects ,", "the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .", "the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death .", "upr : unfolded protein response ; ers : endoplasmic reticulum stress ; a : amyloid  ; 5fad : transgenic mice with five familiar alzheimer 's disease ; p - perk : phosphorylated protein kinase rna - like er kinase ; p - eif2 : phosphorylated eukaryotic translation initiation factor 2 ; atf4 : activating transcription factor 4 ; chop : ccaat / enhancer - binding protein homologous protein ; p - ire-1 : phosphorylated inositol - requiring enzyme 1 ; xbp1s : spliced x box - binding protein 1 ; atf6 : activating transcription factor 6 ; grp 78 : glucose - regulated protein 78 ; erad : endoplasmic reticulum - associated protein degradation ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; traf2 : tumor necrosis factor - receptor - associated factor 2 ; ask1 : apoptosis signal - regulating kinase 1 ; jnk : c - jun amino - terminal kinase . in conclusion ,", "current data reveal that intracellular a aggregation induces obvious ers in neurons at the early stage of 5fad brains .", "imbalanced regulation of grp 78 or syvn1 and chop or cleaved caspase-12 may be involved in the resistance to the restoration of er homeostasis in a-secreting neurons . if the development of ers can be delayed or reduced appropriately or the protective functions of the upr can be enhanced exogenously , the capacity of the er to tolerate abnormal proteins can be increased to delay cell damage and disease progression .", "targeting at ers to control the pathophysiological changes at the early stage of ad may be a better strategy for the prevention and treatment of ad .", "this work was supported by grants from the national natural science foundation of china ( no .", "xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs ."], ["this work was supported by grants from the national natural science foundation of china ( no .", "xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs ."], [""]]}
{"article_id": "PMC4451743", "article_text": ["cvds are the leading causes of death in diabetic patients   and the substantial rise in diabetes prevalence will ultimately lead to an increase in the burden of diabetes - related cvd in coming decades . among the cardiovascular diseases ,", "ischemic heart disease is the most frequent [ 5 , 6 ] and is associated with the highest morbidity and mortality in patients with type 2 dm . despite the advances in the treatment of coronary artery disease ( cad ) , morbidity and mortality are still high , especially in patients with type 2 dm .", "moreover , despite the evolution in the treatment of hyperglycemia , patients with both cad and diabetes have worse clinical outcomes , irrespective of the treatment applied .", "although dm alters the healthy functioning of arteries   and blood compounds , some clinical studies suggest that myocardial responses to ischemic insults may be deficient in diabetic patients [ 11 , 12 ] , leading to higher myocardial damage and risk of complications .", "thus , recent studies   have focused on the understanding of such myocardial responses , aiming at discovering the mechanisms of myocardial protection to achieve less aggression and a better prognosis .", "a substantial effort has been put forth to investigate any promising cardioprotective strategy to effectively reduce myocardial infarct size .", "and this matter is of particular importance to diabetic cad patients . by 1986 , in a landmark experimental study , murry and colleagues demonstrated that a short antecedent period of ischemia could result in a great reduction in myocardial infarct size .", "for the first time , a method for reducing myocardial cell death other than reperfusion had been discovered .", "since its discovery , this phenomenon , termed ischemic preconditioning ( ip ) has been extensively studied .", "currently , ip is the intrinsic myocardial mechanism that provides the greatest protection regarding the reduction in myocardial ischemic damage .", "it has demonstrated a 75  % reduction in the infarcted area in animal models that have undergone the preconditioning protocol .", "ip is assumed as a mechanism of myocardial protection in which brief episodes of sublethal myocardial ischemia followed by reperfusion trigger multiple intracellular pathways that ultimately result in greater myocardial resistance to a subsequent intense ischemic injury .", "experimentally , this phenomenon was demonstrated by the reduction in the infarcted area by short episodes of ischemia prior to a pronounced ischemic insult . although ip was initially thought to result from the opening of collateral vessels and higher coronary flow , some interesting studies have shown that the phenomenon occurs irrespective of coronary flow changes [ 17 , 18 ] .", "such studies also demonstrated that ip can be observed by sequential exercise tests ( sets ) , in which the improvement in ischemic parameters in the second of 2 sets were confirmed by invasive measurements of myocardial oxygen consumption .", "moreover , the clinical observation that patients with chronic ischemic heart disease frequently describe attenuation or even cessation of angina symptoms if they rest and restart the exercise is termed  warm - up  or  walk - through  angina . this phenomenon has been related to ip and documented in studies using sets [ 19 , 20 ] . following the demonstration of ip , the cellular mechanisms underlying the phenomenon began to be investigated .", "although some cellular pathways have not been fully elucidated , there is a growing understanding of some phenomena and currently it is assumed that ip may be modulated by external factors like age , diseases [ 2224 ] , as well as some specific classes of medications [ 2527 ] .", "currently , the opening of k - atp channels plays a fundamental role in the cellular cascades of ip .", "it is also assumed that medications that bind to this channel may interfere with the ip mechanism .", "thus , some classes of medications like the oral hypoglycemic agents may also block such channels in extrapancreatic sites , such as in the heart .", "it has been shown by some authors that these medications may cause the blockage of myocardial ip . however , despite the fact that some oral hypoglycemic agents may interfere with ip , it is still uncertain whether the intrinsic , complex , intracellular alterations of diabetes itself may affect the cellular mechanisms of this cardioprotective phenomenon .", "some of these studies have shown that diabetes does not interfere with ip [ 31 , 32 ] , but others have shown negative influences [ 33 , 34 ] .", "the great variability in these results is especially due to differences in study protocols , most notably the variability in animal models studied , in the protocols to induce diabetes , the duration of the disease , as well as the variability in myocardial injury protocols .", "on the other hand , studies in humans are scarce and their results have also been conflicting [ 12 , 13 , 35 ] .", "thus , in this study , we aimed at identifying ip in symptomatic multivessel cad patients and compared the results among patients with and without type 2 dm .", "this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .", "briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .", "the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .", "multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70  % obstruction in 2 or more coronary artery territories .", "the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was    0.50 .", "treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was    1.0  mm , associated or not with thoracic discomfort .", "exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction  <  0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3  months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study .", "after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5  days before sets .", "diabetic patients were also instructed to stop antidiabetes medications 5  days before the tests .", "patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .", "a telephone contact was available 24  h a day in case of questions or worsening symptoms . before treadmill test initiation ,", "all patients underwent 2 sets , symptom limited , with a 30-min interval between them .", "tests were conducted during the same period each day , 1  h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .", "a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5  min during the recovery phase , at the onset of 1.0  mm st - segment depression , during arrhythmias , and when it was clinically relevant .", "the level of the st - segment deviation was based on visual assessment of the 0.08  s after the j point by 2 independent cardiologists . in case of disagreement", "only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0  mm ) .", "criteria for interrupting the exercise test were st - segment depression    3.0  mm , st segment elevation    2.0  mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .", "the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0  mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0  mm , and exercise duration in seconds .", "the improvement in ischemic parameters ( t-1.0  mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .", "other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .", "both arrhythmias and st - segment morphology were graded according to their density and severity .", "according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia ,", "the quartiles were defined as quartile 1 : < 90  mg / dl , quartile 2 : 90100  mg / dl , quartile 3 : 101125  mg / dl , and quartile 4 :  126  mg / dl . for glycosylated hemoglobin ,", "the quartiles were defined as quartile 1 : < 5.7  % , quartile 2 : 5.76.3  % , quartile 3 : 6.46.9  % , and quartile 4 :  7.0  % .", "briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients   and studies that included only nondiabetic patients . by the differences in t-1.0  mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30  % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group )", ", 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) .", "continuous variables were compared using an unpaired student s t test or mann  whitney test , when appropriate .", "data are expressed as mean    standard deviation or as absolute frequencies and percentages .", "all tests were 2-sided , and a value of p  <  0.05 was considered significant .", "this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .", "briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .", "the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .", "multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70  % obstruction in 2 or more coronary artery territories .", "the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was    0.50 .", "treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was    1.0  mm , associated or not with thoracic discomfort .", "exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction  <  0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3  months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study .", "after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5  days before sets .", "diabetic patients were also instructed to stop antidiabetes medications 5  days before the tests .", "patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .", "a telephone contact was available 24  h a day in case of questions or worsening symptoms . before treadmill test initiation ,", "all patients underwent 2 sets , symptom limited , with a 30-min interval between them . the modified bruce protocol was applied .", "tests were conducted during the same period each day , 1  h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .", "a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5  min during the recovery phase , at the onset of 1.0  mm st - segment depression , during arrhythmias , and when it was clinically relevant .", "the level of the st - segment deviation was based on visual assessment of the 0.08  s after the j point by 2 independent cardiologists . in case of disagreement ,", "only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0  mm ) .", "criteria for interrupting the exercise test were st - segment depression    3.0  mm , st segment elevation    2.0  mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .", "the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0  mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0  mm , and exercise duration in seconds .", "the improvement in ischemic parameters ( t-1.0  mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .", "other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .", "both arrhythmias and st - segment morphology were graded according to their density and severity . according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia , the quartiles were defined as quartile 1 : < 90  mg / dl , quartile 2 : 90100  mg / dl , quartile 3 : 101125  mg / dl , and quartile 4 :  126  mg / dl . for glycosylated hemoglobin ,", "the quartiles were defined as quartile 1 : < 5.7  % , quartile 2 : 5.76.3  % , quartile 3 : 6.46.9  % , and quartile 4 :  7.0  % .", "the sample size calculation has been previously published elsewhere . briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients   and studies that included only nondiabetic patients . by the differences in t-1.0  mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30  % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group ) , 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) .", "continuous variables were compared using an unpaired student s t test or mann  whitney test , when appropriate .", "data are expressed as mean    standard deviation or as absolute frequencies and percentages .", "all tests were 2-sided , and a value of p  <  0.05 was considered significant .", "of 2,140 patients with stable cad followed at our institution , 361 met the inclusion criteria .", "the main reasons for non - inclusion and exclusion are shown in fig .  1 .", "thus , a total of 174 patients completed the 2 sets and had ip assessed .", "the study population comprised 86 patients with dm and 88 without this diagnosis ( fig . ", "the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .", "cad  =  coronary artery disease ; ckd  =  chronic kidney disease ; cva  =  cerebrovascular accident ; ecg  =  electrocardiogram ; ef  =  ejection fraction ; et  =  exercise test ; dm  =  diabetes mellitus ; ip  =  ischemic preconditioning ; set  =  sequential exercise test ( s ) study flow chart . of", "the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .", "cad  =  coronary artery disease ; ckd  =  chronic kidney disease ; cva  =  cerebrovascular accident ; ecg  =  electrocardiogram ; ef  =  ejection fraction ; et  =  exercise test ; dm  =  diabetes mellitus ; ip  =  ischemic preconditioning ; set  =  sequential exercise test ( s ) the main demographic , clinical and biochemical characteristics of the 2 groups are presented in table  1 . despite the higher prevalence of previous myocardial infarction in the diabetic population and lipid profile ( higher ldl - cholesterol levels in nondiabetic patients ) , both groups had homogeneous characteristics .", "the duration of diabetes was 11.5    8.8  years ( mean    sd ) , with a median of 10  years ( interquartiles ranges of 6 and 15  years ) .", "table  2 shows the medications used in the two groups of patients.table 1main demographic , biochemical , and clinical characteristics of the study populationtotal n  =  174diabetics ( n  =  86)non - diabetics ( n  =  88 ) \n p valueage64.1    6.864.3    8.60.84male n ( % ) 73 ( 84.9)76 ( 86.3)0.83hypertension64 ( 80)74 ( 84.1)0.54smokers7 ( 8.8)7 ( 8.6)previous smokers38 ( 48.1)33 ( 40.7)0.6non - smokers34 ( 43.0)41 ( 50.6)previous ami36 ( 46.1)17 ( 22.7)0.004cabg28 ( 34.6)23 ( 26.1)0.25pci24 ( 30.4)28 ( 32.2)0.87ef0.61    0.060.62    0.060.2tri - vessel disease38 ( 52)43 ( 51.2)1.0bi - vessel disease35 ( 48)41 ( 48.8)1.0lad disease63 ( 87.5)74 ( 86.0)1.0collateral circulation34 ( 50.7)38 ( 46.9)0.64collateral grade 2/330 ( 88.2)32 ( 84.2)0.74weight ( kg)74.5    11.073.2    12.70.51height ( m)1.66    8.11.66    9.10.75bmi26.5    2.826.4    3.20.54fasting glycemia143.5    47.099.0    9.40.0001a1c7.35    1.615.63    0.300.0001bun41.9    13.440.1    9.40.32creatinine1.04   ", "0.231.07    0.200.37total cholesterol156.5    34.5168.9    36.30.02ldl cholesterol90.5    27.4102.0    32.70.01hdl cholesterol38.0    9.640.3    9.00.12triglycerides147.2    88.3132.6    72.30.24data are expressed as means    standard deviation or as absolute and relative risks \n ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoproteintable 2classes of medications used by the two groups of diabetic and nondiabetic patientsaspirin / clopidogrelstatinsbeta - blockersacei / arbcalcium blockersdiureticsoadinsulinsdm93.2  % 93.2  % 89.8  % 78.4  % 35.2  % 37.5  % 88.5  % 26.5  % non - dm96.5  % 96.5  % 86.2  % 78.2  % 36.8  % 34.5  % -- \n dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs main demographic , biochemical , and clinical characteristics of the study population data are expressed as means    standard deviation or as absolute and relative risks \n ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoprotein classes of medications used by the two groups of diabetic and nondiabetic patients \n dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs among the 86 diabetic patients , 62 ( 72  % ) had an improvement in t-1.0  mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68  % ) had an ischemic improvement consistent with ip ( fig .  2 , p  =  0.62).fig .", "2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning the t-1.0  mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0  mm between the 2 sets of 79.4    47.6  s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5    36.4  s ( table  3 , p  =  0.12).table 3t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm269.2    117.8320.9    132.351.7    63.20.15non - dm275.6    111.9314.6   ", "126.239.0    52.3 dm / ip+274.8    102.8354.3    115.179.4    47.60.12non - dm / ip+296.9    107.7362.4    108.565.5    36.4 dm / ip -254.7    151.6234.7    137.020.0    36.40.80non - dm / ip -230.1    108.7212.4    98.217.7    31.9data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test .", "* the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011    2,430  bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081    2,139  bpm x mmhg ( table  4 , p  =  0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm / ip+22,841    4,50025,853    4,7423,011    2,4300.01non - dm / ip + 23,094    5,31225,175    5,4852,081    2,139 dm / ip -22,705    4,00022,124    3,686580    2,2500.43non - dm / ip -23,910    5,38022,869    5,3511,050    2,027data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table  5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip + hr ( baseline)79.0    15.186.3    17.07.3    11.0hr ( peak)139.0   ", "18.2149.7    19.36.8    12.6bp ( peak)192.0    25.0191.8    23.90.16    15.4 dm / ip -hr ( baseline)79.0    14.784.4    14.95.4    8.0hr ( peak)141.7    14.3143.0    13.51.3    7.4bp ( baseline)153.3    22.6147.1    19.76.2    14.7bp ( peak)183.7    23.2180.6    22.23.12    14.7non - dm / ip + hr ( baseline)78.0   ", "13.187.1    13.99.1    7.9hr ( peak)139.6    15.4144.3    14.74.7    5.7bp ( baseline)146.7    20.6138.7    18.38.0    11.3bp ( peak)193.5    23.6190.2    24.53.3    12.4non - dm / ip -hr ( baseline)82.5    12.987.1    12.64.6    7.1hr ( peak)139.3    12.4139.6    13.00.35    5.2bp ( baseline)158.6    26.2147.9    26.110.7    14.6bp ( peak)204.6    25.3194.8    28.18.9 ", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means    standard deviation .", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20    39  s versus 17    36  s , respectively , p  =  0.60 ) .", "the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50  % versus 63  % of patients demonstrated improvement in arrhythmias , respectively ; p  =  0.41 ) .", "the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5  % versus 48.3  % , respectively , p  =  0.41 ) .", "when the total group of patients ( n  =  174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table  6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n  =  122)ip - ( n  =  52 ) \n", "p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3    42.6118.9    34.10.72a1c6.63    1.66.32    1.20.25data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig . ", "3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .", "ip  =  ischemic preconditioning ; q  =  quartile ( s ) .", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip  =  ischemic preconditioning ; q  =  quartile ( s ) .", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia", "among the 86 diabetic patients , 62 ( 72  % ) had an improvement in t-1.0  mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68  % ) had an ischemic improvement consistent with ip ( fig .  2 , p  =  0.62).fig .", "2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning the t-1.0  mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0  mm between the 2 sets of 79.4    47.6  s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5    36.4  s ( table  3 , p  =  0.12).table 3t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm269.2    117.8320.9    132.351.7    63.20.15non - dm275.6    111.9314.6    126.239.0    52.3 dm / ip+274.8    102.8354.3    115.179.4    47.60.12non - dm / ip+296.9    107.7362.4   ", "108.565.5    36.4 dm / ip -254.7    151.6234.7    137.020.0    36.40.80non - dm / ip -230.1    108.7212.4    98.217.7    31.9data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation", ". dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011    2,430  bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081    2,139  bpm x mmhg ( table  4 , p  =  0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm / ip+22,841    4,50025,853    4,7423,011    2,4300.01non - dm / ip + 23,094    5,31225,175    5,4852,081    2,139 dm / ip -22,705    4,00022,124    3,686580    2,2500.43non - dm / ip -23,910    5,38022,869    5,3511,050    2,027data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation", ". dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table  5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip", "+ hr ( baseline)79.0    15.186.3    17.07.3    11.0hr ( peak)139.0   ", "15.1142.1    14.73.2    5.6bp ( baseline)156.5    18.2149.7    19.36.8    12.6bp ( peak)192.0    25.0191.8    23.90.16    15.4 dm / ip -hr ( baseline)79.0    14.784.4    14.95.4    8.0hr ( peak)141.7    14.3143.0    13.51.3    7.4bp ( baseline)153.3    22.6147.1    19.76.2    14.7bp ( peak)183.7    23.2180.6    22.23.12   ", "14.7non - dm / ip + hr ( baseline)78.0    13.187.1    13.99.1    7.9hr ( peak)139.6    15.4144.3    14.74.7    5.7bp ( baseline)146.7    20.6138.7    18.38.0    11.3bp ( peak)193.5    23.6190.2    24.53.3    12.4non - dm / ip -hr ( baseline)82.5    12.987.1    12.64.6    7.1hr ( peak)139.3   ", "12.4139.6    13.00.35    5.2bp ( baseline)158.6    26.2147.9    26.110.7    14.6bp ( peak)204.6    25.3194.8    28.18.9    16.5data are expressed as means    standard deviation .", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means    standard deviation .", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test", "the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20    39  s versus 17    36  s , respectively , p  =  0.60 ) .", "the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50  % versus 63  % of patients demonstrated improvement in arrhythmias , respectively ; p  =  0.41 ) .", "the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5  % versus 48.3  % , respectively , p  =  0.41 ) .", "when the total group of patients ( n  =  174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table  6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n  =  122)ip - ( n  =  52 ) \n p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3    42.6118.9    34.10.72a1c6.63    1.66.32    1.20.25data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig", "3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip  =  ischemic preconditioning", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .", "ip  =  ischemic preconditioning ; q  =  quartile ( s ) .", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia", "considering that diabetes is an independent risk factor for the occurrence of major cardiovascular events and mortality [ 5 , 6 ] , it is reasonable to consider that diabetes might damage the protective mechanism of ip in cad patients , leading to worse outcomes .", "however , in this study , the presence of type 2 dm did not have any deleterious effects on the myocardial protective mechanism termed ischemic preconditioning . in this context", ", our study showed that patients with type 2 dm demonstrated ip in similar frequency and intensity compared with nondiabetic patients and , interestingly , our data indicated an improvement in ischemic parameters associated with diabetes .", "the analyses of the data showed a better ischemic response evaluated by rpp in diabetic patients .", "thus , this study adds clinical information on some questions that have emerged from contradictory experimental studies .", "the improvement in myocardial oxygen consumption , observed by the analysis of rpp , was more pronounced in patients with compared to those without diabetes , indicating better adaptation of the myocardium of diabetic patients after an ischemic insult . moreover , diabetic patients had an improvement in t-1.0  mm greater than that in nondiabetic patients , although it did not reach statistical significance .", "analyzes of the total exercise time also confirmed the main results of the study , as times were similar among patients with and without dm .", "similarly , the st - segment deviation morphology did not differ among diabetic and nondiabetic patients . because myocardial ischemia is a frequent cause of arrhythmias during treadmill stress tests", ", we also assessed the occurrence and complexity of arrhythmias and their improvement during set .", "thus , the improvement in the occurrence of arrhythmias also confirmed the main findings of the study , as the occurrence did not differ among diabetic and nondiabetic patients .", "interestingly , analyzes of the percentage of patients who demonstrated ip among different quartiles of fasting glycemia and glycosylated hemoglobin showed similar rates of ip demonstration .", "this analyzes infer that the differences in the intensity of glycemic control in our population did not prevent the demonstration of this cardioprotective phenomenon .", "however , despite our consistent findings , the information from this study is contrary to lee s et al .   and ishihara s et al . , who have also studied ip in humans .", "studied diabetic and nondiabetic cad patients during percutaneous coronary interventions , and evaluated the action of hypoglycemic agents on ip .", "patients underwent consecutive balloon coronary inflations . during the second sequential balloon inflation , patients had less thoracic discomfort , less myocardial lactate production , and lower st - segment deviation .", "moreover , the authors observed that diabetic patients treated with glimepiride had higher lactate production compared to nondiabetic patients treated with glimepiride . despite this finding , an important limitation of this study is that there was not a direct comparison of diabetic and nondiabetic patients who had no drug interference in ip evaluation .", "studied patients hospitalized due to an acute myocardial infarction and evaluated the effects of preinfarct angina , on the release of cardiac markers of necrosis , ventricular function , and in - hospital death and compared the results among diabetic and nondiabetic patients .", "the authors found that in the nondiabetic population , patients with preinfarct angina had a lower release of cardiac markers of necrosis , better recovery of ventricular function , and lower in - hospital mortality , compared to patients with no preinfarct angina . on the other hand , when they analyzed diabetic patients , these variables were not different among patients with and without preinfarct angina .", "thus , the authors inferred that diabetes prevented the appearance of ip . despite their findings ,", "first , this was a retrospective study , which included a small number of diabetic ( n  =  121 ) compared to nondiabetic patients ( n  =  490 ) .", "many studies have shown that some of these drugs may block ip , and it has been speculated that this interference with ip mechanisms may partially explain the worse prognosis of diabetic patients hospitalized due to an acute myocardial infarction . on the other hand ,", "other experimental studies that evaluated  in vitro  human myocardial tissue [ 41 , 42 ] showed results that match those of the present study .", "they evaluated the release of cardiac biomarkers of necrosis and the percentage of tissue viability . among other findings", ", the authors found a similar intensity in myocardial protection among diabetic and nondiabetic patients .", "additionally , cleveland et al .   evaluated the contractile function of isolated right atrial trabeculae from cad patients , resected during coronary artery bypass graft surgery .", "they showed that the diabetic group that underwent the preconditioning stimuli had similar improvement in contractile function compared to the nondiabetic group .", "moreover , a small study conducted by bilinska and colleagues   with diabetic patients treated with glibenclamide , gliclazide , and diet compared the demonstration of ip in these groups with that in nondiabetic patients .", "besides the main findings of the study , which were related to the effects of sulfonylureas in the warm - up phenomenon , they showed that the group of diabetic patients on diet ( n  =  15 ) had similar improvement in ischemic parameters compared to nondiabetic patients ( n  =  17 ) .", "of note , this was a secondary result , and the small sample size did not permit to make a definitive conclusion .", "this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .", "the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control .", ", the response of the myocardium to an ischemic insult may probably play an important role in prognosis .", "one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .", "contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients .", "this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .", "the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control .", ", the response of the myocardium to an ischemic insult may probably play an important role in prognosis .", "one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .", "contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients .", "in this study , diabetes mellitus did not substantially affect myocardial ip in symptomatic cad patients ."], "abstract_text": ["<S> backgroundthe influence of diabetes mellitus on myocardial ischemic preconditioning is not clearly defined . </S>", "<S> experimental studies are conflicting and human studies are scarce and inconclusive.objectivesidentify whether diabetes mellitus intervenes on ischemic preconditioning in symptomatic coronary artery disease patients.methodssymptomatic multivessel coronary artery disease patients with preserved systolic ventricular function and a positive exercise test underwent two sequential exercise tests to demonstrate ischemic preconditioning . </S>", "<S> ischemic parameters were compared among patients with and without type 2 diabetes mellitus . </S>", "<S> ischemic preconditioning was considered present when the time to 1.0  mm st deviation and rate pressure - product were greater in the second of 2 exercise tests . </S>", "<S> sequential exercise tests were analyzed by 2 independent cardiologists.resultsof the 2,140 consecutive coronary artery disease patients screened , 361 met inclusion criteria , and 174 patients ( 64.2    7.6  years ) completed the study protocol . of these , 86 had the diagnosis of type 2 diabetes . </S>", "<S> among diabetic patients , 62 ( 72  % ) manifested an improvement in ischemic parameters consistent with ischemic preconditioning , whereas among nondiabetic patients , 60 ( 68  % ) manifested ischemic preconditioning ( p  =  0.62 ) . </S>", "<S> the analysis of patients who demonstrated ischemic preconditioning showed similar improvement in the time to 1.0  mm st deviation between diabetic and nondiabetic groups ( 79.4    47.6 vs 65.5    36.4  s , respectively , p  =  0.12 ) . </S>", "<S> regarding rate pressure - product , the improvement was greater in diabetic compared to nondiabetic patients ( 3011    2430 vs 2081    2139  bpm x mmhg , respectively , p  =  0.01).conclusionsin this study , diabetes mellitus was not associated with impairment in ischemic preconditioning in symptomatic coronary artery disease patients . </S>", "<S> furthermore , diabetic patients experienced an improvement in this significant mechanism of myocardial protection . </S>"], "labels": null, "section_names": ["Background", "Methods", "Study design", "Patients", "Sequential treadmill exercise tests", "Sample size calculation", "Statistical analysis", "Results", "Demonstration of IP", "Analyses of secondary ergometric variables", "Analysis of glycemic variables", "Discussion", "Study limitations and strengths", "Clinical practice perspectives", "Conclusions"], "sections": [["according to the world health organization , diabetes mellitus ( dm ) is one of the most common chronic diseases in the world , affecting 9.3  % of all adults older than 25  years .", "diabetes poses a high risk for the development of cardiovascular diseases ( cvd ) , because it heralds an accelerated process of atherosclerosis and an increased risk for atherothrombotic complications .", "cvds are the leading causes of death in diabetic patients   and the substantial rise in diabetes prevalence will ultimately lead to an increase in the burden of diabetes - related cvd in coming decades . among the cardiovascular diseases ,", "ischemic heart disease is the most frequent [ 5 , 6 ] and is associated with the highest morbidity and mortality in patients with type 2 dm . despite the advances in the treatment of coronary artery disease ( cad ) , morbidity and mortality are still high , especially in patients with type 2 dm .", "moreover , despite the evolution in the treatment of hyperglycemia , patients with both cad and diabetes have worse clinical outcomes , irrespective of the treatment applied .", "although dm alters the healthy functioning of arteries   and blood compounds , some clinical studies suggest that myocardial responses to ischemic insults may be deficient in diabetic patients [ 11 , 12 ] , leading to higher myocardial damage and risk of complications .", "thus , recent studies   have focused on the understanding of such myocardial responses , aiming at discovering the mechanisms of myocardial protection to achieve less aggression and a better prognosis .", "a substantial effort has been put forth to investigate any promising cardioprotective strategy to effectively reduce myocardial infarct size .", "and this matter is of particular importance to diabetic cad patients . by 1986 , in a landmark experimental study , murry and colleagues demonstrated that a short antecedent period of ischemia could result in a great reduction in myocardial infarct size .", "for the first time , a method for reducing myocardial cell death other than reperfusion had been discovered .", "since its discovery , this phenomenon , termed ischemic preconditioning ( ip ) has been extensively studied .", "currently , ip is the intrinsic myocardial mechanism that provides the greatest protection regarding the reduction in myocardial ischemic damage .", "it has demonstrated a 75  % reduction in the infarcted area in animal models that have undergone the preconditioning protocol .", "ip is assumed as a mechanism of myocardial protection in which brief episodes of sublethal myocardial ischemia followed by reperfusion trigger multiple intracellular pathways that ultimately result in greater myocardial resistance to a subsequent intense ischemic injury .", "experimentally , this phenomenon was demonstrated by the reduction in the infarcted area by short episodes of ischemia prior to a pronounced ischemic insult . although ip was initially thought to result from the opening of collateral vessels and higher coronary flow , some interesting studies have shown that the phenomenon occurs irrespective of coronary flow changes [ 17 , 18 ] .", "such studies also demonstrated that ip can be observed by sequential exercise tests ( sets ) , in which the improvement in ischemic parameters in the second of 2 sets were confirmed by invasive measurements of myocardial oxygen consumption .", "moreover , the clinical observation that patients with chronic ischemic heart disease frequently describe attenuation or even cessation of angina symptoms if they rest and restart the exercise is termed  warm - up  or  walk - through  angina . this phenomenon has been related to ip and documented in studies using sets [ 19 , 20 ] . following the demonstration of ip , the cellular mechanisms underlying the phenomenon began to be investigated .", "although some cellular pathways have not been fully elucidated , there is a growing understanding of some phenomena and currently it is assumed that ip may be modulated by external factors like age , diseases [ 2224 ] , as well as some specific classes of medications [ 2527 ] .", "currently , the opening of k - atp channels plays a fundamental role in the cellular cascades of ip .", "it is also assumed that medications that bind to this channel may interfere with the ip mechanism .", "thus , some classes of medications like the oral hypoglycemic agents may also block such channels in extrapancreatic sites , such as in the heart .", "it has been shown by some authors that these medications may cause the blockage of myocardial ip . however , despite the fact that some oral hypoglycemic agents may interfere with ip , it is still uncertain whether the intrinsic , complex , intracellular alterations of diabetes itself may affect the cellular mechanisms of this cardioprotective phenomenon .", "some of these studies have shown that diabetes does not interfere with ip [ 31 , 32 ] , but others have shown negative influences [ 33 , 34 ] .", "the great variability in these results is especially due to differences in study protocols , most notably the variability in animal models studied , in the protocols to induce diabetes , the duration of the disease , as well as the variability in myocardial injury protocols .", "on the other hand , studies in humans are scarce and their results have also been conflicting [ 12 , 13 , 35 ] .", "thus , in this study , we aimed at identifying ip in symptomatic multivessel cad patients and compared the results among patients with and without type 2 dm ."], ["this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .", "briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .", "the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .", "multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70  % obstruction in 2 or more coronary artery territories .", "the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was    0.50 .", "treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was    1.0  mm , associated or not with thoracic discomfort .", "exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction  <  0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3  months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study .", "after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5  days before sets .", "diabetic patients were also instructed to stop antidiabetes medications 5  days before the tests .", "patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .", "a telephone contact was available 24  h a day in case of questions or worsening symptoms . before treadmill test initiation ,", "all patients underwent 2 sets , symptom limited , with a 30-min interval between them .", "tests were conducted during the same period each day , 1  h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .", "a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5  min during the recovery phase , at the onset of 1.0  mm st - segment depression , during arrhythmias , and when it was clinically relevant .", "the level of the st - segment deviation was based on visual assessment of the 0.08  s after the j point by 2 independent cardiologists . in case of disagreement", "only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0  mm ) .", "criteria for interrupting the exercise test were st - segment depression    3.0  mm , st segment elevation    2.0  mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .", "the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0  mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0  mm , and exercise duration in seconds .", "the improvement in ischemic parameters ( t-1.0  mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .", "other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .", "both arrhythmias and st - segment morphology were graded according to their density and severity .", "according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia ,", "the quartiles were defined as quartile 1 : < 90  mg / dl , quartile 2 : 90100  mg / dl , quartile 3 : 101125  mg / dl , and quartile 4 :  126  mg / dl . for glycosylated hemoglobin ,", "the quartiles were defined as quartile 1 : < 5.7  % , quartile 2 : 5.76.3  % , quartile 3 : 6.46.9  % , and quartile 4 :  7.0  % .", "briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients   and studies that included only nondiabetic patients . by the differences in t-1.0  mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30  % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group )", ", 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) .", "continuous variables were compared using an unpaired student s t test or mann  whitney test , when appropriate .", "data are expressed as mean    standard deviation or as absolute frequencies and percentages .", "all tests were 2-sided , and a value of p  <  0.05 was considered significant ."], ["this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .", "briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .", "the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .", "multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70  % obstruction in 2 or more coronary artery territories .", "the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was    0.50 .", "treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was    1.0  mm , associated or not with thoracic discomfort .", "exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction  <  0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3  months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study ."], ["after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5  days before sets .", "diabetic patients were also instructed to stop antidiabetes medications 5  days before the tests .", "patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .", "a telephone contact was available 24  h a day in case of questions or worsening symptoms . before treadmill test initiation ,"], ["all patients underwent 2 sets , symptom limited , with a 30-min interval between them . the modified bruce protocol was applied .", "tests were conducted during the same period each day , 1  h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .", "a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5  min during the recovery phase , at the onset of 1.0  mm st - segment depression , during arrhythmias , and when it was clinically relevant .", "the level of the st - segment deviation was based on visual assessment of the 0.08  s after the j point by 2 independent cardiologists . in case of disagreement ,", "only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0  mm ) .", "criteria for interrupting the exercise test were st - segment depression    3.0  mm , st segment elevation    2.0  mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .", "the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0  mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0  mm , and exercise duration in seconds .", "the improvement in ischemic parameters ( t-1.0  mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .", "other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .", "both arrhythmias and st - segment morphology were graded according to their density and severity . according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia , the quartiles were defined as quartile 1 : < 90  mg / dl , quartile 2 : 90100  mg / dl , quartile 3 : 101125  mg / dl , and quartile 4 :  126  mg / dl . for glycosylated hemoglobin ,", "the quartiles were defined as quartile 1 : < 5.7  % , quartile 2 : 5.76.3  % , quartile 3 : 6.46.9  % , and quartile 4 :  7.0  % ."], ["the sample size calculation has been previously published elsewhere . briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients   and studies that included only nondiabetic patients . by the differences in t-1.0  mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30  % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group ) , 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) ."], ["continuous variables were compared using an unpaired student s t test or mann  whitney test , when appropriate .", "data are expressed as mean    standard deviation or as absolute frequencies and percentages .", "all tests were 2-sided , and a value of p  <  0.05 was considered significant ."], ["of 2,140 patients with stable cad followed at our institution , 361 met the inclusion criteria .", "the main reasons for non - inclusion and exclusion are shown in fig .  1 .", "thus , a total of 174 patients completed the 2 sets and had ip assessed .", "the study population comprised 86 patients with dm and 88 without this diagnosis ( fig . ", "the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .", "cad  =  coronary artery disease ; ckd  =  chronic kidney disease ; cva  =  cerebrovascular accident ; ecg  =  electrocardiogram ; ef  =  ejection fraction ; et  =  exercise test ; dm  =  diabetes mellitus ; ip  =  ischemic preconditioning ; set  =  sequential exercise test ( s ) study flow chart . of", "the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .", "cad  =  coronary artery disease ; ckd  =  chronic kidney disease ; cva  =  cerebrovascular accident ; ecg  =  electrocardiogram ; ef  =  ejection fraction ; et  =  exercise test ; dm  =  diabetes mellitus ; ip  =  ischemic preconditioning ; set  =  sequential exercise test ( s ) the main demographic , clinical and biochemical characteristics of the 2 groups are presented in table  1 . despite the higher prevalence of previous myocardial infarction in the diabetic population and lipid profile ( higher ldl - cholesterol levels in nondiabetic patients ) , both groups had homogeneous characteristics .", "the duration of diabetes was 11.5    8.8  years ( mean    sd ) , with a median of 10  years ( interquartiles ranges of 6 and 15  years ) .", "table  2 shows the medications used in the two groups of patients.table 1main demographic , biochemical , and clinical characteristics of the study populationtotal n  =  174diabetics ( n  =  86)non - diabetics ( n  =  88 ) \n p valueage64.1    6.864.3    8.60.84male n ( % ) 73 ( 84.9)76 ( 86.3)0.83hypertension64 ( 80)74 ( 84.1)0.54smokers7 ( 8.8)7 ( 8.6)previous smokers38 ( 48.1)33 ( 40.7)0.6non - smokers34 ( 43.0)41 ( 50.6)previous ami36 ( 46.1)17 ( 22.7)0.004cabg28 ( 34.6)23 ( 26.1)0.25pci24 ( 30.4)28 ( 32.2)0.87ef0.61    0.060.62    0.060.2tri - vessel disease38 ( 52)43 ( 51.2)1.0bi - vessel disease35 ( 48)41 ( 48.8)1.0lad disease63 ( 87.5)74 ( 86.0)1.0collateral circulation34 ( 50.7)38 ( 46.9)0.64collateral grade 2/330 ( 88.2)32 ( 84.2)0.74weight ( kg)74.5    11.073.2    12.70.51height ( m)1.66    8.11.66    9.10.75bmi26.5    2.826.4    3.20.54fasting glycemia143.5    47.099.0    9.40.0001a1c7.35    1.615.63    0.300.0001bun41.9    13.440.1    9.40.32creatinine1.04   ", "0.231.07    0.200.37total cholesterol156.5    34.5168.9    36.30.02ldl cholesterol90.5    27.4102.0    32.70.01hdl cholesterol38.0    9.640.3    9.00.12triglycerides147.2    88.3132.6    72.30.24data are expressed as means    standard deviation or as absolute and relative risks \n ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoproteintable 2classes of medications used by the two groups of diabetic and nondiabetic patientsaspirin / clopidogrelstatinsbeta - blockersacei / arbcalcium blockersdiureticsoadinsulinsdm93.2  % 93.2  % 89.8  % 78.4  % 35.2  % 37.5  % 88.5  % 26.5  % non - dm96.5  % 96.5  % 86.2  % 78.2  % 36.8  % 34.5  % -- \n dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs main demographic , biochemical , and clinical characteristics of the study population data are expressed as means    standard deviation or as absolute and relative risks \n ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoprotein classes of medications used by the two groups of diabetic and nondiabetic patients \n dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs among the 86 diabetic patients , 62 ( 72  % ) had an improvement in t-1.0  mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68  % ) had an ischemic improvement consistent with ip ( fig .  2 , p  =  0.62).fig .", "2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning the t-1.0  mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0  mm between the 2 sets of 79.4    47.6  s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5    36.4  s ( table  3 , p  =  0.12).table 3t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm269.2    117.8320.9    132.351.7    63.20.15non - dm275.6    111.9314.6   ", "126.239.0    52.3 dm / ip+274.8    102.8354.3    115.179.4    47.60.12non - dm / ip+296.9    107.7362.4    108.565.5    36.4 dm / ip -254.7    151.6234.7    137.020.0    36.40.80non - dm / ip -230.1    108.7212.4    98.217.7    31.9data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test .", "* the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011    2,430  bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081    2,139  bpm x mmhg ( table  4 , p  =  0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm / ip+22,841    4,50025,853    4,7423,011    2,4300.01non - dm / ip + 23,094    5,31225,175    5,4852,081    2,139 dm / ip -22,705    4,00022,124    3,686580    2,2500.43non - dm / ip -23,910    5,38022,869    5,3511,050    2,027data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table  5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip + hr ( baseline)79.0    15.186.3    17.07.3    11.0hr ( peak)139.0   ", "18.2149.7    19.36.8    12.6bp ( peak)192.0    25.0191.8    23.90.16    15.4 dm / ip -hr ( baseline)79.0    14.784.4    14.95.4    8.0hr ( peak)141.7    14.3143.0    13.51.3    7.4bp ( baseline)153.3    22.6147.1    19.76.2    14.7bp ( peak)183.7    23.2180.6    22.23.12    14.7non - dm / ip + hr ( baseline)78.0   ", "13.187.1    13.99.1    7.9hr ( peak)139.6    15.4144.3    14.74.7    5.7bp ( baseline)146.7    20.6138.7    18.38.0    11.3bp ( peak)193.5    23.6190.2    24.53.3    12.4non - dm / ip -hr ( baseline)82.5    12.987.1    12.64.6    7.1hr ( peak)139.3    12.4139.6    13.00.35    5.2bp ( baseline)158.6    26.2147.9    26.110.7    14.6bp ( peak)204.6    25.3194.8    28.18.9 ", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means    standard deviation .", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20    39  s versus 17    36  s , respectively , p  =  0.60 ) .", "the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50  % versus 63  % of patients demonstrated improvement in arrhythmias , respectively ; p  =  0.41 ) .", "the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5  % versus 48.3  % , respectively , p  =  0.41 ) .", "when the total group of patients ( n  =  174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table  6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n  =  122)ip - ( n  =  52 ) \n", "p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3    42.6118.9    34.10.72a1c6.63    1.66.32    1.20.25data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig . ", "3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .", "ip  =  ischemic preconditioning ; q  =  quartile ( s ) .", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip  =  ischemic preconditioning ; q  =  quartile ( s ) .", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia"], ["among the 86 diabetic patients , 62 ( 72  % ) had an improvement in t-1.0  mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68  % ) had an ischemic improvement consistent with ip ( fig .  2 , p  =  0.62).fig .", "2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .", "ip  =  ischemic preconditioning the t-1.0  mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0  mm between the 2 sets of 79.4    47.6  s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5    36.4  s ( table  3 , p  =  0.12).table 3t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm269.2    117.8320.9    132.351.7    63.20.15non - dm275.6    111.9314.6    126.239.0    52.3 dm / ip+274.8    102.8354.3    115.179.4    47.60.12non - dm / ip+296.9    107.7362.4   ", "108.565.5    36.4 dm / ip -254.7    151.6234.7    137.020.0    36.40.80non - dm / ip -230.1    108.7212.4    98.217.7    31.9data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0  mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation", ". dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011    2,430  bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081    2,139  bpm x mmhg ( table  4 , p  =  0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1 \n p value*dm / ip+22,841    4,50025,853    4,7423,011    2,4300.01non - dm / ip + 23,094    5,31225,175    5,4852,081    2,139 dm / ip -22,705    4,00022,124    3,686580    2,2500.43non - dm / ip -23,910    5,38022,869    5,3511,050    2,027data are expressed as means    standard deviation .", "dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means    standard deviation", ". dm stands for diabetes mellitus , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *", "the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table  5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip", "+ hr ( baseline)79.0    15.186.3    17.07.3    11.0hr ( peak)139.0   ", "15.1142.1    14.73.2    5.6bp ( baseline)156.5    18.2149.7    19.36.8    12.6bp ( peak)192.0    25.0191.8    23.90.16    15.4 dm / ip -hr ( baseline)79.0    14.784.4    14.95.4    8.0hr ( peak)141.7    14.3143.0    13.51.3    7.4bp ( baseline)153.3    22.6147.1    19.76.2    14.7bp ( peak)183.7    23.2180.6    22.23.12   ", "14.7non - dm / ip + hr ( baseline)78.0    13.187.1    13.99.1    7.9hr ( peak)139.6    15.4144.3    14.74.7    5.7bp ( baseline)146.7    20.6138.7    18.38.0    11.3bp ( peak)193.5    23.6190.2    24.53.3    12.4non - dm / ip -hr ( baseline)82.5    12.987.1    12.64.6    7.1hr ( peak)139.3   ", "12.4139.6    13.00.35    5.2bp ( baseline)158.6    26.2147.9    26.110.7    14.6bp ( peak)204.6    25.3194.8    28.18.9    16.5data are expressed as means    standard deviation .", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means    standard deviation .", "dm stands for diabetic patients , non - dm nondiabetic patients , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test"], ["the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20    39  s versus 17    36  s , respectively , p  =  0.60 ) .", "the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50  % versus 63  % of patients demonstrated improvement in arrhythmias , respectively ; p  =  0.41 ) .", "the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5  % versus 48.3  % , respectively , p  =  0.41 ) ."], ["when the total group of patients ( n  =  174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table  6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n  =  122)ip - ( n  =  52 ) \n p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3    42.6118.9    34.10.72a1c6.63    1.66.32    1.20.25data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means    standard deviation and as absolute and relative frequencies \n dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip  +  ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig", "3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip  =  ischemic preconditioning", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .", "ip  =  ischemic preconditioning ; q  =  quartile ( s ) .", "x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia"], ["considering that diabetes is an independent risk factor for the occurrence of major cardiovascular events and mortality [ 5 , 6 ] , it is reasonable to consider that diabetes might damage the protective mechanism of ip in cad patients , leading to worse outcomes .", "however , in this study , the presence of type 2 dm did not have any deleterious effects on the myocardial protective mechanism termed ischemic preconditioning . in this context", ", our study showed that patients with type 2 dm demonstrated ip in similar frequency and intensity compared with nondiabetic patients and , interestingly , our data indicated an improvement in ischemic parameters associated with diabetes .", "the analyses of the data showed a better ischemic response evaluated by rpp in diabetic patients .", "thus , this study adds clinical information on some questions that have emerged from contradictory experimental studies .", "the improvement in myocardial oxygen consumption , observed by the analysis of rpp , was more pronounced in patients with compared to those without diabetes , indicating better adaptation of the myocardium of diabetic patients after an ischemic insult . moreover , diabetic patients had an improvement in t-1.0  mm greater than that in nondiabetic patients , although it did not reach statistical significance .", "analyzes of the total exercise time also confirmed the main results of the study , as times were similar among patients with and without dm .", "similarly , the st - segment deviation morphology did not differ among diabetic and nondiabetic patients . because myocardial ischemia is a frequent cause of arrhythmias during treadmill stress tests", ", we also assessed the occurrence and complexity of arrhythmias and their improvement during set .", "thus , the improvement in the occurrence of arrhythmias also confirmed the main findings of the study , as the occurrence did not differ among diabetic and nondiabetic patients .", "interestingly , analyzes of the percentage of patients who demonstrated ip among different quartiles of fasting glycemia and glycosylated hemoglobin showed similar rates of ip demonstration .", "this analyzes infer that the differences in the intensity of glycemic control in our population did not prevent the demonstration of this cardioprotective phenomenon .", "however , despite our consistent findings , the information from this study is contrary to lee s et al .   and ishihara s et al . , who have also studied ip in humans .", "studied diabetic and nondiabetic cad patients during percutaneous coronary interventions , and evaluated the action of hypoglycemic agents on ip .", "patients underwent consecutive balloon coronary inflations . during the second sequential balloon inflation , patients had less thoracic discomfort , less myocardial lactate production , and lower st - segment deviation .", "moreover , the authors observed that diabetic patients treated with glimepiride had higher lactate production compared to nondiabetic patients treated with glimepiride . despite this finding , an important limitation of this study is that there was not a direct comparison of diabetic and nondiabetic patients who had no drug interference in ip evaluation .", "studied patients hospitalized due to an acute myocardial infarction and evaluated the effects of preinfarct angina , on the release of cardiac markers of necrosis , ventricular function , and in - hospital death and compared the results among diabetic and nondiabetic patients .", "the authors found that in the nondiabetic population , patients with preinfarct angina had a lower release of cardiac markers of necrosis , better recovery of ventricular function , and lower in - hospital mortality , compared to patients with no preinfarct angina . on the other hand , when they analyzed diabetic patients , these variables were not different among patients with and without preinfarct angina .", "thus , the authors inferred that diabetes prevented the appearance of ip . despite their findings ,", "first , this was a retrospective study , which included a small number of diabetic ( n  =  121 ) compared to nondiabetic patients ( n  =  490 ) .", "many studies have shown that some of these drugs may block ip , and it has been speculated that this interference with ip mechanisms may partially explain the worse prognosis of diabetic patients hospitalized due to an acute myocardial infarction . on the other hand ,", "other experimental studies that evaluated  in vitro  human myocardial tissue [ 41 , 42 ] showed results that match those of the present study .", "they evaluated the release of cardiac biomarkers of necrosis and the percentage of tissue viability . among other findings", ", the authors found a similar intensity in myocardial protection among diabetic and nondiabetic patients .", "additionally , cleveland et al .   evaluated the contractile function of isolated right atrial trabeculae from cad patients , resected during coronary artery bypass graft surgery .", "they showed that the diabetic group that underwent the preconditioning stimuli had similar improvement in contractile function compared to the nondiabetic group .", "moreover , a small study conducted by bilinska and colleagues   with diabetic patients treated with glibenclamide , gliclazide , and diet compared the demonstration of ip in these groups with that in nondiabetic patients .", "besides the main findings of the study , which were related to the effects of sulfonylureas in the warm - up phenomenon , they showed that the group of diabetic patients on diet ( n  =  15 ) had similar improvement in ischemic parameters compared to nondiabetic patients ( n  =  17 ) .", "of note , this was a secondary result , and the small sample size did not permit to make a definitive conclusion .", "this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .", "the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control .", ", the response of the myocardium to an ischemic insult may probably play an important role in prognosis .", "one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .", "contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients ."], ["this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .", "the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control ."], [", the response of the myocardium to an ischemic insult may probably play an important role in prognosis .", "one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .", "contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients ."], ["in this study , diabetes mellitus did not substantially affect myocardial ip in symptomatic cad patients ."]]}
{"article_id": "PMC5103823", "article_text": ["arrhythmias can develop from intraatrial invasion , which can also lead to massive pericardial effusion and tamponade .", "if the tumor mass is located in the left atrium or ventricle , embolic infarction might occur .", "a 55-year - old man presented signs of progressive cardiac decompensation during the last 4 months due to tumorous obstruction of the mitral valve . in 2014 the patient underwent resection of a suspected myxoma ( 824124 mm ) in the left atrium .", "however , in the histological workup , the tumor mass , resected from the posterior mitral annulus , was diagnosed as chondrosarcoma . in 2014 ,", "our colleagues in the referring center noticed nodal calcification in the myocardium and the mitral valve .", "the tumor board in 2014 decided on palliative care with poor sensitivity to chemotherapy and radiation and high risk for organ toxicity .", "ct scan and magnetic resonance imaging ( mri ) showed no signs of malignancy outside the cardiac tissue , and the chondrosarcoma found was the primary tumor .", "during the course of 12 months , the patient was in a clinically stable state with combined mild mitral valve regurgitation and stenosis and good left ventricular pump function , with ejection fraction ( ef ) 63% . in january 2016 ,", "the patient first presented with recurring tumor mass on the posterior mitral leaflet ( pml ) max .", "23 mm , with moderately decreasing pump function ( ef 58% ) . in april 2016", ", the patient showed increasing peripheral edema and orthopnea , with no signs of angina pectoris , and nyha iii - iv .", "the patient presented absolute arrhythmia with atrial fibrillation , pulse 109/min , and blood pressure 82/52 mmhg .", "echocardiography showed pleural effusions on both sides and distended vena cava inferior and hepatic veins .", "upon admission to our center , the patient presented a 403532 mm mass , originating from the pml ( figure 1 ) .", "a cardio mri could not be performed , as the patient was unable to lie down flat due to major orthopnea .", "instead , we decided to perform a cardio ct , which showed atrial roof and interatrial septum invasion .", "it was not perfectly clear on the ct scan whether the tumor was infiltrating the ascending aorta .", "a cerebral scan showed no signs of cerebral embolisms or metastasis . due to clinical instability", "serum creatinine levels at that point were 1.56 mg / dl and bilirubin 1.10 mg / dl .", "after careful review of the scan images , we decided on the implantation of a total artificial heart , as the sarcoma had already invaded the whole atrium and the complete mitral valve annulus .", "a mitral valve replacement was not feasible because there would be no myocardium or endocardium to fixate the stitches in .", "implantation of a self - expanding stent valve would also require an intact mitral valve annulus .", "furthermore , autotransplantation was rejected for the same reasons . the severe mitral valve stenosis precluded use of a left ventricular assist device ( lvad ) or biventricular assist device ( bivad ) . the new approach introduced by bruckner et al .  ", "has never been performed in our center and needs experience in the surgical technique as the construction of the new atria presents major challenges because they easily collapse when not prepared properly .", "we decided on the syncardia total artificial heart ( tah ) ( syncardia , tucson , az ) .", "as the ct scan showed no metastases and the sarcoma presented as a recurrence in the left atrium , we hoped that the patient could be listed for htx following recovery after tah implantation . in a similar setting , reich et al .", "successfully transplanted a patient after tah implantation , with an occult intracardial malignancy , after a cancer - free period of 14 months . before the operation", ", the patient and next of kin were informed that it would be an ultimo ratio intervention because we could not 100% foresee the invasion of the myocardium and the intraoperative circumstances .", "zhang et al . demonstrated a 50% survival rate after initial surgical treatment of primary cardiac sarcomas , depending on histological grade of the tumor .", "surgical treatment is the most common approach to improve survival . even as a palliative measure , reducing the tumor mass through an operation relieves orthopnea and ameliorates signs of decompensation if the patient stabilizes after the intervention . after re - sternotomy", "following aortic cross - clamping , the apex , ascending aorta , and pulmonary trunk were distally resected , preserving both the aortic and pulmonary valve .", "the left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve ( figure 2 ) .", "due to extensive left atrial resection , a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture .", "functional valve movements were tested and tah chambers were put in the av - annuli , followed by de - aeration of both tah chambers .", "to allow postoperative tissue edema , we decided to leave the thorax open . after sufficient volume substitution", ", the patient was transferred to the icu under moderate catecholamine support and no ventilation .", "five days after the initial operation , we planned a secondary thorax closure . during the intervention", "cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn , with ongoing hemorrhagic transformation .", "neurological examination showed the patient was comatose , with slight head movement to the left side . a right - sided facial paralysis was diagnosed . under continuous anticoagulation for tah , there was a major risk for secondary bleeding due to the size of the media infarction .", ". the patient showed signs of prolonged low - output syndrome with beginning multiorgan failure .", "neurological status was deemed unfavorable and after consultation with the family , the patient died on postoperative day 16 after withdrawal of support .", "histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components , with dystrophic calcifications .", "chromogenic in situ hybridization could not confirm an amplification of mdm2-gene , and translocation of syt - gene locus was ruled out .", "hence , the classification was undifferentiated high - grade spindle cell sarcoma , grade 2 using the french fdration nationale des centres de lutte contre le cancer ( fnclcc ) grading .", "cardiopulmonary bypass was established via the ascending aorta and venae cavae cannulation . following aortic cross - clamping , the apex , ascending aorta , and pulmonary trunk", "the left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve ( figure 2 ) .", "the atrial cuffs were prepared and broad felt strips for suture support were placed . due to", "extensive left atrial resection , a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture .", "functional valve movements were tested and tah chambers were put in the av - annuli , followed by de - aeration of both tah chambers .", "to allow postoperative tissue edema , we decided to leave the thorax open . after sufficient volume substitution", ", the patient was transferred to the icu under moderate catecholamine support and no ventilation .", "five days after the initial operation , we planned a secondary thorax closure . during the intervention", "cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn , with ongoing hemorrhagic transformation .", "neurological examination showed the patient was comatose , with slight head movement to the left side .", "under continuous anticoagulation for tah , there was a major risk for secondary bleeding due to the size of the media infarction .", "neurological status was deemed unfavorable and after consultation with the family , the patient died on postoperative day 16 after withdrawal of support .", "histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components , with dystrophic calcifications .", "chromogenic in situ hybridization could not confirm an amplification of mdm2-gene , and translocation of syt - gene locus was ruled out .", "hence , the classification was undifferentiated high - grade spindle cell sarcoma , grade 2 using the french fdration nationale des centres de lutte contre le cancer ( fnclcc ) grading .", "chondrosarcomas are cartilaginous tissue sarcomas . our patient was first diagnosed with primary cardiac chondrosarcoma .", "after initial resection , relapse of the cardiac mass in the left atrium was diagnosed as early as 14 months later .", "after excision of the heart for tah implantation , our pathologists found an undifferentiated high - grade spindle cell sarcoma , fnclcc g2 . with histological grading through the fnclcc classification , survival , by major correlation", "cell differentiation , mitotic rate , and percentage of necrosis are evaluated in a systematic score .", "since the 4 edition of the who classification of tumors of soft tissue and bone , there is a new category of tumors that can not be classified in earlier - defined categories .", "there were no signs of assist malfunction during icu stay . with atrial infiltration and major excision of healthy tissue , implantation of tah", "traditional bivad implantation as bridge - to - transplant as a short - term solution was not an option due to severe mitral valve stenosis from the atrial mass and mitral valve infiltration .", "the left atrial tumor was successfully removed . like bruckner et al . , we acknowledge that tah implantation as bridge - to - transplantation is surgically possible even with atrial wall involvement .", "surgical treatment is still the criterion standard for localized cardiac sarcomas , with various perioperative survival rates , ranging from 6 to 17 months . with complete resection of the tumor mass", "there is no valid data on the benefits of adjuvant chemotherapy or radiation therapy on malignant cardiac soft - tissue sarcomas", ". limitations of radiotherapy are cardiac sensitivity to radiation injury and adverse effects on global cardiac functions .", "our patient showed good recovery after initial gross resection in 2014 for 14 months without any adjuvant therapy .", "bleeding complications due to continuous anticoagulation for device functioning remains a major risk factor in circulatory assist device programs . in a study in 13 patients with tah , ramirez et al . found that bleeding complications occur generally between postoperative days 1 and 3 .", "patients with mechanical assist devices are prone to suffer from thrombotic events [ 1316 ] , device malfunctioning , wound infections , and bleeding complications .", "this intervention was the second attempt to help the patient with a long - term solution after first recurrence of an atrial tumor .", "the surgical options and possibilities were discussed at length with the patient and his family before the tah implantation .", "a multidisciplinary team and truthfully informed next of kin are hugely important when taking decisions in such a high - risk case ."], "abstract_text": ["<S> backgroundtotal artificial heart ( tah ) implantation in patients with aggressive tumor infiltration of the heart can be challenging.case reportwe report on a patient with a rare primary undifferentiated high - grade spindle cell sarcoma of the mitral valve and in the left atrium , first diagnosed in 2014 . </S>", "<S> the referring center did a first resection in 2014 . </S>", "<S> in the course of 17 months , computer tomography ( ct ) scan again showed massive invasion of the mitral valve and left atrium . </S>", "<S> partial resection and mitral valve replacement was not an option . </S>", "<S> we did a subtotal heart excision with total artificial heart implantation . in this report </S>", "<S> we discuss complications , risk factors , and perioperative management of this patient.conclusionspatients with aggressive tumors of the heart can be considered for tah implantation . </S>"], "labels": null, "section_names": ["Background", "Case Report", "Operative technique", "Postoperative course", "Discussion", "Conclusions"], "sections": [["most malignant heart tumors are sarcomas : myxosarcoma , liposarcoma , angiosarcoma , fibrosarcoma , leiomyosarcoma , osteosarcoma , synovial sarcoma , rhabdomyosarcoma , undifferentiated sarcoma , reticulum cell sarcoma , neurofibrosarcoma , and malignant fibrous histiocytoma .", "arrhythmias can develop from intraatrial invasion , which can also lead to massive pericardial effusion and tamponade .", "if the tumor mass is located in the left atrium or ventricle , embolic infarction might occur ."], ["a 55-year - old man presented signs of progressive cardiac decompensation during the last 4 months due to tumorous obstruction of the mitral valve . in 2014 the patient underwent resection of a suspected myxoma ( 824124 mm ) in the left atrium .", "however , in the histological workup , the tumor mass , resected from the posterior mitral annulus , was diagnosed as chondrosarcoma . in 2014 ,", "our colleagues in the referring center noticed nodal calcification in the myocardium and the mitral valve .", "the tumor board in 2014 decided on palliative care with poor sensitivity to chemotherapy and radiation and high risk for organ toxicity .", "ct scan and magnetic resonance imaging ( mri ) showed no signs of malignancy outside the cardiac tissue , and the chondrosarcoma found was the primary tumor .", "during the course of 12 months , the patient was in a clinically stable state with combined mild mitral valve regurgitation and stenosis and good left ventricular pump function , with ejection fraction ( ef ) 63% . in january 2016 ,", "the patient first presented with recurring tumor mass on the posterior mitral leaflet ( pml ) max .", "23 mm , with moderately decreasing pump function ( ef 58% ) . in april 2016", ", the patient showed increasing peripheral edema and orthopnea , with no signs of angina pectoris , and nyha iii - iv .", "the patient presented absolute arrhythmia with atrial fibrillation , pulse 109/min , and blood pressure 82/52 mmhg .", "echocardiography showed pleural effusions on both sides and distended vena cava inferior and hepatic veins .", "upon admission to our center , the patient presented a 403532 mm mass , originating from the pml ( figure 1 ) .", "a cardio mri could not be performed , as the patient was unable to lie down flat due to major orthopnea .", "instead , we decided to perform a cardio ct , which showed atrial roof and interatrial septum invasion .", "it was not perfectly clear on the ct scan whether the tumor was infiltrating the ascending aorta .", "a cerebral scan showed no signs of cerebral embolisms or metastasis . due to clinical instability", "serum creatinine levels at that point were 1.56 mg / dl and bilirubin 1.10 mg / dl .", "after careful review of the scan images , we decided on the implantation of a total artificial heart , as the sarcoma had already invaded the whole atrium and the complete mitral valve annulus .", "a mitral valve replacement was not feasible because there would be no myocardium or endocardium to fixate the stitches in .", "implantation of a self - expanding stent valve would also require an intact mitral valve annulus .", "furthermore , autotransplantation was rejected for the same reasons . the severe mitral valve stenosis precluded use of a left ventricular assist device ( lvad ) or biventricular assist device ( bivad ) . the new approach introduced by bruckner et al .  ", "has never been performed in our center and needs experience in the surgical technique as the construction of the new atria presents major challenges because they easily collapse when not prepared properly .", "we decided on the syncardia total artificial heart ( tah ) ( syncardia , tucson , az ) .", "as the ct scan showed no metastases and the sarcoma presented as a recurrence in the left atrium , we hoped that the patient could be listed for htx following recovery after tah implantation . in a similar setting , reich et al .", "successfully transplanted a patient after tah implantation , with an occult intracardial malignancy , after a cancer - free period of 14 months . before the operation", ", the patient and next of kin were informed that it would be an ultimo ratio intervention because we could not 100% foresee the invasion of the myocardium and the intraoperative circumstances .", "zhang et al . demonstrated a 50% survival rate after initial surgical treatment of primary cardiac sarcomas , depending on histological grade of the tumor .", "surgical treatment is the most common approach to improve survival . even as a palliative measure , reducing the tumor mass through an operation relieves orthopnea and ameliorates signs of decompensation if the patient stabilizes after the intervention . after re - sternotomy", "following aortic cross - clamping , the apex , ascending aorta , and pulmonary trunk were distally resected , preserving both the aortic and pulmonary valve .", "the left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve ( figure 2 ) .", "due to extensive left atrial resection , a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture .", "functional valve movements were tested and tah chambers were put in the av - annuli , followed by de - aeration of both tah chambers .", "to allow postoperative tissue edema , we decided to leave the thorax open . after sufficient volume substitution", ", the patient was transferred to the icu under moderate catecholamine support and no ventilation .", "five days after the initial operation , we planned a secondary thorax closure . during the intervention", "cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn , with ongoing hemorrhagic transformation .", "neurological examination showed the patient was comatose , with slight head movement to the left side . a right - sided facial paralysis was diagnosed . under continuous anticoagulation for tah , there was a major risk for secondary bleeding due to the size of the media infarction .", ". the patient showed signs of prolonged low - output syndrome with beginning multiorgan failure .", "neurological status was deemed unfavorable and after consultation with the family , the patient died on postoperative day 16 after withdrawal of support .", "histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components , with dystrophic calcifications .", "chromogenic in situ hybridization could not confirm an amplification of mdm2-gene , and translocation of syt - gene locus was ruled out .", "hence , the classification was undifferentiated high - grade spindle cell sarcoma , grade 2 using the french fdration nationale des centres de lutte contre le cancer ( fnclcc ) grading ."], ["cardiopulmonary bypass was established via the ascending aorta and venae cavae cannulation . following aortic cross - clamping , the apex , ascending aorta , and pulmonary trunk", "the left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve ( figure 2 ) .", "the atrial cuffs were prepared and broad felt strips for suture support were placed . due to", "extensive left atrial resection , a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture .", "functional valve movements were tested and tah chambers were put in the av - annuli , followed by de - aeration of both tah chambers .", "to allow postoperative tissue edema , we decided to leave the thorax open . after sufficient volume substitution", ", the patient was transferred to the icu under moderate catecholamine support and no ventilation ."], ["five days after the initial operation , we planned a secondary thorax closure . during the intervention", "cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn , with ongoing hemorrhagic transformation .", "neurological examination showed the patient was comatose , with slight head movement to the left side .", "under continuous anticoagulation for tah , there was a major risk for secondary bleeding due to the size of the media infarction .", "neurological status was deemed unfavorable and after consultation with the family , the patient died on postoperative day 16 after withdrawal of support .", "histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components , with dystrophic calcifications .", "chromogenic in situ hybridization could not confirm an amplification of mdm2-gene , and translocation of syt - gene locus was ruled out .", "hence , the classification was undifferentiated high - grade spindle cell sarcoma , grade 2 using the french fdration nationale des centres de lutte contre le cancer ( fnclcc ) grading ."], ["chondrosarcomas are cartilaginous tissue sarcomas . our patient was first diagnosed with primary cardiac chondrosarcoma .", "after initial resection , relapse of the cardiac mass in the left atrium was diagnosed as early as 14 months later .", "after excision of the heart for tah implantation , our pathologists found an undifferentiated high - grade spindle cell sarcoma , fnclcc g2 . with histological grading through the fnclcc classification , survival , by major correlation", "cell differentiation , mitotic rate , and percentage of necrosis are evaluated in a systematic score .", "since the 4 edition of the who classification of tumors of soft tissue and bone , there is a new category of tumors that can not be classified in earlier - defined categories .", "there were no signs of assist malfunction during icu stay . with atrial infiltration and major excision of healthy tissue , implantation of tah", "traditional bivad implantation as bridge - to - transplant as a short - term solution was not an option due to severe mitral valve stenosis from the atrial mass and mitral valve infiltration .", "the left atrial tumor was successfully removed . like bruckner et al . , we acknowledge that tah implantation as bridge - to - transplantation is surgically possible even with atrial wall involvement .", "surgical treatment is still the criterion standard for localized cardiac sarcomas , with various perioperative survival rates , ranging from 6 to 17 months . with complete resection of the tumor mass", "there is no valid data on the benefits of adjuvant chemotherapy or radiation therapy on malignant cardiac soft - tissue sarcomas", ". limitations of radiotherapy are cardiac sensitivity to radiation injury and adverse effects on global cardiac functions .", "our patient showed good recovery after initial gross resection in 2014 for 14 months without any adjuvant therapy .", "bleeding complications due to continuous anticoagulation for device functioning remains a major risk factor in circulatory assist device programs . in a study in 13 patients with tah , ramirez et al . found that bleeding complications occur generally between postoperative days 1 and 3 .", "patients with mechanical assist devices are prone to suffer from thrombotic events [ 1316 ] , device malfunctioning , wound infections , and bleeding complications ."], ["this intervention was the second attempt to help the patient with a long - term solution after first recurrence of an atrial tumor .", "the surgical options and possibilities were discussed at length with the patient and his family before the tah implantation .", "a multidisciplinary team and truthfully informed next of kin are hugely important when taking decisions in such a high - risk case ."]]}
{"article_id": "PMC5034357", "article_text": ["cannabis is a prominent political , health , and law enforcement issue in north america that is currently receiving a great deal of attention .", "as we watch the effects of the legalization of cannabis in colorado , washington , alaska , oregon , and the district of columbia , other jurisdictions are debating the future of this illicit drug .", "how can we prevent commercialization promoting increased rates of use within a legalization model ? where does marijuana for medical purposes fit into the picture , if at all ?", "a critical piece of information often missing from these debates or not addressed adequately is the scientific evidence about the effects of cannabis use on a person 's overall health .", "this concern is even more pressing when it comes to the cognitive , physical , and mental health harms to adolescents who use cannabis .", "it is critical we ask these questions because adolescence is a time of rapid development that helps lay the foundation for success later in life .", "conversely , it can also set the stage for experiencing challenges in adulthood , as youth are disproportionately more likely than adults to experience greater harms from drug use .", "their brains are undergoing rapid and extensive development that can be negatively affected by cannabis use .", "there is certainly cause for concern about the amount and frequency of cannabis use among youth . according to the 2014 monitoring the future ( mtf ) survey in the us , 35.1% of high school seniors reported pastyear use of cannabis , with 5.8% reporting daily or near daily use .", "comparable prevalence rates are reported in canada , with 22% of 1519yearolds and 26% of 2024yearolds reporting pastyear use of cannabis in 2013 according to the canadian tobacco , alcohol and drugs survey . moreover", ", a 2013 unicef report noted that canada 's youth are the highest users of cannabis when compared to students in other developed countries .", "the mtf survey also indicates a growing perception among young people that cannabis is a relatively harmless drug , and there is strong evidence that perception of harm is inversely related to rates of use . knowing that youth are disproportionately heavy users of cannabis", ", what specific concerns should this raise about short and longterm consequences and how do we incorporate these concerns in the debate ?", "recent evidence shows that early and frequent use of cannabis has been linked with deficits in shortterm cognitive functioning , reduced iq , impaired school performance , and increased risk of leaving school early  all of which can have significant consequences on a young person 's life trajectory.1 of note , the impact of cannabis use on iq has been the topic of recent debate among experts in the field and further research aimed at understanding this relationship will provide much needed clarity .", "heavy cannabis use in adolescence is also a risk factor for psychosis , a risk that is heightened if users have a preexisting vulnerability to that condition.1 cannabis use can also lead to dependence , with evidence from longitudinal studies in the us estimating the risk of developing cannabis dependence to be one in six among those users who initiated in adolescence,2 a rate higher than that reported among adults .", "recent data from the 2012 canadian community health survey reveal that more than one in 20 young canadians aged 1524 met the criteria for cannabis abuse or dependence during the past year . there is also growing evidence from the human literature that early and frequent use of cannabis can alter structural aspects of the developing brain , including those that are responsible for memory , decisionmaking , and executive functioning.1 however , we do not yet know the full extent of the impact of early cannabis use on changes in brain function and behavior of young people , which underscores the need for further investment in research in this area . beyond longerterm consequences , cannabis can also acutely impair cognitive and motor functions like perception , coordination , and balance for hours after use .", "this impairment presents a safety hazard for drivers getting behind the wheel , as well as for other motorists and pedestrians .", "in fact , the risk of car crashes doubles for drivers who get behind the wheel after using cannabis and this risk is further elevated if cannabis use is combined with other substances , most notably alcohol .", "recent data from the national fatality database indicate that cannabis was the most common illicit drug present among fatally injured drivers aged 1524 in canada between 2000 and 2010 .", "the drug abuse warning network , which monitors the health impact of drugs in the us , estimated that in 2011 , there were nearly 456,000 drugrelated emergency department visits in which cannabis use was mentioned in the medical record .", "data from the canadian institute for health information reveal that from 2006 to 2011 , youth aged 1524 spent the largest number of days in a hospital for a primary diagnosis of mental and behavioral disorders due to the use of cannabinoids . furthermore , the number of days spent in a hospital increased by almost 40% during the same time period.3 \n  these findings are particularly troubling because recent research from canada has revealed that young people are confused about cannabis and do not have the knowledge they need about the risks associated with this drug to make more informed decisions.4 some have expressed questionable beliefs about the effects of cannabis , indicating that it helps to improve their focus at school and can prevent  or even cure  cancer .", "youth also expressed mixed beliefs as to whether cannabis improves or impairs driving performance , and felt that cannabis and driving was not as dangerous as drunk driving .", "youth talked about how cannabis is natural and so they did not really think of it as a drug .", "the findings from this reviewed research highlight the complexity of the issues surrounding youth consumption of cannabis . youth are confused with the mixed messages they are receiving from discussions ranging from legalization to the use of cannabis for medical purposes , which points to the need for a coordinated , comprehensive , factual , and consistent approach to increasing awareness of this drug and its impact .", "as primary care is an important intervention point for youth experiencing harms related to cannabis , there is an urgent need to develop practical , evidenceinformed tools to assist healthcare providers in the early identification of cannabis use and cannabis dependence , as well as tools to deliver interventions to treat potential problems .", "examples of such tools may include screening instruments to screen for cannabis use that warrants clinical intervention , as well as brief intervention strategies aimed at addressing the problematic substance use revealed during the screening process .", "there is also an urgent need for further research aimed at improving the understanding of differences between the short and longterm effects of cannabis use , particularly on the developing adolescent brain .", "such research should include mechanistic studies regarding alteration of cellular structure and function related to cannabis to determine the impact on adolescent brain development .", "of particular concern are the impacts of the dramatic increase in concentrations of tetrahydrocannabinol ( thc ) , the main psychoactive ingredient of the cannabis plant , over the past 25 years .", "the average potency of federally seized cannabis in the us has steadily increased from 3.5% in 1985 to 13.2% in 2012.5 in light of such an increase in potency , some historical findings about the health and developmental effects of cannabis use might not be relevant when trying to predict effects on contemporary users .", "more research is also needed to shed light on the influence of cannabis policy on public health , public safety , and other outcomes .", "the current understanding of the impact of cannabis policy on market forces , such as healthcare costs , lost productivity costs , and tax revenues , for example , is very limited , as is overall understanding of how perception , use , and outcomes interrelate around this drug . in an effort to help  clear the smoke ,  the canadian centre on substance abuse is currently undertaking a comprehensive review and synthesis of the literature on the effects of cannabis use during adolescence that will highlight key areas for action in policy , practice , and research", ". this muchneeded work will provide clarity to misperceptions about cannabis , and make sense of some conflicting evidence about the effects of cannabis use and the impact of early or regular use during a time when the brain is still developing . by integrating neuroscience with the behavioral and social context of cannabis use by youth", ", this report will provide a muchneeded resource for those working with youth and those involved in making decisions about policies , programs , or practices related to cannabis .", "aj porathwaller has provided expert testimony at the standing committee on health 's ( hesa ) recent report on marijuana 's health risks and harms , october 2014 ."], "abstract_text": ["<S> cannabis is the most commonly used illicit substance among youth . </S>", "<S> recent policy developments and ongoing debate related to this drug underscore the urgent need to  clear the smoke  and better understand what the scientific evidence says about the health and behavioral effects of cannabis use , particularly on youth whose brains are undergoing rapid and extensive development . </S>"], "labels": null, "section_names": ["Clearing the smoke", "A call to action", "Conflict of Interest"], "sections": [["cannabis is a prominent political , health , and law enforcement issue in north america that is currently receiving a great deal of attention .", "as we watch the effects of the legalization of cannabis in colorado , washington , alaska , oregon , and the district of columbia , other jurisdictions are debating the future of this illicit drug .", "how can we prevent commercialization promoting increased rates of use within a legalization model ? where does marijuana for medical purposes fit into the picture , if at all ?", "a critical piece of information often missing from these debates or not addressed adequately is the scientific evidence about the effects of cannabis use on a person 's overall health .", "this concern is even more pressing when it comes to the cognitive , physical , and mental health harms to adolescents who use cannabis .", "it is critical we ask these questions because adolescence is a time of rapid development that helps lay the foundation for success later in life .", "conversely , it can also set the stage for experiencing challenges in adulthood , as youth are disproportionately more likely than adults to experience greater harms from drug use .", "their brains are undergoing rapid and extensive development that can be negatively affected by cannabis use .", "there is certainly cause for concern about the amount and frequency of cannabis use among youth . according to the 2014 monitoring the future ( mtf ) survey in the us , 35.1% of high school seniors reported pastyear use of cannabis , with 5.8% reporting daily or near daily use .", "comparable prevalence rates are reported in canada , with 22% of 1519yearolds and 26% of 2024yearolds reporting pastyear use of cannabis in 2013 according to the canadian tobacco , alcohol and drugs survey . moreover", ", a 2013 unicef report noted that canada 's youth are the highest users of cannabis when compared to students in other developed countries .", "the mtf survey also indicates a growing perception among young people that cannabis is a relatively harmless drug , and there is strong evidence that perception of harm is inversely related to rates of use . knowing that youth are disproportionately heavy users of cannabis", ", what specific concerns should this raise about short and longterm consequences and how do we incorporate these concerns in the debate ?", "recent evidence shows that early and frequent use of cannabis has been linked with deficits in shortterm cognitive functioning , reduced iq , impaired school performance , and increased risk of leaving school early  all of which can have significant consequences on a young person 's life trajectory.1 of note , the impact of cannabis use on iq has been the topic of recent debate among experts in the field and further research aimed at understanding this relationship will provide much needed clarity .", "heavy cannabis use in adolescence is also a risk factor for psychosis , a risk that is heightened if users have a preexisting vulnerability to that condition.1 cannabis use can also lead to dependence , with evidence from longitudinal studies in the us estimating the risk of developing cannabis dependence to be one in six among those users who initiated in adolescence,2 a rate higher than that reported among adults .", "recent data from the 2012 canadian community health survey reveal that more than one in 20 young canadians aged 1524 met the criteria for cannabis abuse or dependence during the past year . there is also growing evidence from the human literature that early and frequent use of cannabis can alter structural aspects of the developing brain , including those that are responsible for memory , decisionmaking , and executive functioning.1 however , we do not yet know the full extent of the impact of early cannabis use on changes in brain function and behavior of young people , which underscores the need for further investment in research in this area . beyond longerterm consequences , cannabis can also acutely impair cognitive and motor functions like perception , coordination , and balance for hours after use .", "this impairment presents a safety hazard for drivers getting behind the wheel , as well as for other motorists and pedestrians .", "in fact , the risk of car crashes doubles for drivers who get behind the wheel after using cannabis and this risk is further elevated if cannabis use is combined with other substances , most notably alcohol .", "recent data from the national fatality database indicate that cannabis was the most common illicit drug present among fatally injured drivers aged 1524 in canada between 2000 and 2010 .", "the drug abuse warning network , which monitors the health impact of drugs in the us , estimated that in 2011 , there were nearly 456,000 drugrelated emergency department visits in which cannabis use was mentioned in the medical record .", "data from the canadian institute for health information reveal that from 2006 to 2011 , youth aged 1524 spent the largest number of days in a hospital for a primary diagnosis of mental and behavioral disorders due to the use of cannabinoids . furthermore , the number of days spent in a hospital increased by almost 40% during the same time period.3 \n  these findings are particularly troubling because recent research from canada has revealed that young people are confused about cannabis and do not have the knowledge they need about the risks associated with this drug to make more informed decisions.4 some have expressed questionable beliefs about the effects of cannabis , indicating that it helps to improve their focus at school and can prevent  or even cure  cancer .", "youth also expressed mixed beliefs as to whether cannabis improves or impairs driving performance , and felt that cannabis and driving was not as dangerous as drunk driving .", "youth talked about how cannabis is natural and so they did not really think of it as a drug ."], ["the findings from this reviewed research highlight the complexity of the issues surrounding youth consumption of cannabis . youth are confused with the mixed messages they are receiving from discussions ranging from legalization to the use of cannabis for medical purposes , which points to the need for a coordinated , comprehensive , factual , and consistent approach to increasing awareness of this drug and its impact .", "as primary care is an important intervention point for youth experiencing harms related to cannabis , there is an urgent need to develop practical , evidenceinformed tools to assist healthcare providers in the early identification of cannabis use and cannabis dependence , as well as tools to deliver interventions to treat potential problems .", "examples of such tools may include screening instruments to screen for cannabis use that warrants clinical intervention , as well as brief intervention strategies aimed at addressing the problematic substance use revealed during the screening process .", "there is also an urgent need for further research aimed at improving the understanding of differences between the short and longterm effects of cannabis use , particularly on the developing adolescent brain .", "such research should include mechanistic studies regarding alteration of cellular structure and function related to cannabis to determine the impact on adolescent brain development .", "of particular concern are the impacts of the dramatic increase in concentrations of tetrahydrocannabinol ( thc ) , the main psychoactive ingredient of the cannabis plant , over the past 25 years .", "the average potency of federally seized cannabis in the us has steadily increased from 3.5% in 1985 to 13.2% in 2012.5 in light of such an increase in potency , some historical findings about the health and developmental effects of cannabis use might not be relevant when trying to predict effects on contemporary users .", "more research is also needed to shed light on the influence of cannabis policy on public health , public safety , and other outcomes .", "the current understanding of the impact of cannabis policy on market forces , such as healthcare costs , lost productivity costs , and tax revenues , for example , is very limited , as is overall understanding of how perception , use , and outcomes interrelate around this drug . in an effort to help  clear the smoke ,  the canadian centre on substance abuse is currently undertaking a comprehensive review and synthesis of the literature on the effects of cannabis use during adolescence that will highlight key areas for action in policy , practice , and research", ". this muchneeded work will provide clarity to misperceptions about cannabis , and make sense of some conflicting evidence about the effects of cannabis use and the impact of early or regular use during a time when the brain is still developing . by integrating neuroscience with the behavioral and social context of cannabis use by youth", ", this report will provide a muchneeded resource for those working with youth and those involved in making decisions about policies , programs , or practices related to cannabis ."], ["aj porathwaller has provided expert testimony at the standing committee on health 's ( hesa ) recent report on marijuana 's health risks and harms , october 2014 ."]]}
{"article_id": "PMC3124316", "article_text": ["hypertension ( htn ) is the most common comorbidity in the world with significant public health implications     [ 1 , 2 ] . the overall u.s .", "prevalence of hypertension among adults ages 18 years in 20052008 was 30.9% and was highest among persons ages 65 years ( 69.7% )   and non - hispanic blacks ( 44% )     .  ", "the american heart association estimates that the incurred costs of hypertension are more than $ 93.5  billion per year , and that cardiovascular disease and stroke for which htn is the predominant risk factor , account for 17% of the total annual health expenditures in the united states .", "hypertension and its sequelae , heart failure ( hf ) , are a progressive disease .", "evidence shows that less than half of patients with heart failure survive five years ( after diagnosis ) , and less than a quarter of them live ten years after their initial diagnosis .", "framingham heart study showed that hypertensive patients were more likely to develop heart failure ( 142 cases of hf detected during the first 16 years of followup ) than those who were normotensive .", "the lifetime risk for development of hf among people with blood pressure ( bp ) > 160/90  mm  hg is double that of those with bp < 140/90  mm  hg .  ", "heart failure in comparison to the most prevalent gender malignancies ( bowel cancer in men and breast cancer in women ) , was associated with worse long - term survival     .", "over a million hospitalizations , contributing to more than 250,000 deaths     , and escalating billions of dollars in health care expenditure is the norm .", "the disastrous duo , htn and hf , should be recognized , and a tailored focus on management in special populations , high - risk ethnic minority groups especially blacks who are disproportionately burdened should be developed .   among racial groups ,", "african - american adults have the highest rates ( 44% ) of hypertension in the world      and are more resistant      to treatment .  ", "in particular , black women have the highest prevalence      and the lowest blood pressure control     .  ", "the relative incidence of hf is 50% higher in african americans , 3% of whom have hf , compared with 2% of the general population     [ 13 , 14 ] .  ", "the disease occurs at an earlier age , and usually at a more advanced stage   along with increased hospitalization   and mortality .", "other causes of hf include coronary artery disease , valvular heart disease , diabetes , left ventricular hypertrophy and cardiomyopathies .  ", "overt clinical hf resulting from diastolic left ventricular dysfunction may be clinically indistinguishable from that resulting from systolic dysfunction", ". hf with preserved left ventricular function is observed in 30% to 50% of adult cases of hf .", "coronary artery disease and myocardial infarction is a principal cause of systolic left ventricular dysfunction followed by hypertension .", "a variety of neurohormonal systems , especially the renin - angiotensin aldosterone and sympathetic nervous systems are activated in response to the left ventricular dysfunction and such activation leads to abnormal ventricular remodeling . the inexorable progression to more severe stages of left ventricular dysfunction", "can be significantly reduced by effective therapy with neurohormonal blockade including angiotensin converting enzyme inhibitors ( aceis ) , beta blockers ( bbs ) , and aldosterone antagonists     .", "current understanding of the pathophysiology of hf has revolved around neurohormonal activation     [ 19 , 20 ] .  ", "this endothelial dysfunction may result from insufficient nitric oxide ( no ) secondary to either reduced endothelial production of no or to increase no inactivation [ 17 , 19 , 21 ] . in hf , a pronounced increase in angiotensin ii , with modestly reduced no is seen among whites . however , african americans with hf may exhibit greater no reduction ; conversely , angiotensin ii might be less elevated among whites .", "important studies are investigating gene variation among the races that may influence the risk of developing hypertension and its complications , such as single nucleotide polymorphisms in the genes encoding for 1-adrenergic receptors and a2c - adrenergic receptors .", "african americans who are homozygous for polymorphisms in both the 1-adrenergic and a2c - adrenergic receptor genes are at a 10-fold greater risk for heart failure , which is overexpressed in african - americans .  ", "groups with overexpression of transforming growth factor -1 ( tgf-1 ) , a cytokine involved in cardiac fibrosis and remodeling , vascular and renal disease , production of the vasoconstrictor , endothelin-1 , and stimulation of renin release , have a worse prognosis .", "other genetic polymorphisms in african americans that may influence heart failure risk include elevated production of endothelial nitric oxide synthase , aldosterone synthase , and the g protein 825-t allele .", "  there are two major approaches to classifying the patient 's hf stage or clinical severity .  ", "the most common is the new york heart association ( nyha ) classification , mainly based on functional capacity and subjective level of symptoms severity .  ", "this scheme is fluid and its class fluctuates in the same patient depending on their subjective assessment and clinical status .", "the other approach , adopted by the american college of cardiology / american heart association ( acc / aha ) guidelines , emphasizes disease progression", ".   classified as stages a , b , c , and d , each of these stages has definable characteristics and recommended interventions . to emphasize the progressive nature of hf and the need to prevent its development or progression to end - stage disease ( stage d )     [ 18 , 23 ] ,", "current hf guidelines define patients in stage a as those at increased risk for hf without evidence of structural heart disease or symptoms of hf     .", "stage a represents a critically important group because many of the risk factors including hypertension can be effectively controlled and by doing so may prevent development of cardiovascular diseases including hf .", "patients are in stage b when they present with evidence of structural heart disease but without current or prior symptoms of hf .", "stage c is recognized by the presence of structural heart disease and current or prior symptoms of hf .", "stage d patients are those who have refractory heart failure requiring frequent hospitalization and specialized interventions .", "several studies have shown the efficacy of treatment of hypertension in preventing hf [ 2427 ] .", "treatment of hypertension reduces the incidence of hf by about 50% .   in recent years", ", significant advances have been made in increasing awareness , treatment , and control of blood pressure .", "several large , randomized clinical trials have shown that specific classes of medication have mortality benefit in patients with heart failure     [ 2834 ] . in the most recent guidelines      for the diagnosis and management of hf in adults , the american college of cardiology foundation ( accf)/american heart association ( aha ) recognized the need for specific recommendations for special population .", "lifestyle modifications have been shown to reduce blood pressure , increase drug efficacy , and decrease cardiovascular risk .", "long - term primary prevention of hypertension includes weight reduction in overweight / obese individuals .", "weight loss intervention was associated with a 21% reduction in the incidence of hypertension over a 36-month period     .", "dietary practices , reducing intake of dietary sodium to < 1.5  g nacl [ 3 , 36 ] , low potassium , magnesium , and saturated fat intake , increased calcium intake     , decreased consumption of alcohol , have demonstrated greater bp reduction in blacks    ", ". these practices are most successful when reinforced with comprehensive education and counseling     [ 39 , 40 ] .", "exercise training , regular aerobic physical activity for at least 30 minutes per day most days , has been shown to reduce recurrent cardiac events in patients with lv dysfunction from ischemic heart disease and is recommended under close medical supervision . culturally cognizant and sensitive modalities should be implemented to help guide improved compliance of therapeutic lifestyle changes ( tlc ) in special populations     [ 43 , 44 ] . despite lifestyle modifications ,", "most patients with hypertension need pharmacologic therapy for better bp control . in the united states ,", "pharmacological treatment of hypertension before development of structural heart disease or symptoms of hf is mainly based on studies looking at prevention of end - organ damage .", "specific classes of medication are recommended for patients in stages b - d that have shown benefit in reducing mortality and number of hospitalizations .", "the reader should refer to the major guidelines for full treatment recommendations of hf in the general population     [ 18 , 23 , 46 ] .", ".   however , ethnic minorities who are at greater risk for developing hf remain underrepresented in most clinical trials .   according to a recent paper by mitchell et al . ", "treatment of hf in african americans  a call to action  , most of the available evidence is based on limited representation of african americans in these trials or posthoc analysis of limited evidence .", "these papers once again summarize the available evidence in hf management in african americans and call for more therapeutic trial focused in these high - risk population .  ", "the african american heart failure trial ( a - heft ) was the first heart failure large trial to focus specifically on the african - american population and evidence represented approximately 50% of all african americans ever studied in heart failure trials     .  ", "this is randomized placebo - controlled double blind study of fixed dose isosorbide dinitrate / hydralazine ( isbd / hyd ) ( figure 2 ) . in an effort to summarize efficacy of available treatments , psaty et al", "the database included 192,478 patients from 42 trials randomized to 7 treatment strategies including placebo .", "low - dose diuretics proved the most effective first - line treatment for preventing cardiovascular disease and mortality including hf .", "the allhat trial showed that diuretics are as effective as calcium channel blockers or acei in preventing cardiovascular disease outcomes including hf .", "the allhat study also has suggested that thiazide - diuretic therapy is useful in preventing disease progression     .  ", "although , allhat trials and other trials showed diuretics is the first - line of antihypertensive therapy and is still the most commonly prescribed antihypertensive medications , this is challenged by recent studies as published in february jacc and presented in the european society of htn meetings .  ", "however , the diuretics used in the allhat trials was chlorthalidone a long acting thiazide ( shown to effectively lower bp and improve survival ) , not really the same as hydrochlorothiazide ( hctz ) which is the most commonly prescribed antihypertensive thiazide at a dose of 12.525  mg .  ", "( st luke roosevelt hospital , new york , ny ) , published the analysis of 18 trials on   hctz where the antihypertensive effect of hctz at the dose that is most often used12.5 to 25  mg a day  is consistently inferior to that of most other antihypertensive drug .", "the other limitation of diuretics as first line therapy in african americans is not only not adequate but also , has multiple side effects like dm , electrolyte abnormalities and urecemia , to mention a few .  ", "however , there is still a role of diuretics in combination with neurohormonal ( renin angiotensin aldosterone axis ) blockade and when there is compelling indication .   according to the recent consensus statement of the international society on hypertension in blacks ( ishb ) , chlorthalidone", "furthermore , the ishb consensus statement reinforces , patient above goal bp ( sbp / dbp15/10 ) with ( goal bp < 130/80 ) end organ damage ( hf , lvh , kidney disease ) or without end organ damage ( goal bp < 135/85 ) should be on at least on combination pharmacotherapy .", "use of beta blockers ( bb ) as treatment for hypertension has come under scrutiny .", "conducted a meta - analysis of 13 trials , which included 105,951 patients showing that atenolol did not improve outcomes in terms of stroke and cardiovascular events .", "in stage b chf ( nyha class i ) , defined by the presence of reduced left ventricular ejection fraction ( lvef ) < 40 percent in otherwise asymptomatic individuals , aceis and bbs are recommended .", "iii ) manifest left ventricular dysfunction and overt symptoms ; in these individuals , aceis and bbs are also indicated .", "bbs are also recommended in hf because of clinical studies demonstrating decreased morbidity and mortality , and improvement in hf symptoms .", "aldosterone antagonists may provide additional benefit in patients with severe left ventricular dysfunction , usually late stage c ( nyha class iii ", "low - dose spironolactone ( 12.525  mg daily ) , when added to standard therapy , decreased mortality by 34 percent     . in the eplerenone postacute myocardial infarction heart failure efficacy and survival study ( ephesus ) ,", "eplerenone reduced mortality by 15% in patients following a recent mi with lvef < 40% , 90% of whom had hf symptoms     .  ", "patients were excluded who had a serum creatinine of < 2.5  mg / dl and hyperkalemia .        ", "the v - heft i and ii are a large - scale trial that compared different vasodilators with placebo and among themselves .  ", "v - heft i showed , the mortality rate was significantly lower for the total cohort ( african americans and whites ) in isdn / hyd arm compared to patients treated with prazosin or placebo added to digitalis and diuretics ( figure 1 )     .    in v - heft ii , though isbd / hyd was statistically inferior to enalapril in effect on mortality , patients taking isbd / hyd experienced similar mortality rate in   patients taking isbd / hyd in   v - heft - ii   as those in v - heft - i taking isbd / hyd .  ", "posthoc analysis of v - heft i showed isbd / hyd significantly reduced mortality in african american patients but not in white patients .  ", "the reanalysis of v - heft ii , demonstrated the annual mortality rate african american ( 12.9 ) patients receiving isbd / hyd was 12.9% compared with 12.8% for african americans receiving enalapril .  ", "due to difference in outcomes in african americans in v - heft , the african american heart failure trial ( a - heft ) definitive study was designed .  ", "the a - heft was terminated earlier than anticipated completion date due to a significant ( 43% ) reduction in mortality in patients receiving combination ( isdn / hyd ) compared to the placebo arm .    adding the combination of a fixed - dose of isdn / hyd ( 20  mg/37.5  mg ) to a standard medical regimen for hf , including acei and bbs ,", "is recommended in order to improve outcomes for patients self - described as african americans , with nyha functional class iii or iv chf     .  ", "furthermore , genomic evidence from the grahf ( genetic risk assessment of heart failure in african americans ) arm of the african american heart failure trial ( a - heft ) showed that blacks with the more common tt phenotype ( 61% ) of the aldosterone synthase gene ( cyp11b2 ) showed greater responsiveness to the combination of isosorbide dinitrate and hydralazine     . the vast majority of blacks will have no identified cause for their htn     .", "past studies elucidating the pathophysiological mechanisms have shown conflicting information and further investigations exploring the complex relationships and interactions are warranted     .", "there are , however , several secondary forms of htn in blacks that are prominent and preventable .", "we will focus on the most common , preventable and treatable secondary form of hypertension ,   obstructive sleep apnea ( osa ) .", "the prevalence of osa in hypertensive populations is estimated to range between ( 3040% )   and in heart failure at 53% ; apnea - hypopnea index ( ahi ) 10/h )     .", "it is an independent risk factor for htn , nondipping of blood pressure and increased bp variability   and follows a dose - dependent relationship .", "a prospective study showed that the odds of developing hypertension over 48 years was 2.89 times more likely for participants who had ahi  15/h compared to ahi  0 , independent of known confounders .  ", "cross - sectional data has shown 2.38-times increased likelihood of having heart failure in association with osa , independent of confounders ( n = 6424 ) [ 67 , 68 ] .", "osa has been shown to act through various mechanisms , independent of blood pressure , in promoting left - ventricular ( lv ) hypertrophy , diastolic     , and systolic      dysfunction , and overt heart failure .", "these mechanisms include the hemodynamic consequences of repetitive increases in left - ventricular transmutable wall pressure and over time impaired vigil heart rate modulation leading to lv hypertrophy and ventricular remodeling .", "additionally , osa patients without overt heart failure have more impaired diastolic relaxation than those without osa .", "emerging evidence illustrates patients with osa have more active atherosclerotic disease with a greater degree of vessel involvement and more vulnerable plaques than do patients without the disease , resulting in greater cardiovascular burden .", "the definite treatment for osa , long - term adherence to continuous positive airway pressure ( cpap ) therapy , has shown to suppress sympathetic activity     , lower blood pressure , reduce right ventricular volume     , and improve systolic function in patients with hf     .", "systematic reviews , meta - analyses , and randomized controlled trials have shown statistically significant net reduction in blood pressure with cpap , especially in patients with increased ahi events     [ 7883 ] . a recent 3-year study ( n = 340 ) by durn - cantolla et al . reported that participants assigned to cpap therapy for 3 months had mean 24-hour ambulatory blood pressure decreased by 2.1  mmhg ( 0.4 to 3.7 ) mmhg ( p = .01 ) for systolic and 1.3 ( 0.2 to 2.3 ) mm  hg ( p = .02 ) for diastolic blood pressures     .", "similar evidence from randomized trials showed up to 9% increase in lvef      and increased quality of life ( qol ) and functional capacity among patients treated with 13 months of cpap therapy     .", "furthermore , among patients with no overt failure , cpap therapy has been shown to reverse impaired diastolic relaxation .", "there is a need for further randomized clinical trials with cpap therapy among high - risk ethnic minorities especially blacks to assess whether treatment of osa has similar long - term effects and to evaluate the effect on htn and hf morbidity and mortality .", "taking into consideration , lifestyle and pharmaceutical interventions , our changing health care community needs to emphasize the essential nature of the patient - provider relationship and establish trust and collaboration . in the past , this has been accomplished through physician , patients , family member , and other caretaker awareness , increased education of both provider and patients , and establishment of community outreach programs .", "these measures have contributed to an increase in compliance and a reduction in the risk of hospitalizations [ 23 , 87 ] .", "we recommend an additional emphasis on the implementation of specialty care in patients with heart failure in collaboration with the primary care provider .", "the dramatic improvement in the management of hf and hypertension over the last 50 years has allowed us to start to target specific populations and provide more evidence - based treatment that will lead to an improvement in mortality .  ", "there are disparities in the prevalence , treatment , and control of hypertension and the incidence and morbidity and mortality of heart failure between blacks , a diverse and heterogeneous population , and whites .  ", "the management team needs to take into consideration new evidence and develop tailored strategies for effective treatment , especially for hypertension , one of the main causes of hf and many other cardiovascular complications in african africans ."], "abstract_text": ["<S> hypertension ( htn ) is the most common co - morbidity in the world , and its sequelae , heart failure ( hf ) is one of most common causes of mortality and morbidity in the world . </S>", "<S> current understanding of pathophysiology and management of htn in hf is mainly based on studies , which have mainly included whites . among racial groups , </S>", "<S> african - american adults have the highest rates ( 44% ) of hypertension in the world and are more resistant to treatment . </S>", "<S> there is an emerging consensus on the significance of racial disparities in the pathophysiology and treatment options of hypertension and heart failure . </S>", "<S> however , african americans had been underrepresented in all the trials until the initiation of the a - heft trial . since the recognition of obstructive sleep apnea ( osa ) as an important medical condition , </S>", "<S> large clinical trials have shown benefits of osa treatment among patients with htn and hf . </S>", "<S> this paper focuses on the pathophysiology , causes of secondary hypertension , and treatment of hypertension among african - american patients with heart failure . </S>", "<S> there is increasing need for randomized clinical trials testing innovative treatment options for african - american patients . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Management", "3. Treatment of Hypertension in Heart Failure", "4. Recommendations", "5. Summary"], "sections": [["hypertension ( htn ) is the most common comorbidity in the world with significant public health implications     [ 1 , 2 ] . the overall u.s .", "prevalence of hypertension among adults ages 18 years in 20052008 was 30.9% and was highest among persons ages 65 years ( 69.7% )   and non - hispanic blacks ( 44% )     .  ", "the american heart association estimates that the incurred costs of hypertension are more than $ 93.5  billion per year , and that cardiovascular disease and stroke for which htn is the predominant risk factor , account for 17% of the total annual health expenditures in the united states .", "hypertension and its sequelae , heart failure ( hf ) , are a progressive disease .", "evidence shows that less than half of patients with heart failure survive five years ( after diagnosis ) , and less than a quarter of them live ten years after their initial diagnosis .", "framingham heart study showed that hypertensive patients were more likely to develop heart failure ( 142 cases of hf detected during the first 16 years of followup ) than those who were normotensive .", "the lifetime risk for development of hf among people with blood pressure ( bp ) > 160/90  mm  hg is double that of those with bp < 140/90  mm  hg .  ", "heart failure in comparison to the most prevalent gender malignancies ( bowel cancer in men and breast cancer in women ) , was associated with worse long - term survival     .", "over a million hospitalizations , contributing to more than 250,000 deaths     , and escalating billions of dollars in health care expenditure is the norm .", "the disastrous duo , htn and hf , should be recognized , and a tailored focus on management in special populations , high - risk ethnic minority groups especially blacks who are disproportionately burdened should be developed .   among racial groups ,", "african - american adults have the highest rates ( 44% ) of hypertension in the world      and are more resistant      to treatment .  ", "in particular , black women have the highest prevalence      and the lowest blood pressure control     .  ", "the relative incidence of hf is 50% higher in african americans , 3% of whom have hf , compared with 2% of the general population     [ 13 , 14 ] .  ", "the disease occurs at an earlier age , and usually at a more advanced stage   along with increased hospitalization   and mortality .", "other causes of hf include coronary artery disease , valvular heart disease , diabetes , left ventricular hypertrophy and cardiomyopathies .  ", "overt clinical hf resulting from diastolic left ventricular dysfunction may be clinically indistinguishable from that resulting from systolic dysfunction", ". hf with preserved left ventricular function is observed in 30% to 50% of adult cases of hf .", "coronary artery disease and myocardial infarction is a principal cause of systolic left ventricular dysfunction followed by hypertension .", "a variety of neurohormonal systems , especially the renin - angiotensin aldosterone and sympathetic nervous systems are activated in response to the left ventricular dysfunction and such activation leads to abnormal ventricular remodeling . the inexorable progression to more severe stages of left ventricular dysfunction", "can be significantly reduced by effective therapy with neurohormonal blockade including angiotensin converting enzyme inhibitors ( aceis ) , beta blockers ( bbs ) , and aldosterone antagonists     .", "current understanding of the pathophysiology of hf has revolved around neurohormonal activation     [ 19 , 20 ] .  ", "this endothelial dysfunction may result from insufficient nitric oxide ( no ) secondary to either reduced endothelial production of no or to increase no inactivation [ 17 , 19 , 21 ] . in hf , a pronounced increase in angiotensin ii , with modestly reduced no is seen among whites . however , african americans with hf may exhibit greater no reduction ; conversely , angiotensin ii might be less elevated among whites .", "important studies are investigating gene variation among the races that may influence the risk of developing hypertension and its complications , such as single nucleotide polymorphisms in the genes encoding for 1-adrenergic receptors and a2c - adrenergic receptors .", "african americans who are homozygous for polymorphisms in both the 1-adrenergic and a2c - adrenergic receptor genes are at a 10-fold greater risk for heart failure , which is overexpressed in african - americans .  ", "groups with overexpression of transforming growth factor -1 ( tgf-1 ) , a cytokine involved in cardiac fibrosis and remodeling , vascular and renal disease , production of the vasoconstrictor , endothelin-1 , and stimulation of renin release , have a worse prognosis .", "other genetic polymorphisms in african americans that may influence heart failure risk include elevated production of endothelial nitric oxide synthase , aldosterone synthase , and the g protein 825-t allele ."], ["  there are two major approaches to classifying the patient 's hf stage or clinical severity .  ", "the most common is the new york heart association ( nyha ) classification , mainly based on functional capacity and subjective level of symptoms severity .  ", "this scheme is fluid and its class fluctuates in the same patient depending on their subjective assessment and clinical status .", "the other approach , adopted by the american college of cardiology / american heart association ( acc / aha ) guidelines , emphasizes disease progression", ".   classified as stages a , b , c , and d , each of these stages has definable characteristics and recommended interventions . to emphasize the progressive nature of hf and the need to prevent its development or progression to end - stage disease ( stage d )     [ 18 , 23 ] ,", "current hf guidelines define patients in stage a as those at increased risk for hf without evidence of structural heart disease or symptoms of hf     .", "stage a represents a critically important group because many of the risk factors including hypertension can be effectively controlled and by doing so may prevent development of cardiovascular diseases including hf .", "patients are in stage b when they present with evidence of structural heart disease but without current or prior symptoms of hf .", "stage c is recognized by the presence of structural heart disease and current or prior symptoms of hf .", "stage d patients are those who have refractory heart failure requiring frequent hospitalization and specialized interventions ."], ["several studies have shown the efficacy of treatment of hypertension in preventing hf [ 2427 ] .", "treatment of hypertension reduces the incidence of hf by about 50% .   in recent years", ", significant advances have been made in increasing awareness , treatment , and control of blood pressure .", "several large , randomized clinical trials have shown that specific classes of medication have mortality benefit in patients with heart failure     [ 2834 ] . in the most recent guidelines      for the diagnosis and management of hf in adults , the american college of cardiology foundation ( accf)/american heart association ( aha ) recognized the need for specific recommendations for special population .", "lifestyle modifications have been shown to reduce blood pressure , increase drug efficacy , and decrease cardiovascular risk .", "long - term primary prevention of hypertension includes weight reduction in overweight / obese individuals .", "weight loss intervention was associated with a 21% reduction in the incidence of hypertension over a 36-month period     .", "dietary practices , reducing intake of dietary sodium to < 1.5  g nacl [ 3 , 36 ] , low potassium , magnesium , and saturated fat intake , increased calcium intake     , decreased consumption of alcohol , have demonstrated greater bp reduction in blacks    ", ". these practices are most successful when reinforced with comprehensive education and counseling     [ 39 , 40 ] .", "exercise training , regular aerobic physical activity for at least 30 minutes per day most days , has been shown to reduce recurrent cardiac events in patients with lv dysfunction from ischemic heart disease and is recommended under close medical supervision . culturally cognizant and sensitive modalities should be implemented to help guide improved compliance of therapeutic lifestyle changes ( tlc ) in special populations     [ 43 , 44 ] . despite lifestyle modifications ,", "most patients with hypertension need pharmacologic therapy for better bp control . in the united states ,", "pharmacological treatment of hypertension before development of structural heart disease or symptoms of hf is mainly based on studies looking at prevention of end - organ damage .", "specific classes of medication are recommended for patients in stages b - d that have shown benefit in reducing mortality and number of hospitalizations .", "the reader should refer to the major guidelines for full treatment recommendations of hf in the general population     [ 18 , 23 , 46 ] .", ".   however , ethnic minorities who are at greater risk for developing hf remain underrepresented in most clinical trials .   according to a recent paper by mitchell et al . ", "treatment of hf in african americans  a call to action  , most of the available evidence is based on limited representation of african americans in these trials or posthoc analysis of limited evidence .", "these papers once again summarize the available evidence in hf management in african americans and call for more therapeutic trial focused in these high - risk population .  ", "the african american heart failure trial ( a - heft ) was the first heart failure large trial to focus specifically on the african - american population and evidence represented approximately 50% of all african americans ever studied in heart failure trials     .  ", "this is randomized placebo - controlled double blind study of fixed dose isosorbide dinitrate / hydralazine ( isbd / hyd ) ( figure 2 ) . in an effort to summarize efficacy of available treatments , psaty et al", "the database included 192,478 patients from 42 trials randomized to 7 treatment strategies including placebo .", "low - dose diuretics proved the most effective first - line treatment for preventing cardiovascular disease and mortality including hf .", "the allhat trial showed that diuretics are as effective as calcium channel blockers or acei in preventing cardiovascular disease outcomes including hf .", "the allhat study also has suggested that thiazide - diuretic therapy is useful in preventing disease progression     .  ", "although , allhat trials and other trials showed diuretics is the first - line of antihypertensive therapy and is still the most commonly prescribed antihypertensive medications , this is challenged by recent studies as published in february jacc and presented in the european society of htn meetings .  ", "however , the diuretics used in the allhat trials was chlorthalidone a long acting thiazide ( shown to effectively lower bp and improve survival ) , not really the same as hydrochlorothiazide ( hctz ) which is the most commonly prescribed antihypertensive thiazide at a dose of 12.525  mg .  ", "( st luke roosevelt hospital , new york , ny ) , published the analysis of 18 trials on   hctz where the antihypertensive effect of hctz at the dose that is most often used12.5 to 25  mg a day  is consistently inferior to that of most other antihypertensive drug .", "the other limitation of diuretics as first line therapy in african americans is not only not adequate but also , has multiple side effects like dm , electrolyte abnormalities and urecemia , to mention a few .  ", "however , there is still a role of diuretics in combination with neurohormonal ( renin angiotensin aldosterone axis ) blockade and when there is compelling indication .   according to the recent consensus statement of the international society on hypertension in blacks ( ishb ) , chlorthalidone", "furthermore , the ishb consensus statement reinforces , patient above goal bp ( sbp / dbp15/10 ) with ( goal bp < 130/80 ) end organ damage ( hf , lvh , kidney disease ) or without end organ damage ( goal bp < 135/85 ) should be on at least on combination pharmacotherapy .", "use of beta blockers ( bb ) as treatment for hypertension has come under scrutiny .", "conducted a meta - analysis of 13 trials , which included 105,951 patients showing that atenolol did not improve outcomes in terms of stroke and cardiovascular events .", "in stage b chf ( nyha class i ) , defined by the presence of reduced left ventricular ejection fraction ( lvef ) < 40 percent in otherwise asymptomatic individuals , aceis and bbs are recommended .", "iii ) manifest left ventricular dysfunction and overt symptoms ; in these individuals , aceis and bbs are also indicated .", "bbs are also recommended in hf because of clinical studies demonstrating decreased morbidity and mortality , and improvement in hf symptoms .", "aldosterone antagonists may provide additional benefit in patients with severe left ventricular dysfunction , usually late stage c ( nyha class iii ", "low - dose spironolactone ( 12.525  mg daily ) , when added to standard therapy , decreased mortality by 34 percent     . in the eplerenone postacute myocardial infarction heart failure efficacy and survival study ( ephesus ) ,", "eplerenone reduced mortality by 15% in patients following a recent mi with lvef < 40% , 90% of whom had hf symptoms     .  ", "patients were excluded who had a serum creatinine of < 2.5  mg / dl and hyperkalemia .        ", "the v - heft i and ii are a large - scale trial that compared different vasodilators with placebo and among themselves .  ", "v - heft i showed , the mortality rate was significantly lower for the total cohort ( african americans and whites ) in isdn / hyd arm compared to patients treated with prazosin or placebo added to digitalis and diuretics ( figure 1 )     .    in v - heft ii , though isbd / hyd was statistically inferior to enalapril in effect on mortality , patients taking isbd / hyd experienced similar mortality rate in   patients taking isbd / hyd in   v - heft - ii   as those in v - heft - i taking isbd / hyd .  ", "posthoc analysis of v - heft i showed isbd / hyd significantly reduced mortality in african american patients but not in white patients .  ", "the reanalysis of v - heft ii , demonstrated the annual mortality rate african american ( 12.9 ) patients receiving isbd / hyd was 12.9% compared with 12.8% for african americans receiving enalapril .  ", "due to difference in outcomes in african americans in v - heft , the african american heart failure trial ( a - heft ) definitive study was designed .  ", "the a - heft was terminated earlier than anticipated completion date due to a significant ( 43% ) reduction in mortality in patients receiving combination ( isdn / hyd ) compared to the placebo arm .    adding the combination of a fixed - dose of isdn / hyd ( 20  mg/37.5  mg ) to a standard medical regimen for hf , including acei and bbs ,", "is recommended in order to improve outcomes for patients self - described as african americans , with nyha functional class iii or iv chf     .  ", "furthermore , genomic evidence from the grahf ( genetic risk assessment of heart failure in african americans ) arm of the african american heart failure trial ( a - heft ) showed that blacks with the more common tt phenotype ( 61% ) of the aldosterone synthase gene ( cyp11b2 ) showed greater responsiveness to the combination of isosorbide dinitrate and hydralazine     . the vast majority of blacks will have no identified cause for their htn     .", "past studies elucidating the pathophysiological mechanisms have shown conflicting information and further investigations exploring the complex relationships and interactions are warranted     .", "there are , however , several secondary forms of htn in blacks that are prominent and preventable .", "we will focus on the most common , preventable and treatable secondary form of hypertension ,   obstructive sleep apnea ( osa ) .", "the prevalence of osa in hypertensive populations is estimated to range between ( 3040% )   and in heart failure at 53% ; apnea - hypopnea index ( ahi ) 10/h )     .", "it is an independent risk factor for htn , nondipping of blood pressure and increased bp variability   and follows a dose - dependent relationship .", "a prospective study showed that the odds of developing hypertension over 48 years was 2.89 times more likely for participants who had ahi  15/h compared to ahi  0 , independent of known confounders .  ", "cross - sectional data has shown 2.38-times increased likelihood of having heart failure in association with osa , independent of confounders ( n = 6424 ) [ 67 , 68 ] .", "osa has been shown to act through various mechanisms , independent of blood pressure , in promoting left - ventricular ( lv ) hypertrophy , diastolic     , and systolic      dysfunction , and overt heart failure .", "these mechanisms include the hemodynamic consequences of repetitive increases in left - ventricular transmutable wall pressure and over time impaired vigil heart rate modulation leading to lv hypertrophy and ventricular remodeling .", "additionally , osa patients without overt heart failure have more impaired diastolic relaxation than those without osa .", "emerging evidence illustrates patients with osa have more active atherosclerotic disease with a greater degree of vessel involvement and more vulnerable plaques than do patients without the disease , resulting in greater cardiovascular burden .", "the definite treatment for osa , long - term adherence to continuous positive airway pressure ( cpap ) therapy , has shown to suppress sympathetic activity     , lower blood pressure , reduce right ventricular volume     , and improve systolic function in patients with hf     .", "systematic reviews , meta - analyses , and randomized controlled trials have shown statistically significant net reduction in blood pressure with cpap , especially in patients with increased ahi events     [ 7883 ] . a recent 3-year study ( n = 340 ) by durn - cantolla et al . reported that participants assigned to cpap therapy for 3 months had mean 24-hour ambulatory blood pressure decreased by 2.1  mmhg ( 0.4 to 3.7 ) mmhg ( p = .01 ) for systolic and 1.3 ( 0.2 to 2.3 ) mm  hg ( p = .02 ) for diastolic blood pressures     .", "similar evidence from randomized trials showed up to 9% increase in lvef      and increased quality of life ( qol ) and functional capacity among patients treated with 13 months of cpap therapy     .", "furthermore , among patients with no overt failure , cpap therapy has been shown to reverse impaired diastolic relaxation .", "there is a need for further randomized clinical trials with cpap therapy among high - risk ethnic minorities especially blacks to assess whether treatment of osa has similar long - term effects and to evaluate the effect on htn and hf morbidity and mortality ."], ["taking into consideration , lifestyle and pharmaceutical interventions , our changing health care community needs to emphasize the essential nature of the patient - provider relationship and establish trust and collaboration . in the past , this has been accomplished through physician , patients , family member , and other caretaker awareness , increased education of both provider and patients , and establishment of community outreach programs .", "these measures have contributed to an increase in compliance and a reduction in the risk of hospitalizations [ 23 , 87 ] .", "we recommend an additional emphasis on the implementation of specialty care in patients with heart failure in collaboration with the primary care provider ."], ["the dramatic improvement in the management of hf and hypertension over the last 50 years has allowed us to start to target specific populations and provide more evidence - based treatment that will lead to an improvement in mortality .  ", "there are disparities in the prevalence , treatment , and control of hypertension and the incidence and morbidity and mortality of heart failure between blacks , a diverse and heterogeneous population , and whites .  ", "the management team needs to take into consideration new evidence and develop tailored strategies for effective treatment , especially for hypertension , one of the main causes of hf and many other cardiovascular complications in african africans ."]]}
{"article_id": "PMC4166874", "article_text": ["radiological investigations and therapeutics have become an integral part of the management of critically ill patients in the intensive care unit ( icu ) .", "patients admitted to the icu routinely undergo bedside imaging procedures such as chest radiographs for diagnosing heart , lung and other pathology , and for confirmation of the position of devices like endotracheal tubes , nasogastric tubes , central venous catheters and intercostal drains .", "a review article has suggested that among icu patients , up to 65% of daily chest radiographs and 70 - 75% of chest computerized tomography ( ct ) scans reveal significant or unsuspected abnormalities that may lead to a change in the patient 's management .", "further , critically ill patients are frequently transported to the ct scan as well as digital subtraction angiography suites for diagnostic and therapeutic procedures such as angiography , embolization and stenting . in most situations ,", "the icu team is responsible for the transport and management of the patient in the radiology department for these procedures .", "there is , therefore , potential radiation exposure to healthcare workers , particularly for those working for long periods of time in the icu .", "epidemiological data indicates that the exposure to even low - dose radiation may be a cause for concern because such exposure can result in leukemia , thyroid malignancies and other cancers .", "nonneoplastic effects of radiation include genetic mutation and developmental malformation in children whose mothers were exposed to radiation during pregnancy .", "a number of studies have looked at radiation exposure in critically ill patients undergoing repeated radiological procedures .", "one study has specifically focused on the pediatric age group probably in view of the high ratio of exposure to patient size and the potential for long - term sequelae as the radiation effects have a longer period to become manifest . however , the available literature on the extent of radiation exposure to icu personnel is scarce and relates mainly to the level of scattered radiation within the icu .", "the conclusion of these studies is that the level of radiation exposure is extremely low and does not pose a hazard to icu personnel .", "none of these studies have considered the additional radiation exposure to icu personnel involved in the management of critically ill patients in the radiology department . in our icu in a tertiary care cancer referral center , resident doctors working on 12 h shifts in the icu are also responsible for the transport and management of icu patients when they undergo diagnostic and therapeutic procedures in the radiology department .", "we , therefore , carried out this study to determine the total radiation exposure to icu resident doctors involved in the course of their duties .", "we conducted a prospective , observational study in the icu and postanesthesia care unit ( pacu ) of a 500-bedded tertiary care cancer referral center in mumbai , india from september 2012 to february 2013 .", "all resident doctors who gave voluntary written consent to participate in the study were included .", "since the study did not involve patient contact , the requirement for obtaining consent from patients was waived by the ethics committee .", "the study was carried out in accordance with the principles of good clinical research practice .", "the resident doctors provide 24 h cover , working in 12 h shifts , with four doctors in each shift . in each shift", "the icu has a total of 14 beds as shown in figure 1 . on another floor", "is the pacu with 23 beds , subdivided into 14 postoperative recovery room beds and 9 intensive care beds [ figure 2 ] .", "residents provide care for patients in the icu and pacu and also accompany patients from these locations for radiological procedures in the ct scan or interventional radiology suites .", "location of thermoluminescent dosimeters are shown with filled triangles layout of postanesthesia care unit showing location of thermoluminescent dosimeter in pacu ( tld surrounded by beds ) and other in long lobby as control tld to detect levels of radiation , we used thermoluminescent dosimeters ( tlds ) which are a type of radiation detector .", "the tld measures cumulative dose of ionizing radiation exposure by measuring the amount of visible light emitted from a crystal in the detector when the crystal is heated .", "tlds can measure a wide dosimetric range ( from 10 gray to 10 grays ) of radiation exposure and are routinely used as personal dosimeters because they are small in size , convenient to use and not expensive .", "a tld was given to each of the four resident doctors ; they were handed over to the next team during shift changeover .", "in addition , three tlds were kept in nursing stations in icu , and one tld was placed in the pacu .", "one tld was kept in the doctors ' duty room which was within the premises of the icu , and the last was kept as a control in the office of the department of anesthesia , critical care and pain , remote from icu and pacu , and where no radiological procedures were performed .", "the layout of icu and pacu is represented by figures 1 and 2 respectively and locations of tlds are marked with filled triangles .", "the participating resident doctors were instructed to wear the tlds at all times during their duty hours .", "staff not required to be present during radiographic procedures were kept at least 3 m away from the patient ( a distance at which exposure from scattered radiation is considered negligible ) .", "it was ensured that patients who were shifted from icu to the radiology suite were accompanied by one icu resident and that the resident was wearing his tld throughout .", "residents , if required to be directly involved in the radiologic procedure in suites , wore lead aprons , and the tld was underneath the lead apron . bedside chest", "x - rays in the icu were done by siemens multimobil 2.5 ( manufactured by siemens ltd , goa , india ) , ct and ct guided biopsies were performed on siemens somatom emotion 16 ( manufactured by siemens shanghai medical equipments ltd , shanghai china ) and ge light speed 16 ( manufactured by general electric co. , milwaukee , usa ) respectively .", "interventional radiology procedures were carried out using ge innova 4100 iq ( manufactured by ge medical systems , france ) .", "the study was carried out in two phases of 3 months each . at the end of the first phase ,", "the tlds were handed over to department of nuclear medicine for analysis and another set of tlds was issued at the same time . during the period of the study , we maintained a database of procedures performed both in the icu and in the radiological suites along with the bed number and the patient 's hospital registration number .", "the primary outcome of this study was to quantify the cumulative radiation exposure to the resident doctors working in the icu over a period of 6 months in the course of their duties .", "the secondary outcomes were to measure the cumulative scattered radiation exposure in various areas of the icu calculated as the average of readings of icu tld badges over 6 months and the estimated cumulative radiation exposure to the resident doctors per year .", "the control tld placed in the department of anesthesia served as a measure of baseline atmospheric ionizing radiation , and all other values are reported above this baseline .", "the results of dosimetric analysis of residents tlds during the two phases are shown in table 2 .", "readings from tlds placed in the nursing station and doctors ' duty room during both phases were immeasurable .", "the mean values in the first and last 3 months were 0.052 and 0.069 msv respectively , though the highest individual value approached 0.1 msv .", "since this was recorded in a 3 month period , the projected reading for similar exposure throughout the year , even assuming a 24 h duty period , would be 0.4 msv , which is well below the safety limit of 20 msv / year .", "revolutionary progress in the field of medical imaging has given a big leap to advances in medical diagnostics and therapeutics .", "this development has also infiltrated the field of critical care medicine , and radio - diagnosis and interventional radiological procedures now play a key role in the management of critically ill patients . while this advancement offers the advantages of rapid bedside diagnosis , and cost - effective and minimally invasive treatment options to critically ill patients , it carries the danger of exposure of patient and physician to radiation .", "the detrimental effects of exposure to even low - level ionizing radiation have always been known ; however , there is renewed concern because of its wide - spread use in medical radio - diagnosis and therapeutics in critically ill patients .", "it is , therefore , natural that there may be concerns about the long - term effects of radiation exposure to doctors working for long periods of time in the icu .", "in addition , where icu doctors are responsible for transport of patients to the radiology suite , the extent of radiation exposure is increased .", "it is reassuring that the results of our study confirm that the extent of radiation exposure to critical care physician during the course of his duty is well within acceptable limits .", "there is considerable literature on occupational hazards of radiation exposure among radiologists ; however , it is difficult to directly extrapolate the conclusions of these studies to the icu .", "working conditions in the icu do not mimic those in radiology suites - working hours tend to be long , there is a need to accompany mechanically ventilated and hemodynamically unstable patients inside the radiology suite for procedures , and icu doctors may sometimes take radiation safety norms lightly .", "a study performed in a trauma icu ( ticu ) has concluded that radiation exposure is not a significant occupational hazard for the ticu personnel .", "similarly , another study looked into the radiation exposure to icu nurses and found that the exposure was well below the permissible level .", "the strength of our study is that it was planned in a more pragmatic way - apart from bedside radiological procedures , we also took into account the radiation exposure to icu residents accompanying the patient to radiological suites for diagnostic and therapeutic procedures", ". ionizing radiation from fluoroscopy in the ct scan or interventional radiology suites may be significant and were not considered in the previous studies .", "we also tried to measure the amount of scattered radiation within the icu and the pacu , which contributes to overall radiation exposure .", "even after taking these additional sources of radiation exposure into account , we found that the cumulative radiation exposure was negligible .", "the number and types of bedside and out - of - icu radiological procedures can vary on a day - to - day basis according to the case - mix of the icu population , and this may affect the overall radiation exposure ; however , this study was carried out over a period of 6 months , and the data obtained would have been adequately representative .", "the other limitation relates to the generalizability of the study ; differences in the types of cases , working pattern of resident doctors , quality and maintenance of radiological equipment between hospitals may restrict the applicability of these results to other hospitals .", "however , given the large margin of safety that our study has shown , it is unlikely that exposure levels would be dangerously high in any other setting .", "though , with advances in technology , the number and types of radiological procedures performed on patients are likely to increase . furthermore , there is a growing trend toward using radionuclide - based positron emission tomography scans for diagnostic procedures in critically ill patients especially when they are admitted to icu during their diagnostic work - up .", "some of these patients may continue to emit radiation long after their procedures are completed .", "though none of these patients featured in our study , it will add to the radiation exposure to doctors .", "the results of this study do not in any way underrate the need to follow safety precautions , while carrying out radiological procedures in critically ill patients .", "the levels of exposure found in this study should be interpreted bearing in mind that standard protection norms were used by all personnel involved in the study . with these precautions in place ,", "radiation exposure to doctors managing critically ill patients in the icu is minimal and acceptable .", "literature on radiation exposure among icu doctors is scarce . in addition to bedside radiological procedures , the risk of exposure may be increased if the same doctors accompany icu patients for out - of - icu radiological investigations .", "however , we found that if standard safety precautions were followed , cumulative radiation exposure to icu resident doctor was well within permissible limits and was not the cause of concern and hence routine personal dosimetric monitoring is not needed for residents in icu .", "however , in view of changing practice , there is a need to repeat such audits periodically to monitor radiation exposure ."], "abstract_text": ["<S> background and aims : with the expanding use of diagnostic and therapeutic radiological modalities in critically ill patients , doctors working in intensive care units ( icus ) are increasingly exposed to ionizing radiation . </S>", "<S> this risk of radiation exposure occurs not only during bedside radiologic procedures , but also when icu physicians accompany patients to radiology suites . </S>", "<S> the aim of this study was to quantify levels of radiation exposure among medical professionals working in the icu.materials and methods : the study was carried out prospectively over 6 months in the icu of a tertiary - referral cancer hospital . </S>", "<S> two teams consisting of 4 icu resident doctors each were instructed to wear thermoluminescent dosimeters ( tlds ) during their duty shifts . </S>", "<S> standard radiation protection precautions were used throughout the study period . </S>", "<S> tlds were also placed in selected areas of the icu to measure the amount of scattered radiation . </S>", "<S> tlds were analyzed at the end of every 3 months.results:the readings recorded on tlds placed in the icu were almost immeasurable . </S>", "<S> the mean value of residents ' radiation exposure was 0.059 msv , though the highest individual reading approached 0.1 msv . </S>", "<S> the projected maximum yearly radiation exposure was 0.4 msv.conclusions:if standard radiation safety precautions are followed , the cumulative radiation exposure to icu resident doctors is well within permissible limits and is not a cause of concern . </S>", "<S> however , with the increasing use of radiological procedures in the management of critically ill patients , there is a need to repeat such audits periodically to monitor radiation exposure . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusions"], "sections": [["radiological investigations and therapeutics have become an integral part of the management of critically ill patients in the intensive care unit ( icu ) .", "patients admitted to the icu routinely undergo bedside imaging procedures such as chest radiographs for diagnosing heart , lung and other pathology , and for confirmation of the position of devices like endotracheal tubes , nasogastric tubes , central venous catheters and intercostal drains .", "a review article has suggested that among icu patients , up to 65% of daily chest radiographs and 70 - 75% of chest computerized tomography ( ct ) scans reveal significant or unsuspected abnormalities that may lead to a change in the patient 's management .", "further , critically ill patients are frequently transported to the ct scan as well as digital subtraction angiography suites for diagnostic and therapeutic procedures such as angiography , embolization and stenting . in most situations ,", "the icu team is responsible for the transport and management of the patient in the radiology department for these procedures .", "there is , therefore , potential radiation exposure to healthcare workers , particularly for those working for long periods of time in the icu .", "epidemiological data indicates that the exposure to even low - dose radiation may be a cause for concern because such exposure can result in leukemia , thyroid malignancies and other cancers .", "nonneoplastic effects of radiation include genetic mutation and developmental malformation in children whose mothers were exposed to radiation during pregnancy .", "a number of studies have looked at radiation exposure in critically ill patients undergoing repeated radiological procedures .", "one study has specifically focused on the pediatric age group probably in view of the high ratio of exposure to patient size and the potential for long - term sequelae as the radiation effects have a longer period to become manifest . however , the available literature on the extent of radiation exposure to icu personnel is scarce and relates mainly to the level of scattered radiation within the icu .", "the conclusion of these studies is that the level of radiation exposure is extremely low and does not pose a hazard to icu personnel .", "none of these studies have considered the additional radiation exposure to icu personnel involved in the management of critically ill patients in the radiology department . in our icu in a tertiary care cancer referral center , resident doctors working on 12 h shifts in the icu are also responsible for the transport and management of icu patients when they undergo diagnostic and therapeutic procedures in the radiology department .", "we , therefore , carried out this study to determine the total radiation exposure to icu resident doctors involved in the course of their duties ."], ["we conducted a prospective , observational study in the icu and postanesthesia care unit ( pacu ) of a 500-bedded tertiary care cancer referral center in mumbai , india from september 2012 to february 2013 .", "all resident doctors who gave voluntary written consent to participate in the study were included .", "since the study did not involve patient contact , the requirement for obtaining consent from patients was waived by the ethics committee .", "the study was carried out in accordance with the principles of good clinical research practice .", "the resident doctors provide 24 h cover , working in 12 h shifts , with four doctors in each shift . in each shift", "the icu has a total of 14 beds as shown in figure 1 . on another floor", "is the pacu with 23 beds , subdivided into 14 postoperative recovery room beds and 9 intensive care beds [ figure 2 ] .", "residents provide care for patients in the icu and pacu and also accompany patients from these locations for radiological procedures in the ct scan or interventional radiology suites .", "location of thermoluminescent dosimeters are shown with filled triangles layout of postanesthesia care unit showing location of thermoluminescent dosimeter in pacu ( tld surrounded by beds ) and other in long lobby as control tld to detect levels of radiation , we used thermoluminescent dosimeters ( tlds ) which are a type of radiation detector .", "the tld measures cumulative dose of ionizing radiation exposure by measuring the amount of visible light emitted from a crystal in the detector when the crystal is heated .", "tlds can measure a wide dosimetric range ( from 10 gray to 10 grays ) of radiation exposure and are routinely used as personal dosimeters because they are small in size , convenient to use and not expensive .", "a tld was given to each of the four resident doctors ; they were handed over to the next team during shift changeover .", "in addition , three tlds were kept in nursing stations in icu , and one tld was placed in the pacu .", "one tld was kept in the doctors ' duty room which was within the premises of the icu , and the last was kept as a control in the office of the department of anesthesia , critical care and pain , remote from icu and pacu , and where no radiological procedures were performed .", "the layout of icu and pacu is represented by figures 1 and 2 respectively and locations of tlds are marked with filled triangles .", "the participating resident doctors were instructed to wear the tlds at all times during their duty hours .", "staff not required to be present during radiographic procedures were kept at least 3 m away from the patient ( a distance at which exposure from scattered radiation is considered negligible ) .", "it was ensured that patients who were shifted from icu to the radiology suite were accompanied by one icu resident and that the resident was wearing his tld throughout .", "residents , if required to be directly involved in the radiologic procedure in suites , wore lead aprons , and the tld was underneath the lead apron . bedside chest", "x - rays in the icu were done by siemens multimobil 2.5 ( manufactured by siemens ltd , goa , india ) , ct and ct guided biopsies were performed on siemens somatom emotion 16 ( manufactured by siemens shanghai medical equipments ltd , shanghai china ) and ge light speed 16 ( manufactured by general electric co. , milwaukee , usa ) respectively .", "interventional radiology procedures were carried out using ge innova 4100 iq ( manufactured by ge medical systems , france ) .", "the study was carried out in two phases of 3 months each . at the end of the first phase ,", "the tlds were handed over to department of nuclear medicine for analysis and another set of tlds was issued at the same time . during the period of the study , we maintained a database of procedures performed both in the icu and in the radiological suites along with the bed number and the patient 's hospital registration number .", "the primary outcome of this study was to quantify the cumulative radiation exposure to the resident doctors working in the icu over a period of 6 months in the course of their duties .", "the secondary outcomes were to measure the cumulative scattered radiation exposure in various areas of the icu calculated as the average of readings of icu tld badges over 6 months and the estimated cumulative radiation exposure to the resident doctors per year ."], ["the control tld placed in the department of anesthesia served as a measure of baseline atmospheric ionizing radiation , and all other values are reported above this baseline .", "the results of dosimetric analysis of residents tlds during the two phases are shown in table 2 .", "readings from tlds placed in the nursing station and doctors ' duty room during both phases were immeasurable .", "the mean values in the first and last 3 months were 0.052 and 0.069 msv respectively , though the highest individual value approached 0.1 msv .", "since this was recorded in a 3 month period , the projected reading for similar exposure throughout the year , even assuming a 24 h duty period , would be 0.4 msv , which is well below the safety limit of 20 msv / year ."], ["revolutionary progress in the field of medical imaging has given a big leap to advances in medical diagnostics and therapeutics .", "this development has also infiltrated the field of critical care medicine , and radio - diagnosis and interventional radiological procedures now play a key role in the management of critically ill patients . while this advancement offers the advantages of rapid bedside diagnosis , and cost - effective and minimally invasive treatment options to critically ill patients , it carries the danger of exposure of patient and physician to radiation .", "the detrimental effects of exposure to even low - level ionizing radiation have always been known ; however , there is renewed concern because of its wide - spread use in medical radio - diagnosis and therapeutics in critically ill patients .", "it is , therefore , natural that there may be concerns about the long - term effects of radiation exposure to doctors working for long periods of time in the icu .", "in addition , where icu doctors are responsible for transport of patients to the radiology suite , the extent of radiation exposure is increased .", "it is reassuring that the results of our study confirm that the extent of radiation exposure to critical care physician during the course of his duty is well within acceptable limits .", "there is considerable literature on occupational hazards of radiation exposure among radiologists ; however , it is difficult to directly extrapolate the conclusions of these studies to the icu .", "working conditions in the icu do not mimic those in radiology suites - working hours tend to be long , there is a need to accompany mechanically ventilated and hemodynamically unstable patients inside the radiology suite for procedures , and icu doctors may sometimes take radiation safety norms lightly .", "a study performed in a trauma icu ( ticu ) has concluded that radiation exposure is not a significant occupational hazard for the ticu personnel .", "similarly , another study looked into the radiation exposure to icu nurses and found that the exposure was well below the permissible level .", "the strength of our study is that it was planned in a more pragmatic way - apart from bedside radiological procedures , we also took into account the radiation exposure to icu residents accompanying the patient to radiological suites for diagnostic and therapeutic procedures", ". ionizing radiation from fluoroscopy in the ct scan or interventional radiology suites may be significant and were not considered in the previous studies .", "we also tried to measure the amount of scattered radiation within the icu and the pacu , which contributes to overall radiation exposure .", "even after taking these additional sources of radiation exposure into account , we found that the cumulative radiation exposure was negligible .", "the number and types of bedside and out - of - icu radiological procedures can vary on a day - to - day basis according to the case - mix of the icu population , and this may affect the overall radiation exposure ; however , this study was carried out over a period of 6 months , and the data obtained would have been adequately representative .", "the other limitation relates to the generalizability of the study ; differences in the types of cases , working pattern of resident doctors , quality and maintenance of radiological equipment between hospitals may restrict the applicability of these results to other hospitals .", "however , given the large margin of safety that our study has shown , it is unlikely that exposure levels would be dangerously high in any other setting .", "though , with advances in technology , the number and types of radiological procedures performed on patients are likely to increase . furthermore , there is a growing trend toward using radionuclide - based positron emission tomography scans for diagnostic procedures in critically ill patients especially when they are admitted to icu during their diagnostic work - up .", "some of these patients may continue to emit radiation long after their procedures are completed .", "though none of these patients featured in our study , it will add to the radiation exposure to doctors .", "the results of this study do not in any way underrate the need to follow safety precautions , while carrying out radiological procedures in critically ill patients .", "the levels of exposure found in this study should be interpreted bearing in mind that standard protection norms were used by all personnel involved in the study . with these precautions in place ,", "radiation exposure to doctors managing critically ill patients in the icu is minimal and acceptable ."], ["literature on radiation exposure among icu doctors is scarce . in addition to bedside radiological procedures , the risk of exposure may be increased if the same doctors accompany icu patients for out - of - icu radiological investigations .", "however , we found that if standard safety precautions were followed , cumulative radiation exposure to icu resident doctor was well within permissible limits and was not the cause of concern and hence routine personal dosimetric monitoring is not needed for residents in icu .", "however , in view of changing practice , there is a need to repeat such audits periodically to monitor radiation exposure ."]]}
{"article_id": "PMC4403955", "article_text": ["despite the huge burden of hepatitis b worldwide , with an estimated 350 million people chronically infected , very few patients in low- and middle - income countries are currently receiving antiviral treatment .", "this is largely to blame on lack of access to viral load quantification , because this is considered a mandatory component of the diagnostic work - up in all international liver society guidelines . without a viral load measurement ,", "it is virtually impossible to establish whether a hepatitis b surface antigen ( hbsag ) positive individual has chronic hepatitis b , characterized by a level of hepatitis b virus ( hbv ) dna > 200020  000  iu / ml and continued necro - inflammation in the liver .", "these patients are at high risk of progression to cirrhosis and hepatocellular carcinoma in the absence of antiviral treatment , whereas inactive carriers , characterized by hbv dna levels < 2000  iu / ml , have an excellent prognosis in the absence of treatment .", "the situation for hepatitis c is somewhat different because the main obstacle to treatment has been expensive and toxic treatment regimens . with the release of new direct acting antivirals for hepatitis c", ", opportunities are opening up in less developed countries where the disease burden is often high .", "an estimated 130150 million people are chronically infected with hepatitis c virus ( hcv ) globally , and the prevalence is higher in certain areas such as north africa and central and east asia . however , prior to starting treatment of hepatitis c , hcv rna measurements are required to establish the diagnosis of chronic hcv infection , which is a major obstacle in many places in the world .", "lack of access to viral load testing and antiviral treatment of hbv and hcv in resource - limited settings is a silent epidemic with major consequences .", "the world health organization ( who ) estimates that about 1 million people die from chronic hbv and hcv infection each year , which places viral hepatitis on the top 10 leading causes of mortality globally .", "first , serological tests are used to screen for hepatitis markers and exclude co - infections .", "thereafter , a new battery of tests is needed to distinguish past or inactive infection from chronic hepatitis .", "this diagnostic work - up involves molecular biology laboratories and advanced target amplification methods such as polymerase chain reaction ( pcr ) assays .", "these tests are typically performed at large referral laboratories , because they require sophisticated equipment , highly specialized laboratory personnel and strict quality control measures . in resource - limited settings ,", "furthermore , stringent requirements for storage and shipment of plasma to the referral laboratory are barriers in settings with limited infrastructure .", "the solution to this challenge would be to develop reliable , cheap , and easy - to - use assays that can be performed at the site of patient care (  point - of - care assays ) .", "numerous rapid diagnostic tests ( rdts ) for hepatitis b and c are commercially available , most of which provide a test result within 530 minutes ; however , the diagnostic accuracy varies from excellent to very poor . a recent meta - analysis by shivkumar and colleagues found that the sensitivity of hbsag rdts used to screen for hepatitis b infection varied from 42 to 100% and the specificity from 0 to 100%", ". the same authors also reviewed anti - hcv rdts used for hepatitis c screening and found sensitivities ranging from 0 to 100% and specificities from 81.6 to 100% .", "hence , although high - quality rdts can be used with confidence in screening for hbv and hcv , health authorities should warn against the use of unreliable ( and often very cheap ) rdts with poor sensitivity and specificity .", "even though serological point - of - care assays for hepatitis b and c are widely available , the same is not the case for virological analyses . however ,", "if we look over the fence into the hiv landscape , there has been an active development of point - of - care kits for viral load quantification over the past few years .", "because of major international investments in the fight against hiv / aids , there is an attractive commercial market for such kits .", "the first products were recently launched , including the alere q analyzer ( alere inc . ,", "waltham , ma , usa ) and the samba ( diagnostics for the real world , sunnyvale , ca , usa ) , both of which have undergone field testing in sub - saharan africa and shown excellent performance . as there are no similar funding mechanisms for viral hepatitis in resource - limited settings ,", "commercial actors have not shown the same interest in low - tech diagnostics for hbv and hcv ; however , many of the technological advances for hiv can be modified to detect other viruses . currently , there is one product , truenat ( molbio diagnostics , goa , india ) , for hbv dna quantification which is advertized as a point - of - care assay .", "the truenat kit provides a pcr result within an hour ; however , it seems rather complex , involving several manual steps and multiple reagents , making it less ideal for outside laboratory settings .", ". an independent evaluation of the truenat kit under field conditions would be of major interest .", "the us - based company wave 80 biosciences is developing a point - of - care kit for hbv dna quantification as well as a qualitative assay for hcv rna detection , building on their existing hiv viral load assay eoscape - hiv ( wave 80 biosciences , san francisco , ca , usa ) .", "eoscape - hiv is a fully automated , cartridge format system which is said to be robust and easy to operate .", "however , eoscape - hiv is still not commercially available , so the timing for the release of the hbv and hcv kits is still uncertain .", "the uk biotechnology company epistem received a prestigious grant award in 2013 to develop an hcv point - of - care device for viral load testing and genotyping .", "this novel assay will build on their existing genedrive ( epistem , manchester , uk ) device , which is a rapid , easy to use , sensitive , handheld pcr platform already ce - ivd marked for other genotype tests , such as mycobacterium tuberculosis identification and antibiotic resistance testing .", "far too often advanced technology has been shipped to low - income countries with good intentions , but ended up in a storeroom because of insufficient training , lack of maintenance , and shortage of reagents .", "partly , this has to be blamed on the manufacturers , who rarely offer training or service agreements to low- and middle - income countries .", "it is important , therefore , that point - of - care assays should be designed specifically to operate under basic conditions with minimal maintenance requirements and not just be high - tech solutions forced to fit low - tech settings .", "the international nongovernmental organization mdecins sans frontires published their desired specifications for a point - of - care hiv viral load assay a few years ago , and the same specification would also apply to an ideal hbv or hcv assay : no need for specialized laboratory facilities closed system to avoid contamination long shelf life in tropical climate no need for cold chain transportation or refrigerated storage affordability is an important issue if treatment for viral hepatitis is to be scaled - up globally .", "both the initial cost for the machine , but also the consumables thereafter must be priced reasonably . with regard to hbv , repeated tests over time are usually required , and the accumulated cost of viral load testing can be high . for hepatitis c , on the other hand , one viral load measurement prior to treatment and another 24  weeks after treatment would be sufficient if patients are being treated with the new direct acting antivirals .", "sustainability is another major concern with all technological devices , and it should be a prerequisite that the manufacturers provide adequate training and service agreements locally . furthermore , sensitivity of the assay should be high enough to ensure treatment for those who need it , and even more importantly , specificity must be close to 100% to avoid unnecessary and expensive treatment in uninfected individuals .", "as described by greenman and colleagues in the current issue of jvh , dried blood spots ( dbs ) can be a feasible and reliable alternative to point - of - care assays for viral hepatitis .", "the main advantage of dbs is that it solves the problem of storage and shipment of samples in places with poor infrastructure .", "dbs can be stored for weeks at ambient temperature without clinically significant degradation of nucleic acids .", "a drawback with dbs is the delayed reporting back of results . with viral hepatitis , however , this might be less of a concern , as viral load quantification is part of the pretreatment work - up of each patient and not the day - to - day monitoring of treatment effect ( as in hiv ) .", "and in viral hepatitis , the decision to start treatment is rarely a matter of urgency .", "use of dbs is limited by the small amount of plasma per blood spot and less efficient nucleic acid extraction , which gives a reduced sensitivity in samples with low - level viraemia . with regard to hepatitis c", ", this rarely has any practical consequences , as most untreated patients have viral loads ( far ) above 1000  iu / ml . for hepatitis b ,", "however , the situation is rather different as hepatitis b e - antigen ( hbeag ) negative hepatitis is now the main type of chronic hepatitis b worldwide .", "these patients typically have fluctuating viral load levels in the lower to medium range , and it is often difficult to distinguish them from inactive carriers . a lack of precision in dbs could therefore jeopardize the management of these patients", ". previous studies of hbv dna quantification in dbs have shown inconsistent results . a recent study by mohamed and colleagues showed that dbs yielded viral loads 0.65 log10 lower than plasma , and an older study by jardi and colleagues found that viral load levels were 1 log10 lower in dbs compared to plasma . on the contrary , lira and", "colleagues found no significant difference between dbs and plasma ( 0.21 log10 lower in dbs ) .", "thus , more studies are needed to evaluate the precision of dbs for hbv dna quantification , especially in the lower range around the decision threshold of 200020  000 iu / ml . in conclusion ,", "dbs can be a valuable tool for the pretreatment evaluation of patients with hepatitis c , but might be more troublesome in hepatitis b due to a reduced sensitivity in the lower range of viral loads .", "management of viral hepatitis is going through a revolution with the launch of new direct acting antivirals for hepatitis c treatment .", "the cost of nearly 100  000 usd per patient , however , keeps these drugs out of reach for most patients worldwide .", "recently , the pharmaceutical company gilead announced that they work with generic drug manufactures in india to produce high - quality , low - cost sofosbuvir for developing countries , raising expectations that this game - changing drug might become available in low- and middle - income countries .", "drug availability , however , is not the only issue . in the absence of viral load measurements ,", "doctors in resource - limited settings are left virtually blindfolded in the management of their patients with hepatitis .", "point - of - care viral load assays for hepatitis b and c have the potential to bridge this gap and prove valuable tools for expansion of treatment globally", ". however , assay performance under standardized conditions in europe or north america does not necessarily reflect real - life application in sub - saharan africa , and it is crucial to carry out independent field testing of these instruments before large scale use can be recommended . finally , sustainable funding mechanisms for diagnostics and treatment of viral hepatitis must be established .", "scaling up of hiv treatment globally would have been impossible without major donor programs such as pepfar , unitaid and the global fund to fight aids , tuberculosis , and malaria .", "then our colleagues in resource - limited settings might be able to treat viral hepatitis in the not so distant future ."], "abstract_text": ["<S> viral hepatitis claims one million lives each year . </S>", "<S> scaling up treatment for hepatitis b and c in resource - limited settings is not possible without access to reliable diagnostic tools . </S>", "<S> this article gives an overview of current technologies and the pipeline for easy - to - use assays for serological and virological analyses , which can be performed at the site of patient care (  point - of - care assays ) . </S>", "<S> furthermore , the utility of dried blood spots for hepatitis b and c viral load testing is discussed . </S>", "<S> in addition to simple and reliable diagnostics , there is a need for a sustainable funding scheme and generic production of antiviral drugs to reduce the burden of viral hepatitis worldwide . </S>"], "labels": null, "section_names": ["The Diagnostic Challenge", "Do We Need Point-of-Care Assays?", "The Pipeline for Point-of-Care Viral Load Assays", "What Does an Ideal Point-of-Care Assay Look Like?", "Point-of-Care Assays Versus Dried Blood Spots", "Steps to Action"], "sections": [["despite the huge burden of hepatitis b worldwide , with an estimated 350 million people chronically infected , very few patients in low- and middle - income countries are currently receiving antiviral treatment .", "this is largely to blame on lack of access to viral load quantification , because this is considered a mandatory component of the diagnostic work - up in all international liver society guidelines . without a viral load measurement ,", "it is virtually impossible to establish whether a hepatitis b surface antigen ( hbsag ) positive individual has chronic hepatitis b , characterized by a level of hepatitis b virus ( hbv ) dna > 200020  000  iu / ml and continued necro - inflammation in the liver .", "these patients are at high risk of progression to cirrhosis and hepatocellular carcinoma in the absence of antiviral treatment , whereas inactive carriers , characterized by hbv dna levels < 2000  iu / ml , have an excellent prognosis in the absence of treatment .", "the situation for hepatitis c is somewhat different because the main obstacle to treatment has been expensive and toxic treatment regimens . with the release of new direct acting antivirals for hepatitis c", ", opportunities are opening up in less developed countries where the disease burden is often high .", "an estimated 130150 million people are chronically infected with hepatitis c virus ( hcv ) globally , and the prevalence is higher in certain areas such as north africa and central and east asia . however , prior to starting treatment of hepatitis c , hcv rna measurements are required to establish the diagnosis of chronic hcv infection , which is a major obstacle in many places in the world .", "lack of access to viral load testing and antiviral treatment of hbv and hcv in resource - limited settings is a silent epidemic with major consequences .", "the world health organization ( who ) estimates that about 1 million people die from chronic hbv and hcv infection each year , which places viral hepatitis on the top 10 leading causes of mortality globally ."], ["first , serological tests are used to screen for hepatitis markers and exclude co - infections .", "thereafter , a new battery of tests is needed to distinguish past or inactive infection from chronic hepatitis .", "this diagnostic work - up involves molecular biology laboratories and advanced target amplification methods such as polymerase chain reaction ( pcr ) assays .", "these tests are typically performed at large referral laboratories , because they require sophisticated equipment , highly specialized laboratory personnel and strict quality control measures . in resource - limited settings ,", "furthermore , stringent requirements for storage and shipment of plasma to the referral laboratory are barriers in settings with limited infrastructure .", "the solution to this challenge would be to develop reliable , cheap , and easy - to - use assays that can be performed at the site of patient care (  point - of - care assays ) .", "numerous rapid diagnostic tests ( rdts ) for hepatitis b and c are commercially available , most of which provide a test result within 530 minutes ; however , the diagnostic accuracy varies from excellent to very poor . a recent meta - analysis by shivkumar and colleagues found that the sensitivity of hbsag rdts used to screen for hepatitis b infection varied from 42 to 100% and the specificity from 0 to 100%", ". the same authors also reviewed anti - hcv rdts used for hepatitis c screening and found sensitivities ranging from 0 to 100% and specificities from 81.6 to 100% .", "hence , although high - quality rdts can be used with confidence in screening for hbv and hcv , health authorities should warn against the use of unreliable ( and often very cheap ) rdts with poor sensitivity and specificity ."], ["even though serological point - of - care assays for hepatitis b and c are widely available , the same is not the case for virological analyses . however ,", "if we look over the fence into the hiv landscape , there has been an active development of point - of - care kits for viral load quantification over the past few years .", "because of major international investments in the fight against hiv / aids , there is an attractive commercial market for such kits .", "the first products were recently launched , including the alere q analyzer ( alere inc . ,", "waltham , ma , usa ) and the samba ( diagnostics for the real world , sunnyvale , ca , usa ) , both of which have undergone field testing in sub - saharan africa and shown excellent performance . as there are no similar funding mechanisms for viral hepatitis in resource - limited settings ,", "commercial actors have not shown the same interest in low - tech diagnostics for hbv and hcv ; however , many of the technological advances for hiv can be modified to detect other viruses . currently , there is one product , truenat ( molbio diagnostics , goa , india ) , for hbv dna quantification which is advertized as a point - of - care assay .", "the truenat kit provides a pcr result within an hour ; however , it seems rather complex , involving several manual steps and multiple reagents , making it less ideal for outside laboratory settings .", ". an independent evaluation of the truenat kit under field conditions would be of major interest .", "the us - based company wave 80 biosciences is developing a point - of - care kit for hbv dna quantification as well as a qualitative assay for hcv rna detection , building on their existing hiv viral load assay eoscape - hiv ( wave 80 biosciences , san francisco , ca , usa ) .", "eoscape - hiv is a fully automated , cartridge format system which is said to be robust and easy to operate .", "however , eoscape - hiv is still not commercially available , so the timing for the release of the hbv and hcv kits is still uncertain .", "the uk biotechnology company epistem received a prestigious grant award in 2013 to develop an hcv point - of - care device for viral load testing and genotyping .", "this novel assay will build on their existing genedrive ( epistem , manchester , uk ) device , which is a rapid , easy to use , sensitive , handheld pcr platform already ce - ivd marked for other genotype tests , such as mycobacterium tuberculosis identification and antibiotic resistance testing ."], ["far too often advanced technology has been shipped to low - income countries with good intentions , but ended up in a storeroom because of insufficient training , lack of maintenance , and shortage of reagents .", "partly , this has to be blamed on the manufacturers , who rarely offer training or service agreements to low- and middle - income countries .", "it is important , therefore , that point - of - care assays should be designed specifically to operate under basic conditions with minimal maintenance requirements and not just be high - tech solutions forced to fit low - tech settings .", "the international nongovernmental organization mdecins sans frontires published their desired specifications for a point - of - care hiv viral load assay a few years ago , and the same specification would also apply to an ideal hbv or hcv assay : no need for specialized laboratory facilities closed system to avoid contamination long shelf life in tropical climate no need for cold chain transportation or refrigerated storage affordability is an important issue if treatment for viral hepatitis is to be scaled - up globally .", "both the initial cost for the machine , but also the consumables thereafter must be priced reasonably . with regard to hbv , repeated tests over time are usually required , and the accumulated cost of viral load testing can be high . for hepatitis c , on the other hand , one viral load measurement prior to treatment and another 24  weeks after treatment would be sufficient if patients are being treated with the new direct acting antivirals .", "sustainability is another major concern with all technological devices , and it should be a prerequisite that the manufacturers provide adequate training and service agreements locally . furthermore , sensitivity of the assay should be high enough to ensure treatment for those who need it , and even more importantly , specificity must be close to 100% to avoid unnecessary and expensive treatment in uninfected individuals ."], ["as described by greenman and colleagues in the current issue of jvh , dried blood spots ( dbs ) can be a feasible and reliable alternative to point - of - care assays for viral hepatitis .", "the main advantage of dbs is that it solves the problem of storage and shipment of samples in places with poor infrastructure .", "dbs can be stored for weeks at ambient temperature without clinically significant degradation of nucleic acids .", "a drawback with dbs is the delayed reporting back of results . with viral hepatitis , however , this might be less of a concern , as viral load quantification is part of the pretreatment work - up of each patient and not the day - to - day monitoring of treatment effect ( as in hiv ) .", "and in viral hepatitis , the decision to start treatment is rarely a matter of urgency .", "use of dbs is limited by the small amount of plasma per blood spot and less efficient nucleic acid extraction , which gives a reduced sensitivity in samples with low - level viraemia . with regard to hepatitis c", ", this rarely has any practical consequences , as most untreated patients have viral loads ( far ) above 1000  iu / ml . for hepatitis b ,", "however , the situation is rather different as hepatitis b e - antigen ( hbeag ) negative hepatitis is now the main type of chronic hepatitis b worldwide .", "these patients typically have fluctuating viral load levels in the lower to medium range , and it is often difficult to distinguish them from inactive carriers . a lack of precision in dbs could therefore jeopardize the management of these patients", ". previous studies of hbv dna quantification in dbs have shown inconsistent results . a recent study by mohamed and colleagues showed that dbs yielded viral loads 0.65 log10 lower than plasma , and an older study by jardi and colleagues found that viral load levels were 1 log10 lower in dbs compared to plasma . on the contrary , lira and", "colleagues found no significant difference between dbs and plasma ( 0.21 log10 lower in dbs ) .", "thus , more studies are needed to evaluate the precision of dbs for hbv dna quantification , especially in the lower range around the decision threshold of 200020  000 iu / ml . in conclusion ,", "dbs can be a valuable tool for the pretreatment evaluation of patients with hepatitis c , but might be more troublesome in hepatitis b due to a reduced sensitivity in the lower range of viral loads ."], ["management of viral hepatitis is going through a revolution with the launch of new direct acting antivirals for hepatitis c treatment .", "the cost of nearly 100  000 usd per patient , however , keeps these drugs out of reach for most patients worldwide .", "recently , the pharmaceutical company gilead announced that they work with generic drug manufactures in india to produce high - quality , low - cost sofosbuvir for developing countries , raising expectations that this game - changing drug might become available in low- and middle - income countries .", "drug availability , however , is not the only issue . in the absence of viral load measurements ,", "doctors in resource - limited settings are left virtually blindfolded in the management of their patients with hepatitis .", "point - of - care viral load assays for hepatitis b and c have the potential to bridge this gap and prove valuable tools for expansion of treatment globally", ". however , assay performance under standardized conditions in europe or north america does not necessarily reflect real - life application in sub - saharan africa , and it is crucial to carry out independent field testing of these instruments before large scale use can be recommended . finally , sustainable funding mechanisms for diagnostics and treatment of viral hepatitis must be established .", "scaling up of hiv treatment globally would have been impossible without major donor programs such as pepfar , unitaid and the global fund to fight aids , tuberculosis , and malaria .", "then our colleagues in resource - limited settings might be able to treat viral hepatitis in the not so distant future ."]]}
{"article_id": "PMC3667037", "article_text": ["the patient was a 41-week - gestational - age infant girl with a birth weight of 3300 g. after an uneventful pregnancy , the patient was delivered spontaneously and healthy until this episode . at 35 days of age , the patient was admitted to our hospital with a one - day history of anorexia , vomiting and recurrent diarrhea . on admission , conscious level was glasgow coma scale e2 v2 m2 , and blood pressure was 104/flat . physical examination revealed a distended and tender abdomen .", "laboratory values were significant for a c- reactive protein ( crp ) of 1.7 mg / dl ( normal range was less than 0.3 mg / dl ) .", "the patient was suspected intestinal obstruction due to enteritis , antibiotics were started ( abpc / mcipc and amk ) .", "4 hours after admission , she was pale with a temperature of 39.2 c and 220 per minutes pulse .", "blood pressure was low , and the fevers , vomiting , and diarrhea persisted and worsened despite antibiotics .", "she failed to respond to medical managements and died 27 hours after admission due to multiple organ failures .", "the cyst was unilocular , contained watery fluid , did not communicate with the adjacent intestinal lumen ( fig .", "microscopic finding of the ileocecal duplication cyst had an undefined mucosal lining though it resembled pyrolic glands or duodenal glands .", "duplications of the alimentary tract can be located anywhere between the mouth and the anus.4 they are most commonly found in the ileum ( 30% ) , ileocecal valve ( 30% ) , duodenum ( 10% ) , stomach ( 8% ) , jejunum ( 8% ) , colon ( 7% ) and rectum ( 5% ) with the half of the all lesions may contain ectopic gastric mucosa.5,6 most alimentary tract duplications are cystic ( 80%90% ) , with the remainder being tubular.7 they have three common features , hollow structure that has a wall of smooth muscle often shared with the contiguous intestinal tract , lined by mucous membrane similar to some part of the alimentary canal , and usually attached to some part of the alimentary tube.8 the clinical symptoms are highly variable from minor digestive problems to intestinal obstruction , gastrointestinal bleeding , or perforation.9 most patients present before the age of 2 years with obstructive symptoms , pain , or an abdominal mass .", "the presence of gastric or pancreatic mucosa can lead to peptic ulceration , perforation , and hemorrhage .", "early diagnosis and treatment reduces the risk of dangerous complications , such as bowel perforation , bleeding , obstruction , and malignant alteration .", "resection of duplication alone is the treatment of choice , and in uncomplicated cases laparoscopic resection is advised .", "surgically treated intestinal duplications have good prognosis , however , such duplications can have a fatal consequence if not properly treated .", "possible complications like bowel perforation , bleeding , obstruction , and malignant alterations are the reason why all duplications should be surgically treated at the time of diagnosis.10 alimentary tract duplications can be easily mis - diagnosed as other disorders.5 pyloric and duodenal duplications can mimic hypertrophic pyloric stenosis or choledochal cyst.11,12 ovarian cyst should be considered in female patients , and adolescent patients can sometimes have a presumptive diagnosis of crohn s disease.5 yousefzadeh et al13 showed that duplication of the colon and rectum clinically presented as abdominal pain , vomiting and chronic constipation , a significant number of patients were thought to have hirschsprung s disease . in our fatal case ,", "there are two reasons for this diagnostic difficulty : first , fevers , vomiting , and recurrent diarrhea persisted and worsened despite antibiotics .", "the patient was suspected intestinal obstruction due to enteritis , however , actually the ileocecal duplication cyst led to intestinal obstruction and necrotizing ileum ; second , the patient died in an instant due to multiple organ failures .", "if this patient was considered for exploratory laparotomy , she might be able to be alive .", "however , the patient was getting worse very quickly due to multiple organ failures and showed intractable psvt .", "exploratory laparotomy was impossible . it is demonstrated a high association of other anomalies in these patients including skeletal and urologic abnormalities,13,14 however , in this case , there were no other congenital anomalies .", "sudden unexpected death of infants associated with duplication cysts is extremely rare with only 4 cases reported in medical literature to date ( table 1 ) .", "puligandla ps et al5 and byard rw15 reported the patients presented with group b streptococcal sepsis and clostridium perfringens sepsis , respectively .", "kibayashi et al16 reported the patient died at home after being diagnosed as having gastroenteritis .", "in present case , though blood , stool and cyst cultures were negative , the patient had high fever and crp level elevated .", "infection is suspected to be a risk factor for sudden and unexpected death in infants with duplication cysts . in all cases ,", "this potential diagnosis should be borne in mind for a patient who complains of abdominal symptoms with an unknown cause , and duplication cyst should be recognized as a fatal cause in infant ."], "abstract_text": ["<S> duplications of the alimentary tract are very rare . a one - month - old female presented with symptoms of anorexia , vomiting and continuous watery diarrhea . </S>", "<S> the plain abdominal radiograph showed thickened intestinal wall and signs of small bowel obstruction . </S>", "<S> the fevers , vomiting , and continuous wartery diarrhea persisted despite antibiotics , and worsened . </S>", "<S> the patient failed to respond to medical managements , 27 hours after admission , the patient died due to multiple organ failures . </S>", "<S> the autopsy was performed , small bowel obstruction due to an ileocecal duplication cyst ( 3  3 cm ) was recognized . </S>", "<S> the ileocecal duplication cyst was attached to the ileum which was changed edematous and necrotic . </S>", "<S> this potential diagnosis should be borne in mind for a patient who complains of abdominal symptoms with an unknown cause , and duplication cyst should be recognized as a fatal cause in infant . </S>"], "labels": null, "section_names": ["Report of Case", "Discussion"], "sections": [["the patient was a 41-week - gestational - age infant girl with a birth weight of 3300 g. after an uneventful pregnancy , the patient was delivered spontaneously and healthy until this episode . at 35 days of age , the patient was admitted to our hospital with a one - day history of anorexia , vomiting and recurrent diarrhea . on admission , conscious level was glasgow coma scale e2 v2 m2 , and blood pressure was 104/flat . physical examination revealed a distended and tender abdomen .", "laboratory values were significant for a c- reactive protein ( crp ) of 1.7 mg / dl ( normal range was less than 0.3 mg / dl ) .", "the patient was suspected intestinal obstruction due to enteritis , antibiotics were started ( abpc / mcipc and amk ) .", "4 hours after admission , she was pale with a temperature of 39.2 c and 220 per minutes pulse .", "blood pressure was low , and the fevers , vomiting , and diarrhea persisted and worsened despite antibiotics .", "she failed to respond to medical managements and died 27 hours after admission due to multiple organ failures .", "the cyst was unilocular , contained watery fluid , did not communicate with the adjacent intestinal lumen ( fig .", "microscopic finding of the ileocecal duplication cyst had an undefined mucosal lining though it resembled pyrolic glands or duodenal glands ."], ["duplications of the alimentary tract can be located anywhere between the mouth and the anus.4 they are most commonly found in the ileum ( 30% ) , ileocecal valve ( 30% ) , duodenum ( 10% ) , stomach ( 8% ) , jejunum ( 8% ) , colon ( 7% ) and rectum ( 5% ) with the half of the all lesions may contain ectopic gastric mucosa.5,6 most alimentary tract duplications are cystic ( 80%90% ) , with the remainder being tubular.7 they have three common features , hollow structure that has a wall of smooth muscle often shared with the contiguous intestinal tract , lined by mucous membrane similar to some part of the alimentary canal , and usually attached to some part of the alimentary tube.8 the clinical symptoms are highly variable from minor digestive problems to intestinal obstruction , gastrointestinal bleeding , or perforation.9 most patients present before the age of 2 years with obstructive symptoms , pain , or an abdominal mass .", "the presence of gastric or pancreatic mucosa can lead to peptic ulceration , perforation , and hemorrhage .", "early diagnosis and treatment reduces the risk of dangerous complications , such as bowel perforation , bleeding , obstruction , and malignant alteration .", "resection of duplication alone is the treatment of choice , and in uncomplicated cases laparoscopic resection is advised .", "surgically treated intestinal duplications have good prognosis , however , such duplications can have a fatal consequence if not properly treated .", "possible complications like bowel perforation , bleeding , obstruction , and malignant alterations are the reason why all duplications should be surgically treated at the time of diagnosis.10 alimentary tract duplications can be easily mis - diagnosed as other disorders.5 pyloric and duodenal duplications can mimic hypertrophic pyloric stenosis or choledochal cyst.11,12 ovarian cyst should be considered in female patients , and adolescent patients can sometimes have a presumptive diagnosis of crohn s disease.5 yousefzadeh et al13 showed that duplication of the colon and rectum clinically presented as abdominal pain , vomiting and chronic constipation , a significant number of patients were thought to have hirschsprung s disease . in our fatal case ,", "there are two reasons for this diagnostic difficulty : first , fevers , vomiting , and recurrent diarrhea persisted and worsened despite antibiotics .", "the patient was suspected intestinal obstruction due to enteritis , however , actually the ileocecal duplication cyst led to intestinal obstruction and necrotizing ileum ; second , the patient died in an instant due to multiple organ failures .", "if this patient was considered for exploratory laparotomy , she might be able to be alive .", "however , the patient was getting worse very quickly due to multiple organ failures and showed intractable psvt .", "exploratory laparotomy was impossible . it is demonstrated a high association of other anomalies in these patients including skeletal and urologic abnormalities,13,14 however , in this case , there were no other congenital anomalies .", "sudden unexpected death of infants associated with duplication cysts is extremely rare with only 4 cases reported in medical literature to date ( table 1 ) .", "puligandla ps et al5 and byard rw15 reported the patients presented with group b streptococcal sepsis and clostridium perfringens sepsis , respectively .", "kibayashi et al16 reported the patient died at home after being diagnosed as having gastroenteritis .", "in present case , though blood , stool and cyst cultures were negative , the patient had high fever and crp level elevated .", "infection is suspected to be a risk factor for sudden and unexpected death in infants with duplication cysts . in all cases ,", "this potential diagnosis should be borne in mind for a patient who complains of abdominal symptoms with an unknown cause , and duplication cyst should be recognized as a fatal cause in infant ."]]}
{"article_id": "PMC4804036", "article_text": ["the term  presbyopia  derives from greek for  old eyes    and refers to the age - related loss of natural accommodation and resulting reduction of baseline near vision around the age of 40 years . as people continue to work and stay active later in life than ever before , their need for quality vision at both near and distance vision is also growing .", "in fact , the presbyopic population worldwide is predicted to rise to 1.4 billion by 2020 and to 1.8 billion by 2050 .", "presbyopia can be compensated by glasses or contact lenses , but there is increasing interest in surgical options .", "since presbyopia is caused by progressive elasticity changes in the biological crystalline lens , presbyopic surgeries may either directly replace the lens through an intraocular approach or modify extraocular structures such as the cornea or sclera .", "this review will focus on corneal - based surgical strategies to treat presbyopia and in particular how these methods have been or may be used in pseudophakic patients . to improve uncorrected near vision in presbyopia , the two major techniques to alter the cornea generally utilize either an intracorneal inlay device or laser refractive surgery .", "corneal inlays are devices that are surgically placed within the corneal stroma of the nondominant eye to change the optical properties of the cornea .", "several different types of corneal inlays each take a distinct approach to minimizing presbyopia ( table 1 ) .", "the best studied inlay to date is the kamra inlay by acufocus , which is an opaque polymer ring that employs a pinhole concept to expand the depth of focus .", "the raindrop near vision inlay by revision optics is a clear hydrogel implant that increases the anterior corneal curvature to add optical power , with a refractive index approximating that of the cornea .", "inlays can also confer differing amounts of refractive power as in the flexivue microlens implant by presbia , which creates a multifocal effect via a central plano zone surrounded by circular rings of plus power .", "the icolens by neoptics ag is another corneal inlay with a bifocal design similar in concept to the flexivue .", "corneal inlays are intended to improve uncorrected near vision but may come at the cost of lowered distance vision or increased glare or haloes [ 7 , 8 ] or rarely infectious keratitis .", "however , inlays have been promoted as an additive , removable technology unlike laser refractive surgery which ablates corneal tissue ; patients usually return to within + /1.00 diopter of their preoperative refractive state after corneal inlay removal .", "it should be noted that intrastromal corneal ring segments ( icrs or intacs ) are another type of corneal implant but are indicated for treatment of keratoconus rather than presbyopia .", "laser refractive surgery on the cornea uses an excimer laser to remodel the corneal curvature in order to improve uncorrected vision and reduce dependency on eyeglasses or contact lenses .", "one of the most popular techniques , laser - assisted in situ keratomileusis ( lasik ) , removes corneal stromal tissue under an anterior flap .", "lasik can produce either monovision or multifocality , the latter of which has been nicknamed  presbylasik  when it is used to treat presbyopia .", "conventional monovision lasik corrects the dominant eye for distance vision and the nondominant eye for near vision .", "in central presbylasik , the central area is shaped hyperpositively for near vision , whereas the midperipheral cornea is adjusted for far vision . in peripheral presbylasik , the central area is shaped for far vision and the midperipheral corneal area for near vision . in the third approach ,", "laser blended vision creates a combination of micromonovision and depth of field increase by inducing spherical aberration .", "there are concerns for the decreased contrast sensitivity , visual quality , and irreversibility of these laser ablation procedures .", "laser blended vision treatment seems to provide the best compromise in terms of safety and quality of vision .", "in contrast to the literature on corneal inlays in phakic patients , there are only a handful of published reports in pseudophakic individuals ( table 2 ) . these include only case reports and retrospective case series .", "the largest study of pseudophakic patients undergoing inlay implantation included 13 patients with monofocal intraocular lenses ( iols ) .", "four of these patients also underwent simultaneous lasik to optimize their underlying refractive error prior to insertion of a kamra pinhole inlay .", "the mean uncorrected near visual acuity ( unva ) improved 5 lines from j10 to j4 at 3 months postoperatively , without significant change in mean uncorrected or corrected distance visual acuity ( udva , cdva ) or corrected near visual acuity ( cnva ) . on a postoperative survey ,", "77% of the patients said they would opt to have the surgery again . in an earlier report by the same group ,", "kamra inlays were inserted in the nondominant eye of 3 patients with phakic iol implants .", "these patients saw 25 lines of improvement in unva without change in udva at 3 months postoperatively .", "both of these studies had a short follow - up period of 3 months , at which point the authors assert that operative results would have stabilized .", "nonetheless , longer studies are definitely needed as the mean age of these patients was around 55 years and so the patients would be expected to live with the inlays for several decades .", "two case reports with slightly longer follow - up each described a pseudophakic patient who retained improvement of uncorrected near vision at 624 months after a corneal inlay procedure .", "one young patient at 32 years of age had received a monofocal iol on account of a traumatic cataract in the right eye , and a year later he developed headaches and asthenopia while reading with his right eye .", "given his requirement of a + 3.00 reading add in the right eye , he could not tolerate glasses due to anisometropia .", "after kamra inlay implantation , he reported improved symptoms but also slightly worse reading in dim light compared to bright light conditions .", "his unva improved from j17 to j3 in the operated eye at the 6-month follow - up .", "another pseudophakic patient with a monofocal iol saw a j3 to j1 improvement in unva after implantation of a flexivue microlens inlay 6 months after the cataract extraction .", "this recovery of uncorrected near vision continued to be maintained at a 2-year follow - up after inlay implantation .", "interestingly , this patient developed a symptomatic posterior capsule opacification in the operated eye at 2 years after the cataract surgery , underwent standard neodymium : yag ( nd : yag ) capsulotomy , and was reported to still be satisfied and spectacle free with unva of j1 6 months later .", "the authors asserted that the transparent design of this particular inlay enabled visualization for the nd : yag capsulotomy and also fundoscopy .", "the aforementioned studies demonstrate the efficacy of corneal inlays in presbyopes with a history of prior intraocular and/or refractive surgery .", "similar studies done in phakic patients suggest that the improved uncorrected near vision is achieved for at least 1 to 5 years after corneal inlay implantation .", "a mexican prospective study of 30 phakic patients undergoing inlay implantation ( raindrop near vision inlay ) and lasik demonstrated improvement of mean unva by 3 lines at one year , suggesting that the improved unva could also be stable at one year in pseudophakes", ". a larger retrospective study of 277 eyes in japan showed similar results at one year after simultaneous kamra inlay implantation and lasik , with mean unva improving from j8j10 to j2-j3 depending on patient age .", "the longest published follow - up of inlays involved an austrian prospective cohort of 32 emmetropic phakic patients who retained improved uncorrected near vision at 5 years after kamra inlay implantation .", "of note , the mean unva enhancement declined somewhat from j1 to j3 between the 3- and 5-year time points ; however , cdva did not change .", "it is worth mentioning that , by the 5-year follow - up , there were still no biocompatibility concerns and 84% of the patients said they would opt for the surgery again .", "several complications occurred , including 2 inlays needed to be recentered , 1 eye that required debridement for epithelial ingrowth , 18 eyes that developed iron deposits by year 3 , and 1 inlay that was removed at year 3 due to patient dissatisfaction .", "there are several case reports describing the opposite sequence of events , where patients with prior corneal inlay implantation subsequently undergo cataract extraction with intraocular lens placement , either with or without initially removing the inlay .", "these patients had previously received any one of the three major corneal inlay types , including the kamra inlay , presbia microlens inlay , and the raindrop near vision inlay .", "these anecdotes suggest that cataract surgery can still be successfully performed after inlay implantation , which will be important for those young presbyopes opting for surgical treatment of their presbyopia .", "an extensive review of the literature on laser refractive surgery for presbyopia revealed very few studies done in pseudophakic patients , although there are numerous published accounts in phakic patients .", "similar to the body of literature on corneal inlays , a history of prior intraocular surgery such as cataract extraction typically excluded such patients from studies on laser refractive surgery for presbyopia .", "nonetheless , laser refractive surgery has been performed successfully on pseudophakes to correct for ametropias [ 2225 ] , suggesting that laser refractive surgery to address presbyopia is likely to be equally successful in pseudophakic patients who have monofocal iols . clearly , the application of laser refractive surgery to presbyopia in pseudophakia merits more research .", "various studies examine the efficacy of laser refractive surgery on treating presbyopia in phakic patients .", "a prospective trial of central multifocal presbylasik on 50 hyperopic - presbyopic eyes resulted in spectacle independence at all distances for 72% of the patients after 6 months , although nearly a third lost 1 - 2 lines of cdva .", "this and similar studies have suggested that multifocal laser approaches to improving uncorrected near vision in presbyopia may compromise distance vision to some degree [ 2729 ] .", "another study utilizing peripheral presbylasik improved mean binocular udva and unva with high reported patient satisfaction rates but decreased contrast sensitivity .", "presbylasik treatment for presbyopia may be further modified in pseudophakic patients who are not satisfied with the outcome , as described in a recent case report using a wavefront - guided aspheric treatment to reverse the presbyopic treatment and eliminate dysphotopsia up to 6 months later .", "lasik and photorefractive keratectomy ( prk ) are known to be effective for fine - tuning residual ametropias after presbyopia - compensating iol implantation , which suggests that laser refractive surgery could similarly address presbyopia after iol implantation .", "two subsequent studies showed efficacy of lasik at up to 6 months in treating pseudophakic ametropia after multifocal iol implantation in 53 eyes   and 85 eyes with mixed ametropias .", "these and other similar studies suggest that laser refractive surgery could also be used in pseudophakes to treat presbyopia .", "a general consensus is that lasik should be performed at least 612 weeks after intraocular surgery to reduce possible complications related to cataract incisions , postoperative corneal edema , and refractive and iol stability .", "while prk and lasik have both been performed safely in pseudophakic patients , it has been postulated that neither are as effective as primary refractive surgery .", "conductive keratoplasty ( ck ) , a corneal refractive surgical technique that uses radiofrequency energy to reshape the central cornea by shrinking peripheral collagen , has also been used to treat presbyopia .", "while again the majority of studies only include phakic patients , one chinese study reported that ck improved unva up to one year later in presbyopic pseudophakic patients with monofocal iols .", "studies have noted that myopes and hyperopes may express different levels of satisfaction with presbylasik as well as with other presbyopic treatments .", "although near acuity results tend to be better in myopes , the majority of hyperopes are satisfied whereas the majority of myopes are not . the decreasing visual quality after presbylasik in myopes who have experienced excellent uncorrected near visual acuity preoperatively can lead to dissatisfaction over unmet expectations .", "pseudophakic patients present a unique set of challenges and advantages for presbyopic surgeries as compared to the phakic population .", "some of the challenges stem from the prior intraocular surgery itself , such as navigating corneal scars and residual corneal irregularities from prior incisions .", "additionally , iol aberrations may alter laser refractive measurements and calculations normally used on phakic eyes .", "in general , the age difference of approximately two decades between pseudophakic patients and typical refractive patients also renders keratorefractive treatments less predictable and less effective .", "the older age of most pseudophakic patients correlates with increased prevalence of dry eye symptoms and slower healing rates .", "studies have suggested that the final uncorrected visual acuity after excimer laser or corneal inlay surgery is lower in pseudophakic patients than in typical refractive patients . on the other hand ,", "older patients have reported similar or higher levels of satisfaction after presbyopic surgeries as compared with younger patients who may have had higher postoperative acuity expectations [ 17 , 36 ] .", "presbyopia may continue to progress for phakic individuals who undergo refractive or inlay procedures at a younger age , whereas this is not an issue for patients who have had cataract surgery . at the same time", ", the lack of residual natural accommodative power in pseudophakic patients gives little room for error in presbyopic surgeries .", "it is worth noting , however , that because the iol treats most of the spherical error in pseudophakic patients , there would be less keratorefractive - induced effect on the corneal prolate asphericity and quality of vision than in nonametropic phakic patients . in all patients , regardless of phakic status , there are certain considerations to optimize a satisfactory outcome from corneal - based presbyopic surgeries .", "the importance of a thorough preoperative evaluation can not be understated , and trial frames or contact lenses should be used in various lighting conditions to help develop realistic patient expectations .", "patients should be selected appropriately from baseline characteristics including tear film adequacy , normal corneal shape and thickness , and reasonable expectations .", "there need to be more studies and counseling of patients on possible negative outcomes of presbyopic surgeries , ranging from increased dry eye or glare symptoms to decreased distance and/or night vision and subsequent safety concerns .", "presbyopic surgery may not completely eliminate the need for reading glasses . given the necessity of fundus viewing for diagnosis and treatment of diseases such as age - related macular degeneration , diabetic retinopathy , or retinal detachments", ", it should be considered that corneal inlays may obstruct the view or hinder treatment unless they are first removed .", "although corneal inlays are promoted as removable , long - term follow - up on removed inlays is not available and presbylasik procedures are in general not reversible with the commonly available technology at present . as the first wave of refractive patients begins to require cataract surgery , we may be required to change not only the assessment and technique of cataract surgery for these patients , but also how we counsel these patients who have high expectations for visual acuity and spectacle independence .", "for patients suffering from presbyopia , there are now exciting surgical alternatives to glasses and contact lenses .", "pseudophakic patients who still desire better uncorrected near vision may choose corneal - based surgical therapies , such as corneal inlays or laser refractive surgery .", "nevertheless , presbyopic surgeries will not be ideal for all patients , and appropriately screening patients and managing patient expectations are both key to maximizing satisfactory outcomes .", "many studies on presbyopic surgeries excluded patients with prior ocular surgery , and the few published studies on pseudophakic patients are limited by short - term follow - up , small numbers , and limited lighting conditions .", "undoubtedly , there is a pressing need for more research on presbyopic surgeries in the pseudophakic population ."], "abstract_text": ["<S> \n purpose . </S>", "<S> the purpose of this review is to provide a summary of laser refractive surgery and corneal inlay approaches to treat presbyopia in patients after cataract surgery . </S>", "<S> summary . </S>", "<S> the presbyopic population is growing rapidly along with increasing demands for spectacle independence . </S>", "<S> this review will focus on the corneal - based surgical options to address presbyopia including various types of corneal intrastromal inlays and laser ablation techniques to generate either a multifocal cornea (  presbylasik  ) or monovision . the natural history of presbyopia develops prior to cataracts , and these presbyopic surgeries have been largely studied in phakic patients . </S>", "<S> nevertheless , pseudophakic patients may also undergo these presbyopia - compensating procedures for enhanced quality of life . </S>", "<S> this review examines the published reports that apply these technologies to patients after cataract surgery and discusses unique considerations for this population . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Corneal Inlays for Presbyopia", "3. Laser Refractive Surgery for Presbyopia", "4. Patient Factors and Expectations: Special Considerations for Pseudophakia", "5. Conclusions"], "sections": [["the term  presbyopia  derives from greek for  old eyes    and refers to the age - related loss of natural accommodation and resulting reduction of baseline near vision around the age of 40 years . as people continue to work and stay active later in life than ever before , their need for quality vision at both near and distance vision is also growing .", "in fact , the presbyopic population worldwide is predicted to rise to 1.4 billion by 2020 and to 1.8 billion by 2050 .", "presbyopia can be compensated by glasses or contact lenses , but there is increasing interest in surgical options .", "since presbyopia is caused by progressive elasticity changes in the biological crystalline lens , presbyopic surgeries may either directly replace the lens through an intraocular approach or modify extraocular structures such as the cornea or sclera .", "this review will focus on corneal - based surgical strategies to treat presbyopia and in particular how these methods have been or may be used in pseudophakic patients . to improve uncorrected near vision in presbyopia , the two major techniques to alter the cornea generally utilize either an intracorneal inlay device or laser refractive surgery .", "corneal inlays are devices that are surgically placed within the corneal stroma of the nondominant eye to change the optical properties of the cornea .", "several different types of corneal inlays each take a distinct approach to minimizing presbyopia ( table 1 ) .", "the best studied inlay to date is the kamra inlay by acufocus , which is an opaque polymer ring that employs a pinhole concept to expand the depth of focus .", "the raindrop near vision inlay by revision optics is a clear hydrogel implant that increases the anterior corneal curvature to add optical power , with a refractive index approximating that of the cornea .", "inlays can also confer differing amounts of refractive power as in the flexivue microlens implant by presbia , which creates a multifocal effect via a central plano zone surrounded by circular rings of plus power .", "the icolens by neoptics ag is another corneal inlay with a bifocal design similar in concept to the flexivue .", "corneal inlays are intended to improve uncorrected near vision but may come at the cost of lowered distance vision or increased glare or haloes [ 7 , 8 ] or rarely infectious keratitis .", "however , inlays have been promoted as an additive , removable technology unlike laser refractive surgery which ablates corneal tissue ; patients usually return to within + /1.00 diopter of their preoperative refractive state after corneal inlay removal .", "it should be noted that intrastromal corneal ring segments ( icrs or intacs ) are another type of corneal implant but are indicated for treatment of keratoconus rather than presbyopia .", "laser refractive surgery on the cornea uses an excimer laser to remodel the corneal curvature in order to improve uncorrected vision and reduce dependency on eyeglasses or contact lenses .", "one of the most popular techniques , laser - assisted in situ keratomileusis ( lasik ) , removes corneal stromal tissue under an anterior flap .", "lasik can produce either monovision or multifocality , the latter of which has been nicknamed  presbylasik  when it is used to treat presbyopia .", "conventional monovision lasik corrects the dominant eye for distance vision and the nondominant eye for near vision .", "in central presbylasik , the central area is shaped hyperpositively for near vision , whereas the midperipheral cornea is adjusted for far vision . in peripheral presbylasik , the central area is shaped for far vision and the midperipheral corneal area for near vision . in the third approach ,", "laser blended vision creates a combination of micromonovision and depth of field increase by inducing spherical aberration .", "there are concerns for the decreased contrast sensitivity , visual quality , and irreversibility of these laser ablation procedures .", "laser blended vision treatment seems to provide the best compromise in terms of safety and quality of vision ."], ["in contrast to the literature on corneal inlays in phakic patients , there are only a handful of published reports in pseudophakic individuals ( table 2 ) . these include only case reports and retrospective case series .", "the largest study of pseudophakic patients undergoing inlay implantation included 13 patients with monofocal intraocular lenses ( iols ) .", "four of these patients also underwent simultaneous lasik to optimize their underlying refractive error prior to insertion of a kamra pinhole inlay .", "the mean uncorrected near visual acuity ( unva ) improved 5 lines from j10 to j4 at 3 months postoperatively , without significant change in mean uncorrected or corrected distance visual acuity ( udva , cdva ) or corrected near visual acuity ( cnva ) . on a postoperative survey ,", "77% of the patients said they would opt to have the surgery again . in an earlier report by the same group ,", "kamra inlays were inserted in the nondominant eye of 3 patients with phakic iol implants .", "these patients saw 25 lines of improvement in unva without change in udva at 3 months postoperatively .", "both of these studies had a short follow - up period of 3 months , at which point the authors assert that operative results would have stabilized .", "nonetheless , longer studies are definitely needed as the mean age of these patients was around 55 years and so the patients would be expected to live with the inlays for several decades .", "two case reports with slightly longer follow - up each described a pseudophakic patient who retained improvement of uncorrected near vision at 624 months after a corneal inlay procedure .", "one young patient at 32 years of age had received a monofocal iol on account of a traumatic cataract in the right eye , and a year later he developed headaches and asthenopia while reading with his right eye .", "given his requirement of a + 3.00 reading add in the right eye , he could not tolerate glasses due to anisometropia .", "after kamra inlay implantation , he reported improved symptoms but also slightly worse reading in dim light compared to bright light conditions .", "his unva improved from j17 to j3 in the operated eye at the 6-month follow - up .", "another pseudophakic patient with a monofocal iol saw a j3 to j1 improvement in unva after implantation of a flexivue microlens inlay 6 months after the cataract extraction .", "this recovery of uncorrected near vision continued to be maintained at a 2-year follow - up after inlay implantation .", "interestingly , this patient developed a symptomatic posterior capsule opacification in the operated eye at 2 years after the cataract surgery , underwent standard neodymium : yag ( nd : yag ) capsulotomy , and was reported to still be satisfied and spectacle free with unva of j1 6 months later .", "the authors asserted that the transparent design of this particular inlay enabled visualization for the nd : yag capsulotomy and also fundoscopy .", "the aforementioned studies demonstrate the efficacy of corneal inlays in presbyopes with a history of prior intraocular and/or refractive surgery .", "similar studies done in phakic patients suggest that the improved uncorrected near vision is achieved for at least 1 to 5 years after corneal inlay implantation .", "a mexican prospective study of 30 phakic patients undergoing inlay implantation ( raindrop near vision inlay ) and lasik demonstrated improvement of mean unva by 3 lines at one year , suggesting that the improved unva could also be stable at one year in pseudophakes", ". a larger retrospective study of 277 eyes in japan showed similar results at one year after simultaneous kamra inlay implantation and lasik , with mean unva improving from j8j10 to j2-j3 depending on patient age .", "the longest published follow - up of inlays involved an austrian prospective cohort of 32 emmetropic phakic patients who retained improved uncorrected near vision at 5 years after kamra inlay implantation .", "of note , the mean unva enhancement declined somewhat from j1 to j3 between the 3- and 5-year time points ; however , cdva did not change .", "it is worth mentioning that , by the 5-year follow - up , there were still no biocompatibility concerns and 84% of the patients said they would opt for the surgery again .", "several complications occurred , including 2 inlays needed to be recentered , 1 eye that required debridement for epithelial ingrowth , 18 eyes that developed iron deposits by year 3 , and 1 inlay that was removed at year 3 due to patient dissatisfaction .", "there are several case reports describing the opposite sequence of events , where patients with prior corneal inlay implantation subsequently undergo cataract extraction with intraocular lens placement , either with or without initially removing the inlay .", "these patients had previously received any one of the three major corneal inlay types , including the kamra inlay , presbia microlens inlay , and the raindrop near vision inlay .", "these anecdotes suggest that cataract surgery can still be successfully performed after inlay implantation , which will be important for those young presbyopes opting for surgical treatment of their presbyopia ."], ["an extensive review of the literature on laser refractive surgery for presbyopia revealed very few studies done in pseudophakic patients , although there are numerous published accounts in phakic patients .", "similar to the body of literature on corneal inlays , a history of prior intraocular surgery such as cataract extraction typically excluded such patients from studies on laser refractive surgery for presbyopia .", "nonetheless , laser refractive surgery has been performed successfully on pseudophakes to correct for ametropias [ 2225 ] , suggesting that laser refractive surgery to address presbyopia is likely to be equally successful in pseudophakic patients who have monofocal iols . clearly , the application of laser refractive surgery to presbyopia in pseudophakia merits more research .", "various studies examine the efficacy of laser refractive surgery on treating presbyopia in phakic patients .", "a prospective trial of central multifocal presbylasik on 50 hyperopic - presbyopic eyes resulted in spectacle independence at all distances for 72% of the patients after 6 months , although nearly a third lost 1 - 2 lines of cdva .", "this and similar studies have suggested that multifocal laser approaches to improving uncorrected near vision in presbyopia may compromise distance vision to some degree [ 2729 ] .", "another study utilizing peripheral presbylasik improved mean binocular udva and unva with high reported patient satisfaction rates but decreased contrast sensitivity .", "presbylasik treatment for presbyopia may be further modified in pseudophakic patients who are not satisfied with the outcome , as described in a recent case report using a wavefront - guided aspheric treatment to reverse the presbyopic treatment and eliminate dysphotopsia up to 6 months later .", "lasik and photorefractive keratectomy ( prk ) are known to be effective for fine - tuning residual ametropias after presbyopia - compensating iol implantation , which suggests that laser refractive surgery could similarly address presbyopia after iol implantation .", "two subsequent studies showed efficacy of lasik at up to 6 months in treating pseudophakic ametropia after multifocal iol implantation in 53 eyes   and 85 eyes with mixed ametropias .", "these and other similar studies suggest that laser refractive surgery could also be used in pseudophakes to treat presbyopia .", "a general consensus is that lasik should be performed at least 612 weeks after intraocular surgery to reduce possible complications related to cataract incisions , postoperative corneal edema , and refractive and iol stability .", "while prk and lasik have both been performed safely in pseudophakic patients , it has been postulated that neither are as effective as primary refractive surgery .", "conductive keratoplasty ( ck ) , a corneal refractive surgical technique that uses radiofrequency energy to reshape the central cornea by shrinking peripheral collagen , has also been used to treat presbyopia .", "while again the majority of studies only include phakic patients , one chinese study reported that ck improved unva up to one year later in presbyopic pseudophakic patients with monofocal iols .", "studies have noted that myopes and hyperopes may express different levels of satisfaction with presbylasik as well as with other presbyopic treatments .", "although near acuity results tend to be better in myopes , the majority of hyperopes are satisfied whereas the majority of myopes are not . the decreasing visual quality after presbylasik in myopes who have experienced excellent uncorrected near visual acuity preoperatively can lead to dissatisfaction over unmet expectations ."], ["pseudophakic patients present a unique set of challenges and advantages for presbyopic surgeries as compared to the phakic population .", "some of the challenges stem from the prior intraocular surgery itself , such as navigating corneal scars and residual corneal irregularities from prior incisions .", "additionally , iol aberrations may alter laser refractive measurements and calculations normally used on phakic eyes .", "in general , the age difference of approximately two decades between pseudophakic patients and typical refractive patients also renders keratorefractive treatments less predictable and less effective .", "the older age of most pseudophakic patients correlates with increased prevalence of dry eye symptoms and slower healing rates .", "studies have suggested that the final uncorrected visual acuity after excimer laser or corneal inlay surgery is lower in pseudophakic patients than in typical refractive patients . on the other hand ,", "older patients have reported similar or higher levels of satisfaction after presbyopic surgeries as compared with younger patients who may have had higher postoperative acuity expectations [ 17 , 36 ] .", "presbyopia may continue to progress for phakic individuals who undergo refractive or inlay procedures at a younger age , whereas this is not an issue for patients who have had cataract surgery . at the same time", ", the lack of residual natural accommodative power in pseudophakic patients gives little room for error in presbyopic surgeries .", "it is worth noting , however , that because the iol treats most of the spherical error in pseudophakic patients , there would be less keratorefractive - induced effect on the corneal prolate asphericity and quality of vision than in nonametropic phakic patients . in all patients , regardless of phakic status , there are certain considerations to optimize a satisfactory outcome from corneal - based presbyopic surgeries .", "the importance of a thorough preoperative evaluation can not be understated , and trial frames or contact lenses should be used in various lighting conditions to help develop realistic patient expectations .", "patients should be selected appropriately from baseline characteristics including tear film adequacy , normal corneal shape and thickness , and reasonable expectations .", "there need to be more studies and counseling of patients on possible negative outcomes of presbyopic surgeries , ranging from increased dry eye or glare symptoms to decreased distance and/or night vision and subsequent safety concerns .", "presbyopic surgery may not completely eliminate the need for reading glasses . given the necessity of fundus viewing for diagnosis and treatment of diseases such as age - related macular degeneration , diabetic retinopathy , or retinal detachments", ", it should be considered that corneal inlays may obstruct the view or hinder treatment unless they are first removed .", "although corneal inlays are promoted as removable , long - term follow - up on removed inlays is not available and presbylasik procedures are in general not reversible with the commonly available technology at present . as the first wave of refractive patients begins to require cataract surgery , we may be required to change not only the assessment and technique of cataract surgery for these patients , but also how we counsel these patients who have high expectations for visual acuity and spectacle independence ."], ["for patients suffering from presbyopia , there are now exciting surgical alternatives to glasses and contact lenses .", "pseudophakic patients who still desire better uncorrected near vision may choose corneal - based surgical therapies , such as corneal inlays or laser refractive surgery .", "nevertheless , presbyopic surgeries will not be ideal for all patients , and appropriately screening patients and managing patient expectations are both key to maximizing satisfactory outcomes .", "many studies on presbyopic surgeries excluded patients with prior ocular surgery , and the few published studies on pseudophakic patients are limited by short - term follow - up , small numbers , and limited lighting conditions .", "undoubtedly , there is a pressing need for more research on presbyopic surgeries in the pseudophakic population ."]]}
{"article_id": "PMC5217147", "article_text": ["enteric duplications ( eds ) are uncommon anomalies that can occur at any point of the gastrointestinal tract .", "the small intestine is the most common location ; retroperitoneum is an extremely rare site . in general ,", "diagnosed in the neonatal period or during infancy , they are increasingly diagnosed prenatally ; early prenatal detection is possible .", ", there have been seven reported cases of retroperitoneal ed cyst in the english literature .", "a female newborn , vaginally born at 39 weeks of gestation from a 32-year - old mother , gravid 3 , para 3 .", "prenatal ultrasound at 22 weeks of gestation objectified an abdominal cystic mass located in the left upper abdominal quadrant , associated with fetal pyelectasis .", "birth weight was 4000 g , length was 51 cm , and head circumference was 35 cm .", "postnatal ultrasound found a retroperitoneal para - aortic liquid - filled mass measuring 60 mm  33 mm  22 mm .", "magnetic resonance imaging ( mri ) confirmed the presence of a retroperitoneal cyst occupying the upper left retroperitoneal space ; with mass effect displacing the left kidney down [ figure 1 ] .", "peroperative finding was a retroperitoneal cyst above the left adrenal , displacing the left kidney down , measuring 70 mm  30 mm ; with no communication with any portion of the alimentary tract .", "histopathologic examination revealed an ed cyst lined by small intestinal epithelium , with no ectopic gastric mucosa .", "the postoperative period was uneventful ; the patient was discharged on the 5 postoperative day .", "magnetic resonance imaging showing retroperitoneal cyst occupying the upper left retroperitoneal space ; with mass effect displacing the left kidney", "intraabdominal duplications account for two - thirds of localization , among them , jejunoileal duplications account for 65% .", "eds occur early in intrauterine life ; their pathogenesis remains unclear ; several hypotheses have been proposed .", "reported 38 cases of ed cysts ; only one was retroperitoneal communicating with a nonfunctioning right kidney .", "eds are a part of fetal intraabdominal cysts which evoke several differential diagnoses including ovarian cysts , renal cysts , choledochal cysts , hepatic cysts , and mesenteric cysts .", "marchitelli et al . found high concordance between prenatal and postnatal findings in fetal intra - abdominal cystic lesions ( 90.4% ) .", "double - wall  sign and peristaltism are suggestive of ed and help to differentiate it of differential diagnosis .", "postnatal ultrasound helps diagnosis showing an anechoic fluid - filled mass , rarely an echogenic mass is found due to complication ( hemorrhage , necrosis ) .", "more serious complications may occur : ulceration , perforation , severe hemorrhage , and malignant changes associated with the presence of ectopic gastric mucosa ."], "abstract_text": ["<S> retroperitoneum is a very uncommon site of enteric duplication ( ed ) . </S>", "<S> we report a new case of retroperitoneal ed cyst suspected in utero . </S>", "<S> prenatal ultrasound showed an abdominal cystic mass . </S>", "<S> noncommunicating retroperitoneal ed cyst measuring 70 mm  30 mm was resected . </S>", "<S> histopathologic examination confirmed the diagnosis . </S>"], "labels": null, "section_names": ["I", "C", "D", "None", "F", "C"], "sections": [["enteric duplications ( eds ) are uncommon anomalies that can occur at any point of the gastrointestinal tract .", "the small intestine is the most common location ; retroperitoneum is an extremely rare site . in general ,", "diagnosed in the neonatal period or during infancy , they are increasingly diagnosed prenatally ; early prenatal detection is possible .", ", there have been seven reported cases of retroperitoneal ed cyst in the english literature ."], ["a female newborn , vaginally born at 39 weeks of gestation from a 32-year - old mother , gravid 3 , para 3 .", "prenatal ultrasound at 22 weeks of gestation objectified an abdominal cystic mass located in the left upper abdominal quadrant , associated with fetal pyelectasis .", "birth weight was 4000 g , length was 51 cm , and head circumference was 35 cm .", "postnatal ultrasound found a retroperitoneal para - aortic liquid - filled mass measuring 60 mm  33 mm  22 mm .", "magnetic resonance imaging ( mri ) confirmed the presence of a retroperitoneal cyst occupying the upper left retroperitoneal space ; with mass effect displacing the left kidney down [ figure 1 ] .", "peroperative finding was a retroperitoneal cyst above the left adrenal , displacing the left kidney down , measuring 70 mm  30 mm ; with no communication with any portion of the alimentary tract .", "histopathologic examination revealed an ed cyst lined by small intestinal epithelium , with no ectopic gastric mucosa .", "the postoperative period was uneventful ; the patient was discharged on the 5 postoperative day .", "magnetic resonance imaging showing retroperitoneal cyst occupying the upper left retroperitoneal space ; with mass effect displacing the left kidney"], ["intraabdominal duplications account for two - thirds of localization , among them , jejunoileal duplications account for 65% .", "eds occur early in intrauterine life ; their pathogenesis remains unclear ; several hypotheses have been proposed .", "reported 38 cases of ed cysts ; only one was retroperitoneal communicating with a nonfunctioning right kidney .", "eds are a part of fetal intraabdominal cysts which evoke several differential diagnoses including ovarian cysts , renal cysts , choledochal cysts , hepatic cysts , and mesenteric cysts .", "marchitelli et al . found high concordance between prenatal and postnatal findings in fetal intra - abdominal cystic lesions ( 90.4% ) .", "double - wall  sign and peristaltism are suggestive of ed and help to differentiate it of differential diagnosis .", "postnatal ultrasound helps diagnosis showing an anechoic fluid - filled mass , rarely an echogenic mass is found due to complication ( hemorrhage , necrosis ) .", "more serious complications may occur : ulceration , perforation , severe hemorrhage , and malignant changes associated with the presence of ectopic gastric mucosa ."], [""], [""], [""]]}
{"article_id": "PMC3687909", "article_text": ["albert hoffa first described this fracture in 1904.1 these fractures usually involve the lateral femoral condyle .", "bicondylar hoffa fracture involving both the femoral condyles is a rare injury and has anecdotally been reported in the literature [ table 1].234567 most reported cases of bicondylar hoffa fracture have two separate fracture lines and the two condyles are separated from each other .", "we describe a rare case of conjoint bicondylar hoffa fracture where both the femoral condyles were joined by a bridge of intact bone adjacent to the intercondylar notch . to the best of our knowledge ,", "only one case of such an injury has been reported in a child.7 previously published reports of bicondylar hoffa fractures", "a 17-year - old male labourer presented to orthopaedic emergency with the complaint of acute pain and swelling in his right knee following trauma .", "he had a fall from a 10-m - high ladder with direct impact on his semiflexed right knee .", "local examination revealed a painful swollen right knee with a 1 cm  1 cm lacerated wound over the anterior aspect of the knee [ figure 1 ] .", "plain radiographs revealed fracture distal end femur but were inadequate to define the exact fracture pattern [ figure 2 ] .", "noncontrast computed tomographic ( ct ) scan was performed which established the diagnosis of conjoined bicondylar hoffa fracture [ figure 3 ] .", "cm lacerated wound over the anterior aspect of the knee at the point of direct impact plain anteroposterior and lateral radiographs were inadequate to define the exact fracture pattern axial ct scan cuts showing a bicondylar hoffa fracture joined by a bridge of intact bone the patient was operated in supine position under tourniquet and regional anesthesia .", "the knee joint was then exposed by swashbuckler ( modified anterior ) approach.8 a lateral parapatellar arthrotomy was done .", "the vastus lateralis was retracted laterally after lifting it up from the lateral femoral side .", "the rest of the extensor mechanism along with the patella was retracted medially for unobstructed visualization of both femoral condyles .", "on exposing the knee , a tangential fracture involving both the femoral condyles was noted [ figure 4 ] .", "both the condyles were joined by a thin bridge of intact bone forming the roof of the intercondylar notch [ figure 4 ] .", "however , reduction was achieved and five 6.5 mm cannulated cancellous screws were passed from anterior to posterior direction through the nonarticular part under fluoroscopy control .", "post operatively above knee back splint with 30 of knee flexion was applied for 2 weeks .", "partial weight bearing was started at 6 weeks postoperatively and full weight bearing at 3 months when the fracture had united radiologically . at 18 months , patient had 0145 of range of movements without pain and deformity [ figure 5 ] .", "radiographs showed no signs of avascular necrosis , osteoarthritis , or implant breakage [ figure 6 ] .", "intraoperative photographs showing the swashbuckler approach ( a ) , pattern of fracture ( b ) , provision reduction ( c ) and fixation with cannulated cancellous screws ( d ) clinical photographs at 18 months showing range of motion followup radiographs anteroposterior ( a ) and lateral ( b ) views at 18 months showing radiological union , without any avascular necrosis , osteoarthritis or implant breakage", "hoffa fracture usually results from high - velocity trauma following road traffic accidents or fall from height .", "the specific mechanism of injury that produces hoffa fracture is not known . though a shearing force to the posterior femoral condyle has been postulated ,", "both direct impact and vertical shear with twisting mechanisms have also been proposed and a single mechanism is not agreed upon.9 because of the physiological genu valgum , the lateral femoral condyle is more likely to sustain a direct shearing force , and hence is more likely to get fractured.9 this injury is usually caused when knee is hyperflexed at the time of impact , as during driving motorcycle.9 the exact mechanism of injury of a bicondylar hoffa fracture has also not been described .", "we feel that a bicondylar hoffa fracture occurs when the flexed knee is subjected to a posterior and upward directed force without any varus or valgus component and that the proximity of the fracture line and its obliquity depends upon the degree of knee flexion at the time of impact .", "the greater the degree of knee flexion , more is the distance of fracture line from the posterior femoral cortex [ figures 7 and 8 ] .", "our patient had sustained an injury following a fall from a 10-m - high ladder with direct impact on his semiflexed right knee , as evident by a small lacerated wound , probably without any varus or valgus component .", "therefore , the coronal shear force resulted in a bicondylar hoffa fracture where both the femoral condyles were joined by a bridge of intact bone adjacent to the intercondylar notch as the fracture line was anterior to the intercondylar notch . had the knee been flexed more , it would have resulted in a bicondylar hoffa fracture with two separate fragments with the fracture line being posterior to the intercondylar notch .", "the most widely used classification system developed by muller , updated by the ao group , and adopted by the orthopaedic trauma association ( ota ) classifies distal femoral fractures into three groups [ figure 9].10 we feel that b3.3 should be subclassified into two groups : type b3.3a- conjoint bicondylar hoffa fracture where the two condyles are joined by a bride of intact bone and occurs due to a posterior and upward directed force with a semiflexed knee without any varus or valgus [ figure 7 ] .", "type b3.3b- nonconjoint bicondylar hoffa fracture where the two condyles are separate from each other and occurs due to posterior and upward directed force with a hyperflexed knee without any varus or valgus [ figure 8 ] .", "however , this subclassification needs validation and further evaluation . in view of the rare occurrence of hoffa , further rarer occurrence of bicondylar hoffa , and unreported occurrence of conjoint bicondylar hoffa", "similarly , in view of limited information and followup , it is not possible to provide information about the prognosis of this fracture .", "a line diagram showing conjoint bicondylar hoffa fracture following a posterior and upward directed force ( f ) in a semiflexed knee a line diagram showing nonconjoint bicondylar hoffa fracture following a posterior and upward directed force ( f ) in a hyperflexed knee classification of distal femoral fractures the diagnosis of hoffa fracture on plain radiographs can present difficulties because the fracture is obscured in the anteroposterior view by the intact anterior part of the femoral condyle.5 if it is minimally displaced , the fracture may also be difficult to define in the lateral view .", "if the lateral view is not taken in a standard position , then it is further difficult to interpret whether it is the medial or the lateral condyle that is fractured even if displaced .", "this may result in the injury being missed on initial imaging.6 oblique radiographs have an important role both preoperatively and also intraoperatively for evaluation of the reduction , screw length , and condyle identification .", "however , a ct scan is most helpful not only in defining the fracture pattern but also in deciding the surgical approach . in our case also , the initial radiographs were inadequate to define the exact pattern of the injury .", "the ct scan demonstrated the bicondylar hoffa fracture with both fragments joined to each other by a bridge of bone forming the roof of intercondylar notch .", "it helped us not only in defining the exact nature of the injury but also in the surgical approach .", "therefore , like previous authors , we feel a ct scan with 3d reconstruction is the investigation of choice in such patients to define the exact pattern of the fracture and plan the surgical approach .", "nonoperative treatment in the form of plaster cast or skeletal traction leads to loss of extension , nonunion , instability , joint contracture , and deformity.239 therefore , anatomical reduction of articular surface , stable fixation , and early mobilization should be the aim of treatment.11 for open reduction of bicondylar hoffa fracture , most authors have used a combined medial and lateral approach .", "however , we used a swashbuckler ( modified anterior ) approach as advocated by dua et al.6 this allowed us to expose the back of femoral condyles on both the sides and facilitated unhindered anatomic reduction of the fracture fragments .", "the more anterior position of the skin incision as compared to the classical lateral approach allows unobstructed visualization of both the femoral condyles and obviates the need of two incisions .", "we believe that single incision would result in decreased breach of the quadriceps mechanism , lesser fibrosis , and earlier return of quadriceps strength and range of motion .", "lal et al.7 described arthroscopic assisted reduction and fixation for such injuries . however , we believe that it would be technically challenging in a case like ours where the fragment was large and displaced . although there is no consensus , there is some suggestion that posterior to anterior directed screws may be better.12 however , in our case , the unusual fracture pattern required that we use an approach by which both condyles could be exposed adequately .", "once the exposure was adequate , the best method was to fix the fractures from anterior to posterior direction under direct vision .", "thus , the direction of the screws was determined by the approach required for the fracture exposure . in conclusion", ", we described a rare case of a conjoint bicondylar hoffa fracture managed successfully by open reduction and internal fixation with good clinical outcome at 18 months of followup .", "we feel that a bicondylar hoffa fracture occurs when the flexed knee is subjected to a posterior and upward directed force without any varus or valgus component and that the proximity of the fracture line and its obliquity depends upon the degree of knee flexion at the time of impact .", "ct scan not only helps in defining the exact pattern of injury but also is invaluable in the surgical planning .", "the swashbukler ( modified anterior ) approach allows excellent exposure of both the condyles . anatomic reduction and rigid internal fixation allows early mobilization and excellent long term outcome ."], "abstract_text": ["<S> conjoint bicondylar hoffa fracture is an extremely rare injury . </S>", "<S> only one case has been reported previously in the pediatric age group . </S>", "<S> we describe this injury in a 17-year - old male who presented following a fall with direct impact on his semiflexed right knee . </S>", "<S> plain radiographs were inadequate to define the exact pattern of injury </S>", "<S> . computed tomographic ( ct ) scans demonstrated the coronal fracture involving both the femoral condyles which were joined by a bridge of intact bone . </S>", "<S> the patient was treated with open reduction and internal fixation using swashbuckler ( modified anterior ) approach . </S>", "<S> union occurred within 3 months and at final followup ( at 18 months ) the patient had a good clinical outcome . </S>", "<S> the possible mechanism of injury is discussed . </S>"], "labels": null, "section_names": ["I", "C", "D"], "sections": [["albert hoffa first described this fracture in 1904.1 these fractures usually involve the lateral femoral condyle .", "bicondylar hoffa fracture involving both the femoral condyles is a rare injury and has anecdotally been reported in the literature [ table 1].234567 most reported cases of bicondylar hoffa fracture have two separate fracture lines and the two condyles are separated from each other .", "we describe a rare case of conjoint bicondylar hoffa fracture where both the femoral condyles were joined by a bridge of intact bone adjacent to the intercondylar notch . to the best of our knowledge ,", "only one case of such an injury has been reported in a child.7 previously published reports of bicondylar hoffa fractures"], ["a 17-year - old male labourer presented to orthopaedic emergency with the complaint of acute pain and swelling in his right knee following trauma .", "he had a fall from a 10-m - high ladder with direct impact on his semiflexed right knee .", "local examination revealed a painful swollen right knee with a 1 cm  1 cm lacerated wound over the anterior aspect of the knee [ figure 1 ] .", "plain radiographs revealed fracture distal end femur but were inadequate to define the exact fracture pattern [ figure 2 ] .", "noncontrast computed tomographic ( ct ) scan was performed which established the diagnosis of conjoined bicondylar hoffa fracture [ figure 3 ] .", "cm lacerated wound over the anterior aspect of the knee at the point of direct impact plain anteroposterior and lateral radiographs were inadequate to define the exact fracture pattern axial ct scan cuts showing a bicondylar hoffa fracture joined by a bridge of intact bone the patient was operated in supine position under tourniquet and regional anesthesia .", "the knee joint was then exposed by swashbuckler ( modified anterior ) approach.8 a lateral parapatellar arthrotomy was done .", "the vastus lateralis was retracted laterally after lifting it up from the lateral femoral side .", "the rest of the extensor mechanism along with the patella was retracted medially for unobstructed visualization of both femoral condyles .", "on exposing the knee , a tangential fracture involving both the femoral condyles was noted [ figure 4 ] .", "both the condyles were joined by a thin bridge of intact bone forming the roof of the intercondylar notch [ figure 4 ] .", "however , reduction was achieved and five 6.5 mm cannulated cancellous screws were passed from anterior to posterior direction through the nonarticular part under fluoroscopy control .", "post operatively above knee back splint with 30 of knee flexion was applied for 2 weeks .", "partial weight bearing was started at 6 weeks postoperatively and full weight bearing at 3 months when the fracture had united radiologically . at 18 months , patient had 0145 of range of movements without pain and deformity [ figure 5 ] .", "radiographs showed no signs of avascular necrosis , osteoarthritis , or implant breakage [ figure 6 ] .", "intraoperative photographs showing the swashbuckler approach ( a ) , pattern of fracture ( b ) , provision reduction ( c ) and fixation with cannulated cancellous screws ( d ) clinical photographs at 18 months showing range of motion followup radiographs anteroposterior ( a ) and lateral ( b ) views at 18 months showing radiological union , without any avascular necrosis , osteoarthritis or implant breakage"], ["hoffa fracture usually results from high - velocity trauma following road traffic accidents or fall from height .", "the specific mechanism of injury that produces hoffa fracture is not known . though a shearing force to the posterior femoral condyle has been postulated ,", "both direct impact and vertical shear with twisting mechanisms have also been proposed and a single mechanism is not agreed upon.9 because of the physiological genu valgum , the lateral femoral condyle is more likely to sustain a direct shearing force , and hence is more likely to get fractured.9 this injury is usually caused when knee is hyperflexed at the time of impact , as during driving motorcycle.9 the exact mechanism of injury of a bicondylar hoffa fracture has also not been described .", "we feel that a bicondylar hoffa fracture occurs when the flexed knee is subjected to a posterior and upward directed force without any varus or valgus component and that the proximity of the fracture line and its obliquity depends upon the degree of knee flexion at the time of impact .", "the greater the degree of knee flexion , more is the distance of fracture line from the posterior femoral cortex [ figures 7 and 8 ] .", "our patient had sustained an injury following a fall from a 10-m - high ladder with direct impact on his semiflexed right knee , as evident by a small lacerated wound , probably without any varus or valgus component .", "therefore , the coronal shear force resulted in a bicondylar hoffa fracture where both the femoral condyles were joined by a bridge of intact bone adjacent to the intercondylar notch as the fracture line was anterior to the intercondylar notch . had the knee been flexed more , it would have resulted in a bicondylar hoffa fracture with two separate fragments with the fracture line being posterior to the intercondylar notch .", "the most widely used classification system developed by muller , updated by the ao group , and adopted by the orthopaedic trauma association ( ota ) classifies distal femoral fractures into three groups [ figure 9].10 we feel that b3.3 should be subclassified into two groups : type b3.3a- conjoint bicondylar hoffa fracture where the two condyles are joined by a bride of intact bone and occurs due to a posterior and upward directed force with a semiflexed knee without any varus or valgus [ figure 7 ] .", "type b3.3b- nonconjoint bicondylar hoffa fracture where the two condyles are separate from each other and occurs due to posterior and upward directed force with a hyperflexed knee without any varus or valgus [ figure 8 ] .", "however , this subclassification needs validation and further evaluation . in view of the rare occurrence of hoffa , further rarer occurrence of bicondylar hoffa , and unreported occurrence of conjoint bicondylar hoffa", "similarly , in view of limited information and followup , it is not possible to provide information about the prognosis of this fracture .", "a line diagram showing conjoint bicondylar hoffa fracture following a posterior and upward directed force ( f ) in a semiflexed knee a line diagram showing nonconjoint bicondylar hoffa fracture following a posterior and upward directed force ( f ) in a hyperflexed knee classification of distal femoral fractures the diagnosis of hoffa fracture on plain radiographs can present difficulties because the fracture is obscured in the anteroposterior view by the intact anterior part of the femoral condyle.5 if it is minimally displaced , the fracture may also be difficult to define in the lateral view .", "if the lateral view is not taken in a standard position , then it is further difficult to interpret whether it is the medial or the lateral condyle that is fractured even if displaced .", "this may result in the injury being missed on initial imaging.6 oblique radiographs have an important role both preoperatively and also intraoperatively for evaluation of the reduction , screw length , and condyle identification .", "however , a ct scan is most helpful not only in defining the fracture pattern but also in deciding the surgical approach . in our case also , the initial radiographs were inadequate to define the exact pattern of the injury .", "the ct scan demonstrated the bicondylar hoffa fracture with both fragments joined to each other by a bridge of bone forming the roof of intercondylar notch .", "it helped us not only in defining the exact nature of the injury but also in the surgical approach .", "therefore , like previous authors , we feel a ct scan with 3d reconstruction is the investigation of choice in such patients to define the exact pattern of the fracture and plan the surgical approach .", "nonoperative treatment in the form of plaster cast or skeletal traction leads to loss of extension , nonunion , instability , joint contracture , and deformity.239 therefore , anatomical reduction of articular surface , stable fixation , and early mobilization should be the aim of treatment.11 for open reduction of bicondylar hoffa fracture , most authors have used a combined medial and lateral approach .", "however , we used a swashbuckler ( modified anterior ) approach as advocated by dua et al.6 this allowed us to expose the back of femoral condyles on both the sides and facilitated unhindered anatomic reduction of the fracture fragments .", "the more anterior position of the skin incision as compared to the classical lateral approach allows unobstructed visualization of both the femoral condyles and obviates the need of two incisions .", "we believe that single incision would result in decreased breach of the quadriceps mechanism , lesser fibrosis , and earlier return of quadriceps strength and range of motion .", "lal et al.7 described arthroscopic assisted reduction and fixation for such injuries . however , we believe that it would be technically challenging in a case like ours where the fragment was large and displaced . although there is no consensus , there is some suggestion that posterior to anterior directed screws may be better.12 however , in our case , the unusual fracture pattern required that we use an approach by which both condyles could be exposed adequately .", "once the exposure was adequate , the best method was to fix the fractures from anterior to posterior direction under direct vision .", "thus , the direction of the screws was determined by the approach required for the fracture exposure . in conclusion", ", we described a rare case of a conjoint bicondylar hoffa fracture managed successfully by open reduction and internal fixation with good clinical outcome at 18 months of followup .", "we feel that a bicondylar hoffa fracture occurs when the flexed knee is subjected to a posterior and upward directed force without any varus or valgus component and that the proximity of the fracture line and its obliquity depends upon the degree of knee flexion at the time of impact .", "ct scan not only helps in defining the exact pattern of injury but also is invaluable in the surgical planning .", "the swashbukler ( modified anterior ) approach allows excellent exposure of both the condyles . anatomic reduction and rigid internal fixation allows early mobilization and excellent long term outcome ."]]}
{"article_id": "PMC4837779", "article_text": [", neither buccolingual width nor angulation can be properly visualized on the most traditional radiographs . in the following case report , an innovative simplified method without radiation", "is used to fabricate a three - dimensional model to assess the available bone for implant placement in the mandible .", "even a minor variation in comparison to ideal placement causes difficulties in fabrication of final prostheses .", "the surgical guide is essential to establish a logical continuity among diagnosis , prosthetic planning , and surgical phase .", "a patient of age 27 years reported to the department of prosthodontics for the replacement of missing left first molar .", "following steps were performed to achieve a three - dimensional model : \n intraoral impression was recorded with addition silicone putty and light body [ figure 1]fabrication of acrylic assembly for aligning both intraoral and extraoral impressiona rectangular block of acrylic was made to which impression tray was pressed to make indentations . after applying separating medium", ", the counter block of acrylic was made to which extraoral impression was attached [ figure 2 ] . in prototype ,", "[ figures 24 ] plastic rod was used for supporting the extraoral impressionreplacing the plastic rod with metal plate and key system the plastic rod was replaced by a metal plate in the first generation [ figure 5 ] for better stability and better customization .", "a key system was incorporated to adjust the length and angulation according to each patient 's mandible . on the top of the metal plate ,", "addition silicone putty placed on the acrylic plate can be customized each time to record the extraoral impression .", "the extraoral impression should record the lower border of the mandible to the medial extent as much as possible [ figure 6]fabrication of three - dimensional model the intraoral impression was poured first to get a cast . after the initial setting was over , the entire assembly was poured , and a three dimensional model of the mandible was fabricated [ figure 7 ] . \n ", "intraoral impression was recorded with addition silicone putty and light body [ figure 1 ] fabrication of acrylic assembly for aligning both intraoral and extraoral impression a rectangular block of acrylic was made to which impression tray was pressed to make indentations . after applying separating medium , the counter block of acrylic was made to which extraoral impression was attached [ figure 2 ] . in prototype ,", "[ figures 24 ] plastic rod was used for supporting the extraoral impression replacing the plastic rod with metal plate and key system the plastic rod was replaced by a metal plate in the first generation [ figure 5 ] for better stability and better customization .", "a key system was incorporated to adjust the length and angulation according to each patient 's mandible . on the top of the metal plate , acrylic", "addition silicone putty placed on the acrylic plate can be customized each time to record the extraoral impression .", "the extraoral impression should record the lower border of the mandible to the medial extent as much as possible [ figure 6 ] fabrication of three - dimensional model the intraoral impression was poured first to get a cast . after the initial setting was over , the entire assembly was poured , and a three dimensional model of the mandible was fabricated [ figure 7 ] .", "intraoral impression with acrylic assembly prototype ( extraoral impression ) prototype - extraoral and intraoral impression together with acrylic assembly first generation metal plate assembled metal frame three - dimensional model the accuracy of this method was verified with computed tomography ( ct ) scan .", "the cross section where the mandibular premolar teeth were disappearing from the ct image was taken ( 14 mm depth ) as a reference and measured to get the available bone width buccolingually ( 8.7 mm ) .", "this measurement was same when compared with the available bone in the sectioned cast at the premolar area , at a depth of 14 mm ( premolar root length ) .", "on the three - dimensional model , the surgical guide was fabricated [ figure 8 ]", ". this surgical template can dictate the implant body placement that offers the best combination of support for the repetitive forces of occlusion and esthetics .", "three - dimensional model with surgical guide this article had won the best table clinic award in 35 ips conference conducted in new delhi 2007 by army dental corps ."], "abstract_text": ["<S> the primary factor that determines the success of implant is the amount of available bone in the edentulous site . diagnostic imaging and techniques help to develop and implement a cohesive and comprehensive treatment plan according to the available bone . in the following case </S>", "<S> report an innovative simplified method ( poor man ct ) is used to fabricate a three dimensional model to assess the available bone for implant placement in mandible . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "Financial support and sponsorship", "Conflicts of interest"], "sections": [[", neither buccolingual width nor angulation can be properly visualized on the most traditional radiographs . in the following case report , an innovative simplified method without radiation", "is used to fabricate a three - dimensional model to assess the available bone for implant placement in the mandible .", "even a minor variation in comparison to ideal placement causes difficulties in fabrication of final prostheses .", "the surgical guide is essential to establish a logical continuity among diagnosis , prosthetic planning , and surgical phase ."], ["a patient of age 27 years reported to the department of prosthodontics for the replacement of missing left first molar .", "following steps were performed to achieve a three - dimensional model : \n intraoral impression was recorded with addition silicone putty and light body [ figure 1]fabrication of acrylic assembly for aligning both intraoral and extraoral impressiona rectangular block of acrylic was made to which impression tray was pressed to make indentations . after applying separating medium", ", the counter block of acrylic was made to which extraoral impression was attached [ figure 2 ] . in prototype ,", "[ figures 24 ] plastic rod was used for supporting the extraoral impressionreplacing the plastic rod with metal plate and key system the plastic rod was replaced by a metal plate in the first generation [ figure 5 ] for better stability and better customization .", "a key system was incorporated to adjust the length and angulation according to each patient 's mandible . on the top of the metal plate ,", "addition silicone putty placed on the acrylic plate can be customized each time to record the extraoral impression .", "the extraoral impression should record the lower border of the mandible to the medial extent as much as possible [ figure 6]fabrication of three - dimensional model the intraoral impression was poured first to get a cast . after the initial setting was over , the entire assembly was poured , and a three dimensional model of the mandible was fabricated [ figure 7 ] . \n ", "intraoral impression was recorded with addition silicone putty and light body [ figure 1 ] fabrication of acrylic assembly for aligning both intraoral and extraoral impression a rectangular block of acrylic was made to which impression tray was pressed to make indentations . after applying separating medium , the counter block of acrylic was made to which extraoral impression was attached [ figure 2 ] . in prototype ,", "[ figures 24 ] plastic rod was used for supporting the extraoral impression replacing the plastic rod with metal plate and key system the plastic rod was replaced by a metal plate in the first generation [ figure 5 ] for better stability and better customization .", "a key system was incorporated to adjust the length and angulation according to each patient 's mandible . on the top of the metal plate , acrylic", "addition silicone putty placed on the acrylic plate can be customized each time to record the extraoral impression .", "the extraoral impression should record the lower border of the mandible to the medial extent as much as possible [ figure 6 ] fabrication of three - dimensional model the intraoral impression was poured first to get a cast . after the initial setting was over , the entire assembly was poured , and a three dimensional model of the mandible was fabricated [ figure 7 ] .", "intraoral impression with acrylic assembly prototype ( extraoral impression ) prototype - extraoral and intraoral impression together with acrylic assembly first generation metal plate assembled metal frame three - dimensional model the accuracy of this method was verified with computed tomography ( ct ) scan .", "the cross section where the mandibular premolar teeth were disappearing from the ct image was taken ( 14 mm depth ) as a reference and measured to get the available bone width buccolingually ( 8.7 mm ) .", "this measurement was same when compared with the available bone in the sectioned cast at the premolar area , at a depth of 14 mm ( premolar root length ) .", "on the three - dimensional model , the surgical guide was fabricated [ figure 8 ]", ". this surgical template can dictate the implant body placement that offers the best combination of support for the repetitive forces of occlusion and esthetics .", "three - dimensional model with surgical guide this article had won the best table clinic award in 35 ips conference conducted in new delhi 2007 by army dental corps ."], [""], [""]]}
{"article_id": "PMC3280477", "article_text": ["the use of ergotamines causing ischemia of peripheral vessels has been reported , particularly in patients with peripheral vascular disease .", "ischemic bowel induced by ergotamines is a much rarer event , with few documented cases in the literature .", "while colitis is always on the differential diagnosis of hospital patients with abdominal pain , the majority of cases are secondary to infectious colitis associated with antibiotic therapy .", "when suspected , non - occlusive mesenteric ischemia can be treated with anti - coagulation and blood pressure support with intravenous fluids .", "the predisposition for ischemia in patients with chronic constipation secondary to opioid use is amplified by concomitant use of vasoactive medications to control symptoms of migraine headache .", "while abdominal ct scanning is frequently used to assess colonic pathology , findings often lag behind those identified at the time of colonoscopy .", "patchy areas of ischemia are often managed non - surgically by discontinuing the offending medication .", "widespread necrosis , coupled with clinical deterioration , as in this case , mandated immediate surgical intervention to prevent irreversible septic shock .", "a 48-year - old woman with a long - standing history of intractable migraine headaches was admitted to our institution 's specialty headache unit for aggressive management . she had no known history of peripheral vascular or cardiac disease but", "has been admitted multiple times for treatment of her headaches since her initial diagnosis in 2003 .", "during the course of each of these hospitalizations , she had received one to two courses of dihydroergotamine mesylate ( dhe ) 45 . during this admission she received two courses of dhe 0.5 mg  1 , then 1 mg every 8 h for a total of eight doses , on days 13 and the second course of therapy on days 79 .", "several hours after the final dose of her second round she became obtunded , minimally responsive and hypotensive with a blood pressure of 80/45 mm hg . on physical examination her abdomen was moderately distended with few bowel sounds .", "mmol / l , white blood cell count was 18.0 ( with 17% bands ) , and stool was clostridium difficile negative .", "the patient was transferred to the intensive care unit , given intravenous fluid boluses , started on vasopressors , and given broad spectrum antibiotics .", "her chest radiograph was unremarkable , and her abdominal x - rays showed only mild prominence of the colon . over the next several hours she became progressively acidotic with a worsening bandemia .", "a bedside colonoscopy showed diffuse ischemic colitis with fecal impaction and no pseudomembranes ( fig .", "operative findings showed a peritoneal cavity filled with murky , foul - smelling fluid and gangrene of the descending colon was seen with necrosis in the area immediately distal to the splenic flexure to the sigmoid colon .", "final pathology on the surgical specimen showed 86 cm of patchy , dark green / black large bowel , consistent with ischemic necrosis .", "the final diagnosis was gangrenous large bowel , ischemic colitis with thrombi or emboli noted .", "a hypercoaguable panel was performed following the operation and the patient was negative for clotting disorders .", "the differential diagnosis of ischemic colitis includes arterial occlusion , caused by emboli or thrombus , venous thrombosis , and non - occlusive ischemia caused by systemic hypotension or , as is strongly suspected in this case , ergotamine - induced vasospasm .", "a diagnosis of ergotamine - induced vasospasm is a diagnosis of exclusion , made when all other etiologies of ischemia have been ruled out . in this case ,", "an arterial occlusion is unlikely , because there was no palpable thrombus and there was no clear ischemic demarcation to suggest a major vessel distribution .", "the patient had no history of cardiac arrhythmias , such as atrial fibrillation , to predispose her to emboli .", "furthermore she was a non - smoker and had no other cardiac risk factors such as atherosclerosis or hyperlipidemia .", "emboli to inferior mesenteric artery ( i m a ) are exceedingly rare , and there are no reported cases of spontaneous emboli or thrombosis to i m a .", "all reported cases of i m a thrombosis are subsequent to trauma [ 1 , 2 ] .", "venous thrombosis as a cause of ischemia in this patient is equally unlikely as there were no signs of venous congestion or stasis at the time of surgery .", "the patient had no hypercoagulable disorder and on examination the colon was not edematous and no clot was identified in the vasculature .", "ischemia secondary to systemic hypotension alone would have resulted in a watershed - type distribution of gangrene . in this case", "the most probable cause of this event was non - occlusive mesenteric ischemia in the i m a distribution and more specifically the left colic artery due to intense vasoconstriction induced by high - dose ergotamine use .", "underlying bowel wall distention from the impacted stool in combination with vasoconstriction from ergotamine is the most likely etiology for this patient 's bowel ischemia .", "dhe is indicated for the acute treatment of migraine headaches with or without aura and the acute treatment of cluster headache episodes .", "its therapeutic activity in the treatment of migraine headaches is generally attributed to the agonist effect at 5-ht1d receptors . according to the neurovascular theory of migraine ,", "it is hypothesized that activation of 5-ht1d receptors on intracranial blood vessels leads to vasoconstriction .", "the 5-ht1d receptors on sensory nerve endings of the trigeminal are also activated , causing inhibition of pro - inflammatory neuropeptide release .", "ergotamine and dhe cause vasoconstriction of both arteries and veins due to alpha - adrenergic agonistic action , and they may also have possible interaction with prostaglandins , calcium and serotonin .", "dhe was developed in 1943 for migraine prophylaxis and shown in four studies in 1987 and five studies in 2001 to have less effect on peripheral arteries than ergotamine . as indicated in this case", "a european consensus report in 2000 concluded that ergotamine is not a drug of first choice in the triptan era , but may be useful in longstanding migraine attacks with multiple recurrences .", "ergotamine is known to cause peripheral ischemia , as has been reported in several case studies , most commonly in the lower limbs [ 5 , 6 , 7 , 8 , 9 ] .", "a causal relationship has not however been clearly established and is still the subject of some debate .", "other reports of ergotamine causing ischemic bowel appear in the literature [ 13 , 14 , 15 ] . in previous reported cases of colitis , expectant management with", "this is the first reported cases of ischemia progressing to gangrene such that colon resection was required", ". it should be noted in this case that the patient 's dose of dhe was greater than the recommended amount .", "the recommended dosing of dhe 45 is 1 mg iv / im / sc , repeated once every 24 h for a total dose not to exceed 6 mg in 7 days . in this case , the patient received 17 mg of dhe over the course of 9 days .", "although ischemic colitis is considered a rare side effect of high - dose ergotamine use , it is a very real risk of which clinicians need to remain aware .", "this is the fourth reported case of ischemic colitis secondary to high - dose ergotamine use reported in the literature and the first to require colon resection .", "while dhe can be useful in patients with refractory migraines , patients should be informed of the risks of both peripheral and visceral ischemia before deciding on this treatment .", "finally , ischemic colitis should be considered in the differential diagnosis of all patients taking ergotamines who present with abdominal pain ."], "abstract_text": ["<S> a 48-year - old woman with a history of chronic migraines , initially admitted for inpatient management of intractable migraine headaches , developed new onset abdominal pain , hypotension , and diarrhea on hospital day number ten . in our institution 's headache unit , </S>", "<S> patients are treated by a multidisciplinary approach , including individualized drug therapy based on diagnosis and previous response to therapy . </S>", "<S> given the patient 's hypotension and clinical appearance , she was transferred to the intensive care unit and treated for septic shock and metabolic acidosis . </S>", "<S> a bedside colonscopy revealed diffuse ischemic colitis . </S>", "<S> final pathology after colon resection showed widespread , transmural necrosis of the colonic wall . </S>", "<S> we review the pathophysiology of ergotamine use and its potential association with ischemic colitis . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["the use of ergotamines causing ischemia of peripheral vessels has been reported , particularly in patients with peripheral vascular disease .", "ischemic bowel induced by ergotamines is a much rarer event , with few documented cases in the literature .", "while colitis is always on the differential diagnosis of hospital patients with abdominal pain , the majority of cases are secondary to infectious colitis associated with antibiotic therapy .", "when suspected , non - occlusive mesenteric ischemia can be treated with anti - coagulation and blood pressure support with intravenous fluids .", "the predisposition for ischemia in patients with chronic constipation secondary to opioid use is amplified by concomitant use of vasoactive medications to control symptoms of migraine headache .", "while abdominal ct scanning is frequently used to assess colonic pathology , findings often lag behind those identified at the time of colonoscopy .", "patchy areas of ischemia are often managed non - surgically by discontinuing the offending medication .", "widespread necrosis , coupled with clinical deterioration , as in this case , mandated immediate surgical intervention to prevent irreversible septic shock ."], ["a 48-year - old woman with a long - standing history of intractable migraine headaches was admitted to our institution 's specialty headache unit for aggressive management . she had no known history of peripheral vascular or cardiac disease but", "has been admitted multiple times for treatment of her headaches since her initial diagnosis in 2003 .", "during the course of each of these hospitalizations , she had received one to two courses of dihydroergotamine mesylate ( dhe ) 45 . during this admission she received two courses of dhe 0.5 mg  1 , then 1 mg every 8 h for a total of eight doses , on days 13 and the second course of therapy on days 79 .", "several hours after the final dose of her second round she became obtunded , minimally responsive and hypotensive with a blood pressure of 80/45 mm hg . on physical examination her abdomen was moderately distended with few bowel sounds .", "mmol / l , white blood cell count was 18.0 ( with 17% bands ) , and stool was clostridium difficile negative .", "the patient was transferred to the intensive care unit , given intravenous fluid boluses , started on vasopressors , and given broad spectrum antibiotics .", "her chest radiograph was unremarkable , and her abdominal x - rays showed only mild prominence of the colon . over the next several hours she became progressively acidotic with a worsening bandemia .", "a bedside colonoscopy showed diffuse ischemic colitis with fecal impaction and no pseudomembranes ( fig .", "operative findings showed a peritoneal cavity filled with murky , foul - smelling fluid and gangrene of the descending colon was seen with necrosis in the area immediately distal to the splenic flexure to the sigmoid colon .", "final pathology on the surgical specimen showed 86 cm of patchy , dark green / black large bowel , consistent with ischemic necrosis .", "the final diagnosis was gangrenous large bowel , ischemic colitis with thrombi or emboli noted .", "a hypercoaguable panel was performed following the operation and the patient was negative for clotting disorders ."], ["the differential diagnosis of ischemic colitis includes arterial occlusion , caused by emboli or thrombus , venous thrombosis , and non - occlusive ischemia caused by systemic hypotension or , as is strongly suspected in this case , ergotamine - induced vasospasm .", "a diagnosis of ergotamine - induced vasospasm is a diagnosis of exclusion , made when all other etiologies of ischemia have been ruled out . in this case ,", "an arterial occlusion is unlikely , because there was no palpable thrombus and there was no clear ischemic demarcation to suggest a major vessel distribution .", "the patient had no history of cardiac arrhythmias , such as atrial fibrillation , to predispose her to emboli .", "furthermore she was a non - smoker and had no other cardiac risk factors such as atherosclerosis or hyperlipidemia .", "emboli to inferior mesenteric artery ( i m a ) are exceedingly rare , and there are no reported cases of spontaneous emboli or thrombosis to i m a .", "all reported cases of i m a thrombosis are subsequent to trauma [ 1 , 2 ] .", "venous thrombosis as a cause of ischemia in this patient is equally unlikely as there were no signs of venous congestion or stasis at the time of surgery .", "the patient had no hypercoagulable disorder and on examination the colon was not edematous and no clot was identified in the vasculature .", "ischemia secondary to systemic hypotension alone would have resulted in a watershed - type distribution of gangrene . in this case", "the most probable cause of this event was non - occlusive mesenteric ischemia in the i m a distribution and more specifically the left colic artery due to intense vasoconstriction induced by high - dose ergotamine use .", "underlying bowel wall distention from the impacted stool in combination with vasoconstriction from ergotamine is the most likely etiology for this patient 's bowel ischemia .", "dhe is indicated for the acute treatment of migraine headaches with or without aura and the acute treatment of cluster headache episodes .", "its therapeutic activity in the treatment of migraine headaches is generally attributed to the agonist effect at 5-ht1d receptors . according to the neurovascular theory of migraine ,", "it is hypothesized that activation of 5-ht1d receptors on intracranial blood vessels leads to vasoconstriction .", "the 5-ht1d receptors on sensory nerve endings of the trigeminal are also activated , causing inhibition of pro - inflammatory neuropeptide release .", "ergotamine and dhe cause vasoconstriction of both arteries and veins due to alpha - adrenergic agonistic action , and they may also have possible interaction with prostaglandins , calcium and serotonin .", "dhe was developed in 1943 for migraine prophylaxis and shown in four studies in 1987 and five studies in 2001 to have less effect on peripheral arteries than ergotamine . as indicated in this case", "a european consensus report in 2000 concluded that ergotamine is not a drug of first choice in the triptan era , but may be useful in longstanding migraine attacks with multiple recurrences .", "ergotamine is known to cause peripheral ischemia , as has been reported in several case studies , most commonly in the lower limbs [ 5 , 6 , 7 , 8 , 9 ] .", "a causal relationship has not however been clearly established and is still the subject of some debate .", "other reports of ergotamine causing ischemic bowel appear in the literature [ 13 , 14 , 15 ] . in previous reported cases of colitis , expectant management with", "this is the first reported cases of ischemia progressing to gangrene such that colon resection was required", ". it should be noted in this case that the patient 's dose of dhe was greater than the recommended amount .", "the recommended dosing of dhe 45 is 1 mg iv / im / sc , repeated once every 24 h for a total dose not to exceed 6 mg in 7 days . in this case , the patient received 17 mg of dhe over the course of 9 days .", "although ischemic colitis is considered a rare side effect of high - dose ergotamine use , it is a very real risk of which clinicians need to remain aware ."], ["this is the fourth reported case of ischemic colitis secondary to high - dose ergotamine use reported in the literature and the first to require colon resection .", "while dhe can be useful in patients with refractory migraines , patients should be informed of the risks of both peripheral and visceral ischemia before deciding on this treatment .", "finally , ischemic colitis should be considered in the differential diagnosis of all patients taking ergotamines who present with abdominal pain ."]]}
{"article_id": "PMC2725918", "article_text": ["we analyzed all 140 mrsa isolates we obtained during a previous study ( 2 ) and 50 other isolates selected from our collection of nosocomial mrsa isolates obtained in 2003 .", "characteristics of these 190 isolates are shown in table 1 . * ni , nosocomial infection acquired at national taiwan university hospital ( ntuh ) ; nc , nosocomial colonization at ntuh ; ieoh , mrsa infection detected at ntuh within 48 h after transfer from another hospital . ", "bl , blood ; ur , urine ; sp , sputum ; pu , pus ; wo , wound ; ns , nostril ; ct , catheter tip ; st , stool . ", "pulsed - field gel electrophoresis patterns were interpreted according to procedures previously reported ( 7,8 ) .", "thirty - four isolates belonged to pulsotype a , 49 to pulsotype b , 69 to pulsotype c , 6 to pulsotype d , 2 to pulsotype e , 3 to pulsotype f , 11 to pulsotype k , 5 to pulsotype l , 8 to pulsotype m , and 3 ( all isolated in 2003 ) to 3 minor pulsotypes .", "all isolates were tested by sccmec element typing and multilocus sequence typing ( mlst ) ( 9 ) and were analyzed for the panton - valentine leukocidin ( pvl ) gene ( 10 ) and drug susceptibility to erythromycin , clindamycin , gentamicin , amikacin , ciprofloxacin , levofloxacin , tetracycline , trimethoprim - sulfamethoxazole , rifampin , and vancomycin by using the disk diffusion method ( 11 ) .", "isolates with pulsotype a are sequence type 254 ( st254 ) ; those with pulsotype b are st241 ; those with pulsotypes c , k , and l are st239 ; and those with pulsotypes d , e , f , and m are st59 , st 254 , st30 , and st5 , respectively .", "all mrsa isolates with pulsotypes a , d , e , and f have the type iv sccmec element .", "however , only isolates with pulsotypes d and f , as well as 2 isolates from 2003 with 2 minor pulsotypes , have the pvl gene .", "isolates with pulsotypes b and c have the type iii sccmec element , and isolates with pulsotype m have the type ii sccmec element . *", "p , pulsotype ; scc , staphylococcal cassette chromosome ; mlst , multilocus sequence typing ; pvl , panton - valentine leukocidin ; n , no ; y , yes .  ox , oxacillin ; em , erythromycin ; cl , clinidamycin ; gm , gentamicin ; am , amikacin ; cp , ciprofloxacin ; lv , levofloxacin ; tc , tetracycline ; ts , trimethoprim - sulfamethoxazole ; rf , rifampin ; va , vancomycin . ", "results of mlst and typing of sccmec elements of the 3 isolates with 3 minor pulsotypes obtained in 2003 are shown in table 2 .", "the correlation between sccmec element typing and mlst in this study corresponds to findings of previous reports ( 6,1214 ) .", "enright et al . identified st254-iv mrsa isolates in germany and the united kingdom ( 12 ) , and chongtrakool et al . identified st239-iii and st241-iii mrsa isolates in several asian countries ( 14 ) .", "we demonstrate that the predominant mrsa clone at ntuh in early 1990s had the type iv sccmec element . however , the predominant mrsa clones at ntuh from 1994 to 2003 had the type iii sccmec element .", "these findings differ from those of wisplinghoff et al . , who reported that that the sccmec element in predominant mrsa clones at their institute changed from type iii in 1984 to 1988 to type i in 1989 to 1998 ( 6 ) .", "mrsa isolates of pulsotypes b and c are more resistant than isolates of pulsotype a to certain antimicrobial drugs , especially fluoroquinolones and trimethoprim - sulfamethoxazole ; mrsa isolates with pulsotype c are more resistant to clindamycin but less resistant to rifampin than those with pulsotype b ( table 2 ) . from 1993 through 2000 , annual use of fluoroquinolones increased 3 at ntuh ; however , use of trimethoprim - sulfamethoxazole , clindamycin , and rifampin did not change ( 15 ) .", "therefore , the shift of predominant mrsa clones , which also led to the shift in types of sccmec elements at ntuh , might be caused by selective pressure from antimicrobial drugs , especially fluoroquinolones .", "the mrsa clone ( pulsotype a ) that predominated in 1992 and 1993 at ntuh has the type iv sccmec element .", "although the first study of mrsa with the type iv sccmec element reported that this element was found in community - acquired mrsa ( ca - mrsa ) ( 5 ) , some studies have reported mrsa isolates with this element in a hospital environment ( 12 ) . however , to our knowledge , these reports did not demonstrate that mrsa isolates with the type iv sccmec element became predominant among all mrsa isolates in an institution , especially before the mid-1990s .", "furthermore , 4 st59 mrsa isolates obtained in 1994 and 1996 and 3 st30 mrsa isolates obtained in 1992 and 1993 have the type iv sccmec element and pvl gene .", "recently , st59 mrsa isolates were found to cause ca - mrsa infection in taiwan ( 13 ) . among these st59", "ca - mrsa isolates , some had the type iv sccmec element , and others had the type v sccmec element ( 13 ) . although the type iv sccmec element could be transferred to ca - mrsa clones with other genetic backgrounds , our finding supports the possibility that st59 mrsa isolates with the sccmec element type iv in taiwan may originate from hospital strains but transfer into ca - mrsa strains .", "recently reported the results of sccmec typing of 615 mrsa isolates obtained in 1998 and 1999 from 11 asian countries ( 14 ) .", "the st239-iii , st241-iii , st254-ii , and st5-ii mrsa isolates were prevalent in many asian countries .", "however , the st254-iv , st30-iv , and st59-iv mrsa isolates from our study were not found in other asian countries .", "in addition , st254-iv mrsa isolates have been found in germany and the united kingdom ( 12 ) . whether st254-iv mrsa isolates in our study", "were transmitted from those in germany or the united kingdom by international travel requires further study .", "the first mrsa isolate with the type iv sccmec element in our hospital appeared as early as 1992 . the sccmec element carried by predominant mrsa clones changed from type iv to type iii sccmec element during the period 19922003 at ntuh .", "because the major mrsa clones isolated in 19942003 are more resistant to antimicrobial drugs , especially fluoroquinolones and trimethoprim - sulfamethoxazole , than those obtained in 1992 and 1993 , this shift may be caused by selective pressure from indiscriminate use of antimicrobial drugs ."], "abstract_text": ["<S> to determine the predominant staphylococcal cassette chromosome ( scc ) mec element in methicillin - resistant staphylococcus aureus , we typed 190 isolates from a hospital in taiwan . </S>", "<S> we found a shift from type iv to type iii sccmec element during 19922003 , perhaps caused by selective pressure from indiscriminate use of antimicrobial drugs . </S>"], "labels": null, "section_names": ["The Study", "Conclusions"], "sections": [["we analyzed all 140 mrsa isolates we obtained during a previous study ( 2 ) and 50 other isolates selected from our collection of nosocomial mrsa isolates obtained in 2003 .", "characteristics of these 190 isolates are shown in table 1 . * ni , nosocomial infection acquired at national taiwan university hospital ( ntuh ) ; nc , nosocomial colonization at ntuh ; ieoh , mrsa infection detected at ntuh within 48 h after transfer from another hospital . ", "bl , blood ; ur , urine ; sp , sputum ; pu , pus ; wo , wound ; ns , nostril ; ct , catheter tip ; st , stool . ", "pulsed - field gel electrophoresis patterns were interpreted according to procedures previously reported ( 7,8 ) .", "thirty - four isolates belonged to pulsotype a , 49 to pulsotype b , 69 to pulsotype c , 6 to pulsotype d , 2 to pulsotype e , 3 to pulsotype f , 11 to pulsotype k , 5 to pulsotype l , 8 to pulsotype m , and 3 ( all isolated in 2003 ) to 3 minor pulsotypes .", "all isolates were tested by sccmec element typing and multilocus sequence typing ( mlst ) ( 9 ) and were analyzed for the panton - valentine leukocidin ( pvl ) gene ( 10 ) and drug susceptibility to erythromycin , clindamycin , gentamicin , amikacin , ciprofloxacin , levofloxacin , tetracycline , trimethoprim - sulfamethoxazole , rifampin , and vancomycin by using the disk diffusion method ( 11 ) .", "isolates with pulsotype a are sequence type 254 ( st254 ) ; those with pulsotype b are st241 ; those with pulsotypes c , k , and l are st239 ; and those with pulsotypes d , e , f , and m are st59 , st 254 , st30 , and st5 , respectively .", "all mrsa isolates with pulsotypes a , d , e , and f have the type iv sccmec element .", "however , only isolates with pulsotypes d and f , as well as 2 isolates from 2003 with 2 minor pulsotypes , have the pvl gene .", "isolates with pulsotypes b and c have the type iii sccmec element , and isolates with pulsotype m have the type ii sccmec element . *", "p , pulsotype ; scc , staphylococcal cassette chromosome ; mlst , multilocus sequence typing ; pvl , panton - valentine leukocidin ; n , no ; y , yes .  ox , oxacillin ; em , erythromycin ; cl , clinidamycin ; gm , gentamicin ; am , amikacin ; cp , ciprofloxacin ; lv , levofloxacin ; tc , tetracycline ; ts , trimethoprim - sulfamethoxazole ; rf , rifampin ; va , vancomycin . ", "results of mlst and typing of sccmec elements of the 3 isolates with 3 minor pulsotypes obtained in 2003 are shown in table 2 .", "the correlation between sccmec element typing and mlst in this study corresponds to findings of previous reports ( 6,1214 ) .", "enright et al . identified st254-iv mrsa isolates in germany and the united kingdom ( 12 ) , and chongtrakool et al . identified st239-iii and st241-iii mrsa isolates in several asian countries ( 14 ) ."], ["we demonstrate that the predominant mrsa clone at ntuh in early 1990s had the type iv sccmec element . however , the predominant mrsa clones at ntuh from 1994 to 2003 had the type iii sccmec element .", "these findings differ from those of wisplinghoff et al . , who reported that that the sccmec element in predominant mrsa clones at their institute changed from type iii in 1984 to 1988 to type i in 1989 to 1998 ( 6 ) .", "mrsa isolates of pulsotypes b and c are more resistant than isolates of pulsotype a to certain antimicrobial drugs , especially fluoroquinolones and trimethoprim - sulfamethoxazole ; mrsa isolates with pulsotype c are more resistant to clindamycin but less resistant to rifampin than those with pulsotype b ( table 2 ) . from 1993 through 2000 , annual use of fluoroquinolones increased 3 at ntuh ; however , use of trimethoprim - sulfamethoxazole , clindamycin , and rifampin did not change ( 15 ) .", "therefore , the shift of predominant mrsa clones , which also led to the shift in types of sccmec elements at ntuh , might be caused by selective pressure from antimicrobial drugs , especially fluoroquinolones .", "the mrsa clone ( pulsotype a ) that predominated in 1992 and 1993 at ntuh has the type iv sccmec element .", "although the first study of mrsa with the type iv sccmec element reported that this element was found in community - acquired mrsa ( ca - mrsa ) ( 5 ) , some studies have reported mrsa isolates with this element in a hospital environment ( 12 ) . however , to our knowledge , these reports did not demonstrate that mrsa isolates with the type iv sccmec element became predominant among all mrsa isolates in an institution , especially before the mid-1990s .", "furthermore , 4 st59 mrsa isolates obtained in 1994 and 1996 and 3 st30 mrsa isolates obtained in 1992 and 1993 have the type iv sccmec element and pvl gene .", "recently , st59 mrsa isolates were found to cause ca - mrsa infection in taiwan ( 13 ) . among these st59", "ca - mrsa isolates , some had the type iv sccmec element , and others had the type v sccmec element ( 13 ) . although the type iv sccmec element could be transferred to ca - mrsa clones with other genetic backgrounds , our finding supports the possibility that st59 mrsa isolates with the sccmec element type iv in taiwan may originate from hospital strains but transfer into ca - mrsa strains .", "recently reported the results of sccmec typing of 615 mrsa isolates obtained in 1998 and 1999 from 11 asian countries ( 14 ) .", "the st239-iii , st241-iii , st254-ii , and st5-ii mrsa isolates were prevalent in many asian countries .", "however , the st254-iv , st30-iv , and st59-iv mrsa isolates from our study were not found in other asian countries .", "in addition , st254-iv mrsa isolates have been found in germany and the united kingdom ( 12 ) . whether st254-iv mrsa isolates in our study", "were transmitted from those in germany or the united kingdom by international travel requires further study .", "the first mrsa isolate with the type iv sccmec element in our hospital appeared as early as 1992 . the sccmec element carried by predominant mrsa clones changed from type iv to type iii sccmec element during the period 19922003 at ntuh .", "because the major mrsa clones isolated in 19942003 are more resistant to antimicrobial drugs , especially fluoroquinolones and trimethoprim - sulfamethoxazole , than those obtained in 1992 and 1993 , this shift may be caused by selective pressure from indiscriminate use of antimicrobial drugs ."]]}
{"article_id": "PMC4943822", "article_text": ["acute embolic limb ischemia is an urgent clinical condition in which sudden decrease or worsening in limb perfusion causes a potential threat to extremity viability and to life .", "thorough removal of all occlusive emboli in the arterial tree results in the best outcomes ; hence , some authors favored arteriography after embolectomy for lower limbs . \n ", "angiography is traditionally performed with iodinated contrast material , but carbon dioxide ( co2 ) has been studied as an alternative intravascular contrast agent for patients with iodine allergy or renal function impairment .", "since it is not nephrotoxic , it was considered reasonable to utilize this contrast in a patient with borderline renal function who would be submitted to the noxious stimuli of the reperfused limb after revascularization . \n  to the best of our knowledge ,", "this was the first case assessing the intraoperative use of co2 as a substitute for iodine contrast in a patient with known chronic kidney disease and an acute ischemic limb .", "a 79-year - old woman presented to the emergency department with acute limb ischemia in her left leg with a 2-hour history .", "past medical history was positive for hypertension , atrial fibrillation and non - dialysis - dependent chronic renal failure .", "two years before , she had been operated on for acute aortic embolic occlusion , with bilateral femoral catheter embolectomy .", "upon discharge she presented pedal pulses , but no posterior tibial pulses on either limb .", "medication use was irregular , and her international normalized ratio ( inr ) of prothrombin time upon admission was 1.1 .", "her pulse was arrhythmic ; she was hypertensive ( 160x110mmhg ) and presented with normal femoral , popliteal and pedal pulses on her right leg .", "on palpation in the left lower limb only a weak femoral pulse was observed ; there were no distal pulses .", "she was classified as grade iib limb ischemia ( immediately threatened ) and was immediately referred for surgery .", "preoperative exams revealed a diminished creatinine clearance ( cockroft - gault of 12ml / min ) .", "arteriotomy was performed and a 4-fr embolectomy catheter was utilized ( edwards lifesciences corp , irvine , california , usa ) .", "the catheter progressed more than 60 cm on the superficial artery , but could not be felt neither on pedal artery topography nor on posterior tibial artery topography .", "back bleeding was not significant , and a decision was made to perform an angiography to better assess the infra - popliteal arterial tree .", "a homemade water seal co2 delivery system was used , similar to another previously described , but with one modification to prevent room - air contamination .", "bloomington , indianopolis , usa ) inserted in the arteriotomy while the arteries were clamped . \n", "figure 1 shows a patent popliteal artery , a patent fibular artery and an occlusion on the mid - third of the anterior tibial artery .", "we hypothesized that the embolectomy catheter reached the fibular artery on the first attempt , and thus could not be felt on physical exam . \n", "patent popliteal and fibular arteries , occlusion in the mid - third of the anterior tibial artery \n  a new embolectomy was then performed .", "the catheter progressed to the foot , on the topography of the pedal artery ( i.e. , the catheter could be felt on the projection of the artery ) , and more thrombi were retrieved .", "control angiography ( figure 2 ) showed complete resolution of the anterior tibial artery occlusion , with contrast up to the foot ( figure 3 ) .", "a total of 28ml of co2 was used , and no iodine contrast was needed . \n", "the arteries were unclamped , and the popliteal and pedal pulses were noted to be present on the limb .", "ischemia signs ( e.g. , pallor , hypothermia ) were promptly resolved after arterial flow release .", "the patient remained in the intensive care unit ( icu ) until the 11 postoperative day . during this period ,", "the patient did not receive any nephrotoxic agent , such as vancomycin or vasoactive drugs .", "after icu discharge she developed a urinary tract infection , as well as deteriorating renal function and uremia .", "the patient developed pneumonia , which progressed to septic shock on the 36 day of her admission .", "she required vasoactive drugs for 2 days at the icu and , unfortunately , her renal function was never fully recovered .", "she was discharged from hospital to a hospice care facility with a pedal pulse and no deficit on her limb , though requiring chronic dialysis .", "treatment of embolic arterial occlusion with severe limb ischemia is well defined and involves the use of an embolectomy catheter as the best alternative . \n ", "co2 has been used electively in both diagnostic and therapeutic procedures in the femoropopliteal tree , and is reported to be safe and non - nephrotoxic . \n ", "intraoperative angiography was beneficial because it allowed the diagnosis of the arterial tree occlusion after the initial embolectomy .", "additional removal of clots was performed and the result was documented as complete patency of the anterior tibial artery to the foot .", "our goal , when using co2 , was to decrease the need for postoperative dialysis .", "probably , in this case , the deleterious stimuli ( surgical stress , urinary tract infection and septic shock due to pneumonia ) contributed to renal failure and subsequent need for dialysis . the countermeasure ( such as the use of non - nephrotoxic contrast )", "if we had used the iodinated medium , we might wonder if something could have been done differently to avoid dialysis .", "we consider co2 to be an alternative to iodine contrast when the decision to perform angiography is made in the acutely ischemic limb . to the best of our knowledge", ", this agent has never been described in this clinical setting ; it presents good quality imaging , is not nephrotoxic and does not increase the risk for the limb or the patient in acute arterial occlusions .", "this approach may prove to be fruitful in patients with borderline renal function , thereby reducing the risk of short or long - term need for dialysis .", "a isquemia aguda de membro de origem emblica  uma situao clnica de carter urgente , em que ocorre uma diminuio ou piora sbita na perfuso de um membro , e configura um risco  viabilidade da extremidade e  vida .", "o tratamento consiste em revascularizao imediata com um cateter de embolectomia . a remoo completa de todos os mbolos oclusivos na rvore arterial leva aos melhores desfechos ; por isso", ", alguns autores defendem a arteriografia aps a embolectomia , para os membros inferiores . \n  a angiografia  tradicionalmente realizada com contraste iodado , mas o dixido de carbono ( co2 ) foi estudado como um agente de contraste intravascular alternativo para pacientes com alergia a iodo ou alterao na funo renal .", "como o co2 no  nefrotxico , julgou - se razovel utilizar esse contraste em uma paciente com funo renal limtrofe ( borderline ) que seria submetida aos estmulos nocivos de reperfuso do membro , aps a revascularizao . \n ", "em nosso entender , este foi o primeiro caso que avalia o uso intraoperatrio de co2 como um substituto de contraste iodado em um paciente com doena renal crnica conhecida e isquemia aguda de membro .", "paciente do sexo feminino , 79 anos , chegou ao pronto - socorro com isquemia aguda no membro inferior esquerdo , com durao de 2 horas .", "como antecedentes , apresentava hipertenso , fibrilao atrial e insuficincia renal crnica , no dependente de dilise .", "a paciente tinha sido operada 2 anos antes , por ocluso artica aguda de origem emblica , quando foi realizada embolectomia femoral bilateral com cateter .", " alta , os pulsos pediosos eram palpveis , mas os tibiais posteriores eram ausentes , bilateralmente .", "a paciente fazia uso irregular dos medicamentos , e o tempo de protrombina ( inr , sigla do ingls international normalized ratio ) era de 1,1 . o pulso estava arrtmico ; era hipertensa ( 160x110mmhg ) e apresentava pulsos femoral , poplteo e distal normais , na perna direita .", " palpao do membro inferior esquerdo , havia apenas um pulso femoral fraco ; os pulsos distais estavam ausentes . a paciente apresentava dor e dfice motor .", "ela foi considerada como portadora de isquemia de membro grau iib ( risco eminente ) e foi encaminhada imediatamente para cirurgia .", "o clearance de creatinina estava diminudo nos exames pr - operatrios ( cockroft - gault de 12ml / min ) .", "sob anestesia geral , foi realizado acesso femoral na cicatriz da inciso anterior do membro inferior esquerdo . as artrias femorais comum , superficial e profunda foram dissecadas e clampeadas .", "foi feita arteriotomia e uso de cateter de embolectomia de 4fr ( edwards lifesciences corp , irvine , califrnia , estados unidos ) .", "os trombos foram removidos das artrias femorais superficial e profunda . o cateter avanou mais de 60 cm na artria femoral superficial , mas no pde ser palpado . o fluxo retrgrado no foi significativo e decidiu - se realizar uma angiografia para avaliar melhor a rvore arterial infrapopltea .", "foi escolhido o co2 como substituto ao contraste iodado para a angiografia . utilizou - se um sistema home - made com fornecimento de co2 em selo dgua , semelhante a outro descrito anteriormente , mas com uma modificao para evitar a contaminao do ar ambiente .", "bloomington , indianpolis , estados unidos ) inserido na arteriotomia , quando as artrias foram pinadas .", "a figura 1 mostra uma artria popltea e uma fibular prvias , e uma ocluso no tero mdio da artria tibial anterior .", "levantamos a hiptese de o cateter de embolectomia ter alcanado a artria fibular na primeira tentativa , e por isso no ter sido palpvel ao exame fsico . \n", "artrias popltea e fibular prvias , ocluso no tero mdio da artria tibial anterior \n  realizou - se , ento , nova embolectomia .", "o cateter avanou at o p , na topografia da artria pediosa ( isto  , o cateter pde ser palpado na projeo da artria ) e mais trombos foram removidos . a angiografia de", "controle ( figura 2 ) mostrou resoluo completa da ocluso da artria tibial anterior , com contraste fluindo at o p ( figura 3 ) .", "foram usados 28ml de co2 , e no foi necessrio aplicar o contraste iodado . \n", "resoluo completa da ocluso da artria tibial anterior \n \n figura 3arteriografia com dixido de carbono .", "chegada de contraste na artria tibial anterior at o p \n  foi realizada a arteriorrafia . as artrias foram desclampeadas ,", "os sinais de isquemia ( palidez e hipotermia ) foram imediatamente resolvidos aps a liberao do fluxo arterial .", "a paciente no desenvolveu sinais de sndrome compartimental . a paciente permaneceu na unidade de terapia intensiva ( uti ) at o 11 dia ps - operatrio .", "durante esse perodo , no recebeu nenhum agente nefrotxico , como vancomicina ou drogas vasoativas .", "aps a alta da uti , desenvolveu infeco urinria , piora da funo renal e uremia .", "iniciou dilise no 18 dia ps - operatrio . a paciente teve pneumonia , que evoluiu para choque sptico no 36 dia de internao .", "necessitou de drogas vasoativas por 2 dias na uti e , infelizmente , nunca mais recuperou a funo renal .", "teve alta hospitalar e foi para instituio de cuidados terminais , com pulso distal sem dfice no membro inferior , mas em dilise crnica .", "o tratamento da ocluso arterial emblica com isquemia grave de membro est bem definido , sendo o uso de cateter de embolectomia a melhor alternativa . \n ", "o co2 tem sido empregado de forma eletiva tanto nos procedimentos diagnsticos como nos teraputicos na rvore fmoro - popltea , sendo descrito como seguro e no nefrotxico . \n  a angiografia intraoperatria foi benfica , j que permitiu o diagnstico de ocluso da rvore arterial aps a primeira embolectomia . mais cogulos foram removidos , e o resultado documentado foi a permeabilidade completa da artria tibial anterior at o p .", "nosso objetivo , ao utilizar co2 , foi o de diminuir a necessidade de dilise no ps - operatrio .", "neste caso , provavelmente , os estmulos prejudiciais ( estresse cirrgico , infeco do trato urinrio e choque sptico por pneumonia ) contriburam para a insuficincia renal e para consequente necessidade de dilise . a contramedida ( como o uso de contraste no nefrotxico ) poderia ter prevenido a insuficincia renal , se fosse esta uma situao nica .", "se tivssemos usado o contraste iodado , pensaramos se algo diferente poderia ter sido feito para evitar dilise .", "consideramos o co2 como uma alternativa ao contraste iodado quando  necessria a angiografia para isquemia aguda de membro .", "em nosso entender , esse agente nunca foi descrito nessa situao clnica , proporcionando boa qualidade de imagem , no sendo nefrotxico , nem aumentado o risco do membro ou do paciente , em casos de ocluses arteriais agudas .", "essa abordagem pode se mostrar til em pacientes com funo renal limtrofe ( borderline ) , reduzindo , assim , o risco de precisar de dilise a curto ou longo prazo ."], "abstract_text": ["<S> acute limb ischemia can be potentially harmful to the limb and life threatening . </S>", "<S> renal failure is a possible outcome associated with release of products of ischemic limb reperfusion . </S>", "<S> some authors reported the benefit of performing angiography after embolectomy , even though iodine contrast is also nephrotoxic . </S>", "<S> we report a case of embolectomy on a patient with renal insufficiency in whom carbon dioxide was used as a substitute for iodine contrast . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "INTRODUO", "RELATO DE CASO", "DISCUSSO"], "sections": [["acute embolic limb ischemia is an urgent clinical condition in which sudden decrease or worsening in limb perfusion causes a potential threat to extremity viability and to life .", "thorough removal of all occlusive emboli in the arterial tree results in the best outcomes ; hence , some authors favored arteriography after embolectomy for lower limbs . \n ", "angiography is traditionally performed with iodinated contrast material , but carbon dioxide ( co2 ) has been studied as an alternative intravascular contrast agent for patients with iodine allergy or renal function impairment .", "since it is not nephrotoxic , it was considered reasonable to utilize this contrast in a patient with borderline renal function who would be submitted to the noxious stimuli of the reperfused limb after revascularization . \n  to the best of our knowledge ,", "this was the first case assessing the intraoperative use of co2 as a substitute for iodine contrast in a patient with known chronic kidney disease and an acute ischemic limb ."], ["a 79-year - old woman presented to the emergency department with acute limb ischemia in her left leg with a 2-hour history .", "past medical history was positive for hypertension , atrial fibrillation and non - dialysis - dependent chronic renal failure .", "two years before , she had been operated on for acute aortic embolic occlusion , with bilateral femoral catheter embolectomy .", "upon discharge she presented pedal pulses , but no posterior tibial pulses on either limb .", "medication use was irregular , and her international normalized ratio ( inr ) of prothrombin time upon admission was 1.1 .", "her pulse was arrhythmic ; she was hypertensive ( 160x110mmhg ) and presented with normal femoral , popliteal and pedal pulses on her right leg .", "on palpation in the left lower limb only a weak femoral pulse was observed ; there were no distal pulses .", "she was classified as grade iib limb ischemia ( immediately threatened ) and was immediately referred for surgery .", "preoperative exams revealed a diminished creatinine clearance ( cockroft - gault of 12ml / min ) .", "arteriotomy was performed and a 4-fr embolectomy catheter was utilized ( edwards lifesciences corp , irvine , california , usa ) .", "the catheter progressed more than 60 cm on the superficial artery , but could not be felt neither on pedal artery topography nor on posterior tibial artery topography .", "back bleeding was not significant , and a decision was made to perform an angiography to better assess the infra - popliteal arterial tree .", "a homemade water seal co2 delivery system was used , similar to another previously described , but with one modification to prevent room - air contamination .", "bloomington , indianopolis , usa ) inserted in the arteriotomy while the arteries were clamped . \n", "figure 1 shows a patent popliteal artery , a patent fibular artery and an occlusion on the mid - third of the anterior tibial artery .", "we hypothesized that the embolectomy catheter reached the fibular artery on the first attempt , and thus could not be felt on physical exam . \n", "patent popliteal and fibular arteries , occlusion in the mid - third of the anterior tibial artery \n  a new embolectomy was then performed .", "the catheter progressed to the foot , on the topography of the pedal artery ( i.e. , the catheter could be felt on the projection of the artery ) , and more thrombi were retrieved .", "control angiography ( figure 2 ) showed complete resolution of the anterior tibial artery occlusion , with contrast up to the foot ( figure 3 ) .", "a total of 28ml of co2 was used , and no iodine contrast was needed . \n", "the arteries were unclamped , and the popliteal and pedal pulses were noted to be present on the limb .", "ischemia signs ( e.g. , pallor , hypothermia ) were promptly resolved after arterial flow release .", "the patient remained in the intensive care unit ( icu ) until the 11 postoperative day . during this period ,", "the patient did not receive any nephrotoxic agent , such as vancomycin or vasoactive drugs .", "after icu discharge she developed a urinary tract infection , as well as deteriorating renal function and uremia .", "the patient developed pneumonia , which progressed to septic shock on the 36 day of her admission .", "she required vasoactive drugs for 2 days at the icu and , unfortunately , her renal function was never fully recovered .", "she was discharged from hospital to a hospice care facility with a pedal pulse and no deficit on her limb , though requiring chronic dialysis ."], ["treatment of embolic arterial occlusion with severe limb ischemia is well defined and involves the use of an embolectomy catheter as the best alternative . \n ", "co2 has been used electively in both diagnostic and therapeutic procedures in the femoropopliteal tree , and is reported to be safe and non - nephrotoxic . \n ", "intraoperative angiography was beneficial because it allowed the diagnosis of the arterial tree occlusion after the initial embolectomy .", "additional removal of clots was performed and the result was documented as complete patency of the anterior tibial artery to the foot .", "our goal , when using co2 , was to decrease the need for postoperative dialysis .", "probably , in this case , the deleterious stimuli ( surgical stress , urinary tract infection and septic shock due to pneumonia ) contributed to renal failure and subsequent need for dialysis . the countermeasure ( such as the use of non - nephrotoxic contrast )", "if we had used the iodinated medium , we might wonder if something could have been done differently to avoid dialysis .", "we consider co2 to be an alternative to iodine contrast when the decision to perform angiography is made in the acutely ischemic limb . to the best of our knowledge", ", this agent has never been described in this clinical setting ; it presents good quality imaging , is not nephrotoxic and does not increase the risk for the limb or the patient in acute arterial occlusions .", "this approach may prove to be fruitful in patients with borderline renal function , thereby reducing the risk of short or long - term need for dialysis ."], ["a isquemia aguda de membro de origem emblica  uma situao clnica de carter urgente , em que ocorre uma diminuio ou piora sbita na perfuso de um membro , e configura um risco  viabilidade da extremidade e  vida .", "o tratamento consiste em revascularizao imediata com um cateter de embolectomia . a remoo completa de todos os mbolos oclusivos na rvore arterial leva aos melhores desfechos ; por isso", ", alguns autores defendem a arteriografia aps a embolectomia , para os membros inferiores . \n  a angiografia  tradicionalmente realizada com contraste iodado , mas o dixido de carbono ( co2 ) foi estudado como um agente de contraste intravascular alternativo para pacientes com alergia a iodo ou alterao na funo renal .", "como o co2 no  nefrotxico , julgou - se razovel utilizar esse contraste em uma paciente com funo renal limtrofe ( borderline ) que seria submetida aos estmulos nocivos de reperfuso do membro , aps a revascularizao . \n ", "em nosso entender , este foi o primeiro caso que avalia o uso intraoperatrio de co2 como um substituto de contraste iodado em um paciente com doena renal crnica conhecida e isquemia aguda de membro ."], ["paciente do sexo feminino , 79 anos , chegou ao pronto - socorro com isquemia aguda no membro inferior esquerdo , com durao de 2 horas .", "como antecedentes , apresentava hipertenso , fibrilao atrial e insuficincia renal crnica , no dependente de dilise .", "a paciente tinha sido operada 2 anos antes , por ocluso artica aguda de origem emblica , quando foi realizada embolectomia femoral bilateral com cateter .", " alta , os pulsos pediosos eram palpveis , mas os tibiais posteriores eram ausentes , bilateralmente .", "a paciente fazia uso irregular dos medicamentos , e o tempo de protrombina ( inr , sigla do ingls international normalized ratio ) era de 1,1 . o pulso estava arrtmico ; era hipertensa ( 160x110mmhg ) e apresentava pulsos femoral , poplteo e distal normais , na perna direita .", " palpao do membro inferior esquerdo , havia apenas um pulso femoral fraco ; os pulsos distais estavam ausentes . a paciente apresentava dor e dfice motor .", "ela foi considerada como portadora de isquemia de membro grau iib ( risco eminente ) e foi encaminhada imediatamente para cirurgia .", "o clearance de creatinina estava diminudo nos exames pr - operatrios ( cockroft - gault de 12ml / min ) .", "sob anestesia geral , foi realizado acesso femoral na cicatriz da inciso anterior do membro inferior esquerdo . as artrias femorais comum , superficial e profunda foram dissecadas e clampeadas .", "foi feita arteriotomia e uso de cateter de embolectomia de 4fr ( edwards lifesciences corp , irvine , califrnia , estados unidos ) .", "os trombos foram removidos das artrias femorais superficial e profunda . o cateter avanou mais de 60 cm na artria femoral superficial , mas no pde ser palpado . o fluxo retrgrado no foi significativo e decidiu - se realizar uma angiografia para avaliar melhor a rvore arterial infrapopltea .", "foi escolhido o co2 como substituto ao contraste iodado para a angiografia . utilizou - se um sistema home - made com fornecimento de co2 em selo dgua , semelhante a outro descrito anteriormente , mas com uma modificao para evitar a contaminao do ar ambiente .", "bloomington , indianpolis , estados unidos ) inserido na arteriotomia , quando as artrias foram pinadas .", "a figura 1 mostra uma artria popltea e uma fibular prvias , e uma ocluso no tero mdio da artria tibial anterior .", "levantamos a hiptese de o cateter de embolectomia ter alcanado a artria fibular na primeira tentativa , e por isso no ter sido palpvel ao exame fsico . \n", "artrias popltea e fibular prvias , ocluso no tero mdio da artria tibial anterior \n  realizou - se , ento , nova embolectomia .", "o cateter avanou at o p , na topografia da artria pediosa ( isto  , o cateter pde ser palpado na projeo da artria ) e mais trombos foram removidos . a angiografia de", "controle ( figura 2 ) mostrou resoluo completa da ocluso da artria tibial anterior , com contraste fluindo at o p ( figura 3 ) .", "foram usados 28ml de co2 , e no foi necessrio aplicar o contraste iodado . \n", "resoluo completa da ocluso da artria tibial anterior \n \n figura 3arteriografia com dixido de carbono .", "chegada de contraste na artria tibial anterior at o p \n  foi realizada a arteriorrafia . as artrias foram desclampeadas ,", "os sinais de isquemia ( palidez e hipotermia ) foram imediatamente resolvidos aps a liberao do fluxo arterial .", "a paciente no desenvolveu sinais de sndrome compartimental . a paciente permaneceu na unidade de terapia intensiva ( uti ) at o 11 dia ps - operatrio .", "durante esse perodo , no recebeu nenhum agente nefrotxico , como vancomicina ou drogas vasoativas .", "aps a alta da uti , desenvolveu infeco urinria , piora da funo renal e uremia .", "iniciou dilise no 18 dia ps - operatrio . a paciente teve pneumonia , que evoluiu para choque sptico no 36 dia de internao .", "necessitou de drogas vasoativas por 2 dias na uti e , infelizmente , nunca mais recuperou a funo renal .", "teve alta hospitalar e foi para instituio de cuidados terminais , com pulso distal sem dfice no membro inferior , mas em dilise crnica ."], ["o tratamento da ocluso arterial emblica com isquemia grave de membro est bem definido , sendo o uso de cateter de embolectomia a melhor alternativa . \n ", "o co2 tem sido empregado de forma eletiva tanto nos procedimentos diagnsticos como nos teraputicos na rvore fmoro - popltea , sendo descrito como seguro e no nefrotxico . \n  a angiografia intraoperatria foi benfica , j que permitiu o diagnstico de ocluso da rvore arterial aps a primeira embolectomia . mais cogulos foram removidos , e o resultado documentado foi a permeabilidade completa da artria tibial anterior at o p .", "nosso objetivo , ao utilizar co2 , foi o de diminuir a necessidade de dilise no ps - operatrio .", "neste caso , provavelmente , os estmulos prejudiciais ( estresse cirrgico , infeco do trato urinrio e choque sptico por pneumonia ) contriburam para a insuficincia renal e para consequente necessidade de dilise . a contramedida ( como o uso de contraste no nefrotxico ) poderia ter prevenido a insuficincia renal , se fosse esta uma situao nica .", "se tivssemos usado o contraste iodado , pensaramos se algo diferente poderia ter sido feito para evitar dilise .", "consideramos o co2 como uma alternativa ao contraste iodado quando  necessria a angiografia para isquemia aguda de membro .", "em nosso entender , esse agente nunca foi descrito nessa situao clnica , proporcionando boa qualidade de imagem , no sendo nefrotxico , nem aumentado o risco do membro ou do paciente , em casos de ocluses arteriais agudas .", "essa abordagem pode se mostrar til em pacientes com funo renal limtrofe ( borderline ) , reduzindo , assim , o risco de precisar de dilise a curto ou longo prazo ."]]}
{"article_id": "PMC2693866", "article_text": ["emphysematous gastritis , which was first described by fraenkel in 1889 ( 1 ) , is a rare disease characterized by the presence of air within the wall of the stomach and diffuse gastric wall inflammation by gas - forming bacteria , which may be also life - threatening due to systemic toxicity .", "predisposing factors include the ingestion of corrosives , alcohol abuse , recent abdominal surgery , diabetes , and immunocompromised conditions ( 1 - 3 ) .", "since 1889 , only 51 cases have been reported worldwide ( 1 - 8 ) .", "mucormycosis is a rare fungal infection that usually involves the nasopharynx ( 9 ) . among the several forms ,", "gastrointestinal mucormycosis is very rare , and the manifestations range from colonization of peptic ulcers to infiltrative disease with vascular invasion and dissemination ( 9 ) . commonly associated conditions include diabetes mellitus , lymphoma , leukemia , renal disease , septicemia , malnutrition , and long - term treatment with steroids and antibiotics ( 10 ) . among the reported cases of emphysematous gastritis , fungal organisms such as candida species have been isolated rarely ( 4 , 11 - 15 ) .", "however , emphysematous gastritis associated with mucormycotic infection is extremely rare , and only one case has been reported in the english literature ( 15 ) .", "here we report a case of emphysematous gastritis , diagnosed by computed tomography ( ct ) and confirmed by histopathologic findings associated with invasive gastric mucormycosis that showed angioinvasion , necrosis , ultimately disseminated to the colon and liver , and was fatal despite medical and surgical treatment . to our knowledge , this is the first report of a patient with emphysematous gastritis associated with invasive gastric mucormycosis in korea .", "a 43-yr old man was admitted to the emergency room because of diffuse abdominal pain , indigestion , and poor oral intake for 4 days . he had had a 1-yr history of hypertension ,", "there was also a history of excessive ingestion of alcohol and heavy smoking for 20 yr , but there was no history of peptic ulcer disease .", "his blood pressure was 160/100 mmhg , with a body temperature of 36.2 , pulse rate of 98/min , and respiration rate of 26/min .", "abdominal examination revealed decreased bowel sounds and severe tenderness in the epigastrium , with rigidity and rebound tenderness .", "hematological and biochemical examinations on admission showed hemoglobin 13.9 g / dl ( normal : 13 - 18 ) , white blood cell 11.7710/l ( 3.6 - 11 ) , platelet 21010/l ( 150 - 450 ) , blood sugar 816 mg / dl ( 70 - 110 ) , aspartate aminotransferase 148 iu / l ( 5 - 38 ) , alanine aminotransferase 76", "iu / l ( 4 - 43 ) , lactate dehydrogenase 1,320 iu / l ( 180 - 460 ) , amylase 6 u / l ( 45 - 108 ) , blood urea nitrogen 37.7 mg / dl ( 8 - 23 ) , and creatinine 1.6 mg / dl ( 0.5 - 1.2 ) , and blood acetone - positive .", "chest radiography revealed collapse and consolidation in the basal part of the left lower lobe , suggesting pneumonia .", "abdominal ct scan revealed gastric wall thickening with a collection of dirty air bubbles and a small amount of pneumoperitoneum ( fig .", "operation findings showed nearly total necrosis of stomach wall with dark green colored pus collection around the stomach .", "grossly , the stomach had a large area of necrosis with black discoloration of the mucosa ( fig .", "the remaining viable stomach wall showed large numbers of neutrophilis in the edematous submucosa and muscle layer .", "there were several gas - filled bubbles in the submucosa and muscle layer associated with numerous scattered or colonies of gram - negative bacilli , consistent with emphysematous gastritis ( fig .", "1 ) . at the same time , there were numerous broad - based , non - septate , right angular branched fungal hyphae , morphologically consistent with mucormycosis , detected on hematoxyline and eosin stain as well as silver stains throughout both necrotic and viable stomach wall ( fig .", "culture of pus in the peritoneum revealed klebsiella pneumonia , staphylococcus aureus , and pseudomonas aeruginosa .", "amphotericin b infusion was added after operation . on the fifth hospital day , there was pus discharge from the wound site .", "however , the patient remained to have non - toxic appearance with stable vital signs . on the seventh hospital day", ", he complained of diarrhea and vague abdominal discomfort . over the following 2 days ,", "follow - up ct revealed an approximately 14.012.012.0 cm - sized , irregularly shaped air - containing fluid collection at the gastrectomy site , suggesting air - forming postoperative abscess and anastomotic site leakage .", "reoperation was performed , and the operative findings showed multiple perforations with necrosis in the transverse colon , necrotic change in the liver , hematoma with fluid collection in the peritoneal cavity , and leakage in the anastomotic site .", "rouxen - y esophagojejunostomy with cecocolostomy and segmental resection of the large intestine were performed . the submitted pathological specimen consisted of segments of colon , small intestine , and a portion of liver .", "the gross examination of the segment of colon showed multiple perforations , dirty serosa , and necrotic mucosa with greenish black exudates .", "microscopic examination of the colon and small intestine showed ischemic necrosis of mucosa with acute and chronic inflammation , and foreign body reaction .", "microscopic examination of the liver also showed totally necrotic parenchyma with many angioinvasive mucormycotic fungal hyphae .", "auscultation of the lungs revealed decreased breathing sounds with rale on both lung fields , and saturation of arterial blood gas analysis was decreased .", "he underwent intubation and ventilation . on the 21st - hospital day , the patient expired .", "clinically this condition is divided into gastric emphysema and emphysematous gastritis ( 3 , 5 , 12 ) .", "these two conditions should be differentiated because they are characterized by different clinical symptoms , possible etiology , radiologic findings , treatment , and prognosis ( 3 , 5 , 12 ) . in gastric emphysema , air enters the stomach wall from the lumen , from the peritoneal surface , or from its connections with the esophagus and duodenum , resulting from barotraumas in the absence of bacterial infection ( 12 ) .", "radiologically , in contrast to emphysematous gastritis , a more linear distribution of gas in the gastric wall is characteristic of gastric emphysema ( 3 ) .", "the symptom of acute abdomen as seen in patients with emphysematous gastritis is usually absent in patients with gastric emphysema ( 5 ) . in general ,", "gastric emphysema is asymptomatic ; its course is usually benign and resolves spontaneously without treatment ( 3 , 5 , 12 ) . on the other hand , emphysematous gastritis , the gas formed in situ by gas - forming bacteria invading the gastric wall , results in a necrotizing inflammation of the gastric wall . in 1889", ", fraenkel first reported a young man who died after several attacks of severe abdominal pain , blood vomitus , and diarrhea ( 1 ) . at autopsy ,", "originally , the stomach wall is well protected from bacterial infection by the close connection between cells , acidic ph , and good blood supply ( 3 , 12 ) .", "the predisposing factors , leading to the breakdown of these defenses , are ingestion of corrosives , alcohol abuse , diabetes , recent abdominal surgery , gastroenteritis , immunocompromised conditions , and treatment with non - steroidal anti - inflammatory drugs ( 1 - 3 ) . in the present case ,", "the patient had a history of alcohol abuse , which might have altered the unstirred mucous layer , and diabetes due to chronic pancreatitis , which might have led to a systemic predisposition to infection .", "patients with emphysematous gastritis typically develop acute abdominal pain , as was the case in our patient , usually developing 1 week after initiation , accompanied by diarrhea , nausea , vomiting , and occasionally hematemesis and melena ( 1 , 4 , 12 ) .", "physical examination of the abdomen reveals epigastric tenderness , distension , and decreased bowel sounds ( 4 , 12 ) .", "abdominal computed tomography , the best imaging modality to establish the diagnosis , reveals gastric wall thickening and intramural gas ( 1 , 3 , 12 ) .", "in contrast to gastric emphysema , the gas in the wall is in the form of irregular mottled bubbles or spots , especially around the fundus and the greater curvature , and remains in place despite the position of the body or absorption through the gastric tube ( 3 , 4 , 12 ) .", "organisms that have been cultured from stomach aspirates , blood , and peritoneal fluid include streptococci , e. coli , p. aeruginosa , clostridium .", "perfringens , and s. aureus ( 1 , 4 ) . among the previously reported cases , those associated with fungal infection were rare ( 4", ", 11 - 15 ) . of these , most cases were candida species ( 4 , 11 , 12 , 15 ) .", "pathologically , the gas forming organisms in emphysematous gastritis have been shown to infiltrate the stomach wall diffusely and there are edematous stomach wall - containing gas bubbles ( 1 , 6 ) . the therapeutic approach for emphysematous gastritis included initial stabilization of the septic condition through vigorous fluid resuscitation and early empirical parenteral antibiotic therapy with a broad range of antibiotics covering gram - negative and anaerobic bacteria ( 2 , 3 , 12 , 14 ) .", "specific therapy should be modified according to the results of the gastric fluid culture and sensitivity testing of the isolated organism ( 12 ) .", "indication for emergency surgery include deterioration despite optimal medical management , involvement of a large portion of or the entire stomach , presence of gastric infarction , or perforation ( 3 , 4 ) . despite meticulous treatment ,", "the mortality rate of emphysematous gastritis is about 60% , and the morbidity near 21% ( 2 ) .", "mucormycosis is a rare , opportunistic fungal infection that occurs almost exclusively in immunocompromised hosts such as patients with diabetes , leukemia , lymphoma , renal disease , septicemia , burns , malnutrition , and following long - term treatment with steroids and antibiotics ( 10 ) .", "clinical manifestations of mucormycosis can be categorized as rhinocerebral , pulmonary , disseminated , gastrointestinal , and cutaneous ( 9 ) . among these ,", "rhinocerebral and pulmonary disease are the most common forms , and gastrointestinal involvement is very rare , accounting for only 7% of all cases ( 16 ) .", "other associated conditions are immunosuppressed conditions such as renal failure , hematological malignancies , cirrhosis , and solid organ transplantation ( 16 ) .", "it is believed that infection of the alimentary tract is acquired through direct ingestion of and invasion by fungal spores .", "the gastrointestinal organ most frequently involved is the stomach , followed by the colon , small intestine , and esophagus ( 16 ) .", "when they invade , the lesion extends and marked surrounding induration develops , with a shaggy , velvety discolored surface or large plaqe - like areas of green and blacked eschar ( 16 ) .", "they also invade blood vessels and thus tend to cause extensive thrombosis , necrosis , and , ultimately , dissemination ( 10 ) .", "association with emphysematous gastritis is an extremely rare , and only one such case has been reported ( 15 ) .", "mucormycosis is not gas - forming , therefore this infection is not a direct cause of emphysematous gastritis .", "gastric wall necrosis caused by bacterial infection may be predisposed to the invasion by mucormycosis . alternatively , it is possible that invasive mucormycosis with subsequent gastric wall necrosis led to secondary bacterial invasion and intramural gas production ( 15 ) .", "successful management of mucormycosis includes aggressive metabolic support , amphotericin b , and surgical debridement of all necrotic involved tissue ( 15 , 16 ) . as in emphysematous gastritis , the prognosis of gastrointestinal mucormycosis is poor .", "forty - two cases of gastric mucormycosis have been described in the literature thus for , with a mortality above 98% ( 15 ) .", "we report an extremely rare case of emphysematous gastritis associated with invasive gastric mucormycosis in a 43-yr - old man . heavy alcohol abuse and diabetes mellitus were the predisposing factors .", "ct is the diagnostic procedure of choice of emphysematous gastritis and helps in differentiating with gastric emphysema .", "moreover , the gastric mucormycosis showed angioinvasion , necrosis , and ultimately , disseminate to colon and liver . in the case of mucormycosis , biopsy of involved areas", "this patient also had a fatal outcome despite antibiotic and antifungal therapy and surgery . increased awareness of these disease entities may facilitate early diagnosis , prompt medical therapy , and appropriate surgical intervention , and ultimately improve survival rates ."], "abstract_text": ["<S> emphysematous gastritis is a rare form of phlegmonous gastritis , characterized by air in the wall of the stomach due to invasion by gas - forming microorganisms . </S>", "<S> the most commonly involved microorganisms are streptococci , escherichia coli , pseudomonas aeruginosa , clostrodium perfrigens and staphylococcus aureus . </S>", "<S> gastrointestinal mucormycosis is another rare condition , which is most frequently occurs in the stomach . because emphysematous gastritis associated with invasive gastric mucormycosis is an extremely rare clinical condition and both are </S>", "<S> life - threatening diseases , early precise diagnosis and early treatment should be done to avoid mortality . </S>", "<S> herein we present an extremely rare case of emphysematous gastritis associated with invasive gastric mucormycosis . </S>", "<S> a 43-yr - old man , suffering from alcoholism and diabetes , has experienced diffuse abdominal pain for 4 days . </S>", "<S> abdominal computed tomography scan demonstrated gas within the stomach wall . </S>", "<S> a histologic examination of the total gastrectomy specimen showed several gas - filled bubbles in the wall , along with numerous fungal hyphae throughout the necrotic stomach wall . </S>", "<S> he died of multiorgan failure secondary to disseminated mucormycosis , despite the intensive medical therapy . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["emphysematous gastritis , which was first described by fraenkel in 1889 ( 1 ) , is a rare disease characterized by the presence of air within the wall of the stomach and diffuse gastric wall inflammation by gas - forming bacteria , which may be also life - threatening due to systemic toxicity .", "predisposing factors include the ingestion of corrosives , alcohol abuse , recent abdominal surgery , diabetes , and immunocompromised conditions ( 1 - 3 ) .", "since 1889 , only 51 cases have been reported worldwide ( 1 - 8 ) .", "mucormycosis is a rare fungal infection that usually involves the nasopharynx ( 9 ) . among the several forms ,", "gastrointestinal mucormycosis is very rare , and the manifestations range from colonization of peptic ulcers to infiltrative disease with vascular invasion and dissemination ( 9 ) . commonly associated conditions include diabetes mellitus , lymphoma , leukemia , renal disease , septicemia , malnutrition , and long - term treatment with steroids and antibiotics ( 10 ) . among the reported cases of emphysematous gastritis , fungal organisms such as candida species have been isolated rarely ( 4 , 11 - 15 ) .", "however , emphysematous gastritis associated with mucormycotic infection is extremely rare , and only one case has been reported in the english literature ( 15 ) .", "here we report a case of emphysematous gastritis , diagnosed by computed tomography ( ct ) and confirmed by histopathologic findings associated with invasive gastric mucormycosis that showed angioinvasion , necrosis , ultimately disseminated to the colon and liver , and was fatal despite medical and surgical treatment . to our knowledge , this is the first report of a patient with emphysematous gastritis associated with invasive gastric mucormycosis in korea ."], ["a 43-yr old man was admitted to the emergency room because of diffuse abdominal pain , indigestion , and poor oral intake for 4 days . he had had a 1-yr history of hypertension ,", "there was also a history of excessive ingestion of alcohol and heavy smoking for 20 yr , but there was no history of peptic ulcer disease .", "his blood pressure was 160/100 mmhg , with a body temperature of 36.2 , pulse rate of 98/min , and respiration rate of 26/min .", "abdominal examination revealed decreased bowel sounds and severe tenderness in the epigastrium , with rigidity and rebound tenderness .", "hematological and biochemical examinations on admission showed hemoglobin 13.9 g / dl ( normal : 13 - 18 ) , white blood cell 11.7710/l ( 3.6 - 11 ) , platelet 21010/l ( 150 - 450 ) , blood sugar 816 mg / dl ( 70 - 110 ) , aspartate aminotransferase 148 iu / l ( 5 - 38 ) , alanine aminotransferase 76", "iu / l ( 4 - 43 ) , lactate dehydrogenase 1,320 iu / l ( 180 - 460 ) , amylase 6 u / l ( 45 - 108 ) , blood urea nitrogen 37.7 mg / dl ( 8 - 23 ) , and creatinine 1.6 mg / dl ( 0.5 - 1.2 ) , and blood acetone - positive .", "chest radiography revealed collapse and consolidation in the basal part of the left lower lobe , suggesting pneumonia .", "abdominal ct scan revealed gastric wall thickening with a collection of dirty air bubbles and a small amount of pneumoperitoneum ( fig .", "operation findings showed nearly total necrosis of stomach wall with dark green colored pus collection around the stomach .", "grossly , the stomach had a large area of necrosis with black discoloration of the mucosa ( fig .", "the remaining viable stomach wall showed large numbers of neutrophilis in the edematous submucosa and muscle layer .", "there were several gas - filled bubbles in the submucosa and muscle layer associated with numerous scattered or colonies of gram - negative bacilli , consistent with emphysematous gastritis ( fig .", "1 ) . at the same time , there were numerous broad - based , non - septate , right angular branched fungal hyphae , morphologically consistent with mucormycosis , detected on hematoxyline and eosin stain as well as silver stains throughout both necrotic and viable stomach wall ( fig .", "culture of pus in the peritoneum revealed klebsiella pneumonia , staphylococcus aureus , and pseudomonas aeruginosa .", "amphotericin b infusion was added after operation . on the fifth hospital day , there was pus discharge from the wound site .", "however , the patient remained to have non - toxic appearance with stable vital signs . on the seventh hospital day", ", he complained of diarrhea and vague abdominal discomfort . over the following 2 days ,", "follow - up ct revealed an approximately 14.012.012.0 cm - sized , irregularly shaped air - containing fluid collection at the gastrectomy site , suggesting air - forming postoperative abscess and anastomotic site leakage .", "reoperation was performed , and the operative findings showed multiple perforations with necrosis in the transverse colon , necrotic change in the liver , hematoma with fluid collection in the peritoneal cavity , and leakage in the anastomotic site .", "rouxen - y esophagojejunostomy with cecocolostomy and segmental resection of the large intestine were performed . the submitted pathological specimen consisted of segments of colon , small intestine , and a portion of liver .", "the gross examination of the segment of colon showed multiple perforations , dirty serosa , and necrotic mucosa with greenish black exudates .", "microscopic examination of the colon and small intestine showed ischemic necrosis of mucosa with acute and chronic inflammation , and foreign body reaction .", "microscopic examination of the liver also showed totally necrotic parenchyma with many angioinvasive mucormycotic fungal hyphae .", "auscultation of the lungs revealed decreased breathing sounds with rale on both lung fields , and saturation of arterial blood gas analysis was decreased .", "he underwent intubation and ventilation . on the 21st - hospital day , the patient expired ."], ["clinically this condition is divided into gastric emphysema and emphysematous gastritis ( 3 , 5 , 12 ) .", "these two conditions should be differentiated because they are characterized by different clinical symptoms , possible etiology , radiologic findings , treatment , and prognosis ( 3 , 5 , 12 ) . in gastric emphysema , air enters the stomach wall from the lumen , from the peritoneal surface , or from its connections with the esophagus and duodenum , resulting from barotraumas in the absence of bacterial infection ( 12 ) .", "radiologically , in contrast to emphysematous gastritis , a more linear distribution of gas in the gastric wall is characteristic of gastric emphysema ( 3 ) .", "the symptom of acute abdomen as seen in patients with emphysematous gastritis is usually absent in patients with gastric emphysema ( 5 ) . in general ,", "gastric emphysema is asymptomatic ; its course is usually benign and resolves spontaneously without treatment ( 3 , 5 , 12 ) . on the other hand , emphysematous gastritis , the gas formed in situ by gas - forming bacteria invading the gastric wall , results in a necrotizing inflammation of the gastric wall . in 1889", ", fraenkel first reported a young man who died after several attacks of severe abdominal pain , blood vomitus , and diarrhea ( 1 ) . at autopsy ,", "originally , the stomach wall is well protected from bacterial infection by the close connection between cells , acidic ph , and good blood supply ( 3 , 12 ) .", "the predisposing factors , leading to the breakdown of these defenses , are ingestion of corrosives , alcohol abuse , diabetes , recent abdominal surgery , gastroenteritis , immunocompromised conditions , and treatment with non - steroidal anti - inflammatory drugs ( 1 - 3 ) . in the present case ,", "the patient had a history of alcohol abuse , which might have altered the unstirred mucous layer , and diabetes due to chronic pancreatitis , which might have led to a systemic predisposition to infection .", "patients with emphysematous gastritis typically develop acute abdominal pain , as was the case in our patient , usually developing 1 week after initiation , accompanied by diarrhea , nausea , vomiting , and occasionally hematemesis and melena ( 1 , 4 , 12 ) .", "physical examination of the abdomen reveals epigastric tenderness , distension , and decreased bowel sounds ( 4 , 12 ) .", "abdominal computed tomography , the best imaging modality to establish the diagnosis , reveals gastric wall thickening and intramural gas ( 1 , 3 , 12 ) .", "in contrast to gastric emphysema , the gas in the wall is in the form of irregular mottled bubbles or spots , especially around the fundus and the greater curvature , and remains in place despite the position of the body or absorption through the gastric tube ( 3 , 4 , 12 ) .", "organisms that have been cultured from stomach aspirates , blood , and peritoneal fluid include streptococci , e. coli , p. aeruginosa , clostridium .", "perfringens , and s. aureus ( 1 , 4 ) . among the previously reported cases , those associated with fungal infection were rare ( 4", ", 11 - 15 ) . of these , most cases were candida species ( 4 , 11 , 12 , 15 ) .", "pathologically , the gas forming organisms in emphysematous gastritis have been shown to infiltrate the stomach wall diffusely and there are edematous stomach wall - containing gas bubbles ( 1 , 6 ) . the therapeutic approach for emphysematous gastritis included initial stabilization of the septic condition through vigorous fluid resuscitation and early empirical parenteral antibiotic therapy with a broad range of antibiotics covering gram - negative and anaerobic bacteria ( 2 , 3 , 12 , 14 ) .", "specific therapy should be modified according to the results of the gastric fluid culture and sensitivity testing of the isolated organism ( 12 ) .", "indication for emergency surgery include deterioration despite optimal medical management , involvement of a large portion of or the entire stomach , presence of gastric infarction , or perforation ( 3 , 4 ) . despite meticulous treatment ,", "the mortality rate of emphysematous gastritis is about 60% , and the morbidity near 21% ( 2 ) .", "mucormycosis is a rare , opportunistic fungal infection that occurs almost exclusively in immunocompromised hosts such as patients with diabetes , leukemia , lymphoma , renal disease , septicemia , burns , malnutrition , and following long - term treatment with steroids and antibiotics ( 10 ) .", "clinical manifestations of mucormycosis can be categorized as rhinocerebral , pulmonary , disseminated , gastrointestinal , and cutaneous ( 9 ) . among these ,", "rhinocerebral and pulmonary disease are the most common forms , and gastrointestinal involvement is very rare , accounting for only 7% of all cases ( 16 ) .", "other associated conditions are immunosuppressed conditions such as renal failure , hematological malignancies , cirrhosis , and solid organ transplantation ( 16 ) .", "it is believed that infection of the alimentary tract is acquired through direct ingestion of and invasion by fungal spores .", "the gastrointestinal organ most frequently involved is the stomach , followed by the colon , small intestine , and esophagus ( 16 ) .", "when they invade , the lesion extends and marked surrounding induration develops , with a shaggy , velvety discolored surface or large plaqe - like areas of green and blacked eschar ( 16 ) .", "they also invade blood vessels and thus tend to cause extensive thrombosis , necrosis , and , ultimately , dissemination ( 10 ) .", "association with emphysematous gastritis is an extremely rare , and only one such case has been reported ( 15 ) .", "mucormycosis is not gas - forming , therefore this infection is not a direct cause of emphysematous gastritis .", "gastric wall necrosis caused by bacterial infection may be predisposed to the invasion by mucormycosis . alternatively , it is possible that invasive mucormycosis with subsequent gastric wall necrosis led to secondary bacterial invasion and intramural gas production ( 15 ) .", "successful management of mucormycosis includes aggressive metabolic support , amphotericin b , and surgical debridement of all necrotic involved tissue ( 15 , 16 ) . as in emphysematous gastritis , the prognosis of gastrointestinal mucormycosis is poor .", "forty - two cases of gastric mucormycosis have been described in the literature thus for , with a mortality above 98% ( 15 ) .", "we report an extremely rare case of emphysematous gastritis associated with invasive gastric mucormycosis in a 43-yr - old man . heavy alcohol abuse and diabetes mellitus were the predisposing factors .", "ct is the diagnostic procedure of choice of emphysematous gastritis and helps in differentiating with gastric emphysema .", "moreover , the gastric mucormycosis showed angioinvasion , necrosis , and ultimately , disseminate to colon and liver . in the case of mucormycosis , biopsy of involved areas", "this patient also had a fatal outcome despite antibiotic and antifungal therapy and surgery . increased awareness of these disease entities may facilitate early diagnosis , prompt medical therapy , and appropriate surgical intervention , and ultimately improve survival rates ."]]}
{"article_id": "PMC3125031", "article_text": ["in accordance with the 1997 documents of the world health organization ( who ) , amoebiasis is the infection by the protozoan parasite entamoeba histolytica with or without clinical manifestations .", "the only known natural host of e. histolytica is the human body with the large intestine as the major target organ .", "this parasite has a very simple life cycle in which the infective form is the cyst that is considered a resistant form of the parasite .", "the asymptomatic cyst passers and the intestinal amoebiasis patients are the natural transmitters ; they excrete cysts in their feces , which can contaminate food and water sources .", "the cysts are round structures around 1016 m in diameter . however , estimation of cyst diameter in entamoeba spp . in the old and recent literature", "the cyst has four visible nuclei when mature and only one when immature , and the nuclei are spherical with a membrane displaying small chromatin granules and a central karyosome .", "when in excystation , each cyst produces eight vegetative forms or trophozoites , which are the motile form of the parasite ; they are 2040 m in diameter .", "life cycle and the relevant structures of both forms of parasite are shown in figure 1 .", "cysts are resistant to desiccation in soil and can survive in humid environments and in water for several weeks .", "susceptible hosts exposed to the aforementioned infection sources ingest the cysts , which then undergo excystation during their pass through the gastrointestinal tract .", "amoebiasis is also considered a sexually transmitted disease , particularly in sexual relationships between men and in individuals with sexual anilingus practices", ". clinical and etiological diagnosis of intestinal and extra - intestinal amoebiasis is neither easy or simple in part because of the discovery of two species made from the previously known e. histolytica species , both indistinguishable under microscopy .", "e. histolytica sensu stricto is the potentially pathogenic species and e. dispar the commensal non - pathogenic entamoeba .", "d ) trophozoites of e. histolytica species with phagocyted erythrocytes ( dic 40 ) knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the twentieth century , which gave rise to the rapid development of diagnosis technology based on molecular and immunological strategies.[59 ] during the last 10 years , knowledge of the new epidemiology of amoebiasis in different geographic endemic and non - endemic areas has been obtained through the application of mostly molecular techniques.[1014 ] moreover , these molecular epidemiology studies have unveiled the extraordinary genetic variability[1315 ] of e. histolytica and e. dispar , allowing the discovery of other entamoeba species , such as e. moshkowskii , which can also infect the human intestine with a significant frequency .", "however , much of the epidemiology and its contribution to morbidity of entamoeba infections remain unknown .", "there are excellent recent reviews on the molecular epidemiology and intestinal and extra - intestinal characteristics of amoebiasis in the human host that can be consulted .", "nevertheless , the major purpose of the present work is to highlight the novelties in regard to human infection and the disease that can help the general physician from both endemic and non - endemic countries in their medical practice .", "this is especially critical given that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide .", "the speciation of entamoeba protozoa has been discussed since 1925 when emile brumpt proposed the existence of two distinct species that could infiltrate the human intestine : one associated with symptoms of diarrhea with or without dysentery and the other excreted with feces from asymptomatic individuals .", "while years of scientific discussions have left brump 's theory behind , no molecular technology prior to the 1990s allowed clear differentiation of the currently known e. histolytica and e. dispar species in terms of pathogenic or non - pathogenic phenotypes .", "both species are genetically diverse and this variability allowed for the beginning of studies on molecular epidemiology in different endemic areas .", "the new data on the epidemiology of amoebiasis based on frontier technology suggests that genetic variability could be an important tool in the study of geographic distribution of both species and particular strains of entamoeba , which may determine the morbidity rates of different forms of amoebic infection in different geographic areas .", "some e. histolytica genetic variants ( strains ) have been isolated from asymptomatic cyst passers , patients with invasive intestinal amoebiasis or from samples of amoebic liver abscess material .", "however , e. dispar has been mainly isolated in feces samples resulting from asymptomatic cyst passers and displays high genetic polymorphisms , even more than the e. histolytica species .", "we recently have obtained evidence that in at least two endemic countries ( brazil and mexico ) , e. dispar genetic variants have been detected in patients with invasive amoebiasis . in brazil ,", "the icb - ado e. dispar strain was isolated from a non - dysenteric colitis patient maintained in culture with his own intestinal flora displaying a pathogenic behavior in experimental models of amoebic liver abscess .", "dna extracted from hepatic abscess material obtained from six patients in mexico also clearly showed the presence of e. dispar dna sequences .", "the third species of entamoeba , e. moshkowskii , has been considered a free - living organism since 1940s in contrast to e. histolytica and e. dispar , with a geographic distribution mainly in developing countries .", "e. moshkowskii has been frequently detected in individuals from developed and highly industrialized countries . particularly in regard to this species we are at the beginning of the study of its pathogenic potential and the context in which it is expressed and the epidemiologic significance of infection and its contribution to morbidity rates of diarrheic diseases .", "what doses of cysts are necessary for colonization of large bowel mucosa ? for the three entamoeba species this is not known with any certainty .", "moreover , we do not know which environmental characteristics are permissive for intestinal colonization , and this could be an interesting field for future research . finally , until today , the only species recognized as an etiologic agent of amoebic invasive disease is e. histolytica , which , once in the colon , undergoes excystation and the generation of trophozoites .", "trophozoites multiply by binary fusion and some of them may encyst and be excreted with stools .", "cyst viability under appropriate conditions of humidity may last as long as several weeks and thus they be available for a new susceptible host .", "we have to stress that more than 90% of infections have an asymptomatic course and are frequently auto - limited at different periods of time . after intestinal infection there is no evidence of induction of a long - lasting protective immune response , and in endemic areas , individuals may have several periods during the year of re - infection and clearance of infection . in relation to the susceptibility of hiv and aids patients to the invasive forms of infection ,", "contradictory evidence exists ; however , morbidity seems to be more related to the particular prevalence of the e. histolytica strain with invasive phenotypes than to the specific immunological status of the patient.[2427 ] as for the invasive behavior of e. histolytica , some authors consider this trait not to be typical . on the contrary", "the natural history of invasive intestinal amoebiasis is an acute event , characterized by the presence of diarrhea that occurs days or weeks after exposure  in our personal experience lasting no more than four to five weeks . although there are reports of occurrence years after exposure , in this case we presume the cause and effect relationship is extremely difficult to corroborate .", "intestinal amoebiasis is basically an acute disease in which the most frequent symptoms are abdominal pain ( colic ) and the presence of diarrhea with mucus and/or blood , or a clear dysenteric syndrome .", "however , fever and other systemic symptoms are infrequent . severe forms of invasive amoebiasis can be observed in young children ( < 5 years ) , pregnant woman , the elderly , and particular those with chronic diseases , such as diabetes mellitus , and in individuals being treated with immunosuppressants or those with immunodeficiency disorders .", "those severe forms of amoebiasis are colon ameboma , fulminant necrotizing colitis , and toxic mega colon .", "the appearance of symptoms , such as severe dysentery and pain with signs of peritoneal irritation ( rebound ) , intense tenesmus , fever ( > 38c ) , tachycardia , hypertension , nausea , and anorexia are suggestive of the previously mentioned severe forms of intestinal amoebiasis .", "the mortality rates of dysenteric syndrome due to e. histolytica are less than 1% , but mortality due to complications increases up to 75%.[2931 ] fortunately in the last few decades such complications are uncommon .", "the intestinal amoebiasis form known as chronic non - dysenteric colitis is the most frequent form of amoebiasis in people of all ages , characterized by non - specific symptoms .", "symptoms more relevant in this instance are periods of abdominal pain ( colic ) and auto - limited episodes of diarrhea alternating with constipation . however , both non - dysenteric amoebic colitis and irritable bowel syndrome are controversial themes in the clinical practice . where endoscopy examination is available , colonoscopy can be of great help in clinical diagnosis of invasive intestinal amoebiasis .", "this procedure allows for microscopic examination of samples taken directly from the characteristic flask - shaped ulcer produced by e. histolytica and from other sites of mucosal lesions .", "microscopic observations of this type of material are described in the diagnostics section and in figure 2 . on the other hand", ", colonoscopy detects the presence of lesions related to the mentioned severe forms of intestinal amoebiasis and allows for differential diagnosis of other pathologies , such as inflammatory bowel disease or colon carcinoma .", "d ) intestinal biopsy obtained from the edge of flask - shaped ulcer where large numbers of trophozoites ( he and pas stained , 60 ) are clearly visible .", "e ) biopsy obtained from the edge of amoebic liver abscess ( he and pas stained , 20 ) .", "notice the presence of trophozoites , hepatocytes , and the large number of inflammatory cells .", "what circumstances define the extra - intestinal invasive behavior of some e. histolytica strains ? this remains unknown today .", "for example , we do not know the frequency of extra - intestinal invasion after intestinal colonization with virulent e. histolytica", ". however , this seems to be an infrequent event as suggested by the low morbidity rates of amoebic liver abscess and other extra - intestinal forms of invasive amoebiasis compared with the prevalence rates of asymptomatic infections and intestinal disease . while amoebic liver abscess is a disease that can affect individuals of all ages , in some endemic areas the incidence rates are higher in both children under 5 years and young adults ( 2045 years ) .", "males are also more prone to developing amoebic hepatic abscess than females ( 1 female for 46 males ) . after exposure , 80% of patients display symptoms over a few days to 46 weeks .", "the most common symptoms suggestive of amoebic liver abscess are fever ( 38c ) , chills and diaphoresis , anorexia and abdominal pain in the right upper quadrant that increases during inspiration . pain also frequently radiates to shoulder and back .", "nausea has also been referenced but diarrhea is only occasionally mentioned ( 50% of cases ) .", "hepatomegaly can be detected during digital percussion of the hepatic area and is always related to the dimensions of the abscess ; patients can also display peritoneal signs ( abdominal guarding or rebound ) .", "absence of intestinal noises , jaundice , and pleural or pericardial rub are symptoms that should elicit alarm related to the rupture or imminence of rupture of the hepatic abscess . in general", ", the right hepatic lobule is the most frequently affected due to the portal circulatory system of the right colon .", "laboratory findings suggestive of amoebic liver abscess are the presence of leukocytosis , neutrophilia , increased globular sedimentation velocity , and high levels of alkaline phosphatase .", "thoracic x - ray data useful in the diagnosis of amoebic abscesses , as well as other types of hepatic abscesses , include elevation of the right hemidiaphragm , atelectasis , or pleural effusion [ figure 3a ] .", "ultrasound is the gold standard technique for diagnosis of amoebic liver abscess , as its positive predictive value ( ppv ) is around 95% ( 85100% depending on analyzed series ) .", "although contrast computed tomography ( ct scan ) [ figures 3d and 3e ] have a ppv up to 95% due to a higher definition capacity , ultrasound is considerably less expensive compared to ct scan technology , which is of tremendous importance to countries with limited medical and economic resources .", "ultrasound reveals hypoechoic areas that can be single or multiple with round edges [ figure 3b and 3c ] .", "several authors have mentioned the presence of a large single abscess as a frequent characteristic of a amoebic liver abscess . however , this characteristic is not a sine qua non of amoebic abscesses , and in our medical practice we have seen multiple abscesses more frequently than we had first assumed .", "early lesions due to amoebic invasion of hepatic parenchyma are multifocal in nature ( micro - abscesses ) as a consequence of tissue destruction and necrosis by proteases from e. histolytica and neutrophil recruitment to the site of infection .", "the advantage of ct scans and magnetic resonance is detection of small abscesses and the high definition of the images .", "moreover , other techniques not always available in endemic countries ( e.g. gallium scans ) can help differentiate between amoebic ( cold images ) and pyogenic abscesses ( hot images ) .", "thus , the difference is based on the absence ( amoebic ) or presence ( pyogenic ) of white blood cells in the abscess .", "additionally , we have to mention that in endemic countries the frequency of mixed abscesses ( pyogenic and amoebic ) is considerable ; in our practice this frequency is approximately 17% ( non - reported data ) . another important fact in medical practice is the coincidence of previous symptoms with the presence of high levels of serum anti - amoebic antibodies  more than 90% of amoebic liver abscess patients develop this type of antibody response", ". however , in cases of fast development of amoebic abscess these antibodies may not be present .", "a ) thoracic x - ray of a patient with amoebic liver abscess showing the elevation of the right hemi - diaphragm .", "ultrasound images of : b ) single large amoebic abscess and c ) three amoebic hepatic abscesses .", "d ) contrasted computed tomography ( ct ) scan of a single abscess and e ) three clear amoebic liver abscesses", "etiological diagnosis of intestinal parasitic diseases has been mainly performed using direct or concentration techniques for microscopic examination of fecal specimens . while technically simple in approach ,", "these techniques require the expertise of highly qualified technicians in the morphological identification of ova , cysts , and helminths to be feasible , and the sensitivity and specificity is no more than 80% .", "while this technique can not differentiate between the three entamoeba species already mentioned , in some endemic communities this is the only diagnostic technology available . on the other hand , during", "the last 10 years diagnostics in amoebiasis have changed dramatically , considerably improving sensitivity and specificity .", "some of the current techniques are based on immunological strategies , such as elisa and different modalities of polymerase chain reaction ( pcr ) , with clear advantages in bedside diagnosis and in clinical laboratories of health institutions .", "even though some of these diagnosis procedures are also suitable for large epidemiologic trials , it is mandatory to make a careful selection of the diagnosis test when the test has to be applied in the field . in particular , the election has to be directed to those procedures that do not need special conditions for sample preservation or pretreatment of specimens in the working field . in our experience ,", "immunologically based diagnostics tests for detection of anti - amoebic secretory antibodies in feces or detection of intestinal amoebic antigens through polyclonal or monoclonal specific antibodies ( elisa ) are excellent tests in hospitals where fresh specimens ( feces ) can be obtained .", "results are reproducible and reliable . in contrast , in epidemiological trials , these techniques can be biased when samples are more than 18 hours old .", "tables 1 and 2 have a list of diagnostic tests that have proven to be useful in clinical and laboratory diagnosis of intestinal and extra - intestinal amoebiasis in first and second level health institutions .", "microscopy and immunoassays for e. histolytica detection pcr assays for e. histolytica and/or e. dispar detection", "the who / paho recommendations published in 1997 contain in detail the actions that who country members have to observe with regard to e. histolytica species infection .", "they highlight the characterization ( when possible ) of e. histolytica and e. dispar ( we now add e. moshkowskii ) to be treated properly . in accordance with the who recommendation only e. histolytica", "it is also important to remember that in some endemic countries , mixed infections ( e.g. e. histolytica and e. dispar ) are frequent , and that only those infected with e. dispar should be excluded for anti - amoebic treatment .", "table 3 shows treatment schedules that have proven to be highly effective in both intestinal and extra - intestinal invasive amoebiasis . in cases of large amoebic abscesses", "in which imminence of rupture or distances of less than 1 cm between the abscess wall and liver surface prevail , ultrasound - guided puncture is indicated .", "the procedure allows for the establishment of a differential diagnosis with other liver pathologies , especially pyogenic liver abscess , which is common in clinical practice .", "treatment of amoebiasis disease in our experience , patients with amoebic liver abscess may not excrete e. histolytica / e .", "however , some of these patients are asymptomatic cyst passers after treatment with systemic anti - amoebic drugs , such as metronidazole . in such cases", ", treatment has to include luminal anti - amoebic drugs . at present , evidence of low susceptibility or resistant strains of either e. histolytica or e. dispar species to metronidazole", "gastrointestinal infections are responsible for morbidity and mortality rates of children and young adults worldwide . in africa , diarrhea is responsible for 2575% of all childhood illnesses .", "infection and intestinal diseases with icd10 code a00-a09 are the second cause of disease in children under 10 years old in mexico , and intestinal amoebiasis ranks with some annual variations 5 and 6 in the list of the 20 major causes of disease in mexico ( http://www.dgepi.salud.gob.mx/anuario/index.html # ) .", "causes of diarrhea in endemic areas include a large variety of enteropathogens ( viruses , bacteria , and parasites ) . in mexico ,", "parasitic intestinal infections are multiple infections that constitute approximately 40% of analyzed individuals in which it is possible to detect more than one pathogen together with commensal parasites that are an indicator of fecalism .", "prevalence of parasitic infection in three different communities in the state of morelos , mexico , are shown in \n table 4 , one of which is a strictly rural population ( amacuzac ) and two ( tlaltizapan and xoxocotla ) are suburban communities .", "the relevance of these results lies in the high frequency of mixed parasitic infections detected in the studied populations", ". a remarkable low prevalence of soil - transmitted helminthiasis was also observed and could be the consequence of a biannual anti - parasitic treatment of school children with albendazole .", "this policy was implemented by the health ministry since the 1990s . however , there are emerging parasites with an increasing prevalence in the last 10 years , including blastocystis hominis and in some areas cryptosporidium spp . in south africa intestinal mixed infection is present in 46% of patients with diarrhea and 33% in school children .", "furthermore , mixed intestinal infections due to bacteria , parasites , and viral pathogens are the major forms of intestinal infection in developing countries .", "however , cheun et al recently published a splendid study on diarrheal patients in hospitals in korea .", "the study documented the presence of enteropathogenic bacteria , parasites , and viruses in mixed infections , and highlighted the importance of diarrheal disease associated with protozoan infections .", "the major association of e. histolytica positive samples was with rotavirus type 1[10.3(6.114.6 ) positivity/100 infected individuals , 95% ci ] , astrovirus [ 9.3(5.213.4 ) positivity/100 infected individuals , 95% ci ] , pathogenic escherichia coli [ 7.2 ( 3610.0 ) positivity/100 infected individuals , 95% ci ] and clostridium perfringens [ 10.3(6.014.6 ) positivity/100 infected individuals , 95% ci ] .", "as the authors mentioned , the question is whether mixed infections with protozoa are more likely to induce serious diarrhea .", "prevalence rates of parasite intestinal infections in morelos , mexico efforts in the near future have to be directed on studies focusing the interactions of microorganism in the intestinal environment", ". this knowledge will have a positive impact in clinical and laboratory diagnosis of diarrheic syndrome , its treatment , and thereafter the implementation of more reliable control schedules ."], "abstract_text": ["<S> in accordance with the 1997 documents of the world health organization ( who ) , amoebiasis is defined as the infection by the protozoan parasite entamoeba histolytica with or without clinical manifestations . </S>", "<S> the only known natural host of e. histolytica is the human with the large intestine as major target organ . </S>", "<S> this parasite has a very simple life cycle in which the infective form is the cyst , considered a resistant form of parasite : the asymptomatic cyst passers and the intestinal amoebiasis patients are the transmitters ; they excrete cysts in their feces , which can contaminate food and water sources . </S>", "<S> e. histolytica sensu stricto is the potentially pathogenic species and e. dispar is a commensal non - pathogenic entamoeba . </S>", "<S> both species are biochemical , immunological and genetically distinct . </S>", "<S> the knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the 20th century , which gave place to the rapid development of diagnostics technology based on molecular and immunological strategies . during the last ten years , </S>", "<S> knowledge of the new epidemiology of amoebiasis in different geographic endemic and non - endemic areas has been obtained by applying mostly molecular techniques . in the present work we highlight novelties on human infection and the disease that can help the general physician from both endemic and non - endemic countries in their medical practice , particularly , now that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide . </S>"], "labels": null, "section_names": ["INTRODUCTION", "THE THREE SPECIES OF INTESTINAL ", "INTESTINAL AND EXTRA-INTESTINAL AMOEBIASIS", "HEPATIC INVASION", "CURRENTLY AVAILABLE DIAGNOSTIC TESTS FOR INVASIVE AMOEBIASIS", "AMOEBIASIS TREATMENT", "CONSIDERATIONS OF AMOEBIASIS IN THE CONTEXT OF DIARRHEIC SYNDROME"], "sections": [["in accordance with the 1997 documents of the world health organization ( who ) , amoebiasis is the infection by the protozoan parasite entamoeba histolytica with or without clinical manifestations .", "the only known natural host of e. histolytica is the human body with the large intestine as the major target organ .", "this parasite has a very simple life cycle in which the infective form is the cyst that is considered a resistant form of the parasite .", "the asymptomatic cyst passers and the intestinal amoebiasis patients are the natural transmitters ; they excrete cysts in their feces , which can contaminate food and water sources .", "the cysts are round structures around 1016 m in diameter . however , estimation of cyst diameter in entamoeba spp . in the old and recent literature", "the cyst has four visible nuclei when mature and only one when immature , and the nuclei are spherical with a membrane displaying small chromatin granules and a central karyosome .", "when in excystation , each cyst produces eight vegetative forms or trophozoites , which are the motile form of the parasite ; they are 2040 m in diameter .", "life cycle and the relevant structures of both forms of parasite are shown in figure 1 .", "cysts are resistant to desiccation in soil and can survive in humid environments and in water for several weeks .", "susceptible hosts exposed to the aforementioned infection sources ingest the cysts , which then undergo excystation during their pass through the gastrointestinal tract .", "amoebiasis is also considered a sexually transmitted disease , particularly in sexual relationships between men and in individuals with sexual anilingus practices", ". clinical and etiological diagnosis of intestinal and extra - intestinal amoebiasis is neither easy or simple in part because of the discovery of two species made from the previously known e. histolytica species , both indistinguishable under microscopy .", "e. histolytica sensu stricto is the potentially pathogenic species and e. dispar the commensal non - pathogenic entamoeba .", "d ) trophozoites of e. histolytica species with phagocyted erythrocytes ( dic 40 ) knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the twentieth century , which gave rise to the rapid development of diagnosis technology based on molecular and immunological strategies.[59 ] during the last 10 years , knowledge of the new epidemiology of amoebiasis in different geographic endemic and non - endemic areas has been obtained through the application of mostly molecular techniques.[1014 ] moreover , these molecular epidemiology studies have unveiled the extraordinary genetic variability[1315 ] of e. histolytica and e. dispar , allowing the discovery of other entamoeba species , such as e. moshkowskii , which can also infect the human intestine with a significant frequency .", "however , much of the epidemiology and its contribution to morbidity of entamoeba infections remain unknown .", "there are excellent recent reviews on the molecular epidemiology and intestinal and extra - intestinal characteristics of amoebiasis in the human host that can be consulted .", "nevertheless , the major purpose of the present work is to highlight the novelties in regard to human infection and the disease that can help the general physician from both endemic and non - endemic countries in their medical practice .", "this is especially critical given that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide ."], ["the speciation of entamoeba protozoa has been discussed since 1925 when emile brumpt proposed the existence of two distinct species that could infiltrate the human intestine : one associated with symptoms of diarrhea with or without dysentery and the other excreted with feces from asymptomatic individuals .", "while years of scientific discussions have left brump 's theory behind , no molecular technology prior to the 1990s allowed clear differentiation of the currently known e. histolytica and e. dispar species in terms of pathogenic or non - pathogenic phenotypes .", "both species are genetically diverse and this variability allowed for the beginning of studies on molecular epidemiology in different endemic areas .", "the new data on the epidemiology of amoebiasis based on frontier technology suggests that genetic variability could be an important tool in the study of geographic distribution of both species and particular strains of entamoeba , which may determine the morbidity rates of different forms of amoebic infection in different geographic areas .", "some e. histolytica genetic variants ( strains ) have been isolated from asymptomatic cyst passers , patients with invasive intestinal amoebiasis or from samples of amoebic liver abscess material .", "however , e. dispar has been mainly isolated in feces samples resulting from asymptomatic cyst passers and displays high genetic polymorphisms , even more than the e. histolytica species .", "we recently have obtained evidence that in at least two endemic countries ( brazil and mexico ) , e. dispar genetic variants have been detected in patients with invasive amoebiasis . in brazil ,", "the icb - ado e. dispar strain was isolated from a non - dysenteric colitis patient maintained in culture with his own intestinal flora displaying a pathogenic behavior in experimental models of amoebic liver abscess .", "dna extracted from hepatic abscess material obtained from six patients in mexico also clearly showed the presence of e. dispar dna sequences .", "the third species of entamoeba , e. moshkowskii , has been considered a free - living organism since 1940s in contrast to e. histolytica and e. dispar , with a geographic distribution mainly in developing countries .", "e. moshkowskii has been frequently detected in individuals from developed and highly industrialized countries . particularly in regard to this species we are at the beginning of the study of its pathogenic potential and the context in which it is expressed and the epidemiologic significance of infection and its contribution to morbidity rates of diarrheic diseases ."], ["what doses of cysts are necessary for colonization of large bowel mucosa ? for the three entamoeba species this is not known with any certainty .", "moreover , we do not know which environmental characteristics are permissive for intestinal colonization , and this could be an interesting field for future research . finally , until today , the only species recognized as an etiologic agent of amoebic invasive disease is e. histolytica , which , once in the colon , undergoes excystation and the generation of trophozoites .", "trophozoites multiply by binary fusion and some of them may encyst and be excreted with stools .", "cyst viability under appropriate conditions of humidity may last as long as several weeks and thus they be available for a new susceptible host .", "we have to stress that more than 90% of infections have an asymptomatic course and are frequently auto - limited at different periods of time . after intestinal infection there is no evidence of induction of a long - lasting protective immune response , and in endemic areas , individuals may have several periods during the year of re - infection and clearance of infection . in relation to the susceptibility of hiv and aids patients to the invasive forms of infection ,", "contradictory evidence exists ; however , morbidity seems to be more related to the particular prevalence of the e. histolytica strain with invasive phenotypes than to the specific immunological status of the patient.[2427 ] as for the invasive behavior of e. histolytica , some authors consider this trait not to be typical . on the contrary", "the natural history of invasive intestinal amoebiasis is an acute event , characterized by the presence of diarrhea that occurs days or weeks after exposure  in our personal experience lasting no more than four to five weeks . although there are reports of occurrence years after exposure , in this case we presume the cause and effect relationship is extremely difficult to corroborate .", "intestinal amoebiasis is basically an acute disease in which the most frequent symptoms are abdominal pain ( colic ) and the presence of diarrhea with mucus and/or blood , or a clear dysenteric syndrome .", "however , fever and other systemic symptoms are infrequent . severe forms of invasive amoebiasis can be observed in young children ( < 5 years ) , pregnant woman , the elderly , and particular those with chronic diseases , such as diabetes mellitus , and in individuals being treated with immunosuppressants or those with immunodeficiency disorders .", "those severe forms of amoebiasis are colon ameboma , fulminant necrotizing colitis , and toxic mega colon .", "the appearance of symptoms , such as severe dysentery and pain with signs of peritoneal irritation ( rebound ) , intense tenesmus , fever ( > 38c ) , tachycardia , hypertension , nausea , and anorexia are suggestive of the previously mentioned severe forms of intestinal amoebiasis .", "the mortality rates of dysenteric syndrome due to e. histolytica are less than 1% , but mortality due to complications increases up to 75%.[2931 ] fortunately in the last few decades such complications are uncommon .", "the intestinal amoebiasis form known as chronic non - dysenteric colitis is the most frequent form of amoebiasis in people of all ages , characterized by non - specific symptoms .", "symptoms more relevant in this instance are periods of abdominal pain ( colic ) and auto - limited episodes of diarrhea alternating with constipation . however , both non - dysenteric amoebic colitis and irritable bowel syndrome are controversial themes in the clinical practice . where endoscopy examination is available , colonoscopy can be of great help in clinical diagnosis of invasive intestinal amoebiasis .", "this procedure allows for microscopic examination of samples taken directly from the characteristic flask - shaped ulcer produced by e. histolytica and from other sites of mucosal lesions .", "microscopic observations of this type of material are described in the diagnostics section and in figure 2 . on the other hand", ", colonoscopy detects the presence of lesions related to the mentioned severe forms of intestinal amoebiasis and allows for differential diagnosis of other pathologies , such as inflammatory bowel disease or colon carcinoma .", "d ) intestinal biopsy obtained from the edge of flask - shaped ulcer where large numbers of trophozoites ( he and pas stained , 60 ) are clearly visible .", "e ) biopsy obtained from the edge of amoebic liver abscess ( he and pas stained , 20 ) .", "notice the presence of trophozoites , hepatocytes , and the large number of inflammatory cells ."], ["what circumstances define the extra - intestinal invasive behavior of some e. histolytica strains ? this remains unknown today .", "for example , we do not know the frequency of extra - intestinal invasion after intestinal colonization with virulent e. histolytica", ". however , this seems to be an infrequent event as suggested by the low morbidity rates of amoebic liver abscess and other extra - intestinal forms of invasive amoebiasis compared with the prevalence rates of asymptomatic infections and intestinal disease . while amoebic liver abscess is a disease that can affect individuals of all ages , in some endemic areas the incidence rates are higher in both children under 5 years and young adults ( 2045 years ) .", "males are also more prone to developing amoebic hepatic abscess than females ( 1 female for 46 males ) . after exposure , 80% of patients display symptoms over a few days to 46 weeks .", "the most common symptoms suggestive of amoebic liver abscess are fever ( 38c ) , chills and diaphoresis , anorexia and abdominal pain in the right upper quadrant that increases during inspiration . pain also frequently radiates to shoulder and back .", "nausea has also been referenced but diarrhea is only occasionally mentioned ( 50% of cases ) .", "hepatomegaly can be detected during digital percussion of the hepatic area and is always related to the dimensions of the abscess ; patients can also display peritoneal signs ( abdominal guarding or rebound ) .", "absence of intestinal noises , jaundice , and pleural or pericardial rub are symptoms that should elicit alarm related to the rupture or imminence of rupture of the hepatic abscess . in general", ", the right hepatic lobule is the most frequently affected due to the portal circulatory system of the right colon .", "laboratory findings suggestive of amoebic liver abscess are the presence of leukocytosis , neutrophilia , increased globular sedimentation velocity , and high levels of alkaline phosphatase .", "thoracic x - ray data useful in the diagnosis of amoebic abscesses , as well as other types of hepatic abscesses , include elevation of the right hemidiaphragm , atelectasis , or pleural effusion [ figure 3a ] .", "ultrasound is the gold standard technique for diagnosis of amoebic liver abscess , as its positive predictive value ( ppv ) is around 95% ( 85100% depending on analyzed series ) .", "although contrast computed tomography ( ct scan ) [ figures 3d and 3e ] have a ppv up to 95% due to a higher definition capacity , ultrasound is considerably less expensive compared to ct scan technology , which is of tremendous importance to countries with limited medical and economic resources .", "ultrasound reveals hypoechoic areas that can be single or multiple with round edges [ figure 3b and 3c ] .", "several authors have mentioned the presence of a large single abscess as a frequent characteristic of a amoebic liver abscess . however , this characteristic is not a sine qua non of amoebic abscesses , and in our medical practice we have seen multiple abscesses more frequently than we had first assumed .", "early lesions due to amoebic invasion of hepatic parenchyma are multifocal in nature ( micro - abscesses ) as a consequence of tissue destruction and necrosis by proteases from e. histolytica and neutrophil recruitment to the site of infection .", "the advantage of ct scans and magnetic resonance is detection of small abscesses and the high definition of the images .", "moreover , other techniques not always available in endemic countries ( e.g. gallium scans ) can help differentiate between amoebic ( cold images ) and pyogenic abscesses ( hot images ) .", "thus , the difference is based on the absence ( amoebic ) or presence ( pyogenic ) of white blood cells in the abscess .", "additionally , we have to mention that in endemic countries the frequency of mixed abscesses ( pyogenic and amoebic ) is considerable ; in our practice this frequency is approximately 17% ( non - reported data ) . another important fact in medical practice is the coincidence of previous symptoms with the presence of high levels of serum anti - amoebic antibodies  more than 90% of amoebic liver abscess patients develop this type of antibody response", ". however , in cases of fast development of amoebic abscess these antibodies may not be present .", "a ) thoracic x - ray of a patient with amoebic liver abscess showing the elevation of the right hemi - diaphragm .", "ultrasound images of : b ) single large amoebic abscess and c ) three amoebic hepatic abscesses .", "d ) contrasted computed tomography ( ct ) scan of a single abscess and e ) three clear amoebic liver abscesses"], ["etiological diagnosis of intestinal parasitic diseases has been mainly performed using direct or concentration techniques for microscopic examination of fecal specimens . while technically simple in approach ,", "these techniques require the expertise of highly qualified technicians in the morphological identification of ova , cysts , and helminths to be feasible , and the sensitivity and specificity is no more than 80% .", "while this technique can not differentiate between the three entamoeba species already mentioned , in some endemic communities this is the only diagnostic technology available . on the other hand , during", "the last 10 years diagnostics in amoebiasis have changed dramatically , considerably improving sensitivity and specificity .", "some of the current techniques are based on immunological strategies , such as elisa and different modalities of polymerase chain reaction ( pcr ) , with clear advantages in bedside diagnosis and in clinical laboratories of health institutions .", "even though some of these diagnosis procedures are also suitable for large epidemiologic trials , it is mandatory to make a careful selection of the diagnosis test when the test has to be applied in the field . in particular , the election has to be directed to those procedures that do not need special conditions for sample preservation or pretreatment of specimens in the working field . in our experience ,", "immunologically based diagnostics tests for detection of anti - amoebic secretory antibodies in feces or detection of intestinal amoebic antigens through polyclonal or monoclonal specific antibodies ( elisa ) are excellent tests in hospitals where fresh specimens ( feces ) can be obtained .", "results are reproducible and reliable . in contrast , in epidemiological trials , these techniques can be biased when samples are more than 18 hours old .", "tables 1 and 2 have a list of diagnostic tests that have proven to be useful in clinical and laboratory diagnosis of intestinal and extra - intestinal amoebiasis in first and second level health institutions .", "microscopy and immunoassays for e. histolytica detection pcr assays for e. histolytica and/or e. dispar detection"], ["the who / paho recommendations published in 1997 contain in detail the actions that who country members have to observe with regard to e. histolytica species infection .", "they highlight the characterization ( when possible ) of e. histolytica and e. dispar ( we now add e. moshkowskii ) to be treated properly . in accordance with the who recommendation only e. histolytica", "it is also important to remember that in some endemic countries , mixed infections ( e.g. e. histolytica and e. dispar ) are frequent , and that only those infected with e. dispar should be excluded for anti - amoebic treatment .", "table 3 shows treatment schedules that have proven to be highly effective in both intestinal and extra - intestinal invasive amoebiasis . in cases of large amoebic abscesses", "in which imminence of rupture or distances of less than 1 cm between the abscess wall and liver surface prevail , ultrasound - guided puncture is indicated .", "the procedure allows for the establishment of a differential diagnosis with other liver pathologies , especially pyogenic liver abscess , which is common in clinical practice .", "treatment of amoebiasis disease in our experience , patients with amoebic liver abscess may not excrete e. histolytica / e .", "however , some of these patients are asymptomatic cyst passers after treatment with systemic anti - amoebic drugs , such as metronidazole . in such cases", ", treatment has to include luminal anti - amoebic drugs . at present , evidence of low susceptibility or resistant strains of either e. histolytica or e. dispar species to metronidazole"], ["gastrointestinal infections are responsible for morbidity and mortality rates of children and young adults worldwide . in africa , diarrhea is responsible for 2575% of all childhood illnesses .", "infection and intestinal diseases with icd10 code a00-a09 are the second cause of disease in children under 10 years old in mexico , and intestinal amoebiasis ranks with some annual variations 5 and 6 in the list of the 20 major causes of disease in mexico ( http://www.dgepi.salud.gob.mx/anuario/index.html # ) .", "causes of diarrhea in endemic areas include a large variety of enteropathogens ( viruses , bacteria , and parasites ) . in mexico ,", "parasitic intestinal infections are multiple infections that constitute approximately 40% of analyzed individuals in which it is possible to detect more than one pathogen together with commensal parasites that are an indicator of fecalism .", "prevalence of parasitic infection in three different communities in the state of morelos , mexico , are shown in \n table 4 , one of which is a strictly rural population ( amacuzac ) and two ( tlaltizapan and xoxocotla ) are suburban communities .", "the relevance of these results lies in the high frequency of mixed parasitic infections detected in the studied populations", ". a remarkable low prevalence of soil - transmitted helminthiasis was also observed and could be the consequence of a biannual anti - parasitic treatment of school children with albendazole .", "this policy was implemented by the health ministry since the 1990s . however , there are emerging parasites with an increasing prevalence in the last 10 years , including blastocystis hominis and in some areas cryptosporidium spp . in south africa intestinal mixed infection is present in 46% of patients with diarrhea and 33% in school children .", "furthermore , mixed intestinal infections due to bacteria , parasites , and viral pathogens are the major forms of intestinal infection in developing countries .", "however , cheun et al recently published a splendid study on diarrheal patients in hospitals in korea .", "the study documented the presence of enteropathogenic bacteria , parasites , and viruses in mixed infections , and highlighted the importance of diarrheal disease associated with protozoan infections .", "the major association of e. histolytica positive samples was with rotavirus type 1[10.3(6.114.6 ) positivity/100 infected individuals , 95% ci ] , astrovirus [ 9.3(5.213.4 ) positivity/100 infected individuals , 95% ci ] , pathogenic escherichia coli [ 7.2 ( 3610.0 ) positivity/100 infected individuals , 95% ci ] and clostridium perfringens [ 10.3(6.014.6 ) positivity/100 infected individuals , 95% ci ] .", "as the authors mentioned , the question is whether mixed infections with protozoa are more likely to induce serious diarrhea .", "prevalence rates of parasite intestinal infections in morelos , mexico efforts in the near future have to be directed on studies focusing the interactions of microorganism in the intestinal environment", ". this knowledge will have a positive impact in clinical and laboratory diagnosis of diarrheic syndrome , its treatment , and thereafter the implementation of more reliable control schedules ."]]}
{"article_id": "PMC3306916", "article_text": ["engaging in high levels of physical activity is a key strategy for successful maintenance of weight loss .", "a study of nearly 4,000 participants in the national weight control registry ( nwcr ) , the largest longitudinal study of successful weight loss maintainers , indicated participants expend 2,621  2,252  kcal per week through physical activity , which is equivalent to approximately 60 minutes of moderate - intensity physical activity per day .", "additionally , long - term followup of participants in behavioral weight loss programs has shown that those who are most successful at maintaining their weight loss report activity levels similar to those of nwcr participants [ 3 , 4 ] .", "many individuals , particularly those who are obese , are insufficiently active , and increasing physical activity can be a challenge [ 5 , 6 ] .", "thus , strategies that assist individuals in adopting and sustaining high levels of physical activity to help facilitate a healthy body weight are needed .", "prior studies have identified multiple strategies for increasing physical activity adoption and maintenance , such as providing home or clinic - based exercise programs , increasing access to active behaviors ( e.g. , adding exercise equipment in the home ) , and reducing access to sedentary behaviors ( e.g. , limiting time to watch television ) , using pedometers to track activity and progress toward physical activity goals , and accumulating exercise throughout the day in multiple short bouts ( 10  min ) [ 11 , 12 ] . performing a variety of different types of activities may be another strategy to increase physical activity levels . the national health and nutrition examination survey ( nhanes ) showed that individuals who reported engaging in a variety of different activities ( i.e. , walking + other leisure - time activities ) were more likely to meet national physical activity recommendations compared to those who reported no variety ( i.e. , only walking ) .", "likewise , in an 18-month behavioral weight loss intervention , overweight participants who reported physical activity variety ( i.e. , 2 different activities ) at 6 months had higher self - reported activity - related energy expenditure and a lower body mass index ( bmi ) at 18 months than those who did not report physical activity variety ( i.e. , only 1 activity ) . thus , while the above findings suggest that physical activity variety may contribute to higher physical activity levels within the context of behavioral weight loss treatment , it is unclear whether physical activity variety is associated with higher physical activity in individuals who have achieved long - term success in controlling their body weight .", "in the current study , we examined the relationship between physical activity variety , defined as the number of different types of self - reported moderate - to - vigorous activities performed in one week , and minutes spent in objectively measured moderate - to - vigorous physical activity ( mvpa ) in two groups of individuals who have successfully maintained their body weight long - term : ( 1 ) weight loss maintainers with previous history of overweight / obesity and ( 2 ) normal - weight individuals without a history of overweight .", "normal - weight participants were included as a comparison group given that weight loss maintainers represent a unique group of individuals who report strict adherence to multiple behavioral strategies in order to maintain their body weight . due to their unique characteristics and history ,", "it is possible that the association between physical activity variety and mvpa could be different for weight loss maintainers and normal - weight individuals . however , based on previous research showing a relationship between physical activity variety and higher physical activity levels across diverse groups and settings [ 14 , 15 ] , we predicted that engagement in a greater variety of moderate - to - vigorous activities would be associated with higher mvpa daily minutes in both the weight loss maintainer and normal - weight groups .", "participants were enrollees in the cross - sectional  lite  study that compared weight control behaviors of weight loss maintainers and normal - weight controls .", "a convenience sample of men and women was recruited through advertisements placed in national and local publications intended for a general audience .", "persons interested in participating were asked to either call a toll - free number or to visit a website .", "participants were recruited from across the united states , although most were from new england , california , and the washington , dc area .", "weight loss maintainers had a history of overweight or obesity ( bmi 25 ) but were currently normal weight ( bmi = 18.524.9 ) , having maintained a 10% loss of their lifetime maximum body weight for at least 5 years .", "normal - weight participants had a current bmi between 18.5 and 24.9 and no history of overweight or obesity .", "participants in both groups were weight stable ( 10  lb ) for at least 2 years prior to enrollment . of 813 individuals who responded to advertisements and a brief online screening tool ,", "the study protocol was approved by the miriam hospital institutional review board , providence , ri , usa . participants reported information about age , gender , marital status , ethnicity / race , type of employment , and education . weight and weight history were assessed via self - report methods that have been previously validated .", "questions on the paffenbarger physical activity questionnaire   regarding average number of city blocks walked per day , and weekly frequency and duration of sports and recreational activities performed were used to determine variety or number of different activities performed during the past 7 days .", "only activities that were performed at a moderate or vigorous intensity and for 10 minutes in duration were included given that engagement in these activities is considered necessary for improving health and achieving a healthy body weight [ 1 , 12 ] .", "the intensity of sports and recreational activities was determined using the paffenbarger coding scheme . for walking to be counted as an activity", ", participants had to report walking the equivalent of at least 12 blocks per day ( i.e. , 1 mile at a moderate intensity ) .", "treadmill walking reported as a sports and recreational activity was not distinguished as a separate activity from walking 12 blocks / day . climbing stair flights , walking <", "12 blocks / day , and sports and recreational activities that were performed for < 10 minutes were not considered to contribute to variety .  ", "monrovia , ca , usa ) was used to objectively measure daily minutes spent in mvpa .", "this device converts accelerations or movements from vertical , horizontal , and anterior - posterior planes into counts , with greater magnitude or intensity of acceleration over a given time period generating a higher number of counts .", "participants were sent the device in a postage - paid envelope with instructions on how to activate the device and wear it on their waistband during all waking hours for 7 consecutive days , except while bathing or swimming .", "each device was programmed with the participant 's personal data ( sex , age , height , and weight ) and set to capture movements continuously in 1-minute intervals .", "the rt3 has shown to be a strong predictor of oxygen consumption during sedentary and treadmill activities [ 20 , 21 ] and a more precise measure of physical activity at the group level compared to its tri - trac predecessor .", "consistent with previously documented methods for analyzing rt3 data , a minimum of 4 days on which the device was worn for 10 hours each day was required for data to be considered valid [ 23 , 24 ] .", "rt3 nonwear times , defined as periods of 30 consecutive minutes of zero counts ( permitting intervals of up to 2 consecutive minutes registering 1100 counts / min ) , were deleted from analysis . the remaining time was partitioned according to intensity level . based on previous rt3 validation research   and a recent study that used the rt3 to compare mvpa patterns in weight loss maintainers , normal - weight , and obese groups", ", we computed time spent in mvpa using a threshold of 984 counts / min .", "descriptive statistics are presented in tables as means  sd for continuous measures and percentages for categorical responses . chi", "independent t - tests were conducted to assess differences between the groups on demographic characteristics , weight , accelerometer daily wear time , mvpa minutes / day , and reported variety / number of different types of moderate - to - vigorous activities performed .", "linear regression was used to examine the associations of physical activity variety and group status ( weight loss maintainers versus normal weight ) with objectively measured mvpa minutes / day , adjusting for age , gender , years of education , marital status , bmi , and accelerometer daily wear time .", "logistic regression was used to assess whether physical activity variety and group status were associated with achieving the 250 mvpa minutes / week recommendation for optimal long - term weight maintenance .", "for this analysis , mvpa minutes / week was calculated by multiplying average daily mvpa minutes by 7 ( days ) .", "of 413 participants who were sent an accelerometer and the ppaq , 394 ( 95% ) met valid accelerometer wear requirements and provided complete data on the ppaq .", "the characteristics of these 226 weight loss maintainers and 169 normal - weight participants are presented in table 1 .", "both groups were similar in age ( 48.8  12.9 years ) , gender ( 84% female ) , marital status ( 66% married ) , race / ethnicity ( 94% caucasian ) , employment status ( 82% employed ) , and job type ( 95% in professional or clerical positions ) . a greater proportion of normal - weight participants was college educated , compared to the weight loss maintainers .", "both groups were normal weight , although the weight loss maintainers had a slightly higher bmi . on average ,", "weight loss maintainers had lost nearly 29  kg and maintained 10% weight loss for 13.7  9.6 years . there were no differences in accelerometer wear time between the groups , with the weight loss maintainers and normal - weight participants wearing the accelerometer for an average of 14.8  2.0 hours / day on 7.5  0.9 days .", "as reported previously , weight loss maintainers spent an average of 6 minutes more per day in mvpa , compared to normal - weight participants ( 58 versus 52  min / d ) .", "the variety / number of different activities performed by the weight loss maintainers and normal - weight participants was similar ( 1.8  1.2 versus 1.7  1.2 , p = 0.52 ) . additionally , when participants who reported no moderate - to - vigorous activities were excluded , the variety / number of activities performed by wlm ( n = 180 ) and nw ( n = 141 ) remained similar ( 2.2  1.0 versus 2.0  1.0 , p = 0.10 ) .  ", "we next examined the independent and joint associations of physical activity variety and group status with mvpa minutes per day . given that the physical activity variety  group status interaction was not significant ( p = 0.73 ) , the results of the main effects linear regression model for mvpa minutes per day are shown ( table 2 ) . greater physical activity variety ( p < 0.001 ) and weight loss maintainer status ( p < 0.05 ) were independently related to greater daily time spent in mvpa , after adjustment for demographic characteristics ( age , gender , educational level , marital status , current bmi ) and daily accelerometer wear time .  ", "figure 1 presents estimated mvpa minutes per day in the weight loss maintainer and normal - weight groups by reported number of different moderate - to - vigorous activities performed . across both groups", "combined , the number of moderate - to - vigorous activities ranged from 0 to 4 , with 73 participants ( 18.5% ) reporting 0 activities , 105 reporting 1 activity ( 26.6% ) , 112 ( 28.4% ) reporting 2 activities , 74 reporting 3 activities ( 18.7% ) , and 30 ( 15.4% ) reporting 4 or more activities .", "each 1 unit increase in the number of different moderate - to - vigorous activities performed was associated with an additional 9.4  1.3 daily minutes spent in mvpa , representing an additional 56.2  8.3  kcal expended per day based on rt3-derived estimates .  ", "similarly , logistic regression analyses showed that greater physical activity variety ( or = 1.78 [ 1.372.30 ] , p < 0.001 ) , weight loss maintainer status ( or = 0.23 [ 0.080.69 ] , p = 0.002 ) , and lower bmi ( or = 0.81 [ 0.690.95 ] , p = 0.01 ) were independently associated with meeting the 250 mvpa minutes / week guideline for optimal weight maintenance .", "the physical activityvariety  group status interaction was not significant ( p = 0.25 ) .", "given the continuing obesity epidemic and growing evidence that indicates greater amounts of physical activity are needed for successful long - term weight control , it is important to identify strategies that can assist individuals in adopting and maintaining high levels of physical activity .", "this study examined whether performing a greater variety of different types of moderate - to - vigorous physical activities was related to greater time spent in mvpa among weight loss maintainers and normal - weight individuals without a history of overweight / obesity .", "we found that , independent of group , greater variety was associated with higher daily mvpa minutes and meeting the 250 mvpa minutes per week recommendation for optimal long - term weight maintenance .", "these findings are consistent with previous studies of the general population   and in overweight / obese individuals undergoing behavioral weight loss treatment .   however , the present study is the first to show a relationship between greater physical activity variety and higher objectively measured mvpa duration and energy expenditure in two groups of individuals who have had long - term success in maintaining a normal body weight .", "we found that for each additional different type of moderate - to - vigorous activity performed , participants on average spent an additional 9 minutes in mvpa and expended 56 more kcal per day .", "thus , for example , participants who reported engaging in 3 different activities during the previous week spent on average an additional 18 minutes in mvpa and expended 112 more calories per day compared to participants who reported engaging in only 1 activity .", "while our findings do not imply causation , they do warrant additional longitudinal research to examine whether incorporating variety into a physical activity routine may be an efficacious strategy to achieve higher mvpa levels for enhanced weight control .   ", "the relationship between greater physical activity variety and engagement in higher mvpa levels may be potentially explained by a number of physiological and psychological factors .", "for example , alternating different physical activities that involve different muscle groups and energy systems ( aerobic , anaerobic ) might promote greater exercise consistency by affording more time for recovery and decreasing risk of overuse injuries [ 13 , 26 ] .", "participating in a variety of activities may also facilitate greater exercise adherence via increased enjoyment   and decreased boredom [ 13 , 26 ] .", "greater access to a variety of activities may increase the likelihood that individuals will find an exercise activity or a combination of exercise activities that they like and will perform regularly . additionally ,", "research based on the behavioral economics model suggests that motivation to exercise is enhanced when individuals can choose from a variety of physical activities versus only one physical activity [ 28 , 29 ] .", "consequently , adding a variety component to a physical activity prescription may aid individuals in achieving and maintaining high levels of physical activity .", "future research is needed to investigate potential mechanisms that underlie the relationship between variety and higher mvpa levels .   whereas increased variety of healthy physical activities is associated with greater time spent in mvpa", ", it is also possible that decreased variety of unhealthy sedentary activities might contribute to lesser time spent being sedentary and higher overall physical activity levels . given that sedentary behaviors , independent of physical activity , have shown to be detrimentally associated with bmi and other cardiometabolic risk factors [ 30 , 31 ] , future studies that examine the association between variety and sedentary behaviors are needed .  ", "the cross - sectional nature of our study does not allow us to determine whether greater variety contributes to higher mvpa levels or alternatively whether individuals with higher mvpa levels naturally incorporate more variety into their physical activity routine .", "as variety in physical activity is rarely measured , the importance of factors such as the time frame for assessing variety ( i.e. , a week , month , year ) and frequency of occurrence of different activities within the time frame ( i.e. , once a week , twice a month ) are not known . while the use of an objective measure of physical activity is a strength of this investigation , it is important to note that hip - worn accelerometers like the rt3 used in this study may be limited in their ability to accurately estimate the intensity of activities not performed on flat surfaces .", "thus , it is possible that mvpa was underestimated in individuals who more frequently engaged in activities that involved an incline or greater upper body movement .", "additionally , the accelerometer count threshold we used to identify mvpa was determined in a leaner , younger male sample   and thus may have affected validity in our older , largely female sample . given the homogeneity of our sample with respect to gender ( female ) and race ( white non - hispanic ) , our results may not be generalizable to men or other ethnic populations .", "similarly , participants in this study were all normal weight and had high physical activity levels on average .", "thus , future investigations should examine the potential importance of variety for increasing physical activity in overweight and inactive populations .", "finally , it is important to note that just as altering the variety of different types of activities performed could potentially increase mvpa duration and energy expenditure , so could altering the frequency , intensity , and duration of a single activity , independent of changes in physical activity variety .   in summary", ", this study examined the relationship between physical activity variety and objectively measured mvpa levels in weight loss maintainers and normal - weight individuals who both had long - term success in maintaining their body weight . in both groups ,", "physical activity variety was related to greater engagement in mvpa and likelihood of accumulating 250 mvpa minutes / week , consistent with physical activity guidelines for optimal long - term weight maintenance .", "future studies are needed to test whether incorporating variety or different types of activities ( e.g. , walking and cycling ) can facilitate engagement in higher levels of mvpa within interventions aimed at promoting and maintaining physical activity ."], "abstract_text": ["<S> given the importance of physical activity ( pa ) for weight control , identifying strategies to achieve higher pa levels is imperative . we hypothesized that performing a greater variety of self - reported moderate - to - vigorous activities ( mvpas ) would relate to higher objectively measured mvpa minutes in two groups who were successfully maintaining their body weight : weight loss maintainers ( wlm / n = 226 ) and normal - weight individuals ( nw / n = 169 ) . the paffenbarger questionnaire and rt3 accelerometer were used to determine variety / number of different mvpas performed and mvpa minutes , respectively . </S>", "<S> the variety / number of different activities performed by wlm and nw was similar ( 1.8  1.2 versus 1.7  1.2 , p = 0.52 ) . </S>", "<S> regression analyses showed that greater variety ( p < 0.01 ) and wlm status ( p < 0.05 ) were each positively related to greater mvpa minutes / day and meeting the 250 mvpa minutes / week guideline for long - term weight maintenance . </S>", "<S> the association between greater variety and higher mvpa was similar in nw and wlm . </S>", "<S> future studies should test whether variety can facilitate engagement in higher mvpa levels for more effective weight control . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion "], "sections": [["engaging in high levels of physical activity is a key strategy for successful maintenance of weight loss .", "a study of nearly 4,000 participants in the national weight control registry ( nwcr ) , the largest longitudinal study of successful weight loss maintainers , indicated participants expend 2,621  2,252  kcal per week through physical activity , which is equivalent to approximately 60 minutes of moderate - intensity physical activity per day .", "additionally , long - term followup of participants in behavioral weight loss programs has shown that those who are most successful at maintaining their weight loss report activity levels similar to those of nwcr participants [ 3 , 4 ] .", "many individuals , particularly those who are obese , are insufficiently active , and increasing physical activity can be a challenge [ 5 , 6 ] .", "thus , strategies that assist individuals in adopting and sustaining high levels of physical activity to help facilitate a healthy body weight are needed .", "prior studies have identified multiple strategies for increasing physical activity adoption and maintenance , such as providing home or clinic - based exercise programs , increasing access to active behaviors ( e.g. , adding exercise equipment in the home ) , and reducing access to sedentary behaviors ( e.g. , limiting time to watch television ) , using pedometers to track activity and progress toward physical activity goals , and accumulating exercise throughout the day in multiple short bouts ( 10  min ) [ 11 , 12 ] . performing a variety of different types of activities may be another strategy to increase physical activity levels . the national health and nutrition examination survey ( nhanes ) showed that individuals who reported engaging in a variety of different activities ( i.e. , walking + other leisure - time activities ) were more likely to meet national physical activity recommendations compared to those who reported no variety ( i.e. , only walking ) .", "likewise , in an 18-month behavioral weight loss intervention , overweight participants who reported physical activity variety ( i.e. , 2 different activities ) at 6 months had higher self - reported activity - related energy expenditure and a lower body mass index ( bmi ) at 18 months than those who did not report physical activity variety ( i.e. , only 1 activity ) . thus , while the above findings suggest that physical activity variety may contribute to higher physical activity levels within the context of behavioral weight loss treatment , it is unclear whether physical activity variety is associated with higher physical activity in individuals who have achieved long - term success in controlling their body weight .", "in the current study , we examined the relationship between physical activity variety , defined as the number of different types of self - reported moderate - to - vigorous activities performed in one week , and minutes spent in objectively measured moderate - to - vigorous physical activity ( mvpa ) in two groups of individuals who have successfully maintained their body weight long - term : ( 1 ) weight loss maintainers with previous history of overweight / obesity and ( 2 ) normal - weight individuals without a history of overweight .", "normal - weight participants were included as a comparison group given that weight loss maintainers represent a unique group of individuals who report strict adherence to multiple behavioral strategies in order to maintain their body weight . due to their unique characteristics and history ,", "it is possible that the association between physical activity variety and mvpa could be different for weight loss maintainers and normal - weight individuals . however , based on previous research showing a relationship between physical activity variety and higher physical activity levels across diverse groups and settings [ 14 , 15 ] , we predicted that engagement in a greater variety of moderate - to - vigorous activities would be associated with higher mvpa daily minutes in both the weight loss maintainer and normal - weight groups ."], ["participants were enrollees in the cross - sectional  lite  study that compared weight control behaviors of weight loss maintainers and normal - weight controls .", "a convenience sample of men and women was recruited through advertisements placed in national and local publications intended for a general audience .", "persons interested in participating were asked to either call a toll - free number or to visit a website .", "participants were recruited from across the united states , although most were from new england , california , and the washington , dc area .", "weight loss maintainers had a history of overweight or obesity ( bmi 25 ) but were currently normal weight ( bmi = 18.524.9 ) , having maintained a 10% loss of their lifetime maximum body weight for at least 5 years .", "normal - weight participants had a current bmi between 18.5 and 24.9 and no history of overweight or obesity .", "participants in both groups were weight stable ( 10  lb ) for at least 2 years prior to enrollment . of 813 individuals who responded to advertisements and a brief online screening tool ,", "the study protocol was approved by the miriam hospital institutional review board , providence , ri , usa . participants reported information about age , gender , marital status , ethnicity / race , type of employment , and education . weight and weight history were assessed via self - report methods that have been previously validated .", "questions on the paffenbarger physical activity questionnaire   regarding average number of city blocks walked per day , and weekly frequency and duration of sports and recreational activities performed were used to determine variety or number of different activities performed during the past 7 days .", "only activities that were performed at a moderate or vigorous intensity and for 10 minutes in duration were included given that engagement in these activities is considered necessary for improving health and achieving a healthy body weight [ 1 , 12 ] .", "the intensity of sports and recreational activities was determined using the paffenbarger coding scheme . for walking to be counted as an activity", ", participants had to report walking the equivalent of at least 12 blocks per day ( i.e. , 1 mile at a moderate intensity ) .", "treadmill walking reported as a sports and recreational activity was not distinguished as a separate activity from walking 12 blocks / day . climbing stair flights , walking <", "12 blocks / day , and sports and recreational activities that were performed for < 10 minutes were not considered to contribute to variety .  ", "monrovia , ca , usa ) was used to objectively measure daily minutes spent in mvpa .", "this device converts accelerations or movements from vertical , horizontal , and anterior - posterior planes into counts , with greater magnitude or intensity of acceleration over a given time period generating a higher number of counts .", "participants were sent the device in a postage - paid envelope with instructions on how to activate the device and wear it on their waistband during all waking hours for 7 consecutive days , except while bathing or swimming .", "each device was programmed with the participant 's personal data ( sex , age , height , and weight ) and set to capture movements continuously in 1-minute intervals .", "the rt3 has shown to be a strong predictor of oxygen consumption during sedentary and treadmill activities [ 20 , 21 ] and a more precise measure of physical activity at the group level compared to its tri - trac predecessor .", "consistent with previously documented methods for analyzing rt3 data , a minimum of 4 days on which the device was worn for 10 hours each day was required for data to be considered valid [ 23 , 24 ] .", "rt3 nonwear times , defined as periods of 30 consecutive minutes of zero counts ( permitting intervals of up to 2 consecutive minutes registering 1100 counts / min ) , were deleted from analysis . the remaining time was partitioned according to intensity level . based on previous rt3 validation research   and a recent study that used the rt3 to compare mvpa patterns in weight loss maintainers , normal - weight , and obese groups", ", we computed time spent in mvpa using a threshold of 984 counts / min .", "descriptive statistics are presented in tables as means  sd for continuous measures and percentages for categorical responses . chi", "independent t - tests were conducted to assess differences between the groups on demographic characteristics , weight , accelerometer daily wear time , mvpa minutes / day , and reported variety / number of different types of moderate - to - vigorous activities performed .", "linear regression was used to examine the associations of physical activity variety and group status ( weight loss maintainers versus normal weight ) with objectively measured mvpa minutes / day , adjusting for age , gender , years of education , marital status , bmi , and accelerometer daily wear time .", "logistic regression was used to assess whether physical activity variety and group status were associated with achieving the 250 mvpa minutes / week recommendation for optimal long - term weight maintenance .", "for this analysis , mvpa minutes / week was calculated by multiplying average daily mvpa minutes by 7 ( days ) ."], ["of 413 participants who were sent an accelerometer and the ppaq , 394 ( 95% ) met valid accelerometer wear requirements and provided complete data on the ppaq .", "the characteristics of these 226 weight loss maintainers and 169 normal - weight participants are presented in table 1 .", "both groups were similar in age ( 48.8  12.9 years ) , gender ( 84% female ) , marital status ( 66% married ) , race / ethnicity ( 94% caucasian ) , employment status ( 82% employed ) , and job type ( 95% in professional or clerical positions ) . a greater proportion of normal - weight participants was college educated , compared to the weight loss maintainers .", "both groups were normal weight , although the weight loss maintainers had a slightly higher bmi . on average ,", "weight loss maintainers had lost nearly 29  kg and maintained 10% weight loss for 13.7  9.6 years . there were no differences in accelerometer wear time between the groups , with the weight loss maintainers and normal - weight participants wearing the accelerometer for an average of 14.8  2.0 hours / day on 7.5  0.9 days .", "as reported previously , weight loss maintainers spent an average of 6 minutes more per day in mvpa , compared to normal - weight participants ( 58 versus 52  min / d ) .", "the variety / number of different activities performed by the weight loss maintainers and normal - weight participants was similar ( 1.8  1.2 versus 1.7  1.2 , p = 0.52 ) . additionally , when participants who reported no moderate - to - vigorous activities were excluded , the variety / number of activities performed by wlm ( n = 180 ) and nw ( n = 141 ) remained similar ( 2.2  1.0 versus 2.0  1.0 , p = 0.10 ) .  ", "we next examined the independent and joint associations of physical activity variety and group status with mvpa minutes per day . given that the physical activity variety  group status interaction was not significant ( p = 0.73 ) , the results of the main effects linear regression model for mvpa minutes per day are shown ( table 2 ) . greater physical activity variety ( p < 0.001 ) and weight loss maintainer status ( p < 0.05 ) were independently related to greater daily time spent in mvpa , after adjustment for demographic characteristics ( age , gender , educational level , marital status , current bmi ) and daily accelerometer wear time .  ", "figure 1 presents estimated mvpa minutes per day in the weight loss maintainer and normal - weight groups by reported number of different moderate - to - vigorous activities performed . across both groups", "combined , the number of moderate - to - vigorous activities ranged from 0 to 4 , with 73 participants ( 18.5% ) reporting 0 activities , 105 reporting 1 activity ( 26.6% ) , 112 ( 28.4% ) reporting 2 activities , 74 reporting 3 activities ( 18.7% ) , and 30 ( 15.4% ) reporting 4 or more activities .", "each 1 unit increase in the number of different moderate - to - vigorous activities performed was associated with an additional 9.4  1.3 daily minutes spent in mvpa , representing an additional 56.2  8.3  kcal expended per day based on rt3-derived estimates .  ", "similarly , logistic regression analyses showed that greater physical activity variety ( or = 1.78 [ 1.372.30 ] , p < 0.001 ) , weight loss maintainer status ( or = 0.23 [ 0.080.69 ] , p = 0.002 ) , and lower bmi ( or = 0.81 [ 0.690.95 ] , p = 0.01 ) were independently associated with meeting the 250 mvpa minutes / week guideline for optimal weight maintenance .", "the physical activityvariety  group status interaction was not significant ( p = 0.25 ) ."], ["given the continuing obesity epidemic and growing evidence that indicates greater amounts of physical activity are needed for successful long - term weight control , it is important to identify strategies that can assist individuals in adopting and maintaining high levels of physical activity .", "this study examined whether performing a greater variety of different types of moderate - to - vigorous physical activities was related to greater time spent in mvpa among weight loss maintainers and normal - weight individuals without a history of overweight / obesity .", "we found that , independent of group , greater variety was associated with higher daily mvpa minutes and meeting the 250 mvpa minutes per week recommendation for optimal long - term weight maintenance .", "these findings are consistent with previous studies of the general population   and in overweight / obese individuals undergoing behavioral weight loss treatment .   however , the present study is the first to show a relationship between greater physical activity variety and higher objectively measured mvpa duration and energy expenditure in two groups of individuals who have had long - term success in maintaining a normal body weight .", "we found that for each additional different type of moderate - to - vigorous activity performed , participants on average spent an additional 9 minutes in mvpa and expended 56 more kcal per day .", "thus , for example , participants who reported engaging in 3 different activities during the previous week spent on average an additional 18 minutes in mvpa and expended 112 more calories per day compared to participants who reported engaging in only 1 activity .", "while our findings do not imply causation , they do warrant additional longitudinal research to examine whether incorporating variety into a physical activity routine may be an efficacious strategy to achieve higher mvpa levels for enhanced weight control .   ", "the relationship between greater physical activity variety and engagement in higher mvpa levels may be potentially explained by a number of physiological and psychological factors .", "for example , alternating different physical activities that involve different muscle groups and energy systems ( aerobic , anaerobic ) might promote greater exercise consistency by affording more time for recovery and decreasing risk of overuse injuries [ 13 , 26 ] .", "participating in a variety of activities may also facilitate greater exercise adherence via increased enjoyment   and decreased boredom [ 13 , 26 ] .", "greater access to a variety of activities may increase the likelihood that individuals will find an exercise activity or a combination of exercise activities that they like and will perform regularly . additionally ,", "research based on the behavioral economics model suggests that motivation to exercise is enhanced when individuals can choose from a variety of physical activities versus only one physical activity [ 28 , 29 ] .", "consequently , adding a variety component to a physical activity prescription may aid individuals in achieving and maintaining high levels of physical activity .", "future research is needed to investigate potential mechanisms that underlie the relationship between variety and higher mvpa levels .   whereas increased variety of healthy physical activities is associated with greater time spent in mvpa", ", it is also possible that decreased variety of unhealthy sedentary activities might contribute to lesser time spent being sedentary and higher overall physical activity levels . given that sedentary behaviors , independent of physical activity , have shown to be detrimentally associated with bmi and other cardiometabolic risk factors [ 30 , 31 ] , future studies that examine the association between variety and sedentary behaviors are needed .  ", "the cross - sectional nature of our study does not allow us to determine whether greater variety contributes to higher mvpa levels or alternatively whether individuals with higher mvpa levels naturally incorporate more variety into their physical activity routine .", "as variety in physical activity is rarely measured , the importance of factors such as the time frame for assessing variety ( i.e. , a week , month , year ) and frequency of occurrence of different activities within the time frame ( i.e. , once a week , twice a month ) are not known . while the use of an objective measure of physical activity is a strength of this investigation , it is important to note that hip - worn accelerometers like the rt3 used in this study may be limited in their ability to accurately estimate the intensity of activities not performed on flat surfaces .", "thus , it is possible that mvpa was underestimated in individuals who more frequently engaged in activities that involved an incline or greater upper body movement .", "additionally , the accelerometer count threshold we used to identify mvpa was determined in a leaner , younger male sample   and thus may have affected validity in our older , largely female sample . given the homogeneity of our sample with respect to gender ( female ) and race ( white non - hispanic ) , our results may not be generalizable to men or other ethnic populations .", "similarly , participants in this study were all normal weight and had high physical activity levels on average .", "thus , future investigations should examine the potential importance of variety for increasing physical activity in overweight and inactive populations .", "finally , it is important to note that just as altering the variety of different types of activities performed could potentially increase mvpa duration and energy expenditure , so could altering the frequency , intensity , and duration of a single activity , independent of changes in physical activity variety .   in summary", ", this study examined the relationship between physical activity variety and objectively measured mvpa levels in weight loss maintainers and normal - weight individuals who both had long - term success in maintaining their body weight . in both groups ,", "physical activity variety was related to greater engagement in mvpa and likelihood of accumulating 250 mvpa minutes / week , consistent with physical activity guidelines for optimal long - term weight maintenance .", "future studies are needed to test whether incorporating variety or different types of activities ( e.g. , walking and cycling ) can facilitate engagement in higher levels of mvpa within interventions aimed at promoting and maintaining physical activity ."]]}
{"article_id": "PMC4141698", "article_text": ["recently \n we reported on an analysis of the infrared spectrum of \n c2f4 . by observation \n of many combination and difference bands , including many from two c - containing isotopologues , and by comparison with mp2 and \n density - functional calculations , all 12 fundamentals could be assigned , \n although only five are ir active .", "the geometry \n was deduced from ro - vibrational analysis of strong ir active bands \n and by comparison with theoretical calculations .", "however , it is also \n instructive to correlate anharmonic constants with some peculiarities \n of the reactions of c2f4 .", "accordingly , in this \n work more anharmonic constants ( altogether 54 ) are derived from the \n spectra , and a complete set ( 78 ) is calculated .", "the earlier vibrational \n assignments of c2f4 were reported at a time \n when the unusual properties of this molecule were barely known .", "explained this property by positing that the electron attraction \n of fluorine leads to a preference of sp- over sp - type carbon : radical addition converts both \n c atoms from sp to sp hybridization , which \n stabilizes the primary product , lowers the transition state , and thus \n accelerates the reaction .", "alder reactions , for example , \n which was again explained by stabilization of radical intermediates .", "( borden also presented an alternative but related description based \n on delocalization of unpaired radicalic electrons to accepting  * \n orbitals of cf groups . )", "the preference \n for carbon sp hybridization can also be expected to lower \n the force constants for the pyramidalization ( wagging ) vibrations \n ( 7 , 8 ) and/or increase their anharmonicity . \n", "furthermore , torsion ( 4 ) should be facilitated by \n rehybridization and in fact quantum chemistry confirmed this for a \n 90 twist .", "another spectacular property of c2f4 is how \n readily it can dissociate thermally to two cf2 molecules .", "a c = c bond energy of only 2.95 ev is derived , less than that of a typical c  c single \n bond ( 4 ev ) .", "the low dissociation energy is caused by stabilization \n of the resulting two difluorocarbenes by back - bonding from nonbonding \n fluorine electrons to the empty carbon p - orbital .", "this also explains why cf2 has a singlet \n ground state which is more stable than the triplet ( by 2.458 ev , taken \n from the band origin of the phosphorescence spectrum ) . whereas in the previous work", ", only cc stretching is considered \n as the dissociation coordinate , it was argued in ref ( 16 ) that pyramidalization \n must also be involved .", "this idea is supported in the present work \n by the large value found for the anharmonic constant x18 coupling cc stretch with trans pyramidalization .", "the \n magnitude of x18 as compared to the much \n smaller x17 ( coupling cc stretch with \n cis pyramidalization ) also supports a valence - bond analysis of weak \n double bonds , which was reviewed in ref ( 19 ) .", "the samples in natural isotopic abundance were either taken from \n commercial c2f4 ( hoechst ) , that contained a \n trace of the cyclic dimer , or prepared from c2f4br2 + zn in a high - boiling \n alcohol ( diethylene glycol ) ; the comparison allowed for recognizing \n absorptions of impurities .", "a sample enriched in c to \n 50% was prepared from chclf2 by co2 laser isotope \n separation with enriched cf2 as the primary product .", "ir spectra of c2f4 in natural isotopic \n abundance \n and isotopically enriched were recorded between 370 and 4000 cm with 0.2 cm resolution by a perkinelmer \n ftir spectrometer in garching . at the \n synchrotron lab ,", "the isotopically enriched sample was measured at \n moderate 0.1 cm resolution in the far ir region \n from 100 to 600 cm ( capturing the band at 210 \n cm ) , and at high resolution for the ir active \n cf stretch bands , see ref ( 1 ) .", "the present work focuses on anharmonic constants of c2f4 . only where the constants were \n expected to be different for ccf4 and c2f4 ( e.g. , in the case of \n fermi resonance )", "theoretical values of the anharmonic constants xij were obtained from anharmonic \n vibrational \n frequency calculations performed using gaussian 03 revision c02 at the mp2 level using \n dunning - type correlation - consistent ( cc - pvtz ) orbitals .", "the molecular force field at this level of theory \n was shown in the previous study to reproduce the fundamental wavenumbers \n and the isotopic shifts of c2f4 very satisfactorily \n ( for details , see ref ( 1 ) ) , and hence was an appropriate choice for computing xij values .", "the calculations were performed for each of \n the c2f4 , ccf4 , and c2f4 isotopologues .", "the spectra were measured previously and the \n fundamentals extracted \n from them , using a limited number of anharmonic constants ( from combination \n and hot bands ) where necessary . for convenience \n table 1", "lists the fundamentals again . in this \n work we derived a large set of experimental anharmonic constants from \n combination bands and  in particular for the lower - wavenumber \n vibrations  from hot bands , and report a complete set of theoretically \n computed values .", "symmetry types refer to a d2h molecule with z axis along the cc bond and the y axis perpendicular \n to the plane , as used in ref ( 1 ) for easier comparison with the c2v symmetric ccf4 ; the more conventional designation with x axis along the cc bond and z axis perpendicular \n to the plane is given in parentheses .", "revised value ( instead of 210 cm ) , based on a more definite \n assignment of a hot - band structure and x10,10 , see supporting information .", "band origins from high - resolution \n spectra ; low - resolution values are 1338.4 \n and 1187.0 .", "upright : \n experimental values . \n in parentheses : values calculated by mp2/pvtz . where two values or \n a range are given for the experimental data", ", it may indicate ( a ) a \n dependence of the effective xij on the involved levels due to perturbations ( fermi resonances , \n section 4.4 ) or ( b ) an uncertainty due to error \n limits ( e.g. , where broad bands are involved ) or an uncertain assignment . \n", "a preferred value is indicated by bold face . for further details on \n the derivation of each xij and some isotopic data ,", "the anharmonic constants xij were calculated from observed \n combination and difference bands \n by assuming that anharmonic shifts are additive : for binary combination \n bands ( i  j ) and for overtones \n we used1and2with obvious extensions for more complicated \n combinations .", "( the xij values are mostly negative , as usual . ) the left - hand side of these \n equations should be read as  wavenumber of ( name )  . for hot bands ( satellite transitions near i that originate from a thermally populated \n lower level j ) we used3again \n with obvious extensions .", "one can often \n observe sequences of equidistant q branches , corresponding to hot \n bands starting from higher levels mj , or combination of such sequences ( from mj + nk ) .", "equation 3 implies that the distance of a ( first ) hot band from the fundamental \n directly indicates the corresponding anharmonic constant .", "combination \n bands allow for a direct assignment , whereas hot bands must be identified \n by their intensity ( controlled by the boltzmann factor ) . in a number \n of cases", "the supporting information ( si ) compiles the sources for each xij in table s2 , whereas table s1 lists the observed \n bands ( with hot bands ) and their isotopic shifts .", "two examples of \n hot bands , whose shifts are dominated by the large x18 anharmonicity , are shown in figure 1 .", "absorbance spectra demonstrating the large x18 derived shifts for hot - bands originating in 8 .", "the spectra were recorded \n in the garching laboratory , with pressure and path length of 2 bar \n and 10 cm ( for the lower trace of each panel ) , and 140 mbar and 4 \n m ( upper trace ) , respectively . for levels involving more than two quanta of vibration , anharmonic \n shifts", "this helped to decide between different \n assignments of combination bands in a few cases where the harmonic \n sums were similar to each other .", "however , such additivity need not \n apply when the levels involved are subject to fermi resonance , producing \n shifts that depend on the separation of nearby interacting levels . \n", "we use eqs 13 to \n obtain the experimental values , and hence , table 2 reports phenomenological xij , some of which show a spread in the values suggestive of fermi \n resonances ( in particular x18 , x26 , and x77 ) .", "fermi \n resonances can also be recognized if the apparent xij or the hot - band structure depends \n on the isotopologue . for c2f4 ,", "fermi resonances \n were identified ( see section 4.4 and si ) between:5 and 2+6 ( discussed already in ref ( 1)),1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue),2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77).2 and 23 , identified from the \n mp2 calculations .", "5 and 2+6 ( discussed already in ref ( 1 ) ) , 1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue ) , 2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77 ) . 2 and 23 , identified from the \n mp2 calculations .", "the mp2/cc - pvtz calculations \n reproduced the observed wavenumbers \n and isotopic shifts very well . supporting \n the overall vibrational analysis", "exceptions are 15 ( x22 , x28 , x29 , x2,10 , x2,11 , x36 , x3,10 , x45 , x59 , x5,11 , x66 , x68 , x77 , x88 , x10,11 ) out of 54 cases where an observed value is available \n for comparison . some of these will be discussed below .", "as already \n described in the introduction , the peculiar \n reactions of c2f4 have to do \n with cc stretching and carbon pyramidalization . the discussion will \n focus on these coordinates ( sections 4.14.3 ) . for \n a planar geometry , \n", "cc dissociation correlates c2h4 and c2f4 with a pair of carbenes in their lowest triplet states ( figure 2a ) . because \n the energy of forming these products is practically the same for the \n two molecules", ", one could also expect that the potential energy curves \n coincide and the cc stretch force constants are equal .", "in fact , already \n early force - field calculations showed no substantial difference ( 889 \n n / m for c2f4 and \n 940", "furthermore , \n the short cc distance ( 132 pm , which is 1 pm shorter than in ethylene ) and the high cc stretch wavenumber ( 1873.8 cm , compared to 1625.4 cm   for ethylene ) do not point to a weakened bond . \n as early as 1965 , the low dissociation energy of c2f4 was attributed to switching over to a channel producing two \n singlet carbenes and to the stabilization ( by 2.458 ev , see introduction ) of singlet cf2 by back bonding , so that this channel is energetically favored by 4.9 ev .", "this path hence leads over an avoided \n crossing of potential surfaces ( figure 2a ) . \n", "if the avoidance is strong , one could expect an influence at least \n on the anharmonicity x11 ( 1 is dominated by cc stretching ) , even if not on the force \n constant at the bottom of the potential energy well .", "table 2 shows that the magnitude of x11 ( calculated 7.8 cm ) is not unusually \n large for such a high - frequency vibration , though noticeably larger \n than for ethylene ( x22 = 2.3 cm  ) . that the increase \n in magnitude is only moderate", "can be taken as a sign that the avoidance \n along this coordinate is not very strong .", "hence , the back reaction \n ( recombination ) will have a high barrier in planar geometry . whereas \n the measured recombination barrier is not , in fact , zero , it is only \n minor ( 9 kj / mol = 0.09 ev  ) .", "so , \n a lower - energy path must lead around the barrier , out of the drawing \n plane of figure 2a , that is , with distortion \n different from cc stretch .", "potentials for ground - state cc dissociation \n of c2f4 , leading over an avoided crossing to \n two singlet carbenes . \n in ( a ) it is assumed that the one - sided pyramidalization ( q7 + q8 ) begins only after overcoming the barrier , whereas \n ( b ) shows the preferred path , where this distortion is already fully \n developed on the barrier and is preceded by a trans pyramidalization \n ( q8 ) , while the cc distance is steadily increasing .", "it was argued in ref ( 16 ) that recombination of \n two cf2 molecules should \n be easiest if one of them points with its filled n orbital \n to the empty p orbital of the other .", "hence , the dissociation \n path will change from pure cc stretch to include also pyramidalization \n ( figure 2b ) , and the potential should be lowered \n in energy in the direction of q7 ( the coordinate of 7 ) and/or q8 . in turn", "in fact x18 has an unusually large magnitude , although x17 is in the normal range ( table 2 ) .", "( the magnitude of x18 is not \n caused by secondary effects such as fermi resonance , as we shall see \n in section 4.4.3 . )", "we can conclude that on \n stretching the cc bond the molecule first bends toward trans pyramidalization \n ( q8 ) ( figure 2b ) .", "the one - sided \n nonplanar bending ( figure 2 ) is a superposition \n of q7 and q8 .", "the reason why q8 is \n active earlier than q7 can be understood by a valence - bond \n model for weak double bonds . in this model", "several symmetric arrangements \n of carbenes ( a  c \n of scheme 2 ) were considered : whereas with \n two triplet carbenes all arrangements ( a  d ) lead to bonding \n interaction , with two singlet carbenes bonding occurs only in the \n trans - bent case ( c ) ; this corresponds to a double donor ", "one can add that the cis - bent case with singlet carbenes would \n lead only to antibonding ( scheme 2d ) .", "malrieu \n and trinquier derived a criterion for trans - bending ( deriving even \n the angle ) by comparing the standard double - bond energy with the singlet ", "their findings were confirmed by many examples , in particular with \n heavier - element homologues .", "we can apply \n this rule not only to the equilibrium geometry of c2f4 but also to its dissociation path , if we consider in the \n comparison the double - bond energy that is reduced on extension of \n this bond .", "the rule thus predicts that on cc extension c2f4 is distorted from planar geometry to a trans - bent structure , \n and this rationalizes the large magnitude of x18 . in the triplet case ,", "the interaction \n can be bonding in all arrangements . in the singlet case , ( a )", "is antibonding , \n ( b ) nonbonding , ( c ) bonding , ( d ) antibonding .", "more recently , borisov et al . found evidence \n for trans pyramidalization \n on extension of the cc bond also from molecular orbital calculation \n ( at high level : mller  plesset perturbation theory of \n second , third and fourth order ) : the \n authors found that the onset is relatively sudden on cc extension \n to 142 pm and that the distortion lowers the energy appreciably .", "( the \n large x18 anharmonicity is an indication \n that this pyramidalization already begins not far from the s0 minimum .", "in fact , a cc stretch by 10 pm  from 132 to 142 pm ", "is \n just about the vibrational amplitude in the 1 = \n 1 level . )", "although borisov et al . did not find an energy minimum with \n such a distortion , they suggested that it plays a crucial role in \n the low - energy real intermediate found by buravtsev , kolbanovskii \n et al . in thermal reactions of c2f4 and recombination \n of cf2 ( see ref ( 31 ) and literature quoted therein and section 4.3 ) .", "one could suppose that in order to lower the activation \n energy \n for dissociation the only necessary deviation from simple cc stretching \n is distortion toward the 8 coordinate ( q8 ) .", "however , there is experimental \n evidence that the asymmetric pyramidalization ( corresponding to superposition \n of q7 and q8 as in figure 2b ) is already crucial in the transition state : the pre - exponential \n factor found in the rate of thermal c2f4 dissociation \n ( 2.8  10 s  ) is clearly larger than would be typical for \n a simple bond splitting ( 10 s ) .", "this \n corresponds to a raised entropy of activation , such as that which \n happens if an additional degree of freedom is activated in the transition \n state .", "the most plausible candidate is free internal rotation , as \n it can be expected in the asymmetric structure with a single dative \n bond but not in symmetric geometry with double donor  acceptor \n bond .", "this picture is also consistent with a stepwise cleavage of \n the double donator  acceptor bond on c2f4 dissociation and its stepwise formation on cf2 recombination .", "it is worth noting that the molecule with one - sided pyramidalization \n has an electron distribution ( figure 2b ) that \n is zwitterionic and correlates in planar geometry with the 2ag state with two  electrons excited to  * , as \n already pointed out in ref ( 16 ) .", "a fully analogous dissociation path can also be \n expected for the \n other systems considered by malrieu and trinquier .", "in fact , according \n to ref ( 32 ) ground - state \n ( ir induced ) dissociation of diazomethane ( h2cnn ) follows \n a path with a pyramidalized methylene group to produce n2 + singlet ch2 ; the out - of - plane deformation was experimentally \n confirmed . in the backward reaction ,", "n2 points with its \n n electron pair to the empty p orbital of the singlet \n methylene ( see refs ( 32 and 33 ) for a calculated potential in two dimensions , which would look similar \n for c2f4 ) .", "hence , \n the out - of - plane deformation on dissociation of c2f4 is not an isolated phenomenon . applying \n their energetic criterion to the equilibrium geometry of c2f4 , trinquier and malrieu found that this molecule is \n not far from the limit , where it would become nonplanar , and suggested \n that the 8 force constant may be significantly lower \n than that of ethylene .", "this is in fact \n confirmed in the following way : analytical expressions for a valence \n force field model for planar x2y4 were given \n by herzberg . besides the masses mx and my , bond lengths l1 ( for xx ) and l2 ( xy ) and the angle  ( half of yxy ) , both 7 and 8 only depend on the force constant for pyramidalization \n ( k/l2 ) . with equilibrium distances and angles from ref ( 1 ) and i = ( 2ci )", "one derives this force constant from \n 7 and 8 ( with identical results \n for c2f4 and c2f4):4both values should be identical \n in this valence - force field model .", "in fact for ethylene , the value \n is 22.9 n / m , whether derived from 7 or 8 .", "the pyramidalization force constant \n derived from 7 of c2f4 is \n slightly higher than in ethylene ( by 17% ) , but the same quantity derived \n from 8 is only 49% of the ethylene value .", "( of course , \n these values are not quantitatively meaningful , as the discrepancy \n highlights that a pure valence - force field model is not sufficient \n for wagging of c2f4 . )", "so in fact the restoring \n force for the 8 vibration is significantly smaller \n than in ethylene and than that expected on the basis of the same pyramidalization \n force constant as for 7 .", "this is consistent with \n the prediction of the valence - bond model favoring the trans - bent geometry .", "the slightly raised force constant for 7 could \n be due to repulsive nonbonding interaction in cis pyramidalization . \n", "such a geometry would be involved in the hypothetical transition state \n of the diels ", "alder addition of c2f4 to \n butadiene , a reaction that has not been observed .", "it was argued that \n an increased energy associated with this distortion might contribute \n to inhibiting the reaction .", "however , an \n increase in the force constant of only 17% is hardly sufficient to \n support this explanation , and the other effects discussed in ref ( 7 ) , in particular the competing \n fast radical reactions , are probably more important . applying \n again the force - field formulas of herzberg ,", "the force constant for torsion , k/l2 , is found to be 30.3 \n n / m for both ethylene and tetrafluoroethylene .", "that is , near the potential \n minimum the c = c bond in c2f4 behaves \n just like a normal double bond .", "consequently , the torsional barrier \n at a 90 twist ( which is a measure of the  bond strength ) \n would be identical for both molecules ( 2.8 ev ) if the two c  cf2 groups remained planar ; this was confirmed by a high - level \n calculation .", "however , pyramidalization \n lowers this barrier by 0.57 ev ( at mp2 level , consistent with ref ( 35 ) ) .", "if this stabilization is activated early along q4 , \n it could give rise to substantially negative values for x44 and especially for x47 and x48 .", "however , x44 and x47 are actually slightly positive \n ( 0.8 and 0.3 cm ) and x48 has no unusual magnitude ( 0.7 to 1.05 cm ) .", "evidently , the stabilizing effect of pyramidalization becomes \n effective only at high torsional angles .", "the corresponding values \n for ethylene are 2.4 , 8.9 , and 7.2 cm , respectively .", "the nondiagonal anharmonicities xi4 and x4j are \n generally smaller in magnitude than in ethylene . but this may simply \n have to do with the relative vibrational amplitudes , which are much \n larger for torsion of ch2 than of cf2 due to \n the relative masses . in the twisted - pyramidalized structure \n of c2f4 ,", "the triplet ( t1 ) is only \n 0.013 ev lower than the singlet \n ( s0 ) .", "it \n can therefore act as a real intermediate and undergo radical - type \n reactions , further assisted by its low energy that is calculated at \n 2.1 ev .", "this has been a widespread interpretation \n of some peculiarities of c2f4 reactions ( see , \n for example ref ( 7 ) ) .", "however , buravtsev , kolbanovskii et al . found in shock - tube experiments \n ( with c2f4 diluted by ar and adiabatically compressed ) \n an intermediate at much lower energy ( 0.8 to 1 ev ) with uv and visible \n absorption ( 235 and 500 nm ) ( see refs ( 31,36 , and 37 ) and the \n literature quoted there ) .", "they supposed it to be a singlet diradicaloid \n of c2f4 with a geometry corresponding not to \n an energy minimum but to a point on a slope of the potential ( see , \n for example ref ( 31 ) ) .", "however , as these points are not stationary there are too many \n ( nearly ) isoenergetic locations with shifted electronic transitions , \n so that the electronic absorption would be smeared out .", "it was therefore \n suggested that the observed intermediate \n is another low - energy isomer , tetrafluoro - ethylidene cf3cf ( electronic absorption in an ar matrix : ) .", "however , its energy ( 1.9 ev  ) seems to be nearly as high as that of the twisted - pyramidalized \n triplet .", "( in one study the twisted - pyramidalized c2f4 triplet is calculated at 0.90 ev , but computational details are not given , and previous theoretical \n results are not discussed . )", "some more anharmonicities of table 1 have unusually large magnitudes or show a range \n of values and/or deviate from prediction . in many of them", "fermi resonances \n play a role , as will be explained here . in section4.4.3 , however , it is shown that x18 is hardly influenced by such effects and its large magnitude mainly \n results from the overall shape of the potential energy surface .", "fermi resonances mix two nearby levels of the \n same symmetry , which differ by three units in the quantum numbers . \n", "the fermi - induced \n repulsion of the levels depends in a nonlinear way on both the magnitude \n of the fermi interaction parameter and on the original ( i.e. , unperturbed ) \n energetic distance of the levels .", "this separation will , in general , \n change ( a ) on isotopic substitution and ( b ) on adding a quantum of \n another vibration ( i.e. , in analogous combination bands ; similarly \n also in higher overtones , in which case a quantum number dependence \n must also be taken into account ) .", "another method of characterizing fermi resonance interactions relies \n on intensity borrowing caused by the mixing of levels . according \n to the mp2 calculations , the great majority of xij change by 1 cm on isotopic substitution", "the few exceptions are \n listed in table 3 and are compared to experimental \n values . identified ( fr13 ) and", "the interpretation in the line  influenced by  \n is based on the assumption that an xij is affected by such a resonance if at least one \n of the levels i and j is involved in the fermi interaction .", "the table shows that \n the calculated isotopic shift is sometimes \n much larger than in the experiment , for instance x26 , x27 .", "also x56 for c2f4 is probably \n too large , as the two observed bands 5+6+11 and 5+6+12 are close to their harmonic positions \n ( table s1 in si ) .", "it seems that at the \n present level of theory , the calculation does not reliably reproduce \n the fermi resonance induced shifts , at least the stronger ones .", "this \n is not surprising given the highly sensitive and nonlinear dependence \n of fermi shift on the  unperturbed  level separations . \n", "the same deficiency will also affect those xij connected with at least one level ( or a \n combination thereof ) involved in a resonance listed above ( examples \n in figure 3a ) .", "in fact , 10 of the 15 deviations \n listed at the end of section 3 can be attributed \n in this way to the resonances fr1 and fr2 with possible contributions \n of fr3 .", "the exceptions are x36 , x3,10 , x66 , x68 , and x88 . in \n all 42 other cases", "it is worthwhile to consider \n the two strongest fermi resonances , \n fr1 ( 2/27 ) and fr2 ( 5/2+6 ) , in c2f4 in some detail .", "the effect of these resonances \n is summarized in figure 3 . some levels involved \n in fermi resonances and observed transitions . \n", "solid arrows : ir , broken arrows : raman ; double arrows ( with interaction matrix element ) indicate repulsion \n between level pairs .", "the anharmonic shifts involving x77 are only nominal , because the fermi perturbation is \n strong :  6x77  = 18.1 is \n less than 3 times ", "(= 8.2 ) and  4x77  is only \n 9.9 . ( but  2x77  +  4x77  =  6x77  , as is obvious from the level scheme , and the raman \n anharmonic shifts practically coincide with those from the ir spectra . ) \n ( b ) three - level fermi resonance 5/2+6/27+6 and \n their combinations with 9 for c2f4 and c2f4 . \n", "the indicated level energies are from the spectra except those in \n parentheses , which are calculated .", "the broken horizontal lines are \n the estimated unperturbed positions ( see the si ) , and the signed numbers are the calculated shifts .", "the 16.4 cm magnitude of the interaction term ( with its expected dependence \n on quantum numbers ) has been derived \n from band shifts and intensities for a series of levels built on 2/27 for all three isotopologues .", "bands for \n 27+x ( x = 9 , 11 ) borrow their entire intensity from 2+x . the detailed analysis \n is provided in si .", "it \n is also evident that x77 , x27 and x22 are affected by \n the fermi resonance ( and probably other x2i , if the level separations differ ) and that x77 is level dependent because of the varying \n separation .", "the fermi term is almost entirely responsible for the \n + 8.2 cm shift of 27 for c2f4 and those of the heavier isotopologues \n ( table 3 under fr1 ) . in the latter", "the fermi - induced \n shift is increased , because the levels 2 and 27 ( before perturbation ) are closer together than in c2f4 and are practically degenerate in c2f4 .", "the success of these calculations \n shows that the additional mixing of 2 with 23 ( fr3 ) is apparently a weak perturbation at most .", "the \n fermi resonance fr2 between 5 and 2+6 was qualitatively discussed in ref ( 1 ) .", "a more complete understanding \n of the resonance shifts and pattern of intensity borrowing requires \n the three - level scheme shown in figure 3b ; \n this figure also shows combinations with 9 , as they \n are all ir active and were directly observed .", "analysis based on this \n model has enabled the magnitude of the fermi term to be determined \n as 11 cm ; the fr1 term of 16.4 cm was used without change .", "significantly , the model predicts varying \n fr shifts that even differ in sign ( originating from the different \n repulsions in figure 3b ) and can thus account \n for the unusual isotopic shifts observed between c2f4 and c2f4 . \n", "it explains a substantial deviation of the observed isotopic shift \n 5 = 51.9 from the value of 5 = 43.1 \n expected from the teller  redlich product rule , which is valid \n for harmonic oscillators .", "additionally \n the relative intensities of combination bands and their isotopic dependence \n are well predicted .", "it is also remarkable that according to the model \n the largest part of the anharmonicity x26 is due to fermi resonance .", "as described in ref ( 1 ) , the lower symmetry in ccf4 leads to strong mixing of 5 with 9 ( which are nearly degenerate in c2f4 ) .", "the two levels repel each other \n ( even in the harmonic approximation ) and give rise to two infrared - active \n transitions .", "it is sometimes difficult to disentangle them and their \n combination bands from the spectra .", "if the large magnitude of some \n anharmonicities is caused by fermi resonance , a crucial question is \n whether this might also be the case for x18 .", "if so , the conspicuous value of x18 could be largely a matter of the character of the resonance and \n less a consequence of global features of the potential for dissociation , \n which we considered in section 4.1 .", "in fact , \n weak perturbations of the 1 level and its combinations \n by the 1/5+6 fermi resonance are indicated by the slight x18 dependence on the level and on isotopic substitution and \n the change of the hot - band structure of 1 combinations \n in ccf4 , as explained in the si .", "a quantitative estimate of the strength \n of the 1/5+6 interaction can be deduced from the intensities of 5+6 combinations relative to those of the corresponding \n 1 combinations , assuming that the former borrow \n their full strength from the latter .", "the 5+6+12 band ( 2444.3 cm ) \n has 12% the strength of the 1+12 band ( 2428.7 cm ) , see figure 1a .", "similarly , the 5+6+11 shoulder ( 3072 cm ) is < 1% as intense as 1+11 ( 3056.2 cm ) and clearly visible only with longer \n path length than shown in figure 1b .", "from both \n observations , one can estimate a fermi term ( w ) of \n no more than 2.5 cm , causing a shift ( and \n consequent change in x18 ) of < 0.5 cm .", "this is actually an upper bound , because such weak \n bands can also be expected without intensity borrowing .", "hence , fermi \n resonance affects the magnitude of x18 only by a little ( by < 3% ) , and we can maintain the conclusions \n of section 4.1 on the shape of the potential .", "a large set of anharmonic \n constants of c2f4 was derived from spectroscopic \n data , and a complete set was calculated . \n", "most of the calculated and experimental values agree very well , and \n nearly all deviations can be attributed to fermi resonances . the values \n were used to interpret peculiarities of the potential and reactions \n of this molecule .", "the facile thermal cc dissociation of c2f4 is due to curve crossing of the ground state \n ( 1ag ) with \n the two - electron excited zwitterionic 2ag state .", "the crossing \n barely alters the properties of the potential curve ( cut purely as \n a function of cc stretch ) near the equilibrium geometry ; the force \n constant for cc stretching ( 1 in c2f4 , 2 in c2h4 ) is nearly \n identical for the two molecules .", "only a slightly larger magnitude \n of the anharmonicity x11 compared to the \n corresponding x22 of ethylene provides \n a hint of a weakly avoided crossing in planar geometry of c2f4", ". however , the large magnitude of x18 leads us to conclude that the dissociation path bends \n early toward \n q8 , i.e. to trans pyramidalization of the two carbon atoms \n ( figure 2b ) .", "this is rationalized by a valence - bond \n model , which describes the stretched cc bond as a double donor  acceptor \n bond ( scheme 2c ) .", "it is also consistent with \n the tendency of fluorine to induce sp hybridization at \n carbon , although this preference alone would also allow cis pyramidalization , \n q7 , which is not involved early in dissociation .", "on further \n cc stretching , one of the two donor  acceptor bonds breaks , \n and the pyramidalization becomes fully one - sided already in the transition \n state ( figure 2b ) . in this way , free internal \n rotation of the cf2 groups becomes possible , providing \n an additional degree of freedom that rationalizes the high pre - exponential \n factor in the rate constant of dissociation .", "this structure with perpendicular \n arrangement of the two cf2 groups appears very plausible \n for the back reaction .", "thus , one cf2 group approaches with \n its filled n orbital the empty p orbital of the other \n cf2 group ( figure 2b ) .", "the easy \n trans pyramidalization on minor cc stretching is probably also the \n reason that the corresponding 8 force constant is \n lowered , although all other force constants are similar to those in \n ethylene .", "the preference of fluorine for sp hybridization \n at \n carbon has been invoked to explain not only the easy radical addition \n and other reactions , but also the low - lying , twisted - pyramidalized \n triplet , which is nearly isoenergetic with the singlet that has a \n saddle point there .", "this is indicated by x44 , x47 , and x48 , which have no unusual values .", "very recently , \n high - level theoretical studies investigated \n electronic ground and excited states of c2f4 and their dependence on some coordinates , mainly \n to discover the fate of this molecule after electronic excitation \n and make comparisons with a corresponding time - resolved experiment \n ( ref ( 16 ) ) . for the \n ground state , the stabilizing effect of pyramidalization in the twisted \n molecule was confirmed .", "interestingly , the excited molecule enters \n the s1/s0 conical intersection from the 2ag state , where the molecule has a large one - sided pyramidalization , \n a 90 cc twist and a cc bond length ( 1.51  ) similar to \n that of a single bond . in the present work ,", "the transition state for \n s0 dissociation was said to result from avoided crossing \n of the 2ag and 1ag ( i.e. , s0 ) states , \n also with large one - sided pyramidalization but at longer cc distances \n and perhaps without twist", ". it would be interesting to check whether \n these geometrical differences are large enough to generate an excited - state \n barrier , because evidence was found in ref ( 16 ) that cc dissociation does not begin in the excited \n state ."], "abstract_text": ["<S> compared to ethylene and its nonfluorinated \n derivatives , c2f4 is peculiar in many reactions . \n </S>", "<S> it very easily \n adds to radicals and prefers formation of four - membered rings over \n diels  alder reactions . </S>", "<S> this has been rationalized by the preference \n of fluorine for carbon sp3 hybridization , which is possible \n on opening of the double bond . </S>", "<S> another property , the thermal dissociation \n of the c = c bond , has been explained by the stabilization of \n the product ( cf2 ) by back - bonding . </S>", "<S> here , it is attempted \n to correlate such properties with vibrational constants , in particular \n for c = c stretching and twisting and for carbon pyramidalization . \n </S>", "<S> the only force constant found to be lowered compared to ethylene is \n that for trans pyramidalization ( 8 ) , and cc bond \n softening on 8 distortion is indicated by the conspicuously \n large magnitude of anharmonic constant , x18 . </S>", "<S> both observations can be rationalized by a valence - bond model that \n predicts a trans bent structure on weakening the cc bond . </S>", "<S> conclusions \n are drawn about the dissociation path and peculiarities of the potential . \n </S>", "<S> other anharmonicities , both experimental and calculated and some in 12c13cf4 and 13c2f4 , are also discussed . in particular </S>", "<S> some strong fermi \n resonances are identified and their effects accounted for . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusion"], "sections": [["recently \n we reported on an analysis of the infrared spectrum of \n c2f4 . by observation \n of many combination and difference bands , including many from two c - containing isotopologues , and by comparison with mp2 and \n density - functional calculations , all 12 fundamentals could be assigned , \n although only five are ir active .", "the geometry \n was deduced from ro - vibrational analysis of strong ir active bands \n and by comparison with theoretical calculations .", "however , it is also \n instructive to correlate anharmonic constants with some peculiarities \n of the reactions of c2f4 .", "accordingly , in this \n work more anharmonic constants ( altogether 54 ) are derived from the \n spectra , and a complete set ( 78 ) is calculated .", "the earlier vibrational \n assignments of c2f4 were reported at a time \n when the unusual properties of this molecule were barely known .", "explained this property by positing that the electron attraction \n of fluorine leads to a preference of sp- over sp - type carbon : radical addition converts both \n c atoms from sp to sp hybridization , which \n stabilizes the primary product , lowers the transition state , and thus \n accelerates the reaction .", "alder reactions , for example , \n which was again explained by stabilization of radical intermediates .", "( borden also presented an alternative but related description based \n on delocalization of unpaired radicalic electrons to accepting  * \n orbitals of cf groups . )", "the preference \n for carbon sp hybridization can also be expected to lower \n the force constants for the pyramidalization ( wagging ) vibrations \n ( 7 , 8 ) and/or increase their anharmonicity . \n", "furthermore , torsion ( 4 ) should be facilitated by \n rehybridization and in fact quantum chemistry confirmed this for a \n 90 twist .", "another spectacular property of c2f4 is how \n readily it can dissociate thermally to two cf2 molecules .", "a c = c bond energy of only 2.95 ev is derived , less than that of a typical c  c single \n bond ( 4 ev ) .", "the low dissociation energy is caused by stabilization \n of the resulting two difluorocarbenes by back - bonding from nonbonding \n fluorine electrons to the empty carbon p - orbital .", "this also explains why cf2 has a singlet \n ground state which is more stable than the triplet ( by 2.458 ev , taken \n from the band origin of the phosphorescence spectrum ) . whereas in the previous work", ", only cc stretching is considered \n as the dissociation coordinate , it was argued in ref ( 16 ) that pyramidalization \n must also be involved .", "this idea is supported in the present work \n by the large value found for the anharmonic constant x18 coupling cc stretch with trans pyramidalization .", "the \n magnitude of x18 as compared to the much \n smaller x17 ( coupling cc stretch with \n cis pyramidalization ) also supports a valence - bond analysis of weak \n double bonds , which was reviewed in ref ( 19 ) ."], ["the samples in natural isotopic abundance were either taken from \n commercial c2f4 ( hoechst ) , that contained a \n trace of the cyclic dimer , or prepared from c2f4br2 + zn in a high - boiling \n alcohol ( diethylene glycol ) ; the comparison allowed for recognizing \n absorptions of impurities .", "a sample enriched in c to \n 50% was prepared from chclf2 by co2 laser isotope \n separation with enriched cf2 as the primary product .", "ir spectra of c2f4 in natural isotopic \n abundance \n and isotopically enriched were recorded between 370 and 4000 cm with 0.2 cm resolution by a perkinelmer \n ftir spectrometer in garching . at the \n synchrotron lab ,", "the isotopically enriched sample was measured at \n moderate 0.1 cm resolution in the far ir region \n from 100 to 600 cm ( capturing the band at 210 \n cm ) , and at high resolution for the ir active \n cf stretch bands , see ref ( 1 ) .", "the present work focuses on anharmonic constants of c2f4 . only where the constants were \n expected to be different for ccf4 and c2f4 ( e.g. , in the case of \n fermi resonance )", "theoretical values of the anharmonic constants xij were obtained from anharmonic \n vibrational \n frequency calculations performed using gaussian 03 revision c02 at the mp2 level using \n dunning - type correlation - consistent ( cc - pvtz ) orbitals .", "the molecular force field at this level of theory \n was shown in the previous study to reproduce the fundamental wavenumbers \n and the isotopic shifts of c2f4 very satisfactorily \n ( for details , see ref ( 1 ) ) , and hence was an appropriate choice for computing xij values .", "the calculations were performed for each of \n the c2f4 , ccf4 , and c2f4 isotopologues ."], ["the spectra were measured previously and the \n fundamentals extracted \n from them , using a limited number of anharmonic constants ( from combination \n and hot bands ) where necessary . for convenience \n table 1", "lists the fundamentals again . in this \n work we derived a large set of experimental anharmonic constants from \n combination bands and  in particular for the lower - wavenumber \n vibrations  from hot bands , and report a complete set of theoretically \n computed values .", "symmetry types refer to a d2h molecule with z axis along the cc bond and the y axis perpendicular \n to the plane , as used in ref ( 1 ) for easier comparison with the c2v symmetric ccf4 ; the more conventional designation with x axis along the cc bond and z axis perpendicular \n to the plane is given in parentheses .", "revised value ( instead of 210 cm ) , based on a more definite \n assignment of a hot - band structure and x10,10 , see supporting information .", "band origins from high - resolution \n spectra ; low - resolution values are 1338.4 \n and 1187.0 .", "upright : \n experimental values . \n in parentheses : values calculated by mp2/pvtz . where two values or \n a range are given for the experimental data", ", it may indicate ( a ) a \n dependence of the effective xij on the involved levels due to perturbations ( fermi resonances , \n section 4.4 ) or ( b ) an uncertainty due to error \n limits ( e.g. , where broad bands are involved ) or an uncertain assignment . \n", "a preferred value is indicated by bold face . for further details on \n the derivation of each xij and some isotopic data ,", "the anharmonic constants xij were calculated from observed \n combination and difference bands \n by assuming that anharmonic shifts are additive : for binary combination \n bands ( i  j ) and for overtones \n we used1and2with obvious extensions for more complicated \n combinations .", "( the xij values are mostly negative , as usual . ) the left - hand side of these \n equations should be read as  wavenumber of ( name )  . for hot bands ( satellite transitions near i that originate from a thermally populated \n lower level j ) we used3again \n with obvious extensions .", "one can often \n observe sequences of equidistant q branches , corresponding to hot \n bands starting from higher levels mj , or combination of such sequences ( from mj + nk ) .", "equation 3 implies that the distance of a ( first ) hot band from the fundamental \n directly indicates the corresponding anharmonic constant .", "combination \n bands allow for a direct assignment , whereas hot bands must be identified \n by their intensity ( controlled by the boltzmann factor ) . in a number \n of cases", "the supporting information ( si ) compiles the sources for each xij in table s2 , whereas table s1 lists the observed \n bands ( with hot bands ) and their isotopic shifts .", "two examples of \n hot bands , whose shifts are dominated by the large x18 anharmonicity , are shown in figure 1 .", "absorbance spectra demonstrating the large x18 derived shifts for hot - bands originating in 8 .", "the spectra were recorded \n in the garching laboratory , with pressure and path length of 2 bar \n and 10 cm ( for the lower trace of each panel ) , and 140 mbar and 4 \n m ( upper trace ) , respectively . for levels involving more than two quanta of vibration , anharmonic \n shifts", "this helped to decide between different \n assignments of combination bands in a few cases where the harmonic \n sums were similar to each other .", "however , such additivity need not \n apply when the levels involved are subject to fermi resonance , producing \n shifts that depend on the separation of nearby interacting levels . \n", "we use eqs 13 to \n obtain the experimental values , and hence , table 2 reports phenomenological xij , some of which show a spread in the values suggestive of fermi \n resonances ( in particular x18 , x26 , and x77 ) .", "fermi \n resonances can also be recognized if the apparent xij or the hot - band structure depends \n on the isotopologue . for c2f4 ,", "fermi resonances \n were identified ( see section 4.4 and si ) between:5 and 2+6 ( discussed already in ref ( 1)),1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue),2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77).2 and 23 , identified from the \n mp2 calculations .", "5 and 2+6 ( discussed already in ref ( 1 ) ) , 1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue ) , 2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77 ) . 2 and 23 , identified from the \n mp2 calculations .", "the mp2/cc - pvtz calculations \n reproduced the observed wavenumbers \n and isotopic shifts very well . supporting \n the overall vibrational analysis", "exceptions are 15 ( x22 , x28 , x29 , x2,10 , x2,11 , x36 , x3,10 , x45 , x59 , x5,11 , x66 , x68 , x77 , x88 , x10,11 ) out of 54 cases where an observed value is available \n for comparison . some of these will be discussed below ."], ["as already \n described in the introduction , the peculiar \n reactions of c2f4 have to do \n with cc stretching and carbon pyramidalization . the discussion will \n focus on these coordinates ( sections 4.14.3 ) . for \n a planar geometry , \n", "cc dissociation correlates c2h4 and c2f4 with a pair of carbenes in their lowest triplet states ( figure 2a ) . because \n the energy of forming these products is practically the same for the \n two molecules", ", one could also expect that the potential energy curves \n coincide and the cc stretch force constants are equal .", "in fact , already \n early force - field calculations showed no substantial difference ( 889 \n n / m for c2f4 and \n 940", "furthermore , \n the short cc distance ( 132 pm , which is 1 pm shorter than in ethylene ) and the high cc stretch wavenumber ( 1873.8 cm , compared to 1625.4 cm   for ethylene ) do not point to a weakened bond . \n as early as 1965 , the low dissociation energy of c2f4 was attributed to switching over to a channel producing two \n singlet carbenes and to the stabilization ( by 2.458 ev , see introduction ) of singlet cf2 by back bonding , so that this channel is energetically favored by 4.9 ev .", "this path hence leads over an avoided \n crossing of potential surfaces ( figure 2a ) . \n", "if the avoidance is strong , one could expect an influence at least \n on the anharmonicity x11 ( 1 is dominated by cc stretching ) , even if not on the force \n constant at the bottom of the potential energy well .", "table 2 shows that the magnitude of x11 ( calculated 7.8 cm ) is not unusually \n large for such a high - frequency vibration , though noticeably larger \n than for ethylene ( x22 = 2.3 cm  ) . that the increase \n in magnitude is only moderate", "can be taken as a sign that the avoidance \n along this coordinate is not very strong .", "hence , the back reaction \n ( recombination ) will have a high barrier in planar geometry . whereas \n the measured recombination barrier is not , in fact , zero , it is only \n minor ( 9 kj / mol = 0.09 ev  ) .", "so , \n a lower - energy path must lead around the barrier , out of the drawing \n plane of figure 2a , that is , with distortion \n different from cc stretch .", "potentials for ground - state cc dissociation \n of c2f4 , leading over an avoided crossing to \n two singlet carbenes . \n in ( a ) it is assumed that the one - sided pyramidalization ( q7 + q8 ) begins only after overcoming the barrier , whereas \n ( b ) shows the preferred path , where this distortion is already fully \n developed on the barrier and is preceded by a trans pyramidalization \n ( q8 ) , while the cc distance is steadily increasing .", "it was argued in ref ( 16 ) that recombination of \n two cf2 molecules should \n be easiest if one of them points with its filled n orbital \n to the empty p orbital of the other .", "hence , the dissociation \n path will change from pure cc stretch to include also pyramidalization \n ( figure 2b ) , and the potential should be lowered \n in energy in the direction of q7 ( the coordinate of 7 ) and/or q8 . in turn", "in fact x18 has an unusually large magnitude , although x17 is in the normal range ( table 2 ) .", "( the magnitude of x18 is not \n caused by secondary effects such as fermi resonance , as we shall see \n in section 4.4.3 . )", "we can conclude that on \n stretching the cc bond the molecule first bends toward trans pyramidalization \n ( q8 ) ( figure 2b ) .", "the one - sided \n nonplanar bending ( figure 2 ) is a superposition \n of q7 and q8 .", "the reason why q8 is \n active earlier than q7 can be understood by a valence - bond \n model for weak double bonds . in this model", "several symmetric arrangements \n of carbenes ( a  c \n of scheme 2 ) were considered : whereas with \n two triplet carbenes all arrangements ( a  d ) lead to bonding \n interaction , with two singlet carbenes bonding occurs only in the \n trans - bent case ( c ) ; this corresponds to a double donor ", "one can add that the cis - bent case with singlet carbenes would \n lead only to antibonding ( scheme 2d ) .", "malrieu \n and trinquier derived a criterion for trans - bending ( deriving even \n the angle ) by comparing the standard double - bond energy with the singlet ", "their findings were confirmed by many examples , in particular with \n heavier - element homologues .", "we can apply \n this rule not only to the equilibrium geometry of c2f4 but also to its dissociation path , if we consider in the \n comparison the double - bond energy that is reduced on extension of \n this bond .", "the rule thus predicts that on cc extension c2f4 is distorted from planar geometry to a trans - bent structure , \n and this rationalizes the large magnitude of x18 . in the triplet case ,", "the interaction \n can be bonding in all arrangements . in the singlet case , ( a )", "is antibonding , \n ( b ) nonbonding , ( c ) bonding , ( d ) antibonding .", "more recently , borisov et al . found evidence \n for trans pyramidalization \n on extension of the cc bond also from molecular orbital calculation \n ( at high level : mller  plesset perturbation theory of \n second , third and fourth order ) : the \n authors found that the onset is relatively sudden on cc extension \n to 142 pm and that the distortion lowers the energy appreciably .", "( the \n large x18 anharmonicity is an indication \n that this pyramidalization already begins not far from the s0 minimum .", "in fact , a cc stretch by 10 pm  from 132 to 142 pm ", "is \n just about the vibrational amplitude in the 1 = \n 1 level . )", "although borisov et al . did not find an energy minimum with \n such a distortion , they suggested that it plays a crucial role in \n the low - energy real intermediate found by buravtsev , kolbanovskii \n et al . in thermal reactions of c2f4 and recombination \n of cf2 ( see ref ( 31 ) and literature quoted therein and section 4.3 ) .", "one could suppose that in order to lower the activation \n energy \n for dissociation the only necessary deviation from simple cc stretching \n is distortion toward the 8 coordinate ( q8 ) .", "however , there is experimental \n evidence that the asymmetric pyramidalization ( corresponding to superposition \n of q7 and q8 as in figure 2b ) is already crucial in the transition state : the pre - exponential \n factor found in the rate of thermal c2f4 dissociation \n ( 2.8  10 s  ) is clearly larger than would be typical for \n a simple bond splitting ( 10 s ) .", "this \n corresponds to a raised entropy of activation , such as that which \n happens if an additional degree of freedom is activated in the transition \n state .", "the most plausible candidate is free internal rotation , as \n it can be expected in the asymmetric structure with a single dative \n bond but not in symmetric geometry with double donor  acceptor \n bond .", "this picture is also consistent with a stepwise cleavage of \n the double donator  acceptor bond on c2f4 dissociation and its stepwise formation on cf2 recombination .", "it is worth noting that the molecule with one - sided pyramidalization \n has an electron distribution ( figure 2b ) that \n is zwitterionic and correlates in planar geometry with the 2ag state with two  electrons excited to  * , as \n already pointed out in ref ( 16 ) .", "a fully analogous dissociation path can also be \n expected for the \n other systems considered by malrieu and trinquier .", "in fact , according \n to ref ( 32 ) ground - state \n ( ir induced ) dissociation of diazomethane ( h2cnn ) follows \n a path with a pyramidalized methylene group to produce n2 + singlet ch2 ; the out - of - plane deformation was experimentally \n confirmed . in the backward reaction ,", "n2 points with its \n n electron pair to the empty p orbital of the singlet \n methylene ( see refs ( 32 and 33 ) for a calculated potential in two dimensions , which would look similar \n for c2f4 ) .", "hence , \n the out - of - plane deformation on dissociation of c2f4 is not an isolated phenomenon . applying \n their energetic criterion to the equilibrium geometry of c2f4 , trinquier and malrieu found that this molecule is \n not far from the limit , where it would become nonplanar , and suggested \n that the 8 force constant may be significantly lower \n than that of ethylene .", "this is in fact \n confirmed in the following way : analytical expressions for a valence \n force field model for planar x2y4 were given \n by herzberg . besides the masses mx and my , bond lengths l1 ( for xx ) and l2 ( xy ) and the angle  ( half of yxy ) , both 7 and 8 only depend on the force constant for pyramidalization \n ( k/l2 ) . with equilibrium distances and angles from ref ( 1 ) and i = ( 2ci )", "one derives this force constant from \n 7 and 8 ( with identical results \n for c2f4 and c2f4):4both values should be identical \n in this valence - force field model .", "in fact for ethylene , the value \n is 22.9 n / m , whether derived from 7 or 8 .", "the pyramidalization force constant \n derived from 7 of c2f4 is \n slightly higher than in ethylene ( by 17% ) , but the same quantity derived \n from 8 is only 49% of the ethylene value .", "( of course , \n these values are not quantitatively meaningful , as the discrepancy \n highlights that a pure valence - force field model is not sufficient \n for wagging of c2f4 . )", "so in fact the restoring \n force for the 8 vibration is significantly smaller \n than in ethylene and than that expected on the basis of the same pyramidalization \n force constant as for 7 .", "this is consistent with \n the prediction of the valence - bond model favoring the trans - bent geometry .", "the slightly raised force constant for 7 could \n be due to repulsive nonbonding interaction in cis pyramidalization . \n", "such a geometry would be involved in the hypothetical transition state \n of the diels ", "alder addition of c2f4 to \n butadiene , a reaction that has not been observed .", "it was argued that \n an increased energy associated with this distortion might contribute \n to inhibiting the reaction .", "however , an \n increase in the force constant of only 17% is hardly sufficient to \n support this explanation , and the other effects discussed in ref ( 7 ) , in particular the competing \n fast radical reactions , are probably more important . applying \n again the force - field formulas of herzberg ,", "the force constant for torsion , k/l2 , is found to be 30.3 \n n / m for both ethylene and tetrafluoroethylene .", "that is , near the potential \n minimum the c = c bond in c2f4 behaves \n just like a normal double bond .", "consequently , the torsional barrier \n at a 90 twist ( which is a measure of the  bond strength ) \n would be identical for both molecules ( 2.8 ev ) if the two c  cf2 groups remained planar ; this was confirmed by a high - level \n calculation .", "however , pyramidalization \n lowers this barrier by 0.57 ev ( at mp2 level , consistent with ref ( 35 ) ) .", "if this stabilization is activated early along q4 , \n it could give rise to substantially negative values for x44 and especially for x47 and x48 .", "however , x44 and x47 are actually slightly positive \n ( 0.8 and 0.3 cm ) and x48 has no unusual magnitude ( 0.7 to 1.05 cm ) .", "evidently , the stabilizing effect of pyramidalization becomes \n effective only at high torsional angles .", "the corresponding values \n for ethylene are 2.4 , 8.9 , and 7.2 cm , respectively .", "the nondiagonal anharmonicities xi4 and x4j are \n generally smaller in magnitude than in ethylene . but this may simply \n have to do with the relative vibrational amplitudes , which are much \n larger for torsion of ch2 than of cf2 due to \n the relative masses . in the twisted - pyramidalized structure \n of c2f4 ,", "the triplet ( t1 ) is only \n 0.013 ev lower than the singlet \n ( s0 ) .", "it \n can therefore act as a real intermediate and undergo radical - type \n reactions , further assisted by its low energy that is calculated at \n 2.1 ev .", "this has been a widespread interpretation \n of some peculiarities of c2f4 reactions ( see , \n for example ref ( 7 ) ) .", "however , buravtsev , kolbanovskii et al . found in shock - tube experiments \n ( with c2f4 diluted by ar and adiabatically compressed ) \n an intermediate at much lower energy ( 0.8 to 1 ev ) with uv and visible \n absorption ( 235 and 500 nm ) ( see refs ( 31,36 , and 37 ) and the \n literature quoted there ) .", "they supposed it to be a singlet diradicaloid \n of c2f4 with a geometry corresponding not to \n an energy minimum but to a point on a slope of the potential ( see , \n for example ref ( 31 ) ) .", "however , as these points are not stationary there are too many \n ( nearly ) isoenergetic locations with shifted electronic transitions , \n so that the electronic absorption would be smeared out .", "it was therefore \n suggested that the observed intermediate \n is another low - energy isomer , tetrafluoro - ethylidene cf3cf ( electronic absorption in an ar matrix : ) .", "however , its energy ( 1.9 ev  ) seems to be nearly as high as that of the twisted - pyramidalized \n triplet .", "( in one study the twisted - pyramidalized c2f4 triplet is calculated at 0.90 ev , but computational details are not given , and previous theoretical \n results are not discussed . )", "some more anharmonicities of table 1 have unusually large magnitudes or show a range \n of values and/or deviate from prediction . in many of them", "fermi resonances \n play a role , as will be explained here . in section4.4.3 , however , it is shown that x18 is hardly influenced by such effects and its large magnitude mainly \n results from the overall shape of the potential energy surface .", "fermi resonances mix two nearby levels of the \n same symmetry , which differ by three units in the quantum numbers . \n", "the fermi - induced \n repulsion of the levels depends in a nonlinear way on both the magnitude \n of the fermi interaction parameter and on the original ( i.e. , unperturbed ) \n energetic distance of the levels .", "this separation will , in general , \n change ( a ) on isotopic substitution and ( b ) on adding a quantum of \n another vibration ( i.e. , in analogous combination bands ; similarly \n also in higher overtones , in which case a quantum number dependence \n must also be taken into account ) .", "another method of characterizing fermi resonance interactions relies \n on intensity borrowing caused by the mixing of levels . according \n to the mp2 calculations , the great majority of xij change by 1 cm on isotopic substitution", "the few exceptions are \n listed in table 3 and are compared to experimental \n values . identified ( fr13 ) and", "the interpretation in the line  influenced by  \n is based on the assumption that an xij is affected by such a resonance if at least one \n of the levels i and j is involved in the fermi interaction .", "the table shows that \n the calculated isotopic shift is sometimes \n much larger than in the experiment , for instance x26 , x27 .", "also x56 for c2f4 is probably \n too large , as the two observed bands 5+6+11 and 5+6+12 are close to their harmonic positions \n ( table s1 in si ) .", "it seems that at the \n present level of theory , the calculation does not reliably reproduce \n the fermi resonance induced shifts , at least the stronger ones .", "this \n is not surprising given the highly sensitive and nonlinear dependence \n of fermi shift on the  unperturbed  level separations . \n", "the same deficiency will also affect those xij connected with at least one level ( or a \n combination thereof ) involved in a resonance listed above ( examples \n in figure 3a ) .", "in fact , 10 of the 15 deviations \n listed at the end of section 3 can be attributed \n in this way to the resonances fr1 and fr2 with possible contributions \n of fr3 .", "the exceptions are x36 , x3,10 , x66 , x68 , and x88 . in \n all 42 other cases", "it is worthwhile to consider \n the two strongest fermi resonances , \n fr1 ( 2/27 ) and fr2 ( 5/2+6 ) , in c2f4 in some detail .", "the effect of these resonances \n is summarized in figure 3 . some levels involved \n in fermi resonances and observed transitions . \n", "solid arrows : ir , broken arrows : raman ; double arrows ( with interaction matrix element ) indicate repulsion \n between level pairs .", "the anharmonic shifts involving x77 are only nominal , because the fermi perturbation is \n strong :  6x77  = 18.1 is \n less than 3 times ", "(= 8.2 ) and  4x77  is only \n 9.9 . ( but  2x77  +  4x77  =  6x77  , as is obvious from the level scheme , and the raman \n anharmonic shifts practically coincide with those from the ir spectra . ) \n ( b ) three - level fermi resonance 5/2+6/27+6 and \n their combinations with 9 for c2f4 and c2f4 . \n", "the indicated level energies are from the spectra except those in \n parentheses , which are calculated .", "the broken horizontal lines are \n the estimated unperturbed positions ( see the si ) , and the signed numbers are the calculated shifts .", "the 16.4 cm magnitude of the interaction term ( with its expected dependence \n on quantum numbers ) has been derived \n from band shifts and intensities for a series of levels built on 2/27 for all three isotopologues .", "bands for \n 27+x ( x = 9 , 11 ) borrow their entire intensity from 2+x . the detailed analysis \n is provided in si .", "it \n is also evident that x77 , x27 and x22 are affected by \n the fermi resonance ( and probably other x2i , if the level separations differ ) and that x77 is level dependent because of the varying \n separation .", "the fermi term is almost entirely responsible for the \n + 8.2 cm shift of 27 for c2f4 and those of the heavier isotopologues \n ( table 3 under fr1 ) . in the latter", "the fermi - induced \n shift is increased , because the levels 2 and 27 ( before perturbation ) are closer together than in c2f4 and are practically degenerate in c2f4 .", "the success of these calculations \n shows that the additional mixing of 2 with 23 ( fr3 ) is apparently a weak perturbation at most .", "the \n fermi resonance fr2 between 5 and 2+6 was qualitatively discussed in ref ( 1 ) .", "a more complete understanding \n of the resonance shifts and pattern of intensity borrowing requires \n the three - level scheme shown in figure 3b ; \n this figure also shows combinations with 9 , as they \n are all ir active and were directly observed .", "analysis based on this \n model has enabled the magnitude of the fermi term to be determined \n as 11 cm ; the fr1 term of 16.4 cm was used without change .", "significantly , the model predicts varying \n fr shifts that even differ in sign ( originating from the different \n repulsions in figure 3b ) and can thus account \n for the unusual isotopic shifts observed between c2f4 and c2f4 . \n", "it explains a substantial deviation of the observed isotopic shift \n 5 = 51.9 from the value of 5 = 43.1 \n expected from the teller  redlich product rule , which is valid \n for harmonic oscillators .", "additionally \n the relative intensities of combination bands and their isotopic dependence \n are well predicted .", "it is also remarkable that according to the model \n the largest part of the anharmonicity x26 is due to fermi resonance .", "as described in ref ( 1 ) , the lower symmetry in ccf4 leads to strong mixing of 5 with 9 ( which are nearly degenerate in c2f4 ) .", "the two levels repel each other \n ( even in the harmonic approximation ) and give rise to two infrared - active \n transitions .", "it is sometimes difficult to disentangle them and their \n combination bands from the spectra .", "if the large magnitude of some \n anharmonicities is caused by fermi resonance , a crucial question is \n whether this might also be the case for x18 .", "if so , the conspicuous value of x18 could be largely a matter of the character of the resonance and \n less a consequence of global features of the potential for dissociation , \n which we considered in section 4.1 .", "in fact , \n weak perturbations of the 1 level and its combinations \n by the 1/5+6 fermi resonance are indicated by the slight x18 dependence on the level and on isotopic substitution and \n the change of the hot - band structure of 1 combinations \n in ccf4 , as explained in the si .", "a quantitative estimate of the strength \n of the 1/5+6 interaction can be deduced from the intensities of 5+6 combinations relative to those of the corresponding \n 1 combinations , assuming that the former borrow \n their full strength from the latter .", "the 5+6+12 band ( 2444.3 cm ) \n has 12% the strength of the 1+12 band ( 2428.7 cm ) , see figure 1a .", "similarly , the 5+6+11 shoulder ( 3072 cm ) is < 1% as intense as 1+11 ( 3056.2 cm ) and clearly visible only with longer \n path length than shown in figure 1b .", "from both \n observations , one can estimate a fermi term ( w ) of \n no more than 2.5 cm , causing a shift ( and \n consequent change in x18 ) of < 0.5 cm .", "this is actually an upper bound , because such weak \n bands can also be expected without intensity borrowing .", "hence , fermi \n resonance affects the magnitude of x18 only by a little ( by < 3% ) , and we can maintain the conclusions \n of section 4.1 on the shape of the potential ."], ["a large set of anharmonic \n constants of c2f4 was derived from spectroscopic \n data , and a complete set was calculated . \n", "most of the calculated and experimental values agree very well , and \n nearly all deviations can be attributed to fermi resonances . the values \n were used to interpret peculiarities of the potential and reactions \n of this molecule .", "the facile thermal cc dissociation of c2f4 is due to curve crossing of the ground state \n ( 1ag ) with \n the two - electron excited zwitterionic 2ag state .", "the crossing \n barely alters the properties of the potential curve ( cut purely as \n a function of cc stretch ) near the equilibrium geometry ; the force \n constant for cc stretching ( 1 in c2f4 , 2 in c2h4 ) is nearly \n identical for the two molecules .", "only a slightly larger magnitude \n of the anharmonicity x11 compared to the \n corresponding x22 of ethylene provides \n a hint of a weakly avoided crossing in planar geometry of c2f4", ". however , the large magnitude of x18 leads us to conclude that the dissociation path bends \n early toward \n q8 , i.e. to trans pyramidalization of the two carbon atoms \n ( figure 2b ) .", "this is rationalized by a valence - bond \n model , which describes the stretched cc bond as a double donor  acceptor \n bond ( scheme 2c ) .", "it is also consistent with \n the tendency of fluorine to induce sp hybridization at \n carbon , although this preference alone would also allow cis pyramidalization , \n q7 , which is not involved early in dissociation .", "on further \n cc stretching , one of the two donor  acceptor bonds breaks , \n and the pyramidalization becomes fully one - sided already in the transition \n state ( figure 2b ) . in this way , free internal \n rotation of the cf2 groups becomes possible , providing \n an additional degree of freedom that rationalizes the high pre - exponential \n factor in the rate constant of dissociation .", "this structure with perpendicular \n arrangement of the two cf2 groups appears very plausible \n for the back reaction .", "thus , one cf2 group approaches with \n its filled n orbital the empty p orbital of the other \n cf2 group ( figure 2b ) .", "the easy \n trans pyramidalization on minor cc stretching is probably also the \n reason that the corresponding 8 force constant is \n lowered , although all other force constants are similar to those in \n ethylene .", "the preference of fluorine for sp hybridization \n at \n carbon has been invoked to explain not only the easy radical addition \n and other reactions , but also the low - lying , twisted - pyramidalized \n triplet , which is nearly isoenergetic with the singlet that has a \n saddle point there .", "this is indicated by x44 , x47 , and x48 , which have no unusual values .", "very recently , \n high - level theoretical studies investigated \n electronic ground and excited states of c2f4 and their dependence on some coordinates , mainly \n to discover the fate of this molecule after electronic excitation \n and make comparisons with a corresponding time - resolved experiment \n ( ref ( 16 ) ) . for the \n ground state , the stabilizing effect of pyramidalization in the twisted \n molecule was confirmed .", "interestingly , the excited molecule enters \n the s1/s0 conical intersection from the 2ag state , where the molecule has a large one - sided pyramidalization , \n a 90 cc twist and a cc bond length ( 1.51  ) similar to \n that of a single bond . in the present work ,", "the transition state for \n s0 dissociation was said to result from avoided crossing \n of the 2ag and 1ag ( i.e. , s0 ) states , \n also with large one - sided pyramidalization but at longer cc distances \n and perhaps without twist", ". it would be interesting to check whether \n these geometrical differences are large enough to generate an excited - state \n barrier , because evidence was found in ref ( 16 ) that cc dissociation does not begin in the excited \n state ."]]}
{"article_id": "PMC3671708", "article_text": ["cytomegalovirus ( cmv ) is one of the most common congenital viral infections in many regions . in the united states it occurs in 0.51% of live births , or approximately 40,000 infants annually .", "the manifestations of cmv infection cover a broad spectrum ranging from asymptomatic to severe systemic disease resulting in significant morbidity and mortality .", "90% of infants with congenital cmv are asymptomatic at birth . despite being asymptomatic at birth , up to 7% of these children", "will develop sensorineural hearing loss ( snhl ) that can be unilateral or bilateral , fluctuating or progressive , and range from mild to profound [ 2 , 3 ] .", "approximately 10% of infants with congenital cmv are symptomatic at birth , and 40% of these patients will develop snhl [ 1 , 2 ] .", "hearing loss is the most common manifestation of congenital cmv infection making cmv a leading cause of nonhereditary congenital hearing loss .", "given the relatively large number of children potentially affected by cmv - related hearing loss and the wide range of manifestations of congenital cmv infection , it is difficult to predict how a child with symptomatic cmv will perform with a cochlear implant ( ci ) .", "previous studies suggest that children with symptomatic cmv derive auditory benefit from cis , albeit at a slower rate than other children . the degree to which these children benefit remains somewhat controversial as there is variation in the development of language skills after ci [ 59 ] .", "the objective of our study was to examine the relationships between intracranial radiographic abnormalities and preimplantation developmental assessment ( including head circumference ) with postimplant audiometric and language outcomes in children with symptomatic congenital cmv who have undergone cochlear implantation .", "this study was a retrospective review of children with a diagnosis of symptomatic congenital cmv who underwent cochlear implantation between 2004 and 2010 at a tertiary pediatric cochlear implant center .", "congenital cmv was diagnosed for most children via urine culture of the virus during the first two weeks of life .", "diagnosis was also made based on the identification of hearing loss coupled with brain image findings .", "medical charts of these patients were reviewed for pre- and postimplantation evaluations and central nervous system imaging findings .", "data regarding preimplantation developmental assessments included the diagnoses of developmental delay and/or other disabilities , nonverbal intelligence quotients or developmental quotients ( iq / dq ) , and head circumference ( hc ) at the preoperative visit .", "results from computed tomography ( ct ) and/or magnetic resonance imaging ( mri ) scans of the brain were reviewed , and the findings were classified by type and location of anomaly .", "postimplant audiometric behavioral testing , which was performed using validated and developmentally appropriate testing techniques , was collected .", "this data included frequency - specific thresholds and speech awareness or speech reception thresholds ( sat / srt ) .", "pure tone averages ( pta ) were calculated by an average of 4 frequencies ( 0.5 , 1.0 , 2.0 , and 4.0  khz ) .  ", "children 's developmental disability diagnoses were classified according to the areas of cognition , motor , vision , and language .", "cognitive disability was diagnosed using nonverbal cognitive measures such as the leiter international performance scale   and supported by evaluating adaptive functioning with the vineland adaptive behavior scale .", "these evaluations were completed by psychologists with knowledge of testing deaf / hard of hearing children . if the nonverbal iq was not available , a nonverbal developmental quotient was used .", "this nonverbal or adaptive quotient was obtained from a developmental pediatrician with expertise in hearing loss as a part of the precochlear implant assessment . for this study ,", "the diagnosis of cerebral palsy ( cp ) was made based on neurologic examination , patterns of persistent primitive reflexes , abnormalities in tone and reflexes , and presence of abnormalities on mri of the brain .", "the diagnosis of pervasive developmental disorder not otherwise specified was made by interdisciplinary evaluations with speech pathology and psychology using standardized measures to evaluate autism spectrum disorders .", "standardized language assessment scores , using the preschool language scale ( 4th edition )   were available on eight subjects who returned for speech therapy after implantation .", "this study was approved by the institutional review board at cincinnati children 's hospital medical center .  ", "data distributions were reported using medians with ranges ( for continuous data ) and frequencies with percentages ( for categorical data ) .", "characteristic differences between children with hc < 5th percentile and > 5th percentile were tested using wilcoxon rank sums test for continuous data and chi - square or fisher 's exact test for categorical data .", "biserial correlations were conducted to understand the association between presence and location of brain abnormalities with audiometric and language outcomes .", "the wilcoxon rank sums test was also used to explore statistical differences regarding factors ( e.g. , iq / dq ) that may be associated with presence and location of brain abnormalities . due to the small sample size , exact p values are reported .", "\n table 1 lists the fifteen cmv - positive children ( 9 females and 6 males ) who received a ci during the study timeframe .", "two additional subjects with cmv were excluded from this analysis : one subject had not returned for any postimplant visits and one child had normal cns imaging ( thought not to be symptomatic cmv ) . the median age at time of identification of hearing loss was 5 months ( range 162 months ) , and the median age at implantation was 28 months ( range 1296 months ) ( table 2 ) .", "the median iq / dq for the study population was 65 ( range 2390 ) ; 9 ( 60% ) children had an iq / dq < 70 .", "seventy - three percent ( n = 11 ) of children had a diagnosis of cognitive impairment and 13 children ( 87% ) had a motor delay or disability ; 9 had a diagnosis of cp .", "over half ( 60% , n = 9 ) of these children carried a diagnosis of more than one developmental disability and nearly half ( 47% , n = 7 ) had both a cognitive disability , and cp .  ", "seven children ( 47% ) had a head circumference ( hc ) less than the fifth percentile ( table 2 ) .", "children with a hc < 5th percentile had a significantly lower median iq / dq than children with a hc  5th percentile ( 42 versus 77 , p = 0.003 ) .", "all children with hc < 5th percentile had cognitive disabilities compared to only 50% of children with a hc  5th percentile . additionally , the children with a hc < 5th percentile had slightly poorer postimplant pta ( 38  db versus 27  db , p = 0.02 ) and sat / srt ( 30  db versus 23  db , p = 0.04 ) compared to children with larger heads .", "all children included in the study had central nervous system abnormalities present on imaging studies ( table 3 ) .", "twelve children had mri imaging ( all with identified abnormalities ) and 13  ct imaging of the brain with identified abnormalities .", "eighty percent ( n = 12 ) had calcifications , which occurred most frequently ( 53% ) in the periventricular region ( table 4 ) .", "additional imaging findings included ventriculomegaly in 53% , migrational abnormalities in 33% ( n = 5 ) , and encephalomalacia in 27% .", "all four children with encephalomalacia and all 3 children with cerebellar abnormalities had a diagnosis of cp .", "in addition to cp , 3 of the 4 children with encephalomalacia and all 3 children with cerebellar abnormalities also had a cognitive disability diagnosis .", "in fact , the children with cerebellar abnormalities had a lower nonverbal iq / dq than children without cerebellar abnormalities ( 35 ( range 2350 ) versus 68 ( range 2790 ) , p = 0.048 ) .", "calcifications in the temporal lobes were associated with poorer post - ci pta ( biserial correlation rb = 0.57 , p = 0.04 ) .  ", "the median ( range ) receptive language score was 21 ( 327 ) , and expressive language score was 28 ( 1777 ) .", "the iq / dq was highly correlated with both receptive ( spearman rho = 0.9 , p = 0.002 ) and expressive ( spearman rho = 0.86 , p = 0.007 ) , as having a head circumference < 5th percentile ( point biserial correlation rb = 0.75 and 0.68 resp . ) .", "periventricular calcifications were associated with lower receptive language ( rb = 0.75 , p = 0.03 ) and expressive language ( rb = 0.84 , p = 0.008 ) among children with post - ci assessments .", "because the iq / dq was associated with both periventricular calcifications ( rb = 0.53 , p = 0.04 ) and hc < 5th percentile ( rb = 0.73 , p = 0.002 ) , the relationship between these factors and language appears to be , at least in part , driven by iq / dq scores .", "one of the challenges in children with congenital cmv is predicting the sequelae they will develop and the extent of neurological and developmental deficits they will incur .", "previous studies have shown that brain imaging may be a good predictor of adverse neurodevelopmental outcomes . in one study of children with symptomatic congenital cmv ,", "90% of the children with an abnormal ct scan developed at least one sequela compared to 29% of those with a normal scan .", "greater than two - thirds of all children with congenital cmv had mri abnormalities and , of these , cortical malformations , ventriculomegaly and hippocampal dysplasia correlated highly with poor neurologic outcome .", "found that microcephaly was the most specific predictor , and abnormality on head ct was the most sensitive predictor of poor cognitive outcomes in this patient population . in a study of children with a diagnosis of snhl , 80% of the cmv positive children had abnormal brain mri scans compared with only 33% of cmv negative children .", "children with calcifications had a slightly poorer median post - ci pta compared to children without calcifications .", "interestingly , the location of the abnormalities also seemed to correlate with worse language outcomes .", "the parietal lobe processes sensory information and houses our language abilities , and the temporal lobe regulates emotion , hearing , language , and learning , which could explain why language outcomes are poorer in children with abnormalities in these regions .", "children with cerebellar anomalies also had lower receptive and expressive language levels than the other children .", "although the cerebellum has been largely thought to control balance , there may be other aspects of learning and development which depend on this entry point of sensory information to the higher cortical areas .", "the authors have implicated the cerebellum to be involved in temporal processing , language production and comprehension , spatial reasoning , and visual attention [ 1821 ] . in our 3 patients with cerebellar lesions ,", "it is not clear if it is the location of the lesions in the cerebellum or the severe cognitive delay that resulted in worse outcomes . with only 3 patients ,", "it is not possible to draw additional conclusions regarding cause and effect in this group .  ", "in addition to looking at the associations between cns imaging findings and post - ci outcomes , our study also looked for correlations between preimplantation developmental assessment and cochlear implant performance in these patients .", "although there is a relative paucity of relevant studies in the literature , previous studies have demonstrated that children with cognitive delay seem to derive benefit from ci , albeit at a slower rate than nondelayed children [ 22 , 23 ] .", "although children with cochlear implants who have more significant developmental delay have poorer outcomes than children with only mild delay , the biggest predictor of language development among children with implants and disabilities is a measure of nonverbal cognitive ability . unfortunately , when compared to appropriately matched developmentally delayed children without hearing impairment , children with cis had lower receptive and expressive language skills , which were not commensurate with their cognitive potential .", "although children with multiple handicaps may progress slower and to a lesser extent than typically developing children , these children still derive benefit from the auditory stimulation provided by a ci but may require additional post - ci intervention to optimize benefit [ 2729 ] .  ", "as we found in our study , the majority of children with symptomatic congenital cmv have associated developmental disabilities . the variability of neurocognitive deficits in these children warrants the examination of this group separately from other children with developmental disabilities . as with children with developmental disabilities ,", "children with symptomatic congenital cmv appear to derive benefit from ci albeit at a slower rate .", "ramirez inscoe and nikolopoulos demonstrated mixed results for speech perception and intelligibility with 50% of children with congenital cmv performing more poorly than controls , 31% performing similarly , and 19% performing better than controls .", "these children did , however , derive auditory benefit from ci . in their study of 13 children with symptomatic congenital cmv ,", "73% of implanted children achieved closed - set word recognition , and 63% achieved open - set word recognition .", "children with congenital cmv have also demonstrated both improved pure - tone hearing thresholds and improved language perception and production   as well as useful speech comprehension albeit lower than matched controls .", "another study of 11 children with congenital cmv found that 9 of the 11 carried a diagnosis of psycho - neurological disorders ( including attention deficit hyperactivity disorder , mental retardation , autism , and pervasive developmental disorder ) . although hearing thresholds were similar among children in this group , post - ci performance varied widely depending upon the concomitant diagnosis .", "our findings were consistent with these previous studies in that our patients did demonstrate overall improvement in post - ci thresholds .  ", "our results indicate that head size was significantly related to iq / dq and that children with microcephaly ( hc < 5th percentile ) were more likely to have cognitive disabilities than those without microcephaly .", "the microcephalic children also had significantly poorer post - ci pta and sat / srt as well as worse receptive language .", "these findings indicate that microcephalic children do not achieve the same degree of auditory benefit as the children with normal head size .", "the mean age at last audiometric followup did vary between the microcephalic and nonmicrocephalic groups ( 46 versus 59 months ) .", "this difference would not be expected to affect the results of audiometric testing as all testing was performed using developmentally appropriate behavioral methods that should account for the difference in age .", "given that the majority of the children ( 73% ) had developmental delay and nearly half were considered multiply handicapped , it is notable that head circumference was a better predictor of post - ci outcome than specific developmental delay diagnosis .", "this is not entirely surprising as head circumference relates to brain growth , which can impact developmental outcomes .", "although our study has limitations including its retrospective nature and small sample size , it provides data that may further efforts to identify factors which may help predict which children with congenital symptomatic cmv will benefit from ci . the presence of cerebellar anomalies or greater than one cns abnormality on imaging correlates with poorer outcomes after ci . the location of cns abnormalities , including calcifications , may play a role in audiometric and language outcomes after ci .", "early measurements such as brain imaging findings , head circumference , and iq / dq may allow for more accurate counseling of families regarding anticipated postimplantation performance in children with symptomatic congenital cmv ."], "abstract_text": ["<S> \n objective . to examine the association of intracranial radiographic abnormalities and developmental measures with outcomes in children with congenital symptomatic cytomegalovirus ( cmv ) and cochlear implants ( ci ) . design / methods . </S>", "<S> it was a retrospective review of 15 children implanted from 2004 to 2010 . </S>", "<S> preimplant nonverbal intelligence quotient / developmental quotient ( iq / dq ) and head circumference ( hc ) were obtained . </S>", "<S> computed tomography and magnetic resonance imaging of the brain and post - ci audiometry and language assessments were reviewed . </S>", "<S> results . </S>", "<S> eleven children ( 73% ) had cognitive delay . </S>", "<S> most had > 1 developmental disability . </S>", "<S> median iq / dq was 65 ( 2390 ) . </S>", "<S> all had imaging abnormalities . </S>", "<S> most imaging abnormalities were in parietal ( 60% ) and temporal ( 60% ) lobes . children with hc < 5th percentile had poorer median post - ci pta ( 38  db versus 27  db , p = 0.02 ) . </S>", "<S> periventricular calcifications were associated with lower receptive ( rb = 0.75 , p = 0.03 ) and expressive ( rb = 0.84 , p = 0.008 ) language . because iq / dq was associated with periventricular calcifications ( rb = 0.53 , p = 0.04 ) and small hc \n ( rb = 0.73 , p = 0.002 ) , their relationships with language appear partially driven by iq / dq </S>", "<S> . conclusions . </S>", "<S> the location of brain abnormalities appears to correlate with worse outcomes after ci . </S>", "<S> these findings may allow for more accurate counseling of parents regarding anticipated postimplantation performance . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["cytomegalovirus ( cmv ) is one of the most common congenital viral infections in many regions . in the united states it occurs in 0.51% of live births , or approximately 40,000 infants annually .", "the manifestations of cmv infection cover a broad spectrum ranging from asymptomatic to severe systemic disease resulting in significant morbidity and mortality .", "90% of infants with congenital cmv are asymptomatic at birth . despite being asymptomatic at birth , up to 7% of these children", "will develop sensorineural hearing loss ( snhl ) that can be unilateral or bilateral , fluctuating or progressive , and range from mild to profound [ 2 , 3 ] .", "approximately 10% of infants with congenital cmv are symptomatic at birth , and 40% of these patients will develop snhl [ 1 , 2 ] .", "hearing loss is the most common manifestation of congenital cmv infection making cmv a leading cause of nonhereditary congenital hearing loss .", "given the relatively large number of children potentially affected by cmv - related hearing loss and the wide range of manifestations of congenital cmv infection , it is difficult to predict how a child with symptomatic cmv will perform with a cochlear implant ( ci ) .", "previous studies suggest that children with symptomatic cmv derive auditory benefit from cis , albeit at a slower rate than other children . the degree to which these children benefit remains somewhat controversial as there is variation in the development of language skills after ci [ 59 ] .", "the objective of our study was to examine the relationships between intracranial radiographic abnormalities and preimplantation developmental assessment ( including head circumference ) with postimplant audiometric and language outcomes in children with symptomatic congenital cmv who have undergone cochlear implantation ."], ["this study was a retrospective review of children with a diagnosis of symptomatic congenital cmv who underwent cochlear implantation between 2004 and 2010 at a tertiary pediatric cochlear implant center .", "congenital cmv was diagnosed for most children via urine culture of the virus during the first two weeks of life .", "diagnosis was also made based on the identification of hearing loss coupled with brain image findings .", "medical charts of these patients were reviewed for pre- and postimplantation evaluations and central nervous system imaging findings .", "data regarding preimplantation developmental assessments included the diagnoses of developmental delay and/or other disabilities , nonverbal intelligence quotients or developmental quotients ( iq / dq ) , and head circumference ( hc ) at the preoperative visit .", "results from computed tomography ( ct ) and/or magnetic resonance imaging ( mri ) scans of the brain were reviewed , and the findings were classified by type and location of anomaly .", "postimplant audiometric behavioral testing , which was performed using validated and developmentally appropriate testing techniques , was collected .", "this data included frequency - specific thresholds and speech awareness or speech reception thresholds ( sat / srt ) .", "pure tone averages ( pta ) were calculated by an average of 4 frequencies ( 0.5 , 1.0 , 2.0 , and 4.0  khz ) .  ", "children 's developmental disability diagnoses were classified according to the areas of cognition , motor , vision , and language .", "cognitive disability was diagnosed using nonverbal cognitive measures such as the leiter international performance scale   and supported by evaluating adaptive functioning with the vineland adaptive behavior scale .", "these evaluations were completed by psychologists with knowledge of testing deaf / hard of hearing children . if the nonverbal iq was not available , a nonverbal developmental quotient was used .", "this nonverbal or adaptive quotient was obtained from a developmental pediatrician with expertise in hearing loss as a part of the precochlear implant assessment . for this study ,", "the diagnosis of cerebral palsy ( cp ) was made based on neurologic examination , patterns of persistent primitive reflexes , abnormalities in tone and reflexes , and presence of abnormalities on mri of the brain .", "the diagnosis of pervasive developmental disorder not otherwise specified was made by interdisciplinary evaluations with speech pathology and psychology using standardized measures to evaluate autism spectrum disorders .", "standardized language assessment scores , using the preschool language scale ( 4th edition )   were available on eight subjects who returned for speech therapy after implantation .", "this study was approved by the institutional review board at cincinnati children 's hospital medical center .  ", "data distributions were reported using medians with ranges ( for continuous data ) and frequencies with percentages ( for categorical data ) .", "characteristic differences between children with hc < 5th percentile and > 5th percentile were tested using wilcoxon rank sums test for continuous data and chi - square or fisher 's exact test for categorical data .", "biserial correlations were conducted to understand the association between presence and location of brain abnormalities with audiometric and language outcomes .", "the wilcoxon rank sums test was also used to explore statistical differences regarding factors ( e.g. , iq / dq ) that may be associated with presence and location of brain abnormalities . due to the small sample size , exact p values are reported ."], ["\n table 1 lists the fifteen cmv - positive children ( 9 females and 6 males ) who received a ci during the study timeframe .", "two additional subjects with cmv were excluded from this analysis : one subject had not returned for any postimplant visits and one child had normal cns imaging ( thought not to be symptomatic cmv ) . the median age at time of identification of hearing loss was 5 months ( range 162 months ) , and the median age at implantation was 28 months ( range 1296 months ) ( table 2 ) .", "the median iq / dq for the study population was 65 ( range 2390 ) ; 9 ( 60% ) children had an iq / dq < 70 .", "seventy - three percent ( n = 11 ) of children had a diagnosis of cognitive impairment and 13 children ( 87% ) had a motor delay or disability ; 9 had a diagnosis of cp .", "over half ( 60% , n = 9 ) of these children carried a diagnosis of more than one developmental disability and nearly half ( 47% , n = 7 ) had both a cognitive disability , and cp .  ", "seven children ( 47% ) had a head circumference ( hc ) less than the fifth percentile ( table 2 ) .", "children with a hc < 5th percentile had a significantly lower median iq / dq than children with a hc  5th percentile ( 42 versus 77 , p = 0.003 ) .", "all children with hc < 5th percentile had cognitive disabilities compared to only 50% of children with a hc  5th percentile . additionally , the children with a hc < 5th percentile had slightly poorer postimplant pta ( 38  db versus 27  db , p = 0.02 ) and sat / srt ( 30  db versus 23  db , p = 0.04 ) compared to children with larger heads .", "all children included in the study had central nervous system abnormalities present on imaging studies ( table 3 ) .", "twelve children had mri imaging ( all with identified abnormalities ) and 13  ct imaging of the brain with identified abnormalities .", "eighty percent ( n = 12 ) had calcifications , which occurred most frequently ( 53% ) in the periventricular region ( table 4 ) .", "additional imaging findings included ventriculomegaly in 53% , migrational abnormalities in 33% ( n = 5 ) , and encephalomalacia in 27% .", "all four children with encephalomalacia and all 3 children with cerebellar abnormalities had a diagnosis of cp .", "in addition to cp , 3 of the 4 children with encephalomalacia and all 3 children with cerebellar abnormalities also had a cognitive disability diagnosis .", "in fact , the children with cerebellar abnormalities had a lower nonverbal iq / dq than children without cerebellar abnormalities ( 35 ( range 2350 ) versus 68 ( range 2790 ) , p = 0.048 ) .", "calcifications in the temporal lobes were associated with poorer post - ci pta ( biserial correlation rb = 0.57 , p = 0.04 ) .  ", "the median ( range ) receptive language score was 21 ( 327 ) , and expressive language score was 28 ( 1777 ) .", "the iq / dq was highly correlated with both receptive ( spearman rho = 0.9 , p = 0.002 ) and expressive ( spearman rho = 0.86 , p = 0.007 ) , as having a head circumference < 5th percentile ( point biserial correlation rb = 0.75 and 0.68 resp . ) .", "periventricular calcifications were associated with lower receptive language ( rb = 0.75 , p = 0.03 ) and expressive language ( rb = 0.84 , p = 0.008 ) among children with post - ci assessments .", "because the iq / dq was associated with both periventricular calcifications ( rb = 0.53 , p = 0.04 ) and hc < 5th percentile ( rb = 0.73 , p = 0.002 ) , the relationship between these factors and language appears to be , at least in part , driven by iq / dq scores ."], ["one of the challenges in children with congenital cmv is predicting the sequelae they will develop and the extent of neurological and developmental deficits they will incur .", "previous studies have shown that brain imaging may be a good predictor of adverse neurodevelopmental outcomes . in one study of children with symptomatic congenital cmv ,", "90% of the children with an abnormal ct scan developed at least one sequela compared to 29% of those with a normal scan .", "greater than two - thirds of all children with congenital cmv had mri abnormalities and , of these , cortical malformations , ventriculomegaly and hippocampal dysplasia correlated highly with poor neurologic outcome .", "found that microcephaly was the most specific predictor , and abnormality on head ct was the most sensitive predictor of poor cognitive outcomes in this patient population . in a study of children with a diagnosis of snhl , 80% of the cmv positive children had abnormal brain mri scans compared with only 33% of cmv negative children .", "children with calcifications had a slightly poorer median post - ci pta compared to children without calcifications .", "interestingly , the location of the abnormalities also seemed to correlate with worse language outcomes .", "the parietal lobe processes sensory information and houses our language abilities , and the temporal lobe regulates emotion , hearing , language , and learning , which could explain why language outcomes are poorer in children with abnormalities in these regions .", "children with cerebellar anomalies also had lower receptive and expressive language levels than the other children .", "although the cerebellum has been largely thought to control balance , there may be other aspects of learning and development which depend on this entry point of sensory information to the higher cortical areas .", "the authors have implicated the cerebellum to be involved in temporal processing , language production and comprehension , spatial reasoning , and visual attention [ 1821 ] . in our 3 patients with cerebellar lesions ,", "it is not clear if it is the location of the lesions in the cerebellum or the severe cognitive delay that resulted in worse outcomes . with only 3 patients ,", "it is not possible to draw additional conclusions regarding cause and effect in this group .  ", "in addition to looking at the associations between cns imaging findings and post - ci outcomes , our study also looked for correlations between preimplantation developmental assessment and cochlear implant performance in these patients .", "although there is a relative paucity of relevant studies in the literature , previous studies have demonstrated that children with cognitive delay seem to derive benefit from ci , albeit at a slower rate than nondelayed children [ 22 , 23 ] .", "although children with cochlear implants who have more significant developmental delay have poorer outcomes than children with only mild delay , the biggest predictor of language development among children with implants and disabilities is a measure of nonverbal cognitive ability . unfortunately , when compared to appropriately matched developmentally delayed children without hearing impairment , children with cis had lower receptive and expressive language skills , which were not commensurate with their cognitive potential .", "although children with multiple handicaps may progress slower and to a lesser extent than typically developing children , these children still derive benefit from the auditory stimulation provided by a ci but may require additional post - ci intervention to optimize benefit [ 2729 ] .  ", "as we found in our study , the majority of children with symptomatic congenital cmv have associated developmental disabilities . the variability of neurocognitive deficits in these children warrants the examination of this group separately from other children with developmental disabilities . as with children with developmental disabilities ,", "children with symptomatic congenital cmv appear to derive benefit from ci albeit at a slower rate .", "ramirez inscoe and nikolopoulos demonstrated mixed results for speech perception and intelligibility with 50% of children with congenital cmv performing more poorly than controls , 31% performing similarly , and 19% performing better than controls .", "these children did , however , derive auditory benefit from ci . in their study of 13 children with symptomatic congenital cmv ,", "73% of implanted children achieved closed - set word recognition , and 63% achieved open - set word recognition .", "children with congenital cmv have also demonstrated both improved pure - tone hearing thresholds and improved language perception and production   as well as useful speech comprehension albeit lower than matched controls .", "another study of 11 children with congenital cmv found that 9 of the 11 carried a diagnosis of psycho - neurological disorders ( including attention deficit hyperactivity disorder , mental retardation , autism , and pervasive developmental disorder ) . although hearing thresholds were similar among children in this group , post - ci performance varied widely depending upon the concomitant diagnosis .", "our findings were consistent with these previous studies in that our patients did demonstrate overall improvement in post - ci thresholds .  ", "our results indicate that head size was significantly related to iq / dq and that children with microcephaly ( hc < 5th percentile ) were more likely to have cognitive disabilities than those without microcephaly .", "the microcephalic children also had significantly poorer post - ci pta and sat / srt as well as worse receptive language .", "these findings indicate that microcephalic children do not achieve the same degree of auditory benefit as the children with normal head size .", "the mean age at last audiometric followup did vary between the microcephalic and nonmicrocephalic groups ( 46 versus 59 months ) .", "this difference would not be expected to affect the results of audiometric testing as all testing was performed using developmentally appropriate behavioral methods that should account for the difference in age .", "given that the majority of the children ( 73% ) had developmental delay and nearly half were considered multiply handicapped , it is notable that head circumference was a better predictor of post - ci outcome than specific developmental delay diagnosis .", "this is not entirely surprising as head circumference relates to brain growth , which can impact developmental outcomes ."], ["although our study has limitations including its retrospective nature and small sample size , it provides data that may further efforts to identify factors which may help predict which children with congenital symptomatic cmv will benefit from ci . the presence of cerebellar anomalies or greater than one cns abnormality on imaging correlates with poorer outcomes after ci . the location of cns abnormalities , including calcifications , may play a role in audiometric and language outcomes after ci .", "early measurements such as brain imaging findings , head circumference , and iq / dq may allow for more accurate counseling of families regarding anticipated postimplantation performance in children with symptomatic congenital cmv ."]]}
{"article_id": "PMC4852117", "article_text": [". there have been significant achievements regarding to the millennium development goals ( mdgs ) as a global development target .", "the indicators showed reflection of these achievements , such as decreasing global poverty rate , increasing the number of children going to school , decreasing child death rate , increasing access to clean water , and also increasing malaria , hiv / aids , and tuberculosis control investment .", "mdgs were evaluated in the year 2015 , and there will be remaining new challenges to sustain the achievement at post-2015 era .", "numerous important issues have been raised by united nation ( un ) , which are poverty and hunger elimination , improving health and education , sustainable cities , combating climate changes , and also ocean and forest conservation . in mdgs , hiv / aids , malaria , and major diseases are clearly mentioned as global development goals . in this case , there will be plenty of diseases that are not a major concern of the world .", "questions were raised concerning neglected tropical disease ( ntd ) which is definitely out of the highlight . will these diseases be included in the ambitious post - mdgs sustainable development ? the new sustainable development goals ( sdgs ) , known also as the global goals , had been established after world leaders gathered on 25 september 2015 , at the united nations in new york to adopt the 2030 agenda for sustainable development and the broader and further agenda than the mdgs .", "mdg explicitly stated that hiv / aids , malaria , and tuberculosis will be efficiently controlled at the end of the year 2015 .", "the big three are the diseases that attract priority from sponsors , researchers , and policy makers [ 2 , 3 ] .", "this situation is not the same as ntd , which has not gained any priority from them and even worsened .", "the term of neglected in ntd referred to the fact that these tropical diseases are not being considered as important diseases .", "ntd result in long life 's deformities and handicaps , decrease productivity and economical status , and also end up many social consequences and stigmatization . though ntd is commonly found in tropical countries , it is not identical with tropical diseases .", "poverty as one of ntd determinants frequently occurs in rural area , slums , and marginalized populations living nearby the equator .", "ntd in this area are closely associated with poverty and limited resources , such as clean drinking water access , poor sanitation , and healthy housing .", "ntd may decrease child health , increase the health expenses and risk of ineffective treatment , decrease productivity , and result in education default .", "their potential to spread in developed countries is low , because ntd are closely related with local vector and intermediate host distribution which are specifically associated with geographic region .", "the technology and resource to control , prevent , and eliminate ntd are available , but not for the developing countries . who has included 17 diseases caused by bacteria , virus , and protozoa into ntd ( figure 1 ) .", "helminths caused diseases that dominated ntd : taeniasis , dracunculiasis , echinococcosis , trematodiasis , filariasis , onchocerciasis , schistosomiasis , and soil - transmitted helminthiasis .", "the ministry of health of indonesia reported only five ntd that can be found in indonesia , that is , leprosy , filariasis , schistosomiasis , soil - transmitted helminth , and yaws .", "however , this review describes more than those five diseases and aims to address the magnitude of the problem of ntd in indonesia .", "leprosy is an infectious disease is caused by mycobacterium leprae , a road shaped bacteria classified in the same genus as mycobacterium tuberculosis", "leprosy may be complicated with deformity and handicap , which may decrease the ability of the patient to work in daily life .", "leprosy was a serious problem in indonesia at 1980 , with 126,221 cases in 1985 .", "it was noted that the significant improvement in leprosy control was because of massive promotion of leprosy prevention and multidrug therapy ( mdt ) intervention in more than 5,600 primary health centers in indonesia .", "however , the new leprosy case finding in 2000 was not significantly different with year 2013 ( table 1 ) .", "fourteen provinces and 160 districts , which mostly are located in java island , reported prevalence of > 1 per 10,000 population in 2009 .", "leprosy epidemical indicators were significant , such as deformity grade 2 10.5% , leprosy cases in children 12.01% , and 82.43% multibacillary ( mb ) cases [ 10 , 1315 ] .", "the problems of leprosy in indonesia are summarized as follows : early detection.development of more effective treatment.development of more effective vaccine.understanding immunopathogenesis of peripheral nerve damage.management of chronic erythema nodosum leprosum ( enl).deformity and stigmatization.deformity and stigmatization are serious problems among leprosy patients in indonesia .", "survey conducted in subang district ( west java ) ; gresik and malang district ( east java ) ; bone and gowa district ( south sulawesi ) showed that deformity in leprosy patients occurred in 76.7% of patients .", "almost 60% respondents were experienced handicaps during their life , including participating to social activity .", "mdt has significant impact to decrease leprosy prevalence globally . however , there are various substantial operational and technical problems among countries and territory .", "resistance of m. leprae is a serious problem because of limited effective antimicrobes for leprosy .", "resistance against dapsone , that was applied as monotherapy , was initially reported in 1964 at malaysia . since then , resistance to dapsone and other drugs was reported .", "theoretically , combination of more than two drugs with different action mechanism will reduce the probability of resistant in mycobacteria .", "national surveillance of m. leprae resistance against mdt should be established to provide accurate data .", "sporadic and partial reports were not sufficient to measure the problems and establish the programs to reduce the prevalence and spread of resistance against mdt .", "phenotypic testing is the basic method for susceptibility testing of antimicrobial agents . even in the new proof of concept which combines the classical growth - based phenotypic test and dna based approach , pure culture of bacteria", "efforts were documented to culture m. leprae in artificial media and animal models [ 24 , 25 ] .", "mutations in the folp1 , rpob , and gyra genes are responsible for resistance to dapsone , rifampicin , and ofloxacin , respectively . to test the susceptibility of m. leprae against particular antibiotics ,", "screening in north maluku and north sulawesi showed that resistance to dapsone occurs as 0.8% in new cases and 10% in relapse cases , while rifampicin occurs with 3.3% in new cases and 20% in relapse cases .", "there is higher prevalence of dapsone and rifampicin resistance among relapse cases comparing with new cases .", "it was noted that the incidence of resistance against dapsone and rifampicin in indonesia almost did not change between the time monotherapy was introduced and after mdt was recommended by who [ 29 , 30 ] .", "yaws is a treponema pallidum subspecies pertenue infection , which may develop as chronic and recurrent disease .", "yaws is nonsexually transmitted treponemal infection , which is latent and asymptomatic for years with positive serology result .", "small portion ( 10% ) of the patients will undergo bone destruction leading to deformities .", "diagnosis of yaws is commonly based on the clinical findings and serology [ 31 , 32 ] .", "confirmatory diagnosis of yaws is using serological test , which includes nontreponemal agglutination tests , such as rapid plasma reagin ( rpr ) and venereal disease research laboratory ( vdrl ) test , and also treponemal tests such as treponema pallidum hemagglutination assay ( tpha ) , treponema pallidum particle agglutination tppa , and fluorescent treponemal antibody - absorption ( fta - abs ) .", "the accuracy of yaws serological test is hampered in the area which syphilis , caused by t. pallidum subspecies pallidum , is endemically circulated .", "yaws was reported in 68 ( 14% ) of 497 districts in indonesia , mostly in the eastern part of indonesia .", "east nusa tenggara province reported 2,800 cases in 2012 , which is scattered at 566 small islands in this region .", "however , it gradually decreases to 5,319 cases in 2011 and 3,476 in 2012 [ 10 , 34 ] .", "there are several obstacles to eradicate yaws in indonesia : geographically the yaws patients are in a remote area including rural and small islands which are not easy to handle , difficulty to give benzathine penicillin for children , and also local political condition that is somehow counterproductive to the programs . who planned to eradicate yaws globally in 2020 with adoption of morges strategies .", "this program intends to introduce a mass treatment in the endemic regions by using azithromycin .", "however , benzathine penicillin can be used as back up in case of azithromycin is not indicated , treatment failure , or in places where azithromycin is not available .", "this treatment followed an active survey every six months to detect and treat the remaining cases [ 32 , 34 ] .", "the ministry of health launched yaws eradication program , aimed to eradicate yaws in 2013 , one year later than who target .", "there were several approaches conducted , such as active screening new cases and contacts , empowering community , improving the capacity of health worker to diagnose and manage yaws , and intersectoral collaboration approaches .", "access to health service is limited in the yaws endemic area , which obstacle the passive screening program .", "reported a survey among health workers in primary health center , elementary school teachers , and parents in muna , southeast sulawesi .", "they were asked about yaws to measure their knowledge about sign and symptoms , causative agent , and therapy for yaws .", "another report from southwest sumba showed clean water and healthy behavior is the risk factor for yaws .", "these reports showed that comprehensive intervention is needed to boost the community participation for yaws eradication .", "reporting system for yaws incidence is very important to support the best intervention for yaws eradication .", "learning the fact that their knowledge about yaws is remarkably low , it may diminish the validity of yaws reporting system .", "insufficiency of this sector may result in delay of yaws eradication and increasing yaws contact population .", "yaws eradication problem in indonesia is summarized as follows : early detection by health workers.geographical problem of endemic area.surveillances and reporting system.inadequate confirmatory laboratory test .", "dengue virus consists of four serotypes : denv1 , denv2 , denv3 , and denv4 .", "these viruses are responsible for infectious diseases with width spectrum of clinical manifestation : dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and dengue shock syndrome ( dss ) [ 38 , 39 ] .", "dengue virus infection is an arbovirus disease , which in indonesia is primarily transmitted by aedes aegypti and aedes albopictus .", "the incidence of dhf significantly increased since reported initially in 1968 ( 0.05/100.000 ) to 3540/100,000 in 2013 . however , in 2010 , there was an extreme surge of dhf incidence to 86/100,000 .", "the significant increasing of dhf incidence was parallel with decreasing of case fatality rate ( cfr ) , which is 41% in 1968 to 0.73% in 2013 .", "however , it was noticed that from 1999 there was a trend of shifting to adults .", "dhf incidence described here is believed not to be able to draw a clear picture of all denv infections .", ". however , the proportion of those three clinical manifestations still becomes an enigma , although the awareness to the diseases , surveillance programs , diagnostic tools , and management of denv infection in indonesia have been significantly improved .", "it has been realized that controlling adult mosquitoes is prone to fail reducing denv transmission .", "the vector control approach should aim to interrupt the transmission cycle at an early phase through immature mosquito control , which is including larvae and pupae stages .", "immature mosquitoes control is believed to be more effective . the vector control approach is still considered effective while vaccine and prophylaxis remedies continuously develop .", "it seems that the socioecological approach is the best candidate to be implemented in an endemic country , such as indonesia .", "community and intersectoral approach are significantly important fundamentals of integrated public health strategies for dengue vector control [ 47 , 48 ] .", "as dengue infection has wide spectrum of clinical manifestation , the clinical diagnosis is confusing .", "there are guidelines issued by who to respond to the need of applicable tool for clinician : who classification system   and dengue and control study ( denco ) revised clinical management .", "the study concerning the two guidelines showed that in surabaya , indonesia , the denco revised clinical management guideline superior in detection of severe dengue cases .", "however , in a study in semarang , indonesia , the denco guideline has failed to show its superiority .", "it is true that the denco guideline needs to be adjusted in terms of geographic and age - related variations issues .", "there are many factors that may be related with clinical manifestation of dengue virus infection , including human genetics involvement which is still controversially related with population , race , and geography [ 54 , 55 ] .", "several methods are available for laboratory test confirmation of denv infection : virus isolation , genome detection , antigen detection , and antibody detection .", "the first three methods are direct detection methods which were considered as more specific and able to confirm earlier onset of denv infection .", "the antibody detection is widely used in the field because they are easy to perform and less expensive , though it is not as powerful as direct detection especially in secondary infection [ 50 , 56 ] .", "rabies is an acute , progressive , incurable viral encephalitis disease that is transmitted from animal , mainly dogs , to human .", "it is caused by rabies virus ( rabv ) , a member of the family rhabdoviridae , genus lyssavirus .", "the highest incidence is concentrated in asia and africa . despite the fact that rabies is 100% vaccine - preventable infectious disease , it potentially threatens over 3 billion people in the world .", "however , poor surveillance , underreporting , frequent misdiagnosis , and poor coordination among sectors may lead to underestimation of the burden of the disease .", "ten countries of asean have been declared rabies - free asean by 2020 in the occasion of the thirty - fourth meeting of the asean ministers on agriculture and forestry ( 34th amaf ) , on september 2012 .", "rabies was reported in 24 provinces out of 34 provinces in indonesia . reported human rabies in indonesia is small ( 206 cases in 2010 and significantly decreased to 119 cases in 2013 ) compared to thousands of cases reported in other countries such as india , china , and other asian countries . to control rabies transmission , indonesian", "government uses one health approach which facilitates the multidisciplinary participation in management of zoonosis diseases .", "however , kalimantan and sulawesi , other islands of indonesia , are the most plausible hypothetical origin of the bali rabv strains .", "totally there were 133 reported human deaths because of rabies in bali from 2008 to september 2011 .", "this significantly decrease of rabid human death within 3 years is a good model of rabies control for other regions or countries .", "it was proposed that this success is mainly because of mass vaccination program for dogs , which is the primary ( 98% ) vector of rabies in bali . however , the number of reported humans bitten by dogs remained over 4,000 per month .", "the preparedness to human rabies should be maintained , as the threat of human rabies escalation is still high .", "lymphatic filariasis ( lf ) is caused by worms inhabiting the lymphatics [ 6365 ] .", "approximately 65% of total cases are found in the southeast asian region [ 6668 ] .", "lf cases were found in all provinces of indonesia . in 2009 , there were 11.914 lf cases reported . the number of total cases was steadily reported until 2013 . there were 11.912 cases registered in 2013 .", "lf prevalence in indonesia varied from 0.5 to 27.6% , in which the highest rates were found in maluku , papua , west papua , east nusa tenggara , and north maluku provinces .", "three lymphatic parasites are prevalently circulating in indonesia : wuchereria bancrofti , brugia malayi , and brugia timori [ 69 , 70 ] , which are transmitted by mosquitoes of five genera ", "the site of adult - worm parasitism is within the lymphatic vessels , most commonly involving the extremities and male genitals [ 64 , 65 , 70 ] .", "the disease predominantly afflicts poor people in both urban and rural areas with limited resources condition , where mosquitoes as a vector might be found in high density [ 64 , 68 ] . who launched global program to eliminate lymphatic filariasis ( gpelf ) , which relies on mass drug administration ( mda ) approach [ 68 , 71 ] .", "the main goal of the program is to hamper transmission of disease between mosquitoes and human beings , mainly through mass drug distribution of diethylcarbamazine ( 6  mg / kg ) or ivermectin ( 150200  g / kg ) combined with albendazole ( 400  mg ) [ 68 , 71 ] .", "it is recommended that all people at risk are involved in this program , since patients with asymptomatic infection may have abnormal lymphatics , and that early treatment may prevent subsequent lymphatic damage [ 66 , 69 ] .", "the efficacy of six annual rounds of mda was studied in alor island , eastern part of indonesia .", "microfilaria rates of b. timori decreased significantly after mda intervention , from 26% to 0.17% .", "the prevalence of filarial - specific igg4 antibodies was significantly decreased from 80% to 6% .", "the lf national plan has implemented mda campaigns in 2002 . however , due to financial and human resource constraints , districts often provide only partial coverage of the at - risk population within the district . in 2009 ,", "the challenge of mda implementation in indonesia is not the efficacy of the drugs which were given to the community .", "it seems that the infrastructure is the key of the implementation , which includes the availability of transportation and physical access to the target population , reliable data bases , and competence health workers .", "the other challenge was to gain the trust from the community member to boost the compliance of drug administration .", "currently , mda has been scaled up in a geographically scattered way to address high prevalence areas and political needs .", "medical professionals , donors , universities , and ngos have played a critical role in finding new cases , assessing disease burden and supporting trainings and mda campaigns .", "prevention of lf depends on mosquito vectors control , which has had limited success because development of mosquitoes resistance against insecticides .", "urbanization of vast areas of tropical asia , including indonesia , has resulted in a concomitant rise in the prevalence of both w. bancrofti and b. malayi varieties of filariasis , carried by mosquitoes that breed in nonsylvatic habitats [ 68 , 73 ] .", "schistosomiasis is a chronic water - borne infection caused by trematodes schistosoma , mainly found in developing countries in africa , south america , the caribbean , the middle east , and asia [ 69 , 74 , 75 ] . in indonesia", "it is found in three isolated areas of central sulawesi province , namely , lindu , napu , and bada valleys . except for schistosoma haematobium that is responsible for urinary tract disease , the human schistosomes primarily affect the intestine and liver .", "clinical manifestations of the disease are recognized as fever with dysenteric symptoms and loss of appetite , as well as physical growth and cognitive delay in children [ 69 , 77 , 78 ] .", "disease prevalence fluctuated between 0.3% and 4.8% in napu valley and between 0.8% and 3.6% in lindu valley .", "however , the prevalence of schistosomiasis in both areas tended to increase during the 20082011 period . the parasite transmission cycle involved domestic and wild animals as reservoir [ 76 , 79 ] . in 2012 , who approved the goal of eliminating schistosomiasis in endemic countries .", "it focused on improving sanitary conditions and large - scale distribution of the antiparasitic drug , praziquantel to high - risk target groups , such as school - age children , child bearing age women , and individuals involved in frequent contact with contaminated fresh water .", "praziquantel is well - tolerated , associated with few side effects , and has a very high therapeutic index .", "moreover , a single dose praziquantel administration is usually sufficient to kill all adult worms [ 69 , 78 ] .", "schistosomiasis control in indonesia has faced many difficulties even though the endemic areas are very limited [ 79 , 81 ] .", "the core strategy of mda should be coupled with education to the local community , rat and snail surveillance , and support to the environmental management programs including introduction of latrines and suitable water sources . in the future", ", there should be emphasis to understand the social dynamics and social change related to schistosomiasis , which may provide more information about concrete issues to control transmission of schistosomiasis .", "lack of intersectoral coordination and collaboration may have occurred , possibly leading to increase transmission and reinfection rates , and be prone to control failure .", "soil - transmitted helminthiasis ( sth ) is an infection with one or more intestinal parasitic worms : roundworms ( ascaris lumbricoides ) , whipworms ( trichuris trichiura ) , or hookworms ( necator americanus and ancylostoma duodenale )", ". the tropical climate of indonesia is highly favourable to support the circulation of soil - transmitted helminths in the country .", "there are sporadic reports regarding the prevalence of the sth which come from scattered part of the country .", "the prevalence of sth was reported ranging from 40 to 70% in 80s to 90s .", "however , the estimated prevalence of sth was decreased in 2005 : ascaris ( 15.2% ) , trichuris ( 12.9% ) , and hookworms ( 8.4% ) .", "the survey conducted by ministry of health in elementary schools located in 33 provinces of indonesia showed prevalence of sth was 31.8% .", "india ( 64% ) and indonesia ( 16% ) together are contributing 80% of the regional 's burden .", "drugs used for deworming , albendazole , and mebendazole are effective and inexpensive to control sth transmission and reinfection .", "the mda programme for elimination of lf which is implemented by using combination of albendazole with ivermectin or diethylcarbamazine seems to have synergistic effect to decrease the prevalence of sth .", "the program targeted preschool and elementary school age . however , until this moment the country still struggles to combat sth . in conclusion , there were efforts and programs concerning ntd that were planed and implemented in indonesia .", "many factors may contribute to the rendering of ntd elimination in indonesia , such as high population , wide range geographic of the archipelago , and limited resources . by the end of 2015 ntd"], "abstract_text": ["<S> the world will enter the postmillennium development goals 2015 era . </S>", "<S> the achievements of the millennium development goals ( mdgs ) as a global development target need to be evaluated . </S>", "<S> a sustainable new reasonable target is important for neglected tropical diseases ( ntd ) elimination in indonesia . </S>", "<S> this review describes the ntd situation in indonesia and highlights problems beneath the ntd transmission . </S>", "<S> multidisciplinary approach is a promising strategy to help the marginalized people . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Leprosy", "3. Yaws", "4. Dengue", "5. Rabies", "6. Lymphatic Filariasis", "7. Schistosomiasis", "8. Soil-Transmitted Helminths"], "sections": [[". there have been significant achievements regarding to the millennium development goals ( mdgs ) as a global development target .", "the indicators showed reflection of these achievements , such as decreasing global poverty rate , increasing the number of children going to school , decreasing child death rate , increasing access to clean water , and also increasing malaria , hiv / aids , and tuberculosis control investment .", "mdgs were evaluated in the year 2015 , and there will be remaining new challenges to sustain the achievement at post-2015 era .", "numerous important issues have been raised by united nation ( un ) , which are poverty and hunger elimination , improving health and education , sustainable cities , combating climate changes , and also ocean and forest conservation . in mdgs , hiv / aids , malaria , and major diseases are clearly mentioned as global development goals . in this case , there will be plenty of diseases that are not a major concern of the world .", "questions were raised concerning neglected tropical disease ( ntd ) which is definitely out of the highlight . will these diseases be included in the ambitious post - mdgs sustainable development ? the new sustainable development goals ( sdgs ) , known also as the global goals , had been established after world leaders gathered on 25 september 2015 , at the united nations in new york to adopt the 2030 agenda for sustainable development and the broader and further agenda than the mdgs .", "mdg explicitly stated that hiv / aids , malaria , and tuberculosis will be efficiently controlled at the end of the year 2015 .", "the big three are the diseases that attract priority from sponsors , researchers , and policy makers [ 2 , 3 ] .", "this situation is not the same as ntd , which has not gained any priority from them and even worsened .", "the term of neglected in ntd referred to the fact that these tropical diseases are not being considered as important diseases .", "ntd result in long life 's deformities and handicaps , decrease productivity and economical status , and also end up many social consequences and stigmatization . though ntd is commonly found in tropical countries , it is not identical with tropical diseases .", "poverty as one of ntd determinants frequently occurs in rural area , slums , and marginalized populations living nearby the equator .", "ntd in this area are closely associated with poverty and limited resources , such as clean drinking water access , poor sanitation , and healthy housing .", "ntd may decrease child health , increase the health expenses and risk of ineffective treatment , decrease productivity , and result in education default .", "their potential to spread in developed countries is low , because ntd are closely related with local vector and intermediate host distribution which are specifically associated with geographic region .", "the technology and resource to control , prevent , and eliminate ntd are available , but not for the developing countries . who has included 17 diseases caused by bacteria , virus , and protozoa into ntd ( figure 1 ) .", "helminths caused diseases that dominated ntd : taeniasis , dracunculiasis , echinococcosis , trematodiasis , filariasis , onchocerciasis , schistosomiasis , and soil - transmitted helminthiasis .", "the ministry of health of indonesia reported only five ntd that can be found in indonesia , that is , leprosy , filariasis , schistosomiasis , soil - transmitted helminth , and yaws .", "however , this review describes more than those five diseases and aims to address the magnitude of the problem of ntd in indonesia ."], ["leprosy is an infectious disease is caused by mycobacterium leprae , a road shaped bacteria classified in the same genus as mycobacterium tuberculosis", "leprosy may be complicated with deformity and handicap , which may decrease the ability of the patient to work in daily life .", "leprosy was a serious problem in indonesia at 1980 , with 126,221 cases in 1985 .", "it was noted that the significant improvement in leprosy control was because of massive promotion of leprosy prevention and multidrug therapy ( mdt ) intervention in more than 5,600 primary health centers in indonesia .", "however , the new leprosy case finding in 2000 was not significantly different with year 2013 ( table 1 ) .", "fourteen provinces and 160 districts , which mostly are located in java island , reported prevalence of > 1 per 10,000 population in 2009 .", "leprosy epidemical indicators were significant , such as deformity grade 2 10.5% , leprosy cases in children 12.01% , and 82.43% multibacillary ( mb ) cases [ 10 , 1315 ] .", "the problems of leprosy in indonesia are summarized as follows : early detection.development of more effective treatment.development of more effective vaccine.understanding immunopathogenesis of peripheral nerve damage.management of chronic erythema nodosum leprosum ( enl).deformity and stigmatization.deformity and stigmatization are serious problems among leprosy patients in indonesia .", "survey conducted in subang district ( west java ) ; gresik and malang district ( east java ) ; bone and gowa district ( south sulawesi ) showed that deformity in leprosy patients occurred in 76.7% of patients .", "almost 60% respondents were experienced handicaps during their life , including participating to social activity .", "mdt has significant impact to decrease leprosy prevalence globally . however , there are various substantial operational and technical problems among countries and territory .", "resistance of m. leprae is a serious problem because of limited effective antimicrobes for leprosy .", "resistance against dapsone , that was applied as monotherapy , was initially reported in 1964 at malaysia . since then , resistance to dapsone and other drugs was reported .", "theoretically , combination of more than two drugs with different action mechanism will reduce the probability of resistant in mycobacteria .", "national surveillance of m. leprae resistance against mdt should be established to provide accurate data .", "sporadic and partial reports were not sufficient to measure the problems and establish the programs to reduce the prevalence and spread of resistance against mdt .", "phenotypic testing is the basic method for susceptibility testing of antimicrobial agents . even in the new proof of concept which combines the classical growth - based phenotypic test and dna based approach , pure culture of bacteria", "efforts were documented to culture m. leprae in artificial media and animal models [ 24 , 25 ] .", "mutations in the folp1 , rpob , and gyra genes are responsible for resistance to dapsone , rifampicin , and ofloxacin , respectively . to test the susceptibility of m. leprae against particular antibiotics ,", "screening in north maluku and north sulawesi showed that resistance to dapsone occurs as 0.8% in new cases and 10% in relapse cases , while rifampicin occurs with 3.3% in new cases and 20% in relapse cases .", "there is higher prevalence of dapsone and rifampicin resistance among relapse cases comparing with new cases .", "it was noted that the incidence of resistance against dapsone and rifampicin in indonesia almost did not change between the time monotherapy was introduced and after mdt was recommended by who [ 29 , 30 ] ."], ["yaws is a treponema pallidum subspecies pertenue infection , which may develop as chronic and recurrent disease .", "yaws is nonsexually transmitted treponemal infection , which is latent and asymptomatic for years with positive serology result .", "small portion ( 10% ) of the patients will undergo bone destruction leading to deformities .", "diagnosis of yaws is commonly based on the clinical findings and serology [ 31 , 32 ] .", "confirmatory diagnosis of yaws is using serological test , which includes nontreponemal agglutination tests , such as rapid plasma reagin ( rpr ) and venereal disease research laboratory ( vdrl ) test , and also treponemal tests such as treponema pallidum hemagglutination assay ( tpha ) , treponema pallidum particle agglutination tppa , and fluorescent treponemal antibody - absorption ( fta - abs ) .", "the accuracy of yaws serological test is hampered in the area which syphilis , caused by t. pallidum subspecies pallidum , is endemically circulated .", "yaws was reported in 68 ( 14% ) of 497 districts in indonesia , mostly in the eastern part of indonesia .", "east nusa tenggara province reported 2,800 cases in 2012 , which is scattered at 566 small islands in this region .", "however , it gradually decreases to 5,319 cases in 2011 and 3,476 in 2012 [ 10 , 34 ] .", "there are several obstacles to eradicate yaws in indonesia : geographically the yaws patients are in a remote area including rural and small islands which are not easy to handle , difficulty to give benzathine penicillin for children , and also local political condition that is somehow counterproductive to the programs . who planned to eradicate yaws globally in 2020 with adoption of morges strategies .", "this program intends to introduce a mass treatment in the endemic regions by using azithromycin .", "however , benzathine penicillin can be used as back up in case of azithromycin is not indicated , treatment failure , or in places where azithromycin is not available .", "this treatment followed an active survey every six months to detect and treat the remaining cases [ 32 , 34 ] .", "the ministry of health launched yaws eradication program , aimed to eradicate yaws in 2013 , one year later than who target .", "there were several approaches conducted , such as active screening new cases and contacts , empowering community , improving the capacity of health worker to diagnose and manage yaws , and intersectoral collaboration approaches .", "access to health service is limited in the yaws endemic area , which obstacle the passive screening program .", "reported a survey among health workers in primary health center , elementary school teachers , and parents in muna , southeast sulawesi .", "they were asked about yaws to measure their knowledge about sign and symptoms , causative agent , and therapy for yaws .", "another report from southwest sumba showed clean water and healthy behavior is the risk factor for yaws .", "these reports showed that comprehensive intervention is needed to boost the community participation for yaws eradication .", "reporting system for yaws incidence is very important to support the best intervention for yaws eradication .", "learning the fact that their knowledge about yaws is remarkably low , it may diminish the validity of yaws reporting system .", "insufficiency of this sector may result in delay of yaws eradication and increasing yaws contact population .", "yaws eradication problem in indonesia is summarized as follows : early detection by health workers.geographical problem of endemic area.surveillances and reporting system.inadequate confirmatory laboratory test ."], ["dengue virus consists of four serotypes : denv1 , denv2 , denv3 , and denv4 .", "these viruses are responsible for infectious diseases with width spectrum of clinical manifestation : dengue fever ( df ) , dengue hemorrhagic fever ( dhf ) , and dengue shock syndrome ( dss ) [ 38 , 39 ] .", "dengue virus infection is an arbovirus disease , which in indonesia is primarily transmitted by aedes aegypti and aedes albopictus .", "the incidence of dhf significantly increased since reported initially in 1968 ( 0.05/100.000 ) to 3540/100,000 in 2013 . however , in 2010 , there was an extreme surge of dhf incidence to 86/100,000 .", "the significant increasing of dhf incidence was parallel with decreasing of case fatality rate ( cfr ) , which is 41% in 1968 to 0.73% in 2013 .", "however , it was noticed that from 1999 there was a trend of shifting to adults .", "dhf incidence described here is believed not to be able to draw a clear picture of all denv infections .", ". however , the proportion of those three clinical manifestations still becomes an enigma , although the awareness to the diseases , surveillance programs , diagnostic tools , and management of denv infection in indonesia have been significantly improved .", "it has been realized that controlling adult mosquitoes is prone to fail reducing denv transmission .", "the vector control approach should aim to interrupt the transmission cycle at an early phase through immature mosquito control , which is including larvae and pupae stages .", "immature mosquitoes control is believed to be more effective . the vector control approach is still considered effective while vaccine and prophylaxis remedies continuously develop .", "it seems that the socioecological approach is the best candidate to be implemented in an endemic country , such as indonesia .", "community and intersectoral approach are significantly important fundamentals of integrated public health strategies for dengue vector control [ 47 , 48 ] .", "as dengue infection has wide spectrum of clinical manifestation , the clinical diagnosis is confusing .", "there are guidelines issued by who to respond to the need of applicable tool for clinician : who classification system   and dengue and control study ( denco ) revised clinical management .", "the study concerning the two guidelines showed that in surabaya , indonesia , the denco revised clinical management guideline superior in detection of severe dengue cases .", "however , in a study in semarang , indonesia , the denco guideline has failed to show its superiority .", "it is true that the denco guideline needs to be adjusted in terms of geographic and age - related variations issues .", "there are many factors that may be related with clinical manifestation of dengue virus infection , including human genetics involvement which is still controversially related with population , race , and geography [ 54 , 55 ] .", "several methods are available for laboratory test confirmation of denv infection : virus isolation , genome detection , antigen detection , and antibody detection .", "the first three methods are direct detection methods which were considered as more specific and able to confirm earlier onset of denv infection .", "the antibody detection is widely used in the field because they are easy to perform and less expensive , though it is not as powerful as direct detection especially in secondary infection [ 50 , 56 ] ."], ["rabies is an acute , progressive , incurable viral encephalitis disease that is transmitted from animal , mainly dogs , to human .", "it is caused by rabies virus ( rabv ) , a member of the family rhabdoviridae , genus lyssavirus .", "the highest incidence is concentrated in asia and africa . despite the fact that rabies is 100% vaccine - preventable infectious disease , it potentially threatens over 3 billion people in the world .", "however , poor surveillance , underreporting , frequent misdiagnosis , and poor coordination among sectors may lead to underestimation of the burden of the disease .", "ten countries of asean have been declared rabies - free asean by 2020 in the occasion of the thirty - fourth meeting of the asean ministers on agriculture and forestry ( 34th amaf ) , on september 2012 .", "rabies was reported in 24 provinces out of 34 provinces in indonesia . reported human rabies in indonesia is small ( 206 cases in 2010 and significantly decreased to 119 cases in 2013 ) compared to thousands of cases reported in other countries such as india , china , and other asian countries . to control rabies transmission , indonesian", "government uses one health approach which facilitates the multidisciplinary participation in management of zoonosis diseases .", "however , kalimantan and sulawesi , other islands of indonesia , are the most plausible hypothetical origin of the bali rabv strains .", "totally there were 133 reported human deaths because of rabies in bali from 2008 to september 2011 .", "this significantly decrease of rabid human death within 3 years is a good model of rabies control for other regions or countries .", "it was proposed that this success is mainly because of mass vaccination program for dogs , which is the primary ( 98% ) vector of rabies in bali . however , the number of reported humans bitten by dogs remained over 4,000 per month .", "the preparedness to human rabies should be maintained , as the threat of human rabies escalation is still high ."], ["lymphatic filariasis ( lf ) is caused by worms inhabiting the lymphatics [ 6365 ] .", "approximately 65% of total cases are found in the southeast asian region [ 6668 ] .", "lf cases were found in all provinces of indonesia . in 2009 , there were 11.914 lf cases reported . the number of total cases was steadily reported until 2013 . there were 11.912 cases registered in 2013 .", "lf prevalence in indonesia varied from 0.5 to 27.6% , in which the highest rates were found in maluku , papua , west papua , east nusa tenggara , and north maluku provinces .", "three lymphatic parasites are prevalently circulating in indonesia : wuchereria bancrofti , brugia malayi , and brugia timori [ 69 , 70 ] , which are transmitted by mosquitoes of five genera ", "the site of adult - worm parasitism is within the lymphatic vessels , most commonly involving the extremities and male genitals [ 64 , 65 , 70 ] .", "the disease predominantly afflicts poor people in both urban and rural areas with limited resources condition , where mosquitoes as a vector might be found in high density [ 64 , 68 ] . who launched global program to eliminate lymphatic filariasis ( gpelf ) , which relies on mass drug administration ( mda ) approach [ 68 , 71 ] .", "the main goal of the program is to hamper transmission of disease between mosquitoes and human beings , mainly through mass drug distribution of diethylcarbamazine ( 6  mg / kg ) or ivermectin ( 150200  g / kg ) combined with albendazole ( 400  mg ) [ 68 , 71 ] .", "it is recommended that all people at risk are involved in this program , since patients with asymptomatic infection may have abnormal lymphatics , and that early treatment may prevent subsequent lymphatic damage [ 66 , 69 ] .", "the efficacy of six annual rounds of mda was studied in alor island , eastern part of indonesia .", "microfilaria rates of b. timori decreased significantly after mda intervention , from 26% to 0.17% .", "the prevalence of filarial - specific igg4 antibodies was significantly decreased from 80% to 6% .", "the lf national plan has implemented mda campaigns in 2002 . however , due to financial and human resource constraints , districts often provide only partial coverage of the at - risk population within the district . in 2009 ,", "the challenge of mda implementation in indonesia is not the efficacy of the drugs which were given to the community .", "it seems that the infrastructure is the key of the implementation , which includes the availability of transportation and physical access to the target population , reliable data bases , and competence health workers .", "the other challenge was to gain the trust from the community member to boost the compliance of drug administration .", "currently , mda has been scaled up in a geographically scattered way to address high prevalence areas and political needs .", "medical professionals , donors , universities , and ngos have played a critical role in finding new cases , assessing disease burden and supporting trainings and mda campaigns .", "prevention of lf depends on mosquito vectors control , which has had limited success because development of mosquitoes resistance against insecticides .", "urbanization of vast areas of tropical asia , including indonesia , has resulted in a concomitant rise in the prevalence of both w. bancrofti and b. malayi varieties of filariasis , carried by mosquitoes that breed in nonsylvatic habitats [ 68 , 73 ] ."], ["schistosomiasis is a chronic water - borne infection caused by trematodes schistosoma , mainly found in developing countries in africa , south america , the caribbean , the middle east , and asia [ 69 , 74 , 75 ] . in indonesia", "it is found in three isolated areas of central sulawesi province , namely , lindu , napu , and bada valleys . except for schistosoma haematobium that is responsible for urinary tract disease , the human schistosomes primarily affect the intestine and liver .", "clinical manifestations of the disease are recognized as fever with dysenteric symptoms and loss of appetite , as well as physical growth and cognitive delay in children [ 69 , 77 , 78 ] .", "disease prevalence fluctuated between 0.3% and 4.8% in napu valley and between 0.8% and 3.6% in lindu valley .", "however , the prevalence of schistosomiasis in both areas tended to increase during the 20082011 period . the parasite transmission cycle involved domestic and wild animals as reservoir [ 76 , 79 ] . in 2012 , who approved the goal of eliminating schistosomiasis in endemic countries .", "it focused on improving sanitary conditions and large - scale distribution of the antiparasitic drug , praziquantel to high - risk target groups , such as school - age children , child bearing age women , and individuals involved in frequent contact with contaminated fresh water .", "praziquantel is well - tolerated , associated with few side effects , and has a very high therapeutic index .", "moreover , a single dose praziquantel administration is usually sufficient to kill all adult worms [ 69 , 78 ] .", "schistosomiasis control in indonesia has faced many difficulties even though the endemic areas are very limited [ 79 , 81 ] .", "the core strategy of mda should be coupled with education to the local community , rat and snail surveillance , and support to the environmental management programs including introduction of latrines and suitable water sources . in the future", ", there should be emphasis to understand the social dynamics and social change related to schistosomiasis , which may provide more information about concrete issues to control transmission of schistosomiasis .", "lack of intersectoral coordination and collaboration may have occurred , possibly leading to increase transmission and reinfection rates , and be prone to control failure ."], ["soil - transmitted helminthiasis ( sth ) is an infection with one or more intestinal parasitic worms : roundworms ( ascaris lumbricoides ) , whipworms ( trichuris trichiura ) , or hookworms ( necator americanus and ancylostoma duodenale )", ". the tropical climate of indonesia is highly favourable to support the circulation of soil - transmitted helminths in the country .", "there are sporadic reports regarding the prevalence of the sth which come from scattered part of the country .", "the prevalence of sth was reported ranging from 40 to 70% in 80s to 90s .", "however , the estimated prevalence of sth was decreased in 2005 : ascaris ( 15.2% ) , trichuris ( 12.9% ) , and hookworms ( 8.4% ) .", "the survey conducted by ministry of health in elementary schools located in 33 provinces of indonesia showed prevalence of sth was 31.8% .", "india ( 64% ) and indonesia ( 16% ) together are contributing 80% of the regional 's burden .", "drugs used for deworming , albendazole , and mebendazole are effective and inexpensive to control sth transmission and reinfection .", "the mda programme for elimination of lf which is implemented by using combination of albendazole with ivermectin or diethylcarbamazine seems to have synergistic effect to decrease the prevalence of sth .", "the program targeted preschool and elementary school age . however , until this moment the country still struggles to combat sth . in conclusion , there were efforts and programs concerning ntd that were planed and implemented in indonesia .", "many factors may contribute to the rendering of ntd elimination in indonesia , such as high population , wide range geographic of the archipelago , and limited resources . by the end of 2015 ntd"]]}
{"article_id": "PMC4905913", "article_text": ["walking is one of the recommended modes of exercise for obese individuals because of its \n merits in terms of safety , accessibility , and popularity , as well as proven efficacy in \n weight management1 , 2 .", "however , walking over a terrain of repetitive uphill and downhill \n inclines in activities such as mountain climbing can cause excessive fatigue and muscle \n damage3 .", "in particular , repetitive \n eccentric contractions of the lower limb muscles during downhill walking places sarcomeres \n under excessive tensile stress .", "this stress can destroy sarcomeres by extending them beyond \n their normal length , sometimes involving a local inflammatory reaction , and can thereby lead \n to delayed onset muscle soreness or exercise - induced muscle damage .", "the indexes reflecting \n such conditions are serum creatine kinase ( ck ) and lactate dehydrogenase ( ldh ) levels4 , 5 .", "it \n has been reported that the use of a pole during walking over an uneven terrain could improve \n gait stability and balance , and that its use during downhill terrain walking can reduce the \n load and stress on the lower limbs ( such as the ankle , knee , and hip joints)6,7,8 .", "however , previous studies on the effects of \n a pole during downhill walking have been limited to the kinetic aspects such as the \n reduction of knee joint forces or the load on the lower extremity joints .", "meanwhile , it has been suggested that increases in peripheral cartilage oligomeric matrix \n protein ( comp ) levels after exercise could serve as a biomarker of cartilage degradation and \n damage9 , and pruksakorn et al .", "in addition , according to a comparative study by \n andriacchi et al . that measured the maximum flexion of the lower extremity joints during \n various routine activities , the flexion of the knee joint was approximately 4 times greater \n during walking down stairs than during walking over level ground11 . taken together , such previous reports suggest that the \n use of a pole during downhill walking may alleviate the burden on lower extremity joint \n cartilages ; however , thus far , there has been no report examining this potential \n benefit .", "accordingly , this study aimed to investigate the effect of the use of a trekking pole on \n muscle and cartilage damage and fatigue during downhill walking .", "the study specifically \n examined obese women who had less muscle mass than men and whose heavy weights were likely \n to increase the burden on their lower limb muscles and joints during walking .", "eight obese women ( average age of 21.1  1.8  years , average height of 164.8  4.9  cm , \n average weight of 61.8  7.7  kg , average body fat percentage of 33.9  1.4% , and average \n resting heart rate of 69.6  3.7 beats / min ) with body fat percentages ranging from 30.0 to \n 36.1 volunteered as subjects for this study .", "the study conformed to the standards set by the \n latest revision of the declaration of helsinki , and all subjects read and signed a written \n informed consent statement consistent with the guidelines of the department of physical \n education at yonsei university .", "height and body composition were measured using a stadiometer ( seca213 ; seca , hamburg , \n germany ) and a bioimpedance analysis ( bia ) device ( inbody720 ; biospace , seoul , korea ) , \n respectively .", "resting heart rate was measured in a seated position using a heart rate \n monitor ( polar a5 ; polar , kempele , finland ) .", "each of the eight subjects participated in a total of two trials : one np trial ( walking \n without using a trekking pole ) , and one tp trial ( walking using a trekking pole ) . with the \n treadmill angle set to a decline of 15% based on a study by perrey and fabre12 ,", "the participants were subjected to 30 \n minutes of walking at an exercise intensity of 50% of their heart rate reserve .", "the \n experiment used a crossover design to minimize the adaption from the repetitive exercise \n trials , and each trial was separated by 7 days to avoid any transient effects on the \n physiological and psychological conditions of the subjects .", "an adjustable - length trekking \n pole ( 6342011 ; leki , hamburg , germany ) was used , and the length was initially set at 70% of \n the user s height so that the elbow angle was maintained at 90 when the user was standing \n on level ground . when the pole was used for downhill walking ,", "its length was increased by \n approximately 5  cm . using a 22-gauge needle ,", "a serum separator tube ( becton dickinson , franklin lakes , nj , \n usa ) , and a ethylenediamine tetra - acetic acid tube ( becton dickinson ) , 7  ml of blood was \n collected from the antecubital vein of each subject at the pre - walking baseline ( pwb ) , \n immediately after walking ( iaw ) , and 2 hours post - walking ( 2hpw ) . collected blood samples \n were centrifuged for 15 minutes at 3,000  rpm and then stored at 80  c until analysis .", "the \n serum ck and ldh levels were determined using a clinical chemistry analyzer ( ektachem \n dtscii ; eastman kodak , rochester , ny , usa ) .", "the serum comp levels were determined with an \n enzyme - linked immunosorbent assay ( elisa ) using a commercially available human \n comp elisa kit ( anamar ab , lund , sweden ) .", "the absorbance was measured at \n 450  nm with a microplate reader ( molecular device , palo alto , ca , usa ) .", "the plasma lactate \n levels were determined using a clinical chemistry analyzer ( ektachem dt 60 ; eastman kodak , \n rochester , ny , usa ) .", "data are presented as the mean  standard deviation ( sd ) unless otherwise stated . \n for identifying differences in normally distributed results ,", "when significant group by time interactions occurred , simple \n main effects were assessed using one - way anova and independent t - tests .", "the biomarker levels measured are presented in table \n 1table 1.peripheral biomarkers after downhill walking with the use of trekking poles ( tp ) \n and without their use ( np)variabletrialpwbiaw2hpwck(u / l)np260.3  39.1349.1  37.9302.9  42.0tp256.1  42.0312.5  44.9270.9 ", "34.5ldh(u / l)np351.5  77.8453.4  70.6377.0  84.0tp354.9  79.3385.1  71.2363.5  64.6comp(u / l)np9.6  2.013.6  3.812.0  2.8tp9.9  2.213.2  3.610.0 ", "np : no trekking pole trial ; tp : trekking pole trial ; \n pwb : pre - walking baseline ; iaw : immediately after walking ; 2hpw : 2 hours post - walking ; \n * p<0.05 vs. pwb .. the serum ck , serum ldh , serum comp , and plasma lactate levels were \n significantly increased iaw when compared with those at the pwb in trials np and tp \n ( p<0.05 ) .", "in addition , in trial np , the serum ck , serum ldh , and serum comp levels were \n significantly increased at 2hpw when compared with those at the pwb ( p<0.05 ) .", "np : no trekking pole trial ; tp : trekking pole trial ; \n pwb : pre - walking baseline ; iaw : immediately after walking ; 2hpw : 2 hours post - walking ; \n * p<0.05 vs. pwb .", "exercise - induced muscle damage increases in situations involving eccentric muscle \n contractions , such as during downhill walking and/or running4 .", "this study \n measured the levels of serum ck , ldh , and comp to investigate the effect of the use of a \n trekking pole on muscle and cartilage damage during downhill walking .", "the results \n demonstrated that the levels of ck , ldh , and comp were significantly higher iaw and 2hpw \n than at the pwb in the case of the np trials . in the case of the tp trials , however , the \n levels 2hpw were significantly lower than those for the np trials and actually returned to \n their level at the pwb", ". the rationale could be that the use of a trekking pole alleviated \n the temporary muscle and cartilage damage induced after downhill walking .", "this result is in \n accordance with those of previous studies ; howatson et al . showed significantly lower serum \n ck levels at 24 hours after mountain climbing in an experimental group that used trekking \n poles than in the control group , suggesting that the use of trekking poles could alleviate \n muscle damage during recovery3 . in \n addition , exercise - induced increases in joint load have been shown to cause a temporary \n increase in serum comp levels after walking or running by promoting catabolism , in \n particular , in cartilages such as the articular cartilage in the knee9 , 13 .", "bohne and \n abendroth - smith suggested that the use of a trekking pole during downhill walking could be \n effective for reducing such loads on the lower extremity joints7 . according to the results of some previous studies ,", "the use \n of a trekking pole could reduce lower limb joint forces by as much as 25% , enabling a 13  kg \n reduction of load per stride during downhill walking3 , \n 14 .", "however , as for the plasma lactate levels in this study , no significant difference was \n observed with the use of a trekking pole .", "the rationale is that although the use of a \n trekking pole reduced forces on the lower limbs , it increased the activity of the upper \n limbs and consequently maintained a similar fatigue level in the whole body .", "this \n interpretation is supported by the report that plasma lactate concentration was a biomarker \n reflecting peripheral muscle fatigue during exercise15 , as well as by the suggestion of fritschi et al . that , although the \n use of a pole during walking could reduce vertical knee joint forces , it could increase \n upper limb muscle activation16 . in conclusion ,", "downhill walking can cause muscle and cartilage damage , and it is suggested \n that the use of a trekking pole can reduce temporary muscle and cartilage damage after \n downhill walking ."], "abstract_text": ["<S> [ purpose ] this study investigated the effect of the use of trekking poles on muscle and \n cartilage damage and fatigue during downhill walking in obese women . </S>", "<S> [ subjects and \n methods ] subjects included eight obese women who had a body fat percentage greater than \n 30 . </S>", "<S> subjects performed downhill walking without a trekking pole ( np ) and with a trekking \n pole ( tp ) at 50% heart rate reserve for 30 minutes on a treadmill . the treadmill was set \n at a 15% downhill declination . </S>", "<S> blood samples were collected to examine muscle damage \n ( serum creatine kinase [ ck ] and lactate dehydrogenase [ ldh ] levels ) , cartilage damage \n ( serum cartilage oligomeric matrix protein [ comp ] levels ) , and fatigue ( plasma lactate \n levels ) at the pre - walking baseline ( pwb ) , immediately after walking ( iaw ) , and 2 hours \n post - walking ( 2hpw ) . [ results ] the ck , ldh , comp , and lactate levels were significantly \n increased iaw when compared with those at the pwb in both trials . </S>", "<S> in addition , in the np \n trial , the ck , ldh , and comp levels were significantly increased at 2hpw when compared \n with those at the pwb . </S>", "<S> [ conclusion ] downhill walking can cause muscle and cartilage \n damage , and our results suggest that the use of a trekking pole can reduce temporary \n muscle and cartilage damage after downhill walking . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["walking is one of the recommended modes of exercise for obese individuals because of its \n merits in terms of safety , accessibility , and popularity , as well as proven efficacy in \n weight management1 , 2 .", "however , walking over a terrain of repetitive uphill and downhill \n inclines in activities such as mountain climbing can cause excessive fatigue and muscle \n damage3 .", "in particular , repetitive \n eccentric contractions of the lower limb muscles during downhill walking places sarcomeres \n under excessive tensile stress .", "this stress can destroy sarcomeres by extending them beyond \n their normal length , sometimes involving a local inflammatory reaction , and can thereby lead \n to delayed onset muscle soreness or exercise - induced muscle damage .", "the indexes reflecting \n such conditions are serum creatine kinase ( ck ) and lactate dehydrogenase ( ldh ) levels4 , 5 .", "it \n has been reported that the use of a pole during walking over an uneven terrain could improve \n gait stability and balance , and that its use during downhill terrain walking can reduce the \n load and stress on the lower limbs ( such as the ankle , knee , and hip joints)6,7,8 .", "however , previous studies on the effects of \n a pole during downhill walking have been limited to the kinetic aspects such as the \n reduction of knee joint forces or the load on the lower extremity joints .", "meanwhile , it has been suggested that increases in peripheral cartilage oligomeric matrix \n protein ( comp ) levels after exercise could serve as a biomarker of cartilage degradation and \n damage9 , and pruksakorn et al .", "in addition , according to a comparative study by \n andriacchi et al . that measured the maximum flexion of the lower extremity joints during \n various routine activities , the flexion of the knee joint was approximately 4 times greater \n during walking down stairs than during walking over level ground11 . taken together , such previous reports suggest that the \n use of a pole during downhill walking may alleviate the burden on lower extremity joint \n cartilages ; however , thus far , there has been no report examining this potential \n benefit .", "accordingly , this study aimed to investigate the effect of the use of a trekking pole on \n muscle and cartilage damage and fatigue during downhill walking .", "the study specifically \n examined obese women who had less muscle mass than men and whose heavy weights were likely \n to increase the burden on their lower limb muscles and joints during walking ."], ["eight obese women ( average age of 21.1  1.8  years , average height of 164.8  4.9  cm , \n average weight of 61.8  7.7  kg , average body fat percentage of 33.9  1.4% , and average \n resting heart rate of 69.6  3.7 beats / min ) with body fat percentages ranging from 30.0 to \n 36.1 volunteered as subjects for this study .", "the study conformed to the standards set by the \n latest revision of the declaration of helsinki , and all subjects read and signed a written \n informed consent statement consistent with the guidelines of the department of physical \n education at yonsei university .", "height and body composition were measured using a stadiometer ( seca213 ; seca , hamburg , \n germany ) and a bioimpedance analysis ( bia ) device ( inbody720 ; biospace , seoul , korea ) , \n respectively .", "resting heart rate was measured in a seated position using a heart rate \n monitor ( polar a5 ; polar , kempele , finland ) .", "each of the eight subjects participated in a total of two trials : one np trial ( walking \n without using a trekking pole ) , and one tp trial ( walking using a trekking pole ) . with the \n treadmill angle set to a decline of 15% based on a study by perrey and fabre12 ,", "the participants were subjected to 30 \n minutes of walking at an exercise intensity of 50% of their heart rate reserve .", "the \n experiment used a crossover design to minimize the adaption from the repetitive exercise \n trials , and each trial was separated by 7 days to avoid any transient effects on the \n physiological and psychological conditions of the subjects .", "an adjustable - length trekking \n pole ( 6342011 ; leki , hamburg , germany ) was used , and the length was initially set at 70% of \n the user s height so that the elbow angle was maintained at 90 when the user was standing \n on level ground . when the pole was used for downhill walking ,", "its length was increased by \n approximately 5  cm . using a 22-gauge needle ,", "a serum separator tube ( becton dickinson , franklin lakes , nj , \n usa ) , and a ethylenediamine tetra - acetic acid tube ( becton dickinson ) , 7  ml of blood was \n collected from the antecubital vein of each subject at the pre - walking baseline ( pwb ) , \n immediately after walking ( iaw ) , and 2 hours post - walking ( 2hpw ) . collected blood samples \n were centrifuged for 15 minutes at 3,000  rpm and then stored at 80  c until analysis .", "the \n serum ck and ldh levels were determined using a clinical chemistry analyzer ( ektachem \n dtscii ; eastman kodak , rochester , ny , usa ) .", "the serum comp levels were determined with an \n enzyme - linked immunosorbent assay ( elisa ) using a commercially available human \n comp elisa kit ( anamar ab , lund , sweden ) .", "the absorbance was measured at \n 450  nm with a microplate reader ( molecular device , palo alto , ca , usa ) .", "the plasma lactate \n levels were determined using a clinical chemistry analyzer ( ektachem dt 60 ; eastman kodak , \n rochester , ny , usa ) .", "data are presented as the mean  standard deviation ( sd ) unless otherwise stated . \n for identifying differences in normally distributed results ,", "when significant group by time interactions occurred , simple \n main effects were assessed using one - way anova and independent t - tests ."], ["the biomarker levels measured are presented in table \n 1table 1.peripheral biomarkers after downhill walking with the use of trekking poles ( tp ) \n and without their use ( np)variabletrialpwbiaw2hpwck(u / l)np260.3  39.1349.1  37.9302.9  42.0tp256.1  42.0312.5  44.9270.9 ", "34.5ldh(u / l)np351.5  77.8453.4  70.6377.0  84.0tp354.9  79.3385.1  71.2363.5  64.6comp(u / l)np9.6  2.013.6  3.812.0  2.8tp9.9  2.213.2  3.610.0 ", "np : no trekking pole trial ; tp : trekking pole trial ; \n pwb : pre - walking baseline ; iaw : immediately after walking ; 2hpw : 2 hours post - walking ; \n * p<0.05 vs. pwb .. the serum ck , serum ldh , serum comp , and plasma lactate levels were \n significantly increased iaw when compared with those at the pwb in trials np and tp \n ( p<0.05 ) .", "in addition , in trial np , the serum ck , serum ldh , and serum comp levels were \n significantly increased at 2hpw when compared with those at the pwb ( p<0.05 ) .", "np : no trekking pole trial ; tp : trekking pole trial ; \n pwb : pre - walking baseline ; iaw : immediately after walking ; 2hpw : 2 hours post - walking ; \n * p<0.05 vs. pwb ."], ["exercise - induced muscle damage increases in situations involving eccentric muscle \n contractions , such as during downhill walking and/or running4 .", "this study \n measured the levels of serum ck , ldh , and comp to investigate the effect of the use of a \n trekking pole on muscle and cartilage damage during downhill walking .", "the results \n demonstrated that the levels of ck , ldh , and comp were significantly higher iaw and 2hpw \n than at the pwb in the case of the np trials . in the case of the tp trials , however , the \n levels 2hpw were significantly lower than those for the np trials and actually returned to \n their level at the pwb", ". the rationale could be that the use of a trekking pole alleviated \n the temporary muscle and cartilage damage induced after downhill walking .", "this result is in \n accordance with those of previous studies ; howatson et al . showed significantly lower serum \n ck levels at 24 hours after mountain climbing in an experimental group that used trekking \n poles than in the control group , suggesting that the use of trekking poles could alleviate \n muscle damage during recovery3 . in \n addition , exercise - induced increases in joint load have been shown to cause a temporary \n increase in serum comp levels after walking or running by promoting catabolism , in \n particular , in cartilages such as the articular cartilage in the knee9 , 13 .", "bohne and \n abendroth - smith suggested that the use of a trekking pole during downhill walking could be \n effective for reducing such loads on the lower extremity joints7 . according to the results of some previous studies ,", "the use \n of a trekking pole could reduce lower limb joint forces by as much as 25% , enabling a 13  kg \n reduction of load per stride during downhill walking3 , \n 14 .", "however , as for the plasma lactate levels in this study , no significant difference was \n observed with the use of a trekking pole .", "the rationale is that although the use of a \n trekking pole reduced forces on the lower limbs , it increased the activity of the upper \n limbs and consequently maintained a similar fatigue level in the whole body .", "this \n interpretation is supported by the report that plasma lactate concentration was a biomarker \n reflecting peripheral muscle fatigue during exercise15 , as well as by the suggestion of fritschi et al . that , although the \n use of a pole during walking could reduce vertical knee joint forces , it could increase \n upper limb muscle activation16 . in conclusion ,", "downhill walking can cause muscle and cartilage damage , and it is suggested \n that the use of a trekking pole can reduce temporary muscle and cartilage damage after \n downhill walking ."]]}
{"article_id": "PMC4055825", "article_text": ["periampullary diverticula ( pad ) refer to extraluminal outpouchings of duodenal mucosa that develop within the radius of 2 to 3 cm from the ampulla of vater ( 1 ) .", "pad are largely asymptomatic but they sometimes can cause both pancreaticobiliary and non - pancreaticobiliary complications . rarely , obstructive jaundice can develop secondary to pad in the absence of choledocholithiasis or tumor and is termed lemmel 's syndrome ( 2 ) .", "recently , the authors experienced an unusual case of abdominal pain and obstructive jaundice due to extrinsic compression of mid common bile duct ( cbd ) by distended pad filled with pus - like material as a result of impacted intradiverticular enterolith at the pad orifice .", "an 81-yr - old woman presented to the emergency department on august 3 , 2012 with nausea , vomiting , fever ( 38.4 ) , and diffuse abdominal pain of 4 days ' duration .", "she had undergone subtotal gastrectomy with billroth ii anastomosis due to peptic ulcer perforation 10 yr ago . on physical examination", ", there was tenderness on her right upper quadrant but murphy 's sign was equivocal . her white blood count was increased to 11,170/l ( neutrophil 83.0% ) and crp was elevated to 2.392 mg / dl . the results of her liver function test were as follows : total bilirubin , 2.37 mg / dl ; aspartate aminotransferase , 88", "iu / l ; alkaline phosphatase , 349 iu / l ; and -glutamyl transpeptidase , 571 iu / l . to evaluate the cause of diffuse abdominal pain with liver enzyme elevation in a cholestatic pattern , abdominal ct scan was taken .", "the axial images of the ct scan demonstrated distal cbd stone with upstream bile duct dilatation ( fig .", "however , on coronal reconstructed images , the stone was not located within the bile duct but inside the pad and the distended diverticulum was compressing the mid cbd ( fig .", "1b , 1c ) . magnetic resonance cholangiopancreatography ( mrcp ) also revealed mid cbd compression and absence of choledocholithiasis ( fig .", "these findings were confirmed on endoscopic retrograde cholangiopancreatography ( ercp ) which showed normal biliary orifice ( fig .", "2a ) with impacted dark brown pigment stone ( henceforth enterolith ) at the pad orifice ( fig .", "when the enterolith was pushed into the diverticulum by cannulation catheter and contrast dye was injected ( fig .", "2c , 3a ) , old blood clots and pus - like fluid gushed out from the opening ( fig .", "biliary cannulation combined with endoscopic sphincterotomy ( est ) was also performed to explore the cbd for other possible causes of obstructive jaundice but no stone , stricture or obstruction by tumor could be found . on endoscopic nasobiliary drainage ( enbd )", "tubogram , stenosis at mid cbd was also shown to be resolved , likely because the pad had been decompressed ( fig .", "3b ) . after confirming that no other pathology was present , intradiverticular enterolith was crushed and removed by dormia basket on the following day ( fig .", "the patient no longer complained of abdominal pain , her liver enzyme was normalized , and she was discharged without any complication .", "she had been well until 6 months after enterolith removal , when the patient visited the outpatient clinic with vague abdominal discomfort .", "laboratory examination only revealed slightly increased total bilirubin to 1.96 mg / dl , but a large cbd stone was found on abdominal ct scan ( fig .", "when the cbd stone was removed by ercp , the stone proved to be brown pigment sludge stone that typically forms in the presence of ascending infection ( fig .", "follow - up ercp was performed again after 6 months but there was no recurrence of cbd stone or enterolith .", "diverticula of the gastrointestinal tract are outpouchings of all or part of the intestinal wall which can occur anywhere throughout the alimentary tract .", "the most common site of gastrointestinal diverticula is colon followed by duodenum , which was first described by chomel in 1710 ( 3 ) .", "the detection rate of duodenal diverticula ranges from 1% to 27% depending on the diagnostic modalities used and the average age at the time of diagnosis ( 1 ) . among duodenal diverticula", ", pad is the most common type comprising about 70% to 75% of all duodenal diverticula ( 1 ) .", "most pad are asymptomatic but complications can occur in about 5% of cases and they include bleeding , perforation , diverticulitis , pancreatitis , choledocholithiasis , cholangitis , jaundice , enterolith or bezoar formation , intestinal obstruction , etc . among these complications ,", "hepatocholangiopancreatic disease can seldomly occur in the absence of choledocholithiasis and is termed lemmel 's syndrome ( 2 ) .", "first , diverticulitis or direct mechanical irritation of pad may cause chronic inflammation of ampulla and lead to chronic fibrosis of papilla ( papillitis chronica fibrosa ) ( 4 ) .", "third , distal cbd or ampulla can be directly compressed mechanically by pad that is usually filled with enterolith or bezoar ( 6 , 7 ) . in our case ,", "enterolith that formed within the pad did not directly compress the distal cbd but it obstructed the pad orifice instead .", "this obstruction combined with inflammation of the diverticulum and collection of pus - like material within the obstructed pad seems to have expanded the pad with resultant extrinsic compression of mid cbd ( fig .", "therefore , the enterolith , bezoar , or food material within the pad is frequently evacuated and thus , the symptom could be intermittent .", "however , pad in our case had a narrow opening , likely due to repeated inflammation of the pad , and this seems to have hindered the clearance of entrapped enterolith out into the duodenal lumen .", "enterolith formation within the duodenal diverticula is known to be facilitated in the static environment such as a blind loop after gastrectomy or proximal portion of stricture formed by crohn 's disease or tuberculosis ( 8) . in our case , blind loop created by billroth ii anastomosis seems to have provided a static environment favoring enterolith formation within the pad . during enterolith removal", ", cbd was also explored to search for other possible source of obstructive jaundice such as cbd stone since primary biliary stone is known to occur more frequently in the presence of pad ( 9 , 10 ) .", "one possible mechanism behind increased occurrence of primary cbd stone in these patients involves colonization and overgrowth of -glucuronidase producing bacteria within the pad that spread into the bile duct , which in turn leads to deconjugation of bilirubin glucuronides and eventually results in precipitation of calcium bilirubinate gallstones ( 11 ) .", "although cbd was explored in our case , no other etiology of obstructive jaundice could be identified other than extrinsic compression by distended pad . however , primary bile duct stone newly developed on follow - up ercp performed 6 months later ( fig .", "the most plausible explanation is that est performed during cbd exploration at the time of enterolith removal has permitted the occurrence of ascending infection with resultant brown pigment stone formation .", "diagnosing lemmel 's syndrome could be challenging , but being aware of this condition is important to avoid mismanagement and it begins with identification of pad .", "scan and mrcp , pad appear as thin - walled cavitary lesions situated on the medial wall of the duodenum 2nd portion that typically contain gas .", "however , pad are sometimes filled with fluid and can frequently be mistaken for pancreatic pseudocyst , pancreatic abscess , cystic neoplasm in the pancreas head or even metastatic lymph node ( 12 , 13 ) . therefore , high index of suspicion is mandatory to arrive at a correct diagnosis in these patients . in our case ,", "enterolith within the pad on axial images was at first mistaken for distal cbd stone due to its distal location combined with upstream dilatation of the bile duct ( fig .", "however , upon careful scrutinization of the coronal reconstructed images , it became evident that the stone was located within the pad ( fig .", "treatment is generally not recommended in asymptomatic patients or would be conservative management in pauci - symptomatic patients . nevertheless , since most patients with lemmel 's syndrome present with symptoms related to biliary obstruction ( i.e. jaundice , abdominal pain , and cholangitis ) as a result of extrinsic compression of cbd , some form of treatment is advocated .", "therapeutic options in this situation run the gamut from endoscopic extraction of entrapped material , extracorporeal shock wave lithotripsy to surgery ( diverticulectomy or biliodigestive anastomosis ) ( 7 , 14 , 15 ) .", "the patient in our case was also successfully treated endoscopically by fragmenting and removing enterolith using a dormia basket .", "it should be kept in mind that not all forms of lemmel 's syndrome are caused by extrinsic compression of cbd by bulging pad .", "if the underlying mechanism of lemmel 's syndrome is likely to be due to papillitis chronica fibrosa or sphincter of oddi dysfunction as mentioned above , the simplest and the most appropriate management would be to perform est ( 16 ) . in conclusion , lemmel 's syndrome is a rare cause of obstructive jaundice that should be included in the differential diagnosis of biliary obstruction when pad is present . maintaining a high index of suspicion is imperative to establish an accurate diagnosis since it can mimic other cystic or solid lesions around the pancreas head .", "symptomatic patient can be successfully managed endoscopically in many instances but recourse to surgical management would be necessary in selected cases ."], "abstract_text": ["<S> duodenal diverticula are detected in up to 27% of patients undergoing upper gastrointestinal tract evaluation with periampullary diverticula ( pad ) being the most common type . </S>", "<S> although pad usually do not cause symptoms , it can serve as a source of obstructive jaundice even when choledocholithiasis or tumor is not present . </S>", "<S> this duodenal diverticulum obstructive jaundice syndrome is called lemmel 's syndrome . </S>", "<S> an 81-yr - old woman came to the emergency room with obstructive jaundice and cholangitis . </S>", "<S> abdominal ct scan revealed stony opacity on distal cbd with cbd dilatation . </S>", "<S> ercp was performed to remove the stone . </S>", "<S> however , the stone was not located in the cbd but rather inside the pad . </S>", "<S> after removal of the enterolith within the pad , all her symptoms resolved . </S>", "<S> recognition of this condition is important since misdiagnosis could lead to mismanagement and therapeutic delay . </S>", "<S> lemmel 's syndrome should always be included as one of the differential diagnosis of obstructive jaundice when pad are present.graphical abstract </S>"], "labels": null, "section_names": ["Graphical Abstract", "INTRODUCTION", "CASE DESCRIPTION", "DISCUSSION"], "sections": [[""], ["periampullary diverticula ( pad ) refer to extraluminal outpouchings of duodenal mucosa that develop within the radius of 2 to 3 cm from the ampulla of vater ( 1 ) .", "pad are largely asymptomatic but they sometimes can cause both pancreaticobiliary and non - pancreaticobiliary complications . rarely , obstructive jaundice can develop secondary to pad in the absence of choledocholithiasis or tumor and is termed lemmel 's syndrome ( 2 ) .", "recently , the authors experienced an unusual case of abdominal pain and obstructive jaundice due to extrinsic compression of mid common bile duct ( cbd ) by distended pad filled with pus - like material as a result of impacted intradiverticular enterolith at the pad orifice ."], ["an 81-yr - old woman presented to the emergency department on august 3 , 2012 with nausea , vomiting , fever ( 38.4 ) , and diffuse abdominal pain of 4 days ' duration .", "she had undergone subtotal gastrectomy with billroth ii anastomosis due to peptic ulcer perforation 10 yr ago . on physical examination", ", there was tenderness on her right upper quadrant but murphy 's sign was equivocal . her white blood count was increased to 11,170/l ( neutrophil 83.0% ) and crp was elevated to 2.392 mg / dl . the results of her liver function test were as follows : total bilirubin , 2.37 mg / dl ; aspartate aminotransferase , 88", "iu / l ; alkaline phosphatase , 349 iu / l ; and -glutamyl transpeptidase , 571 iu / l . to evaluate the cause of diffuse abdominal pain with liver enzyme elevation in a cholestatic pattern , abdominal ct scan was taken .", "the axial images of the ct scan demonstrated distal cbd stone with upstream bile duct dilatation ( fig .", "however , on coronal reconstructed images , the stone was not located within the bile duct but inside the pad and the distended diverticulum was compressing the mid cbd ( fig .", "1b , 1c ) . magnetic resonance cholangiopancreatography ( mrcp ) also revealed mid cbd compression and absence of choledocholithiasis ( fig .", "these findings were confirmed on endoscopic retrograde cholangiopancreatography ( ercp ) which showed normal biliary orifice ( fig .", "2a ) with impacted dark brown pigment stone ( henceforth enterolith ) at the pad orifice ( fig .", "when the enterolith was pushed into the diverticulum by cannulation catheter and contrast dye was injected ( fig .", "2c , 3a ) , old blood clots and pus - like fluid gushed out from the opening ( fig .", "biliary cannulation combined with endoscopic sphincterotomy ( est ) was also performed to explore the cbd for other possible causes of obstructive jaundice but no stone , stricture or obstruction by tumor could be found . on endoscopic nasobiliary drainage ( enbd )", "tubogram , stenosis at mid cbd was also shown to be resolved , likely because the pad had been decompressed ( fig .", "3b ) . after confirming that no other pathology was present , intradiverticular enterolith was crushed and removed by dormia basket on the following day ( fig .", "the patient no longer complained of abdominal pain , her liver enzyme was normalized , and she was discharged without any complication .", "she had been well until 6 months after enterolith removal , when the patient visited the outpatient clinic with vague abdominal discomfort .", "laboratory examination only revealed slightly increased total bilirubin to 1.96 mg / dl , but a large cbd stone was found on abdominal ct scan ( fig .", "when the cbd stone was removed by ercp , the stone proved to be brown pigment sludge stone that typically forms in the presence of ascending infection ( fig .", "follow - up ercp was performed again after 6 months but there was no recurrence of cbd stone or enterolith ."], ["diverticula of the gastrointestinal tract are outpouchings of all or part of the intestinal wall which can occur anywhere throughout the alimentary tract .", "the most common site of gastrointestinal diverticula is colon followed by duodenum , which was first described by chomel in 1710 ( 3 ) .", "the detection rate of duodenal diverticula ranges from 1% to 27% depending on the diagnostic modalities used and the average age at the time of diagnosis ( 1 ) . among duodenal diverticula", ", pad is the most common type comprising about 70% to 75% of all duodenal diverticula ( 1 ) .", "most pad are asymptomatic but complications can occur in about 5% of cases and they include bleeding , perforation , diverticulitis , pancreatitis , choledocholithiasis , cholangitis , jaundice , enterolith or bezoar formation , intestinal obstruction , etc . among these complications ,", "hepatocholangiopancreatic disease can seldomly occur in the absence of choledocholithiasis and is termed lemmel 's syndrome ( 2 ) .", "first , diverticulitis or direct mechanical irritation of pad may cause chronic inflammation of ampulla and lead to chronic fibrosis of papilla ( papillitis chronica fibrosa ) ( 4 ) .", "third , distal cbd or ampulla can be directly compressed mechanically by pad that is usually filled with enterolith or bezoar ( 6 , 7 ) . in our case ,", "enterolith that formed within the pad did not directly compress the distal cbd but it obstructed the pad orifice instead .", "this obstruction combined with inflammation of the diverticulum and collection of pus - like material within the obstructed pad seems to have expanded the pad with resultant extrinsic compression of mid cbd ( fig .", "therefore , the enterolith , bezoar , or food material within the pad is frequently evacuated and thus , the symptom could be intermittent .", "however , pad in our case had a narrow opening , likely due to repeated inflammation of the pad , and this seems to have hindered the clearance of entrapped enterolith out into the duodenal lumen .", "enterolith formation within the duodenal diverticula is known to be facilitated in the static environment such as a blind loop after gastrectomy or proximal portion of stricture formed by crohn 's disease or tuberculosis ( 8) . in our case , blind loop created by billroth ii anastomosis seems to have provided a static environment favoring enterolith formation within the pad . during enterolith removal", ", cbd was also explored to search for other possible source of obstructive jaundice such as cbd stone since primary biliary stone is known to occur more frequently in the presence of pad ( 9 , 10 ) .", "one possible mechanism behind increased occurrence of primary cbd stone in these patients involves colonization and overgrowth of -glucuronidase producing bacteria within the pad that spread into the bile duct , which in turn leads to deconjugation of bilirubin glucuronides and eventually results in precipitation of calcium bilirubinate gallstones ( 11 ) .", "although cbd was explored in our case , no other etiology of obstructive jaundice could be identified other than extrinsic compression by distended pad . however , primary bile duct stone newly developed on follow - up ercp performed 6 months later ( fig .", "the most plausible explanation is that est performed during cbd exploration at the time of enterolith removal has permitted the occurrence of ascending infection with resultant brown pigment stone formation .", "diagnosing lemmel 's syndrome could be challenging , but being aware of this condition is important to avoid mismanagement and it begins with identification of pad .", "scan and mrcp , pad appear as thin - walled cavitary lesions situated on the medial wall of the duodenum 2nd portion that typically contain gas .", "however , pad are sometimes filled with fluid and can frequently be mistaken for pancreatic pseudocyst , pancreatic abscess , cystic neoplasm in the pancreas head or even metastatic lymph node ( 12 , 13 ) . therefore , high index of suspicion is mandatory to arrive at a correct diagnosis in these patients . in our case ,", "enterolith within the pad on axial images was at first mistaken for distal cbd stone due to its distal location combined with upstream dilatation of the bile duct ( fig .", "however , upon careful scrutinization of the coronal reconstructed images , it became evident that the stone was located within the pad ( fig .", "treatment is generally not recommended in asymptomatic patients or would be conservative management in pauci - symptomatic patients . nevertheless , since most patients with lemmel 's syndrome present with symptoms related to biliary obstruction ( i.e. jaundice , abdominal pain , and cholangitis ) as a result of extrinsic compression of cbd , some form of treatment is advocated .", "therapeutic options in this situation run the gamut from endoscopic extraction of entrapped material , extracorporeal shock wave lithotripsy to surgery ( diverticulectomy or biliodigestive anastomosis ) ( 7 , 14 , 15 ) .", "the patient in our case was also successfully treated endoscopically by fragmenting and removing enterolith using a dormia basket .", "it should be kept in mind that not all forms of lemmel 's syndrome are caused by extrinsic compression of cbd by bulging pad .", "if the underlying mechanism of lemmel 's syndrome is likely to be due to papillitis chronica fibrosa or sphincter of oddi dysfunction as mentioned above , the simplest and the most appropriate management would be to perform est ( 16 ) . in conclusion , lemmel 's syndrome is a rare cause of obstructive jaundice that should be included in the differential diagnosis of biliary obstruction when pad is present . maintaining a high index of suspicion is imperative to establish an accurate diagnosis since it can mimic other cystic or solid lesions around the pancreas head .", "symptomatic patient can be successfully managed endoscopically in many instances but recourse to surgical management would be necessary in selected cases ."]]}
{"article_id": "PMC3124838", "article_text": ["body mass index is a simple index of weight for height that is frequently used in the assessment of nutritional status .  ", "a low bmi , or underweight status , is often associated with an increased risk of mortality in seriously ill or hospitalized older adults [ 1 , 2 ] .", "conversely , a high bmi , indicative of overweight or obesity , is associated with an exacerbation in age - related physical and cognitive decline [ 3 , 4 ] and with an increased prevalence or risk of many chronic health conditions common in older adults such as diabetes , hypertension , and cardiovascular disease [ 35 ] .  ", "such associations are typically determined across the entire spectrum of older adults ( aged 60 + ) , with no further demarcation within this age classification .", "our finding of a much higher prevalence of several nutritional deficiencies in centenarians as compared with octogenarians [ 6 , 7 ] , suggests that there is considerable heterogeneity in nutrient status in the  older adult  age group .  ", "likewise , there may also be considerable heterogeneity within the older adult age group with regard to chronic health conditions .", "thus , it is not known whether the associations between underweight or overweight / obesity and chronic health conditions as observed in previous studies of older adults extend to the very old .", "dietary intake patterns featuring a high intake of nutrient - dense foods such as cereals , fruits , vegetables , and low - fat meat and dairy products have been associated with a number of favorable health outcomes in adults including a decreased prevalence of obesity [ 8 , 9 ] , lower rates of weight gain over time , and better quality of life and improved survival .", "in contrast , low - nutrient dense dietary patterns with high intakes of sweets , desserts , and high - fat dairy products have been associated with higher rates of obesity and poor nutritional status in older adults .", "studies comparing energy intakes and dietary intake patterns of centenarians to younger older adult cohorts have generally observed lower energy and/or fat intake in the centenarians , while dietary preferences of centenarians are considerably more varied and dependent of the region of study likely reflecting cultural patterns and cohort differences rather than longevity - related differences per se ( reviewed ) .  ", "nonetheless , there can be considerable variation of body weight status within a given group of centenarians , though to our knowledge the extent to which this may be associated with potential differences in dietary intake patterns or selected health conditions has not been explored .   thus , the objectives of this study were to explore associations ( 1 ) between bmi and dietary habits and ( 2 ) between underweight or overweight / obesity and health status in a population - based multiethnic sample of centenarians ( 98 years and above ) from northern georgia in the usa .  ", "it was hypothesized that overweight / obesity , but not underweight , would be associated with poorer dietary habits and that both overweight / obesity and underweight , as well as dietary habits , would be associated with specific health conditions in this population .", "this study was a secondary analysis of data collected by the georgia centenarian study , a population - based multidisciplinary study conducted in 44 counties in northern georgia ( usa ) from 2002 to 2005 .  ", "the original study included 244 centenarians ( defined as age 98 and older ) .   the sampling procedures and data collection methods", "briefly , recruitment of participants from skilled nursing facilities was based on estimates of the institutionalized population of the area according to the 2000 u.s . census tabulations .  ", "the community dwelling participants resided in private residences and personal care homes and were recruited from voter registration lists .  ", "participants were recruited to match census figures for gender and race / ethnicity ( white or black ; all were non - hispanic ) and were interviewed by trained personnel in their place of residence .", "all questionnaires and procedures were approved by the university of georgia institutional review board on human subjects .", "information regarding age , gender , race / ethnicity , living arrangements , health conditions ( cardiovascular disease , diabetes , hypertension , etc . ) , and behaviors ( including tobacco and nutritional supplement use ) were obtained from each participant ( or his / her caregiver ) by self - report .  ", "questions regarding food intake , appetite , and weight change were adapted from the mini - nutritional assessment [ 15 , 16 ] and the response categories for food intake represented current frequency of consumption of food groups , including dairy products ( milk , yogurt , and cheese ) ; meat , fish , or poultry ; orange / yellow vegetables ; green vegetables ; citrus and noncitrus fruit and juice .", "the total food score , ranging from 0 to 5 , was based on comparisons with the dietary guidelines for americans   1,600-calorie meal pattern for sedentary older adults , as previously described .  ", "body weight and height were measured by interviewers , obtained from charts or via self - report .", "in addition , knee height was measured on the right leg , unless contraindicated , to the nearest 0.1 centimeter and used to predict stature as per the formulas of chumlea et al . .  ", "body mass index ( bmi ) was calculated as weight ( kg)/height ( meters ) .", "bmi calculated from observed / recorded height and weight was highly correlated with bmi calculated from predicted stature   and observed / recorded weight ( r = 0.877 ; p < .0001 ) .  ", "bmi calculated from observed / recorded height and weight was used to form three bmi classifications based on the national institutes of health criteria   for overweight / obesity and defined as underweight 20  kg / m , normal weight > 20 and < 25  kg / m , and overweight / obese 25  kg / m .", "triceps skinfold ( tsf ) was measured on the right arm , unless contraindicated , by caliper to the nearest 0.1 millimeter .   mid - arm circumference ( mac )", "was measured on the right arm , unless contraindicated , to the nearest 0.1 centimeter .    ", ", burlington , nc ) and anemia was defined as hemoglobin < 12  g / dl for females or < 13  g / dl for males .", "participants missing data for primary variables of interest were excluded from the present analyses .   from the original sample of centenarians ,", "11 individuals were excluded due to missing data for bmi ( n = 7 ; includes one double - amputee ) , food intake patterns ( n = 1 ) , average grip strength ( n = 1 ) , and/or systolic / diastolic blood pressure ( n = 3 ) .   overall characteristics of the 233 centenarians included in the study are given in table 1 . compared to the included centenarians ( n = 233 )", ", the excluded centenarians tended to be older ( 102.6  3.6 versus 100.5  1.9  yrs ; p = .052 ) , but did not differ in gender ( 90.9% [ excluded ] versus 84.6% female ) , race / ethnicity ( 72.7% versus 79.0% white ) , or place of residence ( 36.4% versus 43.4% skilled nursing facility ) .   means , standard deviations , medians , range of values , and/or frequencies were calculated .  ", "differences between participants with different bmi classifications were assessed with the wilcoxon rank sum test for continuous variables and chi square analysis for categorical variables .  ", "the level of significance was set at p < .05 . because the food groups provide calories , a series of logistic regression analyses were performed with bmi 20 or bmi 25 as the dependent variable and gender , race / ethnicity , living arrangements , and reported intake of specific food groups as the independent variables .  ", "in addition , because bmi can play a causative role as a risk factor for chronic disease , a second series of logistic regression analyses was performed with diabetes , anemia , or other chronic health conditions as the dependent variable and race , gender , residence , and bmi 20 or bmi > 25 as the independent variables ( model 1 ) .  ", "a final series of logistic regression analyses was performed with diabetes , anemia , or other chronic health conditions as the dependent variable and race , gender , residence , total fruit , and vegetable intake and bmi 20 or bmi > 25 as the independent variables ( model 2 ) .", "p - values are unadjusted for multiple comparisons , because all comparisons were preplanned .  ", "approximately one - third ( 31.3% ) of the centenarians included in the final analytical sample had a bmi of 20 ( underweight ) , 43.8% were classified as being in the normal weight range , and 24.9% met the nih classification for overweight / obesity ( bmi 25 ) .  ", "triceps skin fold ( tsf ) and mid - arm circumference ( mac ) of centenarians in the lowest bmi classification averaged below the 5th percentile for females 80 + years in usa based on 20032006 nhanes data .   ", "both parameters increased with bmi classification in a stepwise manner ( data not shown ) and were highly correlated with bmi ( pearson correlation coefficient between bmi and tsf = 0.482 ; and between bmi and mac = 0.624 ; p < .0001 for both ) .  ", "chi - square analysis indicated that those with a bmi 20 were more likely to be women , live in a skilled nursing facility , eat a modified food diet , have experienced a weight change in the past three months , and have anemia as compared to centenarians classified as normal weight or overweight / obese ( table 2 ) .  ", "conversely , those with a bmi 25 were more likely to be black , diabetic , have a higher systolic blood pressure , and/or have a diastolic blood pressure 90  mm as compared to centenarians classified as underweight or normal weight .  ", "there were no differences according to bmi classification with regard to history of cvd , cancer , stroke , depression , or past or current tobacco use .", "bivariate analysis of diet intake patterns suggested that centenarians with bmi 20 had the highest total food scores and were more likely to report eating two or more servings of meat , fish , and poultry per day and three or more total servings of fruit per day as compared with centenarians in the other bmi classifications .", "in contrast , those with a bmi 25 were more likely to report eating less than one serving of citrus or noncitrus fruit per day , less than four servings of orange / yellow vegetables per week , or three total servings of fruit and vegetables per day ( table 2 ) .  ", "a series of logistic regression analyses indicated that when controlled for gender , race , and place of residence the odds of having a bmi 25 were about two to three times higher in centenarians with lower intakes of citrus and noncitrus fruit ( less than one serving per day ) , orange and yellow vegetables ( less than four servings per week ) , or total fruits and vegetables ( less than three servings per day ) , but were not related to intake of the meat group or dairy group ( table 3 ) .  ", "similar analyses with bmi 20 as the dependent variable , failed to show any significant association with dietary intake categories ( table 3 ) .", "finally , associations between bmi classifications and chronic health conditions were determined in a series of logistic regression analyses that included either bmi 20 or bmi 25 as an independent variable ( table 4 ) .   when controlled for gender , race , and place of residence ,", "the odds of having anemia , based on blood hemoglobin values , were over twofold higher in centenarians with bmi 20 versus those with bmi > 20 whereas the odds of self - reported cvd tended to be reduced in those in the underweight classification ( bmi 20 ; p = .053 ) .  ", "the latter finding became significant in regression models further controlled for total fruit and vegetable intake ( model 2 ; p = .048 ) . in analyses controlled for gender , race , and place of residence , the odds of having self - reported diabetes or systolic blood pressure", "140  mmhg were approximately three- and twofold higher , respectively , in centenarians with bmi 25 versus those with bmi < 25 .  ", "being overweight / obese ( bmi 25 ) also tended to increase the odds of having diastolic blood pressure > 90  mmhg ( approximately three - fold ; p = .055 ) or cardiovascular disease ( approximately twofold ; p = .074 ) .  ", "further controlling for total fruit and vegetable intake ( model 2 ) strengthened the associations between bmi 25 and diabetes and systolic blood pressure > 140  mmhg , and resulted in a significant association between bmi 25 and diastolic blood pressure 90  mmhg .  ", "there were no associations between bmi 20 or bmi 25 and stroke , depression , or cancer in any of the regression models .", "to our knowledge , this is the first study to explore associations between dietary patterns and body weight status in the oldest old segment of the population .  ", "prevalence of overweight / obesity in this population - based study of centenarians was approximately 25% , which was considerably below the prevalence for these conditions in the overall population of older adults , aged 60 and above , in the usa at the time of data collection ( 69% ; ) .   in analyses controlled for gender , race , and place of residence ,", "several parameters indicative of a low frequency of fruit and vegetable intake were associated with overweight / obesity ( bmi 25 ) , whereas there were no associations between frequency of intake of meat , dairy , and fruits and vegetables and being underweight ( bmi 20 ) .  ", "other findings include strong associations of underweight with anemia and of overweight / obesity with diabetes and high blood pressure , extending knowledge of such associations to the very old .", "the present research was part of a large , multidisciplinary study across a range of cognitive , mental , physical , and health - associated domains exploring the role of various factors pertinent to the survival and functioning of centenarians . to decrease testing burden for participants , dietary intake data focused on frequency of intake of specific food groups selected based on the dietary guidelines for americans and age - related associations with nutritional deficiencies and chronic diseases [ 16 , 17 ] .  ", "notably , we observed that based on frequency of intake and regardless of bmi classification , a large percentage of centenarians were not meeting the dietary guidelines for many food groups , with the exception of green and orange / yellow vegetables .   ", "this later finding is consistent with an apparent preference for sweet potatoes and green vegetables reported for an earlier convenience sampling of georgia centenarians [ 23 , 24 ] .  ", "such preferences likely are reflective of the traditional diet of the southeastern usa , rather than longevity - related differences in dietary patterns , and may not be replicable in other regions and cultures .", "our initial analysis indicated a greater intake of meat and total fruits in the underweight centenarians , suggesting that they were eating better than those in the normal weight and overweight / obese classifications .  ", "the underweight centenarians also had the highest total food scores , suggesting that they were meeting more of the recommended servings for specific food groups .   however , almost twice as many underweight centenarians lived in skilled nursing facilities as compared to the community , and there was no association between low bmi and dietary groups after controlling for race , residence , and gender .  ", "in a specific comparison between centenarians residing in skilled nursing facilities and in the community , johnson et al .   reported that those in skilled nursing facilities were more likely to eat three or more meals a day and to have a higher frequency of intake of most food groups .  ", "they suggested that such differences may be due to ( 1 ) the requirement that skilled nursing facilities serve meals that meet dietary guidelines and other federal nutrition policies [ 25 , 26 ] , ( 2 ) the inability of the methodology used to distinguish between food that was served and food that was eaten , and ( 3 ) barriers faced by community dwelling centenarians or their caregivers in purchasing , preparing , or consuming appropriate food to meet their nutritional needs .", "thus , associations in centenarians between low bmi and dietary status may be quite complex and influenced considerably by place of residence . in the present study , there was an increase in the relative risk for being overweight / obese in centenarians reporting the lowest frequency of intake of some nutrient dense foods including orange / yellow vegetables and citrus and noncitrus fruits .  ", "these observations are consistent with previous studies finding associations between lower reported or inferred consumption of fruits and/or vegetables and increased prevalence of overweight / obesity in children   and young to middle - age adults [ 5 , 2830 ] .    ", "interestingly , other studies in adults have indicated that increased consumption of fruits and vegetables may be an effective strategy for decreasing energy consumption and for increasing and maintaining weight loss [ 29 , 31 , 32 ] .   ", "in addition to potential beneficial effects on body weight , there is considerable evidence in other age groups that high consumption of fruits and vegetables may offer protective effects against and/or to reduce the relative risk of cardiovascular disease , hypertension , diabetes , and certain cancers [ 3340 ] .", "we observed that centenarians in the highest weight category ( bmi 25 ) reported eating lower amounts of certain types of fruits and vegetables than their nonoverweight / nonobese counterparts and also appeared to be at greater risk for diabetes , high blood pressure , and cardiovascular disease .  ", "this suggests that a high intake of fruits and vegetables may be beneficial for maintaining optimal weight , and perhaps decreasing risk of chronic disease , even at very advanced age .", "however , additional , ideally longitudinal , research collecting more detailed food intake data is needed to support this contention .", "in addition , as social isolation , missing teeth , digestion difficulties , poor self - reported health , cost and preparation issues have been identified as barriers to fruit and vegetable intake in older adults [ 4143 ] , research is needed to determine if and to what degree these or other potential barriers may be influencing the intake of fruit , vegetables , and other low energy , high nutrient dense foods in the very old .", "clinically defined anemia was present in greater than 60% of the centenarians in the lowest bmi grouping .", "after controlling for demographic covariates , a strong association remained between low bmi and anemia but not with other health conditions and indicators .", "accordingly , low bmi had been identified as an independent correlate or risk factor for anemia in some previous studies in older adults and clinical populations [ 4446 ] , but not in others .  ", "although older adults with a low bmi are considered to be at nutritional risk [ 47 , 48 ] , it can not be assumed that the anemia observed in underweight older adults is primarily of dietary or nutritional etiology .  ", "indeed , our previous studies indicate a similar prevalence of anemia in vitamin b12-deficient and vitamin b12-adequate in georgia centenarians   and a high prevalence of inflammatory anemia , either alone or in combination with nutritional deficiencies , in this population .  ", "nonetheless , as anemia is associated with increased mortality in acute and chronic disease states , particularly in those underweight [ 50 , 51 ] , it is important to monitor and treat this condition , as appropriate , in the very old . in summary", ", this secondary analysis provides evidence of an inverse association between fruit and vegetable intake and body weight status in a population - based study of centenarians .  ", "in addition , both underweight and overweight emerged as potential risk factors for various chronic diseases , emphasizing the importance of monitoring weight and of maintaining a healthy weight , even at very advanced ages .    a major strength of the study is the inclusion of a population - based sampling of centenarians with greater diversity in race , place of residence , and functional status than would be typically obtained with a convenience sample", ". limitations of the study include the relatively small sample size , lack of information regarding physical activity , and reliance on data of frequency of intake for only a few food groupings rather than the use of a more extensive food frequency questionnaire including individual foods and serving sizes as per our earlier convenience study of centenarians [ 23 , 24 ] .  ", "absence of intake data on key dietary components including grains / cereals and sweets / desserts necessitated the use a series of nonindependent binary logistic regression models instead of a more complex , single multinomial logistic regression to explore potential associations between bmi and dietary intake patterns .  ", "thus , specifically designed studies including more detailed information of dietary intake , physical activity data , and additional   chronic disease indicators are needed to verify associations , or lack thereof , between dietary intake patterns , weight status , and chronic health conditions in the very old .    furthermore , as dietary habits and other characteristics of this sample from georgia likely", "differ from those of centenarians from other countries and cultures , our findings need replication in other population groups ."], "abstract_text": ["<S> associations between body mass index ( bmi ) and dietary patterns and health conditions were explored in a population - based multiethnic sample of centenarians from northern georgia . </S>", "<S> bmi 20 and 25 was prevalent in 30.9% and 25.3% of study participants , respectively .   in a series of logistic regression analyses controlled for gender and place of residence , </S>", "<S> the probability of having bmi 25 was increased by being black versus white and having a low citrus fruit , noncitrus fruit , orange / yellow vegetable or total fruit and vegetable intake . </S>", "<S> the probability of having bmi 20 was not associated with dietary intake . when controlled for race , gender , residence , and total fruit and vegetable intake , </S>", "<S> bmi 25 was an independent risk factor for diabetes or having a systolic blood pressure 140  mmhg or diastolic blood pressure 90  mmhg , whereas bmi 20 was a risk factor for anemia . given the many potential adverse consequences of under- and overweight , efforts are needed to maintain a healthy weight , even in the oldest old . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["body mass index is a simple index of weight for height that is frequently used in the assessment of nutritional status .  ", "a low bmi , or underweight status , is often associated with an increased risk of mortality in seriously ill or hospitalized older adults [ 1 , 2 ] .", "conversely , a high bmi , indicative of overweight or obesity , is associated with an exacerbation in age - related physical and cognitive decline [ 3 , 4 ] and with an increased prevalence or risk of many chronic health conditions common in older adults such as diabetes , hypertension , and cardiovascular disease [ 35 ] .  ", "such associations are typically determined across the entire spectrum of older adults ( aged 60 + ) , with no further demarcation within this age classification .", "our finding of a much higher prevalence of several nutritional deficiencies in centenarians as compared with octogenarians [ 6 , 7 ] , suggests that there is considerable heterogeneity in nutrient status in the  older adult  age group .  ", "likewise , there may also be considerable heterogeneity within the older adult age group with regard to chronic health conditions .", "thus , it is not known whether the associations between underweight or overweight / obesity and chronic health conditions as observed in previous studies of older adults extend to the very old .", "dietary intake patterns featuring a high intake of nutrient - dense foods such as cereals , fruits , vegetables , and low - fat meat and dairy products have been associated with a number of favorable health outcomes in adults including a decreased prevalence of obesity [ 8 , 9 ] , lower rates of weight gain over time , and better quality of life and improved survival .", "in contrast , low - nutrient dense dietary patterns with high intakes of sweets , desserts , and high - fat dairy products have been associated with higher rates of obesity and poor nutritional status in older adults .", "studies comparing energy intakes and dietary intake patterns of centenarians to younger older adult cohorts have generally observed lower energy and/or fat intake in the centenarians , while dietary preferences of centenarians are considerably more varied and dependent of the region of study likely reflecting cultural patterns and cohort differences rather than longevity - related differences per se ( reviewed ) .  ", "nonetheless , there can be considerable variation of body weight status within a given group of centenarians , though to our knowledge the extent to which this may be associated with potential differences in dietary intake patterns or selected health conditions has not been explored .   thus , the objectives of this study were to explore associations ( 1 ) between bmi and dietary habits and ( 2 ) between underweight or overweight / obesity and health status in a population - based multiethnic sample of centenarians ( 98 years and above ) from northern georgia in the usa .  ", "it was hypothesized that overweight / obesity , but not underweight , would be associated with poorer dietary habits and that both overweight / obesity and underweight , as well as dietary habits , would be associated with specific health conditions in this population ."], ["this study was a secondary analysis of data collected by the georgia centenarian study , a population - based multidisciplinary study conducted in 44 counties in northern georgia ( usa ) from 2002 to 2005 .  ", "the original study included 244 centenarians ( defined as age 98 and older ) .   the sampling procedures and data collection methods", "briefly , recruitment of participants from skilled nursing facilities was based on estimates of the institutionalized population of the area according to the 2000 u.s . census tabulations .  ", "the community dwelling participants resided in private residences and personal care homes and were recruited from voter registration lists .  ", "participants were recruited to match census figures for gender and race / ethnicity ( white or black ; all were non - hispanic ) and were interviewed by trained personnel in their place of residence .", "all questionnaires and procedures were approved by the university of georgia institutional review board on human subjects .", "information regarding age , gender , race / ethnicity , living arrangements , health conditions ( cardiovascular disease , diabetes , hypertension , etc . ) , and behaviors ( including tobacco and nutritional supplement use ) were obtained from each participant ( or his / her caregiver ) by self - report .  ", "questions regarding food intake , appetite , and weight change were adapted from the mini - nutritional assessment [ 15 , 16 ] and the response categories for food intake represented current frequency of consumption of food groups , including dairy products ( milk , yogurt , and cheese ) ; meat , fish , or poultry ; orange / yellow vegetables ; green vegetables ; citrus and noncitrus fruit and juice .", "the total food score , ranging from 0 to 5 , was based on comparisons with the dietary guidelines for americans   1,600-calorie meal pattern for sedentary older adults , as previously described .  ", "body weight and height were measured by interviewers , obtained from charts or via self - report .", "in addition , knee height was measured on the right leg , unless contraindicated , to the nearest 0.1 centimeter and used to predict stature as per the formulas of chumlea et al . .  ", "body mass index ( bmi ) was calculated as weight ( kg)/height ( meters ) .", "bmi calculated from observed / recorded height and weight was highly correlated with bmi calculated from predicted stature   and observed / recorded weight ( r = 0.877 ; p < .0001 ) .  ", "bmi calculated from observed / recorded height and weight was used to form three bmi classifications based on the national institutes of health criteria   for overweight / obesity and defined as underweight 20  kg / m , normal weight > 20 and < 25  kg / m , and overweight / obese 25  kg / m .", "triceps skinfold ( tsf ) was measured on the right arm , unless contraindicated , by caliper to the nearest 0.1 millimeter .   mid - arm circumference ( mac )", "was measured on the right arm , unless contraindicated , to the nearest 0.1 centimeter .    ", ", burlington , nc ) and anemia was defined as hemoglobin < 12  g / dl for females or < 13  g / dl for males .", "participants missing data for primary variables of interest were excluded from the present analyses .   from the original sample of centenarians ,", "11 individuals were excluded due to missing data for bmi ( n = 7 ; includes one double - amputee ) , food intake patterns ( n = 1 ) , average grip strength ( n = 1 ) , and/or systolic / diastolic blood pressure ( n = 3 ) .   overall characteristics of the 233 centenarians included in the study are given in table 1 . compared to the included centenarians ( n = 233 )", ", the excluded centenarians tended to be older ( 102.6  3.6 versus 100.5  1.9  yrs ; p = .052 ) , but did not differ in gender ( 90.9% [ excluded ] versus 84.6% female ) , race / ethnicity ( 72.7% versus 79.0% white ) , or place of residence ( 36.4% versus 43.4% skilled nursing facility ) .   means , standard deviations , medians , range of values , and/or frequencies were calculated .  ", "differences between participants with different bmi classifications were assessed with the wilcoxon rank sum test for continuous variables and chi square analysis for categorical variables .  ", "the level of significance was set at p < .05 . because the food groups provide calories , a series of logistic regression analyses were performed with bmi 20 or bmi 25 as the dependent variable and gender , race / ethnicity , living arrangements , and reported intake of specific food groups as the independent variables .  ", "in addition , because bmi can play a causative role as a risk factor for chronic disease , a second series of logistic regression analyses was performed with diabetes , anemia , or other chronic health conditions as the dependent variable and race , gender , residence , and bmi 20 or bmi > 25 as the independent variables ( model 1 ) .  ", "a final series of logistic regression analyses was performed with diabetes , anemia , or other chronic health conditions as the dependent variable and race , gender , residence , total fruit , and vegetable intake and bmi 20 or bmi > 25 as the independent variables ( model 2 ) .", "p - values are unadjusted for multiple comparisons , because all comparisons were preplanned .  "], ["approximately one - third ( 31.3% ) of the centenarians included in the final analytical sample had a bmi of 20 ( underweight ) , 43.8% were classified as being in the normal weight range , and 24.9% met the nih classification for overweight / obesity ( bmi 25 ) .  ", "triceps skin fold ( tsf ) and mid - arm circumference ( mac ) of centenarians in the lowest bmi classification averaged below the 5th percentile for females 80 + years in usa based on 20032006 nhanes data .   ", "both parameters increased with bmi classification in a stepwise manner ( data not shown ) and were highly correlated with bmi ( pearson correlation coefficient between bmi and tsf = 0.482 ; and between bmi and mac = 0.624 ; p < .0001 for both ) .  ", "chi - square analysis indicated that those with a bmi 20 were more likely to be women , live in a skilled nursing facility , eat a modified food diet , have experienced a weight change in the past three months , and have anemia as compared to centenarians classified as normal weight or overweight / obese ( table 2 ) .  ", "conversely , those with a bmi 25 were more likely to be black , diabetic , have a higher systolic blood pressure , and/or have a diastolic blood pressure 90  mm as compared to centenarians classified as underweight or normal weight .  ", "there were no differences according to bmi classification with regard to history of cvd , cancer , stroke , depression , or past or current tobacco use .", "bivariate analysis of diet intake patterns suggested that centenarians with bmi 20 had the highest total food scores and were more likely to report eating two or more servings of meat , fish , and poultry per day and three or more total servings of fruit per day as compared with centenarians in the other bmi classifications .", "in contrast , those with a bmi 25 were more likely to report eating less than one serving of citrus or noncitrus fruit per day , less than four servings of orange / yellow vegetables per week , or three total servings of fruit and vegetables per day ( table 2 ) .  ", "a series of logistic regression analyses indicated that when controlled for gender , race , and place of residence the odds of having a bmi 25 were about two to three times higher in centenarians with lower intakes of citrus and noncitrus fruit ( less than one serving per day ) , orange and yellow vegetables ( less than four servings per week ) , or total fruits and vegetables ( less than three servings per day ) , but were not related to intake of the meat group or dairy group ( table 3 ) .  ", "similar analyses with bmi 20 as the dependent variable , failed to show any significant association with dietary intake categories ( table 3 ) .", "finally , associations between bmi classifications and chronic health conditions were determined in a series of logistic regression analyses that included either bmi 20 or bmi 25 as an independent variable ( table 4 ) .   when controlled for gender , race , and place of residence ,", "the odds of having anemia , based on blood hemoglobin values , were over twofold higher in centenarians with bmi 20 versus those with bmi > 20 whereas the odds of self - reported cvd tended to be reduced in those in the underweight classification ( bmi 20 ; p = .053 ) .  ", "the latter finding became significant in regression models further controlled for total fruit and vegetable intake ( model 2 ; p = .048 ) . in analyses controlled for gender , race , and place of residence , the odds of having self - reported diabetes or systolic blood pressure", "140  mmhg were approximately three- and twofold higher , respectively , in centenarians with bmi 25 versus those with bmi < 25 .  ", "being overweight / obese ( bmi 25 ) also tended to increase the odds of having diastolic blood pressure > 90  mmhg ( approximately three - fold ; p = .055 ) or cardiovascular disease ( approximately twofold ; p = .074 ) .  ", "further controlling for total fruit and vegetable intake ( model 2 ) strengthened the associations between bmi 25 and diabetes and systolic blood pressure > 140  mmhg , and resulted in a significant association between bmi 25 and diastolic blood pressure 90  mmhg .  ", "there were no associations between bmi 20 or bmi 25 and stroke , depression , or cancer in any of the regression models ."], ["to our knowledge , this is the first study to explore associations between dietary patterns and body weight status in the oldest old segment of the population .  ", "prevalence of overweight / obesity in this population - based study of centenarians was approximately 25% , which was considerably below the prevalence for these conditions in the overall population of older adults , aged 60 and above , in the usa at the time of data collection ( 69% ; ) .   in analyses controlled for gender , race , and place of residence ,", "several parameters indicative of a low frequency of fruit and vegetable intake were associated with overweight / obesity ( bmi 25 ) , whereas there were no associations between frequency of intake of meat , dairy , and fruits and vegetables and being underweight ( bmi 20 ) .  ", "other findings include strong associations of underweight with anemia and of overweight / obesity with diabetes and high blood pressure , extending knowledge of such associations to the very old .", "the present research was part of a large , multidisciplinary study across a range of cognitive , mental , physical , and health - associated domains exploring the role of various factors pertinent to the survival and functioning of centenarians . to decrease testing burden for participants , dietary intake data focused on frequency of intake of specific food groups selected based on the dietary guidelines for americans and age - related associations with nutritional deficiencies and chronic diseases [ 16 , 17 ] .  ", "notably , we observed that based on frequency of intake and regardless of bmi classification , a large percentage of centenarians were not meeting the dietary guidelines for many food groups , with the exception of green and orange / yellow vegetables .   ", "this later finding is consistent with an apparent preference for sweet potatoes and green vegetables reported for an earlier convenience sampling of georgia centenarians [ 23 , 24 ] .  ", "such preferences likely are reflective of the traditional diet of the southeastern usa , rather than longevity - related differences in dietary patterns , and may not be replicable in other regions and cultures .", "our initial analysis indicated a greater intake of meat and total fruits in the underweight centenarians , suggesting that they were eating better than those in the normal weight and overweight / obese classifications .  ", "the underweight centenarians also had the highest total food scores , suggesting that they were meeting more of the recommended servings for specific food groups .   however , almost twice as many underweight centenarians lived in skilled nursing facilities as compared to the community , and there was no association between low bmi and dietary groups after controlling for race , residence , and gender .  ", "in a specific comparison between centenarians residing in skilled nursing facilities and in the community , johnson et al .   reported that those in skilled nursing facilities were more likely to eat three or more meals a day and to have a higher frequency of intake of most food groups .  ", "they suggested that such differences may be due to ( 1 ) the requirement that skilled nursing facilities serve meals that meet dietary guidelines and other federal nutrition policies [ 25 , 26 ] , ( 2 ) the inability of the methodology used to distinguish between food that was served and food that was eaten , and ( 3 ) barriers faced by community dwelling centenarians or their caregivers in purchasing , preparing , or consuming appropriate food to meet their nutritional needs .", "thus , associations in centenarians between low bmi and dietary status may be quite complex and influenced considerably by place of residence . in the present study , there was an increase in the relative risk for being overweight / obese in centenarians reporting the lowest frequency of intake of some nutrient dense foods including orange / yellow vegetables and citrus and noncitrus fruits .  ", "these observations are consistent with previous studies finding associations between lower reported or inferred consumption of fruits and/or vegetables and increased prevalence of overweight / obesity in children   and young to middle - age adults [ 5 , 2830 ] .    ", "interestingly , other studies in adults have indicated that increased consumption of fruits and vegetables may be an effective strategy for decreasing energy consumption and for increasing and maintaining weight loss [ 29 , 31 , 32 ] .   ", "in addition to potential beneficial effects on body weight , there is considerable evidence in other age groups that high consumption of fruits and vegetables may offer protective effects against and/or to reduce the relative risk of cardiovascular disease , hypertension , diabetes , and certain cancers [ 3340 ] .", "we observed that centenarians in the highest weight category ( bmi 25 ) reported eating lower amounts of certain types of fruits and vegetables than their nonoverweight / nonobese counterparts and also appeared to be at greater risk for diabetes , high blood pressure , and cardiovascular disease .  ", "this suggests that a high intake of fruits and vegetables may be beneficial for maintaining optimal weight , and perhaps decreasing risk of chronic disease , even at very advanced age .", "however , additional , ideally longitudinal , research collecting more detailed food intake data is needed to support this contention .", "in addition , as social isolation , missing teeth , digestion difficulties , poor self - reported health , cost and preparation issues have been identified as barriers to fruit and vegetable intake in older adults [ 4143 ] , research is needed to determine if and to what degree these or other potential barriers may be influencing the intake of fruit , vegetables , and other low energy , high nutrient dense foods in the very old .", "clinically defined anemia was present in greater than 60% of the centenarians in the lowest bmi grouping .", "after controlling for demographic covariates , a strong association remained between low bmi and anemia but not with other health conditions and indicators .", "accordingly , low bmi had been identified as an independent correlate or risk factor for anemia in some previous studies in older adults and clinical populations [ 4446 ] , but not in others .  ", "although older adults with a low bmi are considered to be at nutritional risk [ 47 , 48 ] , it can not be assumed that the anemia observed in underweight older adults is primarily of dietary or nutritional etiology .  ", "indeed , our previous studies indicate a similar prevalence of anemia in vitamin b12-deficient and vitamin b12-adequate in georgia centenarians   and a high prevalence of inflammatory anemia , either alone or in combination with nutritional deficiencies , in this population .  ", "nonetheless , as anemia is associated with increased mortality in acute and chronic disease states , particularly in those underweight [ 50 , 51 ] , it is important to monitor and treat this condition , as appropriate , in the very old . in summary", ", this secondary analysis provides evidence of an inverse association between fruit and vegetable intake and body weight status in a population - based study of centenarians .  ", "in addition , both underweight and overweight emerged as potential risk factors for various chronic diseases , emphasizing the importance of monitoring weight and of maintaining a healthy weight , even at very advanced ages .    a major strength of the study is the inclusion of a population - based sampling of centenarians with greater diversity in race , place of residence , and functional status than would be typically obtained with a convenience sample", ". limitations of the study include the relatively small sample size , lack of information regarding physical activity , and reliance on data of frequency of intake for only a few food groupings rather than the use of a more extensive food frequency questionnaire including individual foods and serving sizes as per our earlier convenience study of centenarians [ 23 , 24 ] .  ", "absence of intake data on key dietary components including grains / cereals and sweets / desserts necessitated the use a series of nonindependent binary logistic regression models instead of a more complex , single multinomial logistic regression to explore potential associations between bmi and dietary intake patterns .  ", "thus , specifically designed studies including more detailed information of dietary intake , physical activity data , and additional   chronic disease indicators are needed to verify associations , or lack thereof , between dietary intake patterns , weight status , and chronic health conditions in the very old .    furthermore , as dietary habits and other characteristics of this sample from georgia likely", "differ from those of centenarians from other countries and cultures , our findings need replication in other population groups ."]]}
{"article_id": "PMC5210656", "article_text": ["environmental factors play a major role in the etiology of cancers , cardiovascular disease , and other chronic diseases .", "these factors are diverse in nature and include diet , drugs , cosmetics , household chemicals , pollutants , or infectious agents .", "exposures to these factors vary widely between populations , and between individuals within the same population . therefore , their measurement is essential to : ( i ) study associations in epidemiological studies with disease outcomes and assess their contribution to disease risk , ( ii ) monitor exposures to disease risk factors in population studies and ( iii ) assess subject compliance in clinical trials or large intervention studies ( 13 ) .", "exposure measurements have traditionally relied on the use of questionnaires and self - reporting . however , these methods are known to be error - prone and biased .", "molecular biomarkers , on the other hand , are more direct and objective indicators of exposure .", "indeed , biomarkers of exposure have been increasingly used since the early 1980s , thanks to rapid progress in analytical techniques and the establishment of large cohorts with extensive biospecimen collections .", "biomarkers of exposure can be compounds present in the environment and absorbed in the gut after ingestion , inhaled in lungs , or absorbed through the skin .", "they can also be metabolic end - products derived from environmental compounds that were metabolized by the liver and other tissues , and the microbiota .", "they may also be macromolecular indicators of environmental effects ( e.g. enzymes , proteins or rna transcripts related to the status of a nutrient or toxic agent ) . over the past 30 years", "several hundred biomarkers of exposure have been measured and reported in blood , urine and other biospecimens in various populations . however , this information is scattered over hundreds of publications under many diverse titles and subject headings .", "this makes the identification of these biomarkers along with their comparative performance , their field of application and their concentration ranges in different populations difficult .", "historically , most biomarkers of exposure have been measured individually using compound - specific assays . however , with the emergence of various omics technologies there is an increasing tendency to characterize exposures more comprehensively .", "indeed , modern mass spectrometry techniques now allow the measurement of thousands of compounds in blood , urine or other biospecimens in a single analytical run .", "these developments are leading , increasingly , to the reporting of data from multiple markers of exposure .", "modern omics technologies should also allow the translation into practice of the concept of the exposome ( the totality of exposures of a particular individual over lifetime ( 4,5 ) ) and the development of exposome - wide association studies ( ewas ) ( 2,68 ) .", "these newly emerging trends in exposure science , combined with the growing volume of comprehensive exposure data being published , make the establishment of a centralized , online database on biomarkers of exposure particularly critical . to date", ", relatively little effort has been directed to collecting and organizing data on biomarkers of exposure .", "the expocastdb database contains information on exposure to environmental chemicals such as pahs , pcbs , nonylphenols , or pesticides ( http://actor.epa.gov/actor/faces/expocastdb/home.jsp ) .", "expocastdb contains information on compound concentrations in various environmental matrices but very limited data in biospecimens .", "the comparative toxicogenomics database ( ctd ) is the only online database containing a large number of concentration values in blood , urine and other biospecimens extracted from the scientific literature ( 9 ) .", "the ctd contains about 35 000 concentration values in various biospecimens for 250 organic and inorganic compounds . while expocastdb and ctd are very useful and important databases", "it contains comprehensive information on almost 500 biomarkers of exposure with concentrations , correlations with exposure estimates and temporal reproducibility , as well as other details on study population and analytical methods .", "all data in exposome - explorer was acquired from a careful review and analysis of nearly 500 peer - reviewed publications , with a particular focus on dietary and pollution exposures .", "all data in exposome - explorer was compiled through extensive literature analysis along with manual curation and computer - assisted validation .", "literature searches were conducted using the web of science ( wos ) . only peer - reviewed publications describing original work with biomarker measurements in observational studies conducted in human populations", "publications on intervention studies , analytical method development , associations of biomarker with biological status or disease , biomarkers of biological status ( e.g. inflammation , oxidative stress , disease ) , or animal studies were not considered for this initial stage of development .", "articles not available online were omitted . for pollutants , general rather than occupational exposures were prioritized .", "exposure ,  biological monitoring ) and biospecimens (  blood ,  serum ,  plasma ,", "adduct ) . for dietary biomarkers , data on the correlations between food or food compound intake and biomarker concentrations", "citations were searched for according to several intake synonyms (  intake ,  consumption ,  diet ,  recall , ", "citations were searched for by common pollutant chemical group , such as polycyclic aromatic hydrocarbons ( pah ) , polychlorinated biphenyls ( pcb ) , polybrominated diphenyl ethers ( pbde ) , polybrominated biphenyls ( pbb ) , polychlorinated dibenzodioxins / furans ( pcdd / f ) , heterocyclic amines ( hca ) , phthalates and disinfection byproducts ( dbp ) . to search for biomarker reproducibility values ,", "reliability ,  reproducibility ,  repeatability ,  intra - subject ,  inter - subject ,  within - subject ,  between - subject. full - record citations from the wos were downloaded in the bibtex format ( http://www.bibtex.org/ ) and handled with bibdesk , an open - source bibliography manager for bibtex libraries ( http://bibdesk.sourceforge.net/ ) .", "those rated as relevant were then manually described using a series of attributes (  tags ) related to exposures , study design , type of numerical data , populations , biospecimens , biomarkers and confounding variables to facilitate the selection of references for annotators .", "the bibdesk  smart groups functionality and a combination of criteria based on the tags were used to dynamically generate a priority list of publications for further annotation .", "full - texts from these articles were then retrieved and submitted to annotators for detailed analysis and upload of the data to exposome - explorer using the annotation interface .", "a password protected annotation interface was used for the manual uploading of all data from scientific publications to exposome - explorer .", ", the annotator is guided through successive steps to ensure comprehensive capture of the following information : \n publication with its bibliographic details ( title , authors , year , journal , pubmed id).subject groups studied in the publication , and the populations to which they belong .", "each population is named with a short informative summary sentence ( e.g.  cases and controls in a case - control study on breast cancer ) .", "all ) or several ( e.g.  soy consumers and  soy non - consumers ) subject groups according to different criteria ( e.g.  by gender ,  by ethnicity ,  by smoking status ) .", "a reference to a cohort ( e.g.  epic ) can also be indicated in this data field .", "age , height , weight , bmi , group size , gender , health condition , exclusion of supplement users , smoker proportion , country of origin and ethnicity are also specified when available.samples defined for each subject group .", "these describe number and time of collections ( e.g.  baseline ,  1 year ) of different biospecimens .", "biospecimens include urine , whole blood , serum , plasma , hair , nails , adipose tissue , breast milk , or fractions such as plasma phospholipids or red blood cell membranes.biomarkers .", "chemical information ( e.g. structure , chemical formula , average mass , monoisotopic mass ) is automatically generated by the structure server .", "identifiers and links to external databases such as cas , pubchem ( 10 ) , chebi ( 11 ) , hmdb ( 12 ) and foodb ( www.foodb.ca ) are provided.biomarker measurements . for each sample or biospecimen type ,", "also included is the analytical method , the original measurement units as described in the publication , and specimen - specific concentration value adjustments ( e.g. normalizing to ", "lipid for blood , or a list of regression variables ) . arithmetic or geometric mean , as well as median", "concentration values are compiled along with the minimum and maximum values , standard deviation , percentiles , confidence intervals , measurement size ( number of subjects from which the concentration value was calculated ) , as well as the proportion of subjects for which the biomarker was detected .", "information on the inclusion or exclusion of zero values in the calculated concentration is also given .", "this was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population .", "some authors used a threshold ( e.g.  detected in at least 30% samples ) .", "below this threshold , the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . in certain cases ,", "the compound used to express the concentration is given if it is different from the compound or compound class being measured ( e.g.  polyphenols in urine expressed as gallic acid).correlations . to compile data on the correlations between specific biomarker measurements and food or dietary compound intake ,", "information was collected on the intake of different foods , food groups or dietary compounds together with the method used for dietary assessment ( record or questionnaire , food composition database , food coverage and time period ) .", "food items in exposome - explorer are linked to the foodb database ( www.foodb.ca ) . for food groups ,", "the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication .", "for dietary compounds , the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication .", "correlation coefficients ( pearson 's product moment or spearman 's rank - order ) are collated together with p - value , confidence intervals , statistical significance (  yes/no ) , and correlation size ( number of measurements from which the correlation coefficient was calculated ) .", "adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient ( e.g.  energy intake by residual method or ", "creatinine ) as well as a list of regression variables included in the calculation ( e.g.  age , smoking status , bmi , gender ) , and the use of measurement error de - attenuation are also indicated.temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker .", "a high reproducibility is required when only one sample per subject is available for biomarker measurement , as in most large cohort studies .", "reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval ( generally weeks or months ) .", "reproducibility is principally described by the intraclass correlation coefficient ( icc ) , defined as the ratio of between - subject variance to the sum of within- and between - subject variance .", "the higher the icc value , the higher the reliability of the measurement . in some cases , within- and between - subject coefficient of variation ( cv ) , as well as within- and between - subject", "reproducibility size ( number of measurements from which the reproducibility value was calculated ) and the confidence interval are also indicated in the database .", "publication with its bibliographic details ( title , authors , year , journal , pubmed i d ) .", "each population is named with a short informative summary sentence ( e.g.  cases and controls in a case - control study on breast cancer ) .", "all ) or several ( e.g.  soy consumers and  soy non - consumers ) subject groups according to different criteria ( e.g.  by gender ,  by ethnicity ,  by smoking status ) . a reference to a cohort ( e.g.  epic )", "age , height , weight , bmi , group size , gender , health condition , exclusion of supplement users , smoker proportion , country of origin and ethnicity are also specified when available .", "these describe number and time of collections ( e.g.  baseline ,  1 year ) of different biospecimens .", "biospecimens include urine , whole blood , serum , plasma , hair , nails , adipose tissue , breast milk , or fractions such as plasma phospholipids or red blood cell membranes . biomarkers .", "chemical information ( e.g. structure , chemical formula , average mass , monoisotopic mass ) is automatically generated by the structure server .", "identifiers and links to external databases such as cas , pubchem ( 10 ) , chebi ( 11 ) , hmdb ( 12 ) and foodb ( www.foodb.ca ) are provided .", "also included is the analytical method , the original measurement units as described in the publication , and specimen - specific concentration value adjustments ( e.g. normalizing to ", "lipid for blood , or a list of regression variables ) . arithmetic or geometric mean , as well as median", "concentration values are compiled along with the minimum and maximum values , standard deviation , percentiles , confidence intervals , measurement size ( number of subjects from which the concentration value was calculated ) , as well as the proportion of subjects for which the biomarker was detected .", "information on the inclusion or exclusion of zero values in the calculated concentration is also given .", "this was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population .", "some authors used a threshold ( e.g.  detected in at least 30% samples ) .", "below this threshold , the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . in certain cases ,", "the compound used to express the concentration is given if it is different from the compound or compound class being measured ( e.g.  polyphenols in urine expressed as gallic acid ) .", "correlations . to compile data on the correlations between specific biomarker measurements and food or dietary compound intake ,", "information was collected on the intake of different foods , food groups or dietary compounds together with the method used for dietary assessment ( record or questionnaire , food composition database , food coverage and time period ) .", "food items in exposome - explorer are linked to the foodb database ( www.foodb.ca ) . for food groups ,", "the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication . for dietary compounds ,", "the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication .", "correlation coefficients ( pearson 's product moment or spearman 's rank - order ) are collated together with p - value , confidence intervals , statistical significance (  yes/no ) , and correlation size ( number of measurements from which the correlation coefficient was calculated ) .", "adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient ( e.g.  energy intake by residual method or ", "creatinine ) as well as a list of regression variables included in the calculation ( e.g.  age , smoking status , bmi , gender ) , and the use of measurement error de - attenuation are also indicated .", "temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker .", "a high reproducibility is required when only one sample per subject is available for biomarker measurement , as in most large cohort studies .", "reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval ( generally weeks or months ) .", "reproducibility is principally described by the intraclass correlation coefficient ( icc ) , defined as the ratio of between - subject variance to the sum of within- and between - subject variance .", "the higher the icc value , the higher the reliability of the measurement . in some cases , within- and between - subject coefficient of variation ( cv ) , as well as within- and between - subject variance ( var )", "reproducibility size ( number of measurements from which the reproducibility value was calculated ) and the confidence interval are also indicated in the database .", "exposome - explorer 's annotation interface uses a number of features to help complex , heterogeneous , literature - derived data to be easily and systematically translated into organized electronic data .", "controlled vocabularies for compounds , foods , experimental methods , specimens , cohorts and units can be created and are fully documented in both the annotation interface and the database .", "this helps to avoid the inclusion of duplicate vocabulary items , such as different spellings or synonyms for a same item .", "hierarchical associations of populations , samples and measurements can be represented through the creation of parent - children relations .", "for instance , populations stratified into different sub - groups can be easily described , or the association of several samples taken at different collection times .", "all data uploaded to the database via the exposome - explorer annotation interface is automatically validated , thereby preventing the uploading of erroneous records .", "error checks include the usual database integrity verifications such as the presence of mandatory fields , checks on input text size , checks on allowable values or the uniqueness of specific values .", "the error checking utilities also include application - specific consistency verifications such as  correlations can not be created between measurements of different subject groups or  measurement size can not exceed subject group size. these checks ensure the highest consistency for data collected by different annotators .", "newly uploaded data is also manually and systematically checked by the database 's chief curator .", "repetitive insertion of similar data is facilitated via the support of record duplication and the ability to edit several records at once .", "error messages are displayed to guide annotators toward making correct data entries or fixing erroneous entries .", "exposome - explorer 's public user interface allows intuitive browsing and searching of almost any data type in the database ( figure 1 ) .", "data can be retrieved through the quick search functionality or through specific searches offered on the website 's different menu pages .", "distinct areas are shown : structure and chemical information ( a ) , concentrations ( b ) , correlations ( c ) and reproducibility ( d ) collated from several publications .", "biomarkers menu lists the biomarkers , biospecimens , analytical methods and cohorts documented in the database .", "each item has its own summary page with both general details ( name and classification ) and all data related to this item in the database .", "correlations menu lists the correlation values between different food or dietary compound intakes and the corresponding biomarker measurements .", "these correlations can be searched according to the food type , dietary compound , or biomarker .", "the data can be filtered according to a variety of parameters including the type of biospecimen or the method used to assess the dietary exposure .", "reproducibility menu lists the biomarker reproducibility values , which can also be filtered according to biomarker classification ( as well as other parameters ) and ranked in decreasing or increasing order .", "the  data search menu lists all biomarker concentration values and allows searches over all data fields .", "searches by chemical structure are also possible , as well as browsing biomarker classes and subclasses . the ", "the list can be filtered by title , authors , year or pubmed i d .", "full - text for these publications can be accessed via pubmed links or direct publisher urls . for every annotated publication , the totality of collected data including the bibliographic information , detailed subject descriptions , and biomarker data ( concentration values , dietary intake values , correlation values and reproducibility values ) is displayed in a single page .", "tables on exposome - explorer 's different webpages can be easily adjusted to suit user - specific needs .", "several hidden columns are available and can be shown in order to provide more details for the default display .", "every original value is listed with its corresponding bibliographic citation , making it possible to link each value back to its metadata .", "default conversions from highly diverse to standardized units are possible while original values are preserved .", "tables can be exported in different formats ( e.g. csv , tab ) and reused in other programs ( e.g. excel , r ) in order to conduct more specific analyses .", "ruby on rails is a web framework which employs the model - view - controller ( mvc ) design pattern .", "the exposome - explorer data is stored in a mysql relational database ( http://www.mysql.com/ ) .", "hierarchical associations of records (  trees ) are implemented as a nested set model inside a single database column of the tables using the rails gem ancestry ( https://github.com/stefankroes/ancestry ) .", "highly diverse units and their conversions are transparently handled with the phys - units library ( https://github.com/masa16/phys-units/ ) , which is a ruby implementation of the gnu units software ( http://www.gnu.org/software/units/ ) .", "website global search is implemented using the wishart 's unearth gem , which uses elasticsearch indexing ( https://www.elastic.co/ ) .", "the web interface is built with the bootstrap front - end framework ( http://getbootstrap.com/ ) .", "to date , data from a total of 480 publications have been entered into this first release of exposome - explorer .", "this includes 10 510 concentration values for 692 biomarker entries , among which 8,861 concentrations correspond to 488  single dietary and pollutant biomarkers .", "approximately one third of these biomarkers are dietary biomarkers ( table 1 ) . for dietary biomarkers ,", "almost half of the concentrations are related to fatty acids , followed by carotenoids and polyphenols . with regard to pollutant biomarkers , about two thirds of the concentration data in exposome - explorer", "are related to pcbs and pbdes , followed by pahs , pcdd / fs and phthalates .", "some concentration data are also available for other biospecimens such as hair , nails , adipose tissue or breast milk .", "exposome - explorer currently contains the most complete and comprehensive information on exposure biomarkers ever compiled from peer - reviewed literature .", "it is also the first publicly available , web - enabled database specifically dedicated to exposure biomarkers .", "we believe it provides a good starting point for selecting markers of interest or for defining panels of biomarkers that can be used in exposome - wide association studies . through exposome - explorer ,", "biomarker concentrations can be compared in different cohorts or population groups at different levels of exposure ( e.g. consumers and non - consumers of a particular food ) , or between different geographical areas .", "all of the database 's manually curated information is fully linked with other online databases and with the original publications .", "the high granularity of the data in the database should allow users to conduct very diverse and advanced analyses or comparisons across publications .", "looking both at the range of studied compounds and the number of corresponding studies , exposome - explorer also allows users to quickly identify poorly studied exposures or biomarkers that may require further work for validation .", "plans for future enhancements to exposome - explorer include its extension to other categories of exposures ( nutritional status , pesticides , occupational exposures ) , the addition of other types of biomarkers ( dna and protein adducts ) , and the inclusion of more information characterizing biomarkers such as their half - life and other pharmacokinetic parameters .", "plans are also being made to add putative or non - validated biomarkers identified in ( pre)clinical studies , but never measured in populations .", "overall , we believe exposome - explorer will help in the generation of hypotheses for discovery of new biomarkers to be tested in the laboratory . it should also help in evaluating the performance of existing biomarkers and integrating exposure data based on biomarkers with data collected with other technologies .", "exposome - explorer should contribute to the translation of the exposome into practice in epidemiological research .", "european commission : exposomics fp7-kbbe-2012 ; nutritech fp7-kbbe-2011 - 5 ; joint programming initiative foodball 201417 ."], "abstract_text": ["<S> exposome - explorer ( http://exposome-explorer.iarc.fr ) is the first database dedicated to biomarkers of exposure to environmental risk factors . </S>", "<S> it contains detailed information on the nature of biomarkers , their concentrations in various human biospecimens , the study population where measured and the analytical techniques used for measurement . </S>", "<S> it also contains correlations with external exposure measurements and data on biological reproducibility over time . </S>", "<S> the data in exposome - explorer was manually collected from peer - reviewed publications and organized to make it easily accessible through a web interface for in - depth analyses . </S>", "<S> the database and the web interface were developed using the ruby on rails framework . </S>", "<S> a total of 480 publications were analyzed and 10 510 concentration values in blood , urine and other biospecimens for 692 dietary and pollutant biomarkers were collected . over 8000 correlation values between dietary biomarker levels and food intake as well as 536 values of biological reproducibility over time were also compiled . </S>", "<S> exposome - explorer makes it easy to compare the performance between biomarkers and their fields of application </S>", "<S> . it should be particularly useful for epidemiologists and clinicians wishing to select panels of biomarkers that can be used in biomonitoring studies or in exposome - wide association studies , thereby allowing them to better understand the etiology of chronic diseases . </S>"], "labels": null, "section_names": ["INTRODUCTION", "DATA COLLECTION", "DATA COMPILATION", "QUERYING THE DATABASE", "DATABASE IMPLEMENTATION", "DATA STATISTICS", "CONCLUSIONS AND FUTURE ENHANCEMENTS", "FUNDING"], "sections": [["environmental factors play a major role in the etiology of cancers , cardiovascular disease , and other chronic diseases .", "these factors are diverse in nature and include diet , drugs , cosmetics , household chemicals , pollutants , or infectious agents .", "exposures to these factors vary widely between populations , and between individuals within the same population . therefore , their measurement is essential to : ( i ) study associations in epidemiological studies with disease outcomes and assess their contribution to disease risk , ( ii ) monitor exposures to disease risk factors in population studies and ( iii ) assess subject compliance in clinical trials or large intervention studies ( 13 ) .", "exposure measurements have traditionally relied on the use of questionnaires and self - reporting . however , these methods are known to be error - prone and biased .", "molecular biomarkers , on the other hand , are more direct and objective indicators of exposure .", "indeed , biomarkers of exposure have been increasingly used since the early 1980s , thanks to rapid progress in analytical techniques and the establishment of large cohorts with extensive biospecimen collections .", "biomarkers of exposure can be compounds present in the environment and absorbed in the gut after ingestion , inhaled in lungs , or absorbed through the skin .", "they can also be metabolic end - products derived from environmental compounds that were metabolized by the liver and other tissues , and the microbiota .", "they may also be macromolecular indicators of environmental effects ( e.g. enzymes , proteins or rna transcripts related to the status of a nutrient or toxic agent ) . over the past 30 years", "several hundred biomarkers of exposure have been measured and reported in blood , urine and other biospecimens in various populations . however , this information is scattered over hundreds of publications under many diverse titles and subject headings .", "this makes the identification of these biomarkers along with their comparative performance , their field of application and their concentration ranges in different populations difficult .", "historically , most biomarkers of exposure have been measured individually using compound - specific assays . however , with the emergence of various omics technologies there is an increasing tendency to characterize exposures more comprehensively .", "indeed , modern mass spectrometry techniques now allow the measurement of thousands of compounds in blood , urine or other biospecimens in a single analytical run .", "these developments are leading , increasingly , to the reporting of data from multiple markers of exposure .", "modern omics technologies should also allow the translation into practice of the concept of the exposome ( the totality of exposures of a particular individual over lifetime ( 4,5 ) ) and the development of exposome - wide association studies ( ewas ) ( 2,68 ) .", "these newly emerging trends in exposure science , combined with the growing volume of comprehensive exposure data being published , make the establishment of a centralized , online database on biomarkers of exposure particularly critical . to date", ", relatively little effort has been directed to collecting and organizing data on biomarkers of exposure .", "the expocastdb database contains information on exposure to environmental chemicals such as pahs , pcbs , nonylphenols , or pesticides ( http://actor.epa.gov/actor/faces/expocastdb/home.jsp ) .", "expocastdb contains information on compound concentrations in various environmental matrices but very limited data in biospecimens .", "the comparative toxicogenomics database ( ctd ) is the only online database containing a large number of concentration values in blood , urine and other biospecimens extracted from the scientific literature ( 9 ) .", "the ctd contains about 35 000 concentration values in various biospecimens for 250 organic and inorganic compounds . while expocastdb and ctd are very useful and important databases", "it contains comprehensive information on almost 500 biomarkers of exposure with concentrations , correlations with exposure estimates and temporal reproducibility , as well as other details on study population and analytical methods .", "all data in exposome - explorer was acquired from a careful review and analysis of nearly 500 peer - reviewed publications , with a particular focus on dietary and pollution exposures ."], ["all data in exposome - explorer was compiled through extensive literature analysis along with manual curation and computer - assisted validation .", "literature searches were conducted using the web of science ( wos ) . only peer - reviewed publications describing original work with biomarker measurements in observational studies conducted in human populations", "publications on intervention studies , analytical method development , associations of biomarker with biological status or disease , biomarkers of biological status ( e.g. inflammation , oxidative stress , disease ) , or animal studies were not considered for this initial stage of development .", "articles not available online were omitted . for pollutants , general rather than occupational exposures were prioritized .", "exposure ,  biological monitoring ) and biospecimens (  blood ,  serum ,  plasma ,", "adduct ) . for dietary biomarkers , data on the correlations between food or food compound intake and biomarker concentrations", "citations were searched for according to several intake synonyms (  intake ,  consumption ,  diet ,  recall , ", "citations were searched for by common pollutant chemical group , such as polycyclic aromatic hydrocarbons ( pah ) , polychlorinated biphenyls ( pcb ) , polybrominated diphenyl ethers ( pbde ) , polybrominated biphenyls ( pbb ) , polychlorinated dibenzodioxins / furans ( pcdd / f ) , heterocyclic amines ( hca ) , phthalates and disinfection byproducts ( dbp ) . to search for biomarker reproducibility values ,", "reliability ,  reproducibility ,  repeatability ,  intra - subject ,  inter - subject ,  within - subject ,  between - subject. full - record citations from the wos were downloaded in the bibtex format ( http://www.bibtex.org/ ) and handled with bibdesk , an open - source bibliography manager for bibtex libraries ( http://bibdesk.sourceforge.net/ ) .", "those rated as relevant were then manually described using a series of attributes (  tags ) related to exposures , study design , type of numerical data , populations , biospecimens , biomarkers and confounding variables to facilitate the selection of references for annotators .", "the bibdesk  smart groups functionality and a combination of criteria based on the tags were used to dynamically generate a priority list of publications for further annotation .", "full - texts from these articles were then retrieved and submitted to annotators for detailed analysis and upload of the data to exposome - explorer using the annotation interface ."], ["a password protected annotation interface was used for the manual uploading of all data from scientific publications to exposome - explorer .", ", the annotator is guided through successive steps to ensure comprehensive capture of the following information : \n publication with its bibliographic details ( title , authors , year , journal , pubmed id).subject groups studied in the publication , and the populations to which they belong .", "each population is named with a short informative summary sentence ( e.g.  cases and controls in a case - control study on breast cancer ) .", "all ) or several ( e.g.  soy consumers and  soy non - consumers ) subject groups according to different criteria ( e.g.  by gender ,  by ethnicity ,  by smoking status ) .", "a reference to a cohort ( e.g.  epic ) can also be indicated in this data field .", "age , height , weight , bmi , group size , gender , health condition , exclusion of supplement users , smoker proportion , country of origin and ethnicity are also specified when available.samples defined for each subject group .", "these describe number and time of collections ( e.g.  baseline ,  1 year ) of different biospecimens .", "biospecimens include urine , whole blood , serum , plasma , hair , nails , adipose tissue , breast milk , or fractions such as plasma phospholipids or red blood cell membranes.biomarkers .", "chemical information ( e.g. structure , chemical formula , average mass , monoisotopic mass ) is automatically generated by the structure server .", "identifiers and links to external databases such as cas , pubchem ( 10 ) , chebi ( 11 ) , hmdb ( 12 ) and foodb ( www.foodb.ca ) are provided.biomarker measurements . for each sample or biospecimen type ,", "also included is the analytical method , the original measurement units as described in the publication , and specimen - specific concentration value adjustments ( e.g. normalizing to ", "lipid for blood , or a list of regression variables ) . arithmetic or geometric mean , as well as median", "concentration values are compiled along with the minimum and maximum values , standard deviation , percentiles , confidence intervals , measurement size ( number of subjects from which the concentration value was calculated ) , as well as the proportion of subjects for which the biomarker was detected .", "information on the inclusion or exclusion of zero values in the calculated concentration is also given .", "this was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population .", "some authors used a threshold ( e.g.  detected in at least 30% samples ) .", "below this threshold , the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . in certain cases ,", "the compound used to express the concentration is given if it is different from the compound or compound class being measured ( e.g.  polyphenols in urine expressed as gallic acid).correlations . to compile data on the correlations between specific biomarker measurements and food or dietary compound intake ,", "information was collected on the intake of different foods , food groups or dietary compounds together with the method used for dietary assessment ( record or questionnaire , food composition database , food coverage and time period ) .", "food items in exposome - explorer are linked to the foodb database ( www.foodb.ca ) . for food groups ,", "the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication .", "for dietary compounds , the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication .", "correlation coefficients ( pearson 's product moment or spearman 's rank - order ) are collated together with p - value , confidence intervals , statistical significance (  yes/no ) , and correlation size ( number of measurements from which the correlation coefficient was calculated ) .", "adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient ( e.g.  energy intake by residual method or ", "creatinine ) as well as a list of regression variables included in the calculation ( e.g.  age , smoking status , bmi , gender ) , and the use of measurement error de - attenuation are also indicated.temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker .", "a high reproducibility is required when only one sample per subject is available for biomarker measurement , as in most large cohort studies .", "reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval ( generally weeks or months ) .", "reproducibility is principally described by the intraclass correlation coefficient ( icc ) , defined as the ratio of between - subject variance to the sum of within- and between - subject variance .", "the higher the icc value , the higher the reliability of the measurement . in some cases , within- and between - subject coefficient of variation ( cv ) , as well as within- and between - subject", "reproducibility size ( number of measurements from which the reproducibility value was calculated ) and the confidence interval are also indicated in the database .", "publication with its bibliographic details ( title , authors , year , journal , pubmed i d ) .", "each population is named with a short informative summary sentence ( e.g.  cases and controls in a case - control study on breast cancer ) .", "all ) or several ( e.g.  soy consumers and  soy non - consumers ) subject groups according to different criteria ( e.g.  by gender ,  by ethnicity ,  by smoking status ) . a reference to a cohort ( e.g.  epic )", "age , height , weight , bmi , group size , gender , health condition , exclusion of supplement users , smoker proportion , country of origin and ethnicity are also specified when available .", "these describe number and time of collections ( e.g.  baseline ,  1 year ) of different biospecimens .", "biospecimens include urine , whole blood , serum , plasma , hair , nails , adipose tissue , breast milk , or fractions such as plasma phospholipids or red blood cell membranes . biomarkers .", "chemical information ( e.g. structure , chemical formula , average mass , monoisotopic mass ) is automatically generated by the structure server .", "identifiers and links to external databases such as cas , pubchem ( 10 ) , chebi ( 11 ) , hmdb ( 12 ) and foodb ( www.foodb.ca ) are provided .", "also included is the analytical method , the original measurement units as described in the publication , and specimen - specific concentration value adjustments ( e.g. normalizing to ", "lipid for blood , or a list of regression variables ) . arithmetic or geometric mean , as well as median", "concentration values are compiled along with the minimum and maximum values , standard deviation , percentiles , confidence intervals , measurement size ( number of subjects from which the concentration value was calculated ) , as well as the proportion of subjects for which the biomarker was detected .", "information on the inclusion or exclusion of zero values in the calculated concentration is also given .", "this was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population .", "some authors used a threshold ( e.g.  detected in at least 30% samples ) .", "below this threshold , the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . in certain cases ,", "the compound used to express the concentration is given if it is different from the compound or compound class being measured ( e.g.  polyphenols in urine expressed as gallic acid ) .", "correlations . to compile data on the correlations between specific biomarker measurements and food or dietary compound intake ,", "information was collected on the intake of different foods , food groups or dietary compounds together with the method used for dietary assessment ( record or questionnaire , food composition database , food coverage and time period ) .", "food items in exposome - explorer are linked to the foodb database ( www.foodb.ca ) . for food groups ,", "the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication . for dietary compounds ,", "the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication .", "correlation coefficients ( pearson 's product moment or spearman 's rank - order ) are collated together with p - value , confidence intervals , statistical significance (  yes/no ) , and correlation size ( number of measurements from which the correlation coefficient was calculated ) .", "adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient ( e.g.  energy intake by residual method or ", "creatinine ) as well as a list of regression variables included in the calculation ( e.g.  age , smoking status , bmi , gender ) , and the use of measurement error de - attenuation are also indicated .", "temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker .", "a high reproducibility is required when only one sample per subject is available for biomarker measurement , as in most large cohort studies .", "reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval ( generally weeks or months ) .", "reproducibility is principally described by the intraclass correlation coefficient ( icc ) , defined as the ratio of between - subject variance to the sum of within- and between - subject variance .", "the higher the icc value , the higher the reliability of the measurement . in some cases , within- and between - subject coefficient of variation ( cv ) , as well as within- and between - subject variance ( var )", "reproducibility size ( number of measurements from which the reproducibility value was calculated ) and the confidence interval are also indicated in the database .", "exposome - explorer 's annotation interface uses a number of features to help complex , heterogeneous , literature - derived data to be easily and systematically translated into organized electronic data .", "controlled vocabularies for compounds , foods , experimental methods , specimens , cohorts and units can be created and are fully documented in both the annotation interface and the database .", "this helps to avoid the inclusion of duplicate vocabulary items , such as different spellings or synonyms for a same item .", "hierarchical associations of populations , samples and measurements can be represented through the creation of parent - children relations .", "for instance , populations stratified into different sub - groups can be easily described , or the association of several samples taken at different collection times .", "all data uploaded to the database via the exposome - explorer annotation interface is automatically validated , thereby preventing the uploading of erroneous records .", "error checks include the usual database integrity verifications such as the presence of mandatory fields , checks on input text size , checks on allowable values or the uniqueness of specific values .", "the error checking utilities also include application - specific consistency verifications such as  correlations can not be created between measurements of different subject groups or  measurement size can not exceed subject group size. these checks ensure the highest consistency for data collected by different annotators .", "newly uploaded data is also manually and systematically checked by the database 's chief curator .", "repetitive insertion of similar data is facilitated via the support of record duplication and the ability to edit several records at once .", "error messages are displayed to guide annotators toward making correct data entries or fixing erroneous entries ."], ["exposome - explorer 's public user interface allows intuitive browsing and searching of almost any data type in the database ( figure 1 ) .", "data can be retrieved through the quick search functionality or through specific searches offered on the website 's different menu pages .", "distinct areas are shown : structure and chemical information ( a ) , concentrations ( b ) , correlations ( c ) and reproducibility ( d ) collated from several publications .", "biomarkers menu lists the biomarkers , biospecimens , analytical methods and cohorts documented in the database .", "each item has its own summary page with both general details ( name and classification ) and all data related to this item in the database .", "correlations menu lists the correlation values between different food or dietary compound intakes and the corresponding biomarker measurements .", "these correlations can be searched according to the food type , dietary compound , or biomarker .", "the data can be filtered according to a variety of parameters including the type of biospecimen or the method used to assess the dietary exposure .", "reproducibility menu lists the biomarker reproducibility values , which can also be filtered according to biomarker classification ( as well as other parameters ) and ranked in decreasing or increasing order .", "the  data search menu lists all biomarker concentration values and allows searches over all data fields .", "searches by chemical structure are also possible , as well as browsing biomarker classes and subclasses . the ", "the list can be filtered by title , authors , year or pubmed i d .", "full - text for these publications can be accessed via pubmed links or direct publisher urls . for every annotated publication , the totality of collected data including the bibliographic information , detailed subject descriptions , and biomarker data ( concentration values , dietary intake values , correlation values and reproducibility values ) is displayed in a single page .", "tables on exposome - explorer 's different webpages can be easily adjusted to suit user - specific needs .", "several hidden columns are available and can be shown in order to provide more details for the default display .", "every original value is listed with its corresponding bibliographic citation , making it possible to link each value back to its metadata .", "default conversions from highly diverse to standardized units are possible while original values are preserved .", "tables can be exported in different formats ( e.g. csv , tab ) and reused in other programs ( e.g. excel , r ) in order to conduct more specific analyses ."], ["ruby on rails is a web framework which employs the model - view - controller ( mvc ) design pattern .", "the exposome - explorer data is stored in a mysql relational database ( http://www.mysql.com/ ) .", "hierarchical associations of records (  trees ) are implemented as a nested set model inside a single database column of the tables using the rails gem ancestry ( https://github.com/stefankroes/ancestry ) .", "highly diverse units and their conversions are transparently handled with the phys - units library ( https://github.com/masa16/phys-units/ ) , which is a ruby implementation of the gnu units software ( http://www.gnu.org/software/units/ ) .", "website global search is implemented using the wishart 's unearth gem , which uses elasticsearch indexing ( https://www.elastic.co/ ) .", "the web interface is built with the bootstrap front - end framework ( http://getbootstrap.com/ ) ."], ["to date , data from a total of 480 publications have been entered into this first release of exposome - explorer .", "this includes 10 510 concentration values for 692 biomarker entries , among which 8,861 concentrations correspond to 488  single dietary and pollutant biomarkers .", "approximately one third of these biomarkers are dietary biomarkers ( table 1 ) . for dietary biomarkers ,", "almost half of the concentrations are related to fatty acids , followed by carotenoids and polyphenols . with regard to pollutant biomarkers , about two thirds of the concentration data in exposome - explorer", "are related to pcbs and pbdes , followed by pahs , pcdd / fs and phthalates .", "some concentration data are also available for other biospecimens such as hair , nails , adipose tissue or breast milk ."], ["exposome - explorer currently contains the most complete and comprehensive information on exposure biomarkers ever compiled from peer - reviewed literature .", "it is also the first publicly available , web - enabled database specifically dedicated to exposure biomarkers .", "we believe it provides a good starting point for selecting markers of interest or for defining panels of biomarkers that can be used in exposome - wide association studies . through exposome - explorer ,", "biomarker concentrations can be compared in different cohorts or population groups at different levels of exposure ( e.g. consumers and non - consumers of a particular food ) , or between different geographical areas .", "all of the database 's manually curated information is fully linked with other online databases and with the original publications .", "the high granularity of the data in the database should allow users to conduct very diverse and advanced analyses or comparisons across publications .", "looking both at the range of studied compounds and the number of corresponding studies , exposome - explorer also allows users to quickly identify poorly studied exposures or biomarkers that may require further work for validation .", "plans for future enhancements to exposome - explorer include its extension to other categories of exposures ( nutritional status , pesticides , occupational exposures ) , the addition of other types of biomarkers ( dna and protein adducts ) , and the inclusion of more information characterizing biomarkers such as their half - life and other pharmacokinetic parameters .", "plans are also being made to add putative or non - validated biomarkers identified in ( pre)clinical studies , but never measured in populations .", "overall , we believe exposome - explorer will help in the generation of hypotheses for discovery of new biomarkers to be tested in the laboratory . it should also help in evaluating the performance of existing biomarkers and integrating exposure data based on biomarkers with data collected with other technologies .", "exposome - explorer should contribute to the translation of the exposome into practice in epidemiological research ."], ["european commission : exposomics fp7-kbbe-2012 ; nutritech fp7-kbbe-2011 - 5 ; joint programming initiative foodball 201417 ."]]}
{"article_id": "PMC3184424", "article_text": ["the aspergilli are filamentous fungi , which are multicellular eukaryotes with a relatively simple life cycle .", "many of them have long been used in food production , industrial fermentation , and agriculture . on the other hand , a few , such as a. fumigatus , a. flavus , a. niger , a. nidulans , and a. terreus , are opportunistic fungal pathogens , causing life - threatening invasive aspergillosis ( ia ) in immunosuppressed patients [ 1 , 2 ] , in which a. fumigatus is the predominant pathogen [ 35 ] .", "the crude mortality for ia is 6090% and remains around 2942% even when treatment is given .", "the main reasons for patient death are late diagnosis and the low efficiency of the drug therapies available to treat ia .  ", "it maintains cell shape and provides osmotic protection [ 7 , 8 ] and has therefore been recognized for a long time as an ideal drug target .", "indeed , several cell - wall - targeted drugs , such as echinocandins , caspofungin , micafungin , and anidulafungin , have been introduced as therapies .", "for example , echinocandins , which inhibit synthesis of -1,3-glucan , a crucial component of the cell wall are effective in the treatment of invasive fungal infections including ia .", "unfortunately , the echinocandins also trigger an increase of chitin [ 9 , 10 ] , which partially compensates for the loss of -1,3-glucan and reduces the efficiency of treatment due to the complicated mechanism of cell wall biogenesis in a. fumigatus .", "therefore , a more profound understanding of the mechanisms of cell wall biosynthesis in a. fumigatus would help to improve the efficiency of drug therapies , especially for drugs which target the cell wall .", "the cell wall of a. fumigatus is composed of a unique -1,3/1,4-glucan skeleton with chitin and galactomannan covalently linked to the nonreducing ends of -1,3-glucan . the cell wall is mainly coated with gpi proteins , which contain n- and o - glycans [ 11 , 12 ] . while there is no doubt that glycosylation is involved in cell wall organization , the functional importance of protein glycosylation in cell wall organization has , until recently , remained poorly understood .", "however , during the past few years , it has become increasingly evident that glycosylation is vital for cell wall synthesis and thus vital for growth and morphology of a. fumigatus .  ", "basically , all eukaryotes possess three types of protein glycosylation , n - glycosylation of asparagine residues , o - glycosylation of threonine and serine residues , and glycosylphosphatidylinositol - anchoring ( gpi - anchoring ) of the c - terminus of some proteins .", "humans lacking individual glycosyltransferases suffer from severe congenital diseases , known as carbohydrate - deficient glycoprotein syndromes ( cdgs ) [ 1214 ] .", "clearly , the sugar components of proteins play a major role in embryonic and postembryonic development of humans as well as of all higher eukaryotes .", "however , the molecular details leading to cdgs are only vaguely understood . during the past 20 years", "it is now known that carbohydrates play increasingly important roles in regulating the development of higher organisms . however , the mechanism by which carbohydrates play a role in development and disease is still unclear .", "our knowledge of protein glycosylation comes mainly from investigation of the model yeast s. cerevisiae and of mammalian cells .", "although investigation of the model yeast has been very useful in elucidating the biochemical features of protein glycosylation at the cellular level , they can not reveal the complicated functions of glycosylation in the development of multicellular eukaryotes .", "therefore , investigation of glycosylation in the multicellular fungus a. fumigatus not only helps understand the mechanism of cell wall synthesis in this species but also provides insights into the role of glycosylation in the development of multicellular eukaryotes .", "this paper concentrates on protein glycosylation in a. fumigatus , which will be discussed with respect to the enzymatic pathways involved and their functional importance .", "furthermore , the utility of a. fumigatus as a model for glycosylation during development of multicellular eukaryotes will be outlined .", "the cell wall of a. fumigatus is mainly composed of -1,3-glucans that are highly branched with -1,6 linkages . together", "they constitute a three - dimensional network with a large number of nonreducing ends , to which chitin , galactomannan , and -1,3/1,4-glucan are covalently anchored . the cell wall is mainly coated with gpi proteins , which contain n- and o - glycans derived primarily from the process of glycosylation [ 11 , 17 ] .", "deletion of gel2 leads to slower growth , abnormal conidiogenesis , an altered cell wall composition , and reduced virulence [ 18 , 19 ] .", "it has been proposed that gel2p is responsible for the elongation of -1,3-glucan side chains of -1,3/1,6 branched glucan to provide new nonreducing ends .", "ecm33p is also involved in maintaining proper cell wall architecture though its function is unknown .", "disruption of ecm33 results in morphogenetic aberrations such as defects in conidial separation , increase of chitin in conidial cell walls , rapid conidial germination , and increased virulence [ 20 , 21 ] .", "it is clear that glycoproteins are directly , as structural components of the cell wall , and indirectly , as enzymes required for cell wall synthesis , involved in maintaining proper cell wall architecture in a. fumigatus .", "increased chitin synthesis has been known as an important compensatory response to cell wall stress both in s. cerevisiae and filamentous fungi [ 2226 ] . in s. cerevisiae , the cell wall is required to maintain cell shape , which is essential for the formation of a bud and hence cell division .", "the yeast cell remodels its rigid structure to accommodate cell expansion during vegetative proliferation , mating pheromone - induced morphogenesis , and nutrient - driven filamentation through the cell wall integrity ( cwi ) signaling pathway .", "cell wall defects or damage induces the cells to activate the cwi pathway to survive , and the compensatory mechanism characterized by an increased chitin content is triggered .  ", "the cwi signaling pathway in s. cerevisiae is activated in response to low osmolarity , thermal stress , or mating pheromone and polarized growth .", "it is comprised of a family of cell surface sensors coupled to the small g - protein rho1p , which activates the cwi mapk cascade via protein kinase c ( pkc1p ) .", "this signaling cascade activates the expression of genes encoding for cell wall proteins that stabilize the cell wall .", "meanwhile , activated rho1p also activates a set of additional effectors such as bni1p and bnr1p formin proteins , skn7p transcription factor , and the sec3p exocyst component , which regulate a diverse set of processes including -glucan synthesis at the site of cell wall remodeling , gene expression related to cell wall biogenesis , organization of the actin cytoskeleton , and secretory vesicle targeting to the growth site .  ", "a family of cell surface sensors is responsible for detecting and transmitting the status of the cell wall to rho1p .", "these sensor molecules include wsc1p ( hcs77p / slg1p ) [ 2931 ] , wsc2p and wsc3p , and mid2p and mtl1p [ 32 , 33 ] . among these cell wall stress sensors , wsc1p and mid2p", "appear to be the most important and serve a partially overlapping role in cwi signaling .", "reduced o - mannosylation leads to incorrect proteolytic processing of these proteins , which in turn results in impaired activation of the pkc1 pathway and finally causes cell death in the absence of osmotic stabilization .", "more recently , n - glycan is found to be directly involved in mid2p sensing .", "it has been shown that both the extent of the n - linked glycan and its distance from the plasma membrane affect mid2p function .", "these observations demonstrate that n- and o - glycosylation are important for cwi sensing and thus important for cell wall biogenesis and polarized growth in yeast .  ", "the presence of a. fumigatus genes encoding for proteins homologous to the yeast rho1p , rho3p , and cdc42p suggests a similar mechanism for the cwi pathway .", "indeed , it has been recently shown that afcdc42/cdc42 , afrho1/rho1 , and afrho3/rho3 are highly expressed in the mutant devoid of cwh41p ( glucosidase i ) , which suggests an activation of these genes induced by cell wall damage . also ,", "increased expression and activation of the a. fumigatus mpkap , an ortholog of the s. cerevisiae mpk1p , is also induced by cell wall damage [ 38 , 39 ] .", "it is becoming clear that , as in yeast , defects in cell wall integrity also trigger the cwi mapk cascade in a. fumigatus . on the other hand ,", "in contrast to yeast , little is known about the cell wall stress sensors in a. fumigatus . in the last release of the a. fumigatus genomic database ( http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main ) , only one protein ( afua_5g09020 )", "is annotated as a homologue of the wsc4p , which does not appear to contribute to cwi signaling in yeast .", "therefore , the a. fumigatus cell wall stress sensor molecule remains to be identified and investigated .", "the precursor of all mannose residues found in galactomannan , glycoprotein , and gpi anchor in a. fumigatus is gdp - mannose .", "the activation of mannose initiates from formation of mannose 6-phosphate ( man-6-p ) , which occurs by one of two routes : direct phosphorylation of mannose by hexokinase or interconversion from fructose 6-phosphate ( fru-6-p ) via phosphomannose isomerase ( pmi ) , and the latter pathway requires three enzymes : pmi , phosphomannomutase ( pmm ) , and gdp - mannose pyrophosphorylase ( gmpp ) .", "fru-6-p is converted to man-6-p by pmi , and then man-6-p is converted to mannose 1-phosphate ( man-1-p ) by pmm .", "subsequently , man-1-p is ligated with the guanosine 5-triphosphate molecule ( gtp ) to form gdp - mannose by man-1-p guanylyltransferase [ 4063 ] .  ", "the interconversion of man-6-p and fru-6-p catalysed by pmi is the first committed step in the synthesis of man - containing sugar chains and provides a link between glucose metabolism and mannosylation .", "pmi deficiency is the cause of carbohydrate - deficient glycoprotein syndrome type ib ( cdg - ib , omim 602579 ) in humans , but the clinical symptoms and aberrant glycosylation can be corrected with dietary mannose supplements .  ", "genes encoding for pmis have been investigated in several fungal species , such as s. cerevisiae , candida albicans , a. nidulans , and cryptococcus neoformans [ 4851 ] .", "the pmi   mutant shows a significantly reduced growth rate at high concentrations of mannose .", "biochemical and genome - wide analysis reveals that excess mannose leads to an accumulation of man-6-p , which mainly inhibits the activity of phosphoglucose isomerase ( pgi ) and thus represses glycolysis , protein biosynthesis , and cell wall biogenesis .  ", "the a. nidulans mana1 mutant exhibits abnormal ballooning of hyphal tips and eventually ceases to grow . disrupted man1 mutant of c. neoformans leads to poor capsule formation , reduced polysaccharide secretion , morphological abnormalities , and attenuated virulence .", "in a. fumigatus , pmi activity is essential for viability and plays a central regulatory role in both glycosylation and energy production .", "deletion of the a. fumigatus   pmi1 gene leads to uncoupling of the link between energy production and glycosylation and accumulation of man-6-p , which then results in defects in cell wall integrity , conidiation , and morphology . although extracellular mannose can rescue the growth of pmi deficient mutants in a. fumigatus , both lower and higher concentrations of mannose lead to a reduction in the levels of -glucan in the cell wall and an accumulation of man-6-p .", "the phenotypes associated with the mutant under mannose starvation are mainly due to an insufficient supply of gdp - man required for cell wall synthesis .", "the abnormal morphology associated with the pmi1 mutant under mannose - replete conditions is mainly ascribed to an accumulation of man-6-p , which can not efficiently enter glycolysis , instead becoming trapped in a cycle of dephosphorylation and rephosphorylation resulting in depletion of intracellular atp .", "it should be pointed out that the pmi in a. fumigatus mainly catalyzes the conversion of fru-6-p to man-6-p , and its binding affinity for man-6-p is similar to that of yeasts but different from the ones from bacteria or animals ( table 1 ) .", "this suggests that it may be possible to design a specific inhibitor for fungal pmis .", "several gmpps have been identified and characterized in different species [ 5559 ] . in s. cerevisiae and c. albicans , gmpp is essential [ 60 , 61 ] , while in leishmania mexicana gmpp is not required for viability .", "repression of gmpp in yeast leads to pleiotropic phenotypes including cell lysis , failure of cell separation , impaired budding and hyphal switching , clumping and flocculation , and cell wall defects .", "repression of expression of a. fumigatus gmpp srb1 , a homologue of yeast srb1/psa1/vig9 , leads to hyphal lysis , a defective cell wall , impaired polarity maintenance , and branching site selection , as well as rapid germination and reduced conidiation .", "in contrast to yeast , inducible repression of srb1 expression in a. fumigatus does affect the ability to direct polarity establishment and septation .  ", "these reports imply that mannose activation is specifically crucial for the synthesis and organization of the cell wall and thus essential for survival of fungal species .", "this further suggests that glycosylation is essential for the viability of pathogenic fungal species such as a. fumigates , and inhibitors that specifically block mannose activation in fungi may be potential drugs to treat fungal infections .", "n - glycosylated proteins contain oligosaccharides that are n - glycosidically linked to the -amido group of asparagine .", "this type of glycoprotein has been intensively studied in many model systems from yeast to human cells with respect to their structure , biosynthesis , and function .", "it has been shown that the formation of the highly variable n - linked oligosaccharides is initiated by the assembly of a lipid - linked oligosaccharide glc3man9glcnac2-pp - dol by a series of glycosyltransferases located on the cytoplasmic and luminal faces of the er membrane .", "the most complete understanding of biosynthesis of the lipid bound precursor has been obtained from s. cerevisiae and from mammals . as far as it is known , the corresponding reactions proceed almost identically in other eukaryotes .   subsequently , the dol - pp - linked glc3man9glcnac2 is transferred as a whole to an asparagine residue within an n - x - t / s consensus sequence of a nascent peptide , which is catalyzed by the oligosaccharyltransferase ( ost ) , and then the n - glycosylated proteins are modified in a species - specific manner and transferred through the secretory pathway to the cell surface where they either get exported or anchored to the plasma membrane , to the extracellular matrix , or to the cell wall ( figure 1 ) .  ", "the ost complex consists of at least eight different subunits , including ost1p , ost2p , wbp1 , stt3p , swp1p , ost4p , ost5p , and ost3p / ost6p [ 6467 ] .", "although the function of each subunit is still unclear , stt3p is believed to be the catalytic subunit [ 6870 ] , and its homologues are found in almost all eukaryotes .", "it appears that the a. fumigatus   stt3 is also essential as no viable knockout mutant has been recovered .", "repression of the stt3 gene in a. fumigatus leads to a severe retardation of growth and a slight defect in cell wall integrity .", "further analysis shows that repression of stt3 upregulates expression of the genes responsible for glucan and chitin synthesis , especially gel1 , gel2 , fska , chse , and chsg .", "indeed , an increase of cell wall mannoprotein and chitin was observed following repression of the stt3 gene .", "however , this upregulation of chitin is not accompanied by an activation of the mpka kinase .", "indeed , only the unfolded protein response ( upr ) is induced . as the upr has been shown to be involved in cwi signaling in a. fumigatus ,", "it is likely that upr , instead of the mpka - dependent cwi signaling pathway , is the major compensatory mechanism induced by repression of the n - glycosylation in a. fumigatus .  ", "once glc3man9glcnac2 is transferred to proteins , the n - glycan is processed sequentially in the er and golgi .", "n - glycan processing is initiated by the removal of the glucose residues catalyzed by er glucosidase i and glucosidase ii .   in mammalian cells ,", "n - linked glycan plays a decisive role as a quality control ( qc ) of the folding of secretory proteins , which is composed of calnexin , calreticulin , udp - glucose : glycoprotein glucosyltransferase ( gt ) and glucosidase ii ( gii ) and is essential for cellular survival [ 7577 ] .", "n - glycans initially serve to increase the hydrophilicity of the as - yet - unstructured nascent polypeptides .", "subsequently , the two outermost glucose residues of the n - glycan are removed by the sequential action of glucosidase i ( gi ) and gii to the monoglucosylated form , which is recognized and bound by calnexin , a type i er membrane lectin , and calreticulin , its soluble relative . for many glycoproteins ,", "the interaction with calnexin or calreticulin slows down the rate of folding but increases efficiency .", "gii - catalyzed removal of the third glucose residue follows the dissociation of folding substrates from calnexin and is required for release of native polypeptides from the er and transport to their final destination .", "the folding sensor gt adds back a terminal glucose to promote reassociation of nonnative polypeptides released from calnexin , thus prolonging their retention in the er folding environment .", "cycles of de-/reglucosylation might be protracted until the polypeptide released from calnexin fulfills quality control requirements .", "when correct folding is not achieved , an er - specific n - glycan - dependent pathway of degradation removes the misfolded proteins .", "when n - glycosylation is inhibited , the most commonly observed effect is the generation of misfolded , aggregated proteins that fail to reach a functional state [ 75 , 76 ] .   before entering the qc system ,", "the outermost glucose residue of the n - glycan is trimmed by er glucosidase i .", "a human inherited glucosidase i deficiency has been reported to result in neonatal birth with severe generalized hypotonia and dysmorphic features . unlike mammalian cells ,", "s. cerevisiae lacks a calnexin cycle and gt and only has an effective mannosidase i - dependent erad system [ 80 , 81 ] . the yeast glucosidase i ( cwh1p ) is encoded by the cwh41 gene .", "mutational defects in the cwh41 gene cause severe and selective instability of glycoprotein kre6p , a putative golgi glucan synthase required for -l,6-glucan synthesis [ 23 , 83 , 84 ] .  ", "some filamentous fungi have been proposed to possess n - glycan - dependent qc of glycoprotein folding based on fungal genome sequence data . recently ,", "evidence that filamentous fungi possess an n - glycan - dependent qc system has been reported in a. fumigatus [ 37 , 86 ] .", "indeed , calnexin ( aas68033 ) , glucosidase ii , and gt have been annotated in the last release of the a. fumigatus genomic database ( figure 2 ) .", "zhang et al .   reported that deletion of the cwh41 gene in a. fumigatus results in defective n - glycan processing of the proteins secreted by a. fumigatus .", "although afcwh41 is not essential for hyphal growth and virulence , a severe reduction in conidial formation , abnormalities of polar growth and septation , and a temperature - sensitive deficiency of cell wall integrity were documented .", "also , the genes encoding rho - type gtpases ( rho - type gtpase / cdc42 ) were upregulated , which suggests that the cwi pathway was activated in the mutant .", "after processing by two er -glucosidases , the n - glycan is further processed by the action of various 1,2-mannosidases , which can remove one or more of the four 1,2-linked mannose residues . in mammalian cells , man9glcnac2 is converted to man5glcnac2 by the action of er and golgi -mannosidases , which is the precursor for complex , hybrid , and high - mannose n - glycans . in s. cerevisiae , a specific er 1,2-mannosidase converts man9glcnac2 into man8glcnac2 , which is elongated in the golgi to form an outer chain containing up to 200 residues of mannose [ 89 , 90 ] .", "a. saitoi and trichoderma reesei have been found to produce n - glycan structures containing five mannose units ( man5glcnac2 ) , suggesting further processing of the man9glcnac2 precursor [ 91 , 92 ] .", "the n - glycans on mature secreted glycoprotein produced by a. fumigatus are man6glcnac2 , man7glcnac2 , and man8glcnac2 , in which man6glcnac2 is the major glycoform .", "these observations demonstrate that n - glycan synthesis in filamentous fungi seems to differ from that in yeast and is similar to that in higher eukaryotes ( figure 3 ) .", "although small n - glycans are commonly found on glycoproteins of a. fumigatus , hex5 - 13hexnac2 glycans on the galactomannoproteins , and man5 - 9glcnac2 as well as galf1man5 - 7glcnac2 structures on other secreted glycoproteins have been identified in a. fumigatus [ 93 , 94 ] . the enzyme ( udp - galp mutase ) required to synthesize the requisite udp - galf donor has been shown to be an important factor in biosynthesis of the cell wall in a. fumigatus , while the gene / enzyme responsible for the transfer of galf has not been identified .", "recently , the a. fumigatus och1 , a key mannosyltransferase for synthesis of elaborated protein n - glycans in yeast , has been identified .", "deletion of the och1 gene results in a reduction of sporulation in the presence of high calcium concentrations .", "this evidence suggests that polymannosylated n - glycans exist in a. fumigatus and certain proteins engaged in sporulation require n - glycan outer chains to be fully functional .  ", "the -mannosidases have been classified into two groups : class i and class ii [ 102 , 120 , 121 ] .", "class i -mannosidases include er man9-mannosidase , endomannosidase , and golgi mannosidase i. class ii -mannosidases include the lysosomal mannosidases , golgi mannosidase ii , yeast vascular mannosidase [ 98 , 99 ] , and er -mannosidase ii [ 122 , 123 ] .", "several golgi -mannosidases have been cloned and characterized from penicillium citrinum [ 124 , 125 ] , a. saitoi , a. oryzae , t. reesei , and a. nidulans [ 102 , 129 ] .", "these enzymes are all monomeric with a molecular weight of 5060  kda and show the maximal activity in the semiacidic condition ( ph 46 ) .  ", "class i -mannosidase is known to play an important role in the processing of mannose - containing glycans . in drosophila melanogaster , deletion of the golgi mannosidase i ( mas-1 ) results in viable progeny , and the null organisms synthesize the same range of oligosaccharides as the wild - type ones , albeit at different ratios . in s. cerevisiae , disruption of the er -mannosidase gene does not prevent outer chain synthesis . in the last release of the tigr database ( http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main ) , nine a. fumigatus genes are annotated to encode -mannosidases , including xp_749038.1 , xp_754794.1 , xp_751252.1 , xp_751819.1 , xp_752444.1 , xp_752825.1 , xp_753592.1 , xp_751114.1 , and xp_750572.1 . among them , msdsp ( xp_752825.1 ) has been identified to encode a class i 1 , 2-mannosidase and acts on man8glcnac2 to produce man6glcnac2 .", "deletion of the msds gene leads to a defect in n - glycan processing , as well as a reduction of cell wall components ( including -glucan , -glucan , mannoprotein , and chitin ) and reduced conidiation .", ".   in mammalian cells , free oligosaccharides ( fos ) are generated by ost - mediated hydrolysis of glc3man9glcnac2-pp - dolichol in the lumen of the er or peptide n - glycanase ( pngase)-mediated de - n - glycosylation of newly synthesized glycoproteins either in the er or the cytosol .", "fos that are liberated in the lumen of the er can be transported into the cytosol , where they are trimmed by an endo--d - n - acetylglucosamine h ( endo h)-like enzyme and the -mannosidase man2c1p in order to yield an oligosaccharide , man5glcnac , that can be imported directly into lysosomes to be degraded ( figure 4 ) [ 122 , 123 , 131135 ] . in humans   and cattle [ 137139 ] , a deficiency in -mannosidase results in the lethal disease mannosidosis , a rare lysosomal storage disease with a collection of clinical symptoms including progressive mental retardation , impaired hearing , dysostosis multiplex , immune defects , elevation of serum and urinary oligosaccharide levels , and an enlargement of lysosomes in most cell types resulting from the accumulation of undegraded oligosaccharides .  ", "the rat man2c1p is involved in oligosaccharide catabolism of misfolded glycoproteins in the lumen of the er which have been retrotranslocated into the cytoplasm for proteolytic disposal [ 131133 ] .", "a proteolytically cleaved version of the rat man2c1p has been found in the lumen of the er where it is believed to be involved in the early stages of glycoprotein maturation ( also called er -mannosidase ii ) ( figure 4 ) [ 122 , 123 ] .  ", "the yeast cytosolic -mannosidase ams1p , a counterpart of man2c1p , is also involved in the processing of fos .", "since the yeast png1p is mainly localized to the cytosol , it is proposed that the png1p - generated fos may both be generated and processed in the cytosol .", "the role of the yeast ams1p is to aid in recycling macromolecular components of the cell under nutrient deprivation . interestingly , after its synthesis in the cytosol , the ams1p is translocated into the vacuole by the cytosol - to - vacuole targeting pathway , which suggests a common feature shared by the s. cerevisiae ams1p and its mammalian counterparts .", "however , the yeast ams1p only participates in recycling or utilizing of oligosaccharide but not in processing of n - glycan ( figure 4 ) .", "it should also be noted that no structural studies have been performed on the products that can be generated from man8glcnac2 by ams1p , and the ultimate fate of such products remains obscure . on the other hand , two png1p - independent fos pools , man3glcnac2 and man8glcnac2 , are also seen in s. cerevisiae .", "the pool comprising small fos ( man3glcnac2 ) appears to be disposed of by unknown enzymes in the vacuole .", "the pool containing mainly man8glcnac2 may be generated and disposed of along the secretory pathway .  ", "similarly , a. nidulans -mannosidase iic is also proposed to be involved in oligosaccharide catabolism .", "both a. nidulans-mannosidase iic and s. cerevisiae ams1p are not essential for normal cellular function since disruption of these genes has no visible effect on growth or morphology [ 98 , 99 , 102 ] .  ", "in contrast to its counterpart in yeast or a. nidulans , the a. fumigatus ams1p is required for normal cellular function .", "deletion of the a. fumigatus ams1 leads to a severe defect in conidial formation , especially at a higher temperature .", "these results show that the afams1 gene is required for morphogenesis and cellular function in a. fumigatus .", "the involvement of the afams1 gene in polarized growth demonstrates that the processes involved in fos regulation are important for a. fumigatus .", "it is likely that the ams1p is involved in cell wall synthesis and thus polarity through the cwi pathway .", "therefore , probably the afams1 mutant could serve as a simple model to investigate the mechanism of -mannosidosis .   functional analyses of the genes required for n - glycosylation reveal that protein", "n - glycosylation is important for cell wall synthesis , morphogenesis , and polarized growth in a. fumigatus .", "2-d gel analysis reveals that deletion of the cwh41 gene encoding glucosidase i in a. fumigatus leads to er stress , which induces overexpression of hsp70 and calnexin chaperone and activates the erad .", "meanwhile , the proteins required for actin rearrangement are found to be underexpressed or missing , which is consistent with the observation of random localization of actin fibers in the mutant .", "these observations , for the first time , clearly suggest that n - glycosylation contributes to proper folding and trafficking in a. fumigatus .", "it appears that proteins involved in cell wall biosynthesis in a. fumigatus are more dependent on the n - glycan - dependent folding system . as in yeast", ", cell wall defects also trigger the cwi signaling pathway in a. fumigatus , which activates downstream effectors that regulate cell wall biogenesis and polarized growth .", "zhang et al . [ 37 , 86 ] have proposed that the proteins required for cell wall synthesis or cell wall stress sensing are substrates of a. fumigatus cwh41p and require glucose trimming for their proper localization and function .", "misfolding of these proteins would cause cell wall defects , which then leads to activation of the erad and rho - type gtpases - mediated cwi pathway .", "moreover , activation of cdc42 in the cwi pathway also activates sepa , an upstream organizer of actin ring formation at septation sites , and thus causes abnormal polarized growth associated with the afcwh41 mutant ( figure 5 ) .", "although the phenotypes associated with different n - glycosylation mutants vary , the finding that all of these mutants exhibit phenotypes associated with cell wall defects , abnormal polarization , and morphological changes can all be explained by this proposed model .   obviously , more investigations are needed to identify and characterize all of the proteins affected by n - glycosylation in a. fumigatus .", "this information would be key to understanding the complex compensatory mechanisms participating in cell wall biosynthesis in a. fumigatus , which would serve as a basis to develop new antifungal therapies , as well as help to elucidate the molecular mechanism of human diseases associated with defects in glycosylation .", "o - mannose glycosylated proteins were first discovered in yeast and filamentous fungi , and recently this type of glycoproteins has also been described in mammals .", "the o - mannosylation most likely occurs in all animals , with the exception of nematodes ( e.g. , caenorhabditis elegans ) ; it is also not detected in plants ( arabidopsis thaliana , oryza sativa ) .", "however , it has also been discovered in one bacterial species ( mycobacterium tuberculosis ) . in mammalian cells ,", "the inner o - linked mannose is elongated with the first addition of a n - acetylglucosamine and then various sugars . in the case of yeast ,", "the o - mannose type carbohydrate chain starts with a serine / threonine - linked mannose , which is extended to an oligomannose chain . in a. fumigatus ,", "the o - linked glycans on cell wall mannoproteins are found to be glc1 , 6man , galf1 , 6man1 , 6man , galf1 , 5galf1 , 6man1 , 6man and galf1 , 5[galf1,5]3 galf1 , 6man , while only a single mannose residue was detected on secreted proteins .  ", "a further type of protein o - glycosylation , in which a single -o - linked glcnac residue is linked to serine and threonine occurs in animals , plants , and filamentous fungi , but not in s. cerevisiae . for this type of protein modification ,", "protein o - mannosylation is initiated by a family of protein o - mannosyltransferases ( pmts ) that are evolutionarily conserved from yeast to human [ 145 , 146 ] . in s. cerevisiae", "a total of seven pmt family members ( scpmt17p ) are present [ 104 , 147 ] , which fall into three major groups of homology : ( i ) pmt1/5/7 , ( ii ) pmt2/3/6 , and ( iii ) pmt4 .", "genes with significant homology to pmts have been cloned in humans , mice , and drosophila [ 148150 ] .", "specific protein substrates that are o - mannosylated by scpmt1p , scpmt2p , or scpmt4p have been described in s. cerevisiae [ 151153 ] . in comparison with s. cerevisiae , the pmt family is less redundant in higher eukaryotes . in drosophila", "only two pmt family members are present ( rotated abdomen and twisted ) [ 149 , 150 ] . the same is true for mice and humans ( pomt1 and pomt2 ) [ 148 , 150 ] .", "mutations in human pomt1 , a homologue of the yeast pmt4 , cause walker - warburg syndrome ( wws ) , which is characterized by severe congenital muscular dystrophy , neuronal migration defects , and structural abnormalities of the eye . targeted deletion of pomt1 in mice results in embryonic lethality due to defects in the formation of the reichert 's membrane , the first basement membrane to form in the embryo .", "mutations of the drosophila pmt homologues alter muscle structures and the alignment of adult cuticle . the pmt family is crucial for viability , cell wall integrity , and morphogenesis in several fungal species , such as s. cerevisiae , s. pombe , c. albicans and c. neoformans , a. nidulans , and a. fumigatus [ 108 , 109 , 111115 , 145 , 156 ] . in s. cerevisiae , single pmt1 mutants fail to grow in anaerobic conditions on some media .", "the pmt1,2,3-triple mutants grow only in osmotically stabilized medium , whereas the pmt1,2,4- and pmt2,3,4-triple mutants are not viable in any conditions , indicating that pmt protein activity is essential in s. cerevisiae , although individual genes are dispensable .  \n c. albicans contains five pmt genes .", "the pmt1 mutants are viable , but they are defective in undergoing cellular differentiation from yeast to a true hyphal growth form under some conditions .", "the virulence of the pmt1 null mutant is significantly attenuated , which is likely due to reduced o - glycosylation of the c. albicans adhesin als1p .", "the pmt phenotypes are closely linked to alterations in cell wall components , including cell wall mannoproteins and polysaccharides .   in s. pombe only one member of each pmt subfamily", "is present , namely , oma1 , oma2 , and oma4 . deletion of oma2 , as well as simultaneous deletion of oma1 and oma4 is lethal .", "characterization of the viable s. pombe oma1d and oma4d single mutants shows that reduced o - mannosylation results in abnormal cell wall and septum formation , therefore severely affecting cell morphology and cell - cell separation . in c. neoformans ,", "recently , willger et al . showed that pmt2 is an essential gene , and the double pmt1pmt4 deletion is lethal .", "filamentous fungi , such as a. fumigatus , a. nidulans , neurospora crassa , and fusarium gramineum , contain only three pmt genes that belong to the pmt1 , pmt2 , and pmt4 subfamilies , respectively [ 107 , 108 , 113 ] .", "all single pmt mutants in a. nidulans are viable but showed reduced growth at elevated temperatures and defects in morphogenesis [ 111 , 112 ] .", "the double deletion pmta / pmtc ( orthologues of the pmt2 and pmt4 ) and pmtb / pmtc ( orthologues of pmt1 and pmt4 ) are synthetically lethal .  ", "have shown that a single deletion of a. fumigatus   pmt1 results in temperature - sensitive phenotypes .", "when the a. fumigatus pmt1 mutant was grown on solid complete medium at 37c , no difference was found between the mutant and the wild type .", "a strongly retarded growth , however , was observed when this mutant was grown at 42c and 50c .", "this temperature - sensitive phenotype could be complemented by the addition of 1  m sucrose in the media .", "further analysis shows that the mannoprotein , -glucan , and chitin in the cell wall of the mutant grown at 37c are increased , while -glucan is reduced . when the a. fumigatus pmt1 mutant was cultured at 42c ,", "the -glucan was increased , while the -glucan was decreased , and the mannoprotein and chitin content remained unchanged .", "moreover , deficient conidiation and reduced germination have been documented at 42c . as compared with the s. cerevisiae   pmt1 mutants , the a. fumigatus pmt1 mutant , as well as the c. albicans and s. pombe   pmt1 mutants , shows more severe defects in cell wall integrity .", "this significant phenotype could be explained by the fewer members of pmt family presented in a. fumigatus , c. albicans , and s. pombe .", "however , in a recent study by mouyna et al . , the a. fumigatus pmt1 mutant does not show any visible phenotype . in the report by zhuo et al .", ", the pmt1 deletion mutant was constructed by replacement of the entire coding region of the pmt1 in a. fumigatus strain cea17 ( pyrg ) with a pyrg cassette .", "therefore , the genetic background of the pmt1 null mutant is pyrg  pmt1 , while in the report by mouyna et al .", ", the pmt1 mutant was constructed by transformation of a. fumigatus strain ku80 with a deletion cassette containing the e. coli phleomycin phosphotransferase gene ( phle ) .", "therefore , the major differences may be due to the different genetic background of the strains used in these two reports .", "the single pmt2 or double pmt1pmt4 deletion(s ) are lethal [ 114 , 115 ] .", "fang et al .   reported that reduced expression of pmt2 leads to retarded growth , cell wall defects , abnormal polarity , and reduced conidiation ; however , no temperature - sensitive growth was found .", "interestingly , this is the first time that pmt2p is revealed to be involved in polarized growth .", "these observations suggest that a. fumigatus pmt2p is required for cell wall synthesis and morphogenesis and its function is distinct from that of a. fumigatus pmt1p .  ", "disruption of a. fumigatus pmt4 leads to abnormal mycelial growth , poor conidiation , and abnormal polarity .", "although an increased sensitivity to echinocandin , a 1,3-glucan synthase inhibitor , was observed in the a. fumigatus pmt4 null mutant , glucan synthase activity and 1,3-glucan content were not affected .", "in contrast to its counterpart in c. albicans , a. fumigatus   pmt4 is not required for full virulence .  ", "the different functions associated with different a. fumigatus pmts are likely due to their different substrate specificities .", "further investigation of the pmt mutants will be helpful for understanding their molecular mechanism , which will not only increase our understanding of the function of o - mannosylation in a. fumigatus , but also may deepen our understanding of the molecular basis of the human walker - warburg syndrome ( wws ) which features mutations in pomt1 , a homologue of a. fumigatus pmt4p , and results in a failure of polarized growth during neuronal migration .", "gpi anchoring is a conserved glycosylation process in eukaryotes , which enables many cell surface proteins such as cell surface enzymes , receptors , and adhesion molecules to be covalently anchored to the cell membrane .", "the core structure of the gpi anchor consists of a lipid group , myoinositol , glucosamine , several mannose residues , and a phosphoethanolamine group , which ultimately connects the gpi anchor to the protein via an amide bond .", "although the number of mannose groups and the position of side - chains on the gpi anchors vary widely between species , a common core structure of etnman3glcn - pi is conserved in all gpi - anchored proteins found in protozoa , yeast , plants , and mammals ( figure 6 ) .  ", "gpi anchoring is not essential in mammals at a cellular level as several gpi - deficient cell lines have been established .", "however , an acquired gpi - anchoring deficiency in haematopoietic stem cells causes paroxysmal nocturnal haemoglobinuria , a rare but serious human disease .", "also an overexpression of pig - p , a protein of unknown function required for gpi anchor synthesis , has been noted in fetal down syndrome brain .", "in contrast to mammals , gpi anchor synthesis is essential in s. cerevisiae . in s. cerevisiae , many gpi - anchored proteins are known to be involved in morphogenesis and cell wall organization .", "one type is the gpi - mannoproteins covalently linked to cell wall -1,6-glucan which play important biological functions in filamentation , mating , flocculation , or adhesion to the external matrix [ 161167 ] .", "the second type are the gpi proteins associated with the plasma membrane which possess enzymatic activities able to modify cell wall polymers and are involved in altering cell morphology , such as -glucanase and -glucosyltransferase [ 168170 ] .", "recent studies in a. fumigatus suggest that at least nine gpi - anchored proteins are common to filamentous fungi and yeast .", "five of them are homologues of putative gpi - anchored yeast proteins that have been shown to play a role in cell wall morphogenesis .", "the gpi anchor is assembled at the er in multiple steps catalyzed by the concerted actions of approximately 20 proteins .", "the first step of gpi anchor synthesis is initiated by the transfer of n - acetylglucosamine ( glcnac ) from udp - glcnac to phosphatidylinositol ( pi ) , which is catalyzed by the glycosylphosphatidylinositol - n - acetyl - glucosaminyltransferase ( gpi - gnt ) complex .", "the mammalian gpi - gnt complex consists of seven proteins , including pig - a , pig - h , pig - c , pig - p , gpi1 , pig - y , and dpm2 .", "all except dpm2 have structural and functional counterparts in s. cerevisiae , where they are known as gpi3p , gpi15p , gpi2p , gpi19p , gpi1p , and eri1p , respectively .", "pig - a / gpi3p is believed to possess the catalytic domain because gpi3p binds a photoactivatable udp - glcnac analog and is a member of glycosyltransferase family 4 of retaining glycosyltransferases .", "the roles of the other subunits in the gpi - gnt complex are as yet unclear , but they may mediate regulatory interactions .   in yeast ,", "gpi anchoring is essential for viability and plays an important role in the biosynthesis and organization of the cell wall .", "a gpi3 temperature - sensitive mutant is not viable at 37c [ 116 , 117 ] .", ", it is postulated that the introduction of mutations in gpi3/gpig-1 genes allows for minimal level of product function and survival when growing the mutant cells below the restrictive temperatures .", "however , the mechanism , by which the defect in gpi anchoring leads to a lethal phenotype in these two species , is poorly understood .  ", "it has been shown that a. fumigatus gpi anchors possess five mannose residues with a phosphoethanolamine linked on the first three residues [ 174 , 175 ] .", "the a. fumigatus pig - a gene , the homologue of the gpi3/pig - a gene in yeast , has been investigated .", "also , an increased content of -glucan and mannoprotein was observed in the mycelial cell wall of the afpig - a mutant .", "unlike the temperature - sensitive or conditional lethal phenotype seen in the yeast gpi3 mutant , afpig - a can survive at temperatures from 30c to 50c . completely blocking gpi anchor synthesis in a. fumigatus afpig - a leads to cell wall defects , abnormal hyphal growth , rapid conidial germination , and aberrant conidiation . in vivo assays", "therefore , the gpi anchor seems not essential for viability , but required for cell wall integrity , morphogenesis and virulence in a. fumigatus .", "indeed , this is the first report that a deficiency in gpi - anchor synthesis does not lead to a temperature - sensitive or conditional lethal phenotype in microbes , which provides an opportunity to identify the basic function of gpi anchoring in fungi .", "during the past 50 years , proteins and nucleic acids have dominated the field of biology .", "the enormous advances in the analysis of complex carbohydrates have enabled us to investigate the structure and function of carbohydrates , and the field has developed enormously .", "it is now known that carbohydrates play very important roles , especially in the regulation of development of higher organisms .", "however , the mechanisms by which carbohydrates play a role in development and diseases are still poorly understood .", "our knowledge of protein glycosylation comes mainly from the investigation of s. cerevisiae and mammalian cells .", "although investigations of the model yeast and mammalian cells have been very useful in elucidating the biochemical features of protein glycosylation , these investigations at the cellular level can not reflect the complicated functions of glycosylation in the development of multicellular eukaryotes .", "therefore , more model systems have been introduced , such as caenorhabditis elegans , drosophila melanogaster , and mice . however , these model systems are still too complex since deletion of individual glycosyltransferase genes in these systems sometimes leads to fetal death or nonvisible phenotypes .", "as compared with s. cerevisiae , c. elegans , d. melanogaster , or mice , a. fumigatus seems to be an ideal model for investigation of the function of glycosylation since a. fumigatus is a multicellular eukaryote with a relative simple life cycle , in which it undergoes a series of developmental events that require polarized growth .", "recent progress shows that a. fumigatus has evolved an intact n - glycan - dependent qc system , which is present in mammalian cells but not in yeast . disruption of either processing or degradation of n - glycan in a. fumigatus leads to phenotypes such as cell wall defects and abnormal polarity .  ", "based on investigations of s. cerevisiae and filamentous fungi , it is likely that glycosylation first evolved to ensure synthesis of the fungal cell wall and only later did the n - glycan - dependent qc system evolve to ensure precisely controlled cell wall synthesis and polarized growth which are important for multicellular development . however , this hypothesis is controversial .", ".   showed that the n - glycan - dependent qc system is functional in entamoeba , trichomonas , cryptococcus , and s. pombe , but is not functional in some fungi such as giardia and plasmodium , theileria , encephalitozoon , toxoplasma , cryptosporidium , and tetrahymena .", "they proposed that the n - glycan - dependent qc system was likely present in the common ancestor of extant eukaryotes and was secondarily lost from some eukaryotes .", "for example , the s. cerevisiae kre5 is believed to be the gt ortholog that no longer glucosylates misfolded glycoproteins but is instead thought to be involved in -1,6-glucan synthesis .", "of course , the possibility that the s. cerevisiae kre5 is the ancestor of gt can not be excluded .", "it remains unclear where is the evolutionary origin of glycosylation , what is the basic function of glycosylation at the early stages of evolution , and how glycosylation is regulated .", "definitely , the answers to these questions will enable us to understand the basic function and regulation of glycosylation in the development of multicellular eukaryotes and help to understand more complex functions in higher eukaryotes .", "on the other hand , the investigation of a. fumigatus is also a key to understanding complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development . during the past few years", ", the framework of the biosynthetic pathways of glycosylation in a. fumigatus has been delineated .", "functional analyses of some of the genes in this pathway have shown that glycosylation is required for cell wall synthesis , polarity , morphogenesis , and cellular function in a. fumigatus ( figure 7 and table 2 ) .", "however , a detailed understanding of this pathway remains unknown , such as details regarding the synthesis of the n - glycan precursor , the precise molecular mechanism of n - glycan processing , qc of protein folding , and modification of the gpi anchor .", "moreover , the molecular mechanisms by which glycosylation plays a role in morphogenesis and development of a. fumigatus are vaguely understood .", "therefore , the future direction would be looking for those key proteins that are affected by glycosylation and identifying the signal transduction pathways that link glycosylation and development , through genetic , biochemical , cell biological , and proteomic studies ."], "abstract_text": ["<S> glycosylation is a conserved posttranslational modification that is found in all eukaryotes , which helps generate proteins with multiple functions . </S>", "<S> our knowledge of glycosylation mainly comes from the investigation of the yeast saccharomyces cerevisiae and mammalian cells . </S>", "<S> however , during the last decade , glycosylation in the human pathogenic mold aspergillus fumigatus has drawn significant attention . </S>", "<S> it has been revealed that glycosylation in a. fumigatus is crucial for its growth , cell wall synthesis , and development and that the process is more complicated than that found in the budding yeast s. cerevisiae . </S>", "<S> the present paper implies that the investigation of glycosylation in a. fumigatus is not only vital for elucidating the mechanism of fungal cell wall synthesis , which will benefit the design of new antifungal therapies , but also helps to understand the role of protein glycosylation in the development of multicellular eukaryotes . </S>", "<S> this paper describes the advances in functional analysis of protein glycosylation in a. fumigatus . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Cell Wall Organization and Its Compensatory Mechanism in ", "3. Importance of Glycosylation in ", "4. Biosynthesis and Function of N-Glycosylation in ", "5. Biosynthesis and Function of O-Glycosylation", "6. Biosynthesis and Function of GPI Anchoring in ", "7. Outlook"], "sections": [["the aspergilli are filamentous fungi , which are multicellular eukaryotes with a relatively simple life cycle .", "many of them have long been used in food production , industrial fermentation , and agriculture . on the other hand , a few , such as a. fumigatus , a. flavus , a. niger , a. nidulans , and a. terreus , are opportunistic fungal pathogens , causing life - threatening invasive aspergillosis ( ia ) in immunosuppressed patients [ 1 , 2 ] , in which a. fumigatus is the predominant pathogen [ 35 ] .", "the crude mortality for ia is 6090% and remains around 2942% even when treatment is given .", "the main reasons for patient death are late diagnosis and the low efficiency of the drug therapies available to treat ia .  ", "it maintains cell shape and provides osmotic protection [ 7 , 8 ] and has therefore been recognized for a long time as an ideal drug target .", "indeed , several cell - wall - targeted drugs , such as echinocandins , caspofungin , micafungin , and anidulafungin , have been introduced as therapies .", "for example , echinocandins , which inhibit synthesis of -1,3-glucan , a crucial component of the cell wall are effective in the treatment of invasive fungal infections including ia .", "unfortunately , the echinocandins also trigger an increase of chitin [ 9 , 10 ] , which partially compensates for the loss of -1,3-glucan and reduces the efficiency of treatment due to the complicated mechanism of cell wall biogenesis in a. fumigatus .", "therefore , a more profound understanding of the mechanisms of cell wall biosynthesis in a. fumigatus would help to improve the efficiency of drug therapies , especially for drugs which target the cell wall .", "the cell wall of a. fumigatus is composed of a unique -1,3/1,4-glucan skeleton with chitin and galactomannan covalently linked to the nonreducing ends of -1,3-glucan . the cell wall is mainly coated with gpi proteins , which contain n- and o - glycans [ 11 , 12 ] . while there is no doubt that glycosylation is involved in cell wall organization , the functional importance of protein glycosylation in cell wall organization has , until recently , remained poorly understood .", "however , during the past few years , it has become increasingly evident that glycosylation is vital for cell wall synthesis and thus vital for growth and morphology of a. fumigatus .  ", "basically , all eukaryotes possess three types of protein glycosylation , n - glycosylation of asparagine residues , o - glycosylation of threonine and serine residues , and glycosylphosphatidylinositol - anchoring ( gpi - anchoring ) of the c - terminus of some proteins .", "humans lacking individual glycosyltransferases suffer from severe congenital diseases , known as carbohydrate - deficient glycoprotein syndromes ( cdgs ) [ 1214 ] .", "clearly , the sugar components of proteins play a major role in embryonic and postembryonic development of humans as well as of all higher eukaryotes .", "however , the molecular details leading to cdgs are only vaguely understood . during the past 20 years", "it is now known that carbohydrates play increasingly important roles in regulating the development of higher organisms . however , the mechanism by which carbohydrates play a role in development and disease is still unclear .", "our knowledge of protein glycosylation comes mainly from investigation of the model yeast s. cerevisiae and of mammalian cells .", "although investigation of the model yeast has been very useful in elucidating the biochemical features of protein glycosylation at the cellular level , they can not reveal the complicated functions of glycosylation in the development of multicellular eukaryotes .", "therefore , investigation of glycosylation in the multicellular fungus a. fumigatus not only helps understand the mechanism of cell wall synthesis in this species but also provides insights into the role of glycosylation in the development of multicellular eukaryotes .", "this paper concentrates on protein glycosylation in a. fumigatus , which will be discussed with respect to the enzymatic pathways involved and their functional importance .", "furthermore , the utility of a. fumigatus as a model for glycosylation during development of multicellular eukaryotes will be outlined ."], ["the cell wall of a. fumigatus is mainly composed of -1,3-glucans that are highly branched with -1,6 linkages . together", "they constitute a three - dimensional network with a large number of nonreducing ends , to which chitin , galactomannan , and -1,3/1,4-glucan are covalently anchored . the cell wall is mainly coated with gpi proteins , which contain n- and o - glycans derived primarily from the process of glycosylation [ 11 , 17 ] .", "deletion of gel2 leads to slower growth , abnormal conidiogenesis , an altered cell wall composition , and reduced virulence [ 18 , 19 ] .", "it has been proposed that gel2p is responsible for the elongation of -1,3-glucan side chains of -1,3/1,6 branched glucan to provide new nonreducing ends .", "ecm33p is also involved in maintaining proper cell wall architecture though its function is unknown .", "disruption of ecm33 results in morphogenetic aberrations such as defects in conidial separation , increase of chitin in conidial cell walls , rapid conidial germination , and increased virulence [ 20 , 21 ] .", "it is clear that glycoproteins are directly , as structural components of the cell wall , and indirectly , as enzymes required for cell wall synthesis , involved in maintaining proper cell wall architecture in a. fumigatus .", "increased chitin synthesis has been known as an important compensatory response to cell wall stress both in s. cerevisiae and filamentous fungi [ 2226 ] . in s. cerevisiae , the cell wall is required to maintain cell shape , which is essential for the formation of a bud and hence cell division .", "the yeast cell remodels its rigid structure to accommodate cell expansion during vegetative proliferation , mating pheromone - induced morphogenesis , and nutrient - driven filamentation through the cell wall integrity ( cwi ) signaling pathway .", "cell wall defects or damage induces the cells to activate the cwi pathway to survive , and the compensatory mechanism characterized by an increased chitin content is triggered .  ", "the cwi signaling pathway in s. cerevisiae is activated in response to low osmolarity , thermal stress , or mating pheromone and polarized growth .", "it is comprised of a family of cell surface sensors coupled to the small g - protein rho1p , which activates the cwi mapk cascade via protein kinase c ( pkc1p ) .", "this signaling cascade activates the expression of genes encoding for cell wall proteins that stabilize the cell wall .", "meanwhile , activated rho1p also activates a set of additional effectors such as bni1p and bnr1p formin proteins , skn7p transcription factor , and the sec3p exocyst component , which regulate a diverse set of processes including -glucan synthesis at the site of cell wall remodeling , gene expression related to cell wall biogenesis , organization of the actin cytoskeleton , and secretory vesicle targeting to the growth site .  ", "a family of cell surface sensors is responsible for detecting and transmitting the status of the cell wall to rho1p .", "these sensor molecules include wsc1p ( hcs77p / slg1p ) [ 2931 ] , wsc2p and wsc3p , and mid2p and mtl1p [ 32 , 33 ] . among these cell wall stress sensors , wsc1p and mid2p", "appear to be the most important and serve a partially overlapping role in cwi signaling .", "reduced o - mannosylation leads to incorrect proteolytic processing of these proteins , which in turn results in impaired activation of the pkc1 pathway and finally causes cell death in the absence of osmotic stabilization .", "more recently , n - glycan is found to be directly involved in mid2p sensing .", "it has been shown that both the extent of the n - linked glycan and its distance from the plasma membrane affect mid2p function .", "these observations demonstrate that n- and o - glycosylation are important for cwi sensing and thus important for cell wall biogenesis and polarized growth in yeast .  ", "the presence of a. fumigatus genes encoding for proteins homologous to the yeast rho1p , rho3p , and cdc42p suggests a similar mechanism for the cwi pathway .", "indeed , it has been recently shown that afcdc42/cdc42 , afrho1/rho1 , and afrho3/rho3 are highly expressed in the mutant devoid of cwh41p ( glucosidase i ) , which suggests an activation of these genes induced by cell wall damage . also ,", "increased expression and activation of the a. fumigatus mpkap , an ortholog of the s. cerevisiae mpk1p , is also induced by cell wall damage [ 38 , 39 ] .", "it is becoming clear that , as in yeast , defects in cell wall integrity also trigger the cwi mapk cascade in a. fumigatus . on the other hand ,", "in contrast to yeast , little is known about the cell wall stress sensors in a. fumigatus . in the last release of the a. fumigatus genomic database ( http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main ) , only one protein ( afua_5g09020 )", "is annotated as a homologue of the wsc4p , which does not appear to contribute to cwi signaling in yeast .", "therefore , the a. fumigatus cell wall stress sensor molecule remains to be identified and investigated ."], ["the precursor of all mannose residues found in galactomannan , glycoprotein , and gpi anchor in a. fumigatus is gdp - mannose .", "the activation of mannose initiates from formation of mannose 6-phosphate ( man-6-p ) , which occurs by one of two routes : direct phosphorylation of mannose by hexokinase or interconversion from fructose 6-phosphate ( fru-6-p ) via phosphomannose isomerase ( pmi ) , and the latter pathway requires three enzymes : pmi , phosphomannomutase ( pmm ) , and gdp - mannose pyrophosphorylase ( gmpp ) .", "fru-6-p is converted to man-6-p by pmi , and then man-6-p is converted to mannose 1-phosphate ( man-1-p ) by pmm .", "subsequently , man-1-p is ligated with the guanosine 5-triphosphate molecule ( gtp ) to form gdp - mannose by man-1-p guanylyltransferase [ 4063 ] .  ", "the interconversion of man-6-p and fru-6-p catalysed by pmi is the first committed step in the synthesis of man - containing sugar chains and provides a link between glucose metabolism and mannosylation .", "pmi deficiency is the cause of carbohydrate - deficient glycoprotein syndrome type ib ( cdg - ib , omim 602579 ) in humans , but the clinical symptoms and aberrant glycosylation can be corrected with dietary mannose supplements .  ", "genes encoding for pmis have been investigated in several fungal species , such as s. cerevisiae , candida albicans , a. nidulans , and cryptococcus neoformans [ 4851 ] .", "the pmi   mutant shows a significantly reduced growth rate at high concentrations of mannose .", "biochemical and genome - wide analysis reveals that excess mannose leads to an accumulation of man-6-p , which mainly inhibits the activity of phosphoglucose isomerase ( pgi ) and thus represses glycolysis , protein biosynthesis , and cell wall biogenesis .  ", "the a. nidulans mana1 mutant exhibits abnormal ballooning of hyphal tips and eventually ceases to grow . disrupted man1 mutant of c. neoformans leads to poor capsule formation , reduced polysaccharide secretion , morphological abnormalities , and attenuated virulence .", "in a. fumigatus , pmi activity is essential for viability and plays a central regulatory role in both glycosylation and energy production .", "deletion of the a. fumigatus   pmi1 gene leads to uncoupling of the link between energy production and glycosylation and accumulation of man-6-p , which then results in defects in cell wall integrity , conidiation , and morphology . although extracellular mannose can rescue the growth of pmi deficient mutants in a. fumigatus , both lower and higher concentrations of mannose lead to a reduction in the levels of -glucan in the cell wall and an accumulation of man-6-p .", "the phenotypes associated with the mutant under mannose starvation are mainly due to an insufficient supply of gdp - man required for cell wall synthesis .", "the abnormal morphology associated with the pmi1 mutant under mannose - replete conditions is mainly ascribed to an accumulation of man-6-p , which can not efficiently enter glycolysis , instead becoming trapped in a cycle of dephosphorylation and rephosphorylation resulting in depletion of intracellular atp .", "it should be pointed out that the pmi in a. fumigatus mainly catalyzes the conversion of fru-6-p to man-6-p , and its binding affinity for man-6-p is similar to that of yeasts but different from the ones from bacteria or animals ( table 1 ) .", "this suggests that it may be possible to design a specific inhibitor for fungal pmis .", "several gmpps have been identified and characterized in different species [ 5559 ] . in s. cerevisiae and c. albicans , gmpp is essential [ 60 , 61 ] , while in leishmania mexicana gmpp is not required for viability .", "repression of gmpp in yeast leads to pleiotropic phenotypes including cell lysis , failure of cell separation , impaired budding and hyphal switching , clumping and flocculation , and cell wall defects .", "repression of expression of a. fumigatus gmpp srb1 , a homologue of yeast srb1/psa1/vig9 , leads to hyphal lysis , a defective cell wall , impaired polarity maintenance , and branching site selection , as well as rapid germination and reduced conidiation .", "in contrast to yeast , inducible repression of srb1 expression in a. fumigatus does affect the ability to direct polarity establishment and septation .  ", "these reports imply that mannose activation is specifically crucial for the synthesis and organization of the cell wall and thus essential for survival of fungal species .", "this further suggests that glycosylation is essential for the viability of pathogenic fungal species such as a. fumigates , and inhibitors that specifically block mannose activation in fungi may be potential drugs to treat fungal infections ."], ["n - glycosylated proteins contain oligosaccharides that are n - glycosidically linked to the -amido group of asparagine .", "this type of glycoprotein has been intensively studied in many model systems from yeast to human cells with respect to their structure , biosynthesis , and function .", "it has been shown that the formation of the highly variable n - linked oligosaccharides is initiated by the assembly of a lipid - linked oligosaccharide glc3man9glcnac2-pp - dol by a series of glycosyltransferases located on the cytoplasmic and luminal faces of the er membrane .", "the most complete understanding of biosynthesis of the lipid bound precursor has been obtained from s. cerevisiae and from mammals . as far as it is known , the corresponding reactions proceed almost identically in other eukaryotes .   subsequently , the dol - pp - linked glc3man9glcnac2 is transferred as a whole to an asparagine residue within an n - x - t / s consensus sequence of a nascent peptide , which is catalyzed by the oligosaccharyltransferase ( ost ) , and then the n - glycosylated proteins are modified in a species - specific manner and transferred through the secretory pathway to the cell surface where they either get exported or anchored to the plasma membrane , to the extracellular matrix , or to the cell wall ( figure 1 ) .  ", "the ost complex consists of at least eight different subunits , including ost1p , ost2p , wbp1 , stt3p , swp1p , ost4p , ost5p , and ost3p / ost6p [ 6467 ] .", "although the function of each subunit is still unclear , stt3p is believed to be the catalytic subunit [ 6870 ] , and its homologues are found in almost all eukaryotes .", "it appears that the a. fumigatus   stt3 is also essential as no viable knockout mutant has been recovered .", "repression of the stt3 gene in a. fumigatus leads to a severe retardation of growth and a slight defect in cell wall integrity .", "further analysis shows that repression of stt3 upregulates expression of the genes responsible for glucan and chitin synthesis , especially gel1 , gel2 , fska , chse , and chsg .", "indeed , an increase of cell wall mannoprotein and chitin was observed following repression of the stt3 gene .", "however , this upregulation of chitin is not accompanied by an activation of the mpka kinase .", "indeed , only the unfolded protein response ( upr ) is induced . as the upr has been shown to be involved in cwi signaling in a. fumigatus ,", "it is likely that upr , instead of the mpka - dependent cwi signaling pathway , is the major compensatory mechanism induced by repression of the n - glycosylation in a. fumigatus .  ", "once glc3man9glcnac2 is transferred to proteins , the n - glycan is processed sequentially in the er and golgi .", "n - glycan processing is initiated by the removal of the glucose residues catalyzed by er glucosidase i and glucosidase ii .   in mammalian cells ,", "n - linked glycan plays a decisive role as a quality control ( qc ) of the folding of secretory proteins , which is composed of calnexin , calreticulin , udp - glucose : glycoprotein glucosyltransferase ( gt ) and glucosidase ii ( gii ) and is essential for cellular survival [ 7577 ] .", "n - glycans initially serve to increase the hydrophilicity of the as - yet - unstructured nascent polypeptides .", "subsequently , the two outermost glucose residues of the n - glycan are removed by the sequential action of glucosidase i ( gi ) and gii to the monoglucosylated form , which is recognized and bound by calnexin , a type i er membrane lectin , and calreticulin , its soluble relative . for many glycoproteins ,", "the interaction with calnexin or calreticulin slows down the rate of folding but increases efficiency .", "gii - catalyzed removal of the third glucose residue follows the dissociation of folding substrates from calnexin and is required for release of native polypeptides from the er and transport to their final destination .", "the folding sensor gt adds back a terminal glucose to promote reassociation of nonnative polypeptides released from calnexin , thus prolonging their retention in the er folding environment .", "cycles of de-/reglucosylation might be protracted until the polypeptide released from calnexin fulfills quality control requirements .", "when correct folding is not achieved , an er - specific n - glycan - dependent pathway of degradation removes the misfolded proteins .", "when n - glycosylation is inhibited , the most commonly observed effect is the generation of misfolded , aggregated proteins that fail to reach a functional state [ 75 , 76 ] .   before entering the qc system ,", "the outermost glucose residue of the n - glycan is trimmed by er glucosidase i .", "a human inherited glucosidase i deficiency has been reported to result in neonatal birth with severe generalized hypotonia and dysmorphic features . unlike mammalian cells ,", "s. cerevisiae lacks a calnexin cycle and gt and only has an effective mannosidase i - dependent erad system [ 80 , 81 ] . the yeast glucosidase i ( cwh1p ) is encoded by the cwh41 gene .", "mutational defects in the cwh41 gene cause severe and selective instability of glycoprotein kre6p , a putative golgi glucan synthase required for -l,6-glucan synthesis [ 23 , 83 , 84 ] .  ", "some filamentous fungi have been proposed to possess n - glycan - dependent qc of glycoprotein folding based on fungal genome sequence data . recently ,", "evidence that filamentous fungi possess an n - glycan - dependent qc system has been reported in a. fumigatus [ 37 , 86 ] .", "indeed , calnexin ( aas68033 ) , glucosidase ii , and gt have been annotated in the last release of the a. fumigatus genomic database ( figure 2 ) .", "zhang et al .   reported that deletion of the cwh41 gene in a. fumigatus results in defective n - glycan processing of the proteins secreted by a. fumigatus .", "although afcwh41 is not essential for hyphal growth and virulence , a severe reduction in conidial formation , abnormalities of polar growth and septation , and a temperature - sensitive deficiency of cell wall integrity were documented .", "also , the genes encoding rho - type gtpases ( rho - type gtpase / cdc42 ) were upregulated , which suggests that the cwi pathway was activated in the mutant .", "after processing by two er -glucosidases , the n - glycan is further processed by the action of various 1,2-mannosidases , which can remove one or more of the four 1,2-linked mannose residues . in mammalian cells , man9glcnac2 is converted to man5glcnac2 by the action of er and golgi -mannosidases , which is the precursor for complex , hybrid , and high - mannose n - glycans . in s. cerevisiae , a specific er 1,2-mannosidase converts man9glcnac2 into man8glcnac2 , which is elongated in the golgi to form an outer chain containing up to 200 residues of mannose [ 89 , 90 ] .", "a. saitoi and trichoderma reesei have been found to produce n - glycan structures containing five mannose units ( man5glcnac2 ) , suggesting further processing of the man9glcnac2 precursor [ 91 , 92 ] .", "the n - glycans on mature secreted glycoprotein produced by a. fumigatus are man6glcnac2 , man7glcnac2 , and man8glcnac2 , in which man6glcnac2 is the major glycoform .", "these observations demonstrate that n - glycan synthesis in filamentous fungi seems to differ from that in yeast and is similar to that in higher eukaryotes ( figure 3 ) .", "although small n - glycans are commonly found on glycoproteins of a. fumigatus , hex5 - 13hexnac2 glycans on the galactomannoproteins , and man5 - 9glcnac2 as well as galf1man5 - 7glcnac2 structures on other secreted glycoproteins have been identified in a. fumigatus [ 93 , 94 ] . the enzyme ( udp - galp mutase ) required to synthesize the requisite udp - galf donor has been shown to be an important factor in biosynthesis of the cell wall in a. fumigatus , while the gene / enzyme responsible for the transfer of galf has not been identified .", "recently , the a. fumigatus och1 , a key mannosyltransferase for synthesis of elaborated protein n - glycans in yeast , has been identified .", "deletion of the och1 gene results in a reduction of sporulation in the presence of high calcium concentrations .", "this evidence suggests that polymannosylated n - glycans exist in a. fumigatus and certain proteins engaged in sporulation require n - glycan outer chains to be fully functional .  ", "the -mannosidases have been classified into two groups : class i and class ii [ 102 , 120 , 121 ] .", "class i -mannosidases include er man9-mannosidase , endomannosidase , and golgi mannosidase i. class ii -mannosidases include the lysosomal mannosidases , golgi mannosidase ii , yeast vascular mannosidase [ 98 , 99 ] , and er -mannosidase ii [ 122 , 123 ] .", "several golgi -mannosidases have been cloned and characterized from penicillium citrinum [ 124 , 125 ] , a. saitoi , a. oryzae , t. reesei , and a. nidulans [ 102 , 129 ] .", "these enzymes are all monomeric with a molecular weight of 5060  kda and show the maximal activity in the semiacidic condition ( ph 46 ) .  ", "class i -mannosidase is known to play an important role in the processing of mannose - containing glycans . in drosophila melanogaster , deletion of the golgi mannosidase i ( mas-1 ) results in viable progeny , and the null organisms synthesize the same range of oligosaccharides as the wild - type ones , albeit at different ratios . in s. cerevisiae , disruption of the er -mannosidase gene does not prevent outer chain synthesis . in the last release of the tigr database ( http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main ) , nine a. fumigatus genes are annotated to encode -mannosidases , including xp_749038.1 , xp_754794.1 , xp_751252.1 , xp_751819.1 , xp_752444.1 , xp_752825.1 , xp_753592.1 , xp_751114.1 , and xp_750572.1 . among them , msdsp ( xp_752825.1 ) has been identified to encode a class i 1 , 2-mannosidase and acts on man8glcnac2 to produce man6glcnac2 .", "deletion of the msds gene leads to a defect in n - glycan processing , as well as a reduction of cell wall components ( including -glucan , -glucan , mannoprotein , and chitin ) and reduced conidiation .", ".   in mammalian cells , free oligosaccharides ( fos ) are generated by ost - mediated hydrolysis of glc3man9glcnac2-pp - dolichol in the lumen of the er or peptide n - glycanase ( pngase)-mediated de - n - glycosylation of newly synthesized glycoproteins either in the er or the cytosol .", "fos that are liberated in the lumen of the er can be transported into the cytosol , where they are trimmed by an endo--d - n - acetylglucosamine h ( endo h)-like enzyme and the -mannosidase man2c1p in order to yield an oligosaccharide , man5glcnac , that can be imported directly into lysosomes to be degraded ( figure 4 ) [ 122 , 123 , 131135 ] . in humans   and cattle [ 137139 ] , a deficiency in -mannosidase results in the lethal disease mannosidosis , a rare lysosomal storage disease with a collection of clinical symptoms including progressive mental retardation , impaired hearing , dysostosis multiplex , immune defects , elevation of serum and urinary oligosaccharide levels , and an enlargement of lysosomes in most cell types resulting from the accumulation of undegraded oligosaccharides .  ", "the rat man2c1p is involved in oligosaccharide catabolism of misfolded glycoproteins in the lumen of the er which have been retrotranslocated into the cytoplasm for proteolytic disposal [ 131133 ] .", "a proteolytically cleaved version of the rat man2c1p has been found in the lumen of the er where it is believed to be involved in the early stages of glycoprotein maturation ( also called er -mannosidase ii ) ( figure 4 ) [ 122 , 123 ] .  ", "the yeast cytosolic -mannosidase ams1p , a counterpart of man2c1p , is also involved in the processing of fos .", "since the yeast png1p is mainly localized to the cytosol , it is proposed that the png1p - generated fos may both be generated and processed in the cytosol .", "the role of the yeast ams1p is to aid in recycling macromolecular components of the cell under nutrient deprivation . interestingly , after its synthesis in the cytosol , the ams1p is translocated into the vacuole by the cytosol - to - vacuole targeting pathway , which suggests a common feature shared by the s. cerevisiae ams1p and its mammalian counterparts .", "however , the yeast ams1p only participates in recycling or utilizing of oligosaccharide but not in processing of n - glycan ( figure 4 ) .", "it should also be noted that no structural studies have been performed on the products that can be generated from man8glcnac2 by ams1p , and the ultimate fate of such products remains obscure . on the other hand , two png1p - independent fos pools , man3glcnac2 and man8glcnac2 , are also seen in s. cerevisiae .", "the pool comprising small fos ( man3glcnac2 ) appears to be disposed of by unknown enzymes in the vacuole .", "the pool containing mainly man8glcnac2 may be generated and disposed of along the secretory pathway .  ", "similarly , a. nidulans -mannosidase iic is also proposed to be involved in oligosaccharide catabolism .", "both a. nidulans-mannosidase iic and s. cerevisiae ams1p are not essential for normal cellular function since disruption of these genes has no visible effect on growth or morphology [ 98 , 99 , 102 ] .  ", "in contrast to its counterpart in yeast or a. nidulans , the a. fumigatus ams1p is required for normal cellular function .", "deletion of the a. fumigatus ams1 leads to a severe defect in conidial formation , especially at a higher temperature .", "these results show that the afams1 gene is required for morphogenesis and cellular function in a. fumigatus .", "the involvement of the afams1 gene in polarized growth demonstrates that the processes involved in fos regulation are important for a. fumigatus .", "it is likely that the ams1p is involved in cell wall synthesis and thus polarity through the cwi pathway .", "therefore , probably the afams1 mutant could serve as a simple model to investigate the mechanism of -mannosidosis .   functional analyses of the genes required for n - glycosylation reveal that protein", "n - glycosylation is important for cell wall synthesis , morphogenesis , and polarized growth in a. fumigatus .", "2-d gel analysis reveals that deletion of the cwh41 gene encoding glucosidase i in a. fumigatus leads to er stress , which induces overexpression of hsp70 and calnexin chaperone and activates the erad .", "meanwhile , the proteins required for actin rearrangement are found to be underexpressed or missing , which is consistent with the observation of random localization of actin fibers in the mutant .", "these observations , for the first time , clearly suggest that n - glycosylation contributes to proper folding and trafficking in a. fumigatus .", "it appears that proteins involved in cell wall biosynthesis in a. fumigatus are more dependent on the n - glycan - dependent folding system . as in yeast", ", cell wall defects also trigger the cwi signaling pathway in a. fumigatus , which activates downstream effectors that regulate cell wall biogenesis and polarized growth .", "zhang et al . [ 37 , 86 ] have proposed that the proteins required for cell wall synthesis or cell wall stress sensing are substrates of a. fumigatus cwh41p and require glucose trimming for their proper localization and function .", "misfolding of these proteins would cause cell wall defects , which then leads to activation of the erad and rho - type gtpases - mediated cwi pathway .", "moreover , activation of cdc42 in the cwi pathway also activates sepa , an upstream organizer of actin ring formation at septation sites , and thus causes abnormal polarized growth associated with the afcwh41 mutant ( figure 5 ) .", "although the phenotypes associated with different n - glycosylation mutants vary , the finding that all of these mutants exhibit phenotypes associated with cell wall defects , abnormal polarization , and morphological changes can all be explained by this proposed model .   obviously , more investigations are needed to identify and characterize all of the proteins affected by n - glycosylation in a. fumigatus .", "this information would be key to understanding the complex compensatory mechanisms participating in cell wall biosynthesis in a. fumigatus , which would serve as a basis to develop new antifungal therapies , as well as help to elucidate the molecular mechanism of human diseases associated with defects in glycosylation ."], ["o - mannose glycosylated proteins were first discovered in yeast and filamentous fungi , and recently this type of glycoproteins has also been described in mammals .", "the o - mannosylation most likely occurs in all animals , with the exception of nematodes ( e.g. , caenorhabditis elegans ) ; it is also not detected in plants ( arabidopsis thaliana , oryza sativa ) .", "however , it has also been discovered in one bacterial species ( mycobacterium tuberculosis ) . in mammalian cells ,", "the inner o - linked mannose is elongated with the first addition of a n - acetylglucosamine and then various sugars . in the case of yeast ,", "the o - mannose type carbohydrate chain starts with a serine / threonine - linked mannose , which is extended to an oligomannose chain . in a. fumigatus ,", "the o - linked glycans on cell wall mannoproteins are found to be glc1 , 6man , galf1 , 6man1 , 6man , galf1 , 5galf1 , 6man1 , 6man and galf1 , 5[galf1,5]3 galf1 , 6man , while only a single mannose residue was detected on secreted proteins .  ", "a further type of protein o - glycosylation , in which a single -o - linked glcnac residue is linked to serine and threonine occurs in animals , plants , and filamentous fungi , but not in s. cerevisiae . for this type of protein modification ,", "protein o - mannosylation is initiated by a family of protein o - mannosyltransferases ( pmts ) that are evolutionarily conserved from yeast to human [ 145 , 146 ] . in s. cerevisiae", "a total of seven pmt family members ( scpmt17p ) are present [ 104 , 147 ] , which fall into three major groups of homology : ( i ) pmt1/5/7 , ( ii ) pmt2/3/6 , and ( iii ) pmt4 .", "genes with significant homology to pmts have been cloned in humans , mice , and drosophila [ 148150 ] .", "specific protein substrates that are o - mannosylated by scpmt1p , scpmt2p , or scpmt4p have been described in s. cerevisiae [ 151153 ] . in comparison with s. cerevisiae , the pmt family is less redundant in higher eukaryotes . in drosophila", "only two pmt family members are present ( rotated abdomen and twisted ) [ 149 , 150 ] . the same is true for mice and humans ( pomt1 and pomt2 ) [ 148 , 150 ] .", "mutations in human pomt1 , a homologue of the yeast pmt4 , cause walker - warburg syndrome ( wws ) , which is characterized by severe congenital muscular dystrophy , neuronal migration defects , and structural abnormalities of the eye . targeted deletion of pomt1 in mice results in embryonic lethality due to defects in the formation of the reichert 's membrane , the first basement membrane to form in the embryo .", "mutations of the drosophila pmt homologues alter muscle structures and the alignment of adult cuticle . the pmt family is crucial for viability , cell wall integrity , and morphogenesis in several fungal species , such as s. cerevisiae , s. pombe , c. albicans and c. neoformans , a. nidulans , and a. fumigatus [ 108 , 109 , 111115 , 145 , 156 ] . in s. cerevisiae , single pmt1 mutants fail to grow in anaerobic conditions on some media .", "the pmt1,2,3-triple mutants grow only in osmotically stabilized medium , whereas the pmt1,2,4- and pmt2,3,4-triple mutants are not viable in any conditions , indicating that pmt protein activity is essential in s. cerevisiae , although individual genes are dispensable .  \n c. albicans contains five pmt genes .", "the pmt1 mutants are viable , but they are defective in undergoing cellular differentiation from yeast to a true hyphal growth form under some conditions .", "the virulence of the pmt1 null mutant is significantly attenuated , which is likely due to reduced o - glycosylation of the c. albicans adhesin als1p .", "the pmt phenotypes are closely linked to alterations in cell wall components , including cell wall mannoproteins and polysaccharides .   in s. pombe only one member of each pmt subfamily", "is present , namely , oma1 , oma2 , and oma4 . deletion of oma2 , as well as simultaneous deletion of oma1 and oma4 is lethal .", "characterization of the viable s. pombe oma1d and oma4d single mutants shows that reduced o - mannosylation results in abnormal cell wall and septum formation , therefore severely affecting cell morphology and cell - cell separation . in c. neoformans ,", "recently , willger et al . showed that pmt2 is an essential gene , and the double pmt1pmt4 deletion is lethal .", "filamentous fungi , such as a. fumigatus , a. nidulans , neurospora crassa , and fusarium gramineum , contain only three pmt genes that belong to the pmt1 , pmt2 , and pmt4 subfamilies , respectively [ 107 , 108 , 113 ] .", "all single pmt mutants in a. nidulans are viable but showed reduced growth at elevated temperatures and defects in morphogenesis [ 111 , 112 ] .", "the double deletion pmta / pmtc ( orthologues of the pmt2 and pmt4 ) and pmtb / pmtc ( orthologues of pmt1 and pmt4 ) are synthetically lethal .  ", "have shown that a single deletion of a. fumigatus   pmt1 results in temperature - sensitive phenotypes .", "when the a. fumigatus pmt1 mutant was grown on solid complete medium at 37c , no difference was found between the mutant and the wild type .", "a strongly retarded growth , however , was observed when this mutant was grown at 42c and 50c .", "this temperature - sensitive phenotype could be complemented by the addition of 1  m sucrose in the media .", "further analysis shows that the mannoprotein , -glucan , and chitin in the cell wall of the mutant grown at 37c are increased , while -glucan is reduced . when the a. fumigatus pmt1 mutant was cultured at 42c ,", "the -glucan was increased , while the -glucan was decreased , and the mannoprotein and chitin content remained unchanged .", "moreover , deficient conidiation and reduced germination have been documented at 42c . as compared with the s. cerevisiae   pmt1 mutants , the a. fumigatus pmt1 mutant , as well as the c. albicans and s. pombe   pmt1 mutants , shows more severe defects in cell wall integrity .", "this significant phenotype could be explained by the fewer members of pmt family presented in a. fumigatus , c. albicans , and s. pombe .", "however , in a recent study by mouyna et al . , the a. fumigatus pmt1 mutant does not show any visible phenotype . in the report by zhuo et al .", ", the pmt1 deletion mutant was constructed by replacement of the entire coding region of the pmt1 in a. fumigatus strain cea17 ( pyrg ) with a pyrg cassette .", "therefore , the genetic background of the pmt1 null mutant is pyrg  pmt1 , while in the report by mouyna et al .", ", the pmt1 mutant was constructed by transformation of a. fumigatus strain ku80 with a deletion cassette containing the e. coli phleomycin phosphotransferase gene ( phle ) .", "therefore , the major differences may be due to the different genetic background of the strains used in these two reports .", "the single pmt2 or double pmt1pmt4 deletion(s ) are lethal [ 114 , 115 ] .", "fang et al .   reported that reduced expression of pmt2 leads to retarded growth , cell wall defects , abnormal polarity , and reduced conidiation ; however , no temperature - sensitive growth was found .", "interestingly , this is the first time that pmt2p is revealed to be involved in polarized growth .", "these observations suggest that a. fumigatus pmt2p is required for cell wall synthesis and morphogenesis and its function is distinct from that of a. fumigatus pmt1p .  ", "disruption of a. fumigatus pmt4 leads to abnormal mycelial growth , poor conidiation , and abnormal polarity .", "although an increased sensitivity to echinocandin , a 1,3-glucan synthase inhibitor , was observed in the a. fumigatus pmt4 null mutant , glucan synthase activity and 1,3-glucan content were not affected .", "in contrast to its counterpart in c. albicans , a. fumigatus   pmt4 is not required for full virulence .  ", "the different functions associated with different a. fumigatus pmts are likely due to their different substrate specificities .", "further investigation of the pmt mutants will be helpful for understanding their molecular mechanism , which will not only increase our understanding of the function of o - mannosylation in a. fumigatus , but also may deepen our understanding of the molecular basis of the human walker - warburg syndrome ( wws ) which features mutations in pomt1 , a homologue of a. fumigatus pmt4p , and results in a failure of polarized growth during neuronal migration ."], ["gpi anchoring is a conserved glycosylation process in eukaryotes , which enables many cell surface proteins such as cell surface enzymes , receptors , and adhesion molecules to be covalently anchored to the cell membrane .", "the core structure of the gpi anchor consists of a lipid group , myoinositol , glucosamine , several mannose residues , and a phosphoethanolamine group , which ultimately connects the gpi anchor to the protein via an amide bond .", "although the number of mannose groups and the position of side - chains on the gpi anchors vary widely between species , a common core structure of etnman3glcn - pi is conserved in all gpi - anchored proteins found in protozoa , yeast , plants , and mammals ( figure 6 ) .  ", "gpi anchoring is not essential in mammals at a cellular level as several gpi - deficient cell lines have been established .", "however , an acquired gpi - anchoring deficiency in haematopoietic stem cells causes paroxysmal nocturnal haemoglobinuria , a rare but serious human disease .", "also an overexpression of pig - p , a protein of unknown function required for gpi anchor synthesis , has been noted in fetal down syndrome brain .", "in contrast to mammals , gpi anchor synthesis is essential in s. cerevisiae . in s. cerevisiae , many gpi - anchored proteins are known to be involved in morphogenesis and cell wall organization .", "one type is the gpi - mannoproteins covalently linked to cell wall -1,6-glucan which play important biological functions in filamentation , mating , flocculation , or adhesion to the external matrix [ 161167 ] .", "the second type are the gpi proteins associated with the plasma membrane which possess enzymatic activities able to modify cell wall polymers and are involved in altering cell morphology , such as -glucanase and -glucosyltransferase [ 168170 ] .", "recent studies in a. fumigatus suggest that at least nine gpi - anchored proteins are common to filamentous fungi and yeast .", "five of them are homologues of putative gpi - anchored yeast proteins that have been shown to play a role in cell wall morphogenesis .", "the gpi anchor is assembled at the er in multiple steps catalyzed by the concerted actions of approximately 20 proteins .", "the first step of gpi anchor synthesis is initiated by the transfer of n - acetylglucosamine ( glcnac ) from udp - glcnac to phosphatidylinositol ( pi ) , which is catalyzed by the glycosylphosphatidylinositol - n - acetyl - glucosaminyltransferase ( gpi - gnt ) complex .", "the mammalian gpi - gnt complex consists of seven proteins , including pig - a , pig - h , pig - c , pig - p , gpi1 , pig - y , and dpm2 .", "all except dpm2 have structural and functional counterparts in s. cerevisiae , where they are known as gpi3p , gpi15p , gpi2p , gpi19p , gpi1p , and eri1p , respectively .", "pig - a / gpi3p is believed to possess the catalytic domain because gpi3p binds a photoactivatable udp - glcnac analog and is a member of glycosyltransferase family 4 of retaining glycosyltransferases .", "the roles of the other subunits in the gpi - gnt complex are as yet unclear , but they may mediate regulatory interactions .   in yeast ,", "gpi anchoring is essential for viability and plays an important role in the biosynthesis and organization of the cell wall .", "a gpi3 temperature - sensitive mutant is not viable at 37c [ 116 , 117 ] .", ", it is postulated that the introduction of mutations in gpi3/gpig-1 genes allows for minimal level of product function and survival when growing the mutant cells below the restrictive temperatures .", "however , the mechanism , by which the defect in gpi anchoring leads to a lethal phenotype in these two species , is poorly understood .  ", "it has been shown that a. fumigatus gpi anchors possess five mannose residues with a phosphoethanolamine linked on the first three residues [ 174 , 175 ] .", "the a. fumigatus pig - a gene , the homologue of the gpi3/pig - a gene in yeast , has been investigated .", "also , an increased content of -glucan and mannoprotein was observed in the mycelial cell wall of the afpig - a mutant .", "unlike the temperature - sensitive or conditional lethal phenotype seen in the yeast gpi3 mutant , afpig - a can survive at temperatures from 30c to 50c . completely blocking gpi anchor synthesis in a. fumigatus afpig - a leads to cell wall defects , abnormal hyphal growth , rapid conidial germination , and aberrant conidiation . in vivo assays", "therefore , the gpi anchor seems not essential for viability , but required for cell wall integrity , morphogenesis and virulence in a. fumigatus .", "indeed , this is the first report that a deficiency in gpi - anchor synthesis does not lead to a temperature - sensitive or conditional lethal phenotype in microbes , which provides an opportunity to identify the basic function of gpi anchoring in fungi ."], ["during the past 50 years , proteins and nucleic acids have dominated the field of biology .", "the enormous advances in the analysis of complex carbohydrates have enabled us to investigate the structure and function of carbohydrates , and the field has developed enormously .", "it is now known that carbohydrates play very important roles , especially in the regulation of development of higher organisms .", "however , the mechanisms by which carbohydrates play a role in development and diseases are still poorly understood .", "our knowledge of protein glycosylation comes mainly from the investigation of s. cerevisiae and mammalian cells .", "although investigations of the model yeast and mammalian cells have been very useful in elucidating the biochemical features of protein glycosylation , these investigations at the cellular level can not reflect the complicated functions of glycosylation in the development of multicellular eukaryotes .", "therefore , more model systems have been introduced , such as caenorhabditis elegans , drosophila melanogaster , and mice . however , these model systems are still too complex since deletion of individual glycosyltransferase genes in these systems sometimes leads to fetal death or nonvisible phenotypes .", "as compared with s. cerevisiae , c. elegans , d. melanogaster , or mice , a. fumigatus seems to be an ideal model for investigation of the function of glycosylation since a. fumigatus is a multicellular eukaryote with a relative simple life cycle , in which it undergoes a series of developmental events that require polarized growth .", "recent progress shows that a. fumigatus has evolved an intact n - glycan - dependent qc system , which is present in mammalian cells but not in yeast . disruption of either processing or degradation of n - glycan in a. fumigatus leads to phenotypes such as cell wall defects and abnormal polarity .  ", "based on investigations of s. cerevisiae and filamentous fungi , it is likely that glycosylation first evolved to ensure synthesis of the fungal cell wall and only later did the n - glycan - dependent qc system evolve to ensure precisely controlled cell wall synthesis and polarized growth which are important for multicellular development . however , this hypothesis is controversial .", ".   showed that the n - glycan - dependent qc system is functional in entamoeba , trichomonas , cryptococcus , and s. pombe , but is not functional in some fungi such as giardia and plasmodium , theileria , encephalitozoon , toxoplasma , cryptosporidium , and tetrahymena .", "they proposed that the n - glycan - dependent qc system was likely present in the common ancestor of extant eukaryotes and was secondarily lost from some eukaryotes .", "for example , the s. cerevisiae kre5 is believed to be the gt ortholog that no longer glucosylates misfolded glycoproteins but is instead thought to be involved in -1,6-glucan synthesis .", "of course , the possibility that the s. cerevisiae kre5 is the ancestor of gt can not be excluded .", "it remains unclear where is the evolutionary origin of glycosylation , what is the basic function of glycosylation at the early stages of evolution , and how glycosylation is regulated .", "definitely , the answers to these questions will enable us to understand the basic function and regulation of glycosylation in the development of multicellular eukaryotes and help to understand more complex functions in higher eukaryotes .", "on the other hand , the investigation of a. fumigatus is also a key to understanding complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development . during the past few years", ", the framework of the biosynthetic pathways of glycosylation in a. fumigatus has been delineated .", "functional analyses of some of the genes in this pathway have shown that glycosylation is required for cell wall synthesis , polarity , morphogenesis , and cellular function in a. fumigatus ( figure 7 and table 2 ) .", "however , a detailed understanding of this pathway remains unknown , such as details regarding the synthesis of the n - glycan precursor , the precise molecular mechanism of n - glycan processing , qc of protein folding , and modification of the gpi anchor .", "moreover , the molecular mechanisms by which glycosylation plays a role in morphogenesis and development of a. fumigatus are vaguely understood .", "therefore , the future direction would be looking for those key proteins that are affected by glycosylation and identifying the signal transduction pathways that link glycosylation and development , through genetic , biochemical , cell biological , and proteomic studies ."]]}
{"article_id": "PMC5063136", "article_text": ["in patients with hf , increased raas contributes to the pathogenesis , and ace inhibitors reduce the activity of the raas by inhibiting the production of angiotensin ii .", "two benchmark randomized controlled trials , namely the cooperative north scandinavian enalapril survival study7 and the studies of left ventricular dysfunction treatment trial,8 demonstrated that ace inhibitors reduce mortality and improved new york heart association class , exercise capacity and cardiac function in patients with hf with reduced ef .", "subsequently , the assessment of treatment with lisinopril and survival study5 investigated whether ace inhibitors had favourable effects on the outcome of patients with hf with reduced ef in a dosedependent manner . in this trial ,", "an ace inhibitor , lisinopril , at high dose ( 32.535  mg daily ) significantly decreased death or hospitalization for any cause by 12% ( p  =  0.002 ) compared with that at low dose ( 2.55  mg daily ) , but not significantly decreased death by 8% ( p  =  0.128 ) .", "show that benazepril at supramaximal dose results in prolonged survival by 41% compared with that at low dose [ 95% confidence interval ( ci ) 0.360.98 , p  =  0.042 ] .", "further research should reveal whether ace inhibitors at supramaximal dose reduce the mortality rate in patients with hf compared with those at high dose .", "common side effects of ace inhibitors are hyperkalaemia , hypotension , cough , and impaired renal function . according to the severity of these adverse events ,", "it is crucial to manage when to stop uptitration , reduce dose , or discontinue treatment .", "five ace inhibitors , namely ramipril , enalapril , lisinopril , captopril , and trandolapril , are listed for the treatment of hf with reduced ef , and the starting dose and the target dose are also described in the current guidelines.3 however , it remains unclear which ace inhibitor should be administered or if there are differences between the drugs , because headtohead comparisons are still missing .", "angiotensin receptor blockers would be expected to exert beneficial effects on the treatment of hf through the stronger inhibition of angiotensin ii than ace inhibitors .", "the evaluation of losartan in the elderly study ii study9 showed that losartan was noninferior to captopril in reducing mortality in elderly patients with hf with reduced ef ( hazard ratio 1.13 , 95% ci 0.951.35 , p  =  0.16 ) , and the candesartan in heart failureassessment of reduction in mortality and morbidityalternative trial10 showed that candesartan reduces the risk of cardiovascular death or hospital admission for hf in patients intolerant of ace inhibitors compared to placebo ( hazard ratio 0.70 , 95% ci 0.600.81 , p  <  0.0001 ) .", "subsequently , the heart failure endpoint evaluation of angiotensin ii antagonist losartan study6 revealed that losartan at high dose ( 150  mg daily ) reduced death or hospitalization for hf in patients with hf with reduced ef , compared with losartan at low dose ( 50  mg daily ) ( hazard ratio 0.90 , 95% ci 0.820.99 ; p  =  0.027 ) , indicating the doserelated therapeutic effect of arbs on the outcome of hf as well as ace inhibitors . in the swedish heart failure registry,11 the use of losartan was associated with significant increase in mortality in patients with hf compared with that of candesartan ( hazard ratio 1.43 , 95% ci 1.231.65 , p  <  0.001 ) , suggesting the different clinical effects among arbs .", "common side effects of arbs are well known and include hyperkalaemia , hypotension , and impaired renal function , similar to ace inhibitors ; however , arbs are believed to be better tolerated than ace inhibitors .", "reports that 29 patients ( 29% ) at supramaximaldose benazepril withdrew from this study , as did 12 patients ( 12% ) at supramaximaldose valsartan .", "doserelated increase in the development of adverse events in patients receiving both benazepril and valsartan is observed , which is inconsistent with the findings of previous studies using very highdose arb.12 , 13 \n  in view of these findings , arbs are considered second choice in patients with hf with reduced ef who are not tolerated to ace inhibitors or mineralocorticoid receptor antagonists in the current guidelines .", "because of the different mechanism of action of ace inhibitors and arbs in blocking the raas , combination therapy of an ace inhibitor and an arb was thought to be attractive for the treatment of hf as well as the monotherapy with an arb . in the valsartan heart failure trial,14 the addition of valsartan to conventional therapy for hf resulted in a significant decrease in cardiovascular events compared to placebo ( relative risk 0.87 , 97.5% ci 0.770.97 , p  =  0.009 ) ; however , a post hoc analysis indicated increased mortality and complications in the subgroup taking an ace inhibitor , valsartan , and a betablocker .", "moreover , a recent metaanalysis15 suggested that combination therapy of an ace inhibitor with an arb should not be advocated in patients with hf , because the combination does not seem to reduce mortality or hospitalization and was associated with more adverse events .", "as described earlier , an ace inhibitor , lisinopril , or an arb , losartan , at high dose produced better clinical outcomes in patients with hf compared with each at low dose but did not decrease mortality significantly.5 , 6 on the other hand , he et al .", "reports that supramaximal dose of benazepril or valsartan improves not only cardiac function but also survival in patients with dcm compared with the low dose of each medication,16 which indicates that ace inhibitors or arbs at supramaximaldose might produce better outcomes in patients with ef compared with those at high dose ( target dose ) .", "these unique findings are derived from a singlecentre prospective , randomized , and controlled trial .", "the sample size is small , but the followup period is long enough to detect the statistical difference in the outcomes of hf among the treatment groups . they should be confirmed with doubleblind multicentre randomized controlled studies .", "recently , ivabradine17 and lcz696 , a combination of the new neprilysin inhibitor sacubitril ( ahu377 ) with valsartan,18 have attracted attention for the treatment of hf .", "if the efficacy and safety of supramaximaldose valsartan were well acknowledged in this field , it would be helpful to investigate the appropriate dose of lcz696 more precisely ."], "abstract_text": ["<S> abstractheart failure is a major public issue , and dilated cardiomyopathy ( dcm ) is one of the common etiologies of heart failure . </S>", "<S> dcm is generally progressive , and some patients with dcm need heart transplant despite optimal medical and mechanical therapy . </S>", "<S> current guidelines recommend inhibitors of renin  angiotensin  </S>", "<S> aldosterone system , namely angiotensinconvertingenzyme ( ace ) inhibitor , angiotensin receptor blocker ( arb ) , and mineralocorticoid receptor antagonist as well as betablockers for the medical treatment of heart failure with reduced ejection fraction , including dcm . </S>", "<S> furthermore , because they have beneficial effects on the outcome of heart failure in a doserelated fashion , they should be titrated to the target dose . in clinical practice , </S>", "<S> the underuse and underdose of these agents matter ; however , the efficacy and safety of supramaximal dose of ace inhibitor or arb have never been investigated in the patients with dcm . in this issue of esc heart failure </S>", "<S> , it is demonstrated that benazepril or valsartan at supramaximal dose improved left ventricular function and reduced cardiovascular events compared with each drug at low dose , respectively . in this editorial , </S>", "<S> the current evidence concerning the use of ace inhibitor or arb in patients with hf and future prospective will be discussed . </S>"], "labels": null, "section_names": ["Angiotensinconvertingenzyme inhibitor for the treatment of heart failure", "Angiotensin receptor blocker for the treatment of heart failure", "Combination therapy with angiotensinconvertingenzyme inhibitor and angiotensin receptor blocker, supramaximal dose of angiotensinconvertingenzyme inhibitor or angiotensin receptor blocker and beyond", "Future perspective", "Conflict of interest"], "sections": [["in patients with hf , increased raas contributes to the pathogenesis , and ace inhibitors reduce the activity of the raas by inhibiting the production of angiotensin ii .", "two benchmark randomized controlled trials , namely the cooperative north scandinavian enalapril survival study7 and the studies of left ventricular dysfunction treatment trial,8 demonstrated that ace inhibitors reduce mortality and improved new york heart association class , exercise capacity and cardiac function in patients with hf with reduced ef .", "subsequently , the assessment of treatment with lisinopril and survival study5 investigated whether ace inhibitors had favourable effects on the outcome of patients with hf with reduced ef in a dosedependent manner . in this trial ,", "an ace inhibitor , lisinopril , at high dose ( 32.535  mg daily ) significantly decreased death or hospitalization for any cause by 12% ( p  =  0.002 ) compared with that at low dose ( 2.55  mg daily ) , but not significantly decreased death by 8% ( p  =  0.128 ) .", "show that benazepril at supramaximal dose results in prolonged survival by 41% compared with that at low dose [ 95% confidence interval ( ci ) 0.360.98 , p  =  0.042 ] .", "further research should reveal whether ace inhibitors at supramaximal dose reduce the mortality rate in patients with hf compared with those at high dose .", "common side effects of ace inhibitors are hyperkalaemia , hypotension , cough , and impaired renal function . according to the severity of these adverse events ,", "it is crucial to manage when to stop uptitration , reduce dose , or discontinue treatment .", "five ace inhibitors , namely ramipril , enalapril , lisinopril , captopril , and trandolapril , are listed for the treatment of hf with reduced ef , and the starting dose and the target dose are also described in the current guidelines.3 however , it remains unclear which ace inhibitor should be administered or if there are differences between the drugs , because headtohead comparisons are still missing ."], ["angiotensin receptor blockers would be expected to exert beneficial effects on the treatment of hf through the stronger inhibition of angiotensin ii than ace inhibitors .", "the evaluation of losartan in the elderly study ii study9 showed that losartan was noninferior to captopril in reducing mortality in elderly patients with hf with reduced ef ( hazard ratio 1.13 , 95% ci 0.951.35 , p  =  0.16 ) , and the candesartan in heart failureassessment of reduction in mortality and morbidityalternative trial10 showed that candesartan reduces the risk of cardiovascular death or hospital admission for hf in patients intolerant of ace inhibitors compared to placebo ( hazard ratio 0.70 , 95% ci 0.600.81 , p  <  0.0001 ) .", "subsequently , the heart failure endpoint evaluation of angiotensin ii antagonist losartan study6 revealed that losartan at high dose ( 150  mg daily ) reduced death or hospitalization for hf in patients with hf with reduced ef , compared with losartan at low dose ( 50  mg daily ) ( hazard ratio 0.90 , 95% ci 0.820.99 ; p  =  0.027 ) , indicating the doserelated therapeutic effect of arbs on the outcome of hf as well as ace inhibitors . in the swedish heart failure registry,11 the use of losartan was associated with significant increase in mortality in patients with hf compared with that of candesartan ( hazard ratio 1.43 , 95% ci 1.231.65 , p  <  0.001 ) , suggesting the different clinical effects among arbs .", "common side effects of arbs are well known and include hyperkalaemia , hypotension , and impaired renal function , similar to ace inhibitors ; however , arbs are believed to be better tolerated than ace inhibitors .", "reports that 29 patients ( 29% ) at supramaximaldose benazepril withdrew from this study , as did 12 patients ( 12% ) at supramaximaldose valsartan .", "doserelated increase in the development of adverse events in patients receiving both benazepril and valsartan is observed , which is inconsistent with the findings of previous studies using very highdose arb.12 , 13 \n  in view of these findings , arbs are considered second choice in patients with hf with reduced ef who are not tolerated to ace inhibitors or mineralocorticoid receptor antagonists in the current guidelines ."], ["because of the different mechanism of action of ace inhibitors and arbs in blocking the raas , combination therapy of an ace inhibitor and an arb was thought to be attractive for the treatment of hf as well as the monotherapy with an arb . in the valsartan heart failure trial,14 the addition of valsartan to conventional therapy for hf resulted in a significant decrease in cardiovascular events compared to placebo ( relative risk 0.87 , 97.5% ci 0.770.97 , p  =  0.009 ) ; however , a post hoc analysis indicated increased mortality and complications in the subgroup taking an ace inhibitor , valsartan , and a betablocker .", "moreover , a recent metaanalysis15 suggested that combination therapy of an ace inhibitor with an arb should not be advocated in patients with hf , because the combination does not seem to reduce mortality or hospitalization and was associated with more adverse events .", "as described earlier , an ace inhibitor , lisinopril , or an arb , losartan , at high dose produced better clinical outcomes in patients with hf compared with each at low dose but did not decrease mortality significantly.5 , 6 on the other hand , he et al .", "reports that supramaximal dose of benazepril or valsartan improves not only cardiac function but also survival in patients with dcm compared with the low dose of each medication,16 which indicates that ace inhibitors or arbs at supramaximaldose might produce better outcomes in patients with ef compared with those at high dose ( target dose ) .", "these unique findings are derived from a singlecentre prospective , randomized , and controlled trial .", "the sample size is small , but the followup period is long enough to detect the statistical difference in the outcomes of hf among the treatment groups . they should be confirmed with doubleblind multicentre randomized controlled studies ."], ["recently , ivabradine17 and lcz696 , a combination of the new neprilysin inhibitor sacubitril ( ahu377 ) with valsartan,18 have attracted attention for the treatment of hf .", "if the efficacy and safety of supramaximaldose valsartan were well acknowledged in this field , it would be helpful to investigate the appropriate dose of lcz696 more precisely ."], [""]]}
{"article_id": "PMC2942067", "article_text": ["indian population representing one - sixth of the world population has been the global melting pot of human diversity .", "it has all the world s major linguistic groups and the populations have been shaped by different waves of migrations and admixture ( 1 ,  2 ) .", "further , stringent mating patterns have led to the existence of several endogamous populations , which makes it an important resource for mapping genes ( 3 ) .", "the indian genome variation consortium ( igvc ) project , an initiative of the council for scientific and industrial research ( csir)was set up to develop a database of genomic variations in indian population for predictive marker discovery in complex diseases such as diabetes , asthma , neuropsychiatric , infectious and cardiovascular disorders , response to drugs , etc .", "the phase i of the project was conducted to determine the extent of genetic differentiation in india . toward this genotype data of 405 snps from 75 genes and 4.2 ", "mb contiguous chromosome 22 regions were studied in 55 contrasting populations ( 4 , 5 ) .", "these populations were identified from 4 major linguistic groups namely , austro - asiatic ( aa ) , tibeto - burman ( tb ) , indo - european ( ie ) and dravidian(dr ) spanning 6 geographical regions of habitat ( n , north ; ne , north - east ; w , west ; e , east ; s , south ; c , central ) and different ethnic groups ( lp , large population , caste ; ip , isolated population , tribes ; sp , special population , religious groups ) .", "five genetically distinct clusters were identified and a set of 24 populations that represent these clusters were selected for the phase ii of the project . in the phase ii , 3824 snps from 834 candidate gene as well as 50  000 ( affy 50  k array ) genome wide neutral markers", "this initiative lays the foundation for the integration of global genotype - to - phenotype data ( 6 ) with indian population data and development of a federated database .", "to address the need for an online comprehensive resource that enables users to visualize igvc data with integrated information about snps from different resources we have developed igvbrowser as shown in figure 1 . \n", "distribution of markers in 2.41  mb region in human chromosome 1 from igvc data is displayed along with annotation data from different resources .", "distribution of markers in 2.41  mb region in human chromosome 1 from igvc data is displayed along with annotation data from different resources .", "the database includes ( i ) final validated dataset from 1871 samples in phase i comprising of 405 autosomal snps spanning over 75 genes including 90 snps from 5.2  mb region of chromosome 22 from 55 diverse endogamous indian populations ( 3 ) ; ( ii ) phase ii dataset for 3824 snps spanning from 834 genes in 545 samples from 24 igvdb populations and ( iii ) 50  000 ( affy 50k xbai array ) neutral markers in 26 populations .", "the phase ii populations are a subset of the populations genotyped in the phase i. web - based tool snpper ( http://snpper.chip.org/ ) was used to classify the 4229 markers in phase i and phase ii according to their location in genic regions ( figure 2 ) .", "similarly , david ( http://david.abcc.ncifcrf.gov/ ) was used to classify the genes containing these markers according to gene  disease association class ( figure 3 ) and their mapping in various kegg pathways ( figure 4 ) .", "we report that a large fraction of genes are implicated in cardiovascular , metabolic , cancer and immune system - related diseases .", "thus , the igvc data provide a basal level variation data in indian population to study genetic diseases and pharmacology . \n", "more than 50% of the snps belong to intronic regions and 15% are in coding exons . \n", "figure 3.bar graph shows the functional annotation of candidate genes in igvc according to gene  disease association . \n", "figure 4.bar graph shows the mapping of candidate genes in significant pathways ( after bonferroni correction ) of kegg pathway database .", "more than 50% of the snps belong to intronic regions and 15% are in coding exons .", "bar graph shows the mapping of candidate genes in significant pathways ( after bonferroni correction ) of kegg pathway database .", "igvbrowser also included hapmap snp genotype data from phases i + ii and iii of the hapmap project ( http://hapmap.ncbi.nlm.nih.gov/downloads/gbrowse/2009-02_phaseii+iii/gff/ ) based on ncbi b36 assembly , dbsnp b126 from 4 populations : yoruba from ibadan , nigeria ( yri ) ; japanese in tokyo , japan ( jpt ) ; han chinese in beijing , china ( chb ) ; and ceph ( utah residents with ancestry from northern and western europe ) ( ceu ) .", "additional annotation information including cytogenetic positions , link to pathway annotations in the reactome knowledgebase and mrna sequences were retrieved from hapmap in generic feature finding ( gff ) format .", "annotation data in tab - delimited format for non - coding rna genes and pseudogenes , omim - associated genes , mirbase and snornabase , simple repeats , database of genomic variants were downloaded from ucsc genome annotation database ( http://hgdownload.cse.ucsc.edu/goldenpath/hg18/database ) based on build hg18 . the browser implements one of the widely used platform - independent genome annotation viewer generic genome browser ( gbrowse v1.69 ) , developed by stein et al .", "( 7 ) as a part of the generic model organism system database project ( http://www.gmod.org ) .", "gbrowse is a combination of database and interactive webpage for displaying genomic information along with providing data interoperability across systems running the same software .", "integrated annotation data from primary sources like ncbi , ucsc and hapmap have been linked with variation data from different ethnic populations in india .", "compiled data processed into gff format and complete human genome sequence as plain text files were loaded into mysql relational database management system using a script of gbrowse .", "a user can query chromosomal region of interest , reference snp i d , hgnc symbols , pathway name or any other unique feature recognized by database as a query .", "it allows researchers to upload their own data in gff format and view it along with data available in igvbrowser .", "in addition , the resource is facilitated with sequence analysis servers maintained by ncbi and ucsc .", "online data analysis plugins allows text dumps of visible features using a number of standard formats and also facilitates the download of sequence corresponding to selected region .", "the browser implements one of the widely used platform - independent genome annotation viewer generic genome browser ( gbrowse v1.69 ) , developed by stein et al .", "( 7 ) as a part of the generic model organism system database project ( http://www.gmod.org ) .", "gbrowse is a combination of database and interactive webpage for displaying genomic information along with providing data interoperability across systems running the same software .", "integrated annotation data from primary sources like ncbi , ucsc and hapmap have been linked with variation data from different ethnic populations in india .", "compiled data processed into gff format and complete human genome sequence as plain text files were loaded into mysql relational database management system using a script of gbrowse .", "a user can query chromosomal region of interest , reference snp i d , hgnc symbols , pathway name or any other unique feature recognized by database as a query .", "it allows researchers to upload their own data in gff format and view it along with data available in igvbrowser", "in addition , the resource is facilitated with sequence analysis servers maintained by ncbi and ucsc .", "online data analysis plugins allows text dumps of visible features using a number of standard formats and also facilitates the download of sequence corresponding to selected region .", "indian genome variation data would be enormously useful for the dissection of common complex diseases and in pharmacogenomics studies .", "frequency profiles of markers on disease or drug - related genes that have been generated through the igvc are being used to identify at - risk chromosomes , founders , ld - based mapping , tracing history of diseases in pharmacogenetics as well as reference populations for mapping relatedness ( 3,4,5,819 ) .", "the interactive web browser , igvbrowser , has been created as a central repository for the current and future dataset on indian populations and is being made accessible in the public domain .", "a possible integration of igvbrowser with hgvbaseg2p ( 20 ) can enable researchers for cross study comparison among different populations of the world for disease  gene association study .", "indian genome variation project was funded by the council for scientific and industrial research programme cmm0016 and sip0006 .", "funding for igvbrowser and open access charge is provided by european community 's seventh framework programme ( fp7/2007 - 2013 ) under grant agreement number 200754the gen2phen project ."], "abstract_text": ["<S> the indian genome variation consortium ( igvc ) project , an initiative of the council for scientific and industrial research , has been the first large - scale comprehensive study of the indian population . </S>", "<S> one of the major aims of the project is to study and catalog the variations in nearly thousand candidate genes related to diseases and drug response for predictive marker discovery , founder identification and also to address questions related to ethnic diversity , migrations , extent and relatedness with other world population . </S>", "<S> the phase i of the project aimed at providing a set of reference populations that would represent the entire genetic spectrum of india in terms of language , ethnicity and geography and phase ii in providing variation data on candidate genes and genome wide neutral markers on these reference set of populations . </S>", "<S> we report here development of the igvbrowser that provides allele and genotype frequency data generated in the igvc project . </S>", "<S> the database harbors 4229 snps from more than 900 candidate genes in contrasting indian populations . </S>", "<S> analysis shows that most of the markers are from genic regions . </S>", "<S> further , a large fraction of genes are implicated in cardiovascular , metabolic , cancer and immune system - related diseases . </S>", "<S> thus , the igvc data provide a basal level variation data in indian population to study genetic diseases and pharmacology . </S>", "<S> additionally , it also houses data on 50  000 ( affy 50  k array ) genome wide neutral markers in these reference populations . </S>", "<S> in igvbrowser one can analyze and compare genomic variations in indian population with those reported in hapmap along with annotation information from various primary data sources.database url : http://igvbrowser.igib.res.in </S>"], "labels": null, "section_names": ["Introduction", "Data Source and Organization", "Database structure, implementation and accessibility", "Future directions", "Funding"], "sections": [["indian population representing one - sixth of the world population has been the global melting pot of human diversity .", "it has all the world s major linguistic groups and the populations have been shaped by different waves of migrations and admixture ( 1 ,  2 ) .", "further , stringent mating patterns have led to the existence of several endogamous populations , which makes it an important resource for mapping genes ( 3 ) .", "the indian genome variation consortium ( igvc ) project , an initiative of the council for scientific and industrial research ( csir)was set up to develop a database of genomic variations in indian population for predictive marker discovery in complex diseases such as diabetes , asthma , neuropsychiatric , infectious and cardiovascular disorders , response to drugs , etc .", "the phase i of the project was conducted to determine the extent of genetic differentiation in india . toward this genotype data of 405 snps from 75 genes and 4.2 ", "mb contiguous chromosome 22 regions were studied in 55 contrasting populations ( 4 , 5 ) .", "these populations were identified from 4 major linguistic groups namely , austro - asiatic ( aa ) , tibeto - burman ( tb ) , indo - european ( ie ) and dravidian(dr ) spanning 6 geographical regions of habitat ( n , north ; ne , north - east ; w , west ; e , east ; s , south ; c , central ) and different ethnic groups ( lp , large population , caste ; ip , isolated population , tribes ; sp , special population , religious groups ) .", "five genetically distinct clusters were identified and a set of 24 populations that represent these clusters were selected for the phase ii of the project . in the phase ii , 3824 snps from 834 candidate gene as well as 50  000 ( affy 50  k array ) genome wide neutral markers", "this initiative lays the foundation for the integration of global genotype - to - phenotype data ( 6 ) with indian population data and development of a federated database ."], ["to address the need for an online comprehensive resource that enables users to visualize igvc data with integrated information about snps from different resources we have developed igvbrowser as shown in figure 1 . \n", "distribution of markers in 2.41  mb region in human chromosome 1 from igvc data is displayed along with annotation data from different resources .", "distribution of markers in 2.41  mb region in human chromosome 1 from igvc data is displayed along with annotation data from different resources .", "the database includes ( i ) final validated dataset from 1871 samples in phase i comprising of 405 autosomal snps spanning over 75 genes including 90 snps from 5.2  mb region of chromosome 22 from 55 diverse endogamous indian populations ( 3 ) ; ( ii ) phase ii dataset for 3824 snps spanning from 834 genes in 545 samples from 24 igvdb populations and ( iii ) 50  000 ( affy 50k xbai array ) neutral markers in 26 populations .", "the phase ii populations are a subset of the populations genotyped in the phase i. web - based tool snpper ( http://snpper.chip.org/ ) was used to classify the 4229 markers in phase i and phase ii according to their location in genic regions ( figure 2 ) .", "similarly , david ( http://david.abcc.ncifcrf.gov/ ) was used to classify the genes containing these markers according to gene  disease association class ( figure 3 ) and their mapping in various kegg pathways ( figure 4 ) .", "we report that a large fraction of genes are implicated in cardiovascular , metabolic , cancer and immune system - related diseases .", "thus , the igvc data provide a basal level variation data in indian population to study genetic diseases and pharmacology . \n", "more than 50% of the snps belong to intronic regions and 15% are in coding exons . \n", "figure 3.bar graph shows the functional annotation of candidate genes in igvc according to gene  disease association . \n", "figure 4.bar graph shows the mapping of candidate genes in significant pathways ( after bonferroni correction ) of kegg pathway database .", "more than 50% of the snps belong to intronic regions and 15% are in coding exons .", "bar graph shows the mapping of candidate genes in significant pathways ( after bonferroni correction ) of kegg pathway database .", "igvbrowser also included hapmap snp genotype data from phases i + ii and iii of the hapmap project ( http://hapmap.ncbi.nlm.nih.gov/downloads/gbrowse/2009-02_phaseii+iii/gff/ ) based on ncbi b36 assembly , dbsnp b126 from 4 populations : yoruba from ibadan , nigeria ( yri ) ; japanese in tokyo , japan ( jpt ) ; han chinese in beijing , china ( chb ) ; and ceph ( utah residents with ancestry from northern and western europe ) ( ceu ) .", "additional annotation information including cytogenetic positions , link to pathway annotations in the reactome knowledgebase and mrna sequences were retrieved from hapmap in generic feature finding ( gff ) format .", "annotation data in tab - delimited format for non - coding rna genes and pseudogenes , omim - associated genes , mirbase and snornabase , simple repeats , database of genomic variants were downloaded from ucsc genome annotation database ( http://hgdownload.cse.ucsc.edu/goldenpath/hg18/database ) based on build hg18 . the browser implements one of the widely used platform - independent genome annotation viewer generic genome browser ( gbrowse v1.69 ) , developed by stein et al .", "( 7 ) as a part of the generic model organism system database project ( http://www.gmod.org ) .", "gbrowse is a combination of database and interactive webpage for displaying genomic information along with providing data interoperability across systems running the same software .", "integrated annotation data from primary sources like ncbi , ucsc and hapmap have been linked with variation data from different ethnic populations in india .", "compiled data processed into gff format and complete human genome sequence as plain text files were loaded into mysql relational database management system using a script of gbrowse .", "a user can query chromosomal region of interest , reference snp i d , hgnc symbols , pathway name or any other unique feature recognized by database as a query .", "it allows researchers to upload their own data in gff format and view it along with data available in igvbrowser .", "in addition , the resource is facilitated with sequence analysis servers maintained by ncbi and ucsc .", "online data analysis plugins allows text dumps of visible features using a number of standard formats and also facilitates the download of sequence corresponding to selected region ."], ["the browser implements one of the widely used platform - independent genome annotation viewer generic genome browser ( gbrowse v1.69 ) , developed by stein et al .", "( 7 ) as a part of the generic model organism system database project ( http://www.gmod.org ) .", "gbrowse is a combination of database and interactive webpage for displaying genomic information along with providing data interoperability across systems running the same software .", "integrated annotation data from primary sources like ncbi , ucsc and hapmap have been linked with variation data from different ethnic populations in india .", "compiled data processed into gff format and complete human genome sequence as plain text files were loaded into mysql relational database management system using a script of gbrowse .", "a user can query chromosomal region of interest , reference snp i d , hgnc symbols , pathway name or any other unique feature recognized by database as a query .", "it allows researchers to upload their own data in gff format and view it along with data available in igvbrowser", "in addition , the resource is facilitated with sequence analysis servers maintained by ncbi and ucsc .", "online data analysis plugins allows text dumps of visible features using a number of standard formats and also facilitates the download of sequence corresponding to selected region ."], ["indian genome variation data would be enormously useful for the dissection of common complex diseases and in pharmacogenomics studies .", "frequency profiles of markers on disease or drug - related genes that have been generated through the igvc are being used to identify at - risk chromosomes , founders , ld - based mapping , tracing history of diseases in pharmacogenetics as well as reference populations for mapping relatedness ( 3,4,5,819 ) .", "the interactive web browser , igvbrowser , has been created as a central repository for the current and future dataset on indian populations and is being made accessible in the public domain .", "a possible integration of igvbrowser with hgvbaseg2p ( 20 ) can enable researchers for cross study comparison among different populations of the world for disease  gene association study ."], ["indian genome variation project was funded by the council for scientific and industrial research programme cmm0016 and sip0006 .", "funding for igvbrowser and open access charge is provided by european community 's seventh framework programme ( fp7/2007 - 2013 ) under grant agreement number 200754the gen2phen project ."]]}
{"article_id": "PMC4667018", "article_text": ["coronary artery disease ( cad ) remains the principal cause of mortality and morbidity around the world [ 1 , 2 ] .", "although the mechanisms are still not entirely resolved , previous studies have demonstrated that an imbalance between t - helper 1- ( th1- ) mediated and th2-mediated immune functions contributes to the development of atherosclerosis .", "recently , th17 , which are a newly defined subset different from th1 and th2 , reactive to autoantigens are involved in the pathogenesis of several autoimmune diseases [ 4 , 5 ] .", "in addition to th1 , th2 , and th17 lineage , regulatory t cells ( tregs ) , a special subset of cd4 t cells , inhibit atherosclerosis development by attenuating activated t cell responses [ 6 , 7 ] .", "inspiringly , previous clinical and experimental studies from our laboratory have found that th17/tregs functional imbalance exists during atherogenesis , indicating that the imbalance of th17/tregs plays a critical role in cad [ 8 , 9 ] .", "hence , these findings have showed that cd4 t cell subpopulations play an important role in the initiation and progression of atherosclerosis .", "it is well known that cd4 t cells are divided into different subsets depending on the cytokines they produce .", "interferon- ( ifn- )  , the principal cytokine of th1 cells , is proinflammatory and exacerbates atherosclerotic disease , while the th2 cytokine such as interleukin- ( il- ) 4 is considered to be mainly atheroprotective and can neutralize th1 cytokine activity [ 1012 ] .", "moreover , hashmi and zeng have found that il-17 and il-17 induced cytokines ( il-6 and il-8 ) were significantly increased in acs patients .", "interestingly , tregs exert antiatherosclerosis partly by secretion of anti - inflammatory cytokine transforming growth factor ( tgf-1 ) , which can in turn induce the expression of forkhead box transcription factor p3 ( foxp3 ) and promote differentiation of tregs derived from cd4cd25 t cells [ 15 , 16 ] . therefore , these findings suggest that cytokines as those mentioned above are also essential for the formation and progression of the atherosclerotic plaque", ". the galectin family is characterized by conserved carbohydrate recognition domains ( crd ) that can bind glycosylated proteins .", "galectins are involved in a wide range of biologic processes such as cell adhesion and migration , proliferation and apoptosis , tumor biology , and regulation of the immune system .", "galectin- ( gal- ) 9 is a member of the galectin family of carbohydrate - binding proteins comprising two carbohydrate recognition domains connected by a linker peptide .", "gal-9 is mainly expressed by eosinophils , endothelial cells , macrophages , dcs , kupffer cells , vascular endothelial cells , intestinal epithelial cells , and in particular t lymphocytes .", "furthermore , gal-9 is believed to function predominantly as a multifaceted player in adaptive and innate immunity , especially in t cell development and homeostasis [ 2023 ] .", "accumulating evidence shows that gal-9 induces apoptosis in subsets of differentiated t cells , particularly in th1 and th17 cells , and stimulates the activity of tregs [ 19 , 2428 ] .", "indeed , treatment with recombinant gal-9 moderated the progression of experimental autoimmune encephalomyelitis ( eae ) , arthritis , and diabetes in animal models [ 19 , 2931 ] , by reducing the number of th1 and th17 cells and downregulating circulating ifn- levels .", "t cell immunoglobulin mucin- ( tim- ) 3 has been identified as a receptor for gal-9 .", "although gal-9 can function in a tim-3-independent fashion , the immune - regulatory effects of gal-9 are largely attributed to gal-9-tim-3 pathway [ 3235 ] .", "more recently , foks et al . demonstrated that anti - tim-3-ab administration promoted atherosclerotic plaque formation , which indicated that tim-3-gal-9 pathway may be concerned with the development of atherosclerosis .", "thus , it is reasonable to postulate that gal-9 may be involved in atherosclerosis based cad .", "in addition , several findings strongly support the original experimental observations that gal-3 plays an important role in the underlying heart failure ( hf ) processes and that elevation of gal-3 is associated with disease progression and poor outcome in hf [ 3739 ] .", "herein , we investigate serum gal-9 levels in chinese patients with cad , and the severity of coronary arteries stenosis was evaluated by gensini score .", "furthermore , ifn- , il-4 , il-17 , tgf-1 , high - sensitivity c - reactive protein ( hs - crp ) , and the classical atherosclerosis risk factors were evaluated .", "additionally , th1 , th2 , th17 , and cd4cd25foxp3 tregs differentiation were detected in pbmc cultures exposed to gal-9 .", "232 patients presenting at the department of cardiology of huazhong university of science and technology affiliated union hospital between jan .", "2013 with suspected or established cad were recruited , including 149 males and 83 females .", "they were divided into four groups : ( 1 ) the control group , which consisted of 50 subjects with nca ( 28 men and 22 women , mean age 56.7  11.7 ) ; ( 2 ) sap group ( 25 men and 15 women , mean age 62.4  10.0 , inclusion criteria : typical chest discomfort that was associated with horizontal or downsloping st - segment depression > 1  mm in an exercise test ) ; ( 3 ) nsteacs group ( 60 men and 30 women , mean age 60.6  9.9 , which included unstable angina pectoris ( uap ) and non - st - segment elevation myocardial infarction ( nstemi ) , inclusion criteria : chest pain at rest with definite ischemic electrocardiographic changes , t - wave inversions and/or st - segment changes , or typical ischemic chest pain which persists for more than 20  min , elevated serum myocardial necrosis markers concentration and dynamic evolution , but not the typical st - elevation electrocardiogram ) ; ( 4 ) stemi group ( 36 men and 16 women , mean age 54.1  12.3 , inclusion criteria : myocardial infarction that was confirmed by a significant increase of serum creatine kinase mb , troponin i levels , and elevation of st - segment )", ". furthermore , all participants underwent coronary angiography after admission and completed a standardized questionnaire about previous and present illness , medication history , and smoking status .", "cardiovascular - interrelated factors , such as age , body mass index ( bmi ) , gender , and ejection fraction ( ef ) were estimated via physical examination , ultrasonic cardiography ( ucg ) , and electrocardiogram ( ecg ) , respectively .", "patients with the following characteristics were excluded from study enrollment : valvular heart disease , thromboembolism , autoimmune diseases ( systemic lupus erythematosus , rheumatoid arthritis , etc . ) , collagen disease , disseminated intravascular coagulation , severe liver and kidney disease , symptomatic heart failure , trauma or surgery , or malignant disease .", "the study was approved by the ethics committee of tongji medical college of huazhong university of science and technology , and all participants were provided written informed consent prior to study entry .", "two experienced cardiologists , who were not aware of the patients ' clinical and biochemical results , visually examined angiographic images to assess the extent and severity of cad .", "cad diagnosis was made according to the presence of 50% stenosis in 1 main coronary artery .", "the stenosis severity of cad was assessed by gensini score as previously described . in the ami group ,", "the blood was drawn with a 21-gauge needle for clean venipuncture of an antecubital vein and the samples were collected into sodium heparin vacutainers ( becton - dickinson ) .", "after centrifugation , the serum obtained was stored at 80c until further use to measure the concentration of gal-9 .", "the level of gal-9 was measured by elisa according to the manufacturer 's instructions ( r&d systems ) .", "in addition , the levels of ifn- , il-17 ( bolingkewei biotechnology , china ) , il-4 , hs - crp ( huijia biotechnology , china ) , and tgf-1 ( fumeng gene biotechnology , china ) were measured by elisa , following the manufacturer 's instructions .", "the minimal detectable concentrations were 28  pg / ml for gal-9 , 1.8  pg / ml for ifn- , 1.8  pg / ml for il-17 , 0.4  pg / ml for il-4 , 22  pg / ml for hs - crp , and 8  pg / ml for tgf-1 .", "the intra - assay and interassay coefficients of variation for all elisa were < 5% and < 10% , respectively .", "all the other biochemical measurements , including serum total cholesterol ( tc ) , triglyceride ( tg ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , apolipoprotein a - i ( apoa - i ) , apolipoprotein b ( apob ) , lipoprotein(a ) , creatinine , fasting plasma glucose ( fpg ) , uric acid , and cardiac troponin i ( ctni ) , were carried out by the biochemical laboratory of our cardiovascular institute using standard methods . peripheral blood mononuclear cells ( pbmcs ) from nca ( excluded from hypertension , diabetes , and dyslipidemia ) were isolated by ficoll - hypaque density gradient centrifugation ( pharmacia lkb technology , uppsala , sweden ) .", "cells from the interphase were collected and washed twice with dulbecco 's phosphate buffered saline ( d - pbs ) .", "remaining erythrocytes were removed using lysis buffer ( 4.14  g nh4cl , 0.5  g khco3 , and 18.6  mg na2edta in 500  ml water , ph = 7.4 and sterilized ) for 5  min on ice .", "pbmcs were resuspended in rpmi1640 ( lonza , verviers , belgium ) supplemented with 2.5% fcs , penicillin ( 100  u / ml)/streptomycin ( 100  u / ml ) , and sodium pyruvate ( 1  mm , sigma ) .", "pbmcs were stimulated with anti - human cd3 and anti - human cd28 antibodies ( both 2  g / ml , sanquin ) in the presence of recombinant gal-9 ( m ) ( 0.010.1  m , prospec - tany technogene ltd .", ", israel ) for 24  h. cells were allocated into tubes and washed once in phosphate buffered saline ( pbs ) . for tregs analysis", ", the cells were incubated with anti - cd4-fitc - hab and anti - cd25-apc - hab ( bd pharmingen ) . after the surface staining", ", the cells were stained with anti - foxp3-pe - hab ( bd pharmingen ) after fixation and permeabilization according to the manufacturer 's instructions . for analysis of th1 , th2 , and th17 cells ,", "the cells were stimulated with phorbol myristate acetate ( pma , 20  ng / ml , alexis biochemicals , san diego , ca ) and ionomycin ( 1  g / ml , alexis biochemicals ) for 4  h in the presence of 2  mol / ml monensin ( alexis biochemicals ) . the incubator was set at 37c under a 5% co2 environment .", "after culture for 4 hours , the cells were collected for staining according to the instructions .", "fixation and permeabilization were necessary before staining with anti - ifn--pe- , anti - il-4-pe- , or anti - il-17-pe- hab ( bd pharmingen ) .", "continuous variables are summarized as mean  standard deviation ( sd ) , and categorical data are presented as percentages . the shapiro - wilk test was used to assess the normality of distribution of continuous variables . in order to compare two groups ,", "continuous variables were tested using the independent samples t - test for normally distributed data and the mann - whitney u test for nonnormally distributed data ; the chi - square test was used for categorical variables . when three or more groups were compared , one - way anova was used .", "if significance was found , newman - keuls test was performed for post hoc analysis to detect the difference among groups .", "multiple stepwise regression analysis was used to evaluate the influence of different variables on gal-9 and to adjust for covariates .", "independent factors were sex , age , ctni , and the metabolic - related variables including bmi , fpg , lipid profiles , and hs - crp . to determine the independent predictors for the presence and severity of cad , all the conventional risk factors associated with cad were tested in multiple stepwise regression analysis .", "the prevalence of smoking and the levels of tg , lipoprotein(a ) , fpg , creatinine , hs - crp , and ctni were significantly higher in patients with cad compared to patients with nca group ( all p < 0.05 ) .", "however , other biochemical results , including tc , hdl - c , ldl - c , and uric acid , were similar between nca and cad patients .", "compared with stemi group , patients in sap and nsteacs groups showed markedly higher hdl - c levels and age and lower levels of lipoprotein(a ) , fpg , hs - crp , and ctni ( all p < 0.01 ) . compared to patients with sap ,", "the use of aspirin , -blockers , and statins was rare in patients with acs ( all p < 0.05 ) , whereas the levels of lipoprotein(a ) and hs - crp were markedly higher in patients with acs ( all p < 0.01 ) .", "a significant increase of creatinine levels was observed in patients with stemi compared with nsteacs group ( p < 0.05 ) and an obvious decrease of uric acid levels was found in patients with stemi compared to sap group ( p < 0.01 ) .", "unexpectedly , the distribution of hypertension , diabetes mellitus , dyslipidemia , and family history was similar among patients with acs and sap . among the total 232 study participants , serum gal-9 levels ranged from 1733.86 to 5259.39  pg / ml .", "compared with the nca group , patients with cad had significantly lower levels of gal-9 ( 3283.55  587.59 versus 3565.97  544.37  pg / ml , p < 0.05 ; figure 1(a ) ) .", "in addition , we found that serum gal-9 levels were significantly lower in the stemi ( 3126.36  637.7 ", "pg / ml ) than those in the sap group ( 3607.91  541.35  pg / ml ) or the nca group ( stemi versus sap and nsteacs versus sap , all p < 0.01 ; stemi versus nca and nsteacs versus nca , all p < 0.01 ; figure 1(b ) ) .", "interestingly , serum gal-9 levels did not differ significantly between patients with nsteacs and stemi ( p > 0.05 ) , nor was there a difference between the sap and nca groups ( p > 0.05 ; figure 1(b ) ) .", "as shown in figure 2 , serum il-17 , il-4 , ifn- , and tgf-1 were detected in each group .", "the il-17 and ifn- concentrations in patients with stemi ( il-17 , 57.16  16.14  pg / ml ; ifn- , 13.43  5.29 ", "pg / ml ) and nsteacs ( il-17 , 47.68  14.46  pg / ml ; ifn- , 11.28  4.23 ", "pg / ml ) were significantly higher compared with those in patients with sap ( il-17 , 26.94  9.09  pg / ml ; ifn- , 6.05  2.41  pg / ml ) and nca groups ( il-17 , 24.00  10.14  pg / ml ; ifn- , 5.83  2.12  pg / ml ) ( all p < 0.01 ; figures 2(a ) and 2(c ) ) .", "il-4 concentrations showed no difference in any of the groups ( stemi , 7.25  3.82  pg / ml ; nsteacs , 7.22  3.87  pg / ml ; sap , 8.12  4.48  pg / ml ; nca , 6.83  3.25 ", "tgf-1 concentrations in patients with stemi ( 4.08  2.13  ng / ml ) and nsteacs ( 4.73  2.71  ng / ml ) were significantly lower than those in patients with sap ( 8.03  3.88  ng / ml ) and nca ( 9.02  4.86  ng / ml ) ( all p < 0.01 ; figure 2(d ) ) . importantly , gal-9 levels were negatively correlated with il-17 ( r = 0.45 , p < 0.001 ; figure 3(a ) ) and ifn- ( r = 0.53 , p < 0.001 ; figure 3(c ) ) but positively associated with tgf-1 ( r = 0.58 , p < 0.001 ; figure 3(d ) ) . however , gal-9 levels showed no correlation with il-4 concentrations ( r = 0.04 , p = 0.528 ; figure 3(b ) ) . to detect the involvement of gal-9 in the induction of differentiation of human cd4 t cells", "gal-9 expanded the number of cd4cd25foxp3 tregs ( control , 6.99  1.61% ; gal-9 ( 0.01  m ) , 9.24  2.04% ; gal-9 ( 0.03  m ) , 10.27  3.04% ; gal-9 ( 0.1  m ) , 11.24  3.23% ) and decreased the development of th17 ( control , 0.628  0.239% ; gal-9 ( 0.01  m ) , 0.418  0.171% ; gal-9 ( 0.03  m ) , 0.214  0.064% ; gal-9 ( 0.1  m ) , 0.059  0.015% ) dose dependently ( figures", "4(c ) and 4(d ) ) , with resulting increase in secretion of tgf-1 ( control , 165.3  48.6  pg / ml ; gal-9 ( 0.01  m ) , 227.8  72.4  pg / ml ; gal-9 ( 0.03  m ) , 312.9  81.7  pg / ml ; gal-9 ( 0.1  m ) , 528.1  97.4 ", "pg / ml ) and suppressed il-17 production ( control , 132.4  23.6  pg / ml ; gal-9 ( 0.01  m ) , 110.3  25.7 ", "pg / ml ; gal-9 ( 0.03  m ) , 81.9  18.0  pg / ml ; gal-9 ( 0.1  m ) , 56.6  13.8 ", "however , the development of th1 and th2 cells and secretion of ifn- and il-4 in the supernatant were similar in the gal-9 untreated and treated pbmcs ( figures 4(a ) , 4(b ) , and 4(e ) ) .", "as we expected , gal-9 levels were found to be negatively correlated with hs - crp ( r = 0.37 , p < 0.001 ; figure 5(a ) ) , the gensini score ( r = 0.23 , p = 0.001 ; figure 5(b ) ) , fpg ( r = 0.15 , p = 0.027 ; figure 5(c ) ) , and lipoprotein(a ) ( r = 0.16 , p = 0.012 ; figure 5(d ) ) but positively associated with creatinine ( r = 0.21 , p = 0.002 ; figure 5(e ) ) and age ( r = 0.14 , p = 0.028 ; figure 5(f ) ) . to determine", "which variables were independently associated with gal-9 , multiple stepwise regression analysis using gal-9 as the dependent variable identified sex , age , bmi , fpg , tc , tg , hdl - c , ldl - c , hs - crp , lipoprotein(a ) , creatinine , uric acid , ctni , and therapeutic use of statins as independent variables . after adjustments , only hs - crp ( b = 72.158 , p < 0.001 ) , lipoprotein(a ) ( b = 8.333 , p = 0.045 ) , creatinine ( b = 5.228 , p < 0.001 ) , and use of therapeutic statins ( b = 362.29 , p < 0.001 ) retained significance for independently predicting gal-9 ( table 3 ) . in order to determine which variables", "gensini score was the dependent variable , and sex , age , bmi , fpg , lipid profiles , gal-9 , hs - crp , uric acid , traditional risk factors , and statins were independent variables .", "after adjustments , only gal-9 ( b = 0.145 , p = 0.011 ) , age ( b = 0.276 , p < 0.001 ) , and lipoprotein(a ) ( b = 0.280 , p <", "in the present study , we found that participants with cad had lower serum gal-9 levels compared with nca . furthermore , serum gal-9 levels in the acs group were significantly lower than those in the sap or nca group .", "moreover , gal-9 was found to be independently associated with hs - crp , lipoprotein(a ) , and creatinine .", "notably , gal-9 suppressed t - helper 17 ( th17 ) and expanded regulatory t cells ( tregs ) as analyzed within the activated cd4 t cell population , resulting in decreased il-17 production and increased secretion of tgf-1 .", "to the best of our knowledge , this is the first study to investigate the relation of serum gal-9 levels with the presence and the severity of coronary arteries stenosis .", "mor et al . have reported that cd4cd25 tregs deficiency enhanced atherosclerotic lesion development in ldlr mice , and adoptive transfer of cd4cd25 tregs attenuated the initiation and progression of atherosclerosis in apoe mice [ 42 , 43 ] . moreover", ", our laboratory has detected that th17/treg functional imbalance exists in patients with acs , suggesting a potential role for th17/treg imbalance in plaque destabilization and the onset of acs [ 8 , 9 ] .", "of note , gal-9 is a multifunctional modulator of t cell immunity with apoptotic effects on th17 cells and stimulatory activity on tregs in autoimmune diseases . in line with this publication", ", we found that gal-9 was able to induce cd4cd25foxp3 tregs development and inhibit th17 differentiation in activated pbmcs of nca .", "therefore , we speculated that gal-9 plays an immune - regulatory role in atherosclerosis via its effects on tregs and th17 cells .", "acs patients have significantly higher il-17 and il-17 induced cytokines ( il-6 and il-8 ) , while the pleiotropic cytokine ifn- is a proinflammatory regulator that is expressed at high levels in atherosclerotic lesions .", "in addition , it has been demonstrated that increased expression of tgf-1 is a stabilizing factor in human atherosclerotic plaques . in this study", ", we also found that il-17 and ifn- concentrations in patients with stemi and nsteacs were significantly higher compared with those in patients with sap and nca , while tgf-1 concentrations in patients with stemi and nsteacs were significantly lower than those in patients with sap and nca .", "these results again confirmed that inflammatory cytokines mentioned above have differential effects in atherosclerosis based cad .", "interestingly , gal-9 levels were showed to be negatively correlated with il-17 and ifn- but positively associated with tgf-1 .", "furthermore , we found that gal-9 was able to increase secretion of tgf-1 and decrease il-17 secretion in activated pbmcs .", "consequently , our data in vitro imply a protective role for gal-9 in atherosclerosis via expanding tgf-1 and suppressing il-17 secretion .", "however , the precise effect and mechanism of gal-9 in atherosclerosis are still to be elucidated in atherosclerosis animal models .", "surprisingly , we found that the development of th1 cells and secretion of ifn- in the supernatant were similar in gal-9 untreated and treated pbmcs , since zhu et al . reported that gal-9 induces apoptosis in th1 cells .", "this discrepancy may be ascribed to the diverse concentrations of gal-9 and different incubation periods . additionally , il-4 concentrations in the supernatant showed no difference in gal-9 untreated and treated pbmcs .", "this result further confirms that the effect of il-4 is still controversial [ 45 , 46 ] , reflecting its dual pro- and anti - inflammatory character .", "moreover , there are numerous publications showing that gal-9 has proinflammatory effects in addition to its immune - suppressive effects , especially through its actions on cells of the innate immune system like dc and nk cells [ 47 , 48 ] .", "thus , these results indicate that gal-9 has bidirectional effects like many immune - regulatory molecules .", "our data showed that cad patients had lower serum gal-9 levels than those without cad , and gal-9 was independently associated with the gensini score in patients with cad .", "these results suggest that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis .", "previous studies have elucidated that serum biomarkers are recognized as important tools for prediction , diagnosis , risk stratification , and therapeutic decision - marking for patients with cad .", "accumulating studies demonstrated that atherosclerotic lesion instability and rupture induced by inflammation are the major mechanisms of acs [ 50 , 51 ] .", "recently , a large number of clinical experiments have reported that hs - crp is not only a biomarker of inflammations and atherosclerosis , but also a marker of atheromatous plaque vulnerability [ 5254 ] . in our study , patients with acs had higher serum hs - crp levels compared to patients with sap . of note , gal-9 was found to be independently associated with hs - crp .", "hence , low serum concentrations of gal-9 may take part in the onset of acs and may act as a predictor of atheromatous plaque vulnerability .", "however , whether a causal relationship exists between the two processes is still to be studied .", "several lines of evidence indicate that lipoprotein(a ) is an independent risk factor for patients with cad [ 55 , 56 ] .", "all these findings together suggest that gal-9 might be a potential independent biomarker of cad .", "interestingly , our data showed that serum gal-9 levels were higher in patients of cad complicated with t2 dm than those of cad without t2 dm ( data not shown ) .", "'s study reporting that serum gal-9 levels are elevated in the patients with type 2 diabetes ( t2 dm ) and chronic kidney disease ( ckd ) .", "however , serum gal-9 levels were decreased in cad and elevated in the patients with t2 dm .", "this paradox could be attributed to the fact that atherosclerosis is highly multifactorial , tregs and gal-9 being only some factors among many others .", "the exact mechanism is still to be elucidated but different diseases may have divergent immune states .", "worth noting is the fact that the serum gal-9 level seems relatively higher in our study than in the previous studies [ 22 , 57 ] .", "the differences among these studies might be explained , at least partly , by different experimental designs , different number and characteristics of participants recruited , ethnical differences , medical treatments , lack of standardization of methods for the determination of serum gal-9 levels , and different ways in which samples were handled . nevertheless , several limitations of the present study should be considered .", "firstly , the number of study participants ( 232 ) was relatively small . secondly , given the cross - sectional design of the study , the causal relationship between gal-9 level and the severity of coronary artery stenosis can not be determined .", "thirdly , different methods of assessment of the coronary stenosis and the criterion for diagnosis of the diseased artery may lead to another result . finally , although we accounted for confounding of traditional cardiovascular risk factors , a potential for uncontrolled or residual confounding that could influence the relationship between gal-9 and cad would be plausible . in conclusion", ", we have shown that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis .", "importantly , gal-9 can expand cd4cd25foxp3 tregs and inhibit th17 development in activated pbmcs , leading to increased secretion of tgf-1 and decreased il-17 secretion .", "our observations provide evidence of the role of gal-9 in cad and that gal-9 is an independent negative predictor of acs , which may also at least partially explain the protection of gal-9 against coronary atherosclerotic plaque vulnerability .", "large - scale population - based clinical studies and further experimental studies are needed to elucidate the exact effect and potential biological mechanisms of gal-9 in the pathogenesis of cad and to evaluate whether gal-9 is a potential novel biomarker of cad ."], "abstract_text": ["<S> \n background . </S>", "<S> recently , several studies suggest that galectin-9 ( gal-9 ) might play a pivotal role in the pathogenesis of autoimmune diseases . </S>", "<S> however , the exact role of gal-9 in atherosclerosis remains to be elucidated . </S>", "<S> methods . </S>", "<S> serum gal-9 , high - sensitivity c - reactive protein ( hs - crp ) , interferon- ( ifn- )  , interleukin- ( il- ) 4 , il-17 , and transforming growth factor- ( tgf- ) 1 were measured . </S>", "<S> the effect of gal-9 on peripheral blood mononuclear cells ( pbmc ) was investigated in patients with normal coronary artery ( nca ) . </S>", "<S> results . the lowest level of gal-9 was found in the st - segment elevation myocardial infarction ( stemi ) group , followed by the non - st - segment elevation acs ( nsteacs ) , the nca , and the stable angina pectoris ( sap ) groups , respectively . </S>", "<S> additionally , gal-9 was found to be independently associated with hs - crp , lipoprotein(a ) , and creatinine . </S>", "<S> notably , gal-9 was also noted to be an independent predictor of the gensini score . </S>", "<S> moreover , gal-9 suppressed t - helper 17 ( th17 ) and expanded regulatory t cells ( tregs ) , resulting in decreased il-17 production and increased secretion of tgf-1 . </S>", "<S> conclusions . </S>", "<S> serum gal-9 is associated with not only coronary artery disease ( cad ) , but also the severity of coronary arteries stenosis . </S>", "<S> gal-9 can expand tregs and suppress th17 development in activated pbmc , implying that gal-9 has the potential to dampen the development of atherosclerosis and may be a new therapy for cad . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["coronary artery disease ( cad ) remains the principal cause of mortality and morbidity around the world [ 1 , 2 ] .", "although the mechanisms are still not entirely resolved , previous studies have demonstrated that an imbalance between t - helper 1- ( th1- ) mediated and th2-mediated immune functions contributes to the development of atherosclerosis .", "recently , th17 , which are a newly defined subset different from th1 and th2 , reactive to autoantigens are involved in the pathogenesis of several autoimmune diseases [ 4 , 5 ] .", "in addition to th1 , th2 , and th17 lineage , regulatory t cells ( tregs ) , a special subset of cd4 t cells , inhibit atherosclerosis development by attenuating activated t cell responses [ 6 , 7 ] .", "inspiringly , previous clinical and experimental studies from our laboratory have found that th17/tregs functional imbalance exists during atherogenesis , indicating that the imbalance of th17/tregs plays a critical role in cad [ 8 , 9 ] .", "hence , these findings have showed that cd4 t cell subpopulations play an important role in the initiation and progression of atherosclerosis .", "it is well known that cd4 t cells are divided into different subsets depending on the cytokines they produce .", "interferon- ( ifn- )  , the principal cytokine of th1 cells , is proinflammatory and exacerbates atherosclerotic disease , while the th2 cytokine such as interleukin- ( il- ) 4 is considered to be mainly atheroprotective and can neutralize th1 cytokine activity [ 1012 ] .", "moreover , hashmi and zeng have found that il-17 and il-17 induced cytokines ( il-6 and il-8 ) were significantly increased in acs patients .", "interestingly , tregs exert antiatherosclerosis partly by secretion of anti - inflammatory cytokine transforming growth factor ( tgf-1 ) , which can in turn induce the expression of forkhead box transcription factor p3 ( foxp3 ) and promote differentiation of tregs derived from cd4cd25 t cells [ 15 , 16 ] . therefore , these findings suggest that cytokines as those mentioned above are also essential for the formation and progression of the atherosclerotic plaque", ". the galectin family is characterized by conserved carbohydrate recognition domains ( crd ) that can bind glycosylated proteins .", "galectins are involved in a wide range of biologic processes such as cell adhesion and migration , proliferation and apoptosis , tumor biology , and regulation of the immune system .", "galectin- ( gal- ) 9 is a member of the galectin family of carbohydrate - binding proteins comprising two carbohydrate recognition domains connected by a linker peptide .", "gal-9 is mainly expressed by eosinophils , endothelial cells , macrophages , dcs , kupffer cells , vascular endothelial cells , intestinal epithelial cells , and in particular t lymphocytes .", "furthermore , gal-9 is believed to function predominantly as a multifaceted player in adaptive and innate immunity , especially in t cell development and homeostasis [ 2023 ] .", "accumulating evidence shows that gal-9 induces apoptosis in subsets of differentiated t cells , particularly in th1 and th17 cells , and stimulates the activity of tregs [ 19 , 2428 ] .", "indeed , treatment with recombinant gal-9 moderated the progression of experimental autoimmune encephalomyelitis ( eae ) , arthritis , and diabetes in animal models [ 19 , 2931 ] , by reducing the number of th1 and th17 cells and downregulating circulating ifn- levels .", "t cell immunoglobulin mucin- ( tim- ) 3 has been identified as a receptor for gal-9 .", "although gal-9 can function in a tim-3-independent fashion , the immune - regulatory effects of gal-9 are largely attributed to gal-9-tim-3 pathway [ 3235 ] .", "more recently , foks et al . demonstrated that anti - tim-3-ab administration promoted atherosclerotic plaque formation , which indicated that tim-3-gal-9 pathway may be concerned with the development of atherosclerosis .", "thus , it is reasonable to postulate that gal-9 may be involved in atherosclerosis based cad .", "in addition , several findings strongly support the original experimental observations that gal-3 plays an important role in the underlying heart failure ( hf ) processes and that elevation of gal-3 is associated with disease progression and poor outcome in hf [ 3739 ] .", "herein , we investigate serum gal-9 levels in chinese patients with cad , and the severity of coronary arteries stenosis was evaluated by gensini score .", "furthermore , ifn- , il-4 , il-17 , tgf-1 , high - sensitivity c - reactive protein ( hs - crp ) , and the classical atherosclerosis risk factors were evaluated .", "additionally , th1 , th2 , th17 , and cd4cd25foxp3 tregs differentiation were detected in pbmc cultures exposed to gal-9 ."], ["232 patients presenting at the department of cardiology of huazhong university of science and technology affiliated union hospital between jan .", "2013 with suspected or established cad were recruited , including 149 males and 83 females .", "they were divided into four groups : ( 1 ) the control group , which consisted of 50 subjects with nca ( 28 men and 22 women , mean age 56.7  11.7 ) ; ( 2 ) sap group ( 25 men and 15 women , mean age 62.4  10.0 , inclusion criteria : typical chest discomfort that was associated with horizontal or downsloping st - segment depression > 1  mm in an exercise test ) ; ( 3 ) nsteacs group ( 60 men and 30 women , mean age 60.6  9.9 , which included unstable angina pectoris ( uap ) and non - st - segment elevation myocardial infarction ( nstemi ) , inclusion criteria : chest pain at rest with definite ischemic electrocardiographic changes , t - wave inversions and/or st - segment changes , or typical ischemic chest pain which persists for more than 20  min , elevated serum myocardial necrosis markers concentration and dynamic evolution , but not the typical st - elevation electrocardiogram ) ; ( 4 ) stemi group ( 36 men and 16 women , mean age 54.1  12.3 , inclusion criteria : myocardial infarction that was confirmed by a significant increase of serum creatine kinase mb , troponin i levels , and elevation of st - segment )", ". furthermore , all participants underwent coronary angiography after admission and completed a standardized questionnaire about previous and present illness , medication history , and smoking status .", "cardiovascular - interrelated factors , such as age , body mass index ( bmi ) , gender , and ejection fraction ( ef ) were estimated via physical examination , ultrasonic cardiography ( ucg ) , and electrocardiogram ( ecg ) , respectively .", "patients with the following characteristics were excluded from study enrollment : valvular heart disease , thromboembolism , autoimmune diseases ( systemic lupus erythematosus , rheumatoid arthritis , etc . ) , collagen disease , disseminated intravascular coagulation , severe liver and kidney disease , symptomatic heart failure , trauma or surgery , or malignant disease .", "the study was approved by the ethics committee of tongji medical college of huazhong university of science and technology , and all participants were provided written informed consent prior to study entry .", "two experienced cardiologists , who were not aware of the patients ' clinical and biochemical results , visually examined angiographic images to assess the extent and severity of cad .", "cad diagnosis was made according to the presence of 50% stenosis in 1 main coronary artery .", "the stenosis severity of cad was assessed by gensini score as previously described . in the ami group ,", "the blood was drawn with a 21-gauge needle for clean venipuncture of an antecubital vein and the samples were collected into sodium heparin vacutainers ( becton - dickinson ) .", "after centrifugation , the serum obtained was stored at 80c until further use to measure the concentration of gal-9 .", "the level of gal-9 was measured by elisa according to the manufacturer 's instructions ( r&d systems ) .", "in addition , the levels of ifn- , il-17 ( bolingkewei biotechnology , china ) , il-4 , hs - crp ( huijia biotechnology , china ) , and tgf-1 ( fumeng gene biotechnology , china ) were measured by elisa , following the manufacturer 's instructions .", "the minimal detectable concentrations were 28  pg / ml for gal-9 , 1.8  pg / ml for ifn- , 1.8  pg / ml for il-17 , 0.4  pg / ml for il-4 , 22  pg / ml for hs - crp , and 8  pg / ml for tgf-1 .", "the intra - assay and interassay coefficients of variation for all elisa were < 5% and < 10% , respectively .", "all the other biochemical measurements , including serum total cholesterol ( tc ) , triglyceride ( tg ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , apolipoprotein a - i ( apoa - i ) , apolipoprotein b ( apob ) , lipoprotein(a ) , creatinine , fasting plasma glucose ( fpg ) , uric acid , and cardiac troponin i ( ctni ) , were carried out by the biochemical laboratory of our cardiovascular institute using standard methods . peripheral blood mononuclear cells ( pbmcs ) from nca ( excluded from hypertension , diabetes , and dyslipidemia ) were isolated by ficoll - hypaque density gradient centrifugation ( pharmacia lkb technology , uppsala , sweden ) .", "cells from the interphase were collected and washed twice with dulbecco 's phosphate buffered saline ( d - pbs ) .", "remaining erythrocytes were removed using lysis buffer ( 4.14  g nh4cl , 0.5  g khco3 , and 18.6  mg na2edta in 500  ml water , ph = 7.4 and sterilized ) for 5  min on ice .", "pbmcs were resuspended in rpmi1640 ( lonza , verviers , belgium ) supplemented with 2.5% fcs , penicillin ( 100  u / ml)/streptomycin ( 100  u / ml ) , and sodium pyruvate ( 1  mm , sigma ) .", "pbmcs were stimulated with anti - human cd3 and anti - human cd28 antibodies ( both 2  g / ml , sanquin ) in the presence of recombinant gal-9 ( m ) ( 0.010.1  m , prospec - tany technogene ltd .", ", israel ) for 24  h. cells were allocated into tubes and washed once in phosphate buffered saline ( pbs ) . for tregs analysis", ", the cells were incubated with anti - cd4-fitc - hab and anti - cd25-apc - hab ( bd pharmingen ) . after the surface staining", ", the cells were stained with anti - foxp3-pe - hab ( bd pharmingen ) after fixation and permeabilization according to the manufacturer 's instructions . for analysis of th1 , th2 , and th17 cells ,", "the cells were stimulated with phorbol myristate acetate ( pma , 20  ng / ml , alexis biochemicals , san diego , ca ) and ionomycin ( 1  g / ml , alexis biochemicals ) for 4  h in the presence of 2  mol / ml monensin ( alexis biochemicals ) . the incubator was set at 37c under a 5% co2 environment .", "after culture for 4 hours , the cells were collected for staining according to the instructions .", "fixation and permeabilization were necessary before staining with anti - ifn--pe- , anti - il-4-pe- , or anti - il-17-pe- hab ( bd pharmingen ) .", "continuous variables are summarized as mean  standard deviation ( sd ) , and categorical data are presented as percentages . the shapiro - wilk test was used to assess the normality of distribution of continuous variables . in order to compare two groups ,", "continuous variables were tested using the independent samples t - test for normally distributed data and the mann - whitney u test for nonnormally distributed data ; the chi - square test was used for categorical variables . when three or more groups were compared , one - way anova was used .", "if significance was found , newman - keuls test was performed for post hoc analysis to detect the difference among groups .", "multiple stepwise regression analysis was used to evaluate the influence of different variables on gal-9 and to adjust for covariates .", "independent factors were sex , age , ctni , and the metabolic - related variables including bmi , fpg , lipid profiles , and hs - crp . to determine the independent predictors for the presence and severity of cad , all the conventional risk factors associated with cad were tested in multiple stepwise regression analysis ."], ["the prevalence of smoking and the levels of tg , lipoprotein(a ) , fpg , creatinine , hs - crp , and ctni were significantly higher in patients with cad compared to patients with nca group ( all p < 0.05 ) .", "however , other biochemical results , including tc , hdl - c , ldl - c , and uric acid , were similar between nca and cad patients .", "compared with stemi group , patients in sap and nsteacs groups showed markedly higher hdl - c levels and age and lower levels of lipoprotein(a ) , fpg , hs - crp , and ctni ( all p < 0.01 ) . compared to patients with sap ,", "the use of aspirin , -blockers , and statins was rare in patients with acs ( all p < 0.05 ) , whereas the levels of lipoprotein(a ) and hs - crp were markedly higher in patients with acs ( all p < 0.01 ) .", "a significant increase of creatinine levels was observed in patients with stemi compared with nsteacs group ( p < 0.05 ) and an obvious decrease of uric acid levels was found in patients with stemi compared to sap group ( p < 0.01 ) .", "unexpectedly , the distribution of hypertension , diabetes mellitus , dyslipidemia , and family history was similar among patients with acs and sap . among the total 232 study participants , serum gal-9 levels ranged from 1733.86 to 5259.39  pg / ml .", "compared with the nca group , patients with cad had significantly lower levels of gal-9 ( 3283.55  587.59 versus 3565.97  544.37  pg / ml , p < 0.05 ; figure 1(a ) ) .", "in addition , we found that serum gal-9 levels were significantly lower in the stemi ( 3126.36  637.7 ", "pg / ml ) than those in the sap group ( 3607.91  541.35  pg / ml ) or the nca group ( stemi versus sap and nsteacs versus sap , all p < 0.01 ; stemi versus nca and nsteacs versus nca , all p < 0.01 ; figure 1(b ) ) .", "interestingly , serum gal-9 levels did not differ significantly between patients with nsteacs and stemi ( p > 0.05 ) , nor was there a difference between the sap and nca groups ( p > 0.05 ; figure 1(b ) ) .", "as shown in figure 2 , serum il-17 , il-4 , ifn- , and tgf-1 were detected in each group .", "the il-17 and ifn- concentrations in patients with stemi ( il-17 , 57.16  16.14  pg / ml ; ifn- , 13.43  5.29 ", "pg / ml ) and nsteacs ( il-17 , 47.68  14.46  pg / ml ; ifn- , 11.28  4.23 ", "pg / ml ) were significantly higher compared with those in patients with sap ( il-17 , 26.94  9.09  pg / ml ; ifn- , 6.05  2.41  pg / ml ) and nca groups ( il-17 , 24.00  10.14  pg / ml ; ifn- , 5.83  2.12  pg / ml ) ( all p < 0.01 ; figures 2(a ) and 2(c ) ) .", "il-4 concentrations showed no difference in any of the groups ( stemi , 7.25  3.82  pg / ml ; nsteacs , 7.22  3.87  pg / ml ; sap , 8.12  4.48  pg / ml ; nca , 6.83  3.25 ", "tgf-1 concentrations in patients with stemi ( 4.08  2.13  ng / ml ) and nsteacs ( 4.73  2.71  ng / ml ) were significantly lower than those in patients with sap ( 8.03  3.88  ng / ml ) and nca ( 9.02  4.86  ng / ml ) ( all p < 0.01 ; figure 2(d ) ) . importantly , gal-9 levels were negatively correlated with il-17 ( r = 0.45 , p < 0.001 ; figure 3(a ) ) and ifn- ( r = 0.53 , p < 0.001 ; figure 3(c ) ) but positively associated with tgf-1 ( r = 0.58 , p < 0.001 ; figure 3(d ) ) . however , gal-9 levels showed no correlation with il-4 concentrations ( r = 0.04 , p = 0.528 ; figure 3(b ) ) . to detect the involvement of gal-9 in the induction of differentiation of human cd4 t cells", "gal-9 expanded the number of cd4cd25foxp3 tregs ( control , 6.99  1.61% ; gal-9 ( 0.01  m ) , 9.24  2.04% ; gal-9 ( 0.03  m ) , 10.27  3.04% ; gal-9 ( 0.1  m ) , 11.24  3.23% ) and decreased the development of th17 ( control , 0.628  0.239% ; gal-9 ( 0.01  m ) , 0.418  0.171% ; gal-9 ( 0.03  m ) , 0.214  0.064% ; gal-9 ( 0.1  m ) , 0.059  0.015% ) dose dependently ( figures", "4(c ) and 4(d ) ) , with resulting increase in secretion of tgf-1 ( control , 165.3  48.6  pg / ml ; gal-9 ( 0.01  m ) , 227.8  72.4  pg / ml ; gal-9 ( 0.03  m ) , 312.9  81.7  pg / ml ; gal-9 ( 0.1  m ) , 528.1  97.4 ", "pg / ml ) and suppressed il-17 production ( control , 132.4  23.6  pg / ml ; gal-9 ( 0.01  m ) , 110.3  25.7 ", "pg / ml ; gal-9 ( 0.03  m ) , 81.9  18.0  pg / ml ; gal-9 ( 0.1  m ) , 56.6  13.8 ", "however , the development of th1 and th2 cells and secretion of ifn- and il-4 in the supernatant were similar in the gal-9 untreated and treated pbmcs ( figures 4(a ) , 4(b ) , and 4(e ) ) .", "as we expected , gal-9 levels were found to be negatively correlated with hs - crp ( r = 0.37 , p < 0.001 ; figure 5(a ) ) , the gensini score ( r = 0.23 , p = 0.001 ; figure 5(b ) ) , fpg ( r = 0.15 , p = 0.027 ; figure 5(c ) ) , and lipoprotein(a ) ( r = 0.16 , p = 0.012 ; figure 5(d ) ) but positively associated with creatinine ( r = 0.21 , p = 0.002 ; figure 5(e ) ) and age ( r = 0.14 , p = 0.028 ; figure 5(f ) ) . to determine", "which variables were independently associated with gal-9 , multiple stepwise regression analysis using gal-9 as the dependent variable identified sex , age , bmi , fpg , tc , tg , hdl - c , ldl - c , hs - crp , lipoprotein(a ) , creatinine , uric acid , ctni , and therapeutic use of statins as independent variables . after adjustments , only hs - crp ( b = 72.158 , p < 0.001 ) , lipoprotein(a ) ( b = 8.333 , p = 0.045 ) , creatinine ( b = 5.228 , p < 0.001 ) , and use of therapeutic statins ( b = 362.29 , p < 0.001 ) retained significance for independently predicting gal-9 ( table 3 ) . in order to determine which variables", "gensini score was the dependent variable , and sex , age , bmi , fpg , lipid profiles , gal-9 , hs - crp , uric acid , traditional risk factors , and statins were independent variables .", "after adjustments , only gal-9 ( b = 0.145 , p = 0.011 ) , age ( b = 0.276 , p < 0.001 ) , and lipoprotein(a ) ( b = 0.280 , p <"], ["in the present study , we found that participants with cad had lower serum gal-9 levels compared with nca . furthermore , serum gal-9 levels in the acs group were significantly lower than those in the sap or nca group .", "moreover , gal-9 was found to be independently associated with hs - crp , lipoprotein(a ) , and creatinine .", "notably , gal-9 suppressed t - helper 17 ( th17 ) and expanded regulatory t cells ( tregs ) as analyzed within the activated cd4 t cell population , resulting in decreased il-17 production and increased secretion of tgf-1 .", "to the best of our knowledge , this is the first study to investigate the relation of serum gal-9 levels with the presence and the severity of coronary arteries stenosis .", "mor et al . have reported that cd4cd25 tregs deficiency enhanced atherosclerotic lesion development in ldlr mice , and adoptive transfer of cd4cd25 tregs attenuated the initiation and progression of atherosclerosis in apoe mice [ 42 , 43 ] . moreover", ", our laboratory has detected that th17/treg functional imbalance exists in patients with acs , suggesting a potential role for th17/treg imbalance in plaque destabilization and the onset of acs [ 8 , 9 ] .", "of note , gal-9 is a multifunctional modulator of t cell immunity with apoptotic effects on th17 cells and stimulatory activity on tregs in autoimmune diseases . in line with this publication", ", we found that gal-9 was able to induce cd4cd25foxp3 tregs development and inhibit th17 differentiation in activated pbmcs of nca .", "therefore , we speculated that gal-9 plays an immune - regulatory role in atherosclerosis via its effects on tregs and th17 cells .", "acs patients have significantly higher il-17 and il-17 induced cytokines ( il-6 and il-8 ) , while the pleiotropic cytokine ifn- is a proinflammatory regulator that is expressed at high levels in atherosclerotic lesions .", "in addition , it has been demonstrated that increased expression of tgf-1 is a stabilizing factor in human atherosclerotic plaques . in this study", ", we also found that il-17 and ifn- concentrations in patients with stemi and nsteacs were significantly higher compared with those in patients with sap and nca , while tgf-1 concentrations in patients with stemi and nsteacs were significantly lower than those in patients with sap and nca .", "these results again confirmed that inflammatory cytokines mentioned above have differential effects in atherosclerosis based cad .", "interestingly , gal-9 levels were showed to be negatively correlated with il-17 and ifn- but positively associated with tgf-1 .", "furthermore , we found that gal-9 was able to increase secretion of tgf-1 and decrease il-17 secretion in activated pbmcs .", "consequently , our data in vitro imply a protective role for gal-9 in atherosclerosis via expanding tgf-1 and suppressing il-17 secretion .", "however , the precise effect and mechanism of gal-9 in atherosclerosis are still to be elucidated in atherosclerosis animal models .", "surprisingly , we found that the development of th1 cells and secretion of ifn- in the supernatant were similar in gal-9 untreated and treated pbmcs , since zhu et al . reported that gal-9 induces apoptosis in th1 cells .", "this discrepancy may be ascribed to the diverse concentrations of gal-9 and different incubation periods . additionally , il-4 concentrations in the supernatant showed no difference in gal-9 untreated and treated pbmcs .", "this result further confirms that the effect of il-4 is still controversial [ 45 , 46 ] , reflecting its dual pro- and anti - inflammatory character .", "moreover , there are numerous publications showing that gal-9 has proinflammatory effects in addition to its immune - suppressive effects , especially through its actions on cells of the innate immune system like dc and nk cells [ 47 , 48 ] .", "thus , these results indicate that gal-9 has bidirectional effects like many immune - regulatory molecules .", "our data showed that cad patients had lower serum gal-9 levels than those without cad , and gal-9 was independently associated with the gensini score in patients with cad .", "these results suggest that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis .", "previous studies have elucidated that serum biomarkers are recognized as important tools for prediction , diagnosis , risk stratification , and therapeutic decision - marking for patients with cad .", "accumulating studies demonstrated that atherosclerotic lesion instability and rupture induced by inflammation are the major mechanisms of acs [ 50 , 51 ] .", "recently , a large number of clinical experiments have reported that hs - crp is not only a biomarker of inflammations and atherosclerosis , but also a marker of atheromatous plaque vulnerability [ 5254 ] . in our study , patients with acs had higher serum hs - crp levels compared to patients with sap . of note , gal-9 was found to be independently associated with hs - crp .", "hence , low serum concentrations of gal-9 may take part in the onset of acs and may act as a predictor of atheromatous plaque vulnerability .", "however , whether a causal relationship exists between the two processes is still to be studied .", "several lines of evidence indicate that lipoprotein(a ) is an independent risk factor for patients with cad [ 55 , 56 ] .", "all these findings together suggest that gal-9 might be a potential independent biomarker of cad .", "interestingly , our data showed that serum gal-9 levels were higher in patients of cad complicated with t2 dm than those of cad without t2 dm ( data not shown ) .", "'s study reporting that serum gal-9 levels are elevated in the patients with type 2 diabetes ( t2 dm ) and chronic kidney disease ( ckd ) .", "however , serum gal-9 levels were decreased in cad and elevated in the patients with t2 dm .", "this paradox could be attributed to the fact that atherosclerosis is highly multifactorial , tregs and gal-9 being only some factors among many others .", "the exact mechanism is still to be elucidated but different diseases may have divergent immune states .", "worth noting is the fact that the serum gal-9 level seems relatively higher in our study than in the previous studies [ 22 , 57 ] .", "the differences among these studies might be explained , at least partly , by different experimental designs , different number and characteristics of participants recruited , ethnical differences , medical treatments , lack of standardization of methods for the determination of serum gal-9 levels , and different ways in which samples were handled . nevertheless , several limitations of the present study should be considered .", "firstly , the number of study participants ( 232 ) was relatively small . secondly , given the cross - sectional design of the study , the causal relationship between gal-9 level and the severity of coronary artery stenosis can not be determined .", "thirdly , different methods of assessment of the coronary stenosis and the criterion for diagnosis of the diseased artery may lead to another result . finally , although we accounted for confounding of traditional cardiovascular risk factors , a potential for uncontrolled or residual confounding that could influence the relationship between gal-9 and cad would be plausible . in conclusion", ", we have shown that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis .", "importantly , gal-9 can expand cd4cd25foxp3 tregs and inhibit th17 development in activated pbmcs , leading to increased secretion of tgf-1 and decreased il-17 secretion .", "our observations provide evidence of the role of gal-9 in cad and that gal-9 is an independent negative predictor of acs , which may also at least partially explain the protection of gal-9 against coronary atherosclerotic plaque vulnerability .", "large - scale population - based clinical studies and further experimental studies are needed to elucidate the exact effect and potential biological mechanisms of gal-9 in the pathogenesis of cad and to evaluate whether gal-9 is a potential novel biomarker of cad ."]]}
{"article_id": "PMC4326345", "article_text": ["sedation is often required in young children in order to obtain good diagnostic cross - sectional imaging like computed tomography scan ( ct scan ) and magnetic resonance imaging ( mri ) .", "sometimes sedation is also needed for older children with complex neurodevelopmental disorders , such as autism and attention deficit hyperactivity disorder .", "moderate sedation is unable to guarantee patient compliance ; therefore , a deeper level of sedation is required [ 1 , 2 ] .", "the success of sedation for mri has typically been measured by two factors : the safety of sedation procedure ( lack of adverse events ) and its effectiveness ( completion of diagnostic examination ) .", "it can be challenging to obtain the deep sedation level required to prevent the patient 's movement while maintaining respiratory and hemodynamic stability .", "therefore , it is very important to select the appropriate drugs and dosage to achieve those objectives .", "dexmedetomidine is a highly selective adrenoceptor agonist with a distribution half - life of six minutes and an elimination half - life of two hours .", "five prospective trials have evaluated the efficacy of dexmedetomidine for sedation during noninvasive radiologic imaging [ 812 ] .", "dexmedetomidine can be associated with side effects of hypotension , bradycardia , and transient hypertension with loading dose .", "dexmedetomidine has been used at our institution since 2006 for mri and other noninvasive radiologic procedures .", "the purpose of this retrospective study is to present our institutional experience with dexmedetomidine in relation to efficacy and related side effects .", "collection of quality assurance data includes patient demographics , adverse events , physiologic variables , drug dosages , the time required to sedate the patient , time needed to obtain the imaging study , and recovery time .", "after approval by the institutional review board , we conducted a retrospective analysis of all patients who received sedation for mri from july 2007 to december 2012 .", "institutional sedation policies are based on guidelines by the joint commission on accreditation of health care organization and american academy of pediatrics and were closely followed [ 13 , 14 ] .", "vital signs including pulse oximetry , heart rate , noninvasive blood pressure monitoring , and nasal capnography ( with concomitant oxygen delivery via the nasal cannula ) are continuously monitored and documented every five minutes throughout sedation . at the start of sedation", ", intravenous dexmedetomidine was bolused at 2  g / kg over 10 minutes followed by a continuous infusion of 1  g / kg / h . most of the patients were sedated by the end of the bolus ; if not , a second bolus of 2  g / kg was repeated over another 10 minutes prior to the start of the maintenance infusion .", "patients who continue to move after the second bolus or while the procedure is in progress , can compromise the quality of imaging and were given additional medications like midazolam or fentanyl as per physician discretion .", "mg / kg to a max of 2  mg and for fentanyl was 0.5  mcg / kg1  mcg / kg to a max of 50  mcg .", "the goal was to achieve a minimum ramsay sedation score ( rss ) of 4 as assessed by a sedation nurse .", "rss is a clinically derived sedation score generally accepted as a tool for assessing the depth of sedation .", "usually , a score of 4 - 5 is targeted to ensure appropriate sedation for diagnostic imaging studies [ 11 , 16 ] .", "peak onset of the sedation was defined as the time from the start of the loading dose to achievement of a ramsay score of 4 .", "procedure time was the time of achieving the required ramsay score to the end of the procedure ( stoppage of drug administration ) .", "discharge time was defined as time from the end of the procedure to actual time when the patient was discharged home .", "normal ranges for heart rates , blood pressure , and respiratory rates for data analysis were based on the published normal of fleming et al . [ 17 , 18 ] .", "a trained registered nurse provided continuous assessment and monitoring while the procedure was in progress under the direct supervision of a pediatric intensivist .", "monitoring was continued until the patient was awake with a minimum aldrete score of 9 points , and the patient has tolerated clear liquids prior to discharge [ 19 , 20 ] . all the parents were provided with written discharge instructions and a direct phone number for further assistance or to report any adverse effects .", "database records were analyzed using dedicated statistical software sas v9.3 ( sas institute , cary , nc ) .", "changes in the vital signs associated with the use of dexmedetomidine from the baseline were evaluated and compared using student 's t - test and mann - whitney rank - sum test , depending on the distribution of the data .", "the entire study cohort was divided into three groups depending on dexmedetomidine bolus and additional medications received .", "dexmedetomidine group a received one bolus ; dexmedetomidine group b received two boluses ; dexmedetomidine group c received one or two boluses and additional medications .", "three groups were compared with respect to age , weight , blood pressure , heart rate , procedure time , and discharge time using analysis of variance .", "incidences of bradycardia and hypotension were analyzed with fisher 's exact test , due to low cell counts .", "discharge time of bradycardia and hypotensive patients was compared with normal cohorts using the t - test .", "the most common indications included seizure disorder , developmental delay and behavioral disorder , autism , and neoplasia .", "382 ( 70.2% ) of the total patients received one - time bolus , while two boluses were given to 162 patients ( 29.8% ) . in the population of 544 patients , additional medications ( fentanyl or midazolam )", "were required in 117 ( 21.5% ) for spontaneous movements to avoid motion artifacts that can compromise mri quality ( 54 patients with one - time bolus and 43 patients with two boluses ) .", "dexmedetomidine - induced vital sign changes from baseline for the whole group are shown in figure 1 .", "hypotension ( systolic blood pressure 20% below the normal limits ) was observed in 100 patients ( 18.4% ) and transient hypertension ( systolic blood pressure > 20% above age - specific high limits ) was observed in 137 of the patients ( 25.2% ) .", "there were 210 ( 38.6% ) patients who had a significant decrease in respiratory rate , that is , > 20% from the baseline , but no desaturation , upper airway obstruction , or apnea events were observed and the decrease was minimal in 403 patients ( 74% ) .", "most of the patients received prophylactic supplemental oxygen to maintain oxygen saturation > 95% as per protocol , which makes it impossible to determine the actual incidence of desaturation at room air .", "patients were divided into three groups based on numbers of dexmedetomidine boluses and additional medications received .", "comparisons of blood pressure , heart rate , and other clinical measures between these groups of patients are shown in table 2 .", "procedure time was significantly shorter in dexmedetomidine group a ( 40.31  19.40  min , p < 0.0001 ) compared to the other two groups .", "oxygen , dose , maximum dbp , maximum respiration rate , and initial spo2 had statistically significant ( but clinically insignificant ) differences between groups .", "the occurrence of hypotension among all three groups did not reach a statistically significant value ( p = 0.82 ) .", "decrease in blood pressure from the baseline was frequently observed ; however , medical intervention was not needed to correct it .", "the incidence of hypotension was usually noticed to be towards the completion of procedure or after stopping the infusion , with a mean procedure time of 48.73  25.46 minutes .", "initial transient hypertension occurred in 137 patients approximately 20 minutes after the dexmedetomidine bolus and lasted for < 15 minutes . in the population of 544 patients , 165 children ( 30.9% ) had heart rates below the age - specific normal awake range during sedation ( based on the published normal of fleming et al . ) . only in 21 children ( 3.9% of total cohort ) did the lowest recorded heart rate fall > 20% below the given baseline average range .", "bradycardia ( hr < 60/min ) , as defined according to pediatric advance life support ( pals ) guidelines , was observed in 10 children ( 4.5% ) mostly in the age range of 13 years ( figure 2 ) .", "all the patients with bradycardia were continuously monitored and assessed by the supervising intensivist for normal blood pressure and oxygen saturation of 95% or above . no patient required treatment .", "the incidence of bradycardia was high in patients in the age range of 13 years compared with the rest of the cohort .", "the precise etiology of the bradycardia observed in this analysis can not be delineated as electrocardiogram monitoring is known to be significantly distorted by the magnetohydrodynamic ( mhd ) effect and is nondiagnostic within the bore of any mri magnet .", "time to discharge when dexmedetomidine was used alone was 92  25 versus 94  37  min when additional medications were used ( p = 0.46 ) .", "average discharge time among 165 children with bradycardia was longer and statistically significant compared to the other 372 children in the study population without bradycardia ( 99.08  32.57  min versus 89.74  25.54  min , p = 0.0012 , student 's t - test ) .", "gender was not associated with any change in discharge time ( p = 0.30 ) ( table 3 ) .", "the ideal drug would allow optimal imaging , while maintaining hemodynamic and respiratory stability . as reported in previous studies ,", "this occurred more frequently in hyperactive , uncooperative , and older children [ 4 , 22 ] .", "dexmedetomidine , a selective alpha 2 adrenoceptor agonist , has a very safe therapeutic window with respect to respiratory depression .", "this quality offers a distinct advantage in procedural sedations , where the patient is not immediately accessible to the medical team .", "there are various studies demonstrating that dexmedetomidine is a good option for procedural sedation [ 10 , 12 ] .", "interestingly , the dose used varies greatly indicating an open debate regarding the best dosage .", "previous studies indicate that infusion of relatively low dose dexmedetomidine 0.10.7  /kg / h provides effective sedation [ 2327 ] . in a study conducted on eight healthy volunteers receiving dexmedetomidine infusion of 0.20.6  /kg / h , the visual analog sedation scores and bispectral index scores dropped by 3060% . however , this level of sedation will likely not be conducive to pediatric mri sedation , where higher doses of dexmedetomidine will be needed to accomplish the necessary sedation level .", "while dosing started low in initial reports , a recent one found that higher doses are required and have been well tolerated .", "a comparison of pentobarbital and chloral hydrate was performed by rooks et al . in 498 children for mri sedation .", "several clinical trials have compared dexmedetomidine to propofol for pediatric procedural sedation . in a trial of 60 children undergoing mri ,", "onset , recovery , and discharge time were all significantly shorter in the propofol group .", "adverse effects such as lower blood pressure and heart rate and respiratory rate were also found more in the propofol group .", "dexmedetomidine safety and efficacy for sedation during noninvasive radiologic procedures have been evaluated in four prospective trials [ 1 , 810 ] .", "all of these studies reported that the use of high dose dexmedetomidine as a sole sedative resulted in high quality radiologic imaging with less use of additional rescue medicines .", "our study also demonstrated that the use of high dose dexmedetomidine as sole agent is effective for mri sedation .", "we used fentanyl and midazolam as rescue medicines in 117 ( 21.5% ) of our patients to avoid imaging artifacts , suggesting that these agents make a significant contribution to the sedation strategy for a sizeable minority of patients . in our study , the heart rate decreased significantly from baseline during sedation .", "this is an expected effect of anxiolysis and reflects higher baseline heart rate values from anxiety .", "other studies reported a decrease in heart rate < 20% from the baseline that was considered to be clinically insignificant in 86 ( 26% ) patients .", "to have a true comparison with previously published data , we use the same definition of bradycardia ; that is , the lowest recorded heart rate during sedation fell > 20% below the given baseline average range . using that definition ,", "the incidence of bradycardia in our study was 3.9% ( 21 children ) in total cohort comparable to 4% as reported by mason et al .", "more importantly , during those periods of bradycardia , all patients maintained normal blood pressure and normal oxygen saturation ( 95% or higher ) .", "treating bradycardia in normotensive children with glycopyrrolate has been reported to be associated with hypertensive episode , which could be beneficial to children who are hypotensive and bradycardic at the same time .", "similar to our findings , previous investigators have also reported the occurrence of transient hypertension with bolus administration of dexmedetomidine . in our study ,", "the occurrence of hypotension associated with high doses of dexmedetomidine was about 18% , observed immediately after stopping the infusion or towards the end of the procedure .", "none of the children needed any intervention , consistent with observation reported in other studies [ 12 , 31 ] .", "respiratory events make up a large proportion ( 5.5% ) of sedation complications in children . in some studies", ", rapid administration of large loading doses has been described to cause respiratory complications [ 3234 ] .", "a loading dose of dexmedetomidine given over 2 minutes has been reported to cause irregular respiration , apnea , slight hypoxemia , and hypercapnia .", "however , similar to ours , several other studies have reported trivial effect of dexmedetomidine on respiration [ 12 , 34 , 36 ] which is consistent with the notion that the risk of respiratory depression is minimal with careful dexmedetomidine sedation . despite these supporting lines of evidence , monitoring of respiratory function during the administration of dexmedetomidine in those receiving midazolam or fentanyl , which may depress respiratory function , appears warranted .", "our reported discharge time of 92 minutes with dexmedetomidine only and 94 minutes when additional medicines were used is comparable to the 90 minutes ' discharge time of heard et al . .", "observed a recovery time of 47 minutes   and mason et al .   reported recovery times ranging from 24.8 minutes to 35.2 minutes , depending on the dose of dexmedetomidine used .", "in some studies , recovery time is the time lapsed until the patient meets the discharge criteria , while in our study , like few others , discharged time is defined as the actual time of leaving the recovery room to go home .", "discharge time of children experiencing bradycardia was longer than that for those who did not .", "in our study , 165 children with bradycardia had a mean discharge time which was statistically significant and longer than the other 372 children in the study without bradycardia ( 99.08 minutes versus 89.74 minutes ; p = 0.0012 ) .", "this probably could be due to the prolonged monitoring of these patients until the bradycardia resolved .", "recovery time previously reported after sedation with propofol for mri has been 17  8 minutes , almost half of the recovery time when dexmedetomidine was used as a sole agent . in a second trial", ", forty children between the ages of 1 and 10 years were randomized to receive either a combination of midazolam + dexmedetomidine or propofol only for sedation during mri .", "the fast recovery times of propofol must be weighed against the fact that it induced deeper sedation with significant hypotension and oxygen desaturation . taking the results of these comparison trials as a whole", ", dexmedetomidine appears to provide a useful alternative to propofol for procedural sedation in children , with a longer time of recovery but a lower incidence of adverse effects .", "this is a good alternative to propofol for patients with soy and egg white allergy and also in institutions where use of propofol is strictly limited to be used by anesthesiologists .", "it could be asserted that the reported efficacy is due to the use of an intensivist based specialized sedation team rather than to dexmedetomidine itself .", "this is reasonably true to some extent as specialization and experience should increase both success and efficiency . in spite of that , this can be stated with confidence ; much of the reported success is specifically a function of dexmedetomidine .", "this is a descriptive study and few , if any , conclusions can be drawn about safety because the occurrence of serious sedation related side effects is fortunately rare .", "additional prospective studies of the sedation for mri in children using a greater number of patients are warranted to provide a true idea of safety . in summary ,", "high dose dexmedetomidine is an attractive and effective medication in children for mri sedation . when using high dose dexmedetomidine as the only agent for pediatric mri , it is not unusual to observe heart rate and blood pressure outside the established ", "awake  normal values . in our experience , these changes were pretty benign and were not associated with any adverse event .", "we conclude and recommend that , from hemodynamics and respiratory perspective , higher dose dexmedetomidine was well tolerated and is effective to use for successful completion of mri , in the majority of pediatric patients ."], "abstract_text": ["<S> \n objective . to determine the efficacy and safety of high dose dexmedetomidine as a sole sedative agent for mri . </S>", "<S> we report our institution 's experience . design . a retrospective institutional review of dexmedetomidine usage for pediatric mri over 5.5 years . </S>", "<S> protocol included a dexmedetomidine bolus of 2  g / kg intravenously over ten minutes followed by 1  g / kg / hr infusion . </S>", "<S> 544 patients received high dose dexmedetomidine for mri . </S>", "<S> a second bolus was used in 103 ( 18.9% ) patients . </S>", "<S> 117 ( 21.5% ) required additional medications . </S>", "<S> efficacy , side effects , and use of additional medicines to complete the mri were reviewed . </S>", "<S> data was analyzed using student 's t - test , fisher 's exact test , and analysis of variance ( anova ) . </S>", "<S> main results . </S>", "<S> dexmedetomidine infusion was associated with bradycardia ( 3.9% ) and hypotension ( 18.4% ) . </S>", "<S> none of the patients required any intervention . </S>", "<S> vital signs were not significantly different among the subgroup of patients receiving one or two boluses of dexmedetomidine or additional medications . </S>", "<S> procedure time was significantly shorter in the group receiving only one dexmedetomidine bolus and increased with second bolus or additional medications ( p < 0.0001 ) . </S>", "<S> discharge time was longer for children experiencing bradycardia ( p = 0.0012 ) . </S>", "<S> conclusion . </S>", "<S> high dose dexmedetomidine was effective in 78.5% of cases ; 21.5% of patients required additional medications . </S>", "<S> side effects occurred in approximately 25% of cases , resolving spontaneously . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["sedation is often required in young children in order to obtain good diagnostic cross - sectional imaging like computed tomography scan ( ct scan ) and magnetic resonance imaging ( mri ) .", "sometimes sedation is also needed for older children with complex neurodevelopmental disorders , such as autism and attention deficit hyperactivity disorder .", "moderate sedation is unable to guarantee patient compliance ; therefore , a deeper level of sedation is required [ 1 , 2 ] .", "the success of sedation for mri has typically been measured by two factors : the safety of sedation procedure ( lack of adverse events ) and its effectiveness ( completion of diagnostic examination ) .", "it can be challenging to obtain the deep sedation level required to prevent the patient 's movement while maintaining respiratory and hemodynamic stability .", "therefore , it is very important to select the appropriate drugs and dosage to achieve those objectives .", "dexmedetomidine is a highly selective adrenoceptor agonist with a distribution half - life of six minutes and an elimination half - life of two hours .", "five prospective trials have evaluated the efficacy of dexmedetomidine for sedation during noninvasive radiologic imaging [ 812 ] .", "dexmedetomidine can be associated with side effects of hypotension , bradycardia , and transient hypertension with loading dose .", "dexmedetomidine has been used at our institution since 2006 for mri and other noninvasive radiologic procedures .", "the purpose of this retrospective study is to present our institutional experience with dexmedetomidine in relation to efficacy and related side effects ."], ["collection of quality assurance data includes patient demographics , adverse events , physiologic variables , drug dosages , the time required to sedate the patient , time needed to obtain the imaging study , and recovery time .", "after approval by the institutional review board , we conducted a retrospective analysis of all patients who received sedation for mri from july 2007 to december 2012 .", "institutional sedation policies are based on guidelines by the joint commission on accreditation of health care organization and american academy of pediatrics and were closely followed [ 13 , 14 ] .", "vital signs including pulse oximetry , heart rate , noninvasive blood pressure monitoring , and nasal capnography ( with concomitant oxygen delivery via the nasal cannula ) are continuously monitored and documented every five minutes throughout sedation . at the start of sedation", ", intravenous dexmedetomidine was bolused at 2  g / kg over 10 minutes followed by a continuous infusion of 1  g / kg / h . most of the patients were sedated by the end of the bolus ; if not , a second bolus of 2  g / kg was repeated over another 10 minutes prior to the start of the maintenance infusion .", "patients who continue to move after the second bolus or while the procedure is in progress , can compromise the quality of imaging and were given additional medications like midazolam or fentanyl as per physician discretion .", "mg / kg to a max of 2  mg and for fentanyl was 0.5  mcg / kg1  mcg / kg to a max of 50  mcg .", "the goal was to achieve a minimum ramsay sedation score ( rss ) of 4 as assessed by a sedation nurse .", "rss is a clinically derived sedation score generally accepted as a tool for assessing the depth of sedation .", "usually , a score of 4 - 5 is targeted to ensure appropriate sedation for diagnostic imaging studies [ 11 , 16 ] .", "peak onset of the sedation was defined as the time from the start of the loading dose to achievement of a ramsay score of 4 .", "procedure time was the time of achieving the required ramsay score to the end of the procedure ( stoppage of drug administration ) .", "discharge time was defined as time from the end of the procedure to actual time when the patient was discharged home .", "normal ranges for heart rates , blood pressure , and respiratory rates for data analysis were based on the published normal of fleming et al . [ 17 , 18 ] .", "a trained registered nurse provided continuous assessment and monitoring while the procedure was in progress under the direct supervision of a pediatric intensivist .", "monitoring was continued until the patient was awake with a minimum aldrete score of 9 points , and the patient has tolerated clear liquids prior to discharge [ 19 , 20 ] . all the parents were provided with written discharge instructions and a direct phone number for further assistance or to report any adverse effects .", "database records were analyzed using dedicated statistical software sas v9.3 ( sas institute , cary , nc ) .", "changes in the vital signs associated with the use of dexmedetomidine from the baseline were evaluated and compared using student 's t - test and mann - whitney rank - sum test , depending on the distribution of the data .", "the entire study cohort was divided into three groups depending on dexmedetomidine bolus and additional medications received .", "dexmedetomidine group a received one bolus ; dexmedetomidine group b received two boluses ; dexmedetomidine group c received one or two boluses and additional medications .", "three groups were compared with respect to age , weight , blood pressure , heart rate , procedure time , and discharge time using analysis of variance .", "incidences of bradycardia and hypotension were analyzed with fisher 's exact test , due to low cell counts .", "discharge time of bradycardia and hypotensive patients was compared with normal cohorts using the t - test ."], ["the most common indications included seizure disorder , developmental delay and behavioral disorder , autism , and neoplasia .", "382 ( 70.2% ) of the total patients received one - time bolus , while two boluses were given to 162 patients ( 29.8% ) . in the population of 544 patients , additional medications ( fentanyl or midazolam )", "were required in 117 ( 21.5% ) for spontaneous movements to avoid motion artifacts that can compromise mri quality ( 54 patients with one - time bolus and 43 patients with two boluses ) .", "dexmedetomidine - induced vital sign changes from baseline for the whole group are shown in figure 1 .", "hypotension ( systolic blood pressure 20% below the normal limits ) was observed in 100 patients ( 18.4% ) and transient hypertension ( systolic blood pressure > 20% above age - specific high limits ) was observed in 137 of the patients ( 25.2% ) .", "there were 210 ( 38.6% ) patients who had a significant decrease in respiratory rate , that is , > 20% from the baseline , but no desaturation , upper airway obstruction , or apnea events were observed and the decrease was minimal in 403 patients ( 74% ) .", "most of the patients received prophylactic supplemental oxygen to maintain oxygen saturation > 95% as per protocol , which makes it impossible to determine the actual incidence of desaturation at room air .", "patients were divided into three groups based on numbers of dexmedetomidine boluses and additional medications received .", "comparisons of blood pressure , heart rate , and other clinical measures between these groups of patients are shown in table 2 .", "procedure time was significantly shorter in dexmedetomidine group a ( 40.31  19.40  min , p < 0.0001 ) compared to the other two groups .", "oxygen , dose , maximum dbp , maximum respiration rate , and initial spo2 had statistically significant ( but clinically insignificant ) differences between groups .", "the occurrence of hypotension among all three groups did not reach a statistically significant value ( p = 0.82 ) .", "decrease in blood pressure from the baseline was frequently observed ; however , medical intervention was not needed to correct it .", "the incidence of hypotension was usually noticed to be towards the completion of procedure or after stopping the infusion , with a mean procedure time of 48.73  25.46 minutes .", "initial transient hypertension occurred in 137 patients approximately 20 minutes after the dexmedetomidine bolus and lasted for < 15 minutes . in the population of 544 patients , 165 children ( 30.9% ) had heart rates below the age - specific normal awake range during sedation ( based on the published normal of fleming et al . ) . only in 21 children ( 3.9% of total cohort ) did the lowest recorded heart rate fall > 20% below the given baseline average range .", "bradycardia ( hr < 60/min ) , as defined according to pediatric advance life support ( pals ) guidelines , was observed in 10 children ( 4.5% ) mostly in the age range of 13 years ( figure 2 ) .", "all the patients with bradycardia were continuously monitored and assessed by the supervising intensivist for normal blood pressure and oxygen saturation of 95% or above . no patient required treatment .", "the incidence of bradycardia was high in patients in the age range of 13 years compared with the rest of the cohort .", "the precise etiology of the bradycardia observed in this analysis can not be delineated as electrocardiogram monitoring is known to be significantly distorted by the magnetohydrodynamic ( mhd ) effect and is nondiagnostic within the bore of any mri magnet .", "time to discharge when dexmedetomidine was used alone was 92  25 versus 94  37  min when additional medications were used ( p = 0.46 ) .", "average discharge time among 165 children with bradycardia was longer and statistically significant compared to the other 372 children in the study population without bradycardia ( 99.08  32.57  min versus 89.74  25.54  min , p = 0.0012 , student 's t - test ) .", "gender was not associated with any change in discharge time ( p = 0.30 ) ( table 3 ) ."], ["the ideal drug would allow optimal imaging , while maintaining hemodynamic and respiratory stability . as reported in previous studies ,", "this occurred more frequently in hyperactive , uncooperative , and older children [ 4 , 22 ] .", "dexmedetomidine , a selective alpha 2 adrenoceptor agonist , has a very safe therapeutic window with respect to respiratory depression .", "this quality offers a distinct advantage in procedural sedations , where the patient is not immediately accessible to the medical team .", "there are various studies demonstrating that dexmedetomidine is a good option for procedural sedation [ 10 , 12 ] .", "interestingly , the dose used varies greatly indicating an open debate regarding the best dosage .", "previous studies indicate that infusion of relatively low dose dexmedetomidine 0.10.7  /kg / h provides effective sedation [ 2327 ] . in a study conducted on eight healthy volunteers receiving dexmedetomidine infusion of 0.20.6  /kg / h , the visual analog sedation scores and bispectral index scores dropped by 3060% . however , this level of sedation will likely not be conducive to pediatric mri sedation , where higher doses of dexmedetomidine will be needed to accomplish the necessary sedation level .", "while dosing started low in initial reports , a recent one found that higher doses are required and have been well tolerated .", "a comparison of pentobarbital and chloral hydrate was performed by rooks et al . in 498 children for mri sedation .", "several clinical trials have compared dexmedetomidine to propofol for pediatric procedural sedation . in a trial of 60 children undergoing mri ,", "onset , recovery , and discharge time were all significantly shorter in the propofol group .", "adverse effects such as lower blood pressure and heart rate and respiratory rate were also found more in the propofol group .", "dexmedetomidine safety and efficacy for sedation during noninvasive radiologic procedures have been evaluated in four prospective trials [ 1 , 810 ] .", "all of these studies reported that the use of high dose dexmedetomidine as a sole sedative resulted in high quality radiologic imaging with less use of additional rescue medicines .", "our study also demonstrated that the use of high dose dexmedetomidine as sole agent is effective for mri sedation .", "we used fentanyl and midazolam as rescue medicines in 117 ( 21.5% ) of our patients to avoid imaging artifacts , suggesting that these agents make a significant contribution to the sedation strategy for a sizeable minority of patients . in our study , the heart rate decreased significantly from baseline during sedation .", "this is an expected effect of anxiolysis and reflects higher baseline heart rate values from anxiety .", "other studies reported a decrease in heart rate < 20% from the baseline that was considered to be clinically insignificant in 86 ( 26% ) patients .", "to have a true comparison with previously published data , we use the same definition of bradycardia ; that is , the lowest recorded heart rate during sedation fell > 20% below the given baseline average range . using that definition ,", "the incidence of bradycardia in our study was 3.9% ( 21 children ) in total cohort comparable to 4% as reported by mason et al .", "more importantly , during those periods of bradycardia , all patients maintained normal blood pressure and normal oxygen saturation ( 95% or higher ) .", "treating bradycardia in normotensive children with glycopyrrolate has been reported to be associated with hypertensive episode , which could be beneficial to children who are hypotensive and bradycardic at the same time .", "similar to our findings , previous investigators have also reported the occurrence of transient hypertension with bolus administration of dexmedetomidine . in our study ,", "the occurrence of hypotension associated with high doses of dexmedetomidine was about 18% , observed immediately after stopping the infusion or towards the end of the procedure .", "none of the children needed any intervention , consistent with observation reported in other studies [ 12 , 31 ] .", "respiratory events make up a large proportion ( 5.5% ) of sedation complications in children . in some studies", ", rapid administration of large loading doses has been described to cause respiratory complications [ 3234 ] .", "a loading dose of dexmedetomidine given over 2 minutes has been reported to cause irregular respiration , apnea , slight hypoxemia , and hypercapnia .", "however , similar to ours , several other studies have reported trivial effect of dexmedetomidine on respiration [ 12 , 34 , 36 ] which is consistent with the notion that the risk of respiratory depression is minimal with careful dexmedetomidine sedation . despite these supporting lines of evidence , monitoring of respiratory function during the administration of dexmedetomidine in those receiving midazolam or fentanyl , which may depress respiratory function , appears warranted .", "our reported discharge time of 92 minutes with dexmedetomidine only and 94 minutes when additional medicines were used is comparable to the 90 minutes ' discharge time of heard et al . .", "observed a recovery time of 47 minutes   and mason et al .   reported recovery times ranging from 24.8 minutes to 35.2 minutes , depending on the dose of dexmedetomidine used .", "in some studies , recovery time is the time lapsed until the patient meets the discharge criteria , while in our study , like few others , discharged time is defined as the actual time of leaving the recovery room to go home .", "discharge time of children experiencing bradycardia was longer than that for those who did not .", "in our study , 165 children with bradycardia had a mean discharge time which was statistically significant and longer than the other 372 children in the study without bradycardia ( 99.08 minutes versus 89.74 minutes ; p = 0.0012 ) .", "this probably could be due to the prolonged monitoring of these patients until the bradycardia resolved .", "recovery time previously reported after sedation with propofol for mri has been 17  8 minutes , almost half of the recovery time when dexmedetomidine was used as a sole agent . in a second trial", ", forty children between the ages of 1 and 10 years were randomized to receive either a combination of midazolam + dexmedetomidine or propofol only for sedation during mri .", "the fast recovery times of propofol must be weighed against the fact that it induced deeper sedation with significant hypotension and oxygen desaturation . taking the results of these comparison trials as a whole", ", dexmedetomidine appears to provide a useful alternative to propofol for procedural sedation in children , with a longer time of recovery but a lower incidence of adverse effects .", "this is a good alternative to propofol for patients with soy and egg white allergy and also in institutions where use of propofol is strictly limited to be used by anesthesiologists .", "it could be asserted that the reported efficacy is due to the use of an intensivist based specialized sedation team rather than to dexmedetomidine itself .", "this is reasonably true to some extent as specialization and experience should increase both success and efficiency . in spite of that , this can be stated with confidence ; much of the reported success is specifically a function of dexmedetomidine .", "this is a descriptive study and few , if any , conclusions can be drawn about safety because the occurrence of serious sedation related side effects is fortunately rare .", "additional prospective studies of the sedation for mri in children using a greater number of patients are warranted to provide a true idea of safety . in summary ,", "high dose dexmedetomidine is an attractive and effective medication in children for mri sedation . when using high dose dexmedetomidine as the only agent for pediatric mri , it is not unusual to observe heart rate and blood pressure outside the established ", "awake  normal values . in our experience , these changes were pretty benign and were not associated with any adverse event .", "we conclude and recommend that , from hemodynamics and respiratory perspective , higher dose dexmedetomidine was well tolerated and is effective to use for successful completion of mri , in the majority of pediatric patients ."]]}
{"article_id": "PMC2935156", "article_text": ["exposure of ultraviolet ( uv ) irradiation to the skin causes acute and chronic detrimental cutaneous effects , which may result in photocarcinogenesis [ 17 ] .", "native human melanin includes eumelanin and pheomelanin that contains sulfur , and eumelanin has been found in almost every type of human skin [ 8 , 9 ] . in the skin , melanin synthesized in melanocytes located in the basal layer and hair bulbs transfers to keratinocytes .", "melanin in keratinocytes acts as a photoprotector through body coloration and scavenging reactive oxygen species [ 1015 ] . in spite of photoprotective role of melanin , there are many cosmetics developed to prevent melanin formation in the skin because of aesthetic satisfaction by whitening ability . of these ,", "inhibitor of tyrosinase , which is a pivotal enzyme for melanin synthesis , has become major ingredient of cosmetics [ 1721 ] .", "tyrosinase is an enzyme which catalyzes the biological conversion of tyrosine to dopaquinone with dioxygen at the dinuclear copper active site under physiological conditions [ 2224 ] .", "other than oxidative catalysis of substrates by tyrosinase , a few studies on radical formation by tyrosinase have been reported .", "for instance , it was reported that tyrosinase - dependent activation of hydroxybenzenes formed reactive compounds including free radical , and some radicals were produced during the tyrosinase reaction and dopa - melanin formation . however , in these few studies , radical species have not been determined .", "we have studied the tyrosine - tyrosinase reaction in terms of melanin formation and ros scavenging ability of melanin . in a battery of studies", ", we found radical formation through the enzyme reaction , and discuss the formation mechanism in this paper .", "reagents were purchased from the following sources : l - tyrosine , phosphate buffer solution ( pb , ph  6.5 ) and catalase ( from bovine liver ) from wako pure chemicals ( osaka , japan ) ; tyrosinase ( from mushroom ) , and superoxide dismutase ( sod from bovine erythrocytes ) from sigma - aldrich corp .", "( st . louis , mo ) ; 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec ( tokyo , japan ) ; deuterium water ( d2o ) from tokyo chemical industry co. , ltd ( tokyo , japan ) .", "then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of 0.2  m pb . dmpo was dissolved in ultrapure water to be 4.5  m. the reaction mixture was prepared to contain different activity of tyrosinase , 20  l of 4.5  m dmpo , 60  l of 1  mm tyrosine and 0.2  m pb which was added to adjust a total volume of 200  l . immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 45  s. the measurement conditions of esr ( jes - fa-100 , jeol , tokyo , japan ) were as follows : field sweep , 330.80340.80  mt ; field modulation frequency , 100  khz ; filed modulation width , 0.07  mt ; amplitude , 400 ; sweep time , 1  min ; time constant , 0.1  s ; microwave frequency , 9.430  ghz ; microwave power , 45  mw .", "then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of ultrapure water or d2o .", "tyrosinase was dissolved in ultrapure water to be 100  u/l . immediately before the measurement tyrosinase was diluted to be 10  u/l with ultrapure water or d2o .", "dmpo ( 8.9  m ) was diluted to be 4.5  m with ultrapure water or d2o .", "the reaction mixture was prepared to contain 100  l of d2o , 20  l of 10  u/l tyrosinase ( 90% d2o solution ) , 20  l of 4.5  m dmpo ( 50% d2o solution ) , and 60  l of 1  mm tyrosine ( 99% d2o solution ) .", "immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 60  s. as a control , the reaction mixture was prepared to contain 100  l of h2o ( ultrapure water ) , 20  l of 10  u/l tyrosinase dissolved in h2o , 20  l of 4.5  m dmpo dissolved in h2o , and 60  l of 1  mm tyrosine dissolved in h2o , and was similarly subjected to esr measurement .", "reagents were purchased from the following sources : l - tyrosine , phosphate buffer solution ( pb , ph  6.5 ) and catalase ( from bovine liver ) from wako pure chemicals ( osaka , japan ) ; tyrosinase ( from mushroom ) , and superoxide dismutase ( sod from bovine erythrocytes ) from sigma - aldrich corp .", "( st . louis , mo ) ; 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec ( tokyo , japan ) ; deuterium water ( d2o ) from tokyo chemical industry co. , ltd ( tokyo , japan ) .", "then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of 0.2  m pb . dmpo was dissolved in ultrapure water to be 4.5  m. the reaction mixture was prepared to contain different activity of tyrosinase , 20  l of 4.5  m dmpo , 60  l of 1  mm tyrosine and 0.2  m pb which was added to adjust a total volume of 200  l . immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 45  s. the measurement conditions of esr ( jes - fa-100 , jeol , tokyo , japan ) were as follows : field sweep , 330.80340.80  mt ; field modulation frequency , 100  khz ; filed modulation width , 0.07  mt ; amplitude , 400 ; sweep time , 1  min ; time constant , 0.1  s ; microwave frequency , 9.430  ghz ; microwave power , 45  mw .", "then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of ultrapure water or d2o .", "tyrosinase was dissolved in ultrapure water to be 100  u/l . immediately before the measurement tyrosinase was diluted to be 10  u/l with ultrapure water or d2o .", "dmpo ( 8.9  m ) was diluted to be 4.5  m with ultrapure water or d2o .", "the reaction mixture was prepared to contain 100  l of d2o , 20  l of 10  u/l tyrosinase ( 90% d2o solution ) , 20  l of 4.5  m dmpo ( 50% d2o solution ) , and 60  l of 1  mm tyrosine ( 99% d2o solution ) .", "immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 60  s. as a control , the reaction mixture was prepared to contain 100  l of h2o ( ultrapure water ) , 20  l of 10  u/l tyrosinase dissolved in h2o , 20  l of 4.5  m dmpo dissolved in h2o , and 60  l of 1  mm tyrosine dissolved in h2o , and was similarly subjected to esr measurement .", "representative esr spectra obtained from tyrosine - tyrosinase reaction with different activity of tyrosinase are summarized in fig .  1", ". each spectrum consists of quartet segments ( intensity ratio , 1:2:2:1 ) and triplet of triplet segments ( intensity ratio , 1:1:2:1:2:1:2:1:1 ) .", "the former segment was assigned to dmpo - oh , a spin adduct derived from oh ( hyperfine coupling constant , an  =  1.49 ; ah  = 1.49  mt ) .", "the latter segment was assigned to dmpo - h , a spin adduct derived from h ( hyperfine coupling constant , an  =  1.63 ; ah  =  2.25  mt ) as reported in a previous study .", "the ratio of dmpo - h and dmpo - oh generated in the tyrosine - tyrosinase reaction was approximately 1:2 . to examine", "if o2 and h2o2 are involved in a downstream of the generation process of h and oh , esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction were analyzed in the presence of sod , a scavenger for o2 , and catalase , a cleaving enzyme for h2o2 .", "neither sod ( 1  u/l ) nor catalase ( 1  u/l ) affected the amounts of dmpo - h and dmpo - oh ( data not shown ) , suggesting that neither o2 nor h2o2 is involved in the generation process of h and oh . then to further examine if h2o is a source of h and oh , esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction where d2o was used as a major solvent ( 94% d2o solution ) were compared with those where h2o was used as a solvent . as shown in fig .  2 ,", "the amount of dmpo - h was reduced by 70% , suggesting that approximately 70% of h is derived from h2o .", "thus , we assume that the electron derived from the ligand of tyrosinase reacts with h2o or h to form h. that is , h2o  +  e ", "oh  +  h or h  +  e  h. on the other hand , the amount of dmpo - oh was not affected by the replacement of h2o to d2o , suggesting that oh was derived from the catalytic reaction of tyrosine to dopaquinone by tyrosinase . since catalase had no effect on the generation of two radicals as described above , fenton - like reaction in which h2o2 reacts with transient metal copper in the structures of tyrosinase and its intermediates", "is least likely involved in the generation mechanism of oh . as reported in the previous study on dinuclear copper complexes , a rather stable dicopper - peroxide intermediate in aqueous solution decays through an internal electron transfer from the ligand to produce oh . since tyrosinase is an enzyme which contains dinuclear copper ions at the active site , [ 2224 ] , dicopper - peroxide intermediates formed during the catalytic process of tyrosine to dopaquinone possibly decay to produce oh through an internal electron transfer from the ligand .", "the proposed mechanism by which h and oh are generated in tyrosine - tyrosinase reaction is illustrated in fig . ", "although tyrosine - tyrosinase reaction is a key step of melanin formation as a photoprotection , it also produces not only h but oh which might be a causative factor of oxidative damage of the skin .", "since there are many cosmetics developed for whitening ability by inhibiting tyrosine - tyrosinase reaction , our study revealed that they also might contribute to alleviate the oxidative damage of the skin by inhibiting oh generation via the enzyme reaction ."], "abstract_text": ["<S> alleviated melanin formation in the skin through inhibition of tyrosine - tyrosinase reaction is one of the major targets of cosmetics for whitening ability . </S>", "<S> since melanin has a pivotal role for photoprotection , there are pros and cons of inhibition of melanin formation . </S>", "<S> this study applying electron spin resonance ( esr)-spin trapping method revealed that h and oh are generated through tyrosine - tyrosinase reaction . when deuterium water was used instead of h2o , the signal of 5,5-dimethyl-1-pyrroline n - oxide ( dmpo)-h ( a spin adduct of dmpo and h ) greatly decreased , whilst dmpo - oh ( a spin adduct of dmpo and oh ) did not . </S>", "<S> thus , it is suggested that h was derived from h2o , and oh through oxidative catalytic process of tyrosine to dopaquinone . </S>", "<S> our study suggests that tyrosinase inhibitors might contribute to alleviate the oxidative damage of the skin by inhibiting oh generation via the enzyme reaction . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Test materials and reagents", "Electron spin resonance (ESR)-spin trapping determinations of ROS generated by tyrosine-tyrosinase reaction", "Deuterium water (D", "Results and Discussion"], "sections": [["exposure of ultraviolet ( uv ) irradiation to the skin causes acute and chronic detrimental cutaneous effects , which may result in photocarcinogenesis [ 17 ] .", "native human melanin includes eumelanin and pheomelanin that contains sulfur , and eumelanin has been found in almost every type of human skin [ 8 , 9 ] . in the skin , melanin synthesized in melanocytes located in the basal layer and hair bulbs transfers to keratinocytes .", "melanin in keratinocytes acts as a photoprotector through body coloration and scavenging reactive oxygen species [ 1015 ] . in spite of photoprotective role of melanin , there are many cosmetics developed to prevent melanin formation in the skin because of aesthetic satisfaction by whitening ability . of these ,", "inhibitor of tyrosinase , which is a pivotal enzyme for melanin synthesis , has become major ingredient of cosmetics [ 1721 ] .", "tyrosinase is an enzyme which catalyzes the biological conversion of tyrosine to dopaquinone with dioxygen at the dinuclear copper active site under physiological conditions [ 2224 ] .", "other than oxidative catalysis of substrates by tyrosinase , a few studies on radical formation by tyrosinase have been reported .", "for instance , it was reported that tyrosinase - dependent activation of hydroxybenzenes formed reactive compounds including free radical , and some radicals were produced during the tyrosinase reaction and dopa - melanin formation . however , in these few studies , radical species have not been determined .", "we have studied the tyrosine - tyrosinase reaction in terms of melanin formation and ros scavenging ability of melanin . in a battery of studies", ", we found radical formation through the enzyme reaction , and discuss the formation mechanism in this paper ."], ["reagents were purchased from the following sources : l - tyrosine , phosphate buffer solution ( pb , ph  6.5 ) and catalase ( from bovine liver ) from wako pure chemicals ( osaka , japan ) ; tyrosinase ( from mushroom ) , and superoxide dismutase ( sod from bovine erythrocytes ) from sigma - aldrich corp .", "( st . louis , mo ) ; 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec ( tokyo , japan ) ; deuterium water ( d2o ) from tokyo chemical industry co. , ltd ( tokyo , japan ) .", "then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of 0.2  m pb . dmpo was dissolved in ultrapure water to be 4.5  m. the reaction mixture was prepared to contain different activity of tyrosinase , 20  l of 4.5  m dmpo , 60  l of 1  mm tyrosine and 0.2  m pb which was added to adjust a total volume of 200  l . immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 45  s. the measurement conditions of esr ( jes - fa-100 , jeol , tokyo , japan ) were as follows : field sweep , 330.80340.80  mt ; field modulation frequency , 100  khz ; filed modulation width , 0.07  mt ; amplitude , 400 ; sweep time , 1  min ; time constant , 0.1  s ; microwave frequency , 9.430  ghz ; microwave power , 45  mw .", "then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of ultrapure water or d2o .", "tyrosinase was dissolved in ultrapure water to be 100  u/l . immediately before the measurement tyrosinase was diluted to be 10  u/l with ultrapure water or d2o .", "dmpo ( 8.9  m ) was diluted to be 4.5  m with ultrapure water or d2o .", "the reaction mixture was prepared to contain 100  l of d2o , 20  l of 10  u/l tyrosinase ( 90% d2o solution ) , 20  l of 4.5  m dmpo ( 50% d2o solution ) , and 60  l of 1  mm tyrosine ( 99% d2o solution ) .", "immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 60  s. as a control , the reaction mixture was prepared to contain 100  l of h2o ( ultrapure water ) , 20  l of 10  u/l tyrosinase dissolved in h2o , 20  l of 4.5  m dmpo dissolved in h2o , and 60  l of 1  mm tyrosine dissolved in h2o , and was similarly subjected to esr measurement ."], ["reagents were purchased from the following sources : l - tyrosine , phosphate buffer solution ( pb , ph  6.5 ) and catalase ( from bovine liver ) from wako pure chemicals ( osaka , japan ) ; tyrosinase ( from mushroom ) , and superoxide dismutase ( sod from bovine erythrocytes ) from sigma - aldrich corp .", "( st . louis , mo ) ; 5,5-dimethyl-1-pyrroline - n - oxide ( dmpo ) from labotec ( tokyo , japan ) ; deuterium water ( d2o ) from tokyo chemical industry co. , ltd ( tokyo , japan ) ."], ["then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of 0.2  m pb . dmpo was dissolved in ultrapure water to be 4.5  m. the reaction mixture was prepared to contain different activity of tyrosinase , 20  l of 4.5  m dmpo , 60  l of 1  mm tyrosine and 0.2  m pb which was added to adjust a total volume of 200  l . immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 45  s. the measurement conditions of esr ( jes - fa-100 , jeol , tokyo , japan ) were as follows : field sweep , 330.80340.80  mt ; field modulation frequency , 100  khz ; filed modulation width , 0.07  mt ; amplitude , 400 ; sweep time , 1  min ; time constant , 0.1  s ; microwave frequency , 9.430  ghz ; microwave power , 45  mw ."], ["then 1  mm tyrosine solution was prepared by mixing 5  l of 200  mm tyrosine solution with 5  l of 1  m naoh and 990  l of ultrapure water or d2o .", "tyrosinase was dissolved in ultrapure water to be 100  u/l . immediately before the measurement tyrosinase was diluted to be 10  u/l with ultrapure water or d2o .", "dmpo ( 8.9  m ) was diluted to be 4.5  m with ultrapure water or d2o .", "the reaction mixture was prepared to contain 100  l of d2o , 20  l of 10  u/l tyrosinase ( 90% d2o solution ) , 20  l of 4.5  m dmpo ( 50% d2o solution ) , and 60  l of 1  mm tyrosine ( 99% d2o solution ) .", "immediately after mixing the mixture was transferred to an esr spectrometry cell , and the esr measurement was started after 60  s. as a control , the reaction mixture was prepared to contain 100  l of h2o ( ultrapure water ) , 20  l of 10  u/l tyrosinase dissolved in h2o , 20  l of 4.5  m dmpo dissolved in h2o , and 60  l of 1  mm tyrosine dissolved in h2o , and was similarly subjected to esr measurement ."], ["representative esr spectra obtained from tyrosine - tyrosinase reaction with different activity of tyrosinase are summarized in fig .  1", ". each spectrum consists of quartet segments ( intensity ratio , 1:2:2:1 ) and triplet of triplet segments ( intensity ratio , 1:1:2:1:2:1:2:1:1 ) .", "the former segment was assigned to dmpo - oh , a spin adduct derived from oh ( hyperfine coupling constant , an  =  1.49 ; ah  = 1.49  mt ) .", "the latter segment was assigned to dmpo - h , a spin adduct derived from h ( hyperfine coupling constant , an  =  1.63 ; ah  =  2.25  mt ) as reported in a previous study .", "the ratio of dmpo - h and dmpo - oh generated in the tyrosine - tyrosinase reaction was approximately 1:2 . to examine", "if o2 and h2o2 are involved in a downstream of the generation process of h and oh , esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction were analyzed in the presence of sod , a scavenger for o2 , and catalase , a cleaving enzyme for h2o2 .", "neither sod ( 1  u/l ) nor catalase ( 1  u/l ) affected the amounts of dmpo - h and dmpo - oh ( data not shown ) , suggesting that neither o2 nor h2o2 is involved in the generation process of h and oh . then to further examine if h2o is a source of h and oh , esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction where d2o was used as a major solvent ( 94% d2o solution ) were compared with those where h2o was used as a solvent . as shown in fig .  2 ,", "the amount of dmpo - h was reduced by 70% , suggesting that approximately 70% of h is derived from h2o .", "thus , we assume that the electron derived from the ligand of tyrosinase reacts with h2o or h to form h. that is , h2o  +  e ", "oh  +  h or h  +  e  h. on the other hand , the amount of dmpo - oh was not affected by the replacement of h2o to d2o , suggesting that oh was derived from the catalytic reaction of tyrosine to dopaquinone by tyrosinase . since catalase had no effect on the generation of two radicals as described above , fenton - like reaction in which h2o2 reacts with transient metal copper in the structures of tyrosinase and its intermediates", "is least likely involved in the generation mechanism of oh . as reported in the previous study on dinuclear copper complexes , a rather stable dicopper - peroxide intermediate in aqueous solution decays through an internal electron transfer from the ligand to produce oh . since tyrosinase is an enzyme which contains dinuclear copper ions at the active site , [ 2224 ] , dicopper - peroxide intermediates formed during the catalytic process of tyrosine to dopaquinone possibly decay to produce oh through an internal electron transfer from the ligand .", "the proposed mechanism by which h and oh are generated in tyrosine - tyrosinase reaction is illustrated in fig . ", "although tyrosine - tyrosinase reaction is a key step of melanin formation as a photoprotection , it also produces not only h but oh which might be a causative factor of oxidative damage of the skin .", "since there are many cosmetics developed for whitening ability by inhibiting tyrosine - tyrosinase reaction , our study revealed that they also might contribute to alleviate the oxidative damage of the skin by inhibiting oh generation via the enzyme reaction ."]]}
{"article_id": "PMC5201070", "article_text": ["bezoars are masses formed by the condensation of debris or stomach contents in the gastrointestinal tract", ". there are four major types of bezoars : a trichobezoar consists of hair , a phytobezoar of vegetable and fruit residues , a lactobezoar is formed from dairy products , a pharmacobezoar is caused by medications .", "a high - fiber diet , incomplete mastication , reduced gastric secretion , and decreased gastrointestinal motility from autonomic neuropathy can predispose patients to phytobezoar formation .", "it occurs more frequently in the small intestine , particularly in the terminal ileum and jejunum .", "the complications caused by bezoars are mechanical irritation , gastrointestinal obstruction , and , in severe cases , peritonitis caused by bowel perforation .", "bezoars are responsible for 0.44% of cases of mechanical intestinal obstruction , which occurs mainly in the stomach and small intestine .", "contrast - enhanced computed tomography ( ct ) is the method of choice to identify the location , severity , and etiology of intestinal obstruction .", "we describe our experience regarding a large - bowel obstruction caused by phytobezoar in a patient with a transplanted kidney .", "a 39-year - old man was admitted to our hospital for constipation , distension , and severe acute abdominal pain in the lower left quadrant .", "his medical history included alport syndrome resulting in progressive chronic kidney disease ( ckd ) , hypertension , and iron deficiency anemia under medical treatment .", "twelve years ago , he received a successful deceased - donor kidney transplant [ figure 1 ] . at the time of admission , vitals were stable , and he was afebrile .", "chest x - ray was normal and abdominal x - ray did not show signs of bowel obstruction .", "coronal computed tomography scan shows transplanted kidney in left iliac fossa (  ) non - contrast ct ( ncct ) of the abdomen was done ( toshiba  aquilion 64-tsx-101a / hc ) as the patient had ckd ( serum creatinine level = 7.71 mg / dl ) .", "ct scan revealed a focal circumferential thickening of the sigmoid colon with an ovoid and encapsulated intraluminal mass measuring 4.7  3.6 cm [ figure 2 ] .", "dilatation of the proximal segment with abundant fecal material was seen [ figure 2 ] .", "based on the ct findings , particularly mottled gas pattern and encapsulated wall of the mass , we made the diagnosis of phytobezoar . hence , because of acute intestinal obstruction and to avoid peritonitis , the patient underwent urgent exploratory laparotomy .", "( a ) coronal computed tomography scan shows an ovoid intraluminal mass containing mottled gas pattern with encapsulating wall and associated wall thickening ( arrows ) ; note some dilated bowel loops ( stars ) and an abundant fecal material ( arrowhead ) .", "( b ) the mean attenuation value of the intraluminal mass is about 10.21 hu the obstructive intraluminal mass in the sigmoid colon was identified ( approximately 4  4 cm in size ) and manually broken down with evacuation of the obstructive material along with large amounts of liquid fecal material .", "the postoperative course was uneventful and the patient was discharged on the seventh postoperative day . at 3-month follow - up ,", "phytobezoars are concretions of poorly digested fruit and vegetable fibres that are found in the alimentary track and are mostly composed of indigestible cellulose , tannin , and lignin from ingested vegetables and fruits .", "normally , they are found in the stomach and may enter into the small bowel .", "the most important risk factors for phytobezoar formation are excessive consumption of fruits rich in fibers , poor dental health , insufficient mastication , diabetic gastroparesis , kidney failure , hypothyroidism , use of drugs which affect gastric motility , and previous gastrointestinal surgery . in our case ,", "other symptoms include abdominal distension , vomiting , constipation , diarrhoea , anorexia , and weight loss .", "if treatment is delayed , colonic bezoars can be complicated by intestinal obstruction and perforation with consequent peritonitis .", "plain abdominal radiography and ct scan are used for radiological evaluation of the patients presenting with intestinal obstruction .", "simple abdominal x - ray may reveal air - fluid levels associated with mechanical obstruction , and occasionally it is possible to see the outline of bezoar , which is difficult to differentiate from abscess or feces within the colon . on abdominal ct , 89% of bezoars appear as spherically - shaped masses containing air - fluid levels .", "ct is also helpful in discriminating bezoars from other causes of bowel obstruction such as gastrointestinal malignancies , gallstone ileus , etc . in our case", "indeed , many authors have highlighted some ct features to allow easier differential diagnosis between these two entities .", "small bowel feces tends to be more amorphous and tubular in shape without evidence of an encapsulating wall , whereas phytobezoars have a well - defined ovoid shape and an encapsulating wall , such as in our case .", "chen et al . showed that a higher grade of obstruction is commonly associated with phytobezoar impaction without mesenteric fatty stranding and intraperitoneal fluid ; moreover , they found that a combination of the food debris length < 9.5 cm and the mean attenuation value < 11.75 hu of the obstructed bowel at the transition point was suggestive for phytobezoar , such as in our patient .", "although these features are typically found in the small bowel , they may also be considered very useful in the large bowel .", "conservative management is considered in uncomplicated cases of colonic bezoars and consist of digital evacuation , enemas and manual disimpaction .", "if conservative approach fails , a colonoscopy can be attempted for bezoar removal with contextual mechanical fragmentation and extraction using several instruments .", "although endoscopy has an acceptable success rate and fewer complications than surgery , surgical approach is mandatory in case of endoscopic treatment failure , sigmoid volvulus , hematochezia , intestinal obstructions , perforations , or peritonitis .", "when surgical approach is chosen , the first step is intestinal decompression and fluid - electrolyte replacement ; later , open or laparoscopic abdominal exploration may be performed . in our case , the patient underwent laparotomic surgery and phytobezoar was manually broken down with evacuation .", "bezoar is a rare disorder , however , it must be suspected in case of mechanical intestinal obstruction .", "our case underlined the importance of early detection and removal of a bezoar to prevent complications such as obstruction , perforation , or peritonitis ."], "abstract_text": ["<S> bezoars are masses formed by the concretion of stomach contents or debris within the gastrointestinal tract . </S>", "<S> bezoars are rare and account for only 0.44% of all cases of gastrointestinal obstruction and mainly occur in the stomach or small intestine . intestinal obstruction caused by colonic bezoars </S>", "<S> is extremely rare . </S>", "<S> a 39-year - old man with a transplanted kidney came to the hospital because of abdominal pain , constipation , and distension . </S>", "<S> we performed an abdominal computed tomography scan and found an ovoid intraluminal mass with a mottled gas pattern in the distal sigmoid colon . </S>", "<S> subsequently , the patient underwent laparotomic surgery and removal of the bezoar . </S>", "<S> we report a rare case of large bowel obstruction due to colonic phytobezoar , which was confirmed intraoperatively . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusions", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["bezoars are masses formed by the condensation of debris or stomach contents in the gastrointestinal tract", ". there are four major types of bezoars : a trichobezoar consists of hair , a phytobezoar of vegetable and fruit residues , a lactobezoar is formed from dairy products , a pharmacobezoar is caused by medications .", "a high - fiber diet , incomplete mastication , reduced gastric secretion , and decreased gastrointestinal motility from autonomic neuropathy can predispose patients to phytobezoar formation .", "it occurs more frequently in the small intestine , particularly in the terminal ileum and jejunum .", "the complications caused by bezoars are mechanical irritation , gastrointestinal obstruction , and , in severe cases , peritonitis caused by bowel perforation .", "bezoars are responsible for 0.44% of cases of mechanical intestinal obstruction , which occurs mainly in the stomach and small intestine .", "contrast - enhanced computed tomography ( ct ) is the method of choice to identify the location , severity , and etiology of intestinal obstruction .", "we describe our experience regarding a large - bowel obstruction caused by phytobezoar in a patient with a transplanted kidney ."], ["a 39-year - old man was admitted to our hospital for constipation , distension , and severe acute abdominal pain in the lower left quadrant .", "his medical history included alport syndrome resulting in progressive chronic kidney disease ( ckd ) , hypertension , and iron deficiency anemia under medical treatment .", "twelve years ago , he received a successful deceased - donor kidney transplant [ figure 1 ] . at the time of admission , vitals were stable , and he was afebrile .", "chest x - ray was normal and abdominal x - ray did not show signs of bowel obstruction .", "coronal computed tomography scan shows transplanted kidney in left iliac fossa (  ) non - contrast ct ( ncct ) of the abdomen was done ( toshiba  aquilion 64-tsx-101a / hc ) as the patient had ckd ( serum creatinine level = 7.71 mg / dl ) .", "ct scan revealed a focal circumferential thickening of the sigmoid colon with an ovoid and encapsulated intraluminal mass measuring 4.7  3.6 cm [ figure 2 ] .", "dilatation of the proximal segment with abundant fecal material was seen [ figure 2 ] .", "based on the ct findings , particularly mottled gas pattern and encapsulated wall of the mass , we made the diagnosis of phytobezoar . hence , because of acute intestinal obstruction and to avoid peritonitis , the patient underwent urgent exploratory laparotomy .", "( a ) coronal computed tomography scan shows an ovoid intraluminal mass containing mottled gas pattern with encapsulating wall and associated wall thickening ( arrows ) ; note some dilated bowel loops ( stars ) and an abundant fecal material ( arrowhead ) .", "( b ) the mean attenuation value of the intraluminal mass is about 10.21 hu the obstructive intraluminal mass in the sigmoid colon was identified ( approximately 4  4 cm in size ) and manually broken down with evacuation of the obstructive material along with large amounts of liquid fecal material .", "the postoperative course was uneventful and the patient was discharged on the seventh postoperative day . at 3-month follow - up ,"], ["phytobezoars are concretions of poorly digested fruit and vegetable fibres that are found in the alimentary track and are mostly composed of indigestible cellulose , tannin , and lignin from ingested vegetables and fruits .", "normally , they are found in the stomach and may enter into the small bowel .", "the most important risk factors for phytobezoar formation are excessive consumption of fruits rich in fibers , poor dental health , insufficient mastication , diabetic gastroparesis , kidney failure , hypothyroidism , use of drugs which affect gastric motility , and previous gastrointestinal surgery . in our case ,", "other symptoms include abdominal distension , vomiting , constipation , diarrhoea , anorexia , and weight loss .", "if treatment is delayed , colonic bezoars can be complicated by intestinal obstruction and perforation with consequent peritonitis .", "plain abdominal radiography and ct scan are used for radiological evaluation of the patients presenting with intestinal obstruction .", "simple abdominal x - ray may reveal air - fluid levels associated with mechanical obstruction , and occasionally it is possible to see the outline of bezoar , which is difficult to differentiate from abscess or feces within the colon . on abdominal ct , 89% of bezoars appear as spherically - shaped masses containing air - fluid levels .", "ct is also helpful in discriminating bezoars from other causes of bowel obstruction such as gastrointestinal malignancies , gallstone ileus , etc . in our case", "indeed , many authors have highlighted some ct features to allow easier differential diagnosis between these two entities .", "small bowel feces tends to be more amorphous and tubular in shape without evidence of an encapsulating wall , whereas phytobezoars have a well - defined ovoid shape and an encapsulating wall , such as in our case .", "chen et al . showed that a higher grade of obstruction is commonly associated with phytobezoar impaction without mesenteric fatty stranding and intraperitoneal fluid ; moreover , they found that a combination of the food debris length < 9.5 cm and the mean attenuation value < 11.75 hu of the obstructed bowel at the transition point was suggestive for phytobezoar , such as in our patient .", "although these features are typically found in the small bowel , they may also be considered very useful in the large bowel .", "conservative management is considered in uncomplicated cases of colonic bezoars and consist of digital evacuation , enemas and manual disimpaction .", "if conservative approach fails , a colonoscopy can be attempted for bezoar removal with contextual mechanical fragmentation and extraction using several instruments .", "although endoscopy has an acceptable success rate and fewer complications than surgery , surgical approach is mandatory in case of endoscopic treatment failure , sigmoid volvulus , hematochezia , intestinal obstructions , perforations , or peritonitis .", "when surgical approach is chosen , the first step is intestinal decompression and fluid - electrolyte replacement ; later , open or laparoscopic abdominal exploration may be performed . in our case , the patient underwent laparotomic surgery and phytobezoar was manually broken down with evacuation ."], ["bezoar is a rare disorder , however , it must be suspected in case of mechanical intestinal obstruction .", "our case underlined the importance of early detection and removal of a bezoar to prevent complications such as obstruction , perforation , or peritonitis ."], [""], [""]]}
{"article_id": "PMC2600385", "article_text": ["mosquito samples were collected in vietnam in 2002 . each species was separated and pooled , and each mosquito pool was then homogenized and centrifuged .", "the supernatant was filtrated through a 0.22-m filter and then inoculated onto a monolayer c6/36 mosquito cell culture and subsequently incubated at 28c for 7 days .", "virus amplification in c6/36 cells was repeated twice , and the culture fluids were stored at 80c for further analysis .", "rna was extracted from the second cell culture fluid by trizol ls reagent ( invitrogen , carlsbad , ca , usa ) .", "reverse transcription was performed by using superscript iii reverse transcriptase ( invitrogen ) and random hexamers , after rna denaturation at a temperature of 95c for 5 minutes in the presence of 15% dimethyl sulfoxide .", "pcr was conducted by using takara la taq dna polymerase ( takara bio , inc . ,", "otsu , japan ) with bav targeting primers ( for segment 5 , 5-cagctgcagtggttattgga-3 and 5accgtgcatcttaacccttg-3 ; for segment 8 , 5-ttgcagtcgctgagctttta-3 and 5-cgcatttgatcgtatgcttg-3 ) .", "these targeting primers were designed from sequences of bav strains from china and java ( genbank accession nos .", "af134519af134527 , ay549307ay549309 , af052024af052035 , ay568287ay568290 , nc_004211 , nc_004217-nc_004221 , nc_004200-nc_004204 , nc_004198 , and af052008af052013 ) . the amplified cdnas were sequenced in the 3100-avant genetic analyzer ( applied biosystems , foster city , ca , usa ) .", "the genomic sequences of all 12 genome segments for the 5 strains were determined ( genbank accession nos .", "the sequences were translated to protein sequences by using the transeq program of the emboss package ( 8) , version 5.00 .", "the protein sequences were aligned with the t - coffee program version 5.05 ( 9 ) with chinese and javanese bavs .", "nucleotide sequences were then aligned , on the basis of protein alignment , with the tranalign program of the emboss package .", "maximum likelihood phylogenetic trees were constructed from the protein - guided alignments of nucleotide sequences with dnaml software of the phylip package ( 10 ) , version 3.67 .", "five bav strains were isolated in ha tay and quang bing provinces ( figure 1 ) in vietnam from culex annulus and cx .", "phylogenetic analysis showed diversity within the phylogenetic clustering of each segment ( table 2 ) .", "segments 7 and 9 formed 5 independent clusters , segment 12 formed 3 clusters , and the remaining segments formed 4 clusters .", "phylogenetic trees for some representative segments ( segments 2 , 6 , 9 , and 12 ) are shown in figure 2 .", "javanese strains formed a single cluster for segments 1 , 2 , 6 , 8 , 10 , and 12 . for segments 7 , 9 , and 11 , however , they diverged into 2 clusters , i and ii ( figure 2 , panel c ) .", "segments 11 and 12 of the 2 vietnamese strains 02vn178b and 02vn018b were included in cluster iii with chinese bavs .", "segments 6 and 9 of isolate 02vn178b belonged to cluster v , although segments 1 , 2 , 3 , 4 , and 8 belonged to cluster iv .", "moreover , segments 5 and 7 of isolate 02vn178b and segments 8 and 11 of isolate 02vn009b showed a mixed electrogram pattern of clusters v and iv or iii .", "mosquito collection sites in vietnam . * length , length of each segment in the reference sequence of banna virus ( bav ) from refseq ( nc_004198 , nc_004200nc_004204 , nc_004211 , nc_004217nc_004221 ) ; aligned area , the fragment used to make the alignment and phylogenetic tree .", "the numbers are set following the sequences of strain jht-6423 , which is the reference strain in national center for biotechnology information reference sequences .", "i , the cluster including the javanese jkt-6423 strain ; ii , the cluster specific to the jkt-6969 and jkt-7043 strains ; iii , the cluster including chinese bavs ; iv , the cluster including the vietnamese 02vn018b strain ; v , the cluster including the vietnamese 02vn180b strain .", "we report isolation of bav in vietnam . the nucleotide sequences of the vietnamese isolates genomic rna segments diverged into 2 phylogenetically distant clusters .", "our data indicate that 2 bav populations exist in the country and that both evolved independently .", "two strains were clearly different from chinese and javanese bavs ( 02vn078b and 02vn180b ) .", "two other strains ( 02vn018b and 02vn178b ) were phylogenetically close to chinese bavs when 2 shorter segments , segments 11 and 12 , were analyzed but not when the remaining 10 , longer segments were considered .", "this finding implies that a recent reassortment event has occurred and that segments of chinese or vietnamese strains were replaced .", "in addition , although some vietnamese strains were phylogenetically distant , they could produce a viable progeny by reassortment of segments 5 , 6 , 7 , 8 , 9 , and 11 .", "this finding is clearly illustrated by the diversity in the phylogenetic clustering pattern and the mixed electrogram pattern of these segments ( table 2 ) .", "analysis of segment 12 showed that cluster iii ( table 2 ) contained 2 vietnamese strains ( 02vn178b and 02vn018b ) and the chinese strains af052030 and yn-6 ( isolated in yunnan province ) and bj95 - 75 ( isolated in beijing ) .", "this finding suggests the possibility of wide circulation of bavs containing segment 12 type iii not only in southeast asia but also in east asia . a regionwide genotype shift of japanese encephalitis virus ( jev ) , which is also carried by culex spp .", "mosquitoes , from genotype iii to genotype i , was witnessed in east asia during the 1990s ( 11,12 ) .", "these results and phylogenetic analysis of old jev strains in japan and southeast asia ( 11 ) strongly suggested that jev could be transferred frequently from southeast asia to east asia .", "similarly , bavs with segment 12 belonging to cluster type iii were also distributed in both southeast and east asia .", "therefore , we can speculate that bavs are actively circulating within the asian continent . to achieve a better understanding of the distribution dynamics of bavs in asia , more isolates are needed . in vietnam ,", "a seasonal increase of viral encephalitis and meningitis cases is reported during the rainy season every year . among these cases ,", "50%70% are diagnosed as japanese encephalitis , while the rest of them remain idiopathic ( p.t .", "nga et al . , unpub . data ) . a clear association between bav and human diseases , as well as the prevalence of bav infection in humans , has not yet been established in vietnam .", "we also believe that bav is a good candidate for the differential diagnosis of viral encephalitis and meningitis cases of unknown origin in tropical and subtropical asia , where culex mosquitoes are abundant ."], "abstract_text": ["<S> we isolated and characterized a banna virus from mosquitoes in vietnam ; 5 strains were isolated from field - caught mosquitoes at various locations ; banna virus was previously isolated from encephalitis patients in yunnan , china , in 1987 . </S>", "<S> together , these findings suggest widespread distribution of this virus throughout southeast asia . </S>"], "labels": null, "section_names": ["The Study", "Conclusions"], "sections": [["mosquito samples were collected in vietnam in 2002 . each species was separated and pooled , and each mosquito pool was then homogenized and centrifuged .", "the supernatant was filtrated through a 0.22-m filter and then inoculated onto a monolayer c6/36 mosquito cell culture and subsequently incubated at 28c for 7 days .", "virus amplification in c6/36 cells was repeated twice , and the culture fluids were stored at 80c for further analysis .", "rna was extracted from the second cell culture fluid by trizol ls reagent ( invitrogen , carlsbad , ca , usa ) .", "reverse transcription was performed by using superscript iii reverse transcriptase ( invitrogen ) and random hexamers , after rna denaturation at a temperature of 95c for 5 minutes in the presence of 15% dimethyl sulfoxide .", "pcr was conducted by using takara la taq dna polymerase ( takara bio , inc . ,", "otsu , japan ) with bav targeting primers ( for segment 5 , 5-cagctgcagtggttattgga-3 and 5accgtgcatcttaacccttg-3 ; for segment 8 , 5-ttgcagtcgctgagctttta-3 and 5-cgcatttgatcgtatgcttg-3 ) .", "these targeting primers were designed from sequences of bav strains from china and java ( genbank accession nos .", "af134519af134527 , ay549307ay549309 , af052024af052035 , ay568287ay568290 , nc_004211 , nc_004217-nc_004221 , nc_004200-nc_004204 , nc_004198 , and af052008af052013 ) . the amplified cdnas were sequenced in the 3100-avant genetic analyzer ( applied biosystems , foster city , ca , usa ) .", "the genomic sequences of all 12 genome segments for the 5 strains were determined ( genbank accession nos .", "the sequences were translated to protein sequences by using the transeq program of the emboss package ( 8) , version 5.00 .", "the protein sequences were aligned with the t - coffee program version 5.05 ( 9 ) with chinese and javanese bavs .", "nucleotide sequences were then aligned , on the basis of protein alignment , with the tranalign program of the emboss package .", "maximum likelihood phylogenetic trees were constructed from the protein - guided alignments of nucleotide sequences with dnaml software of the phylip package ( 10 ) , version 3.67 .", "five bav strains were isolated in ha tay and quang bing provinces ( figure 1 ) in vietnam from culex annulus and cx .", "phylogenetic analysis showed diversity within the phylogenetic clustering of each segment ( table 2 ) .", "segments 7 and 9 formed 5 independent clusters , segment 12 formed 3 clusters , and the remaining segments formed 4 clusters .", "phylogenetic trees for some representative segments ( segments 2 , 6 , 9 , and 12 ) are shown in figure 2 .", "javanese strains formed a single cluster for segments 1 , 2 , 6 , 8 , 10 , and 12 . for segments 7 , 9 , and 11 , however , they diverged into 2 clusters , i and ii ( figure 2 , panel c ) .", "segments 11 and 12 of the 2 vietnamese strains 02vn178b and 02vn018b were included in cluster iii with chinese bavs .", "segments 6 and 9 of isolate 02vn178b belonged to cluster v , although segments 1 , 2 , 3 , 4 , and 8 belonged to cluster iv .", "moreover , segments 5 and 7 of isolate 02vn178b and segments 8 and 11 of isolate 02vn009b showed a mixed electrogram pattern of clusters v and iv or iii .", "mosquito collection sites in vietnam . * length , length of each segment in the reference sequence of banna virus ( bav ) from refseq ( nc_004198 , nc_004200nc_004204 , nc_004211 , nc_004217nc_004221 ) ; aligned area , the fragment used to make the alignment and phylogenetic tree .", "the numbers are set following the sequences of strain jht-6423 , which is the reference strain in national center for biotechnology information reference sequences .", "i , the cluster including the javanese jkt-6423 strain ; ii , the cluster specific to the jkt-6969 and jkt-7043 strains ; iii , the cluster including chinese bavs ; iv , the cluster including the vietnamese 02vn018b strain ; v , the cluster including the vietnamese 02vn180b strain ."], ["we report isolation of bav in vietnam . the nucleotide sequences of the vietnamese isolates genomic rna segments diverged into 2 phylogenetically distant clusters .", "our data indicate that 2 bav populations exist in the country and that both evolved independently .", "two strains were clearly different from chinese and javanese bavs ( 02vn078b and 02vn180b ) .", "two other strains ( 02vn018b and 02vn178b ) were phylogenetically close to chinese bavs when 2 shorter segments , segments 11 and 12 , were analyzed but not when the remaining 10 , longer segments were considered .", "this finding implies that a recent reassortment event has occurred and that segments of chinese or vietnamese strains were replaced .", "in addition , although some vietnamese strains were phylogenetically distant , they could produce a viable progeny by reassortment of segments 5 , 6 , 7 , 8 , 9 , and 11 .", "this finding is clearly illustrated by the diversity in the phylogenetic clustering pattern and the mixed electrogram pattern of these segments ( table 2 ) .", "analysis of segment 12 showed that cluster iii ( table 2 ) contained 2 vietnamese strains ( 02vn178b and 02vn018b ) and the chinese strains af052030 and yn-6 ( isolated in yunnan province ) and bj95 - 75 ( isolated in beijing ) .", "this finding suggests the possibility of wide circulation of bavs containing segment 12 type iii not only in southeast asia but also in east asia . a regionwide genotype shift of japanese encephalitis virus ( jev ) , which is also carried by culex spp .", "mosquitoes , from genotype iii to genotype i , was witnessed in east asia during the 1990s ( 11,12 ) .", "these results and phylogenetic analysis of old jev strains in japan and southeast asia ( 11 ) strongly suggested that jev could be transferred frequently from southeast asia to east asia .", "similarly , bavs with segment 12 belonging to cluster type iii were also distributed in both southeast and east asia .", "therefore , we can speculate that bavs are actively circulating within the asian continent . to achieve a better understanding of the distribution dynamics of bavs in asia , more isolates are needed . in vietnam ,", "a seasonal increase of viral encephalitis and meningitis cases is reported during the rainy season every year . among these cases ,", "50%70% are diagnosed as japanese encephalitis , while the rest of them remain idiopathic ( p.t .", "nga et al . , unpub . data ) . a clear association between bav and human diseases , as well as the prevalence of bav infection in humans , has not yet been established in vietnam .", "we also believe that bav is a good candidate for the differential diagnosis of viral encephalitis and meningitis cases of unknown origin in tropical and subtropical asia , where culex mosquitoes are abundant ."]]}
{"article_id": "PMC4351436", "article_text": ["clozapine remains an ultimate option for patients with treatment resistant schizophrenia ( kane 2012 ) .", "efficacy of clozapine against positive symptoms of schizophrenia was confirmed in numerous studies and meta - analyses ( chakos et al .", "2001 ) , including analysis published by the cochrane library ( asenjo lobos et al .", "however , treatment with clozapine is associated with very severe metabolic side - effects ( newcomer 2005 ) .", "clozapine - induced weight gain is very common ( wetterling 2001 ) , and so is impaired fasting plasma glucose levels . in patients with schizophrenia weight gain", "is associated with impaired physical functioning and negative body appraisal ( bachmann et al . 2012 ) , both of which affect quality of life . while treatment with clozapine may reduce mortality by reducing suicide rate , mortality due to clozapine - associated weight gain will diminish reduction in the suicide rate almost entirely over 10  years by the increased mortality associated with weight gain of 10  kg ( fontaine et al .", "also , obesity is linked with numerous secondary health problems ( pressure overload on lungs , joints and bones ) and is an important risk factor for life - threatening diseases , such as cardiovascular disease , type 2 diabetes and certain cancers .", "several mechanisms are considered as taking role in clozapine - induced weight gain , including antagonism at histamine h1 receptors ( kroeze et al .", "various receptors , including aforementioned 5-ht1b and 5-ht1c , regulate activity of the hypothalamic nuclei ( particularly the arcuate nucleus ( arc ) , which plays the key role in appetite regulation ) .", "activity of arc is also regulated by several hormones of anorexigenic properties : leptin , pancreatic polypeptide ( pp ) , cholecystokinin ( cck ) , glucagon - like peptide-1 ( glp-1 ) , oxyntomodulin ( oxm ) , peptide yy ( pyy ) and the first discovered orexigenic substance - ghrelin ( druce et al . 2004 ) .", "agouti - related peptide ( agrp ) is one of the hypothalamic hormones that works by increasing appetite and decreasing metabolism and energy expenditure , thus leading to weight gain .", "it was identified in 1997 based on its sequence similarity with agouti signaling peptide ( asip ) , a protein synthesized in the skin that controls coat color .", "compared with asip , agrp is physiologically expressed in the hypothalamus ( shutter et al .", "agrp is also expressed in the adrenal gland , testes , kidneys , and lungs .", "agrp induces obesity by antagonism of the melanocortin receptors ( mc3-r and mc4-r , subtypes implicated in weight regulation , via metabolism and appetite control ) ( ollmann et al .", "these neurons make peptides that potently stimulate food intake not only by increasing neuropeptide y ( npy ) signaling , but also by reducing melanocortin signaling via the release of agrp ( morton and schwartz 2001 ) .", "activity of the agrp / npy neurons is modulated by leptin , released from the adipose tissue , and insulin .", "the adiposity signals ( insulin and leptin ) are secreted in proportion to body fat content and act in the hypothalamus to inhibit anabolic and stimulate catabolic , effector pathways ( schwartz et al .", "the increase of food intake following a single central administration of agrp is sustained for up to a week ( hagan et al . 2000 ) , while the response to npy is sustained over hours , rather than days .", "this study was undertaken with the purpose to determine if patients with schizophrenia on clozapine monotherapy have higher fasting levels of agrp compared with healthy control . in order to provide more accurate measurements ,", "biochemical and anthropometric measurements were combined with body composition determined using bioelectric impedance analysis ( bia ) , which provides accurate measurements of body fat , lean mass and body water ( bosy - westphal et al .", "this is the first study to investigate such combination of these parameters in subjects with schizophrenia treated with clozapine .", "data for 24 european caucasian adult hospitalized patients with paranoid schizophrenia ( 295.30 according to dsm - iv , f20.0 according to icd-10 ) was included into the study .", "these subjects were on clozapine monotherapy for at least two months prior the assessments with a minimum dose of 100  mg / day .", "most patients was in stable phase of the disease ( i.e. no acute psychosis ) .", "control group was 24 healthy subjects and was gender- and age - matched with patients in the clozapine group .", "health status of the control subjects was determined on the basis of basic physical examination , including vital signs and an interview .", "all patients and volunteers included in the study expressed their written informed consent for participation in this study .", "the blood samples for the chemistry panel were collected between 7  am and 8  am , after ensuring at least 8  h of overnight fasting .", "glucose , lipids , calcium and uric acid levels were measured using a dirui cs-400 analyzer ( dirui , china ) .", "homocysteine chemiluminescence assessments were performed using an immulite 2,000 analyzer ( siemens , germany ) , insulin immunochemistry assessments were performed using a cobas e411 analyzer ( roche diagnostics , switzerland ) and albumin levels were assessed using a cobas integra 800 analyzer ( roche diagnostics , switzerland ) .", "levels of agrp were measured in blood serum using elisa ( enzyme - linked immunosorbent assay ) method . prior to assays ,", "serum samples were stored at  80  c for up to 6  months .", "elisa assays were performed using commercial kits ( intra - assay : cv < 10  % , inter - assay : cv < 12  % ) manufactured by raybiotech ( usa ) , according to protocol provided by its manufacturer ( all samples were 2-fold diluted ) . metabolic syndrome and abdominal obesity were defined according to international diabetes foundation ( idf ) criteria ( alberti et al .", "kg / m , 2530  kg / m and 30  kg / m were defined as normal weight , overweight and obesity , respectively . raised triglycerides ( tga ) level 150  mg / dl and/or total cholesterol ( tc ) 200  mg / dl and/or reduced hdl cholesterol level < 40  mg / dl for men and < 50  mg / dl for women and/or raised ldl cholesterol level 135  mg / dl were interpreted as dyslipidemia .", "corrected calcium was calculated using the formula : corrected calcium [ mg / dl ]  = measured total calcium [ mg / dl ]  + 0.8 ( 4.0  serum albumin [ g / dl ] ) .", "insulin resistance was estimated from fasting glucose and insulin results by homeostasis model assessment and quicki index , using the formula : homa1-ir = ( fasting plasma glucose [ mg / dl ]  insulin [ mu / l])/405 . homa2-ir index was calculated using a calculator downloaded from http://www.dtu.ox.ac.uk .", "quicki index ( lower numbers reflect greater insulin resistance ) was calculated using the formula : 1/(log ( fasting insulin [ mu / l ] ) + log ( fasting plasma glucose [ mg / dl ] ) ) .", "height was measured with a wall - mounted height measure to the nearest 0.5  cm .", "weight was measured with a spring balance that was kept on a firm horizontal surface .", "subjects wore light clothing , stood upright without shoes and weight was recorded to the nearest 0.5  kg .", "body mass index ( bmi ) was calculated as body weight in kilogram divided by the height in meter squared ( kg / m ) .", "waist , abdominal and hip circumference was measured using a non - stretchable fiber measuring tape .", "waist - to - hip ratio ( whr ) was calculated as waist circumference divided by hip circumference .", "whr cut - off points were defined according to who recommendations ( 0.85 for women and 0.9 for men ) .", "fat mass index ( fmi ) was calculated as total body fat in kilogram divided by the height in meter squared ( kg / m ) .", "excess body fat according to fmi classification ranges were defined as fmi > 6 for men and fmi > 9 for women", "body composition was measured using a maltron bf-906 body fat analyzer ( maltron , uk ) , single frequency bioelectrical impedance analyzer to determine resistance and reactance at 50  hz .", "standard operating conditions were observed by a trained operator including preparation of the participant , electrode placement and operation .", "the measurement using bia was taken immediately prior to anthropometry measurements with participants lying supine , in a rested state .", "simple descriptive statistics ( means , standard deviations , 95  % confidence interval [ ci ] ) were generated for all continuous variables . for discrete variables number of patients and percentages are given .", "skewed variables were transformed to follow normal distribution using log , square root , inverse square root or square transformation .", "means , standard deviations , and confidence intervals are reported for non - transformed variables , results of tests are reported for transformed or non - transformed variables . if transformation resulted in normal distribution , two - tailed t - test", "was used to assess inter - group differences , otherwise variables were analyzed using mann ", "associations were tested by pearson s ( for variables with normal distribution ) or spearman 's ( for other variables ) correlation coefficients . the significant level was set at p < 0.05 .", "for group of patients treated with clozapine the mean age was 38.812.6 and 39.912.3 for the control group ( p  = 0.62 ) . in both groups", "there were 12 men and 12 women . in the clozapine group 12 subjects smoked cigarettes and 8 in the control group ( p  = 0.38 ) .", "the mean duration of monotherapy with clozapine was 60.579.4 [ 95  % ci : 27.094.1 ] months and mean clozapine dose was 341.1148.6 [ 95  % ci : 278.4403.8 ] mg / day .", "detailed results for anthropometric measurements and laboratory tests are shown in table  1 . \n", "table 1results of anthropometric measurements and laboratory testsclozapine \n n  = 24control \n n  = 24weight [ kg]78.114.2(72.184.1)72.615.1(66.278.9 ) \n p  = 0.08 \n t  = 1.38bmi [ kg / m]27.13.6(25.528.6)24.83.5(23.326.2 ) \n", "p  = 0.01 \n t  = 2.25fmi [ kg / m]8.93.1(7.610.2)7.73.0(6.49.0 ) \n p  = 0.09 \n t  = 1.38abdominal circumference [ cm]96.59.4(92.5100.4)85.511.6(80.690.3 ) \n p <", "0.001 \n z  = 3.58waist circumference [ cm]91.112.1(86.096.2)82.410.6(77.886.8 ) \n p  = 0.005 \n z  = 2.66hip circumference [ cm]99.08.4(95.5102.5)95.97.1(92.998.9 ) \n p  = 0.08 \n t  = 1.39whr0.920.08(0.880.95)0.860.08(0.820.89 ) \n p  = 0.005 \n z  = 2.66sbp [ mm hg]121.713.6(115.9127.4)136.717.9(129.1144.2 ) \n p  = 0.001 \n z  = 3.26dbp [ mm hg]81.28.5(77.684.8)82.812.1(77.687.9 ) \n p  = 0.30 \n t  = 0.50uric acid [ mg / dl]4.51.4(3.95.1)4.31.3(3.74.8 ) \n", "t  = 0.57homocysteine [ mol / l]14.5    4.4(12.616.4)13.6    5.0(11.515.7 ) \n p  =  0.25 \n t  =  0.67tc [ mg / dl]194.2    53.2(171.7216.6)216.6    65.3(189.0244.1 ) \n", "p  =  0.06 \n t  =  1.54hdl [ mg / dl]43.5    12.6(38.248.8)55.1    14.3(49.161.1 ) \n p ", "41.9(104.8140.3)128.3    39.7(111.5145.0 ) \n p  =  0.29 \n t  = 0.54tga [ mg / dl]140.3    120.4(89.4191.1)104.3    81.4(69.9138.6 ) \n", "p  =  0.07 \n t  =  1.50fpg [ mg / dl]103.5    31.7(90.1116.8)87.8    11.7(82.892.7 ) \n p  =  0.03 \n", "t  = 1.88insulin [ u / ml]11.8    8.2(8.3 - 15.3)7.7    3.2(6.3 - 9.1 ) \n p  =  0.02 \n t  = 2.08homa1-ir3.3    3.4(1.84.7)1.7    0.8(1.32.0 ) \n p  =  0.009 \n t  = 2.46homa2-ir1.6    1.1(1.12.1)1.0    0.4(0.81.2 ) \n p  =  0.01 \n t  = 2.26quicki0.15    0.01(0.140.15)0.16 ", "  0.01(0.150.16 ) \n p  =  0.007 \n t  = 2.52corrected calcium [ mg / dl]8.6    0.9(8.29.0)8.7 ", "p  =  0.37 \n t  = 0.33data given as : mean    standard deviation ( 95  % ci)bmi  =  body mass index ; fmi  =  fat mass index ; whr  =  waist - to - hip ratio ; sbp  =  systolic blood pressure ; dbp  =  diastolic blood pressure ; tc  =  total cholesterol ; hdl  =  high density lipoproteins ; ldl  =  low density lipoproteins ; tga  =  triglycerides ; fpg  =  fasting plasma glucose ; homa1-ir  =  homoeostasis model assessment of insulin resistance 1 ; homa2-ir  =  homoeostasis model assessment of insulin resistance 2 ; quicki  =  quantitative insulin sensitivity check index results of anthropometric measurements and laboratory tests data given as : mean    standard deviation ( 95  % ci ) bmi  =  body mass index ; fmi  =  fat mass index ; whr  =  waist - to - hip ratio ; sbp  =  systolic blood pressure ; dbp  =  diastolic blood pressure ; tc  =  total cholesterol ; hdl  =  high density lipoproteins ; ldl  =  low density lipoproteins ; tga  =  triglycerides ; fpg  =  fasting plasma glucose ; homa1-ir  =  homoeostasis model assessment of insulin resistance 1 ; homa2-ir  =  homoeostasis model assessment of insulin resistance 2 ; quicki  =  quantitative insulin sensitivity check index we have found no inter - group differences for body composition analysis . detailed results for bia analysis are shown in table  2 . lean body mass was higher in men in the whole study sample ( 60.16.4 [ 95  % ci : 57.4 - 62.8 ] vs. 43.85.4 [ 95  % ci : 41.546.1 ] kg , t = 9.56 , p < 0.001 ) and in the clozapine group ( 59.65.7 [ 95  % ci : 56.063.3 ] vs. 45.37.0 [ 95  % ci : 40.9 - 49.8 ] kg , t = 5.48 , p < 0.001 ) .", "similarly , basal metabolic rate was higher in men in the whole study sample ( 1,707.7182.3 [ 95  % ci : 1,630.71,784.6 ] vs. 1,337.3138.4 [ 95  % ci : 1,278.8 - 1,395.7 ] kcal / day , t  = 7.92 , p < 0.001 ) and in the clozapine group ( 1,701.2138.2 [ 95  % ci : 1,613.41,789.0 ] vs. 1,362.7173.0 [ 95  % ci : 1,252.71,472.5 ] kcal / day , t = 5.29 , p < 0.001 ) . \n", "n  =  24control \n n  =  24total body fat [ kg]25.6    8.8(21.829.2)22.4    8.7(18.726.1 ) \n p  =  0.10 \n t  =  1.25total body fat [ % ] 32.6    8.4(29.136.2)28.9    7.1(25.831.8 ) \n p  =  0.06 \n t  =  1.67lean body mass [ kg]52.5    9.6(48.456.5)51.4    10.8(46.855.9 ) \n p  =  0.36 \n t  =  0.36lean body weight [ % ] 67.6    8.1(64.271.0)71.1    7.1(68.174.1 ) \n p  =  0.07 \n t  = 1.61total body water [ l]38.4    7.0(35.441.3)37.7    7.9(34.340.9)p  =  0.36 t  =  0.35total body water [ % ] 49.9    5.4(47.652.1)52.1    5.2(49.854.2 ) \n p  =  0.09 \n t  =  1.42basal metabolic rate [ kcal / day]1,532.0 ", "p  =  0.39 \n t  =  0.26data given as : mean    standard deviation ( 95  % ci ) results of body composition analysis data given as : mean    standard deviation ( 95  % ci ) there were no significant difference for fasting serum levels of agrp between the clozapine group and the control group ( 15.008.65 [ 95  % ci : 11.3418.65 ] vs. 15.336.82 [ 95  % ci : 12.4518.22 ] pg / ml , t  = 0.32 , p  = 0.37 ) , see fig .  1 .", "women had significantly lower levels of agrp in the whole study group ( 12.535.65 [ 95  % ci : 10.1414.92 ] vs. 17.808.66 [ 95  % ci : 14.1421.45 ] pg / ml , t  = 2.47 , p  = 0.009 ) and in the control group ( 12.905.77 [ 95  % ci : 9.2216.57 ] vs. 17.777.14 [ 95  % ci : 13.2322.31 ] pg / ml , t  = 1.82 , p  = 0.04 ) , but the difference was not significant for the clozapine group ( 12.175.75 [ 95  % ci : 8.5115.83 ] vs. 17.8210.28 [ 95  % ci : 11.28 - 24.36 ] pg / ml , t  = 1.64 , p  = 0.06).fig .", "1mean fasting agrp serum levels [ pg / ml ] in subjects on clozapine and in the control group ( p  = 0.37 ; horizontal bars indicate means ) .", "mean fasting agrp serum levels [ pg / ml ] in subjects on clozapine and in the control group ( p  = 0.37 ; horizontal bars indicate means ) .", "for the whole study group significant correlations of agrp levels were found for total body fat [ % ] ( r  = 0.34 , p  = 0.02 ) , lean body mass [ kg ] ( r  = 0.38 , p  = 0.006 ) , lean body mass [ % ] ( r  = 0.34 , p  = 0.02 ) , body water [ l ] ( r  = 0.38 , p  = 0.006 ) , body water [ % ] ( r  = 0.34 , p  = 0.02 ) and homocysteine levels ( r  = 0.29 , p  = 0.04 ) .", "for the clozapine group significant correlations of agrp levels were found for total body fat [ % ] ( r  = 0.48 , p  = 0.02 ) , basal metabolic rate ( r  = 0.42 , p  = 0.04 ) , lean body mass [ % ] ( r  = 0.49 , p  = 0.01 ) , body water [ % ] ( r  = 0.49 , p  = 0.01 ) . for the control group", "significant correlations of agrp levels were found only for basal metabolic rate ( r  = 0.42 , p  = 0.04 ) . in the clozapine group", "there were no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p  = 0.59 ) , idf - defined metabolic syndrome ( p  = 0.33 ) , smokers and non - smokers ( p  = 0.15 ) , subjects with bmi < 25  kg / m and with bmi 25  kg / m ( p  = 0.09 ) , subjects with and without impaired fasting glucose ( p  = 0.16 ) , subjects with and without abdominal obesity ( p  = 0.11 ) , subjects with and without dyslipidemia ( p  = 0.09 ) , and subjects with and without homa-1ir defined insulin resistance ( p  = 0.07 ) . in the control group there were no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p  =  0.20 ) , idf - defined metabolic syndrome ( p  =  0.44 ) , smokers and non - smokers ( p  =  0.35 ) , subjects with bmi < 25  kg / m and with bmi 25  kg / m ( p  = 0.12 ) , subjects with and without impaired fasting glucose ( p  = 0.36 ) , subjects with and without abdominal obesity ( p  = 0.10 ) , subjects with and without dyslipidemia ( p  = 0.37 ) , and subjects with and without homa-1ir defined insulin resistance ( p  = 0.13 ) . in the whole study group there was no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p  = 0.30 ) , idf - defined metabolic syndrome ( p  = 0.43 ) , smokers and non - smokers ( p  = 0.29 ) , subjects with bmi < 25  kg / m and with bmi 25  kg / m ( p  = 0.39 ) , subjects with and without impaired fasting glucose ( p  = 0.29 ) , subjects with and without dyslipidemia ( p  = 0.11 ) , and subjects with and without homa-1ir defined insulin resistance ( p  = 0.33 ) . there was a significant difference for agrp levels between subjects with or without abdominal obesity ( 13.466.50 [ 95  % ci : 9.0917.83 ] vs. 16.926.93 [ 95  % ci : 12.7321.11 ] pg / ml , t  = 1.80 , p  = 0.04 ) .", "the main objective of the present study was to find out whether there is a significant difference in fasting serum levels of agrp peptide between patients with schizophrenia on clozapine monotherapy and age- and sex - matched healthy controls .", "we have found the difference was not significant ( clozapine : 15.008.65 , control : 15.336.82  pg / ml , p  = 0.37 ) .", "we do not know pre - treatment values , so we can not determine whether and how this parameter changed during therapy with clozapine . to our knowledge ,", "no data are available on the effect of clozapine on blood agrp concentrations in humans .", "limited data are available for olanzapine , which has clinical and pharmacological properties similar to clozapine .", "results of these studies show that treatment with olanzapine does not affect agrp levels ( basoglu et al .", "however , in animal study it was demonstrated that administration of olanzapine or clozapine upregulates npy and agrp and downregulates proopiomelanocortin in the arcuate nucleus of the hypothalamus ( ferno et al .", "we do not know studies demonstrating correlation between blood levels of agrp and levels in the hypothalamus , but since it was showed recently that agrp neurons are unique among hypothalamic neurons by being the predominant neuronal subtype situated outside the blood ", "brain barrier ( olofsson et al . 2013 ) , we may assume that changes in hypothalamic expression are reflected in changes in blood levels .", "the finding that women had significantly lower levels of agrp in the whole study group ( 12.535.65 vs. 17.808.66  pg / ml , p  = 0.009 ) and in the control group ( 12.905.77 vs. 17.777.14  pg / ml , p  = 0.04 ) , but not in the clozapine group ( 12.175.75 vs. 17.8210.28  pg / ml , p  = 0.057 ) , might indicate that the testes are important source of agrp , as shown previously ( shutter et al .", "however , several more recent studies showed no difference between men and ( gavrila et al .", "2001 ) , therefore it may indicate that plasma agrp levels reflect central agrp concentrations .", "it was previously reported that total level of agrp is higher in obese subjects ( katsuki et al .", "2001 ) , although in another study it has been demonstrated that agrp levels were lower in normal weight group compared with women with anorexia , probably due to increased leptin levels ( moriya et al .", "we did not measure levels of leptin or acylated ghrelin , but we found in the same group of patients that there were no differences for desacyl ghrelin levels between patients taking clozapine and control group ( wysokiski et al .", "while there was a significant difference in bmi values between the clozapine group and the control group , we found no differences between both groups for fmi values . compared with bmi ,", "fmi is a better indicator of central obesity since it is much more sensitive to body fat content ( kelly et al .", "moreover , there were no differences for any of the bia results between both groups .", "these indicate that there was a substantial similarity between both groups in terms of body fat content , which is important considering that agrp levels may affect or result more from the amount of adipose tissue than from body weight per se .", "on the other hand , we have found negative correlations between agrp levels and total body fat ( r  = 0.34 and  0.48 in the whole study group and clozapine group , respectively ) .", "these may indicate the effect of leptin on the expression of agrp , but require further studies .", "additionally , there were positive correlations with lean body mass ( r  = 0.38 and 0.49 in the whole study group and clozapine group , respectively ) , body water ( which amount is negatively correlated with the amount of body fat : r  = 0.96 , p  <  0.001 ) ( r  = 0.34 and 0.49 in the whole study group and clozapine group , respectively ) and basal metabolic rate ( r  = 0.42 both in the clozapine group and control group ) , also being in line with the above - mentioned mechanism . in animal studies", "it has been found that high - fat diet leads to decreased expression of agrp in the hypothalamus , probably in the mechanism moderated by leptin ( staszkiewicz et al .", "we have no detailed data on diet patterns of our study subjects , but reports are consistent that patients with schizophrenia consume higher amounts of high - fat foods compared with healthy population ( dipasquale et al .", ". this might be one of the reasons why we have found no differences between our study groups . also ,", "level of physical activity ( usually lower in hospitalized patients ) might affect mechanisms regulating agrp levels .", "finally , another important factor is the effect of treatment mediated by other anabolic or catabolic neuropeptides . here , two most important peptides are leptin ( which decreases agrp levels ) and ghrelin ( which has opposite effect ) and have also been studied most extensively ( sentissi et al .", "2008 ) . since both leptin and ghrelin ( andrews 2011 ) may regulate activity of agrp / npy neurons in the arcuate nucleus , effects of antipsychotics on these neuropeptides must be taken into consideration . therefore", ", a study focused on interactions within this regulatory system would be very important for better understanding of this problem . based on our results", "however , the low number of study subjects limited the probability of finding inter - group differences due to lack of statistical power . due to the cross - sectional study design causal relationships", "can not be established and the effect of previous antipsychotic treatment can not be excluded .", "dual - energy x - ray absorptiometry ( dxa ) should be used to measure body composition and adipose tissue mass more accurately .", "due to complex structure of interactions between anabolic and catabolic neuropeptides , a longitudinal study comparing these interactions are crucial for understanding mechanisms of treatment - induced weight gain ."], "abstract_text": ["<S> aim : agouti - related peptide ( agrp ) is one of the hypothalamic hormones that works by increasing appetite and decreasing metabolism , thus leading to weight gain . </S>", "<S> the aim of the study was to find out if agrp level in subjects with schizophrenia on clozapine monotherapy is higher compared with healthy controls . </S>", "<S> methodology : we determined fasting serum agrp levels in 24 subjects with schizophrenia on clozapine monotherapy and 24 healthy , age- and sex - matched controls . </S>", "<S> biochemical and anthropometric measurements were combined with body composition analysis . </S>", "<S> results : there was no difference for agrp levels between patients taking clozapine and control group ( 15.008.65 vs. 15.336.82  pg / ml , p  = 0.37 ) . </S>", "<S> we found negative correlations between agrp levels and total body fat ( r  = 0.34 and  0.48 in the whole study group and clozapine group , respectively ) and positive correlations with lean body mass ( r  = 0.38 and 0.49 in the whole study group and clozapine group , respectively ) , body water ( r  = 0.34 and 0.49 in the whole study group and clozapine group , respectively ) and basal metabolic rate ( r  = 0.42 both in the clozapine and control groups ) . </S>", "<S> there were no correlations with age , height , weight , body mass index , fat mass index , abdominal , waist or hip circumferences , waist - hip ratio , blood pressure , total cholesterol , hdl , ldl , triglycerides , uric acid , glucose , insulin , clozapine dose or treatment duration , duration of treatment with antipsychotics and markers for insulin resistance . </S>", "<S> conclusion : we can not conclude that treatment with clozapine is associated with increased level of agrp . </S>", "<S> we did not find previously described differences in agrp levels between obese and non - obese subjects or associations between agrp and various metabolic parameters . </S>"], "labels": null, "section_names": ["Introduction", "Material and methods", "Results", "Discussion"], "sections": [["clozapine remains an ultimate option for patients with treatment resistant schizophrenia ( kane 2012 ) .", "efficacy of clozapine against positive symptoms of schizophrenia was confirmed in numerous studies and meta - analyses ( chakos et al .", "2001 ) , including analysis published by the cochrane library ( asenjo lobos et al .", "however , treatment with clozapine is associated with very severe metabolic side - effects ( newcomer 2005 ) .", "clozapine - induced weight gain is very common ( wetterling 2001 ) , and so is impaired fasting plasma glucose levels . in patients with schizophrenia weight gain", "is associated with impaired physical functioning and negative body appraisal ( bachmann et al . 2012 ) , both of which affect quality of life . while treatment with clozapine may reduce mortality by reducing suicide rate , mortality due to clozapine - associated weight gain will diminish reduction in the suicide rate almost entirely over 10  years by the increased mortality associated with weight gain of 10  kg ( fontaine et al .", "also , obesity is linked with numerous secondary health problems ( pressure overload on lungs , joints and bones ) and is an important risk factor for life - threatening diseases , such as cardiovascular disease , type 2 diabetes and certain cancers .", "several mechanisms are considered as taking role in clozapine - induced weight gain , including antagonism at histamine h1 receptors ( kroeze et al .", "various receptors , including aforementioned 5-ht1b and 5-ht1c , regulate activity of the hypothalamic nuclei ( particularly the arcuate nucleus ( arc ) , which plays the key role in appetite regulation ) .", "activity of arc is also regulated by several hormones of anorexigenic properties : leptin , pancreatic polypeptide ( pp ) , cholecystokinin ( cck ) , glucagon - like peptide-1 ( glp-1 ) , oxyntomodulin ( oxm ) , peptide yy ( pyy ) and the first discovered orexigenic substance - ghrelin ( druce et al . 2004 ) .", "agouti - related peptide ( agrp ) is one of the hypothalamic hormones that works by increasing appetite and decreasing metabolism and energy expenditure , thus leading to weight gain .", "it was identified in 1997 based on its sequence similarity with agouti signaling peptide ( asip ) , a protein synthesized in the skin that controls coat color .", "compared with asip , agrp is physiologically expressed in the hypothalamus ( shutter et al .", "agrp is also expressed in the adrenal gland , testes , kidneys , and lungs .", "agrp induces obesity by antagonism of the melanocortin receptors ( mc3-r and mc4-r , subtypes implicated in weight regulation , via metabolism and appetite control ) ( ollmann et al .", "these neurons make peptides that potently stimulate food intake not only by increasing neuropeptide y ( npy ) signaling , but also by reducing melanocortin signaling via the release of agrp ( morton and schwartz 2001 ) .", "activity of the agrp / npy neurons is modulated by leptin , released from the adipose tissue , and insulin .", "the adiposity signals ( insulin and leptin ) are secreted in proportion to body fat content and act in the hypothalamus to inhibit anabolic and stimulate catabolic , effector pathways ( schwartz et al .", "the increase of food intake following a single central administration of agrp is sustained for up to a week ( hagan et al . 2000 ) , while the response to npy is sustained over hours , rather than days .", "this study was undertaken with the purpose to determine if patients with schizophrenia on clozapine monotherapy have higher fasting levels of agrp compared with healthy control . in order to provide more accurate measurements ,", "biochemical and anthropometric measurements were combined with body composition determined using bioelectric impedance analysis ( bia ) , which provides accurate measurements of body fat , lean mass and body water ( bosy - westphal et al .", "this is the first study to investigate such combination of these parameters in subjects with schizophrenia treated with clozapine ."], ["data for 24 european caucasian adult hospitalized patients with paranoid schizophrenia ( 295.30 according to dsm - iv , f20.0 according to icd-10 ) was included into the study .", "these subjects were on clozapine monotherapy for at least two months prior the assessments with a minimum dose of 100  mg / day .", "most patients was in stable phase of the disease ( i.e. no acute psychosis ) .", "control group was 24 healthy subjects and was gender- and age - matched with patients in the clozapine group .", "health status of the control subjects was determined on the basis of basic physical examination , including vital signs and an interview .", "all patients and volunteers included in the study expressed their written informed consent for participation in this study .", "the blood samples for the chemistry panel were collected between 7  am and 8  am , after ensuring at least 8  h of overnight fasting .", "glucose , lipids , calcium and uric acid levels were measured using a dirui cs-400 analyzer ( dirui , china ) .", "homocysteine chemiluminescence assessments were performed using an immulite 2,000 analyzer ( siemens , germany ) , insulin immunochemistry assessments were performed using a cobas e411 analyzer ( roche diagnostics , switzerland ) and albumin levels were assessed using a cobas integra 800 analyzer ( roche diagnostics , switzerland ) .", "levels of agrp were measured in blood serum using elisa ( enzyme - linked immunosorbent assay ) method . prior to assays ,", "serum samples were stored at  80  c for up to 6  months .", "elisa assays were performed using commercial kits ( intra - assay : cv < 10  % , inter - assay : cv < 12  % ) manufactured by raybiotech ( usa ) , according to protocol provided by its manufacturer ( all samples were 2-fold diluted ) . metabolic syndrome and abdominal obesity were defined according to international diabetes foundation ( idf ) criteria ( alberti et al .", "kg / m , 2530  kg / m and 30  kg / m were defined as normal weight , overweight and obesity , respectively . raised triglycerides ( tga ) level 150  mg / dl and/or total cholesterol ( tc ) 200  mg / dl and/or reduced hdl cholesterol level < 40  mg / dl for men and < 50  mg / dl for women and/or raised ldl cholesterol level 135  mg / dl were interpreted as dyslipidemia .", "corrected calcium was calculated using the formula : corrected calcium [ mg / dl ]  = measured total calcium [ mg / dl ]  + 0.8 ( 4.0  serum albumin [ g / dl ] ) .", "insulin resistance was estimated from fasting glucose and insulin results by homeostasis model assessment and quicki index , using the formula : homa1-ir = ( fasting plasma glucose [ mg / dl ]  insulin [ mu / l])/405 . homa2-ir index was calculated using a calculator downloaded from http://www.dtu.ox.ac.uk .", "quicki index ( lower numbers reflect greater insulin resistance ) was calculated using the formula : 1/(log ( fasting insulin [ mu / l ] ) + log ( fasting plasma glucose [ mg / dl ] ) ) .", "height was measured with a wall - mounted height measure to the nearest 0.5  cm .", "weight was measured with a spring balance that was kept on a firm horizontal surface .", "subjects wore light clothing , stood upright without shoes and weight was recorded to the nearest 0.5  kg .", "body mass index ( bmi ) was calculated as body weight in kilogram divided by the height in meter squared ( kg / m ) .", "waist , abdominal and hip circumference was measured using a non - stretchable fiber measuring tape .", "waist - to - hip ratio ( whr ) was calculated as waist circumference divided by hip circumference .", "whr cut - off points were defined according to who recommendations ( 0.85 for women and 0.9 for men ) .", "fat mass index ( fmi ) was calculated as total body fat in kilogram divided by the height in meter squared ( kg / m ) .", "excess body fat according to fmi classification ranges were defined as fmi > 6 for men and fmi > 9 for women", "body composition was measured using a maltron bf-906 body fat analyzer ( maltron , uk ) , single frequency bioelectrical impedance analyzer to determine resistance and reactance at 50  hz .", "standard operating conditions were observed by a trained operator including preparation of the participant , electrode placement and operation .", "the measurement using bia was taken immediately prior to anthropometry measurements with participants lying supine , in a rested state .", "simple descriptive statistics ( means , standard deviations , 95  % confidence interval [ ci ] ) were generated for all continuous variables . for discrete variables number of patients and percentages are given .", "skewed variables were transformed to follow normal distribution using log , square root , inverse square root or square transformation .", "means , standard deviations , and confidence intervals are reported for non - transformed variables , results of tests are reported for transformed or non - transformed variables . if transformation resulted in normal distribution , two - tailed t - test", "was used to assess inter - group differences , otherwise variables were analyzed using mann ", "associations were tested by pearson s ( for variables with normal distribution ) or spearman 's ( for other variables ) correlation coefficients . the significant level was set at p < 0.05 ."], ["for group of patients treated with clozapine the mean age was 38.812.6 and 39.912.3 for the control group ( p  = 0.62 ) . in both groups", "there were 12 men and 12 women . in the clozapine group 12 subjects smoked cigarettes and 8 in the control group ( p  = 0.38 ) .", "the mean duration of monotherapy with clozapine was 60.579.4 [ 95  % ci : 27.094.1 ] months and mean clozapine dose was 341.1148.6 [ 95  % ci : 278.4403.8 ] mg / day .", "detailed results for anthropometric measurements and laboratory tests are shown in table  1 . \n", "table 1results of anthropometric measurements and laboratory testsclozapine \n n  = 24control \n n  = 24weight [ kg]78.114.2(72.184.1)72.615.1(66.278.9 ) \n p  = 0.08 \n t  = 1.38bmi [ kg / m]27.13.6(25.528.6)24.83.5(23.326.2 ) \n", "p  = 0.01 \n t  = 2.25fmi [ kg / m]8.93.1(7.610.2)7.73.0(6.49.0 ) \n p  = 0.09 \n t  = 1.38abdominal circumference [ cm]96.59.4(92.5100.4)85.511.6(80.690.3 ) \n p <", "0.001 \n z  = 3.58waist circumference [ cm]91.112.1(86.096.2)82.410.6(77.886.8 ) \n p  = 0.005 \n z  = 2.66hip circumference [ cm]99.08.4(95.5102.5)95.97.1(92.998.9 ) \n p  = 0.08 \n t  = 1.39whr0.920.08(0.880.95)0.860.08(0.820.89 ) \n p  = 0.005 \n z  = 2.66sbp [ mm hg]121.713.6(115.9127.4)136.717.9(129.1144.2 ) \n p  = 0.001 \n z  = 3.26dbp [ mm hg]81.28.5(77.684.8)82.812.1(77.687.9 ) \n p  = 0.30 \n t  = 0.50uric acid [ mg / dl]4.51.4(3.95.1)4.31.3(3.74.8 ) \n", "t  = 0.57homocysteine [ mol / l]14.5    4.4(12.616.4)13.6    5.0(11.515.7 ) \n p  =  0.25 \n t  =  0.67tc [ mg / dl]194.2    53.2(171.7216.6)216.6    65.3(189.0244.1 ) \n", "p  =  0.06 \n t  =  1.54hdl [ mg / dl]43.5    12.6(38.248.8)55.1    14.3(49.161.1 ) \n p ", "41.9(104.8140.3)128.3    39.7(111.5145.0 ) \n p  =  0.29 \n t  = 0.54tga [ mg / dl]140.3    120.4(89.4191.1)104.3    81.4(69.9138.6 ) \n", "p  =  0.07 \n t  =  1.50fpg [ mg / dl]103.5    31.7(90.1116.8)87.8    11.7(82.892.7 ) \n p  =  0.03 \n", "t  = 1.88insulin [ u / ml]11.8    8.2(8.3 - 15.3)7.7    3.2(6.3 - 9.1 ) \n p  =  0.02 \n t  = 2.08homa1-ir3.3    3.4(1.84.7)1.7    0.8(1.32.0 ) \n p  =  0.009 \n t  = 2.46homa2-ir1.6    1.1(1.12.1)1.0    0.4(0.81.2 ) \n p  =  0.01 \n t  = 2.26quicki0.15    0.01(0.140.15)0.16 ", "  0.01(0.150.16 ) \n p  =  0.007 \n t  = 2.52corrected calcium [ mg / dl]8.6    0.9(8.29.0)8.7 ", "p  =  0.37 \n t  = 0.33data given as : mean    standard deviation ( 95  % ci)bmi  =  body mass index ; fmi  =  fat mass index ; whr  =  waist - to - hip ratio ; sbp  =  systolic blood pressure ; dbp  =  diastolic blood pressure ; tc  =  total cholesterol ; hdl  =  high density lipoproteins ; ldl  =  low density lipoproteins ; tga  =  triglycerides ; fpg  =  fasting plasma glucose ; homa1-ir  =  homoeostasis model assessment of insulin resistance 1 ; homa2-ir  =  homoeostasis model assessment of insulin resistance 2 ; quicki  =  quantitative insulin sensitivity check index results of anthropometric measurements and laboratory tests data given as : mean    standard deviation ( 95  % ci ) bmi  =  body mass index ; fmi  =  fat mass index ; whr  =  waist - to - hip ratio ; sbp  =  systolic blood pressure ; dbp  =  diastolic blood pressure ; tc  =  total cholesterol ; hdl  =  high density lipoproteins ; ldl  =  low density lipoproteins ; tga  =  triglycerides ; fpg  =  fasting plasma glucose ; homa1-ir  =  homoeostasis model assessment of insulin resistance 1 ; homa2-ir  =  homoeostasis model assessment of insulin resistance 2 ; quicki  =  quantitative insulin sensitivity check index we have found no inter - group differences for body composition analysis . detailed results for bia analysis are shown in table  2 . lean body mass was higher in men in the whole study sample ( 60.16.4 [ 95  % ci : 57.4 - 62.8 ] vs. 43.85.4 [ 95  % ci : 41.546.1 ] kg , t = 9.56 , p < 0.001 ) and in the clozapine group ( 59.65.7 [ 95  % ci : 56.063.3 ] vs. 45.37.0 [ 95  % ci : 40.9 - 49.8 ] kg , t = 5.48 , p < 0.001 ) .", "similarly , basal metabolic rate was higher in men in the whole study sample ( 1,707.7182.3 [ 95  % ci : 1,630.71,784.6 ] vs. 1,337.3138.4 [ 95  % ci : 1,278.8 - 1,395.7 ] kcal / day , t  = 7.92 , p < 0.001 ) and in the clozapine group ( 1,701.2138.2 [ 95  % ci : 1,613.41,789.0 ] vs. 1,362.7173.0 [ 95  % ci : 1,252.71,472.5 ] kcal / day , t = 5.29 , p < 0.001 ) . \n", "n  =  24control \n n  =  24total body fat [ kg]25.6    8.8(21.829.2)22.4    8.7(18.726.1 ) \n p  =  0.10 \n t  =  1.25total body fat [ % ] 32.6    8.4(29.136.2)28.9    7.1(25.831.8 ) \n p  =  0.06 \n t  =  1.67lean body mass [ kg]52.5    9.6(48.456.5)51.4    10.8(46.855.9 ) \n p  =  0.36 \n t  =  0.36lean body weight [ % ] 67.6    8.1(64.271.0)71.1    7.1(68.174.1 ) \n p  =  0.07 \n t  = 1.61total body water [ l]38.4    7.0(35.441.3)37.7    7.9(34.340.9)p  =  0.36 t  =  0.35total body water [ % ] 49.9    5.4(47.652.1)52.1    5.2(49.854.2 ) \n p  =  0.09 \n t  =  1.42basal metabolic rate [ kcal / day]1,532.0 ", "p  =  0.39 \n t  =  0.26data given as : mean    standard deviation ( 95  % ci ) results of body composition analysis data given as : mean    standard deviation ( 95  % ci ) there were no significant difference for fasting serum levels of agrp between the clozapine group and the control group ( 15.008.65 [ 95  % ci : 11.3418.65 ] vs. 15.336.82 [ 95  % ci : 12.4518.22 ] pg / ml , t  = 0.32 , p  = 0.37 ) , see fig .  1 .", "women had significantly lower levels of agrp in the whole study group ( 12.535.65 [ 95  % ci : 10.1414.92 ] vs. 17.808.66 [ 95  % ci : 14.1421.45 ] pg / ml , t  = 2.47 , p  = 0.009 ) and in the control group ( 12.905.77 [ 95  % ci : 9.2216.57 ] vs. 17.777.14 [ 95  % ci : 13.2322.31 ] pg / ml , t  = 1.82 , p  = 0.04 ) , but the difference was not significant for the clozapine group ( 12.175.75 [ 95  % ci : 8.5115.83 ] vs. 17.8210.28 [ 95  % ci : 11.28 - 24.36 ] pg / ml , t  = 1.64 , p  = 0.06).fig .", "1mean fasting agrp serum levels [ pg / ml ] in subjects on clozapine and in the control group ( p  = 0.37 ; horizontal bars indicate means ) .", "mean fasting agrp serum levels [ pg / ml ] in subjects on clozapine and in the control group ( p  = 0.37 ; horizontal bars indicate means ) .", "for the whole study group significant correlations of agrp levels were found for total body fat [ % ] ( r  = 0.34 , p  = 0.02 ) , lean body mass [ kg ] ( r  = 0.38 , p  = 0.006 ) , lean body mass [ % ] ( r  = 0.34 , p  = 0.02 ) , body water [ l ] ( r  = 0.38 , p  = 0.006 ) , body water [ % ] ( r  = 0.34 , p  = 0.02 ) and homocysteine levels ( r  = 0.29 , p  = 0.04 ) .", "for the clozapine group significant correlations of agrp levels were found for total body fat [ % ] ( r  = 0.48 , p  = 0.02 ) , basal metabolic rate ( r  = 0.42 , p  = 0.04 ) , lean body mass [ % ] ( r  = 0.49 , p  = 0.01 ) , body water [ % ] ( r  = 0.49 , p  = 0.01 ) . for the control group", "significant correlations of agrp levels were found only for basal metabolic rate ( r  = 0.42 , p  = 0.04 ) . in the clozapine group", "there were no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p  = 0.59 ) , idf - defined metabolic syndrome ( p  = 0.33 ) , smokers and non - smokers ( p  = 0.15 ) , subjects with bmi < 25  kg / m and with bmi 25  kg / m ( p  = 0.09 ) , subjects with and without impaired fasting glucose ( p  = 0.16 ) , subjects with and without abdominal obesity ( p  = 0.11 ) , subjects with and without dyslipidemia ( p  = 0.09 ) , and subjects with and without homa-1ir defined insulin resistance ( p  = 0.07 ) . in the control group there were no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p  =  0.20 ) , idf - defined metabolic syndrome ( p  =  0.44 ) , smokers and non - smokers ( p  =  0.35 ) , subjects with bmi < 25  kg / m and with bmi 25  kg / m ( p  = 0.12 ) , subjects with and without impaired fasting glucose ( p  = 0.36 ) , subjects with and without abdominal obesity ( p  = 0.10 ) , subjects with and without dyslipidemia ( p  = 0.37 ) , and subjects with and without homa-1ir defined insulin resistance ( p  = 0.13 ) . in the whole study group there was no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p  = 0.30 ) , idf - defined metabolic syndrome ( p  = 0.43 ) , smokers and non - smokers ( p  = 0.29 ) , subjects with bmi < 25  kg / m and with bmi 25  kg / m ( p  = 0.39 ) , subjects with and without impaired fasting glucose ( p  = 0.29 ) , subjects with and without dyslipidemia ( p  = 0.11 ) , and subjects with and without homa-1ir defined insulin resistance ( p  = 0.33 ) . there was a significant difference for agrp levels between subjects with or without abdominal obesity ( 13.466.50 [ 95  % ci : 9.0917.83 ] vs. 16.926.93 [ 95  % ci : 12.7321.11 ] pg / ml , t  = 1.80 , p  = 0.04 ) ."], ["the main objective of the present study was to find out whether there is a significant difference in fasting serum levels of agrp peptide between patients with schizophrenia on clozapine monotherapy and age- and sex - matched healthy controls .", "we have found the difference was not significant ( clozapine : 15.008.65 , control : 15.336.82  pg / ml , p  = 0.37 ) .", "we do not know pre - treatment values , so we can not determine whether and how this parameter changed during therapy with clozapine . to our knowledge ,", "no data are available on the effect of clozapine on blood agrp concentrations in humans .", "limited data are available for olanzapine , which has clinical and pharmacological properties similar to clozapine .", "results of these studies show that treatment with olanzapine does not affect agrp levels ( basoglu et al .", "however , in animal study it was demonstrated that administration of olanzapine or clozapine upregulates npy and agrp and downregulates proopiomelanocortin in the arcuate nucleus of the hypothalamus ( ferno et al .", "we do not know studies demonstrating correlation between blood levels of agrp and levels in the hypothalamus , but since it was showed recently that agrp neurons are unique among hypothalamic neurons by being the predominant neuronal subtype situated outside the blood ", "brain barrier ( olofsson et al . 2013 ) , we may assume that changes in hypothalamic expression are reflected in changes in blood levels .", "the finding that women had significantly lower levels of agrp in the whole study group ( 12.535.65 vs. 17.808.66  pg / ml , p  = 0.009 ) and in the control group ( 12.905.77 vs. 17.777.14  pg / ml , p  = 0.04 ) , but not in the clozapine group ( 12.175.75 vs. 17.8210.28  pg / ml , p  = 0.057 ) , might indicate that the testes are important source of agrp , as shown previously ( shutter et al .", "however , several more recent studies showed no difference between men and ( gavrila et al .", "2001 ) , therefore it may indicate that plasma agrp levels reflect central agrp concentrations .", "it was previously reported that total level of agrp is higher in obese subjects ( katsuki et al .", "2001 ) , although in another study it has been demonstrated that agrp levels were lower in normal weight group compared with women with anorexia , probably due to increased leptin levels ( moriya et al .", "we did not measure levels of leptin or acylated ghrelin , but we found in the same group of patients that there were no differences for desacyl ghrelin levels between patients taking clozapine and control group ( wysokiski et al .", "while there was a significant difference in bmi values between the clozapine group and the control group , we found no differences between both groups for fmi values . compared with bmi ,", "fmi is a better indicator of central obesity since it is much more sensitive to body fat content ( kelly et al .", "moreover , there were no differences for any of the bia results between both groups .", "these indicate that there was a substantial similarity between both groups in terms of body fat content , which is important considering that agrp levels may affect or result more from the amount of adipose tissue than from body weight per se .", "on the other hand , we have found negative correlations between agrp levels and total body fat ( r  = 0.34 and  0.48 in the whole study group and clozapine group , respectively ) .", "these may indicate the effect of leptin on the expression of agrp , but require further studies .", "additionally , there were positive correlations with lean body mass ( r  = 0.38 and 0.49 in the whole study group and clozapine group , respectively ) , body water ( which amount is negatively correlated with the amount of body fat : r  = 0.96 , p  <  0.001 ) ( r  = 0.34 and 0.49 in the whole study group and clozapine group , respectively ) and basal metabolic rate ( r  = 0.42 both in the clozapine group and control group ) , also being in line with the above - mentioned mechanism . in animal studies", "it has been found that high - fat diet leads to decreased expression of agrp in the hypothalamus , probably in the mechanism moderated by leptin ( staszkiewicz et al .", "we have no detailed data on diet patterns of our study subjects , but reports are consistent that patients with schizophrenia consume higher amounts of high - fat foods compared with healthy population ( dipasquale et al .", ". this might be one of the reasons why we have found no differences between our study groups . also ,", "level of physical activity ( usually lower in hospitalized patients ) might affect mechanisms regulating agrp levels .", "finally , another important factor is the effect of treatment mediated by other anabolic or catabolic neuropeptides . here , two most important peptides are leptin ( which decreases agrp levels ) and ghrelin ( which has opposite effect ) and have also been studied most extensively ( sentissi et al .", "2008 ) . since both leptin and ghrelin ( andrews 2011 ) may regulate activity of agrp / npy neurons in the arcuate nucleus , effects of antipsychotics on these neuropeptides must be taken into consideration . therefore", ", a study focused on interactions within this regulatory system would be very important for better understanding of this problem . based on our results", "however , the low number of study subjects limited the probability of finding inter - group differences due to lack of statistical power . due to the cross - sectional study design causal relationships", "can not be established and the effect of previous antipsychotic treatment can not be excluded .", "dual - energy x - ray absorptiometry ( dxa ) should be used to measure body composition and adipose tissue mass more accurately .", "due to complex structure of interactions between anabolic and catabolic neuropeptides , a longitudinal study comparing these interactions are crucial for understanding mechanisms of treatment - induced weight gain ."]]}
{"article_id": "PMC4330268", "article_text": ["it is widely accepted that mutation is the main causal factor for diverse tumorigenesis [ 1 , 2 ] .", "the recent development of next - generation sequencing ( ngs ) technologies has revolutionized the speed and throughput of dna sequencing , which facilitates the discovery of new driver mutations .", "for example , the arid1a gene is mutated in 83% of gastric cancers with microsatellite instability and the sf3b1 gene is somatically mutated in 9.7% of chronic lymphocytic leukemia patients [ 4 , 5 ] . in colorectal cancers , although the majority of recurrent mutations have been previously known , some novel mutations , such as the mutations in the sox9 and fam123b genes , were also identified by ngs technology , which may have implications for colorectal tumorigenesis . in melanoma , approximately 40% was found to have causal mutations affecting b - raf function .", "along with the well - known driver mutations , the novel mutations identified by ngs will be useful for predicting the prognosis and molecular characterization of cancers [ 8 , 9 ] .", "recently , a number of mutation calling tools , such as varscan , genome analysis toolkit , and mutect , have been developed to detect single - nucleotide variants ( snvs ) or indels from ngs data . however , snv calling from a single cancer case is not the final step for defining clinically meaningful mutations . to define the pathogenic snvs in human cancers ,", "a commonly used approach is to identify the phenotype - associated recurrent mutations in the study group . due to the data size burden of ngs output , profiling the mutations associated with a certain phenotype or prognosis in a study group by a researcher who does not have a bioinformatics background or facilities", "is very difficult . currently , there is no user - friendly tool supporting methods for the detection of recurrent or phenotype - specific mutations . in this study , in order to support the definition of recurrent mutations or phenotype - specific mutations from ngs data of a group of cancers with diverse phenotypes , we aimed to develop a user - friendly tool , named mutation arranger for defining phenotype - related snv ( map ) .", "map software was designed as a standalone program , compatible in microsoft windows environments with a user - friendly graphic interface , and compiled codes of map can be easily installed .", "both variant call format   and its annotation files , such as annovar output , can be used as input files for map .", "when a user loads the input file , called sample descriptor file ( gsf ) , map software displays the sample set information and filter options ( fig .", "1 ) . once data are loaded , the user can filter the data using the ' analysis options ' tab in map software based on annotation information . by using the sample set and analysis options", "summary metrics include the mutation status for each sample , total mutation frequency , p - values for differences between phenotype groups based on clinical information , genomic position , reference / observed sequence , and additional information , such as read depth by default . map software also displays more detailed information in summary metrics based on the user - selected ' analysis options . ' in the ' analysis result ' tab , map software provides the frequency- or p - value - based filter function .", "this functionality supports detection of the recurrent mutations in study subjects and identification of the phenotype - specific mutation that occurs significantly in a certain phenotypic subclass .", "then , map software represents the user - selected mutations graphically in the ' visualization ' tab . to sort the variants by genomic position , p - value , or frequency , users can use the sort function in the ' analysis result ' tab .", "details of the program download , installation , and analysis procedures are available in the user manual .", "the major steps of map are as follows . \n - generating mutation summary metrics in study subjects- determination of recurrent mutations by frequency- determination of phenotype - specific mutations by association test- graphical illustration of user - selected mutations \n  - generating mutation summary metrics in study subjects - determination of recurrent mutations by frequency - determination of phenotype - specific mutations by association test - graphical illustration of user - selected mutations", "mutation calling files from genome analysis toolkit   can be used directly as input files for ' sample descriptor . ' alternatively , manually prepared standard variant call format   is also available .", "annotation information files , such as annovar outputs , can be used as input files for sample descriptor .", "gsf is a comma - separated values text file that describes the input files , such as gatk output files and annovar output files , and phenotype information .", "when users load the input file , map software displays the sample set information and filter options in the ' source ' tab .", "if any file is not prepared properly , map software informs the user in the ' sample set files ' tab .", "after data are loaded , the user can filter the data using the ' analysis option ' tab based on annotation information .", "based on user - selected filters in the ' analysis options ' tab , summary metrics represent additional annotation information , such as gene name , function , known variant , and amino acid change .", "the additional annotations can be hidden using the snp filter option in the ' analysis option ' tab .", "these mutation metrics are exported into a comma - separated values text file for further investigation .", "in the ' analysis result ' tab , map software defines the recurrent mutations in study subjects by frequency .", "the frequency threshold can be determined arbitrarily by the user in the ' frequency filter ' function .", "map also defines the phenotype - specific mutations using the mutation metrics and phenotype information .", "phenotype - specific mutations that occur more frequently in one of two clinical conditions ( e.g. , drug - responder vs. non - responder ) are determined by chi - square test under the user - defined significance level .", "the graphic user interface is implemented in the software for users to easily handle large - scale data or access the analysis result ( fig . 2b and 2c ) .", "the recurrent or phenotype - specific mutations can also be demonstrated visually in heat - map style .", "for example , the left and right of the heat - map represent the sample name and mutational spectrum , respectively .", "the bottom of the heat - map represents the gene name by color for mutational spectrum . frequency or p - value plots", "are represented on top of the heat - map ( fig . 2b and 2c ) .", "map is a user - friendly program with multiple functions that supports the determination of recurrent or phenotype - specific mutations and provides graphic illustration images to the users .", "its operation environment , microsoft windows , enables more researchers who can not operate linux to define clinically meaningful mutations with ngs data from cancer cohorts ."], "abstract_text": ["<S> next - generation sequencing ( ngs ) is widely used to identify the causative mutations underlying diverse human diseases , including cancers , which can be useful for discovering the diagnostic and therapeutic targets . currently , a number of single - nucleotide variant ( snv)-calling algorithms are available ; however , there is no tool for visualizing the recurrent and phenotype - specific mutations for general researchers . in this study , in order to support defining the recurrent mutations or phenotype - specific mutations from ngs data of a group of cancers with diverse phenotypes , we aimed to develop a user - friendly tool , named mutation arranger for defining phenotype - related snv ( map ) . </S>", "<S> map is a user - friendly program with multiple functions that supports the determination of recurrent or phenotype - specific mutations and provides graphic illustration images to the users . </S>", "<S> its operation environment , the microsoft windows environment , enables more researchers who can not operate linux to define clinically meaningful mutations with ngs data from cancer cohorts . </S>"], "labels": null, "section_names": ["Introduction", "Description", "Prerequisites", "Generation of summary metrics ", "Determination of recurrent or phenotype-specific mutations ", "Visualization ", "Conclusion"], "sections": [["it is widely accepted that mutation is the main causal factor for diverse tumorigenesis [ 1 , 2 ] .", "the recent development of next - generation sequencing ( ngs ) technologies has revolutionized the speed and throughput of dna sequencing , which facilitates the discovery of new driver mutations .", "for example , the arid1a gene is mutated in 83% of gastric cancers with microsatellite instability and the sf3b1 gene is somatically mutated in 9.7% of chronic lymphocytic leukemia patients [ 4 , 5 ] . in colorectal cancers , although the majority of recurrent mutations have been previously known , some novel mutations , such as the mutations in the sox9 and fam123b genes , were also identified by ngs technology , which may have implications for colorectal tumorigenesis . in melanoma , approximately 40% was found to have causal mutations affecting b - raf function .", "along with the well - known driver mutations , the novel mutations identified by ngs will be useful for predicting the prognosis and molecular characterization of cancers [ 8 , 9 ] .", "recently , a number of mutation calling tools , such as varscan , genome analysis toolkit , and mutect , have been developed to detect single - nucleotide variants ( snvs ) or indels from ngs data . however , snv calling from a single cancer case is not the final step for defining clinically meaningful mutations . to define the pathogenic snvs in human cancers ,", "a commonly used approach is to identify the phenotype - associated recurrent mutations in the study group . due to the data size burden of ngs output , profiling the mutations associated with a certain phenotype or prognosis in a study group by a researcher who does not have a bioinformatics background or facilities", "is very difficult . currently , there is no user - friendly tool supporting methods for the detection of recurrent or phenotype - specific mutations . in this study , in order to support the definition of recurrent mutations or phenotype - specific mutations from ngs data of a group of cancers with diverse phenotypes , we aimed to develop a user - friendly tool , named mutation arranger for defining phenotype - related snv ( map ) ."], ["map software was designed as a standalone program , compatible in microsoft windows environments with a user - friendly graphic interface , and compiled codes of map can be easily installed .", "both variant call format   and its annotation files , such as annovar output , can be used as input files for map .", "when a user loads the input file , called sample descriptor file ( gsf ) , map software displays the sample set information and filter options ( fig .", "1 ) . once data are loaded , the user can filter the data using the ' analysis options ' tab in map software based on annotation information . by using the sample set and analysis options", "summary metrics include the mutation status for each sample , total mutation frequency , p - values for differences between phenotype groups based on clinical information , genomic position , reference / observed sequence , and additional information , such as read depth by default . map software also displays more detailed information in summary metrics based on the user - selected ' analysis options . ' in the ' analysis result ' tab , map software provides the frequency- or p - value - based filter function .", "this functionality supports detection of the recurrent mutations in study subjects and identification of the phenotype - specific mutation that occurs significantly in a certain phenotypic subclass .", "then , map software represents the user - selected mutations graphically in the ' visualization ' tab . to sort the variants by genomic position , p - value , or frequency , users can use the sort function in the ' analysis result ' tab .", "details of the program download , installation , and analysis procedures are available in the user manual .", "the major steps of map are as follows . \n - generating mutation summary metrics in study subjects- determination of recurrent mutations by frequency- determination of phenotype - specific mutations by association test- graphical illustration of user - selected mutations \n  - generating mutation summary metrics in study subjects - determination of recurrent mutations by frequency - determination of phenotype - specific mutations by association test - graphical illustration of user - selected mutations"], ["mutation calling files from genome analysis toolkit   can be used directly as input files for ' sample descriptor . ' alternatively , manually prepared standard variant call format   is also available .", "annotation information files , such as annovar outputs , can be used as input files for sample descriptor .", "gsf is a comma - separated values text file that describes the input files , such as gatk output files and annovar output files , and phenotype information ."], ["when users load the input file , map software displays the sample set information and filter options in the ' source ' tab .", "if any file is not prepared properly , map software informs the user in the ' sample set files ' tab .", "after data are loaded , the user can filter the data using the ' analysis option ' tab based on annotation information .", "based on user - selected filters in the ' analysis options ' tab , summary metrics represent additional annotation information , such as gene name , function , known variant , and amino acid change .", "the additional annotations can be hidden using the snp filter option in the ' analysis option ' tab .", "these mutation metrics are exported into a comma - separated values text file for further investigation ."], ["in the ' analysis result ' tab , map software defines the recurrent mutations in study subjects by frequency .", "the frequency threshold can be determined arbitrarily by the user in the ' frequency filter ' function .", "map also defines the phenotype - specific mutations using the mutation metrics and phenotype information .", "phenotype - specific mutations that occur more frequently in one of two clinical conditions ( e.g. , drug - responder vs. non - responder ) are determined by chi - square test under the user - defined significance level ."], ["the graphic user interface is implemented in the software for users to easily handle large - scale data or access the analysis result ( fig . 2b and 2c ) .", "the recurrent or phenotype - specific mutations can also be demonstrated visually in heat - map style .", "for example , the left and right of the heat - map represent the sample name and mutational spectrum , respectively .", "the bottom of the heat - map represents the gene name by color for mutational spectrum . frequency or p - value plots", "are represented on top of the heat - map ( fig . 2b and 2c ) ."], ["map is a user - friendly program with multiple functions that supports the determination of recurrent or phenotype - specific mutations and provides graphic illustration images to the users .", "its operation environment , microsoft windows , enables more researchers who can not operate linux to define clinically meaningful mutations with ngs data from cancer cohorts ."]]}
{"article_id": "PMC3357236", "article_text": ["echocardiographic measurements were performed in 19992001 ( baseline ) and 20072009 ( follow - up ) examinations of the hoorn study .", "the hoorn study is a population - based cohort study on glucose metabolism and cardiovascular diseases , previously described in detail ( 10 ) . at baseline , 290 individuals with normal glucose metabolism ( ngm ) , 187 with impaired glucose metabolism ( igm ) , and 345 with type 2 diabetes participated . at follow - up ,", "167 ( 20% ) individuals were excluded a priori because of incomplete baseline data ( n = 26 ) , mental incompetence to participate ( n = 12 ) , or death ( n = 129 ) . of the remaining 655 individuals , 441 ( 67% ) participated .", "thirteen ( 3% ) of those were excluded in the present analyses because of missing echocardiography data at follow - up . to restrict the study population to individuals at risk of developing lv", "systolic and/or diastolic dysfunction , we also excluded 34 individuals ( 8% ) with an lv ejection fraction < 50% or an la volume index > 40 ml / m at baseline .", "the local ethics committee approved the study , and written informed consent was obtained from all participants .", "echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system ( 24 mhz transducer ) according to a standardized protocol consisting of two - dimensional , m - mode , and pulsed wave doppler assessments as previously described ( 13 ) . at follow - up , this protocol was expanded with tissue doppler assessments of mitral annular velocities .", "a set of three markers of lv diastolic dysfunction was determined : la volume index , lv mass indexed to height to the power of 2.7 ( 14 ) , and the ratio of early ( e ) mitral valve flow to early ( e ) diastolic lengthening velocity ( e / e ) using tissue and pulsed wave doppler assessments .", "incident heart failure was considered present if signs and symptoms were accompanied by lv systolic ( lv ejection fraction < 35% ) and/or diastolic dysfunction ( 15 ) .", "presence of incident lv diastolic dysfunction ( grade 2 or 3 ) was assessed according to international recommendations ( 16 ) . in the present analyses ,", "we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid , brachial , and femoral arteries to assess stiffness of the arterial wall ( 10 ) .", "additionally , systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure ( 17 ) .", "augmentation index was determined using applanation tonometry . in a subset of the study population ,", "carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time , adjusted for height in the statistical analyses .", "reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described ( 10 ) . at follow - up ,", "intraobserver intersession coefficients of variation in 10 individuals ( aged 70.3  3.6 years ) were comparable : 4.2 , 13.1 , 4.1 , and 6.1% for lv ejection fraction , la volume , lv mass , and e / e , respectively .", "for assessment of glucose status , all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm , igm ( impaired fasting glucose and/or impaired glucose tolerance ) , and type 2 diabetes groups according to the 1999 world health organization criteria ( 18 ) .", "health status , medical history , medication use , and smoking habits were assessed by questionnaires ( 10 ) .", "waist circumference , blood pressures ( systolic , mean arterial , and pulse pressures ) , and levels of cholesterol , insulin , hba1c , and c - reactive protein were determined as previously described ( 10 ) .", "descriptive statistics are presented as means  sd or , in the case of a skewed distribution , as median ( interquartile range ) .", "the f test for anova in the case of continuous variables or  tests in the case of proportions were performed to test for differences between groups of glucose status . in the case of skewed distributions ,", "the above tests were done on log - transformed data . for every variable that had >", "10% missing data , the number missing is presented in the legends of tables 13 .", "linear regression analyses , adjusted for age and sex , were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up .", "regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure .", "for investigation of changes in markers , lv systolic and diastolic dysfunction according to glucose status or arterial stiffness , we adjusted all models for baseline values of these markers ( models assessing e / e adjusted for baseline la volume index ) .", "lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors , like overweight , hypertension , insulin resistance , or previous cardiovascular events ( 19 ) .", "therefore , we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models ( supplementary data ) .", "a secondary purpose of these analyses was to investigate potential residual confounding by these factors .", "product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease .", "associations of arterial distensibility coefficients , systemic arterial compliance , and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness .", "additional analyses , adjusted for age and sex , were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function .", "p values < 0.05 , or < 0.10 in case of interaction analyses , were considered statistically significant .", "statistical analyses were performed with spss for windows ( version 15.0 ; spss , chicago , il ) .", "echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system ( 24 mhz transducer ) according to a standardized protocol consisting of two - dimensional , m - mode , and pulsed wave doppler assessments as previously described ( 13 ) . at follow - up , this protocol was expanded with tissue doppler assessments of mitral annular velocities .", "a set of three markers of lv diastolic dysfunction was determined : la volume index , lv mass indexed to height to the power of 2.7 ( 14 ) , and the ratio of early ( e ) mitral valve flow to early ( e ) diastolic lengthening velocity ( e / e ) using tissue and pulsed wave doppler assessments .", "incident heart failure was considered present if signs and symptoms were accompanied by lv systolic ( lv ejection fraction < 35% ) and/or diastolic dysfunction ( 15 ) .", "presence of incident lv diastolic dysfunction ( grade 2 or 3 ) was assessed according to international recommendations ( 16 ) .", "in the present analyses , we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid , brachial , and femoral arteries to assess stiffness of the arterial wall ( 10 ) .", "additionally , systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure ( 17 ) .", "augmentation index was determined using applanation tonometry . in a subset of the study population ,", "carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time , adjusted for height in the statistical analyses .", "reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described ( 10 ) . at follow - up ,", "intraobserver intersession coefficients of variation in 10 individuals ( aged 70.3  3.6 years ) were comparable : 4.2 , 13.1 , 4.1 , and 6.1% for lv ejection fraction , la volume , lv mass , and e / e , respectively .", "for assessment of glucose status , all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm , igm ( impaired fasting glucose and/or impaired glucose tolerance ) , and type 2 diabetes groups according to the 1999 world health organization criteria ( 18 ) .", "health status , medical history , medication use , and smoking habits were assessed by questionnaires ( 10 ) .", "waist circumference , blood pressures ( systolic , mean arterial , and pulse pressures ) , and levels of cholesterol , insulin , hba1c , and c - reactive protein were determined as previously described ( 10 ) .", "descriptive statistics are presented as means  sd or , in the case of a skewed distribution , as median ( interquartile range ) . the f test for anova in the case of continuous variables or  tests in the case of proportions", "were performed to test for differences between groups of glucose status . in the case of skewed distributions ,", "the above tests were done on log - transformed data . for every variable that had >", "10% missing data , the number missing is presented in the legends of tables 13 .", "linear regression analyses , adjusted for age and sex , were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up .", "regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure . for investigation of changes in markers , lv systolic and diastolic dysfunction according to glucose status or arterial stiffness , we adjusted all models for baseline values of these markers ( models assessing e / e adjusted for baseline la volume index ) .", "lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors , like overweight , hypertension , insulin resistance , or previous cardiovascular events ( 19 ) .", "therefore , we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models ( supplementary data ) .", "a secondary purpose of these analyses was to investigate potential residual confounding by these factors .", "product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease .", "associations of arterial distensibility coefficients , systemic arterial compliance , and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness .", "additional analyses , adjusted for age and sex , were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function .", "p values < 0.05 , or < 0.10 in case of interaction analyses , were considered statistically significant .", "statistical analyses were performed with spss for windows ( version 15.0 ; spss , chicago , il ) .", "a total of 394 individuals in the age range of 5087 years were included in the present analyses , 87 ( 22% ) of whom had igm and 128 ( 32% ) of whom had type 2 diabetes . compared with individuals who were not included ,", "these individuals were younger and less likely to have type 2 diabetes and had more favorable levels of arterial stiffness and lv systolic and diastolic dysfunction at baseline ( data not shown ) .", "individuals with type 2 diabetes were younger , had higher bmi and blood pressure , and had greater arterial stiffness and more severe lv systolic and diastolic dysfunction at baseline compared with individuals without type 2 diabetes ( table 1 ) .", "heart failure incidence was not significantly different : 23 ( 21% ) in type 2 diabetes versus 15 ( 19% ) in igm and 24 ( 15% ) in ngm .", "incidence of lv diastolic dysfunction grade 2 or 3 was higher with deteriorating glucose status : 88 ( 72% ) in type 2 diabetes versus 52 ( 61% ) in igm and 55 ( 31% ) in ngm .", "characteristics according to glucose status the presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction : lv ejection fraction , la volume index , lv mass index , and e / e ( table 2 ) .", "after adjustment for age and sex , lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm .", "individuals with type 2 diabetes had a 3.71 ml / m higher la volume index , 5.86 g / m higher lv mass index , and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction , but type 2 diabetes was still significantly associated with a higher lv mass index ( 3.41 g / m ) and e / e ( 1.43 ) .", "individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index , independent of baseline lv mass index .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status ( ngm = reference ) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure , lipid levels , renal function , c - reactive protein , and coronary artery disease ( supplementary data table a ) .", "adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index .", "associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference .", "interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd . a longer diabetes duration ( per year )", "was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function .", "greater stiffness of carotid , brachial , and femoral arteries , measured as lower distensibility coefficients , was associated with higher levels of la volume index , lv mass index , and e / e ( table 3 ) .", "after adjustment for age and sex , every 10  kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index .", "every 10  kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10  kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index , a 0.91 g / m higher lv mass index , and a 0.09 higher e / e. these associations were independent of mean arterial pressure , except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction , 10  kpa lower distensibility coefficients of carotid , brachial , and femoral arteries remained significantly associated with a 0.29 , 0.38 , and 0.73 ml / m higher la volume index , respectively .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure ( supplementary data table a ) .", "lower compliance coefficients of brachial and femoral arteries , as well as a higher young elastic modulus or pulse pressure , were similarly associated with more severe lv diastolic dysfunction ( supplementary data table b ) .", "a higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction : lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index .", "carotid - femoral transit time was measured in a subset of 178 individuals , and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time , though not significantly ( p = 0.13 ) .", "there was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction ( p values for interaction > 0.10 ) .", "figure 1 shows the associations of glucose status and sd scores of arterial stiffness ( measured as femoral artery distensibility coefficients ) with sd scores for all four markers of lv systolic and diastolic dysfunction , adjusted for age and sex .", "the right half of each histogram shows that  values hardly changed if glucose status and arterial stiffness were combined in one model .", "for instance , the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex .", "after subsequent adjustment for arterial stiffness , this  value remained similar , despite a loss of statistical significance : 0.25 ( p = 0.08 ) .", "associations of glucose status with la volume index , lv mass index , and e / e hardly changed either after adjustment for arterial stiffness .", "furthermore , each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex .", "after subsequent adjustment for glucose status , this  value was 0.12 ( p = 0.02 ) .", "associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status , despite a loss of statistical significance .", "standardized associations of impaired glucose metabolism ( black bars ) , type 2 diabetes ( light gray bars ) , and lower femoral artery distensibility coefficients ( dark gray bars ) with markers of lv systolic and diastolic dysfunction , adjusted for age and sex ( left half of each histogram ) and , additionally , for each other ( right half of each histogram ) . *", "there was no significant effect modification by sex , use of ace inhibitors , or lipid - lowering , glucose - lowering , or blood pressure  lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction ( p > 0.10 , data not shown ) .", "the presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction : lv ejection fraction , la volume index , lv mass index , and e / e ( table 2 ) .", "after adjustment for age and sex , lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm .", "individuals with type 2 diabetes had a 3.71 ml / m higher la volume index , 5.86 g / m higher lv mass index , and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction , but type 2 diabetes was still significantly associated with a higher lv mass index ( 3.41 g / m ) and e / e ( 1.43 ) . individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index , independent of baseline lv mass index .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status ( ngm = reference ) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure , lipid levels , renal function , c - reactive protein , and coronary artery disease ( supplementary data table a ) .", "adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index . associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference .", "interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd .", "a longer diabetes duration ( per year ) was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function .", "greater stiffness of carotid , brachial , and femoral arteries , measured as lower distensibility coefficients , was associated with higher levels of la volume index , lv mass index , and e / e ( table 3 ) .", "after adjustment for age and sex , every 10  kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index .", "every 10  kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10  kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index , a 0.91 g / m higher lv mass index , and a 0.09 higher e / e. these associations were independent of mean arterial pressure , except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction , 10  kpa lower distensibility coefficients of carotid , brachial , and femoral arteries remained significantly associated with a 0.29 , 0.38 , and 0.73 ml / m higher la volume index , respectively .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure ( supplementary data table a ) .", "lower compliance coefficients of brachial and femoral arteries , as well as a higher young elastic modulus or pulse pressure , were similarly associated with more severe lv diastolic dysfunction ( supplementary data table b ) .", "a higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction : lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index .", "carotid - femoral transit time was measured in a subset of 178 individuals , and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time , though not significantly ( p = 0.13 ) .", "there was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction ( p values for interaction > 0.10 ) .", "figure 1 shows the associations of glucose status and sd scores of arterial stiffness ( measured as femoral artery distensibility coefficients ) with sd scores for all four markers of lv systolic and diastolic dysfunction , adjusted for age and sex .", "the right half of each histogram shows that  values hardly changed if glucose status and arterial stiffness were combined in one model .", "for instance , the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex .", "after subsequent adjustment for arterial stiffness , this  value remained similar , despite a loss of statistical significance : 0.25 ( p = 0.08 ) .", "associations of glucose status with la volume index , lv mass index , and e / e hardly changed either after adjustment for arterial stiffness . furthermore , each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex .", "after subsequent adjustment for glucose status , this  value was 0.12 ( p = 0.02 ) .", "associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status , despite a loss of statistical significance .", "standardized associations of impaired glucose metabolism ( black bars ) , type 2 diabetes ( light gray bars ) , and lower femoral artery distensibility coefficients ( dark gray bars ) with markers of lv systolic and diastolic dysfunction , adjusted for age and sex ( left half of each histogram ) and , additionally , for each other ( right half of each histogram ) .", "there was no significant effect modification by sex , use of ace inhibitors , or lipid - lowering , glucose - lowering , or blood pressure  lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction ( p > 0.10 , data not shown ) .", "this study shows that both glucose status and arterial distensibility coefficients were prospectively associated with more severe lv diastolic dysfunction .", "furthermore , associations of glucose status and arterial stiffness with lv diastolic dysfunction were largely independent of each other and indicated a deterioration of lv diastolic dysfunction compared with baseline .", "strengths include the population - based design and comprehensive assessment of glucose status , arterial stiffness , and lv systolic and diastolic dysfunction .", "these data provide a unique insight into many different aspects of arterial stiffness and their influence on lv systolic and diastolic dysfunction .", "lv diastolic dysfunction at baseline was therefore based on la volume index only , and this might have been subject to some misclassification .", "furthermore , differences between attendees and nonattendees suggest that a  healthy cohort bias  may have occurred in this study .", "we also had missing data for some echocardiographic markers , predominantly in individuals with high bmi .", "our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction ( 20,21 ) . the type 2 diabetes  associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline .", "these results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes .", "there was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well , but this was not statistically significant .", "individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up .", "peripheral markers of arterial stiffness , like arterial distensibility and compliance coefficients , and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction .", "associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure , since adjustment for mean arterial pressure only resulted in a small reduction of the associations .", "associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index .", "this might imply that 1 ) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2 ) stiffening of arteries and lv walls occurs simultaneously .", "the first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion ( 6 ) .", "the second might be due to coinciding structural changes in arterial and lv walls ( 9 ) . in the current study", ", wave reflections seemed especially harmful to lv systolic function , since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction , followed by carotid - femoral transit time .", "lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness .", "this might support the second theory : that stiffening of arteries and lv walls occurs simultaneously .", "type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study .", "more severe lv systolic and diastolic dysfunction in type 2 diabetes therefore can not be explained or can or just partly be explained by increased arterial stiffness", ". other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension ( 11,2224 ) .", "this might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c .", "overweight and obesity are known to be associated with a higher lv mass index , and indeed , waist circumference seemed to play a role in type 2 diabetes  related higher lv mass index ( 25 ) .", "lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline , the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining .", "to conclude , the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction .", "it is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways , since these associations were independent of each other .", "a better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure .", "our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction ( 20,21 ) . the type 2 diabetes  associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline .", "these results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes .", "there was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well , but this was not statistically significant .", "individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up .", "peripheral markers of arterial stiffness , like arterial distensibility and compliance coefficients , and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction .", "associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure , since adjustment for mean arterial pressure only resulted in a small reduction of the associations .", "associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index .", "this might imply that 1 ) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2 ) stiffening of arteries and lv walls occurs simultaneously .", "the first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion ( 6 ) .", "the second might be due to coinciding structural changes in arterial and lv walls ( 9 ) . in the current study", ", wave reflections seemed especially harmful to lv systolic function , since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction , followed by carotid - femoral transit time .", "lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness .", "this might support the second theory : that stiffening of arteries and lv walls occurs simultaneously .", "type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study .", "more severe lv systolic and diastolic dysfunction in type 2 diabetes therefore can not be explained or can or just partly be explained by increased arterial stiffness", ". other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension ( 11,2224 ) .", "this might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c .", "overweight and obesity are known to be associated with a higher lv mass index , and indeed , waist circumference seemed to play a role in type 2 diabetes  related higher lv mass index ( 25 ) .", "lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline , the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining .", "to conclude , the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction .", "it is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways , since these associations were independent of each other .", "a better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure ."], "abstract_text": ["<S> objectiveto investigate relative contributions of glucose status and arterial stiffness to markers of left ventricular ( lv ) systolic and diastolic dysfunction after 8 years of follow-up.research design and methodsin the population - based prospective hoorn study , 394 individuals with preserved lv systolic and diastolic function participated , of whom 87 had impaired glucose metabolism and 128 had type 2 diabetes . </S>", "<S> measurements including arterial ultrasound and echocardiography were performed according to standardized protocols.resultsthe presence of type 2 diabetes was associated with more severe lv systolic and diastolic dysfunction 8 years later : lv ejection fraction was 2.98% ( 95% ci 0.465.51 ) lower , and left atrial ( la ) volume index , lv mass index , and tissue doppler - derived e / e were 3.71 ml / m2 ( 1.206.22 ) , 5.86 g / m2.7 ( 2.948.78 ) , and 1.64 units ( 0.952.33 ) higher , respectively . </S>", "<S> furthermore , presence of impaired glucose metabolism or type 2 diabetes was associated with 8-year increases in lv mass index . </S>", "<S> more arterial stiffness ( measured as a lower distensibility ) was associated with lv diastolic dysfunction 8 years later : la volume index , lv mass index , and e / e at follow - up were higher . </S>", "<S> subsequent adjustments for baseline mean arterial pressure and/or lv diastolic dysfunction did not eliminate these associations . </S>", "<S> associations of type 2 diabetes and arterial stiffness with markers of lv diastolic dysfunction were largely independent of each other.conclusionsboth glucose status and arterial distensibility are independently associated with more severe lv diastolic dysfunction 8 years later and with deterioration of lv diastolic dysfunction . </S>", "<S> therefore , type 2 diabetes and arterial stiffness may relate to lv diastolic dysfunction through different pathways . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Echocardiography", "Arterial stiffness", "Reproducibility", "Other baseline measurements", "Statistical analysis", "RESULTS", "Glucose status", "Arterial stiffness", "Combined influence of glucose status and arterial stiffness", "CONCLUSIONS", "Glucose status", "Arterial stiffness", "Combined influence of glucose status and arterial stiffness"], "sections": [["echocardiographic measurements were performed in 19992001 ( baseline ) and 20072009 ( follow - up ) examinations of the hoorn study .", "the hoorn study is a population - based cohort study on glucose metabolism and cardiovascular diseases , previously described in detail ( 10 ) . at baseline , 290 individuals with normal glucose metabolism ( ngm ) , 187 with impaired glucose metabolism ( igm ) , and 345 with type 2 diabetes participated . at follow - up ,", "167 ( 20% ) individuals were excluded a priori because of incomplete baseline data ( n = 26 ) , mental incompetence to participate ( n = 12 ) , or death ( n = 129 ) . of the remaining 655 individuals , 441 ( 67% ) participated .", "thirteen ( 3% ) of those were excluded in the present analyses because of missing echocardiography data at follow - up . to restrict the study population to individuals at risk of developing lv", "systolic and/or diastolic dysfunction , we also excluded 34 individuals ( 8% ) with an lv ejection fraction < 50% or an la volume index > 40 ml / m at baseline .", "the local ethics committee approved the study , and written informed consent was obtained from all participants .", "echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system ( 24 mhz transducer ) according to a standardized protocol consisting of two - dimensional , m - mode , and pulsed wave doppler assessments as previously described ( 13 ) . at follow - up , this protocol was expanded with tissue doppler assessments of mitral annular velocities .", "a set of three markers of lv diastolic dysfunction was determined : la volume index , lv mass indexed to height to the power of 2.7 ( 14 ) , and the ratio of early ( e ) mitral valve flow to early ( e ) diastolic lengthening velocity ( e / e ) using tissue and pulsed wave doppler assessments .", "incident heart failure was considered present if signs and symptoms were accompanied by lv systolic ( lv ejection fraction < 35% ) and/or diastolic dysfunction ( 15 ) .", "presence of incident lv diastolic dysfunction ( grade 2 or 3 ) was assessed according to international recommendations ( 16 ) . in the present analyses ,", "we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid , brachial , and femoral arteries to assess stiffness of the arterial wall ( 10 ) .", "additionally , systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure ( 17 ) .", "augmentation index was determined using applanation tonometry . in a subset of the study population ,", "carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time , adjusted for height in the statistical analyses .", "reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described ( 10 ) . at follow - up ,", "intraobserver intersession coefficients of variation in 10 individuals ( aged 70.3  3.6 years ) were comparable : 4.2 , 13.1 , 4.1 , and 6.1% for lv ejection fraction , la volume , lv mass , and e / e , respectively .", "for assessment of glucose status , all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm , igm ( impaired fasting glucose and/or impaired glucose tolerance ) , and type 2 diabetes groups according to the 1999 world health organization criteria ( 18 ) .", "health status , medical history , medication use , and smoking habits were assessed by questionnaires ( 10 ) .", "waist circumference , blood pressures ( systolic , mean arterial , and pulse pressures ) , and levels of cholesterol , insulin , hba1c , and c - reactive protein were determined as previously described ( 10 ) .", "descriptive statistics are presented as means  sd or , in the case of a skewed distribution , as median ( interquartile range ) .", "the f test for anova in the case of continuous variables or  tests in the case of proportions were performed to test for differences between groups of glucose status . in the case of skewed distributions ,", "the above tests were done on log - transformed data . for every variable that had >", "10% missing data , the number missing is presented in the legends of tables 13 .", "linear regression analyses , adjusted for age and sex , were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up .", "regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure .", "for investigation of changes in markers , lv systolic and diastolic dysfunction according to glucose status or arterial stiffness , we adjusted all models for baseline values of these markers ( models assessing e / e adjusted for baseline la volume index ) .", "lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors , like overweight , hypertension , insulin resistance , or previous cardiovascular events ( 19 ) .", "therefore , we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models ( supplementary data ) .", "a secondary purpose of these analyses was to investigate potential residual confounding by these factors .", "product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease .", "associations of arterial distensibility coefficients , systemic arterial compliance , and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness .", "additional analyses , adjusted for age and sex , were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function .", "p values < 0.05 , or < 0.10 in case of interaction analyses , were considered statistically significant .", "statistical analyses were performed with spss for windows ( version 15.0 ; spss , chicago , il ) ."], ["echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system ( 24 mhz transducer ) according to a standardized protocol consisting of two - dimensional , m - mode , and pulsed wave doppler assessments as previously described ( 13 ) . at follow - up , this protocol was expanded with tissue doppler assessments of mitral annular velocities .", "a set of three markers of lv diastolic dysfunction was determined : la volume index , lv mass indexed to height to the power of 2.7 ( 14 ) , and the ratio of early ( e ) mitral valve flow to early ( e ) diastolic lengthening velocity ( e / e ) using tissue and pulsed wave doppler assessments .", "incident heart failure was considered present if signs and symptoms were accompanied by lv systolic ( lv ejection fraction < 35% ) and/or diastolic dysfunction ( 15 ) .", "presence of incident lv diastolic dysfunction ( grade 2 or 3 ) was assessed according to international recommendations ( 16 ) ."], ["in the present analyses , we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid , brachial , and femoral arteries to assess stiffness of the arterial wall ( 10 ) .", "additionally , systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure ( 17 ) .", "augmentation index was determined using applanation tonometry . in a subset of the study population ,", "carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time , adjusted for height in the statistical analyses ."], ["reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described ( 10 ) . at follow - up ,", "intraobserver intersession coefficients of variation in 10 individuals ( aged 70.3  3.6 years ) were comparable : 4.2 , 13.1 , 4.1 , and 6.1% for lv ejection fraction , la volume , lv mass , and e / e , respectively ."], ["for assessment of glucose status , all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm , igm ( impaired fasting glucose and/or impaired glucose tolerance ) , and type 2 diabetes groups according to the 1999 world health organization criteria ( 18 ) .", "health status , medical history , medication use , and smoking habits were assessed by questionnaires ( 10 ) .", "waist circumference , blood pressures ( systolic , mean arterial , and pulse pressures ) , and levels of cholesterol , insulin , hba1c , and c - reactive protein were determined as previously described ( 10 ) ."], ["descriptive statistics are presented as means  sd or , in the case of a skewed distribution , as median ( interquartile range ) . the f test for anova in the case of continuous variables or  tests in the case of proportions", "were performed to test for differences between groups of glucose status . in the case of skewed distributions ,", "the above tests were done on log - transformed data . for every variable that had >", "10% missing data , the number missing is presented in the legends of tables 13 .", "linear regression analyses , adjusted for age and sex , were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up .", "regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure . for investigation of changes in markers , lv systolic and diastolic dysfunction according to glucose status or arterial stiffness , we adjusted all models for baseline values of these markers ( models assessing e / e adjusted for baseline la volume index ) .", "lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors , like overweight , hypertension , insulin resistance , or previous cardiovascular events ( 19 ) .", "therefore , we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models ( supplementary data ) .", "a secondary purpose of these analyses was to investigate potential residual confounding by these factors .", "product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease .", "associations of arterial distensibility coefficients , systemic arterial compliance , and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness .", "additional analyses , adjusted for age and sex , were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function .", "p values < 0.05 , or < 0.10 in case of interaction analyses , were considered statistically significant .", "statistical analyses were performed with spss for windows ( version 15.0 ; spss , chicago , il ) ."], ["a total of 394 individuals in the age range of 5087 years were included in the present analyses , 87 ( 22% ) of whom had igm and 128 ( 32% ) of whom had type 2 diabetes . compared with individuals who were not included ,", "these individuals were younger and less likely to have type 2 diabetes and had more favorable levels of arterial stiffness and lv systolic and diastolic dysfunction at baseline ( data not shown ) .", "individuals with type 2 diabetes were younger , had higher bmi and blood pressure , and had greater arterial stiffness and more severe lv systolic and diastolic dysfunction at baseline compared with individuals without type 2 diabetes ( table 1 ) .", "heart failure incidence was not significantly different : 23 ( 21% ) in type 2 diabetes versus 15 ( 19% ) in igm and 24 ( 15% ) in ngm .", "incidence of lv diastolic dysfunction grade 2 or 3 was higher with deteriorating glucose status : 88 ( 72% ) in type 2 diabetes versus 52 ( 61% ) in igm and 55 ( 31% ) in ngm .", "characteristics according to glucose status the presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction : lv ejection fraction , la volume index , lv mass index , and e / e ( table 2 ) .", "after adjustment for age and sex , lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm .", "individuals with type 2 diabetes had a 3.71 ml / m higher la volume index , 5.86 g / m higher lv mass index , and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction , but type 2 diabetes was still significantly associated with a higher lv mass index ( 3.41 g / m ) and e / e ( 1.43 ) .", "individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index , independent of baseline lv mass index .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status ( ngm = reference ) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure , lipid levels , renal function , c - reactive protein , and coronary artery disease ( supplementary data table a ) .", "adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index .", "associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference .", "interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd . a longer diabetes duration ( per year )", "was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function .", "greater stiffness of carotid , brachial , and femoral arteries , measured as lower distensibility coefficients , was associated with higher levels of la volume index , lv mass index , and e / e ( table 3 ) .", "after adjustment for age and sex , every 10  kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index .", "every 10  kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10  kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index , a 0.91 g / m higher lv mass index , and a 0.09 higher e / e. these associations were independent of mean arterial pressure , except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction , 10  kpa lower distensibility coefficients of carotid , brachial , and femoral arteries remained significantly associated with a 0.29 , 0.38 , and 0.73 ml / m higher la volume index , respectively .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure ( supplementary data table a ) .", "lower compliance coefficients of brachial and femoral arteries , as well as a higher young elastic modulus or pulse pressure , were similarly associated with more severe lv diastolic dysfunction ( supplementary data table b ) .", "a higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction : lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index .", "carotid - femoral transit time was measured in a subset of 178 individuals , and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time , though not significantly ( p = 0.13 ) .", "there was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction ( p values for interaction > 0.10 ) .", "figure 1 shows the associations of glucose status and sd scores of arterial stiffness ( measured as femoral artery distensibility coefficients ) with sd scores for all four markers of lv systolic and diastolic dysfunction , adjusted for age and sex .", "the right half of each histogram shows that  values hardly changed if glucose status and arterial stiffness were combined in one model .", "for instance , the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex .", "after subsequent adjustment for arterial stiffness , this  value remained similar , despite a loss of statistical significance : 0.25 ( p = 0.08 ) .", "associations of glucose status with la volume index , lv mass index , and e / e hardly changed either after adjustment for arterial stiffness .", "furthermore , each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex .", "after subsequent adjustment for glucose status , this  value was 0.12 ( p = 0.02 ) .", "associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status , despite a loss of statistical significance .", "standardized associations of impaired glucose metabolism ( black bars ) , type 2 diabetes ( light gray bars ) , and lower femoral artery distensibility coefficients ( dark gray bars ) with markers of lv systolic and diastolic dysfunction , adjusted for age and sex ( left half of each histogram ) and , additionally , for each other ( right half of each histogram ) . *", "there was no significant effect modification by sex , use of ace inhibitors , or lipid - lowering , glucose - lowering , or blood pressure  lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction ( p > 0.10 , data not shown ) ."], ["the presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction : lv ejection fraction , la volume index , lv mass index , and e / e ( table 2 ) .", "after adjustment for age and sex , lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm .", "individuals with type 2 diabetes had a 3.71 ml / m higher la volume index , 5.86 g / m higher lv mass index , and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction , but type 2 diabetes was still significantly associated with a higher lv mass index ( 3.41 g / m ) and e / e ( 1.43 ) . individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index , independent of baseline lv mass index .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status ( ngm = reference ) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure , lipid levels , renal function , c - reactive protein , and coronary artery disease ( supplementary data table a ) .", "adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index . associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference .", "interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd .", "a longer diabetes duration ( per year ) was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function ."], ["greater stiffness of carotid , brachial , and femoral arteries , measured as lower distensibility coefficients , was associated with higher levels of la volume index , lv mass index , and e / e ( table 3 ) .", "after adjustment for age and sex , every 10  kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index .", "every 10  kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10  kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index , a 0.91 g / m higher lv mass index , and a 0.09 higher e / e. these associations were independent of mean arterial pressure , except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction , 10  kpa lower distensibility coefficients of carotid , brachial , and femoral arteries remained significantly associated with a 0.29 , 0.38 , and 0.73 ml / m higher la volume index , respectively .", "markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure ( supplementary data table a ) .", "lower compliance coefficients of brachial and femoral arteries , as well as a higher young elastic modulus or pulse pressure , were similarly associated with more severe lv diastolic dysfunction ( supplementary data table b ) .", "a higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction : lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index .", "carotid - femoral transit time was measured in a subset of 178 individuals , and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time , though not significantly ( p = 0.13 ) ."], ["there was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction ( p values for interaction > 0.10 ) .", "figure 1 shows the associations of glucose status and sd scores of arterial stiffness ( measured as femoral artery distensibility coefficients ) with sd scores for all four markers of lv systolic and diastolic dysfunction , adjusted for age and sex .", "the right half of each histogram shows that  values hardly changed if glucose status and arterial stiffness were combined in one model .", "for instance , the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex .", "after subsequent adjustment for arterial stiffness , this  value remained similar , despite a loss of statistical significance : 0.25 ( p = 0.08 ) .", "associations of glucose status with la volume index , lv mass index , and e / e hardly changed either after adjustment for arterial stiffness . furthermore , each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex .", "after subsequent adjustment for glucose status , this  value was 0.12 ( p = 0.02 ) .", "associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status , despite a loss of statistical significance .", "standardized associations of impaired glucose metabolism ( black bars ) , type 2 diabetes ( light gray bars ) , and lower femoral artery distensibility coefficients ( dark gray bars ) with markers of lv systolic and diastolic dysfunction , adjusted for age and sex ( left half of each histogram ) and , additionally , for each other ( right half of each histogram ) .", "there was no significant effect modification by sex , use of ace inhibitors , or lipid - lowering , glucose - lowering , or blood pressure  lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction ( p > 0.10 , data not shown ) ."], ["this study shows that both glucose status and arterial distensibility coefficients were prospectively associated with more severe lv diastolic dysfunction .", "furthermore , associations of glucose status and arterial stiffness with lv diastolic dysfunction were largely independent of each other and indicated a deterioration of lv diastolic dysfunction compared with baseline .", "strengths include the population - based design and comprehensive assessment of glucose status , arterial stiffness , and lv systolic and diastolic dysfunction .", "these data provide a unique insight into many different aspects of arterial stiffness and their influence on lv systolic and diastolic dysfunction .", "lv diastolic dysfunction at baseline was therefore based on la volume index only , and this might have been subject to some misclassification .", "furthermore , differences between attendees and nonattendees suggest that a  healthy cohort bias  may have occurred in this study .", "we also had missing data for some echocardiographic markers , predominantly in individuals with high bmi .", "our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction ( 20,21 ) . the type 2 diabetes  associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline .", "these results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes .", "there was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well , but this was not statistically significant .", "individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up .", "peripheral markers of arterial stiffness , like arterial distensibility and compliance coefficients , and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction .", "associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure , since adjustment for mean arterial pressure only resulted in a small reduction of the associations .", "associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index .", "this might imply that 1 ) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2 ) stiffening of arteries and lv walls occurs simultaneously .", "the first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion ( 6 ) .", "the second might be due to coinciding structural changes in arterial and lv walls ( 9 ) . in the current study", ", wave reflections seemed especially harmful to lv systolic function , since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction , followed by carotid - femoral transit time .", "lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness .", "this might support the second theory : that stiffening of arteries and lv walls occurs simultaneously .", "type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study .", "more severe lv systolic and diastolic dysfunction in type 2 diabetes therefore can not be explained or can or just partly be explained by increased arterial stiffness", ". other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension ( 11,2224 ) .", "this might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c .", "overweight and obesity are known to be associated with a higher lv mass index , and indeed , waist circumference seemed to play a role in type 2 diabetes  related higher lv mass index ( 25 ) .", "lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline , the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining .", "to conclude , the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction .", "it is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways , since these associations were independent of each other .", "a better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure ."], ["our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction ( 20,21 ) . the type 2 diabetes  associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline .", "these results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes .", "there was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well , but this was not statistically significant .", "individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up ."], ["peripheral markers of arterial stiffness , like arterial distensibility and compliance coefficients , and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction .", "associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure , since adjustment for mean arterial pressure only resulted in a small reduction of the associations .", "associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index .", "this might imply that 1 ) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2 ) stiffening of arteries and lv walls occurs simultaneously .", "the first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion ( 6 ) .", "the second might be due to coinciding structural changes in arterial and lv walls ( 9 ) . in the current study", ", wave reflections seemed especially harmful to lv systolic function , since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction , followed by carotid - femoral transit time .", "lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness .", "this might support the second theory : that stiffening of arteries and lv walls occurs simultaneously ."], ["type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study .", "more severe lv systolic and diastolic dysfunction in type 2 diabetes therefore can not be explained or can or just partly be explained by increased arterial stiffness", ". other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension ( 11,2224 ) .", "this might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c .", "overweight and obesity are known to be associated with a higher lv mass index , and indeed , waist circumference seemed to play a role in type 2 diabetes  related higher lv mass index ( 25 ) .", "lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline , the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining .", "to conclude , the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction .", "it is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways , since these associations were independent of each other .", "a better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure ."]]}
{"article_id": "PMC4943429", "article_text": ["primitive neuroectodermal tumor ( pnet ) , a highly aggressive tumor of the kidney is a rare malignancy with only few cases being reported in literature .", "boys and men are more likely to suffer rpnet ( renal pnet ) , and the sex ratio ( male : female ) is about 3:1 .", "it usually presents at an advanced stage , with distant metastasis and is associated with a poor prognosis .", "we report in this paper , a case of primary renal pnet in a young male .", "a 39-year - old male presented with complaints of hematuria and heaviness in the right flank since 15 days . physical examination revealed a soft lump in the right hypochondrium .", "ultrasound examination of the abdomen showed an irregular cystic structure in the right kidney at lower pole with few internal echoes .", "ct scan of the abdomen demonstrated a large significantly enhancing right renal lower polar mass with necrosis with thrombosis in right renal vein . on the basis of these clinico - radiological findings , diagnosis of right side renal cell cancer", "gross examination revealed a friable , grayish - white , lobulated mass ( 7  5 cm ) with multiple foci of hemorrhage and necrosis .", "the mass extended from mid to lower pole of the right kidney and infiltrated into the renal pelvis and was limited to the renal capsule the renal vein and ureter were free of the tumor .", "there was a thrombus inside the renal vein , which was not adherent to intima of renal vein .", "microscopically , the tumor was composed of monotonous sheets of round cells with scant cytoplasm , hyperchromatic vesicular nuclei , coarse chromatin and few conspicuous nucleoli , transverse by thin fibrous bands infiltrating and entrapping the renal glomeruli and tubules [ figure 1 ] .", "on immunohistochemistry , the tumor cells were positive for cd99 [ figure 2a ] , fli-1 [ figure 2b ] , nse [ figure 2c ] , and synaptophysin [ figure 2d ] , but negative for desmin , ck , ema , and lca .", "a diagnosis of pnet was made based on the above findings , and patient was started on vac ( vincristine , adriamycin , and cyclophosphamide ) alternating with ie ( ifosfamide and etoposide)-based adjuvant chemotherapy regimen which he tolerated well .", "after 6 cycles of chemotherapy , a pet scan showed absence of any metabolically active focal uptake in whole body .", "chemotherapy with vac ( vincristine , actinomycin d , and cyclophosphamide ) alternating with ie ( ifosfamide and etoposide ) for a year was planned .", "section showing malignant round cells arranged in pattern sheets infiltrating and entrapping the renal glomeruli and tubules { figure 1a : h&e ; 100 , figure 1b : h&e ; 200 } ( a ) the tumor cells express membranous expression of cd99 { dab ; 200 } ( b ) the tumor cells express nuclear positivity for fli-1{dab ; 200 } ( c ) the tumor cells express cytoplasmic positivity for neuron - specific enolase ( nse ) { dab ; 200 } ( d ) the tumor cells expressing cytoplasmic positivity for synaptophysin { dab ; 200 }", "the primitive neuroectodermal tumor ( pnet ) , was first recognized by arthur purdy stout in 1918 , and is a member of the family of  small round - cell tumors .  pnet arising in the kidney was first reported by mor in 1994 .", "primitive neuroectodermal tumors occurs preferentially in the soft - tissues of the paravertebral region and chest wall , less frequently in extremities , with a slight male predominance .", "rpnet appears to be an unique clinical entity that behaves more aggressively than pnet arising at other sites .", "the presenting symptoms and images of rpnet are non - specific and similar to other renal tumors .", "rpnet show a male predominance with 85% cases being diagnosed during the second to fourth decade .", "it is an aggressive tumor that tends to recur locally and to metastasize to lymph nodes , lung , liver , bone , and bone marrow , which entail a worse prognosis .", "the differential diagnosis of rpnet includes other small round cell tumors like neuroblastoma , adult nephroblastoma , malignant lymphoma , rhabdoid tumor , small cell carcinoma and monophasic , poorly differentiated , round cell variant of synovial sarcoma , carcinoid , rhabdomyosarcoma , clear cell sarcoma of the kidney , the small cell variant of osteosarcoma , and desmoplastic small round cell tumor .", "grossly , rpnet are typically very large tumors with about 65% measuring > 10 cm in diameter .", "they tend to be grayish in color , encapsulated , and contain focal areas of hemorrhage or necrosis .", "microscopically , the tumor is composed of sheets of round cells ; individual cells have a round nucleus with a distinct nuclear membrane , fine powdery chromatin , and 1 or 2 small nucleoli .", "the cytoplasm is ill - defined , scanty , usually pas - positive , and mitosis are variable .", "perivascular pseudo rosettes and homer - wright rosettes are common . to better address the diagnosis ,", "immunohistochemical markers to be used to confirm diagnosis are cytokeratin ( for nephroblastoma , small cell carcinoma , and synovial sarcoma ) , lca ( for lymphoma ) , nse / chromogranin a ( for neuroblastoma ) , and cd-99 for pnet .", "cd99 ( the product of mic2 gene ) greatly aids in recognizing the es / pnet family of tumors .", "many pnet also stain for neural markers , including nse , leu-7 , s-100 protein , synaptophysin , and pgp9.5 .", "the most frequent translocation in pnet is t(11;22)(q24;q12 ) , detected in approximately 90% of cases , resulting in ews - fli fusion gene .", "the overall survival in patients who had localized rpent was longer than that in the patients who had rpent with regional nodes or distant metastases .", "the role of radiotherapy is not clear , but it may be advocated in locally advanced disease and involvement of gerota 's fascia .", "post - operative chemotherapy for rpent is usually used and can improve the prognosis of rpnet .", "the most commonly used chemotherapeutics are adriamycin , etoposide , dactinomycin , vincristine , cyclophosphamide , and ifosfamide . a multimodality management is recommended ."], "abstract_text": ["<S> primitive neuroectodermal tumor of the kidney is a rare entity . </S>", "<S> very few cases of primary renal pnet have been reported to date . </S>", "<S> most literature about rpnet is isolated case reports . </S>", "<S> we report a case of rpnet in a 39-year - old male with a pre - operative diagnosis of renal cell carcinoma with renal vein thrombosis . </S>", "<S> the patient underwent radical nephrectomy with thrombolectomy , and histopathological examination revealed a highly aggressive tumor composed of monotonous sheets of round cells . </S>", "<S> tumor cells were positive for cd 99 and fli-1 , hence confirming the diagnosis of primitive neuroectodermal tumor . </S>", "<S> post - surgery , patient was given vac / ie - based adjuvant chemotherapy . in view of highly aggressive nature of this tumor , </S>", "<S> prompt diagnosis and imparting effective chemotherapy regimen to the patient is required , and it is important to differentiate pnet from other small round - cell tumors because of different therapeutic approach . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["primitive neuroectodermal tumor ( pnet ) , a highly aggressive tumor of the kidney is a rare malignancy with only few cases being reported in literature .", "boys and men are more likely to suffer rpnet ( renal pnet ) , and the sex ratio ( male : female ) is about 3:1 .", "it usually presents at an advanced stage , with distant metastasis and is associated with a poor prognosis .", "we report in this paper , a case of primary renal pnet in a young male ."], ["a 39-year - old male presented with complaints of hematuria and heaviness in the right flank since 15 days . physical examination revealed a soft lump in the right hypochondrium .", "ultrasound examination of the abdomen showed an irregular cystic structure in the right kidney at lower pole with few internal echoes .", "ct scan of the abdomen demonstrated a large significantly enhancing right renal lower polar mass with necrosis with thrombosis in right renal vein . on the basis of these clinico - radiological findings , diagnosis of right side renal cell cancer", "gross examination revealed a friable , grayish - white , lobulated mass ( 7  5 cm ) with multiple foci of hemorrhage and necrosis .", "the mass extended from mid to lower pole of the right kidney and infiltrated into the renal pelvis and was limited to the renal capsule the renal vein and ureter were free of the tumor .", "there was a thrombus inside the renal vein , which was not adherent to intima of renal vein .", "microscopically , the tumor was composed of monotonous sheets of round cells with scant cytoplasm , hyperchromatic vesicular nuclei , coarse chromatin and few conspicuous nucleoli , transverse by thin fibrous bands infiltrating and entrapping the renal glomeruli and tubules [ figure 1 ] .", "on immunohistochemistry , the tumor cells were positive for cd99 [ figure 2a ] , fli-1 [ figure 2b ] , nse [ figure 2c ] , and synaptophysin [ figure 2d ] , but negative for desmin , ck , ema , and lca .", "a diagnosis of pnet was made based on the above findings , and patient was started on vac ( vincristine , adriamycin , and cyclophosphamide ) alternating with ie ( ifosfamide and etoposide)-based adjuvant chemotherapy regimen which he tolerated well .", "after 6 cycles of chemotherapy , a pet scan showed absence of any metabolically active focal uptake in whole body .", "chemotherapy with vac ( vincristine , actinomycin d , and cyclophosphamide ) alternating with ie ( ifosfamide and etoposide ) for a year was planned .", "section showing malignant round cells arranged in pattern sheets infiltrating and entrapping the renal glomeruli and tubules { figure 1a : h&e ; 100 , figure 1b : h&e ; 200 } ( a ) the tumor cells express membranous expression of cd99 { dab ; 200 } ( b ) the tumor cells express nuclear positivity for fli-1{dab ; 200 } ( c ) the tumor cells express cytoplasmic positivity for neuron - specific enolase ( nse ) { dab ; 200 } ( d ) the tumor cells expressing cytoplasmic positivity for synaptophysin { dab ; 200 }"], ["the primitive neuroectodermal tumor ( pnet ) , was first recognized by arthur purdy stout in 1918 , and is a member of the family of  small round - cell tumors .  pnet arising in the kidney was first reported by mor in 1994 .", "primitive neuroectodermal tumors occurs preferentially in the soft - tissues of the paravertebral region and chest wall , less frequently in extremities , with a slight male predominance .", "rpnet appears to be an unique clinical entity that behaves more aggressively than pnet arising at other sites .", "the presenting symptoms and images of rpnet are non - specific and similar to other renal tumors .", "rpnet show a male predominance with 85% cases being diagnosed during the second to fourth decade .", "it is an aggressive tumor that tends to recur locally and to metastasize to lymph nodes , lung , liver , bone , and bone marrow , which entail a worse prognosis .", "the differential diagnosis of rpnet includes other small round cell tumors like neuroblastoma , adult nephroblastoma , malignant lymphoma , rhabdoid tumor , small cell carcinoma and monophasic , poorly differentiated , round cell variant of synovial sarcoma , carcinoid , rhabdomyosarcoma , clear cell sarcoma of the kidney , the small cell variant of osteosarcoma , and desmoplastic small round cell tumor .", "grossly , rpnet are typically very large tumors with about 65% measuring > 10 cm in diameter .", "they tend to be grayish in color , encapsulated , and contain focal areas of hemorrhage or necrosis .", "microscopically , the tumor is composed of sheets of round cells ; individual cells have a round nucleus with a distinct nuclear membrane , fine powdery chromatin , and 1 or 2 small nucleoli .", "the cytoplasm is ill - defined , scanty , usually pas - positive , and mitosis are variable .", "perivascular pseudo rosettes and homer - wright rosettes are common . to better address the diagnosis ,", "immunohistochemical markers to be used to confirm diagnosis are cytokeratin ( for nephroblastoma , small cell carcinoma , and synovial sarcoma ) , lca ( for lymphoma ) , nse / chromogranin a ( for neuroblastoma ) , and cd-99 for pnet .", "cd99 ( the product of mic2 gene ) greatly aids in recognizing the es / pnet family of tumors .", "many pnet also stain for neural markers , including nse , leu-7 , s-100 protein , synaptophysin , and pgp9.5 .", "the most frequent translocation in pnet is t(11;22)(q24;q12 ) , detected in approximately 90% of cases , resulting in ews - fli fusion gene .", "the overall survival in patients who had localized rpent was longer than that in the patients who had rpent with regional nodes or distant metastases .", "the role of radiotherapy is not clear , but it may be advocated in locally advanced disease and involvement of gerota 's fascia .", "post - operative chemotherapy for rpent is usually used and can improve the prognosis of rpnet .", "the most commonly used chemotherapeutics are adriamycin , etoposide , dactinomycin , vincristine , cyclophosphamide , and ifosfamide . a multimodality management is recommended ."]]}
{"article_id": "PMC4963560", "article_text": ["female sex workers ( fsws ) are at high risk of hiv infection and transmission .", "a systematic review conducted in over 25 countries with medium and high hiv prevalence indicated that 36.9% of sex workers in sub - saharan africa were hiv positive with some countries having hiv prevalence as high as 70.7% . despite this high burden of hiv infection ,", "only 58% of sex workers have access to hiv prevention and care programmes   and less than half of hiv positive sex workers are enrolled into hiv care .", "unaids reported that , in 2013 , art averted 6.3 million aids related deaths worldwide as a result of the increased number of people receiving art . in 2015 , who released revised guidelines for hiv treatment and recommended initiation of art among all hiv infected individuals irrespective of their cd4 count , for improved treatment outcomes and prevention of hiv transmission .", "sex workers are among the priority populations for expansion of hiv services including art . however , despite the existence of established targeted sex worker interventions , lots of challenges still exist that have hindered many from enrolling into care .", "several studies have highlighted barriers to linkage to care among female sex workers including discrimination by hospital staff [ 3 , 7 ] , delays at the health facility [ 8 , 9 ] , lack of money for transport and food , fear of drugs , stigma , use of drugs and alcohol , fear to be seen by clients , lack of knowledge about the treatment center [ 10 , 11 ] , and delays in accessing treatment among those who felt they were still healthy .", "however , while these barriers have been reported , there is still limited information on linkage to hiv care among sex workers in sub - saharan africa , particularly on effective linkage models to inform scale up of hiv interventions among sex workers . in 2012 , reach out mbuya hiv / aids initiative ( rom ) , a faith - based community non - government organization in uganda , introduced mobile outreach services to promote hiv testing and treatment among fsws and their clients .", "although the program has over 200 fsws enrolled in hiv care , some of the fsws who tested hiv positive did not enrol for treatment .", "we conducted a study to identify the facilitators and barriers to linkage to hiv care among the fsws who tested positive in order to design appropriate hiv interventions for this key population group .", "this was a cross - sectional qualitative study that involved in - depth and key informant interviews .", "this study was part of a larger study that explored the facilitators and barriers to linkage to hiv care among fsws in kampala and wakiso districts in central uganda .", "rom promotes hiv prevention through peer educators and provides hiv counselling and testing ( hct ) services through established outreach clinics that provide care and treatment for hiv positive fsws .", "mobile hct outreaches are conducted in locations surrounding mbuya parish in kampala and wakiso districts in central uganda .", "in - depth interview ( idi ) participants were selected from 301 hiv positive female sex workers who were registered in the hct registers at the different outreach points between may 2012 and december 2013 .", "of the 301 registered clients , 144 were traced and interviewed as part of the larger study where 28 of these were purposively selected to participate in the in - depth interviews .", "these included 14 fsws who were in hiv care and 14 who had not yet enrolled into care .", "the selected fsws were contacted by counsellors by phone to explain the study and to ask them to participate in the study .", "fsws with missing phone contact information or those who were unavailable after a minimum of three telephone attempts were tracked through their peer educators and friends .", "key informant interview ( kii ) participants were selected from peer educators who were trained by rom and were involved in following up fsws to encourage them to access hiv care .", "in addition , five rom staffs who were involved in the implementation of the outreach program were interviewed as key informants .", "rom provides static and bi - weekly outreach clinics for fsws using staff specifically trained on how to deal with key populations including fsws . at the time of the study ,", "hiv testing and linkage to hiv care services were offered using bi - weekly mobile outreach clinics , brothel based testing , and nocturnal mobile vans at places where the fsws reside or conduct business .", "hiv testing was conducted following the national rapid testing algorithm   and posttest counselling was provided to hiv infected women who were advised on the available health facilities for follow - up care .", "hiv infected women were advised to begin care immediately either at the static or mobile clinics operated by rom or other partners and were followed up with phone calls or by peer educators a week after testing and once every quarter thereafter , to ascertain linkage to care .", "those who were enrolled at the static clinic were subjected to a home assessment by the community art and tb treatment supporters ( catts ) to enable adequate follow - up .", "the information collected using the home assessment tool included the names and number of household members , water and sanitation , economic status , property owned , distance to the facility , and acceptable mode for communication .", "sex workers , who agreed to participate in the study , provided written informed consent and were interviewed in the language of their choice using unstructured , pretested questionnaires .", "the pretesting was conducted in the nearby project area two weeks before the commencement of the study . to ensure anonymity ,", "data were collected on the facilitators and barriers to linkage to hiv care by trained research assistants with experience in handling fsws .", "data were collected on age , education , and linkage status to aid proper categorization of the study participants ( linked or not linked to care ) .", "we defined linkage to care as having registered within an hiv clinic . the interview themes ( i.e. , sociodemographic , socioeconomic , structural , and hiv - related factors )", "were informed by theoretical constructs from the health belief model   and the socio - ecological model   and further informed by literature on barriers and facilitators to hiv service access among fsws .  ", "figure 1 presents a summary of the key variables and their interrelationships in informing linkage to hiv care among fsws .", "interviews were conducted at selected brothels , fsws homes , and static and outreach clinics .", "key informant interviews were conducted at the workplace or residence of the respondents , in english and luganda , using unstructured pretested questionnaires .", "the themes for the key informant interviews included hiv treatment seeking behaviour of the fsws , the challenges of enrolling into care , and the interventions to enhance linkage to hiv care in this population .", "additional data , including age , education , job category , residence , and the time allocated to working with fsws , was collected .", "the review of transcripts was guided by a priori themes pertaining to linkage to care , loss to follow - up , stopping treatment , and facilitators and barriers to linkage .", "issues that cut across the different themes which were captured by both authors were coded and categorized while those that were captured by one but not the other were subjected to further analysis until they were resolved and used to support the overriding themes or dropped .", "the study was approved by makerere university school of public health higher degrees research and ethics committee ( irb # : irb00011353 ) and the uganda national council of science and technology .", "of the 28 fsws interviewed , 14 ( 50% ) were enrolled in hiv care ( nine at rom ) , 8 had not yet enrolled , and 6 had previously enrolled but were either lost to follow - up or had stopped treatment .", "of the 28 women interviewed , 24 ( 85% ) were aged 2030 years while the rest where > 30 years .", "half ( 14 ) had at least secondary or higher education and the other half ( 14 ) had at least primary education .", "the fsws who were lost to follow - up or had stopped treatment were aged between 15 and 36 years with their education level ranging from none to secondary .", "the respondents included fsws who operate in bars ( 6 ) , on the streets ( 8) , at home ( 3 ) , and in brothels ( 11 ) ( table 1 ) .", "the findings have been grouped into two themes : ( a ) facilitators of linkage to hiv care and ( b ) barriers to linkage to hiv care among fsws , as presented below .", "the facilitators have been further grouped into two main themes : health system and social network factors ( table 2 ) .", "majority of the participants associated their linkage to care with the good quality of the services including good attitude and friendliness exhibited by the counsellors towards them .", "they noted that the counsellors talked to them with ease and were not judgmental : the counsellors are good , they talk to us very well and are almost like our parents .", "when i was told my results i got so scared as though i would die there and then .", "i failed to breath for 30 minutes ; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment . ", "( fsw , 29  years , enrolled  in  care ) the counsellors are good , they talk to us very well and are almost like our parents .", "when i was told my results i got so scared as though i would die there and then .", "i failed to breath for 30 minutes ; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment . ", "( fsw , 29  years , enrolled  in  care ) participants reported that the counsellors provided detailed information and encouragement to help them start treatment .", "the counsellors told them that testing hiv positive was not the end of life and that with medication they would be helped to stay healthy and live longer .", "in addition , participants appreciated the good follow - up support for those who tested positive .", "some said the counsellors called them and followed them up to their homes , which made them feel cared for : i started treatment due to counselor x 's advice , she was so caring and always followed me up , she could visit me ; otherwise on my own i had even disappeared but she was on my case . ", "( fsw , 24  years , enrolled  in  care ) i started treatment due to counselor x 's advice , she was so caring and always followed me up , she could visit me ; otherwise on my own i had even disappeared but she was on my case . ", "( fsw , 24  years , enrolled  in  care ) some participants acknowledged that it was easy for them to enrol in care because of the same - day results and immediate initiation of treatment .", "the peer educators and fellow sex workers enabled them to start treatment by encouraging them and helping to allay their fears regarding hiv treatment and occasionally escorting them to the clinics to start medication .", "some respondents said they would forget the clinic days because the clinic did not run every day but the peer educators reminded them . during the key informant interviews the peer educators mentioned how tirelessly they worked to follow up the hiv positive fsws ; they were sometimes abused by the sex workers but they never gave up on them : i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine . ", "( peer  educator , 29  years )  they come and tell me that on such and such a date i will be going to the clinic so remind me , i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic . ", "( peer  educator , 25  years ) i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine . ", "( peer  educator , 29  years )    they come and tell me that on such and such a date i will be going to the clinic so remind me , i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic . ", "( peer  educator , 25  years ) the proximity of the clinic to their residence was another enabling factor for linkage to care .", "some fsws did not want to move long distances for hiv care and others did not have transport so they thought that enrolling at rom would help reduce such burdens . across all interviews fsws who were enrolled in savings and", "they noted that the members of the support groups like village savings groups started by rom for sex workers only or mixed with other community members helped them to enrol into care .", "the members of the savings group reportedly advised them to start on treatment and live longer .", "being part of a savings group also created hope that money would be available for food and they would have capital to start their own business and live longer : i thought i was alone but when i realized we were many , i stopped fearing and also started medicine .", "my group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more . ", "( fsw , 25  years , enrolled  in  care ) i thought i was alone but when i realized we were many , i stopped fearing and also started medicine .", "my group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more . ", "( fsw , 25  years , enrolled  in  care ) across all the interviews the participants cited the need to remain healthy as one of their motivators for linkage to care . in order to continue doing their work , they wanted to start medication to remain healthy and not show any signs of opportunistic infections , which would scare away their customers .", "others wanted to remain healthy so as to live longer and take care of their children : it is because of my children ; i have to live for them because i am a widow , if i do n't take care of myself and die who will take care of them . ", "( fsw  with  3  children , 24  years , in  care ) it is because of my children ; i have to live for them because i am a widow , if i do n't take care of myself and die who will take care of them . ", "( fsw  with  3  children , 24  years , in  care ) several participants reported that seeing their colleagues improve after starting treatment motivated them to start on treatment while others did so because many of their friends were dying , which compelled them to start treatment so as to survive : i had a friend who stopped treatment and died , when we took her to the hospital the nurses said if only she continued with treatment she would be alive .", "when they ( providers ) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment .", "( fsw  20  years , enrolled  in  care ) i had a friend who stopped treatment and died , when we took her to the hospital the nurses said if only she continued with treatment she would be alive .", "when they ( providers ) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment .", "( fsw  20  years , enrolled  in  care ) barriers were similarly categorized into two themes : health systems and social network factors ( table 3 ) .", "the fsws enrolled at the government facilities mentioned discrimination from the health workers who openly exhibited a negative attitude towards sex workers .", "they said the health workers reacted differently after realizing that they were dealing with a sex worker and , upon telling their friends , those who had not yet registered for care feared to enrol : the counselor asked me what i do and i said sex work .", "she called four other counselors to ask me why i do that kind of work ; i wanted to ask them , ", "what do you expect me to do ? '  but the counselor stood up and asked me rudely ;  is that also work to mention among people ? '", "that counselor annoyed me so much i walked off and never wanted to see her again . ", "( peer  educator  who  is  a  fsw , 29  years ) the counselor asked me what i do and i said sex work .", "she called four other counselors to ask me why i do that kind of work ; i wanted to ask them ,  what do you expect me to do ? '  but the counselor stood up and asked me rudely ;  is that also work to mention among people ? '", "that counselor annoyed me so much i walked off and never wanted to see her again . ", "( peer  educator  who  is  a  fsw , 29  years ) the four fsws who were enrolled at government facilities noted that they were discouraged by the requirement to present a treatment supporter coupled with the long procedures for enrolment that involved several visits to the facility : it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get .", "may be i do not want someone else to know that i am positive . ", "( fsw , 29  years , enrolled  in  care ) it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get .", "i felt this was not right because i must go for treatment by myself  may be i do not want someone else to know that i am positive . ", "( fsw , 29  years , enrolled  in  care ) almost all the participants cited stigma as a major barrier to linkage to care .", "the majority feared to be seen by their clients at the clinic . although the close proximity of the clinic was mentioned as a motivator , for some fsws it was a hindrance .", "participants were afraid of being seen at the hiv clinic by customers , their friends , and other people in the village and were particularly concerned about rumours : i can not go to that clinic because everyone here goes there and they will see me which will spoil my business . right now", "the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick . ", "( fsw  22  years , not  enrolled ) i can not go to that clinic because everyone here goes there and they will see me which will spoil my business . right now", "the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick . ", "( fsw  22  years , not  enrolled ) due to lack of information , many participants had many myths about arvs ( e.g. , that they kill fast and need a lot of care ) .", "they avoided the arvs by not going to the clinic while some opted to wait until they got other jobs because of the perception that arvs could weaken them and disrupt their work .", "some respondents feared drugs and could not imagine swallowing them for life so they opted out . on the other hand , some participants feared death after testing and lost hope for living ; they did not see the need for starting on art : madam is n't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs  ( fsw , 20  years , not  in  care  and  never  went  to  school ) madam is n't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs  ( fsw , 20  years , not  in  care  and  never  went  to  school ) some fsws did not know what hiv is while others encountered difficulties in identifying an hiv clinic because they lacked proper information about the location of the clinics .", "this was mainly a challenge to the young girls ( aged between 20 and 25 years ) who lived in the brothels as indicated by the project staff : some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is . ", "( key  informant ) some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is . ", "( key  informant ) some fsws mentioned that they could not enrol in an hiv clinic since they did not believe their hiv results .", "some thought it was just a prick performed hurriedly in the night and wondered how it translated into an hiv positive result so they waited and retested many times : when i reached home , i started doubting the results and decided to go for another test in a different place .", "i continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin . ", "( fsw  29  yrs , not  enrolled  in  care ) when i reached home , i started doubting the results and decided to go for another test in a different place .", "i continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin . ", "( fsw  29  yrs , not  enrolled  in  care ) a third of the fsws mentioned that they were using herbs as a substitute for arvs and therefore saw no need of being registered in an hiv clinic .", "besides many of the fsws said they used local herbs from traditional healers to attract customers and did not have to line up for herbs like they did in the hiv clinic : no .", "i have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [ meaning arvs ] . even with the herbs now am on medication ; i do take the local herbs and am fine with the treatment , i do n't want to be forced to take arvs . ", "( fsw , 24  yrs , not  enrolled ) no . i have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [ meaning arvs ] . even with the herbs now am on medication ; i do take the local herbs and am fine with the treatment , i do n't want to be forced to take arvs . ", "( fsw , 24  yrs , not  enrolled ) some participants bought painkillers from general clinics instead of enrolling in an hiv clinic ( self - medication ) .", "even when the nurses in these clinics suspected that they were hiv infected , they did not tell them because they wanted business through treating their on and off sickness . because they are always travelling in different places looking for clients , fsws cited lack of time as one of the barriers to linkage to care : i am still very busy right now looking for rent and food .", "i can not lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries . ", "( fsw  23  years , not  enrolled ) i am still very busy right now looking for rent and food .", "i can not lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries . ", "( fsw  23  years , not  enrolled ) the kis felt that the street based sex workers had more challenges with linkage to hiv care compared to those in brothels because they were difficult to trace given that they only relied on phone numbers which were off most of the time : those on the streets do not want to access care  .", "( key  informant ) those on the streets do not want to access care  .", "( key  informant ) across all interviews , the fsws mentioned lack of finances as a major hindrance to linkage to care .", "they were concerned about requirements such as good food and frequent transport to the health facility : it 's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking . ", "( fsw  22  years , not  enrolled ) it 's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking . ", "our study of facilitators and barriers to linkage to hiv care among female sex workers in kampala and wakiso districts in uganda shows that health system and social network factors are the major facilitators and barriers to linkage to hiv care .", "good quality health services ( especially polite and caring providers , strong follow - up structures using peer educators , and provider telephone calls ) and social network factors ( encouragement from peers and membership of savings group and the need to maintain good health ) were the primary facilitators of linkage to hiv care among fsws . on the other hand , perceived stigma ,", "various forms of misinformation , and prohibitive clinic policies were major barriers to linkage to hiv care .", "contrary to the experience of fsws at rom , those who were registered in the public health facilities encountered negative staff attitudes , a major barrier that has been documented in several studies [ 8 , 16 , 17 ] .", "these findings suggest that efforts to improve linkage and retention in hiv care among fsws should focus on quality of services and improving social support networks . ensuring good quality services , particularly providers who can deliver services in a respectful and no - judgmental manner   without undue delays and restrictions and adequate community education and mobilization as well as support structures for retention are key health system issues that can improve the quality of services . using peers to educate communities and follow - up of those who need treatment reduce the burden on health workers and strengthens community engagement . as reported elsewhere , the requirement of a treatment supporter before initiating care and treatment was a barrier to treatment that should no longer be strictly enforced .", "the training of providers and peer educators prior to implementation of this program could have contributed to delivery of quality services at rom and are a key intervention area .", "given the overriding concerns about being seen at hiv facilities , training for providers should emphasize the need for privacy and confidentiality .", "further , providing an integrated package of services for sex workers could reduce stigma around accessing hiv and other services .", "training should also emphasize flexibility with certain clinic policies that could hinder access to hiv services among sex workers .", "social network issues were also strong facilitators and barriers to linkage and require interventions particularly targeted at enhancing support by peers and reducing misinformation and stigma . enacted stigma , myths , and fears about arvs coupled with lack of correct information about hiv continue to be major barriers to linkage to hiv care .", "financial constraints were a prominent barrier to linkage to care . the savings groups were a good avenue for social support and financial stability and could be integrated into health programs for low - income sex workers [ 20 , 21 ] .", "collectively , these findings suggest that programs targeting fsws should integrate continuous sensitization and provision of tailor made services that address the individual level , community , and health system needs of fsw .", "this study is based on interviews with 14 fsws who were in hiv care and 14 others who were not . by all counts ,", "the numbers are too small to represent the views of all fsws who are enrolled or not enrolled in hiv care .", "however , our study findings provide good insights into the facilitators and barriers to linkage that can inform interventions to improving linkage for fsws .", "future research is needed to further understand how the fsws can be maintained in care once they have been linked .", "our study shows that fsws ability to be linked to hiv care is largely influenced by good quality friendly services and community support systems especially from their peers .", "hiv programs for fsw should focus on enhancing these as well as dealing with the barriers that mainly stem from stigma , ignorance , and work - related challenges .", "these findings call for a need to design interventions that utilize a multichannel , multipronged approach to increase access to hiv services among fsws ."], "abstract_text": ["<S> \n introduction . </S>", "<S> while four in ten female sex workers ( fsws ) in sub - saharan africa are infected with hiv , only a small proportion is enrolled in hiv care . </S>", "<S> we explored facilitators and barriers to linkage to hiv care among fsws receiving hiv testing services at a community - based organization in periurban uganda . </S>", "<S> methods . </S>", "<S> the cross - sectional qualitative study was conducted among 28 hiv positive fsws from may to july 2014 . </S>", "<S> key informant interviews were conducted with five project staff and eleven peer educators . </S>", "<S> data were collected on facilitators for and barriers to linkage to hiv care and manually analyzed following a thematic framework approach . results . </S>", "<S> facilitators for linkage to hiv care included the perceived good quality of health services with same - day results and immediate initiation of treatment , community peer support systems , individual 's need to remain healthy , and having alternative sources of income . </S>", "<S> linkage barriers included perceived stigma , fear to be seen at outreach hiv clinics , fear and myths about antiretroviral therapy , lack of time to attend clinic , and financial constraints . </S>", "<S> conclusion . </S>", "<S> linkage to hiv care among fsws is influenced by good quality friendly services and peer support . </S>", "<S> hiv service delivery programs for fsws should focus on enhancing these and dealing with barriers stemming from stigma and misinformation . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["female sex workers ( fsws ) are at high risk of hiv infection and transmission .", "a systematic review conducted in over 25 countries with medium and high hiv prevalence indicated that 36.9% of sex workers in sub - saharan africa were hiv positive with some countries having hiv prevalence as high as 70.7% . despite this high burden of hiv infection ,", "only 58% of sex workers have access to hiv prevention and care programmes   and less than half of hiv positive sex workers are enrolled into hiv care .", "unaids reported that , in 2013 , art averted 6.3 million aids related deaths worldwide as a result of the increased number of people receiving art . in 2015 , who released revised guidelines for hiv treatment and recommended initiation of art among all hiv infected individuals irrespective of their cd4 count , for improved treatment outcomes and prevention of hiv transmission .", "sex workers are among the priority populations for expansion of hiv services including art . however , despite the existence of established targeted sex worker interventions , lots of challenges still exist that have hindered many from enrolling into care .", "several studies have highlighted barriers to linkage to care among female sex workers including discrimination by hospital staff [ 3 , 7 ] , delays at the health facility [ 8 , 9 ] , lack of money for transport and food , fear of drugs , stigma , use of drugs and alcohol , fear to be seen by clients , lack of knowledge about the treatment center [ 10 , 11 ] , and delays in accessing treatment among those who felt they were still healthy .", "however , while these barriers have been reported , there is still limited information on linkage to hiv care among sex workers in sub - saharan africa , particularly on effective linkage models to inform scale up of hiv interventions among sex workers . in 2012 , reach out mbuya hiv / aids initiative ( rom ) , a faith - based community non - government organization in uganda , introduced mobile outreach services to promote hiv testing and treatment among fsws and their clients .", "although the program has over 200 fsws enrolled in hiv care , some of the fsws who tested hiv positive did not enrol for treatment .", "we conducted a study to identify the facilitators and barriers to linkage to hiv care among the fsws who tested positive in order to design appropriate hiv interventions for this key population group ."], ["this was a cross - sectional qualitative study that involved in - depth and key informant interviews .", "this study was part of a larger study that explored the facilitators and barriers to linkage to hiv care among fsws in kampala and wakiso districts in central uganda .", "rom promotes hiv prevention through peer educators and provides hiv counselling and testing ( hct ) services through established outreach clinics that provide care and treatment for hiv positive fsws .", "mobile hct outreaches are conducted in locations surrounding mbuya parish in kampala and wakiso districts in central uganda .", "in - depth interview ( idi ) participants were selected from 301 hiv positive female sex workers who were registered in the hct registers at the different outreach points between may 2012 and december 2013 .", "of the 301 registered clients , 144 were traced and interviewed as part of the larger study where 28 of these were purposively selected to participate in the in - depth interviews .", "these included 14 fsws who were in hiv care and 14 who had not yet enrolled into care .", "the selected fsws were contacted by counsellors by phone to explain the study and to ask them to participate in the study .", "fsws with missing phone contact information or those who were unavailable after a minimum of three telephone attempts were tracked through their peer educators and friends .", "key informant interview ( kii ) participants were selected from peer educators who were trained by rom and were involved in following up fsws to encourage them to access hiv care .", "in addition , five rom staffs who were involved in the implementation of the outreach program were interviewed as key informants .", "rom provides static and bi - weekly outreach clinics for fsws using staff specifically trained on how to deal with key populations including fsws . at the time of the study ,", "hiv testing and linkage to hiv care services were offered using bi - weekly mobile outreach clinics , brothel based testing , and nocturnal mobile vans at places where the fsws reside or conduct business .", "hiv testing was conducted following the national rapid testing algorithm   and posttest counselling was provided to hiv infected women who were advised on the available health facilities for follow - up care .", "hiv infected women were advised to begin care immediately either at the static or mobile clinics operated by rom or other partners and were followed up with phone calls or by peer educators a week after testing and once every quarter thereafter , to ascertain linkage to care .", "those who were enrolled at the static clinic were subjected to a home assessment by the community art and tb treatment supporters ( catts ) to enable adequate follow - up .", "the information collected using the home assessment tool included the names and number of household members , water and sanitation , economic status , property owned , distance to the facility , and acceptable mode for communication .", "sex workers , who agreed to participate in the study , provided written informed consent and were interviewed in the language of their choice using unstructured , pretested questionnaires .", "the pretesting was conducted in the nearby project area two weeks before the commencement of the study . to ensure anonymity ,", "data were collected on the facilitators and barriers to linkage to hiv care by trained research assistants with experience in handling fsws .", "data were collected on age , education , and linkage status to aid proper categorization of the study participants ( linked or not linked to care ) .", "we defined linkage to care as having registered within an hiv clinic . the interview themes ( i.e. , sociodemographic , socioeconomic , structural , and hiv - related factors )", "were informed by theoretical constructs from the health belief model   and the socio - ecological model   and further informed by literature on barriers and facilitators to hiv service access among fsws .  ", "figure 1 presents a summary of the key variables and their interrelationships in informing linkage to hiv care among fsws .", "interviews were conducted at selected brothels , fsws homes , and static and outreach clinics .", "key informant interviews were conducted at the workplace or residence of the respondents , in english and luganda , using unstructured pretested questionnaires .", "the themes for the key informant interviews included hiv treatment seeking behaviour of the fsws , the challenges of enrolling into care , and the interventions to enhance linkage to hiv care in this population .", "additional data , including age , education , job category , residence , and the time allocated to working with fsws , was collected .", "the review of transcripts was guided by a priori themes pertaining to linkage to care , loss to follow - up , stopping treatment , and facilitators and barriers to linkage .", "issues that cut across the different themes which were captured by both authors were coded and categorized while those that were captured by one but not the other were subjected to further analysis until they were resolved and used to support the overriding themes or dropped .", "the study was approved by makerere university school of public health higher degrees research and ethics committee ( irb # : irb00011353 ) and the uganda national council of science and technology ."], ["of the 28 fsws interviewed , 14 ( 50% ) were enrolled in hiv care ( nine at rom ) , 8 had not yet enrolled , and 6 had previously enrolled but were either lost to follow - up or had stopped treatment .", "of the 28 women interviewed , 24 ( 85% ) were aged 2030 years while the rest where > 30 years .", "half ( 14 ) had at least secondary or higher education and the other half ( 14 ) had at least primary education .", "the fsws who were lost to follow - up or had stopped treatment were aged between 15 and 36 years with their education level ranging from none to secondary .", "the respondents included fsws who operate in bars ( 6 ) , on the streets ( 8) , at home ( 3 ) , and in brothels ( 11 ) ( table 1 ) .", "the findings have been grouped into two themes : ( a ) facilitators of linkage to hiv care and ( b ) barriers to linkage to hiv care among fsws , as presented below .", "the facilitators have been further grouped into two main themes : health system and social network factors ( table 2 ) .", "majority of the participants associated their linkage to care with the good quality of the services including good attitude and friendliness exhibited by the counsellors towards them .", "they noted that the counsellors talked to them with ease and were not judgmental : the counsellors are good , they talk to us very well and are almost like our parents .", "when i was told my results i got so scared as though i would die there and then .", "i failed to breath for 30 minutes ; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment . ", "( fsw , 29  years , enrolled  in  care ) the counsellors are good , they talk to us very well and are almost like our parents .", "when i was told my results i got so scared as though i would die there and then .", "i failed to breath for 30 minutes ; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment . ", "( fsw , 29  years , enrolled  in  care ) participants reported that the counsellors provided detailed information and encouragement to help them start treatment .", "the counsellors told them that testing hiv positive was not the end of life and that with medication they would be helped to stay healthy and live longer .", "in addition , participants appreciated the good follow - up support for those who tested positive .", "some said the counsellors called them and followed them up to their homes , which made them feel cared for : i started treatment due to counselor x 's advice , she was so caring and always followed me up , she could visit me ; otherwise on my own i had even disappeared but she was on my case . ", "( fsw , 24  years , enrolled  in  care ) i started treatment due to counselor x 's advice , she was so caring and always followed me up , she could visit me ; otherwise on my own i had even disappeared but she was on my case . ", "( fsw , 24  years , enrolled  in  care ) some participants acknowledged that it was easy for them to enrol in care because of the same - day results and immediate initiation of treatment .", "the peer educators and fellow sex workers enabled them to start treatment by encouraging them and helping to allay their fears regarding hiv treatment and occasionally escorting them to the clinics to start medication .", "some respondents said they would forget the clinic days because the clinic did not run every day but the peer educators reminded them . during the key informant interviews the peer educators mentioned how tirelessly they worked to follow up the hiv positive fsws ; they were sometimes abused by the sex workers but they never gave up on them : i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine . ", "( peer  educator , 29  years )  they come and tell me that on such and such a date i will be going to the clinic so remind me , i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic . ", "( peer  educator , 25  years ) i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine . ", "( peer  educator , 29  years )    they come and tell me that on such and such a date i will be going to the clinic so remind me , i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic . ", "( peer  educator , 25  years ) the proximity of the clinic to their residence was another enabling factor for linkage to care .", "some fsws did not want to move long distances for hiv care and others did not have transport so they thought that enrolling at rom would help reduce such burdens . across all interviews fsws who were enrolled in savings and", "they noted that the members of the support groups like village savings groups started by rom for sex workers only or mixed with other community members helped them to enrol into care .", "the members of the savings group reportedly advised them to start on treatment and live longer .", "being part of a savings group also created hope that money would be available for food and they would have capital to start their own business and live longer : i thought i was alone but when i realized we were many , i stopped fearing and also started medicine .", "my group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more . ", "( fsw , 25  years , enrolled  in  care ) i thought i was alone but when i realized we were many , i stopped fearing and also started medicine .", "my group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more . ", "( fsw , 25  years , enrolled  in  care ) across all the interviews the participants cited the need to remain healthy as one of their motivators for linkage to care . in order to continue doing their work , they wanted to start medication to remain healthy and not show any signs of opportunistic infections , which would scare away their customers .", "others wanted to remain healthy so as to live longer and take care of their children : it is because of my children ; i have to live for them because i am a widow , if i do n't take care of myself and die who will take care of them . ", "( fsw  with  3  children , 24  years , in  care ) it is because of my children ; i have to live for them because i am a widow , if i do n't take care of myself and die who will take care of them . ", "( fsw  with  3  children , 24  years , in  care ) several participants reported that seeing their colleagues improve after starting treatment motivated them to start on treatment while others did so because many of their friends were dying , which compelled them to start treatment so as to survive : i had a friend who stopped treatment and died , when we took her to the hospital the nurses said if only she continued with treatment she would be alive .", "when they ( providers ) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment .", "( fsw  20  years , enrolled  in  care ) i had a friend who stopped treatment and died , when we took her to the hospital the nurses said if only she continued with treatment she would be alive .", "when they ( providers ) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment .", "( fsw  20  years , enrolled  in  care ) barriers were similarly categorized into two themes : health systems and social network factors ( table 3 ) .", "the fsws enrolled at the government facilities mentioned discrimination from the health workers who openly exhibited a negative attitude towards sex workers .", "they said the health workers reacted differently after realizing that they were dealing with a sex worker and , upon telling their friends , those who had not yet registered for care feared to enrol : the counselor asked me what i do and i said sex work .", "she called four other counselors to ask me why i do that kind of work ; i wanted to ask them , ", "what do you expect me to do ? '  but the counselor stood up and asked me rudely ;  is that also work to mention among people ? '", "that counselor annoyed me so much i walked off and never wanted to see her again . ", "( peer  educator  who  is  a  fsw , 29  years ) the counselor asked me what i do and i said sex work .", "she called four other counselors to ask me why i do that kind of work ; i wanted to ask them ,  what do you expect me to do ? '  but the counselor stood up and asked me rudely ;  is that also work to mention among people ? '", "that counselor annoyed me so much i walked off and never wanted to see her again . ", "( peer  educator  who  is  a  fsw , 29  years ) the four fsws who were enrolled at government facilities noted that they were discouraged by the requirement to present a treatment supporter coupled with the long procedures for enrolment that involved several visits to the facility : it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get .", "may be i do not want someone else to know that i am positive . ", "( fsw , 29  years , enrolled  in  care ) it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get .", "i felt this was not right because i must go for treatment by myself  may be i do not want someone else to know that i am positive . ", "( fsw , 29  years , enrolled  in  care ) almost all the participants cited stigma as a major barrier to linkage to care .", "the majority feared to be seen by their clients at the clinic . although the close proximity of the clinic was mentioned as a motivator , for some fsws it was a hindrance .", "participants were afraid of being seen at the hiv clinic by customers , their friends , and other people in the village and were particularly concerned about rumours : i can not go to that clinic because everyone here goes there and they will see me which will spoil my business . right now", "the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick . ", "( fsw  22  years , not  enrolled ) i can not go to that clinic because everyone here goes there and they will see me which will spoil my business . right now", "the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick . ", "( fsw  22  years , not  enrolled ) due to lack of information , many participants had many myths about arvs ( e.g. , that they kill fast and need a lot of care ) .", "they avoided the arvs by not going to the clinic while some opted to wait until they got other jobs because of the perception that arvs could weaken them and disrupt their work .", "some respondents feared drugs and could not imagine swallowing them for life so they opted out . on the other hand , some participants feared death after testing and lost hope for living ; they did not see the need for starting on art : madam is n't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs  ( fsw , 20  years , not  in  care  and  never  went  to  school ) madam is n't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs  ( fsw , 20  years , not  in  care  and  never  went  to  school ) some fsws did not know what hiv is while others encountered difficulties in identifying an hiv clinic because they lacked proper information about the location of the clinics .", "this was mainly a challenge to the young girls ( aged between 20 and 25 years ) who lived in the brothels as indicated by the project staff : some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is . ", "( key  informant ) some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is . ", "( key  informant ) some fsws mentioned that they could not enrol in an hiv clinic since they did not believe their hiv results .", "some thought it was just a prick performed hurriedly in the night and wondered how it translated into an hiv positive result so they waited and retested many times : when i reached home , i started doubting the results and decided to go for another test in a different place .", "i continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin . ", "( fsw  29  yrs , not  enrolled  in  care ) when i reached home , i started doubting the results and decided to go for another test in a different place .", "i continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin . ", "( fsw  29  yrs , not  enrolled  in  care ) a third of the fsws mentioned that they were using herbs as a substitute for arvs and therefore saw no need of being registered in an hiv clinic .", "besides many of the fsws said they used local herbs from traditional healers to attract customers and did not have to line up for herbs like they did in the hiv clinic : no .", "i have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [ meaning arvs ] . even with the herbs now am on medication ; i do take the local herbs and am fine with the treatment , i do n't want to be forced to take arvs . ", "( fsw , 24  yrs , not  enrolled ) no . i have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [ meaning arvs ] . even with the herbs now am on medication ; i do take the local herbs and am fine with the treatment , i do n't want to be forced to take arvs . ", "( fsw , 24  yrs , not  enrolled ) some participants bought painkillers from general clinics instead of enrolling in an hiv clinic ( self - medication ) .", "even when the nurses in these clinics suspected that they were hiv infected , they did not tell them because they wanted business through treating their on and off sickness . because they are always travelling in different places looking for clients , fsws cited lack of time as one of the barriers to linkage to care : i am still very busy right now looking for rent and food .", "i can not lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries . ", "( fsw  23  years , not  enrolled ) i am still very busy right now looking for rent and food .", "i can not lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries . ", "( fsw  23  years , not  enrolled ) the kis felt that the street based sex workers had more challenges with linkage to hiv care compared to those in brothels because they were difficult to trace given that they only relied on phone numbers which were off most of the time : those on the streets do not want to access care  .", "( key  informant ) those on the streets do not want to access care  .", "( key  informant ) across all interviews , the fsws mentioned lack of finances as a major hindrance to linkage to care .", "they were concerned about requirements such as good food and frequent transport to the health facility : it 's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking . ", "( fsw  22  years , not  enrolled ) it 's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking . "], ["our study of facilitators and barriers to linkage to hiv care among female sex workers in kampala and wakiso districts in uganda shows that health system and social network factors are the major facilitators and barriers to linkage to hiv care .", "good quality health services ( especially polite and caring providers , strong follow - up structures using peer educators , and provider telephone calls ) and social network factors ( encouragement from peers and membership of savings group and the need to maintain good health ) were the primary facilitators of linkage to hiv care among fsws . on the other hand , perceived stigma ,", "various forms of misinformation , and prohibitive clinic policies were major barriers to linkage to hiv care .", "contrary to the experience of fsws at rom , those who were registered in the public health facilities encountered negative staff attitudes , a major barrier that has been documented in several studies [ 8 , 16 , 17 ] .", "these findings suggest that efforts to improve linkage and retention in hiv care among fsws should focus on quality of services and improving social support networks . ensuring good quality services , particularly providers who can deliver services in a respectful and no - judgmental manner   without undue delays and restrictions and adequate community education and mobilization as well as support structures for retention are key health system issues that can improve the quality of services . using peers to educate communities and follow - up of those who need treatment reduce the burden on health workers and strengthens community engagement . as reported elsewhere , the requirement of a treatment supporter before initiating care and treatment was a barrier to treatment that should no longer be strictly enforced .", "the training of providers and peer educators prior to implementation of this program could have contributed to delivery of quality services at rom and are a key intervention area .", "given the overriding concerns about being seen at hiv facilities , training for providers should emphasize the need for privacy and confidentiality .", "further , providing an integrated package of services for sex workers could reduce stigma around accessing hiv and other services .", "training should also emphasize flexibility with certain clinic policies that could hinder access to hiv services among sex workers .", "social network issues were also strong facilitators and barriers to linkage and require interventions particularly targeted at enhancing support by peers and reducing misinformation and stigma . enacted stigma , myths , and fears about arvs coupled with lack of correct information about hiv continue to be major barriers to linkage to hiv care .", "financial constraints were a prominent barrier to linkage to care . the savings groups were a good avenue for social support and financial stability and could be integrated into health programs for low - income sex workers [ 20 , 21 ] .", "collectively , these findings suggest that programs targeting fsws should integrate continuous sensitization and provision of tailor made services that address the individual level , community , and health system needs of fsw .", "this study is based on interviews with 14 fsws who were in hiv care and 14 others who were not . by all counts ,", "the numbers are too small to represent the views of all fsws who are enrolled or not enrolled in hiv care .", "however , our study findings provide good insights into the facilitators and barriers to linkage that can inform interventions to improving linkage for fsws .", "future research is needed to further understand how the fsws can be maintained in care once they have been linked ."], ["our study shows that fsws ability to be linked to hiv care is largely influenced by good quality friendly services and community support systems especially from their peers .", "hiv programs for fsw should focus on enhancing these as well as dealing with the barriers that mainly stem from stigma , ignorance , and work - related challenges .", "these findings call for a need to design interventions that utilize a multichannel , multipronged approach to increase access to hiv services among fsws ."]]}
{"article_id": "PMC3349961", "article_text": ["the acquired alexias may be categorized into posterior , anterior , central and deep alexias .", "analagous to semantic paraphasias , semantic paralexias constitute the substitution of content related words during reading . unlike the analogous subcortical aphasias such as thalamic aphasia , subcortical syndromes of alexia have rarely been described .", "this report depicts a patient with thalamic alexia with features of deep alexia ( or paralexia ) , the latter which has not been described after review of the english based literature .", "a 57 year old white , right handed , english speaking woman with 13 years of education , presented to our stroke center with mild right sided numbness and weakness ( graded + 4/5 medical research council grading scale ) with the family reporting transient confused conversation and speech difficulty .", "the clinical evaluation at our center encompasses a cognitive screening evaluation in all patients followed by psychometric assessment in selected patients described in detail elsewhere .", "no dysnomia was documented . specifically visual acuity and visual fields were normal and there was no object agnosia , dyschromatopsia , dyscalculia , finger agnosia , right left disorientation , or hemineglect syndrome .", "the substitution of semantically related words was documented with only mild slowing in her reading ( family corroboration ) .", "cerebrovascular risk factors included a significant smoking history and on investigation hyperhomocyteinemia ( 15.6 mmol / l ) . within 4 days , the reading difficulty had normalized but she remained with mild speech dysfluency , right hand ideomotor apraxia and developed a dejerine roussey or post stroke thalamic pain syndrome .", "multimodality magnetic resonance imaging investigations revealed a left lateral posterior thalamic infarct , well circumscribed on t2 weighted and diffusion weighted imaging without other brain parenchymal lesion ( figure 1 ) .", "magnetic resonance angiography revealed a left internal carotid artery stenosis graded 5074% and basilar artery fenestration . \n", "figure 1left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) . \n  left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) . in view of the isolated reading impairment ,", "additional psychometric examination was performed on day 3 after the stroke onset , with the boston diagnostic aphasia examination ( third edition - short form ) .", "in addition , the test for semantic paralexia prone words were tested with the standard version of the boston diagnostic aphasia examination ( version iii ) ( table 1 ) . in comparison to normative data ,", "notable deficiencies were noted only in speech fluency , articulation , oral word and sentence reading , picture word matching and writing . \n", "table 1boston diagnostic aphasia test - version iii.subtests%fluency  phrase length30  melodic line60  grammatical form70conversation expository speech100auditory comprehension  basic word discrimination100 ", "commands100  complex ideational material100articulation ( agility)70recitation ( automatized sequences)100repetition  words60  sentences100naming  responsive naming100  boston naming test100  special categories100paraphasia100reading  matching case and scripts100  number matching100  picture word matching40  oral word reading40  oral sentence reading80  oral sentence comprehension100  sentence / paragraph100  comprehension100wiriting  form20  letter choice40  motor facility40 \n", "a 57 year old white , right handed , english speaking woman with 13 years of education , presented to our stroke center with mild right sided numbness and weakness ( graded + 4/5 medical research council grading scale ) with the family reporting transient confused conversation and speech difficulty .", "the clinical evaluation at our center encompasses a cognitive screening evaluation in all patients followed by psychometric assessment in selected patients described in detail elsewhere .", "no dysnomia was documented . specifically visual acuity and visual fields were normal and there was no object agnosia , dyschromatopsia , dyscalculia , finger agnosia , right left disorientation , or hemineglect syndrome .", "the substitution of semantically related words was documented with only mild slowing in her reading ( family corroboration ) .", "cerebrovascular risk factors included a significant smoking history and on investigation hyperhomocyteinemia ( 15.6 mmol / l ) .", "within 4 days , the reading difficulty had normalized but she remained with mild speech dysfluency , right hand ideomotor apraxia and developed a dejerine roussey or post stroke thalamic pain syndrome .", "multimodality magnetic resonance imaging investigations revealed a left lateral posterior thalamic infarct , well circumscribed on t2 weighted and diffusion weighted imaging without other brain parenchymal lesion ( figure 1 ) .", "magnetic resonance angiography revealed a left internal carotid artery stenosis graded 5074% and basilar artery fenestration . \n", "figure 1left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) . \n  left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) .", "in view of the isolated reading impairment , additional psychometric examination was performed on day 3 after the stroke onset , with the boston diagnostic aphasia examination ( third edition - short form ) .", "in addition , the test for semantic paralexia prone words were tested with the standard version of the boston diagnostic aphasia examination ( version iii ) ( table 1 ) . in comparison to normative data ,", "notable deficiencies were noted only in speech fluency , articulation , oral word and sentence reading , picture word matching and writing . \n", "table 1boston diagnostic aphasia test - version iii.subtests%fluency  phrase length30  melodic line60  grammatical form70conversation expository speech100auditory comprehension  basic word discrimination100 ", "commands100  complex ideational material100articulation ( agility)70recitation ( automatized sequences)100repetition  words60  sentences100naming  responsive naming100  boston naming test100  special categories100paraphasia100reading  matching case and scripts100  number matching100  picture word matching40  oral word reading40  oral sentence reading80  oral sentence comprehension100  sentence / paragraph100  comprehension100wiriting  form20  letter choice40  motor facility40 \n", "the commonly referred to syndromic classification of benson and geschwind of posterior alexia ( alexia without agraphia ) , central alexia ( alexia with agraphia ) , anterior alexia ( alexia in association with expressive dysphasia ) and deep dyslexia ( primarily semantic paralexia disorder ) aptly describes most acquired reading deficiencies .", "alexia secondary to isolated thalamic lesion has not been reported other than in combination with left occipital lesions , the latter lesions the usual focus .", "semantic paralexia due to thalamic lesions has not been reported previously with neuroanatomical lesions mostly referred to as large perisylvian and even right hemisphere lesions implicated .", "similar to subcortical aphasias , the subcortical ( thalamic ) alexia reported here is characterized by atypical alexia syndrome components .", "these include relatively mild deficit , transience , a mixture of syndromic components with rapid recovery . the lack of hemispheric lesions on magnetic resonance imaging using standard t2 weighted images diffusion weighted imaging for infarct exclusion , fluid attenuation inversion recovery sequences for covert white matter lesions and gradient echo sequences to exclude minor hemorrhagic lesions , implicated the thalamic lesion alone as the critical lesion in the syndrome described .", "although not a routine stroke investigative tool , functional magnetic imaging ( f - mri ) would have been an important additional neuroimaging modality to assess whether the right hemisphere had or had not played a role", ". however , the rapidity of recovery precluded functional magnetic imaging , that may have helped ascertain whether right hemisphere linguistic processing was operative , the contemporary hypothesis of semantic paralexia ."], "abstract_text": ["<S> alexia may be divided into different subtypes , with semantic paralexia being particularly rare . a 57 year old woman with a discreet left thalamic </S>", "<S> stroke and semantic paralexia is described . </S>", "<S> language evalution with the boston diagnostic aphasia battery confirmed the semantic paralexia ( deep alexia ) . </S>", "<S> multimodality magnetic resonance imaging brain scanning excluded other cerebral lesions . a good recovery ensued . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Index patient", "Neuroimaging", "Cognitive testing", "Discussion"], "sections": [["the acquired alexias may be categorized into posterior , anterior , central and deep alexias .", "analagous to semantic paraphasias , semantic paralexias constitute the substitution of content related words during reading . unlike the analogous subcortical aphasias such as thalamic aphasia , subcortical syndromes of alexia have rarely been described .", "this report depicts a patient with thalamic alexia with features of deep alexia ( or paralexia ) , the latter which has not been described after review of the english based literature ."], ["a 57 year old white , right handed , english speaking woman with 13 years of education , presented to our stroke center with mild right sided numbness and weakness ( graded + 4/5 medical research council grading scale ) with the family reporting transient confused conversation and speech difficulty .", "the clinical evaluation at our center encompasses a cognitive screening evaluation in all patients followed by psychometric assessment in selected patients described in detail elsewhere .", "no dysnomia was documented . specifically visual acuity and visual fields were normal and there was no object agnosia , dyschromatopsia , dyscalculia , finger agnosia , right left disorientation , or hemineglect syndrome .", "the substitution of semantically related words was documented with only mild slowing in her reading ( family corroboration ) .", "cerebrovascular risk factors included a significant smoking history and on investigation hyperhomocyteinemia ( 15.6 mmol / l ) . within 4 days , the reading difficulty had normalized but she remained with mild speech dysfluency , right hand ideomotor apraxia and developed a dejerine roussey or post stroke thalamic pain syndrome .", "multimodality magnetic resonance imaging investigations revealed a left lateral posterior thalamic infarct , well circumscribed on t2 weighted and diffusion weighted imaging without other brain parenchymal lesion ( figure 1 ) .", "magnetic resonance angiography revealed a left internal carotid artery stenosis graded 5074% and basilar artery fenestration . \n", "figure 1left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) . \n  left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) . in view of the isolated reading impairment ,", "additional psychometric examination was performed on day 3 after the stroke onset , with the boston diagnostic aphasia examination ( third edition - short form ) .", "in addition , the test for semantic paralexia prone words were tested with the standard version of the boston diagnostic aphasia examination ( version iii ) ( table 1 ) . in comparison to normative data ,", "notable deficiencies were noted only in speech fluency , articulation , oral word and sentence reading , picture word matching and writing . \n", "table 1boston diagnostic aphasia test - version iii.subtests%fluency  phrase length30  melodic line60  grammatical form70conversation expository speech100auditory comprehension  basic word discrimination100 ", "commands100  complex ideational material100articulation ( agility)70recitation ( automatized sequences)100repetition  words60  sentences100naming  responsive naming100  boston naming test100  special categories100paraphasia100reading  matching case and scripts100  number matching100  picture word matching40  oral word reading40  oral sentence reading80  oral sentence comprehension100  sentence / paragraph100  comprehension100wiriting  form20  letter choice40  motor facility40 \n"], ["a 57 year old white , right handed , english speaking woman with 13 years of education , presented to our stroke center with mild right sided numbness and weakness ( graded + 4/5 medical research council grading scale ) with the family reporting transient confused conversation and speech difficulty .", "the clinical evaluation at our center encompasses a cognitive screening evaluation in all patients followed by psychometric assessment in selected patients described in detail elsewhere .", "no dysnomia was documented . specifically visual acuity and visual fields were normal and there was no object agnosia , dyschromatopsia , dyscalculia , finger agnosia , right left disorientation , or hemineglect syndrome .", "the substitution of semantically related words was documented with only mild slowing in her reading ( family corroboration ) .", "cerebrovascular risk factors included a significant smoking history and on investigation hyperhomocyteinemia ( 15.6 mmol / l ) .", "within 4 days , the reading difficulty had normalized but she remained with mild speech dysfluency , right hand ideomotor apraxia and developed a dejerine roussey or post stroke thalamic pain syndrome ."], ["multimodality magnetic resonance imaging investigations revealed a left lateral posterior thalamic infarct , well circumscribed on t2 weighted and diffusion weighted imaging without other brain parenchymal lesion ( figure 1 ) .", "magnetic resonance angiography revealed a left internal carotid artery stenosis graded 5074% and basilar artery fenestration . \n", "figure 1left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) . \n  left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan ( arrows ) ."], ["in view of the isolated reading impairment , additional psychometric examination was performed on day 3 after the stroke onset , with the boston diagnostic aphasia examination ( third edition - short form ) .", "in addition , the test for semantic paralexia prone words were tested with the standard version of the boston diagnostic aphasia examination ( version iii ) ( table 1 ) . in comparison to normative data ,", "notable deficiencies were noted only in speech fluency , articulation , oral word and sentence reading , picture word matching and writing . \n", "table 1boston diagnostic aphasia test - version iii.subtests%fluency  phrase length30  melodic line60  grammatical form70conversation expository speech100auditory comprehension  basic word discrimination100 ", "commands100  complex ideational material100articulation ( agility)70recitation ( automatized sequences)100repetition  words60  sentences100naming  responsive naming100  boston naming test100  special categories100paraphasia100reading  matching case and scripts100  number matching100  picture word matching40  oral word reading40  oral sentence reading80  oral sentence comprehension100  sentence / paragraph100  comprehension100wiriting  form20  letter choice40  motor facility40 \n"], ["the commonly referred to syndromic classification of benson and geschwind of posterior alexia ( alexia without agraphia ) , central alexia ( alexia with agraphia ) , anterior alexia ( alexia in association with expressive dysphasia ) and deep dyslexia ( primarily semantic paralexia disorder ) aptly describes most acquired reading deficiencies .", "alexia secondary to isolated thalamic lesion has not been reported other than in combination with left occipital lesions , the latter lesions the usual focus .", "semantic paralexia due to thalamic lesions has not been reported previously with neuroanatomical lesions mostly referred to as large perisylvian and even right hemisphere lesions implicated .", "similar to subcortical aphasias , the subcortical ( thalamic ) alexia reported here is characterized by atypical alexia syndrome components .", "these include relatively mild deficit , transience , a mixture of syndromic components with rapid recovery . the lack of hemispheric lesions on magnetic resonance imaging using standard t2 weighted images diffusion weighted imaging for infarct exclusion , fluid attenuation inversion recovery sequences for covert white matter lesions and gradient echo sequences to exclude minor hemorrhagic lesions , implicated the thalamic lesion alone as the critical lesion in the syndrome described .", "although not a routine stroke investigative tool , functional magnetic imaging ( f - mri ) would have been an important additional neuroimaging modality to assess whether the right hemisphere had or had not played a role", ". however , the rapidity of recovery precluded functional magnetic imaging , that may have helped ascertain whether right hemisphere linguistic processing was operative , the contemporary hypothesis of semantic paralexia ."]]}
{"article_id": "PMC4939352", "article_text": ["proliferative vitreoretinopathy ( pvr ) remains the major complication after retinal detachment surgery [ 13 ] .", "pvr was identified as an independent clinical entity in 1983 by the retina society terminology committee and a classification was created , based on the condition formerly named  massive vitreous traction  or  massive periretinal proliferation  [ 57 ] .", "this classification divided pvr into four stages , a , b , c , and d , apparently by increasing its severity , from minimal to massive pvr ( table 1 ) . nevertheless , this classification had numerous limitations .", "it did not consider the location of the vitreoretinal traction and the magnitude of the contraction .", "in addition , some of the stages provide a false idea of severity ; for instance , d4 caused by a localized epiretinal membrane could be more easily treated by surgery than a c1 caused by intraretinal changes [ 8 , 9 ] . in 1989 , the silicone study group introduced a new classification   including some characteristics , such as the location , anterior or posterior , and the type of contraction ( table 2 ) .", "classification was then updated in 1991   according to modifications proposed by the silicone study group and also by other authors   ( table 3 ) .", "this classification appears to be difficult to use in clinical practice and may not offer any special advantage for decision - making in relation to the treatment of the disease .", "moreover , the classifications might have prevented advances in the understanding of the disease pathogenesis .", "for instance , because the retina society committee defined pvr as a  proliferative disease ,  many treatments based on the inhibition of cell proliferation were developed for more than 20 years , none of which appears to have produced a significant clinical advance .", "therefore , a review of both the classification and the pathogenesis of pvr appears to be appropriate to aid the development of new treatments .", "there is a clinical impression that over the last 15 years clinicians have abandoned current pvr classifications .", "thus , the purpose of this study has been to evaluate the current use of pvr classifications in papers dealing with clinical practice and therapy as an indirect measure of the degree of usefulness of existing classifications .", "a search in pubmed for papers published between january 2000 and january 2014 was undertaken by two independent researchers .", "inclusion criteria comprised human studies published in english , french , and spanish on pvr or retinal detachment with specific mention to pvr .", "prospective randomized and nonrandomized clinical studies , retrospective clinical studies , short series , and pilot studies were included .", "a manual search of related articles was also performed through references reported in each article .", "we also analyzed how the different authors interpreted the classifications , comparing the results they provided with the original description .", "therefore , we investigated if they used all the characteristics ( grade [ a , b , c ] , pvr localization , extension in hours , and type ) of the updated retina society classification or if they were used only partially .", "furthermore , it was also noted whether classifications were used both before and after an eventual pvr pharmacological or surgical treatment .", "data were extracted using a custom - made data sheet performed with agreement between authors .", "preliminary search identified a total of 219 publications , 81 of which were eliminated as they did not fulfill the inclusion criteria .", "35 articles ( 25,4% ) did not undergo detailed analysis because there was no reference to the type of pvr classification used by the authors or because no classification was used ( generic terms such as initial or severe pvr or simply pvr were adopted , without using any grading system ) .", "103 publications ( 74,6% ) used standardized pvr classifications and they were analyzed in detail .", "the most used classification was the updated retina society classification   ( 58 cites ; 56,3% ) , followed by the first retina society classification   ( 35 cites ; 33,9% ) .", "in addition , 3 authors ( 2,9% ) used modified or customized classifications [ 1315 ] . in 4 publications ( 3,8% ) , errors in the stated classification", "were identified [ 13 , 1618 ] : in two papers [ 16 , 18 ] , the authors cite the updated retina society (  91 ) but in fact they used the first retina society classification (  83 ) , koerner et al . , after citing the updated retina society classification used a customized one , and roldn - pallars et al .", "cite the two retina society classifications at the same time . among the papers using the updated retina society classification , the most documented was grade c pvr ( 48 articles , 83% ) , while grades b and a were less frequently documented ( 15 ( 26% ) and 7 ( 12% ) articles , resp . ) .", "of the publications documenting only c grade , the classification of subtypes was infrequently used . in 5 of 48 publications ( 10,4% )", ", there was a full c grade description in terms of localization ( anterior or posterior ) , extension ( in clock hours ) , and type ( focal , diffuse , subretinal , circumferential , or anterior displacement ) .", "a similar finding was observed when authors used the first retina society classification ( 35 papers ) : only c and d grades were frequently reported ( 27 and 10 papers , resp . ) , while grades a and b were infrequently used ( 4 and 7 articles , resp . ) .", "when the silicone study classification was used , only grade c was mentioned ( in 2 out of 4 articles ) .", "finally , there were 2 publications ( 1,9% ) in which the standard classifications were used to assess changes in pvr status after any treatment , reporting pvr grade before and after it [ 19 , 20 ] .", "the remaining authors used the classifications only to describe pvr status , rather than using them to analyze the improvement or worsening of the previous stage after the applied treatment .", "classifications of pvr were developed to provide clinicians with a useful tool to compare results of treatments .", "they are , to date , purely descriptive and do not reflect the pathobiology of this complex vitreoretinal disease .", "the original classification of 1983 , which was relatively simple to use , induces errors in the estimating severity in some cases .", "as mentioned , d grades were based on the ophthalmoscopic appearance of the detached retina , and experience demonstrated that some of these cases could be relatively easily solved by peeling localized epiretinal membranes although , in some cases , classified as c , intraretinal severe changes prevent reattachment , unless complicated surgical techniques were used .", "subsequent classifications [ 10 , 11 ] were more detailed and therefore more complicated to use on a routine basis .", "they incorporated the anterior pvr forms , which add severity to the case , but until now no one has incorporated the intraretinal changes , observed in many surgical retinal samples [ 8 , 9 ] , which may prevent the anatomical reattachment of the retina and require retinectomy .", "another important limitation is that they do not provide information on the activity of the process , although it has been observed that pvr progresses through several stages [ 13 ] .", "this may be crucial in estimating the risk of reproliferation after surgery or when surgeons schedule removing silicone oil , and no data on the chronology of events is included in any classification .", "furthermore , current classifications are not prognostic and do not correlate with visual prognosis or anatomical success after surgical treatment", ". moreover , some of these classifications can be difficult to use in clinical practice , due to their complexity ( grade , localization , extension , type , etc . ) and because they may not provide useful information they have been largely abandoned by clinicians .", "our findings in this study demonstrate that , in clinical research , investigators are using them inconsistently and reporting limited observations of stages ( mostly grade c ) .", "some authors simply do not use any , preferring generic terms , such as minimal , moderate , and severe pvr [ 21 , 22 ] .", "furthermore , early stages of pvr ( named as grades a and b ) , which are common to all classifications , are not used , and most authors refer only to most advanced stages , basically grade c [ 20 , 23 ] . moreover", ", the presence of classification errors could indicate that there is confusion concerning their use [ 13 , 1618 ] .", "an important relevant finding is that only a limited number of clinicians appropriately and fully use the current updated classification of 1991 [ 2427 ] , while the majority of authors avoid using the added characteristics of localization , extension , and type , limiting the description to the grade .", "in addition , many colleagues still use the first reported classification , probably because although it presents many limitations , it is easier to use compared to the last version [ 23 , 28 ] .", "additionally , some authors decided to use alternative classification , probably to avoid problems evaluating pvr stages .", "the use of multiple classifications makes the efficient communication between clinicians and the comparison of different studies very problematic .", "as mentioned , one of the aims of this study was to evaluate the usefulness of these classifications for pvr management with surgery and other adjunctive treatments .", "unfortunately , many ophthalmologists did not use the normalized classifications for comparing the outcomes using non - clearly defined terms such as pvr recurrence [ 29 , 30 ] .", "our findings showed the inconsistent and limited use of the current pvr classifications suggesting that it may be of benefit to produce a revised classification incorporating , if possible , the new knowledge on pvr which has been published since 1991 , pointing out new potential targets for therapeutic agents distinct from those , mainly proliferative agents , targeted by the original description of this disease .", "thus , it is possible that we could reduce its prevalence after retinal detachment surgery and to improve the anatomical and functional results of this disease which resists the attempts of both basic researchers and clinicians for more than 30 years ."], "abstract_text": ["<S> \n purpose . to evaluate the current and suitable use of current proliferative vitreoretinopathy ( pvr ) classifications in clinical publications related to treatment </S>", "<S> . methods . </S>", "<S> a pubmed search was undertaken using the term  proliferative vitreoretinopathy therapy  . </S>", "<S> outcome parameters were the reported pvr classification and pvr grades . </S>", "<S> the way the classifications were used in comparison to the original description was analyzed . </S>", "<S> classification errors were also included . </S>", "<S> it was also noted whether classifications were used for comparison before and after pharmacological or surgical treatment . </S>", "<S> results . </S>", "<S> 138 papers were included . </S>", "<S> 35 of them ( 25.4% ) presented no classification reference or did not use any one . </S>", "<S> 103 publications ( 74.6% ) used a standardized classification . </S>", "<S> the updated retina society classification , the first retina society classification , and the silicone study classification were cited in 56.3% , 33.9% , and 3.8% papers , respectively . </S>", "<S> furthermore , 3 authors ( 2.9% ) used modified - customized classifications and 4 ( 3.8% ) classification errors were identified . </S>", "<S> when the updated retina society classification was used , only 10.4% of authors used a full c grade description . </S>", "<S> finally , only 2 authors reported pvr grade before and after treatment . conclusions . </S>", "<S> our findings suggest that current classifications are of limited value in clinical practice due to the inconsistent and limited use and that it may be of benefit to produce a revised classification . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["proliferative vitreoretinopathy ( pvr ) remains the major complication after retinal detachment surgery [ 13 ] .", "pvr was identified as an independent clinical entity in 1983 by the retina society terminology committee and a classification was created , based on the condition formerly named  massive vitreous traction  or  massive periretinal proliferation  [ 57 ] .", "this classification divided pvr into four stages , a , b , c , and d , apparently by increasing its severity , from minimal to massive pvr ( table 1 ) . nevertheless , this classification had numerous limitations .", "it did not consider the location of the vitreoretinal traction and the magnitude of the contraction .", "in addition , some of the stages provide a false idea of severity ; for instance , d4 caused by a localized epiretinal membrane could be more easily treated by surgery than a c1 caused by intraretinal changes [ 8 , 9 ] . in 1989 , the silicone study group introduced a new classification   including some characteristics , such as the location , anterior or posterior , and the type of contraction ( table 2 ) .", "classification was then updated in 1991   according to modifications proposed by the silicone study group and also by other authors   ( table 3 ) .", "this classification appears to be difficult to use in clinical practice and may not offer any special advantage for decision - making in relation to the treatment of the disease .", "moreover , the classifications might have prevented advances in the understanding of the disease pathogenesis .", "for instance , because the retina society committee defined pvr as a  proliferative disease ,  many treatments based on the inhibition of cell proliferation were developed for more than 20 years , none of which appears to have produced a significant clinical advance .", "therefore , a review of both the classification and the pathogenesis of pvr appears to be appropriate to aid the development of new treatments .", "there is a clinical impression that over the last 15 years clinicians have abandoned current pvr classifications .", "thus , the purpose of this study has been to evaluate the current use of pvr classifications in papers dealing with clinical practice and therapy as an indirect measure of the degree of usefulness of existing classifications ."], ["a search in pubmed for papers published between january 2000 and january 2014 was undertaken by two independent researchers .", "inclusion criteria comprised human studies published in english , french , and spanish on pvr or retinal detachment with specific mention to pvr .", "prospective randomized and nonrandomized clinical studies , retrospective clinical studies , short series , and pilot studies were included .", "a manual search of related articles was also performed through references reported in each article .", "we also analyzed how the different authors interpreted the classifications , comparing the results they provided with the original description .", "therefore , we investigated if they used all the characteristics ( grade [ a , b , c ] , pvr localization , extension in hours , and type ) of the updated retina society classification or if they were used only partially .", "furthermore , it was also noted whether classifications were used both before and after an eventual pvr pharmacological or surgical treatment .", "data were extracted using a custom - made data sheet performed with agreement between authors ."], ["preliminary search identified a total of 219 publications , 81 of which were eliminated as they did not fulfill the inclusion criteria .", "35 articles ( 25,4% ) did not undergo detailed analysis because there was no reference to the type of pvr classification used by the authors or because no classification was used ( generic terms such as initial or severe pvr or simply pvr were adopted , without using any grading system ) .", "103 publications ( 74,6% ) used standardized pvr classifications and they were analyzed in detail .", "the most used classification was the updated retina society classification   ( 58 cites ; 56,3% ) , followed by the first retina society classification   ( 35 cites ; 33,9% ) .", "in addition , 3 authors ( 2,9% ) used modified or customized classifications [ 1315 ] . in 4 publications ( 3,8% ) , errors in the stated classification", "were identified [ 13 , 1618 ] : in two papers [ 16 , 18 ] , the authors cite the updated retina society (  91 ) but in fact they used the first retina society classification (  83 ) , koerner et al . , after citing the updated retina society classification used a customized one , and roldn - pallars et al .", "cite the two retina society classifications at the same time . among the papers using the updated retina society classification , the most documented was grade c pvr ( 48 articles , 83% ) , while grades b and a were less frequently documented ( 15 ( 26% ) and 7 ( 12% ) articles , resp . ) .", "of the publications documenting only c grade , the classification of subtypes was infrequently used . in 5 of 48 publications ( 10,4% )", ", there was a full c grade description in terms of localization ( anterior or posterior ) , extension ( in clock hours ) , and type ( focal , diffuse , subretinal , circumferential , or anterior displacement ) .", "a similar finding was observed when authors used the first retina society classification ( 35 papers ) : only c and d grades were frequently reported ( 27 and 10 papers , resp . ) , while grades a and b were infrequently used ( 4 and 7 articles , resp . ) .", "when the silicone study classification was used , only grade c was mentioned ( in 2 out of 4 articles ) .", "finally , there were 2 publications ( 1,9% ) in which the standard classifications were used to assess changes in pvr status after any treatment , reporting pvr grade before and after it [ 19 , 20 ] .", "the remaining authors used the classifications only to describe pvr status , rather than using them to analyze the improvement or worsening of the previous stage after the applied treatment ."], ["classifications of pvr were developed to provide clinicians with a useful tool to compare results of treatments .", "they are , to date , purely descriptive and do not reflect the pathobiology of this complex vitreoretinal disease .", "the original classification of 1983 , which was relatively simple to use , induces errors in the estimating severity in some cases .", "as mentioned , d grades were based on the ophthalmoscopic appearance of the detached retina , and experience demonstrated that some of these cases could be relatively easily solved by peeling localized epiretinal membranes although , in some cases , classified as c , intraretinal severe changes prevent reattachment , unless complicated surgical techniques were used .", "subsequent classifications [ 10 , 11 ] were more detailed and therefore more complicated to use on a routine basis .", "they incorporated the anterior pvr forms , which add severity to the case , but until now no one has incorporated the intraretinal changes , observed in many surgical retinal samples [ 8 , 9 ] , which may prevent the anatomical reattachment of the retina and require retinectomy .", "another important limitation is that they do not provide information on the activity of the process , although it has been observed that pvr progresses through several stages [ 13 ] .", "this may be crucial in estimating the risk of reproliferation after surgery or when surgeons schedule removing silicone oil , and no data on the chronology of events is included in any classification .", "furthermore , current classifications are not prognostic and do not correlate with visual prognosis or anatomical success after surgical treatment", ". moreover , some of these classifications can be difficult to use in clinical practice , due to their complexity ( grade , localization , extension , type , etc . ) and because they may not provide useful information they have been largely abandoned by clinicians .", "our findings in this study demonstrate that , in clinical research , investigators are using them inconsistently and reporting limited observations of stages ( mostly grade c ) .", "some authors simply do not use any , preferring generic terms , such as minimal , moderate , and severe pvr [ 21 , 22 ] .", "furthermore , early stages of pvr ( named as grades a and b ) , which are common to all classifications , are not used , and most authors refer only to most advanced stages , basically grade c [ 20 , 23 ] . moreover", ", the presence of classification errors could indicate that there is confusion concerning their use [ 13 , 1618 ] .", "an important relevant finding is that only a limited number of clinicians appropriately and fully use the current updated classification of 1991 [ 2427 ] , while the majority of authors avoid using the added characteristics of localization , extension , and type , limiting the description to the grade .", "in addition , many colleagues still use the first reported classification , probably because although it presents many limitations , it is easier to use compared to the last version [ 23 , 28 ] .", "additionally , some authors decided to use alternative classification , probably to avoid problems evaluating pvr stages .", "the use of multiple classifications makes the efficient communication between clinicians and the comparison of different studies very problematic .", "as mentioned , one of the aims of this study was to evaluate the usefulness of these classifications for pvr management with surgery and other adjunctive treatments .", "unfortunately , many ophthalmologists did not use the normalized classifications for comparing the outcomes using non - clearly defined terms such as pvr recurrence [ 29 , 30 ] ."], ["our findings showed the inconsistent and limited use of the current pvr classifications suggesting that it may be of benefit to produce a revised classification incorporating , if possible , the new knowledge on pvr which has been published since 1991 , pointing out new potential targets for therapeutic agents distinct from those , mainly proliferative agents , targeted by the original description of this disease .", "thus , it is possible that we could reduce its prevalence after retinal detachment surgery and to improve the anatomical and functional results of this disease which resists the attempts of both basic researchers and clinicians for more than 30 years ."]]}
{"article_id": "PMC5367521", "article_text": ["the number of total joint arthroplasties ( tjas ) performed has increased steadily in recent years , with projected numbers for the coming years rising further . consequently", "according to recent data , peri - prosthetic joint infection ( pji ) incidence constitutes between approximately 0.3% and 1.7% of all total hip arthroplasties ( tha ) , and between 0.8% and 1.9% of all total knee arthroplasties ( tka ) .", "pji can be classified in intra - operative , early post - operative , acute haematogenous and chronic infections , according to both timing and cause of infection .", "the risk factors potentially correlated with acute pji infection can be divided into pre - operative ( usually related to patient comorbidities ) , peri - operative and post - operative , which are mainly linked to the behaviours of the surgeon and the hospital staff .", "conversely , chronic infections are less influenced by the conduct of the surgeon , as they are most often related to haematogenous diffusion of bacteria .", "the aim of this paper is to review the recent literature , summarising the most relevant risk factors that the surgeon can modify in order to reduce the incidence of peri - prosthetic joint infection .", "several studies demonstrated that some comorbidity can be associated with an increased risk of pji .", "the american academy of orthopaedic surgeons ( aaos ) reported on the different risk factors for pji and developed a guideline for pji prevention and treatment . in 2013", "consensus for definition , prevention and management of pji , as an update to the previous guidelines .", "potential risk factors for development of surgical site infection or peri - prosthetic joint infection after elective total joint arthroplasty , according to the international consensus on periprosthetic joint infection bmi , body mass index ; hba1c , glycated haemoglobin .", "although the majority of these conditions must be considered as non - modifiable factors , many preventive measures may be adopted to reduce their impact on the development of pji .", "table 2 summarises the modifiable and non - modifiable factors related to pji , including some preventive measures to manage reversible medical comorbidities , according to the existing literature .", "pre - operative modifiable and non - modifiable risk factors ; measures the surgeon can adopt to reduce impact of risk factors on development of pji pji , peri - prosthetic joint infections ; bmi , body mass index ; hba1c , glycated haemoglobin ; mrsa , methicillin - resistant staphylococcus aureus ; s. aureus , staphylococcus aureus other potential pre - operative risk factors are intra - articular corticosteroid injections and any infectious disease , particularly urinary tract infection ( uti ) and nasal colonisation with staphylococcus ( s. ) aureus .", "the relationship between steroid injections and post - operative pji was evaluated in several studies .", "papavasiliou et al reported an incidence of only 2% of infections in a series of 114 tkas , but all of the infected tkas had previously been treated with an intra - articular corticosteroid injection within the 11-month period prior to surgery .", "conversely , desai et al stated that the incidence of infection did not increase in patients with prior steroid injection treatment .", "the correlation between post - operative utis and pji has been demonstrated . however , the association between pre - operative bacteriuria and early deep infections remains uncertain .", "the presence of symptoms of a uti in association with urinary leukocyte counts greater than 1  10/ml and a bacterial count greater than 1  10/ml should be the only indication for surgical delay .", "conversely , in asymptomatic patients it is still possible to proceed with tja by treating those patients with urine colony counts greater than 1  10/ml .", "different authors confirmed that being a high - level nasal carrier of s. aureus is an important and significant independent risk factor for developing ssi with s. aureus .", "nasal application of mupirocin is widely accepted as treatment for nasal carriers of s. aureus . in a recent randomised controlled trial ( rct ) ,", "mupirocin treatment resulted in a simple , safe and cost - effective intervention that can reduce the risk of ssi .", "different intra - operative components may play an important role as risk factors for developing pji ( table 3).the first six hours following surgery are the most important regarding infection , as during those hours the numbers of bacteria multiply exponentially .", "maintaining a low blood level of bacteria in this period is critical , and for this reason prophylactic antibiotics are infused to decrease bacterial multiplication and to extend this  golden period .", "the pre - operative dose of antibiotics should be administered within one hour before the surgical incision ; this can be extended to two hours for vancomycin and fluoroquinolones .", "most authors agree that a single pre - operative prophylactic infusion of cefazolin ( 1 gr if < 80 kg ; 2 gr if > 80 kg ) is a good choice .", "however , recent studies have demonstrated that targeted use of vancomycin and cefazolin among patients undergoing revision tka significantly reduced the rate of overall infections , in particular of mrsa .", "surgeons should consider additional antibiotic administration if the surgery time is twice the length of the half - life of the antibiotic , or whenever the blood loss exceeds 2000 ml and fluid resuscitation is over 2000 ml . to reduce pji infection rate , some authors advocate using antibiotic - loaded bone cement ( albc ) for the cementation .", "however , it was demonstrated that routine use of albc does not change the pji rate , though it may be useful in the reduction of the pji rate in high - risk patients ( for example those with diabetes or immunosuppression ) .", "intra - operative factors potentially associated with pjis pji , peri - prosthetic joint infections surgical site preparation also plays a role in reducing pji rate .", "some authors have confirmed the reduction of pji in patients who underwent pre - admission surgical site preparation using chlorhexidine washing .", "different studies have evaluated the best solutions for surgical site preparation to reduce pji . in a rct conducted by darouiche et al , it was demonstrated that a chlorhexidine  alcohol solution was more protective than povidone  iodine against both superficial and deep infections .", "this is probably due to the more rapid action , persistent activity despite exposure to bodily fluids , and residual effect of chlorexidine compared with povidone .", "the preparation of the surgical site often includes using plastic adhesive drapes and sterile stockinette .", "however , a recent cochrane review showed no evidence that adhesive drapes reduce surgical site infection rates .", "the bactericidal action of incision drapes containing iodine is inferior to conventional skin preparation solutions , so using incision drapes as a substitute for conventional skin preparation is not recommended .", "furthermore , boekel et al concluded that the surgical field for tka can be contaminated by proximal microbial spread from the unprepared foot with the use of a sterile stockinette drape .", "the risk of pji is also directly correlated with the length of the surgery , which should be less than 2.5 hours as a reasonable cut - off point .", "zhu et al , in their meta - analysis , concluded that increased operative time is associated with a higher risk of pji development ( or = 2.18 , ci 95% 1.39 - 3.42 , p = 0.003 ) .", "however , these data may also be correlated with the high complexity of long procedures .", "furthermore , the surgeon s surgical volume may be directly associated with pji : surgeons with low volumes may have higher rates of infection .", "hand care is another crucial point in reducing pji infection ; hand surgical scrub recommendations were previously published by the centers for disease control and prevention . in particular , surgeons should remove debris from underneath the fingernails using a nail cleaner under running water , and either an antimicrobial soap or an alcohol - based hand rub should be used persistently for at least five minutes .", "different studies evaluated the number of glove changes necessary to reduce the risk of pji .", "of note , beldame et al recommended : renewing outer gloves after draping ( before placing a cutaneous adhesive ) ; opening the instrumentation secondarily , with a new glove change after handling instruments which may cause perforations ; renewing outer gloves after each surgical stage .", "no strong evidence is available in the literature regarding the appropriate number of glove changes .", "different authors recommended double gloving to reduce the risk of inner glove perforation , but no correlation with pji has been demonstrated .", "the role of the personal protection system ( pps ) in preventing pji is still debated .", "kearns et al demonstrated that the external surface of the pps can not be assumed to be sterile after removal from the original packaging , and they suggested the need to change gloves if the pps is touched or adjusted during the procedure .", "other authors agree with the consideration of pps as more a personal  protection than equipment specifically to reduce pji .", "light handles can be a source of contamination , and surgeons must minimise their handling as far as possible .", "furthermore a limited number of portable devices such as mobile telephones and tablet computers in the operating room is recommended , although no evidence in the literature is able to link their use to increased risk . as airborne pathogens are a potential source of infection , the location and length of time that surgical instruments remain exposed", "for this reason , all instruments should be opened in operating rooms with clean air systems .", "these basins are repeatedly used during a surgical procedure and should therefore be considered a potential source of contamination .", "anto et al demonstrated that 23.8% of specimens from splash basins tested positive for bacterial contamination , and they suggested that surgeons should stop using them .", "a recent systematic review showed no conclusive results regarding the utility of laminar flow in reducing pji rate .", "other authors agree that , despite the number of previous studies demonstrating the efficacy of laminar flow , more recent research has failed to prove this efficacy .", "ultraviolet light seems to be more effective when compared with laminar flow in reducing pji ; however it is characterised by potentially unacceptable health costs to operative personnel .", "a level iii study showed that the number of door openings had a role in increased infection rate .", "surgeons may also play a role in reducing the rate of pji using power - pulsed lavage or wound lavage at surgery . in a level iv study ,", "different post - operative variables may also play a role as risk factors for pji development .", "antibiotic prophylaxis for other surgical procedures before and after tja , such as dental care or urological procedures , seems to play a role as a ", "despite the lack of literature demonstrating the relationship between dental procedures and pji , the current recommendation of the aaos is to use antibiotic prophylaxis in patients with a tja who are undergoing dental procedures , as well as any other invasive procedure .", "furthermore , patients should be aware that any infection is a potential source of haematogenous dissemination . as previously reported by different authors , patients with tja who have an active infection anywhere in the body are at risk of developing a pji .", "for this reason , a prompt diagnosis and management of those infections is a mandatory prevention mechanism .", "there is still debate regarding the association between blood transfusion and pji . in a level", "ii study , pulido et al demonstrated that transfusion with allogenic blood is an independent risk factor for pji .", "patients receiving allogenic transfusions were 2.1 times more likely to develop pji compared with patients receiving no transfusion . in their study ,", "innerhofer et al concluded that allogenic filtered transfusion is an independent variable for pji prediction ( or 23.65 ; ci 95% , 1.3 - 422.1 ; p = 0.01 ) .", "furthermore , the centers for disease control and prevention guidelines defined peri - operative allogeneic transfusion as a potential risk factor for developing pji , but concluded that the interpretation of the existing literature is difficult due to variations in assessment criteria .", "however , different measures can be adopted to reduce the need for blood transfusion , such as pre - operative screening for anaemia and its treatment , intra - operative accurate haemostasis , minimisation of surgical time and use of tranexamic acid .", "haematoma and persistent wound drainage were also related to an increased pji rate ; these conditions should be treated promptly with antibiotic prophylaxis , a decrease in anticoagulation dose , surgical evacuation of the haematoma , irrigation and debridement and modular component exchange .", "recently , more advanced surgical bandages such as hydrofibre absorbent dressings were proposed , with the aim of reducing the medication to allow for better wound healing and to prevent bacteria from entering the wound site from the external environment . in a level ii", "study , cai et al concluded that advanced surgical dressings such as hydrofibre may contribute to a reduction in the incidence of acute pji .", "infection represents a major challenge in tja , and is costly and demanding to manage for both surgeons and patients .", "the main risk factors involved in pji development are divided into pre - operative , intra - operative and post - operative factors .", "the surgeon can act to reduce the impact of some reversible comorbidities , for example controlling glycaemia in diabetic patients or improving malnutrition .", "various intra - operative risk factors such as operating theatre traffic , use of light handles , pulsed lavage or number of glove changes may also be related to infection .", "post - operative risk factors include transient bacteraemia related to dental procedures or other infections , wound care and blood transfusion as well as haematoma , and wound drainage should be controlled with care .", "no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article ."], "abstract_text": ["<S> total joint arthroplasty ( tja ) is one of the most common orthopaedic procedures . </S>", "<S> nevertheless , several complications can lead to implant failure.peri-prosthetic joint infections ( pji ) certainly represent a significant challenge in tja , constituting a major cause of prosthetic revision . </S>", "<S> the surgeon may have an important role in reducing the pji rate by limiting the impact of significant risk factors associated to either the patient , the operative environment or the post - operative care.in the pre - operative period , several preventive measures may be adopted to manage reversible medical comorbidities . </S>", "<S> other recognised pre - operative risk factors are urinary tract infections , intra - articular corticosteroid injections and nasal colonisation with staphylococcus ( s. ) aureus , particularly the methicillin - resistant strain ( mrsa).in the intra - operative setting , protective measures for pji include antibiotic prophylaxis , surgical - site antisepsis and use of pre - admission chlorhexidine washing and pulsed lavage during surgery . in this </S>", "<S> setting , the use of plastic adhesive drapes and sterile stockinette , as well as using personal protection systems , do not clearly reduce the risk of infection . </S>", "<S> on the contrary , using sterile theatre light handles and splash basins as well as an increased traffic in the operating room are all associated with an increased risk for pji.in the post - operative period , other infections causing transient bacteraemia , blood transfusion and poor wound care are considered as risk factors for pji.cite this article : ratto n , arrigoni c , rosso f , bruzzone m , dettoni f , bonasia de , rossi r. total knee arthroplasty and infection : how surgeons can reduce the risks . </S>", "<S> efort open rev 2016;1 : 339 - 344 doi : 10.1302/2058 - 5241.1.000032 . </S>"], "labels": null, "section_names": ["Introduction", "Pre-operative factors", "Intra-operative factors", "Post-operative factors", "Conclusions", "Funding"], "sections": [["the number of total joint arthroplasties ( tjas ) performed has increased steadily in recent years , with projected numbers for the coming years rising further . consequently", "according to recent data , peri - prosthetic joint infection ( pji ) incidence constitutes between approximately 0.3% and 1.7% of all total hip arthroplasties ( tha ) , and between 0.8% and 1.9% of all total knee arthroplasties ( tka ) .", "pji can be classified in intra - operative , early post - operative , acute haematogenous and chronic infections , according to both timing and cause of infection .", "the risk factors potentially correlated with acute pji infection can be divided into pre - operative ( usually related to patient comorbidities ) , peri - operative and post - operative , which are mainly linked to the behaviours of the surgeon and the hospital staff .", "conversely , chronic infections are less influenced by the conduct of the surgeon , as they are most often related to haematogenous diffusion of bacteria .", "the aim of this paper is to review the recent literature , summarising the most relevant risk factors that the surgeon can modify in order to reduce the incidence of peri - prosthetic joint infection ."], ["several studies demonstrated that some comorbidity can be associated with an increased risk of pji .", "the american academy of orthopaedic surgeons ( aaos ) reported on the different risk factors for pji and developed a guideline for pji prevention and treatment . in 2013", "consensus for definition , prevention and management of pji , as an update to the previous guidelines .", "potential risk factors for development of surgical site infection or peri - prosthetic joint infection after elective total joint arthroplasty , according to the international consensus on periprosthetic joint infection bmi , body mass index ; hba1c , glycated haemoglobin .", "although the majority of these conditions must be considered as non - modifiable factors , many preventive measures may be adopted to reduce their impact on the development of pji .", "table 2 summarises the modifiable and non - modifiable factors related to pji , including some preventive measures to manage reversible medical comorbidities , according to the existing literature .", "pre - operative modifiable and non - modifiable risk factors ; measures the surgeon can adopt to reduce impact of risk factors on development of pji pji , peri - prosthetic joint infections ; bmi , body mass index ; hba1c , glycated haemoglobin ; mrsa , methicillin - resistant staphylococcus aureus ; s. aureus , staphylococcus aureus other potential pre - operative risk factors are intra - articular corticosteroid injections and any infectious disease , particularly urinary tract infection ( uti ) and nasal colonisation with staphylococcus ( s. ) aureus .", "the relationship between steroid injections and post - operative pji was evaluated in several studies .", "papavasiliou et al reported an incidence of only 2% of infections in a series of 114 tkas , but all of the infected tkas had previously been treated with an intra - articular corticosteroid injection within the 11-month period prior to surgery .", "conversely , desai et al stated that the incidence of infection did not increase in patients with prior steroid injection treatment .", "the correlation between post - operative utis and pji has been demonstrated . however , the association between pre - operative bacteriuria and early deep infections remains uncertain .", "the presence of symptoms of a uti in association with urinary leukocyte counts greater than 1  10/ml and a bacterial count greater than 1  10/ml should be the only indication for surgical delay .", "conversely , in asymptomatic patients it is still possible to proceed with tja by treating those patients with urine colony counts greater than 1  10/ml .", "different authors confirmed that being a high - level nasal carrier of s. aureus is an important and significant independent risk factor for developing ssi with s. aureus .", "nasal application of mupirocin is widely accepted as treatment for nasal carriers of s. aureus . in a recent randomised controlled trial ( rct ) ,", "mupirocin treatment resulted in a simple , safe and cost - effective intervention that can reduce the risk of ssi ."], ["different intra - operative components may play an important role as risk factors for developing pji ( table 3).the first six hours following surgery are the most important regarding infection , as during those hours the numbers of bacteria multiply exponentially .", "maintaining a low blood level of bacteria in this period is critical , and for this reason prophylactic antibiotics are infused to decrease bacterial multiplication and to extend this  golden period .", "the pre - operative dose of antibiotics should be administered within one hour before the surgical incision ; this can be extended to two hours for vancomycin and fluoroquinolones .", "most authors agree that a single pre - operative prophylactic infusion of cefazolin ( 1 gr if < 80 kg ; 2 gr if > 80 kg ) is a good choice .", "however , recent studies have demonstrated that targeted use of vancomycin and cefazolin among patients undergoing revision tka significantly reduced the rate of overall infections , in particular of mrsa .", "surgeons should consider additional antibiotic administration if the surgery time is twice the length of the half - life of the antibiotic , or whenever the blood loss exceeds 2000 ml and fluid resuscitation is over 2000 ml . to reduce pji infection rate , some authors advocate using antibiotic - loaded bone cement ( albc ) for the cementation .", "however , it was demonstrated that routine use of albc does not change the pji rate , though it may be useful in the reduction of the pji rate in high - risk patients ( for example those with diabetes or immunosuppression ) .", "intra - operative factors potentially associated with pjis pji , peri - prosthetic joint infections surgical site preparation also plays a role in reducing pji rate .", "some authors have confirmed the reduction of pji in patients who underwent pre - admission surgical site preparation using chlorhexidine washing .", "different studies have evaluated the best solutions for surgical site preparation to reduce pji . in a rct conducted by darouiche et al , it was demonstrated that a chlorhexidine  alcohol solution was more protective than povidone  iodine against both superficial and deep infections .", "this is probably due to the more rapid action , persistent activity despite exposure to bodily fluids , and residual effect of chlorexidine compared with povidone .", "the preparation of the surgical site often includes using plastic adhesive drapes and sterile stockinette .", "however , a recent cochrane review showed no evidence that adhesive drapes reduce surgical site infection rates .", "the bactericidal action of incision drapes containing iodine is inferior to conventional skin preparation solutions , so using incision drapes as a substitute for conventional skin preparation is not recommended .", "furthermore , boekel et al concluded that the surgical field for tka can be contaminated by proximal microbial spread from the unprepared foot with the use of a sterile stockinette drape .", "the risk of pji is also directly correlated with the length of the surgery , which should be less than 2.5 hours as a reasonable cut - off point .", "zhu et al , in their meta - analysis , concluded that increased operative time is associated with a higher risk of pji development ( or = 2.18 , ci 95% 1.39 - 3.42 , p = 0.003 ) .", "however , these data may also be correlated with the high complexity of long procedures .", "furthermore , the surgeon s surgical volume may be directly associated with pji : surgeons with low volumes may have higher rates of infection .", "hand care is another crucial point in reducing pji infection ; hand surgical scrub recommendations were previously published by the centers for disease control and prevention . in particular , surgeons should remove debris from underneath the fingernails using a nail cleaner under running water , and either an antimicrobial soap or an alcohol - based hand rub should be used persistently for at least five minutes .", "different studies evaluated the number of glove changes necessary to reduce the risk of pji .", "of note , beldame et al recommended : renewing outer gloves after draping ( before placing a cutaneous adhesive ) ; opening the instrumentation secondarily , with a new glove change after handling instruments which may cause perforations ; renewing outer gloves after each surgical stage .", "no strong evidence is available in the literature regarding the appropriate number of glove changes .", "different authors recommended double gloving to reduce the risk of inner glove perforation , but no correlation with pji has been demonstrated .", "the role of the personal protection system ( pps ) in preventing pji is still debated .", "kearns et al demonstrated that the external surface of the pps can not be assumed to be sterile after removal from the original packaging , and they suggested the need to change gloves if the pps is touched or adjusted during the procedure .", "other authors agree with the consideration of pps as more a personal  protection than equipment specifically to reduce pji .", "light handles can be a source of contamination , and surgeons must minimise their handling as far as possible .", "furthermore a limited number of portable devices such as mobile telephones and tablet computers in the operating room is recommended , although no evidence in the literature is able to link their use to increased risk . as airborne pathogens are a potential source of infection , the location and length of time that surgical instruments remain exposed", "for this reason , all instruments should be opened in operating rooms with clean air systems .", "these basins are repeatedly used during a surgical procedure and should therefore be considered a potential source of contamination .", "anto et al demonstrated that 23.8% of specimens from splash basins tested positive for bacterial contamination , and they suggested that surgeons should stop using them .", "a recent systematic review showed no conclusive results regarding the utility of laminar flow in reducing pji rate .", "other authors agree that , despite the number of previous studies demonstrating the efficacy of laminar flow , more recent research has failed to prove this efficacy .", "ultraviolet light seems to be more effective when compared with laminar flow in reducing pji ; however it is characterised by potentially unacceptable health costs to operative personnel .", "a level iii study showed that the number of door openings had a role in increased infection rate .", "surgeons may also play a role in reducing the rate of pji using power - pulsed lavage or wound lavage at surgery . in a level iv study ,"], ["different post - operative variables may also play a role as risk factors for pji development .", "antibiotic prophylaxis for other surgical procedures before and after tja , such as dental care or urological procedures , seems to play a role as a ", "despite the lack of literature demonstrating the relationship between dental procedures and pji , the current recommendation of the aaos is to use antibiotic prophylaxis in patients with a tja who are undergoing dental procedures , as well as any other invasive procedure .", "furthermore , patients should be aware that any infection is a potential source of haematogenous dissemination . as previously reported by different authors , patients with tja who have an active infection anywhere in the body are at risk of developing a pji .", "for this reason , a prompt diagnosis and management of those infections is a mandatory prevention mechanism .", "there is still debate regarding the association between blood transfusion and pji . in a level", "ii study , pulido et al demonstrated that transfusion with allogenic blood is an independent risk factor for pji .", "patients receiving allogenic transfusions were 2.1 times more likely to develop pji compared with patients receiving no transfusion . in their study ,", "innerhofer et al concluded that allogenic filtered transfusion is an independent variable for pji prediction ( or 23.65 ; ci 95% , 1.3 - 422.1 ; p = 0.01 ) .", "furthermore , the centers for disease control and prevention guidelines defined peri - operative allogeneic transfusion as a potential risk factor for developing pji , but concluded that the interpretation of the existing literature is difficult due to variations in assessment criteria .", "however , different measures can be adopted to reduce the need for blood transfusion , such as pre - operative screening for anaemia and its treatment , intra - operative accurate haemostasis , minimisation of surgical time and use of tranexamic acid .", "haematoma and persistent wound drainage were also related to an increased pji rate ; these conditions should be treated promptly with antibiotic prophylaxis , a decrease in anticoagulation dose , surgical evacuation of the haematoma , irrigation and debridement and modular component exchange .", "recently , more advanced surgical bandages such as hydrofibre absorbent dressings were proposed , with the aim of reducing the medication to allow for better wound healing and to prevent bacteria from entering the wound site from the external environment . in a level ii", "study , cai et al concluded that advanced surgical dressings such as hydrofibre may contribute to a reduction in the incidence of acute pji ."], ["infection represents a major challenge in tja , and is costly and demanding to manage for both surgeons and patients .", "the main risk factors involved in pji development are divided into pre - operative , intra - operative and post - operative factors .", "the surgeon can act to reduce the impact of some reversible comorbidities , for example controlling glycaemia in diabetic patients or improving malnutrition .", "various intra - operative risk factors such as operating theatre traffic , use of light handles , pulsed lavage or number of glove changes may also be related to infection .", "post - operative risk factors include transient bacteraemia related to dental procedures or other infections , wound care and blood transfusion as well as haematoma , and wound drainage should be controlled with care ."], ["no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article ."]]}
{"article_id": "PMC5005790", "article_text": ["\n the use of sodium hypochlorite as toilet detergent is related to a red - brown urine discolouration in patients taking mesalamine.it seems that the red - brown discolouration relates to a harmless reaction.polymerisation of mesalamine and/or metabolites could be a theoretical mechanism of this discolouration . \n", "the exact working mechanism of mesalamine is not fully understood , but it appears to have anti - inflammatory properties .", "the drug blocks interleukin-1 and tumour necrosis factor-. mesalamine also inhibits the cyclo - oxygenase pathway , leading to inhibition of prostaglandin e2 in inflamed intestine .", "the most common adverse drug reactions ( adrs ) , as described in the summary of product characteristics ( smpc ) , are headache , rash and gastrointestinal symptoms , including diarrhoea , nausea , vomiting and abdominal pain .", "mesalamine is also associated with the renal adrs of renal failure , interstitial nephritis and haematuria [ 1 , 3 ] . in the period from august 2009 to april 2015 , the netherlands pharmacovigilance centre lareb received two case reports of red - brown urine discolouration in association with mesalamine .", "both cases were reported by patients and the urine discoloured after contact with the toilet bowl .", "this case concerned a 38-year - old male with a history of crohn s disease .", "the patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 ( pentasa slow - release 500  mg tablet ) .", "the latest episode occurred in april 2015 , 2  days after the use of mesalamine .", "the patient had a mild form of crohn s disease that had been stable for 68  years and only used mesalamine during episodes of exacerbations .", "due to an exacerbation in september 2014 , the daily dose of mesalamine was increased from 2 to 4  g. during this dose escalation , the patient recognised the same red - brown deposit that had occurred during previous use . in january 2015 , the mesalamine was tapered over a period of 2  months to 2  g once daily and he remained stable .", "he observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl .", "most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days , despite flushing the toilet .", "of note , the deposit in the toilet bowl was more intense during the use of an increased dose of 4  g a day .", "the patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . in the evening", "a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . as the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent , he reported it to lareb .", "after the report had been assessed , the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test , to which he agreed ( fig .  1 ) .", "the urine ( 40  ml ) was mixed with 20  ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . after shaking the mixture ,", "1 \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) .", "agitation discoloured the urine completely ( right ) \n \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) .", "agitation discoloured the urine completely ( right ) this case concerned a 36-year - old woman with ulcerative colitis , for which she is treated with 2  g of mesalamine ( pentasa sachet 2  g prolonged - release granules ) twice daily .", "since she started taking mesalamine in june 2009 , her urine appeared red - brown in the toilet bowl , but only when she had recently cleaned the toilet with sodium hypochlorite detergent .", "when the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet , the same red - brown discolouration occurred immediately .", "the patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet .", "this case concerned a 38-year - old male with a history of crohn s disease .", "the patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 ( pentasa slow - release 500  mg tablet ) .", "the latest episode occurred in april 2015 , 2  days after the use of mesalamine .", "the patient had a mild form of crohn s disease that had been stable for 68  years and only used mesalamine during episodes of exacerbations .", "due to an exacerbation in september 2014 , the daily dose of mesalamine was increased from 2 to 4  g. during this dose escalation , the patient recognised the same red - brown deposit that had occurred during previous use . in january 2015 , the mesalamine was tapered over a period of 2  months to 2  g once daily and he remained stable .", "he observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl .", "most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days , despite flushing the toilet .", "of note , the deposit in the toilet bowl was more intense during the use of an increased dose of 4  g a day .", "the patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . in the evening", "a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . as the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent , he reported it to lareb .", "after the report had been assessed , the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test , to which he agreed ( fig .  1 ) .", "the urine ( 40  ml ) was mixed with 20  ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . after shaking the mixture ,", "1 \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) .", "agitation discoloured the urine completely ( right ) \n \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) .", "this case concerned a 36-year - old woman with ulcerative colitis , for which she is treated with 2  g of mesalamine ( pentasa sachet 2  g prolonged - release granules ) twice daily .", "since she started taking mesalamine in june 2009 , her urine appeared red - brown in the toilet bowl , but only when she had recently cleaned the toilet with sodium hypochlorite detergent .", "when the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet , the same red - brown discolouration occurred immediately .", "the patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet .", "likewise , the disordered haem production in patients with porphyria can result in red - brown discolouration .", "a red - brown urine discolouration could be perceived as a sign of possible renal impairment or other diseases ; however , neither of the described patients are known to have an impaired renal function or physical abnormalities .", "furthermore , various drugs are associated with red and/or brown discolouration , e.g. rifampicin , doxorubicin , metronidazole , methyldopa , levodopa and sulfasalazine .", "a notable difference from the described mesalamine cases is that the urine of patients taking the above - mentioned drugs , such as sulfasalazine , is already discoloured at micturition .", "a pubmed search was conducted and it was found that two cases of red - brown urine associated with mesalamine have been reported previously by sacks and davis .", "both of the male adolescents in these reports experienced the same red - brown urine discolouration after contact with sodium hypochlorite bleach .", "a similar experiment was performed to that performed by patient  a , and after adding sodium hypochlorite bleach to the collected urine the discolouration developed spontaneously . to our knowledge", ", there are no other published reports of this association after a chemical reaction with drugs structurally related to mesalamine ( sulfasalazine , olsalazine and 4-aminosalicylic acid ) .", "notably , there has been discussion regarding this phenomenon in several patient forums and blogs .", "case reports from altmann and mansell   do , however , describe urine discolouration in patients using methyldopa or levodopa after contact with lavatory bleach and exposure to light .", "normal biotransformation of methyldopa produces a variety of metabolites ; presumably , these metabolites have properties that form melanin by spontaneous polymerisation , which results in darkening of the urine .", "melanins are polymers of phenolic compounds ( e.g. dopaquinone , benzothiazole ) that are formed by an extensive bio pathway .", "melanin is an aggregate of smaller component molecules such as pheomelanin and eumelanin ; both of these are initially formed by oxidation of tyrosine and then undergo cysteinylation and cyclisation , respectively . the end product is ultimately formed by polymerisation of several different derivatives .", "altmann and mansell   found that urine samples that were made alkaline in patients treated with methyldopa or levodopa turned black on exposure to sunlight .", "mesalamine is acetylated by the enzyme n - acetyltransferase  1 ( nat1 ) in the liver and gut mucosal wall into the metabolite n - acetyl-5-aminosalicylic acid ( n - ac-5asa ) .", "evidence from the literature indicates that nat1 is polymorphic , but that these polymorphisms are associated with relatively minor effects on acetylation function .", "it is unknown whether the patients in our reports have a nat1 polymorphism , but if so we would expect no significant effect on the propensity of discolouring .", "furthermore , this acetylation is minimal when mesalamine and n - ac-5asa are both secreted back into the lumen by the drug efflux pump p - glycoprotein , excreted in the faeces , absorbed via the colon into the blood and , lastly , eliminated in the urine .", "the theory is that n - ac-5asa and/or mesalamine react with the sodium hypochlorite - containing bleach .", "sodium hypochlorite usually has a ph of 1113 and could therefore initiate the reaction as described by altmann and mansell . according to the cases reported by altmann and mansell ,", "it is also possible that a similar polymerisation of mesalamine and/or n - ac-5asa could cause the discolouration of the urine . perhaps due to the alkaline environment", ", partial deprotonation of n - ac-5asa and mesalamine could also contribute to this reaction .", "since the introduction of the national spontaneous reporting system in 2003 to the general public , consumers have now become the largest group of reporters in the netherlands .", "the quality of consumer reports are generally good and these reports highlight via first - hand information the real - life experience of an adr .", "being unconstrained with regards to the probability of causality , consumers may report relevant adrs that healthcare professionals may not recognise initially or consider to be unlikely .", "patient  b s gastroenterologist recognised the phenomenon , but it is conceivable that not every gastroenterologist will establish a direct relationship between urine discolouration , mesalamine therapy and the use of a specific detergent .", "the two reports received provide important information about a harmless reaction as this phenomenon probably concerns a lot of other patients using mesalamine .", "based on the described reports and the case reports in the literature , we suggest a causal relationship between red  brown discolouration of urine after contact with sodium hypochlorite detergent and the use of mesalamine .", "the naranjo assessment scores for cases a and b were 9 and 6 , indicating a certain and probable relationship , respectively .", "this seems to be a harmless reaction as the patients experienced no physical complaints and in both the lareb cases and those described by sacks and davis , the patients were under the care of a gastroenterologist . in the differential diagnosis it is important to be aware that the use of sodium hypochlorite as a toilet detergent could be the cause of coloured deposits in and on the toilet bowl .", "the presence of sodium hypochlorite does not exclude the possibility of renal impairment ; therefore , it is important to distinguish already discoloured urine from normal - coloured urine that only discoloured after contacting the toilet bowl .", "thus , to inform and reassure patients , it might be helpful to mention red - brown discolouration in the patient leaflet for mesalamine .", "no financial support was received for the conduct of this study or preparation of this manuscript ."], "abstract_text": ["<S> a 38-year - old male and a 36-year - old female experienced red - brown urine discolouration after 2 and 3  days , respectively , during the use of mesalamine for inflammatory bowel disease . </S>", "<S> both patients mentioned that the urine discoloured after contact with sodium hypochlorite detergent in toilet water . </S>", "<S> mesalamine and the inactive metabolite n - acetyl-5-aminosalicylic acid are primarily excreted in the urine . </S>", "<S> we hypothesised a possible reaction with sodium hypochlorite and/or light . </S>", "<S> naranjo assessment scores of 9 and 6 were obtained for the reports , indicating a certain and probable relationship , respectively , between the red - brown urine discolouration and the use of the suspect drug mesalamine . </S>", "<S> knowledge of this harmless reaction is desirable to avoid unnecessary physical examination and worry . </S>"], "labels": null, "section_names": ["Key Points", "Introduction", "Case Reports", "Patient A (Report Date April 2015)", "Patient B (Report Date August 2009)", "Discussion", "Conclusion", "Competing interests/funding"], "sections": [["\n the use of sodium hypochlorite as toilet detergent is related to a red - brown urine discolouration in patients taking mesalamine.it seems that the red - brown discolouration relates to a harmless reaction.polymerisation of mesalamine and/or metabolites could be a theoretical mechanism of this discolouration . \n"], ["the exact working mechanism of mesalamine is not fully understood , but it appears to have anti - inflammatory properties .", "the drug blocks interleukin-1 and tumour necrosis factor-. mesalamine also inhibits the cyclo - oxygenase pathway , leading to inhibition of prostaglandin e2 in inflamed intestine .", "the most common adverse drug reactions ( adrs ) , as described in the summary of product characteristics ( smpc ) , are headache , rash and gastrointestinal symptoms , including diarrhoea , nausea , vomiting and abdominal pain .", "mesalamine is also associated with the renal adrs of renal failure , interstitial nephritis and haematuria [ 1 , 3 ] . in the period from august 2009 to april 2015 , the netherlands pharmacovigilance centre lareb received two case reports of red - brown urine discolouration in association with mesalamine .", "both cases were reported by patients and the urine discoloured after contact with the toilet bowl ."], ["this case concerned a 38-year - old male with a history of crohn s disease .", "the patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 ( pentasa slow - release 500  mg tablet ) .", "the latest episode occurred in april 2015 , 2  days after the use of mesalamine .", "the patient had a mild form of crohn s disease that had been stable for 68  years and only used mesalamine during episodes of exacerbations .", "due to an exacerbation in september 2014 , the daily dose of mesalamine was increased from 2 to 4  g. during this dose escalation , the patient recognised the same red - brown deposit that had occurred during previous use . in january 2015 , the mesalamine was tapered over a period of 2  months to 2  g once daily and he remained stable .", "he observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl .", "most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days , despite flushing the toilet .", "of note , the deposit in the toilet bowl was more intense during the use of an increased dose of 4  g a day .", "the patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . in the evening", "a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . as the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent , he reported it to lareb .", "after the report had been assessed , the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test , to which he agreed ( fig .  1 ) .", "the urine ( 40  ml ) was mixed with 20  ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . after shaking the mixture ,", "1 \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) .", "agitation discoloured the urine completely ( right ) \n \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) .", "agitation discoloured the urine completely ( right ) this case concerned a 36-year - old woman with ulcerative colitis , for which she is treated with 2  g of mesalamine ( pentasa sachet 2  g prolonged - release granules ) twice daily .", "since she started taking mesalamine in june 2009 , her urine appeared red - brown in the toilet bowl , but only when she had recently cleaned the toilet with sodium hypochlorite detergent .", "when the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet , the same red - brown discolouration occurred immediately .", "the patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet ."], ["this case concerned a 38-year - old male with a history of crohn s disease .", "the patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 ( pentasa slow - release 500  mg tablet ) .", "the latest episode occurred in april 2015 , 2  days after the use of mesalamine .", "the patient had a mild form of crohn s disease that had been stable for 68  years and only used mesalamine during episodes of exacerbations .", "due to an exacerbation in september 2014 , the daily dose of mesalamine was increased from 2 to 4  g. during this dose escalation , the patient recognised the same red - brown deposit that had occurred during previous use . in january 2015 , the mesalamine was tapered over a period of 2  months to 2  g once daily and he remained stable .", "he observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl .", "most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days , despite flushing the toilet .", "of note , the deposit in the toilet bowl was more intense during the use of an increased dose of 4  g a day .", "the patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . in the evening", "a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . as the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent , he reported it to lareb .", "after the report had been assessed , the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test , to which he agreed ( fig .  1 ) .", "the urine ( 40  ml ) was mixed with 20  ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . after shaking the mixture ,", "1 \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) .", "agitation discoloured the urine completely ( right ) \n \n red  brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine ( left , right ) ."], ["this case concerned a 36-year - old woman with ulcerative colitis , for which she is treated with 2  g of mesalamine ( pentasa sachet 2  g prolonged - release granules ) twice daily .", "since she started taking mesalamine in june 2009 , her urine appeared red - brown in the toilet bowl , but only when she had recently cleaned the toilet with sodium hypochlorite detergent .", "when the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet , the same red - brown discolouration occurred immediately .", "the patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet ."], ["likewise , the disordered haem production in patients with porphyria can result in red - brown discolouration .", "a red - brown urine discolouration could be perceived as a sign of possible renal impairment or other diseases ; however , neither of the described patients are known to have an impaired renal function or physical abnormalities .", "furthermore , various drugs are associated with red and/or brown discolouration , e.g. rifampicin , doxorubicin , metronidazole , methyldopa , levodopa and sulfasalazine .", "a notable difference from the described mesalamine cases is that the urine of patients taking the above - mentioned drugs , such as sulfasalazine , is already discoloured at micturition .", "a pubmed search was conducted and it was found that two cases of red - brown urine associated with mesalamine have been reported previously by sacks and davis .", "both of the male adolescents in these reports experienced the same red - brown urine discolouration after contact with sodium hypochlorite bleach .", "a similar experiment was performed to that performed by patient  a , and after adding sodium hypochlorite bleach to the collected urine the discolouration developed spontaneously . to our knowledge", ", there are no other published reports of this association after a chemical reaction with drugs structurally related to mesalamine ( sulfasalazine , olsalazine and 4-aminosalicylic acid ) .", "notably , there has been discussion regarding this phenomenon in several patient forums and blogs .", "case reports from altmann and mansell   do , however , describe urine discolouration in patients using methyldopa or levodopa after contact with lavatory bleach and exposure to light .", "normal biotransformation of methyldopa produces a variety of metabolites ; presumably , these metabolites have properties that form melanin by spontaneous polymerisation , which results in darkening of the urine .", "melanins are polymers of phenolic compounds ( e.g. dopaquinone , benzothiazole ) that are formed by an extensive bio pathway .", "melanin is an aggregate of smaller component molecules such as pheomelanin and eumelanin ; both of these are initially formed by oxidation of tyrosine and then undergo cysteinylation and cyclisation , respectively . the end product is ultimately formed by polymerisation of several different derivatives .", "altmann and mansell   found that urine samples that were made alkaline in patients treated with methyldopa or levodopa turned black on exposure to sunlight .", "mesalamine is acetylated by the enzyme n - acetyltransferase  1 ( nat1 ) in the liver and gut mucosal wall into the metabolite n - acetyl-5-aminosalicylic acid ( n - ac-5asa ) .", "evidence from the literature indicates that nat1 is polymorphic , but that these polymorphisms are associated with relatively minor effects on acetylation function .", "it is unknown whether the patients in our reports have a nat1 polymorphism , but if so we would expect no significant effect on the propensity of discolouring .", "furthermore , this acetylation is minimal when mesalamine and n - ac-5asa are both secreted back into the lumen by the drug efflux pump p - glycoprotein , excreted in the faeces , absorbed via the colon into the blood and , lastly , eliminated in the urine .", "the theory is that n - ac-5asa and/or mesalamine react with the sodium hypochlorite - containing bleach .", "sodium hypochlorite usually has a ph of 1113 and could therefore initiate the reaction as described by altmann and mansell . according to the cases reported by altmann and mansell ,", "it is also possible that a similar polymerisation of mesalamine and/or n - ac-5asa could cause the discolouration of the urine . perhaps due to the alkaline environment", ", partial deprotonation of n - ac-5asa and mesalamine could also contribute to this reaction .", "since the introduction of the national spontaneous reporting system in 2003 to the general public , consumers have now become the largest group of reporters in the netherlands .", "the quality of consumer reports are generally good and these reports highlight via first - hand information the real - life experience of an adr .", "being unconstrained with regards to the probability of causality , consumers may report relevant adrs that healthcare professionals may not recognise initially or consider to be unlikely .", "patient  b s gastroenterologist recognised the phenomenon , but it is conceivable that not every gastroenterologist will establish a direct relationship between urine discolouration , mesalamine therapy and the use of a specific detergent .", "the two reports received provide important information about a harmless reaction as this phenomenon probably concerns a lot of other patients using mesalamine ."], ["based on the described reports and the case reports in the literature , we suggest a causal relationship between red  brown discolouration of urine after contact with sodium hypochlorite detergent and the use of mesalamine .", "the naranjo assessment scores for cases a and b were 9 and 6 , indicating a certain and probable relationship , respectively .", "this seems to be a harmless reaction as the patients experienced no physical complaints and in both the lareb cases and those described by sacks and davis , the patients were under the care of a gastroenterologist . in the differential diagnosis it is important to be aware that the use of sodium hypochlorite as a toilet detergent could be the cause of coloured deposits in and on the toilet bowl .", "the presence of sodium hypochlorite does not exclude the possibility of renal impairment ; therefore , it is important to distinguish already discoloured urine from normal - coloured urine that only discoloured after contacting the toilet bowl .", "thus , to inform and reassure patients , it might be helpful to mention red - brown discolouration in the patient leaflet for mesalamine ."], ["no financial support was received for the conduct of this study or preparation of this manuscript ."]]}
{"article_id": "PMC4906216", "article_text": ["diabetic retinopathy ( dr ) is a leading cause of visual impairment and blindness in developed countries .", "the decrease in vision is due to diabetic macular edema ( dme ) and proliferative diabetic retinopathy ( pdr ) [ 1 , 2 ] .", "the control of the blood glucose level , vitreous surgery , and photocoagulation are the major treatments used to prevent dr from progressing to the pdr stage .", "currently , intravitreal injections of antivascular endothelium growth factor ( vegf ) or steroids have become the primary therapy for dme [ 35 ] .", "however , the use of a single therapeutic agent is not effective in all cases , and additional treatments or different agents are needed . the blood retinal barrier ( brb )", "consists of two anatomical parts ; the inner brb is located within the endothelial cells of the retinal capillaries , and the outer brb is located between the retinal pigment epithelial ( rpe ) cells . an intact brb is required for an efficient and regulated control of fluids in the subretinal space and for the maintenance of healthy rpe and retinal cells .", "a breakdown of the outer brb results in an increase in the paracellular permeability between the rpe cells , and the leakage can cause retinal edema .", "the tight junctions ( tjs ) of the rpe cells are intercellular junctions located at the apical ends of the rpe cells , and they are integral structural components of the brb .", "tjs are made up of three tj related proteins , for example , the zonula occludens-1 ( zo-1 ) , occludin , and claudin .", "recent studies have shown that interactions between inflammatory cells and retinal cells are critical for the development of intraocular neovascularization [ 1012 ] .", "the results of these studies also demonstrated that , in eyes with pdr , an elevation of vegf was significantly correlated with the levels of several cytokines including interleukin- ( il- ) 6 , il-8 , and the monocyte chemoattractant protein-1 ( mcp-1 ) .", "another study showed that the levels of these cytokines were also strongly correlated with each other which suggest that there are common pathways involved in the inflammatory processes .", "in addition , various cytokines have been shown to be related to the maintenance of the conformation of the tj proteins .", "lecithin - bound iodine ( lbi , jolethin , daiichi pharmaceutical co. , tokyo , japan ) has been used clinically to reduce the antigen - induced immune responses in children with bronchial asthma .", "lbi acts on the peripheral blood mononuclear cells and downregulates the il-4-induced ige synthesis , which suggests that lbi have anti - inflammatory properties [ 15 , 16 ] . in patients with eye diseases ,", "lbi has been used for the absorption of retinal or vitreous bleeding , vitreous opacities , and improvement of central serous choroidopathy .", "the purpose of this study was to determine whether lbi will alter the integrity of the tjs of arpe-19 cells in culture .", "lbi ( jolethin , daiichi pharmaceutical co. , tokyo , japan ) was dissolved in distilled water and diluted to the appropriate concentration for each experiment .", "an earlier study showed that lbi solutions contain 48.250.3% lecithin - iodine , approximately 10% free lecithin , and 40% phosphatidylinositol .", "rabbit polyclonal anti - zo-1 antibody ( sc-10804 ) was purchased from santa cruz biotechnology ( santa cruz , ca ) , and alexa 594 anti - rabbit igg was purchased from invitrogen ( molecular probs , eugene , or ) .", "rabbit anti - mcp-1 antibody ( # 2029 ) was purchased from cell signaling technology ( danvers , ma ) and rabbit anti - eotaxin ( ccl-11 ) antibody ( ab133604 ) was purchased from abcam ( cambridge , ma ) .", "cells from a human retinal pigment epithelium cell line , arpe-19 , were grown in dulbecco 's modified eagle 's medium / ham 's f-12 supplemented with 10% fetal bovine serum ( fbs , hyclone laboratories , inc . ,", "logan , ut ) , 50  units / ml penicillin , and 50  g / ml streptomycin in an air-5% co2 atmosphere with constant humidity .", "prior to the experiments , cells were placed in serum - free media and pretreated with 50  g / ml of lbi or 250500  pg / ml of antibody ( anti - mcp-1 antibody and anti - chemokine ( c - c motif ) ligand-11 ( ccl-11 ) antibody ) for 24  h. to mimic hypoxic conditions , arpe-19 cells were incubated for 4  h with 100  m of cocl2 ( sigma , st .", "louis , mo ) , which is a chemical hypoxia - inducing agent . after the incubation ,", "optimum concentration of lbi was defined as 50  g / ml because over 100  g / ml of lbi seems to be toxic ( data not shown ) . for the immunofluorescence studies , cells", "were grown to a density of 2  10 cells / ml on 12  mm cover slips and fixed in 10% tricarboxylic acid for 10  min at 4c .", "they were then treated with 0.5% triton x-100 for 15  min . to detect the presence of zo-1 , the cultured rpe cells were exposed to rabbit polyclonal anti - zo-1 antibody as the primary antibody and alexa 594 anti - rabbit igg as the secondary antibody .", "samples were examined and photographed with a fluorescence microscope ( bz-9000 ; keyence , osaka , japan , and eclipse 50i , nikon , tokyo , japan ) , and the intensity of fluorescence was quantified with a bz - ii analyzer ( keyence ) .", "the levels of the bioactive molecules in the conditioned medium were determined by human il-6 , il-8 , and mcp-1 elisa kits ( r&d systems , minneapolis , mn ) and ccl-11 elisa kit ( biosensis , thebarton , south australia , australia ) .", "all experiments were repeated at least three times and values are presented as the means  standard deviations .", "data were analyzed by two - way nonrepeated analysis of variance ( anova ) followed by bonferroni post hoc tests for the comparison of the means .", "we first determined whether the lbi pretreatment affected the changes in the conformation of the tj proteins caused by the hypoxic stress induced by cocl2 .", "immunofluorescence microscopy showed that there appeared to be a disruption of tjs with cocl2 treatment ( figures 1(a ) and 1(b ) ) .", "when the arpe-19 cells were pretreated with lbi before the addition of cocl2 , the disruption of the tight junctions was not detected ( figures 1(c ) and 1(d ) ) .", "the signal intensity measurements showed that there was a significant decrease with the cocl2 addition compared to the control .", "but there was no significant change in the lbi pretreated group ( 29244.6  2981.2 in controls ; 5787.7  4126.4 with cocl2 ; and 27189.0  11231.1 with cocl2 after lbi pretreatment ; p < 0.05 , nonrepeated anova , n = 5 , figure 1(e ) ) . because disruption of tjs is detected as a decrease of signal intensity , these results indicate that lbi pretreatment can protect the tjs of the outer brb from hypoxic stress .", "because lbi pretreatment protected the conformation of the tj proteins from hypoxic stress , we hypothesized that lbi will block different inflammatory molecules that are secreted from cells during hypoxia .", "the results of the elisa measurements of the conditioned culture media ( n = 5 ) indicated that il-8 was not significantly changed after the addition of cocl2 or after pretreatment with lbi ( control , 6.9  0.1 ; with cocl2 , 6.8  0.1 ; and with cocl2 after lbi pretreatment , 6.9  0.1  pg / ml ; figure 2(a ) ) .", "although the level of il-6 was increased after addition of cocl2 which was decreased by lbi pretreatment , these changes were not significant ( control , 10.4  1.6 ; cocl2 addition , 15.0  6.7 ; and cocl2 addition after lbi pretreatment , 13.3  7.6  pg / ml ; figure 2(b ) ) .", "the level of mcp-1 increased significantly after the addition of cocl2 , and it was significantly decreased after the addition of cocl2 after lbi pretreatment ( control , 279.7  68.3 ; with cocl2 , 340.8  43.3 ; and with cocl2 after lbi pretreatment , 182.6  23.8  pg / ml , p < 0.05 ; nonrepeated anova ; figure 2(c ) ) .", "a similar tendency was observed for ccl-11 ( control , 12.5  6.1 ; with cocl2 , 15.2  12.9 ; and with cocl2 after lbi pretreatment , 5.46  1.9  pg / ml ; p < 0.05 ; nonrepeated anova ; figure 2(d ) ) .", "these results indicated that there is an increase of the mcp-1 and ccl-11 secretion under hypoxic stress , and all are suppressed by lbi pretreatment .   to confirm that lbi effectiveness is related to suppression of mcp-1 or ccl-11", ", cells were pretreated with anti - mcp-1 or anti - ccl-11 antibody for 24  hrs .", "the tj disruption from hypoxic stress was reduced with pretreatment ( figures 3(e ) and 3(f ) ) .", "these results indicated that both mcp-1 and ccl-11 were important to protect tjs from hypoxic stress and support the effectiveness of lbi .", "vegf is a major antigen factor and is biogenic permeability factor which can increase the vascular permeability endothelial cell - cell junctions . finally , to confirm that lbi pretreatment can protect tjs from direct vegf induced stress , after lbi pretreatment", "but after pretreatment of lbi , this disruption was decreased which indicates that lbi can also protect tjs from vegf induced damage .", "but there still remain patients who do not respond to the anti - vegf therapy   and who had a reduction of their vision even after therapy .", "it must also be noted that the cost of anti - vegf therapy is quite high and continuous injections to control the dme can become an economic issue .", "combination therapies have also been proposed that can enhance the efficacy   and may minimize the cost of anti - vegf therapies .", "for example , photocoagulation can extend the interval of anti - vegf injections resulting in a reduction in the number of injections [ 21 , 22 ] .", "if new drugs or treatments can be approved , this will make it easier and less expensive to use combination therapies .", "moreover , adjunctive therapies that include oral kallidinogenase could also be used for combined therapy [ 23 , 24 ] .", "our results showed that the hypoxic stress induced by exposure of arpe-19 cells to cocl2 caused a disruption of the tight junctions and that lbi pretreatment can protect the tjs from the hypoxic stress .", "hypoxic stress also enhanced the secretion of mcp-1 and ccl-11 , and this enhancement can be suppressed by lbi pretreatment .", "hypoxia has been reported to produce alterations in the tight junction proteins that are correlated with the vascular permeability increases .", "the integrity of the brb is dependent on tj - associated proteins such as occludin , claudin , and zo-1 .", "the function of zo-1 is to link the transmembrane protein , occludin , to the actin cytoskeleton . under hypoxic conditions ,", "the phosphorylation of zo-1 is enhanced which is related to the damage of the tight junctions .", "mcp-1 is associated with a breakdown of the brb [ 3032 ] , and it also induces a disruption of the tjs by the caveolae that are dedicated to the internalization of the tj proteins , such as zo-1 .", "because mcp-1 is also increased in the vitreous of patients with dr , it probably plays an important role in the progression of dr and dme . in the photocoagulation - induced mouse model of retinal neovascularization , it was reported that an increase in mcp-1 contributes to the postischemic inflammation and dr progression [ 34 , 35 ] .", "mcp-1 has been recently postulated to have a direct effect on angiogenesis even though there is no link with macrophage recruitment .", "ccl-11 also promotes the recruitment of vascular endothelial cells and relates the conformation of the tight junctions .", "interestingly , there is also an increase of ccl-11 in the proliferative membranes obtained from eyes with pdr .", "so these cytokines relate to zo-1 and maintain tight junctions on diabetic retina . in the photocoagulation - induced retinal neovascularization or light - exposed mouse models ,", "the levels of not only mcp-1 but also ccl-11 are increased in the rpe [ 3941 ] .", "these increases were related to the remodeling of f - actin that occurred after light damage and caused proinflammatory changes in the rpe cells .", "these findings support our results that showed that the hypoxia - induced stress affected the expression of cytokines especially mcp-1 and ccl-11 .", "the suppression of these cytokines by lbi leads to the protection of these cells from disruption of the tight junctions .", "steroids are also commonly used for dme therapy because their anti - inflammatory property helps to protect the tissues from neovascularization or vascular permeability increases .", "similarly , the effect of lbi on the suppression of the postischemic inflammation may be an additional contributor when used as either a primary or adjunctive dme therapy . mainly in japan and china ,", "lbi has been approved for the treatment of various retinal diseases including central serous chorioretinopathy , vitreous hemorrhage , or vitreous opacity for a long time .", "in addition , both lecithin and iodine are commonly used in food materials indicating the safety of lbi .", "thus , the possibility exists that lbi can be even used as a safe adjunctive therapy with an anti - vegf drug to treat dme .", "further investigations into the use of lbi in conjunction with anti - vegf therapy need to be undertaken to determine the effectiveness of lbi in preventing the development or progression of dme .", "our findings showed that lbi pretreatment reduced the levels of mcp-1 and ccl-11 and blockage of them also results in protection of tjs .", "however , no significant changes were detected in the levels of il-6 and il-8 , which are the major cytokines related to neovascularization . in ischemic", "retinas , il-8 causes increases in the vegf and mcp-1 levels but it is dependent on tnf- .", "because il-8 has been shown to be secreted from either monocytes or macrophages   and because our experiments were limited to arpe-19 cells in culture , there is the possibility that the results may not hold under in situ conditions . though we observed that vascular permeability improved after oral lbi administration for diabetic mice model ( data not shown ) , it is not clear whether this is the result of hypoxia improvement the same as we show here using arpe-19 .", "in addition , lbi pretreatment can also protect tjs from direct vegf induced damage which results in tj disruption .", "further investigations will need to be undertaken to definitively explain these phenomena . in conclusion ,", "our data demonstrate the therapeutic potential of lbi for protecting the integrity of the tight junctions from hypoxia - induced stress .", "these data suggest a new aspect on the clinical use of lbi in the treatment of dme ."], "abstract_text": ["<S> \n aim . we investigated </S>", "<S> whether lecithin - bound iodine ( lbi ) can protect the integrity of tight junctions of retinal pigment epithelial cells from hypoxia . method . </S>", "<S> cultured human retinal pigment epithelial ( arpe-19 ) cells were pretreated with lbi . </S>", "<S> to mimic hypoxic conditions , cells were incubated with cocl2 . </S>", "<S> we compared the integrity of the tight junctions ( tjs ) of control to cells with either lbi alone , cocl2 alone , or lbi + cocl2 . </S>", "<S> the levels of cytokines in the conditioned media were also determined . </S>", "<S> results . </S>", "<S> significant decrease in the zonula occludens-1 ( zo-1 ) intensity in the cocl2 group compared to the control ( 5787.7  4126.4 in cocl2 group versus 29244.6  2981.2 in control ; average  standard deviation ) . </S>", "<S> but the decrease was not significant in the lbi + cocl2 ( 27189.0  11231.1 ) . </S>", "<S> the levels of monocyte chemoattractant protein-1 ( mcp-1 ) and chemokine ( c - c motif ) ligand 11 ( ccl-11 ) were significantly higher in the cocl2 than in the control ( 340.8  43.3 versus 279.7  68.3  pg / ml for mcp-1 , and 15.2  12.9 versus 12.5  6.1  pg / ml for ccl-11 . </S>", "<S> with lbi pretreatment , the levels of both cytokines were decreased to 182.6  23.8 ( mcp-1 ) and 5.46  1.9  pg / ml for ccl-11 ) . </S>", "<S> blockade of mcp-1 or ccl-11 also shows similar result representing tj protection from hypoxic stress </S>", "<S> . conclusions . </S>", "<S> lbi results in a protective action from hypoxia . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["diabetic retinopathy ( dr ) is a leading cause of visual impairment and blindness in developed countries .", "the decrease in vision is due to diabetic macular edema ( dme ) and proliferative diabetic retinopathy ( pdr ) [ 1 , 2 ] .", "the control of the blood glucose level , vitreous surgery , and photocoagulation are the major treatments used to prevent dr from progressing to the pdr stage .", "currently , intravitreal injections of antivascular endothelium growth factor ( vegf ) or steroids have become the primary therapy for dme [ 35 ] .", "however , the use of a single therapeutic agent is not effective in all cases , and additional treatments or different agents are needed . the blood retinal barrier ( brb )", "consists of two anatomical parts ; the inner brb is located within the endothelial cells of the retinal capillaries , and the outer brb is located between the retinal pigment epithelial ( rpe ) cells . an intact brb is required for an efficient and regulated control of fluids in the subretinal space and for the maintenance of healthy rpe and retinal cells .", "a breakdown of the outer brb results in an increase in the paracellular permeability between the rpe cells , and the leakage can cause retinal edema .", "the tight junctions ( tjs ) of the rpe cells are intercellular junctions located at the apical ends of the rpe cells , and they are integral structural components of the brb .", "tjs are made up of three tj related proteins , for example , the zonula occludens-1 ( zo-1 ) , occludin , and claudin .", "recent studies have shown that interactions between inflammatory cells and retinal cells are critical for the development of intraocular neovascularization [ 1012 ] .", "the results of these studies also demonstrated that , in eyes with pdr , an elevation of vegf was significantly correlated with the levels of several cytokines including interleukin- ( il- ) 6 , il-8 , and the monocyte chemoattractant protein-1 ( mcp-1 ) .", "another study showed that the levels of these cytokines were also strongly correlated with each other which suggest that there are common pathways involved in the inflammatory processes .", "in addition , various cytokines have been shown to be related to the maintenance of the conformation of the tj proteins .", "lecithin - bound iodine ( lbi , jolethin , daiichi pharmaceutical co. , tokyo , japan ) has been used clinically to reduce the antigen - induced immune responses in children with bronchial asthma .", "lbi acts on the peripheral blood mononuclear cells and downregulates the il-4-induced ige synthesis , which suggests that lbi have anti - inflammatory properties [ 15 , 16 ] . in patients with eye diseases ,", "lbi has been used for the absorption of retinal or vitreous bleeding , vitreous opacities , and improvement of central serous choroidopathy .", "the purpose of this study was to determine whether lbi will alter the integrity of the tjs of arpe-19 cells in culture ."], ["lbi ( jolethin , daiichi pharmaceutical co. , tokyo , japan ) was dissolved in distilled water and diluted to the appropriate concentration for each experiment .", "an earlier study showed that lbi solutions contain 48.250.3% lecithin - iodine , approximately 10% free lecithin , and 40% phosphatidylinositol .", "rabbit polyclonal anti - zo-1 antibody ( sc-10804 ) was purchased from santa cruz biotechnology ( santa cruz , ca ) , and alexa 594 anti - rabbit igg was purchased from invitrogen ( molecular probs , eugene , or ) .", "rabbit anti - mcp-1 antibody ( # 2029 ) was purchased from cell signaling technology ( danvers , ma ) and rabbit anti - eotaxin ( ccl-11 ) antibody ( ab133604 ) was purchased from abcam ( cambridge , ma ) .", "cells from a human retinal pigment epithelium cell line , arpe-19 , were grown in dulbecco 's modified eagle 's medium / ham 's f-12 supplemented with 10% fetal bovine serum ( fbs , hyclone laboratories , inc . ,", "logan , ut ) , 50  units / ml penicillin , and 50  g / ml streptomycin in an air-5% co2 atmosphere with constant humidity .", "prior to the experiments , cells were placed in serum - free media and pretreated with 50  g / ml of lbi or 250500  pg / ml of antibody ( anti - mcp-1 antibody and anti - chemokine ( c - c motif ) ligand-11 ( ccl-11 ) antibody ) for 24  h. to mimic hypoxic conditions , arpe-19 cells were incubated for 4  h with 100  m of cocl2 ( sigma , st .", "louis , mo ) , which is a chemical hypoxia - inducing agent . after the incubation ,", "optimum concentration of lbi was defined as 50  g / ml because over 100  g / ml of lbi seems to be toxic ( data not shown ) . for the immunofluorescence studies , cells", "were grown to a density of 2  10 cells / ml on 12  mm cover slips and fixed in 10% tricarboxylic acid for 10  min at 4c .", "they were then treated with 0.5% triton x-100 for 15  min . to detect the presence of zo-1 , the cultured rpe cells were exposed to rabbit polyclonal anti - zo-1 antibody as the primary antibody and alexa 594 anti - rabbit igg as the secondary antibody .", "samples were examined and photographed with a fluorescence microscope ( bz-9000 ; keyence , osaka , japan , and eclipse 50i , nikon , tokyo , japan ) , and the intensity of fluorescence was quantified with a bz - ii analyzer ( keyence ) .", "the levels of the bioactive molecules in the conditioned medium were determined by human il-6 , il-8 , and mcp-1 elisa kits ( r&d systems , minneapolis , mn ) and ccl-11 elisa kit ( biosensis , thebarton , south australia , australia ) .", "all experiments were repeated at least three times and values are presented as the means  standard deviations .", "data were analyzed by two - way nonrepeated analysis of variance ( anova ) followed by bonferroni post hoc tests for the comparison of the means ."], ["we first determined whether the lbi pretreatment affected the changes in the conformation of the tj proteins caused by the hypoxic stress induced by cocl2 .", "immunofluorescence microscopy showed that there appeared to be a disruption of tjs with cocl2 treatment ( figures 1(a ) and 1(b ) ) .", "when the arpe-19 cells were pretreated with lbi before the addition of cocl2 , the disruption of the tight junctions was not detected ( figures 1(c ) and 1(d ) ) .", "the signal intensity measurements showed that there was a significant decrease with the cocl2 addition compared to the control .", "but there was no significant change in the lbi pretreated group ( 29244.6  2981.2 in controls ; 5787.7  4126.4 with cocl2 ; and 27189.0  11231.1 with cocl2 after lbi pretreatment ; p < 0.05 , nonrepeated anova , n = 5 , figure 1(e ) ) . because disruption of tjs is detected as a decrease of signal intensity , these results indicate that lbi pretreatment can protect the tjs of the outer brb from hypoxic stress .", "because lbi pretreatment protected the conformation of the tj proteins from hypoxic stress , we hypothesized that lbi will block different inflammatory molecules that are secreted from cells during hypoxia .", "the results of the elisa measurements of the conditioned culture media ( n = 5 ) indicated that il-8 was not significantly changed after the addition of cocl2 or after pretreatment with lbi ( control , 6.9  0.1 ; with cocl2 , 6.8  0.1 ; and with cocl2 after lbi pretreatment , 6.9  0.1  pg / ml ; figure 2(a ) ) .", "although the level of il-6 was increased after addition of cocl2 which was decreased by lbi pretreatment , these changes were not significant ( control , 10.4  1.6 ; cocl2 addition , 15.0  6.7 ; and cocl2 addition after lbi pretreatment , 13.3  7.6  pg / ml ; figure 2(b ) ) .", "the level of mcp-1 increased significantly after the addition of cocl2 , and it was significantly decreased after the addition of cocl2 after lbi pretreatment ( control , 279.7  68.3 ; with cocl2 , 340.8  43.3 ; and with cocl2 after lbi pretreatment , 182.6  23.8  pg / ml , p < 0.05 ; nonrepeated anova ; figure 2(c ) ) .", "a similar tendency was observed for ccl-11 ( control , 12.5  6.1 ; with cocl2 , 15.2  12.9 ; and with cocl2 after lbi pretreatment , 5.46  1.9  pg / ml ; p < 0.05 ; nonrepeated anova ; figure 2(d ) ) .", "these results indicated that there is an increase of the mcp-1 and ccl-11 secretion under hypoxic stress , and all are suppressed by lbi pretreatment .   to confirm that lbi effectiveness is related to suppression of mcp-1 or ccl-11", ", cells were pretreated with anti - mcp-1 or anti - ccl-11 antibody for 24  hrs .", "the tj disruption from hypoxic stress was reduced with pretreatment ( figures 3(e ) and 3(f ) ) .", "these results indicated that both mcp-1 and ccl-11 were important to protect tjs from hypoxic stress and support the effectiveness of lbi .", "vegf is a major antigen factor and is biogenic permeability factor which can increase the vascular permeability endothelial cell - cell junctions . finally , to confirm that lbi pretreatment can protect tjs from direct vegf induced stress , after lbi pretreatment", "but after pretreatment of lbi , this disruption was decreased which indicates that lbi can also protect tjs from vegf induced damage ."], ["but there still remain patients who do not respond to the anti - vegf therapy   and who had a reduction of their vision even after therapy .", "it must also be noted that the cost of anti - vegf therapy is quite high and continuous injections to control the dme can become an economic issue .", "combination therapies have also been proposed that can enhance the efficacy   and may minimize the cost of anti - vegf therapies .", "for example , photocoagulation can extend the interval of anti - vegf injections resulting in a reduction in the number of injections [ 21 , 22 ] .", "if new drugs or treatments can be approved , this will make it easier and less expensive to use combination therapies .", "moreover , adjunctive therapies that include oral kallidinogenase could also be used for combined therapy [ 23 , 24 ] .", "our results showed that the hypoxic stress induced by exposure of arpe-19 cells to cocl2 caused a disruption of the tight junctions and that lbi pretreatment can protect the tjs from the hypoxic stress .", "hypoxic stress also enhanced the secretion of mcp-1 and ccl-11 , and this enhancement can be suppressed by lbi pretreatment .", "hypoxia has been reported to produce alterations in the tight junction proteins that are correlated with the vascular permeability increases .", "the integrity of the brb is dependent on tj - associated proteins such as occludin , claudin , and zo-1 .", "the function of zo-1 is to link the transmembrane protein , occludin , to the actin cytoskeleton . under hypoxic conditions ,", "the phosphorylation of zo-1 is enhanced which is related to the damage of the tight junctions .", "mcp-1 is associated with a breakdown of the brb [ 3032 ] , and it also induces a disruption of the tjs by the caveolae that are dedicated to the internalization of the tj proteins , such as zo-1 .", "because mcp-1 is also increased in the vitreous of patients with dr , it probably plays an important role in the progression of dr and dme . in the photocoagulation - induced mouse model of retinal neovascularization , it was reported that an increase in mcp-1 contributes to the postischemic inflammation and dr progression [ 34 , 35 ] .", "mcp-1 has been recently postulated to have a direct effect on angiogenesis even though there is no link with macrophage recruitment .", "ccl-11 also promotes the recruitment of vascular endothelial cells and relates the conformation of the tight junctions .", "interestingly , there is also an increase of ccl-11 in the proliferative membranes obtained from eyes with pdr .", "so these cytokines relate to zo-1 and maintain tight junctions on diabetic retina . in the photocoagulation - induced retinal neovascularization or light - exposed mouse models ,", "the levels of not only mcp-1 but also ccl-11 are increased in the rpe [ 3941 ] .", "these increases were related to the remodeling of f - actin that occurred after light damage and caused proinflammatory changes in the rpe cells .", "these findings support our results that showed that the hypoxia - induced stress affected the expression of cytokines especially mcp-1 and ccl-11 .", "the suppression of these cytokines by lbi leads to the protection of these cells from disruption of the tight junctions .", "steroids are also commonly used for dme therapy because their anti - inflammatory property helps to protect the tissues from neovascularization or vascular permeability increases .", "similarly , the effect of lbi on the suppression of the postischemic inflammation may be an additional contributor when used as either a primary or adjunctive dme therapy . mainly in japan and china ,", "lbi has been approved for the treatment of various retinal diseases including central serous chorioretinopathy , vitreous hemorrhage , or vitreous opacity for a long time .", "in addition , both lecithin and iodine are commonly used in food materials indicating the safety of lbi .", "thus , the possibility exists that lbi can be even used as a safe adjunctive therapy with an anti - vegf drug to treat dme .", "further investigations into the use of lbi in conjunction with anti - vegf therapy need to be undertaken to determine the effectiveness of lbi in preventing the development or progression of dme .", "our findings showed that lbi pretreatment reduced the levels of mcp-1 and ccl-11 and blockage of them also results in protection of tjs .", "however , no significant changes were detected in the levels of il-6 and il-8 , which are the major cytokines related to neovascularization . in ischemic", "retinas , il-8 causes increases in the vegf and mcp-1 levels but it is dependent on tnf- .", "because il-8 has been shown to be secreted from either monocytes or macrophages   and because our experiments were limited to arpe-19 cells in culture , there is the possibility that the results may not hold under in situ conditions . though we observed that vascular permeability improved after oral lbi administration for diabetic mice model ( data not shown ) , it is not clear whether this is the result of hypoxia improvement the same as we show here using arpe-19 .", "in addition , lbi pretreatment can also protect tjs from direct vegf induced damage which results in tj disruption .", "further investigations will need to be undertaken to definitively explain these phenomena . in conclusion ,", "our data demonstrate the therapeutic potential of lbi for protecting the integrity of the tight junctions from hypoxia - induced stress .", "these data suggest a new aspect on the clinical use of lbi in the treatment of dme ."]]}
{"article_id": "PMC5089152", "article_text": ["a 21-year - old caucasian woman presented to an outside facility with altered mental status after being found lethargic by family . on arrival to the emergency department , plasma glucose was 20 mg / dl .", "the patient was hospitalized and started on dextrose infusion , including 10% dextrose in water but remained hypoglycemic with capillary glucose ranging between 50 and 60 mg / dl .", "she was transferred to our facility on suspicion of an insulinoma as the patient was unable to recall her prior diagnosis .", "the patient reported repeated episodes of seizures , syncope , dizziness , headaches , palpitations , and sweating around age 12 ; however , symptoms were never formally investigated as she did not seek expert care .", "these symptoms were also reported to have been present since birth but had been intermittent and of varying severity .", "she remained seizure - free and without syncopal episodes until 17 when she experienced another syncopal episode . at that time", ", she underwent extensive inpatient hypoglycemia evaluation with the following results : blood glucose of 47 mg / dl after 2 h of fasting , proinsulin levels varying from 10.4 to 84.1 pmol / l ( normal range 010", "pmol / l ) , c - peptide level 3.1 ng / ml ( normal range 1.14.4 ng / ml ) , negative insulin antibodies and sulfonylurea screen .", "magnetic resonance imaging of the abdomen showed no insulinoma , but genetic studies revealed val452 leu activating mutation of the gck gene .", "she was successfully treated with diazoxide and discharged home on oral diazoxide 250 mg daily , which she discontinued due to side effects of hirsutism and fluid retention . on current admission ,", "her blood glucose level remained low in spite of a continuous infusion of 5% dextrose necessitating transfer to an intensive care unit .", "as review of her medication list did not show any implicating drugs , no additional work - up was pursued .", "she was then placed on octreotide 200 g subcutaneously twice daily and diazoxide suspension 100 mg three times a day on consultation with the pediatric endocrinologist .", "the patient 's hypoglycemia improved with capillary glucose of 55110 mg / dl by the time of discharge .", "importance of compliance with the treatment and follow - up with an endocrinologist was emphasized .", "when encountered in the inpatient setting , it is easily recognized as patients already have risk factors such as diabetes mellitus ( dm ) on treatment , sepsis , and end - organ failure . however , for patients presenting emergently with hypoglycemia alone , appropriate investigations are warranted to treat and prevent recurrence . after establishing a diagnosis of hypoglycemia by whipple 's triad ( low plasma glucose , hypoglycemic symptoms , and resolution of adrenergic / neuroglycopenic symptoms with correction of the blood sugar ) , interim treatment and search for risk / causative factors should ensue ( table 1 ) .", "ideally , treatment is only given after obtaining a plasma sample to ensure accuracy of diagnosis .", "medical history of ethanol use , surreptitious insulin ingestion , medication use and interaction , autoimmune disease , gastric bypass ( 4 ) , and subtle risk factors such as undiagnosed psychiatric disorder and polypharmacy should be excluded ( 5 ) . in seemingly well patients ,", "the timing of hypoglycemic spells ( fasting / postabsorptive or postprandial / reactive ) is required to determine diagnostic studies .", "a mixed meal tolerance test under supervision is done for postprandial hypoglycemia while a 72-h fast should be done for fasting hypoglycemia .", "hepatic or renal disease , endocrine disorders of growth hormone , cortisol , adrenal insufficiency , and hypopituitarism should be excluded .", "plasma insulin , proinsulin , c - peptide , and beta - hydroxybutyrate levels should be assessed to exclude surreptitious use of insulin or insulin receptor antibodies .", "intravenous glucagon can be given to distinguish disorders of gluconeogenesis from glycogenolysis ( 5 ) .", "a 48-h supervised fast should be done to exclude insulinoma , with plasma insulin and proinsulin levels .", "pro - igf ii to igf ii ratio can be measured to look for non - beta - cell tumors ( 5 ) .", "negative findings suggest non - insulinoma pancreatogenous hypoglycemia syndrome ( niphs ) , which encompasses chi ( 4 , 6 ) . a selective pancreatic arterial calcium stimulation test ( spaci ) done at this time can detect hyperfunctioning of b - cells and confirm the diagnosis of niphs .", "causes of hypoglycemia in adults chi is uncommon in the pediatric population with an estimated incidence of 1 in 40,000 to 50,000 live births annually in europe ( 7 ) .", "gck mutations account for 7% of all causes of chi ( 8 , 9 ) ; gck facilitates phosphorylation of glucose to glucose-6-phosphate and is the main glucose sensor of the pancreatic beta cell and hepatocytes - controlling glucose - stimulated insulin secretion and glycogenesis ( 10 ) . activating mutations of the gck gene", "are typically inherited in an autosomal dominant pattern , showing variable phenotypic penetrance and expressivity ranging from asymptomatic to marked hypoglycemia ( 11 , 12 ) .", "inactivating forms of the mutation cause varying severity of persistent hyperglycemia depending on zygosity ( 13 ) .", "de novo gck mutations are very rare and exhibit marked variation in phenotypic expression ( 14 ) .", "over 600 heterozygous inactivating mutations have been reported , causing mild persistent hyperglycemia known as maturity onset diabetes of the young ( mody ) as opposed to only 13 known activating mutations of the gene ( 15 , 16 ) .", "activating mutations are usually clustered around the allosteric activator site of the enzyme and led to hyperinsulinemic hypoglycemia by lowering the glucose set point and threshold for glucose stimulated insulin release .", "the sheer numbers of existing mutations of the gck gene show it to be a highly mutable gene with varied disease outcomes depending on the type of activation .", "it is key to blood glucose homeostasis and has been identified as a potential target for anti - diabetic therapy .", "trials manipulating gck regulatory protein an allosteric switch for gck , which indirectly restores gck activity , are currently underway to evaluate its effect on blood glucose in patients with type 2 dm ( 17 , 18 ) .", "treatment of niphs involves lifelong dietary modification consisting of frequent , low carbohydrate meals ( 5 ) , which prevent surges in plasma glucose level , insulin level , and the consequent hypoglycemic spells .", "medications such as diazoxide , octreotide , glucagon , verapamil , and somatostatin , whose actions all involve inhibition of calcium channels , have been employed with varying degrees of success ( 7 ) . diazoxide binds to and activates the sulfonylurea receptor 1 subunit of the potassium adenosine triphosphate [ k(atp ) ] channel , causing calcium channel closure .", "response to diazoxide therefore varies with genetic mutations [ ineffective in mutations of the k(atp ) channel genes ] ( 8) and with pattern of inheritance ( 7 ) .", "octreotide , a somatostatin analogue ( secretin and insulin inhibitor ) , has been used in diazoxide unresponsive chi with improved blood glucose in these patients ; however , side effects such as gastrointestinal symptoms , dilated gallbladder , necrotizing enterocolitis , and qt prolongation sometimes limit its use ( 7 ) .", "long - term use of glucagon is effective in diazoxide - unresponsive cases or severe / diffuse chi but gastrointestinal side effects and crystallization in infusion tubing have been reported ( 19 ) .", "glucagon - like protein 1 receptor agonists such as exendin-4 are newer drugs that improve hypoglycemia in diazoxide - unresponsive patients by inhibiting insulin release ( 7 ) .", "partial pancreatectomy is indicated for medical treatment failure [ suggesting a k(atp ) defect ] and is only curative for focal lesions while extensive resection is reserved for diffuse chi to limit pancreatic burden ( 7 , 8 , 11 , 20 ) .", "affected patients can present in adulthood ; de novo mutations should be considered given the following:-  positive family history of unremitting hypoglycemia-  lifelong symptoms of low plasma glucose , even after eating-  increased bmi-  negative sulfonylurea and autoimmune work up-  positive glycemic response to octreotide \n  -  positive family history of unremitting hypoglycemia -  lifelong symptoms of low plasma glucose , even after eating -  negative sulfonylurea and autoimmune work up -  positive glycemic response to octreotide to our knowledge , only three families with affected adults have been reported .", "our case is unique because it expounds on genetic causes of hypoglycemia in adults . in addition", ", this case highlights the pediatric - to - adulthood transition for rare diseases and the key role that coordination and transition of care plays in timely management .", "after establishing a diagnosis of hypoglycemia by whipple 's triad ( low plasma glucose , hypoglycemic symptoms , and resolution of adrenergic / neuroglycopenic symptoms with correction of the blood sugar ) , interim treatment and search for risk / causative factors should ensue ( table 1 ) .", "ideally , treatment is only given after obtaining a plasma sample to ensure accuracy of diagnosis .", "medical history of ethanol use , surreptitious insulin ingestion , medication use and interaction , autoimmune disease , gastric bypass ( 4 ) , and subtle risk factors such as undiagnosed psychiatric disorder and polypharmacy should be excluded ( 5 ) . in seemingly well patients ,", "the timing of hypoglycemic spells ( fasting / postabsorptive or postprandial / reactive ) is required to determine diagnostic studies .", "a mixed meal tolerance test under supervision is done for postprandial hypoglycemia while a 72-h fast should be done for fasting hypoglycemia .", "hepatic or renal disease , endocrine disorders of growth hormone , cortisol , adrenal insufficiency , and hypopituitarism should be excluded .", "plasma insulin , proinsulin , c - peptide , and beta - hydroxybutyrate levels should be assessed to exclude surreptitious use of insulin or insulin receptor antibodies .", "intravenous glucagon can be given to distinguish disorders of gluconeogenesis from glycogenolysis ( 5 ) .", "a 48-h supervised fast should be done to exclude insulinoma , with plasma insulin and proinsulin levels .", "pro - igf ii to igf ii ratio can be measured to look for non - beta - cell tumors ( 5 ) .", "negative findings suggest non - insulinoma pancreatogenous hypoglycemia syndrome ( niphs ) , which encompasses chi ( 4 , 6 ) . a selective pancreatic arterial calcium stimulation test ( spaci ) done at this time can detect hyperfunctioning of b - cells and confirm the diagnosis of niphs .", "chi is uncommon in the pediatric population with an estimated incidence of 1 in 40,000 to 50,000 live births annually in europe ( 7 ) .", "gck mutations account for 7% of all causes of chi ( 8 , 9 ) ; gck facilitates phosphorylation of glucose to glucose-6-phosphate and is the main glucose sensor of the pancreatic beta cell and hepatocytes - controlling glucose - stimulated insulin secretion and glycogenesis ( 10 ) . activating mutations of the gck gene", "are typically inherited in an autosomal dominant pattern , showing variable phenotypic penetrance and expressivity ranging from asymptomatic to marked hypoglycemia ( 11 , 12 ) .", "inactivating forms of the mutation cause varying severity of persistent hyperglycemia depending on zygosity ( 13 ) .", "de novo gck mutations are very rare and exhibit marked variation in phenotypic expression ( 14 ) .", "over 600 heterozygous inactivating mutations have been reported , causing mild persistent hyperglycemia known as maturity onset diabetes of the young ( mody ) as opposed to only 13 known activating mutations of the gene ( 15 , 16 ) .", "activating mutations are usually clustered around the allosteric activator site of the enzyme and led to hyperinsulinemic hypoglycemia by lowering the glucose set point and threshold for glucose stimulated insulin release .", "the sheer numbers of existing mutations of the gck gene show it to be a highly mutable gene with varied disease outcomes depending on the type of activation .", "it is key to blood glucose homeostasis and has been identified as a potential target for anti - diabetic therapy .", "trials manipulating gck regulatory protein an allosteric switch for gck , which indirectly restores gck activity , are currently underway to evaluate its effect on blood glucose in patients with type 2 dm ( 17 , 18 ) .", "treatment of niphs involves lifelong dietary modification consisting of frequent , low carbohydrate meals ( 5 ) , which prevent surges in plasma glucose level , insulin level , and the consequent hypoglycemic spells .", "medications such as diazoxide , octreotide , glucagon , verapamil , and somatostatin , whose actions all involve inhibition of calcium channels , have been employed with varying degrees of success ( 7 ) . diazoxide binds to and activates the sulfonylurea receptor 1 subunit of the potassium adenosine triphosphate [ k(atp ) ] channel , causing calcium channel closure .", "response to diazoxide therefore varies with genetic mutations [ ineffective in mutations of the k(atp ) channel genes ] ( 8) and with pattern of inheritance ( 7 ) .", "octreotide , a somatostatin analogue ( secretin and insulin inhibitor ) , has been used in diazoxide unresponsive chi with improved blood glucose in these patients ; however , side effects such as gastrointestinal symptoms , dilated gallbladder , necrotizing enterocolitis , and qt prolongation sometimes limit its use ( 7 ) .", "long - term use of glucagon is effective in diazoxide - unresponsive cases or severe / diffuse chi but gastrointestinal side effects and crystallization in infusion tubing have been reported ( 19 ) .", "glucagon - like protein 1 receptor agonists such as exendin-4 are newer drugs that improve hypoglycemia in diazoxide - unresponsive patients by inhibiting insulin release ( 7 ) .", "partial pancreatectomy is indicated for medical treatment failure [ suggesting a k(atp ) defect ] and is only curative for focal lesions while extensive resection is reserved for diffuse chi to limit pancreatic burden ( 7 , 8 , 11 , 20 ) .", "affected patients can present in adulthood ; de novo mutations should be considered given the following:-  positive family history of unremitting hypoglycemia-  lifelong symptoms of low plasma glucose , even after eating-  increased bmi-  negative sulfonylurea and autoimmune work up-  positive glycemic response to octreotide \n  -  positive family history of unremitting hypoglycemia -  lifelong symptoms of low plasma glucose , even after eating -  negative sulfonylurea and autoimmune work up -  positive glycemic response to octreotide to our knowledge , only three families with affected adults have been reported .", "in addition , this case highlights the pediatric - to - adulthood transition for rare diseases and the key role that coordination and transition of care plays in timely management .", "despite the congenital nature of gck mutations , some patients can go undiagnosed till early adulthood .", "future research into a cost - effective and prompt scheme of patient information dissemination is warranted for uncommon diseases .", "the authors have not received any funding or benefits from industry or elsewhere to conduct this study ."], "abstract_text": ["<S> backgroundhypoglycemia occurs frequently in patients both in the inpatient and outpatient settings . while most hypoglycemia unrelated to diabetes treatment results from excessive endogenous insulin action , rare cases involve functional and congenital mutations in glycolytic enzymes of insulin regulation.casea 21-year - old obese woman presented to the emergency department with complaints of repeated episodes of lethargy , syncope , dizziness , and sweating . </S>", "<S> she was referred from an outside facility on suspicion of insulinoma , with severe hypoglycemia unresponsive to repeated dextrose infusions . </S>", "<S> her plasma glucose was 20 mg / dl at presentation , 44 mg / dl on arrival at our facility , and remained low in spite of multiple dextrose infusions . </S>", "<S> the patient had been treated for persistent hyperinsulinemic hypoglycemia of infancy at our neonatal facility and 4 years ago was diagnosed as having an activating glucokinase ( gck ) mutation . </S>", "<S> she was then treated with octreotide and diazoxide with improvement in symptoms and blood glucose levels.conclusionimproved diagnostication and management of uncommon genetic mutations as typified in this patient with an activating mutation of the gck gene has expanded the spectrum of disease in adult medicine . </S>", "<S> this calls for improved patient information dissemination across different levels and aspects of the health care delivery system to ensure cost - effective and timely health care . </S>"], "labels": null, "section_names": ["Case presentation", "Discussion", "Diagnostic work up of hypoglycemia", "Pathophysiology of glucokinase mutations", "Therapeutic utility", "Treatment of underlying cause", "Clinical implication", "Conclusion", "Conflict of interest and funding"], "sections": [["a 21-year - old caucasian woman presented to an outside facility with altered mental status after being found lethargic by family . on arrival to the emergency department , plasma glucose was 20 mg / dl .", "the patient was hospitalized and started on dextrose infusion , including 10% dextrose in water but remained hypoglycemic with capillary glucose ranging between 50 and 60 mg / dl .", "she was transferred to our facility on suspicion of an insulinoma as the patient was unable to recall her prior diagnosis .", "the patient reported repeated episodes of seizures , syncope , dizziness , headaches , palpitations , and sweating around age 12 ; however , symptoms were never formally investigated as she did not seek expert care .", "these symptoms were also reported to have been present since birth but had been intermittent and of varying severity .", "she remained seizure - free and without syncopal episodes until 17 when she experienced another syncopal episode . at that time", ", she underwent extensive inpatient hypoglycemia evaluation with the following results : blood glucose of 47 mg / dl after 2 h of fasting , proinsulin levels varying from 10.4 to 84.1 pmol / l ( normal range 010", "pmol / l ) , c - peptide level 3.1 ng / ml ( normal range 1.14.4 ng / ml ) , negative insulin antibodies and sulfonylurea screen .", "magnetic resonance imaging of the abdomen showed no insulinoma , but genetic studies revealed val452 leu activating mutation of the gck gene .", "she was successfully treated with diazoxide and discharged home on oral diazoxide 250 mg daily , which she discontinued due to side effects of hirsutism and fluid retention . on current admission ,", "her blood glucose level remained low in spite of a continuous infusion of 5% dextrose necessitating transfer to an intensive care unit .", "as review of her medication list did not show any implicating drugs , no additional work - up was pursued .", "she was then placed on octreotide 200 g subcutaneously twice daily and diazoxide suspension 100 mg three times a day on consultation with the pediatric endocrinologist .", "the patient 's hypoglycemia improved with capillary glucose of 55110 mg / dl by the time of discharge .", "importance of compliance with the treatment and follow - up with an endocrinologist was emphasized ."], ["when encountered in the inpatient setting , it is easily recognized as patients already have risk factors such as diabetes mellitus ( dm ) on treatment , sepsis , and end - organ failure . however , for patients presenting emergently with hypoglycemia alone , appropriate investigations are warranted to treat and prevent recurrence . after establishing a diagnosis of hypoglycemia by whipple 's triad ( low plasma glucose , hypoglycemic symptoms , and resolution of adrenergic / neuroglycopenic symptoms with correction of the blood sugar ) , interim treatment and search for risk / causative factors should ensue ( table 1 ) .", "ideally , treatment is only given after obtaining a plasma sample to ensure accuracy of diagnosis .", "medical history of ethanol use , surreptitious insulin ingestion , medication use and interaction , autoimmune disease , gastric bypass ( 4 ) , and subtle risk factors such as undiagnosed psychiatric disorder and polypharmacy should be excluded ( 5 ) . in seemingly well patients ,", "the timing of hypoglycemic spells ( fasting / postabsorptive or postprandial / reactive ) is required to determine diagnostic studies .", "a mixed meal tolerance test under supervision is done for postprandial hypoglycemia while a 72-h fast should be done for fasting hypoglycemia .", "hepatic or renal disease , endocrine disorders of growth hormone , cortisol , adrenal insufficiency , and hypopituitarism should be excluded .", "plasma insulin , proinsulin , c - peptide , and beta - hydroxybutyrate levels should be assessed to exclude surreptitious use of insulin or insulin receptor antibodies .", "intravenous glucagon can be given to distinguish disorders of gluconeogenesis from glycogenolysis ( 5 ) .", "a 48-h supervised fast should be done to exclude insulinoma , with plasma insulin and proinsulin levels .", "pro - igf ii to igf ii ratio can be measured to look for non - beta - cell tumors ( 5 ) .", "negative findings suggest non - insulinoma pancreatogenous hypoglycemia syndrome ( niphs ) , which encompasses chi ( 4 , 6 ) . a selective pancreatic arterial calcium stimulation test ( spaci ) done at this time can detect hyperfunctioning of b - cells and confirm the diagnosis of niphs .", "causes of hypoglycemia in adults chi is uncommon in the pediatric population with an estimated incidence of 1 in 40,000 to 50,000 live births annually in europe ( 7 ) .", "gck mutations account for 7% of all causes of chi ( 8 , 9 ) ; gck facilitates phosphorylation of glucose to glucose-6-phosphate and is the main glucose sensor of the pancreatic beta cell and hepatocytes - controlling glucose - stimulated insulin secretion and glycogenesis ( 10 ) . activating mutations of the gck gene", "are typically inherited in an autosomal dominant pattern , showing variable phenotypic penetrance and expressivity ranging from asymptomatic to marked hypoglycemia ( 11 , 12 ) .", "inactivating forms of the mutation cause varying severity of persistent hyperglycemia depending on zygosity ( 13 ) .", "de novo gck mutations are very rare and exhibit marked variation in phenotypic expression ( 14 ) .", "over 600 heterozygous inactivating mutations have been reported , causing mild persistent hyperglycemia known as maturity onset diabetes of the young ( mody ) as opposed to only 13 known activating mutations of the gene ( 15 , 16 ) .", "activating mutations are usually clustered around the allosteric activator site of the enzyme and led to hyperinsulinemic hypoglycemia by lowering the glucose set point and threshold for glucose stimulated insulin release .", "the sheer numbers of existing mutations of the gck gene show it to be a highly mutable gene with varied disease outcomes depending on the type of activation .", "it is key to blood glucose homeostasis and has been identified as a potential target for anti - diabetic therapy .", "trials manipulating gck regulatory protein an allosteric switch for gck , which indirectly restores gck activity , are currently underway to evaluate its effect on blood glucose in patients with type 2 dm ( 17 , 18 ) .", "treatment of niphs involves lifelong dietary modification consisting of frequent , low carbohydrate meals ( 5 ) , which prevent surges in plasma glucose level , insulin level , and the consequent hypoglycemic spells .", "medications such as diazoxide , octreotide , glucagon , verapamil , and somatostatin , whose actions all involve inhibition of calcium channels , have been employed with varying degrees of success ( 7 ) . diazoxide binds to and activates the sulfonylurea receptor 1 subunit of the potassium adenosine triphosphate [ k(atp ) ] channel , causing calcium channel closure .", "response to diazoxide therefore varies with genetic mutations [ ineffective in mutations of the k(atp ) channel genes ] ( 8) and with pattern of inheritance ( 7 ) .", "octreotide , a somatostatin analogue ( secretin and insulin inhibitor ) , has been used in diazoxide unresponsive chi with improved blood glucose in these patients ; however , side effects such as gastrointestinal symptoms , dilated gallbladder , necrotizing enterocolitis , and qt prolongation sometimes limit its use ( 7 ) .", "long - term use of glucagon is effective in diazoxide - unresponsive cases or severe / diffuse chi but gastrointestinal side effects and crystallization in infusion tubing have been reported ( 19 ) .", "glucagon - like protein 1 receptor agonists such as exendin-4 are newer drugs that improve hypoglycemia in diazoxide - unresponsive patients by inhibiting insulin release ( 7 ) .", "partial pancreatectomy is indicated for medical treatment failure [ suggesting a k(atp ) defect ] and is only curative for focal lesions while extensive resection is reserved for diffuse chi to limit pancreatic burden ( 7 , 8 , 11 , 20 ) .", "affected patients can present in adulthood ; de novo mutations should be considered given the following:-  positive family history of unremitting hypoglycemia-  lifelong symptoms of low plasma glucose , even after eating-  increased bmi-  negative sulfonylurea and autoimmune work up-  positive glycemic response to octreotide \n  -  positive family history of unremitting hypoglycemia -  lifelong symptoms of low plasma glucose , even after eating -  negative sulfonylurea and autoimmune work up -  positive glycemic response to octreotide to our knowledge , only three families with affected adults have been reported .", "our case is unique because it expounds on genetic causes of hypoglycemia in adults . in addition", ", this case highlights the pediatric - to - adulthood transition for rare diseases and the key role that coordination and transition of care plays in timely management ."], ["after establishing a diagnosis of hypoglycemia by whipple 's triad ( low plasma glucose , hypoglycemic symptoms , and resolution of adrenergic / neuroglycopenic symptoms with correction of the blood sugar ) , interim treatment and search for risk / causative factors should ensue ( table 1 ) .", "ideally , treatment is only given after obtaining a plasma sample to ensure accuracy of diagnosis .", "medical history of ethanol use , surreptitious insulin ingestion , medication use and interaction , autoimmune disease , gastric bypass ( 4 ) , and subtle risk factors such as undiagnosed psychiatric disorder and polypharmacy should be excluded ( 5 ) . in seemingly well patients ,", "the timing of hypoglycemic spells ( fasting / postabsorptive or postprandial / reactive ) is required to determine diagnostic studies .", "a mixed meal tolerance test under supervision is done for postprandial hypoglycemia while a 72-h fast should be done for fasting hypoglycemia .", "hepatic or renal disease , endocrine disorders of growth hormone , cortisol , adrenal insufficiency , and hypopituitarism should be excluded .", "plasma insulin , proinsulin , c - peptide , and beta - hydroxybutyrate levels should be assessed to exclude surreptitious use of insulin or insulin receptor antibodies .", "intravenous glucagon can be given to distinguish disorders of gluconeogenesis from glycogenolysis ( 5 ) .", "a 48-h supervised fast should be done to exclude insulinoma , with plasma insulin and proinsulin levels .", "pro - igf ii to igf ii ratio can be measured to look for non - beta - cell tumors ( 5 ) .", "negative findings suggest non - insulinoma pancreatogenous hypoglycemia syndrome ( niphs ) , which encompasses chi ( 4 , 6 ) . a selective pancreatic arterial calcium stimulation test ( spaci ) done at this time can detect hyperfunctioning of b - cells and confirm the diagnosis of niphs ."], ["chi is uncommon in the pediatric population with an estimated incidence of 1 in 40,000 to 50,000 live births annually in europe ( 7 ) .", "gck mutations account for 7% of all causes of chi ( 8 , 9 ) ; gck facilitates phosphorylation of glucose to glucose-6-phosphate and is the main glucose sensor of the pancreatic beta cell and hepatocytes - controlling glucose - stimulated insulin secretion and glycogenesis ( 10 ) . activating mutations of the gck gene", "are typically inherited in an autosomal dominant pattern , showing variable phenotypic penetrance and expressivity ranging from asymptomatic to marked hypoglycemia ( 11 , 12 ) .", "inactivating forms of the mutation cause varying severity of persistent hyperglycemia depending on zygosity ( 13 ) .", "de novo gck mutations are very rare and exhibit marked variation in phenotypic expression ( 14 ) .", "over 600 heterozygous inactivating mutations have been reported , causing mild persistent hyperglycemia known as maturity onset diabetes of the young ( mody ) as opposed to only 13 known activating mutations of the gene ( 15 , 16 ) .", "activating mutations are usually clustered around the allosteric activator site of the enzyme and led to hyperinsulinemic hypoglycemia by lowering the glucose set point and threshold for glucose stimulated insulin release .", "the sheer numbers of existing mutations of the gck gene show it to be a highly mutable gene with varied disease outcomes depending on the type of activation ."], ["it is key to blood glucose homeostasis and has been identified as a potential target for anti - diabetic therapy .", "trials manipulating gck regulatory protein an allosteric switch for gck , which indirectly restores gck activity , are currently underway to evaluate its effect on blood glucose in patients with type 2 dm ( 17 , 18 ) ."], ["treatment of niphs involves lifelong dietary modification consisting of frequent , low carbohydrate meals ( 5 ) , which prevent surges in plasma glucose level , insulin level , and the consequent hypoglycemic spells .", "medications such as diazoxide , octreotide , glucagon , verapamil , and somatostatin , whose actions all involve inhibition of calcium channels , have been employed with varying degrees of success ( 7 ) . diazoxide binds to and activates the sulfonylurea receptor 1 subunit of the potassium adenosine triphosphate [ k(atp ) ] channel , causing calcium channel closure .", "response to diazoxide therefore varies with genetic mutations [ ineffective in mutations of the k(atp ) channel genes ] ( 8) and with pattern of inheritance ( 7 ) .", "octreotide , a somatostatin analogue ( secretin and insulin inhibitor ) , has been used in diazoxide unresponsive chi with improved blood glucose in these patients ; however , side effects such as gastrointestinal symptoms , dilated gallbladder , necrotizing enterocolitis , and qt prolongation sometimes limit its use ( 7 ) .", "long - term use of glucagon is effective in diazoxide - unresponsive cases or severe / diffuse chi but gastrointestinal side effects and crystallization in infusion tubing have been reported ( 19 ) .", "glucagon - like protein 1 receptor agonists such as exendin-4 are newer drugs that improve hypoglycemia in diazoxide - unresponsive patients by inhibiting insulin release ( 7 ) .", "partial pancreatectomy is indicated for medical treatment failure [ suggesting a k(atp ) defect ] and is only curative for focal lesions while extensive resection is reserved for diffuse chi to limit pancreatic burden ( 7 , 8 , 11 , 20 ) ."], ["affected patients can present in adulthood ; de novo mutations should be considered given the following:-  positive family history of unremitting hypoglycemia-  lifelong symptoms of low plasma glucose , even after eating-  increased bmi-  negative sulfonylurea and autoimmune work up-  positive glycemic response to octreotide \n  -  positive family history of unremitting hypoglycemia -  lifelong symptoms of low plasma glucose , even after eating -  negative sulfonylurea and autoimmune work up -  positive glycemic response to octreotide to our knowledge , only three families with affected adults have been reported .", "in addition , this case highlights the pediatric - to - adulthood transition for rare diseases and the key role that coordination and transition of care plays in timely management ."], ["despite the congenital nature of gck mutations , some patients can go undiagnosed till early adulthood .", "future research into a cost - effective and prompt scheme of patient information dissemination is warranted for uncommon diseases ."], ["the authors have not received any funding or benefits from industry or elsewhere to conduct this study ."]]}
{"article_id": "PMC4830364", "article_text": ["obstructive sleep apnea ( osa ) is a common public health problem worldwide and it has been shown to be associated with an increased incidence of cardiovascular complications such as death , stroke , or myocardial infarction .", "the 2014 american heart association / american stroke association guideline recommends that patients with stroke / transient ischemic disorder ( tia ) showed be worked up for osa . in this study , patients who had osa and stroke had an improved outcome with continuous positive airway pressure .", "extrapolating data from western population and implementing in the asian populations may not be possible as there are differences in genetic and anatomical factors .", "for example , the cutoff point for obesity based on body mass index ( bmi ) criterion for western population is 30 kg / m meanwhile the bmi for asian population is 25 kg / m .", "it is also evident that there are differences in anatomical factors between asian and caucasian population such as retrognathia and oropharyngeal width which are main risk factors for osa .", "alternatively , the stop - bang questionnaire developed by chung et al . has been widely used as a sensitive screening tool for osa .", "the stop - bang acronym stands for : snoring history , tired during the day , observed stop breathing while sleep , high blood pressure , bmi more than 35 kg / m , age more than 50 years , neck circumference more than 40 cm and male gender .", "the authors recommended that if a patient had 3 or more criteria mentioned above , it is strongly suggestive for osa .", "however , the cut - off values of this questionnaire are based on the data from caucasians , therefore we hypothesized that to use it in asian population , some adjustments are needed .", "we compared the clinical features in the stop - bang questionnaire between osa - induced hypertension patients and healthy control subjects .", "the study was conducted in the faculty of medicine , khon kaen university ( thailand ) .", "the study protocol was approved by the ethics committee for human research , khon kaen university .", "osa induced - hypertension patients were diagnosed by : i ) met the criteria of hypertension ; ii ) having average apnea - hypopnea index ( ahi ) by polysomnography more than or equal 5 times / hours and iii ) no evidence of other secondary hypertension .", "the control subjects filled up the modified berlin questionnaire and epworth sleepiness scale less than 10 and defined as low risk for osa with low scores .", "demographic data and clinical features of both osa - induced hypertension patients and healthy subjects were recorded and compared .", "the clinical features included age , gender , bmi , neck circumference , mallampati classification , torus palatinus , and torus mandibularis .", "the mallampati classification was defined by asking the subjects to protrude their tongue as much as possible and classified as class 1 to 4 .", "the sample size of the study population was calculated by using the proportion comparison between osa and healthy subjects using winpepi program .", "the proportion of osa was 0.35 and the healthy control was 0.07 with deviation of 5% , power of 90% , and missing data of 10% .", "the sample size was calculated to be 102 subjects ( osa 34 subjects and healthy control 68 subjects ) . due to the incompleteness of the database of medical students ,", "120 healthy controls were selected by systematic sampling from the database ( total of 1174 medical students ) .", "baseline and clinical characteristics of the participants in both groups were compared using descriptive statistics .", "univariate logistic regression analyses were applied to calculate the crude odds ratios of individual variables for having osa .", "all clinically significant variables or p<0.20 by the univariate analyses were included in subsequent multivariate logistic regression analyses .", "analytical results were presented as crude odds ratios ( or ) , adjusted or , and 95% confidence intervals ( ci ) .", "significant risk factors were calculated for the best cut - off points by the receiver operator characteristic curve ( roc curve ) .", "data analyses were performed with stata software ( college station , tx , usa ) and spss software ( chicago , il , usa ) .", "in this study , 42 osa - induced hypertension patients and 82 control subjects who had a complete set of clinical data were included . all clinical features of both groups", "the osa - induced hypertension patients were significantly older ( 59.5 vs 21.0 years ) , with higher proportion of males ( 64.3 vs 59.8% ) , more obese ( 78.6 vs 6.1% ) , higher incidence of mallampati class 3 or more ( 54.8 vs 24.4% ) , larger neck circumference ( 41.3 vs 32.0 cm ) , higher incidence of torus palatinus ( 26.6 vs 0% ) and of torus mandibularis ( 9.5 vs 0% ) . by multiple logistic regression analysis , only two factors bmi and neck circumference ,", "the adjusted odds ratios for both factors were 1.49 ( 95% ci : 1.06 , 2.09 ) and 1.67 ( 95% ci : 1.11 , 2.51 ) , respectively . by the roc curve analyses ,", "the best cut - off points for the bmi and the neck circumference were 24.5 kg / m ( figure 1a ) and 36 cm ( figure 1b ) .", "the sensitivity and the specificity for bmi cut - off point were 97.2% and 91.4% , whereas those for the neck circumference were 94.7% and 82.9% .", "the present results showed that the stop - bang questionnaire needed to adjust the cutoff values of the bmi and neck circumference suitable for thai population .", "subjects should referred for polysomnography if positive at least 3 questions : s , snoring history ; t , tired during the day ; o , observed stop breathing while sleep ; h , high blood pressure ; b , bmi more than 25 kg / m ; a , age more than 50 years ; n , neck circumference more than 36 cm ; g , male gender ( modified from chung et al . ) . for", "the criteria of obesity for asians , the cut - off of the bmi should lower from 35 kg / m to 24.5 or round up to 25 kg / m .", "the cut - off of the neck circumference also should be lowered from 40 cm to 36 cm for asians .", "proposed the cutoff points of 24.1 kg / m for the bmi and 35.5 cm for the neck circumference for japanese population .", "using stop - bang questionnaire is very suitable for thailand and other developing countries due to limited availability of polysomnography , the standard diagnostic tool for osa .", "this low cost tool can select appropriate patients for referral to sleep center for further polysomnography .", "healthy control subjects were not performed polysomnography to exclude osa . however , both the berlin questionnaire and the epworth sleepiness scale were used to exclude osa , which has a sensitivity and specificity of 0.86 , 0.95 and 0.49 , 0.80 , respectively .", "even though the age is a significant factor by univariate logistic regression , it was not included in the subsequent multivariate regression .", "this is because the difference of age group composition between the osa and the healthy control subjects .", "further studies , therefore , are needed to confirm the results of this study and to identify the appropriate cut - off point using the age- and sex - matched controls . also , to increase their sensitivity and specificity , osa patients with other complications than hypertension should be included and compared . whether simple measurement of the neck circumference and the bmi calculation are sufficient to identify osa patients in routine practice by primary health care personnel including physicians , nurses and volunteers should be examined in further study .", "osa has been proved to be a contributing factor for major cardiovascular diseases ; stroke , hypertension , sudden death , and also coronary artery disease .", "treatment of osa may reduce large economic burden from prevention of the morbidity and mortality from stroke and acute coronary syndrome . in conclusion ,", "the appropriate cut - off points for the bmi and the neck circumference for stop - bang questionnaire were 25 kg / m and 36 cm for asian people .", "all hypertensive patients should have their bmi and neck circumference measured to detect the risk factors for osa .", "public health campaign for osa screening is also needed to reduce morbidity and mortality from osa complications ."], "abstract_text": ["<S> obstructive sleep apnea ( osa ) is a common public health issue . if left untreated , osa may cause a large health economic burden from cardiovascular complications particularly stroke . </S>", "<S> the diagnosis of osa can be made by polysomnography , but its availability is limited in the developing countries in asia . </S>", "<S> stop - bang questionnaire is a good screening tool but may need some adjustment for asian population . </S>", "<S> stop - bang stands for : snoring history , tired during the day , observed stop breathing while sleep , high blood pressure , body mass index ( bmi ) more than 35 kg / m2 , age more than 50 years , neck circumference more than 40 cm and male gender . </S>", "<S> we compared clinical features in stop - bang questionnaire between 42 osa induced hypertension patients and 82 healthy control subjects in the faculty of medicine , khon kaen university , thailand . </S>", "<S> the best cutoff point for the bmi and the neck circumference were 24.5 kg / m2 and 36 cm , respectively . </S>", "<S> the sensitivity and specificity of the bmi cutoff point were 97.2% and 91.40 , while those of the neck circumference were 94.7% and 82.9% . in conclusion , the appropriate cutoff points of bmi and neck circumference for thai stop - bang questionnaire were 25 kg / m2 and 36 cm . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion"], "sections": [["obstructive sleep apnea ( osa ) is a common public health problem worldwide and it has been shown to be associated with an increased incidence of cardiovascular complications such as death , stroke , or myocardial infarction .", "the 2014 american heart association / american stroke association guideline recommends that patients with stroke / transient ischemic disorder ( tia ) showed be worked up for osa . in this study , patients who had osa and stroke had an improved outcome with continuous positive airway pressure .", "extrapolating data from western population and implementing in the asian populations may not be possible as there are differences in genetic and anatomical factors .", "for example , the cutoff point for obesity based on body mass index ( bmi ) criterion for western population is 30 kg / m meanwhile the bmi for asian population is 25 kg / m .", "it is also evident that there are differences in anatomical factors between asian and caucasian population such as retrognathia and oropharyngeal width which are main risk factors for osa .", "alternatively , the stop - bang questionnaire developed by chung et al . has been widely used as a sensitive screening tool for osa .", "the stop - bang acronym stands for : snoring history , tired during the day , observed stop breathing while sleep , high blood pressure , bmi more than 35 kg / m , age more than 50 years , neck circumference more than 40 cm and male gender .", "the authors recommended that if a patient had 3 or more criteria mentioned above , it is strongly suggestive for osa .", "however , the cut - off values of this questionnaire are based on the data from caucasians , therefore we hypothesized that to use it in asian population , some adjustments are needed ."], ["we compared the clinical features in the stop - bang questionnaire between osa - induced hypertension patients and healthy control subjects .", "the study was conducted in the faculty of medicine , khon kaen university ( thailand ) .", "the study protocol was approved by the ethics committee for human research , khon kaen university .", "osa induced - hypertension patients were diagnosed by : i ) met the criteria of hypertension ; ii ) having average apnea - hypopnea index ( ahi ) by polysomnography more than or equal 5 times / hours and iii ) no evidence of other secondary hypertension .", "the control subjects filled up the modified berlin questionnaire and epworth sleepiness scale less than 10 and defined as low risk for osa with low scores .", "demographic data and clinical features of both osa - induced hypertension patients and healthy subjects were recorded and compared .", "the clinical features included age , gender , bmi , neck circumference , mallampati classification , torus palatinus , and torus mandibularis .", "the mallampati classification was defined by asking the subjects to protrude their tongue as much as possible and classified as class 1 to 4 .", "the sample size of the study population was calculated by using the proportion comparison between osa and healthy subjects using winpepi program .", "the proportion of osa was 0.35 and the healthy control was 0.07 with deviation of 5% , power of 90% , and missing data of 10% .", "the sample size was calculated to be 102 subjects ( osa 34 subjects and healthy control 68 subjects ) . due to the incompleteness of the database of medical students ,", "120 healthy controls were selected by systematic sampling from the database ( total of 1174 medical students ) .", "baseline and clinical characteristics of the participants in both groups were compared using descriptive statistics .", "univariate logistic regression analyses were applied to calculate the crude odds ratios of individual variables for having osa .", "all clinically significant variables or p<0.20 by the univariate analyses were included in subsequent multivariate logistic regression analyses .", "analytical results were presented as crude odds ratios ( or ) , adjusted or , and 95% confidence intervals ( ci ) .", "significant risk factors were calculated for the best cut - off points by the receiver operator characteristic curve ( roc curve ) .", "data analyses were performed with stata software ( college station , tx , usa ) and spss software ( chicago , il , usa ) ."], ["in this study , 42 osa - induced hypertension patients and 82 control subjects who had a complete set of clinical data were included . all clinical features of both groups", "the osa - induced hypertension patients were significantly older ( 59.5 vs 21.0 years ) , with higher proportion of males ( 64.3 vs 59.8% ) , more obese ( 78.6 vs 6.1% ) , higher incidence of mallampati class 3 or more ( 54.8 vs 24.4% ) , larger neck circumference ( 41.3 vs 32.0 cm ) , higher incidence of torus palatinus ( 26.6 vs 0% ) and of torus mandibularis ( 9.5 vs 0% ) . by multiple logistic regression analysis , only two factors bmi and neck circumference ,", "the adjusted odds ratios for both factors were 1.49 ( 95% ci : 1.06 , 2.09 ) and 1.67 ( 95% ci : 1.11 , 2.51 ) , respectively . by the roc curve analyses ,", "the best cut - off points for the bmi and the neck circumference were 24.5 kg / m ( figure 1a ) and 36 cm ( figure 1b ) .", "the sensitivity and the specificity for bmi cut - off point were 97.2% and 91.4% , whereas those for the neck circumference were 94.7% and 82.9% ."], ["the present results showed that the stop - bang questionnaire needed to adjust the cutoff values of the bmi and neck circumference suitable for thai population .", "subjects should referred for polysomnography if positive at least 3 questions : s , snoring history ; t , tired during the day ; o , observed stop breathing while sleep ; h , high blood pressure ; b , bmi more than 25 kg / m ; a , age more than 50 years ; n , neck circumference more than 36 cm ; g , male gender ( modified from chung et al . ) . for", "the criteria of obesity for asians , the cut - off of the bmi should lower from 35 kg / m to 24.5 or round up to 25 kg / m .", "the cut - off of the neck circumference also should be lowered from 40 cm to 36 cm for asians .", "proposed the cutoff points of 24.1 kg / m for the bmi and 35.5 cm for the neck circumference for japanese population .", "using stop - bang questionnaire is very suitable for thailand and other developing countries due to limited availability of polysomnography , the standard diagnostic tool for osa .", "this low cost tool can select appropriate patients for referral to sleep center for further polysomnography .", "healthy control subjects were not performed polysomnography to exclude osa . however , both the berlin questionnaire and the epworth sleepiness scale were used to exclude osa , which has a sensitivity and specificity of 0.86 , 0.95 and 0.49 , 0.80 , respectively .", "even though the age is a significant factor by univariate logistic regression , it was not included in the subsequent multivariate regression .", "this is because the difference of age group composition between the osa and the healthy control subjects .", "further studies , therefore , are needed to confirm the results of this study and to identify the appropriate cut - off point using the age- and sex - matched controls . also , to increase their sensitivity and specificity , osa patients with other complications than hypertension should be included and compared . whether simple measurement of the neck circumference and the bmi calculation are sufficient to identify osa patients in routine practice by primary health care personnel including physicians , nurses and volunteers should be examined in further study .", "osa has been proved to be a contributing factor for major cardiovascular diseases ; stroke , hypertension , sudden death , and also coronary artery disease .", "treatment of osa may reduce large economic burden from prevention of the morbidity and mortality from stroke and acute coronary syndrome . in conclusion ,", "the appropriate cut - off points for the bmi and the neck circumference for stop - bang questionnaire were 25 kg / m and 36 cm for asian people .", "all hypertensive patients should have their bmi and neck circumference measured to detect the risk factors for osa .", "public health campaign for osa screening is also needed to reduce morbidity and mortality from osa complications ."]]}
{"article_id": "PMC3551390", "article_text": ["triple - negative breast cancer ( tnbc ) is characterized by absence of expression of the estrogen ( er ) and progesterone receptors ( pr ) and no human epidermal growth factor receptor 2 ( her2)/neu gene amplification [ 1 , 2 ] . unlike hormone receptor - positive and her2-overexpressing breast cancers , tnbc is unresponsive to endocrine therapy and her2-targeted agents , respectively , thereby limiting available systemic treatment options to conventional cytotoxic chemotherapy [ 1 , 2 ] .", "patients with tnbc are more likely to have visceral or brain metastases and short relapse - free survival compared with those with other breast cancer subtypes [ 3 , 4 , 5 , 6 ] . while chemotherapies have been effective for treating early - stage disease , with pathologic complete response ( cr ) rates exceeding those of hormone receptor - positive subtypes , patients with metastatic disease have shorter disease - free survival progressing rapidly through several lines of chemotherapy .", "thus , response to chemotherapy has not translated to improvements in progression - free ( pfs ) or overall survival ( os ) in the metastatic setting , and the overall prognosis for tnbc remains poor [ 3 , 7 ] .", "the median os of patients with metastatic disease is relatively short , with reports between 9 and 13.3 months [ 3 , 4 , 5 , 6 ] .", "an unmet clinical need exists for more effective therapies for tnbc , particularly in the metastatic setting .", "taxane - based chemotherapy has demonstrated activity in patients with tnbc [ 1 , 2 ] . in the phase iii eastern cooperative oncology group 2100 trial in which", "91% of the patients had her2-negative disease , 51% of the patients were pr negative and 37% were er negative , the addition of bevacizumab to solvent - based paclitaxel demonstrated significantly improved median pfs compared with solvent - based paclitaxel alone ( 11.8 vs. 5.9 months , respectively ) ; however , this did not translate into a significant improvement in median os for the combination arm .", "nab - paclitaxel , a novel formulation albumin - bound paclitaxel with a mean particle size of 130 nm , was initially designed with the intent to improve the therapeutic index of solvent - based taxanes [ 9 , 10 ] .", "currently , nab - paclitaxel is indicated for the treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy .", "a phase ii study examined the combination of nab - paclitaxel , bevacizumab , and gemcitabine as first - line therapy for patients with her2-negative metastatic breast cancer .", "in this small cohort of patients with stage iv tnbc , the clinical benefit rate as defined by cr , partial response , and stabilization of disease was 85% .", "additionally , in this study , there were no significant differences in pfs or os between triple - negative and hormone receptor - positive patients .", "this work reports on a patient with metastatic tnbc enrolled in this phase ii study of nab - paclitaxel , bevacizumab , and gemcitabine who experienced a long - term complete remission lasting just under 2 years and a pfs from diagnosis of nearly 3 years .", "in june 2007 , a 52-year - old woman was diagnosed with invasive ductal carcinoma .", "the patient initially presented with chest pain , and a subsequent breast examination revealed a mass in the left breast and a palpable sternal mass .", "a lumpectomy and axillary lymph node dissection were performed at an outside institution and results revealed a moderately differentiated , infiltrating ductal carcinoma , high nuclear grade , measuring 4 cm in maximal diameter , with metastases to 2 of 24 axillary nodes .", "further staging also revealed sternal and mediastinal metastases , and a computed tomography ( ct ) scan revealed bilateral pulmonary nodules ( fig .", "tumor samples were negative for er and pr , and positive ( + + ) for her2 by immunohistochemistry .", "however , fluorescent in situ hybridization results were negative for her2 gene amplification , and the patient was diagnosed with triple - negative primary metastatic / stage iv breast cancer .", "the patient was initially seen at the university of miami sylvester comprehensive cancer center and enrolled in the phase ii study of nab - paclitaxel , bevacizumab , and gemcitabine for her2-negative metastatic breast cancer .", "she was treated on study with gemcitabine 1,500 mg / m by 30-min infusion , followed by nab - paclitaxel 150 mg / m by 30-min infusion , then bevacizumab 10 mg / kg by 30-min infusion once every 2 weeks in a 4-week cycle .", "the patient experienced pain relief after 5 weeks on the study ( 2 infusions ) .", "serial ct scans , performed every 2 months , demonstrated progressive reduction in the size of all target lesions ( fig .", "she showed a complete clearance of lesions in all imaging studies , which was confirmed on at least 2 separate occasions approximately 2 months apart according to response evaluation criteria in solid tumors .", "one dose reduction , as required by the study protocol , occurred in november 2008 on cycle 12 , due to treatment - related fatigue .", "the dose of gemcitabine was reduced to 1,250 mg / m , nab - paclitaxel to 125 mg / m , with no modification of the bevacizumab dose , and treatment was continued once every 2 weeks on a 4 week cycle . throughout her treatment ,", "she experienced no major side effects and continued to work uninterrupted except for visits for chemotherapy and evaluation , and her treatment - related fatigue was successfully managed with the dose reduction .", "the patient maintained her response and continued to receive treatment for just over 2 years .", "after 12 months on study , follow - up was performed every 3 months . in may 2010 , the patient had documented disease progression by ct scan .", "metastases were found in the lung , bone , and lymph nodes . as specified in the study protocol , the patient discontinued study treatment due to disease progression .", "she received her last dose of nab - paclitaxel , bevacizumab , and gemcitabine on may 28 , 2010 .", "three weeks after progressing , the patient received capecitabine at a dose of 2,000 mg / m orally twice a day for 2 weeks in 3-week cycles and showed minor response .", "however , therapy was stopped due to unacceptable toxicity , including diarrhea , dehydration , and severe palmar plantar erythrodysesthesia . after 2 months", ", she initiated therapy with ixabepilone , with a minor radiographic response . from august 2010", "to december 2010 , she received 8 cycles of treatment with ixabepilone ( 30 mg / m ) administered intravenously every 3 weeks . in december 2010 , the patient requested a chemotherapy holiday .", "beginning in november 2011 , the patient was treated with eribulin 1.1 mg / m intravenously on days 1 and 8 of a 3-week cycle .", "treatment was discontinued in march 2012 due to rapid disease progression with development of central nervous system metastases .", "she was transferred to hospice care where she died a month later . throughout the course of her disease ,", "she received zoledronic acid at 4 mg intravenously every 4 weeks beginning in november 2007 when she began the phase ii study . in may 2010 ,", "she continued to receive denosumab throughout her chemotherapy holiday from december 2010 to november 2011 until discontinuing therapy in march 2012 after being transitioned to hospice care .", "patients with metastatic tnbc have a very poor prognosis . in a large analysis of 255 patients diagnosed with tnbc at the md anderson cancer center between 1985 and 2004 ,", "the median os of patients with recurrent disease was only 1.0 year ( 95% ci , 0.81.2 years ) for tnbc patients , significantly shorter than the median os of 2.3 years ( 95% ci , 1.92.7 years ) for those with other breast cancer subtypes ( hr , 2.5 ; 95% ci , 1.83.5 ; p < 0.001 ) .", "other studies have reported median os times for patients with recurrent or metastatic tnbc between 9 and13.3 months [ 4 , 5 , 6 ] .", "while median os has not been specifically assessed in a large patient population initially presenting with metastatic tnbc , it is likely to be similarly poor .", "additionally , patients with metastatic tnbc exhibit progressively shorter response durations to successive lines of therapy . in a retrospective analysis of 111 patients with tnbc ( 14%", "of whom presented with metastatic disease at diagnosis ) , the median duration of response was only 12 weeks ( range , 073.1 weeks ) to first - line therapy , 9 weeks ( range , 0120.9 weeks ) to second - line chemotherapy , and 4 weeks ( range , 059 weeks ) to third - line chemotherapy .", "there is an unmet clinical need for effective therapies that prolong survival for patients with metastatic tnbc . in the absence of curative therapies for metastatic disease ,", "the goal of therapy is primarily palliative in nature , leveraging systemic treatment with minimal toxicity to both prolong os and at the same time enhance quality of life by delaying the onset of cancer - related symptoms .", "numerous studies have shown that the combination of gemcitabine and a taxane is an effective regimen that is well tolerated with good response rates .", "the case described here is of a patient with metastatic tnbc treated with first - line combination chemotherapy consisting of nab - paclitaxel , bevacizumab , and gemcitabine every other week . after only 5 weeks and 2 infusions , the patient experienced significant symptom relief from her pain from bone metastases , including the sternum . following 7 months of treatment , she achieved a complete remission that , with continuous therapy , persisted for nearly 2 years for an overall progression - free period from initial diagnosis of 2 years and 7 months .", "following progression , subsequent lines of therapy were able to produce only limited efficacy in disease control .", "following her initial progression - free interval of almost 2 years in cr with nab - paclitaxel , bevacizumab , and gemcitabine therapy , subsequent treatment with capecitabine then ixabepilone resulted in short progression - free intervals of only 2 months and 4 months , respectively . while ixabepilone did provide a pfs benefit , it was very modest . from her initial diagnosis in june 2007 ,", "the patient survived nearly 5 years , which is greatly improved compared to reported median survival of 913.3 months for stage 4 tnbc [ 3 , 4 , 5 , 6 ] . with the aim to enhance quality of life , systemic treatments with minimal toxicity", "while the patient did experience treatment - related fatigue while on nab - paclitaxel , bevacizumab , and gemcitabine combination therapy , this was easily managed with a dose reduction .", "in addition to a lengthy complete remission , the patient was able to work uninterrupted throughout her treatment period , a factor that may significantly elevate quality of life for many patients .", "this patient initially received treatment as a participant in a phase ii study of nab - paclitaxel , bevacizumab , and gemcitabine as first - line therapy for her2-negative metastatic breast cancer . a large proportion ( 13 of 29 ) of the patients enrolled in this study had triple - negative disease .", "overall , this subset of patients demonstrated promising outcomes , with 38% achieving a complete radiographic response and 85% experiencing clinical benefit .", "most notably , survival outcomes were almost identical between tnbc patients and those with hormone receptor - positive disease . additionally , severe adverse events appeared to be less frequent in the overall study population relative to results in other published chemotherapy studies in patients with metastatic breast cancer .", "this case study demonstrates the potential for triplet chemotherapy with nab - paclitaxel , bevacizumab , and gemcitabine to elicit prolonged responses , potentially prolong os , and improve quality of life for patients with metastatic tnbc .", "this combination shows promise for the treatment of tnbc and should be the subject of further investigation in larger randomized trials ."], "abstract_text": ["<S> this is a case study of a 52-year - old female patient diagnosed in june 2007 with primary metastatic invasive ductal carcinoma of the left breast and synchronous metastases in the bone , lymph nodes , and lung . </S>", "<S> biopsy results of the tumor tissue were negative for the estrogen receptor , progesterone receptor , and human epidermal growth factor receptor 2 ( her2 ) . in november 2007 , she participated in a phase ii study of metastatic her2-negative breast cancer . </S>", "<S> treatment consisted of systemic chemotherapy with gemcitabine 1,500 mg / m2 , nab - paclitaxel 150 mg / m2 , and bevacizumab 10 mg / kg once every other week . </S>", "<S> the patient experienced pain relief in her sternum after 5 weeks of chemotherapy , and her analgesic therapy was discontinued . </S>", "<S> after 7 months , the patient achieved a complete radiographic response , which was maintained for nearly 2 additional years . </S>", "<S> she continued receiving treatment throughout this period , requiring 1 dose reduction due to fatigue . </S>", "<S> the patient experienced no other adverse events , including neuropathy , and continued working uninterrupted throughout her treatment . </S>", "<S> the patient was discontinued from the study in may 2010 after disease progression , almost a full 3 years after diagnosis . </S>", "<S> the patient showed minimal response to subsequent therapies but had disease stabilization and died from her disease in april 2012 . </S>", "<S> median overall survival for patients with metastatic triple - negative breast cancer is between 12 and 13.3 months . </S>", "<S> this patient survived nearly 5 years following diagnosis . </S>", "<S> this case exemplifies how therapy with nab - paclitaxel , bevacizumab , and gemcitabine may prolong survival , with minimal toxicity , in select patients with triple - negative metastatic breast cancer . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["triple - negative breast cancer ( tnbc ) is characterized by absence of expression of the estrogen ( er ) and progesterone receptors ( pr ) and no human epidermal growth factor receptor 2 ( her2)/neu gene amplification [ 1 , 2 ] . unlike hormone receptor - positive and her2-overexpressing breast cancers , tnbc is unresponsive to endocrine therapy and her2-targeted agents , respectively , thereby limiting available systemic treatment options to conventional cytotoxic chemotherapy [ 1 , 2 ] .", "patients with tnbc are more likely to have visceral or brain metastases and short relapse - free survival compared with those with other breast cancer subtypes [ 3 , 4 , 5 , 6 ] . while chemotherapies have been effective for treating early - stage disease , with pathologic complete response ( cr ) rates exceeding those of hormone receptor - positive subtypes , patients with metastatic disease have shorter disease - free survival progressing rapidly through several lines of chemotherapy .", "thus , response to chemotherapy has not translated to improvements in progression - free ( pfs ) or overall survival ( os ) in the metastatic setting , and the overall prognosis for tnbc remains poor [ 3 , 7 ] .", "the median os of patients with metastatic disease is relatively short , with reports between 9 and 13.3 months [ 3 , 4 , 5 , 6 ] .", "an unmet clinical need exists for more effective therapies for tnbc , particularly in the metastatic setting .", "taxane - based chemotherapy has demonstrated activity in patients with tnbc [ 1 , 2 ] . in the phase iii eastern cooperative oncology group 2100 trial in which", "91% of the patients had her2-negative disease , 51% of the patients were pr negative and 37% were er negative , the addition of bevacizumab to solvent - based paclitaxel demonstrated significantly improved median pfs compared with solvent - based paclitaxel alone ( 11.8 vs. 5.9 months , respectively ) ; however , this did not translate into a significant improvement in median os for the combination arm .", "nab - paclitaxel , a novel formulation albumin - bound paclitaxel with a mean particle size of 130 nm , was initially designed with the intent to improve the therapeutic index of solvent - based taxanes [ 9 , 10 ] .", "currently , nab - paclitaxel is indicated for the treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy .", "a phase ii study examined the combination of nab - paclitaxel , bevacizumab , and gemcitabine as first - line therapy for patients with her2-negative metastatic breast cancer .", "in this small cohort of patients with stage iv tnbc , the clinical benefit rate as defined by cr , partial response , and stabilization of disease was 85% .", "additionally , in this study , there were no significant differences in pfs or os between triple - negative and hormone receptor - positive patients .", "this work reports on a patient with metastatic tnbc enrolled in this phase ii study of nab - paclitaxel , bevacizumab , and gemcitabine who experienced a long - term complete remission lasting just under 2 years and a pfs from diagnosis of nearly 3 years ."], ["in june 2007 , a 52-year - old woman was diagnosed with invasive ductal carcinoma .", "the patient initially presented with chest pain , and a subsequent breast examination revealed a mass in the left breast and a palpable sternal mass .", "a lumpectomy and axillary lymph node dissection were performed at an outside institution and results revealed a moderately differentiated , infiltrating ductal carcinoma , high nuclear grade , measuring 4 cm in maximal diameter , with metastases to 2 of 24 axillary nodes .", "further staging also revealed sternal and mediastinal metastases , and a computed tomography ( ct ) scan revealed bilateral pulmonary nodules ( fig .", "tumor samples were negative for er and pr , and positive ( + + ) for her2 by immunohistochemistry .", "however , fluorescent in situ hybridization results were negative for her2 gene amplification , and the patient was diagnosed with triple - negative primary metastatic / stage iv breast cancer .", "the patient was initially seen at the university of miami sylvester comprehensive cancer center and enrolled in the phase ii study of nab - paclitaxel , bevacizumab , and gemcitabine for her2-negative metastatic breast cancer .", "she was treated on study with gemcitabine 1,500 mg / m by 30-min infusion , followed by nab - paclitaxel 150 mg / m by 30-min infusion , then bevacizumab 10 mg / kg by 30-min infusion once every 2 weeks in a 4-week cycle .", "the patient experienced pain relief after 5 weeks on the study ( 2 infusions ) .", "serial ct scans , performed every 2 months , demonstrated progressive reduction in the size of all target lesions ( fig .", "she showed a complete clearance of lesions in all imaging studies , which was confirmed on at least 2 separate occasions approximately 2 months apart according to response evaluation criteria in solid tumors .", "one dose reduction , as required by the study protocol , occurred in november 2008 on cycle 12 , due to treatment - related fatigue .", "the dose of gemcitabine was reduced to 1,250 mg / m , nab - paclitaxel to 125 mg / m , with no modification of the bevacizumab dose , and treatment was continued once every 2 weeks on a 4 week cycle . throughout her treatment ,", "she experienced no major side effects and continued to work uninterrupted except for visits for chemotherapy and evaluation , and her treatment - related fatigue was successfully managed with the dose reduction .", "the patient maintained her response and continued to receive treatment for just over 2 years .", "after 12 months on study , follow - up was performed every 3 months . in may 2010 , the patient had documented disease progression by ct scan .", "metastases were found in the lung , bone , and lymph nodes . as specified in the study protocol , the patient discontinued study treatment due to disease progression .", "she received her last dose of nab - paclitaxel , bevacizumab , and gemcitabine on may 28 , 2010 .", "three weeks after progressing , the patient received capecitabine at a dose of 2,000 mg / m orally twice a day for 2 weeks in 3-week cycles and showed minor response .", "however , therapy was stopped due to unacceptable toxicity , including diarrhea , dehydration , and severe palmar plantar erythrodysesthesia . after 2 months", ", she initiated therapy with ixabepilone , with a minor radiographic response . from august 2010", "to december 2010 , she received 8 cycles of treatment with ixabepilone ( 30 mg / m ) administered intravenously every 3 weeks . in december 2010 , the patient requested a chemotherapy holiday .", "beginning in november 2011 , the patient was treated with eribulin 1.1 mg / m intravenously on days 1 and 8 of a 3-week cycle .", "treatment was discontinued in march 2012 due to rapid disease progression with development of central nervous system metastases .", "she was transferred to hospice care where she died a month later . throughout the course of her disease ,", "she received zoledronic acid at 4 mg intravenously every 4 weeks beginning in november 2007 when she began the phase ii study . in may 2010 ,", "she continued to receive denosumab throughout her chemotherapy holiday from december 2010 to november 2011 until discontinuing therapy in march 2012 after being transitioned to hospice care ."], ["patients with metastatic tnbc have a very poor prognosis . in a large analysis of 255 patients diagnosed with tnbc at the md anderson cancer center between 1985 and 2004 ,", "the median os of patients with recurrent disease was only 1.0 year ( 95% ci , 0.81.2 years ) for tnbc patients , significantly shorter than the median os of 2.3 years ( 95% ci , 1.92.7 years ) for those with other breast cancer subtypes ( hr , 2.5 ; 95% ci , 1.83.5 ; p < 0.001 ) .", "other studies have reported median os times for patients with recurrent or metastatic tnbc between 9 and13.3 months [ 4 , 5 , 6 ] .", "while median os has not been specifically assessed in a large patient population initially presenting with metastatic tnbc , it is likely to be similarly poor .", "additionally , patients with metastatic tnbc exhibit progressively shorter response durations to successive lines of therapy . in a retrospective analysis of 111 patients with tnbc ( 14%", "of whom presented with metastatic disease at diagnosis ) , the median duration of response was only 12 weeks ( range , 073.1 weeks ) to first - line therapy , 9 weeks ( range , 0120.9 weeks ) to second - line chemotherapy , and 4 weeks ( range , 059 weeks ) to third - line chemotherapy .", "there is an unmet clinical need for effective therapies that prolong survival for patients with metastatic tnbc . in the absence of curative therapies for metastatic disease ,", "the goal of therapy is primarily palliative in nature , leveraging systemic treatment with minimal toxicity to both prolong os and at the same time enhance quality of life by delaying the onset of cancer - related symptoms .", "numerous studies have shown that the combination of gemcitabine and a taxane is an effective regimen that is well tolerated with good response rates .", "the case described here is of a patient with metastatic tnbc treated with first - line combination chemotherapy consisting of nab - paclitaxel , bevacizumab , and gemcitabine every other week . after only 5 weeks and 2 infusions , the patient experienced significant symptom relief from her pain from bone metastases , including the sternum . following 7 months of treatment , she achieved a complete remission that , with continuous therapy , persisted for nearly 2 years for an overall progression - free period from initial diagnosis of 2 years and 7 months .", "following progression , subsequent lines of therapy were able to produce only limited efficacy in disease control .", "following her initial progression - free interval of almost 2 years in cr with nab - paclitaxel , bevacizumab , and gemcitabine therapy , subsequent treatment with capecitabine then ixabepilone resulted in short progression - free intervals of only 2 months and 4 months , respectively . while ixabepilone did provide a pfs benefit , it was very modest . from her initial diagnosis in june 2007 ,", "the patient survived nearly 5 years , which is greatly improved compared to reported median survival of 913.3 months for stage 4 tnbc [ 3 , 4 , 5 , 6 ] . with the aim to enhance quality of life , systemic treatments with minimal toxicity", "while the patient did experience treatment - related fatigue while on nab - paclitaxel , bevacizumab , and gemcitabine combination therapy , this was easily managed with a dose reduction .", "in addition to a lengthy complete remission , the patient was able to work uninterrupted throughout her treatment period , a factor that may significantly elevate quality of life for many patients .", "this patient initially received treatment as a participant in a phase ii study of nab - paclitaxel , bevacizumab , and gemcitabine as first - line therapy for her2-negative metastatic breast cancer . a large proportion ( 13 of 29 ) of the patients enrolled in this study had triple - negative disease .", "overall , this subset of patients demonstrated promising outcomes , with 38% achieving a complete radiographic response and 85% experiencing clinical benefit .", "most notably , survival outcomes were almost identical between tnbc patients and those with hormone receptor - positive disease . additionally , severe adverse events appeared to be less frequent in the overall study population relative to results in other published chemotherapy studies in patients with metastatic breast cancer .", "this case study demonstrates the potential for triplet chemotherapy with nab - paclitaxel , bevacizumab , and gemcitabine to elicit prolonged responses , potentially prolong os , and improve quality of life for patients with metastatic tnbc .", "this combination shows promise for the treatment of tnbc and should be the subject of further investigation in larger randomized trials ."]]}
{"article_id": "PMC3670274", "article_text": ["in humans , type i interferons are encoded by ~16  genes and individual genes encoding  ,  ,  and . their gene products are highly structurally related and all bind to a single receptor ( ifnar ) consisting of heterodimeric r1 and r2 subunits .", "in contrast , ifn- consists of 3 genes , ifn-1 , 2 and 3 ( formally , il-29 , il-28a and il-28b ) that are more related to the il-10 family than to interferon . ifn- binds a receptor ( ifnlr ) composed of a unique ifnlr1 and a shared il-10r2 subunit .", "while stats 1 and 3 are promiscuously activated by a variety of other cytokines and growth factor receptors , stat2 is selectively recruited to the ifnar and ifnlr .", "stat2 recruitment and activation by both receptors involves tyrosine phosphorylation of stat2 by jak kinases and subsequent oligomerization with stat1 and irf-9 .", "although a fraction of stat1:stat2 heterodimers can translocate to the nucleus and bind atypical gamma activated sequence ( gas)-like elements , the canonical interferon - stimulated gene factor-3 ( isgf3 ) complex of stat2:stat1:irf-9 regulates a large fraction of the interferon pathway genes .", "thus , stat2 is central to the induction of these genes in response to both ifn-/ and ifn-. in some cases , type ii interferon , ifn- , can mobilize the antiviral response in a stat2-dependent manner .", "although there is no evidence for direct recruitment of stat2 to the ifn-r , ifn- signaling can drive the formation of an isgf3-like complex containing stat2 , inhibit viral replication and induce expression of ifn-/ target genes , perhaps through the pairing of phosphorylated stat1 with latent stat2 .", "regardless of its mode and route of activation , stat2 acts as the gatekeeper to the antiviral response , which is underscored by the severe susceptibility of stat2 knockout mice to viruses ranging from influenza to dengue .", "type i and iii interferon , while produced in different amounts by distinct cell populations , regulate the expression of an overlapping set of interferon stimulated genes ( isgs ) .", "some examples include 25-oligoadenylate synthase ( oas ) , which decorates viral rnas with branched polyadenosine , and rna endonuclease l , which promptly degrades rnas containing these polyadenosine modifications .", "most isgs , however , have not been well characterized , and some of these genes are so enigmatic that they do not contain any canonical secondary structures that could aid in predicting their function .", "over 300 isgs have been identified by microarray and genomic analysis , which is curious given the magnitude of antiviral activity exhibited by select individual isgs .", "for example , mxa potently inhibits the replication of a wide range of viruses and can do so in the absence of other isgs or interferon signaling if ectopically expressed .", "not all isgs exhibit such robust effects individually ; nonetheless , when combined with other isgs , the host can mount an impressive arsenal of anti - viral proteins that could conceptually block every step in the viral life cycle .", "however , like oas and mxa , most isg expression requires interferon signaling through stat2 .", "thus , while there are three major nodes to drive interferon secretion ( tlrs , rig - i and nlrs ) and two pathways available to drive stat2 activation ( ifn-/ and ifn- ) , there is only one stat2 .", "in addition to its role in interferon mediated antiviral effect , stat2 also confers anti - proliferative and apoptotic activities on a variety of cell types in response to both type i and iii interferon .", "this activity is perhaps one of the most fundamental actions of interferon to inhibit viral spread by blocking the replication of virally infected cells .", "however , much attention has been focused on the use of ifn-/ to inhibit cancer outgrowth and metastasis given its unique ability to inhibit proliferation of a variety of tumor cell types .", "stat2 is a critical signaling intermediate that regulates much of the apoptotic and anti - proliferative effects of ifn-/ and ifn-. further , mutations within stat2 that protract its ligand - mediated phosphorylation can drive cells down the apoptotic pathway .", "a variety of genetic mutations have been identified in humans that affect ifn-/ and ifn- production , including mutations in nemo , tyk2 and tlr3 .", "further , functional defects in the human unc93b1 gene , which controls signaling via tlr3 , 7 , 8 and 9 , results in dramatic reductions in ifn-/ and ifn- secretion in response to ligands that activate these receptors . however , these subjects are susceptible to only a few types of viral infections , mainly encephalitis caused by herpes simplex virus .", "this restriction in viral susceptibility may reflect the inherent redundancy in driving ifn-/ and ifn- production through alternative pathways such as rig - i and nlrs .", "in contrast , to date , no mutations have been found that block responsiveness to type i or iii interferons in humans , particularly within either the ifn-/ receptors or stat2 . yet", ", defects in a variety of genes that affect both ifn- production and responsiveness have been characterized including ifngr1 , ifngr2 , stat1 and il-12rb1 .", "thus , it is interesting to speculate that mutations that affect ifn- responsiveness , while debilitating , can be tolerated ; yet , mutations that cripple ifn-/ or ifn- responsiveness such as stat2 are perhaps lethal .", "a cascade of cytokines regulate processes of inflammation that drive both local and systemic activation of immune cells , and this process can be initiated by both pathogen sensing as well as sterile tissue injury . when discussing the role of stats in inflammation , it is perhaps more accurate to discuss their function in the context of distinct cytokine receptors , as most of the stat family members can be activated by multiple cytokines .", "for example , stat3 can be activated by both il-6 and il-10 , yet their biological activities are diametrically opposed and counterregulatory with il-6 contributing to inflammation and il-10 blocking inflammation .", "while ifn-/ can induce local inflammation under certain circumstances , it does not mediate the intense forms of inflammation seen with tnf- or il-1. initially , ifn-/ was thought to act similarly to il-12 in driving th1 development through the recruitment and activation of stat4 , thereby driving th1-mediated inflammation .", "indeed , stat2 was shown to be involved in recruiting stat4 to the human ifnar .", "however , our recent findings demonstrated that although stat4 is transiently activated by the human ifnar , it is not sufficient to promote th1 development .", "further , distinct ifn- subtypes can drive stat4 activation to the murine ifnar , which can occur in mouse cells even in the absence of stat2 , yet still does not promote either th1 or tc1 development compared with the effects of il-12 .", "nonetheless , ifn-/ may regulate some aspects of inflammation that play a more supportive rather than direct role .", "ifn-/ ( as well as ifn- ) is a potent regulator of the chemokine cxcl10 ( ip-10 ) , which recruits activated effector t and nk cells to sites of infection .", "thus , direct pathogen sensing by epithelial and endothelial cells can lead to recruitment of inflammatory cells via autocrine responses to ifn-/. ifn-/ also activates nk cells directly by driving lytic activity and ifn- secretion .", "further , recent studies by tracey and colleagues demonstrated that the ifn-/-induced kinase , pkr , regulates an alternative pathway for inflammasome activation . as a result", ", the induction of pkr by ifn-/ can lead to potent secretion of hmgb1 and other leaderless cytokines that are dependent upon inflammasome - mediated cleavage .", "although ifn-/ regulates these processes , a formal test for the direct role stat2 in driving these responses has not been performed in stat2 knockout mice . however , a recent study by gamero and colleagues demonstrated reduced skin and colonic chemokine and cytokine expression in stat2 knockout mice in two models of chemically induced carcinogenesis .", "these results suggest that stat2 may directly contribute to carcinogenesis by promoting expression of proinflammatory cytokines and chemokines . in summary ,", "stat2 participates in some aspects of inflammation by autocrine responses to ifn-/ and downstream induction of inflammatory chemokines and cytokines .", "given the critical nature of the interferon pathway in viral defense , it is not surprising that viruses have evolved to antagonize the antiviral state induced by type i and type iii interferons .", "the different antagonistic mechanisms include inhibition of interferon production , interferon signaling and inhibition of interferon induced antiviral proteins .", "stat2 in particular is a target of many viral proteins , which in some cases determines viral host range .", "here we discuss the viruses that have particularly acquired the ability to subvert the interferon signaling pathway by targeting stat2 ( summarized in table 1 ) .", "viruses from a broad array of genera have developed unique ways to subvert the interferon response , and such examples include herpes simplex virus 2 ( hsv-2 ) , mouse cytomegalovirus ( mcmv ) and lymphocytic choreomeningitis virus ( lcmv ) .", "however , the paramyxoviridae family of viruses contains perhaps the most replete examples of how viruses have subverted the interferon response pathway by targeting stat2 .", "the henipavirus ( nipah and hendra virus ) are newly emerging zoonotic pathogens causing acute respiratory disease in both human and other animal populations .", "each of these viruses along with other members of the paramyxoviridae family express a v protein , which is encoded by a polycistronic gene segment ( p gene ) that utilizes unique overlapping reading frames .", "the v protein from measles , nipah and hendra viruses can bind to stat2 and prevent the interferon response via sequestration of stat2 in the cytoplasm .", "however , the modes of interaction of the v protein from measles mediate this interference by distinct mechanisms from that of henipahviruses .", "while the v proteins of these viruses contain a conserved c - terminal domain ( ctd ) , only the measles virus requires the ctd for binding and targeting stat2 as means of interference with interferon signaling . in contrast , the nipah virus v protein can also bind stat2 in a stat1 dependent manner through distinct regions of the v protein mapping outside the boundary of the ctd .", "these complexes are unable to translocate to the nucleus , thus sequestering stat2 to the cytoplasm and inhibiting efficient interferon signaling .", "the hendravirus v protein also operates in a similar manner to nipah virus , although the distinct interaction with stats have not been mapped .", "in addition , the nipah virus v protein has been shown to contain a nuclear export signal ( nes ) that enables the v protein to shuttle between the cytoplasm and nucleus .", "nonetheless , binding of stat1:stat2 complexes by nipah v protein is sufficient to prevent ifn-/ signaling even in the absence of the nes .", "therefore viruses belonging to the same family have evolved to target stat2 using diverse mechanisms in order to evade interferon response in the host .", "respiratory syncytial virus ( rsv ) is a member of the paramyxovirus family and causes severe flu and cold like symptoms in humans , particularly in children .", "rsv antagonizes the interferon - signaling pathway through two virally encoded nonstructural proteins , ns1 and ns2 .", "together , ns1/2 form a complex with stat2 and drive ubiquitin - mediated proteasomal degradation .", "binding of stat2 with this complex requires the c - terminus of ns2 , while ns1 acts as an e3 ligase that targets stat2 for degradation . in a similar manner to rsv , the rodent pathogen sendai virus ( sev )", "encodes a c protein that antagonizes ifn-/ signaling at all phases of the infection process by interacting with stat1 and blocking activation of stat2 .", "finally , the human parainfluenza virus 2 ( hpiv2 ) is an rna virus of the paramyxoviridae family that causes upper and lower respiratory infections . in human ,", "hpiv2 blocks type i interferon signaling by selectively inducing degradation of stat2 but not stat1 .", "the degradation of stat2 is independent of ifn-/ signaling , and like other members of this family of viruses , the v protein mediates the degradation of stat2 .", "post - translational degradation of stat2 protein by hpiv2 v protein was blocked by proteasome inhibitors indicating a role for proteasomal degradation .", "in contrast , the v protein of parainfluenza virus 5 [ piv5 , previously known as simian virus v ( sv5 ) ] , another member of the same family of viruses , preferentially induces stat1 degradation in a stat2-dependent manner in human .", "the c - terminal domain of this v protein is highly conserved across the members of rubulavirus genus of the paramyxoviridae family that includes both hpiv2 and piv5 .", "although piv5 can target human stat1 via stat2 , mouse stat2 does not interact with the piv5 v protein .", "thus , mouse stat2 restricts piv5 v protein interaction leaving the interferon response pathway intact .", "as such , piv5 is unable to efficiently replicate in mice due to an intact interferon response .", "transgenic mice expressing human stat2 restores the ability of piv5 v protein to mediated degradation of stat1 .", "likewise , expression of human stat2 in mouse embryonic fibroblasts permits antagonism of endogenous murine ifn-/ signaling by both piv5 and hpiv2 v proteins .", "thus , the requirement for stat2 interaction for antagonism of interferon response limits the host range of piv5 . dengue virus ( denv ) is another example of how host - pathogen co - evolution has become a host range - determining factor .", "dengue virus is a mosquito - borne rna virus that causes dengue fever , dengue hemorrhagic fever and dengue shock syndrome in humans .", "denv belongs to the genus flavivirus and has been shown to replicate efficiently in a variety of human immune cells including monocytes , macrophages , b cells and dendritic cells ( reviewed in ref .", "initial studies with denv replicons expressing all of the non - structural proteins demonstrated reduced ifn- stimulated genes concomitant with a reduction in endogenous stat2 protein levels . in later studies ,", "denv ns5 was found to directly bind to and promote ubiquitin - mediated degradation of human , but not mouse stat2 .", "mouse stat2 expression in human cell lines restored the ifn - dependent block in virus production .", "ns5 interaction with human stat2 was mapped to the coiled - coil domain of human stat2 containing a species - specific sequence between residues 181301 .", "as ns5 failed to bind mouse stat2 , replacement of this region of mouse sequence with the human counterpart restored ns5 binding to the chimerized mouse molecule .", "in parallel studies , denv ns5 was found to specifically antagonize ifn- , but not ifn- signaling by binding to and inhibiting stat2 phosphorylation .", "recently , schoggins et al . have demonstrated that overexpression of stat2 in stat1-deficient fibroblast can limit denv replication without any significant isg expression .", "collectively these studies demonstrate that denv is able to establish infection in human due to its ability to surmount human antiviral response via stat2 degradation and blocking phosphorylation that may be circumvented in humans through augmented stat2 expression .", "however , murine stat2 has evolved to disengage this mechanism of stat2 antagonism by denv and thus restricting denv host range to humans .", "given the central role of stat2 in the induction of isgs , it is perhaps surprising that only a restricted subset of viruses have adapted their mechanism of innate subversion to target stat2 .", "for some viruses , stat2 determines the host range across species , and this is particularly evident with dengue virus , discussed above", ". however , closer examination of the stat2 gene and protein product reveals some important insights into how viruses shape the evolution of their hosts . in this section", ", we will explore how stat2 has emerged as a highly divergent member of the stat family while retaining its ability to exclusively regulate the interferon response in each species .", "the stat family can be traced as far back to rudimentary stat - like genes that exist in slime molds ( d. discoideum ) , thus suggesting that the current members that exist in mammals ( stats 1 , 2 , 3 , 4 5a , 5b and 6 ) arose from a common ancestral gene through duplication . in mammals ,", "the seven stat family members are located as clusters in 3 genomic locations : in humans , stats 1 and 4 on chr .", "17 and stats 2 and 6 on chr . 12 . considering their syntenic arrangement across species ,", "how conserved are the various stat members in mammals ? within the ensemble database , there are seven species , including human , for which the full - length sequences are available for six of the seven stat family members .", "an alignment of these sequences reveals remarkable similarity ( > 95% for most pair - wise comparisons ) for each of the stat genes except stat2 ( fig .  1 ) .", "stat3 is the most highly conserved member , retaining > 98% sequence similarity comparing human with both mouse and rat .", "however , human stat2 , while being relatively well conserved with macaque and chimpanzee , diverges significantly when paired with mouse , rat , horse and elephant .", "a phylogenetic analysis further reveals the degree to which stat2 has diverged even within the non - human primate species when compared with the human counterpart ( fig .  2 ) .", "moreover , stat2 s dimerization partner stat1 displays roughly 5-fold greater sequence similarity than stat2 within primates ( based on sequence identity within the phylogenetic analysis in fig . ", "the full length protein sequences of stats 1 , 2 , 3 , 4 , 5a and 6 were aligned against the human sequence for each of the species shown in the radar plot .", "the clustal alignment algorithm within macvector was used for the alignment , and the percent sequence similarity to human in pair - wise comparisons is plotted in the graph .", "the full length sequences of stat1 and stat2 for each species shown were aligned with the clustal algorithm within macvector .", "phylogenetic analysis was performed with this alignment , and the scale indicates the absolute numbers of sequence differences across the length of the dendrogram . within each stat family member ,", "the sh2 domain is the most highly conserved segment of the gene across species , and stat2 is no exception to this ( fig . ", "however , just beyond the sh2 domain , the c - terminal transactivation domain of stat2 diverges so significantly that only small segments of conservation are retained at the distal end of the protein .", "most striking is the disruption of the structure and insertion of a  mini - satellite  segment within the c - terminus of mouse stat2 that contains a repetitive element papqvlle .", "this element has no significant similarity to any other protein in the database , and mouse is the only species that harbors this sequence .", "the sequences of stat2 from the indicated species were aligned beginning with residue 651 of human stat2 .", "the sh2 and c - terminus of stat2 is indicated along with the conserved tyrosine residue ( y690 ) , which is phosphorylated in responses to ifn-/ signaling .", "the divergence in stat2 was first appreciated when mouse stat2 was cloned and sequenced over 12 years ago . at that time", ", it was known that stat2 interacted with other transcription factors to regulate expression of various isgs , and one of those factors was cbp .", "demonstrated that while the c - terminus of mouse was significantly different than human , cbp could still interact with the c - terminus of both mouse and human stat2 and functionally cooperate to regulate transcription .", "in addition , stat2 was also found to be required for recruitment of stat4 to the human , but not mouse ifnar .", "further , the human stat2 c - terminus was found to be required for this recruitment in human cells , and the significant divergence in this region partially explained the difference in signaling between the two species .", "however , the interaction was perhaps indirect as replacement of the mouse c - terminus with the human counterpart via knock - in failed to restore significant ifn-/-dependent stat4 activation in mouse t cells . nonetheless , these animals still responded normally to ifn-/ by activating isgf3 and by driving the expression of isgs .", "these types of domain - swapping experiments reveal some very interesting aspects of stat2 biology . despite drastic differences within the c - terminus ,", "this domain still maintains a physical interaction with cbp and perhaps many other transcription factors whose evolution has remained relatively constant compared with stat2 .", "indeed , the c - terminus of stat2 is relatively unstructured compared with other domains within the protein .", "however , when bound to the transcriptional activator zinc binding ( taz ) domain of cbp , the human stat2 c - terminus undergoes dynamic folding to create a stable tertiary structure , thereby stabilizing its bound conformation to the taz domain .", "unfortunately , the structure of the c - terminus from other species such as mouse with the cbp taz domain has not been solved .", "nonetheless , stat2 displays remarkable plasticity in both its proximal receptor activation and downstream gene transactivation capabilities regardless of the remarkable structural differences that exist , particularly within the c - terminus .", "thus , even though stat2 has diverged at a much greater rate than other stat family members , the interactions that stat2 must maintain to mediate its function are still preserved and presumably contain enough inherent flexibility to accommodate large changes in stat2 s structure .", "considering the central role that stat2 plays in regulating the intracellular anti - viral response , it represents an important target for viral interference . while speculative , it is likely that lethal pandemic viruses drive stat2 evolution , in some cases , by targeting stat2 directly .", "perhaps the unique haplotypes that have been identified in stat2 among east asians and shared with neanderthal point to recent selection events in human evolution .", "regardless , the survival of each species , including humans , to viral assaults thus depends upon the flexibility of stat2 to maintain distinct physical interactions , which preserve its function in driving the interferon response ."], "abstract_text": ["<S> stat2 is unique among the stat family of transcription factors in that its activation is driven predominantly by only two classes of cell surface receptors : type i and iii interferon receptors . as such </S>", "<S> , stat2 plays a critical role in host defenses against viral infections . </S>", "<S> viruses have evolved to target stat2 by either inhibiting its expression , blocking its activity , or by targeting it for degradation . </S>", "<S> consequently , these viral onslaughts have driven remarkable divergence in the stat2 gene across species that is not observed in other stat family members . </S>", "<S> thus , the evolution of stat2 may preserve its activity and protect each species in the face of an ever - changing viral community . </S>"], "labels": null, "section_names": ["Receptor-Mediated STAT2 Activation and the Interferon Response", "STAT2 in Inflammation", "Viruses Target STAT2", "STAT2 Evolution"], "sections": [["in humans , type i interferons are encoded by ~16  genes and individual genes encoding  ,  ,  and . their gene products are highly structurally related and all bind to a single receptor ( ifnar ) consisting of heterodimeric r1 and r2 subunits .", "in contrast , ifn- consists of 3 genes , ifn-1 , 2 and 3 ( formally , il-29 , il-28a and il-28b ) that are more related to the il-10 family than to interferon . ifn- binds a receptor ( ifnlr ) composed of a unique ifnlr1 and a shared il-10r2 subunit .", "while stats 1 and 3 are promiscuously activated by a variety of other cytokines and growth factor receptors , stat2 is selectively recruited to the ifnar and ifnlr .", "stat2 recruitment and activation by both receptors involves tyrosine phosphorylation of stat2 by jak kinases and subsequent oligomerization with stat1 and irf-9 .", "although a fraction of stat1:stat2 heterodimers can translocate to the nucleus and bind atypical gamma activated sequence ( gas)-like elements , the canonical interferon - stimulated gene factor-3 ( isgf3 ) complex of stat2:stat1:irf-9 regulates a large fraction of the interferon pathway genes .", "thus , stat2 is central to the induction of these genes in response to both ifn-/ and ifn-. in some cases , type ii interferon , ifn- , can mobilize the antiviral response in a stat2-dependent manner .", "although there is no evidence for direct recruitment of stat2 to the ifn-r , ifn- signaling can drive the formation of an isgf3-like complex containing stat2 , inhibit viral replication and induce expression of ifn-/ target genes , perhaps through the pairing of phosphorylated stat1 with latent stat2 .", "regardless of its mode and route of activation , stat2 acts as the gatekeeper to the antiviral response , which is underscored by the severe susceptibility of stat2 knockout mice to viruses ranging from influenza to dengue .", "type i and iii interferon , while produced in different amounts by distinct cell populations , regulate the expression of an overlapping set of interferon stimulated genes ( isgs ) .", "some examples include 25-oligoadenylate synthase ( oas ) , which decorates viral rnas with branched polyadenosine , and rna endonuclease l , which promptly degrades rnas containing these polyadenosine modifications .", "most isgs , however , have not been well characterized , and some of these genes are so enigmatic that they do not contain any canonical secondary structures that could aid in predicting their function .", "over 300 isgs have been identified by microarray and genomic analysis , which is curious given the magnitude of antiviral activity exhibited by select individual isgs .", "for example , mxa potently inhibits the replication of a wide range of viruses and can do so in the absence of other isgs or interferon signaling if ectopically expressed .", "not all isgs exhibit such robust effects individually ; nonetheless , when combined with other isgs , the host can mount an impressive arsenal of anti - viral proteins that could conceptually block every step in the viral life cycle .", "however , like oas and mxa , most isg expression requires interferon signaling through stat2 .", "thus , while there are three major nodes to drive interferon secretion ( tlrs , rig - i and nlrs ) and two pathways available to drive stat2 activation ( ifn-/ and ifn- ) , there is only one stat2 .", "in addition to its role in interferon mediated antiviral effect , stat2 also confers anti - proliferative and apoptotic activities on a variety of cell types in response to both type i and iii interferon .", "this activity is perhaps one of the most fundamental actions of interferon to inhibit viral spread by blocking the replication of virally infected cells .", "however , much attention has been focused on the use of ifn-/ to inhibit cancer outgrowth and metastasis given its unique ability to inhibit proliferation of a variety of tumor cell types .", "stat2 is a critical signaling intermediate that regulates much of the apoptotic and anti - proliferative effects of ifn-/ and ifn-. further , mutations within stat2 that protract its ligand - mediated phosphorylation can drive cells down the apoptotic pathway .", "a variety of genetic mutations have been identified in humans that affect ifn-/ and ifn- production , including mutations in nemo , tyk2 and tlr3 .", "further , functional defects in the human unc93b1 gene , which controls signaling via tlr3 , 7 , 8 and 9 , results in dramatic reductions in ifn-/ and ifn- secretion in response to ligands that activate these receptors . however , these subjects are susceptible to only a few types of viral infections , mainly encephalitis caused by herpes simplex virus .", "this restriction in viral susceptibility may reflect the inherent redundancy in driving ifn-/ and ifn- production through alternative pathways such as rig - i and nlrs .", "in contrast , to date , no mutations have been found that block responsiveness to type i or iii interferons in humans , particularly within either the ifn-/ receptors or stat2 . yet", ", defects in a variety of genes that affect both ifn- production and responsiveness have been characterized including ifngr1 , ifngr2 , stat1 and il-12rb1 .", "thus , it is interesting to speculate that mutations that affect ifn- responsiveness , while debilitating , can be tolerated ; yet , mutations that cripple ifn-/ or ifn- responsiveness such as stat2 are perhaps lethal ."], ["a cascade of cytokines regulate processes of inflammation that drive both local and systemic activation of immune cells , and this process can be initiated by both pathogen sensing as well as sterile tissue injury . when discussing the role of stats in inflammation , it is perhaps more accurate to discuss their function in the context of distinct cytokine receptors , as most of the stat family members can be activated by multiple cytokines .", "for example , stat3 can be activated by both il-6 and il-10 , yet their biological activities are diametrically opposed and counterregulatory with il-6 contributing to inflammation and il-10 blocking inflammation .", "while ifn-/ can induce local inflammation under certain circumstances , it does not mediate the intense forms of inflammation seen with tnf- or il-1. initially , ifn-/ was thought to act similarly to il-12 in driving th1 development through the recruitment and activation of stat4 , thereby driving th1-mediated inflammation .", "indeed , stat2 was shown to be involved in recruiting stat4 to the human ifnar .", "however , our recent findings demonstrated that although stat4 is transiently activated by the human ifnar , it is not sufficient to promote th1 development .", "further , distinct ifn- subtypes can drive stat4 activation to the murine ifnar , which can occur in mouse cells even in the absence of stat2 , yet still does not promote either th1 or tc1 development compared with the effects of il-12 .", "nonetheless , ifn-/ may regulate some aspects of inflammation that play a more supportive rather than direct role .", "ifn-/ ( as well as ifn- ) is a potent regulator of the chemokine cxcl10 ( ip-10 ) , which recruits activated effector t and nk cells to sites of infection .", "thus , direct pathogen sensing by epithelial and endothelial cells can lead to recruitment of inflammatory cells via autocrine responses to ifn-/. ifn-/ also activates nk cells directly by driving lytic activity and ifn- secretion .", "further , recent studies by tracey and colleagues demonstrated that the ifn-/-induced kinase , pkr , regulates an alternative pathway for inflammasome activation . as a result", ", the induction of pkr by ifn-/ can lead to potent secretion of hmgb1 and other leaderless cytokines that are dependent upon inflammasome - mediated cleavage .", "although ifn-/ regulates these processes , a formal test for the direct role stat2 in driving these responses has not been performed in stat2 knockout mice . however , a recent study by gamero and colleagues demonstrated reduced skin and colonic chemokine and cytokine expression in stat2 knockout mice in two models of chemically induced carcinogenesis .", "these results suggest that stat2 may directly contribute to carcinogenesis by promoting expression of proinflammatory cytokines and chemokines . in summary ,", "stat2 participates in some aspects of inflammation by autocrine responses to ifn-/ and downstream induction of inflammatory chemokines and cytokines ."], ["given the critical nature of the interferon pathway in viral defense , it is not surprising that viruses have evolved to antagonize the antiviral state induced by type i and type iii interferons .", "the different antagonistic mechanisms include inhibition of interferon production , interferon signaling and inhibition of interferon induced antiviral proteins .", "stat2 in particular is a target of many viral proteins , which in some cases determines viral host range .", "here we discuss the viruses that have particularly acquired the ability to subvert the interferon signaling pathway by targeting stat2 ( summarized in table 1 ) .", "viruses from a broad array of genera have developed unique ways to subvert the interferon response , and such examples include herpes simplex virus 2 ( hsv-2 ) , mouse cytomegalovirus ( mcmv ) and lymphocytic choreomeningitis virus ( lcmv ) .", "however , the paramyxoviridae family of viruses contains perhaps the most replete examples of how viruses have subverted the interferon response pathway by targeting stat2 .", "the henipavirus ( nipah and hendra virus ) are newly emerging zoonotic pathogens causing acute respiratory disease in both human and other animal populations .", "each of these viruses along with other members of the paramyxoviridae family express a v protein , which is encoded by a polycistronic gene segment ( p gene ) that utilizes unique overlapping reading frames .", "the v protein from measles , nipah and hendra viruses can bind to stat2 and prevent the interferon response via sequestration of stat2 in the cytoplasm .", "however , the modes of interaction of the v protein from measles mediate this interference by distinct mechanisms from that of henipahviruses .", "while the v proteins of these viruses contain a conserved c - terminal domain ( ctd ) , only the measles virus requires the ctd for binding and targeting stat2 as means of interference with interferon signaling . in contrast , the nipah virus v protein can also bind stat2 in a stat1 dependent manner through distinct regions of the v protein mapping outside the boundary of the ctd .", "these complexes are unable to translocate to the nucleus , thus sequestering stat2 to the cytoplasm and inhibiting efficient interferon signaling .", "the hendravirus v protein also operates in a similar manner to nipah virus , although the distinct interaction with stats have not been mapped .", "in addition , the nipah virus v protein has been shown to contain a nuclear export signal ( nes ) that enables the v protein to shuttle between the cytoplasm and nucleus .", "nonetheless , binding of stat1:stat2 complexes by nipah v protein is sufficient to prevent ifn-/ signaling even in the absence of the nes .", "therefore viruses belonging to the same family have evolved to target stat2 using diverse mechanisms in order to evade interferon response in the host .", "respiratory syncytial virus ( rsv ) is a member of the paramyxovirus family and causes severe flu and cold like symptoms in humans , particularly in children .", "rsv antagonizes the interferon - signaling pathway through two virally encoded nonstructural proteins , ns1 and ns2 .", "together , ns1/2 form a complex with stat2 and drive ubiquitin - mediated proteasomal degradation .", "binding of stat2 with this complex requires the c - terminus of ns2 , while ns1 acts as an e3 ligase that targets stat2 for degradation . in a similar manner to rsv , the rodent pathogen sendai virus ( sev )", "encodes a c protein that antagonizes ifn-/ signaling at all phases of the infection process by interacting with stat1 and blocking activation of stat2 .", "finally , the human parainfluenza virus 2 ( hpiv2 ) is an rna virus of the paramyxoviridae family that causes upper and lower respiratory infections . in human ,", "hpiv2 blocks type i interferon signaling by selectively inducing degradation of stat2 but not stat1 .", "the degradation of stat2 is independent of ifn-/ signaling , and like other members of this family of viruses , the v protein mediates the degradation of stat2 .", "post - translational degradation of stat2 protein by hpiv2 v protein was blocked by proteasome inhibitors indicating a role for proteasomal degradation .", "in contrast , the v protein of parainfluenza virus 5 [ piv5 , previously known as simian virus v ( sv5 ) ] , another member of the same family of viruses , preferentially induces stat1 degradation in a stat2-dependent manner in human .", "the c - terminal domain of this v protein is highly conserved across the members of rubulavirus genus of the paramyxoviridae family that includes both hpiv2 and piv5 .", "although piv5 can target human stat1 via stat2 , mouse stat2 does not interact with the piv5 v protein .", "thus , mouse stat2 restricts piv5 v protein interaction leaving the interferon response pathway intact .", "as such , piv5 is unable to efficiently replicate in mice due to an intact interferon response .", "transgenic mice expressing human stat2 restores the ability of piv5 v protein to mediated degradation of stat1 .", "likewise , expression of human stat2 in mouse embryonic fibroblasts permits antagonism of endogenous murine ifn-/ signaling by both piv5 and hpiv2 v proteins .", "thus , the requirement for stat2 interaction for antagonism of interferon response limits the host range of piv5 . dengue virus ( denv ) is another example of how host - pathogen co - evolution has become a host range - determining factor .", "dengue virus is a mosquito - borne rna virus that causes dengue fever , dengue hemorrhagic fever and dengue shock syndrome in humans .", "denv belongs to the genus flavivirus and has been shown to replicate efficiently in a variety of human immune cells including monocytes , macrophages , b cells and dendritic cells ( reviewed in ref .", "initial studies with denv replicons expressing all of the non - structural proteins demonstrated reduced ifn- stimulated genes concomitant with a reduction in endogenous stat2 protein levels . in later studies ,", "denv ns5 was found to directly bind to and promote ubiquitin - mediated degradation of human , but not mouse stat2 .", "mouse stat2 expression in human cell lines restored the ifn - dependent block in virus production .", "ns5 interaction with human stat2 was mapped to the coiled - coil domain of human stat2 containing a species - specific sequence between residues 181301 .", "as ns5 failed to bind mouse stat2 , replacement of this region of mouse sequence with the human counterpart restored ns5 binding to the chimerized mouse molecule .", "in parallel studies , denv ns5 was found to specifically antagonize ifn- , but not ifn- signaling by binding to and inhibiting stat2 phosphorylation .", "recently , schoggins et al . have demonstrated that overexpression of stat2 in stat1-deficient fibroblast can limit denv replication without any significant isg expression .", "collectively these studies demonstrate that denv is able to establish infection in human due to its ability to surmount human antiviral response via stat2 degradation and blocking phosphorylation that may be circumvented in humans through augmented stat2 expression .", "however , murine stat2 has evolved to disengage this mechanism of stat2 antagonism by denv and thus restricting denv host range to humans ."], ["given the central role of stat2 in the induction of isgs , it is perhaps surprising that only a restricted subset of viruses have adapted their mechanism of innate subversion to target stat2 .", "for some viruses , stat2 determines the host range across species , and this is particularly evident with dengue virus , discussed above", ". however , closer examination of the stat2 gene and protein product reveals some important insights into how viruses shape the evolution of their hosts . in this section", ", we will explore how stat2 has emerged as a highly divergent member of the stat family while retaining its ability to exclusively regulate the interferon response in each species .", "the stat family can be traced as far back to rudimentary stat - like genes that exist in slime molds ( d. discoideum ) , thus suggesting that the current members that exist in mammals ( stats 1 , 2 , 3 , 4 5a , 5b and 6 ) arose from a common ancestral gene through duplication . in mammals ,", "the seven stat family members are located as clusters in 3 genomic locations : in humans , stats 1 and 4 on chr .", "17 and stats 2 and 6 on chr . 12 . considering their syntenic arrangement across species ,", "how conserved are the various stat members in mammals ? within the ensemble database , there are seven species , including human , for which the full - length sequences are available for six of the seven stat family members .", "an alignment of these sequences reveals remarkable similarity ( > 95% for most pair - wise comparisons ) for each of the stat genes except stat2 ( fig .  1 ) .", "stat3 is the most highly conserved member , retaining > 98% sequence similarity comparing human with both mouse and rat .", "however , human stat2 , while being relatively well conserved with macaque and chimpanzee , diverges significantly when paired with mouse , rat , horse and elephant .", "a phylogenetic analysis further reveals the degree to which stat2 has diverged even within the non - human primate species when compared with the human counterpart ( fig .  2 ) .", "moreover , stat2 s dimerization partner stat1 displays roughly 5-fold greater sequence similarity than stat2 within primates ( based on sequence identity within the phylogenetic analysis in fig . ", "the full length protein sequences of stats 1 , 2 , 3 , 4 , 5a and 6 were aligned against the human sequence for each of the species shown in the radar plot .", "the clustal alignment algorithm within macvector was used for the alignment , and the percent sequence similarity to human in pair - wise comparisons is plotted in the graph .", "the full length sequences of stat1 and stat2 for each species shown were aligned with the clustal algorithm within macvector .", "phylogenetic analysis was performed with this alignment , and the scale indicates the absolute numbers of sequence differences across the length of the dendrogram . within each stat family member ,", "the sh2 domain is the most highly conserved segment of the gene across species , and stat2 is no exception to this ( fig . ", "however , just beyond the sh2 domain , the c - terminal transactivation domain of stat2 diverges so significantly that only small segments of conservation are retained at the distal end of the protein .", "most striking is the disruption of the structure and insertion of a  mini - satellite  segment within the c - terminus of mouse stat2 that contains a repetitive element papqvlle .", "this element has no significant similarity to any other protein in the database , and mouse is the only species that harbors this sequence .", "the sequences of stat2 from the indicated species were aligned beginning with residue 651 of human stat2 .", "the sh2 and c - terminus of stat2 is indicated along with the conserved tyrosine residue ( y690 ) , which is phosphorylated in responses to ifn-/ signaling .", "the divergence in stat2 was first appreciated when mouse stat2 was cloned and sequenced over 12 years ago . at that time", ", it was known that stat2 interacted with other transcription factors to regulate expression of various isgs , and one of those factors was cbp .", "demonstrated that while the c - terminus of mouse was significantly different than human , cbp could still interact with the c - terminus of both mouse and human stat2 and functionally cooperate to regulate transcription .", "in addition , stat2 was also found to be required for recruitment of stat4 to the human , but not mouse ifnar .", "further , the human stat2 c - terminus was found to be required for this recruitment in human cells , and the significant divergence in this region partially explained the difference in signaling between the two species .", "however , the interaction was perhaps indirect as replacement of the mouse c - terminus with the human counterpart via knock - in failed to restore significant ifn-/-dependent stat4 activation in mouse t cells . nonetheless , these animals still responded normally to ifn-/ by activating isgf3 and by driving the expression of isgs .", "these types of domain - swapping experiments reveal some very interesting aspects of stat2 biology . despite drastic differences within the c - terminus ,", "this domain still maintains a physical interaction with cbp and perhaps many other transcription factors whose evolution has remained relatively constant compared with stat2 .", "indeed , the c - terminus of stat2 is relatively unstructured compared with other domains within the protein .", "however , when bound to the transcriptional activator zinc binding ( taz ) domain of cbp , the human stat2 c - terminus undergoes dynamic folding to create a stable tertiary structure , thereby stabilizing its bound conformation to the taz domain .", "unfortunately , the structure of the c - terminus from other species such as mouse with the cbp taz domain has not been solved .", "nonetheless , stat2 displays remarkable plasticity in both its proximal receptor activation and downstream gene transactivation capabilities regardless of the remarkable structural differences that exist , particularly within the c - terminus .", "thus , even though stat2 has diverged at a much greater rate than other stat family members , the interactions that stat2 must maintain to mediate its function are still preserved and presumably contain enough inherent flexibility to accommodate large changes in stat2 s structure .", "considering the central role that stat2 plays in regulating the intracellular anti - viral response , it represents an important target for viral interference . while speculative , it is likely that lethal pandemic viruses drive stat2 evolution , in some cases , by targeting stat2 directly .", "perhaps the unique haplotypes that have been identified in stat2 among east asians and shared with neanderthal point to recent selection events in human evolution .", "regardless , the survival of each species , including humans , to viral assaults thus depends upon the flexibility of stat2 to maintain distinct physical interactions , which preserve its function in driving the interferon response ."]]}
{"article_id": "PMC3262558", "article_text": ["for patients with operable breast cancer , the major prognostic determinant is whether there has or has not been spread to the axilla and the number of involved axillary nodes .", "initially it was suggested that breast cancer first spreads locoregionally via lymphatics to the axillary lymph nodes and then metastasises more distantly . in accordance with this concept ,", "subsequently fisher postulated that the extent of micrometastases at diagnosis of breast cancer is an indicator of outcome , with biological behaviour of cancer predetermining the likelihood of progression of the disease . nowadays gene expression profiling arrays can delineate tumour types with different prognoses .", "the surgical approach for breast cancer treatment evolved from the extensive radical mastectomy and the patey modified radical mastectomy   to breast conserving and minimally invasive techniques .", "traditionally the surgical management of breast cancer comprised wide local resection of the primary tumour and axillary lymph node dissection ( alnd ) .", "axillary status is the most important prognostic factor in breast cancer providing staging information and therefore largely defining treatment strategy .", "diagnostic imaging modalities such as ultrasound , magnetic resonance mammography , positron emission tomography , and 99  m technetium ( tc ) sestamibi scintimammography are not reliable for staging the axilla , particularly with lymph node metastases < 0.5  cm [ 8 , 9 ] .", "clinically palpable lymph nodes prove to be false positive in 2530% of patients   and about 40% have positive results after ultrasound with or without fine - needle aspiration node negativity .", "studying 24,740 women with invasive breast cancer , carter et al . showed that approximately 80% with tumour size < 1  cm , 50% with up to 5  cm , and 30% with > 5% had negative axilla , a fact suggesting that metastases do not occur exclusively via the axillary lymph nodes , but rather lymph node status serves as an indicator of the tumour 's ability to spread . additionally , it has been recently shown that the molecular profile of the primary tumour is a more significant prognostic indicator in terms of disease - free survival ( dfs ) and overall survival ( os ) than lymph node metastases .", "the combination of the introduction of population - based mammographic screening for breast cancer , modern imaging methods , and increased public awareness resulted in patients being diagnosed more often with smaller - size tumours and less likelihood of axillary lymph node metastases .", "it is evident now that 6070% of patients with early breast cancer are node negative at the time of diagnosis   and alnd puts them at significant risk of short- and long - term morbidities without benefit .", "alnd is associated with acute complication rates of 2030% including seroma formation , local swelling , numbness , impaired shoulder movement , neuropathy , infection , and chronic lymphoedema rates of 737% . in a prospective study by petrek et al . evaluating a cohort of 923 women with 20 years", "follow up , it was shown that breast - cancer - related lymphoedema following alnd occurred maximally in the first 3 years following surgery ; however , up to 23% of patients may still develop arm swelling during the rest of their lives .", "several randomised studies have established that sentinel node biopsy ( snb ) is a safe and accurate procedure for detecting tumour cells in sln and predicting the status of the other axillary nodes ( non - sln ) .", "although accuracy and appropriateness of snb were disputed by the finding of 510% false - negative cases when snb was followed by axillary dissection at high - risk patients for axillary nodal disease [ 13 , 18 ] , false - negative snb results seem to have decreased with the increasing experience of surgeons , and it is expected that the utilisation of snb in the future will be increased . a meta - analysis of seven prospective randomised controlled trials by kell et al . demonstrated that snb is equivalent to alnd for the detection of lymph node metastasis with the additional advantage of reduction of up to 75% in morbidity in patients with early stage breast cancer .  ", "furthermore , a trend towards an improved detection of ln metastases was shown when snb is used .", "patients undergoing snb have a 22% higher odds ratio of having a positive sln , due to the more intensive pathological examination which utilises multiple sections and immunohistochemistry ( ihc ) .", "in contrast , the false - negative cases seen after axillary dissection are probably due to the inability of the pathologist to perform serial sections and ihc on the 2030 lymph nodes found in a complete axillary clearance specimen .", "studies have shown that sln is the only positive lymph node in 3867% of patients when alnc followed .", "interestingly , it has been reported that only 48% of patients with negative alns have internal mammary lymph node involvement ( imn ) whereas 2550% of patients with affected alns have also imn metastases [ 23 , 24 ] .", "dissection of imn is not recommended because of the high morbidity and the uncertain benefit on survival .", "the recently published outcomes of nsabp b-32 trial established the efficacy of sln biopsy alone with no further alnd in 5611 breast cancer patients with clinically negative lymph nodes .", "women with invasive breast cancer who were randomly assigned to either sln resection plus alnd ( group 1 ) or to sln resection alone with alnd only if the slns were positive ( group 2 ) , after 8 years of followup , showed statistically equivalent overall survival , disease - free survival , and regional control .  ", "patient followup is still continuing for longer - term assessment of survival and regional control .", "moreover , a closer look into mature studies focused on axillary relapses and overall survival is in agreement with current findings favouring slb .", "the national surgical adjuvant breast and bowel project ( nsabp ) b04 randomised study compared breast cancer patients with clinically negative alns managed either by radical mastectomy , total mastectomy with axillary radiation , or total mastectomy alone .", "the results clearly defined that alnd decreases the risk of locoregional relaps ; however , no significant differences in survival were found among the treatment groups .", "this study has been criticised because of the variability of numbers of lymph nodes resected in the total mastectomy alone arm and the lack of statistical power to detect a small difference in outcome . indeed , a meta - analysis has suggested that inadequate axillary treatment may lead to not only an increased risk of local relapse but also a 5% reduction in survival . as a result of increasing detection of early breast cancer and the high rate of micrometastases and itcs ( itcs ) found in the detailed pathological examination of sln", ", a new debate has opened about the consequent necessity of alnd in these patients .", "this has arisen because of better understanding of breast cancer behaviour and improved efficacy of combined therapeutic modalities . in this paper", "we report the current guidelines concerning the management of the axilla after slnb and review the different aspects arising from recent studies on the role of micrometastases and itc clusters in sln on decision making .", "the american society of clinical oncology ( asco ) expert panel conducted a systematic review of the literature available through february 2004 on the use of snb in early - stage breast cancer in order to develop guidelines for the management of the axilla ( http://jop.ascopubs.org/content/1/4/134 ) , and these are similar to those of the national institute of health and clinical excellence ( nice ) recommendations in uk ( http://www.nice.org.uk/nicemedia/live/12132/43413/43413.pdf ) .  ", "alnd is the standard of care in those with a macrometastatic or micrometastatic positive sln to maximise local control .", "if the sln is negative , a calnd ( calnd ) is not necessary .  ", "itcs detected by ihc are of unknown clinical significance , and when identified , the sln is regarded as negative and no further alnd is required . although ihc is often used , it is not included in routine sln evaluation for breast cancer at this time .", "asco and nice recommendations for slnb , alnd alone and managing of the axilla after slnb are summarised in table 1 .  ", "in contrast the german guidelines do not recommend axillary clearance for 1 - 2 sln positive in patients with t1 and t2 tumours ( http://www.ago-online.de ) .  ", "it has been suggested that slnb should be carried out by an experienced team in order to minimise false negativity and improve the predictive value of the procedure .  ", "the american joint committee on cancer ( ajcc ) in the sixth edition of the cancer staging manual defined a lymph node metastatic tumour with maximum diameter      > 2  mm as macrometastasis ( pn1 ) , when the diameter of deposit is 0.22  mm as micrometastasis ( pnmi ) , and a lesion of single tumour cells or small cell clusters with diameter < 0.2  mm as itcs [ pn0(i+ ) ] .", "itcs are not distinguishable by h&e staining but detected only with immunohistochemistry ( ihc ) or molecular methods .  ", "moore et al . suggested that the presence of itcs was unrelated to known prognostic variables and partly the result of instrumentation and manipulation of the tumour .", "the management of patients with minimal sln involvement is problematic . in a meta - analysis of 25 studies of patients with sln micrometastases , in approximately 20% there was nonsentinel node disease falling to 9% when the sln involvement was detected by ihc .", "furthermore , the consequent effect on dfs and os remains controversial , so the biological relevance and clinical significance is a matter of debate .  ", "amaros investigates the benefit of a calnd in comparison to treatment with axillary radiotherapy ( art ) in patients with sln - positive breast cancer .", "a recently published substudy evaluated the identification rate and the nodal involvement of the first 2,000 patients between 2001 and 2005 who entered from 26 european institutions .", "the sentinel node identification rate was 97% which is high considering the relatively early days of this procedure .  ", "34% were sln positive of whom 63% had macrometastases , 25% had micrometastases , and 12% had itcs .   in the calnd arm non - sln involvement", "was identified in 41% of patients with macrometastases and in 18% of patients with either micrometastases or itcs .", "several studies have investigated the significance of occult metastases , such as micrometastases or small clusters of tumour cells in association with non - sln involvement and the impact of calnd on disease - free survival and overall survival , and the larger ones are summarised in table 2 [ 3743 ] .", "although the majority show no prognostic impact of itcs in the sentinel node , a large dutch investigation with 5-year followup indicated that women with itcs who received adjuvant chemotherapy had a significantly better event - free survival compared with untreated cases with itcs .", "furthermore , a finnish study showed a worse 5-year breast - cancer - specific survival for those with itc compared with node negative cases . in the", "largest published multicenter retrospective study of 187 sln - itcs patients undergoing calnd , houvenaeghel et al . reported an incidence of 16% non - sln involvement .", "the difference in the risk of non - sln involvement between sentinel nodes with itcs ( 16% ) and those with micrometastases ( 14% ) was not statistically significant .", "however it was not apparent whether the presence of non - sln metastases should affect the therapeutic decision in these patients .", "the authors proposed that calnd could be avoided in patients with tubular , colloid , or medullary small primary tumours ( pt1 ) with a risk of non - sln involvement approximately 5% .", "mirror is a large dutch cohort retrospective study which assesses the impact of sln - itcs and micrometastases on 5-year disease - free survival in patients with favourable primary tumour characteristics .   according to recent published data , both patients with snb micrometastases and those with itcs who did not undergo calnd experienced a far higher 5-year axillary recurrence rate , 6% in comparison to 1% of snb - negative patients who did not undergo calnd .   additionally , both patients with sln micrometastases and itcs had approximately 5-year disease - free survival improved by 10% with adjuvant systemic therapy .", "mirror findings support an aggressive treatment approach in patients with either sln micrometastases or itcs .", "many investigators have studied the incidence of non - sln involvement in patients with sln micrometastases to define which patients may need further axillary treatment . wada and imoto . collected 22 studies from 1999 until 2006 referring to the frequency of sln micrometastases in patients with breast cancer and the prevalence of non - sln involvement in those patients after alnd .", "the frequency of sln micrometastases was 38% with non - sln micrometastases ranging from 0 to 57% .", "additionally , a wide range of non - sln macrometastases was found ( 018% ) . because the prevalence of non - sln micrometastases was low , the prognostic impact was unclear .", "the wide range of results arose from the different numbers of patients involved , variations in number of pathological sections examined , and differences in tumour stage and grade .", "results of studies in which patients with micrometastases in snb and who were not treated by completion axillary node clearance are summarised in table 3 [ 38 , 4854 ] .", "most of the studies had small numbers and relatively short followup and tended to conclude that there was no benefit from completion axillary node clearance .  ", "the largest study ; however , found a significantly worse disease - free survival for women with micrometastases who did not undergo calnd . .", "de boer et al . conducted a systematic review of 58 studies conducted from 1977 to 2008 included 297,533 patients , aiming to define the prognostic relevance of micrometastases and itcs in patients with breast cancer . using random - effect meta - analysis they showed that the presence of aln metastases < 2  mm in diameter detected on single - section examination was associated with poorer overall survival .  ", "moreover the presence of occult metastases on retrospective examination of aln - negative patients by step sectioning and/or immunohistochemistry ( n = 7740 patients ) was associated with poorer 5-year disease - free and overall survival .", "outcomes from sentinel lymph node biopsy studies were not assessable due to small patient groups and short followup .", "the international breast cancer study group trial ibcsg-23 - 01 is a randomised multicentre study designed to determine the significance of minimal ln metastasis in patients with breast cancer .", "the trial was initiated in april 2001 , and it compares survival between patients with sln micrometastases who undergo slnb alone with those who receive calnd .", "it is known that the extent of macrometastases in sln is strongly correlated with non - sln involvement .", "the long - term effect of the residual axillary disease in the sentinel - lymph - node - positive patient on local and systemic recurrence has not been clearly defined for patients receiving modern radiotherapy and chemotherapy .", "older studies of patients with symptomatic breast cancers have shown that inadequate axillary surgery does lead to reduced overall survival [ 5760 ] .", "the study set out to randomise 1900  women with breast cancer and 15 involved snln to either calnd or observation .  ", "all had a lumpectomy and tangential breast irradiation , but systemic therapy was at the discretion of the treating centre .  ", "after a median followup of 6.3  years , the relapse - free survival for the alnd group was 82% compared with 84% for the observation group , and the overall survival was 92% in both groups .  ", "unfortunately the trial stopped accrual after 891 cases had been entered which makes it underpowered to detect a 5% difference in outcome .  ", "an additional aspect to be considered is that the guy 's wide excision studies showed no difference between the wide excision group and the radical mastectomy group at 10 years whereas after 25 years there was a significantly worse relapse - free and overall survival in the wide excision group with inadequately treated axillae . in a retrospective study ,", "takei et al . confirmed the importance of calnd in sln - positive patients with high nuclear grade and hormone - negative breast cancer .", "it was noticed that of 459 patients with macrometastatic disease treated with calnd , after a median follow - up period of 34  months , the axillary recurrence rate was only 0.6%      .", "bilimoria et al . studied a cohort of 403,167 patients with clinically node - negative breast cancer that underwent slnb from the us national cancer data base ( 19982005 ) .", "of the 97,314 ( 24% ) patients identified with nodal metastases , 28% had no further surgical intervention in the axilla and 72% underwent calnd .  ", "after a median followup of 63  months , it was found that in all patients and separately in those with macroscopic and microscopic nodal disease , the unadjusted axillary recurrence rate and overall survival were comparable .  ", "after adjustment for clinicopathological differences , there was a trend towards a lower risk of axillary recurrence and death in patients with macroscopic nodal involvement undergoing calnd .   for those with micrometastases , recurrence rates were similar in those undergoing either slnb alone or calnd .", "of 26,986 patients with slnb - positive breast cancer from the seer database ( surveillance , epidemiology , and end results ) , 16% had no further axillary treatment and 84% had calnd .", "after a median followup of 50 months , although a higher rate of ipsilateral regional recurrence was noticed in patients who underwent slnb alone , no statistically significant differences in overall survival ( os ) between patients who underwent slnb alone versus complete alnd were found .", "the investigators suggested that in patients with small , low - grade primary tumours , positive er status , older age and who have received segmental mastectomy , calnd may be omitted .  ", "hwang et al . reviewed the outcome of 3,366 patients with invasive breast cancer who underwent slnb from 1993 to 2005 .", "of 750 sln - positive patients , 65% , 45.9% , and 34.2% were pn1 , pn1mi , and pn0 ( i+ ) , respectively .", "of these patients , 196 had no further axillary surgery due to clinician and patient preference . according to clinicopathological variables ,", "adjuvant treatment was applied and locoregional and distant recurrence and survival were studied . after a median followup of 29.5 months , no patient had an axillary recurrence , one had supraclavicular lymph node recurrence , and three patients developed metastatic disease to the lung or bone . the median time to recurrence was 32 months .", "notably the patients with distant metastases had t3 grade iii invasive carcinoma . despite the low axillary recurrence rate", ", authors suggested that it is not possible from these results to conclude definitively that calnd should be abandoned for these patients .", "several factors correlated with the likelihood of additional non - sln metastasis have been investigated in an effort to distinguish which patients could avoid extensive axillary surgery .", "characteristics of the primary tumour , such as size [ 40 , 66 ] , grade , hormone receptor and her2 profile , tumour type , multifocality , mean proliferative fraction , and lymphovascular invasion [ 68 , 69 ] , have all been studied .", "additional features of the involved slns , such as size of metastases , number of positive slns , ratio of positive to resected slns , and the extracapsular spread have been also examined [ 21 , 61 , 7072 ] .", "particularly patients with minimal sln metastases are at a significantly lower risk to have further non - sln invasion than those with sln macrometastases ( 1324% versus 4579% ) . however", ", none of these characteristics individually can determine a subset of patients for whom alnd is unnecessary .  ", "molecular profiling of metastatic foci different from the primary tumour could be used as indicator for the selection of patients who might benefit of completion axillary dissection .", "the most important prognostic factors for the presence of non - sln metastases in patients with minimal sln involvement are presented in table 4 .", "several mathematical models have been developed to predict the risk of non - sln involvement in patients with sln - positive breast cancer .", "these include four nomograms : the memorial sloan - kettering cancer center ( mskcc ) , ( https://www.mskcc.org/mskcc/html/15938.cfm ) , mayo   ( https://www.mayoclinic.org/breast-cancer/sentinelbiopsy.html ) , cambridge , and stanford   ( https://www3-hrpdcc.stanford.edu/nsln-calculator/ ) .  ", "there are three scoring systems , the tenon , mda , and saidi , and two recursive partitioning ( rp ) tools developed by kohrt et al . .  ", "the institut curie studied 588 consecutive patients with positive slns who underwent alnd to compare the actual rate of non - sln metastases with those predicted by breast cancer nomogram of memorial sloan - kettering cancer center ( mskcc ) .   while the predicted rate in non - sln macrometastases was relatively accurate , when the nomogram was applied to the 213 slns that contained only micrometastases , the predicted rate 59% was far away from the actual rate 44% of non - sln micrometastases detected by ihc .  ", "consequently , the authors concluded that a different predictive model should   be created for patients with micrometastases .", "molecular tests based on technology such as oncotype dx ( genomic health , redwood city , calif , usa ) or other multigene arrays developed prognostic and predictive markers aiming to personalize surgical and adjuvant treatment of early breast cancer .", "an accurate , intraoperative sentinel lymph node test probably could help in avoidance of delayed axillary dissections .", "molecular tests may be proved more sensitive than current intraoperative tests but have not yet been validated .", "osna is an automated assay for the detection of cytokeratin message , ck19 mrna , present in approximately 98% of breast cancers .", "it provides an opportunity to make an intraoperative diagnosis of sentinel node involvement within 30  minutes , avoiding frozen section and allowing a one - stage procedure .  ", "larger verification studies in which half of the bisected sentinel node was sent for histology and the other half homogenised for osna are shown in table 5 [ 71 , 72 , 7981 ] .", "the usual reason for discordance between histopathology and osna was an uneven distribution of nodal metastases ( tissue allocation bias ) .  ", "the studies indicate a good concordance between results of histopathology and osna .   indeed in a study conducted by osaka , comparing osna with frozen section ,", "it is likely that in time osna will replace histopathological examination of sentinel lymph nodes because of its ease , accuracy , and potential for enabling almost all patients to have a one - stage operation for early breast cancer .", "recent studies aiming to determine if calnd is beneficial in patients with sln - positive breast cancer and even more in patients with minimal sln involvement have reached contrary conclusions .", "limitations in the published studies include the methods of pathological evaluation of lymph nodes and that the number of additional non - sln - positive nodes is usually not known", "thus lymph node step sectioning and ihc lead to increased identification of minimal metastases upstaging 9% to 25% of patients who initially were considered node negative [ 83 , 84 ] and did not have further axillary surgery .   ", "the different rates of recurrence may be due to the molecular type of cancers and so that patients with sln metastases may also have different risks of metastatic involvement .", "finally it is possible that not all minor tumour foci in axillary lymph nodes progress to local recurrences . according to al - hajj et al .", ", only a minority of cancer cells potentially give metastases and most itcs are not viable and do not have the ability to form new tumours .", "there may be two different breast cancer cell populations , true stem cells that have the capacity to develop metastases and the nonstem cells that never grow and are finally destroyed .   at the level of everyday clinical practice , with both promising and disappointing results of the published studies", ", most breast surgeons will hardly ever take the risk of avoiding completion axillary dissection in breast cancer patients even with minimal sentinel lymph node metastases .  ", "many are seeking to find a balance between the needs of the majority to have minimal axillary surgery with minimal postoperative morbidity against the possibility that a minority will suffer relapse , morbidity , and possible increased mortality from undertreatment .  ", "results from ongoing phase iii trials will perhaps provide new guidelines for the treatment of patients with micrometastases or itcs .  ", "a predictive model which estimates accurately the likelihood of additional disease in the axilla might help tailor surgical therapy to the needs of the individual patient and identify those most likely to benefit from completion or alnd .   genetic assays defining prognostic markers and new intraoperative tests detecting accurately sln involvement will help in early therapeutic decision making in the future .  ", "it is important that premature decisions to restrict axillary surgery are not made on a basis of early results from underpowered clinical trials ."], "abstract_text": ["<S> sentinel lymph node biopsy ( slnb ) is a safe and accurate minimally invasive method for detecting axillary lymph node ( aln ) involvement in the clinically negative axilla thereby reducing morbidity in patients who avoid unnecessary axillary lymph node dissection ( alnd ) . </S>", "<S> although current guidelines recommend completion alnd when macro- and micrometastatic diseases are identified by slnb , the benefit of this surgical intervention is under debate . </S>", "<S> additionally , the management of the axilla in the presence of isolated tumour cells ( itcs ) in slnb is questioned . </S>", "<S> particularly controversial is the prognostic significance of minimal slnb metastasis in relation to local recurrence and overall survival . </S>", "<S> preliminary results of the recently published z0011 trial suggest similar outcomes after snb or alnd when the sn is positive , but this finding has to be interpreted with caution . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Current Guidelines ", "3. Micrometastases and Isolated Tumour Cells (ITCs)", "4. Completion ALND and Micrometastases", "5. Completion Axillary Lymph Node Dissection in SLN Macrometastases", "6. Predictive Models", "7. One-Step Nucleic Acid Amplification (OSNA)", "8. Conclusions "], "sections": [["for patients with operable breast cancer , the major prognostic determinant is whether there has or has not been spread to the axilla and the number of involved axillary nodes .", "initially it was suggested that breast cancer first spreads locoregionally via lymphatics to the axillary lymph nodes and then metastasises more distantly . in accordance with this concept ,", "subsequently fisher postulated that the extent of micrometastases at diagnosis of breast cancer is an indicator of outcome , with biological behaviour of cancer predetermining the likelihood of progression of the disease . nowadays gene expression profiling arrays can delineate tumour types with different prognoses .", "the surgical approach for breast cancer treatment evolved from the extensive radical mastectomy and the patey modified radical mastectomy   to breast conserving and minimally invasive techniques .", "traditionally the surgical management of breast cancer comprised wide local resection of the primary tumour and axillary lymph node dissection ( alnd ) .", "axillary status is the most important prognostic factor in breast cancer providing staging information and therefore largely defining treatment strategy .", "diagnostic imaging modalities such as ultrasound , magnetic resonance mammography , positron emission tomography , and 99  m technetium ( tc ) sestamibi scintimammography are not reliable for staging the axilla , particularly with lymph node metastases < 0.5  cm [ 8 , 9 ] .", "clinically palpable lymph nodes prove to be false positive in 2530% of patients   and about 40% have positive results after ultrasound with or without fine - needle aspiration node negativity .", "studying 24,740 women with invasive breast cancer , carter et al . showed that approximately 80% with tumour size < 1  cm , 50% with up to 5  cm , and 30% with > 5% had negative axilla , a fact suggesting that metastases do not occur exclusively via the axillary lymph nodes , but rather lymph node status serves as an indicator of the tumour 's ability to spread . additionally , it has been recently shown that the molecular profile of the primary tumour is a more significant prognostic indicator in terms of disease - free survival ( dfs ) and overall survival ( os ) than lymph node metastases .", "the combination of the introduction of population - based mammographic screening for breast cancer , modern imaging methods , and increased public awareness resulted in patients being diagnosed more often with smaller - size tumours and less likelihood of axillary lymph node metastases .", "it is evident now that 6070% of patients with early breast cancer are node negative at the time of diagnosis   and alnd puts them at significant risk of short- and long - term morbidities without benefit .", "alnd is associated with acute complication rates of 2030% including seroma formation , local swelling , numbness , impaired shoulder movement , neuropathy , infection , and chronic lymphoedema rates of 737% . in a prospective study by petrek et al . evaluating a cohort of 923 women with 20 years", "follow up , it was shown that breast - cancer - related lymphoedema following alnd occurred maximally in the first 3 years following surgery ; however , up to 23% of patients may still develop arm swelling during the rest of their lives .", "several randomised studies have established that sentinel node biopsy ( snb ) is a safe and accurate procedure for detecting tumour cells in sln and predicting the status of the other axillary nodes ( non - sln ) .", "although accuracy and appropriateness of snb were disputed by the finding of 510% false - negative cases when snb was followed by axillary dissection at high - risk patients for axillary nodal disease [ 13 , 18 ] , false - negative snb results seem to have decreased with the increasing experience of surgeons , and it is expected that the utilisation of snb in the future will be increased . a meta - analysis of seven prospective randomised controlled trials by kell et al . demonstrated that snb is equivalent to alnd for the detection of lymph node metastasis with the additional advantage of reduction of up to 75% in morbidity in patients with early stage breast cancer .  ", "furthermore , a trend towards an improved detection of ln metastases was shown when snb is used .", "patients undergoing snb have a 22% higher odds ratio of having a positive sln , due to the more intensive pathological examination which utilises multiple sections and immunohistochemistry ( ihc ) .", "in contrast , the false - negative cases seen after axillary dissection are probably due to the inability of the pathologist to perform serial sections and ihc on the 2030 lymph nodes found in a complete axillary clearance specimen .", "studies have shown that sln is the only positive lymph node in 3867% of patients when alnc followed .", "interestingly , it has been reported that only 48% of patients with negative alns have internal mammary lymph node involvement ( imn ) whereas 2550% of patients with affected alns have also imn metastases [ 23 , 24 ] .", "dissection of imn is not recommended because of the high morbidity and the uncertain benefit on survival .", "the recently published outcomes of nsabp b-32 trial established the efficacy of sln biopsy alone with no further alnd in 5611 breast cancer patients with clinically negative lymph nodes .", "women with invasive breast cancer who were randomly assigned to either sln resection plus alnd ( group 1 ) or to sln resection alone with alnd only if the slns were positive ( group 2 ) , after 8 years of followup , showed statistically equivalent overall survival , disease - free survival , and regional control .  ", "patient followup is still continuing for longer - term assessment of survival and regional control .", "moreover , a closer look into mature studies focused on axillary relapses and overall survival is in agreement with current findings favouring slb .", "the national surgical adjuvant breast and bowel project ( nsabp ) b04 randomised study compared breast cancer patients with clinically negative alns managed either by radical mastectomy , total mastectomy with axillary radiation , or total mastectomy alone .", "the results clearly defined that alnd decreases the risk of locoregional relaps ; however , no significant differences in survival were found among the treatment groups .", "this study has been criticised because of the variability of numbers of lymph nodes resected in the total mastectomy alone arm and the lack of statistical power to detect a small difference in outcome . indeed , a meta - analysis has suggested that inadequate axillary treatment may lead to not only an increased risk of local relapse but also a 5% reduction in survival . as a result of increasing detection of early breast cancer and the high rate of micrometastases and itcs ( itcs ) found in the detailed pathological examination of sln", ", a new debate has opened about the consequent necessity of alnd in these patients .", "this has arisen because of better understanding of breast cancer behaviour and improved efficacy of combined therapeutic modalities . in this paper", "we report the current guidelines concerning the management of the axilla after slnb and review the different aspects arising from recent studies on the role of micrometastases and itc clusters in sln on decision making ."], ["the american society of clinical oncology ( asco ) expert panel conducted a systematic review of the literature available through february 2004 on the use of snb in early - stage breast cancer in order to develop guidelines for the management of the axilla ( http://jop.ascopubs.org/content/1/4/134 ) , and these are similar to those of the national institute of health and clinical excellence ( nice ) recommendations in uk ( http://www.nice.org.uk/nicemedia/live/12132/43413/43413.pdf ) .  ", "alnd is the standard of care in those with a macrometastatic or micrometastatic positive sln to maximise local control .", "if the sln is negative , a calnd ( calnd ) is not necessary .  ", "itcs detected by ihc are of unknown clinical significance , and when identified , the sln is regarded as negative and no further alnd is required . although ihc is often used , it is not included in routine sln evaluation for breast cancer at this time .", "asco and nice recommendations for slnb , alnd alone and managing of the axilla after slnb are summarised in table 1 .  ", "in contrast the german guidelines do not recommend axillary clearance for 1 - 2 sln positive in patients with t1 and t2 tumours ( http://www.ago-online.de ) .  ", "it has been suggested that slnb should be carried out by an experienced team in order to minimise false negativity and improve the predictive value of the procedure .  "], ["the american joint committee on cancer ( ajcc ) in the sixth edition of the cancer staging manual defined a lymph node metastatic tumour with maximum diameter      > 2  mm as macrometastasis ( pn1 ) , when the diameter of deposit is 0.22  mm as micrometastasis ( pnmi ) , and a lesion of single tumour cells or small cell clusters with diameter < 0.2  mm as itcs [ pn0(i+ ) ] .", "itcs are not distinguishable by h&e staining but detected only with immunohistochemistry ( ihc ) or molecular methods .  ", "moore et al . suggested that the presence of itcs was unrelated to known prognostic variables and partly the result of instrumentation and manipulation of the tumour .", "the management of patients with minimal sln involvement is problematic . in a meta - analysis of 25 studies of patients with sln micrometastases , in approximately 20% there was nonsentinel node disease falling to 9% when the sln involvement was detected by ihc .", "furthermore , the consequent effect on dfs and os remains controversial , so the biological relevance and clinical significance is a matter of debate .  ", "amaros investigates the benefit of a calnd in comparison to treatment with axillary radiotherapy ( art ) in patients with sln - positive breast cancer .", "a recently published substudy evaluated the identification rate and the nodal involvement of the first 2,000 patients between 2001 and 2005 who entered from 26 european institutions .", "the sentinel node identification rate was 97% which is high considering the relatively early days of this procedure .  ", "34% were sln positive of whom 63% had macrometastases , 25% had micrometastases , and 12% had itcs .   in the calnd arm non - sln involvement", "was identified in 41% of patients with macrometastases and in 18% of patients with either micrometastases or itcs .", "several studies have investigated the significance of occult metastases , such as micrometastases or small clusters of tumour cells in association with non - sln involvement and the impact of calnd on disease - free survival and overall survival , and the larger ones are summarised in table 2 [ 3743 ] .", "although the majority show no prognostic impact of itcs in the sentinel node , a large dutch investigation with 5-year followup indicated that women with itcs who received adjuvant chemotherapy had a significantly better event - free survival compared with untreated cases with itcs .", "furthermore , a finnish study showed a worse 5-year breast - cancer - specific survival for those with itc compared with node negative cases . in the", "largest published multicenter retrospective study of 187 sln - itcs patients undergoing calnd , houvenaeghel et al . reported an incidence of 16% non - sln involvement .", "the difference in the risk of non - sln involvement between sentinel nodes with itcs ( 16% ) and those with micrometastases ( 14% ) was not statistically significant .", "however it was not apparent whether the presence of non - sln metastases should affect the therapeutic decision in these patients .", "the authors proposed that calnd could be avoided in patients with tubular , colloid , or medullary small primary tumours ( pt1 ) with a risk of non - sln involvement approximately 5% .", "mirror is a large dutch cohort retrospective study which assesses the impact of sln - itcs and micrometastases on 5-year disease - free survival in patients with favourable primary tumour characteristics .   according to recent published data , both patients with snb micrometastases and those with itcs who did not undergo calnd experienced a far higher 5-year axillary recurrence rate , 6% in comparison to 1% of snb - negative patients who did not undergo calnd .   additionally , both patients with sln micrometastases and itcs had approximately 5-year disease - free survival improved by 10% with adjuvant systemic therapy .", "mirror findings support an aggressive treatment approach in patients with either sln micrometastases or itcs ."], ["many investigators have studied the incidence of non - sln involvement in patients with sln micrometastases to define which patients may need further axillary treatment . wada and imoto . collected 22 studies from 1999 until 2006 referring to the frequency of sln micrometastases in patients with breast cancer and the prevalence of non - sln involvement in those patients after alnd .", "the frequency of sln micrometastases was 38% with non - sln micrometastases ranging from 0 to 57% .", "additionally , a wide range of non - sln macrometastases was found ( 018% ) . because the prevalence of non - sln micrometastases was low , the prognostic impact was unclear .", "the wide range of results arose from the different numbers of patients involved , variations in number of pathological sections examined , and differences in tumour stage and grade .", "results of studies in which patients with micrometastases in snb and who were not treated by completion axillary node clearance are summarised in table 3 [ 38 , 4854 ] .", "most of the studies had small numbers and relatively short followup and tended to conclude that there was no benefit from completion axillary node clearance .  ", "the largest study ; however , found a significantly worse disease - free survival for women with micrometastases who did not undergo calnd . .", "de boer et al . conducted a systematic review of 58 studies conducted from 1977 to 2008 included 297,533 patients , aiming to define the prognostic relevance of micrometastases and itcs in patients with breast cancer . using random - effect meta - analysis they showed that the presence of aln metastases < 2  mm in diameter detected on single - section examination was associated with poorer overall survival .  ", "moreover the presence of occult metastases on retrospective examination of aln - negative patients by step sectioning and/or immunohistochemistry ( n = 7740 patients ) was associated with poorer 5-year disease - free and overall survival .", "outcomes from sentinel lymph node biopsy studies were not assessable due to small patient groups and short followup .", "the international breast cancer study group trial ibcsg-23 - 01 is a randomised multicentre study designed to determine the significance of minimal ln metastasis in patients with breast cancer .", "the trial was initiated in april 2001 , and it compares survival between patients with sln micrometastases who undergo slnb alone with those who receive calnd ."], ["it is known that the extent of macrometastases in sln is strongly correlated with non - sln involvement .", "the long - term effect of the residual axillary disease in the sentinel - lymph - node - positive patient on local and systemic recurrence has not been clearly defined for patients receiving modern radiotherapy and chemotherapy .", "older studies of patients with symptomatic breast cancers have shown that inadequate axillary surgery does lead to reduced overall survival [ 5760 ] .", "the study set out to randomise 1900  women with breast cancer and 15 involved snln to either calnd or observation .  ", "all had a lumpectomy and tangential breast irradiation , but systemic therapy was at the discretion of the treating centre .  ", "after a median followup of 6.3  years , the relapse - free survival for the alnd group was 82% compared with 84% for the observation group , and the overall survival was 92% in both groups .  ", "unfortunately the trial stopped accrual after 891 cases had been entered which makes it underpowered to detect a 5% difference in outcome .  ", "an additional aspect to be considered is that the guy 's wide excision studies showed no difference between the wide excision group and the radical mastectomy group at 10 years whereas after 25 years there was a significantly worse relapse - free and overall survival in the wide excision group with inadequately treated axillae . in a retrospective study ,", "takei et al . confirmed the importance of calnd in sln - positive patients with high nuclear grade and hormone - negative breast cancer .", "it was noticed that of 459 patients with macrometastatic disease treated with calnd , after a median follow - up period of 34  months , the axillary recurrence rate was only 0.6%      .", "bilimoria et al . studied a cohort of 403,167 patients with clinically node - negative breast cancer that underwent slnb from the us national cancer data base ( 19982005 ) .", "of the 97,314 ( 24% ) patients identified with nodal metastases , 28% had no further surgical intervention in the axilla and 72% underwent calnd .  ", "after a median followup of 63  months , it was found that in all patients and separately in those with macroscopic and microscopic nodal disease , the unadjusted axillary recurrence rate and overall survival were comparable .  ", "after adjustment for clinicopathological differences , there was a trend towards a lower risk of axillary recurrence and death in patients with macroscopic nodal involvement undergoing calnd .   for those with micrometastases , recurrence rates were similar in those undergoing either slnb alone or calnd .", "of 26,986 patients with slnb - positive breast cancer from the seer database ( surveillance , epidemiology , and end results ) , 16% had no further axillary treatment and 84% had calnd .", "after a median followup of 50 months , although a higher rate of ipsilateral regional recurrence was noticed in patients who underwent slnb alone , no statistically significant differences in overall survival ( os ) between patients who underwent slnb alone versus complete alnd were found .", "the investigators suggested that in patients with small , low - grade primary tumours , positive er status , older age and who have received segmental mastectomy , calnd may be omitted .  ", "hwang et al . reviewed the outcome of 3,366 patients with invasive breast cancer who underwent slnb from 1993 to 2005 .", "of 750 sln - positive patients , 65% , 45.9% , and 34.2% were pn1 , pn1mi , and pn0 ( i+ ) , respectively .", "of these patients , 196 had no further axillary surgery due to clinician and patient preference . according to clinicopathological variables ,", "adjuvant treatment was applied and locoregional and distant recurrence and survival were studied . after a median followup of 29.5 months , no patient had an axillary recurrence , one had supraclavicular lymph node recurrence , and three patients developed metastatic disease to the lung or bone . the median time to recurrence was 32 months .", "notably the patients with distant metastases had t3 grade iii invasive carcinoma . despite the low axillary recurrence rate", ", authors suggested that it is not possible from these results to conclude definitively that calnd should be abandoned for these patients ."], ["several factors correlated with the likelihood of additional non - sln metastasis have been investigated in an effort to distinguish which patients could avoid extensive axillary surgery .", "characteristics of the primary tumour , such as size [ 40 , 66 ] , grade , hormone receptor and her2 profile , tumour type , multifocality , mean proliferative fraction , and lymphovascular invasion [ 68 , 69 ] , have all been studied .", "additional features of the involved slns , such as size of metastases , number of positive slns , ratio of positive to resected slns , and the extracapsular spread have been also examined [ 21 , 61 , 7072 ] .", "particularly patients with minimal sln metastases are at a significantly lower risk to have further non - sln invasion than those with sln macrometastases ( 1324% versus 4579% ) . however", ", none of these characteristics individually can determine a subset of patients for whom alnd is unnecessary .  ", "molecular profiling of metastatic foci different from the primary tumour could be used as indicator for the selection of patients who might benefit of completion axillary dissection .", "the most important prognostic factors for the presence of non - sln metastases in patients with minimal sln involvement are presented in table 4 .", "several mathematical models have been developed to predict the risk of non - sln involvement in patients with sln - positive breast cancer .", "these include four nomograms : the memorial sloan - kettering cancer center ( mskcc ) , ( https://www.mskcc.org/mskcc/html/15938.cfm ) , mayo   ( https://www.mayoclinic.org/breast-cancer/sentinelbiopsy.html ) , cambridge , and stanford   ( https://www3-hrpdcc.stanford.edu/nsln-calculator/ ) .  ", "there are three scoring systems , the tenon , mda , and saidi , and two recursive partitioning ( rp ) tools developed by kohrt et al . .  ", "the institut curie studied 588 consecutive patients with positive slns who underwent alnd to compare the actual rate of non - sln metastases with those predicted by breast cancer nomogram of memorial sloan - kettering cancer center ( mskcc ) .   while the predicted rate in non - sln macrometastases was relatively accurate , when the nomogram was applied to the 213 slns that contained only micrometastases , the predicted rate 59% was far away from the actual rate 44% of non - sln micrometastases detected by ihc .  ", "consequently , the authors concluded that a different predictive model should   be created for patients with micrometastases .", "molecular tests based on technology such as oncotype dx ( genomic health , redwood city , calif , usa ) or other multigene arrays developed prognostic and predictive markers aiming to personalize surgical and adjuvant treatment of early breast cancer .", "an accurate , intraoperative sentinel lymph node test probably could help in avoidance of delayed axillary dissections .", "molecular tests may be proved more sensitive than current intraoperative tests but have not yet been validated ."], ["osna is an automated assay for the detection of cytokeratin message , ck19 mrna , present in approximately 98% of breast cancers .", "it provides an opportunity to make an intraoperative diagnosis of sentinel node involvement within 30  minutes , avoiding frozen section and allowing a one - stage procedure .  ", "larger verification studies in which half of the bisected sentinel node was sent for histology and the other half homogenised for osna are shown in table 5 [ 71 , 72 , 7981 ] .", "the usual reason for discordance between histopathology and osna was an uneven distribution of nodal metastases ( tissue allocation bias ) .  ", "the studies indicate a good concordance between results of histopathology and osna .   indeed in a study conducted by osaka , comparing osna with frozen section ,", "it is likely that in time osna will replace histopathological examination of sentinel lymph nodes because of its ease , accuracy , and potential for enabling almost all patients to have a one - stage operation for early breast cancer ."], ["recent studies aiming to determine if calnd is beneficial in patients with sln - positive breast cancer and even more in patients with minimal sln involvement have reached contrary conclusions .", "limitations in the published studies include the methods of pathological evaluation of lymph nodes and that the number of additional non - sln - positive nodes is usually not known", "thus lymph node step sectioning and ihc lead to increased identification of minimal metastases upstaging 9% to 25% of patients who initially were considered node negative [ 83 , 84 ] and did not have further axillary surgery .   ", "the different rates of recurrence may be due to the molecular type of cancers and so that patients with sln metastases may also have different risks of metastatic involvement .", "finally it is possible that not all minor tumour foci in axillary lymph nodes progress to local recurrences . according to al - hajj et al .", ", only a minority of cancer cells potentially give metastases and most itcs are not viable and do not have the ability to form new tumours .", "there may be two different breast cancer cell populations , true stem cells that have the capacity to develop metastases and the nonstem cells that never grow and are finally destroyed .   at the level of everyday clinical practice , with both promising and disappointing results of the published studies", ", most breast surgeons will hardly ever take the risk of avoiding completion axillary dissection in breast cancer patients even with minimal sentinel lymph node metastases .  ", "many are seeking to find a balance between the needs of the majority to have minimal axillary surgery with minimal postoperative morbidity against the possibility that a minority will suffer relapse , morbidity , and possible increased mortality from undertreatment .  ", "results from ongoing phase iii trials will perhaps provide new guidelines for the treatment of patients with micrometastases or itcs .  ", "a predictive model which estimates accurately the likelihood of additional disease in the axilla might help tailor surgical therapy to the needs of the individual patient and identify those most likely to benefit from completion or alnd .   genetic assays defining prognostic markers and new intraoperative tests detecting accurately sln involvement will help in early therapeutic decision making in the future .  ", "it is important that premature decisions to restrict axillary surgery are not made on a basis of early results from underpowered clinical trials ."]]}
{"article_id": "PMC4355841", "article_text": ["labor wellbeing refers to all efforts of employers , trade unions , voluntary organizations , and governmental agencies , which help employees feel better and perform ( work ) better .", "it is a comprehensive term that refers to the physical , mental , moral , and emotional wellbeing of an individual including intra and extra mural facilities .", "although wellbeing includes a wide range of programs and services in organizations , the focus of this study is on the employee assistance programs and employee group services , which are provided by employers according to the real needs of the employees .", "hence , it is necessary for organizations to identify the real needs , as a base to provide a wellbeing package for their employees through wellbeing need assessment techniques .", "wellbeing need assessment is a systematic process of gathering information to determine and address the  needs or  gaps between the current conditions and the desired conditions .", "a need can be a desire to improve the current performance or to correct a deficiency .", "the wellbeing needs assessment includes two main targets ; exploring the effectiveness of the delivered services and exploring those wellbeing needs that are not fulfilled by an organization .", "davis and gibson emphasize on the importance of a comprehensive wellbeing needs assessment , both for obtaining the required information needed to design the appropriate interventions and also for providing the basic required information to evaluate the effectiveness of the wellbeing program .", "wellbeing assessments are vital for the determination of the economic and social needs of employees and for the best decisions to be taken in response to such needs . reviewing", "the human resources management literature showed an absence of attention given to the employee 's benefits .", "moreover , various expectations of employees in the field of wellbeing services caused organizations , especially the human resources ( hr ) offices , to face many challenges ( chen et al . ) .", "therefore , for keeping employees satisfied with the wellbeing services , all challenges had to be truly and effectively met ( hyde et al . ) .", "research indicated that satisfaction of the employees was not positively correlated with the benefits of the welfare package ( e.g. dreher , ash , and bretz ) .", "dencker , joshi , and martocchio argued that individual differences in benefit preferences emerged from particular employment relationships , with individuals having greater experience .", "the tehran university of medical sciences ( tums ) , including 69 research centers , 25 hospitals , 11 educational and health care centers , three health networks , and 10 faculties with more than 2020 faculty members and 19,000 students ( tums 2012 ) , requires an improved comprehensive system for delivering the wellbeing services ( financial , insurance , healthcare services , educational and training services , etc . ) .", "taking a look at the functions of the university 's wellbeing services system ; it uncovers a gap between the employees real needs and what is delivered to meet their needs .", "it is because of the lack of a participatory strategic wellbeing delivery plan that is based on prioritized wellbeing needs ( requirements ) .", "as the first step to make a strategic plan is identifying the needs , the main purpose of this study is to assess the wellbeing services of the staff working in tums  first to see if they are satisfactory enough or they need to be changed , and second , to help decision - makers develop services that are essential to improve the well - being of employees , as also the quality and effectiveness of the tums 's wellbeing delivery system .", "what are the services used and to what extent they have been satisfactory? this question aims to explore the reasons why they are not delivered .", "the second question is ,  what type of services are essential to be provided by the university? with respect to the main purpose of this research , the wellbeing delivery system of tums is assessed with a thorough answering of the questions mentioned above .", "the research design was a cross - sectional survey ( 2008 - 2009 ) using a structured non - disguised questionnaire .", "the sample size was 98 with a 95% level of confidence and absolute error of 0/01 .", "cluster sampling based on the  occupational scope as the main variable , was used as the sampling method , with regard to the existence of different occupational scopes .", "data was collected from 98 officers working at different departments of central organizations of tums ( tehran university of medical sciences ) including logistics , education , research and technology , student and cultural affairs departments .", "the research participants were assured of the confidentiality of their personal information at the beginning of the questionnaire .", "the questionnaire included 45 questions ( 43 close - ended questions and 2 open - ended questions1 ) organized into two separate parts ( cranach alpha = 0.85 ) : according to table 1 , within the first part , the respondents were supposed to mark the wellbeing services if they had ever used any of them . those who used the services were supposed to show their level of satisfaction by choosing from ", "almost satisfied ,  almost dissatisfied, and  full dissatisfied , and those who had never used the services , were supposed to express their reasons by choosing from ", "poor announcement ,  unwillingness to receive the service ,  undesirability of the service, and  undesirability of the receiving process of the service. main parts and the main purpose of asking questions for each part in the second part , the respondents were provided with a list of not delivered wellbeing services to choose which of them is ", "essential ,  neither essential nor non - essential or  non - essential. for making sure if the questionnaire was valid content - wise , first a comprehensive collection of wellbeing services delivered both in tums and in other organizations was identified according to the comprehensive definition of wellbeing services .", "next , according to the main purpose of the research , they were put into classified groups to form the research tool .", "furthermore , to make sure about the appropriateness and transparency of each question and also the whole tool , in addition to its comprehensiveness , the questionnaire was sent to five professionals and experts in the relevant field .", "the reliability of the questionnaire was determined after thoroughly calculating the cronbach alpha , which was reported to be around 0.85 .", "subsequently , after improving and clarifying the instrument , the participants of sample group were asked to fill in the questionnaire .", "the data was gathered by means of filled questionnaires , and were analyzed with the excel software and spss , by calculating the frequency rate of the staff that used the welfare services and those who did not use the services .", "in addition , the t - test was used to calculate the satisfaction score for each service used by the staff .", "among all the 98 respondents , almost 46 employees with a mean satisfactory score of 48.46 used the existing services , for which the satisfactory rate was distributed as shown in table 2 .", "satisfactory scores of categorized wellbeing services the result shows that financial services , including loans for buying a car , emergency loan , loan for buying commodities , awards for staffs children , and two other kinds of non - profit loans in comparison with other categories is considered as the most satisfactory service ( average t = 61.83 ) . among respondents who did not use the categorized services ( 52 persons on an average ) , as shown in the table 3 , the most frequents reasons for not receiving services was ,  staff 's unwillingness to receive the service ( 38.75 in average ) and  poor announcement ( unawareness on the wellbeing services ) ( 37.6 in average )", ". frequency rate of reasons for not receiving welfare services table 4 presents the  frequency of wellbeing services and  necessity ( delivered by other public / private organizations ) , which were asked to be judged by respondents ( to be added or replaced by the delivered wellbeing services list ) , as follows : frequency distribution of wellbeing services  necessity", "educational subvention for children of the staff , who were under their patronage is the only non - financial service , with the highest frequency ( 92.85 ) , identified as the most necessary wellbeing service among all . among other services that were mentioned by respondents ( 61 persons of the sample group ) , the provision of discount cards for using other organizations services ( frequency = 12 ) , providing employees with free health care services ( frequency = 7 ) , and contracting with adult higher education institutions ( frequency = 6 ) had the most frequency among other items .", "as mentioned , the objectives of the study were to measure the staff 's satisfaction with the wellbeing services received , identify the reasons for not receiving the existing wellbeing services , identify the wellbeing services that were not delivered , but were essential as per the staff , and then ask the staff about other wellbeing services that were possible to be delivered and sort them according to their priority . with regard to the first purpose , the results showed that almost half of the respondents used the wellbeing services provided by the wellbeing office of the university , among which almost half of them were satisfied with the services .", "the results suggested that not only were most of the staff not interested in using the provided services , but also more than half of those respondents who used them were not really happy . with regard to the second purpose", ", the results showed that among those who did not use the services , most of them decided not to use it by themselves .", "this was recommended to be surveyed in a separate study in the future . as was evident", ", it can be concluded that the functional desirability of the wellbeing service delivery system of tums was not evaluated as satisfactory enough , because of the almost high dissatisfaction level of services that were used . with regard to the third purpose", ", the results indicated a big gap between the required wellbeing services ( by employees ) and what was provided by the office , as almost all the respondents agreed with the necessity of services such as health , education , and safety .", "the existing gap can result from lack of managerial attention to differences in the demographical characteristics and expectations of employees .", "consistent with the arguments of boudreau and ramstad , dencker et al . argued that market segmentation of employees ( offering market segments have different benefits ) may strengthen the benefits  return - on - investment. one of the most significant reasons for the dissatisfaction and unwillingness of employees to use the services refers to the different priorities and type of services they prefer to include , according to their individual differences . ", "need assessment as a useful technique can help planners to pay more attention to employee 's priority differences in wellbeing services and what they really need to include , by designing the appropriate interventions , as davis and gibson emphasized .", "considering the employees as the most important organizational stakeholders makes them to participate in the wellbeing decision - making process , such as , asking them to add what services they prefer to be included within the wellbeing package .", "although the wellbeing service delivery system of tums has not been effective enough to meet the real needs of the staff by delivering the appropriate services , the most prioritized service type preference of most respondents can be identified . as in the first place the satisfactory score of educational and training services among those who used them was rather high , and second", ", the most necessary identified services were related to educational services , and of course , among the identified wellbeing service priorities , provision of educational opportunity was one of the preferences with high frequency , therefore , it was clear that educational and training opportunities were important for a majority of the respondents .", "the findings show that the functional desirability of tums wellbeing services is not satisfactory enough by the staff 's view point .", "the following conclusions can be drawn from the present study ; the first major finding is the existence of a big gap between the required wellbeing services ( by employees ) and what is provided by the wellbeing office of tums .", "the second and the most obvious finding of this study is the importance of the educational and training opportunities from the point of view of the staff , which has not been paid attention to by the wellbeing service provider in tums .", "although we tried to assess the overall quality and functional desirability of the wellbeing service delivery system of tums , some further studies are required in the researcher 's view .", "it is recommended that the factors that cause dissatisfaction and unwillingness to use services in employees be identified , and also the real needs and wellbeing requirements ( based on employees preferences , job categories , genders , age , and other considerable individual and social factors ) as a strategic basis for redesigning the current wellbeing service delivery system that appears not to be desirable enough in the employees view , be determined ."], "abstract_text": ["<S> background : reviewing the human resources management literature shows an absence of attention given to the employee 's benefits . taking a look at functions of the tehran university of medical sciences wellbeing services system </S>", "<S> , it uncovers a gap between employees real needs and what is delivered to meet their needs . </S>", "<S> so it requires an improved comprehensive system for delivering wellbeing services ( financial , insurance , health care services , educational and training services , etc ) . </S>", "<S> wellbeing need assessment can helps planners to identify vital needs of employee and response to them effectively . </S>", "<S> moreover it can be used to evaluate the effectiveness of the current services which are delivered . </S>", "<S> thus , the aim of this study is to assess wellbeing services of staffs working in tums to ( 1 ) evaluate the satisfactory rate of services which are delivered , and ( 2 ) exploring those wellbeing needs which were not fulfilled by the organization.material and methods : being a cross - sectional and analytic - descriptive survey including 98 responding participants , it is conducted by a questionnaire collecting employees demographic information , their satisfactory rate of the implemented services , and determines unfulfilled wellbeing needs which were not already covered.result:results indicated that services related to financial , educational , non - financial , insurance , occupational health and tourism / recreational services were the most satisfactory services successively .  </S>", "<S> staff 's unwillingness to receive services and  poor announcement ( unawareness on the wellbeing services), were found to be the most frequent reasons for not receiving the existing wellbeing services.conclusion:to increase the satisfaction rate and responsiveness to the real needs of the staff , the current delivery system of wellbeing services in the tums should be redesigned by defining new wellbeing packages . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["labor wellbeing refers to all efforts of employers , trade unions , voluntary organizations , and governmental agencies , which help employees feel better and perform ( work ) better .", "it is a comprehensive term that refers to the physical , mental , moral , and emotional wellbeing of an individual including intra and extra mural facilities .", "although wellbeing includes a wide range of programs and services in organizations , the focus of this study is on the employee assistance programs and employee group services , which are provided by employers according to the real needs of the employees .", "hence , it is necessary for organizations to identify the real needs , as a base to provide a wellbeing package for their employees through wellbeing need assessment techniques .", "wellbeing need assessment is a systematic process of gathering information to determine and address the  needs or  gaps between the current conditions and the desired conditions .", "a need can be a desire to improve the current performance or to correct a deficiency .", "the wellbeing needs assessment includes two main targets ; exploring the effectiveness of the delivered services and exploring those wellbeing needs that are not fulfilled by an organization .", "davis and gibson emphasize on the importance of a comprehensive wellbeing needs assessment , both for obtaining the required information needed to design the appropriate interventions and also for providing the basic required information to evaluate the effectiveness of the wellbeing program .", "wellbeing assessments are vital for the determination of the economic and social needs of employees and for the best decisions to be taken in response to such needs . reviewing", "the human resources management literature showed an absence of attention given to the employee 's benefits .", "moreover , various expectations of employees in the field of wellbeing services caused organizations , especially the human resources ( hr ) offices , to face many challenges ( chen et al . ) .", "therefore , for keeping employees satisfied with the wellbeing services , all challenges had to be truly and effectively met ( hyde et al . ) .", "research indicated that satisfaction of the employees was not positively correlated with the benefits of the welfare package ( e.g. dreher , ash , and bretz ) .", "dencker , joshi , and martocchio argued that individual differences in benefit preferences emerged from particular employment relationships , with individuals having greater experience .", "the tehran university of medical sciences ( tums ) , including 69 research centers , 25 hospitals , 11 educational and health care centers , three health networks , and 10 faculties with more than 2020 faculty members and 19,000 students ( tums 2012 ) , requires an improved comprehensive system for delivering the wellbeing services ( financial , insurance , healthcare services , educational and training services , etc . ) .", "taking a look at the functions of the university 's wellbeing services system ; it uncovers a gap between the employees real needs and what is delivered to meet their needs .", "it is because of the lack of a participatory strategic wellbeing delivery plan that is based on prioritized wellbeing needs ( requirements ) .", "as the first step to make a strategic plan is identifying the needs , the main purpose of this study is to assess the wellbeing services of the staff working in tums  first to see if they are satisfactory enough or they need to be changed , and second , to help decision - makers develop services that are essential to improve the well - being of employees , as also the quality and effectiveness of the tums 's wellbeing delivery system .", "what are the services used and to what extent they have been satisfactory? this question aims to explore the reasons why they are not delivered .", "the second question is ,  what type of services are essential to be provided by the university? with respect to the main purpose of this research , the wellbeing delivery system of tums is assessed with a thorough answering of the questions mentioned above ."], ["the research design was a cross - sectional survey ( 2008 - 2009 ) using a structured non - disguised questionnaire .", "the sample size was 98 with a 95% level of confidence and absolute error of 0/01 .", "cluster sampling based on the  occupational scope as the main variable , was used as the sampling method , with regard to the existence of different occupational scopes .", "data was collected from 98 officers working at different departments of central organizations of tums ( tehran university of medical sciences ) including logistics , education , research and technology , student and cultural affairs departments .", "the research participants were assured of the confidentiality of their personal information at the beginning of the questionnaire .", "the questionnaire included 45 questions ( 43 close - ended questions and 2 open - ended questions1 ) organized into two separate parts ( cranach alpha = 0.85 ) : according to table 1 , within the first part , the respondents were supposed to mark the wellbeing services if they had ever used any of them . those who used the services were supposed to show their level of satisfaction by choosing from ", "almost satisfied ,  almost dissatisfied, and  full dissatisfied , and those who had never used the services , were supposed to express their reasons by choosing from ", "poor announcement ,  unwillingness to receive the service ,  undesirability of the service, and  undesirability of the receiving process of the service. main parts and the main purpose of asking questions for each part in the second part , the respondents were provided with a list of not delivered wellbeing services to choose which of them is ", "essential ,  neither essential nor non - essential or  non - essential. for making sure if the questionnaire was valid content - wise , first a comprehensive collection of wellbeing services delivered both in tums and in other organizations was identified according to the comprehensive definition of wellbeing services .", "next , according to the main purpose of the research , they were put into classified groups to form the research tool .", "furthermore , to make sure about the appropriateness and transparency of each question and also the whole tool , in addition to its comprehensiveness , the questionnaire was sent to five professionals and experts in the relevant field .", "the reliability of the questionnaire was determined after thoroughly calculating the cronbach alpha , which was reported to be around 0.85 .", "subsequently , after improving and clarifying the instrument , the participants of sample group were asked to fill in the questionnaire .", "the data was gathered by means of filled questionnaires , and were analyzed with the excel software and spss , by calculating the frequency rate of the staff that used the welfare services and those who did not use the services .", "in addition , the t - test was used to calculate the satisfaction score for each service used by the staff ."], ["among all the 98 respondents , almost 46 employees with a mean satisfactory score of 48.46 used the existing services , for which the satisfactory rate was distributed as shown in table 2 .", "satisfactory scores of categorized wellbeing services the result shows that financial services , including loans for buying a car , emergency loan , loan for buying commodities , awards for staffs children , and two other kinds of non - profit loans in comparison with other categories is considered as the most satisfactory service ( average t = 61.83 ) . among respondents who did not use the categorized services ( 52 persons on an average ) , as shown in the table 3 , the most frequents reasons for not receiving services was ,  staff 's unwillingness to receive the service ( 38.75 in average ) and  poor announcement ( unawareness on the wellbeing services ) ( 37.6 in average )", ". frequency rate of reasons for not receiving welfare services table 4 presents the  frequency of wellbeing services and  necessity ( delivered by other public / private organizations ) , which were asked to be judged by respondents ( to be added or replaced by the delivered wellbeing services list ) , as follows : frequency distribution of wellbeing services  necessity", "educational subvention for children of the staff , who were under their patronage is the only non - financial service , with the highest frequency ( 92.85 ) , identified as the most necessary wellbeing service among all . among other services that were mentioned by respondents ( 61 persons of the sample group ) , the provision of discount cards for using other organizations services ( frequency = 12 ) , providing employees with free health care services ( frequency = 7 ) , and contracting with adult higher education institutions ( frequency = 6 ) had the most frequency among other items ."], ["as mentioned , the objectives of the study were to measure the staff 's satisfaction with the wellbeing services received , identify the reasons for not receiving the existing wellbeing services , identify the wellbeing services that were not delivered , but were essential as per the staff , and then ask the staff about other wellbeing services that were possible to be delivered and sort them according to their priority . with regard to the first purpose , the results showed that almost half of the respondents used the wellbeing services provided by the wellbeing office of the university , among which almost half of them were satisfied with the services .", "the results suggested that not only were most of the staff not interested in using the provided services , but also more than half of those respondents who used them were not really happy . with regard to the second purpose", ", the results showed that among those who did not use the services , most of them decided not to use it by themselves .", "this was recommended to be surveyed in a separate study in the future . as was evident", ", it can be concluded that the functional desirability of the wellbeing service delivery system of tums was not evaluated as satisfactory enough , because of the almost high dissatisfaction level of services that were used . with regard to the third purpose", ", the results indicated a big gap between the required wellbeing services ( by employees ) and what was provided by the office , as almost all the respondents agreed with the necessity of services such as health , education , and safety .", "the existing gap can result from lack of managerial attention to differences in the demographical characteristics and expectations of employees .", "consistent with the arguments of boudreau and ramstad , dencker et al . argued that market segmentation of employees ( offering market segments have different benefits ) may strengthen the benefits  return - on - investment. one of the most significant reasons for the dissatisfaction and unwillingness of employees to use the services refers to the different priorities and type of services they prefer to include , according to their individual differences . ", "need assessment as a useful technique can help planners to pay more attention to employee 's priority differences in wellbeing services and what they really need to include , by designing the appropriate interventions , as davis and gibson emphasized .", "considering the employees as the most important organizational stakeholders makes them to participate in the wellbeing decision - making process , such as , asking them to add what services they prefer to be included within the wellbeing package .", "although the wellbeing service delivery system of tums has not been effective enough to meet the real needs of the staff by delivering the appropriate services , the most prioritized service type preference of most respondents can be identified . as in the first place the satisfactory score of educational and training services among those who used them was rather high , and second", ", the most necessary identified services were related to educational services , and of course , among the identified wellbeing service priorities , provision of educational opportunity was one of the preferences with high frequency , therefore , it was clear that educational and training opportunities were important for a majority of the respondents .", "the findings show that the functional desirability of tums wellbeing services is not satisfactory enough by the staff 's view point ."], ["the following conclusions can be drawn from the present study ; the first major finding is the existence of a big gap between the required wellbeing services ( by employees ) and what is provided by the wellbeing office of tums .", "the second and the most obvious finding of this study is the importance of the educational and training opportunities from the point of view of the staff , which has not been paid attention to by the wellbeing service provider in tums .", "although we tried to assess the overall quality and functional desirability of the wellbeing service delivery system of tums , some further studies are required in the researcher 's view .", "it is recommended that the factors that cause dissatisfaction and unwillingness to use services in employees be identified , and also the real needs and wellbeing requirements ( based on employees preferences , job categories , genders , age , and other considerable individual and social factors ) as a strategic basis for redesigning the current wellbeing service delivery system that appears not to be desirable enough in the employees view , be determined ."]]}
{"article_id": "PMC2688588", "article_text": ["the clinical manifestation is typical and it is characterized by abduction of the affected arm , flexion of the elbow and pronation of the forearm .", "bilateral luxatio erecta represents an extremely rare condition , and just a few cases were reported in medical literature . because of a high incidence of related complications such as neurovascular damages early recognition and treatment of this type of dislocation is required by physicians . in this article", "we describe a case of a bilateral luxatio erecta that was successfully treated without long - term complications .", "a 70-year - old female was accepted in our emergency department because of a bilateral shoulder pain that onsets after a sudden fall happened in her house .", "the anamnestic research showed that the patient tumbled downstairs ; while clinging to both lateral banisters , she slipped down banging her face and chest to the ground . at physical examination", "both shoulders were abducted and elevated approximately 90100 from the horizontal plane , with flexed elbows and with hands below the head .", "the patient was unable to adduct and lower the arms , and there was immediate pain when attempting passive movements .", "no vascular deficit was found , and the patient complained of slight paresthesias on both arms , with superficial and deep sensation deficit on both arms .", "an x - ray examination was performed showing a bilateral inferior dislocation of humeral heads in relation with the glenoid fossa ; no fracture was associated ( fig .  1 ) .", "1bilateral luxatio erecta bilateral luxatio erecta close reduction under sedation was achieved without difficulty by pushing the humeral head superiorly with an axial traction - countertraction maneuver on both shoulders .", "x - ray control revealed a correct anatomic reduction of dislocations and after the reduction no vascular damage were found while paresthesias were still present on the left arm ( fig .  2 ) .", "both arms were immobilized with bilateral slings in total adduction , intrarotation with the supinated arms .", "2post - reduction x - ray the progressive mobilization of the shoulders started after 2  weeks with passive movements and pendulum codman s exercises .", "after 3  weeks the patient gave up complaining paresthesias on the left arm and active movements were allowed .", "after 8  weeks she recovered the complete function of both shoulders and after a one - year follow - up treatment the patient showed a complete range of motion of both arms .", "inferior dislocations are rare ( 0.5% of all shoulder dislocations ) and bilateral inferior shoulder dislocations are exceptional : only 10 cases have been described so far in the literature . according to the literature , luxatio erecta is due to an indirect mechanism such as a violent abduction force on abducted limb resulting in impingement of the proximal shaft of the humerus against acromion .", "direct axial loading from superior direction is another different mechanism , though less common . in our case", "the first mechanism has to be taken into consideration ; in fact , while the patient was sliding in a sitting position downstairs , her hands were holding onto both banisters and the arms were abduced .", "when she reached the ground floor the hands were blocked around the end of the banisters and her head and body were thrown forward by gravity .", "the clinical presentation of an inferior shoulder dislocation is typical : the extremities are held over the head or abduced in a fixed position with the elbow flexed .", "any passive movement is possible , and the humeral head is palpable on the chest wall .", "x - ray shows the humeral head located below the rim of the glenoid and the humeral shaft is parallel to the scapular spine . in order to reduce inferior glenohumeral dislocation ,", "it is recommended a traction counter - traction maneuver under sedation followed by immobilization in a desault bandage for at least 2  weeks .", "there is a high incidence of associate tears of the rotator cuff ( 12% of patients )   and a slap lesion has been described by schai and hintermann   after a post - reduction arthroscopic assessment view .", "fracture of the acromion , clavicle , inferior glenoid fossa and greater tuberosity represent a complication that should need a surgical treatment .", "neurovascular damages are also commonly associated with inferior shoulder dislocation [ 5 , 7 ] ; garcia et al .   described a case of bilateral luxatio erecta complicated by an axillary vein thrombosis . in general , neurologic injuries ( 60% of cases ) of brachial plexus , axillar , radial and ulnar nerves", "are more common than vascular injuries but they tend to resolve after reduction with an excellent prognosis , suggesting the neuropraxia as the main mechanism of the injury [ 5 , 9 , 10 ] ."], "abstract_text": ["<S> luxatio erecta is an uncommon form of glenohumeral dislocation </S>", "<S> . cases of bilateral inferior shoulder dislocation ( luxatio erecta ) are very rare , and only ten cases have been described in literature . </S>", "<S> we describe a case of a woman with bilateral luxatio erecta of the shoulders treated with close reduction , immobilization and rehabilitation . </S>", "<S> the patient had fallen downstairs while clinging to both lateral banisters . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["the clinical manifestation is typical and it is characterized by abduction of the affected arm , flexion of the elbow and pronation of the forearm .", "bilateral luxatio erecta represents an extremely rare condition , and just a few cases were reported in medical literature . because of a high incidence of related complications such as neurovascular damages early recognition and treatment of this type of dislocation is required by physicians . in this article", "we describe a case of a bilateral luxatio erecta that was successfully treated without long - term complications ."], ["a 70-year - old female was accepted in our emergency department because of a bilateral shoulder pain that onsets after a sudden fall happened in her house .", "the anamnestic research showed that the patient tumbled downstairs ; while clinging to both lateral banisters , she slipped down banging her face and chest to the ground . at physical examination", "both shoulders were abducted and elevated approximately 90100 from the horizontal plane , with flexed elbows and with hands below the head .", "the patient was unable to adduct and lower the arms , and there was immediate pain when attempting passive movements .", "no vascular deficit was found , and the patient complained of slight paresthesias on both arms , with superficial and deep sensation deficit on both arms .", "an x - ray examination was performed showing a bilateral inferior dislocation of humeral heads in relation with the glenoid fossa ; no fracture was associated ( fig .  1 ) .", "1bilateral luxatio erecta bilateral luxatio erecta close reduction under sedation was achieved without difficulty by pushing the humeral head superiorly with an axial traction - countertraction maneuver on both shoulders .", "x - ray control revealed a correct anatomic reduction of dislocations and after the reduction no vascular damage were found while paresthesias were still present on the left arm ( fig .  2 ) .", "both arms were immobilized with bilateral slings in total adduction , intrarotation with the supinated arms .", "2post - reduction x - ray the progressive mobilization of the shoulders started after 2  weeks with passive movements and pendulum codman s exercises .", "after 3  weeks the patient gave up complaining paresthesias on the left arm and active movements were allowed .", "after 8  weeks she recovered the complete function of both shoulders and after a one - year follow - up treatment the patient showed a complete range of motion of both arms ."], ["inferior dislocations are rare ( 0.5% of all shoulder dislocations ) and bilateral inferior shoulder dislocations are exceptional : only 10 cases have been described so far in the literature . according to the literature , luxatio erecta is due to an indirect mechanism such as a violent abduction force on abducted limb resulting in impingement of the proximal shaft of the humerus against acromion .", "direct axial loading from superior direction is another different mechanism , though less common . in our case", "the first mechanism has to be taken into consideration ; in fact , while the patient was sliding in a sitting position downstairs , her hands were holding onto both banisters and the arms were abduced .", "when she reached the ground floor the hands were blocked around the end of the banisters and her head and body were thrown forward by gravity .", "the clinical presentation of an inferior shoulder dislocation is typical : the extremities are held over the head or abduced in a fixed position with the elbow flexed .", "any passive movement is possible , and the humeral head is palpable on the chest wall .", "x - ray shows the humeral head located below the rim of the glenoid and the humeral shaft is parallel to the scapular spine . in order to reduce inferior glenohumeral dislocation ,", "it is recommended a traction counter - traction maneuver under sedation followed by immobilization in a desault bandage for at least 2  weeks .", "there is a high incidence of associate tears of the rotator cuff ( 12% of patients )   and a slap lesion has been described by schai and hintermann   after a post - reduction arthroscopic assessment view .", "fracture of the acromion , clavicle , inferior glenoid fossa and greater tuberosity represent a complication that should need a surgical treatment .", "neurovascular damages are also commonly associated with inferior shoulder dislocation [ 5 , 7 ] ; garcia et al .   described a case of bilateral luxatio erecta complicated by an axillary vein thrombosis . in general , neurologic injuries ( 60% of cases ) of brachial plexus , axillar , radial and ulnar nerves", "are more common than vascular injuries but they tend to resolve after reduction with an excellent prognosis , suggesting the neuropraxia as the main mechanism of the injury [ 5 , 9 , 10 ] ."]]}
{"article_id": "PMC2687696", "article_text": ["a biloma is a rare abnormal accumulation of intrahepatic or extrahepatic bile caused by traumatic or spontaneous rupture of the biliary tree1 , 2 ) .", "it is most commonly caused by surgery , percutaneous transhepatic cholangiography ( ptc ) , percutaneous transhepatic biliary drainage ( ptbd ) , and abdominal trauma11 , 12 ) .", "we report here a patient who complained of chills , fever and right upper quadrant abdominal discomfort .", "a 78-year - old woman presented to the emergency room with her guardian , with acute fever and chills that developed the day before admission .", "the patient was diagnosed with diabetes a few years previously , but this was not being treated .", "on admission to the emergency room , the vital signs were as follows : blood pressure 70/40 mmhg , heart rate 118/min , respiratory rate 30/min and temperature 40.3. the patient appeared to be in acute distress ; she was lethargic , the sclerae were slightly yellow and the conjunctivae were pale .", "the abdominal examination revealed slight tenderness in the right upper quadrant , but there was no palpable mass .", "the laboratory studies were as follows : wbc 52,400/mm , hb 12.3 g / dl , platelets 90,000/mm , ast 153 iu / l ,", "alt 135 iu / l , alkaline phosphatase 255 iu / l , total bilirubin 2.07 mg / dl , total protein 5.9 g / dl , albumin 3.0 g / dl , bun / cr 25/1.9 mg / dl , esr 25 mm / hr , and crp 20.4 mg / dl .", "the serum electrolyte levels were normal except for an elevated potassium 7.0 mmol / l .", "the abga data were as follows : ph 7.24 , pao271 mmhg , paco2 19 mmhg , hco3 15.1", "the hepatitis viral marker tests , including hbsag , anti - hbs ab and anti - hcv , were all negative .", "standard x - rays on the day of admission revealed lung infiltrates and no specific findings in the abdomen .", "the abdominal computed tomography ( ct ) revealed a 9.58.5 cm subcapsular cyst with a clear boundary in the left hepatic lobe and enlargement of the intrahepatic bile duct around the cyst .", "although the left hepatic lobe had some atrophic findings , there were no significant signs of an enlarged bile duct or abnormal gallbladder ( figure 1 ) . on the day of admission after the computed tomography ,", "the abga revealed a ph 7.05 , pco2 64.8 mmhg , po2 73.3 mmhg , hco3", "the patient was intubated and put on a respirator ; she was treated with antibiotics and vasopressors .", "on the second day of admission , percutaneous drainage with an 8.5 fr pigtail catheter , under the guidance of an abdominal ultrasound was performed for diagnosis and treatment .", "we removed 565 ml of fluid that was dark brown in color . for a diagnosis ,", "biochemical tests were carried out on the bile , and these tests revealed a total bilirubin of 22.3 mg / dl and a direct bilirubin of 18.9 mg / dl .", "the leukocyte count was 4,560/mm , and gram staining showed a gram negative bacillus . on the third day of admission ,", "her vital signs were as follows : blood pressure 100/65 mmhg , heart rate 110/min and temperature 37.2. there was 200 ml of drained bile , the s - fibrinogen was 894 mg / dl ( 200 - 415 ) , the d - dimer was 9,040 ng / ml ( 0 - 255 ) , and the fdp was elevated up to 97 ug / ml ( normal range : 0 - 5 ) . on the eighth day of admission , the patient 's mental status improved , the vital signs were stable and voluntary respirations recovered .", "on the thirteenth day of admission , pancreatico - biliary magnetic resonance imaging ( mri ) revealed a biloma that was connected to the intrahepatic bile duct ( figure 2 ) . on the sixteenth day of admission , the biloma that was connected to the intrahepatic duct and common bile duct", "was noted again via a tubogram performed with a catheter . over time , the amount of bile drainage decreased . on the twentieth day of admission ,", "64 ml of bile was drained , the leukocyte count was 340/mm and there were no signs of bacteria on culture or gram staining of the bile .", "the follow - up ct showed that the size of the cyst had decreased to 5.44.6 cm and the density around the cyst had increased .", "the amount of drainage did not decrease afterward , and the connection between the biloma and the bile duct was still noted . on the 25th day of admission ,", "the drainage tube was blocked and follow - up of the bilirubin in blood and the size of the biloma were assessed . on the 29th day of admission ,", "follow - up ct showed a marked decrease in the size of the cyst ( figure 3 ) , and the serum bilirubin was normal ( 0.22 mg / dl ) . on the 34th day of admission ,", "the drainage tube was removed after evaluating the size and connection of the biloma via a catheter tubogram ( figure 4 ) .", "they reported a case with extrahepatic bile leakage after trauma to the upper right quadrant of the abdomen ; the bile did not cause peritonitis , but it accumulated in an encapsulated form .", "this concept was later applied to all cases with biliary tract lacerations caused by bile leaks that formed a capsule inside or outside of the liver1 ) .", "the mechanism of encapsulation is explained by a large amount of bile leaking at a fast pace that causes biliary peritonitis , or a small amount of bile leaking at a slow pace that causes mild inflammation .", "this leaked fluid is simultaneously trapped by the greater omentum and mesentery , where it becomes encapsulated2 ) .", "although the amount of bile leaked may be minor , the occurrence of an inflammatory reaction contributes to the appearance of a growth in size of the biloma3 ) . as diagnostic techniques", "continue to develop , more cases of biloma are being reported , but cases of spontaneous biloma and biloma accompanied by other diseases have rarely been reported to date . from 1979 to 1997 , 25 cases of spontaneous biloma were analyzed by fujiwara ; the underlying causes included choledocholithiasis ( 16 cases ) , cholangiocarcinoma ( 2 cases ) , acute cholecystitis ( 1 case ) , liver infarction ( 1 case ) , hepatic abscess ( 1 case ) , nephrotic syndrome ( 1 case ) , obstructive jaundice ( 1 case ) , sickle cell anemia ( 1 case ) , and tuberculosis ( 1 case)4 ) . in korea ,", "there have been two cases of choledocholithiasis accompanied by a biloma , and they were treated by nonsurgical methods5 , 6 ) .", "there is one report of a case of biloma after trauma7 ) , and 1 case of a biloma after endoscopic retrograde cholangiopancreatography ( ercp)8 ) .", "one of the patients with a choledocholithiasis who suffered a nontraumatic intrahepatic rupture of the bile duct , had a surgical repair9 ) .", "the causes of biloma include trauma to the liver , which is the most common cause , abdominal surgery , endoscopic surgery and percutaneous catheter drainage10 - 13 ) .", "currently , the term biloma is generally used to describe intrahepatic or intraperitoneal focal biliary stasis14 ) .", "the clinical symptoms of a biloma are nonspecific , and they can range from no symptoms to abdominal pain and distention , jaundice and fever ; leukocytosis can also be present15 ) . in the case", "there were complaints of sudden chills , fever , upper abdominal discomfort and sensations of abdominal distention .", "physical examinations showed tenderness in the upper right area of the abdomen , slight jaundice , hypotension , leukocytosis and fever over 40. the size and location of the biloma are determined by the cause of the rupture of the bile duct , the location and speed of bile leakage and the rate the bile is reabsorbed by the peritoneum2 ) .", "fujiwara reported that the locations for bile leakage included the left hepatobiliary duct ( 8 cases ) , the gallbladder ( 5 cases ) , the right hepatobiliary tract ( 2 cases ) , the common bile duct ( 1 case ) , and unknown ( 9 cases ) ; the locations where the bilomas formed were the left lobe ( 11 cases ) , the right lobe ( 10 cases ) , and the upper abdomen ( 4 cases ) .", "vazquez analyzed 21 cases of biloma and found that 16 cases occurred in the upper abdominal area near the liver and five in the left upper abdominal area4 ) .", "the diagnosis of a biloma can be assisted by surgery , a history of trauma with abdominal ultrasonography , abdominal ct and mri .", "a definite diagnosis can be made by a 99mtc - disida scan , percutaneous aspiration or by ercp1 , 2 ) .", "abdominal ultrasonography shows a mass of low echogenicity with a clear border in the liver , and the follow up tests show changes in size16 ) .", "the abdominal ct will give a more accurate view of the location of the biloma and show its relationship to the surrounding organs . a standard ct view", "will show a low - density mass in the liver at a ct number lower than 20 .", "ultrasonography and abdominal ct are sometimes unable to differentiate bilomas from seromas , lymphoceles and angiomas .", "mris or hepatobiliary scans can be useful when continuous bile leakage is present , but they are not diagnostic when continuous leakage is not present7 , 17 ) . radiological image - guided aspiration tests , when testing for bilirubin , can be diagnostic18 ) .", "when the aspirated substance is a clear yellow liquid , then microbiological tests must be done to rule out an infection .", "ercp can be used to determine the location and severity of an active bile leak .", "however , the presence of small biliary cysts or bilomas , located in the lower areas of the liver that can be hidden by gastrointestinal shadows , can be difficult to diagnosis19 ) .", "we were able to identify a cystic lesion by abdominal ct ; a tubogram and magnetic resonance cholangiopancreatography ( mrcp ) were used to confirm a bile leak from the common bile duct .", "the fluid obtained by the transabdominal ultrasonography assisted percutaneous cyst drainage containing 22.3 mg / dl of total bilirubin and 18.9 mg / dl of direct bilirubin ; these findings confirmed the diagnosis of a biloma .", "treatment for bilomas that have a diameter of only a few centimeters is not always necessary ; these lesions can be watched . however , most bilomas require treatment . in the past , surgery was the main approach to treatment . currently , there are a much wider variety of options such as percutaneous catheter drainage , endoscopic sphincterotomy ( est ) , endoscopic nasobiliary drainage ( enbd ) and endoscopic drainage4 , 6 , 15 , 19 , 20 ) .", "surgery is now performed only in cases with a persistent bile leak or for treatment of underlying disease . in this case , a spontaneous biloma with bacterial infection was found after the patient presented in septic shock .", "after percutaneous drainage was performed , the amount of bile excreted did not decrease to lower than 64 ml per day . as there was no difficulty in flow through the common bile duct , the biochemical tests performed after removal of the drainage catheter showed no increase in total bilirubin or any increase in the size of the biloma , thus demonstrating a rare case of recovery ."], "abstract_text": ["<S> a \" biloma \" is a loculated collection of bile located outside of the biliary tree . </S>", "<S> it can be caused by traumatic , iatrogenic or spontaneous rupture of the biliary tree . </S>", "<S> prior reports have documented an association of biloma with abdominal trauma , surgery and other primary causes , but spontaneous bile leakage has rarely been reported . </S>", "<S> a spontaneous infected biloma , without any underlying disease , is a very rare finding . </S>", "<S> we recently diagnosed a spontaneous infected biloma by abdominal computed tomography and sonographically guided percutaneous aspiration . </S>", "<S> the patient was successfully managed with percutaneous drainage and intravenous antibiotics . </S>", "<S> we report here a case of infected biloma caused by spontaneous rupture of the intrahepatic duct , and review the relevant medical literature . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["a biloma is a rare abnormal accumulation of intrahepatic or extrahepatic bile caused by traumatic or spontaneous rupture of the biliary tree1 , 2 ) .", "it is most commonly caused by surgery , percutaneous transhepatic cholangiography ( ptc ) , percutaneous transhepatic biliary drainage ( ptbd ) , and abdominal trauma11 , 12 ) .", "we report here a patient who complained of chills , fever and right upper quadrant abdominal discomfort ."], ["a 78-year - old woman presented to the emergency room with her guardian , with acute fever and chills that developed the day before admission .", "the patient was diagnosed with diabetes a few years previously , but this was not being treated .", "on admission to the emergency room , the vital signs were as follows : blood pressure 70/40 mmhg , heart rate 118/min , respiratory rate 30/min and temperature 40.3. the patient appeared to be in acute distress ; she was lethargic , the sclerae were slightly yellow and the conjunctivae were pale .", "the abdominal examination revealed slight tenderness in the right upper quadrant , but there was no palpable mass .", "the laboratory studies were as follows : wbc 52,400/mm , hb 12.3 g / dl , platelets 90,000/mm , ast 153 iu / l ,", "alt 135 iu / l , alkaline phosphatase 255 iu / l , total bilirubin 2.07 mg / dl , total protein 5.9 g / dl , albumin 3.0 g / dl , bun / cr 25/1.9 mg / dl , esr 25 mm / hr , and crp 20.4 mg / dl .", "the serum electrolyte levels were normal except for an elevated potassium 7.0 mmol / l .", "the abga data were as follows : ph 7.24 , pao271 mmhg , paco2 19 mmhg , hco3 15.1", "the hepatitis viral marker tests , including hbsag , anti - hbs ab and anti - hcv , were all negative .", "standard x - rays on the day of admission revealed lung infiltrates and no specific findings in the abdomen .", "the abdominal computed tomography ( ct ) revealed a 9.58.5 cm subcapsular cyst with a clear boundary in the left hepatic lobe and enlargement of the intrahepatic bile duct around the cyst .", "although the left hepatic lobe had some atrophic findings , there were no significant signs of an enlarged bile duct or abnormal gallbladder ( figure 1 ) . on the day of admission after the computed tomography ,", "the abga revealed a ph 7.05 , pco2 64.8 mmhg , po2 73.3 mmhg , hco3", "the patient was intubated and put on a respirator ; she was treated with antibiotics and vasopressors .", "on the second day of admission , percutaneous drainage with an 8.5 fr pigtail catheter , under the guidance of an abdominal ultrasound was performed for diagnosis and treatment .", "we removed 565 ml of fluid that was dark brown in color . for a diagnosis ,", "biochemical tests were carried out on the bile , and these tests revealed a total bilirubin of 22.3 mg / dl and a direct bilirubin of 18.9 mg / dl .", "the leukocyte count was 4,560/mm , and gram staining showed a gram negative bacillus . on the third day of admission ,", "her vital signs were as follows : blood pressure 100/65 mmhg , heart rate 110/min and temperature 37.2. there was 200 ml of drained bile , the s - fibrinogen was 894 mg / dl ( 200 - 415 ) , the d - dimer was 9,040 ng / ml ( 0 - 255 ) , and the fdp was elevated up to 97 ug / ml ( normal range : 0 - 5 ) . on the eighth day of admission , the patient 's mental status improved , the vital signs were stable and voluntary respirations recovered .", "on the thirteenth day of admission , pancreatico - biliary magnetic resonance imaging ( mri ) revealed a biloma that was connected to the intrahepatic bile duct ( figure 2 ) . on the sixteenth day of admission , the biloma that was connected to the intrahepatic duct and common bile duct", "was noted again via a tubogram performed with a catheter . over time , the amount of bile drainage decreased . on the twentieth day of admission ,", "64 ml of bile was drained , the leukocyte count was 340/mm and there were no signs of bacteria on culture or gram staining of the bile .", "the follow - up ct showed that the size of the cyst had decreased to 5.44.6 cm and the density around the cyst had increased .", "the amount of drainage did not decrease afterward , and the connection between the biloma and the bile duct was still noted . on the 25th day of admission ,", "the drainage tube was blocked and follow - up of the bilirubin in blood and the size of the biloma were assessed . on the 29th day of admission ,", "follow - up ct showed a marked decrease in the size of the cyst ( figure 3 ) , and the serum bilirubin was normal ( 0.22 mg / dl ) . on the 34th day of admission ,", "the drainage tube was removed after evaluating the size and connection of the biloma via a catheter tubogram ( figure 4 ) ."], ["they reported a case with extrahepatic bile leakage after trauma to the upper right quadrant of the abdomen ; the bile did not cause peritonitis , but it accumulated in an encapsulated form .", "this concept was later applied to all cases with biliary tract lacerations caused by bile leaks that formed a capsule inside or outside of the liver1 ) .", "the mechanism of encapsulation is explained by a large amount of bile leaking at a fast pace that causes biliary peritonitis , or a small amount of bile leaking at a slow pace that causes mild inflammation .", "this leaked fluid is simultaneously trapped by the greater omentum and mesentery , where it becomes encapsulated2 ) .", "although the amount of bile leaked may be minor , the occurrence of an inflammatory reaction contributes to the appearance of a growth in size of the biloma3 ) . as diagnostic techniques", "continue to develop , more cases of biloma are being reported , but cases of spontaneous biloma and biloma accompanied by other diseases have rarely been reported to date . from 1979 to 1997 , 25 cases of spontaneous biloma were analyzed by fujiwara ; the underlying causes included choledocholithiasis ( 16 cases ) , cholangiocarcinoma ( 2 cases ) , acute cholecystitis ( 1 case ) , liver infarction ( 1 case ) , hepatic abscess ( 1 case ) , nephrotic syndrome ( 1 case ) , obstructive jaundice ( 1 case ) , sickle cell anemia ( 1 case ) , and tuberculosis ( 1 case)4 ) . in korea ,", "there have been two cases of choledocholithiasis accompanied by a biloma , and they were treated by nonsurgical methods5 , 6 ) .", "there is one report of a case of biloma after trauma7 ) , and 1 case of a biloma after endoscopic retrograde cholangiopancreatography ( ercp)8 ) .", "one of the patients with a choledocholithiasis who suffered a nontraumatic intrahepatic rupture of the bile duct , had a surgical repair9 ) .", "the causes of biloma include trauma to the liver , which is the most common cause , abdominal surgery , endoscopic surgery and percutaneous catheter drainage10 - 13 ) .", "currently , the term biloma is generally used to describe intrahepatic or intraperitoneal focal biliary stasis14 ) .", "the clinical symptoms of a biloma are nonspecific , and they can range from no symptoms to abdominal pain and distention , jaundice and fever ; leukocytosis can also be present15 ) . in the case", "there were complaints of sudden chills , fever , upper abdominal discomfort and sensations of abdominal distention .", "physical examinations showed tenderness in the upper right area of the abdomen , slight jaundice , hypotension , leukocytosis and fever over 40. the size and location of the biloma are determined by the cause of the rupture of the bile duct , the location and speed of bile leakage and the rate the bile is reabsorbed by the peritoneum2 ) .", "fujiwara reported that the locations for bile leakage included the left hepatobiliary duct ( 8 cases ) , the gallbladder ( 5 cases ) , the right hepatobiliary tract ( 2 cases ) , the common bile duct ( 1 case ) , and unknown ( 9 cases ) ; the locations where the bilomas formed were the left lobe ( 11 cases ) , the right lobe ( 10 cases ) , and the upper abdomen ( 4 cases ) .", "vazquez analyzed 21 cases of biloma and found that 16 cases occurred in the upper abdominal area near the liver and five in the left upper abdominal area4 ) .", "the diagnosis of a biloma can be assisted by surgery , a history of trauma with abdominal ultrasonography , abdominal ct and mri .", "a definite diagnosis can be made by a 99mtc - disida scan , percutaneous aspiration or by ercp1 , 2 ) .", "abdominal ultrasonography shows a mass of low echogenicity with a clear border in the liver , and the follow up tests show changes in size16 ) .", "the abdominal ct will give a more accurate view of the location of the biloma and show its relationship to the surrounding organs . a standard ct view", "will show a low - density mass in the liver at a ct number lower than 20 .", "ultrasonography and abdominal ct are sometimes unable to differentiate bilomas from seromas , lymphoceles and angiomas .", "mris or hepatobiliary scans can be useful when continuous bile leakage is present , but they are not diagnostic when continuous leakage is not present7 , 17 ) . radiological image - guided aspiration tests , when testing for bilirubin , can be diagnostic18 ) .", "when the aspirated substance is a clear yellow liquid , then microbiological tests must be done to rule out an infection .", "ercp can be used to determine the location and severity of an active bile leak .", "however , the presence of small biliary cysts or bilomas , located in the lower areas of the liver that can be hidden by gastrointestinal shadows , can be difficult to diagnosis19 ) .", "we were able to identify a cystic lesion by abdominal ct ; a tubogram and magnetic resonance cholangiopancreatography ( mrcp ) were used to confirm a bile leak from the common bile duct .", "the fluid obtained by the transabdominal ultrasonography assisted percutaneous cyst drainage containing 22.3 mg / dl of total bilirubin and 18.9 mg / dl of direct bilirubin ; these findings confirmed the diagnosis of a biloma .", "treatment for bilomas that have a diameter of only a few centimeters is not always necessary ; these lesions can be watched . however , most bilomas require treatment . in the past , surgery was the main approach to treatment . currently , there are a much wider variety of options such as percutaneous catheter drainage , endoscopic sphincterotomy ( est ) , endoscopic nasobiliary drainage ( enbd ) and endoscopic drainage4 , 6 , 15 , 19 , 20 ) .", "surgery is now performed only in cases with a persistent bile leak or for treatment of underlying disease . in this case , a spontaneous biloma with bacterial infection was found after the patient presented in septic shock .", "after percutaneous drainage was performed , the amount of bile excreted did not decrease to lower than 64 ml per day . as there was no difficulty in flow through the common bile duct , the biochemical tests performed after removal of the drainage catheter showed no increase in total bilirubin or any increase in the size of the biloma , thus demonstrating a rare case of recovery ."]]}
{"article_id": "PMC4106956", "article_text": ["the construct of unintended pregnancies ( ups ) is multifactorial and broadly encompasses pregnancies that are either unwanted or mistimed.1 in many instances , ups are likely to end by induced abortion ; worldwide estimates suggest that 50% will be voluntarily terminated.2 in 2008 , it was estimated that 43.8 million abortions occurred worldwide , of which 86% occurred in low-/middle - income countries.3 furthermore , between 2003 and 2008 , the number of induced abortions was found to have decreased in high - income countries but to have increased in low-/middle - income countries . during this same period of time , the proportion of unsafe abortions increased and they were believed to account for 13% of maternal deaths , with the majority of these concentrated in countries with restrictive laws on abortion.4 elective abortion in brazil is considered non - legal , unless the pregnancy resulted from a rape , would cause a life - threatening condition to the mother , or the fetus has anencephaly or any other malformation that is incompatible with the extrauterine life . as a result ,", "data on prevalence and associated costs of abortion are limited and remain a challenge to collect .", "however , it is recognized worldwide that direct health costs of up and resulting abortions can significantly impact local health services and the families affected.5 health system and societal costs can also arise from ups that do not end in abortion and are carried to parturition . in the united states ,", "the public health costs attributed to unintended births in a single year cost taxpayers $ 11.1 billion ( 2006).6 furthermore , previous studies have reported that ups are more likely to result in preterm births and low - birth - weight babies , which would increase health costs for neonatal care and costs associated with long - term disabilities.7 this is particularly relevant for ups in adolescence , which are more likely to result in low - birth - weight births , which can increase health costs.8 over the past few decades , brazil , like many fast - emerging economies , has experienced a fertility transition noted by a dramatic reduction in the total fertility rate , which currently sits at 1.8 births per woman.9,10 furthermore , during the fertility transition , the rate of contraception has also increased.11 despite increased contraception use , the cumulative rate of spontaneous and induced abortion changed very little between 1996 and 2006 , for which population survey estimates are available.12,13 furthermore , a survey of more than 1,000 women aged 1024 years in a single postnatal unit in brazil reported that more than 50% of all births were unintended , suggesting that possibly significant unmet contraception need still exists.8 similarly , for the whole country , the data from the last demographic and health survey ( dhs ) performed in 2006 indicates that 55% of all births were unintended.14 prevention of up using publicly funded programs in high - income countries has proven to generate significant cost savings for health services and public services.15 previous economic studies evaluating the consequences of up have mostly been performed in high - income countries where publicly funded fertility services and abortion legislation are different from those in low-/middle - income countries.15 the aim of this study was to evaluate the cost consequences of up in brazil , an emerging economy with vastly different fertility planning services than most advanced economies and with restrictive abortion laws . to the best of our knowledge ,", "this is the first published study that describes the economics of up , and it is believed that this work can aid decision makers regarding access to contraceptives .", "a model was developed to evaluate the humanistic burden and financial impact of ups in brazil .", "the analysis factored in costs and outcomes for 1 year post - delivery of ups .", "possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth .", "all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil .", "available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries .", "complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy .", "data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used .", "a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) .", "the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% .", "the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births .", "differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy .", "all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30 resource use and unit costs were identified from published sources and the dhs in brazil . in brazil", "the public national health system ( sus ) pays for around 70%75% of all reproductive procedures .", "however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately .", "costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system .", "costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage .", "the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 .", "a model was developed to evaluate the humanistic burden and financial impact of ups in brazil .", "the analysis factored in costs and outcomes for 1 year post - delivery of ups .", "possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth .", "all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil .", "available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries .", "complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy .", "data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used .", "a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) .", "the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% .", "the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births .", "differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy .", "all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30", "resource use and unit costs were identified from published sources and the dhs in brazil . in brazil", "the public national health system ( sus ) pays for around 70%75% of all reproductive procedures .", "however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately .", "costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system .", "costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage .", "the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 .", "based on the modeled parameters we estimate 1.79 million annual ups and 1.47 million annual planned pregnancies .", "we estimate annual maternal deaths of 351 , of which 49 ( 14% ) are attributed to abortions and 302 to complications from miscarriages and deliveries .", "the number of infant deaths within the 12 months following birth was estimated at 32,864 .", "the number of estimated neonatal admissions for the year 2010 associated with up was 224,631 which included all preterm deliveries and 7.6% of all term deliveries .", "the disaggregated values for different pregnancy outcomes attributed to up are described in table 2 .", "costs of abortions are not reimbursed in the public health system and are not included in the analysis .", "the total costs attributed to up are estimated to be r$4.1 billion annually , of which approximately r$32.9 million ( 0.8% ) was attributed to miscarriage and r$4.07 billion ( 99.2% ) to births and resulting complications . from the direct birth - related costs , antenatal care accounted for approximately r$233 million ( 5.7% ) of birth costs , with labor and delivery costs responsible for r$988 million ( 24.3% ) .", "the remainder of birth costs were attributed to infant complications , estimated to be r$2.8 billion ( 70.0% ) , with neonatal costs accountable for approximately r$628 million ( 15.4% ) and hospital readmission in the first year costs of r$2.1 billion ( 52.9% ) of all birth costs annually . based on the national cost estimates and the number of annual ups", "our analysis estimates the costs and outcomes associated with unintended pregnancies to derive a cost per up of r$2,293 .", "the cost per up factors in a range of health - related costs attributed to those resulting in abortion and resulting live births for those carried to parturition .", "this figure is broadly aligned with previous analyses in the united states of $ 1,609 per up after factoring in the purchasing power parity between the two countries.35 it is also worth noting that the estimates provided here are likely an underestimate of all costs .", "for example , the analysis described here focused on the costs that occur within the public health systems .", "consequently , costs of elective abortions paid for by individuals are not represented in this analysis .", "furthermore , because elective abortion is illegal in brazil except in extreme and rare cases , hidden costs from elective abortions may filter into the public health system .", "for instance , costs associated with post - abortive care are likely to be classified as miscarriages when reported to national authorities . additionally , the long - term societal costs that arise from up and reduced educational attainment and lost productivity of young mothers has not been accounted for in our analysis.3639 our analysis highlights that considerable cost savings can be achieved by reducing up , which are thought to represent 55% of all pregnancies in brazil.20 the analysis accounted for both untimed and unwanted births to estimate the up rate and associated costs , in which live births resulting from unwanted pregnancies represented the largest share of health costs , representing 99% of all costs .", "this is also attributed to the fact that abortion is a relatively inexpensive procedure that is paid for outside of the health service . because a substantial proportion of pregnancies are attributed to mistimed pregnancies , ie , pregnancies that would have occurred at some point in the future , but occurred", "sooner due to mistimed pregnancy , the actual cost savings to be realized by reducing up is less than described here and would mostly be associated with averting those pregnancies considered to be unwanted . to put the potential cost savings into perspective", ", an analysis conducted for the state of california , usa estimated that preventing many up in a single year will offer savings of $ 1.1 billion up to 2 years of age of the child , and that every dollar spent on averting up offers $ 2.76 of saving in 2 years and $ 5.33 at 5 years post - delivery.15 a national - level analysis that focused on the federally financed medicaid program in the united states noted that taxpayer savings of $ 4.7$6.2 billion could be achieved by reducing unintended pregnancies.40 based on the cost savings attributed to up , it is anticipated that future health researchers can build on the research described here to better understand the cost savings that may be achieved through improved contraceptive use .", "the analysis sought to incorporate a broad range of costs associated with up to illustrate that costs extend beyond the point of delivery .", "for instance , ups often occur in adolescent and young adults and can be predictive of low birth weight , preterm birth , and neonatal admission.8,4143 the precise mechanism by which up influences preterm birth is likely to be multifactorial .", "however , some studies have observed that women with ups are less likely to receive adequate prenatal care44 and are also more likely to smoke and drink alcohol during pregnancy , which contributes to adverse outcomes.44,45 it is important to highlight these costs because it suggests that ups place unnecessary demand on health services that are often stretched to capacity . since up , for the most part , can be avoided , this illustrates the health service benefits and health service capacity that could be released by reducing up .", "much of the humanistic burden attributed to up relates to maternal mortality and morbidity of women who pursue unsafe abortions . in our analysis", ", we estimate 49 annual deaths from abortions representing 13% of the estimated maternal deaths .", "it has been suggested that access to improved contraceptive services could prevent maternal mortality by 25%35%.46 furthermore , estimates from the world health organization reported that unsafe abortion disables approximately 5 million women each year , suggesting reductions in morbidity could also be achieved through improved contraception use.4 empirical evidence and clinical guidelines agree that the most effective approach to preventing up is through education and contraceptive use , of which long - acting contraceptive ( larc ) methods are the most effective intervention.4751 in particular , among adolescents , prevailing evidence suggests that education and contraception are the main interventions for reducing up.49 in advanced economies , the percentage of women using larc methods has been steadily increasing .", "a recent study noted that the proportion of women using larc methods has increased from 2.4% of women aged 1544 years since 2002 to 8.5% of women in 2009.52 in the united states , the american college of obstetricians and gynecologists ( acog ) advocates use of intrauterine contraceptives ( both copper - intrauterine device and the levonorgestrel - releasing intrauterine system ) and implants as first - line therapy for adolescent women because they contribute to reduce the number of ups.48 larc has also proven to be cost - saving compared with combined oral contraceptives in the united kingdom .", "investigators reported that this finding was influenced almost entirely by the lower failure rates associated with larc.50,53 an analysis conducted in the united states also noted that cost savings could be achieved by switching women from oral contraceptives to larcs , and approximately 50% of costs of up were attributed to contraceptive adherence.54 despite the positive benefits of larc , use in brazil lags behind that of other countries . a survey conducted in 2008 in northern brazil reported that only 1% of women attending a post - abortion clinic elected for an intrauterine device , despite 93% of women having knowledge of the product.55 several barriers have been identified , which often limit uptake of larc methods.56 these include health care provider lack of knowledge of risks , myths , misconceptions , and lack of training for insertion of the various devices .", "also , misinformation and fear of pain at insertion among women can limit demand for larc methods .", "furthermore , larc can often involve considerable up - front expense , although evidence does suggest that these methods are cost - effective compared with alternative contraceptive methods .", "because of the expense , reimbursement can influence the rate of uptake . even recognizing the importance of abortion , and especially induced and unsafe abortion , for maternal mortality and morbidity in brazil , there are no reliable and available data confirming a pandemic of such conditions as commonly reported by media and gray literature .", "data extracted from the 2006 brazilian dhs indicated that only 1.5% self - reported induced abortions among all pregnancies for the whole period of the survey.14,19 a probable contribution for this scenario is the high prevalence of and easy access to contraceptive use among women in reproductive age in the country during the last 2 decades .", "although all induced abortions are technically classified as unsafe , this is not the impression of the majority of obstetricians working in the field .", "there is a general belief that complications due to unsafe induced abortions are less and less frequent and that there was recently a marked drop in their occurrence . with no direct information to confirm this , one indirect point of evidence is the decrease in the proportional contribution of abortion as cause of maternal mortality in the country .", "in fact , official brazilian data confirm that it was 16.4% in 1990 ( representing the third cause of maternal mortality ) , while only 8.9% in 2000 and 9.0% in 2010 ( the fifth cause , behind even the indirect obstetric causes).20 in the meantime , there was a spread of the use of misoprostol as an agent for inducing abortion , not only in health facilities for situations where abortion is allowed , but mainly for situations not legally permitted , wherein the women obtain misoprostol in the black market and self - administer it vaginally .", "this is believed to be the current most common way of inducing medical abortion and also to be associated with the low rates of severe complications due to abortion , especially infection and hemorrhage , in brazil .", "there are also at least two possible limitations for the external validity of the current cost estimates .", "the first refers to the assumption that all miscarriages are really diagnosed and  treated  with dilation and curettage .", "however , it is possible that a proportion of individuals will never reach health facilities and receive no intervention .", "currently , in brazil , the majority of medical abortions are performed by the woman through self - administration of misoprostol acquired off - label or in the black market . with these procedures ,", "the rate of complications due to medical abortions has dropped significantly during the last decade , with some of these cases arriving at hospitals already as complete abortions that need no complementary procedures .", "although insufficient data are available to support this scenario , this would probably impact strongly on the estimates of post - abortion care , taking into account the correspondent reduced morbidity and mortality .", "the cost consequences of live births attributed to up in adolescents can extend beyond the observed costs of increased prenatal care , preterm births , and abortions .", "studies have noted that adolescent women are more likely to dropout from school following pregnancy .", "furthermore , others have suggested that future generations will follow the reproductive pattern of their parents , which could perpetuate the likelihood of up in the offspring of these mothers.57 as reported , education has been shown to break the association of adolescent fertility across generations , suggesting the longer - term benefits to be achieved by reducing up .", "our analysis illustrates the cost consequences associated with up in brazil . in the past 2 decades , brazil has made considerable progress toward reducing abortion rates , but , despite these efforts , the humanistic and direct economic costs associated with up remain high.58 to achieve economic savings from reducing the up rate will require education , training of providers , and improved access to effective contraceptive measures .", "it is believed that the research described herein can inform the burden of up and the benefits of improved fertility planning .", "the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report .", "it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries .", "the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report .", "it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries ."], "abstract_text": ["<S> backgroundunintended pregnancy ( up ) is an unmet medical need with consequences worldwide . </S>", "<S> we evaluate the costs of up based on pregnancies in brazil from for the year 2010.methodsthe consequences of up were evaluated using decision analysis based on pregnancy rates and outcomes as miscarriage , induced abortion , and live birth , which were factored into the analysis . the model discriminated between maternal and child outcomes and accounted for costs ( in brazilian currency [ real$ , r$ ] ) within the brazilian public health service attributed to preterm birth , neonatal admission , cerebral palsy , and neonatal and maternal mortality . </S>", "<S> event probabilities were obtained from local resources.resultswe estimate that 1.8 million ups resulted in 159,151 miscarriages , 48,769 induced abortions , 1.58 million live births , and 312 maternal deaths , including ten ( 3% ) attributed to unsafe abortions . </S>", "<S> the total estimated costs attributed to up are r$4.1 billion annually , including r$32 million ( 0.8% ) and r$4.07 billion ( 99.2% ) attributed to miscarriages and births and complications , respectively . </S>", "<S> direct birth costs accounted for approximately r$1.22 billion ( 30.0% ) , with labor and delivery responsible for most costs ( r$988 million ; 24.3% ) for the year 2010 . </S>", "<S> the remainder of costs were for infant complications ( r$2.84 billion ; 72.3% ) with hospital readmission during the first year accounting for approximately r$2.15 billion ( 52.9% ) . </S>", "<S> based on the national cost , we estimate the cost per up to be r$2,293.conclusiondespite weaknesses in precise estimates in annual pregnancies and induced abortions , our estimates reflect the costs of up for different pregnancy outcomes . </S>", "<S> the main costs associated with up are in those carried to parturition . </S>", "<S> the health cost of abortion represents a small proportion of total costs as these are paid for outside of the public health system . </S>", "<S> consequently , reductions in up will generate not only cost savings , but reductions in woman and child morbidity and mortality . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Model description", "Data source and analysis", "Cost resources", "Results", "Discussion", "Conclusion", "Implications"], "sections": [["the construct of unintended pregnancies ( ups ) is multifactorial and broadly encompasses pregnancies that are either unwanted or mistimed.1 in many instances , ups are likely to end by induced abortion ; worldwide estimates suggest that 50% will be voluntarily terminated.2 in 2008 , it was estimated that 43.8 million abortions occurred worldwide , of which 86% occurred in low-/middle - income countries.3 furthermore , between 2003 and 2008 , the number of induced abortions was found to have decreased in high - income countries but to have increased in low-/middle - income countries . during this same period of time , the proportion of unsafe abortions increased and they were believed to account for 13% of maternal deaths , with the majority of these concentrated in countries with restrictive laws on abortion.4 elective abortion in brazil is considered non - legal , unless the pregnancy resulted from a rape , would cause a life - threatening condition to the mother , or the fetus has anencephaly or any other malformation that is incompatible with the extrauterine life . as a result ,", "data on prevalence and associated costs of abortion are limited and remain a challenge to collect .", "however , it is recognized worldwide that direct health costs of up and resulting abortions can significantly impact local health services and the families affected.5 health system and societal costs can also arise from ups that do not end in abortion and are carried to parturition . in the united states ,", "the public health costs attributed to unintended births in a single year cost taxpayers $ 11.1 billion ( 2006).6 furthermore , previous studies have reported that ups are more likely to result in preterm births and low - birth - weight babies , which would increase health costs for neonatal care and costs associated with long - term disabilities.7 this is particularly relevant for ups in adolescence , which are more likely to result in low - birth - weight births , which can increase health costs.8 over the past few decades , brazil , like many fast - emerging economies , has experienced a fertility transition noted by a dramatic reduction in the total fertility rate , which currently sits at 1.8 births per woman.9,10 furthermore , during the fertility transition , the rate of contraception has also increased.11 despite increased contraception use , the cumulative rate of spontaneous and induced abortion changed very little between 1996 and 2006 , for which population survey estimates are available.12,13 furthermore , a survey of more than 1,000 women aged 1024 years in a single postnatal unit in brazil reported that more than 50% of all births were unintended , suggesting that possibly significant unmet contraception need still exists.8 similarly , for the whole country , the data from the last demographic and health survey ( dhs ) performed in 2006 indicates that 55% of all births were unintended.14 prevention of up using publicly funded programs in high - income countries has proven to generate significant cost savings for health services and public services.15 previous economic studies evaluating the consequences of up have mostly been performed in high - income countries where publicly funded fertility services and abortion legislation are different from those in low-/middle - income countries.15 the aim of this study was to evaluate the cost consequences of up in brazil , an emerging economy with vastly different fertility planning services than most advanced economies and with restrictive abortion laws . to the best of our knowledge ,", "this is the first published study that describes the economics of up , and it is believed that this work can aid decision makers regarding access to contraceptives ."], ["a model was developed to evaluate the humanistic burden and financial impact of ups in brazil .", "the analysis factored in costs and outcomes for 1 year post - delivery of ups .", "possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth .", "all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil .", "available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries .", "complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy .", "data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used .", "a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) .", "the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% .", "the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births .", "differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy .", "all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30 resource use and unit costs were identified from published sources and the dhs in brazil . in brazil", "the public national health system ( sus ) pays for around 70%75% of all reproductive procedures .", "however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately .", "costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system .", "costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage .", "the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 ."], ["a model was developed to evaluate the humanistic burden and financial impact of ups in brazil .", "the analysis factored in costs and outcomes for 1 year post - delivery of ups .", "possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth .", "all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil .", "available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries .", "complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy .", "data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used .", "a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) ."], ["the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% .", "the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births .", "differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy .", "all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30"], ["resource use and unit costs were identified from published sources and the dhs in brazil . in brazil", "the public national health system ( sus ) pays for around 70%75% of all reproductive procedures .", "however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately .", "costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system .", "costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage .", "the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 ."], ["based on the modeled parameters we estimate 1.79 million annual ups and 1.47 million annual planned pregnancies .", "we estimate annual maternal deaths of 351 , of which 49 ( 14% ) are attributed to abortions and 302 to complications from miscarriages and deliveries .", "the number of infant deaths within the 12 months following birth was estimated at 32,864 .", "the number of estimated neonatal admissions for the year 2010 associated with up was 224,631 which included all preterm deliveries and 7.6% of all term deliveries .", "the disaggregated values for different pregnancy outcomes attributed to up are described in table 2 .", "costs of abortions are not reimbursed in the public health system and are not included in the analysis .", "the total costs attributed to up are estimated to be r$4.1 billion annually , of which approximately r$32.9 million ( 0.8% ) was attributed to miscarriage and r$4.07 billion ( 99.2% ) to births and resulting complications . from the direct birth - related costs , antenatal care accounted for approximately r$233 million ( 5.7% ) of birth costs , with labor and delivery costs responsible for r$988 million ( 24.3% ) .", "the remainder of birth costs were attributed to infant complications , estimated to be r$2.8 billion ( 70.0% ) , with neonatal costs accountable for approximately r$628 million ( 15.4% ) and hospital readmission in the first year costs of r$2.1 billion ( 52.9% ) of all birth costs annually . based on the national cost estimates and the number of annual ups"], ["our analysis estimates the costs and outcomes associated with unintended pregnancies to derive a cost per up of r$2,293 .", "the cost per up factors in a range of health - related costs attributed to those resulting in abortion and resulting live births for those carried to parturition .", "this figure is broadly aligned with previous analyses in the united states of $ 1,609 per up after factoring in the purchasing power parity between the two countries.35 it is also worth noting that the estimates provided here are likely an underestimate of all costs .", "for example , the analysis described here focused on the costs that occur within the public health systems .", "consequently , costs of elective abortions paid for by individuals are not represented in this analysis .", "furthermore , because elective abortion is illegal in brazil except in extreme and rare cases , hidden costs from elective abortions may filter into the public health system .", "for instance , costs associated with post - abortive care are likely to be classified as miscarriages when reported to national authorities . additionally , the long - term societal costs that arise from up and reduced educational attainment and lost productivity of young mothers has not been accounted for in our analysis.3639 our analysis highlights that considerable cost savings can be achieved by reducing up , which are thought to represent 55% of all pregnancies in brazil.20 the analysis accounted for both untimed and unwanted births to estimate the up rate and associated costs , in which live births resulting from unwanted pregnancies represented the largest share of health costs , representing 99% of all costs .", "this is also attributed to the fact that abortion is a relatively inexpensive procedure that is paid for outside of the health service . because a substantial proportion of pregnancies are attributed to mistimed pregnancies , ie , pregnancies that would have occurred at some point in the future , but occurred", "sooner due to mistimed pregnancy , the actual cost savings to be realized by reducing up is less than described here and would mostly be associated with averting those pregnancies considered to be unwanted . to put the potential cost savings into perspective", ", an analysis conducted for the state of california , usa estimated that preventing many up in a single year will offer savings of $ 1.1 billion up to 2 years of age of the child , and that every dollar spent on averting up offers $ 2.76 of saving in 2 years and $ 5.33 at 5 years post - delivery.15 a national - level analysis that focused on the federally financed medicaid program in the united states noted that taxpayer savings of $ 4.7$6.2 billion could be achieved by reducing unintended pregnancies.40 based on the cost savings attributed to up , it is anticipated that future health researchers can build on the research described here to better understand the cost savings that may be achieved through improved contraceptive use .", "the analysis sought to incorporate a broad range of costs associated with up to illustrate that costs extend beyond the point of delivery .", "for instance , ups often occur in adolescent and young adults and can be predictive of low birth weight , preterm birth , and neonatal admission.8,4143 the precise mechanism by which up influences preterm birth is likely to be multifactorial .", "however , some studies have observed that women with ups are less likely to receive adequate prenatal care44 and are also more likely to smoke and drink alcohol during pregnancy , which contributes to adverse outcomes.44,45 it is important to highlight these costs because it suggests that ups place unnecessary demand on health services that are often stretched to capacity . since up , for the most part , can be avoided , this illustrates the health service benefits and health service capacity that could be released by reducing up .", "much of the humanistic burden attributed to up relates to maternal mortality and morbidity of women who pursue unsafe abortions . in our analysis", ", we estimate 49 annual deaths from abortions representing 13% of the estimated maternal deaths .", "it has been suggested that access to improved contraceptive services could prevent maternal mortality by 25%35%.46 furthermore , estimates from the world health organization reported that unsafe abortion disables approximately 5 million women each year , suggesting reductions in morbidity could also be achieved through improved contraception use.4 empirical evidence and clinical guidelines agree that the most effective approach to preventing up is through education and contraceptive use , of which long - acting contraceptive ( larc ) methods are the most effective intervention.4751 in particular , among adolescents , prevailing evidence suggests that education and contraception are the main interventions for reducing up.49 in advanced economies , the percentage of women using larc methods has been steadily increasing .", "a recent study noted that the proportion of women using larc methods has increased from 2.4% of women aged 1544 years since 2002 to 8.5% of women in 2009.52 in the united states , the american college of obstetricians and gynecologists ( acog ) advocates use of intrauterine contraceptives ( both copper - intrauterine device and the levonorgestrel - releasing intrauterine system ) and implants as first - line therapy for adolescent women because they contribute to reduce the number of ups.48 larc has also proven to be cost - saving compared with combined oral contraceptives in the united kingdom .", "investigators reported that this finding was influenced almost entirely by the lower failure rates associated with larc.50,53 an analysis conducted in the united states also noted that cost savings could be achieved by switching women from oral contraceptives to larcs , and approximately 50% of costs of up were attributed to contraceptive adherence.54 despite the positive benefits of larc , use in brazil lags behind that of other countries . a survey conducted in 2008 in northern brazil reported that only 1% of women attending a post - abortion clinic elected for an intrauterine device , despite 93% of women having knowledge of the product.55 several barriers have been identified , which often limit uptake of larc methods.56 these include health care provider lack of knowledge of risks , myths , misconceptions , and lack of training for insertion of the various devices .", "also , misinformation and fear of pain at insertion among women can limit demand for larc methods .", "furthermore , larc can often involve considerable up - front expense , although evidence does suggest that these methods are cost - effective compared with alternative contraceptive methods .", "because of the expense , reimbursement can influence the rate of uptake . even recognizing the importance of abortion , and especially induced and unsafe abortion , for maternal mortality and morbidity in brazil , there are no reliable and available data confirming a pandemic of such conditions as commonly reported by media and gray literature .", "data extracted from the 2006 brazilian dhs indicated that only 1.5% self - reported induced abortions among all pregnancies for the whole period of the survey.14,19 a probable contribution for this scenario is the high prevalence of and easy access to contraceptive use among women in reproductive age in the country during the last 2 decades .", "although all induced abortions are technically classified as unsafe , this is not the impression of the majority of obstetricians working in the field .", "there is a general belief that complications due to unsafe induced abortions are less and less frequent and that there was recently a marked drop in their occurrence . with no direct information to confirm this , one indirect point of evidence is the decrease in the proportional contribution of abortion as cause of maternal mortality in the country .", "in fact , official brazilian data confirm that it was 16.4% in 1990 ( representing the third cause of maternal mortality ) , while only 8.9% in 2000 and 9.0% in 2010 ( the fifth cause , behind even the indirect obstetric causes).20 in the meantime , there was a spread of the use of misoprostol as an agent for inducing abortion , not only in health facilities for situations where abortion is allowed , but mainly for situations not legally permitted , wherein the women obtain misoprostol in the black market and self - administer it vaginally .", "this is believed to be the current most common way of inducing medical abortion and also to be associated with the low rates of severe complications due to abortion , especially infection and hemorrhage , in brazil .", "there are also at least two possible limitations for the external validity of the current cost estimates .", "the first refers to the assumption that all miscarriages are really diagnosed and  treated  with dilation and curettage .", "however , it is possible that a proportion of individuals will never reach health facilities and receive no intervention .", "currently , in brazil , the majority of medical abortions are performed by the woman through self - administration of misoprostol acquired off - label or in the black market . with these procedures ,", "the rate of complications due to medical abortions has dropped significantly during the last decade , with some of these cases arriving at hospitals already as complete abortions that need no complementary procedures .", "although insufficient data are available to support this scenario , this would probably impact strongly on the estimates of post - abortion care , taking into account the correspondent reduced morbidity and mortality .", "the cost consequences of live births attributed to up in adolescents can extend beyond the observed costs of increased prenatal care , preterm births , and abortions .", "studies have noted that adolescent women are more likely to dropout from school following pregnancy .", "furthermore , others have suggested that future generations will follow the reproductive pattern of their parents , which could perpetuate the likelihood of up in the offspring of these mothers.57 as reported , education has been shown to break the association of adolescent fertility across generations , suggesting the longer - term benefits to be achieved by reducing up ."], ["our analysis illustrates the cost consequences associated with up in brazil . in the past 2 decades , brazil has made considerable progress toward reducing abortion rates , but , despite these efforts , the humanistic and direct economic costs associated with up remain high.58 to achieve economic savings from reducing the up rate will require education , training of providers , and improved access to effective contraceptive measures .", "it is believed that the research described herein can inform the burden of up and the benefits of improved fertility planning .", "the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report .", "it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries ."], ["the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report .", "it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries ."]]}
{"article_id": "PMC4276263", "article_text": ["pmds is a rare form of male pseudo - hermaphroditism characterized by the presence of mullerian duct structures in an otherwise phenotypically , as well as genotypically , normal man .", "it is characterized by the persistence of the uterus , fallopian tubes and upper vagina in otherwise normally virilized boys . despite the normal male genotype ( 46 xy ) and the subsequent normal development of fetal testes , mllerian structures do not regress either due to absence of mllerian inhibiting substance ( mis ) or lack of response to it .", "the persistence of a large uterus - like paramesonephric duct in a man is in itself clinically unusual , but when it forms a part of the contents of a hernial sac , it must be considered a rarity .", "we report the case of a 70 year old man with unilateral cryptorchidism on the right side and left obstructed inguinal hernia containing uterus and fallopian tube ( that is , hernia uteri inguinalis ; type i male form of pmds ) coincidentally detected during an operation for an obstructed left inguinal hernia with right cryptorchidism .", "a 70 year old man presented to our hospital with a painful left - sided inguinal swelling of one day duration . the patient gave history of asymptomatic left inguinal swelling from past 20 years and absence of the right testis since birth .", "he had been married for fifty years and was having 4 children , the youngest one being 38 year old female .", "general physical examination revealed a man of sub - average built with well developed secondary sexual characters . his urethra and penis", "were fully developed with a poorly developed right hemi - scrotum and no palpable right testis in the scrotum or inguinal canal .", "there was a non - reducible , tender swelling measuring approximately 10    8  cm in the left inguinal region with absent cough impulse .", "exploration of the inguinal canal revealed an indirect inguinal hernia containing a globular structure resembling uterus , fallopian tubes with an atrophic right testis embedded in the broad ligament and attached to pelvis with a thick fibrous band [ figs . 1 and 2 ] .", "total excision of the uterus with fallopian tubes and atrophic right testis was performed and the operation was completed with left inguinal hernioplasty [ figs . 3 and 4 ] .", "grossly , the specimens removed were identified as a uterus with patent endometrial and endocervical linings and two fallopian tubes .", "the right testis measuring 2    1    1  cm , was atrophic and embedded in the right broad ligament .", "the specimen was sent for histopathological examination which revealed uterine muscular tissue with its cavity lined by endometrial tissue and congested fallopian tubes .", "male pseudo - hermaphroditism is a condition in which the gonads are testes but the internal genitalia are not completely virilized .", "pmds is a rare form of internal male pseudo - hermaphroditism in which mullerian duct derivatives are seen in men .", "the exact cause of pmds is not known , however it is thought to result from a defect of the synthesis or release of mif , or from defects in the mif receptor .", "defects in the mif gene lead to the persistence of a uterus and fallopian tube in males .", "it is likely that remnant mullerian structures lead to cryptorchidism by hindering the normal testicular descent mechanism .", "patients with pmds usually have normal development of external genitalia and secondary sexual characteristics . the typical patient with pmds", "has unilateral or bilateral cryptorchidism and is assigned to the male sex at birth without hesitation , as they have normal male genotypes and phenotypes .", "the male form is encountered in 8090% of cases , characterized by unilateral cryptorchidism with contralateral inguinal hernia , and can be one of the two types : the first type is hernia uteri inguinalis , which is characterized by one descended testis and herniation of the ipsilateral corner of uterus and fallopian tube into the inguinal canal .", "the second type is crossed testicular ectopia , which is characterized by herniation of both testes and the entire uterus with both fallopian tubes .", "clinically , the persistence of a uterus and fallopian tubes leads to either cryptorchidism or inguinal hernia depending on whether or not mullerian derivatives can be mobilized during testicular descent .", "if the uterus and fallopian tube are mobile , they may descend into the inguinal canal during testicular descent .", "pmds is usually coincidently detected during surgical operation , as in our patient 's case .", "however pre - operative ultrasonography , computerized tomography and mri allow possible pre - operative diagnosis .", "an element of deciding on intervention is largely based on reducing risk of malignancy , while maintaining maximum reproductive function . in pmds patients with undescended testes ,", "the rate of testicular cancer is about 12% which is comparable to the rate seen in undescended testes not associated with pmds .", "several authors have asserted that the mullerian structures should not be removed as there is minimal risk associated with their retention , and excision of the mullerian structures risks damaging primary blood supply to the pmds testis via the internal spermatic and deferential arteries .", "however most recent reports have demonstrated rare malignancies including adenocarcinomas of the mullerian duct associated with retained mullerian structures .", "pmds is a rare form of male pseudo - hermaphroditism characterized by the presence of mullerian duct structures in an otherwise phenotypically , as well as genotypically , normal man .", "since patients are phenotypically male , the diagnosis is usually not suspected until surgery is performed for cryptorchidism or hernia repair .", "hernia uteri inguinalis is type i of the male form of pmds , characterized by one descended testis and the herniation of the ipsilateral corner of the uterus and fallopian tube into the inguinal canal . in order to prevent further complications such as infertility and malignant change", ", the surgeon should be aware of pmds while dealing with patients who present with unilateral or bilateral cryptorchidism . in summary , in cases of unilateral or bilateral cryptorchidism associated with hernia , as in our patient 's case ,", "a written informed consent was obtained from the patient for publication of the case report and various images which may be used with the same .", "the patient being a major gave a written consent of the same after assuring him that name and hospital mrd no of the patient will not appear in the case report .", "all the authors have contributed either as being part of surgical and anaesthesia team or helping in the photography , paper writing , submission etc . afak yusuf sherwani , abdul qayoom shah , abdul majeed wani , farooq ahmad sofi and ashfaq amin wani were in the surgical team .", "bashir a chalkoo , bashir a khan and ab hamid sherwani in the anaesthesia team .", "wasim lone , mehmood a sheikh and raj reshi sharma did photography and paper writing ."], "abstract_text": ["<S> introductionpersistent mullerian duct syndrome is a rare form of male pseudo - hermaphroditism characterized by the presence of mullerian duct structures in an otherwise phenotypically , as well as genotypically , normal man ; only a few cases have been reported in the worldwide literature . </S>", "<S> a great variety of organs have been found in indirect inguinal hernial sacs.presentation of casewe report a case of 70 year old man , father of 4 children with unilateral cryptorchidism on the right side and left - sided obstructed inguinal hernia containing uterus and fallopian tube ( that is , hernia uteri inguinalis ; type i male form of persistent mullerian duct syndrome ) coincidentally detected during an operation for an obstructed left inguinal hernia.discussionpmds is usually coincidently detected during surgical operation , as was in our case . </S>", "<S> however pre - operative ultrasonography , computerized tomography and mri allow possible pre - operative diagnosis.3conclusionin cases of unilateral or bilateral cryptorchidism associated with hernia , as in our patient 's case , the possibility of pmds should be kept in mind . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion", "Conclusion", "Conflict of interest", "Funding", "Ethical approval", "Author contributions"], "sections": [["pmds is a rare form of male pseudo - hermaphroditism characterized by the presence of mullerian duct structures in an otherwise phenotypically , as well as genotypically , normal man .", "it is characterized by the persistence of the uterus , fallopian tubes and upper vagina in otherwise normally virilized boys . despite the normal male genotype ( 46 xy ) and the subsequent normal development of fetal testes , mllerian structures do not regress either due to absence of mllerian inhibiting substance ( mis ) or lack of response to it .", "the persistence of a large uterus - like paramesonephric duct in a man is in itself clinically unusual , but when it forms a part of the contents of a hernial sac , it must be considered a rarity .", "we report the case of a 70 year old man with unilateral cryptorchidism on the right side and left obstructed inguinal hernia containing uterus and fallopian tube ( that is , hernia uteri inguinalis ; type i male form of pmds ) coincidentally detected during an operation for an obstructed left inguinal hernia with right cryptorchidism ."], ["a 70 year old man presented to our hospital with a painful left - sided inguinal swelling of one day duration . the patient gave history of asymptomatic left inguinal swelling from past 20 years and absence of the right testis since birth .", "he had been married for fifty years and was having 4 children , the youngest one being 38 year old female .", "general physical examination revealed a man of sub - average built with well developed secondary sexual characters . his urethra and penis", "were fully developed with a poorly developed right hemi - scrotum and no palpable right testis in the scrotum or inguinal canal .", "there was a non - reducible , tender swelling measuring approximately 10    8  cm in the left inguinal region with absent cough impulse .", "exploration of the inguinal canal revealed an indirect inguinal hernia containing a globular structure resembling uterus , fallopian tubes with an atrophic right testis embedded in the broad ligament and attached to pelvis with a thick fibrous band [ figs . 1 and 2 ] .", "total excision of the uterus with fallopian tubes and atrophic right testis was performed and the operation was completed with left inguinal hernioplasty [ figs . 3 and 4 ] .", "grossly , the specimens removed were identified as a uterus with patent endometrial and endocervical linings and two fallopian tubes .", "the right testis measuring 2    1    1  cm , was atrophic and embedded in the right broad ligament .", "the specimen was sent for histopathological examination which revealed uterine muscular tissue with its cavity lined by endometrial tissue and congested fallopian tubes ."], ["male pseudo - hermaphroditism is a condition in which the gonads are testes but the internal genitalia are not completely virilized .", "pmds is a rare form of internal male pseudo - hermaphroditism in which mullerian duct derivatives are seen in men .", "the exact cause of pmds is not known , however it is thought to result from a defect of the synthesis or release of mif , or from defects in the mif receptor .", "defects in the mif gene lead to the persistence of a uterus and fallopian tube in males .", "it is likely that remnant mullerian structures lead to cryptorchidism by hindering the normal testicular descent mechanism .", "patients with pmds usually have normal development of external genitalia and secondary sexual characteristics . the typical patient with pmds", "has unilateral or bilateral cryptorchidism and is assigned to the male sex at birth without hesitation , as they have normal male genotypes and phenotypes .", "the male form is encountered in 8090% of cases , characterized by unilateral cryptorchidism with contralateral inguinal hernia , and can be one of the two types : the first type is hernia uteri inguinalis , which is characterized by one descended testis and herniation of the ipsilateral corner of uterus and fallopian tube into the inguinal canal .", "the second type is crossed testicular ectopia , which is characterized by herniation of both testes and the entire uterus with both fallopian tubes .", "clinically , the persistence of a uterus and fallopian tubes leads to either cryptorchidism or inguinal hernia depending on whether or not mullerian derivatives can be mobilized during testicular descent .", "if the uterus and fallopian tube are mobile , they may descend into the inguinal canal during testicular descent .", "pmds is usually coincidently detected during surgical operation , as in our patient 's case .", "however pre - operative ultrasonography , computerized tomography and mri allow possible pre - operative diagnosis .", "an element of deciding on intervention is largely based on reducing risk of malignancy , while maintaining maximum reproductive function . in pmds patients with undescended testes ,", "the rate of testicular cancer is about 12% which is comparable to the rate seen in undescended testes not associated with pmds .", "several authors have asserted that the mullerian structures should not be removed as there is minimal risk associated with their retention , and excision of the mullerian structures risks damaging primary blood supply to the pmds testis via the internal spermatic and deferential arteries .", "however most recent reports have demonstrated rare malignancies including adenocarcinomas of the mullerian duct associated with retained mullerian structures ."], ["pmds is a rare form of male pseudo - hermaphroditism characterized by the presence of mullerian duct structures in an otherwise phenotypically , as well as genotypically , normal man .", "since patients are phenotypically male , the diagnosis is usually not suspected until surgery is performed for cryptorchidism or hernia repair .", "hernia uteri inguinalis is type i of the male form of pmds , characterized by one descended testis and the herniation of the ipsilateral corner of the uterus and fallopian tube into the inguinal canal . in order to prevent further complications such as infertility and malignant change", ", the surgeon should be aware of pmds while dealing with patients who present with unilateral or bilateral cryptorchidism . in summary , in cases of unilateral or bilateral cryptorchidism associated with hernia , as in our patient 's case ,"], [""], [""], ["a written informed consent was obtained from the patient for publication of the case report and various images which may be used with the same .", "the patient being a major gave a written consent of the same after assuring him that name and hospital mrd no of the patient will not appear in the case report ."], ["all the authors have contributed either as being part of surgical and anaesthesia team or helping in the photography , paper writing , submission etc . afak yusuf sherwani , abdul qayoom shah , abdul majeed wani , farooq ahmad sofi and ashfaq amin wani were in the surgical team .", "bashir a chalkoo , bashir a khan and ab hamid sherwani in the anaesthesia team .", "wasim lone , mehmood a sheikh and raj reshi sharma did photography and paper writing ."]]}
{"article_id": "PMC4606583", "article_text": ["spider nevus ( also known as spider angioma or vascular spider ) is a common benign vascular anomaly that may appear as solitary or multiple lesions .", "spider nevi are represented by telangiectases that consist of a large arteriole from which radiate numerous small vessels that resemble spider 's legs .", "they are found in the distribution of the superior vena cava , that is , on the face , neck , arms , and upper trunk .", "spider nevi are usually < 2 cm in diameter and rarely grow to assume large size more than 2 cm . we hereby report a case of very large spider nevus in a patient with hepatitis c virus ( hcv ) related liver cirrhosis .", "a 48-year - old - female presented with a history of abdominal distension and jaundice of 3-month duration . physical examination revealed pallor , icterus , pedal edema , hepatospleenomegaly and tense ascites .", "she had multiple classical spider nevi over the chest . also , she had a large spider nevi situated on the forehead , measuring 8 cm in largest dimension with palpable dilated vessels and venous hum [ figure 1 ] .", "deep compression on the central area of the lesion produced complete disappearance with blanching and when released refilling of vessels occurred .", "laboratory tests revealed a hemoglobin level of 9.8 g / dl , a white - cell count of 4240/mm , and a platelet count of 77,000/mm .", "anti - hcv antibody for hcv was positive with hcv rna of 1.6  10 iu / ml .", "an ultrasound abdomen was suggestive of liver cirrhosis with portal hypertension ( portal vein diameter of 14 mm ) , ascites and splenomegaly .", "thus , a diagnosis of cirrhosis liver with portal hypertension was made ( ctp-9 , class b , and meld  11 ) .", "she was put on diuretics for the control of ascites but developed hepatic encephalopathy and renal dysfunction .", "diuretics were stopped and in view of refractory tense ascites , she was taken up for a transjugular intrahepatic portosystemic shunt . however , during the procedure she had massive intrahepatic bleed and in spite of intensive resuscitative measures she died 48 hours later .", "spider nevi can be seen in pregnancy , in patients with thyrotoxicosis , rheumatoid arthritis , oral contraceptive use and most commonly , liver cirrhosis .", "these frequently appear in alcoholic cirrhotic or when liver function deteriorates in patients of the chronic liver disease .", "found elevated levels of vascular endothelial growth factor and basic fibroblast growth factor in patients with liver cirrhosis , especially in those with spider nevi . in patients with nonalcoholic cirrhosis ,", "the levels of substance p are elevated which may play an important role in the pathogenesis of spider nevi by causing vasodilatation .", "most remain small in size and rarely do they assume the size as seen in our patient .", "the present case is unique because of its very large size , rare presentation over the forehead and palpable mass like vascular lesion in a patient with nonalcoholic cirrhosis .", "electrodesiccation and laser treatments under local anesthesia are effective therapeutic procedures for facial spider nevi .", "spider nevi , however , usually do not require any treatment in cirrhotic patients due to a high risk of bleeding from these vascular lesions ."], "abstract_text": ["<S> spider nevi are benign vascular lesions mostly seen in patients with decompensated liver cirrhosis . mostly , these are seen in the superior vena cava distribution and are small with pinhead size central vessel . </S>", "<S> giant spider nevus is rarely seen and hence this report . </S>"], "labels": null, "section_names": ["I", "C", "D"], "sections": [["spider nevus ( also known as spider angioma or vascular spider ) is a common benign vascular anomaly that may appear as solitary or multiple lesions .", "spider nevi are represented by telangiectases that consist of a large arteriole from which radiate numerous small vessels that resemble spider 's legs .", "they are found in the distribution of the superior vena cava , that is , on the face , neck , arms , and upper trunk .", "spider nevi are usually < 2 cm in diameter and rarely grow to assume large size more than 2 cm . we hereby report a case of very large spider nevus in a patient with hepatitis c virus ( hcv ) related liver cirrhosis ."], ["a 48-year - old - female presented with a history of abdominal distension and jaundice of 3-month duration . physical examination revealed pallor , icterus , pedal edema , hepatospleenomegaly and tense ascites .", "she had multiple classical spider nevi over the chest . also , she had a large spider nevi situated on the forehead , measuring 8 cm in largest dimension with palpable dilated vessels and venous hum [ figure 1 ] .", "deep compression on the central area of the lesion produced complete disappearance with blanching and when released refilling of vessels occurred .", "laboratory tests revealed a hemoglobin level of 9.8 g / dl , a white - cell count of 4240/mm , and a platelet count of 77,000/mm .", "anti - hcv antibody for hcv was positive with hcv rna of 1.6  10 iu / ml .", "an ultrasound abdomen was suggestive of liver cirrhosis with portal hypertension ( portal vein diameter of 14 mm ) , ascites and splenomegaly .", "thus , a diagnosis of cirrhosis liver with portal hypertension was made ( ctp-9 , class b , and meld  11 ) .", "she was put on diuretics for the control of ascites but developed hepatic encephalopathy and renal dysfunction .", "diuretics were stopped and in view of refractory tense ascites , she was taken up for a transjugular intrahepatic portosystemic shunt . however , during the procedure she had massive intrahepatic bleed and in spite of intensive resuscitative measures she died 48 hours later ."], ["spider nevi can be seen in pregnancy , in patients with thyrotoxicosis , rheumatoid arthritis , oral contraceptive use and most commonly , liver cirrhosis .", "these frequently appear in alcoholic cirrhotic or when liver function deteriorates in patients of the chronic liver disease .", "found elevated levels of vascular endothelial growth factor and basic fibroblast growth factor in patients with liver cirrhosis , especially in those with spider nevi . in patients with nonalcoholic cirrhosis ,", "the levels of substance p are elevated which may play an important role in the pathogenesis of spider nevi by causing vasodilatation .", "most remain small in size and rarely do they assume the size as seen in our patient .", "the present case is unique because of its very large size , rare presentation over the forehead and palpable mass like vascular lesion in a patient with nonalcoholic cirrhosis .", "electrodesiccation and laser treatments under local anesthesia are effective therapeutic procedures for facial spider nevi .", "spider nevi , however , usually do not require any treatment in cirrhotic patients due to a high risk of bleeding from these vascular lesions ."]]}
{"article_id": "PMC2844605", "article_text": ["primary hyperhidrosis is a common disease that is characterized by excessive sweating of face , palms , or axilla , occurring in 0.6 - 3% of a population ( 1 , 2 ) .", "since kux performed endoscopic thoracic sympathetic surgery ( 3 ) , it has been popular during the past few decades as the surgical treatment of choice for facial , palmar , and axillary hyperhidrosis ( 4 - 6 ) .", "although endoscopic thoracic sympathetic surgery offers permanent cure for hyperhidrosis , it is often accompanied by serious complications , such as compensatory hyperhidrosis . in order to decrease compensatory hyperhidrosis , endoscopic thoracic", "sympathetic surgery has attempted to reduce the extent of resection of the sympathetic nerve , but these procedures did not significantly decrease the occurrence of compensatory hyperhidrosis ( 7 ) .", "we performed sympathetic nerve reconstruction surgery using intercostal nerve in 19 patients with severe compensatory hyperhidrosis after endoscopic thoracic sympathetic surgery and evaluated the results of the procedure .", "from february 2004 to august 2007 , we performed endoscopic thoracic sympathetic surgery in 184 patients with primary hyperhidrosis . among theses", "patients , reconstruction surgery of sympathetic nerve using intercostal nerve was performed in 19 patients with severe compensatory hyperhidrosis after endoscopic thoracic sympathetic surgery .", "fourteen patients were male and 5 were female ( median age : 28 yr , range : 19 - 61 ) .", "this study was approved by the institutional review board of our hospital ( irb no : 3 - 20080021 ) .", "all procedures of reconstruction surgery were performed under general anesthesia with a single lumen endotracheal tube .", "the patients were in a semi - fowler position with their arms extended . in 18 patients except for one patient with severe pleural adhesion ,", "starting on the left side , two separate skin incisions were made along the previous thoracoscopic scars and thoracoscopic ports were placed .", "after co2 gas insufflation into the thoracic cavity with less than 10 mmhg of pressure to deflate the lung , the pleural space was inspected with 5-mm thoracoscope .", "the intercostal neurovascular bundle was dissected in lengths of 5 - 7 cm and the distal part was resected .", "after the proximal and distal part of previously operated sympathetic nerve was exposed , the nerve sheaths of proximal and distal part of exposed sympathetic nerve and harvested intercostal nerve end were removed with an electro - surgical tip cleaner ( surgisite , ethicon , gargrave , skipton , uk ) .", "the intercostal nerve end was placed between the proximal and distal part of the exposed sympathetic nerve and the fibrin glue was applied to contact surface of the sympathetic and intercostal nerve ( fig .", "the same procedure was repeated on the right side . in cases of sympathetic nerve clipping", ", this procedure was performed after clip removal . in one patient with severe pleural adhesion", "we reviewed the clinical charts of all patients who underwent sympathetic nerve reconstruction surgery using the intercostal nerve .", "patients were followed by telephone questionnaire on the effects of the surgery and postoperative complications .", "the degree of improvement of compensatory hyperhidrosis was graded as \" definite \" , \" mild \" , or \" absent \" .", "\" definite \" means that patients felt fully satisfied after the reconstruction surgery , \" mild \" means that patients felt satisfied to a certain extent , and \" absent \" means that patients felt no improvement .", "median interval between the sympathetic nerve reconstruction surgery and questionnaire was 22 ( range : 1 - 45 ) months .", "in one patient ( patient number 17 in table 1 ) , digital infrared thermographic imaging was performed preoperatively and postoperatively .", "primary hyperhidrosis patients were composed of 9 facial , 8 palmar , and 1 axillary , and one patient had both facial and palmar hyperhidrosis .", "initial endoscopic thoracic sympathetic surgery for primary hyperhidrosis was t3 sympathicotomy in 5 patients ; t2 sympathicotomy in 8 ; t2 , 3 sympathicotomy in 1 ; t2 , 3 , 4 sympathicotomy in 1 ; t2 clipping in 2 ; t2 , 3 clipping in 1 ; and t2 clipping with t3 sympathicotomy in 1 patient .", "the median interval between the first endoscopic thoracic sympathetic surgery for primary hyperhidrosis and sympathetic nerve reconstruction surgery was 47 ( range , 4 - 111 ) months .", "r3 intercostal nerve was used for sympathetic nerve reconstruction surgery in 15 patients , r4 intercostal nerve in 2 , r2 intercostal nerve in 1 , and r3 with r5 intercostal nerve in 1 patient .", "three patients replied that the effects of reconstruction were \" definite \" , 6 responded with \" mild \" , and 8 said \" absent \" .", "postoperative complications were numbness of the chest wall in 2 patients , chest wall pain in 2 patients , and temporary ptosis in 1 patient in whom ptosis spontaneously resolved after 3 months .", "preoperative and postoperative digital infrared thermographic imagings performed in one patient showed the thermal change in chest and back .", "since kux advocated thoracoscopic sympathetic surgery ( 3 ) , recent developments in thoracoscopy and specialized instruments have facilitated the procedure . and , it became the treatment of choice for hyperhidrosis because of its low morbidity , short hospital stay , and excellent cosmetic results ( 6 ) .", "although it has many advantages , some patients suffer from compensatory hyperhidrosis , which is by far the most common and disagreeable complication after endoscopic thoracic sympathetic surgery . according to the previous reports", ", compensatory hyperhidrosis occurs in 59.8 - 90% of patients after sympathetic surgery ( 8 - 10 ) .", "the mechanism of compensatory hyperhidrosis is not clear , but it seems to be associated with compensation for thermoregulatory function ( 11 , 12 ) . because the incidence and degree of compensatory hyperhidrosis appear to be related to the extent of resection of the sympathetic chain , some clinicians have suggested that the extent of resection should be limited ( 13 ) . for these reasons , many treatment methods , such as different level sympathectomy or sympathicotomy , ramicotomy , and clipping ,", "have been attempted to reduce the extent of resection ( 14 - 18 ) , although the effects of these methods remain controversial . in mild compensatory symptoms , antiperspirants including aluminum - based compounds , iontophoresis , and systemic or topical anticholinergic drugs can be used . however ,", "if the symptoms are severe , the management is more difficult and results are unsatisfactory .", "since philipeaux and vulpian reported the first experimental nerve graft in 1870 , many successful nerve grafts were reported in the field of orthopedic surgery . in the field of thoracic surgery ,", "schoeller et al . ( 19 ) reported successful phrenic nerve reconstruction using sural nerve in patient with mediastinal tumor resection .", "telaranta reported that reconstruction of the sympathetic chain using sural nerve graft diminished compensatory sweating in a male patient who underwent sympathicotomy for palmar hyperhidrosis ( 20 ) .", "miura et al . ( 21 ) reported that sympathetic nerve reconstruction surgery using the intercostal nerve was useful after resection of the sympathetic nerve involved by tumor .", "although the sural nerve is the most commonly used for nerve graft , the intercostal nerve has several advantages over the sural nerve .", "first , the intercostal nerve has more sympathetic nerve fibers than the sural nerve , hence the intercostal nerve is more appropriate for sympathetic nerve reconstruction surgery .", "second , the sural nerve can be used only as free graft but the intercostal nerve can be used as pedicled graft and harvested as a neurovascular bundle . therefore , a sufficient blood supply in the graft can be maintained .", "so , an additional incision is not needed and donor site morbidity decreases . in our experience ,", "thoracoscopic intercostal nerve could be harvested in all patients except 1 patient with severe pleural adhesion , leading us to believe that the intercostal nerve is a useful graft for sympathetic nerve reconstruction surgery .", "epineural and fascicular sutures are the most used for nerve anastomosis , but the foreign body reaction caused by suture material to nerve is another possible problem ( 22 , 23 ) .", "some reports demonstrated the successful nerve anastomosis using fibrin sealant without suture technique ( 24 , 25 ) .", "if microscopic suture technique is used in sympathetic nerve reconstruction surgery , it inevitably needs thoracotomy .", "the type of anastomosis performed in this procedure is the modification of end - to - side neurorrhaphy where axonal sprouting occurs ( 26 ) . the nerve is generally anastomosed in the original direction , but the free graft should be prepared to be anastomosed in the original direction in this operation .", "some reports revealed the anastomosis in reverse orientation did not influence the nerve conduction ( 27 , 28 ) . in the questionnaire on the effects of sympathetic nerve reconstruction surgery , nine patients replied \" definite \" or \" mild \" . in 3 patients who replied that the results were \" definite \" ,", "compensatory sweating decreased and anhidrosis of the affected areas after endoscopic thoracic sympathetic surgery improved .", "patients , who replied mild improvement , complained still uncomfortable compensatory hyperhidrosis , although amount and frequency of perspiration decreased .", "these results of the reconstruction surgery were not different between variables such as age , sex , affected area , and interval from endoscopic thoracic sympathetic surgery due to the small number of cases in this study .", "the digital infrared thermographic imaging is a useful tool in evaluation of body temperature distribution . in the image ,", "before and after sympathetic nerve reconstruction , we performed the digital infrared thermographic imagings in 22 yr - old man complaining of compensatory hyperhidrosis in chest after t3 sympathicotomy .", "after the reconstruction surgery , trunk temperature has increased , and this result corresponded with patient 's symptom . even though the number of patients is not sufficient for data analysis , outcome of the reconstruction surgery did not correlate with the interval between the sympathetic surgery and reconstructive surgery . to evaluate the exact effects of sympathetic nerve reconstruction , more cases and longer", "postoperative complications of the reconstruction surgery were seen in 5 patients ; prolonged chest wall pain which was tolerable in 2 patients , and numbness of chest wall in 2 patients .", "in addition , because compensatory hyperhidrosis depends on climate and season , the timing of a questionnaire survey is important . in this study , the questionnaire was performed on october when it is relatively cool and dry in korea .", ", our results suggest that sympathetic nerve reconstruction with intercostal nerve may be one of the useful surgical methods in severe compensatory hyperhidrosis patients .", "the reconstruction surgery must be decided very carefully in highly selected patients with severe compensatory hyperhidrosis ."], "abstract_text": ["<S> we performed sympathetic nerve reconstruction using intercostal nerve in patients with severe compensatory hyperhidrosis after sympathetic surgery for primary hyperhidrosis , and analyzed the surgical results . from february 2004 to august 2007 , sympathetic nerve reconstruction using intercostal nerve was performed in 19 patients . </S>", "<S> the subjected patients presented severe compensatory hyperhidrosis after thoracoscopic sympathetic surgery for primary hyperhidrosis . </S>", "<S> reconstruction of sympathetic nerve was performed by thoracoscopic surgery except in 1 patient with severe pleural adhesion . </S>", "<S> the median interval between the initial sympathetic surgery and sympathetic nerve reconstruction was 47.2 ( range : 3.5 - 110.7 ) months . </S>", "<S> compensatory sweating after the reconstruction surgery improved in 9 patients , and 3 out of them had markedly improved symptoms . </S>", "<S> sympathetic nerve reconstruction using intercostal nerve may be one of the useful surgical options for severe compensatory hyperhidrosis following sympathetic surgery for primary hyperhidrosis . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["primary hyperhidrosis is a common disease that is characterized by excessive sweating of face , palms , or axilla , occurring in 0.6 - 3% of a population ( 1 , 2 ) .", "since kux performed endoscopic thoracic sympathetic surgery ( 3 ) , it has been popular during the past few decades as the surgical treatment of choice for facial , palmar , and axillary hyperhidrosis ( 4 - 6 ) .", "although endoscopic thoracic sympathetic surgery offers permanent cure for hyperhidrosis , it is often accompanied by serious complications , such as compensatory hyperhidrosis . in order to decrease compensatory hyperhidrosis , endoscopic thoracic", "sympathetic surgery has attempted to reduce the extent of resection of the sympathetic nerve , but these procedures did not significantly decrease the occurrence of compensatory hyperhidrosis ( 7 ) .", "we performed sympathetic nerve reconstruction surgery using intercostal nerve in 19 patients with severe compensatory hyperhidrosis after endoscopic thoracic sympathetic surgery and evaluated the results of the procedure ."], ["from february 2004 to august 2007 , we performed endoscopic thoracic sympathetic surgery in 184 patients with primary hyperhidrosis . among theses", "patients , reconstruction surgery of sympathetic nerve using intercostal nerve was performed in 19 patients with severe compensatory hyperhidrosis after endoscopic thoracic sympathetic surgery .", "fourteen patients were male and 5 were female ( median age : 28 yr , range : 19 - 61 ) .", "this study was approved by the institutional review board of our hospital ( irb no : 3 - 20080021 ) .", "all procedures of reconstruction surgery were performed under general anesthesia with a single lumen endotracheal tube .", "the patients were in a semi - fowler position with their arms extended . in 18 patients except for one patient with severe pleural adhesion ,", "starting on the left side , two separate skin incisions were made along the previous thoracoscopic scars and thoracoscopic ports were placed .", "after co2 gas insufflation into the thoracic cavity with less than 10 mmhg of pressure to deflate the lung , the pleural space was inspected with 5-mm thoracoscope .", "the intercostal neurovascular bundle was dissected in lengths of 5 - 7 cm and the distal part was resected .", "after the proximal and distal part of previously operated sympathetic nerve was exposed , the nerve sheaths of proximal and distal part of exposed sympathetic nerve and harvested intercostal nerve end were removed with an electro - surgical tip cleaner ( surgisite , ethicon , gargrave , skipton , uk ) .", "the intercostal nerve end was placed between the proximal and distal part of the exposed sympathetic nerve and the fibrin glue was applied to contact surface of the sympathetic and intercostal nerve ( fig .", "the same procedure was repeated on the right side . in cases of sympathetic nerve clipping", ", this procedure was performed after clip removal . in one patient with severe pleural adhesion", "we reviewed the clinical charts of all patients who underwent sympathetic nerve reconstruction surgery using the intercostal nerve .", "patients were followed by telephone questionnaire on the effects of the surgery and postoperative complications .", "the degree of improvement of compensatory hyperhidrosis was graded as \" definite \" , \" mild \" , or \" absent \" .", "\" definite \" means that patients felt fully satisfied after the reconstruction surgery , \" mild \" means that patients felt satisfied to a certain extent , and \" absent \" means that patients felt no improvement .", "median interval between the sympathetic nerve reconstruction surgery and questionnaire was 22 ( range : 1 - 45 ) months .", "in one patient ( patient number 17 in table 1 ) , digital infrared thermographic imaging was performed preoperatively and postoperatively ."], ["primary hyperhidrosis patients were composed of 9 facial , 8 palmar , and 1 axillary , and one patient had both facial and palmar hyperhidrosis .", "initial endoscopic thoracic sympathetic surgery for primary hyperhidrosis was t3 sympathicotomy in 5 patients ; t2 sympathicotomy in 8 ; t2 , 3 sympathicotomy in 1 ; t2 , 3 , 4 sympathicotomy in 1 ; t2 clipping in 2 ; t2 , 3 clipping in 1 ; and t2 clipping with t3 sympathicotomy in 1 patient .", "the median interval between the first endoscopic thoracic sympathetic surgery for primary hyperhidrosis and sympathetic nerve reconstruction surgery was 47 ( range , 4 - 111 ) months .", "r3 intercostal nerve was used for sympathetic nerve reconstruction surgery in 15 patients , r4 intercostal nerve in 2 , r2 intercostal nerve in 1 , and r3 with r5 intercostal nerve in 1 patient .", "three patients replied that the effects of reconstruction were \" definite \" , 6 responded with \" mild \" , and 8 said \" absent \" .", "postoperative complications were numbness of the chest wall in 2 patients , chest wall pain in 2 patients , and temporary ptosis in 1 patient in whom ptosis spontaneously resolved after 3 months .", "preoperative and postoperative digital infrared thermographic imagings performed in one patient showed the thermal change in chest and back ."], ["since kux advocated thoracoscopic sympathetic surgery ( 3 ) , recent developments in thoracoscopy and specialized instruments have facilitated the procedure . and , it became the treatment of choice for hyperhidrosis because of its low morbidity , short hospital stay , and excellent cosmetic results ( 6 ) .", "although it has many advantages , some patients suffer from compensatory hyperhidrosis , which is by far the most common and disagreeable complication after endoscopic thoracic sympathetic surgery . according to the previous reports", ", compensatory hyperhidrosis occurs in 59.8 - 90% of patients after sympathetic surgery ( 8 - 10 ) .", "the mechanism of compensatory hyperhidrosis is not clear , but it seems to be associated with compensation for thermoregulatory function ( 11 , 12 ) . because the incidence and degree of compensatory hyperhidrosis appear to be related to the extent of resection of the sympathetic chain , some clinicians have suggested that the extent of resection should be limited ( 13 ) . for these reasons , many treatment methods , such as different level sympathectomy or sympathicotomy , ramicotomy , and clipping ,", "have been attempted to reduce the extent of resection ( 14 - 18 ) , although the effects of these methods remain controversial . in mild compensatory symptoms , antiperspirants including aluminum - based compounds , iontophoresis , and systemic or topical anticholinergic drugs can be used . however ,", "if the symptoms are severe , the management is more difficult and results are unsatisfactory .", "since philipeaux and vulpian reported the first experimental nerve graft in 1870 , many successful nerve grafts were reported in the field of orthopedic surgery . in the field of thoracic surgery ,", "schoeller et al . ( 19 ) reported successful phrenic nerve reconstruction using sural nerve in patient with mediastinal tumor resection .", "telaranta reported that reconstruction of the sympathetic chain using sural nerve graft diminished compensatory sweating in a male patient who underwent sympathicotomy for palmar hyperhidrosis ( 20 ) .", "miura et al . ( 21 ) reported that sympathetic nerve reconstruction surgery using the intercostal nerve was useful after resection of the sympathetic nerve involved by tumor .", "although the sural nerve is the most commonly used for nerve graft , the intercostal nerve has several advantages over the sural nerve .", "first , the intercostal nerve has more sympathetic nerve fibers than the sural nerve , hence the intercostal nerve is more appropriate for sympathetic nerve reconstruction surgery .", "second , the sural nerve can be used only as free graft but the intercostal nerve can be used as pedicled graft and harvested as a neurovascular bundle . therefore , a sufficient blood supply in the graft can be maintained .", "so , an additional incision is not needed and donor site morbidity decreases . in our experience ,", "thoracoscopic intercostal nerve could be harvested in all patients except 1 patient with severe pleural adhesion , leading us to believe that the intercostal nerve is a useful graft for sympathetic nerve reconstruction surgery .", "epineural and fascicular sutures are the most used for nerve anastomosis , but the foreign body reaction caused by suture material to nerve is another possible problem ( 22 , 23 ) .", "some reports demonstrated the successful nerve anastomosis using fibrin sealant without suture technique ( 24 , 25 ) .", "if microscopic suture technique is used in sympathetic nerve reconstruction surgery , it inevitably needs thoracotomy .", "the type of anastomosis performed in this procedure is the modification of end - to - side neurorrhaphy where axonal sprouting occurs ( 26 ) . the nerve is generally anastomosed in the original direction , but the free graft should be prepared to be anastomosed in the original direction in this operation .", "some reports revealed the anastomosis in reverse orientation did not influence the nerve conduction ( 27 , 28 ) . in the questionnaire on the effects of sympathetic nerve reconstruction surgery , nine patients replied \" definite \" or \" mild \" . in 3 patients who replied that the results were \" definite \" ,", "compensatory sweating decreased and anhidrosis of the affected areas after endoscopic thoracic sympathetic surgery improved .", "patients , who replied mild improvement , complained still uncomfortable compensatory hyperhidrosis , although amount and frequency of perspiration decreased .", "these results of the reconstruction surgery were not different between variables such as age , sex , affected area , and interval from endoscopic thoracic sympathetic surgery due to the small number of cases in this study .", "the digital infrared thermographic imaging is a useful tool in evaluation of body temperature distribution . in the image ,", "before and after sympathetic nerve reconstruction , we performed the digital infrared thermographic imagings in 22 yr - old man complaining of compensatory hyperhidrosis in chest after t3 sympathicotomy .", "after the reconstruction surgery , trunk temperature has increased , and this result corresponded with patient 's symptom . even though the number of patients is not sufficient for data analysis , outcome of the reconstruction surgery did not correlate with the interval between the sympathetic surgery and reconstructive surgery . to evaluate the exact effects of sympathetic nerve reconstruction , more cases and longer", "postoperative complications of the reconstruction surgery were seen in 5 patients ; prolonged chest wall pain which was tolerable in 2 patients , and numbness of chest wall in 2 patients .", "in addition , because compensatory hyperhidrosis depends on climate and season , the timing of a questionnaire survey is important . in this study , the questionnaire was performed on october when it is relatively cool and dry in korea .", ", our results suggest that sympathetic nerve reconstruction with intercostal nerve may be one of the useful surgical methods in severe compensatory hyperhidrosis patients .", "the reconstruction surgery must be decided very carefully in highly selected patients with severe compensatory hyperhidrosis ."]]}
{"article_id": "PMC4554115", "article_text": ["endometrial cancer ( ec ) is one of the most common gynecologic malignancies , occupying the sixth place in malignant tumor throughout the world with an increasing trend .", "the initial treatment of ec is comprehensive staging surgery , which consists of total hysterectomy , bilateral salpingo - oophorectomy , pelvic and para - aortic lymphadenectomy and peritoneal cytology .", "laparotomy is the traditional approach , but laparoscopic surgery is widely proposed recent years since it was first reported using for the treatment of ec in 1992 by childers and surwit . in lots of retrospective and prospective studies ,", "laparoscopy has been showed with many advantages compared to laparotomy , such as smaller incision , less pain , and faster recovery .", "it seems that laparoscopic surgery for ec also has similar recurrence rate and survival outcome with laparotomy procedure .", "however , most of these studies have focused on patients with early stage or low - risk disease and few specifically on patients with advanced stage or high risk .", "what 's more , data of long - term survival outcomes about laparoscopic surgery remain limited .", "willis et al have published an article about laparoscopic hysterectomy in a high - risk series of patients with ec , showed that laparoscopy provide all the benefits inherent in minimal access surgery with no compromise in terms of outcomes , but there was no laparotomy data as control .", "therefore , to compare the long - term safety and efficacy between laparoscopic surgery and laparotomy in patients with high - risk ec , we conducted a retrospective analysis based on our decade of clinical data using propensity score matching ( psm ) in this research .", "a total of about 400 medical case notes of patients with histologically confirmed ec who has been received surgical treatment in beijing chao - yang hospital affiliated capital medical university from 2000 to april 2014 were reviewed .", "we selected subjects according to the following criteria : high - risk ( grade 2 , histologic nonendometrioid , myometrial invasion 1/2 , positive pelvic or para - aortic nodes or stages ii ", "iv ) patients who had undergone comprehensive surgical staging in the form of peritoneal cytology , total hysterectomy , bilateral salpingo - oophorectomy , pelvic lymphadenectomy and para - aortic lymphadenectomy or sampling , + / cytoreductive surgery by the same experienced surgical team via laparotomy ( total abdominal hysterectomy , tah ) or laparoscopy ( total laparoscopic hysterectomy , tlh ) .", "para - aortic lymphadenectomy was performed when para - aortic lymph nodes sampled positive and nonendometrioid carcinomas .", "patients who were enrolled ( excepted staged i ag2 with histologic endometrioid ) received postoperative adjuvant therapy including 3 to 6 cycles of chemotherapy ( paclitaxel , 175  mg / m  +  carboplatin , area under the concentration ", "time curve [ auc ] 5 or adriamycin , 45  mg / m  +  cisplatin , 50  mg / m  +  paclitaxel , 160  mg / m ) and vaginal cuff brachytherapy or pelvic irradiation ( 45  gy over 5 weeks ) .", "patients had antibiotic prophylaxis ( cephalosporins or quinolones ) half an hour before operation and venous thrombosis prophylaxis after operation by using molecular weight enoxaparin ( 0.4  ml every day for 45 days ) and lower extremity sequential compression device or graduated compression stocking .", "patients associated with severe cardiopulmonary disease ( myocardial infarction , heart failure , or cerebral infarction ) , other malignancies or a bulky uterus 12 weeks of gestation were excluded . because our data source was case notes and the missing data were few , we deleted the individuals with missing data .", "the collected data of patients included age , body mass index ( bmi ) , the scope of operation , operating time , blood loss , intraoperative and postoperative complications , first flatus time , postoperative hospital stay , surgical stage , histologic type and grade , number of lymph nodes dissection , and clinical outcome .", "the staging of patients was done according to the international federation of gynecology and obstetrics ( figo ) 2009 staging classification .", "intraoperative complications included injuries to bladder , ureter , intestine or large vessels , bleeding requiring blood transfusion , conversion from laparoscopy to laparotomy and transferred to intensive care unit ( icu ) for any unintended reasons .", "postoperative complications included fever ( > 38c ) after 24  hours postoperatively , wound infection or dehiscence , deep - vein thrombosis ( dvt ) , and pulmonary embolism ( pe ) within 30 days after operation .", "the overall survival ( os ) was defined as the period from the date of surgery to death or the date of last contact for living people .", "follow - up is completed by telephone , e - mail , or outpatient service .", "all procedures were approved by the ethics committee for human experiments of the capital medical university . because parts of the baseline characteristics were statistical different between patients received laparoscopic surgery and laparotomy", "eight covariates entered in the propensity model , including age , bmi , the scope of operation , myometrial invasion , pathologic type and grade , lymph node metastasis , and surgical stage .", "propensity scores were calculated using a nonparsimonious multivariable logistic regression model to estimate the conditional probability of a patient receiving a surgery approach .", "then a 1:1 match between the laparoscopy and laparotomy groups was performed by using the nearest available neighbor matching . after matching ,", "data of patient baseline characteristics , operation outcome and incidence of intraoperative and postoperative complication were compared using t test , chi - square test , or fisher exact test .", "survival data were estimated using kaplan  meier curves and compared between groups by log - rank statistics . the 5- and 10-year survival rate and os were reported .", "all of the statistical analysis was completed by spss 19.0 ( spss version 19.0 ; spss , inc . , chicago , il ) . statistical significance was defined as p  <  0.05 .", "three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . among these 225 patients ,", "31 individuals with severe cardiopulmonary disease , other malignancies or uterus 12 weeks of gestation were excluded ; 4 individuals with missing surgical or pathological data were deleted .", "finally , a total of 220 patients with high - risk ec met the study criteria , 107 were treated by laparotomy and 113 by laparoscopy .", "table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . before matching", ", significant differences were not found in age , myometrial invasion , pathological type , and grade and lymph node metastasis between 2 groups but in bmi , scope of operation , and figo stage which could be the confounding factors . after matching , 81 pairs of patients were matched successfully . in the matched cohorts", "baseline patient characteristics pre- and postpropensity score matching the mean operative time was similar between 2 groups , 253  minutes in laparoscopy group and 258  minutes in laparotomy group .", "the mean blood loss were significant less in laparoscopy group ( 107  ml ) than laparotomy group ( 414  ml ) ( p  <  0.01 ) .", "the laparoscopy cohort also has significant shorter first flatus time ( 2.0 days vs. 2.4 days , p  <  0.01 ) and postoperative hospital stay ( 14.7 days vs. 17.7 days , p  =  0.02 ) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy .", "the mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference ( p  <  0.01 ) .", "however , the mean numbers of para - aortic lymph nodes were comparable between 2 groups ( p  =  0.11 ) ( table 2 ) .", "comparison of operation outcome intraoperative complications were significant different between the 2 groups ( 6.2% for laparoscopy and 25.9% for laparotomy , p  <  0.01 ) .", "the patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy ( p  <  0.01 ) . for the incidence of transferred to icu on account of poor cardiopulmonary resuscitation , it was also statistically higher in patients treated by laparotomy than by laparoscopy . moreover , no injury to bladder , ureter , or large vessels occurred in both 2 groups , only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair .", "there was no significant difference in postoperative complications between the 2 groups ( 19.8% for laparoscopy and 29.6% for laparotomy , p  =  0.14 ) .", "dvt occurred more often in laparotomy patients than laparoscopy patients , 14.8% versus 3.7% ( p  =  0.01 ) .", "six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference ( table 3 ) .", "comparison of intraoperative and postoperative complications clinical outcome of 2 groups was shown in table 4 .", "the median follow - up was 45 months ( rang 5176 ) . during the follow - up time", ", there were 16 ( 9.9% ) recurrences which were similar between the 2 treatment cohorts , 7 ( 8.6% ) were observed in laparotomy group at peritoneal , liver , lung , vaginal , and abdominal incision versus 9 ( 11.1% ) in laparoscopy group at peritoneal , liver , lung , and vaginal .", "twenty patients died , 11 in the laparotomy group and 9 in the laparoscopy group .", "the kaplan  meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2% , 85.3% and at 10 years was 75.8% , 85.3% , respectively .", "no statistically significant difference was found between the 2 groups when os was compared ( p  =  0.97 ) ( figure 1 ) .", "it showed that no significant difference between laparotomy and laparoscopy cohorts ( 75.8% vs. 85.3% , p  =  0.97 ) .", "three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . among these 225 patients ,", "31 individuals with severe cardiopulmonary disease , other malignancies or uterus 12 weeks of gestation were excluded ; 4 individuals with missing surgical or pathological data were deleted .", "finally , a total of 220 patients with high - risk ec met the study criteria , 107 were treated by laparotomy and 113 by laparoscopy .", "table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . before matching", ", significant differences were not found in age , myometrial invasion , pathological type , and grade and lymph node metastasis between 2 groups but in bmi , scope of operation , and figo stage which could be the confounding factors . after matching , 81 pairs of patients were matched successfully . in the matched cohorts", "the mean operative time was similar between 2 groups , 253  minutes in laparoscopy group and 258  minutes in laparotomy group .", "the mean blood loss were significant less in laparoscopy group ( 107  ml ) than laparotomy group ( 414  ml ) ( p  <  0.01 ) .", "the laparoscopy cohort also has significant shorter first flatus time ( 2.0 days vs. 2.4 days , p  <  0.01 ) and postoperative hospital stay ( 14.7 days vs. 17.7 days , p  =  0.02 ) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy .", "the mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference ( p  <  0.01 ) .", "however , the mean numbers of para - aortic lymph nodes were comparable between 2 groups ( p  =  0.11 ) ( table 2 ) .", "intraoperative complications were significant different between the 2 groups ( 6.2% for laparoscopy and 25.9% for laparotomy , p  <  0.01 ) . none of the laparoscopic cases was converted into open procedures .", "the patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy ( p  <  0.01 ) . for the incidence of transferred to icu on account of poor cardiopulmonary resuscitation", ", it was also statistically higher in patients treated by laparotomy than by laparoscopy . moreover , no injury to bladder , ureter , or large vessels occurred in both 2 groups , only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair .", "there was no significant difference in postoperative complications between the 2 groups ( 19.8% for laparoscopy and 29.6% for laparotomy , p  =  0.14 ) .", "dvt occurred more often in laparotomy patients than laparoscopy patients , 14.8% versus 3.7% ( p  =  0.01 ) .", "six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference ( table 3 ) .", "the median follow - up was 45 months ( rang 5176 ) . during the follow - up time", ", there were 16 ( 9.9% ) recurrences which were similar between the 2 treatment cohorts , 7 ( 8.6% ) were observed in laparotomy group at peritoneal , liver , lung , vaginal , and abdominal incision versus 9 ( 11.1% ) in laparoscopy group at peritoneal , liver , lung , and vaginal .", "twenty patients died , 11 in the laparotomy group and 9 in the laparoscopy group .", "the kaplan  meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2% , 85.3% and at 10 years was 75.8% , 85.3% , respectively .", "no statistically significant difference was found between the 2 groups when os was compared ( p  =  0.97 ) ( figure 1 ) .", "it showed that no significant difference between laparotomy and laparoscopy cohorts ( 75.8% vs. 85.3% , p  =  0.97 ) .", "for accurately estimating the effect of intervention , randomized controlled trial ( rct ) is considered the optimal approach but often limited by ethic and other problems .", "psm can eliminate the imbalance between groups and reduce the effects of confounding to achieve a random effect in observational studies . in the present study ,", "the patient baseline characteristics of laparotomy and laparoscopy group were adequately balanced after matching , and then , the surgery outcomes achieved comparability .", "this retrospective study demonstrated that laparoscopic technique is effective and even safer compared with laparotomy for patients with high - risk ec . in 2009 , gynecologic oncology group ( gog ) compared laparoscopy and laparotomy for comprehensive surgical staging of ec in a randomized multicenter trial which involved 2616 patients and reported that laparoscopic surgery resulted in fewer complications and shorter hospital stay but longer operation time . in another large randomized trial published in 2010 , laparoscopic surgery was associated with a shorter hospital stay and less amount of blood loss , but no beneficial effect was observed in terms of major complications and operation time .", "however , our data showed evidence of less blood loss , lower proportion of major complications and shorter hospital stay with laparoscopy versus laparotomy and a similar operation time .", "operation time is much related to the experience of the surgical team and learning curve .", "as the improvement of surgical technique , it is a trend that laparoscopic operation time becomes shorter .", "moreover , our study indicated that the first flatus time was shorter in laparoscopy group than in laparotomy .", "patients treated with laparoscopic surgery had smaller incision and less pain compared to those treated with laparotomy .", "laparoscopy can also bring out an earlier resumption of bowel function and shorter hospital stays .", "we found that the postoperative hospital stays in our data were longer than that reported in most of the previously studies and just similar to kong 's study in korea .", "kong reported a mean hospitalization of 16.4 days for laparoscopy and 23.3 days for laparotomy .", "but in gog study , the median hospital stays was 3 days for laparoscopy and 4 days for laparotomy .", "patients in china are accustomed to a longer hospitalization , which is mainly decided by the social factors . in chinese traditional opinion , patients are willing to discharge from hospital only when they have complete recovery ( after removing drainage tube and urinary catheter or taking out stitches ) , what 's more , they need not worry about the medical expense because of the coverage of insurance .", "so , some patients even have been in hospital until the end of the first chemotherapy . in the present study ,", "intraoperative complications were significantly lower in the laparoscopy group and no injuries to bladder , ureter , or large vessels occurred .", "the incidence of major complications can be highly reduced when the procedure was performed by experience surgeon .", "however , it seems that laparoscopy had no benefit in the incidence of postoperative complications .", "but we found dvt occurred significantly often in patients treated by laparotomy than those treated by laparoscopy . in gog study , there was no significant difference in incidence of venous thromboembolism ( vte ) and pe between different approaches .", "in contrast , fader 's study showed that the laparoscopy cohort had significantly fewer episodes of vte .", "lots of studies indicate that the combination of underlying malignancy and pelvic surgery is the risk for developing a vte . in a japanese large research about risk factors for vte , the age ( 50 or 55 years ) , a diagnosis of cancer , operating time ( 4  hours ) , blood loss ( 1000  ml ) , and blood transfusion ( 2000  ml ) were reported significantly related to postoperative vte .", "it may account for the difference of dvt incidence between the 2 groups that laparotomy had longer operating time and more blood loss in our study . what 's more , trendelenburg position during laparoscopic surgery may play a role in preventing vte .", "most of the previous studies demonstrate that the pelvic or para - aotic lymph nodes count removed by laparoscopy approach were similar to those dissected by laparotomy .", "laparoscopy has been proven to be even more efficient in lymphadenectomy because of its clearer vision .", "however , obermair and kalogiannidis reported lower number of lymph node dissections in the laparoscopy group . in the present study , the mean number of dissected para - aotic lymph nodes was comparable between the 2 groups , but the pelvic lymph nodes were significant fewer in laparoscopy cohort than laparotomy .", "two large rcts reported by benedett and kitchener showed no evidence that lymphadenectomy could decrease the risk of recurrence or mortality rate in patients with early ec .", "conversely , another retrospective research gave a result that lymphadenectomy improved the survival of patients with high - risk ec and stage i b to stage iv .", "moreover , an article published in lancet in 2010 reported that para - aotic had a significantly benefit in terms of os for patients with ec at mediate or high risk .", "it seems that the difference of pelvic lymph nodes count between the laparoscopy and laparotomy groups had not influenced the os in our study .", "lots of previous studies have proven that laparoscopy surgery was similar to laparotomy in short - term survival . a meta - analysis reported in 2012", "based on 8 rcts showed that there was no significant difference in disease - free survival , cancer - related survival , or os .", "cho et al reported a similar 5-year os and progression - free survival between laparoscopy and laparotomy groups based on 10-year experience . in 2012 , the gog published the survival data of their large rct in 2009 that the 5-year os was almost identical in both laparoscopy and laparotomy cohorts at 89.8% . between 2000 and 2010", ", we conducted a randomized trial and reported that the 5-year os rates were 91% of laparotomy and 96% of laparoscopy with no significantly difference . in another retrospective study focus on high - grade ec in 2012 , progression - free and os were not significantly different between the surgical cohorts at 44-month median follow - up . in the present study , parts of patients were follow - up more than 10 years . with a median follow - up time of 45 months ,", "the 5- and 10-year survival rate for the laparotomy were 89.2% and 75.8% compared with 85.3% and 85.3% for the laparoscopy .", "there was no significant difference in os between 2 cohorts and also the recurrence rate .", "although the patients enrolled were not assigned to different approaches randomly which was a limitation of our study , we performed a psm and successfully to control a certain confounding factors yet .", "when randomized trial was limited by the objective conditions , psm might be a feasible alternative to reduce selection bias . in conclusion", ", it seems that laparoscopic surgery is as effective as laparotomy in the long term and can be safely carried out in patients with high - risk ec for surgery treatment .", "furthermore , other long - term follow - up data on high - risk ec of randomized trials is still needed to confirm these results ."], "abstract_text": ["<S> abstractthe aim of this study was to compare the long - term safety and efficacy of laparoscopic surgery and laparotomy for high - risk endometrial cancer ( ec).a retrospective analysis based on our decade of clinical data of patients with high - risk ec who were comprehensively surgically staged by laparotomy or laparoscopy was performed . </S>", "<S> the surgical outcomes were compared between different approaches using propensity score matching ( psm).eighty - one pairs of patients from the initial 220 enrolled ones were matched by psm . </S>", "<S> the mean operative time is similar between laparotomy and laparoscopy groups ( 258  minutes vs. 253  minutes ) . </S>", "<S> the laparoscopy cohort has less blood loss ( 107  ml vs.414  ml , p  <  0.01 ) , shorter hospital stay ( 14.7 days vs. 17.7 days , p  =  0.02 ) and significant fewer intraoperative complications ( 6.2% vs. 25.9% , p  <  0.01 ) . </S>", "<S> the pelvic lymph nodes dissected by laparoscopy ( 16.4 ) were significant less than that dissected by laparotomy ( 21.9 ) . </S>", "<S> the 5- and 10-year survival rate for laparotomy were 89.2% and 75.8% compared with 85.3% and 85.3% for the laparoscopy . </S>", "<S> there was no significant difference in overall survival ( p  =  0.97).laparoscopy is as effective as laparotomy in the long term and can be safely carried out in patients with high - risk ec for surgery treatment . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "Baseline Characteristics", "Operation Outcome", "Intraoperative and Postoperative Complication", "Clinical Outcome", "DISCUSSION"], "sections": [["endometrial cancer ( ec ) is one of the most common gynecologic malignancies , occupying the sixth place in malignant tumor throughout the world with an increasing trend .", "the initial treatment of ec is comprehensive staging surgery , which consists of total hysterectomy , bilateral salpingo - oophorectomy , pelvic and para - aortic lymphadenectomy and peritoneal cytology .", "laparotomy is the traditional approach , but laparoscopic surgery is widely proposed recent years since it was first reported using for the treatment of ec in 1992 by childers and surwit . in lots of retrospective and prospective studies ,", "laparoscopy has been showed with many advantages compared to laparotomy , such as smaller incision , less pain , and faster recovery .", "it seems that laparoscopic surgery for ec also has similar recurrence rate and survival outcome with laparotomy procedure .", "however , most of these studies have focused on patients with early stage or low - risk disease and few specifically on patients with advanced stage or high risk .", "what 's more , data of long - term survival outcomes about laparoscopic surgery remain limited .", "willis et al have published an article about laparoscopic hysterectomy in a high - risk series of patients with ec , showed that laparoscopy provide all the benefits inherent in minimal access surgery with no compromise in terms of outcomes , but there was no laparotomy data as control .", "therefore , to compare the long - term safety and efficacy between laparoscopic surgery and laparotomy in patients with high - risk ec , we conducted a retrospective analysis based on our decade of clinical data using propensity score matching ( psm ) in this research ."], ["a total of about 400 medical case notes of patients with histologically confirmed ec who has been received surgical treatment in beijing chao - yang hospital affiliated capital medical university from 2000 to april 2014 were reviewed .", "we selected subjects according to the following criteria : high - risk ( grade 2 , histologic nonendometrioid , myometrial invasion 1/2 , positive pelvic or para - aortic nodes or stages ii ", "iv ) patients who had undergone comprehensive surgical staging in the form of peritoneal cytology , total hysterectomy , bilateral salpingo - oophorectomy , pelvic lymphadenectomy and para - aortic lymphadenectomy or sampling , + / cytoreductive surgery by the same experienced surgical team via laparotomy ( total abdominal hysterectomy , tah ) or laparoscopy ( total laparoscopic hysterectomy , tlh ) .", "para - aortic lymphadenectomy was performed when para - aortic lymph nodes sampled positive and nonendometrioid carcinomas .", "patients who were enrolled ( excepted staged i ag2 with histologic endometrioid ) received postoperative adjuvant therapy including 3 to 6 cycles of chemotherapy ( paclitaxel , 175  mg / m  +  carboplatin , area under the concentration ", "time curve [ auc ] 5 or adriamycin , 45  mg / m  +  cisplatin , 50  mg / m  +  paclitaxel , 160  mg / m ) and vaginal cuff brachytherapy or pelvic irradiation ( 45  gy over 5 weeks ) .", "patients had antibiotic prophylaxis ( cephalosporins or quinolones ) half an hour before operation and venous thrombosis prophylaxis after operation by using molecular weight enoxaparin ( 0.4  ml every day for 45 days ) and lower extremity sequential compression device or graduated compression stocking .", "patients associated with severe cardiopulmonary disease ( myocardial infarction , heart failure , or cerebral infarction ) , other malignancies or a bulky uterus 12 weeks of gestation were excluded . because our data source was case notes and the missing data were few , we deleted the individuals with missing data .", "the collected data of patients included age , body mass index ( bmi ) , the scope of operation , operating time , blood loss , intraoperative and postoperative complications , first flatus time , postoperative hospital stay , surgical stage , histologic type and grade , number of lymph nodes dissection , and clinical outcome .", "the staging of patients was done according to the international federation of gynecology and obstetrics ( figo ) 2009 staging classification .", "intraoperative complications included injuries to bladder , ureter , intestine or large vessels , bleeding requiring blood transfusion , conversion from laparoscopy to laparotomy and transferred to intensive care unit ( icu ) for any unintended reasons .", "postoperative complications included fever ( > 38c ) after 24  hours postoperatively , wound infection or dehiscence , deep - vein thrombosis ( dvt ) , and pulmonary embolism ( pe ) within 30 days after operation .", "the overall survival ( os ) was defined as the period from the date of surgery to death or the date of last contact for living people .", "follow - up is completed by telephone , e - mail , or outpatient service .", "all procedures were approved by the ethics committee for human experiments of the capital medical university . because parts of the baseline characteristics were statistical different between patients received laparoscopic surgery and laparotomy", "eight covariates entered in the propensity model , including age , bmi , the scope of operation , myometrial invasion , pathologic type and grade , lymph node metastasis , and surgical stage .", "propensity scores were calculated using a nonparsimonious multivariable logistic regression model to estimate the conditional probability of a patient receiving a surgery approach .", "then a 1:1 match between the laparoscopy and laparotomy groups was performed by using the nearest available neighbor matching . after matching ,", "data of patient baseline characteristics , operation outcome and incidence of intraoperative and postoperative complication were compared using t test , chi - square test , or fisher exact test .", "survival data were estimated using kaplan  meier curves and compared between groups by log - rank statistics . the 5- and 10-year survival rate and os were reported .", "all of the statistical analysis was completed by spss 19.0 ( spss version 19.0 ; spss , inc . , chicago , il ) . statistical significance was defined as p  <  0.05 ."], ["three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . among these 225 patients ,", "31 individuals with severe cardiopulmonary disease , other malignancies or uterus 12 weeks of gestation were excluded ; 4 individuals with missing surgical or pathological data were deleted .", "finally , a total of 220 patients with high - risk ec met the study criteria , 107 were treated by laparotomy and 113 by laparoscopy .", "table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . before matching", ", significant differences were not found in age , myometrial invasion , pathological type , and grade and lymph node metastasis between 2 groups but in bmi , scope of operation , and figo stage which could be the confounding factors . after matching , 81 pairs of patients were matched successfully . in the matched cohorts", "baseline patient characteristics pre- and postpropensity score matching the mean operative time was similar between 2 groups , 253  minutes in laparoscopy group and 258  minutes in laparotomy group .", "the mean blood loss were significant less in laparoscopy group ( 107  ml ) than laparotomy group ( 414  ml ) ( p  <  0.01 ) .", "the laparoscopy cohort also has significant shorter first flatus time ( 2.0 days vs. 2.4 days , p  <  0.01 ) and postoperative hospital stay ( 14.7 days vs. 17.7 days , p  =  0.02 ) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy .", "the mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference ( p  <  0.01 ) .", "however , the mean numbers of para - aortic lymph nodes were comparable between 2 groups ( p  =  0.11 ) ( table 2 ) .", "comparison of operation outcome intraoperative complications were significant different between the 2 groups ( 6.2% for laparoscopy and 25.9% for laparotomy , p  <  0.01 ) .", "the patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy ( p  <  0.01 ) . for the incidence of transferred to icu on account of poor cardiopulmonary resuscitation , it was also statistically higher in patients treated by laparotomy than by laparoscopy . moreover , no injury to bladder , ureter , or large vessels occurred in both 2 groups , only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair .", "there was no significant difference in postoperative complications between the 2 groups ( 19.8% for laparoscopy and 29.6% for laparotomy , p  =  0.14 ) .", "dvt occurred more often in laparotomy patients than laparoscopy patients , 14.8% versus 3.7% ( p  =  0.01 ) .", "six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference ( table 3 ) .", "comparison of intraoperative and postoperative complications clinical outcome of 2 groups was shown in table 4 .", "the median follow - up was 45 months ( rang 5176 ) . during the follow - up time", ", there were 16 ( 9.9% ) recurrences which were similar between the 2 treatment cohorts , 7 ( 8.6% ) were observed in laparotomy group at peritoneal , liver , lung , vaginal , and abdominal incision versus 9 ( 11.1% ) in laparoscopy group at peritoneal , liver , lung , and vaginal .", "twenty patients died , 11 in the laparotomy group and 9 in the laparoscopy group .", "the kaplan  meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2% , 85.3% and at 10 years was 75.8% , 85.3% , respectively .", "no statistically significant difference was found between the 2 groups when os was compared ( p  =  0.97 ) ( figure 1 ) .", "it showed that no significant difference between laparotomy and laparoscopy cohorts ( 75.8% vs. 85.3% , p  =  0.97 ) ."], ["three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . among these 225 patients ,", "31 individuals with severe cardiopulmonary disease , other malignancies or uterus 12 weeks of gestation were excluded ; 4 individuals with missing surgical or pathological data were deleted .", "finally , a total of 220 patients with high - risk ec met the study criteria , 107 were treated by laparotomy and 113 by laparoscopy .", "table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . before matching", ", significant differences were not found in age , myometrial invasion , pathological type , and grade and lymph node metastasis between 2 groups but in bmi , scope of operation , and figo stage which could be the confounding factors . after matching , 81 pairs of patients were matched successfully . in the matched cohorts"], ["the mean operative time was similar between 2 groups , 253  minutes in laparoscopy group and 258  minutes in laparotomy group .", "the mean blood loss were significant less in laparoscopy group ( 107  ml ) than laparotomy group ( 414  ml ) ( p  <  0.01 ) .", "the laparoscopy cohort also has significant shorter first flatus time ( 2.0 days vs. 2.4 days , p  <  0.01 ) and postoperative hospital stay ( 14.7 days vs. 17.7 days , p  =  0.02 ) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy .", "the mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference ( p  <  0.01 ) .", "however , the mean numbers of para - aortic lymph nodes were comparable between 2 groups ( p  =  0.11 ) ( table 2 ) ."], ["intraoperative complications were significant different between the 2 groups ( 6.2% for laparoscopy and 25.9% for laparotomy , p  <  0.01 ) . none of the laparoscopic cases was converted into open procedures .", "the patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy ( p  <  0.01 ) . for the incidence of transferred to icu on account of poor cardiopulmonary resuscitation", ", it was also statistically higher in patients treated by laparotomy than by laparoscopy . moreover , no injury to bladder , ureter , or large vessels occurred in both 2 groups , only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair .", "there was no significant difference in postoperative complications between the 2 groups ( 19.8% for laparoscopy and 29.6% for laparotomy , p  =  0.14 ) .", "dvt occurred more often in laparotomy patients than laparoscopy patients , 14.8% versus 3.7% ( p  =  0.01 ) .", "six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference ( table 3 ) ."], ["the median follow - up was 45 months ( rang 5176 ) . during the follow - up time", ", there were 16 ( 9.9% ) recurrences which were similar between the 2 treatment cohorts , 7 ( 8.6% ) were observed in laparotomy group at peritoneal , liver , lung , vaginal , and abdominal incision versus 9 ( 11.1% ) in laparoscopy group at peritoneal , liver , lung , and vaginal .", "twenty patients died , 11 in the laparotomy group and 9 in the laparoscopy group .", "the kaplan  meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2% , 85.3% and at 10 years was 75.8% , 85.3% , respectively .", "no statistically significant difference was found between the 2 groups when os was compared ( p  =  0.97 ) ( figure 1 ) .", "it showed that no significant difference between laparotomy and laparoscopy cohorts ( 75.8% vs. 85.3% , p  =  0.97 ) ."], ["for accurately estimating the effect of intervention , randomized controlled trial ( rct ) is considered the optimal approach but often limited by ethic and other problems .", "psm can eliminate the imbalance between groups and reduce the effects of confounding to achieve a random effect in observational studies . in the present study ,", "the patient baseline characteristics of laparotomy and laparoscopy group were adequately balanced after matching , and then , the surgery outcomes achieved comparability .", "this retrospective study demonstrated that laparoscopic technique is effective and even safer compared with laparotomy for patients with high - risk ec . in 2009 , gynecologic oncology group ( gog ) compared laparoscopy and laparotomy for comprehensive surgical staging of ec in a randomized multicenter trial which involved 2616 patients and reported that laparoscopic surgery resulted in fewer complications and shorter hospital stay but longer operation time . in another large randomized trial published in 2010 , laparoscopic surgery was associated with a shorter hospital stay and less amount of blood loss , but no beneficial effect was observed in terms of major complications and operation time .", "however , our data showed evidence of less blood loss , lower proportion of major complications and shorter hospital stay with laparoscopy versus laparotomy and a similar operation time .", "operation time is much related to the experience of the surgical team and learning curve .", "as the improvement of surgical technique , it is a trend that laparoscopic operation time becomes shorter .", "moreover , our study indicated that the first flatus time was shorter in laparoscopy group than in laparotomy .", "patients treated with laparoscopic surgery had smaller incision and less pain compared to those treated with laparotomy .", "laparoscopy can also bring out an earlier resumption of bowel function and shorter hospital stays .", "we found that the postoperative hospital stays in our data were longer than that reported in most of the previously studies and just similar to kong 's study in korea .", "kong reported a mean hospitalization of 16.4 days for laparoscopy and 23.3 days for laparotomy .", "but in gog study , the median hospital stays was 3 days for laparoscopy and 4 days for laparotomy .", "patients in china are accustomed to a longer hospitalization , which is mainly decided by the social factors . in chinese traditional opinion , patients are willing to discharge from hospital only when they have complete recovery ( after removing drainage tube and urinary catheter or taking out stitches ) , what 's more , they need not worry about the medical expense because of the coverage of insurance .", "so , some patients even have been in hospital until the end of the first chemotherapy . in the present study ,", "intraoperative complications were significantly lower in the laparoscopy group and no injuries to bladder , ureter , or large vessels occurred .", "the incidence of major complications can be highly reduced when the procedure was performed by experience surgeon .", "however , it seems that laparoscopy had no benefit in the incidence of postoperative complications .", "but we found dvt occurred significantly often in patients treated by laparotomy than those treated by laparoscopy . in gog study , there was no significant difference in incidence of venous thromboembolism ( vte ) and pe between different approaches .", "in contrast , fader 's study showed that the laparoscopy cohort had significantly fewer episodes of vte .", "lots of studies indicate that the combination of underlying malignancy and pelvic surgery is the risk for developing a vte . in a japanese large research about risk factors for vte , the age ( 50 or 55 years ) , a diagnosis of cancer , operating time ( 4  hours ) , blood loss ( 1000  ml ) , and blood transfusion ( 2000  ml ) were reported significantly related to postoperative vte .", "it may account for the difference of dvt incidence between the 2 groups that laparotomy had longer operating time and more blood loss in our study . what 's more , trendelenburg position during laparoscopic surgery may play a role in preventing vte .", "most of the previous studies demonstrate that the pelvic or para - aotic lymph nodes count removed by laparoscopy approach were similar to those dissected by laparotomy .", "laparoscopy has been proven to be even more efficient in lymphadenectomy because of its clearer vision .", "however , obermair and kalogiannidis reported lower number of lymph node dissections in the laparoscopy group . in the present study , the mean number of dissected para - aotic lymph nodes was comparable between the 2 groups , but the pelvic lymph nodes were significant fewer in laparoscopy cohort than laparotomy .", "two large rcts reported by benedett and kitchener showed no evidence that lymphadenectomy could decrease the risk of recurrence or mortality rate in patients with early ec .", "conversely , another retrospective research gave a result that lymphadenectomy improved the survival of patients with high - risk ec and stage i b to stage iv .", "moreover , an article published in lancet in 2010 reported that para - aotic had a significantly benefit in terms of os for patients with ec at mediate or high risk .", "it seems that the difference of pelvic lymph nodes count between the laparoscopy and laparotomy groups had not influenced the os in our study .", "lots of previous studies have proven that laparoscopy surgery was similar to laparotomy in short - term survival . a meta - analysis reported in 2012", "based on 8 rcts showed that there was no significant difference in disease - free survival , cancer - related survival , or os .", "cho et al reported a similar 5-year os and progression - free survival between laparoscopy and laparotomy groups based on 10-year experience . in 2012 , the gog published the survival data of their large rct in 2009 that the 5-year os was almost identical in both laparoscopy and laparotomy cohorts at 89.8% . between 2000 and 2010", ", we conducted a randomized trial and reported that the 5-year os rates were 91% of laparotomy and 96% of laparoscopy with no significantly difference . in another retrospective study focus on high - grade ec in 2012 , progression - free and os were not significantly different between the surgical cohorts at 44-month median follow - up . in the present study , parts of patients were follow - up more than 10 years . with a median follow - up time of 45 months ,", "the 5- and 10-year survival rate for the laparotomy were 89.2% and 75.8% compared with 85.3% and 85.3% for the laparoscopy .", "there was no significant difference in os between 2 cohorts and also the recurrence rate .", "although the patients enrolled were not assigned to different approaches randomly which was a limitation of our study , we performed a psm and successfully to control a certain confounding factors yet .", "when randomized trial was limited by the objective conditions , psm might be a feasible alternative to reduce selection bias . in conclusion", ", it seems that laparoscopic surgery is as effective as laparotomy in the long term and can be safely carried out in patients with high - risk ec for surgery treatment .", "furthermore , other long - term follow - up data on high - risk ec of randomized trials is still needed to confirm these results ."]]}
{"article_id": "PMC4872557", "article_text": ["syringomyelia is the development of a fluid - filled cavity within the spinal cord . despite different etiologies that cause syringomyelia", ", it is largely agreed that the alteration of cerebrospinal fluid ( csf ) flow dynamics plays an important role in the development of a syrinx .", "known causes of syringomyelia include chiari 1 malformation and basal arachnoiditis [ postinfectious , inflammatory , postirradiation , blood in subarachnoid space ( sas ) amongst others ] . primary spinal syringomyelia refers to syrinx formation due to a spinal pathology , e.g. , spinal cord tumor , spinal infection , trauma , and surgery . the term  idiopathic syringomyelia  has been used to describe syrinx formation in cases where causative mechanism remains undetermined after clinical and routine radiological work - up .", "the term does not imply the absence of a cause but just the absence of a cause that can be detected using standard diagnostic methods .", "there are very few reports in the literature describing arachnoid bands / webs as causes of  idiopathic syringomyelia .", " we present our experience in the management of cases of syringomyelia due to dorsal spinal arachnoid bands and review the current available evidence of the diagnostic work - up and management of this etiology .", "a 43-year - old male was referred with a 2-year history of progressive dissociative sensory loss in his lower limbs with myoclonic twitches .", "this was associated with neck and low back pain in addition to left - sided radiating leg pain .", "there was no history of previous trauma , central nervous system ( cns ) infection , or spinal surgery . on examination", ", there was non - dermatomal numbness in left lower limb but normal power in all four limbs .", "he had been investigated previously in another neurosurgical center and the magnetic resonance imaging ( mri ) scan revealed a cervicothoracic syrinx but in the absence of any apparent cause ; the patient was managed conservatively for 2 years till he was eventually referred to our center for a second opinion .", "mri scan [ figure 1 ] showed twin syrinx cavities in the cervical and upper thoracic regions with an unusual distribution and no apparent etiology .", "the patient underwent a computed tomography ( ct ) myelogram [ figure 2 ] that showed csf block at t1/t2 suggestive of arachnoid pathology . during the ct myelogram", ", a small air bubble was inadvertently introduced that ascended superiorly and was trapped at the level of the csf block .", "he was now experiencing increasing dysesthetic pain down both arms along with left flank numbness and gait disturbance . in view of progressive deterioration", ", he underwent a t1/t2 thoracic laminectomy for intradural exploration that revealed a thickened dorsal band of arachnoid .", "preoperative ( top row ) and postoperative ( bottom row ) mri for case 1 showing cervical significant syrinx prior to surgery and collapsed down following surgery . the postoperative scan was done 6 months after the surgery preoperative ct myelogram and axial mri for case 1 . the abrupt change in cord caliber / displacement demonstrated by the myelogram was not evident on mri studies .", "note inadvertent bubble of air that ascended superiorly to the level of arachnoid web , which was useful in confirming the presence and level of the web postoperatively , the patient recovered well from the operation and there was gradual improvement in his symptoms with normal gait and return of normal function of hands .", "six months after the surgery , the patient reported complete symptom resolution and follow - up mri scan [ figure 1 ] showed significant reduction in the size of the syrinx .", "a 45-year - old male was referred following an episode of legs giving way that was followed by paraparesis for a few hours that recovered fully . on examination", "mri scan [ figure 3 ] revealed a lower thoracic syrinx with some signal change in spinal cord .", "follow - up imaging after 18 months showed no change . however , 4 months later the patient reported increasing discomfort and numbness in both the arms with subjective lower limb weakness and gait disturbance .", "a ct myelogram [ figure 4 ] was performed that revealed displacement of the spinal cord at t3 to t5 and compression to the left likely secondary to either arachnoid band or cyst .", "pre- ( top row ) and postoperative ( bottom row ) mri for case 2 showing significant syrinx prior to surgery and collapsed down following surgery .", "the postoperative scan was done 12 months after the surgery preoperative ct myelogram for case 2 demonstrating the cord displacement and change in caliber by now , the patient had developed right lower limb spastic weakness along with left radiating leg pain .", "on examination he had brisk reflexes , positive hoffmann 's sign bilaterally , positive ankle clonus , reduced sensation in the right leg , and upgoing plantars .", "intradurally , we found a dorsal arachnoid band that was excised and histology confirmed arachnoid tissue .", "postoperatively , the patient recovered well from the operation with improvement in right leg strength and the left leg pain . at 6-month follow - up , he reported complete symptomatic resolution , and neurological examination was normal .", "his follow - up mri [ figure 3 ] at 6 months postoperatively revealed significant reduction in the syrinx size .", "a 43-year - old male was referred with a 2-year history of progressive dissociative sensory loss in his lower limbs with myoclonic twitches .", "this was associated with neck and low back pain in addition to left - sided radiating leg pain .", "there was no history of previous trauma , central nervous system ( cns ) infection , or spinal surgery . on examination", ", there was non - dermatomal numbness in left lower limb but normal power in all four limbs .", "he had been investigated previously in another neurosurgical center and the magnetic resonance imaging ( mri ) scan revealed a cervicothoracic syrinx but in the absence of any apparent cause ; the patient was managed conservatively for 2 years till he was eventually referred to our center for a second opinion .", "mri scan [ figure 1 ] showed twin syrinx cavities in the cervical and upper thoracic regions with an unusual distribution and no apparent etiology .", "the patient underwent a computed tomography ( ct ) myelogram [ figure 2 ] that showed csf block at t1/t2 suggestive of arachnoid pathology . during the ct myelogram", ", a small air bubble was inadvertently introduced that ascended superiorly and was trapped at the level of the csf block .", "he was now experiencing increasing dysesthetic pain down both arms along with left flank numbness and gait disturbance . in view of progressive deterioration", ", he underwent a t1/t2 thoracic laminectomy for intradural exploration that revealed a thickened dorsal band of arachnoid .", "preoperative ( top row ) and postoperative ( bottom row ) mri for case 1 showing cervical significant syrinx prior to surgery and collapsed down following surgery . the postoperative scan was done 6 months after the surgery preoperative ct myelogram and axial mri for case 1 . the abrupt change in cord caliber / displacement demonstrated by the myelogram was not evident on mri studies .", "note inadvertent bubble of air that ascended superiorly to the level of arachnoid web , which was useful in confirming the presence and level of the web postoperatively , the patient recovered well from the operation and there was gradual improvement in his symptoms with normal gait and return of normal function of hands .", "six months after the surgery , the patient reported complete symptom resolution and follow - up mri scan [ figure 1 ] showed significant reduction in the size of the syrinx .", "a 45-year - old male was referred following an episode of legs giving way that was followed by paraparesis for a few hours that recovered fully . on examination", "mri scan [ figure 3 ] revealed a lower thoracic syrinx with some signal change in spinal cord .", "follow - up imaging after 18 months showed no change . however , 4 months later the patient reported increasing discomfort and numbness in both the arms with subjective lower limb weakness and gait disturbance .", "a ct myelogram [ figure 4 ] was performed that revealed displacement of the spinal cord at t3 to t5 and compression to the left likely secondary to either arachnoid band or cyst .", "pre- ( top row ) and postoperative ( bottom row ) mri for case 2 showing significant syrinx prior to surgery and collapsed down following surgery .", "the postoperative scan was done 12 months after the surgery preoperative ct myelogram for case 2 demonstrating the cord displacement and change in caliber by now , the patient had developed right lower limb spastic weakness along with left radiating leg pain .", "on examination he had brisk reflexes , positive hoffmann 's sign bilaterally , positive ankle clonus , reduced sensation in the right leg , and upgoing plantars .", "intradurally , we found a dorsal arachnoid band that was excised and histology confirmed arachnoid tissue .", "postoperatively , the patient recovered well from the operation with improvement in right leg strength and the left leg pain . at 6-month follow - up , he reported complete symptomatic resolution , and neurological examination was normal .", "his follow - up mri [ figure 3 ] at 6 months postoperatively revealed significant reduction in the syrinx size .", "we report a very uncommon and hence under - recognized cause of symptomatic syringomyelia . whilst arachnoid bands / webs have previously been reported as the etiologic factor for idiopathic syringomyelia", "first reported the importance of recognizing occult arachnoid webs , pouches , and cysts in idiopathic syringomyelia . in a large series of 28 patients with thoracic arachnoid pathology ,", "6 cases of ventral or dorsal arachnoid bands were reported . however , there is uncertainty not only regarding the pathophysiology of syrinx formation with arachnoid webs but also regarding the ideal radiological investigations .", "arachnoid adhesions due to trauma / infection / previous surgery are well - recognized causes of syringomyelia .", "idiopathic arachnoid bands have been hypothesized to develop from the septum posticum , the membrane dividing the midline dorsal arachnoid space of the cervicothoracic spinal canal .", "it is however equally possible that they represent localized arachnoiditis due to factors that are not always elicited in the history .", "frequently cited theories in syrinx formation are those of gardner , williams , and oldfield .", "greitz proposed that increased intramedullary pulse pressure in the spinal cord relative to the nearby sas is the distending force in the production of syringomyelia and that syrinx consists of extracellular fluid accumulated in the distended spinal cord .", "directly addressed the pathophysiology of primary spinal syringomyelia in their prospective study of 36 patients .", "their findings were consistent with their theory that a spinal subarachnoid block increases subarachnoid pulse pressure above the block , producing a pressure differential across the obstructed segment of the sas , which results in syrinx formation and progression .", "they reported that their findings were similar to that of their previous studies , which examined the pathophysiology of syringomyelia associated with obstruction of sas at the foramen magnum , inferring a common mechanism for both .", "three - dimensional computational models of the spinal sas have been used to study csf flow either in unobstructed sas or with sas obstructed by a porous region simulating dorsal and circumferential arachnoiditis .", "the findings suggest that syrinx formation may be related to a change of temporal csf pulse pressure dynamics .", "consequently , it would appear that restoring csf flow across the arachnoid webs / bands should result in syrinx resolution . alternatively , syrinx formation may occur due to resonance in a mechanical system with a pathology - induced nodal point .", "however , the recognition of a secondary imaging finding  the scalpel sign  can suggest the presence of an arachnoid web . this is a focal indentation of the thoracic spinal cord , resembling a scalpel on sagittal mri images with the scalpel blade pointing posteriorly . however , none of our patients had any clear evidence suggesting an arachnoid web on conventional mri .", "in all the early reported series of arachnoid webs , ct myelography was the investigation of choice .", "indeed , it has been reported that cardiac gated mri techniques were less successful in studying csf flow in the spine than at the craniovertebral junction . however , in a large series of 125 patients with idiopathic syringomyelia , mauer et al . performed cardiac gated phase contrast mri of csf flow in the median sagittal plane in the spine and reported that these were more reliable compared to myelography .", "cardiac gated phase contrast cine - mode mri in multiple axial planes has also been used to quantitatively analyze csf flow in those planes for the diagnosis of arachnoid webs .", "whilst in these papers , arachnoid webs were indirectly diagnosed using flow studies ; direct visualization of arachnoid membranes has been reported with high resolution using retrospectively cardiac gated cine steady - state free precession mri .", "however , all these mri techniques are susceptible to movement artifacts and also require a 3tesla mri that may not always be available .", "arachnoid webs may be missed even with mri csf flow sequences , hence ct myelography remains useful .", "it is worth noting that the inadvertent air bubble introduced during the ct myelogram of our first patient provided an excellent clue about the location of the csf block .", "the location of the syrinx is not a reliable clue regarding the level of arachnoid web / csf block .", "the arachnoid web was just caudal to the syrinx in our first patient and at the cranial end of the syrinx in the second patient .", "we recognize that myelography is an invasive procedure with potential morbidity and with recent significant advancements in mr technology , it may be prudent to investigate symptomatic idiopathic syringomyelia with mri csf flow studies in the first instance and failing that , to proceed to myelography .", "we believe that restoring csf flow by excision of the arachnoid band and thereby improving sas compliance offers the best possible outcome in these symptomatic cases . in the earliest reported series ,", "all patients treated by web excision improved whereas both patients treated with syringo - pleural shunts worsened .", "mixed result were obtained with shunting in other series but excision of the webs was almost always associated with successful outcome . shunting syrinxes", "is associated with high rates of recurrence , poor long - term outcomes , and sometimes worsening of neurological status .", "in fact , most patients with any short segment arachnoid pathology causing primary spinal syringomyelia , generally have good outcomes after surgical decompression / detethering procedures .", "it is the patient group with long segment pathology that often requires multiple surgeries , frequently for csf diversion .", "this was corroborated in a recent systematic review , which suggested that arachnolysis rather than shunting was the only initial surgical treatment that is likely to have a statistically significant effect on lowering recurrence rates .", "this once again highlights the importance of exhaustively looking for a rectifiable surgical targets , such as arachnoid webs and cysts in the presumed idiopathic syrinxes , which would otherwise have be treated with shunts .", "arachnoid webs / bands are a rare and hence often underdiagnosed cause of idiopathic syringomyelia , leading to delayed diagnosis and treatment . in symptomatic", "/ deteriorating patients with idiopathic syringomyelia , imaging studies should be closely inspected for the presence of a transverse arachnoid web .", "conventional mri should always be augmented with mri csf flow studies in the sagittal plane and if needed in multiple axial planes to aid in the diagnosis .", "ct myelography should be performed if these studies do not yield answers or also if the surgeon feels further information is needed prior to definitive treatment , especially as myelography provides more robust localization of the level of block .", "focussed surgical decompression and excision of the offending arachnoid tissue usually leads to good clinical and radiological outcomes ."], "abstract_text": ["<S> in a certain group of patients with syringomyelia , even with the advent of sophisticated magnetic resonance imaging ( mri ) , no associated abnormality or cerebrospinal fluid ( csf ) block is easily identified . </S>", "<S> this type of syringomyelia is often termed idiopathic . </S>", "<S> current literature has less than 10 reports of arachnoid webs to be the causative factor . </S>", "<S> we present our experience in the management of two cases of syringomyelia secondary to arachnoid webs . both our patients presented with progressive neurological deterioration with mri scans demonstrating cervical / thoracic syrinx without chiari malformation or low - lying cord . </S>", "<S> there was no history of previous meningitis or trauma . </S>", "<S> both patients underwent myelography that demonstrated dorsal flow block implying csf obstruction . </S>", "<S> cord displacement / change in caliber was also noted and this was not evident on mri scans . both patients underwent thoracic laminectomy . </S>", "<S> after opening the dura , thickened / abnormal arachnoid tissue was found that was resected thus widely communicating the dorsal subarachnoid space . </S>", "<S> postoperatively at 6 months , both patients had significant symptomatic improvement with follow - up mri scans demonstrating significant resolution of the syrinx . in patients with presumed idiopathic </S>", "<S> syringomyelia , imaging studies should be closely inspected for the presence of a transverse arachnoid web . </S>", "<S> we believe that all patients with idiopathic symptomatic syringomyelia should have mri csf flow studies and/or computed tomography ( ct ) myelography to identify such arachnoid abnormalities that are often underdiagnosed . </S>", "<S> subsequent surgery should be directed at the establishment of normal csf flow by laminectomy and excision of the offending arachnoid tissue . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORTS", "Case 1", "Case 2", "DISCUSSION", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["syringomyelia is the development of a fluid - filled cavity within the spinal cord . despite different etiologies that cause syringomyelia", ", it is largely agreed that the alteration of cerebrospinal fluid ( csf ) flow dynamics plays an important role in the development of a syrinx .", "known causes of syringomyelia include chiari 1 malformation and basal arachnoiditis [ postinfectious , inflammatory , postirradiation , blood in subarachnoid space ( sas ) amongst others ] . primary spinal syringomyelia refers to syrinx formation due to a spinal pathology , e.g. , spinal cord tumor , spinal infection , trauma , and surgery . the term  idiopathic syringomyelia  has been used to describe syrinx formation in cases where causative mechanism remains undetermined after clinical and routine radiological work - up .", "the term does not imply the absence of a cause but just the absence of a cause that can be detected using standard diagnostic methods .", "there are very few reports in the literature describing arachnoid bands / webs as causes of  idiopathic syringomyelia .", " we present our experience in the management of cases of syringomyelia due to dorsal spinal arachnoid bands and review the current available evidence of the diagnostic work - up and management of this etiology ."], ["a 43-year - old male was referred with a 2-year history of progressive dissociative sensory loss in his lower limbs with myoclonic twitches .", "this was associated with neck and low back pain in addition to left - sided radiating leg pain .", "there was no history of previous trauma , central nervous system ( cns ) infection , or spinal surgery . on examination", ", there was non - dermatomal numbness in left lower limb but normal power in all four limbs .", "he had been investigated previously in another neurosurgical center and the magnetic resonance imaging ( mri ) scan revealed a cervicothoracic syrinx but in the absence of any apparent cause ; the patient was managed conservatively for 2 years till he was eventually referred to our center for a second opinion .", "mri scan [ figure 1 ] showed twin syrinx cavities in the cervical and upper thoracic regions with an unusual distribution and no apparent etiology .", "the patient underwent a computed tomography ( ct ) myelogram [ figure 2 ] that showed csf block at t1/t2 suggestive of arachnoid pathology . during the ct myelogram", ", a small air bubble was inadvertently introduced that ascended superiorly and was trapped at the level of the csf block .", "he was now experiencing increasing dysesthetic pain down both arms along with left flank numbness and gait disturbance . in view of progressive deterioration", ", he underwent a t1/t2 thoracic laminectomy for intradural exploration that revealed a thickened dorsal band of arachnoid .", "preoperative ( top row ) and postoperative ( bottom row ) mri for case 1 showing cervical significant syrinx prior to surgery and collapsed down following surgery . the postoperative scan was done 6 months after the surgery preoperative ct myelogram and axial mri for case 1 . the abrupt change in cord caliber / displacement demonstrated by the myelogram was not evident on mri studies .", "note inadvertent bubble of air that ascended superiorly to the level of arachnoid web , which was useful in confirming the presence and level of the web postoperatively , the patient recovered well from the operation and there was gradual improvement in his symptoms with normal gait and return of normal function of hands .", "six months after the surgery , the patient reported complete symptom resolution and follow - up mri scan [ figure 1 ] showed significant reduction in the size of the syrinx .", "a 45-year - old male was referred following an episode of legs giving way that was followed by paraparesis for a few hours that recovered fully . on examination", "mri scan [ figure 3 ] revealed a lower thoracic syrinx with some signal change in spinal cord .", "follow - up imaging after 18 months showed no change . however , 4 months later the patient reported increasing discomfort and numbness in both the arms with subjective lower limb weakness and gait disturbance .", "a ct myelogram [ figure 4 ] was performed that revealed displacement of the spinal cord at t3 to t5 and compression to the left likely secondary to either arachnoid band or cyst .", "pre- ( top row ) and postoperative ( bottom row ) mri for case 2 showing significant syrinx prior to surgery and collapsed down following surgery .", "the postoperative scan was done 12 months after the surgery preoperative ct myelogram for case 2 demonstrating the cord displacement and change in caliber by now , the patient had developed right lower limb spastic weakness along with left radiating leg pain .", "on examination he had brisk reflexes , positive hoffmann 's sign bilaterally , positive ankle clonus , reduced sensation in the right leg , and upgoing plantars .", "intradurally , we found a dorsal arachnoid band that was excised and histology confirmed arachnoid tissue .", "postoperatively , the patient recovered well from the operation with improvement in right leg strength and the left leg pain . at 6-month follow - up , he reported complete symptomatic resolution , and neurological examination was normal .", "his follow - up mri [ figure 3 ] at 6 months postoperatively revealed significant reduction in the syrinx size ."], ["a 43-year - old male was referred with a 2-year history of progressive dissociative sensory loss in his lower limbs with myoclonic twitches .", "this was associated with neck and low back pain in addition to left - sided radiating leg pain .", "there was no history of previous trauma , central nervous system ( cns ) infection , or spinal surgery . on examination", ", there was non - dermatomal numbness in left lower limb but normal power in all four limbs .", "he had been investigated previously in another neurosurgical center and the magnetic resonance imaging ( mri ) scan revealed a cervicothoracic syrinx but in the absence of any apparent cause ; the patient was managed conservatively for 2 years till he was eventually referred to our center for a second opinion .", "mri scan [ figure 1 ] showed twin syrinx cavities in the cervical and upper thoracic regions with an unusual distribution and no apparent etiology .", "the patient underwent a computed tomography ( ct ) myelogram [ figure 2 ] that showed csf block at t1/t2 suggestive of arachnoid pathology . during the ct myelogram", ", a small air bubble was inadvertently introduced that ascended superiorly and was trapped at the level of the csf block .", "he was now experiencing increasing dysesthetic pain down both arms along with left flank numbness and gait disturbance . in view of progressive deterioration", ", he underwent a t1/t2 thoracic laminectomy for intradural exploration that revealed a thickened dorsal band of arachnoid .", "preoperative ( top row ) and postoperative ( bottom row ) mri for case 1 showing cervical significant syrinx prior to surgery and collapsed down following surgery . the postoperative scan was done 6 months after the surgery preoperative ct myelogram and axial mri for case 1 . the abrupt change in cord caliber / displacement demonstrated by the myelogram was not evident on mri studies .", "note inadvertent bubble of air that ascended superiorly to the level of arachnoid web , which was useful in confirming the presence and level of the web postoperatively , the patient recovered well from the operation and there was gradual improvement in his symptoms with normal gait and return of normal function of hands .", "six months after the surgery , the patient reported complete symptom resolution and follow - up mri scan [ figure 1 ] showed significant reduction in the size of the syrinx ."], ["a 45-year - old male was referred following an episode of legs giving way that was followed by paraparesis for a few hours that recovered fully . on examination", "mri scan [ figure 3 ] revealed a lower thoracic syrinx with some signal change in spinal cord .", "follow - up imaging after 18 months showed no change . however , 4 months later the patient reported increasing discomfort and numbness in both the arms with subjective lower limb weakness and gait disturbance .", "a ct myelogram [ figure 4 ] was performed that revealed displacement of the spinal cord at t3 to t5 and compression to the left likely secondary to either arachnoid band or cyst .", "pre- ( top row ) and postoperative ( bottom row ) mri for case 2 showing significant syrinx prior to surgery and collapsed down following surgery .", "the postoperative scan was done 12 months after the surgery preoperative ct myelogram for case 2 demonstrating the cord displacement and change in caliber by now , the patient had developed right lower limb spastic weakness along with left radiating leg pain .", "on examination he had brisk reflexes , positive hoffmann 's sign bilaterally , positive ankle clonus , reduced sensation in the right leg , and upgoing plantars .", "intradurally , we found a dorsal arachnoid band that was excised and histology confirmed arachnoid tissue .", "postoperatively , the patient recovered well from the operation with improvement in right leg strength and the left leg pain . at 6-month follow - up , he reported complete symptomatic resolution , and neurological examination was normal .", "his follow - up mri [ figure 3 ] at 6 months postoperatively revealed significant reduction in the syrinx size ."], ["we report a very uncommon and hence under - recognized cause of symptomatic syringomyelia . whilst arachnoid bands / webs have previously been reported as the etiologic factor for idiopathic syringomyelia", "first reported the importance of recognizing occult arachnoid webs , pouches , and cysts in idiopathic syringomyelia . in a large series of 28 patients with thoracic arachnoid pathology ,", "6 cases of ventral or dorsal arachnoid bands were reported . however , there is uncertainty not only regarding the pathophysiology of syrinx formation with arachnoid webs but also regarding the ideal radiological investigations .", "arachnoid adhesions due to trauma / infection / previous surgery are well - recognized causes of syringomyelia .", "idiopathic arachnoid bands have been hypothesized to develop from the septum posticum , the membrane dividing the midline dorsal arachnoid space of the cervicothoracic spinal canal .", "it is however equally possible that they represent localized arachnoiditis due to factors that are not always elicited in the history .", "frequently cited theories in syrinx formation are those of gardner , williams , and oldfield .", "greitz proposed that increased intramedullary pulse pressure in the spinal cord relative to the nearby sas is the distending force in the production of syringomyelia and that syrinx consists of extracellular fluid accumulated in the distended spinal cord .", "directly addressed the pathophysiology of primary spinal syringomyelia in their prospective study of 36 patients .", "their findings were consistent with their theory that a spinal subarachnoid block increases subarachnoid pulse pressure above the block , producing a pressure differential across the obstructed segment of the sas , which results in syrinx formation and progression .", "they reported that their findings were similar to that of their previous studies , which examined the pathophysiology of syringomyelia associated with obstruction of sas at the foramen magnum , inferring a common mechanism for both .", "three - dimensional computational models of the spinal sas have been used to study csf flow either in unobstructed sas or with sas obstructed by a porous region simulating dorsal and circumferential arachnoiditis .", "the findings suggest that syrinx formation may be related to a change of temporal csf pulse pressure dynamics .", "consequently , it would appear that restoring csf flow across the arachnoid webs / bands should result in syrinx resolution . alternatively , syrinx formation may occur due to resonance in a mechanical system with a pathology - induced nodal point .", "however , the recognition of a secondary imaging finding  the scalpel sign  can suggest the presence of an arachnoid web . this is a focal indentation of the thoracic spinal cord , resembling a scalpel on sagittal mri images with the scalpel blade pointing posteriorly . however , none of our patients had any clear evidence suggesting an arachnoid web on conventional mri .", "in all the early reported series of arachnoid webs , ct myelography was the investigation of choice .", "indeed , it has been reported that cardiac gated mri techniques were less successful in studying csf flow in the spine than at the craniovertebral junction . however , in a large series of 125 patients with idiopathic syringomyelia , mauer et al . performed cardiac gated phase contrast mri of csf flow in the median sagittal plane in the spine and reported that these were more reliable compared to myelography .", "cardiac gated phase contrast cine - mode mri in multiple axial planes has also been used to quantitatively analyze csf flow in those planes for the diagnosis of arachnoid webs .", "whilst in these papers , arachnoid webs were indirectly diagnosed using flow studies ; direct visualization of arachnoid membranes has been reported with high resolution using retrospectively cardiac gated cine steady - state free precession mri .", "however , all these mri techniques are susceptible to movement artifacts and also require a 3tesla mri that may not always be available .", "arachnoid webs may be missed even with mri csf flow sequences , hence ct myelography remains useful .", "it is worth noting that the inadvertent air bubble introduced during the ct myelogram of our first patient provided an excellent clue about the location of the csf block .", "the location of the syrinx is not a reliable clue regarding the level of arachnoid web / csf block .", "the arachnoid web was just caudal to the syrinx in our first patient and at the cranial end of the syrinx in the second patient .", "we recognize that myelography is an invasive procedure with potential morbidity and with recent significant advancements in mr technology , it may be prudent to investigate symptomatic idiopathic syringomyelia with mri csf flow studies in the first instance and failing that , to proceed to myelography .", "we believe that restoring csf flow by excision of the arachnoid band and thereby improving sas compliance offers the best possible outcome in these symptomatic cases . in the earliest reported series ,", "all patients treated by web excision improved whereas both patients treated with syringo - pleural shunts worsened .", "mixed result were obtained with shunting in other series but excision of the webs was almost always associated with successful outcome . shunting syrinxes", "is associated with high rates of recurrence , poor long - term outcomes , and sometimes worsening of neurological status .", "in fact , most patients with any short segment arachnoid pathology causing primary spinal syringomyelia , generally have good outcomes after surgical decompression / detethering procedures .", "it is the patient group with long segment pathology that often requires multiple surgeries , frequently for csf diversion .", "this was corroborated in a recent systematic review , which suggested that arachnolysis rather than shunting was the only initial surgical treatment that is likely to have a statistically significant effect on lowering recurrence rates .", "this once again highlights the importance of exhaustively looking for a rectifiable surgical targets , such as arachnoid webs and cysts in the presumed idiopathic syrinxes , which would otherwise have be treated with shunts ."], ["arachnoid webs / bands are a rare and hence often underdiagnosed cause of idiopathic syringomyelia , leading to delayed diagnosis and treatment . in symptomatic", "/ deteriorating patients with idiopathic syringomyelia , imaging studies should be closely inspected for the presence of a transverse arachnoid web .", "conventional mri should always be augmented with mri csf flow studies in the sagittal plane and if needed in multiple axial planes to aid in the diagnosis .", "ct myelography should be performed if these studies do not yield answers or also if the surgeon feels further information is needed prior to definitive treatment , especially as myelography provides more robust localization of the level of block .", "focussed surgical decompression and excision of the offending arachnoid tissue usually leads to good clinical and radiological outcomes ."], [""], [""]]}
{"article_id": "PMC3539612", "article_text": ["approximately 30% of women have sustained at least 1 vertebral fracture by the age of 75 .", "these fractures are an important and common cause of morbidity in osteoporotic patients ; moreover , fractures evidenced both clinically and at radiographic examination are associated with an increased mortality rate .", "back pain owing to vertebral fractures has a significant affect on osteoporotic patients [ 46 ] , and the number and severity of these fractures also increases the risk of developing chronic back pain .", "this has a marked negative impact on the quality of life ( qol ) and functional impairment of the affected patients .", "conventional treatments for osteoporosis including bisphosphonates , selective estrogen receptor modulators ( serms ) , and estrogen have been shown to reduce the rate of bone resorption and to preserve bone mass .", "another therapeutic option includes rhpth , an agent that has been shown to increase both bone mass and bone strength .", "when injected , teriparatide ( rdna origin ) , the amino - terminal fragment of human pth ( rhpth 134 ) , is a potent bone formation agent for the treatment of severe osteoporosis .", "it increases osteoblast production / growth and prevents osteoblast apoptosis ; at the same time , it enhances absorption of calcium from the intestine , renal reabsorption of calcium , while decreasing the excretion of phosphates in the kidney [ 1114 ] .", "when administered once daily by subcutaneous injection , rhpth increases bone density and improves trabecular architecture , cortical geometry , and strength .", "not only does rhpth increase trabecular bone density by stimulating bone formation , but it also stimulates osteoclastic bone resorption [ 1719 ] . in several studies , rhpth has been shown to increase bone mineral density in postmenopausal osteoporosis , in senile osteoporosis in men , and in glucocorticoid - induced osteoporosis .", "teriparatide acts via the pth-1 receptor on osteoblasts and bone marrow stromal cells to induce osteoblastic bone formation , that is , osteoid synthesis and accelerated mineralization .", "this , in turn , results in reductions in skeletal fractures to levels equivalent to , or over , those obtained by using antiresorptive agents .", "the increase in bone mineral density induced by rhpth is substantial , ranging 10% to 15% over 2 to 3 years in most studies [ 10,2024 ] . moreover , rhpth can cause demonstrable increases in bone mineral density and changes in markers of bone turnover within 3 months since the start of treatment .", "quality of life ( qol ) can be measured to compare the effect of different treatments for osteoporosis .", "measuring pain scores only for these patients would be inadequate , because apart from acute and chronic back pain , patients with vertebral fractures also experience anxiety , constant fear of falling , or suffering another fracture while their daily living activities are impaired .", "most information has been collected thanks to the efforts made by some researchers to develop specific instruments to test the physical and emotional disability generated by the disease .", "generic tools available for measuring qol are useful to evaluate general health but they lack disease specificity .", "more recently , some specific instruments have been developed to measure the qol in osteoporosis more accurately .", "the most widely used are the osteoporosis quality of life questionnaire ( oqlq ) , the osteoporosis assessment questionnaire ( opaq ) , the osteoporosis - targeted quality of life questionnaire ( optqol ) , and the quality of life questionnaire of the european foundation for osteoporosis ( qualeffo-41 ) .", "one of the first ones was qualeffo-41 , which has been translated and validated in different languages including italian .", "this questionnaire has proved to be repeatable , coherent , and able to discriminate between patients and controls . in the last few years , other specific questionnaires", "have been developed , but not all of them have been as extensively used and validated in different countries as qualeffo-41 .", "the goal of this study was firstly to assess the validity of rhpth treatment in a cohort of postmenopausal women with severe osteoporosis ; secondly , to evaluate the improvement in qol and pain symptoms after several months of rhpth therapy .", "a follow - up qualeffo-41 questionnaire was used to quantify the patient s pain and the affect on qol after rhpth therapy .", "the study was a 18 months , randomized prospective cohort study conducted at department of molecular and clinical endocrinology and oncology , university of naples federico ii , naples , italy .", "inclusion criteria for this study consisted of back pain , postmenopausal osteoporosis ( t - score 2.5 at lumbar spine or femoral neck ) , the presence of 2 osteoporotic vertebral fractures , previous treatment for osteoporosis .", "the exclusion criteria were : an increased risk of osteosarcoma ( ie , patients with paget disease bone , previous skeletal exposure to external beam radiotherapy , or previous malignant neoplasm involving the skeleton ) , hypercalcemia , malignant neoplasms , impaired renal function , liver disease , history of diseases other than postmenopausal osteoporosis that affect bone metabolism , nephrolithiasis , alcohol or drug abuse .", "secondary osteoporosis was excluded in order to avoid the interference of the primitive disease with the patient s quality of life .", "informed consent was obtained from all subjects and the study protocol was approved by the hospital / science s ethical committee .", "eighty - one postmenopausal women were enrolled and divided in two groups with no statistically significant differences in any of the considered variables : group a  forty - two women ( mean age 659 yrs ; mean body mass index ", "bmi 24.52.6 kg / m ) , with severe postmenopausal osteoporosis ( mean lumbar bmd 3.880.70 , mean femoral neck bmd 3.070.60 and with 2 vertebral atraumatic fractures ) , persistent back pain and previous treatment with biphosphonates for osteoporosis ; group b  thirty - nine women matched for age ( 6014.4 yrs ) , bmi ( 22.88.8 kg / m ) , menopausal status , affected by back pain , severe postmenopausal osteoporosis ( lumbar spine bmd 3.900.73 , mean femoral neck bmd 3.020.61 and with 2 vertebral atraumatic fractures ) previously treated for osteoporosis with biphosphonates .", "bmi , age at menopause , lifestyle habits ( i.e. , smoking , drinking , nutrition style ) , nutrition anamnesis ( calcium intake ) , history of diseases other than osteoporosis , family history of osteoporosis were considered .", "groups were randomized to daily treatment with 20 g s.c . of recombinant human parathyroid hormone ( rhpth 134 ) , self - administered injections ( group a ) or 70 mg per os of alendronate every week ( group b ) .", "all women received 1,000 mg elemental calcium daily and 800 iu of vitamin d daily for 18 months .", "biochemical markers of bone turnover were dosed in all the selected female patients : alkaline phosphatase ( alp : 35104", "u / l ) , n - terminal propeptide of type i procollagen ( pinp : 19102 g / l ) , n - telopeptide crosslinks ( ntx : 565 nmol / mmol crea ) were assessed at baseline , 3 , 12 and 18 months ( t0 , t3 , t12 , t18 ) .", "the bmd of the lumbar spine and the proximal femur was measured by dual - energy x - ray absorptiometry ( dual - energy x - ray absorptiometry qdr 1000 ; hologic , waltham , ma , usa ) at baseline and at 18 months ( t0 , t18 ) .", "all women underwent anteroposterior and lateral radiography of thoracic and lumbar spine at t0 and t18 .", "the qol questionnaire of the european foundation for osteoporosis ( qualeffo ) was administered at baseline and at the end of the study to evaluate the impact of rhpth on health - related qol .", "originally , the questionnaire consisted of 48 questions and 6 visual analogue scales . in the qualeffo validation study ,", "this resulted in the qualeffo-41 , which consisted of 41 questions in 5 domains : pain , physical function , social function , general health perception , and mental function .", "all answers were recorded so that all items range from 1 to 5 , and all answers were standardized so that 1 represents the best and 5 the worst qol , with the exception of questions e23 - 25 ( questions with 3 answer options ) , questions e24 - 26 - 27 - 28 ( 4 answer options ) , and questions g33 - 34 - 35 - 37 - 39 - 40 ( answer with reverse scores : 1 is the worst while 5 is the best ) .", "domain scores are calculated by averaging the answers of 1 domain and transforming the scores to a score from 0 to 100 .", "data are expressed as mean  sd or percentage ; moreover , to assess the affect of teriparatide treatment on markers of bone turnover , bone mineral density , and on health - related qol , pearson correlation coefficients were computed in the 2 study groups .", "therefore , percentage changes of biomarkers , bone mineral density , and qualeffo results were calculated .", "there was no significant difference in these characteristics between the 2 groups . in the group a , 39 ( 93% ) out of the 42 recruited women completed the study : 2 patients discontinued the drug therapy because of lack of compliance with the study treatment ; 1 patient discontinued because of a new vertebral atraumatic fracture .", "the aim of this study was to evaluate percentage changes from baseline in biochemical markers of bone turnover , values of bone mineral density ( measured at lumbar spine and proximal femur ) , and measurements of qol .", "follow - up checks , where we also evaluated clinical conditions , adverse events , compliance to treatment and use of non - steroid anti - inflammatory drugs , were carried out at 3 , 12 and 18 months since the beginning of treatment . in group a serum levels of pinp increased of 90% , 145% and 127% at t3 , t12 , t18 , respectively ; bone alp levels increased of 57% , 79% and 65% ; ntx levels increased of 53% at t3 , of 100% at t12 , of 110% at t18 . in group", "b percentage changes from baseline of serum levels of pinp were 50% , 70% and 74% at t3 , t12 , t18 , respectively ; bone alp levels decreased of 30% , 48% and 41% ; ntx levels were reduced by 55% at t3 , of 69% at t12 , of 72% at t18 .", "mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 .", "mean pinp values were 426 g / l at t0 , 8012 g / l at t3 , 10334 g / l at t12 , 9526 g / l at t18 in group a ( t0 vs t3 r : 0.81 , p<0.001 ; t0 vs t12 r : 0.88 , p<0.001 ; t0 vs t18 r : 0.86 , p<0.001 ) and", "7816 g / l , 395 g / l , 2313 g / l , 208 g / l at t0 , t3 , t12 , t18 respectively ( t0 vs t3 r : 0.67 , p<0.001 ; t0 vs t12 r : 0.76 , p<0.001 ; t0 vs t18 r : 0.82 , p<0.001 ) . in group", "u / l , 10720 u / l at t3 , 12245 u / l at t12 and", "11235 u / l at t18 ( t0 vs t3 r : 0.72 , p<0.001 ; t0 vs t12 r : 0.46 , p<0.01 ; t0 vs t18 r : 0.85 , p<0.001 ) ; instead , in group b mean alp was 72 15 u / l at baseline , 5015 after 3 months , 3717 u / l at t12 and 427 u / l at the end of the study ( t0 vs t3 r : 0.91 , p<0.001 ; t0 vs t12 r : 0.53 , p<0.001 ; t0 vs t18 r : 0.65 , p<0.001 ) .", "ntx mean values in group a were 317 nmol / mmol crea , 4721 nmol / mmol crea , 6211 nmol / mmol crea , 6523 nmol / mmol crea at t0 , t3 , t12 and t18 respectively ( t0 vs t3 r : 0.22 , p>0.1 ; t0 vs t12 r : 0.57 , p<0.001 ; t0 vs t18 r : 0.49 , p<0.01 ) while in group b ntx mean values were 534 nmol / mmol crea at t0 , 29 11 nmol / mmol crea at t3 , 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 ( t0 vs t3 r : 0.74 , p<0.001 ; t0 vs t12 r : 0.58 , p<0.001 ; t0 vs t18 r : 0.69 , p<0.001 ) . by the end of the study period ,", "the bmd values expressed in terms of t - score in our total pool displayed important changes ( figure 3 ) . at month 18 ,", "lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% .", "specifically , in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 ( r : 0.88 ; p<0.001 ) ; instead , in group b , mean t - score at t0 was 3.900.73 and mean t - score at t18 : 3.750.72 ( r : 0.98 ; p<0.001 ) .", "the bmd in the femur increased from baseline at month 18 , in group a , by 5.2% and by 1.99% in group b. in group a , mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study ( r : 0.87 ; p<0.001 ) ; in group b , mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 ( r : 0.99 ; p<0.001 ) . at t0 ,", "patients treated with teriparatide were more protected against new fractures , compared with patients treated with bisphosphonates ; in fact , only 1 new vertebral fracture occurred in group a ( 2.4% ) at study endpoint ( t18 ) vs 6 new vertebral fractures that occurred in group b ( 15.7% ) at t18 .", "the most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women ; nausea , reported by 10% ; headache and dizziness that were reported by only 1 and 2 women .", "the reported adverse effects were abdominal pain in 9 patients , arthralgia in 4 patients , and dyspepsia in 1 patient .", "we evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 ( figure 4 ) .", "first domain ( domain a ) result indicated a serious reduction in pain ( 22% ) after treatment with rhpth : mean scores measured was 729.2 at start and 5614 after 18 months ( r : 0.61 ; p<.001 ) compared with group b ( 9.7% ; 718.7 at t0 and 6511 at t18 ; r : 0.53 ; p<.001 ) .", "for everyday activities ( domain b ) , an average of 5513.6 was measured at t0 and of 4022.9 at t18", "( r : 0.44 ; p<.01 ) in group a which had a total improvement of 27.3% , while the improvement was of 11% in group b ( 61.118 at t0 and 54.315.7 at t18 ; r : 0.68 ; p<.001 ) .", "the performed domestic job domain ( domain c ) showed an improvement of 29% in group a ( 6010.7 at t0 ; 42.613.3 at t18 ; r : 0.68 ; p<.001 ) and of 2.9% in group b ( 62.113.3 at t0 ; 60.313.7 at t18 ; r : 0.88 ; p<.001 ) .", "the mean score of domain d ( locomotor function ) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% ( 46.88.6 at t0 ; 41.410.05 at t18 ; r : 0.69 ; p<.001 ) .", "the quality of free time and of the social activities ( domain e ) at the 2 time points they were 36.28.6 and 25.98.6 , indicating a percentage change of 28.4% in group a ( r : 0.50 ; p<.01 ) ; the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only ( r : 0.87 ; p<.001 ) .", "patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health ( domain f ) ( 5925 at t0 ; 3916.7 at t18 ; r : 0.84 ; p<.001 ) versus 12.8% improvement of group b ( 63.228.2 at t0 ; 55.124.8 at t18 ; r : 0.90 ; p<.001 ) . in the mood domain ( domain g ) ,", "the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a ( r : 0.71 ; p<.001 ) .", "these data demonstrated a considerable improvement ( 29.7% ) compared with group b ( 1.8% ; 32.59.8 at t0 ; 33.19.9 at t18 ; r : 0.18 ; p>.1 ) .", "as a consequence of pain relief , the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a , while it did not decrease in group b.", "in group a serum levels of pinp increased of 90% , 145% and 127% at t3 , t12 , t18 , respectively ; bone alp levels increased of 57% , 79% and 65% ; ntx levels increased of 53% at t3 , of 100% at t12 , of 110% at t18 . in group", "b percentage changes from baseline of serum levels of pinp were 50% , 70% and 74% at t3 , t12 , t18 , respectively ; bone alp levels decreased of 30% , 48% and 41% ; ntx levels were reduced by 55% at t3 , of 69% at t12 , of 72% at t18 .", "mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 .", "mean pinp values were 426 g / l at t0 , 8012 g / l at t3 , 10334 g / l at t12 , 9526 g / l at t18 in group a ( t0 vs t3 r : 0.81 , p<0.001 ; t0 vs t12 r : 0.88 , p<0.001 ; t0 vs t18 r : 0.86 , p<0.001 ) and", "7816 g / l , 395 g / l , 2313 g / l , 208 g / l at t0 , t3 , t12 , t18 respectively ( t0 vs t3 r : 0.67 , p<0.001 ; t0 vs t12 r : 0.76 , p<0.001 ; t0 vs t18 r : 0.82 , p<0.001 ) .", "u / l , 10720 u / l at t3 , 12245 u / l at t12 and", "11235 u / l at t18 ( t0 vs t3 r : 0.72 , p<0.001 ; t0 vs t12 r : 0.46 , p<0.01 ; t0 vs t18 r : 0.85 , p<0.001 ) ; instead , in group b mean alp was 72 15 u / l at baseline , 5015 after 3 months , 3717 u / l at t12 and 427 u / l at the end of the study ( t0 vs t3 r : 0.91 , p<0.001 ; t0 vs t12 r : 0.53 , p<0.001 ; t0 vs t18 r : 0.65 , p<0.001 ) .", "ntx mean values in group a were 317 nmol / mmol crea , 4721 nmol / mmol crea , 6211 nmol / mmol crea , 6523 nmol / mmol crea at t0 , t3 , t12 and t18 respectively ( t0 vs t3 r : 0.22 , p>0.1 ; t0 vs t12 r : 0.57 , p<0.001 ; t0 vs t18 r : 0.49 , p<0.01 ) while in group b ntx mean values were 534 nmol / mmol crea at t0 , 29 11 nmol / mmol crea at t3 , 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 ( t0 vs t3 r : 0.74 , p<0.001 ; t0 vs t12 r : 0.58 , p<0.001 ; t0 vs t18 r : 0.69 , p<0.001 ) .", "by the end of the study period , the bmd values expressed in terms of t - score in our total pool displayed important changes ( figure 3 ) . at month 18 ,", "lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% .", "specifically , in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 ( r : 0.88 ; p<0.001 ) ; instead , in group b , mean t - score at t0 was 3.900.73 and mean t - score at t18 : 3.750.72 ( r : 0.98 ; p<0.001 ) .", "the bmd in the femur increased from baseline at month 18 , in group a , by 5.2% and by 1.99% in group b. in group a , mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study ( r : 0.87 ; p<0.001 ) ; in group b , mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 ( r : 0.99 ; p<0.001 ) .", "at t0 , all patients of groups a and b showed baseline vertebral fractures . patients treated with teriparatide were more protected against new fractures , compared with patients treated with bisphosphonates ; in fact , only 1 new vertebral fracture occurred in group a ( 2.4% ) at study endpoint ( t18 ) vs 6 new vertebral fractures that occurred in group b ( 15.7% ) at t18 .", "the most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women ; nausea , reported by 10% ; headache and dizziness that were reported by only 1 and 2 women .", "the reported adverse effects were abdominal pain in 9 patients , arthralgia in 4 patients , and dyspepsia in 1 patient .", "we evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 ( figure 4 ) . first domain ( domain", "a ) result indicated a serious reduction in pain ( 22% ) after treatment with rhpth : mean scores measured was 729.2 at start and 5614 after 18 months ( r : 0.61 ; p<.001 ) compared with group b ( 9.7% ; 718.7 at t0 and 6511 at t18 ; r : 0.53 ; p<.001 ) . for everyday activities ( domain b ) , an average of 5513.6 was measured at t0 and of 4022.9 at t18 ( r : 0.44 ; p<.01 ) in group a which had a total improvement of 27.3% , while the improvement was of 11% in group b ( 61.118 at t0 and 54.315.7 at t18 ; r : 0.68 ; p<.001 ) .", "the performed domestic job domain ( domain c ) showed an improvement of 29% in group a ( 6010.7 at t0 ; 42.613.3 at t18 ; r : 0.68 ; p<.001 ) and of 2.9% in group b ( 62.113.3 at t0 ; 60.313.7 at t18 ; r : 0.88 ; p<.001 ) .", "the mean score of domain d ( locomotor function ) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% ( 46.88.6 at t0 ; 41.410.05 at t18 ; r : 0.69 ; p<.001 ) .", "the quality of free time and of the social activities ( domain e ) at the 2 time points they were 36.28.6 and 25.98.6 , indicating a percentage change of 28.4% in group a ( r : 0.50 ; p<.01 ) ; the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only ( r : 0.87 ; p<.001 ) .", "patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health ( domain f ) ( 5925 at t0 ; 3916.7 at t18 ; r : 0.84 ; p<.001 ) versus 12.8% improvement of group b ( 63.228.2 at t0 ; 55.124.8 at t18 ; r : 0.90 ; p<.001 ) . in the mood domain ( domain g ) , the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a ( r : 0.71 ; p<.001 ) .", "these data demonstrated a considerable improvement ( 29.7% ) compared with group b ( 1.8% ; 32.59.8 at t0 ; 33.19.9 at t18 ; r : 0.18 ; p>.1 ) .", "as a consequence of pain relief , the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a , while it did not decrease in group b.", "our results demonstrate that daily injections of rhpth for 18 months are an efficacious and generally well - tolerated therapy in postmenopausal women with severe osteoporosis . more importantly , in our experience rhpth therapy results in decreased fractures and pain symptoms in patients with osteoporosis , and", "teriparatide is a bone - forming agent and its effect is demonstrated by increases in biochemical markers of bone turnover over the study period : bone formation markers showed more rapid and higher increases than resorption ones , suggesting an early imbalance of bone turnover in favor of formation ; these data are in agreement with previous studies .", "treatment with teriparatide resulted in a greater increase in bone mineral density ( bmd ) .", "after 18 months of therapy with rhpth , bone mineral density in the lumbar spine and of the proximal femur increased by 12.4% and 5.2% ; these reported percentage increases are consistent with results of other studies . at month 18 in patients treated with bisphosphonates , instead , lumbar spine increased only by 3.85% and the bone mineral density in the femur increased by 1.99% .", "the differences in the percentage increases between the 2 groups of patients can be explained with substantial differences between antiresorptive and anabolic therapeutic effects on bone mass and architecture as well as on bone mineral density .", "moreover , rhpth increases trabecular connectivity , whereas the majority of bone mineral density increases observed with bisphosphonate treatment are a result of increased mineralization of existing bone matrix .", "teriparatide has been referred to be an efficacious treatment against new fractures , making a significant change in the course of severe osteoporosis , which can lead rapidly not only to varying degrees of disability , the loss of self - sufficiency , and to institutionalization , but also to death [ 3436 ] . in this study , we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms .", "in addition , use of teriparatide caused a considerable decrease of pain , accompanied by a consequent reduction of the need for nonsteroidal anti - inflammatory drugs by the patients ; this was the main factor that assured an absolute compliance of the patients .", "in severe osteoporosis vertebral fractures are an important cause of back pain owing to muscle weakness and altered posture , resulting in serious acute and chronic pain that contributes to further disabilities ; in fact , vertebral fractures are the top health condition accounting for length of hospitalization , and added significantly to the length of hospitalization in patients admitted for other medical problems . apart from the physical disabilities had by these patients", "in fact , patients with vertebral fractures often experience impaired physical functions , limited activities of daily living , limited leisure and recreational activities , and significant emotional distress with loss of self - esteem and depression [ 3436 ] . in this study", ", we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms . to analyze all these variables and to measure the values of qol at baseline and at the end of the study , we used the qualeffo - test , developed by lips and associates in 1997 .", "specifically , we used qualeffo-41 , that represents a qol questionnaire which is brief , easy to administer , and with adequate preliminary psychometric properties .", "we preferred this test to the generic ones ( such as nhp , sip , sf-36 , eq - sd ) because of its specificity in the evaluation of qol in people affected by osteoporosis with vertebral deformities ; in fact , disadvantages of generic instruments are that they usually include irrelevant questions and that these questionnaires are less able to investigate those aspects that mostly influence qol of osteoporotic patients [ 2628 ] .", "this makes the qualeffo-41 potentially useful during routine clinical practice and research for the treatment and the follow - up of postmenopausal women with severe osteoporosis . by using this test", ", our study evidences that the use of rhpth influences all considered domains of qol ; in fact , pain , physical and social functions , mood significantly improved in nearly all patients treated with rhpth at the end of the study .", "thus , the reduction of back pain observed from baseline of active treatment through posttreatment follow - up is consistent with the reduction of new vertebral painful fractures , that means improved qol for this kind of patients .", "our data clearly demonstrate that rhpth is more effective than bisphosphonates in acting on back pain and all the domains of qol .", "adverse events ( back pain , headache , nausea , dizziness ) in our patients receiving rhpth were mild and transient , with a percentage lower than in previous studies .", "the adverse effects attributed to teriparatide did not stop the patients from continuing with the treatment .", "our results demonstrate that rhpth increases bone mineral density considerably , reduces the occurrence of new fractures , and the need for analgesic therapy .", "therefore , this study evidences that this anabolic agent represents a valid therapeutic option in severe postmenopausal osteoporosis ; moreover , patients after this treatment experience , improvements not only in pain relief , but also in certain aspects of emotional functioning , activities of daily living , and leisure activities , improve qol considerably ."], "abstract_text": ["<S> summarybackgroundwe studied the use of teriparatide in postmenopausal women with severe osteoporosis.material/methodstwo groups ( a and b ) of patients affected by severe osteoporosis ( t - score 2.5 at bone mineral density were analyzed and 2 vertebral fractures on radiograph).group a was treated for 18 months with 20 g / day of teriparatide . </S>", "<S> group b was treated with bisphosphonates 70 mg / week . </S>", "<S> every woman assumed 1 g of calcium and 800 iu of vitamin d3 daily . </S>", "<S> we evaluated the effects of therapy after 18 months ( t18 ) from the beginning with bone turnover markers ( alkaline phosphatase , procollagen type 1 n - terminal propeptide , and n - telopeptide cross - links ) and dual - energy x - ray absorptiometry.resultsgroup a , at t18 procollagen type 1 n - terminal propeptide levels , increased 127% ; bone alkaline phosphatase levels increased to 65% ; n - telopeptide cross - links levels increased to 110%.group b , at t18 procollagen type 1 n - terminal propeptide levels , decreased to 74% ; bone alkaline phosphatase levels decreased to 41% ; n - telopeptide cross - links levels decreased to 72%.after 18 months , lumbar bone mineral density increased to 12.4% and femoral bone mineral density increased to 5.2% in group a. group b lumbar bone mineral density increased to 3.85% and femoral bone mineral density increased to 1.99% . </S>", "<S> only a new vertebral fracture occurred in group a ( 2.4% ) , whereas 6 fractures occurred in group b ( 15.7%).the quality of life questionnaire of the european foundation for osteoporosis ( qualeffo ) revealed a significant improvement in daily living , performed domestic jobs , and locomotor function in groups a and b.conclusionsthe use of rhpth in patients with severe osteoporosis offers more protection against fractures and improves the qol more than bisphosphonates . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Results", "Markers of bone turnover", "Bone mineral density", "X-ray evaluation", "Adverse events", "Quality of life", "Discussion", "Conclusions"], "sections": [["osteoporosis is a systemic disease leading to progressive decrease in bone mineral density , decreased bone strength , and increased risk of skeletal fractures .", "approximately 30% of women have sustained at least 1 vertebral fracture by the age of 75 .", "these fractures are an important and common cause of morbidity in osteoporotic patients ; moreover , fractures evidenced both clinically and at radiographic examination are associated with an increased mortality rate .", "back pain owing to vertebral fractures has a significant affect on osteoporotic patients [ 46 ] , and the number and severity of these fractures also increases the risk of developing chronic back pain .", "this has a marked negative impact on the quality of life ( qol ) and functional impairment of the affected patients .", "conventional treatments for osteoporosis including bisphosphonates , selective estrogen receptor modulators ( serms ) , and estrogen have been shown to reduce the rate of bone resorption and to preserve bone mass .", "another therapeutic option includes rhpth , an agent that has been shown to increase both bone mass and bone strength .", "when injected , teriparatide ( rdna origin ) , the amino - terminal fragment of human pth ( rhpth 134 ) , is a potent bone formation agent for the treatment of severe osteoporosis .", "it increases osteoblast production / growth and prevents osteoblast apoptosis ; at the same time , it enhances absorption of calcium from the intestine , renal reabsorption of calcium , while decreasing the excretion of phosphates in the kidney [ 1114 ] .", "when administered once daily by subcutaneous injection , rhpth increases bone density and improves trabecular architecture , cortical geometry , and strength .", "not only does rhpth increase trabecular bone density by stimulating bone formation , but it also stimulates osteoclastic bone resorption [ 1719 ] . in several studies , rhpth has been shown to increase bone mineral density in postmenopausal osteoporosis , in senile osteoporosis in men , and in glucocorticoid - induced osteoporosis .", "teriparatide acts via the pth-1 receptor on osteoblasts and bone marrow stromal cells to induce osteoblastic bone formation , that is , osteoid synthesis and accelerated mineralization .", "this , in turn , results in reductions in skeletal fractures to levels equivalent to , or over , those obtained by using antiresorptive agents .", "the increase in bone mineral density induced by rhpth is substantial , ranging 10% to 15% over 2 to 3 years in most studies [ 10,2024 ] . moreover , rhpth can cause demonstrable increases in bone mineral density and changes in markers of bone turnover within 3 months since the start of treatment .", "quality of life ( qol ) can be measured to compare the effect of different treatments for osteoporosis .", "measuring pain scores only for these patients would be inadequate , because apart from acute and chronic back pain , patients with vertebral fractures also experience anxiety , constant fear of falling , or suffering another fracture while their daily living activities are impaired .", "most information has been collected thanks to the efforts made by some researchers to develop specific instruments to test the physical and emotional disability generated by the disease .", "generic tools available for measuring qol are useful to evaluate general health but they lack disease specificity .", "more recently , some specific instruments have been developed to measure the qol in osteoporosis more accurately .", "the most widely used are the osteoporosis quality of life questionnaire ( oqlq ) , the osteoporosis assessment questionnaire ( opaq ) , the osteoporosis - targeted quality of life questionnaire ( optqol ) , and the quality of life questionnaire of the european foundation for osteoporosis ( qualeffo-41 ) .", "one of the first ones was qualeffo-41 , which has been translated and validated in different languages including italian .", "this questionnaire has proved to be repeatable , coherent , and able to discriminate between patients and controls . in the last few years , other specific questionnaires", "have been developed , but not all of them have been as extensively used and validated in different countries as qualeffo-41 .", "the goal of this study was firstly to assess the validity of rhpth treatment in a cohort of postmenopausal women with severe osteoporosis ; secondly , to evaluate the improvement in qol and pain symptoms after several months of rhpth therapy .", "a follow - up qualeffo-41 questionnaire was used to quantify the patient s pain and the affect on qol after rhpth therapy ."], ["the study was a 18 months , randomized prospective cohort study conducted at department of molecular and clinical endocrinology and oncology , university of naples federico ii , naples , italy .", "inclusion criteria for this study consisted of back pain , postmenopausal osteoporosis ( t - score 2.5 at lumbar spine or femoral neck ) , the presence of 2 osteoporotic vertebral fractures , previous treatment for osteoporosis .", "the exclusion criteria were : an increased risk of osteosarcoma ( ie , patients with paget disease bone , previous skeletal exposure to external beam radiotherapy , or previous malignant neoplasm involving the skeleton ) , hypercalcemia , malignant neoplasms , impaired renal function , liver disease , history of diseases other than postmenopausal osteoporosis that affect bone metabolism , nephrolithiasis , alcohol or drug abuse .", "secondary osteoporosis was excluded in order to avoid the interference of the primitive disease with the patient s quality of life .", "informed consent was obtained from all subjects and the study protocol was approved by the hospital / science s ethical committee .", "eighty - one postmenopausal women were enrolled and divided in two groups with no statistically significant differences in any of the considered variables : group a  forty - two women ( mean age 659 yrs ; mean body mass index ", "bmi 24.52.6 kg / m ) , with severe postmenopausal osteoporosis ( mean lumbar bmd 3.880.70 , mean femoral neck bmd 3.070.60 and with 2 vertebral atraumatic fractures ) , persistent back pain and previous treatment with biphosphonates for osteoporosis ; group b  thirty - nine women matched for age ( 6014.4 yrs ) , bmi ( 22.88.8 kg / m ) , menopausal status , affected by back pain , severe postmenopausal osteoporosis ( lumbar spine bmd 3.900.73 , mean femoral neck bmd 3.020.61 and with 2 vertebral atraumatic fractures ) previously treated for osteoporosis with biphosphonates .", "bmi , age at menopause , lifestyle habits ( i.e. , smoking , drinking , nutrition style ) , nutrition anamnesis ( calcium intake ) , history of diseases other than osteoporosis , family history of osteoporosis were considered .", "groups were randomized to daily treatment with 20 g s.c . of recombinant human parathyroid hormone ( rhpth 134 ) , self - administered injections ( group a ) or 70 mg per os of alendronate every week ( group b ) .", "all women received 1,000 mg elemental calcium daily and 800 iu of vitamin d daily for 18 months .", "biochemical markers of bone turnover were dosed in all the selected female patients : alkaline phosphatase ( alp : 35104", "u / l ) , n - terminal propeptide of type i procollagen ( pinp : 19102 g / l ) , n - telopeptide crosslinks ( ntx : 565 nmol / mmol crea ) were assessed at baseline , 3 , 12 and 18 months ( t0 , t3 , t12 , t18 ) .", "the bmd of the lumbar spine and the proximal femur was measured by dual - energy x - ray absorptiometry ( dual - energy x - ray absorptiometry qdr 1000 ; hologic , waltham , ma , usa ) at baseline and at 18 months ( t0 , t18 ) .", "all women underwent anteroposterior and lateral radiography of thoracic and lumbar spine at t0 and t18 .", "the qol questionnaire of the european foundation for osteoporosis ( qualeffo ) was administered at baseline and at the end of the study to evaluate the impact of rhpth on health - related qol .", "originally , the questionnaire consisted of 48 questions and 6 visual analogue scales . in the qualeffo validation study ,", "this resulted in the qualeffo-41 , which consisted of 41 questions in 5 domains : pain , physical function , social function , general health perception , and mental function .", "all answers were recorded so that all items range from 1 to 5 , and all answers were standardized so that 1 represents the best and 5 the worst qol , with the exception of questions e23 - 25 ( questions with 3 answer options ) , questions e24 - 26 - 27 - 28 ( 4 answer options ) , and questions g33 - 34 - 35 - 37 - 39 - 40 ( answer with reverse scores : 1 is the worst while 5 is the best ) .", "domain scores are calculated by averaging the answers of 1 domain and transforming the scores to a score from 0 to 100 .", "data are expressed as mean  sd or percentage ; moreover , to assess the affect of teriparatide treatment on markers of bone turnover , bone mineral density , and on health - related qol , pearson correlation coefficients were computed in the 2 study groups .", "therefore , percentage changes of biomarkers , bone mineral density , and qualeffo results were calculated ."], ["there was no significant difference in these characteristics between the 2 groups . in the group a , 39 ( 93% ) out of the 42 recruited women completed the study : 2 patients discontinued the drug therapy because of lack of compliance with the study treatment ; 1 patient discontinued because of a new vertebral atraumatic fracture .", "the aim of this study was to evaluate percentage changes from baseline in biochemical markers of bone turnover , values of bone mineral density ( measured at lumbar spine and proximal femur ) , and measurements of qol .", "follow - up checks , where we also evaluated clinical conditions , adverse events , compliance to treatment and use of non - steroid anti - inflammatory drugs , were carried out at 3 , 12 and 18 months since the beginning of treatment . in group a serum levels of pinp increased of 90% , 145% and 127% at t3 , t12 , t18 , respectively ; bone alp levels increased of 57% , 79% and 65% ; ntx levels increased of 53% at t3 , of 100% at t12 , of 110% at t18 . in group", "b percentage changes from baseline of serum levels of pinp were 50% , 70% and 74% at t3 , t12 , t18 , respectively ; bone alp levels decreased of 30% , 48% and 41% ; ntx levels were reduced by 55% at t3 , of 69% at t12 , of 72% at t18 .", "mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 .", "mean pinp values were 426 g / l at t0 , 8012 g / l at t3 , 10334 g / l at t12 , 9526 g / l at t18 in group a ( t0 vs t3 r : 0.81 , p<0.001 ; t0 vs t12 r : 0.88 , p<0.001 ; t0 vs t18 r : 0.86 , p<0.001 ) and", "7816 g / l , 395 g / l , 2313 g / l , 208 g / l at t0 , t3 , t12 , t18 respectively ( t0 vs t3 r : 0.67 , p<0.001 ; t0 vs t12 r : 0.76 , p<0.001 ; t0 vs t18 r : 0.82 , p<0.001 ) . in group", "u / l , 10720 u / l at t3 , 12245 u / l at t12 and", "11235 u / l at t18 ( t0 vs t3 r : 0.72 , p<0.001 ; t0 vs t12 r : 0.46 , p<0.01 ; t0 vs t18 r : 0.85 , p<0.001 ) ; instead , in group b mean alp was 72 15 u / l at baseline , 5015 after 3 months , 3717 u / l at t12 and 427 u / l at the end of the study ( t0 vs t3 r : 0.91 , p<0.001 ; t0 vs t12 r : 0.53 , p<0.001 ; t0 vs t18 r : 0.65 , p<0.001 ) .", "ntx mean values in group a were 317 nmol / mmol crea , 4721 nmol / mmol crea , 6211 nmol / mmol crea , 6523 nmol / mmol crea at t0 , t3 , t12 and t18 respectively ( t0 vs t3 r : 0.22 , p>0.1 ; t0 vs t12 r : 0.57 , p<0.001 ; t0 vs t18 r : 0.49 , p<0.01 ) while in group b ntx mean values were 534 nmol / mmol crea at t0 , 29 11 nmol / mmol crea at t3 , 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 ( t0 vs t3 r : 0.74 , p<0.001 ; t0 vs t12 r : 0.58 , p<0.001 ; t0 vs t18 r : 0.69 , p<0.001 ) . by the end of the study period ,", "the bmd values expressed in terms of t - score in our total pool displayed important changes ( figure 3 ) . at month 18 ,", "lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% .", "specifically , in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 ( r : 0.88 ; p<0.001 ) ; instead , in group b , mean t - score at t0 was 3.900.73 and mean t - score at t18 : 3.750.72 ( r : 0.98 ; p<0.001 ) .", "the bmd in the femur increased from baseline at month 18 , in group a , by 5.2% and by 1.99% in group b. in group a , mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study ( r : 0.87 ; p<0.001 ) ; in group b , mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 ( r : 0.99 ; p<0.001 ) . at t0 ,", "patients treated with teriparatide were more protected against new fractures , compared with patients treated with bisphosphonates ; in fact , only 1 new vertebral fracture occurred in group a ( 2.4% ) at study endpoint ( t18 ) vs 6 new vertebral fractures that occurred in group b ( 15.7% ) at t18 .", "the most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women ; nausea , reported by 10% ; headache and dizziness that were reported by only 1 and 2 women .", "the reported adverse effects were abdominal pain in 9 patients , arthralgia in 4 patients , and dyspepsia in 1 patient .", "we evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 ( figure 4 ) .", "first domain ( domain a ) result indicated a serious reduction in pain ( 22% ) after treatment with rhpth : mean scores measured was 729.2 at start and 5614 after 18 months ( r : 0.61 ; p<.001 ) compared with group b ( 9.7% ; 718.7 at t0 and 6511 at t18 ; r : 0.53 ; p<.001 ) .", "for everyday activities ( domain b ) , an average of 5513.6 was measured at t0 and of 4022.9 at t18", "( r : 0.44 ; p<.01 ) in group a which had a total improvement of 27.3% , while the improvement was of 11% in group b ( 61.118 at t0 and 54.315.7 at t18 ; r : 0.68 ; p<.001 ) .", "the performed domestic job domain ( domain c ) showed an improvement of 29% in group a ( 6010.7 at t0 ; 42.613.3 at t18 ; r : 0.68 ; p<.001 ) and of 2.9% in group b ( 62.113.3 at t0 ; 60.313.7 at t18 ; r : 0.88 ; p<.001 ) .", "the mean score of domain d ( locomotor function ) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% ( 46.88.6 at t0 ; 41.410.05 at t18 ; r : 0.69 ; p<.001 ) .", "the quality of free time and of the social activities ( domain e ) at the 2 time points they were 36.28.6 and 25.98.6 , indicating a percentage change of 28.4% in group a ( r : 0.50 ; p<.01 ) ; the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only ( r : 0.87 ; p<.001 ) .", "patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health ( domain f ) ( 5925 at t0 ; 3916.7 at t18 ; r : 0.84 ; p<.001 ) versus 12.8% improvement of group b ( 63.228.2 at t0 ; 55.124.8 at t18 ; r : 0.90 ; p<.001 ) . in the mood domain ( domain g ) ,", "the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a ( r : 0.71 ; p<.001 ) .", "these data demonstrated a considerable improvement ( 29.7% ) compared with group b ( 1.8% ; 32.59.8 at t0 ; 33.19.9 at t18 ; r : 0.18 ; p>.1 ) .", "as a consequence of pain relief , the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a , while it did not decrease in group b."], ["in group a serum levels of pinp increased of 90% , 145% and 127% at t3 , t12 , t18 , respectively ; bone alp levels increased of 57% , 79% and 65% ; ntx levels increased of 53% at t3 , of 100% at t12 , of 110% at t18 . in group", "b percentage changes from baseline of serum levels of pinp were 50% , 70% and 74% at t3 , t12 , t18 , respectively ; bone alp levels decreased of 30% , 48% and 41% ; ntx levels were reduced by 55% at t3 , of 69% at t12 , of 72% at t18 .", "mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 .", "mean pinp values were 426 g / l at t0 , 8012 g / l at t3 , 10334 g / l at t12 , 9526 g / l at t18 in group a ( t0 vs t3 r : 0.81 , p<0.001 ; t0 vs t12 r : 0.88 , p<0.001 ; t0 vs t18 r : 0.86 , p<0.001 ) and", "7816 g / l , 395 g / l , 2313 g / l , 208 g / l at t0 , t3 , t12 , t18 respectively ( t0 vs t3 r : 0.67 , p<0.001 ; t0 vs t12 r : 0.76 , p<0.001 ; t0 vs t18 r : 0.82 , p<0.001 ) .", "u / l , 10720 u / l at t3 , 12245 u / l at t12 and", "11235 u / l at t18 ( t0 vs t3 r : 0.72 , p<0.001 ; t0 vs t12 r : 0.46 , p<0.01 ; t0 vs t18 r : 0.85 , p<0.001 ) ; instead , in group b mean alp was 72 15 u / l at baseline , 5015 after 3 months , 3717 u / l at t12 and 427 u / l at the end of the study ( t0 vs t3 r : 0.91 , p<0.001 ; t0 vs t12 r : 0.53 , p<0.001 ; t0 vs t18 r : 0.65 , p<0.001 ) .", "ntx mean values in group a were 317 nmol / mmol crea , 4721 nmol / mmol crea , 6211 nmol / mmol crea , 6523 nmol / mmol crea at t0 , t3 , t12 and t18 respectively ( t0 vs t3 r : 0.22 , p>0.1 ; t0 vs t12 r : 0.57 , p<0.001 ; t0 vs t18 r : 0.49 , p<0.01 ) while in group b ntx mean values were 534 nmol / mmol crea at t0 , 29 11 nmol / mmol crea at t3 , 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 ( t0 vs t3 r : 0.74 , p<0.001 ; t0 vs t12 r : 0.58 , p<0.001 ; t0 vs t18 r : 0.69 , p<0.001 ) ."], ["by the end of the study period , the bmd values expressed in terms of t - score in our total pool displayed important changes ( figure 3 ) . at month 18 ,", "lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% .", "specifically , in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 ( r : 0.88 ; p<0.001 ) ; instead , in group b , mean t - score at t0 was 3.900.73 and mean t - score at t18 : 3.750.72 ( r : 0.98 ; p<0.001 ) .", "the bmd in the femur increased from baseline at month 18 , in group a , by 5.2% and by 1.99% in group b. in group a , mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study ( r : 0.87 ; p<0.001 ) ; in group b , mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 ( r : 0.99 ; p<0.001 ) ."], ["at t0 , all patients of groups a and b showed baseline vertebral fractures . patients treated with teriparatide were more protected against new fractures , compared with patients treated with bisphosphonates ; in fact , only 1 new vertebral fracture occurred in group a ( 2.4% ) at study endpoint ( t18 ) vs 6 new vertebral fractures that occurred in group b ( 15.7% ) at t18 ."], ["the most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women ; nausea , reported by 10% ; headache and dizziness that were reported by only 1 and 2 women .", "the reported adverse effects were abdominal pain in 9 patients , arthralgia in 4 patients , and dyspepsia in 1 patient ."], ["we evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 ( figure 4 ) . first domain ( domain", "a ) result indicated a serious reduction in pain ( 22% ) after treatment with rhpth : mean scores measured was 729.2 at start and 5614 after 18 months ( r : 0.61 ; p<.001 ) compared with group b ( 9.7% ; 718.7 at t0 and 6511 at t18 ; r : 0.53 ; p<.001 ) . for everyday activities ( domain b ) , an average of 5513.6 was measured at t0 and of 4022.9 at t18 ( r : 0.44 ; p<.01 ) in group a which had a total improvement of 27.3% , while the improvement was of 11% in group b ( 61.118 at t0 and 54.315.7 at t18 ; r : 0.68 ; p<.001 ) .", "the performed domestic job domain ( domain c ) showed an improvement of 29% in group a ( 6010.7 at t0 ; 42.613.3 at t18 ; r : 0.68 ; p<.001 ) and of 2.9% in group b ( 62.113.3 at t0 ; 60.313.7 at t18 ; r : 0.88 ; p<.001 ) .", "the mean score of domain d ( locomotor function ) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% ( 46.88.6 at t0 ; 41.410.05 at t18 ; r : 0.69 ; p<.001 ) .", "the quality of free time and of the social activities ( domain e ) at the 2 time points they were 36.28.6 and 25.98.6 , indicating a percentage change of 28.4% in group a ( r : 0.50 ; p<.01 ) ; the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only ( r : 0.87 ; p<.001 ) .", "patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health ( domain f ) ( 5925 at t0 ; 3916.7 at t18 ; r : 0.84 ; p<.001 ) versus 12.8% improvement of group b ( 63.228.2 at t0 ; 55.124.8 at t18 ; r : 0.90 ; p<.001 ) . in the mood domain ( domain g ) , the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a ( r : 0.71 ; p<.001 ) .", "these data demonstrated a considerable improvement ( 29.7% ) compared with group b ( 1.8% ; 32.59.8 at t0 ; 33.19.9 at t18 ; r : 0.18 ; p>.1 ) .", "as a consequence of pain relief , the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a , while it did not decrease in group b."], ["our results demonstrate that daily injections of rhpth for 18 months are an efficacious and generally well - tolerated therapy in postmenopausal women with severe osteoporosis . more importantly , in our experience rhpth therapy results in decreased fractures and pain symptoms in patients with osteoporosis , and", "teriparatide is a bone - forming agent and its effect is demonstrated by increases in biochemical markers of bone turnover over the study period : bone formation markers showed more rapid and higher increases than resorption ones , suggesting an early imbalance of bone turnover in favor of formation ; these data are in agreement with previous studies .", "treatment with teriparatide resulted in a greater increase in bone mineral density ( bmd ) .", "after 18 months of therapy with rhpth , bone mineral density in the lumbar spine and of the proximal femur increased by 12.4% and 5.2% ; these reported percentage increases are consistent with results of other studies . at month 18 in patients treated with bisphosphonates , instead , lumbar spine increased only by 3.85% and the bone mineral density in the femur increased by 1.99% .", "the differences in the percentage increases between the 2 groups of patients can be explained with substantial differences between antiresorptive and anabolic therapeutic effects on bone mass and architecture as well as on bone mineral density .", "moreover , rhpth increases trabecular connectivity , whereas the majority of bone mineral density increases observed with bisphosphonate treatment are a result of increased mineralization of existing bone matrix .", "teriparatide has been referred to be an efficacious treatment against new fractures , making a significant change in the course of severe osteoporosis , which can lead rapidly not only to varying degrees of disability , the loss of self - sufficiency , and to institutionalization , but also to death [ 3436 ] . in this study , we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms .", "in addition , use of teriparatide caused a considerable decrease of pain , accompanied by a consequent reduction of the need for nonsteroidal anti - inflammatory drugs by the patients ; this was the main factor that assured an absolute compliance of the patients .", "in severe osteoporosis vertebral fractures are an important cause of back pain owing to muscle weakness and altered posture , resulting in serious acute and chronic pain that contributes to further disabilities ; in fact , vertebral fractures are the top health condition accounting for length of hospitalization , and added significantly to the length of hospitalization in patients admitted for other medical problems . apart from the physical disabilities had by these patients", "in fact , patients with vertebral fractures often experience impaired physical functions , limited activities of daily living , limited leisure and recreational activities , and significant emotional distress with loss of self - esteem and depression [ 3436 ] . in this study", ", we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms . to analyze all these variables and to measure the values of qol at baseline and at the end of the study , we used the qualeffo - test , developed by lips and associates in 1997 .", "specifically , we used qualeffo-41 , that represents a qol questionnaire which is brief , easy to administer , and with adequate preliminary psychometric properties .", "we preferred this test to the generic ones ( such as nhp , sip , sf-36 , eq - sd ) because of its specificity in the evaluation of qol in people affected by osteoporosis with vertebral deformities ; in fact , disadvantages of generic instruments are that they usually include irrelevant questions and that these questionnaires are less able to investigate those aspects that mostly influence qol of osteoporotic patients [ 2628 ] .", "this makes the qualeffo-41 potentially useful during routine clinical practice and research for the treatment and the follow - up of postmenopausal women with severe osteoporosis . by using this test", ", our study evidences that the use of rhpth influences all considered domains of qol ; in fact , pain , physical and social functions , mood significantly improved in nearly all patients treated with rhpth at the end of the study .", "thus , the reduction of back pain observed from baseline of active treatment through posttreatment follow - up is consistent with the reduction of new vertebral painful fractures , that means improved qol for this kind of patients .", "our data clearly demonstrate that rhpth is more effective than bisphosphonates in acting on back pain and all the domains of qol .", "adverse events ( back pain , headache , nausea , dizziness ) in our patients receiving rhpth were mild and transient , with a percentage lower than in previous studies .", "the adverse effects attributed to teriparatide did not stop the patients from continuing with the treatment ."], ["our results demonstrate that rhpth increases bone mineral density considerably , reduces the occurrence of new fractures , and the need for analgesic therapy .", "therefore , this study evidences that this anabolic agent represents a valid therapeutic option in severe postmenopausal osteoporosis ; moreover , patients after this treatment experience , improvements not only in pain relief , but also in certain aspects of emotional functioning , activities of daily living , and leisure activities , improve qol considerably ."]]}
{"article_id": "PMC3631788", "article_text": ["a 22 years old female patient was admitted to our outpatient clinic with a complaint of dyspnea for the previous three years .", "a right bundle branch block was detected on her electrocardiogram . in the transthorasic echocardiography ( tte ) ,", "a 22 mm secundum type atrial septal defect ( asd ) , right heart dilatation and pulmonary arterial hypertension ( pap 45 mmhg ) were detected .", "transesophageal echocardiography ( tee ) showed that the defect was 25 mm , the superior rim was 16 mm and the aortic rim was 8 mm in size .", "the defect was closed through a right femoral route with a 30 mm asd occluder after balloon sizing with tte . in the cineangiography and tte", "early after the procedure , the device was observed to be in the correct position ( fig .", "the tte revealed that the device was not in the defect and it had embolized to the pulmonary artery .", "the patient was referred to the cardiovascular surgery unit and underwent surgery promptly . under general anaesthesia", "the pulmonary artery was incised along its vertical axis and the occluder device was removed ( fig .", "the right atrium was opened and the defect was then closed with a pericardial patch .", "secundum asd is the most common type , which accounts for 50% to 70% of all asds and located in the fossa ovalis .", "sinus venosus type asd is located at the upper portion of the interatrial septum where the vena cava superior opens .", "a shunt between atria leads to chronic right ventricular volume overload , which in turn causes lower filling rates of left ventricle .", "surgical or percutaneous closure in asymptomatic patients is indicated in the case of right ventricular volume overload and if the pulmonary to systemic flow ratio increases beyond 1.5 . in adult patients ,", "the defect should be repaired as soon as possible after it is detected because patients who undergo repair after 25 years of age have a shorter life span than a control population .", "the most important factor for success in percutaneous closure is the correct patient selection . in patients with secundum asd , percutaneous", "closure can be performed safely with careful evaluation of anatomical factors like defect size and sufficiency of the rims . in a previous report", ", it was shown that atrial defects up to 43 mm in diameter can be closed percutaneously .", "a sufficient anterior rim is not necessary to bring the device into a stable position .", "conversely , deploying the device in patients with deficient cranial rims can be difficult and may lead to an unstable position of the device and increased risk of device embolization .", "the most common type of complications in percutaneous closure is device embolization , with a rate of 0.5% to 1 % . in a study with 450 patients , seven cases of device embolization into pulmonary artery in the first 12 hours", "one hour after intervention we detected device embolization into the pulmonary artery after performing echocardiography since the patient had dyspnea .", "after that , emergency surgery was performed within one hour with a median sternotomy and under cardiopulmonary bypass .", ", snares can be used to take out the device , but most of the time , surgical extraction of the device and asd patch closure is needed .", "percutaneous asd closure is a widely used and has been performed successfully throughout the world in recent years .", "however , it should be kept in mind that improper patient and device selection may result in deadly complications like device embolization , cardiac perforation and tamponade ."], "abstract_text": ["<S> an atrial septal defect is the most common type of congenital heart disease among adults . </S>", "<S> surgical repair or percutaneous closure of the defect is the treatment options . </S>", "<S> even though percutaneous closure seems to be less risky than surgical repair , it may result in fatal complications like device embolism , cardiac perforation and tamponade . </S>", "<S> herein we report a case of the embolism of a device into the pulmonary artery after one hour of percutaneous closure in which the embolized device was surgically removed and the defect was closed with a pericardial patch . </S>"], "labels": null, "section_names": ["CASE REPORT", "DISCUSSION"], "sections": [["a 22 years old female patient was admitted to our outpatient clinic with a complaint of dyspnea for the previous three years .", "a right bundle branch block was detected on her electrocardiogram . in the transthorasic echocardiography ( tte ) ,", "a 22 mm secundum type atrial septal defect ( asd ) , right heart dilatation and pulmonary arterial hypertension ( pap 45 mmhg ) were detected .", "transesophageal echocardiography ( tee ) showed that the defect was 25 mm , the superior rim was 16 mm and the aortic rim was 8 mm in size .", "the defect was closed through a right femoral route with a 30 mm asd occluder after balloon sizing with tte . in the cineangiography and tte", "early after the procedure , the device was observed to be in the correct position ( fig .", "the tte revealed that the device was not in the defect and it had embolized to the pulmonary artery .", "the patient was referred to the cardiovascular surgery unit and underwent surgery promptly . under general anaesthesia", "the pulmonary artery was incised along its vertical axis and the occluder device was removed ( fig .", "the right atrium was opened and the defect was then closed with a pericardial patch ."], ["secundum asd is the most common type , which accounts for 50% to 70% of all asds and located in the fossa ovalis .", "sinus venosus type asd is located at the upper portion of the interatrial septum where the vena cava superior opens .", "a shunt between atria leads to chronic right ventricular volume overload , which in turn causes lower filling rates of left ventricle .", "surgical or percutaneous closure in asymptomatic patients is indicated in the case of right ventricular volume overload and if the pulmonary to systemic flow ratio increases beyond 1.5 . in adult patients ,", "the defect should be repaired as soon as possible after it is detected because patients who undergo repair after 25 years of age have a shorter life span than a control population .", "the most important factor for success in percutaneous closure is the correct patient selection . in patients with secundum asd , percutaneous", "closure can be performed safely with careful evaluation of anatomical factors like defect size and sufficiency of the rims . in a previous report", ", it was shown that atrial defects up to 43 mm in diameter can be closed percutaneously .", "a sufficient anterior rim is not necessary to bring the device into a stable position .", "conversely , deploying the device in patients with deficient cranial rims can be difficult and may lead to an unstable position of the device and increased risk of device embolization .", "the most common type of complications in percutaneous closure is device embolization , with a rate of 0.5% to 1 % . in a study with 450 patients , seven cases of device embolization into pulmonary artery in the first 12 hours", "one hour after intervention we detected device embolization into the pulmonary artery after performing echocardiography since the patient had dyspnea .", "after that , emergency surgery was performed within one hour with a median sternotomy and under cardiopulmonary bypass .", ", snares can be used to take out the device , but most of the time , surgical extraction of the device and asd patch closure is needed .", "percutaneous asd closure is a widely used and has been performed successfully throughout the world in recent years .", "however , it should be kept in mind that improper patient and device selection may result in deadly complications like device embolization , cardiac perforation and tamponade ."]]}
{"article_id": "PMC4852366", "article_text": ["extracellular vesicles ( evs ) are a heterogeneous group of vesicles that can be subdivided based on their size , biogenesis , and molecular composition . using the biogenesis as a classification tool", ", evs can be divided into three major groups , namely , exosomes , microvesicles ( mvs ) , and apoptotic bodies [ 13 ] . even though the molecular composition of these three subsets of evs is different , several markers overlap .", "so far , identification of a specific marker that with certainty can distinguish or identify the particular ev subset still awaits .", "it can be expected that the different ev subsets may cover different biological roles , but the function of evs has also been described to depend both on the cellular source and on the recipient tissue / cell .", "nevertheless , it is now recognized that evs are involved in numerous physiological processes , including intercellular communication and delivery of proteins , lipids , and genetic material to recipient cells [ 2 , 3 , 57 ] .", "in addition , evs have been associated with the development and progression of different pathological conditions , including cancer and infectious diseases [ 815 ] .", "the immunological effects of evs comprise a broad range of mechanisms , including immune activation , immune suppression , and modulation of immune surveillance .", "cells from both the innate and the adaptive immune system have been shown to release evs , such as t and b cells , dendritic cells ( dcs ) , macrophages , mast cells , and natural killer ( nk ) cells [ 1624 ] .", "the effect of evs is directly related to their molecular composition and several studies have ascribed an immunostimulatory effect of evs to the presence of a very specific molecular content [ 20 , 23 , 2531 ] ; for instance , cd56-positive and perforin - containing evs from nk cells can mediate ev - induced cytotoxicity .", "several immunosuppressive effects of evs have also been reported [ 21 , 32 , 33 ] ; for instance , fas ligand ( fas - l ) evs released from regulatory t cells are able to inhibit dc - induced cytotoxic t - lymphocyte ( ctl ) responses .", "furthermore , inhibitory roles of evs derived from immature dcs have been observed in relation to transplant tolerance [ 3436 ] .", "thus , identification of a specific molecular signature of evs released by immune cells can provide knowledge that can lead to the use of evs in a therapeutic setting .", "numerous studies have investigated the effects of evs released from different leukocytes , leading to an incipient understanding of the physiological functions of these evs .", "nevertheless , several basic questions concerning specific characteristics , like the protein composition of the different types of leukocyte - derived evs , remain unclear .", "the present study investigated the expression of selected immunological lineage - specific markers and selected vesicle - related markers on five major leukocyte subpopulations , namely , cd4 t cells , cd8 t cells , nk cells , b cells , and monocytes .", "the expression was determined for leukocytes present in freshly isolated whole blood and on cultured isolated leukocyte subpopulations and compared to the presentation of these markers on the corresponding leukocyte - derived evs .", "the ev array , used for phenotyping of evs , is optimized for detection of small evs with a size below 150  nm that present cd9 , cd63 , and/or cd81 , such as exosomes and exosome - like vesicles .", "however , as these markers may be present on several types of evs and as the intracellular origin of the characterized evs was not determined , the ev subset investigated in the current study was denoted by small evs ( sevs ) .", "blood samples were obtained from ten healthy volunteers at the department of clinical immunology , aalborg university hospital . from each donor two blood samples were collected , one tube containing edta ( k3edta , vacuette , greiner bio - one , austria ) for immediate analysis of noncultured leukocytes and one tube containing cpda ( vacuette , greiner bio - one , austria ) for the vesicle analysis ( plasma ) .", "plasma was isolated by centrifugation at 1800  g for 6  min at room temperature ( rt ) , after which the plasma supernatant was aliquoted and stored at 40c until analysis .", "buffy coats were obtained from three healthy donors at the department of clinical immunology , aalborg university hospital , and used for isolation of peripheral blood mononuclear cells ( pbmcs ) .", "buffy coats were diluted ( 1  :  4 ) in sterile pbs ( spbs ) and pbmcs were isolated by density gradient centrifugation using lymphoprep ( axis - shield , oslo , norway ) .", "pbmcs were subsequently washed twice in growth medium ( rpmi1640 ( gibco , life technologies , carlsbad , ca , usa ) , 10% heat - inactivated fetal calf serum ( fsc ) ( gibco ) , 100  u / ml penicillin , and 10  g / ml streptomycin ( amplicon , odense , denmark ) ) and counted in trypan blue and resuspended in isolation buffer ( ca- and mg - free pbs , 2  mm edta , and 0.1% bovine serum albumin ( bsa ) ) . magnetic dynabeads were used for isolation of human cd4 and cd8 t cells according to the manufacturer 's instructions ( dynabeads cd4 positive isolation kit and dynabeads cd8 positive isolation kit , life technologies ) .", "briefly , 1  10  cells / ml were mixed with washed beads and incubated for 20  min at 4c with gentle rotation .", "the cell suspension was subsequently placed in a magnet for 3 - 4  min .", "the supernatant was removed and the bead - bound cells were incubated with detachabeads for 45  min at rt with gentle rotation . the cell suspension was placed in a magnet for 3 - 4  min and the supernatant containing the detached cells was collected .", "the detached cells were washed once in growth medium and adjusted to 3  10  cells / ml . from the cd4 depleted pbmcs , b cells were isolated using the dynabeads untouched human b cells kit ( life technologies ) according to the manufacturer 's instructions . from the cd8 depleted pbmcs , monocytes were isolated using the dynabeads untouched human monocytes kit ( life technologies ) according to the manufacturer 's instructions .", "human nk cells were isolated from pbmcs by negative selection according to the manufacturer 's instructions ( dynabeads untouched human nk cells kit , life technologies ) .", "briefly , 1  10  cells / ml were mixed with the antibody cocktail and incubated for 20  min at 4c followed by washing and incubation with dynabeads for 15  min at 4c while rotating .", "the cell suspension was subsequently placed in a magnet for 3 - 4  min .", "the supernatant , containing the cells of interest , was removed and centrifuged at 500  g , for 5  min at rt .", "the pellet was washed once in growth medium and adjusted to 3  10  cells / ml . for each isolation , the purity of the cells was determined by flow cytometry .", "isolated leukocytes were adjusted to 3  10  cells / ml and cultured in either 12-well ( 3  10  cells / well , 1.5  ml / well ) or 24-well plates ( 1.5  10  cells / well , 1  ml / well ) ( nunc , thermo scientific , carlsbad , ca , usa ) for 4448 hours at 37c and 5% co2 .", "following incubation , the plates were centrifuged at 600  g for 10  min at rt and the supernatants containing the cell - derived evs were harvested from the plates .", "complete protease inhibitor , edta - free ( roche , de , usa ) , was added to the ev - rich supernatants , which were subsequently upconcentrated using amicon ultra 100k spin columns according to the manufacturer 's instructions .", "briefly , pbs was added to the harvested supernatants ( total volume of 5  ml ) and the supernatants were subsequently centrifuged at 600  g for 7  min at rt .", "the supernatants were transferred to 50  ml spin columns and centrifuged at 3200  g for 10  min at rt . prior to adding the supernatants , the spin columns were washed once in 5  ml pbs ( 3200  g , 10  min , rt ) . following the first centrifugation of the supernatant", ", 5  ml of pbs was added to the retention volume and centrifuged again .", "the retention volume , containing the upconcentrated supernatants , was harvested and the filter unit was washed in 50100  l pbs . due to varying cell counts between the subpopulations and across the experiments ,", "every supernatant was adjusted to a volume corresponding to 5.5  10  cells / ml . no further purification of the evs was performed and the upconcentrated supernatants were aliquoted and stored at 40c until analysis by the ev array .", "the cells were harvested in pbs , washed , and resuspended in pbs with 0.5% bsa and 0.09% nan3 , followed by a subsequent surface marker staining . for the ev array ,", "the following antibodies were used for capturing : cd11a ( hi111 ) from ab biotech ( san diego , ca , usa ) ; flotillin-1 and tsg101 ( 5b7 ) from abcam ( cambridge , ma , usa ) ; cd3 ( hit3a ) , cd14 ( m5e2 ) , cd16 ( 3g8 ) , cd28 ( l293 ) , cd49d ( l25 ) , and cd56 ( 3g8 ) from bd biosciences ( mountain view , ca , usa ) ; cd41 ( hip8 ) , cd63 ( mem-259 ) , hla - abc ( w6/32 ) , and alix ( 3a9 ) from biolegend ( san diego , ca , usa ) ; icam-1 ( r6.5 ) from ebiosciences ( san diego , ca , usa ) ; cd9 , cd42a , cd81 , and ctla-4 ( anc152.2/8h5 ) from lifespan biosciences ( seattle , wa , usa ) ; annexin v ( af399 ) , cd4 ( 34930 ) , cd8 ( 37006 ) , cd19 ( 4g7 - 2e3 ) , cd37 ( 424925 ) , cd45 ( 2d1 ) , cd80 ( 37711 ) , cd82 ( 423524 ) , cd83 ( hb15e ) , mic a / b ( 159207 ) , tnf ri , and tnf rii from r&d systems ( minneapolis , mn , usa ) ; tlr3 ( 3.7 ) from santa cruz biotechnologies ( dallas , tx , usa ) ; hla - dr / dp / dq ( hb-145 ) from loke diagnostics ( aarhus , denmark ) ; fas ligand ( 10f2 ) from serotec ( oxford , uk ) ; and pd - l1 from sino biological ( beijing , china ) . the following antibodies were used for detection ( biotinylated ) : cd9 , cd63 , and cd81 from lifespan biosciences . for flow cytometry", "the following antibodies were used : cd45 apc ( t29/33 ) , cd45 fitc ( t29/33 ) , and cd3 fitc ( ucht1 ) from dakocytomation ( glostrup , denmark ) ; cd3 apc ( ucht1 ) , cd4 fitc ( rpa - t4 ) , cd4 apc - h7 ( sk3 ) , cd8 apc - h7 ( sk1 ) , cd9 percp - cy5.5 ( m - l13 ) , cd14 fitc ( m5e2 ) , cd16 fitc ( 3g8 ) , cd16 pe - cy7 ( 3g8 ) , cd19 apc ( hib19 ) , cd56 apc ( b159 ) , and cd56 pe - cy7 ( b159 ) from bd biosciences ( mountain view , ca , usa ) ; cd63 pe - cy7 ( h5c6 ) from ebiosciences ; and cd81 pe from lifespan biosciences .", "in addition , isotype- and fluorophore - matched control antibodies were included . for analysis of noncultured leukocytes , 100  l of freshly drawn whole blood was labeled with fluorescence - conjugated antibodies for 30  min at rt , red blood cells were lysed , and the remaining leukocytes were washed and resuspended in bd facsflow sheath fluid ( bd biosciences ) with 1% paraformaldehyde .", "the cultured leukocytes were labeled with fluorescence - conjugated antibodies for 30  min at rt .", "the leukocytes were washed twice in pbs with 0.5% bsa and 0.09% nan3 and resuspended in bd facsflow sheath fluid ( bd biosciences ) with 1% paraformaldehyde .", "cells were analyzed on a facscanto ii flow cytometer ( bd biosciences ) using the bd facsdiva software version 6.1.3 ( bd biosciences ) .", "the acquired data files were analyzed by flowjo vx.0.7 ( treestar , ashland , usa ) , first by adding a leukocyte - gate based on morphologic characteristics ( fsc / ssc ) and subsequently by the use of the lineage - specific markers ; cd3 and cd4 for cd4 t cells ; cd3 and cd8 for cd8 t cells ; cd16 and cd56 for nk cells ; cd19 for b cells ; and cd14 for monocytes . for the production of the protein microarray , microarray printing was performed on a spotbot extreme protein edition microarray printer with a 946mp4 pin ( arrayit , sunnyvale , ca , usa ) .", "epoxy coated slides ( 75.6  mm  25.0  mm ; schott nexterion , jena , germany ) were used as microarray basis .", "the antibodies listed above were diluted in pbs containing 5% glycerol and printed at a concentration of 180200  g / ml . as a positive control , 100  g / ml of biotinylated human igg in pbs with 5% glycerol was printed . as a negative control ,", "for catching , visualization , and data analysis , the procedures were performed as previously described . in short", ", the slides were incubated with 100  l plasma , prediluted 1  :  10 , or 100  l upconcentrated cell culture supernatant , prediluted 1  :  2 in wash buffer ( pbs with 0.05% tween-20 ) .", "after overnight sample incubation and a subsequent wash , the slides were incubated with a cocktail of biotinylated detection antibodies ( anti - human cd9 , cd63 , and cd81 ) , diluted 1  :  1500 in wash buffer .", "after incubation , cy5-labeled streptavidin ( invitrogen , frederick , md , usa ) diluted 1  :  1500 was used for detection . prior to scanning , the slides were washed and dried using a microarray high - speed centrifuge ( arrayit ) .", "briefly , the intensity of the antibody signal was calculated by subtracting the mean of the background ( without sample / blank ) from the mean of the triplicate antibody spots .", "this signal was then divided by the signal from the mean of the triplicate negative spots ( without capture antibody , with sample ) .", "leukocytes can be subdivided based on morphological characteristics upon staining into polymorphonuclear cells ( the granulocytes ) and mononuclear cells ( the lymphocytes and the monocytes ) .", "the present study investigated the sevs produced by the different subpopulations of mononuclear cells found in peripheral blood and compared the vesicular phenotype to the cellular phenotype .", "in addition , sevs present in plasma were phenotyped for the same panel of markers .", "the purity of the different subpopulations throughout the three individual experiments was 9799.5% for the cd4 and the cd8 t cells , 8295% for the b cells , 6894% for the monocytes , and 8597% for the nk cells .", "the isolated subpopulations of leukocytes were cultured for 4448 hours and the supernatants , containing the leukocyte - produced evs , were investigated for the presence of a panel of selected immunological and ev - related markers using the ev array ( table 1 ) .", "it was investigated whether sevs from isolated leukocyte subpopulations presented the lineage - specific markers that define the parent cells .", "the results demonstrated that not all lineage - specific markers could be observed on the corresponding sev subpopulations ( figure 1(a ) ) .", "for instance , cd19 , which is a well - defined lineage marker for b cells , could not be detected on sevs in supernatant from cultured b cells .", "likewise , cd14 , cd16 , cd56 , and cd3 could not be detected on sevs from cultured monocytes , nk cells , and t cells , respectively ( figure 1(a ) ) .", "in contrast , cd4 and cd8 , which are lineage markers for two different populations of t cells , were detected on sevs from the supernatants of these two t cell subpopulations .", "in addition , sevs from cultured pbmcs presented cd8 , but none of the other markers . aside from the parental lineage - specific markers ,", "likewise , cd45 and cd16 could be detected in one of the experiments with sevs from cd4 t cells .", "a panel of ev - related markers was assayed on the different subsets of the leukocyte - derived evs ( figure 1(b ) ) .", "cd9 was detected on all sev subsets , though at very low levels on sevs from t cells and nk cells .", "similarly , cd81 was detected on all sevs but at very low levels on sevs from b cells and nk cells .", "cd82 was detected at high levels on sevs from monocytes and pbmcs and at very low levels on sevs from b cells and t cells but was absent on sevs from nk cells .", "furthermore , alix was detected at very low levels on sevs from the cultured cd8 t cells .", "in contrast , annexin v , tsg101 , and flotillin-1 could not be detected on the leukocyte - derived sevs .", "furthermore , the presence of more general immunological markers was investigated on the different sev populations .", "a total of 18 markers were chosen based on their relevance for the function of leukocytes ( table 1 ) .", "the majority of markers could not be detected on the sev subpopulations using the ev array ( figure 1(c ) ) .", "however , cd49d was found on sevs from all the different leukocyte subpopulations and cd41 was detected on sevs from all subpopulations besides sevs from nk cells .", "additionally , sevs from cultured cd4 and cd8 t cells presented several of the immunological markers , including cd11a , tlr3 , cd28 , ctla-4 , and the fas - l .", "the data presented in the heat maps were from three individual experiments and illustrated a degree of variation between the individuals .", "the presence of a natural variation in the phenotype of sevs between healthy individuals was also observed upon phenotyping of the sevs present in plasma from 10 healthy individuals ( figure 2 ) .", "plasma sevs presented several of the markers that were detected on the leukocyte - derived sevs .", "in addition , plasma sevs presented some lineage - specific markers , including cd3 , cd14 , and cd19 that were not detected on sevs from cultured leukocytes .", "furthermore , tnf ri , tlr3 , cd42a , cd80 , and cd83 as well as annexin v , flotillin-1 , and alix were detected on the majority of the plasma sevs .", "the cellular expression pattern of cd9 , cd63 , and cd81 was determined for all the leukocyte subpopulations by flow cytometry ( figure 3 ) .", "the expression of these vesicle - related markers was investigated for leukocytes present in freshly drawn whole blood ( n = 10 ) as well as for isolated subpopulations of leukocytes following two days of culture ( n = 3 ) .", "figures 3(a ) and 3(b ) demonstrate the expression levels in histograms from two representative donors .", "none of the leukocyte subsets from whole blood presented cd9 on their surface ( figure 3(a ) ) .", "in contrast , both cd63 and cd81 were expressed on all leukocytes , but the expression pattern varied between the different subsets . the lymphocytes , including the cd4 t cells , the cd8 t cells , the b cells , and the nk cells , expressed cd81 at high levels , while they all expressed cd63 at low - to - intermediate levels .", "the opposite situation was observed for the monocytes that presented high levels of cd63 on their surface , but only low - to - intermediate levels of cd81 . regarding the cultured leukocytes ,", "each subpopulation was isolated from three different donors and cultured for 4448 hours ( figure 3(b ) ) .", "the results showed that , in contrast to the freshly isolated leukocytes , the cultured leukocytes presented cd9 on their surface .", "for the lymphocyte populations , the expression of cd9 was low , while the monocyte population expressed intermediate levels of cd9 .", "all cultured lymphocyte subpopulations expressed cd63 and cd81 at low - to - intermediate levels , while the monocytes expressed high levels of both markers . even though the levels varied , the expression patterns of cd63 and cd81 on the cultured leukocyte subpopulations were similar to the patterns observed on leukocytes present in freshly isolated blood . based on the obtained minimum and maximum mfi values , it was clear that the expression level of the three markers varied between the individuals illustrating a natural variation . in order to investigate the phenotypic homogeneity between the sevs and the parent cells , the cellular expression patterns of selected lineage - specific and vesicle - related markers were compared to the vesicular presentation of these markers ( figure 4 ) . in relation to the selected lineage - specific markers , cd3 , cd14 , cd16 , and cd19", "could not be detected on sevs from cultured t cells , monocytes , nk cells , and b cells , respectively , indicating that not all cell surface molecules are transferred to the sev surface .", "for cd9 , cd63 , and cd81 , the majority of the subpopulations presented vesicular levels that overall correlated with the cellular expression .", "exceptions were observed with cd63 , in which case the cellular expression on monocytes was high , but the vesicular presentation was low .", "similarly , the cellular expression of cd63 was intermediate or low for both nk cells and b cells , but no cd63 could be detected on the corresponding sevs .", "several studies have reported that evs are released from different leukocyte subsets ; however , a thorough simultaneous investigation of the major leukocyte subsets with focus on lineage - specific markers has not yet been described . in the current study", "the phenotype of five different subsets of leukocyte - derived sevs was determined and related to both the phenotype of sevs present in plasma and the cellular phenotype of the leukocyte subpopulations .", "the ev array , used for the investigation of the phenotype of sevs , is a protein microarray technique that provides the opportunity to detect and characterize sevs for up to 60 markers in a high - throughput manner and with high sensitivity [ 37 , 38 ] .", "one major advantage of the ev array is the ability to phenotype sevs from unpurified material without a requirement for preanalytical purification .", "isolation of specific ev subsets is in many cases highly warranted , but for phenotyping of evs using the ev array , crude plasma or cell - free supernatants are applicable . in order to investigate whether lineage - specific proteins present on leukocytes could be detected on the corresponding sevs", ", leukocytes were phenotyped for selected lineage - specific markers using flow cytometry , while the sevs were phenotyped using the ev array .", "the results showed that none of the included lineage - specific markers could be detected on the sevs released from b cells , nk cells , and monocytes in monocultures ( cd19 , cd56 , and cd14 , resp . ) .", "in contrast , sevs from monocultures of cd4 and cd8 t cells did present cd4 and cd45 or cd8 and cd45 , respectively .", "none of the t cell - derived sevs presented cd3 . a study by kornek et al . investigated evs from jurkat t cells activated with phytohemagglutinin ( pha ) and observed that the fraction of large evs ( sedimented at 10.000 ", "g ) only presented low levels of cd3 , while the fraction of sevs ( sedimented at 100.000  g ) presented high levels of cd3 .", "regarding cd19 , a study by admyre and coworkers from 2007 showed that evs from a b cell line presented cd19 , which differs from the observations obtained in the study at hand .", "similarly , another study has described that evs from resting nk cells present typical nk cell markers like cd56 and fas - l , but not cd16 . in terms of cd14 , a study by aharon et al . observed this marker on large evs ( mvs ) released from a monocytic cell line .", "the discrepancy between some of the results may be due to investigations of different preparations of evs and/or may be explained by the fact that the ev - producing cells were different , for example , cells isolated from peripheral blood versus cell lines .", "in addition , differences in the activation state of the ev - producing cells are of importance for the outcome . upon stimulation ,", "cells change their phenotype and this phenotypic change depends on the specific stimulus given to the cells [ 28 , 42 ] . in the present study , leukocyte subpopulations were isolated and cultured without any activation signal , while several of the other studies investigated the phenotype of the evs released upon adding a stimulus to the cells .", "the presented results obtained with sevs released from cultured pbmcs support this phenomenon as these sevs apparently displayed fewer markers than sevs from monocultures .", "overall , these data indicate that the phenotype of the vesicle subset is context - dependent", ". however , during the current conditions , several of the leukocyte - derived sevs did not present the lineage - specific markers found on the parent cell type ( figure 1(a ) ) .", "variations in the molecular content between cells and evs were also observed in a study by hunter et al . that reported significant differences in the presence of mirnas between plasma evs and pbmcs . for comparison , the presentation of lineage - specific markers on plasma sevs was determined .", "in contrast to the leukocyte - derived sevs , plasma sevs presented several lineage - specific markers , including cd3 , cd14 , and cd19 .", "compared to the evs present in cell supernatants , evs in plasma are a mixture of evs produced by many different cell types .", "thus , plasma can act as a liquid biopsy and will present a very heterogeneous ev phenotype that provides information about numerous cellular processes .", "the detection of cd3 , cd14 , and cd19 on plasma sevs , but not on sevs from cultured leukocyte subpopulations , emphasizes that the biological context has a great impact on the phenotype of the released evs .", "interestingly , some of the sev subsets presented other markers , for example , sevs from cd8 t cells presenting cd16 .", "several studies have described the expression of cd16 on a small portion of different types of t cells and even shown a functional role of cd16 on t cells [ 44 , 45 ] .", "thus , the observations of cd16 on sevs released from cultured t cells combined with the fact that functional molecules , such as perforin and lytic granules , can be stored inside evs may indicate a specific functionality of these evs , for example , surrogates or extensions of the effector cell in antibody - dependent , cell - mediated cytotoxicity .", "apart from the selected lineage - specific markers , the sevs were phenotyped for the presence of 26 different immunological or vesicle - related markers .", "regarding the leukocyte - derived sevs , cd49d was the only marker detected on all sev subsets ( figure 1(c ) ) .", "cd49d is an 4 integrin that is expressed as a heterodimer on t , b , and nk cells and monocytes as well as on other immune cells .", "together with cd29 , cd49d forms the very late antigen-4 ( vla-4 ) that is involved in leukocyte trafficking , adhesion , and extravasation [ 46 , 47 ] .", "so far , two studies have reported on the presence of cd49d / vla-4 on evs from b cells [ 48 , 49 ] , and these results can be confirmed by the present study . in terms of plasma sevs ,", "surprisingly , cd41 was observed on sevs from t cells , b cells , monocytes , and pbmcs as well as on plasma sevs from half of the healthy donors ( figure 1(c ) ) .", "however , according to the protein atlas , cd41 is expressed by pbmcs   and even though the function is unclear , the presence of cd41 on sevs released from these cells , either in monocultures or as pbmcs , is possible .", "in addition , sevs from cultured t cells presented an array of other immunological markers , ranging from more general markers , like cd45 , to specific markers like cd28 , and the tlr3 .", "moreover , sevs from cd8 t cells presented ctla-4 and the fas - l . even though the mechanisms are quite different , both of these molecules play a role in downregulation of an immune response , either by transmitting an inhibitory signal in activated t cells or by inducing activation - induced cell death , respectively .", "the presence of such regulatory molecules on evs suggests that t cell - derived evs can be involved in immune regulation .", "the presence of fas - l on evs from t cells has also been observed in other studies [ 5254 ] , but to the best of our knowledge , studies observing ctla-4 on evs from leukocytes have not previously been published . upon evaluating the presence of markers on evs , it is important to consider the natural variation in the molecular composition of the ev pool that exists between healthy individuals as visualized by the ten plasma samples .", "thus , the present results are a snapshot providing insight about the current vesicular presentation of selected markers .", "the sevs investigated in this study were identified by the presence of cd9 , cd63 , and/or cd81 .", "thus , in order to investigate the similarity of the leukocytes and the corresponding sevs , the cellular expression patterns of cd9 , cd63 , and cd81 were determined .", "the expression pattern was determined for both leukocytes present in whole blood and cultured leukocyte subpopulations .", "the results demonstrated a lack of cd9 on the surface of leukocytes present in freshly drawn whole blood ( figure 3(a ) ) .", "in contrast , cd9 was present on every leukocyte subpopulation following two days of culture .", "in accordance with the present results , a recently published study also observed cd9 on the surface of isolated populations of cd3 , cd14 , and cd19 cells .", "the expression patterns of cd63 and cd81 were quite similar between the two cell preparations but with minor differences in the expressions levels .", "overall , these findings are in agreement with previously described results [ 5661 ] . when looking at both the cellular and the vesicular presentation of these markers , it is clear that , for the majority of the cultured subpopulations , the presentation of these markers was comparable . however , for the nk cells , the b cells , and the monocytes , the cellular expression of cd63 was very different from the presentation observed on sevs . a comparison between the presentation of cd9 , cd63 , and cd81 on whole blood leukocytes and plasma evs is a more complex procedure that needs to take into account that plasma evs are a very heterogeneous group of evs that emanate from multiple cell types .", "the tetraspanin signals observed on the plasma sevs represent all tetraspanin - positive sevs irrespective of their origin .", "thus , the presentation of tetraspanins on plasma sevs can not be expected to correlate with the expression of tetraspanin on leukocytes .", "nevertheless , it is clear that the cd63 signals in plasma generally were low , while the cellular expression of cd63 for several of the leukocyte subpopulations is medium to high , indicating that the presentation of cd63 is different from sevs to cells .", "overall , the vesicular signal intensities for cd63 were much lower than the intensities observed for cd9 and cd81 , suggesting that cd63 might be a poor marker for sevs in general , which has also been observed in other studies [ 38 , 6264 ] .", "the results underline the importance of detecting evs with a cocktail of antibodies against tetraspanins , as detection with a single marker may overlook some subsets of evs .", "surface molecules on evs are responsible for the biodistribution and the ligation to target cells .", "thus , the protein composition is related to the functionality of evs , why phenotyping of evs can be used to gain knowledge about the functionality . in summary , the presented data regarding the lineage - specific markers and the tetraspanins support the accumulating observations suggesting that the transfer of molecular cargo into evs is tightly controlled .", "a tightly regulated sampling of molecules to evs would match with the fact that evs play an important role as systemic regulators , traveling through tissues providing key intercellular communication as well as transfer of biologically active components ."], "abstract_text": ["<S> extracellular vesicles ( evs ) have a demonstrated involvement in modulating the immune system . </S>", "<S> it has been proposed that evs could be used as biomarkers for detection of inflammatory and immunological disorders . </S>", "<S> consequently , it is of great interest to investigate evs in more detail with focus on immunological markers . in this study , </S>", "<S> five major leukocyte subpopulations and the corresponding leukocyte - derived evs were phenotyped with focus on selected immunological lineage - specific markers and selected vesicle - related markers . </S>", "<S> the leukocyte - derived evs displayed phenotypic differences in the 34 markers investigated . </S>", "<S> the majority of the lineage - specific markers used for identification of the parent cell types could not be detected on evs released from monocultures of the associated cell types . </S>", "<S> in contrast , the vesicular presentation of cd9 , cd63 , and cd81 correlated to the cell surface expression of these markers , however , with few exceptions . furthermore , the cellular expression of cd9 , cd63 , and cd81 varied between leukocytes present in whole blood and cultured leukocytes . in summary , these data demonstrate that the cellular and vesicular presentation of selected lineage - specific and vesicle - related markers may differ , supporting the accumulating observations that sorting of molecular cargo into evs is tightly controlled . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["extracellular vesicles ( evs ) are a heterogeneous group of vesicles that can be subdivided based on their size , biogenesis , and molecular composition . using the biogenesis as a classification tool", ", evs can be divided into three major groups , namely , exosomes , microvesicles ( mvs ) , and apoptotic bodies [ 13 ] . even though the molecular composition of these three subsets of evs is different , several markers overlap .", "so far , identification of a specific marker that with certainty can distinguish or identify the particular ev subset still awaits .", "it can be expected that the different ev subsets may cover different biological roles , but the function of evs has also been described to depend both on the cellular source and on the recipient tissue / cell .", "nevertheless , it is now recognized that evs are involved in numerous physiological processes , including intercellular communication and delivery of proteins , lipids , and genetic material to recipient cells [ 2 , 3 , 57 ] .", "in addition , evs have been associated with the development and progression of different pathological conditions , including cancer and infectious diseases [ 815 ] .", "the immunological effects of evs comprise a broad range of mechanisms , including immune activation , immune suppression , and modulation of immune surveillance .", "cells from both the innate and the adaptive immune system have been shown to release evs , such as t and b cells , dendritic cells ( dcs ) , macrophages , mast cells , and natural killer ( nk ) cells [ 1624 ] .", "the effect of evs is directly related to their molecular composition and several studies have ascribed an immunostimulatory effect of evs to the presence of a very specific molecular content [ 20 , 23 , 2531 ] ; for instance , cd56-positive and perforin - containing evs from nk cells can mediate ev - induced cytotoxicity .", "several immunosuppressive effects of evs have also been reported [ 21 , 32 , 33 ] ; for instance , fas ligand ( fas - l ) evs released from regulatory t cells are able to inhibit dc - induced cytotoxic t - lymphocyte ( ctl ) responses .", "furthermore , inhibitory roles of evs derived from immature dcs have been observed in relation to transplant tolerance [ 3436 ] .", "thus , identification of a specific molecular signature of evs released by immune cells can provide knowledge that can lead to the use of evs in a therapeutic setting .", "numerous studies have investigated the effects of evs released from different leukocytes , leading to an incipient understanding of the physiological functions of these evs .", "nevertheless , several basic questions concerning specific characteristics , like the protein composition of the different types of leukocyte - derived evs , remain unclear .", "the present study investigated the expression of selected immunological lineage - specific markers and selected vesicle - related markers on five major leukocyte subpopulations , namely , cd4 t cells , cd8 t cells , nk cells , b cells , and monocytes .", "the expression was determined for leukocytes present in freshly isolated whole blood and on cultured isolated leukocyte subpopulations and compared to the presentation of these markers on the corresponding leukocyte - derived evs .", "the ev array , used for phenotyping of evs , is optimized for detection of small evs with a size below 150  nm that present cd9 , cd63 , and/or cd81 , such as exosomes and exosome - like vesicles .", "however , as these markers may be present on several types of evs and as the intracellular origin of the characterized evs was not determined , the ev subset investigated in the current study was denoted by small evs ( sevs ) ."], ["blood samples were obtained from ten healthy volunteers at the department of clinical immunology , aalborg university hospital . from each donor two blood samples were collected , one tube containing edta ( k3edta , vacuette , greiner bio - one , austria ) for immediate analysis of noncultured leukocytes and one tube containing cpda ( vacuette , greiner bio - one , austria ) for the vesicle analysis ( plasma ) .", "plasma was isolated by centrifugation at 1800  g for 6  min at room temperature ( rt ) , after which the plasma supernatant was aliquoted and stored at 40c until analysis .", "buffy coats were obtained from three healthy donors at the department of clinical immunology , aalborg university hospital , and used for isolation of peripheral blood mononuclear cells ( pbmcs ) .", "buffy coats were diluted ( 1  :  4 ) in sterile pbs ( spbs ) and pbmcs were isolated by density gradient centrifugation using lymphoprep ( axis - shield , oslo , norway ) .", "pbmcs were subsequently washed twice in growth medium ( rpmi1640 ( gibco , life technologies , carlsbad , ca , usa ) , 10% heat - inactivated fetal calf serum ( fsc ) ( gibco ) , 100  u / ml penicillin , and 10  g / ml streptomycin ( amplicon , odense , denmark ) ) and counted in trypan blue and resuspended in isolation buffer ( ca- and mg - free pbs , 2  mm edta , and 0.1% bovine serum albumin ( bsa ) ) . magnetic dynabeads were used for isolation of human cd4 and cd8 t cells according to the manufacturer 's instructions ( dynabeads cd4 positive isolation kit and dynabeads cd8 positive isolation kit , life technologies ) .", "briefly , 1  10  cells / ml were mixed with washed beads and incubated for 20  min at 4c with gentle rotation .", "the cell suspension was subsequently placed in a magnet for 3 - 4  min .", "the supernatant was removed and the bead - bound cells were incubated with detachabeads for 45  min at rt with gentle rotation . the cell suspension was placed in a magnet for 3 - 4  min and the supernatant containing the detached cells was collected .", "the detached cells were washed once in growth medium and adjusted to 3  10  cells / ml . from the cd4 depleted pbmcs , b cells were isolated using the dynabeads untouched human b cells kit ( life technologies ) according to the manufacturer 's instructions . from the cd8 depleted pbmcs , monocytes were isolated using the dynabeads untouched human monocytes kit ( life technologies ) according to the manufacturer 's instructions .", "human nk cells were isolated from pbmcs by negative selection according to the manufacturer 's instructions ( dynabeads untouched human nk cells kit , life technologies ) .", "briefly , 1  10  cells / ml were mixed with the antibody cocktail and incubated for 20  min at 4c followed by washing and incubation with dynabeads for 15  min at 4c while rotating .", "the cell suspension was subsequently placed in a magnet for 3 - 4  min .", "the supernatant , containing the cells of interest , was removed and centrifuged at 500  g , for 5  min at rt .", "the pellet was washed once in growth medium and adjusted to 3  10  cells / ml . for each isolation , the purity of the cells was determined by flow cytometry .", "isolated leukocytes were adjusted to 3  10  cells / ml and cultured in either 12-well ( 3  10  cells / well , 1.5  ml / well ) or 24-well plates ( 1.5  10  cells / well , 1  ml / well ) ( nunc , thermo scientific , carlsbad , ca , usa ) for 4448 hours at 37c and 5% co2 .", "following incubation , the plates were centrifuged at 600  g for 10  min at rt and the supernatants containing the cell - derived evs were harvested from the plates .", "complete protease inhibitor , edta - free ( roche , de , usa ) , was added to the ev - rich supernatants , which were subsequently upconcentrated using amicon ultra 100k spin columns according to the manufacturer 's instructions .", "briefly , pbs was added to the harvested supernatants ( total volume of 5  ml ) and the supernatants were subsequently centrifuged at 600  g for 7  min at rt .", "the supernatants were transferred to 50  ml spin columns and centrifuged at 3200  g for 10  min at rt . prior to adding the supernatants , the spin columns were washed once in 5  ml pbs ( 3200  g , 10  min , rt ) . following the first centrifugation of the supernatant", ", 5  ml of pbs was added to the retention volume and centrifuged again .", "the retention volume , containing the upconcentrated supernatants , was harvested and the filter unit was washed in 50100  l pbs . due to varying cell counts between the subpopulations and across the experiments ,", "every supernatant was adjusted to a volume corresponding to 5.5  10  cells / ml . no further purification of the evs was performed and the upconcentrated supernatants were aliquoted and stored at 40c until analysis by the ev array .", "the cells were harvested in pbs , washed , and resuspended in pbs with 0.5% bsa and 0.09% nan3 , followed by a subsequent surface marker staining . for the ev array ,", "the following antibodies were used for capturing : cd11a ( hi111 ) from ab biotech ( san diego , ca , usa ) ; flotillin-1 and tsg101 ( 5b7 ) from abcam ( cambridge , ma , usa ) ; cd3 ( hit3a ) , cd14 ( m5e2 ) , cd16 ( 3g8 ) , cd28 ( l293 ) , cd49d ( l25 ) , and cd56 ( 3g8 ) from bd biosciences ( mountain view , ca , usa ) ; cd41 ( hip8 ) , cd63 ( mem-259 ) , hla - abc ( w6/32 ) , and alix ( 3a9 ) from biolegend ( san diego , ca , usa ) ; icam-1 ( r6.5 ) from ebiosciences ( san diego , ca , usa ) ; cd9 , cd42a , cd81 , and ctla-4 ( anc152.2/8h5 ) from lifespan biosciences ( seattle , wa , usa ) ; annexin v ( af399 ) , cd4 ( 34930 ) , cd8 ( 37006 ) , cd19 ( 4g7 - 2e3 ) , cd37 ( 424925 ) , cd45 ( 2d1 ) , cd80 ( 37711 ) , cd82 ( 423524 ) , cd83 ( hb15e ) , mic a / b ( 159207 ) , tnf ri , and tnf rii from r&d systems ( minneapolis , mn , usa ) ; tlr3 ( 3.7 ) from santa cruz biotechnologies ( dallas , tx , usa ) ; hla - dr / dp / dq ( hb-145 ) from loke diagnostics ( aarhus , denmark ) ; fas ligand ( 10f2 ) from serotec ( oxford , uk ) ; and pd - l1 from sino biological ( beijing , china ) . the following antibodies were used for detection ( biotinylated ) : cd9 , cd63 , and cd81 from lifespan biosciences . for flow cytometry", "the following antibodies were used : cd45 apc ( t29/33 ) , cd45 fitc ( t29/33 ) , and cd3 fitc ( ucht1 ) from dakocytomation ( glostrup , denmark ) ; cd3 apc ( ucht1 ) , cd4 fitc ( rpa - t4 ) , cd4 apc - h7 ( sk3 ) , cd8 apc - h7 ( sk1 ) , cd9 percp - cy5.5 ( m - l13 ) , cd14 fitc ( m5e2 ) , cd16 fitc ( 3g8 ) , cd16 pe - cy7 ( 3g8 ) , cd19 apc ( hib19 ) , cd56 apc ( b159 ) , and cd56 pe - cy7 ( b159 ) from bd biosciences ( mountain view , ca , usa ) ; cd63 pe - cy7 ( h5c6 ) from ebiosciences ; and cd81 pe from lifespan biosciences .", "in addition , isotype- and fluorophore - matched control antibodies were included . for analysis of noncultured leukocytes , 100  l of freshly drawn whole blood was labeled with fluorescence - conjugated antibodies for 30  min at rt , red blood cells were lysed , and the remaining leukocytes were washed and resuspended in bd facsflow sheath fluid ( bd biosciences ) with 1% paraformaldehyde .", "the cultured leukocytes were labeled with fluorescence - conjugated antibodies for 30  min at rt .", "the leukocytes were washed twice in pbs with 0.5% bsa and 0.09% nan3 and resuspended in bd facsflow sheath fluid ( bd biosciences ) with 1% paraformaldehyde .", "cells were analyzed on a facscanto ii flow cytometer ( bd biosciences ) using the bd facsdiva software version 6.1.3 ( bd biosciences ) .", "the acquired data files were analyzed by flowjo vx.0.7 ( treestar , ashland , usa ) , first by adding a leukocyte - gate based on morphologic characteristics ( fsc / ssc ) and subsequently by the use of the lineage - specific markers ; cd3 and cd4 for cd4 t cells ; cd3 and cd8 for cd8 t cells ; cd16 and cd56 for nk cells ; cd19 for b cells ; and cd14 for monocytes . for the production of the protein microarray , microarray printing was performed on a spotbot extreme protein edition microarray printer with a 946mp4 pin ( arrayit , sunnyvale , ca , usa ) .", "epoxy coated slides ( 75.6  mm  25.0  mm ; schott nexterion , jena , germany ) were used as microarray basis .", "the antibodies listed above were diluted in pbs containing 5% glycerol and printed at a concentration of 180200  g / ml . as a positive control , 100  g / ml of biotinylated human igg in pbs with 5% glycerol was printed . as a negative control ,", "for catching , visualization , and data analysis , the procedures were performed as previously described . in short", ", the slides were incubated with 100  l plasma , prediluted 1  :  10 , or 100  l upconcentrated cell culture supernatant , prediluted 1  :  2 in wash buffer ( pbs with 0.05% tween-20 ) .", "after overnight sample incubation and a subsequent wash , the slides were incubated with a cocktail of biotinylated detection antibodies ( anti - human cd9 , cd63 , and cd81 ) , diluted 1  :  1500 in wash buffer .", "after incubation , cy5-labeled streptavidin ( invitrogen , frederick , md , usa ) diluted 1  :  1500 was used for detection . prior to scanning , the slides were washed and dried using a microarray high - speed centrifuge ( arrayit ) .", "briefly , the intensity of the antibody signal was calculated by subtracting the mean of the background ( without sample / blank ) from the mean of the triplicate antibody spots .", "this signal was then divided by the signal from the mean of the triplicate negative spots ( without capture antibody , with sample ) ."], ["leukocytes can be subdivided based on morphological characteristics upon staining into polymorphonuclear cells ( the granulocytes ) and mononuclear cells ( the lymphocytes and the monocytes ) .", "the present study investigated the sevs produced by the different subpopulations of mononuclear cells found in peripheral blood and compared the vesicular phenotype to the cellular phenotype .", "in addition , sevs present in plasma were phenotyped for the same panel of markers .", "the purity of the different subpopulations throughout the three individual experiments was 9799.5% for the cd4 and the cd8 t cells , 8295% for the b cells , 6894% for the monocytes , and 8597% for the nk cells .", "the isolated subpopulations of leukocytes were cultured for 4448 hours and the supernatants , containing the leukocyte - produced evs , were investigated for the presence of a panel of selected immunological and ev - related markers using the ev array ( table 1 ) .", "it was investigated whether sevs from isolated leukocyte subpopulations presented the lineage - specific markers that define the parent cells .", "the results demonstrated that not all lineage - specific markers could be observed on the corresponding sev subpopulations ( figure 1(a ) ) .", "for instance , cd19 , which is a well - defined lineage marker for b cells , could not be detected on sevs in supernatant from cultured b cells .", "likewise , cd14 , cd16 , cd56 , and cd3 could not be detected on sevs from cultured monocytes , nk cells , and t cells , respectively ( figure 1(a ) ) .", "in contrast , cd4 and cd8 , which are lineage markers for two different populations of t cells , were detected on sevs from the supernatants of these two t cell subpopulations .", "in addition , sevs from cultured pbmcs presented cd8 , but none of the other markers . aside from the parental lineage - specific markers ,", "likewise , cd45 and cd16 could be detected in one of the experiments with sevs from cd4 t cells .", "a panel of ev - related markers was assayed on the different subsets of the leukocyte - derived evs ( figure 1(b ) ) .", "cd9 was detected on all sev subsets , though at very low levels on sevs from t cells and nk cells .", "similarly , cd81 was detected on all sevs but at very low levels on sevs from b cells and nk cells .", "cd82 was detected at high levels on sevs from monocytes and pbmcs and at very low levels on sevs from b cells and t cells but was absent on sevs from nk cells .", "furthermore , alix was detected at very low levels on sevs from the cultured cd8 t cells .", "in contrast , annexin v , tsg101 , and flotillin-1 could not be detected on the leukocyte - derived sevs .", "furthermore , the presence of more general immunological markers was investigated on the different sev populations .", "a total of 18 markers were chosen based on their relevance for the function of leukocytes ( table 1 ) .", "the majority of markers could not be detected on the sev subpopulations using the ev array ( figure 1(c ) ) .", "however , cd49d was found on sevs from all the different leukocyte subpopulations and cd41 was detected on sevs from all subpopulations besides sevs from nk cells .", "additionally , sevs from cultured cd4 and cd8 t cells presented several of the immunological markers , including cd11a , tlr3 , cd28 , ctla-4 , and the fas - l .", "the data presented in the heat maps were from three individual experiments and illustrated a degree of variation between the individuals .", "the presence of a natural variation in the phenotype of sevs between healthy individuals was also observed upon phenotyping of the sevs present in plasma from 10 healthy individuals ( figure 2 ) .", "plasma sevs presented several of the markers that were detected on the leukocyte - derived sevs .", "in addition , plasma sevs presented some lineage - specific markers , including cd3 , cd14 , and cd19 that were not detected on sevs from cultured leukocytes .", "furthermore , tnf ri , tlr3 , cd42a , cd80 , and cd83 as well as annexin v , flotillin-1 , and alix were detected on the majority of the plasma sevs .", "the cellular expression pattern of cd9 , cd63 , and cd81 was determined for all the leukocyte subpopulations by flow cytometry ( figure 3 ) .", "the expression of these vesicle - related markers was investigated for leukocytes present in freshly drawn whole blood ( n = 10 ) as well as for isolated subpopulations of leukocytes following two days of culture ( n = 3 ) .", "figures 3(a ) and 3(b ) demonstrate the expression levels in histograms from two representative donors .", "none of the leukocyte subsets from whole blood presented cd9 on their surface ( figure 3(a ) ) .", "in contrast , both cd63 and cd81 were expressed on all leukocytes , but the expression pattern varied between the different subsets . the lymphocytes , including the cd4 t cells , the cd8 t cells , the b cells , and the nk cells , expressed cd81 at high levels , while they all expressed cd63 at low - to - intermediate levels .", "the opposite situation was observed for the monocytes that presented high levels of cd63 on their surface , but only low - to - intermediate levels of cd81 . regarding the cultured leukocytes ,", "each subpopulation was isolated from three different donors and cultured for 4448 hours ( figure 3(b ) ) .", "the results showed that , in contrast to the freshly isolated leukocytes , the cultured leukocytes presented cd9 on their surface .", "for the lymphocyte populations , the expression of cd9 was low , while the monocyte population expressed intermediate levels of cd9 .", "all cultured lymphocyte subpopulations expressed cd63 and cd81 at low - to - intermediate levels , while the monocytes expressed high levels of both markers . even though the levels varied , the expression patterns of cd63 and cd81 on the cultured leukocyte subpopulations were similar to the patterns observed on leukocytes present in freshly isolated blood . based on the obtained minimum and maximum mfi values , it was clear that the expression level of the three markers varied between the individuals illustrating a natural variation . in order to investigate the phenotypic homogeneity between the sevs and the parent cells , the cellular expression patterns of selected lineage - specific and vesicle - related markers were compared to the vesicular presentation of these markers ( figure 4 ) . in relation to the selected lineage - specific markers , cd3 , cd14 , cd16 , and cd19", "could not be detected on sevs from cultured t cells , monocytes , nk cells , and b cells , respectively , indicating that not all cell surface molecules are transferred to the sev surface .", "for cd9 , cd63 , and cd81 , the majority of the subpopulations presented vesicular levels that overall correlated with the cellular expression .", "exceptions were observed with cd63 , in which case the cellular expression on monocytes was high , but the vesicular presentation was low .", "similarly , the cellular expression of cd63 was intermediate or low for both nk cells and b cells , but no cd63 could be detected on the corresponding sevs ."], ["several studies have reported that evs are released from different leukocyte subsets ; however , a thorough simultaneous investigation of the major leukocyte subsets with focus on lineage - specific markers has not yet been described . in the current study", "the phenotype of five different subsets of leukocyte - derived sevs was determined and related to both the phenotype of sevs present in plasma and the cellular phenotype of the leukocyte subpopulations .", "the ev array , used for the investigation of the phenotype of sevs , is a protein microarray technique that provides the opportunity to detect and characterize sevs for up to 60 markers in a high - throughput manner and with high sensitivity [ 37 , 38 ] .", "one major advantage of the ev array is the ability to phenotype sevs from unpurified material without a requirement for preanalytical purification .", "isolation of specific ev subsets is in many cases highly warranted , but for phenotyping of evs using the ev array , crude plasma or cell - free supernatants are applicable . in order to investigate whether lineage - specific proteins present on leukocytes could be detected on the corresponding sevs", ", leukocytes were phenotyped for selected lineage - specific markers using flow cytometry , while the sevs were phenotyped using the ev array .", "the results showed that none of the included lineage - specific markers could be detected on the sevs released from b cells , nk cells , and monocytes in monocultures ( cd19 , cd56 , and cd14 , resp . ) .", "in contrast , sevs from monocultures of cd4 and cd8 t cells did present cd4 and cd45 or cd8 and cd45 , respectively .", "none of the t cell - derived sevs presented cd3 . a study by kornek et al . investigated evs from jurkat t cells activated with phytohemagglutinin ( pha ) and observed that the fraction of large evs ( sedimented at 10.000 ", "g ) only presented low levels of cd3 , while the fraction of sevs ( sedimented at 100.000  g ) presented high levels of cd3 .", "regarding cd19 , a study by admyre and coworkers from 2007 showed that evs from a b cell line presented cd19 , which differs from the observations obtained in the study at hand .", "similarly , another study has described that evs from resting nk cells present typical nk cell markers like cd56 and fas - l , but not cd16 . in terms of cd14 , a study by aharon et al . observed this marker on large evs ( mvs ) released from a monocytic cell line .", "the discrepancy between some of the results may be due to investigations of different preparations of evs and/or may be explained by the fact that the ev - producing cells were different , for example , cells isolated from peripheral blood versus cell lines .", "in addition , differences in the activation state of the ev - producing cells are of importance for the outcome . upon stimulation ,", "cells change their phenotype and this phenotypic change depends on the specific stimulus given to the cells [ 28 , 42 ] . in the present study , leukocyte subpopulations were isolated and cultured without any activation signal , while several of the other studies investigated the phenotype of the evs released upon adding a stimulus to the cells .", "the presented results obtained with sevs released from cultured pbmcs support this phenomenon as these sevs apparently displayed fewer markers than sevs from monocultures .", "overall , these data indicate that the phenotype of the vesicle subset is context - dependent", ". however , during the current conditions , several of the leukocyte - derived sevs did not present the lineage - specific markers found on the parent cell type ( figure 1(a ) ) .", "variations in the molecular content between cells and evs were also observed in a study by hunter et al . that reported significant differences in the presence of mirnas between plasma evs and pbmcs . for comparison , the presentation of lineage - specific markers on plasma sevs was determined .", "in contrast to the leukocyte - derived sevs , plasma sevs presented several lineage - specific markers , including cd3 , cd14 , and cd19 .", "compared to the evs present in cell supernatants , evs in plasma are a mixture of evs produced by many different cell types .", "thus , plasma can act as a liquid biopsy and will present a very heterogeneous ev phenotype that provides information about numerous cellular processes .", "the detection of cd3 , cd14 , and cd19 on plasma sevs , but not on sevs from cultured leukocyte subpopulations , emphasizes that the biological context has a great impact on the phenotype of the released evs .", "interestingly , some of the sev subsets presented other markers , for example , sevs from cd8 t cells presenting cd16 .", "several studies have described the expression of cd16 on a small portion of different types of t cells and even shown a functional role of cd16 on t cells [ 44 , 45 ] .", "thus , the observations of cd16 on sevs released from cultured t cells combined with the fact that functional molecules , such as perforin and lytic granules , can be stored inside evs may indicate a specific functionality of these evs , for example , surrogates or extensions of the effector cell in antibody - dependent , cell - mediated cytotoxicity .", "apart from the selected lineage - specific markers , the sevs were phenotyped for the presence of 26 different immunological or vesicle - related markers .", "regarding the leukocyte - derived sevs , cd49d was the only marker detected on all sev subsets ( figure 1(c ) ) .", "cd49d is an 4 integrin that is expressed as a heterodimer on t , b , and nk cells and monocytes as well as on other immune cells .", "together with cd29 , cd49d forms the very late antigen-4 ( vla-4 ) that is involved in leukocyte trafficking , adhesion , and extravasation [ 46 , 47 ] .", "so far , two studies have reported on the presence of cd49d / vla-4 on evs from b cells [ 48 , 49 ] , and these results can be confirmed by the present study . in terms of plasma sevs ,", "surprisingly , cd41 was observed on sevs from t cells , b cells , monocytes , and pbmcs as well as on plasma sevs from half of the healthy donors ( figure 1(c ) ) .", "however , according to the protein atlas , cd41 is expressed by pbmcs   and even though the function is unclear , the presence of cd41 on sevs released from these cells , either in monocultures or as pbmcs , is possible .", "in addition , sevs from cultured t cells presented an array of other immunological markers , ranging from more general markers , like cd45 , to specific markers like cd28 , and the tlr3 .", "moreover , sevs from cd8 t cells presented ctla-4 and the fas - l . even though the mechanisms are quite different , both of these molecules play a role in downregulation of an immune response , either by transmitting an inhibitory signal in activated t cells or by inducing activation - induced cell death , respectively .", "the presence of such regulatory molecules on evs suggests that t cell - derived evs can be involved in immune regulation .", "the presence of fas - l on evs from t cells has also been observed in other studies [ 5254 ] , but to the best of our knowledge , studies observing ctla-4 on evs from leukocytes have not previously been published . upon evaluating the presence of markers on evs , it is important to consider the natural variation in the molecular composition of the ev pool that exists between healthy individuals as visualized by the ten plasma samples .", "thus , the present results are a snapshot providing insight about the current vesicular presentation of selected markers .", "the sevs investigated in this study were identified by the presence of cd9 , cd63 , and/or cd81 .", "thus , in order to investigate the similarity of the leukocytes and the corresponding sevs , the cellular expression patterns of cd9 , cd63 , and cd81 were determined .", "the expression pattern was determined for both leukocytes present in whole blood and cultured leukocyte subpopulations .", "the results demonstrated a lack of cd9 on the surface of leukocytes present in freshly drawn whole blood ( figure 3(a ) ) .", "in contrast , cd9 was present on every leukocyte subpopulation following two days of culture .", "in accordance with the present results , a recently published study also observed cd9 on the surface of isolated populations of cd3 , cd14 , and cd19 cells .", "the expression patterns of cd63 and cd81 were quite similar between the two cell preparations but with minor differences in the expressions levels .", "overall , these findings are in agreement with previously described results [ 5661 ] . when looking at both the cellular and the vesicular presentation of these markers , it is clear that , for the majority of the cultured subpopulations , the presentation of these markers was comparable . however , for the nk cells , the b cells , and the monocytes , the cellular expression of cd63 was very different from the presentation observed on sevs . a comparison between the presentation of cd9 , cd63 , and cd81 on whole blood leukocytes and plasma evs is a more complex procedure that needs to take into account that plasma evs are a very heterogeneous group of evs that emanate from multiple cell types .", "the tetraspanin signals observed on the plasma sevs represent all tetraspanin - positive sevs irrespective of their origin .", "thus , the presentation of tetraspanins on plasma sevs can not be expected to correlate with the expression of tetraspanin on leukocytes .", "nevertheless , it is clear that the cd63 signals in plasma generally were low , while the cellular expression of cd63 for several of the leukocyte subpopulations is medium to high , indicating that the presentation of cd63 is different from sevs to cells .", "overall , the vesicular signal intensities for cd63 were much lower than the intensities observed for cd9 and cd81 , suggesting that cd63 might be a poor marker for sevs in general , which has also been observed in other studies [ 38 , 6264 ] .", "the results underline the importance of detecting evs with a cocktail of antibodies against tetraspanins , as detection with a single marker may overlook some subsets of evs ."], ["surface molecules on evs are responsible for the biodistribution and the ligation to target cells .", "thus , the protein composition is related to the functionality of evs , why phenotyping of evs can be used to gain knowledge about the functionality . in summary , the presented data regarding the lineage - specific markers and the tetraspanins support the accumulating observations suggesting that the transfer of molecular cargo into evs is tightly controlled .", "a tightly regulated sampling of molecules to evs would match with the fact that evs play an important role as systemic regulators , traveling through tissues providing key intercellular communication as well as transfer of biologically active components ."]]}
{"article_id": "PMC4613737", "article_text": ["in march 2014 , an unprecedented \n outbreak of ebola virus disease \n ( evd ) began in western africa that as of july 2015 is ongoing and \n has claimed more than 11000 lives .", "the \n current outbreak is caused by viruses belonging to the species of \n the zaire ebolavirus , a member of the filoviridae family .", "filoviridae are enveloped , filamentous viruses with lengths that \n may reach > 1000 nm .", "ebola virus \n can be excreted in bodily fluids , including vomit , stool , blood , saliva , \n semen , and breast milk .", "ebola virus loads of up to 10 genome \n copies ml have been reported in blood , 10 genome copies ml in stool , and 10 genome copies ml in urine ; however , the conversion between genome copies \n and infectious units is unknown .", "once infected , individuals may produce up to 9 l of liquid waste \n per day , primarily watery diarrhea .", "ebola virus is considered a potential bioterrorism \n agent . in response to the evd epidemic , both \n the world health organization \n ( who ) and the u.s .", "centers for disease control and prevention advised \n direct disposal of ebola - contaminated liquid waste into sewage systems \n ( wastewater collection and treatment systems ) and latrines without \n disinfection .", "initial recommendations \n were made on the basis of an expected limited persistence of ebola \n virus in the environment , as ebola virus is an enveloped virus , and \n a lack of strong evidence for a waterborne transmission route . as \n stated by a who guidance document , ", "ebola virus is likely to \n inactivate significantly faster in the environment than enteric viruses \n with known waterborne transmission ( e.g. , norovirus , hepatitis a virus )  .", "however , as has been noted in a recent review , \n the persistence of enveloped viruses in the water environment varies \n by > 2 orders of magnitude .", "recommendations \n for ebola virus - contaminated wastewater disposal were met with debate \n ( e.g. , refs ( 1618 ) ) because of uncertainty \n about ebola virus persistence within wastewater matrices and the lack \n of a risk - based analysis for waste handling .", "wastewater handling recommendations \n have since been revised to recognize uncertainty in this area and \n to recommend disinfection of latrines and holding of wastewater prior \n to handling to allow ebola virus inactivation .", "additionally , \n some facilities have opted to provide additional disinfection prior \n to disposal of liquid waste into sewer systems .", "recent research found both ethanol and hypochlorite to \n be effective disinfectants for ebola virus dried on surfaces ; however , the disinfection kinetics of ebola \n virus within liquid matrices remains unknown .", "various wastewater disinfection \n approaches have been recently suggested for pathogen control in an \n outbreak setting .", "currently , no \n data on ebola virus persistence in wastewater exist , \n hindering risk estimation and examination of potential environmental \n exposure routes .", "the necessity of evaluating ebola virus persistence \n in wastewater matrices has previously been highlighted , as wastewater \n in ebola virus outbreak settings may be temporarily held in open containers \n or disposed of in open sewers .", "historically , \n the transmission of ebola virus via environmental routes ( droplets , \n aerosols , or fomites ) has been thought to be unlikely due to epidemiological \n evidence and environmental sampling .", "the \n primary ebola virus transmission route is via direct contact with \n bodily fluids .", "transmission has previously \n occurred without known direct contact with infected individuals , providing supporting evidence that ebola virus \n transmission may be possible via large droplets .", "the potential for transmission of ebola virus via wastewater \n is currently unknown . to address uncertainties regarding ebola \n virus persistence in wastewater", ", \n we have conducted an initial evaluation of ebola virus persistence \n within wastewater to address uncertainty and inform ongoing risk assessments . \n a current ebola virus outbreak strain from guinea ( makona - wpgc07 ) \n", "was spiked to two end concentrations ( 10 and 10 tcid50 ml ) into a domestic wastewater \n ( untreated sewage ) sample .", "the ebola virus - containing wastewater was \n sampled for 8 days , and the viability of ebola virus was determined \n in these samples .", "study results are presented , and study limitations \n and implications are discussed , as well as recommendations for an \n ongoing research agenda .", "untreated wastewater was \n collected from an anonymous regional wastewater \n treatment facility in western pennsylvania that receives wastewater \n from seven communities ( combined population of approximately 60000 ) . \n", "the total raw wastewater flow to the treatment facility is < 10 \n mgd ( million gallons per day ) .", "the wastewater \n was frozen at 80 c to minimize compositional changes \n prior to analysis .", "wastewater characteristics ( table 1 ) were determined at an epa - certified analysis \n facility ( microbac laboratories , marietta , oh ) .", "the region from which \n the sample was collected uses a combined sewer system that experiences \n significant infiltration , and the determined composition is typical \n for the region .", "ebola virus cultivation experiments \n were conducted at rocky mountain \n laboratories under bsl4 conditions .", "wastewater samples were shipped \n to rocky mountain laboratories overnight on ice . upon receipt , samples \n were sterilized with 5 mrad of -irradiation .", "sterilization \n was performed to limit cell culture death due to wastewater microbial \n activity leading to a false positive .", "stock virus ( ebola virus guinea \n makona - wpgc07 , 10 tcid50 ml ) was handled as described previously .", "ebola virus was diluted in wastewater to achieve \n two separate virus titers ( 10 and 10 tcid50 ml ) , and both experiments were completed \n in triplicate .", "the ebola virus concentration in sewage has not been \n previously measured or estimated ; thus , two separate concentrations \n were utilized to cover possible concentration scenarios .", "tcid50 is an end point dilution series that is used to determine \n at what dilution 50% of the infected wells produce cell death .", "an approximation of focus - forming \n units ( ffus ) can be made from a poisson distribution utilizing the \n formula tcid50  0.69 , assuming each ffu is formed \n from a single virus .", "spiked wastewater was then distributed into three \n labeled vials for each concentration , and samples were taken daily \n for 8 days .", "tests were conducted at 20 c and 40% relative humidity . \n at each time point , including the time zero measurement , 50 l \n of wastewater from the bulk wastewater vial was added into 450 l \n of dulbecco s modified eagle s medium ( dmem , sigma ) \n supplemented with heat - inactivated fetal bovine serum ( fbs , gibco ) \n to a final concentration of 2% , pen / strep ( gibco ) to a final concentration \n of 50 units / ml penicillin and 50 g / ml streptomycin , and l - glutamine ( gibco ) to a final concentration of 2 mm in an appropriately \n labeled 2 ml screw top vial and frozen at 80 c .", "negative \n controls were 50 l of nonspiked wastewater in 450 l \n of dmem . to perform the titrations ,", "a 96-well 1.1 ml well dilution \n plate ( axygen , corning , ny ) was prepared . the thawed sample ( 400 l ) \n", "was placed in the top row of the plate and a 10-fold dilution conducted \n by passing 40 l of sample into 360 l of dmem .", "next , \n 100 l from each well of the dilution plate was transferred \n to a 96-well cell culture plate seeded with vero cells .", "the cells \n were incubated with virus dilutions for 1 h ; then the medium was removed \n from the two highest concentrations and rinsed two times with pbs , \n and 200 l of fresh culture medium was added .", "fresh culture \n medium ( 100 l ) was also added to the remaining wells in the \n plate .", "the plates were incubated at 37 c for 7 days , inspected \n for the cytopathic effect ( cpe ) , and scored .", "the natural \n logarithm of c / co tcid50 was plotted and fit with a linear trendline for estimation \n of the inactivation constant ( k ) .", "a literature review \n was then performed to compare observed inactivation to other environmental \n matrices .", "ebola virus was \n spiked into wastewater at two concentrations and assayed for 8 days \n to determine persistence in a wastewater matrix .", "the time zero time \n point was measured immediately following addition of ebola virus to \n the wastewater .", "no viable ebola virus was recovered from samples spiked \n with 10 ebola virus tcid50 ml after the initial time zero sampling .", "virus \n viabilities from the initial 10 ebola virus tcid50 ml concentration are shown in figure 1 and detailed in table s1 .", "ebola virus titer was rapidly reduced ( approximately \n 99% ) within the first day of the test , consistent with an inability \n to identify infectious ebola virus from the initial sample with 10 ebola virus tcid50 ml on day \n 1 .", "persistence of an initial ebola virus concentration of 10 tcid50 ml in domestic wastewater \n ( untreated sewage ) ( a ) including the time zero time point and ( b ) \n excluding the time zero time point to mitigate potential aggregation \n effects .", "fit inactivation constants ( k ) were determined to be 1.08 when including time \n zero and 0.35 when excluding time zero .", "error bars are 1 \n standard deviation . there was a rapid decrease \n ( approximately 99% ) in ebola virus titer \n within the first day of the test .", "in addition to inactivation , viral \n particle aggregation or adsorption to wastewater particles may play \n a role in the apparent rapid viral decrease and enhanced viral persistence . \n in this case", "first , aggregated \n particles would not be detected as multiple infectious units , resulting \n in an apparent rapid decrease .", "second , it has been previously recognized \n that aggregation increases viral persistence and resistance to inactivation \n stressors .", "similarly , viral association with particles \n has been recognized to provide protection from inactivation , including association of viral particles with \n wastewater solids .", "mechanistically , particle \n association is believed to protect viral particles from inactivation \n by shielding them from environmental stressors and is dependent on \n the organism and particle type . utilizing the current assay , it can not be determined if the initial \n rapid decrease in viral titer was due to viral inactivation or aggregation . \n", "aggregation would result in an apparent viral titer decrease as each \n viral aggregate would function as an infectious unit in the cell culture \n assay . to address this uncertainty , we plotted two inactivation curves , \n both including ( figure 1a ) and excluding the measured time zero point ( figure 1b ) .", "a linear trendline , as has been previously \n suggested for viral and ebola virus persistence , showed a lower fit ( r = 0.59 including the time zero time point , and r = 0.67 excluding the time zero time point ) \n than that previously observed for ebola virus in deionized water ( r > 0.91 ) , but a fit better than that previously \n observed for ebola virus in human blood ( r < 0.29 ) .", "the inactivation constant ( k ) was determined \n to be 1.08 when including the time zero time point and 0.35 \n when excluding the time zero time point .", "on the basis of the model \n fit , the t90 ( time for 90% inactivation ) \n would be 2.1 days including the time zero time point and 6.6 days \n excluding the time zero time point .", "the observed ebola virus inactivation \n in wastewater was slower than that observed for deionized water , which \n required 1.8 days for 90% inactivation at 21 c .", "the observed ebola virus inactivation in wastewater was \n more rapid than that reported for human blood , which required 20 days \n for a 90% inactivation , and results are \n consistent with recent studies that identified viable ebola virus \n to persist in infected macaque blood for > 8 days . in general , ebola virus was found to be less persistent \n in wastewater than model enteric viruses . while the t90 for ebola virus in wastewater was found to be < 1 \n day , the t90 for hepatitis a is greater \n than 17 days and the t90 for enteric adenovirus is 33 days ; however , the t90 for poliovirus \n is 5 days , which is between the observed t90 values including or excluding the time zero \n time point .", "the results demonstrate a more rapid initial viral titer \n decrease but overall enhanced persistence of ebola virus in wastewater \n compared to the proposed enveloped surrogate bacteriophage phi6 .", "this \n study has two primary limitations \n that may alter the persistence of ebola virus compared to what may \n be observed in the field .", "first , the tested wastewater was more dilute \n than would be expected in typical latrine waste . in general , interaction with constituents within the wastewater \n ( e.g. , ammonia ) would be expected to contribute to more rapid inactivation \n of viruses ; however , the true effect \n of these constituents on ebola virus persistence is unknown .", "second , \n the wastewater was frozen to minimize compositional changes and disinfected \n ( -irradiation ) prior to utilization to limit microbial activity \n resulting in false positive viral cell culture . microbial activity \n within wastewater matrices would be expected to contribute to more \n rapid inactivation of infectious viral particles ; however , the true effect of microbial activity on ebola virus persistence \n is unknown .", "microbial activity reduces viral persistence through both \n the production of metabolites detrimental to viral persistence and \n direct usage of the viral particles as a nutrient source .", "additionally , the influence of other environmental \n characteristics ( e.g. , temperature , ph , and mixing ) on ebola virus \n persistence is unknown and may contribute to altered environmental \n behavior . as such", ", we believe these results to be an upper bound for \n ebola virus persistence in wastewater matrices .", "subsequently , we caution \n extrapolation of these results without a holistic assessment of all \n factors , including wastewater composition , dilution , and potential \n exposure routes .", "the results of this \n study suggest a potential \n exposure route to infectious ebola virus via wastewater ; however , \n any assessment of potential exposure routes must consider the effects \n of wastewater composition , dilution of contaminated wastewater , and \n inactivation of ebola virus during treatment and holding .", "additionally , \n the possibility for ebola virus transmission via wastewater and subsequent \n infection remains unknown .", "the who updated guidelines in january 2015 \n to recommend holding of latrine waste for 1 week prior to further \n handling or transport .", "the objective \n of this holding period is to allow ebola virus die - off . on the basis \n of these results", ", it would be reasonable to approximate a three - log \n ( i.e. , 99.9% ) removal of ebola virus due to this holding period . the \n resulting risk would ultimately depend upon the initial ebola virus \n concentration in wastewater , potential for exposure , and susceptibility \n to infection via wastewater exposure .", "the wastewater travel times \n for wastewater via a sewer system to a centralized sewage treatment \n works would typically be < 1 day , depending on system dynamics .", "further assessment is necessary to determine \n ebola inactivation and dilution within this period and potential human \n exposure routes , including workers within the sewer system and ebola \n virus persistence within wastewater sludges .", "the greatest exposure \n risk would be expected for persons in contact with contaminated wastewater \n prior to significant dilution , treatment , or holding .", "these \n results demonstrate a persistence of ebola virus in wastewater greater \n than what has previously been suggested and the potential of a wastewater \n exposure route to infectious ebola virus .", "while ebola virus was found \n to be generally less persistent than enteric viruses in wastewater , \n the identified survival period suggests value in a nuanced evaluation \n of wastewater exposure risks during an epidemic response .", "specifically , \n these findings highlight the value of a precautionary approach to \n wastewater handling within an outbreak scenario , in response to both \n ebola virus and other emerging viruses ."], "abstract_text": ["<S> in the wake of the ongoing 2014/2015 \n ebola virus outbreak , significant \n questions regarding the appropriate handling of ebola virus - contaminated \n liquid waste remain , including the persistence of ebola virus in wastewater . \n </S>", "<S> to address these uncertainties , we evaluated the persistence of ebola \n virus spiked in sterilized domestic sewage . </S>", "<S> the viral titer decreased \n approximately 99% within the first test day from an initial viral \n titer of 106 tcid50 ml1 ; \n however , it could not be determined if this initial rapid decrease \n was due to aggregation or inactivation of the viral particles . </S>", "<S> the \n subsequent viral titer decrease was less rapid , and infectious ebola \n virus particles persisted for all 8 days of the test . </S>", "<S> the inactivation \n constant ( k ) was determined to be 1.08 ( 2.1 \n days for a 90% viral titer decrease ) . </S>", "<S> due to experimental conditions , \n we believe these results to be an upper bound for ebola virus persistence \n in wastewater . </S>", "<S> wastewater composition is inherently heterogeneous ; \n subsequently , we caution that interpretation of these results should \n be made within a holistic assessment , including the effects of wastewater \n composition , dilution , and potential exposure routes within wastewater \n infrastructure . </S>", "<S> while it remains unknown if ebola virus may be transmitted \n via wastewater , these data demonstrate a potential exposure route \n to infectious ebola virus via wastewater and emphasize the value of \n a precautionary approach to wastewater handling in an epidemic response . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results and Discussion"], "sections": [["in march 2014 , an unprecedented \n outbreak of ebola virus disease \n ( evd ) began in western africa that as of july 2015 is ongoing and \n has claimed more than 11000 lives .", "the \n current outbreak is caused by viruses belonging to the species of \n the zaire ebolavirus , a member of the filoviridae family .", "filoviridae are enveloped , filamentous viruses with lengths that \n may reach > 1000 nm .", "ebola virus \n can be excreted in bodily fluids , including vomit , stool , blood , saliva , \n semen , and breast milk .", "ebola virus loads of up to 10 genome \n copies ml have been reported in blood , 10 genome copies ml in stool , and 10 genome copies ml in urine ; however , the conversion between genome copies \n and infectious units is unknown .", "once infected , individuals may produce up to 9 l of liquid waste \n per day , primarily watery diarrhea .", "ebola virus is considered a potential bioterrorism \n agent . in response to the evd epidemic , both \n the world health organization \n ( who ) and the u.s .", "centers for disease control and prevention advised \n direct disposal of ebola - contaminated liquid waste into sewage systems \n ( wastewater collection and treatment systems ) and latrines without \n disinfection .", "initial recommendations \n were made on the basis of an expected limited persistence of ebola \n virus in the environment , as ebola virus is an enveloped virus , and \n a lack of strong evidence for a waterborne transmission route . as \n stated by a who guidance document , ", "ebola virus is likely to \n inactivate significantly faster in the environment than enteric viruses \n with known waterborne transmission ( e.g. , norovirus , hepatitis a virus )  .", "however , as has been noted in a recent review , \n the persistence of enveloped viruses in the water environment varies \n by > 2 orders of magnitude .", "recommendations \n for ebola virus - contaminated wastewater disposal were met with debate \n ( e.g. , refs ( 1618 ) ) because of uncertainty \n about ebola virus persistence within wastewater matrices and the lack \n of a risk - based analysis for waste handling .", "wastewater handling recommendations \n have since been revised to recognize uncertainty in this area and \n to recommend disinfection of latrines and holding of wastewater prior \n to handling to allow ebola virus inactivation .", "additionally , \n some facilities have opted to provide additional disinfection prior \n to disposal of liquid waste into sewer systems .", "recent research found both ethanol and hypochlorite to \n be effective disinfectants for ebola virus dried on surfaces ; however , the disinfection kinetics of ebola \n virus within liquid matrices remains unknown .", "various wastewater disinfection \n approaches have been recently suggested for pathogen control in an \n outbreak setting .", "currently , no \n data on ebola virus persistence in wastewater exist , \n hindering risk estimation and examination of potential environmental \n exposure routes .", "the necessity of evaluating ebola virus persistence \n in wastewater matrices has previously been highlighted , as wastewater \n in ebola virus outbreak settings may be temporarily held in open containers \n or disposed of in open sewers .", "historically , \n the transmission of ebola virus via environmental routes ( droplets , \n aerosols , or fomites ) has been thought to be unlikely due to epidemiological \n evidence and environmental sampling .", "the \n primary ebola virus transmission route is via direct contact with \n bodily fluids .", "transmission has previously \n occurred without known direct contact with infected individuals , providing supporting evidence that ebola virus \n transmission may be possible via large droplets .", "the potential for transmission of ebola virus via wastewater \n is currently unknown . to address uncertainties regarding ebola \n virus persistence in wastewater", ", \n we have conducted an initial evaluation of ebola virus persistence \n within wastewater to address uncertainty and inform ongoing risk assessments . \n a current ebola virus outbreak strain from guinea ( makona - wpgc07 ) \n", "was spiked to two end concentrations ( 10 and 10 tcid50 ml ) into a domestic wastewater \n ( untreated sewage ) sample .", "the ebola virus - containing wastewater was \n sampled for 8 days , and the viability of ebola virus was determined \n in these samples .", "study results are presented , and study limitations \n and implications are discussed , as well as recommendations for an \n ongoing research agenda ."], ["untreated wastewater was \n collected from an anonymous regional wastewater \n treatment facility in western pennsylvania that receives wastewater \n from seven communities ( combined population of approximately 60000 ) . \n", "the total raw wastewater flow to the treatment facility is < 10 \n mgd ( million gallons per day ) .", "the wastewater \n was frozen at 80 c to minimize compositional changes \n prior to analysis .", "wastewater characteristics ( table 1 ) were determined at an epa - certified analysis \n facility ( microbac laboratories , marietta , oh ) .", "the region from which \n the sample was collected uses a combined sewer system that experiences \n significant infiltration , and the determined composition is typical \n for the region .", "ebola virus cultivation experiments \n were conducted at rocky mountain \n laboratories under bsl4 conditions .", "wastewater samples were shipped \n to rocky mountain laboratories overnight on ice . upon receipt , samples \n were sterilized with 5 mrad of -irradiation .", "sterilization \n was performed to limit cell culture death due to wastewater microbial \n activity leading to a false positive .", "stock virus ( ebola virus guinea \n makona - wpgc07 , 10 tcid50 ml ) was handled as described previously .", "ebola virus was diluted in wastewater to achieve \n two separate virus titers ( 10 and 10 tcid50 ml ) , and both experiments were completed \n in triplicate .", "the ebola virus concentration in sewage has not been \n previously measured or estimated ; thus , two separate concentrations \n were utilized to cover possible concentration scenarios .", "tcid50 is an end point dilution series that is used to determine \n at what dilution 50% of the infected wells produce cell death .", "an approximation of focus - forming \n units ( ffus ) can be made from a poisson distribution utilizing the \n formula tcid50  0.69 , assuming each ffu is formed \n from a single virus .", "spiked wastewater was then distributed into three \n labeled vials for each concentration , and samples were taken daily \n for 8 days .", "tests were conducted at 20 c and 40% relative humidity . \n at each time point , including the time zero measurement , 50 l \n of wastewater from the bulk wastewater vial was added into 450 l \n of dulbecco s modified eagle s medium ( dmem , sigma ) \n supplemented with heat - inactivated fetal bovine serum ( fbs , gibco ) \n to a final concentration of 2% , pen / strep ( gibco ) to a final concentration \n of 50 units / ml penicillin and 50 g / ml streptomycin , and l - glutamine ( gibco ) to a final concentration of 2 mm in an appropriately \n labeled 2 ml screw top vial and frozen at 80 c .", "negative \n controls were 50 l of nonspiked wastewater in 450 l \n of dmem . to perform the titrations ,", "a 96-well 1.1 ml well dilution \n plate ( axygen , corning , ny ) was prepared . the thawed sample ( 400 l ) \n", "was placed in the top row of the plate and a 10-fold dilution conducted \n by passing 40 l of sample into 360 l of dmem .", "next , \n 100 l from each well of the dilution plate was transferred \n to a 96-well cell culture plate seeded with vero cells .", "the cells \n were incubated with virus dilutions for 1 h ; then the medium was removed \n from the two highest concentrations and rinsed two times with pbs , \n and 200 l of fresh culture medium was added .", "fresh culture \n medium ( 100 l ) was also added to the remaining wells in the \n plate .", "the plates were incubated at 37 c for 7 days , inspected \n for the cytopathic effect ( cpe ) , and scored .", "the natural \n logarithm of c / co tcid50 was plotted and fit with a linear trendline for estimation \n of the inactivation constant ( k ) .", "a literature review \n was then performed to compare observed inactivation to other environmental \n matrices ."], ["ebola virus was \n spiked into wastewater at two concentrations and assayed for 8 days \n to determine persistence in a wastewater matrix .", "the time zero time \n point was measured immediately following addition of ebola virus to \n the wastewater .", "no viable ebola virus was recovered from samples spiked \n with 10 ebola virus tcid50 ml after the initial time zero sampling .", "virus \n viabilities from the initial 10 ebola virus tcid50 ml concentration are shown in figure 1 and detailed in table s1 .", "ebola virus titer was rapidly reduced ( approximately \n 99% ) within the first day of the test , consistent with an inability \n to identify infectious ebola virus from the initial sample with 10 ebola virus tcid50 ml on day \n 1 .", "persistence of an initial ebola virus concentration of 10 tcid50 ml in domestic wastewater \n ( untreated sewage ) ( a ) including the time zero time point and ( b ) \n excluding the time zero time point to mitigate potential aggregation \n effects .", "fit inactivation constants ( k ) were determined to be 1.08 when including time \n zero and 0.35 when excluding time zero .", "error bars are 1 \n standard deviation . there was a rapid decrease \n ( approximately 99% ) in ebola virus titer \n within the first day of the test .", "in addition to inactivation , viral \n particle aggregation or adsorption to wastewater particles may play \n a role in the apparent rapid viral decrease and enhanced viral persistence . \n in this case", "first , aggregated \n particles would not be detected as multiple infectious units , resulting \n in an apparent rapid decrease .", "second , it has been previously recognized \n that aggregation increases viral persistence and resistance to inactivation \n stressors .", "similarly , viral association with particles \n has been recognized to provide protection from inactivation , including association of viral particles with \n wastewater solids .", "mechanistically , particle \n association is believed to protect viral particles from inactivation \n by shielding them from environmental stressors and is dependent on \n the organism and particle type . utilizing the current assay , it can not be determined if the initial \n rapid decrease in viral titer was due to viral inactivation or aggregation . \n", "aggregation would result in an apparent viral titer decrease as each \n viral aggregate would function as an infectious unit in the cell culture \n assay . to address this uncertainty , we plotted two inactivation curves , \n both including ( figure 1a ) and excluding the measured time zero point ( figure 1b ) .", "a linear trendline , as has been previously \n suggested for viral and ebola virus persistence , showed a lower fit ( r = 0.59 including the time zero time point , and r = 0.67 excluding the time zero time point ) \n than that previously observed for ebola virus in deionized water ( r > 0.91 ) , but a fit better than that previously \n observed for ebola virus in human blood ( r < 0.29 ) .", "the inactivation constant ( k ) was determined \n to be 1.08 when including the time zero time point and 0.35 \n when excluding the time zero time point .", "on the basis of the model \n fit , the t90 ( time for 90% inactivation ) \n would be 2.1 days including the time zero time point and 6.6 days \n excluding the time zero time point .", "the observed ebola virus inactivation \n in wastewater was slower than that observed for deionized water , which \n required 1.8 days for 90% inactivation at 21 c .", "the observed ebola virus inactivation in wastewater was \n more rapid than that reported for human blood , which required 20 days \n for a 90% inactivation , and results are \n consistent with recent studies that identified viable ebola virus \n to persist in infected macaque blood for > 8 days . in general , ebola virus was found to be less persistent \n in wastewater than model enteric viruses . while the t90 for ebola virus in wastewater was found to be < 1 \n day , the t90 for hepatitis a is greater \n than 17 days and the t90 for enteric adenovirus is 33 days ; however , the t90 for poliovirus \n is 5 days , which is between the observed t90 values including or excluding the time zero \n time point .", "the results demonstrate a more rapid initial viral titer \n decrease but overall enhanced persistence of ebola virus in wastewater \n compared to the proposed enveloped surrogate bacteriophage phi6 .", "this \n study has two primary limitations \n that may alter the persistence of ebola virus compared to what may \n be observed in the field .", "first , the tested wastewater was more dilute \n than would be expected in typical latrine waste . in general , interaction with constituents within the wastewater \n ( e.g. , ammonia ) would be expected to contribute to more rapid inactivation \n of viruses ; however , the true effect \n of these constituents on ebola virus persistence is unknown .", "second , \n the wastewater was frozen to minimize compositional changes and disinfected \n ( -irradiation ) prior to utilization to limit microbial activity \n resulting in false positive viral cell culture . microbial activity \n within wastewater matrices would be expected to contribute to more \n rapid inactivation of infectious viral particles ; however , the true effect of microbial activity on ebola virus persistence \n is unknown .", "microbial activity reduces viral persistence through both \n the production of metabolites detrimental to viral persistence and \n direct usage of the viral particles as a nutrient source .", "additionally , the influence of other environmental \n characteristics ( e.g. , temperature , ph , and mixing ) on ebola virus \n persistence is unknown and may contribute to altered environmental \n behavior . as such", ", we believe these results to be an upper bound for \n ebola virus persistence in wastewater matrices .", "subsequently , we caution \n extrapolation of these results without a holistic assessment of all \n factors , including wastewater composition , dilution , and potential \n exposure routes .", "the results of this \n study suggest a potential \n exposure route to infectious ebola virus via wastewater ; however , \n any assessment of potential exposure routes must consider the effects \n of wastewater composition , dilution of contaminated wastewater , and \n inactivation of ebola virus during treatment and holding .", "additionally , \n the possibility for ebola virus transmission via wastewater and subsequent \n infection remains unknown .", "the who updated guidelines in january 2015 \n to recommend holding of latrine waste for 1 week prior to further \n handling or transport .", "the objective \n of this holding period is to allow ebola virus die - off . on the basis \n of these results", ", it would be reasonable to approximate a three - log \n ( i.e. , 99.9% ) removal of ebola virus due to this holding period . the \n resulting risk would ultimately depend upon the initial ebola virus \n concentration in wastewater , potential for exposure , and susceptibility \n to infection via wastewater exposure .", "the wastewater travel times \n for wastewater via a sewer system to a centralized sewage treatment \n works would typically be < 1 day , depending on system dynamics .", "further assessment is necessary to determine \n ebola inactivation and dilution within this period and potential human \n exposure routes , including workers within the sewer system and ebola \n virus persistence within wastewater sludges .", "the greatest exposure \n risk would be expected for persons in contact with contaminated wastewater \n prior to significant dilution , treatment , or holding .", "these \n results demonstrate a persistence of ebola virus in wastewater greater \n than what has previously been suggested and the potential of a wastewater \n exposure route to infectious ebola virus .", "while ebola virus was found \n to be generally less persistent than enteric viruses in wastewater , \n the identified survival period suggests value in a nuanced evaluation \n of wastewater exposure risks during an epidemic response .", "specifically , \n these findings highlight the value of a precautionary approach to \n wastewater handling within an outbreak scenario , in response to both \n ebola virus and other emerging viruses ."]]}
{"article_id": "PMC1805066", "article_text": ["pulmonary nodules and lung tumours move substantially during respiration with significant differences according to the tumour localization .", "respiratory tumour motion is a big challenge for radiotherapy , especially for high precision techniques .", "an additional target dose escalation which is deemed necessary for a curative approach of lung cancer is limited as so far as tumour motion is not sufficiently quantified and integrated into modern treatment planning .", "thus , the risk of side effects within the surrounding normal tissue is unacceptably high .", "radiation pneumonitis and the development of lung fibrosis are the physiological reactions of the lung which are not acceptable in severely ill patients .", "thus modern techniques of adaptive radiotherapy will greatly benefit from devices which take lung and tumour motion into account in radiotherapy planning .", "the final objective is an additional increase in the radiation dose within the tumour volume together with maximum protection of the adjacent normal lung parenchyma .", "a basic requirement is the accurate measurement and quantification of the mobility of pulmonary tumours . but also for follow - up examinations of patients with pulmonary tumours , a confident registration of the respiratory level is important in order to achieve easy identification and valid measurements .", "integration of the knowledge about lung and lung tumour motion is essential for planning of high precision radiotherapy .", "four - dimensional imaging using computed tomography ( 4d - ct ) is under development for optimization of radiotherapy by integration of motion [ 24 ] .", "these developments also include online imaging with a linac - integrated cone beam ct . the particular advantages of magnetic resonance imaging ( mri ) are the high soft - tissue contrast , differentiation of tumour and atelectasis , the possibility to choose the optimal plane for motion quantification and the possibility to integrate further dynamic parameters , e.g. lung perfusion or o 2 \n quantification , into one imaging session .", "the temporal resolution is already reasonably high for monitoring lung tumour motion in real - time . \n", "novel mri techniques allow for non - invasive acquisition of the motion of lung and pulmonary tumours   during the respiratory cycle with high spatial and temporal resolution [ 8 , 9 ] .", "continuous improvements and new developments in mr scanner technology , such as high performance gradient systems and pulse sequences , have made this possible and address the inherent challenges of mri of the chest , such as low proton density with an unfavourable signal to noise ratio and short t2   relaxation times with \n substantial susceptibility artefacts .", "parallel imaging techniques make use of the different spatial sensitivities for the receiver coils in order to employ them for the simultaneous acquisition of image data , significantly reduce the phase encoding steps and achieve a marked improvement in spatial and temporal resolution .", "thus , important requirements were met to measure the respiratory movement of the lung quantitatively .", "fast - low - angle - shot ( flash ) sequences with a temporal resolution of one image per second demonstrate chest wall motion per se and postoperative changes of respiratory motion , i.e. changes of the cranio - caudal distance before and after lung volume reduction surgery .", "modified true - fast - imaging - with - steady - state - precession ( truefisp ) sequences , widely used in cardiac mri , were successfully applied in volunteers ( fig .", "1 ) . by adding a mathematical model , a continuous quantitative measurement of lung volume was achieved , which again showed a high correlation with spirometry . \n\n", "after successful tests in volunteers , dynamic mri was transferred to patients with solitary pulmonary tumours in order to visualise lung tumour motion by mri in two dimensions ( fig .", "quantification was performed by measuring the cranio - caudal distance and the diaphragmatic length over time .", "again , the results showed good correlations with the forced expiratory capacity in 1 s .", "the diaphragmatic length was particularly useful in the assessment of respiratory mechanics and the respective effects of pulmonary tumours .", "in such studies the truefisp sequence was superior to a flash sequence due to its higher t2 signal .", "the respiratory motion of patients with a pulmonary tumour showed a significant difference between the tumour bearing and the non - tumour bearing hemithorax .", "in addition , the location of the nodule had a marked effect on the regional mobility of the lung .", "thus , mri is capable of providing important complementary quantitative regional information , which might be especially useful during follow - up . depending on size and location of the tumour , differences in motion", "were observed in the three axes ( x- , \n y - and \n z - direction ) . for this assessment", "pulmonary nodules were grouped as cranial , middle and caudal according to giraud et al . \n", "especially , the cranio - caudal motion ( y -direction ) showed a significant dependence on tumour size and location .", "small tumours in the caudal lung area can \n move up to 5 cm during maximum respiration . but also during shallow breathing", ", nodules in the lower lung zone exhibited a mean dislocation of 9.5 mm in the cranio - caudal direction and 6.6 mm in the anterior  posterior direction ( fig .", "theoretical calculations for 20 patients with pulmonary nodules demonstrated that this information for treatment planning might already lead to a marked increase of the target dose .", "a sufficient safety margin might not be larger than 3.4 mm for the upper , 4.5 mm for the middle , and 7.2 mm for the lower lung zone .", "this is substantially smaller than the conventional safety margin of 5 or 10 mm , respectively , and might allow for an additional dose escalation especially in the upper lung zone .", "but it has to be mentioned that an individual quantification of the tumour motion is recommended as the interindividual differences in tumour motion are high .", "simultaneous application of markers on the external chest wall demonstrated correlation and influencing factors between internal and external motion .", "further developments in parallel imaging technology , especially view sharing techniques , have resulted in a further improvement in temporal resolution .", "this advantage can be applied to establish a three - dimensional flash sequence for a continuous volumetric visualisation of respiratory motion of lung and tumours . in \n fig .", "4 , the 3d - flash sequence is compared to the 2d - truefisp sequence .", "a 3d - flash data set contains 52 slices with isotropic 3 mm voxels and a temporal resolution of one data set per second .", "motion of pulmonary nodules can be visualised and quantified in all three axes in order to be transferred in potential treatment concepts .", "segmentation of such data sets , however , is still demanding and time - consuming and can not be recommended for clinical use , yet . \n  besides the mere information about tumour motion , three - dimensional techniques also allow for quantification of changes in tumour volume during the respiratory cycle .", "changes in tumour volume are associated with deformations and changes of the main rotation axes of the nodules .", "although this is not real rotation , the rotation of the axes can add up to 30 and impressively \n reflects the degree of deformation .", "however , this information seems to be highly important , e.g. for further improvements in position and adaptation of collimators in radiotherapy .", "in addition , the knowledge about deformation of nodules and changes in volume during the respiratory cycle has substantial impact on the accuracy of volume measurement during the follow - up of pulmonary nodules and attempts to calculate nodule doubling times . \n", "radiotherapy very quickly leads to a significant , often temporary reduction of motion of the treated hemithorax when compared to the situation prior to radiotherapy .", "this ipsilateral reduction is compensated for by an increased motion range of the contralateral lung .", "obviously , such changes between right and left lung can not be detected by global lung function tests .", "further studies are warranted to demonstrate which patients might benefit from preventive treatment , e.g. to prevent clinical aggravation . \n", "additional split lung analysis is feasible revealing important insights into differences between right and left lungs undetectable by global pulmonary function tests .", "motion of pulmonary tumours can be measured in two or three dimensions , effects of respiration on volume and shape can be calculated .", "however , presently the evaluation of such data sets is extremely time - consuming . \n", "substantial portions of this paper and the figures were originally published in plathow c , meinzer hp , kauczor h - u .", "respiratory cycle from maximum expiration to maximum inspiration within 1 s. top row shows a healthy volunteer with synchronous movement of both lungs .", "bottom row shows a patient with lung cancer on the left and subsequently reduced motion range of the left lung . \n ", "lung motion is reflected by measuring the distance from the apex to the top of the diaphragm ( l ) .", "cranio - caudal lung tumour motion is measured with regard to the intervertebral space t 6/7 ( 1 ) ; antero - posterior motion with regard central anterior margin of the spine ( 2 ) ; and medio - lateral motion with regard to the centre line of the spinal processus ( 3 ) . \n  maximum lung tumour motion ( mm ) during shallow breathing in cranio - caudal ( cc ) , medio - lateral ( ml ) and antero - posterior ( ap ) direction depending on tumour localization in the upper , middle or lower lung zone . \n ", "top row , 2d - truefisp sequence ; middle row , 3d - flash sequence with a temporal resolution of one volume data set per second ; bottom row , volume segmentation of lung and tumour from the 3d - flash data set . \n"], "abstract_text": ["<S> novel technology has made dynamic magnetic resonance imaging ( mri ) of lung motion and lung tumour mobility during continuous respiration feasible . </S>", "<S> this might be beneficial for planning of radiotherapy of lung tumours , especially when using high precision techniques . </S>", "<S> this paper describes the recent developments to analyze and visualize pulmonary nodules during continuous respiration using mri . besides recent dynamic two - dimensional approaches to quantify motion of pulmonary nodules during respiration novel three - dimensional techniques are presented . beyond good correlation to pulmonary function tests mri also provides regional information about differences between tumour - bearing and non - tumour bearing lung and the restrictive effects of radiotherapy as well as the compensation by the contralateral lung . \n </S>"], "labels": null, "section_names": ["Introduction", "MRI techniques", "Visualisation of lung tumour motion", "Therapy monitoring", "Summary", "Acknowledgement", "Figures and Tables"], "sections": [["pulmonary nodules and lung tumours move substantially during respiration with significant differences according to the tumour localization .", "respiratory tumour motion is a big challenge for radiotherapy , especially for high precision techniques .", "an additional target dose escalation which is deemed necessary for a curative approach of lung cancer is limited as so far as tumour motion is not sufficiently quantified and integrated into modern treatment planning .", "thus , the risk of side effects within the surrounding normal tissue is unacceptably high .", "radiation pneumonitis and the development of lung fibrosis are the physiological reactions of the lung which are not acceptable in severely ill patients .", "thus modern techniques of adaptive radiotherapy will greatly benefit from devices which take lung and tumour motion into account in radiotherapy planning .", "the final objective is an additional increase in the radiation dose within the tumour volume together with maximum protection of the adjacent normal lung parenchyma .", "a basic requirement is the accurate measurement and quantification of the mobility of pulmonary tumours . but also for follow - up examinations of patients with pulmonary tumours , a confident registration of the respiratory level is important in order to achieve easy identification and valid measurements .", "integration of the knowledge about lung and lung tumour motion is essential for planning of high precision radiotherapy .", "four - dimensional imaging using computed tomography ( 4d - ct ) is under development for optimization of radiotherapy by integration of motion [ 24 ] .", "these developments also include online imaging with a linac - integrated cone beam ct . the particular advantages of magnetic resonance imaging ( mri ) are the high soft - tissue contrast , differentiation of tumour and atelectasis , the possibility to choose the optimal plane for motion quantification and the possibility to integrate further dynamic parameters , e.g. lung perfusion or o 2 \n quantification , into one imaging session .", "the temporal resolution is already reasonably high for monitoring lung tumour motion in real - time . \n"], ["novel mri techniques allow for non - invasive acquisition of the motion of lung and pulmonary tumours   during the respiratory cycle with high spatial and temporal resolution [ 8 , 9 ] .", "continuous improvements and new developments in mr scanner technology , such as high performance gradient systems and pulse sequences , have made this possible and address the inherent challenges of mri of the chest , such as low proton density with an unfavourable signal to noise ratio and short t2   relaxation times with \n substantial susceptibility artefacts .", "parallel imaging techniques make use of the different spatial sensitivities for the receiver coils in order to employ them for the simultaneous acquisition of image data , significantly reduce the phase encoding steps and achieve a marked improvement in spatial and temporal resolution .", "thus , important requirements were met to measure the respiratory movement of the lung quantitatively .", "fast - low - angle - shot ( flash ) sequences with a temporal resolution of one image per second demonstrate chest wall motion per se and postoperative changes of respiratory motion , i.e. changes of the cranio - caudal distance before and after lung volume reduction surgery .", "modified true - fast - imaging - with - steady - state - precession ( truefisp ) sequences , widely used in cardiac mri , were successfully applied in volunteers ( fig .", "1 ) . by adding a mathematical model , a continuous quantitative measurement of lung volume was achieved , which again showed a high correlation with spirometry . \n\n"], ["after successful tests in volunteers , dynamic mri was transferred to patients with solitary pulmonary tumours in order to visualise lung tumour motion by mri in two dimensions ( fig .", "quantification was performed by measuring the cranio - caudal distance and the diaphragmatic length over time .", "again , the results showed good correlations with the forced expiratory capacity in 1 s .", "the diaphragmatic length was particularly useful in the assessment of respiratory mechanics and the respective effects of pulmonary tumours .", "in such studies the truefisp sequence was superior to a flash sequence due to its higher t2 signal .", "the respiratory motion of patients with a pulmonary tumour showed a significant difference between the tumour bearing and the non - tumour bearing hemithorax .", "in addition , the location of the nodule had a marked effect on the regional mobility of the lung .", "thus , mri is capable of providing important complementary quantitative regional information , which might be especially useful during follow - up . depending on size and location of the tumour , differences in motion", "were observed in the three axes ( x- , \n y - and \n z - direction ) . for this assessment", "pulmonary nodules were grouped as cranial , middle and caudal according to giraud et al . \n", "especially , the cranio - caudal motion ( y -direction ) showed a significant dependence on tumour size and location .", "small tumours in the caudal lung area can \n move up to 5 cm during maximum respiration . but also during shallow breathing", ", nodules in the lower lung zone exhibited a mean dislocation of 9.5 mm in the cranio - caudal direction and 6.6 mm in the anterior  posterior direction ( fig .", "theoretical calculations for 20 patients with pulmonary nodules demonstrated that this information for treatment planning might already lead to a marked increase of the target dose .", "a sufficient safety margin might not be larger than 3.4 mm for the upper , 4.5 mm for the middle , and 7.2 mm for the lower lung zone .", "this is substantially smaller than the conventional safety margin of 5 or 10 mm , respectively , and might allow for an additional dose escalation especially in the upper lung zone .", "but it has to be mentioned that an individual quantification of the tumour motion is recommended as the interindividual differences in tumour motion are high .", "simultaneous application of markers on the external chest wall demonstrated correlation and influencing factors between internal and external motion .", "further developments in parallel imaging technology , especially view sharing techniques , have resulted in a further improvement in temporal resolution .", "this advantage can be applied to establish a three - dimensional flash sequence for a continuous volumetric visualisation of respiratory motion of lung and tumours . in \n fig .", "4 , the 3d - flash sequence is compared to the 2d - truefisp sequence .", "a 3d - flash data set contains 52 slices with isotropic 3 mm voxels and a temporal resolution of one data set per second .", "motion of pulmonary nodules can be visualised and quantified in all three axes in order to be transferred in potential treatment concepts .", "segmentation of such data sets , however , is still demanding and time - consuming and can not be recommended for clinical use , yet . \n  besides the mere information about tumour motion , three - dimensional techniques also allow for quantification of changes in tumour volume during the respiratory cycle .", "changes in tumour volume are associated with deformations and changes of the main rotation axes of the nodules .", "although this is not real rotation , the rotation of the axes can add up to 30 and impressively \n reflects the degree of deformation .", "however , this information seems to be highly important , e.g. for further improvements in position and adaptation of collimators in radiotherapy .", "in addition , the knowledge about deformation of nodules and changes in volume during the respiratory cycle has substantial impact on the accuracy of volume measurement during the follow - up of pulmonary nodules and attempts to calculate nodule doubling times . \n"], ["radiotherapy very quickly leads to a significant , often temporary reduction of motion of the treated hemithorax when compared to the situation prior to radiotherapy .", "this ipsilateral reduction is compensated for by an increased motion range of the contralateral lung .", "obviously , such changes between right and left lung can not be detected by global lung function tests .", "further studies are warranted to demonstrate which patients might benefit from preventive treatment , e.g. to prevent clinical aggravation . \n"], ["additional split lung analysis is feasible revealing important insights into differences between right and left lungs undetectable by global pulmonary function tests .", "motion of pulmonary tumours can be measured in two or three dimensions , effects of respiration on volume and shape can be calculated .", "however , presently the evaluation of such data sets is extremely time - consuming . \n"], ["substantial portions of this paper and the figures were originally published in plathow c , meinzer hp , kauczor h - u ."], ["respiratory cycle from maximum expiration to maximum inspiration within 1 s. top row shows a healthy volunteer with synchronous movement of both lungs .", "bottom row shows a patient with lung cancer on the left and subsequently reduced motion range of the left lung . \n ", "lung motion is reflected by measuring the distance from the apex to the top of the diaphragm ( l ) .", "cranio - caudal lung tumour motion is measured with regard to the intervertebral space t 6/7 ( 1 ) ; antero - posterior motion with regard central anterior margin of the spine ( 2 ) ; and medio - lateral motion with regard to the centre line of the spinal processus ( 3 ) . \n  maximum lung tumour motion ( mm ) during shallow breathing in cranio - caudal ( cc ) , medio - lateral ( ml ) and antero - posterior ( ap ) direction depending on tumour localization in the upper , middle or lower lung zone . \n ", "top row , 2d - truefisp sequence ; middle row , 3d - flash sequence with a temporal resolution of one volume data set per second ; bottom row , volume segmentation of lung and tumour from the 3d - flash data set . \n"]]}
{"article_id": "PMC2908231", "article_text": ["a 21-month - old male patient , 75 cm in height and 10 kg in weight , experienced tricuspid valve regurgitation ( tvr ) and was scheduled to undergo tricuspid valve plasty . the tvr was discovered in a follow - up test 1 year after he had undergone ventricular septal defect repair surgery . before tricuspid valve plasty", ", a tee was performed , which showed a level 3 - 4 tvr and severe right ventricular hypertrophy .", "atropine , thiopental sodium , and vecuronium were administered to induce anesthesia and muscle relaxation .", "a catheter was placed at the left radial artery to measure the constant blood pressure and analyze the abga ( table 1 ) .", "there were complications due to the adhesion of the svc and ivc near the surgical site .", "therefore , a 20 fr and 22 fr cannula was placed on them , respectively . 2 u of packed red blood cells ( prc ) , albumin , and mannitol was used as the priming solution . immediately after activating the cardiopulmonary bypass , the measured hematocrit level was 27.5% .", "the acid - base balance management mid - surgery was performed with an -stat . during extracorporeal circulation ,", "the body temperature was aimed at a moderately low degree of 25. the perfusion rate was kept at 0.5 - 1.0 l / min .", "after tricuspid valve surgery , the patient was weaned from the cardiopulmonary bypass by reducing the perfusion rate of the machine and starting mechanical ventilation .", "the total anesthesia , surgery and extracorporeal circulation time was 510 minutes , 420 minutes and 180 minutes , respectively .", "the amount of blood loss was estimated to be 800 ml . during surgery , 600 ml of a crystalloid solution , 3 u prc and 2 u platelets were administered .", "however the patient 's eyes could not focus , and his upper and lower limbs experienced intermittent convulsions .", "brain computed tomography revealed findings indicative of a cerebral infarction in the occipital lobe ( fig .", "eeg was performed , which showed no abnormal findings of alpha rhythm loss , which is customary in cortical blindness .", "phenobarbital was administered until 19 days after surgery under the suspicion of a connection between the ocular deviation and the convulsions .", "the convulsions did not return , but the reaction to light or objects in front of the eyes did not trigger an avoidance response from the patient .", "there were no hopeful clinical signs of a visual recovery but fundoscopy showed normal findings .", "on the postoperative day 28 , when the flash visual evoked potential ( fvep ) and mri had been planned , the patient 's eyes refocused .", "he was able to recognize the objects in front of his eyes , could walk and avoid things in his path , and was able to recognize his guardians .", "post surgery visual loss or reduction can be caused by a variety of factors , and occurs 0.1% to 1% of cases .", "visual loss is caused by damage to any connecting striate area between the cornea and occipital cortex .", "direct pressure on the eyeball , indirect increase in ocular tension due to improper positioning and blood vessel damage can cause central retinal artery occlusion or ischemic optic nerve damage , which can lead to visual loss . in addition , a severe decrease in blood pressure , emboli , and thrombus mid- or post - surgery can cause an infarction in the occipital cortex , which can lead to cortical blindness .", "visual loss after a cpb might be caused primarily due to emboli , severe low blood pressure , acute anemia , hypoxia or a combination of these factors . in the present case", "twenty seven cases were cortical blindness , of which 22 cases occurred after cardiac surgery ( 81% ) .", "aldrich et al .   reported a retrospective study of 25 patients , where the causes of cortical blindness were natural ischemic stroke in 8 patients ( 32% ) , cardiac surgery in 5 patients ( 20% ) , cerebral angiography in 3 patients ( 12% ) , non - surgery in 4 patients ( 16% ) , seizure in 2 patients ( 8% ) , and other factors , such as damage to the head and peritoneal dialysis , in 3 patients ( 12% ) .", "there were also cases of temporary cortical blindness reported after post - cardiac surgery angiography . in the case of children ,", "the early indicators of brain damage after surgery are age , the complexity of surgery , metabolic acidosis , increase in lactate , and artery acidosis . diagnosing cortical blindness is simple when the patient makes a complaint but as in our case , a pediatric patient has limited ability to communicate . therefore , conducting an eeg , fvep , angiography , and funduscopy can assist in making a diagnosis . in this case , the eeg did not show the abnormal finding of a loss in alpha rhythm . however , computed laminography revealed an infarction in the occipital cortex", "we checked for visual loss with light and objects to test the patient 's avoidance response but he did not respond properly .", "however , microemboli caused by extracorporeal circulation can obstruct the blood flow in the cerebral vessels , particularly in the watershed area .", "braekken et al .   reported that cardiac surgery using extracorporeal circulation causes microemboli in most cases .", "in addition , in patients with neural damage , there are more cases of transcranial doppler detection of microembolic signals in the right middle cerebral artery than in patients without .", "microemboli mid - surgery occurs most often when blocking the aorta ( 18% ) and unblocking the aorta ( 13% ) .", "rodriguez and belway   stated that in pediatric cardiac surgery , the likelihood of microemboli can increase with increasing time of extracorporeal circulation depending on the oxygenator , circuit , adnexa parts , and components .", "the surgery in this case was not complicated , but may have increased the likelihood of microembolization due to the long extracorporeal circulation time .", "however , we should have been more careful in the selecting the -stat and ph - stat method .", "nevertheless , the strength of the ph - stat should have been considered regarding neural sequelae .", "the cpb priming solution in pediatric patients , unlike adults , can cause blood thinning , which can lead to possible ischemic organ failure .", "aldrich et al .   reported that conditions , such as the age below 40 , no diabetes or high blood pressure , and no impairment in cognitive skills , language skills or memory , indicate a good prognosis for visual recovery .", "in addition , eeg tests can assist in a diagnosis but are not helpful in accurately predicting the prognosis . in this study ,", "two weeks after the surgery , he recovered his speech and cognitive skills , so we did expected a good prognosis .", "the patient 's vision recovered rapidly from postoperative day 28 and he could be discharged .", "other tests such as a fvep were not performed , so the precise extent of the recovery rate is unknown .", "considerable effort is required to reduce neural sequelae after pediatric cardiac surgery , such as visual loss .", "the cpb machine 's circuit components and type choice should be made on what can best prevent the likelihood of emboli .", "a pediatric transesophageal scan and cerebral oxymeter can help in the early identification of neural side - effects .", "a transesophageal scan plays an important role in eliminating emboli , so it should be prepared for all suitable pediatric patients .", "the pathological difference between an adult and pediatric cardiopulmonary bypass should be recognized , and proper measures should be followed .", "when cortical blindness is suspected , the ophthalmologist and neurologist should perform a combined examination , being cautious of permanent sequelae . in conclusion , with the reported visual loss in our pediatric patient , surgeons should consider the possibility of visual loss and cerebrovascular injury after a cardiac surgery using cardiopulmonary bypass ."], "abstract_text": ["<S> visual loss occurring after pediatric cardiac surgery employing cardiopulmonary bypass ( cpb ) is relatively rare but the risk is substantial . </S>", "<S> compromised cerebral perfusion due to a cpb related micro - embolization and inflammatory vascular changes as well as reduced oxygen carrying capacity in hemodilution and hypothermia during cpb might be major contributing factors to the development of postoperative visual loss after cardiac surgery with cpb . </S>", "<S> a case of immediate but transient postoperative visual loss was encountered in a 21-month - old male who underwent tricuspid valve surgery . despite routine intraoperative measures to maintain an adequate perfusion pressure throughout the procedure , postoperative computed tomography revealed a subacute infarct in his occipital lobe . </S>", "<S> recovery began on postoperative day 28 , and the patient 's vision was restored by 31 days . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 21-month - old male patient , 75 cm in height and 10 kg in weight , experienced tricuspid valve regurgitation ( tvr ) and was scheduled to undergo tricuspid valve plasty . the tvr was discovered in a follow - up test 1 year after he had undergone ventricular septal defect repair surgery . before tricuspid valve plasty", ", a tee was performed , which showed a level 3 - 4 tvr and severe right ventricular hypertrophy .", "atropine , thiopental sodium , and vecuronium were administered to induce anesthesia and muscle relaxation .", "a catheter was placed at the left radial artery to measure the constant blood pressure and analyze the abga ( table 1 ) .", "there were complications due to the adhesion of the svc and ivc near the surgical site .", "therefore , a 20 fr and 22 fr cannula was placed on them , respectively . 2 u of packed red blood cells ( prc ) , albumin , and mannitol was used as the priming solution . immediately after activating the cardiopulmonary bypass , the measured hematocrit level was 27.5% .", "the acid - base balance management mid - surgery was performed with an -stat . during extracorporeal circulation ,", "the body temperature was aimed at a moderately low degree of 25. the perfusion rate was kept at 0.5 - 1.0 l / min .", "after tricuspid valve surgery , the patient was weaned from the cardiopulmonary bypass by reducing the perfusion rate of the machine and starting mechanical ventilation .", "the total anesthesia , surgery and extracorporeal circulation time was 510 minutes , 420 minutes and 180 minutes , respectively .", "the amount of blood loss was estimated to be 800 ml . during surgery , 600 ml of a crystalloid solution , 3 u prc and 2 u platelets were administered .", "however the patient 's eyes could not focus , and his upper and lower limbs experienced intermittent convulsions .", "brain computed tomography revealed findings indicative of a cerebral infarction in the occipital lobe ( fig .", "eeg was performed , which showed no abnormal findings of alpha rhythm loss , which is customary in cortical blindness .", "phenobarbital was administered until 19 days after surgery under the suspicion of a connection between the ocular deviation and the convulsions .", "the convulsions did not return , but the reaction to light or objects in front of the eyes did not trigger an avoidance response from the patient .", "there were no hopeful clinical signs of a visual recovery but fundoscopy showed normal findings .", "on the postoperative day 28 , when the flash visual evoked potential ( fvep ) and mri had been planned , the patient 's eyes refocused .", "he was able to recognize the objects in front of his eyes , could walk and avoid things in his path , and was able to recognize his guardians ."], ["post surgery visual loss or reduction can be caused by a variety of factors , and occurs 0.1% to 1% of cases .", "visual loss is caused by damage to any connecting striate area between the cornea and occipital cortex .", "direct pressure on the eyeball , indirect increase in ocular tension due to improper positioning and blood vessel damage can cause central retinal artery occlusion or ischemic optic nerve damage , which can lead to visual loss . in addition , a severe decrease in blood pressure , emboli , and thrombus mid- or post - surgery can cause an infarction in the occipital cortex , which can lead to cortical blindness .", "visual loss after a cpb might be caused primarily due to emboli , severe low blood pressure , acute anemia , hypoxia or a combination of these factors . in the present case", "twenty seven cases were cortical blindness , of which 22 cases occurred after cardiac surgery ( 81% ) .", "aldrich et al .   reported a retrospective study of 25 patients , where the causes of cortical blindness were natural ischemic stroke in 8 patients ( 32% ) , cardiac surgery in 5 patients ( 20% ) , cerebral angiography in 3 patients ( 12% ) , non - surgery in 4 patients ( 16% ) , seizure in 2 patients ( 8% ) , and other factors , such as damage to the head and peritoneal dialysis , in 3 patients ( 12% ) .", "there were also cases of temporary cortical blindness reported after post - cardiac surgery angiography . in the case of children ,", "the early indicators of brain damage after surgery are age , the complexity of surgery , metabolic acidosis , increase in lactate , and artery acidosis . diagnosing cortical blindness is simple when the patient makes a complaint but as in our case , a pediatric patient has limited ability to communicate . therefore , conducting an eeg , fvep , angiography , and funduscopy can assist in making a diagnosis . in this case , the eeg did not show the abnormal finding of a loss in alpha rhythm . however , computed laminography revealed an infarction in the occipital cortex", "we checked for visual loss with light and objects to test the patient 's avoidance response but he did not respond properly .", "however , microemboli caused by extracorporeal circulation can obstruct the blood flow in the cerebral vessels , particularly in the watershed area .", "braekken et al .   reported that cardiac surgery using extracorporeal circulation causes microemboli in most cases .", "in addition , in patients with neural damage , there are more cases of transcranial doppler detection of microembolic signals in the right middle cerebral artery than in patients without .", "microemboli mid - surgery occurs most often when blocking the aorta ( 18% ) and unblocking the aorta ( 13% ) .", "rodriguez and belway   stated that in pediatric cardiac surgery , the likelihood of microemboli can increase with increasing time of extracorporeal circulation depending on the oxygenator , circuit , adnexa parts , and components .", "the surgery in this case was not complicated , but may have increased the likelihood of microembolization due to the long extracorporeal circulation time .", "however , we should have been more careful in the selecting the -stat and ph - stat method .", "nevertheless , the strength of the ph - stat should have been considered regarding neural sequelae .", "the cpb priming solution in pediatric patients , unlike adults , can cause blood thinning , which can lead to possible ischemic organ failure .", "aldrich et al .   reported that conditions , such as the age below 40 , no diabetes or high blood pressure , and no impairment in cognitive skills , language skills or memory , indicate a good prognosis for visual recovery .", "in addition , eeg tests can assist in a diagnosis but are not helpful in accurately predicting the prognosis . in this study ,", "two weeks after the surgery , he recovered his speech and cognitive skills , so we did expected a good prognosis .", "the patient 's vision recovered rapidly from postoperative day 28 and he could be discharged .", "other tests such as a fvep were not performed , so the precise extent of the recovery rate is unknown .", "considerable effort is required to reduce neural sequelae after pediatric cardiac surgery , such as visual loss .", "the cpb machine 's circuit components and type choice should be made on what can best prevent the likelihood of emboli .", "a pediatric transesophageal scan and cerebral oxymeter can help in the early identification of neural side - effects .", "a transesophageal scan plays an important role in eliminating emboli , so it should be prepared for all suitable pediatric patients .", "the pathological difference between an adult and pediatric cardiopulmonary bypass should be recognized , and proper measures should be followed .", "when cortical blindness is suspected , the ophthalmologist and neurologist should perform a combined examination , being cautious of permanent sequelae . in conclusion , with the reported visual loss in our pediatric patient , surgeons should consider the possibility of visual loss and cerebrovascular injury after a cardiac surgery using cardiopulmonary bypass ."]]}
{"article_id": "PMC4223965", "article_text": ["neonatal period ( the first 28 days after birth ) , which is the period of various physiological adaptations to the extrauterine life , is a vulnerable time .", "more than 10 million children die before the age of 5 years , 8 million of whom die before the age of 1 year and more than half of these deaths occur in the first 4 weeks after birth .", "about 98% of the neonatal deaths occur in developing countries . according to the world health organization ( who )", ", the rate of deaths is 30 times more in the countries with the highest rate compared to the countries with the lowest rate . according to the reports of ministry of health and medical education", ", the index of neonatal death in iran is 16 - 20 deaths out of 1000 survived births , which is higher than that in developed countries . who has urged iran to halve its neonatal death rate by 2015 .", "risk factors in studying neonatal death are categorized into three groups : prenatal , intrapartum , and postnatal factors . all over the world , infection , preterm delivery , and birth asphyxia contribute to about 87% of neonatal deaths . in most societies , congenital malformations and premature birth", "babies with low birth weight ( lbw ) and extremely lbw account for 6 - 7% and 1% of babies death , respectively .", "these causes , however , are the second and third reasons , respectively , which are responsible for one half of the neonatal deaths .", "prematurity is responsible for about 60 - 80% of neonatal deaths all over the world in babies without congenital anomalies . in many cases ,", "neonatal diseases are caused by respiratory problems , of which hyaline membrane disease ( hmd ) can be mentioned as the foremost .", "about 1.3% of all neonatal deaths are caused by hmd or its complications in premature newborns .", "have mentioned five main causes of neonatal death , including prematurity , internal bleeding , septicemia , respiratory distress syndrome , and congenital anomalies , while gheibi et al .", "have reported prematurity ( 68% ) , hmds ( 51% ) , asphyxia ( 13% ) , sepsis ( 13% ) , and congenital cardiac malformations ( 8% ) as the most important causes of neonatal death .", "the baby 's gender has also been considered as an important factor in his or her death .", "one of the most important issues in babies death is suitable management and timely referral of newborn babies with high - risk levels and being hospitalized in neonatal intensive care unit ( nicu ) .", "nicu is a critical section in a hospital , dealing with infections ( e.g. septicemia , pneumonia , surgical infections , and other infections ) that are difficult to control . in the usa ,", "management , appropriate care , and supportive cares are the important factors in preventing babies death .", "rostami et al . have reported that the rescue chances of the babies who have received preventive nasal continuous positive airflow pressure ( ncpap ) are three times higher than the other babies . considering that an analytical and comprehensive study has not been done about the role of neonatal risk factors and the therapy given for hospitalized neonatal death in west azerbaijan ,", "the study is case  control study , wherein the case group included 250 died babies who were hospitalized in the nicu during 2007 - 2009 .", "the control group also included 250 newborn babies who were hospitalized in the same unit and were discharged alive and healthy after they were given appropriate treatment . only the newborn babies from shahid motahhari therapy - training center", "were selected in order to homogenize the sample and eliminate some threatening factors ( factors involved in referral , death , etc . ) .", "data collection was carried out through a researcher - designed questionnaire which was designed based on other studies and scholars points of view .", "inclusion criteria included the babies complete profiles , and the criteria for exclusion included their incomplete profiles .", "the questionnaire contained six sections ( mother 's and baby 's demographic information , causes of hospitalization , during - treatment complications , therapeutic actions , and causes of death ) for alive babies and five sections for dead ones .", "chi - square test , odds ratio ( or ) , and logistic regression were applied to analyze how the variables were related .", "based on single - variable analysis , neonatal risk factors affecting the newborn babies hospitalized in the nicu are as follows : 51.6% of case babies and 18.3% of control babies had first - minute apgar score of less than 6 , which proved to have a significant correlation with babies death ( p < 0.001 ) .", "about 9.6% of babies of case group and 2.4% of babies in the control group had during - birth asphyxia which proved to have a significant correlation with neonatal death ( p = 0.001 ) [ table 1 ] .", "the distribution of independent variables by independent variables ; p value based on chi - square test in this study , one of the factors causing death of babies hospitalized in the nicu was hmd ; 77.6% of case babies and 44.8% of control babies suffered from this disease ( p < 0.001 ) .", "fourteen percent of case babies and 2% of control babies had sepsis which showed a significant relation with babies death ( p < 0.001 ) . moreover ,", "58.8% of case babies and 71.5% of control babies were born through cesarean section which also proved to have a significant effect ( p < 0.001 ) [ table 1 ] .", "congenital anomalies have been recognized as one of the significant risk factors of death of babies hospitalized in the nicu .", "eight percent of case babies and 2.8% of control babies were reported to have congenital anomalies ( p < 0.001 ) [ table 1 ] . regarding therapeutic actions", ", the results of the study also showed that having ncpap played a significant role in decreasing death of babies hospitalized in the nicu ( p < 0.001 ) .", "there was also a significant relation between supportive cares and neonatal death ( p < 0.001 ) .", "increased number of pregnancies also had a significant effect on neonatal death ( p < 0.001 ) [ table 1 ] .", "variables like lbw , baby 's gender , blood group , history of preterm labor , and consanguinity had no significant role in the death of neonates hospitalized in the nicu . in order to gauge the key effects of the mentioned factors ,", "the adjusted or calculated from advanced logistic regression model was measured through advance selection method .", "the results showed that the or of neonatal death was 4.02 times greater for neonates with first - minute apgar score of less than 6 compared to those with first - minute apgar score of 6 and more , for asphyxia , the or was 6.16 , for hmd it was 4.08 , and for sepsis it was 6.42 . applying ncpap and", "multiparity led to a decrease in the death of neonates hospitalized in the nicu [ table 1 ] .", "in the present study , the most important neonatal risk factors affecting neonatal death were found to be sepsis , birth asphyxia , hmd , first - minute apgar score of less than 6 , congenital anomalies , and delivery method . in our study ,", "the most common risk factor for neonatal deaths was sepsis which raised the risk of death by infection to 6.42 times more than the other causes .", ", sepsis and its complications were reported to be the first cause of death , but in the investigations of nayeri et al . and amin et al . , sepsis had higher ratings .", "this may be due to the factor that premature babies are prone to infection , but it should be more investigated because of the high number of deaths resulting from it .", "this spread of sepsis has been reported to be in 1 - 10 cases out of 1000 live births , but 10 - 50% of deaths occur because of it . the second risk factor in the present study was at birth asphyxia , which is in line with tariq and kundi 's study which mentioned sepsis and neonatal asphyxia as the main causes of death , but in the studies carried out by sereshtedari et al . , it was the cause of the 16% deaths in 18 provinces of the country , in bangladesh approximately 26% of neonatal deaths are caused by asphyxia .", "the other serious clinical problem is respiratory difficulty , and in this study , it manifested as respiratory distress and hmd . in our study , it was the third risk factor of infant death .", "the study of abdul in malaysia showed that this problem with sepsis is the most important cause of infants death .", "there is also a second leading cause of infant death in tariq and kundi 's study , and respiratory distress syndrome which were studied by kumar et al . in india ,", "jehan et al . in pakistan , naghavi in 18 provinces of iran , javanmardi et al . in isfahan , amani et al . in ardebil , and in the studies of nayeri et al . in tehran and sereshtedari", "one of the causes of infant death , low apgar score at birth , is increasingly mentioned in many studies to be a risk factor .", "low apgar score at birth could be correlated to factors such as neonatal infections , birth asphyxia , meconium aspiration , respiratory distress syndrome , and other causes which may result in infant mortality . in our study , ncpap was applied in the control group two times more than in the case group , which resulted in a decrease of babies death by 0.43 .", "in their study , rostami et al . also reported that applying preventive ncpap to newborn babies will increase their survival chances by three times .", "congenital anomalies was found to be in the second position in the studies of sharifi , hematyar and yarjo , javanmardi et al . in esfahan , and naghavi 's study in 18 provinces .", "it was mentioned as the second and first cause in the studies of sareshtedati et al . and", "'s study , congenital anomalies and infection were mentioned as the leading factors of infant death .", "the rate of abnormalities in our study compared with other studies , which shows that it is a less important factor in the incidence of neonatal death .", "this may be due to high morbidity and mortality caused by other factors such as immaturity , lack of attention and careful examination at the time of birth , lack of screening for neonatal abnormalities , and not performing autopsy in dead neonates .", "death rate in babies with lbw is about 40 times higher than in babies with normal weight . unlike these studies", "this finding is in correlation with that of nayeri et al . in the present study ,", "normal vaginal delivery was higher in the case group than that in the control group .", "this finding shows that cesarean section can be better when there is an emergency case or prematurity .", "although emergency cesarean section is performed to save the lives of the mother and baby , the selection is not always uncomplicated . in a study , it has been reported that two - third of babies who died were born through cesarean section . in our study , despite the high level of infant mortality in males , there was no significant relation between gender and the cause of death .", "an important risk factor that was investigated in this study was the number of pregnancies .", "the results of this investigation showed that death rate decreased as the number of pregnancies increased .", "number of births higher than 5 , pregnancy number above 3 , and high rate of birth were mentioned as effective factors causing babies death in studies conducted by shirvani and khosravi , chaman et al .", ", and titaley et al . , respectively . appropriately carrying out care and supportive cares can be effective factors in preventing babies death . in our study ,", "51.6% of case babies and 2.3% of control babies had received supportive care from birth .", "higher death rate in case babies can be due to bad physical conditions and other threatening factors .", "the results of the study show that incorrect methods of transfer can increase the death rate . in this study , special attention was devoted to increasing the care standards in order to decrease the death rate . according to the results of the study that took into consideration factors such as age , occupation , consanguinity , blood group , preterm delivery background , and sex in two groups with equal distribution , it can be concluded that factors related to accompanying diseases and care during delivery play a significant role in neonatal death .", "therefore , regarding the fourth goal of millennium development , decreasing babies death to two - fourths by 2015 in countries with high rate of mortality , it seems to be better to spot pregnancies with high risk of babies death through pre - delivery care and to transfer mothers who are likely to bear premature or sick babies to centers that have nicus . in cases where it is impossible to specify whether a delivery is risky before birth ,", "the subsequent hospitalization problems can be prevented by providing appropriate conditions of transfer and therapeutic actions in intensive care units .", "the major risk factors in this study ( sepsis , asphyxia during birth , hmd , congenital anomaly , and apgar score less than 6 in the first minute of birth ) are considered as the major complications of preterm labor and preterm infants .", "so , strict preventive programs should be undertaken for premature preterm labor and preterm infants ; taking care of the infants in four stages ( before conception , during pregnancy , during delivery , and thereafter ) can be highly effective as well ."], "abstract_text": ["<S> background : a newborn is highly fragile whose death can be caused by embryonic , intrapartum , and postpartum factors . identifying the causes of death is the first step to be taken in order to decrease the death rate . </S>", "<S> the present study was aimed at finding out how neonatal factors and therapeutic actions affect death of newborn babies.materials and methods : the is a case - control study where the case group included 250 dead neonates who were hospitalized in the neonatal intensive care unit during 2007 - 2009 . </S>", "<S> the control group also included 250 newborn neonates who were hospitalized in the same unit and were discharged alive and healthy after they were treated appropriately . </S>", "<S> researcher - designed questionnaires including demographic characteristics of mothers and infants , causes of hospitalization , treatment and its complications , and causes of death were used to collect the required data . </S>", "<S> chi - square test , odds ratio ( or ) , and logistic regression were applied to analyze how the variables were related . </S>", "<S> p < 0.05 were considered significant . </S>", "<S> statistical analyses were carried out using software spss 16.0.results:the results of advanced logistic regression model showed that first - minute apgar less than 6 ( or = 4.02 ) , existence of birth asphyxia ( or = 6.16 ) , hyaline membrane disease ( or = 4.08 ) , and sepsis ( or = 6.42 ) increased the death rate of neonates hospitalized in the intensive care unit , and applying nasal continuous positive airway pressure and multiparity decreased the death rate of newborn babies . </S>", "<S> there was no significant correlation between variables such as low birth weight , consanguinity , blood group , pr eterm labor history , and baby 's gender , and the death rate of neonates.conclusions:the results of the study show that factors such as low apgar score , asphyxia , hyaline membrane disease , sepsis , prematurity and congenital anomalies , and method of delivery have a positive effect on the death of neonates . </S>", "<S> they also prove that suitable management and carrying out appropriate therapeutic actions and intensive care decrease the death rate of newborn babies . </S>"], "labels": null, "section_names": ["I", "M", "R", "D", "C"], "sections": [["neonatal period ( the first 28 days after birth ) , which is the period of various physiological adaptations to the extrauterine life , is a vulnerable time .", "more than 10 million children die before the age of 5 years , 8 million of whom die before the age of 1 year and more than half of these deaths occur in the first 4 weeks after birth .", "about 98% of the neonatal deaths occur in developing countries . according to the world health organization ( who )", ", the rate of deaths is 30 times more in the countries with the highest rate compared to the countries with the lowest rate . according to the reports of ministry of health and medical education", ", the index of neonatal death in iran is 16 - 20 deaths out of 1000 survived births , which is higher than that in developed countries . who has urged iran to halve its neonatal death rate by 2015 .", "risk factors in studying neonatal death are categorized into three groups : prenatal , intrapartum , and postnatal factors . all over the world , infection , preterm delivery , and birth asphyxia contribute to about 87% of neonatal deaths . in most societies , congenital malformations and premature birth", "babies with low birth weight ( lbw ) and extremely lbw account for 6 - 7% and 1% of babies death , respectively .", "these causes , however , are the second and third reasons , respectively , which are responsible for one half of the neonatal deaths .", "prematurity is responsible for about 60 - 80% of neonatal deaths all over the world in babies without congenital anomalies . in many cases ,", "neonatal diseases are caused by respiratory problems , of which hyaline membrane disease ( hmd ) can be mentioned as the foremost .", "about 1.3% of all neonatal deaths are caused by hmd or its complications in premature newborns .", "have mentioned five main causes of neonatal death , including prematurity , internal bleeding , septicemia , respiratory distress syndrome , and congenital anomalies , while gheibi et al .", "have reported prematurity ( 68% ) , hmds ( 51% ) , asphyxia ( 13% ) , sepsis ( 13% ) , and congenital cardiac malformations ( 8% ) as the most important causes of neonatal death .", "the baby 's gender has also been considered as an important factor in his or her death .", "one of the most important issues in babies death is suitable management and timely referral of newborn babies with high - risk levels and being hospitalized in neonatal intensive care unit ( nicu ) .", "nicu is a critical section in a hospital , dealing with infections ( e.g. septicemia , pneumonia , surgical infections , and other infections ) that are difficult to control . in the usa ,", "management , appropriate care , and supportive cares are the important factors in preventing babies death .", "rostami et al . have reported that the rescue chances of the babies who have received preventive nasal continuous positive airflow pressure ( ncpap ) are three times higher than the other babies . considering that an analytical and comprehensive study has not been done about the role of neonatal risk factors and the therapy given for hospitalized neonatal death in west azerbaijan ,"], ["the study is case  control study , wherein the case group included 250 died babies who were hospitalized in the nicu during 2007 - 2009 .", "the control group also included 250 newborn babies who were hospitalized in the same unit and were discharged alive and healthy after they were given appropriate treatment . only the newborn babies from shahid motahhari therapy - training center", "were selected in order to homogenize the sample and eliminate some threatening factors ( factors involved in referral , death , etc . ) .", "data collection was carried out through a researcher - designed questionnaire which was designed based on other studies and scholars points of view .", "inclusion criteria included the babies complete profiles , and the criteria for exclusion included their incomplete profiles .", "the questionnaire contained six sections ( mother 's and baby 's demographic information , causes of hospitalization , during - treatment complications , therapeutic actions , and causes of death ) for alive babies and five sections for dead ones .", "chi - square test , odds ratio ( or ) , and logistic regression were applied to analyze how the variables were related ."], ["based on single - variable analysis , neonatal risk factors affecting the newborn babies hospitalized in the nicu are as follows : 51.6% of case babies and 18.3% of control babies had first - minute apgar score of less than 6 , which proved to have a significant correlation with babies death ( p < 0.001 ) .", "about 9.6% of babies of case group and 2.4% of babies in the control group had during - birth asphyxia which proved to have a significant correlation with neonatal death ( p = 0.001 ) [ table 1 ] .", "the distribution of independent variables by independent variables ; p value based on chi - square test in this study , one of the factors causing death of babies hospitalized in the nicu was hmd ; 77.6% of case babies and 44.8% of control babies suffered from this disease ( p < 0.001 ) .", "fourteen percent of case babies and 2% of control babies had sepsis which showed a significant relation with babies death ( p < 0.001 ) . moreover ,", "58.8% of case babies and 71.5% of control babies were born through cesarean section which also proved to have a significant effect ( p < 0.001 ) [ table 1 ] .", "congenital anomalies have been recognized as one of the significant risk factors of death of babies hospitalized in the nicu .", "eight percent of case babies and 2.8% of control babies were reported to have congenital anomalies ( p < 0.001 ) [ table 1 ] . regarding therapeutic actions", ", the results of the study also showed that having ncpap played a significant role in decreasing death of babies hospitalized in the nicu ( p < 0.001 ) .", "there was also a significant relation between supportive cares and neonatal death ( p < 0.001 ) .", "increased number of pregnancies also had a significant effect on neonatal death ( p < 0.001 ) [ table 1 ] .", "variables like lbw , baby 's gender , blood group , history of preterm labor , and consanguinity had no significant role in the death of neonates hospitalized in the nicu . in order to gauge the key effects of the mentioned factors ,", "the adjusted or calculated from advanced logistic regression model was measured through advance selection method .", "the results showed that the or of neonatal death was 4.02 times greater for neonates with first - minute apgar score of less than 6 compared to those with first - minute apgar score of 6 and more , for asphyxia , the or was 6.16 , for hmd it was 4.08 , and for sepsis it was 6.42 . applying ncpap and", "multiparity led to a decrease in the death of neonates hospitalized in the nicu [ table 1 ] ."], ["in the present study , the most important neonatal risk factors affecting neonatal death were found to be sepsis , birth asphyxia , hmd , first - minute apgar score of less than 6 , congenital anomalies , and delivery method . in our study ,", "the most common risk factor for neonatal deaths was sepsis which raised the risk of death by infection to 6.42 times more than the other causes .", ", sepsis and its complications were reported to be the first cause of death , but in the investigations of nayeri et al . and amin et al . , sepsis had higher ratings .", "this may be due to the factor that premature babies are prone to infection , but it should be more investigated because of the high number of deaths resulting from it .", "this spread of sepsis has been reported to be in 1 - 10 cases out of 1000 live births , but 10 - 50% of deaths occur because of it . the second risk factor in the present study was at birth asphyxia , which is in line with tariq and kundi 's study which mentioned sepsis and neonatal asphyxia as the main causes of death , but in the studies carried out by sereshtedari et al . , it was the cause of the 16% deaths in 18 provinces of the country , in bangladesh approximately 26% of neonatal deaths are caused by asphyxia .", "the other serious clinical problem is respiratory difficulty , and in this study , it manifested as respiratory distress and hmd . in our study , it was the third risk factor of infant death .", "the study of abdul in malaysia showed that this problem with sepsis is the most important cause of infants death .", "there is also a second leading cause of infant death in tariq and kundi 's study , and respiratory distress syndrome which were studied by kumar et al . in india ,", "jehan et al . in pakistan , naghavi in 18 provinces of iran , javanmardi et al . in isfahan , amani et al . in ardebil , and in the studies of nayeri et al . in tehran and sereshtedari", "one of the causes of infant death , low apgar score at birth , is increasingly mentioned in many studies to be a risk factor .", "low apgar score at birth could be correlated to factors such as neonatal infections , birth asphyxia , meconium aspiration , respiratory distress syndrome , and other causes which may result in infant mortality . in our study , ncpap was applied in the control group two times more than in the case group , which resulted in a decrease of babies death by 0.43 .", "in their study , rostami et al . also reported that applying preventive ncpap to newborn babies will increase their survival chances by three times .", "congenital anomalies was found to be in the second position in the studies of sharifi , hematyar and yarjo , javanmardi et al . in esfahan , and naghavi 's study in 18 provinces .", "it was mentioned as the second and first cause in the studies of sareshtedati et al . and", "'s study , congenital anomalies and infection were mentioned as the leading factors of infant death .", "the rate of abnormalities in our study compared with other studies , which shows that it is a less important factor in the incidence of neonatal death .", "this may be due to high morbidity and mortality caused by other factors such as immaturity , lack of attention and careful examination at the time of birth , lack of screening for neonatal abnormalities , and not performing autopsy in dead neonates .", "death rate in babies with lbw is about 40 times higher than in babies with normal weight . unlike these studies", "this finding is in correlation with that of nayeri et al . in the present study ,", "normal vaginal delivery was higher in the case group than that in the control group .", "this finding shows that cesarean section can be better when there is an emergency case or prematurity .", "although emergency cesarean section is performed to save the lives of the mother and baby , the selection is not always uncomplicated . in a study , it has been reported that two - third of babies who died were born through cesarean section . in our study , despite the high level of infant mortality in males , there was no significant relation between gender and the cause of death .", "an important risk factor that was investigated in this study was the number of pregnancies .", "the results of this investigation showed that death rate decreased as the number of pregnancies increased .", "number of births higher than 5 , pregnancy number above 3 , and high rate of birth were mentioned as effective factors causing babies death in studies conducted by shirvani and khosravi , chaman et al .", ", and titaley et al . , respectively . appropriately carrying out care and supportive cares can be effective factors in preventing babies death . in our study ,", "51.6% of case babies and 2.3% of control babies had received supportive care from birth .", "higher death rate in case babies can be due to bad physical conditions and other threatening factors .", "the results of the study show that incorrect methods of transfer can increase the death rate . in this study , special attention was devoted to increasing the care standards in order to decrease the death rate . according to the results of the study that took into consideration factors such as age , occupation , consanguinity , blood group , preterm delivery background , and sex in two groups with equal distribution , it can be concluded that factors related to accompanying diseases and care during delivery play a significant role in neonatal death .", "therefore , regarding the fourth goal of millennium development , decreasing babies death to two - fourths by 2015 in countries with high rate of mortality , it seems to be better to spot pregnancies with high risk of babies death through pre - delivery care and to transfer mothers who are likely to bear premature or sick babies to centers that have nicus . in cases where it is impossible to specify whether a delivery is risky before birth ,", "the subsequent hospitalization problems can be prevented by providing appropriate conditions of transfer and therapeutic actions in intensive care units ."], ["the major risk factors in this study ( sepsis , asphyxia during birth , hmd , congenital anomaly , and apgar score less than 6 in the first minute of birth ) are considered as the major complications of preterm labor and preterm infants .", "so , strict preventive programs should be undertaken for premature preterm labor and preterm infants ; taking care of the infants in four stages ( before conception , during pregnancy , during delivery , and thereafter ) can be highly effective as well ."]]}
{"article_id": "PMC4749813", "article_text": ["lung cancer remains the leading type of cancer worldwide and in latin america [ 1 , 2 ] .", "the disease burden is significantly high , with around 2.5 million new cases per year and 1.5 million deaths worldwide .", "the two main histological subtypes of lung cancer are small - cell lung cancer ( sclc ) , which comprises 15% of cases , and non - small - cell lung cancer ( nsclc ) accounting for 85% of cases   which include adenocarcinoma , squamous cell carcinoma , and large cell carcinoma . among all newly diagnosed nsclc cases ,", "adenocarcinomas are the most frequent subgroup following by squamous cell carcinomas [ 6 , 7 ] .", "cigarette smoking is the major risk factor for lung cancer but around 1020% of cases are found in never smokers ; also wood - smoke is a major risk factor in countries like mexico [ 811 ] .", "surgery is the selected treatment for early stage nsclc with the greatest probability of long - term survival in such patients . in advanced nsclc ,", "conventional therapies are based on chemotherapy and radiotherapy but with low efficacy . over the last decade , there have been advances in the study of molecular pathways underlying tumor development leading to the development of targeted therapies such as tyrosine kinase inhibitors ( tkis ) and antibodies directed against the two main actionable genes in nsclc up to now : mutations in the epidermal growth factor receptor ( egfr ) gene targeted by tkis like gefitinib [ 13 , 14 ] , erlotinib [ 9 , 15 , 16 ] , and afatinib [ 1719 ] and translocations involving the anaplastic lymphoma kinase ( alk ) gene treated with the tki crizotinib , alectinib , and ceritinib .", "benefits have been shown in a subset of 1520% of patients harboring egfr mutations which correlate with definite clinical characteristics : adenocarcinoma histology , female sex , asian ethnicity , and nonsmokers [ 2325 ] . despite these improvements in therapeutic strategies ,", "early diagnosis is very difficult ; most cases are diagnosed at an advanced stage and cancer metastasis is very frequent ; therefore , there is still an exceedingly low 5-year survival rate of 1124% [ 2628 ] .", "the immunotherapy approach has opened new therapeutic options in advanced nsclc with the advent of antibodies against immune checkpoints [ 29 , 30 ] .", "recently , the anti - programmed death-1 ( pd-1 ) antibodies nivolumab and pembrolizumab have been approved in the treatment of advanced metastatic nsclc based on results from clinical trials after prior chemotherapy [ 31 , 32 ] .", "both antibodies block signaling through pd-1 and may restore antitumor immunity with benefits in overall survival [ 33 , 34 ] .", "for example , nivolumab , a fully human monoclonal antibody , has recently shown greater overall survival than docetaxel .", "these antibodies exhibit a reasonable toxicity profile but they should be administered in selected patient populations based on biomarkers such as pd - l1 expression to avoid serious immune - mediated adverse effects .", "although these checkpoint inhibitors have proven efficacy in patients , their mechanism of action implies side effects as the onset of autoimmune diseases and a series of endocrine disorders [ 37 , 38 ] .", "this is the rationale for further research into other molecular and cellular factors of the immune system that could be effectively targeted to develop novel therapeutic strategies for the management of advanced nsclc .", "recent findings indicate that inflammation plays a key role in tumor progression and survival across several cancer types .", "cancer related inflammation affects many aspects of malignancy including proliferation , survival , angiogenesis , and tumor metastasis .", "inflammatory components in the development of the neoplasm include diverse leukocytes populations , like macrophages and neutrophils , which respond immediately to inflammatory stimulus .", "immunoregulatory cytokines secreted in a proinflammatory environment also contribute to tumor growth and metastases and identify patient subsets in advanced nsclc with differential prognosis .", "both macrophages and neutrophils are increased in patients with lung cancer ; this is associated with poor clinical outcomes , suggesting that these cells might have important tumor - promoting activities [ 43 , 44 ] .", "tumors escape phagocytosis and immune response through overexpressing cd47 that interacts with the signal regulatory protein alpha ( sirp ) preventing engulfment .", "macrophages within the tumor microenvironment are called tumor - associated macrophages ( tams ) . tams", "have a complex relationship with tumor cells ; at an early stage they attack tumor cells avoiding tumor spread ; however , over time they begin producing reciprocal growth factors and establish a symbiotic relationship with tumor cells .", "macrophages are polarized into two functionally distinct forms m1 and m2 , mirroring the th1 and th2 nomenclature of t cells .", "differentiation of the m1 macrophages is induced by interferon- , lipopolysaccharides , tumor necrosis factor ( tnf )  , and granulocyte - monocyte colony - stimulating factor .", "the m1 macrophages produce high levels of interleukin- ( il- ) 12 , il-23 , tnf , il-1 , il-6 , cxc ligand 10 ( cxcl10 ) , inducible nitric oxide synthase ( inos ) , human leukocyte antigen- ( hla- ) dr , and reactive oxygen and nitrogen intermediates [ 47 , 48 ] .", "differentiation of the m2 macrophages is induced by il-4 , il-10 , il-13 , il-21 , activin a , immune complexes , and glucocorticoid .", "the m2 macrophages express high levels of il-10 , il-1 receptor antagonist , cc ligand 22 ( ccl22 ) , scavenger , mannose receptor , galactose receptor , arginase i , and cd163 antigen , reduce the expression of inos , and inhibit antigen presentation and t cell proliferation [ 47 , 49 ] . factors that shift tams towards a m2 phenotype include the location of tams within the tumor microenvironment , tumor stage , and type of cancer .", "nevertheless , it is still not fully defined whether the diversity within the tam population is due to the maturation of unique monocytic precursors or due to various factors within the local tumor microenvironment .", "the m2 macrophages have been found to encourage the growth of various tumour cells in vitro and to increase tumor cell survival [ 51 , 52 ] .", "studies suggest that in solid tumors established and progressively growing tams are reprogrammed to induce immune suppression in situ in the host through cytokines , prostanoids , and other humoral mediators [ 54 , 55 ] .", "il-1 and il-6 expression in tams differs in ovarian cancer compared to peripheral blood monocytes .", "tams in the ovary produce low levels of il1 and increase the release of il-6 , which contributes to elevated acute phase proteins and increased malignancy .", "there is an association between the number of macrophages and prognosis in a variety of human tumors .", "tam infiltration increased in carcinomas of breast , cervix , and bladder and correlates with a poor prognosis .", "however , in prostate , lung , and brain , increasing tams is associated with regression of tumors .", "tams can regulate the development of new blood vessels within tumors . in hypoxic sites , they stimulate the production of enzymes and extracellular matrix molecules that regulate endothelial cell activity by stimulating factors such as vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) , tumor necrosis factor- ( tnf- ) , transforming growth factors-  and  ( tgf-  ,  ) , interferons , thrombospondin , il-8 , and epidermal growth factor ( egf ) .", "innate immunity in lung involves alveolar macrophages ( ams ) which act as a barrier avoiding penetration of pathogens .", "conversely , macrophages contribute in part to the pathogenesis of lung disease due to toxic particles ingestion , releasing lysosomal enzymes that can kill the macrophage itself , or contribute to the recruitment of new macrophages inducing chronic inflammation . clinical evidence has indicated that the activation of alveolar macrophages by sio2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1 , which , in turn , induces fibrosis .", "exposure to cigarette smoke activates nf - e2-related factor 2 ( nrf2 ) in macrophages and reduces neutrophil recruitment , reduces ams phagocytic ability and expression of several important recognition molecules , and impairs clearance of apoptotic cells through oxidant - dependent activation of rhoa [ 60 , 61 ] . in current smokers", ", the exposure to cigarette smoke affects several important recognition molecules on ams and downregulates cd31 , cd91 , cd44 , and cd71 on these cells .", "ams with defective phagocytosis lead to chronic inflammation and significantly increase the likelihood of developing chronic obstructive pulmonary disease , lung injury , and cancer   ( figure 1 ) .", "the infiltration of alveolar macrophages promotes the death of tumor cells in those sites of primary tumor growth and/or metastasis in lung .", "the antitumor activity of alveolar macrophages from lung cancer patients decreases with increased metastasis , tumor size , and development of pleural invasion .", "the onset of malignant disease triggers the immune response recruiting tams into the tumor site .", "high numbers of intratumor tams have been linked with invasion , angiogenesis , hypoxia , and early occurrence of metastasis in different tumor types including lung cancer [ 48 , 50 ] ( figure 2 ) . in patients with nsclc ,", "the m1 macrophage phenotype has been associated with the expression of il-1 , il-12 , tumor necrosis factor- ( tnf- ) , and inos and also has been correlated with extended survival time . in a study", ", m1 tams were identified using cd68 and inos markers in tumor compared to nontumor tissue in nsclc patients .", "results indicate that inos expression is lower in tissues from patients with adenocarcinoma and squamous cell carcinoma compared to nontumor tissues but surprisingly this was not the case in large cell lung carcinomas .", "the classically activated m1 macrophages produce effector molecules such as reactive oxygen intermediates , reactive nitrogen intermediates , and tnf , to limit tumor growth .", "overall there is an association of m1 tams with better lung cancer prognosis . at the other end", "are the alternatively activated m2 macrophages which have been correlated with tumor initiation , progression , metastases , by secretion of matrix - degrading enzymes , angiogenic factors , and immunosuppressive cytokines chemokines , inhibiting inflammation [ 65 , 67 , 68 ] .", "m2 macrophages polarized by cigarette smoke lead to proliferation , migration , and invasion of alveolar basal epithelial cells , and exposition to these cigarette smoke - induced m2 macrophages also significantly increased the cell population in g2/m phase causing proliferation in lung cancer cells .", "in nsclc , the concentration of macrophages m2 was 70% in comparison with 30% m1 .", "density of macrophages m1 in the tumor islets , stroma , or islets and stroma was positively associated with patient 's survival time . also ,", "neutrophils are also polarized into n1/n2 subgroups , n1 being proinflammatory , while n2 is anti - inflammatory . n1 and n2 represent a dichotomy in neutrophil subpopulations present in patients and animal models with cancer where they play distinctive roles in the pathogenesis of disease .", "they displayed an activated phenotype that included chemokine receptors as ccr5 , ccr7 , cxcr3 , and cxcr4 .", "also , tans produced proinflammatory factors mcp-1 , il-8 , mip-1 , and il-6 , as well as the anti - inflammatory il-1r antagonist . also , tans exhibit high activated phenotype compared with peripheral neutrophils . in cancer patients", ", tans could drive antitumoral immunity through regulating cytotoxic t lymphocytes . in early stages of lung cancer disease , tans increased t cell ifn- production and activation and increase t cell proliferation .", "the blockage of tgf- is able to polarize n2 tans to n1 tans in murine models of mesothelioma and lung cancer .", "if apoptotic neutrophils within the tissues are not removed in an efficient and timely manner , they will become necrotic and release cytotoxic granule proteins that may perpetuate host tissue damage .", "thus , neutrophils apoptosis and clearance is a critical limiting factor for the successful resolution of inflammation . in colon adenocarcinoma cell line ,", "so far , the possible mechanisms by which neutrophils are increased in nsclc patients have not been described ; despite this , these cells are dysfunctional ; increased levels of il-8 could explain this accumulation ; however , the mechanisms by which this occurs are not known .", "neutrophils are recruited to tumor sites through transendothelial migration involving the cd47:sirp recognition ( signal regulatory protein alpha ) creating an inflammatory environment .", "malignant cells escape phagocytosis displaying high levels of cd47 on their surface which binds to sirp in macrophages and dendritic cells . after binding to sirp", ", cd47 induces a dephosphorylation cascade preventing phagocytosis through impaired synaptic myosin accumulation . in this way", ", cd47 can regulate the function of cells in the monocyte / macrophage lineage [ 8082 ] .", "cd47 is a ubiquitous cell - surface molecule from the immunoglobulin ( ig ) superfamily that interacts with sirp , thrombospondins , and integrins .", "cd47 was first isolated in association with integrin in neutrophil granulocytes and was later shown to regulate integrin function [ 84 , 85 ] .", "it plays a role in cellular processes like proliferation , apoptosis , adhesion , and migration   and in immunological processes such as inflammatory response , immune response , and tumor immunity [ 87 , 88 ] .", "this receptor is recognized as a marker of  self    highly expressed by circulating hematopoietic stem cells , red blood cells , macrophages , macrophages neutrophils , and many cancer types .", "cd47 has also been identified as a tumor marker , and its dysregulation contributes to cancer progression and evasion of antitumor immunity [ 9194 ] .", "cd47 is expressed ubiquitously whereas its counter - receptor sirp is more abundant in myeloid - lineage cells such as macrophages , neutrophils , and dendritic cells .", "several processes are regulated through the cd47:sirp signaling system of macrophages , including phagocytosis mature red blood cells ( rbcs ) in the spleen , phagocytosis of senescent cells and apoptotic bodies , rejection of transplants of hematopoietic stem cells ( hscs ) , and immunosurveillance thereby preserving tissue integrity and function [ 9699 ] .", "remarkably , there are many factors positively regulating phagocytosis while sirp-cd47 is the only negative regulator preventing self - phagocytosis .", "cd47 is critical for transepithelial and transendothelial migration of neutrophils or polymorphonuclear leukocytes ( pmn ) facilitating diapedesis through endothelial cells while targeted cd47 deletion decreases neutrophil extravasation [ 100 , 101 ] .", "the sirp-cd47 interaction initially recruits pmns to tumor sites or sites of injury but later negatively regulates these cells to end the inflammatory response .", "however , in a postacute stage of inflammation , neutrophils experience cleavage of the cytoplasmic signaling domains of sirp , correlating with increased recruitment and neutrophil - associated damage .", "truncated sirp acts like a decoy , able to bind cd47 but not signaling intracellularly therefore maintaining the inflammatory microenvironment and being a caveat for cd47 targeted therapies [ 102105 ] .", "additionally , sirp binding to cd47 in vitro downregulates cd18 as marker of neutrophil activation thus playing a role in the inflammatory activation state of pmns [ 106 , 107 ] .", "the dual role of cd47 in promoting inflammation through neutrophil migration and recognition of self through blocking phagocytosis in macrophages plays a role in the development of cancer and later in tumor immune evasion .", "loss of cd47 induces phagocytosis by macrophages in vitro and blocks tumor development and metastasis in vivo .", "this receptor is strongly overexpressed in several cancer types including both hematological and solid tumors [ 80 , 91 , 109 , 110 ] .", "a high cd47 expression has been a poor prognostic factor for patients with these diseases [ 80 , 111 , 112 ] .", "cd47 is also highly expressed in tumor initiating cells ( tics ) or cancer stem cells ( csc ) where it is a marker of more aggressive tumor cells , with higher metastatic potential , and less sensitive to engulfment by macrophages , thereby escaping from immune surveillance while increasing cell proliferation through activation of the pi3k / akt pathway [ 92 , 113116 ] .", "therefore , cd47 becomes an attractive target for therapeutic approaches with both antitumor and anti - inflammatory properties and anti - cd47 antibodies are being tested with positive results in preclinical and clinical settings [ 80 , 111 , 112 , 117 ] . in lung cancer and in several types of cancers including breast , bladder , colon , pancreatic , and hematological cancers , blocking cd47 in tumor cells", "the cd47:sirp interaction is involved in the pathogenesis of lung cancer and other cancer types when tumors release cytokines promoting tumor growth and stimulating the conversion of macrophages from m1 to m2 phenotype .", "systemic administration of nanoparticles with anti - cd47 sirna showed efficient inhibition of lung metastasis to about 30% of controls . in patients with lung metastasis , the number of circulating tumor cells ( ctc ) with the phenotype epcam(+)cd44(+)cd47(+)met(+", ") were associated with poor overall survival and increased metastasis and cd47 was a marker associated with the fraction of metastasis - initiating cells within the pool of ctcs .", "antisense suppression of cd47 in squamous lung tumors prior to irradiation showed benefit obtaining a 71% tumor size reduction .", "this protection could possibly be exerted through thrombospondin-1 signaling to recover from radiation stress , revealing a strategy to protect normal tissues from radiation damage using anti - cd47 antibodies which could be useful in the application of combined radiation with targeted therapies in lung cancer .", "there is a close relationship between macrophage , neutrophil infiltration , and upregulation or cd47 with poor prognosis and lack response to treatment .", "nowadays , therapies are developed to block the interaction of tumor cells with macrophages through cd47 , thereby offering an opportunity to turn tams against nsclc cells by allowing the phagocytic behavior of resident macrophages .", "also , anti - cd47 could regulate the recruitment of neutrophils into tumor and diminish the chronic inflammation figure 3 .", "inhibition of csf-1 receptor , which is essential for macrophage differentiation significantly increased survival and suppressed established tumors , accompanied by decreased m2-like tam .", "treatment with metformin is able to reduce the metastases in vivo , through blocked matrix metalloproteinase-9 and expression of mmp-2 , maintaining the components of the extracellular matrix , avoiding the separation of tumor cells , inhibiting the growth and metastasis of tumors .", "also , metformin prevented m2-polarization of macrophages regulated ampk1 and , besides , inhibited il-1 induced release of the proinflammatory cytokines il-6 and il-8 in macrophages [ 124 , 125 ] .", "glycodelin ( gene name paep ) is a proliferation suppressor and apoptosis inducer of t cells , monocytes , b cells , nk , and regulated pulmonary immune response in asthmatic inflammation .", "however , atypical expression is observed in squamous cell carcinomas and adenocarcinomas of nsclc . in vitro , silencing by sirna - transfection of paep in two nsclc cell lines resulted in significant upregulation of immune system modulatory factors such as pdl1 , cxcl5 , cxcl16 , mica / b , and cd83 as well as proliferation stimulators edn1 and hbegf .", "this kind of therapy provides a mechanism to overcome tumor immunosurveillance . as mentioned above ,", "currently the only fda - approved immunotherapies for the treatment of nsclc are nivolumab and pembrolizumab .", "these antibodies inhibit checkpoint molecules such as ctl-4 and pdl-1 , improving the survival and response to treatment .", "ctla-4 is thought to regulate t cell proliferation early in an immune response , primarily in lymph nodes , whereas pd-1 is upregulated in current smokers and suppresses t cells .", "these antibodies switch on immune system cells mediated by t cells , increasing their ability to recognize and destroy cancer cells [ 128 , 130 ] .", "monoclonal antibodies specific for tumor cell antigens , coupled with appropriate cytokines , may provide rational basis for designing trials to employ the neutrophil cytotoxic potential as adjuvant therapy in cancer patients .", "chronic inflammation seems to play a major role in the onset and development of cancer . understanding the interaction between the cellular and molecular factors that mediate inflammation in nsclc , including", "the rather unexplored components of innate immunity such as macrophages and neutrophils , can elucidate novel targets affecting key oncogenic pathways in this malignancy and allow preventing cancer cell proliferation , angiogenesis , and metastasis . inhibiting cd47 as promoter of neutrophil extravasation and migration", "may reduce inflammation thereby preventing cancer , and blocking the antiphagocytic signal of cd47 on the surface of tumor cells can overcome immune suppression , harnessing the immune system to target malignant cells more effectively .", "on the other hand , the potential side effects should be addressed by careful selection of patient populations based on biomarkers such as tumor cd47 overexpression ."], "abstract_text": ["<S> inflammation is a component of the tumor microenvironment and represents the 7th hallmark of cancer . </S>", "<S> chronic inflammation plays a critical role in tumorigenesis . </S>", "<S> tumor infiltrating inflammatory cells mediate processes associated with progression , immune suppression , promotion of neoangiogenesis and lymphangiogenesis , remodeling of extracellular matrix , invasion and metastasis , and , lastly , the inhibition of vaccine - induced antitumor t cell response . </S>", "<S> accumulating evidence indicates a critical role of myeloid cells in the pathophysiology of human cancers . </S>", "<S> in contrast to the well - characterized tumor - associated macrophages ( tams ) , the significance of granulocytes in cancer has only recently begun to emerge with the characterization of tumor - associated neutrophils ( tans ) . </S>", "<S> recent studies show the importance of cd47 in the interaction with macrophages inhibiting phagocytosis and promoting the migration of neutrophils , increasing inflammation which can lead to recurrence and progression in lung cancer . </S>", "<S> currently , therapies are targeted towards blocking cd47 and enhancing macrophage - mediated phagocytosis . </S>", "<S> however , antibody - based therapies may have adverse effects that limit its use . </S>"], "labels": null, "section_names": ["1. Non-Small Cell Lung Cancer (NSCLC)", "2. Tumor-Associated Macrophages (TAMs)", "3. Tumor-Macrophage Interactions in Lung", "4. Tumor-Associated Neutrophils (TANs)", "5. CD47 and Immune Evasion", "6. Therapeutic Approaches: TAMs and TANs", "7. Conclusion"], "sections": [["lung cancer remains the leading type of cancer worldwide and in latin america [ 1 , 2 ] .", "the disease burden is significantly high , with around 2.5 million new cases per year and 1.5 million deaths worldwide .", "the two main histological subtypes of lung cancer are small - cell lung cancer ( sclc ) , which comprises 15% of cases , and non - small - cell lung cancer ( nsclc ) accounting for 85% of cases   which include adenocarcinoma , squamous cell carcinoma , and large cell carcinoma . among all newly diagnosed nsclc cases ,", "adenocarcinomas are the most frequent subgroup following by squamous cell carcinomas [ 6 , 7 ] .", "cigarette smoking is the major risk factor for lung cancer but around 1020% of cases are found in never smokers ; also wood - smoke is a major risk factor in countries like mexico [ 811 ] .", "surgery is the selected treatment for early stage nsclc with the greatest probability of long - term survival in such patients . in advanced nsclc ,", "conventional therapies are based on chemotherapy and radiotherapy but with low efficacy . over the last decade , there have been advances in the study of molecular pathways underlying tumor development leading to the development of targeted therapies such as tyrosine kinase inhibitors ( tkis ) and antibodies directed against the two main actionable genes in nsclc up to now : mutations in the epidermal growth factor receptor ( egfr ) gene targeted by tkis like gefitinib [ 13 , 14 ] , erlotinib [ 9 , 15 , 16 ] , and afatinib [ 1719 ] and translocations involving the anaplastic lymphoma kinase ( alk ) gene treated with the tki crizotinib , alectinib , and ceritinib .", "benefits have been shown in a subset of 1520% of patients harboring egfr mutations which correlate with definite clinical characteristics : adenocarcinoma histology , female sex , asian ethnicity , and nonsmokers [ 2325 ] . despite these improvements in therapeutic strategies ,", "early diagnosis is very difficult ; most cases are diagnosed at an advanced stage and cancer metastasis is very frequent ; therefore , there is still an exceedingly low 5-year survival rate of 1124% [ 2628 ] .", "the immunotherapy approach has opened new therapeutic options in advanced nsclc with the advent of antibodies against immune checkpoints [ 29 , 30 ] .", "recently , the anti - programmed death-1 ( pd-1 ) antibodies nivolumab and pembrolizumab have been approved in the treatment of advanced metastatic nsclc based on results from clinical trials after prior chemotherapy [ 31 , 32 ] .", "both antibodies block signaling through pd-1 and may restore antitumor immunity with benefits in overall survival [ 33 , 34 ] .", "for example , nivolumab , a fully human monoclonal antibody , has recently shown greater overall survival than docetaxel .", "these antibodies exhibit a reasonable toxicity profile but they should be administered in selected patient populations based on biomarkers such as pd - l1 expression to avoid serious immune - mediated adverse effects .", "although these checkpoint inhibitors have proven efficacy in patients , their mechanism of action implies side effects as the onset of autoimmune diseases and a series of endocrine disorders [ 37 , 38 ] .", "this is the rationale for further research into other molecular and cellular factors of the immune system that could be effectively targeted to develop novel therapeutic strategies for the management of advanced nsclc .", "recent findings indicate that inflammation plays a key role in tumor progression and survival across several cancer types .", "cancer related inflammation affects many aspects of malignancy including proliferation , survival , angiogenesis , and tumor metastasis .", "inflammatory components in the development of the neoplasm include diverse leukocytes populations , like macrophages and neutrophils , which respond immediately to inflammatory stimulus .", "immunoregulatory cytokines secreted in a proinflammatory environment also contribute to tumor growth and metastases and identify patient subsets in advanced nsclc with differential prognosis .", "both macrophages and neutrophils are increased in patients with lung cancer ; this is associated with poor clinical outcomes , suggesting that these cells might have important tumor - promoting activities [ 43 , 44 ] .", "tumors escape phagocytosis and immune response through overexpressing cd47 that interacts with the signal regulatory protein alpha ( sirp ) preventing engulfment ."], ["macrophages within the tumor microenvironment are called tumor - associated macrophages ( tams ) . tams", "have a complex relationship with tumor cells ; at an early stage they attack tumor cells avoiding tumor spread ; however , over time they begin producing reciprocal growth factors and establish a symbiotic relationship with tumor cells .", "macrophages are polarized into two functionally distinct forms m1 and m2 , mirroring the th1 and th2 nomenclature of t cells .", "differentiation of the m1 macrophages is induced by interferon- , lipopolysaccharides , tumor necrosis factor ( tnf )  , and granulocyte - monocyte colony - stimulating factor .", "the m1 macrophages produce high levels of interleukin- ( il- ) 12 , il-23 , tnf , il-1 , il-6 , cxc ligand 10 ( cxcl10 ) , inducible nitric oxide synthase ( inos ) , human leukocyte antigen- ( hla- ) dr , and reactive oxygen and nitrogen intermediates [ 47 , 48 ] .", "differentiation of the m2 macrophages is induced by il-4 , il-10 , il-13 , il-21 , activin a , immune complexes , and glucocorticoid .", "the m2 macrophages express high levels of il-10 , il-1 receptor antagonist , cc ligand 22 ( ccl22 ) , scavenger , mannose receptor , galactose receptor , arginase i , and cd163 antigen , reduce the expression of inos , and inhibit antigen presentation and t cell proliferation [ 47 , 49 ] . factors that shift tams towards a m2 phenotype include the location of tams within the tumor microenvironment , tumor stage , and type of cancer .", "nevertheless , it is still not fully defined whether the diversity within the tam population is due to the maturation of unique monocytic precursors or due to various factors within the local tumor microenvironment .", "the m2 macrophages have been found to encourage the growth of various tumour cells in vitro and to increase tumor cell survival [ 51 , 52 ] .", "studies suggest that in solid tumors established and progressively growing tams are reprogrammed to induce immune suppression in situ in the host through cytokines , prostanoids , and other humoral mediators [ 54 , 55 ] .", "il-1 and il-6 expression in tams differs in ovarian cancer compared to peripheral blood monocytes .", "tams in the ovary produce low levels of il1 and increase the release of il-6 , which contributes to elevated acute phase proteins and increased malignancy .", "there is an association between the number of macrophages and prognosis in a variety of human tumors .", "tam infiltration increased in carcinomas of breast , cervix , and bladder and correlates with a poor prognosis .", "however , in prostate , lung , and brain , increasing tams is associated with regression of tumors .", "tams can regulate the development of new blood vessels within tumors . in hypoxic sites , they stimulate the production of enzymes and extracellular matrix molecules that regulate endothelial cell activity by stimulating factors such as vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) , tumor necrosis factor- ( tnf- ) , transforming growth factors-  and  ( tgf-  ,  ) , interferons , thrombospondin , il-8 , and epidermal growth factor ( egf ) ."], ["innate immunity in lung involves alveolar macrophages ( ams ) which act as a barrier avoiding penetration of pathogens .", "conversely , macrophages contribute in part to the pathogenesis of lung disease due to toxic particles ingestion , releasing lysosomal enzymes that can kill the macrophage itself , or contribute to the recruitment of new macrophages inducing chronic inflammation . clinical evidence has indicated that the activation of alveolar macrophages by sio2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1 , which , in turn , induces fibrosis .", "exposure to cigarette smoke activates nf - e2-related factor 2 ( nrf2 ) in macrophages and reduces neutrophil recruitment , reduces ams phagocytic ability and expression of several important recognition molecules , and impairs clearance of apoptotic cells through oxidant - dependent activation of rhoa [ 60 , 61 ] . in current smokers", ", the exposure to cigarette smoke affects several important recognition molecules on ams and downregulates cd31 , cd91 , cd44 , and cd71 on these cells .", "ams with defective phagocytosis lead to chronic inflammation and significantly increase the likelihood of developing chronic obstructive pulmonary disease , lung injury , and cancer   ( figure 1 ) .", "the infiltration of alveolar macrophages promotes the death of tumor cells in those sites of primary tumor growth and/or metastasis in lung .", "the antitumor activity of alveolar macrophages from lung cancer patients decreases with increased metastasis , tumor size , and development of pleural invasion .", "the onset of malignant disease triggers the immune response recruiting tams into the tumor site .", "high numbers of intratumor tams have been linked with invasion , angiogenesis , hypoxia , and early occurrence of metastasis in different tumor types including lung cancer [ 48 , 50 ] ( figure 2 ) . in patients with nsclc ,", "the m1 macrophage phenotype has been associated with the expression of il-1 , il-12 , tumor necrosis factor- ( tnf- ) , and inos and also has been correlated with extended survival time . in a study", ", m1 tams were identified using cd68 and inos markers in tumor compared to nontumor tissue in nsclc patients .", "results indicate that inos expression is lower in tissues from patients with adenocarcinoma and squamous cell carcinoma compared to nontumor tissues but surprisingly this was not the case in large cell lung carcinomas .", "the classically activated m1 macrophages produce effector molecules such as reactive oxygen intermediates , reactive nitrogen intermediates , and tnf , to limit tumor growth .", "overall there is an association of m1 tams with better lung cancer prognosis . at the other end", "are the alternatively activated m2 macrophages which have been correlated with tumor initiation , progression , metastases , by secretion of matrix - degrading enzymes , angiogenic factors , and immunosuppressive cytokines chemokines , inhibiting inflammation [ 65 , 67 , 68 ] .", "m2 macrophages polarized by cigarette smoke lead to proliferation , migration , and invasion of alveolar basal epithelial cells , and exposition to these cigarette smoke - induced m2 macrophages also significantly increased the cell population in g2/m phase causing proliferation in lung cancer cells .", "in nsclc , the concentration of macrophages m2 was 70% in comparison with 30% m1 .", "density of macrophages m1 in the tumor islets , stroma , or islets and stroma was positively associated with patient 's survival time . also ,"], ["neutrophils are also polarized into n1/n2 subgroups , n1 being proinflammatory , while n2 is anti - inflammatory . n1 and n2 represent a dichotomy in neutrophil subpopulations present in patients and animal models with cancer where they play distinctive roles in the pathogenesis of disease .", "they displayed an activated phenotype that included chemokine receptors as ccr5 , ccr7 , cxcr3 , and cxcr4 .", "also , tans produced proinflammatory factors mcp-1 , il-8 , mip-1 , and il-6 , as well as the anti - inflammatory il-1r antagonist . also , tans exhibit high activated phenotype compared with peripheral neutrophils . in cancer patients", ", tans could drive antitumoral immunity through regulating cytotoxic t lymphocytes . in early stages of lung cancer disease , tans increased t cell ifn- production and activation and increase t cell proliferation .", "the blockage of tgf- is able to polarize n2 tans to n1 tans in murine models of mesothelioma and lung cancer .", "if apoptotic neutrophils within the tissues are not removed in an efficient and timely manner , they will become necrotic and release cytotoxic granule proteins that may perpetuate host tissue damage .", "thus , neutrophils apoptosis and clearance is a critical limiting factor for the successful resolution of inflammation . in colon adenocarcinoma cell line ,", "so far , the possible mechanisms by which neutrophils are increased in nsclc patients have not been described ; despite this , these cells are dysfunctional ; increased levels of il-8 could explain this accumulation ; however , the mechanisms by which this occurs are not known ."], ["neutrophils are recruited to tumor sites through transendothelial migration involving the cd47:sirp recognition ( signal regulatory protein alpha ) creating an inflammatory environment .", "malignant cells escape phagocytosis displaying high levels of cd47 on their surface which binds to sirp in macrophages and dendritic cells . after binding to sirp", ", cd47 induces a dephosphorylation cascade preventing phagocytosis through impaired synaptic myosin accumulation . in this way", ", cd47 can regulate the function of cells in the monocyte / macrophage lineage [ 8082 ] .", "cd47 is a ubiquitous cell - surface molecule from the immunoglobulin ( ig ) superfamily that interacts with sirp , thrombospondins , and integrins .", "cd47 was first isolated in association with integrin in neutrophil granulocytes and was later shown to regulate integrin function [ 84 , 85 ] .", "it plays a role in cellular processes like proliferation , apoptosis , adhesion , and migration   and in immunological processes such as inflammatory response , immune response , and tumor immunity [ 87 , 88 ] .", "this receptor is recognized as a marker of  self    highly expressed by circulating hematopoietic stem cells , red blood cells , macrophages , macrophages neutrophils , and many cancer types .", "cd47 has also been identified as a tumor marker , and its dysregulation contributes to cancer progression and evasion of antitumor immunity [ 9194 ] .", "cd47 is expressed ubiquitously whereas its counter - receptor sirp is more abundant in myeloid - lineage cells such as macrophages , neutrophils , and dendritic cells .", "several processes are regulated through the cd47:sirp signaling system of macrophages , including phagocytosis mature red blood cells ( rbcs ) in the spleen , phagocytosis of senescent cells and apoptotic bodies , rejection of transplants of hematopoietic stem cells ( hscs ) , and immunosurveillance thereby preserving tissue integrity and function [ 9699 ] .", "remarkably , there are many factors positively regulating phagocytosis while sirp-cd47 is the only negative regulator preventing self - phagocytosis .", "cd47 is critical for transepithelial and transendothelial migration of neutrophils or polymorphonuclear leukocytes ( pmn ) facilitating diapedesis through endothelial cells while targeted cd47 deletion decreases neutrophil extravasation [ 100 , 101 ] .", "the sirp-cd47 interaction initially recruits pmns to tumor sites or sites of injury but later negatively regulates these cells to end the inflammatory response .", "however , in a postacute stage of inflammation , neutrophils experience cleavage of the cytoplasmic signaling domains of sirp , correlating with increased recruitment and neutrophil - associated damage .", "truncated sirp acts like a decoy , able to bind cd47 but not signaling intracellularly therefore maintaining the inflammatory microenvironment and being a caveat for cd47 targeted therapies [ 102105 ] .", "additionally , sirp binding to cd47 in vitro downregulates cd18 as marker of neutrophil activation thus playing a role in the inflammatory activation state of pmns [ 106 , 107 ] .", "the dual role of cd47 in promoting inflammation through neutrophil migration and recognition of self through blocking phagocytosis in macrophages plays a role in the development of cancer and later in tumor immune evasion .", "loss of cd47 induces phagocytosis by macrophages in vitro and blocks tumor development and metastasis in vivo .", "this receptor is strongly overexpressed in several cancer types including both hematological and solid tumors [ 80 , 91 , 109 , 110 ] .", "a high cd47 expression has been a poor prognostic factor for patients with these diseases [ 80 , 111 , 112 ] .", "cd47 is also highly expressed in tumor initiating cells ( tics ) or cancer stem cells ( csc ) where it is a marker of more aggressive tumor cells , with higher metastatic potential , and less sensitive to engulfment by macrophages , thereby escaping from immune surveillance while increasing cell proliferation through activation of the pi3k / akt pathway [ 92 , 113116 ] .", "therefore , cd47 becomes an attractive target for therapeutic approaches with both antitumor and anti - inflammatory properties and anti - cd47 antibodies are being tested with positive results in preclinical and clinical settings [ 80 , 111 , 112 , 117 ] . in lung cancer and in several types of cancers including breast , bladder , colon , pancreatic , and hematological cancers , blocking cd47 in tumor cells", "the cd47:sirp interaction is involved in the pathogenesis of lung cancer and other cancer types when tumors release cytokines promoting tumor growth and stimulating the conversion of macrophages from m1 to m2 phenotype .", "systemic administration of nanoparticles with anti - cd47 sirna showed efficient inhibition of lung metastasis to about 30% of controls . in patients with lung metastasis , the number of circulating tumor cells ( ctc ) with the phenotype epcam(+)cd44(+)cd47(+)met(+", ") were associated with poor overall survival and increased metastasis and cd47 was a marker associated with the fraction of metastasis - initiating cells within the pool of ctcs .", "antisense suppression of cd47 in squamous lung tumors prior to irradiation showed benefit obtaining a 71% tumor size reduction .", "this protection could possibly be exerted through thrombospondin-1 signaling to recover from radiation stress , revealing a strategy to protect normal tissues from radiation damage using anti - cd47 antibodies which could be useful in the application of combined radiation with targeted therapies in lung cancer .", "there is a close relationship between macrophage , neutrophil infiltration , and upregulation or cd47 with poor prognosis and lack response to treatment .", "nowadays , therapies are developed to block the interaction of tumor cells with macrophages through cd47 , thereby offering an opportunity to turn tams against nsclc cells by allowing the phagocytic behavior of resident macrophages .", "also , anti - cd47 could regulate the recruitment of neutrophils into tumor and diminish the chronic inflammation figure 3 ."], ["inhibition of csf-1 receptor , which is essential for macrophage differentiation significantly increased survival and suppressed established tumors , accompanied by decreased m2-like tam .", "treatment with metformin is able to reduce the metastases in vivo , through blocked matrix metalloproteinase-9 and expression of mmp-2 , maintaining the components of the extracellular matrix , avoiding the separation of tumor cells , inhibiting the growth and metastasis of tumors .", "also , metformin prevented m2-polarization of macrophages regulated ampk1 and , besides , inhibited il-1 induced release of the proinflammatory cytokines il-6 and il-8 in macrophages [ 124 , 125 ] .", "glycodelin ( gene name paep ) is a proliferation suppressor and apoptosis inducer of t cells , monocytes , b cells , nk , and regulated pulmonary immune response in asthmatic inflammation .", "however , atypical expression is observed in squamous cell carcinomas and adenocarcinomas of nsclc . in vitro , silencing by sirna - transfection of paep in two nsclc cell lines resulted in significant upregulation of immune system modulatory factors such as pdl1 , cxcl5 , cxcl16 , mica / b , and cd83 as well as proliferation stimulators edn1 and hbegf .", "this kind of therapy provides a mechanism to overcome tumor immunosurveillance . as mentioned above ,", "currently the only fda - approved immunotherapies for the treatment of nsclc are nivolumab and pembrolizumab .", "these antibodies inhibit checkpoint molecules such as ctl-4 and pdl-1 , improving the survival and response to treatment .", "ctla-4 is thought to regulate t cell proliferation early in an immune response , primarily in lymph nodes , whereas pd-1 is upregulated in current smokers and suppresses t cells .", "these antibodies switch on immune system cells mediated by t cells , increasing their ability to recognize and destroy cancer cells [ 128 , 130 ] .", "monoclonal antibodies specific for tumor cell antigens , coupled with appropriate cytokines , may provide rational basis for designing trials to employ the neutrophil cytotoxic potential as adjuvant therapy in cancer patients ."], ["chronic inflammation seems to play a major role in the onset and development of cancer . understanding the interaction between the cellular and molecular factors that mediate inflammation in nsclc , including", "the rather unexplored components of innate immunity such as macrophages and neutrophils , can elucidate novel targets affecting key oncogenic pathways in this malignancy and allow preventing cancer cell proliferation , angiogenesis , and metastasis . inhibiting cd47 as promoter of neutrophil extravasation and migration", "may reduce inflammation thereby preventing cancer , and blocking the antiphagocytic signal of cd47 on the surface of tumor cells can overcome immune suppression , harnessing the immune system to target malignant cells more effectively .", "on the other hand , the potential side effects should be addressed by careful selection of patient populations based on biomarkers such as tumor cd47 overexpression ."]]}
{"article_id": "PMC4720067", "article_text": ["gastroenteropancreatic neuroendocrine tumors ( gepnets ) are rare malignant neoplasm with an incidence ranging from two to five cases/100,000/yr .", "because it originates from the enterochromaffin serotonin - producing cell , it has a unique feature of hormone secretion and expression of distinctive differentiation markers [ 1 - 6 ] .", "some patients remain asymptomatic for several years , or complain of episodic flushing , abdominal pain , nausea , vomiting , and diarrhea . in most cases , due to the vagueness of symptoms , a diagnosis is delayed ( 3 - 10 years on average ) , with an increased risk of developing metastases .", "the ability of the imaging method to localize primary or metastatic site of gep - net also has some limitations .", "most gep - nets are highly vascular , thus could easily be detected by a contrast enhanced computed tomography ( ct ) ; however , approximately 6% to 20% are hypovascular and often difficult to be evaluated by a ct scan or other imaging methods .", "gep - nets originating from jejunum and ileum are also often difficult to identify on an image due to their small size .", "therefore , non - invasive parameters , indicating gep - pet , are needed for diagnosis , following - ups and prognosis .", "chromogranin a ( cga ) , a glycoprotein contained in secretion granules of neuroendocrine cells , is the most abundant granin in gep - nets and widely used as a circulating tumor marker , but only few studies have been published on the role of cga in patients with gep - pet , and the range of sensitivity were variously reported [ 8 - 10 ] .", "we investigated to evaluate whether biochemical response using serial plasma cga is reliable in concordance with the tumor response based on response evaluation criteria in solid tumors ( recist ) criteria in gep - nets irrespective of chemotherapeutic agents .", "a total of 27 cases in 18 patients , who were pathologically diagnosed in gep - nets , were analysed between march 2011 and september 2013 . for all cases ,", "non - functioning was defined to the absence of clinical syndromes of hormonal hypersecretion , such as hypoglycemia , peptic ulcer , diarrhea , steatorrhea , acromegaly , cushing s syndrome , and gallstone .", "the following clinicopathological characteristics of all 18 patients were collected : age , sex , primary site , tumor grade in accordance to the 2010 world health organization ( who ) classification , liver metastasis , number of metastatic site , site of metastasis , and information of chemotherapy .", "systemic chemotherapies for gep - net included octreotide , vip ( vincristine , ifosfamide , cisplatin ) , xelox ( capecitabine , oxaliplatin ) , ep ( etoposide , cisplatin ) , pazopanib , sunitinib , everolimus , and xeliri ( capecitabine , irinotecan ) .", "biochemical efficacy was estimated according to the criteria proposed by the italian trials in medical oncology ( itmo ) group   for evaluating markers ( biochemical response ) .", "partial response ( pr ) was defined as  50% decrease in plasma cga compared to the baseline cga ; stable disease ( sd ) was defined as a decrease < 50% or as an increase < 25% ; and progressive disease ( pd ) was defined as an increase  25% .", "the level of cga was determined from venous blood samples drawn into edta - containing tube after overnight fasting and collected before systemic treatment .", "the plasma cga level was measured by cga - ria ( chromoa assay , cis bio international , saclay , france ) with a normal range of 27 - 94 ng / ml .", "chromoa is based on sandwich enzyme - linked immunosorbent assay , using two monoclonal antibodies ( the same antibodies as cga - riact ) directed against the central domain of the molecule ( 145 - 245 ) , which is less sensitive to proteolysis . the tumor size was measured by using a ct or magnetic resonance imaging by recist criteria ver . 1.1 .", "group comparisons were performed using a nonparametric test of mann - whitney or kruskal - wallis , followed by a dunn multiple comparison test , as appropriate .", "comparisons of paired values were performed using a nonparametric test of wilcoxon . the chi - square test and the fisher exact test ( for value less than 5 )", "were employed to compare the sensitivity and rate of concordance in different groups . in all statistical tests ,", "progression - free survival ( pfs ) was measured as the time from the date of chemotherapy to the date of first documented disease progression or death .", "the pfs were estimated using the kaplan - meier method with log - rank analysis .", "a total of 27 cases in 18 patients , who were pathologically diagnosed in gep - nets , were analysed between march 2011 and september 2013 . for all cases ,", "non - functioning was defined to the absence of clinical syndromes of hormonal hypersecretion , such as hypoglycemia , peptic ulcer , diarrhea , steatorrhea , acromegaly , cushing s syndrome , and gallstone .", "the following clinicopathological characteristics of all 18 patients were collected : age , sex , primary site , tumor grade in accordance to the 2010 world health organization ( who ) classification , liver metastasis , number of metastatic site , site of metastasis , and information of chemotherapy .", "systemic chemotherapies for gep - net included octreotide , vip ( vincristine , ifosfamide , cisplatin ) , xelox ( capecitabine , oxaliplatin ) , ep ( etoposide , cisplatin ) , pazopanib , sunitinib , everolimus , and xeliri ( capecitabine , irinotecan ) .", "biochemical efficacy was estimated according to the criteria proposed by the italian trials in medical oncology ( itmo ) group   for evaluating markers ( biochemical response ) .", "partial response ( pr ) was defined as  50% decrease in plasma cga compared to the baseline cga ; stable disease ( sd ) was defined as a decrease < 50% or as an increase < 25% ; and progressive disease ( pd ) was defined as an increase  25% .", "the level of cga was determined from venous blood samples drawn into edta - containing tube after overnight fasting and collected before systemic treatment .", "the plasma cga level was measured by cga - ria ( chromoa assay , cis bio international , saclay , france ) with a normal range of 27 - 94 ng / ml .", "chromoa is based on sandwich enzyme - linked immunosorbent assay , using two monoclonal antibodies ( the same antibodies as cga - riact ) directed against the central domain of the molecule ( 145 - 245 ) , which is less sensitive to proteolysis .", "the tumor size was measured by using a ct or magnetic resonance imaging by recist criteria ver .", "group comparisons were performed using a nonparametric test of mann - whitney or kruskal - wallis , followed by a dunn multiple comparison test , as appropriate .", "comparisons of paired values were performed using a nonparametric test of wilcoxon . the chi - square test and the fisher exact test ( for value less than 5 )", "were employed to compare the sensitivity and rate of concordance in different groups . in all statistical tests ,", "progression - free survival ( pfs ) was measured as the time from the date of chemotherapy to the date of first documented disease progression or death .", "the pfs were estimated using the kaplan - meier method with log - rank analysis .", "the rectum was the second most common site of gep - net ( 22.2% ) in this study . according to who classification , ten of the 27 cases ( 37% ) were grade 3 neuroendocrine carcinoma .", "grades 1 and 2 nets were nine ( 33.3% ) and eight ( 29.6% ) , respectively .", "measurable lesions were found on ct images in 18 out of the 27 cases . among the 27 cases included in this study , no difference in the basal cga level", "was observed in terms of gender , primary tumor site , tumor grade ( who classification ) , liver metastasis , number of metastatic site , and line of chemotherapy with serial cga monitoring .", "the plasma cga level ranged from 33.18 to 895 ng / ml ( table 1 ) .", "the overall response rate ( rr ) by recist criteria was six out of the 27 cases ( 22.2% ) and eight out of the 27 cases ( 29.6% ) for biochemical rr .", "the concordance of response between recist criteria and biochemical criteria were 74% ( table 3 ) . compared with the baseline values , a decrease of  50% in the cga level", "were observed in three out of six cases ( 50% ) with pr by recist criteria ( figs .", "1a , 2 ) . in only grades 1 and 2 gep - nets cases ( n=17 ) ,", "the concordance of disease control between recist criteria and biochemical criteria were 94.1% ( fig .", "there was a significant difference for pfs between responders and non - responders ( 35.73 months vs. 5.93 months , p=0.05 ) based on the biochemical criteria ( fig .", "3 ) . a subgroup analysis of pfs between responders and non - responders , in accordance to recist response , tumor grade , and primary site , were not statistically significant , but showed longer pfs in the biochemical responder group ( fig .", "the rectum was the second most common site of gep - net ( 22.2% ) in this study . according to who classification , ten of the 27 cases ( 37% ) were grade 3 neuroendocrine carcinoma .", "grades 1 and 2 nets were nine ( 33.3% ) and eight ( 29.6% ) , respectively .", "among the 27 cases included in this study , no difference in the basal cga level was observed in terms of gender , primary tumor site , tumor grade ( who classification ) , liver metastasis , number of metastatic site , and line of chemotherapy with serial cga monitoring .", "the plasma cga level ranged from 33.18 to 895 ng / ml ( table 1 ) .", "the overall response rate ( rr ) by recist criteria was six out of the 27 cases ( 22.2% ) and eight out of the 27 cases ( 29.6% ) for biochemical rr .", "the concordance of response between recist criteria and biochemical criteria were 74% ( table 3 ) . compared with the baseline values , a decrease of  50% in the cga level", "were observed in three out of six cases ( 50% ) with pr by recist criteria ( figs .", "1a , 2 ) . in only grades 1 and 2 gep - nets cases ( n=17 ) ,", "the concordance of disease control between recist criteria and biochemical criteria were 94.1% ( fig .", "there was a significant difference for pfs between responders and non - responders ( 35.73 months vs. 5.93 months , p=0.05 ) based on the biochemical criteria ( fig .", "3 ) . a subgroup analysis of pfs between responders and non - responders , in accordance to recist response , tumor grade , and primary site , were not statistically significant , but showed longer pfs in the biochemical responder group ( fig .", "cga is the most abundant granin in gep - nets and represents the best general marker in the tissue and blood .", "cga expression generally correlates with the number of dense core granules in the neuroendocrine cells .", "thus , cga monitoring may be helpful in assessing the response to the different therapeutic options . in some studies ,", "however , it is still controversial whether serial cga changes reflect tumor response for treatment .", "this study showed that the change of the cga level was correlated with tumor response in nonfunctioning gepnets .", "additionally , biochemical response based on serial cga may be a predictive marker for pfs in gep - nets .", "changes in circulating cga have been reported to represent tumor burden and treatment response . to our knowledge", ", there were no definite measuring criteria of plasma cga for tumor response and conflicting result of sensitivity and specificity in accordance to treatment response were reported in the literature .", "previous studies [ 2 - 10,18 ] showed acceptable sensitivity ( 54%-78% ) and specificity ( 60%-86% ) for regression and sd ; jensen et al .   also reported that plasma cga concentration is important to disclose tumor progression with specificity and sensitivity , 86% and 86% , respectively . in our study , the concordance of response between recist criteria and biochemical criteria was 74% . compared with the baseline values , a decrease of  25% in the cga level was observed in four out of six patients ( 66.7% ) with pr based on recist criteria and an increased cga levels was shown in all 4 patients with pd ( 100% ) .", "among 11 patients available for serial cga levels , all five patients with sd or partial remission had a more than 20% decrease in the cga levels compared to the baseline value .", "all six patients with pd showed a less than 20% decrease or increase in the cga levels .", "several other studies also showed the possibility of cga as a biochemical marker of treatment response ( table 4 ) .", "high - baseline cga value has been considered to be an independent poor prognostic marker for gep - nets in previous studies [ 21 - 25 ] . however", ", whether changes of the cga levels compared to the baseline values could predict the prognosis has not been established . in this study", ", there was a significant difference for pfs between the responders and non - responders for biochemical criteria ( p=0.05 ) .", "it suggested that a decrease in the cga levels from the baseline levels may be an important predictive marker for survival in gep - nets .", "this study was a retrospective analysis with small sample size , and heterogeneous patient population .", "a recognized international standard for cga assay is not available and variations in assay types may influence results . nevertheless , this analysis identified the usefulness of serial cga monitoring as a biomarker that predicts treatment response and survival .", "this study revealed that the changes of the plasma cga levels were associated with tumour response . additionally , biochemical response based on serial cga may be a predictive marker for pfs in gep - nets ."], "abstract_text": ["<S> purposechromogranin a ( cga ) has been considered to be valuable not only in the diagnosis but also in monitoring the disease response to treatment . however , only a few studies have been published on this issue . we purposed to evaluate whether biochemical response using plasma cga level is reliable in concordance with the clinical response of grade 1 - 3 nonfunctiong gastroenteropancreatic neuroendocrine tumors ( gep - nets).materials and methodsbetween march 2011 and september 2013 , a total of 27 cases in 18 patients were analysed , clinically and radiologically while serial cga tests were also conducted during treatment . </S>", "<S> tumor responses were defined by both response evaluation criteria in solid tumors ( recist ) criteria ver . </S>", "<S> 1.1 and biochemical criteria based on the cga level.resultsamong the 27 cases analysed , no difference in the basal cga level was observed with regard to gender , primary tumor site , tumor grade ( world health organization classification ) , liver metastasis , number of metastatic site , and line of chemotherapy . </S>", "<S> the overall response rate ( rr ) by recist criteria ver . </S>", "<S> 1.1 was six out of the 27 cases ( 22.2% ) and eight out of the 27 cases ( 29.6% ) for biochemical rr . </S>", "<S> the overall concordance rates of the response based on recist and biochemical criteria were 74% . in grades 1 and 2 gep - nets ( n=17 ) , </S>", "<S> the concordance rate of the disease control was 94.1% . </S>", "<S> there was a significant difference for progression - free survival ( pfs ) between responders and non - responder in accordance to biochemical criteria ( 35.73 months vs. 5.93 months , p=0.05).conclusionthis study revealed that changes of the plasma cga levels were associated with tumour response . </S>", "<S> additionally , biochemical response based on serial cga may be a predictive marker for pfs in gep - nets . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "1. Patient", "2. Efficacy assessment", "3. Statistical analysis", "Results", "1. Charateristics of cases", "2. CgA measurement at baseline", "3. Correlation of treatment response and survival with changes of CgA levels", "Discussion", "Conclusion"], "sections": [["gastroenteropancreatic neuroendocrine tumors ( gepnets ) are rare malignant neoplasm with an incidence ranging from two to five cases/100,000/yr .", "because it originates from the enterochromaffin serotonin - producing cell , it has a unique feature of hormone secretion and expression of distinctive differentiation markers [ 1 - 6 ] .", "some patients remain asymptomatic for several years , or complain of episodic flushing , abdominal pain , nausea , vomiting , and diarrhea . in most cases , due to the vagueness of symptoms , a diagnosis is delayed ( 3 - 10 years on average ) , with an increased risk of developing metastases .", "the ability of the imaging method to localize primary or metastatic site of gep - net also has some limitations .", "most gep - nets are highly vascular , thus could easily be detected by a contrast enhanced computed tomography ( ct ) ; however , approximately 6% to 20% are hypovascular and often difficult to be evaluated by a ct scan or other imaging methods .", "gep - nets originating from jejunum and ileum are also often difficult to identify on an image due to their small size .", "therefore , non - invasive parameters , indicating gep - pet , are needed for diagnosis , following - ups and prognosis .", "chromogranin a ( cga ) , a glycoprotein contained in secretion granules of neuroendocrine cells , is the most abundant granin in gep - nets and widely used as a circulating tumor marker , but only few studies have been published on the role of cga in patients with gep - pet , and the range of sensitivity were variously reported [ 8 - 10 ] .", "we investigated to evaluate whether biochemical response using serial plasma cga is reliable in concordance with the tumor response based on response evaluation criteria in solid tumors ( recist ) criteria in gep - nets irrespective of chemotherapeutic agents ."], ["a total of 27 cases in 18 patients , who were pathologically diagnosed in gep - nets , were analysed between march 2011 and september 2013 . for all cases ,", "non - functioning was defined to the absence of clinical syndromes of hormonal hypersecretion , such as hypoglycemia , peptic ulcer , diarrhea , steatorrhea , acromegaly , cushing s syndrome , and gallstone .", "the following clinicopathological characteristics of all 18 patients were collected : age , sex , primary site , tumor grade in accordance to the 2010 world health organization ( who ) classification , liver metastasis , number of metastatic site , site of metastasis , and information of chemotherapy .", "systemic chemotherapies for gep - net included octreotide , vip ( vincristine , ifosfamide , cisplatin ) , xelox ( capecitabine , oxaliplatin ) , ep ( etoposide , cisplatin ) , pazopanib , sunitinib , everolimus , and xeliri ( capecitabine , irinotecan ) .", "biochemical efficacy was estimated according to the criteria proposed by the italian trials in medical oncology ( itmo ) group   for evaluating markers ( biochemical response ) .", "partial response ( pr ) was defined as  50% decrease in plasma cga compared to the baseline cga ; stable disease ( sd ) was defined as a decrease < 50% or as an increase < 25% ; and progressive disease ( pd ) was defined as an increase  25% .", "the level of cga was determined from venous blood samples drawn into edta - containing tube after overnight fasting and collected before systemic treatment .", "the plasma cga level was measured by cga - ria ( chromoa assay , cis bio international , saclay , france ) with a normal range of 27 - 94 ng / ml .", "chromoa is based on sandwich enzyme - linked immunosorbent assay , using two monoclonal antibodies ( the same antibodies as cga - riact ) directed against the central domain of the molecule ( 145 - 245 ) , which is less sensitive to proteolysis . the tumor size was measured by using a ct or magnetic resonance imaging by recist criteria ver . 1.1 .", "group comparisons were performed using a nonparametric test of mann - whitney or kruskal - wallis , followed by a dunn multiple comparison test , as appropriate .", "comparisons of paired values were performed using a nonparametric test of wilcoxon . the chi - square test and the fisher exact test ( for value less than 5 )", "were employed to compare the sensitivity and rate of concordance in different groups . in all statistical tests ,", "progression - free survival ( pfs ) was measured as the time from the date of chemotherapy to the date of first documented disease progression or death .", "the pfs were estimated using the kaplan - meier method with log - rank analysis ."], ["a total of 27 cases in 18 patients , who were pathologically diagnosed in gep - nets , were analysed between march 2011 and september 2013 . for all cases ,", "non - functioning was defined to the absence of clinical syndromes of hormonal hypersecretion , such as hypoglycemia , peptic ulcer , diarrhea , steatorrhea , acromegaly , cushing s syndrome , and gallstone .", "the following clinicopathological characteristics of all 18 patients were collected : age , sex , primary site , tumor grade in accordance to the 2010 world health organization ( who ) classification , liver metastasis , number of metastatic site , site of metastasis , and information of chemotherapy .", "systemic chemotherapies for gep - net included octreotide , vip ( vincristine , ifosfamide , cisplatin ) , xelox ( capecitabine , oxaliplatin ) , ep ( etoposide , cisplatin ) , pazopanib , sunitinib , everolimus , and xeliri ( capecitabine , irinotecan ) ."], ["biochemical efficacy was estimated according to the criteria proposed by the italian trials in medical oncology ( itmo ) group   for evaluating markers ( biochemical response ) .", "partial response ( pr ) was defined as  50% decrease in plasma cga compared to the baseline cga ; stable disease ( sd ) was defined as a decrease < 50% or as an increase < 25% ; and progressive disease ( pd ) was defined as an increase  25% .", "the level of cga was determined from venous blood samples drawn into edta - containing tube after overnight fasting and collected before systemic treatment .", "the plasma cga level was measured by cga - ria ( chromoa assay , cis bio international , saclay , france ) with a normal range of 27 - 94 ng / ml .", "chromoa is based on sandwich enzyme - linked immunosorbent assay , using two monoclonal antibodies ( the same antibodies as cga - riact ) directed against the central domain of the molecule ( 145 - 245 ) , which is less sensitive to proteolysis .", "the tumor size was measured by using a ct or magnetic resonance imaging by recist criteria ver ."], ["group comparisons were performed using a nonparametric test of mann - whitney or kruskal - wallis , followed by a dunn multiple comparison test , as appropriate .", "comparisons of paired values were performed using a nonparametric test of wilcoxon . the chi - square test and the fisher exact test ( for value less than 5 )", "were employed to compare the sensitivity and rate of concordance in different groups . in all statistical tests ,", "progression - free survival ( pfs ) was measured as the time from the date of chemotherapy to the date of first documented disease progression or death .", "the pfs were estimated using the kaplan - meier method with log - rank analysis ."], ["the rectum was the second most common site of gep - net ( 22.2% ) in this study . according to who classification , ten of the 27 cases ( 37% ) were grade 3 neuroendocrine carcinoma .", "grades 1 and 2 nets were nine ( 33.3% ) and eight ( 29.6% ) , respectively .", "measurable lesions were found on ct images in 18 out of the 27 cases . among the 27 cases included in this study , no difference in the basal cga level", "was observed in terms of gender , primary tumor site , tumor grade ( who classification ) , liver metastasis , number of metastatic site , and line of chemotherapy with serial cga monitoring .", "the plasma cga level ranged from 33.18 to 895 ng / ml ( table 1 ) .", "the overall response rate ( rr ) by recist criteria was six out of the 27 cases ( 22.2% ) and eight out of the 27 cases ( 29.6% ) for biochemical rr .", "the concordance of response between recist criteria and biochemical criteria were 74% ( table 3 ) . compared with the baseline values , a decrease of  50% in the cga level", "were observed in three out of six cases ( 50% ) with pr by recist criteria ( figs .", "1a , 2 ) . in only grades 1 and 2 gep - nets cases ( n=17 ) ,", "the concordance of disease control between recist criteria and biochemical criteria were 94.1% ( fig .", "there was a significant difference for pfs between responders and non - responders ( 35.73 months vs. 5.93 months , p=0.05 ) based on the biochemical criteria ( fig .", "3 ) . a subgroup analysis of pfs between responders and non - responders , in accordance to recist response , tumor grade , and primary site , were not statistically significant , but showed longer pfs in the biochemical responder group ( fig ."], ["the rectum was the second most common site of gep - net ( 22.2% ) in this study . according to who classification , ten of the 27 cases ( 37% ) were grade 3 neuroendocrine carcinoma .", "grades 1 and 2 nets were nine ( 33.3% ) and eight ( 29.6% ) , respectively ."], ["among the 27 cases included in this study , no difference in the basal cga level was observed in terms of gender , primary tumor site , tumor grade ( who classification ) , liver metastasis , number of metastatic site , and line of chemotherapy with serial cga monitoring .", "the plasma cga level ranged from 33.18 to 895 ng / ml ( table 1 ) ."], ["the overall response rate ( rr ) by recist criteria was six out of the 27 cases ( 22.2% ) and eight out of the 27 cases ( 29.6% ) for biochemical rr .", "the concordance of response between recist criteria and biochemical criteria were 74% ( table 3 ) . compared with the baseline values , a decrease of  50% in the cga level", "were observed in three out of six cases ( 50% ) with pr by recist criteria ( figs .", "1a , 2 ) . in only grades 1 and 2 gep - nets cases ( n=17 ) ,", "the concordance of disease control between recist criteria and biochemical criteria were 94.1% ( fig .", "there was a significant difference for pfs between responders and non - responders ( 35.73 months vs. 5.93 months , p=0.05 ) based on the biochemical criteria ( fig .", "3 ) . a subgroup analysis of pfs between responders and non - responders , in accordance to recist response , tumor grade , and primary site , were not statistically significant , but showed longer pfs in the biochemical responder group ( fig ."], ["cga is the most abundant granin in gep - nets and represents the best general marker in the tissue and blood .", "cga expression generally correlates with the number of dense core granules in the neuroendocrine cells .", "thus , cga monitoring may be helpful in assessing the response to the different therapeutic options . in some studies ,", "however , it is still controversial whether serial cga changes reflect tumor response for treatment .", "this study showed that the change of the cga level was correlated with tumor response in nonfunctioning gepnets .", "additionally , biochemical response based on serial cga may be a predictive marker for pfs in gep - nets .", "changes in circulating cga have been reported to represent tumor burden and treatment response . to our knowledge", ", there were no definite measuring criteria of plasma cga for tumor response and conflicting result of sensitivity and specificity in accordance to treatment response were reported in the literature .", "previous studies [ 2 - 10,18 ] showed acceptable sensitivity ( 54%-78% ) and specificity ( 60%-86% ) for regression and sd ; jensen et al .   also reported that plasma cga concentration is important to disclose tumor progression with specificity and sensitivity , 86% and 86% , respectively . in our study , the concordance of response between recist criteria and biochemical criteria was 74% . compared with the baseline values , a decrease of  25% in the cga level was observed in four out of six patients ( 66.7% ) with pr based on recist criteria and an increased cga levels was shown in all 4 patients with pd ( 100% ) .", "among 11 patients available for serial cga levels , all five patients with sd or partial remission had a more than 20% decrease in the cga levels compared to the baseline value .", "all six patients with pd showed a less than 20% decrease or increase in the cga levels .", "several other studies also showed the possibility of cga as a biochemical marker of treatment response ( table 4 ) .", "high - baseline cga value has been considered to be an independent poor prognostic marker for gep - nets in previous studies [ 21 - 25 ] . however", ", whether changes of the cga levels compared to the baseline values could predict the prognosis has not been established . in this study", ", there was a significant difference for pfs between the responders and non - responders for biochemical criteria ( p=0.05 ) .", "it suggested that a decrease in the cga levels from the baseline levels may be an important predictive marker for survival in gep - nets .", "this study was a retrospective analysis with small sample size , and heterogeneous patient population .", "a recognized international standard for cga assay is not available and variations in assay types may influence results . nevertheless , this analysis identified the usefulness of serial cga monitoring as a biomarker that predicts treatment response and survival ."], ["this study revealed that the changes of the plasma cga levels were associated with tumour response . additionally , biochemical response based on serial cga may be a predictive marker for pfs in gep - nets ."]]}
{"article_id": "PMC3830842", "article_text": ["subjective cognitive complaints ( scc ) are quite prevalent among older adults , with some estimates suggesting that between 25% and 50% of all older adults have self - perceived memory impairment [ 1 , 2 ] . in clinical practice , it is often difficult to assess the veracity and severity of subjective cognitive complaints , primarily because such complaints vary widely from individual to individual . as a result", ", clinicians and caregivers perhaps do not consider subjective complaints to have the same weight as objective findings .", "however , studies have shown that subjective complaints may be valid indicators of current and future cognitive impairment . a recent study by amariglio and colleagues showed that certain subjective complaints , such as  i have trouble finding my way around familiar streets ,  are correlated with impairment in delayed recall , naming , and semantic fluency .", "a review conducted by jonker and colleagues showed that memory complaints may be predictive of dementia or alzheimer 's disease onset within two to four years , especially in individuals with a diagnosis of mild cognitive impairment ( mci ) .", "subjective cognitive complaints also have clinical implications that are outside the realm of cognitive function . a review by mol and colleagues   found that scc correlated with depression , anxiety [ 4 , 5 ] , and low level of well - being , even in the absence of objective cognitive impairment .", "scc and objective cognitive function in older patients with and without major depression and found no changes in objective cognitive function between the two groups , but significantly more scc in the depressed group .", "scc have also been correlated with decreased functional ability , even when depression is controlled for .", "although the direction of causality between many of these variables remains unclear , scc should be considered in a clinical setting because of their associations with objective impairment , dementia , depression , low quality of life , and functional ability .", "previous research in the area of scc has largely focused on memory complaints [ 13 , 7 , 8 ] . in the current study , we have broadened the scope of scc in order to examine other areas of cognition that have shown impairment following complaints .", "the most common method appears to be a single , dichotomous ,  yes or no  assessment of complaints ( e.g. ,  do you find that you have trouble with your memory ?  ) [ 713 ] , or a combination of similar  yes or no  questions [ 3 , 14 , 15 ] .", "furthermore , most of the existing literature on scc examines community - dwelling , healthy , and older populations [ 3 , 7 , 9 , 12 , 16 ] .  ", "subjective cognitive complaints , in addition to predicting the onset of a neurodegenerative process , may yield important information about areas of impairment , specifically in patients with mild disease .", "two such disorders are mild cognitive impairment ( mci ) and its vascular equivalent , vascular mild cognitive impairment ( vamci ) .", "differences in functional impairment between individuals with mci and those with vamci , which may be overlooked by objective cognitive testing , may be predicted by an analysis of significance of scc . in the current study , we compared the profile of scc in individuals with a diagnosis of mci to those with vamci using the neuropsychological impairment scale ( nis ) .", "the nis is a comprehensive scale - based questionnaire , which may elicit differences in presence and severity of scc between the two groups .", "we hypothesized that the severity of subjective cognitive complaints would not differ between the two groups , because objective cognitive functioning is similar .", "however , we hypothesized that the specific types of scc might differ between mci and vamci , due to the differing pathologies of these two disorders .", "twenty patients with mci and twenty patients with vamci matched on demographic characteristics ( age , education ) were recruited from the memory disorders clinic at st .", "patients are referred to the clinic by their primary or secondary care physicians , for assessment and diagnosis of cognitive impairment .", "patients typically undergo a standardized history and a standardized cognitive exam as part of the workup .", "in addition , routine blood work including tsh , b12 , and rbc folate is done to rule out reversible causes of dementia . as well", ", most patients receive structural imaging ( either computed tomography ( ct ) or magnetic resonance imaging ( mri ) ) and in some cases functional imaging ( single photon emission computed tomography ( spect ) ) . for this study , only patients clinically diagnosed with either mci or vamci were recruited .", "the diagnostic criteria for mci describe cognitive impairment which is more severe than normal aging but less severe than dementia and typically not associated with serious impairment in everyday functions .", "vamci is described as the vascular equivalent of mci , thereby indicating a similar level of objective cognitive performance despite possible differences in mechanism and etiology .", "currently , there are no specific neuroimaging or vascular criteria that are necessary for a diagnosis of vamci .", "some patients underwent mri scans instead of ct scans as part of their clinical visit .", "as we used different imaging modalities we could not compare degree of white matter disease across study subjects in a quantifiable manner .", "rather , patients were assigned to the vamci group if scans showed evidence of white matter lesions , lacunar infarcts , and/or moderate to severe microangiopathic change .", "cognitive testing and data intake occurred during the patients ' initial visits to the clinic .", "follow - up visits to the memory disorders clinic were not considered for this cross - sectional analysis .", "subjective cognitive complaints were assessed using the neuropsychological impairment scale ( nis ) [ 20 , 21 ] .", "the nis is a questionnaire consisting of 95 complaints , such as  i am forgetful  and  i am easily distracted .", " subjects rated these statements on a five - point likert scale according to applicability and intensity .", "when scored , the nis divides complaints into seven domains : critical items ( e.g. , head injury , stroke , and dizziness ) , cognitive efficiency ( e.g. , confusion , mental slowness ) , and attention , memory , frustration tolerance , verbal learning , and academic skills ( e.g. , counting change , learning new tasks ) .", "the nis also includes three scores that give a general , comprehensive picture of complaints : the global measure of impairment ( gmi ) score is a simple sum of all domain subscores ; the total items circled ( tic ) score indicates how many complaints are reported ; and the symptom intensity measure ( sim ) score indicates the average intensity of complaints .", "the nis also includes three validity checks : defensiveness , inconsistency , and affective disturbance .", "thus , the nis can detect if subjects are overly defensive or inconsistent ; and the affective disturbance validity check controls for levels of depression and other emotional disturbances .", "the mini - mental state examination ( mmse )   was used to measure cognitive performance from a general perspective .", "the bna consists of five subcategories : memory , attention , naming , visuospatial function , and executive function .", "functional ability was objectively assessed using the older american resources and services ( oars ) activities of daily living questionnaire .", "this questionnaire assesses fourteen activities of daily living ( adls ) : seven basic adls ( eating , dressing and undressing , grooming , walking , getting in and out of bed , bathing , and going to the bathroom ) and seven instrumental adls ( using the telephone , traveling , shopping , preparing meals , doing housework , taking medications , and handling money ) .", "subjects were asked about their own ability to perform the above adls ; where possible , informants who knew the subjects well were asked to supplement and verify responses .  ", "mean scores for each group ( mci and vamci ) were compared via an independent samples t - test .", "age , education , and other demographic factors were also compared between the two groups .", "the mci and vamci groups were matched on demographic measures such as age , years of education , and hollingshead two - factor index of social position ( table 1 ) .", "functional ability , as measured by the oars questionnaire , was also similar between the two groups ( p = 0.919 ) ( table 2 ) .", "objective cognition function was measured by the mmse and the bna ( table 2 ) .", "mmse scores were similar between the two groups ( p = 0.330 ) , which implies similar levels of general cognitive function .", "overall bna scores were also similar between the mci and vamci groups ( p = 0.177 )", ". scores from subcategories of the bna were also compared between groups to look for specific areas of cognitive impairment ( table 2 ) .", "performance on all subcategories of the bna ( attention , memory , naming , visuospatial function , and executive function ) was similar between mci and vamci groups .", "performance on the individual tasks which comprise the above five subcategories was also similar between groups , with the exception of the  explaining proverbs  task ( p = 0.034 ) . on this task , which falls into the subcategory of executive function", ", mci subjects averaged a better score than vamci subjects ( 3.15 compared to 2.30 ) . despite similar performance on objective functional and cognitive measures ( oars , mmse , and bna ) ,", "the mci and vamci groups achieved markedly different results when subjective cognitive complaints were assessed by the nis . in general ,", "vamci subjects had significantly higher scores on the nis than mci subjects ( table 3 ) ; higher scores indicate greater scc .", "the nis global measure of impairment ( gmi ) score gives a broad , comprehensive view of complaints .", "the vamci group averaged a gmi score of 117.0 , compared to just 79.3 for the mci group ( p = 0.050 ) .", "a qualitative analysis of the total items circled ( tic ) scores shows that vamci subjects had a greater number of complaints than mci subjects ( 52.8 compared to 44.2 ) ; however this difference was not statistically significant ( p = 0.136 ) .", "the symptom intensity measure ( sim ) scores reveal that vamci subjects ' complaints were greater in severity than those of mci subjects ( 2.0 compared to 1.7 ) ; however , again , this difference was not statistically significant ( p = 0.065 ) .", "thus , we can not attribute the difference in gmi scores to just one cause .", "rather , it is likely that difference in gmi scores stems from differences in both complaint quantity and complaint severity .", "quantitatively , the vamci group had higher scores in all seven domains of the nis ( table 3 ) .", "four of these domains yielded differences in scores that were statistically significant : critical items ( p = 0.017 ) , cognitive efficiency ( p = 0.043 ) , memory ( p = 0.046 ) , and verbal learning ( p = 0.027 ) .", "the nis also includes three validity checks to test for defensiveness , affective disturbance , and inconsistency . in our sample of subjects", ", there was no statistical difference in any of the above variables between the mci and vamci groups ( table 3 ) , confirming that the complaints were valid .", "although vamci subjects had qualitatively higher scores on the three validity measures , their scores were not significantly different from those of the mci group .", "mci and vamci are clinical conditions that precede the development of neurodegenerative disorders including alzheimer 's disease and vascular dementia , respectively .", "apart from differences on neuroimaging there has been very little study of how these two clinical conditions differ in terms of their subjective and objective clinical profiles . taken altogether , our findings from the mmse , bna , oars , and nis show that despite similar levels of functional ability and objective cognitive function , vamci subjects have significantly more scc than mci subjects .", "our findings support the existing view of mci and vamci as disorders with similar presentations with regard to objective function , but ultimately different etiologies .", "as previously discussed , mci and vamci subjects were matched on age , education , and social status and achieved statistically similar scores on the mmse and bna .", "the one component of the bna that differed between the two groups was the  explaining proverbs  task ; however , this seeming disparity may have resulted from differences in cultural background . in our sample of patients from the diverse city of toronto , 17 out of 40 subjects (", "unfamiliarity with english proverbs certainly confounded this result and may explain this difference observed on the bna . leaving aside this single anomaly , our findings show that mci and vamci subjects had comparable levels of objective cognitive function .", "however , our findings from the nis questionnaire show that vamci subjects had more subjective cognitive complaints than their mci counterparts .", "this discrepancy suggests that vamci patients are exhibiting particular cognitive deficits , which are not identified by the bna , or that vamci patients are more sensitive to and aware of mild deficits in these particular areas .", "the nature of vamci - specific deficits may be determined from the domains in which vamci patients report greater complaints : critical items , cognitive efficiency , memory , and verbal learning .", "the critical items domain references events in a patient 's history that are evident predictors of cognitive difficulty : dizziness , concussion , head trauma , stroke , and so forth .", "vamci subjects reported greater complaints in this domain than mci subjects ; this finding may be explained by simply considering the diagnostic criteria of vamci compared to those of mci . as mentioned above , history of clinical stroke can differentiate vamci from mci .", "a vamci patient is more likely than an mci patient to have experienced stroke or stroke - like symptoms , so he / she is more likely to have complaints in the critical items domain .", "vamci subjects ' greater complaints in the memory , verbal learning , and cognitive efficiency domains may be explained by examining the pathology of vamci , which differs from that of mci upon neuroimaging . a study by vannorsdall et al .", "found that white matter hyperintensities in both periventricular and subcortical regions correlated with poorer working memory .", "found that deep white matter hyperintensities correlated with decreased performance on verbal fluency tasks , while periventricular hyperintensities were not associated with any particular domain .", "white matter lesions have also been correlated with slowed information processing [ 25 , 27 ] .", "recent research has demonstrated that this decrease in processing speed may be specific to white matter lesions in the anterior thalamic radiation .  ", "our analysis may be hampered by the limitations of our cognitive testing methods . in this study , the", "mmse and bna may have suffered from ceiling effects , which would have masked any differences in cognitive function between the mci and vamci groups .", "however , the advantages of the mmse and bna are numerous and informed our decision to use these tests .", "in addition , more sensitive cognitive testing would not have been possible at the study facility , given the amount of time allotted for a clinical visit .", "the differences we have found in scc between mci and vamci groups may suggest a need for alternate cognitive testing methods , which , unlike the mmse or bna , would elicit different results between the two groups and would not suffer from ceiling effects .", "our study has demonstrated that vamci patients may be exhibiting particular cognitive deficits in memory , verbal learning , and cognitive efficiency , separate from those exhibited by mci patients .", "these deficits may be explained by the greater white matter burden in vamci patients , as compared to age - matched mci patients .", "further research should be done to elaborate on the nature of vamci - specific deficits , particularly in the domains we have identified in this study .", "as well , vamci - specific deficits should be related to neuroimaging findings , in order to associate deficits with particular locations and severities of white matter disease .", "finally , tools for more sensitive cognitive testing must be developed , so that we may identify vamci - specific cognitive deficits ."], "abstract_text": ["<S> \n objective . vascular mild cognitive impairment ( vamci ) is differentiated from mild cognitive impairment ( mci ) by the presence of vascular events such as stroke or small vessel disease . </S>", "<S> typically , mci and vamci patients present with subjective complaints regarding cognition ; however , little is known about the specific nature of these complaints . </S>", "<S> we aimed to create a profile of subjective cognitive complaints in mci and vamci patients with similar levels of objective cognitive performance . </S>", "<S> methods . </S>", "<S> twenty mci and twenty vamci patients were recruited from a memory disorders clinic in toronto . </S>", "<S> subjective cognitive complaints were assessed and categorized using the neuropsychological impairment scale . results . </S>", "<S> mci and vamci patients achieved similar scores on measures of objective cognitive function ( p > 0.100 ) . </S>", "<S> however , the vamci group had more subjective complaints than the mci group ( p = 0.050 ) , particularly in the critical items , cognitive efficiency , memory , and verbal learning domains of the neuropsychological impairment scale . conclusions . </S>", "<S> our findings support the idea that vamci and mci differ in their clinical profiles , independent of neuroimaging . </S>", "<S> vamci patients have significantly more subjective cognitive complaints and may be exhibiting particular deficits in memory , verbal learning , and cognitive efficiency . </S>", "<S> our findings promote the need for further research into vamci - specific cognitive deficits . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["subjective cognitive complaints ( scc ) are quite prevalent among older adults , with some estimates suggesting that between 25% and 50% of all older adults have self - perceived memory impairment [ 1 , 2 ] . in clinical practice , it is often difficult to assess the veracity and severity of subjective cognitive complaints , primarily because such complaints vary widely from individual to individual . as a result", ", clinicians and caregivers perhaps do not consider subjective complaints to have the same weight as objective findings .", "however , studies have shown that subjective complaints may be valid indicators of current and future cognitive impairment . a recent study by amariglio and colleagues showed that certain subjective complaints , such as  i have trouble finding my way around familiar streets ,  are correlated with impairment in delayed recall , naming , and semantic fluency .", "a review conducted by jonker and colleagues showed that memory complaints may be predictive of dementia or alzheimer 's disease onset within two to four years , especially in individuals with a diagnosis of mild cognitive impairment ( mci ) .", "subjective cognitive complaints also have clinical implications that are outside the realm of cognitive function . a review by mol and colleagues   found that scc correlated with depression , anxiety [ 4 , 5 ] , and low level of well - being , even in the absence of objective cognitive impairment .", "scc and objective cognitive function in older patients with and without major depression and found no changes in objective cognitive function between the two groups , but significantly more scc in the depressed group .", "scc have also been correlated with decreased functional ability , even when depression is controlled for .", "although the direction of causality between many of these variables remains unclear , scc should be considered in a clinical setting because of their associations with objective impairment , dementia , depression , low quality of life , and functional ability .", "previous research in the area of scc has largely focused on memory complaints [ 13 , 7 , 8 ] . in the current study , we have broadened the scope of scc in order to examine other areas of cognition that have shown impairment following complaints .", "the most common method appears to be a single , dichotomous ,  yes or no  assessment of complaints ( e.g. ,  do you find that you have trouble with your memory ?  ) [ 713 ] , or a combination of similar  yes or no  questions [ 3 , 14 , 15 ] .", "furthermore , most of the existing literature on scc examines community - dwelling , healthy , and older populations [ 3 , 7 , 9 , 12 , 16 ] .  ", "subjective cognitive complaints , in addition to predicting the onset of a neurodegenerative process , may yield important information about areas of impairment , specifically in patients with mild disease .", "two such disorders are mild cognitive impairment ( mci ) and its vascular equivalent , vascular mild cognitive impairment ( vamci ) .", "differences in functional impairment between individuals with mci and those with vamci , which may be overlooked by objective cognitive testing , may be predicted by an analysis of significance of scc . in the current study , we compared the profile of scc in individuals with a diagnosis of mci to those with vamci using the neuropsychological impairment scale ( nis ) .", "the nis is a comprehensive scale - based questionnaire , which may elicit differences in presence and severity of scc between the two groups .", "we hypothesized that the severity of subjective cognitive complaints would not differ between the two groups , because objective cognitive functioning is similar .", "however , we hypothesized that the specific types of scc might differ between mci and vamci , due to the differing pathologies of these two disorders ."], ["twenty patients with mci and twenty patients with vamci matched on demographic characteristics ( age , education ) were recruited from the memory disorders clinic at st .", "patients are referred to the clinic by their primary or secondary care physicians , for assessment and diagnosis of cognitive impairment .", "patients typically undergo a standardized history and a standardized cognitive exam as part of the workup .", "in addition , routine blood work including tsh , b12 , and rbc folate is done to rule out reversible causes of dementia . as well", ", most patients receive structural imaging ( either computed tomography ( ct ) or magnetic resonance imaging ( mri ) ) and in some cases functional imaging ( single photon emission computed tomography ( spect ) ) . for this study , only patients clinically diagnosed with either mci or vamci were recruited .", "the diagnostic criteria for mci describe cognitive impairment which is more severe than normal aging but less severe than dementia and typically not associated with serious impairment in everyday functions .", "vamci is described as the vascular equivalent of mci , thereby indicating a similar level of objective cognitive performance despite possible differences in mechanism and etiology .", "currently , there are no specific neuroimaging or vascular criteria that are necessary for a diagnosis of vamci .", "some patients underwent mri scans instead of ct scans as part of their clinical visit .", "as we used different imaging modalities we could not compare degree of white matter disease across study subjects in a quantifiable manner .", "rather , patients were assigned to the vamci group if scans showed evidence of white matter lesions , lacunar infarcts , and/or moderate to severe microangiopathic change .", "cognitive testing and data intake occurred during the patients ' initial visits to the clinic .", "follow - up visits to the memory disorders clinic were not considered for this cross - sectional analysis .", "subjective cognitive complaints were assessed using the neuropsychological impairment scale ( nis ) [ 20 , 21 ] .", "the nis is a questionnaire consisting of 95 complaints , such as  i am forgetful  and  i am easily distracted .", " subjects rated these statements on a five - point likert scale according to applicability and intensity .", "when scored , the nis divides complaints into seven domains : critical items ( e.g. , head injury , stroke , and dizziness ) , cognitive efficiency ( e.g. , confusion , mental slowness ) , and attention , memory , frustration tolerance , verbal learning , and academic skills ( e.g. , counting change , learning new tasks ) .", "the nis also includes three scores that give a general , comprehensive picture of complaints : the global measure of impairment ( gmi ) score is a simple sum of all domain subscores ; the total items circled ( tic ) score indicates how many complaints are reported ; and the symptom intensity measure ( sim ) score indicates the average intensity of complaints .", "the nis also includes three validity checks : defensiveness , inconsistency , and affective disturbance .", "thus , the nis can detect if subjects are overly defensive or inconsistent ; and the affective disturbance validity check controls for levels of depression and other emotional disturbances .", "the mini - mental state examination ( mmse )   was used to measure cognitive performance from a general perspective .", "the bna consists of five subcategories : memory , attention , naming , visuospatial function , and executive function .", "functional ability was objectively assessed using the older american resources and services ( oars ) activities of daily living questionnaire .", "this questionnaire assesses fourteen activities of daily living ( adls ) : seven basic adls ( eating , dressing and undressing , grooming , walking , getting in and out of bed , bathing , and going to the bathroom ) and seven instrumental adls ( using the telephone , traveling , shopping , preparing meals , doing housework , taking medications , and handling money ) .", "subjects were asked about their own ability to perform the above adls ; where possible , informants who knew the subjects well were asked to supplement and verify responses .  ", "mean scores for each group ( mci and vamci ) were compared via an independent samples t - test .", "age , education , and other demographic factors were also compared between the two groups ."], ["the mci and vamci groups were matched on demographic measures such as age , years of education , and hollingshead two - factor index of social position ( table 1 ) .", "functional ability , as measured by the oars questionnaire , was also similar between the two groups ( p = 0.919 ) ( table 2 ) .", "objective cognition function was measured by the mmse and the bna ( table 2 ) .", "mmse scores were similar between the two groups ( p = 0.330 ) , which implies similar levels of general cognitive function .", "overall bna scores were also similar between the mci and vamci groups ( p = 0.177 )", ". scores from subcategories of the bna were also compared between groups to look for specific areas of cognitive impairment ( table 2 ) .", "performance on all subcategories of the bna ( attention , memory , naming , visuospatial function , and executive function ) was similar between mci and vamci groups .", "performance on the individual tasks which comprise the above five subcategories was also similar between groups , with the exception of the  explaining proverbs  task ( p = 0.034 ) . on this task , which falls into the subcategory of executive function", ", mci subjects averaged a better score than vamci subjects ( 3.15 compared to 2.30 ) . despite similar performance on objective functional and cognitive measures ( oars , mmse , and bna ) ,", "the mci and vamci groups achieved markedly different results when subjective cognitive complaints were assessed by the nis . in general ,", "vamci subjects had significantly higher scores on the nis than mci subjects ( table 3 ) ; higher scores indicate greater scc .", "the nis global measure of impairment ( gmi ) score gives a broad , comprehensive view of complaints .", "the vamci group averaged a gmi score of 117.0 , compared to just 79.3 for the mci group ( p = 0.050 ) .", "a qualitative analysis of the total items circled ( tic ) scores shows that vamci subjects had a greater number of complaints than mci subjects ( 52.8 compared to 44.2 ) ; however this difference was not statistically significant ( p = 0.136 ) .", "the symptom intensity measure ( sim ) scores reveal that vamci subjects ' complaints were greater in severity than those of mci subjects ( 2.0 compared to 1.7 ) ; however , again , this difference was not statistically significant ( p = 0.065 ) .", "thus , we can not attribute the difference in gmi scores to just one cause .", "rather , it is likely that difference in gmi scores stems from differences in both complaint quantity and complaint severity .", "quantitatively , the vamci group had higher scores in all seven domains of the nis ( table 3 ) .", "four of these domains yielded differences in scores that were statistically significant : critical items ( p = 0.017 ) , cognitive efficiency ( p = 0.043 ) , memory ( p = 0.046 ) , and verbal learning ( p = 0.027 ) .", "the nis also includes three validity checks to test for defensiveness , affective disturbance , and inconsistency . in our sample of subjects", ", there was no statistical difference in any of the above variables between the mci and vamci groups ( table 3 ) , confirming that the complaints were valid .", "although vamci subjects had qualitatively higher scores on the three validity measures , their scores were not significantly different from those of the mci group ."], ["mci and vamci are clinical conditions that precede the development of neurodegenerative disorders including alzheimer 's disease and vascular dementia , respectively .", "apart from differences on neuroimaging there has been very little study of how these two clinical conditions differ in terms of their subjective and objective clinical profiles . taken altogether , our findings from the mmse , bna , oars , and nis show that despite similar levels of functional ability and objective cognitive function , vamci subjects have significantly more scc than mci subjects .", "our findings support the existing view of mci and vamci as disorders with similar presentations with regard to objective function , but ultimately different etiologies .", "as previously discussed , mci and vamci subjects were matched on age , education , and social status and achieved statistically similar scores on the mmse and bna .", "the one component of the bna that differed between the two groups was the  explaining proverbs  task ; however , this seeming disparity may have resulted from differences in cultural background . in our sample of patients from the diverse city of toronto , 17 out of 40 subjects (", "unfamiliarity with english proverbs certainly confounded this result and may explain this difference observed on the bna . leaving aside this single anomaly , our findings show that mci and vamci subjects had comparable levels of objective cognitive function .", "however , our findings from the nis questionnaire show that vamci subjects had more subjective cognitive complaints than their mci counterparts .", "this discrepancy suggests that vamci patients are exhibiting particular cognitive deficits , which are not identified by the bna , or that vamci patients are more sensitive to and aware of mild deficits in these particular areas .", "the nature of vamci - specific deficits may be determined from the domains in which vamci patients report greater complaints : critical items , cognitive efficiency , memory , and verbal learning .", "the critical items domain references events in a patient 's history that are evident predictors of cognitive difficulty : dizziness , concussion , head trauma , stroke , and so forth .", "vamci subjects reported greater complaints in this domain than mci subjects ; this finding may be explained by simply considering the diagnostic criteria of vamci compared to those of mci . as mentioned above , history of clinical stroke can differentiate vamci from mci .", "a vamci patient is more likely than an mci patient to have experienced stroke or stroke - like symptoms , so he / she is more likely to have complaints in the critical items domain .", "vamci subjects ' greater complaints in the memory , verbal learning , and cognitive efficiency domains may be explained by examining the pathology of vamci , which differs from that of mci upon neuroimaging . a study by vannorsdall et al .", "found that white matter hyperintensities in both periventricular and subcortical regions correlated with poorer working memory .", "found that deep white matter hyperintensities correlated with decreased performance on verbal fluency tasks , while periventricular hyperintensities were not associated with any particular domain .", "white matter lesions have also been correlated with slowed information processing [ 25 , 27 ] .", "recent research has demonstrated that this decrease in processing speed may be specific to white matter lesions in the anterior thalamic radiation .  ", "our analysis may be hampered by the limitations of our cognitive testing methods . in this study , the", "mmse and bna may have suffered from ceiling effects , which would have masked any differences in cognitive function between the mci and vamci groups .", "however , the advantages of the mmse and bna are numerous and informed our decision to use these tests .", "in addition , more sensitive cognitive testing would not have been possible at the study facility , given the amount of time allotted for a clinical visit .", "the differences we have found in scc between mci and vamci groups may suggest a need for alternate cognitive testing methods , which , unlike the mmse or bna , would elicit different results between the two groups and would not suffer from ceiling effects .", "our study has demonstrated that vamci patients may be exhibiting particular cognitive deficits in memory , verbal learning , and cognitive efficiency , separate from those exhibited by mci patients .", "these deficits may be explained by the greater white matter burden in vamci patients , as compared to age - matched mci patients .", "further research should be done to elaborate on the nature of vamci - specific deficits , particularly in the domains we have identified in this study .", "as well , vamci - specific deficits should be related to neuroimaging findings , in order to associate deficits with particular locations and severities of white matter disease .", "finally , tools for more sensitive cognitive testing must be developed , so that we may identify vamci - specific cognitive deficits ."]]}
{"article_id": "PMC2600377", "article_text": ["the 42 participating spanish hospitals were selected according to earss criteria ( 1,7 ) . the total catchment population was 9 million people , or 22.5% of the spanish population .", "the first blood e. coli isolates obtained from each patient between 2003 and 2006 were included .", "each laboratory identified the strains and tested their susceptibilities according to standard microbiologic procedures ; all used commercial microdilution systems .", "susceptibility data were interpreted according to clinical laboratory standards institute criteria ( 8) . for epidemiologic purposes , intermediate susceptibility to amc was considered as resistance .", "multidrug resistance was defined as resistance to > 3 of the following antimicrobial agents : ciprofloxacin , gentamicin , cotrimoxazole , and cefotaxime . to assess", "the comparability of susceptibility test results , an external quality assurance exercise ( uk national external quality assessment scheme ) was performed yearly .", "hospital - acquired infections were defined as infections acquired at least 48 hours after hospital admission .", "outpatient consumption of penicillin/-lactamase inhibitors ( world health organization code j01cr02 ) for the period 20022006 was assessed from the especialidades consumo de medicamentos database , which showed retail pharmacy sales of all medicines acquired with national health system prescriptions and covered nearly 100% of the spanish population ( 5 ) .", "the information was tabulated , and the number of units was converted into defined daily doses ( ddd ) of active drug ingredients according to who methodology ( 9 ) .", "the number of ddd per 1,000 inhabitants per day ( dids ) was calculated for each active drug ingredient .", "differences in the antimicrobial resistance prevalence between different groups were assessed by fisher exact test .", "association was determined by calculation of the odds ratio ( or ) with 95% confidence intervals ( ci ) .", "statistical analyses were performed by using graphpad prism version 3.02 software ( graphpad software , inc .", "participating hospitals reported data on 9,090 cases of e. coli bacteremia during the study period , corresponding to the same number of patients ; 4,526 ( 49.8% ) were male patients and 4,564 ( 50.2% ) were female patients .", "a total of 1,531 cases ( 16.8% ) were diagnosed in 2003 ; 2,526 ( 27.8% ) in 2004 ; 2,438 ( 26.8% ) in 2005 ; and 2,597 ( 28.6% ) in 2006 . of the total number of isolates , 328 ( 3.6% ) were obtained from children < 14 years of age ; 2,857 ( 31.4% ) were obtained from patients > 15 and < 64 years of age ; and 5,909 ( 65% ) were obtained from patients > 64 years of age .", "there were 3,384 ( 37.9% ) isolates implicated in hospital - acquired infections and 5,540 ( 62.1% ) in community - acquired infections ; information was missing for 166 cases .", "of the 9,090 e. coli isolates tested , 1,136 ( 12.5% ) were nonsusceptible to amc , 5.1% were resistant , and 7.4% were intermediate .", "the prevalence of amoxicillin / clavulanic acid nonsusceptibility in relation to gender , age , infection origin , and resistance to other antibimicrobial drugs is detailed in the table . *", "the most prevalent phenotypes included multidrug resistance to ciprofloxacin , cotrimoxazole , and gentamicin , which was detected in 73 nonsusceptible amc isolates ( 36.9% of multiresistant isolates and 6.4% of isolates overall ) , and resistance to ciprofloxacin , cotrimoxazole , and cefotaxime was detected in 55 isolates ( 27.8% of multiresistant isolates ; 4.9% of isolates overall ) .", "multidrug resistance was more prevalent in nosocomial ( 23.5% ) than in community - acquired isolates ( 15.1% ; or 1.99 , 95% ci 1.462.72 ; p<0.0001 ) . among nonsusceptible", "amc isolates , susceptibility to other antimicrobial drugs , including ciprofloxacin , gentamicin , cefotaxime , and cotrimoxazole , was more frequent in community- ( 28.7% ) than in hospital - acquired isolates ( 13.3% ; or 1.68 , 95% ci 1.272.22 ; p = 0.0003 ) .", "the overall rate of invasive e. coli nonsusceptibility to amc increased from 9.3% ( 2003 ) to 15.4% ( 2006 ) (  test for trend 36.51 ; p<0.0001 ) ( figure 1 ) ; this increase was observed in 64.3% of the participant hospitals .", "this increase was also detected in both intermediate and resistant isolates , with annual distributions of 5.6% and 3.8% , respectively , in 2003 ; 6.8% and 4.8% in 2004 ; 7.5% and 5.4% in 2005 ; and 9.4% and 6% in 2006 .", "evolution of amoxicillin - clavulanic acid nonsusceptibility of escherichia coli from blood isolates , spain , 20032006 .", "amc nonsusceptibility according to age groups increased over the study period as follows : children < 14 years of age ( 10.6% in 2003 , 14.6% in 2004 , 14.3% in 2005 , and 16.3% in 2006 ) ; patients", "> 15 and < 64 years 9.6% in 2003 , 11.2% in 2004 , 11.7% in 2005 , and 13.3% in 2006 ) ; patients > 64 years ( 8.8% in 2002 , 11.3% in 2004 , 15.9% in 2005 , and 16.3% in 2006 ) .", "the prevalence of amc nonsusceptibility in community - acquired infections increased from 8.9% ( 2003 ) to 15.6% ( 2006 ) (  test for trend 29.43 ; p<0.0001 ) .", "amc nonsusceptibility in nosocomial infections increased from 9.2% ( 2003 ) to 15.2% ( 2006 ) (  test for trend 11.94 ; p = 0.0006 ) . in the final 2 years of the study period ( 20052006 )", "this increase was due to community - acquired e. coli isolates only ; the nonsusceptible proportion varied from 11.5% ( 2005 ) to 15.6% ( 2006 ) ( or 1.42 , 95% ci 1.161.74 ; p = 0.0009 ) in community - acquired isolates compared with 15.4% ( 2005 ) to 15.2% ( 2006 ) in hospital - acquired isolates ( figure 1 ) .", "community - acquired infection probably included healthcare - associated infections , a recently described epidemiologic category distinct from both community - acquired and nosocomial status . in this study , the number of blood isolates of e. coli producing extended - spectrum -lactamase ( esbl ) was 614 ( 6.7% ) ; 188 of them ( 30.6% ) were nonsusceptible to amc .", "when esbl - producing e. coli isolates were excluded from analysis , amc nonsusceptibility increased from 8.4% ( 2003 ) to 14.3% ( 2006 ) (  test for trend 34.39 ; p<0.0001 ) in total isolates ; from 8.2% ( 2003 ) to 14.5% ( 2006 ) (  test or trend 25.23 ; p<0.0001 ) in community - acquired isolates ; and from 8.9% ( 2003 ) to 13.3% ( 2006 ) (  test for trend 6.35 ; p = 0.012 ) in hospital - acquired isolates ( figure 1 ) . the proportion of isolates highly susceptible to amc ( mic < 4 mg / l ) steadily decreased over the study period as follows : 70.2% ( 2003 ) , 70% ( 2004 ) , 64.8% ( 2005 ) , and 57.4% ( 2006 ) (  test for trend 99.36 ; p<0.0001 ) .", "community consumption of penicillin/-lactamase inhibitors , predominantly amc , increased 34.7% from 2000 to 2006 ( figure 2 ) , whereas total antimicrobial drug consumption remained relatively constant ( 19.6 dids in 2000 compared with 19.1 dids in 2006 ) .", "after amc , the most used -lactam antimicrobial agents in the community in spain were amoxicillin , cefuroxime , and cefixime ; their consumption did not vary or slightly decreased from 2002 through 2005 ( 6 ) .", "evolution of consumption of outpatient penicillin/-lactamase inhibitors ( world health organization code j01cr02 ) , spain , 20002006 .", "the increased amc resistance of e. coli isolates from blood observed in this study is of serious concern from clinical and epidemiologic standpoints because amc is the first - choice antimicrobial treatment for many invasive e. coli infections .", "increased amc resistance coincided with growing amc consumption at the community level . in urinary infections ,", "previous treatment with amc is a risk factor for the development of amc resistance ( 10 ) .", "amc resistance mechanisms ( -lactamase overproduction , ampc cephalosporinase hyperproduction , and inhibitor - resistant penicillinases ) ( 11 ) might be favored by strong amc consumption ."], "abstract_text": ["<S> to determine the evolution and trends of amoxicillin  clavulanic acid resistance among escherichia coli isolates in spain , we tested 9,090 blood isolates from 42 spanish hospitals and compared resistance with trends in outpatient consumption . </S>", "<S> these isolates were collected by spanish hospitals that participated in the european antimicrobial resistance surveillance system network from april 2003 through december 2006 . </S>"], "labels": null, "section_names": ["The Study", "Conclusions"], "sections": [["the 42 participating spanish hospitals were selected according to earss criteria ( 1,7 ) . the total catchment population was 9 million people , or 22.5% of the spanish population .", "the first blood e. coli isolates obtained from each patient between 2003 and 2006 were included .", "each laboratory identified the strains and tested their susceptibilities according to standard microbiologic procedures ; all used commercial microdilution systems .", "susceptibility data were interpreted according to clinical laboratory standards institute criteria ( 8) . for epidemiologic purposes , intermediate susceptibility to amc was considered as resistance .", "multidrug resistance was defined as resistance to > 3 of the following antimicrobial agents : ciprofloxacin , gentamicin , cotrimoxazole , and cefotaxime . to assess", "the comparability of susceptibility test results , an external quality assurance exercise ( uk national external quality assessment scheme ) was performed yearly .", "hospital - acquired infections were defined as infections acquired at least 48 hours after hospital admission .", "outpatient consumption of penicillin/-lactamase inhibitors ( world health organization code j01cr02 ) for the period 20022006 was assessed from the especialidades consumo de medicamentos database , which showed retail pharmacy sales of all medicines acquired with national health system prescriptions and covered nearly 100% of the spanish population ( 5 ) .", "the information was tabulated , and the number of units was converted into defined daily doses ( ddd ) of active drug ingredients according to who methodology ( 9 ) .", "the number of ddd per 1,000 inhabitants per day ( dids ) was calculated for each active drug ingredient .", "differences in the antimicrobial resistance prevalence between different groups were assessed by fisher exact test .", "association was determined by calculation of the odds ratio ( or ) with 95% confidence intervals ( ci ) .", "statistical analyses were performed by using graphpad prism version 3.02 software ( graphpad software , inc .", "participating hospitals reported data on 9,090 cases of e. coli bacteremia during the study period , corresponding to the same number of patients ; 4,526 ( 49.8% ) were male patients and 4,564 ( 50.2% ) were female patients .", "a total of 1,531 cases ( 16.8% ) were diagnosed in 2003 ; 2,526 ( 27.8% ) in 2004 ; 2,438 ( 26.8% ) in 2005 ; and 2,597 ( 28.6% ) in 2006 . of the total number of isolates , 328 ( 3.6% ) were obtained from children < 14 years of age ; 2,857 ( 31.4% ) were obtained from patients > 15 and < 64 years of age ; and 5,909 ( 65% ) were obtained from patients > 64 years of age .", "there were 3,384 ( 37.9% ) isolates implicated in hospital - acquired infections and 5,540 ( 62.1% ) in community - acquired infections ; information was missing for 166 cases .", "of the 9,090 e. coli isolates tested , 1,136 ( 12.5% ) were nonsusceptible to amc , 5.1% were resistant , and 7.4% were intermediate .", "the prevalence of amoxicillin / clavulanic acid nonsusceptibility in relation to gender , age , infection origin , and resistance to other antibimicrobial drugs is detailed in the table . *", "the most prevalent phenotypes included multidrug resistance to ciprofloxacin , cotrimoxazole , and gentamicin , which was detected in 73 nonsusceptible amc isolates ( 36.9% of multiresistant isolates and 6.4% of isolates overall ) , and resistance to ciprofloxacin , cotrimoxazole , and cefotaxime was detected in 55 isolates ( 27.8% of multiresistant isolates ; 4.9% of isolates overall ) .", "multidrug resistance was more prevalent in nosocomial ( 23.5% ) than in community - acquired isolates ( 15.1% ; or 1.99 , 95% ci 1.462.72 ; p<0.0001 ) . among nonsusceptible", "amc isolates , susceptibility to other antimicrobial drugs , including ciprofloxacin , gentamicin , cefotaxime , and cotrimoxazole , was more frequent in community- ( 28.7% ) than in hospital - acquired isolates ( 13.3% ; or 1.68 , 95% ci 1.272.22 ; p = 0.0003 ) .", "the overall rate of invasive e. coli nonsusceptibility to amc increased from 9.3% ( 2003 ) to 15.4% ( 2006 ) (  test for trend 36.51 ; p<0.0001 ) ( figure 1 ) ; this increase was observed in 64.3% of the participant hospitals .", "this increase was also detected in both intermediate and resistant isolates , with annual distributions of 5.6% and 3.8% , respectively , in 2003 ; 6.8% and 4.8% in 2004 ; 7.5% and 5.4% in 2005 ; and 9.4% and 6% in 2006 .", "evolution of amoxicillin - clavulanic acid nonsusceptibility of escherichia coli from blood isolates , spain , 20032006 .", "amc nonsusceptibility according to age groups increased over the study period as follows : children < 14 years of age ( 10.6% in 2003 , 14.6% in 2004 , 14.3% in 2005 , and 16.3% in 2006 ) ; patients", "> 15 and < 64 years 9.6% in 2003 , 11.2% in 2004 , 11.7% in 2005 , and 13.3% in 2006 ) ; patients > 64 years ( 8.8% in 2002 , 11.3% in 2004 , 15.9% in 2005 , and 16.3% in 2006 ) .", "the prevalence of amc nonsusceptibility in community - acquired infections increased from 8.9% ( 2003 ) to 15.6% ( 2006 ) (  test for trend 29.43 ; p<0.0001 ) .", "amc nonsusceptibility in nosocomial infections increased from 9.2% ( 2003 ) to 15.2% ( 2006 ) (  test for trend 11.94 ; p = 0.0006 ) . in the final 2 years of the study period ( 20052006 )", "this increase was due to community - acquired e. coli isolates only ; the nonsusceptible proportion varied from 11.5% ( 2005 ) to 15.6% ( 2006 ) ( or 1.42 , 95% ci 1.161.74 ; p = 0.0009 ) in community - acquired isolates compared with 15.4% ( 2005 ) to 15.2% ( 2006 ) in hospital - acquired isolates ( figure 1 ) .", "community - acquired infection probably included healthcare - associated infections , a recently described epidemiologic category distinct from both community - acquired and nosocomial status . in this study , the number of blood isolates of e. coli producing extended - spectrum -lactamase ( esbl ) was 614 ( 6.7% ) ; 188 of them ( 30.6% ) were nonsusceptible to amc .", "when esbl - producing e. coli isolates were excluded from analysis , amc nonsusceptibility increased from 8.4% ( 2003 ) to 14.3% ( 2006 ) (  test for trend 34.39 ; p<0.0001 ) in total isolates ; from 8.2% ( 2003 ) to 14.5% ( 2006 ) (  test or trend 25.23 ; p<0.0001 ) in community - acquired isolates ; and from 8.9% ( 2003 ) to 13.3% ( 2006 ) (  test for trend 6.35 ; p = 0.012 ) in hospital - acquired isolates ( figure 1 ) . the proportion of isolates highly susceptible to amc ( mic < 4 mg / l ) steadily decreased over the study period as follows : 70.2% ( 2003 ) , 70% ( 2004 ) , 64.8% ( 2005 ) , and 57.4% ( 2006 ) (  test for trend 99.36 ; p<0.0001 ) .", "community consumption of penicillin/-lactamase inhibitors , predominantly amc , increased 34.7% from 2000 to 2006 ( figure 2 ) , whereas total antimicrobial drug consumption remained relatively constant ( 19.6 dids in 2000 compared with 19.1 dids in 2006 ) .", "after amc , the most used -lactam antimicrobial agents in the community in spain were amoxicillin , cefuroxime , and cefixime ; their consumption did not vary or slightly decreased from 2002 through 2005 ( 6 ) .", "evolution of consumption of outpatient penicillin/-lactamase inhibitors ( world health organization code j01cr02 ) , spain , 20002006 ."], ["the increased amc resistance of e. coli isolates from blood observed in this study is of serious concern from clinical and epidemiologic standpoints because amc is the first - choice antimicrobial treatment for many invasive e. coli infections .", "increased amc resistance coincided with growing amc consumption at the community level . in urinary infections ,", "previous treatment with amc is a risk factor for the development of amc resistance ( 10 ) .", "amc resistance mechanisms ( -lactamase overproduction , ampc cephalosporinase hyperproduction , and inhibitor - resistant penicillinases ) ( 11 ) might be favored by strong amc consumption ."]]}
{"article_id": "PMC3756163", "article_text": ["a 51-year - old caucasian female was referred to our institution because of an abnormal nodule detected in her right lung .", "this had at first been detected four months earlier when she underwent an urgent hartmann procedure due to refractory adenocarcinoma of the sigmoid colon ( pt4n1 stage ) . due to the urgency of the surgery ,", "the patient was not staged prior to the surgery but was initially staged after the operation .", "the only pathologic finding in the computed tomography ( ct ) of the lung was a nodule in the lower right lobe of 2.5 cm at its longest dimension ( fig .", "she received 4 cycles of chemotherapy for the sigmoid adenocarcinoma before she was referred to us for excision of what was believed to be a single metastatic nodule . although the radiologic findings supported a benign tumor , neither positron emission tomography - ct nor any other procedure to attempt to diagnose the nodule", "was performed due to the patient 's willingness to undergo complete removal of the mass even if it were benign .", "the patient underwent video - assisted thoracoscopic ( vats ) wedge excision of the tumor .", "the specimen of the resected lung contained a firm intrapulmonary white - colored well - defined mass with dimensions of 2.52.32.2 cm .", "microscopically , the tumor had a \" patternless pattern , \" with proliferation of bland spindle cells in alternating hypocellular and hypercellular areas , accompanied by a collagenous stroma ( fig .", "the tumor had no evidence of increased mitotic activity ( 0 to 1 mitoses per 10 high power fields using an olympus bh-2 microscope , with a 40 field , 0.5 mm diameter , and area of 0.196 mm ) , any significant atypia , or necrosis .", "immunohistocemically , the tumor cells were positive for vimentin , cd34 ( fig . 2c ) , and bcl-2 .", "the index of cellular proliferation with the antibody ki-67 is low and estimated at < 1% of the cellular population .", "immunohistochemistry staining of the tumor cells was negative for alfa - smooth muscle actin ( sma ) , muscle specific actin ( hhf-35 ) , h - caldesmon , cd117 ( c - kit ) , s-100 protein , epithelial antigens ( wide spectrum cytokeratins [ ae1/ae3 ] and epithelial membrane antigen ) , and thyroid transcription factor-1 .", "solitary fibrous tumors ( sfts ) were first pathologically described by klemperer and rabin in 1931 . to the present ,", "sft is the preferred term for an uncommon , but histomorphologically distinctive spindle cell neoplasm , that was identified in the past as fibrous mesothelioma , localized fibrous mesothelioma , localized fibrous tumor , localized mesothelioma , pleural fibroma , solitary fibrous mesothelioma , or submesothelial fibroma .", "there are reports of sfts arising either from mediastinal , diaphragmatic , or parietal pleura , or from within a lung fissure and in various extrapleural locations , such as the retroperitoneum , mediastinum , thyroid gland , nasal cavities , meninges , or parietal surfaces of the intra - abdominal viscera .", "less than 20 cases of intraparenchymal sfts have been reported in the english literature [ 1 - 5 ] . to prove and establish the fact that these lesions are indeed of pulmonary origin , it is important to consider the clinical , radiologic , and pathologic findings to demonstrate the lack of continuity with the visceral pleura and to exclude an endophytic growth in a pleural - based lesion .", "hypoglycemia and seizures can be encountered in some cases due to secretion of insulin - like growth factor ii by the tumor [ 1 - 3,5 ] .", "hemoptysis due to sft as well as hypertrophic osteoarthropathy secondary to sft of the lung has also been reported .", "there is no association with smoking or asbestos exposure . due to its non - specific symptoms", ", it can potentially mimic solitary pulmonary metastasis , as was the case with our patient . on gross examination ,", "histologically , it is associated with variable patterns , the most common being the \" patternless pattern . \"", "the cytologic features consist of cellular areas composed of spindled to ovoid cells with scanty cytoplasm , occurring either solely or in groups .", "immunohistochemistry plays a substantial role in supporting the diagnosis made by morphologic features and is also helpful in differentiating these tumors from mesothelioma , neurofibroma , and other spindle - cell lesions .", "a differential diagnosis of intrapulmonary sft includes , among other lesions , pulmonary adenofibroma , benign neural neoplasms , leiomyoma and leiomyosarcoma , synovial sarcoma , spindle cell thymoma , spindle cell carcinoid tumor , nerve sheath tumor , fibrosarcoma , sarcomatoid carcinoma , and sarcomatoid mesothelioma .", "the morphologic features and immunohistochemical profile of sft are sufficiently distinct to allow separation from such conditions in the majority of the cases .", "several studies have reported positivity of tumor cells with cd34 antibody in almost 100% of cases , and with cd99 antibody in 70% of cases , whereas bcl-2 , sma , and epithelial membrane antigen are positive in 20% to 35% of the cases .", "the histologic criteria for classifying the malignant variants of sft of the lung and pleura were described by england et al . in 1989 and vallat - decouvelaere et al . in 1998 .", "they established the following features suggestive of malignancy : 1 ) more than 4 mitoses per 10 high - power fields , 2 ) presence of necrosis , 3 ) hemorrhage , 4 ) hypercellularity as detected by nuclear crowding and overlapping , 5 ) nuclear atypia , 6 ) pleomorphism , 7 ) stromal or vascular invasion , and 8) size exceeding 10 cm .", ". the absence of these characteristics does not exclude malignant behavior . on the other hand , encapsulation , pedunculated and", "resectability with free surgical margins are considered to be favorable prognostic factors even in histologically malignant variants .", "positive margins are associated with an aggressive clinical course and high rates of local recurrence and metastasis .", "resection with free margins is considered to be the treatment for sft located either in the lung or on the pleura .", "wedge resection may be accomplished by vats or standard thoracotomy , according to the anatomic position and size of the lesion and the experience of the surgeon .", "adjuvant therapy may have a place in recurrent or systemic disease , but its benefit is undefined . in general , sfts have an unpredictable course , depending on their potential for malignancy . in large series , the recurrence rate of benign sfts", "is reported to be low ( 1.4% ) , while the recurrence rate of malignant variants is reported to be higher ( range , 9% to 19% ) .", "a staging system based on pedunculated versus sessile attachment and malignant versus benign histology that predicts recurrence has been proposed by perrot et al .  ", "( table 1 ) . due to the possibility of local recurrence and/or distal metastasis after surgical removal of the primary sft , long - term follow - up is recommended .", "local recurrence detected early is amenable to reoperation and resection , with good long - term results . the long - term survival rate for both benign and malignant variants", "is reported to be more than 90% [ 1 - 3,6 ] . in conclusion , sfts are neoplasms that usually arise from the pleura . an intraparenchymal or endobronchial location is a rare occurrence .", "resection with clear margins is curative and final diagnosis and prognosis can be defined only after surgical resection .", "the prognosis of patients with rare sfts of the lung depends on the completeness of the tumor resection .", "variants of sfts of the lung that are malignant or suspected to be malignant should be managed as lung cancer with regard to the surgical resection of the tumor and follow - up strategy ."], "abstract_text": ["<S> solitary fibrous tumor is a rare spindle cell mesenchymal tumor entity , with either benign or malignant behavior that can not be accurately predicted by histological findings . </S>", "<S> an intrapulmonary site of origin is even rarer . </S>", "<S> we report a case of a 51-year - old woman in whom an abnormal nodule in the lower right lung was detected during staging for sigmoid adenocarcinoma . </S>", "<S> the nodule was excised and pathological examination revealed an intrapulmonary solitary fibrous tumor . </S>"], "labels": null, "section_names": ["CASE REPORT", "DISCUSSION"], "sections": [["a 51-year - old caucasian female was referred to our institution because of an abnormal nodule detected in her right lung .", "this had at first been detected four months earlier when she underwent an urgent hartmann procedure due to refractory adenocarcinoma of the sigmoid colon ( pt4n1 stage ) . due to the urgency of the surgery ,", "the patient was not staged prior to the surgery but was initially staged after the operation .", "the only pathologic finding in the computed tomography ( ct ) of the lung was a nodule in the lower right lobe of 2.5 cm at its longest dimension ( fig .", "she received 4 cycles of chemotherapy for the sigmoid adenocarcinoma before she was referred to us for excision of what was believed to be a single metastatic nodule . although the radiologic findings supported a benign tumor , neither positron emission tomography - ct nor any other procedure to attempt to diagnose the nodule", "was performed due to the patient 's willingness to undergo complete removal of the mass even if it were benign .", "the patient underwent video - assisted thoracoscopic ( vats ) wedge excision of the tumor .", "the specimen of the resected lung contained a firm intrapulmonary white - colored well - defined mass with dimensions of 2.52.32.2 cm .", "microscopically , the tumor had a \" patternless pattern , \" with proliferation of bland spindle cells in alternating hypocellular and hypercellular areas , accompanied by a collagenous stroma ( fig .", "the tumor had no evidence of increased mitotic activity ( 0 to 1 mitoses per 10 high power fields using an olympus bh-2 microscope , with a 40 field , 0.5 mm diameter , and area of 0.196 mm ) , any significant atypia , or necrosis .", "immunohistocemically , the tumor cells were positive for vimentin , cd34 ( fig . 2c ) , and bcl-2 .", "the index of cellular proliferation with the antibody ki-67 is low and estimated at < 1% of the cellular population .", "immunohistochemistry staining of the tumor cells was negative for alfa - smooth muscle actin ( sma ) , muscle specific actin ( hhf-35 ) , h - caldesmon , cd117 ( c - kit ) , s-100 protein , epithelial antigens ( wide spectrum cytokeratins [ ae1/ae3 ] and epithelial membrane antigen ) , and thyroid transcription factor-1 ."], ["solitary fibrous tumors ( sfts ) were first pathologically described by klemperer and rabin in 1931 . to the present ,", "sft is the preferred term for an uncommon , but histomorphologically distinctive spindle cell neoplasm , that was identified in the past as fibrous mesothelioma , localized fibrous mesothelioma , localized fibrous tumor , localized mesothelioma , pleural fibroma , solitary fibrous mesothelioma , or submesothelial fibroma .", "there are reports of sfts arising either from mediastinal , diaphragmatic , or parietal pleura , or from within a lung fissure and in various extrapleural locations , such as the retroperitoneum , mediastinum , thyroid gland , nasal cavities , meninges , or parietal surfaces of the intra - abdominal viscera .", "less than 20 cases of intraparenchymal sfts have been reported in the english literature [ 1 - 5 ] . to prove and establish the fact that these lesions are indeed of pulmonary origin , it is important to consider the clinical , radiologic , and pathologic findings to demonstrate the lack of continuity with the visceral pleura and to exclude an endophytic growth in a pleural - based lesion .", "hypoglycemia and seizures can be encountered in some cases due to secretion of insulin - like growth factor ii by the tumor [ 1 - 3,5 ] .", "hemoptysis due to sft as well as hypertrophic osteoarthropathy secondary to sft of the lung has also been reported .", "there is no association with smoking or asbestos exposure . due to its non - specific symptoms", ", it can potentially mimic solitary pulmonary metastasis , as was the case with our patient . on gross examination ,", "histologically , it is associated with variable patterns , the most common being the \" patternless pattern . \"", "the cytologic features consist of cellular areas composed of spindled to ovoid cells with scanty cytoplasm , occurring either solely or in groups .", "immunohistochemistry plays a substantial role in supporting the diagnosis made by morphologic features and is also helpful in differentiating these tumors from mesothelioma , neurofibroma , and other spindle - cell lesions .", "a differential diagnosis of intrapulmonary sft includes , among other lesions , pulmonary adenofibroma , benign neural neoplasms , leiomyoma and leiomyosarcoma , synovial sarcoma , spindle cell thymoma , spindle cell carcinoid tumor , nerve sheath tumor , fibrosarcoma , sarcomatoid carcinoma , and sarcomatoid mesothelioma .", "the morphologic features and immunohistochemical profile of sft are sufficiently distinct to allow separation from such conditions in the majority of the cases .", "several studies have reported positivity of tumor cells with cd34 antibody in almost 100% of cases , and with cd99 antibody in 70% of cases , whereas bcl-2 , sma , and epithelial membrane antigen are positive in 20% to 35% of the cases .", "the histologic criteria for classifying the malignant variants of sft of the lung and pleura were described by england et al . in 1989 and vallat - decouvelaere et al . in 1998 .", "they established the following features suggestive of malignancy : 1 ) more than 4 mitoses per 10 high - power fields , 2 ) presence of necrosis , 3 ) hemorrhage , 4 ) hypercellularity as detected by nuclear crowding and overlapping , 5 ) nuclear atypia , 6 ) pleomorphism , 7 ) stromal or vascular invasion , and 8) size exceeding 10 cm .", ". the absence of these characteristics does not exclude malignant behavior . on the other hand , encapsulation , pedunculated and", "resectability with free surgical margins are considered to be favorable prognostic factors even in histologically malignant variants .", "positive margins are associated with an aggressive clinical course and high rates of local recurrence and metastasis .", "resection with free margins is considered to be the treatment for sft located either in the lung or on the pleura .", "wedge resection may be accomplished by vats or standard thoracotomy , according to the anatomic position and size of the lesion and the experience of the surgeon .", "adjuvant therapy may have a place in recurrent or systemic disease , but its benefit is undefined . in general , sfts have an unpredictable course , depending on their potential for malignancy . in large series , the recurrence rate of benign sfts", "is reported to be low ( 1.4% ) , while the recurrence rate of malignant variants is reported to be higher ( range , 9% to 19% ) .", "a staging system based on pedunculated versus sessile attachment and malignant versus benign histology that predicts recurrence has been proposed by perrot et al .  ", "( table 1 ) . due to the possibility of local recurrence and/or distal metastasis after surgical removal of the primary sft , long - term follow - up is recommended .", "local recurrence detected early is amenable to reoperation and resection , with good long - term results . the long - term survival rate for both benign and malignant variants", "is reported to be more than 90% [ 1 - 3,6 ] . in conclusion , sfts are neoplasms that usually arise from the pleura . an intraparenchymal or endobronchial location is a rare occurrence .", "resection with clear margins is curative and final diagnosis and prognosis can be defined only after surgical resection .", "the prognosis of patients with rare sfts of the lung depends on the completeness of the tumor resection .", "variants of sfts of the lung that are malignant or suspected to be malignant should be managed as lung cancer with regard to the surgical resection of the tumor and follow - up strategy ."]]}
{"article_id": "PMC4805152", "article_text": ["synthesis of metal nanoparticles with the help of plant extracts is an emerging field of nanotechnology , due to their novel properties , terrific applications in biomedicine and its eco - friendly nature . in recent past", "great efforts were made for synthesis of environment benign and eco - friendly nanoparticle from plants such as iron oxide nanoparticles from medicago sativa , copper nanoparticles from magnolia kobus , calcium nanoparticles from boswellia ovalifoliolata , gold nanoparticles from avena sativa , zinc oxide nanoparticles from catharanthus roseus , and silver nanoparticles ( agnps ) from syzygium alternifolium .", "silver is one among the metal nanoparticles focused much interest due to its wide variety of applications .", "it has different biological activities such as antimicrobial , anthelmintic , antilarvicidic , antioxidant , anticancer , anti - inflammatory , hepatoprotective , and wound healing activity .", "conventional methods for synthesizing agnps are mainly by chemical , physical , and microbe - mediated synthesis . in these chemical and physical methods , usage of hazardous chemicals , high energy requirements ,", "difficult and wasteful materials generate potential and biological hazards to the environment . whereas in the case of microbe - mediated synthesis", ". therefore biological synthesis of agnps by using plant materials is easy , efficient , and eco - friendliness in comparison to chemical mediated or microbe - mediated synthesis   and they possess secondary metabolites having the redox capacity to reduce metal nanoparticles in an easiest way .", "adansonia digitata l. belongs to the family malvaceae is a large tree indigenous to africa and found in many countries also .", "traditionally , the fruit pulp dissolved in water or milk is used as a drink or sauce for food in africa .", "fruit pulp and powdered seeds are used for the treatment of diarrhea and dysentery in india .", "the high content of vitamin c in fruit pulp is recommended to pregnant women s for daily intake . in recent times , different scientists prove different activities of pulp such as hepatoprotective , antimicrobial , antiviral , antioxidant , antidiarrheal , hypoglycemic , anti - inflammatory , and antioxidant   activities . due to high medicinal values and mythological significance of this plant", "is known as  kalpavriksha  ( a tree which fulfill all desires ) in india . in our previous studies ,", "synthesis of agnps from stem bark and leaf extract of a. digitata acts as excellent reducing agents and show potential antimicrobial activity against different microorganisms .", "however , the potentiality of fruit pulp mediated synthesis of agnps is not carried out so far .", "hence , the present study is aimed to synthesize agnps from a. digitata fruit pulp extract , characterize and to know the potentiality of agnps against different microbial pathogens .", "2 to 4 kg weight mature fruits possess a great amount of pulp is collected from acharya ranga agricultural university , tirupati and cross checked by herbarium ( voucher no : svu362 ) deposited in department of botany , sri venkateswara university , tirupati .", "cut open the fruit and separate the seeds adhesive to pulp . grounded the collected pulp with the help of mortar and pestle , sieved the content for the synthesis of nanoparticles . for synthesis , 25 g of fruit pulp is extracted with 100 ml of distilled water on boiling water bath for 30 min , filter the content with whatman no .", "1 filter paper and stored at room temperature . from this , 5 ml of the extract is taken into 250 ml of erlenmeyer conical flask and titrated against with the solution of 1 mm agno3 at a 60 - 80c temperature . the contents are cooled and centrifuged at 10,000 rpm for 20 min to avoid the presence of any biological impurities , and it is used for further characterization and antimicrobial studies .", "confirmation of synthesized nanoparticles is agnps by ultraviolet ( uv)-vis spectrophotometry absorption spectra using a spectro uv 2080 double beam , between the wavelength scan range of 190 - 700 nm , 1200 l / mm spectrophotometer , analytical technologies , india . to know the possible bio - molecules responsible for reduction and stabilization of agnps by fourier transform infra - red ( ftir ) spectra in the scan range of 4,000 to 500 cm transmittance with an alpha interferometer , bruker , ettlingen , karlsruhe , germany by kbr pellet method .", "x - ray diffraction of synthesized nanoparticles is examined by an x - ray diffractometer ( xrd ) ( shimadzu , xrd-6000 ) equipped with cu ka radiation source using ni as a filter at a setting of 30 kv/30 ma to know the crystalline nature of agnps .", "purity of agnps was analyzed by fei quanta 200 feg high resolution ( hr)-scanning electron microscopy ( sem ) machine equipped with edax instrument . to know the size , shape , agglomeration pattern , and dispersed nature of the nanoparticles", "are done by atomic force microscopy ( afm ) by nova nt - mdt solver next , russia .", "transmission electron microscopy ( tem ) using hf-3300 advanced 300 kv tem / stem from hitachi .", "the antimicrobial activity of biologically synthesized nanoparticles are analyzed on seven pathogenic bacteria such as bacillus subtilis atcc 6633 , staphylococcus aureus atcc 6538 ( gram - positive ) , escherichia coli atcc 25922 , klebsiella pneumonia atcc 43816 , proteus vulgaris atcc 13315 , pseudomonas aeruginosa atcc 15442 , salmonella typhimurium atcc 14028", "( gram - negative ) , and five fungal pathogens such as alternaria solani atcc 32904 , aspergillus flavus atcc 9643 , aspergillus niger atcc 16404 , penicillium chrysogenum atcc 11709 , and trichoderma harzianum atcc 20476 procured from department of microbiology , sri venkateswara university , tirupati .", "disc diffusion method   was followed for testing antimicrobial activity against biologically synthesized agnps , and comparative studies were made with plant pulp extract , 1 mm agno3 as negative controls and streptomycin or fluconazole as a standard for bacteria and fungi , respectively .", "1 filter paper and 20 l of plant extract , 1 mm agno3 solutions , and 10 g / disc streptomycin / fluconazole are loaded on separate discs and allowed to air dry for 1 h at sterile conditions .", "apart from these , a 5 , 10 , 20 , 40 , 60 , 80 g / ml concentrations of synthesized agnps are tested separately to know the minimum inhibitory concentration .", "20 and 40 g / ml concentration of agnps show minimum inhibitory effect and 80 g / ml concentrations of agnps show maximum inhibitory concentration . due to this , we prefer 80 g / ml concentrations of agnps to check the antimicrobial activity on different microbial organisms .", "freshly prepared nutrient agar media for bacteria and potato dextrose agar media for fungi are poured into sterile petri plates , allowed to 30 min for solidification .", "the plates are swabbed with 100 l of microbial cultures and placed the previously prepared discs ; the experiment is carried out in triplicates .", "the plates are incubated at 37c for 24 - 48 h , and then the zone of inhibition is measured with the help of a scale and tabulated the results .", "2 to 4 kg weight mature fruits possess a great amount of pulp is collected from acharya ranga agricultural university , tirupati and cross checked by herbarium ( voucher no : svu362 ) deposited in department of botany , sri venkateswara university , tirupati .", "cut open the fruit and separate the seeds adhesive to pulp . grounded the collected pulp with the help of mortar and pestle , sieved the content for the synthesis of nanoparticles", "for synthesis , 25 g of fruit pulp is extracted with 100 ml of distilled water on boiling water bath for 30 min , filter the content with whatman no .", "1 filter paper and stored at room temperature . from this , 5 ml of the extract is taken into 250 ml of erlenmeyer conical flask and titrated against with the solution of 1 mm agno3 at a 60 - 80c temperature . the contents are cooled and centrifuged at 10,000 rpm for 20 min to avoid the presence of any biological impurities , and it is used for further characterization and antimicrobial studies .", "confirmation of synthesized nanoparticles is agnps by ultraviolet ( uv)-vis spectrophotometry absorption spectra using a spectro uv 2080 double beam , between the wavelength scan range of 190 - 700 nm , 1200 l / mm spectrophotometer , analytical technologies , india . to know the possible bio - molecules responsible for reduction and stabilization of agnps by fourier transform infra - red ( ftir ) spectra in the scan range of 4,000 to 500 cm transmittance with an alpha interferometer , bruker , ettlingen , karlsruhe , germany by kbr pellet method .", "x - ray diffraction of synthesized nanoparticles is examined by an x - ray diffractometer ( xrd ) ( shimadzu , xrd-6000 ) equipped with cu ka radiation source using ni as a filter at a setting of 30 kv/30 ma to know the crystalline nature of agnps .", "purity of agnps was analyzed by fei quanta 200 feg high resolution ( hr)-scanning electron microscopy ( sem ) machine equipped with edax instrument . to know the size , shape , agglomeration pattern , and dispersed nature of the nanoparticles", "are done by atomic force microscopy ( afm ) by nova nt - mdt solver next , russia .", "transmission electron microscopy ( tem ) using hf-3300 advanced 300 kv tem / stem from hitachi .", "the antimicrobial activity of biologically synthesized nanoparticles are analyzed on seven pathogenic bacteria such as bacillus subtilis atcc 6633 , staphylococcus aureus atcc 6538 ( gram - positive ) , escherichia coli atcc 25922 , klebsiella pneumonia atcc 43816 , proteus vulgaris atcc 13315 , pseudomonas aeruginosa atcc 15442 , salmonella typhimurium atcc 14028 ( gram - negative ) , and five fungal pathogens such as alternaria solani atcc 32904 ,", "aspergillus flavus atcc 9643 , aspergillus niger atcc 16404 , penicillium chrysogenum atcc 11709 , and trichoderma harzianum atcc 20476 procured from department of microbiology , sri venkateswara university , tirupati .", "disc diffusion method   was followed for testing antimicrobial activity against biologically synthesized agnps , and comparative studies were made with plant pulp extract , 1 mm agno3 as negative controls and streptomycin or fluconazole as a standard for bacteria and fungi , respectively .", "7 mm sterile discs are prepared from whatman no . 1 filter paper and 20 l of plant extract , 1 mm agno3 solutions , and 10 g / disc streptomycin / fluconazole are loaded on separate discs and allowed to air dry for 1 h at sterile conditions .", "apart from these , a 5 , 10 , 20 , 40 , 60 , 80 g / ml concentrations of synthesized agnps are tested separately to know the minimum inhibitory concentration .", "20 and 40 g / ml concentration of agnps show minimum inhibitory effect and 80 g / ml concentrations of agnps show maximum inhibitory concentration . due to this , we prefer 80 g / ml concentrations of agnps to check the antimicrobial activity on different microbial organisms . freshly prepared nutrient agar media for bacteria and potato dextrose agar media for fungi", "the plates are swabbed with 100 l of microbial cultures and placed the previously prepared discs ; the experiment is carried out in triplicates .", "the plates are incubated at 37c for 24 - 48 h , and then the zone of inhibition is measured with the help of a scale and tabulated the results .", "reduction of ag into ag nanoparticles was observed visually by means of a color change pattern of the reaction medium .", "a. digitata fruit pulp having thick cream color , upon synthesis the color change from creamy to light yellow indicates the formation of nanoparticles .", "this color changed nanoparticles solution was analyzed by uv - vis spectrophotometry shows a broad absorption peak at 434 nm further confirms the formation of nanoparticles are agnps [ figure 1 ] .", "ftir spectroscopic studies of these synthesized nanoparticles show broad transmittance peaks at 3322 cm assigned for an o - h bond of phenols and 1636 cm assigned for an n - h bond of primary amines [ figure 2 ] .", "xrd spectrum of synthesized agnps shows the crystalline nature of agnps and gives four intensive peaks at 38.1 , 46.2 , 64.5 , and 77.3 of 2 degrees of x - axis corresponds to 111 , 200 , 220 , and 311 bragg reflections of y - axis [ figure 3 ] .", "the mean particle diameter of synthesized agnps is 44 nm , calculated according to debye - scherrer equation ( d = k/ cos  ) and it coincides with powder diffraction file of joint committee on powder diffraction standards file no .", "the full width at half maximum values , i.e. k = 0.44 was derived from 38 , 46 , 64 , and 77 bragg reflections of the x - axis . 2 m resolution studies of biologically synthesized agnps with afm reveal the particles are polydispersed , spherical in shape , having the size range from 25 to 57 nm , and there is no agglomeration observed between the particles [ figure 4a ] .", "raw data obtained from this afm microscope is treated with a specially designed image processing software ( nova - tx ) to further exploit the three - dimensional ( 3d ) image of nanoparticles [ figure 4b ] .", "500 nm resolution studies with 20 kv electron energy passing through the nanoparticles coated thin films on a clean glass slide reveals the nanoparticles are spherical in shape , polydispersed and having the size range from 18 to 32 nm [ figure 5a ] . the same sample was analyzed with the help of edax instrument shows 33.28% presence of ag in the sample [ figure 5b ] along with 05.29% of carbon , 03.28% of nitrogen , 31.80% of oxygen , 08.37% of sodium , 02.57% of magnesium , 01.01% of aluminum , 00.78% of silicon , 07.87% of aurum , and 05.74% of calcium [ table 1 ] .", "crystalline nature of synthesized agnps was analyzed using selected area electron diffraction ( saed ) by directing the electron beam perpendicular to nanoparticles .", "the saed pattern of synthesized agnps shows the spots had been corresponding to 111 , 200 , 220 , and 311 of the crystallographic nature of face - centered cubic structures [ figure 6a ] . 20 nm resolution studies with 300 kv electron energy passing through nanoparticles coated thin films on copper grid show the nanoparticles are polydispersed , spherical in shape and size range from 3 to 7 nm [ figure 6b ] . to know the antimicrobial potency of biologically synthesized agnps from fruit pulp of a. digitata", "was analyzed against two gram - positive , five gram - negative , and five fungal pathogens by disc diffusion method . among the bacterial pathogens ,", "the highest inhibition zones were observed in p. vulgaris followed by k. pneumoniae , p. aeruginosa , s. typhimurium , e. coli , b. subtilis , and s. aureus . whereas in the case of fungi , the highest inhibition zones were observed in t. harzianum , followed by a. niger , a. flavus , p. chrysogenum , and a. solani .", "ultraviolet - vis spectrum of synthesized silver nanoparticles shows broad peak at 434 , 280 , and 247 nm of narrow peaks is due interference of phytoconstituents in the medium .", "inset figure shows color change pattern from cream to light yellow fourier transform infra - red spectrum of synthesized silver nanoparticles shows broad peaks at 3322 cm and 1636 cm x - ray diffractometer pattern of synthesized silver nanoparticles shows four intensive peaks atomic force microscopy micrograph of synthesized silver nanoparticles ( agnps ) , ( a ) 2 m resolution studies 25 - 57 nm size , spherical shaped , polydispersed particles , ( b ) three - dimensional image of agnps analyzed by nova - tx software scanning electron microscopy micrograph of synthesized silver nanoparticles ( agnps ) ( a ) 500 nm resolution studies shows 18 - 32 nm size , spherical shaped particles , ( b ) edax spectrum of synthesized agnps shows 33.28 weight percent of ag metal in the sample metal analysis by edax spectrum shows different weight percentages of sample transmission electron microscopy micrograph of synthesized silver nanoparticles ( agnps ) , ( a ) selected area electron diffraction pattern shows characteristic crystal spots of elemental silver , ( b ) 20 nm resolution studies of agnps shows 3 - 7 nm size , spherical shaped nanoparticles antimicrobial studies of biologically synthesized silver nanoparticles ( agnps ) from adansonia digitata pulp extract , ( a ) bacillus subtilis , ( b ) staphylococcus aureus , ( c ) escherichia coli , ( d ) klebsiella pneumoniae , ( e ) proteus vulgaris , ( f ) pseudomonas aeruginosa , ( g ) salmonella typhimurium , ( h ) alternaria solani , ( i ) aspergillus flavus , ( j ) aspergillus niger , ( k ) penicillium chrysogenum , ( l ) trichoderma harzianum , ( 1 ) plant extract , ( 2 ) agno3 , ( 3 ) agnps , ( 4 ) streptomycin / fluconazole zone of inhibition ( mm ) of agnps on microbial pathogens and comparative studies with different controls", "when the addition of agno3 solution to the plant extract the color of the plant extract is changed gradually according to the quantitative addition of agno3 solution .", "this color change is due to the reduction of nanoparticles with the help of electrons present in the fruit pulp extract coming from nad and ascorbic acid as electron donors present in the plant extracts .", "significantly higher amount of ascorbic acid , i.e. , > 300 mg/100 g was reported in fruit pulp of a. digitata .", "this solution was subjected to uv - vis spectrophotometry between the scan ranges of 190 - 750 nm , a broad peak is obtained at 434 nm . the same type of results was observed in leaf extract mediated synthesis of agnps from couroupita guianensis .", "the color change pattern and broad peak obtained in uv - vis spectrophotometry are due to surface plasmon resonance nature of agnps present in the medium .", "these nanoparticles absorb light at different wavelengths and excited due to charge density at the interface between conductor and insulator to give a respective peak on uv - vis spectrophotometry .", "ft - ir is a sensitive tool to analyze functional groups present in the biological samples .", "it relies on the light absorbance between 4000 cm to 500 cm of the electromagnetic , infrared region . in our case ,", "the same type of results was observed in myristica fragrans seed extract mediated synthesis of agnps . from this ftir study , clearly reveals hydroxyl groups of phenols and amide groups of proteins from plant extract forming a layer to the nanoparticles and acting as a capping agent to prevent agglomeration and providing stability in the medium .", "based on these ftir studies , we suggest that the bio - molecules present in plant extracts play dual role in formation and stabilization to agnps .", "afm is a primary tool for analyzing size , shape , agglomeration pattern and also offers visualizations of 3d views of the nanoparticles , unlike the electron microscopes .", "it has an advantage over combination of hr , samples do not have to be conductive and does not require the high - pressure vacuum conditions .", "better resolution and percentage presence of nanoparticles were demonstrated by sem with edax instrument provides reliable characterization and morphology of the particles when compare to afm .", "the four intensive bragg reflections are appear in xrd pattern [ figure 3 ] is correlated with saed pattern [ figure 6a ] of agnps , clearly indicates the nanoparticles are crystalline in nature .", "higher resolution studies with tem analysis shows size , shape , and agglomeration pattern of nanoparticles .", "it achieves better resolution than sem due to electron energies higher than 20 kv used in sem .", "finally , all the microscopic studies reveal the biologically synthesized agnps are spherical in shape , having the size range from 3 to 57 nm polydispersed and no agglomerations were found between the particles . to test the inhibitory effect of biologically synthesized nanoparticles on different microorganisms", "bacterial species show the highest inhibitory activity when compare to fungi because the cell walls of fungi are made up of chitin is more rigid when compare to bacterial cell walls containing peptidoglycans . among the bacteria ,", "gram - negative species show the highest inhibitory zones when to compare to gram - positive species , because the gram - negative species containing a less amount of peptidoglycans .", "silver is a precious metal used as an effective antimicrobial agent before the advent of agnps . due to overuse of silver products ,", "decreased the efficiency of silver agents as antibiotic . in recent times with the advancement of nanotechnology , the interest in the use of agnps as antibacterial agents had been rekindled .", "small sized nanoparticles have higher antimicrobial activity than larger particles because they have large surface area to interact bacteria efficiently . in recent times , the scientists produce 30 to 40 nm size , spherical shaped agnps from fruit extract of vitis vinifera shows excellent antimicrobial activity against b. subtilis and k. planticola . 10 to 70", "nm size , spherical shaped agnps synthesized from emblica officinalis fruit extract shows potential antimicrobial activity against s. aureus , b. subtilis , e. coli , and k. pneumoniae .", "euphorbia hirta leaf mediated synthesis of agnps having a spherical shape with 40 - 45 nm size shows good antifungal efficacy . in our studies also 3 - 57 nm size , spherical shaped agnps produced from a. digitata pulp extract confirmed by hr microscopic studies with afm , sem , and tem proved these agnps are eco - friendly antimicrobial agents against different microorganisms .", "in the present study , we report an eco - friendly , non - toxic , cost - effective method for synthesis of agnps from a. digitata pulp extract as reducing agent . in this method , naturally occurring materials are acts as reducing agents such as bio - molecules such as phenols and proteins present in plant extract as a simple and alternative to complex physical or chemical synthetic procedures", ". 3 to 57 nm size , spherical shaped , polydispersed nanoparticles are produced from a. digitata pulp extract confirmed by afm , sem , and tem prove this plant extract as an effective reducing agent for the production of narrow range of nanoparticles by industrial scale .", "further antimicrobial studies reveal that small size , spherical shaped particles have immense activity against different microbial pathogens and acts as eco - friendly antimicrobial agents ."], "abstract_text": ["<S> aim : in the present study , we report a cost - effective , eco - friendly , and an efficient alternative method for large scale production of silver nanoparticles ( agnps ) from adansonia digitata fruit pulp extract . </S>", "<S> the study mainly focused on the synthesis , characterization , and antimicrobial properties of agnps.materials and methods : synthesis of agnps with the help of standard protocol and characterized by ultraviolet ( uv)-vis spectrophotometry , fourier transform infra - red ( ftir ) , x - ray diffractometer ( xrd ) , atomic force microscopy ( afm ) , scanning electron microscopy ( sem ) with edax , transmission electron microscopy ( tem ) and explore their potential growth inhibitory effect on 07 bacterial and 05 fungal pathogens.results:the synthesized agnps are characterized by uv - vis spectrophotometry shows a broad peak at 434 nm . </S>", "<S> the ftir spectroscopic analysis clearly reveals phenols and proteins are main responsible for reduction and stabilization of nanoparticles . </S>", "<S> xrd studies show the nanoparticles are crystalline in nature owing 44 nm in size . </S>", "<S> edax spectrum shows a 33.28 weight percentage of ag metal in the reaction medium confirms the purity of agnps . </S>", "<S> high resolution and magnification studies with afm , sem , and tem reveal the nanoparticles are polydispersed , spherical in shape , having the size range from 3 to 57 nm without any agglomeration between the particles . </S>", "<S> further , the antimicrobial studies reveal the potentiality of nanoparticles against different microbial pathogens.conclusion:the present study is mainly focused on the synthesis of agnps from a. digitata fruit pulp extract . here , we succeed to synthesize a narrow range of particles and validate its potential antimicrobial activity on different microorganisms . based on this </S>", "<S> , we conclude that a. digitata pulp extract is a good source toward the reduction of agnps and acts as environment benign antimicrobial agents . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Collection of Plant Material", "Extract Preparation and Synthesis of AgNPs", "Characterization of AgNPs", "Antimicrobial Studies of AgNPs", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["synthesis of metal nanoparticles with the help of plant extracts is an emerging field of nanotechnology , due to their novel properties , terrific applications in biomedicine and its eco - friendly nature . in recent past", "great efforts were made for synthesis of environment benign and eco - friendly nanoparticle from plants such as iron oxide nanoparticles from medicago sativa , copper nanoparticles from magnolia kobus , calcium nanoparticles from boswellia ovalifoliolata , gold nanoparticles from avena sativa , zinc oxide nanoparticles from catharanthus roseus , and silver nanoparticles ( agnps ) from syzygium alternifolium .", "silver is one among the metal nanoparticles focused much interest due to its wide variety of applications .", "it has different biological activities such as antimicrobial , anthelmintic , antilarvicidic , antioxidant , anticancer , anti - inflammatory , hepatoprotective , and wound healing activity .", "conventional methods for synthesizing agnps are mainly by chemical , physical , and microbe - mediated synthesis . in these chemical and physical methods , usage of hazardous chemicals , high energy requirements ,", "difficult and wasteful materials generate potential and biological hazards to the environment . whereas in the case of microbe - mediated synthesis", ". therefore biological synthesis of agnps by using plant materials is easy , efficient , and eco - friendliness in comparison to chemical mediated or microbe - mediated synthesis   and they possess secondary metabolites having the redox capacity to reduce metal nanoparticles in an easiest way .", "adansonia digitata l. belongs to the family malvaceae is a large tree indigenous to africa and found in many countries also .", "traditionally , the fruit pulp dissolved in water or milk is used as a drink or sauce for food in africa .", "fruit pulp and powdered seeds are used for the treatment of diarrhea and dysentery in india .", "the high content of vitamin c in fruit pulp is recommended to pregnant women s for daily intake . in recent times , different scientists prove different activities of pulp such as hepatoprotective , antimicrobial , antiviral , antioxidant , antidiarrheal , hypoglycemic , anti - inflammatory , and antioxidant   activities . due to high medicinal values and mythological significance of this plant", "is known as  kalpavriksha  ( a tree which fulfill all desires ) in india . in our previous studies ,", "synthesis of agnps from stem bark and leaf extract of a. digitata acts as excellent reducing agents and show potential antimicrobial activity against different microorganisms .", "however , the potentiality of fruit pulp mediated synthesis of agnps is not carried out so far .", "hence , the present study is aimed to synthesize agnps from a. digitata fruit pulp extract , characterize and to know the potentiality of agnps against different microbial pathogens ."], ["2 to 4 kg weight mature fruits possess a great amount of pulp is collected from acharya ranga agricultural university , tirupati and cross checked by herbarium ( voucher no : svu362 ) deposited in department of botany , sri venkateswara university , tirupati .", "cut open the fruit and separate the seeds adhesive to pulp . grounded the collected pulp with the help of mortar and pestle , sieved the content for the synthesis of nanoparticles . for synthesis , 25 g of fruit pulp is extracted with 100 ml of distilled water on boiling water bath for 30 min , filter the content with whatman no .", "1 filter paper and stored at room temperature . from this , 5 ml of the extract is taken into 250 ml of erlenmeyer conical flask and titrated against with the solution of 1 mm agno3 at a 60 - 80c temperature . the contents are cooled and centrifuged at 10,000 rpm for 20 min to avoid the presence of any biological impurities , and it is used for further characterization and antimicrobial studies .", "confirmation of synthesized nanoparticles is agnps by ultraviolet ( uv)-vis spectrophotometry absorption spectra using a spectro uv 2080 double beam , between the wavelength scan range of 190 - 700 nm , 1200 l / mm spectrophotometer , analytical technologies , india . to know the possible bio - molecules responsible for reduction and stabilization of agnps by fourier transform infra - red ( ftir ) spectra in the scan range of 4,000 to 500 cm transmittance with an alpha interferometer , bruker , ettlingen , karlsruhe , germany by kbr pellet method .", "x - ray diffraction of synthesized nanoparticles is examined by an x - ray diffractometer ( xrd ) ( shimadzu , xrd-6000 ) equipped with cu ka radiation source using ni as a filter at a setting of 30 kv/30 ma to know the crystalline nature of agnps .", "purity of agnps was analyzed by fei quanta 200 feg high resolution ( hr)-scanning electron microscopy ( sem ) machine equipped with edax instrument . to know the size , shape , agglomeration pattern , and dispersed nature of the nanoparticles", "are done by atomic force microscopy ( afm ) by nova nt - mdt solver next , russia .", "transmission electron microscopy ( tem ) using hf-3300 advanced 300 kv tem / stem from hitachi .", "the antimicrobial activity of biologically synthesized nanoparticles are analyzed on seven pathogenic bacteria such as bacillus subtilis atcc 6633 , staphylococcus aureus atcc 6538 ( gram - positive ) , escherichia coli atcc 25922 , klebsiella pneumonia atcc 43816 , proteus vulgaris atcc 13315 , pseudomonas aeruginosa atcc 15442 , salmonella typhimurium atcc 14028", "( gram - negative ) , and five fungal pathogens such as alternaria solani atcc 32904 , aspergillus flavus atcc 9643 , aspergillus niger atcc 16404 , penicillium chrysogenum atcc 11709 , and trichoderma harzianum atcc 20476 procured from department of microbiology , sri venkateswara university , tirupati .", "disc diffusion method   was followed for testing antimicrobial activity against biologically synthesized agnps , and comparative studies were made with plant pulp extract , 1 mm agno3 as negative controls and streptomycin or fluconazole as a standard for bacteria and fungi , respectively .", "1 filter paper and 20 l of plant extract , 1 mm agno3 solutions , and 10 g / disc streptomycin / fluconazole are loaded on separate discs and allowed to air dry for 1 h at sterile conditions .", "apart from these , a 5 , 10 , 20 , 40 , 60 , 80 g / ml concentrations of synthesized agnps are tested separately to know the minimum inhibitory concentration .", "20 and 40 g / ml concentration of agnps show minimum inhibitory effect and 80 g / ml concentrations of agnps show maximum inhibitory concentration . due to this , we prefer 80 g / ml concentrations of agnps to check the antimicrobial activity on different microbial organisms .", "freshly prepared nutrient agar media for bacteria and potato dextrose agar media for fungi are poured into sterile petri plates , allowed to 30 min for solidification .", "the plates are swabbed with 100 l of microbial cultures and placed the previously prepared discs ; the experiment is carried out in triplicates .", "the plates are incubated at 37c for 24 - 48 h , and then the zone of inhibition is measured with the help of a scale and tabulated the results ."], ["2 to 4 kg weight mature fruits possess a great amount of pulp is collected from acharya ranga agricultural university , tirupati and cross checked by herbarium ( voucher no : svu362 ) deposited in department of botany , sri venkateswara university , tirupati .", "cut open the fruit and separate the seeds adhesive to pulp . grounded the collected pulp with the help of mortar and pestle , sieved the content for the synthesis of nanoparticles"], ["for synthesis , 25 g of fruit pulp is extracted with 100 ml of distilled water on boiling water bath for 30 min , filter the content with whatman no .", "1 filter paper and stored at room temperature . from this , 5 ml of the extract is taken into 250 ml of erlenmeyer conical flask and titrated against with the solution of 1 mm agno3 at a 60 - 80c temperature . the contents are cooled and centrifuged at 10,000 rpm for 20 min to avoid the presence of any biological impurities , and it is used for further characterization and antimicrobial studies ."], ["confirmation of synthesized nanoparticles is agnps by ultraviolet ( uv)-vis spectrophotometry absorption spectra using a spectro uv 2080 double beam , between the wavelength scan range of 190 - 700 nm , 1200 l / mm spectrophotometer , analytical technologies , india . to know the possible bio - molecules responsible for reduction and stabilization of agnps by fourier transform infra - red ( ftir ) spectra in the scan range of 4,000 to 500 cm transmittance with an alpha interferometer , bruker , ettlingen , karlsruhe , germany by kbr pellet method .", "x - ray diffraction of synthesized nanoparticles is examined by an x - ray diffractometer ( xrd ) ( shimadzu , xrd-6000 ) equipped with cu ka radiation source using ni as a filter at a setting of 30 kv/30 ma to know the crystalline nature of agnps .", "purity of agnps was analyzed by fei quanta 200 feg high resolution ( hr)-scanning electron microscopy ( sem ) machine equipped with edax instrument . to know the size , shape , agglomeration pattern , and dispersed nature of the nanoparticles", "are done by atomic force microscopy ( afm ) by nova nt - mdt solver next , russia .", "transmission electron microscopy ( tem ) using hf-3300 advanced 300 kv tem / stem from hitachi ."], ["the antimicrobial activity of biologically synthesized nanoparticles are analyzed on seven pathogenic bacteria such as bacillus subtilis atcc 6633 , staphylococcus aureus atcc 6538 ( gram - positive ) , escherichia coli atcc 25922 , klebsiella pneumonia atcc 43816 , proteus vulgaris atcc 13315 , pseudomonas aeruginosa atcc 15442 , salmonella typhimurium atcc 14028 ( gram - negative ) , and five fungal pathogens such as alternaria solani atcc 32904 ,", "aspergillus flavus atcc 9643 , aspergillus niger atcc 16404 , penicillium chrysogenum atcc 11709 , and trichoderma harzianum atcc 20476 procured from department of microbiology , sri venkateswara university , tirupati .", "disc diffusion method   was followed for testing antimicrobial activity against biologically synthesized agnps , and comparative studies were made with plant pulp extract , 1 mm agno3 as negative controls and streptomycin or fluconazole as a standard for bacteria and fungi , respectively .", "7 mm sterile discs are prepared from whatman no . 1 filter paper and 20 l of plant extract , 1 mm agno3 solutions , and 10 g / disc streptomycin / fluconazole are loaded on separate discs and allowed to air dry for 1 h at sterile conditions .", "apart from these , a 5 , 10 , 20 , 40 , 60 , 80 g / ml concentrations of synthesized agnps are tested separately to know the minimum inhibitory concentration .", "20 and 40 g / ml concentration of agnps show minimum inhibitory effect and 80 g / ml concentrations of agnps show maximum inhibitory concentration . due to this , we prefer 80 g / ml concentrations of agnps to check the antimicrobial activity on different microbial organisms . freshly prepared nutrient agar media for bacteria and potato dextrose agar media for fungi", "the plates are swabbed with 100 l of microbial cultures and placed the previously prepared discs ; the experiment is carried out in triplicates .", "the plates are incubated at 37c for 24 - 48 h , and then the zone of inhibition is measured with the help of a scale and tabulated the results ."], ["reduction of ag into ag nanoparticles was observed visually by means of a color change pattern of the reaction medium .", "a. digitata fruit pulp having thick cream color , upon synthesis the color change from creamy to light yellow indicates the formation of nanoparticles .", "this color changed nanoparticles solution was analyzed by uv - vis spectrophotometry shows a broad absorption peak at 434 nm further confirms the formation of nanoparticles are agnps [ figure 1 ] .", "ftir spectroscopic studies of these synthesized nanoparticles show broad transmittance peaks at 3322 cm assigned for an o - h bond of phenols and 1636 cm assigned for an n - h bond of primary amines [ figure 2 ] .", "xrd spectrum of synthesized agnps shows the crystalline nature of agnps and gives four intensive peaks at 38.1 , 46.2 , 64.5 , and 77.3 of 2 degrees of x - axis corresponds to 111 , 200 , 220 , and 311 bragg reflections of y - axis [ figure 3 ] .", "the mean particle diameter of synthesized agnps is 44 nm , calculated according to debye - scherrer equation ( d = k/ cos  ) and it coincides with powder diffraction file of joint committee on powder diffraction standards file no .", "the full width at half maximum values , i.e. k = 0.44 was derived from 38 , 46 , 64 , and 77 bragg reflections of the x - axis . 2 m resolution studies of biologically synthesized agnps with afm reveal the particles are polydispersed , spherical in shape , having the size range from 25 to 57 nm , and there is no agglomeration observed between the particles [ figure 4a ] .", "raw data obtained from this afm microscope is treated with a specially designed image processing software ( nova - tx ) to further exploit the three - dimensional ( 3d ) image of nanoparticles [ figure 4b ] .", "500 nm resolution studies with 20 kv electron energy passing through the nanoparticles coated thin films on a clean glass slide reveals the nanoparticles are spherical in shape , polydispersed and having the size range from 18 to 32 nm [ figure 5a ] . the same sample was analyzed with the help of edax instrument shows 33.28% presence of ag in the sample [ figure 5b ] along with 05.29% of carbon , 03.28% of nitrogen , 31.80% of oxygen , 08.37% of sodium , 02.57% of magnesium , 01.01% of aluminum , 00.78% of silicon , 07.87% of aurum , and 05.74% of calcium [ table 1 ] .", "crystalline nature of synthesized agnps was analyzed using selected area electron diffraction ( saed ) by directing the electron beam perpendicular to nanoparticles .", "the saed pattern of synthesized agnps shows the spots had been corresponding to 111 , 200 , 220 , and 311 of the crystallographic nature of face - centered cubic structures [ figure 6a ] . 20 nm resolution studies with 300 kv electron energy passing through nanoparticles coated thin films on copper grid show the nanoparticles are polydispersed , spherical in shape and size range from 3 to 7 nm [ figure 6b ] . to know the antimicrobial potency of biologically synthesized agnps from fruit pulp of a. digitata", "was analyzed against two gram - positive , five gram - negative , and five fungal pathogens by disc diffusion method . among the bacterial pathogens ,", "the highest inhibition zones were observed in p. vulgaris followed by k. pneumoniae , p. aeruginosa , s. typhimurium , e. coli , b. subtilis , and s. aureus . whereas in the case of fungi , the highest inhibition zones were observed in t. harzianum , followed by a. niger , a. flavus , p. chrysogenum , and a. solani .", "ultraviolet - vis spectrum of synthesized silver nanoparticles shows broad peak at 434 , 280 , and 247 nm of narrow peaks is due interference of phytoconstituents in the medium .", "inset figure shows color change pattern from cream to light yellow fourier transform infra - red spectrum of synthesized silver nanoparticles shows broad peaks at 3322 cm and 1636 cm x - ray diffractometer pattern of synthesized silver nanoparticles shows four intensive peaks atomic force microscopy micrograph of synthesized silver nanoparticles ( agnps ) , ( a ) 2 m resolution studies 25 - 57 nm size , spherical shaped , polydispersed particles , ( b ) three - dimensional image of agnps analyzed by nova - tx software scanning electron microscopy micrograph of synthesized silver nanoparticles ( agnps ) ( a ) 500 nm resolution studies shows 18 - 32 nm size , spherical shaped particles , ( b ) edax spectrum of synthesized agnps shows 33.28 weight percent of ag metal in the sample metal analysis by edax spectrum shows different weight percentages of sample transmission electron microscopy micrograph of synthesized silver nanoparticles ( agnps ) , ( a ) selected area electron diffraction pattern shows characteristic crystal spots of elemental silver , ( b ) 20 nm resolution studies of agnps shows 3 - 7 nm size , spherical shaped nanoparticles antimicrobial studies of biologically synthesized silver nanoparticles ( agnps ) from adansonia digitata pulp extract , ( a ) bacillus subtilis , ( b ) staphylococcus aureus , ( c ) escherichia coli , ( d ) klebsiella pneumoniae , ( e ) proteus vulgaris , ( f ) pseudomonas aeruginosa , ( g ) salmonella typhimurium , ( h ) alternaria solani , ( i ) aspergillus flavus , ( j ) aspergillus niger , ( k ) penicillium chrysogenum , ( l ) trichoderma harzianum , ( 1 ) plant extract , ( 2 ) agno3 , ( 3 ) agnps , ( 4 ) streptomycin / fluconazole zone of inhibition ( mm ) of agnps on microbial pathogens and comparative studies with different controls"], ["when the addition of agno3 solution to the plant extract the color of the plant extract is changed gradually according to the quantitative addition of agno3 solution .", "this color change is due to the reduction of nanoparticles with the help of electrons present in the fruit pulp extract coming from nad and ascorbic acid as electron donors present in the plant extracts .", "significantly higher amount of ascorbic acid , i.e. , > 300 mg/100 g was reported in fruit pulp of a. digitata .", "this solution was subjected to uv - vis spectrophotometry between the scan ranges of 190 - 750 nm , a broad peak is obtained at 434 nm . the same type of results was observed in leaf extract mediated synthesis of agnps from couroupita guianensis .", "the color change pattern and broad peak obtained in uv - vis spectrophotometry are due to surface plasmon resonance nature of agnps present in the medium .", "these nanoparticles absorb light at different wavelengths and excited due to charge density at the interface between conductor and insulator to give a respective peak on uv - vis spectrophotometry .", "ft - ir is a sensitive tool to analyze functional groups present in the biological samples .", "it relies on the light absorbance between 4000 cm to 500 cm of the electromagnetic , infrared region . in our case ,", "the same type of results was observed in myristica fragrans seed extract mediated synthesis of agnps . from this ftir study , clearly reveals hydroxyl groups of phenols and amide groups of proteins from plant extract forming a layer to the nanoparticles and acting as a capping agent to prevent agglomeration and providing stability in the medium .", "based on these ftir studies , we suggest that the bio - molecules present in plant extracts play dual role in formation and stabilization to agnps .", "afm is a primary tool for analyzing size , shape , agglomeration pattern and also offers visualizations of 3d views of the nanoparticles , unlike the electron microscopes .", "it has an advantage over combination of hr , samples do not have to be conductive and does not require the high - pressure vacuum conditions .", "better resolution and percentage presence of nanoparticles were demonstrated by sem with edax instrument provides reliable characterization and morphology of the particles when compare to afm .", "the four intensive bragg reflections are appear in xrd pattern [ figure 3 ] is correlated with saed pattern [ figure 6a ] of agnps , clearly indicates the nanoparticles are crystalline in nature .", "higher resolution studies with tem analysis shows size , shape , and agglomeration pattern of nanoparticles .", "it achieves better resolution than sem due to electron energies higher than 20 kv used in sem .", "finally , all the microscopic studies reveal the biologically synthesized agnps are spherical in shape , having the size range from 3 to 57 nm polydispersed and no agglomerations were found between the particles . to test the inhibitory effect of biologically synthesized nanoparticles on different microorganisms", "bacterial species show the highest inhibitory activity when compare to fungi because the cell walls of fungi are made up of chitin is more rigid when compare to bacterial cell walls containing peptidoglycans . among the bacteria ,", "gram - negative species show the highest inhibitory zones when to compare to gram - positive species , because the gram - negative species containing a less amount of peptidoglycans .", "silver is a precious metal used as an effective antimicrobial agent before the advent of agnps . due to overuse of silver products ,", "decreased the efficiency of silver agents as antibiotic . in recent times with the advancement of nanotechnology , the interest in the use of agnps as antibacterial agents had been rekindled .", "small sized nanoparticles have higher antimicrobial activity than larger particles because they have large surface area to interact bacteria efficiently . in recent times , the scientists produce 30 to 40 nm size , spherical shaped agnps from fruit extract of vitis vinifera shows excellent antimicrobial activity against b. subtilis and k. planticola . 10 to 70", "nm size , spherical shaped agnps synthesized from emblica officinalis fruit extract shows potential antimicrobial activity against s. aureus , b. subtilis , e. coli , and k. pneumoniae .", "euphorbia hirta leaf mediated synthesis of agnps having a spherical shape with 40 - 45 nm size shows good antifungal efficacy . in our studies also 3 - 57 nm size , spherical shaped agnps produced from a. digitata pulp extract confirmed by hr microscopic studies with afm , sem , and tem proved these agnps are eco - friendly antimicrobial agents against different microorganisms ."], ["in the present study , we report an eco - friendly , non - toxic , cost - effective method for synthesis of agnps from a. digitata pulp extract as reducing agent . in this method , naturally occurring materials are acts as reducing agents such as bio - molecules such as phenols and proteins present in plant extract as a simple and alternative to complex physical or chemical synthetic procedures", ". 3 to 57 nm size , spherical shaped , polydispersed nanoparticles are produced from a. digitata pulp extract confirmed by afm , sem , and tem prove this plant extract as an effective reducing agent for the production of narrow range of nanoparticles by industrial scale .", "further antimicrobial studies reveal that small size , spherical shaped particles have immense activity against different microbial pathogens and acts as eco - friendly antimicrobial agents ."]]}
{"article_id": "PMC3383148", "article_text": ["malakoplakia is a rare chronic inflammatory disease that occurs most commonly in the genitourinary tract , especially the urinary bladder .", "most patients have associated conditions characterized by some degree of immunosuppression , as seen in solid - organ transplants , autoimmune diseases requiring steroid use , chemotherapy , chronic systemic diseases , alcohol abuse and poorly controlled diabetes.1 ) despite some characteristic histological features including the presence of large histiocytes with clusters of michaelis - gutmann ( mg ) bodies , malakoplakia of the kidney is difficult to diagnose because of its rarity and varying histological pictures.2 ) to our knowledge , there have been no case reports of malakoplakia of the kidney in a secondary adrenal insufficiency from korea . here", ", we report an unusual case of renal malakoplakia involving the perirenal space and extending to the descending colon in a 65-year - old korean woman with secondary adrenal insufficiency and diabetes mellitus .", "in march 2010 , a 65-year - old woman was transferred from the national police hospital ( nph ) to our facility , shinchon severance hospital ( seoul , korea ) via the emergency room ( er ) , presenting with a decreased level of consciousness . according to her medical history", ", she had hypertension , diabetes mellitus for 10 years , recurrent cystitis , and degenerative arthritis .", "she had been taking dexamethasone ( 5 mg , once a day ) for the degenerative arthritis for approximately five years up to the time of admission .", "three weeks before the admission , she had been hospitalized at nph due to drowsy mentality for 3 or 4 hours . on the day of admission at nph , she presented with a fever ( 38.8 ) and dyspnea .", "the patient was given antibiotics ( ceftriaxone and azithromycin ) for 14 days , on suspicion of community - acquired pneumonia .", "thereafter , her presenting symptoms were improved , but she complained of consistent pain in the left lower quadrant of the abdomen .", "abdomen - pelvis computed tomography ( ct ) scan was performed , which showed the mass - like lesion in the left renal area suspecting abscess . while being treated with ciprofloxacin , she developed a decreased level of consciousness . upon physical examination on the day of the admission to our hospital ,", "her initial vital signs were as follows : blood pressure 134/71 mm hg , pulse rate 95 bits per minute , body temperature 36.4 , and respiration rate 18 breaths per minute .", "her breathing sound was clear , and her heart sound was normal without any murmurs .", "laboratory studies had the following results ( reference values ) : low sodium ( na ) levels of 112 mmol / l ( 135.0 - 145.0 ) , potassium ( k ) 3.4 mmol / l ( 3.5 - 5.5 ) , chloride ( cl ) 77.0 mmol / l ( 98 - 110 ) , total co2 21.0", "mmol / l ( 24 - 30 ) , low serum osmolality 240.0 mosm ( 289.0 - 308.0 ) , and normal urine osmolality 315.0 ( 50.0 - 1200 ) .", "initial urinalysis results showed 3 - 5 white blood cells / high - power field and yeast - like organisms . in the radiological examination", ", there was no acute ischemic lesion in the brain magnetic re sonance imaging and angiography with neck angiography ( 3d ) , and no specific finding on the chest x - ray .", "sodium replacement therapy for hypotonic euvolemic hyponatremia had been started from the day of admission . at that stage ,", "secondary adrenal insufficiency due to exogenous steroid was suspected . a low dose adrenocorticotropic hormone ( 1 g cosynthropin ) stimulation test was conducted to diagnose the secondary adrenal insufficiency .", "the stimulation test showed that there was adrenal insufficiency ; basal level of cortisol 8.56 g / dl , peak level during the hour - long test 13.94 g / dl ( table 2 ) .", "steroid hormone ( hydrocortisone sodium succinate ; solucortef ) was replaced . on the third day after admission , her general condition including clear consciousness was improved , with a normal level of serum sodium ( 136 mmol / l ) , and her steroid hormone dosage had been gradually tapered down and was maintained at 7.5 mg ( 5 mg , 2.5 mg two times a day ) of prednisolone acetate .", "however , the patient complained of myalgia , which was accompanied with leukocytosis ( white blood cell count 13,650 g / l [ neutrophil 84.9 % ] ) .", "radiological reading of the abdomen - pelvic ct conducted on the second day after admission showed that there was an ill - defined inflammatory lesion with multifocal abscess pockets in the left perirenal space , which was suspected of actinomycosis of the descending colon , extended to the left kidney ( figure 1a ) . compared to the abdomen - pelvic ct finding performed outside , the lesion seemed to have decreased slightly in size .", "urinalysis was repeated and showed many white blood cells per high - power field and yeast - like organisms , which was confirmed as yeast in the urine culture .", "considering the laboratory and radiologic findings , intravenous antibiotic treatment was started ( ubacillin 1,500 mg a day ) for suspected actinomycosis .", "no specific lesion was found at the colonoscopy performed to confirm the diagnosis of actinomycosis . on the 12th day after admission", "the histology of the lesion showed a collection of foamy histiocytes also known as von hansemann cells with von - kossa stain positive spheres ( michelis - gutmann body ) ( figure 2 ) .", "the patient presented dysuria and urgency , and antibiotic treatment was changed to oral ciprofloxacin ( 500 mg a day ) .", "on the 15th day after admission , she was discharged , as her presenting symptoms and laboratory findings were improved .", "about 4 months after discharge , the patient was readmitted presenting with general weakness for one month .", "the findings of the abdomen - pelvic ct conducted upon readmission ( figure 1b ) showed that the extent of the malakoplakia in the perirenal space had decreased since 9 week prior , as she had been taking the oral antibiotics .", "she has had follow - up in our outpatient clinic ever since , taking oral trimethoprim - sulfamethoxazole .", "malakoplakia is an uncommon but distinctive type of chronic granulomatous inflammation . upon microscopic examination ,", "malakoplakia is characterized by the presence of sheets of histiocytes with granular cytoplasm ( von hansemann cells ) and the formation of intracellular bacterial inclusions called michaelis - gutmann bodies.3 - 7 ) the pathogenesis is poorly understood , but it is thought to result from acquired bactericidal defects in macrophages occurring mostly in patients with chronic debility or immunosuppression , a kidney transplant requiring steroids , or azathioprine.8,9 ) these observations suggest that an altered immune response may play a role in the pathogenesis of the disease .", "individuals with diabetes mellitus have a greater frequency and severity of infection . from the earliest reports on studies of adrenocortical function", ", there has been impressive evidence that adrenocortical hormone , and more specifically cortisol , is essential for normal immunity .", "otherwise , prednisone , triamcinolone , dexamethasone and similar commercially popular steroids have the dangerous side - effect of lowering resistance to infection , which is common to all known glucocorticoids.10 ) according to previous studies , gram - negative bacteria , particularly escherichia coli , have been isolated from involved sites of genitourinary malakoplakia in more than two - thirds of patients.11 ) bacteria other than yeast were not isolated in the urine culture in the present case with cystitis because the patient was on antibiotic therapy before admission to our hospital .", "although we did not carry out a detailed investigation of the patient 's immunity , we believe that unilateral renal malakoplakia extending to the perirenal space and the descending colon , as observed in the present case , may result from an interaction between various factors such as bacterial infection , impaired host immune response and diminished leukocyte function under the influence of a systemic illness such as secondary adrenal insufficiency and diabetes mellitus .", "the common presenting features of malakoplakia of the kidney are high fever , loin tenderness , and a palpable mass with a history of urinary tract infection.8 ) based on the reported studies , the degree of decreased kidney function varies up to acute renal failure.12,13 ) ultrasound and ct features are variable and only briefly mentioned in many reviews .", "features include enlarged kidneys,14 ) focal hypoechoic renal masses that simulate abscesses15 ) and heterogeneous masses that calcify , show central areas of necrosis and are mistaken for tumors.16 ) the imaging appearance of malakoplakia is therefore non - specific , and malignant renal tumor is a common misdiagnosis .", "the final diagnosis of malakoplakia is usually attained only upon histopathologic examination of the excised specimen .", "there is no established treatment for patients with malakoplakia . however , there are treatment options for malakoplakia that include antibiotics , surgical excision , or a combination of both , depending on the site and extent of involvement .", "some researchers have suggested that such antibiotics as trimethoprim - sulfamethoxazole and ciprofloxacin were beneficial because of their ability to penetrate intracellularly.8,17 - 20 ) we started the treatment with ciprofloxacin . during follow - up in the outpatient clinic , we changed the antibiotic treatment to trimethoprim - sulfamethoxazole due to the patient 's continued complaint of cystitis symptoms . in conclusion", ", renal malakoplakia should be taken into account in the differential diagnosis of a patient who presents with urinary tract infection , fever , abdominal pain , and abscess - like lesion in ultrasonography or ct , especially if the patient concerned has diabetes mellitus and a long history of use of exogenous steroids .", "it is considered that family physicians ' early diagnosis would enable prompt treatment and regular monitoring , leading to avoidance of long - term morbidity and mortality ."], "abstract_text": ["<S> malakoplakia is an uncommon but distinctive type of chronic granulomatous inflammation that occurs most commonly in the genitourinary tract , especially the urinary bladder . </S>", "<S> most patients have associated conditions characterized by some degree of immunosuppression , as seen in solid - organ transplants , autoimmune diseases requiring steroid use , chemotherapy , chronic systemic diseases , alcohol abuse and poorly controlled diabetes . </S>", "<S> we report an unusual case of the renal malakoplakia that involved the perirenal space , extending to the descending colon in a 65-year - old korean woman with secondary adrenal insufficiency and diabetes mellitus . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["malakoplakia is a rare chronic inflammatory disease that occurs most commonly in the genitourinary tract , especially the urinary bladder .", "most patients have associated conditions characterized by some degree of immunosuppression , as seen in solid - organ transplants , autoimmune diseases requiring steroid use , chemotherapy , chronic systemic diseases , alcohol abuse and poorly controlled diabetes.1 ) despite some characteristic histological features including the presence of large histiocytes with clusters of michaelis - gutmann ( mg ) bodies , malakoplakia of the kidney is difficult to diagnose because of its rarity and varying histological pictures.2 ) to our knowledge , there have been no case reports of malakoplakia of the kidney in a secondary adrenal insufficiency from korea . here", ", we report an unusual case of renal malakoplakia involving the perirenal space and extending to the descending colon in a 65-year - old korean woman with secondary adrenal insufficiency and diabetes mellitus ."], ["in march 2010 , a 65-year - old woman was transferred from the national police hospital ( nph ) to our facility , shinchon severance hospital ( seoul , korea ) via the emergency room ( er ) , presenting with a decreased level of consciousness . according to her medical history", ", she had hypertension , diabetes mellitus for 10 years , recurrent cystitis , and degenerative arthritis .", "she had been taking dexamethasone ( 5 mg , once a day ) for the degenerative arthritis for approximately five years up to the time of admission .", "three weeks before the admission , she had been hospitalized at nph due to drowsy mentality for 3 or 4 hours . on the day of admission at nph , she presented with a fever ( 38.8 ) and dyspnea .", "the patient was given antibiotics ( ceftriaxone and azithromycin ) for 14 days , on suspicion of community - acquired pneumonia .", "thereafter , her presenting symptoms were improved , but she complained of consistent pain in the left lower quadrant of the abdomen .", "abdomen - pelvis computed tomography ( ct ) scan was performed , which showed the mass - like lesion in the left renal area suspecting abscess . while being treated with ciprofloxacin , she developed a decreased level of consciousness . upon physical examination on the day of the admission to our hospital ,", "her initial vital signs were as follows : blood pressure 134/71 mm hg , pulse rate 95 bits per minute , body temperature 36.4 , and respiration rate 18 breaths per minute .", "her breathing sound was clear , and her heart sound was normal without any murmurs .", "laboratory studies had the following results ( reference values ) : low sodium ( na ) levels of 112 mmol / l ( 135.0 - 145.0 ) , potassium ( k ) 3.4 mmol / l ( 3.5 - 5.5 ) , chloride ( cl ) 77.0 mmol / l ( 98 - 110 ) , total co2 21.0", "mmol / l ( 24 - 30 ) , low serum osmolality 240.0 mosm ( 289.0 - 308.0 ) , and normal urine osmolality 315.0 ( 50.0 - 1200 ) .", "initial urinalysis results showed 3 - 5 white blood cells / high - power field and yeast - like organisms . in the radiological examination", ", there was no acute ischemic lesion in the brain magnetic re sonance imaging and angiography with neck angiography ( 3d ) , and no specific finding on the chest x - ray .", "sodium replacement therapy for hypotonic euvolemic hyponatremia had been started from the day of admission . at that stage ,", "secondary adrenal insufficiency due to exogenous steroid was suspected . a low dose adrenocorticotropic hormone ( 1 g cosynthropin ) stimulation test was conducted to diagnose the secondary adrenal insufficiency .", "the stimulation test showed that there was adrenal insufficiency ; basal level of cortisol 8.56 g / dl , peak level during the hour - long test 13.94 g / dl ( table 2 ) .", "steroid hormone ( hydrocortisone sodium succinate ; solucortef ) was replaced . on the third day after admission , her general condition including clear consciousness was improved , with a normal level of serum sodium ( 136 mmol / l ) , and her steroid hormone dosage had been gradually tapered down and was maintained at 7.5 mg ( 5 mg , 2.5 mg two times a day ) of prednisolone acetate .", "however , the patient complained of myalgia , which was accompanied with leukocytosis ( white blood cell count 13,650 g / l [ neutrophil 84.9 % ] ) .", "radiological reading of the abdomen - pelvic ct conducted on the second day after admission showed that there was an ill - defined inflammatory lesion with multifocal abscess pockets in the left perirenal space , which was suspected of actinomycosis of the descending colon , extended to the left kidney ( figure 1a ) . compared to the abdomen - pelvic ct finding performed outside , the lesion seemed to have decreased slightly in size .", "urinalysis was repeated and showed many white blood cells per high - power field and yeast - like organisms , which was confirmed as yeast in the urine culture .", "considering the laboratory and radiologic findings , intravenous antibiotic treatment was started ( ubacillin 1,500 mg a day ) for suspected actinomycosis .", "no specific lesion was found at the colonoscopy performed to confirm the diagnosis of actinomycosis . on the 12th day after admission", "the histology of the lesion showed a collection of foamy histiocytes also known as von hansemann cells with von - kossa stain positive spheres ( michelis - gutmann body ) ( figure 2 ) .", "the patient presented dysuria and urgency , and antibiotic treatment was changed to oral ciprofloxacin ( 500 mg a day ) .", "on the 15th day after admission , she was discharged , as her presenting symptoms and laboratory findings were improved .", "about 4 months after discharge , the patient was readmitted presenting with general weakness for one month .", "the findings of the abdomen - pelvic ct conducted upon readmission ( figure 1b ) showed that the extent of the malakoplakia in the perirenal space had decreased since 9 week prior , as she had been taking the oral antibiotics .", "she has had follow - up in our outpatient clinic ever since , taking oral trimethoprim - sulfamethoxazole ."], ["malakoplakia is an uncommon but distinctive type of chronic granulomatous inflammation . upon microscopic examination ,", "malakoplakia is characterized by the presence of sheets of histiocytes with granular cytoplasm ( von hansemann cells ) and the formation of intracellular bacterial inclusions called michaelis - gutmann bodies.3 - 7 ) the pathogenesis is poorly understood , but it is thought to result from acquired bactericidal defects in macrophages occurring mostly in patients with chronic debility or immunosuppression , a kidney transplant requiring steroids , or azathioprine.8,9 ) these observations suggest that an altered immune response may play a role in the pathogenesis of the disease .", "individuals with diabetes mellitus have a greater frequency and severity of infection . from the earliest reports on studies of adrenocortical function", ", there has been impressive evidence that adrenocortical hormone , and more specifically cortisol , is essential for normal immunity .", "otherwise , prednisone , triamcinolone , dexamethasone and similar commercially popular steroids have the dangerous side - effect of lowering resistance to infection , which is common to all known glucocorticoids.10 ) according to previous studies , gram - negative bacteria , particularly escherichia coli , have been isolated from involved sites of genitourinary malakoplakia in more than two - thirds of patients.11 ) bacteria other than yeast were not isolated in the urine culture in the present case with cystitis because the patient was on antibiotic therapy before admission to our hospital .", "although we did not carry out a detailed investigation of the patient 's immunity , we believe that unilateral renal malakoplakia extending to the perirenal space and the descending colon , as observed in the present case , may result from an interaction between various factors such as bacterial infection , impaired host immune response and diminished leukocyte function under the influence of a systemic illness such as secondary adrenal insufficiency and diabetes mellitus .", "the common presenting features of malakoplakia of the kidney are high fever , loin tenderness , and a palpable mass with a history of urinary tract infection.8 ) based on the reported studies , the degree of decreased kidney function varies up to acute renal failure.12,13 ) ultrasound and ct features are variable and only briefly mentioned in many reviews .", "features include enlarged kidneys,14 ) focal hypoechoic renal masses that simulate abscesses15 ) and heterogeneous masses that calcify , show central areas of necrosis and are mistaken for tumors.16 ) the imaging appearance of malakoplakia is therefore non - specific , and malignant renal tumor is a common misdiagnosis .", "the final diagnosis of malakoplakia is usually attained only upon histopathologic examination of the excised specimen .", "there is no established treatment for patients with malakoplakia . however , there are treatment options for malakoplakia that include antibiotics , surgical excision , or a combination of both , depending on the site and extent of involvement .", "some researchers have suggested that such antibiotics as trimethoprim - sulfamethoxazole and ciprofloxacin were beneficial because of their ability to penetrate intracellularly.8,17 - 20 ) we started the treatment with ciprofloxacin . during follow - up in the outpatient clinic , we changed the antibiotic treatment to trimethoprim - sulfamethoxazole due to the patient 's continued complaint of cystitis symptoms . in conclusion", ", renal malakoplakia should be taken into account in the differential diagnosis of a patient who presents with urinary tract infection , fever , abdominal pain , and abscess - like lesion in ultrasonography or ct , especially if the patient concerned has diabetes mellitus and a long history of use of exogenous steroids .", "it is considered that family physicians ' early diagnosis would enable prompt treatment and regular monitoring , leading to avoidance of long - term morbidity and mortality ."]]}
{"article_id": "PMC2220042", "article_text": ["ghrelin , a 28-amino - acide peptide , is   a potent stimulator of growth hormone release that \n has been implicated in the control of food intake and energy homeostasis in \n human begins and rodents \n [ 15 ] .", "ghrelin is not secreted into the gastrointestinal tract like digestive enzymes but into blood vessels to circulate throughout the body .", "ghrelin causes weight gain by increasing food intake and reducing fat use [ 7 , 8 ] .", "ghrelin has effects on nutrient intake and growth hormone ( gh ) release , subsequently on physical development and growth .", "tumor necrosis factor  ( tnf- ) and interleukin-6 ( il-6 ) are pleiotropic cytokines with numerous immunologic and metabolic actions [ 10 , 11 ] .", "il-6 is generally considered to be an important cytokine in the network of cytokines that regulate immune reactions and acute phase responses .", "the relationship between congenital heart disease ( chd ) , malnutrition , and growth \n retardation is well documented .", "infants with congenital heart disease are prone to malnutrition for several reasons including decreased energy intake , \n increased energy requirements , or both . different types of cardiac malformations can affect nutrition and growth to varying degrees .", "although \n nutritional and growth status were investigated in children with cyanotic and acyanotic heart disease , serum ghrelin levels have not been established .", "the objective of this study is to investigate and compare the functional role of ghrelin on the regulation of energy balance in children with cyanotic and acyanotic congenital heart disease and the association of ghrelin with tnf- , il-6 , that were not entirely confirmed in literature by now .", "the study was conducted on 47 children with acyanotic chd , 21 children \n with cyanotic chd , and 25 healthy children .", "all patients ' cardiac diagnoses were \n made on the basis of clinical and laboratory examinations .", "body mass index ( bmi ) \n was calculated as the ratio of body weight ( kg ) and squared height ( m ) .", "all blood samples were drawn at 08 - 09 am and stored 20c \n until the procedure .", "serum ghrelin , tnf- , and il-6 levels were analyzed with \n elisa kits ( tnf- , il-6 kit was purchased from bio - source   international inc .", "( camarillo , calif , usa ) ; ghrelin kit from phoenix international , inc , usa ) .", "the given data were compared between groups using one - way anova , followed by post - hoc ; bonferroni test .", "in 47 acyanotic patients , mean age was 30.518.4 months , in 21 cyanotic patients was 28.415.6 months and in 25 control subjects was 31.115.1 months .", "age and anthropometric data of the patients and the control subjects are shown in table 1 .", "there was no significant difference between groups ( the acyanotic patients , the cyanotic patients ) in terms of mean age , weight , height , bmi .", "serum ghrelin levels were significantly higher than in acyanotic and cyanotic groups compared to in the control group   ( p<.0001 ) ( table 3 ) . \n", "serum ghrelin levels in the acyanotic patients were significantly higher than in the cyanotic patients \n ( p<.0001 ) . \n", "tnf- \n levels were significantly higher \n than in cyanotic and acyanotic patients with chd compared to in the control groups   ( p<.001 , p<.0001 , resp . ) .", "serum tnf- values were higher in the acyanotic patients compared to the cyanotic patients with chd ( p<.001 ) .", "serum il-6 levels were higher than in cyanotic and acyanotic patients with chd compared to in the control groups   ( p<.0001 , p<.001 , resp . ) . in both acyanotic and cyanotic groups ,", "serum ghrelin levels were negatively \n correlated with bmi ( r=.549 , p<.05 and r=.688 , p<.01 , resp . ) \n\t\t\t\t", "tnf- levels were not related \n to bmi in the acyanotic and cyanotic patients with chd .", "ghrelin levels were also correlated with tnf- in the acyanotic and cyanotic groups ( r=.485 , p<.05 and r=.573 , p<.01 , resp . )", "ghrelin levels were not related to il-6 in the acyanotic and cyanotic patients with chd ( r=.263 , p>.05 and r=.398 , p>.05 , resp . ) .", "inadequate caloric intake , \n malabsorption , and increased energy requirements caused by increased metabolism \n may all contribute .", "however , inadequate caloric intake appears to be the most \n important cause of growth failure in chd [ 13 , 15 , 16 ] .", "patients with acyanotic \n heart disease had a greater growth deficit in weight , and those with cyanotic \n heart disease had a greater growth deficit in stature as demonstrated by both \n decreased height and weight .", "although growth impairment is most pronounced in \n infants with cyanotic chd , growth failure does not correlate well with the \n degree of hypoxia . in this study ,", "the cyanotic patients had a more pronounced \n retardation in both height and weight than in the acyanotic patients [ 13 , 17 ] .", "ghrelin is   accepted as a good marker of the nutritional state , mainly in situations of \n malnutrition , like anorexia nervosa , owing its fast recovery after weight gain . the inverse correlation between ghrelin levels and bmi", "we observed the mentioned correlation , both in children with cyanotic heart disease and in children with acyanotic heart disease .", "although the cyanotic patients had a more pronounced retardation in both height and \n weight than in the acyanotic patients , we found that serum ghrelin levels \n significantly elevated in the acyanotic patients than in the cyanotic patients \n ( p<.0001 ) .", "growth failure in cyanotic children has not been shown \n to be proportional to the severity of cyanosis , suggesting that multiple \n factors are involved in the pathogenesis of their growth disturbance .", "alteration of endocrine mediators of growth has been implicated as a possible \n mechanism of growth failure in cyanotic patients .", "cyanotic newborn lambs have \n decreased levels of serum insulin - like growth factor i without a corresponding \n decrease in growth hormone or hepatic growth factor receptors .", "weintraub et al .   reported that while insulin - like growth factor i levels were linearly related to height and weight in patients with cyanotic lesions , no \n such correlation was found in their cyanotic patients .", "these studies suggest \n that chronic tissue hypoxia may have independent role in growth failure .", "we found that serum tnf- significantly increased in the cyanotic patients and in \n the acyanotic patients .", "similarly , serum il-6 was increased in both groups but the change was more distinctive in the cyanotic patients . tnf- and il-6 appear to be important cachectic process mediators , although this association is not completely established [ 23 , 24 ] .", "cachexia results in decreased muscle \n strength and function and compromised immune function [ 25 , 26 ] .", "this syndrome is likely to occur in children who have chronic congestive heart failure , chronic shunt hypoxemia .", "in addition to inadequate calorie and protein intake , there is evidence that this syndrome may be caused by circulating tumor necrosis factor , which stimulates catabolism . in the present study , ghrelin correlated to positively with tnf- , in acyanotic patients and cyanotic patients with chd .", "the relation of ghrelin with tnf- raises the possibility \n of the direct effect of tnf- upon ghrelin or the impact of heart failure \n severity upon both ghrelin and tnf-. nagaya et al .  ", "have shown that plasma ghrelin level is increased in cachectic patients with congestive heart \n failure as a compensatory mechanism in response to anabolic - catabolic \n imbalance . in conclusion ,", "serum ghrelin level is elevated in cyanotic and acyanotic patients \n with chd . increased ghrelin levels", "additionally , the relation of ghrelin with cytokines \n may be explained by the possible effect of chronic congestive heart failure and chronic shunt \n hypoxemia ."], "abstract_text": ["<S> background / aim . </S>", "<S> ghrelin has effects on nutrient intake and growth . </S>", "<S> the cause of growth retardation in congenital heart disease is multifactorial . </S>", "<S> the aim of the present study is to investigate the ghrelin in congenital heart disease and the association of ghrelin with tnf- and il-6 . \n </S>", "<S> materials and methods . </S>", "<S> we measured serum ghrelin , tnf- , and il-6 levels using spesific immunoassay in 68 patients ( 47 acyanotic , 21 cyanotic with congenital heart disease ) and in 25 control subjects . \n results . in comparison to controls , serum ghrelin , </S>", "<S> tnf- levels were significantly higher in acyanotic patients and cyanotic patients with congenital heart disease ( p<.0001 ) . </S>", "<S> in acyanotic and cyanotic patients with congenital heart disease , there was a positive correlation between ghrelin and tnf- ( r=.485 , p<.05 and r=.573 , p<.01 , resp . ) . </S>", "<S> conclusion . </S>", "<S> serum ghrelin levels is elevated in acyanotic and cyanotic patients with congenital heart disease . increased ghrelin levels </S>", "<S> represents malnutrition and growth retardation in these patients . </S>", "<S> the relation of ghrelin with cytokines may be explained by the possible effect of chronic congestive heart failure and chronic shunt hypoxemia . </S>"], "labels": null, "section_names": ["1. INTRODUCTION", "2. MATERIAL AND METHODS", "3. RESULTS", "4. DISCUSSION"], "sections": [["ghrelin , a 28-amino - acide peptide , is   a potent stimulator of growth hormone release that \n has been implicated in the control of food intake and energy homeostasis in \n human begins and rodents \n [ 15 ] .", "ghrelin is not secreted into the gastrointestinal tract like digestive enzymes but into blood vessels to circulate throughout the body .", "ghrelin causes weight gain by increasing food intake and reducing fat use [ 7 , 8 ] .", "ghrelin has effects on nutrient intake and growth hormone ( gh ) release , subsequently on physical development and growth .", "tumor necrosis factor  ( tnf- ) and interleukin-6 ( il-6 ) are pleiotropic cytokines with numerous immunologic and metabolic actions [ 10 , 11 ] .", "il-6 is generally considered to be an important cytokine in the network of cytokines that regulate immune reactions and acute phase responses .", "the relationship between congenital heart disease ( chd ) , malnutrition , and growth \n retardation is well documented .", "infants with congenital heart disease are prone to malnutrition for several reasons including decreased energy intake , \n increased energy requirements , or both . different types of cardiac malformations can affect nutrition and growth to varying degrees .", "although \n nutritional and growth status were investigated in children with cyanotic and acyanotic heart disease , serum ghrelin levels have not been established .", "the objective of this study is to investigate and compare the functional role of ghrelin on the regulation of energy balance in children with cyanotic and acyanotic congenital heart disease and the association of ghrelin with tnf- , il-6 , that were not entirely confirmed in literature by now ."], ["the study was conducted on 47 children with acyanotic chd , 21 children \n with cyanotic chd , and 25 healthy children .", "all patients ' cardiac diagnoses were \n made on the basis of clinical and laboratory examinations .", "body mass index ( bmi ) \n was calculated as the ratio of body weight ( kg ) and squared height ( m ) .", "all blood samples were drawn at 08 - 09 am and stored 20c \n until the procedure .", "serum ghrelin , tnf- , and il-6 levels were analyzed with \n elisa kits ( tnf- , il-6 kit was purchased from bio - source   international inc .", "( camarillo , calif , usa ) ; ghrelin kit from phoenix international , inc , usa ) .", "the given data were compared between groups using one - way anova , followed by post - hoc ; bonferroni test ."], ["in 47 acyanotic patients , mean age was 30.518.4 months , in 21 cyanotic patients was 28.415.6 months and in 25 control subjects was 31.115.1 months .", "age and anthropometric data of the patients and the control subjects are shown in table 1 .", "there was no significant difference between groups ( the acyanotic patients , the cyanotic patients ) in terms of mean age , weight , height , bmi .", "serum ghrelin levels were significantly higher than in acyanotic and cyanotic groups compared to in the control group   ( p<.0001 ) ( table 3 ) . \n", "serum ghrelin levels in the acyanotic patients were significantly higher than in the cyanotic patients \n ( p<.0001 ) . \n", "tnf- \n levels were significantly higher \n than in cyanotic and acyanotic patients with chd compared to in the control groups   ( p<.001 , p<.0001 , resp . ) .", "serum tnf- values were higher in the acyanotic patients compared to the cyanotic patients with chd ( p<.001 ) .", "serum il-6 levels were higher than in cyanotic and acyanotic patients with chd compared to in the control groups   ( p<.0001 , p<.001 , resp . ) . in both acyanotic and cyanotic groups ,", "serum ghrelin levels were negatively \n correlated with bmi ( r=.549 , p<.05 and r=.688 , p<.01 , resp . ) \n\t\t\t\t", "tnf- levels were not related \n to bmi in the acyanotic and cyanotic patients with chd .", "ghrelin levels were also correlated with tnf- in the acyanotic and cyanotic groups ( r=.485 , p<.05 and r=.573 , p<.01 , resp . )", "ghrelin levels were not related to il-6 in the acyanotic and cyanotic patients with chd ( r=.263 , p>.05 and r=.398 , p>.05 , resp . ) ."], ["inadequate caloric intake , \n malabsorption , and increased energy requirements caused by increased metabolism \n may all contribute .", "however , inadequate caloric intake appears to be the most \n important cause of growth failure in chd [ 13 , 15 , 16 ] .", "patients with acyanotic \n heart disease had a greater growth deficit in weight , and those with cyanotic \n heart disease had a greater growth deficit in stature as demonstrated by both \n decreased height and weight .", "although growth impairment is most pronounced in \n infants with cyanotic chd , growth failure does not correlate well with the \n degree of hypoxia . in this study ,", "the cyanotic patients had a more pronounced \n retardation in both height and weight than in the acyanotic patients [ 13 , 17 ] .", "ghrelin is   accepted as a good marker of the nutritional state , mainly in situations of \n malnutrition , like anorexia nervosa , owing its fast recovery after weight gain . the inverse correlation between ghrelin levels and bmi", "we observed the mentioned correlation , both in children with cyanotic heart disease and in children with acyanotic heart disease .", "although the cyanotic patients had a more pronounced retardation in both height and \n weight than in the acyanotic patients , we found that serum ghrelin levels \n significantly elevated in the acyanotic patients than in the cyanotic patients \n ( p<.0001 ) .", "growth failure in cyanotic children has not been shown \n to be proportional to the severity of cyanosis , suggesting that multiple \n factors are involved in the pathogenesis of their growth disturbance .", "alteration of endocrine mediators of growth has been implicated as a possible \n mechanism of growth failure in cyanotic patients .", "cyanotic newborn lambs have \n decreased levels of serum insulin - like growth factor i without a corresponding \n decrease in growth hormone or hepatic growth factor receptors .", "weintraub et al .   reported that while insulin - like growth factor i levels were linearly related to height and weight in patients with cyanotic lesions , no \n such correlation was found in their cyanotic patients .", "these studies suggest \n that chronic tissue hypoxia may have independent role in growth failure .", "we found that serum tnf- significantly increased in the cyanotic patients and in \n the acyanotic patients .", "similarly , serum il-6 was increased in both groups but the change was more distinctive in the cyanotic patients . tnf- and il-6 appear to be important cachectic process mediators , although this association is not completely established [ 23 , 24 ] .", "cachexia results in decreased muscle \n strength and function and compromised immune function [ 25 , 26 ] .", "this syndrome is likely to occur in children who have chronic congestive heart failure , chronic shunt hypoxemia .", "in addition to inadequate calorie and protein intake , there is evidence that this syndrome may be caused by circulating tumor necrosis factor , which stimulates catabolism . in the present study , ghrelin correlated to positively with tnf- , in acyanotic patients and cyanotic patients with chd .", "the relation of ghrelin with tnf- raises the possibility \n of the direct effect of tnf- upon ghrelin or the impact of heart failure \n severity upon both ghrelin and tnf-. nagaya et al .  ", "have shown that plasma ghrelin level is increased in cachectic patients with congestive heart \n failure as a compensatory mechanism in response to anabolic - catabolic \n imbalance . in conclusion ,", "serum ghrelin level is elevated in cyanotic and acyanotic patients \n with chd . increased ghrelin levels", "additionally , the relation of ghrelin with cytokines \n may be explained by the possible effect of chronic congestive heart failure and chronic shunt \n hypoxemia ."]]}
{"article_id": "PMC4430648", "article_text": ["enteric viruses are major etiologic agents of acute gastroenteritis among infants and young children worldwide .", "rotavirus , norovirus , adenovirus , and astrovirus are the recognized viral causes of pediatric gastroenteritis .", "the world health organization ( who ) data showed that each child practically has viral diarrhea irrespective of race and socioeconomic status within the first 5 years of life and this has great economic burden for the system of public health services and all society [ 2 , 3 ] .", "viral intestinal infections are the most common cause of acute infectious diarrhea in the pediatric group and accounted for approximately 70% of episodes of acute infectious diarrhea in children .", "there is paucity of information as regards viral enteropathogens of diarrhea in many developing countries including nigeria .", "the reason(s ) attributed to this may be as a result of the poor health care system in the country where important health issues are taken for granted such as the aforementioned case .", "thus , as a result of the nonperformance of viral tests for diarrheal patients , information on viral enteropathogens is lost and thus measures to implement control strategies become difficult . against this background", ", this study was carried out to ascertain the prevalence of rotavirus , adenovirus , and norovirus infection in young children with diarrhea in two primary health centers in edo state , nigeria .", "it also aimed at assessing the coinfection rates of the viral agents , age , seasonal distribution of infection , and the association between clinical symptoms and viral diarrhea .", "this study was carried out in two primary health centers located in ikpoba - okha local government area of edo state , nigeria .", "the health centers attend to the primary health needs of the people within and around the locality .", "cases attended to include malaria , diarrhea , immunization of infants and children , antenatal and postnatal cases , and other minor health issues within the scope of the health personnel .", "the two primary health centers are aduwawa and evbomodu primary health centers and they are neighboring communities with a fast growing population made up of indigenes with new residents from the main city alongside other inhabitants from other parts of the country . a total of two hundred and eighty - two ( 282 ) stool specimens comprising 223 diarrhea and 59 nondiarrhea stool specimens were collected from children aged between 0 and 36 months attending two primary health centers . as regards children with diarrhea , males were 121 while females were 102 .", "verbal informed consent was obtained from patients or guardian of the children prior to sample collection .", "the samples were collected from patients at the time of clinic visit as well as other times when the child defecates .", "sterile wide mouth specimen containers were used for specimen collection and they were processed within 6 hours of collection .", "rotavirus , adenovirus , and norovirus were detected by the immunochromatographic technique ( ict ) .", "rotavirus and adenovirus were detected using vikia rota - adeno rapid test device manufactured by biomerieux , france .", "briefly , 2 drops of liquid stool specimen was added to the specimen dilution buffer and shaken vigorously to homogenize .", "two drops of the diluted sample was transferred to the sample well of the test device ( cassette ) and was timed for 10 minutes .", "similarly , norovirus was detected using rida quick norovirus ( ni403 ) test device manufactured by r - biopharm ag , germany .", "briefly , 1  ml of sample dilution buffer ( diluent ) was placed in a separate labeled test tube and 100  ml of liquid stool was added to it and shaken vigorously to homogenize .", "this was allowed to settle for 2 minutes and 250  l of the supernatant was placed in a clean labeled test tube .", "six drops of conjugate 1 was added to the test tube and was shaken vigorously to homogenize .", "the mixture was emptied into the sample well of the test device and incubated for 10 minutes at room temperature .", "four drops of conjugate 2 was added to the reaction window of the test device and incubated for 1 minute at room temperature .", "10 drops of wash buffer was added to the reaction window and was allowed to stay until the buffer was completely absorbed .", "six drops of substrate was added to the reaction window and timed for 3 minutes .", "statistical analysis was carried out using odd ratio and chi - square (  ) tests .", "a total of 223 children with diarrhea were tested for three viral agents ( rotavirus , adenovirus , and norovirus ) .", "a total of 95 ( 42.6% ) were positive for at least one viral agent while none of the 59 children without diarrhea was positive for any viral agent .", "the overall result showed that rotavirus had a prevalence of 63 ( 28.3% ) ( table 1 ) .", "the sex distribution of enteric viruses showed that males had 54 ( 44.6% ) positive cases while females had 41 ( 40.2% ) , and this was not statistically significant ( p = 0.60 ) .", "age group distribution of infection showed that 712 months had the highest infection rate with 48 ( 58.5% ) and was closely followed by 06 months which had 32 ( 50.8% ) .", "there was a statistical significance between age group and infection ( p < 0.0001 ) ( table 2 ) .", "the pattern of coinfecting viruses showed rotavirus - adenovirus mixed infection as the most prevalent with 12 ( 5.4% ) ( table 3 ) .", "the seasonal pattern of enteric viruses showed that rainy season had 60 ( 46.9% ) while dry season had 35 ( 36.8% ) , and this was not statistically significant ( p = 0.17 ) .", "the distribution of enteric viruses according to health centers was not statistically significant ( p = 0.89 ) ( table 4 ) .", "the distribution of viral agents with respect to clinical symptoms is showed in table 5 .", "the prevalence of rotavirus , adenovirus , and norovirus infections was investigated among children with diarrhea .", "the results of this study when compared to other studies carried out in nigeria and other parts of the world showed that the incidence of viral agents varied from one locality to another .", "the 28.3% of rotavirus in this study is lower than 55.9% in ilorin , nigeria , 35% in jos , nigeria , 33.3% in two districts in nigeria , and 39% in ghana . it is higher than 27% in zaria , nigeria , 28.1% in edo state , nigeria , 9.2% in botswana , and 16.9% in korea .", "the 19.3% of adenovirus in this study is lower than 22.3% in northwestern nigeria   and 23.0% in tanzania . it is higher than 6.7% in nigeria , 3.0% in south africa , and 7.8% in botswana .", "the 3.6% of norovirus in this study is lower than 37.3% in nigeria , 16.4% in ghana , 39% in brazil , 11.6% in korea , and 15% in ghana .", "the differences among studies reporting viral infections in different countries might be explained by the different age group , seasonal variations , and viral detection methods used .", "there was a statistical significance between infection and age group ( p < 0.0001 ) .", "age group of 712 months had the highest prevalence of viral diarrhea with 48 ( 58.8% ) .", "this is consistent with the report of moyo et al . , who also found 712 months as the group with the highest infection .", "the reason for this may be due to the fact that it is the period of activities for many children .", "crawling and walking stages are at this period and in the process , children could pick harmful materials into their mouth especially in unhygienic conditions .", "it was observed in this study that , within the first year of life , viral infection rate was 84.2% and in the second year , it was 97.9% .", "this is in accordance with other studies where infection was most prevalent in children within the first 2 years of life [ 11 , 16 , 19 , 20 ]", ". the relatively low prevalence of viruses among older children could be partly due to immunity acquired through previous exposures . in this study ,", "coinfection rate was 7.6% , and rotavirus - adenovirus coinfection was the highest with 12 ( 5.4% ) .", "this , however , did not agree with the reports of chung et al .   and koh et al . , who had rotavirus - norovirus mixed infection as the most prevalent .", "the seasonal pattern of viral infection was not statistically significant ( p = 0.17 ) , though infection was more during the rainy season ( 46.9% ) than in the dry season ( 36.8% ) .", "reports from nigeria have showed that viral diarrhea occurs throughout the year but with variations with respect to seasons [ 8 , 13 , 16 ] .", "viral diarrhea with respect to the two primary health centers did not show statistical significance ( p = 0.89 ) , as infection rates for evbomodu and aduwawa health centers were 43.0% and 42.1% , respectively . the most commonly associated clinical symptom observed in this study with rotavirus , adenovirus , and norovirus positive cases was  vomiting  with 55.6% , 55.8% , and 75.0% , respectively .", "this finding is similar to other studies [ 2325 ] where vomiting was the most commonly associated symptom with viral diarrhea .", "rotavirus positive patients with respect to clinical symptoms such as fever , dehydration , and abdominal pain were low in this study compared to other studies [ 23 , 26 , 27 ] which reported high percentage of clinical symptoms .", "similarly , for adenovirus positive patients , fever , dehydration , and abdominal pain were low compared to other studies with moderate symptoms [ 13 , 24 , 28 , 29 ] .", "however , as regards norovirus positive patients , fever , dehydration , and abdominal pain were also low , but other studies with norovirus showed moderate to high clinical symptoms [ 25 , 30 , 31 ] .", "thus , the differences in clinical symptoms with viral diarrhea as seen from the different studies may be attributed to seasonal variations , geographical location , nutritional status of the patients , and type of viral pathogens causing the infection .", "coinfection of viral agents was found to be 7.6% and the most common clinical symptom was vomiting .", "this is worrisome considering the burden of these viruses on the young children in this locality .", "the fact that viral diagnostic tests are not routinely done or rarely done in any of the hospitals in this locality means that such vital information on viral diarrhea is missed out due to the poor attention given to health care in the country .", "thus , there is need to test stool specimens of clinically confirmed diarrheal patients for enteric viruses as this will go a long way in reducing the wasteful use of antibiotics which are used as blind treatment for persistent diarrhea that may be of viral origin .", "finally , the primary health care centers should be provided with all the necessary diagnostic test materials to address cases of viral diarrhea ."], "abstract_text": ["<S> enteric viruses have been shown to be responsible for diarrhea among children during their early childhood . </S>", "<S> this study was carried out to determine the prevalence of rotavirus , adenovirus , and norovirus infection in young children with diarrhea in two primary health centers in edo state , nigeria . </S>", "<S> a total of 223 stool specimens were collected from children aged 036 months with clinical signs of diarrhea and 59 apparently healthy age - matched children as control . </S>", "<S> these specimens were investigated for three viral agents using immunochromatographic technique ( ict ) . </S>", "<S> the overall results showed that at least one viral agent was detected in 95/223 ( 42.6% ) of the children with diarrhea while the control had none . </S>", "<S> the prevalence of rotavirus was 28.3% , adenovirus 19.3% , and norovirus 3.6% . </S>", "<S> there was a significant association between age group and infection ( p < 0.0001 ) . </S>", "<S> seasonal pattern of enteric viruses was not statistically significant ( p = 0.17 ) . </S>", "<S> the overall coinfection rate was 7.6% and rotavirus - adenovirus coinfection had the highest with 5.4% . </S>", "<S> rotavirus was the most prevalent viral agent . </S>", "<S> coinfections are not uncommon among the population studied . </S>", "<S> the most commonly associated clinical symptom of viral diarrhea in this study was vomiting . </S>", "<S> viral diagnostic tests are advocated for primary health care facilities in this locality . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Patients and Methods", "3. Results and Discussion", "4. Conclusion"], "sections": [["enteric viruses are major etiologic agents of acute gastroenteritis among infants and young children worldwide .", "rotavirus , norovirus , adenovirus , and astrovirus are the recognized viral causes of pediatric gastroenteritis .", "the world health organization ( who ) data showed that each child practically has viral diarrhea irrespective of race and socioeconomic status within the first 5 years of life and this has great economic burden for the system of public health services and all society [ 2 , 3 ] .", "viral intestinal infections are the most common cause of acute infectious diarrhea in the pediatric group and accounted for approximately 70% of episodes of acute infectious diarrhea in children .", "there is paucity of information as regards viral enteropathogens of diarrhea in many developing countries including nigeria .", "the reason(s ) attributed to this may be as a result of the poor health care system in the country where important health issues are taken for granted such as the aforementioned case .", "thus , as a result of the nonperformance of viral tests for diarrheal patients , information on viral enteropathogens is lost and thus measures to implement control strategies become difficult . against this background", ", this study was carried out to ascertain the prevalence of rotavirus , adenovirus , and norovirus infection in young children with diarrhea in two primary health centers in edo state , nigeria .", "it also aimed at assessing the coinfection rates of the viral agents , age , seasonal distribution of infection , and the association between clinical symptoms and viral diarrhea ."], ["this study was carried out in two primary health centers located in ikpoba - okha local government area of edo state , nigeria .", "the health centers attend to the primary health needs of the people within and around the locality .", "cases attended to include malaria , diarrhea , immunization of infants and children , antenatal and postnatal cases , and other minor health issues within the scope of the health personnel .", "the two primary health centers are aduwawa and evbomodu primary health centers and they are neighboring communities with a fast growing population made up of indigenes with new residents from the main city alongside other inhabitants from other parts of the country . a total of two hundred and eighty - two ( 282 ) stool specimens comprising 223 diarrhea and 59 nondiarrhea stool specimens were collected from children aged between 0 and 36 months attending two primary health centers . as regards children with diarrhea , males were 121 while females were 102 .", "verbal informed consent was obtained from patients or guardian of the children prior to sample collection .", "the samples were collected from patients at the time of clinic visit as well as other times when the child defecates .", "sterile wide mouth specimen containers were used for specimen collection and they were processed within 6 hours of collection .", "rotavirus , adenovirus , and norovirus were detected by the immunochromatographic technique ( ict ) .", "rotavirus and adenovirus were detected using vikia rota - adeno rapid test device manufactured by biomerieux , france .", "briefly , 2 drops of liquid stool specimen was added to the specimen dilution buffer and shaken vigorously to homogenize .", "two drops of the diluted sample was transferred to the sample well of the test device ( cassette ) and was timed for 10 minutes .", "similarly , norovirus was detected using rida quick norovirus ( ni403 ) test device manufactured by r - biopharm ag , germany .", "briefly , 1  ml of sample dilution buffer ( diluent ) was placed in a separate labeled test tube and 100  ml of liquid stool was added to it and shaken vigorously to homogenize .", "this was allowed to settle for 2 minutes and 250  l of the supernatant was placed in a clean labeled test tube .", "six drops of conjugate 1 was added to the test tube and was shaken vigorously to homogenize .", "the mixture was emptied into the sample well of the test device and incubated for 10 minutes at room temperature .", "four drops of conjugate 2 was added to the reaction window of the test device and incubated for 1 minute at room temperature .", "10 drops of wash buffer was added to the reaction window and was allowed to stay until the buffer was completely absorbed .", "six drops of substrate was added to the reaction window and timed for 3 minutes .", "statistical analysis was carried out using odd ratio and chi - square (  ) tests ."], ["a total of 223 children with diarrhea were tested for three viral agents ( rotavirus , adenovirus , and norovirus ) .", "a total of 95 ( 42.6% ) were positive for at least one viral agent while none of the 59 children without diarrhea was positive for any viral agent .", "the overall result showed that rotavirus had a prevalence of 63 ( 28.3% ) ( table 1 ) .", "the sex distribution of enteric viruses showed that males had 54 ( 44.6% ) positive cases while females had 41 ( 40.2% ) , and this was not statistically significant ( p = 0.60 ) .", "age group distribution of infection showed that 712 months had the highest infection rate with 48 ( 58.5% ) and was closely followed by 06 months which had 32 ( 50.8% ) .", "there was a statistical significance between age group and infection ( p < 0.0001 ) ( table 2 ) .", "the pattern of coinfecting viruses showed rotavirus - adenovirus mixed infection as the most prevalent with 12 ( 5.4% ) ( table 3 ) .", "the seasonal pattern of enteric viruses showed that rainy season had 60 ( 46.9% ) while dry season had 35 ( 36.8% ) , and this was not statistically significant ( p = 0.17 ) .", "the distribution of enteric viruses according to health centers was not statistically significant ( p = 0.89 ) ( table 4 ) .", "the distribution of viral agents with respect to clinical symptoms is showed in table 5 .", "the prevalence of rotavirus , adenovirus , and norovirus infections was investigated among children with diarrhea .", "the results of this study when compared to other studies carried out in nigeria and other parts of the world showed that the incidence of viral agents varied from one locality to another .", "the 28.3% of rotavirus in this study is lower than 55.9% in ilorin , nigeria , 35% in jos , nigeria , 33.3% in two districts in nigeria , and 39% in ghana . it is higher than 27% in zaria , nigeria , 28.1% in edo state , nigeria , 9.2% in botswana , and 16.9% in korea .", "the 19.3% of adenovirus in this study is lower than 22.3% in northwestern nigeria   and 23.0% in tanzania . it is higher than 6.7% in nigeria , 3.0% in south africa , and 7.8% in botswana .", "the 3.6% of norovirus in this study is lower than 37.3% in nigeria , 16.4% in ghana , 39% in brazil , 11.6% in korea , and 15% in ghana .", "the differences among studies reporting viral infections in different countries might be explained by the different age group , seasonal variations , and viral detection methods used .", "there was a statistical significance between infection and age group ( p < 0.0001 ) .", "age group of 712 months had the highest prevalence of viral diarrhea with 48 ( 58.8% ) .", "this is consistent with the report of moyo et al . , who also found 712 months as the group with the highest infection .", "the reason for this may be due to the fact that it is the period of activities for many children .", "crawling and walking stages are at this period and in the process , children could pick harmful materials into their mouth especially in unhygienic conditions .", "it was observed in this study that , within the first year of life , viral infection rate was 84.2% and in the second year , it was 97.9% .", "this is in accordance with other studies where infection was most prevalent in children within the first 2 years of life [ 11 , 16 , 19 , 20 ]", ". the relatively low prevalence of viruses among older children could be partly due to immunity acquired through previous exposures . in this study ,", "coinfection rate was 7.6% , and rotavirus - adenovirus coinfection was the highest with 12 ( 5.4% ) .", "this , however , did not agree with the reports of chung et al .   and koh et al . , who had rotavirus - norovirus mixed infection as the most prevalent .", "the seasonal pattern of viral infection was not statistically significant ( p = 0.17 ) , though infection was more during the rainy season ( 46.9% ) than in the dry season ( 36.8% ) .", "reports from nigeria have showed that viral diarrhea occurs throughout the year but with variations with respect to seasons [ 8 , 13 , 16 ] .", "viral diarrhea with respect to the two primary health centers did not show statistical significance ( p = 0.89 ) , as infection rates for evbomodu and aduwawa health centers were 43.0% and 42.1% , respectively . the most commonly associated clinical symptom observed in this study with rotavirus , adenovirus , and norovirus positive cases was  vomiting  with 55.6% , 55.8% , and 75.0% , respectively .", "this finding is similar to other studies [ 2325 ] where vomiting was the most commonly associated symptom with viral diarrhea .", "rotavirus positive patients with respect to clinical symptoms such as fever , dehydration , and abdominal pain were low in this study compared to other studies [ 23 , 26 , 27 ] which reported high percentage of clinical symptoms .", "similarly , for adenovirus positive patients , fever , dehydration , and abdominal pain were low compared to other studies with moderate symptoms [ 13 , 24 , 28 , 29 ] .", "however , as regards norovirus positive patients , fever , dehydration , and abdominal pain were also low , but other studies with norovirus showed moderate to high clinical symptoms [ 25 , 30 , 31 ] .", "thus , the differences in clinical symptoms with viral diarrhea as seen from the different studies may be attributed to seasonal variations , geographical location , nutritional status of the patients , and type of viral pathogens causing the infection ."], ["coinfection of viral agents was found to be 7.6% and the most common clinical symptom was vomiting .", "this is worrisome considering the burden of these viruses on the young children in this locality .", "the fact that viral diagnostic tests are not routinely done or rarely done in any of the hospitals in this locality means that such vital information on viral diarrhea is missed out due to the poor attention given to health care in the country .", "thus , there is need to test stool specimens of clinically confirmed diarrheal patients for enteric viruses as this will go a long way in reducing the wasteful use of antibiotics which are used as blind treatment for persistent diarrhea that may be of viral origin .", "finally , the primary health care centers should be provided with all the necessary diagnostic test materials to address cases of viral diarrhea ."]]}
{"article_id": "PMC3788228", "article_text": ["alpha ( )2-adrenoceptor agonists have been used as adjuvant to anesthetic agents in peri - operative period for its several beneficial actions . these drugs improve hemodynamic stability during endotracheal intubation and surgical stress by its central sympatholytic action , and thus reduce anesthetic and opioids requirements .", "dexmedetomidine , the pharmacologically active d - isomer of medetomidine , is highly selective and specific 2-adrenoceptor agonist .", "the analgesic effect of dexmedetomidine is qualitatively different as compared to opioids and can be used as an alternative to opioids in general anesthesia .", "the anesthetic sparing effect of dexmedetomidine demonstrated in earlier studies is confounded by the use of opioids .", "opioids , when used as analgesic in general anesthesia , are known to decrease mac value of sevoflurane .", "inhalation agents were titrated based on either hemodynamic criteria and/or bispectral index values in studies done earlier , and none of them demonstrated the effect of dexmedetomidine alone on requirement of sevoflurane with the use of entropy as a measure of depth of anesthesia .", "the primary objective of our study was to evaluate the effect of continuous infusion of dexmedetomidine alone , without use of opioids , on sevoflurane requirement during general anesthesia with continuous monitoring of depth of anesthesia by entropy analysis .", "the study protocol was approved by ethical committee of the institute , and written informed consent was obtained from all patients included in the study .", "the study population comprised of 60 patients , aged 18 - 55 years , with american society of anesthesiologists ( asa ) status of 1 and 2 , undergoing major elective surgical procedures .", "we excluded patients older than 55 years , those with a history of psychiatric / neurological illness , cardiovascular disease , hypertensive patients , morbid obese patients , pregnant and nursing women , with known allergic reaction to any of the study medication , recent use of sedatives or analgesics , and with significant laboratory abnormalities .", "the consenting patients were randomly allocated into one of the two anesthetic groups based on the simple randomization table generated : group a ( control group ) : sevoflurane - fentanyl and group b ( test group ) : sevoflurane - dexmedetomidine .", "after arrival in the operating room , glycopyrrolate 0.004 mg / kg and ondansetron 0.15 mg / kg intravenous ( iv ) were given as pre - medication in both the groups .", "the patients in group b received dexmedetomidine(2 ml diluted in 48 ml of saline ) iv in a dose of 1 mcg / kg over10 minutes through infusion pump prior to induction .", "monitoring consisted of continuous electrokardiogram ( ekg ) , non - invasive blood pressure ( nibp ) , pulse oximetry ( spo2 ) , co2 expired fraction ( etco2 ) , sevoflurane inspired fraction ( fisevo ) , sevoflurane expired fraction ( etsevo ) , and electroencephalograph ( eeg ) analysis by entropy ( response and state entropy ) . after the start of drug infusion , heart rate ( hr ) , blood pressure", "( bp ) , respiratory rate ( rr ) , spo2 , and entropy were measured at 5 and 10 minute .", "patients in group a received fentanyl 2 g / kg intravenous slowly , 2 minutes prior to induction .", "after pre - oxygenation for 10 minutes , general anesthesia was induced with thiopentone sodium 5 mg / kg iv slowly .", ", dexmedetomidine infusion was continued between 0.2 and 0.8 mcg / kg / h depending on hr , bp , and entropy value changes .", "anesthesia was maintained with sevoflurane to a maximum of 2.5% end tidal to maintain heart rate and blood pressure within 20% of baseline value and entropy value between 40 and 60 .", "the anesthesiologist was permitted to treat hemodynamic events , defined as heart rate and blood pressure more than 20% of baseline , in spite of increasing sevoflurane concentration to 2.5% and dexmedetomidine infusion to 0.8 mg / kg with supplemental analgesia in the form of fentanyl 1 mcg / kg . if rise in heart rate and blood pressure persisted further after supplementation with fentanyl , anesthesiologist was permitted to administer incremental doses of metoprolol 2 - 5 mg .", "ephedrine 5 mg iv was given for fall in heart rate and blood pressure more than 20% of baseline .", "in the group b , dexmedetomidine was stopped approximately 15 - 20 minutes before completion of surgery , diclofenac sodium aqueous 1 mg / kg was given iv in both groups at the time of skin closure .", "reversal of neuromuscular blockade was achieved with neostigmine 0.05 mg / kg and glycopyrrolate 0.008 mg / kg iv slowly .", "tracheal extubation was done when respiration was satisfactory and adequate muscle tone was achieved . \n", "hr and bp were recorded before induction , at the time of induction , intubation , and then at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation.depth of anesthesia was evaluated by entropy ( state entropy , response entropy ) analysis ( datex - ohmeda s/5 avance workstation , ge healthcare , helsinki , finland ) at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation.end tidal concentration of sevoflurane was assessed with anesthesia gas assessment module e - caiovx at same interval as entropy analysis . \n ", "hr and bp were recorded before induction , at the time of induction , intubation , and then at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation .", "depth of anesthesia was evaluated by entropy ( state entropy , response entropy ) analysis ( datex - ohmeda s/5 avance workstation , ge healthcare , helsinki , finland ) at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation .", "end tidal concentration of sevoflurane was assessed with anesthesia gas assessment module e - caiovx at same interval as entropy analysis .", "statistical analysis was conducted with epi info software ( version 3.5.3 , 2011 , centers for disease control and prevention , atlanta , ga , usa ) for windows statistical package using unpaired t - test for continuous variables with normal distribution .", "the non - parametric kruskal - wallis test was used for variables not normally distributed . for categorical variables , chi - square test was used .", "the two groups were similar regarding age , sex , weight , and asa physical status [ table 1 ] .", "pre - induction heart rate , systolic and diastolic blood pressures were similar between two groups ( p > 0.05 ) .", "the depth of anesthesia as assessed by response entropy ( re ) and state entropy ( se ) was comparable between two groups at all time points during maintenance period ( p > 0.05 ) .", "the re and se was maintained between 40 and 60 during the period of observation .", "the fisevo concentration was significantly less in group b as compared to the group a at all time points ( p < 0.05 ) [ figure 1 ] .", "the average etsevo concentration during anesthetic maintenance was 1.35% and 1.72% in the group b and a , respectively .", "a significant decrease of 13% to 33% of etsevo concentration was seen with group b from 5 min to 60 minutes post - intubation ( p < 0.05 ) during surgery [ figure 2 ] .", "fraction of inspired sevoflurane at different time interval ( pi - post - intubation , fi - fraction of inspired ) end tidal sevoflurane at different time intervals ( pi - post - intubation ) during anesthesia maintenance , group b showed a statistical significant decrease in heart rate , systolic and diastolic blood pressure from baseline at all time points as compared to the group a ( p < 0.05 ) .", "an average 6.5% fall in heart rate from baseline in group b as compared to 3.7% rise in the group a , 8% fall in systolic blood pressure from baseline as compared to 3.6% rise in the group a , and 8.16% fall in diastolic blood pressure from baseline as compared to 3.3% rise in the group a was observed [ figures 3 and 4 ] .", "changes in sbp and dbp at various time points post - intubation ( pi - post - intubation , sbp - systolic blood pressure , dbp - diastolic blood pressure ) changes in hr at various time points post - intubation ( pi - post intubation , hr - heart rate ) in our study , none of the patients in either group required supplemental analgesia or anti - hypertensive drugs intra - operatively .", "bradycardia was observed in 2 patients in group b within 10 mins post - extubation , which promptly responded to atropine 0.6 mg iv .", "the major observation in our study was that dexmedetomidine infusion as an adjuvant in general anesthesia causes decreased requirement of sevoflurane without compromising adequate depth of anesthesia , thus it has anesthetic - sparing property .", "a study done on patients undergoing hysterectomy showed a 30% reduction of maintenance concentration of isoflurane .", "similarly , a reduction in 35% to 50% in isoflurane concentration with low or high dose of dexmedetomidine was found in a study on healthy human volunteers .", "this larger reduction in isoflurane requirement seen as compared to our study might be due to difference in study population ( healthy human volunteers vs. patients posted for elective surgery ) and the type of stimulus ( tetanus nerve stimulus vs. surgical stimulus ) .", "the results of our study results are consistent with that of the study done by fragen , et al . in elderly patients , which showed a 17% reduction .", "dexmedetomidine by its sympatholytic action decreases heart rate and blood pressure , thus assessing the depth of anesthesia by hemodynamic parameters would be unreliable in evaluating its effect on requirement of inhalational agent . several electroencephalogram - dependent indices such as bispectral index and entropy have been used to measure the depth of anesthesia .", "entropy is a useful monitor for measuring the electroencephalographic effects of increasing and decreasing sevoflurane concentration and assessing the depth of anesthesia .", "analogous to the bispectral index , entropy displays a high degree of specificity and sensitivity in assessing consciousness during anesthesia . using bispectral index to assess the depth of anesthesia , magalhes et al", ". showed decreased requirement of sevoflurane with continuous infusion of dexmedetomidine during general anesthesia . in our study , we used entropy to measure the depth of anesthesia , thereby eliminating the bias of evaluation by hemodynamic parameters as in earlier studies .", "use of dexmedetomidine produces intra - operative and post - operative opioids - sparing effect .", "dexmedetomidine by its sympatholytic action attenuates symapthoadrenal response to tracheal intubation . in patients undergoing laparoscopic tubal ligation , a 33% decrease in morphine", "use post - operatively was observed when dexmedetomidine was used at a dose of 0.4 mcg / kg .", "dexmedetomidine when administered as infusion at a dose of 0.5 mcg / kg / h has specific analgesic effect and provides visceral pain relief . in morbidly obese ,", "dexmedetomidine produces a greater decrease in sympathovagal balance intra - operatively than fentanyl along with better post - operative analgesia .", "dexmedetomidine , when used as sole substitute for remifentanil in ambulatory gynecologic laparoscopic surgery , provides better peri - operative hemodynamic stability and post - operative analgesia .", "dexmedetomidine provides similar intra - operative hemodynamic response and better post - operative analgesia compared to remifentanil in patients undergoing supratentorial craniotomy .", "use of opioids along with dexmedetomidine would confound its effect on requirement of inhalation agent .", "hence , in our study , fentanyl was not administered in dexmedetomidine group b , and we found a 21.5% decrease in the etsevo , in contrast to 33.12% decrease in study done by magalhes e et al . where fentanyl was used .", "one limitation of our study was that we included both laparoscopic as well as open surgeries .", "to conclude , the continuous infusion of dexmedetomidine , as adjuvant in general anesthesia , significantly decreases the requirement of sevoflurane for maintaining adequate depth of anesthesia .", "studies measuring plasma concentration of dexmedetomidine should be undertaken to establish the precise correlation between its dose and inhalational agent 's requirements ."], "abstract_text": ["<S> background : dexmedetomidine , a 2 agonist as an adjuvant in general anesthesia , has anesthetic and analgesic - sparing property.aims:to evaluate the effect of continuous infusion of dexmedetomidine alone , without use of opioids , on requirement of sevoflurane during general anesthesia with continuous monitoring of depth of anesthesia by entropy analysis.materials and methods : sixty patients were randomly divided into 2 groups of 30 each . in group </S>", "<S> a , fentanyl 2 mcg / kg was given while in group b , dexmedetomidine was given intravenously as loading dose of 1 mcg / kg over 10 min prior to induction . </S>", "<S> after induction with thiopentone in group b , dexmedetomidine was given as infusion at a dose of 0.2 - 0.8 mcg / kg . </S>", "<S> sevoflurane was used as inhalation agent in both groups . </S>", "<S> hemodynamic variables , sevoflurane inspired fraction ( fisevo ) , sevoflurane expired fraction ( etsevo ) , and entropy ( response entropy and state entropy ) were continuously recorded . </S>", "<S> statistical analysis was done by unpaired student 's t - test and chi - square test for continuous and categorical variables , respectively . </S>", "<S> a p - value < 0.05 was considered significant.results:the use of dexmedetomidine with sevoflurane was associated with a statistical significant decrease in etsevo at 5 minutes post - intubation ( 1.49  0.11 ) and 60 minutes post - intubation ( 1.11 0.28 ) as compared to the group a [ 1.73 0.30 ( 5 minutes ) ; 1.68 0.50 ( 60 minutes ) ] . </S>", "<S> there was an average 21.5% decrease in etsevo in group b as compared to group a.conclusions:dexmedetomidine , as an adjuvant in general anesthesia , decreases requirement of sevoflurane for maintaining adequate depth of anesthesia . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion"], "sections": [["alpha ( )2-adrenoceptor agonists have been used as adjuvant to anesthetic agents in peri - operative period for its several beneficial actions . these drugs improve hemodynamic stability during endotracheal intubation and surgical stress by its central sympatholytic action , and thus reduce anesthetic and opioids requirements .", "dexmedetomidine , the pharmacologically active d - isomer of medetomidine , is highly selective and specific 2-adrenoceptor agonist .", "the analgesic effect of dexmedetomidine is qualitatively different as compared to opioids and can be used as an alternative to opioids in general anesthesia .", "the anesthetic sparing effect of dexmedetomidine demonstrated in earlier studies is confounded by the use of opioids .", "opioids , when used as analgesic in general anesthesia , are known to decrease mac value of sevoflurane .", "inhalation agents were titrated based on either hemodynamic criteria and/or bispectral index values in studies done earlier , and none of them demonstrated the effect of dexmedetomidine alone on requirement of sevoflurane with the use of entropy as a measure of depth of anesthesia .", "the primary objective of our study was to evaluate the effect of continuous infusion of dexmedetomidine alone , without use of opioids , on sevoflurane requirement during general anesthesia with continuous monitoring of depth of anesthesia by entropy analysis ."], ["the study protocol was approved by ethical committee of the institute , and written informed consent was obtained from all patients included in the study .", "the study population comprised of 60 patients , aged 18 - 55 years , with american society of anesthesiologists ( asa ) status of 1 and 2 , undergoing major elective surgical procedures .", "we excluded patients older than 55 years , those with a history of psychiatric / neurological illness , cardiovascular disease , hypertensive patients , morbid obese patients , pregnant and nursing women , with known allergic reaction to any of the study medication , recent use of sedatives or analgesics , and with significant laboratory abnormalities .", "the consenting patients were randomly allocated into one of the two anesthetic groups based on the simple randomization table generated : group a ( control group ) : sevoflurane - fentanyl and group b ( test group ) : sevoflurane - dexmedetomidine .", "after arrival in the operating room , glycopyrrolate 0.004 mg / kg and ondansetron 0.15 mg / kg intravenous ( iv ) were given as pre - medication in both the groups .", "the patients in group b received dexmedetomidine(2 ml diluted in 48 ml of saline ) iv in a dose of 1 mcg / kg over10 minutes through infusion pump prior to induction .", "monitoring consisted of continuous electrokardiogram ( ekg ) , non - invasive blood pressure ( nibp ) , pulse oximetry ( spo2 ) , co2 expired fraction ( etco2 ) , sevoflurane inspired fraction ( fisevo ) , sevoflurane expired fraction ( etsevo ) , and electroencephalograph ( eeg ) analysis by entropy ( response and state entropy ) . after the start of drug infusion , heart rate ( hr ) , blood pressure", "( bp ) , respiratory rate ( rr ) , spo2 , and entropy were measured at 5 and 10 minute .", "patients in group a received fentanyl 2 g / kg intravenous slowly , 2 minutes prior to induction .", "after pre - oxygenation for 10 minutes , general anesthesia was induced with thiopentone sodium 5 mg / kg iv slowly .", ", dexmedetomidine infusion was continued between 0.2 and 0.8 mcg / kg / h depending on hr , bp , and entropy value changes .", "anesthesia was maintained with sevoflurane to a maximum of 2.5% end tidal to maintain heart rate and blood pressure within 20% of baseline value and entropy value between 40 and 60 .", "the anesthesiologist was permitted to treat hemodynamic events , defined as heart rate and blood pressure more than 20% of baseline , in spite of increasing sevoflurane concentration to 2.5% and dexmedetomidine infusion to 0.8 mg / kg with supplemental analgesia in the form of fentanyl 1 mcg / kg . if rise in heart rate and blood pressure persisted further after supplementation with fentanyl , anesthesiologist was permitted to administer incremental doses of metoprolol 2 - 5 mg .", "ephedrine 5 mg iv was given for fall in heart rate and blood pressure more than 20% of baseline .", "in the group b , dexmedetomidine was stopped approximately 15 - 20 minutes before completion of surgery , diclofenac sodium aqueous 1 mg / kg was given iv in both groups at the time of skin closure .", "reversal of neuromuscular blockade was achieved with neostigmine 0.05 mg / kg and glycopyrrolate 0.008 mg / kg iv slowly .", "tracheal extubation was done when respiration was satisfactory and adequate muscle tone was achieved . \n", "hr and bp were recorded before induction , at the time of induction , intubation , and then at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation.depth of anesthesia was evaluated by entropy ( state entropy , response entropy ) analysis ( datex - ohmeda s/5 avance workstation , ge healthcare , helsinki , finland ) at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation.end tidal concentration of sevoflurane was assessed with anesthesia gas assessment module e - caiovx at same interval as entropy analysis . \n ", "hr and bp were recorded before induction , at the time of induction , intubation , and then at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation .", "depth of anesthesia was evaluated by entropy ( state entropy , response entropy ) analysis ( datex - ohmeda s/5 avance workstation , ge healthcare , helsinki , finland ) at 5 , 10 , 15 , 30 , 45 , and 60 minute after intubation and at extubation .", "end tidal concentration of sevoflurane was assessed with anesthesia gas assessment module e - caiovx at same interval as entropy analysis .", "statistical analysis was conducted with epi info software ( version 3.5.3 , 2011 , centers for disease control and prevention , atlanta , ga , usa ) for windows statistical package using unpaired t - test for continuous variables with normal distribution .", "the non - parametric kruskal - wallis test was used for variables not normally distributed . for categorical variables , chi - square test was used ."], ["the two groups were similar regarding age , sex , weight , and asa physical status [ table 1 ] .", "pre - induction heart rate , systolic and diastolic blood pressures were similar between two groups ( p > 0.05 ) .", "the depth of anesthesia as assessed by response entropy ( re ) and state entropy ( se ) was comparable between two groups at all time points during maintenance period ( p > 0.05 ) .", "the re and se was maintained between 40 and 60 during the period of observation .", "the fisevo concentration was significantly less in group b as compared to the group a at all time points ( p < 0.05 ) [ figure 1 ] .", "the average etsevo concentration during anesthetic maintenance was 1.35% and 1.72% in the group b and a , respectively .", "a significant decrease of 13% to 33% of etsevo concentration was seen with group b from 5 min to 60 minutes post - intubation ( p < 0.05 ) during surgery [ figure 2 ] .", "fraction of inspired sevoflurane at different time interval ( pi - post - intubation , fi - fraction of inspired ) end tidal sevoflurane at different time intervals ( pi - post - intubation ) during anesthesia maintenance , group b showed a statistical significant decrease in heart rate , systolic and diastolic blood pressure from baseline at all time points as compared to the group a ( p < 0.05 ) .", "an average 6.5% fall in heart rate from baseline in group b as compared to 3.7% rise in the group a , 8% fall in systolic blood pressure from baseline as compared to 3.6% rise in the group a , and 8.16% fall in diastolic blood pressure from baseline as compared to 3.3% rise in the group a was observed [ figures 3 and 4 ] .", "changes in sbp and dbp at various time points post - intubation ( pi - post - intubation , sbp - systolic blood pressure , dbp - diastolic blood pressure ) changes in hr at various time points post - intubation ( pi - post intubation , hr - heart rate ) in our study , none of the patients in either group required supplemental analgesia or anti - hypertensive drugs intra - operatively .", "bradycardia was observed in 2 patients in group b within 10 mins post - extubation , which promptly responded to atropine 0.6 mg iv ."], ["the major observation in our study was that dexmedetomidine infusion as an adjuvant in general anesthesia causes decreased requirement of sevoflurane without compromising adequate depth of anesthesia , thus it has anesthetic - sparing property .", "a study done on patients undergoing hysterectomy showed a 30% reduction of maintenance concentration of isoflurane .", "similarly , a reduction in 35% to 50% in isoflurane concentration with low or high dose of dexmedetomidine was found in a study on healthy human volunteers .", "this larger reduction in isoflurane requirement seen as compared to our study might be due to difference in study population ( healthy human volunteers vs. patients posted for elective surgery ) and the type of stimulus ( tetanus nerve stimulus vs. surgical stimulus ) .", "the results of our study results are consistent with that of the study done by fragen , et al . in elderly patients , which showed a 17% reduction .", "dexmedetomidine by its sympatholytic action decreases heart rate and blood pressure , thus assessing the depth of anesthesia by hemodynamic parameters would be unreliable in evaluating its effect on requirement of inhalational agent . several electroencephalogram - dependent indices such as bispectral index and entropy have been used to measure the depth of anesthesia .", "entropy is a useful monitor for measuring the electroencephalographic effects of increasing and decreasing sevoflurane concentration and assessing the depth of anesthesia .", "analogous to the bispectral index , entropy displays a high degree of specificity and sensitivity in assessing consciousness during anesthesia . using bispectral index to assess the depth of anesthesia , magalhes et al", ". showed decreased requirement of sevoflurane with continuous infusion of dexmedetomidine during general anesthesia . in our study , we used entropy to measure the depth of anesthesia , thereby eliminating the bias of evaluation by hemodynamic parameters as in earlier studies .", "use of dexmedetomidine produces intra - operative and post - operative opioids - sparing effect .", "dexmedetomidine by its sympatholytic action attenuates symapthoadrenal response to tracheal intubation . in patients undergoing laparoscopic tubal ligation , a 33% decrease in morphine", "use post - operatively was observed when dexmedetomidine was used at a dose of 0.4 mcg / kg .", "dexmedetomidine when administered as infusion at a dose of 0.5 mcg / kg / h has specific analgesic effect and provides visceral pain relief . in morbidly obese ,", "dexmedetomidine produces a greater decrease in sympathovagal balance intra - operatively than fentanyl along with better post - operative analgesia .", "dexmedetomidine , when used as sole substitute for remifentanil in ambulatory gynecologic laparoscopic surgery , provides better peri - operative hemodynamic stability and post - operative analgesia .", "dexmedetomidine provides similar intra - operative hemodynamic response and better post - operative analgesia compared to remifentanil in patients undergoing supratentorial craniotomy .", "use of opioids along with dexmedetomidine would confound its effect on requirement of inhalation agent .", "hence , in our study , fentanyl was not administered in dexmedetomidine group b , and we found a 21.5% decrease in the etsevo , in contrast to 33.12% decrease in study done by magalhes e et al . where fentanyl was used .", "one limitation of our study was that we included both laparoscopic as well as open surgeries .", "to conclude , the continuous infusion of dexmedetomidine , as adjuvant in general anesthesia , significantly decreases the requirement of sevoflurane for maintaining adequate depth of anesthesia .", "studies measuring plasma concentration of dexmedetomidine should be undertaken to establish the precise correlation between its dose and inhalational agent 's requirements ."]]}
{"article_id": "PMC4138544", "article_text": ["rkshv.219 virus is latently infected in islk.rkshv219 cell line , derived from islk cell line , as previously described by .", "rkshv.219 cells were seeded at 5    10 cells into 150-mm dish ( one day prior to induction ) . for induction of kshv reactivation ,", "the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2  g / ml doxycycline .", "kshv reactivation was induced at 0 , 6 , 12 , 24 , and 48  h. the 0  h sample was treated identically to other timepoints , however doxycycline was not added .", "two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay .", "following doxycycline induction of kshv reactivation , islk.rkshv219 cells were harvested at 0 , 6 , 12 , 24 , and 48  h at 2.5    10 cells .", "media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10  min at room temperature , to cross - link the chromatin structure .", "125  mm glycine was then added to stop the formaldehyde cross - linking reaction .", "nucleus isolation buffer , 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , and ph  7.8 were then added to islk.rkshv219 cross - linked cells .", "the cells were scraped and placed within a 50  ml conical tube , and the nuclei were isolated by centrifugation at 1000  g for 5  min at 4  c .", "micrococcal nuclease , mnase , is a internucleosomal cleavage reagent , which does not cleave the dna protected by a nucleosome .", "islk.rkshv219 nuclei were digested with a titration of mnase : 4  u / ml , 2  u / ml , 1  u / ml , and 0.5  u / ml ( worthington biochemical corp . ) in mnase cleavage buffer ( 5  mm kcl , 4  mm mgcl2 , 1  mm cacl2 , 12.5% glycerol , and 50  mm tris  cl ( ph  7.4 ) ) .", "a titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented .", "the nuclei , following mnase digestion , were treated with 0.2  g/l proteinase k and 1% sodium dodecyl sulfate .", "mnase digested dna for all samples was resolved on a 2% agarose gel , and mononucleosomally protected dna , ~  150  bp , was isolated by gel excision .", "following gel excision , the mononucleosomally protected dna was purified by electroelution , and for each sample 's mnase titration the mononucleosomally protected dna was combined .", "the mononucleosomally protected dna was further purified with phenol  chloroform extraction and precipitated with alcohol .", "islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55  c overnight for bare genomic dna isolation .", "we utilized a high - resolution dna microarray , which we customized to contain 1  kb upstream and downstream of the transcription start sites of 462 immunity - related genes .", "nimblegen 's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array .", "sequences were repeat - masked around the tsss , and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray .", "oligonucleotide probes , sized at 60  bp , were tiled on each subarray with a mean spacing of 47  bp overlap .", "approximately 180 probes of spacing of 12.5  bp were printed on the microarray for each locus .", "the samples were fluorescently labeled : nucleosomally protected dna with cy3 and bare genomic dna ( reference ) with cy5 .", "samples were hybridized to the dna microarray according to nimblegen 's instructions ( http://www.nimblegen.com/technology/index.html ) .", "the resulting .tiff files were used for microarray data feature extraction , primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow .", "specifically , loess spatial correction was applied to the raw data ; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes ; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal .", "the ratio between nucleosomal ( cy3 ) and bare genomic cy5 signals was then calculated and stored as a general feature format , .gff", "subsequent analysis was performed using a software developed in our laboratory dennislab.r , which runs in the r environment for statistical computing .", "dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome .", "finally , we analyzed the nucleosome distribution changes at the 24  hour timepoint on a per chromosome basis , by calculating the t - statistic and p - value ( assuming unequal variance ) for the pairwise comparison of the range of values between all chromosomes . in our previous work", "we demonstrated that nucleosome redistributions are widespread , transient , and dna - directed , .", "we next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner .", "we first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint ( fig . ", "these results comport with our previous observation that the greatest positioning occurs at the 24  hour timepoint , however do not indicate chromosome specificity with respect to increased positioning .", "we next wanted to investigate if the nucleosome redistribution changes at the 24  hour timepoint reflected increased or decreased nucleosomal occupancy .", "we calculated the median nucleosome occupancy values on each chromosome for all of the loci measured .", "we discovered that nucleosome occupancy values change in a chromosome specific manner ( fig . ", "chromosomes 18 , 19 , and 22 show the greatest median nucleosome occupancy values at the 24  hour timepoint .", "conversely , chromosomes 1 , 2 , 9 , 10 , 12 , 15 , and 17 show the lower median nucleosome occupancy values at the 24  hour timepoint .", "we were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24  hour timepoint .", "most of the ranges of nucleosome occupancy values are not statistically significantly different from one another ( fig .  1c , yellow highlights ) .", "these two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . at the other end of the scale , chromosomes", "these results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint , nucleosomal occupancy changes in a chromosome specific manner .", "rkshv.219 virus is latently infected in islk.rkshv219 cell line , derived from islk cell line , as previously described by .", "rkshv.219 cells were seeded at 5    10 cells into 150-mm dish ( one day prior to induction ) . for induction of kshv reactivation ,", "the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2  g / ml doxycycline .", "kshv reactivation was induced at 0 , 6 , 12 , 24 , and 48  h. the 0  h sample was treated identically to other timepoints , however doxycycline was not added .", "two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay .", "following doxycycline induction of kshv reactivation , islk.rkshv219 cells were harvested at 0 , 6 , 12 , 24 , and 48  h at 2.5    10 cells .", "media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10  min at room temperature , to cross - link the chromatin structure .", "125  mm glycine was then added to stop the formaldehyde cross - linking reaction .", "nucleus isolation buffer , 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , and ph  7.8 were then added to islk.rkshv219 cross - linked cells . the cells were scraped and placed within a 50  ml conical tube , and the nuclei were isolated by centrifugation at 1000  g for 5  min at 4  c .", "micrococcal nuclease , mnase , is a internucleosomal cleavage reagent , which does not cleave the dna protected by a nucleosome .", "islk.rkshv219 nuclei were digested with a titration of mnase : 4  u / ml , 2  u / ml , 1  u / ml , and 0.5  u / ml ( worthington biochemical corp . ) in mnase cleavage buffer ( 5  mm kcl , 4  mm mgcl2 , 1  mm cacl2 , 12.5% glycerol , and 50  mm tris  cl ( ph  7.4 ) ) .", "a titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented .", "the nuclei , following mnase digestion , were treated with 0.2  g/l proteinase k and 1% sodium dodecyl sulfate .", "mnase digested dna for all samples was resolved on a 2% agarose gel , and mononucleosomally protected dna , ~  150  bp , was isolated by gel excision .", "following gel excision , the mononucleosomally protected dna was purified by electroelution , and for each sample 's mnase titration the mononucleosomally protected dna was combined .", "the mononucleosomally protected dna was further purified with phenol  chloroform extraction and precipitated with alcohol .", "islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55  c overnight for bare genomic dna isolation .", "we utilized a high - resolution dna microarray , which we customized to contain 1  kb upstream and downstream of the transcription start sites of 462 immunity - related genes .", "nimblegen 's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array .", "sequences were repeat - masked around the tsss , and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray .", "oligonucleotide probes , sized at 60  bp , were tiled on each subarray with a mean spacing of 47  bp overlap .", "approximately 180 probes of spacing of 12.5  bp were printed on the microarray for each locus .", "the samples were fluorescently labeled : nucleosomally protected dna with cy3 and bare genomic dna ( reference ) with cy5 .", "samples were hybridized to the dna microarray according to nimblegen 's instructions ( http://www.nimblegen.com/technology/index.html ) .", "the resulting .tiff files were used for microarray data feature extraction , primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow .", "specifically , loess spatial correction was applied to the raw data ; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes ; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal .", "the ratio between nucleosomal ( cy3 ) and bare genomic cy5 signals was then calculated and stored as a general feature format , .gff", "subsequent analysis was performed using a software developed in our laboratory dennislab.r , which runs in the r environment for statistical computing .", "dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome .", "finally , we analyzed the nucleosome distribution changes at the 24  hour timepoint on a per chromosome basis , by calculating the t - statistic and p - value ( assuming unequal variance ) for the pairwise comparison of the range of values between all chromosomes .", "in our previous work we demonstrated that nucleosome redistributions are widespread , transient , and dna - directed , .", "we next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner .", "we first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint ( fig . ", "these results comport with our previous observation that the greatest positioning occurs at the 24  hour timepoint , however do not indicate chromosome specificity with respect to increased positioning .", "we next wanted to investigate if the nucleosome redistribution changes at the 24  hour timepoint reflected increased or decreased nucleosomal occupancy .", "we calculated the median nucleosome occupancy values on each chromosome for all of the loci measured .", "we discovered that nucleosome occupancy values change in a chromosome specific manner ( fig . ", "chromosomes 18 , 19 , and 22 show the greatest median nucleosome occupancy values at the 24  hour timepoint .", "conversely , chromosomes 1 , 2 , 9 , 10 , 12 , 15 , and 17 show the lower median nucleosome occupancy values at the 24  hour timepoint .", "we were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24  hour timepoint .", "most of the ranges of nucleosome occupancy values are not statistically significantly different from one another ( fig .  1c , yellow highlights ) .", "these two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . at the other end of the scale , chromosomes 1 and 12 are statistically indistinguishable , yet different from all other chromosomes .", "these results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint , nucleosomal occupancy changes in a chromosome specific manner ."], "abstract_text": ["<S> in the eukaryotic nucleus , dna is packaged into chromatin . </S>", "<S> the fundamental subunit of chromatin is the nucleosome , dna is wrapped 1.6 times around a histone octamer core . </S>", "<S> nuclear processes in eukaryotes are impacted by whether regulatory dna is occupied by nucleosomes . </S>", "<S> we used microarrays to measure nucleosome occupancy in human cells post - kaposi 's sarcoma - associated herpesvirus ( kshv ) reactivation at hundreds of immunity - related loci . </S>", "<S> the detailed analysis of these technologies can be found in recent publications from our lab [ 1,3 ] . </S>", "<S> we found that nucleosome redistributions displayed chromosome specific nucleosome occupancy . </S>", "<S> this resource can be used to map nucleosome distributions in a variety of biological contexts . </S>"], "labels": null, "section_names": ["Direct link to deposited data", "Experimental design, materials and methods", "Cell culture and KSHV reactivation in iSLK.rKSHV219 cells", "Post-KSHV reactivation cell harvest and nucleus purification", "MNase digestion and purification of mononucleosomal protected DNA", "Microarray design and processing", "Data processing and analysis", "Nucleosome occupancy changes occur in a chromosome specific manner"], "sections": [[""], ["rkshv.219 virus is latently infected in islk.rkshv219 cell line , derived from islk cell line , as previously described by .", "rkshv.219 cells were seeded at 5    10 cells into 150-mm dish ( one day prior to induction ) . for induction of kshv reactivation ,", "the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2  g / ml doxycycline .", "kshv reactivation was induced at 0 , 6 , 12 , 24 , and 48  h. the 0  h sample was treated identically to other timepoints , however doxycycline was not added .", "two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay .", "following doxycycline induction of kshv reactivation , islk.rkshv219 cells were harvested at 0 , 6 , 12 , 24 , and 48  h at 2.5    10 cells .", "media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10  min at room temperature , to cross - link the chromatin structure .", "125  mm glycine was then added to stop the formaldehyde cross - linking reaction .", "nucleus isolation buffer , 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , and ph  7.8 were then added to islk.rkshv219 cross - linked cells .", "the cells were scraped and placed within a 50  ml conical tube , and the nuclei were isolated by centrifugation at 1000  g for 5  min at 4  c .", "micrococcal nuclease , mnase , is a internucleosomal cleavage reagent , which does not cleave the dna protected by a nucleosome .", "islk.rkshv219 nuclei were digested with a titration of mnase : 4  u / ml , 2  u / ml , 1  u / ml , and 0.5  u / ml ( worthington biochemical corp . ) in mnase cleavage buffer ( 5  mm kcl , 4  mm mgcl2 , 1  mm cacl2 , 12.5% glycerol , and 50  mm tris  cl ( ph  7.4 ) ) .", "a titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented .", "the nuclei , following mnase digestion , were treated with 0.2  g/l proteinase k and 1% sodium dodecyl sulfate .", "mnase digested dna for all samples was resolved on a 2% agarose gel , and mononucleosomally protected dna , ~  150  bp , was isolated by gel excision .", "following gel excision , the mononucleosomally protected dna was purified by electroelution , and for each sample 's mnase titration the mononucleosomally protected dna was combined .", "the mononucleosomally protected dna was further purified with phenol  chloroform extraction and precipitated with alcohol .", "islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55  c overnight for bare genomic dna isolation .", "we utilized a high - resolution dna microarray , which we customized to contain 1  kb upstream and downstream of the transcription start sites of 462 immunity - related genes .", "nimblegen 's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array .", "sequences were repeat - masked around the tsss , and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray .", "oligonucleotide probes , sized at 60  bp , were tiled on each subarray with a mean spacing of 47  bp overlap .", "approximately 180 probes of spacing of 12.5  bp were printed on the microarray for each locus .", "the samples were fluorescently labeled : nucleosomally protected dna with cy3 and bare genomic dna ( reference ) with cy5 .", "samples were hybridized to the dna microarray according to nimblegen 's instructions ( http://www.nimblegen.com/technology/index.html ) .", "the resulting .tiff files were used for microarray data feature extraction , primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow .", "specifically , loess spatial correction was applied to the raw data ; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes ; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal .", "the ratio between nucleosomal ( cy3 ) and bare genomic cy5 signals was then calculated and stored as a general feature format , .gff", "subsequent analysis was performed using a software developed in our laboratory dennislab.r , which runs in the r environment for statistical computing .", "dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome .", "finally , we analyzed the nucleosome distribution changes at the 24  hour timepoint on a per chromosome basis , by calculating the t - statistic and p - value ( assuming unequal variance ) for the pairwise comparison of the range of values between all chromosomes . in our previous work", "we demonstrated that nucleosome redistributions are widespread , transient , and dna - directed , .", "we next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner .", "we first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint ( fig . ", "these results comport with our previous observation that the greatest positioning occurs at the 24  hour timepoint , however do not indicate chromosome specificity with respect to increased positioning .", "we next wanted to investigate if the nucleosome redistribution changes at the 24  hour timepoint reflected increased or decreased nucleosomal occupancy .", "we calculated the median nucleosome occupancy values on each chromosome for all of the loci measured .", "we discovered that nucleosome occupancy values change in a chromosome specific manner ( fig . ", "chromosomes 18 , 19 , and 22 show the greatest median nucleosome occupancy values at the 24  hour timepoint .", "conversely , chromosomes 1 , 2 , 9 , 10 , 12 , 15 , and 17 show the lower median nucleosome occupancy values at the 24  hour timepoint .", "we were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24  hour timepoint .", "most of the ranges of nucleosome occupancy values are not statistically significantly different from one another ( fig .  1c , yellow highlights ) .", "these two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . at the other end of the scale , chromosomes", "these results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint , nucleosomal occupancy changes in a chromosome specific manner ."], ["rkshv.219 virus is latently infected in islk.rkshv219 cell line , derived from islk cell line , as previously described by .", "rkshv.219 cells were seeded at 5    10 cells into 150-mm dish ( one day prior to induction ) . for induction of kshv reactivation ,", "the previous medium is replaced with 1% fbs dmem media containing a final concentration of 0.2  g / ml doxycycline .", "kshv reactivation was induced at 0 , 6 , 12 , 24 , and 48  h. the 0  h sample was treated identically to other timepoints , however doxycycline was not added .", "two independent biological replicates for each timepoint in islk.rkshv219 cell lines were processed and behaved similar for the nucleosome distribution assay ."], ["following doxycycline induction of kshv reactivation , islk.rkshv219 cells were harvested at 0 , 6 , 12 , 24 , and 48  h at 2.5    10 cells .", "media was aspirated and then 1% formaldehyde in pbs was incubated with the cells for 10  min at room temperature , to cross - link the chromatin structure .", "125  mm glycine was then added to stop the formaldehyde cross - linking reaction .", "nucleus isolation buffer , 0.3  m sucrose , 2  mm mgoac2 , 1  mm cacl2 , 1% nonidet p-40 , 10  mm hepes , and ph  7.8 were then added to islk.rkshv219 cross - linked cells . the cells were scraped and placed within a 50  ml conical tube , and the nuclei were isolated by centrifugation at 1000  g for 5  min at 4  c ."], ["micrococcal nuclease , mnase , is a internucleosomal cleavage reagent , which does not cleave the dna protected by a nucleosome .", "islk.rkshv219 nuclei were digested with a titration of mnase : 4  u / ml , 2  u / ml , 1  u / ml , and 0.5  u / ml ( worthington biochemical corp . ) in mnase cleavage buffer ( 5  mm kcl , 4  mm mgcl2 , 1  mm cacl2 , 12.5% glycerol , and 50  mm tris  cl ( ph  7.4 ) ) .", "a titration of mnase concentrations was utilized to ensure nucleosomes that are more and less readily cleaved are equally represented .", "the nuclei , following mnase digestion , were treated with 0.2  g/l proteinase k and 1% sodium dodecyl sulfate .", "mnase digested dna for all samples was resolved on a 2% agarose gel , and mononucleosomally protected dna , ~  150  bp , was isolated by gel excision .", "following gel excision , the mononucleosomally protected dna was purified by electroelution , and for each sample 's mnase titration the mononucleosomally protected dna was combined .", "the mononucleosomally protected dna was further purified with phenol  chloroform extraction and precipitated with alcohol .", "islk.rkshv219 cells were resuspended in 0.2-g/l proteinase k and 1% sds solution and incubated at 55  c overnight for bare genomic dna isolation ."], ["we utilized a high - resolution dna microarray , which we customized to contain 1  kb upstream and downstream of the transcription start sites of 462 immunity - related genes .", "nimblegen 's 12-plex and hd2 design were utilized for the custom dna microarray 100319_hg19_immunity_jd_cgh array .", "sequences were repeat - masked around the tsss , and only probes containing unique sequences for both the forward and reverse dna strands were printed on the microarray .", "oligonucleotide probes , sized at 60  bp , were tiled on each subarray with a mean spacing of 47  bp overlap .", "approximately 180 probes of spacing of 12.5  bp were printed on the microarray for each locus .", "the samples were fluorescently labeled : nucleosomally protected dna with cy3 and bare genomic dna ( reference ) with cy5 .", "samples were hybridized to the dna microarray according to nimblegen 's instructions ( http://www.nimblegen.com/technology/index.html ) ."], ["the resulting .tiff files were used for microarray data feature extraction , primary data analysis and generation of initial data files using the nimblegen deva software version 1.2 cgh workflow .", "specifically , loess spatial correction was applied to the raw data ; background correction was used to determine and adjust the signal data for noise using specifically designed background control probes ; and qspline normalization was applied to compensate for inherent differences in cy3 and cy5 signal .", "the ratio between nucleosomal ( cy3 ) and bare genomic cy5 signals was then calculated and stored as a general feature format , .gff", "subsequent analysis was performed using a software developed in our laboratory dennislab.r , which runs in the r environment for statistical computing .", "dennislab.r was used to generate summary statistics on the range of nucleosome distribution values for each individual chromosome .", "finally , we analyzed the nucleosome distribution changes at the 24  hour timepoint on a per chromosome basis , by calculating the t - statistic and p - value ( assuming unequal variance ) for the pairwise comparison of the range of values between all chromosomes ."], ["in our previous work we demonstrated that nucleosome redistributions are widespread , transient , and dna - directed , .", "we next wanted to determine if nucleosome redistributions showed chromosome specific changes in a time dependent manner .", "we first calculated the range of nucleosome occupancy values for all loci studied on a per chromosome basis for each post - kshv reactivation timepoint ( fig . ", "these results comport with our previous observation that the greatest positioning occurs at the 24  hour timepoint , however do not indicate chromosome specificity with respect to increased positioning .", "we next wanted to investigate if the nucleosome redistribution changes at the 24  hour timepoint reflected increased or decreased nucleosomal occupancy .", "we calculated the median nucleosome occupancy values on each chromosome for all of the loci measured .", "we discovered that nucleosome occupancy values change in a chromosome specific manner ( fig . ", "chromosomes 18 , 19 , and 22 show the greatest median nucleosome occupancy values at the 24  hour timepoint .", "conversely , chromosomes 1 , 2 , 9 , 10 , 12 , 15 , and 17 show the lower median nucleosome occupancy values at the 24  hour timepoint .", "we were next interested in calculating the statistical significance of the chromosome specific nucleosome occupancy changes that we had identified at the 24  hour timepoint .", "most of the ranges of nucleosome occupancy values are not statistically significantly different from one another ( fig .  1c , yellow highlights ) .", "these two chromosomes show clear increases in the nucleosome occupancy relative to the other chromosomes . at the other end of the scale , chromosomes 1 and 12 are statistically indistinguishable , yet different from all other chromosomes .", "these results demonstrate that while nucleosomal positioning remains relatively constant between chromosomes at each timepoint , nucleosomal occupancy changes in a chromosome specific manner ."]]}
{"article_id": "PMC5118976", "article_text": ["primary pleuropulmonary synovial sarcoma ( ppss ) is a rare soft tissue malignancy representing only 0.1% - 0.5% of all primary lung malignancies .", "the frequency of cardiac metastasis from soft tissue sarcomas ranges from 3.5% to 35% with only two cases of metastatic synovial sarcoma of the heart reported : one involving the right ventricle and the other the left ventricle .", "both cases had a primary tumor in the extremities rather than in the lungs without focusing on the imaging findings of these rare lesions ( 2 - 4 ) .", "we report a case of ppss that eventually metastasized to the heart in a 17-year - old female .", "a 17-year - old female presented with dull pain in the left upper chest alternating with brief periods of chest discomfort lasting a few months .", "she had no serious medical or surgical history and grew up in normal residential and social environments .", "all laboratory data including blood tests , standard biochemical tests , and urinalysis were within normal limits .", "chest radiographs ( figure 1a and b ) revealed a well - defined round mass in the left upper hemithorax .", "the mass was at an obtuse angle to the chest wall and was approximately 8 cm long .", "chest ct ( figure 1c - e ) confirmed a well - defined oval - shaped mass with heterogeneous enhancement in the left upper hemithorax abutting the pleura . in the pre - contrast image ,", "the lesion showed homogeneous soft tissue attenuation and a lack of calcification , but a small amount of high - attenuation fluid was present in the left pleural space , suggestive of hemothorax .", "after contrast enhancement , the mass showed heterogeneous enhancement , with attenuation similar to that of the back muscles , and contained subtle low densities suggestive of necrosis or hemorrhage .", "the mediastinal and hilar lymph nodes were not enlarged . in the scanned portion of the abdomen", "f-18 fluorodeoxyglucose positron emission tomography - ct ( 18f fdg pet / ct ) revealed increased fdg uptake in the mass , with a maximum standardized uptake value of 31.7 ( figure 1f ) . from these imaging findings ,", "the diagnoses of a localized fibrous tumor of the pleura , malignant mesothelioma , metastatic pleural malignancy , and rare primary pulmonary sarcoma ( e.g. , pleuropulmonary synovial sarcoma , fibrosarcoma , leimyosarcoma , sarcomatoid carcinoma , malignant nerve sheath tumor , hemangiopericytoma , and malignant fibrous histiocytoma ) were considered .", "these diagnoses were based on the tumor s appearance as a sharply marginated , heterogeneously enhanced mass with low attenuation foci and no involvement of the bone or calcifications .", "ct - guided core needle biopsy of the mass yielded two samples of yellowish gelatinous material .", "microscopic examination showed relatively uniform spindle - shaped cells with occasional mitoses arranged in tight fascicles .", "the tumor cells were immunoreactive to vimentin and cd99 and were focally positive for epithelial membrane antigen ( ema ) , suggesting a monophasic synovial sarcoma .", "medical and surgical oncologists agreed to treat the tumor with surgical resection and to initiate a combination chemotherapy regimen consisting of five cycles of vincristine , doxorubicin , and cyclophosphamide , alternating with four cycles of ifosfamide and etoposide .", "a thoracotomy was performed , and the mass was found to arise from the visceral pleura and to adhere to the left first to third intercostal spaces , with invasion into the left first intercostal muscle with adjacent hemothorax . the surgical specimen consisted of an 8.0  6.5  5.5 cm well - circumscribed but unencapsulated tumor .", "the tumor was whitish - yellow , soft , and fleshy with cystic degenerative changes and hemorrhage .", "histologically , the tumor was composed of densely packed , cellular sheets of spindle cells , and some of the tumor cells were arranged in intersecting fascicles in a herringbone pattern , suggesting fibrosarcoma - like changes ( figure 1 g - h ) .", "the tumor cells were relatively uniform with ovoid nuclei , scant cytoplasm , and occasional mitotic figures .", "immunohistochemical staining revealed immunoreactivity of the tumor cells to cytokeratin , ema , and cd99 but not to cd34 or desmin ( figure 1i - j ) .", "a final diagnosis of a monophasic synovial sarcoma from the visceral pleura was made based on the histological and immunohistochemical findings .", "she was discharged after surgical resection without complications , after which she underwent adjuvant chemotherapy and radiotherapy .", "the patient remained stable for 28 months until follow - up contrast - enhanced chest ct revealed a 1.6 cm low attenuation nodule with poor enhancement in the left pericardial area and a small amount of left pericardial and pleural fluid ( figure 2a ) .", "subsequent 18f fdg pet / ct showed slightly increased fdg uptake in the developing nodule with suspected metastasis .", "five months after discovery , follow up contrast - enhanced chest ct demonstrated a well - circumscribed polycyclic marginated mass with heterogeneous enhancement in the left pericardial region that had enlarged from 1.6 cm to 5.1 cm ( figure 2b ) .", "although mri is the most accurate technique for demonstrating cardiac mass , oncologists found the following mri might be ineffective for choosing further treatment for the pericardial mass due to high cost .", "they had no choice but to do surgery for the mass whether to perform mri or not .", "therefore , they agreed to operate surgical resection to treat pericardial mass without obtaining cardiac mri .", "the tumor specimen was a 6.0  5.0  2.0 cm well - circumscribed , whitish - yellowish , fleshy to rubbery mass with zones of necrosis and hemorrhage .", "the tumor was lateral to the left ventricular wall with invasion into the pericardium , myoendocardium , and endocardium of the left ventricle .", "microscopically , the tumor contained tight clusters and fascicles of spindle cells with scant cytoplasm , identical to the ppss , confirming a metastatic synovial sarcoma with left pericardial , myoendocardial , and endocardial involvement .", "after metastatectomy , the patient received adjuvant chemotherapy and radiotherapy . the patient s postoperative period was complicated by recurrent pneumonia .", "ppss is a rare mesenchymal spindle cell malignancy representing a type of pulmonary sarcoma that comprises 0.1% - 0.5% of all primary lung malignancies .", "typically , it arises from the chest wall , lung parenchyma , pleura , or mediastinum , but rarely at the level of the bronchial tree , and during the fifth decade of the patient s life ( 1 ) .", "the 5-year survival rate varies from 36% to 76% ( 6 ) . a small but increasing number of cases of ppss", "have been reported over the past decades ( 5 ) , but very few of these cases have involved cardiac metastasis .", "cardiac metastasis from soft tissue sarcomas varies from 3.5% to 35% , with only two cases of metastatic synovial sarcoma of the heart reported : one involving the right ventricle and the other the left ventricle .", "both of those cases had a primary tumor in the extremities but not in the lung without focusing on the imaging findings of these rare lesions ( 2 - 4 ) .", "additionally , a literature review of soft tissue sarcomas included only 105 cases of cardiac metastasis . in that review ,", "the predominant histological type was leiomyosarcoma ( 22% ) followed by rhabdomyosarcoma ( 19% ) , and liposarcoma ( 9% ) ; 27% of the cases were unspecified .", "that report suggested that the clinical manifestation of cardiac metastasis can be subtle , as one - third presented with congestive cardiac failure , which led to other complications such as embolism , tumor thrombus , cardiac tamponade , and arrhythmia ( 2 ) . in our case , the patient was initially asymptomatic except for dull chest pains with only pericardial effusion on follow - up chest ct .", "symptoms frequently associated with these tumors are chest pain , dyspnea , and cough ; up to 40% of patients are asymptomatic ( 7 ) . the radiological features of ppss are summarized as follows : chest radiographs typically demonstrate a well - defined pleural - based or parenchymal mass - like lesion occasionally associated with ipsilateral pleural effusion . generally , the contralateral lung is not involved .", "ct and magnetic resonance imaging ( mri ) are relatively useful tools for evaluating ppss , although the findings are nonspecific .", "the most common ct finding of ppss is a well - defined , heterogeneously enhancing mass often with combined ipsilateral pleural effusion .", "the inner low - density area often contains necrosis , hemorrhage , or fibrous components . associated lymphadenopathy as well as calcifications , cortical bone destruction , or tumor infiltration of the chest wall and musculature are infrequent .", "ppss often exhibits heterogeneous high signal intensity on t2-weighted images and iso- or low signal intensity on t1-weighted images . after administering gadolinium", "differential diagnoses for ppss include metastasis from the extrapulmonary synovial sarcoma , a primary tumor such as a pleural fibroma or mesothelioma , or other primary sarcomatous or sarcomatoid neoplasms .", "the typical clinical and radiologic features can help narrow down the differential diagnosis of ppss .", "a history of asbestos exposure and the presence of pleural plaques are indicative of malignant mesothelioma .", "metastatic disease rarely manifests as a large solitary pulmonary lesion , although many other rare primary sarcomatous or sarcomatoid neoplasms can not be distinguished reliably from ppss based on clinical and radiologic features alone ( 5 ) .", "monophasic synovial sarcomas are much more common , representing 94% of ppss , and are composed of uniform spindle cells .", "the surrounding plump spindle cells have hyperchromatic nuclei with scant cytoplasm resembling the tumor cells of monophasic synovial sarcoma .", "immunohistochemically , most synovial sarcomas show focal expression of cytokeratin and ema . furthermore , 30% are protein s-100 positive , 60% - 70% cd99 positive , and 75% - 100%", "bcl-2 positive ( 8) . the prognosis of ppss remains uncertain but is usually poor .", "the 5-year survival rate varies from 36% to 76% depending on the patient s age , tumor size , and tumor resectability ( 6 ) .", "there is no recommended treatment for ppss , but the mainstay of treatment for all soft tissue sarcomas is surgical resection .", "aggressive surgery can reduce the tumor size before or after chemotherapy , and complete surgical excision seems to improve the survival of patients with ppss .", "combination chemotherapy consisting of doxorubicin and ifosfamide has been associated with higher rates of survival in patients with synovial sarcoma ( 6 ) . in summary , ppss is a very rare , aggressive malignancy that must be considered in the differential diagnosis of lung and pleural malignancies .", "the imaging findings of ppss are nonspecific , and ppss is difficult to confirm based on imaging ; detailed immunohistochemical staining is required for definitive diagnosis .", "ppss with cardiac metastasis is also extremely rare , and only two such cases have been reported .", "however , cardiac metastasis of ppss can lead to immediate or delayed death ; therefore , early detection and appropriate management are very important to extend survival ."], "abstract_text": ["<S> primary pleuropulmonary synovial sarcomas are rare soft tissue malignancies ; combined metastatic involvement of the heart is extremely rare . in this case report , a 17-year - old female presented with a history of chest pain . </S>", "<S> chest radiographs revealed a round mass in the left upper hemithorax , and computed tomography ( ct ) showed a well - defined heterogeneous enhancing mass abutting the pleura . </S>", "<S> a core needle biopsy revealed malignant spindle cells . </S>", "<S> surgical resection was performed , and a final diagnosis of primary pleural synovial sarcoma , monophasic fibrous type , was made . </S>", "<S> the patient underwent radical irradiation and chemotherapy and remained stable for 28 months until a follow - up chest ct showed a poorly enhancing nodule in the left pericardial region that enlarged after 5 months . </S>", "<S> surgical resection was performed . </S>", "<S> histological examination confirmed metastatic cardiac involvement from a primary pleural synovial sarcoma . </S>", "<S> we report this unusual case of a primary pleural synovial sarcoma metastasis to the heart . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Case Presentation", "3. Discussion"], "sections": [["primary pleuropulmonary synovial sarcoma ( ppss ) is a rare soft tissue malignancy representing only 0.1% - 0.5% of all primary lung malignancies .", "the frequency of cardiac metastasis from soft tissue sarcomas ranges from 3.5% to 35% with only two cases of metastatic synovial sarcoma of the heart reported : one involving the right ventricle and the other the left ventricle .", "both cases had a primary tumor in the extremities rather than in the lungs without focusing on the imaging findings of these rare lesions ( 2 - 4 ) .", "we report a case of ppss that eventually metastasized to the heart in a 17-year - old female ."], ["a 17-year - old female presented with dull pain in the left upper chest alternating with brief periods of chest discomfort lasting a few months .", "she had no serious medical or surgical history and grew up in normal residential and social environments .", "all laboratory data including blood tests , standard biochemical tests , and urinalysis were within normal limits .", "chest radiographs ( figure 1a and b ) revealed a well - defined round mass in the left upper hemithorax .", "the mass was at an obtuse angle to the chest wall and was approximately 8 cm long .", "chest ct ( figure 1c - e ) confirmed a well - defined oval - shaped mass with heterogeneous enhancement in the left upper hemithorax abutting the pleura . in the pre - contrast image ,", "the lesion showed homogeneous soft tissue attenuation and a lack of calcification , but a small amount of high - attenuation fluid was present in the left pleural space , suggestive of hemothorax .", "after contrast enhancement , the mass showed heterogeneous enhancement , with attenuation similar to that of the back muscles , and contained subtle low densities suggestive of necrosis or hemorrhage .", "the mediastinal and hilar lymph nodes were not enlarged . in the scanned portion of the abdomen", "f-18 fluorodeoxyglucose positron emission tomography - ct ( 18f fdg pet / ct ) revealed increased fdg uptake in the mass , with a maximum standardized uptake value of 31.7 ( figure 1f ) . from these imaging findings ,", "the diagnoses of a localized fibrous tumor of the pleura , malignant mesothelioma , metastatic pleural malignancy , and rare primary pulmonary sarcoma ( e.g. , pleuropulmonary synovial sarcoma , fibrosarcoma , leimyosarcoma , sarcomatoid carcinoma , malignant nerve sheath tumor , hemangiopericytoma , and malignant fibrous histiocytoma ) were considered .", "these diagnoses were based on the tumor s appearance as a sharply marginated , heterogeneously enhanced mass with low attenuation foci and no involvement of the bone or calcifications .", "ct - guided core needle biopsy of the mass yielded two samples of yellowish gelatinous material .", "microscopic examination showed relatively uniform spindle - shaped cells with occasional mitoses arranged in tight fascicles .", "the tumor cells were immunoreactive to vimentin and cd99 and were focally positive for epithelial membrane antigen ( ema ) , suggesting a monophasic synovial sarcoma .", "medical and surgical oncologists agreed to treat the tumor with surgical resection and to initiate a combination chemotherapy regimen consisting of five cycles of vincristine , doxorubicin , and cyclophosphamide , alternating with four cycles of ifosfamide and etoposide .", "a thoracotomy was performed , and the mass was found to arise from the visceral pleura and to adhere to the left first to third intercostal spaces , with invasion into the left first intercostal muscle with adjacent hemothorax . the surgical specimen consisted of an 8.0  6.5  5.5 cm well - circumscribed but unencapsulated tumor .", "the tumor was whitish - yellow , soft , and fleshy with cystic degenerative changes and hemorrhage .", "histologically , the tumor was composed of densely packed , cellular sheets of spindle cells , and some of the tumor cells were arranged in intersecting fascicles in a herringbone pattern , suggesting fibrosarcoma - like changes ( figure 1 g - h ) .", "the tumor cells were relatively uniform with ovoid nuclei , scant cytoplasm , and occasional mitotic figures .", "immunohistochemical staining revealed immunoreactivity of the tumor cells to cytokeratin , ema , and cd99 but not to cd34 or desmin ( figure 1i - j ) .", "a final diagnosis of a monophasic synovial sarcoma from the visceral pleura was made based on the histological and immunohistochemical findings .", "she was discharged after surgical resection without complications , after which she underwent adjuvant chemotherapy and radiotherapy .", "the patient remained stable for 28 months until follow - up contrast - enhanced chest ct revealed a 1.6 cm low attenuation nodule with poor enhancement in the left pericardial area and a small amount of left pericardial and pleural fluid ( figure 2a ) .", "subsequent 18f fdg pet / ct showed slightly increased fdg uptake in the developing nodule with suspected metastasis .", "five months after discovery , follow up contrast - enhanced chest ct demonstrated a well - circumscribed polycyclic marginated mass with heterogeneous enhancement in the left pericardial region that had enlarged from 1.6 cm to 5.1 cm ( figure 2b ) .", "although mri is the most accurate technique for demonstrating cardiac mass , oncologists found the following mri might be ineffective for choosing further treatment for the pericardial mass due to high cost .", "they had no choice but to do surgery for the mass whether to perform mri or not .", "therefore , they agreed to operate surgical resection to treat pericardial mass without obtaining cardiac mri .", "the tumor specimen was a 6.0  5.0  2.0 cm well - circumscribed , whitish - yellowish , fleshy to rubbery mass with zones of necrosis and hemorrhage .", "the tumor was lateral to the left ventricular wall with invasion into the pericardium , myoendocardium , and endocardium of the left ventricle .", "microscopically , the tumor contained tight clusters and fascicles of spindle cells with scant cytoplasm , identical to the ppss , confirming a metastatic synovial sarcoma with left pericardial , myoendocardial , and endocardial involvement .", "after metastatectomy , the patient received adjuvant chemotherapy and radiotherapy . the patient s postoperative period was complicated by recurrent pneumonia ."], ["ppss is a rare mesenchymal spindle cell malignancy representing a type of pulmonary sarcoma that comprises 0.1% - 0.5% of all primary lung malignancies .", "typically , it arises from the chest wall , lung parenchyma , pleura , or mediastinum , but rarely at the level of the bronchial tree , and during the fifth decade of the patient s life ( 1 ) .", "the 5-year survival rate varies from 36% to 76% ( 6 ) . a small but increasing number of cases of ppss", "have been reported over the past decades ( 5 ) , but very few of these cases have involved cardiac metastasis .", "cardiac metastasis from soft tissue sarcomas varies from 3.5% to 35% , with only two cases of metastatic synovial sarcoma of the heart reported : one involving the right ventricle and the other the left ventricle .", "both of those cases had a primary tumor in the extremities but not in the lung without focusing on the imaging findings of these rare lesions ( 2 - 4 ) .", "additionally , a literature review of soft tissue sarcomas included only 105 cases of cardiac metastasis . in that review ,", "the predominant histological type was leiomyosarcoma ( 22% ) followed by rhabdomyosarcoma ( 19% ) , and liposarcoma ( 9% ) ; 27% of the cases were unspecified .", "that report suggested that the clinical manifestation of cardiac metastasis can be subtle , as one - third presented with congestive cardiac failure , which led to other complications such as embolism , tumor thrombus , cardiac tamponade , and arrhythmia ( 2 ) . in our case , the patient was initially asymptomatic except for dull chest pains with only pericardial effusion on follow - up chest ct .", "symptoms frequently associated with these tumors are chest pain , dyspnea , and cough ; up to 40% of patients are asymptomatic ( 7 ) . the radiological features of ppss are summarized as follows : chest radiographs typically demonstrate a well - defined pleural - based or parenchymal mass - like lesion occasionally associated with ipsilateral pleural effusion . generally , the contralateral lung is not involved .", "ct and magnetic resonance imaging ( mri ) are relatively useful tools for evaluating ppss , although the findings are nonspecific .", "the most common ct finding of ppss is a well - defined , heterogeneously enhancing mass often with combined ipsilateral pleural effusion .", "the inner low - density area often contains necrosis , hemorrhage , or fibrous components . associated lymphadenopathy as well as calcifications , cortical bone destruction , or tumor infiltration of the chest wall and musculature are infrequent .", "ppss often exhibits heterogeneous high signal intensity on t2-weighted images and iso- or low signal intensity on t1-weighted images . after administering gadolinium", "differential diagnoses for ppss include metastasis from the extrapulmonary synovial sarcoma , a primary tumor such as a pleural fibroma or mesothelioma , or other primary sarcomatous or sarcomatoid neoplasms .", "the typical clinical and radiologic features can help narrow down the differential diagnosis of ppss .", "a history of asbestos exposure and the presence of pleural plaques are indicative of malignant mesothelioma .", "metastatic disease rarely manifests as a large solitary pulmonary lesion , although many other rare primary sarcomatous or sarcomatoid neoplasms can not be distinguished reliably from ppss based on clinical and radiologic features alone ( 5 ) .", "monophasic synovial sarcomas are much more common , representing 94% of ppss , and are composed of uniform spindle cells .", "the surrounding plump spindle cells have hyperchromatic nuclei with scant cytoplasm resembling the tumor cells of monophasic synovial sarcoma .", "immunohistochemically , most synovial sarcomas show focal expression of cytokeratin and ema . furthermore , 30% are protein s-100 positive , 60% - 70% cd99 positive , and 75% - 100%", "bcl-2 positive ( 8) . the prognosis of ppss remains uncertain but is usually poor .", "the 5-year survival rate varies from 36% to 76% depending on the patient s age , tumor size , and tumor resectability ( 6 ) .", "there is no recommended treatment for ppss , but the mainstay of treatment for all soft tissue sarcomas is surgical resection .", "aggressive surgery can reduce the tumor size before or after chemotherapy , and complete surgical excision seems to improve the survival of patients with ppss .", "combination chemotherapy consisting of doxorubicin and ifosfamide has been associated with higher rates of survival in patients with synovial sarcoma ( 6 ) . in summary , ppss is a very rare , aggressive malignancy that must be considered in the differential diagnosis of lung and pleural malignancies .", "the imaging findings of ppss are nonspecific , and ppss is difficult to confirm based on imaging ; detailed immunohistochemical staining is required for definitive diagnosis .", "ppss with cardiac metastasis is also extremely rare , and only two such cases have been reported .", "however , cardiac metastasis of ppss can lead to immediate or delayed death ; therefore , early detection and appropriate management are very important to extend survival ."]]}
{"article_id": "PMC4701856", "article_text": ["the work is self - sponsored by the first author who conceptualized and designed and wrote the manuscript . the second author assisted in the surgery provided some literature review and made necessary correction which lead to the final manuscript .", "the ethical committee of first choice specialist hospital where the authors carried out the procedure approved the work .", "he understood that part of his images will be published and also understood that due to the rarity of the case , that it is worthwhile publishing it for education and for health care delivery .", "the second author assisted during the surgery , provided immediate care , provided some literature review and made necessary correction which lead to the final manuscript ."], "abstract_text": ["<S> highlightsremoval of an entrapped penile ring where possible should be done in a simple non - invasive way.milking the oedematous penis through the thick metallic ring in a slippery field complemented by levering with a blunt end paediatric bone lever , was the technique used to remove the ring.the ring was extricated without any morbidity and the patient went home on the same day . </S>"], "labels": null, "section_names": ["Conflict of interest", "Funding", "Ethical approval", "Consent", "Author contribution", "Guarantor"], "sections": [[""], ["the work is self - sponsored by the first author who conceptualized and designed and wrote the manuscript . the second author assisted in the surgery provided some literature review and made necessary correction which lead to the final manuscript ."], ["the ethical committee of first choice specialist hospital where the authors carried out the procedure approved the work ."], ["he understood that part of his images will be published and also understood that due to the rarity of the case , that it is worthwhile publishing it for education and for health care delivery ."], ["the second author assisted during the surgery , provided immediate care , provided some literature review and made necessary correction which lead to the final manuscript ."], [""]]}
{"article_id": "PMC4211479", "article_text": ["a number of skin disorders have been described in patients with acquired immunodeficiency syndrome ( aids ) . among them , secondary infections are common , but their incidence has decreased considerably following the introduction of combined therapies targeted to the human immunodeficiency virus ( hiv ) .", "some clinical presentations may be puzzling , particularly in secondary syphilis exhibiting lesions showing a marked tendency to polymorphism .", "a 29-year - old woman with a three - year history of aids presented with polymorphic papules on the face and abdomen .", "the skin lesions were asymptomatic but the patient complained of discrete malaise , stiff neck , myalgia headache , and mild fever . a biopsy specimen was taken from a papule on the abdomen .", "a series of 5-m thick sections were cut from the formalin - fixed paraffin - embedded biopsy .", "an immunohistochemical assessment was performed using a rabbit polyclonal antibody directed to treponema pallidum ( 1:200 biocare medical , walnut creek , ca , usa ) .", "the envision ( dakopatts , glostrup , denmark ) polymer - based revelation system and fast red ( dakopatts ) staining were used .", "negative immunohistochemical controls were performed by omitting or substituting the primary and the secondary antibodies in the laboratory procedure .", "the dermoepidermal junction contained a band - like infiltrate composed mostly of lymphocytes , histiocytes , and plasma cells .", "a deeper cell infiltrate of similar composition extended along the microvasculature , hair follicles , and sweat glands .", "the typical spiral , corkscrew , and threadlike spirochetes were highlighted by the red chromogen , and the contrast with the clear background was striking ( figure 1a , b , c ) .", "their presence inside the lichenoid infiltrate was associated with a dense superficial and deep perivascular cuff of spirochetes .", "the latter slender spirochetes were clustered in the dermal stroma ( figure 1a ) and in rims confined to the perivascular areas ( figure 1b , c ) .", "in addition , some t. pallidum were evident in the cytoplasm of cells , particularly endothelial cells ( figure 1c ) .", "dermal homing of t. pallidum on immunohistochemistry : ( a ) multiple interstitial clumps of spirochetes ( 200 ) ; ( b ) vascular trapping of spirochetes ( 400 ) ; ( c ) prominent accumulation of spirochetes in the microvasculature wall ( 400 ) . early syphilis .", "dermal homing of t. pallidum on immunohistochemistry : ( a ) multiple interstitial clumps of spirochetes ( 200 ) ; ( b ) vascular trapping of spirochetes ( 400 ) ; ( c ) prominent accumulation of spirochetes in the microvasculature wall ( 400 ) .", "in the present case , skin immunohistochemistry shed some light on the diagnosis of syphilis in an aids patient and showed the dermal homing and the microvascular tropism of t. pallidum .", "it is acknowledged that during a five - year period after inoculation , t. pallidum spreads to every organ .", "a long time later , it commonly re - emerges as a chronic and deadly illness . at any stage in its evolution , syphilis may mimic a number of other unrelated diseases .", "during the past decade , a sizable proportion of the population with syphilis corresponded to gay men coinfected with hiv . when the clinical diagnosis of syphilis is not established , a skin biopsy sometimes is submitted to the dermatopathologist without any relevant information . at the conventional histological examination ,", "the diagnostic clues for syphilis are not always obvious because the disease presentation depends on both the host immunological response to the infection and the diverse angioinvasive propensity of the t. pallidum strains", "indeed , the histochemical silver stain may be difficult to interpret owing to heavy background staining .", "immunohistochemistry using an antibody directed to t. pallidum was reported to improve the histological diagnostic accuracy of syphilis .", "the present finding was assumed to illustrate the migration of t. pallidum toward the microvasculature during early syphilis . in summary , t. pallidum were abundant and heavily clustered in some specific portions of the skin .", "the peculiar homing of t. pallidum in the skin appears quite specific as it has not been reported for any other infectious microorganism ."], "abstract_text": ["<S> during the past decade or so , the incidence of syphilis has increased in most parts of the world . in some urban regions , a coinfection with human immunodeficiency virus </S>", "<S> is disclosed in nearly 50% of the cases . </S>", "<S> owing to the polymorphism of the lesions , the clinical diagnosis may be puzzling . </S>", "<S> the homing patterns and migration paths of treponema pallidum in the skin during early syphilis represent the preliminary steps preceding dissemination to other organs . </S>", "<S> immunohistochemistry directed to t. pallidum is a convenient means for reaching the diagnosis and for exploring the dissemination process . </S>", "<S> the present case illustrates the dermal clustering and the vascular spread of t. pallidum in a woman with acquired immunodeficiency syndrome . </S>"], "labels": null, "section_names": ["Introduction", "Design and Methods", "Results", "Discussion"], "sections": [["a number of skin disorders have been described in patients with acquired immunodeficiency syndrome ( aids ) . among them , secondary infections are common , but their incidence has decreased considerably following the introduction of combined therapies targeted to the human immunodeficiency virus ( hiv ) .", "some clinical presentations may be puzzling , particularly in secondary syphilis exhibiting lesions showing a marked tendency to polymorphism ."], ["a 29-year - old woman with a three - year history of aids presented with polymorphic papules on the face and abdomen .", "the skin lesions were asymptomatic but the patient complained of discrete malaise , stiff neck , myalgia headache , and mild fever . a biopsy specimen was taken from a papule on the abdomen .", "a series of 5-m thick sections were cut from the formalin - fixed paraffin - embedded biopsy .", "an immunohistochemical assessment was performed using a rabbit polyclonal antibody directed to treponema pallidum ( 1:200 biocare medical , walnut creek , ca , usa ) .", "the envision ( dakopatts , glostrup , denmark ) polymer - based revelation system and fast red ( dakopatts ) staining were used .", "negative immunohistochemical controls were performed by omitting or substituting the primary and the secondary antibodies in the laboratory procedure ."], ["the dermoepidermal junction contained a band - like infiltrate composed mostly of lymphocytes , histiocytes , and plasma cells .", "a deeper cell infiltrate of similar composition extended along the microvasculature , hair follicles , and sweat glands .", "the typical spiral , corkscrew , and threadlike spirochetes were highlighted by the red chromogen , and the contrast with the clear background was striking ( figure 1a , b , c ) .", "their presence inside the lichenoid infiltrate was associated with a dense superficial and deep perivascular cuff of spirochetes .", "the latter slender spirochetes were clustered in the dermal stroma ( figure 1a ) and in rims confined to the perivascular areas ( figure 1b , c ) .", "in addition , some t. pallidum were evident in the cytoplasm of cells , particularly endothelial cells ( figure 1c ) .", "dermal homing of t. pallidum on immunohistochemistry : ( a ) multiple interstitial clumps of spirochetes ( 200 ) ; ( b ) vascular trapping of spirochetes ( 400 ) ; ( c ) prominent accumulation of spirochetes in the microvasculature wall ( 400 ) . early syphilis .", "dermal homing of t. pallidum on immunohistochemistry : ( a ) multiple interstitial clumps of spirochetes ( 200 ) ; ( b ) vascular trapping of spirochetes ( 400 ) ; ( c ) prominent accumulation of spirochetes in the microvasculature wall ( 400 ) ."], ["in the present case , skin immunohistochemistry shed some light on the diagnosis of syphilis in an aids patient and showed the dermal homing and the microvascular tropism of t. pallidum .", "it is acknowledged that during a five - year period after inoculation , t. pallidum spreads to every organ .", "a long time later , it commonly re - emerges as a chronic and deadly illness . at any stage in its evolution , syphilis may mimic a number of other unrelated diseases .", "during the past decade , a sizable proportion of the population with syphilis corresponded to gay men coinfected with hiv . when the clinical diagnosis of syphilis is not established , a skin biopsy sometimes is submitted to the dermatopathologist without any relevant information . at the conventional histological examination ,", "the diagnostic clues for syphilis are not always obvious because the disease presentation depends on both the host immunological response to the infection and the diverse angioinvasive propensity of the t. pallidum strains", "indeed , the histochemical silver stain may be difficult to interpret owing to heavy background staining .", "immunohistochemistry using an antibody directed to t. pallidum was reported to improve the histological diagnostic accuracy of syphilis .", "the present finding was assumed to illustrate the migration of t. pallidum toward the microvasculature during early syphilis . in summary , t. pallidum were abundant and heavily clustered in some specific portions of the skin .", "the peculiar homing of t. pallidum in the skin appears quite specific as it has not been reported for any other infectious microorganism ."]]}
{"article_id": "PMC3705988", "article_text": ["hadrontherapy is an innovative form of radiotherapy , based on high - technology equipment using proton or carbon ions beams to destroy tumours [ 1 , 2 ] .", "this treatment method enables significantly higher ballistic precision to be achieved , compared to photons ( x - rays ) with , as expected therapeutic benefit , an improvement of quality of life and chances of recovery .", "carbon ions are also specifically characterised by superior biological efficacy ( relative biological effectiveness from 1.5 to 3 ) , overcoming the radiation resistance of certain cancers to photons and even protons .", "indeed , carbon ion beams when compared to x - rays represent a distinct advantage for the treatment of highly radiation - resistant tumours .", "an initial study assessing recruitment potential for proton therapy was conducted in 1998 in italy , showing an incidence of 10  825 cases / year .", "one year later , a second study was carried out in the context of the medaustron project for the construction of a carbon ions therapy centre in austria . considering patients living in austria and neighbouring countries ,", "the patient recruitment potential ( proton and carbon ) was estimated with an incidence of 13  145 cases / year . in france , in 2002 , a third study was conducted in the context of the etoile medical project .", "this study revealed an annual incidence of approximately 5320 cases / year for carbon ions .", "concomitantly , a fourth study was conducted , again in the context of the medaustron project , estimating the incidence of cases eligible for hadrontherapy in austria at 2044 cases / year .", "finally , again in 2002 , in the context of the project for the construction of the cnao centre in italy , an epidemiological study was conducted , estimating the incidence of cases eligible for carbon therapy at 3694 cases / year and proton therapy at 1885 cases / year . .", "since 2004 , no other epidemiological studies have been published for hadrontherapy . in 2011 , however , the japanese team from the national institute for radiological sciences ( nirs ) , pioneer in carbon therapy , published the results of its 1994 to 2011 clinical activity .", "patients were included in clinical trials and the number of patients receiving carbon therapy was 6157 .", "this value is the incidence of treated cases , over 17 years without any notion of recruitment potential and with a rather slow ramp up of the activity .  ", "a list of priority indications that can be treated by hadrontherapy ( table 1 ) was drawn up by the gcs etoile medical group , in collaboration with national and international experts , based on published data ranked using an  evidence - based medicine", "this literature review was based on provided or expected medical service criteria ( survival and quality of life ) . according to the classification used by the european survey group of rare diseases , the eligible tumours that can be treated by hadrontherapy conform to the criteria of rare diseases based on the following thresholds of epidemiological indicators : prevalence  50 cases/100  000 persons within a given population and incidence  6 cases/100  000 persons / year .", "indeed , for all tumor types identified during the previous hadrontherapy epidemiological studies [ 58 ] , the calculated and cumulative incidences are all below these thresholds ( table 2 ) . amongst the hadrontherapy indications , 25 are tumours listed in the orphanet international database of rare diseases   ( cf .", "the exact incidence of each disease eligible for hadrontherapy is difficult to determine from the available resources .", "indeed , the published data [ 513 ] pertain to series of patients for which the aim was not to assess the level of demand for treatment .", "these were rather series of patients who had received the therapy , but whose recruitment had not been accounted for in tumour registries .", "considering the scarcity and specific nature of the tumours concerned by hadrontherapy , it is difficult to extrapolate and assess the treatment demand from these results .", "hadrontherapy indications are defined according to anatomical location of the tumours , clinical stage , pathology , therapeutic alternatives ( e.g. , surgical contraindications ) , and patient characteristics ( general condition and comorbidities ) .", "french cancer registries record data required to estimate cancer incidence but fail to provide sufficient detail of the tumour stage or of therapeutic data . moreover , the registries cover only approximately 15% of the french population and mainly for the most frequent cancers .", "therefore , these registries do not allow the incidence of these tumours to be estimated .", "this regional study will provide us with more detailed figures concerning the potential number of new cases eligible for hadrontherapy , allowing us to better adapt the future care offer delivered by the national hadrontherapy centre .", "this survey should also provide us with information on certain medical indicators to assist in the design of future hadrontherapy clinical trials ( detailed incidence data , patient population characteristics , tumour stages , recruitment pools , etc . ) .", "finally , this epidemiological field study is also eventually intended to be used to organise a network of highly specialised cares . according to the first epidemiological study carried out in france , in association with ( i ) the results of the literature review that defined the list of hadrontherapy indications and ( ii ) the indications chosen for proton therapy in france", ", the number of cases in the rhne - alpes region eligible for hadrontherapy is estimated at 200 per year ( the rhne - alpes region is home to 10% of the french population ) .", "the main goal is to asses , within the rhne - alpes region , the incidence of cancers with hadrontherapy indication .", "the secondary goals are ( 1 ) to characterise the affected population , ( 2 ) to describe the characteristics of the observed cancers , and ( 3 ) to describe the characteristics of the treatments implemented when hadrontherapy is unavailable .", "this is a prospective , multicentre incidence study , lasting 24 months , conducted with the rcp ( runion de concertation pluridisciplinaire ", "multidisciplinary tumor board ) groups of the rhne - alpes region in france , that offer a therapeutic strategy for cancers for which hadrontherapy is an alternative . \n", "this study required the involvement of a part - time consultant epidemiology engineer , who totalled one woman - year of work to provide ( 1 ) phone canvassing to identify and involve regional cancer research players ; ( 2 ) the protocol construction and testing phase , along with the data collection tools ; ( 3 ) meetings with 3c ( cancer coordination centres ) coordinators and rcps ; ( 4 ) investigator centres inclusion meetings .", "these included year of birth ; gender ; patient 's department of residence ; world health organization ( who ) performance status , patient 's therapeutic status : initial stage or recurrence ; initial staging ( uicc tnm classification : international union against cancer ) ; treatments received before recurrence ; time to recurrence ; staging at time of current tumour management ; icd-10 ( international classification of diseases ) ; icdo ( international classification of diseases for oncology ) histological type ; site of surgery and margin quality ; postsurgical histopathological stage ( ptnm stage ) ; prior radiotherapy ; proposed treatment . \n", "the criteria included adults and children , no age limit , and patients presenting with hadrontherapy indications listed in table 1 , whose medical files have been discussed by experts during an rcp .", "compatible general and psychological condition refers to patients not suffering from life - threatening comorbidities ( no acute or chronic diseases whose short - term lethal risk is dominant relative to the cancer ) , capable of adhering to a disease monitoring protocol and of understanding and accepting a complex treatment requiring close cooperation and staying for several days away from home .  \n  tumour  inclusion criteria .", "these include unresectable tumour , belonging to a known radiation - resistant pathological group , mainly in a locoregional development stage , recurrent or local relapse , with low metastatic potential or m+ dissemination ( presence of one or more remote metastases at the time of diagnosis ) presenting a low threat ( slow development and/or metastases that can be readily treated ) .", "these inclusion criteria are the results of a consensus synthesis of a systematic literature review performed during 8 years , pathology by pathology with the participation of about one hundred of european medical , surgical , and radiation oncologists , managed by physicians trained in hadrontherapy ( mainly protontherapy ) .", "the review was carried out by an academic laboratory specialized in clinical trial setup and analysis and was made according to the  evidence - based medicine ", "definition of  tumour  inclusion criteria \n\n radiation resistance : tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent ( mpde ) for surrounding organs necessarily irradiated under applicable technical conditions , assessed by a radiation oncologist ( guide des procdures de radiothrapie externe 2007 ( guidelines for external radiotherapy procedures ) ; joint effort by the french society for oncology radiotherapy ( socit franaise de radiothrapie oncologique  sfro ) , and the french society for medical physics ( socit franaise de physique mdicale  sfpm ) , conducted in collaboration with the representatives of the french association of electroradiology paramedical workers ( association franaise du personnel paramdical d'electroradiologie  afppe ) ) . \n", "unresectable : tumour which local or locoregional expansion stage renders excision either is technically impossible ( opinion of an experienced surgical team ) or surgically unacceptable due to the irreparable damage that would be necessary ( cancer : principles & practice of oncology 2005 ; devita , v. general articles on treatment strategies in cancer ) . \n", "locoregional : it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion ( t of tnm ) and/or by lymphatic diffusion , maintaining a rank of n in tnm staging , in other words , any tumour at stage m0 ( latest edition of the tumour tnm classification published by the ( uicc ) international union against cancer or the ( ajc ) american joint committee for cancer staging ) . \n", "low metastatic potential : m0 situations whose medium - term ( 5-year ) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means .", "this threshold is difficult to determine ; a level greater than 50% seems unreasonable . as an example ,", "nonthreatening m+ dissemination or controlled by medical treatments : this means the presence of one or more remote metastases at the time of diagnosis , presenting no immediate threat as displaying slow development and/or accessible to effective treatment ( typical case of some low - grade sarcomas lung metastases ) .", "these situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy . \n", "local relapse or local recurrence ( these two terms are synonymous ) : it means the redevelopment , at the same site as initially , of a previously effectively treated tumour ( dictionary of medical terms , garnier - delamare ) . \n \n", "radiation resistance : tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent ( mpde ) for surrounding organs necessarily irradiated under applicable technical conditions , assessed by a radiation oncologist ( guide des procdures de radiothrapie externe 2007 ( guidelines for external radiotherapy procedures ) ; joint effort by the french society for oncology radiotherapy ( socit franaise de radiothrapie oncologique  sfro ) , and the french society for medical physics ( socit franaise de physique mdicale  sfpm ) , conducted in collaboration with the representatives of the french association of electroradiology paramedical workers ( association franaise du personnel paramdical d'electroradiologie  afppe ) ) . \n", "unresectable : tumour which local or locoregional expansion stage renders excision either is technically impossible ( opinion of an experienced surgical team ) or surgically unacceptable due to the irreparable damage that would be necessary ( cancer : principles & practice of oncology 2005 ; devita , v. general articles on treatment strategies in cancer ) . \n", "locoregional : it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion ( t of tnm ) and/or by lymphatic diffusion , maintaining a rank of n in tnm staging , in other words , any tumour at stage m0 ( latest edition of the tumour tnm classification published by the ( uicc ) international union against cancer or the ( ajc ) american joint committee for cancer staging ) . \n", "low metastatic potential : m0 situations whose medium - term ( 5-year ) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means .", "this threshold is difficult to determine ; a level greater than 50% seems unreasonable . as an example ,", "nonthreatening m+ dissemination or controlled by medical treatments : this means the presence of one or more remote metastases at the time of diagnosis , presenting no immediate threat as displaying slow development and/or accessible to effective treatment ( typical case of some low - grade sarcomas lung metastases ) .", "these situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy .  \n", "local relapse or local recurrence ( these two terms are synonymous ) : it means the redevelopment , at the same site as initially , of a previously effectively treated tumour ( dictionary of medical terms , garnier - delamare ) . \n", "they are the description of the tumours and population characteristics , along with the treatments proposed and implemented by the rcps .  \n", "those were healthcare establishments ( private and public ) in the rhne - alpes region hosting rcp groups and having radiotherapy departments . \n", "they consisted of groups of oncology specialists with expertise in the following diseases : musculoskeletal sarcomas and tumours , head and neck ( h and n ) , gastroenterology , paediatrics , dermatology , endocrine and central nervous system tumours . \n", "case screening is performed continuously by the physicians and clinical research technician in light of the list of indications ( table 1 ) , during each rcp . \n", "the questionnaire , along with the organisation of the investigation , was assessed beforehand by a sample population of investigators ( 3c coordinators and cras ) via a semiguided telephone interview .", "for the questionnaire , the evaluation focused on general understanding , the form and length of the questionnaire , time required for completion , amount of data to collect , and the relevance of the selected criteria .", "the evaluation of the investigation method pertained to the choice of individuals involved in data collection organisation , the data recording method , the organisation for data recording , and the identification source for clinical cases .  \n", "variables are encoded in a uniform and standard manner in an investigator 's guide book , which contains detailed information concerning the definitions of each indicator to collect .", "dual computer data input is performed , along with checks for missing or improbable data ( encoding errors , date inconsistencies ) . \n statistical methods .", "the incidence of cancers is expressed as the number of cases reported from the demographic figures of the rhne - alpes region over the studied period and expressed per 100,000 inhabitants .", "  the study involves 13 clinical research technicians , 50 rcp coordinating physicians and 12 cancer coordination centres ( see table 3 ) .", "the study mobilises 27 rhne - alpes region healthcare centres , of which 8 university hospitals , 1 regional cancer centre , 1 public - private cancer institute , 12 general hospitals , the lyon paediatric oncology hospital institute , and 4 private clinics .", "this led to a sense of ownership and motivation on the part of the centres . as a whole , the study covered the region 's 11 hospital areas and involved 52 groups , out of some sixty listed , of cancer specialists with expertise in the diseases that were to identify . during the study ,", "mean patient age was 43 years [ 289 ] , and men were more numerous ( or = 1.52 ) .", "the highest number of cases ( 39.6% ) was in the 218 years age group .", "the number of patients with recurrence was slightly lower ( 45.3% ) than the number of patients receiving initial care ( 54.7% ) .", "carcinomas , sarcomas ( table 4 ) , and paediatric tumours ( table 5 ) were the most frequently identified diseases , and their locations are varied . during the rcp , radiotherapy , chemotherapy , and surgery were prescribed , respectively , in 58% , 57% , and 57% of cases .", "previous treatments were chemotherapy , surgery , or conventional radiotherapy in 62.5% , 58.3% , and 58.3% of cases , respectively .", "the number of cases identified during the study was approximately four times lower than the potential estimate .", "( i.e. , 2.5 cases / rcp ) , and the rhne department identified 56.6% ( i.e. , 1.2 cases / rcp ) .", "isre identified twice as many cases as rhne , even though it only has nine expert groups , that is , 2.6 times less than in rhne , which has 24 .", "the geographic areas not covered by the study correspond to the location of 15 private healthcare centres and to the medical activity of six general and four specialist rcp groups .", "the first six groups had no recruitment potential according to the statement of their coordinators .", "this preliminary data collection , conducted over a one - year period , reported 53 cases of hadrontherapy indications , compared to the estimated cap of 200 cases .", "the estimated incidence of cancers eligible for hadrontherapy in the rhne - alpes region in 2010 , regardless of disease , is of 8.5/100,000 inhabitants .", "the diseases to identify in this study are little known , their distribution has not been studied , and we are unaware of any epidemic outbreaks . moreover , considering that the cancers to identify are rare , it is likely that the number of cases to identify per investigator centre will be very low ( estimated incidence of at the most 1 to 5 cases / year ) . finally , the primary goal of the rcp groups is to provide an immediate therapeutic solution .", "case identification for an observational study is thus unusual in the context of an rcp . because of this", ", sustaining team vigilance and motivation for case identification is difficult to obtain and requires regular refreshing throughout the study .", "this step also served to circulate hadrontherapy information and to facilitate adhesion , ownership , and mobilisation around the project .", "the individuals involved in study organisation were in charge of systematically directing each cancer patient to the elective experts .", "patients suffering from diseases eligible for hadrontherapy were frequently in relapse condition and rather at the end of their therapeutic options .", "this is due to the fact that these diseases have usually a slow and long evolution ; therefore , this study had more chance to catch them somewhere in an advanced stage than at the beginning .", "it can also be considered that the lack of satisfactory treatment means as hadrontherapy makes these populations have more chance to show more advanced disease than it will be expected to be seen in the future .", "the identification and decision to include a patient in the study were made by the rcp groups including these experts . under these conditions , we have confidence in the case orientation and identification system .", "selection criterion is one of the criteria used to identify patients eligible for hadrontherapy . in our current state of knowledge", "this can be explained in part by the fact that it is easier to create multidisciplinary expert groups in public hospitals and to set up clinical research . due to their structure , these establishments benefit from human resources with more varied medical specialities than in private radiotherapy practices . in all likelihood , cases eligible for hadrontherapy could have been treated by these private centres during the study and ignored .  ", "one should be very cautious when comparing the results of this study with those of published incidence studies .", "the methodologies employed in these previous studies are heterogeneous , and/or their flow is insufficiently documented .", "moreover , their goal was to estimate a recruitment potential based on the extrapolation of the results .", "the methodologies used were not intended to provide information concerning an exhaustive incidence , as proposed by our study .", "finally , the published studies were conducted in attractive institutions , specialising in cancer care and selected for this reason ; this may bias the assessment of prevalence . under these conditions , it is easy to confuse incidence and prevalence .", "these elements could , in part , explain the observed difference between the preliminary results obtained in our study and the estimate calculated from the epidemiological results published in the literature .", "only the publication of results obtained from other exhaustive epidemiological studies conducted in the same areas could support this hypothesis .  ", "it is unlikely that the lack of case recording associated with the medical activity of the four groups not involved in this data collection could alone explain the observed difference between the calculated incidence estimates and the preliminary results obtained . for several clinicians involved in the study ,", "the prognosis and risk of metastatic development were difficult to estimate considering the lack of knowledge for these rare diseases .", "moreover , the novelty of hadrontherapy , which can potentially modify usual treatment strategies , could have led to the omission of this indication in some cases , in particular conditions unusually treated by radiotherapy as hepatic and biliary tumours .", "these difficulties , along with the more sustained vigilance effort made by some groups relative to others , could account for the nonhomogeneous data collection and the observed difference between predictions and obtained results , despite the high motivation of the investigators .", "furthermore , the reorganisations ( case of two major rcp clusters ) , changes of persons involved and the low frequency of cases to report , lead to a constant erosion of vigilance , requiring continuous action by the organiser .", "however , some important institutions with their own recruitment targets did not wish to participate , which reduces the scope of our results . about the detailed conditions of the recruited cases , there are a large proportion of recurrent diseases as explained above , and some metastatic diseases have also been included according to defined characteristics : ( i ) the number of metastasis was limited ( 1 to 3 metastasis ) ; ( ii ) their very slow growing speed was not immediately threatening ; ( iii ) the possibility of effective treatment of these  pauci - metastatic  conditions , essentially by ablative procedures .", "actually , the principle to treat , with curative intend , patients in such situation is more and more accepted by expert oncologists .", "the estimated incidence of cancers , irrespective of type and location , eligible for treatment by hadrontherapy , for the study , that is , 8.5/100,000 individuals , is mildly higher than the reference incidence threshold of 6 cases/100,000 individuals , as defined in the orphanet international database of  rare diseases  .", "this figure can be obviously explained by the fact that the incidence was calculated by adding the five major cancer categories and 34 different locations .", "if one was to calculate the incidence for each of the disease types , their respective incidence would be significantly below the reference threshold of 6 cases/100,000 .", "indeed , for our study , the minimum number of cases listed per disease is of 1 case and the maximum is of 7 cases .", "table 4 . considering all of the difficulties and limitations of this preliminary phase of the study ,", "the yield of approximately 25% notification , compared to the expected maximum , appears satisfactory . in the perspective of a ( necessary ) ramp up of a future hadrontherapy centre", ", there is thus a sufficient population to initiate operation and to progressively establish recruitment for this centre , without immediately generating an insufficient offer effect .", "future epidemiological studies will benefit from focusing on the characterisation of the metastatic development of diseases eligible for hadrontherapy .", "all cancers have their own development pattern , and , in this respect , the literature provides few elements concerning diseases eligible for hadrontherapy .", "this information should serve both to improve diagnosis procedures , medical care , and the surveillance of these rare diseases .  ", "this study of hadrontherapy healthcare decision networks in the rhne - alpes region , able to identify areas not properly covered by rcps , should enable us to analyse the potential effect of this lack on the outcome of the patients of these areas .  ", "they shed no light on posttreatment medical activity : follow - up visits , medical imaging , and other care beyond the irradiated activity per se .", "one of the most valuable outcomes of this study has been to make the existence of some particularly rare diseases known and to provide information concerning an innovative treatment : hadrontherapy , whose existence will doubtless have a favourable effect on the very knowledge of these diseases .", "an additional benefit of this approach has been to open the way for a new treatment system in a region of france that is home to approximately 10% of the country 's population . at last", ", these elective indications will have to be validated by health authorities to register them as part of the good professional practices of oncology and give an equal chance to each patient to receive hadrontherapy through the well - established decision process of the rcps ."], "abstract_text": ["<S> \n context . </S>", "<S> hadrontherapy is an innovative form of radiotherapy using beams of protons or carbon ions able to destroy some radio - resistant tumours . </S>", "<S> because these tumours are highly specific amongst all cancerous tumours , it is impossible to determine the incidence of these diseases from surveillance registries . goal . to assess , within the rhne - alpes region , the incidence of cancers being hadrontherapy indications </S>", "<S> . method . </S>", "<S> prospective , multicentre continuous data collection during 1 year , by practitioners participating to multidisciplinary tumor board . </S>", "<S> tumours are inoperable , radio resistant , at primary stage of development , or locally recurrent , with low metastatic potential </S>", "<S> . results . </S>", "<S> study involved 27 healthcare centres , 52 groups of specialist practitioners . </S>", "<S> the estimated incidence of cancers eligible for hadrontherapy in the rhne - alpes region in 2010 , that is , for 34 locations in all , is of 8.5/100  000 inhabitants . </S>", "<S> appraisal of the low potential of metastatic progression is impeded , because these are rare diseases , whose outcome is unfamiliar to investigators . </S>", "<S> conclusion . </S>", "<S> future epidemiological studies will need to focus on prognosis and on the metastatic progression rate of these diseases . </S>", "<S> indeed , there are few information available on this subject in the literature that could be used to improve preventive measures , medical care , and the surveillance of these rare cancers . </S>"], "labels": null, "section_names": ["1. Scientific Context", "2. Goals", "3. Method", "4. Results", "5. Discussion", "6. Conclusion"], "sections": [["hadrontherapy is an innovative form of radiotherapy , based on high - technology equipment using proton or carbon ions beams to destroy tumours [ 1 , 2 ] .", "this treatment method enables significantly higher ballistic precision to be achieved , compared to photons ( x - rays ) with , as expected therapeutic benefit , an improvement of quality of life and chances of recovery .", "carbon ions are also specifically characterised by superior biological efficacy ( relative biological effectiveness from 1.5 to 3 ) , overcoming the radiation resistance of certain cancers to photons and even protons .", "indeed , carbon ion beams when compared to x - rays represent a distinct advantage for the treatment of highly radiation - resistant tumours .", "an initial study assessing recruitment potential for proton therapy was conducted in 1998 in italy , showing an incidence of 10  825 cases / year .", "one year later , a second study was carried out in the context of the medaustron project for the construction of a carbon ions therapy centre in austria . considering patients living in austria and neighbouring countries ,", "the patient recruitment potential ( proton and carbon ) was estimated with an incidence of 13  145 cases / year . in france , in 2002 , a third study was conducted in the context of the etoile medical project .", "this study revealed an annual incidence of approximately 5320 cases / year for carbon ions .", "concomitantly , a fourth study was conducted , again in the context of the medaustron project , estimating the incidence of cases eligible for hadrontherapy in austria at 2044 cases / year .", "finally , again in 2002 , in the context of the project for the construction of the cnao centre in italy , an epidemiological study was conducted , estimating the incidence of cases eligible for carbon therapy at 3694 cases / year and proton therapy at 1885 cases / year . .", "since 2004 , no other epidemiological studies have been published for hadrontherapy . in 2011 , however , the japanese team from the national institute for radiological sciences ( nirs ) , pioneer in carbon therapy , published the results of its 1994 to 2011 clinical activity .", "patients were included in clinical trials and the number of patients receiving carbon therapy was 6157 .", "this value is the incidence of treated cases , over 17 years without any notion of recruitment potential and with a rather slow ramp up of the activity .  ", "a list of priority indications that can be treated by hadrontherapy ( table 1 ) was drawn up by the gcs etoile medical group , in collaboration with national and international experts , based on published data ranked using an  evidence - based medicine", "this literature review was based on provided or expected medical service criteria ( survival and quality of life ) . according to the classification used by the european survey group of rare diseases , the eligible tumours that can be treated by hadrontherapy conform to the criteria of rare diseases based on the following thresholds of epidemiological indicators : prevalence  50 cases/100  000 persons within a given population and incidence  6 cases/100  000 persons / year .", "indeed , for all tumor types identified during the previous hadrontherapy epidemiological studies [ 58 ] , the calculated and cumulative incidences are all below these thresholds ( table 2 ) . amongst the hadrontherapy indications , 25 are tumours listed in the orphanet international database of rare diseases   ( cf .", "the exact incidence of each disease eligible for hadrontherapy is difficult to determine from the available resources .", "indeed , the published data [ 513 ] pertain to series of patients for which the aim was not to assess the level of demand for treatment .", "these were rather series of patients who had received the therapy , but whose recruitment had not been accounted for in tumour registries .", "considering the scarcity and specific nature of the tumours concerned by hadrontherapy , it is difficult to extrapolate and assess the treatment demand from these results .", "hadrontherapy indications are defined according to anatomical location of the tumours , clinical stage , pathology , therapeutic alternatives ( e.g. , surgical contraindications ) , and patient characteristics ( general condition and comorbidities ) .", "french cancer registries record data required to estimate cancer incidence but fail to provide sufficient detail of the tumour stage or of therapeutic data . moreover , the registries cover only approximately 15% of the french population and mainly for the most frequent cancers .", "therefore , these registries do not allow the incidence of these tumours to be estimated .", "this regional study will provide us with more detailed figures concerning the potential number of new cases eligible for hadrontherapy , allowing us to better adapt the future care offer delivered by the national hadrontherapy centre .", "this survey should also provide us with information on certain medical indicators to assist in the design of future hadrontherapy clinical trials ( detailed incidence data , patient population characteristics , tumour stages , recruitment pools , etc . ) .", "finally , this epidemiological field study is also eventually intended to be used to organise a network of highly specialised cares . according to the first epidemiological study carried out in france , in association with ( i ) the results of the literature review that defined the list of hadrontherapy indications and ( ii ) the indications chosen for proton therapy in france", ", the number of cases in the rhne - alpes region eligible for hadrontherapy is estimated at 200 per year ( the rhne - alpes region is home to 10% of the french population ) ."], ["the main goal is to asses , within the rhne - alpes region , the incidence of cancers with hadrontherapy indication .", "the secondary goals are ( 1 ) to characterise the affected population , ( 2 ) to describe the characteristics of the observed cancers , and ( 3 ) to describe the characteristics of the treatments implemented when hadrontherapy is unavailable ."], ["this is a prospective , multicentre incidence study , lasting 24 months , conducted with the rcp ( runion de concertation pluridisciplinaire ", "multidisciplinary tumor board ) groups of the rhne - alpes region in france , that offer a therapeutic strategy for cancers for which hadrontherapy is an alternative . \n", "this study required the involvement of a part - time consultant epidemiology engineer , who totalled one woman - year of work to provide ( 1 ) phone canvassing to identify and involve regional cancer research players ; ( 2 ) the protocol construction and testing phase , along with the data collection tools ; ( 3 ) meetings with 3c ( cancer coordination centres ) coordinators and rcps ; ( 4 ) investigator centres inclusion meetings .", "these included year of birth ; gender ; patient 's department of residence ; world health organization ( who ) performance status , patient 's therapeutic status : initial stage or recurrence ; initial staging ( uicc tnm classification : international union against cancer ) ; treatments received before recurrence ; time to recurrence ; staging at time of current tumour management ; icd-10 ( international classification of diseases ) ; icdo ( international classification of diseases for oncology ) histological type ; site of surgery and margin quality ; postsurgical histopathological stage ( ptnm stage ) ; prior radiotherapy ; proposed treatment . \n", "the criteria included adults and children , no age limit , and patients presenting with hadrontherapy indications listed in table 1 , whose medical files have been discussed by experts during an rcp .", "compatible general and psychological condition refers to patients not suffering from life - threatening comorbidities ( no acute or chronic diseases whose short - term lethal risk is dominant relative to the cancer ) , capable of adhering to a disease monitoring protocol and of understanding and accepting a complex treatment requiring close cooperation and staying for several days away from home .  \n  tumour  inclusion criteria .", "these include unresectable tumour , belonging to a known radiation - resistant pathological group , mainly in a locoregional development stage , recurrent or local relapse , with low metastatic potential or m+ dissemination ( presence of one or more remote metastases at the time of diagnosis ) presenting a low threat ( slow development and/or metastases that can be readily treated ) .", "these inclusion criteria are the results of a consensus synthesis of a systematic literature review performed during 8 years , pathology by pathology with the participation of about one hundred of european medical , surgical , and radiation oncologists , managed by physicians trained in hadrontherapy ( mainly protontherapy ) .", "the review was carried out by an academic laboratory specialized in clinical trial setup and analysis and was made according to the  evidence - based medicine ", "definition of  tumour  inclusion criteria \n\n radiation resistance : tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent ( mpde ) for surrounding organs necessarily irradiated under applicable technical conditions , assessed by a radiation oncologist ( guide des procdures de radiothrapie externe 2007 ( guidelines for external radiotherapy procedures ) ; joint effort by the french society for oncology radiotherapy ( socit franaise de radiothrapie oncologique  sfro ) , and the french society for medical physics ( socit franaise de physique mdicale  sfpm ) , conducted in collaboration with the representatives of the french association of electroradiology paramedical workers ( association franaise du personnel paramdical d'electroradiologie  afppe ) ) . \n", "unresectable : tumour which local or locoregional expansion stage renders excision either is technically impossible ( opinion of an experienced surgical team ) or surgically unacceptable due to the irreparable damage that would be necessary ( cancer : principles & practice of oncology 2005 ; devita , v. general articles on treatment strategies in cancer ) . \n", "locoregional : it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion ( t of tnm ) and/or by lymphatic diffusion , maintaining a rank of n in tnm staging , in other words , any tumour at stage m0 ( latest edition of the tumour tnm classification published by the ( uicc ) international union against cancer or the ( ajc ) american joint committee for cancer staging ) . \n", "low metastatic potential : m0 situations whose medium - term ( 5-year ) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means .", "this threshold is difficult to determine ; a level greater than 50% seems unreasonable . as an example ,", "nonthreatening m+ dissemination or controlled by medical treatments : this means the presence of one or more remote metastases at the time of diagnosis , presenting no immediate threat as displaying slow development and/or accessible to effective treatment ( typical case of some low - grade sarcomas lung metastases ) .", "these situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy . \n", "local relapse or local recurrence ( these two terms are synonymous ) : it means the redevelopment , at the same site as initially , of a previously effectively treated tumour ( dictionary of medical terms , garnier - delamare ) . \n \n", "radiation resistance : tumour which estimated local dose required for local control that is higher that the maximum acceptable dose equivalent ( mpde ) for surrounding organs necessarily irradiated under applicable technical conditions , assessed by a radiation oncologist ( guide des procdures de radiothrapie externe 2007 ( guidelines for external radiotherapy procedures ) ; joint effort by the french society for oncology radiotherapy ( socit franaise de radiothrapie oncologique  sfro ) , and the french society for medical physics ( socit franaise de physique mdicale  sfpm ) , conducted in collaboration with the representatives of the french association of electroradiology paramedical workers ( association franaise du personnel paramdical d'electroradiologie  afppe ) ) . \n", "unresectable : tumour which local or locoregional expansion stage renders excision either is technically impossible ( opinion of an experienced surgical team ) or surgically unacceptable due to the irreparable damage that would be necessary ( cancer : principles & practice of oncology 2005 ; devita , v. general articles on treatment strategies in cancer ) . \n", "locoregional : it refers to the tumour expansion state corresponding to the anatomical diffusion region of the primary tumour by contiguous expansion ( t of tnm ) and/or by lymphatic diffusion , maintaining a rank of n in tnm staging , in other words , any tumour at stage m0 ( latest edition of the tumour tnm classification published by the ( uicc ) international union against cancer or the ( ajc ) american joint committee for cancer staging ) . \n", "low metastatic potential : m0 situations whose medium - term ( 5-year ) metastatic risk is considered to be sufficiently low to justify curative locoregional treatment involving significant means .", "this threshold is difficult to determine ; a level greater than 50% seems unreasonable . as an example ,", "nonthreatening m+ dissemination or controlled by medical treatments : this means the presence of one or more remote metastases at the time of diagnosis , presenting no immediate threat as displaying slow development and/or accessible to effective treatment ( typical case of some low - grade sarcomas lung metastases ) .", "these situations are indicative of locoregional disease treatment using complex and expensive techniques such as hadrontherapy .  \n", "local relapse or local recurrence ( these two terms are synonymous ) : it means the redevelopment , at the same site as initially , of a previously effectively treated tumour ( dictionary of medical terms , garnier - delamare ) . \n", "they are the description of the tumours and population characteristics , along with the treatments proposed and implemented by the rcps .  \n", "those were healthcare establishments ( private and public ) in the rhne - alpes region hosting rcp groups and having radiotherapy departments . \n", "they consisted of groups of oncology specialists with expertise in the following diseases : musculoskeletal sarcomas and tumours , head and neck ( h and n ) , gastroenterology , paediatrics , dermatology , endocrine and central nervous system tumours . \n", "case screening is performed continuously by the physicians and clinical research technician in light of the list of indications ( table 1 ) , during each rcp . \n", "the questionnaire , along with the organisation of the investigation , was assessed beforehand by a sample population of investigators ( 3c coordinators and cras ) via a semiguided telephone interview .", "for the questionnaire , the evaluation focused on general understanding , the form and length of the questionnaire , time required for completion , amount of data to collect , and the relevance of the selected criteria .", "the evaluation of the investigation method pertained to the choice of individuals involved in data collection organisation , the data recording method , the organisation for data recording , and the identification source for clinical cases .  \n", "variables are encoded in a uniform and standard manner in an investigator 's guide book , which contains detailed information concerning the definitions of each indicator to collect .", "dual computer data input is performed , along with checks for missing or improbable data ( encoding errors , date inconsistencies ) . \n statistical methods .", "the incidence of cancers is expressed as the number of cases reported from the demographic figures of the rhne - alpes region over the studied period and expressed per 100,000 inhabitants ."], ["  the study involves 13 clinical research technicians , 50 rcp coordinating physicians and 12 cancer coordination centres ( see table 3 ) .", "the study mobilises 27 rhne - alpes region healthcare centres , of which 8 university hospitals , 1 regional cancer centre , 1 public - private cancer institute , 12 general hospitals , the lyon paediatric oncology hospital institute , and 4 private clinics .", "this led to a sense of ownership and motivation on the part of the centres . as a whole , the study covered the region 's 11 hospital areas and involved 52 groups , out of some sixty listed , of cancer specialists with expertise in the diseases that were to identify . during the study ,", "mean patient age was 43 years [ 289 ] , and men were more numerous ( or = 1.52 ) .", "the highest number of cases ( 39.6% ) was in the 218 years age group .", "the number of patients with recurrence was slightly lower ( 45.3% ) than the number of patients receiving initial care ( 54.7% ) .", "carcinomas , sarcomas ( table 4 ) , and paediatric tumours ( table 5 ) were the most frequently identified diseases , and their locations are varied . during the rcp , radiotherapy , chemotherapy , and surgery were prescribed , respectively , in 58% , 57% , and 57% of cases .", "previous treatments were chemotherapy , surgery , or conventional radiotherapy in 62.5% , 58.3% , and 58.3% of cases , respectively .", "the number of cases identified during the study was approximately four times lower than the potential estimate .", "( i.e. , 2.5 cases / rcp ) , and the rhne department identified 56.6% ( i.e. , 1.2 cases / rcp ) .", "isre identified twice as many cases as rhne , even though it only has nine expert groups , that is , 2.6 times less than in rhne , which has 24 .", "the geographic areas not covered by the study correspond to the location of 15 private healthcare centres and to the medical activity of six general and four specialist rcp groups .", "the first six groups had no recruitment potential according to the statement of their coordinators .", "this preliminary data collection , conducted over a one - year period , reported 53 cases of hadrontherapy indications , compared to the estimated cap of 200 cases .", "the estimated incidence of cancers eligible for hadrontherapy in the rhne - alpes region in 2010 , regardless of disease , is of 8.5/100,000 inhabitants ."], ["the diseases to identify in this study are little known , their distribution has not been studied , and we are unaware of any epidemic outbreaks . moreover , considering that the cancers to identify are rare , it is likely that the number of cases to identify per investigator centre will be very low ( estimated incidence of at the most 1 to 5 cases / year ) . finally , the primary goal of the rcp groups is to provide an immediate therapeutic solution .", "case identification for an observational study is thus unusual in the context of an rcp . because of this", ", sustaining team vigilance and motivation for case identification is difficult to obtain and requires regular refreshing throughout the study .", "this step also served to circulate hadrontherapy information and to facilitate adhesion , ownership , and mobilisation around the project .", "the individuals involved in study organisation were in charge of systematically directing each cancer patient to the elective experts .", "patients suffering from diseases eligible for hadrontherapy were frequently in relapse condition and rather at the end of their therapeutic options .", "this is due to the fact that these diseases have usually a slow and long evolution ; therefore , this study had more chance to catch them somewhere in an advanced stage than at the beginning .", "it can also be considered that the lack of satisfactory treatment means as hadrontherapy makes these populations have more chance to show more advanced disease than it will be expected to be seen in the future .", "the identification and decision to include a patient in the study were made by the rcp groups including these experts . under these conditions , we have confidence in the case orientation and identification system .", "selection criterion is one of the criteria used to identify patients eligible for hadrontherapy . in our current state of knowledge", "this can be explained in part by the fact that it is easier to create multidisciplinary expert groups in public hospitals and to set up clinical research . due to their structure , these establishments benefit from human resources with more varied medical specialities than in private radiotherapy practices . in all likelihood , cases eligible for hadrontherapy could have been treated by these private centres during the study and ignored .  ", "one should be very cautious when comparing the results of this study with those of published incidence studies .", "the methodologies employed in these previous studies are heterogeneous , and/or their flow is insufficiently documented .", "moreover , their goal was to estimate a recruitment potential based on the extrapolation of the results .", "the methodologies used were not intended to provide information concerning an exhaustive incidence , as proposed by our study .", "finally , the published studies were conducted in attractive institutions , specialising in cancer care and selected for this reason ; this may bias the assessment of prevalence . under these conditions , it is easy to confuse incidence and prevalence .", "these elements could , in part , explain the observed difference between the preliminary results obtained in our study and the estimate calculated from the epidemiological results published in the literature .", "only the publication of results obtained from other exhaustive epidemiological studies conducted in the same areas could support this hypothesis .  ", "it is unlikely that the lack of case recording associated with the medical activity of the four groups not involved in this data collection could alone explain the observed difference between the calculated incidence estimates and the preliminary results obtained . for several clinicians involved in the study ,", "the prognosis and risk of metastatic development were difficult to estimate considering the lack of knowledge for these rare diseases .", "moreover , the novelty of hadrontherapy , which can potentially modify usual treatment strategies , could have led to the omission of this indication in some cases , in particular conditions unusually treated by radiotherapy as hepatic and biliary tumours .", "these difficulties , along with the more sustained vigilance effort made by some groups relative to others , could account for the nonhomogeneous data collection and the observed difference between predictions and obtained results , despite the high motivation of the investigators .", "furthermore , the reorganisations ( case of two major rcp clusters ) , changes of persons involved and the low frequency of cases to report , lead to a constant erosion of vigilance , requiring continuous action by the organiser .", "however , some important institutions with their own recruitment targets did not wish to participate , which reduces the scope of our results . about the detailed conditions of the recruited cases , there are a large proportion of recurrent diseases as explained above , and some metastatic diseases have also been included according to defined characteristics : ( i ) the number of metastasis was limited ( 1 to 3 metastasis ) ; ( ii ) their very slow growing speed was not immediately threatening ; ( iii ) the possibility of effective treatment of these  pauci - metastatic  conditions , essentially by ablative procedures .", "actually , the principle to treat , with curative intend , patients in such situation is more and more accepted by expert oncologists .", "the estimated incidence of cancers , irrespective of type and location , eligible for treatment by hadrontherapy , for the study , that is , 8.5/100,000 individuals , is mildly higher than the reference incidence threshold of 6 cases/100,000 individuals , as defined in the orphanet international database of  rare diseases  .", "this figure can be obviously explained by the fact that the incidence was calculated by adding the five major cancer categories and 34 different locations .", "if one was to calculate the incidence for each of the disease types , their respective incidence would be significantly below the reference threshold of 6 cases/100,000 .", "indeed , for our study , the minimum number of cases listed per disease is of 1 case and the maximum is of 7 cases .", "table 4 . considering all of the difficulties and limitations of this preliminary phase of the study ,", "the yield of approximately 25% notification , compared to the expected maximum , appears satisfactory . in the perspective of a ( necessary ) ramp up of a future hadrontherapy centre", ", there is thus a sufficient population to initiate operation and to progressively establish recruitment for this centre , without immediately generating an insufficient offer effect ."], ["future epidemiological studies will benefit from focusing on the characterisation of the metastatic development of diseases eligible for hadrontherapy .", "all cancers have their own development pattern , and , in this respect , the literature provides few elements concerning diseases eligible for hadrontherapy .", "this information should serve both to improve diagnosis procedures , medical care , and the surveillance of these rare diseases .  ", "this study of hadrontherapy healthcare decision networks in the rhne - alpes region , able to identify areas not properly covered by rcps , should enable us to analyse the potential effect of this lack on the outcome of the patients of these areas .  ", "they shed no light on posttreatment medical activity : follow - up visits , medical imaging , and other care beyond the irradiated activity per se .", "one of the most valuable outcomes of this study has been to make the existence of some particularly rare diseases known and to provide information concerning an innovative treatment : hadrontherapy , whose existence will doubtless have a favourable effect on the very knowledge of these diseases .", "an additional benefit of this approach has been to open the way for a new treatment system in a region of france that is home to approximately 10% of the country 's population . at last", ", these elective indications will have to be validated by health authorities to register them as part of the good professional practices of oncology and give an equal chance to each patient to receive hadrontherapy through the well - established decision process of the rcps ."]]}
{"article_id": "PMC4015425", "article_text": ["type  2 diabetes continues to present a public health burden across the world , and asia is considered to be at the epicenter of this diabetes epidemic , with 60% of the disease burden borne by this region1 .", "the increase in diabetes prevalence across asia has been rapid , fuelled by increasing obesity , aging populations and urbanization , which in turn have been driven by rapid economic development , a sedentary lifestyle and increased consumption of refined foods3 .", "these socioeconomic changes have been particularly significant in countries across southeast asia4 , and reflected by dramatic rises in type  2 diabetes prevalence over the past decade ( figure  1 ) .", "indeed , indonesia and the philippines are projected to be among the top 10 countries in the world with the highest number of estimated cases by 20301 .", "another issue curtailing the type  2 diabetes rise in asia is the high number of undiagnosed individuals .", "for instance , in hong kong and taiwan it was reported that almost 53% of diabetic patients remain undiagnosed5 .", "furthermore , the prevalence of prediabetes ( impaired glucose tolerance ) further adds to the already burgeoning burden of overt diabetes and its consequences in this region ( figure  1 ) .", "comparative prevalence of diabetes in countries across southeast asia in 2011 ( calculated by assuming that every country and region has the same age profile ) .", "these data are based on figures compiled by the international diabetes federation in the international diabetes atlas , 2011 ( available at : http://www.idf.org/atlasmap/atlasmap ) .", "it is widely recognized that diabetes is a major source of morbidity , mortality and economic burden6 .", "prolonged and poor glucose control increases the risk of developing macrovascular ( coronary artery disease , stroke ) and microvascular ( nephropathy , retinopathy and neuropathy ) complications7 , which can greatly reduce life expectancy and quality of life8 .", "in fact , the risk of allcause mortality ( cardiovascular and noncardiovascular ) is higher in diabetic than nondiabetic patients9 . consequently , the rapidly growing prevalence of diabetes in asia is likely to result in large increases in diabetesassociated mortality10 . from an economic perspective", ", the impact of diabetes on healthcare resources and personal expenditure , as well as the financial implications associated with loss in productivity and disability , is enormous11 . in taiwan ,", "the direct healthcare cost associated with diabetes was 11.5% of the total national healthcare cost and was 4.3fold higher than the care of nondiabetic individuals12 . in hong kong , the annual total cost of type  2 diabetes equated to usd  1,725    2,044 per patient .", "furthermore , complications increased treatment costs associated with inpatient hospital stay by between 6 and 300% compared with treatment of type  2 diabetes patients without complications14 .", "of note is the considerable heterogeneity in healthcare spending for diabetes between countries across southeast asia .", "the international diabetes federation estimated that , in 2010 , treatment of diabetes accounted for 5 and 7% of the total healthcare expenditure in vietnam and indonesia , respectively , compared with 15 and 16% in singapore and malaysia , respectively11 .", "taken together , it is clear that early diagnosis and disease control are important healthcare initiatives to improve the lives , outcomes and economies of these atrisk countries . in determining the optimal management approach , recognizing the differences in clinical characteristics and risk of diabetes between asian and caucasian populations is important to understand .", "for instance in asians , diabetes develops over a shorter time , in younger people and in those with a lower body mass index ( bmi)15 .", "furthermore , asians have a higher proportion of body fat and abdominal obesity , putting them at a higher risk of developing insulin resistance16 .", "reports that asians show early cell dysfunction might further explain their increased susceptibility to type  2 diabetes16 .", "the thrifty gene and thrifty phenotype hypotheses have been proposed as plausible explanations for the increasing diabetes epidemic seen across asia18 .", "although considered protective during periods of famine or food shortage , thrifty genes have rendered individuals highly predisposed to obesity in times of plenty .", "alternatively , the thrifty phenotype relates to the impact of an unfavorable intrauterine environment ( e.g. undernutrition ) and low birth weight on the subsequent development of diabetes and metabolic syndrome in adults18 .", "several diabetesassociated genes have been implicated to account for the heightened predilection in asians , especially asian indians , and include polymorphisms of the peroxisome proliferatoractivated receptor coactivator1 alpha ( pgc1 alpha ) gene , plasma cell glycoprotein ( pc1 ) gene and insulin receptor substrate ( irs2 ) gene ( reviewed in radha and mohan)19 .", "given the increased prevalence of diabetes among younger populations in asia and early exposure to hyperglycemia , it has been suggested that they are also at a high risk of endorgan damage and are likely to have to live with complications for longer20 .", "indeed , the asia  pacific cohort studies collaboration ( apcsc ) , which assessed the impact of diabetes on cardiovascular outcomes and mortality , found a greater risk of fatal coronary heart disease , hemorrhagic and ischemic stroke among younger compared with older individuals21 .", "apcsc also showed an association between diabetes and increased risk of death as a result of renal disease , cancer , respiratory infections , and other infective and inflammatory causes21 .", "although optimal blood glucose control has been shown to reduce the risk of both macrovascular and microvascular complications6 , the marked prevalence of renal dysfunction in asia reflects , in part , the continuing challenge in achieving this goal22 .", "the aim of the present article was to review the barriers to achieving optimal blood glucose control in asian patients with type  2 diabetes who have declining renal function and to discuss the treatment modalities that might be appropriate for the management of these special patient populations .", "of the diabetesassociated complications , the most debilitating is chronic kidney disease ( ckd ) and its pathogenic progression to endstage renal disease ( esrd ) that requires dialysis or transplantation23 .", "defining and diagnosing ckd in clinical practice has evolved since its first classification by the national foundation kidney disease outcomes quality initiative ( nkfkdoqi ) in 200224 . in these guidelines ,", "ckd was categorized into five stages according to the severity of disease as defined by decreasing glomerular filtration rates ( gfr ) alone24 . in the more recent update ,", "the assessment criteria have been modified to encompass both gfr and albuminuria measures as correlates of a patient 's prognosis .", "in addition , a subdivision of the previously described gfr stage  3 has been proposed to help further define the severity of kidney dysfunction in patients with ckd25 .", "risk factors for the development of reduced kidney function in southeast asian populations have been identified and include the presence of diabetes , systolic hypertension ( > 159  mmhg ) , hyperuricemia ( > 6.29  mg / dl ) , elevated bmi ( > 24.9  kg / m ) and hypercholesterolemia ( > 248.3  mg / dl)26 .", "microalbuminuria is the earliest clinical manifestation of ckd in type  2 diabetes and persistent microalbuminuria represents an early clinical marker for the development of diabetic nephropathy ( dn)27 . although the number of studies exploring the epidemiology of microalbuminuria across asia has been limited , the microalbuminuria prevalence study ( maps ) of 6,800 hypertensive diabetic patients from 10 countries in the region reported an overall prevalence rate of 39.8% ( 95% confidence interval [ ci ] 39.240.5)22 .", "these data show how a high proportion of hypertensive , type  2 diabetes asian patients are showing early signs of declining renal function , and further support a need for early intervention so as to reduce accelerated progression of cardiorenal complications and the associated healthcare burden28 . with the exception of taiwan ( chiang sc et  al .", "jcma 2011 ; 74 : 310 ) , these data are based on subanalyses of the microalbuminuria prevalence study of 6,800 hypertensive diabetic patients from 10 countries across asia ( wu ay et  al .", "2005 ; 48 : 1726 . ) in recognition of the utility of measuring microalbuminuria , guidelines recommend annual screening of this clinical marker for identifying atrisk populations32 .", "patients identified with microalbuminuria early in the course of disease might require stricter glycemic and blood pressure control targets .", "indeed , several prospective studies have shown the importance of tight glycemic control alone and combined with blood pressure lowering in the prevention of microvascular complications .", "for instance , the uk prospective diabetes study compared the effects of intensive bloodglucose control with either sulfonylureas or insulin and conventional treatment ( including diet ) on the risk of microvascular and macrovascular complications in type  2 diabetes patients .", "intensive glucose control with sulfonylureas or insulin resulted in a 33% relative risk reduction in the development of microalbuminuria .", "furthermore , this reduction in risk was sustained in the 10year followup study during which longterm benefits on cardiovascular disease ( cvd ) risk also emerged33 . in the kumamoto study ,", "the frequency and severity of diabetic microvascular complications were assessed in japanese type  2 diabetes patients after treatment with either intensive glucose control ( multiple insulin injections of three or more ) or conventional therapy ( insulin injections administered once or twice daily )", ". there was a significant reduction in the progression of nephropathy and other microvascular complications ( retinopathy ) after intensive insulin therapy ( p  <  0.05)35 .", "the effect of an intensive , stepwise multifactorial intervention , targeting hyperglycemia , hypertension , dyslipidemia and microalbuminuria , on the initiation and progression of microvascular complications in type  2 diabetes patients with microalbuminuria was assessed in the steno2 study .", "type  2 diabetes patients receiving early intensive diabetes management including tight glucose control and angiotensin converting enzyme inhibitors ( acei ) to reduce blood pressure were significantly less likely to develop nephropathy than patients receiving standard therapy ( odds ratio 0.27 ; 95% ci 0.100.75 ) .", "this reduction in risk was maintained in an observational follow up of 5.5  years36 . in the most recent analysis of the action in diabetes and vascular disease : preterax and diamicronmodified release controlled evaluation ( advance ) trial , which included a large number of asian type  2 diabetes patients , further support for the benefits of combining bloodpressure lowering with intensive blood glucose control was provided38 .", "patients received either intensive glucose control ( target glycated hemoglobin [ hba1c ]   6.5% ) or standard glucose control ( defined by local guidelines ) with placebo or acei plus indapamide therapy .", "a combination of intensive glucose control plus acei plus indapamide therapy led to a significant percentage in risk reduction in new or worsening nephropathy ( 95% ci 1250% ; p  =  0.005 ) ; a 54% reduction in the development of newonset macroalbuminuria ( 95% ci 3568% ; p  <  0.001 ) , and a 25% reduction in the risk of newonset microalbuminuria ( 95% ci 1633% ; p  <  0.001 ) .", "furthermore , the effects of blood pressure lowering and intensive glucose control were independent of each other , so that on combining these agents , additive benefits were observed , particularly on renal outcomes38 . indeed , the benefits of early and tight target control strategies on the prevention of dn in chinese type  2 diabetes patients have been observed . in this asian cohort with normoalbuminuria , achievement of american diabetes association ( ada)recommended targets of hba1c ( < 7% ) , systolic blood pressure ( < 130  mmhg ) , diastolic blood pressure ( < 80  mmhg ) and lipid profiles ( lowdensity lipoprotein [ ldl ] < 100  mg / dl ; triglycerides < 150  mg / dl ; highdensity lipoprotein > 4050  mg / dl ) reduced the risk of developing newonset microalbuminuria compared with those who were unable to attain these targets39 .", "interestingly , in a separate study of taiwanese type  2 diabetes patients with microalbuminuria , almost 36% of patients achieved remission to normoalbuminuria following a tight , multifactorial target control approach40 .", "worldwide , rates of ckd have been increasing , and have accompanied the increasing prevalence of type  2 diabetes , hypertension and cvd .", "for instance , in the usa , it is estimated that 30% of patients with type  2 diabetes will develop some form of kidney disease as reflected by protein leakage , microalbuminuria , macroalbuminuria or a decline in the glomerular filtration rate ( gfr)41 .", "nephropathy is a common complication of asians with type  2 diabetes , as reflected by the accelerating prevalence and incidence rates of observed ckd23 .", "indeed , incidence rates of esrd across asia have shown increases over a 6year period ( 20032008 ) of 15.8% in hong kong , 22% in malaysia and thailand , 24% in singapore , and up to 31% in the philippines43 .", "although incidence rates of esrd appear to have declined in taiwan since 2005 , this country has the third highest rate of esrd in the world , with an incidence rate of 384 per million population ( pmp ) , highlighting the disparity between the rate of kidney disease and a country 's economic status42 .", "furthermore , it has become apparent that a high proportion of esrd cases are as a result of diabetes , ranging from 41% to 62% in individual countries across southeast asia ( figure  2)43 .", "incidence of endstage renal disease ( esrd ) as a result of diabetes in selected countries across southeast asia in 2008 .", "these data are based on figures compiled by the us renal disease system ( figure adapted from 2010 us renal disease system annual report chapter 12 international comparisons ; volume 2 ; pages 383396 ) . for singapore ,", "the 2008 data have been extracted from the national registry of diseases office , released 1 march 2011 ( inp111 ) .", "the presence of ckd is also associated with a high cvd risk , which is further exacerbated by the presence of diabetes and hypertension45 .", "indeed , cvd remains the main cause of death in patients with ckd rather than kidney failure , and it is estimated that cvdassociated mortality is 1030fold higher in patients with advanced ckd receiving dialysis than the general population46 .", "given that the prevalence of hypertension in asia is increasing , and that diabetes and hypertension commonly coexist ( type  2 diabetes patients are twice as likely to have elevated blood pressure than nontype  2 diabetes patients ) , screening these highrisk patients for ckd early might help slow the course of renal function decline and reduce the greater cardiovascular mortality risk observed in such individuals47 .", "microalbuminuria is the earliest clinical manifestation of ckd in type  2 diabetes and persistent microalbuminuria represents an early clinical marker for the development of diabetic nephropathy ( dn)27 . although the number of studies exploring the epidemiology of microalbuminuria across asia has been limited , the microalbuminuria prevalence study ( maps ) of 6,800 hypertensive diabetic patients from 10 countries in the region reported an overall prevalence rate of 39.8% ( 95% confidence interval [ ci ] 39.240.5)22 .", "these data show how a high proportion of hypertensive , type  2 diabetes asian patients are showing early signs of declining renal function , and further support a need for early intervention so as to reduce accelerated progression of cardiorenal complications and the associated healthcare burden28 . with the exception of taiwan ( chiang sc et  al .", "jcma 2011 ; 74 : 310 ) , these data are based on subanalyses of the microalbuminuria prevalence study of 6,800 hypertensive diabetic patients from 10 countries across asia ( wu ay et  al . diabetologia .", "2005 ; 48 : 1726 . ) in recognition of the utility of measuring microalbuminuria , guidelines recommend annual screening of this clinical marker for identifying atrisk populations32 .", "patients identified with microalbuminuria early in the course of disease might require stricter glycemic and blood pressure control targets .", "indeed , several prospective studies have shown the importance of tight glycemic control alone and combined with blood pressure lowering in the prevention of microvascular complications .", "for instance , the uk prospective diabetes study compared the effects of intensive bloodglucose control with either sulfonylureas or insulin and conventional treatment ( including diet ) on the risk of microvascular and macrovascular complications in type  2 diabetes patients .", "intensive glucose control with sulfonylureas or insulin resulted in a 33% relative risk reduction in the development of microalbuminuria .", "furthermore , this reduction in risk was sustained in the 10year followup study during which longterm benefits on cardiovascular disease ( cvd ) risk also emerged33 . in the kumamoto study ,", "the frequency and severity of diabetic microvascular complications were assessed in japanese type  2 diabetes patients after treatment with either intensive glucose control ( multiple insulin injections of three or more ) or conventional therapy ( insulin injections administered once or twice daily ) .", "there was a significant reduction in the progression of nephropathy and other microvascular complications ( retinopathy ) after intensive insulin therapy ( p  <  0.05)35 .", "the effect of an intensive , stepwise multifactorial intervention , targeting hyperglycemia , hypertension , dyslipidemia and microalbuminuria , on the initiation and progression of microvascular complications in type  2 diabetes patients with microalbuminuria was assessed in the steno2 study .", "type  2 diabetes patients receiving early intensive diabetes management including tight glucose control and angiotensin converting enzyme inhibitors ( acei ) to reduce blood pressure were significantly less likely to develop nephropathy than patients receiving standard therapy ( odds ratio 0.27 ; 95% ci 0.100.75 ) .", "this reduction in risk was maintained in an observational follow up of 5.5  years36 . in the most recent analysis of the action in diabetes and vascular disease : preterax and diamicronmodified release controlled evaluation ( advance ) trial , which included a large number of asian type  2 diabetes patients , further support for the benefits of combining bloodpressure lowering with intensive blood glucose control was provided38 .", "patients received either intensive glucose control ( target glycated hemoglobin [ hba1c ]   6.5% ) or standard glucose control ( defined by local guidelines ) with placebo or acei plus indapamide therapy .", "a combination of intensive glucose control plus acei plus indapamide therapy led to a significant percentage in risk reduction in new or worsening nephropathy ( 95% ci 1250% ; p  =  0.005 ) ; a 54% reduction in the development of newonset macroalbuminuria ( 95% ci 3568% ; p  <  0.001 ) , and a 25% reduction in the risk of newonset microalbuminuria ( 95% ci 1633% ; p  <  0.001 ) .", "furthermore , the effects of blood pressure lowering and intensive glucose control were independent of each other , so that on combining these agents , additive benefits were observed , particularly on renal outcomes38 . indeed , the benefits of early and tight target control strategies on the prevention of dn in chinese type  2 diabetes patients have been observed . in this asian cohort with normoalbuminuria , achievement of american diabetes association ( ada)recommended targets of hba1c ( < 7% ) , systolic blood pressure ( < 130  mmhg ) , diastolic blood pressure ( < 80  mmhg ) and lipid profiles ( lowdensity lipoprotein [ ldl ] < 100  mg / dl ; triglycerides < 150  mg / dl ; highdensity lipoprotein > 4050  mg / dl ) reduced the risk of developing newonset microalbuminuria compared with those who were unable to attain these targets39 .", "interestingly , in a separate study of taiwanese type  2 diabetes patients with microalbuminuria , almost 36% of patients achieved remission to normoalbuminuria following a tight , multifactorial target control approach40 .", "worldwide , rates of ckd have been increasing , and have accompanied the increasing prevalence of type  2 diabetes , hypertension and cvd .", "for instance , in the usa , it is estimated that 30% of patients with type  2 diabetes will develop some form of kidney disease as reflected by protein leakage , microalbuminuria , macroalbuminuria or a decline in the glomerular filtration rate ( gfr)41 .", "nephropathy is a common complication of asians with type  2 diabetes , as reflected by the accelerating prevalence and incidence rates of observed ckd23 .", "indeed , incidence rates of esrd across asia have shown increases over a 6year period ( 20032008 ) of 15.8% in hong kong , 22% in malaysia and thailand , 24% in singapore , and up to 31% in the philippines43 .", "although incidence rates of esrd appear to have declined in taiwan since 2005 , this country has the third highest rate of esrd in the world , with an incidence rate of 384 per million population ( pmp ) , highlighting the disparity between the rate of kidney disease and a country 's economic status42 .", "furthermore , it has become apparent that a high proportion of esrd cases are as a result of diabetes , ranging from 41% to 62% in individual countries across southeast asia ( figure  2)43 .", "incidence of endstage renal disease ( esrd ) as a result of diabetes in selected countries across southeast asia in 2008 .", "these data are based on figures compiled by the us renal disease system ( figure adapted from 2010 us renal disease system annual report chapter 12 international comparisons ; volume 2 ; pages 383396 ) . for singapore ,", "the 2008 data have been extracted from the national registry of diseases office , released 1 march 2011 ( inp111 ) .", "the presence of ckd is also associated with a high cvd risk , which is further exacerbated by the presence of diabetes and hypertension45 .", "indeed , cvd remains the main cause of death in patients with ckd rather than kidney failure , and it is estimated that cvdassociated mortality is 1030fold higher in patients with advanced ckd receiving dialysis than the general population46 .", "given that the prevalence of hypertension in asia is increasing , and that diabetes and hypertension commonly coexist ( type  2 diabetes patients are twice as likely to have elevated blood pressure than nontype  2 diabetes patients ) , screening these highrisk patients for ckd early might help slow the course of renal function decline and reduce the greater cardiovascular mortality risk observed in such individuals47 .", "despite the advent and availability of a range of antidiabetic and blood pressure lowering agents , many patients in asia remain suboptimally controlled and therefore at risk of developing complications . in the maps study , just 10.6% of type  2 diabetes asian patients with microalbuminuria achieved blood pressure targets below 130/80  mmhg and mean hba1c levels of 7.9%22 .", "similarly , in a crosssectional survey in china , the majority of type  2 diabetes patients with nephropathy ( 66.9% ) had a mean hba1c level of > 7.5% , indicating poor glycemic control in patients with complications50 .", "these data are supported by the recent international diabetes management practice study ( idmps ) that explored the barriers to achieving optimal glycemic control across asia , latin america and eastern europe51 . in asia , of the 5,372 type  2", "diabetes patients assessed , 35.8% had microalbuminuria and just 37.3% achieved the hba1c target of < 7.0% ; 21.8% achieved the blood pressure target of < 130/80  mmhg ; and 37% achieved the ldl cholesterol target of < 100  mg / dl .", "another significant and worrying finding was that just 4.7% of patients in asia attained all three treatment targets51 .", "factors that might be contributing to such low control rates have been reported to include : difficult medical access by patients in some developing countries , physician perception of target levels , prescribing habits , as well as knowledge of guidelines ", "an aging population provides additional challenges in managing type  2 diabetes , as aging itself is associated with changes in kidney structure and function , and these agerelated renal changes can be accelerated by the presence of comorbid conditions52 .", "furthermore , a study of the hong kong diabetes registry revealed how long disease duration and complexity of treatment regimens might also play a role in suboptimal glycemic control54 .", "poor control might also result in type  2 diabetes patients who present and commence treatment when their renal function is normal , but subsequently go on to develop kidney disease . without appropriate monitoring of gfr levels and diagnosis of kidney disease or its progression , the need for dose adjustments or reevaluation of prescribed therapies might be missed and , consequently , adverse sideeffects might arise55 .", "hypoglycemia is a particular challenge in the management of type  2 diabetes , especially in patients with a decline in renal function .", "for instance , diabetic patients with ckd are twice as likely to experience hypoglycemic events than those without kidney disease56 .", "this might be a result of reduced insulin clearance , reduced gluconeogenesis by the kidney and increased accumulation of drugs excreted by the kidney57 .", "the reduction in drug clearance results in prolonged exposure to the drug itself or its metabolites , which can lead to adverse sideeffects .", "this is a major issue in moderate to severe ckd ( stages 35 ) when renal dysfunction is particularly pronounced55 .", "the american diabetes association guidelines recommend target hba1c levels of < 8% for patients with more advanced microvascular complications and increased risk of hypoglycemia , such as those with more advanced ckd .", "however , in practice , the use of antidiabetic medications in achieving these glycemic targets is often challenging58 .", "there are now an array of antihyperglycemic agents available that include both injectable , such as insulin and the incretin mimetics ", "glp1 analogs ( exenatide , liraglutide ) , and oral antidiabetics ( oads ) . among the oads are biguanides ( metformin ) , second generation sulfonylureas ( glimepiride , glipizide , glicazide ) , meglitinides ( nateglinide , repaglinide ) glucosidase inhibitors ( acarbose ) , thiazolidinediones ( tzd ; pioglitazone ) and incretinbased therapies that include dipeptidylpeptidase  iv ( dpp4 ) inhibitors ( linagliptin , sitagliptin , vildagliptin , saxagliptin ) .", "in addition , oral sodiumglucose transporter  2 ( inhibitors are currently in development ( empagliflozin , dapagliflozin ) . despite the range of medications available for the treatment of type  2 diabetes and its complications , many of these require dose modification or avoidance and close monitoring55 .", "this poses a particular challenge in more advanced disease ( ckd stages  35 ) , where contraindications or the increased risk of hypoglycemia serve to limit the number of treatment options ( table  2)55 .", "as all these agents differ in their mechanism of action , efficacy , sideeffects and costs , the choice of therapy should be guided by several factors .", "patient demographics , presence of comorbid conditions , risk or severity of renal function decline , tolerability , and risk of developing adverse events , such as hypoglycemia , weight gain and edema , should help in the treatment decision process57 .", "with most of the oads currently available for the treatment of type  2 diabetes , their use in patients with kidney dysfunction is compromised55 .", "however , sulfonylureas and their metabolites are affected by kidney function by increasing in potency as renal function declines . furthermore , in type  2 diabetes patients with esrd receiving dialysis , treatment with sulfonylureas might lead to severe and prolonged episodes of hypoglycemia61 .", "however , secondgeneration sulfonylureas , such as gliclazide and glipizide , which have inactive metabolites , carry less risk of hypoglycemia than the firstgeneration sulfonylureas and might therefore be used in type  2 diabetes patients with declining renal function , but with caution .", "similarly , the meglitinide , nateglinide , undergoes hepatic metabolism that gives rise to weakly active metabolites that are excreted predominantly through the kidneys . furthermore , 15% of the drug", "is excreted unchanged in urine . given the reduced clearance in ckd and the likelihood of hypoglycemic episodes , cautious use in these special populations , particularly in more advanced stages of ckd , is warranted .", "in contrast , repaglinide appears to have a lower risk of hypoglycemia and might be of use in ckd stages  3 and 4 , but requires low initial dosing and careful dose titration in these patients62 .", "the use of metformin , the standard firstline therapy of type  2 diabetes , also poses a risk for diabetic patients with ckd as a result of lactic acidosis , and is therefore contraindicated in patients with advanced renal dysfunction57 . whether this medication provides an additional risk to the underlying comorbidity or acts directly to bring about lactic acidosis requires further investigation63 .", "treatment with glycosidase inhibitors in type  2 diabetes patients with ckd ( stage  35 ) is also not recommended , because of the potential risk of hepatic damage as a result of cumulated dose effects57 .", "in contrast , a few small , shortterm studies have implicated a renoprotective role for tzd , as it resulted in greater reductions in albuminuria than metformin or sulfonylureas .", "whether this translates into direct prevention of renal function decline , however , requires further study64 .", "nevertheless , tzd undergo hepatic metabolism and have a low risk of hypoglycemia , making them appropriate for use in ckd . of note ,", "fluid retention is a known sideeffect of tzd , and in ckd patients this outcome could be exacerbated .", "dpp4 inhibitors are the most recent addition to the type  2 diabetes treatment paradigm and have been developed in recognition of the multifactorial nature of type  2 diabetes pathology .", "this drug class mediates its antihyperglycemic effects by preventing the degradation of the incretin hormones ", "these hormones are released in the intestine after a meal , and target the pancreas by increasing glucosedependent insulin secretion and suppressing glucagon secretion , which subsequently improves both fasting plasma glucose ( fpg ) and postprandial glucose ( ppg ) levels .", "dpp4 inhibitors are therefore important in regulating glucose homeostasis67 . furthermore , beneficial effects on pancreatic cells have also been reported with this drug class and therefore it might play a role in preventing or slowing the progression of cell dysfunction68 .", "there are currently five dpp4 inhibitors approved for type  2 diabetes : sitagliptin , vildagliptin , saxagliptin , alogliptin ( japan ) and linagliptin ; and their efficacy and tolerability have been studied in several clinical trials72 .", "although there are limited direct headtohead studies between dpp4 inhibitors that have been carried out to date , observations across studies suggest that all five agents have comparable efficacy as reflected in their levels of hba1c reduction .", "when compared with other antihyperglycemic agents , headtohead studies showed dpp4 inhibitors to have similar efficacy to metformin , sulfonylureas , tzd and glycosidase inhibitors73 . in terms of tolerability", ", all five dpp4 inhibitors have been shown to be well tolerated with low rates of adverse effects and , in general , comparable with placebo or comparator72 .", "furthermore , the issues of hypoglycemia and weight gain frequently seen with the more traditional antihyperglycemic therapies are not readily observed with dpp4 inhibitors .", "although there is little to differentiate between these agents in terms of observed efficacy , each differs in their chemistry and pharmacokinetic profile , which might translate into differences in their clinical applicability79 .", "indeed , dpp4 inhibitors differ in their halflives : sitagliptin , alogliptin and linagliptin have longhalf lives , allowing for oncedaily administration ; vildagliptin has a short halflife and twicedaily dosing is recommended .", "saxagliptin has a short halflife , but given its active metabolite , oncedaily dosing is adequate79 .", "furthermore , on examining their metabolism and excretion profiles , differences between the dpp4 inhibitors have emerged .", "sitagliptin , vildagliptin , alogliptin and saxagliptin are predominantly excreted through the kidneys72 . despite the renal excretion profiles of these agents , their favorable tolerability in patients with various severity of renal dysfunction still permits their use in such patients ,", "although dose modifications are recommended in line with increasing kidney dysfunction ( table  2 ) .", "in contrast , linagliptin is excreted through the bile and with approximately 5% being renally excreted80 .", "furthermore , recent data suggest that dose modifications of linagliptin are not required irrespective of the severity of renal dysfunction ( table  2)81", ". linagliptin might therefore represent an important addition to the currently limited therapeutic armamentarium for achieving glycemic control in type  2 diabetes patients with declining renal function . in recognition of the favorable clinical profile of dpp4 inhibitors ,", "recent international guidelines recommend dpp4 inhibitors as an option in both firstline ( when fpg and ppg levels are elevated ) and combination therapy82 .", "thus , type  2 diabetes patients , particularly special populations like those who are renally impaired , benefit from the emergence and evolution of newer antidiabetic agents , such as the dpp4 inhibitors , that address the limitations of older therapies , and allow for a more tailored and effective approach to disease control .", "longterm studies to assess the impact of dpp4 inhibitors on reducing the risk of diabetes complications are awaited .", "dpp4 inhibitors represent a good choice of oad for patients with type  2 diabetes .", "within this drug class , linagliptin , a novel dpp4 inhibitor , possesses favorable and comparable profiles in terms of both efficacy and adverse events , and is indicated for use in all patients with type  2 diabetes .", "furthermore , linagliptin is the only dpp4 inhibitor that is not predominantly dependent on renal metabolism .", "many antidiabetic therapies are often of little use in renallyimpaired type  2 diabetes patients because of contraindications or increased risk of hypoglycemia .", "the favorable pharmacokinetic profile of linagliptin has advantageous implications given the importance of addressing the decline in renal function in asian patients with type  2 diabetes early .", "in addition , the lack of dose modification requirements and ease of dosing ( one dose , once daily ) further support the suitability of linagliptin in these difficulttotreat populations ."], "abstract_text": ["<S> abstractit is well recognised that asia is at the epicenter of the global type  2 diabetes epidemic . driven by socioeconomic changes involving industrialization , urbanization and adoption of western lifestyles , </S>", "<S> the unprecedented increases in the prevalence of diabetes are particularly evident in southeast asia . </S>", "<S> the impact of diabetes is immense , and despite evidence of the benefit of optimal glucose control in reducing the risk of disease progression and development of macrovascular and microvascular complications , many individuals in this region remain poorly controlled . </S>", "<S> chronic kidney disease ( ckd ) is an increasingly common diabetesassociated complication in asian patients . </S>", "<S> furthermore , southeast asia has one of the highest rates of endstage renal disease ( esrd ) in the world . </S>", "<S> consequently , ckd in diabetes is associated with considerable morbidity and cardiovascularrelated mortality , highlighting the need to screen and assess patients early in the course of the disease . </S>", "<S> the management of type  2 diabetes patients with declining renal function represents a significant challenge . </S>", "<S> many of the older antidiabetic agents , such as metformin and sulfonylureas , are limited in their utility in ckd as a result of contraindications or hypoglycemic episodes . </S>", "<S> in contrast , dipeptidylpeptidase  iv inhibitors have provided a welcome addition to the therapeutic armamentarium for achieving glycemic control in these special populations . with comparable efficacy to and more favorable pharmacokinetic and sideeffect profiles than traditional therapies , </S>", "<S> agents in this drug class , such as linagliptin , offer a more tailored approach to disease control in type  2 diabetes patients with declining renal function . </S>"], "labels": null, "section_names": ["Current Burden of Type2 Diabetes in Southeast Asia", "Diabetes aND Chronic Kidney Disease Complications in Asia", "Microalbuminuria and Type2 Diabetes", "Impact of CKD in Type2 Diabetes", "Impact of Declining Renal Function on Type2 Diabetes Control", "Current Oral AntiDiabetics in Diabetic Patients with CKD", "Conclusions"], "sections": [["type  2 diabetes continues to present a public health burden across the world , and asia is considered to be at the epicenter of this diabetes epidemic , with 60% of the disease burden borne by this region1 .", "the increase in diabetes prevalence across asia has been rapid , fuelled by increasing obesity , aging populations and urbanization , which in turn have been driven by rapid economic development , a sedentary lifestyle and increased consumption of refined foods3 .", "these socioeconomic changes have been particularly significant in countries across southeast asia4 , and reflected by dramatic rises in type  2 diabetes prevalence over the past decade ( figure  1 ) .", "indeed , indonesia and the philippines are projected to be among the top 10 countries in the world with the highest number of estimated cases by 20301 .", "another issue curtailing the type  2 diabetes rise in asia is the high number of undiagnosed individuals .", "for instance , in hong kong and taiwan it was reported that almost 53% of diabetic patients remain undiagnosed5 .", "furthermore , the prevalence of prediabetes ( impaired glucose tolerance ) further adds to the already burgeoning burden of overt diabetes and its consequences in this region ( figure  1 ) .", "comparative prevalence of diabetes in countries across southeast asia in 2011 ( calculated by assuming that every country and region has the same age profile ) .", "these data are based on figures compiled by the international diabetes federation in the international diabetes atlas , 2011 ( available at : http://www.idf.org/atlasmap/atlasmap ) .", "it is widely recognized that diabetes is a major source of morbidity , mortality and economic burden6 .", "prolonged and poor glucose control increases the risk of developing macrovascular ( coronary artery disease , stroke ) and microvascular ( nephropathy , retinopathy and neuropathy ) complications7 , which can greatly reduce life expectancy and quality of life8 .", "in fact , the risk of allcause mortality ( cardiovascular and noncardiovascular ) is higher in diabetic than nondiabetic patients9 . consequently , the rapidly growing prevalence of diabetes in asia is likely to result in large increases in diabetesassociated mortality10 . from an economic perspective", ", the impact of diabetes on healthcare resources and personal expenditure , as well as the financial implications associated with loss in productivity and disability , is enormous11 . in taiwan ,", "the direct healthcare cost associated with diabetes was 11.5% of the total national healthcare cost and was 4.3fold higher than the care of nondiabetic individuals12 . in hong kong , the annual total cost of type  2 diabetes equated to usd  1,725    2,044 per patient .", "furthermore , complications increased treatment costs associated with inpatient hospital stay by between 6 and 300% compared with treatment of type  2 diabetes patients without complications14 .", "of note is the considerable heterogeneity in healthcare spending for diabetes between countries across southeast asia .", "the international diabetes federation estimated that , in 2010 , treatment of diabetes accounted for 5 and 7% of the total healthcare expenditure in vietnam and indonesia , respectively , compared with 15 and 16% in singapore and malaysia , respectively11 .", "taken together , it is clear that early diagnosis and disease control are important healthcare initiatives to improve the lives , outcomes and economies of these atrisk countries . in determining the optimal management approach , recognizing the differences in clinical characteristics and risk of diabetes between asian and caucasian populations is important to understand .", "for instance in asians , diabetes develops over a shorter time , in younger people and in those with a lower body mass index ( bmi)15 .", "furthermore , asians have a higher proportion of body fat and abdominal obesity , putting them at a higher risk of developing insulin resistance16 .", "reports that asians show early cell dysfunction might further explain their increased susceptibility to type  2 diabetes16 .", "the thrifty gene and thrifty phenotype hypotheses have been proposed as plausible explanations for the increasing diabetes epidemic seen across asia18 .", "although considered protective during periods of famine or food shortage , thrifty genes have rendered individuals highly predisposed to obesity in times of plenty .", "alternatively , the thrifty phenotype relates to the impact of an unfavorable intrauterine environment ( e.g. undernutrition ) and low birth weight on the subsequent development of diabetes and metabolic syndrome in adults18 .", "several diabetesassociated genes have been implicated to account for the heightened predilection in asians , especially asian indians , and include polymorphisms of the peroxisome proliferatoractivated receptor coactivator1 alpha ( pgc1 alpha ) gene , plasma cell glycoprotein ( pc1 ) gene and insulin receptor substrate ( irs2 ) gene ( reviewed in radha and mohan)19 .", "given the increased prevalence of diabetes among younger populations in asia and early exposure to hyperglycemia , it has been suggested that they are also at a high risk of endorgan damage and are likely to have to live with complications for longer20 .", "indeed , the asia  pacific cohort studies collaboration ( apcsc ) , which assessed the impact of diabetes on cardiovascular outcomes and mortality , found a greater risk of fatal coronary heart disease , hemorrhagic and ischemic stroke among younger compared with older individuals21 .", "apcsc also showed an association between diabetes and increased risk of death as a result of renal disease , cancer , respiratory infections , and other infective and inflammatory causes21 .", "although optimal blood glucose control has been shown to reduce the risk of both macrovascular and microvascular complications6 , the marked prevalence of renal dysfunction in asia reflects , in part , the continuing challenge in achieving this goal22 .", "the aim of the present article was to review the barriers to achieving optimal blood glucose control in asian patients with type  2 diabetes who have declining renal function and to discuss the treatment modalities that might be appropriate for the management of these special patient populations ."], ["of the diabetesassociated complications , the most debilitating is chronic kidney disease ( ckd ) and its pathogenic progression to endstage renal disease ( esrd ) that requires dialysis or transplantation23 .", "defining and diagnosing ckd in clinical practice has evolved since its first classification by the national foundation kidney disease outcomes quality initiative ( nkfkdoqi ) in 200224 . in these guidelines ,", "ckd was categorized into five stages according to the severity of disease as defined by decreasing glomerular filtration rates ( gfr ) alone24 . in the more recent update ,", "the assessment criteria have been modified to encompass both gfr and albuminuria measures as correlates of a patient 's prognosis .", "in addition , a subdivision of the previously described gfr stage  3 has been proposed to help further define the severity of kidney dysfunction in patients with ckd25 .", "risk factors for the development of reduced kidney function in southeast asian populations have been identified and include the presence of diabetes , systolic hypertension ( > 159  mmhg ) , hyperuricemia ( > 6.29  mg / dl ) , elevated bmi ( > 24.9  kg / m ) and hypercholesterolemia ( > 248.3  mg / dl)26 .", "microalbuminuria is the earliest clinical manifestation of ckd in type  2 diabetes and persistent microalbuminuria represents an early clinical marker for the development of diabetic nephropathy ( dn)27 . although the number of studies exploring the epidemiology of microalbuminuria across asia has been limited , the microalbuminuria prevalence study ( maps ) of 6,800 hypertensive diabetic patients from 10 countries in the region reported an overall prevalence rate of 39.8% ( 95% confidence interval [ ci ] 39.240.5)22 .", "these data show how a high proportion of hypertensive , type  2 diabetes asian patients are showing early signs of declining renal function , and further support a need for early intervention so as to reduce accelerated progression of cardiorenal complications and the associated healthcare burden28 . with the exception of taiwan ( chiang sc et  al .", "jcma 2011 ; 74 : 310 ) , these data are based on subanalyses of the microalbuminuria prevalence study of 6,800 hypertensive diabetic patients from 10 countries across asia ( wu ay et  al .", "2005 ; 48 : 1726 . ) in recognition of the utility of measuring microalbuminuria , guidelines recommend annual screening of this clinical marker for identifying atrisk populations32 .", "patients identified with microalbuminuria early in the course of disease might require stricter glycemic and blood pressure control targets .", "indeed , several prospective studies have shown the importance of tight glycemic control alone and combined with blood pressure lowering in the prevention of microvascular complications .", "for instance , the uk prospective diabetes study compared the effects of intensive bloodglucose control with either sulfonylureas or insulin and conventional treatment ( including diet ) on the risk of microvascular and macrovascular complications in type  2 diabetes patients .", "intensive glucose control with sulfonylureas or insulin resulted in a 33% relative risk reduction in the development of microalbuminuria .", "furthermore , this reduction in risk was sustained in the 10year followup study during which longterm benefits on cardiovascular disease ( cvd ) risk also emerged33 . in the kumamoto study ,", "the frequency and severity of diabetic microvascular complications were assessed in japanese type  2 diabetes patients after treatment with either intensive glucose control ( multiple insulin injections of three or more ) or conventional therapy ( insulin injections administered once or twice daily )", ". there was a significant reduction in the progression of nephropathy and other microvascular complications ( retinopathy ) after intensive insulin therapy ( p  <  0.05)35 .", "the effect of an intensive , stepwise multifactorial intervention , targeting hyperglycemia , hypertension , dyslipidemia and microalbuminuria , on the initiation and progression of microvascular complications in type  2 diabetes patients with microalbuminuria was assessed in the steno2 study .", "type  2 diabetes patients receiving early intensive diabetes management including tight glucose control and angiotensin converting enzyme inhibitors ( acei ) to reduce blood pressure were significantly less likely to develop nephropathy than patients receiving standard therapy ( odds ratio 0.27 ; 95% ci 0.100.75 ) .", "this reduction in risk was maintained in an observational follow up of 5.5  years36 . in the most recent analysis of the action in diabetes and vascular disease : preterax and diamicronmodified release controlled evaluation ( advance ) trial , which included a large number of asian type  2 diabetes patients , further support for the benefits of combining bloodpressure lowering with intensive blood glucose control was provided38 .", "patients received either intensive glucose control ( target glycated hemoglobin [ hba1c ]   6.5% ) or standard glucose control ( defined by local guidelines ) with placebo or acei plus indapamide therapy .", "a combination of intensive glucose control plus acei plus indapamide therapy led to a significant percentage in risk reduction in new or worsening nephropathy ( 95% ci 1250% ; p  =  0.005 ) ; a 54% reduction in the development of newonset macroalbuminuria ( 95% ci 3568% ; p  <  0.001 ) , and a 25% reduction in the risk of newonset microalbuminuria ( 95% ci 1633% ; p  <  0.001 ) .", "furthermore , the effects of blood pressure lowering and intensive glucose control were independent of each other , so that on combining these agents , additive benefits were observed , particularly on renal outcomes38 . indeed , the benefits of early and tight target control strategies on the prevention of dn in chinese type  2 diabetes patients have been observed . in this asian cohort with normoalbuminuria , achievement of american diabetes association ( ada)recommended targets of hba1c ( < 7% ) , systolic blood pressure ( < 130  mmhg ) , diastolic blood pressure ( < 80  mmhg ) and lipid profiles ( lowdensity lipoprotein [ ldl ] < 100  mg / dl ; triglycerides < 150  mg / dl ; highdensity lipoprotein > 4050  mg / dl ) reduced the risk of developing newonset microalbuminuria compared with those who were unable to attain these targets39 .", "interestingly , in a separate study of taiwanese type  2 diabetes patients with microalbuminuria , almost 36% of patients achieved remission to normoalbuminuria following a tight , multifactorial target control approach40 .", "worldwide , rates of ckd have been increasing , and have accompanied the increasing prevalence of type  2 diabetes , hypertension and cvd .", "for instance , in the usa , it is estimated that 30% of patients with type  2 diabetes will develop some form of kidney disease as reflected by protein leakage , microalbuminuria , macroalbuminuria or a decline in the glomerular filtration rate ( gfr)41 .", "nephropathy is a common complication of asians with type  2 diabetes , as reflected by the accelerating prevalence and incidence rates of observed ckd23 .", "indeed , incidence rates of esrd across asia have shown increases over a 6year period ( 20032008 ) of 15.8% in hong kong , 22% in malaysia and thailand , 24% in singapore , and up to 31% in the philippines43 .", "although incidence rates of esrd appear to have declined in taiwan since 2005 , this country has the third highest rate of esrd in the world , with an incidence rate of 384 per million population ( pmp ) , highlighting the disparity between the rate of kidney disease and a country 's economic status42 .", "furthermore , it has become apparent that a high proportion of esrd cases are as a result of diabetes , ranging from 41% to 62% in individual countries across southeast asia ( figure  2)43 .", "incidence of endstage renal disease ( esrd ) as a result of diabetes in selected countries across southeast asia in 2008 .", "these data are based on figures compiled by the us renal disease system ( figure adapted from 2010 us renal disease system annual report chapter 12 international comparisons ; volume 2 ; pages 383396 ) . for singapore ,", "the 2008 data have been extracted from the national registry of diseases office , released 1 march 2011 ( inp111 ) .", "the presence of ckd is also associated with a high cvd risk , which is further exacerbated by the presence of diabetes and hypertension45 .", "indeed , cvd remains the main cause of death in patients with ckd rather than kidney failure , and it is estimated that cvdassociated mortality is 1030fold higher in patients with advanced ckd receiving dialysis than the general population46 .", "given that the prevalence of hypertension in asia is increasing , and that diabetes and hypertension commonly coexist ( type  2 diabetes patients are twice as likely to have elevated blood pressure than nontype  2 diabetes patients ) , screening these highrisk patients for ckd early might help slow the course of renal function decline and reduce the greater cardiovascular mortality risk observed in such individuals47 ."], ["microalbuminuria is the earliest clinical manifestation of ckd in type  2 diabetes and persistent microalbuminuria represents an early clinical marker for the development of diabetic nephropathy ( dn)27 . although the number of studies exploring the epidemiology of microalbuminuria across asia has been limited , the microalbuminuria prevalence study ( maps ) of 6,800 hypertensive diabetic patients from 10 countries in the region reported an overall prevalence rate of 39.8% ( 95% confidence interval [ ci ] 39.240.5)22 .", "these data show how a high proportion of hypertensive , type  2 diabetes asian patients are showing early signs of declining renal function , and further support a need for early intervention so as to reduce accelerated progression of cardiorenal complications and the associated healthcare burden28 . with the exception of taiwan ( chiang sc et  al .", "jcma 2011 ; 74 : 310 ) , these data are based on subanalyses of the microalbuminuria prevalence study of 6,800 hypertensive diabetic patients from 10 countries across asia ( wu ay et  al . diabetologia .", "2005 ; 48 : 1726 . ) in recognition of the utility of measuring microalbuminuria , guidelines recommend annual screening of this clinical marker for identifying atrisk populations32 .", "patients identified with microalbuminuria early in the course of disease might require stricter glycemic and blood pressure control targets .", "indeed , several prospective studies have shown the importance of tight glycemic control alone and combined with blood pressure lowering in the prevention of microvascular complications .", "for instance , the uk prospective diabetes study compared the effects of intensive bloodglucose control with either sulfonylureas or insulin and conventional treatment ( including diet ) on the risk of microvascular and macrovascular complications in type  2 diabetes patients .", "intensive glucose control with sulfonylureas or insulin resulted in a 33% relative risk reduction in the development of microalbuminuria .", "furthermore , this reduction in risk was sustained in the 10year followup study during which longterm benefits on cardiovascular disease ( cvd ) risk also emerged33 . in the kumamoto study ,", "the frequency and severity of diabetic microvascular complications were assessed in japanese type  2 diabetes patients after treatment with either intensive glucose control ( multiple insulin injections of three or more ) or conventional therapy ( insulin injections administered once or twice daily ) .", "there was a significant reduction in the progression of nephropathy and other microvascular complications ( retinopathy ) after intensive insulin therapy ( p  <  0.05)35 .", "the effect of an intensive , stepwise multifactorial intervention , targeting hyperglycemia , hypertension , dyslipidemia and microalbuminuria , on the initiation and progression of microvascular complications in type  2 diabetes patients with microalbuminuria was assessed in the steno2 study .", "type  2 diabetes patients receiving early intensive diabetes management including tight glucose control and angiotensin converting enzyme inhibitors ( acei ) to reduce blood pressure were significantly less likely to develop nephropathy than patients receiving standard therapy ( odds ratio 0.27 ; 95% ci 0.100.75 ) .", "this reduction in risk was maintained in an observational follow up of 5.5  years36 . in the most recent analysis of the action in diabetes and vascular disease : preterax and diamicronmodified release controlled evaluation ( advance ) trial , which included a large number of asian type  2 diabetes patients , further support for the benefits of combining bloodpressure lowering with intensive blood glucose control was provided38 .", "patients received either intensive glucose control ( target glycated hemoglobin [ hba1c ]   6.5% ) or standard glucose control ( defined by local guidelines ) with placebo or acei plus indapamide therapy .", "a combination of intensive glucose control plus acei plus indapamide therapy led to a significant percentage in risk reduction in new or worsening nephropathy ( 95% ci 1250% ; p  =  0.005 ) ; a 54% reduction in the development of newonset macroalbuminuria ( 95% ci 3568% ; p  <  0.001 ) , and a 25% reduction in the risk of newonset microalbuminuria ( 95% ci 1633% ; p  <  0.001 ) .", "furthermore , the effects of blood pressure lowering and intensive glucose control were independent of each other , so that on combining these agents , additive benefits were observed , particularly on renal outcomes38 . indeed , the benefits of early and tight target control strategies on the prevention of dn in chinese type  2 diabetes patients have been observed . in this asian cohort with normoalbuminuria , achievement of american diabetes association ( ada)recommended targets of hba1c ( < 7% ) , systolic blood pressure ( < 130  mmhg ) , diastolic blood pressure ( < 80  mmhg ) and lipid profiles ( lowdensity lipoprotein [ ldl ] < 100  mg / dl ; triglycerides < 150  mg / dl ; highdensity lipoprotein > 4050  mg / dl ) reduced the risk of developing newonset microalbuminuria compared with those who were unable to attain these targets39 .", "interestingly , in a separate study of taiwanese type  2 diabetes patients with microalbuminuria , almost 36% of patients achieved remission to normoalbuminuria following a tight , multifactorial target control approach40 ."], ["worldwide , rates of ckd have been increasing , and have accompanied the increasing prevalence of type  2 diabetes , hypertension and cvd .", "for instance , in the usa , it is estimated that 30% of patients with type  2 diabetes will develop some form of kidney disease as reflected by protein leakage , microalbuminuria , macroalbuminuria or a decline in the glomerular filtration rate ( gfr)41 .", "nephropathy is a common complication of asians with type  2 diabetes , as reflected by the accelerating prevalence and incidence rates of observed ckd23 .", "indeed , incidence rates of esrd across asia have shown increases over a 6year period ( 20032008 ) of 15.8% in hong kong , 22% in malaysia and thailand , 24% in singapore , and up to 31% in the philippines43 .", "although incidence rates of esrd appear to have declined in taiwan since 2005 , this country has the third highest rate of esrd in the world , with an incidence rate of 384 per million population ( pmp ) , highlighting the disparity between the rate of kidney disease and a country 's economic status42 .", "furthermore , it has become apparent that a high proportion of esrd cases are as a result of diabetes , ranging from 41% to 62% in individual countries across southeast asia ( figure  2)43 .", "incidence of endstage renal disease ( esrd ) as a result of diabetes in selected countries across southeast asia in 2008 .", "these data are based on figures compiled by the us renal disease system ( figure adapted from 2010 us renal disease system annual report chapter 12 international comparisons ; volume 2 ; pages 383396 ) . for singapore ,", "the 2008 data have been extracted from the national registry of diseases office , released 1 march 2011 ( inp111 ) .", "the presence of ckd is also associated with a high cvd risk , which is further exacerbated by the presence of diabetes and hypertension45 .", "indeed , cvd remains the main cause of death in patients with ckd rather than kidney failure , and it is estimated that cvdassociated mortality is 1030fold higher in patients with advanced ckd receiving dialysis than the general population46 .", "given that the prevalence of hypertension in asia is increasing , and that diabetes and hypertension commonly coexist ( type  2 diabetes patients are twice as likely to have elevated blood pressure than nontype  2 diabetes patients ) , screening these highrisk patients for ckd early might help slow the course of renal function decline and reduce the greater cardiovascular mortality risk observed in such individuals47 ."], ["despite the advent and availability of a range of antidiabetic and blood pressure lowering agents , many patients in asia remain suboptimally controlled and therefore at risk of developing complications . in the maps study , just 10.6% of type  2 diabetes asian patients with microalbuminuria achieved blood pressure targets below 130/80  mmhg and mean hba1c levels of 7.9%22 .", "similarly , in a crosssectional survey in china , the majority of type  2 diabetes patients with nephropathy ( 66.9% ) had a mean hba1c level of > 7.5% , indicating poor glycemic control in patients with complications50 .", "these data are supported by the recent international diabetes management practice study ( idmps ) that explored the barriers to achieving optimal glycemic control across asia , latin america and eastern europe51 . in asia , of the 5,372 type  2", "diabetes patients assessed , 35.8% had microalbuminuria and just 37.3% achieved the hba1c target of < 7.0% ; 21.8% achieved the blood pressure target of < 130/80  mmhg ; and 37% achieved the ldl cholesterol target of < 100  mg / dl .", "another significant and worrying finding was that just 4.7% of patients in asia attained all three treatment targets51 .", "factors that might be contributing to such low control rates have been reported to include : difficult medical access by patients in some developing countries , physician perception of target levels , prescribing habits , as well as knowledge of guidelines ", "an aging population provides additional challenges in managing type  2 diabetes , as aging itself is associated with changes in kidney structure and function , and these agerelated renal changes can be accelerated by the presence of comorbid conditions52 .", "furthermore , a study of the hong kong diabetes registry revealed how long disease duration and complexity of treatment regimens might also play a role in suboptimal glycemic control54 .", "poor control might also result in type  2 diabetes patients who present and commence treatment when their renal function is normal , but subsequently go on to develop kidney disease . without appropriate monitoring of gfr levels and diagnosis of kidney disease or its progression , the need for dose adjustments or reevaluation of prescribed therapies might be missed and , consequently , adverse sideeffects might arise55 .", "hypoglycemia is a particular challenge in the management of type  2 diabetes , especially in patients with a decline in renal function .", "for instance , diabetic patients with ckd are twice as likely to experience hypoglycemic events than those without kidney disease56 .", "this might be a result of reduced insulin clearance , reduced gluconeogenesis by the kidney and increased accumulation of drugs excreted by the kidney57 .", "the reduction in drug clearance results in prolonged exposure to the drug itself or its metabolites , which can lead to adverse sideeffects .", "this is a major issue in moderate to severe ckd ( stages 35 ) when renal dysfunction is particularly pronounced55 ."], ["the american diabetes association guidelines recommend target hba1c levels of < 8% for patients with more advanced microvascular complications and increased risk of hypoglycemia , such as those with more advanced ckd .", "however , in practice , the use of antidiabetic medications in achieving these glycemic targets is often challenging58 .", "there are now an array of antihyperglycemic agents available that include both injectable , such as insulin and the incretin mimetics ", "glp1 analogs ( exenatide , liraglutide ) , and oral antidiabetics ( oads ) . among the oads are biguanides ( metformin ) , second generation sulfonylureas ( glimepiride , glipizide , glicazide ) , meglitinides ( nateglinide , repaglinide ) glucosidase inhibitors ( acarbose ) , thiazolidinediones ( tzd ; pioglitazone ) and incretinbased therapies that include dipeptidylpeptidase  iv ( dpp4 ) inhibitors ( linagliptin , sitagliptin , vildagliptin , saxagliptin ) .", "in addition , oral sodiumglucose transporter  2 ( inhibitors are currently in development ( empagliflozin , dapagliflozin ) . despite the range of medications available for the treatment of type  2 diabetes and its complications , many of these require dose modification or avoidance and close monitoring55 .", "this poses a particular challenge in more advanced disease ( ckd stages  35 ) , where contraindications or the increased risk of hypoglycemia serve to limit the number of treatment options ( table  2)55 .", "as all these agents differ in their mechanism of action , efficacy , sideeffects and costs , the choice of therapy should be guided by several factors .", "patient demographics , presence of comorbid conditions , risk or severity of renal function decline , tolerability , and risk of developing adverse events , such as hypoglycemia , weight gain and edema , should help in the treatment decision process57 .", "with most of the oads currently available for the treatment of type  2 diabetes , their use in patients with kidney dysfunction is compromised55 .", "however , sulfonylureas and their metabolites are affected by kidney function by increasing in potency as renal function declines . furthermore , in type  2 diabetes patients with esrd receiving dialysis , treatment with sulfonylureas might lead to severe and prolonged episodes of hypoglycemia61 .", "however , secondgeneration sulfonylureas , such as gliclazide and glipizide , which have inactive metabolites , carry less risk of hypoglycemia than the firstgeneration sulfonylureas and might therefore be used in type  2 diabetes patients with declining renal function , but with caution .", "similarly , the meglitinide , nateglinide , undergoes hepatic metabolism that gives rise to weakly active metabolites that are excreted predominantly through the kidneys . furthermore , 15% of the drug", "is excreted unchanged in urine . given the reduced clearance in ckd and the likelihood of hypoglycemic episodes , cautious use in these special populations , particularly in more advanced stages of ckd , is warranted .", "in contrast , repaglinide appears to have a lower risk of hypoglycemia and might be of use in ckd stages  3 and 4 , but requires low initial dosing and careful dose titration in these patients62 .", "the use of metformin , the standard firstline therapy of type  2 diabetes , also poses a risk for diabetic patients with ckd as a result of lactic acidosis , and is therefore contraindicated in patients with advanced renal dysfunction57 . whether this medication provides an additional risk to the underlying comorbidity or acts directly to bring about lactic acidosis requires further investigation63 .", "treatment with glycosidase inhibitors in type  2 diabetes patients with ckd ( stage  35 ) is also not recommended , because of the potential risk of hepatic damage as a result of cumulated dose effects57 .", "in contrast , a few small , shortterm studies have implicated a renoprotective role for tzd , as it resulted in greater reductions in albuminuria than metformin or sulfonylureas .", "whether this translates into direct prevention of renal function decline , however , requires further study64 .", "nevertheless , tzd undergo hepatic metabolism and have a low risk of hypoglycemia , making them appropriate for use in ckd . of note ,", "fluid retention is a known sideeffect of tzd , and in ckd patients this outcome could be exacerbated .", "dpp4 inhibitors are the most recent addition to the type  2 diabetes treatment paradigm and have been developed in recognition of the multifactorial nature of type  2 diabetes pathology .", "this drug class mediates its antihyperglycemic effects by preventing the degradation of the incretin hormones ", "these hormones are released in the intestine after a meal , and target the pancreas by increasing glucosedependent insulin secretion and suppressing glucagon secretion , which subsequently improves both fasting plasma glucose ( fpg ) and postprandial glucose ( ppg ) levels .", "dpp4 inhibitors are therefore important in regulating glucose homeostasis67 . furthermore , beneficial effects on pancreatic cells have also been reported with this drug class and therefore it might play a role in preventing or slowing the progression of cell dysfunction68 .", "there are currently five dpp4 inhibitors approved for type  2 diabetes : sitagliptin , vildagliptin , saxagliptin , alogliptin ( japan ) and linagliptin ; and their efficacy and tolerability have been studied in several clinical trials72 .", "although there are limited direct headtohead studies between dpp4 inhibitors that have been carried out to date , observations across studies suggest that all five agents have comparable efficacy as reflected in their levels of hba1c reduction .", "when compared with other antihyperglycemic agents , headtohead studies showed dpp4 inhibitors to have similar efficacy to metformin , sulfonylureas , tzd and glycosidase inhibitors73 . in terms of tolerability", ", all five dpp4 inhibitors have been shown to be well tolerated with low rates of adverse effects and , in general , comparable with placebo or comparator72 .", "furthermore , the issues of hypoglycemia and weight gain frequently seen with the more traditional antihyperglycemic therapies are not readily observed with dpp4 inhibitors .", "although there is little to differentiate between these agents in terms of observed efficacy , each differs in their chemistry and pharmacokinetic profile , which might translate into differences in their clinical applicability79 .", "indeed , dpp4 inhibitors differ in their halflives : sitagliptin , alogliptin and linagliptin have longhalf lives , allowing for oncedaily administration ; vildagliptin has a short halflife and twicedaily dosing is recommended .", "saxagliptin has a short halflife , but given its active metabolite , oncedaily dosing is adequate79 .", "furthermore , on examining their metabolism and excretion profiles , differences between the dpp4 inhibitors have emerged .", "sitagliptin , vildagliptin , alogliptin and saxagliptin are predominantly excreted through the kidneys72 . despite the renal excretion profiles of these agents , their favorable tolerability in patients with various severity of renal dysfunction still permits their use in such patients ,", "although dose modifications are recommended in line with increasing kidney dysfunction ( table  2 ) .", "in contrast , linagliptin is excreted through the bile and with approximately 5% being renally excreted80 .", "furthermore , recent data suggest that dose modifications of linagliptin are not required irrespective of the severity of renal dysfunction ( table  2)81", ". linagliptin might therefore represent an important addition to the currently limited therapeutic armamentarium for achieving glycemic control in type  2 diabetes patients with declining renal function . in recognition of the favorable clinical profile of dpp4 inhibitors ,", "recent international guidelines recommend dpp4 inhibitors as an option in both firstline ( when fpg and ppg levels are elevated ) and combination therapy82 .", "thus , type  2 diabetes patients , particularly special populations like those who are renally impaired , benefit from the emergence and evolution of newer antidiabetic agents , such as the dpp4 inhibitors , that address the limitations of older therapies , and allow for a more tailored and effective approach to disease control .", "longterm studies to assess the impact of dpp4 inhibitors on reducing the risk of diabetes complications are awaited ."], ["dpp4 inhibitors represent a good choice of oad for patients with type  2 diabetes .", "within this drug class , linagliptin , a novel dpp4 inhibitor , possesses favorable and comparable profiles in terms of both efficacy and adverse events , and is indicated for use in all patients with type  2 diabetes .", "furthermore , linagliptin is the only dpp4 inhibitor that is not predominantly dependent on renal metabolism .", "many antidiabetic therapies are often of little use in renallyimpaired type  2 diabetes patients because of contraindications or increased risk of hypoglycemia .", "the favorable pharmacokinetic profile of linagliptin has advantageous implications given the importance of addressing the decline in renal function in asian patients with type  2 diabetes early .", "in addition , the lack of dose modification requirements and ease of dosing ( one dose , once daily ) further support the suitability of linagliptin in these difficulttotreat populations ."]]}
{"article_id": "PMC3931155", "article_text": ["pott 's disease accounts for 50% of the cases of skeletal tuberculosis ( tb ) , 15% of the cases of extrapulmonary tuberculosis ( eptb ) and 2% of all cases of tb.1 the diagnosis of pott 's disease is mostly based on clinicoradiological observations substantiated by staining and culture methods to detect the causative organism .", "the typical tubercular lesions of the spine can be diagnosed by radiological methods as their sensitivity is increased by the advent of newer imaging techniques like computed tomography and magnetic resonance imaging ( ct and mri ) .", "these techniques give sufficient information about lesions in the bone and tissues ; although the definite diagnosis is based on tissue / pus examination.2 the conventional bacteriological examination includes ziehl - neelsen ( zn ) microscopy and culture for acid fast bacilli ( afb ) .", "zn microscopy method is a popular technique routinely used in the clinical laboratories worldwide , due to its simplicity , cost effectiveness and rapidity , but it suffers with low sensitivity and requires 10 to 10 bacilli / ml in the clinical specimens to be positive.34 the culture of mycobacterium tuberculosis ( m. tuberculosis ) is a gold standard method for diagnosis but it also has limitations like that required 6 to 8 weeks due to the slow growth of m. tuberculosis and is often negative , it still needs 10 -10 bacilli / ml ( live bacilli ) in clinical specimens for culture recovery and also stringent test conditions that is difficult to implement at primary or secondary clinical laboratories.56 moreover , histopathological examination plays a valuable role in the diagnosis of pott 's disease but sometime it may be inconclusive and in addition need high expertise and the final reporting also takes more than 1 week.6 recently , the molecular biology technique , polymerase chain reaction ( pcr ) represents a major advance in the diagnosis of tb.6 with the use of amplification systems , nucleic acid sequences unique to m. tuberculosis can be detected directly in clinical specimens , offering better accuracy than zn microscopy and greater speed than culture .", "the pcr has shown very promising results for early and rapid diagnosis of the disease due to its detection limit of one to 10 bacilli in various clinical specimens.6 the present study was undertaken to evaluate the efficiency and effectiveness of different laboratory diagnostic modalities along with the role of pcr in the diagnosis of clinicoradiological suspected cases of pott 's disease .", "62 clinicoradiological suspected cases of pott 's disease with neurological complications [ table 1 ] were prospectively enrolled in this study , from 2008 to 2011 in the department of neurosurgery , at a tertiary care hospital , india .", "the specimens of these patients such as pus and tissue were obtained either during surgery or under ct guidance .", "clinicoradiological evidence of suspected pott 's disease we included clinicoradiological suspected cases of pott 's spine who underwent either open biopsy or ct guided aspiration at our institute .", "we excluded those subjects who did not give consent for biopsy or ct guided aspiration or biopsy showed neoplastic pathology .", "the specimens were examined by following methods : \n histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . \n ", "histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer .", "dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 .", "mb505 bacterial and yeast genomic dna miniprep purification spin kit , hi - media laboratories private limited mumbai , india .", "the reaction mixture contained 10 l pyrostart fast pcr master mix 2x ( dntp , taq polymerase with mgcl2 , fermentas , india ) , 1 l ( 10 pmole ) of each primer , 3 l water ( nuclease free ) and 5 l of extracted dna .", "the oligonucleotide primers11 were used forward and reverse sequence : cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively ( sbs gene tech co. ltd . ) .", "these primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc .", "it was performed in a programmable thermal cycler ( mj research , ptc-100 , gmi , inc .", "each cycle comprised denaturation at 94c for 2 min , annealing at 68c for 2 min and primer extension at 72c for 1 min .", "after the 40 cycles , the additional extension for 10 min at 72c was carried out .", "the amplified products were separated on 2% agarose gel , visualized on an ultraviolet - transilluminator ( bangalore genei , bangalore , india ) .", "the presence of 123 bp fragment indicated as a positive test for mtbc [ figure 1 ] .", "the positive controls included the dna of h37rv strain and negative control included pcr grade water .", "polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel .", "lane 1 ( l1 ) and l9 ladder 100 base pairs , l2 positive and l3 negative control , l4 , 5 , 6 , 8 showed positive and l7 showed negative specimens a definite treatment either by surgery ( 46 cases ) or by only anti tubercular treatment ( att ) ( 9 cases ) was offered .", "we followed the following major indications for surgical intervention in our setup : ( 1 ) advanced cases of neurological involvement such as marked sensory or sphincter disturbances , flaccid paralysis or severe flexor spasms .", "( 2 ) prevertebral cervical abscess with difficulty in respiration and deglutition , seemingly difficult resolution on chemotherapy during conservative treatment . ( 3 ) worsening of already present neurological complications during the treatment .", "( 5 ) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att . a four drug regimen chemotherapy consisting of isoniazid , rifampicin , pyrazinamide and ethambutol ( hrze ) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol , following or along with surgery .", "four - drug regimen for at least 4 - 6 weeks to gauge the response was given ( 8 - 12 mg / kg / day rifampicin , 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose ; and pyrazinamide 15 - 20 mg / kg / day in two divided doses ) , if not contraindicated the drug therapy was continued for 18 months12 . in cases of no improvements or hepatotoxicity ,", "the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them .", "the therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity , sensory loss of limbs , autonomic dysfunctions and constitutional symptoms [ table 2 ] .", "the liver function test was done as per protocol to rule out the subclinical hepatotoxcity .", "the radiological assessment was done by using followup mri , the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute .", "criteria for clinical improvement : ( first ( pain ) is mandatory and any other two improved symptoms / sign ) this study was approved by 41 institutional ethics committee ( a-04 pgi / imp / ec/41/28/2/2008 ) .", "the final diagnosis was established by using the collective results of zn microscopy , bactec culture , histopathological findings and response to att .", "the efficiency of test tools was calculated as ( total positive cases / total number of cases ) 100 .", "the positive concordance between traditional tools and pcr were assessed using the kappa coefficient (  > 0.75 , excellent agreement ; < 0.4 , poor agreement ; 0.4 and 0.75 , good to a fair agreement)13 and significance difference was analyzed by the chi square ( 2 ) test with the help of the spss 15.1 version .", "the cases were labeled as definite tb if at least one performed test ( zn microscopy / bactec culture / or histopathology ) was found positive .", "the specimens were examined by following methods : \n histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . \n ", "histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer .", "dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 .", "mb505 bacterial and yeast genomic dna miniprep purification spin kit , hi - media laboratories private limited mumbai , india .", "the reaction mixture contained 10 l pyrostart fast pcr master mix 2x ( dntp , taq polymerase with mgcl2 , fermentas , india ) , 1 l ( 10 pmole ) of each primer , 3 l water ( nuclease free ) and 5 l of extracted dna .", "the oligonucleotide primers11 were used forward and reverse sequence : cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively ( sbs gene tech co. ltd . ) .", "these primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc .", "it was performed in a programmable thermal cycler ( mj research , ptc-100 , gmi , inc .", "each cycle comprised denaturation at 94c for 2 min , annealing at 68c for 2 min and primer extension at 72c for 1 min .", "after the 40 cycles , the additional extension for 10 min at 72c was carried out .", "the amplified products were separated on 2% agarose gel , visualized on an ultraviolet - transilluminator ( bangalore genei , bangalore , india ) .", "the presence of 123 bp fragment indicated as a positive test for mtbc [ figure 1 ] .", "the positive controls included the dna of h37rv strain and negative control included pcr grade water .", "polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel .", "lane 1 ( l1 ) and l9 ladder 100 base pairs , l2 positive and l3 negative control , l4 , 5 , 6 , 8 showed positive and l7 showed negative specimens a definite treatment either by surgery ( 46 cases ) or by only anti tubercular treatment ( att ) ( 9 cases ) was offered .", "we followed the following major indications for surgical intervention in our setup : ( 1 ) advanced cases of neurological involvement such as marked sensory or sphincter disturbances , flaccid paralysis or severe flexor spasms .", "( 2 ) prevertebral cervical abscess with difficulty in respiration and deglutition , seemingly difficult resolution on chemotherapy during conservative treatment . ( 3 ) worsening of already present neurological complications during the treatment .", "( 5 ) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att .", "a four drug regimen chemotherapy consisting of isoniazid , rifampicin , pyrazinamide and ethambutol ( hrze ) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol , following or along with surgery .", "four - drug regimen for at least 4 - 6 weeks to gauge the response was given ( 8 - 12 mg / kg / day rifampicin , 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose ; and pyrazinamide 15 - 20 mg / kg / day in two divided doses ) , if not contraindicated the drug therapy was continued for 18 months12 . in cases of no improvements or hepatotoxicity ,", "the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them .", "the therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity , sensory loss of limbs , autonomic dysfunctions and constitutional symptoms [ table 2 ] .", "the liver function test was done as per protocol to rule out the subclinical hepatotoxcity .", "the radiological assessment was done by using followup mri , the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute .", "criteria for clinical improvement : ( first ( pain ) is mandatory and any other two improved symptoms / sign ) this study was approved by 41 institutional ethics committee ( a-04 pgi / imp / ec/41/28/2/2008 ) .", "the final diagnosis was established by using the collective results of zn microscopy , bactec culture , histopathological findings and response to att .", "the efficiency of test tools was calculated as ( total positive cases / total number of cases ) 100 .", "the positive concordance between traditional tools and pcr were assessed using the kappa coefficient (  > 0.75 , excellent agreement ; < 0.4 , poor agreement ; 0.4 and 0.75 , good to a fair agreement)13 and significance difference was analyzed by the chi square ( 2 ) test with the help of the spss 15.1 version .", "the cases were labeled as definite tb if at least one performed test ( zn microscopy / bactec culture / or histopathology ) was found positive .", "the study group consisted of 62 specimens from suspected pott 's spine individuals . on the basis of histopathological examination ,", "7 cases ( 5 specimens from open biopsy and 2 from fine needle aspiration [ fna ] ) were excluded from the study as they were diagnosed to be neoplastic . out of rest 55 cases ,", "46 specimens were obtained through open biopsy during surgery and 9 were ct guided fna .", "further , in all ( 46 ) open biopsy specimens , 7 were positive for epithelioid cell granulomas with afb , 27 revealed granulomatous lesions suggestive of tb with or without the presence of the langerhans giant cell .", "however , the histological diagnosis could not be reached in 12 specimens because of either suspicious granulomatous lesions or on account of inconclusive histology .", "five of nine ct guided fna specimens were diagnosed as tubercular and 4 specimens showed no granulomatous / no malignant lesions .", "the received specimens included 31 ( 56.4% ) pus and 15 ( 27.3% ) tissues through open biopsy along with 6 ( 11% ) pus and 3 ( 0.54% ) tissues were obtained through ct guided fna .", "the overall collected specimens were consisted 37 pus and 18 tissue specimens ( either open biopsy or by ct fna ) were analyzed .", "the zn microscopy was positive in total 20 ( 36.4% ) of 55 cases ( 18/37 cases in pus and 2/18 cases in tissue specimens ) .", "the bactec culture was positive 27 ( 49% ) of 55 cases ( 22/37 in pus and 5/18 in tissue specimens ) .", "the desired amplification of pcr is6110 was positive in 37 ( 67.5% ) of 55 cases ( 30/37 cases in pus and 7/18 in tissue specimens ) [ table 3 ] .", "the kappa coefficient of pcr with zn microscopy (  = 0.37 , [ p = 0.001 ] ) showed poor agreement and with the bactec culture (  = 0.57 , [ p < 0.001 ] ) it showed a good agreement [ figure 2 ] .", "result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools .", "( znm = zn microscopy , bc = bactec culture and hp = histopathology ) on histopathological results ; 39 ( 71% ) cases were positive and 16 ( 29% ) were negative for tb", ". out of 39 positive cases , 31 ( 56.4% ) were positive on pcr also .", "thus , the false negative and positive of pcr was found to be 8 ( 14.4% ) and 6 ( 11% ) respectively .", "therefore , the kappa coefficient of pcr with histopathology was (  = 0.4 [ p = 0 . 004 ] ) slightly good [ figure 2 ] . in 55 cases", ", it was possible to reach a final diagnosis through the combined result of all performed tests ( without consideration of pcr results ) and with att response .", "hence , among all 55 cases , 45 ( 82% ) cases were tb positive test and 10 ( 18% ) were negative by all tests carried out ( zn microscopy , bactec culture and histopathology ) .", "although pcr showed 67.3% ( 37/55 ) sensitivity at the final diagnosis , while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests", ". therefore , the kappa coefficient of pcr with the combined results of all traditional tests (  = 0.63 [ p < 0.001 ] ) ultimately reached to a fair positive agreement [ figure 2 ] .", "the present study , the combination of ( a ) zn microscopy with bactec , the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture .", "( b ) zn microscopy with histopathology , the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy .", "( c ) bactec culture with histopathology , the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture , where the histopathology was negative .", "( d ) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology .", "( e ) zn microscopy with pcr , the total positivity was 38 cases , in which 18 more cases were positive on pcr method .", "( f ) bactec culture with pcr , the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . (", "g ) histopathology with pcr , the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr .", "( h ) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [ table 4 ] .", "the received specimens included 31 ( 56.4% ) pus and 15 ( 27.3% ) tissues through open biopsy along with 6 ( 11% ) pus and 3 ( 0.54% ) tissues were obtained through ct guided fna .", "the overall collected specimens were consisted 37 pus and 18 tissue specimens ( either open biopsy or by ct fna ) were analyzed .", "the zn microscopy was positive in total 20 ( 36.4% ) of 55 cases ( 18/37 cases in pus and 2/18 cases in tissue specimens ) .", "the bactec culture was positive 27 ( 49% ) of 55 cases ( 22/37 in pus and 5/18 in tissue specimens ) .", "the desired amplification of pcr is6110 was positive in 37 ( 67.5% ) of 55 cases ( 30/37 cases in pus and 7/18 in tissue specimens ) [ table 3 ] .", "the kappa coefficient of pcr with zn microscopy (  = 0.37 , [ p = 0.001 ] ) showed poor agreement and with the bactec culture (  = 0.57 , [ p < 0.001 ] ) it showed a good agreement [ figure 2 ] .", "result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools .", "on histopathological results ; 39 ( 71% ) cases were positive and 16 ( 29% ) were negative for tb", ". out of 39 positive cases , 31 ( 56.4% ) were positive on pcr also .", "thus , the false negative and positive of pcr was found to be 8 ( 14.4% ) and 6 ( 11% ) respectively .", "therefore , the kappa coefficient of pcr with histopathology was (  = 0.4 [ p = 0 . 004 ] ) slightly good [ figure 2 ] .", "in 55 cases , it was possible to reach a final diagnosis through the combined result of all performed tests ( without consideration of pcr results ) and with att response .", "hence , among all 55 cases , 45 ( 82% ) cases were tb positive test and 10 ( 18% ) were negative by all tests carried out ( zn microscopy , bactec culture and histopathology ) .", "although pcr showed 67.3% ( 37/55 ) sensitivity at the final diagnosis , while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests .", "therefore , the kappa coefficient of pcr with the combined results of all traditional tests (  = 0.63 [ p < 0.001 ] ) ultimately reached to a fair positive agreement [ figure 2 ] .", "the present study , the combination of ( a ) zn microscopy with bactec , the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture .", "( b ) zn microscopy with histopathology , the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy .", "( c ) bactec culture with histopathology , the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture , where the histopathology was negative .", "( d ) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology .", "( e ) zn microscopy with pcr , the total positivity was 38 cases , in which 18 more cases were positive on pcr method .", "( f ) bactec culture with pcr , the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . (", "g ) histopathology with pcr , the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr .", "( h ) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [ table 4 ] .", "pott 's disease , accounts for 2% of all tb infections.1 the diagnosis of pott 's disease is principally based on classical clinical manifestations of spinal infection supplemented by modern imaging like ct and mri.1415 the ct and mri in particular can detect the subtle changes in the spine , from initial changes in the intensity pattern of the vertebrae to the extreme changes like deformity of the spine including soft tissue shadow , granulation tissue and pus etc.16 however , the various pathologies including neoplastic and other inflammatory conditions simulate with pott 's diseases on imaging .", "the current study excluded 7 of 62 cases ; they were diagnosed to be neoplastic by the histopathological examination .", "it signifies that even strong clinicoradiological diagnosis based on classical or nonclassical radiological and clinical findings can be wrong on histopathological evaluation .", "the remaining 55 cases , 20 ( 36.3% ) were tested positive with zn microscopy and 27 ( 49% ) with bactec culture .", "an earlier report by jain et al.17 found 14.8% positivity on zn microscopy and 11.11% positivity on culture method in cases of spinal tb .", "the zn microscopy and bactec culture reports revealed higher positivity in relation to the earlier published series .", "our higher detection rate was because of the fact that we used the liquid culture medium bactec 12b medium .", "it is the rapid , sensitive and efficient method for the isolation of mycobacterium in a clinical laboratory as compared to solid medium lowenstein ", "jensen.19 moreover , it is established today that all the traditional tests need to have a minimum concentration of bacilli for yielding positive results as mentioned in the introduction . hence , if the specimens , which are naturally having more concentration of bacilli , like sputum and pus it may become easy to detect m. tuberculosis.20 similarly , in our study the pus specimens had higher recognition of tubercular bacilli on zn microscopy and bactec culture methods [ table 3 ] .", "the study quoted the detection rate of eptb including pott 's spine varying from 53% to 81%.182122 the histopathological diagnosis could be reached in 71% ( 39 out of 55 ) of the cases which were either confirmed by the presence of afb in 12.7% or strongly suggestive of tubercular pathology . in our series , we found 16 cases were negative on histology . among these negative cases ,", "10 out of 16 cases had been under anti tubercular therapy for more than 6 months and another reason of histology negative might be a possible sampling error during the collection of the representative tissues for processing .", "the positivity of fna specimens seems to higher than surgical specimens , however , we require a long sample size for definite conclusion .", "molecular diagnostic tool , such as pcr has been shown to have higher efficiency in the diagnosis of pulmonary tb as well as eptb.4 it has been studied on clinical specimens of sputum , cerebrospinal fluid , edta - blood , pleural fluid , fluid from fistulae and pus from wounds.2324 in an attempt to increase diagnostic precision , we performed pcr is6110 , which was detected in 37/55 ( 67.3% ) cases .", "various studies documented the increased positivity by pcr targeting is6110 in specimens of eptb i.e. , sekar et al.25 found 63% positivity , negi et al.26 73% and tiwari et al.27 62% positivity among clinical specimens of eptb .", "the overall positive agreement shows a fair agreement (  = 0.63 ) between pcr and definite tb .", "the false negative results were observed in 14.5% cases out of 45 cases diagnosed by all performed traditional tests .", "the reasons of false negative results of pcr may be on account of insufficient specimens , irregular distribution of mycobacterium bacilli in specimens , the presence of extensive necrosis and presence of inhibitors.28 the limitation of our study are a low positive correlation (  = 0.4 ) of pcr with histopathological findings .", "this can be explained up to some extent by the specimen variation as that is different specimens ( such as pus , tissue ) from the same subjects .", "surgeons prefer to send tissue specimens for histology and pus for zn microscopy , bactec culture and pcr . in this study histology", "was done on tissue specimens ( all 55 cases ) , while zn microscopy , bactec culture and pcr were done in 37 pus and 18 tissue specimens . in our diagnostic battery ,", "all performed traditional tests were diagnosed 45/55 cases and pcr was alone detected 37/55 cases .", "thus , positive results of pcr with the clinicoradiological assumption it would be possible to make a decision in suspected pott 's disease to start early treatment to prevent irreversible complications . in the present study , it is observed that no single diagnostic modality is having a good positivity rate .", "hence , a combination of diagnostic battery is required for rapid diagnosis and better results .", "the combination of bactec culture and histopathology has detected a maximum of 45 cases . while the combination of pcr and histopathology has also detected same number of cases table 4 .", "as culture takes long incubation time for results , the combination of pcr and histopathology can be applied for rapid detection of pott 's disease . to conclude , for diagnosis of pott 's disease , the combined results of zn microscopy , bactec culture and histopathology are most appropriate . however , for rapid diagnosis the combination of pcr amplification and histopathology offer a better prospect ."], "abstract_text": ["<S> background : the diagnosis of pott 's disease is mostly based on clinicoradiological observations substantiated by the bacterial culture , staining and histopathology . since , no single technique is enough to conclude pott 's disease in diagnosis , the present study was undertaken to correlate the clinicoradiological , microbiological , histopathological and molecular method to evaluate the effectiveness in diagnosis of pott 's disease.materials and methods:62 clinicoradiologically suspected cases of pott 's disease </S>", "<S> were included in this study . </S>", "<S> the specimens for diagnostic work up were collected either during surgery or by computed tomography guided fine needle aspiration . </S>", "<S> all these specimens were tested for tuberculosis ( tb ) through ziehl - neelsen ( zn ) microscopy , bactec culture , histopathology and polymerase chain reaction ( pcr ) . </S>", "<S> the final diagnosis was established by the results of performed tests and clinicoradiological improvement of cases at the end of 6 months on anti tubercular treatment.results:out of 62 cases , 7 were excluded from this study as these were turned out to be neoplastic lesions on histopathology . amongst remaining 55 cases , </S>", "<S> the tb was diagnosed in 39 ( 71% ) on histopathology , 37 ( 67.5% ) on pcr , 27 ( 49% ) on bactec culture and 20 ( 36.3% ) on zn microscopy . </S>", "<S> ultimately 45 cases were tested as positive and 10 were detected as negative for tb in combination of zn microscopy , bactec culture and histopathology . </S>", "<S> pcr was positive in 37 of 45 cases and 10/55 cases remained negative . on clinical analysis of these 10 cases , it was noted that these were cases of relapse / poor compliance . </S>", "<S> the combination of pcr and histopathology was also shown positive for tb in 45 cases . </S>", "<S> hence , the pcr showed a fair positive agreement ( c = 0.63 ) against the combined results of all performed traditional methods.conclusions:the combination of pcr and histopathology is a rapid and efficient tool for diagnosis of pott 's disease . </S>"], "labels": null, "section_names": ["I", "M", "Laboratory examinations", "Statistical analysis", "R", "Specimens characteristic for microbiological and PCR ", "Comparison and correlation of histopathological results with molecular method (PCR ", "Efficiency of PCR ", "Possible diagnostic battery", "D"], "sections": [["pott 's disease accounts for 50% of the cases of skeletal tuberculosis ( tb ) , 15% of the cases of extrapulmonary tuberculosis ( eptb ) and 2% of all cases of tb.1 the diagnosis of pott 's disease is mostly based on clinicoradiological observations substantiated by staining and culture methods to detect the causative organism .", "the typical tubercular lesions of the spine can be diagnosed by radiological methods as their sensitivity is increased by the advent of newer imaging techniques like computed tomography and magnetic resonance imaging ( ct and mri ) .", "these techniques give sufficient information about lesions in the bone and tissues ; although the definite diagnosis is based on tissue / pus examination.2 the conventional bacteriological examination includes ziehl - neelsen ( zn ) microscopy and culture for acid fast bacilli ( afb ) .", "zn microscopy method is a popular technique routinely used in the clinical laboratories worldwide , due to its simplicity , cost effectiveness and rapidity , but it suffers with low sensitivity and requires 10 to 10 bacilli / ml in the clinical specimens to be positive.34 the culture of mycobacterium tuberculosis ( m. tuberculosis ) is a gold standard method for diagnosis but it also has limitations like that required 6 to 8 weeks due to the slow growth of m. tuberculosis and is often negative , it still needs 10 -10 bacilli / ml ( live bacilli ) in clinical specimens for culture recovery and also stringent test conditions that is difficult to implement at primary or secondary clinical laboratories.56 moreover , histopathological examination plays a valuable role in the diagnosis of pott 's disease but sometime it may be inconclusive and in addition need high expertise and the final reporting also takes more than 1 week.6 recently , the molecular biology technique , polymerase chain reaction ( pcr ) represents a major advance in the diagnosis of tb.6 with the use of amplification systems , nucleic acid sequences unique to m. tuberculosis can be detected directly in clinical specimens , offering better accuracy than zn microscopy and greater speed than culture .", "the pcr has shown very promising results for early and rapid diagnosis of the disease due to its detection limit of one to 10 bacilli in various clinical specimens.6 the present study was undertaken to evaluate the efficiency and effectiveness of different laboratory diagnostic modalities along with the role of pcr in the diagnosis of clinicoradiological suspected cases of pott 's disease ."], ["62 clinicoradiological suspected cases of pott 's disease with neurological complications [ table 1 ] were prospectively enrolled in this study , from 2008 to 2011 in the department of neurosurgery , at a tertiary care hospital , india .", "the specimens of these patients such as pus and tissue were obtained either during surgery or under ct guidance .", "clinicoradiological evidence of suspected pott 's disease we included clinicoradiological suspected cases of pott 's spine who underwent either open biopsy or ct guided aspiration at our institute .", "we excluded those subjects who did not give consent for biopsy or ct guided aspiration or biopsy showed neoplastic pathology .", "the specimens were examined by following methods : \n histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . \n ", "histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer .", "dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 .", "mb505 bacterial and yeast genomic dna miniprep purification spin kit , hi - media laboratories private limited mumbai , india .", "the reaction mixture contained 10 l pyrostart fast pcr master mix 2x ( dntp , taq polymerase with mgcl2 , fermentas , india ) , 1 l ( 10 pmole ) of each primer , 3 l water ( nuclease free ) and 5 l of extracted dna .", "the oligonucleotide primers11 were used forward and reverse sequence : cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively ( sbs gene tech co. ltd . ) .", "these primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc .", "it was performed in a programmable thermal cycler ( mj research , ptc-100 , gmi , inc .", "each cycle comprised denaturation at 94c for 2 min , annealing at 68c for 2 min and primer extension at 72c for 1 min .", "after the 40 cycles , the additional extension for 10 min at 72c was carried out .", "the amplified products were separated on 2% agarose gel , visualized on an ultraviolet - transilluminator ( bangalore genei , bangalore , india ) .", "the presence of 123 bp fragment indicated as a positive test for mtbc [ figure 1 ] .", "the positive controls included the dna of h37rv strain and negative control included pcr grade water .", "polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel .", "lane 1 ( l1 ) and l9 ladder 100 base pairs , l2 positive and l3 negative control , l4 , 5 , 6 , 8 showed positive and l7 showed negative specimens a definite treatment either by surgery ( 46 cases ) or by only anti tubercular treatment ( att ) ( 9 cases ) was offered .", "we followed the following major indications for surgical intervention in our setup : ( 1 ) advanced cases of neurological involvement such as marked sensory or sphincter disturbances , flaccid paralysis or severe flexor spasms .", "( 2 ) prevertebral cervical abscess with difficulty in respiration and deglutition , seemingly difficult resolution on chemotherapy during conservative treatment . ( 3 ) worsening of already present neurological complications during the treatment .", "( 5 ) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att . a four drug regimen chemotherapy consisting of isoniazid , rifampicin , pyrazinamide and ethambutol ( hrze ) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol , following or along with surgery .", "four - drug regimen for at least 4 - 6 weeks to gauge the response was given ( 8 - 12 mg / kg / day rifampicin , 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose ; and pyrazinamide 15 - 20 mg / kg / day in two divided doses ) , if not contraindicated the drug therapy was continued for 18 months12 . in cases of no improvements or hepatotoxicity ,", "the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them .", "the therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity , sensory loss of limbs , autonomic dysfunctions and constitutional symptoms [ table 2 ] .", "the liver function test was done as per protocol to rule out the subclinical hepatotoxcity .", "the radiological assessment was done by using followup mri , the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute .", "criteria for clinical improvement : ( first ( pain ) is mandatory and any other two improved symptoms / sign ) this study was approved by 41 institutional ethics committee ( a-04 pgi / imp / ec/41/28/2/2008 ) .", "the final diagnosis was established by using the collective results of zn microscopy , bactec culture , histopathological findings and response to att .", "the efficiency of test tools was calculated as ( total positive cases / total number of cases ) 100 .", "the positive concordance between traditional tools and pcr were assessed using the kappa coefficient (  > 0.75 , excellent agreement ; < 0.4 , poor agreement ; 0.4 and 0.75 , good to a fair agreement)13 and significance difference was analyzed by the chi square ( 2 ) test with the help of the spss 15.1 version .", "the cases were labeled as definite tb if at least one performed test ( zn microscopy / bactec culture / or histopathology ) was found positive ."], ["the specimens were examined by following methods : \n histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer . \n ", "histopathology - the tissues stained with hematoxylin and eosin and zn stain were analyzed under the microscope for epithelioid cell granulomas with or without the presence of langerhans giant cell and afb.7 periodic acid - schiff ( pas ) stain - fungal examination was performed by pas stain according to standard laboratory procedure.7 zn microscopy - smears were stained using the zn method and examination for afb were done under light microscopy.8 bactec 12b culture - bactec vials were incubated and interpreted as per becton dickinson ( bd , sparks , md , usa ) manual instructions.9 p - nitro--acetylamino--hydroxy propiophenone ( nap ) test - the nap was done for identification and differentiation of mycobacterium tuberculosis complex ( mtbc ) and nontubercular mycobacterium from culture isolates.9 molecular diagnosis - pcr for tb was done using a mtbc specific sequence is6110 ( 123 base pairs [ bp ] ) primer .", "dna extractions from tissues were done with hipura genomic dna extraction kit according to the manufacturer protocol3 .", "mb505 bacterial and yeast genomic dna miniprep purification spin kit , hi - media laboratories private limited mumbai , india .", "the reaction mixture contained 10 l pyrostart fast pcr master mix 2x ( dntp , taq polymerase with mgcl2 , fermentas , india ) , 1 l ( 10 pmole ) of each primer , 3 l water ( nuclease free ) and 5 l of extracted dna .", "the oligonucleotide primers11 were used forward and reverse sequence : cct gcg agc gta ggc gtc gg and ctc gtc cag cgc cgc ttc gg respectively ( sbs gene tech co. ltd . ) .", "these primers amplified a target fragment of 123 bp from the insertion like sequence element is6110 of mtbc .", "it was performed in a programmable thermal cycler ( mj research , ptc-100 , gmi , inc .", "each cycle comprised denaturation at 94c for 2 min , annealing at 68c for 2 min and primer extension at 72c for 1 min .", "after the 40 cycles , the additional extension for 10 min at 72c was carried out .", "the amplified products were separated on 2% agarose gel , visualized on an ultraviolet - transilluminator ( bangalore genei , bangalore , india ) .", "the presence of 123 bp fragment indicated as a positive test for mtbc [ figure 1 ] .", "the positive controls included the dna of h37rv strain and negative control included pcr grade water .", "polymerase chain reaction is6110 gene for detection of mycobacterium tuberculosis complex at 2% agarose gel .", "lane 1 ( l1 ) and l9 ladder 100 base pairs , l2 positive and l3 negative control , l4 , 5 , 6 , 8 showed positive and l7 showed negative specimens a definite treatment either by surgery ( 46 cases ) or by only anti tubercular treatment ( att ) ( 9 cases ) was offered .", "we followed the following major indications for surgical intervention in our setup : ( 1 ) advanced cases of neurological involvement such as marked sensory or sphincter disturbances , flaccid paralysis or severe flexor spasms .", "( 2 ) prevertebral cervical abscess with difficulty in respiration and deglutition , seemingly difficult resolution on chemotherapy during conservative treatment . ( 3 ) worsening of already present neurological complications during the treatment .", "( 5 ) no improvement in neurological complications / morbidity even after 4 - 6 weeks of att .", "a four drug regimen chemotherapy consisting of isoniazid , rifampicin , pyrazinamide and ethambutol ( hrze ) were given to all patients in their appropriate doses according to the weight of patients as per departmental protocol , following or along with surgery .", "four - drug regimen for at least 4 - 6 weeks to gauge the response was given ( 8 - 12 mg / kg / day rifampicin , 4 - 6 mg / kg / day isoniazid and 15 - 20 mg / kg / day ethambutol in a single daily dose ; and pyrazinamide 15 - 20 mg / kg / day in two divided doses ) , if not contraindicated the drug therapy was continued for 18 months12 . in cases of no improvements or hepatotoxicity ,", "the patients were switched to modified att with the addition of ofloxacin and streptomycin and modification in doses of isoniazid and rifampicin or discontinuation of them .", "the therapeutic response was observed by clinical improvement in the form of healing sign that is an improvement in motor power / spasticity , sensory loss of limbs , autonomic dysfunctions and constitutional symptoms [ table 2 ] .", "the liver function test was done as per protocol to rule out the subclinical hepatotoxcity .", "the radiological assessment was done by using followup mri , the first at the end of 6 months of the commencement of the att and was compared with pretreatment images by the same senior radiologist of our institute .", "criteria for clinical improvement : ( first ( pain ) is mandatory and any other two improved symptoms / sign ) this study was approved by 41 institutional ethics committee ( a-04 pgi / imp / ec/41/28/2/2008 ) ."], ["the final diagnosis was established by using the collective results of zn microscopy , bactec culture , histopathological findings and response to att .", "the efficiency of test tools was calculated as ( total positive cases / total number of cases ) 100 .", "the positive concordance between traditional tools and pcr were assessed using the kappa coefficient (  > 0.75 , excellent agreement ; < 0.4 , poor agreement ; 0.4 and 0.75 , good to a fair agreement)13 and significance difference was analyzed by the chi square ( 2 ) test with the help of the spss 15.1 version .", "the cases were labeled as definite tb if at least one performed test ( zn microscopy / bactec culture / or histopathology ) was found positive ."], ["the study group consisted of 62 specimens from suspected pott 's spine individuals . on the basis of histopathological examination ,", "7 cases ( 5 specimens from open biopsy and 2 from fine needle aspiration [ fna ] ) were excluded from the study as they were diagnosed to be neoplastic . out of rest 55 cases ,", "46 specimens were obtained through open biopsy during surgery and 9 were ct guided fna .", "further , in all ( 46 ) open biopsy specimens , 7 were positive for epithelioid cell granulomas with afb , 27 revealed granulomatous lesions suggestive of tb with or without the presence of the langerhans giant cell .", "however , the histological diagnosis could not be reached in 12 specimens because of either suspicious granulomatous lesions or on account of inconclusive histology .", "five of nine ct guided fna specimens were diagnosed as tubercular and 4 specimens showed no granulomatous / no malignant lesions .", "the received specimens included 31 ( 56.4% ) pus and 15 ( 27.3% ) tissues through open biopsy along with 6 ( 11% ) pus and 3 ( 0.54% ) tissues were obtained through ct guided fna .", "the overall collected specimens were consisted 37 pus and 18 tissue specimens ( either open biopsy or by ct fna ) were analyzed .", "the zn microscopy was positive in total 20 ( 36.4% ) of 55 cases ( 18/37 cases in pus and 2/18 cases in tissue specimens ) .", "the bactec culture was positive 27 ( 49% ) of 55 cases ( 22/37 in pus and 5/18 in tissue specimens ) .", "the desired amplification of pcr is6110 was positive in 37 ( 67.5% ) of 55 cases ( 30/37 cases in pus and 7/18 in tissue specimens ) [ table 3 ] .", "the kappa coefficient of pcr with zn microscopy (  = 0.37 , [ p = 0.001 ] ) showed poor agreement and with the bactec culture (  = 0.57 , [ p < 0.001 ] ) it showed a good agreement [ figure 2 ] .", "result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools .", "( znm = zn microscopy , bc = bactec culture and hp = histopathology ) on histopathological results ; 39 ( 71% ) cases were positive and 16 ( 29% ) were negative for tb", ". out of 39 positive cases , 31 ( 56.4% ) were positive on pcr also .", "thus , the false negative and positive of pcr was found to be 8 ( 14.4% ) and 6 ( 11% ) respectively .", "therefore , the kappa coefficient of pcr with histopathology was (  = 0.4 [ p = 0 . 004 ] ) slightly good [ figure 2 ] . in 55 cases", ", it was possible to reach a final diagnosis through the combined result of all performed tests ( without consideration of pcr results ) and with att response .", "hence , among all 55 cases , 45 ( 82% ) cases were tb positive test and 10 ( 18% ) were negative by all tests carried out ( zn microscopy , bactec culture and histopathology ) .", "although pcr showed 67.3% ( 37/55 ) sensitivity at the final diagnosis , while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests", ". therefore , the kappa coefficient of pcr with the combined results of all traditional tests (  = 0.63 [ p < 0.001 ] ) ultimately reached to a fair positive agreement [ figure 2 ] .", "the present study , the combination of ( a ) zn microscopy with bactec , the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture .", "( b ) zn microscopy with histopathology , the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy .", "( c ) bactec culture with histopathology , the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture , where the histopathology was negative .", "( d ) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology .", "( e ) zn microscopy with pcr , the total positivity was 38 cases , in which 18 more cases were positive on pcr method .", "( f ) bactec culture with pcr , the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . (", "g ) histopathology with pcr , the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr .", "( h ) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [ table 4 ] ."], ["the received specimens included 31 ( 56.4% ) pus and 15 ( 27.3% ) tissues through open biopsy along with 6 ( 11% ) pus and 3 ( 0.54% ) tissues were obtained through ct guided fna .", "the overall collected specimens were consisted 37 pus and 18 tissue specimens ( either open biopsy or by ct fna ) were analyzed .", "the zn microscopy was positive in total 20 ( 36.4% ) of 55 cases ( 18/37 cases in pus and 2/18 cases in tissue specimens ) .", "the bactec culture was positive 27 ( 49% ) of 55 cases ( 22/37 in pus and 5/18 in tissue specimens ) .", "the desired amplification of pcr is6110 was positive in 37 ( 67.5% ) of 55 cases ( 30/37 cases in pus and 7/18 in tissue specimens ) [ table 3 ] .", "the kappa coefficient of pcr with zn microscopy (  = 0.37 , [ p = 0.001 ] ) showed poor agreement and with the bactec culture (  = 0.57 , [ p < 0.001 ] ) it showed a good agreement [ figure 2 ] .", "result of laboratory findings bar diagram showing kappa coefficient concordance between pcr and traditional tools ."], ["on histopathological results ; 39 ( 71% ) cases were positive and 16 ( 29% ) were negative for tb", ". out of 39 positive cases , 31 ( 56.4% ) were positive on pcr also .", "thus , the false negative and positive of pcr was found to be 8 ( 14.4% ) and 6 ( 11% ) respectively .", "therefore , the kappa coefficient of pcr with histopathology was (  = 0.4 [ p = 0 . 004 ] ) slightly good [ figure 2 ] ."], ["in 55 cases , it was possible to reach a final diagnosis through the combined result of all performed tests ( without consideration of pcr results ) and with att response .", "hence , among all 55 cases , 45 ( 82% ) cases were tb positive test and 10 ( 18% ) were negative by all tests carried out ( zn microscopy , bactec culture and histopathology ) .", "although pcr showed 67.3% ( 37/55 ) sensitivity at the final diagnosis , while pcr showed 37/45 positive and no any false positive out of 10 negative cases by all traditional tests .", "therefore , the kappa coefficient of pcr with the combined results of all traditional tests (  = 0.63 [ p < 0.001 ] ) ultimately reached to a fair positive agreement [ figure 2 ] ."], ["the present study , the combination of ( a ) zn microscopy with bactec , the total 31 positive cases due to only 20 cases positive on zn microscopy and an additional 11 cases were positive on bactec culture .", "( b ) zn microscopy with histopathology , the total positivity showed 43 cases due to 23 cases more positive on histopathology against zn microscopy .", "( c ) bactec culture with histopathology , the total 45 cases to be positive due to 39 cases positive on histopathology and an additional 6 cases were positive on bactec culture , where the histopathology was negative .", "( d ) the total 45 cases were positive in combination of zn microscopy + bactec culture + histopathology .", "( e ) zn microscopy with pcr , the total positivity was 38 cases , in which 18 more cases were positive on pcr method .", "( f ) bactec culture with pcr , the total 38 positive cases due to only 27 cases positive on bactec culture and additional 11 cases were positive on pcr . (", "g ) histopathology with pcr , the total 45 cases positive on this possible battery due to 39 cases positive on histopathology and an additional 6 cases were positive on pcr .", "( h ) the battery of zn microscopy + bactec culture + histopathology + pcr showed ultimately 45 cases were positive because pcr could not give any more positive against the mentioned battery d and g [ table 4 ] ."], ["pott 's disease , accounts for 2% of all tb infections.1 the diagnosis of pott 's disease is principally based on classical clinical manifestations of spinal infection supplemented by modern imaging like ct and mri.1415 the ct and mri in particular can detect the subtle changes in the spine , from initial changes in the intensity pattern of the vertebrae to the extreme changes like deformity of the spine including soft tissue shadow , granulation tissue and pus etc.16 however , the various pathologies including neoplastic and other inflammatory conditions simulate with pott 's diseases on imaging .", "the current study excluded 7 of 62 cases ; they were diagnosed to be neoplastic by the histopathological examination .", "it signifies that even strong clinicoradiological diagnosis based on classical or nonclassical radiological and clinical findings can be wrong on histopathological evaluation .", "the remaining 55 cases , 20 ( 36.3% ) were tested positive with zn microscopy and 27 ( 49% ) with bactec culture .", "an earlier report by jain et al.17 found 14.8% positivity on zn microscopy and 11.11% positivity on culture method in cases of spinal tb .", "the zn microscopy and bactec culture reports revealed higher positivity in relation to the earlier published series .", "our higher detection rate was because of the fact that we used the liquid culture medium bactec 12b medium .", "it is the rapid , sensitive and efficient method for the isolation of mycobacterium in a clinical laboratory as compared to solid medium lowenstein ", "jensen.19 moreover , it is established today that all the traditional tests need to have a minimum concentration of bacilli for yielding positive results as mentioned in the introduction . hence , if the specimens , which are naturally having more concentration of bacilli , like sputum and pus it may become easy to detect m. tuberculosis.20 similarly , in our study the pus specimens had higher recognition of tubercular bacilli on zn microscopy and bactec culture methods [ table 3 ] .", "the study quoted the detection rate of eptb including pott 's spine varying from 53% to 81%.182122 the histopathological diagnosis could be reached in 71% ( 39 out of 55 ) of the cases which were either confirmed by the presence of afb in 12.7% or strongly suggestive of tubercular pathology . in our series , we found 16 cases were negative on histology . among these negative cases ,", "10 out of 16 cases had been under anti tubercular therapy for more than 6 months and another reason of histology negative might be a possible sampling error during the collection of the representative tissues for processing .", "the positivity of fna specimens seems to higher than surgical specimens , however , we require a long sample size for definite conclusion .", "molecular diagnostic tool , such as pcr has been shown to have higher efficiency in the diagnosis of pulmonary tb as well as eptb.4 it has been studied on clinical specimens of sputum , cerebrospinal fluid , edta - blood , pleural fluid , fluid from fistulae and pus from wounds.2324 in an attempt to increase diagnostic precision , we performed pcr is6110 , which was detected in 37/55 ( 67.3% ) cases .", "various studies documented the increased positivity by pcr targeting is6110 in specimens of eptb i.e. , sekar et al.25 found 63% positivity , negi et al.26 73% and tiwari et al.27 62% positivity among clinical specimens of eptb .", "the overall positive agreement shows a fair agreement (  = 0.63 ) between pcr and definite tb .", "the false negative results were observed in 14.5% cases out of 45 cases diagnosed by all performed traditional tests .", "the reasons of false negative results of pcr may be on account of insufficient specimens , irregular distribution of mycobacterium bacilli in specimens , the presence of extensive necrosis and presence of inhibitors.28 the limitation of our study are a low positive correlation (  = 0.4 ) of pcr with histopathological findings .", "this can be explained up to some extent by the specimen variation as that is different specimens ( such as pus , tissue ) from the same subjects .", "surgeons prefer to send tissue specimens for histology and pus for zn microscopy , bactec culture and pcr . in this study histology", "was done on tissue specimens ( all 55 cases ) , while zn microscopy , bactec culture and pcr were done in 37 pus and 18 tissue specimens . in our diagnostic battery ,", "all performed traditional tests were diagnosed 45/55 cases and pcr was alone detected 37/55 cases .", "thus , positive results of pcr with the clinicoradiological assumption it would be possible to make a decision in suspected pott 's disease to start early treatment to prevent irreversible complications . in the present study , it is observed that no single diagnostic modality is having a good positivity rate .", "hence , a combination of diagnostic battery is required for rapid diagnosis and better results .", "the combination of bactec culture and histopathology has detected a maximum of 45 cases . while the combination of pcr and histopathology has also detected same number of cases table 4 .", "as culture takes long incubation time for results , the combination of pcr and histopathology can be applied for rapid detection of pott 's disease . to conclude , for diagnosis of pott 's disease , the combined results of zn microscopy , bactec culture and histopathology are most appropriate . however , for rapid diagnosis the combination of pcr amplification and histopathology offer a better prospect ."]]}
{"article_id": "PMC1538841", "article_text": ["such proteins perform many basic functions including cell signalling , transcription regulation , energy conservation and transformation , and ion exchange .", "membrane proteins are difficult to study , since they are inserted into lipid bilayers and expose to the polar outer and inner environments portions of different sizes .", "it is therefore difficult to purify them in the native , functional form and even more difficult to crystallize them . for such technical reasons , only a small fraction of the protein data bank structures", "are membrane proteins ( < 1% of the total number of structures and far less than their estimated abundance in cells ) ( 1 ) .", "there are a number of computational methods available to predict the topology of membrane proteins , which consists of two basic features : ( i ) the location of transmembrane domains along the protein chain and ( ii ) the location of the n- and c - termini with respect to the lipid membrane .", "topological models are sufficient in many instances to design simple experiments in order to prove the location of the n- and c - protein termini , that of the inner and outer loops with respect to the membrane plane , and concomitantly the number of transmembrane segments in the chain", ". however , the best - performing methods are offered at different servers and are endowed with different graphical interfaces .", "this hampers the direct comparison of predictions , especially for experimentalists interested in comparing their results with computational methods .", "currently , two other web servers of which we are aware ( 2,3 ) are available and separately comprise two of the predictors that we implement tmhmm2.0 ( 4 ) and memsat ( 5 ) .", "the novelty of our web server is to include in the same framework ensemble ( 6 ) and prodiv ( 7 ) , two powerful methods that became available only recently .", "furthermore the available web servers render only the consensus prediction , without allowing a critical discrimination among different predictions that may be useful , considering that different predictors may highlight different properties , depending on the different implementation .", "automatic topology annotation for membrane proteins has been included among the efforts of the biosapiens european network of excellence ( ) with the specific aim of taking into consideration different predictors for annotating membrane proteins in the human genome .", "the common platform for these efforts is the biosapiens distributed annotation servers ( das ) ( ) . in this context", ", the pongo - das server ( ) provides topology annotation for the all - alpha membrane proteins of the human genome .", "this is done using the das protocol ( ) to answer at das queries that can be seen using specific visualizers such as dasty ( 8) or ensembl ( 9 ) . in order to allow users to browse directly the pre - computed transmembrane annotations", "the annotation is carried out using four selected predictors , namely ensemble1.0 ( 6 ) and prodiv ( 7 ) , in order to allow a direct comparison of the topology prediction for the sequence at hand .", "the server has also been set up to make it possible to predict the topology of any putative membrane proteins of interest regardless of provenance .", "the topological models computed by the different predictors can be directly obtained simply by pasting in a box the sequence of interest and looking at the results .", "recently developed web technologies ( e.g. ajax ) are used to improve the user interface .", "the website implements the following predictors : memsat is a new version of the memsat predictor of transmembrane helices in proteins ( 4 ) .", "tmhmm2.0 which is a predictor of transmembrane helices in proteins based on hidden markov models ( 5 ) .", "it has been shown that it performs quite well taking into account that it uses only single sequence information .", "ensemble1.0 is an ensemble of two hidden markov models and one neural network ( 6 ) .", "ensemble takes also advantage of the evolutionary information derived by multiple sequence alignment , both for the neural network and the hidden markov model systems .", "prodiv_tmhmm_0.91 is a recent predictor of transmembrane helices in proteins ( 7 ) which uses a hidden markov model similar to tmhmm , but exploits the evolutionary information derived by multiple sequence alignment .", "spep is a signal peptide predictor based on combination of neural networks ( 10 ) .", "this predictor has performance similar to the most widely used signalp ( 11 ) , and it has been included since it is quite common that signal peptides are mispredicted as transmembrane helices . in the context of the biosapiens project , we downloaded the uniprot dataset ( september 22 , 2004 ) which consists of 33  135 human protein sequences , and for future use also the ipi dataset ( august 5 , 2004 ) which consists of 46  782 human proteins .", "the union of the two datasets comprises 50  600 sequences from the human genome .", "it is worth noticing that these 50 600 sequences do not include the known splice variants , since those variants are not presently included as unique uniprot codes .", "the choice of annotating uniprot sequences is well justified from the fact that ensembl gene products ( in contrast to the uniprot entries ) are not stable ; for instance , only 60% of the sequences are common ( and conserved ) between the ensembl releases 34 and 35 , respectively .", "we then use four state - of - the - art predictors already described in the literature in order to identify the most probable integral membrane proteins . in this way", "the union of the predictions obtained with the four methods gives a set of likely membrane proteins , while the intersection contains those chains on whose annotation as membrane proteins all the predictors agree upon .", "the pre - computed annotations are filtered with spep before being processed by the transmembrane predictors .", "this is done since it is quite common that signal peptides are mispredicted as transmembrane helices by all the predictors implemented in our web server . in the case of a positive spep answer", ", the system cuts the corresponding predicted segment and processes the remaining part of the sequence with the four transmembrane predictors .", "the website implements the following predictors : memsat is a new version of the memsat predictor of transmembrane helices in proteins ( 4 ) .", "tmhmm2.0 which is a predictor of transmembrane helices in proteins based on hidden markov models ( 5 ) .", "it has been shown that it performs quite well taking into account that it uses only single sequence information .", "ensemble1.0 is an ensemble of two hidden markov models and one neural network ( 6 ) .", "ensemble takes also advantage of the evolutionary information derived by multiple sequence alignment , both for the neural network and the hidden markov model systems .", "prodiv_tmhmm_0.91 is a recent predictor of transmembrane helices in proteins ( 7 ) which uses a hidden markov model similar to tmhmm , but exploits the evolutionary information derived by multiple sequence alignment .", "spep is a signal peptide predictor based on combination of neural networks ( 10 ) .", "this predictor has performance similar to the most widely used signalp ( 11 ) , and it has been included since it is quite common that signal peptides are mispredicted as transmembrane helices .", "in the context of the biosapiens project , we downloaded the uniprot dataset ( september 22 , 2004 ) which consists of 33  135 human protein sequences , and for future use also the ipi dataset ( august 5 , 2004 ) which consists of 46  782 human proteins .", "the union of the two datasets comprises 50  600 sequences from the human genome .", "it is worth noticing that these 50 600 sequences do not include the known splice variants , since those variants are not presently included as unique uniprot codes .", "the choice of annotating uniprot sequences is well justified from the fact that ensembl gene products ( in contrast to the uniprot entries ) are not stable ; for instance , only 60% of the sequences are common ( and conserved ) between the ensembl releases 34 and 35 , respectively .", "we then use four state - of - the - art predictors already described in the literature in order to identify the most probable integral membrane proteins . in this way", "the union of the predictions obtained with the four methods gives a set of likely membrane proteins , while the intersection contains those chains on whose annotation as membrane proteins all the predictors agree upon .", "the pre - computed annotations are filtered with spep before being processed by the transmembrane predictors .", "this is done since it is quite common that signal peptides are mispredicted as transmembrane helices by all the predictors implemented in our web server . in the case of a positive spep answer ,", "the system cuts the corresponding predicted segment and processes the remaining part of the sequence with the four transmembrane predictors .", "pongo has two different usage options : \n pongo - das accessing the pre - computed annotations using keywords or das queries;pongo - pred that is an enhanced and modern version of a standard  through - the - web server application .", "pongo - das accessing the pre - computed annotations using keywords or das queries ; pongo - pred that is an enhanced and modern version of a standard  through - the - web server application . in the case of pongo - das we implemented two types of result visualization : \n by means of a das , which is a client  server system in which a single client integrates information from multiple servers .", "it allows a single machine to gather up genome annotation information from multiple distant web sites , collate the information , and display it to the user in a single view .", "little coordination is needed among the various information providers , anda user - friendly interface that allows the search for a specific prediction.through the das protocol a web client like ensembl can obtain the list of the annotations for each human protein using its uniprot code as requested for the das queries ( ) ; in order to check this url the client needs a das client such as the one that will be embedded in ensembl pages at ebi and a uniprot code for the sequence .", "a user can directly query the database , without using the das infrastructure , using the web interface available at   ( figure 1 ) the user can then use a crc64 ( the sequence hash code ) , or the sequence uniprot or ipi code to get the predictions in a graphical view .", "an example of a sequence filtered with the different predictors is presented together with the detailed sequence annotation ( figure 2 ) .", "a colour code is used to quickly identify the transmembrane segments . by means of a das , which is a client  server system in which a single client integrates information from multiple servers .", "it allows a single machine to gather up genome annotation information from multiple distant web sites , collate the information , and display it to the user in a single view .", "little coordination is needed among the various information providers , and a user - friendly interface that allows the search for a specific prediction .", "conversely , pongo - pred provides the user with a unique framework for membrane protein topology and signal peptide predictions .", "another interesting feature of pongo - pred is the javascript - enabled portlet that in real time refreshes the queue status ( without reloading of the submission page and with very lightweight xml http requests ) . in particular , on the left - side of the page the user can realise whether her / his submission is a new call , whether it is running ( in this case the starting date and an absolute link is provided for bookmarking ) , or whether it has been processed .", "the pongo homepage . on the left - side it is possible to follow the status of the different queries and the starting of a new action ."], "abstract_text": ["<S> the annotation efforts of the biosapiens european network of excellence have generated several distributed annotation systems ( das ) with the aim of integrating bioinformatics resources and annotating metazoan genomes ( ) . in this context </S>", "<S> , the pongo das server ( ) provides the annotation on predictive basis for the all - alpha membrane proteins in the human genome , not only through das queries , but also directly using a simple web interface . in order to produce a more comprehensive analysis of the sequence at hand , </S>", "<S> this annotation is carried out with four selected and high scoring predictors : tmhmm2.0 , memsat , prodiv and ensemble1.0 . </S>", "<S> the stored and pre - computed predictions for the human proteins can be searched and displayed in a graphical view . </S>", "<S> however the web service allows the prediction of the topology of any kind of putative membrane proteins , regardless of the organism and more importantly with the same sequence profile for a given sequence when required . here </S>", "<S> we present a new web server that incorporates the state - of - the - art topology predictors in a single framework , so that putative users can interactively compare and evaluate four predictions simultaneously for a given sequence . </S>", "<S> together with the predicted topology , the server also displays a signal peptide prediction determined with spep . </S>", "<S> the pongo web server is available at . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "The predictors", "Pre-computed transmembrane annotations for the human proteome", "RESULTS", "Figures and Tables"], "sections": [["such proteins perform many basic functions including cell signalling , transcription regulation , energy conservation and transformation , and ion exchange .", "membrane proteins are difficult to study , since they are inserted into lipid bilayers and expose to the polar outer and inner environments portions of different sizes .", "it is therefore difficult to purify them in the native , functional form and even more difficult to crystallize them . for such technical reasons , only a small fraction of the protein data bank structures", "are membrane proteins ( < 1% of the total number of structures and far less than their estimated abundance in cells ) ( 1 ) .", "there are a number of computational methods available to predict the topology of membrane proteins , which consists of two basic features : ( i ) the location of transmembrane domains along the protein chain and ( ii ) the location of the n- and c - termini with respect to the lipid membrane .", "topological models are sufficient in many instances to design simple experiments in order to prove the location of the n- and c - protein termini , that of the inner and outer loops with respect to the membrane plane , and concomitantly the number of transmembrane segments in the chain", ". however , the best - performing methods are offered at different servers and are endowed with different graphical interfaces .", "this hampers the direct comparison of predictions , especially for experimentalists interested in comparing their results with computational methods .", "currently , two other web servers of which we are aware ( 2,3 ) are available and separately comprise two of the predictors that we implement tmhmm2.0 ( 4 ) and memsat ( 5 ) .", "the novelty of our web server is to include in the same framework ensemble ( 6 ) and prodiv ( 7 ) , two powerful methods that became available only recently .", "furthermore the available web servers render only the consensus prediction , without allowing a critical discrimination among different predictions that may be useful , considering that different predictors may highlight different properties , depending on the different implementation .", "automatic topology annotation for membrane proteins has been included among the efforts of the biosapiens european network of excellence ( ) with the specific aim of taking into consideration different predictors for annotating membrane proteins in the human genome .", "the common platform for these efforts is the biosapiens distributed annotation servers ( das ) ( ) . in this context", ", the pongo - das server ( ) provides topology annotation for the all - alpha membrane proteins of the human genome .", "this is done using the das protocol ( ) to answer at das queries that can be seen using specific visualizers such as dasty ( 8) or ensembl ( 9 ) . in order to allow users to browse directly the pre - computed transmembrane annotations", "the annotation is carried out using four selected predictors , namely ensemble1.0 ( 6 ) and prodiv ( 7 ) , in order to allow a direct comparison of the topology prediction for the sequence at hand .", "the server has also been set up to make it possible to predict the topology of any putative membrane proteins of interest regardless of provenance .", "the topological models computed by the different predictors can be directly obtained simply by pasting in a box the sequence of interest and looking at the results .", "recently developed web technologies ( e.g. ajax ) are used to improve the user interface ."], ["the website implements the following predictors : memsat is a new version of the memsat predictor of transmembrane helices in proteins ( 4 ) .", "tmhmm2.0 which is a predictor of transmembrane helices in proteins based on hidden markov models ( 5 ) .", "it has been shown that it performs quite well taking into account that it uses only single sequence information .", "ensemble1.0 is an ensemble of two hidden markov models and one neural network ( 6 ) .", "ensemble takes also advantage of the evolutionary information derived by multiple sequence alignment , both for the neural network and the hidden markov model systems .", "prodiv_tmhmm_0.91 is a recent predictor of transmembrane helices in proteins ( 7 ) which uses a hidden markov model similar to tmhmm , but exploits the evolutionary information derived by multiple sequence alignment .", "spep is a signal peptide predictor based on combination of neural networks ( 10 ) .", "this predictor has performance similar to the most widely used signalp ( 11 ) , and it has been included since it is quite common that signal peptides are mispredicted as transmembrane helices . in the context of the biosapiens project , we downloaded the uniprot dataset ( september 22 , 2004 ) which consists of 33  135 human protein sequences , and for future use also the ipi dataset ( august 5 , 2004 ) which consists of 46  782 human proteins .", "the union of the two datasets comprises 50  600 sequences from the human genome .", "it is worth noticing that these 50 600 sequences do not include the known splice variants , since those variants are not presently included as unique uniprot codes .", "the choice of annotating uniprot sequences is well justified from the fact that ensembl gene products ( in contrast to the uniprot entries ) are not stable ; for instance , only 60% of the sequences are common ( and conserved ) between the ensembl releases 34 and 35 , respectively .", "we then use four state - of - the - art predictors already described in the literature in order to identify the most probable integral membrane proteins . in this way", "the union of the predictions obtained with the four methods gives a set of likely membrane proteins , while the intersection contains those chains on whose annotation as membrane proteins all the predictors agree upon .", "the pre - computed annotations are filtered with spep before being processed by the transmembrane predictors .", "this is done since it is quite common that signal peptides are mispredicted as transmembrane helices by all the predictors implemented in our web server . in the case of a positive spep answer", ", the system cuts the corresponding predicted segment and processes the remaining part of the sequence with the four transmembrane predictors ."], ["the website implements the following predictors : memsat is a new version of the memsat predictor of transmembrane helices in proteins ( 4 ) .", "tmhmm2.0 which is a predictor of transmembrane helices in proteins based on hidden markov models ( 5 ) .", "it has been shown that it performs quite well taking into account that it uses only single sequence information .", "ensemble1.0 is an ensemble of two hidden markov models and one neural network ( 6 ) .", "ensemble takes also advantage of the evolutionary information derived by multiple sequence alignment , both for the neural network and the hidden markov model systems .", "prodiv_tmhmm_0.91 is a recent predictor of transmembrane helices in proteins ( 7 ) which uses a hidden markov model similar to tmhmm , but exploits the evolutionary information derived by multiple sequence alignment .", "spep is a signal peptide predictor based on combination of neural networks ( 10 ) .", "this predictor has performance similar to the most widely used signalp ( 11 ) , and it has been included since it is quite common that signal peptides are mispredicted as transmembrane helices ."], ["in the context of the biosapiens project , we downloaded the uniprot dataset ( september 22 , 2004 ) which consists of 33  135 human protein sequences , and for future use also the ipi dataset ( august 5 , 2004 ) which consists of 46  782 human proteins .", "the union of the two datasets comprises 50  600 sequences from the human genome .", "it is worth noticing that these 50 600 sequences do not include the known splice variants , since those variants are not presently included as unique uniprot codes .", "the choice of annotating uniprot sequences is well justified from the fact that ensembl gene products ( in contrast to the uniprot entries ) are not stable ; for instance , only 60% of the sequences are common ( and conserved ) between the ensembl releases 34 and 35 , respectively .", "we then use four state - of - the - art predictors already described in the literature in order to identify the most probable integral membrane proteins . in this way", "the union of the predictions obtained with the four methods gives a set of likely membrane proteins , while the intersection contains those chains on whose annotation as membrane proteins all the predictors agree upon .", "the pre - computed annotations are filtered with spep before being processed by the transmembrane predictors .", "this is done since it is quite common that signal peptides are mispredicted as transmembrane helices by all the predictors implemented in our web server . in the case of a positive spep answer ,", "the system cuts the corresponding predicted segment and processes the remaining part of the sequence with the four transmembrane predictors ."], ["pongo has two different usage options : \n pongo - das accessing the pre - computed annotations using keywords or das queries;pongo - pred that is an enhanced and modern version of a standard  through - the - web server application .", "pongo - das accessing the pre - computed annotations using keywords or das queries ; pongo - pred that is an enhanced and modern version of a standard  through - the - web server application . in the case of pongo - das we implemented two types of result visualization : \n by means of a das , which is a client  server system in which a single client integrates information from multiple servers .", "it allows a single machine to gather up genome annotation information from multiple distant web sites , collate the information , and display it to the user in a single view .", "little coordination is needed among the various information providers , anda user - friendly interface that allows the search for a specific prediction.through the das protocol a web client like ensembl can obtain the list of the annotations for each human protein using its uniprot code as requested for the das queries ( ) ; in order to check this url the client needs a das client such as the one that will be embedded in ensembl pages at ebi and a uniprot code for the sequence .", "a user can directly query the database , without using the das infrastructure , using the web interface available at   ( figure 1 ) the user can then use a crc64 ( the sequence hash code ) , or the sequence uniprot or ipi code to get the predictions in a graphical view .", "an example of a sequence filtered with the different predictors is presented together with the detailed sequence annotation ( figure 2 ) .", "a colour code is used to quickly identify the transmembrane segments . by means of a das , which is a client  server system in which a single client integrates information from multiple servers .", "it allows a single machine to gather up genome annotation information from multiple distant web sites , collate the information , and display it to the user in a single view .", "little coordination is needed among the various information providers , and a user - friendly interface that allows the search for a specific prediction .", "conversely , pongo - pred provides the user with a unique framework for membrane protein topology and signal peptide predictions .", "another interesting feature of pongo - pred is the javascript - enabled portlet that in real time refreshes the queue status ( without reloading of the submission page and with very lightweight xml http requests ) . in particular , on the left - side of the page the user can realise whether her / his submission is a new call , whether it is running ( in this case the starting date and an absolute link is provided for bookmarking ) , or whether it has been processed ."], ["the pongo homepage . on the left - side it is possible to follow the status of the different queries and the starting of a new action ."]]}
{"article_id": "PMC3913132", "article_text": ["the epstein  barr virus ( ebv ) infects b cells . in most cases , ebv - positive patients develop a severe form of infectious mononucleosis which typically manifests with fever , tonsillopharyngitis , lymphadenopathy and hepatosplenomegaly .", "deficiencies may be characterized by abnormal activity of lymphocyte function leading to hemophagocytosis and multi - organ failure .", "although ebv - associated hlh carries a high mortality rate , quick diagnosis and appropriate treatment can subside the disease .", "we describe here a patient who developed hepatosplenomegaly and pancytopenia at 2 years of age but achieved automatic recovery .", ", there was a clinical suspicion of ebv - induced hlh and the patient was given the option of treatment with rituximab .", "she received antibiotic treatment due to fever and chills . during the hospitalization period , the patient recovered and developed hemophagocytosis with pancytopenia as well as sores on the lip .", "the patient underwent bma and nothing abnormal was found . in spite of treatment , the disease did not subside and she developed fever , abdominal distention and edema in her lower extremities .", "physical examination showed that the patient had decreased deep - tendon reflexes ( dtr ) and was unable to walk .", "clindamycin , ceftazidime , dexamethasone , ivig ( intravenous immunoglobulin ) and g - csf were administered .", "since the patient did not achieve full recovery , she was transferred to our center .", "based on clinical symptoms and lab tests ( table 1 ) , bma was performed once again and the result raised suspicion of ebv - induced secondary hlh .", "rituximab was injected once and the patient developed a blood ebv load of zero within one week . in order to control abnormal stimulation of the immune system ,", "the patient was investigated for mutations in il-2-inducible t cell kinase ( itk ) and negative test results were received .", "( the patient was negative for mutations in il-2-inducible t cell kinase ( itk ) ) .", "she is now considered to be an acceptable candidate for transplantation from an hla matched sibling donor . \n ", "hlh can be divided into two subgroups : primary or familial hlh and secondary hlh .", "the primary form is an inflammatory disease which is similar to secondary one on the basis of symptoms .", "primary hlh usually arises in young children less than one year old ( 70% cases ) ; however , in rare cases it can also occur in adults ( perforin gene mutations have been identified in patients with primary hlh ) .", "for example , type 2 is caused by mutations in genes 21 - 22 and type 5 is due to mutations in the munc18 - 20 that may be accompanied by hypogammaglobulinemia .", "hypogammaglobulinemia was not confirmed in the patient because it was not possible to check mutation ( the related tests were sent to germany to examine the type 5 disease ) .", "ebv may also be accompanied by hypogammaglobulinemia for a certain period secondary hlh is associated with immunologic stimulation caused by malignancies and bacterial or congenital infections .", "the most common causes of secondary hlh are viral infections by ebv , cmv , prob19 and hiv .", "however , it may also be seen in patients with immune system defects . , 4 secondary hlh is a known complication of ebv infection , particularly in patients with x - linked lymphoproliferative disease .", "the incidence of x - linked lymphoproliferative syndrome is 1 in 1,000,000 male infants . \n ", "diagnostic criteria for hlh disease diagnosis of hlh requires the presence of all 5 major criteria .", "if the patient meets only 4criteria but the clinical suspicion for hlh is high , one should initiate treatment , because delays may be fatal .", "alternative criteria 1 or a combination of 2 and 3 may substitute for 1 major criterion . adapted from henter ji , elinder g , ost a.semin oncol . 1991 ; 18:29 - 33 .", "it means that ebv - infected b cells stimulate cytotoxic t lymphocytes leading to hypercytokinemia and stimulation of histolytic cells .", "more recently , it has been found that chronic stimulation by ebv may cause chronic hlh in the patient . on the other hand", ", ebv causes stimulation , generation and uncontrolled secretion of t - and nk- cells , as well as generation of il2 , infa and il6 .", "there is another mechanism by which ebv stimulates membrane protein ( lmp-1 ) in cells .", "similar to xlp , lmp-1 may cause acquired immune deficiency , leading to hlh accordingly . \n ", "treatment follows two goals : first , to suppress immune response using ivig , steroid and hlh protocol .", "second , to inhibit undesirable influence of immune response and cytokine - stimulated cell activation using immunochemotherapy . in cases with neurological symptoms", "the level of viral dna which is measured by quantitative pcr may be useful to demonstrate treatment response predicting mortality .", "the dna level reached zero in our patient . using ivig prior to start of protocol 2004", "it has also been recommended in the treatment of ebv - induced secondary hlh monthly . \n ", "decrease of b cells , which have been contaminated by ebv , actually causes decrease of viruses , as it may be useful for our patient as well .", "finally , treatment - refractory patients , transplant candidates , patients with familial type of hlh , or recurrent disease undergo chemotherapy for 8 weeks .", "patients who respond favorably to treatment do not undergo transplantation but those who experience relapse and exhibit neurological symptoms are considered for transplantation .", "severe infection of ebv is not associated with life - threatening condition , but our case study needs urgent treatment . in patients with long - term ebv infection , pancytopenia , coagulopathy and hepatosplenomegaly ,", "bma will be performed and the serum ferritin level will be measured as well . for patients with secondary hlh ,", "rituximab can be used when hlh is associated with ebv to inhibit the immune response .", "thus , the main aim of treatment is to reduce the rate of mortality in patients with hlh ."], "abstract_text": ["<S> hemophagocytic lymphohistiocytosis ( hlh ) is one of the complications of epstein - barr virus ( ebv ) infection . </S>", "<S> although the patients who have developed hlh following ebv have normal immune system , there are a few patients with ebv - induced immune deficiency who develop hlh as well . here </S>", "<S> , we describe the case of a 10-year - old girl with neurological complications caused by ebv - induced hlh . </S>", "<S> the patient received rituximab , leading to weakening inflammation associated with ebv infection and suppression of disease through quick treatment . </S>"], "labels": null, "section_names": ["INTRODUCTION", "INTRODUCING THE CASE", "DISCUSSION", "CONCLUSION"], "sections": [["the epstein  barr virus ( ebv ) infects b cells . in most cases , ebv - positive patients develop a severe form of infectious mononucleosis which typically manifests with fever , tonsillopharyngitis , lymphadenopathy and hepatosplenomegaly .", "deficiencies may be characterized by abnormal activity of lymphocyte function leading to hemophagocytosis and multi - organ failure .", "although ebv - associated hlh carries a high mortality rate , quick diagnosis and appropriate treatment can subside the disease .", "we describe here a patient who developed hepatosplenomegaly and pancytopenia at 2 years of age but achieved automatic recovery .", ", there was a clinical suspicion of ebv - induced hlh and the patient was given the option of treatment with rituximab ."], ["she received antibiotic treatment due to fever and chills . during the hospitalization period , the patient recovered and developed hemophagocytosis with pancytopenia as well as sores on the lip .", "the patient underwent bma and nothing abnormal was found . in spite of treatment , the disease did not subside and she developed fever , abdominal distention and edema in her lower extremities .", "physical examination showed that the patient had decreased deep - tendon reflexes ( dtr ) and was unable to walk .", "clindamycin , ceftazidime , dexamethasone , ivig ( intravenous immunoglobulin ) and g - csf were administered .", "since the patient did not achieve full recovery , she was transferred to our center .", "based on clinical symptoms and lab tests ( table 1 ) , bma was performed once again and the result raised suspicion of ebv - induced secondary hlh .", "rituximab was injected once and the patient developed a blood ebv load of zero within one week . in order to control abnormal stimulation of the immune system ,", "the patient was investigated for mutations in il-2-inducible t cell kinase ( itk ) and negative test results were received .", "( the patient was negative for mutations in il-2-inducible t cell kinase ( itk ) ) .", "she is now considered to be an acceptable candidate for transplantation from an hla matched sibling donor . \n "], ["hlh can be divided into two subgroups : primary or familial hlh and secondary hlh .", "the primary form is an inflammatory disease which is similar to secondary one on the basis of symptoms .", "primary hlh usually arises in young children less than one year old ( 70% cases ) ; however , in rare cases it can also occur in adults ( perforin gene mutations have been identified in patients with primary hlh ) .", "for example , type 2 is caused by mutations in genes 21 - 22 and type 5 is due to mutations in the munc18 - 20 that may be accompanied by hypogammaglobulinemia .", "hypogammaglobulinemia was not confirmed in the patient because it was not possible to check mutation ( the related tests were sent to germany to examine the type 5 disease ) .", "ebv may also be accompanied by hypogammaglobulinemia for a certain period secondary hlh is associated with immunologic stimulation caused by malignancies and bacterial or congenital infections .", "the most common causes of secondary hlh are viral infections by ebv , cmv , prob19 and hiv .", "however , it may also be seen in patients with immune system defects . , 4 secondary hlh is a known complication of ebv infection , particularly in patients with x - linked lymphoproliferative disease .", "the incidence of x - linked lymphoproliferative syndrome is 1 in 1,000,000 male infants . \n ", "diagnostic criteria for hlh disease diagnosis of hlh requires the presence of all 5 major criteria .", "if the patient meets only 4criteria but the clinical suspicion for hlh is high , one should initiate treatment , because delays may be fatal .", "alternative criteria 1 or a combination of 2 and 3 may substitute for 1 major criterion . adapted from henter ji , elinder g , ost a.semin oncol . 1991 ; 18:29 - 33 .", "it means that ebv - infected b cells stimulate cytotoxic t lymphocytes leading to hypercytokinemia and stimulation of histolytic cells .", "more recently , it has been found that chronic stimulation by ebv may cause chronic hlh in the patient . on the other hand", ", ebv causes stimulation , generation and uncontrolled secretion of t - and nk- cells , as well as generation of il2 , infa and il6 .", "there is another mechanism by which ebv stimulates membrane protein ( lmp-1 ) in cells .", "similar to xlp , lmp-1 may cause acquired immune deficiency , leading to hlh accordingly . \n ", "treatment follows two goals : first , to suppress immune response using ivig , steroid and hlh protocol .", "second , to inhibit undesirable influence of immune response and cytokine - stimulated cell activation using immunochemotherapy . in cases with neurological symptoms", "the level of viral dna which is measured by quantitative pcr may be useful to demonstrate treatment response predicting mortality .", "the dna level reached zero in our patient . using ivig prior to start of protocol 2004", "it has also been recommended in the treatment of ebv - induced secondary hlh monthly . \n ", "decrease of b cells , which have been contaminated by ebv , actually causes decrease of viruses , as it may be useful for our patient as well .", "finally , treatment - refractory patients , transplant candidates , patients with familial type of hlh , or recurrent disease undergo chemotherapy for 8 weeks .", "patients who respond favorably to treatment do not undergo transplantation but those who experience relapse and exhibit neurological symptoms are considered for transplantation ."], ["severe infection of ebv is not associated with life - threatening condition , but our case study needs urgent treatment . in patients with long - term ebv infection , pancytopenia , coagulopathy and hepatosplenomegaly ,", "bma will be performed and the serum ferritin level will be measured as well . for patients with secondary hlh ,", "rituximab can be used when hlh is associated with ebv to inhibit the immune response .", "thus , the main aim of treatment is to reduce the rate of mortality in patients with hlh ."]]}
{"article_id": "PMC2732136", "article_text": ["we used data from the hoorn study , a population - based cohort study on glucose metabolism and other cardiovascular risk factors in a general caucasian population , which has been described in detail previously ( 12 ) . in brief", ", men and women aged 5075 years were randomly selected from the population register of the town of hoorn , the netherlands ; 2,484 subjects participated ( response rate 71% ) .", "all subjects had a 75-g oral glucose tolerance test , except those in whom type 2 diabetes had previously been diagnosed .", "an extensive metabolic and cardiovascular investigation was performed in an age- , sex- , and glucose tolerance  stratified , random subsample of 631 participants ( 89% of those invited ) , which is used in the present study ( 12 ) .", "participants without representative urine samples available ( n = 45 ) and/or who did not complete at least 7 of the 10 autonomic function tests ( n = 31 ) , had a history of neurological disease ( n = 5 ) , and were using ace inhibitors ( n = 33 ) and/or drugs known to influence autonomic nerve function ( namely antiparkinson drugs , phenytoin , antihistamines , or parasympatholytic , parasympathomimetic , and sympathomimetic drugs ) ( n = 49 ) were excluded from the analyses . the present study , therefore , consisted of 490 individuals : 305 with normal glucose metabolism , 71 with igm ( including those with impaired fasting glucose and/or impaired glucose tolerance ) , and 114 with type 2 diabetes .", "the hoorn study was approved by the ethical review committee of vu university medical centre , amsterdam , the netherlands .", "urinary albumin concentration was measured in a first voided sample by rate nephelometry ( array protein system , beckman coulter , galway , ireland ) with a detection threshold of 6.2 mg / l ( intra- and interassay coefficients of variation of 5 and 8% , respectively ) .", "microalbuminuria was present if the albumin - to - creatinine ratio ( acr ) was in the range of 2.030 mg / mmol ( 5 ) .", "the average value of the acr was used if two representative samples ( n = 154 ) were available . excluding subjects with an acr > 30 mg / mmol ( i.e. , macroalbuminuria , n = 4 ) did not materially affect any of the results , which are therefore presented for the entire group . cardiovascular autonomic function tests were performed as described in detail elsewhere ( 9 ) . in brief ,", "participants were asked to refrain from smoking and drinking coffee for at least 2 h before the assessments .", "tests took place between 8:30 a.m. and 4:00 p.m. in a quiet location with room temperature of 1922c after a resting period of at least 10 min .", "ten parameters of cardiac autonomic function were derived from the r - r interval and finger systolic blood pressure ( sbp ) continuous recordings by a computerized data analyses system developed locally by the department of medical physics , vu university , amsterdam , the netherlands ( 13 ) .", "tests were performed under three conditions : during spontaneous breathing over 3 min in the supine position ( mean of all normal to normal r - r intervals [ meannn ] , sd of all normal to normal r - r intervals [ sdnn ] , low - frequency [ lf ] power , high - frequency [ hf ] power , and lf/(lf + hf ) ) , during six deep breaths over 1 min in the supine position ( expiration - inspiration [ ei ] difference and baroreflex sensitivity [ brs ] ) , and during an active change from lying to standing ( sbp difference , r - r interval maximum [ rrmax ] , and r - r interval maximum divided by minimum [ rrmax / min ] ) ( for further explanation of measurements , see supplementary table a1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1544/dc1 ) .", "we thus obtained results in four tests that reflect heart rate or blood pressure changes due to certain  maneuvers ,  in this case deep breathing or standing up ( i.e. , ei difference , rrmax , rrmax / min , and sbp difference ) ( 14 ) , five tests of spectral analyses of heart rate variability ( hrv ) [ i.e. , meannn , sdnn , lf power , hf power , and lf/(lf + hf ) ] ( 15 ) , and one brs measurement ( 16 ) .", "in addition , these tests were grouped into those representing predominantly parasympathetic ( i.e. , ei difference , rrmax , and hf power ) , sympathetic ( i.e. , sbp difference ) , or both functions ( i.e. , mean - nn , sdnn , lf power , lf/(lf + hf ) , brs , and rrmax / min ) ( 15,16 ) .", "bmi , waist - to - hip ratio ( whr ) , sbp and diastolic blood pressure ( dbp ) , levels of fasting plasma glucose ; a1c ; insulin ; total , hdl , and ldl cholesterol ; triglycerides ; creatinine ; and smoking status were measured as described elsewhere ( 5,9,12 ) .", "glomerular filtration rate was estimated by the short modification of diet in renal disease equation .", "hypertension was defined as sbp 140 mmhg and/or dbp 90 mmhg and/or the use of antihypertensive drugs .", "prior cardiovascular disease ( cvd ) was defined when individuals had ( any of the following ) : a history of myocardial infarction , abnormalities on a resting electrocardiogram ( minnesota codes 1.11.3 , 4.14.3 , 5.15.3 , or 7.1 ) , previous coronary bypass surgery or angioplasty , peripheral arterial bypass , or nontraumatic amputation , or an ankle - brachial index of < 0.9 in either leg .", "data on the participants ' vital status up to 1 january 2005 were collected from the mortality register of the municipality of hoorn .", "information on cause of death was extracted from the medical records of the general practitioners and the local hospital and coded according to the icd-9 .", "cardiovascular mortality , including sudden death , was defined by icd-9 codes 390459 and 798 .", "information on cause of death could not be obtained for 20 of the deceased individuals .", "all subjects were followed until death or end of follow - up , at which time they were censored .", "baseline characteristics between survivors and nonsurvivors were compared with the use of student 's t or  tests .", "we computed z scores ( [ individual 's observed value  population mean]/sd ) for each of the 10 measurements of c - ad .", "all z scores ( except sbp difference ) were inverted and averaged into a total score (  c - ad total score  ) , so that higher values reflect greater c - ad .", "the association between microalbuminuria and c - ad total score was investigated with multiple linear regression analyses .", "kaplan - meier survival curves were plotted for cardiovascular mortality among individuals with normoalbuminuria versus microalbuminuria and similarly across tertiles of the c - ad total score , and differences between groups were tested with a log - rank test .", "cox proportional hazards regression models were used to calculate the crude and adjusted relative risks ( rrs ) and respective 95% ci of microalbuminuria ( versus normoalbuminuria ) and of the c - ad total score ( per sd [ = 0.605 ] increase ) for cardiovascular and all - cause mortality .", "urinary albumin concentration was measured in a first voided sample by rate nephelometry ( array protein system , beckman coulter , galway , ireland ) with a detection threshold of 6.2 mg / l ( intra- and interassay coefficients of variation of 5 and 8% , respectively ) .", "microalbuminuria was present if the albumin - to - creatinine ratio ( acr ) was in the range of 2.030 mg / mmol ( 5 ) .", "the average value of the acr was used if two representative samples ( n = 154 ) were available . excluding subjects with an acr > 30 mg / mmol ( i.e. , macroalbuminuria , n = 4 ) did not materially affect any of the results , which are therefore presented for the entire group .", "cardiovascular autonomic function tests were performed as described in detail elsewhere ( 9 ) . in brief , participants were asked to refrain from smoking and drinking coffee for at least 2 h before the assessments . a light meal > 1 h before the measurements was allowed .", "tests took place between 8:30 a.m. and 4:00 p.m. in a quiet location with room temperature of 1922c after a resting period of at least 10 min .", "ten parameters of cardiac autonomic function were derived from the r - r interval and finger systolic blood pressure ( sbp ) continuous recordings by a computerized data analyses system developed locally by the department of medical physics , vu university , amsterdam , the netherlands ( 13 ) .", "tests were performed under three conditions : during spontaneous breathing over 3 min in the supine position ( mean of all normal to normal r - r intervals [ meannn ] , sd of all normal to normal r - r intervals [ sdnn ] , low - frequency [ lf ] power , high - frequency [ hf ] power , and lf/(lf + hf ) ) , during six deep breaths over 1 min in the supine position ( expiration - inspiration [ ei ] difference and baroreflex sensitivity [ brs ] ) , and during an active change from lying to standing ( sbp difference , r - r interval maximum [ rrmax ] , and r - r interval maximum divided by minimum [ rrmax / min ] ) ( for further explanation of measurements , see supplementary table a1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1544/dc1 ) .", "we thus obtained results in four tests that reflect heart rate or blood pressure changes due to certain  maneuvers ,  in this case deep breathing or standing up ( i.e. , ei difference , rrmax , rrmax / min , and sbp difference ) ( 14 ) , five tests of spectral analyses of heart rate variability ( hrv ) [ i.e. , meannn , sdnn , lf power , hf power , and lf/(lf + hf ) ] ( 15 ) , and one brs measurement ( 16 ) .", "in addition , these tests were grouped into those representing predominantly parasympathetic ( i.e. , ei difference , rrmax , and hf power ) , sympathetic ( i.e. , sbp difference ) , or both functions ( i.e. , mean - nn , sdnn , lf power , lf/(lf + hf ) , brs , and rrmax / min ) ( 15,16 ) .", "bmi , waist - to - hip ratio ( whr ) , sbp and diastolic blood pressure ( dbp ) , levels of fasting plasma glucose ; a1c ; insulin ; total , hdl , and ldl cholesterol ; triglycerides ; creatinine ; and smoking status were measured as described elsewhere ( 5,9,12 ) .", "glomerular filtration rate was estimated by the short modification of diet in renal disease equation .", "hypertension was defined as sbp 140 mmhg and/or dbp 90 mmhg and/or the use of antihypertensive drugs .", "prior cardiovascular disease ( cvd ) was defined when individuals had ( any of the following ) : a history of myocardial infarction , abnormalities on a resting electrocardiogram ( minnesota codes 1.11.3 , 4.14.3 , 5.15.3 , or 7.1 ) , previous coronary bypass surgery or angioplasty , peripheral arterial bypass , or nontraumatic amputation , or an ankle - brachial index of < 0.9 in either leg .", "data on the participants ' vital status up to 1 january 2005 were collected from the mortality register of the municipality of hoorn .", "information on cause of death was extracted from the medical records of the general practitioners and the local hospital and coded according to the icd-9 .", "cardiovascular mortality , including sudden death , was defined by icd-9 codes 390459 and 798 .", "information on cause of death could not be obtained for 20 of the deceased individuals .", "all subjects were followed until death or end of follow - up , at which time they were censored .", "baseline characteristics between survivors and nonsurvivors were compared with the use of student 's t or  tests .", "we computed z scores ( [ individual 's observed value  population mean]/sd ) for each of the 10 measurements of c - ad .", "all z scores ( except sbp difference ) were inverted and averaged into a total score (  c - ad total score  ) , so that higher values reflect greater c - ad .", "the association between microalbuminuria and c - ad total score was investigated with multiple linear regression analyses .", "kaplan - meier survival curves were plotted for cardiovascular mortality among individuals with normoalbuminuria versus microalbuminuria and similarly across tertiles of the c - ad total score , and differences between groups were tested with a log - rank test .", "cox proportional hazards regression models were used to calculate the crude and adjusted relative risks ( rrs ) and respective 95% ci of microalbuminuria ( versus normoalbuminuria ) and of the c - ad total score ( per sd [ = 0.605 ] increase ) for cardiovascular and all - cause mortality .", "subjects excluded from the analyses ( n = 141 ) were older , more often had type 2 diabetes , and had a worse risk profile than the subjects included ( n = 490 ) ( data not shown ) .", "median duration of the follow - up was 13.6 ( range 0.5215.19 ) years . during the follow - up period , 141 ( 28.8% ) participants died , of whom 53 ( 37.6% ) died of cvd . at baseline", ", individuals who died had a worse c - ad total score , more often had microalbuminuria and were older , more often were men and had type 2 diabetes and hypertension , and had a higher a1c and whr as compared with the survivors ( table 1 ) . baseline characteristics of survivors versus nonsurvivors data are presented as frequencies ( % ) , means  sd , or medians ( interquartile range ) . for a detailed explanation of autonomic function tests ,", "these include diuretics , -blockers , -blockers , calcium channel blockers , and other blood pressure  lowering drugs but exclude ace inhibitors ( because subjects using these drugs were excluded from the analyses ) .", "c - ad total score was associated with microalbuminuria (  = 0.33 [ 95% ci 0.160.51 ] ) ( table 2 , model 1 ) .", "after adjustment for potential confounding factors , c - ad total score was still borderline associated with microalbuminuria ( 0.16 [ 0.01 to 0.33 ] ) ( table 2 , model 3 ) .", "comparable results were obtained when tests were combined on the basis of their methodology or on the part of the autonomic nervous system they predominately represent .", "all individual c - ad tests , except lf/(lf + hf ) , were positively associated with microalbuminuria ( although not all statistically significantly so ) ( data not shown ) .", "association between microalbuminuria and c - ad * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking , and prior cvd .", " indicates the difference in c - ad score between individuals with microalbuminuria vs. normoalbuminuria . both microalbuminuria ( rr 3.49 [ 95% ci 1.876.53 ] ) and c - ad total score ( 2.54 [ 1.604.04 ] )", "these associations were attenuated after adjustment for age , sex , and glucose tolerance status ( gts ) ( 2.23 [ 1.164.29 ] and 1.81 [ 1.112.94 ] , respectively ; model 2 ) . further adjustment for other cardiovascular risk factors only slightly further attenuated these rrs ( model 3 ) , and when the c - ad total score or microalbuminuria was added to these models , the rr attributable to microalbuminuria remained practically unchanged and both microalbuminuria ( 2.13 [ 1.094.17 ] ) and c - ad total score ( 1.76 [ 1.052.94 ] ) were independently associated with cardiovascular mortality ( model 4 ) .", "association of microalbuminuria or c - ad with cardiovascular and all - cause mortality * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking and prior cvd ; model 4 : model 3 , with additional adjustment for , respectively , c - ad or microalbuminuria .", "rr of microalbuminuria vs. normoalbuminuria or per sd ( = 0.605 ) increase in c - ad total score .", "kaplan - meier survival plots for cardiovascular mortality . among individuals with normo- versus microalbuminuria ( a ) and across tertiles of c - ad total score ( b ) .", "both microalbuminuria and c - ad total score were also associated with all - cause mortality ( table 3 , model 1 ) . however , after adjustment for age , sex , gts , and other cardiovascular risk factors , microalbuminuria ( rr 1.33 [ 95% ci 0.832.13 ] ) , in contrast to c - ad total score ( 1.52 [ 1.112.09 ] ) , was not independently associated with all - cause mortality ( model 3 ) .", "the associations described above were also investigated by examining autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent ( supplementary table a2 , available in an online appendix ) .", "these additional analyses showed that overall all different groups of tests were associated with increased mortality risk , that these associations were attenuated when further adjusted for age , sex , gts , and other risk factors ; and that none of the specific group of tests explained the association between microalbuminuria and mortality .", "the associations reported in table 3 did not materially change after additional adjustment for use of lipid- or blood pressure  lowering drugs and the presence of leukocytes in urine ( tested by microscopy and scored as positive whenever there were more than five leukocytes per high - power field ) or when the dichotomous variable hypertension used in those models was replaced by either sbp or pulse pressure ( a marker of arterial stiffness ) or when analyses were adjusted for prior coronary heart disease instead of prior cvd ( data not shown ) .", "finally , we found no evidence of effect modification by gts in the association between cardiovascular mortality and c - ad total score or microalbuminuria ( pinteraction > 0.4 in both cases ) .", "c - ad total score was associated with microalbuminuria (  = 0.33 [ 95% ci 0.160.51 ] ) ( table 2 , model 1 ) . after adjustment for potential confounding factors ,", "c - ad total score was still borderline associated with microalbuminuria ( 0.16 [ 0.01 to 0.33 ] ) ( table 2 , model 3 ) .", "comparable results were obtained when tests were combined on the basis of their methodology or on the part of the autonomic nervous system they predominately represent .", "all individual c - ad tests , except lf/(lf + hf ) , were positively associated with microalbuminuria ( although not all statistically significantly so ) ( data not shown ) .", "association between microalbuminuria and c - ad * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking , and prior cvd .", " indicates the difference in c - ad score between individuals with microalbuminuria vs. normoalbuminuria .", "both microalbuminuria ( rr 3.49 [ 95% ci 1.876.53 ] ) and c - ad total score ( 2.54 [ 1.604.04 ] ) were associated with cardiovascular mortality ( table 3 , model 1 ; fig .", "these associations were attenuated after adjustment for age , sex , and glucose tolerance status ( gts ) ( 2.23 [ 1.164.29 ] and 1.81 [ 1.112.94 ] , respectively ; model 2 ) . further adjustment for other cardiovascular risk factors only slightly further attenuated these rrs ( model 3 ) , and when the c - ad total score or microalbuminuria was added to these models , the rr attributable to microalbuminuria remained practically unchanged and both microalbuminuria ( 2.13 [ 1.094.17 ] ) and c - ad total score ( 1.76 [ 1.052.94 ] ) were independently associated with cardiovascular mortality ( model 4 ) .", "association of microalbuminuria or c - ad with cardiovascular and all - cause mortality * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking and prior cvd ; model 4 : model 3 , with additional adjustment for , respectively , c - ad or microalbuminuria .", "rr of microalbuminuria vs. normoalbuminuria or per sd ( = 0.605 ) increase in c - ad total score .", "kaplan - meier survival plots for cardiovascular mortality . among individuals with normo- versus microalbuminuria ( a ) and across tertiles of c - ad total score ( b ) . both microalbuminuria and c - ad total score", "were also associated with all - cause mortality ( table 3 , model 1 ) . however , after adjustment for age , sex , gts , and other cardiovascular risk factors , microalbuminuria ( rr 1.33 [ 95% ci 0.832.13 ] ) , in contrast to c - ad total score ( 1.52 [ 1.112.09 ] ) , was not independently associated with all - cause mortality ( model 3 ) .", "the associations described above were also investigated by examining autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent ( supplementary table a2 , available in an online appendix ) .", "these additional analyses showed that overall all different groups of tests were associated with increased mortality risk , that these associations were attenuated when further adjusted for age , sex , gts , and other risk factors ; and that none of the specific group of tests explained the association between microalbuminuria and mortality .", "the associations reported in table 3 did not materially change after additional adjustment for use of lipid- or blood pressure  lowering drugs and the presence of leukocytes in urine ( tested by microscopy and scored as positive whenever there were more than five leukocytes per high - power field ) or when the dichotomous variable hypertension used in those models was replaced by either sbp or pulse pressure ( a marker of arterial stiffness ) or when analyses were adjusted for prior coronary heart disease instead of prior cvd ( data not shown ) .", "finally , we found no evidence of effect modification by gts in the association between cardiovascular mortality and c - ad total score or microalbuminuria ( pinteraction > 0.4 in both cases ) .", "in this prospective study we found that both microalbuminuria and c - ad ( estimated from the mean of 10 standardized tests ) were independently associated with cardiovascular mortality in a cohort of elderly , caucasian subjects with and without diabetes . therefore , c", "- ad does not explain why microalbuminuria is related to cardiovascular mortality in either the general population or in diabetic subjects .", "recently , hrv was found not to predict the decline of gfr but was independently associated with cardiovascular mortality in middle - aged type 1 diabetic subjects with overt nephropathy ( 17 ) . in another study ,", "investigating the independent predictive role of gfr , microalbuminuria , and cardiovascular autonomic neuropathy showed the latter to also be associated with all - cause and cardiovascular mortality in subjects with diabetes ( 18 ) .", "hitherto , no other prospective studies examining the relation of microalbuminuria and c - ad with cardiovascular mortality have been reported .", "the independent association of both microalbuminuria and c - ad with cardiovascular mortality reported herein indicates that they are linked to cardiovascular mortality by different biological pathways .", "several mechanisms linking c - ad to cardiovascular mortality have been proposed ( e.g. , qt interval prolongation and silent myocardial ischemia ) ( 19 ) . microalbuminuria may be linked to cvd by a common pathophysiological process ( such as endothelial dysfunction or chronic low - grade inflammation ) ( 3 ) .", "however , the possibility that microalbuminuria reflects the presence of a set of undiscovered factors that are causally related to cvd remains to be further elucidated .", "furthermore , aggressive treatment of microalbuminuria by inhibiting the renin - angiotensin - aldosterone system has been shown to decrease renal and cardiovascular risk in individuals with diabetes ( 20 ) .", "there is some evidence that c - ad is also a useful risk indicator for cvd , especially in subjects at high risk ( type 2 diabetes , hypertension , or prior cvd ) ( 9,21 ) , and this study reinforces that observation .", "however , whether the improvement in c - ad , e.g. , with exercise training or blood pressure or lipid - lowering drugs ( 2224 ) , leads to a better prognosis is unknown . in our study ,", "additional adjustment for use of -blockers or lipid - lowering drugs had no effect on the associations reported .", "therefore , the clinical relevance of treating c - ad in the general population remains unclear and needs to be further examined .", "the strengths of our study are the relatively large , truly population - based and prospective design with a long follow - up period .", "in addition , we characterized subjects ' c - ad as comprehensively as possible by conducting 10 autonomic function tests , which are thought to reflect all aspects of cardiovascular autonomic function .", "previous cross - sectional studies evaluating the relationship between microalbuminuria and c - ad used less extensive autonomic function tests ( 4,6 ) . because we included all three groups of gts", ", we were able to analyze possible effect modification of gts in the relationship between the main determinants , c - ad and microalbuminuria and cardiovascular mortality ( but found none ) .", "first , each of the test parameters of autonomic function was measured with moderate levels of reproducibility ( reliability coefficient 50% ) ( 13 ) .", "therefore , the results were combined into a c - ad total score , which enabled us to circumvent , at least partially , this problem .", "specifically , the results herein reported with the c - ad total score were more powerful than results obtained on the basis of each individual test ( separately ) , because these results would be more strongly affected by misclassification ( likely to be random ) , causing an underestimation of the strength of the associations .", "indeed , the associations between each individual test result with cardiovascular mortality were in accordance , although with lower strength , with those as reported herein with the c - ad total score ( data not shown ) .", "in addition , this approach had the advantage of circumventing the problem of multiple testing ( type i errors ) that would have occurred when the associations with each test were analyzed separately . however , because each test may represent different parts of autonomic function , some overlapping but others complementary , our c - ad total score does not allow the distinction of potential different contributions of sympathetic and parasympathetic functions to cardiovascular mortality .", "therefore , we examined the autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent in the additional analyses .", "the results of these alternative combinations of autonomic function tests were comparable to those of the c - ad total score ( supplementary table a2 ) , but we acknowledge that these analyses may not have been specific enough , as most authors agree that a clear distinction between parasympathetic or sympathetic dysfunction can not be made on the basis of these tests .", "altogether , we feel that the advantages ( i.e. , reduced misclassification and avoidance of type i errors without impairment of etiological validity ) of the approach used by us outweigh the disadvantages .", "measurement of hrv during a longer period may be more reliable , although measurements obtained in time periods as short as 2 min correlate highly with 24-h measurements ( 25 ) .", "third , evaluation of subjects ' c - ad was conducted at baseline only , and , therefore , we were not able to evaluate deterioration of autonomic function during follow - up .", "fourth , for most participants microalbuminuria was defined on an acr measured in one urine sample only .", "because microalbuminuria is quite variable from day to day we can not fully exclude the possibility that some subjects were misclassified .", "however , we tried to avoid this problem by collecting urine overnight and expressing albumin excretion as acr .", "in addition , analyses in the 154 participants for whom two samples were available yielded essentially similar results ( data not shown ) .", "finally , we studied an elderly , caucasian population , and it is not known whether our results can be generalized to other ethnic groups or to younger individuals . in summary", ", we have shown that both microalbuminuria and c - ad are independently associated with cardiovascular mortality in an elderly , caucasian population of individuals with normal glucose metabolism , igm , and diabetes .", "these results suggest that microalbuminuria and c - ad are related to cardiovascular mortality by different biological pathways .", "therefore , it may be useful to treat not only microalbuminuria but also c - ad in populations at high risk of cardiovascular mortality ."], "abstract_text": ["<S> objectivemicroalbuminuria is associated with cardiovascular mortality , particularly among individuals with type 2 diabetes , but the mechanisms underlying this association are not completely understood . </S>", "<S> microalbuminuria is known to be associated with cardiovascular autonomic dysfunction ( c - ad ) , and c - ad in turn is associated with cardiovascular mortality . the purpose of this study , therefore , was to investigate whether c - ad can explain the relationship between microalbuminuria and cardiovascular mortality.research design and methodswe studied 490 individuals from a population - based cohort of individuals aged 5075 years who were followed for a median period of 13.6 years . </S>", "<S> microalbuminuria was defined as an albumin - to - creatinine ratio 2.0 mg / mmol in an early - morning spot - urine sample . </S>", "<S> ten parameters reflecting different aspects of cardiovascular autonomic function were measured and compiled into a total score of c - ad ( mean of separate z scores ) . </S>", "<S> the association between c - ad and microalbuminuria was estimated by multiple linear regression , and relative risks ( rrs ) for cardiovascular mortality were estimated by cox proportional hazards analyses.resultsafter adjustments for age , sex , glucose tolerance status , and other risk factors , c - ad was associated with microalbuminuria (  = 0.16 [ 95% ci 0.010.33 ] ) , and both microalbuminuria ( rr 2.09 [ 1.074.08 ] ) and c - ad ( 1.74 [ 1.042.89 ] ) were associated with cardiovascular mortality . </S>", "<S> these associations did not change after further mutual adjustment for c - ad ( 2.13 [ 1.094.17 ] ) or microalbuminuria ( 1.76 [ 1.052.94 ] ) , respectively.conclusionsboth microalbuminuria and c - ad are independently associated with cardiovascular mortality , and the excess mortality attributable to microalbuminuria can not be explained by c - ad . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Baseline measurements", "Follow-up", "Statistical analyses", "RESULTS", "Association between microalbuminuria and C-AD", "Association of microalbuminuria and C-AD total score with cardiovascular and all-cause mortality", "Additional analyses", "CONCLUSIONS", "Supplementary Material"], "sections": [["we used data from the hoorn study , a population - based cohort study on glucose metabolism and other cardiovascular risk factors in a general caucasian population , which has been described in detail previously ( 12 ) . in brief", ", men and women aged 5075 years were randomly selected from the population register of the town of hoorn , the netherlands ; 2,484 subjects participated ( response rate 71% ) .", "all subjects had a 75-g oral glucose tolerance test , except those in whom type 2 diabetes had previously been diagnosed .", "an extensive metabolic and cardiovascular investigation was performed in an age- , sex- , and glucose tolerance  stratified , random subsample of 631 participants ( 89% of those invited ) , which is used in the present study ( 12 ) .", "participants without representative urine samples available ( n = 45 ) and/or who did not complete at least 7 of the 10 autonomic function tests ( n = 31 ) , had a history of neurological disease ( n = 5 ) , and were using ace inhibitors ( n = 33 ) and/or drugs known to influence autonomic nerve function ( namely antiparkinson drugs , phenytoin , antihistamines , or parasympatholytic , parasympathomimetic , and sympathomimetic drugs ) ( n = 49 ) were excluded from the analyses . the present study , therefore , consisted of 490 individuals : 305 with normal glucose metabolism , 71 with igm ( including those with impaired fasting glucose and/or impaired glucose tolerance ) , and 114 with type 2 diabetes .", "the hoorn study was approved by the ethical review committee of vu university medical centre , amsterdam , the netherlands .", "urinary albumin concentration was measured in a first voided sample by rate nephelometry ( array protein system , beckman coulter , galway , ireland ) with a detection threshold of 6.2 mg / l ( intra- and interassay coefficients of variation of 5 and 8% , respectively ) .", "microalbuminuria was present if the albumin - to - creatinine ratio ( acr ) was in the range of 2.030 mg / mmol ( 5 ) .", "the average value of the acr was used if two representative samples ( n = 154 ) were available . excluding subjects with an acr > 30 mg / mmol ( i.e. , macroalbuminuria , n = 4 ) did not materially affect any of the results , which are therefore presented for the entire group . cardiovascular autonomic function tests were performed as described in detail elsewhere ( 9 ) . in brief ,", "participants were asked to refrain from smoking and drinking coffee for at least 2 h before the assessments .", "tests took place between 8:30 a.m. and 4:00 p.m. in a quiet location with room temperature of 1922c after a resting period of at least 10 min .", "ten parameters of cardiac autonomic function were derived from the r - r interval and finger systolic blood pressure ( sbp ) continuous recordings by a computerized data analyses system developed locally by the department of medical physics , vu university , amsterdam , the netherlands ( 13 ) .", "tests were performed under three conditions : during spontaneous breathing over 3 min in the supine position ( mean of all normal to normal r - r intervals [ meannn ] , sd of all normal to normal r - r intervals [ sdnn ] , low - frequency [ lf ] power , high - frequency [ hf ] power , and lf/(lf + hf ) ) , during six deep breaths over 1 min in the supine position ( expiration - inspiration [ ei ] difference and baroreflex sensitivity [ brs ] ) , and during an active change from lying to standing ( sbp difference , r - r interval maximum [ rrmax ] , and r - r interval maximum divided by minimum [ rrmax / min ] ) ( for further explanation of measurements , see supplementary table a1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1544/dc1 ) .", "we thus obtained results in four tests that reflect heart rate or blood pressure changes due to certain  maneuvers ,  in this case deep breathing or standing up ( i.e. , ei difference , rrmax , rrmax / min , and sbp difference ) ( 14 ) , five tests of spectral analyses of heart rate variability ( hrv ) [ i.e. , meannn , sdnn , lf power , hf power , and lf/(lf + hf ) ] ( 15 ) , and one brs measurement ( 16 ) .", "in addition , these tests were grouped into those representing predominantly parasympathetic ( i.e. , ei difference , rrmax , and hf power ) , sympathetic ( i.e. , sbp difference ) , or both functions ( i.e. , mean - nn , sdnn , lf power , lf/(lf + hf ) , brs , and rrmax / min ) ( 15,16 ) .", "bmi , waist - to - hip ratio ( whr ) , sbp and diastolic blood pressure ( dbp ) , levels of fasting plasma glucose ; a1c ; insulin ; total , hdl , and ldl cholesterol ; triglycerides ; creatinine ; and smoking status were measured as described elsewhere ( 5,9,12 ) .", "glomerular filtration rate was estimated by the short modification of diet in renal disease equation .", "hypertension was defined as sbp 140 mmhg and/or dbp 90 mmhg and/or the use of antihypertensive drugs .", "prior cardiovascular disease ( cvd ) was defined when individuals had ( any of the following ) : a history of myocardial infarction , abnormalities on a resting electrocardiogram ( minnesota codes 1.11.3 , 4.14.3 , 5.15.3 , or 7.1 ) , previous coronary bypass surgery or angioplasty , peripheral arterial bypass , or nontraumatic amputation , or an ankle - brachial index of < 0.9 in either leg .", "data on the participants ' vital status up to 1 january 2005 were collected from the mortality register of the municipality of hoorn .", "information on cause of death was extracted from the medical records of the general practitioners and the local hospital and coded according to the icd-9 .", "cardiovascular mortality , including sudden death , was defined by icd-9 codes 390459 and 798 .", "information on cause of death could not be obtained for 20 of the deceased individuals .", "all subjects were followed until death or end of follow - up , at which time they were censored .", "baseline characteristics between survivors and nonsurvivors were compared with the use of student 's t or  tests .", "we computed z scores ( [ individual 's observed value  population mean]/sd ) for each of the 10 measurements of c - ad .", "all z scores ( except sbp difference ) were inverted and averaged into a total score (  c - ad total score  ) , so that higher values reflect greater c - ad .", "the association between microalbuminuria and c - ad total score was investigated with multiple linear regression analyses .", "kaplan - meier survival curves were plotted for cardiovascular mortality among individuals with normoalbuminuria versus microalbuminuria and similarly across tertiles of the c - ad total score , and differences between groups were tested with a log - rank test .", "cox proportional hazards regression models were used to calculate the crude and adjusted relative risks ( rrs ) and respective 95% ci of microalbuminuria ( versus normoalbuminuria ) and of the c - ad total score ( per sd [ = 0.605 ] increase ) for cardiovascular and all - cause mortality ."], ["urinary albumin concentration was measured in a first voided sample by rate nephelometry ( array protein system , beckman coulter , galway , ireland ) with a detection threshold of 6.2 mg / l ( intra- and interassay coefficients of variation of 5 and 8% , respectively ) .", "microalbuminuria was present if the albumin - to - creatinine ratio ( acr ) was in the range of 2.030 mg / mmol ( 5 ) .", "the average value of the acr was used if two representative samples ( n = 154 ) were available . excluding subjects with an acr > 30 mg / mmol ( i.e. , macroalbuminuria , n = 4 ) did not materially affect any of the results , which are therefore presented for the entire group .", "cardiovascular autonomic function tests were performed as described in detail elsewhere ( 9 ) . in brief , participants were asked to refrain from smoking and drinking coffee for at least 2 h before the assessments . a light meal > 1 h before the measurements was allowed .", "tests took place between 8:30 a.m. and 4:00 p.m. in a quiet location with room temperature of 1922c after a resting period of at least 10 min .", "ten parameters of cardiac autonomic function were derived from the r - r interval and finger systolic blood pressure ( sbp ) continuous recordings by a computerized data analyses system developed locally by the department of medical physics , vu university , amsterdam , the netherlands ( 13 ) .", "tests were performed under three conditions : during spontaneous breathing over 3 min in the supine position ( mean of all normal to normal r - r intervals [ meannn ] , sd of all normal to normal r - r intervals [ sdnn ] , low - frequency [ lf ] power , high - frequency [ hf ] power , and lf/(lf + hf ) ) , during six deep breaths over 1 min in the supine position ( expiration - inspiration [ ei ] difference and baroreflex sensitivity [ brs ] ) , and during an active change from lying to standing ( sbp difference , r - r interval maximum [ rrmax ] , and r - r interval maximum divided by minimum [ rrmax / min ] ) ( for further explanation of measurements , see supplementary table a1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1544/dc1 ) .", "we thus obtained results in four tests that reflect heart rate or blood pressure changes due to certain  maneuvers ,  in this case deep breathing or standing up ( i.e. , ei difference , rrmax , rrmax / min , and sbp difference ) ( 14 ) , five tests of spectral analyses of heart rate variability ( hrv ) [ i.e. , meannn , sdnn , lf power , hf power , and lf/(lf + hf ) ] ( 15 ) , and one brs measurement ( 16 ) .", "in addition , these tests were grouped into those representing predominantly parasympathetic ( i.e. , ei difference , rrmax , and hf power ) , sympathetic ( i.e. , sbp difference ) , or both functions ( i.e. , mean - nn , sdnn , lf power , lf/(lf + hf ) , brs , and rrmax / min ) ( 15,16 ) .", "bmi , waist - to - hip ratio ( whr ) , sbp and diastolic blood pressure ( dbp ) , levels of fasting plasma glucose ; a1c ; insulin ; total , hdl , and ldl cholesterol ; triglycerides ; creatinine ; and smoking status were measured as described elsewhere ( 5,9,12 ) .", "glomerular filtration rate was estimated by the short modification of diet in renal disease equation .", "hypertension was defined as sbp 140 mmhg and/or dbp 90 mmhg and/or the use of antihypertensive drugs .", "prior cardiovascular disease ( cvd ) was defined when individuals had ( any of the following ) : a history of myocardial infarction , abnormalities on a resting electrocardiogram ( minnesota codes 1.11.3 , 4.14.3 , 5.15.3 , or 7.1 ) , previous coronary bypass surgery or angioplasty , peripheral arterial bypass , or nontraumatic amputation , or an ankle - brachial index of < 0.9 in either leg ."], ["data on the participants ' vital status up to 1 january 2005 were collected from the mortality register of the municipality of hoorn .", "information on cause of death was extracted from the medical records of the general practitioners and the local hospital and coded according to the icd-9 .", "cardiovascular mortality , including sudden death , was defined by icd-9 codes 390459 and 798 .", "information on cause of death could not be obtained for 20 of the deceased individuals .", "all subjects were followed until death or end of follow - up , at which time they were censored ."], ["baseline characteristics between survivors and nonsurvivors were compared with the use of student 's t or  tests .", "we computed z scores ( [ individual 's observed value  population mean]/sd ) for each of the 10 measurements of c - ad .", "all z scores ( except sbp difference ) were inverted and averaged into a total score (  c - ad total score  ) , so that higher values reflect greater c - ad .", "the association between microalbuminuria and c - ad total score was investigated with multiple linear regression analyses .", "kaplan - meier survival curves were plotted for cardiovascular mortality among individuals with normoalbuminuria versus microalbuminuria and similarly across tertiles of the c - ad total score , and differences between groups were tested with a log - rank test .", "cox proportional hazards regression models were used to calculate the crude and adjusted relative risks ( rrs ) and respective 95% ci of microalbuminuria ( versus normoalbuminuria ) and of the c - ad total score ( per sd [ = 0.605 ] increase ) for cardiovascular and all - cause mortality ."], ["subjects excluded from the analyses ( n = 141 ) were older , more often had type 2 diabetes , and had a worse risk profile than the subjects included ( n = 490 ) ( data not shown ) .", "median duration of the follow - up was 13.6 ( range 0.5215.19 ) years . during the follow - up period , 141 ( 28.8% ) participants died , of whom 53 ( 37.6% ) died of cvd . at baseline", ", individuals who died had a worse c - ad total score , more often had microalbuminuria and were older , more often were men and had type 2 diabetes and hypertension , and had a higher a1c and whr as compared with the survivors ( table 1 ) . baseline characteristics of survivors versus nonsurvivors data are presented as frequencies ( % ) , means  sd , or medians ( interquartile range ) . for a detailed explanation of autonomic function tests ,", "these include diuretics , -blockers , -blockers , calcium channel blockers , and other blood pressure  lowering drugs but exclude ace inhibitors ( because subjects using these drugs were excluded from the analyses ) .", "c - ad total score was associated with microalbuminuria (  = 0.33 [ 95% ci 0.160.51 ] ) ( table 2 , model 1 ) .", "after adjustment for potential confounding factors , c - ad total score was still borderline associated with microalbuminuria ( 0.16 [ 0.01 to 0.33 ] ) ( table 2 , model 3 ) .", "comparable results were obtained when tests were combined on the basis of their methodology or on the part of the autonomic nervous system they predominately represent .", "all individual c - ad tests , except lf/(lf + hf ) , were positively associated with microalbuminuria ( although not all statistically significantly so ) ( data not shown ) .", "association between microalbuminuria and c - ad * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking , and prior cvd .", " indicates the difference in c - ad score between individuals with microalbuminuria vs. normoalbuminuria . both microalbuminuria ( rr 3.49 [ 95% ci 1.876.53 ] ) and c - ad total score ( 2.54 [ 1.604.04 ] )", "these associations were attenuated after adjustment for age , sex , and glucose tolerance status ( gts ) ( 2.23 [ 1.164.29 ] and 1.81 [ 1.112.94 ] , respectively ; model 2 ) . further adjustment for other cardiovascular risk factors only slightly further attenuated these rrs ( model 3 ) , and when the c - ad total score or microalbuminuria was added to these models , the rr attributable to microalbuminuria remained practically unchanged and both microalbuminuria ( 2.13 [ 1.094.17 ] ) and c - ad total score ( 1.76 [ 1.052.94 ] ) were independently associated with cardiovascular mortality ( model 4 ) .", "association of microalbuminuria or c - ad with cardiovascular and all - cause mortality * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking and prior cvd ; model 4 : model 3 , with additional adjustment for , respectively , c - ad or microalbuminuria .", "rr of microalbuminuria vs. normoalbuminuria or per sd ( = 0.605 ) increase in c - ad total score .", "kaplan - meier survival plots for cardiovascular mortality . among individuals with normo- versus microalbuminuria ( a ) and across tertiles of c - ad total score ( b ) .", "both microalbuminuria and c - ad total score were also associated with all - cause mortality ( table 3 , model 1 ) . however , after adjustment for age , sex , gts , and other cardiovascular risk factors , microalbuminuria ( rr 1.33 [ 95% ci 0.832.13 ] ) , in contrast to c - ad total score ( 1.52 [ 1.112.09 ] ) , was not independently associated with all - cause mortality ( model 3 ) .", "the associations described above were also investigated by examining autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent ( supplementary table a2 , available in an online appendix ) .", "these additional analyses showed that overall all different groups of tests were associated with increased mortality risk , that these associations were attenuated when further adjusted for age , sex , gts , and other risk factors ; and that none of the specific group of tests explained the association between microalbuminuria and mortality .", "the associations reported in table 3 did not materially change after additional adjustment for use of lipid- or blood pressure  lowering drugs and the presence of leukocytes in urine ( tested by microscopy and scored as positive whenever there were more than five leukocytes per high - power field ) or when the dichotomous variable hypertension used in those models was replaced by either sbp or pulse pressure ( a marker of arterial stiffness ) or when analyses were adjusted for prior coronary heart disease instead of prior cvd ( data not shown ) .", "finally , we found no evidence of effect modification by gts in the association between cardiovascular mortality and c - ad total score or microalbuminuria ( pinteraction > 0.4 in both cases ) ."], ["c - ad total score was associated with microalbuminuria (  = 0.33 [ 95% ci 0.160.51 ] ) ( table 2 , model 1 ) . after adjustment for potential confounding factors ,", "c - ad total score was still borderline associated with microalbuminuria ( 0.16 [ 0.01 to 0.33 ] ) ( table 2 , model 3 ) .", "comparable results were obtained when tests were combined on the basis of their methodology or on the part of the autonomic nervous system they predominately represent .", "all individual c - ad tests , except lf/(lf + hf ) , were positively associated with microalbuminuria ( although not all statistically significantly so ) ( data not shown ) .", "association between microalbuminuria and c - ad * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking , and prior cvd .", " indicates the difference in c - ad score between individuals with microalbuminuria vs. normoalbuminuria ."], ["both microalbuminuria ( rr 3.49 [ 95% ci 1.876.53 ] ) and c - ad total score ( 2.54 [ 1.604.04 ] ) were associated with cardiovascular mortality ( table 3 , model 1 ; fig .", "these associations were attenuated after adjustment for age , sex , and glucose tolerance status ( gts ) ( 2.23 [ 1.164.29 ] and 1.81 [ 1.112.94 ] , respectively ; model 2 ) . further adjustment for other cardiovascular risk factors only slightly further attenuated these rrs ( model 3 ) , and when the c - ad total score or microalbuminuria was added to these models , the rr attributable to microalbuminuria remained practically unchanged and both microalbuminuria ( 2.13 [ 1.094.17 ] ) and c - ad total score ( 1.76 [ 1.052.94 ] ) were independently associated with cardiovascular mortality ( model 4 ) .", "association of microalbuminuria or c - ad with cardiovascular and all - cause mortality * model 1 : univariate analysis ; model 2 : adjusted for sex , age , and gts ; model 3 : model 2 plus adjustments for hypertension , whr , estimated glomerular filtration rate , ldl , hdl , triglycerides , smoking and prior cvd ; model 4 : model 3 , with additional adjustment for , respectively , c - ad or microalbuminuria .", "rr of microalbuminuria vs. normoalbuminuria or per sd ( = 0.605 ) increase in c - ad total score .", "kaplan - meier survival plots for cardiovascular mortality . among individuals with normo- versus microalbuminuria ( a ) and across tertiles of c - ad total score ( b ) . both microalbuminuria and c - ad total score", "were also associated with all - cause mortality ( table 3 , model 1 ) . however , after adjustment for age , sex , gts , and other cardiovascular risk factors , microalbuminuria ( rr 1.33 [ 95% ci 0.832.13 ] ) , in contrast to c - ad total score ( 1.52 [ 1.112.09 ] ) , was not independently associated with all - cause mortality ( model 3 ) ."], ["the associations described above were also investigated by examining autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent ( supplementary table a2 , available in an online appendix ) .", "these additional analyses showed that overall all different groups of tests were associated with increased mortality risk , that these associations were attenuated when further adjusted for age , sex , gts , and other risk factors ; and that none of the specific group of tests explained the association between microalbuminuria and mortality .", "the associations reported in table 3 did not materially change after additional adjustment for use of lipid- or blood pressure  lowering drugs and the presence of leukocytes in urine ( tested by microscopy and scored as positive whenever there were more than five leukocytes per high - power field ) or when the dichotomous variable hypertension used in those models was replaced by either sbp or pulse pressure ( a marker of arterial stiffness ) or when analyses were adjusted for prior coronary heart disease instead of prior cvd ( data not shown ) .", "finally , we found no evidence of effect modification by gts in the association between cardiovascular mortality and c - ad total score or microalbuminuria ( pinteraction > 0.4 in both cases ) ."], ["in this prospective study we found that both microalbuminuria and c - ad ( estimated from the mean of 10 standardized tests ) were independently associated with cardiovascular mortality in a cohort of elderly , caucasian subjects with and without diabetes . therefore , c", "- ad does not explain why microalbuminuria is related to cardiovascular mortality in either the general population or in diabetic subjects .", "recently , hrv was found not to predict the decline of gfr but was independently associated with cardiovascular mortality in middle - aged type 1 diabetic subjects with overt nephropathy ( 17 ) . in another study ,", "investigating the independent predictive role of gfr , microalbuminuria , and cardiovascular autonomic neuropathy showed the latter to also be associated with all - cause and cardiovascular mortality in subjects with diabetes ( 18 ) .", "hitherto , no other prospective studies examining the relation of microalbuminuria and c - ad with cardiovascular mortality have been reported .", "the independent association of both microalbuminuria and c - ad with cardiovascular mortality reported herein indicates that they are linked to cardiovascular mortality by different biological pathways .", "several mechanisms linking c - ad to cardiovascular mortality have been proposed ( e.g. , qt interval prolongation and silent myocardial ischemia ) ( 19 ) . microalbuminuria may be linked to cvd by a common pathophysiological process ( such as endothelial dysfunction or chronic low - grade inflammation ) ( 3 ) .", "however , the possibility that microalbuminuria reflects the presence of a set of undiscovered factors that are causally related to cvd remains to be further elucidated .", "furthermore , aggressive treatment of microalbuminuria by inhibiting the renin - angiotensin - aldosterone system has been shown to decrease renal and cardiovascular risk in individuals with diabetes ( 20 ) .", "there is some evidence that c - ad is also a useful risk indicator for cvd , especially in subjects at high risk ( type 2 diabetes , hypertension , or prior cvd ) ( 9,21 ) , and this study reinforces that observation .", "however , whether the improvement in c - ad , e.g. , with exercise training or blood pressure or lipid - lowering drugs ( 2224 ) , leads to a better prognosis is unknown . in our study ,", "additional adjustment for use of -blockers or lipid - lowering drugs had no effect on the associations reported .", "therefore , the clinical relevance of treating c - ad in the general population remains unclear and needs to be further examined .", "the strengths of our study are the relatively large , truly population - based and prospective design with a long follow - up period .", "in addition , we characterized subjects ' c - ad as comprehensively as possible by conducting 10 autonomic function tests , which are thought to reflect all aspects of cardiovascular autonomic function .", "previous cross - sectional studies evaluating the relationship between microalbuminuria and c - ad used less extensive autonomic function tests ( 4,6 ) . because we included all three groups of gts", ", we were able to analyze possible effect modification of gts in the relationship between the main determinants , c - ad and microalbuminuria and cardiovascular mortality ( but found none ) .", "first , each of the test parameters of autonomic function was measured with moderate levels of reproducibility ( reliability coefficient 50% ) ( 13 ) .", "therefore , the results were combined into a c - ad total score , which enabled us to circumvent , at least partially , this problem .", "specifically , the results herein reported with the c - ad total score were more powerful than results obtained on the basis of each individual test ( separately ) , because these results would be more strongly affected by misclassification ( likely to be random ) , causing an underestimation of the strength of the associations .", "indeed , the associations between each individual test result with cardiovascular mortality were in accordance , although with lower strength , with those as reported herein with the c - ad total score ( data not shown ) .", "in addition , this approach had the advantage of circumventing the problem of multiple testing ( type i errors ) that would have occurred when the associations with each test were analyzed separately . however , because each test may represent different parts of autonomic function , some overlapping but others complementary , our c - ad total score does not allow the distinction of potential different contributions of sympathetic and parasympathetic functions to cardiovascular mortality .", "therefore , we examined the autonomic function tests combined on the basis of their methodology or on the part of the autonomic nervous system they predominantly represent in the additional analyses .", "the results of these alternative combinations of autonomic function tests were comparable to those of the c - ad total score ( supplementary table a2 ) , but we acknowledge that these analyses may not have been specific enough , as most authors agree that a clear distinction between parasympathetic or sympathetic dysfunction can not be made on the basis of these tests .", "altogether , we feel that the advantages ( i.e. , reduced misclassification and avoidance of type i errors without impairment of etiological validity ) of the approach used by us outweigh the disadvantages .", "measurement of hrv during a longer period may be more reliable , although measurements obtained in time periods as short as 2 min correlate highly with 24-h measurements ( 25 ) .", "third , evaluation of subjects ' c - ad was conducted at baseline only , and , therefore , we were not able to evaluate deterioration of autonomic function during follow - up .", "fourth , for most participants microalbuminuria was defined on an acr measured in one urine sample only .", "because microalbuminuria is quite variable from day to day we can not fully exclude the possibility that some subjects were misclassified .", "however , we tried to avoid this problem by collecting urine overnight and expressing albumin excretion as acr .", "in addition , analyses in the 154 participants for whom two samples were available yielded essentially similar results ( data not shown ) .", "finally , we studied an elderly , caucasian population , and it is not known whether our results can be generalized to other ethnic groups or to younger individuals . in summary", ", we have shown that both microalbuminuria and c - ad are independently associated with cardiovascular mortality in an elderly , caucasian population of individuals with normal glucose metabolism , igm , and diabetes .", "these results suggest that microalbuminuria and c - ad are related to cardiovascular mortality by different biological pathways .", "therefore , it may be useful to treat not only microalbuminuria but also c - ad in populations at high risk of cardiovascular mortality ."], [""]]}
{"article_id": "PMC4453897", "article_text": ["cervical cancer is the third most common cancer after breast and colorectal cancer and the fourth leading cause of death among women in malaysia(1 - 3 ) .", "it remains to be one of the major cancers that burden worldwide particularly in under - developed and developing countries ( cervical cancer incidence rate increased with age after 30 years and has its peak at ages 65 - 69 years . according to ethnicity in malaysia , women of indian ethnic group have the highest incidence for cervical cancer followed by chinese and malay ( 3 ) .", "majority malays are muslims , thus lower incidence of this cancer was observed . in iran ,", "there are almost no extramarital sexual relations and they depend strongly on family - based traditions ( 7 )", "the five - year survival exceeded 70% in korea ( 810 ) , 55% in turkey ( 11 ) and it can be as low as 17% which was in uganda ( 12 ) .", "there were various prognostic factors established in many studies such as stage , age , lymph node involvement and tumor size ( 1316 ) . in kentucky , between january 2001 and may 2010 ,", "a study of 381 cervical cancer patients who were referred to tertiary care centre found that stage of disease was a significant prognostic factor for overall survival ( 17 ) .", "meanwhile , a study of 44,182 patients diagnosed with cervical cancer between 1993 and 2002 in korea , found that both stage and histological type of cervical cancer were important factors for survival ( 18 ) . to our knowledge ,", "the analysis from published studies was often limited to the frequently used cox proportional hazard regression model when examining the relationship of the survival distribution to covariates ( 17 , 1921 ) .", "this is perhaps due to the fact that although baseline hazard is not specified in cox model , the parameter can still be estimated .", "the objective of this study was to determine the effects of explanatory variables on the survival of cervical cancer patients using parametric regression model . in some cases ,", "parametric models are more informative than the cox model such as the baseline hazard and survival estimates are known ( can be estimated ) .", "several examples of parametric models can be found in the following studies ( 2227 ) .", "zhu et al . have compared the cox and weibull model in modeling the gastric cancer data and found that the weibull model gave more a precise results in than the cox model ( 28 ) .", "the weibull model has gained popularity in modeling survival data due to its flexibility in the shape parameter that can accommodate a decreasing , constant and increasing hazard .", "furthermore , the suitability of the weibull model can be easily assessed using a log - cumulative hazard plot . in this study ,", "a stratified weibull model was used since there was a time - dependent covariate that caused the proportional hazard assumption vio - lated .", "the study design was retrospective in nature in which patients records were reviewed retrospectively to obtain the data .", "the husm , located in kubang kerian , kelantan has been long regarded as the referral centre for the east coast region of malaysia .", "the inclusion criteria were histopathologically and clinically diagnosed with cancer of cervix between 1 july 1995 and 30 june 2007 , and received at least one treatment related to cervical cancer in husm .", "patients who were died due to other competing causes of death ( not cervical cancer ) , or with incomplete data were excluded from this study .", "ethical clearance was obtained from research and ethics committee of university sains malaysia ( reference number : usmkk / ppp / jepem [ 205.4 ( 2.4 ) ] ) .", "the variable of interest was time ( in months ) which was measured from the patient was diagnosed with cervical cancer up to the time of death .", "factors that were considered in the analysis were ethnicity ( non malay , malay ) , lymph node involvement ( negative , positive ) , metastasis ( with metastasis , without metastasis ) , histology ( squamous cell carcinoma , adeno cell carcinoma ) , primary treatment ( surgery , radiotherapy and/or chemotherapy ) , age at diagnosis ( < 40 , 40  59 ,  60 ) and stage ( stage i & ii , stage iii & iv ) .", "international federation of gynecology and obstetrics ( figo ) system was used in staging patients with cervical cancer .", "data analysis was performed using the statistical package tibco spotfire s - plus version 8.1 .", "the suitability of the weibull model for the data was assessed using a plot of the log of the negative log of the estimated survivor function against log time or log - cumulative hazard plot .", "univariate analysis was conducted using the simple weibull regression analysis to identify the possible prognostic factors individually .", "significant factors from the univariate analysis were further analyzed by the weibull multivariate analysis to model the prognostic factors .", "model selection procedure was based on the forward variable selection method with statistical significance set at  = 0.10 .", "the proportionality of the hazards was assessed using the test based on schoenfeld ( 29 ) .", "as the proportional hazard assumption was not satisfied , a stratified model was used instead ( 30 ) and in measuring the goodness of fit of the model , deviance residuals were used .", "1 shows a study flow diagram which summarizes the steps of the statistical analysis performed . the study flow diagram", "the log - cumulative hazard plotshows a straight line which suggests that the distribution of survival time considered follows weibull distribution ( fig .", "patients characteristics and the incidence rate ratio were tabulated in table 1 . about 74% of total patients", "the ratio of the risk of death for patients who were diagnosed in stage iii & iv compared to the risk of death for patients in group stage i & ii was 1.44 .", "meanwhile , the risk of dying of patients in with metastasis group was 1.55 , greater than patients who had no metastasis .", "the results from the univariate analysis are presented in table 2 . from this analysis ,", "based on the forward selection procedure in multivariate analysis , two variables namely stage and metastasis was found to be significant factors ( table 3 ) .", "however , it was noted that metastasis ( p=0.032 ) did not satisfy the proportional hazard assumption and a stratified model was then considered .", "it is worthwhile to note that while the stratification allows separate baseline hazard function to each metastasis group , the coefficient of the predictor variables are assumed to be the same within each metastasis group ( 29 ) .", "the first stratum consists of patients without metastasis and the second stratum were patients who do have metastasis .", "parameter estimates of the stratified model were performed and the output is given in table 4 .", "the log - cumulative hazard plot characteristics of patients treated in husm ( n=120 ) univariate analysis of weibull model with prognostic factors multivariate analysis of weibull model with prognostic factors output of weibull model under stratification the hazard ratio for variable stage stratified on metastasis the estimated scale parameters (  ) for the first and second stratum were 1 = 1.11 and 2 = 0.733 respectively .", "the risks of death for cervical cancer patients were estimated by finding the regression coefficient ( j ) \n j=j , \n and the hazard ratio ( j ) \n j = exp(j ) where j = 1,2 denotes the stratum .", "for the first stratum , patients who were diagnosed at stage iii & iv are at 2.30 times the risk of death as those in stage i & ii . meanwhile , for the second stratum , patients in stage iii & iv group were more likely to die ( 3.53 times ) than patients in stage i & ii .", "the index plot of deviance residuals ( rdi ) was performed to identify the presence of subjects who was poorly predicted by the model ( 31 ) .", "3 shows that the residuals are roughly symmetrically distributed around zero and most of the residuals are between -2 to 2 .", "outliers and influential observations are not observed in the plot thus implying a good fit of the model .", "in this study , the weibull model was used instead of the cox model because the log - cumulative hazard plot has confirmed that the survival time followed the weibull distribution . multivariate analysis through forward selection method found that the most feasible model to describe the survival of cervical cancer patients was dependent upon the covariates namely , stage and metastasis .", "as the metastasis variable did not meet the proportional hazard assumption , the stratified weibull model was applied .", "this study found that patients who were diagnosed at stage iii & iv have greater risk of death compared to those who were diagnosed at early stage , stage i & ii for both stratum , with and without metastasis .", "it is worthwhile to note that a study of 515 cervical cancer patients by dueas - gonzlez et al", ". showed a significant result for advance stage iii & iv with adjusted hazard ratio of 1.54 ( 95% ci= 1.11- 2.14 ) ( 16 ) , indicating that patients with advanced stage of disease had a 54% higher risk of progression or death at any time than earlier stage patients .", "after applying the stratified model , this study found that the prognosis of cervical cancer patients was dependent upon the stage at diagnosis .", "these findings provide useful knowledge on the understanding of the survival of cervical cancer patients .", "besides , this study also demonstrates the solution to time - dependent covariates problem , or when the proportional hazards assumption is violated .", "cervical cancer dataset that comprise of the national data may be obtained to give a more general prognosis and a better description of the survival pattern of all cervical cancer patients in malaysia .", "ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors ."], "abstract_text": ["<S> abstractbackgroundcervical cancer is the third most common cancer among women in malaysia . </S>", "<S> the objective of this study was to estimate the effect of explanatory variables on survival time of cervical cancer patients receiving treatment at a hospital in malaysia.methodsin this retrospective record review study , cervical cancer data obtained from hospital universitisains malaysia ( husm ) was analysed . </S>", "<S> the data comprises of 120 patients who had been diagnosed as cervical cancer between 1st july 1995 and 30th june 2007 , and obtained treatment from the hospital . the outcome variable was survival time ( in months ) from cervical cancer diagnosis to death . </S>", "<S> a stratified weibull model was applied to study the effect of explanatory variable on survival time when there was time - dependent covariate in the model.resultsstage of disease and metastases were important prognostic variables . </S>", "<S> however , metastasis had been stratified because this variable did not satisfy the proportional hazard assumption . in without metastasis stratum , patients who were diagnosed at stage iii & iv are at 2.30 times the risk of death as those in stage i & ii . meanwhile , in with metastasis stratum , patients in stage iii & iv group had 3.53 times the hazard faced by patients in stage i & ii.conclusionthe prognosis of cervical cancer patients was dependent upon the stage at diagnosis , after the stratification of the metastasis variable . a poorer prognosis on survival </S>", "<S> was observed for patients in stage iii & iv than those in stage i & ii . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion", "Ethical considerations"], "sections": [["cervical cancer is the third most common cancer after breast and colorectal cancer and the fourth leading cause of death among women in malaysia(1 - 3 ) .", "it remains to be one of the major cancers that burden worldwide particularly in under - developed and developing countries ( cervical cancer incidence rate increased with age after 30 years and has its peak at ages 65 - 69 years . according to ethnicity in malaysia , women of indian ethnic group have the highest incidence for cervical cancer followed by chinese and malay ( 3 ) .", "majority malays are muslims , thus lower incidence of this cancer was observed . in iran ,", "there are almost no extramarital sexual relations and they depend strongly on family - based traditions ( 7 )", "the five - year survival exceeded 70% in korea ( 810 ) , 55% in turkey ( 11 ) and it can be as low as 17% which was in uganda ( 12 ) .", "there were various prognostic factors established in many studies such as stage , age , lymph node involvement and tumor size ( 1316 ) . in kentucky , between january 2001 and may 2010 ,", "a study of 381 cervical cancer patients who were referred to tertiary care centre found that stage of disease was a significant prognostic factor for overall survival ( 17 ) .", "meanwhile , a study of 44,182 patients diagnosed with cervical cancer between 1993 and 2002 in korea , found that both stage and histological type of cervical cancer were important factors for survival ( 18 ) . to our knowledge ,", "the analysis from published studies was often limited to the frequently used cox proportional hazard regression model when examining the relationship of the survival distribution to covariates ( 17 , 1921 ) .", "this is perhaps due to the fact that although baseline hazard is not specified in cox model , the parameter can still be estimated .", "the objective of this study was to determine the effects of explanatory variables on the survival of cervical cancer patients using parametric regression model . in some cases ,", "parametric models are more informative than the cox model such as the baseline hazard and survival estimates are known ( can be estimated ) .", "several examples of parametric models can be found in the following studies ( 2227 ) .", "zhu et al . have compared the cox and weibull model in modeling the gastric cancer data and found that the weibull model gave more a precise results in than the cox model ( 28 ) .", "the weibull model has gained popularity in modeling survival data due to its flexibility in the shape parameter that can accommodate a decreasing , constant and increasing hazard .", "furthermore , the suitability of the weibull model can be easily assessed using a log - cumulative hazard plot . in this study ,", "a stratified weibull model was used since there was a time - dependent covariate that caused the proportional hazard assumption vio - lated ."], ["the study design was retrospective in nature in which patients records were reviewed retrospectively to obtain the data .", "the husm , located in kubang kerian , kelantan has been long regarded as the referral centre for the east coast region of malaysia .", "the inclusion criteria were histopathologically and clinically diagnosed with cancer of cervix between 1 july 1995 and 30 june 2007 , and received at least one treatment related to cervical cancer in husm .", "patients who were died due to other competing causes of death ( not cervical cancer ) , or with incomplete data were excluded from this study .", "ethical clearance was obtained from research and ethics committee of university sains malaysia ( reference number : usmkk / ppp / jepem [ 205.4 ( 2.4 ) ] ) .", "the variable of interest was time ( in months ) which was measured from the patient was diagnosed with cervical cancer up to the time of death .", "factors that were considered in the analysis were ethnicity ( non malay , malay ) , lymph node involvement ( negative , positive ) , metastasis ( with metastasis , without metastasis ) , histology ( squamous cell carcinoma , adeno cell carcinoma ) , primary treatment ( surgery , radiotherapy and/or chemotherapy ) , age at diagnosis ( < 40 , 40  59 ,  60 ) and stage ( stage i & ii , stage iii & iv ) .", "international federation of gynecology and obstetrics ( figo ) system was used in staging patients with cervical cancer .", "data analysis was performed using the statistical package tibco spotfire s - plus version 8.1 .", "the suitability of the weibull model for the data was assessed using a plot of the log of the negative log of the estimated survivor function against log time or log - cumulative hazard plot .", "univariate analysis was conducted using the simple weibull regression analysis to identify the possible prognostic factors individually .", "significant factors from the univariate analysis were further analyzed by the weibull multivariate analysis to model the prognostic factors .", "model selection procedure was based on the forward variable selection method with statistical significance set at  = 0.10 .", "the proportionality of the hazards was assessed using the test based on schoenfeld ( 29 ) .", "as the proportional hazard assumption was not satisfied , a stratified model was used instead ( 30 ) and in measuring the goodness of fit of the model , deviance residuals were used .", "1 shows a study flow diagram which summarizes the steps of the statistical analysis performed . the study flow diagram"], ["the log - cumulative hazard plotshows a straight line which suggests that the distribution of survival time considered follows weibull distribution ( fig .", "patients characteristics and the incidence rate ratio were tabulated in table 1 . about 74% of total patients", "the ratio of the risk of death for patients who were diagnosed in stage iii & iv compared to the risk of death for patients in group stage i & ii was 1.44 .", "meanwhile , the risk of dying of patients in with metastasis group was 1.55 , greater than patients who had no metastasis .", "the results from the univariate analysis are presented in table 2 . from this analysis ,", "based on the forward selection procedure in multivariate analysis , two variables namely stage and metastasis was found to be significant factors ( table 3 ) .", "however , it was noted that metastasis ( p=0.032 ) did not satisfy the proportional hazard assumption and a stratified model was then considered .", "it is worthwhile to note that while the stratification allows separate baseline hazard function to each metastasis group , the coefficient of the predictor variables are assumed to be the same within each metastasis group ( 29 ) .", "the first stratum consists of patients without metastasis and the second stratum were patients who do have metastasis .", "parameter estimates of the stratified model were performed and the output is given in table 4 .", "the log - cumulative hazard plot characteristics of patients treated in husm ( n=120 ) univariate analysis of weibull model with prognostic factors multivariate analysis of weibull model with prognostic factors output of weibull model under stratification the hazard ratio for variable stage stratified on metastasis the estimated scale parameters (  ) for the first and second stratum were 1 = 1.11 and 2 = 0.733 respectively .", "the risks of death for cervical cancer patients were estimated by finding the regression coefficient ( j ) \n j=j , \n and the hazard ratio ( j ) \n j = exp(j ) where j = 1,2 denotes the stratum .", "for the first stratum , patients who were diagnosed at stage iii & iv are at 2.30 times the risk of death as those in stage i & ii . meanwhile , for the second stratum , patients in stage iii & iv group were more likely to die ( 3.53 times ) than patients in stage i & ii .", "the index plot of deviance residuals ( rdi ) was performed to identify the presence of subjects who was poorly predicted by the model ( 31 ) .", "3 shows that the residuals are roughly symmetrically distributed around zero and most of the residuals are between -2 to 2 .", "outliers and influential observations are not observed in the plot thus implying a good fit of the model ."], ["in this study , the weibull model was used instead of the cox model because the log - cumulative hazard plot has confirmed that the survival time followed the weibull distribution . multivariate analysis through forward selection method found that the most feasible model to describe the survival of cervical cancer patients was dependent upon the covariates namely , stage and metastasis .", "as the metastasis variable did not meet the proportional hazard assumption , the stratified weibull model was applied .", "this study found that patients who were diagnosed at stage iii & iv have greater risk of death compared to those who were diagnosed at early stage , stage i & ii for both stratum , with and without metastasis .", "it is worthwhile to note that a study of 515 cervical cancer patients by dueas - gonzlez et al", ". showed a significant result for advance stage iii & iv with adjusted hazard ratio of 1.54 ( 95% ci= 1.11- 2.14 ) ( 16 ) , indicating that patients with advanced stage of disease had a 54% higher risk of progression or death at any time than earlier stage patients ."], ["after applying the stratified model , this study found that the prognosis of cervical cancer patients was dependent upon the stage at diagnosis .", "these findings provide useful knowledge on the understanding of the survival of cervical cancer patients .", "besides , this study also demonstrates the solution to time - dependent covariates problem , or when the proportional hazards assumption is violated .", "cervical cancer dataset that comprise of the national data may be obtained to give a more general prognosis and a better description of the survival pattern of all cervical cancer patients in malaysia ."], ["ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors ."]]}
{"article_id": "PMC2798785", "article_text": ["according to the european society of endodontology ( 1994 ) , the assessment of endodontic treatment requires clinical as well as radiological follow - ups at regular intervals .", "the radiographic evidence of success is the presence of a normal periodontal ligament space around the root .", "if radiographs reveal that a lesion has remained the same or has only diminished in size , the treatment is not considered a success.1,2 it is generally accepted that the outcome of endodontic treatment is positively correlated with the technical quality of the root filling , expected to provide an hermetic seal against bacterial ingress.3,4 however , it has been suggested that the quality of the coronal restoration may also have an impact on the periapical health of root - filled teeth;5,6 when the restoration quality is good , this may allow for a favorable outcome even when the root filling quality is poor.7 attention has been focused on the prevalence and the technical quality of root fillings through the evaluation of intraoral810 or panoramic radiographs.2,11,12 epidemiologic studies have been performed on the root canal morphology in turkish populations.13,14 information about the prevalence and technical standard of root - canal treatment , and the occurence of periapical lesions in turkey are scarce.12,15 moreover , there is only one investigation about quality of root fillings and coronal restorations in turkish population.16 the aim of the present study was to relate the quality of root canal treatment and coronal restorations to the periapical status of root filled teeth in adult turkish population based on radiographic examination .", "the sample consisted of 400 subjects , aged 38.7013.80 years , 138 males ( 34.5% ) and 262 females ( 65.5% ) , presenting consecutively as new patients seeking routine dental care in department of oral diagnosis and radiology , faculty of dentistry , marmara university , istanbul , turkey , between 2005 and 2006 .", "the criteria for inclusion in the study were that the patients should be attending for the first time .", "patients younger than 20 years and patients having less than eight remaining teeth were excluded . all the patients gave written informed consent for the study .", "all participants underwent a panoramic radiograph and additional periapical radiographs of endodontically treated teeth were processed .", "all panoramic and periapical radiographs were taken with a veraviewpocs hi - sped ( j. morita mfg .", "kyoto , japan ) and evolution x 3000 - 2c x - ray unit ( new life radiology srl , italy ) , respectively .", "two radiographers using the long - cone paralleling technique , setting of 70 kv , 10 ma , a film - focus distance of 30 cm took all periapical radiographs .", ", kg , bissingen , germany ) , kodak medical x - ray processor ( eastman kodak company , rochester , ny , usa ) , general rapid medical developer and fixer ( general a. . ,", "the viewing conditions were standardized , using a view - box with fixed light intensity .", "the incidence of root fillings was recorded along with the periapical status of all teeth with the exception of third molars .", "adequate if the root filling was 2 mm or less from the radiographic apex , when it was more than 2 mm from the apex , it was considered as an ", "underfilling and when excess filling was beyond the radiographic apex , it was categorized as an ", "adequate if the radiodensity of fill was uniform and appeared to be radiographically adapted to the root - canal walls", " periapical index ( pai ) score ( orstavik et al18 ) , recorded for each one of the roots .", "pai scores 1 and 2 signified a nondiseased apical periodontium , and pai scores 3 , 4 , and 5 signified the presence of an apical radiolucency .", "the worst score of all roots was taken to represent pai score for multirooted teeth . in case of a root filling", "was present , the type of coronal restoration ( crown or filling ) was recorded .", "the quality of the restoration was assessed radiographically and classified as inadequate if the restoration was absent or if open margins , overhangs or secondary caries were detectable .", "evaluation of the periapical status and the quality of root filling in the present study was made independently by two examiners ( fnp , bg ) using the same examination procedure after a calibration session . for calibration 20", "radiographs were used and not included in main study . the cohen s kappa value for inter - examiner agreement of pai - values for all scored teeth was 0.81 .", "the inter - examiner agreement scores gave cohen s kappa of 0.68 for quality of filling and crown restoration , 0.83 for adaptation of fillings to canal walls , and 0.86 for length of root fillings .", "the data were analyzed with spss ( statistical package for social sciences ) for windows 10.0 statistical package software .", "descriptive statistical methods ( mean and standard deviation ) were used for the evaluation of the data .", "p values of less than .05 were interpreted as significant , and the level in confidence intervals was 95% .", "the sample consisted of 400 subjects , aged 38.7013.80 years , 138 males ( 34.5% ) and 262 females ( 65.5% ) , presenting consecutively as new patients seeking routine dental care in department of oral diagnosis and radiology , faculty of dentistry , marmara university , istanbul , turkey , between 2005 and 2006 .", "the criteria for inclusion in the study were that the patients should be attending for the first time .", "patients younger than 20 years and patients having less than eight remaining teeth were excluded . all the patients gave written informed consent for the study .", "all participants underwent a panoramic radiograph and additional periapical radiographs of endodontically treated teeth were processed .", "all panoramic and periapical radiographs were taken with a veraviewpocs hi - sped ( j. morita mfg .", "kyoto , japan ) and evolution x 3000 - 2c x - ray unit ( new life radiology srl , italy ) , respectively .", "two radiographers using the long - cone paralleling technique , setting of 70 kv , 10 ma , a film - focus distance of 30 cm took all periapical radiographs .", ", kg , bissingen , germany ) , kodak medical x - ray processor ( eastman kodak company , rochester , ny , usa ) , general rapid medical developer and fixer ( general a. . , istanbul , turkey ) ) were used in this study .", "the viewing conditions were standardized , using a view - box with fixed light intensity .", "the incidence of root fillings was recorded along with the periapical status of all teeth with the exception of third molars .", "adequate if the root filling was 2 mm or less from the radiographic apex , when it was more than 2 mm from the apex , it was considered as an ", "underfilling and when excess filling was beyond the radiographic apex , it was categorized as an ", "adequate if the radiodensity of fill was uniform and appeared to be radiographically adapted to the root - canal walls .", "periapical index ( pai ) score ( orstavik et al18 ) , recorded for each one of the roots .", "pai scores 1 and 2 signified a nondiseased apical periodontium , and pai scores 3 , 4 , and 5 signified the presence of an apical radiolucency .", "the worst score of all roots was taken to represent pai score for multirooted teeth . in case of a root filling", "was present , the type of coronal restoration ( crown or filling ) was recorded .", "the quality of the restoration was assessed radiographically and classified as inadequate if the restoration was absent or if open margins , overhangs or secondary caries were detectable .", "evaluation of the periapical status and the quality of root filling in the present study was made independently by two examiners ( fnp , bg ) using the same examination procedure after a calibration session . for calibration 20", "the cohen s kappa value for inter - examiner agreement of pai - values for all scored teeth was 0.81 .", "the inter - examiner agreement scores gave cohen s kappa of 0.68 for quality of filling and crown restoration , 0.83 for adaptation of fillings to canal walls , and 0.86 for length of root fillings .", "the data were analyzed with spss ( statistical package for social sciences ) for windows 10.0 statistical package software .", "descriptive statistical methods ( mean and standard deviation ) were used for the evaluation of the data .", "p values of less than .05 were interpreted as significant , and the level in confidence intervals was 95% .", "a total of 9460 teeth were examined , an average number of 23.634.35 remaining teeth per subject .", "the total number of root - filled teeth was 890 ( 9.39% ) ; of which 658 ( 73.9% ) teeth had ap .", "apical periodontitis was found in 75.5% of teeth with adequate length of root filling , whereas if the filling was too short or long , periapical lesions were present in 72.8% and 96.2% of teeth , respectively .", "there was a significant correlation between the presence of periapical pathology and inadequate root canal fillings ( p<.05 ) .", "the relationship between the adaptation of root filling to canal walls and periapical status is shown table 2 .", "root filled teeth without voids had ap in 72.9% of cases , whereas if voids were detected , ap was present in 74.7% of the teeth ( p : 0.54 , odds ratio : 1.09 , ci : 0.811.48 ) .", "the relationship between the quality of the prosthetic restoration and periapical status is presented in table 3 .", "75.9% of the teeth with adequate coronal restorations had ap , whereas if the coronal restoration was inadequate , ap was present in 74.2% of the teeth ( p : 0.70 , odds ratio : 0.91 , ci : 0.581.45 ) .", "the relationship between the quality of the filling restoration and periapical status is presented in table 4 .", "72.8% of the teeth with adequate filling restoration had ap , whereas if the filling restoration was inadequate , ap was present in 72.3% of the teeth ( p : 0.92 , odds ratio : 0.97 , ci : 0.621.54 ) .", "the subjects included in this study were adult patients attending for general dental treatment for the first time .", "the recruitment of subjects was the same as those used by others.8,11,17,19,20 there is no information available for this patient population , which makes it difficult to extrapolate the data obtained into the turkish population .", "however , the dental faculty attracts a patient population from numerous parts of the city and its surroundings , which eliminates the risk of only including patients previously treated by a limited number of practitioners .", "some patients sought care because of the expense of prosthetic treatment , which in general is less at the dental faculty than in the private sector in turkey .", "in addition , extraction of teeth due to low socio - economic status gives rise to a limited number of root filled teeth epidemiologically .", "patients with eight or fewer remaining teeth were excluded because they often had periodontal disease and it was impossible to determine the role played by the endodontic treatment in the occurrence of a radiographic periapical lesion.17 although studies comparing panoramic and periapical radiographs for detecting periapical lesions indicate similar overall diagnostic accuracy,21,22 panoramic radiographs are claimed to show lower sensitivity when detecting periapical lesions of the anterior teeth and mandibular molars compared with periapical radiographs.23 therefore , there is a possibility for loss of information when endodontic conditions are recorded based on panoramic radiographs only in some studies.2,11,17,2426 however , other epidemiological studies have used a combination of panoramic and periapical radiographs.4,8,9,27 as panoramic radiographs do not reveal details and inter - observer variability is greater with panoramic radiographs.28 in this investigation , therefore , all participants underwent a panoramic and additional periapical radiographs of affected teeth were processed .", "criteria for ap vary among studies.2,10,29,30 in recent years , most of the studies on the prevalence of ap have used this index scoring to assess periapical status , so the results of this study can be more appropriately compared with them.4,18,25 therefore , in the present study , the pai was used to assess the periapical status .", "the reproducibility of the observer ( cohen s kappa=0.81 ) was acceptable , probably because of prior calibration .", "a total of 890 teeth had root fillings , accounting for 9.39% of all teeth .", "the prevalence of root - filled teeth in the turkish population was found lower than previously done other studies about different countries ( 34%87%).4,5,9,10,19,27,29 this phenomenon can be explained by the fact that first , the survey population was not representative of the whole country , and secondly , the differences in health care services and socioeconomical factors in the various countries could account for these discrepancies .", "in contrast , other studies found the prevalence of root - filled teeth to range between 1.3% and 4.8% , similar to that found in this study.8,17,3032 the quality of endodontic treatment was disappointing .", "root filling length was found to be adequate in 24.5% of the teeth and the adaptation of fillings to the canal walls in 27.1% of the teeth . in this respect ,", "the data confirm the findings of several other investigators showing poor quality of root canal treatment.8,17,19 apical periodontitis was found in 75.5% of teeth with adequate length of root filling , whereas if the filling was too short or long , periapical lesions were present in 72.8% and 96.2% of teeth , respectively .", "tsuneishi et al29 observed apical radiolucency most often in overfilled teeth ( 79.8% ) , followed by adequately filled teeth ( 41.6% ) and then underfilled teeth ( 34.5% ) .", "siquera et al33 indicated that poor outcome in case of overfillings is usually associated with concurrent endodontic infection , or as a result of previous overinstrumentation of an infected root canal that propelled infected dentin chips to the periradicular tissues or can be due to a lack of apical seal that allows the traffic of tissue fluids into the canal and of microorganisms and their products towards the periradicular tissues .", "in contrast , sunay et al12 reported 90.8% of root - filled teeth with apical periodontitis had inadequate root canal fillings , the majority of them being short of the radiographic apex .", "dugas et al4 reported the prevalence of apical periodontitis close to 60% when the root filling was inadequate , regardless of whether the filling was too short or too long consistent with the observations in other studies.17,19,27 in the present study , the relationship between the type of coronal restoration within root filled teeth and the presence of apical periodontitis were investigated .", "it is indicated that the technical quality of the coronal restoration may be more important for periapical health than the technical quality of the root filling.34 although some researchers found a significant correlation between the radiographic quality of the coronal restoration and the periapical status of root filled teeth.4,5 on the other hand , hommez et al35 reported that the quality of the coronal restoration did not have a significant influence on the periradicular status when it was combined with the quality of endodontic treatment .", "the results of the present study revealed that the quality of the restoration did not significantly affect the treatment outcome .", "in the present study , the frequency of endodontically treated teeth was found lower than other studies .", "the technical quality of root fillings and restoration in a turkish subpopulation was poor and was consistent with a high prevalence of apical periodontitis ."], "abstract_text": ["<S> objectives : to investigate the quality of root fillings and coronal restorations and their association with periapical status in an adult turkish subpopulation.methods:a total of 400 subjects were examined . </S>", "<S> panoramic radiographs of all participants and additional periapical radiographs of affected teeth were processed . </S>", "<S> the frequency of root canal treatment and the periapical status of all root filled teeth were evaluated . </S>", "<S> the relationship between the radiographic quality of root fillings and coronal restorations was examined by chi - squared statistical analysis.results:a total of 9460 teeth were examined . </S>", "<S> the total number of root filled teeth was 890 ( 9.39% ) , and 658 ( 73.9% ) had apical periodontitis ( ap ) . </S>", "<S> there was a significant correlation between the presence of periapical pathology and inadequate root canal fillings ( p<.05 ) . </S>", "<S> presence of ap in root filled teeth was associated with inadequate adaptation of the filling ( or=1.097 ; p=0.54 ) , and with poor radiographic quality of the coronal restoration ( or=0.91 ; p=0.70 ) , and with poor radiographic quality of the filling restoration ( or=0.97 ; p=0.92 ) . </S>", "<S> only 24.5% of the root fillings were adequate . </S>", "<S> the highest percentage of root fillings was found in maxillary right first molars ( 6.9%).conclusions : the results demonstrate a low prevalence of root - filled teeth and poor quality of coronal restorations and root fillings consistent with a high prevalence of apical periodontitis in a turkish subpopulation . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Patient selection", "Radiographic examination", "Radiographic evaluation", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["according to the european society of endodontology ( 1994 ) , the assessment of endodontic treatment requires clinical as well as radiological follow - ups at regular intervals .", "the radiographic evidence of success is the presence of a normal periodontal ligament space around the root .", "if radiographs reveal that a lesion has remained the same or has only diminished in size , the treatment is not considered a success.1,2 it is generally accepted that the outcome of endodontic treatment is positively correlated with the technical quality of the root filling , expected to provide an hermetic seal against bacterial ingress.3,4 however , it has been suggested that the quality of the coronal restoration may also have an impact on the periapical health of root - filled teeth;5,6 when the restoration quality is good , this may allow for a favorable outcome even when the root filling quality is poor.7 attention has been focused on the prevalence and the technical quality of root fillings through the evaluation of intraoral810 or panoramic radiographs.2,11,12 epidemiologic studies have been performed on the root canal morphology in turkish populations.13,14 information about the prevalence and technical standard of root - canal treatment , and the occurence of periapical lesions in turkey are scarce.12,15 moreover , there is only one investigation about quality of root fillings and coronal restorations in turkish population.16 the aim of the present study was to relate the quality of root canal treatment and coronal restorations to the periapical status of root filled teeth in adult turkish population based on radiographic examination ."], ["the sample consisted of 400 subjects , aged 38.7013.80 years , 138 males ( 34.5% ) and 262 females ( 65.5% ) , presenting consecutively as new patients seeking routine dental care in department of oral diagnosis and radiology , faculty of dentistry , marmara university , istanbul , turkey , between 2005 and 2006 .", "the criteria for inclusion in the study were that the patients should be attending for the first time .", "patients younger than 20 years and patients having less than eight remaining teeth were excluded . all the patients gave written informed consent for the study .", "all participants underwent a panoramic radiograph and additional periapical radiographs of endodontically treated teeth were processed .", "all panoramic and periapical radiographs were taken with a veraviewpocs hi - sped ( j. morita mfg .", "kyoto , japan ) and evolution x 3000 - 2c x - ray unit ( new life radiology srl , italy ) , respectively .", "two radiographers using the long - cone paralleling technique , setting of 70 kv , 10 ma , a film - focus distance of 30 cm took all periapical radiographs .", ", kg , bissingen , germany ) , kodak medical x - ray processor ( eastman kodak company , rochester , ny , usa ) , general rapid medical developer and fixer ( general a. . ,", "the viewing conditions were standardized , using a view - box with fixed light intensity .", "the incidence of root fillings was recorded along with the periapical status of all teeth with the exception of third molars .", "adequate if the root filling was 2 mm or less from the radiographic apex , when it was more than 2 mm from the apex , it was considered as an ", "underfilling and when excess filling was beyond the radiographic apex , it was categorized as an ", "adequate if the radiodensity of fill was uniform and appeared to be radiographically adapted to the root - canal walls", " periapical index ( pai ) score ( orstavik et al18 ) , recorded for each one of the roots .", "pai scores 1 and 2 signified a nondiseased apical periodontium , and pai scores 3 , 4 , and 5 signified the presence of an apical radiolucency .", "the worst score of all roots was taken to represent pai score for multirooted teeth . in case of a root filling", "was present , the type of coronal restoration ( crown or filling ) was recorded .", "the quality of the restoration was assessed radiographically and classified as inadequate if the restoration was absent or if open margins , overhangs or secondary caries were detectable .", "evaluation of the periapical status and the quality of root filling in the present study was made independently by two examiners ( fnp , bg ) using the same examination procedure after a calibration session . for calibration 20", "radiographs were used and not included in main study . the cohen s kappa value for inter - examiner agreement of pai - values for all scored teeth was 0.81 .", "the inter - examiner agreement scores gave cohen s kappa of 0.68 for quality of filling and crown restoration , 0.83 for adaptation of fillings to canal walls , and 0.86 for length of root fillings .", "the data were analyzed with spss ( statistical package for social sciences ) for windows 10.0 statistical package software .", "descriptive statistical methods ( mean and standard deviation ) were used for the evaluation of the data .", "p values of less than .05 were interpreted as significant , and the level in confidence intervals was 95% ."], ["the sample consisted of 400 subjects , aged 38.7013.80 years , 138 males ( 34.5% ) and 262 females ( 65.5% ) , presenting consecutively as new patients seeking routine dental care in department of oral diagnosis and radiology , faculty of dentistry , marmara university , istanbul , turkey , between 2005 and 2006 .", "the criteria for inclusion in the study were that the patients should be attending for the first time .", "patients younger than 20 years and patients having less than eight remaining teeth were excluded . all the patients gave written informed consent for the study ."], ["all participants underwent a panoramic radiograph and additional periapical radiographs of endodontically treated teeth were processed .", "all panoramic and periapical radiographs were taken with a veraviewpocs hi - sped ( j. morita mfg .", "kyoto , japan ) and evolution x 3000 - 2c x - ray unit ( new life radiology srl , italy ) , respectively .", "two radiographers using the long - cone paralleling technique , setting of 70 kv , 10 ma , a film - focus distance of 30 cm took all periapical radiographs .", ", kg , bissingen , germany ) , kodak medical x - ray processor ( eastman kodak company , rochester , ny , usa ) , general rapid medical developer and fixer ( general a. . , istanbul , turkey ) ) were used in this study ."], ["the viewing conditions were standardized , using a view - box with fixed light intensity .", "the incidence of root fillings was recorded along with the periapical status of all teeth with the exception of third molars .", "adequate if the root filling was 2 mm or less from the radiographic apex , when it was more than 2 mm from the apex , it was considered as an ", "underfilling and when excess filling was beyond the radiographic apex , it was categorized as an ", "adequate if the radiodensity of fill was uniform and appeared to be radiographically adapted to the root - canal walls .", "periapical index ( pai ) score ( orstavik et al18 ) , recorded for each one of the roots .", "pai scores 1 and 2 signified a nondiseased apical periodontium , and pai scores 3 , 4 , and 5 signified the presence of an apical radiolucency .", "the worst score of all roots was taken to represent pai score for multirooted teeth . in case of a root filling", "was present , the type of coronal restoration ( crown or filling ) was recorded .", "the quality of the restoration was assessed radiographically and classified as inadequate if the restoration was absent or if open margins , overhangs or secondary caries were detectable .", "evaluation of the periapical status and the quality of root filling in the present study was made independently by two examiners ( fnp , bg ) using the same examination procedure after a calibration session . for calibration 20", "the cohen s kappa value for inter - examiner agreement of pai - values for all scored teeth was 0.81 .", "the inter - examiner agreement scores gave cohen s kappa of 0.68 for quality of filling and crown restoration , 0.83 for adaptation of fillings to canal walls , and 0.86 for length of root fillings ."], ["the data were analyzed with spss ( statistical package for social sciences ) for windows 10.0 statistical package software .", "descriptive statistical methods ( mean and standard deviation ) were used for the evaluation of the data .", "p values of less than .05 were interpreted as significant , and the level in confidence intervals was 95% ."], ["a total of 9460 teeth were examined , an average number of 23.634.35 remaining teeth per subject .", "the total number of root - filled teeth was 890 ( 9.39% ) ; of which 658 ( 73.9% ) teeth had ap .", "apical periodontitis was found in 75.5% of teeth with adequate length of root filling , whereas if the filling was too short or long , periapical lesions were present in 72.8% and 96.2% of teeth , respectively .", "there was a significant correlation between the presence of periapical pathology and inadequate root canal fillings ( p<.05 ) .", "the relationship between the adaptation of root filling to canal walls and periapical status is shown table 2 .", "root filled teeth without voids had ap in 72.9% of cases , whereas if voids were detected , ap was present in 74.7% of the teeth ( p : 0.54 , odds ratio : 1.09 , ci : 0.811.48 ) .", "the relationship between the quality of the prosthetic restoration and periapical status is presented in table 3 .", "75.9% of the teeth with adequate coronal restorations had ap , whereas if the coronal restoration was inadequate , ap was present in 74.2% of the teeth ( p : 0.70 , odds ratio : 0.91 , ci : 0.581.45 ) .", "the relationship between the quality of the filling restoration and periapical status is presented in table 4 .", "72.8% of the teeth with adequate filling restoration had ap , whereas if the filling restoration was inadequate , ap was present in 72.3% of the teeth ( p : 0.92 , odds ratio : 0.97 , ci : 0.621.54 ) ."], ["the subjects included in this study were adult patients attending for general dental treatment for the first time .", "the recruitment of subjects was the same as those used by others.8,11,17,19,20 there is no information available for this patient population , which makes it difficult to extrapolate the data obtained into the turkish population .", "however , the dental faculty attracts a patient population from numerous parts of the city and its surroundings , which eliminates the risk of only including patients previously treated by a limited number of practitioners .", "some patients sought care because of the expense of prosthetic treatment , which in general is less at the dental faculty than in the private sector in turkey .", "in addition , extraction of teeth due to low socio - economic status gives rise to a limited number of root filled teeth epidemiologically .", "patients with eight or fewer remaining teeth were excluded because they often had periodontal disease and it was impossible to determine the role played by the endodontic treatment in the occurrence of a radiographic periapical lesion.17 although studies comparing panoramic and periapical radiographs for detecting periapical lesions indicate similar overall diagnostic accuracy,21,22 panoramic radiographs are claimed to show lower sensitivity when detecting periapical lesions of the anterior teeth and mandibular molars compared with periapical radiographs.23 therefore , there is a possibility for loss of information when endodontic conditions are recorded based on panoramic radiographs only in some studies.2,11,17,2426 however , other epidemiological studies have used a combination of panoramic and periapical radiographs.4,8,9,27 as panoramic radiographs do not reveal details and inter - observer variability is greater with panoramic radiographs.28 in this investigation , therefore , all participants underwent a panoramic and additional periapical radiographs of affected teeth were processed .", "criteria for ap vary among studies.2,10,29,30 in recent years , most of the studies on the prevalence of ap have used this index scoring to assess periapical status , so the results of this study can be more appropriately compared with them.4,18,25 therefore , in the present study , the pai was used to assess the periapical status .", "the reproducibility of the observer ( cohen s kappa=0.81 ) was acceptable , probably because of prior calibration .", "a total of 890 teeth had root fillings , accounting for 9.39% of all teeth .", "the prevalence of root - filled teeth in the turkish population was found lower than previously done other studies about different countries ( 34%87%).4,5,9,10,19,27,29 this phenomenon can be explained by the fact that first , the survey population was not representative of the whole country , and secondly , the differences in health care services and socioeconomical factors in the various countries could account for these discrepancies .", "in contrast , other studies found the prevalence of root - filled teeth to range between 1.3% and 4.8% , similar to that found in this study.8,17,3032 the quality of endodontic treatment was disappointing .", "root filling length was found to be adequate in 24.5% of the teeth and the adaptation of fillings to the canal walls in 27.1% of the teeth . in this respect ,", "the data confirm the findings of several other investigators showing poor quality of root canal treatment.8,17,19 apical periodontitis was found in 75.5% of teeth with adequate length of root filling , whereas if the filling was too short or long , periapical lesions were present in 72.8% and 96.2% of teeth , respectively .", "tsuneishi et al29 observed apical radiolucency most often in overfilled teeth ( 79.8% ) , followed by adequately filled teeth ( 41.6% ) and then underfilled teeth ( 34.5% ) .", "siquera et al33 indicated that poor outcome in case of overfillings is usually associated with concurrent endodontic infection , or as a result of previous overinstrumentation of an infected root canal that propelled infected dentin chips to the periradicular tissues or can be due to a lack of apical seal that allows the traffic of tissue fluids into the canal and of microorganisms and their products towards the periradicular tissues .", "in contrast , sunay et al12 reported 90.8% of root - filled teeth with apical periodontitis had inadequate root canal fillings , the majority of them being short of the radiographic apex .", "dugas et al4 reported the prevalence of apical periodontitis close to 60% when the root filling was inadequate , regardless of whether the filling was too short or too long consistent with the observations in other studies.17,19,27 in the present study , the relationship between the type of coronal restoration within root filled teeth and the presence of apical periodontitis were investigated .", "it is indicated that the technical quality of the coronal restoration may be more important for periapical health than the technical quality of the root filling.34 although some researchers found a significant correlation between the radiographic quality of the coronal restoration and the periapical status of root filled teeth.4,5 on the other hand , hommez et al35 reported that the quality of the coronal restoration did not have a significant influence on the periradicular status when it was combined with the quality of endodontic treatment .", "the results of the present study revealed that the quality of the restoration did not significantly affect the treatment outcome ."], ["in the present study , the frequency of endodontically treated teeth was found lower than other studies .", "the technical quality of root fillings and restoration in a turkish subpopulation was poor and was consistent with a high prevalence of apical periodontitis ."]]}
{"article_id": "PMC3433144", "article_text": ["the neighborhood has emerged as a prominent level of analysis in studies of contextual influences on health and wellbeing in human development .", "this owes to the social organization of life within neighborhoods , and to the availability of data at neighborhood - like levels of analysis ( i.e. , census tracts ) .", "neighborhood context has also received greater attention due to ecological psychology 's conceptualization of human development as the product of a dynamic interaction between the individual and multiple nested environments , of which the neighborhood is one of the most immediate environments .", "contemporary scholarship on neighborhood factors has also highlighted the importance of understanding whether neighborhood variables add explanatory variance above and beyond individual differences .  ", "neighborhood socioeconomic status ( ses ) is the most consistently reported neighborhood - level predictor of cognitive outcomes  certainly in childhood , and potentially across the life span .", "ses is also the strongest and most consistently reported neighborhood - level predictor of health outcomes among older adults  a relationship which may have a cumulative effect across the life span .", "the association of neighborhood - level ses with health is in fact stronger among adults aged 6069 than among young and middle - aged adults , and during these years its association with health is comparable to or stronger than the relationship of individual ses to health .", "in addition to predicting poorer physical health , lower neighborhood ses is also associated with reduced rates of physical activity , increased incidence of depression and psychosocial stress , and less neighborhood - based social support and social engagement .", "many of these outcomes have also been identified as correlates of late life cognition , yet few studies to date have specifically investigated the role of the neighborhood in cognitive aging .   while neighborhood effects on late life cognition have received less attention , neighborhood effects on early - life cognitive development are relatively well documented .", "neighborhood ses is positively correlated with cognitive development across the entire spectrum of child development , from the prenatal stages ( in the form of reduced rates of congenital anomalies and neural tube defects among higher - ses neighborhoods , presumably due to reduced risk of exposure to environmental toxins ; [ 6 , 7 ] ) and preschool years [ 8 , 9 ] to academic achievement in the school age [ 10 , 11 ] adolescent , and young adulthood years ( e.g. , high school graduation rates and college attendance ) .   four recent studies explored relationships between late life cognition and the neighborhood context on a broad scale .", "wight and colleagues   found that after controlling for individual - level education and area - level median household income , elders living in areas with low neighborhood - level educational attainment ( defined by census tract area ) achieved lower cognitive status scores compared to elders living in areas with high neighborhood - level educational attainment ( as assessed using the mini - mental state examination or mmse ) .", "a united kingdom study   reported a clear and significant downward trend in cognitive performance on the mmse and tests of verbal fluency and memory for older individuals living in neighborhoods with greater deprivation , after controlling for individual - level wealth , income , and education , across all age and gender groups .", "these effects were also robust after adjusting for the effects of individual systolic blood pressure , history of stroke , and duration of residence in the current neighborhood .", "sheffield and peek   moved beyond cross - sectional analysis to examine how neighborhood ses and ethnic composition ( proportion mexican american ) predicted 5-year change in mmse score in a us national sample .", "independent of individual - level risk factors , odds and rate of incident cognitive decline increased as a function of lower neighborhood ses and decreased with the proportion of mexican american neighborhood residents .", "finally , recent findings from the baltimore memory study suggest that the relationship between neighborhood and late life cognition may also depend on genotype .", "however , apolipoprotein e4 genotype was found to interact with high neighborhood psychosocial hazards , resulting in poorer cognitive performance on measures of processing speed and executive functioning after controlling for individual - level covariates .", "this suggests evidence of a gene - environment interaction in neighborhood 's relation to late life cognition .", "the existing studies focused on broad differences in late life cognitive status ( i.e. , mmse score ) , but did not investigate the effect of neighborhood on different cognitive domains ( see recommendations by cullum et al . ) .", "furthermore , the use of the mmse in these studies was not an ideal measure of cognition , as they targeted samples of initially healthy , independent elders .", "the mmse is not sensitive to differences among healthy older adults , having been designed to screen for dementia .", "cognitive aging research has instead focused increasingly on the use of confirmatory factor analysis ( cfa ) and structural equation modeling ( sem ) to explore whether differences in late life cognitive performance are related to general or specific processes .", "it has also been noted that conceptualizing late life cognition using a single general factor (  g  ) is a simplistic approach to a complex and dynamic cognitive system .", "this suggests neighborhood influences should be investigated at both general and domain - specific levels .", "neighborhood effects may support some dimensions of cognition more strongly than others , in addition to influencing general cognitive status .   the present study aimed to identify whether neighborhood effects on cognition were global , or specifically related to particular cognitive domains such as memory , reasoning , processing speed , everyday cognition , or vocabulary .", "another innovation of the present study is the capacity to address whether neighborhood effects might also influence the magnitude of potential benefit from cognitive intervention .", "no study , in part because of a lack of data availability , has examined this question . elucidating both the specificity of neighborhood effects on particular domains of cognition and", "the extent to which neighborhood affects cognitive training response may provide additional hints toward potential mechanisms by which the neighborhood exerts influence .", "for example , effects on the fluid cognitive abilities such as processing speed , memory , and reasoning ( and their response to cognitive training ) would suggest a more biologically mediated influence given their sensitivity to brain integrity .", "in contrast , effects on measures of crystallized cognitive abilities , especially vocabulary , might hint toward a more socioculturally mediated , and possibly lifespan accumulative influence , as crystallized cognitive abilities are developed with the accumulation of verbal knowledge , developed over a lifetime of experience in engaging with culture . as this lifelong accumulation", "is strongly influenced during childhood , adolescence , and young adulthood , effects of current neighborhood sep on crystallized abilities may also reflect , in part , effects of early - life neighborhood sep .", "the advanced cognitive training for independent and vital elderly ( active ) study was a randomized , controlled clinical trial examining the effects of three cognitive interventions for community - dwelling older adults .", "in active , older adults from six us catchment areas ( baltimore / coastal maryland ; the metropolitan areas of birmingham , boston , detroit , and indianapolis ; central pennsylvania ) completed a baseline assessment including multiple cognitive measures .", "participants were then randomized to one of three ten - session cognitive intervention programs or a no - contact control condition .  ", "the present study sought to examine whether neighborhood socioeconomic position ( sep ) shows bivariate relationships with baseline cognitive level and immediate response to cognitive training , beyond individual - level predictors of cognition .", "if so , we aimed to discern whether neighborhood effects were significant for general cognitive ability (  g  ) , and whether effects were differential for specific cognitive abilities ( memory , reasoning , speed , everyday cognition , and vocabulary ) .", "we hypothesized that the potential effect of neighborhood sep may most likely occur by way of sociocultural processes , and therefore would have significant positive associations with general cognitive ability and with crystallized cognitive domains , including vocabulary .", "neighborhood sep was hypothesized to have relatively weaker , if detectable , associations with fluid cognitive domains , including memory , reasoning , speed , and everyday cognition .", "the primary objective of active was to test the effectiveness and durability of three cognitive interventions .", "the initial trial randomized individuals to one of three 10-session cognitive interventions designed to improve memory , reasoning , or processing speed performance or to a no - contact control condition . following training", "there was an immediate posttest and follow - up assessments at 1- , 2- , 3- , and 5-years after intervention .", "the current study sought to examine only the immediate pretest - posttest data , focusing on the proximal outcome measures used .", "information on potential covariates was collected , including demographic variables , mmse , recent depressive symptoms , and general health .", "testers at all six sites were trained in standardized assessment protocols and quality control by both study investigators , and the coordinating center ensured fidelity to testing procedures .", "the initial sample of 2,802 participants was recruited through on - site presentations , letters to interested persons , newspaper advertisements , introductory letters , and follow - up telephone calls .", "participants were cognitively healthy , community - dwelling older adults aged 65 to 94 years ( mean age = 73.6 years , mean years of education = 13.5 , mean mmse = 27 , 75.8% female , 72% caucasian , 26% african american , and 2% other minority groups ) .", "the majority of participants ( 64% ) were not married , and most reported good to excellent health ( 84.3% ) .", "efforts were aimed at recruiting older adults independent of care at enrollment , as well as recruiting a diverse sample especially inclusive of african americans , who were previously under - represented in most cognitive training research .", "exclusion criteria included ( a ) being under age 65 at the start of the study , ( b ) significant functional and/or cognitive decline at enrollment ( e.g. , impaired activities of daily living , mmse < 23 , and diagnosis of alzheimer 's disease ) , ( c ) having a medical condition disposing the participant to imminent cognitive decline or to mortality within the next 2 years , ( d ) severe sensory or communicative difficulties precluding participation in assessments and training , ( e ) having had cognitive training , or ( f ) planning to be unavailable during the testing and training periods of the study .  ", "active included multiple assessments of participants , but the present paper restricted itself to baseline cognitive performance and the immediate post - test .", "neighborhood - level socioeconomic data from publicly available geographic datasets were then merged with active data at the individual level .", "active did not aim a priori to include multiple measures of individual - level socioeconomic position .", "education ( highest level achieved ) was assessed for all participants , but other person - level socioeconomic indices ( e.g. , income , occupation ) were not collected at baseline ; occupational status was collected at the second annual follow - up occasion .  ", "the cognitive domains measured in actve included memory , inductive reasoning , processing speed , everyday cognition , and vocabulary ; descriptive statistics on the sample performance for measures within each domain are illustrated in table 1 .", "memory was measured using the hopkins verbal learning test ( hvlt , ) , a 12-word list memory task assessing immediate and delayed recall over 3 learning trials , the rey auditory verbal learning test ( avlt , ) , a 15-word list memory task assessing immediate and delayed recall over 5 consecutive learning trials , and the rivermead behavioral memory test , a paragraph recall task assessing immediate verbal episodic memory . reasoning was measured using letter series , a task requiring accurate identification of the next logical letter group in a series of letters ( e.g. , a m b a n b a o b ) , letter sets , an inductive reasoning task requiring participants to identify which of 4 sets of letters is unrelated to the others ( e.g. , eef hhi llm ysy ) , and word series , a task requiring accurate identification of the next logical word in a series .", "processing speed was measured using the useful field of view test ( ufov , ) , a computer - administered task measuring visual sustained , selective , and divided attention through four subtasks , and the complex reaction time test , a computer - administered task measuring the time taken to perform various motor behaviors and to complete each task . everyday cognition was measured using the everyday problems test , a performance - based test of ability to solve everyday problems including medications , nutrition , phone use , shopping , and management of finances and household , the observed tasks of daily living ( otdl ) , a timed task of problem solving in medication use , telephone use , and financial management , and the timed instrumental activities of daily living ( tiadl ) , a timed test of ability to complete daily living tasks ( e.g , find telephone number , make change , find and read ingredients on a can , find food items on a shelf , read instructions on medicine bottle ) .", "vocabulary was measured using a multiple - choice measure of vocabulary attainment in which the participant is presented with a word and must choose one of four possible synonyms .", "geocoding   is a process by which a location ( e.g. , street address ) is assigned cartesian mathematical coordinates , allowing for other levels of geographic information to be linked with that location .", "geolytics , a commercial provider of geocoding services and census demographic data , ( 1 ) geocoded the active participant addresses , and ( 2 ) appended to these addresses data from the 2000 u.s . census and 2002 economic census , creating a dataset that could be used to characterize the neighborhood environment .", "neighborhood research , active participant addresses were geocoded and linked with their associated census tract numbers , which allowed for census tract - level data from the 2000 u.s . census to be appended to the individual - level active data .", "participants receiving mail by post office boxes were excluded as such addresses can not be verified to represent the participant 's actual place of residence .", "addresses with invalid house numbers , street names , and zip codes were flagged for followup ; invalid addresses or poorly matched addresses were dropped from the analysis ( 93% of the active addresses accurately matched to a us geological survey ( usgs ) geocode ; final sample size = 2,521 ) .  ", "census tracts are subdivisions of a county , with an average size of approximately 4,000 residents designed to capture collectively agreed - upon areas approximating neighborhoods .", "that is , the boundaries of census tracts were agreed upon by local officials knowledgeable of the area and were intended to be homogenous with respect to population characteristics , economic status , and living conditions .", "smaller measurement units of area - level ses ( e.g. , block group ) have been shown to correlate highly with census - level measures of ses , and variability in ses among block groups is small relative to variability within their census tract .", "the association of area - level ses with the cognitive measures was tested at three different levels of area measurement ( block group , census tract , and zip code ) , with no significant difference in the strength of associations across levels . because the census tract has traditionally been the most frequently used and most strongly recommended unit of measurement for area - level effects in health research , especially in terms of sep [ 3 , 35 ] , and because it differed very little in model fit or regression coefficients from the other geographic levels , the census tract level of aggregation was selected for all final analyses involving sep .", "neighborhood socioeconomic position was measured by creating a socioeconomic position ( sep ) index . because ses variables such as income , education , and occupation are often strongly correlated and have been found to load onto a common factor at the census level , census - level data on these variables were combined into a weighted factor score to create a neighborhood sep index that parsimoniously represented multiple socioeconomic variables .   the analytical framework for the study involved exploratory and confirmatory factor analysis , structural equation modeling , and repeated measures mixed effects modeling . in keeping with", "best practice  in conducting neighborhood research , individual- and area - level effects would ideally have been examined using a multi - level modeling ( mlm ) approach , with samples of individuals clustered in neighborhoods allowing formal assessment of random variation within neighborhoods .", "however because active was not originally designed to sample data stratified by neighborhood but sampled widely across each region , the insufficient sample size within each neighborhood measured precluded the use of mlm . in keeping with recent scholarship", ", the current study can instead be classified as ecologic , in which neighborhood variables are measured for each person .", "statistical package for the social sciences ( spss ) 18.0 and analysis of moment structures ( amos ) 18.0 were used to conduct all statistical analyses . prior", "to analysis , all variables were inspected for consistency with the assumption of multivariate normality ( to permit maximum likelihood estimation ) .", "where that assumption was not met , blom transformations   were applied to those variables to improve the normality of their distributions . while no data were missing for any neighborhood - level variables , in a few instances data were missing from the active dataset 's cognitive variables ( e.g. , due to failure to complete a task or attrition between the baseline and posttraining testing occasions ) .", "models were estimated using full information maximum likelihood ( fiml ) , which uses all available data ( thereby not eliminating participants with incomplete data ) .  ", "preparatory to examining the relationship of neighborhood sep to active cognitive outcomes , factor analytic constructs representing neighborhood sep and baseline cognition were developed .", "criteria for acceptable model fit indices included root mean square error of approximation ( rmsea ) < 0.06 , comparative fit index ( cfi ) > 0.95 , normed fit index ( nfi ) > 0.95 , and tucker - lewis index ( tli ) > 0.95 .", "exploratory factor analyses were first conducted on the neighborhood sep variables , using promax rotation to allow optimal fit to the data . a weighted composite measure of sep", "was then estimated and optimized in amos based on a common factor identified in the exploratory factor analysis .", "the cognitive measurement model for the present study dimensionalized cognition into several domains , for which the active dataset was well designed , having captured at least three measures each of specific cognitive domains including memory , reasoning , processing speed , and everyday cognition , or measures of cognition related to everyday abilities .", "as stated in the introduction , cognitive aging research has increasingly emphasized conceptualization of cognition as both a general ability (  g  ) , and a multifaceted system of specific cognitive domains which may respond differently to neighborhood effects .", "therefore the model also estimated effects on each of the above cognitive domains , as well as effects on a higher - order factor representing general cognition or ", "g  , which captures the shared variance among the five cognitive domains in the measurement model .", "the sep and cognition factor models were combined in a structural equation model estimating regression paths from sep to the each of the cognitive factors .", "model covariates included age , quadratic age , years of education , gender , and race ( white , african american ) .", "measures of individual wealth and duration of residence in neighborhood were not collected in active , and could not be included as covariates .", "neighborhood sep as a predictor of training gains was estimated as repeated - measures mixed effects models   rather than as structural equation models because this provided a more flexible interface for the analysis of training gains , which was operationalized as a two - occasion difference score ( and thus would not significantly benefit from a latent variable approach ) while still permitting fiml estimation for maximum power in the presence of missing data ( i.e. , using all available cases ) .", "separate models were estimated for each domain trained ( memory , reasoning , or speed ) to examine the gains of each training group relative to other participants who did not receive that training ( i.e. , the other two training groups and the no - contact controls ) .", "each model tested sep as a continuous predictor and occasion ( baseline , posttest ) and training group ( received training on the ability being examined or not ) each as class variables .", "each model also tested all possible two - way interactions and the three - way interaction of sep , occasion , and group .", "interactions were estimated as residualized interaction terms to correct for collinearity with the model 's main effects .", "neighborhood sep was initially developed using exploratory factor analysis ( efa ) to combine 8 indicators of area - level ses : median household income , % households with income $150,000 , % persons in poverty , % with 9th grade education , % with  high school education , % with  bachelor 's degree , % unemployed , and % working in management or higher .", "the efa indicated all 8 variables loaded strongly onto one factor ( eigenvalue = 5.97 ) , explaining 71% of the variance ( factor loadings ranged from 0.71  0.94 ) .", "all indicators were then included in a confirmatory factor analysis , where modification indices ( mi ) suggested high intercorrelations of error variances among variables ( e.g. , ranging up to mi = 1325.42 ) . to derive an optimally - fit , parsimonious set of variables capturing the shared variance of characteristics on which sep is typically based ( e.g. , income , education , and occupational attainment ) ,", "redundant variables were progressively removed from the model .   the final sep factor model ( figure 1 ) consisted of four variables measuring socioeconomic advantage in income , education , and occupation ( median household income , % with income  $ 150,000 , % with  bachelor 's degree , and % in management or higher ) . as two indices of area - level income", "would be expected to correlate , a correlation path was estimated between the error variances of median household income and % income  $ 150,000 and was retained in all subsequent models .", "model fit was good ( cfi = 0.999 , nfi = 0.999 , tli = 0.996 , /df ratio = 8.23 ( p = 0.004 ) , and rmsea = 0.05 ( p = 0.36 ) ) .", "notably , the variables loading on the latent sep factor are all indicators of neighborhood affluence or advantage rather than disadvantage ; prior neighborhood research has suggested that measures of social advantage , compared to social disadvantage , may be especially important protective factors in neighborhood influences on development .", "mean sep factor scores were found to differ across active catchment sites ( f(5,2515 ) = 184.30 , p < .001 ; table 2 ) . reestimating the models with catchment site as an additional covariate did not alter the pattern of effects , but reduced the fit of the model to an unacceptable extent .", "thus , active catchment site was tested but not retained in the study models as a covariate .", "the present paper sought to characterize both general and specific domains of late life cognition .", "the measured variables ( figure 2 ) related to memory were combined to represent a memory factor ; measures of reasoning combined to form a reasoning factor ; guided by preliminary exploratory analyses , measures of processing speed optimally first combined into separate factors based on test , which were then combined to form a speed factor , and individual measures of everyday cognition combined on a factor representing everyday cognition . because these cognitive domains were represented by latent factors ( making them ", "purer  representations of their domains ) , the vocabulary measure was also represented as a factor by using odd and even scores as indicators loading on a single vocabulary factor ; this effectively transforms that factor into a construct representing the odd - even split half reliable variance of the measure .", "model covariates included linear age , the quadratic effect of age , years of education , gender , and race .", "the error variances of the ufov 2 and 4 subtests , and the everyday problems test and vocabulary were also allowed to correlate in the process of optimizing model fit ( table 3 ) . as the first - order cognitive factors all correlated with one another ( p < 0.001 ) , and as discussed earlier", "g  , capturing the variance shared by the cognitive factors ( table 4 ) .", "model fit was acceptable ( cfi = 0.98 , nfi = 0.97 , rfi = 0.96 , tli = 0.97 , rmsea = 0.046 , ( p = 1.00 ) , /df ratio = 6.40 , and ( p < 0.001 ) ) .  ", "the sep and cognitive measurement models were combined in a structural equation model ( figure 2 ) estimating regression paths from neighborhood - level sep to the cognitive constructs of ", "g  , reasoning , speed , everyday cognition , and vocabulary ( a path could not be estimated for sep or any covariates to memory ) .", "model covariates included linear age , the quadratic effect of age , years of education , gender , and race .", "model fit was adequate ( cfi = 0.96 , nfi = 0.96 , tli = 0.95 , /df ratio = 6.52 , ( p < 0.001 ) , and rmsea = 0.05 ( p = 0.99 ) ) .", "after controlling for individual - level predictors , sep remained a significant predictor of vocabulary alone ( p < 0.01 ; table 5 ) .", "repeated - measures mixed effects analyses of variance were used to estimate whether neighborhood sep predicted differences in training - related gains , beyond practice - related gains , on posttraining measures of memory , reasoning , and speed .", "three separate models examined the gains of each training group relative to all other participants ( control group plus members of other training groups ) who did not receive that particular training ( i.e. , for the reasoning outcome , the reasoning training group was compared to the control plus memory plus speed training groups ) .", "each model tested sep as a continuous predictor and occasion ( baseline , posttest ) and training group ( received training on the ability being examined or not ) , all possible two - way interactions , and the three - way interaction of sep , occasion , and group .", "significant effects for occasion , training group , and their interaction were observed as previously reported in the parent study .", "a main effect was found for sep , but for sep to predict differences in response to active training the three - way interaction would have to be significant ; this was not found for any of the trained abilities ( memory :  f(1 , 4594 ) = 0.66 , p = 0.42 ; reasoning : f(1 , 4847 ) = 0.001 , p = 0.98 ; speed : f(1 , 4793 ) = 0.001 , p = 0.97 ) .", "the present study sought to examine the relationship between current neighborhood - level socioeconomic position ( sep ) and cognitive level , including several specific cognitive domains in the active study , and response to cognitive training .", "the findings demonstrated that , after controlling for individual - level demographic predictors , census - defined neighborhood socioeconomic position independently predicted differences in late life vocabulary as measured in active , but not differences in general cognition (  g  ) , reasoning , everyday cognition , or speed .", "these findings differed from the similar prior study by brandt , which found neighborhood ses effects on a composite cognitive measure capturing cognitive status , verbal fluency , verbal learning , and prospective memory .", "the results also demonstrated that neighborhood sep does not predict individual differences in the immediate response to cognitive training in memory , reasoning , or processing speed .", "the lack of effect for neighborhood sep on any fluid cognitive ability ( memory , reasoning , speed , and everyday cognition ) or on cognitive plasticity ( i.e. , response to training ) is somewhat surprising given the extensive research documenting neighborhood effects on factors affecting brain health , such as cardiovascular fitness and chronic diseases ( e.g. , [ 43 , 44 ] )", ". it would be reasonable to hypothesize that neighborhood could indirectly affect fluid cognitive abilities and training gains , which are more sensitive to compromised brain health than vocabulary , through differences in access to health care , nutrition , and opportunities for exercise ; however , results suggest that if these indirect effects are present they may be relatively weak .", "given the relatively good health of this cohort at baseline testing , the majority of respondents reported  good  or ", "health , and all were independent of care , there also may not have been sufficient variability in baseline fluid cognition scores for neighborhood effects to be detectable .", "the specific association of neighborhood sep with vocabulary suggests neighborhood influences cognition more through sociocultural mechanisms , as vocabulary captures crystallized cognitive abilities , the dimension of cognition related to stored verbal knowledge , developed over a lifetime of engaging with culture .", "crystallized knowledge is also the only domain that continues to improve in the presence of advancing age , compared to fluid cognitive abilities which become less efficient with age .", "a review of vocabulary in aging   reported increasing vocabulary scores with advancing age , as well as with higher education ( although in the present study neighborhood sep predicted vocabulary independent of education ) .", "vocabulary measures like the one used in this study , requiring multiple - choice word recognition rather than production of word meanings , are especially robust to the effects of aging .  ", "neighborhood may influence vocabulary by facilitating or constraining one 's capacity for , and influencing the results of , sociocultural engagement in the community .", "it may do so by providing resources or facilities   encouraging cultural interaction or by encouraging acculturation through modeling and social comparisons .", "that is , high - sep neighborhoods may support vocabulary because more neighbors with high educational and occupational attainment provide more social models of high achievement", ". this modeling may foster upward social comparisons [ 48 , 49 ] , pressuring or evoking desire in an individual to be more like his or her neighbors , resulting in greater engagement in activities enhancing cognitive skills and abilities .", "certainly , other researchers have hinted toward social processes ( i.e. , social norms , interactions and ties , and collective efficacy ) as the mechanism linking neighborhoods with developmental outcomes [ 2 , 47 ] . while vocabulary is related to current neighborhood sep , it is important to consider that vocabulary is also highly correlated with childhood cognitive level .", "vocabulary often reflects both cognitive reserve and premorbid ability level , as it is most robust to not only aging but physical insults to the brain , including head trauma , medical conditions , and exposure to neurotoxins . expressed differently , when cognition is measured in late life , vocabulary is the strongest index of early life cognitive ability", "therefore , the current neighborhood - vocabulary association found in this study may reflect both historical and current relationships between neighborhood context and cognition ; the well - established relationship between neighborhood and cognition in childhood supports this hypothesis .", "thus , a part of the late life neighborhood effects on cognition observed here may represent an ", "berkman and glymour   found that the county of residence during primary schooling , by way of the laws guiding educational requirements in that county , predicted individual differences in both educational attainment and late life cognitive performance .", "similarly , wilson and colleagues   reported that parental education during childhood independently predicted cognitive activity across the lifespan and into old age .", "therefore , it is likely that childhood sep , had it been collected in active , may be associated with both late life vocabulary and late life neighborhood sep .", "finally , it was reported in the results that there were catchment site - level differences in mean sep .", "clearly , an individual 's neighborhood sep is also part of the general sep of the region in which that individual lived ; the interaction between neighborhood and regional sep may influence how a neighborhood 's sep relates to cognition .", "normal  to live in a lower - sep neighborhood ; this experience might differ from the experience of living in a low - sep neighborhood within a high - sep region , and might differently affect cognition .", "such issues have been explored on the level of individual ses - to - neighborhood ses interactions ( e.g. , low individual education predicts worse cognition for those living in low - education neighborhoods versus high - education neighborhoods , ) , but neighborhood - region sep interactions affecting cognition have not been described to our knowledge .  ", "this study was limited in several ways due to its nature as a secondary analysis .", "several covariates would ideally have been included had they been collected in the parent study , including individual - level income and occupation ( although this may later be examined in for the subset present at the 2nd - annual followup ) and the length of time lived at current residence . as a consequence ,", "this is an important limitation , as the observed neighborhood association may be attributable , partially or completely , to unmeasured differences in individual - level socioeconomic status .", "the possibility that the relationship between vocabulary and neighborhood sep can be explained by childhood neighborhood sep must also remain speculation , as this data was not originally collected in active .", "this study therefore was unable to examine measures of childhood socioeconomic status ( e.g. , mother 's or father 's education and income , neighborhood sep in childhood ) .", "it is likely that childhood sep is associated with both vocabulary and late life sep , in which case vocabulary 's relationship with current neighborhood sep would be expected to diminish or disappear if childhood sep was accounted for in the study .  ", "the cross - sectional nature of effects also limits discussion to relationships among variables at a particular time rather than to causal or temporal relationships between variables .", "longitudinal assessment of these relationships is an important next step that will be attempted in future studies . at the time this study was conducted", ", active collected its 10th annual follow - up testing occasion , allowing examination of the interaction between 2000 neighborhood and cognition with 2010 neighborhood and cognition .", "future studies will also attempt to examine ( a ) neighborhood effects on change trajectories , and ( b ) whether there were neighborhood moves for participants , and whether such moves were associated with functional changes .", "furthermore , at this follow - up data was collected documenting participant 's county of primary schooling .", "national historical data will allow investigators to use county information to explore whether and how distal environmental influences ( i.e. , county - level sep and educational laws during primary schooling years ) might predict contextual neighborhood characteristics and cognitive outcomes later in life .", "this paper adds to the body of research examining neighborhood - cognition associations in late life , and extends previous findings by looking beyond general cognition or cognitive status to examine effects of neighborhood across specific cognitive domains .", "the finding that neighborhood sep predicts crystallized cognitive abilities ( specifically , vocabulary ) suggests that neighborhood effects may be most related to sociocultural influences on cognitive development .", "there was a lack of association between neighborhood sep and fluid cognitive abilities , as well as between neighborhood sep and immediate cognitive change following training .", "as discussed above , future research should investigate how associations between early life neighborhood context and cognitive development may influence cognitive function and neighborhood selection in late life ."], "abstract_text": ["<S> low neighborhood - level socioeconomic status has been associated with poorer health , reduced physical activity , increased psychological stress , and less neighborhood - based social support . </S>", "<S> these outcomes are correlates of late life cognition , but few studies have specifically investigated the neighborhood as a unique source of explanatory variance in cognitive aging . </S>", "<S> this study supplemented baseline cognitive data from the active ( advanced cognitive training for independent and vital elderly ) study with neighborhood - level data to investigate ( 1 ) whether neighborhood socioeconomic position ( sep ) predicts cognitive level , and if so , whether it differentially predicts performance in general and specific domains of cognition and ( 2 ) whether neighborhood sep predicts differences in response to short - term cognitive intervention for memory , reasoning , or processing speed . </S>", "<S> neighborhood sep positively predicted vocabulary , but did not predict other general or specific measures of cognitive level , and did not predict individual differences in response to cognitive intervention . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Method", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["the neighborhood has emerged as a prominent level of analysis in studies of contextual influences on health and wellbeing in human development .", "this owes to the social organization of life within neighborhoods , and to the availability of data at neighborhood - like levels of analysis ( i.e. , census tracts ) .", "neighborhood context has also received greater attention due to ecological psychology 's conceptualization of human development as the product of a dynamic interaction between the individual and multiple nested environments , of which the neighborhood is one of the most immediate environments .", "contemporary scholarship on neighborhood factors has also highlighted the importance of understanding whether neighborhood variables add explanatory variance above and beyond individual differences .  ", "neighborhood socioeconomic status ( ses ) is the most consistently reported neighborhood - level predictor of cognitive outcomes  certainly in childhood , and potentially across the life span .", "ses is also the strongest and most consistently reported neighborhood - level predictor of health outcomes among older adults  a relationship which may have a cumulative effect across the life span .", "the association of neighborhood - level ses with health is in fact stronger among adults aged 6069 than among young and middle - aged adults , and during these years its association with health is comparable to or stronger than the relationship of individual ses to health .", "in addition to predicting poorer physical health , lower neighborhood ses is also associated with reduced rates of physical activity , increased incidence of depression and psychosocial stress , and less neighborhood - based social support and social engagement .", "many of these outcomes have also been identified as correlates of late life cognition , yet few studies to date have specifically investigated the role of the neighborhood in cognitive aging .   while neighborhood effects on late life cognition have received less attention , neighborhood effects on early - life cognitive development are relatively well documented .", "neighborhood ses is positively correlated with cognitive development across the entire spectrum of child development , from the prenatal stages ( in the form of reduced rates of congenital anomalies and neural tube defects among higher - ses neighborhoods , presumably due to reduced risk of exposure to environmental toxins ; [ 6 , 7 ] ) and preschool years [ 8 , 9 ] to academic achievement in the school age [ 10 , 11 ] adolescent , and young adulthood years ( e.g. , high school graduation rates and college attendance ) .   four recent studies explored relationships between late life cognition and the neighborhood context on a broad scale .", "wight and colleagues   found that after controlling for individual - level education and area - level median household income , elders living in areas with low neighborhood - level educational attainment ( defined by census tract area ) achieved lower cognitive status scores compared to elders living in areas with high neighborhood - level educational attainment ( as assessed using the mini - mental state examination or mmse ) .", "a united kingdom study   reported a clear and significant downward trend in cognitive performance on the mmse and tests of verbal fluency and memory for older individuals living in neighborhoods with greater deprivation , after controlling for individual - level wealth , income , and education , across all age and gender groups .", "these effects were also robust after adjusting for the effects of individual systolic blood pressure , history of stroke , and duration of residence in the current neighborhood .", "sheffield and peek   moved beyond cross - sectional analysis to examine how neighborhood ses and ethnic composition ( proportion mexican american ) predicted 5-year change in mmse score in a us national sample .", "independent of individual - level risk factors , odds and rate of incident cognitive decline increased as a function of lower neighborhood ses and decreased with the proportion of mexican american neighborhood residents .", "finally , recent findings from the baltimore memory study suggest that the relationship between neighborhood and late life cognition may also depend on genotype .", "however , apolipoprotein e4 genotype was found to interact with high neighborhood psychosocial hazards , resulting in poorer cognitive performance on measures of processing speed and executive functioning after controlling for individual - level covariates .", "this suggests evidence of a gene - environment interaction in neighborhood 's relation to late life cognition .", "the existing studies focused on broad differences in late life cognitive status ( i.e. , mmse score ) , but did not investigate the effect of neighborhood on different cognitive domains ( see recommendations by cullum et al . ) .", "furthermore , the use of the mmse in these studies was not an ideal measure of cognition , as they targeted samples of initially healthy , independent elders .", "the mmse is not sensitive to differences among healthy older adults , having been designed to screen for dementia .", "cognitive aging research has instead focused increasingly on the use of confirmatory factor analysis ( cfa ) and structural equation modeling ( sem ) to explore whether differences in late life cognitive performance are related to general or specific processes .", "it has also been noted that conceptualizing late life cognition using a single general factor (  g  ) is a simplistic approach to a complex and dynamic cognitive system .", "this suggests neighborhood influences should be investigated at both general and domain - specific levels .", "neighborhood effects may support some dimensions of cognition more strongly than others , in addition to influencing general cognitive status .   the present study aimed to identify whether neighborhood effects on cognition were global , or specifically related to particular cognitive domains such as memory , reasoning , processing speed , everyday cognition , or vocabulary .", "another innovation of the present study is the capacity to address whether neighborhood effects might also influence the magnitude of potential benefit from cognitive intervention .", "no study , in part because of a lack of data availability , has examined this question . elucidating both the specificity of neighborhood effects on particular domains of cognition and", "the extent to which neighborhood affects cognitive training response may provide additional hints toward potential mechanisms by which the neighborhood exerts influence .", "for example , effects on the fluid cognitive abilities such as processing speed , memory , and reasoning ( and their response to cognitive training ) would suggest a more biologically mediated influence given their sensitivity to brain integrity .", "in contrast , effects on measures of crystallized cognitive abilities , especially vocabulary , might hint toward a more socioculturally mediated , and possibly lifespan accumulative influence , as crystallized cognitive abilities are developed with the accumulation of verbal knowledge , developed over a lifetime of experience in engaging with culture . as this lifelong accumulation", "is strongly influenced during childhood , adolescence , and young adulthood , effects of current neighborhood sep on crystallized abilities may also reflect , in part , effects of early - life neighborhood sep .", "the advanced cognitive training for independent and vital elderly ( active ) study was a randomized , controlled clinical trial examining the effects of three cognitive interventions for community - dwelling older adults .", "in active , older adults from six us catchment areas ( baltimore / coastal maryland ; the metropolitan areas of birmingham , boston , detroit , and indianapolis ; central pennsylvania ) completed a baseline assessment including multiple cognitive measures .", "participants were then randomized to one of three ten - session cognitive intervention programs or a no - contact control condition .  ", "the present study sought to examine whether neighborhood socioeconomic position ( sep ) shows bivariate relationships with baseline cognitive level and immediate response to cognitive training , beyond individual - level predictors of cognition .", "if so , we aimed to discern whether neighborhood effects were significant for general cognitive ability (  g  ) , and whether effects were differential for specific cognitive abilities ( memory , reasoning , speed , everyday cognition , and vocabulary ) .", "we hypothesized that the potential effect of neighborhood sep may most likely occur by way of sociocultural processes , and therefore would have significant positive associations with general cognitive ability and with crystallized cognitive domains , including vocabulary .", "neighborhood sep was hypothesized to have relatively weaker , if detectable , associations with fluid cognitive domains , including memory , reasoning , speed , and everyday cognition ."], ["the primary objective of active was to test the effectiveness and durability of three cognitive interventions .", "the initial trial randomized individuals to one of three 10-session cognitive interventions designed to improve memory , reasoning , or processing speed performance or to a no - contact control condition . following training", "there was an immediate posttest and follow - up assessments at 1- , 2- , 3- , and 5-years after intervention .", "the current study sought to examine only the immediate pretest - posttest data , focusing on the proximal outcome measures used .", "information on potential covariates was collected , including demographic variables , mmse , recent depressive symptoms , and general health .", "testers at all six sites were trained in standardized assessment protocols and quality control by both study investigators , and the coordinating center ensured fidelity to testing procedures .", "the initial sample of 2,802 participants was recruited through on - site presentations , letters to interested persons , newspaper advertisements , introductory letters , and follow - up telephone calls .", "participants were cognitively healthy , community - dwelling older adults aged 65 to 94 years ( mean age = 73.6 years , mean years of education = 13.5 , mean mmse = 27 , 75.8% female , 72% caucasian , 26% african american , and 2% other minority groups ) .", "the majority of participants ( 64% ) were not married , and most reported good to excellent health ( 84.3% ) .", "efforts were aimed at recruiting older adults independent of care at enrollment , as well as recruiting a diverse sample especially inclusive of african americans , who were previously under - represented in most cognitive training research .", "exclusion criteria included ( a ) being under age 65 at the start of the study , ( b ) significant functional and/or cognitive decline at enrollment ( e.g. , impaired activities of daily living , mmse < 23 , and diagnosis of alzheimer 's disease ) , ( c ) having a medical condition disposing the participant to imminent cognitive decline or to mortality within the next 2 years , ( d ) severe sensory or communicative difficulties precluding participation in assessments and training , ( e ) having had cognitive training , or ( f ) planning to be unavailable during the testing and training periods of the study .  ", "active included multiple assessments of participants , but the present paper restricted itself to baseline cognitive performance and the immediate post - test .", "neighborhood - level socioeconomic data from publicly available geographic datasets were then merged with active data at the individual level .", "active did not aim a priori to include multiple measures of individual - level socioeconomic position .", "education ( highest level achieved ) was assessed for all participants , but other person - level socioeconomic indices ( e.g. , income , occupation ) were not collected at baseline ; occupational status was collected at the second annual follow - up occasion .  ", "the cognitive domains measured in actve included memory , inductive reasoning , processing speed , everyday cognition , and vocabulary ; descriptive statistics on the sample performance for measures within each domain are illustrated in table 1 .", "memory was measured using the hopkins verbal learning test ( hvlt , ) , a 12-word list memory task assessing immediate and delayed recall over 3 learning trials , the rey auditory verbal learning test ( avlt , ) , a 15-word list memory task assessing immediate and delayed recall over 5 consecutive learning trials , and the rivermead behavioral memory test , a paragraph recall task assessing immediate verbal episodic memory . reasoning was measured using letter series , a task requiring accurate identification of the next logical letter group in a series of letters ( e.g. , a m b a n b a o b ) , letter sets , an inductive reasoning task requiring participants to identify which of 4 sets of letters is unrelated to the others ( e.g. , eef hhi llm ysy ) , and word series , a task requiring accurate identification of the next logical word in a series .", "processing speed was measured using the useful field of view test ( ufov , ) , a computer - administered task measuring visual sustained , selective , and divided attention through four subtasks , and the complex reaction time test , a computer - administered task measuring the time taken to perform various motor behaviors and to complete each task . everyday cognition was measured using the everyday problems test , a performance - based test of ability to solve everyday problems including medications , nutrition , phone use , shopping , and management of finances and household , the observed tasks of daily living ( otdl ) , a timed task of problem solving in medication use , telephone use , and financial management , and the timed instrumental activities of daily living ( tiadl ) , a timed test of ability to complete daily living tasks ( e.g , find telephone number , make change , find and read ingredients on a can , find food items on a shelf , read instructions on medicine bottle ) .", "vocabulary was measured using a multiple - choice measure of vocabulary attainment in which the participant is presented with a word and must choose one of four possible synonyms .", "geocoding   is a process by which a location ( e.g. , street address ) is assigned cartesian mathematical coordinates , allowing for other levels of geographic information to be linked with that location .", "geolytics , a commercial provider of geocoding services and census demographic data , ( 1 ) geocoded the active participant addresses , and ( 2 ) appended to these addresses data from the 2000 u.s . census and 2002 economic census , creating a dataset that could be used to characterize the neighborhood environment .", "neighborhood research , active participant addresses were geocoded and linked with their associated census tract numbers , which allowed for census tract - level data from the 2000 u.s . census to be appended to the individual - level active data .", "participants receiving mail by post office boxes were excluded as such addresses can not be verified to represent the participant 's actual place of residence .", "addresses with invalid house numbers , street names , and zip codes were flagged for followup ; invalid addresses or poorly matched addresses were dropped from the analysis ( 93% of the active addresses accurately matched to a us geological survey ( usgs ) geocode ; final sample size = 2,521 ) .  ", "census tracts are subdivisions of a county , with an average size of approximately 4,000 residents designed to capture collectively agreed - upon areas approximating neighborhoods .", "that is , the boundaries of census tracts were agreed upon by local officials knowledgeable of the area and were intended to be homogenous with respect to population characteristics , economic status , and living conditions .", "smaller measurement units of area - level ses ( e.g. , block group ) have been shown to correlate highly with census - level measures of ses , and variability in ses among block groups is small relative to variability within their census tract .", "the association of area - level ses with the cognitive measures was tested at three different levels of area measurement ( block group , census tract , and zip code ) , with no significant difference in the strength of associations across levels . because the census tract has traditionally been the most frequently used and most strongly recommended unit of measurement for area - level effects in health research , especially in terms of sep [ 3 , 35 ] , and because it differed very little in model fit or regression coefficients from the other geographic levels , the census tract level of aggregation was selected for all final analyses involving sep .", "neighborhood socioeconomic position was measured by creating a socioeconomic position ( sep ) index . because ses variables such as income , education , and occupation are often strongly correlated and have been found to load onto a common factor at the census level , census - level data on these variables were combined into a weighted factor score to create a neighborhood sep index that parsimoniously represented multiple socioeconomic variables .   the analytical framework for the study involved exploratory and confirmatory factor analysis , structural equation modeling , and repeated measures mixed effects modeling . in keeping with", "best practice  in conducting neighborhood research , individual- and area - level effects would ideally have been examined using a multi - level modeling ( mlm ) approach , with samples of individuals clustered in neighborhoods allowing formal assessment of random variation within neighborhoods .", "however because active was not originally designed to sample data stratified by neighborhood but sampled widely across each region , the insufficient sample size within each neighborhood measured precluded the use of mlm . in keeping with recent scholarship", ", the current study can instead be classified as ecologic , in which neighborhood variables are measured for each person .", "statistical package for the social sciences ( spss ) 18.0 and analysis of moment structures ( amos ) 18.0 were used to conduct all statistical analyses . prior", "to analysis , all variables were inspected for consistency with the assumption of multivariate normality ( to permit maximum likelihood estimation ) .", "where that assumption was not met , blom transformations   were applied to those variables to improve the normality of their distributions . while no data were missing for any neighborhood - level variables , in a few instances data were missing from the active dataset 's cognitive variables ( e.g. , due to failure to complete a task or attrition between the baseline and posttraining testing occasions ) .", "models were estimated using full information maximum likelihood ( fiml ) , which uses all available data ( thereby not eliminating participants with incomplete data ) .  ", "preparatory to examining the relationship of neighborhood sep to active cognitive outcomes , factor analytic constructs representing neighborhood sep and baseline cognition were developed .", "criteria for acceptable model fit indices included root mean square error of approximation ( rmsea ) < 0.06 , comparative fit index ( cfi ) > 0.95 , normed fit index ( nfi ) > 0.95 , and tucker - lewis index ( tli ) > 0.95 .", "exploratory factor analyses were first conducted on the neighborhood sep variables , using promax rotation to allow optimal fit to the data . a weighted composite measure of sep", "was then estimated and optimized in amos based on a common factor identified in the exploratory factor analysis .", "the cognitive measurement model for the present study dimensionalized cognition into several domains , for which the active dataset was well designed , having captured at least three measures each of specific cognitive domains including memory , reasoning , processing speed , and everyday cognition , or measures of cognition related to everyday abilities .", "as stated in the introduction , cognitive aging research has increasingly emphasized conceptualization of cognition as both a general ability (  g  ) , and a multifaceted system of specific cognitive domains which may respond differently to neighborhood effects .", "therefore the model also estimated effects on each of the above cognitive domains , as well as effects on a higher - order factor representing general cognition or ", "g  , which captures the shared variance among the five cognitive domains in the measurement model .", "the sep and cognition factor models were combined in a structural equation model estimating regression paths from sep to the each of the cognitive factors .", "model covariates included age , quadratic age , years of education , gender , and race ( white , african american ) .", "measures of individual wealth and duration of residence in neighborhood were not collected in active , and could not be included as covariates .", "neighborhood sep as a predictor of training gains was estimated as repeated - measures mixed effects models   rather than as structural equation models because this provided a more flexible interface for the analysis of training gains , which was operationalized as a two - occasion difference score ( and thus would not significantly benefit from a latent variable approach ) while still permitting fiml estimation for maximum power in the presence of missing data ( i.e. , using all available cases ) .", "separate models were estimated for each domain trained ( memory , reasoning , or speed ) to examine the gains of each training group relative to other participants who did not receive that training ( i.e. , the other two training groups and the no - contact controls ) .", "each model tested sep as a continuous predictor and occasion ( baseline , posttest ) and training group ( received training on the ability being examined or not ) each as class variables .", "each model also tested all possible two - way interactions and the three - way interaction of sep , occasion , and group .", "interactions were estimated as residualized interaction terms to correct for collinearity with the model 's main effects ."], ["neighborhood sep was initially developed using exploratory factor analysis ( efa ) to combine 8 indicators of area - level ses : median household income , % households with income $150,000 , % persons in poverty , % with 9th grade education , % with  high school education , % with  bachelor 's degree , % unemployed , and % working in management or higher .", "the efa indicated all 8 variables loaded strongly onto one factor ( eigenvalue = 5.97 ) , explaining 71% of the variance ( factor loadings ranged from 0.71  0.94 ) .", "all indicators were then included in a confirmatory factor analysis , where modification indices ( mi ) suggested high intercorrelations of error variances among variables ( e.g. , ranging up to mi = 1325.42 ) . to derive an optimally - fit , parsimonious set of variables capturing the shared variance of characteristics on which sep is typically based ( e.g. , income , education , and occupational attainment ) ,", "redundant variables were progressively removed from the model .   the final sep factor model ( figure 1 ) consisted of four variables measuring socioeconomic advantage in income , education , and occupation ( median household income , % with income  $ 150,000 , % with  bachelor 's degree , and % in management or higher ) . as two indices of area - level income", "would be expected to correlate , a correlation path was estimated between the error variances of median household income and % income  $ 150,000 and was retained in all subsequent models .", "model fit was good ( cfi = 0.999 , nfi = 0.999 , tli = 0.996 , /df ratio = 8.23 ( p = 0.004 ) , and rmsea = 0.05 ( p = 0.36 ) ) .", "notably , the variables loading on the latent sep factor are all indicators of neighborhood affluence or advantage rather than disadvantage ; prior neighborhood research has suggested that measures of social advantage , compared to social disadvantage , may be especially important protective factors in neighborhood influences on development .", "mean sep factor scores were found to differ across active catchment sites ( f(5,2515 ) = 184.30 , p < .001 ; table 2 ) . reestimating the models with catchment site as an additional covariate did not alter the pattern of effects , but reduced the fit of the model to an unacceptable extent .", "thus , active catchment site was tested but not retained in the study models as a covariate .", "the present paper sought to characterize both general and specific domains of late life cognition .", "the measured variables ( figure 2 ) related to memory were combined to represent a memory factor ; measures of reasoning combined to form a reasoning factor ; guided by preliminary exploratory analyses , measures of processing speed optimally first combined into separate factors based on test , which were then combined to form a speed factor , and individual measures of everyday cognition combined on a factor representing everyday cognition . because these cognitive domains were represented by latent factors ( making them ", "purer  representations of their domains ) , the vocabulary measure was also represented as a factor by using odd and even scores as indicators loading on a single vocabulary factor ; this effectively transforms that factor into a construct representing the odd - even split half reliable variance of the measure .", "model covariates included linear age , the quadratic effect of age , years of education , gender , and race .", "the error variances of the ufov 2 and 4 subtests , and the everyday problems test and vocabulary were also allowed to correlate in the process of optimizing model fit ( table 3 ) . as the first - order cognitive factors all correlated with one another ( p < 0.001 ) , and as discussed earlier", "g  , capturing the variance shared by the cognitive factors ( table 4 ) .", "model fit was acceptable ( cfi = 0.98 , nfi = 0.97 , rfi = 0.96 , tli = 0.97 , rmsea = 0.046 , ( p = 1.00 ) , /df ratio = 6.40 , and ( p < 0.001 ) ) .  ", "the sep and cognitive measurement models were combined in a structural equation model ( figure 2 ) estimating regression paths from neighborhood - level sep to the cognitive constructs of ", "g  , reasoning , speed , everyday cognition , and vocabulary ( a path could not be estimated for sep or any covariates to memory ) .", "model covariates included linear age , the quadratic effect of age , years of education , gender , and race .", "model fit was adequate ( cfi = 0.96 , nfi = 0.96 , tli = 0.95 , /df ratio = 6.52 , ( p < 0.001 ) , and rmsea = 0.05 ( p = 0.99 ) ) .", "after controlling for individual - level predictors , sep remained a significant predictor of vocabulary alone ( p < 0.01 ; table 5 ) .", "repeated - measures mixed effects analyses of variance were used to estimate whether neighborhood sep predicted differences in training - related gains , beyond practice - related gains , on posttraining measures of memory , reasoning , and speed .", "three separate models examined the gains of each training group relative to all other participants ( control group plus members of other training groups ) who did not receive that particular training ( i.e. , for the reasoning outcome , the reasoning training group was compared to the control plus memory plus speed training groups ) .", "each model tested sep as a continuous predictor and occasion ( baseline , posttest ) and training group ( received training on the ability being examined or not ) , all possible two - way interactions , and the three - way interaction of sep , occasion , and group .", "significant effects for occasion , training group , and their interaction were observed as previously reported in the parent study .", "a main effect was found for sep , but for sep to predict differences in response to active training the three - way interaction would have to be significant ; this was not found for any of the trained abilities ( memory :  f(1 , 4594 ) = 0.66 , p = 0.42 ; reasoning : f(1 , 4847 ) = 0.001 , p = 0.98 ; speed : f(1 , 4793 ) = 0.001 , p = 0.97 ) ."], ["the present study sought to examine the relationship between current neighborhood - level socioeconomic position ( sep ) and cognitive level , including several specific cognitive domains in the active study , and response to cognitive training .", "the findings demonstrated that , after controlling for individual - level demographic predictors , census - defined neighborhood socioeconomic position independently predicted differences in late life vocabulary as measured in active , but not differences in general cognition (  g  ) , reasoning , everyday cognition , or speed .", "these findings differed from the similar prior study by brandt , which found neighborhood ses effects on a composite cognitive measure capturing cognitive status , verbal fluency , verbal learning , and prospective memory .", "the results also demonstrated that neighborhood sep does not predict individual differences in the immediate response to cognitive training in memory , reasoning , or processing speed .", "the lack of effect for neighborhood sep on any fluid cognitive ability ( memory , reasoning , speed , and everyday cognition ) or on cognitive plasticity ( i.e. , response to training ) is somewhat surprising given the extensive research documenting neighborhood effects on factors affecting brain health , such as cardiovascular fitness and chronic diseases ( e.g. , [ 43 , 44 ] )", ". it would be reasonable to hypothesize that neighborhood could indirectly affect fluid cognitive abilities and training gains , which are more sensitive to compromised brain health than vocabulary , through differences in access to health care , nutrition , and opportunities for exercise ; however , results suggest that if these indirect effects are present they may be relatively weak .", "given the relatively good health of this cohort at baseline testing , the majority of respondents reported  good  or ", "health , and all were independent of care , there also may not have been sufficient variability in baseline fluid cognition scores for neighborhood effects to be detectable .", "the specific association of neighborhood sep with vocabulary suggests neighborhood influences cognition more through sociocultural mechanisms , as vocabulary captures crystallized cognitive abilities , the dimension of cognition related to stored verbal knowledge , developed over a lifetime of engaging with culture .", "crystallized knowledge is also the only domain that continues to improve in the presence of advancing age , compared to fluid cognitive abilities which become less efficient with age .", "a review of vocabulary in aging   reported increasing vocabulary scores with advancing age , as well as with higher education ( although in the present study neighborhood sep predicted vocabulary independent of education ) .", "vocabulary measures like the one used in this study , requiring multiple - choice word recognition rather than production of word meanings , are especially robust to the effects of aging .  ", "neighborhood may influence vocabulary by facilitating or constraining one 's capacity for , and influencing the results of , sociocultural engagement in the community .", "it may do so by providing resources or facilities   encouraging cultural interaction or by encouraging acculturation through modeling and social comparisons .", "that is , high - sep neighborhoods may support vocabulary because more neighbors with high educational and occupational attainment provide more social models of high achievement", ". this modeling may foster upward social comparisons [ 48 , 49 ] , pressuring or evoking desire in an individual to be more like his or her neighbors , resulting in greater engagement in activities enhancing cognitive skills and abilities .", "certainly , other researchers have hinted toward social processes ( i.e. , social norms , interactions and ties , and collective efficacy ) as the mechanism linking neighborhoods with developmental outcomes [ 2 , 47 ] . while vocabulary is related to current neighborhood sep , it is important to consider that vocabulary is also highly correlated with childhood cognitive level .", "vocabulary often reflects both cognitive reserve and premorbid ability level , as it is most robust to not only aging but physical insults to the brain , including head trauma , medical conditions , and exposure to neurotoxins . expressed differently , when cognition is measured in late life , vocabulary is the strongest index of early life cognitive ability", "therefore , the current neighborhood - vocabulary association found in this study may reflect both historical and current relationships between neighborhood context and cognition ; the well - established relationship between neighborhood and cognition in childhood supports this hypothesis .", "thus , a part of the late life neighborhood effects on cognition observed here may represent an ", "berkman and glymour   found that the county of residence during primary schooling , by way of the laws guiding educational requirements in that county , predicted individual differences in both educational attainment and late life cognitive performance .", "similarly , wilson and colleagues   reported that parental education during childhood independently predicted cognitive activity across the lifespan and into old age .", "therefore , it is likely that childhood sep , had it been collected in active , may be associated with both late life vocabulary and late life neighborhood sep .", "finally , it was reported in the results that there were catchment site - level differences in mean sep .", "clearly , an individual 's neighborhood sep is also part of the general sep of the region in which that individual lived ; the interaction between neighborhood and regional sep may influence how a neighborhood 's sep relates to cognition .", "normal  to live in a lower - sep neighborhood ; this experience might differ from the experience of living in a low - sep neighborhood within a high - sep region , and might differently affect cognition .", "such issues have been explored on the level of individual ses - to - neighborhood ses interactions ( e.g. , low individual education predicts worse cognition for those living in low - education neighborhoods versus high - education neighborhoods , ) , but neighborhood - region sep interactions affecting cognition have not been described to our knowledge .  ", "this study was limited in several ways due to its nature as a secondary analysis .", "several covariates would ideally have been included had they been collected in the parent study , including individual - level income and occupation ( although this may later be examined in for the subset present at the 2nd - annual followup ) and the length of time lived at current residence . as a consequence ,", "this is an important limitation , as the observed neighborhood association may be attributable , partially or completely , to unmeasured differences in individual - level socioeconomic status .", "the possibility that the relationship between vocabulary and neighborhood sep can be explained by childhood neighborhood sep must also remain speculation , as this data was not originally collected in active .", "this study therefore was unable to examine measures of childhood socioeconomic status ( e.g. , mother 's or father 's education and income , neighborhood sep in childhood ) .", "it is likely that childhood sep is associated with both vocabulary and late life sep , in which case vocabulary 's relationship with current neighborhood sep would be expected to diminish or disappear if childhood sep was accounted for in the study .  ", "the cross - sectional nature of effects also limits discussion to relationships among variables at a particular time rather than to causal or temporal relationships between variables .", "longitudinal assessment of these relationships is an important next step that will be attempted in future studies . at the time this study was conducted", ", active collected its 10th annual follow - up testing occasion , allowing examination of the interaction between 2000 neighborhood and cognition with 2010 neighborhood and cognition .", "future studies will also attempt to examine ( a ) neighborhood effects on change trajectories , and ( b ) whether there were neighborhood moves for participants , and whether such moves were associated with functional changes .", "furthermore , at this follow - up data was collected documenting participant 's county of primary schooling .", "national historical data will allow investigators to use county information to explore whether and how distal environmental influences ( i.e. , county - level sep and educational laws during primary schooling years ) might predict contextual neighborhood characteristics and cognitive outcomes later in life ."], ["this paper adds to the body of research examining neighborhood - cognition associations in late life , and extends previous findings by looking beyond general cognition or cognitive status to examine effects of neighborhood across specific cognitive domains .", "the finding that neighborhood sep predicts crystallized cognitive abilities ( specifically , vocabulary ) suggests that neighborhood effects may be most related to sociocultural influences on cognitive development .", "there was a lack of association between neighborhood sep and fluid cognitive abilities , as well as between neighborhood sep and immediate cognitive change following training .", "as discussed above , future research should investigate how associations between early life neighborhood context and cognitive development may influence cognitive function and neighborhood selection in late life ."]]}
{"article_id": "PMC3202508", "article_text": ["cocaine and other amphetamine - like psychostimulants have been a significant part of the human pharmacopoeia for thousands of years .", "however , the appearance of these substances in western societies has been relatively recent , cocaine having debuted as both a local anesthetic and a psychostimulant in the 19th century . over the course of the next century , it became increasingly clear that the amphetamine - like psychostimulants carried serious abuse liability , as well as producing a prominent paranoia - like syndrome among many individuals who chronically used this class of drugs .", "the abuse liability of these drugs has resulted in sociological use patterns that have been described as epidemics , such as the methamphetamine epidemic in japan in the 1950s , the cocaine epidemic in the united states in the 1980s , and the crack cocaine epidemic of the 1990s . \n ", "the high abuse liability of this class of drugs relies on both pharmacological properties and the sociological characteristics of how the drugs are introduced into various societies around the world .", "this article will not significantly address the sociology of psychostimulant abuse , which involves diverse events ranging from the use of amphetamines by japanese soldiers in world war ii , to the formulation of crack as a less expensive version of cocaine in the united states , to the introduction of prescription formulations to regulate eating habits or to treat attention deficit - hyperactivity disorder .  ", "rather , we will review the basic pharmacology of amphetamine - like drugs , integrate these molecular mechanisms into the brain circuitry of reward , and describe how these drugs are thought to create pathological changes in reward and learning circuitry finally , this knowledge will be amalgamated into a vision of future pharmacotherapies for treating psychostimulant addiction .", "the defining mechanism of action of amphetamine4ike psychostimulants as a class of drugs with high abuse liability is the ability to bind to dopamine transporters ( dat ) .", "dopamine transporters are a member of a class of proteins that eliminate monoamines , including dopamine , from the synaptic cleft after neuronal release .", "this protein has a high affinity for dopamine relative to other monoamines , such as norepinephrine or serotonin , and while all the readily abused psychostimulants bind to dat , they may also bind to the other monoamine transporters with greater or lesser affinity . to some extent ,", "the relative profile of binding by individual drugs to the different transporter proteins explains different characteristics of the drugs .", "most striking , for example , is 3,4-methylenedioxymethamphetamine ( mdma ) which has a relatively higher affinity for serotonin transporters , and is thereby a mild hallucinogen and neurotoxic to serotonin axon terminals , while methamphetamine binds more avidly to dat , which explains its greater toxicity at dopamine terminals , as well as its propensity to induce paranoid psychosis - like symptoms . while the binding to other monoamine transporters contributes to the antidepressant and hallucinogenic characteristics of some psychostimulants , it is the binding to dat that provides the major influence on abuse liability , which is the focus of this review .", "there are two major categories of interaction by ampetamine - like psychostimulants with dat , but in all cases the end result is to inhibit the elimination of dopamine from the synapse and thereby increase the quantity and half - life of synaptic and extrasynaptic dopamine levels .", "the first mechanism is typified by cocaine and methylphenidate that bind to dat , but are not transported into the presynaptic terminal as surrogate dopamine .", "therefore , when these drugs bind to dat the increase in extracellular dopamine relies primarily on normal synaptic release , which is more amenable to physiological feedback regulation .", "the second mechanism is typified by amphetamines , and involves not only binding to dat , but also translocation into the cell in place of dopamine .", "in addition , these drugs enter dopamine synaptic vesicles , where the fact that these compounds are basically charged degrades the ph gradient necessary to sequester dopamine into the vesicle . this in turn results in a large buildup of dopamine in the cytosol , thereby reversing the direction of dat to release dopamine into the extracellular space rather than facilitating its removal regardless of the precise interaction with dat by individual amphetamine - like psychostimulants", ", this class of drugs dramatically elevates extracellular dopamine , and this action is thought to be the initiating molecular event that reinforces drugseeking behaviors , ultimately culminating in addiction . \n", "thus , when an organism encounters a novel stimulus , whether a positive stimulus such as a food reward or a negative stimulus such as a stressor , the activity of dopamine cells in the ventral tegmental area , and dopamine release in axon terminal fields in the prefrontal cortex , nucleus accumbens , and/or amygdale , are altered .", "an important characteristic of this brain - environment interaction is that the ability of a given stimulus to increase dopamine cell firing and release decreases with repeated presentation of the stimulus .", "however , it can be shown that if a motivationally neutral stimulus ( such as a light or tone ) is associated with the motivational event in such a manner that the neutral stimulus predicts arrival of the motivational event , the ability of the motivational stimulus to release dopamine is transferred to the neutral stimulus .", "thus , the neutral stimulus now causes dopamine release in a manner predicting arrival of a motivationally relevant event . based upon these data , the role for dopamine release in the mesocorticolimbic brain regions is twofold : ( i ) to cue the organism that a novel motivationally relevant event is occurring and that adaptive behavioral responses need to be engaged ( eg , approach a reward or avoid a stress ) ; ( ii ) once the behavioral response is established , dopamine release is antecedent to the appearance of the motivationally relevant event and is triggered by environmental associations that the organism has made with the event as part of learning the adaptive behavioral response . in this way , dopamine serves to alert and thereby prepare the organism for an impending important event .", "the primary differences between psychostimulant - induced dopamine release and release associated with normal learning about important environmental events such as rewards and stressors is : ( i ) since psychostimulants block the elimination of dopamine through dat , the level of dopamine achieved far exceeds what is possible from a biological stimulus ; ( ii ) in contrast to biological stimuli that cease to release dopamine once an approach or avoidance response to that stimulus has been learned , psychostimulants continue to release large amounts of dopamine upon every administration ( with the possible exception of extended binging that can temporarily deplete dopamine stores ) .", "thus , with psychostimulants , each administration releases dopamine into mesocorticolimbic regions , causing further associations to be made between the drug experience and the environment . in this way", ", it is thought that the more a psychostimulant is administered , the more learned associations are made with the environment and the more effective the environment becomes at triggering craving and drug - seeking .", "it is this  overlearning  of drugseeking behaviors by progressive associations formed between repeated drug - induced dopamine release and the environment that is thought to lead to increased vulnerability to relapse .", "as outlined above , psychostimulant - induced dopamine release is responsible for reinforcing behaviors designed to seek and administer the drugs .", "the dopamine projections involved in this process are outlined in figure 1a , and as indicated , the most critical projection in this regard is the projection from the ventral tegmental area dopamine cells to the nucleus accumbens .", "for example , if psychostimulant - induced release of dopamine in the nucleus accumbens is impaired , this affects the acquisition of drug - seeking behaviors , and can markedly influence the amount of drug taken in a well - trained subject .", "thus , the learning of a task to obtain the drug and the amount of drug taken in a given session is strongly regulated by dopamine release in the accumbens . however ,", "when an animal has been withdrawn from repeated psychostimulant use , and drug - seeking is initiated by an environmental stimulus such as a cue previously paired with drug delivery , or a novel stressor , it is dopamine release in the prefrontal cortex and amygdala , respectively , that mediates the reinstatement of drug - seeking .", "thus , relapse can be induced by dopamine release in prefrontal and allocortical brain regions , and reflects the aforementioned physiological role of dopamine release as a predictive antecendent to stimulus ( drug ) delivery .", "what this implies is that chronic release of dopamine by repeated psychostimulant administration may be modifying cortical and allocortical regulation of behavior . \n", "figure 1b shows that the cortical and allocortical regulation of behavior is primarily mediated by glutamatergic projections .", "these projections are to subcortical structures , such as the nucleus accumbens and dopamine cells in the ventral tegmental area , as well as between the cortical and allocortical regions .", "thus , when dopamine is released into the prefrontal cortex or amygdala by a drug - associated cue or stressor , this is thought to stimulate glutamatergic projections between the prefrontal cortex and amygdala , as well as glutamatergic outputs to the accumbens and ventral tegmental area .", "a variety of studies have linked this activation of corticofugal glutamate transmission with craving in psychostimulant addicts or drug - seeking in animal models of addiction .", "the neuroimaging literature clearly shows metabolic activation of regions of the prefrontal cortex , including portions of the anterior cingulate and ventral orbital cortices , and the amygdala during cue - induced craving for amphetamine - like psychostimulants .", "interestingly , while a cue or low dose of psychostimlant markedly increases metabolic activity in the prefrontal cortex and amygdala , in the absence of a learned drug association the prefrontal cortex is hypoactive .", "the reduction in basal metabolic activity is taken to indicate a potential deficit in cognitive ability to regulate relapse , and recent cognitive testing in psychostimulant addicts confirms the presence of certain cognitive dysfunctions related to impulse control and switching behaviors in an adaptive manner to changing environmental circumstances . a strong role for activation of both the prefrontal cortex and amygdala", "thus , pharmacological inhibition of either of these regions prevents the reinstatement of drug - seeking in animals withdrawn from drugs that have undergone extinction training .", "moreover , a marked release of glutamate is measured in the nucleus accumbens of animals initiating drug - seeking in response to a stressor , and this glutamate is derived from increased activity in the projection from the prefrontal cortex to the nucleus accumbens .", "one final set of studies to be considered regarding cortical glutamate is the recent evidence that as drug - seeking becomes more compulsive there is a gradual shift to greater reliance on corticostriatal habit circuitry , and less involvement of prefrontal to accumbens circuitry .", "this possibility is supported by animal models in two ways : ( i ) if animals that have been trained to self - administer cocaine are left in abstinence for an extended period , drug - seeking is augmented , and in this case inhibition of the prefrontal cortex or amygdala no longer inhibits drug - seeking induced by drug - associated stimuli . however , inhibition of the dorsolateral striatum is still effective at blocking drug - seeking ; ( ii ) as training of an animal in drug - seeking paradigms progresses it is possible to show a gradual increase in dopamine released into the caudate in favor of release into the nucleus accumbens .", "this is illustrated in figure 1a , showing that dopamine release into the caudate can regulate habitual behaviors .", "on one hand , these data point to the possibility that in treating compulsive relapse we should be focusing on regulation of corticostriatal habit circuitry , including glutamate input to the caudate from sensorymotor cortex and dopamine input from the substantia nigra .", "however , these studies have been conducted in rats in whom the frontal cortex is poorly evolved , and given the marked activation produced in the prefrontal cortex and amygdala by drug - associated stimuli in psychostimulant addicts , the conclusion that compulsive relapse is entirely derived from corticostriatal habit circuitry may be an oversimplification . indeed , it has been argued that a primary role for therapy in treating addiction is to strengthen prefrontal regulation of drug - seeking behaviors , whether through psychosocial interventions or pharmacotherapy . \n", "given the apparent critical role played by glutamatergic affrents to the nucleus accumbens in initiating drugseeking or craving , recent studies have identified a number of enduring cellular changes in glutamate transmission that may be critical pathological neuroadaptations to psychostimulant use , and may serve as targets for pharmcotherapeutic intervention . in general", "the neuroplasticity can be categorized as postsynaptic , presynaptic and nonsynaptic ( ie , residing predominantly in glia ) . however , since these processes are intimately related to each other , it is perhaps best to consider all the adaptations as changes in glutamate homeostasis , the end result of which is a psychostimulant - induced enduring change in the fidelity of communication between the prefrontal cortex and the nucleus accumbens , and the regulation by this projection of corticostriatal habit circuitry .", "it has been proposed that this loss of fidelity results in a weakening or loss in the capacity of psychostimulant addicts to cognitively intervene in habitual behaviors , thereby making drug - seeking more difficult to control and increasing the vulnerability to relapse . \n ", "as mentioned above , drug - seeking is associated with a large release of prefrontal glutamate into the nucleus accumbens .", "the large release of glutamate during drugseeking is all the more remarkable because it was discovered using microdialysis . which is not a very sensitive measure of glutamate transmission . indeed , when animals are trained to seek a biological reward , such as food , microdialysis can not measure glutamate release .", "thus , the large psychostimulant - induced release of glutamate has been hypothesized to be a pathological and perhaps critical mediator of relapse .", "this hypothesis is supported by the fact that treatments interrupting synaptic glutamate release also inhibit drug - seeking .", "this includes a variety of pharmacological treatments that have the potential to be developed into pharmacotherapeutic agents , as outlined below . perhaps in part a consequence of the massive synaptic glutamate release occurring during psychostimulantseeking behavior , a number of marked changes in postsynaptic glutamate transmission have been measured in animals withdrawn from chronic cocaine or amphetamine administration . perhaps among the most dramatic", "importantly , this appears to be accompanied by an increase in the insertion of a - amino3-hydroxy-5-methylisoxazole-4-propionic acid ( ampa ) glutamate receptors into the membrane of spiny neurons in the accumbens , and is associated with an increase in electrophysiological sensitivity to ampa receptor stimulation ( as measured by the ampa : n - methyl d - aspartate [ nmda ] ratio ) .", "moreover , a number of other proteins regulating the fidelity of postsynaptic glutamate transmission are altered after chronic cocaine use , including proteins that regulate the structure and function of the protein scaffolding in which the glutamate receptors are embedded , including postsynaptic density ( psd)-95 and homer proteins , among others .", "also , in addition to ampa ionotropic glutamate receptors , signaling through metabotropic glutamate receptors is downregulated .", "finally , this psychostimulant - induced postsynaptic neuroplasticity is associated with changes in the biochemical machinery regulating spine formation , notably an increase in actin cycling and formation of factin ( a primary structural protein regulating spine morphology and the insertion of proteins into and out of the membrane ) .", "taken together , these findings indicate that significant changes have been produced by psychostimulants in the way that synaptically released glutamate will be interpreted by postsynaptic cells .", "thus , there remain many apparent contradictions in the literature regarding changes in specific proteins , and in the overall direction of synaptic grading ( ie , is postsynaptic glutamate transmission augmented or inhibited by chronic psychostimulant administration ) .", "therefore , for now it is probably not prudent to speculate on the type of drug development that may arise from this particular direction of research into psychostimulant - induced changes in glutamate signaling .", "as outlined above , given our current state of knowledge it is more likely that pharmacotherapeutic restoration of normal glutamate release may be a more successful approach than manipulating postsynaptic proteins responsible for and/or associated with changes in the fidelity of postsynaptic glutamate transmission . in part , this is due to the relatively contradictory status of the emerging literature on postsynaptic plasticity .", "moreover , it has been hypothesized that the adaptations in presynaptic glutamate release may be at least partly causal in the postsynaptic adaptations , posing the possibility that if the pathological release of glutamate can be successfully ameliorated , postsynaptic normalization may follow . \n ", "pharmacotherapeutic targets for regulating the pathological synaptic glutamate release seen in the accumbens of psychostimulant - seeking animals can be placed into two categories : ( i ) targets based upon psychostimulant induced changes in proteins regulating synaptic glutamate release ; ( ii ) proteins that produce a general decrease in excitatory transmission .", "compounds in the first category are likely to be the most specific for psychostimulant addiction , and perhaps carry the least number of unwanted side effects , while the latter category may be less selective not only regarding effects on other addictive drugs , but also in terms of unwanted side effects .", "neuroplasticity produced by chronic cocaine administration that could potentially contribute to pathological glutamate release includes downregulation of cystine - glutamate exchange , downregulation of glial glutamate transporters , and downregulation of release - regulating presynaptic metabotropic glutamate receptors ( mglur2/3 ) .", "importantly , these three changes are interrelated due to the cystine - glutamate exchanger and glutamate transporter regulating extrasynaptic glutamate tone on release regulating mglur2/3 .", "drugs have been examined in animal models of psychostimulant addiction , and to a lesser extent in clinical trials with cocaine addicts that regulate one or more of these processes .", "for example , n - acetylcysteine upregulates cystine glutamate exchange , and has been shown in animal models to prevent synaptic glutamate release associated with drug - seeking , restore inhibitory tone on synaptic release through activation of mglur2/3 , and to inhibit the desire for cocaine in a double - blind cue - reactivity trial in non - treatment - seeking cocaine addicts .", "also , mglur2/3 agonists have proven effective at inhibiting cocaine seeking in animal models ; however , unlike nacetylcysteine , food - seeking was inhibited at only a 3- to 10-fold increase in dose relative to inhibiting cocaineseeking . although no studies have yet evaluated regulating glutamate transport in drug - seeking models of psychostimulant addiction , recent reports of the use of -lactam antibiotics to increase glutamate transporter membrane insertion poses an interesting possibility for pharmacologically overcoming the cocaine - induced downregulation of glutamate transporters .", "finally , while the mechanism is not clear , modafinil has been reported to increase extracellular glutamate levels , which would restore tone on release inhibiting mglur2/3 .", "notably , modafinil has been found to successfully decrease cocaine relapse in a number of clinical trials . \n ", "the primary drugs in the category of nonspecific inhibitors of synaptic glutamate release include a variety of -aminobutyric acid ( gaba)-mimetic compounds .", "these range from relatively specific agonists at gabab receptors , such as baclofen , which inhibit synaptic glutamate release to a host of less selective compounds known to increase gaba transmission via interactions with synthetic or elimination mechanisms , such as topiramate or vigabatrin .", "for all of these compounds there is preclinical and clinical data to support some potential efficacy .", "however , as predicted , especially for the nonselective gabamimetics untoward side effects , such as sedation , are reported .", "this review has endeavored to transport the reader from the initiating molecular actions of amphetamine - like psychostimulants on dopamine systems in the brain to enduring neuroplasticity produced in glutamate transmission responsible for communicating from prefrontal and allocortical brain regions through the nucleus accumbens to motor regulatory systems . moreover , by examining molecular neuroplasticity produced in excitatory synapses by chronic psychostimulant administration , it is possible to make some deductions about potential pharmacotherapeutic interventions .", "indeed , there already exists an emerging literature supporting this approach in developing potential pharmacotherapies for treating psychostimulant addiction .", "importantly , this is a nascent and emerging science , and while much has been discovered , the cutting edge of discovery into the neuroplasticity produced by psychostimulants is understandably contradictory . as further discoveries are made that allow us to understand the nature of these contradictions , it should follow that additional targets will emerge to provide potential novel pharmacotherapies for treating psychostimulant addiction ."], "abstract_text": ["<S> although the pharmacology of amphetamine - like psychostimulants at dopamine transporters is well understood , addiction to this class of drugs has proven difficult to deal with . the reason for this disconnection </S>", "<S> is that while the molecular mechanism of amphetamine action is critical to reinforce drug use , it is only the first step in a sequence of widespread neuroplastic events in brain circuitry . </S>", "<S> this review outlines the affect of psychostimulants on mesocorticolimbic dopamine projections that mediate their reinforcing effect , and how this action ultimately leads to enduring pathological neuroplasticity in glutamatergic projections from the prefrontal cortex to the nucleus accumbens . </S>", "<S> molecular neuroadaptations induced by psychostimulant abuse are described in glutamate neurotransmission , and from this information potential pharmacotherapeutic targets are identified , based upon reversing or countermanding psychostimulant - induced neuroplasticity . </S>"], "labels": null, "section_names": ["Background", "Basic pharmacology of amphetamine-like psychostimulants", "How release of dopamine by psychostimulants initiates addiction", "How psychostimulant-induced dopamine release creates pathological neuroplasticity in cortical regulation of behavior", "Enduring psychostimulant-induced neuroplasticity in the prefrontal to accumbens glutamate projection", "Ideas for pharmacotherapies based upon psychostimulant-induced plasticity in glutamate transmission", "Conclusions"], "sections": [["cocaine and other amphetamine - like psychostimulants have been a significant part of the human pharmacopoeia for thousands of years .", "however , the appearance of these substances in western societies has been relatively recent , cocaine having debuted as both a local anesthetic and a psychostimulant in the 19th century . over the course of the next century , it became increasingly clear that the amphetamine - like psychostimulants carried serious abuse liability , as well as producing a prominent paranoia - like syndrome among many individuals who chronically used this class of drugs .", "the abuse liability of these drugs has resulted in sociological use patterns that have been described as epidemics , such as the methamphetamine epidemic in japan in the 1950s , the cocaine epidemic in the united states in the 1980s , and the crack cocaine epidemic of the 1990s . \n ", "the high abuse liability of this class of drugs relies on both pharmacological properties and the sociological characteristics of how the drugs are introduced into various societies around the world .", "this article will not significantly address the sociology of psychostimulant abuse , which involves diverse events ranging from the use of amphetamines by japanese soldiers in world war ii , to the formulation of crack as a less expensive version of cocaine in the united states , to the introduction of prescription formulations to regulate eating habits or to treat attention deficit - hyperactivity disorder .  ", "rather , we will review the basic pharmacology of amphetamine - like drugs , integrate these molecular mechanisms into the brain circuitry of reward , and describe how these drugs are thought to create pathological changes in reward and learning circuitry finally , this knowledge will be amalgamated into a vision of future pharmacotherapies for treating psychostimulant addiction ."], ["the defining mechanism of action of amphetamine4ike psychostimulants as a class of drugs with high abuse liability is the ability to bind to dopamine transporters ( dat ) .", "dopamine transporters are a member of a class of proteins that eliminate monoamines , including dopamine , from the synaptic cleft after neuronal release .", "this protein has a high affinity for dopamine relative to other monoamines , such as norepinephrine or serotonin , and while all the readily abused psychostimulants bind to dat , they may also bind to the other monoamine transporters with greater or lesser affinity . to some extent ,", "the relative profile of binding by individual drugs to the different transporter proteins explains different characteristics of the drugs .", "most striking , for example , is 3,4-methylenedioxymethamphetamine ( mdma ) which has a relatively higher affinity for serotonin transporters , and is thereby a mild hallucinogen and neurotoxic to serotonin axon terminals , while methamphetamine binds more avidly to dat , which explains its greater toxicity at dopamine terminals , as well as its propensity to induce paranoid psychosis - like symptoms . while the binding to other monoamine transporters contributes to the antidepressant and hallucinogenic characteristics of some psychostimulants , it is the binding to dat that provides the major influence on abuse liability , which is the focus of this review .", "there are two major categories of interaction by ampetamine - like psychostimulants with dat , but in all cases the end result is to inhibit the elimination of dopamine from the synapse and thereby increase the quantity and half - life of synaptic and extrasynaptic dopamine levels .", "the first mechanism is typified by cocaine and methylphenidate that bind to dat , but are not transported into the presynaptic terminal as surrogate dopamine .", "therefore , when these drugs bind to dat the increase in extracellular dopamine relies primarily on normal synaptic release , which is more amenable to physiological feedback regulation .", "the second mechanism is typified by amphetamines , and involves not only binding to dat , but also translocation into the cell in place of dopamine .", "in addition , these drugs enter dopamine synaptic vesicles , where the fact that these compounds are basically charged degrades the ph gradient necessary to sequester dopamine into the vesicle . this in turn results in a large buildup of dopamine in the cytosol , thereby reversing the direction of dat to release dopamine into the extracellular space rather than facilitating its removal regardless of the precise interaction with dat by individual amphetamine - like psychostimulants", ", this class of drugs dramatically elevates extracellular dopamine , and this action is thought to be the initiating molecular event that reinforces drugseeking behaviors , ultimately culminating in addiction . \n"], ["thus , when an organism encounters a novel stimulus , whether a positive stimulus such as a food reward or a negative stimulus such as a stressor , the activity of dopamine cells in the ventral tegmental area , and dopamine release in axon terminal fields in the prefrontal cortex , nucleus accumbens , and/or amygdale , are altered .", "an important characteristic of this brain - environment interaction is that the ability of a given stimulus to increase dopamine cell firing and release decreases with repeated presentation of the stimulus .", "however , it can be shown that if a motivationally neutral stimulus ( such as a light or tone ) is associated with the motivational event in such a manner that the neutral stimulus predicts arrival of the motivational event , the ability of the motivational stimulus to release dopamine is transferred to the neutral stimulus .", "thus , the neutral stimulus now causes dopamine release in a manner predicting arrival of a motivationally relevant event . based upon these data , the role for dopamine release in the mesocorticolimbic brain regions is twofold : ( i ) to cue the organism that a novel motivationally relevant event is occurring and that adaptive behavioral responses need to be engaged ( eg , approach a reward or avoid a stress ) ; ( ii ) once the behavioral response is established , dopamine release is antecedent to the appearance of the motivationally relevant event and is triggered by environmental associations that the organism has made with the event as part of learning the adaptive behavioral response . in this way , dopamine serves to alert and thereby prepare the organism for an impending important event .", "the primary differences between psychostimulant - induced dopamine release and release associated with normal learning about important environmental events such as rewards and stressors is : ( i ) since psychostimulants block the elimination of dopamine through dat , the level of dopamine achieved far exceeds what is possible from a biological stimulus ; ( ii ) in contrast to biological stimuli that cease to release dopamine once an approach or avoidance response to that stimulus has been learned , psychostimulants continue to release large amounts of dopamine upon every administration ( with the possible exception of extended binging that can temporarily deplete dopamine stores ) .", "thus , with psychostimulants , each administration releases dopamine into mesocorticolimbic regions , causing further associations to be made between the drug experience and the environment . in this way", ", it is thought that the more a psychostimulant is administered , the more learned associations are made with the environment and the more effective the environment becomes at triggering craving and drug - seeking .", "it is this  overlearning  of drugseeking behaviors by progressive associations formed between repeated drug - induced dopamine release and the environment that is thought to lead to increased vulnerability to relapse ."], ["as outlined above , psychostimulant - induced dopamine release is responsible for reinforcing behaviors designed to seek and administer the drugs .", "the dopamine projections involved in this process are outlined in figure 1a , and as indicated , the most critical projection in this regard is the projection from the ventral tegmental area dopamine cells to the nucleus accumbens .", "for example , if psychostimulant - induced release of dopamine in the nucleus accumbens is impaired , this affects the acquisition of drug - seeking behaviors , and can markedly influence the amount of drug taken in a well - trained subject .", "thus , the learning of a task to obtain the drug and the amount of drug taken in a given session is strongly regulated by dopamine release in the accumbens . however ,", "when an animal has been withdrawn from repeated psychostimulant use , and drug - seeking is initiated by an environmental stimulus such as a cue previously paired with drug delivery , or a novel stressor , it is dopamine release in the prefrontal cortex and amygdala , respectively , that mediates the reinstatement of drug - seeking .", "thus , relapse can be induced by dopamine release in prefrontal and allocortical brain regions , and reflects the aforementioned physiological role of dopamine release as a predictive antecendent to stimulus ( drug ) delivery .", "what this implies is that chronic release of dopamine by repeated psychostimulant administration may be modifying cortical and allocortical regulation of behavior . \n", "figure 1b shows that the cortical and allocortical regulation of behavior is primarily mediated by glutamatergic projections .", "these projections are to subcortical structures , such as the nucleus accumbens and dopamine cells in the ventral tegmental area , as well as between the cortical and allocortical regions .", "thus , when dopamine is released into the prefrontal cortex or amygdala by a drug - associated cue or stressor , this is thought to stimulate glutamatergic projections between the prefrontal cortex and amygdala , as well as glutamatergic outputs to the accumbens and ventral tegmental area .", "a variety of studies have linked this activation of corticofugal glutamate transmission with craving in psychostimulant addicts or drug - seeking in animal models of addiction .", "the neuroimaging literature clearly shows metabolic activation of regions of the prefrontal cortex , including portions of the anterior cingulate and ventral orbital cortices , and the amygdala during cue - induced craving for amphetamine - like psychostimulants .", "interestingly , while a cue or low dose of psychostimlant markedly increases metabolic activity in the prefrontal cortex and amygdala , in the absence of a learned drug association the prefrontal cortex is hypoactive .", "the reduction in basal metabolic activity is taken to indicate a potential deficit in cognitive ability to regulate relapse , and recent cognitive testing in psychostimulant addicts confirms the presence of certain cognitive dysfunctions related to impulse control and switching behaviors in an adaptive manner to changing environmental circumstances . a strong role for activation of both the prefrontal cortex and amygdala", "thus , pharmacological inhibition of either of these regions prevents the reinstatement of drug - seeking in animals withdrawn from drugs that have undergone extinction training .", "moreover , a marked release of glutamate is measured in the nucleus accumbens of animals initiating drug - seeking in response to a stressor , and this glutamate is derived from increased activity in the projection from the prefrontal cortex to the nucleus accumbens .", "one final set of studies to be considered regarding cortical glutamate is the recent evidence that as drug - seeking becomes more compulsive there is a gradual shift to greater reliance on corticostriatal habit circuitry , and less involvement of prefrontal to accumbens circuitry .", "this possibility is supported by animal models in two ways : ( i ) if animals that have been trained to self - administer cocaine are left in abstinence for an extended period , drug - seeking is augmented , and in this case inhibition of the prefrontal cortex or amygdala no longer inhibits drug - seeking induced by drug - associated stimuli . however , inhibition of the dorsolateral striatum is still effective at blocking drug - seeking ; ( ii ) as training of an animal in drug - seeking paradigms progresses it is possible to show a gradual increase in dopamine released into the caudate in favor of release into the nucleus accumbens .", "this is illustrated in figure 1a , showing that dopamine release into the caudate can regulate habitual behaviors .", "on one hand , these data point to the possibility that in treating compulsive relapse we should be focusing on regulation of corticostriatal habit circuitry , including glutamate input to the caudate from sensorymotor cortex and dopamine input from the substantia nigra .", "however , these studies have been conducted in rats in whom the frontal cortex is poorly evolved , and given the marked activation produced in the prefrontal cortex and amygdala by drug - associated stimuli in psychostimulant addicts , the conclusion that compulsive relapse is entirely derived from corticostriatal habit circuitry may be an oversimplification . indeed , it has been argued that a primary role for therapy in treating addiction is to strengthen prefrontal regulation of drug - seeking behaviors , whether through psychosocial interventions or pharmacotherapy . \n"], ["given the apparent critical role played by glutamatergic affrents to the nucleus accumbens in initiating drugseeking or craving , recent studies have identified a number of enduring cellular changes in glutamate transmission that may be critical pathological neuroadaptations to psychostimulant use , and may serve as targets for pharmcotherapeutic intervention . in general", "the neuroplasticity can be categorized as postsynaptic , presynaptic and nonsynaptic ( ie , residing predominantly in glia ) . however , since these processes are intimately related to each other , it is perhaps best to consider all the adaptations as changes in glutamate homeostasis , the end result of which is a psychostimulant - induced enduring change in the fidelity of communication between the prefrontal cortex and the nucleus accumbens , and the regulation by this projection of corticostriatal habit circuitry .", "it has been proposed that this loss of fidelity results in a weakening or loss in the capacity of psychostimulant addicts to cognitively intervene in habitual behaviors , thereby making drug - seeking more difficult to control and increasing the vulnerability to relapse . \n ", "as mentioned above , drug - seeking is associated with a large release of prefrontal glutamate into the nucleus accumbens .", "the large release of glutamate during drugseeking is all the more remarkable because it was discovered using microdialysis . which is not a very sensitive measure of glutamate transmission . indeed , when animals are trained to seek a biological reward , such as food , microdialysis can not measure glutamate release .", "thus , the large psychostimulant - induced release of glutamate has been hypothesized to be a pathological and perhaps critical mediator of relapse .", "this hypothesis is supported by the fact that treatments interrupting synaptic glutamate release also inhibit drug - seeking .", "this includes a variety of pharmacological treatments that have the potential to be developed into pharmacotherapeutic agents , as outlined below . perhaps in part a consequence of the massive synaptic glutamate release occurring during psychostimulantseeking behavior , a number of marked changes in postsynaptic glutamate transmission have been measured in animals withdrawn from chronic cocaine or amphetamine administration . perhaps among the most dramatic", "importantly , this appears to be accompanied by an increase in the insertion of a - amino3-hydroxy-5-methylisoxazole-4-propionic acid ( ampa ) glutamate receptors into the membrane of spiny neurons in the accumbens , and is associated with an increase in electrophysiological sensitivity to ampa receptor stimulation ( as measured by the ampa : n - methyl d - aspartate [ nmda ] ratio ) .", "moreover , a number of other proteins regulating the fidelity of postsynaptic glutamate transmission are altered after chronic cocaine use , including proteins that regulate the structure and function of the protein scaffolding in which the glutamate receptors are embedded , including postsynaptic density ( psd)-95 and homer proteins , among others .", "also , in addition to ampa ionotropic glutamate receptors , signaling through metabotropic glutamate receptors is downregulated .", "finally , this psychostimulant - induced postsynaptic neuroplasticity is associated with changes in the biochemical machinery regulating spine formation , notably an increase in actin cycling and formation of factin ( a primary structural protein regulating spine morphology and the insertion of proteins into and out of the membrane ) .", "taken together , these findings indicate that significant changes have been produced by psychostimulants in the way that synaptically released glutamate will be interpreted by postsynaptic cells .", "thus , there remain many apparent contradictions in the literature regarding changes in specific proteins , and in the overall direction of synaptic grading ( ie , is postsynaptic glutamate transmission augmented or inhibited by chronic psychostimulant administration ) .", "therefore , for now it is probably not prudent to speculate on the type of drug development that may arise from this particular direction of research into psychostimulant - induced changes in glutamate signaling ."], ["as outlined above , given our current state of knowledge it is more likely that pharmacotherapeutic restoration of normal glutamate release may be a more successful approach than manipulating postsynaptic proteins responsible for and/or associated with changes in the fidelity of postsynaptic glutamate transmission . in part , this is due to the relatively contradictory status of the emerging literature on postsynaptic plasticity .", "moreover , it has been hypothesized that the adaptations in presynaptic glutamate release may be at least partly causal in the postsynaptic adaptations , posing the possibility that if the pathological release of glutamate can be successfully ameliorated , postsynaptic normalization may follow . \n ", "pharmacotherapeutic targets for regulating the pathological synaptic glutamate release seen in the accumbens of psychostimulant - seeking animals can be placed into two categories : ( i ) targets based upon psychostimulant induced changes in proteins regulating synaptic glutamate release ; ( ii ) proteins that produce a general decrease in excitatory transmission .", "compounds in the first category are likely to be the most specific for psychostimulant addiction , and perhaps carry the least number of unwanted side effects , while the latter category may be less selective not only regarding effects on other addictive drugs , but also in terms of unwanted side effects .", "neuroplasticity produced by chronic cocaine administration that could potentially contribute to pathological glutamate release includes downregulation of cystine - glutamate exchange , downregulation of glial glutamate transporters , and downregulation of release - regulating presynaptic metabotropic glutamate receptors ( mglur2/3 ) .", "importantly , these three changes are interrelated due to the cystine - glutamate exchanger and glutamate transporter regulating extrasynaptic glutamate tone on release regulating mglur2/3 .", "drugs have been examined in animal models of psychostimulant addiction , and to a lesser extent in clinical trials with cocaine addicts that regulate one or more of these processes .", "for example , n - acetylcysteine upregulates cystine glutamate exchange , and has been shown in animal models to prevent synaptic glutamate release associated with drug - seeking , restore inhibitory tone on synaptic release through activation of mglur2/3 , and to inhibit the desire for cocaine in a double - blind cue - reactivity trial in non - treatment - seeking cocaine addicts .", "also , mglur2/3 agonists have proven effective at inhibiting cocaine seeking in animal models ; however , unlike nacetylcysteine , food - seeking was inhibited at only a 3- to 10-fold increase in dose relative to inhibiting cocaineseeking . although no studies have yet evaluated regulating glutamate transport in drug - seeking models of psychostimulant addiction , recent reports of the use of -lactam antibiotics to increase glutamate transporter membrane insertion poses an interesting possibility for pharmacologically overcoming the cocaine - induced downregulation of glutamate transporters .", "finally , while the mechanism is not clear , modafinil has been reported to increase extracellular glutamate levels , which would restore tone on release inhibiting mglur2/3 .", "notably , modafinil has been found to successfully decrease cocaine relapse in a number of clinical trials . \n ", "the primary drugs in the category of nonspecific inhibitors of synaptic glutamate release include a variety of -aminobutyric acid ( gaba)-mimetic compounds .", "these range from relatively specific agonists at gabab receptors , such as baclofen , which inhibit synaptic glutamate release to a host of less selective compounds known to increase gaba transmission via interactions with synthetic or elimination mechanisms , such as topiramate or vigabatrin .", "for all of these compounds there is preclinical and clinical data to support some potential efficacy .", "however , as predicted , especially for the nonselective gabamimetics untoward side effects , such as sedation , are reported ."], ["this review has endeavored to transport the reader from the initiating molecular actions of amphetamine - like psychostimulants on dopamine systems in the brain to enduring neuroplasticity produced in glutamate transmission responsible for communicating from prefrontal and allocortical brain regions through the nucleus accumbens to motor regulatory systems . moreover , by examining molecular neuroplasticity produced in excitatory synapses by chronic psychostimulant administration , it is possible to make some deductions about potential pharmacotherapeutic interventions .", "indeed , there already exists an emerging literature supporting this approach in developing potential pharmacotherapies for treating psychostimulant addiction .", "importantly , this is a nascent and emerging science , and while much has been discovered , the cutting edge of discovery into the neuroplasticity produced by psychostimulants is understandably contradictory . as further discoveries are made that allow us to understand the nature of these contradictions , it should follow that additional targets will emerge to provide potential novel pharmacotherapies for treating psychostimulant addiction ."]]}
{"article_id": "PMC3979193", "article_text": ["the roots of zapoteca portricensis is a common remedy in the treatment gastrointestinal disorders used by tradomedical practitioners in eastern nigeria .", "this study was aimed at evaluating the possible antiulcer activity of the root of this plant in experimental rats .", "different groups of albino rats of male sex were given three doses ( 50 , 100 , 200 mg / kg ) of the extract .", "the ethanol model produced an average ulceration in rats with reduction of ulcer ( 50% , 75% and 90% ) seen in all the extract treatment .", "a dose dependent inhibition of ulcer was seen in all doses of the extract with doses 100 and 200 mg / kg produced a significant reduction compared with control . in the indomethacin model ,", "inhibition of ulcer ( 57.1% , 65.7% and 80.0% ) was seen in the treatments with the extracts in a dose dependent manner .", "this study has shown that roots of this plant ( zapoteca portoricensis ) possess potent antiulcer activity ."], "abstract_text": ["<S> background : the roots of zapoteca portricensis is a common remedy in the treatment gastrointestinal disorders used by tradomedical practitioners in eastern nigeria.aim:this study was aimed at evaluating the possible antiulcer activity of the root of this plant in experimental rats.methods:a methanolic root extract was prepared by cold maceration . </S>", "<S> antiulcer activity was tested using absolute ethanol and indomethacin induced ulcer models . </S>", "<S> sucralfate ( 100 mg / kg oral ) was used as the reference drug . </S>", "<S> different groups of albino rats of male sex were given three doses ( 50 , 100 , 200 mg / kg ) of the extract . </S>", "<S> phytochemical analysis of the freshly dried roots was also done.results:phytochemical results revealed presence of alkaloids , terpenoids , glycosides and flavonoids . </S>", "<S> the ethanol model produced an average ulceration in rats with reduction of ulcer ( 50% , 75% and 90% ) seen in all the extract treatment . </S>", "<S> a dose dependent inhibition of ulcer was seen in all doses of the extract with doses 100 and 200 mg / kg produced a significant reduction compared with control . in the indomethacin model , </S>", "<S> an absolute ulceration was produced in all the animals . </S>", "<S> inhibition of ulcer ( 57.1% , 65.7% and 80.0% ) was seen in the treatments with the extracts in a dose dependent manner . </S>", "<S> all the three doses of the extracts produced significant ulcer protection compared with control.conclusion:this study has shown that roots of this plant ( zapoteca portoricensis ) possess potent antiulcer activity . </S>"], "labels": null, "section_names": ["Background:", "Aim:", "Methods:", "Results:", "Conclusion:"], "sections": [["the roots of zapoteca portricensis is a common remedy in the treatment gastrointestinal disorders used by tradomedical practitioners in eastern nigeria ."], ["this study was aimed at evaluating the possible antiulcer activity of the root of this plant in experimental rats ."], ["different groups of albino rats of male sex were given three doses ( 50 , 100 , 200 mg / kg ) of the extract ."], ["the ethanol model produced an average ulceration in rats with reduction of ulcer ( 50% , 75% and 90% ) seen in all the extract treatment .", "a dose dependent inhibition of ulcer was seen in all doses of the extract with doses 100 and 200 mg / kg produced a significant reduction compared with control . in the indomethacin model ,", "inhibition of ulcer ( 57.1% , 65.7% and 80.0% ) was seen in the treatments with the extracts in a dose dependent manner ."], ["this study has shown that roots of this plant ( zapoteca portoricensis ) possess potent antiulcer activity ."]]}
{"article_id": "PMC3463667", "article_text": ["the online version of this article ( doi:10.1007/s10815 - 012 - 9815-x ) contains supplementary material , which is available to authorized users .", "the development of assisted reproductive technology ( art ) has recently enabled the direct observation of human oocytes , revealing various mysterious phenomena involving the beginning of life . however , it is undeniable that frequent microscopic examinations of human early embryos may have negative effects on them , making it difficult to obtain reliable detailed information of human embryonic development from still images .", "we therefore developed an in vitro culture system for time - lapse cinematography ( tlc ) , based on payne et al .", "this system enables non - invasive and continuous imaging of human oocyte fertilization and embryonic development .", "our previous dynamic analyses of the fertilization process in human oocytes and of human embryonic development using the in vitro tlc system   confirmed for the first time the detailed time course of sequential events during embryonic development and revealed novel phenomena [ fertilization cone , cytoplasmic strand , and splitting of the inner cell mass ( icm ) ] under culture conditions in vitro [ supplementary movie 1 ( online resource 1 ) ] .", "our further observation of the movie revealed another novel phenomenon that is likely to be involved in the mechanism of polyspermy block , wherein once the leading sperm has penetrated the zona pellucida ( zp ) and attached to the oocyte membrane , any following sperm within the zp stop penetrating immediately . to date , two types of polyspermy block were thought to exist in marine animals and mammals , including human (  oocyte membrane block  and  zona reaction  ) .", "in addition , the  oocyte membrane block  occurs in seconds , while an oocyte membrane depolarization block has not been detected in studies of mammalian oocytes .", "we therefore consider that the phenomenon described herein differs from previously proposed mechanisms for polyspermy block , prompting us to re - analyze all of our tlc data covering the fertilization process .", "herein , we provide the results of this second detailed tlc analysis , which we believe confirms the existence of a novel mechanism for the prevention of polyspermy .", "the in vitro culture system for tlc has been described in detail elsewhere . in brief", ", we used an inverted microscope ( ix-71 ; olympus , tokyo , japan ) with nomarski differential interference contrast optics ( olympus ) and a micromanipulator ( narishige , japan ) , which was covered by a handcrafted acrylic chamber .", "our system also contained a small acrylic chamber surrounded by a small waterbath on the stage of the microscope , into which a glass petri dish containing a microdrop of culture medium ( 5  l ) was placed .", "the volume of flowing co2 and temperature within the chamber were adjusted to give the optimal values ( temperature , 37.0    0.3  c ; ph , 7.37    0.03 ) .", "the inverted microscope was equipped with a ccd digital camera ( roper scientific photometrics , tucson , az ) connected to the computer and display by metamorph ( universal imaging co , downingtown , pa ) ( fig . ", "the upper two images are from the tlc system , and the lower two tables indicate imaging and culture conditions of the tlc time - lapse cinematography ( tlc ) .", "the upper two images are from the tlc system , and the lower two tables indicate imaging and culture conditions of the tlc digital images of the cultured embryos were acquired for approximately 40  h by using an exposure time of 50  s . in total , approximately 2,0002,800 frames were taken during the observation period .", "we displayed movies at 30 frames per second to analyze the fertilization process and elucidate the mechanism of polyspermy block . before the commencement of tlc observation", ", we mechanically and gently removed the cumulus cells from around the oocytes , so as not to damage the tails of the sperm that penetrated into the zp , at 1  h after the in vitro insemination ( approximately 50    10 sperm per oocyte ) .", "images were acquired at 10-s intervals for the first 2  h , and thereafter at 2-min intervals , based on our preliminary study that showed most of the sperm penetrating the zp within 3  h after the in vitro insemination .", "oocytes were collected from 122 couples after receiving informed consent , from july 2004 to december 2011 .", "one oocyte was randomly selected from each couple and 122 oocytes were tested . in the tlc images obtained from these oocytes , penetration of the leading sperm into the zp and attachment to the oocyte membrane was confirmed in the 22 oocytes . among these , only three oocytes showed both the leading and following sperms within the zp in the same frame , and these tlc data were therefore used to evaluate the dynamic changes in both sperms until the leading sperm attached to the oocyte membrane during the fertilization process .", "the ethics committee of the japanese institution for standardizing art ( jisart ) approved our study protocol .", "of the 22 imaged oocytes , in which penetration into the zp and attachment to the oocyte membrane of the leading sperm were confirmed , the leading sperm attached to the oocyte membrane within an average of 96  min after insemination , and the sperm head disappeared an average of 37  min after attachment of the sperm to the oocyte membrane .", "there was no difference in the time course of the fertilization process between the three oocytes subsequently chosen to analyze the mechanism for prevention of polyspermy and the remaining 19 oocytes .", "figure  2 shows the results of analyzing each tlc frame of the selected oocytes ( oocyte 1 , 2 and 3 ) , in which the following sperm penetrated the zp together with the leading sperm .", "penetration of the following sperm into the zp was arrested within 10  s after the leading sperm attached to the oocyte membrane , even though the tail of the following sperm was still actively moving in all three oocytes ( fig .  2 ) .", "additional data are given in supplementary movies 2 to 4 ( online resource 2 to 4).fig .", "the sperm fertilizing the oocyte ( leading sperm ) is indicated by a blue circle , while the sperm following the fertilizing sperm ( following sperm ) is indicated by a red circle among three oocytes ( a ) .", "sections of the images containing both leading and following sperms were magnified to facilitate observation of the process ( b , c , d ) . the images were acquired in 10-s intervals . with regard to oocyte 1 shown in the upper panel , both the leading and following sperm penetrated into the zona pellucida from the beginning of imaging to shortly prior to the attachment of the leading sperm to the oocyte membrane ( a , b ) .", "the leading sperm attached to the oocyte membrane 1,850  s ( 30.8  min ) after the beginning of imaging ( c ) , and the penetration of the following sperm was inhibited at 1,860  s ( d ) , which is within 10  s after the attachment of the leading sperm to the oocyte membrane . with regard to oocyte 2 shown in the middle panel and oocyte 3 shown in the lower panel ,", "the penetration of the following sperm was also inhibited within 10  s after the attachment of the leading sperm to the oocyte membrane ( c , d ) dynamics of the leading sperm and following sperm .", "the sperm fertilizing the oocyte ( leading sperm ) is indicated by a blue circle , while the sperm following the fertilizing sperm ( following sperm ) is indicated by a red circle among three oocytes ( a ) .", "sections of the images containing both leading and following sperms were magnified to facilitate observation of the process ( b , c , d ) .", "the images were acquired in 10-s intervals . with regard to oocyte 1 shown in the upper panel , both the leading and following sperm penetrated into the zona pellucida from the beginning of imaging to shortly prior to the attachment of the leading sperm to the oocyte membrane ( a , b ) . the leading sperm attached to the oocyte membrane 1,850  s ( 30.8  min ) after the beginning of imaging ( c ) , and the penetration of the following sperm was inhibited at 1,860  s ( d ) , which is within 10  s after the attachment of the leading sperm to the oocyte membrane . with regard to oocyte 2 shown in the middle panel and oocyte 3 shown in the lower panel ,", "the penetration of the following sperm was also inhibited within 10  s after the attachment of the leading sperm to the oocyte membrane ( c , d ) next , we analyzed the time course of the distance that the leading and following sperms traveled in the zp ( fig .  3 ) .", "the following sperm traveled at a similar velocity to the leading sperm , until the leading sperm attached to the oocyte membrane after penetrating the zp .", "however , once the leading sperm reached the oocyte membrane across the perivitelline space , the following sperm immediately ceased further penetration , within 10  s. the behaviors of the leading and following sperm were identical among the three oocytes.fig .", "3change in the distance between the surface of the zona pellucida and leading or following sperm .", "the time course of the change in zona pellucida penetration distance of the leading sperm ( blue line ) and the following sperm ( red line ) shows that the zona pellucida penetration of the following sperm was inhibited when the leading sperm attached to the oocyte membrane ( indicated by the arrow ) after zona pellucida penetration in all oocytes ( oocyte 1 , 2 and 3 ) used in this analysis change in the distance between the surface of the zona pellucida and leading or following sperm .", "the time course of the change in zona pellucida penetration distance of the leading sperm ( blue line ) and the following sperm ( red line ) shows that the zona pellucida penetration of the following sperm was inhibited when the leading sperm attached to the oocyte membrane ( indicated by the arrow ) after zona pellucida penetration in all oocytes ( oocyte 1 , 2 and 3 ) used in this analysis", "we revealed a novel phenomenon that is likely to be associated with the polyspermy block in human oocytes .", "this new mechanism involved cessation of the following sperm penetrating the zp within 10  s of the leading sperm penetrating the zp and attaching to the oocyte membrane .", "this was despite the following sperm retaining active tail movement and both sperm traveling at a similar velocity within the zp before penetration .", "the behaviors of the leading and following sperm were identical among the three oocytes imaged and analyzed .", "the difficulty in obtaining tlc images in which both the leading and following sperm within the zp were clearly identified precluded analysis of their respective behaviors in more than three oocytes .", "achieving normal fertilization of a single egg by a single sperm is core to life in many sexually reproducing animals , including humans . in most mammalian reproduction processes ,", "huge numbers of sperm are ejaculated into the female reproductive tract ( ratio of sperm to oocyte is approximately 10:1 ) ; however , the vast majority of sperm are rapidly eliminated from the female tract .", "it is thought that polyspermy is usually prevented by a decreased number of sperm reaching the fallopian tube and a block mechanism in the fertilized oocyte . in this context", ", it is also thought that in vitro fertilization ( ivf ) involves different mechanisms from the normal in vivo process in mammals .", "in particular , successful fertilization in vitro of a single oocyte requires a large number of motile sperms , and thus there is always the possibility that more than one sperm could penetrate the zp and reach the oocyte .", "however , between a healthy mature ovum and a healthy sperm , normal fertilization of a single egg by a single sperm occurs at a relatively high rate , which can also be confirmed easily in the environment of ivf .", "there are also many clinical cases of polyspermy that are attributable to the penetration of excess sperm after ivf .", "hundreds of articles have been published on the process of fertilization and polyspermy block in marine animals and mammals including human [ 24 ] .", "two types of mechanisms for polyspermy block have been reported : the  oocyte membrane block  to sperm penetration and the  zona reaction  .", "the former involves a depolarization of the oocyte membrane caused by the influx of na , which changes the potential of the oocyte membrane from negative to positive .", "this change in potential prevents any following sperm from attaching to the oocyte membrane , transiently contributing to polyspermy block .", "the  oocyte membrane block  or so - called  fast block  to polyspermy occurs in seconds [ 68 ] ; however , it was unlikely to be involved in  fast block  to polyspermy in the fertilization process in mammals .", "the second proposed mechanism involves a ca oscillation event activated by attachment of the sperm to the oocyte membrane .", "the subsequent increase in intracellular ca concentration triggers the exocytosis of cortical granules ( approximately 1  m in diameter ) from just below the oocyte membrane into the perivitelline space .", "enzymes such as hydrolase , proteinase , and peroxidase , which are contained in the cortical granules , prevent the penetration of following sperm by modifying the structure of the sperm receptors such as zp2 and zp3 (  slow block  to polyspermy ) and by hardening the zp .", "this  slow block  occurs within approximately 5 to 8  min of oocyte activation and is considered the main mechanism of polyspermy block in humans ; however , the mechanistic details of this process remain largely unclear in human .", "the entire fertilization process is extremely delicate and precise , and there is no doubt that multiple layers of safety mechanisms exist to ensure the achievement of normal fertilization .", "a failure of this safety mechanism is believed to instantly trigger abnormal fertilization , such as polyspermy . however , because it is so difficult to establish laboratory models of the human in vivo fertilization process and few such studies have been conducted , fertilization safety mechanisms operating in human oocytes remain unclear . in addition , dynamic morphological tactics such as tlc have not been applied to analyze the possible mechanisms of polyspermy block .", "it is true that the tlc analysis presented here still does not exactly represent the in vivo situation .", "nevertheless , we believe that these morphological data reveal a novel system for polyspermy block that could occur in human oocytes , and that the images analyzed represent the true dynamic physiology of the human fertilization process .", "we believe that a novel mechanism of polyspermy block takes place in the zp , which differs from both the oocyte membrane block to sperm penetration (  fast block  to polyspermy ) and the zona reaction (  slow block  to polyspermy ) ( fig . ", "4a proposed mechanism for a fast zona - block to polyspermy during the human fertilization process .", "the findings of our tlc analysis indicate the existence of a novel mechanism of polyspermy block , which takes place in the zona pellucida and differs from the oocyte membrane block to sperm penetration ( fast block to polyspermy ) and the zona reaction ( slow block to polyspermy ) a proposed mechanism for a fast zona - block to polyspermy during the human fertilization process . the findings of our tlc analysis indicate the existence of a novel mechanism of polyspermy block , which takes place in the zona pellucida and differs from the oocyte membrane block to sperm penetration ( fast block to polyspermy ) and the zona reaction ( slow block to polyspermy ) we are planning to elucidate the details of this novel mechanism by further observing the fertilization process of human oocytes using our tlc system .", "by use of our original tlc system , we have succeeded in demonstrating the possible existence of a novel mechanism of polyspermy block in human oocytes .", "however , the details of this mechanism of polyspermy block are not yet fully understood , and the mechanism we have revealed in this study may be a small part of a highly complicated system . we will continue to analyze the fertilization process of human oocytes by use of the tlc system to reveal the details of this novel mechanism of polyspermy block in human oocytes .", "movie 1this is the first movie to successfully demonstrate the dynamic process of fertilization in human oocytes in vitro   whereby once the leading sperm was attached to the oocyte membrane , the following sperm stopped further penetration within the zp .", "this phenomenon prompted our hypothesis that a novel mechanism could exist for the polyspermy block in the human fertilization process .", "( mpg 12897 kb ) this is the first movie to successfully demonstrate the dynamic process of fertilization in human oocytes in vitro   whereby once the leading sperm was attached to the oocyte membrane , the following sperm stopped further penetration within the zp .", "this phenomenon prompted our hypothesis that a novel mechanism could exist for the polyspermy block in the human fertilization process .", "( mpg 12897 kb ) movie 2this movie represents the same tlc imaging used to make movie 1 . while the leading sperm ( blue circle ) traveled within the zp and moved across the perivitelline space , the following sperm ( red circle ) was also steadily penetrating within the zp .", "however , once the leading sperm was attached to the oocyte membrane , the following sperm stopped penetrating within the zp even though the following sperm was far away from the leading sperm attachment site .", "( mpg 2680 kb ) this movie represents the same tlc imaging used to make movie 1 . while the leading sperm ( blue circle ) traveled within the zp and moved across the perivitelline space , the following sperm ( red circle ) was also steadily penetrating within the zp .", "however , once the leading sperm was attached to the oocyte membrane , the following sperm stopped penetrating within the zp even though the following sperm was far away from the leading sperm attachment site .", "( mpg 2680 kb ) movie 3the penetration of the following sperm in this case was also inhibited within 10  s after the attachment of the leading sperm to the oocyte .", "( mpg 2243 kb ) the penetration of the following sperm in this case was also inhibited within 10  s after the attachment of the leading sperm to the oocyte .", "( mpg 2243 kb ) movie 4this movie represents the same tlc imaging data used to make movies 2 and 3 .", "it shows that even though the tail of the following sperm was still moving actively , the sperm did not penetrate further .", "( mpg 2680 kb ) this movie represents the same tlc imaging data used to make movies 2 and 3 .", "it shows that even though the tail of the following sperm was still moving actively , the sperm did not penetrate further .", "this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited ."], "abstract_text": ["<S> purposeto analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time - lapse cinematography.methodswe had 122 oocytes donated for this study from couples that provided informed consent . </S>", "<S> we recorded human oocytes at 2,000 to 2,800 frames every 10  s during the fertilization process and thereafter every 2  min using a new in vitro culture system originally developed by the authors for time - lapse cinematography . </S>", "<S> we displayed 30 frames per second for analysis of the polyspermy block during fertilization.resultsthree oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field . </S>", "<S> the dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane , a following sperm was arrested from further penetration into the zona pellucida within 10  s.conclusionsthe present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization </S>", "<S> . these findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction . </S>", "<S> the finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.electronic supplementary materialthe online version of this article ( doi:10.1007/s10815 - 012 - 9815-x ) contains supplementary material , which is available to authorized users . </S>"], "labels": null, "section_names": ["Electronic supplementary material", "Introduction", "Materials and methods", "Results", "Discussion", "Conclusions", "Electronic supplementary materials", "Open Access"], "sections": [["the online version of this article ( doi:10.1007/s10815 - 012 - 9815-x ) contains supplementary material , which is available to authorized users ."], ["the development of assisted reproductive technology ( art ) has recently enabled the direct observation of human oocytes , revealing various mysterious phenomena involving the beginning of life . however , it is undeniable that frequent microscopic examinations of human early embryos may have negative effects on them , making it difficult to obtain reliable detailed information of human embryonic development from still images .", "we therefore developed an in vitro culture system for time - lapse cinematography ( tlc ) , based on payne et al .", "this system enables non - invasive and continuous imaging of human oocyte fertilization and embryonic development .", "our previous dynamic analyses of the fertilization process in human oocytes and of human embryonic development using the in vitro tlc system   confirmed for the first time the detailed time course of sequential events during embryonic development and revealed novel phenomena [ fertilization cone , cytoplasmic strand , and splitting of the inner cell mass ( icm ) ] under culture conditions in vitro [ supplementary movie 1 ( online resource 1 ) ] .", "our further observation of the movie revealed another novel phenomenon that is likely to be involved in the mechanism of polyspermy block , wherein once the leading sperm has penetrated the zona pellucida ( zp ) and attached to the oocyte membrane , any following sperm within the zp stop penetrating immediately . to date , two types of polyspermy block were thought to exist in marine animals and mammals , including human (  oocyte membrane block  and  zona reaction  ) .", "in addition , the  oocyte membrane block  occurs in seconds , while an oocyte membrane depolarization block has not been detected in studies of mammalian oocytes .", "we therefore consider that the phenomenon described herein differs from previously proposed mechanisms for polyspermy block , prompting us to re - analyze all of our tlc data covering the fertilization process .", "herein , we provide the results of this second detailed tlc analysis , which we believe confirms the existence of a novel mechanism for the prevention of polyspermy ."], ["the in vitro culture system for tlc has been described in detail elsewhere . in brief", ", we used an inverted microscope ( ix-71 ; olympus , tokyo , japan ) with nomarski differential interference contrast optics ( olympus ) and a micromanipulator ( narishige , japan ) , which was covered by a handcrafted acrylic chamber .", "our system also contained a small acrylic chamber surrounded by a small waterbath on the stage of the microscope , into which a glass petri dish containing a microdrop of culture medium ( 5  l ) was placed .", "the volume of flowing co2 and temperature within the chamber were adjusted to give the optimal values ( temperature , 37.0    0.3  c ; ph , 7.37    0.03 ) .", "the inverted microscope was equipped with a ccd digital camera ( roper scientific photometrics , tucson , az ) connected to the computer and display by metamorph ( universal imaging co , downingtown , pa ) ( fig . ", "the upper two images are from the tlc system , and the lower two tables indicate imaging and culture conditions of the tlc time - lapse cinematography ( tlc ) .", "the upper two images are from the tlc system , and the lower two tables indicate imaging and culture conditions of the tlc digital images of the cultured embryos were acquired for approximately 40  h by using an exposure time of 50  s . in total , approximately 2,0002,800 frames were taken during the observation period .", "we displayed movies at 30 frames per second to analyze the fertilization process and elucidate the mechanism of polyspermy block . before the commencement of tlc observation", ", we mechanically and gently removed the cumulus cells from around the oocytes , so as not to damage the tails of the sperm that penetrated into the zp , at 1  h after the in vitro insemination ( approximately 50    10 sperm per oocyte ) .", "images were acquired at 10-s intervals for the first 2  h , and thereafter at 2-min intervals , based on our preliminary study that showed most of the sperm penetrating the zp within 3  h after the in vitro insemination .", "oocytes were collected from 122 couples after receiving informed consent , from july 2004 to december 2011 .", "one oocyte was randomly selected from each couple and 122 oocytes were tested . in the tlc images obtained from these oocytes , penetration of the leading sperm into the zp and attachment to the oocyte membrane was confirmed in the 22 oocytes . among these , only three oocytes showed both the leading and following sperms within the zp in the same frame , and these tlc data were therefore used to evaluate the dynamic changes in both sperms until the leading sperm attached to the oocyte membrane during the fertilization process .", "the ethics committee of the japanese institution for standardizing art ( jisart ) approved our study protocol ."], ["of the 22 imaged oocytes , in which penetration into the zp and attachment to the oocyte membrane of the leading sperm were confirmed , the leading sperm attached to the oocyte membrane within an average of 96  min after insemination , and the sperm head disappeared an average of 37  min after attachment of the sperm to the oocyte membrane .", "there was no difference in the time course of the fertilization process between the three oocytes subsequently chosen to analyze the mechanism for prevention of polyspermy and the remaining 19 oocytes .", "figure  2 shows the results of analyzing each tlc frame of the selected oocytes ( oocyte 1 , 2 and 3 ) , in which the following sperm penetrated the zp together with the leading sperm .", "penetration of the following sperm into the zp was arrested within 10  s after the leading sperm attached to the oocyte membrane , even though the tail of the following sperm was still actively moving in all three oocytes ( fig .  2 ) .", "additional data are given in supplementary movies 2 to 4 ( online resource 2 to 4).fig .", "the sperm fertilizing the oocyte ( leading sperm ) is indicated by a blue circle , while the sperm following the fertilizing sperm ( following sperm ) is indicated by a red circle among three oocytes ( a ) .", "sections of the images containing both leading and following sperms were magnified to facilitate observation of the process ( b , c , d ) . the images were acquired in 10-s intervals . with regard to oocyte 1 shown in the upper panel , both the leading and following sperm penetrated into the zona pellucida from the beginning of imaging to shortly prior to the attachment of the leading sperm to the oocyte membrane ( a , b ) .", "the leading sperm attached to the oocyte membrane 1,850  s ( 30.8  min ) after the beginning of imaging ( c ) , and the penetration of the following sperm was inhibited at 1,860  s ( d ) , which is within 10  s after the attachment of the leading sperm to the oocyte membrane . with regard to oocyte 2 shown in the middle panel and oocyte 3 shown in the lower panel ,", "the penetration of the following sperm was also inhibited within 10  s after the attachment of the leading sperm to the oocyte membrane ( c , d ) dynamics of the leading sperm and following sperm .", "the sperm fertilizing the oocyte ( leading sperm ) is indicated by a blue circle , while the sperm following the fertilizing sperm ( following sperm ) is indicated by a red circle among three oocytes ( a ) .", "sections of the images containing both leading and following sperms were magnified to facilitate observation of the process ( b , c , d ) .", "the images were acquired in 10-s intervals . with regard to oocyte 1 shown in the upper panel , both the leading and following sperm penetrated into the zona pellucida from the beginning of imaging to shortly prior to the attachment of the leading sperm to the oocyte membrane ( a , b ) . the leading sperm attached to the oocyte membrane 1,850  s ( 30.8  min ) after the beginning of imaging ( c ) , and the penetration of the following sperm was inhibited at 1,860  s ( d ) , which is within 10  s after the attachment of the leading sperm to the oocyte membrane . with regard to oocyte 2 shown in the middle panel and oocyte 3 shown in the lower panel ,", "the penetration of the following sperm was also inhibited within 10  s after the attachment of the leading sperm to the oocyte membrane ( c , d ) next , we analyzed the time course of the distance that the leading and following sperms traveled in the zp ( fig .  3 ) .", "the following sperm traveled at a similar velocity to the leading sperm , until the leading sperm attached to the oocyte membrane after penetrating the zp .", "however , once the leading sperm reached the oocyte membrane across the perivitelline space , the following sperm immediately ceased further penetration , within 10  s. the behaviors of the leading and following sperm were identical among the three oocytes.fig .", "3change in the distance between the surface of the zona pellucida and leading or following sperm .", "the time course of the change in zona pellucida penetration distance of the leading sperm ( blue line ) and the following sperm ( red line ) shows that the zona pellucida penetration of the following sperm was inhibited when the leading sperm attached to the oocyte membrane ( indicated by the arrow ) after zona pellucida penetration in all oocytes ( oocyte 1 , 2 and 3 ) used in this analysis change in the distance between the surface of the zona pellucida and leading or following sperm .", "the time course of the change in zona pellucida penetration distance of the leading sperm ( blue line ) and the following sperm ( red line ) shows that the zona pellucida penetration of the following sperm was inhibited when the leading sperm attached to the oocyte membrane ( indicated by the arrow ) after zona pellucida penetration in all oocytes ( oocyte 1 , 2 and 3 ) used in this analysis"], ["we revealed a novel phenomenon that is likely to be associated with the polyspermy block in human oocytes .", "this new mechanism involved cessation of the following sperm penetrating the zp within 10  s of the leading sperm penetrating the zp and attaching to the oocyte membrane .", "this was despite the following sperm retaining active tail movement and both sperm traveling at a similar velocity within the zp before penetration .", "the behaviors of the leading and following sperm were identical among the three oocytes imaged and analyzed .", "the difficulty in obtaining tlc images in which both the leading and following sperm within the zp were clearly identified precluded analysis of their respective behaviors in more than three oocytes .", "achieving normal fertilization of a single egg by a single sperm is core to life in many sexually reproducing animals , including humans . in most mammalian reproduction processes ,", "huge numbers of sperm are ejaculated into the female reproductive tract ( ratio of sperm to oocyte is approximately 10:1 ) ; however , the vast majority of sperm are rapidly eliminated from the female tract .", "it is thought that polyspermy is usually prevented by a decreased number of sperm reaching the fallopian tube and a block mechanism in the fertilized oocyte . in this context", ", it is also thought that in vitro fertilization ( ivf ) involves different mechanisms from the normal in vivo process in mammals .", "in particular , successful fertilization in vitro of a single oocyte requires a large number of motile sperms , and thus there is always the possibility that more than one sperm could penetrate the zp and reach the oocyte .", "however , between a healthy mature ovum and a healthy sperm , normal fertilization of a single egg by a single sperm occurs at a relatively high rate , which can also be confirmed easily in the environment of ivf .", "there are also many clinical cases of polyspermy that are attributable to the penetration of excess sperm after ivf .", "hundreds of articles have been published on the process of fertilization and polyspermy block in marine animals and mammals including human [ 24 ] .", "two types of mechanisms for polyspermy block have been reported : the  oocyte membrane block  to sperm penetration and the  zona reaction  .", "the former involves a depolarization of the oocyte membrane caused by the influx of na , which changes the potential of the oocyte membrane from negative to positive .", "this change in potential prevents any following sperm from attaching to the oocyte membrane , transiently contributing to polyspermy block .", "the  oocyte membrane block  or so - called  fast block  to polyspermy occurs in seconds [ 68 ] ; however , it was unlikely to be involved in  fast block  to polyspermy in the fertilization process in mammals .", "the second proposed mechanism involves a ca oscillation event activated by attachment of the sperm to the oocyte membrane .", "the subsequent increase in intracellular ca concentration triggers the exocytosis of cortical granules ( approximately 1  m in diameter ) from just below the oocyte membrane into the perivitelline space .", "enzymes such as hydrolase , proteinase , and peroxidase , which are contained in the cortical granules , prevent the penetration of following sperm by modifying the structure of the sperm receptors such as zp2 and zp3 (  slow block  to polyspermy ) and by hardening the zp .", "this  slow block  occurs within approximately 5 to 8  min of oocyte activation and is considered the main mechanism of polyspermy block in humans ; however , the mechanistic details of this process remain largely unclear in human .", "the entire fertilization process is extremely delicate and precise , and there is no doubt that multiple layers of safety mechanisms exist to ensure the achievement of normal fertilization .", "a failure of this safety mechanism is believed to instantly trigger abnormal fertilization , such as polyspermy . however , because it is so difficult to establish laboratory models of the human in vivo fertilization process and few such studies have been conducted , fertilization safety mechanisms operating in human oocytes remain unclear . in addition , dynamic morphological tactics such as tlc have not been applied to analyze the possible mechanisms of polyspermy block .", "it is true that the tlc analysis presented here still does not exactly represent the in vivo situation .", "nevertheless , we believe that these morphological data reveal a novel system for polyspermy block that could occur in human oocytes , and that the images analyzed represent the true dynamic physiology of the human fertilization process .", "we believe that a novel mechanism of polyspermy block takes place in the zp , which differs from both the oocyte membrane block to sperm penetration (  fast block  to polyspermy ) and the zona reaction (  slow block  to polyspermy ) ( fig . ", "4a proposed mechanism for a fast zona - block to polyspermy during the human fertilization process .", "the findings of our tlc analysis indicate the existence of a novel mechanism of polyspermy block , which takes place in the zona pellucida and differs from the oocyte membrane block to sperm penetration ( fast block to polyspermy ) and the zona reaction ( slow block to polyspermy ) a proposed mechanism for a fast zona - block to polyspermy during the human fertilization process . the findings of our tlc analysis indicate the existence of a novel mechanism of polyspermy block , which takes place in the zona pellucida and differs from the oocyte membrane block to sperm penetration ( fast block to polyspermy ) and the zona reaction ( slow block to polyspermy ) we are planning to elucidate the details of this novel mechanism by further observing the fertilization process of human oocytes using our tlc system ."], ["by use of our original tlc system , we have succeeded in demonstrating the possible existence of a novel mechanism of polyspermy block in human oocytes .", "however , the details of this mechanism of polyspermy block are not yet fully understood , and the mechanism we have revealed in this study may be a small part of a highly complicated system . we will continue to analyze the fertilization process of human oocytes by use of the tlc system to reveal the details of this novel mechanism of polyspermy block in human oocytes ."], ["movie 1this is the first movie to successfully demonstrate the dynamic process of fertilization in human oocytes in vitro   whereby once the leading sperm was attached to the oocyte membrane , the following sperm stopped further penetration within the zp .", "this phenomenon prompted our hypothesis that a novel mechanism could exist for the polyspermy block in the human fertilization process .", "( mpg 12897 kb ) this is the first movie to successfully demonstrate the dynamic process of fertilization in human oocytes in vitro   whereby once the leading sperm was attached to the oocyte membrane , the following sperm stopped further penetration within the zp .", "this phenomenon prompted our hypothesis that a novel mechanism could exist for the polyspermy block in the human fertilization process .", "( mpg 12897 kb ) movie 2this movie represents the same tlc imaging used to make movie 1 . while the leading sperm ( blue circle ) traveled within the zp and moved across the perivitelline space , the following sperm ( red circle ) was also steadily penetrating within the zp .", "however , once the leading sperm was attached to the oocyte membrane , the following sperm stopped penetrating within the zp even though the following sperm was far away from the leading sperm attachment site .", "( mpg 2680 kb ) this movie represents the same tlc imaging used to make movie 1 . while the leading sperm ( blue circle ) traveled within the zp and moved across the perivitelline space , the following sperm ( red circle ) was also steadily penetrating within the zp .", "however , once the leading sperm was attached to the oocyte membrane , the following sperm stopped penetrating within the zp even though the following sperm was far away from the leading sperm attachment site .", "( mpg 2680 kb ) movie 3the penetration of the following sperm in this case was also inhibited within 10  s after the attachment of the leading sperm to the oocyte .", "( mpg 2243 kb ) the penetration of the following sperm in this case was also inhibited within 10  s after the attachment of the leading sperm to the oocyte .", "( mpg 2243 kb ) movie 4this movie represents the same tlc imaging data used to make movies 2 and 3 .", "it shows that even though the tail of the following sperm was still moving actively , the sperm did not penetrate further .", "( mpg 2680 kb ) this movie represents the same tlc imaging data used to make movies 2 and 3 .", "it shows that even though the tail of the following sperm was still moving actively , the sperm did not penetrate further ."], ["this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited ."]]}
{"article_id": "PMC5444548", "article_text": ["primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c .", "confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells .", "cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested .", "total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) .", "approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr )", "reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) .", "we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested .", "schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate ,", "0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization .", "lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay .", "cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp . , st .", "louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) .", "proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) .", "membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk .", "blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) .", "secondary antibodies used were horseradish peroxidase  linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) .", "antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) .", "chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals .", "each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc . , long beach , ca , usa ) .", "the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh . all animal studies and treatment protocols were approved by the mayo clinic ( rochester , mn , usa ) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research .", "we obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory , baylor college of medicine , and bred at mayo clinic .", "a handheld rebound tonometer ( icare tonolab ; colonial medical supply , franconia , nh , usa ) was used to measure iop in conscious mice .", "for iop measurements , the tonometer was held perpendicular to the cornea according to the manufacturer 's instructions", ". the tonometer records six readings from the same eye , discards the highest and lowest values , and shows the average of the remaining four values as a single iop reading .", "three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye .", "after 1 week of baseline iop measurements , stc-1 mice were treated with latanoprost ( n = 10 ) or rho kinase inhibitor y27632 ( enzo life sciences , farmingdale , ny ; n = 10 ) .", "congenic wild - type controls were treated with latanoprost ( n = 8) or y27632 ( n = 10 ) .", "treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid ( 100 m dissolved in 1:1000 dmso in pbs ) or 10 mm y27632 ( dissolved in phosphate - buffered saline ) . in the contralateral eye , vehicle was added daily in the same proportion as the treated eye for 7 consecutive days .", "additionally , wild - type mice ( n = 7 ; charles rivers laboratories , wilmington , ma , usa ) were treated with 5 l of topically administered recombinant human stc-1 ( 0.5 mg / ml ; biovendor research & diagnostic products , asheville , nc , usa ) or vehicle ( phosphate - buffered saline ) daily for 7 days to examine the effect of stc-1 on iop . in all animals ,", "the right eye served as the vehicle control eye while the left eye received study drug ( latanoprost , y27632 , or stc-1 ) .", "we recorded iop in both eyes three times daily at 1 , 4 , and 23 hours following treatment .", "anterior segments from human donor eyes ( age 75.5  17.5 years , range : 51 to 98 years ; n = 8) were perfused in culture with dmem within 10.2  4.4 hours of death as previously described . after achieving a stable baseline pressure ,", "one anterior segment from each pair received recombinant human stc-1 at concentrations of 5 , 50 , or 500 ng / ml ( dissolved in h2o ) , while the fellow eye received vehicle and served as the control .", "we added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion .", "hourly pressure readings were obtained from the average of 60 , one - minute pressure measurements using a custom - designed software program .", "the experimental eye was typically the right eye and the control was the left eye . for human anterior segments ,", "selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm 's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) .", "tissue wedges were dehydrated in a series of ascending ethanol concentrations , cleared with 100% acetone , infiltrated and embedded in epon araldite , and sectioned at 0.5 m .", "eye tissue sections were stained with toluidine blue and examined using a light microscope ( nikon corp . ,", "additional tissue wedges were sectioned at 100 nm , placed on copper film grids , and stained with uranyl acetate and lead citrate .", "tissue sections on copper film grids were examined using a transmission electron microscope ( jeol-1400 ; jeol usa , inc . , peabody , ma , usa ) . for mouse eye histopathology", "eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) overnight , and processed for light microscopy as described above for human anterior segment wedges .", "additional 100-nm sections were placed on copper film grids , and stained with uranyl acetate and lead citrate .", "processed sections were obtained following examination under a light microscope ( nikon corp . ) and a transmission electron microscope ( jeol usa , inc . ) .", "prior to initiation of statistical analysis , all data sets were evaluated for distribution assessment using the shapiro - wilk test .", "for animal studies , significance of iop change was assessed between experimental and vehicle - treated control eyes using student 's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets .", "variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes .", "statistical calculations were performed using a statistical software package ( jmp ; sas institute , inc . , cary , nc , usa ) . for human anterior segment studies ,", "the effect of latanoprost was expressed as the change in outflow facility ( c ) for each anterior segment .", "results from each pair of anterior segments were combined into a group mean for each drug , and statistical significance was analyzed using a student 's 2-tailed paired t test .", "primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c .", "confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells .", "cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested .", "total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) .", "approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr )", "reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) .", "we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested .", "schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate ,", "0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization .", "lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay .", "cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp . , st .", "louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) .", "proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) .", "membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk .", "blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) .", "secondary antibodies used were horseradish peroxidase  linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) .", "antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) .", "chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals .", "each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc . , long beach , ca , usa ) .", "the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh .", "primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c .", "confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells .", "cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested .", "total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) .", "approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr )", "reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) .", "we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested .", "schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate , 0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization .", "lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay .", "cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp .", "st . louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) .", "proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) .", "membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk .", "blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) .", "secondary antibodies used were horseradish peroxidase  linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) .", "antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) .", "chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals .", "each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc .", "the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh .", "all animal studies and treatment protocols were approved by the mayo clinic ( rochester , mn , usa ) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research .", "we obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory , baylor college of medicine , and bred at mayo clinic .", "mice , aged 5 to 8 months , were utilized in the experiments . a handheld rebound tonometer ( icare tonolab ; colonial medical supply , franconia ,", "nh , usa ) was used to measure iop in conscious mice . for iop measurements ,", ". the tonometer records six readings from the same eye , discards the highest and lowest values , and shows the average of the remaining four values as a single iop reading .", "three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye .", "after 1 week of baseline iop measurements , stc-1 mice were treated with latanoprost ( n = 10 ) or rho kinase inhibitor y27632 ( enzo life sciences , farmingdale , ny ; n = 10 ) .", "congenic wild - type controls were treated with latanoprost ( n = 8) or y27632 ( n = 10 ) .", "treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid ( 100 m dissolved in 1:1000 dmso in pbs ) or 10 mm y27632 ( dissolved in phosphate - buffered saline ) . in the contralateral eye , vehicle was added daily in the same proportion as the treated eye for 7 consecutive days .", "additionally , wild - type mice ( n = 7 ; charles rivers laboratories , wilmington , ma , usa ) were treated with 5 l of topically administered recombinant human stc-1 ( 0.5 mg / ml ; biovendor research & diagnostic products , asheville , nc , usa ) or vehicle ( phosphate - buffered saline ) daily for 7 days to examine the effect of stc-1 on iop . in all animals ,", "the right eye served as the vehicle control eye while the left eye received study drug ( latanoprost , y27632 , or stc-1 ) .", "we recorded iop in both eyes three times daily at 1 , 4 , and 23 hours following treatment .", "anterior segments from human donor eyes ( age 75.5  17.5 years , range : 51 to 98 years ; n = 8) were perfused in culture with dmem within 10.2  4.4 hours of death as previously described .", "after achieving a stable baseline pressure , one anterior segment from each pair received recombinant human stc-1 at concentrations of 5 , 50 , or 500 ng / ml ( dissolved in h2o ) , while the fellow eye received vehicle and served as the control .", "we added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion .", "hourly pressure readings were obtained from the average of 60 , one - minute pressure measurements using a custom - designed software program .", "the experimental eye was typically the right eye and the control was the left eye .", "for human anterior segments , selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm 's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) .", "tissue wedges were dehydrated in a series of ascending ethanol concentrations , cleared with 100% acetone , infiltrated and embedded in epon araldite , and sectioned at 0.5 m .", "eye tissue sections were stained with toluidine blue and examined using a light microscope ( nikon corp . ,", "additional tissue wedges were sectioned at 100 nm , placed on copper film grids , and stained with uranyl acetate and lead citrate .", "tissue sections on copper film grids were examined using a transmission electron microscope ( jeol-1400 ; jeol usa , inc .", ", peabody , ma , usa ) . for mouse eye histopathology , whole eyes were enucleated from euthanized mice following termination of the experiment .", "eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) overnight , and processed for light microscopy as described above for human anterior segment wedges .", "additional 100-nm sections were placed on copper film grids , and stained with uranyl acetate and lead citrate .", "processed sections were obtained following examination under a light microscope ( nikon corp . ) and a transmission electron microscope ( jeol usa , inc . ) .", "prior to initiation of statistical analysis , all data sets were evaluated for distribution assessment using the shapiro - wilk test . for animal studies ,", "significance of iop change was assessed between experimental and vehicle - treated control eyes using student 's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets .", "variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes .", "statistical calculations were performed using a statistical software package ( jmp ; sas institute , inc . , cary , nc , usa ) . for human anterior segment studies ,", "the effect of latanoprost was expressed as the change in outflow facility ( c ) for each anterior segment .", "results from each pair of anterior segments were combined into a group mean for each drug , and statistical significance was analyzed using a student 's 2-tailed paired t test .", "preliminary findings from confluent primary human schlemm 's canal cell lines treated with latanoprost identified stc-1 , a secreted phosphoglycoprotein hormone with multiple functions , as a gene consistently upregulated following latanoprost treatment .", "quantitative pcr studies in human schlemm 's canal cells showed an 18.1  4.3-fold ( n = 3 ) induction .", "cell lysates isolated from latanoprost - treated primary human schlemm 's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours , correlating with increased mrna expression levels identified by qpcr ( fig .", "1 ) . induction of stc-1 in schlemm 's canal cells following treatment with latanoprost .", "because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs , we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls .", "assessment of baseline iops for 6 days showed no significant difference between stc-1 mice ( 16.6  0.5 , n = 10 ) and wild - type controls ( 16.2  0.3 , n = 8 ; p = 0.1 ) .", "histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy , an open angle with appropriate trabecula within the trabecular meshwork , and intact inner and outer walls of schlemm 's canal , all similar to wild - type controls ( fig .", ", we proceeded to assess the effect of latanoprost treatment on iop in these mice .", "topical eye treatment with latanoprost ( 100 nm ) reduced iop in wild - type controls by 3.8 mm hg ( p < 0.001 , n = 8) , which correlated to a 22.0%  1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone ( fig .", "consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1 , 4 , and 23 hours after treatment ( table ) .", "in contrast , stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment ( 0.5%  0.7% , p = 0.34 , n = 10 ) at any time point throughout the 7-day treatment regime . to determine if this was unique to latanoprost , we treated stc-1 with y27632 , a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway .", "treatment of either wild - type ( n = 10 ) or stc-1 ( n = 10 ) mice with y27632 resulted in significant reductions of iop , 12.5%  1.2% and 13.1%  2.8% , respectively ( p < 0.0001 , n = 10 ; fig . 4 ; table ) .", "these data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction .", "top : light microscopy images of whole eye sections from wild - type and stc-1 mice .", "wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . middle : light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula , trabecular meshwork ( tm ) cells , and schlemm 's canal ( sc ) volume .", "bottom : transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm 's canal inner walls ( iw ) and outer walls ( ow ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following latanoprost treatment .", "while congenic control mice show iop reduction with latanoprost , stc-1 do not , suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction .", "intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice .", "n = 10 ) and congenic controls ( n = 10 ) were treated once daily with 10 mm y27632 .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following y27632 treatment .", "both stc-1 mice and congenic controls showed iop reduction following treatment with y27632 . to determine if stc-1 would influence iop reduction by itself , we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours .", "perfusion with either 5 ng / ml ( 0.15  0.04 to 0.15  0.04 l / min / mm hg , n = 2 , p = 0.20 ) or 50 ng / ml ( 0.14  0.05 to 0.18  0.07 l / min / mm hg , n = 4 , p = 0.20 ) had no significant effect on outflow facility .", "however , all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility ( 0.15  0.03 to 0.27  0.09 l / min / mm hg , n = 5 , p = 0.02 ) compared to baseline ( fig . 5 ) .", "paired controls treated with vehicle showed no change in outflow facility from their baseline values . to determine whether the changes in pressure could be secondary to morphologic changes", "morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm 's canal of control and treated eyes ( fig .", "no major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm 's canal inner and outer walls were observed .", "these results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop .", "( a ) outflow facility of human anterior segments ( n = 8) following perfusion with 5 , 50 , or 500 ng / ml of recombinant human stc-1 .", "( b ) representative graph of an eye pair perfused with 500 ng / ml stc-1 .", "( a , b ) representative sections ( 3 m ) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . ( c , d ) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes .", "recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment .", "ac , anterior chamber ; jct , juxtacanalicular region ; tm , trabecular meshwork ; sc , schlemm 's canal . to determine", "if stc-1 has ocular hypotensive activity in vivo , wild - type c57bl/6 mice were treated with recombinant stc-1 ( 0.5 mg / ml ) or vehicle daily for 7 days ( fig .", "stanniocalcin-1 reduced iop by 15.2%  3.0% when compared to vehicle - treated contralateral eyes ( range of iop reduction from 5.1%  5.2%", "these results suggest that stc-1 has iop - lowering properties when used as a standalone agent .", "wild - type mice ( n = 10 ) were treated topically with recombinant stc-1 ( 0.5 mg / ml ) or vehicle ( phosphate buffered saline ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following stc-1 treatment .", "preliminary findings from confluent primary human schlemm 's canal cell lines treated with latanoprost identified stc-1 , a secreted phosphoglycoprotein hormone with multiple functions , as a gene consistently upregulated following latanoprost treatment .", "quantitative pcr studies in human schlemm 's canal cells showed an 18.1  4.3-fold ( n = 3 ) induction .", "cell lysates isolated from latanoprost - treated primary human schlemm 's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours , correlating with increased mrna expression levels identified by qpcr ( fig .", "1 ) . induction of stc-1 in schlemm 's canal cells following treatment with latanoprost .", "because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs , we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls .", "assessment of baseline iops for 6 days showed no significant difference between stc-1 mice ( 16.6  0.5 , n = 10 ) and wild - type controls ( 16.2  0.3 , n = 8 ; p = 0.1 ) .", "histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy , an open angle with appropriate trabecula within the trabecular meshwork , and intact inner and outer walls of schlemm 's canal , all similar to wild - type controls ( fig .", ", we proceeded to assess the effect of latanoprost treatment on iop in these mice .", "topical eye treatment with latanoprost ( 100 nm ) reduced iop in wild - type controls by 3.8 mm hg ( p < 0.001 , n = 8) , which correlated to a 22.0%  1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone ( fig .", "consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1 , 4 , and 23 hours after treatment ( table ) .", "in contrast , stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment ( 0.5%  0.7% , p = 0.34 , n = 10 ) at any time point throughout the 7-day treatment regime . to determine if this was unique to latanoprost , we treated stc-1 with y27632 , a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway .", "treatment of either wild - type ( n = 10 ) or stc-1 ( n = 10 ) mice with y27632 resulted in significant reductions of iop , 12.5%  1.2% and 13.1%  2.8% , respectively ( p < 0.0001 , n = 10 ; fig . 4 ; table ) .", "these data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction .", "top : light microscopy images of whole eye sections from wild - type and stc-1 mice .", "wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . middle : light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula , trabecular meshwork ( tm ) cells , and schlemm 's canal ( sc ) volume .", "bottom : transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm 's canal inner walls ( iw ) and outer walls ( ow ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following latanoprost treatment .", "while congenic control mice show iop reduction with latanoprost , stc-1 do not , suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction .", "intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice .", "stc-1 mice ( n = 10 ) and congenic controls ( n = 10 ) were treated once daily with 10 mm y27632 . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following y27632 treatment .", "to determine if stc-1 would influence iop reduction by itself , we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours .", "perfusion with either 5 ng / ml ( 0.15  0.04 to 0.15  0.04 l / min / mm hg , n = 2 , p = 0.20 ) or 50 ng / ml ( 0.14  0.05 to 0.18  0.07 l / min / mm hg , n = 4 , p = 0.20 ) had no significant effect on outflow facility .", "however , all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility ( 0.15  0.03 to 0.27  0.09 l / min / mm hg , n = 5 , p = 0.02 ) compared to baseline ( fig . 5 ) .", "paired controls treated with vehicle showed no change in outflow facility from their baseline values . to determine whether the changes in pressure could be secondary to morphologic changes", "morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm 's canal of control and treated eyes ( fig .", "6 ) . no major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm 's canal inner and outer walls were observed .", "these results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop .", "( a ) outflow facility of human anterior segments ( n = 8) following perfusion with 5 , 50 , or 500 ng / ml of recombinant human stc-1 .", "( b ) representative graph of an eye pair perfused with 500 ng / ml stc-1 .", "p  0.05 . histologic analysis of human anterior segment ocular tissue following treatment with recombinant stc-1 .", "( a , b ) representative sections ( 3 m ) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . ( c , d ) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes .", "recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment .", "ac , anterior chamber ; jct , juxtacanalicular region ; tm , trabecular meshwork ; sc , schlemm 's canal .", "to determine if stc-1 has ocular hypotensive activity in vivo , wild - type c57bl/6 mice were treated with recombinant stc-1 ( 0.5 mg / ml ) or vehicle daily for 7 days ( fig .", "stanniocalcin-1 reduced iop by 15.2%  3.0% when compared to vehicle - treated contralateral eyes ( range of iop reduction from 5.1%  5.2% [ day 1 ] to 21.4%  5.3%", "these results suggest that stc-1 has iop - lowering properties when used as a standalone agent .", "wild - type mice ( n = 10 ) were treated topically with recombinant stc-1 ( 0.5 mg / ml ) or vehicle ( phosphate buffered saline ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following stc-1 treatment .", "prostaglandin analogs like latanoprost are a first - line medical therapy for iop reduction due to their once daily dosing , greater response rates , and greater amount of iop reduction compared to other classes of pressure - lowering medications .", "identifying the critical effector molecules in the signaling pathway of latanoprost will provide insights into additional molecules to target for iop reduction , potentially with fewer side effects . in the current study ,", "we identified stc-1 as a transcript and protein that is highly induced following treatment of human schlemm 's canal cells with latanoprost .", "topical latanoprost significantly reduced iop in wild - type controls while stc-1 mice demonstrated no iop reduction to topical latanoprost . in contrast , rho kinase inhibitor y27632 demonstrated iop reduction in both wild - type and stc-1 mice , indicating that stc-1 is a unique and important downstream signaling molecule necessary for the ocular hypotensive properties of latanoprost .", "additionally , recombinant stc-1 by itself also increased outflow facility in human anterior segments and reduced iop in vivo in wild - type mice .", "together , these results suggest that stc-1 is a critical and unique effector molecule for the latanoprost signaling pathway that is necessary for latanoprost - induced iop reduction and that stc-1 by itself can act as an ocular hypotensive agent .", "stanniocalcin was first described in fish as a 50-kda homodimeric glycoprotein that is secreted from the corpuscles of stannius into the bloodstream in the setting of hypercalcemia to regulate calcium excretion at the gills and gut . in mammals , two homologues of stc", "stc-1 is the most studied of the two mammalian forms , having a 50% amino acid homology with its fish counterpart .", "stc-1 is a secreted , homodimeric phosphoglycoprotein that has preserved protein structure similarity between mammals and fish including the conservation of 11 cysteine residues .", "it is expressed in a wide variety of tissues most notably bone , skeletal muscle , heart , thymus , and spleen .", "functionally , stc-1 has been associated with calcium uptake , hypoxic preconditioning , and antioxidative stress properties through suppression of reactive oxygen species .", "additionally , stc-1 has also been shown to be neuroprotective for neurons , photoreceptors , and retinal ganglion cells , and has been linked to anti - inflammatory effects by inhibiting macrophage chemotaxis , modulating transendothelial migration of leukocytes and reducing t cell infiltration . in treating wild - type mice with topical latanoprost", ", we observed a reduction in iop by approximately 22.0%  1.9% , consistent with previous reports , while the stc-1 mice showed no response ( 0.55%  0.7% ) .", "mice that were stc-1 were responsive to rho kinase inhibitor y27632 , which confirmed that stc-1 mice have normal functioning outflow pathways , but that elimination of stc-1 expression in these mice renders topical latanoprost ineffective .", "the current study adds schlemm 's canal cells to the list of cells that express stc-1 and shows that its expression is highly influenced by latanoprost .", "this is an important finding as little is known about the molecular events that connect latanoprost treatment with iop reduction .", "latanoprost has been shown to phosphorylate myosin light chain kinase in the iris , indicating a role for latanoprost in cell relaxation .", "however , stc-1 is the first individual molecule that has been identified as a key downstream effector of latanoprost signaling .", "in addition to its variety of functional activities , our results also suggest that stc-1 has ocular hypotensive properties , since addition of recombinant stc-1 to human anterior segment cultures decreased pressure and increased outflow facility , and when applied topically to wild - type mice reduced iop in vivo . despite the benefits of latanoprost in lowering iop", ", long - term treatment with latanoprost can often cause significant side effects , which can be contraindicative for its prolonged usage .", "identification of stc-1 as a downstream signaling molecule through which latanoprost executes its iop - lowering effects , and the fact that recombinant stc-1 shows ocular hypotensive properties , makes this an attractive therapeutic target .", "utilization of a specific downstream molecule such as stc-1 within the latanoprost signaling cascade may eliminate some of the major side effects while providing a specialized therapeutic strategy . in summary ,", "additionally , stc-1 demonstrates ocular hypotensive properties when used by itself , mimicking the effect of latanoprost . given its novel role in latanoprost - mediated iop reduction", ", stc-1 may be considered a promising candidate molecule for devising future therapeutic regiments to reduce iop ."], "abstract_text": ["<S> purposeto identify downstream signaling molecules through which intraocular pressure ( iop ) is lowered following treatment with the prostaglandin analog latanoprost.methodstotal rna and protein isolated from primary human schlemm 's canal cells ( n = 3 ) treated with latanoprost ( free acid ; 100 nm ) were processed for quantitative pcr and western blot analysis . </S>", "<S> iop was evaluated in stanniocalcin-1 ( stc-1/ ) and wild - type mice following treatment with latanoprost or rho kinase inhibitor y27632 . </S>", "<S> human anterior segment pairs ( n = 8) were treated with recombinant stc-1 ( 5 , 50 , or 500 ng / ml ) and pressure was recorded using custom - designed software . </S>", "<S> the effect of recombinant stc-1 ( 0.5 mg / ml ) on iop was evaluated in wild - type mice . </S>", "<S> tissue morphology was evaluated by light and transmission electron microscopy.resultsincreased stc-1 mrna ( 4.0- to 25.2-fold ) and protein expression ( 1.9- to 5.1-fold ) was observed within 12 hours following latanoprost treatment . </S>", "<S> latanoprost reduced iop in wild - type mice ( 22.0%  1.9% ) , but had no effect on stc-1/ mice ( 0.5%  0.7% ) . </S>", "<S> in contrast , y27632 reduced iop in both wild - type ( 12.5%  1.2% ) and in stc-1/ mice ( 13.1%  2.8% ) . </S>", "<S> human anterior segments treated with stc-1 ( 500 ng / ml ) showed an increase in outflow facility ( 0.15  0.03 to 0.27  0.09 l / min / mm hg ) while no change was observed in paired vehicle - treated controls . </S>", "<S> recombinant stc-1 reduced iop in wild - type mice by 15.2%  3.0% . </S>", "<S> no observable morphologic changes were identified between treatment groups when evaluated by microscopy.conclusionslatanoprost-induced reduction of iop is mediated through the downstream signaling molecule stc-1 . when used by itself , stc-1 exhibits ocular hypotensive properties . </S>"], "labels": null, "section_names": ["Methods", "Validation of STC-1 Expression", "Cell Culture.", "RNA Analysis.", "Protein Analysis.", "Animal Experiments", "Ex Vivo Human Anterior Segment Culture", "Tissue Preparation and Microscopy", "Statistics", "Results", "STC-1 Is Upregulated in Latanoprost-Treated Primary Human Schlemm's Canal Cells", "IOP Recordings in STC-1 Knockout Mice Following Treatment With Latanoprost", "STC-1 Perfusion in Ex Vivo Human Anterior Segment Culture", "Effect of Recombinant STC-1 on IOP", "Discussion"], "sections": [["primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c .", "confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells .", "cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested .", "total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) .", "approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr )", "reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) .", "we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested .", "schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate ,", "0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization .", "lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay .", "cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp . , st .", "louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) .", "proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) .", "membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk .", "blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) .", "secondary antibodies used were horseradish peroxidase  linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) .", "antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) .", "chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals .", "each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc . , long beach , ca , usa ) .", "the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh . all animal studies and treatment protocols were approved by the mayo clinic ( rochester , mn , usa ) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research .", "we obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory , baylor college of medicine , and bred at mayo clinic .", "a handheld rebound tonometer ( icare tonolab ; colonial medical supply , franconia , nh , usa ) was used to measure iop in conscious mice .", "for iop measurements , the tonometer was held perpendicular to the cornea according to the manufacturer 's instructions", ". the tonometer records six readings from the same eye , discards the highest and lowest values , and shows the average of the remaining four values as a single iop reading .", "three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye .", "after 1 week of baseline iop measurements , stc-1 mice were treated with latanoprost ( n = 10 ) or rho kinase inhibitor y27632 ( enzo life sciences , farmingdale , ny ; n = 10 ) .", "congenic wild - type controls were treated with latanoprost ( n = 8) or y27632 ( n = 10 ) .", "treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid ( 100 m dissolved in 1:1000 dmso in pbs ) or 10 mm y27632 ( dissolved in phosphate - buffered saline ) . in the contralateral eye , vehicle was added daily in the same proportion as the treated eye for 7 consecutive days .", "additionally , wild - type mice ( n = 7 ; charles rivers laboratories , wilmington , ma , usa ) were treated with 5 l of topically administered recombinant human stc-1 ( 0.5 mg / ml ; biovendor research & diagnostic products , asheville , nc , usa ) or vehicle ( phosphate - buffered saline ) daily for 7 days to examine the effect of stc-1 on iop . in all animals ,", "the right eye served as the vehicle control eye while the left eye received study drug ( latanoprost , y27632 , or stc-1 ) .", "we recorded iop in both eyes three times daily at 1 , 4 , and 23 hours following treatment .", "anterior segments from human donor eyes ( age 75.5  17.5 years , range : 51 to 98 years ; n = 8) were perfused in culture with dmem within 10.2  4.4 hours of death as previously described . after achieving a stable baseline pressure ,", "one anterior segment from each pair received recombinant human stc-1 at concentrations of 5 , 50 , or 500 ng / ml ( dissolved in h2o ) , while the fellow eye received vehicle and served as the control .", "we added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion .", "hourly pressure readings were obtained from the average of 60 , one - minute pressure measurements using a custom - designed software program .", "the experimental eye was typically the right eye and the control was the left eye . for human anterior segments ,", "selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm 's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) .", "tissue wedges were dehydrated in a series of ascending ethanol concentrations , cleared with 100% acetone , infiltrated and embedded in epon araldite , and sectioned at 0.5 m .", "eye tissue sections were stained with toluidine blue and examined using a light microscope ( nikon corp . ,", "additional tissue wedges were sectioned at 100 nm , placed on copper film grids , and stained with uranyl acetate and lead citrate .", "tissue sections on copper film grids were examined using a transmission electron microscope ( jeol-1400 ; jeol usa , inc . , peabody , ma , usa ) . for mouse eye histopathology", "eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) overnight , and processed for light microscopy as described above for human anterior segment wedges .", "additional 100-nm sections were placed on copper film grids , and stained with uranyl acetate and lead citrate .", "processed sections were obtained following examination under a light microscope ( nikon corp . ) and a transmission electron microscope ( jeol usa , inc . ) .", "prior to initiation of statistical analysis , all data sets were evaluated for distribution assessment using the shapiro - wilk test .", "for animal studies , significance of iop change was assessed between experimental and vehicle - treated control eyes using student 's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets .", "variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes .", "statistical calculations were performed using a statistical software package ( jmp ; sas institute , inc . , cary , nc , usa ) . for human anterior segment studies ,", "the effect of latanoprost was expressed as the change in outflow facility ( c ) for each anterior segment .", "results from each pair of anterior segments were combined into a group mean for each drug , and statistical significance was analyzed using a student 's 2-tailed paired t test ."], ["primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c .", "confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells .", "cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested .", "total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) .", "approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr )", "reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) .", "we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c .", "primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested .", "schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate ,", "0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization .", "lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay .", "cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp . , st .", "louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) .", "proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) .", "membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk .", "blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) .", "secondary antibodies used were horseradish peroxidase  linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) .", "antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) .", "chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals .", "each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc . , long beach , ca , usa ) .", "the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh ."], ["primary human schlemm 's canal cell lines were grown to confluence in six - well plates ( bd falcon , franklin lakes , nj , usa ) containing dulbecco 's modified eagle 's medium ( dmem ; life technologies , grand island , ny , usa ) ; 10% fetal bovine serum ( mediatech , manassas , va , usa ) ; and 1% penicillin / streptomycin ( life technologies ) in 5% co2 at 37c .", "confluent cells were washed twice with phosphate - buffered saline and incubated in serum - free dmem for 24 hours to synchronize the growth potential of the cells .", "cells were incubated with latanoprost ( de - esterified free - acid form at 100 nm final concentration ; cayman chemical , ann arbor , mi , usa ) or vehicle ( ethanol , final dilution 1:1000 ) in dmem containing 1% penicillin / streptomycin ."], ["primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 6 hours , and harvested .", "total rna was extracted using an rna isolation kit ( rneasy total rna isolation kit ; qiagen , hilden , germany ) .", "approximately 250 ng of total rna was reverse transcribed into cdna using a synthesis kit ( iscript cdna ; bio - rad laboratories , inc . , hercules , ca , usa ) . quantitative real - time polymerase chain ( qpcr )", "reaction using stc-1 ( forward , 5-aggcggagcagaatgactc-3 ; reverse , 5-gttgaggcaacgaaccactt-3 ) and glyceraldehyde 3-phosphate dehydrogenase ( gapdh : forward , 5-cctctgacttcaacagc-3 ; reverse , 5-gctgtagccaaattcgt-3 ) primers were performed on a pcr system using a master mix ( roche light cycler 480 with sybr green i ; roche , indianapolis , in , usa ) .", "we performed qpcr amplification with a predenaturation step at 95c followed by 45 cycles of denaturation at 95c , annealing at 63c , and extension at 72c ."], ["primary schlemm 's canal cells were grown to confluence , treated with 100 nm latanoprost for 15 minutes , 1 , 2 , 4 , or 6 hours , and harvested .", "schlemm 's canal cell pellets were suspended in ice - cold lysis buffer ( 50 mm tris ph 8.0 , 0.5% sodium dodecyl sulfate , 0.5% triton x-100 , 137 mm nacl , 3 mm kcl , 8 mm na2hpo4 - 7h2o , 1 mm kh2po4 , protease inhibitors ; roche ) and passed repeatedly through a 21-gauge needle for homogenization .", "lysate was centrifuged at 13,000 g for 10 minutes , and total protein was quantified by the bradford assay .", "cell lysates containing 20 g total protein were mixed with reducing lane marker sample buffer ( thermo fisher scientific , waltham , ma , usa ) containing 15% 2-mercaptoethanol ( sigma - aldrich corp .", "st . louis , mo , usa ) , heated and separated on a 4% to 15% sds - page gradient gel ( bio - rad laboratories , inc . ) .", "proteins were transferred to polyvinylidene difluoride membrane ( millipore corp . , billerica , ma , usa ) in 1x transfer buffer ( 50 mm tris , 384 mm glycine , 0.01% sds , 20% methanol ) .", "membranes were blocked in 20 mm tris ( ph 7.5 ) , 150 mm nacl , 0.05% tween-20 , and 2% instant nonfat dry milk .", "blots were probed with rabbit monoclonal anti - human stc-1 ( novus biologicals , littleton , co , usa ) and mouse monoclonal anti - human gapdh ( novus biologicals ) .", "secondary antibodies used were horseradish peroxidase  linked anti - rabbit or anti - mouse , respectively ( ge healthcare , piscataway , nj , usa ) .", "antibody / antigen complexes were detected using ecl western blot signal detection reagent ( ge healthcare ) .", "chemiluminescence film ( biomax xar ; eastman kodak , rochester , ny , usa ) was used to visualize protein signals .", "each film was digitized with a photographic scanner ( epson perfection 2400 ; epson america , inc .", "the band intensities for western blot analysis were quantified using imagej software ( http://rsb.info.nih.gov/ij/index.html in the public domain by the national institutes of health , bethesda , md , usa ) and normalized to gapdh ."], ["all animal studies and treatment protocols were approved by the mayo clinic ( rochester , mn , usa ) institutional animal care and use committee and adhered to the arvo statement for the use of animals in ophthalmic and vision research .", "we obtained stc-1 and littermate wild - type mice from the sheikh - hamad laboratory , baylor college of medicine , and bred at mayo clinic .", "mice , aged 5 to 8 months , were utilized in the experiments . a handheld rebound tonometer ( icare tonolab ; colonial medical supply , franconia ,", "nh , usa ) was used to measure iop in conscious mice . for iop measurements ,", ". the tonometer records six readings from the same eye , discards the highest and lowest values , and shows the average of the remaining four values as a single iop reading .", "three independent measurements were obtained daily at similar time points and were averaged to obtain the daily iop value for each eye .", "after 1 week of baseline iop measurements , stc-1 mice were treated with latanoprost ( n = 10 ) or rho kinase inhibitor y27632 ( enzo life sciences , farmingdale , ny ; n = 10 ) .", "congenic wild - type controls were treated with latanoprost ( n = 8) or y27632 ( n = 10 ) .", "treatments were daily in one eye for 7 consecutive days with 5 l of latanoprost - free acid ( 100 m dissolved in 1:1000 dmso in pbs ) or 10 mm y27632 ( dissolved in phosphate - buffered saline ) . in the contralateral eye , vehicle was added daily in the same proportion as the treated eye for 7 consecutive days .", "additionally , wild - type mice ( n = 7 ; charles rivers laboratories , wilmington , ma , usa ) were treated with 5 l of topically administered recombinant human stc-1 ( 0.5 mg / ml ; biovendor research & diagnostic products , asheville , nc , usa ) or vehicle ( phosphate - buffered saline ) daily for 7 days to examine the effect of stc-1 on iop . in all animals ,", "the right eye served as the vehicle control eye while the left eye received study drug ( latanoprost , y27632 , or stc-1 ) .", "we recorded iop in both eyes three times daily at 1 , 4 , and 23 hours following treatment ."], ["anterior segments from human donor eyes ( age 75.5  17.5 years , range : 51 to 98 years ; n = 8) were perfused in culture with dmem within 10.2  4.4 hours of death as previously described .", "after achieving a stable baseline pressure , one anterior segment from each pair received recombinant human stc-1 at concentrations of 5 , 50 , or 500 ng / ml ( dissolved in h2o ) , while the fellow eye received vehicle and served as the control .", "we added stc-1 and vehicle using a gravity - driven constant pressure method of anterior chamber exchange followed by continuous perfusion .", "hourly pressure readings were obtained from the average of 60 , one - minute pressure measurements using a custom - designed software program .", "the experimental eye was typically the right eye and the control was the left eye ."], ["for human anterior segments , selected wedges of tissue 180 apart that included the trabecular meshwork and schlemm 's canal were isolated and fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) .", "tissue wedges were dehydrated in a series of ascending ethanol concentrations , cleared with 100% acetone , infiltrated and embedded in epon araldite , and sectioned at 0.5 m .", "eye tissue sections were stained with toluidine blue and examined using a light microscope ( nikon corp . ,", "additional tissue wedges were sectioned at 100 nm , placed on copper film grids , and stained with uranyl acetate and lead citrate .", "tissue sections on copper film grids were examined using a transmission electron microscope ( jeol-1400 ; jeol usa , inc .", ", peabody , ma , usa ) . for mouse eye histopathology , whole eyes were enucleated from euthanized mice following termination of the experiment .", "eyes were placed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) overnight , and processed for light microscopy as described above for human anterior segment wedges .", "additional 100-nm sections were placed on copper film grids , and stained with uranyl acetate and lead citrate .", "processed sections were obtained following examination under a light microscope ( nikon corp . ) and a transmission electron microscope ( jeol usa , inc . ) ."], ["prior to initiation of statistical analysis , all data sets were evaluated for distribution assessment using the shapiro - wilk test . for animal studies ,", "significance of iop change was assessed between experimental and vehicle - treated control eyes using student 's paired t - test for data sets with normal distribution and wilcoxon sign - rank test for nonparametric data sets .", "variations in daily iop are graphically presented as the mean daily iop of the vehicle and treated eyes .", "statistical calculations were performed using a statistical software package ( jmp ; sas institute , inc . , cary , nc , usa ) . for human anterior segment studies ,", "the effect of latanoprost was expressed as the change in outflow facility ( c ) for each anterior segment .", "results from each pair of anterior segments were combined into a group mean for each drug , and statistical significance was analyzed using a student 's 2-tailed paired t test ."], ["preliminary findings from confluent primary human schlemm 's canal cell lines treated with latanoprost identified stc-1 , a secreted phosphoglycoprotein hormone with multiple functions , as a gene consistently upregulated following latanoprost treatment .", "quantitative pcr studies in human schlemm 's canal cells showed an 18.1  4.3-fold ( n = 3 ) induction .", "cell lysates isolated from latanoprost - treated primary human schlemm 's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours , correlating with increased mrna expression levels identified by qpcr ( fig .", "1 ) . induction of stc-1 in schlemm 's canal cells following treatment with latanoprost .", "because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs , we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls .", "assessment of baseline iops for 6 days showed no significant difference between stc-1 mice ( 16.6  0.5 , n = 10 ) and wild - type controls ( 16.2  0.3 , n = 8 ; p = 0.1 ) .", "histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy , an open angle with appropriate trabecula within the trabecular meshwork , and intact inner and outer walls of schlemm 's canal , all similar to wild - type controls ( fig .", ", we proceeded to assess the effect of latanoprost treatment on iop in these mice .", "topical eye treatment with latanoprost ( 100 nm ) reduced iop in wild - type controls by 3.8 mm hg ( p < 0.001 , n = 8) , which correlated to a 22.0%  1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone ( fig .", "consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1 , 4 , and 23 hours after treatment ( table ) .", "in contrast , stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment ( 0.5%  0.7% , p = 0.34 , n = 10 ) at any time point throughout the 7-day treatment regime . to determine if this was unique to latanoprost , we treated stc-1 with y27632 , a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway .", "treatment of either wild - type ( n = 10 ) or stc-1 ( n = 10 ) mice with y27632 resulted in significant reductions of iop , 12.5%  1.2% and 13.1%  2.8% , respectively ( p < 0.0001 , n = 10 ; fig . 4 ; table ) .", "these data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction .", "top : light microscopy images of whole eye sections from wild - type and stc-1 mice .", "wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . middle : light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula , trabecular meshwork ( tm ) cells , and schlemm 's canal ( sc ) volume .", "bottom : transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm 's canal inner walls ( iw ) and outer walls ( ow ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following latanoprost treatment .", "while congenic control mice show iop reduction with latanoprost , stc-1 do not , suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction .", "intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice .", "n = 10 ) and congenic controls ( n = 10 ) were treated once daily with 10 mm y27632 .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following y27632 treatment .", "both stc-1 mice and congenic controls showed iop reduction following treatment with y27632 . to determine if stc-1 would influence iop reduction by itself , we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours .", "perfusion with either 5 ng / ml ( 0.15  0.04 to 0.15  0.04 l / min / mm hg , n = 2 , p = 0.20 ) or 50 ng / ml ( 0.14  0.05 to 0.18  0.07 l / min / mm hg , n = 4 , p = 0.20 ) had no significant effect on outflow facility .", "however , all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility ( 0.15  0.03 to 0.27  0.09 l / min / mm hg , n = 5 , p = 0.02 ) compared to baseline ( fig . 5 ) .", "paired controls treated with vehicle showed no change in outflow facility from their baseline values . to determine whether the changes in pressure could be secondary to morphologic changes", "morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm 's canal of control and treated eyes ( fig .", "no major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm 's canal inner and outer walls were observed .", "these results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop .", "( a ) outflow facility of human anterior segments ( n = 8) following perfusion with 5 , 50 , or 500 ng / ml of recombinant human stc-1 .", "( b ) representative graph of an eye pair perfused with 500 ng / ml stc-1 .", "( a , b ) representative sections ( 3 m ) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . ( c , d ) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes .", "recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment .", "ac , anterior chamber ; jct , juxtacanalicular region ; tm , trabecular meshwork ; sc , schlemm 's canal . to determine", "if stc-1 has ocular hypotensive activity in vivo , wild - type c57bl/6 mice were treated with recombinant stc-1 ( 0.5 mg / ml ) or vehicle daily for 7 days ( fig .", "stanniocalcin-1 reduced iop by 15.2%  3.0% when compared to vehicle - treated contralateral eyes ( range of iop reduction from 5.1%  5.2%", "these results suggest that stc-1 has iop - lowering properties when used as a standalone agent .", "wild - type mice ( n = 10 ) were treated topically with recombinant stc-1 ( 0.5 mg / ml ) or vehicle ( phosphate buffered saline ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following stc-1 treatment ."], ["preliminary findings from confluent primary human schlemm 's canal cell lines treated with latanoprost identified stc-1 , a secreted phosphoglycoprotein hormone with multiple functions , as a gene consistently upregulated following latanoprost treatment .", "quantitative pcr studies in human schlemm 's canal cells showed an 18.1  4.3-fold ( n = 3 ) induction .", "cell lysates isolated from latanoprost - treated primary human schlemm 's canal cells at various time points showed an increase of stc-1 protein expression of 1.9-fold at 2 hours and approximately 5.0-fold at 4 and 6 hours , correlating with increased mrna expression levels identified by qpcr ( fig .", "1 ) . induction of stc-1 in schlemm 's canal cells following treatment with latanoprost ."], ["because of its multifunctional properties and ability to act in an autocrine and paracrine fashion similar to prostaglandin analogs , we examined the role of stc-1 in downstream signaling following latanoprost treatment in vivo using stc-1 mice and congenic wild - type controls .", "assessment of baseline iops for 6 days showed no significant difference between stc-1 mice ( 16.6  0.5 , n = 10 ) and wild - type controls ( 16.2  0.3 , n = 8 ; p = 0.1 ) .", "histologic examination of stc-1 mice by light and transmission electron microscopy showed a normal - appearing ocular anatomy , an open angle with appropriate trabecula within the trabecular meshwork , and intact inner and outer walls of schlemm 's canal , all similar to wild - type controls ( fig .", ", we proceeded to assess the effect of latanoprost treatment on iop in these mice .", "topical eye treatment with latanoprost ( 100 nm ) reduced iop in wild - type controls by 3.8 mm hg ( p < 0.001 , n = 8) , which correlated to a 22.0%  1.9% decrease in iop when compared to the fellow contralateral eyes treated with vehicle alone ( fig .", "consistent iop change was identified throughout the once daily dose as determined by iop pressure monitoring at 1 , 4 , and 23 hours after treatment ( table ) .", "in contrast , stc-1 mice did not show any significant reduction in iop after topical latanoprost treatment ( 0.5%  0.7% , p = 0.34 , n = 10 ) at any time point throughout the 7-day treatment regime . to determine if this was unique to latanoprost , we treated stc-1 with y27632 , a rho kinase inhibitor that increases outflow through both the uveoscleral and the conventional outflow pathway .", "treatment of either wild - type ( n = 10 ) or stc-1 ( n = 10 ) mice with y27632 resulted in significant reductions of iop , 12.5%  1.2% and 13.1%  2.8% , respectively ( p < 0.0001 , n = 10 ; fig . 4 ; table ) .", "these data suggest that stc-1 has a unique and key role in latanoprost signaling mediated iop reduction .", "top : light microscopy images of whole eye sections from wild - type and stc-1 mice .", "wild - type and stc-1 mice have similar - sized lens with both wild - type and stc-1 mice showing similar retinal morphology . middle : light microscopy images of wild - type and stc-1 mice showing conventional outflow pathway with open angles and similar number of trabecula , trabecular meshwork ( tm ) cells , and schlemm 's canal ( sc ) volume .", "bottom : transmission electron micrographs of wild - type and stc-1 mice showing similar appearing trabecular meshwork and intact schlemm 's canal inner walls ( iw ) and outer walls ( ow ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following latanoprost treatment .", "while congenic control mice show iop reduction with latanoprost , stc-1 do not , suggesting stc-1 is an important effector molecule for latanoprost - induced iop reduction .", "intraocular pressure in wild - type and stc-1 mice following treatment with latanoprost and rho kinase inhibitor rho kinase inhibitor y27632 reduces iop in stc-1 mice .", "stc-1 mice ( n = 10 ) and congenic controls ( n = 10 ) were treated once daily with 10 mm y27632 . the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following y27632 treatment ."], ["to determine if stc-1 would influence iop reduction by itself , we perfused human anterior segments with several concentrations of recombinant stc-1 for 24 hours .", "perfusion with either 5 ng / ml ( 0.15  0.04 to 0.15  0.04 l / min / mm hg , n = 2 , p = 0.20 ) or 50 ng / ml ( 0.14  0.05 to 0.18  0.07 l / min / mm hg , n = 4 , p = 0.20 ) had no significant effect on outflow facility .", "however , all anterior segments perfused with stc-1 at 500 ng / ml had decreased pressure and increased outflow facility ( 0.15  0.03 to 0.27  0.09 l / min / mm hg , n = 5 , p = 0.02 ) compared to baseline ( fig . 5 ) .", "paired controls treated with vehicle showed no change in outflow facility from their baseline values . to determine whether the changes in pressure could be secondary to morphologic changes", "morphologic analysis showed viable and healthy cells in the trabecular meshwork and schlemm 's canal of control and treated eyes ( fig .", "6 ) . no major disruptions of the juxtacanalicular tissue or the basement membrane of schlemm 's canal inner and outer walls were observed .", "these results indicate that stc-1 may be a molecule within the latanoprost signaling pathway that can be therapeutically targeted to lower iop .", "( a ) outflow facility of human anterior segments ( n = 8) following perfusion with 5 , 50 , or 500 ng / ml of recombinant human stc-1 .", "( b ) representative graph of an eye pair perfused with 500 ng / ml stc-1 .", "p  0.05 . histologic analysis of human anterior segment ocular tissue following treatment with recombinant stc-1 .", "( a , b ) representative sections ( 3 m ) of recombinant stc-1 and vehicle - treated eyes that were stained with toluidine blue . ( c , d ) transmission electron micrographs showing ultrastructure of recombinant stc-1 and vehicle - treated eyes .", "recombinant human stc-1 and vehicle - treated eyes had similar morphology and ultrastructural appearance suggesting no apparent detrimental side effects of recombinant stc-1 treatment .", "ac , anterior chamber ; jct , juxtacanalicular region ; tm , trabecular meshwork ; sc , schlemm 's canal ."], ["to determine if stc-1 has ocular hypotensive activity in vivo , wild - type c57bl/6 mice were treated with recombinant stc-1 ( 0.5 mg / ml ) or vehicle daily for 7 days ( fig .", "stanniocalcin-1 reduced iop by 15.2%  3.0% when compared to vehicle - treated contralateral eyes ( range of iop reduction from 5.1%  5.2% [ day 1 ] to 21.4%  5.3%", "these results suggest that stc-1 has iop - lowering properties when used as a standalone agent .", "wild - type mice ( n = 10 ) were treated topically with recombinant stc-1 ( 0.5 mg / ml ) or vehicle ( phosphate buffered saline ) .", "the daily iop was recorded as the average of iop measurements at 1 , 4 , and 23 hours following stc-1 treatment ."], ["prostaglandin analogs like latanoprost are a first - line medical therapy for iop reduction due to their once daily dosing , greater response rates , and greater amount of iop reduction compared to other classes of pressure - lowering medications .", "identifying the critical effector molecules in the signaling pathway of latanoprost will provide insights into additional molecules to target for iop reduction , potentially with fewer side effects . in the current study ,", "we identified stc-1 as a transcript and protein that is highly induced following treatment of human schlemm 's canal cells with latanoprost .", "topical latanoprost significantly reduced iop in wild - type controls while stc-1 mice demonstrated no iop reduction to topical latanoprost . in contrast , rho kinase inhibitor y27632 demonstrated iop reduction in both wild - type and stc-1 mice , indicating that stc-1 is a unique and important downstream signaling molecule necessary for the ocular hypotensive properties of latanoprost .", "additionally , recombinant stc-1 by itself also increased outflow facility in human anterior segments and reduced iop in vivo in wild - type mice .", "together , these results suggest that stc-1 is a critical and unique effector molecule for the latanoprost signaling pathway that is necessary for latanoprost - induced iop reduction and that stc-1 by itself can act as an ocular hypotensive agent .", "stanniocalcin was first described in fish as a 50-kda homodimeric glycoprotein that is secreted from the corpuscles of stannius into the bloodstream in the setting of hypercalcemia to regulate calcium excretion at the gills and gut . in mammals , two homologues of stc", "stc-1 is the most studied of the two mammalian forms , having a 50% amino acid homology with its fish counterpart .", "stc-1 is a secreted , homodimeric phosphoglycoprotein that has preserved protein structure similarity between mammals and fish including the conservation of 11 cysteine residues .", "it is expressed in a wide variety of tissues most notably bone , skeletal muscle , heart , thymus , and spleen .", "functionally , stc-1 has been associated with calcium uptake , hypoxic preconditioning , and antioxidative stress properties through suppression of reactive oxygen species .", "additionally , stc-1 has also been shown to be neuroprotective for neurons , photoreceptors , and retinal ganglion cells , and has been linked to anti - inflammatory effects by inhibiting macrophage chemotaxis , modulating transendothelial migration of leukocytes and reducing t cell infiltration . in treating wild - type mice with topical latanoprost", ", we observed a reduction in iop by approximately 22.0%  1.9% , consistent with previous reports , while the stc-1 mice showed no response ( 0.55%  0.7% ) .", "mice that were stc-1 were responsive to rho kinase inhibitor y27632 , which confirmed that stc-1 mice have normal functioning outflow pathways , but that elimination of stc-1 expression in these mice renders topical latanoprost ineffective .", "the current study adds schlemm 's canal cells to the list of cells that express stc-1 and shows that its expression is highly influenced by latanoprost .", "this is an important finding as little is known about the molecular events that connect latanoprost treatment with iop reduction .", "latanoprost has been shown to phosphorylate myosin light chain kinase in the iris , indicating a role for latanoprost in cell relaxation .", "however , stc-1 is the first individual molecule that has been identified as a key downstream effector of latanoprost signaling .", "in addition to its variety of functional activities , our results also suggest that stc-1 has ocular hypotensive properties , since addition of recombinant stc-1 to human anterior segment cultures decreased pressure and increased outflow facility , and when applied topically to wild - type mice reduced iop in vivo . despite the benefits of latanoprost in lowering iop", ", long - term treatment with latanoprost can often cause significant side effects , which can be contraindicative for its prolonged usage .", "identification of stc-1 as a downstream signaling molecule through which latanoprost executes its iop - lowering effects , and the fact that recombinant stc-1 shows ocular hypotensive properties , makes this an attractive therapeutic target .", "utilization of a specific downstream molecule such as stc-1 within the latanoprost signaling cascade may eliminate some of the major side effects while providing a specialized therapeutic strategy . in summary ,", "additionally , stc-1 demonstrates ocular hypotensive properties when used by itself , mimicking the effect of latanoprost . given its novel role in latanoprost - mediated iop reduction", ", stc-1 may be considered a promising candidate molecule for devising future therapeutic regiments to reduce iop ."]]}
{"article_id": "PMC2789864", "article_text": ["single - crystalline kinked nanowires were synthesized by the nanocluster - catalyzed vls method described previously18,19 in quartz tube connected to gas manifold and vacuum pump and heated by a temperature controlled tube furnace .", "monodisperse gold nanoparticles ( ted pella ) were dispersed on sio2/si or sapphire growth substrates ( au surface coverage : 0.010.1 particles/m ) , which were placed within the central region of the quartz tube reactor .", "the silicon ( si ) nanowires were synthesized at 450460 c using silane ( sih4 ) as the silicon reactant source , hydrogen ( h2 ) as the carrier gas , and phosphine ( ph3 , 1,000 p.p.m . in h2 ) and diborane ( b2h6,100 p.p.m . in h2 ) as the n- and p - type dopants . in a typical synthesis of uniform n - type , 80 nm kinked silicon nanowires ,", "the flow rates of sih4 , ph3 and h2 were 12 , 210 and 60 standard cubic centimetres per minute , respectively , and the total pressure was 40 torr and purge duration was 15 s ; the minimum pressure during the purge cycle was ca . 3  10 torr . the dopant feed - in ratios ( silicon : boron / phosphorus ) in kinked", "p - n silicon nanowires were 500:1 for both p - and n - type segments . in n - kink - n - kink-(n - n ) dopant modulated silicon nanowires , the silicon - phosphorus feed - in ratios were 200:1 and 10000:1 for n- and n - type segments , respectively , and the n- segment was grown for 30 s. germanium nanowires were synthesized at 270290 c , 40 torr , with germane ( geh4 , 10 % in h2 ) and h2 as the reactant and carrier gas , respectively .", "cadmium sulphide nanowires were grown in a three - zone furnace by evaporating cds power at 650720 c , with nanowire growth by gold nanocluster catalyzed vls method at 550 - 500 c .", "the purge cycle used to form kinks in the germanium and cadmium sulphide nanowires was typically 15 s. zeiss ultra55/supra55vp field - emission sems and jeol 2010 field emission tem were used to carry out sem and tem analyses , respectively . for sample preparation , kinked nanowires were gently sonicated in isopropyl alcohol and dispersed onto heavily doped silicon substrates ( 100 nm oxide/200 nm nitride , 110 cm resistivity , nova electronic materials , carrollton , tx ) or lacey carbon grids ( ted pella ) .", "devices were fabricated on silicon substrates ( nova electronic materials , n - type 0.005 cm ) with 100 nm thermal oxide and 200 nm silicon nitride at the surface .", "devices were defined by electron - beam lithography followed by ti / pd ( 1.5 nm/100 nm ) contact deposition in a thermal evaporator .", "current - voltage ( i  v ) data were recorded using an agilent semiconductor parameter analyzer ( model 4156c ) with contacts to devices made using a probe station ( desert cryogenics , model ttp4 ) .", "electrostatic force microscopy and scanned gate microscopy measurements were carried out with a digital instruments nanoscope iiia multimode afm and metal coated tips ( nanosensors , ppp - nchpt ) . the electrostatic force microscopy surface potential maps and scanned gate microscopy conductance maps were acquired in lift mode with lift heights of 40 and 20 nm , respectively . in the surface potential measurements , the p - n diode was reverse - biased at 5 v and the tip voltage was modulated by 3v at the resonance frequency . in scanned gate measurements , the tip functions as a local gate vtip", "= 10 v , and the conductance versus position provides a measure of local accumulation or depletion of carriers in the device .", "single - crystalline kinked nanowires were synthesized by the nanocluster - catalyzed vls method described previously18,19 in quartz tube connected to gas manifold and vacuum pump and heated by a temperature controlled tube furnace .", "monodisperse gold nanoparticles ( ted pella ) were dispersed on sio2/si or sapphire growth substrates ( au surface coverage : 0.010.1 particles/m ) , which were placed within the central region of the quartz tube reactor .", "the silicon ( si ) nanowires were synthesized at 450460 c using silane ( sih4 ) as the silicon reactant source , hydrogen ( h2 ) as the carrier gas , and phosphine ( ph3 , 1,000 p.p.m . in h2 ) and diborane ( b2h6,100 p.p.m . in h2 ) as the n- and p - type dopants . in a typical synthesis of uniform n - type , 80 nm kinked silicon nanowires ,", "the flow rates of sih4 , ph3 and h2 were 12 , 210 and 60 standard cubic centimetres per minute , respectively , and the total pressure was 40 torr and purge duration was 15 s ; the minimum pressure during the purge cycle was ca . 3  10 torr . the dopant feed - in ratios ( silicon : boron / phosphorus ) in kinked", "p - n silicon nanowires were 500:1 for both p - and n - type segments . in n - kink - n - kink-(n - n ) dopant modulated silicon nanowires , the silicon - phosphorus feed - in ratios were 200:1 and 10000:1 for n- and n - type segments , respectively , and the n- segment was grown for 30 s. germanium nanowires were synthesized at 270290 c , 40 torr , with germane ( geh4 , 10 % in h2 ) and h2 as the reactant and carrier gas , respectively .", "cadmium sulphide nanowires were grown in a three - zone furnace by evaporating cds power at 650720 c , with nanowire growth by gold nanocluster catalyzed vls method at 550 - 500 c .", "the purge cycle used to form kinks in the germanium and cadmium sulphide nanowires was typically 15 s.", "zeiss ultra55/supra55vp field - emission sems and jeol 2010 field emission tem were used to carry out sem and tem analyses , respectively . for sample preparation , kinked nanowires were gently sonicated in isopropyl alcohol and dispersed onto heavily doped silicon substrates ( 100 nm oxide/200 nm nitride , 110 cm resistivity , nova electronic materials , carrollton , tx ) or lacey carbon grids ( ted pella ) .", "devices were fabricated on silicon substrates ( nova electronic materials , n - type 0.005 cm ) with 100 nm thermal oxide and 200 nm silicon nitride at the surface .", "devices were defined by electron - beam lithography followed by ti / pd ( 1.5 nm/100 nm ) contact deposition in a thermal evaporator .", "current - voltage ( i  v ) data were recorded using an agilent semiconductor parameter analyzer ( model 4156c ) with contacts to devices made using a probe station ( desert cryogenics , model ttp4 ) .", "electrostatic force microscopy and scanned gate microscopy measurements were carried out with a digital instruments nanoscope iiia multimode afm and metal coated tips ( nanosensors , ppp - nchpt ) . the electrostatic force microscopy surface potential maps and scanned gate microscopy conductance maps were acquired in lift mode with lift heights of 40 and 20 nm , respectively . in the surface", "potential measurements , the p - n diode was reverse - biased at 5 v and the tip voltage was modulated by 3v at the resonance frequency . in scanned gate measurements , the tip functions as a local gate vtip", "= 10 v , and the conductance versus position provides a measure of local accumulation or depletion of carriers in the device ."], "abstract_text": ["<S> the ability to control and modulate the composition14 , doping1,35 , crystal structure68 and morphology9,10 of semiconductor nanowires during the synthesis process has allowed researchers to explore various applications of nanowires1115 . however , despite advances in nanowire synthesis , progress towards the ab initio design and growth of hierarchical nanostructures has been limited . here </S>", "<S> we demonstrate a  </S>", "<S> nanotectonic approach that provides iterative control over the nucleation and growth of nanowires and use it to grow kinked or zigzag nanowires in which the straight sections are separated by triangular joints . </S>", "<S> moreover , the lengths of the straight sections can be controlled and the growth direction remains coherent along the nanowire . </S>", "<S> we also grow dopant - modulated structures in which specific device functions , including p - n diodes and field - effect transistors , can be precisely localized at the kinked junctions in the nanowires . </S>"], "labels": null, "section_names": ["Methods", "Nanowire synthesis", "Structure characterization", "Device fabrication and measurement", "Supplementary Material"], "sections": [["single - crystalline kinked nanowires were synthesized by the nanocluster - catalyzed vls method described previously18,19 in quartz tube connected to gas manifold and vacuum pump and heated by a temperature controlled tube furnace .", "monodisperse gold nanoparticles ( ted pella ) were dispersed on sio2/si or sapphire growth substrates ( au surface coverage : 0.010.1 particles/m ) , which were placed within the central region of the quartz tube reactor .", "the silicon ( si ) nanowires were synthesized at 450460 c using silane ( sih4 ) as the silicon reactant source , hydrogen ( h2 ) as the carrier gas , and phosphine ( ph3 , 1,000 p.p.m . in h2 ) and diborane ( b2h6,100 p.p.m . in h2 ) as the n- and p - type dopants . in a typical synthesis of uniform n - type , 80 nm kinked silicon nanowires ,", "the flow rates of sih4 , ph3 and h2 were 12 , 210 and 60 standard cubic centimetres per minute , respectively , and the total pressure was 40 torr and purge duration was 15 s ; the minimum pressure during the purge cycle was ca . 3  10 torr . the dopant feed - in ratios ( silicon : boron / phosphorus ) in kinked", "p - n silicon nanowires were 500:1 for both p - and n - type segments . in n - kink - n - kink-(n - n ) dopant modulated silicon nanowires , the silicon - phosphorus feed - in ratios were 200:1 and 10000:1 for n- and n - type segments , respectively , and the n- segment was grown for 30 s. germanium nanowires were synthesized at 270290 c , 40 torr , with germane ( geh4 , 10 % in h2 ) and h2 as the reactant and carrier gas , respectively .", "cadmium sulphide nanowires were grown in a three - zone furnace by evaporating cds power at 650720 c , with nanowire growth by gold nanocluster catalyzed vls method at 550 - 500 c .", "the purge cycle used to form kinks in the germanium and cadmium sulphide nanowires was typically 15 s. zeiss ultra55/supra55vp field - emission sems and jeol 2010 field emission tem were used to carry out sem and tem analyses , respectively . for sample preparation , kinked nanowires were gently sonicated in isopropyl alcohol and dispersed onto heavily doped silicon substrates ( 100 nm oxide/200 nm nitride , 110 cm resistivity , nova electronic materials , carrollton , tx ) or lacey carbon grids ( ted pella ) .", "devices were fabricated on silicon substrates ( nova electronic materials , n - type 0.005 cm ) with 100 nm thermal oxide and 200 nm silicon nitride at the surface .", "devices were defined by electron - beam lithography followed by ti / pd ( 1.5 nm/100 nm ) contact deposition in a thermal evaporator .", "current - voltage ( i  v ) data were recorded using an agilent semiconductor parameter analyzer ( model 4156c ) with contacts to devices made using a probe station ( desert cryogenics , model ttp4 ) .", "electrostatic force microscopy and scanned gate microscopy measurements were carried out with a digital instruments nanoscope iiia multimode afm and metal coated tips ( nanosensors , ppp - nchpt ) . the electrostatic force microscopy surface potential maps and scanned gate microscopy conductance maps were acquired in lift mode with lift heights of 40 and 20 nm , respectively . in the surface potential measurements , the p - n diode was reverse - biased at 5 v and the tip voltage was modulated by 3v at the resonance frequency . in scanned gate measurements , the tip functions as a local gate vtip", "= 10 v , and the conductance versus position provides a measure of local accumulation or depletion of carriers in the device ."], ["single - crystalline kinked nanowires were synthesized by the nanocluster - catalyzed vls method described previously18,19 in quartz tube connected to gas manifold and vacuum pump and heated by a temperature controlled tube furnace .", "monodisperse gold nanoparticles ( ted pella ) were dispersed on sio2/si or sapphire growth substrates ( au surface coverage : 0.010.1 particles/m ) , which were placed within the central region of the quartz tube reactor .", "the silicon ( si ) nanowires were synthesized at 450460 c using silane ( sih4 ) as the silicon reactant source , hydrogen ( h2 ) as the carrier gas , and phosphine ( ph3 , 1,000 p.p.m . in h2 ) and diborane ( b2h6,100 p.p.m . in h2 ) as the n- and p - type dopants . in a typical synthesis of uniform n - type , 80 nm kinked silicon nanowires ,", "the flow rates of sih4 , ph3 and h2 were 12 , 210 and 60 standard cubic centimetres per minute , respectively , and the total pressure was 40 torr and purge duration was 15 s ; the minimum pressure during the purge cycle was ca . 3  10 torr . the dopant feed - in ratios ( silicon : boron / phosphorus ) in kinked", "p - n silicon nanowires were 500:1 for both p - and n - type segments . in n - kink - n - kink-(n - n ) dopant modulated silicon nanowires , the silicon - phosphorus feed - in ratios were 200:1 and 10000:1 for n- and n - type segments , respectively , and the n- segment was grown for 30 s. germanium nanowires were synthesized at 270290 c , 40 torr , with germane ( geh4 , 10 % in h2 ) and h2 as the reactant and carrier gas , respectively .", "cadmium sulphide nanowires were grown in a three - zone furnace by evaporating cds power at 650720 c , with nanowire growth by gold nanocluster catalyzed vls method at 550 - 500 c .", "the purge cycle used to form kinks in the germanium and cadmium sulphide nanowires was typically 15 s."], ["zeiss ultra55/supra55vp field - emission sems and jeol 2010 field emission tem were used to carry out sem and tem analyses , respectively . for sample preparation , kinked nanowires were gently sonicated in isopropyl alcohol and dispersed onto heavily doped silicon substrates ( 100 nm oxide/200 nm nitride , 110 cm resistivity , nova electronic materials , carrollton , tx ) or lacey carbon grids ( ted pella ) ."], ["devices were fabricated on silicon substrates ( nova electronic materials , n - type 0.005 cm ) with 100 nm thermal oxide and 200 nm silicon nitride at the surface .", "devices were defined by electron - beam lithography followed by ti / pd ( 1.5 nm/100 nm ) contact deposition in a thermal evaporator .", "current - voltage ( i  v ) data were recorded using an agilent semiconductor parameter analyzer ( model 4156c ) with contacts to devices made using a probe station ( desert cryogenics , model ttp4 ) .", "electrostatic force microscopy and scanned gate microscopy measurements were carried out with a digital instruments nanoscope iiia multimode afm and metal coated tips ( nanosensors , ppp - nchpt ) . the electrostatic force microscopy surface potential maps and scanned gate microscopy conductance maps were acquired in lift mode with lift heights of 40 and 20 nm , respectively . in the surface", "potential measurements , the p - n diode was reverse - biased at 5 v and the tip voltage was modulated by 3v at the resonance frequency . in scanned gate measurements , the tip functions as a local gate vtip", "= 10 v , and the conductance versus position provides a measure of local accumulation or depletion of carriers in the device ."], [""]]}
{"article_id": "PMC4877559", "article_text": ["arachnoid cysts are benign , non - neoplastic and extra - axial lesions ; most of them are clinically silent and unchanged in size . in previous studies112131524 ) , the incidence of arachnoid cysts has been estimated to range between 0.3% to 1.7% .", "however , the advanced neuroimaging techniques have facilitated the diagnosis of arachnoid cysts , which , in turn , has led to an increase in the number of patients2121415 ) . in some cases ,", "an arachnoid cyst arouses symptoms and its clinical presentations vary according to the size and location of the cyst .", "the most frequent symptom is headache , which is caused by the local mass effect , increased intracranial pressure ( icp ) , and/or hydrocephalus15 ) . above this", ", seizures , cognitive dysfunction , developmental delay , and intracranial hemorrhage can be also presented . in this report", ", we describe a case where a patient with a posterior fossa arachnoid cyst complained of visual field defect , visual disturbance , and mild headache ; no ataxia and unsteady gait were reported .", "the patient 's visual symptoms rapidly deteriorated in a week and , consequently , the patient 's vision got worse .", "a 39-year - old male patient presented at our emergency room after a traffic accident and the brain computed tomography ( ct ) scan indicated an incidental arachnoid cyst on posterior fossa . at that time , the patient complained only of facial abrasions and the left knee pain , but not of headache or visual disturbance . in eight months , however , he revisited our department of ophthalmology to check the decreased visual acuity , visual field defect , and headache . on the ophthalmologic examination , bilateral papilledema ( fig .", "2a ) were found . after the transfer to the department of neurosurgery , we rechecked brain ct scans ( fig .", "the diameter of the patient 's arachnoid cyst did not change compared to the previous ct scans ; however , the slightly enlarged third ventricle was compressing the optic chiasm and the suprasellar area . since visual complications were expected , we recommended an emergency operation .", "one week later , he came back to the hospital with nearly complete visual loss .", "his right eye was completely blind , whereas his left eye recognized only light ( fig .", "a conventional suboccipital craniectomy was performed on the patient lying on the operating table in the prone position .", "after the dural incision , the arachnoid cyst was exposed and cystic fluid was expelled .", "arachnoid membrane was carefully dissected and the arachnoid cyst was fenestrated to cisterna magna for connecting with the cerebrospinal fluid ( csf ) flows .", "arachnoid membrane which covering the foramen of magendie was also fenestrated and the csf flows was running properly ( fig .", "4 ) . excluding the brain tumor , the collected membrane was sent to the pathologic laboratory . in high power field microscopy under the hematoxylin and eosin stain , the cystic membrane was composed by membranous fibrocollagenous tissues without tumor cells .", "one week afterwards , we checked the brain ct scans and the removal of the cyst and the decreased ventricle size were confirmed . on the ophthalmologic examination ,", "the papilledema and visual field were slightly improved ; however , the patient 's visual acuity did not improve . upon the tenth day after the operation", ", the patient was discharged and , during the next one month , he was subject to the steroid therapy . in five months after the operation , papilledema ( fig .", "nevertheless , visual acuity and visual field improved slowly and slightly ( fig . 2c ) .", "the blind right eye improved to recognize the light and the blurred left eye improved to acknowledge the silhouette of an object ; thus , it did not fully recovered to the prior , normal condition .", "their prevalence has been reported to amount to approximately 1% of all intracranial mass lesions112152430 ) ; however , advanced neuroimaging techniques and the widespread use of ct and mri imaging have led to an even higher incidence of report of these lesions1249121619203031 ) .", "one recent study , al - holou et al.1 ) , reviewed 48417 brain images and identified 661 ( 1.4% ) arachnoid cysts .", "this report found that the prevalence of arachnoid cyst in children was 2.6% , slightly higher than in adults , and men ( 1.8% ) have a high prevalence than women ( 1.1% ) .", "the most common locations were middle fossa ( 34% ) , retrocerebellar ( 33% ) , and cerebral convexity ( 14%)124 ) . despite the increased identification of arachnoid cysts , the natural history and mechanism of enlargement has not been well defined1131524 ) .", "nevertheless , when arachnoid cysts give rise to any symptom , headache is the most common complaint .", "in addition to headache , gait imbalance , seizure , and visual changes can also be presented according to the location and size of the arachnoid cyst .", "various operation techniques , such as cyst excision1517 ) , stereotactic aspiration1825 ) , endoscopic cyst fenestration512 ) , ventriculocystostomy26 ) , and cystoperitoneal shunt810152728 ) , have been introduced and the main purpose of such operations is similar , namely , a direct decompression of the arachnoid cyst or making the connection between the cyst and the csf flows for spontaneous resolution .", "generally , visual symptoms occur in the patients with suprasellar arachnoid cysts , because the arachnoid cyst is adjacent to the optic nerves7 ) .", "the patients with posterior fossa arachnoid cysts usually do not complain of visual symptoms , as the cyst is not adjacent to the optic nerves .", "however , a posterior fossa arachnoid cyst can indirectly disrupt the csf flows and , in turn , the disruption increases the whole ventricle size and the icp . in our case", ", the enlarged third ventricle was compressing the optic chiasm with cephalocaudal direction on the ct scan ( fig .", "the compressed superior part of optic chiasm led to the defect of inferior visual field ( fig .", ", visual acuity was affected by the papilledema , which was caused by the increased icp .", "consequently , both visual acuity and visual field were influenced by the posterior fossa arachnoid cyst .", "when the patient first visited our department of ophthalmology , his visual symptoms and headache were not severe and the patient could walk and drive . besides , the brain ct scan showed no changes as compared to previous ct scans , so we could not predict a risk of rapid deterioration .", "the patient 's refusal to have an operation and the surgeon 's wrong prediction delayed the appropriate on - time treatment ; ultimately , the patient 's vision was severely damaged during the critical seven days . after the operation , the patient 's visual acuity , visual field , and papilledema were slightly improved ; however , we regard that the patient 's vision will unfortunately not fully recover .", "unlike gait disturbance and ataxia , visual symptoms do not frequently occur in the patients with posterior fossa arachnoid cysts ; this is so because the cyst is located away from the optic nerves .", "however , the disruption of csf flows can indirectly induce visual symptoms and these symptoms can rapidly deteriorate .", "considering the results of our case , it should be emphasized that the patients with posterior fossa arachnoid cysts could develop a rapid neurologic deterioration .", "therefore , in the event of the development of symptoms , an early intervention or close observation are crucial ."], "abstract_text": ["<S> posterior fossa is a site next to the middle fossa where arachnoid cyst frequently occurs . </S>", "<S> generally , most arachnoid cysts are asymptomatic and are found incidentally in most cases . </S>", "<S> although arachnoid cysts are benign and asymptomatic lesions , patients with posterior fossa arachnoid cysts often complain of headaches , gait disturbance , and ataxia due to the local mass effects on the cerebellum . </S>", "<S> we observed a patient with a posterior fossa arachnoid cyst who had visual symptoms and a headache , but did not have gait disturbance and ataxia . </S>", "<S> we recommended an emergency operation for decompression , but the patient refused for personal reasons . </S>", "<S> after 7 days , the patient revisited our hospital in a state of near - blindness . </S>", "<S> we suspected that the arachnoid cyst induced the hydrocephalus and thereby the enlarged third ventricle directly compressed optic nerves . </S>", "<S> compressed optic nerves were rapidly aggravated during the critical seven days ; consequently , the patient 's vision was damaged despite the operation . </S>", "<S> considering the results of our case , it is important to keep in mind that the aggravation of symptoms can not be predicted ; therefore , symptomatic arachnoid cysts should be treated without undue delay . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["arachnoid cysts are benign , non - neoplastic and extra - axial lesions ; most of them are clinically silent and unchanged in size . in previous studies112131524 ) , the incidence of arachnoid cysts has been estimated to range between 0.3% to 1.7% .", "however , the advanced neuroimaging techniques have facilitated the diagnosis of arachnoid cysts , which , in turn , has led to an increase in the number of patients2121415 ) . in some cases ,", "an arachnoid cyst arouses symptoms and its clinical presentations vary according to the size and location of the cyst .", "the most frequent symptom is headache , which is caused by the local mass effect , increased intracranial pressure ( icp ) , and/or hydrocephalus15 ) . above this", ", seizures , cognitive dysfunction , developmental delay , and intracranial hemorrhage can be also presented . in this report", ", we describe a case where a patient with a posterior fossa arachnoid cyst complained of visual field defect , visual disturbance , and mild headache ; no ataxia and unsteady gait were reported .", "the patient 's visual symptoms rapidly deteriorated in a week and , consequently , the patient 's vision got worse ."], ["a 39-year - old male patient presented at our emergency room after a traffic accident and the brain computed tomography ( ct ) scan indicated an incidental arachnoid cyst on posterior fossa . at that time , the patient complained only of facial abrasions and the left knee pain , but not of headache or visual disturbance . in eight months , however , he revisited our department of ophthalmology to check the decreased visual acuity , visual field defect , and headache . on the ophthalmologic examination , bilateral papilledema ( fig .", "2a ) were found . after the transfer to the department of neurosurgery , we rechecked brain ct scans ( fig .", "the diameter of the patient 's arachnoid cyst did not change compared to the previous ct scans ; however , the slightly enlarged third ventricle was compressing the optic chiasm and the suprasellar area . since visual complications were expected , we recommended an emergency operation .", "one week later , he came back to the hospital with nearly complete visual loss .", "his right eye was completely blind , whereas his left eye recognized only light ( fig .", "a conventional suboccipital craniectomy was performed on the patient lying on the operating table in the prone position .", "after the dural incision , the arachnoid cyst was exposed and cystic fluid was expelled .", "arachnoid membrane was carefully dissected and the arachnoid cyst was fenestrated to cisterna magna for connecting with the cerebrospinal fluid ( csf ) flows .", "arachnoid membrane which covering the foramen of magendie was also fenestrated and the csf flows was running properly ( fig .", "4 ) . excluding the brain tumor , the collected membrane was sent to the pathologic laboratory . in high power field microscopy under the hematoxylin and eosin stain , the cystic membrane was composed by membranous fibrocollagenous tissues without tumor cells .", "one week afterwards , we checked the brain ct scans and the removal of the cyst and the decreased ventricle size were confirmed . on the ophthalmologic examination ,", "the papilledema and visual field were slightly improved ; however , the patient 's visual acuity did not improve . upon the tenth day after the operation", ", the patient was discharged and , during the next one month , he was subject to the steroid therapy . in five months after the operation , papilledema ( fig .", "nevertheless , visual acuity and visual field improved slowly and slightly ( fig . 2c ) .", "the blind right eye improved to recognize the light and the blurred left eye improved to acknowledge the silhouette of an object ; thus , it did not fully recovered to the prior , normal condition ."], ["their prevalence has been reported to amount to approximately 1% of all intracranial mass lesions112152430 ) ; however , advanced neuroimaging techniques and the widespread use of ct and mri imaging have led to an even higher incidence of report of these lesions1249121619203031 ) .", "one recent study , al - holou et al.1 ) , reviewed 48417 brain images and identified 661 ( 1.4% ) arachnoid cysts .", "this report found that the prevalence of arachnoid cyst in children was 2.6% , slightly higher than in adults , and men ( 1.8% ) have a high prevalence than women ( 1.1% ) .", "the most common locations were middle fossa ( 34% ) , retrocerebellar ( 33% ) , and cerebral convexity ( 14%)124 ) . despite the increased identification of arachnoid cysts , the natural history and mechanism of enlargement has not been well defined1131524 ) .", "nevertheless , when arachnoid cysts give rise to any symptom , headache is the most common complaint .", "in addition to headache , gait imbalance , seizure , and visual changes can also be presented according to the location and size of the arachnoid cyst .", "various operation techniques , such as cyst excision1517 ) , stereotactic aspiration1825 ) , endoscopic cyst fenestration512 ) , ventriculocystostomy26 ) , and cystoperitoneal shunt810152728 ) , have been introduced and the main purpose of such operations is similar , namely , a direct decompression of the arachnoid cyst or making the connection between the cyst and the csf flows for spontaneous resolution .", "generally , visual symptoms occur in the patients with suprasellar arachnoid cysts , because the arachnoid cyst is adjacent to the optic nerves7 ) .", "the patients with posterior fossa arachnoid cysts usually do not complain of visual symptoms , as the cyst is not adjacent to the optic nerves .", "however , a posterior fossa arachnoid cyst can indirectly disrupt the csf flows and , in turn , the disruption increases the whole ventricle size and the icp . in our case", ", the enlarged third ventricle was compressing the optic chiasm with cephalocaudal direction on the ct scan ( fig .", "the compressed superior part of optic chiasm led to the defect of inferior visual field ( fig .", ", visual acuity was affected by the papilledema , which was caused by the increased icp .", "consequently , both visual acuity and visual field were influenced by the posterior fossa arachnoid cyst .", "when the patient first visited our department of ophthalmology , his visual symptoms and headache were not severe and the patient could walk and drive . besides , the brain ct scan showed no changes as compared to previous ct scans , so we could not predict a risk of rapid deterioration .", "the patient 's refusal to have an operation and the surgeon 's wrong prediction delayed the appropriate on - time treatment ; ultimately , the patient 's vision was severely damaged during the critical seven days . after the operation , the patient 's visual acuity , visual field , and papilledema were slightly improved ; however , we regard that the patient 's vision will unfortunately not fully recover ."], ["unlike gait disturbance and ataxia , visual symptoms do not frequently occur in the patients with posterior fossa arachnoid cysts ; this is so because the cyst is located away from the optic nerves .", "however , the disruption of csf flows can indirectly induce visual symptoms and these symptoms can rapidly deteriorate .", "considering the results of our case , it should be emphasized that the patients with posterior fossa arachnoid cysts could develop a rapid neurologic deterioration .", "therefore , in the event of the development of symptoms , an early intervention or close observation are crucial ."]]}
{"article_id": "PMC5228694", "article_text": ["these techniques include closed reduction , percutaneous pin reduction , intramedullary pin reduction with elastic stable intramedullary nailing or kirschner - wires ( k - wires ) , and open reduction with or without internal fixation .", "our goals for treatment are to avoid incision , reduce the fracture adequately with no reduction loss , and achieve good postoperative function .", "we performed a closed reduction with a percutaneous leverage technique and internal fixation method with k - wires to treat angulated radial neck fractures in children .", "from january 2011 to april 2013 , we treated 16 cases of angulated radial neck fractures in 12 boys and 4 girls .", "the types of fractures categorized by the obrien classification as follows : type i  angulation < 30 ; type ii ", "angulation > 60. among our patients , 5 had type ii and 11 had type iii fractures .", "the left side was affected in 9 cases , right side was affected in 7 cases .", "complications with ulnar olecranon fracture and medial epicondyle fracture were present in 3 cases and 2 cases , respectively . no blood vessel or nerve trauma was evident .", "the shortest time from trauma to surgery was 4  hours and the longest time was 6 days .", "a 1.6- or 2.0-mm k - wire was inserted from distal to proximal of the elbow percutaneously . in this procedure , the k - wire was inserted into the fracture site .", "the k - wire was used to lever the fracture and to orient the proximal part horizontally with the lateral condyle plane .", "was attained , two 1.2  mm k - wires were percutaneously inserted from the articular surface through the fracture site to the opposite cortex for fixation ( fig .", "the 3 cases with ulnar olecranon fractures were not treated because there was no obvious displacement .", "our research was approved by the biomedical research ethics committee of hong hui hospital , xian jiaotong university college of medicine .", "( a ) the kirschner wire ( k - wire ) was inserted percutaneously into the fracture site .", "( b ) the inserted k - wire was used as a lever to reduce the fracture .", "( c ) one 1.2  mm k - wires was percutaneously inserted from the articular surface passing through the fracture site to the opposite cortex .", "typical case : femail , 12 years old , and right side . a long - arm cast or brace was used for 3 to 4 weeks after the procedures , with the elbow in 90 of flexion and the forearm in supination .", "a 1.6- or 2.0-mm k - wire was inserted from distal to proximal of the elbow percutaneously . in this procedure , the k - wire was inserted into the fracture site .", "the k - wire was used to lever the fracture and to orient the proximal part horizontally with the lateral condyle plane .", "was attained , two 1.2  mm k - wires were percutaneously inserted from the articular surface through the fracture site to the opposite cortex for fixation ( fig .", "the 3 cases with ulnar olecranon fractures were not treated because there was no obvious displacement .", "our research was approved by the biomedical research ethics committee of hong hui hospital , xian jiaotong university college of medicine .", "( a ) the kirschner wire ( k - wire ) was inserted percutaneously into the fracture site .", "( b ) the inserted k - wire was used as a lever to reduce the fracture .", "( c ) one 1.2  mm k - wires was percutaneously inserted from the articular surface passing through the fracture site to the opposite cortex .", "typical case : femail , 12 years old , and right side . a long - arm cast or brace was used for 3 to 4 weeks after the procedures , with the elbow in 90 of flexion and the forearm in supination .", "cases were followed up for an average of 3 years 7 months ( range 2 years 6 months to 4 years 10 months ) .", "the postoperative reduction was assessed by roentgenograph within 1 week after surgery . no infection or nerve damage occurred in the short - term follow - up .", "no radial head necrosis or synostosis of the proximal ulna and radius was seen in the long - term follow - up .", "there was no disclosure of the epiphysis or valgus of the elbow during the follow - up . according to the metaizeau reduction classification ,", "12 cases were excellent , and 4 cases were good . according to the metaizeau clinical classification , 14 cases were excellent , and 2 cases were good .", "( a ) the preoperative x - ray showed the severe tilt and displacement of radial neck .", "( c ) the 2 years later follow - up x - ray picture showed the radial head normal .", "most authors agree that fractures with slight angulation should be treated conservatively , but severely angulated fractures ( > 60 ) require further treatment with different methods .", "the complications of these fractures reported in children include avascular necrosis , early physeal closure , elbow stiffness , periarticular ossification , and overgrowth of the radius head .", "the prognosis was not only related with the angulation of the radial neck fracture , but also with the age of the patient and extent of trauma .", "metaizeau suggested that a residual tilt above 10 to 15 at 10 to 12 years of age or 20 to 30 at a younger age could not be remodeled by growth .", "additionally , bernstein found that the determination of what constituted a significant angulation vari with the child 's age , with greater angulation degrees acceptable for younger children .", "the compression and impaction extends to the lateral side of radial neck upon radial neck fracture .", "the redisplacement will be accomplished easily only if it is immobilized by cast as there is no support from the lateral portion of the radial neck for the radius head after reduction .", "steinberg reported that 22 cases which occurred redisplacement in whole 28 cases which were immobilized by cast after reduction .", "one method uses an elastic stable intramedullary nailing to indirectly reduce and fix the fracture site .", "the other method uses an intramedullary k - wire to perform indirect reduction and internal fixation .", "the 3rd method uses a percutaneous k - wire to perform direct reduction and internal fixation . in comparing the 3 different methods , some authors recommended closed reduction and elastic intramedullary fixation to treat a fracture of the radius in children .", "however , we found that it was difficult to reduce the displaced radius head through rotating the distal part of the elastic intramedullary nail .", "this reduction approach usually requires an assistant to manipulate the percutaneous k - wire . it is challenging to fix the radial head with elastic intramedullary nail after a good reduction .", "the radial head was easily separated from the fracture site when penetrating with and rotating the nail .", "the functional lever arm was too long when the elastic intramedullary nail or intramedullary pin was used to fix the radial head . it was challenging to maintain a good position .", "there was no disclosure of the epiphysis or valgus of the elbow during the follow - up .", "trauma was minor and there was no interference with the blood supply of the radial head", ". there was no postoperative scarring and the patients and their parents were satisfied with outcome .", "the insertion site should be carefully selected to avoid damage to the deep branch of the radial nerve .", "the k - wires should be appropriately sized , with a diameter no greater than 1.5  mm .", "we usually choose 1.2  mm k - wires to prevent damage to the epiphysis .", "we recommend the percutaneous leverage technique during closed reduction and internal fixation using k - wires for the treatment of angulated radial neck fractures in children .", "this is a simple method with little trauma to the patient and a satisfactory clinical outcome ."], "abstract_text": ["<S> abstractpediatric radial neck fractures are uncommon . severely displaced and angulated fractures usually require treatment . our goals for treatment are to avoid incision , reduce the fracture adequately with no reduction loss , and achieve good postoperative function . </S>", "<S> we aimed to observe the clinical outcomes of closed reduction with the percutaneous leverage technique and internal fixation with kirschner - wires ( k - wires ) to treat angulated radial neck fractures in children.from january 2011 to april 2013 , we treated 16 cases of angulated radial neck fracture in 12 boys and 4 girls . </S>", "<S> five fractures were type ii and 11 fractures were type iii using the obrien classification . </S>", "<S> one k - wire was percutaneously introduced into the fracture site using the leverage technique to attain good reduction . </S>", "<S> two k - wires were introduced from the proximal to the distal areas of the fracture site . </S>", "<S> the elbow was immobilized by cast in 90 of flexion and the forearm in supination for 3 to 4 weeks . </S>", "<S> the k - wires were removed at 3 to 4 weeks postoperatively . </S>", "<S> all cases were followed up for a mean duration of 3 years 6 months.according to the metaizeau reduction classification , 12 cases were excellent , and 4 cases were good . according to the metaizeau clinical classification , 14 cases were excellent , and 2 cases were good . </S>", "<S> there was no necrosis of the radial head . </S>", "<S> there was no infection , radioulnar synostosis , and damage of the radial nerve deep branch . </S>", "<S> there was no limitation in the pronation and supination functions of the forearm.closed reduction using the percutaneous leverage technique and internal fixation using k - wires is easy to perform . </S>", "<S> it is encouraged to use this approach as the clinical outcome is good.level of evidence : level iv - retrospective case , treatment study . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Surgical technique", "Results", "Discussion", "Conclusions"], "sections": [["these techniques include closed reduction , percutaneous pin reduction , intramedullary pin reduction with elastic stable intramedullary nailing or kirschner - wires ( k - wires ) , and open reduction with or without internal fixation .", "our goals for treatment are to avoid incision , reduce the fracture adequately with no reduction loss , and achieve good postoperative function .", "we performed a closed reduction with a percutaneous leverage technique and internal fixation method with k - wires to treat angulated radial neck fractures in children ."], ["from january 2011 to april 2013 , we treated 16 cases of angulated radial neck fractures in 12 boys and 4 girls .", "the types of fractures categorized by the obrien classification as follows : type i  angulation < 30 ; type ii ", "angulation > 60. among our patients , 5 had type ii and 11 had type iii fractures .", "the left side was affected in 9 cases , right side was affected in 7 cases .", "complications with ulnar olecranon fracture and medial epicondyle fracture were present in 3 cases and 2 cases , respectively . no blood vessel or nerve trauma was evident .", "the shortest time from trauma to surgery was 4  hours and the longest time was 6 days .", "a 1.6- or 2.0-mm k - wire was inserted from distal to proximal of the elbow percutaneously . in this procedure , the k - wire was inserted into the fracture site .", "the k - wire was used to lever the fracture and to orient the proximal part horizontally with the lateral condyle plane .", "was attained , two 1.2  mm k - wires were percutaneously inserted from the articular surface through the fracture site to the opposite cortex for fixation ( fig .", "the 3 cases with ulnar olecranon fractures were not treated because there was no obvious displacement .", "our research was approved by the biomedical research ethics committee of hong hui hospital , xian jiaotong university college of medicine .", "( a ) the kirschner wire ( k - wire ) was inserted percutaneously into the fracture site .", "( b ) the inserted k - wire was used as a lever to reduce the fracture .", "( c ) one 1.2  mm k - wires was percutaneously inserted from the articular surface passing through the fracture site to the opposite cortex .", "typical case : femail , 12 years old , and right side . a long - arm cast or brace was used for 3 to 4 weeks after the procedures , with the elbow in 90 of flexion and the forearm in supination ."], ["a 1.6- or 2.0-mm k - wire was inserted from distal to proximal of the elbow percutaneously . in this procedure , the k - wire was inserted into the fracture site .", "the k - wire was used to lever the fracture and to orient the proximal part horizontally with the lateral condyle plane .", "was attained , two 1.2  mm k - wires were percutaneously inserted from the articular surface through the fracture site to the opposite cortex for fixation ( fig .", "the 3 cases with ulnar olecranon fractures were not treated because there was no obvious displacement .", "our research was approved by the biomedical research ethics committee of hong hui hospital , xian jiaotong university college of medicine .", "( a ) the kirschner wire ( k - wire ) was inserted percutaneously into the fracture site .", "( b ) the inserted k - wire was used as a lever to reduce the fracture .", "( c ) one 1.2  mm k - wires was percutaneously inserted from the articular surface passing through the fracture site to the opposite cortex .", "typical case : femail , 12 years old , and right side . a long - arm cast or brace was used for 3 to 4 weeks after the procedures , with the elbow in 90 of flexion and the forearm in supination ."], ["cases were followed up for an average of 3 years 7 months ( range 2 years 6 months to 4 years 10 months ) .", "the postoperative reduction was assessed by roentgenograph within 1 week after surgery . no infection or nerve damage occurred in the short - term follow - up .", "no radial head necrosis or synostosis of the proximal ulna and radius was seen in the long - term follow - up .", "there was no disclosure of the epiphysis or valgus of the elbow during the follow - up . according to the metaizeau reduction classification ,", "12 cases were excellent , and 4 cases were good . according to the metaizeau clinical classification , 14 cases were excellent , and 2 cases were good .", "( a ) the preoperative x - ray showed the severe tilt and displacement of radial neck .", "( c ) the 2 years later follow - up x - ray picture showed the radial head normal ."], ["most authors agree that fractures with slight angulation should be treated conservatively , but severely angulated fractures ( > 60 ) require further treatment with different methods .", "the complications of these fractures reported in children include avascular necrosis , early physeal closure , elbow stiffness , periarticular ossification , and overgrowth of the radius head .", "the prognosis was not only related with the angulation of the radial neck fracture , but also with the age of the patient and extent of trauma .", "metaizeau suggested that a residual tilt above 10 to 15 at 10 to 12 years of age or 20 to 30 at a younger age could not be remodeled by growth .", "additionally , bernstein found that the determination of what constituted a significant angulation vari with the child 's age , with greater angulation degrees acceptable for younger children .", "the compression and impaction extends to the lateral side of radial neck upon radial neck fracture .", "the redisplacement will be accomplished easily only if it is immobilized by cast as there is no support from the lateral portion of the radial neck for the radius head after reduction .", "steinberg reported that 22 cases which occurred redisplacement in whole 28 cases which were immobilized by cast after reduction .", "one method uses an elastic stable intramedullary nailing to indirectly reduce and fix the fracture site .", "the other method uses an intramedullary k - wire to perform indirect reduction and internal fixation .", "the 3rd method uses a percutaneous k - wire to perform direct reduction and internal fixation . in comparing the 3 different methods , some authors recommended closed reduction and elastic intramedullary fixation to treat a fracture of the radius in children .", "however , we found that it was difficult to reduce the displaced radius head through rotating the distal part of the elastic intramedullary nail .", "this reduction approach usually requires an assistant to manipulate the percutaneous k - wire . it is challenging to fix the radial head with elastic intramedullary nail after a good reduction .", "the radial head was easily separated from the fracture site when penetrating with and rotating the nail .", "the functional lever arm was too long when the elastic intramedullary nail or intramedullary pin was used to fix the radial head . it was challenging to maintain a good position .", "there was no disclosure of the epiphysis or valgus of the elbow during the follow - up .", "trauma was minor and there was no interference with the blood supply of the radial head", ". there was no postoperative scarring and the patients and their parents were satisfied with outcome .", "the insertion site should be carefully selected to avoid damage to the deep branch of the radial nerve .", "the k - wires should be appropriately sized , with a diameter no greater than 1.5  mm .", "we usually choose 1.2  mm k - wires to prevent damage to the epiphysis ."], ["we recommend the percutaneous leverage technique during closed reduction and internal fixation using k - wires for the treatment of angulated radial neck fractures in children .", "this is a simple method with little trauma to the patient and a satisfactory clinical outcome ."]]}
{"article_id": "PMC4481042", "article_text": ["cities around the world can look very different if you compare the living conditions for the residents .", "however , it is not only different cities that can have completely diverse standards of infrastructure and social security .", "living in the slums compared to more wealthy neighbourhoods , will expose the inhabitants to different risks .", "traditionally cities can offer many advantages compared to rural settings , but under certain circumstances they can rather be a health hazard .", "the rapid migrations of people to cities can lead to overcrowding , which can generate slums or shanty towns .", "these slums are characterized by poor housing , lack of fresh water , and bad sanitation facilities ( 2 ) .", "all of these shortages can be a threat to the residents health and be a possible breeding ground for infectious diseases .", "the location of slums are often outside of the city centres , in more hazardous locations and the population feels a lack of social and economic opportunities compared with other residents . in sub - saharan africa , 62% of the urban population in 2012 lived in shanty towns ( 2 ) . for example , in 2009 , 96% of the urban population in central african republic lived in these slums ( 6 ) . in kenya 's capital nairobi ,", "60% of the population lives in slums , and child mortality there is 2.5 times greater than other parts of the city ( 9 ) . the community and health care services have great challenges to provide the entire population with equal and adequate service . the collected parties need to be aware of the differences in threats with respect to infectious diseases , both at the local and governmental levels .", "infections have been linked to slums in dar es salaam , tanzania , with high population density and low income ( 10 ) . in several other countries ,", "cholera incidence is the highest in urban regions with high population density ( 11 , 12 ) .", "differences in prevalence of asymptomatic carriers of antimicrobial drug - resistant diarrhoeagenic escherichia coli have also been found in brazil between slum settlements and more wealthy parts of the community 13 ) .", "the poor infrastructure in the slum can be a barrier for improvement , but at the same time targeted interventions for safer water and better sanitation facilities could potentially have a profound effect of the overall health .", "overcrowded housing in high - density populations in the slums can be a breeding ground for infectious diseases such as tuberculosis .", "the rate of tuberculosis has traditionally been higher in urban centres compared to rural ( 14 , 15 ) .", "studies in slum settlements in dhaka city , bangladesh , indicate a high prevalence of tuberculosis , which was almost twice as high compared to the overall national average and four times higher than the overall urban levels ( 16 .", "however , different patterns can be seen in different countries ; for example , in poland the rates of tuberculosis have shown only slightly lower incidence in rural population compared to urban , 21.9 per 100,000 versus 22.4 per 100,000 respectively ( 17 ) .", "tuberculosis in the united states has declined in the twentieth century , and several factors such as improved nutrition status , socioeconomic status , overall public health , and new drug regimens have been thought to play a major role . however , in the mid-1980s a resurgence occurred which reached its peak in 1992 , especially in urban areas among the homeless and incarcerated population ( 18 ) .", "the knowledge regarding symptoms , transmission , and prevention has been shown to be greater among the urban population in pakistan 's punjab province compared to the rural population .", "health - seeking behaviour was also better among the urban population , in the aspect of when to seek medical advice for early diagnosis and potential treatment ( 19 ) .", "information about infectious diseases and how they spread in the community can help the individuals to protect themselves , but knowledge about the slums and the infectious diseases panorama is also crucial for local physicians .", "they need to know how to look for the correct diagnosis , even if their diagnostic tools might be limited .", "the rapid influx of migrants can lead to overcrowding and local governments might not be able to provide safe housing , drinking water , and adequate sewage facilities , all of which are potential health hazards and must be taken into account for safe city planning . today more than half of the world 's population , almost 4 billion people , have access to piped water connected to their homes . since 1990 , well over 2 billion people have gained improved drinking water facilities , and almost 2 billion people have access to improved sanitation . however , more than 700 million people still lack access to improved sources of safe drinking water , and in sub - saharan africa half of the population lack such facilities . globally", "however , 1 billion people in the world still practice open defecation . in this group ,", "90% live in rural areas , but the actual amount of residents from urban settings is gradually increasing . between 1990 and 2012 ,", "the group in urban settings which lacked sanitation actually significantly increased from 215 million to 756 million , which could be explained by population growth ( 20 ) .", "much of the hard work to improve sanitation facilities has benefited large population groups , but the rapid influx of new urban residents shows that there is still much hard work to be done .", "residents who are subject to overcrowding and who lack access to safe drinking water or proper sanitation can be more susceptible to soil - transmitted helminths ( 21 ) .", "these infections are among the most important causes of physical and intellectual growth retardation in the world and have a major impact on public health ( 22 ) .", "good hygiene practices and good sanitary conditions have lowered the prevalent levels of contamination . in the brazilian city of salvador , with a population of 2.5 million , an improvement of sewerage coverage from 26 to 80% of the households led to an estimated overall reduction of diarrhoeal diseases of 22% ( 23 ) . neglected tropical diseases can cause substantial health problems in developing countries , and some of these diseases have a faecal - oral transmission pathway .", "examples of such diseases could be schistosomiasis , trachoma , and soil - transmitted helminthiases .", "in many countries , however , the focus is on treatment by medication and not improved sanitation", ". the reason could be that it would be much more expensive to carry out the necessary infrastructural improvements ( 24 ) .", "safe drinking water and proper sanitary facilities must be taken into account in city planning .", "factors like this can potentially have a profound positive effect in lowering infectious diseases with a faecal  oral route .", "one example of this is for chagas disease , which is a parasitic infection caused by the protozoan trypanosoma cruzi", ". an important mode of transmission is vectorial infected bites of triatomine bugs . living in close contact to domestic animals and poor hygienic habits", "chagas disease affects an estimated 8 million people every year , and is an important health challenge in latin america . in recent decades , progress has been made to reduce the burden of disease , by vector control , screening blood donors , improved housing , and epidemiological surveillance .", "chagas disease is a growing health problem in non - endemic areas because of population movements ( 26 ) .", "it is estimated that 300,000 individuals in the united states are infected ( 27 ) and the most affected country in europe , spain , is thought to have 45,00067,000 cases ( 28 ) .", "the example of chagas disease shows that physicians who practice in countries where the disease is not present must be aware of the travel history of the patient to connect the potential symptoms to the correct diagnosis .", "the environment in urban cities has proven to be favourable for the rat population ( rattus spp . ) and close encounters between rats and humans can lead to transmission of zoonotic infectious diseases .", ", rickettsia typhi , streptobacillus moniliformis , bartonella spp . , seoul hantavirus , and angiostrogylus cantonensis \n ( 29 ) .", "new york city has one of the largest populations of rats in the united states .", "it has been shown that encounters between rats and humans have been linked to proximity to open public spaces and subway lines , the presence of vacant housing units , and low education of the population ( 30 ) .", "information like this can be useful for health officials when they launch specific control initiatives .", "the changes in human population with increased urbanization and urban poverty has also altered our perception of some zoonoses linked to the rat population .", "leptospirosis has traditionally been perceived as a primarily rural disease , but the incidence in urban centres is increasing ( 31 , 32 ) . in chinese cities , the incidence of seoul hantavirus haemorrhagic fever with renal syndrome has been linked to urban growth , growing rat population , and increase rat  human contact ( 33 ) .", "large megacities all over the world have large rat populations , but the surveillance and local knowledge seem to be inadequate", ". a better understanding of how to prevent uncontrolled growth in rat population can potentially lead to a decline of these zoonotic diseases .", "the growing trend of urbanization around the world has shifted some infectious diseases , which have traditionally been perceived as rural , to urban settings . the world health organization ( who ) has published a list of 17 neglected tropical diseases .", "several of them have now become a reality in the urban environment , these diseases are something the practicing physicians in these areas have to be aware of ( 34 ) .", "many of the diseases on the list are present in the developing world , which sometimes lack the opportunity to solve these problems by themselves .", "one of the neglected infectious diseases is lymphatic filariasis ( lf ) with 2 billion people at risk , and 394.5 million in urban areas .", "one of the main reasons is the lack of proper sanitation facilities ( 35 ) .", "lf still has its major impact in rural settings , but the increasing urbanization in the developing world has made lf an infectious disease that also has to be considered elsewhere .", "one of the parasite species wuchereria bancrofti has been located in many urban areas and has the potential for transmission in this environment .", "moreover , one of the vectors for the parasite is the mosquito culex quinquefasciatus , which thrives in these surroundings , especially in overcrowded areas with poor sanitary and draining facilities .", "however , within one city the transmission can vary substantially depending on the standard of the sanitary conditions . the mosquito vector culex spp", "can be found in large parts of central and south america , east africa , and asia ( 36 ) .", "another vector which has adapted to urban surroundings is the mosquito aedes aegypti , which is a key component for dengue transmission .", "dengue is on who 's list of neglected tropical diseases , and is on the rise worldwide .", "the number of infections has drastically increased in the tropical regions of the world in the last 40 years .", "recent studies have estimated 390 million cases each year , and the burden is the highest in india with one - third of all the new infections ( 37 ) .", "several factors have played a big role in the escalation , such as urbanization , globalization , and lack of mosquito control .", "aedes aegypti lay their eggs in artificial water containers made by humans , which is a key component in the urban transmission cycle . the adaption of dengue through its vector has made dengue an infectious disease on the clear rise ( 38 ) .", "thailand is a country with all four serotypes of dengue virus , and the epidemics of dengue haemorrhagic fever have shown a possible correlation to originate from the urban capital of bangkok and then spread geographically in an outward manner to more rural settlements and provinces . a model to understand this mechanism could lead to more effective use of the health systems in the affected areas ( 39 ) .", "dengue has become a global problem and is no longer restricted to the developing world . despite better knowledge , it seems tough to control the vector , which has adapted to the urban environment and living close to people .", "an efficient vaccine is not yet commercially available , but could be a powerful factor in the fight against the global dengue epidemic .", "often several different factors need to be favourable for a vector - borne disease to adapt to the conditions in an urban environment .", "for example , west nile virus ( wnv ) infection is an infectious disease which has become a reality in the urban environment .", "the primary vector is the mosquito culex pipens , which lay their eggs in water resources which are often man - made .", "however , for a successful transmission cycle wnv also need the american robin ( turdus migratorius ) , which has several broods per season and hatchlings are more susceptible to wnv infection than adult birds ( 40 ) .", "the county of dallas , texas , experienced an epidemic of wnv infections in 2012 .", "surveillance reports revealed 25% of the cases in the united states were found in dallas county ( 41 ) .", "it shows for a vector - borne disease to have a successful transmission cycle several different factors need to be in place to affect the human population . \n", "leishmaniasis is a disease caused by the protozoa leishmania , which affects 12 million and threatens 350 million people in 88 different countries .", "when rural migrants bring their domesticated animals to urban settings , often slums , they create favourable conditions for an urban transmission ( 43 ) .", "it has been shown that it is a growing health problem and the ongoing urbanization has contributed to the increase ( 44 ) . if the different vectors can adapt to the urban environment and man - made resources , the potential health implications can be of major concern .", "control programs and adequate surveillance is of importance , but in rapidly growing cities and slums it can be tough to implement such measures . emerging infectious diseases can also make the jump to stable transmission in the urban surroundings and surveillance of these can potentially prevent major health concerns and high cost for the health care services . who can play a major role in the fight for better control and knowledge .", "many of the countries in the developing world do not have the proper resources and the problem is not concentrated to one region , but is a global concern .", "numerous of the neglected tropical diseases play a major role in the developing world , which is currently experiencing a much faster pace of urbanization compared to the developed world", ". the who 's call for help is important and , for example , dengue is now turning into a global crisis .", "safe and targeted assistance can be a huge factor for overall health ; such assistance could be an effective vaccine or safe and easy vector control programs .", "the basis of large population groups in a restricted area can provide the perfect conditions for different epidemics .", "international travel has connected the world in the last century , and this mobility creates a potential threat of many emerging diseases .", "international tourist arrivals have shown an exceptional growth from 25 million in 1950 to 1,087 million in 2013 . according to the latest forecast from the world tourism organization", ", international tourism arrivals will continue to increase , and in 2030 the figure is expected to be 1.8 billion ( 45 ) . with the pace of modern travel , highly contagious infectious diseases can be a potential threat in a completely different setting compared to the original outbreak .", "urban population and the density of residents can meet the criteria for a new epidemic and create a public health disaster , if not taken seriously .", "international trade and travel can potentially also contribute to the occurrence of a worldwide pandemic .", "sars is thought to originate from the sars - like coronavirus ( scov ) of bats and reached the human host in china due to hunting and trading of bats for food ( 46 ) .", "investigations have found this scov from the himalayan palm civets in live - animal markets in the region .", "the first cases of sars reportedly occurred in individuals who handled these animals to prepare exotic food , and the virus is thought to have crossed over to their human host ( 47 ) .", "it spread in urban dwellings in large cities and in well - equipped city hospitals .", "public fear of travelling led to considerable economic losses that affected entire countries ( 48 ) .", "the example of sars shows that food markets in southern china can be the origin of a worldwide health crisis .", "travel routes around the world have connected the urban world and large megacities like never before .", "accordingly it is important to take necessary preventive measures before the epidemic gets out of control , and here big organizations like who , but also governments , play an important role .", "early action is of utmost importance , and functional surveillance programs needs to be in place .", "the zoonotic disease dengue is endemic in most tropical and subtropical regions , which often are also popular tourist destinations .", "the burden of disease is on the rise , and estimations are that in returning travellers from southeast asia , dengue is now a more frequent cause of febrile illness compared to malaria ( 49 ) .", "dengue is now an urban health problem , which is one of the major reasons why the rise is exceptional .", "the worldwide spread of certain antibiotic resistant staphylococcus aureus has been linked to tourism , which shows the potential impact on international health ( 50 , 51 ) .", "faecal colonization with esbl - producing enterobacteriaceae has also been linked to international travellers in several studies ( 5254 ) .", "the physician needs to take into account the recent travel activities of the patient to better evaluate the current condition and need for potential treatment and care .", "global travel shows no signs of decline and the interconnected megacities around the world make global surveillance even more important when it comes to contagious infectious diseases .", "measurements to stop the spread need to be taken at the original location , but knowledge about the specific disease needs to be passed on to the global community and local health workers in other parts of the world .", "this global surveillance and alert system needs to be fast and efficient to , if possible , reduce the impact .", "the expected rise of travel makes it critical for the future global health and the possibility to react in time for possible threats .", "rapid and sometimes uncontrolled urbanization can , in certain circumstances , lead to closer encounters with wildlife .", "human influence on the ecosystems creates meeting points for new and potential zoonotic diseases , which could have a profound impact for both local and global health .", "new housing in the outskirts of big cities can potentially be meeting points for new and already known zoonotic diseases .", "of 335 emerging infectious diseases , which have been recognized between 1940 and 2004 , more than 60% have been zoonotic diseases ( 55 ) . living in close contact to domesticated animals and hunt for  bush - meat", "can also be risk factors for an infectious disease to make the jump from the animal host to humans .", "major deforestation creates closer contact between humans and bats and even primates , who can potentially be host for  new viruses .", "a better understanding , surveillance , and prevention of zoonotic diseases would be of great value , to both prevent and manage this upcoming threat for global health .", "hot spots for this transmission have been found and they often correlate where the process of urbanization is on the clear rise ( 56 ) .", "even if it is not always the urban population who is at the front of new encounters with wildlife , it can still have an effect on urban health .", "the trend of people moving to cities are at the highest , where many of these new encounters with ecosystems take place , and infectious diseases can be introduced to these growing urban environments .", "the sometimes uncontrolled growth of cities pushes residents to untouched ecosystems when new housing expands .", "ebola virus disease ( evd ) has had a profound impact on the world in 2014 . since the spring of 2014 ,", "the hub of the epidemic has been the three countries in western africa : sierra leone , liberia , and guinea .", "it all began in december 2013 in guinea , in the providence of guckdou , in the eastern rainforest region .", "the disease transmission in the capital of conakry is thought to be the first major urban setting for evd ( 57 ) . who was first notified of the evd outbreak in march 2014 , and on august 8 , the who declared the current situation as ", "evd outbreaks in central africa had been limited in size and geographical spread to a few hundred persons , mostly in remote areas and not large urban settings ( 59 ) .", "the centre of the epidemic ( guinea , liberia , and sierra leone ) has , as many of their neighbouring countries , a large population living in rural settings ; only 36 , 49 , and 40% of their population live in urban centres ( 6062 ) .", "the population is , however , highly interconnected in these countries with travel and cross - border traffic , with good road access between rural and urban settings .", "these communications have made the magnitude of the evd epidemic possible . despite cases of evd in nigeria and lagos , a megacity with 20 million inhabitants ,", "the transmission has been limited , which proves that implementation of control measures can limit the transmission ( 63 ) .", "the mortality rate has been high in previous outbreaks , up to 90% ( 64 ) .", "the fatality rate in the west africa epidemic has been estimated to around 70% for guinea , liberia , and sierra leone when data for patients with recorded definitive clinical outcomes ( 63 ) .", "this unprecedented epidemic points out the importance of better surveillance , understanding , and preventions measures for this potentially deadly virus .", "ebola virus ( ebov ) is thought to be a zoonotic disease , and fruit bats are under investigation to be the natural reservoir .", "ebov sequences have been found in these animals near the human outbreaks which implies where the virus might originate from ( 65 , 66 ) .", "closer contact with humans and fruit bats are thus risks for a new global health crisis and the severity of an ebola epidemic has already been witnessed .", "the high costs , both from an economic and overall health perspective , have affected entire countries and have even cost lives on the other side of the earth .", "different factors , such as education , direct primary care services , and the governments capacity for rapid response to upcoming health threats , can contribute to the opportunities in a city .", "however , in many cities the poor can find it difficult to access proper health care , due to the cost of such services . in more rural areas", ", the problem can instead be the distance to the nearest clinic , which in reality makes it impossible for prompt and efficient treatment ( 2 ) .", "malaria has historically been and is still a major health concern in large parts of the world . who estimates 198 million cases ( 124283 million ) of malaria and 584,000 deaths ( 367,000755,000 ) in 2013 .", "the highest mortality rates have been shown to be closely linked to poor countries with a low gross national income ( gni ) per capita ( 67 ) .", "estimations have been made that nearly 25% of the total african population , 200 million , currently live in urban settings where malaria transmission is a reality .", "the annual incidence is estimated at 24.8103.2 million cases of clinical malaria among the urban population in africa ( 68 ) .", "the relationship between the malaria mosquito vector and the human host determines the burden of morbidity and mortality .", "this interface is dependent on many different factors and the degree of urbanization is an important one .", "increased urbanization and decreased transmission have correlated in several different studies ( 69 ) . however , whether it was the increased urbanization that led to a reduction in transmission or the malaria reduction that led to development that promoted urbanization of societies is a challenge to determine ( 70 ) .", "a clear connection has been shown between reduced transmission of plasmodium falciparum and urbanization ; however , for plasmodium vivax it is less obvious . for p. vivax , a connection has been found globally and in asia and africa ; inconsistent results , however , were found in the americas .", "several possibilities could explain these incoherent results , such as more widespread transmission of p. vivax , lower transmission intensity , the wide distribution in asia , and high prevalence of duffy negativity in africa , which protects against p. vivax \n ( 71 ) .", "the overall decrease of the burden of malaria has been a positive effect of urbanization , but the exact mechanisms are not yet known .", "coverage of measles vaccination in indonesia have shown to be 68.5% in rural areas , compared with 80.1% in urban regions ( 72 ) .", "studies in nigeria have shown that sometimes the coverage can actually be better in more rural areas , and it might be explained by better mobilization and participation in the delivery of immunization services ( 73 ) . in a study in uganda ,", "58% of the urban group compared to 53% in the rural areas were fully immunized , but polio vaccine was given to 51% in the urban group and 52% in the rural group ( 74 ) .", "immunization coverage can also vary considerably among different settings , not only between rural and urban surroundings , but also between urban , rural , and slum settlements . in changdigarh ,", "a union territory of india , full immunization of children at the age of 2 was 30% in slums , 74% in urban , and 62.5% in rural settings ( 75 ) .", "it shows that there can be a wide variety of reasons for immunization status among the population in different regions and countries of the world .", "high coverage can prevent epidemics in large cities and save many lives ; however , immunization needs to be available both for the rural and urban population to achieve the greatest benefit .", "a study in tanzania has compared the knowledge about certain zoonotic diseases among general practitioners in urban and rural areas .", "the rural practitioners had poor knowledge of how sleeping sickness is transmitted and clinical features of anthrax and rabies . laboratories in rural areas are often poorly equipped and can not always diagnose certain zoonotic diseases , which could limit the doctors capability for correct diagnosis and treatment ( 76 ) .", "the knowledge about sexually transmitted diseases ( stis ) among bangladeshi adolescents was higher among people in urban areas compared to rural , both in general and hiv and aids ( 77 ) .", "the same results about hiv and aids have been found among a canadian population ( 78 ) .", "studies in chengdu and shanghai , china , have shown risk perception about stis and hiv and aids is profoundly changed in rural - to - urban migrants ( 79 , 80 ) .", "the same result has been shown in a study among rural - to - urban migrants in ethiopia 81 ) .", "the rapid influx of migrants moving to cities makes it hard to get adequate information to all the different groups in the society . to educate the public is one of the many challenges for local governments and health officials .", "campaigns to improve the public knowledge are useful to fight the threat of infectious diseases .", "residents need to be aware of symptoms of infectious diseases to gain knowledge about when to seek health care and when it is safe to treat yourself .", "knowledge about food storage , waste management , vector control , and sanitary facilities are all aspects that can lower the burden of communicable diseases .", "these campaigns can sometimes be easier in the urban environment because of the density of the population .", "urbanization is an ongoing process in the world at the moment , but the pace of the process is not universal . the developed countries , which have traditionally been thought of as high - income countries , are already urbanized , and it is in the developing world that the rapid rise is taking place .", "infectious diseases still have a big impact on the global health , and urbanization is now altering the characteristics of these diseases . living conditions in cities", "are overall better in urban environments compared to rural settings ; better housing , sanitation , ventilation , and social services all play an important role in this improvement . certain pathogens can , however , adapt to the different conditions and thus create a new challenge for both local governments and the global community .", "the capacity for surveillance , control programs , prevention , and public knowledge programs is far better in cities .", "but some countries do not have the resources and because these diseases can be of global concern , it is also the international community 's responsibility to help and support with knowledge and resources . the rapid urbanization has also interfered in previously untouched ecosystems .", "these new settlements create new and closer encounters with wildlife , which can be a potential source of zoonotic diseases . these can be both previously known or new pathogens , which make the shift from their animal host to generate infections in humans .", "surveillance is of primary importance to monitor the burden of disease and will give both local authorities and the global community a chance for a quick response to public health threats .", "the author have not received any funding or benefits from industry or elsewhere to conduct this study ."], "abstract_text": ["<S> the world is becoming more urban every day , and the process has been ongoing since the industrial revolution in the 18th century . </S>", "<S> the united nations now estimates that 3.9 billion people live in urban centres . </S>", "<S> the rapid influx of residents is however not universal and the developed countries are already urban , but the big rise in urban population in the next 30 years is expected to be in asia and africa . </S>", "<S> urbanization leads to many challenges for global health and the epidemiology of infectious diseases . </S>", "<S> new megacities can be incubators for new epidemics , and zoonotic diseases can spread in a more rapid manner and become worldwide threats </S>", "<S> . adequate city planning and surveillance can be powerful tools to improve the global health and decrease the burden of communicable diseases . </S>"], "labels": null, "section_names": ["Urban population  a heterogeneous group with different living conditions", "City planning", "Traditional rural infectious diseases turn urban", "Modern cities a catalyst for rapid spread of infectious diseases", "Zoonotic disease a challenge for the future", "Urbanization and the urban environment a cause of better health", "Conclusion", "Conflict of interest and funding"], "sections": [["cities around the world can look very different if you compare the living conditions for the residents .", "however , it is not only different cities that can have completely diverse standards of infrastructure and social security .", "living in the slums compared to more wealthy neighbourhoods , will expose the inhabitants to different risks .", "traditionally cities can offer many advantages compared to rural settings , but under certain circumstances they can rather be a health hazard .", "the rapid migrations of people to cities can lead to overcrowding , which can generate slums or shanty towns .", "these slums are characterized by poor housing , lack of fresh water , and bad sanitation facilities ( 2 ) .", "all of these shortages can be a threat to the residents health and be a possible breeding ground for infectious diseases .", "the location of slums are often outside of the city centres , in more hazardous locations and the population feels a lack of social and economic opportunities compared with other residents . in sub - saharan africa , 62% of the urban population in 2012 lived in shanty towns ( 2 ) . for example , in 2009 , 96% of the urban population in central african republic lived in these slums ( 6 ) . in kenya 's capital nairobi ,", "60% of the population lives in slums , and child mortality there is 2.5 times greater than other parts of the city ( 9 ) . the community and health care services have great challenges to provide the entire population with equal and adequate service . the collected parties need to be aware of the differences in threats with respect to infectious diseases , both at the local and governmental levels .", "infections have been linked to slums in dar es salaam , tanzania , with high population density and low income ( 10 ) . in several other countries ,", "cholera incidence is the highest in urban regions with high population density ( 11 , 12 ) .", "differences in prevalence of asymptomatic carriers of antimicrobial drug - resistant diarrhoeagenic escherichia coli have also been found in brazil between slum settlements and more wealthy parts of the community 13 ) .", "the poor infrastructure in the slum can be a barrier for improvement , but at the same time targeted interventions for safer water and better sanitation facilities could potentially have a profound effect of the overall health .", "overcrowded housing in high - density populations in the slums can be a breeding ground for infectious diseases such as tuberculosis .", "the rate of tuberculosis has traditionally been higher in urban centres compared to rural ( 14 , 15 ) .", "studies in slum settlements in dhaka city , bangladesh , indicate a high prevalence of tuberculosis , which was almost twice as high compared to the overall national average and four times higher than the overall urban levels ( 16 .", "however , different patterns can be seen in different countries ; for example , in poland the rates of tuberculosis have shown only slightly lower incidence in rural population compared to urban , 21.9 per 100,000 versus 22.4 per 100,000 respectively ( 17 ) .", "tuberculosis in the united states has declined in the twentieth century , and several factors such as improved nutrition status , socioeconomic status , overall public health , and new drug regimens have been thought to play a major role . however , in the mid-1980s a resurgence occurred which reached its peak in 1992 , especially in urban areas among the homeless and incarcerated population ( 18 ) .", "the knowledge regarding symptoms , transmission , and prevention has been shown to be greater among the urban population in pakistan 's punjab province compared to the rural population .", "health - seeking behaviour was also better among the urban population , in the aspect of when to seek medical advice for early diagnosis and potential treatment ( 19 ) .", "information about infectious diseases and how they spread in the community can help the individuals to protect themselves , but knowledge about the slums and the infectious diseases panorama is also crucial for local physicians .", "they need to know how to look for the correct diagnosis , even if their diagnostic tools might be limited ."], ["the rapid influx of migrants can lead to overcrowding and local governments might not be able to provide safe housing , drinking water , and adequate sewage facilities , all of which are potential health hazards and must be taken into account for safe city planning . today more than half of the world 's population , almost 4 billion people , have access to piped water connected to their homes . since 1990 , well over 2 billion people have gained improved drinking water facilities , and almost 2 billion people have access to improved sanitation . however , more than 700 million people still lack access to improved sources of safe drinking water , and in sub - saharan africa half of the population lack such facilities . globally", "however , 1 billion people in the world still practice open defecation . in this group ,", "90% live in rural areas , but the actual amount of residents from urban settings is gradually increasing . between 1990 and 2012 ,", "the group in urban settings which lacked sanitation actually significantly increased from 215 million to 756 million , which could be explained by population growth ( 20 ) .", "much of the hard work to improve sanitation facilities has benefited large population groups , but the rapid influx of new urban residents shows that there is still much hard work to be done .", "residents who are subject to overcrowding and who lack access to safe drinking water or proper sanitation can be more susceptible to soil - transmitted helminths ( 21 ) .", "these infections are among the most important causes of physical and intellectual growth retardation in the world and have a major impact on public health ( 22 ) .", "good hygiene practices and good sanitary conditions have lowered the prevalent levels of contamination . in the brazilian city of salvador , with a population of 2.5 million , an improvement of sewerage coverage from 26 to 80% of the households led to an estimated overall reduction of diarrhoeal diseases of 22% ( 23 ) . neglected tropical diseases can cause substantial health problems in developing countries , and some of these diseases have a faecal - oral transmission pathway .", "examples of such diseases could be schistosomiasis , trachoma , and soil - transmitted helminthiases .", "in many countries , however , the focus is on treatment by medication and not improved sanitation", ". the reason could be that it would be much more expensive to carry out the necessary infrastructural improvements ( 24 ) .", "safe drinking water and proper sanitary facilities must be taken into account in city planning .", "factors like this can potentially have a profound positive effect in lowering infectious diseases with a faecal  oral route .", "one example of this is for chagas disease , which is a parasitic infection caused by the protozoan trypanosoma cruzi", ". an important mode of transmission is vectorial infected bites of triatomine bugs . living in close contact to domestic animals and poor hygienic habits", "chagas disease affects an estimated 8 million people every year , and is an important health challenge in latin america . in recent decades , progress has been made to reduce the burden of disease , by vector control , screening blood donors , improved housing , and epidemiological surveillance .", "chagas disease is a growing health problem in non - endemic areas because of population movements ( 26 ) .", "it is estimated that 300,000 individuals in the united states are infected ( 27 ) and the most affected country in europe , spain , is thought to have 45,00067,000 cases ( 28 ) .", "the example of chagas disease shows that physicians who practice in countries where the disease is not present must be aware of the travel history of the patient to connect the potential symptoms to the correct diagnosis .", "the environment in urban cities has proven to be favourable for the rat population ( rattus spp . ) and close encounters between rats and humans can lead to transmission of zoonotic infectious diseases .", ", rickettsia typhi , streptobacillus moniliformis , bartonella spp . , seoul hantavirus , and angiostrogylus cantonensis \n ( 29 ) .", "new york city has one of the largest populations of rats in the united states .", "it has been shown that encounters between rats and humans have been linked to proximity to open public spaces and subway lines , the presence of vacant housing units , and low education of the population ( 30 ) .", "information like this can be useful for health officials when they launch specific control initiatives .", "the changes in human population with increased urbanization and urban poverty has also altered our perception of some zoonoses linked to the rat population .", "leptospirosis has traditionally been perceived as a primarily rural disease , but the incidence in urban centres is increasing ( 31 , 32 ) . in chinese cities , the incidence of seoul hantavirus haemorrhagic fever with renal syndrome has been linked to urban growth , growing rat population , and increase rat  human contact ( 33 ) .", "large megacities all over the world have large rat populations , but the surveillance and local knowledge seem to be inadequate", ". a better understanding of how to prevent uncontrolled growth in rat population can potentially lead to a decline of these zoonotic diseases ."], ["the growing trend of urbanization around the world has shifted some infectious diseases , which have traditionally been perceived as rural , to urban settings . the world health organization ( who ) has published a list of 17 neglected tropical diseases .", "several of them have now become a reality in the urban environment , these diseases are something the practicing physicians in these areas have to be aware of ( 34 ) .", "many of the diseases on the list are present in the developing world , which sometimes lack the opportunity to solve these problems by themselves .", "one of the neglected infectious diseases is lymphatic filariasis ( lf ) with 2 billion people at risk , and 394.5 million in urban areas .", "one of the main reasons is the lack of proper sanitation facilities ( 35 ) .", "lf still has its major impact in rural settings , but the increasing urbanization in the developing world has made lf an infectious disease that also has to be considered elsewhere .", "one of the parasite species wuchereria bancrofti has been located in many urban areas and has the potential for transmission in this environment .", "moreover , one of the vectors for the parasite is the mosquito culex quinquefasciatus , which thrives in these surroundings , especially in overcrowded areas with poor sanitary and draining facilities .", "however , within one city the transmission can vary substantially depending on the standard of the sanitary conditions . the mosquito vector culex spp", "can be found in large parts of central and south america , east africa , and asia ( 36 ) .", "another vector which has adapted to urban surroundings is the mosquito aedes aegypti , which is a key component for dengue transmission .", "dengue is on who 's list of neglected tropical diseases , and is on the rise worldwide .", "the number of infections has drastically increased in the tropical regions of the world in the last 40 years .", "recent studies have estimated 390 million cases each year , and the burden is the highest in india with one - third of all the new infections ( 37 ) .", "several factors have played a big role in the escalation , such as urbanization , globalization , and lack of mosquito control .", "aedes aegypti lay their eggs in artificial water containers made by humans , which is a key component in the urban transmission cycle . the adaption of dengue through its vector has made dengue an infectious disease on the clear rise ( 38 ) .", "thailand is a country with all four serotypes of dengue virus , and the epidemics of dengue haemorrhagic fever have shown a possible correlation to originate from the urban capital of bangkok and then spread geographically in an outward manner to more rural settlements and provinces . a model to understand this mechanism could lead to more effective use of the health systems in the affected areas ( 39 ) .", "dengue has become a global problem and is no longer restricted to the developing world . despite better knowledge , it seems tough to control the vector , which has adapted to the urban environment and living close to people .", "an efficient vaccine is not yet commercially available , but could be a powerful factor in the fight against the global dengue epidemic .", "often several different factors need to be favourable for a vector - borne disease to adapt to the conditions in an urban environment .", "for example , west nile virus ( wnv ) infection is an infectious disease which has become a reality in the urban environment .", "the primary vector is the mosquito culex pipens , which lay their eggs in water resources which are often man - made .", "however , for a successful transmission cycle wnv also need the american robin ( turdus migratorius ) , which has several broods per season and hatchlings are more susceptible to wnv infection than adult birds ( 40 ) .", "the county of dallas , texas , experienced an epidemic of wnv infections in 2012 .", "surveillance reports revealed 25% of the cases in the united states were found in dallas county ( 41 ) .", "it shows for a vector - borne disease to have a successful transmission cycle several different factors need to be in place to affect the human population . \n", "leishmaniasis is a disease caused by the protozoa leishmania , which affects 12 million and threatens 350 million people in 88 different countries .", "when rural migrants bring their domesticated animals to urban settings , often slums , they create favourable conditions for an urban transmission ( 43 ) .", "it has been shown that it is a growing health problem and the ongoing urbanization has contributed to the increase ( 44 ) . if the different vectors can adapt to the urban environment and man - made resources , the potential health implications can be of major concern .", "control programs and adequate surveillance is of importance , but in rapidly growing cities and slums it can be tough to implement such measures . emerging infectious diseases can also make the jump to stable transmission in the urban surroundings and surveillance of these can potentially prevent major health concerns and high cost for the health care services . who can play a major role in the fight for better control and knowledge .", "many of the countries in the developing world do not have the proper resources and the problem is not concentrated to one region , but is a global concern .", "numerous of the neglected tropical diseases play a major role in the developing world , which is currently experiencing a much faster pace of urbanization compared to the developed world", ". the who 's call for help is important and , for example , dengue is now turning into a global crisis .", "safe and targeted assistance can be a huge factor for overall health ; such assistance could be an effective vaccine or safe and easy vector control programs ."], ["the basis of large population groups in a restricted area can provide the perfect conditions for different epidemics .", "international travel has connected the world in the last century , and this mobility creates a potential threat of many emerging diseases .", "international tourist arrivals have shown an exceptional growth from 25 million in 1950 to 1,087 million in 2013 . according to the latest forecast from the world tourism organization", ", international tourism arrivals will continue to increase , and in 2030 the figure is expected to be 1.8 billion ( 45 ) . with the pace of modern travel , highly contagious infectious diseases can be a potential threat in a completely different setting compared to the original outbreak .", "urban population and the density of residents can meet the criteria for a new epidemic and create a public health disaster , if not taken seriously .", "international trade and travel can potentially also contribute to the occurrence of a worldwide pandemic .", "sars is thought to originate from the sars - like coronavirus ( scov ) of bats and reached the human host in china due to hunting and trading of bats for food ( 46 ) .", "investigations have found this scov from the himalayan palm civets in live - animal markets in the region .", "the first cases of sars reportedly occurred in individuals who handled these animals to prepare exotic food , and the virus is thought to have crossed over to their human host ( 47 ) .", "it spread in urban dwellings in large cities and in well - equipped city hospitals .", "public fear of travelling led to considerable economic losses that affected entire countries ( 48 ) .", "the example of sars shows that food markets in southern china can be the origin of a worldwide health crisis .", "travel routes around the world have connected the urban world and large megacities like never before .", "accordingly it is important to take necessary preventive measures before the epidemic gets out of control , and here big organizations like who , but also governments , play an important role .", "early action is of utmost importance , and functional surveillance programs needs to be in place .", "the zoonotic disease dengue is endemic in most tropical and subtropical regions , which often are also popular tourist destinations .", "the burden of disease is on the rise , and estimations are that in returning travellers from southeast asia , dengue is now a more frequent cause of febrile illness compared to malaria ( 49 ) .", "dengue is now an urban health problem , which is one of the major reasons why the rise is exceptional .", "the worldwide spread of certain antibiotic resistant staphylococcus aureus has been linked to tourism , which shows the potential impact on international health ( 50 , 51 ) .", "faecal colonization with esbl - producing enterobacteriaceae has also been linked to international travellers in several studies ( 5254 ) .", "the physician needs to take into account the recent travel activities of the patient to better evaluate the current condition and need for potential treatment and care .", "global travel shows no signs of decline and the interconnected megacities around the world make global surveillance even more important when it comes to contagious infectious diseases .", "measurements to stop the spread need to be taken at the original location , but knowledge about the specific disease needs to be passed on to the global community and local health workers in other parts of the world .", "this global surveillance and alert system needs to be fast and efficient to , if possible , reduce the impact .", "the expected rise of travel makes it critical for the future global health and the possibility to react in time for possible threats ."], ["rapid and sometimes uncontrolled urbanization can , in certain circumstances , lead to closer encounters with wildlife .", "human influence on the ecosystems creates meeting points for new and potential zoonotic diseases , which could have a profound impact for both local and global health .", "new housing in the outskirts of big cities can potentially be meeting points for new and already known zoonotic diseases .", "of 335 emerging infectious diseases , which have been recognized between 1940 and 2004 , more than 60% have been zoonotic diseases ( 55 ) . living in close contact to domesticated animals and hunt for  bush - meat", "can also be risk factors for an infectious disease to make the jump from the animal host to humans .", "major deforestation creates closer contact between humans and bats and even primates , who can potentially be host for  new viruses .", "a better understanding , surveillance , and prevention of zoonotic diseases would be of great value , to both prevent and manage this upcoming threat for global health .", "hot spots for this transmission have been found and they often correlate where the process of urbanization is on the clear rise ( 56 ) .", "even if it is not always the urban population who is at the front of new encounters with wildlife , it can still have an effect on urban health .", "the trend of people moving to cities are at the highest , where many of these new encounters with ecosystems take place , and infectious diseases can be introduced to these growing urban environments .", "the sometimes uncontrolled growth of cities pushes residents to untouched ecosystems when new housing expands .", "ebola virus disease ( evd ) has had a profound impact on the world in 2014 . since the spring of 2014 ,", "the hub of the epidemic has been the three countries in western africa : sierra leone , liberia , and guinea .", "it all began in december 2013 in guinea , in the providence of guckdou , in the eastern rainforest region .", "the disease transmission in the capital of conakry is thought to be the first major urban setting for evd ( 57 ) . who was first notified of the evd outbreak in march 2014 , and on august 8 , the who declared the current situation as ", "evd outbreaks in central africa had been limited in size and geographical spread to a few hundred persons , mostly in remote areas and not large urban settings ( 59 ) .", "the centre of the epidemic ( guinea , liberia , and sierra leone ) has , as many of their neighbouring countries , a large population living in rural settings ; only 36 , 49 , and 40% of their population live in urban centres ( 6062 ) .", "the population is , however , highly interconnected in these countries with travel and cross - border traffic , with good road access between rural and urban settings .", "these communications have made the magnitude of the evd epidemic possible . despite cases of evd in nigeria and lagos , a megacity with 20 million inhabitants ,", "the transmission has been limited , which proves that implementation of control measures can limit the transmission ( 63 ) .", "the mortality rate has been high in previous outbreaks , up to 90% ( 64 ) .", "the fatality rate in the west africa epidemic has been estimated to around 70% for guinea , liberia , and sierra leone when data for patients with recorded definitive clinical outcomes ( 63 ) .", "this unprecedented epidemic points out the importance of better surveillance , understanding , and preventions measures for this potentially deadly virus .", "ebola virus ( ebov ) is thought to be a zoonotic disease , and fruit bats are under investigation to be the natural reservoir .", "ebov sequences have been found in these animals near the human outbreaks which implies where the virus might originate from ( 65 , 66 ) .", "closer contact with humans and fruit bats are thus risks for a new global health crisis and the severity of an ebola epidemic has already been witnessed .", "the high costs , both from an economic and overall health perspective , have affected entire countries and have even cost lives on the other side of the earth ."], ["different factors , such as education , direct primary care services , and the governments capacity for rapid response to upcoming health threats , can contribute to the opportunities in a city .", "however , in many cities the poor can find it difficult to access proper health care , due to the cost of such services . in more rural areas", ", the problem can instead be the distance to the nearest clinic , which in reality makes it impossible for prompt and efficient treatment ( 2 ) .", "malaria has historically been and is still a major health concern in large parts of the world . who estimates 198 million cases ( 124283 million ) of malaria and 584,000 deaths ( 367,000755,000 ) in 2013 .", "the highest mortality rates have been shown to be closely linked to poor countries with a low gross national income ( gni ) per capita ( 67 ) .", "estimations have been made that nearly 25% of the total african population , 200 million , currently live in urban settings where malaria transmission is a reality .", "the annual incidence is estimated at 24.8103.2 million cases of clinical malaria among the urban population in africa ( 68 ) .", "the relationship between the malaria mosquito vector and the human host determines the burden of morbidity and mortality .", "this interface is dependent on many different factors and the degree of urbanization is an important one .", "increased urbanization and decreased transmission have correlated in several different studies ( 69 ) . however , whether it was the increased urbanization that led to a reduction in transmission or the malaria reduction that led to development that promoted urbanization of societies is a challenge to determine ( 70 ) .", "a clear connection has been shown between reduced transmission of plasmodium falciparum and urbanization ; however , for plasmodium vivax it is less obvious . for p. vivax , a connection has been found globally and in asia and africa ; inconsistent results , however , were found in the americas .", "several possibilities could explain these incoherent results , such as more widespread transmission of p. vivax , lower transmission intensity , the wide distribution in asia , and high prevalence of duffy negativity in africa , which protects against p. vivax \n ( 71 ) .", "the overall decrease of the burden of malaria has been a positive effect of urbanization , but the exact mechanisms are not yet known .", "coverage of measles vaccination in indonesia have shown to be 68.5% in rural areas , compared with 80.1% in urban regions ( 72 ) .", "studies in nigeria have shown that sometimes the coverage can actually be better in more rural areas , and it might be explained by better mobilization and participation in the delivery of immunization services ( 73 ) . in a study in uganda ,", "58% of the urban group compared to 53% in the rural areas were fully immunized , but polio vaccine was given to 51% in the urban group and 52% in the rural group ( 74 ) .", "immunization coverage can also vary considerably among different settings , not only between rural and urban surroundings , but also between urban , rural , and slum settlements . in changdigarh ,", "a union territory of india , full immunization of children at the age of 2 was 30% in slums , 74% in urban , and 62.5% in rural settings ( 75 ) .", "it shows that there can be a wide variety of reasons for immunization status among the population in different regions and countries of the world .", "high coverage can prevent epidemics in large cities and save many lives ; however , immunization needs to be available both for the rural and urban population to achieve the greatest benefit .", "a study in tanzania has compared the knowledge about certain zoonotic diseases among general practitioners in urban and rural areas .", "the rural practitioners had poor knowledge of how sleeping sickness is transmitted and clinical features of anthrax and rabies . laboratories in rural areas are often poorly equipped and can not always diagnose certain zoonotic diseases , which could limit the doctors capability for correct diagnosis and treatment ( 76 ) .", "the knowledge about sexually transmitted diseases ( stis ) among bangladeshi adolescents was higher among people in urban areas compared to rural , both in general and hiv and aids ( 77 ) .", "the same results about hiv and aids have been found among a canadian population ( 78 ) .", "studies in chengdu and shanghai , china , have shown risk perception about stis and hiv and aids is profoundly changed in rural - to - urban migrants ( 79 , 80 ) .", "the same result has been shown in a study among rural - to - urban migrants in ethiopia 81 ) .", "the rapid influx of migrants moving to cities makes it hard to get adequate information to all the different groups in the society . to educate the public is one of the many challenges for local governments and health officials .", "campaigns to improve the public knowledge are useful to fight the threat of infectious diseases .", "residents need to be aware of symptoms of infectious diseases to gain knowledge about when to seek health care and when it is safe to treat yourself .", "knowledge about food storage , waste management , vector control , and sanitary facilities are all aspects that can lower the burden of communicable diseases .", "these campaigns can sometimes be easier in the urban environment because of the density of the population ."], ["urbanization is an ongoing process in the world at the moment , but the pace of the process is not universal . the developed countries , which have traditionally been thought of as high - income countries , are already urbanized , and it is in the developing world that the rapid rise is taking place .", "infectious diseases still have a big impact on the global health , and urbanization is now altering the characteristics of these diseases . living conditions in cities", "are overall better in urban environments compared to rural settings ; better housing , sanitation , ventilation , and social services all play an important role in this improvement . certain pathogens can , however , adapt to the different conditions and thus create a new challenge for both local governments and the global community .", "the capacity for surveillance , control programs , prevention , and public knowledge programs is far better in cities .", "but some countries do not have the resources and because these diseases can be of global concern , it is also the international community 's responsibility to help and support with knowledge and resources . the rapid urbanization has also interfered in previously untouched ecosystems .", "these new settlements create new and closer encounters with wildlife , which can be a potential source of zoonotic diseases . these can be both previously known or new pathogens , which make the shift from their animal host to generate infections in humans .", "surveillance is of primary importance to monitor the burden of disease and will give both local authorities and the global community a chance for a quick response to public health threats ."], ["the author have not received any funding or benefits from industry or elsewhere to conduct this study ."]]}
{"article_id": "PMC4215526", "article_text": ["inadequate blood flow to the brain , termed cerebral ischemia , will produce brain injury and a progression of pathophysiological responses associated with energy depletion that include cell swelling , cytotoxic edema , vasogenic edema and if sufficiently severe or prolonged , neuronal cell death .", "when brain regions are directly damaged by ischemia this can lead to secondary injury or degeneration in associated axonal connections such as the corticospinal tracts .", "detection of such changes on early subacute diffusion mri have been described as  early  or", "such early signs of wallerian degenerative injury are predictive of specific functional outcomes and may improve prognostication , guide rehabilitation , and could represent both a selection criteria and potential novel target for clinical trials", ". in particular , if there is diagnostic evidence for early degenerative injury , such as increases in the intensity of the corticospinal tract in diffusion weighted magnetic resonance ( mr ) images , then prognosis for a good recovery in such infants has consistently been poor ( domi et al . , 2009 ;", "understanding better the cellular responses that evolve with axonal degeneration following stroke are important considering both their potential contributions to diagnosis and the potential for providing novel targets for therapeutic intervention .", "currently , the tissue alterations underlying these mr biomarkers are unknown and speculative because histopathology , if available , is usually performed at single and relatively chronic time points many weeks to months after an ischemic insult", ". studies of the evolution of ischemic brain damage or infarction at subacute and chronic times has been assessed using standard mr imaging sequences such as t2 or diffusion weighted imaging ( latchaw et al . , 2009 ) .", "we recently demonstrated that following neonatal hypoxia with unilateral transient ischemia there is evidence for early wallerian degeneration visible as either decreases in the apparent diffusion coefficient ( adc ) and magnetization transfer ratio or increases in dw or t2 ( lama et al .", "the early corticospinal axonal changes ( e.g. in the cerebral peduncle ) reflected several cellular alterations such as a reduced staining for phosphorylated neurofilament h and increased vacuolation in hematoxylin and eosin stained sections .", "dti holds additional promise for detecting specific degenerative responses because of its potential sensitivity to certain ultrastructural cellular changes depending on the measured dti parameter  i.e. fractional anisotropy , mean diffusivity and parallel or radial diffusivity . of the extensive cellular responses produced by ischemia , those considered most likely to affect the diffusion properties of water are those that involve morphological tissue changes such as necrosis , astrogliosis , loss of myelin , loss of axonal neurofilaments or cellular inflammation ( i.e. microglial / macrophage activation ) ( nucifora et al . , 2007 ; zhang et al . , 2012 ) .", "our group and others have shown that corticospinal tract dti alterations are strongly correlated with motor outcome in children with perinatal stroke ( hodge , 2013 ; roze et al .", "however the progression of dti changes and whether there are differences between the evolution of cellular responses in direct ischemically injured brain versus associated connected tracts is not known .", "differences are expected considering the different cellular compositions , for example neuronal rich cerebral cortex compared to axonal bundles within the descending corticospinal tract .", "a differential timing of dti alterations is also expected considering direct ischemic injury is thought to be followed by a delayed wallerian degeneration ( lama et al . , 2011 ; tuor et al . , 2013 ) .", "in order to better understand the unique cellular and dti imaging alterations associated with secondary wallerian degeneration relative to the onset and progression of direct ischemically damaged brain , it is necessary to compare their progression directly at multiple time points . in the current study we hypothesized that dti imaging would provide measures of progressive tissue changes within axonal tracts distal to the ischemic injury distinct from those in directly injured brain ( e.g. cerebral cortex ) and these differences would correspond with specific tissue morphological alterations in response to ischemia", "this was investigated by using a neonatal rat model of unilateral transient cerebral hypoxia  ischemia and measuring dti changes in the parietal cerebral cortex compared with its associated descending corticospinal tract axon fibers within the cerebral peduncle .", "dti measurements were made at acute , subacute and chronic times after hypoxia  ischemia along with processing of brains at each of these time points to assess immunohistochemically potential contributions of morphological modifications .", "we focused on using markers for detecting cell death in neurons , loss of myelin and loss of neurofilaments in axons , and , activation of astrocytes and microglia / macrophages .", "fifty one pups delivered from 9 different pregnant wistar female rats ( charles river laboratories , montreal , canada ) were used in the study .", "experiments followed the canadian council on animal care guidelines and were approved by a university of calgary animal care committee .", "a moderate unilateral ischemic lesion with hypoxia ( vannucci and vannucci , 2005 ) was produced as described previously ( lama et al .", "briefly , pups ( n  =  38 ) on their 7th day of life had their right common carotid artery ligated under isoflurane anesthesia followed by a 60  minute exposure to hypoxia in a chamber containing 8% o2 and 92% n2 at 35.5  c .", "sham control animals ( n  =  13 ) experienced a similar surgical procedure without carotid artery ligation and hypoxia exposure .", "the vannucci model of neonatal cerebral hypoxia  ischemia ( vannucci and vannucci , 2005 ) produces ischemic damage and infarction within the distribution of the middle cerebral artery territory .", "thus , the parietal cortex was selected as a representative brain region of direct hypoxic  ischemic damage or infarction .", "the posterior cerebral peduncle supplied by the posterior circulation was selected as a region of secondary injury remote to the hypoxia ", "brain near the aqueduct in the posterior pons was selected as a control region generally unaffected by the hypoxia  ischemia .", "sham animals or animals subjected to cerebral hypoxia  ischemia were anesthetized ( 1.52% isoflurane ) and dti images in addition to anatomical scans were acquired at 3  h , 1  d , 2  d , 1  w or 4  w post - insult .", "anatomical images were also acquired at 1  d post insult in the chronic animals ( 1 or 4  w ) to confirm the extent of ischemic damage .", "mr images were acquired using a 9.4  t bruker biospin mr imaging system and paravision 5.1 software . throughout the scanning ,", "respiration was monitored and maintained by adjustments in anesthesia and body temperature was maintained using a feedback heated air system ( small instruments inc . , stony brook , ny ) .", "images were acquired using a 3.5  cm diameter quadrature volume coil for radiofrequency transmission and reception .", "the head and body was restrained using custom designed swaddling and a head band or ear pins . depending on the age of the animal ,", "each mr imaging scan consisted of 2530 slices of 0.50.55  mm thick covering the cerebrum and medulla , a 2    2  cm or 2.5    2.5  cm field of view and a data matrix size of 128    128 .", "anatomical t2 maps were first generated using a t2 imaging sequence consisting of a set of t2 weighted spin echo images with 32 echoes , repetition time of 10  s and echo time of 10  ms between echoes .", "for dti , a four - shot echo - planar imaging sequence was used to acquire four averages of sets of diffusion weighted images .", "these were acquired with b values of 0 ( 5 images ) and 1000  s / mm ( 30 images in non - collinear directions ) using a repetition time of 6500  ms and an echo time of 35  ms . artifacts associated with imperfections in the radio frequency pulse , gradient stability , and gradient echo currents were removed using a navigator - echo phase correction .", "nyquist ghost artifacts were suppressed in the image reconstruction using information acquired during the initial automatic receiver gain adjustment .", "dti image acquisition time was approximately 1  h. t2 weighted images were visualized using local mr analysis software ( marevisi , national research council of canada , winnipeg , canada ) .", "these images were used to assess the extent of ischemic damage and measure brain volumes ( atrophy ) .", "inspection of t2 images was also used to exclude from further analysis animals with no cortical t2 lesions or very large hyperintense lesions extending into the pons .", "they were also used to identify anatomical landmarks for selection of the regions of interest in subsequent dti analysis and histological analysis as identified using a rat brain atlas ( paxinos and watson , 1998 ) .", "these regions of interest were manually defined in the mr images and histological sections and included the cerebral peduncle ( 0.30.4  mm ) , pons ( within the central gray and mesencephalic trigeminal nucleus regions , 0.60.9  mm ) and parietal cortex ( 0.71.0  mm ) for areas both ipsilateral and contralateral to the lesion containing hemisphere . once the ischemic infarct had progressed towards cavitation and cyst formation ( i.e. 1 and 4  w post - hypoxia  ischemia ) , homologous measures of cortical tissue were not possible and therefore were taken adjacent to the cyst  a peri - cyst cortical region ( 0.40.6  mm ) .", "sizes and shapes were adjusted using the contralateral region ( e.g. cerebral peduncle ) as a guide and areas of interest were adjusted if there was atrophy in order to minimize partial volume effects .", "diffusion tensor images were imported into dti studio ( jiang et al . , 2006 ) through the dti mapping function as philips rec files .", "entire scans were excluded if less than eighteen artifact free images were available per slice .", "the minimum of eighteen directions was selected empirically as the cut - off following an analysis of 12 test data sets with repeatedly fewer b directions ( e.g. 25 , 22 , 20 , 18 , 16 and 15 compared to 30 ) which demonstrated no significant effect of a reduction in the number of b directions except for fa and l3 ; and , those were altered only once the number of directions was 16 or less ( p  <  0.05 , repeated anova ) . for each image slice", "the dti program calculated the over - determined matrix , the adc vector and the pseudo - inverse of the matrix using singular value decomposition in order to compute the tensor for each pixel ( jiang et al . , 2006 ) .", "imaging maps were then calculated for the mean diffusivity ( adc ; ( 1  +  2  +  3 )  /  3 ) , the parallel diffusivity ( 1 ) , the radial diffusivity ( ( 2  +  3 )  /  2 ) and the fractional anisotropy ( fa ) . measures of the dti variables were obtained within regions of interest manually drawn ipsilateral and contralateral ( non - ischemic damaged side ) of the brain slice .", "following their last mr scan , subgroups of animals were perfused transcardially with 10% formalin under deep pentobarbital anesthesia and their brains were embedded in paraffin .", "brains from 44 rats were processed and stained for 5 different cellular or injury markers within sections containing the parietal cortex , posterior cerebral peduncle and central gray region of the pons .", "thirteen of these animals were sham controls and 31 were animals subjected to hypoxia  ischemia and euthanized at the 5 different times post hypoxia  ischemia ( n  =  47/group ) .", "sections ( 6  m thick ) were cut at the level of the posterior cerebral peduncle and parietal cortex using the brain atlas and anatomical landmarks to match sections to the mr slices and measure staining changes in equivalent regions of interest to those for the mr images .", "sections from each region were stained for a marker of cell death or degenerating neurons using fluorojade b ( schmued and hopkins , 2000 ) according to the manufacturer 's instructions ( millipore , billerica , ma , usa ) . briefly , deparaffinized sections were exposed to 0.06% potassium permanganate for 15  min washed in distilled water and then stained with 0.0004% fluorojade solution .", "sections at each level were also processed immunohistochemically to stain for the following : neurofilaments using a pan - axonal neurofilament marker ( monoclonal smi312 ) , reactive astrocytes using anti - glial fibrillary acidic protein ( gfap ) , activated microglia / macrophages using anti - cd68/ed1 , and , myelin using an antibody against myelin basic protein ( mbp ) .", "sections were deparaffinized and processed for antigen retrieval by boiling in sodium citrate ( ph  =  6.0 , 90  c ) for 10  min then washed in pbs .", "slides were subsequently blocked with 1% h2o2 in methanol for 5  min followed by 10% goat serum in 0.1  m pbs and then a pbs wash .", "slides were then incubated for 1  h at room temperature with one of 4 different primary antibodies : smi312 antibody ( 1:1000 in bsa , covance , berkeley , ca , usa ) , polyclonal rabbit anti - gfap ( 1:500 in bsa , diagnostics canada inc .", ", on , canada ) , mouse anti - rat cd68/ed1 ( 1:500 in bsa , abd serotec , raleigh , nc , usa ) or rabbit anti - mbp ( 1:500 in bsa , chemicon international , ca , usa ) . following a pbs wash", ", slides were incubated overnight at 4  c with the appropriate secondary antibody , either biotinylated goat anti - rabbit igg or biotinylated goat anti - mouse igg ( 1:400 in bsa , jackson immunoresearch lab inc .", "this was followed by incubation for half an hour with horseradish peroxidase - streptavidin ( 1:400 in pbs , diagnostics canada inc . , on , canada ) . finally , positive cells were visualized following an application of 3,3-diaminobenzidine ( sigma aldrich , mo , usa ) dissolved in 0.3% h2o2 in distilled water for approximately 3  min .", "the histological sections were assessed semi - quantitatively for changes in staining within the regions of interest either by measuring optical density or by quantifying numbers of positively stained cells per field of view . for optical density measures , sections stained with either smi312 , gfap or mbp", "were first captured digitally using a scanner ( nikon scan , version 4 , nikon inc . ) .", "these images were converted to gray images and mean gray levels ( imagej , nih ) were measured within the same regions of interest as those drawn for the mr brain slices .", "regions with less intense staining had higher mean gray values . the ipsilateral versus contralateral differences in gray levels were converted to a percentage of the contralateral value .", "the cd68/ed1 and fluorojade stained sections were analyzed using digitized microscopy ( microbrightfield , olympus , 40 magnification ) .", "the numbers of positively stained cells per field of view were estimated within regions corresponding to those selected from mr images .", "three different fields of view were counted for positive staining cells by an investigator ( m.s . )", "all group data is reported as mean    sd and statistical comparisons were considered significant at p  <  0.05 . measured values within different brain regions", "a two way repeated analysis was used to determine significant region and time interactions and these were followed by a holm ", "statistical tests were performed using sigmaplot ( systat software inc , san jose , ca ) . to convey clearly the differences for the cerebral peduncle , we noted in the graphs only the significant differences for this region .", "the correlation between histological changes and dti parameters was assessed using a pearson product correlation analysis within each time point or all time points with a bonferroni adjustment for multiple comparisons .", "despite intensive investigation of dti 's ability to detect brain injury , the interpretation of alterations in dti measures is usually speculative because of a lack of direct documentation of the corresponding histological changes . in the present study", "we report detailed differences in the progression and magnitude of dti and histological changes that provide a reference for identifying the potential contribution of various cellular responses to fa and axial , radial and mean diffusivity .", "the results are first to characterize the correspondence of the progression of cellular and dti changes within the cerebral peduncle of the descending corticospinal tract following hypoxic  ischemic damage to the cortex .", "novel is the demonstration of the differing progression of dti alterations in a directly damaged ischemic region such as parietal cortex versus that in associated subcortical tracts .", "dti changes were less severe or delayed in the cerebral peduncle of the corticospinal tract and this in general corresponded to less severe and delayed cellular responses consistent with early wallerian degeneration .", "as discussed in more detail , the tissue alterations following ischemia and axonal degeneration had a complex relationship with changes in fa , parallel diffusivity and radial diffusivity .", "regarding our assessment of progressive changes in the dti parameters , it is important to realize that superimposed on the effects of hi injury are well recognized developmental changes in the dti parameters that occur during development and aging ( dubois et al . , 2013 ) .", "indeed , the contralateral hemisphere was similar to those in sham animals but developmental changes may have been differentially affected in the left and right hemispheres by the ischemic insult .", "right differences needs to include an awareness that these can be accounted for by an injury modulation of cellular maturation processes in white matter , which we assessed in our histological sections .", "the region of cerebral peduncle measured was approx 0.30.4  mm consisting of approx 10 pixels of sizes 0.024  mm in the neonatal scans and 0.038  mm in 5  wk old animals .", "partial volume effects could be greater in neonatal brain than older brain . however , because we compared left and right differences in values , we expect that there were similar ipsilateral and contralateral partial volume effects in the cerebral peduncle at each time point .", "right differences but this did not appear to be an issue considering that many of our measures had the maximal changes in our neonates ( e.g. 1  d post insult ) . there have been numerous studies regarding the evolution of adc or mean diffusivity following transient cerebral ischemia in regions of stroke .", "adc alterations reported previously are comparable to those observed presently ( e.g. lama et al . , 2011 ; liu et al . , 2007 ) .", "notably , there were striking similarities between the adc and parallel diffusivity changes suggesting that both reflect similar cellular responses to injury . very early", "adc and parallel diffusivity changes in ischemic cortex are likely due to a greater restriction of water diffusion as a result of cytotoxic edema , reduced extracellular space and increased tortuosity which in the present study was also accompanied by decreased neurofilament staining and increased neuronal cell death . as vasogenic edema and damage to cellular membranes proceeded , which histologically corresponded to a continuing cell death and microglial / macrophage activation and astrogliosis , the reductions in adc and parallel diffusivity normalized and this process occurred over several days .", "by 14  w , phagocytosis produced cysts in the infarct region and such cystic regions had an extremely large adc in all directions whereas peri - infarct regions generally had normalized adc and normalized parallel diffusivity despite the presence of inflammatory cells .", "in contrast , in the cerebral peduncle with indirect ischemic injury , by the first 3  h there were modest but significant adc and parallel diffusivity reductions .", "the hyperacute changes in adc and parallel diffusivity correspond to our previous observation of a reduction in smi31 , demonstrating a decrease in phosphorylated heavy neurofilament subunits already hours after insult .", "subsequently , the adc and parallel diffusivity reductions increased in magnitude and were maximal in the first few days .", "this corresponded well with the diminished staining observed for the pan - axonal ( heavy , medium and light ) neurofilament marker smi312 used presently .", "loss of phosphorylation of neurofilaments promotes their disassembly and this corresponded to a decreased radial diffusivity as well as a decreased parallel and mean diffusivity of water . at chronic time points post - insult ( 14  w ) ,", "a similar greater sensitivity for parallel diffusivity than adc in detecting wallerian degenerative changes in the corticospinal tract has been reported after an excision lesion of the feline cortex ( qin et al . , 2012 ) .", "microglial / macrophage cell activation likely did not contribute greatly to the parallel diffusivity alterations observed , considering the rather modest positive ed1/cd68 staining observed in the cerebral peduncle .", "the occurrence of astrogliosis coincided with normalization of adc potentially contributing to some extent to these changes .", "however , as discussed below , there were also ipsilateral increases in radial diffusivity that likely contributed to the adc normalization .", "reductions in radial diffusivity have been proposed to reflect well increases in myelin whereas elevations in radial diffusivity have been found to be associated with a loss of myelin ( song et al . , 2002 , 2004 ;", "the current study provides some support for this if one examines only chronic recovery times  i.e. times when the axons in the contralateral cerebral peduncle have become well myelinated . at 1  w and beyond , myelin within the cerebral peduncle", "ipsilaterally was reduced reflecting less ontogenic myelination of fewer axons following their degeneration and atrophy .", "this coincided with relative increases in radial diffusivity ipsilaterally and is consistent with increases in radial diffusivity reported in the corticospinal or pyramidal tracts at relatively chronic times ( e.g. 1  w to 3  y ) following an ischemic insult in children or adults ( roze et al .", ", 2012 ; yu et al . , 2009 ; zanier et al . , 2013 ) . at acute times post - insult we observed ipsilateral reductions in radial diffusivity within the cerebral peduncle indicating that in the absence of effects on myelination , early axonal injury such as that detected by decreases in phosphorylated neurofilaments were also associated with reduced radial diffusivity", "it is noteworthy that the neurophysiological correlates of alterations in parallel and radial diffusivity are potentially more complex in humans than rodents .", "a diffusion tensor analysis has an implicit assumption that the white matter fibers are cylindrical and when fibers have complicated microstructures or crossing fibers as occurs in human developing brain ( dubois et al . , 2013 ;", "2013 ) then in particular the directions of orientation for tractography require careful interpretation . despite crossing fibers potentially complicating the interpretation of parallel diffusivity changes ,", "transverse or radial diffusivity appears to consistently decrease with all maturational processes within white matter , including myelination ( dubois et al . , 2013 ) .", "furthermore , emerging imaging methods exploring diffusion parameters beyond the tensor model may provide more in depth assessments to facilitate translation from animals to humans ( dubois et al .", "the fa measures provided a sensitive measure of direct ischemic injury . in the cortex at hyperacute times", ", there was an increase in fa , as has been observed previously in rats and non - human primates , however , usually soon after permanent rather than transient occlusion ( kim et al .", ", 2013 ; liu et al . , 2007 ; yang et al . , 1999 ) .", "this suggests that the very early cortical ischemic changes were relatively severe and similar to those with permanent ischemia .", "cellular changes likely involve previously established hyperacute cytotoxic cell swelling and reductions in extracellular space leading to cell death considering our histological evidence for hyperacute neuronal cell death in the cortex . by the first day post - insult ,", "the fa increase became a decrease that persisted at subacute and chronic time points , confirming previous reports of a reduced fa commonly observed in damaged brain post - stroke ( e.g. kim et al .", ", 2013 ; liu et al . , 2007 ; yang et al . , 1999 ) .", "this decrease in fa , reflecting diminished preferential diffusivity , is likely associated with a loss of membrane integrity and intracellular structure due to necrosis as supported histologically by continuing cell death and loss of neurofilament staining .", "the increase in astrogliosis between 3 and 1  d appears rather isotropic considering that there is a decline in fa between these times .", "the fa also provided a rather robust measure of degenerative changes in the cerebral peduncle .", "in contrast to ischemic cortex , fa in cerebral peduncle was decreased at all time points .", "indeed , fa appeared to be the most sensitive dti parameter for detecting axonal degenerative changes in the cerebral peduncle independent of time post - insult and this corresponded well to the persistent decreases in axonal neurofilament staining .", "similar reductions in fa have been reported at various select times in the corticospinal tract following neonatal stroke ( ludeman et al . , 2008 ; puig et al . , 2013 ; tusor et al . , 2012 ) .", "it is currently clear that at chronic times , fa is superior to adc or other standard imaging methods such as t2 imaging because lesions detected with these other sequences tended to pseudo - normalize at times post - insult ( lama et al . , 2011 ; tuor et al . ,", "the results demonstrate that in general , diffusivity changes and histological responses within directly damaged ischemic brain precede and are often larger in magnitude than corresponding alterations in a region of secondary axonal degeneration , such as the cerebral peduncle . with the progression of damage over one or more weeks , dti components such as fa and radial diffusivity are better than mean adc for detecting ischemia associated axonal injury indicative of wallerian degeneration .", "also , differences in radial diffusivity in brain injured perinatally are likely related to a lack of ontogenic myelination on the injured side .", "thus , the specific progression of dti alterations following an ischemic insult not only depends on the maturity of the subject but also appears dependent on other factors such as the type of insult ( e.g. spinal cord injury or brain resection ) ( brennan et al . , 2013 ; liu et al . , 2013 ; qin et al . , 2012 ) .", "the inflammatory response investigated ( microglial or astroglial activation ) appears to have modest contributions to the evolution of axonal changes in diffusion tensor parameters .", "thus , this systematic study is informative regarding the optimal dti mr imaging parameters to probe axonal injury following hypoxic / ischemic brain damage in developing brain in addition to aiding our interpretation of dti parameters for detecting various stages of wallerian degeneration ."], "abstract_text": ["<S> ischemically damaged brain can be accompanied by secondary degeneration of associated axonal connections e.g. wallerian degeneration . diffusion tensor imaging ( dti ) </S>", "<S> is widely used to investigate axonal injury but the cellular correlates of many of the degenerative changes remain speculative . </S>", "<S> we investigated the relationship of dti of directly damaged cerebral cortex and secondary axonal degeneration in the cerebral peduncle with cellular alterations in pan - axonal neurofilament staining , myelination , reactive astrocytes , activation of microglia / macrophages and neuronal cell death . </S>", "<S> dti measures ( axial , radial and mean diffusivity , and fractional anisotropy ( fa ) ) were acquired at hyperacute ( 3  h ) , acute ( 1 and 2  d ) and chronic ( 1 and 4  week ) times after transient cerebral hypoxia with unilateral ischemia in neonatal rats . the tissue pathology underlying ischemic and degenerative responses had a complex relationship with dti parameters . </S>", "<S> dti changes at hyperacute and subacute times were smaller in magnitude and tended to be transient and/or delayed in cerebral peduncle compared to cerebral cortex . in cerebral peduncle by 1  d post - insult , there were reductions in neurofilament staining corresponding with decreases in parallel diffusivity which were more sensitive than mean diffusivity in detecting axonal changes . </S>", "<S> ipsilesional reductions in fa within cerebral peduncle were robust in detecting both early and chronic degenerative responses . at one or four weeks post - insult , radial diffusivity was increased ipsilaterally in the cerebral peduncle corresponding to pathological evidence of a lack of ontogenic myelination in this region . </S>", "<S> the detailed differences in progression and magnitude of dti and histological changes reported provide a reference for identifying the potential contribution of various cellular responses to fa , and , parallel , radial , and mean diffusivity . </S>"], "labels": null, "section_names": ["Introduction", "Material and methods", "Discussion"], "sections": [["inadequate blood flow to the brain , termed cerebral ischemia , will produce brain injury and a progression of pathophysiological responses associated with energy depletion that include cell swelling , cytotoxic edema , vasogenic edema and if sufficiently severe or prolonged , neuronal cell death .", "when brain regions are directly damaged by ischemia this can lead to secondary injury or degeneration in associated axonal connections such as the corticospinal tracts .", "detection of such changes on early subacute diffusion mri have been described as  early  or", "such early signs of wallerian degenerative injury are predictive of specific functional outcomes and may improve prognostication , guide rehabilitation , and could represent both a selection criteria and potential novel target for clinical trials", ". in particular , if there is diagnostic evidence for early degenerative injury , such as increases in the intensity of the corticospinal tract in diffusion weighted magnetic resonance ( mr ) images , then prognosis for a good recovery in such infants has consistently been poor ( domi et al . , 2009 ;", "understanding better the cellular responses that evolve with axonal degeneration following stroke are important considering both their potential contributions to diagnosis and the potential for providing novel targets for therapeutic intervention .", "currently , the tissue alterations underlying these mr biomarkers are unknown and speculative because histopathology , if available , is usually performed at single and relatively chronic time points many weeks to months after an ischemic insult", ". studies of the evolution of ischemic brain damage or infarction at subacute and chronic times has been assessed using standard mr imaging sequences such as t2 or diffusion weighted imaging ( latchaw et al . , 2009 ) .", "we recently demonstrated that following neonatal hypoxia with unilateral transient ischemia there is evidence for early wallerian degeneration visible as either decreases in the apparent diffusion coefficient ( adc ) and magnetization transfer ratio or increases in dw or t2 ( lama et al .", "the early corticospinal axonal changes ( e.g. in the cerebral peduncle ) reflected several cellular alterations such as a reduced staining for phosphorylated neurofilament h and increased vacuolation in hematoxylin and eosin stained sections .", "dti holds additional promise for detecting specific degenerative responses because of its potential sensitivity to certain ultrastructural cellular changes depending on the measured dti parameter  i.e. fractional anisotropy , mean diffusivity and parallel or radial diffusivity . of the extensive cellular responses produced by ischemia , those considered most likely to affect the diffusion properties of water are those that involve morphological tissue changes such as necrosis , astrogliosis , loss of myelin , loss of axonal neurofilaments or cellular inflammation ( i.e. microglial / macrophage activation ) ( nucifora et al . , 2007 ; zhang et al . , 2012 ) .", "our group and others have shown that corticospinal tract dti alterations are strongly correlated with motor outcome in children with perinatal stroke ( hodge , 2013 ; roze et al .", "however the progression of dti changes and whether there are differences between the evolution of cellular responses in direct ischemically injured brain versus associated connected tracts is not known .", "differences are expected considering the different cellular compositions , for example neuronal rich cerebral cortex compared to axonal bundles within the descending corticospinal tract .", "a differential timing of dti alterations is also expected considering direct ischemic injury is thought to be followed by a delayed wallerian degeneration ( lama et al . , 2011 ; tuor et al . , 2013 ) .", "in order to better understand the unique cellular and dti imaging alterations associated with secondary wallerian degeneration relative to the onset and progression of direct ischemically damaged brain , it is necessary to compare their progression directly at multiple time points . in the current study we hypothesized that dti imaging would provide measures of progressive tissue changes within axonal tracts distal to the ischemic injury distinct from those in directly injured brain ( e.g. cerebral cortex ) and these differences would correspond with specific tissue morphological alterations in response to ischemia", "this was investigated by using a neonatal rat model of unilateral transient cerebral hypoxia  ischemia and measuring dti changes in the parietal cerebral cortex compared with its associated descending corticospinal tract axon fibers within the cerebral peduncle .", "dti measurements were made at acute , subacute and chronic times after hypoxia  ischemia along with processing of brains at each of these time points to assess immunohistochemically potential contributions of morphological modifications .", "we focused on using markers for detecting cell death in neurons , loss of myelin and loss of neurofilaments in axons , and , activation of astrocytes and microglia / macrophages ."], ["fifty one pups delivered from 9 different pregnant wistar female rats ( charles river laboratories , montreal , canada ) were used in the study .", "experiments followed the canadian council on animal care guidelines and were approved by a university of calgary animal care committee .", "a moderate unilateral ischemic lesion with hypoxia ( vannucci and vannucci , 2005 ) was produced as described previously ( lama et al .", "briefly , pups ( n  =  38 ) on their 7th day of life had their right common carotid artery ligated under isoflurane anesthesia followed by a 60  minute exposure to hypoxia in a chamber containing 8% o2 and 92% n2 at 35.5  c .", "sham control animals ( n  =  13 ) experienced a similar surgical procedure without carotid artery ligation and hypoxia exposure .", "the vannucci model of neonatal cerebral hypoxia  ischemia ( vannucci and vannucci , 2005 ) produces ischemic damage and infarction within the distribution of the middle cerebral artery territory .", "thus , the parietal cortex was selected as a representative brain region of direct hypoxic  ischemic damage or infarction .", "the posterior cerebral peduncle supplied by the posterior circulation was selected as a region of secondary injury remote to the hypoxia ", "brain near the aqueduct in the posterior pons was selected as a control region generally unaffected by the hypoxia  ischemia .", "sham animals or animals subjected to cerebral hypoxia  ischemia were anesthetized ( 1.52% isoflurane ) and dti images in addition to anatomical scans were acquired at 3  h , 1  d , 2  d , 1  w or 4  w post - insult .", "anatomical images were also acquired at 1  d post insult in the chronic animals ( 1 or 4  w ) to confirm the extent of ischemic damage .", "mr images were acquired using a 9.4  t bruker biospin mr imaging system and paravision 5.1 software . throughout the scanning ,", "respiration was monitored and maintained by adjustments in anesthesia and body temperature was maintained using a feedback heated air system ( small instruments inc . , stony brook , ny ) .", "images were acquired using a 3.5  cm diameter quadrature volume coil for radiofrequency transmission and reception .", "the head and body was restrained using custom designed swaddling and a head band or ear pins . depending on the age of the animal ,", "each mr imaging scan consisted of 2530 slices of 0.50.55  mm thick covering the cerebrum and medulla , a 2    2  cm or 2.5    2.5  cm field of view and a data matrix size of 128    128 .", "anatomical t2 maps were first generated using a t2 imaging sequence consisting of a set of t2 weighted spin echo images with 32 echoes , repetition time of 10  s and echo time of 10  ms between echoes .", "for dti , a four - shot echo - planar imaging sequence was used to acquire four averages of sets of diffusion weighted images .", "these were acquired with b values of 0 ( 5 images ) and 1000  s / mm ( 30 images in non - collinear directions ) using a repetition time of 6500  ms and an echo time of 35  ms . artifacts associated with imperfections in the radio frequency pulse , gradient stability , and gradient echo currents were removed using a navigator - echo phase correction .", "nyquist ghost artifacts were suppressed in the image reconstruction using information acquired during the initial automatic receiver gain adjustment .", "dti image acquisition time was approximately 1  h. t2 weighted images were visualized using local mr analysis software ( marevisi , national research council of canada , winnipeg , canada ) .", "these images were used to assess the extent of ischemic damage and measure brain volumes ( atrophy ) .", "inspection of t2 images was also used to exclude from further analysis animals with no cortical t2 lesions or very large hyperintense lesions extending into the pons .", "they were also used to identify anatomical landmarks for selection of the regions of interest in subsequent dti analysis and histological analysis as identified using a rat brain atlas ( paxinos and watson , 1998 ) .", "these regions of interest were manually defined in the mr images and histological sections and included the cerebral peduncle ( 0.30.4  mm ) , pons ( within the central gray and mesencephalic trigeminal nucleus regions , 0.60.9  mm ) and parietal cortex ( 0.71.0  mm ) for areas both ipsilateral and contralateral to the lesion containing hemisphere . once the ischemic infarct had progressed towards cavitation and cyst formation ( i.e. 1 and 4  w post - hypoxia  ischemia ) , homologous measures of cortical tissue were not possible and therefore were taken adjacent to the cyst  a peri - cyst cortical region ( 0.40.6  mm ) .", "sizes and shapes were adjusted using the contralateral region ( e.g. cerebral peduncle ) as a guide and areas of interest were adjusted if there was atrophy in order to minimize partial volume effects .", "diffusion tensor images were imported into dti studio ( jiang et al . , 2006 ) through the dti mapping function as philips rec files .", "entire scans were excluded if less than eighteen artifact free images were available per slice .", "the minimum of eighteen directions was selected empirically as the cut - off following an analysis of 12 test data sets with repeatedly fewer b directions ( e.g. 25 , 22 , 20 , 18 , 16 and 15 compared to 30 ) which demonstrated no significant effect of a reduction in the number of b directions except for fa and l3 ; and , those were altered only once the number of directions was 16 or less ( p  <  0.05 , repeated anova ) . for each image slice", "the dti program calculated the over - determined matrix , the adc vector and the pseudo - inverse of the matrix using singular value decomposition in order to compute the tensor for each pixel ( jiang et al . , 2006 ) .", "imaging maps were then calculated for the mean diffusivity ( adc ; ( 1  +  2  +  3 )  /  3 ) , the parallel diffusivity ( 1 ) , the radial diffusivity ( ( 2  +  3 )  /  2 ) and the fractional anisotropy ( fa ) . measures of the dti variables were obtained within regions of interest manually drawn ipsilateral and contralateral ( non - ischemic damaged side ) of the brain slice .", "following their last mr scan , subgroups of animals were perfused transcardially with 10% formalin under deep pentobarbital anesthesia and their brains were embedded in paraffin .", "brains from 44 rats were processed and stained for 5 different cellular or injury markers within sections containing the parietal cortex , posterior cerebral peduncle and central gray region of the pons .", "thirteen of these animals were sham controls and 31 were animals subjected to hypoxia  ischemia and euthanized at the 5 different times post hypoxia  ischemia ( n  =  47/group ) .", "sections ( 6  m thick ) were cut at the level of the posterior cerebral peduncle and parietal cortex using the brain atlas and anatomical landmarks to match sections to the mr slices and measure staining changes in equivalent regions of interest to those for the mr images .", "sections from each region were stained for a marker of cell death or degenerating neurons using fluorojade b ( schmued and hopkins , 2000 ) according to the manufacturer 's instructions ( millipore , billerica , ma , usa ) . briefly , deparaffinized sections were exposed to 0.06% potassium permanganate for 15  min washed in distilled water and then stained with 0.0004% fluorojade solution .", "sections at each level were also processed immunohistochemically to stain for the following : neurofilaments using a pan - axonal neurofilament marker ( monoclonal smi312 ) , reactive astrocytes using anti - glial fibrillary acidic protein ( gfap ) , activated microglia / macrophages using anti - cd68/ed1 , and , myelin using an antibody against myelin basic protein ( mbp ) .", "sections were deparaffinized and processed for antigen retrieval by boiling in sodium citrate ( ph  =  6.0 , 90  c ) for 10  min then washed in pbs .", "slides were subsequently blocked with 1% h2o2 in methanol for 5  min followed by 10% goat serum in 0.1  m pbs and then a pbs wash .", "slides were then incubated for 1  h at room temperature with one of 4 different primary antibodies : smi312 antibody ( 1:1000 in bsa , covance , berkeley , ca , usa ) , polyclonal rabbit anti - gfap ( 1:500 in bsa , diagnostics canada inc .", ", on , canada ) , mouse anti - rat cd68/ed1 ( 1:500 in bsa , abd serotec , raleigh , nc , usa ) or rabbit anti - mbp ( 1:500 in bsa , chemicon international , ca , usa ) . following a pbs wash", ", slides were incubated overnight at 4  c with the appropriate secondary antibody , either biotinylated goat anti - rabbit igg or biotinylated goat anti - mouse igg ( 1:400 in bsa , jackson immunoresearch lab inc .", "this was followed by incubation for half an hour with horseradish peroxidase - streptavidin ( 1:400 in pbs , diagnostics canada inc . , on , canada ) . finally , positive cells were visualized following an application of 3,3-diaminobenzidine ( sigma aldrich , mo , usa ) dissolved in 0.3% h2o2 in distilled water for approximately 3  min .", "the histological sections were assessed semi - quantitatively for changes in staining within the regions of interest either by measuring optical density or by quantifying numbers of positively stained cells per field of view . for optical density measures , sections stained with either smi312 , gfap or mbp", "were first captured digitally using a scanner ( nikon scan , version 4 , nikon inc . ) .", "these images were converted to gray images and mean gray levels ( imagej , nih ) were measured within the same regions of interest as those drawn for the mr brain slices .", "regions with less intense staining had higher mean gray values . the ipsilateral versus contralateral differences in gray levels were converted to a percentage of the contralateral value .", "the cd68/ed1 and fluorojade stained sections were analyzed using digitized microscopy ( microbrightfield , olympus , 40 magnification ) .", "the numbers of positively stained cells per field of view were estimated within regions corresponding to those selected from mr images .", "three different fields of view were counted for positive staining cells by an investigator ( m.s . )", "all group data is reported as mean    sd and statistical comparisons were considered significant at p  <  0.05 . measured values within different brain regions", "a two way repeated analysis was used to determine significant region and time interactions and these were followed by a holm ", "statistical tests were performed using sigmaplot ( systat software inc , san jose , ca ) . to convey clearly the differences for the cerebral peduncle , we noted in the graphs only the significant differences for this region .", "the correlation between histological changes and dti parameters was assessed using a pearson product correlation analysis within each time point or all time points with a bonferroni adjustment for multiple comparisons ."], ["despite intensive investigation of dti 's ability to detect brain injury , the interpretation of alterations in dti measures is usually speculative because of a lack of direct documentation of the corresponding histological changes . in the present study", "we report detailed differences in the progression and magnitude of dti and histological changes that provide a reference for identifying the potential contribution of various cellular responses to fa and axial , radial and mean diffusivity .", "the results are first to characterize the correspondence of the progression of cellular and dti changes within the cerebral peduncle of the descending corticospinal tract following hypoxic  ischemic damage to the cortex .", "novel is the demonstration of the differing progression of dti alterations in a directly damaged ischemic region such as parietal cortex versus that in associated subcortical tracts .", "dti changes were less severe or delayed in the cerebral peduncle of the corticospinal tract and this in general corresponded to less severe and delayed cellular responses consistent with early wallerian degeneration .", "as discussed in more detail , the tissue alterations following ischemia and axonal degeneration had a complex relationship with changes in fa , parallel diffusivity and radial diffusivity .", "regarding our assessment of progressive changes in the dti parameters , it is important to realize that superimposed on the effects of hi injury are well recognized developmental changes in the dti parameters that occur during development and aging ( dubois et al . , 2013 ) .", "indeed , the contralateral hemisphere was similar to those in sham animals but developmental changes may have been differentially affected in the left and right hemispheres by the ischemic insult .", "right differences needs to include an awareness that these can be accounted for by an injury modulation of cellular maturation processes in white matter , which we assessed in our histological sections .", "the region of cerebral peduncle measured was approx 0.30.4  mm consisting of approx 10 pixels of sizes 0.024  mm in the neonatal scans and 0.038  mm in 5  wk old animals .", "partial volume effects could be greater in neonatal brain than older brain . however , because we compared left and right differences in values , we expect that there were similar ipsilateral and contralateral partial volume effects in the cerebral peduncle at each time point .", "right differences but this did not appear to be an issue considering that many of our measures had the maximal changes in our neonates ( e.g. 1  d post insult ) . there have been numerous studies regarding the evolution of adc or mean diffusivity following transient cerebral ischemia in regions of stroke .", "adc alterations reported previously are comparable to those observed presently ( e.g. lama et al . , 2011 ; liu et al . , 2007 ) .", "notably , there were striking similarities between the adc and parallel diffusivity changes suggesting that both reflect similar cellular responses to injury . very early", "adc and parallel diffusivity changes in ischemic cortex are likely due to a greater restriction of water diffusion as a result of cytotoxic edema , reduced extracellular space and increased tortuosity which in the present study was also accompanied by decreased neurofilament staining and increased neuronal cell death . as vasogenic edema and damage to cellular membranes proceeded , which histologically corresponded to a continuing cell death and microglial / macrophage activation and astrogliosis , the reductions in adc and parallel diffusivity normalized and this process occurred over several days .", "by 14  w , phagocytosis produced cysts in the infarct region and such cystic regions had an extremely large adc in all directions whereas peri - infarct regions generally had normalized adc and normalized parallel diffusivity despite the presence of inflammatory cells .", "in contrast , in the cerebral peduncle with indirect ischemic injury , by the first 3  h there were modest but significant adc and parallel diffusivity reductions .", "the hyperacute changes in adc and parallel diffusivity correspond to our previous observation of a reduction in smi31 , demonstrating a decrease in phosphorylated heavy neurofilament subunits already hours after insult .", "subsequently , the adc and parallel diffusivity reductions increased in magnitude and were maximal in the first few days .", "this corresponded well with the diminished staining observed for the pan - axonal ( heavy , medium and light ) neurofilament marker smi312 used presently .", "loss of phosphorylation of neurofilaments promotes their disassembly and this corresponded to a decreased radial diffusivity as well as a decreased parallel and mean diffusivity of water . at chronic time points post - insult ( 14  w ) ,", "a similar greater sensitivity for parallel diffusivity than adc in detecting wallerian degenerative changes in the corticospinal tract has been reported after an excision lesion of the feline cortex ( qin et al . , 2012 ) .", "microglial / macrophage cell activation likely did not contribute greatly to the parallel diffusivity alterations observed , considering the rather modest positive ed1/cd68 staining observed in the cerebral peduncle .", "the occurrence of astrogliosis coincided with normalization of adc potentially contributing to some extent to these changes .", "however , as discussed below , there were also ipsilateral increases in radial diffusivity that likely contributed to the adc normalization .", "reductions in radial diffusivity have been proposed to reflect well increases in myelin whereas elevations in radial diffusivity have been found to be associated with a loss of myelin ( song et al . , 2002 , 2004 ;", "the current study provides some support for this if one examines only chronic recovery times  i.e. times when the axons in the contralateral cerebral peduncle have become well myelinated . at 1  w and beyond , myelin within the cerebral peduncle", "ipsilaterally was reduced reflecting less ontogenic myelination of fewer axons following their degeneration and atrophy .", "this coincided with relative increases in radial diffusivity ipsilaterally and is consistent with increases in radial diffusivity reported in the corticospinal or pyramidal tracts at relatively chronic times ( e.g. 1  w to 3  y ) following an ischemic insult in children or adults ( roze et al .", ", 2012 ; yu et al . , 2009 ; zanier et al . , 2013 ) . at acute times post - insult we observed ipsilateral reductions in radial diffusivity within the cerebral peduncle indicating that in the absence of effects on myelination , early axonal injury such as that detected by decreases in phosphorylated neurofilaments were also associated with reduced radial diffusivity", "it is noteworthy that the neurophysiological correlates of alterations in parallel and radial diffusivity are potentially more complex in humans than rodents .", "a diffusion tensor analysis has an implicit assumption that the white matter fibers are cylindrical and when fibers have complicated microstructures or crossing fibers as occurs in human developing brain ( dubois et al . , 2013 ;", "2013 ) then in particular the directions of orientation for tractography require careful interpretation . despite crossing fibers potentially complicating the interpretation of parallel diffusivity changes ,", "transverse or radial diffusivity appears to consistently decrease with all maturational processes within white matter , including myelination ( dubois et al . , 2013 ) .", "furthermore , emerging imaging methods exploring diffusion parameters beyond the tensor model may provide more in depth assessments to facilitate translation from animals to humans ( dubois et al .", "the fa measures provided a sensitive measure of direct ischemic injury . in the cortex at hyperacute times", ", there was an increase in fa , as has been observed previously in rats and non - human primates , however , usually soon after permanent rather than transient occlusion ( kim et al .", ", 2013 ; liu et al . , 2007 ; yang et al . , 1999 ) .", "this suggests that the very early cortical ischemic changes were relatively severe and similar to those with permanent ischemia .", "cellular changes likely involve previously established hyperacute cytotoxic cell swelling and reductions in extracellular space leading to cell death considering our histological evidence for hyperacute neuronal cell death in the cortex . by the first day post - insult ,", "the fa increase became a decrease that persisted at subacute and chronic time points , confirming previous reports of a reduced fa commonly observed in damaged brain post - stroke ( e.g. kim et al .", ", 2013 ; liu et al . , 2007 ; yang et al . , 1999 ) .", "this decrease in fa , reflecting diminished preferential diffusivity , is likely associated with a loss of membrane integrity and intracellular structure due to necrosis as supported histologically by continuing cell death and loss of neurofilament staining .", "the increase in astrogliosis between 3 and 1  d appears rather isotropic considering that there is a decline in fa between these times .", "the fa also provided a rather robust measure of degenerative changes in the cerebral peduncle .", "in contrast to ischemic cortex , fa in cerebral peduncle was decreased at all time points .", "indeed , fa appeared to be the most sensitive dti parameter for detecting axonal degenerative changes in the cerebral peduncle independent of time post - insult and this corresponded well to the persistent decreases in axonal neurofilament staining .", "similar reductions in fa have been reported at various select times in the corticospinal tract following neonatal stroke ( ludeman et al . , 2008 ; puig et al . , 2013 ; tusor et al . , 2012 ) .", "it is currently clear that at chronic times , fa is superior to adc or other standard imaging methods such as t2 imaging because lesions detected with these other sequences tended to pseudo - normalize at times post - insult ( lama et al . , 2011 ; tuor et al . ,", "the results demonstrate that in general , diffusivity changes and histological responses within directly damaged ischemic brain precede and are often larger in magnitude than corresponding alterations in a region of secondary axonal degeneration , such as the cerebral peduncle . with the progression of damage over one or more weeks , dti components such as fa and radial diffusivity are better than mean adc for detecting ischemia associated axonal injury indicative of wallerian degeneration .", "also , differences in radial diffusivity in brain injured perinatally are likely related to a lack of ontogenic myelination on the injured side .", "thus , the specific progression of dti alterations following an ischemic insult not only depends on the maturity of the subject but also appears dependent on other factors such as the type of insult ( e.g. spinal cord injury or brain resection ) ( brennan et al . , 2013 ; liu et al . , 2013 ; qin et al . , 2012 ) .", "the inflammatory response investigated ( microglial or astroglial activation ) appears to have modest contributions to the evolution of axonal changes in diffusion tensor parameters .", "thus , this systematic study is informative regarding the optimal dti mr imaging parameters to probe axonal injury following hypoxic / ischemic brain damage in developing brain in addition to aiding our interpretation of dti parameters for detecting various stages of wallerian degeneration ."]]}
{"article_id": "PMC2923417", "article_text": ["the effect of family on workforce is an important though often overlooked issue for work organizations , with implications for the morale , stability and productivity of the workforce .", "presumably men and women do not shed their family roles , relationships and experiences the moment they don their work clothes .", "indeed , the logic underlying many corporations ' decisions to offer employer - based family support , such as child care and flexible working schedules , may be that such benefits will enhance employees ' abilities to handle family matters and in so doing will enhance their work performance , commitment and satisfaction .", "this line of reasoning views the relationship between work and family as a reciprocal process in which work and family influence each other in a circular or feedback fashion .", "indian families are undergoing change , which in turn influences the environment in the basic unit .", "the family environment has been bombarded with new expectations , the media , high cost of living and a striving for better quality of life .", "although not much literature is available on the aspect of how family stress has influenced the productivity of the indian workforce , the author is of the opinion that absenteeism , alcoholism , gambling , heavy debts among employees are a reflection of bruised familial systems .", "the dual - earning couple is a new prototype that reflects the increasing educational and career aspirations of women .", "a significant proportion of these women in the workforce comprises of wives and mothers whose employment status demands a radical change in their pattern , activities , commitments and responsibilities , requiring a reassessment of the family environment . in india", "the proportion of women in the workforce in 1981 was 19.67% and it rose to 22.73% in 1991 , further rising to 25.68% in 2001 . on the flipside , psychosocial studies have shown that dual - earning couples in india have a poorer quality of marital life compared to single - earning couples,[46 ] in their study found that work - family conflict was expressed in 63% of thematic appreciation test ( tat ) stories written by women , whereas men 's stories did not reflect this conflict .", "sources of stress in the lives of working women emerged from a lack of time to attend to multiple roles , presence of young children ( 6 - 12 years ) in the family and additional responsibility at work in the form of promotions .", "the most common outcome of stress for the working woman was found to be poor mental and physical health resulting in depression , anxiety , asthma and colitis.[911 ] rajadhyaksha[79 ] studied dual - career as opposed to dual - earning couples in two metropolitan cities in india and found that in a dual - career family where husbands and wives were presumably matched in terms of their career involvement , work - family role conflict and organizational role stress were not significantly different among husbands and wives .", "for instance , women experienced more conflict between their job and home roles , while men experienced more conflict between their job and spousal roles . in the present day", ", the young indian adult is exposed to an entirely new pattern of living and a new set of mores , values and standards that are being widely accepted but which stand in contrast to those which were promoted by their parents and grandparents .", "the ambiguous values children and adolescents observe today in india , coupled with the increasing gap between aspirations and possible achievement , have led to a greater sense of ' alienation . '", "parents too appear ill - prepared to cope with rapid social change , having grown up in hierarchically structured and interdependent social groups that included the extended family and kinship network , as well as caste groups that provided stability and solidarity .", "parents who seem modern in their child - rearing practices get anxious when their offspring breach established social codes .", "inter - generation conflicts related to marriage , career choice or separate living arrangements result in the tendency to fall back on tradition .", "subtle changes in family patterns , especially with regard to the use of authority within the family , as well as an increased focus on individual autonomy,[1617 ] are also likely to influence members ' expectations with regard to marriage and their choice of a spouse .", "educated middle class families are now more hesitant to make decisions for their offspring with regard to marriage , education and employment .", "changes have also been noticed with regard to a greater focus on the husband - wife relationship rather than the parent - child relationship . with an increased onus of responsibility falling on the individual rather than on the entire family ,", "gore calls ' choice anxiety' increased autonomy and increased choice that have led to increased anxiety .", "ambiguity with regard to issues like respect for elders , role of the eldest son , love marriages , working wives , etc . , is an inevitable fallout of the changes within the indian family .", "relationship - focused education for young adults in the workforce has not been on top of the agenda for india 's policy makers .", "india 's policies on family and child welfare have been focused more in terms of health care , nutrition , reproductive health , sexuality and venereal disease .", "however , one can not forget that young adults of marriageable age from ' first - generation urban - educated families ' are facing relationship - based problems due to changing dynamics within the indian family .", "these include shifts in loyalty from filial obligations to lean more towards conjugal ties and issues of power and gender role allocations .", "marriage is traditionally seen as means for procreation , but there are slow changes in perception among adolescents towards viewing it as a means of companionship . with couples migrating to cities for better job prospects , there is a decreased availability of the informal support provided by the extended family in the past , resulting in young couples having to develop greater problem - solving and coping skills on their own .", "this in turn weighs heavily on an employee 's ability to focus completely on tasks at work .", "it is essential to bridge the gap between what their families of origin considered as an ideal or happy marriage and what they now desire from a spouse and family life .", "the author conducted individual interviews with family - intervention experts in bangalore city in order to better understand the changing family patterns that they were observing . among the professionals , there were two clinical psychologists , two psychiatric social workers , two psychiatrists and one divorce lawyer .", "the key person interview ( kpi ) cue statements to initiate discussions with the experts were as follows [ table 1 ] : description of key person interview participants \n changes that you are seeing in the nature of problems that families are coming to you with in comparison to the nature of problems brought up a decade agoname some culture - specific areas that need to be kept in mind while preparing the family life education programsome areas that you feel need to be essentially covered by a family life education program for single young adults . \n ", "changes that you are seeing in the nature of problems that families are coming to you with in comparison to the nature of problems brought up a decade ago name some culture - specific areas that need to be kept in mind while preparing the family life education program some areas that you feel need to be essentially covered by a family life education program for single young adults .", "it is interesting to note that among the changes noticed by family experts in relation to the aspect of family issues , is the ability to recognize issues early on in the marriage and to seek help for the same .", "another important change noticed by the experts was that a larger number of young couples who were either engaged or in a relationship were now seeking professional help in order to help them make the right choice of a partner or to resolve issues that they were afraid would snowball into a larger issue post - marriage .", "an interesting perspective put forward by the legal expert in family law was that he was seeing an increasing number of divorces that were occurring as early as the third or sixth month of marriage , with both partners willing to go in for a divorce by mutual consent .", "this , he said , was in stark contrast to what he encountered some 10 years ago , when most couples would go through a long - drawn - out battle with much mud - slinging or would want to keep trying to make things work .", "the recognition of sexual dissatisfaction by , and the unwillingness to stay in a marriage on the part of , the female spouse was also another prominent change that experts were noticing .", "a long - distance relationship , with spouses staying in different cities and meeting on the weekends , was also another trend that was being noticed .", "themes from key person interview with family experts leadership and decision - making issues in the joint family largely arose due to the inability to face up to , or communicate with , authority figures within the family , thus leading to frustration and passive aggression .", "talking about one 's sexual needs was another cultural peculiarity that often created rifts within couples or forced couples to try and work things out .", "it is also interesting to note that the family experts were able to clearly point out the need for any family life education program to be sensitive to indian culture in terms of how it viewed the role of the joint family .", "experts also pointed out the program had to be careful about how it portrayed communication within the family and had to keep in mind the factor of ' respect ' that indian families often prescribe to .", "the third column clearly gives the areas that these family experts felt a family life education program should cover .", "the experts felt that adaptability was an important skill that the new indian spouse was quickly losing .", "gender sensitivity in terms of acceptance of changing work and family roles of the female spouse by the male spouse was another important area that the experts talked about .", "discussions with these family - intervention experts brought out the following areas as possible aspects to be included in family life education program for the younger - generation workforce : \n gender sensitivityparenting skillsconflict resolution skillsadaptability to changebalancing work and the familycommunication skillssocialization and social supportmoney managementchoosing a life partnersexuality and intimacydealing with violence and addiction \n  conflict resolution skills adaptability to change balancing work and the family socialization and social support choosing a life partner sexuality and intimacy dealing with violence and addiction the experts definitely seemed to sense the need for some form of education or training for the young indian adult who is either contemplating family life or is a new entrant into marriage or parenthood .", "family issues that eat into employees ' ability to perform or affect his cognitive and emotional state of being need to be addressed . a promotive and preventive strategy rather than a curative one would have to be adopted for this purpose .", "relationship - focused family life education programs for the target group of employees at the workplace may actually serve to fulfill this purpose , whereby employees , through group interaction and experiential learning , can be taught skills to deftly handle family - life conflicts either by themselves or through the support of mental health professionals .", "thus a mental health professional need not be there merely to diffuse a difficult situation arising out of deviance of an employee .", "he or she may act as a preventive agent of avoiding such deviance from arising , by tackling the problem at the root by pre - equipping the family with the necessary psychoeducation and skills ."], "abstract_text": ["<S> the article presents the premises for the need to develop a relationship - focused family life education program for young adult employees . </S>", "<S> the article explores the changing trends in the indian family unit and their impact on the workforce . </S>", "<S> the author also presents the findings from interviews with family - intervention experts and their recommendations for the contents of such a program . </S>"], "labels": null, "section_names": ["INTRODUCTION", "Changing family trends and needs observed by family-intervention experts", "RESULTS", "DISCUSSION"], "sections": [["the effect of family on workforce is an important though often overlooked issue for work organizations , with implications for the morale , stability and productivity of the workforce .", "presumably men and women do not shed their family roles , relationships and experiences the moment they don their work clothes .", "indeed , the logic underlying many corporations ' decisions to offer employer - based family support , such as child care and flexible working schedules , may be that such benefits will enhance employees ' abilities to handle family matters and in so doing will enhance their work performance , commitment and satisfaction .", "this line of reasoning views the relationship between work and family as a reciprocal process in which work and family influence each other in a circular or feedback fashion .", "indian families are undergoing change , which in turn influences the environment in the basic unit .", "the family environment has been bombarded with new expectations , the media , high cost of living and a striving for better quality of life .", "although not much literature is available on the aspect of how family stress has influenced the productivity of the indian workforce , the author is of the opinion that absenteeism , alcoholism , gambling , heavy debts among employees are a reflection of bruised familial systems .", "the dual - earning couple is a new prototype that reflects the increasing educational and career aspirations of women .", "a significant proportion of these women in the workforce comprises of wives and mothers whose employment status demands a radical change in their pattern , activities , commitments and responsibilities , requiring a reassessment of the family environment . in india", "the proportion of women in the workforce in 1981 was 19.67% and it rose to 22.73% in 1991 , further rising to 25.68% in 2001 . on the flipside , psychosocial studies have shown that dual - earning couples in india have a poorer quality of marital life compared to single - earning couples,[46 ] in their study found that work - family conflict was expressed in 63% of thematic appreciation test ( tat ) stories written by women , whereas men 's stories did not reflect this conflict .", "sources of stress in the lives of working women emerged from a lack of time to attend to multiple roles , presence of young children ( 6 - 12 years ) in the family and additional responsibility at work in the form of promotions .", "the most common outcome of stress for the working woman was found to be poor mental and physical health resulting in depression , anxiety , asthma and colitis.[911 ] rajadhyaksha[79 ] studied dual - career as opposed to dual - earning couples in two metropolitan cities in india and found that in a dual - career family where husbands and wives were presumably matched in terms of their career involvement , work - family role conflict and organizational role stress were not significantly different among husbands and wives .", "for instance , women experienced more conflict between their job and home roles , while men experienced more conflict between their job and spousal roles . in the present day", ", the young indian adult is exposed to an entirely new pattern of living and a new set of mores , values and standards that are being widely accepted but which stand in contrast to those which were promoted by their parents and grandparents .", "the ambiguous values children and adolescents observe today in india , coupled with the increasing gap between aspirations and possible achievement , have led to a greater sense of ' alienation . '", "parents too appear ill - prepared to cope with rapid social change , having grown up in hierarchically structured and interdependent social groups that included the extended family and kinship network , as well as caste groups that provided stability and solidarity .", "parents who seem modern in their child - rearing practices get anxious when their offspring breach established social codes .", "inter - generation conflicts related to marriage , career choice or separate living arrangements result in the tendency to fall back on tradition .", "subtle changes in family patterns , especially with regard to the use of authority within the family , as well as an increased focus on individual autonomy,[1617 ] are also likely to influence members ' expectations with regard to marriage and their choice of a spouse .", "educated middle class families are now more hesitant to make decisions for their offspring with regard to marriage , education and employment .", "changes have also been noticed with regard to a greater focus on the husband - wife relationship rather than the parent - child relationship . with an increased onus of responsibility falling on the individual rather than on the entire family ,", "gore calls ' choice anxiety' increased autonomy and increased choice that have led to increased anxiety .", "ambiguity with regard to issues like respect for elders , role of the eldest son , love marriages , working wives , etc . , is an inevitable fallout of the changes within the indian family .", "relationship - focused education for young adults in the workforce has not been on top of the agenda for india 's policy makers .", "india 's policies on family and child welfare have been focused more in terms of health care , nutrition , reproductive health , sexuality and venereal disease .", "however , one can not forget that young adults of marriageable age from ' first - generation urban - educated families ' are facing relationship - based problems due to changing dynamics within the indian family .", "these include shifts in loyalty from filial obligations to lean more towards conjugal ties and issues of power and gender role allocations .", "marriage is traditionally seen as means for procreation , but there are slow changes in perception among adolescents towards viewing it as a means of companionship . with couples migrating to cities for better job prospects , there is a decreased availability of the informal support provided by the extended family in the past , resulting in young couples having to develop greater problem - solving and coping skills on their own .", "this in turn weighs heavily on an employee 's ability to focus completely on tasks at work .", "it is essential to bridge the gap between what their families of origin considered as an ideal or happy marriage and what they now desire from a spouse and family life ."], ["the author conducted individual interviews with family - intervention experts in bangalore city in order to better understand the changing family patterns that they were observing . among the professionals , there were two clinical psychologists , two psychiatric social workers , two psychiatrists and one divorce lawyer .", "the key person interview ( kpi ) cue statements to initiate discussions with the experts were as follows [ table 1 ] : description of key person interview participants \n changes that you are seeing in the nature of problems that families are coming to you with in comparison to the nature of problems brought up a decade agoname some culture - specific areas that need to be kept in mind while preparing the family life education programsome areas that you feel need to be essentially covered by a family life education program for single young adults . \n ", "changes that you are seeing in the nature of problems that families are coming to you with in comparison to the nature of problems brought up a decade ago name some culture - specific areas that need to be kept in mind while preparing the family life education program some areas that you feel need to be essentially covered by a family life education program for single young adults ."], ["it is interesting to note that among the changes noticed by family experts in relation to the aspect of family issues , is the ability to recognize issues early on in the marriage and to seek help for the same .", "another important change noticed by the experts was that a larger number of young couples who were either engaged or in a relationship were now seeking professional help in order to help them make the right choice of a partner or to resolve issues that they were afraid would snowball into a larger issue post - marriage .", "an interesting perspective put forward by the legal expert in family law was that he was seeing an increasing number of divorces that were occurring as early as the third or sixth month of marriage , with both partners willing to go in for a divorce by mutual consent .", "this , he said , was in stark contrast to what he encountered some 10 years ago , when most couples would go through a long - drawn - out battle with much mud - slinging or would want to keep trying to make things work .", "the recognition of sexual dissatisfaction by , and the unwillingness to stay in a marriage on the part of , the female spouse was also another prominent change that experts were noticing .", "a long - distance relationship , with spouses staying in different cities and meeting on the weekends , was also another trend that was being noticed .", "themes from key person interview with family experts leadership and decision - making issues in the joint family largely arose due to the inability to face up to , or communicate with , authority figures within the family , thus leading to frustration and passive aggression .", "talking about one 's sexual needs was another cultural peculiarity that often created rifts within couples or forced couples to try and work things out .", "it is also interesting to note that the family experts were able to clearly point out the need for any family life education program to be sensitive to indian culture in terms of how it viewed the role of the joint family .", "experts also pointed out the program had to be careful about how it portrayed communication within the family and had to keep in mind the factor of ' respect ' that indian families often prescribe to .", "the third column clearly gives the areas that these family experts felt a family life education program should cover .", "the experts felt that adaptability was an important skill that the new indian spouse was quickly losing .", "gender sensitivity in terms of acceptance of changing work and family roles of the female spouse by the male spouse was another important area that the experts talked about .", "discussions with these family - intervention experts brought out the following areas as possible aspects to be included in family life education program for the younger - generation workforce : \n gender sensitivityparenting skillsconflict resolution skillsadaptability to changebalancing work and the familycommunication skillssocialization and social supportmoney managementchoosing a life partnersexuality and intimacydealing with violence and addiction \n  conflict resolution skills adaptability to change balancing work and the family socialization and social support choosing a life partner sexuality and intimacy dealing with violence and addiction the experts definitely seemed to sense the need for some form of education or training for the young indian adult who is either contemplating family life or is a new entrant into marriage or parenthood ."], ["family issues that eat into employees ' ability to perform or affect his cognitive and emotional state of being need to be addressed . a promotive and preventive strategy rather than a curative one would have to be adopted for this purpose .", "relationship - focused family life education programs for the target group of employees at the workplace may actually serve to fulfill this purpose , whereby employees , through group interaction and experiential learning , can be taught skills to deftly handle family - life conflicts either by themselves or through the support of mental health professionals .", "thus a mental health professional need not be there merely to diffuse a difficult situation arising out of deviance of an employee .", "he or she may act as a preventive agent of avoiding such deviance from arising , by tackling the problem at the root by pre - equipping the family with the necessary psychoeducation and skills ."]]}
{"article_id": "PMC2920003", "article_text": ["macroscopically , renal cell carcinomas ( rccs ) generally present as either solid or cystic mass . among cystic rccs ,", "multilocular cystic rcc represents a rare entity that was recognized in 1982 , with a reported incidence of 1 - 4% of all rccs .", "renal leiomyomas are benign tumors of the kidney originating from smooth muscle cells of the renal capsule , pelvis , calices , or blood vessels .", "although small renal leiomyomas can be found in about 5% of autopsy specimens , clinical incidence of these lesions is much lower . in this report , we discuss the first case of simultaneous occurrence of a multilocular cystic rcc and a leiomyoma in the same kidney .", "a 38-year - old man was referred for treatment of a right renal cystic mass found incidentally .", "physical examination on admission revealed no palpable mass , and laboratory data were negative . computed tomography of the abdomen showed a about 5.4  4.6 cm poorly enhancing cystic mass with enhancing septa in the polar area of the right kidney ( fig .", "since multilocular cystic rcc could not be excluded , the patient underwent right laparoscopic radical nephrectomy .", "macroscopically , in the mid pole , a well - circumscribed mass showing a multicystic feature with thin fibrous septa and without expansile tumor nodules was noted .", "rccs show cystic changes on imaging studies in 4 - 15% of cases [ 4 , 5 ] .", "since it is often difficult to differentiate cystic rccs from benign cystic lesions , a definite diagnosis can in most cases only be established by histopathologic examination .", "these include : ( 1 ) intrinsic multiloculated growth ; ( 2 ) intrinsic unilocular growth ; ( 3 ) cystic necrosis , and ( 4 ) origin from the epithelial lining in a pre - existing cyst .", "because multilocular cystic rcc are more likely to be discovered incidentally , this feature may contribute to their excellent prognosis compared to that of conventional rccs .", "since the majority of rccs originate from the proximal tubules , the characteristics and/or environment of the proximal tubules are considered to play an important role in the pathogenesis of rcc .", "recently , imura et al . carried out a detailed immunohistochemical analysis of multilocular cystic rcc cases .", "they found that in a high proportion of cases , multilocular cystic rcc reacted strongly with the distal nephron markers , but none reacted preferentially with proximal nephron markers .", "these results illustrate that multilocular cystic rcc originates from the distal nephron , although the precise pathogenesis of cystic formation in multilocular cystic rcc has not been elucidated clearly .", "renal leiomyomas are benign tumors arising from the mesenchymal ( or connective ) tissue of the kidney .", "tumors may be subcapsular ( 53% ) , capsular ( 37% ) , or located in the renal pelvis ( 10% ) .", "a variety of structural patterns have been described at imaging : solid , cystic , and both cystic and solid [ 9 , 10 ] . in 1990 , steiner et al .", "the first group comprises small cortical or subcortical neoplasms that are usually asymptomatic , less than 2 cm in size , and are often detected incidentally during autopsy or surgery .", "the second group consists of larger neoplasms that arise from the renal capsule or blood vessels and may be symptomatic . unfortunately , it is almost impossible to clinically distinguish renal leiomyomas from their malignant counterparts . because of their small size ( usually less than 2 cm ) , these smooth muscle - containing tumors are commonly diagnosed during autopsy , and clinical manifestations become apparent only when they grow larger , hemorrhage , or undergo cystic or sarcomatous degeneration . since the finding of primary synchronous renal neoplasms is very uncommon , especially when they have a different histogenesis , up to now , only a few primary , synchronous rccs with different histotypes have been reported . the coexistence of multilocular cystic rcc and leiomyoma has not been documented .", "this present case is the first report of synchronous renal neoplasms with different histogenesis ( multilocular cystic rcc and leiomyoma ) in a patient without any symptoms .", "the significance of the relationship between multilocular cystic rccs and leiomyomas is not well understood and needs further exploration ."], "abstract_text": ["<S> we present an unusual case of concurrent occurrence of a multilocular cystic renal cell carcinoma and a leiomyoma in the same kidney of a patient with no evident clinical symptoms . </S>", "<S> a 38-year - old man was found incidentally to have a cystic right renal mass on computed tomography . </S>", "<S> laparoscopic radical nephrectomy was performed under a preoperative diagnosis of cystic renal cell carcinoma . </S>", "<S> histology revealed a multilocular cystic renal cell carcinoma and a leiomyoma . </S>", "<S> this is the first report of this kind of presentation . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["macroscopically , renal cell carcinomas ( rccs ) generally present as either solid or cystic mass . among cystic rccs ,", "multilocular cystic rcc represents a rare entity that was recognized in 1982 , with a reported incidence of 1 - 4% of all rccs .", "renal leiomyomas are benign tumors of the kidney originating from smooth muscle cells of the renal capsule , pelvis , calices , or blood vessels .", "although small renal leiomyomas can be found in about 5% of autopsy specimens , clinical incidence of these lesions is much lower . in this report , we discuss the first case of simultaneous occurrence of a multilocular cystic rcc and a leiomyoma in the same kidney ."], ["a 38-year - old man was referred for treatment of a right renal cystic mass found incidentally .", "physical examination on admission revealed no palpable mass , and laboratory data were negative . computed tomography of the abdomen showed a about 5.4  4.6 cm poorly enhancing cystic mass with enhancing septa in the polar area of the right kidney ( fig .", "since multilocular cystic rcc could not be excluded , the patient underwent right laparoscopic radical nephrectomy .", "macroscopically , in the mid pole , a well - circumscribed mass showing a multicystic feature with thin fibrous septa and without expansile tumor nodules was noted ."], ["rccs show cystic changes on imaging studies in 4 - 15% of cases [ 4 , 5 ] .", "since it is often difficult to differentiate cystic rccs from benign cystic lesions , a definite diagnosis can in most cases only be established by histopathologic examination .", "these include : ( 1 ) intrinsic multiloculated growth ; ( 2 ) intrinsic unilocular growth ; ( 3 ) cystic necrosis , and ( 4 ) origin from the epithelial lining in a pre - existing cyst .", "because multilocular cystic rcc are more likely to be discovered incidentally , this feature may contribute to their excellent prognosis compared to that of conventional rccs .", "since the majority of rccs originate from the proximal tubules , the characteristics and/or environment of the proximal tubules are considered to play an important role in the pathogenesis of rcc .", "recently , imura et al . carried out a detailed immunohistochemical analysis of multilocular cystic rcc cases .", "they found that in a high proportion of cases , multilocular cystic rcc reacted strongly with the distal nephron markers , but none reacted preferentially with proximal nephron markers .", "these results illustrate that multilocular cystic rcc originates from the distal nephron , although the precise pathogenesis of cystic formation in multilocular cystic rcc has not been elucidated clearly .", "renal leiomyomas are benign tumors arising from the mesenchymal ( or connective ) tissue of the kidney .", "tumors may be subcapsular ( 53% ) , capsular ( 37% ) , or located in the renal pelvis ( 10% ) .", "a variety of structural patterns have been described at imaging : solid , cystic , and both cystic and solid [ 9 , 10 ] . in 1990 , steiner et al .", "the first group comprises small cortical or subcortical neoplasms that are usually asymptomatic , less than 2 cm in size , and are often detected incidentally during autopsy or surgery .", "the second group consists of larger neoplasms that arise from the renal capsule or blood vessels and may be symptomatic . unfortunately , it is almost impossible to clinically distinguish renal leiomyomas from their malignant counterparts . because of their small size ( usually less than 2 cm ) , these smooth muscle - containing tumors are commonly diagnosed during autopsy , and clinical manifestations become apparent only when they grow larger , hemorrhage , or undergo cystic or sarcomatous degeneration . since the finding of primary synchronous renal neoplasms is very uncommon , especially when they have a different histogenesis , up to now , only a few primary , synchronous rccs with different histotypes have been reported . the coexistence of multilocular cystic rcc and leiomyoma has not been documented .", "this present case is the first report of synchronous renal neoplasms with different histogenesis ( multilocular cystic rcc and leiomyoma ) in a patient without any symptoms .", "the significance of the relationship between multilocular cystic rccs and leiomyomas is not well understood and needs further exploration ."]]}
{"article_id": "PMC4838586", "article_text": ["the ganoderma strains listed in table 1 were obtained from the korean collection for type culture ( ktct , jeongeup , korea ) , the american type culture collection ( atcc , rockville , md , usa ) , the korean agricultural culture collection ( kacc , suwon , korea ) , the mushroom division of the rural development administration ( eumseong , korea ) , the centraalbureau voor schimmelcultures ( cbs , utrecht , netherlands ) , incheon university ( incheon , korea ) , and konkuk university ( seoul , korea ) .", "ganoderma species were cultured on potato dextrose broth ( difco , detroit , mi , usa ) and incubated at 30 for 2 wk .", "cultured mycelia , filtered through 2 layers of miracloth ( calbiochem , la jolla , ca , usa ) , were ground in liquid nitrogen , and genomic dna was extracted using the cetyltrimethylammonium bromide method .", "pcrs were performed using a premixed polymerase kit ( taq premix ; tnt research , anyang , korea ) in a 20 l reaction mixture containing 1 l of dna .", "amplification of the its region was carried out using a thermal cycler ( takara , tokyo , japan ) at the following conditions : 5 min at 94 for initial denaturation , followed by 30 cycles of 30 sec at 94 for denaturation , 30 sec at 56 for primer annealing , and 1 min at 72 for extension , and 10 min at 72 for a final extension .", "pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer .", "pcr products were ligated into the pgem - t easy vector ( promega , madison , wi , usa ) according to the manufacturer 's instructions .", "ligation products were transformed into the escherichia coli dh5 competent cell ( rbc , taiwan ) by heat shock .", "plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit ( geneall , seoul , korea ) .", "recombinant clones were identified , and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing ( using the sp6 and t7 promoters ; genotech , daejeon , korea ) .", "nucleotide sequences were deposited in the national center for biotechnology information genbank database ( table 1 ) .", "the sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) .", "the phylogenetic trees were constructed by using the mega5 program   and the neighbor - joining method .", "confidence levels for individual branches of the resulting tree were assessed using the bootstrap test   in which 1,000 replicate trees were generated from resampled data .", "the size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope ( axio observer a1 ; carl zeiss , jena , germany ) and a 5% koh solution as a mounting medium .", "the size of each spore was calculated , and the mean value was used in the description .", "the ganoderma strains listed in table 1 were obtained from the korean collection for type culture ( ktct , jeongeup , korea ) , the american type culture collection ( atcc , rockville , md , usa ) , the korean agricultural culture collection ( kacc , suwon , korea ) , the mushroom division of the rural development administration ( eumseong , korea ) , the centraalbureau voor schimmelcultures ( cbs , utrecht , netherlands ) , incheon university ( incheon , korea ) , and konkuk university ( seoul , korea ) .", "ganoderma species were cultured on potato dextrose broth ( difco , detroit , mi , usa ) and incubated at 30 for 2 wk .", "cultured mycelia , filtered through 2 layers of miracloth ( calbiochem , la jolla , ca , usa ) , were ground in liquid nitrogen , and genomic dna was extracted using the cetyltrimethylammonium bromide method .", "pcrs were performed using a premixed polymerase kit ( taq premix ; tnt research , anyang , korea ) in a 20 l reaction mixture containing 1 l of dna .", "amplification of the its region was carried out using a thermal cycler ( takara , tokyo , japan ) at the following conditions : 5 min at 94 for initial denaturation , followed by 30 cycles of 30 sec at 94 for denaturation , 30 sec at 56 for primer annealing , and 1 min at 72 for extension , and 10 min at 72 for a final extension .", "pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer .", "pcr products were ligated into the pgem - t easy vector ( promega , madison , wi , usa ) according to the manufacturer 's instructions .", "ligation products were transformed into the escherichia coli dh5 competent cell ( rbc , taiwan ) by heat shock .", "plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit ( geneall , seoul , korea ) .", "recombinant clones were identified , and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing ( using the sp6 and t7 promoters ; genotech , daejeon , korea ) .", "nucleotide sequences were deposited in the national center for biotechnology information genbank database ( table 1 ) .", "the sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) .", "the phylogenetic trees were constructed by using the mega5 program   and the neighbor - joining method .", "confidence levels for individual branches of the resulting tree were assessed using the bootstrap test   in which 1,000 replicate trees were generated from resampled data .", "the size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope ( axio observer a1 ; carl zeiss , jena , germany ) and a 5% koh solution as a mounting medium .", "the size of each spore was calculated , and the mean value was used in the description .", "the phylogenetic analysis of ganoderma species in this study was generally consistent with the findings reported by the group from china .", "the 62 ganoderma strains were divided into 7 groups , a to g ( fig .", "in addition , ganoderma lucidum were largely divided into two groups ( groups a and g ) .", "group a included all the korean g. lucidum strains , as well as the g. lucidum strains from bangladesh and japan , and chinese g. sichuanense and g. lingzhi .", "g. meredithae ( usa and unknown sources ) in group b was closely related to g. lucidum in group a as evidenced by a high bootstrap value of 98% .", "g. multipileum ( china and taiwan ) was grouped within group c and g. tropicum ( china and taiwan ) within group d , with 96% and 99% bootstrap support , respectively .", "group e includes three strains of g. resinaceum from the czech republic and uk ( two strains ) , and was supported by very high bootstrap values of 99% .", "strains of g. weberianum ( australia , china , and philippines ) and two other chinese g. sichuanense strains ( cui 7691 and hmas 86597 ; holotype ) were grouped within group f with 99% bootstrap support . groups e and f were closely related with bootstrap values of 96% .", "it is worth noting that other g. lucidum strains from europe ( france , finland , italy , sweden , and uk ) and canada could be clustered into group g with g. tsugae strains from north america ( canada and usa ) , supported by a very high bootstrap value of 99% .", "the findings of the current study are consistent with those of park et al .   who reported that g. lucidum strains from europe and north america could be clustered into one group together with g. tsugae based on both analyses of the its rdna gene and partial -tubulin gene sequences .", "interestingly , korean g. lucidum strains could be clustered into a group together with g. sichuanense and g. lingzhi strains from china .", "reported that g. lucidum is incorrectly recorded in china , as well as in other countries , and suggested that the name ' g. lucidum ' as used for the chinese species should be corrected as g. sichuanense .", "however , they did not obtain sequences from type specimens of g. sichuanense ( the holotype ; hmas 42798 ) .", "reported that korean g. lucidum ( asi-7004 ) could be clustered into one group together with chinese g. lucidum and g. lingzhi based on the its2 sequences and rna secondary structures .", "furthermore , cao et al .   reported that g. sichuanense ( included the holotype ; hmas 42798 ) was distantly phylogenetically related to g. lingzhi ( included the holotype ; wu 1006 - 38 ) , but it was closely related to g. resinaceum . with regard to the morphological characteristics , it has been reported that chinese g. lucidum ( g. lingzhi ) has a yellow pore surface , european g. lucidum has a white pore surface , and korean g. lucidum has pale brownish thread - like tissues in the middle of the context .", "cao et al .   found that the morphological characteristics of ' g. lucidum ' from east asia were consistent with those of g. lingzhi .", "thus , they concluded that g. sichuanense was a distinct species since they display obvious morphological differences from g. lingzhi and suggested that the name ' g. lucidum ' should be corrected as g. lingzhi not g. sichuanense .", "it is commonly called \" yeongji \" in korea , \" lingzhi \" in china , and \" reishi \" or \" mannentake \" in japan .", "however , the ganoderma species from various countries including africa , oceania , america , asia ( china , korea , and japan ) , and europe   have been incorrectly reported as g. lucidum .", "in addition , moncalvo et al .   classified g. lucidum collections from different regions ( asia and europe ) into different species based on its and partial nuclear large subunit ribosomal dna sequences .", "this is similar to the results of our its nucleotide sequence analysis of g. lucidum .", "likewise , hong and jung   reported that the g. lucidum from korea and japan were monophyletic , and were distinguished from the g. lucidum from europe and north america based on sequence analysis of mitochondrial small - subunit ribosomal dna .", "as described above , the taxonomy of g. lucidum has been reported by many researchers .", "nevertheless , the name g. lucidum has been misapplied to various species around the world .", "the results of this study also reveal that korean g. lucidum was clustered into one group together with chinese g. sichuanense and chinese g. lingzhi ( both formerly g. lucidum ) , and it was clearly separated from g. lucidum from europe and north america .", "in addition , g. sichuanense were divided into two groups ( groups a and f ) .", "g. sichuanense strains ( cui 7691 and hmas 86597 ; holotype ) , including the holotype , could be clustered into group f together with g. weberianum , and it was closely related to g. resinaceum .", "cao et al .   also reported that the size of basidiospores of g. lingzhi [ ( 9.85  0.85 )  ( 6.4  0.6 ) m ] differs from that of g. sichuanense [ ( 8.3  0.9 )  ( 5.8  0.8 ) m ] .", "interestingly , the average size of the basidiospores from the korean cultivated yeongji strains [ ( 10.65  0.65 )  ( 6.6  0.6 ) m for asi-7004 , ( 10  1 )  ( 6.4  0.3 ) m for asi-7071 ] were similar to that of g. lingzhi ( fig .", "the comparison of the its rdna sequences and the estimation of the basidiospores size presented in this study confirm previous findings and contribute additional evidence that suggests the naming korean cultivated yeongji strains of ' g. lucidum ' should be renamed as g. lingzhi ."], "abstract_text": ["<S> ganoderma lucidum has a long history of use as a traditional medicine in asian countries . </S>", "<S> however , the taxonomy of ganoderma species remains controversial , since they were initially classified on the basis of their morphological characteristics . </S>", "<S> recently , it was proposed that g. lucidum from china be renamed as g. sichuanense or g. lingzhi . in the present study , </S>", "<S> phylogenetic analysis using the internal transcribed spacer region rdna sequences of the ganoderma species indicated that all strains of the korean ' g. lucidum ' clustered into one group together with g. sichuanense and g. lingzhi from china . </S>", "<S> however , strains from europe and north american , which were regarded as true g. lucidum , were positioned in a clearly different group . in addition , the average size of the basidiospores from the korean cultivated yeongji strains was similar to that of g. lingzhi . based on these results , </S>", "<S> we propose that the korean cultivated yeongji strains of ' g. lucidum ' should be renamed as g. lingzhi . </S>"], "labels": null, "section_names": ["MATERIALS AND METHODS", "None", "Genomic DNA extraction and PCR amplification", "Cloning and sequencing", "Sequence analysis", "Basidiospore observation", "RESULTS AND DISCUSSION"], "sections": [["the ganoderma strains listed in table 1 were obtained from the korean collection for type culture ( ktct , jeongeup , korea ) , the american type culture collection ( atcc , rockville , md , usa ) , the korean agricultural culture collection ( kacc , suwon , korea ) , the mushroom division of the rural development administration ( eumseong , korea ) , the centraalbureau voor schimmelcultures ( cbs , utrecht , netherlands ) , incheon university ( incheon , korea ) , and konkuk university ( seoul , korea ) .", "ganoderma species were cultured on potato dextrose broth ( difco , detroit , mi , usa ) and incubated at 30 for 2 wk .", "cultured mycelia , filtered through 2 layers of miracloth ( calbiochem , la jolla , ca , usa ) , were ground in liquid nitrogen , and genomic dna was extracted using the cetyltrimethylammonium bromide method .", "pcrs were performed using a premixed polymerase kit ( taq premix ; tnt research , anyang , korea ) in a 20 l reaction mixture containing 1 l of dna .", "amplification of the its region was carried out using a thermal cycler ( takara , tokyo , japan ) at the following conditions : 5 min at 94 for initial denaturation , followed by 30 cycles of 30 sec at 94 for denaturation , 30 sec at 56 for primer annealing , and 1 min at 72 for extension , and 10 min at 72 for a final extension .", "pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer .", "pcr products were ligated into the pgem - t easy vector ( promega , madison , wi , usa ) according to the manufacturer 's instructions .", "ligation products were transformed into the escherichia coli dh5 competent cell ( rbc , taiwan ) by heat shock .", "plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit ( geneall , seoul , korea ) .", "recombinant clones were identified , and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing ( using the sp6 and t7 promoters ; genotech , daejeon , korea ) .", "nucleotide sequences were deposited in the national center for biotechnology information genbank database ( table 1 ) .", "the sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) .", "the phylogenetic trees were constructed by using the mega5 program   and the neighbor - joining method .", "confidence levels for individual branches of the resulting tree were assessed using the bootstrap test   in which 1,000 replicate trees were generated from resampled data .", "the size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope ( axio observer a1 ; carl zeiss , jena , germany ) and a 5% koh solution as a mounting medium .", "the size of each spore was calculated , and the mean value was used in the description ."], ["the ganoderma strains listed in table 1 were obtained from the korean collection for type culture ( ktct , jeongeup , korea ) , the american type culture collection ( atcc , rockville , md , usa ) , the korean agricultural culture collection ( kacc , suwon , korea ) , the mushroom division of the rural development administration ( eumseong , korea ) , the centraalbureau voor schimmelcultures ( cbs , utrecht , netherlands ) , incheon university ( incheon , korea ) , and konkuk university ( seoul , korea ) .", "ganoderma species were cultured on potato dextrose broth ( difco , detroit , mi , usa ) and incubated at 30 for 2 wk ."], ["cultured mycelia , filtered through 2 layers of miracloth ( calbiochem , la jolla , ca , usa ) , were ground in liquid nitrogen , and genomic dna was extracted using the cetyltrimethylammonium bromide method .", "pcrs were performed using a premixed polymerase kit ( taq premix ; tnt research , anyang , korea ) in a 20 l reaction mixture containing 1 l of dna .", "amplification of the its region was carried out using a thermal cycler ( takara , tokyo , japan ) at the following conditions : 5 min at 94 for initial denaturation , followed by 30 cycles of 30 sec at 94 for denaturation , 30 sec at 56 for primer annealing , and 1 min at 72 for extension , and 10 min at 72 for a final extension .", "pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer ."], ["pcr products were ligated into the pgem - t easy vector ( promega , madison , wi , usa ) according to the manufacturer 's instructions .", "ligation products were transformed into the escherichia coli dh5 competent cell ( rbc , taiwan ) by heat shock .", "plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit ( geneall , seoul , korea ) .", "recombinant clones were identified , and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing ( using the sp6 and t7 promoters ; genotech , daejeon , korea ) ."], ["nucleotide sequences were deposited in the national center for biotechnology information genbank database ( table 1 ) .", "the sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) .", "the phylogenetic trees were constructed by using the mega5 program   and the neighbor - joining method .", "confidence levels for individual branches of the resulting tree were assessed using the bootstrap test   in which 1,000 replicate trees were generated from resampled data ."], ["the size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope ( axio observer a1 ; carl zeiss , jena , germany ) and a 5% koh solution as a mounting medium .", "the size of each spore was calculated , and the mean value was used in the description ."], ["the phylogenetic analysis of ganoderma species in this study was generally consistent with the findings reported by the group from china .", "the 62 ganoderma strains were divided into 7 groups , a to g ( fig .", "in addition , ganoderma lucidum were largely divided into two groups ( groups a and g ) .", "group a included all the korean g. lucidum strains , as well as the g. lucidum strains from bangladesh and japan , and chinese g. sichuanense and g. lingzhi .", "g. meredithae ( usa and unknown sources ) in group b was closely related to g. lucidum in group a as evidenced by a high bootstrap value of 98% .", "g. multipileum ( china and taiwan ) was grouped within group c and g. tropicum ( china and taiwan ) within group d , with 96% and 99% bootstrap support , respectively .", "group e includes three strains of g. resinaceum from the czech republic and uk ( two strains ) , and was supported by very high bootstrap values of 99% .", "strains of g. weberianum ( australia , china , and philippines ) and two other chinese g. sichuanense strains ( cui 7691 and hmas 86597 ; holotype ) were grouped within group f with 99% bootstrap support . groups e and f were closely related with bootstrap values of 96% .", "it is worth noting that other g. lucidum strains from europe ( france , finland , italy , sweden , and uk ) and canada could be clustered into group g with g. tsugae strains from north america ( canada and usa ) , supported by a very high bootstrap value of 99% .", "the findings of the current study are consistent with those of park et al .   who reported that g. lucidum strains from europe and north america could be clustered into one group together with g. tsugae based on both analyses of the its rdna gene and partial -tubulin gene sequences .", "interestingly , korean g. lucidum strains could be clustered into a group together with g. sichuanense and g. lingzhi strains from china .", "reported that g. lucidum is incorrectly recorded in china , as well as in other countries , and suggested that the name ' g. lucidum ' as used for the chinese species should be corrected as g. sichuanense .", "however , they did not obtain sequences from type specimens of g. sichuanense ( the holotype ; hmas 42798 ) .", "reported that korean g. lucidum ( asi-7004 ) could be clustered into one group together with chinese g. lucidum and g. lingzhi based on the its2 sequences and rna secondary structures .", "furthermore , cao et al .   reported that g. sichuanense ( included the holotype ; hmas 42798 ) was distantly phylogenetically related to g. lingzhi ( included the holotype ; wu 1006 - 38 ) , but it was closely related to g. resinaceum . with regard to the morphological characteristics , it has been reported that chinese g. lucidum ( g. lingzhi ) has a yellow pore surface , european g. lucidum has a white pore surface , and korean g. lucidum has pale brownish thread - like tissues in the middle of the context .", "cao et al .   found that the morphological characteristics of ' g. lucidum ' from east asia were consistent with those of g. lingzhi .", "thus , they concluded that g. sichuanense was a distinct species since they display obvious morphological differences from g. lingzhi and suggested that the name ' g. lucidum ' should be corrected as g. lingzhi not g. sichuanense .", "it is commonly called \" yeongji \" in korea , \" lingzhi \" in china , and \" reishi \" or \" mannentake \" in japan .", "however , the ganoderma species from various countries including africa , oceania , america , asia ( china , korea , and japan ) , and europe   have been incorrectly reported as g. lucidum .", "in addition , moncalvo et al .   classified g. lucidum collections from different regions ( asia and europe ) into different species based on its and partial nuclear large subunit ribosomal dna sequences .", "this is similar to the results of our its nucleotide sequence analysis of g. lucidum .", "likewise , hong and jung   reported that the g. lucidum from korea and japan were monophyletic , and were distinguished from the g. lucidum from europe and north america based on sequence analysis of mitochondrial small - subunit ribosomal dna .", "as described above , the taxonomy of g. lucidum has been reported by many researchers .", "nevertheless , the name g. lucidum has been misapplied to various species around the world .", "the results of this study also reveal that korean g. lucidum was clustered into one group together with chinese g. sichuanense and chinese g. lingzhi ( both formerly g. lucidum ) , and it was clearly separated from g. lucidum from europe and north america .", "in addition , g. sichuanense were divided into two groups ( groups a and f ) .", "g. sichuanense strains ( cui 7691 and hmas 86597 ; holotype ) , including the holotype , could be clustered into group f together with g. weberianum , and it was closely related to g. resinaceum .", "cao et al .   also reported that the size of basidiospores of g. lingzhi [ ( 9.85  0.85 )  ( 6.4  0.6 ) m ] differs from that of g. sichuanense [ ( 8.3  0.9 )  ( 5.8  0.8 ) m ] .", "interestingly , the average size of the basidiospores from the korean cultivated yeongji strains [ ( 10.65  0.65 )  ( 6.6  0.6 ) m for asi-7004 , ( 10  1 )  ( 6.4  0.3 ) m for asi-7071 ] were similar to that of g. lingzhi ( fig .", "the comparison of the its rdna sequences and the estimation of the basidiospores size presented in this study confirm previous findings and contribute additional evidence that suggests the naming korean cultivated yeongji strains of ' g. lucidum ' should be renamed as g. lingzhi ."]]}
{"article_id": "PMC4743679", "article_text": ["successfully conducted the first simultaneous liver - kidney transplant ( slkt ) in 1983 on a patient with chronic kidney allograft rejection and liver cirrhosis , this procedure has become the most effective method for the treatment of combined end - stage liver disease and renal failure .", "the number of patients with end - stage liver disease and renal insufficiency receiving slkt has increased remarkably , particularly since the introduction of the model for end - stage liver disease ( meld ) scoring system by the united network for organ sharing ( unos ) in 2002 .", "statistics showed that the number of slkt recipients in the united states has quadrupled from 1998 to 2006 .", "in addition , the survival rate of slkt recipients has significantly increased following the adoption of meld .", "the primary causes of end - stage liver disease are cirrhosis caused by viral hepatitis , alcoholic cirrhosis , liver cancer , and congenital liver diseases .", "while hepatitis c is the leading cause in western countries , hepatitis b is the most prevalent type in china .", "epidemiological studies have shown that more than 800 million people have been infected with hepatitis b in china .", "up to 10.31% of the chinese population is hepatitis b surface antigen ( hbsag)-positive . in china , up to 80% of patients with liver failure requiring liver transplant are due to hepatitis b - associated causes .", "relapse of hepatitis b after liver transplant can reduce the patient s probability of survival by inducing graft dysfunction , recurrence of liver cancer , and lymphatic proliferation .", "a previous study reported that slkt for hepatitis c , non - alcoholic steatosis and hepatocellular cancer could be associated with worst outcomes compared with other slkt indications .", "however , few studies have reported the effectiveness of slkt and hepatitis b recurrence in hepatitis b carriers receiving slkt .", "previous studies have compared the outcome of slkt mainly to outcomes of liver transplant ( lt ) patients with normal renal function as controls [ 1216 ] , while comparison to lt patients with renal insufficiency has been largely ignored .", "a comparison between slkt recipients with hepatitis b infection and lt recipients with renal insufficiency and hepatitis b infection could provide a more objective assessment of the outcome of slkt associated with hepatitis b. in addition , which indicators can predict the long - term outcomes of liver transplant patients still need to be assessed .", "therefore , this comparative study analyzed rates of postoperative infection , rejection , long - term survival , and relapse of hepatitis b in hepatitis b carriers receiving slkt at the organ transplant center of the shanghai first people s hospital .", "this was a retrospective analyses in 21 patients ( male : 19 , female : 2 ) who underwent slkt at the organ transplant center of shanghai first people s hospital , shanghai jiaotong university , between january 2001 and may 2005 .", "recipients of slkt were selected based on the following criteria : 1 ) irreversibly compromised renal function confirmed by preoperative examination , including serum creatinine levels > 133 mol / l lasting for 1 month continuously ; 2 ) high - risk factors for kidney diseases , such as diabetes and hypertension ; and 3 ) massive proteinuria and/or requiring renal replacement therapy for more than 3 weeks .", "liver and kidney allografts for the same patient came from the same donor , whom was not necessarily a relative .", "panel reactive antibody ( pra ) class i of recipients was 028% , and class ii was 017% .", "the study period also included 25 patients who underwent lt alone with renal insufficiency ( preoperative serum creatinine levels > 133 mol / l ) .", "twenty - five recipients developed renal insufficiency due to hepatorenal syndrome without acute tubular necrosis and renal parenchymal disease .", "this study was approved by the ethical committee of the shanghai first people s hospital , and written informed consent was obtained from each patient . written consent has been obtained from all donors at the time of their donation .", "criteria for slkt : 1 ) end - stage liver disease and irreversible kidney failure ; 2 ) congenital diseases involving the liver and kidney ; 3 ) liver failure and chronic kidney disease ( ckd ) with gfr 30 ml / min ; 4 ) acute kidney injury ( aki ) with creatinine levels 176.8 mol / l and dialysis 8 weeks ; or 5 ) liver failure and ckd and biopsy demonstrating > 30% glomerulosclerosis or fibrosis .", "if there was no evidence to prove irreversible renal damage , liver transplantation alone was preferred .", "whether patients with hepatorenal syndrome should undergo slkt was controversial , and each patient was discussed .", "when patients with hepatorenal syndrome were on dialysis for < 4 weeks , liver transplant alone was performed .", "biochemical indicators ( including serum creatinine , total bilirubin , and aspartate aminotransferase ( ast ) levels ) were followed up for 5 years in all transplant recipients .", "lt patients were stratified according to the severity of aki as described by the risk , injury , failure , loss and endstage kidney disease ( rifle ) classification : risk , injury or failure .", "early- and late - stage postoperative complications , postoperative infections , rejection , and long - term survival were also recorded .", "the diagnosis of acute renal allograft rejection was primarily based on recipients clinical presentation , biochemical indicators ( a 25% increase of serum creatinine levels or more ) , and histopathological biopsy .", "the diagnosis of acute hepatic allograft rejection required increases in serum ast levels and/or in total bile acids , and was confirmed by liver biopsy .", "treatment of acute allograft rejection mainly relied on methylprednisolone sodium succinate pulse therapy , an increase in the dose of immunosuppressants , and/or change of types of immunosuppressive agent .", "the induction regimen of anti - cd25 monoclonal antibody ( daclizumab or basiliximab ) was prescribed to all patients .", "slkt recipients received a triple maintenance therapy of calcineurin inhibitor ( cni ; cyclosporine and tacrolimus ) , mycophenolate mofetil ( mmf ) and prednisolone ( pred ) , while lt recipients received the steroid - free regimen of cni + mmf .", "nucleoside analogues coupled with low - dose hepatitis b immunoglobulin ( hbig ) were given to prevent hepatitis b relapse .", "preoperative nucleoside analogue therapy was followed by intraoperative hbig 2000 u. postoperative combined therapy was administered with maintenance hbig twice per week . within 6 months of transplantation ,", "survival rates of recipients and allografts were analyzed using the kaplan - meier and log - rank methods .", "continuous data were analyzed using the student s t - test , and categorical variables using the chi - square test .", "this was a retrospective analyses in 21 patients ( male : 19 , female : 2 ) who underwent slkt at the organ transplant center of shanghai first people s hospital , shanghai jiaotong university , between january 2001 and may 2005 .", "recipients of slkt were selected based on the following criteria : 1 ) irreversibly compromised renal function confirmed by preoperative examination , including serum creatinine levels > 133 mol / l lasting for 1 month continuously ; 2 ) high - risk factors for kidney diseases , such as diabetes and hypertension ; and 3 ) massive proteinuria and/or requiring renal replacement therapy for more than 3 weeks .", "liver and kidney allografts for the same patient came from the same donor , whom was not necessarily a relative .", "panel reactive antibody ( pra ) class i of recipients was 028% , and class ii was 017% .", "the study period also included 25 patients who underwent lt alone with renal insufficiency ( preoperative serum creatinine levels > 133 mol / l ) .", "twenty - five recipients developed renal insufficiency due to hepatorenal syndrome without acute tubular necrosis and renal parenchymal disease .", "this study was approved by the ethical committee of the shanghai first people s hospital , and written informed consent was obtained from each patient . written consent has been obtained from all donors at the time of their donation .", "criteria for slkt : 1 ) end - stage liver disease and irreversible kidney failure ; 2 ) congenital diseases involving the liver and kidney ; 3 ) liver failure and chronic kidney disease ( ckd ) with gfr 30 ml / min ; 4 ) acute kidney injury ( aki ) with creatinine levels 176.8 mol / l and dialysis 8 weeks ; or 5 ) liver failure and ckd and biopsy demonstrating > 30% glomerulosclerosis or fibrosis .", "if there was no evidence to prove irreversible renal damage , liver transplantation alone was preferred .", "whether patients with hepatorenal syndrome should undergo slkt was controversial , and each patient was discussed .", "when patients with hepatorenal syndrome were on dialysis for < 4 weeks , liver transplant alone was performed .", "biochemical indicators ( including serum creatinine , total bilirubin , and aspartate aminotransferase ( ast ) levels ) were followed up for 5 years in all transplant recipients .", "lt patients were stratified according to the severity of aki as described by the risk , injury , failure , loss and endstage kidney disease ( rifle ) classification : risk , injury or failure .", "early- and late - stage postoperative complications , postoperative infections , rejection , and long - term survival were also recorded .", "the diagnosis of acute renal allograft rejection was primarily based on recipients clinical presentation , biochemical indicators ( a 25% increase of serum creatinine levels or more ) , and histopathological biopsy .", "the diagnosis of acute hepatic allograft rejection required increases in serum ast levels and/or in total bile acids , and was confirmed by liver biopsy .", "treatment of acute allograft rejection mainly relied on methylprednisolone sodium succinate pulse therapy , an increase in the dose of immunosuppressants , and/or change of types of immunosuppressive agent .", "the induction regimen of anti - cd25 monoclonal antibody ( daclizumab or basiliximab ) was prescribed to all patients .", "slkt recipients received a triple maintenance therapy of calcineurin inhibitor ( cni ; cyclosporine and tacrolimus ) , mycophenolate mofetil ( mmf ) and prednisolone ( pred ) , while lt recipients received the steroid - free regimen of cni + mmf .", "nucleoside analogues coupled with low - dose hepatitis b immunoglobulin ( hbig ) were given to prevent hepatitis b relapse .", "preoperative nucleoside analogue therapy was followed by intraoperative hbig 2000 u. postoperative combined therapy was administered with maintenance hbig twice per week . within 6 months of transplantation ,", "survival rates of recipients and allografts were analyzed using the kaplan - meier and log - rank methods .", "continuous data were analyzed using the student s t - test , and categorical variables using the chi - square test .", "meld scores , child - pugh scores , and incidence of preoperative dialysis were not significantly different between slkt and lt recipients . however , slkt recipients had lower egfr ( p=0.003 ) and higher preoperative serum creatinine levels ( p=0.004 ) , lower serum ast levels , and lower total serum bilirubin levels than lt recipients ( table 2 ) .", "four slkt recipients died : 2 died due to hepatitis b relapse and graft - versus - host disease within 2 months of surgery ; 1 discontinued lamivudine against medical advice and died due to hepatitis b relapse and subsequent acute liver failure 14 months after surgery ; and 1 died due to systemic infection 24 months after surgery .", "recipients had a 1-year survival rate of 90.5% , a 3-year survival rate of 81.0% , and a 5-year survival rate of 81.0% ( table 2 ) .", "eleven recipients died ( table 3 ) : 6 died due to fulminant hepatic failure and subsequent renal failure within 1 month of surgery , which is defined as a clinical syndrome developing as a result of massive necrosis of liver cells or following any other cause of sudden and severe impairment of hepatic function occurring in patients without pre - existing or at least well - compensated liver disease ; 1 died due to primary graft dysfunction ; 1 died due to portal vein thrombosis ; and 1 died due to bleeding of an inferior vena cava aneurysm .", "another 2 patients died due to tumor recurrence during long - term follow - up .", "recipients had a 1-year survival rate of 60.0% , a 3-year survival rate of 56.0% , and a 5-year survival rate of 56.0% ( table 2 ) .", "kaplan - meier survival analysis showed that slkt recipients had a higher survival rate than that of lt recipients ( p=0.025 ) , particularly at 1 year post - transplant ( figure 1 ) .", "seven cases of hepatic allograft rejection occurred among the slkt recipients ; 4 developed rejection within 1 month of surgery , and the others developed rejection at 12 months , 18 months , and 37 months after surgery , respectively .", "four lt recipients developed allograft rejection ( table 2 ) : 2 cases of allograft rejection occurred within 1 month of surgery , while the other 2 cases occurred at 6 months and 12 months of surgery , respectively .", "univariate analyses revealed that slkt recipients had a higher but insignificant incidence of infection at an early postoperative stage , compared with lt recipients .", "slkt recipients had significantly longer stay in the intensive care unit ( icu ) and longer total length of hospital stay , compared with lt recipients .", "on the other hand , lt recipients demonstrated higher incidence of renal failure and higher mortality at an early postoperative stage compared to slkt recipients ( p=0.005 ) ( table 2 ) .", "slkt recipients and lt recipients demonstrated sero - clearance of hbsag within 3 months after surgery . however , slkt recipients experienced a significantly higher rate of hepatitis b relapse ( 38.1% , p=0.007 ) compared with lt recipients .", "steroid therapy for other subjects was discontinued , and nucleoside analogues coupled with high - dose hbig ( hbig 20 000 u / d14d ) were given . following the treatment ,", "the patients had serologic hepatitis b virus ( hbv ) dna test results that showed undetectable levels of hbv ( table 4 ) .", "risk factors for high mortality ( 32% ) at an early stage after lt were analyzed using univariate and multivariate analyses . according to univariate analyses ( table 5 ) , patients who died at an early stage after surgery had significantly higher preoperative serum creatinine levels ( 239.5107.7 mol / l ) than survivors ( 146.021.6 mol / l ) , and had significantly lower egfr levels ( 30.812.7 ml / min/1.73 m ) than survivors ( 47.18.7 ml / min/1.73 m ) .", "the early death recipients after lt had higher rifle stage than survivors ( 75% vs. 11.8 , p=0.004 ) . compared with survivors , patients who died during the study had significantly higher preoperative meld scores and child - pugh scores . in addition , a higher incidence rate of postoperative renal failure ( 24% ) was associated with mortality compared with that of survivors ( 4% , p=0.001 ) .", "multivariate analysis revealed that postoperative renal failure ( p=0.003 ; or 48 ; 95%ci : 3.65631.76 ) and higher rifle stage ( p=0.012 , or 8 ; 95%ci : 1.5638.22 ) were independent risk factors for mortality after liver transplant ( table 6 ) .", "meld scores , child - pugh scores , and incidence of preoperative dialysis were not significantly different between slkt and lt recipients", ". however , slkt recipients had lower egfr ( p=0.003 ) and higher preoperative serum creatinine levels ( p=0.004 ) , lower serum ast levels , and lower total serum bilirubin levels than lt recipients ( table 2 ) .", "four slkt recipients died : 2 died due to hepatitis b relapse and graft - versus - host disease within 2 months of surgery ; 1 discontinued lamivudine against medical advice and died due to hepatitis b relapse and subsequent acute liver failure 14 months after surgery ; and 1 died due to systemic infection 24 months after surgery .", "recipients had a 1-year survival rate of 90.5% , a 3-year survival rate of 81.0% , and a 5-year survival rate of 81.0% ( table 2 ) .", "eleven recipients died ( table 3 ) : 6 died due to fulminant hepatic failure and subsequent renal failure within 1 month of surgery , which is defined as a clinical syndrome developing as a result of massive necrosis of liver cells or following any other cause of sudden and severe impairment of hepatic function occurring in patients without pre - existing or at least well - compensated liver disease ; 1 died due to primary graft dysfunction ; 1 died due to portal vein thrombosis ; and 1 died due to bleeding of an inferior vena cava aneurysm .", "another 2 patients died due to tumor recurrence during long - term follow - up .", "recipients had a 1-year survival rate of 60.0% , a 3-year survival rate of 56.0% , and a 5-year survival rate of 56.0% ( table 2 ) .", "kaplan - meier survival analysis showed that slkt recipients had a higher survival rate than that of lt recipients ( p=0.025 ) , particularly at 1 year post - transplant ( figure 1 ) .", "seven cases of hepatic allograft rejection occurred among the slkt recipients ; 4 developed rejection within 1 month of surgery , and the others developed rejection at 12 months , 18 months , and 37 months after surgery , respectively .", "four lt recipients developed allograft rejection ( table 2 ) : 2 cases of allograft rejection occurred within 1 month of surgery , while the other 2 cases occurred at 6 months and 12 months of surgery , respectively .", "univariate analyses revealed that slkt recipients had a higher but insignificant incidence of infection at an early postoperative stage , compared with lt recipients .", "slkt recipients had significantly longer stay in the intensive care unit ( icu ) and longer total length of hospital stay , compared with lt recipients . on the other hand", ", lt recipients demonstrated higher incidence of renal failure and higher mortality at an early postoperative stage compared to slkt recipients ( p=0.005 ) ( table 2 ) .", "slkt recipients and lt recipients demonstrated sero - clearance of hbsag within 3 months after surgery . however , slkt recipients experienced a significantly higher rate of hepatitis b relapse ( 38.1% , p=0.007 ) compared with lt recipients .", "steroid therapy for other subjects was discontinued , and nucleoside analogues coupled with high - dose hbig ( hbig 20 000 u / d14d ) were given .", "following the treatment , the patients had serologic hepatitis b virus ( hbv ) dna test results that showed undetectable levels of hbv ( table 4 ) .", "risk factors for high mortality ( 32% ) at an early stage after lt were analyzed using univariate and multivariate analyses . according to univariate analyses ( table 5 ) ,", "patients who died at an early stage after surgery had significantly higher preoperative serum creatinine levels ( 239.5107.7 mol / l ) than survivors ( 146.021.6 mol / l ) , and had significantly lower egfr levels ( 30.812.7 ml / min/1.73 m ) than survivors ( 47.18.7 ml / min/1.73 m ) .", "the early death recipients after lt had higher rifle stage than survivors ( 75% vs. 11.8 , p=0.004 ) . compared with survivors , patients who died during the study had significantly higher preoperative meld scores and child - pugh scores . in addition , a higher incidence rate of postoperative renal failure ( 24% ) was associated with mortality compared with that of survivors ( 4% , p=0.001 ) .", "multivariate analysis revealed that postoperative renal failure ( p=0.003 ; or 48 ; 95%ci : 3.65631.76 ) and higher rifle stage ( p=0.012 , or 8 ; 95%ci : 1.5638.22 ) were independent risk factors for mortality after liver transplant ( table 6 ) .", "the effectiveness of slkt in patients with hepatitis viral infection had not been thoroughly examined previously . in western countries ,", "hepatitis c virus is the primary cause of end - stage liver disease and subsequently renal impairment .", "hepatitis c carriers who received slkt have demonstrated a 5-year survival rate of 68% . however , in asian countries , including china , most liver transplant recipients are hepatitis b virus carriers , in whom the outcome of slkt is less understood .", "this study reported the largest number of hepatitis b patients receiving slkt , in which the 1- , 3- , and 5-year survival rates were 90.5% , 81.0% , and 81.0% , respectively .", "singal et al . have reported that slkt for hepatitis c , non - alcoholic steatosis and hepatocellular cancer could be associated with worst outcomes compared with other slkt indications ; however , they did not include patients with hepatitis b , which is the most common indication for liver transplantation due to liver failure in china .", "slkt recipients had significantly higher 1-year survival rate ( 90.5% ) , compared with lt recipients with hepatitis b infection and renal failure .", "this finding suggested that slkt was effective in improving the survival rate of transplant recipients with renal insufficiency . compared with lt alone ,", "slkt was associated with significantly longer icu stay , longer total hospital stay , and higher incidence of postoperative early - stage infection ( 38.1% vs. 20% ) , which might be related to the greater surgical trauma and higher incidence of complications .", "in addition , the healthcare system in china requires that the patients are completely symptom - free at discharge , which could explain , at least in part , this longer hospital stay .", "the incidence of acute allograft rejection between slkt recipients ( 33.3% ) and lt recipients ( 16% ) did not reach statistical significance , suggesting that slkt failed to decrease the incidence of acute hepatic allograft rejection .", "these results are comparable with previous studies , but this comparison should be made with caution since these previous studies evaluated patients without hepatitis b virus infection [ 12,1416 ] .", "high mortality was observed in the lt group , which could be attributable to the fact that these patients were with kidney dysfunction , as shown by the multivariate analysis .", "in addition , even if the meld score was similar between the 2 groups , creatinine and bilirubin levels were higher in the lt group , which could be another part of the reason for the high mortality observed in this group .", "in addition , some other articles have reported a mortality rate from fulminant hepatic failure as high as 90% after transplantation .", "a number of factors may also be associated with death after liver transplantation , including infections and rejection .", "a previous study showed that primary liver graft dysfunction could predict kidney failure , but the present study was not designed to address this relationship .", "our study revealed that slkt was associated with a higher rate of hepatitis b recurrence than lt ( 38.1% vs. 4% ) .", "this finding might be due to the stronger and longer immunosuppressive therapy , particularly long - term steroids , administered to slkt recipients compared with steroid - free treatment for lt recipients .", "the use of steroids has been reported as a risk factor for hepatitis b relapse   and for poor survival and hepatitis c relapse .", "glucocorticoids can directly stimulate an enhancer region and a glucocorticoid response element in hepatitis b virus dna , thereby inducing viral transcription .", "glucocorticoid administration also suppresses the immune response by reducing the hbv - specific cytotoxic t cell response , and therefore hbv - dna titer increases during glucocorticoid treatment .", "alternatively , slkt recipients higher hepatitis b relapse rate might be related to the effect of double transplantation on recipients immune function .", "several previous studies have indicated that hbv can influence the survival of hepatic and renal allografts and lead to fulminant hepatic failure and renal allograft dysfunction . however , 2 subjects in our study died due to hepatic failure ; they discontinued anti - hbv medicine against medical advice and died due to fulminant hepatic failure .", "for the 6 slkt recipients with hepatitis b relapse , hormone therapy was discontinued and replaced by nucleoside analogues and high - dose hbig ( 20 000 u / d14d ) immediately after the relapse .", "therefore , slkt recipients with hbv infection should be advised of the risks for hepatitis b relapse .", "reasonable treatment can achieve hbv - dna sero - clearance in patients with hepatitis b relapse without affecting long - term survival .", "criteria for slkt for potential lt recipients with renal insufficiency are still unclear . according to the study by hanish et al .   on transplant recipients with dialysis history ,", "slkt generates better outcome than single kidney transplantation , liver transplantation followed by dialysis , or staged liver and kidney transplantation .", "reached similar conclusions . in the present study , lt recipients with renal insufficiency had higher mortality at an early stage after surgery than slkt recipients ( lt vs. slkt : 32% vs. 0% ) .", "risk factors for death in lt recipients included lower egfr and higher rifle stage , preoperative serum creatinine levels , meld score , child - pugh score , and postoperative renal failure .", "although gfr is considered the best estimate of renal function than serum creatinine levels , gfr was not an independent risk factor for postoperative deaths .", "therefore , a gfr less than 30 ml / min is not enough to qualify for slkt .", "postoperative renal failure ( or=48 ) and rifle stage ( or=8 ) among lt recipients were an independent risk factor for postoperative deaths .", "it is reported that rifle criteria has been shown to predict clinical outcomes with a progressive increase in mortality with worsening rifle class .", "therefore , patients who might inevitably develop postoperative renal failure or be in need of dialysis should opt for slkt when possible .", "it is essential that patients with liver failure with worsening rifle class should be considered for slkt .", "indeed , this was a retrospective study that suffers from all the limitations inherent to this type of study .", "furthermore , these patients were from a single center , and the sample size was relatively small .", "the small number of death events in the slkt group ( 4/21 ) prevented a multivariate analysis in this group .", "despite longer hospital stay and higher infection rate at an early stage after surgery , slkt demonstrated satisfactory long - term survival rates in patients with hepatitis b infection . despite a higher rate of hepatitis b relapse following slkt ,", "timely and reasonable treatments were able to prevent hepatitis b relapse from affecting long - term prognosis .", "therefore , slkt can achieve good long - term outcomes in patients with hepatitis b , end - stage liver disease , and renal insufficiency .", "for lt recipients with renal failure , postoperative renal failure and rifle stage were independent risk factors for postoperative deaths ."], "abstract_text": ["<S> backgroundthe number and survival rate of simultaneous liver - kidney transplant ( slkt ) recipients have increased dramatically since 2002 . however , the long - term effectiveness of slkt in patients with hepatitis b is unknown.material/methodsforty-six patients who visited the organ transplant center of the shanghai first people s hospital between january 2001 and may 2005 had hepatitis b virus infection and renal failure ( any degree ) , and underwent organ transplantation : 21 patients underwent slkt and 25 patients underwent liver transplant ( lt ) alone.resultsthe 1- , 3- , and 5-year survival rates of slkt recipients were 90.5% , 81.0% , and 81.0% , respectively . </S>", "<S> incidence of acute hepatic allograft rejection between slkt recipients and lt recipients ( 33% vs. 16% ) did not reach significance ( p=0.170 ) . despite higher infection rate , more prevalent hepatitis b relapse , and longer </S>", "<S> stay in the intensive care unit , slkt recipients experienced significantly higher 1-year survival rate ( 90.5% ) compared with lt recipients ( 60% , p=0.019 ) . </S>", "<S> multivariate regression analysis revealed that postoperative renal failure ( odds ratio ( or)=48 , p=0.003 ) and risk / injury / failure / loss / end - stage ( rifle ) stage ( or=8 , p=0.012 ) were independent risk factors for postoperative death after lt.conclusionsslkt in patients with hepatitis b had higher early - stage infection rate , but had a higher long - term survival rate compared with the lt group . </S>", "<S> although the incidence of postoperative hepatitis b relapse in slkt recipients was higher , timely and reasonable treatment can ensure long - term survival of patients . worsening </S>", "<S> rifle stage of recipients can predict high mortality when only given lt . </S>", "<S> slkt might be a better choice for rifle stage 2 or 3 patients than lt alone . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Patients", "Operative indication", "Observation indicators", "Diagnosis and treatment of acute allograft rejection", "Immunosuppressive regimen and treatment of hepatitis B relapse", "Statistical analysis", "Results", "Baseline characteristics", "Survival", "Postoperative rejection, infection, and other complications", "Postoperative hepatitis B relapse", "Risk factors for mortality", "Discussion", "Conclusions"], "sections": [["successfully conducted the first simultaneous liver - kidney transplant ( slkt ) in 1983 on a patient with chronic kidney allograft rejection and liver cirrhosis , this procedure has become the most effective method for the treatment of combined end - stage liver disease and renal failure .", "the number of patients with end - stage liver disease and renal insufficiency receiving slkt has increased remarkably , particularly since the introduction of the model for end - stage liver disease ( meld ) scoring system by the united network for organ sharing ( unos ) in 2002 .", "statistics showed that the number of slkt recipients in the united states has quadrupled from 1998 to 2006 .", "in addition , the survival rate of slkt recipients has significantly increased following the adoption of meld .", "the primary causes of end - stage liver disease are cirrhosis caused by viral hepatitis , alcoholic cirrhosis , liver cancer , and congenital liver diseases .", "while hepatitis c is the leading cause in western countries , hepatitis b is the most prevalent type in china .", "epidemiological studies have shown that more than 800 million people have been infected with hepatitis b in china .", "up to 10.31% of the chinese population is hepatitis b surface antigen ( hbsag)-positive . in china , up to 80% of patients with liver failure requiring liver transplant are due to hepatitis b - associated causes .", "relapse of hepatitis b after liver transplant can reduce the patient s probability of survival by inducing graft dysfunction , recurrence of liver cancer , and lymphatic proliferation .", "a previous study reported that slkt for hepatitis c , non - alcoholic steatosis and hepatocellular cancer could be associated with worst outcomes compared with other slkt indications .", "however , few studies have reported the effectiveness of slkt and hepatitis b recurrence in hepatitis b carriers receiving slkt .", "previous studies have compared the outcome of slkt mainly to outcomes of liver transplant ( lt ) patients with normal renal function as controls [ 1216 ] , while comparison to lt patients with renal insufficiency has been largely ignored .", "a comparison between slkt recipients with hepatitis b infection and lt recipients with renal insufficiency and hepatitis b infection could provide a more objective assessment of the outcome of slkt associated with hepatitis b. in addition , which indicators can predict the long - term outcomes of liver transplant patients still need to be assessed .", "therefore , this comparative study analyzed rates of postoperative infection , rejection , long - term survival , and relapse of hepatitis b in hepatitis b carriers receiving slkt at the organ transplant center of the shanghai first people s hospital ."], ["this was a retrospective analyses in 21 patients ( male : 19 , female : 2 ) who underwent slkt at the organ transplant center of shanghai first people s hospital , shanghai jiaotong university , between january 2001 and may 2005 .", "recipients of slkt were selected based on the following criteria : 1 ) irreversibly compromised renal function confirmed by preoperative examination , including serum creatinine levels > 133 mol / l lasting for 1 month continuously ; 2 ) high - risk factors for kidney diseases , such as diabetes and hypertension ; and 3 ) massive proteinuria and/or requiring renal replacement therapy for more than 3 weeks .", "liver and kidney allografts for the same patient came from the same donor , whom was not necessarily a relative .", "panel reactive antibody ( pra ) class i of recipients was 028% , and class ii was 017% .", "the study period also included 25 patients who underwent lt alone with renal insufficiency ( preoperative serum creatinine levels > 133 mol / l ) .", "twenty - five recipients developed renal insufficiency due to hepatorenal syndrome without acute tubular necrosis and renal parenchymal disease .", "this study was approved by the ethical committee of the shanghai first people s hospital , and written informed consent was obtained from each patient . written consent has been obtained from all donors at the time of their donation .", "criteria for slkt : 1 ) end - stage liver disease and irreversible kidney failure ; 2 ) congenital diseases involving the liver and kidney ; 3 ) liver failure and chronic kidney disease ( ckd ) with gfr 30 ml / min ; 4 ) acute kidney injury ( aki ) with creatinine levels 176.8 mol / l and dialysis 8 weeks ; or 5 ) liver failure and ckd and biopsy demonstrating > 30% glomerulosclerosis or fibrosis .", "if there was no evidence to prove irreversible renal damage , liver transplantation alone was preferred .", "whether patients with hepatorenal syndrome should undergo slkt was controversial , and each patient was discussed .", "when patients with hepatorenal syndrome were on dialysis for < 4 weeks , liver transplant alone was performed .", "biochemical indicators ( including serum creatinine , total bilirubin , and aspartate aminotransferase ( ast ) levels ) were followed up for 5 years in all transplant recipients .", "lt patients were stratified according to the severity of aki as described by the risk , injury , failure , loss and endstage kidney disease ( rifle ) classification : risk , injury or failure .", "early- and late - stage postoperative complications , postoperative infections , rejection , and long - term survival were also recorded .", "the diagnosis of acute renal allograft rejection was primarily based on recipients clinical presentation , biochemical indicators ( a 25% increase of serum creatinine levels or more ) , and histopathological biopsy .", "the diagnosis of acute hepatic allograft rejection required increases in serum ast levels and/or in total bile acids , and was confirmed by liver biopsy .", "treatment of acute allograft rejection mainly relied on methylprednisolone sodium succinate pulse therapy , an increase in the dose of immunosuppressants , and/or change of types of immunosuppressive agent .", "the induction regimen of anti - cd25 monoclonal antibody ( daclizumab or basiliximab ) was prescribed to all patients .", "slkt recipients received a triple maintenance therapy of calcineurin inhibitor ( cni ; cyclosporine and tacrolimus ) , mycophenolate mofetil ( mmf ) and prednisolone ( pred ) , while lt recipients received the steroid - free regimen of cni + mmf .", "nucleoside analogues coupled with low - dose hepatitis b immunoglobulin ( hbig ) were given to prevent hepatitis b relapse .", "preoperative nucleoside analogue therapy was followed by intraoperative hbig 2000 u. postoperative combined therapy was administered with maintenance hbig twice per week . within 6 months of transplantation ,", "survival rates of recipients and allografts were analyzed using the kaplan - meier and log - rank methods .", "continuous data were analyzed using the student s t - test , and categorical variables using the chi - square test ."], ["this was a retrospective analyses in 21 patients ( male : 19 , female : 2 ) who underwent slkt at the organ transplant center of shanghai first people s hospital , shanghai jiaotong university , between january 2001 and may 2005 .", "recipients of slkt were selected based on the following criteria : 1 ) irreversibly compromised renal function confirmed by preoperative examination , including serum creatinine levels > 133 mol / l lasting for 1 month continuously ; 2 ) high - risk factors for kidney diseases , such as diabetes and hypertension ; and 3 ) massive proteinuria and/or requiring renal replacement therapy for more than 3 weeks .", "liver and kidney allografts for the same patient came from the same donor , whom was not necessarily a relative .", "panel reactive antibody ( pra ) class i of recipients was 028% , and class ii was 017% .", "the study period also included 25 patients who underwent lt alone with renal insufficiency ( preoperative serum creatinine levels > 133 mol / l ) .", "twenty - five recipients developed renal insufficiency due to hepatorenal syndrome without acute tubular necrosis and renal parenchymal disease .", "this study was approved by the ethical committee of the shanghai first people s hospital , and written informed consent was obtained from each patient . written consent has been obtained from all donors at the time of their donation ."], ["criteria for slkt : 1 ) end - stage liver disease and irreversible kidney failure ; 2 ) congenital diseases involving the liver and kidney ; 3 ) liver failure and chronic kidney disease ( ckd ) with gfr 30 ml / min ; 4 ) acute kidney injury ( aki ) with creatinine levels 176.8 mol / l and dialysis 8 weeks ; or 5 ) liver failure and ckd and biopsy demonstrating > 30% glomerulosclerosis or fibrosis .", "if there was no evidence to prove irreversible renal damage , liver transplantation alone was preferred .", "whether patients with hepatorenal syndrome should undergo slkt was controversial , and each patient was discussed .", "when patients with hepatorenal syndrome were on dialysis for < 4 weeks , liver transplant alone was performed ."], ["biochemical indicators ( including serum creatinine , total bilirubin , and aspartate aminotransferase ( ast ) levels ) were followed up for 5 years in all transplant recipients .", "lt patients were stratified according to the severity of aki as described by the risk , injury , failure , loss and endstage kidney disease ( rifle ) classification : risk , injury or failure .", "early- and late - stage postoperative complications , postoperative infections , rejection , and long - term survival were also recorded ."], ["the diagnosis of acute renal allograft rejection was primarily based on recipients clinical presentation , biochemical indicators ( a 25% increase of serum creatinine levels or more ) , and histopathological biopsy .", "the diagnosis of acute hepatic allograft rejection required increases in serum ast levels and/or in total bile acids , and was confirmed by liver biopsy .", "treatment of acute allograft rejection mainly relied on methylprednisolone sodium succinate pulse therapy , an increase in the dose of immunosuppressants , and/or change of types of immunosuppressive agent ."], ["the induction regimen of anti - cd25 monoclonal antibody ( daclizumab or basiliximab ) was prescribed to all patients .", "slkt recipients received a triple maintenance therapy of calcineurin inhibitor ( cni ; cyclosporine and tacrolimus ) , mycophenolate mofetil ( mmf ) and prednisolone ( pred ) , while lt recipients received the steroid - free regimen of cni + mmf .", "nucleoside analogues coupled with low - dose hepatitis b immunoglobulin ( hbig ) were given to prevent hepatitis b relapse .", "preoperative nucleoside analogue therapy was followed by intraoperative hbig 2000 u. postoperative combined therapy was administered with maintenance hbig twice per week . within 6 months of transplantation ,"], ["survival rates of recipients and allografts were analyzed using the kaplan - meier and log - rank methods .", "continuous data were analyzed using the student s t - test , and categorical variables using the chi - square test ."], ["meld scores , child - pugh scores , and incidence of preoperative dialysis were not significantly different between slkt and lt recipients . however , slkt recipients had lower egfr ( p=0.003 ) and higher preoperative serum creatinine levels ( p=0.004 ) , lower serum ast levels , and lower total serum bilirubin levels than lt recipients ( table 2 ) .", "four slkt recipients died : 2 died due to hepatitis b relapse and graft - versus - host disease within 2 months of surgery ; 1 discontinued lamivudine against medical advice and died due to hepatitis b relapse and subsequent acute liver failure 14 months after surgery ; and 1 died due to systemic infection 24 months after surgery .", "recipients had a 1-year survival rate of 90.5% , a 3-year survival rate of 81.0% , and a 5-year survival rate of 81.0% ( table 2 ) .", "eleven recipients died ( table 3 ) : 6 died due to fulminant hepatic failure and subsequent renal failure within 1 month of surgery , which is defined as a clinical syndrome developing as a result of massive necrosis of liver cells or following any other cause of sudden and severe impairment of hepatic function occurring in patients without pre - existing or at least well - compensated liver disease ; 1 died due to primary graft dysfunction ; 1 died due to portal vein thrombosis ; and 1 died due to bleeding of an inferior vena cava aneurysm .", "another 2 patients died due to tumor recurrence during long - term follow - up .", "recipients had a 1-year survival rate of 60.0% , a 3-year survival rate of 56.0% , and a 5-year survival rate of 56.0% ( table 2 ) .", "kaplan - meier survival analysis showed that slkt recipients had a higher survival rate than that of lt recipients ( p=0.025 ) , particularly at 1 year post - transplant ( figure 1 ) .", "seven cases of hepatic allograft rejection occurred among the slkt recipients ; 4 developed rejection within 1 month of surgery , and the others developed rejection at 12 months , 18 months , and 37 months after surgery , respectively .", "four lt recipients developed allograft rejection ( table 2 ) : 2 cases of allograft rejection occurred within 1 month of surgery , while the other 2 cases occurred at 6 months and 12 months of surgery , respectively .", "univariate analyses revealed that slkt recipients had a higher but insignificant incidence of infection at an early postoperative stage , compared with lt recipients .", "slkt recipients had significantly longer stay in the intensive care unit ( icu ) and longer total length of hospital stay , compared with lt recipients .", "on the other hand , lt recipients demonstrated higher incidence of renal failure and higher mortality at an early postoperative stage compared to slkt recipients ( p=0.005 ) ( table 2 ) .", "slkt recipients and lt recipients demonstrated sero - clearance of hbsag within 3 months after surgery . however , slkt recipients experienced a significantly higher rate of hepatitis b relapse ( 38.1% , p=0.007 ) compared with lt recipients .", "steroid therapy for other subjects was discontinued , and nucleoside analogues coupled with high - dose hbig ( hbig 20 000 u / d14d ) were given . following the treatment ,", "the patients had serologic hepatitis b virus ( hbv ) dna test results that showed undetectable levels of hbv ( table 4 ) .", "risk factors for high mortality ( 32% ) at an early stage after lt were analyzed using univariate and multivariate analyses . according to univariate analyses ( table 5 ) , patients who died at an early stage after surgery had significantly higher preoperative serum creatinine levels ( 239.5107.7 mol / l ) than survivors ( 146.021.6 mol / l ) , and had significantly lower egfr levels ( 30.812.7 ml / min/1.73 m ) than survivors ( 47.18.7 ml / min/1.73 m ) .", "the early death recipients after lt had higher rifle stage than survivors ( 75% vs. 11.8 , p=0.004 ) . compared with survivors , patients who died during the study had significantly higher preoperative meld scores and child - pugh scores . in addition , a higher incidence rate of postoperative renal failure ( 24% ) was associated with mortality compared with that of survivors ( 4% , p=0.001 ) .", "multivariate analysis revealed that postoperative renal failure ( p=0.003 ; or 48 ; 95%ci : 3.65631.76 ) and higher rifle stage ( p=0.012 , or 8 ; 95%ci : 1.5638.22 ) were independent risk factors for mortality after liver transplant ( table 6 ) ."], ["meld scores , child - pugh scores , and incidence of preoperative dialysis were not significantly different between slkt and lt recipients", ". however , slkt recipients had lower egfr ( p=0.003 ) and higher preoperative serum creatinine levels ( p=0.004 ) , lower serum ast levels , and lower total serum bilirubin levels than lt recipients ( table 2 ) ."], ["four slkt recipients died : 2 died due to hepatitis b relapse and graft - versus - host disease within 2 months of surgery ; 1 discontinued lamivudine against medical advice and died due to hepatitis b relapse and subsequent acute liver failure 14 months after surgery ; and 1 died due to systemic infection 24 months after surgery .", "recipients had a 1-year survival rate of 90.5% , a 3-year survival rate of 81.0% , and a 5-year survival rate of 81.0% ( table 2 ) .", "eleven recipients died ( table 3 ) : 6 died due to fulminant hepatic failure and subsequent renal failure within 1 month of surgery , which is defined as a clinical syndrome developing as a result of massive necrosis of liver cells or following any other cause of sudden and severe impairment of hepatic function occurring in patients without pre - existing or at least well - compensated liver disease ; 1 died due to primary graft dysfunction ; 1 died due to portal vein thrombosis ; and 1 died due to bleeding of an inferior vena cava aneurysm .", "another 2 patients died due to tumor recurrence during long - term follow - up .", "recipients had a 1-year survival rate of 60.0% , a 3-year survival rate of 56.0% , and a 5-year survival rate of 56.0% ( table 2 ) .", "kaplan - meier survival analysis showed that slkt recipients had a higher survival rate than that of lt recipients ( p=0.025 ) , particularly at 1 year post - transplant ( figure 1 ) ."], ["seven cases of hepatic allograft rejection occurred among the slkt recipients ; 4 developed rejection within 1 month of surgery , and the others developed rejection at 12 months , 18 months , and 37 months after surgery , respectively .", "four lt recipients developed allograft rejection ( table 2 ) : 2 cases of allograft rejection occurred within 1 month of surgery , while the other 2 cases occurred at 6 months and 12 months of surgery , respectively .", "univariate analyses revealed that slkt recipients had a higher but insignificant incidence of infection at an early postoperative stage , compared with lt recipients .", "slkt recipients had significantly longer stay in the intensive care unit ( icu ) and longer total length of hospital stay , compared with lt recipients . on the other hand", ", lt recipients demonstrated higher incidence of renal failure and higher mortality at an early postoperative stage compared to slkt recipients ( p=0.005 ) ( table 2 ) ."], ["slkt recipients and lt recipients demonstrated sero - clearance of hbsag within 3 months after surgery . however , slkt recipients experienced a significantly higher rate of hepatitis b relapse ( 38.1% , p=0.007 ) compared with lt recipients .", "steroid therapy for other subjects was discontinued , and nucleoside analogues coupled with high - dose hbig ( hbig 20 000 u / d14d ) were given .", "following the treatment , the patients had serologic hepatitis b virus ( hbv ) dna test results that showed undetectable levels of hbv ( table 4 ) ."], ["risk factors for high mortality ( 32% ) at an early stage after lt were analyzed using univariate and multivariate analyses . according to univariate analyses ( table 5 ) ,", "patients who died at an early stage after surgery had significantly higher preoperative serum creatinine levels ( 239.5107.7 mol / l ) than survivors ( 146.021.6 mol / l ) , and had significantly lower egfr levels ( 30.812.7 ml / min/1.73 m ) than survivors ( 47.18.7 ml / min/1.73 m ) .", "the early death recipients after lt had higher rifle stage than survivors ( 75% vs. 11.8 , p=0.004 ) . compared with survivors , patients who died during the study had significantly higher preoperative meld scores and child - pugh scores . in addition , a higher incidence rate of postoperative renal failure ( 24% ) was associated with mortality compared with that of survivors ( 4% , p=0.001 ) .", "multivariate analysis revealed that postoperative renal failure ( p=0.003 ; or 48 ; 95%ci : 3.65631.76 ) and higher rifle stage ( p=0.012 , or 8 ; 95%ci : 1.5638.22 ) were independent risk factors for mortality after liver transplant ( table 6 ) ."], ["the effectiveness of slkt in patients with hepatitis viral infection had not been thoroughly examined previously . in western countries ,", "hepatitis c virus is the primary cause of end - stage liver disease and subsequently renal impairment .", "hepatitis c carriers who received slkt have demonstrated a 5-year survival rate of 68% . however , in asian countries , including china , most liver transplant recipients are hepatitis b virus carriers , in whom the outcome of slkt is less understood .", "this study reported the largest number of hepatitis b patients receiving slkt , in which the 1- , 3- , and 5-year survival rates were 90.5% , 81.0% , and 81.0% , respectively .", "singal et al . have reported that slkt for hepatitis c , non - alcoholic steatosis and hepatocellular cancer could be associated with worst outcomes compared with other slkt indications ; however , they did not include patients with hepatitis b , which is the most common indication for liver transplantation due to liver failure in china .", "slkt recipients had significantly higher 1-year survival rate ( 90.5% ) , compared with lt recipients with hepatitis b infection and renal failure .", "this finding suggested that slkt was effective in improving the survival rate of transplant recipients with renal insufficiency . compared with lt alone ,", "slkt was associated with significantly longer icu stay , longer total hospital stay , and higher incidence of postoperative early - stage infection ( 38.1% vs. 20% ) , which might be related to the greater surgical trauma and higher incidence of complications .", "in addition , the healthcare system in china requires that the patients are completely symptom - free at discharge , which could explain , at least in part , this longer hospital stay .", "the incidence of acute allograft rejection between slkt recipients ( 33.3% ) and lt recipients ( 16% ) did not reach statistical significance , suggesting that slkt failed to decrease the incidence of acute hepatic allograft rejection .", "these results are comparable with previous studies , but this comparison should be made with caution since these previous studies evaluated patients without hepatitis b virus infection [ 12,1416 ] .", "high mortality was observed in the lt group , which could be attributable to the fact that these patients were with kidney dysfunction , as shown by the multivariate analysis .", "in addition , even if the meld score was similar between the 2 groups , creatinine and bilirubin levels were higher in the lt group , which could be another part of the reason for the high mortality observed in this group .", "in addition , some other articles have reported a mortality rate from fulminant hepatic failure as high as 90% after transplantation .", "a number of factors may also be associated with death after liver transplantation , including infections and rejection .", "a previous study showed that primary liver graft dysfunction could predict kidney failure , but the present study was not designed to address this relationship .", "our study revealed that slkt was associated with a higher rate of hepatitis b recurrence than lt ( 38.1% vs. 4% ) .", "this finding might be due to the stronger and longer immunosuppressive therapy , particularly long - term steroids , administered to slkt recipients compared with steroid - free treatment for lt recipients .", "the use of steroids has been reported as a risk factor for hepatitis b relapse   and for poor survival and hepatitis c relapse .", "glucocorticoids can directly stimulate an enhancer region and a glucocorticoid response element in hepatitis b virus dna , thereby inducing viral transcription .", "glucocorticoid administration also suppresses the immune response by reducing the hbv - specific cytotoxic t cell response , and therefore hbv - dna titer increases during glucocorticoid treatment .", "alternatively , slkt recipients higher hepatitis b relapse rate might be related to the effect of double transplantation on recipients immune function .", "several previous studies have indicated that hbv can influence the survival of hepatic and renal allografts and lead to fulminant hepatic failure and renal allograft dysfunction . however , 2 subjects in our study died due to hepatic failure ; they discontinued anti - hbv medicine against medical advice and died due to fulminant hepatic failure .", "for the 6 slkt recipients with hepatitis b relapse , hormone therapy was discontinued and replaced by nucleoside analogues and high - dose hbig ( 20 000 u / d14d ) immediately after the relapse .", "therefore , slkt recipients with hbv infection should be advised of the risks for hepatitis b relapse .", "reasonable treatment can achieve hbv - dna sero - clearance in patients with hepatitis b relapse without affecting long - term survival .", "criteria for slkt for potential lt recipients with renal insufficiency are still unclear . according to the study by hanish et al .   on transplant recipients with dialysis history ,", "slkt generates better outcome than single kidney transplantation , liver transplantation followed by dialysis , or staged liver and kidney transplantation .", "reached similar conclusions . in the present study , lt recipients with renal insufficiency had higher mortality at an early stage after surgery than slkt recipients ( lt vs. slkt : 32% vs. 0% ) .", "risk factors for death in lt recipients included lower egfr and higher rifle stage , preoperative serum creatinine levels , meld score , child - pugh score , and postoperative renal failure .", "although gfr is considered the best estimate of renal function than serum creatinine levels , gfr was not an independent risk factor for postoperative deaths .", "therefore , a gfr less than 30 ml / min is not enough to qualify for slkt .", "postoperative renal failure ( or=48 ) and rifle stage ( or=8 ) among lt recipients were an independent risk factor for postoperative deaths .", "it is reported that rifle criteria has been shown to predict clinical outcomes with a progressive increase in mortality with worsening rifle class .", "therefore , patients who might inevitably develop postoperative renal failure or be in need of dialysis should opt for slkt when possible .", "it is essential that patients with liver failure with worsening rifle class should be considered for slkt .", "indeed , this was a retrospective study that suffers from all the limitations inherent to this type of study .", "furthermore , these patients were from a single center , and the sample size was relatively small .", "the small number of death events in the slkt group ( 4/21 ) prevented a multivariate analysis in this group ."], ["despite longer hospital stay and higher infection rate at an early stage after surgery , slkt demonstrated satisfactory long - term survival rates in patients with hepatitis b infection . despite a higher rate of hepatitis b relapse following slkt ,", "timely and reasonable treatments were able to prevent hepatitis b relapse from affecting long - term prognosis .", "therefore , slkt can achieve good long - term outcomes in patients with hepatitis b , end - stage liver disease , and renal insufficiency .", "for lt recipients with renal failure , postoperative renal failure and rifle stage were independent risk factors for postoperative deaths ."]]}
{"article_id": "PMC1351369", "article_text": ["although dinoflagellates are best known as the notorious cause of toxic red tides , they are also important contributors to the ocean 's primary production .", "photosynthesis in these organisms is typically carried out in plastids surrounded by three membranes ( 1 ) , an evolutionary footprint reflecting their origin through secondary endosymbiosis ( 2 ) .", "the evolutionary ancestor of the peridinin - containing plastids is suggested from molecular phylogenetic reconstructions using plastid - encoded genes to be a red alga ( 3,4 ) , a conclusion supported by phylogeny of nuclear - encoded plastid - directed genes ( 5 ) . in general , these findings are also consistent with phylogenetic reconstructions of the host cells as determined from non - plastid - directed genes ( 6,7 ) . despite widespread acceptance of a red - algal origin for the peridinin - containing plastid", ", these organelles display a number of peculiar characteristics that share no homology with any known extant plastids .", "for example , the carotenoid peridinin itself ( 8) and the unusual light harvesting peridinin - chlorophyll a - protein to which it binds ( 9 ) are found in no other organisms .", "furthermore , the rubisco in peridinin - containing plastids , an unusual form ii enzyme , is dissimilar from the form i protein employed by all other plastids ( 10 ) .", "the evolutionary provenance of these proteins is thus unknown , and as they are derived from nuclear - encoded genes , it seems possible that their history may be distinct from that the plastid itself .", "an additional issue having bearing on plastid evolution , and one more likely to reflect the plastids themselves , is the number and arrangement of the genes within the genome .", "genome architecture may be different from the phylogeny of the plastid genes themselves , and in the case of the dinoflagellates , quite remarkably so .", "indeed , the only known plastid genes so far identified have been located on generally unigenic plasmid - like minicircles .", "these minicircles have been found in at least five genera of peridinin - containing dinoflagellates including amphidinium ( 11 ) , ceratium ( 12 ) , heterocapsa ( 13 ) , protoceratium ( 14 ) and symbiodinium ( 15 ) .", "each minicircle has regions conserved within a species , and extensive pcr amplification of the genes located between these conserved regions has been performed in several species . taken together , these studies have led to the conclusion that the known minicircle gene complement has reached saturation ( 16 ) with a total of sixteen protein encoding genes ( atpa - b ; petb and petd ; psaa - b ; psba - e and psbi ; ycf16 and ycf24 ; rpl28 and rpl33 ) in addition to the large ( 23s ) and small ( 16s ) ribosomal rna .", "the identification of this highly reduced set of plastid genes as comprising the plastid genome is also supported by recent results demonstrating that at least some of the missing genes ( i.e. ones normally found in plastids ) are instead nuclear - encoded in several species ( 1719 ) .", "these experiments suggest that dinoflagellates do not obey for the rules normally governing plastid gene transfer to the nucleus ( 20 ) . however , it is still a formal possibility that additional genes may be encoded in the plastid in a form different from the minicircle format found so far .", "one method potentially applicable to the characterization of the plastid genome is to determine the spectrum of genes expressed , as gene expression should be independent from the form in which the genes are found .", "however , it is generally difficult to discriminate between organelle- and nuclear - encoded transcripts , as both can be modified by addition of a poly(a ) tail at their 3 termini ( 21 ) .", "we have found that , unlike other transcripts in the dinoflagellates , those encoded by known minicircle genes carry a homogenous polyuridine tract at their 3 termini .", "we have taken advantage of this unusual feature to characterize the dinoflagellate plastid transcriptome , and find that our analysis fully supports a highly reduced plastid genome for the peridinin - containing dinoflagellates . furthermore ,", "as it seems likely that polyuridylylation may be common in dinoflagellate plastids , it may be possible to rapidly characterize the transcriptome of many different species using the method described here .", "amphidinium carterae ( ccmp 1314 ) and lingulodinium polyedrum ( ccmp 1936 , formerly gonyaulax polyedra ) were obtained from the provasoli  guillard culture center for marine phytoplancton ( boothbay harbor , maine ) and cultured as described ( 22 ) .", "poly(a ) rna was purified from lingulodinium and amphidinium using oligo(dt ) chromatography ( 23 ) and hybridized to a psba and 23s probes as described ( 22 ) .", "the 16s probe was prepared against an est sequence from our library ( see below ) , while the atpb probe was prepared using previously described amino acid microsequence data ( 22 ) to design two degenerate oligonucleotides 5-ttyticargciggiwsigargt-3 and 5-acytcigciacraaraaiggytg-3 ; the 500 bp pcr product was confirmed as atpb by sequence analysis .", "the 3 end sequences of atpb and psba transcripts were obtained from cdna synthesized from poly(a ) rna tailed in vitro with rgtp by poly(a ) polymerase .", "specific sequences were amplified using a d(c)10 oligonucleotide and 5-tatccaattggacaaggaag-3 ( lingulodinium psba ) 5-gtgtagcacaagatgtaagc-3 ( lingulodinium atpb ) , 5-gtattcggtcaagaggatg-3 ( amphidinium psba ) and 5-ctatctcagccgttctttg-3 ( amphidinium atpb ) .", "the genomic psba sequences from lingulodinium were obtained using tail - pcr ( 24 ) using three nested internal psba oligonucleotides ( 5-gctgcttggccagttattggtatctg-3 ; 5-tctggtttacagcacttggtgttag-3 ; 5-gtccataattgattcatcaggtcatc-3 ) to amplify a fragment from at - rich dna purified by bisbenzimide - cscl gradients . to obtain the 3 end of genomic sequences from amphidinium ,", "minicircle dna was amplified by inverse pcr using outwardly directed oligonucleotides 5-gtattcggtcaagaggatg-3 and 5-caggagcaaggaagaaag-3 ( psba ) or 5-gtggtcgtaagattgagagg-3 and 5-gatgagagcgttggcatac-3 ( atpb ) . for cdna preparation , 10 g poly(a)-enriched rna was used as a template for first strand synthesis using a commercial kit ( stratagene ) but replacing the usual oligo(dt ) primer with 5-(ga)10actagtctcgag(a)18 - 3. sequences were identified using the blast algorithm ( ) and have been deposited in genbank under the accession nos . dq264844 through dq264867 .", "all sequence alignments and analyses were performed using macvector ( accelrys ) except for rna secondary predictions that were made using a web server ( ) . statistical analysis using rarefaction ( 25 ) to determine the likelihood that our sample size was sufficient to detect all different cdnas in the library was made on a web server ( ) .", "we began our analysis of the dinoflagellate plastid transcriptome by fractionation of rna into poly(a)-enriched and depleted fractions by oligo(dt ) chromatography .", "the plastid - encoded psba ( 22 ) in rna extracted from the dinoflagellates lingulodinium ( figure 1a ) and amphidinium ( figure 1b ) , was found enriched by 10-fold in poly(a ) fractions , while 23s rrna remained in the poly(a ) fraction .", "the small size of the 23s rrna signal is suggestive of processing and has been previously observed in dinoflagellates ( 13 ) .", "the 16s rna was similarly processed , but the full - length form was found to a greater extent in the polyadenylated fraction than was the 23s rna .", "the abundance of these transcripts in a poly(a ) rich fraction was unexpected , as usually only a small fraction of plastid messages are polyadenylated ( 21 ) .", "furthermore , it seemed at odds with the lack of minicircle genes found in dinoflagellate est libraries ( 1719 ) .", "we originally thought that the tails might be heterogeneous , similar to the 3 termini in chloroplasts and cyanobacteria rna formed by polynucleotide phosphorylase ( pnp ) ( 21 ) , since the presence of nucleotides other than adenine in the 3 tails might inhibit cdna synthesis more than oligo(dt ) chromatography . to test this , we guanylated our poly(a)-enriched rna using poly(a ) polymerase , and performed rt  pcr with a primer pair allowing specific amplification of the psba 3 end ( figure 2a ) .", "fourteen different psba clones were sequenced , and all contained a homogeneous 3 terminal stretch of thymidine residues .", "at least five of the sequences obtained were independent clones based on differences in tail length , which varied between 25 and 40 thymidine residues ( figure 2b ) . a comparison to the genomic dna sequence , obtained by thermal asymmetric interlaced ( tail ) pcr ( 24 ) , shows that these residues are added post - transcriptionally , and all appeared to have been added at the same site ( between arrows , figure 2b ) .", "poly(t ) tracts of similar length were found on the 3 termini of atpb transcripts from lingulodinium ( data not shown ) and on atpb transcripts from the dinoflagellate amphidinium ( figure 2c ) .", "we propose that binding of these polyuridylylated mrnas to the longer and more prevalent poly(a ) tails of nuclear transcripts is responsible for their presence in poly(a)-enriched rna fractions obtained by oligo(dt ) cellulose chromatography .", "the unusual 3 terminal polyuridylylation , if a common feature of dinoflagellate plastid transcripts , suggested that analysis of cdna synthesized using an oligo(da ) instead of the usual oligo(dt ) primer would provide a straightforward method to catalog the plastid gene complement . roughly 60 ng of cdna was synthesized from 10 g poly(a)-enriched rna , a yield 30-fold less than that obtained with an oligo(dt ) primer in similar experiments .", "polyuridylylated transcripts are thus a small but significant proportion of cellular rna . to characterize the library , several hundred clones were selected at random and sequenced .", "some gc - rich and unidentified sequences were found , but none were polyuridylylated and were presumably derived from hairpin priming of the predominant gc - rich nuclear - encoded transcripts .", "in contrast , all polyuridylylated sequences were at - rich and were identified as transcripts from the minicircle genes found in other dinoflagellates ( 16 ) ( table 1 ) .", "the majority of the transcripts correspond to photosystem ii components ( psba - d ) and 16s rna .", "this latter may appear more frequently in our oligo(da)-primed library than the 23s rna because the full - length16s rna appears more abundant in a poly(a ) enriched sample ( figure 1a ) . to assess the likelihood that other low abundance polyuridylylated transcripts might be present in the library , we performed rarefaction analysis ( 25 ) .", "originally developed to compare species richness in biodiversity collections of different sizes , this technique estimates the number of different cdnas that would be obtained if smaller sample sizes were taken .", "the analysis of the data in table 1 indicates that the identification of twelve different sequences could have been possible with only 120 random clones sequenced ; the three hundred clones reported here represent a significant excess of this minimum value .", "the calculations can also be used to illustrate the progression in the number of random sequences required to reveal each additional sequence : while 10 different cdnas are expected in 65 random sequences , almost twice as many sequences are required to uncover the eleventh sequence , and roughly six times more sequences to yield the twelfth cdna ( figure 3 inset ) .", "this analysis suggests that to recover a potential new plastid transcript , well over a thousand random clones would have to be sequenced . to further test the contention the twelve genes recovered are likely to represent saturation coverage of the oligo(da )", ", roughly a thousand different cdna clones were randomly selected and streaked on a new petri plate in a grid configuration .", "colony lifts were then hybridized with a probe prepared against a mixture of psba - d or 16s sequences . from this , 50 cdnas that hybridized weakly or not at were selected and sequenced .", "only 14 sequences among the 50 sequenced were at - rich and polyuridylylated , and these included two atpa , four 23s rna , a petb , three psbb , three psbc and a psbd .", "these later three photosystem ii components may have been recovered following the virtual subtraction because of poor bacterial growth or poor transfer to the membrane .", "more importantly , no new polyuridylylated genes were identified , again suggesting that our coverage of the library had reached saturation . as it seems unlikely that alternative 3 modifications might be found within the same organelle", ", we conclude that the genome of this photosynthetically active chloroplast is likely to encode only the transcripts identified here . to address the mechanism underlying site selection for poly(u ) addition", "secondary structure is the principal determinant of polyadenylation site selection in prokaryotes ( 27 ) .", "however , computer generated secondary structure predictions from rna complementary to the genomic sequence surrounding the polyuridylylation site of either lingulodinium psba or amphidinium atpb did not show any reasonably stable stem  loop structures ( data not shown ) .", "however , two loosely conserved primary structure motifs are located within 50 bp of the modification site in all twelve transcripts ( figure 4 ) .", "these motifs ( agaaa and aauua ) might thus constitute primary structure elements signalling the polyuridylylation site in a manner similar to the use of aauaaa in determining polyadenylation sites in nuclear encoded transcripts ( 28 ) .", "we were also curious about the identity of the enzyme that might be used to catalyze the polyuridylylation reaction .", "as polyuridylylation of protein coding transcripts has been observed in organelles undergoing extensive uridine insertion editing ( 29,30 ) , we thus checked for uridine insertion in comparisons of genomic and cdna sequences using lingulodinium psba , petb and 16s rna ( 2.5 kb total sequence ) .", "our data reveals no evidence for uridine insertion although numerous examples of substitutional editing in dinoflagellate plastid transcripts ( principally a to g ) were observed ( table 2 , supplementary figures s1 and s2 ) .", "these results agree with a previous report of substitutional editing in the dinoflagellate ceratium ( 31 ) .", "we have found that the plastid transcripts in two species of peridinin - containing dinoflagellates are characterized by an unusual 3 polyuridylylation .", "this modification differs dramatically from the poly(a ) tails of nuclear - encoded transcripts , and so provides a facile method for cataloging the plastid transcriptome .", "we report here that an oligo(da)-primed cdna library has a remarkably low complexity , with only 12 different clones identified in 300 randomly selected polyuridylylated sequences ( table 1 ) .", "interestingly , all the sequences identified from the library were previously identified as minicircle genes in other dinoflagellate species .", "this concordance between two independent methods ( characterization of the minicircular genome and the transcriptome analysis reported here ) suggests that the minicircular gene format is likely to be the only genome architecture in the plastid .", "our results thus strongly support the contention that the dinoflagellate genome is the smallest of any functional chloroplast . furthermore , since the dinoflagellate amphidinium also contains polyuridylylated transcripts , it is possible that the technique described here could be used to catalog the plastid transcriptome from a range of other species .", "is it likely that the transcriptome contains other low abundance transcripts that were undetected in our relatively small sample size ?", "the chance of finding a specific transcript in a single random sample is a function of its relative proportion , or frequency of occurrence within the bank . however , if many other sequences were present , then the chance of finding any other sequence would also depend on the number of additional sequences present and on their relative proportions within the library . to estimate our coverage of the library , we used rarefaction analysis to determine if smaller sample sizes would also have recovered the same twelve genes .", "our analysis suggests that it is likely the same twelve genes would have been recovered using a smaller sample size , indicating that the number of clones sequence was in excess of that required .", "furthermore , the progression in the number of clones sequenced as a function of the number of different clones identified ( figure 3 ) suggests that well over a thousand random clones would have to be sequenced to find an additional clone if it were indeed present in the library .", "taken together with the virtual subtraction of psba - d and 16s rna sequences , these results strongly suggest that coverage of the library has reached saturation .", "the selective forces that serve to maintain genes in the chloroplast are hotly debated , and thought to reflect either difficulties in targeting or transport of proteins that are extremely hydrophobic or a relationship between control of gene expression and the redox state of the organelle ( 32 ) . with respect to the former , the twelve protein encoding genes found in our library are not the most hydrophobic of known thylakoid proteins . indeed , a better explanation for the retention of this particular gene set in the plastid may lie in the length of the protein rather than hydrophobicity . as recently shown by analysis of arabidopsis thylakoid proteins ( 33 ) ,", "the proteins encoded by the dinoflagellate plastid genes are generally the longest of the thylakoid proteins . however , even a combination of length and hydrophobicity is insufficient to explain transfer of some genes to the nucleus , such as the shorter yet relatively hydrophobic protein encoded by the atpi gene recently reported in an oligo(dt ) primed est bank from the dinoflagellate alexandrium .", "if instead of hydrophobicity , the redox control of gene expression were the determining factor , this would suggest the set of proteins encoded by the dinoflagellate plastids are most sensitive to changes in redox potential .", "the proposal that it is genes requiring editing may that are conserved in plastids ( 34 ) is supported by analysis of the petb gene of lingulodinium , as editing removes a stop codon in the middle of the derived protein sequence ( supplementary figure s2 ) . with respect to the unusual nature of the modification itself", ", there is precedent for polyuridylylation in organelles experiencing extensive editing , such as the mitochondria of trypanosomes or myxomycetes . in some cases , 3 polyuridine tails", "have been observed not only for the guide rnas used to determine the site of uridine insertion but for rrna and mrna as well ( 29,30 ) .", "however , a comparison of 2.5 kb genomic and cdna sequences showed only substitutional editing ( table 2 ) .", "our experiments thus provide the first example of extensive polyuridylylation occurring in the absence of rna editing .", "it is tempting to speculate that the poly(u ) tracts may result from a terminal uridylyltransferase ( tut ) ( 35 ) acting in the absence of guide rna to define a site for uridine insertion .", "however , an alternative possibility is that the plastids may contain a type poly(a ) polymerase with specificity for uridine residues instead of adenine .", "recently , it has also been shown that microrna - directed cleavage can result in addition of non - encoded oligonucleotides ( mostly uridine ) to 3 termini ( 36 ) . however , in this case the polyuridylylated transcripts are intermediates in an rna degradation pathway .", "it seems unlikely that the extensive polyuridylylation of the plastid transcripts is used as a signal for rna turnover , since the majority of psba transcripts appear modified as judged by their co - purification on oligo(dt ) chromatography .", "in contrast to the full - length protein coding transcripts , the small fragments hybridizing to 16s and 23s rna on northern analyses appear unmodified by these criteria ( figure 1 ) .", "interestingly , the apparent stability of the polyuridylylated plastid transcripts thus suggests that this particular 3 end modification has a different function from that in either cyanobacteria or the plastids of higher plants , where transcripts are marked for degradation by 3 end polyadenylation ( 21 ) . despite extensive molecular phylogenetic studies pointing to a common evolutionary origin for both host cells ( 7 ) and plastids ( 37 ) of the chromalveolates , no other plastids or cyanobacteria", "thus the mechanism and function of the dinoflagellate plastid transcript 3 end modification are as unique as their form ii rubisco ( 10 ) and peridinin  chlorophyll a - protein ( 9 ) . the major challenge for plastid phylogeny underscored by our results is to reconcile the many unique features of the dinoflagellate plastids with their phylogenetic relationships to red algae . in addition , given the concurrence of several lines of evidence supporting the highly reduced nature of the dinoflagellate plastid genome , it will be of interest to reinvestigate the nature of the selective forces maintaining the current plastid genome size in higher plants .", "dinoflagellate plastid messages are located in poly(a)-enriched rna . total rna samples ( t ) from the dinoflagellates lingulodinium ( a ) and amphidinium ( b ) were resolved into fractions enriched ( a+ ) and depleted ( a ) for poly(a ) rna by chromatography on oligo(dt ) cellulose .", "rna blots were challenged with gene probes for either psba , 23s rna or 16s .", "( a ) lingulodinium rna samples enriched for poly(a ) rna were tailed with guanine residues , and transcript 3 end sequences amplified using a specific internal oligonucleotide and an oligo(dc ) oligonucleotide .", "( b ) sequences of the 3 end of fourteen psba cdnas yielded five clones with different numbers of thymidine residues .", "the polyuridylylation site is defined to the stretch of thymidine residues encoded by the genomic sequence ( arrows ) .", "( c ) amphidinium rna samples were treated similarly except that atpb specific primers were used in the amplification .", "estimates of the number of different sequences expected for different numbers of random clones sequenced were made by rarefaction analysis of the data in table 1 .", "the estimated number of different cdna sequences expected with smaller sample sizes shows a statistical possibility that the twelve different clones could have been identified with only 120 different sequences .", "the progression of the number of random sequences as a function of the number of different clones ( inset ) suggests over a thousand sequences would be required to identify any potentially new clone in the library .", "all different polyuridylylated 3 terminal sequences corresponding to sequences identified in the library were aligned at the site of the poly(u ) modification and at each of two potential conserved sequences ( underlined ) .", "only transcripts from known minicircle genes are polyuridylylated in lingulodinium various patterns of substitutional editing are found in dinoflagellate plastid transcripts"], "abstract_text": ["<S> dinoflagellate plastid genes are believed to be encoded on small generally unigenic plasmid - like minicircles . </S>", "<S> the minicircle gene complement has reached saturation with an incomplete set of plastid genes ( 18 ) compared with typical functional plastids ( 60200 ) . while some of the missing plastid genes have recently been found in the nucleus , it is still unknown if additional genes , not located on minicircles , might also contribute to the plastid genome . </S>", "<S> sequencing of tailed rna showed that transcripts derived from the known minicircle genes psba and atpb contained a homogenous 3 polyuridine tract of 2540 residues . </S>", "<S> this unusual modification suggested that random sequencing of a poly(da ) primed cdna library could be used to characterize the plastid transcriptome . </S>", "<S> we have recovered only 12 different polyuridylylated transcripts from our library , all of which are encoded on minicircles in several dinoflagellate species . </S>", "<S> the correspondence of all polyuridylylated transcripts with previously described minicircle genes thus supports the dinoflagellate plastid as harbouring the smallest genome of any functional chloroplast . </S>", "<S> interestingly , northern blots indicate that the majority of transcripts are modified , suggesting that polyuridylylation is unlikely to act as a degradation signal as do the heterogeneous poly(a)-rich extensions of transcripts in cyanobacteria and other plastids . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "SUPPLEMENTARY DATA", "Supplementary Material", "Figures and Tables"], "sections": [["although dinoflagellates are best known as the notorious cause of toxic red tides , they are also important contributors to the ocean 's primary production .", "photosynthesis in these organisms is typically carried out in plastids surrounded by three membranes ( 1 ) , an evolutionary footprint reflecting their origin through secondary endosymbiosis ( 2 ) .", "the evolutionary ancestor of the peridinin - containing plastids is suggested from molecular phylogenetic reconstructions using plastid - encoded genes to be a red alga ( 3,4 ) , a conclusion supported by phylogeny of nuclear - encoded plastid - directed genes ( 5 ) . in general , these findings are also consistent with phylogenetic reconstructions of the host cells as determined from non - plastid - directed genes ( 6,7 ) . despite widespread acceptance of a red - algal origin for the peridinin - containing plastid", ", these organelles display a number of peculiar characteristics that share no homology with any known extant plastids .", "for example , the carotenoid peridinin itself ( 8) and the unusual light harvesting peridinin - chlorophyll a - protein to which it binds ( 9 ) are found in no other organisms .", "furthermore , the rubisco in peridinin - containing plastids , an unusual form ii enzyme , is dissimilar from the form i protein employed by all other plastids ( 10 ) .", "the evolutionary provenance of these proteins is thus unknown , and as they are derived from nuclear - encoded genes , it seems possible that their history may be distinct from that the plastid itself .", "an additional issue having bearing on plastid evolution , and one more likely to reflect the plastids themselves , is the number and arrangement of the genes within the genome .", "genome architecture may be different from the phylogeny of the plastid genes themselves , and in the case of the dinoflagellates , quite remarkably so .", "indeed , the only known plastid genes so far identified have been located on generally unigenic plasmid - like minicircles .", "these minicircles have been found in at least five genera of peridinin - containing dinoflagellates including amphidinium ( 11 ) , ceratium ( 12 ) , heterocapsa ( 13 ) , protoceratium ( 14 ) and symbiodinium ( 15 ) .", "each minicircle has regions conserved within a species , and extensive pcr amplification of the genes located between these conserved regions has been performed in several species . taken together , these studies have led to the conclusion that the known minicircle gene complement has reached saturation ( 16 ) with a total of sixteen protein encoding genes ( atpa - b ; petb and petd ; psaa - b ; psba - e and psbi ; ycf16 and ycf24 ; rpl28 and rpl33 ) in addition to the large ( 23s ) and small ( 16s ) ribosomal rna .", "the identification of this highly reduced set of plastid genes as comprising the plastid genome is also supported by recent results demonstrating that at least some of the missing genes ( i.e. ones normally found in plastids ) are instead nuclear - encoded in several species ( 1719 ) .", "these experiments suggest that dinoflagellates do not obey for the rules normally governing plastid gene transfer to the nucleus ( 20 ) . however , it is still a formal possibility that additional genes may be encoded in the plastid in a form different from the minicircle format found so far .", "one method potentially applicable to the characterization of the plastid genome is to determine the spectrum of genes expressed , as gene expression should be independent from the form in which the genes are found .", "however , it is generally difficult to discriminate between organelle- and nuclear - encoded transcripts , as both can be modified by addition of a poly(a ) tail at their 3 termini ( 21 ) .", "we have found that , unlike other transcripts in the dinoflagellates , those encoded by known minicircle genes carry a homogenous polyuridine tract at their 3 termini .", "we have taken advantage of this unusual feature to characterize the dinoflagellate plastid transcriptome , and find that our analysis fully supports a highly reduced plastid genome for the peridinin - containing dinoflagellates . furthermore ,", "as it seems likely that polyuridylylation may be common in dinoflagellate plastids , it may be possible to rapidly characterize the transcriptome of many different species using the method described here ."], ["amphidinium carterae ( ccmp 1314 ) and lingulodinium polyedrum ( ccmp 1936 , formerly gonyaulax polyedra ) were obtained from the provasoli  guillard culture center for marine phytoplancton ( boothbay harbor , maine ) and cultured as described ( 22 ) .", "poly(a ) rna was purified from lingulodinium and amphidinium using oligo(dt ) chromatography ( 23 ) and hybridized to a psba and 23s probes as described ( 22 ) .", "the 16s probe was prepared against an est sequence from our library ( see below ) , while the atpb probe was prepared using previously described amino acid microsequence data ( 22 ) to design two degenerate oligonucleotides 5-ttyticargciggiwsigargt-3 and 5-acytcigciacraaraaiggytg-3 ; the 500 bp pcr product was confirmed as atpb by sequence analysis .", "the 3 end sequences of atpb and psba transcripts were obtained from cdna synthesized from poly(a ) rna tailed in vitro with rgtp by poly(a ) polymerase .", "specific sequences were amplified using a d(c)10 oligonucleotide and 5-tatccaattggacaaggaag-3 ( lingulodinium psba ) 5-gtgtagcacaagatgtaagc-3 ( lingulodinium atpb ) , 5-gtattcggtcaagaggatg-3 ( amphidinium psba ) and 5-ctatctcagccgttctttg-3 ( amphidinium atpb ) .", "the genomic psba sequences from lingulodinium were obtained using tail - pcr ( 24 ) using three nested internal psba oligonucleotides ( 5-gctgcttggccagttattggtatctg-3 ; 5-tctggtttacagcacttggtgttag-3 ; 5-gtccataattgattcatcaggtcatc-3 ) to amplify a fragment from at - rich dna purified by bisbenzimide - cscl gradients . to obtain the 3 end of genomic sequences from amphidinium ,", "minicircle dna was amplified by inverse pcr using outwardly directed oligonucleotides 5-gtattcggtcaagaggatg-3 and 5-caggagcaaggaagaaag-3 ( psba ) or 5-gtggtcgtaagattgagagg-3 and 5-gatgagagcgttggcatac-3 ( atpb ) . for cdna preparation , 10 g poly(a)-enriched rna was used as a template for first strand synthesis using a commercial kit ( stratagene ) but replacing the usual oligo(dt ) primer with 5-(ga)10actagtctcgag(a)18 - 3. sequences were identified using the blast algorithm ( ) and have been deposited in genbank under the accession nos . dq264844 through dq264867 .", "all sequence alignments and analyses were performed using macvector ( accelrys ) except for rna secondary predictions that were made using a web server ( ) . statistical analysis using rarefaction ( 25 ) to determine the likelihood that our sample size was sufficient to detect all different cdnas in the library was made on a web server ( ) ."], ["we began our analysis of the dinoflagellate plastid transcriptome by fractionation of rna into poly(a)-enriched and depleted fractions by oligo(dt ) chromatography .", "the plastid - encoded psba ( 22 ) in rna extracted from the dinoflagellates lingulodinium ( figure 1a ) and amphidinium ( figure 1b ) , was found enriched by 10-fold in poly(a ) fractions , while 23s rrna remained in the poly(a ) fraction .", "the small size of the 23s rrna signal is suggestive of processing and has been previously observed in dinoflagellates ( 13 ) .", "the 16s rna was similarly processed , but the full - length form was found to a greater extent in the polyadenylated fraction than was the 23s rna .", "the abundance of these transcripts in a poly(a ) rich fraction was unexpected , as usually only a small fraction of plastid messages are polyadenylated ( 21 ) .", "furthermore , it seemed at odds with the lack of minicircle genes found in dinoflagellate est libraries ( 1719 ) .", "we originally thought that the tails might be heterogeneous , similar to the 3 termini in chloroplasts and cyanobacteria rna formed by polynucleotide phosphorylase ( pnp ) ( 21 ) , since the presence of nucleotides other than adenine in the 3 tails might inhibit cdna synthesis more than oligo(dt ) chromatography . to test this , we guanylated our poly(a)-enriched rna using poly(a ) polymerase , and performed rt  pcr with a primer pair allowing specific amplification of the psba 3 end ( figure 2a ) .", "fourteen different psba clones were sequenced , and all contained a homogeneous 3 terminal stretch of thymidine residues .", "at least five of the sequences obtained were independent clones based on differences in tail length , which varied between 25 and 40 thymidine residues ( figure 2b ) . a comparison to the genomic dna sequence , obtained by thermal asymmetric interlaced ( tail ) pcr ( 24 ) , shows that these residues are added post - transcriptionally , and all appeared to have been added at the same site ( between arrows , figure 2b ) .", "poly(t ) tracts of similar length were found on the 3 termini of atpb transcripts from lingulodinium ( data not shown ) and on atpb transcripts from the dinoflagellate amphidinium ( figure 2c ) .", "we propose that binding of these polyuridylylated mrnas to the longer and more prevalent poly(a ) tails of nuclear transcripts is responsible for their presence in poly(a)-enriched rna fractions obtained by oligo(dt ) cellulose chromatography .", "the unusual 3 terminal polyuridylylation , if a common feature of dinoflagellate plastid transcripts , suggested that analysis of cdna synthesized using an oligo(da ) instead of the usual oligo(dt ) primer would provide a straightforward method to catalog the plastid gene complement . roughly 60 ng of cdna was synthesized from 10 g poly(a)-enriched rna , a yield 30-fold less than that obtained with an oligo(dt ) primer in similar experiments .", "polyuridylylated transcripts are thus a small but significant proportion of cellular rna . to characterize the library , several hundred clones were selected at random and sequenced .", "some gc - rich and unidentified sequences were found , but none were polyuridylylated and were presumably derived from hairpin priming of the predominant gc - rich nuclear - encoded transcripts .", "in contrast , all polyuridylylated sequences were at - rich and were identified as transcripts from the minicircle genes found in other dinoflagellates ( 16 ) ( table 1 ) .", "the majority of the transcripts correspond to photosystem ii components ( psba - d ) and 16s rna .", "this latter may appear more frequently in our oligo(da)-primed library than the 23s rna because the full - length16s rna appears more abundant in a poly(a ) enriched sample ( figure 1a ) . to assess the likelihood that other low abundance polyuridylylated transcripts might be present in the library , we performed rarefaction analysis ( 25 ) .", "originally developed to compare species richness in biodiversity collections of different sizes , this technique estimates the number of different cdnas that would be obtained if smaller sample sizes were taken .", "the analysis of the data in table 1 indicates that the identification of twelve different sequences could have been possible with only 120 random clones sequenced ; the three hundred clones reported here represent a significant excess of this minimum value .", "the calculations can also be used to illustrate the progression in the number of random sequences required to reveal each additional sequence : while 10 different cdnas are expected in 65 random sequences , almost twice as many sequences are required to uncover the eleventh sequence , and roughly six times more sequences to yield the twelfth cdna ( figure 3 inset ) .", "this analysis suggests that to recover a potential new plastid transcript , well over a thousand random clones would have to be sequenced . to further test the contention the twelve genes recovered are likely to represent saturation coverage of the oligo(da )", ", roughly a thousand different cdna clones were randomly selected and streaked on a new petri plate in a grid configuration .", "colony lifts were then hybridized with a probe prepared against a mixture of psba - d or 16s sequences . from this , 50 cdnas that hybridized weakly or not at were selected and sequenced .", "only 14 sequences among the 50 sequenced were at - rich and polyuridylylated , and these included two atpa , four 23s rna , a petb , three psbb , three psbc and a psbd .", "these later three photosystem ii components may have been recovered following the virtual subtraction because of poor bacterial growth or poor transfer to the membrane .", "more importantly , no new polyuridylylated genes were identified , again suggesting that our coverage of the library had reached saturation . as it seems unlikely that alternative 3 modifications might be found within the same organelle", ", we conclude that the genome of this photosynthetically active chloroplast is likely to encode only the transcripts identified here . to address the mechanism underlying site selection for poly(u ) addition", "secondary structure is the principal determinant of polyadenylation site selection in prokaryotes ( 27 ) .", "however , computer generated secondary structure predictions from rna complementary to the genomic sequence surrounding the polyuridylylation site of either lingulodinium psba or amphidinium atpb did not show any reasonably stable stem  loop structures ( data not shown ) .", "however , two loosely conserved primary structure motifs are located within 50 bp of the modification site in all twelve transcripts ( figure 4 ) .", "these motifs ( agaaa and aauua ) might thus constitute primary structure elements signalling the polyuridylylation site in a manner similar to the use of aauaaa in determining polyadenylation sites in nuclear encoded transcripts ( 28 ) .", "we were also curious about the identity of the enzyme that might be used to catalyze the polyuridylylation reaction .", "as polyuridylylation of protein coding transcripts has been observed in organelles undergoing extensive uridine insertion editing ( 29,30 ) , we thus checked for uridine insertion in comparisons of genomic and cdna sequences using lingulodinium psba , petb and 16s rna ( 2.5 kb total sequence ) .", "our data reveals no evidence for uridine insertion although numerous examples of substitutional editing in dinoflagellate plastid transcripts ( principally a to g ) were observed ( table 2 , supplementary figures s1 and s2 ) .", "these results agree with a previous report of substitutional editing in the dinoflagellate ceratium ( 31 ) ."], ["we have found that the plastid transcripts in two species of peridinin - containing dinoflagellates are characterized by an unusual 3 polyuridylylation .", "this modification differs dramatically from the poly(a ) tails of nuclear - encoded transcripts , and so provides a facile method for cataloging the plastid transcriptome .", "we report here that an oligo(da)-primed cdna library has a remarkably low complexity , with only 12 different clones identified in 300 randomly selected polyuridylylated sequences ( table 1 ) .", "interestingly , all the sequences identified from the library were previously identified as minicircle genes in other dinoflagellate species .", "this concordance between two independent methods ( characterization of the minicircular genome and the transcriptome analysis reported here ) suggests that the minicircular gene format is likely to be the only genome architecture in the plastid .", "our results thus strongly support the contention that the dinoflagellate genome is the smallest of any functional chloroplast . furthermore , since the dinoflagellate amphidinium also contains polyuridylylated transcripts , it is possible that the technique described here could be used to catalog the plastid transcriptome from a range of other species .", "is it likely that the transcriptome contains other low abundance transcripts that were undetected in our relatively small sample size ?", "the chance of finding a specific transcript in a single random sample is a function of its relative proportion , or frequency of occurrence within the bank . however , if many other sequences were present , then the chance of finding any other sequence would also depend on the number of additional sequences present and on their relative proportions within the library . to estimate our coverage of the library , we used rarefaction analysis to determine if smaller sample sizes would also have recovered the same twelve genes .", "our analysis suggests that it is likely the same twelve genes would have been recovered using a smaller sample size , indicating that the number of clones sequence was in excess of that required .", "furthermore , the progression in the number of clones sequenced as a function of the number of different clones identified ( figure 3 ) suggests that well over a thousand random clones would have to be sequenced to find an additional clone if it were indeed present in the library .", "taken together with the virtual subtraction of psba - d and 16s rna sequences , these results strongly suggest that coverage of the library has reached saturation .", "the selective forces that serve to maintain genes in the chloroplast are hotly debated , and thought to reflect either difficulties in targeting or transport of proteins that are extremely hydrophobic or a relationship between control of gene expression and the redox state of the organelle ( 32 ) . with respect to the former , the twelve protein encoding genes found in our library are not the most hydrophobic of known thylakoid proteins . indeed , a better explanation for the retention of this particular gene set in the plastid may lie in the length of the protein rather than hydrophobicity . as recently shown by analysis of arabidopsis thylakoid proteins ( 33 ) ,", "the proteins encoded by the dinoflagellate plastid genes are generally the longest of the thylakoid proteins . however , even a combination of length and hydrophobicity is insufficient to explain transfer of some genes to the nucleus , such as the shorter yet relatively hydrophobic protein encoded by the atpi gene recently reported in an oligo(dt ) primed est bank from the dinoflagellate alexandrium .", "if instead of hydrophobicity , the redox control of gene expression were the determining factor , this would suggest the set of proteins encoded by the dinoflagellate plastids are most sensitive to changes in redox potential .", "the proposal that it is genes requiring editing may that are conserved in plastids ( 34 ) is supported by analysis of the petb gene of lingulodinium , as editing removes a stop codon in the middle of the derived protein sequence ( supplementary figure s2 ) . with respect to the unusual nature of the modification itself", ", there is precedent for polyuridylylation in organelles experiencing extensive editing , such as the mitochondria of trypanosomes or myxomycetes . in some cases , 3 polyuridine tails", "have been observed not only for the guide rnas used to determine the site of uridine insertion but for rrna and mrna as well ( 29,30 ) .", "however , a comparison of 2.5 kb genomic and cdna sequences showed only substitutional editing ( table 2 ) .", "our experiments thus provide the first example of extensive polyuridylylation occurring in the absence of rna editing .", "it is tempting to speculate that the poly(u ) tracts may result from a terminal uridylyltransferase ( tut ) ( 35 ) acting in the absence of guide rna to define a site for uridine insertion .", "however , an alternative possibility is that the plastids may contain a type poly(a ) polymerase with specificity for uridine residues instead of adenine .", "recently , it has also been shown that microrna - directed cleavage can result in addition of non - encoded oligonucleotides ( mostly uridine ) to 3 termini ( 36 ) . however , in this case the polyuridylylated transcripts are intermediates in an rna degradation pathway .", "it seems unlikely that the extensive polyuridylylation of the plastid transcripts is used as a signal for rna turnover , since the majority of psba transcripts appear modified as judged by their co - purification on oligo(dt ) chromatography .", "in contrast to the full - length protein coding transcripts , the small fragments hybridizing to 16s and 23s rna on northern analyses appear unmodified by these criteria ( figure 1 ) .", "interestingly , the apparent stability of the polyuridylylated plastid transcripts thus suggests that this particular 3 end modification has a different function from that in either cyanobacteria or the plastids of higher plants , where transcripts are marked for degradation by 3 end polyadenylation ( 21 ) . despite extensive molecular phylogenetic studies pointing to a common evolutionary origin for both host cells ( 7 ) and plastids ( 37 ) of the chromalveolates , no other plastids or cyanobacteria", "thus the mechanism and function of the dinoflagellate plastid transcript 3 end modification are as unique as their form ii rubisco ( 10 ) and peridinin  chlorophyll a - protein ( 9 ) . the major challenge for plastid phylogeny underscored by our results is to reconcile the many unique features of the dinoflagellate plastids with their phylogenetic relationships to red algae . in addition , given the concurrence of several lines of evidence supporting the highly reduced nature of the dinoflagellate plastid genome , it will be of interest to reinvestigate the nature of the selective forces maintaining the current plastid genome size in higher plants ."], [""], [""], ["dinoflagellate plastid messages are located in poly(a)-enriched rna . total rna samples ( t ) from the dinoflagellates lingulodinium ( a ) and amphidinium ( b ) were resolved into fractions enriched ( a+ ) and depleted ( a ) for poly(a ) rna by chromatography on oligo(dt ) cellulose .", "rna blots were challenged with gene probes for either psba , 23s rna or 16s .", "( a ) lingulodinium rna samples enriched for poly(a ) rna were tailed with guanine residues , and transcript 3 end sequences amplified using a specific internal oligonucleotide and an oligo(dc ) oligonucleotide .", "( b ) sequences of the 3 end of fourteen psba cdnas yielded five clones with different numbers of thymidine residues .", "the polyuridylylation site is defined to the stretch of thymidine residues encoded by the genomic sequence ( arrows ) .", "( c ) amphidinium rna samples were treated similarly except that atpb specific primers were used in the amplification .", "estimates of the number of different sequences expected for different numbers of random clones sequenced were made by rarefaction analysis of the data in table 1 .", "the estimated number of different cdna sequences expected with smaller sample sizes shows a statistical possibility that the twelve different clones could have been identified with only 120 different sequences .", "the progression of the number of random sequences as a function of the number of different clones ( inset ) suggests over a thousand sequences would be required to identify any potentially new clone in the library .", "all different polyuridylylated 3 terminal sequences corresponding to sequences identified in the library were aligned at the site of the poly(u ) modification and at each of two potential conserved sequences ( underlined ) .", "only transcripts from known minicircle genes are polyuridylylated in lingulodinium various patterns of substitutional editing are found in dinoflagellate plastid transcripts"]]}
{"article_id": "PMC3519406", "article_text": ["several prospective , randomized , controlled clinical trials regarding cervical artificial disc replacement ( c - adr ) have been published . however , the number of c - adr patients with long - term follow - up for more than 4 years is still sparse .", "the purpose of this systematic review is to provide a summary of the available literature reporting long - term follow - up of c - adr and to elucidate whether the favorable outcomes seen in the short - term continue after 4 to 5 years .", "does single - level unconstrained , semiconstrained , or fully constrained c - adr improve health outcomes compared with single - level acdf in the long - term ?", "search : pubmed , cochrane collaboration database , and national guideline clearinghouse databases ; bibliographies of key articles .", "inclusion criteria : ( 1 ) fda trials comparing c - adr with anterior cervical discectomy and fusion ( acdf ) ; ( 2 ) follow - up  4 years .", "exclusion criteria : ( 1 ) non - fda trials comparing c - adr with acdf ; ( 2 ) follow - up < 4 years . outcomes : neck disability index ( ndi ) , pain in the neck and arm ( visual analog scale [ vas ] ) , quality of life ( sf-36 physical component score [ pcs ] ) , adjacent segment disease ( asd ) , neurological success , subsequent surgeries , and complications .", "study design : systematic review . search : pubmed , cochrane collaboration database , and national guideline clearinghouse databases ; bibliographies of key articles .", "inclusion criteria : ( 1 ) fda trials comparing c - adr with anterior cervical discectomy and fusion ( acdf ) ; ( 2 ) follow - up  4 years .", "exclusion criteria : ( 1 ) non - fda trials comparing c - adr with acdf ; ( 2 ) follow - up < 4 years . outcomes : neck disability index ( ndi ) , pain in the neck and arm ( visual analog scale [ vas ] ) , quality of life ( sf-36 physical component score [ pcs ] ) , adjacent segment disease ( asd ) , neurological success , subsequent surgeries , and complications .", "two randomized , multicenter fda trials comparing outcomes following c - adr and acdf with follow - up > 48 months were found ( fig .", "1 ) . inclusion and exclusion criteria and demographic information for each study are listed in table 1 .", "overall , a total of 1004 adult patients ( 47% male ) with a mean age of 44 years were included .", "all patients were diagnosed with single - level degenerative disc disease between c3 and c7 and had failed a minimum of 6 weeks conservative treatment .", "function , pain , and health - related quality of life ( table 2 ) at 48 months in the bryan trial , patients in the c - adr group showed significantly greater mean improvement in ndi , vas neck and arm pain , and sf-36 pcs measured at 48 months compared with patients in the acdf group . at 60 months in the prestige trial , only mean improvement in ndi scores was significantly greater in the c - adr group ( 38.4 vs 34.1 , p  =  .022 ) ; however , for the remaining three outcomes , mean improvements were slighter greater following c - adr versus fusion .", "success , asd , and return to work ( table 3 ) at 48 months in the bryan trial , overall success and ndi success , were achieved in a significantly greater proportion of c - adr patients compared with acdf patients ( p  =  .004 and .003 , respectively ) . at both 48 months in the bryan trial and 60 months in the prestige trial ,", "more patients achieved overall neurological success ( maintenance or improvement ) and were working following c - adr compared with acdf , although these differences were not significant .", "the rate of asd at 48 months in the bryan trial was identical between groups ( 4.1% ) ; at 60 months in the prestige trial the rate was 2.9% in the c - adr group versus 4.9% in the acdf group , however these differences were not statistically significant . both studies reported preserved segmental range of motion in the cervical spine following c - adr compared with acdf : 8.5 versus 1.1 ( 48 months , bryan ) and 6.5 versus 0.4 ( 60 months , prestige ) .", "subsequent operations ( table 4 ) no significant differences between groups were reported for rates of revisions , hardware removal , supplemental fixation , use of bone growth stimulators , or reoperation at 48 months postoperatively in the bryan trial . at 60 months , a significant difference was seen between the prestige c - adr and acdf groups , respectively , in revisions ( 0% vs 1.9% ; p  =  .028 ) , supplemental fixation ( 0% vs 1.9% ; p  =  .028 ) , and the use of external bone growth stimulator ( 0% vs 2.6% ; p  =  .007 ) .", "the bryan study only reported more severe who grade 3 or 4 events that occurred after 24 months and up to 48 months follow - up in the c - adr and acdf groups , respectively : any , 24.3% vs 26.1% ; severe arm and neck pain , 1.7% vs 3.6% ; and new neurological deficits , 0% vs 1.4% . in patients with complete radiographic follow - up at 60 months in the prestige trial , subsidence ( loss of > than 2 mm in functional spinal unit height )", "was seen in 2.8% and 1.4% of patients in the c - adr and acdf groups , respectively ; bridging bone was reported in 3.2% of the c - adr patients .", "one guideline was found , published by the north american spine society ( nass ) in 2010 , entitled  diagnosis and treatment of cervical radiculopathy from degenerative disorders .  among the major recommendations listed were the following statements relevant to the topic of this review :  acdf and total disc arthroplasty ( tda ) are suggested as comparable treatments , resulting in similarly successful short term outcomes , for single level degenerative cervical radiculopathy .  ( grade : b ; fair evidence  level ii or iii studies )  surgery is an option for the treatment of single level degenerative cervical radiculopathy to produce and maintain favourable long term ( > 4 years ) outcomes . ", "( grade c ; poor quality evidence  level iv or v studies )   results of literature search .", "adults > 18 years of age single - level symptomatic ddd between c3 - 7 intractable radiculopathy , myelopathy or both vas neck pain scores  20 preserved motion at the symptomatic level found in all included patients unresponsive to  6 weeks conservative treatment or progressive neurological worsening despite conservative treatment no previous procedures at the operative level negative for several radiographic findings , medications , and diagnoses multilevel symptomatic ddd or evidence of cervical instability sagittal plane translation of > 3.5 mm or sagittal plane angulation of > 20 at a single level symptomatic c2-c3 or c7-t1 disc disease previous surgery at the involved level severe facet joint disease at the involved level medical condition that required long - term use of medication , such as steroid or nonsteroidal antiinflammatory drugs that could affect bone quality and fusion rates ddd at single level between c3 and c7 disc herniation with radiculopathy , spondylotic radiculopathy , disc herniation with myelopathy , or spondylotic myelopathy 6 weeks minimum unsuccessful conservative unless myelopathy requiring immediate treatment ct , myelography and ct , and/or mri demonstration of need for surgical treatment preoperative ndi  30 and minimum one clinical sign associated with level to be treated willing to sign informed consent and comply with protocol significant cervical anatomical deformity moderate to advanced spondylosis any combination of bridging osteophytes , marked reduction , or absence of motion collapse of intervertebral disc space of > 50% normal height , radiographic signs of subluxation > 3.5 mm , angulation of disc space > 11 greater than adjacent segments , significant kyphotic deformity or reversal or lordosis axial neck pain as solitary symptom previous cervical spine surgery metabolic bone disease active systemic infection or infection at operative site known allergy to components of titanium , polyurethane , ethylene oxide residuals concomitant conditions requiring steroid treatment daily insulin management medical condition which may interfere with postoperative management program or may result in death before study completion current or recent alcohol and/or drug abuser signs of being geographically unstable fda indicates us food and drug administration ; ddd , degenerative disc disease ; ndi , neck disability index ; vas , visual analog scale ; ct , computed tomography ; and mri , magnetic resonance scan .", "c - adr indicates cervical artificial disc replacement ; fda , us food and drug administration ; ndi , neck disability index ; sf-36 , short - form 36 questionnaire ; and pcs , physical component score .", "p values compare the mean improvement in scores from baseline to each follow - up time - point between c - adr and fusion .", "asd indicates adjacent segment disease ; c - adr , cervical artificial disc replacement ; fda , us food and drug administration ; ndi , neck disability index ; and ns , not statistically significant .", "composite measure in which patients had to achieve all the following : an improvement of 15 points on ndi , neurological improvement , no serious ( who grade 3 or 4 ) adverse events related to the implant or surgical implantation procedure , and no subsequent surgery or intervention that would be classified as treatment failure .", "improvement of 15 points in ndi from baseline . defined as maintenance or improvement of all three neurological parameters ( motor and sensory function , and reflexes ) .", "c - adr indicates cervical artificial disc replacement ; fda , us food and drug administration ; and ns , not statistically significant .", "a 43-year - old woman presented with cervical myelo - radiculopathy that did not respond to medical treatment for 6 weeks .", "the magnetic resonance images demonstrated a large herniated disc at the level of c5/6 , eccentric to the right side ( fig .", "her symptoms improved significantly after surgery and x - rays taken 2 years postoperatively demonstrated very good range of motion at the index level ( fig .", "her vas arm pain score improved from a preoperative score of 8 to a postoperative score of 1 at 2-year follow - up .  ", "preoperative axial ( a ) and sagittal ( b ) magnetic resonance images of a 43-year - old woman with myeloradiculopathy due to a c5/6 disc herniation .  ", "postoperative flexion ( a ) and extension ( b ) x - rays of the patient 24 months after surgery .", "limitations : a small number of studies with long - term data comparing c - adr with cervical acdf were available . in both prospective studies ,", "the c - adr cohort maintained statistical improvement in validated clinical outcome measurements at 48 and 60 months and preserved segmental motion at the operated level .", "rates of revision and supplemental fixation surgeries were lower in the c - adr group .", "these studies demonstrate the durability of the c - adr procedure ; however , it may still be too early to detect implant - related failures .", "future studies should examine issues such as wear - related failures , device fatigue , or delayed spinal instability .", "the follow - up rates were 68.7% ( 318/463 ) in the bryan disc study at 4 years and 50.1% ( 271/541 ) in the prestige st study at 5 years .", "these high rates of lost to follow - up may alter the study results if those patients lost to follow - up had late - onset clinical or radiographic issues .", "significantly greater mean improvement from baseline in all outcomes in the bryan c - adr group compared with the acdf group at 48 months .", "only the ndi showed greater mean improvement from baseline in the prestige c - adr group compared with the acdf group at 60 months .", "overall success and ndi success , which were only reported by the bryan trial , were achieved in a significantly greater proportion of c - adr patients compared with acdf patients at 48 months . at both 48 ( bryan ) and 60 months ( prestige )", "the rates of asd at 48 ( bryan ) and 60 months ( prestige ) were not statistically different between treatment groups . at both 48 ( bryan ) and 60 months ( prestige ) , more patients were working following c - adr compared with acdf . significantly fewer revisions , supplemental fixations , and use of external bone growth stimulators were reported in the prestige c - adr compared with the acdf group at 60 months .", "rates of who grade 3 or 4 adverse events were similar between groups at 48 months in the bryan trial .", "cervical arthroplasty is a viable treatment option for cervical herniated disc / spondylosis with radiculopathy . the inclusion / exclusion criteria of us fda trials should be followed .", "c - adr achieves neural decompression and preserves normal segmental motion at the operated level at 4 to 5 years follow - up .", "adjacent level degeneration may be decreased with arthroplasty versus acdf , but further study is warranted on this topic ."], "abstract_text": ["<S> study design :  systematic review.clinical question :  does single - level unconstrained , semiconstrained , or fully constrained cervical artificial disc replacement ( c - adr ) improve health outcomes compared with single - level anterior cervical discectomy and fusion ( acdf ) in the long - term?methods :  a systematic review was undertaken for articles published up to october 2011 . electronic databases and reference lists of key articles were searched to identify us food and drug administration ( fda ) studies reporting long - term (  48 months ) follow - up results of c - adr compared with acdf . </S>", "<S> non - fda trials and fda trials reporting outcomes at short - term or mid - term follow - up periods were excluded . </S>", "<S> two independent reviewers assessed the strength of evidence using the grade criteria and disagreements were resolved by consensus.results :  two fda trials reporting outcomes following c - adr ( bryan disc , prestige disc ) versus acdf at follow - up periods of 48 months and 60 months were found ( follow - up rates are 68.7% [ 318/463 ] and 50.1% [ 271/541 ] , respectively ) . </S>", "<S> patients in the c - adr group showed a higher rate of overall success , greater improvements in neck disability index , neck and arm pain scores , and sf-36 physicalcomponent scores at long - term follow - up compared with those in the acdf group . </S>", "<S> the rate of adjacent segment disease was less in the c - adr group versus the acdf group at 60 months ( 2.9% vs 4.9% ) . </S>", "<S> normal segmental motion was maintained in the c - adr group . </S>", "<S> furthermore , rates of revision and supplemental fixation surgical procedures were lower in the arthroplasty group.conclusions :  c - adr is a viable treatment option for cervical herniated disc / spondylosis with radiculopathy resulting in improved clinical outcomes , maintenance of normal segmental motion , and low rates of subsequent surgical procedures at 4 to 5 years follow - up . more studies with long - term follow - up are warranted . </S>"], "labels": null, "section_names": ["Study Rationale and Context", "Clinical Question", "Materials and Methods", "Results", "Case Study", "Discussion (", "Evidence Summary", "Summary and Conclusions"], "sections": [["several prospective , randomized , controlled clinical trials regarding cervical artificial disc replacement ( c - adr ) have been published . however , the number of c - adr patients with long - term follow - up for more than 4 years is still sparse .", "the purpose of this systematic review is to provide a summary of the available literature reporting long - term follow - up of c - adr and to elucidate whether the favorable outcomes seen in the short - term continue after 4 to 5 years ."], ["does single - level unconstrained , semiconstrained , or fully constrained c - adr improve health outcomes compared with single - level acdf in the long - term ?", "search : pubmed , cochrane collaboration database , and national guideline clearinghouse databases ; bibliographies of key articles .", "inclusion criteria : ( 1 ) fda trials comparing c - adr with anterior cervical discectomy and fusion ( acdf ) ; ( 2 ) follow - up  4 years .", "exclusion criteria : ( 1 ) non - fda trials comparing c - adr with acdf ; ( 2 ) follow - up < 4 years . outcomes : neck disability index ( ndi ) , pain in the neck and arm ( visual analog scale [ vas ] ) , quality of life ( sf-36 physical component score [ pcs ] ) , adjacent segment disease ( asd ) , neurological success , subsequent surgeries , and complications ."], ["study design : systematic review . search : pubmed , cochrane collaboration database , and national guideline clearinghouse databases ; bibliographies of key articles .", "inclusion criteria : ( 1 ) fda trials comparing c - adr with anterior cervical discectomy and fusion ( acdf ) ; ( 2 ) follow - up  4 years .", "exclusion criteria : ( 1 ) non - fda trials comparing c - adr with acdf ; ( 2 ) follow - up < 4 years . outcomes : neck disability index ( ndi ) , pain in the neck and arm ( visual analog scale [ vas ] ) , quality of life ( sf-36 physical component score [ pcs ] ) , adjacent segment disease ( asd ) , neurological success , subsequent surgeries , and complications ."], ["two randomized , multicenter fda trials comparing outcomes following c - adr and acdf with follow - up > 48 months were found ( fig .", "1 ) . inclusion and exclusion criteria and demographic information for each study are listed in table 1 .", "overall , a total of 1004 adult patients ( 47% male ) with a mean age of 44 years were included .", "all patients were diagnosed with single - level degenerative disc disease between c3 and c7 and had failed a minimum of 6 weeks conservative treatment .", "function , pain , and health - related quality of life ( table 2 ) at 48 months in the bryan trial , patients in the c - adr group showed significantly greater mean improvement in ndi , vas neck and arm pain , and sf-36 pcs measured at 48 months compared with patients in the acdf group . at 60 months in the prestige trial , only mean improvement in ndi scores was significantly greater in the c - adr group ( 38.4 vs 34.1 , p  =  .022 ) ; however , for the remaining three outcomes , mean improvements were slighter greater following c - adr versus fusion .", "success , asd , and return to work ( table 3 ) at 48 months in the bryan trial , overall success and ndi success , were achieved in a significantly greater proportion of c - adr patients compared with acdf patients ( p  =  .004 and .003 , respectively ) . at both 48 months in the bryan trial and 60 months in the prestige trial ,", "more patients achieved overall neurological success ( maintenance or improvement ) and were working following c - adr compared with acdf , although these differences were not significant .", "the rate of asd at 48 months in the bryan trial was identical between groups ( 4.1% ) ; at 60 months in the prestige trial the rate was 2.9% in the c - adr group versus 4.9% in the acdf group , however these differences were not statistically significant . both studies reported preserved segmental range of motion in the cervical spine following c - adr compared with acdf : 8.5 versus 1.1 ( 48 months , bryan ) and 6.5 versus 0.4 ( 60 months , prestige ) .", "subsequent operations ( table 4 ) no significant differences between groups were reported for rates of revisions , hardware removal , supplemental fixation , use of bone growth stimulators , or reoperation at 48 months postoperatively in the bryan trial . at 60 months , a significant difference was seen between the prestige c - adr and acdf groups , respectively , in revisions ( 0% vs 1.9% ; p  =  .028 ) , supplemental fixation ( 0% vs 1.9% ; p  =  .028 ) , and the use of external bone growth stimulator ( 0% vs 2.6% ; p  =  .007 ) .", "the bryan study only reported more severe who grade 3 or 4 events that occurred after 24 months and up to 48 months follow - up in the c - adr and acdf groups , respectively : any , 24.3% vs 26.1% ; severe arm and neck pain , 1.7% vs 3.6% ; and new neurological deficits , 0% vs 1.4% . in patients with complete radiographic follow - up at 60 months in the prestige trial , subsidence ( loss of > than 2 mm in functional spinal unit height )", "was seen in 2.8% and 1.4% of patients in the c - adr and acdf groups , respectively ; bridging bone was reported in 3.2% of the c - adr patients .", "one guideline was found , published by the north american spine society ( nass ) in 2010 , entitled  diagnosis and treatment of cervical radiculopathy from degenerative disorders .  among the major recommendations listed were the following statements relevant to the topic of this review :  acdf and total disc arthroplasty ( tda ) are suggested as comparable treatments , resulting in similarly successful short term outcomes , for single level degenerative cervical radiculopathy .  ( grade : b ; fair evidence  level ii or iii studies )  surgery is an option for the treatment of single level degenerative cervical radiculopathy to produce and maintain favourable long term ( > 4 years ) outcomes . ", "( grade c ; poor quality evidence  level iv or v studies )   results of literature search .", "adults > 18 years of age single - level symptomatic ddd between c3 - 7 intractable radiculopathy , myelopathy or both vas neck pain scores  20 preserved motion at the symptomatic level found in all included patients unresponsive to  6 weeks conservative treatment or progressive neurological worsening despite conservative treatment no previous procedures at the operative level negative for several radiographic findings , medications , and diagnoses multilevel symptomatic ddd or evidence of cervical instability sagittal plane translation of > 3.5 mm or sagittal plane angulation of > 20 at a single level symptomatic c2-c3 or c7-t1 disc disease previous surgery at the involved level severe facet joint disease at the involved level medical condition that required long - term use of medication , such as steroid or nonsteroidal antiinflammatory drugs that could affect bone quality and fusion rates ddd at single level between c3 and c7 disc herniation with radiculopathy , spondylotic radiculopathy , disc herniation with myelopathy , or spondylotic myelopathy 6 weeks minimum unsuccessful conservative unless myelopathy requiring immediate treatment ct , myelography and ct , and/or mri demonstration of need for surgical treatment preoperative ndi  30 and minimum one clinical sign associated with level to be treated willing to sign informed consent and comply with protocol significant cervical anatomical deformity moderate to advanced spondylosis any combination of bridging osteophytes , marked reduction , or absence of motion collapse of intervertebral disc space of > 50% normal height , radiographic signs of subluxation > 3.5 mm , angulation of disc space > 11 greater than adjacent segments , significant kyphotic deformity or reversal or lordosis axial neck pain as solitary symptom previous cervical spine surgery metabolic bone disease active systemic infection or infection at operative site known allergy to components of titanium , polyurethane , ethylene oxide residuals concomitant conditions requiring steroid treatment daily insulin management medical condition which may interfere with postoperative management program or may result in death before study completion current or recent alcohol and/or drug abuser signs of being geographically unstable fda indicates us food and drug administration ; ddd , degenerative disc disease ; ndi , neck disability index ; vas , visual analog scale ; ct , computed tomography ; and mri , magnetic resonance scan .", "c - adr indicates cervical artificial disc replacement ; fda , us food and drug administration ; ndi , neck disability index ; sf-36 , short - form 36 questionnaire ; and pcs , physical component score .", "p values compare the mean improvement in scores from baseline to each follow - up time - point between c - adr and fusion .", "asd indicates adjacent segment disease ; c - adr , cervical artificial disc replacement ; fda , us food and drug administration ; ndi , neck disability index ; and ns , not statistically significant .", "composite measure in which patients had to achieve all the following : an improvement of 15 points on ndi , neurological improvement , no serious ( who grade 3 or 4 ) adverse events related to the implant or surgical implantation procedure , and no subsequent surgery or intervention that would be classified as treatment failure .", "improvement of 15 points in ndi from baseline . defined as maintenance or improvement of all three neurological parameters ( motor and sensory function , and reflexes ) .", "c - adr indicates cervical artificial disc replacement ; fda , us food and drug administration ; and ns , not statistically significant ."], ["a 43-year - old woman presented with cervical myelo - radiculopathy that did not respond to medical treatment for 6 weeks .", "the magnetic resonance images demonstrated a large herniated disc at the level of c5/6 , eccentric to the right side ( fig .", "her symptoms improved significantly after surgery and x - rays taken 2 years postoperatively demonstrated very good range of motion at the index level ( fig .", "her vas arm pain score improved from a preoperative score of 8 to a postoperative score of 1 at 2-year follow - up .  ", "preoperative axial ( a ) and sagittal ( b ) magnetic resonance images of a 43-year - old woman with myeloradiculopathy due to a c5/6 disc herniation .  ", "postoperative flexion ( a ) and extension ( b ) x - rays of the patient 24 months after surgery ."], ["limitations : a small number of studies with long - term data comparing c - adr with cervical acdf were available . in both prospective studies ,", "the c - adr cohort maintained statistical improvement in validated clinical outcome measurements at 48 and 60 months and preserved segmental motion at the operated level .", "rates of revision and supplemental fixation surgeries were lower in the c - adr group .", "these studies demonstrate the durability of the c - adr procedure ; however , it may still be too early to detect implant - related failures .", "future studies should examine issues such as wear - related failures , device fatigue , or delayed spinal instability .", "the follow - up rates were 68.7% ( 318/463 ) in the bryan disc study at 4 years and 50.1% ( 271/541 ) in the prestige st study at 5 years .", "these high rates of lost to follow - up may alter the study results if those patients lost to follow - up had late - onset clinical or radiographic issues ."], ["significantly greater mean improvement from baseline in all outcomes in the bryan c - adr group compared with the acdf group at 48 months .", "only the ndi showed greater mean improvement from baseline in the prestige c - adr group compared with the acdf group at 60 months .", "overall success and ndi success , which were only reported by the bryan trial , were achieved in a significantly greater proportion of c - adr patients compared with acdf patients at 48 months . at both 48 ( bryan ) and 60 months ( prestige )", "the rates of asd at 48 ( bryan ) and 60 months ( prestige ) were not statistically different between treatment groups . at both 48 ( bryan ) and 60 months ( prestige ) , more patients were working following c - adr compared with acdf . significantly fewer revisions , supplemental fixations , and use of external bone growth stimulators were reported in the prestige c - adr compared with the acdf group at 60 months .", "rates of who grade 3 or 4 adverse events were similar between groups at 48 months in the bryan trial ."], ["cervical arthroplasty is a viable treatment option for cervical herniated disc / spondylosis with radiculopathy . the inclusion / exclusion criteria of us fda trials should be followed .", "c - adr achieves neural decompression and preserves normal segmental motion at the operated level at 4 to 5 years follow - up .", "adjacent level degeneration may be decreased with arthroplasty versus acdf , but further study is warranted on this topic ."]]}
{"article_id": "PMC4897427", "article_text": ["zeolite is a natural porous mineral described as hydrated aluminosilicates containing exchangeable alkali and earth alkaline cations ( na , k , ca , or mg ) and could be synthesised from the aluminium silicate materials as clay minerals and pumice by alkali attack [ 13 ] .", "zeolites have a large surface area , microporous structure , and high affinity for metal ions , providing an efficient scavenging pathway for heavy metals in toxic systems [ 4 , 5 ] .", "some of the attractive applications of natural zeolites are listed by taffarel and rubio   and babel and kurniawan .", "most of these research efforts are undertaken on laboratory scale and are at early stages of investigations to develop the synthesis methods .", "synthetic zeolites are carried out in many industrialized countries in europe , east asia , and north america .", "na - p1 zeolite is one of the synthetic zeolites which are a class of highly porous materials", ". the unique structural features of these crystalline microporous solids that contain pores and cavities in the order of molecular dimensions ( 310   ) are the main reason for their application in the realms of catalysis , separation , purification , ion exchange , and radioactive waste clean - up .", "more novel applications for na - p1 zeolites are expected in electrochemistry , photochemistry , pharmaceutical , engineering , membrane science , and nanotechnology as their structures are expanded and more suitably engineered in a foreseeable future .", "it is well established that these materials that are equivalently called molecular sieves are mainly hydrated aluminosilicates .", "zeolite na - p1 contains two - dimensional pore system with two intersecting 8-ring channels .", "a synthetic zeolite na - p1 has similar pore structure to the natural zeolite phillipsite .", "na - p1 zeolite is classified as high silica zeolites where it has si / al ratio equal to or greater than about 3 .", "it is considered as a hydrated sodium aluminosilicate zeolite that could be synthesised from the naturally occurring alkaline volcano with naoh attack of varying concentrations at different volcanic temperatures .", "pumice is a natural rock of volcanic origin , formed by gases released during the solidification of acidic lava . the vesicular structure of pumice is formed during the eruption of gases contained in the molten viscous lava on cooling . in egypt ,", "pumice occasionally occurs in few locations , at the northern coast of the mediterranean sea at el - arish , north sinai .", "the heavy metals contamination of water by the discharge of industrial wastewater of electroplating , metallurgy , chemical manufacturing , mining , and battery manufacturing is a worldwide environmental problem .", "adsorption process provides an attractive alternative treatment to other removal techniques because it is more economical and readily available [ 1012 ] .", "a good number of adsorbents have been used for cd(ii ) , fe(ii ) , cu(ii ) , and co(ii ) removal from water .", "the adsorption of cd(ii ) on red mud fitted langmuir isotherm model having a maximum adsorption capacity of 10.57  mg  g .", "wang   used maize cobs for adsorption of fe(iii ) from aqueous solution with the langmuir adsorption capacity and the freundlich adsorption capacity being 2.53  mg  g and 0.104  l  g , respectively . the maximum adsorption capacity for cu(ii ) on red mud", "gupta et al .   used spirogyra species for removal of cu(ii ) with the maximum adsorption capacity of 133.3  mg  g. use of adsorbents like mg pellets has also been reported for co(ii ) removal from water   and the adsorption capacity has been found to be 15.8  mg  g. the aim of this work is to study the best condition of temperature , sodium hydroxide concentration , and time required for synthesis of na - p1 zeolite from the local pumice raw material and utilize the synthetic zeolite as an adsorbent for removing ( cd , fe , cu , and co ) ions from synthetic solutions in a batch laboratory system .", "also , the effect of experimental conditions such as contact time and initial zeolite dose was described .", "the main material used in this work is pumice grains received from el - nile mining company from the mediterranean sea coast at el - arish province , north sinai , egypt .", "in addition , commercial naoh is used for alkaline hydrothermal attack for synthesis of zeolite from pumice .", "hydrated chloride salts of cu , co , fe , and cd of analytical grade ( ar ) supplied by merck as analytical - grade reagents and deionized water are used .", "all adsorption experiments were carried out using an aqueous solution of cucl22h2o , cocl26h2o , fecl2 , and cdcl2h2o .", "the stock solution was prepared by dissolving accurately weighted salt in distilled water to the concentration of 500  mg / l .", "the initial concentrations of solution were done by diluting the stock solutions in appropriate proportions to different initial concentrations .", "na - p1 has been prepared from pumice , which is naturally occurring alkaline volcanic rock with naoh attack .", "a series of experiments were conducted to synthesis na - p1 zeolite from locally produced pumice raw material at temperatures ( 80 to 120c ) , naoh solution concentrations ( 13 molar ) , and crystallization periods ( 12  h , one week , and up to one month .", "pumice powder was mixed with naoh solution in a well - sealed teflon cylindrical vessel , heated , and stirred for 12 hours at 80c , and then the vessel was kept in the dryer at 8090c for one week .", "the produced gel was filtered and washed well till becoming free from sodium hydroxide ; the wet powder was dried for 24  h in the dryer at 110c .", "the optimum conditions required for synthesis of na - p1 zeolite from locally produced pumice raw material are one molar naoh concentration , temperature at 80c , and one week as a crystallization time .", "several investigations for the adsorption of the heavy metal were carried out , including effect of contact zeolite dose , effect of time , and effect of metals concentration .", "the physical and chemical characteristics of prepared adsorbents such as slurry ph , point of zero charge ( phpzc ) , bulk density , and moisture were performed to determine the capability of prepared adsorbents in removing the metals co , cu , fe , and cd from an aqueous media . for ph determination , 0.2  g of the dry , grinded adsorbent was mixed with 25  ml of distilled water and allowed to boil for 30  min in stoppered glass bottle . after cooling ,", "the ph of the adsorbent was measured using a digital ph meter ( hanna , model ph20 ) , allowing 5  min for the ph probe to equilibrate .", "also , the point of zero charge ( pzc ) for the samples was determined by the following procedure : 200  ml of deionized water was added to an erlenmeyer flask , which was then capped with stoppered glass .", "the deionized water was heated until boiling for 20  min to eliminate the co2 dissolved in the water .", "the co2-free water was cooled down as soon as possible and the flask was immediately capped . on the other hand ,", "0.5  g of each sample was weighed and placed in a 25  ml erlenmeyer flask to which 20  ml of co2-free water was added .", "the flask was sealed with a rubber stopper and left in continuous agitation for 72  h at 25c .", "then the solution ph was measured and this value is the point of zero charge .", "the bulk density was estimated by a standard procedure by weighing a known volume of gently tapped adsorbent granules .", "the apparent density was calculated from the volume of the graduated cylinder closely packed with the powdered sample and from the sample weight in triplicate . for moisture measurement , about 0.5 ", "g of powdered air - dried adsorbent sample is weighed and taken in a crucible .", "the crucible is placed inside an electric hot - air oven and heated at 150c for 3 hours .", "it is then taken out , cooled in a desiccator , and weighed . from this , the percentage of moisture can be calculated as follows : percentage of moisture = ( loss in weight of adsorbent / weight of air - dried adsorbent taken )  100 .", "surface characteristics of prepared zeolite sample were characterized by scanning electron microscope ( sem ) using sem model philips xl 30 attached with edx unit , with accelerating voltage 30  k.v . ,", "magnification 10x up to 400.000x , and resolution for w. ( 3.5  nm ) .", "x - ray powder diffractograms , xrd , of the samples were recorded using a bruker diffractometer ( bruker d8 advance target ) and the scan rate was fixed at 8 in 2  min for phase identification .", "the patterns were run with cu k and secondly with monochromator (  = 0.1545  nm ) at 40  kv and 40  ma .", "mineralogical and chemical study of the pumice were done by using x - ray fluorescence spectrometer ( xrf ) to identify the chemical composition .", "xrf , a spectrometer with wavelength dispersion pw 2400 philips , and centrifuge hermle were used .", "xrf shows that quartz , cristobalite , and albite are the main constituents in the pumice ( figure 2 ) .", "sio2 , al2o3 , and na2o are the main oxides in the pumice under investigation ( 93.69% ) confirming the mineralogical constituents , whereas tio2 , mno , mgo , fe2o3 , and cao analyzed in the pumice under are calculated as 3.88% ( table 1 ) .", "the concentrations of heavy metals in all samples were determined according to apha using atomic absorption spectrometer unicam model 939 with graphite furnace accessory , equipped with deuterium arc background corrector .", "precision of the metal measurement was determined by analyzing ( in triplicate ) the metal concentration of all samples and for each series of measurements an absorption calibration curve was constructed , composed of a blank and three or more standards . accuracy and precision of the metals measurement were confirmed using external reference standards from merck .", "the adsorption isotherm shows the equilibrium relationship of concentration in the adsorbate - adsorbent system at constant temperature .", "such adsorption isotherms may be used for scaling up batch type processes with moderate success .", "the shape of the equilibrium adsorption isotherm provides us with information about the adsorbent surface whether it is homogeneous or heterogeneous .", "also , its study is helpful in evaluating the maximum adsorption capacity of adsorbate for the given adsorbent . for adsorption isotherms models", "they differ in their assumption , shape of the isotherm , and nature of the adsorbent surface .", "langmuir ( 1918 ) model assumes that uptake of metal ion occurs on a homogeneous surface by monolayer adsorption without any interaction between adsorbed ions .", "the model is also based on the assumption that all the sorption sites are energetically identical and sorption occurs on a structurally homogeneous sorbent .", "thus , a linear plot of ce / qe versus ce confirms the validity of the langmuir giving correlation coefficients ( r ) close to unity , and kl is a constant related to the free energy of adsorption as expressed ( kle ) .", "deviations from the basic assumption of the langmuir model do limit interpretation of the values for qm and kl in terms of absolute surface areas and sorption free energies . the free energy of adsorption , g , can also be evaluated from the parameter kl according to the expression g = rtlnkl .", "values of qm and kl are obtained from slope and intercept of ce / qe versus ce plot , respectively .", "a further analysis of the langmuir equation can be made on the basis of a dimensionless equilibrium parameter , rl ( known as the separation factor which is considered as a more reliable indicator of adsorptions ) .", "this parameter ( rl ) can be expressed by weber and chakkravorti ( 1974 ) : \n ( 2)rl=11+bci , \n where ci is the initial concentration metal ion ( mg / l ) and b ( l  mg ) is the langmuir constant described above . also , the influence of the isotherm shape on whether the adsorption is ", "unfavorable  at highest initial concentration of the dye , co , can be described by a term  rl  , a dimensionless constant separation factor in equation ; calculated value for rl indicates the nature of adsorption process as given : irreversible ( rl = 0 ) , favorable ( 0 < rl < 1 ) , linear ( rl = 1 ) , and favorable ( rl > 1 ) .", "freundlich isotherm is an empirical equation that can be described as the reversible adsorption onto heterogeneous surface at sites with different energy of adsorption and is not restricted to the formation of the monolayer of adsorbate .", "the nonlinear form of this model is expressed as \n ( 3)qe = kfce1/n , \n where kf is the freundlich constant ( mg / g)/(l / mg)1/n and also referred to as adsorption capacity , while n is the heterogeneity factor and related to adsorption intensity .", "the parameter n gives an indication of the adsorption type whether is linear ( n = 1 ) or a physical process ( n > 1 ) is favorable , or a chemical process ( n < 1 ) . on the other hand", ", the value of 1/n < 1 or 1/n > 1 indicates a normal langmuir isotherm and cooperative adsorption , respectively .", "freundlich model can be represented by linear form as follows : \n ( 4)lnqe = lnkf+1nlnce . a plot of lnqe versus lnce gives a straight line , where the values of kf and 1/n are determined from the intercept and the slope , respectively .", "the kinetic tests were carried out following the same procedure used for the equilibrium tests .", "aqueous samples were taken at different intervals of time and the concentrations of adsorbent were measured at the same intervals .", "the amount of absorbed onto the developed adsorbents at time t ( min ) , qt ( mg / g ) was calculated by means of the expression below : \n ( 5)qe=(c0ct)vm , \n where co and ct are the liquid - phase concentrations at an initial and predetermined time t ( mg / l ) , respectively , v is the volume of solution ( l ) , and m is the dry weight of the added adsorbent ( g ) .", "the kinetic data were then carried out for the pseudo - first - order and pseudo - second - order . the rate constant of adsorption was determined from the pseudo - first - order equation : \n ( 6)logqeqt = logqek1t2.303 , \n where qe and qt ( mg / g ) are the amounts of adsorbed ( mg / g ) at equilibrium and at time t ( min ) , respectively , and kt is the adsorption rate constant ( min ) . the pseudo - second - order equation based on the equilibrium adsorption", "is expressed as \n ( 7)tqt=1k2qe2+tqe , where k2 ( g / mgmin ) is the rate constant of second - order adsorption .", "some physical and chemical features of pumice and na - zeolite p1 are listed in table 2 .", "it is evident that the treatment with naoh has a significant efficiency on the physicochemical properties of synthesized adsorbent na - zeolite p1 .", "the ph of the aqueous slurry and phpzc of adsorbents may give a good indication about the surface oxygen complexes and the electronic surface charges of adsorbents .", "the complexes on adsorbent surface are generally classified as acidic , basic , or neutral . based on the slurry ph , the nature of surface oxygen groups on the support and the dominant complexes can be deduced . from table 2 , it is clear that the ph is higher than the phpzc ; the surface of the adsorbent is negatively charged , favoring the adsorption of cationic species ( co , cu , fe , and cd ) .", "bulk density of the pumice and na - zeolite p1 decreased from 0.85 to 0.50  mg / g and the moisture is 0% .", "this finding is due to the generation of porous structure and the increase in binding sites after chemical modification .", "chemical and phase composition as well as microstructure of the pumice raw material have been studied .", "the data obtained from the xrf and xrd are discussed before . table 1 illustrates that sio2 is the major component ( ~71.0% ) with about 14.20% al2o3 as well as a total of about 12.4% fluxing oxides .", "xrd pattern in figure 1 confirms the glassy phase in addition to minor crystalline quartz phase , which is the only phase .", "the amorphous nature of the silicate pumice as well as the high content of the pore size with variable distributions is ensured by the sem in figure 3(b ) .", "phase composition and microstructure of the synthetic zeolite studied by xrd ( figure 2 ) and sem reveal that the zeolite p1 is the only phase formed during these conditions of alkali treatment during the alkaline hydrothermal attack of locally produced pumice raw material at the optimum condition of synthesis .", "the micrograph of synthetic zeolite shows very fine agglomerated crystalline grains ( gray ) figure 3(a ) .", "xrd analysis was used to prove the formation of na - zeolite p1 before and after treatment with naoh .", "the x - ray diffractogram of pumice shows that it was considered a glass because it has no crystal structure .", "pumice is an unusually light rock due to the many bubbles inside it , figure 3(b ) .", "pumice varies in density according to the thickness of the solid material between the bubbles ; many samples float in water .", "it is formed by volcanic eruptions when molten lava is shot in the air with many bubbles of gas in it .", "the treatment with naoh leads to the formation of na - zeolite p1 which has higher degree of crystallinity , figure 3(a ) .", "xrd analysis was used to prove the formation of na - zeolite p1 before and after treatment with naoh .", "adsorption isotherm and kinetics of co , cu , fe , and cd on the synthetic zeolite were investigated using batch experiments .", "equilibrium and kinetic studies were carried out on the removal of co , cu , fe , and cd from aqueous solution using synthetic zeolite p1 .", "adsorption test on prepared adsorbent was studied using a batch process . for this purpose , the effects of adsorbents dose ( 0.11  g ) , contact time ( 18  min ) , and metal concentration ( 300700  mg / l ) on the adsorption of co , cu , fe , and cd ( 25  ml of 100 ", "for all the above - mentioned equilibrium and kinetic studies , the mixture of adsorbent and solution was magnetically stirred at 150  rpm for 30  min as determined from kinetic tests .", "batch adsorption experiments were conducted by mixing 50  cm solutions of 500  ppm containing one metal of co , cu , fe , and cd with a constant dose of na - zeolite p1 ( 0.1  g , 0.25  g , 0.5  g , 0.75  g , or 1  g ) in 100  cm pyrex erlenmeyer flasks with cap .", "flasks were shaken ( 150  rpm ) at 25  1c using a brunswick scientific g25kc incubator orbital shaker .", "it is observed that the percentage adsorption for co , cu , fe , and cd ions increases with increasing zeolite weight in aqueous solutions as illustrated in figure 4 .", "the maximal exchange levels attained were as follows : cd > fe > cu > co. the heavy metal uptake may be attributed to different mechanisms of ion - exchange processes as well as to the adsorption process [ 1820 ] . during the ion - exchange process ,", "metal ions had to move not only through the pores of the na - zeolite p1 mass , but also through channels of the lattice , and they had to replace exchangeable cations ( mainly sodium ) .", "diffusion was faster through the pores and was retarded when the ions moved through the smaller diameter channels .", "in this case the metal ion uptake 6 could mainly be attributed to ion - exchange reactions in the microporous minerals of the na - zeolite p1 samples . by varying the contact time ( 1 , 2 , 4 , 6 , and 8  min ) while keeping all other parameters constant with respect to ph , temperature , and dose of na - zeolite p1 , 0.25 gm , the influence of contact time on the zeolite capacity for different metal ions is shown in figure 5 .", "removal efficiency increases for cadmium from 95 to 98% , for copper from 71 to 76% , for iron from 64 to 76% , and for cobalt from 58 to 61% as the time increases from 1 to 8  min .  ", "figure 6 represents the variation in metal ion adsorption with time ; however the adsorption rate becomes slower with passage of time up to 4  min .", "the initial faster rate of removal of each metal ion may be due to the availability of the uncovered surface area of the adsorbents .", "the slight increase in the percentage of adsorption of each metal adsorbent is cd > fe > cu > co. this may be attributed to the difference in nature between metal ions with respect to na - zeolite p1 surface .", "that may be attributed to different mechanisms of ion - exchange processes as well as the adsorption process [ 1820 ] .", "the uptake of cd(ii ) , cu(ii ) , ni(ii ) , pb(ii ) , and zn(ii ) onto na - zeolite p1 as a function of their concentrations was studied at 25  0.1c by varying the metal concentrations 300 , 400 , 500 , and 700 ", "mg / l while keeping all other parameters constant with respect to dose and time . for adsorption isotherms models were used in this work , langmuir and freundlich isotherms .", "they differ in their assumption , shape of the isotherm , and nature of the adsorbent surface .", "langmuir ( 1918 ) isotherm which models the monolayer coverage of the sorbent surface assumes that sorption occurs at specific homogeneous sorption site .", "the model is also based on the assumption that all the sorption sites are energetically identical and sorption occurs on a structurally homogeneous sorbent [ 1820 ] .  ", "figure 7 shows that the langmuir model effectively and significantly described the sorption data with all r values 0.96 .  ", "table 3 shows the relation between the ionic radius , electronegativity , and langmuir constant .", "the maximum sorption capacity ( kl ) represents monolayer coverage of sorbent ; according to the kl ( mg / g ) parameter , monolayer capacities of zeolite are arranged in the following sequence : cd > fe > cu > co. these metals seem to reach saturation , which means that the metal had clogged possible available sites in zeolite and further adsorption could take place only at new surfaces .", "the sequence of adsorption of several heavy metals are not correlated either with the sequence of ionic radii or with the sequence of electronegativity .", "there is , however , a parallel relation between the adsorption sequence and the hydrolysis properties of the heavy metal cations , as pointed out by several investigators [ 2224 ] .", "this isotherm developed by freundlich ( 1926 ) describes the equilibrium on heterogeneous surfaces and does not assume monolayer capacity .", "this isotherm gives an expression encompassing the surface heterogeneity and the exponential distribution of active sites and their energies .", "a linear plot is obtained when log  ce is plotted against log  qe over the entire concentration range of metal ions investigated and the values of kf ( mg / g ) and n can be calculated from the intercept and the slope of this straight line , respectively ( figure 8) .", "the arrangement of heavy metals according to dose required for the reduction of metal concentrations in freundlich equation is cd > fe > cu > co. the adsorption isotherm data were fitted well to the langmuir model from langmuir model equations . in order to investigate the adsorption kinetics of heavy metals on the prepared zeolite adsorbent", "the calculated parameters from pseudo - first - order and pseudo - second - order models ( figures 9 and 10 ) are summarized in table 4 . according to the pseudo - first - order model ,", "the values of k1 and qe were calculated from the slope and intercept from the plots of log  ( qe  qt ) versus t. it was found that the experimental values were not adequate with the calculated ones ( table 4 ) , indicating that the first - order model does not reproduce the adsorption kinetic of heavy metals on the prepared adsorbents . in case of the pseudo - second - order model , the calculated values of kinetic studies showed that the zeolite is a good potential as adsorbent for ( co , cu , fe , and cd ) ions follows the pseudo - second - order , and , hence , this model is more likely to predict the behavior over the whole experimental range of h m adsorption more than pseudo - first - order model .", "also , the values of correlation coefficients ( 2 ) are close to unity for pseudo - second - order model rather than the pseudo - first - order one ; that is , the kinetic removal of h m is quite described by pseudo - second - order model .", "the alkaline treatment of very cheap local pumice leads to the formation of na - zeolite p1 which has the high degree of crystallinity , the ph is higher than the phpzc , and the surface of the adsorbent is negatively charged , favoring the adsorption of cationic species ( co , cu , fe , and cd ) .", "the adsorption kinetics study of the heavy metals followed a pseudo - second - order model .", "both the langmuir and freundlich models fit equilibrium data well implying the existence of a monolayer adsorption and a heterogeneous surface existence in na - zeolite p1 , showing a strong adsorption capacity for ( co , cu , fe , and cd ) ions reaching a maximum capacity for cd .", "heavy metal ions uptake is the result of a combination of several interfacial reactions , namely , ion exchange , chemisorption , and adsorption as potential determining ions .", "results found showed that the modified zeolite shows a good potential as adsorbent for ( co , cu , fe , and cd ) ions ."], "abstract_text": ["<S> wastewater treatment of some heavy metals was carried out by synthetic zeolite p1 , which was prepared by alkaline hydrothermal treatment of the pumice . </S>", "<S> both of the pumice raw materials and synthetic zeolite were investigated for their chemical phase composition , physical properties , and microstructure . </S>", "<S> the adsorption behavior of na - zeolite p1 with respect to co+2 , cu+2 , fe+2 , and cd+2 has been studied to be applied in the industrial wastewater treatment . </S>", "<S> metal removal was investigated using synthetic solutions at different ions concentrations , time , and na - p1 zeolite doses as well as constant temperature and ph . </S>", "<S> it is concluded that the optimum conditions for synthesis of highly active na - p1 zeolite from natural pumice raw material are one molar naoh concentration , temperature at 80c , and one week as a crystallization time . </S>", "<S> in addition to the effect of time and zeolite dose as well as the ion concentration of the reaction efficiency for metals removals are recorded . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussions", "4. Conclusion"], "sections": [["zeolite is a natural porous mineral described as hydrated aluminosilicates containing exchangeable alkali and earth alkaline cations ( na , k , ca , or mg ) and could be synthesised from the aluminium silicate materials as clay minerals and pumice by alkali attack [ 13 ] .", "zeolites have a large surface area , microporous structure , and high affinity for metal ions , providing an efficient scavenging pathway for heavy metals in toxic systems [ 4 , 5 ] .", "some of the attractive applications of natural zeolites are listed by taffarel and rubio   and babel and kurniawan .", "most of these research efforts are undertaken on laboratory scale and are at early stages of investigations to develop the synthesis methods .", "synthetic zeolites are carried out in many industrialized countries in europe , east asia , and north america .", "na - p1 zeolite is one of the synthetic zeolites which are a class of highly porous materials", ". the unique structural features of these crystalline microporous solids that contain pores and cavities in the order of molecular dimensions ( 310   ) are the main reason for their application in the realms of catalysis , separation , purification , ion exchange , and radioactive waste clean - up .", "more novel applications for na - p1 zeolites are expected in electrochemistry , photochemistry , pharmaceutical , engineering , membrane science , and nanotechnology as their structures are expanded and more suitably engineered in a foreseeable future .", "it is well established that these materials that are equivalently called molecular sieves are mainly hydrated aluminosilicates .", "zeolite na - p1 contains two - dimensional pore system with two intersecting 8-ring channels .", "a synthetic zeolite na - p1 has similar pore structure to the natural zeolite phillipsite .", "na - p1 zeolite is classified as high silica zeolites where it has si / al ratio equal to or greater than about 3 .", "it is considered as a hydrated sodium aluminosilicate zeolite that could be synthesised from the naturally occurring alkaline volcano with naoh attack of varying concentrations at different volcanic temperatures .", "pumice is a natural rock of volcanic origin , formed by gases released during the solidification of acidic lava . the vesicular structure of pumice is formed during the eruption of gases contained in the molten viscous lava on cooling . in egypt ,", "pumice occasionally occurs in few locations , at the northern coast of the mediterranean sea at el - arish , north sinai .", "the heavy metals contamination of water by the discharge of industrial wastewater of electroplating , metallurgy , chemical manufacturing , mining , and battery manufacturing is a worldwide environmental problem .", "adsorption process provides an attractive alternative treatment to other removal techniques because it is more economical and readily available [ 1012 ] .", "a good number of adsorbents have been used for cd(ii ) , fe(ii ) , cu(ii ) , and co(ii ) removal from water .", "the adsorption of cd(ii ) on red mud fitted langmuir isotherm model having a maximum adsorption capacity of 10.57  mg  g .", "wang   used maize cobs for adsorption of fe(iii ) from aqueous solution with the langmuir adsorption capacity and the freundlich adsorption capacity being 2.53  mg  g and 0.104  l  g , respectively . the maximum adsorption capacity for cu(ii ) on red mud", "gupta et al .   used spirogyra species for removal of cu(ii ) with the maximum adsorption capacity of 133.3  mg  g. use of adsorbents like mg pellets has also been reported for co(ii ) removal from water   and the adsorption capacity has been found to be 15.8  mg  g. the aim of this work is to study the best condition of temperature , sodium hydroxide concentration , and time required for synthesis of na - p1 zeolite from the local pumice raw material and utilize the synthetic zeolite as an adsorbent for removing ( cd , fe , cu , and co ) ions from synthetic solutions in a batch laboratory system .", "also , the effect of experimental conditions such as contact time and initial zeolite dose was described ."], ["the main material used in this work is pumice grains received from el - nile mining company from the mediterranean sea coast at el - arish province , north sinai , egypt .", "in addition , commercial naoh is used for alkaline hydrothermal attack for synthesis of zeolite from pumice .", "hydrated chloride salts of cu , co , fe , and cd of analytical grade ( ar ) supplied by merck as analytical - grade reagents and deionized water are used .", "all adsorption experiments were carried out using an aqueous solution of cucl22h2o , cocl26h2o , fecl2 , and cdcl2h2o .", "the stock solution was prepared by dissolving accurately weighted salt in distilled water to the concentration of 500  mg / l .", "the initial concentrations of solution were done by diluting the stock solutions in appropriate proportions to different initial concentrations .", "na - p1 has been prepared from pumice , which is naturally occurring alkaline volcanic rock with naoh attack .", "a series of experiments were conducted to synthesis na - p1 zeolite from locally produced pumice raw material at temperatures ( 80 to 120c ) , naoh solution concentrations ( 13 molar ) , and crystallization periods ( 12  h , one week , and up to one month .", "pumice powder was mixed with naoh solution in a well - sealed teflon cylindrical vessel , heated , and stirred for 12 hours at 80c , and then the vessel was kept in the dryer at 8090c for one week .", "the produced gel was filtered and washed well till becoming free from sodium hydroxide ; the wet powder was dried for 24  h in the dryer at 110c .", "the optimum conditions required for synthesis of na - p1 zeolite from locally produced pumice raw material are one molar naoh concentration , temperature at 80c , and one week as a crystallization time .", "several investigations for the adsorption of the heavy metal were carried out , including effect of contact zeolite dose , effect of time , and effect of metals concentration .", "the physical and chemical characteristics of prepared adsorbents such as slurry ph , point of zero charge ( phpzc ) , bulk density , and moisture were performed to determine the capability of prepared adsorbents in removing the metals co , cu , fe , and cd from an aqueous media . for ph determination , 0.2  g of the dry , grinded adsorbent was mixed with 25  ml of distilled water and allowed to boil for 30  min in stoppered glass bottle . after cooling ,", "the ph of the adsorbent was measured using a digital ph meter ( hanna , model ph20 ) , allowing 5  min for the ph probe to equilibrate .", "also , the point of zero charge ( pzc ) for the samples was determined by the following procedure : 200  ml of deionized water was added to an erlenmeyer flask , which was then capped with stoppered glass .", "the deionized water was heated until boiling for 20  min to eliminate the co2 dissolved in the water .", "the co2-free water was cooled down as soon as possible and the flask was immediately capped . on the other hand ,", "0.5  g of each sample was weighed and placed in a 25  ml erlenmeyer flask to which 20  ml of co2-free water was added .", "the flask was sealed with a rubber stopper and left in continuous agitation for 72  h at 25c .", "then the solution ph was measured and this value is the point of zero charge .", "the bulk density was estimated by a standard procedure by weighing a known volume of gently tapped adsorbent granules .", "the apparent density was calculated from the volume of the graduated cylinder closely packed with the powdered sample and from the sample weight in triplicate . for moisture measurement , about 0.5 ", "g of powdered air - dried adsorbent sample is weighed and taken in a crucible .", "the crucible is placed inside an electric hot - air oven and heated at 150c for 3 hours .", "it is then taken out , cooled in a desiccator , and weighed . from this , the percentage of moisture can be calculated as follows : percentage of moisture = ( loss in weight of adsorbent / weight of air - dried adsorbent taken )  100 .", "surface characteristics of prepared zeolite sample were characterized by scanning electron microscope ( sem ) using sem model philips xl 30 attached with edx unit , with accelerating voltage 30  k.v . ,", "magnification 10x up to 400.000x , and resolution for w. ( 3.5  nm ) .", "x - ray powder diffractograms , xrd , of the samples were recorded using a bruker diffractometer ( bruker d8 advance target ) and the scan rate was fixed at 8 in 2  min for phase identification .", "the patterns were run with cu k and secondly with monochromator (  = 0.1545  nm ) at 40  kv and 40  ma .", "mineralogical and chemical study of the pumice were done by using x - ray fluorescence spectrometer ( xrf ) to identify the chemical composition .", "xrf , a spectrometer with wavelength dispersion pw 2400 philips , and centrifuge hermle were used .", "xrf shows that quartz , cristobalite , and albite are the main constituents in the pumice ( figure 2 ) .", "sio2 , al2o3 , and na2o are the main oxides in the pumice under investigation ( 93.69% ) confirming the mineralogical constituents , whereas tio2 , mno , mgo , fe2o3 , and cao analyzed in the pumice under are calculated as 3.88% ( table 1 ) .", "the concentrations of heavy metals in all samples were determined according to apha using atomic absorption spectrometer unicam model 939 with graphite furnace accessory , equipped with deuterium arc background corrector .", "precision of the metal measurement was determined by analyzing ( in triplicate ) the metal concentration of all samples and for each series of measurements an absorption calibration curve was constructed , composed of a blank and three or more standards . accuracy and precision of the metals measurement were confirmed using external reference standards from merck .", "the adsorption isotherm shows the equilibrium relationship of concentration in the adsorbate - adsorbent system at constant temperature .", "such adsorption isotherms may be used for scaling up batch type processes with moderate success .", "the shape of the equilibrium adsorption isotherm provides us with information about the adsorbent surface whether it is homogeneous or heterogeneous .", "also , its study is helpful in evaluating the maximum adsorption capacity of adsorbate for the given adsorbent . for adsorption isotherms models", "they differ in their assumption , shape of the isotherm , and nature of the adsorbent surface .", "langmuir ( 1918 ) model assumes that uptake of metal ion occurs on a homogeneous surface by monolayer adsorption without any interaction between adsorbed ions .", "the model is also based on the assumption that all the sorption sites are energetically identical and sorption occurs on a structurally homogeneous sorbent .", "thus , a linear plot of ce / qe versus ce confirms the validity of the langmuir giving correlation coefficients ( r ) close to unity , and kl is a constant related to the free energy of adsorption as expressed ( kle ) .", "deviations from the basic assumption of the langmuir model do limit interpretation of the values for qm and kl in terms of absolute surface areas and sorption free energies . the free energy of adsorption , g , can also be evaluated from the parameter kl according to the expression g = rtlnkl .", "values of qm and kl are obtained from slope and intercept of ce / qe versus ce plot , respectively .", "a further analysis of the langmuir equation can be made on the basis of a dimensionless equilibrium parameter , rl ( known as the separation factor which is considered as a more reliable indicator of adsorptions ) .", "this parameter ( rl ) can be expressed by weber and chakkravorti ( 1974 ) : \n ( 2)rl=11+bci , \n where ci is the initial concentration metal ion ( mg / l ) and b ( l  mg ) is the langmuir constant described above . also , the influence of the isotherm shape on whether the adsorption is ", "unfavorable  at highest initial concentration of the dye , co , can be described by a term  rl  , a dimensionless constant separation factor in equation ; calculated value for rl indicates the nature of adsorption process as given : irreversible ( rl = 0 ) , favorable ( 0 < rl < 1 ) , linear ( rl = 1 ) , and favorable ( rl > 1 ) .", "freundlich isotherm is an empirical equation that can be described as the reversible adsorption onto heterogeneous surface at sites with different energy of adsorption and is not restricted to the formation of the monolayer of adsorbate .", "the nonlinear form of this model is expressed as \n ( 3)qe = kfce1/n , \n where kf is the freundlich constant ( mg / g)/(l / mg)1/n and also referred to as adsorption capacity , while n is the heterogeneity factor and related to adsorption intensity .", "the parameter n gives an indication of the adsorption type whether is linear ( n = 1 ) or a physical process ( n > 1 ) is favorable , or a chemical process ( n < 1 ) . on the other hand", ", the value of 1/n < 1 or 1/n > 1 indicates a normal langmuir isotherm and cooperative adsorption , respectively .", "freundlich model can be represented by linear form as follows : \n ( 4)lnqe = lnkf+1nlnce . a plot of lnqe versus lnce gives a straight line , where the values of kf and 1/n are determined from the intercept and the slope , respectively .", "the kinetic tests were carried out following the same procedure used for the equilibrium tests .", "aqueous samples were taken at different intervals of time and the concentrations of adsorbent were measured at the same intervals .", "the amount of absorbed onto the developed adsorbents at time t ( min ) , qt ( mg / g ) was calculated by means of the expression below : \n ( 5)qe=(c0ct)vm , \n where co and ct are the liquid - phase concentrations at an initial and predetermined time t ( mg / l ) , respectively , v is the volume of solution ( l ) , and m is the dry weight of the added adsorbent ( g ) .", "the kinetic data were then carried out for the pseudo - first - order and pseudo - second - order . the rate constant of adsorption was determined from the pseudo - first - order equation : \n ( 6)logqeqt = logqek1t2.303 , \n where qe and qt ( mg / g ) are the amounts of adsorbed ( mg / g ) at equilibrium and at time t ( min ) , respectively , and kt is the adsorption rate constant ( min ) . the pseudo - second - order equation based on the equilibrium adsorption", "is expressed as \n ( 7)tqt=1k2qe2+tqe , where k2 ( g / mgmin ) is the rate constant of second - order adsorption ."], ["some physical and chemical features of pumice and na - zeolite p1 are listed in table 2 .", "it is evident that the treatment with naoh has a significant efficiency on the physicochemical properties of synthesized adsorbent na - zeolite p1 .", "the ph of the aqueous slurry and phpzc of adsorbents may give a good indication about the surface oxygen complexes and the electronic surface charges of adsorbents .", "the complexes on adsorbent surface are generally classified as acidic , basic , or neutral . based on the slurry ph , the nature of surface oxygen groups on the support and the dominant complexes can be deduced . from table 2 , it is clear that the ph is higher than the phpzc ; the surface of the adsorbent is negatively charged , favoring the adsorption of cationic species ( co , cu , fe , and cd ) .", "bulk density of the pumice and na - zeolite p1 decreased from 0.85 to 0.50  mg / g and the moisture is 0% .", "this finding is due to the generation of porous structure and the increase in binding sites after chemical modification .", "chemical and phase composition as well as microstructure of the pumice raw material have been studied .", "the data obtained from the xrf and xrd are discussed before . table 1 illustrates that sio2 is the major component ( ~71.0% ) with about 14.20% al2o3 as well as a total of about 12.4% fluxing oxides .", "xrd pattern in figure 1 confirms the glassy phase in addition to minor crystalline quartz phase , which is the only phase .", "the amorphous nature of the silicate pumice as well as the high content of the pore size with variable distributions is ensured by the sem in figure 3(b ) .", "phase composition and microstructure of the synthetic zeolite studied by xrd ( figure 2 ) and sem reveal that the zeolite p1 is the only phase formed during these conditions of alkali treatment during the alkaline hydrothermal attack of locally produced pumice raw material at the optimum condition of synthesis .", "the micrograph of synthetic zeolite shows very fine agglomerated crystalline grains ( gray ) figure 3(a ) .", "xrd analysis was used to prove the formation of na - zeolite p1 before and after treatment with naoh .", "the x - ray diffractogram of pumice shows that it was considered a glass because it has no crystal structure .", "pumice is an unusually light rock due to the many bubbles inside it , figure 3(b ) .", "pumice varies in density according to the thickness of the solid material between the bubbles ; many samples float in water .", "it is formed by volcanic eruptions when molten lava is shot in the air with many bubbles of gas in it .", "the treatment with naoh leads to the formation of na - zeolite p1 which has higher degree of crystallinity , figure 3(a ) .", "xrd analysis was used to prove the formation of na - zeolite p1 before and after treatment with naoh .", "adsorption isotherm and kinetics of co , cu , fe , and cd on the synthetic zeolite were investigated using batch experiments .", "equilibrium and kinetic studies were carried out on the removal of co , cu , fe , and cd from aqueous solution using synthetic zeolite p1 .", "adsorption test on prepared adsorbent was studied using a batch process . for this purpose , the effects of adsorbents dose ( 0.11  g ) , contact time ( 18  min ) , and metal concentration ( 300700  mg / l ) on the adsorption of co , cu , fe , and cd ( 25  ml of 100 ", "for all the above - mentioned equilibrium and kinetic studies , the mixture of adsorbent and solution was magnetically stirred at 150  rpm for 30  min as determined from kinetic tests .", "batch adsorption experiments were conducted by mixing 50  cm solutions of 500  ppm containing one metal of co , cu , fe , and cd with a constant dose of na - zeolite p1 ( 0.1  g , 0.25  g , 0.5  g , 0.75  g , or 1  g ) in 100  cm pyrex erlenmeyer flasks with cap .", "flasks were shaken ( 150  rpm ) at 25  1c using a brunswick scientific g25kc incubator orbital shaker .", "it is observed that the percentage adsorption for co , cu , fe , and cd ions increases with increasing zeolite weight in aqueous solutions as illustrated in figure 4 .", "the maximal exchange levels attained were as follows : cd > fe > cu > co. the heavy metal uptake may be attributed to different mechanisms of ion - exchange processes as well as to the adsorption process [ 1820 ] . during the ion - exchange process ,", "metal ions had to move not only through the pores of the na - zeolite p1 mass , but also through channels of the lattice , and they had to replace exchangeable cations ( mainly sodium ) .", "diffusion was faster through the pores and was retarded when the ions moved through the smaller diameter channels .", "in this case the metal ion uptake 6 could mainly be attributed to ion - exchange reactions in the microporous minerals of the na - zeolite p1 samples . by varying the contact time ( 1 , 2 , 4 , 6 , and 8  min ) while keeping all other parameters constant with respect to ph , temperature , and dose of na - zeolite p1 , 0.25 gm , the influence of contact time on the zeolite capacity for different metal ions is shown in figure 5 .", "removal efficiency increases for cadmium from 95 to 98% , for copper from 71 to 76% , for iron from 64 to 76% , and for cobalt from 58 to 61% as the time increases from 1 to 8  min .  ", "figure 6 represents the variation in metal ion adsorption with time ; however the adsorption rate becomes slower with passage of time up to 4  min .", "the initial faster rate of removal of each metal ion may be due to the availability of the uncovered surface area of the adsorbents .", "the slight increase in the percentage of adsorption of each metal adsorbent is cd > fe > cu > co. this may be attributed to the difference in nature between metal ions with respect to na - zeolite p1 surface .", "that may be attributed to different mechanisms of ion - exchange processes as well as the adsorption process [ 1820 ] .", "the uptake of cd(ii ) , cu(ii ) , ni(ii ) , pb(ii ) , and zn(ii ) onto na - zeolite p1 as a function of their concentrations was studied at 25  0.1c by varying the metal concentrations 300 , 400 , 500 , and 700 ", "mg / l while keeping all other parameters constant with respect to dose and time . for adsorption isotherms models were used in this work , langmuir and freundlich isotherms .", "they differ in their assumption , shape of the isotherm , and nature of the adsorbent surface .", "langmuir ( 1918 ) isotherm which models the monolayer coverage of the sorbent surface assumes that sorption occurs at specific homogeneous sorption site .", "the model is also based on the assumption that all the sorption sites are energetically identical and sorption occurs on a structurally homogeneous sorbent [ 1820 ] .  ", "figure 7 shows that the langmuir model effectively and significantly described the sorption data with all r values 0.96 .  ", "table 3 shows the relation between the ionic radius , electronegativity , and langmuir constant .", "the maximum sorption capacity ( kl ) represents monolayer coverage of sorbent ; according to the kl ( mg / g ) parameter , monolayer capacities of zeolite are arranged in the following sequence : cd > fe > cu > co. these metals seem to reach saturation , which means that the metal had clogged possible available sites in zeolite and further adsorption could take place only at new surfaces .", "the sequence of adsorption of several heavy metals are not correlated either with the sequence of ionic radii or with the sequence of electronegativity .", "there is , however , a parallel relation between the adsorption sequence and the hydrolysis properties of the heavy metal cations , as pointed out by several investigators [ 2224 ] .", "this isotherm developed by freundlich ( 1926 ) describes the equilibrium on heterogeneous surfaces and does not assume monolayer capacity .", "this isotherm gives an expression encompassing the surface heterogeneity and the exponential distribution of active sites and their energies .", "a linear plot is obtained when log  ce is plotted against log  qe over the entire concentration range of metal ions investigated and the values of kf ( mg / g ) and n can be calculated from the intercept and the slope of this straight line , respectively ( figure 8) .", "the arrangement of heavy metals according to dose required for the reduction of metal concentrations in freundlich equation is cd > fe > cu > co. the adsorption isotherm data were fitted well to the langmuir model from langmuir model equations . in order to investigate the adsorption kinetics of heavy metals on the prepared zeolite adsorbent", "the calculated parameters from pseudo - first - order and pseudo - second - order models ( figures 9 and 10 ) are summarized in table 4 . according to the pseudo - first - order model ,", "the values of k1 and qe were calculated from the slope and intercept from the plots of log  ( qe  qt ) versus t. it was found that the experimental values were not adequate with the calculated ones ( table 4 ) , indicating that the first - order model does not reproduce the adsorption kinetic of heavy metals on the prepared adsorbents . in case of the pseudo - second - order model , the calculated values of kinetic studies showed that the zeolite is a good potential as adsorbent for ( co , cu , fe , and cd ) ions follows the pseudo - second - order , and , hence , this model is more likely to predict the behavior over the whole experimental range of h m adsorption more than pseudo - first - order model .", "also , the values of correlation coefficients ( 2 ) are close to unity for pseudo - second - order model rather than the pseudo - first - order one ; that is , the kinetic removal of h m is quite described by pseudo - second - order model ."], ["the alkaline treatment of very cheap local pumice leads to the formation of na - zeolite p1 which has the high degree of crystallinity , the ph is higher than the phpzc , and the surface of the adsorbent is negatively charged , favoring the adsorption of cationic species ( co , cu , fe , and cd ) .", "the adsorption kinetics study of the heavy metals followed a pseudo - second - order model .", "both the langmuir and freundlich models fit equilibrium data well implying the existence of a monolayer adsorption and a heterogeneous surface existence in na - zeolite p1 , showing a strong adsorption capacity for ( co , cu , fe , and cd ) ions reaching a maximum capacity for cd .", "heavy metal ions uptake is the result of a combination of several interfacial reactions , namely , ion exchange , chemisorption , and adsorption as potential determining ions .", "results found showed that the modified zeolite shows a good potential as adsorbent for ( co , cu , fe , and cd ) ions ."]]}
{"article_id": "PMC5413586", "article_text": ["surgical resection of tumors is a common therapeutic procedure , especially for early - stage localized , and potentially curative , disease .", "while surgery can ultimately cure many patients , such as those with early - stage disease , distant macroscopic metastasis can emerge in others months to years later ( demicheli et  al .", "it has been reported that 25%30% of colorectal cancer patients who have no visible  metastasis at the time of diagnosis will develop distant metastases within 5 years after primary tumor resection , which  in some cases may be related to the effects of the surgery ( van der bij et  al . , 2009 ) .", "similarly , high risk of recurrence for early - stage breast cancer patients following mastectomy has been reported in an analysis of 1,173 patients who underwent mastectomy with no subsequent adjuvant systemic therapy ( demicheli et  al . , 1996 ) .", "mechanisms to explain distant metastasis following primary tumor resection include ( 1 ) the presence of residual tumor cells or tissue at the resected site ( ando et  al . , 2003 , minsky et  al . , 1988 ) ,", "( 2 ) the recruitment of inflammatory cells and platelets to the resected site that promote wound healing and cell proliferation ( ceelen et  al . , 2014 , hofer et  al . , 1999 , retsky et  al . ,", "2012 ) , and ( 3 ) increased local and systemic effects that can induce an angiogenic switch in remote dormant tumors ( bono et  al . , 2010 , retsky et  al . , 2012 , takemoto et  al . , 2012 ) .", "previous studies have revealed that hypoxic tumor cells stimulate angiogenic - related factors via hif1- , leading to increased tumor invasion ( paraskeva et  al . , 2006 ,", "hif1- expression in tumors can also upregulate lysyl oxidase ( lox ) ( erler et  al . ,", "2006 ) , a member of the secreted copper - dependent amine oxidases known to covalently crosslink collagens and elastin in the extracellular matrix ( ecm ) ( barker et  al . , 2012 ) .", "lox is expressed by different cell types , including tumor cells and stromal cells within the tumor microenvironment ( decitre et  al . , 1998 ) .", "it has been shown that increased lox expression in tumors accounts for the recruitment of cd11b bone - marrow - derived cells ( bmdcs ) at distant organs , contributing to the formation of a niche and facilitating a pre - metastatic microenvironment for tumor cell seeding ( erler et  al . , 2009 ) .", "however , the contribution of lox to tumor cell seeding and subsequently to metastasis soon after surgery is unknown . the host response to anti - cancer therapy and its contribution to tumor ( re)growth and metastasis has been evaluated following chemotherapy ( daenen et  al . , 2011 , gingis - velitski et  al . , 2011 ) , radiotherapy ( barcellos - hoff et  al . , 2005 ,", "2015 ) , and various molecularly targeted drugs ( beyar - katz et  al . , 2016 ) ( for a review , see shaked , 2016 ) . here", ", we evaluated remote ( pulmonary ) changes in lox expression and activity in response to surgery and their contribution to tumor cell seeding and metastasis .", "increased metastases after localized tumor resection in some cases could be due to systemic changes that affect various host tissues in response to surgery .", "previous clinical studies indicated that both systemic and local angiogenesis are induced in response to surgery when compared to laparoscopy ( bono et  al . , 2010 ) .", "to test whether our surgical mouse model induces angiogenesis , we performed a surgical procedure in non - tumor - bearing mice involving a 1  cm incision in the peritoneum followed by suturing .", "thereafter , we evaluated the levels of circulating bone - marrow - derived proangiogenic cells over time and the extent of local angiogenesis following surgery . a significant increase in the number of viable circulating endothelial cells ( cecs ) and endothelial progenitor cells ( ceps )", "likewise , increases in microvessel density in matrigel plugs , in  vitro human umbilical vein endothelial cell ( huvec ) microvessel tube formation , and microvessel sprouting from murine aortic rings were observed in the presence of plasma obtained from post - surgery mice compared to control ( figure  s1b ", "the surgical procedure in our mouse model mimics host angiogenic effects reported in certain clinical circumstances .", "we next evaluated the host - derived effects of surgery on tumor cell seeding and growth in metastatic sites .", "we employed an experimental lung metastasis assay using the murine emt/6-gfp+ breast cancer cell line .", "control mice and mice that previously underwent surgery were intravenously injected with tumor cells , which subsequently seeded in the lungs .", "mortality rates were increased in post - surgery mice compared to controls ( figure  1a ) .", "similarly , recipient mice injected with 100  l plasma from donor mice that had undergone surgery exhibited an increased mortality rate in comparison to mice injected with control plasma ( figure  1b ) , indicating that a host effect , expressed within the plasma , in response to surgery rather than the actual surgical procedure is responsible for the observed effects . to directly assess tumor cell seeding in the lungs , regardless of any host responses that may affect tumor cells per se while seeding and proliferating at the metastatic sites , a pulmonary metastatic assay ( puma ) was performed using the emt/6-gfp+ cell line . in this assay ,", "the potential of tumor cell seeding is solely dependent on tumor cells binding to the lungs and not systemic effects that could affect tumor cell proliferation .", "the number of gfp+ tumor cells present in the lungs of mice that underwent surgery was substantially elevated in comparison to control mice ( figures 1c and 1d ) . taken together , our results suggest that enhanced tumor cell seeding is a result of remote changes occurring , in part , in the pre - metastatic ( lung ) tissue in response to surgery .", "ecm remodeling is one of the main factors that can facilitate tumor cell seeding ; lox mediates collagen crosslinking and therefore is a key regulator of ecm remodeling ( barker et  al . , 2012 ) .", "recent studies have shown that lox is expressed by tumor cells , and serves as a key enzyme promoting metastasis by contributing to a pre - metastatic niche ( cox et  al . , 2015 , erler et  al . , 2006 , erler et  al . , 2009 ) . in our model", ", we found that lox expression and activity were significantly upregulated at the hypoxic surgical site in the wounded peritoneum in comparison to control peritoneum ( figures 2a2c ) .", "specifically , high - magnification images of the surgical site revealed intracellular lox expression of peritoneal myofibers ( figure  s2a ) . furthermore , lox expression and activity , quantity of newly synthesized collagen , and fibrillar collagen expression were all significantly higher in the lungs of mice that underwent surgery than in the lungs of control mice ( figures 2d2i ) .", "notably , high lox extracellular staining was located mainly in the lung stroma surrounding the bronchioles in post - surgery lungs when compared to control lungs ( figure  2j ) .", "however , high - magnification images detected intracellular lox expression in different cell types , with no noticeable differences in expression pattern between control and post - surgery lungs , suggesting that the major source of lox in the lungs of post - surgery  mice is from the remote surgical site ( figure  s2b ) . in addition , a significant decrease in lox expression and activity was found in the liver of mice that underwent surgery compared to control mice , whereas no significant changes were observed in the spleen ( figures s2c and s2d ) .", "importantly , 24  hr after surgery , the percentage of cd11b cells in the pre - metastatic lungs of post - surgery mice was comparable to that in control mice ( figures s2e and s2f ) , ruling out the possibility of the formation of a reported pre - metastatic niche at this early time point ( erler et  al . , 2009 , kaplan et  al . , 2005 ,", "these results indicate that the immediate effects of surgery are primarily associated with ecm remodeling . in order to strengthen the hypothesis that lox is a major contributor to metastatic seeding in the lungs within hours of surgery , a puma , which measures cell seeding in the lungs ,", "was carried out on mice 24  hr after they were injected with recombinant lox , the function of which was evaluated by a lox activity assay ( figure  s2 g ) .", "an increased number of metastatic cells was found in the lungs of mice injected with recombinant lox compared to control mice injected with pbs ( figures 2k and 2l ) .", "the seeding of cells in collagen and ecm is regulated in part by integrin signaling pathways ( larsen et  al . , 2006 ) .", "one of the major readouts of integrin signaling is paxillin , a scaffold protein connecting focal adhesion kinase to the actin cytoskeleton ( turner et  al . , 1990 , zaidel - bar et  al . , 2007 ) .", "we therefore sought to determine the level of phospho - paxillin ( p - pax ) in mcf-7 breast carcinoma cells , a cell line with minimal lox expression ( kraft - sheleg et  al . , 2016 ) , in response to plasma from control or post - surgery mice .", "mcf-7 cells were seeded on collagen , laminin , or fibronectin , all of which are ligands of integrins ( heino and kpyl , 2009 ) , in the presence or absence of plasma from control or post - surgery mice . increased expression of p - pax", "was observed with all three ligands in the presence of plasma from post - surgery mice compared to control , with laminin producing the weakest effect ( figures 3a and 3b ) .", "in addition , when the same experiment was performed using plasma from post - surgery mice depleted of lox , the expression of p - pax was found to be lower than the post - surgery non - depleted plasma group ( figures 3c and 3d ) , suggesting that lox expression mediates focal adhesion signaling .", "furthermore , expression of p - pax was higher in the lungs of mice that underwent surgery than in control mice , an effect that could be inhibited by treating the mice with -aminoproprionitrile ( bapn ) , an irreversible competitive inhibitor of all lox family members ( bondareva et  al . , 2009 )", "it should be noted that all three ligands are substrates of lox as assessed by the oxidation assay , whereas laminin was the least effective substrate , in line with the results shown in figures 3a3d ( figure  s3a ) .", "taken together , increased lox activity in the lungs soon after the mice undergo surgery contributes to tumor cell seeding via focal adhesion signaling , suggesting interactions between integrins and lox ligands in the remodeled ecm .", "as lox contributes to the seeding of tumor cells in the lungs , we next studied the effects of lox inhibition in mice using bapn .", "plasma from control mice or mice that underwent surgery was injected into recipient naive mice .", "after 24  hr , the recipient mice were injected with emt/6-gfp+ cells to obtain the experimental lung colonization metastasis assay described above .", "bapn monotherapy did not affect the percentage of gfp+ cells in the lungs of control mice .", "in contrast , the increase in percentage of gfp+ cells in the lungs of mice injected with plasma obtained from the post - surgery mice was completely abolished by the bapn therapy ( figures 4a and 4b ) , and their mortality rate was  reduced , as assessed in a parallel experiment ( figure  4c ) .", "no significant changes in mortality rate and percentage of gfp+ cells in the lungs of control mice treated with bapn or pbs control were found ( figures 4b and 4c ) .", "importantly , escalating doses of bapn in a range of 010  mg / ml did not contribute to tumor cell apoptosis ( figure  s3b ) , suggesting that the therapeutic effect of bapn is related to the inhibition of lox activity .", "furthermore , inhibiting lox using specific anti - lox activity - inhibiting antibodies similarly reduced tumor cell seeding in the lungs of mice injected with plasma from post - surgery mice , indicating that the effect is largely regulated by lox .", "of note , it is not clear why blocking lox in control mice increased metastatic cell seeding ( figures 4d4f ) .", "it is possible that other bypass pathways may play a role similar to mmp9 inhibition in control mice as previously described ( gingis - velitski et  al . , 2011 ,", "moreover , fibrillar collagen and newly synthesized collagen quantities in the lungs of mice 3  days after localized peritoneal surgery were significantly increased when compared to lungs obtained from post - surgery mice treated with bapn or anti - lox antibodies ( figures 4g4i ) . of note ,", "both anti - lox and bapn reduced lox activity in the lungs , while control rabbit immunoglobulin g ( igg ) had no effect ( figures s3c and s4 , respectively ) .", "to further strengthen our results , we wished to rule out the possibility that inflammation - induced pulmonary vessel permeability mediates the increased tumor cell seeding in the lungs at this early time point .", "evans blue assay performed on mice 24  hr after they underwent surgery revealed that pulmonary vessel permeability was not significantly different in post - surgery mice compared to control mice ( figure  s5a ) , suggesting that tumor cell seeding was not affected by inflammation . additionally , the number and percentage of tumor cells ( emt/6-gfp+ ) seeded in the lungs 20  min after tail vein injection of 2   10 cells ( 10-fold higher than the number of cells injected in the puma ) were significantly higher in post - surgery mice than in control mice .", "this effect was dramatically reduced when the mice were also treated with bapn as assessed by microscopic imaging of lung specimens and flow cytometry of single - cell suspensions from dissociated lungs ( figures s5b and s5c ) .", "taken together , lox - induced ecm remodeling accounts for tumor cell seeding in the lungs of post - surgery mice .", "the fact that plasma derived from post - surgery mice had the same effect on tumor cell seeding as surgery itself suggested it entails the factor mediating this activity .", "therefore , lox levels were evaluated in the plasma of control and post - surgery mice .", "the plasma of post - surgery mice exhibited significantly elevated lox levels compared to plasma from control mice , suggesting that lox upregulation in the plasma may affect tumor cell seeding in the lungs ( figure  5a ) . to test this possibility , we depleted lox from plasma of control mice and mice that underwent surgery and assessed tumor cell seeding using puma in recipient mice injected with the different plasma samples .", "whereas lox depletion did not affect tumor cell seeding in the control group , it significantly decreased tumor cell seeding in the surgery group ( figures 5b and 5c ) . in a parallel experiment", ", plasma was obtained from untreated wild - type mice or bapn - treated lox heterozygous mice ( mki et  al . ,", "the plasma was then injected into mice , and survival was assessed in an experimental lung metastasis assay .", "however , in the surgery group , survival times were increased when lox was inhibited ( figures 5d and 5e ; statistical significance [ p  = 0.043 ] was reached when comparing surgery with surgery  + lox depletion groups ) . collectively , our results suggest that lox in the plasma from post - surgery mice promotes tumor cell seeding , and its depletion inhibits collagen synthesis and ecm remodeling . to bolster the possibility that our results may be clinically relevant , we employed two different experimental approaches . in the first , we used a more clinically relevant tumor mouse model of metastasis , in which spontaneous metastases appear in the lungs after resection of a primary orthotopic 4t1 murine breast cancer . to inhibit lox ,", "bapn - treated mice exhibited significantly extended survival and reduced lung metastasis in comparison to mice that did not receive treatment after surgery ( figures 6a6d ) . in the second approach , plasma was drawn from colorectal cancer patients at baseline and 24  hr after they underwent abdominal surgery ( n  = 6 ) .", "the plasma was injected into mice , and metastases were evaluated using emt/6-gfp+ cells in an experimental lung metastasis assay . in five out of six cases , the post - surgery plasma induced higher numbers of lung metastases in comparison to baseline plasma ( figures 6e and 6f ) .", "such post - surgical plasma also induced ecm remodeling and increased fibrillar collagen expression as well as lox activity in the lungs in a manner similar to that found in mice that underwent abdominal surgery ( figures 6 g , s6a , and s6b ) .", "importantly , pooled plasma obtained from patients that underwent surgery that was depleted of lox did not increase metastatic seeding in the lungs as assessed by puma ( figures 6h and s6c ) .", "the notion that tumor cells remaining at the site of a surgical resection can re - grow and sometimes even spread may contribute to inferior outcomes in both clinical and preclinical scenarios ( coffey et  al . , 2003 , delisser et  al .", "we find that the host , in response to surgery , conditions potential metastatic sites to facilitate tumor cell seeding .", "we demonstrate that mice that undergo abdominal surgery and are subsequently injected with tumor cells via the tail vein succumb to extensive metastatic lesions earlier than controls .", "these mice exhibit structural changes in the ecm at the site of metastasis due to an increase in newly synthesized collagen .", "lox activity and expression are substantially enhanced in the lungs of mice following surgery , as well as in mice pre - conditioned with plasma from donor mice that underwent surgery .", "accordingly , we find that the surgery is associated with release of lox to the circulation from the hypoxic wounded site , which in turn induces the formation of the pre - metastatic sites .", "such pro - metastatic effects are abolished following lox inhibition ( e.g. , using bapn , neutralizing antibodies for lox , or lox depletion from plasma ) .", "notably , antibodies only neutralize extracellular lox and therefore act primarily on the ecm rather than on lox intracellular targets .", "lox has been implicated as a key enzyme contributing to the pre - metastatic niche promoting the recruitment of cd11b cells to future metastatic sites measured within weeks after primary tumor cell implantation ( erler et  al . , 2009 ) . furthermore , increased activity of lox and collagen crosslinking", "have been shown to promote fibrosis and subsequent metastasis ( cox et  al . , 2013 ) .", "in our study , we focused on the immediate contribution of lox to metastasis by means of ecm remodeling , which promotes the seeding of tumor cells in the lungs within hours following surgery . at this time point , no changes in cd11b cells colonizing the pre - metastatic sites were observed , and there was no significant difference in pulmonary permeability , suggesting that inflammation at this early time point did not contribute to tumor cell seeding .", "however , it should be noted that clinically , the use of ketorolac ( an analgesic drug with non - steroidal anti - inflammatory properties ) in surgery resulted in superior disease - free survival of breast cancer patients who underwent mastectomy compared to patients who underwent mastectomy but were not treated with ketorolac ( retsky et  al . , 2012 ) .", "have demonstrated that lox is a key regulator of metastasis that is induced by hypoxia .", "lox expression in hypoxic tumors correlates with increased incidence of metastasis in patients with breast and head and neck cancers .", "accordingly , the blockade of lox in mice bearing orthotopic breast cancers results in decreased tumor cell invasiveness and metastasis ( erler et  al . , 2006 ) .", "in addition , bone metastasis of triple negative breast cancer cells is associated with hypoxia and lox activity ( cox et  al . , 2015 ) .", "family members promote endothelial cell activity and angiogenesis in tumors and support ecm remodeling ( baker et  al .", "therefore , antiangiogenic therapy , which induces hypoxia in tumors ( blagosklonny , 2001 ) , may lead to increased lox expression . as a result", ", alterations in ecm may take place in distant sites ( e.g. , lungs or liver ) that can decrease the therapeutic response to therapy .", "these effects may also explain why , at least pre - clinically , antiangiogenic drugs sometimes promote metastasis ( ebos et  al . , 2009 , pez - ribes et  al . , 2009 ,", "the question remains why lox has a significant effect on ecm remodeling in the lungs while its expression and activity in other organs are reduced or do not change in post - surgery mice when compared to control mice .", "it is possible that the high oxygen concentration in lung tissue contributes to lox enzymatic activity .", "abundant microvessel networks in the lungs contribute to increased oxygen availability , which in turn promotes lox enzymatic activity ( barker et  al .", "this may explain the increased lox enzymatic activity in the lungs compared to other organs .", "thus , the level of ecm remodeling could be higher in lung stroma than in other tissues .", "mice bearing tumors that spontaneously metastasize exhibit a reduced mortality rate when lox is inhibited following surgery .", "additionally , mice preconditioned with plasma from post - surgery colorectal cancer patients exhibit an increased number of metastatic cells in the lungs compared to mice injected with the same plasma that was depleted of lox or plasma at baseline ( before surgery ) . taken together , in addition to the known metastatic effects of lox ( barker et  al . , 2012 )", ", our study reveals that lox induction contributes to the rapid formation of a permissive niche for metastatic cell seeding in response to surgery .", "murine lewis lung carcinoma ( llc ) , emt/6 and 4t1 mammary adenocarcinoma , and human mcf7 breast carcinoma cell lines were purchased from the american type culture collection ( atcc ) .", "all cells were grown in dmem , supplemented with 10% fetal calf serum , 1% l - glutamine , 1% sodium pyruvate , and 1% streptomycin , penicillin , and neomycin in solution ( 10  mg / ml , biological industries ) .", "some of the cell lines ( as indicated below ) were stably transfected with gfp or mcherry vectors ( clontech laboratories , 632379 and 631985 , respectively ) .", "the cells were passaged in culture for no more than 4  months after being thawed from authentic stocks and were regularly tested and found to be mycoplasma - free .", "the use of animals and experimental protocols were approved by the animal care and use committee of the technion .", "balb / c female mice ( harlan ) , 810  weeks of age , were orthotopically injected with 0.5   10 mcherry - expressing 4t1 ( 4t1-mcherry+ ) to the mammary fat pad .", "tumor size was assessed regularly with vernier calipers using the formula , width  length  0.5 .", "an experimental pulmonary metastatic model was obtained using female balb / c or c57bl/6 mice , 810  weeks of age , intravenously injected through the tail vein with 2.5   10 gfp - expressing emt/6 ( emt/6-gfp+ ) or gfp - expressing llc ( llc - gfp+ ) cells , respectively .", "mice were not randomized after surgery except in the case of the clinically relevant tumor model ( 4t1 mouse model ) .", "the experiments were not blinded to the investigator ; however , they were blinded to the mouse health care attendant who carried out daily health checks and reported on mice at endpoint .", "10-week - old female lox c57bl/6 heterozygous mice described previously ( mki et  al . , 2002 ) were used in some experiments .", "mice were anesthetized using an induction of 4% isoflurane , followed by maintenance anesthesia with 1.5% isoflurane .", "a 11.5  cm incision in the peritoneum was then performed , followed by suturing with silk . for resection of primary 4t1 tumors , when tumors reached 150200  mm ,", ". of note , all mice ( control and surgery groups ) received anesthesia and analgesic buprenorphine at a concentration of 0.04  mg / kg for three sequential days according to the institutional ethical protocols .", "8- to 10-week - old balb / c or c57bl lox mice underwent the surgical procedure ( as above ) or left as control .", "after 24  hr , blood was drawn by cardiac puncture using citrate tubes , and plasma was separated .", "plasma was injected intraperitoneally into recipient mice at a volume of 100  l / mouse . in some experiments ,", "bapn ( sigma - aldrich ) was injected intraperitoneally at a dose of 100  mg / kg daily for seven consecutive days , or as indicated in the text , as previously described ( bondareva et  al . , 2009 ) .", "the antibodies were intraperitoneally injected at a dose of 20  mg / kg as previously described ( erler et  al . , 2006 ) .", "control rabbit igg antibodies ( jackson immunoresearch laboratories ) were injected intraperitoneally at a dose of 20  mg / kg .", "recombinant lox ( origene ) was injected intraperitoneally at a dose of 25  g / kg , as previously described ( cox et  al . , 2015 ) .", "the human study was approved by the ethic committee at the european institute of oncology ( eio ) , milan , italy , and all patients signed an informed consent .", "plasma from colorectal cancer patients , who underwent open abdominal surgery , was provided by the department of pathology at the eio .", "blood from the patients ( n  = 6 ) was collected at baseline ( before surgery ) and 24  hr after surgery .", "blood was drawn from anaesthetized mice by cardiac puncture and collected in edta tubes , and red blood cells were lysed .", "lung samples or matrigel plugs were prepared as single cell suspensions as previously described ( adini et  al . , 2009 , gingis - velitski et  al . , 2011 ) .", "cecs , ceps , emt/6-gfp , and 4t1-mcherry+ were analyzed by flow cytometry as previously described ( shaked et  al . , 2005 ) .", "frozen lung sections or matrigel plugs were immunostained as previously described ( gingis - velitski et  al . , 2011 ) .", "plates coated with collagen ( 20  g / ml ) , fibronectin ( 20  g / ml ) , or laminin ( 20  g / ml ) were incubated with plasma from control , post - surgery mice or plasma from post - surgery mice depleted of lox . after 4  hr , the plasma was washed and mcf7 cells ( 7   10/well ) were seeded and cultured for 24  hr . mcf7 cells were then removed , and lysates were prepared to evaluate paxillin and p - pax expression by western blotting .", "the assay was performed as previously described ( mendoza et  al . , 2010 ) .", "collagen production in lung lysates was quantified using the sircol collagen assay kit ( biocolor ) in accordance with the manufacturer s instructions .", "details are provided in supplemental experimental procedures . to visualize ecm and collagen , a two - photon microscopy and a second harmonics generation system", "lungs from the different groups as indicated in the text were frozen in optimal cutting temperature ( oct ) and sliced at a thickness of 150  m in pbs .", "the slices were imaged using a two - photon microscope 2pm : zeiss lsm 510 meta nlo , equipped with a broadband mai tai - hp - femtosecond single box tunable ti - sapphire oscillator , with automated broadband wavelength tuning 7001,020  nm from spectraphysics , for two - photon excitation . for second harmonic imaging ( shg ) to detect collagen , a wavelength of 800  nm was used ( detection at 400  nm ) .", "data were collected ( n  = 3 mice / group ) . for quantification measurements ,", "images were analyzed using imagej 1.41k . to avoid edge effects ( attenuation of the shg signal at the top and bottom of the section ) , only the central image of each z", "mean gray value limited to threshold of each image was calculated for each image and averaged over a set of at least five fields of view .", "lox activity was evaluated as previously described ( siegel , 1974 , siegel et  al . , 1970 ) .", "briefly , a reaction solution containing 50  mm sodium borate ( ph  = 8.2 ) and 4  u / ml horseradish peroxidase was mixed with lung extracts to obtain a protein concentration of 250  g / ml . the lysate extracts were obtained from mice treated with bapn , anti - lox , recombinant lox , or rabbit polyclonal antibody using the same concentrations as above . furthermore , lysate extracts of lungs or peritoneum obtained from control mice or mice that underwent surgery were treated with bapn , anti - lox antibodies , recombinant lox , or rabbit polyclonal antibody and used as controls for the lox activity assay .", "the enzymatic reaction was started by adding substrate mixture containing 50  mm sodium borate ( ph  = 8.2 ) , 100  mm n - acetyl-3,7-dihydroxyphenoxazine ( amplex red ; molecular probes , invitrogen ) , and 20  mm 1,5-diaminopentane . in some experiments , 500  m bapn was added to the mixture as a negative inhibitory control . the production of h2o2 by lox results in fluorescent resorfurin production , which can be measured ( excitation at 540  nm and emission at 580  nm wavelengths ) .", "the fluorescent reaction was measured every 5  min for 1.5  hr at 37c using a fluorimeter ( fluo star galaxy ) .", "lox expression in plasma and organ lysates ( 50  g protein ) was evaluated by elisa ( cloud - clone - corp .", "sec580mu ) in accordance with the manufacturer s instructions . to analyze the activity of lox on different substrates ,", "the oxidation assay was performed as previously described ( kraft - sheleg et  al . , 2016 , zaffryar - eilot et  al . , 2013 ) .", "briefly , the lox activity assay was performed as above using lung lysates from control mice at a concentration of 200  g / ml .", "the only modification is that the substrate ( 1,5-diaminopentane ) used in the lox activity assay was replaced with collagen ( 1  mg / ml ) , fibronectin ( 1  mg / ml ) , or laminin ( 1  mg / ml ) .", "the fluorescent reaction was measured every 5  min for 1.5  hr at 37c using a fluorimeter ( fluo star galaxy ) . a representative plot out", "statistical significance of the in  vitro experiments was determined by either two - tailed student s t test for a comparison between two groups or one - way anova for multiple groups followed by tukey ad hoc statistical test using graphpad prism 5.0 . for the lox activity assay and human experiment results , a comparison between control / baseline and the related group was calculated based on paired student s t test .", "the number of replicates for each experiment is provided in supplemental information and/or figure legends . in the in  vitro experiments , estimate of variance", "was performed and parameters for the statistical test were adjusted accordingly . in the in  vivo / ex  vivo studies ,", "n  = 37 mice / group ( as specified in the figures ) was used to reach statistical significance . the sample size for each experiment", "was designed to have 80% power at a two - sided  of 0.05 . for the calculation of mouse survival", ", a kaplan - meier survival curve statistical analysis was performed in which the uncertainty of the fractional survival of 95% confidence intervals was calculated .", "c.r .- t . , p.h . , and y.s . conceived and designed all experiments ."], "abstract_text": ["<S> summarysurgery remains the most successful curative treatment for cancer . </S>", "<S> however , some patients with early - stage disease who undergo surgery eventually succumb to distant metastasis . here </S>", "<S> , we show that in response to surgery , the lungs become more vulnerable to metastasis due to extracellular matrix remodeling . </S>", "<S> mice that undergo surgery or that are preconditioned with plasma from donor mice that underwent surgery succumb to lung metastases earlier than controls . increased lysyl oxidase ( lox ) activity and expression , fibrillary collagen crosslinking , and focal adhesion signaling contribute to this effect , with the hypoxic surgical site serving as the source of lox . </S>", "<S> furthermore , the lungs of recipient mice injected with plasma from post - surgical colorectal cancer patients are more prone to metastatic seeding than mice injected with baseline plasma . </S>", "<S> downregulation of lox activity or levels reduces lung metastasis after surgery and increases survival , highlighting the potential of lox inhibition in reducing the risk of metastasis following surgery . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Discussion", "Experimental Procedures", "Author Contributions"], "sections": [["surgical resection of tumors is a common therapeutic procedure , especially for early - stage localized , and potentially curative , disease .", "while surgery can ultimately cure many patients , such as those with early - stage disease , distant macroscopic metastasis can emerge in others months to years later ( demicheli et  al .", "it has been reported that 25%30% of colorectal cancer patients who have no visible  metastasis at the time of diagnosis will develop distant metastases within 5 years after primary tumor resection , which  in some cases may be related to the effects of the surgery ( van der bij et  al . , 2009 ) .", "similarly , high risk of recurrence for early - stage breast cancer patients following mastectomy has been reported in an analysis of 1,173 patients who underwent mastectomy with no subsequent adjuvant systemic therapy ( demicheli et  al . , 1996 ) .", "mechanisms to explain distant metastasis following primary tumor resection include ( 1 ) the presence of residual tumor cells or tissue at the resected site ( ando et  al . , 2003 , minsky et  al . , 1988 ) ,", "( 2 ) the recruitment of inflammatory cells and platelets to the resected site that promote wound healing and cell proliferation ( ceelen et  al . , 2014 , hofer et  al . , 1999 , retsky et  al . ,", "2012 ) , and ( 3 ) increased local and systemic effects that can induce an angiogenic switch in remote dormant tumors ( bono et  al . , 2010 , retsky et  al . , 2012 , takemoto et  al . , 2012 ) .", "previous studies have revealed that hypoxic tumor cells stimulate angiogenic - related factors via hif1- , leading to increased tumor invasion ( paraskeva et  al . , 2006 ,", "hif1- expression in tumors can also upregulate lysyl oxidase ( lox ) ( erler et  al . ,", "2006 ) , a member of the secreted copper - dependent amine oxidases known to covalently crosslink collagens and elastin in the extracellular matrix ( ecm ) ( barker et  al . , 2012 ) .", "lox is expressed by different cell types , including tumor cells and stromal cells within the tumor microenvironment ( decitre et  al . , 1998 ) .", "it has been shown that increased lox expression in tumors accounts for the recruitment of cd11b bone - marrow - derived cells ( bmdcs ) at distant organs , contributing to the formation of a niche and facilitating a pre - metastatic microenvironment for tumor cell seeding ( erler et  al . , 2009 ) .", "however , the contribution of lox to tumor cell seeding and subsequently to metastasis soon after surgery is unknown . the host response to anti - cancer therapy and its contribution to tumor ( re)growth and metastasis has been evaluated following chemotherapy ( daenen et  al . , 2011 , gingis - velitski et  al . , 2011 ) , radiotherapy ( barcellos - hoff et  al . , 2005 ,", "2015 ) , and various molecularly targeted drugs ( beyar - katz et  al . , 2016 ) ( for a review , see shaked , 2016 ) . here", ", we evaluated remote ( pulmonary ) changes in lox expression and activity in response to surgery and their contribution to tumor cell seeding and metastasis ."], ["increased metastases after localized tumor resection in some cases could be due to systemic changes that affect various host tissues in response to surgery .", "previous clinical studies indicated that both systemic and local angiogenesis are induced in response to surgery when compared to laparoscopy ( bono et  al . , 2010 ) .", "to test whether our surgical mouse model induces angiogenesis , we performed a surgical procedure in non - tumor - bearing mice involving a 1  cm incision in the peritoneum followed by suturing .", "thereafter , we evaluated the levels of circulating bone - marrow - derived proangiogenic cells over time and the extent of local angiogenesis following surgery . a significant increase in the number of viable circulating endothelial cells ( cecs ) and endothelial progenitor cells ( ceps )", "likewise , increases in microvessel density in matrigel plugs , in  vitro human umbilical vein endothelial cell ( huvec ) microvessel tube formation , and microvessel sprouting from murine aortic rings were observed in the presence of plasma obtained from post - surgery mice compared to control ( figure  s1b ", "the surgical procedure in our mouse model mimics host angiogenic effects reported in certain clinical circumstances .", "we next evaluated the host - derived effects of surgery on tumor cell seeding and growth in metastatic sites .", "we employed an experimental lung metastasis assay using the murine emt/6-gfp+ breast cancer cell line .", "control mice and mice that previously underwent surgery were intravenously injected with tumor cells , which subsequently seeded in the lungs .", "mortality rates were increased in post - surgery mice compared to controls ( figure  1a ) .", "similarly , recipient mice injected with 100  l plasma from donor mice that had undergone surgery exhibited an increased mortality rate in comparison to mice injected with control plasma ( figure  1b ) , indicating that a host effect , expressed within the plasma , in response to surgery rather than the actual surgical procedure is responsible for the observed effects . to directly assess tumor cell seeding in the lungs , regardless of any host responses that may affect tumor cells per se while seeding and proliferating at the metastatic sites , a pulmonary metastatic assay ( puma ) was performed using the emt/6-gfp+ cell line . in this assay ,", "the potential of tumor cell seeding is solely dependent on tumor cells binding to the lungs and not systemic effects that could affect tumor cell proliferation .", "the number of gfp+ tumor cells present in the lungs of mice that underwent surgery was substantially elevated in comparison to control mice ( figures 1c and 1d ) . taken together , our results suggest that enhanced tumor cell seeding is a result of remote changes occurring , in part , in the pre - metastatic ( lung ) tissue in response to surgery .", "ecm remodeling is one of the main factors that can facilitate tumor cell seeding ; lox mediates collagen crosslinking and therefore is a key regulator of ecm remodeling ( barker et  al . , 2012 ) .", "recent studies have shown that lox is expressed by tumor cells , and serves as a key enzyme promoting metastasis by contributing to a pre - metastatic niche ( cox et  al . , 2015 , erler et  al . , 2006 , erler et  al . , 2009 ) . in our model", ", we found that lox expression and activity were significantly upregulated at the hypoxic surgical site in the wounded peritoneum in comparison to control peritoneum ( figures 2a2c ) .", "specifically , high - magnification images of the surgical site revealed intracellular lox expression of peritoneal myofibers ( figure  s2a ) . furthermore , lox expression and activity , quantity of newly synthesized collagen , and fibrillar collagen expression were all significantly higher in the lungs of mice that underwent surgery than in the lungs of control mice ( figures 2d2i ) .", "notably , high lox extracellular staining was located mainly in the lung stroma surrounding the bronchioles in post - surgery lungs when compared to control lungs ( figure  2j ) .", "however , high - magnification images detected intracellular lox expression in different cell types , with no noticeable differences in expression pattern between control and post - surgery lungs , suggesting that the major source of lox in the lungs of post - surgery  mice is from the remote surgical site ( figure  s2b ) . in addition , a significant decrease in lox expression and activity was found in the liver of mice that underwent surgery compared to control mice , whereas no significant changes were observed in the spleen ( figures s2c and s2d ) .", "importantly , 24  hr after surgery , the percentage of cd11b cells in the pre - metastatic lungs of post - surgery mice was comparable to that in control mice ( figures s2e and s2f ) , ruling out the possibility of the formation of a reported pre - metastatic niche at this early time point ( erler et  al . , 2009 , kaplan et  al . , 2005 ,", "these results indicate that the immediate effects of surgery are primarily associated with ecm remodeling . in order to strengthen the hypothesis that lox is a major contributor to metastatic seeding in the lungs within hours of surgery , a puma , which measures cell seeding in the lungs ,", "was carried out on mice 24  hr after they were injected with recombinant lox , the function of which was evaluated by a lox activity assay ( figure  s2 g ) .", "an increased number of metastatic cells was found in the lungs of mice injected with recombinant lox compared to control mice injected with pbs ( figures 2k and 2l ) .", "the seeding of cells in collagen and ecm is regulated in part by integrin signaling pathways ( larsen et  al . , 2006 ) .", "one of the major readouts of integrin signaling is paxillin , a scaffold protein connecting focal adhesion kinase to the actin cytoskeleton ( turner et  al . , 1990 , zaidel - bar et  al . , 2007 ) .", "we therefore sought to determine the level of phospho - paxillin ( p - pax ) in mcf-7 breast carcinoma cells , a cell line with minimal lox expression ( kraft - sheleg et  al . , 2016 ) , in response to plasma from control or post - surgery mice .", "mcf-7 cells were seeded on collagen , laminin , or fibronectin , all of which are ligands of integrins ( heino and kpyl , 2009 ) , in the presence or absence of plasma from control or post - surgery mice . increased expression of p - pax", "was observed with all three ligands in the presence of plasma from post - surgery mice compared to control , with laminin producing the weakest effect ( figures 3a and 3b ) .", "in addition , when the same experiment was performed using plasma from post - surgery mice depleted of lox , the expression of p - pax was found to be lower than the post - surgery non - depleted plasma group ( figures 3c and 3d ) , suggesting that lox expression mediates focal adhesion signaling .", "furthermore , expression of p - pax was higher in the lungs of mice that underwent surgery than in control mice , an effect that could be inhibited by treating the mice with -aminoproprionitrile ( bapn ) , an irreversible competitive inhibitor of all lox family members ( bondareva et  al . , 2009 )", "it should be noted that all three ligands are substrates of lox as assessed by the oxidation assay , whereas laminin was the least effective substrate , in line with the results shown in figures 3a3d ( figure  s3a ) .", "taken together , increased lox activity in the lungs soon after the mice undergo surgery contributes to tumor cell seeding via focal adhesion signaling , suggesting interactions between integrins and lox ligands in the remodeled ecm .", "as lox contributes to the seeding of tumor cells in the lungs , we next studied the effects of lox inhibition in mice using bapn .", "plasma from control mice or mice that underwent surgery was injected into recipient naive mice .", "after 24  hr , the recipient mice were injected with emt/6-gfp+ cells to obtain the experimental lung colonization metastasis assay described above .", "bapn monotherapy did not affect the percentage of gfp+ cells in the lungs of control mice .", "in contrast , the increase in percentage of gfp+ cells in the lungs of mice injected with plasma obtained from the post - surgery mice was completely abolished by the bapn therapy ( figures 4a and 4b ) , and their mortality rate was  reduced , as assessed in a parallel experiment ( figure  4c ) .", "no significant changes in mortality rate and percentage of gfp+ cells in the lungs of control mice treated with bapn or pbs control were found ( figures 4b and 4c ) .", "importantly , escalating doses of bapn in a range of 010  mg / ml did not contribute to tumor cell apoptosis ( figure  s3b ) , suggesting that the therapeutic effect of bapn is related to the inhibition of lox activity .", "furthermore , inhibiting lox using specific anti - lox activity - inhibiting antibodies similarly reduced tumor cell seeding in the lungs of mice injected with plasma from post - surgery mice , indicating that the effect is largely regulated by lox .", "of note , it is not clear why blocking lox in control mice increased metastatic cell seeding ( figures 4d4f ) .", "it is possible that other bypass pathways may play a role similar to mmp9 inhibition in control mice as previously described ( gingis - velitski et  al . , 2011 ,", "moreover , fibrillar collagen and newly synthesized collagen quantities in the lungs of mice 3  days after localized peritoneal surgery were significantly increased when compared to lungs obtained from post - surgery mice treated with bapn or anti - lox antibodies ( figures 4g4i ) . of note ,", "both anti - lox and bapn reduced lox activity in the lungs , while control rabbit immunoglobulin g ( igg ) had no effect ( figures s3c and s4 , respectively ) .", "to further strengthen our results , we wished to rule out the possibility that inflammation - induced pulmonary vessel permeability mediates the increased tumor cell seeding in the lungs at this early time point .", "evans blue assay performed on mice 24  hr after they underwent surgery revealed that pulmonary vessel permeability was not significantly different in post - surgery mice compared to control mice ( figure  s5a ) , suggesting that tumor cell seeding was not affected by inflammation . additionally , the number and percentage of tumor cells ( emt/6-gfp+ ) seeded in the lungs 20  min after tail vein injection of 2   10 cells ( 10-fold higher than the number of cells injected in the puma ) were significantly higher in post - surgery mice than in control mice .", "this effect was dramatically reduced when the mice were also treated with bapn as assessed by microscopic imaging of lung specimens and flow cytometry of single - cell suspensions from dissociated lungs ( figures s5b and s5c ) .", "taken together , lox - induced ecm remodeling accounts for tumor cell seeding in the lungs of post - surgery mice .", "the fact that plasma derived from post - surgery mice had the same effect on tumor cell seeding as surgery itself suggested it entails the factor mediating this activity .", "therefore , lox levels were evaluated in the plasma of control and post - surgery mice .", "the plasma of post - surgery mice exhibited significantly elevated lox levels compared to plasma from control mice , suggesting that lox upregulation in the plasma may affect tumor cell seeding in the lungs ( figure  5a ) . to test this possibility , we depleted lox from plasma of control mice and mice that underwent surgery and assessed tumor cell seeding using puma in recipient mice injected with the different plasma samples .", "whereas lox depletion did not affect tumor cell seeding in the control group , it significantly decreased tumor cell seeding in the surgery group ( figures 5b and 5c ) . in a parallel experiment", ", plasma was obtained from untreated wild - type mice or bapn - treated lox heterozygous mice ( mki et  al . ,", "the plasma was then injected into mice , and survival was assessed in an experimental lung metastasis assay .", "however , in the surgery group , survival times were increased when lox was inhibited ( figures 5d and 5e ; statistical significance [ p  = 0.043 ] was reached when comparing surgery with surgery  + lox depletion groups ) . collectively , our results suggest that lox in the plasma from post - surgery mice promotes tumor cell seeding , and its depletion inhibits collagen synthesis and ecm remodeling . to bolster the possibility that our results may be clinically relevant , we employed two different experimental approaches . in the first , we used a more clinically relevant tumor mouse model of metastasis , in which spontaneous metastases appear in the lungs after resection of a primary orthotopic 4t1 murine breast cancer . to inhibit lox ,", "bapn - treated mice exhibited significantly extended survival and reduced lung metastasis in comparison to mice that did not receive treatment after surgery ( figures 6a6d ) . in the second approach , plasma was drawn from colorectal cancer patients at baseline and 24  hr after they underwent abdominal surgery ( n  = 6 ) .", "the plasma was injected into mice , and metastases were evaluated using emt/6-gfp+ cells in an experimental lung metastasis assay . in five out of six cases , the post - surgery plasma induced higher numbers of lung metastases in comparison to baseline plasma ( figures 6e and 6f ) .", "such post - surgical plasma also induced ecm remodeling and increased fibrillar collagen expression as well as lox activity in the lungs in a manner similar to that found in mice that underwent abdominal surgery ( figures 6 g , s6a , and s6b ) .", "importantly , pooled plasma obtained from patients that underwent surgery that was depleted of lox did not increase metastatic seeding in the lungs as assessed by puma ( figures 6h and s6c ) ."], ["the notion that tumor cells remaining at the site of a surgical resection can re - grow and sometimes even spread may contribute to inferior outcomes in both clinical and preclinical scenarios ( coffey et  al . , 2003 , delisser et  al .", "we find that the host , in response to surgery , conditions potential metastatic sites to facilitate tumor cell seeding .", "we demonstrate that mice that undergo abdominal surgery and are subsequently injected with tumor cells via the tail vein succumb to extensive metastatic lesions earlier than controls .", "these mice exhibit structural changes in the ecm at the site of metastasis due to an increase in newly synthesized collagen .", "lox activity and expression are substantially enhanced in the lungs of mice following surgery , as well as in mice pre - conditioned with plasma from donor mice that underwent surgery .", "accordingly , we find that the surgery is associated with release of lox to the circulation from the hypoxic wounded site , which in turn induces the formation of the pre - metastatic sites .", "such pro - metastatic effects are abolished following lox inhibition ( e.g. , using bapn , neutralizing antibodies for lox , or lox depletion from plasma ) .", "notably , antibodies only neutralize extracellular lox and therefore act primarily on the ecm rather than on lox intracellular targets .", "lox has been implicated as a key enzyme contributing to the pre - metastatic niche promoting the recruitment of cd11b cells to future metastatic sites measured within weeks after primary tumor cell implantation ( erler et  al . , 2009 ) . furthermore , increased activity of lox and collagen crosslinking", "have been shown to promote fibrosis and subsequent metastasis ( cox et  al . , 2013 ) .", "in our study , we focused on the immediate contribution of lox to metastasis by means of ecm remodeling , which promotes the seeding of tumor cells in the lungs within hours following surgery . at this time point , no changes in cd11b cells colonizing the pre - metastatic sites were observed , and there was no significant difference in pulmonary permeability , suggesting that inflammation at this early time point did not contribute to tumor cell seeding .", "however , it should be noted that clinically , the use of ketorolac ( an analgesic drug with non - steroidal anti - inflammatory properties ) in surgery resulted in superior disease - free survival of breast cancer patients who underwent mastectomy compared to patients who underwent mastectomy but were not treated with ketorolac ( retsky et  al . , 2012 ) .", "have demonstrated that lox is a key regulator of metastasis that is induced by hypoxia .", "lox expression in hypoxic tumors correlates with increased incidence of metastasis in patients with breast and head and neck cancers .", "accordingly , the blockade of lox in mice bearing orthotopic breast cancers results in decreased tumor cell invasiveness and metastasis ( erler et  al . , 2006 ) .", "in addition , bone metastasis of triple negative breast cancer cells is associated with hypoxia and lox activity ( cox et  al . , 2015 ) .", "family members promote endothelial cell activity and angiogenesis in tumors and support ecm remodeling ( baker et  al .", "therefore , antiangiogenic therapy , which induces hypoxia in tumors ( blagosklonny , 2001 ) , may lead to increased lox expression . as a result", ", alterations in ecm may take place in distant sites ( e.g. , lungs or liver ) that can decrease the therapeutic response to therapy .", "these effects may also explain why , at least pre - clinically , antiangiogenic drugs sometimes promote metastasis ( ebos et  al . , 2009 , pez - ribes et  al . , 2009 ,", "the question remains why lox has a significant effect on ecm remodeling in the lungs while its expression and activity in other organs are reduced or do not change in post - surgery mice when compared to control mice .", "it is possible that the high oxygen concentration in lung tissue contributes to lox enzymatic activity .", "abundant microvessel networks in the lungs contribute to increased oxygen availability , which in turn promotes lox enzymatic activity ( barker et  al .", "this may explain the increased lox enzymatic activity in the lungs compared to other organs .", "thus , the level of ecm remodeling could be higher in lung stroma than in other tissues .", "mice bearing tumors that spontaneously metastasize exhibit a reduced mortality rate when lox is inhibited following surgery .", "additionally , mice preconditioned with plasma from post - surgery colorectal cancer patients exhibit an increased number of metastatic cells in the lungs compared to mice injected with the same plasma that was depleted of lox or plasma at baseline ( before surgery ) . taken together , in addition to the known metastatic effects of lox ( barker et  al . , 2012 )", ", our study reveals that lox induction contributes to the rapid formation of a permissive niche for metastatic cell seeding in response to surgery ."], ["murine lewis lung carcinoma ( llc ) , emt/6 and 4t1 mammary adenocarcinoma , and human mcf7 breast carcinoma cell lines were purchased from the american type culture collection ( atcc ) .", "all cells were grown in dmem , supplemented with 10% fetal calf serum , 1% l - glutamine , 1% sodium pyruvate , and 1% streptomycin , penicillin , and neomycin in solution ( 10  mg / ml , biological industries ) .", "some of the cell lines ( as indicated below ) were stably transfected with gfp or mcherry vectors ( clontech laboratories , 632379 and 631985 , respectively ) .", "the cells were passaged in culture for no more than 4  months after being thawed from authentic stocks and were regularly tested and found to be mycoplasma - free .", "the use of animals and experimental protocols were approved by the animal care and use committee of the technion .", "balb / c female mice ( harlan ) , 810  weeks of age , were orthotopically injected with 0.5   10 mcherry - expressing 4t1 ( 4t1-mcherry+ ) to the mammary fat pad .", "tumor size was assessed regularly with vernier calipers using the formula , width  length  0.5 .", "an experimental pulmonary metastatic model was obtained using female balb / c or c57bl/6 mice , 810  weeks of age , intravenously injected through the tail vein with 2.5   10 gfp - expressing emt/6 ( emt/6-gfp+ ) or gfp - expressing llc ( llc - gfp+ ) cells , respectively .", "mice were not randomized after surgery except in the case of the clinically relevant tumor model ( 4t1 mouse model ) .", "the experiments were not blinded to the investigator ; however , they were blinded to the mouse health care attendant who carried out daily health checks and reported on mice at endpoint .", "10-week - old female lox c57bl/6 heterozygous mice described previously ( mki et  al . , 2002 ) were used in some experiments .", "mice were anesthetized using an induction of 4% isoflurane , followed by maintenance anesthesia with 1.5% isoflurane .", "a 11.5  cm incision in the peritoneum was then performed , followed by suturing with silk . for resection of primary 4t1 tumors , when tumors reached 150200  mm ,", ". of note , all mice ( control and surgery groups ) received anesthesia and analgesic buprenorphine at a concentration of 0.04  mg / kg for three sequential days according to the institutional ethical protocols .", "8- to 10-week - old balb / c or c57bl lox mice underwent the surgical procedure ( as above ) or left as control .", "after 24  hr , blood was drawn by cardiac puncture using citrate tubes , and plasma was separated .", "plasma was injected intraperitoneally into recipient mice at a volume of 100  l / mouse . in some experiments ,", "bapn ( sigma - aldrich ) was injected intraperitoneally at a dose of 100  mg / kg daily for seven consecutive days , or as indicated in the text , as previously described ( bondareva et  al . , 2009 ) .", "the antibodies were intraperitoneally injected at a dose of 20  mg / kg as previously described ( erler et  al . , 2006 ) .", "control rabbit igg antibodies ( jackson immunoresearch laboratories ) were injected intraperitoneally at a dose of 20  mg / kg .", "recombinant lox ( origene ) was injected intraperitoneally at a dose of 25  g / kg , as previously described ( cox et  al . , 2015 ) .", "the human study was approved by the ethic committee at the european institute of oncology ( eio ) , milan , italy , and all patients signed an informed consent .", "plasma from colorectal cancer patients , who underwent open abdominal surgery , was provided by the department of pathology at the eio .", "blood from the patients ( n  = 6 ) was collected at baseline ( before surgery ) and 24  hr after surgery .", "blood was drawn from anaesthetized mice by cardiac puncture and collected in edta tubes , and red blood cells were lysed .", "lung samples or matrigel plugs were prepared as single cell suspensions as previously described ( adini et  al . , 2009 , gingis - velitski et  al . , 2011 ) .", "cecs , ceps , emt/6-gfp , and 4t1-mcherry+ were analyzed by flow cytometry as previously described ( shaked et  al . , 2005 ) .", "frozen lung sections or matrigel plugs were immunostained as previously described ( gingis - velitski et  al . , 2011 ) .", "plates coated with collagen ( 20  g / ml ) , fibronectin ( 20  g / ml ) , or laminin ( 20  g / ml ) were incubated with plasma from control , post - surgery mice or plasma from post - surgery mice depleted of lox . after 4  hr , the plasma was washed and mcf7 cells ( 7   10/well ) were seeded and cultured for 24  hr . mcf7 cells were then removed , and lysates were prepared to evaluate paxillin and p - pax expression by western blotting .", "the assay was performed as previously described ( mendoza et  al . , 2010 ) .", "collagen production in lung lysates was quantified using the sircol collagen assay kit ( biocolor ) in accordance with the manufacturer s instructions .", "details are provided in supplemental experimental procedures . to visualize ecm and collagen , a two - photon microscopy and a second harmonics generation system", "lungs from the different groups as indicated in the text were frozen in optimal cutting temperature ( oct ) and sliced at a thickness of 150  m in pbs .", "the slices were imaged using a two - photon microscope 2pm : zeiss lsm 510 meta nlo , equipped with a broadband mai tai - hp - femtosecond single box tunable ti - sapphire oscillator , with automated broadband wavelength tuning 7001,020  nm from spectraphysics , for two - photon excitation . for second harmonic imaging ( shg ) to detect collagen , a wavelength of 800  nm was used ( detection at 400  nm ) .", "data were collected ( n  = 3 mice / group ) . for quantification measurements ,", "images were analyzed using imagej 1.41k . to avoid edge effects ( attenuation of the shg signal at the top and bottom of the section ) , only the central image of each z", "mean gray value limited to threshold of each image was calculated for each image and averaged over a set of at least five fields of view .", "lox activity was evaluated as previously described ( siegel , 1974 , siegel et  al . , 1970 ) .", "briefly , a reaction solution containing 50  mm sodium borate ( ph  = 8.2 ) and 4  u / ml horseradish peroxidase was mixed with lung extracts to obtain a protein concentration of 250  g / ml . the lysate extracts were obtained from mice treated with bapn , anti - lox , recombinant lox , or rabbit polyclonal antibody using the same concentrations as above . furthermore , lysate extracts of lungs or peritoneum obtained from control mice or mice that underwent surgery were treated with bapn , anti - lox antibodies , recombinant lox , or rabbit polyclonal antibody and used as controls for the lox activity assay .", "the enzymatic reaction was started by adding substrate mixture containing 50  mm sodium borate ( ph  = 8.2 ) , 100  mm n - acetyl-3,7-dihydroxyphenoxazine ( amplex red ; molecular probes , invitrogen ) , and 20  mm 1,5-diaminopentane . in some experiments , 500  m bapn was added to the mixture as a negative inhibitory control . the production of h2o2 by lox results in fluorescent resorfurin production , which can be measured ( excitation at 540  nm and emission at 580  nm wavelengths ) .", "the fluorescent reaction was measured every 5  min for 1.5  hr at 37c using a fluorimeter ( fluo star galaxy ) .", "lox expression in plasma and organ lysates ( 50  g protein ) was evaluated by elisa ( cloud - clone - corp .", "sec580mu ) in accordance with the manufacturer s instructions . to analyze the activity of lox on different substrates ,", "the oxidation assay was performed as previously described ( kraft - sheleg et  al . , 2016 , zaffryar - eilot et  al . , 2013 ) .", "briefly , the lox activity assay was performed as above using lung lysates from control mice at a concentration of 200  g / ml .", "the only modification is that the substrate ( 1,5-diaminopentane ) used in the lox activity assay was replaced with collagen ( 1  mg / ml ) , fibronectin ( 1  mg / ml ) , or laminin ( 1  mg / ml ) .", "the fluorescent reaction was measured every 5  min for 1.5  hr at 37c using a fluorimeter ( fluo star galaxy ) . a representative plot out", "statistical significance of the in  vitro experiments was determined by either two - tailed student s t test for a comparison between two groups or one - way anova for multiple groups followed by tukey ad hoc statistical test using graphpad prism 5.0 . for the lox activity assay and human experiment results , a comparison between control / baseline and the related group was calculated based on paired student s t test .", "the number of replicates for each experiment is provided in supplemental information and/or figure legends . in the in  vitro experiments , estimate of variance", "was performed and parameters for the statistical test were adjusted accordingly . in the in  vivo / ex  vivo studies ,", "n  = 37 mice / group ( as specified in the figures ) was used to reach statistical significance . the sample size for each experiment", "was designed to have 80% power at a two - sided  of 0.05 . for the calculation of mouse survival", ", a kaplan - meier survival curve statistical analysis was performed in which the uncertainty of the fractional survival of 95% confidence intervals was calculated ."], ["c.r .- t . , p.h . , and y.s . conceived and designed all experiments ."]]}
{"article_id": "PMC3585693", "article_text": ["these conditions may be life - threatening ( e.g. oral cancers ) or not , progressing ( caries , periodontitis , etc . ) or not , dealing with aesthetics ( staining in anterior teeth such as molar ", "incisor hypomineralisation ( mih ) ) or pain ( pulpitis , mih in posterior teeth , etc . ) .", "ohrqol is highly subjective and has to be assessed within the framework of patients conditions , sociocultural environments and own experiences and states of mind : because ohrqol is related to daily life and is unique to each individual , even patients with severe conditions can report having good quality of life .", "furthermore , quality of life is by itself multi - faceted , showing variation over time for each individual .", "ohrqol should therefore be assessed longitudinally to take into account changes over time , using versatile tools .", "this means having relevant questions with well - defined items and being able to analyse answers in a good way .", "many limitations can be found to the current validation testing , including relevance of the questions , validity and sensitivity to change , risk of misinterpretations ( role of the ethnocultural environment ) , problems of translation of english questionnaires and difficulty to interpret the significance of a psychometric measurement when reported simply as a numerical score or a mean [ 1 , 2 ] .", "this latter point is of importance since the same score can be obtained from people answering in a different way to a majority of questions . finally , patient - based outcome measures ( as named by fitzpatrick et al . )", "should provide the opportunity to measure the extent or intensity of the changes in ohrqol .", "various psychometric instruments have also been used to measure ohrqol ( tables  1 and 2 ) [ 1 , 4 ] .", "these are based on different criteria that enable them to be more or less patient- or expert - centred .", "some are generic ( ohip1 - 49 , ohip-14 , oidp , oh - qol , sf-362 ) and can be considered as core indicators ; others are adapted to specific conditions / domains ( orthognathic qol questionnaire , sooq for orthodontic surgery , ohip - aesthetic,3 ohrqol for dental hygiene ) or populations ( cohqol and child - oidp for children , gohai for elderly people , etc.).table 1conceptual and structural basis of psychometric instruments used in dentistry ( adapted from brondani and mcentee ) instrumentsacronymstructural originsempirically basedconnotation of questionsnumber of questionssocial impacts of dental diseasesiddsipyesn14oral health impact profileohipicidhyesn49geriatric ( generic ) oral health assessment indexgohaiicidh and sipyesn and p12oral health - related qol instrumentohrqlmultiplenon36oral impact on daily performancesoidpicidhnon8dental impact on daily livingdidlsipyesn and nt and p36dental impact profiledipsipyesn and nt and p25oral health - related quality of life measureohqolmultiplenon3oral health quality of life inventoryoh - qolsipunclearp15rand dental questionnaireunspecifiedsipnon3oral health questionnaireunspecifiedicidhunclearn and nt and p70oral health quality of life ukohqol - ukicidh2yesn and p16subjective oral health status indicatorssohsimultiplenon and nt34liverpool oral rehabilitation questionnairelorqunclearnon40self - rated oral healthsrohicidhnon and p3dentaldentalunclearnon15dental health status quality of life questionnaireds - qolgeneric qol instrumentnon and punclearn 1/4 negative , nt neutral , p positive , sip sickness impact profile , icidh international classification of impairments , disabilities and handicapsinformation derived from open - ended interviewssome indicators present shorter or extended forms other than the original versionhealth - related models : natural history of disease model and sipdeveloped from existing measures ( rand , oral facial pain index , etc.).table 2oral health outcome measures developed before 2007 ( adapted from locker and allen ) pre-1997 ( presented at the 1997 conference ) social impacts of dental diseasegeneral ( geriatric ) oral health assessment index ( gohai)dental impact profile ( dip)oral health impact profile ( ohip)oral impacts on daily performances ( oidp)subjective oral health status indicators ( sohsi)oral health - related quality of life measuredental impact on daily living ( didls)oral health quality of life inventoryrand dental questionspost-1997ohqol - ukchild oral health quality of life questionnaire ( cohqol)child oidpohrqol for dental hygieneorthognathic qol questionnairesurgical orthodontic outcome questionnaire ( sooq ) conceptual and structural basis of psychometric instruments used in dentistry ( adapted from brondani and mcentee ) n 1/4 negative , nt neutral , p positive , sip sickness impact profile , icidh international classification of impairments , disabilities and handicaps information derived from open - ended interviews some indicators present shorter or extended forms other than the original version health - related models : natural history of disease model and sip developed from existing measures ( rand , oral facial pain index , etc . ) .", "oral health outcome measures developed before 2007 ( adapted from locker and allen ) the ohip , also called ohip-49 , is the most widely used , and this has enabled investigators to modify forms that can be subsequently adapted to populations or conditions .", "the initial 49-question form was constructed to assess the  social impact of oral disorders .", "each of the set of 49 statements represented one of seven domains : it is mainly expert - centred and constructed to select items according to their fit with a conceptual framework rather than on the basis of their importance to the patients from whom they were derived .", "a shorter version of ohip restricted to 14 items ( ohip-14 ) was later proposed .", "one major question is to know if we need to use either a generic questionnaire , an adapted form of a generic questionnaire or to construct a new questionnaire specific to the population or condition to be studied .", "constructing or using one of these specific questionnaires may lead to many questions , for example , ( 1 ) is it made specifically for the purpose of research or for clinical practice ? or ( 2 ) how to adapt each questionnaire to local languages and cultures ?", "this may subsequently lead us to consider the impact of dentin hypersensitivity ( dhs ) or exposed cervical dentin ( ecd ) on ohrqol of those individuals being assessed .", "very few studies have been devoted to this aspect of dhs / ecd as recently shown , with only two papers written in english specifically dedicated to the evaluation of ohqol in dhs / ecd patients .", "one paper provided results using a generic questionnaire   and the second paper constructed a specific questionnaire to evaluate ohqol in dhs / ecd patients but provided no epidemiological results . these studies are more extensively described in an accompanying paper . in the future ,", "studies using validated questionnaires specifically constructed to evaluate the impact of the condition on ohqol should be employed .", "these questionnaires should be patient - centred and derived from interviews with patients who are expected to complete the questionnaire [ 4 , 10 ] . furthermore , if these studies also attempt to evaluate the efficacy of desensitising agents in reducing dhs / ecd and its subsequent impact on ohqol , then it is imperative that the condition should be clearly diagnosed by trained and calibrated dentists experienced in conducting clinical studies using recognised and accepted clinical criteria for the evaluation of dhs / ecd . due to the cultural and language differences between countries ,", "there is also a need of norm or reference value(s ) for each population to be studied .", "for example , when constructing a questionnaire for a non - english - speaking population , the questionnaire should be initially written in english , then translated by two people of the designated native ( foreign ) language and subsequently translated back into english by two native english - speaking people to identify any potential issues that may have arisen from the translation . finally , as indicated above , any future study attempting to evaluate the efficacy of a desensitising agent in reducing dhs / ecd and its subsequent impact on ohqol", "should be conducted by experienced and calibrated examiners using established guidelines for conducting dhs / ecd clinical studies .", "such studies should also be based on a randomised clinical study design and include both placebo or control groups .", "patients suffering from dhs / ecd have been reported to have a significantly impaired ohrqol ; this may however be improved following treatment with a desensitising agent as reported by several authors .", "it is therefore of the upmost importance that the use of the ohrqol as a quality control tool in the dental office be established in robust clinical studies .", "furthermore , because of its ability to reflect a patient s satisfaction with any proposed treatment , it may prove to be a valuable asset for practitioners when assessing their patients quality of life before , during and after treatment of various clinical conditions such as dhs / ecd ."], "abstract_text": ["<S> objectivesoral health - related quality of life ( ohrqol ) can be considered as the scientific expression of that part of a person s well - being that is affected by his / her oral health . </S>", "<S> the aim of this paper was to evaluate how to use the data available in the field of research to make a link between ohrqol and dentin hypersensitivity ( dhs ) in the dental office.materials and methodsresearch papers in the field of ohrqol and dhs and reviews and research papers about ohrqol were used for analysis in this short review , with a particular insight on the instruments used to evaluate ohrqol.resultsvarious psychometric instruments have been used to measure ohrqol that are more or less patient- or expert - centred . </S>", "<S> some are generic , others are adapted to specific conditions / domains or populations . </S>", "<S> the impact of dhs or exposed cervical dentin ( ecd ) on ohrqol has been assessed in very few studies . </S>", "<S> it is therefore of the upmost importance that the use of the ohrqol as a quality control tool be established in robust clinical studies.conclusions/clinical relevancefuture studies evaluating the impact of the dhs / ecd on ohqol or evaluating the efficacy of desensitising agents should respect some key points , including study design ( randomization , placebo / control group , etc . ) , validated specific questionnaires and trained calibrated practitioners . </S>"], "labels": null, "section_names": ["Why is it difficult to evaluate OHRQoL?", "How to assess OHRQoL?", "DHS/ECD and OHRQoL: what is known and where are the problems?", "What are the recommendations for daily dental practice?", "Conflict of interest"], "sections": [["these conditions may be life - threatening ( e.g. oral cancers ) or not , progressing ( caries , periodontitis , etc . ) or not , dealing with aesthetics ( staining in anterior teeth such as molar ", "incisor hypomineralisation ( mih ) ) or pain ( pulpitis , mih in posterior teeth , etc . ) .", "ohrqol is highly subjective and has to be assessed within the framework of patients conditions , sociocultural environments and own experiences and states of mind : because ohrqol is related to daily life and is unique to each individual , even patients with severe conditions can report having good quality of life .", "furthermore , quality of life is by itself multi - faceted , showing variation over time for each individual .", "ohrqol should therefore be assessed longitudinally to take into account changes over time , using versatile tools ."], ["this means having relevant questions with well - defined items and being able to analyse answers in a good way .", "many limitations can be found to the current validation testing , including relevance of the questions , validity and sensitivity to change , risk of misinterpretations ( role of the ethnocultural environment ) , problems of translation of english questionnaires and difficulty to interpret the significance of a psychometric measurement when reported simply as a numerical score or a mean [ 1 , 2 ] .", "this latter point is of importance since the same score can be obtained from people answering in a different way to a majority of questions . finally , patient - based outcome measures ( as named by fitzpatrick et al . )", "should provide the opportunity to measure the extent or intensity of the changes in ohrqol .", "various psychometric instruments have also been used to measure ohrqol ( tables  1 and 2 ) [ 1 , 4 ] .", "these are based on different criteria that enable them to be more or less patient- or expert - centred .", "some are generic ( ohip1 - 49 , ohip-14 , oidp , oh - qol , sf-362 ) and can be considered as core indicators ; others are adapted to specific conditions / domains ( orthognathic qol questionnaire , sooq for orthodontic surgery , ohip - aesthetic,3 ohrqol for dental hygiene ) or populations ( cohqol and child - oidp for children , gohai for elderly people , etc.).table 1conceptual and structural basis of psychometric instruments used in dentistry ( adapted from brondani and mcentee ) instrumentsacronymstructural originsempirically basedconnotation of questionsnumber of questionssocial impacts of dental diseasesiddsipyesn14oral health impact profileohipicidhyesn49geriatric ( generic ) oral health assessment indexgohaiicidh and sipyesn and p12oral health - related qol instrumentohrqlmultiplenon36oral impact on daily performancesoidpicidhnon8dental impact on daily livingdidlsipyesn and nt and p36dental impact profiledipsipyesn and nt and p25oral health - related quality of life measureohqolmultiplenon3oral health quality of life inventoryoh - qolsipunclearp15rand dental questionnaireunspecifiedsipnon3oral health questionnaireunspecifiedicidhunclearn and nt and p70oral health quality of life ukohqol - ukicidh2yesn and p16subjective oral health status indicatorssohsimultiplenon and nt34liverpool oral rehabilitation questionnairelorqunclearnon40self - rated oral healthsrohicidhnon and p3dentaldentalunclearnon15dental health status quality of life questionnaireds - qolgeneric qol instrumentnon and punclearn 1/4 negative , nt neutral , p positive , sip sickness impact profile , icidh international classification of impairments , disabilities and handicapsinformation derived from open - ended interviewssome indicators present shorter or extended forms other than the original versionhealth - related models : natural history of disease model and sipdeveloped from existing measures ( rand , oral facial pain index , etc.).table 2oral health outcome measures developed before 2007 ( adapted from locker and allen ) pre-1997 ( presented at the 1997 conference ) social impacts of dental diseasegeneral ( geriatric ) oral health assessment index ( gohai)dental impact profile ( dip)oral health impact profile ( ohip)oral impacts on daily performances ( oidp)subjective oral health status indicators ( sohsi)oral health - related quality of life measuredental impact on daily living ( didls)oral health quality of life inventoryrand dental questionspost-1997ohqol - ukchild oral health quality of life questionnaire ( cohqol)child oidpohrqol for dental hygieneorthognathic qol questionnairesurgical orthodontic outcome questionnaire ( sooq ) conceptual and structural basis of psychometric instruments used in dentistry ( adapted from brondani and mcentee ) n 1/4 negative , nt neutral , p positive , sip sickness impact profile , icidh international classification of impairments , disabilities and handicaps information derived from open - ended interviews some indicators present shorter or extended forms other than the original version health - related models : natural history of disease model and sip developed from existing measures ( rand , oral facial pain index , etc . ) .", "oral health outcome measures developed before 2007 ( adapted from locker and allen ) the ohip , also called ohip-49 , is the most widely used , and this has enabled investigators to modify forms that can be subsequently adapted to populations or conditions .", "the initial 49-question form was constructed to assess the  social impact of oral disorders .", "each of the set of 49 statements represented one of seven domains : it is mainly expert - centred and constructed to select items according to their fit with a conceptual framework rather than on the basis of their importance to the patients from whom they were derived .", "a shorter version of ohip restricted to 14 items ( ohip-14 ) was later proposed .", "one major question is to know if we need to use either a generic questionnaire , an adapted form of a generic questionnaire or to construct a new questionnaire specific to the population or condition to be studied .", "constructing or using one of these specific questionnaires may lead to many questions , for example , ( 1 ) is it made specifically for the purpose of research or for clinical practice ? or ( 2 ) how to adapt each questionnaire to local languages and cultures ?", "this may subsequently lead us to consider the impact of dentin hypersensitivity ( dhs ) or exposed cervical dentin ( ecd ) on ohrqol of those individuals being assessed ."], ["very few studies have been devoted to this aspect of dhs / ecd as recently shown , with only two papers written in english specifically dedicated to the evaluation of ohqol in dhs / ecd patients .", "one paper provided results using a generic questionnaire   and the second paper constructed a specific questionnaire to evaluate ohqol in dhs / ecd patients but provided no epidemiological results . these studies are more extensively described in an accompanying paper . in the future ,", "studies using validated questionnaires specifically constructed to evaluate the impact of the condition on ohqol should be employed .", "these questionnaires should be patient - centred and derived from interviews with patients who are expected to complete the questionnaire [ 4 , 10 ] . furthermore , if these studies also attempt to evaluate the efficacy of desensitising agents in reducing dhs / ecd and its subsequent impact on ohqol , then it is imperative that the condition should be clearly diagnosed by trained and calibrated dentists experienced in conducting clinical studies using recognised and accepted clinical criteria for the evaluation of dhs / ecd . due to the cultural and language differences between countries ,", "there is also a need of norm or reference value(s ) for each population to be studied .", "for example , when constructing a questionnaire for a non - english - speaking population , the questionnaire should be initially written in english , then translated by two people of the designated native ( foreign ) language and subsequently translated back into english by two native english - speaking people to identify any potential issues that may have arisen from the translation . finally , as indicated above , any future study attempting to evaluate the efficacy of a desensitising agent in reducing dhs / ecd and its subsequent impact on ohqol", "should be conducted by experienced and calibrated examiners using established guidelines for conducting dhs / ecd clinical studies .", "such studies should also be based on a randomised clinical study design and include both placebo or control groups ."], ["patients suffering from dhs / ecd have been reported to have a significantly impaired ohrqol ; this may however be improved following treatment with a desensitising agent as reported by several authors .", "it is therefore of the upmost importance that the use of the ohrqol as a quality control tool in the dental office be established in robust clinical studies .", "furthermore , because of its ability to reflect a patient s satisfaction with any proposed treatment , it may prove to be a valuable asset for practitioners when assessing their patients quality of life before , during and after treatment of various clinical conditions such as dhs / ecd ."], [""]]}
{"article_id": "PMC4980927", "article_text": ["male sprague - dawley rats ( n = 24 ) weighing approximately 250 g were used for the study .", "permission for the experimental work was obtained from the institutional animal ethics committee ( 767/iaec/13 dated 1 - 1 - 2014 ) .", "rats were divided into 4 equal groups and injected physiological saline ( twice daily subcutaneously in morning and evening ; group i ) , morphine sulfate ( 10 mg / kg twice daily subcutaneously ; group ii ) , fosaprepitant ( 30 mg / kg once by intraperitoneal route ; group iii ) , and finally , the last group was co - administered both morphine and fosaprepitant ( group iv ) in the same doses as in group ii and iii , respectively , for a week . in the last group , fosaprepitant was injected 30 min before the morphine injection . the dose of fosaprepitant ( 30 mg / kg ) was higher than that used in an earlier study ( 25 mg / kg ) .", "the rationale for selecting a higher dose was the rapid conversion of fosaprepitant to aprepitant ( half - life ~30 min ) in the rat compared to dogs and humans .", "hot plate latency or the thermal escape behaviour was determined in the morning , 40 min after saline ( group i ) , fosaprepitant ( group iii ) , or morphine injection ( group ii and iv ) .", "the hot plate test is commonly employed for screening the putative antinociceptive property of drugs .", "the advantages of this test are the brief nature of the nociceptive stimulus , which does not produce any tissue damage and that multiple testing can be done in the same animal .", "the predictability of this test to clinical situations is high for morphine and related opioid substances .", "testing was done in a quiet room with the ambient temperature between 22c and 25c . on the day of the experiment ,", "the testing platform of the hot plate apparatus ( stoelting , usa ) was set at a constant temperature of 52.5c .", "the rats were placed on the hot plate , and the behavioral end points were either licking of the hindpaw or jumping .", "the test was repeated thrice at 57 min intervals and the average of these values was the latency period ( sec ) .", "a 40 s , cut - off was fixed to prevent damage to the completely analgesic paw after morphine injection .", "transformation of these values was done by calculating the percent maximum possible effect ( % mpe ) as follows : ( [ drug induced latency  basal response time]/[40 s  basal response time ] ) 100 on day 8 ( morning ) , rats were anesthetized by pentobarbital injection ( 100 mg / kg intraperitoneal ) .", "this was followed by intracardiac perfusion with 4% paraformaldehyde solution in 0.1 m phosphate buffer saline .", "transverse sections of the spinal cord ( 20 m thick ) were obtained in a cryostat and processed for immunohistochemical localization of sp and cgrp using specific antibodies ( anti - sp antibody , abcam , uk ; anti - cgrp antibody , calbiochem , usa ; 1:500 titer ) using the avidin - biotin complex method ( vector labs , usa ) .", "representative sections ( 3/rat ; systematic random sampling ) were later viewed under a microscope and the images captured .", "the expression of sp and cgrp in the superficial laminae ( rexed 's laminae i - ii ) of the gray matter was quantitated by image j software ( nih , usa ) .", "specific expression was obtained after deducting background staining in the white matter ( lateral funiculus ) from the total value obtained from the superficial laminae .", "some of the cryostat sections were stained with 0.5% cresyl violet for localization of neurons in the dorsal horn .", "statistical evaluation of data was done by graphpad prism version 5 ( graphpad software , la jolla , san diego , usa ) .", "values of latency period of the different groups of animals were independently analyzed at each time point by one - way analysis of variance followed by bonferroni multiple comparison test .", "the various groups of rats had different baseline values , which affected the subsequent comparison of the antinociceptive effect of the drugs [ figure 1a ] .", "hence , normalization of the data was done by calculating the % mpe [ figure 1b ] .", "the % mpe values of control and the fosaprepitant - treated groups did not show any statistically significant change during the experiment .", "morphine injection produced significantly higher antinociceptive effect on day 1 ( p < 0.001 ) and to a lesser extent day 4 ( p < 0.05 ) compared to the control group .", "the sharp decrease in the antinociceptive effect from day 2 in the morphine treated group indicated the development of tolerance .", "morphine + fosaprepitant combination delayed the onset of tolerance in comparison to morphine treated group on days 1 ( p < 0.01 ) and 3 ( p < 0.001 ) . compared to control , antinociception was higher between days 1 and 4 ( p < 0.001 ) for this group .", "( a ) please note the variability of basal latency period and hence these values were not considered in the experimental results ( b ) % maximum possible effect shows rapid development of tolerance after morphine treatment .", "however , co - administration of fosaprepitant with morphine increased the antinociceptive effect of morphine ( days 14 ) and also attenuated tolerance ( days 1 and 3 ) .", "values are represented as mean  standard error of mean p < 0.05 , * * p < 0.01 , * * * , ,p < 0.001 cresyl violet staining showed the dorsal horn neurons arranged in various laminae [ figure 2 ] .", "immunohistochemical study revealed the expression of sp and cgrp over the superficial laminae ( laminae i - ii ) of the dorsal horn [ figure 2 ] .", "quantitative image analysis revealed increased sp expression in the morphine + fosaprepitant treated group compared to others [ figure 3 ] .", "statistically significant difference was present between morphine and morphine + fosaprepitant co - treatment groups ( p < 0.001 ) .", "cresyl violet staining showed the different laminae of the gray matter of the cervical region of the spinal cord ( top panel ) .", "location of the box showed the area of interest ( laminae i - ii ) .", "expression of both substance p and calcitonin gene - related peptide was noted over the superficial laminae ( i - ii ) .", "each group had 6 animals quantitative image analysis of the expression of substance p and calcitonin gene - related peptide ( cgrp ) shows that morphine + fosaprepitant treatment led to higher substance p expression compared to all other groups including the morphine treated group . p", "the a and c groups of nerve fibers carry pain from the periphery to the spinal cord .", "the peptidergic subgroup of these nerve fibers contains neuropeptides such as sp and cgrp , which are released into the synaptic cleft following noxious stimulation .", "these neuropeptides diffuse to the postsynaptic terminal , where they bind to the corresponding receptors .", "morphine - induced antinociception is linked to decreased release of glutamate , sp , cgrp , etc . , from presynaptic terminals and greater postsynaptic hyperpolarization due to the activation of inwardly rectifying potassium channels .", "co - administration of fosaprepitant possibly interfered with the binding of sp to the nk1rs expressed by postsynaptic neurons in the spinal cord .", "the results of this study show that combining morphine with fosaprepitant ( group iv ) delayed the development of morphine tolerance and concurrently increased the antinociceptive effect compared to the control group . as mentioned earlier , this could be due to decreased binding of sp to the nk1r .", "evidence from electrophysiological experiments indicates that noxious stimuli produce slow and prolonged excitatory potentials in postsynaptic dorsal horn neurons , which are inhibited by nk1r antagonist . since fosaprepitant treatment ( group iii ) alone", "did not result in an antinociceptive effect , the interaction between morphine and fosaprepitant is likely synergistic in nature .", "previously , injection of a bifunctional peptide having both -opioid receptor agonist and nk1r antagonist properties relieved pain in neuropathic rats without producing tolerance . in a different study ,", "tumati et al . reported that intrathecal administration of both morphine and a nk1r antagonist reduced subsequent opioid withdrawal - induced hyperalgesia .", "pharmacokinetic interaction between fosaprepitant / aprepitant and morphine could also contribute to the enhanced antinociceptive effect .", "hence , morphine concentration in the nervous system could be increased by concurrent administration of these drugs .", "significant alterations in the expression of sp and cgrp were not observed except in the group treated with morphine + fosaprepitant combination ( group iv ) .", "this was unexpected because prevailing evidence suggests that nk1r does not regulate the release of sp in the spinal cord .", "however , contrary to this , nk1r has been recently reported to be crucial for release of sp from dorsal root ganglion neurons .", "moreover , it has been speculated that there could be sp autoreceptors on presynaptic terminals , which can modulate sp release .", "but , fosaprepitant treatment alone did not increase sp immunoreactivity in the present work . as noted earlier", ", morphine inhibits the release of sp under acute conditions but rats chronically treated with morphine in our study did not show a statistically significant increase in sp expression .", "presumably , both nk1 autoreceptors as well as morphine - induced inhibition of sp release might have contributed to the increased sp expression in group iv [ figure 4 ] .", "a limitation of this study was that the expression of sp and cgrp was evaluated at the end of the observation period and not between days 1 and 4 , when the antinociceptive effect was maximum .", "another limitation was the use of a fixed - dose combination of morphine and fosaprepitant .", "diagrammatic representation of the dorsal horn of the spinal cord ( a ) primary sensory afferents carrying nociceptive sensation ends in the dorsal horn of the spinal cord .", "( b ) the presynaptic terminals of the primary sensory afferents contain excess substance p following morphine + fosaprepitant treatment ( red ) .", "- receptor is present on both the presynaptic and postsynaptic terminals the antinociceptive effect of morphine decreased rapidly following daily administration , indicating development of tolerance .", "the antinociceptive effect of morphine is likely due to decreased release of pronociceptive neurotransmitters such as glutamate and neuropeptides such as sp from presynaptic nerve terminals which could be lost during tolerance .", "one of the factors responsible for tolerance could be counter - adapting processes , which maintains the status quo in the spinal cord .", "similarly , the potentiation of the antinociceptive effect of morphine by fosaprepitant is also lost on continued administration ( day 5 onward ) .", "identical result was reported with a chimera possessing both opioid agonist and nk1r antagonist properties .", "moreover , the factors can differ depending upon the specific -opioid receptor agonist used for producing the tolerance .", "the results indicate that addition of fosaprepitant to morphine can delay morphine tolerance for a limited period of time .", "to the best of our knowledge , this is the first report on the novel antinociceptive effect of morphine + fosaprepitant combination .", "further studies are required to further elucidate this novel antinociceptive effect of morphine + fosaprepitant combination ."], "abstract_text": ["<S> objectives : opioids such as morphine form the cornerstone in the treatment of moderate to severe pain </S>", "<S> . however , opioids also produce serious side effects such as tolerance . </S>", "<S> fosaprepitant is a substance p ( sp ) receptor antagonist , which is used for treating chemotherapy - induced nausea and vomiting . </S>", "<S> sp is an important neuropeptide mediating transmission of pain at the spinal level . </S>", "<S> thus , it was hypothesized that combining morphine with fosaprepitant would increase the antinociceptive effect of morphine . </S>", "<S> the objectives were to evaluate the effect of fosaprepitant on morphine - induced antinociception in rats and to investigate its mechanism of action.methods:sprague-dawley rats were injected with morphine ( 10 mg / kg twice daily ) and/or fosaprepitant ( 30 mg / kg once daily ) for 7 days . </S>", "<S> pain threshold was assessed by the hot plate test . </S>", "<S> expression of sp and calcitonin gene - related peptide ( cgrp ) in the spinal cords of these rats was evaluated by immunohistochemistry.results:morphine administration resulted in an antinociceptive effect compared to the control group ( day 1 and to a lesser extent on day 4 ) . </S>", "<S> the decreased antinociception despite continued morphine treatment indicated development of tolerance . </S>", "<S> co - administration of fosaprepitant attenuated tolerance to morphine ( days 1 and 3 ) and increased the antinociceptive effect compared to control group ( days 14 ) . </S>", "<S> expression of sp was increased in the morphine + fosaprepitant group.conclusions:the results show that fosaprepitant attenuates the development of tolerance to morphine and thereby , increases the antinociceptive effect . </S>", "<S> this is likely linked to decreased release of sp from presynaptic terminals . </S>"], "labels": null, "section_names": ["Methods", "Results", "Discussion", "Conclusion", "Financial Support and Sponsorship", "Conflicts of Interest"], "sections": [["male sprague - dawley rats ( n = 24 ) weighing approximately 250 g were used for the study .", "permission for the experimental work was obtained from the institutional animal ethics committee ( 767/iaec/13 dated 1 - 1 - 2014 ) .", "rats were divided into 4 equal groups and injected physiological saline ( twice daily subcutaneously in morning and evening ; group i ) , morphine sulfate ( 10 mg / kg twice daily subcutaneously ; group ii ) , fosaprepitant ( 30 mg / kg once by intraperitoneal route ; group iii ) , and finally , the last group was co - administered both morphine and fosaprepitant ( group iv ) in the same doses as in group ii and iii , respectively , for a week . in the last group , fosaprepitant was injected 30 min before the morphine injection . the dose of fosaprepitant ( 30 mg / kg ) was higher than that used in an earlier study ( 25 mg / kg ) .", "the rationale for selecting a higher dose was the rapid conversion of fosaprepitant to aprepitant ( half - life ~30 min ) in the rat compared to dogs and humans .", "hot plate latency or the thermal escape behaviour was determined in the morning , 40 min after saline ( group i ) , fosaprepitant ( group iii ) , or morphine injection ( group ii and iv ) .", "the hot plate test is commonly employed for screening the putative antinociceptive property of drugs .", "the advantages of this test are the brief nature of the nociceptive stimulus , which does not produce any tissue damage and that multiple testing can be done in the same animal .", "the predictability of this test to clinical situations is high for morphine and related opioid substances .", "testing was done in a quiet room with the ambient temperature between 22c and 25c . on the day of the experiment ,", "the testing platform of the hot plate apparatus ( stoelting , usa ) was set at a constant temperature of 52.5c .", "the rats were placed on the hot plate , and the behavioral end points were either licking of the hindpaw or jumping .", "the test was repeated thrice at 57 min intervals and the average of these values was the latency period ( sec ) .", "a 40 s , cut - off was fixed to prevent damage to the completely analgesic paw after morphine injection .", "transformation of these values was done by calculating the percent maximum possible effect ( % mpe ) as follows : ( [ drug induced latency  basal response time]/[40 s  basal response time ] ) 100 on day 8 ( morning ) , rats were anesthetized by pentobarbital injection ( 100 mg / kg intraperitoneal ) .", "this was followed by intracardiac perfusion with 4% paraformaldehyde solution in 0.1 m phosphate buffer saline .", "transverse sections of the spinal cord ( 20 m thick ) were obtained in a cryostat and processed for immunohistochemical localization of sp and cgrp using specific antibodies ( anti - sp antibody , abcam , uk ; anti - cgrp antibody , calbiochem , usa ; 1:500 titer ) using the avidin - biotin complex method ( vector labs , usa ) .", "representative sections ( 3/rat ; systematic random sampling ) were later viewed under a microscope and the images captured .", "the expression of sp and cgrp in the superficial laminae ( rexed 's laminae i - ii ) of the gray matter was quantitated by image j software ( nih , usa ) .", "specific expression was obtained after deducting background staining in the white matter ( lateral funiculus ) from the total value obtained from the superficial laminae .", "some of the cryostat sections were stained with 0.5% cresyl violet for localization of neurons in the dorsal horn .", "statistical evaluation of data was done by graphpad prism version 5 ( graphpad software , la jolla , san diego , usa ) .", "values of latency period of the different groups of animals were independently analyzed at each time point by one - way analysis of variance followed by bonferroni multiple comparison test ."], ["the various groups of rats had different baseline values , which affected the subsequent comparison of the antinociceptive effect of the drugs [ figure 1a ] .", "hence , normalization of the data was done by calculating the % mpe [ figure 1b ] .", "the % mpe values of control and the fosaprepitant - treated groups did not show any statistically significant change during the experiment .", "morphine injection produced significantly higher antinociceptive effect on day 1 ( p < 0.001 ) and to a lesser extent day 4 ( p < 0.05 ) compared to the control group .", "the sharp decrease in the antinociceptive effect from day 2 in the morphine treated group indicated the development of tolerance .", "morphine + fosaprepitant combination delayed the onset of tolerance in comparison to morphine treated group on days 1 ( p < 0.01 ) and 3 ( p < 0.001 ) . compared to control , antinociception was higher between days 1 and 4 ( p < 0.001 ) for this group .", "( a ) please note the variability of basal latency period and hence these values were not considered in the experimental results ( b ) % maximum possible effect shows rapid development of tolerance after morphine treatment .", "however , co - administration of fosaprepitant with morphine increased the antinociceptive effect of morphine ( days 14 ) and also attenuated tolerance ( days 1 and 3 ) .", "values are represented as mean  standard error of mean p < 0.05 , * * p < 0.01 , * * * , ,p < 0.001 cresyl violet staining showed the dorsal horn neurons arranged in various laminae [ figure 2 ] .", "immunohistochemical study revealed the expression of sp and cgrp over the superficial laminae ( laminae i - ii ) of the dorsal horn [ figure 2 ] .", "quantitative image analysis revealed increased sp expression in the morphine + fosaprepitant treated group compared to others [ figure 3 ] .", "statistically significant difference was present between morphine and morphine + fosaprepitant co - treatment groups ( p < 0.001 ) .", "cresyl violet staining showed the different laminae of the gray matter of the cervical region of the spinal cord ( top panel ) .", "location of the box showed the area of interest ( laminae i - ii ) .", "expression of both substance p and calcitonin gene - related peptide was noted over the superficial laminae ( i - ii ) .", "each group had 6 animals quantitative image analysis of the expression of substance p and calcitonin gene - related peptide ( cgrp ) shows that morphine + fosaprepitant treatment led to higher substance p expression compared to all other groups including the morphine treated group . p"], ["the a and c groups of nerve fibers carry pain from the periphery to the spinal cord .", "the peptidergic subgroup of these nerve fibers contains neuropeptides such as sp and cgrp , which are released into the synaptic cleft following noxious stimulation .", "these neuropeptides diffuse to the postsynaptic terminal , where they bind to the corresponding receptors .", "morphine - induced antinociception is linked to decreased release of glutamate , sp , cgrp , etc . , from presynaptic terminals and greater postsynaptic hyperpolarization due to the activation of inwardly rectifying potassium channels .", "co - administration of fosaprepitant possibly interfered with the binding of sp to the nk1rs expressed by postsynaptic neurons in the spinal cord .", "the results of this study show that combining morphine with fosaprepitant ( group iv ) delayed the development of morphine tolerance and concurrently increased the antinociceptive effect compared to the control group . as mentioned earlier , this could be due to decreased binding of sp to the nk1r .", "evidence from electrophysiological experiments indicates that noxious stimuli produce slow and prolonged excitatory potentials in postsynaptic dorsal horn neurons , which are inhibited by nk1r antagonist . since fosaprepitant treatment ( group iii ) alone", "did not result in an antinociceptive effect , the interaction between morphine and fosaprepitant is likely synergistic in nature .", "previously , injection of a bifunctional peptide having both -opioid receptor agonist and nk1r antagonist properties relieved pain in neuropathic rats without producing tolerance . in a different study ,", "tumati et al . reported that intrathecal administration of both morphine and a nk1r antagonist reduced subsequent opioid withdrawal - induced hyperalgesia .", "pharmacokinetic interaction between fosaprepitant / aprepitant and morphine could also contribute to the enhanced antinociceptive effect .", "hence , morphine concentration in the nervous system could be increased by concurrent administration of these drugs .", "significant alterations in the expression of sp and cgrp were not observed except in the group treated with morphine + fosaprepitant combination ( group iv ) .", "this was unexpected because prevailing evidence suggests that nk1r does not regulate the release of sp in the spinal cord .", "however , contrary to this , nk1r has been recently reported to be crucial for release of sp from dorsal root ganglion neurons .", "moreover , it has been speculated that there could be sp autoreceptors on presynaptic terminals , which can modulate sp release .", "but , fosaprepitant treatment alone did not increase sp immunoreactivity in the present work . as noted earlier", ", morphine inhibits the release of sp under acute conditions but rats chronically treated with morphine in our study did not show a statistically significant increase in sp expression .", "presumably , both nk1 autoreceptors as well as morphine - induced inhibition of sp release might have contributed to the increased sp expression in group iv [ figure 4 ] .", "a limitation of this study was that the expression of sp and cgrp was evaluated at the end of the observation period and not between days 1 and 4 , when the antinociceptive effect was maximum .", "another limitation was the use of a fixed - dose combination of morphine and fosaprepitant .", "diagrammatic representation of the dorsal horn of the spinal cord ( a ) primary sensory afferents carrying nociceptive sensation ends in the dorsal horn of the spinal cord .", "( b ) the presynaptic terminals of the primary sensory afferents contain excess substance p following morphine + fosaprepitant treatment ( red ) .", "- receptor is present on both the presynaptic and postsynaptic terminals the antinociceptive effect of morphine decreased rapidly following daily administration , indicating development of tolerance .", "the antinociceptive effect of morphine is likely due to decreased release of pronociceptive neurotransmitters such as glutamate and neuropeptides such as sp from presynaptic nerve terminals which could be lost during tolerance .", "one of the factors responsible for tolerance could be counter - adapting processes , which maintains the status quo in the spinal cord .", "similarly , the potentiation of the antinociceptive effect of morphine by fosaprepitant is also lost on continued administration ( day 5 onward ) .", "identical result was reported with a chimera possessing both opioid agonist and nk1r antagonist properties .", "moreover , the factors can differ depending upon the specific -opioid receptor agonist used for producing the tolerance ."], ["the results indicate that addition of fosaprepitant to morphine can delay morphine tolerance for a limited period of time .", "to the best of our knowledge , this is the first report on the novel antinociceptive effect of morphine + fosaprepitant combination .", "further studies are required to further elucidate this novel antinociceptive effect of morphine + fosaprepitant combination ."], [""], [""]]}
{"article_id": "PMC3829681", "article_text": ["the direct conversion \n of solar energy to a chemical fuel is an \n essential part of future sustainable energy systems that are independent \n of fossil reserves .", "hydrogen is an environmentally \n benign energy carrier of high - energy density and can be produced by \n photocatalytic water reduction .", "platinum \n and other noble metals can serve as heterogeneous hydrogen evolution \n catalysts ; however , their limited earth abundance and cost precludes \n further development and/or large - scale applications . on the other hand ,", "organometallic compounds are attractive catalysts \n for this transformation considering the variety of complexes that \n can be prepared , the synthetic ease with which electronic properties \n ( and hence catalyst activity ) can be modulated , and the ability to \n study their mechanisms in detail . in nature , hydrogenase enzymes , \n especially those containing fe2 active sites , efficiently \n catalyze proton reduction to molecular dihydrogen .", "models \n of the [ fefe]-hydrogenases can act as biomimetic hydrogen evolution \n catalysts , although they often suffer \n from limited stability , especially when catalysis is driven photochemically \n in conjunction with photosensitizers .", "the \n necessity of an external matrix to stabilize the catalyst is thus \n evident .", "metal  organic frameworks ( mofs ) , also referred \n to as porous \n coordination polymers , have emerged as an intriguing class of microporous \n crystalline materials due to their intrinsic topology and porosity and have been studied for a range of applications \n in gas storage / separation , chemical sensing , drug delivery , and \n catalysis . unlike other porous materials \n such as zeolites , the organic ligand component of mofs allows for \n functionalization of internal channels and/or cavities either through \n direct solvothermal synthesis or by postsynthetic \n modification reactions that include the metathesis of metal ions and \n organic linkers under relatively mild conditions .", "hupp , cohen , and others have reported on this postsynthetic exchange \n ( pse ) phenomenon ( also termed solvent - assisted linker exchange , sale ) , \n including in highly robust mofs , such as the materials of the institute \n lavoisier , zeolitic imidaozolate framework , and university of oslo \n ( uio ) materials .", "all of these materials are considered to be  inert  \n with exceptionally high thermal and chemical stability , and can provide \n a robust platform for the incorporation of potentially labile molecular \n catalysts .", "incorporation of catalytic sites into mofs has resulted \n in heterogeneous \n catalysts that promote a wide range of organic reactions .", "the heterogeneous \n nature of mof catalysts allows for their easy separation , reusability , \n and enhanced stability . in the context of light - to - fuel \n conversion schemes , homogeneous photosensitizers such as ir polypyridine \n complexes and porphyrins", "have been incorporated in mofs and were shown to drive \n photochemical hydrogen production catalyzed by pt nanoparticles .", "organometallic ir and re catalysts have been \n incorporated as the ligand linker part of the mof and were shown to \n catalyze ce - promoted water oxidation and photochemical co2 reduction , respectively .", "although excellent proof - of - concept studies , \n in both cases , resource - limited \n precious metal catalytic sites were used . moreover ,", "the scope of these \n reports is somewhat limited , as the solvothermal procedures that were \n used for the synthesis of the mofs require organometallic units that \n are thermally robust .", "herein , we describe the incorporation of an \n organometallic fe2 complex that bears structural resemblance \n to the active site of [ fefe ] h2ases into a mof .", "[ fefe](bdt)(co)6 ( 2 ) ( bdt = benzenedithiolate ) has previously \n been shown to be an effective proton reduction catalyst in electro- \n and photochemical schemes .", "decoration of complex 2 at the bdt ligand with two carboxylates results in complex 1 which can be introduced into mofs by pse of 1,4-benzenedicarboxylate \n ( bdc ) ligands , which is a common ligand linker in many mofs ( figure 1 ) .", "pse thus allowed for the introduction of a thermally \n unstable [ fefe](bdt)(co)6 moiety into the thermally stable \n zr(iv)-based uio-66 mof ( figure 2 ) .", "x - ray absorption \n spectroscopy ( xas ) was used to confirm the coordination environment \n of the fe2 site in the mof .", "importantly , uio-66-[fefe](dcbdt)(co)6 was found to be a highly active hydrogen production catalyst \n in photochemical arrays with [ ru(bpy)3 ] as \n a photosensitizer and ascorbate as an electron donor .", "the catalytic \n performance of the mof exceeds that of an analogous homogeneous reference \n system .", "[ fefe ] hydrogenase active site model complexes [ fefe](dcbdt)(co)61 and [ fefe](bdt)(co)62 and the bdc ligand", "2,3-dimercaptoterephthalic \n acid was prepared from benzene-1,2-dithiole \n via lithiation and carboxylation and directly \n converted to complex 1 ( [ fefe](dcbdt)(co)6 , dcbdt = 1,4-dicarboxylbenzene-2,3-ditiolate ) by combining the ligand \n with fe3(co)12 in thf .", "single crystal x - ray \n analysis of complex 1 shows the usual distorted octahedral \n coordination sphere around the fe ions , with the dcbdt ligand perpendicular \n to the fe  fe bond vector ( see also esi ) .", "the uio-66 framework , \n consisting of zr(iv)-based secondary building \n units ( sbus ) and bdc ligand ( zr6o4(oh)4(bdc)6 ) , was chosen for the incorporation of complex 1 because of its exceptionally high structural stability with \n respect to water and weak acids .", "highly crystalline uio-66 was synthesized \n under solvothermal conditions using zrcl4 , bdc , and benzoic \n acid ( as a crystal growth modulator ) in dmf for 24 h , followed by \n washing with meoh and activation under vacuum .", "field - emission scanning \n electron microscopy ( fe - sem ) shows an octahedral morphology of the \n resultant uio-66 crystals with a particle size ranging from 200 \n to 500 nm ( figure 3 ) .", "fe - sem image of ( a ) uio-66 \n and ( b ) uio-66-[fefe](dcbdt)(co)6 .", "scale bar is 1 m . attempts to directly include 1 during solvothermal \n synthesis ( > 50 c ) resulted in decomposition of the cluster , \n presumably due to the labile bonds between the fe centers and the \n highly electron - deficient dcbdt ligand .", "taking advantage of the structural \n homology of the bdc and dcbdt ligands in complex 1 ( figure 1 ) , we employed pse as a mild functionalization approach \n to introduce complex 1 into uio-66 ( figure 2 ) .", "optimization of the pse conditions revealed that the use \n of deoxygenated , ultrapure water ( room temperature for 24", "organic solvents , including meoh , dmf , \n and chcl3 , gave lower incorporation , consistent with previous \n observations on the solvent dependence of pse .", "as expected from the attempted solvothermal syntheses , \n pse at elevated temperatures ( > 50 c ) gave results that also \n suggested partial decomposition of 1 .", "interestingly , \n it was found that pse was facilitated by using a highly microcrystalline \n form of uio-66 that was synthesized in the presence of a benzoic acid \n modulator .", "in contrast , conventionally synthesized uio-66 ( without \n modulator ) resulted in a less crystalline material and a less efficient \n exchange process .", "the linker - exchanged material , uio-66-[fefe](dcbdt)(co)6 , was isolated as an orange microcrystalline powder after \n washing \n thoroughly with fresh meoh and activation under vacuum .", "g / cm , measured \n with nitrogen at 77 k. this value is close to the bet surface area \n of pristine uio-66 ( 1475  89", "g / cm ) , suggesting \n a true pse process between 1 and the framework , and not \n simple trapping of the iron complex in the mof pores ( which would \n be expected to produce a much lower surface area ) .", "n2 absorption / desorption \n isotherms of uio-66 and uio-66-[fefe]dcbdt(co)6 do indicate \n a modest decrease in pore size distribution ( figure \n s1 ) .", "uio-66 is known to possess two pore types , tetrahedral \n and octahedral cages , with pore widths of 8 and 11 \n  , respectively ( figure s2 ) .", "pristine uio-66 gave a median pore width of 11.8  , \n while after incorporation of 1 , a reduction in the median \n pore width to 10.9  was observed , consistent with incorporation \n of the [ fe2s2 ] functionality in uio-66-[fefe]dcbdt(co)6 .", "powder x - ray diffraction ( pxrd ) patterns before and after \n pse confirmed the retention of the crystalline uio-66 framework ( figure 4a ) .", "fe - sem showed that uio-66-[fefe](dcbdt)(co)6 possesses a nearly identical particle size and octahedral \n particle morphology compared to uio-66 , again indicative of a pse \n mechanism ( figure 3 ) .", "asterisks \n indicate remaining benzoic acid ( modulator ) and the black square indicates \n dcbdt .", "the degree of pse was characterized \n by energy - dispersed x - ray spectroscopy \n ( edx ) , elemental analysis ( ea ) , thermogravimetric analysis ( tga ) , \n and proton nuclear magnetic resonance spectroscopy ( h \n nmr ) .", "the ratio of heavy elements in uio-66-[fefe](dcbdt)(co)6 was determined to be 3.52:1:1.01 ( zr : fe : s , normalized to \n fe ) via edx , which suggests that 14% of bdc in uio-66 was \n exchanged for [ fefe](dcbdt)(co)6 ( figure \n s3 ) .", "the expected 1:1 fe / s ratio determined by edx also further \n supports that the cluster is intact within the mof .", "treatment of uio-66-[fefe](dcbdt)(co)6 with dilute hf / d - dmso was used to digest the mof but also decomposed [ fefe](dcbdt)(co)6 to dcbdt .", "integration of the proton resonances for bdc and \n dcbdt in the h nmr confirmed the degree of pse at 14% \n ( figure 4 ) , giving an overall formula for uio-66-[fefe](dcbdt)(co)6 as zr6o4(oh)4(bdc)5.1([fefe](dcbdt)(co)6)0.92ch3oh .", "based on this given formula , c / h / n / s elemental analysis also \n confirmed the degree of functionalization [ c(% ) : 32.75 ( obs ) , 32.80 \n ( calcd ) ; h(% ) : 1.75 ( obs ) , 1.68 ( calcd ) ; n(% ) : 0.00 ( obs ) , 0.00 ( calcd ) ; \n s(% ) : 2.97 ( obs ) , 2.84 ( calcd ) ] . unlike pristine uio-66 , which displays \n only one major decomposition step at 400 c , the tga \n trace of uio-66-[fefe](dcbdt)(co)6 exhibits two decomposition \n steps at 80200 and 350400 c , \n respectively ( figure 4 ) .", "the first mass loss \n is likely due to partial thermal liberation of the carbonyl ligands \n attached to fe centers ( obs : 7% ; calcd : 7.2% ) .", "both bdc and [ fefe](dcbdt)(co)6 start to decompose at 350 c , leading to a combination \n of zro2 and fe2o phases ( obs : 46.3% ; calcd : \n 43.6% , percent weight residual mass ) . to confirm that compound 1 was being incorporated \n into the framework lattice to give uio-66-[fefe]dcbdt(co)6 ,", "additional experiments were performed to exclude the possibility \n that compound 1 was merely trapped in the pores of the \n mof . in one experiment ,", "compound 2 ( figure 1 ) contains the same cluster core but lacks the coordinating \n carboxylate ligands required for mof formation and hence pse .", "incubation \n of uio-66 with compound 2 showed no evidence of substantial \n incorporation into the mof as shown by a lack of color change ( figure s4 ) and a low iron content in the edx \n analysis ( figure s5 ) . in a second experiment , \n", "pse between uio-66 and 1 was performed in d2o , and the presence of bdc was observed in the reaction solution , \n as determined by h nmr ( figure s6 ) , indicative of displacement of bdc by 1 .", "importantly , \n uio-66 in d2o in the absence of 1 does not \n show release of free bdc into solution .", "these nmr observations are \n also indicative of a ligand metathesis process and argue against simple \n inclusion of 1 into the pores of the mof . finally , as \n stated above , performing pse between uio-66 and 1 in \n other solvents ( meoh , dmf , and chcl3 ) was not efficient , \n achieving negligible incorporation ( < 2% ) , consistent with the known \n solvent dependence of pse processes .", "if \n complex 1 was only being included into uio-66 via sorption \n into the pores , then inclusion would not be expected to be strongly \n solvent dependent . taken together , these experiments provide strong \n evidence , consistent with reported pse studies , that the iron cluster \n is becoming part of the uio-66 framework via a ligand pse process \n and that the data do not support simple inclusion of the cluster into \n the pores of the mof . in order to further demonstrate the incorporation \n of the intact \n fe2s2 dinuclear cluster into the mof , we employed \n fourier - transformed infrared spectroscopy ( ftir ) and diffuse reflectance \n uv  vis spectroscopy .", "ftir of uio-66-[fefe](dcbdt)(co)6 exhibited three prominent co stretching vibration bands at 2078 , \n 2038 , and 2001 cm , while no such absorption bands \n were observed for pristine uio-66 material between 2100 and 2000 cm ( figure 4 ) .", "moreover , the \n relative intensity of these three characteristic bands was identical \n to that of free 1 , suggesting the dinuclear cluster is \n intact in the mof .", "solid - state uv  vis spectroscopy of uio-66-[fefe](dcbdt)(co)6 also showed a characteristic absorption at 350 nm , \n which is consistent with the spectral features of 1 ( figure s7 ) . due to the potentially labile \n nature of [ fefe](dcbdt)(co)6 , we sought to provide data \n to confirm the coordination environment \n of fe2s2 core in the mof .", "fe k - edge extended \n x - ray absorption fine structure spectroscopy ( exafs ) was performed \n on both 1 and uio-66-[fefe](dcbdt)(co)6 .", "as \n shown in figure 5 , fourier - transformed exafs \n in r space revealed nearly identical coordination environments of \n the fe centers in [ fefe](dcbdt)(co)6 before and after pse \n into the uio-66 framework .", "both sets of data were best fit using the \n first and second neighboring atoms of fe from the single - crystal x - ray \n structure obtained for [ fefe](dcbdt)(co)6 , where fe centers \n occupy a distorted octahedral geometry ( see esi ) .", "exafs of uio-66-[fefe](dcbdt)(co)6 suggests three carbon atoms from carbonyl groups and two \n sulfur atoms bridging the dinuclear fe2 center at distances \n of 1.7961.814 and 2.2832.285  , \n respectively ( table 1 ) .", "importantly , these \n bond lengths are in good agreement with the crystallographic data \n of 1 ( see esi ) , showing 1.7971.811  \n ( fe  c ) and 2.2552.257  ( fe  s ) \n .", "in addition , x - ray absorption near - edge structure indicates a common \n fe(i ) oxidation state of the cluster within the framework and in 1 ( figure s8 ) . fe k - edge exafs fourier \n transforms and exafs spectra ( inset ) for \n ( a ) 1 and ( b ) uio-66-[fefe](dcbdt)(co)6 .", "solid \n black lines show the experimental data , dashed red lines show the \n fits based on crystallographic data of 1 , and dotted \n gray lines show the fitting \n window . fitting \n data", "having observed that pse could be used to incorporate \n complex 1 into a robust mof , we explored the suitability \n of this material \n as a catalyst in photochemical hydrogen production schemes .", "thus , \n uio-66-[fefe](dcbdt)(co)6 was suspended in a 1.0 m acetate \n buffer solution of [ ru(bpy)3 ] photosensitizer \n ( 0.5 mm ) , and ascorbate electron donor ( 100 mm ) at ph 5 . as depicted \n in figure 6 , the projected sequence for photocatalytic \n proton reduction by uio-66-[fefe](dcbdt)(co)6 commences \n with the reductive quenching of photoexcited [ ru(bpy)3 ] by the electron donor ascorbate with a rate constant of \n 1  10 m s. following the analysis of schmehl \n et al .", ", 14% of * [ ru(bpy)3 ] excited states can be expected to form the [ ru(bpy)2(bpy ) ] reductant \n ( [ ascorbate ] = 0.1 m ; (*[ru(bpy)3 ] ) \n = 500 ns ) .", "charge recombination between photogenerated [ ru(bpy)3 ] and oxidized ascorbate can be expected to occur \n close to the diffusion limit and will \n compete with the productive heterogeneous electron transfer ( et ) between \n [ ru(bpy)3 ] and uio-66-[fefe](dcbdt)(co)6 .", "the driving force for the et can be estimated at 300 \n mv from the electrochemically obtained reduction potentials , assuming \n that the reduction potential of the fe2 site in uio-66-[fefe](dcbdt)(co)6 is similar to that obtained for complex 1 in \n solution . from solution studies", ", it is well established that the electrochemical \n reduction of 1 , and its bdt derivative 2 , is a two - electron process due to inverted electrochemical potentials . \n", "assuming that the fe2 complex in uio-66-[fefe](dcbdt)(co)6 shows similar reductive chemistry as complex 2 in solution , the driving force for electron transfer from a second \n photogenerated [ ru(bpy)3 ] to the previously \n produced monoreduced fe2 site in the mof will be even > 300 \n mv . the dianionic fe2 site 1", "a second plausible \n pathway to 1 is through disproportionation \n of two singly reduced 1 in the mof . \n", "this disproportionation is thermodynamically feasible as evidenced \n by the inverted electrochemical potentials of the 1/1 and 1/1 couples .", "reaction scheme for the \n photocatalytic reduction of protons . as demonstrated in figure 7 , uio-66-[fefe](dcbdt)(co)6 is indeed a proton reduction catalyst . under the reaction \n conditions described above , hydrogen production is observed and can \n be quantified with a hydrogen specific solid - state sensor ( see esi \n for details ) .", "it is thus clear that heterogeneous electron transfer \n between photogenerated [ ru(bpy)3 ] and uio-66-[fefe](dcbdt)(co)6 can compete with homogeneous charge recombination with oxidized \n ascorbate .", "the fe2 sites within the mof can be reduced \n in a light - driven reaction and are themselves catalysts for the reduction \n of protons to molecular hydrogen . as the rate of electron transfer \n from photogenerated [ ru(bpy)3 ] decreases exponentially \n with distance , it can be assumed that only fe2 sites that \n reside within a few nm from the surface of the mof particles will \n be viable acceptor sites .", "the de facto concentration of operating \n catalysts in the mof may thus be substantially smaller than the total \n concentration of 1 in the mof . comparing the activity \n of uio-66-[fefe](dcbdt)(co)6 with that of a homogeneous \n reference system that contains complex 1 at concentrations \n similar to the total amount of fe2", "complex in uio-66-[fefe](dcbdt)(co)6 shows that the activity of the former is not only preserved \n but actually exceeds that of the latter , both in terms of initial \n rate as well as overall amount of produced hydrogen .", "control experiments without uio-66-[fefe](dcbdt)(co)6 or with unmodified uio-66 ( which does not contain [ fefe](dcbdt)(co)6 ) do not show meaningful amounts of hydrogen generation ( figure 7 ) .", "photocatalytic hydrogen production in the presence of \n uio-66-[fefe](dcbdt)(co)6 ( blue trace , 5 mg mof , 0.59 \n mol catalyst ) , \n complex 1 ( red , 0.59 mol ) , uio-66 ( black , \n 5 mg mof ) , and background ( gray ) .", "conditions : 1 m acetate buffer ph \n 5 , 100 mm ascorbic acid , 0.5 mm [ ru(bpy)3 ] .", "quantitative comparison between the homogeneous and heterogeneous \n hydrogen production systems must be done with great care , as hydrogen \n production in both systems is not limited by an intrinsic step of \n the catalytic cycle but by insufficient photoproduction of the [ ru(bpy)2(bpy ) ] reductant .", "nevertheless , figure 7 clearly shows that the heterogeneous system outperforms \n the homogeneous one both in overall hydrogen production as well as \n initial rate .", "as shown in a recent study , the photoproduction of [ ru(bpy)2(bpy ) ] is not strongly influenced by homogeneous complex 2(39 ) and probably also not by uio-66-[fefe](dcbdt)(co)6 .", "the amount of available reductant can thus considered to \n be comparable in both systems . also , the heterogeneous et rate constant ket , het in figure 6 is \n presumably not higher than the corresponding ket , hom in the homogeneous reference system .", "therefore , the \n reasons for the superior catalytic performance of uio-66-[fefe](dcbdt)(co)6 compared to that of the homogeneous reference system must \n be due to differences in the catalyst .", "a trivial but nevertheless \n important rationale for the increased hydrogen production yield in \n the mof is the stabilization of the catalyst when inside the framework . \n supporting this notion ,", "it was found that uio-66-[fefe](dcbdt)(co)6 recovered after 1 h of photocatalysis still shows the characteristic \n co bands in the ir spectrum ( figure s10 ) .", "in contrast , and in accordance with published work , complex 1 decomposes under identical \n photocatalysis conditions , as evidenced by the lack of any ir signals \n in the typical co region .", "as for all photochemical reduction \n schemes based on [ ru(bpy)3 ] , one - electron \n photochemistry needs to be coupled \n to a two - electron catalytic process . with the limited availability \n of reductant ,", "most productive et events will produce singly reduced \n fe2 sites ( 1 ) , while dianionic \n fe2 species ( 1 ) are unlikely \n to be formed by an encounter with a second equivalent of [ ru(bpy)2(bpy ) ] due \n to the low concentrations of both species . as discussed above , it \n is thermodynamically feasible that two 1 sites disproportionate to form the catalytically active 1 site that reacts with two protons to form hydrogen . \n", "disproportionation as well as protonation needs to occur before the \n reduced species recombine with oxidized ascorbate .", "here , the fe2 site in uio-66-[fefe](dcbdt)(co)6 has an undisputable \n advantage over the homogeneous system , as its incorporation into the \n mof spatially protects from unproductive charge recombination .", "moreover , \n the presence of many fe2 sites within the mof may promote \n disproportionation as soon as two monoreduced sites are present .", "it \n is these kinetic advantages that explain the higher initial hydrogen \n production rates in uio-66-[fefe](dcbdt)(co)6 .", "we employed pse as an efficient and mild approach to obtain the \n first mof that contains a multinuclear , organometallic , nonprecious - metal - based \n proton reduction catalyst .", "the resulting uio-66-[fefe](dcbdt)(co)6 is a hybrid material that combines the advantages of molecular \n catalysts with a highly ordered and stable inorganic support .", "[ fefe](dcbdt)(co)6 ( 1 ) is among the most complex structure that \n has ever been introduced into a mof , and its presence and molecular \n integrity within the uio-66 framework could be confirmed by exafs , \n ftir , and other methods .", "uio-66-[fefe](dcbdt)(co)6 exhibits \n high efficiency for photochemical hydrogen evolution , exceeding that \n of the homogeneous reference system in terms of rate and total hydrogen \n production yield .", "incorporation of the fe2 complex in uio-66-[fefe](dcbdt)(co)6 results in a higher stability under the photocatalysis conditions , \n protects reduced species from nonproductive charge recombination , \n and may promote disproportionation reactions to produce catalytically \n active dianion 1 ."], "abstract_text": ["<S> a molecular \n proton reduction catalyst [ fefe](dcbdt)(co)6 ( 1 , dcbdt = 1,4-dicarboxylbenzene-2,3-dithiolate ) with \n structural similarities to [ fefe]-hydrogenase active sites has been \n incorporated into a highly robust zr(iv)-based metal  organic \n framework ( mof ) by postsynthetic exchange ( pse ) . </S>", "<S> the pse protocol \n is crucial as direct solvothermal synthesis fails to produce the functionalized \n mof . </S>", "<S> the molecular integrity of the organometallic site within the \n mof is demonstrated by a variety of techniques , including x - ray absorption \n spectroscopy . in conjunction with [ ru(bpy)3]2 + as a photosensitizer and ascorbate as an electron donor </S>", "<S> , mof-[fefe](dcbdt)(co)6 catalyzes photochemical hydrogen evolution in water at ph \n 5 . </S>", "<S> the immobilized catalyst shows substantially improved initial rates \n and overall hydrogen production when compared to a reference system \n of complex 1 in solution . improved catalytic performance \n </S>", "<S> is ascribed to structural stabilization of the complex when incorporated \n in the mof as well as the protection of reduced catalysts 1 and 12 from undesirable \n charge recombination with oxidized ascorbate . </S>"], "labels": null, "section_names": ["Introduction", "Results and Discussion", "Conclusion"], "sections": [["the direct conversion \n of solar energy to a chemical fuel is an \n essential part of future sustainable energy systems that are independent \n of fossil reserves .", "hydrogen is an environmentally \n benign energy carrier of high - energy density and can be produced by \n photocatalytic water reduction .", "platinum \n and other noble metals can serve as heterogeneous hydrogen evolution \n catalysts ; however , their limited earth abundance and cost precludes \n further development and/or large - scale applications . on the other hand ,", "organometallic compounds are attractive catalysts \n for this transformation considering the variety of complexes that \n can be prepared , the synthetic ease with which electronic properties \n ( and hence catalyst activity ) can be modulated , and the ability to \n study their mechanisms in detail . in nature , hydrogenase enzymes , \n especially those containing fe2 active sites , efficiently \n catalyze proton reduction to molecular dihydrogen .", "models \n of the [ fefe]-hydrogenases can act as biomimetic hydrogen evolution \n catalysts , although they often suffer \n from limited stability , especially when catalysis is driven photochemically \n in conjunction with photosensitizers .", "the \n necessity of an external matrix to stabilize the catalyst is thus \n evident .", "metal  organic frameworks ( mofs ) , also referred \n to as porous \n coordination polymers , have emerged as an intriguing class of microporous \n crystalline materials due to their intrinsic topology and porosity and have been studied for a range of applications \n in gas storage / separation , chemical sensing , drug delivery , and \n catalysis . unlike other porous materials \n such as zeolites , the organic ligand component of mofs allows for \n functionalization of internal channels and/or cavities either through \n direct solvothermal synthesis or by postsynthetic \n modification reactions that include the metathesis of metal ions and \n organic linkers under relatively mild conditions .", "hupp , cohen , and others have reported on this postsynthetic exchange \n ( pse ) phenomenon ( also termed solvent - assisted linker exchange , sale ) , \n including in highly robust mofs , such as the materials of the institute \n lavoisier , zeolitic imidaozolate framework , and university of oslo \n ( uio ) materials .", "all of these materials are considered to be  inert  \n with exceptionally high thermal and chemical stability , and can provide \n a robust platform for the incorporation of potentially labile molecular \n catalysts .", "incorporation of catalytic sites into mofs has resulted \n in heterogeneous \n catalysts that promote a wide range of organic reactions .", "the heterogeneous \n nature of mof catalysts allows for their easy separation , reusability , \n and enhanced stability . in the context of light - to - fuel \n conversion schemes , homogeneous photosensitizers such as ir polypyridine \n complexes and porphyrins", "have been incorporated in mofs and were shown to drive \n photochemical hydrogen production catalyzed by pt nanoparticles .", "organometallic ir and re catalysts have been \n incorporated as the ligand linker part of the mof and were shown to \n catalyze ce - promoted water oxidation and photochemical co2 reduction , respectively .", "although excellent proof - of - concept studies , \n in both cases , resource - limited \n precious metal catalytic sites were used . moreover ,", "the scope of these \n reports is somewhat limited , as the solvothermal procedures that were \n used for the synthesis of the mofs require organometallic units that \n are thermally robust .", "herein , we describe the incorporation of an \n organometallic fe2 complex that bears structural resemblance \n to the active site of [ fefe ] h2ases into a mof .", "[ fefe](bdt)(co)6 ( 2 ) ( bdt = benzenedithiolate ) has previously \n been shown to be an effective proton reduction catalyst in electro- \n and photochemical schemes .", "decoration of complex 2 at the bdt ligand with two carboxylates results in complex 1 which can be introduced into mofs by pse of 1,4-benzenedicarboxylate \n ( bdc ) ligands , which is a common ligand linker in many mofs ( figure 1 ) .", "pse thus allowed for the introduction of a thermally \n unstable [ fefe](bdt)(co)6 moiety into the thermally stable \n zr(iv)-based uio-66 mof ( figure 2 ) .", "x - ray absorption \n spectroscopy ( xas ) was used to confirm the coordination environment \n of the fe2 site in the mof .", "importantly , uio-66-[fefe](dcbdt)(co)6 was found to be a highly active hydrogen production catalyst \n in photochemical arrays with [ ru(bpy)3 ] as \n a photosensitizer and ascorbate as an electron donor .", "the catalytic \n performance of the mof exceeds that of an analogous homogeneous reference \n system .", "[ fefe ] hydrogenase active site model complexes [ fefe](dcbdt)(co)61 and [ fefe](bdt)(co)62 and the bdc ligand"], ["2,3-dimercaptoterephthalic \n acid was prepared from benzene-1,2-dithiole \n via lithiation and carboxylation and directly \n converted to complex 1 ( [ fefe](dcbdt)(co)6 , dcbdt = 1,4-dicarboxylbenzene-2,3-ditiolate ) by combining the ligand \n with fe3(co)12 in thf .", "single crystal x - ray \n analysis of complex 1 shows the usual distorted octahedral \n coordination sphere around the fe ions , with the dcbdt ligand perpendicular \n to the fe  fe bond vector ( see also esi ) .", "the uio-66 framework , \n consisting of zr(iv)-based secondary building \n units ( sbus ) and bdc ligand ( zr6o4(oh)4(bdc)6 ) , was chosen for the incorporation of complex 1 because of its exceptionally high structural stability with \n respect to water and weak acids .", "highly crystalline uio-66 was synthesized \n under solvothermal conditions using zrcl4 , bdc , and benzoic \n acid ( as a crystal growth modulator ) in dmf for 24 h , followed by \n washing with meoh and activation under vacuum .", "field - emission scanning \n electron microscopy ( fe - sem ) shows an octahedral morphology of the \n resultant uio-66 crystals with a particle size ranging from 200 \n to 500 nm ( figure 3 ) .", "fe - sem image of ( a ) uio-66 \n and ( b ) uio-66-[fefe](dcbdt)(co)6 .", "scale bar is 1 m . attempts to directly include 1 during solvothermal \n synthesis ( > 50 c ) resulted in decomposition of the cluster , \n presumably due to the labile bonds between the fe centers and the \n highly electron - deficient dcbdt ligand .", "taking advantage of the structural \n homology of the bdc and dcbdt ligands in complex 1 ( figure 1 ) , we employed pse as a mild functionalization approach \n to introduce complex 1 into uio-66 ( figure 2 ) .", "optimization of the pse conditions revealed that the use \n of deoxygenated , ultrapure water ( room temperature for 24", "organic solvents , including meoh , dmf , \n and chcl3 , gave lower incorporation , consistent with previous \n observations on the solvent dependence of pse .", "as expected from the attempted solvothermal syntheses , \n pse at elevated temperatures ( > 50 c ) gave results that also \n suggested partial decomposition of 1 .", "interestingly , \n it was found that pse was facilitated by using a highly microcrystalline \n form of uio-66 that was synthesized in the presence of a benzoic acid \n modulator .", "in contrast , conventionally synthesized uio-66 ( without \n modulator ) resulted in a less crystalline material and a less efficient \n exchange process .", "the linker - exchanged material , uio-66-[fefe](dcbdt)(co)6 , was isolated as an orange microcrystalline powder after \n washing \n thoroughly with fresh meoh and activation under vacuum .", "g / cm , measured \n with nitrogen at 77 k. this value is close to the bet surface area \n of pristine uio-66 ( 1475  89", "g / cm ) , suggesting \n a true pse process between 1 and the framework , and not \n simple trapping of the iron complex in the mof pores ( which would \n be expected to produce a much lower surface area ) .", "n2 absorption / desorption \n isotherms of uio-66 and uio-66-[fefe]dcbdt(co)6 do indicate \n a modest decrease in pore size distribution ( figure \n s1 ) .", "uio-66 is known to possess two pore types , tetrahedral \n and octahedral cages , with pore widths of 8 and 11 \n  , respectively ( figure s2 ) .", "pristine uio-66 gave a median pore width of 11.8  , \n while after incorporation of 1 , a reduction in the median \n pore width to 10.9  was observed , consistent with incorporation \n of the [ fe2s2 ] functionality in uio-66-[fefe]dcbdt(co)6 .", "powder x - ray diffraction ( pxrd ) patterns before and after \n pse confirmed the retention of the crystalline uio-66 framework ( figure 4a ) .", "fe - sem showed that uio-66-[fefe](dcbdt)(co)6 possesses a nearly identical particle size and octahedral \n particle morphology compared to uio-66 , again indicative of a pse \n mechanism ( figure 3 ) .", "asterisks \n indicate remaining benzoic acid ( modulator ) and the black square indicates \n dcbdt .", "the degree of pse was characterized \n by energy - dispersed x - ray spectroscopy \n ( edx ) , elemental analysis ( ea ) , thermogravimetric analysis ( tga ) , \n and proton nuclear magnetic resonance spectroscopy ( h \n nmr ) .", "the ratio of heavy elements in uio-66-[fefe](dcbdt)(co)6 was determined to be 3.52:1:1.01 ( zr : fe : s , normalized to \n fe ) via edx , which suggests that 14% of bdc in uio-66 was \n exchanged for [ fefe](dcbdt)(co)6 ( figure \n s3 ) .", "the expected 1:1 fe / s ratio determined by edx also further \n supports that the cluster is intact within the mof .", "treatment of uio-66-[fefe](dcbdt)(co)6 with dilute hf / d - dmso was used to digest the mof but also decomposed [ fefe](dcbdt)(co)6 to dcbdt .", "integration of the proton resonances for bdc and \n dcbdt in the h nmr confirmed the degree of pse at 14% \n ( figure 4 ) , giving an overall formula for uio-66-[fefe](dcbdt)(co)6 as zr6o4(oh)4(bdc)5.1([fefe](dcbdt)(co)6)0.92ch3oh .", "based on this given formula , c / h / n / s elemental analysis also \n confirmed the degree of functionalization [ c(% ) : 32.75 ( obs ) , 32.80 \n ( calcd ) ; h(% ) : 1.75 ( obs ) , 1.68 ( calcd ) ; n(% ) : 0.00 ( obs ) , 0.00 ( calcd ) ; \n s(% ) : 2.97 ( obs ) , 2.84 ( calcd ) ] . unlike pristine uio-66 , which displays \n only one major decomposition step at 400 c , the tga \n trace of uio-66-[fefe](dcbdt)(co)6 exhibits two decomposition \n steps at 80200 and 350400 c , \n respectively ( figure 4 ) .", "the first mass loss \n is likely due to partial thermal liberation of the carbonyl ligands \n attached to fe centers ( obs : 7% ; calcd : 7.2% ) .", "both bdc and [ fefe](dcbdt)(co)6 start to decompose at 350 c , leading to a combination \n of zro2 and fe2o phases ( obs : 46.3% ; calcd : \n 43.6% , percent weight residual mass ) . to confirm that compound 1 was being incorporated \n into the framework lattice to give uio-66-[fefe]dcbdt(co)6 ,", "additional experiments were performed to exclude the possibility \n that compound 1 was merely trapped in the pores of the \n mof . in one experiment ,", "compound 2 ( figure 1 ) contains the same cluster core but lacks the coordinating \n carboxylate ligands required for mof formation and hence pse .", "incubation \n of uio-66 with compound 2 showed no evidence of substantial \n incorporation into the mof as shown by a lack of color change ( figure s4 ) and a low iron content in the edx \n analysis ( figure s5 ) . in a second experiment , \n", "pse between uio-66 and 1 was performed in d2o , and the presence of bdc was observed in the reaction solution , \n as determined by h nmr ( figure s6 ) , indicative of displacement of bdc by 1 .", "importantly , \n uio-66 in d2o in the absence of 1 does not \n show release of free bdc into solution .", "these nmr observations are \n also indicative of a ligand metathesis process and argue against simple \n inclusion of 1 into the pores of the mof . finally , as \n stated above , performing pse between uio-66 and 1 in \n other solvents ( meoh , dmf , and chcl3 ) was not efficient , \n achieving negligible incorporation ( < 2% ) , consistent with the known \n solvent dependence of pse processes .", "if \n complex 1 was only being included into uio-66 via sorption \n into the pores , then inclusion would not be expected to be strongly \n solvent dependent . taken together , these experiments provide strong \n evidence , consistent with reported pse studies , that the iron cluster \n is becoming part of the uio-66 framework via a ligand pse process \n and that the data do not support simple inclusion of the cluster into \n the pores of the mof . in order to further demonstrate the incorporation \n of the intact \n fe2s2 dinuclear cluster into the mof , we employed \n fourier - transformed infrared spectroscopy ( ftir ) and diffuse reflectance \n uv  vis spectroscopy .", "ftir of uio-66-[fefe](dcbdt)(co)6 exhibited three prominent co stretching vibration bands at 2078 , \n 2038 , and 2001 cm , while no such absorption bands \n were observed for pristine uio-66 material between 2100 and 2000 cm ( figure 4 ) .", "moreover , the \n relative intensity of these three characteristic bands was identical \n to that of free 1 , suggesting the dinuclear cluster is \n intact in the mof .", "solid - state uv  vis spectroscopy of uio-66-[fefe](dcbdt)(co)6 also showed a characteristic absorption at 350 nm , \n which is consistent with the spectral features of 1 ( figure s7 ) . due to the potentially labile \n nature of [ fefe](dcbdt)(co)6 , we sought to provide data \n to confirm the coordination environment \n of fe2s2 core in the mof .", "fe k - edge extended \n x - ray absorption fine structure spectroscopy ( exafs ) was performed \n on both 1 and uio-66-[fefe](dcbdt)(co)6 .", "as \n shown in figure 5 , fourier - transformed exafs \n in r space revealed nearly identical coordination environments of \n the fe centers in [ fefe](dcbdt)(co)6 before and after pse \n into the uio-66 framework .", "both sets of data were best fit using the \n first and second neighboring atoms of fe from the single - crystal x - ray \n structure obtained for [ fefe](dcbdt)(co)6 , where fe centers \n occupy a distorted octahedral geometry ( see esi ) .", "exafs of uio-66-[fefe](dcbdt)(co)6 suggests three carbon atoms from carbonyl groups and two \n sulfur atoms bridging the dinuclear fe2 center at distances \n of 1.7961.814 and 2.2832.285  , \n respectively ( table 1 ) .", "importantly , these \n bond lengths are in good agreement with the crystallographic data \n of 1 ( see esi ) , showing 1.7971.811  \n ( fe  c ) and 2.2552.257  ( fe  s ) \n .", "in addition , x - ray absorption near - edge structure indicates a common \n fe(i ) oxidation state of the cluster within the framework and in 1 ( figure s8 ) . fe k - edge exafs fourier \n transforms and exafs spectra ( inset ) for \n ( a ) 1 and ( b ) uio-66-[fefe](dcbdt)(co)6 .", "solid \n black lines show the experimental data , dashed red lines show the \n fits based on crystallographic data of 1 , and dotted \n gray lines show the fitting \n window . fitting \n data", "having observed that pse could be used to incorporate \n complex 1 into a robust mof , we explored the suitability \n of this material \n as a catalyst in photochemical hydrogen production schemes .", "thus , \n uio-66-[fefe](dcbdt)(co)6 was suspended in a 1.0 m acetate \n buffer solution of [ ru(bpy)3 ] photosensitizer \n ( 0.5 mm ) , and ascorbate electron donor ( 100 mm ) at ph 5 . as depicted \n in figure 6 , the projected sequence for photocatalytic \n proton reduction by uio-66-[fefe](dcbdt)(co)6 commences \n with the reductive quenching of photoexcited [ ru(bpy)3 ] by the electron donor ascorbate with a rate constant of \n 1  10 m s. following the analysis of schmehl \n et al .", ", 14% of * [ ru(bpy)3 ] excited states can be expected to form the [ ru(bpy)2(bpy ) ] reductant \n ( [ ascorbate ] = 0.1 m ; (*[ru(bpy)3 ] ) \n = 500 ns ) .", "charge recombination between photogenerated [ ru(bpy)3 ] and oxidized ascorbate can be expected to occur \n close to the diffusion limit and will \n compete with the productive heterogeneous electron transfer ( et ) between \n [ ru(bpy)3 ] and uio-66-[fefe](dcbdt)(co)6 .", "the driving force for the et can be estimated at 300 \n mv from the electrochemically obtained reduction potentials , assuming \n that the reduction potential of the fe2 site in uio-66-[fefe](dcbdt)(co)6 is similar to that obtained for complex 1 in \n solution . from solution studies", ", it is well established that the electrochemical \n reduction of 1 , and its bdt derivative 2 , is a two - electron process due to inverted electrochemical potentials . \n", "assuming that the fe2 complex in uio-66-[fefe](dcbdt)(co)6 shows similar reductive chemistry as complex 2 in solution , the driving force for electron transfer from a second \n photogenerated [ ru(bpy)3 ] to the previously \n produced monoreduced fe2 site in the mof will be even > 300 \n mv . the dianionic fe2 site 1", "a second plausible \n pathway to 1 is through disproportionation \n of two singly reduced 1 in the mof . \n", "this disproportionation is thermodynamically feasible as evidenced \n by the inverted electrochemical potentials of the 1/1 and 1/1 couples .", "reaction scheme for the \n photocatalytic reduction of protons . as demonstrated in figure 7 , uio-66-[fefe](dcbdt)(co)6 is indeed a proton reduction catalyst . under the reaction \n conditions described above , hydrogen production is observed and can \n be quantified with a hydrogen specific solid - state sensor ( see esi \n for details ) .", "it is thus clear that heterogeneous electron transfer \n between photogenerated [ ru(bpy)3 ] and uio-66-[fefe](dcbdt)(co)6 can compete with homogeneous charge recombination with oxidized \n ascorbate .", "the fe2 sites within the mof can be reduced \n in a light - driven reaction and are themselves catalysts for the reduction \n of protons to molecular hydrogen . as the rate of electron transfer \n from photogenerated [ ru(bpy)3 ] decreases exponentially \n with distance , it can be assumed that only fe2 sites that \n reside within a few nm from the surface of the mof particles will \n be viable acceptor sites .", "the de facto concentration of operating \n catalysts in the mof may thus be substantially smaller than the total \n concentration of 1 in the mof . comparing the activity \n of uio-66-[fefe](dcbdt)(co)6 with that of a homogeneous \n reference system that contains complex 1 at concentrations \n similar to the total amount of fe2", "complex in uio-66-[fefe](dcbdt)(co)6 shows that the activity of the former is not only preserved \n but actually exceeds that of the latter , both in terms of initial \n rate as well as overall amount of produced hydrogen .", "control experiments without uio-66-[fefe](dcbdt)(co)6 or with unmodified uio-66 ( which does not contain [ fefe](dcbdt)(co)6 ) do not show meaningful amounts of hydrogen generation ( figure 7 ) .", "photocatalytic hydrogen production in the presence of \n uio-66-[fefe](dcbdt)(co)6 ( blue trace , 5 mg mof , 0.59 \n mol catalyst ) , \n complex 1 ( red , 0.59 mol ) , uio-66 ( black , \n 5 mg mof ) , and background ( gray ) .", "conditions : 1 m acetate buffer ph \n 5 , 100 mm ascorbic acid , 0.5 mm [ ru(bpy)3 ] .", "quantitative comparison between the homogeneous and heterogeneous \n hydrogen production systems must be done with great care , as hydrogen \n production in both systems is not limited by an intrinsic step of \n the catalytic cycle but by insufficient photoproduction of the [ ru(bpy)2(bpy ) ] reductant .", "nevertheless , figure 7 clearly shows that the heterogeneous system outperforms \n the homogeneous one both in overall hydrogen production as well as \n initial rate .", "as shown in a recent study , the photoproduction of [ ru(bpy)2(bpy ) ] is not strongly influenced by homogeneous complex 2(39 ) and probably also not by uio-66-[fefe](dcbdt)(co)6 .", "the amount of available reductant can thus considered to \n be comparable in both systems . also , the heterogeneous et rate constant ket , het in figure 6 is \n presumably not higher than the corresponding ket , hom in the homogeneous reference system .", "therefore , the \n reasons for the superior catalytic performance of uio-66-[fefe](dcbdt)(co)6 compared to that of the homogeneous reference system must \n be due to differences in the catalyst .", "a trivial but nevertheless \n important rationale for the increased hydrogen production yield in \n the mof is the stabilization of the catalyst when inside the framework . \n supporting this notion ,", "it was found that uio-66-[fefe](dcbdt)(co)6 recovered after 1 h of photocatalysis still shows the characteristic \n co bands in the ir spectrum ( figure s10 ) .", "in contrast , and in accordance with published work , complex 1 decomposes under identical \n photocatalysis conditions , as evidenced by the lack of any ir signals \n in the typical co region .", "as for all photochemical reduction \n schemes based on [ ru(bpy)3 ] , one - electron \n photochemistry needs to be coupled \n to a two - electron catalytic process . with the limited availability \n of reductant ,", "most productive et events will produce singly reduced \n fe2 sites ( 1 ) , while dianionic \n fe2 species ( 1 ) are unlikely \n to be formed by an encounter with a second equivalent of [ ru(bpy)2(bpy ) ] due \n to the low concentrations of both species . as discussed above , it \n is thermodynamically feasible that two 1 sites disproportionate to form the catalytically active 1 site that reacts with two protons to form hydrogen . \n", "disproportionation as well as protonation needs to occur before the \n reduced species recombine with oxidized ascorbate .", "here , the fe2 site in uio-66-[fefe](dcbdt)(co)6 has an undisputable \n advantage over the homogeneous system , as its incorporation into the \n mof spatially protects from unproductive charge recombination .", "moreover , \n the presence of many fe2 sites within the mof may promote \n disproportionation as soon as two monoreduced sites are present .", "it \n is these kinetic advantages that explain the higher initial hydrogen \n production rates in uio-66-[fefe](dcbdt)(co)6 ."], ["we employed pse as an efficient and mild approach to obtain the \n first mof that contains a multinuclear , organometallic , nonprecious - metal - based \n proton reduction catalyst .", "the resulting uio-66-[fefe](dcbdt)(co)6 is a hybrid material that combines the advantages of molecular \n catalysts with a highly ordered and stable inorganic support .", "[ fefe](dcbdt)(co)6 ( 1 ) is among the most complex structure that \n has ever been introduced into a mof , and its presence and molecular \n integrity within the uio-66 framework could be confirmed by exafs , \n ftir , and other methods .", "uio-66-[fefe](dcbdt)(co)6 exhibits \n high efficiency for photochemical hydrogen evolution , exceeding that \n of the homogeneous reference system in terms of rate and total hydrogen \n production yield .", "incorporation of the fe2 complex in uio-66-[fefe](dcbdt)(co)6 results in a higher stability under the photocatalysis conditions , \n protects reduced species from nonproductive charge recombination , \n and may promote disproportionation reactions to produce catalytically \n active dianion 1 ."]]}
{"article_id": "PMC4490931", "article_text": ["aging is part of the natural process of human development , and it brings multiple biopsychosocial changes that cause a slow and progressive deterioration of body functions that are essential to life . among these functions that undergo important mostly irreversible changes is hearing.1 hearing loss is considered one of the three most common conditions in the elderly,2 with occurrence of 5 to 20% in 60-year - olds , increasing to 60% in people over 65 years old.1 \n 3 in brazil , 60% of the elderly have some degree of hearing loss.4 \n  the term used to describe hearing loss associated with aging is presbycusis , which causes a decrease in hearing sensitivity and a reduction in the ability to understand speech .", "this occurs due to several degenerative and physiologic changes that affect the inner ear , impairing hearing at high frequencies and affecting communication , especially in noisy environments.1 \n 2 \n 4 \n 5 \n 6 \n 7 other factors associated with the natural degeneration process , such as exposure to loud noise , ototoxic agents , and sequelae of otitis media caused by medical problems and treatments , can contribute to lifelong aggravation of the loss.4 \n 8 \n 9 \n  difficult communication associated with hearing loss can lead to social and emotional consequences , especially for the elderly , who have several limitations related to aging that can impact their quality of life , increasing social isolation in addition to causing emotional disorders such as depression.1 \n 4 \n 5 \n 8 \n 10 \n 11 hearing loss  associated depression can be even more harmful in the elderly .", "thus , recommended intervention by hearing aid adaptation contributes to the prevention of emotional disorders such as depression.12 \n depression is defined as a mental disorder that causes feelings of sadness , pessimism , hopelessness , difficulty concentrating , and difficulty making decisions and initiating tasks , among other symptoms.13 despite being very common in the elderly , it can not be attributed solely to aging .", "studies in so paulo ( brazil)14 and florianpolis ( santa catarina , brazil)15 showed that the factors associated with depression in the elderly are : female , unmarried or widowed , altered cognitive function , dependence , use of many medicines , very old ( over 80 years ) , low education , poor economic status , cognitive impairment , fair or poor self - rated health , functional dependency , and chronic pain .", "other causes include smoking , comorbidities ( endocrine , vascular , neurologic , oncological ) , and negative changes in relationships with family and friends.15 \n  hearing loss may be associated with depression to be a debilitating , chronic disorder.16 \n 17 \n 18 the main goal of an auditory rehabilitation program in elderly patients is to minimize the effects caused by sensory deprivation of hearing and reinstate the patients in their family and society , helping them cope with the disadvantages and limitations caused by hearing loss.3 \n  monaural adaptations are commonly performed even in cases of bilateral hearing loss , whereas the most appropriate should be binaural adaptation .", "the reasons for that may be related to factors such as rejection to the use of two hearing aids , financial issues , aesthetic reasons , problems with manual dexterity , very asymmetric losses , and central processing issues.19 \n  given such reality , this work intends to analyze the effects of unilateral adaptation of hearing aids on the symptoms of depression and the social activity constraints of elderly subjects with hearing impairment .", "this is a prospective interventional study of elderly individuals of both sexes with recommended use of hearing aids at a hearing center in porto alegre .", "inclusion criteria were signature of the free and informed consent ; 60 years or older ; good health ( seniors able to commute to the research location , conduct tests , and answer questionnaires ) ; new hearing aid users with monaural fitting ; and participation in all phases of the research . exclusion criteria were initiation of antidepressant therapy during the study and a history of cognitive and/or neurologic disorders .", "the study was divided into two phases . in phase 1 , subjects responded to an interview with questions on demographics and health ( general and hearing ) .", "pure tone audiometry was performed to determine the airway tone thresholds ( frequencies 250 to 8,000 hz ) and bone conduction ( frequencies 500 to 4,000 hz ) in a vocal booth , using an ad-229e interacoustics audiometer ( assens , denmark ) .", "the presence and degree of hearing loss were classified according to the world health organization by analyzing mean airway tone threshold in the frequencies of 500 , 1,000 , 2,000 , and 4,000 hz.20 \n  after that , seniors were asked to answer two questionnaires individually , without the intervention of third parties : the geriatric depression scale ( gds ) and the hearing handicap inventory for elderly  short version ( hhie - s ) .", "the gds , an instrument validated for portuguese and composed of 15 yes - or - no questions , is used to detect depression symptoms in the elderly .", "thus , scores less than 5 points indicate absence of symptoms , scores of 5 to 10 indicate mild to moderate symptoms , and scores of 11 or more indicate severe symptoms of depression.21 \n  after completing the first phase , participants answered a summarized version of the hhie - s .", "the questionnaire was prepared in 198222 and translated and adapted to brazilian portuguese.23 it contains 10 questions that aim to assess the impact of hearing loss on the emotional and social status of elderly people .", "all 10 questions provide three choices of answers : for each  yes  answer , 4 points is assigned ; each  sometimes ,  2 points ; and each  no ,  0 points . the total score ranges from 0 to 40 points , and the higher the score , the greater subjects ' self - perception of social activity constraints : 0 to 8 points indicate no social activity constraints ; 10 to 23 points , mild to moderate social activity constraint ; and 24 to 40 points , significant social activity constraints.8 \n 22 \n  after testing , the participants were subjected to the selection and unilaterally hearing aid adaptation processes", "thirty days after purchasing the device , they returned to the hearing center to participate in the second phase of the research . in phase 2", "the research project was approved by the research committee and by the ethics and research committee of the institution ( protocol 266.060 ) .", "patients who agreed to participate signed an informed consent and had their rights guaranteed for confidentiality , nonidentification , and withdrawal of participation .", "the results were analyzed using the statistical package for social science version 20.0 ( ibm , ny , usa ) . to examine associations of categorical data , the chi - square and student t test for paired samples were used .", "all statistical tests were nonparametric tests , and always observed exact p value ( not asymptotic ) , which are the best measures to statistically evaluate a study when the sample is restricted .", "the chi - square test considered the p value of fisher exact test , and for the kappa coefficient of agreement , the exact p value ( not asymptotic ) was observed .", "the sample consisted of 13 elderly patients with hearing loss , ages from 60 to 90 years ( mean 72.85    11.05 years ) , 10 women ( 76.9% ) and 3 men ( 23.1% ) .", "table 1 shows data on the characterization of sample in terms of age , gender , mean thresholds , and degree of hearing loss . \n", "table 2 depicts an association analysis between the gds classification of phases 1 and 2 , indicating that there was no significant association between the variables in the periods before and after the adaptation ( p  =  0.615 ) .", "thus , of the 6 patients ( 46.2% ) who initially had mild symptoms of depression , only 2 ( 15.4% ) of them continued to have mild symptoms .", "all seniors who initially had severe symptoms of depression ( 15.4% ) also started to lose some symptoms of depression in phase 2 .", "hhie classification analysis in phases 1 and 2 of the study are presented in table 3 .", "no significant association was found for these variables in the periods before and after the adaptation ( p  >  0.999 ) .", "thus , the only senior who had mild to moderate social activity constraints in the first phase showed reduction in the second phase .", "after prosthetization , participants who had severe social activity constraints in phase 1 started to show no social activity constraints or mild to moderate social activity constraints . only 1 ( 7.7% )", "the lack of association between the classification of the gds and hhie on the two phases is an important point , because it shows that there were different results in both assessments ( before and after hearing aid adaptation)that is , on the sample studied , unilateral prosthetization resulted in benefits for patients . \n", "table 4 shows the scores obtained by the subjects in the instruments used for evaluation in the two phases of the research .", "there was significant difference between the gds ( p  =  0.031 ) and hhie ( p  < ", "abbreviations : gds , geriatric depression scale ; hhie - s , hearing handicap inventory for elderly  short version . \n", "the analysis of the survey data showed that the age of the individuals analyzed varied from 60 to 90 years .", "another study that examined the degeneration of the auditory system in the course of aging had a sample with similar age and results.4 \n  we had a greater number of female participants . although it has been found that men often have more problems related to hearing loss than women,24 they also have a lower perception of their disability compared with women,1 \n 25 \n 26 which explains why women seek health services more often26 \n 27 and why there is a greater number of female participants in this study .", "another fact to be noted is that , in terms of population , there is a greater number of older women in brazil , a fact known as feminization of aging.28 \n  there was a prevalence of moderate hearing loss in both the right and the left ears , agreeing with other published studies that refer to this as being the most commonly found degree of hearing impairment in the elderly.2 \n 9 \n 25 \n  the data show that many seniors had some degree of depression symptoms before prosthetization ( table 2 ) .", "after unilateral hearing aid adoption , there was improvement in symptoms for most of them .", "thus , a hearing aid , even when unilateral , promoted elimination or reduction of symptoms of depression in the sample studied .", "this result had already been described in the literature,2 but we emphasize that the result was obtained with bilateral prosthesis .", "thus , the adaptation of a single amplifying device can also generate benefit for elderly patients , which is important for the reduction of one of the most prevalent problems in the aging population , depression .", "the data show that all participants had some degree of social activity constraints before using a unilateral hearing aid ( table 3 ) .", "after 30 days of hearing aid use , 12 ( 93.3% ) and 13 ( 100% ) participants showed absence or reduction of social activity constraints , respectively .", "results show evidence of the positive effects of hearing aid use , even if unilateral , helping to reduce the impact that hearing loss has on social relationships and on the quality of life of the elderly.9 results are consistent with another study that examined the short - term benefits of amplification in new users.29 \n  the data also show a significant difference in scores obtained in phases 1 and 2 of the gds ( p  =  0.031 ) and hhie - s ( p  <  0.001 ) , confirming a reduction or elimination of depression symptoms and social activity constraints in elderly patients , agreeing with other published studies ( table 4).2 \n 8 \n 11 \n 29 \n 30 again , it is important to highlight that in the studied group , unilateral prosthesis resulted in substantial benefits not only regarding hearing but also in other matters essential for the well - being and quality of life of individuals .", "the findings reinforce the importance of hearing aid use in elderly patients , because many times family and social relationships are affected by the hearing impairment .", "the adaptation to amplification devices , albeit unilaterally , has proved to be effective in reducing symptoms of depression and social activity constraints .", "moreover , the length of time between assessments was 30 days , shorter than that considered ideal for auditory acclimatization , and even then , benefits occurred , confirming the importance of auditory rehabilitation in elderly .", "results show that unilateral hearing aid contributed to the elimination or reduction of depression symptoms and of social activity constraints in elderly participants in this research sample group ."], "abstract_text": ["<S> \n introduction  hearing loss is one of the most common problems in the elderly population . besides compromising oral communication , it directly affects social relations and prevents elderly patients from living actively in society , possibly leading to the onset of depression or other conditions . \n objective  to analyze the effects of unilateral adaptation of hearing aids on symptoms of depression and the social activity constraints of elderly subjects with hearing impairment . \n </S>", "<S> methods  the sample consisted of elderly subjects with hearing loss who did not use hearing aids . </S>", "<S> data were collected in two phases . </S>", "<S> initially , all participants underwent an audiological assessment and answered the hearing handicap inventory for elderly ( summarized version ) and the geriatric depression scale . </S>", "<S> all subjects participated in the selection and hearing aid adaptation processes and became monaural hearing aid users . after 30 days of hearing aid use , they were assessed with the same instruments . </S>", "<S> the results of the questionnaires before and after hearing aid adaptation were compared . \n </S>", "<S> results  the sample consisted of 13 individuals , between 60 and 90 years old ( mean 72.85    11.05 years ) . </S>", "<S> data analysis showed that there was significant improvement in social activity constraints ( p  <  0.001 ) and in symptoms of depression ( p  =  0.031 ) . \n </S>", "<S> conclusion  results show that , in the sample studied , unilateral hearing aid adaptation reduced social activity constraints and depression symptoms . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusion"], "sections": [["aging is part of the natural process of human development , and it brings multiple biopsychosocial changes that cause a slow and progressive deterioration of body functions that are essential to life . among these functions that undergo important mostly irreversible changes is hearing.1 hearing loss is considered one of the three most common conditions in the elderly,2 with occurrence of 5 to 20% in 60-year - olds , increasing to 60% in people over 65 years old.1 \n 3 in brazil , 60% of the elderly have some degree of hearing loss.4 \n  the term used to describe hearing loss associated with aging is presbycusis , which causes a decrease in hearing sensitivity and a reduction in the ability to understand speech .", "this occurs due to several degenerative and physiologic changes that affect the inner ear , impairing hearing at high frequencies and affecting communication , especially in noisy environments.1 \n 2 \n 4 \n 5 \n 6 \n 7 other factors associated with the natural degeneration process , such as exposure to loud noise , ototoxic agents , and sequelae of otitis media caused by medical problems and treatments , can contribute to lifelong aggravation of the loss.4 \n 8 \n 9 \n  difficult communication associated with hearing loss can lead to social and emotional consequences , especially for the elderly , who have several limitations related to aging that can impact their quality of life , increasing social isolation in addition to causing emotional disorders such as depression.1 \n 4 \n 5 \n 8 \n 10 \n 11 hearing loss  associated depression can be even more harmful in the elderly .", "thus , recommended intervention by hearing aid adaptation contributes to the prevention of emotional disorders such as depression.12 \n depression is defined as a mental disorder that causes feelings of sadness , pessimism , hopelessness , difficulty concentrating , and difficulty making decisions and initiating tasks , among other symptoms.13 despite being very common in the elderly , it can not be attributed solely to aging .", "studies in so paulo ( brazil)14 and florianpolis ( santa catarina , brazil)15 showed that the factors associated with depression in the elderly are : female , unmarried or widowed , altered cognitive function , dependence , use of many medicines , very old ( over 80 years ) , low education , poor economic status , cognitive impairment , fair or poor self - rated health , functional dependency , and chronic pain .", "other causes include smoking , comorbidities ( endocrine , vascular , neurologic , oncological ) , and negative changes in relationships with family and friends.15 \n  hearing loss may be associated with depression to be a debilitating , chronic disorder.16 \n 17 \n 18 the main goal of an auditory rehabilitation program in elderly patients is to minimize the effects caused by sensory deprivation of hearing and reinstate the patients in their family and society , helping them cope with the disadvantages and limitations caused by hearing loss.3 \n  monaural adaptations are commonly performed even in cases of bilateral hearing loss , whereas the most appropriate should be binaural adaptation .", "the reasons for that may be related to factors such as rejection to the use of two hearing aids , financial issues , aesthetic reasons , problems with manual dexterity , very asymmetric losses , and central processing issues.19 \n  given such reality , this work intends to analyze the effects of unilateral adaptation of hearing aids on the symptoms of depression and the social activity constraints of elderly subjects with hearing impairment ."], ["this is a prospective interventional study of elderly individuals of both sexes with recommended use of hearing aids at a hearing center in porto alegre .", "inclusion criteria were signature of the free and informed consent ; 60 years or older ; good health ( seniors able to commute to the research location , conduct tests , and answer questionnaires ) ; new hearing aid users with monaural fitting ; and participation in all phases of the research . exclusion criteria were initiation of antidepressant therapy during the study and a history of cognitive and/or neurologic disorders .", "the study was divided into two phases . in phase 1 , subjects responded to an interview with questions on demographics and health ( general and hearing ) .", "pure tone audiometry was performed to determine the airway tone thresholds ( frequencies 250 to 8,000 hz ) and bone conduction ( frequencies 500 to 4,000 hz ) in a vocal booth , using an ad-229e interacoustics audiometer ( assens , denmark ) .", "the presence and degree of hearing loss were classified according to the world health organization by analyzing mean airway tone threshold in the frequencies of 500 , 1,000 , 2,000 , and 4,000 hz.20 \n  after that , seniors were asked to answer two questionnaires individually , without the intervention of third parties : the geriatric depression scale ( gds ) and the hearing handicap inventory for elderly  short version ( hhie - s ) .", "the gds , an instrument validated for portuguese and composed of 15 yes - or - no questions , is used to detect depression symptoms in the elderly .", "thus , scores less than 5 points indicate absence of symptoms , scores of 5 to 10 indicate mild to moderate symptoms , and scores of 11 or more indicate severe symptoms of depression.21 \n  after completing the first phase , participants answered a summarized version of the hhie - s .", "the questionnaire was prepared in 198222 and translated and adapted to brazilian portuguese.23 it contains 10 questions that aim to assess the impact of hearing loss on the emotional and social status of elderly people .", "all 10 questions provide three choices of answers : for each  yes  answer , 4 points is assigned ; each  sometimes ,  2 points ; and each  no ,  0 points . the total score ranges from 0 to 40 points , and the higher the score , the greater subjects ' self - perception of social activity constraints : 0 to 8 points indicate no social activity constraints ; 10 to 23 points , mild to moderate social activity constraint ; and 24 to 40 points , significant social activity constraints.8 \n 22 \n  after testing , the participants were subjected to the selection and unilaterally hearing aid adaptation processes", "thirty days after purchasing the device , they returned to the hearing center to participate in the second phase of the research . in phase 2", "the research project was approved by the research committee and by the ethics and research committee of the institution ( protocol 266.060 ) .", "patients who agreed to participate signed an informed consent and had their rights guaranteed for confidentiality , nonidentification , and withdrawal of participation .", "the results were analyzed using the statistical package for social science version 20.0 ( ibm , ny , usa ) . to examine associations of categorical data , the chi - square and student t test for paired samples were used .", "all statistical tests were nonparametric tests , and always observed exact p value ( not asymptotic ) , which are the best measures to statistically evaluate a study when the sample is restricted .", "the chi - square test considered the p value of fisher exact test , and for the kappa coefficient of agreement , the exact p value ( not asymptotic ) was observed ."], ["the sample consisted of 13 elderly patients with hearing loss , ages from 60 to 90 years ( mean 72.85    11.05 years ) , 10 women ( 76.9% ) and 3 men ( 23.1% ) .", "table 1 shows data on the characterization of sample in terms of age , gender , mean thresholds , and degree of hearing loss . \n", "table 2 depicts an association analysis between the gds classification of phases 1 and 2 , indicating that there was no significant association between the variables in the periods before and after the adaptation ( p  =  0.615 ) .", "thus , of the 6 patients ( 46.2% ) who initially had mild symptoms of depression , only 2 ( 15.4% ) of them continued to have mild symptoms .", "all seniors who initially had severe symptoms of depression ( 15.4% ) also started to lose some symptoms of depression in phase 2 .", "hhie classification analysis in phases 1 and 2 of the study are presented in table 3 .", "no significant association was found for these variables in the periods before and after the adaptation ( p  >  0.999 ) .", "thus , the only senior who had mild to moderate social activity constraints in the first phase showed reduction in the second phase .", "after prosthetization , participants who had severe social activity constraints in phase 1 started to show no social activity constraints or mild to moderate social activity constraints . only 1 ( 7.7% )", "the lack of association between the classification of the gds and hhie on the two phases is an important point , because it shows that there were different results in both assessments ( before and after hearing aid adaptation)that is , on the sample studied , unilateral prosthetization resulted in benefits for patients . \n", "table 4 shows the scores obtained by the subjects in the instruments used for evaluation in the two phases of the research .", "there was significant difference between the gds ( p  =  0.031 ) and hhie ( p  < ", "abbreviations : gds , geriatric depression scale ; hhie - s , hearing handicap inventory for elderly  short version . \n"], ["the analysis of the survey data showed that the age of the individuals analyzed varied from 60 to 90 years .", "another study that examined the degeneration of the auditory system in the course of aging had a sample with similar age and results.4 \n  we had a greater number of female participants . although it has been found that men often have more problems related to hearing loss than women,24 they also have a lower perception of their disability compared with women,1 \n 25 \n 26 which explains why women seek health services more often26 \n 27 and why there is a greater number of female participants in this study .", "another fact to be noted is that , in terms of population , there is a greater number of older women in brazil , a fact known as feminization of aging.28 \n  there was a prevalence of moderate hearing loss in both the right and the left ears , agreeing with other published studies that refer to this as being the most commonly found degree of hearing impairment in the elderly.2 \n 9 \n 25 \n  the data show that many seniors had some degree of depression symptoms before prosthetization ( table 2 ) .", "after unilateral hearing aid adoption , there was improvement in symptoms for most of them .", "thus , a hearing aid , even when unilateral , promoted elimination or reduction of symptoms of depression in the sample studied .", "this result had already been described in the literature,2 but we emphasize that the result was obtained with bilateral prosthesis .", "thus , the adaptation of a single amplifying device can also generate benefit for elderly patients , which is important for the reduction of one of the most prevalent problems in the aging population , depression .", "the data show that all participants had some degree of social activity constraints before using a unilateral hearing aid ( table 3 ) .", "after 30 days of hearing aid use , 12 ( 93.3% ) and 13 ( 100% ) participants showed absence or reduction of social activity constraints , respectively .", "results show evidence of the positive effects of hearing aid use , even if unilateral , helping to reduce the impact that hearing loss has on social relationships and on the quality of life of the elderly.9 results are consistent with another study that examined the short - term benefits of amplification in new users.29 \n  the data also show a significant difference in scores obtained in phases 1 and 2 of the gds ( p  =  0.031 ) and hhie - s ( p  <  0.001 ) , confirming a reduction or elimination of depression symptoms and social activity constraints in elderly patients , agreeing with other published studies ( table 4).2 \n 8 \n 11 \n 29 \n 30 again , it is important to highlight that in the studied group , unilateral prosthesis resulted in substantial benefits not only regarding hearing but also in other matters essential for the well - being and quality of life of individuals .", "the findings reinforce the importance of hearing aid use in elderly patients , because many times family and social relationships are affected by the hearing impairment .", "the adaptation to amplification devices , albeit unilaterally , has proved to be effective in reducing symptoms of depression and social activity constraints .", "moreover , the length of time between assessments was 30 days , shorter than that considered ideal for auditory acclimatization , and even then , benefits occurred , confirming the importance of auditory rehabilitation in elderly ."], ["results show that unilateral hearing aid contributed to the elimination or reduction of depression symptoms and of social activity constraints in elderly participants in this research sample group ."]]}
{"article_id": "PMC3578664", "article_text": ["ameloblastomas are rare benign odontogenic tumours , most commonly found in a patient s lower jaw .", "although rarely metastatic , the growth of these lesions can result in defects in the jaw easily destroying surrounding bony tissues .", "for this reason , the treatment is often excision of the lesion , with wide excision margins , resulting in the absence of a part , or all , of the mandible .", "the surgical approach for correction of this mandibular defect is often reconstruction of the mandible in view of inserting dental implants at a later date . despite surgical advancements ,", ", bone grafting with non - vascularized bone remains a good option where facilities for soft tissue flaps are not readily accessible .", "creating an effective wound closure and reducing the incidence of infection is an important stage in the success of the operation , and has been combated here by tissue expanding the submucosa , using osmed pellets , prior to surgery .", "this is a relatively new technique to the mandibular region and has the potential to revolutionize the success of microsurgeries in this area . in this report", ", i describe a woman who underwent a repair of her mandibular defect by a combination of innovative techniques , in order to reduce the risk of complications and get her back to an aesthetically pleasing level .", "a 35-year - old lady presented to the craniofacial surgeons looking for a permanent fixture to the bony defect in her lower - right mandible . in 1991", "she had her first operation to remove the ameloblastoma and then had it further excised 6 years later due to a reoccurrence .", "this resulted in an absence of teeth from the canines back and removal of bone down to the inferior alveolar nerve .", "the patient has since been clear of any further reoccurrences and wears lower dentures for aesthetic reasons .", "she has no significant past medical history nor family history , nor does she have any known drug allergies .", "she does not smoke or drink alcohol . on clinical examination , the patient experiences mild parasthesia in the distribution of her right inferior dental nerve , particularly the lateral aspect of the lower lip . on examination ,", "there is clear absence of teeth on the lower - right side from the canines back ( to include the premolar , molar and wisdom teeth ) and presence of healthy oral mucosa with laxity to move the submucosa medially .", "there is a loss of the vertical height of the posterior body of the mandible of about 50% and the vestigial remnants of the lingual plate are just palpable and visible on plain radiograph and ct .", "scans show that the superior margin of the resection is well corticated under the inferior dental nerve ( fig . ", ", the patient wishes to get dental implants . the surgical approach taken , prior to the implants ,", "( b ) ct scan of head imaging of the patient 's mandibular defect pre - surgically .", "( a ) radiolocal image of patient 's jaw . ( b ) ct scan of head the patient was initially given the option between a bone graft or vascularized tissue ( free fibular flap ) and informed of the complications  a bone graft carries a risk of rejection and vascularized tissue needs an increased amount of post - operative care to ensure that the tissue remains vascularized .", "the patient was not keen on a free flap and given the fact that the bony deformity was < 5 cm , non - vascularized iliac bone graft was deemed to be sufficient to carry out this operation .", "the first step was achieving significant tissue expansion ( > 2 cm ) using osmed pellets , guided into the submucosa and kept over the alveolar ridge for several months ( fig . ", "( c ) osmed pellets before insertion into the submucosa image of patient 's oral cavity during insertion of osmed pellets . ( a ) incisions made in the submucosa .", "( c ) osmed pellets before insertion into the submucosa two months later , the patient underwent the main bulk of her operation .", "the osmed pellets were removed from the subperiosteum ( fig .  3a and b ) and the iliac bone graft", "was taken , made up of cortical and cancellous bone and placed to one side ( fig . ", "an epidural catheter was placed inside the soft tissue of the graft site and left overnight .", "it had a continuous fusion of a local anaesthetic to improve post - operative pain and encourage early mobilization . \n", "( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone submucosa after adequate tissue expansion .", "( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone previously , using ct scans , a titanium construct was made to guide the outline of the bone graft and complete the missing height and width of the mandible .", "once fitted to the patients jaw , it was packed with the bone graft and screwed down into position ( fig . ", "a slight groove was made on the mandible to allow space for the inferior dental nerve to lay , so that the construct did not compact the nerve and cause functional problems . \n", "figure  5:titanium construct packed with iliac bone graft and screwed into position on the lower mandible titanium construct packed with iliac bone graft and screwed into position on the lower mandible to complete the surgery , the periosteum was sutured over the construct and then the buccal muscosa was sutured over again to form a double - layered closure ( fig . ", "6a  c).the purpose of this was to prevent infection and rejection , by ensuring that there was an increased distance between the foreign object ( titanium construct ) and the outside environment .", "the dental implants are scheduled to be inserted several months after this operation . \n figure  6:image of patient 's surgical site .", "( c ) image of post - surgical site image of patient 's surgical site .", "bony defects in the maxillofacial area are a huge problem in clinical orthodontics , caused by a wide range of injuries and diseases presenting as either cosmetic or functional defects and in some instances both .", "it is the importance of accounting for the anatomical , functional and aesthetic aspects that make the reconstruction so challenging .", "often patients suffering from ameloblastomas undergo resection as treatment and are left with bony defects that require reconstruction . in this case , the intention was to realize an aesthetically pleasing appearance for the patient and it has been shown that bone grafting with subsequent dental implants is a successful method . as in this case , it is recommended that non - vascularized autogenous bone grafting can be used when a two - layer watertight closure is attainable .", "sources of bone could have been harvested from either local or distant sites but in this case , an extra - oral site ( iliac bone ) was used as a moderate amount of bone was needed .", "the surgery lasted 5 h and incorporated several novel techniques to improve the outcome of the operation .", "the use of osmed pellets was innovative , as pellets of this size have only recently been experimented on goats maxilla .", "as well as allowing for space to put the construct , the pellets allowed for a tension - free closure .", "otherwise , the result would have been a high - tension closure , leading to rapid breakdown of the graft providing an entrance for infection , a common complication occurring when titanium plates are exposed .", "advantages of the osmed tissue expanders are that the material is safe with a low complication rate and low risk of infection due to small incision site and minimal trauma .", "the titanium construct is another relatively novel approach and was invaluable in the success of this surgery , as it guided reconstruction of the mandible , allowing for dental implants to be put in at a later date . at present , an alloplastic material such as titanium is often used for mandibular reconstruction as it is resistant to corrosion , adaptable and biocompatible .", "although the insertion of dental implants is the end goal for this surgery , the importance of this case report is to highlight the novel techniques involved in making this surgery a success . the use of small osmed pellets in the submucosa of the mouth to cause sufficient tissue expansion and create a tension - free closure , combined with the titanium construct packed with grafted iliac bone to rebuild the jaw was an approach recommended by the doctors for the repair of bony defects of < 5 cm .", "i suggest prudent management of future cases post - operatively to ensure a continued success of the graft ."], "abstract_text": ["<S> this case report describes a 35-year - old caucasian radiographer who presented with a significant mandibular bony defect following multiple excisions of an ameloblastoma . as a result , there was an absence of teeth on the lower - right mandible and a clear defect in the mandible . </S>", "<S> the treatment objectives were to rebuild the mandibular defect , with a long - term view of inserting dental implants . in a novel approach outlined in this presentation , tissue expansion of the submucosa , a titanium construct and an iliac bone graft </S>", "<S> were used to rebuild the patient s jaw . </S>", "<S> this surgical technique is recommended for the reconstruction of bony defects . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["ameloblastomas are rare benign odontogenic tumours , most commonly found in a patient s lower jaw .", "although rarely metastatic , the growth of these lesions can result in defects in the jaw easily destroying surrounding bony tissues .", "for this reason , the treatment is often excision of the lesion , with wide excision margins , resulting in the absence of a part , or all , of the mandible .", "the surgical approach for correction of this mandibular defect is often reconstruction of the mandible in view of inserting dental implants at a later date . despite surgical advancements ,", ", bone grafting with non - vascularized bone remains a good option where facilities for soft tissue flaps are not readily accessible .", "creating an effective wound closure and reducing the incidence of infection is an important stage in the success of the operation , and has been combated here by tissue expanding the submucosa , using osmed pellets , prior to surgery .", "this is a relatively new technique to the mandibular region and has the potential to revolutionize the success of microsurgeries in this area . in this report", ", i describe a woman who underwent a repair of her mandibular defect by a combination of innovative techniques , in order to reduce the risk of complications and get her back to an aesthetically pleasing level ."], ["a 35-year - old lady presented to the craniofacial surgeons looking for a permanent fixture to the bony defect in her lower - right mandible . in 1991", "she had her first operation to remove the ameloblastoma and then had it further excised 6 years later due to a reoccurrence .", "this resulted in an absence of teeth from the canines back and removal of bone down to the inferior alveolar nerve .", "the patient has since been clear of any further reoccurrences and wears lower dentures for aesthetic reasons .", "she has no significant past medical history nor family history , nor does she have any known drug allergies .", "she does not smoke or drink alcohol . on clinical examination , the patient experiences mild parasthesia in the distribution of her right inferior dental nerve , particularly the lateral aspect of the lower lip . on examination ,", "there is clear absence of teeth on the lower - right side from the canines back ( to include the premolar , molar and wisdom teeth ) and presence of healthy oral mucosa with laxity to move the submucosa medially .", "there is a loss of the vertical height of the posterior body of the mandible of about 50% and the vestigial remnants of the lingual plate are just palpable and visible on plain radiograph and ct .", "scans show that the superior margin of the resection is well corticated under the inferior dental nerve ( fig . ", ", the patient wishes to get dental implants . the surgical approach taken , prior to the implants ,", "( b ) ct scan of head imaging of the patient 's mandibular defect pre - surgically .", "( a ) radiolocal image of patient 's jaw . ( b ) ct scan of head the patient was initially given the option between a bone graft or vascularized tissue ( free fibular flap ) and informed of the complications  a bone graft carries a risk of rejection and vascularized tissue needs an increased amount of post - operative care to ensure that the tissue remains vascularized .", "the patient was not keen on a free flap and given the fact that the bony deformity was < 5 cm , non - vascularized iliac bone graft was deemed to be sufficient to carry out this operation .", "the first step was achieving significant tissue expansion ( > 2 cm ) using osmed pellets , guided into the submucosa and kept over the alveolar ridge for several months ( fig . ", "( c ) osmed pellets before insertion into the submucosa image of patient 's oral cavity during insertion of osmed pellets . ( a ) incisions made in the submucosa .", "( c ) osmed pellets before insertion into the submucosa two months later , the patient underwent the main bulk of her operation .", "the osmed pellets were removed from the subperiosteum ( fig .  3a and b ) and the iliac bone graft", "was taken , made up of cortical and cancellous bone and placed to one side ( fig . ", "an epidural catheter was placed inside the soft tissue of the graft site and left overnight .", "it had a continuous fusion of a local anaesthetic to improve post - operative pain and encourage early mobilization . \n", "( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone submucosa after adequate tissue expansion .", "( b ) open site where iliac bone graft is harvested from ( c ) cortical and cancellous iliac bone previously , using ct scans , a titanium construct was made to guide the outline of the bone graft and complete the missing height and width of the mandible .", "once fitted to the patients jaw , it was packed with the bone graft and screwed down into position ( fig . ", "a slight groove was made on the mandible to allow space for the inferior dental nerve to lay , so that the construct did not compact the nerve and cause functional problems . \n", "figure  5:titanium construct packed with iliac bone graft and screwed into position on the lower mandible titanium construct packed with iliac bone graft and screwed into position on the lower mandible to complete the surgery , the periosteum was sutured over the construct and then the buccal muscosa was sutured over again to form a double - layered closure ( fig . ", "6a  c).the purpose of this was to prevent infection and rejection , by ensuring that there was an increased distance between the foreign object ( titanium construct ) and the outside environment .", "the dental implants are scheduled to be inserted several months after this operation . \n figure  6:image of patient 's surgical site .", "( c ) image of post - surgical site image of patient 's surgical site ."], ["bony defects in the maxillofacial area are a huge problem in clinical orthodontics , caused by a wide range of injuries and diseases presenting as either cosmetic or functional defects and in some instances both .", "it is the importance of accounting for the anatomical , functional and aesthetic aspects that make the reconstruction so challenging .", "often patients suffering from ameloblastomas undergo resection as treatment and are left with bony defects that require reconstruction . in this case , the intention was to realize an aesthetically pleasing appearance for the patient and it has been shown that bone grafting with subsequent dental implants is a successful method . as in this case , it is recommended that non - vascularized autogenous bone grafting can be used when a two - layer watertight closure is attainable .", "sources of bone could have been harvested from either local or distant sites but in this case , an extra - oral site ( iliac bone ) was used as a moderate amount of bone was needed .", "the surgery lasted 5 h and incorporated several novel techniques to improve the outcome of the operation .", "the use of osmed pellets was innovative , as pellets of this size have only recently been experimented on goats maxilla .", "as well as allowing for space to put the construct , the pellets allowed for a tension - free closure .", "otherwise , the result would have been a high - tension closure , leading to rapid breakdown of the graft providing an entrance for infection , a common complication occurring when titanium plates are exposed .", "advantages of the osmed tissue expanders are that the material is safe with a low complication rate and low risk of infection due to small incision site and minimal trauma .", "the titanium construct is another relatively novel approach and was invaluable in the success of this surgery , as it guided reconstruction of the mandible , allowing for dental implants to be put in at a later date . at present , an alloplastic material such as titanium is often used for mandibular reconstruction as it is resistant to corrosion , adaptable and biocompatible .", "although the insertion of dental implants is the end goal for this surgery , the importance of this case report is to highlight the novel techniques involved in making this surgery a success . the use of small osmed pellets in the submucosa of the mouth to cause sufficient tissue expansion and create a tension - free closure , combined with the titanium construct packed with grafted iliac bone to rebuild the jaw was an approach recommended by the doctors for the repair of bony defects of < 5 cm .", "i suggest prudent management of future cases post - operatively to ensure a continued success of the graft ."]]}
{"article_id": "PMC4015428", "article_text": ["glucagonlike peptide1 ( glp1 ) , the most potent incretin hormone , stimulates glucoseinduced insulin secretion and inhibits glucagon secretion , which consequently leads to a decrease in hepatic glucose production and blood glucose levels .", "in addition to its ability to modulate insulin and glucagon secretion , glp1 inhibits gastric emptying and gastric acid secretion , which suppress appetite and energy intake in obese individuals , and in patients with type  2 diabetes .", "glp1 is secreted from intestinal l  cells in response to ingestion of nutrients , including carbohydrate and lipids .", "a previous study showed that alphalinolelic acid ( la ) promoted glp1 secretion through the stimulation of gproteincoupled receptor ( gpr ) 120 , which is abundantly expressed in the intestine , in mice .", "furthermore , a recent study has shown that eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , metabolites of la , stimulate endogenous glp1 secretion in mice .", "these reports show that 3 polyunsaturated fatty acids ( pufa ) , including la , epa and dha , increase glp1 secretion in mice .", "however , no evidence that 3 pufa ingestion increases glp1 secretion is available in humans .", "as compared with americans and europeans , japanese eat more fish , which is a major source of epa and dha . hence , serum epa or dha concentrations are higher in japanese than those in americans or europeans . evidence that fish intake prevents atherosclerosisrelated cardiovascular disease have been accumulating .", "a recent study has shown a significant inverse correlation between serum epa or dha concentrations and cardiovascular risk .", "furthermore , some studies showed that fish intake reduced the rates of incidence for diabetes in patients with metabolic syndrome , and reduced the rates of death in patients with type  2 diabetes .", "however , the direct effects of epa or dha consumption on glycemic control in patients with type  2 diabetes have remained uncertain .", "dpp4 inhibitor increases endogenous active glp1 levels through inhibition of dpp4 enzyme activity , leading to increased circulating insulin levels and decreased blood glucose levels .", "the circulating glp1 levels were reported to be lower in patients with type  2 diabetes than in nondiabetic patients .", "therefore , dpp4 inhibitor might be more effective in increasing circulating glp1 levels in patients with type  2 diabetes if they have low glp1 levels . based on these lines of evidence , we hypothesized that dpp4 inhibitor would be effective in patients with type  2 diabetes with high serum 3 pufa concentrations . in the present study , therefore , we investigated the relationship between serum epa or dha concentrations and a decrease in hemoglobin a1c in patients with type  2 diabetes prescribed dpp4 inhibitors .", "serum epa and dha concentrations were measured in 62 consecutive patients with type  2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine , kyoto , japan , who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents ( oha ) .", "patients with advanced renal dysfunction ( serum creatinine concentration was equal to or more than 2.0  mg / dl ) were excluded from the present study .", "sitagliptin at a daily dose of 50  mg , which was the standard dose in japanese patients , was continuously given once daily during the observation period .", "the prior diet , exercise program and dosage of oha in the study patients were maintained during the observation period .", "after 24  weeks of treatment with dpp4 inhibitor , we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations , as well as age , sex , body mass index ( bmi ) , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs .", "type  2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus .", "approval for the study was obtained from the local research ethics committee , and informed consent was obtained from all patients .", "serum total cholesterol , highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods .", "hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society", "skewed variables , such as epa and dha , are presented as the median ( interquartile range ) , and continuous variables are presented as the mean    standard deviation ( sd ) .", "paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment , and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software ( version 5.0 ; sas institute , cary , nc , usa ) . because epa and dha showed skewed distributions , logarithm ( log ) transformation was carried out before performing correlation and regression analysis . the relationship between log epa , log dha or log ( epa  +  dha ) and a decrease in hemoglobin a1c", "was examined by linear regression analysis . in multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c , we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses .", "serum epa and dha concentrations were measured in 62 consecutive patients with type  2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine , kyoto , japan , who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents ( oha ) .", "patients with advanced renal dysfunction ( serum creatinine concentration was equal to or more than 2.0  mg / dl ) were excluded from the present study .", "sitagliptin at a daily dose of 50  mg , which was the standard dose in japanese patients , was continuously given once daily during the observation period .", "the prior diet , exercise program and dosage of oha in the study patients were maintained during the observation period .", "after 24  weeks of treatment with dpp4 inhibitor , we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations , as well as age , sex , body mass index ( bmi ) , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs .", "type  2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus .", "approval for the study was obtained from the local research ethics committee , and informed consent was obtained from all patients .", "serum total cholesterol , highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods .", "hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society .", "means , medians or frequencies of potential confounding variables were calculated . skewed variables , such as epa and dha ,", "are presented as the median ( interquartile range ) , and continuous variables are presented as the mean    standard deviation ( sd ) .", "paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment , and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software ( version 5.0 ; sas institute , cary , nc , usa ) . because epa and dha showed skewed distributions , logarithm ( log ) transformation was carried out before performing correlation and regression analysis . the relationship between log epa , log dha or log ( epa  +  dha ) and a decrease in hemoglobin a1c", "was examined by linear regression analysis . in multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c", ", we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses .", "characteristics of the 62 patients with type  2 diabetes enrolled in the present study are shown in table  1 .", "the median ( interquartile range ) serum epa concentration was 62.6  g / ml ( 39.588.0 ) and the median ( interquartile range ) serum dha concentration was 139.1  g / ml ( 109.4165.7 ) . among 62 patients", ", 16 patients were given dpp4 inhibitor as a monotherapy and 46 patients were given it as an addon therapy ( 34 sulfonylurea , 25 metformin , 8 pioglitazones , 5 alphaglucosidase inhibitor ) .", "data are n , mean    sd or median ( interquartile range ) .", "mean hemoglobin a1c was significantly decreased from 8.1    1.1% at baseline to 7.2    1.0% at 24  weeks after administration of dpp4 inhibitor .", "correlation between log epa , log dha or log ( epa  + dha ) and other variables are shown in table  2 .", "log epa and log ( epa  +  dha ) correlated with a decrease in hemoglobin a1c .", "log dha tended to correlate with a decrease in hemoglobin a1c , but it did not reach statistical significance .", "dha , docosahexaenoic acid ; epa , eicosapentaenoic acid . a decrease in hemoglobin a1c correlated with bmi ( r  =  0.396 , p  =  0.0013 ) , age ( r  =  0.275 p  =  0.0032 ) , hemoglobin a1c at baseline ( r  =  0.490 , p  <  0.0001 ) , log epa ( r  =  0.285 , p  =  0.0246 ) and log ( epa  +  dha ) ( r  =  0.260 , p  =  0.0411 ) . there were no significant differences in decrease in hemoglobin a1c between male and female patients ( 1.0    0.8% vs 1.0    0.9% , p  =  0.9758 ) . decrease in hemoglobin a1c was greater in patients treated with antidiabetic concomitant drugs compared with that in patients without those ( 1.1    0.9% vs 0.6    0.4% , p  =  0.0342 ) . in the multiple regression analysis to examine the effects of variables on the decrease in hemoglobin a1c , we included the following independent variables , which were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses : age , bmi , hemoglobin a1c at baseline , usage of antidiabetic concomitant drugs and log epa .", "multiple regression analysis showed that bmi , hemoglobin a1c at baseline and log epa were independent determinants of a decrease in hemoglobin a1c ( table  3 ) .", "presence of antidiabetic concomitant drugs ( no  =  0 , yes  =  1 ) .", "in the present study , serum epa concentrations correlated with a decrease in hemoglobin a1c .", "this correlation remained significant after adjustment for age , bmi , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs .", "the median ( interquartile range ) serum epa concentration was 62.6  g / ml ( 39.588.0 ; the mean serum epa concentration was 68.6    34.1  g / ml ) , which was almost consistent with previous reports of the epa concentration in general japanese .", "serum epa concentrations increase with age in apparently healthy japanese . in the present study , however , there was no significant correlation between log epa and age in patients with type  2 diabetes .", "dpp4 inhibitor increases endogenous active glp1 levels through prevention of glp1 degradation , leading to an increase in circulating insulin levels and a decrease in blood glucose levels . because of the pharmacological action of dpp4 inhibitor on insulin secretion , dpp4 inhibitor is effective in type  2 diabetic patients with impaired insulin secretion , which is a characteristic of japanese patients with type  2 diabetes . indeed , as compared with american and european patients with type  2 diabetes , japanese patients with type  2 diabetes might be higher responders to dpp4 inhibitor .", "interestingly , the present study has shown a strong correlation between bmi and decrease in hemoglobin a1c , suggesting that dpp4 inhibitor is more effective in lean patients who generally have impaired insulin secretion , rather than in obese patients with insulin resistance .", "this is consistent with a recent study reporting the association of bmi with efficacy of the dpp4 inhibitor , sitagliptin .", "previous studies showed that dpp4 inhibitor was effective as an addon therapy to some antidiabetic drugs , including metformin , pioglitazone and sulfonylurea .", "the profile of diabetic therapy before dpp4 inhibitor administration was as follows : 16 used diet , 15 received sulfonylurea monotherapy , 12 received metformin monotherapy , eight received sulfonylurea and metformin , five received sulfonylurea and pioglitazone , two received sulfonylurea and alphaglucosidase inhibitor , and four took three drugs .", "we analyzed the correlations between serum epa concentrations and a decrease in hemoglobin a1c in patients with dpp4 inhibitor monotherapy , dpp4 inhibitor addon therapy to sulfonylurea , to metformin , and to sulfonylurea and metformin .", "log epa tended to correlate with a decrease in hemoglobin a1c only in patients with dpp4 inhibitor addon therapy to metformin , although it did not reach statistical significance ( r  =  0.526 , p  =  0.0796 ) .", "there were no significant correlations between log epa and decrease in hemoglobin a1c in other treatment groups .", "it seems to be difficult to show these results because of the small sample size in each treatment group . in basic studies", "have shown that gpr120 functioned as a receptor for unstructured longchain fatty acids , and that the stimulation of gpr120 with la promoted glp1 secretion in  vitro and in  vivo .", "furthermore , gpr120 has been shown to be abundantly expressed in the intestine both in humans and mice .", ". however , the mechanism of glp1 secretion by 3 pufa has been less clear . in the present study , furthermore , serum dha concentrations did not show a significant association with the efficacy of dpp4 inhibitor , although they tended to positively correlate with a decrease in hemoglobin a1c .", "further studies are required to clarify the mechanism of 3 pufastimulated glp1 secretion . taken together with these previous studies , and the ability of dpp4 inhibitor to increase endogenous active glp1 levels , the present result that serum epa concentrations correlated with the efficacy of dpp4 inhibitor might provide indirect evidence that epa has a potential to promote glp1 secretion in humans .", "limitations of the present study included a small sample size and a short observation period .", "therefore , the direct link between serum epa concentration and glp1 or insulin concentrations could not be assessed .", "furthermore , because ingested fatty acids are generally absorbed within the small intestine , epa might be expected to act on gpr120 inadequately after oral administration .", "there are no reports of a linear relationship between epa intake and serum epa concentrations . however , serum epa concentrations positively correlated with epa intake assessed by a selfadministered diet history questionnaire in a subgroup of patients with type  2 diabetes ( n  =  40 , r  =  0.419 , p  =  0.0066 ) .", "therefore , we hope that serum epa concentrations could be a marker of epa intake . to our knowledge , this is the first report of the relationship between serum epa concentrations and the efficacy of dpp4 inhibitor in patients with type  2 diabetes , and suggests new avenues for research into incretin therapies .", "large interventional studies are required to better assess the contributions of epa to the efficacy of dpp4 inhibitor in patients with type  2 diabetes . in conclusion ,", "dpp4 inhibitor is effective in patients with type  2 diabetes with high serum epa concentrations ."], "abstract_text": ["<S> abstractaims / introduction :   eicosapentaenoic acid ( epa ) stimulates glucagonlike peptide1 ( glp1 ) secretion in mice . </S>", "<S> we investigated the relationship between serum epa concentrations and the efficacy of dipeptidylpeptidase  iv ( dpp4 ) inhibitor in patients with type  2 diabetes.materials and methods :   serum epa concentrations were measured in 62 consecutive patients with type  2 diabetes who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents . </S>", "<S> the dosage of oral hypoglycemic agents was maintained during the observation period . </S>", "<S> after 24  weeks of treatment with dpp4 inhibitor , we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa concentrations , as well as age , sex , body mass index ( bmi ) , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs.results :   hemoglobin a1c was significantly decreased from 8.1    1.1% to 7.2    1.0% by dpp4 inhibitor . a decrease in hemoglobin a1c correlated with bmi ( r  =  0.396 , p  =  0.0013 ) , age ( r  =  0.275 , p  =  0.0032 ) , hemoglobin a1c at baseline ( r  =  0.490 , p  <  0.0001 ) and log epa ( r  =  0.285 , p  =  0.0246 ) . </S>", "<S> multiple regression analysis showed that bmi (   =  0.419 , p  =  0.0002 ) , hemoglobin a1c at baseline (   =  0.579 , p  <  0.0001 ) and log epa (   =  0.220 , p  </S>", "<S> =  0.0228 ) were independent determinants of decrease in hemoglobin a1c.conclusions :   dpp4 inhibitor is effective in patients with type  2 diabetes with high serum epa concentrations . </S>", "<S> ( j diabetes invest , doi : 10.1111/j.20401124.2012.00220.x , 2012 ) </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Patients", "Biochemical Analyses", "Statistical Analysis", "Results", "Discussion"], "sections": [["glucagonlike peptide1 ( glp1 ) , the most potent incretin hormone , stimulates glucoseinduced insulin secretion and inhibits glucagon secretion , which consequently leads to a decrease in hepatic glucose production and blood glucose levels .", "in addition to its ability to modulate insulin and glucagon secretion , glp1 inhibits gastric emptying and gastric acid secretion , which suppress appetite and energy intake in obese individuals , and in patients with type  2 diabetes .", "glp1 is secreted from intestinal l  cells in response to ingestion of nutrients , including carbohydrate and lipids .", "a previous study showed that alphalinolelic acid ( la ) promoted glp1 secretion through the stimulation of gproteincoupled receptor ( gpr ) 120 , which is abundantly expressed in the intestine , in mice .", "furthermore , a recent study has shown that eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) , metabolites of la , stimulate endogenous glp1 secretion in mice .", "these reports show that 3 polyunsaturated fatty acids ( pufa ) , including la , epa and dha , increase glp1 secretion in mice .", "however , no evidence that 3 pufa ingestion increases glp1 secretion is available in humans .", "as compared with americans and europeans , japanese eat more fish , which is a major source of epa and dha . hence , serum epa or dha concentrations are higher in japanese than those in americans or europeans . evidence that fish intake prevents atherosclerosisrelated cardiovascular disease have been accumulating .", "a recent study has shown a significant inverse correlation between serum epa or dha concentrations and cardiovascular risk .", "furthermore , some studies showed that fish intake reduced the rates of incidence for diabetes in patients with metabolic syndrome , and reduced the rates of death in patients with type  2 diabetes .", "however , the direct effects of epa or dha consumption on glycemic control in patients with type  2 diabetes have remained uncertain .", "dpp4 inhibitor increases endogenous active glp1 levels through inhibition of dpp4 enzyme activity , leading to increased circulating insulin levels and decreased blood glucose levels .", "the circulating glp1 levels were reported to be lower in patients with type  2 diabetes than in nondiabetic patients .", "therefore , dpp4 inhibitor might be more effective in increasing circulating glp1 levels in patients with type  2 diabetes if they have low glp1 levels . based on these lines of evidence , we hypothesized that dpp4 inhibitor would be effective in patients with type  2 diabetes with high serum 3 pufa concentrations . in the present study , therefore , we investigated the relationship between serum epa or dha concentrations and a decrease in hemoglobin a1c in patients with type  2 diabetes prescribed dpp4 inhibitors ."], ["serum epa and dha concentrations were measured in 62 consecutive patients with type  2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine , kyoto , japan , who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents ( oha ) .", "patients with advanced renal dysfunction ( serum creatinine concentration was equal to or more than 2.0  mg / dl ) were excluded from the present study .", "sitagliptin at a daily dose of 50  mg , which was the standard dose in japanese patients , was continuously given once daily during the observation period .", "the prior diet , exercise program and dosage of oha in the study patients were maintained during the observation period .", "after 24  weeks of treatment with dpp4 inhibitor , we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations , as well as age , sex , body mass index ( bmi ) , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs .", "type  2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus .", "approval for the study was obtained from the local research ethics committee , and informed consent was obtained from all patients .", "serum total cholesterol , highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods .", "hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society", "skewed variables , such as epa and dha , are presented as the median ( interquartile range ) , and continuous variables are presented as the mean    standard deviation ( sd ) .", "paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment , and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software ( version 5.0 ; sas institute , cary , nc , usa ) . because epa and dha showed skewed distributions , logarithm ( log ) transformation was carried out before performing correlation and regression analysis . the relationship between log epa , log dha or log ( epa  +  dha ) and a decrease in hemoglobin a1c", "was examined by linear regression analysis . in multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c , we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses ."], ["serum epa and dha concentrations were measured in 62 consecutive patients with type  2 diabetes recruited from the outpatient clinic at kyoto prefectural university of medicine , kyoto , japan , who were newly given dpp4 inhibitor as a monotherapy or as an addon therapy to oral hypoglycemic agents ( oha ) .", "patients with advanced renal dysfunction ( serum creatinine concentration was equal to or more than 2.0  mg / dl ) were excluded from the present study .", "sitagliptin at a daily dose of 50  mg , which was the standard dose in japanese patients , was continuously given once daily during the observation period .", "the prior diet , exercise program and dosage of oha in the study patients were maintained during the observation period .", "after 24  weeks of treatment with dpp4 inhibitor , we evaluated the relationships between a decrease in hemoglobin a1c from baseline and serum epa or dha concentrations , as well as age , sex , body mass index ( bmi ) , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs .", "type  2 diabetes was diagnosed according to the report of the expert committee on the diagnosis and classification of diabetes mellitus .", "approval for the study was obtained from the local research ethics committee , and informed consent was obtained from all patients ."], ["serum total cholesterol , highdensity lipoprotein cholesterol and triglyceride concentrations were assessed using standard enzymatic methods .", "hemoglobin a1c was assayed using highperformance liquid chromatography and was expressed as a national glycohemoglobin standardization program unit as recommended by the japan diabetes society ."], ["means , medians or frequencies of potential confounding variables were calculated . skewed variables , such as epa and dha ,", "are presented as the median ( interquartile range ) , and continuous variables are presented as the mean    standard deviation ( sd ) .", "paired ttest was carried out to assess the statistical significance of difference in hemoglobin a1c between baseline and after dpp4 inhibitor treatment , and unpaired ttest was carried out to assess the statistical significance of difference in a decrease in hemoglobin a1c between groups using stat view software ( version 5.0 ; sas institute , cary , nc , usa ) . because epa and dha showed skewed distributions , logarithm ( log ) transformation was carried out before performing correlation and regression analysis . the relationship between log epa , log dha or log ( epa  +  dha ) and a decrease in hemoglobin a1c", "was examined by linear regression analysis . in multiple regression analysis to assess the effects of various factors on decrease in hemoglobin a1c", ", we included independent variables that were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses ."], ["characteristics of the 62 patients with type  2 diabetes enrolled in the present study are shown in table  1 .", "the median ( interquartile range ) serum epa concentration was 62.6  g / ml ( 39.588.0 ) and the median ( interquartile range ) serum dha concentration was 139.1  g / ml ( 109.4165.7 ) . among 62 patients", ", 16 patients were given dpp4 inhibitor as a monotherapy and 46 patients were given it as an addon therapy ( 34 sulfonylurea , 25 metformin , 8 pioglitazones , 5 alphaglucosidase inhibitor ) .", "data are n , mean    sd or median ( interquartile range ) .", "mean hemoglobin a1c was significantly decreased from 8.1    1.1% at baseline to 7.2    1.0% at 24  weeks after administration of dpp4 inhibitor .", "correlation between log epa , log dha or log ( epa  + dha ) and other variables are shown in table  2 .", "log epa and log ( epa  +  dha ) correlated with a decrease in hemoglobin a1c .", "log dha tended to correlate with a decrease in hemoglobin a1c , but it did not reach statistical significance .", "dha , docosahexaenoic acid ; epa , eicosapentaenoic acid . a decrease in hemoglobin a1c correlated with bmi ( r  =  0.396 , p  =  0.0013 ) , age ( r  =  0.275 p  =  0.0032 ) , hemoglobin a1c at baseline ( r  =  0.490 , p  <  0.0001 ) , log epa ( r  =  0.285 , p  =  0.0246 ) and log ( epa  +  dha ) ( r  =  0.260 , p  =  0.0411 ) . there were no significant differences in decrease in hemoglobin a1c between male and female patients ( 1.0    0.8% vs 1.0    0.9% , p  =  0.9758 ) . decrease in hemoglobin a1c was greater in patients treated with antidiabetic concomitant drugs compared with that in patients without those ( 1.1    0.9% vs 0.6    0.4% , p  =  0.0342 ) . in the multiple regression analysis to examine the effects of variables on the decrease in hemoglobin a1c , we included the following independent variables , which were significantly correlated with a decrease in hemoglobin a1c in the univariate analyses : age , bmi , hemoglobin a1c at baseline , usage of antidiabetic concomitant drugs and log epa .", "multiple regression analysis showed that bmi , hemoglobin a1c at baseline and log epa were independent determinants of a decrease in hemoglobin a1c ( table  3 ) .", "presence of antidiabetic concomitant drugs ( no  =  0 , yes  =  1 ) ."], ["in the present study , serum epa concentrations correlated with a decrease in hemoglobin a1c .", "this correlation remained significant after adjustment for age , bmi , hemoglobin a1c at baseline and usage of antidiabetic concomitant drugs .", "the median ( interquartile range ) serum epa concentration was 62.6  g / ml ( 39.588.0 ; the mean serum epa concentration was 68.6    34.1  g / ml ) , which was almost consistent with previous reports of the epa concentration in general japanese .", "serum epa concentrations increase with age in apparently healthy japanese . in the present study , however , there was no significant correlation between log epa and age in patients with type  2 diabetes .", "dpp4 inhibitor increases endogenous active glp1 levels through prevention of glp1 degradation , leading to an increase in circulating insulin levels and a decrease in blood glucose levels . because of the pharmacological action of dpp4 inhibitor on insulin secretion , dpp4 inhibitor is effective in type  2 diabetic patients with impaired insulin secretion , which is a characteristic of japanese patients with type  2 diabetes . indeed , as compared with american and european patients with type  2 diabetes , japanese patients with type  2 diabetes might be higher responders to dpp4 inhibitor .", "interestingly , the present study has shown a strong correlation between bmi and decrease in hemoglobin a1c , suggesting that dpp4 inhibitor is more effective in lean patients who generally have impaired insulin secretion , rather than in obese patients with insulin resistance .", "this is consistent with a recent study reporting the association of bmi with efficacy of the dpp4 inhibitor , sitagliptin .", "previous studies showed that dpp4 inhibitor was effective as an addon therapy to some antidiabetic drugs , including metformin , pioglitazone and sulfonylurea .", "the profile of diabetic therapy before dpp4 inhibitor administration was as follows : 16 used diet , 15 received sulfonylurea monotherapy , 12 received metformin monotherapy , eight received sulfonylurea and metformin , five received sulfonylurea and pioglitazone , two received sulfonylurea and alphaglucosidase inhibitor , and four took three drugs .", "we analyzed the correlations between serum epa concentrations and a decrease in hemoglobin a1c in patients with dpp4 inhibitor monotherapy , dpp4 inhibitor addon therapy to sulfonylurea , to metformin , and to sulfonylurea and metformin .", "log epa tended to correlate with a decrease in hemoglobin a1c only in patients with dpp4 inhibitor addon therapy to metformin , although it did not reach statistical significance ( r  =  0.526 , p  =  0.0796 ) .", "there were no significant correlations between log epa and decrease in hemoglobin a1c in other treatment groups .", "it seems to be difficult to show these results because of the small sample size in each treatment group . in basic studies", "have shown that gpr120 functioned as a receptor for unstructured longchain fatty acids , and that the stimulation of gpr120 with la promoted glp1 secretion in  vitro and in  vivo .", "furthermore , gpr120 has been shown to be abundantly expressed in the intestine both in humans and mice .", ". however , the mechanism of glp1 secretion by 3 pufa has been less clear . in the present study , furthermore , serum dha concentrations did not show a significant association with the efficacy of dpp4 inhibitor , although they tended to positively correlate with a decrease in hemoglobin a1c .", "further studies are required to clarify the mechanism of 3 pufastimulated glp1 secretion . taken together with these previous studies , and the ability of dpp4 inhibitor to increase endogenous active glp1 levels , the present result that serum epa concentrations correlated with the efficacy of dpp4 inhibitor might provide indirect evidence that epa has a potential to promote glp1 secretion in humans .", "limitations of the present study included a small sample size and a short observation period .", "therefore , the direct link between serum epa concentration and glp1 or insulin concentrations could not be assessed .", "furthermore , because ingested fatty acids are generally absorbed within the small intestine , epa might be expected to act on gpr120 inadequately after oral administration .", "there are no reports of a linear relationship between epa intake and serum epa concentrations . however , serum epa concentrations positively correlated with epa intake assessed by a selfadministered diet history questionnaire in a subgroup of patients with type  2 diabetes ( n  =  40 , r  =  0.419 , p  =  0.0066 ) .", "therefore , we hope that serum epa concentrations could be a marker of epa intake . to our knowledge , this is the first report of the relationship between serum epa concentrations and the efficacy of dpp4 inhibitor in patients with type  2 diabetes , and suggests new avenues for research into incretin therapies .", "large interventional studies are required to better assess the contributions of epa to the efficacy of dpp4 inhibitor in patients with type  2 diabetes . in conclusion ,", "dpp4 inhibitor is effective in patients with type  2 diabetes with high serum epa concentrations ."]]}
{"article_id": "PMC4614659", "article_text": ["pregnancy course was regular , except for maternal consumption of mebendazol at 5 weeks ' gestation to treat a pinworm infection .", "maternal history was negative for consumption of any known teratogenic class drug during the whole pregnancy .", "clinical examination at birth was normal , except for complete congenital anonychia of left middle finger ( fig.1 ) .", "x - ray was performed , showing an absence of the left middle finger distal phalanx ( fig.2).the rest of the nails on the fingers and toes were normal .", "parents were not consanguineous and have normal fingernails and toenails . the baby was discharged on the third day of life , and was found healthy and thriving at three - month follow - up visit .", "final diagnosis was that of a sporadic co. x - ray showing absence of left middle finger distal phalanx .", "congenital onychodystrophy ( co ) , also called as iso  kikuchi syndrome , was first described by iso in 1969 1 and later by kikuchi in 1974 2 as a clinical syndrome involving dysplasia / absence of fingernails with underlying bone abnormalities . since these first observations", ", clinical criteria have been expanded to include a number of additional associated conditions derived from small series , case reports , and retrospective reviews over the next 30 years .", "co clinical criteria are the following : \n\n unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 ( up to total anonychia of hands and feet ) ; \n\n radiographic abnormalities of the distal bony phalanx of the affected fingers ; \n\n congenital occurrence , which can be both sporadic or hereditary 4,5 . \n \n", "unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 ( up to total anonychia of hands and feet ) ; \n \n radiographic abnormalities of the distal bony phalanx of the affected fingers ; \n \n congenital occurrence , which can be both sporadic or hereditary 4,5 . \n ", "genetic loci responsible for the condition are still under investigation : linkage to the known keratin gene clusters on 12q12 and 17q21 has been excluded by krebsova et  al . in 2000 ; 7 a putative isolated congenital nail dysplasia locus , designated ndic , has been identified on 17p13 , although the identified region harbors no genes known to be involved in skin or nail abnormalities 7 .", "several acquired isolated nail disorders which may represent differential diagnoses of co are presented in table1 \n 8 . differently from all these conditions", ", co presents as a congenital hypoplasia , dysplasia , or absence of one or more fingernails , and is typically accompanied by underlying phalanx bone disease .", "acquired isolated nail disorders 8 several heterogeneous multisystem pathologies may also come with ungueal abnormalities ; these are summarized in table2 \n 813 .", "813 \n triangular nail lunula \n \n fingernails and toenails hypoplasia 8 \n \n absent or hypoplastic patellae \n \n elbow dysplasia , often involving posterior subluxation of the radial head \n \n iliac horns dysplasia \n \n arrested or reduced nail growth \n \n thickened and over - curved nails with absence of cuticle \n  differently from the above - mentioned conditions , classic co does not come with involvement of systems other than nails and relative phalanges .", "kikuchi originally suggested fetal grip as a causative mechanism of nail and phalanx ischemia , leading to dysplasia or even complete resorption of such structures 14 .", "this theory , however , is not consistent with the recent acquisitions on developmental biology stating that limbs and bones develop early in fetal life , prior to fetus ' ability to exert a significant grip .", "more recently , several pathogenetic mechanisms have been proposed for this condition : \n\n selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury : the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15 , which should be more protected than the ulnar one;16 \n\n in utero dysplastic change in the crescent - shaped cap of the distal phalanx , a theory that would account for the frequent  y  shape of the dysplastic distal phalanx , when present;17 \n\n genetic mutations causing impairment of the wnt signaling pathway , an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development , growth regulation of multiple tissues , and cancer development;18 \n\n fetal exposure to teratogens , particularly antiepilectic drugs : phenytoin 19 , valproate , and carbamazepine20 \n \n selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury : the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15 , which should be more protected than the ulnar one;16 \n \n in utero dysplastic change in the crescent - shaped cap of the distal phalanx , a theory that would account for the frequent  y  shape of the dysplastic distal phalanx , when present;17 \n \n genetic mutations causing impairment of the wnt signaling pathway , an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development , growth regulation of multiple tissues , and cancer development;18 \n \n fetal exposure to teratogens , particularly antiepilectic drugs : phenytoin 19 , valproate , and carbamazepine20 \n  a literature research showed no proven association between the use of mebendazole during pregnancy and co. conversely , helminth infection during pregnancy is associated with poor cognitive and gross motor outcomes in infants , so that measures to prevent helminth infection during pregnancy should be reinforced;21 anthelminthic therapy is actually recommended in infected pregnant women , and it has been associated to a decreased rate of maternal anemia and low birth weight 22 .", "nevertheless , clinical follow - up is needed in order to exclude other conditions associated with multisystem pathology ; a  complete family history may also be warranted to determine sporadic or hereditary transmission of such a condition ."], "abstract_text": ["<S> key clinical messageclassic co ( also called iso  kikuchi syndrome ) represents a benign , isolated condition associated with normal patient outcome . </S>", "<S> nevertheless , clinical follow - up and/or further clinically - based tests are needed to exclude other nail diseases associated with multisystem pathology ; complete family history is also important to determine sporadic or hereditary transmission of such condition . </S>"], "labels": null, "section_names": ["Case Report", "Discussion", "Conclusions and final remarks", "Conflict of interest"], "sections": [["pregnancy course was regular , except for maternal consumption of mebendazol at 5 weeks ' gestation to treat a pinworm infection .", "maternal history was negative for consumption of any known teratogenic class drug during the whole pregnancy .", "clinical examination at birth was normal , except for complete congenital anonychia of left middle finger ( fig.1 ) .", "x - ray was performed , showing an absence of the left middle finger distal phalanx ( fig.2).the rest of the nails on the fingers and toes were normal .", "parents were not consanguineous and have normal fingernails and toenails . the baby was discharged on the third day of life , and was found healthy and thriving at three - month follow - up visit .", "final diagnosis was that of a sporadic co. x - ray showing absence of left middle finger distal phalanx ."], ["congenital onychodystrophy ( co ) , also called as iso  kikuchi syndrome , was first described by iso in 1969 1 and later by kikuchi in 1974 2 as a clinical syndrome involving dysplasia / absence of fingernails with underlying bone abnormalities . since these first observations", ", clinical criteria have been expanded to include a number of additional associated conditions derived from small series , case reports , and retrospective reviews over the next 30 years .", "co clinical criteria are the following : \n\n unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 ( up to total anonychia of hands and feet ) ; \n\n radiographic abnormalities of the distal bony phalanx of the affected fingers ; \n\n congenital occurrence , which can be both sporadic or hereditary 4,5 . \n \n", "unilateral or bilateral hypoplasia of the index fingernails and/or other fingers including toenails 3 ( up to total anonychia of hands and feet ) ; \n \n radiographic abnormalities of the distal bony phalanx of the affected fingers ; \n \n congenital occurrence , which can be both sporadic or hereditary 4,5 . \n ", "genetic loci responsible for the condition are still under investigation : linkage to the known keratin gene clusters on 12q12 and 17q21 has been excluded by krebsova et  al . in 2000 ; 7 a putative isolated congenital nail dysplasia locus , designated ndic , has been identified on 17p13 , although the identified region harbors no genes known to be involved in skin or nail abnormalities 7 .", "several acquired isolated nail disorders which may represent differential diagnoses of co are presented in table1 \n 8 . differently from all these conditions", ", co presents as a congenital hypoplasia , dysplasia , or absence of one or more fingernails , and is typically accompanied by underlying phalanx bone disease .", "acquired isolated nail disorders 8 several heterogeneous multisystem pathologies may also come with ungueal abnormalities ; these are summarized in table2 \n 813 .", "813 \n triangular nail lunula \n \n fingernails and toenails hypoplasia 8 \n \n absent or hypoplastic patellae \n \n elbow dysplasia , often involving posterior subluxation of the radial head \n \n iliac horns dysplasia \n \n arrested or reduced nail growth \n \n thickened and over - curved nails with absence of cuticle \n  differently from the above - mentioned conditions , classic co does not come with involvement of systems other than nails and relative phalanges .", "kikuchi originally suggested fetal grip as a causative mechanism of nail and phalanx ischemia , leading to dysplasia or even complete resorption of such structures 14 .", "this theory , however , is not consistent with the recent acquisitions on developmental biology stating that limbs and bones develop early in fetal life , prior to fetus ' ability to exert a significant grip .", "more recently , several pathogenetic mechanisms have been proposed for this condition : \n\n selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury : the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15 , which should be more protected than the ulnar one;16 \n\n in utero dysplastic change in the crescent - shaped cap of the distal phalanx , a theory that would account for the frequent  y  shape of the dysplastic distal phalanx , when present;17 \n\n genetic mutations causing impairment of the wnt signaling pathway , an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development , growth regulation of multiple tissues , and cancer development;18 \n\n fetal exposure to teratogens , particularly antiepilectic drugs : phenytoin 19 , valproate , and carbamazepine20 \n \n selective abnormal fetal vascular supply from palmar digital artery causing in utero ischemic injury : the ischemic damage would mainly be seen on the radial side of the affected finger due to the smaller caliber of the artery on that side 15 , which should be more protected than the ulnar one;16 \n \n in utero dysplastic change in the crescent - shaped cap of the distal phalanx , a theory that would account for the frequent  y  shape of the dysplastic distal phalanx , when present;17 \n \n genetic mutations causing impairment of the wnt signaling pathway , an evolutionarily conserved signal transduction pathway that plays a pivotal role in embryonic development , growth regulation of multiple tissues , and cancer development;18 \n \n fetal exposure to teratogens , particularly antiepilectic drugs : phenytoin 19 , valproate , and carbamazepine20 \n  a literature research showed no proven association between the use of mebendazole during pregnancy and co. conversely , helminth infection during pregnancy is associated with poor cognitive and gross motor outcomes in infants , so that measures to prevent helminth infection during pregnancy should be reinforced;21 anthelminthic therapy is actually recommended in infected pregnant women , and it has been associated to a decreased rate of maternal anemia and low birth weight 22 ."], ["nevertheless , clinical follow - up is needed in order to exclude other conditions associated with multisystem pathology ; a  complete family history may also be warranted to determine sporadic or hereditary transmission of such a condition ."], [""]]}
{"article_id": "PMC3152742", "article_text": ["glycogen synthase kinase-3 ( gsk-3 ) , an evolutionarily conserved ubiquitous serine  threonine kinase consisting of  and  isoforms , is a multifaceted protein with diverse cellular and neurophysiological functions .", "the main structural difference between gsk-3 and gsk-3 isoforms lies in the n- and c - terminal regions , while their sequences within the kinase domain are highly homologous .", "a growing body of evidence indicates that gsk-3 is pro - apoptotic and that its dysfunction may be linked to the pathophysiology of mood disorders , schizophrenia , diabetes , and various neurological / neurodegenerative diseases , among others ( for review , meijer et al . , 2004 ;", ", 2007 ; chiu and chuang , 2010 ; li and jope , 2010 ) .", "gsk-3 inhibition has attracted widespread attention as one of the critical therapeutic targets whereby lithium exerts its effects on mood stabilization , neurogenesis , neurotrophicity , neuroprotection , anti - inflammation , and others ( for review , rowe and chuang , 2004 ; rowe et al .", ", 2007 ; beurel et al . , 2010 ) . pharmacological inhibition or gene knockout / knockdown of this kinase mimics the anti - depressant and anti - manic effects of lithium observed in rodent models ( gould et al . , 2004 ; kaidanovich - beilin et al", ", 2004 , 2009 ; obrien et al . , 2004 ; rosa et al . , 2008 ; omata et al . , 2011 ) .", "the activities of gsk-3 are negatively regulated by phosphorylation of gsk-3 at ser21 and gsk-3 at ser9 .", "gsk-3 can be inhibited by lithium through direct binding to the atp - dependent magnesium - sensitive catalytic site of the enzyme ( klein and melton , 1996 ; stambolic et al .", ", 1996 ) , and/or indirectly through enhanced serine phosphorylation of gsk-3 isoforms by multiple mechanisms ( figure 1 ) .", "lithium has been shown to enhance gsk-3 serine phosphorylation by activation of protein kinase a ( pka ; jope , 1999 ; liang et al . , 2008 ) , or phosphatidylinositol 3-kinase ( pi3-kinase)-dependent akt ( chalecka - franaszek and chuang , 1999 ) and protein kinase c- ( kirshenboim et al . , 2004", "it has also been reported that lithium can disrupt the -arrestin-2pp2a  akt complex that dephosphorylates / inactivates akt , thereby enhancing gsk-3 serine phosphorylation ( beaulieu et al . , 2005 ) .", "moreover , it has been proposed that lithium can interrupt auto - regulation of gsk-3 via disinhibition of the inhibitory action of inhibitor-2 complex on protein phosphatase-1 ( pp-1 ; zhang et al . , 2003 ) .", "this article reviews the findings supporting the role of gsk-3 inhibition in mediating lithium s neuroprotective effects against excitotoxicity in both cultured neurons and animal models of ischemic stroke .", "lithium , a competitive inhibitor of magnesium , directly inhibits atp  magnesium - dependent catalytic activity of gsk-3 .", "lithium can indirectly increase this serine phosphorylation of gsk-3 through pi3-kinase - mediated phosphorylation / activation of akt , pi3-kinase - mediated activation of pkc , and camp - dependent activation of pka .", "lithium can also increase the serine phosphorylation of gsk-3 by disrupting the -arrestin-2 ( arr2)pp2a  akt complex that dephosphorylates and inactivates akt .", "in addition , by disinhibiting the inhibitory action of inhibitor-2 ( i-2 ) on protein phosphatase - l ( pp-1 ) that dephosphorylates gsk-3 at serine residues , lithium s direct inhibition of gsk-3 interrupts this auto - regulation of gsk-3 and further decreases gsk-3 activity .", "lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections .", "we have designed isoform - specific small interfering rnas ( sirnas ) to distinguish the functional and regulatory differences between the two gsk-3 isoforms in rat cerebral cortical neuronal cultures ( liang and chuang , 2007 ) .", "transfection with sirna for gsk-3 or gsk-3 or with dominant - negative mutants specific for either isoform produced almost complete protection against glutamate - induced , n - methyl - d - aspartate ( nmda ) receptor - mediated excitotoxicity .", "the sirna - induced neuroprotection was associated with enhanced n - terminal phosphorylation in both gsk-3 isoforms .", "moreover , transfection with  or  isoform - specific dominant - negative mutants of gsk-3 mimicked lithium - induced neuroprotection against glutamate excitotoxicity .", "these results strongly suggest that both gsk-3 and  are involved in glutamate - induced neuronal death and that both isoforms are the initial targets of lithium - elicited neuroprotection .", "gsk-3 has also been implicated in neuronal development , maturation / differentiation , and aging in the mammalian cns ( spittaels et al . , 2002 ; kim et al .", "substrates phosphorylated by gsk-3 include metabolic , signaling , and structural proteins as well as transcription factors .", "it is known that inhibition of gsk-3 results in activation , and sometimes suppression , of an array of transcription factors ( for review , grimes and jope , 2001 ; jope and roh , 2006 ; chiu and chuang , 2010 ) . among the long list of transcription factors regulated by gsk-3", "are cyclic amp response element binding protein ( creb ) , nuclear factor-b ( nf-b ) , activating protein-1 ( ap-1 ) , heat - shock factor-1 ( hsf-1 ) , -catenin , t - cell factor ( tcf)/lymphoid enhancer factor ( lef ) , and p53 .", "dysfunction of gsk-3-mediated phosphorylation of transcription factors is believed to relate with the pathophysiology of various pathological conditions ( for review , chiu and chuang , 2010 ) .", "we found that gsk-3 silencing activated camp response element ( cre)- and nf-b - responsive transcription more robustly than gsk-3 silencing ( liang and chuang , 2006 ) .", "dna array further identified two novel gsk-3-regulated transcription factors , early growth response-1 ( egr-1 ) and smad3/4 , both of which play important roles in growth , differentiation , survival , and plasticity of brain cells ( harada et al .", ", 2001 ; derynck and zhang , 2003 ; lee and kim , 2004 ; droguett et al . , 2010 ) . specifically , the binding activity of egr-1 was down - regulated by sirna for gsk-3 , but was up - regulated by sirna for gsk-3 ( liang and chuang , 2006 ) . by using sirnas or dominant - negative mutants specific to gsk-3 isoforms , inhibition of gsk-3", "the differential roles of gsk-3 isoforms are further supported by the opposite effects of gsk-3 and  sirnas on the protein levels of plasminogen activator inhibitor type-1 ( pai-1 ) , a smad3/4-regulated gene product .", "these results demonstrate that selective silencing or inhibition of the two gsk-3 isoforms could produce different and sometimes opposite effects on the regulation of certain transcription factors including novel gsk-3 targets ( liang and chuang , 2006 ) .", "for example , the disruption of gsk-3 in mice is embryonic lethal , despite the normal expression of gsk-3 , indicating that the presence of  isoform can not compensate for the loss of  isoform ( hoeflich et al . , 2000 ) .", "transfection and sirna studies suggested that gsk-3 inhibition decreased the processing of -amyloid ( a ) precursor protein to form a140 and a142 , while gsk-3 appeared to have a lesser role ( phiel et al . , 2003 ) .", "in addition , gsk-3 , but not gsk-3 , is required for interferon--induced activation of signal transducer and activator of transcription-3 ( beurel and jope , 2009 ) .", "together , these findings underscore important similarities and differences between the roles of gsk-3 isoforms  and  in cell survival as well as transcription , and suggest that the development of isoform - specific inhibitors may be essential for therapeutic intervention of gsk-3-related neuropathological conditions .", "we have also explored lithium s effects on smad3/4-dependent transcriptional activity and the underlying mechanisms .", "smad3/4 is a down - stream mediator of the signaling pathway triggered by transforming growth factor- ( tgf- ) , and plays a prominent role in regulating the expression of proteins involved in neuronal survival , differentiation , and synaptic plasticity ( for review , gomes et al . , 2005 ) .", "treating cultured cortical neurons with therapeutically relevant concentrations of lithium significantly decreased smad3/4-dependent transactivation and protein levels of pai-1 , a tgf--responsive smad3/4-dependent gene product ( liang et al . , 2008 ) .", "of particular relevance to the therapeutic efficacy of lithium , pai-1 has been implicated in the etiology and progression of neurodegenerative diseases and mood disorders ( for review , pawlak et al . , 2003 ;", "lithium s effects on smad3/4 likely result from cross - talk of signaling pathways between camp / pka and pi3-kinase / akt / gsk-3. we have shown that lithium - induced smad3/4 suppression involved gsk-3 inhibition through the activation of pka and cell survival factor akt followed by the phosphorylation of gsk-3 at ser9 and creb at ser133 ( liang et al . , 2008 ) .", "creb binding protein ( cbp ) and p300 are known to be co - activators of creb .", "our data further demonstrated that over - expression of p300 , but not cbp , completely antagonized lithium - induced reduction of pai-1 promoter activity .", "a series of experimental data support the notion that , in smad3/4 signaling , the inhibitory effects of lithium are due to complex formation of activated creb and p300 , which results in limited interactions of p300 with the transcription factors / smad complexes . this in turn prevents efficient smad3/4-dependent transcription of smad3/4-dependent genes such as pai-1 and p21 ( figure 2 ) .", "transcriptional activations triggered by stimulation of cell surface tgf- and bdnf receptors are mediated by smad3/4- and pi3-kinase / akt - dependent pathways , respectively .", "lithium treatment - induced inhibition of gsk-3 , directly and indirectly via camp - dependent activation of pka as well as bdnf - stimulated activation of pi3-kinase / akt pathways , potentiates bdnf - induced phosphorylation / activation of creb .", "this in turn increases cre - mediated transactivation and expression of survival factors such as bdnf and bcl-2 .", "enhanced gene transcription triggered by bdnf , via sequestration of transcriptional co - activator p300 , suppresses smad3/4-dependent transactivation and subsequently decreases the expression of tgf--responsive genes , pai-1 , and p21 .", "lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections . dashed lines represent pathways with reduced activity as a result of lithium treatment .", "lithium - induced neuroprotection against glutamate excitotoxicity was first noted in rodent primary neuronal cultures of cerebellar granule cells ( cgcs ) , cerebral cortical neurons , and hippocampal neurons ( nonaka et al . , 1998 ) . this experimental paradigm was selected because glutamate - related excitotoxicity has been implicated in many neurodegenerative diseases including stroke ( for review , chuang , 2004 ; chiu and chuang , 2010 ) .", "our pioneering studies have shown that glutamate - induced , nmda receptor - mediated excitotoxicity was robustly reduced by extended lithium chloride pretreatment ( 57  days ) in cultured rat cgcs and cortical neurons , partly via inhibition of nmda receptor - mediated calcium influx ( nonaka et al . , 1998 ; hashimoto et al . , 2002a ) . moreover ,", "these effects of lithium were likely due to the attenuation of constitutive phosphorylation at tyr1472 of the nr2b subunit of nmda receptors , possibly as a result of inhibiting src tyrosine kinase ( hashimoto et al . , 2002a , 2003 ) .", "although glutamate - induced excitotoxicity in cultured cortical neurons was blocked by treatment with either lithium or mk-801 ( an nmda receptor antagonist ) , the src kinase inhibitor su6656 only partially diminished this toxicity ( hashimoto et al . , 2003 ) , suggesting that other components are involved . in cgcs , lithium - induced neuroprotection against glutamate excitotoxicity was associated with up - regulation of the anti - apoptotic protein bcl-2 , down - regulation of the pro - apoptotic proteins p53 and bax , and suppressed release of cytochrome c from mitochondria ( chen and chuang , 1999 ) , whereas the involvement of gsk-3 in the regulation of nmda signaling by lithium treatment is currently unclear and requires further investigations .", "cyclin - dependent kinase 5 ( cdk5 ) also regulates signaling mediated by nmda receptors , either directly through phosphorylation of the nr2b subunit or indirectly through phosphorylation of psd-95 ( morabito et al . , 2004 ; zhang et al . , 2008", "when it binds to p25 ( the product of calpain - mediated cleavage of p35 ) , cdk5 becomes pro - apoptotic and its activity is dysregulated ( lee et al .", ", p25 accumulation was observed in neurons in response to glutamate or oxidative stress , and also in the brains of several animal models of neurodegenerative diseases . sustained activation of cdk5 in neurons has been implicated in many neurodegenerative diseases ( cruz and tsai , 2004 ; dhariwala and rajadhyaksha , 2008 ) . in cultured rat cgcs ,", "lithium pretreatment prevented colchicine - induced apoptosis and associated increase in cdk5 expression and fragmentation of p35 into p25 ( jorda et al .", ". additionally , pretreatment with lithium also attenuated intracellular calcium increase , calpain activity , cdk5 activation , and cellular death in primary cultured hippocampal neurons and rat striatum following the treatment of 3-nitropropionic acid ( crespo - biel et al . , 2009 ) , a succinate dehydrogenase inhibitor ( for review , brouillet et al . , 1999 ) .", "therefore , lithium - induced inhibition of calpain and cdk5 activation may also contribute to protection against glutamate excitotoxicity .", "prior to changes in gene expression , lithium rapidly and transiently activated the cell survival pi3-kinase and its down - stream target , akt-1 , through phosphorylation at ser473 , thereby reversing glutamate - induced inactivation of this signaling pathway in cgcs ( chalecka - franaszek and chuang , 1999 ) .", "activated akt is known to affect several anti - apoptotic targets including bcl-2 associated death promoter ( bad ) , creb , members of the forkhead family , and procaspase-9 ( for review , neri et al . , 2002 ; nicholson and anderson , 2002 ; huang and reichardt , 2003 ) .", "in addition , lithium also triggered ser21 phosphorylation of the  isoform of gsk-3 ( and hence resulted in inhibition ) , and this effect was prevented by a pi3-kinase inhibitor ( chalecka - franaszek and chuang , 1999 ) .", "another signaling pathway affected by lithium is the mitogen - activated protein ( map ) kinase pathway .", "one of the down - stream targets of map kinase is creb , a transcription factor that is involved in learning and memory , and promotes the expression of bcl-2 as well as brain - derived neurotrophic factor ( bdnf ; for review , finkbeiner , 2000 ) . in cgcs , toxic concentrations of glutamate - induced an nmda receptor - dependent decrease in creb phosphorylation at ser133 and creb - driven transcriptional activity ( kopnisky et al . , 2003 ) .", "concurrent with its neuroprotective effects , long - term ( but not acute ) lithium treatment suppressed glutamate - induced dephosphorylation of creb .", "we also found that glutamate rapidly activated c - jun - n - terminal kinase ( jnk ) and p38 kinase in cgcs , resulting in a robust increase in ap-1 binding ( chen et al . , 2003a ) .", "these two kinases are also activated by a variety of apoptotic insults ( for review , mielke and herdegen , 2000 ) , and ap-1 has been known to be activated by different stress factors as well .", "experiments using lithium and curcumin , a selective ap-1 inhibitor , suggest that nmda receptor - mediated apoptotic death requires concerted action of jnk and p38 to enhance ap-1 binding , and that lithium s neuroprotection is mediated , at least in part , by suppressing the jnk and p38 kinase pathways .", "as one of the major neurotrophins , bdnf is essential for cortical development , synaptic plasticity , and neural survival , and is likely a key mediator of the clinical efficacy of anti - depressants and anxiolytic drugs ( for review , woo and lu , 2006 ) .", "the notion that bdnf plays a key role in neuronal survival is supported by our observation that bdnf and neurotrophin-4 ( nt-4 ) , but not nt-3 , completely protected immature cgcs from apoptosis induced by cytosine arabinoside ( leeds et al . , 2005 ) .", "it was first reported that chronic treatment with lithium increased the expression of bdnf in the rat brain ( fukumoto et al . , 2001 ) , and we have documented that bdnf protein levels were increased in cortical neurons following lithium treatment ( hashimoto et al . , 2002b ) .", "we hypothesized that this bdnf up - regulation and subsequent activation of its receptor trkb might play a critical role in mediating the neuroprotective effects of lithium . in confirmation of this hypothesis", ", we found that lithium s neuroprotection against glutamate excitotoxicity was blocked by a trkb inhibitor , k252a , or by a neutralizing antibody against bdnf , and was mimicked by exogenous bdnf in rat cortical neurons .", "in addition , lithium increased intracellular levels of bdnf and this was followed by activation of trkb .", "furthermore , lithium - induced neuroprotection was prevented in cortical neurons from heterozygous ( + / ) or homozygous ( / ) bdnf knockout mice ( hashimoto et al . , 2002b ) .", "rodent bdnf has a complex genomic structure that makes it an ideal target for multiple and complex regulation .", "we found that treatment of rat cortical neurons with therapeutic concentrations of lithium ( e.g. , 1  mm ) caused a significant increase in the levels of bdnf exon iv - containing mrna , while levels of exon i , ii , or vi - containing mrna remained unchanged ( yasuda et al . , 2009 ) .", "it is known that exon iv - containing bdnf transcripts are expressed in response to kcl - induced depolarization in rat cortical neurons ( tao et al . , 2002 ) .", "this transcriptional activation requires utilization of the promoter region 80  bp up - stream from the transcription initiation site of exon iv - containing three calcium responsive elements ( cares ; chen et al . , 2003b ) .", "we generated various bdnf promoter iv deletion constructs to investigate whether lithium treatment causes an increase in bdnf promoter iv activity , and , if so , which region of promoter iv confers the sensitivity to this drug .", "we identified that the drug - induced up - regulation of exon iv - containing bdnf transcript was associated with a significant increase in the activity of bdnf promoter iv and total bdnf protein . to our surprise , the lithium - responsive element(s ) in promoter iv resides in a region up - stream from the cares responsible for depolarization - induced bdnf induction ( 170 to 704  bp ) .", "moreover , activation of bdnf promoter iv occurred in cortical neurons depolarized with kcl and depletion of these three cares failed to abolish lithium - induced activation .", "importantly , we found that lithium - induced activation of promoter iv was mimicked by pharmacological inhibitors of gsk-3 ( sb216763 , sb415286 , inhibitor i , and inhibitor vii ) or by transfection with specific sirna for gsk-3 or gsk-3. additionally , their dominant - negative mutants also mimicked lithium - induced activation of promoter iv .", "these results demonstrate that gsk-3 is the initial target of lithium to selectively activate bdnf promoter iv and that bdnf induction by lithium involves a novel responsive region in promoter iv of the bdnf gene .", "lithium - induced , gsk-3-dependent bdnf promoter iv activation could be a part of the molecular mechanisms underlying its neuroprotective effects and as such , possibly accounts for the therapeutic actions in bipolar patients .", "it should be noted that in addition to lithium , other gsk-3 inhibitors have been shown to almost completely block glutamate - induced excitotoxicity in rat cortical neuronal cultures ( liang and chuang , 2007 ) .", "these include atp - competitive inhibitors , sb216763 and sb415286 , and atp - non - competitive inhibitors , inhibitor i and vii .", "as mentioned in the preceding section , glutamate - induced death of cortical neurons was mitigated by silencing of gsk-3 and/or  , or both isoforms , or inhibition of gsk-3 activity via transfection with dominant - negative mutants of gsk-3/ isoforms ( liang and chuang , 2007 ) .", "studies from other laboratories also supported the roles of gsk-3 inhibition in protecting neurons from glutamate neurotoxicity .", "for example , stimulation of nmda receptors in cultured rat hippocampal or cortical neurons activated gsk-3 by pp-1-mediated serine dephosphorylation of gsk-3 ( szatmari et al . , 2005 ) .", "treatment of primary rat cortical neurons with -amino-3-hydroxy-5-methyl-4-isoxazole propionate ( ampa ) , lithium or sb216763 blocked glutamate - induced caspase-3 activation and excitotoxicity , and the protective effects of ampa required pi3-kinase  akt - dependent serine phosphorylation of gsk-3 ( nishimoto et al . , 2008 ) .", "further , in organotypic cultures of chick embryo spinal cord , lithium prevented kainate - induced excitotoxic death of motoneurons by targeting gsk-3 , and this neuroprotection was associated with cytopathological changes ( caldero et al . , 2010 ) .", "stroke is the third leading cause of death in the united states and a major global cause of serious long - term disability in adults .", "ischemic strokes represent approximately 87% of all cases , while the rest are hemorrhagic strokes ( roger et al . , 2011 ) .", "in addition to physical deficits , stroke victims also suffer from vascular depression and dementia , both of which are difficult to treat with conventional medicine .", "it is becoming clear that there is a substantial increase in extracellular glutamate in the brain following cerebral ischemia , and that a significant portion of ischemia - induced brain damage is mediated by over - stimulation of nmda receptors .", "shortly after ischemia , the interruption of cerebral blood flow depletes oxygen and glucose and subsequently prevents atp production .", "inadequate atp supply will cause the malfunction of atp - dependent ion pumps and alter the ion concentration gradient across the neuronal membranes .", "the resulting failure to transport glutamate leads to an accumulation of glutamate in the extracellular space and over - stimulates nmda receptors , which leads to a toxic influx of calcium and in turn drives the activation of damaging calcium - mediated intracellular enzymes .", "this cascade of events ultimately results in mitochondrial failure , production of reactive oxygen species , neuroinflammation , and cell necrosis and apoptosis ( allen and bayraktutan , 2009 ; deb et al . , 2010 ) .", "gsk-3 has been strongly implicated in the neuronal cell death caused by cerebral ischemic insult .", "one study in rats subjected to transient middle cerebral artery occlusion ( mcao ) demonstrated a rapid increase in the expression of cytoplasmic and nuclear gsk-3 protein in ipsilateral lamina i , ii , v , and vi in young rat brains , whereas in lamina v and vi in old rat brains ( sasaki et al .", "although the phosphorylation status of gsk-3 was not mentioned , these findings implicate a role of gsk-3 in cerebral ischemic injury .", "it is well known that gsk-3 can be phosphorylated at serine and tyrosine residues in which ser9 phosphorylation renders it inactive , while tyr216 phosphorylation is necessary for its functional activity ( hughes et al . , 1993 ) .", "discrepancies exist in the literature regarding changes of ser9 and tyr216 phosphorylation levels following cerebral ischemia .", "the phosphorylation levels of gsk-3 at ser9 and akt at ser473 were reported to be markedly enhanced in the vulnerable hippocampal ca1 region , but not in the ischemia - resistant ca3 region in rats subjected to transient global cerebral ischemia , while there was no change in levels of tyr216 phosphorylation or total gsk-3 ( endo et al . , 2006 ) .", "levels of gsk-3 ser9 phosphorylation were also increased shortly after permanent focal cerebral ischemia and decreased to basal levels or even lower 24  h after ischemic onset ( sasaki et al . , 2006 ; gao et al . , 2008", "however , it has also been reported that transient focal cerebral ischemia in rats caused an increase in gsk-3 tyr216 phosphorylation in degenerating cortical neurons with no alteration in ser9 phosphorylation ( bhat et al . , 2000 ) .", "this discrepancy may stem , in part , from the difference of ischemic severity and ischemic models across various studies .", "it appears that transient focal cerebral ischemia tends to activate gsk-3 and subsequently to induce apoptotic cell death .", "in contrast , gsk-3 is inactivated shortly after permanent focal cerebral ischemia or global cerebral ischemia , which in turn may promote survival of vulnerable neurons .", "ischemia both gsk-3 and gsk-3 mediated the expression of a lethal protein , neuronal pentraxin 1 ( russell et al . , 2011 ) . in light of these findings", "the beneficial effects of lithium in rodent cerebral ischemic models demonstrated by us and others support this notion . in an initial study ,", "long - term lithium pretreatment at therapeutically relevant doses decreased brain infarct volume , reduced apoptotic cell death and improved behavioral performance after permanent cerebral ischemia - induced by mcao ( nonaka and chuang , 1998 ; xu et al . , 2003 ) . in a subsequent study , we demonstrated that subcutaneous injection of rats with lithium at therapeutic doses ( e.g. , 0.5 and 1.0  meq / kg ) after the onset of transient mcao markedly decreased infarct volume , reduced tunel - positive dna damage , and suppressed neurological deficits measured by sensory , motor , and reflex tests ( ren et al . , 2003 ) .", "the time window for these beneficial effects was at least 3  h after the onset of ischemia .", "heat - shock protein 70 ( hsp70 ) , a well - established cytoprotective factor against apoptosis , was induced in the ischemic penumbra where neuronal recovery takes place .", "post - insult treatment with lithium increased the dna binding activity of hsf-1 to the heat - shock element , superinducing hsp70 which inhibits brain ischemia - induced apoptosis ( ren et al . , 2003 ) .", "lithium - elicited gsk-3 inhibition is likely associated with hsf-1 activation and hsp70 induction ( bijur and jope , 2000 ) .", "notably , post - insult lithium treatment mitigated apoptosis and brain damage by preventing gsk-3 and erk dephosphorylation , suppressing calpain and caspase-3 activation , and inhibiting mitochondrial release of cytochrome c and apoptosis - inducing factor in a neonatal hypoxic ", "these findings suggest that lithium - induced gsk-3 inhibition contributes to its anti - apoptotic effects under ischemic conditions .", "in addition , it was found that lithium pretreatment largely suppressed ischemia - induced exploratory behavioral changes and memory impairments in gerbils after global cerebral ischemia ( bian et al . , 2007 ) .", "these behavioral benefits were associated with an increase in the number of viable cells and a decrease in apoptotic cells in the ca1 hippocampal area of ischemic gerbils . moreover , lithium - induced neuroprotection in the ischemic brain was accompanied by down - regulation of pro - apoptotic p53 in the ca1 , and up - regulation of anti - apoptotic bcl-2 and hsp70 , both of which are targets of gsk-3 .", "it is likely that lithium protection against ischemia - induced injury involves multiple mechanisms . in the rat hippocampus ,", "lithium was reported to inhibit ischemia - induced nmda receptor hyperactivation by inhibiting nmda subunit 2a tyrosine phosphorylation and its interactions with src and fyn through psd-95 ( ma and zhang , 2003 ) .", "lithium also attenuated hypoxia - induced serine dephosphorylation of gsk-3 and  in the mouse brain ( roh et al . , 2005 ) .", "additionally , in organotypic cultures of rat hippocampus subjected to oxygen and glucose deprivation , lithium showed neuroprotection in conjunction with hsp27 activation ( cimarosti et al . , 2001 ) .", "post - ischemic inflammation is a dynamic process involving a complicated set of interactions between inflammatory cells and molecules ( iadecola and alexander , 2001 ) . a recent study documented the anti - inflammatory effects of lithium in a neonatal rat hypoxic  ischemic model .", "post - insult lithium treatment significantly reduced total tissue loss following hypoxia  ischemia , and this beneficial effect of lithium was associated with inhibiting microglia activation and attenuating levels of pro - inflammatory cytokines or chemokines , such as interleukin-1 and chemokine ligand 2 ( li et al . , 2011 ) .", "hsp70 over - expression can inactivate the key inflammatory transcription factor nf-b by stabilizing the nf-b - ib complex , and thereby preventing nuclear translocation of activated nf-b subunits in a mouse mcao model ( zheng et al . , 2008 ) . besides anti - inflammation", ", lithium also increased proliferation and differentiation of hippocampal neural progenitor cells in both non - ischemic and ischemic brains without altering the relative levels of neuronal and astrocytic differentiation , and this effect lasted at least 7  weeks after hypoxia  ischemia in neonatal rats ( li et al . ,", "in line with this finding , chronic lithium pretreatment was found to increase the generation and survival of newborn cells in the hippocampal dentate gyrus , and did not affect the neuronal or astrocytic differentiation of these newborn cells in a transient four - vessel occlusion model ( yan et al .", "erk1/2 phosphorylation following ischemia was enhanced by lithium treatment , while erk1/2 inhibitor u0126 prevented the effects of lithium in increasing brdu - positive cells and improving spatial learning and memory ( yan et al . , 2007 ) .", "in fact , chronic lithium treatment has been demonstrated to increase activity in the mek / erk pathway in vivo , and lithium s neuroprotection has been suggested to depend on the induction of this signaling pathway ( einat et al .", "in addition , it was reported that activation of erk associates with and phosphorylates gsk-3 at the thr43 residue , which primes this kinase for its subsequent phosphorylation at ser9 by p90rsk , resulting in inactivation of gsk-3 and up - regulation of -catenin ( ding et al . , 2005 ) .", "therefore , lithium might affect gsk-3 phosphorylation through the mek / erk pathway , which in turn inhibits this kinase via rsk .", "gsk-3 is also negatively regulated by the wnt/-catenin signaling pathway and , accordingly , activating the canonical wnt pathway has been shown to contribute to adult hippocampal neural progenitor cell proliferation triggered by lithium treatment ( wexler et al . , 2008 ) . in a collaborative study ,", "the neurohemodynamic aspects of recovery induced by delayed chronic lithium treatment were assessed using functional magnetic resonance imaging ( mri ; kim et al . , 2008 ) .", "rats were subjected to transient mcao and then injected with lithium ( licl , 1  meq / kg , s.c . )", "this delayed lithium injection was followed by daily injections , and on day 15 , an mri scan was performed to monitor changes in blood oxygen level dependence ( bold ) and functional cerebral blood volume ( fcbv ) responses using electric stimulation of forelimbs .", "the mean activated volume ratio and total activation magnitude ratio between ipsilateral and contralateral cortices for both bold and fcbv were significantly higher in the lithium - treated than in the saline - treated rats .", "the lithium - induced increase in fcbv in the peri - infarct regions suggests a possible vascular transformation .", "indeed , the size and distribution of immunohistochemical staining of cd31 , a microvasculature marker , were enhanced by lithium treatment in the peri - infarct regions .", "co - localized with cd31 , the tissue staining of matrix metalloproteinase-9 ( mmp-9 ) was also much more pronounced following lithium treatment , suggesting mmp-9-dependent neurovascular remodeling in the recovering brain area .", "moreover , treatment of cultured rat brain endothelial cells with lithium in a follow - up study was also found to increase the protein levels of vascular endothelial growth factor ( vegf ) via a mechanism involving the pi3-kinase and gsk-3 signaling pathways ( guo et al . ,", "since vegf has been linked to angiogenesis , neurogenesis , and neuroprotection ( for review , fan and yang , 2007 ) , vegf over - expression may contribute to lithium s ability to promote neurovascular remodeling and to induce functional recovery after ischemic stroke .", "ample evidence supports the therapeutic potential of mesenchymal stem cells ( mscs ) in several human diseases including stroke .", "however , it is increasingly recognized that the effectiveness of msc transplantation is limited by their poor migration toward disease target sites such as ischemic brain regions . in a recent study", ", we investigated whether treatment of mscs with lithium and another mood stabilizing drug , valproic acid ( vpa ) , would enhance cell migration ( tsai et al . , 2010 ) .", "we found that treatment of mscs with lithium ( 2.5  mm for 1  day ) selectively elevated the transcript and protein levels of mmp-9 and its enzymatic activity .", "these effects were mimicked by pharmacological inhibition or gene silencing of gsk-3. lithium treatment also potentiated stromal cell - derived factor-1 ( sdf-1)-dependent msc migration across the extracellular matrix , which was suppressed by two mmp-9 inhibitors , doxycycline and gm6001 .", "short - term ( 3  h ) exposure of mscs to a relatively high concentration ( 2.5  mm ) of vpa markedly increased the transcript and protein levels of cxc chemokine receptor 4 ( cxcr4 ) .", "vpa - induced cxcr4 expression required its ability to inhibit histone deacetylases ( hdacs ) , including the hdac1 isoform , and involved histone hyperacetylation at the cxcr4 gene promoter .", "vpa treatment enhanced sdf-1-mediated msc migration , which was completely blocked by amd3100 , a cxcr4 antagonist .", "notably , combining lithium and vpa treatment further increased msc migration , and the additive enhancement of migration was completely blocked by the co - presence of amd3100 and gm6001 .", "our results suggest that lithium and vpa stimulate msc migration through distinct targets and mediators : gsk-3mmp-9 and hdac  cxcr4 , respectively ( tsai et al . , 2010 ) .", "in a follow - up in vivo study , mscs were primed with lithium and/or vpa and then injected into the tail vein of transient mcao rats 24  h after ischemic onset . priming with lithium or vpa increased the number of mscs homing to the cerebral infarcted regions such as the cortex and striatum 2  weeks after transplantation , and co - priming with lithium and vpa further enhanced this migratory effect ( tsai et al . , 2011 ) .", "mcao rats receiving lithium- and/or vpa - primed mscs showed improved functional recovery , reduced infarct volume , and enhanced angiogenesis in the infarcted penumbra regions .", "these beneficial effects of lithium and vpa priming were reversed by pharmacological inhibition of mmp-9 and cxcr4 , respectively , suggesting that these effects were likely mediated by lithium - induced mmp-9 up - regulation and vpa - induced cxcr4 over - expression .", "together , these findings raise the potential utility of using mscs primed with inhibitors of gsk-3 and hdac to enhance the migration and homing capacity for transplantation into stroke victims .", "in addition to lithium , other pharmacological gsk-3 inhibitors have been shown to exert neuroprotective effects against cerebral ischemia by various groups .", "a specific gsk-3 inhibitor , chir025 , was demonstrated to protect cultured hippocampal neurons from glutamate excitotoxicity and to attenuate death of cortical neurons following oxygen ", "glucose deprivation , an in vitro model of cerebral ischemia ( kelly et al . , 2004 ) .", "moreover , chir025 reduced infarct size in focal cerebral ischemic rats , but did not affect tunel - positive neurons or caspase-3/9 activities , although bcl-2 expression was increased .", "gsk-3 enzymatic activity was markedly elevated after transient mcao in rats , and this gsk-3 activation was blocked by jugular vein injection of gsk-3 inhibitor viii ( koh et al . , 2008 ) .", "pre- or post- ( up to 2  h ) mcao injection with inhibitor viii also reduced blood glucose levels , infarct size , caspase-3 activity , and water content in the ipsilateral brain hemisphere .", "furthermore , ischemia - induced inflammation - related signals such as cox-2 over - expression and neutrophil infiltration were alleviated by this gsk-3 inhibitor .", "prophylactic or therapeutic administration of a gsk-3 inhibitor tdzd-8 reduced infarct volume and cerebral injury in the rat hippocampus after transient ischemia ( collino et al . , 2008 ) .", "this was accompanied by suppression of ischemia - induced oxidative stress , apoptosis , and neuroinflammation .", "delayed treatment with compound i , a gsk-3 and cdk inhibitor , decreased tunel - positive cells in the ipsilateral hippocampus and striatum of adult ( but not juvenile ) mice subjected to hypoxic ", "these neuroprotective effects of compound i were associated with long - lasting functional recovery . finally , gsk-3 inhibition by sb216763 counteracted oxygen ", "glucose deprivation - induced mitochondrial biogenesis impairment and reduced mitochondrial reactive oxygen species generation in primary cortical neurons ( valerio et al . , 2011 ) .", "when systematically administrated to permanent mcao mice , sb216763 decreased infarct volume and restored the loss of mitochondrial dna , thus supporting a novel role of gsk-3 inhibitors in stimulating the renewal of functional mitochondria following ischemic stroke .", "a growing body of evidence supports that lithium , a mood stabilizer used to treat bipolar disorder , has neuroprotective properties in both cellular and in vivo experimental settings .", "one of the major targets of lithium is gsk-3 , a serine / threonine kinase implicated in the pathogenesis of diverse cns disorders .", "lithium inhibits gsk-3 activity by direct binding to the enzyme or indirectly by enhancing serine phosphorylation of both  and  isoforms through multiple mechanisms .", "lithium has been used as a prototype drug to seek evidence for the involvement of gsk-3 inhibition in lithium - induced protection against excitotoxicity in cultured neurons and animal models of cerebral ischemic stroke .", "lithium at therapeutically relevant concentrations robustly protected primary brain neurons from glutamate - induced , nmda receptor - mediated excitotoxicity .", "the neuroprotective effects of lithium were associated with gsk-3 inhibition , and were mimicked by other pharmacological gsk-3 inhibitors , by silencing gsk-3 and/or  isoforms , or by expression of isoform - specific dominant - negative mutants .", "these results support the roles of gsk-3 inhibition in lithium - elicited protection against excitotoxicity .", "lithium rapidly activated the cell survival pi3-kinase  akt signaling pathway to enhance gsk-3 serine phosphorylation and to block glutamate - induced akt inactivation as well as apoptosis .", "lithium also caused an increase in the expression of cytoprotective bcl-2 and suppressed glutamate - induced up - regulation of pro - apoptotic p53 and bax , resulting in blocking cytochrome c release from mitochondria .", "bdnf promoter iv was selectively activated by gsk-3 inhibition using lithium or other drugs or through gene silencing / inactivation of either isoform . this effect on", "however , there is a gap in the understanding of how gsk-3 inhibition causes an increase in bdnf promoter activity .", "in addition , lithium s neuroprotective effects were associated with inhibition of nmda receptor - mediated calcium influx and suppression of p38/jnk and ap-1 activation , thus reducing apoptosis .", "this effect appears to stem from inhibition of src / fyn kinase to suppress nr2b tyr1472 phosphorylation of the receptor .", "it remains to be explored as to whether this lithium - induced action on nmda receptors is related to gsk-3 inhibition .", "the potential roles of these other targets in mediating the neuroprotective effects of this drug also deserve future investigation .", "it is well known that glutamate overflow and nmda receptor hyper - stimulation are early events following cerebral ischemia . in rodent", "ischemic models , pre- or post - insult treatment with therapeutic doses of lithium decreased infarct volume , caspase-3 activity and apoptotic cells in the injured brain .", "the beneficial time window of lithium is at least 3  h after the ischemic onset .", "up - regulation of hsp70 and bcl-2 as well as down - regulation of p53 likely contributed to the protective effects of lithium in the ischemic conditions , thus supporting similar underlying neuroprotective mechanisms in the excitotoxic cellular models and animal models of ischemic stroke .", "limited data suggested that lithium might also display anti - inflammatory effects by inhibiting ischemia - induced microglia activation and pro - inflammatory factors release .", "delayed and chronic injections of lithium improved functional mri responses such as increases in bold and fcbv .", "indeed , lithium was found to induce two pro - angiogenic factors , mmp-9 and vegf in a gsk-3-dependent manner .", "lithium has also been reported to stimulate erk1/2 activity and to enhance proliferation of hippocampal neural progenitor cells and memory performance after ischemia . finally , lithium promoted migration of mscs in vitro by up - regulation of mmp-9 through gsk-3 inhibition and this migratory effect was potentiated by co - treatment with vpa , another mood stabilizer .", "notably , transplantation of lithium  vpa co - primed mscs into ischemic rats markedly increased msc migration to the injured brain regions , decreased infarct size and improved the neurological performance .", "lithium - induced stem cell migration , neurogenesis , and angiogenesis all likely contribute to functional recovery .", "figure 3 illustrates proposed molecular events leading to lithium - induced beneficial effects following cerebral ischemia .", "it should be noted that several other gsk-3 inhibitors have also been reported to exert beneficial effects in rodent ischemic models and their actions were accompanied by suppression of ischemia - increased gsk-3 activity .", "accordingly , gsk-3 inhibitors have therapeutic potential to treat stroke and other excitotoxicity - related neurodegenerative diseases .", "lithium has been used in bipolar patients over 60  years and its clinical profiles are well understood .", "therefore , lithium is a prime candidate for use in clinical trials of new therapies for stroke victims .", "the neuroprotective effects of lithium against cerebral ischemia are proposed to result from its interactions with cell survival and apoptotic machinery .", "a significant portion of brain damage following cerebral ischemia is caused by an increase in extracellular glutamate and subsequent over - stimulation of nmda receptor - mediated toxic increase in intracellular calcium .", "this signaling pathway plays a critical role in mediating glutamate - induced caspase activation and apoptosis .", "lithium at therapeutically relevant concentrations inhibits nmda receptor - mediated calcium influx , which in turn decreases subsequent activation of jnk , p38 kinase , and transcription factor ap-1 .", "inhibition of intracellular calcium increase also attenuates the activity of calpain and calpain - mediated activation of pro - apoptotic cdk5/p25 kinase . on the other hand ,", "lithium can directly and indirectly reduce the activity of constitutively activated gsk-3 by multiple mechanisms , leading to disinhibition of several transcription factors , such as creb and hsf-1 , and resulting in induction of major cytoprotective proteins such as bdnf , vegf , mmp-9 , hsp70 , and bcl-2 . a decrease in gsk-3 activity", "further reduces the activity of pro - apoptotic protein p53 and its downregulating effect on bcl-2 .", "bdnf , via activating its cell surface receptor and the down - stream erk and pi3-kinase / akt pathways , induces neuroprotective effects in part by inhibiting gsk-3 and stimulating creb .", "induction of bdnf is an early and essential step for neuroprotection and is involved in lithium - induced neurogenesis .", "in addition , superinduction of hsp70 by lithium treatment not only inhibits brain ischemia - induced apoptosis , but also contributes to the anti - inflammatory effects of lithium through inactivation of nf-b .", "counteraction of gsk-3 inhibition of vegf and mmp-9 by lithium enhances angiogenesis and neurovascular remodeling .", "taken together , these effects of lithium in reducing apoptosis , suppressing inflammation , enhancing angiogenesis and neurogenesis , contribute to behavioral improvement and functional recovery after ischemia . lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S> the mood stabilizer lithium inhibits glycogen synthase kinase-3 ( gsk-3 ) directly or indirectly by enhancing serine phosphorylation of both  and  isoforms . </S>", "<S> lithium robustly protected primary brain neurons from glutamate - induced excitotoxicity ; these actions were mimicked by other gsk-3 inhibitors or silencing / inhibiting gsk-3 and/or  isoforms . </S>", "<S> lithium rapidly activated akt to enhance gsk-3 serine phosphorylation and to block glutamate - induced akt inactivation . </S>", "<S> lithium also up - regulated bcl-2 and suppressed glutamate - induced p53 and bax . </S>", "<S> induction of brain - derived neurotrophic factor ( bdnf ) was required for lithium s neuroprotection to occur . </S>", "<S> bdnf promoter iv was activated by gsk-3 inhibition using lithium or other drugs , or through gene silencing / inactivation of either isoform . </S>", "<S> further , lithium s neuroprotective effects were associated with inhibition of nmda receptor - mediated calcium influx and down - stream signaling . in rodent </S>", "<S> ischemic models , post - insult treatment with lithium decreased infarct volume , ameliorated neurological deficits , and improved functional recovery . </S>", "<S> up - regulation of heat - shock protein 70 and bcl-2 as well as down - regulation of p53 likely contributed to lithium s protective effects . </S>", "<S> delayed treatment with lithium improved functional mri responses , which was accompanied by enhanced angiogenesis . </S>", "<S> two gsk-3-regulated pro - angiogenic factors , matrix metalloproteinase-9 ( mmp-9 ) and vascular endothelial growth factor were induced by lithium . finally , lithium promoted migration of mesenchymal stem cells ( mscs ) by up - regulation of mmp-9 through gsk-3 inhibition . </S>", "<S> notably , transplantation of lithium - primed mscs into ischemic rats enhanced msc migration to the injured brain regions and improved the neurological performance . </S>", "<S> several other gsk-3 inhibitors have also been reported to be beneficial in rodent ischemic models . </S>", "<S> together , gsk-3 inhibition is a rational strategy to combat ischemic stroke and other excitotoxicity - related brain disorders . </S>"], "labels": null, "section_names": ["Introduction", "Regulation and Function of GSK-3 Isoforms", "Involvement of GSK-3 and Other Molecules in Lithium-Elicited Neuroprotection Against Glutamate-Induced Excitotoxicity in Cellular Models", "Neuroprotective Effects of Lithium in Preclinical Models of Ischemic Stroke", "Conclusion", "Conflict of Interest Statement"], "sections": [["glycogen synthase kinase-3 ( gsk-3 ) , an evolutionarily conserved ubiquitous serine  threonine kinase consisting of  and  isoforms , is a multifaceted protein with diverse cellular and neurophysiological functions .", "the main structural difference between gsk-3 and gsk-3 isoforms lies in the n- and c - terminal regions , while their sequences within the kinase domain are highly homologous .", "a growing body of evidence indicates that gsk-3 is pro - apoptotic and that its dysfunction may be linked to the pathophysiology of mood disorders , schizophrenia , diabetes , and various neurological / neurodegenerative diseases , among others ( for review , meijer et al . , 2004 ;", ", 2007 ; chiu and chuang , 2010 ; li and jope , 2010 ) .", "gsk-3 inhibition has attracted widespread attention as one of the critical therapeutic targets whereby lithium exerts its effects on mood stabilization , neurogenesis , neurotrophicity , neuroprotection , anti - inflammation , and others ( for review , rowe and chuang , 2004 ; rowe et al .", ", 2007 ; beurel et al . , 2010 ) . pharmacological inhibition or gene knockout / knockdown of this kinase mimics the anti - depressant and anti - manic effects of lithium observed in rodent models ( gould et al . , 2004 ; kaidanovich - beilin et al", ", 2004 , 2009 ; obrien et al . , 2004 ; rosa et al . , 2008 ; omata et al . , 2011 ) .", "the activities of gsk-3 are negatively regulated by phosphorylation of gsk-3 at ser21 and gsk-3 at ser9 .", "gsk-3 can be inhibited by lithium through direct binding to the atp - dependent magnesium - sensitive catalytic site of the enzyme ( klein and melton , 1996 ; stambolic et al .", ", 1996 ) , and/or indirectly through enhanced serine phosphorylation of gsk-3 isoforms by multiple mechanisms ( figure 1 ) .", "lithium has been shown to enhance gsk-3 serine phosphorylation by activation of protein kinase a ( pka ; jope , 1999 ; liang et al . , 2008 ) , or phosphatidylinositol 3-kinase ( pi3-kinase)-dependent akt ( chalecka - franaszek and chuang , 1999 ) and protein kinase c- ( kirshenboim et al . , 2004", "it has also been reported that lithium can disrupt the -arrestin-2pp2a  akt complex that dephosphorylates / inactivates akt , thereby enhancing gsk-3 serine phosphorylation ( beaulieu et al . , 2005 ) .", "moreover , it has been proposed that lithium can interrupt auto - regulation of gsk-3 via disinhibition of the inhibitory action of inhibitor-2 complex on protein phosphatase-1 ( pp-1 ; zhang et al . , 2003 ) .", "this article reviews the findings supporting the role of gsk-3 inhibition in mediating lithium s neuroprotective effects against excitotoxicity in both cultured neurons and animal models of ischemic stroke .", "lithium , a competitive inhibitor of magnesium , directly inhibits atp  magnesium - dependent catalytic activity of gsk-3 .", "lithium can indirectly increase this serine phosphorylation of gsk-3 through pi3-kinase - mediated phosphorylation / activation of akt , pi3-kinase - mediated activation of pkc , and camp - dependent activation of pka .", "lithium can also increase the serine phosphorylation of gsk-3 by disrupting the -arrestin-2 ( arr2)pp2a  akt complex that dephosphorylates and inactivates akt .", "in addition , by disinhibiting the inhibitory action of inhibitor-2 ( i-2 ) on protein phosphatase - l ( pp-1 ) that dephosphorylates gsk-3 at serine residues , lithium s direct inhibition of gsk-3 interrupts this auto - regulation of gsk-3 and further decreases gsk-3 activity .", "lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections ."], ["we have designed isoform - specific small interfering rnas ( sirnas ) to distinguish the functional and regulatory differences between the two gsk-3 isoforms in rat cerebral cortical neuronal cultures ( liang and chuang , 2007 ) .", "transfection with sirna for gsk-3 or gsk-3 or with dominant - negative mutants specific for either isoform produced almost complete protection against glutamate - induced , n - methyl - d - aspartate ( nmda ) receptor - mediated excitotoxicity .", "the sirna - induced neuroprotection was associated with enhanced n - terminal phosphorylation in both gsk-3 isoforms .", "moreover , transfection with  or  isoform - specific dominant - negative mutants of gsk-3 mimicked lithium - induced neuroprotection against glutamate excitotoxicity .", "these results strongly suggest that both gsk-3 and  are involved in glutamate - induced neuronal death and that both isoforms are the initial targets of lithium - elicited neuroprotection .", "gsk-3 has also been implicated in neuronal development , maturation / differentiation , and aging in the mammalian cns ( spittaels et al . , 2002 ; kim et al .", "substrates phosphorylated by gsk-3 include metabolic , signaling , and structural proteins as well as transcription factors .", "it is known that inhibition of gsk-3 results in activation , and sometimes suppression , of an array of transcription factors ( for review , grimes and jope , 2001 ; jope and roh , 2006 ; chiu and chuang , 2010 ) . among the long list of transcription factors regulated by gsk-3", "are cyclic amp response element binding protein ( creb ) , nuclear factor-b ( nf-b ) , activating protein-1 ( ap-1 ) , heat - shock factor-1 ( hsf-1 ) , -catenin , t - cell factor ( tcf)/lymphoid enhancer factor ( lef ) , and p53 .", "dysfunction of gsk-3-mediated phosphorylation of transcription factors is believed to relate with the pathophysiology of various pathological conditions ( for review , chiu and chuang , 2010 ) .", "we found that gsk-3 silencing activated camp response element ( cre)- and nf-b - responsive transcription more robustly than gsk-3 silencing ( liang and chuang , 2006 ) .", "dna array further identified two novel gsk-3-regulated transcription factors , early growth response-1 ( egr-1 ) and smad3/4 , both of which play important roles in growth , differentiation , survival , and plasticity of brain cells ( harada et al .", ", 2001 ; derynck and zhang , 2003 ; lee and kim , 2004 ; droguett et al . , 2010 ) . specifically , the binding activity of egr-1 was down - regulated by sirna for gsk-3 , but was up - regulated by sirna for gsk-3 ( liang and chuang , 2006 ) . by using sirnas or dominant - negative mutants specific to gsk-3 isoforms , inhibition of gsk-3", "the differential roles of gsk-3 isoforms are further supported by the opposite effects of gsk-3 and  sirnas on the protein levels of plasminogen activator inhibitor type-1 ( pai-1 ) , a smad3/4-regulated gene product .", "these results demonstrate that selective silencing or inhibition of the two gsk-3 isoforms could produce different and sometimes opposite effects on the regulation of certain transcription factors including novel gsk-3 targets ( liang and chuang , 2006 ) .", "for example , the disruption of gsk-3 in mice is embryonic lethal , despite the normal expression of gsk-3 , indicating that the presence of  isoform can not compensate for the loss of  isoform ( hoeflich et al . , 2000 ) .", "transfection and sirna studies suggested that gsk-3 inhibition decreased the processing of -amyloid ( a ) precursor protein to form a140 and a142 , while gsk-3 appeared to have a lesser role ( phiel et al . , 2003 ) .", "in addition , gsk-3 , but not gsk-3 , is required for interferon--induced activation of signal transducer and activator of transcription-3 ( beurel and jope , 2009 ) .", "together , these findings underscore important similarities and differences between the roles of gsk-3 isoforms  and  in cell survival as well as transcription , and suggest that the development of isoform - specific inhibitors may be essential for therapeutic intervention of gsk-3-related neuropathological conditions .", "we have also explored lithium s effects on smad3/4-dependent transcriptional activity and the underlying mechanisms .", "smad3/4 is a down - stream mediator of the signaling pathway triggered by transforming growth factor- ( tgf- ) , and plays a prominent role in regulating the expression of proteins involved in neuronal survival , differentiation , and synaptic plasticity ( for review , gomes et al . , 2005 ) .", "treating cultured cortical neurons with therapeutically relevant concentrations of lithium significantly decreased smad3/4-dependent transactivation and protein levels of pai-1 , a tgf--responsive smad3/4-dependent gene product ( liang et al . , 2008 ) .", "of particular relevance to the therapeutic efficacy of lithium , pai-1 has been implicated in the etiology and progression of neurodegenerative diseases and mood disorders ( for review , pawlak et al . , 2003 ;", "lithium s effects on smad3/4 likely result from cross - talk of signaling pathways between camp / pka and pi3-kinase / akt / gsk-3. we have shown that lithium - induced smad3/4 suppression involved gsk-3 inhibition through the activation of pka and cell survival factor akt followed by the phosphorylation of gsk-3 at ser9 and creb at ser133 ( liang et al . , 2008 ) .", "creb binding protein ( cbp ) and p300 are known to be co - activators of creb .", "our data further demonstrated that over - expression of p300 , but not cbp , completely antagonized lithium - induced reduction of pai-1 promoter activity .", "a series of experimental data support the notion that , in smad3/4 signaling , the inhibitory effects of lithium are due to complex formation of activated creb and p300 , which results in limited interactions of p300 with the transcription factors / smad complexes . this in turn prevents efficient smad3/4-dependent transcription of smad3/4-dependent genes such as pai-1 and p21 ( figure 2 ) .", "transcriptional activations triggered by stimulation of cell surface tgf- and bdnf receptors are mediated by smad3/4- and pi3-kinase / akt - dependent pathways , respectively .", "lithium treatment - induced inhibition of gsk-3 , directly and indirectly via camp - dependent activation of pka as well as bdnf - stimulated activation of pi3-kinase / akt pathways , potentiates bdnf - induced phosphorylation / activation of creb .", "this in turn increases cre - mediated transactivation and expression of survival factors such as bdnf and bcl-2 .", "enhanced gene transcription triggered by bdnf , via sequestration of transcriptional co - activator p300 , suppresses smad3/4-dependent transactivation and subsequently decreases the expression of tgf--responsive genes , pai-1 , and p21 .", "lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections . dashed lines represent pathways with reduced activity as a result of lithium treatment ."], ["lithium - induced neuroprotection against glutamate excitotoxicity was first noted in rodent primary neuronal cultures of cerebellar granule cells ( cgcs ) , cerebral cortical neurons , and hippocampal neurons ( nonaka et al . , 1998 ) . this experimental paradigm was selected because glutamate - related excitotoxicity has been implicated in many neurodegenerative diseases including stroke ( for review , chuang , 2004 ; chiu and chuang , 2010 ) .", "our pioneering studies have shown that glutamate - induced , nmda receptor - mediated excitotoxicity was robustly reduced by extended lithium chloride pretreatment ( 57  days ) in cultured rat cgcs and cortical neurons , partly via inhibition of nmda receptor - mediated calcium influx ( nonaka et al . , 1998 ; hashimoto et al . , 2002a ) . moreover ,", "these effects of lithium were likely due to the attenuation of constitutive phosphorylation at tyr1472 of the nr2b subunit of nmda receptors , possibly as a result of inhibiting src tyrosine kinase ( hashimoto et al . , 2002a , 2003 ) .", "although glutamate - induced excitotoxicity in cultured cortical neurons was blocked by treatment with either lithium or mk-801 ( an nmda receptor antagonist ) , the src kinase inhibitor su6656 only partially diminished this toxicity ( hashimoto et al . , 2003 ) , suggesting that other components are involved . in cgcs , lithium - induced neuroprotection against glutamate excitotoxicity was associated with up - regulation of the anti - apoptotic protein bcl-2 , down - regulation of the pro - apoptotic proteins p53 and bax , and suppressed release of cytochrome c from mitochondria ( chen and chuang , 1999 ) , whereas the involvement of gsk-3 in the regulation of nmda signaling by lithium treatment is currently unclear and requires further investigations .", "cyclin - dependent kinase 5 ( cdk5 ) also regulates signaling mediated by nmda receptors , either directly through phosphorylation of the nr2b subunit or indirectly through phosphorylation of psd-95 ( morabito et al . , 2004 ; zhang et al . , 2008", "when it binds to p25 ( the product of calpain - mediated cleavage of p35 ) , cdk5 becomes pro - apoptotic and its activity is dysregulated ( lee et al .", ", p25 accumulation was observed in neurons in response to glutamate or oxidative stress , and also in the brains of several animal models of neurodegenerative diseases . sustained activation of cdk5 in neurons has been implicated in many neurodegenerative diseases ( cruz and tsai , 2004 ; dhariwala and rajadhyaksha , 2008 ) . in cultured rat cgcs ,", "lithium pretreatment prevented colchicine - induced apoptosis and associated increase in cdk5 expression and fragmentation of p35 into p25 ( jorda et al .", ". additionally , pretreatment with lithium also attenuated intracellular calcium increase , calpain activity , cdk5 activation , and cellular death in primary cultured hippocampal neurons and rat striatum following the treatment of 3-nitropropionic acid ( crespo - biel et al . , 2009 ) , a succinate dehydrogenase inhibitor ( for review , brouillet et al . , 1999 ) .", "therefore , lithium - induced inhibition of calpain and cdk5 activation may also contribute to protection against glutamate excitotoxicity .", "prior to changes in gene expression , lithium rapidly and transiently activated the cell survival pi3-kinase and its down - stream target , akt-1 , through phosphorylation at ser473 , thereby reversing glutamate - induced inactivation of this signaling pathway in cgcs ( chalecka - franaszek and chuang , 1999 ) .", "activated akt is known to affect several anti - apoptotic targets including bcl-2 associated death promoter ( bad ) , creb , members of the forkhead family , and procaspase-9 ( for review , neri et al . , 2002 ; nicholson and anderson , 2002 ; huang and reichardt , 2003 ) .", "in addition , lithium also triggered ser21 phosphorylation of the  isoform of gsk-3 ( and hence resulted in inhibition ) , and this effect was prevented by a pi3-kinase inhibitor ( chalecka - franaszek and chuang , 1999 ) .", "another signaling pathway affected by lithium is the mitogen - activated protein ( map ) kinase pathway .", "one of the down - stream targets of map kinase is creb , a transcription factor that is involved in learning and memory , and promotes the expression of bcl-2 as well as brain - derived neurotrophic factor ( bdnf ; for review , finkbeiner , 2000 ) . in cgcs , toxic concentrations of glutamate - induced an nmda receptor - dependent decrease in creb phosphorylation at ser133 and creb - driven transcriptional activity ( kopnisky et al . , 2003 ) .", "concurrent with its neuroprotective effects , long - term ( but not acute ) lithium treatment suppressed glutamate - induced dephosphorylation of creb .", "we also found that glutamate rapidly activated c - jun - n - terminal kinase ( jnk ) and p38 kinase in cgcs , resulting in a robust increase in ap-1 binding ( chen et al . , 2003a ) .", "these two kinases are also activated by a variety of apoptotic insults ( for review , mielke and herdegen , 2000 ) , and ap-1 has been known to be activated by different stress factors as well .", "experiments using lithium and curcumin , a selective ap-1 inhibitor , suggest that nmda receptor - mediated apoptotic death requires concerted action of jnk and p38 to enhance ap-1 binding , and that lithium s neuroprotection is mediated , at least in part , by suppressing the jnk and p38 kinase pathways .", "as one of the major neurotrophins , bdnf is essential for cortical development , synaptic plasticity , and neural survival , and is likely a key mediator of the clinical efficacy of anti - depressants and anxiolytic drugs ( for review , woo and lu , 2006 ) .", "the notion that bdnf plays a key role in neuronal survival is supported by our observation that bdnf and neurotrophin-4 ( nt-4 ) , but not nt-3 , completely protected immature cgcs from apoptosis induced by cytosine arabinoside ( leeds et al . , 2005 ) .", "it was first reported that chronic treatment with lithium increased the expression of bdnf in the rat brain ( fukumoto et al . , 2001 ) , and we have documented that bdnf protein levels were increased in cortical neurons following lithium treatment ( hashimoto et al . , 2002b ) .", "we hypothesized that this bdnf up - regulation and subsequent activation of its receptor trkb might play a critical role in mediating the neuroprotective effects of lithium . in confirmation of this hypothesis", ", we found that lithium s neuroprotection against glutamate excitotoxicity was blocked by a trkb inhibitor , k252a , or by a neutralizing antibody against bdnf , and was mimicked by exogenous bdnf in rat cortical neurons .", "in addition , lithium increased intracellular levels of bdnf and this was followed by activation of trkb .", "furthermore , lithium - induced neuroprotection was prevented in cortical neurons from heterozygous ( + / ) or homozygous ( / ) bdnf knockout mice ( hashimoto et al . , 2002b ) .", "rodent bdnf has a complex genomic structure that makes it an ideal target for multiple and complex regulation .", "we found that treatment of rat cortical neurons with therapeutic concentrations of lithium ( e.g. , 1  mm ) caused a significant increase in the levels of bdnf exon iv - containing mrna , while levels of exon i , ii , or vi - containing mrna remained unchanged ( yasuda et al . , 2009 ) .", "it is known that exon iv - containing bdnf transcripts are expressed in response to kcl - induced depolarization in rat cortical neurons ( tao et al . , 2002 ) .", "this transcriptional activation requires utilization of the promoter region 80  bp up - stream from the transcription initiation site of exon iv - containing three calcium responsive elements ( cares ; chen et al . , 2003b ) .", "we generated various bdnf promoter iv deletion constructs to investigate whether lithium treatment causes an increase in bdnf promoter iv activity , and , if so , which region of promoter iv confers the sensitivity to this drug .", "we identified that the drug - induced up - regulation of exon iv - containing bdnf transcript was associated with a significant increase in the activity of bdnf promoter iv and total bdnf protein . to our surprise , the lithium - responsive element(s ) in promoter iv resides in a region up - stream from the cares responsible for depolarization - induced bdnf induction ( 170 to 704  bp ) .", "moreover , activation of bdnf promoter iv occurred in cortical neurons depolarized with kcl and depletion of these three cares failed to abolish lithium - induced activation .", "importantly , we found that lithium - induced activation of promoter iv was mimicked by pharmacological inhibitors of gsk-3 ( sb216763 , sb415286 , inhibitor i , and inhibitor vii ) or by transfection with specific sirna for gsk-3 or gsk-3. additionally , their dominant - negative mutants also mimicked lithium - induced activation of promoter iv .", "these results demonstrate that gsk-3 is the initial target of lithium to selectively activate bdnf promoter iv and that bdnf induction by lithium involves a novel responsive region in promoter iv of the bdnf gene .", "lithium - induced , gsk-3-dependent bdnf promoter iv activation could be a part of the molecular mechanisms underlying its neuroprotective effects and as such , possibly accounts for the therapeutic actions in bipolar patients .", "it should be noted that in addition to lithium , other gsk-3 inhibitors have been shown to almost completely block glutamate - induced excitotoxicity in rat cortical neuronal cultures ( liang and chuang , 2007 ) .", "these include atp - competitive inhibitors , sb216763 and sb415286 , and atp - non - competitive inhibitors , inhibitor i and vii .", "as mentioned in the preceding section , glutamate - induced death of cortical neurons was mitigated by silencing of gsk-3 and/or  , or both isoforms , or inhibition of gsk-3 activity via transfection with dominant - negative mutants of gsk-3/ isoforms ( liang and chuang , 2007 ) .", "studies from other laboratories also supported the roles of gsk-3 inhibition in protecting neurons from glutamate neurotoxicity .", "for example , stimulation of nmda receptors in cultured rat hippocampal or cortical neurons activated gsk-3 by pp-1-mediated serine dephosphorylation of gsk-3 ( szatmari et al . , 2005 ) .", "treatment of primary rat cortical neurons with -amino-3-hydroxy-5-methyl-4-isoxazole propionate ( ampa ) , lithium or sb216763 blocked glutamate - induced caspase-3 activation and excitotoxicity , and the protective effects of ampa required pi3-kinase  akt - dependent serine phosphorylation of gsk-3 ( nishimoto et al . , 2008 ) .", "further , in organotypic cultures of chick embryo spinal cord , lithium prevented kainate - induced excitotoxic death of motoneurons by targeting gsk-3 , and this neuroprotection was associated with cytopathological changes ( caldero et al . , 2010 ) ."], ["stroke is the third leading cause of death in the united states and a major global cause of serious long - term disability in adults .", "ischemic strokes represent approximately 87% of all cases , while the rest are hemorrhagic strokes ( roger et al . , 2011 ) .", "in addition to physical deficits , stroke victims also suffer from vascular depression and dementia , both of which are difficult to treat with conventional medicine .", "it is becoming clear that there is a substantial increase in extracellular glutamate in the brain following cerebral ischemia , and that a significant portion of ischemia - induced brain damage is mediated by over - stimulation of nmda receptors .", "shortly after ischemia , the interruption of cerebral blood flow depletes oxygen and glucose and subsequently prevents atp production .", "inadequate atp supply will cause the malfunction of atp - dependent ion pumps and alter the ion concentration gradient across the neuronal membranes .", "the resulting failure to transport glutamate leads to an accumulation of glutamate in the extracellular space and over - stimulates nmda receptors , which leads to a toxic influx of calcium and in turn drives the activation of damaging calcium - mediated intracellular enzymes .", "this cascade of events ultimately results in mitochondrial failure , production of reactive oxygen species , neuroinflammation , and cell necrosis and apoptosis ( allen and bayraktutan , 2009 ; deb et al . , 2010 ) .", "gsk-3 has been strongly implicated in the neuronal cell death caused by cerebral ischemic insult .", "one study in rats subjected to transient middle cerebral artery occlusion ( mcao ) demonstrated a rapid increase in the expression of cytoplasmic and nuclear gsk-3 protein in ipsilateral lamina i , ii , v , and vi in young rat brains , whereas in lamina v and vi in old rat brains ( sasaki et al .", "although the phosphorylation status of gsk-3 was not mentioned , these findings implicate a role of gsk-3 in cerebral ischemic injury .", "it is well known that gsk-3 can be phosphorylated at serine and tyrosine residues in which ser9 phosphorylation renders it inactive , while tyr216 phosphorylation is necessary for its functional activity ( hughes et al . , 1993 ) .", "discrepancies exist in the literature regarding changes of ser9 and tyr216 phosphorylation levels following cerebral ischemia .", "the phosphorylation levels of gsk-3 at ser9 and akt at ser473 were reported to be markedly enhanced in the vulnerable hippocampal ca1 region , but not in the ischemia - resistant ca3 region in rats subjected to transient global cerebral ischemia , while there was no change in levels of tyr216 phosphorylation or total gsk-3 ( endo et al . , 2006 ) .", "levels of gsk-3 ser9 phosphorylation were also increased shortly after permanent focal cerebral ischemia and decreased to basal levels or even lower 24  h after ischemic onset ( sasaki et al . , 2006 ; gao et al . , 2008", "however , it has also been reported that transient focal cerebral ischemia in rats caused an increase in gsk-3 tyr216 phosphorylation in degenerating cortical neurons with no alteration in ser9 phosphorylation ( bhat et al . , 2000 ) .", "this discrepancy may stem , in part , from the difference of ischemic severity and ischemic models across various studies .", "it appears that transient focal cerebral ischemia tends to activate gsk-3 and subsequently to induce apoptotic cell death .", "in contrast , gsk-3 is inactivated shortly after permanent focal cerebral ischemia or global cerebral ischemia , which in turn may promote survival of vulnerable neurons .", "ischemia both gsk-3 and gsk-3 mediated the expression of a lethal protein , neuronal pentraxin 1 ( russell et al . , 2011 ) . in light of these findings", "the beneficial effects of lithium in rodent cerebral ischemic models demonstrated by us and others support this notion . in an initial study ,", "long - term lithium pretreatment at therapeutically relevant doses decreased brain infarct volume , reduced apoptotic cell death and improved behavioral performance after permanent cerebral ischemia - induced by mcao ( nonaka and chuang , 1998 ; xu et al . , 2003 ) . in a subsequent study , we demonstrated that subcutaneous injection of rats with lithium at therapeutic doses ( e.g. , 0.5 and 1.0  meq / kg ) after the onset of transient mcao markedly decreased infarct volume , reduced tunel - positive dna damage , and suppressed neurological deficits measured by sensory , motor , and reflex tests ( ren et al . , 2003 ) .", "the time window for these beneficial effects was at least 3  h after the onset of ischemia .", "heat - shock protein 70 ( hsp70 ) , a well - established cytoprotective factor against apoptosis , was induced in the ischemic penumbra where neuronal recovery takes place .", "post - insult treatment with lithium increased the dna binding activity of hsf-1 to the heat - shock element , superinducing hsp70 which inhibits brain ischemia - induced apoptosis ( ren et al . , 2003 ) .", "lithium - elicited gsk-3 inhibition is likely associated with hsf-1 activation and hsp70 induction ( bijur and jope , 2000 ) .", "notably , post - insult lithium treatment mitigated apoptosis and brain damage by preventing gsk-3 and erk dephosphorylation , suppressing calpain and caspase-3 activation , and inhibiting mitochondrial release of cytochrome c and apoptosis - inducing factor in a neonatal hypoxic ", "these findings suggest that lithium - induced gsk-3 inhibition contributes to its anti - apoptotic effects under ischemic conditions .", "in addition , it was found that lithium pretreatment largely suppressed ischemia - induced exploratory behavioral changes and memory impairments in gerbils after global cerebral ischemia ( bian et al . , 2007 ) .", "these behavioral benefits were associated with an increase in the number of viable cells and a decrease in apoptotic cells in the ca1 hippocampal area of ischemic gerbils . moreover , lithium - induced neuroprotection in the ischemic brain was accompanied by down - regulation of pro - apoptotic p53 in the ca1 , and up - regulation of anti - apoptotic bcl-2 and hsp70 , both of which are targets of gsk-3 .", "it is likely that lithium protection against ischemia - induced injury involves multiple mechanisms . in the rat hippocampus ,", "lithium was reported to inhibit ischemia - induced nmda receptor hyperactivation by inhibiting nmda subunit 2a tyrosine phosphorylation and its interactions with src and fyn through psd-95 ( ma and zhang , 2003 ) .", "lithium also attenuated hypoxia - induced serine dephosphorylation of gsk-3 and  in the mouse brain ( roh et al . , 2005 ) .", "additionally , in organotypic cultures of rat hippocampus subjected to oxygen and glucose deprivation , lithium showed neuroprotection in conjunction with hsp27 activation ( cimarosti et al . , 2001 ) .", "post - ischemic inflammation is a dynamic process involving a complicated set of interactions between inflammatory cells and molecules ( iadecola and alexander , 2001 ) . a recent study documented the anti - inflammatory effects of lithium in a neonatal rat hypoxic  ischemic model .", "post - insult lithium treatment significantly reduced total tissue loss following hypoxia  ischemia , and this beneficial effect of lithium was associated with inhibiting microglia activation and attenuating levels of pro - inflammatory cytokines or chemokines , such as interleukin-1 and chemokine ligand 2 ( li et al . , 2011 ) .", "hsp70 over - expression can inactivate the key inflammatory transcription factor nf-b by stabilizing the nf-b - ib complex , and thereby preventing nuclear translocation of activated nf-b subunits in a mouse mcao model ( zheng et al . , 2008 ) . besides anti - inflammation", ", lithium also increased proliferation and differentiation of hippocampal neural progenitor cells in both non - ischemic and ischemic brains without altering the relative levels of neuronal and astrocytic differentiation , and this effect lasted at least 7  weeks after hypoxia  ischemia in neonatal rats ( li et al . ,", "in line with this finding , chronic lithium pretreatment was found to increase the generation and survival of newborn cells in the hippocampal dentate gyrus , and did not affect the neuronal or astrocytic differentiation of these newborn cells in a transient four - vessel occlusion model ( yan et al .", "erk1/2 phosphorylation following ischemia was enhanced by lithium treatment , while erk1/2 inhibitor u0126 prevented the effects of lithium in increasing brdu - positive cells and improving spatial learning and memory ( yan et al . , 2007 ) .", "in fact , chronic lithium treatment has been demonstrated to increase activity in the mek / erk pathway in vivo , and lithium s neuroprotection has been suggested to depend on the induction of this signaling pathway ( einat et al .", "in addition , it was reported that activation of erk associates with and phosphorylates gsk-3 at the thr43 residue , which primes this kinase for its subsequent phosphorylation at ser9 by p90rsk , resulting in inactivation of gsk-3 and up - regulation of -catenin ( ding et al . , 2005 ) .", "therefore , lithium might affect gsk-3 phosphorylation through the mek / erk pathway , which in turn inhibits this kinase via rsk .", "gsk-3 is also negatively regulated by the wnt/-catenin signaling pathway and , accordingly , activating the canonical wnt pathway has been shown to contribute to adult hippocampal neural progenitor cell proliferation triggered by lithium treatment ( wexler et al . , 2008 ) . in a collaborative study ,", "the neurohemodynamic aspects of recovery induced by delayed chronic lithium treatment were assessed using functional magnetic resonance imaging ( mri ; kim et al . , 2008 ) .", "rats were subjected to transient mcao and then injected with lithium ( licl , 1  meq / kg , s.c . )", "this delayed lithium injection was followed by daily injections , and on day 15 , an mri scan was performed to monitor changes in blood oxygen level dependence ( bold ) and functional cerebral blood volume ( fcbv ) responses using electric stimulation of forelimbs .", "the mean activated volume ratio and total activation magnitude ratio between ipsilateral and contralateral cortices for both bold and fcbv were significantly higher in the lithium - treated than in the saline - treated rats .", "the lithium - induced increase in fcbv in the peri - infarct regions suggests a possible vascular transformation .", "indeed , the size and distribution of immunohistochemical staining of cd31 , a microvasculature marker , were enhanced by lithium treatment in the peri - infarct regions .", "co - localized with cd31 , the tissue staining of matrix metalloproteinase-9 ( mmp-9 ) was also much more pronounced following lithium treatment , suggesting mmp-9-dependent neurovascular remodeling in the recovering brain area .", "moreover , treatment of cultured rat brain endothelial cells with lithium in a follow - up study was also found to increase the protein levels of vascular endothelial growth factor ( vegf ) via a mechanism involving the pi3-kinase and gsk-3 signaling pathways ( guo et al . ,", "since vegf has been linked to angiogenesis , neurogenesis , and neuroprotection ( for review , fan and yang , 2007 ) , vegf over - expression may contribute to lithium s ability to promote neurovascular remodeling and to induce functional recovery after ischemic stroke .", "ample evidence supports the therapeutic potential of mesenchymal stem cells ( mscs ) in several human diseases including stroke .", "however , it is increasingly recognized that the effectiveness of msc transplantation is limited by their poor migration toward disease target sites such as ischemic brain regions . in a recent study", ", we investigated whether treatment of mscs with lithium and another mood stabilizing drug , valproic acid ( vpa ) , would enhance cell migration ( tsai et al . , 2010 ) .", "we found that treatment of mscs with lithium ( 2.5  mm for 1  day ) selectively elevated the transcript and protein levels of mmp-9 and its enzymatic activity .", "these effects were mimicked by pharmacological inhibition or gene silencing of gsk-3. lithium treatment also potentiated stromal cell - derived factor-1 ( sdf-1)-dependent msc migration across the extracellular matrix , which was suppressed by two mmp-9 inhibitors , doxycycline and gm6001 .", "short - term ( 3  h ) exposure of mscs to a relatively high concentration ( 2.5  mm ) of vpa markedly increased the transcript and protein levels of cxc chemokine receptor 4 ( cxcr4 ) .", "vpa - induced cxcr4 expression required its ability to inhibit histone deacetylases ( hdacs ) , including the hdac1 isoform , and involved histone hyperacetylation at the cxcr4 gene promoter .", "vpa treatment enhanced sdf-1-mediated msc migration , which was completely blocked by amd3100 , a cxcr4 antagonist .", "notably , combining lithium and vpa treatment further increased msc migration , and the additive enhancement of migration was completely blocked by the co - presence of amd3100 and gm6001 .", "our results suggest that lithium and vpa stimulate msc migration through distinct targets and mediators : gsk-3mmp-9 and hdac  cxcr4 , respectively ( tsai et al . , 2010 ) .", "in a follow - up in vivo study , mscs were primed with lithium and/or vpa and then injected into the tail vein of transient mcao rats 24  h after ischemic onset . priming with lithium or vpa increased the number of mscs homing to the cerebral infarcted regions such as the cortex and striatum 2  weeks after transplantation , and co - priming with lithium and vpa further enhanced this migratory effect ( tsai et al . , 2011 ) .", "mcao rats receiving lithium- and/or vpa - primed mscs showed improved functional recovery , reduced infarct volume , and enhanced angiogenesis in the infarcted penumbra regions .", "these beneficial effects of lithium and vpa priming were reversed by pharmacological inhibition of mmp-9 and cxcr4 , respectively , suggesting that these effects were likely mediated by lithium - induced mmp-9 up - regulation and vpa - induced cxcr4 over - expression .", "together , these findings raise the potential utility of using mscs primed with inhibitors of gsk-3 and hdac to enhance the migration and homing capacity for transplantation into stroke victims .", "in addition to lithium , other pharmacological gsk-3 inhibitors have been shown to exert neuroprotective effects against cerebral ischemia by various groups .", "a specific gsk-3 inhibitor , chir025 , was demonstrated to protect cultured hippocampal neurons from glutamate excitotoxicity and to attenuate death of cortical neurons following oxygen ", "glucose deprivation , an in vitro model of cerebral ischemia ( kelly et al . , 2004 ) .", "moreover , chir025 reduced infarct size in focal cerebral ischemic rats , but did not affect tunel - positive neurons or caspase-3/9 activities , although bcl-2 expression was increased .", "gsk-3 enzymatic activity was markedly elevated after transient mcao in rats , and this gsk-3 activation was blocked by jugular vein injection of gsk-3 inhibitor viii ( koh et al . , 2008 ) .", "pre- or post- ( up to 2  h ) mcao injection with inhibitor viii also reduced blood glucose levels , infarct size , caspase-3 activity , and water content in the ipsilateral brain hemisphere .", "furthermore , ischemia - induced inflammation - related signals such as cox-2 over - expression and neutrophil infiltration were alleviated by this gsk-3 inhibitor .", "prophylactic or therapeutic administration of a gsk-3 inhibitor tdzd-8 reduced infarct volume and cerebral injury in the rat hippocampus after transient ischemia ( collino et al . , 2008 ) .", "this was accompanied by suppression of ischemia - induced oxidative stress , apoptosis , and neuroinflammation .", "delayed treatment with compound i , a gsk-3 and cdk inhibitor , decreased tunel - positive cells in the ipsilateral hippocampus and striatum of adult ( but not juvenile ) mice subjected to hypoxic ", "these neuroprotective effects of compound i were associated with long - lasting functional recovery . finally , gsk-3 inhibition by sb216763 counteracted oxygen ", "glucose deprivation - induced mitochondrial biogenesis impairment and reduced mitochondrial reactive oxygen species generation in primary cortical neurons ( valerio et al . , 2011 ) .", "when systematically administrated to permanent mcao mice , sb216763 decreased infarct volume and restored the loss of mitochondrial dna , thus supporting a novel role of gsk-3 inhibitors in stimulating the renewal of functional mitochondria following ischemic stroke ."], ["a growing body of evidence supports that lithium , a mood stabilizer used to treat bipolar disorder , has neuroprotective properties in both cellular and in vivo experimental settings .", "one of the major targets of lithium is gsk-3 , a serine / threonine kinase implicated in the pathogenesis of diverse cns disorders .", "lithium inhibits gsk-3 activity by direct binding to the enzyme or indirectly by enhancing serine phosphorylation of both  and  isoforms through multiple mechanisms .", "lithium has been used as a prototype drug to seek evidence for the involvement of gsk-3 inhibition in lithium - induced protection against excitotoxicity in cultured neurons and animal models of cerebral ischemic stroke .", "lithium at therapeutically relevant concentrations robustly protected primary brain neurons from glutamate - induced , nmda receptor - mediated excitotoxicity .", "the neuroprotective effects of lithium were associated with gsk-3 inhibition , and were mimicked by other pharmacological gsk-3 inhibitors , by silencing gsk-3 and/or  isoforms , or by expression of isoform - specific dominant - negative mutants .", "these results support the roles of gsk-3 inhibition in lithium - elicited protection against excitotoxicity .", "lithium rapidly activated the cell survival pi3-kinase  akt signaling pathway to enhance gsk-3 serine phosphorylation and to block glutamate - induced akt inactivation as well as apoptosis .", "lithium also caused an increase in the expression of cytoprotective bcl-2 and suppressed glutamate - induced up - regulation of pro - apoptotic p53 and bax , resulting in blocking cytochrome c release from mitochondria .", "bdnf promoter iv was selectively activated by gsk-3 inhibition using lithium or other drugs or through gene silencing / inactivation of either isoform . this effect on", "however , there is a gap in the understanding of how gsk-3 inhibition causes an increase in bdnf promoter activity .", "in addition , lithium s neuroprotective effects were associated with inhibition of nmda receptor - mediated calcium influx and suppression of p38/jnk and ap-1 activation , thus reducing apoptosis .", "this effect appears to stem from inhibition of src / fyn kinase to suppress nr2b tyr1472 phosphorylation of the receptor .", "it remains to be explored as to whether this lithium - induced action on nmda receptors is related to gsk-3 inhibition .", "the potential roles of these other targets in mediating the neuroprotective effects of this drug also deserve future investigation .", "it is well known that glutamate overflow and nmda receptor hyper - stimulation are early events following cerebral ischemia . in rodent", "ischemic models , pre- or post - insult treatment with therapeutic doses of lithium decreased infarct volume , caspase-3 activity and apoptotic cells in the injured brain .", "the beneficial time window of lithium is at least 3  h after the ischemic onset .", "up - regulation of hsp70 and bcl-2 as well as down - regulation of p53 likely contributed to the protective effects of lithium in the ischemic conditions , thus supporting similar underlying neuroprotective mechanisms in the excitotoxic cellular models and animal models of ischemic stroke .", "limited data suggested that lithium might also display anti - inflammatory effects by inhibiting ischemia - induced microglia activation and pro - inflammatory factors release .", "delayed and chronic injections of lithium improved functional mri responses such as increases in bold and fcbv .", "indeed , lithium was found to induce two pro - angiogenic factors , mmp-9 and vegf in a gsk-3-dependent manner .", "lithium has also been reported to stimulate erk1/2 activity and to enhance proliferation of hippocampal neural progenitor cells and memory performance after ischemia . finally , lithium promoted migration of mscs in vitro by up - regulation of mmp-9 through gsk-3 inhibition and this migratory effect was potentiated by co - treatment with vpa , another mood stabilizer .", "notably , transplantation of lithium  vpa co - primed mscs into ischemic rats markedly increased msc migration to the injured brain regions , decreased infarct size and improved the neurological performance .", "lithium - induced stem cell migration , neurogenesis , and angiogenesis all likely contribute to functional recovery .", "figure 3 illustrates proposed molecular events leading to lithium - induced beneficial effects following cerebral ischemia .", "it should be noted that several other gsk-3 inhibitors have also been reported to exert beneficial effects in rodent ischemic models and their actions were accompanied by suppression of ischemia - increased gsk-3 activity .", "accordingly , gsk-3 inhibitors have therapeutic potential to treat stroke and other excitotoxicity - related neurodegenerative diseases .", "lithium has been used in bipolar patients over 60  years and its clinical profiles are well understood .", "therefore , lithium is a prime candidate for use in clinical trials of new therapies for stroke victims .", "the neuroprotective effects of lithium against cerebral ischemia are proposed to result from its interactions with cell survival and apoptotic machinery .", "a significant portion of brain damage following cerebral ischemia is caused by an increase in extracellular glutamate and subsequent over - stimulation of nmda receptor - mediated toxic increase in intracellular calcium .", "this signaling pathway plays a critical role in mediating glutamate - induced caspase activation and apoptosis .", "lithium at therapeutically relevant concentrations inhibits nmda receptor - mediated calcium influx , which in turn decreases subsequent activation of jnk , p38 kinase , and transcription factor ap-1 .", "inhibition of intracellular calcium increase also attenuates the activity of calpain and calpain - mediated activation of pro - apoptotic cdk5/p25 kinase . on the other hand ,", "lithium can directly and indirectly reduce the activity of constitutively activated gsk-3 by multiple mechanisms , leading to disinhibition of several transcription factors , such as creb and hsf-1 , and resulting in induction of major cytoprotective proteins such as bdnf , vegf , mmp-9 , hsp70 , and bcl-2 . a decrease in gsk-3 activity", "further reduces the activity of pro - apoptotic protein p53 and its downregulating effect on bcl-2 .", "bdnf , via activating its cell surface receptor and the down - stream erk and pi3-kinase / akt pathways , induces neuroprotective effects in part by inhibiting gsk-3 and stimulating creb .", "induction of bdnf is an early and essential step for neuroprotection and is involved in lithium - induced neurogenesis .", "in addition , superinduction of hsp70 by lithium treatment not only inhibits brain ischemia - induced apoptosis , but also contributes to the anti - inflammatory effects of lithium through inactivation of nf-b .", "counteraction of gsk-3 inhibition of vegf and mmp-9 by lithium enhances angiogenesis and neurovascular remodeling .", "taken together , these effects of lithium in reducing apoptosis , suppressing inflammation , enhancing angiogenesis and neurogenesis , contribute to behavioral improvement and functional recovery after ischemia . lines with solid arrows represent stimulatory connections ; lines with flattened ends represent inhibitory connections ."], ["the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC3519055", "article_text": ["studies have shown the incidence of injury from squash to range from 35.5 to 80.9 per 100,000 players .", "trauma to the head ( including the eyes ) is the most common cause of squas - related hospital presentation , comprising 48.7% of all emergency department presentations .", "craniofacial and spinal fractures typically result from high - energy blunt force to the skeleton , as seen in motor vehicle accidents .", "this is the first case reported of a patient sustaining such extensive head , neck and spinal injuries from a squash - related injury .", "a 55-year - old man was brought in by ambulance after colliding with the corner of the wall and floor of a squash court , without the use of protective eye - glasses or mouth - guard .", "he was chasing a ball toward the corner of the court when he fell and hit the junction of the wall and floor with his head .", "on examination in the emergency department , the patient had a glasgow coma scale ( gcs ) of 14 , with confusion to time and place .", "initially , the patient reported diplopia at primary position but examination by the surgical team elicited normal eye movements with nil complaint of diplopia .", "there was significant boggyness to palpation of the forehead with a palpable defect of the frontal bone .", "the cervical spine was slightly tender over / c1 - 2 and c5 - 6 .", "upper limb examination was limited by pain maximal over the distal radii but bilateral arm weakness was noted with nil sensory dysfunction .", "primary and secondary surveys did not elicit any further findings . a non - contrast computed tomography ( ct )", "imaging of the facial bones [ figure 1 ] showed a le fort type ii fracture , with comminuted fractures of the roofs of the orbit bilaterally and a depressed fracture of the left orbital floor .", "there was also fracture of the right maxilla at the junction of the maxilla and zygomatic arch , extending towards but not involve the right orbital floor . on the left , there was fracture of the anterior part of the left zygomatic arch which extended into the anterolateral wall of the left maxillary sinus .", "a large , depressed fracture of the frontal bone was also evident , extending superiorly from the superior lateral wall of the right orbit and extending across the frontal bone and inferiorly toward the mid - portion of the roof of the left orbit .", "the fracture involved the inner and outer tables of the frontal bone and was depressed by at least 3.5 mm .", "there was also a comminuted fracture of the nasal bone , which was not significantly displaced .", "anterior and anterolateral views of three - dimensional ct reconstructions demonstrating lefort type ii , frontal , orbital and zygomatic fractures ct brain showed tiny subarachnoid or intraparenchymal haemorrhages but there was no evidence of extensive intra- or extra - axial haemorrhage .", "imaging of the cervical spine [ figure 2 ] showed an undisplaced fracture of the posterior arch of c1 on the right side and a type 3 dens fracture with posterior displacement of 4 mm but no contact with the spinal cord .", "anterolateral view 3 dimensional ct reconstruction demonstrating type 3 dens fracture with posterior displacement and fracture of c7 spinous process subsequent ct of the thoracic spine showed a comminuted t6 vertebral body fracture with loss of vertebral height and extension into the left costovertebral joint .", "there was a small bony fragment projecting posteriorly into the spinal canal and making contact with the cord , but there was nil evidence of compression .", "associated with this finding were two to three hyperdense foci within the spinal cord likely to represent small contusions .", "the above findings were confirmed on magnetic resonance imaging ( mri ) of the spine , which also demonstrated a small cortical buckle at the t6 level without significant cord impingement .", "the patient underwent craniotomy , obliteration of the frontal sinus and open reduction internal fixation ( orif ) of the frontal bone .", "orif of the maxilla bilaterally , orif of the infra - orbital rim and supra - orbital rim bilaterally and inter - maxillary screw fixation was also performed .", "the patient was admitted to the neurosurgical intensive care unit post - operatively and underwent cervical fusion with odontoid screw fixation of the c2 fracture one week after surgical correction of the head injuries .", "post - operative ct scans demonstrated complete bony union in the c2 fracture site [ figure 3 ] and uncomplicated fixation of the facial bone fractures [ figure 4 ] .", "post - operative lateral x - ray of the cervical spine in extension demonstrating union of dens fracture with overall alignment in - tact post - operative coronal ct demonstrating surgical correction of multiple craniofacial fracture sites", "our patient suffered injuries to multiple sites in the cranium , facial bones and the vertebrae that have previously not reported in the literature .", "the thinnest and weakest segment of the orbit is the floor , which is the most common site of fracture .", "our patient suffered a le fort type ii fracture , which is pyramid shaped and passes through the posterior alveolar ridge , lateral walls of maxillary sinuses , inferior orbital rim and nasal bones .", "he also suffered a depressed frontal bone fracture involving the inner and outer tables , an extremely rare complication of sport seen in only 1.3% of sports related craniofacial fractures .", "spinal injury is associated with sports in approximately 8.7% of cases , usually in relation to contact sports such as wrestling .", "our patient had a fracture of the posterior arch of c1 on the right side and a type 3 dens fracture , which required cervical fusion .", "there was also fracture of the c7 cervical process , from forceful flexion of the cervical spine .", "we report the first case of extensive head and spinal injury resulting from collision with the wall of a squash court .", "the injuries ranged from the frontal bone to the thoracic vertebra and are usually seen in high velocity trauma ."], "abstract_text": ["<S> squash is a popular racquet sport not usually associated with severe head or spinal injury . </S>", "<S> the incidence of squash - related injury ranges from 35.5 to 80.9 per 100,000 players , with the most common sites being the lower - limbs and eyes . </S>", "<S> we present a case of extensive traumatic craniomaxillofacial and vertebral injury resulting from collision on a squash court , without use of protective gear . </S>", "<S> the patient sustained fractures of the frontal bone , orbits , maxillae , zygomas , the first and second cervical vertebrae and the spinous process of the seventh cervical vertrebra . </S>", "<S> this is the first case of squash - related injury with such extensive craniofacial and vertebral involvement . </S>", "<S> this unique case required multiple surgical procedures as well as an extensive admission to the intensive care unit and highlights the risk of significant craniomaxillofacial trauma in sports not usually associated with such injuries . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["studies have shown the incidence of injury from squash to range from 35.5 to 80.9 per 100,000 players .", "trauma to the head ( including the eyes ) is the most common cause of squas - related hospital presentation , comprising 48.7% of all emergency department presentations .", "craniofacial and spinal fractures typically result from high - energy blunt force to the skeleton , as seen in motor vehicle accidents .", "this is the first case reported of a patient sustaining such extensive head , neck and spinal injuries from a squash - related injury ."], ["a 55-year - old man was brought in by ambulance after colliding with the corner of the wall and floor of a squash court , without the use of protective eye - glasses or mouth - guard .", "he was chasing a ball toward the corner of the court when he fell and hit the junction of the wall and floor with his head .", "on examination in the emergency department , the patient had a glasgow coma scale ( gcs ) of 14 , with confusion to time and place .", "initially , the patient reported diplopia at primary position but examination by the surgical team elicited normal eye movements with nil complaint of diplopia .", "there was significant boggyness to palpation of the forehead with a palpable defect of the frontal bone .", "the cervical spine was slightly tender over / c1 - 2 and c5 - 6 .", "upper limb examination was limited by pain maximal over the distal radii but bilateral arm weakness was noted with nil sensory dysfunction .", "primary and secondary surveys did not elicit any further findings . a non - contrast computed tomography ( ct )", "imaging of the facial bones [ figure 1 ] showed a le fort type ii fracture , with comminuted fractures of the roofs of the orbit bilaterally and a depressed fracture of the left orbital floor .", "there was also fracture of the right maxilla at the junction of the maxilla and zygomatic arch , extending towards but not involve the right orbital floor . on the left , there was fracture of the anterior part of the left zygomatic arch which extended into the anterolateral wall of the left maxillary sinus .", "a large , depressed fracture of the frontal bone was also evident , extending superiorly from the superior lateral wall of the right orbit and extending across the frontal bone and inferiorly toward the mid - portion of the roof of the left orbit .", "the fracture involved the inner and outer tables of the frontal bone and was depressed by at least 3.5 mm .", "there was also a comminuted fracture of the nasal bone , which was not significantly displaced .", "anterior and anterolateral views of three - dimensional ct reconstructions demonstrating lefort type ii , frontal , orbital and zygomatic fractures ct brain showed tiny subarachnoid or intraparenchymal haemorrhages but there was no evidence of extensive intra- or extra - axial haemorrhage .", "imaging of the cervical spine [ figure 2 ] showed an undisplaced fracture of the posterior arch of c1 on the right side and a type 3 dens fracture with posterior displacement of 4 mm but no contact with the spinal cord .", "anterolateral view 3 dimensional ct reconstruction demonstrating type 3 dens fracture with posterior displacement and fracture of c7 spinous process subsequent ct of the thoracic spine showed a comminuted t6 vertebral body fracture with loss of vertebral height and extension into the left costovertebral joint .", "there was a small bony fragment projecting posteriorly into the spinal canal and making contact with the cord , but there was nil evidence of compression .", "associated with this finding were two to three hyperdense foci within the spinal cord likely to represent small contusions .", "the above findings were confirmed on magnetic resonance imaging ( mri ) of the spine , which also demonstrated a small cortical buckle at the t6 level without significant cord impingement .", "the patient underwent craniotomy , obliteration of the frontal sinus and open reduction internal fixation ( orif ) of the frontal bone .", "orif of the maxilla bilaterally , orif of the infra - orbital rim and supra - orbital rim bilaterally and inter - maxillary screw fixation was also performed .", "the patient was admitted to the neurosurgical intensive care unit post - operatively and underwent cervical fusion with odontoid screw fixation of the c2 fracture one week after surgical correction of the head injuries .", "post - operative ct scans demonstrated complete bony union in the c2 fracture site [ figure 3 ] and uncomplicated fixation of the facial bone fractures [ figure 4 ] .", "post - operative lateral x - ray of the cervical spine in extension demonstrating union of dens fracture with overall alignment in - tact post - operative coronal ct demonstrating surgical correction of multiple craniofacial fracture sites"], ["our patient suffered injuries to multiple sites in the cranium , facial bones and the vertebrae that have previously not reported in the literature .", "the thinnest and weakest segment of the orbit is the floor , which is the most common site of fracture .", "our patient suffered a le fort type ii fracture , which is pyramid shaped and passes through the posterior alveolar ridge , lateral walls of maxillary sinuses , inferior orbital rim and nasal bones .", "he also suffered a depressed frontal bone fracture involving the inner and outer tables , an extremely rare complication of sport seen in only 1.3% of sports related craniofacial fractures .", "spinal injury is associated with sports in approximately 8.7% of cases , usually in relation to contact sports such as wrestling .", "our patient had a fracture of the posterior arch of c1 on the right side and a type 3 dens fracture , which required cervical fusion .", "there was also fracture of the c7 cervical process , from forceful flexion of the cervical spine ."], ["we report the first case of extensive head and spinal injury resulting from collision with the wall of a squash court .", "the injuries ranged from the frontal bone to the thoracic vertebra and are usually seen in high velocity trauma ."]]}
{"article_id": "PMC4404966", "article_text": ["lung cancer is a leading cause of cancer mortality in the usa and other developed countries.1 the two major forms of the disease are non - small cell lung cancer and small cell lung cancer , which account for 85% and 15% of all lung cancers , respectively.2 despite advances in preliminary detection and standard treatment with combination chemotherapy and radiotherapy , the prognosis for patients with lung cancer has not improved significantly in the last 20 years,3,4 and survival rates have changed little over the past 2 decades .", "currently , an enormous amount of research is aimed at understanding the molecular and cellular biology of lung cancer ; however , much more work is needed to better understand the correlation between gene regulation and lung cancer .", "growing evidence shows that aberrant hypermethylation in 5-cpg islands in the promoter regions is a major mechanism for silencing tumor suppressor or other cancer - associated genes in many kinds of human cancer.58 loss of function in cancer suppressor genes may hinder inhibition of growth of cancer cells , which leads to malignant transcription and translation during replication of dna . a number of genes , including the cyclin - dependent kinase inhibitor ( p16 ) , the tumor suppressor gene ras association domain family protein 1a , kelch - like ech - associating protein 1 , the dna repair gene mgmt , and the cystic fibrosis transmembrane conductance regulator are demonstratively methylated in lung cancer.912 cdh1", ", a member of the transmembrane glycoprotein family , also known as cadherin-1 , cam 120/80 , epithelial cadherin ( e - cadherin ) , or uvomorulin , is encoded by the cdh1 gene ( 16q22.1).13 cdh1 is a calcium - dependent cell - cell adhesion glycoprotein containing three domains , ie , five extracellular cadherin repeats , a transmembrane region , and a highly conserved cytoplasmic tail.14 \n cdh1 as a tumor suppressor gene plays an essential role in maintaining cell adhesion and adherent junctions in normal tissues .", "cdh1 expression is frequently absent in a variety of epithelial tumors , and loss of normal intercellular junctions results in promotion of cancer invasion and metastasis and is correlated with several types of cancers.1517 however , the association between and clinicopathological significance of cdh1 promoter hypermethylation and lung cancer remains unclear . in this study , we systematically investigate studies of cdh1 promoter hypermethylation and lung cancer , and validate the association between cdh1 promoter hypermethylation and lung cancer using meta - analysis methods .", "in addition , we summarize these findings and discuss tumor suppressor function , as well as the clinicopathological significance of cdh1 in lung cancer .", "a systematic literature searching was performed using pubmed , embase , and the web of science up to august 13 , 2014 without any language restrictions .", "the following keywords and terms were used : [ methylation or dna methylation or hypermethylation or de - methylation ] and [ cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin ( e - cadherin ) or uvomorulin ] and [ lung cancer or lung carcinoma or lung tumor ] .", "the published scientific articles were restricted to english language , and conference abstracts were excluded due to lack of sufficient data .", "titles , abstracts , and key words in the articles were initially evaluated for inclusion criteria .", "details and additional information were identified and collected from the full text of these articles .", "a study included for meta - analysis needed to have : evaluated the correlation between cdh1 methylation and lung cancer ; included a clinical cohort and controls ; included at least three patients and controls ; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression ; and used tissue data rather than blood data .", "studies that did not meet our inclusion criteria were excluded . when the same groups of patients were reported in multiple papers ,", "two researchers independently collected the information and extracted the data regarding authorship , year , source of publication , inclusion criteria , cdh1 methylation frequency , sex status , smoking history , pathological type , clinical staging , degree of differentiation , lymph node metastasis , epidermal growth factor receptor status , and prognosis in patients and controls .", "methodological evaluation was assessed by the researchers according to the remark ( reporting recommendations for tumor marker prognostic studies ) guidelines and elcwp ( european lung cancer working party ) score.18,19 the meta - analysis was performed using reviewer manager 5 ( cochrane collaboration , oxford , uk ) . pooled odds ratios ( ors ) and confidence intervals ( cis ) were calculated to assess the correlation between cdh1 methylation and lung cancer .", "cochran s q test and i \n 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i \n 2 test > 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters .", "otherwise , a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 ( statacorp , college station , tx , usa ) .", "a systematic literature searching was performed using pubmed , embase , and the web of science up to august 13 , 2014 without any language restrictions .", "the following keywords and terms were used : [ methylation or dna methylation or hypermethylation or de - methylation ] and [ cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin ( e - cadherin ) or uvomorulin ] and [ lung cancer or lung carcinoma or lung tumor ] .", "the published scientific articles were restricted to english language , and conference abstracts were excluded due to lack of sufficient data .", "titles , abstracts , and key words in the articles were initially evaluated for inclusion criteria .", "details and additional information were identified and collected from the full text of these articles .", "a study included for meta - analysis needed to have : evaluated the correlation between cdh1 methylation and lung cancer ; included a clinical cohort and controls ; included at least three patients and controls ; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression ; and used tissue data rather than blood data .", "studies that did not meet our inclusion criteria were excluded . when the same groups of patients were reported in multiple papers , only the most recent and complete paper was selected to avoid overlap .", "two researchers independently collected the information and extracted the data regarding authorship , year , source of publication , inclusion criteria , cdh1 methylation frequency , sex status , smoking history , pathological type , clinical staging , degree of differentiation , lymph node metastasis , epidermal growth factor receptor status , and prognosis in patients and controls .", "methodological evaluation was assessed by the researchers according to the remark ( reporting recommendations for tumor marker prognostic studies ) guidelines and elcwp ( european lung cancer working party ) score.18,19", "the meta - analysis was performed using reviewer manager 5 ( cochrane collaboration , oxford , uk ) .", "pooled odds ratios ( ors ) and confidence intervals ( cis ) were calculated to assess the correlation between cdh1 methylation and lung cancer .", "cochran s q test and i \n 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i \n 2 test > 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters .", "otherwise , a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 ( statacorp , college station , tx , usa ) .", "the process followed to select the papers used in this report is shown in figure 1 .", "ninety papers were identified by electronic database searching and 20 further papers by manual searching .", "eighty - five papers were excluded for being duplicate publications , being an irrelevant title or abstract , or being a relevant title and abstract but not published in the english language .", "after retrieval of the full - text articles , eleven papers were excluded for not having a large enough study population or for being reviews .", "thirteen further studies were screened out ( involving 866 cases and 757 controls ) based on the inclusion and exclusion criteria in the pooled analysis .", "the characteristics of the studies are shown in table 1.2436 six papers were used to study the correlation between cdh1 methylation and sex status .", "five papers provided smoking history data , allowing an investigation of the influence of smoking on cdh1 methylation .", "three studies investigated the effect of clinical stage , ie , stage i , ii , iii , or iv . one paper discussed lymph node metastasis and another discussed degree of differentiation . analyzing tissue samples from 657 patients and 593 controls , the mean frequency of cdh1 methylation was 32% ( range 8.33% to 66.32% ) in tumor tissue and 9% ( range 0.00%27.78% ) in tissue from controls .", "this result indicates that the occurrence of cdh1 methylation is higher in tumor tissue than in normal tissue . using the fixed model , meta - analysis showed that 657 cases and 593 controls from 12 studies were pooled or as shown in figure 2 ( or 3.89 , 95% ci 2.875.27 , p<0.00001 ) .", "these findings indicate that cdh1 methylation is a key molecular event in tumor tissue but not in normal tissue .", "the results also show heterogeneity across the included studies ( i \n 2 is 61% , ie , more than 50% ) . given this significant heterogeneity , a subgroup analysis was performed to investigate sex status , smoking history , pathological type , clinical stage , differentiation degree , and lymph node metastasis to observe the relationship between cdh1 methylation and clinical characteristics ( see figure 3 ) .", "however , there was no correlation between cdh1 promoter methylation and any of these factors ( sex status , or 0.78 , 95% ci 0.411.50 , p=0.46 ; smoking history , or 0.97 , 95% ci 0.531.79 , p=0.93 ; pathological type , or 0.97 , 95% ci 0.591.60 , p=0.91 ; clinical stage , or 1.48 , 95% ci 0.812.68 , p=0.2 ; lymph node metastasis , or 0.68 , 95% ci 0.133.63 , p=0.65 ; and differentiation degree , or 1.01 , 95% ci 0.343.02 , p=0.99 ) .", "the or ranged from 0.78 to 3.89 , which was not a significant change , suggesting that the results of our meta - analysis were not significantly unstable .", "the funnel plot shown in figure 4 is partially symmetric , indicating low publication bias regarding cdh1 methylation in lung cancer .", "hypermethylation of tumor suppressor genes and hypomethylation of oncogenes are two essential components of the molecular mechanism involved in the epigenomic regulation of initiation and progression of cancer . as a tumor suppressor gene ,", "cdh1 maintains cell - cell adhesion and keeps epithelial cells arranged in normal arrangement and layer . in vitro studies", "demonstrate that loss of expression or function of cdh1 can activate transcription factors associated with epithelial - mesenchymal transition , leading to metastasis of cancer cells.37 \n cdh1 methylation has been detected in several types of carcinoma , including breast cancer , gastric cancer , and lung cancer.3840 a comprehensive evaluation of markers of methylation in lung cancer is needed to better understand the relationship between cdh1 methylation and lung cancer .", "although a large number of studies have demonstrated a possible relationship between cdh1 methylation and lung cancer , a meta - analysis can summarize the relevant studies and compare different subgroup characteristics . in this meta - analysis", "we analyzed data from 13 studies that included 657 tumor tissue samples and 593 control samples .", "the results show that the cdh1 methylation level in the cancer group was significantly higher than in the control group . the pooled or using the fixed - effect model was 3.89 ( 95% ci 2.875.27 versus the control group ) .", "cdh1 methylation plays a key role in the induction of lung cancer due to silencing of the tumor suppressor gene cdh1 .", "this conclusion is consistent with that of a previous study.36 since changes in cdh1 promoter hypermethylation are reversible , drug treatment promoting demethylation may be useful for delaying carcinogenesis and progression . treatment with 5-aza-2-deoxycytidine showed that migration of a549 cells decreased markedly upon restoration of cdh1.41 these preclinical studies show the therapeutic potential of restoration of tumor suppressor expression via epigenetic modulation .", "we further determined the clinicopathological significance of cdh1 promoter hypermethylation in patients with lung cancer . for smoking history ,", "the summary or was 0.97 ( 95% ci 0.531.79 ) in the 65 cases and 27 controls .", "smoking may target other specific genes for methylation or mutation , for example , methylenetetrahydrofolate reductase.33 other subgroup meta - analysis was performed , including for sex status ( or 0.78 , 95% ci 0.411.50 ) , pathological type ( or 0.97 , 95% ci 0.591.60 ) , clinical stage ( or 1.48 , 95% ci 0.812.68 ) , lymph node metastasis ( or 0.68 , 95% ci 0.133.63 ) , and degree of differentiation ( or 1.01 , 95% ci 0.343.02 ) .", "cdh1 methylation determined by clinical staging shows a slightly more significant association than the other subgroups .", "interestingly , cdh1 methylation was detected much more frequently in stages iii and iv than in stages i and ii .", "however , cdh1 methylation itself does not correlate with pathological type , sex status , lymph node metastasis , or degree of differentiation .", "possible reasons for this finding might be the widely heterogeneous results for the subgroups or the lack of cases and controls in the subgroups .", "other potentially significant factors may need to be investigated , such as patient age , tumor size , and biopsy sample control.42", "cdh1 methylation might be a biomarker of lung cancer , with potential value in predicting the prognosis of the disease , and warrants further studies involving more clinical cases for meta - analysis in the future .", "in addition , the potential variables on cdh1 methylation from different group database are still not clear due to the limitation of the statistical power of meta - analysis ."], "abstract_text": ["<S> backgroundcdh1 is a protein encoded by the cdh1 gene in humans . </S>", "<S> mutations in this gene are linked with several types of cancer . </S>", "<S> loss of cdh1 function contributes to the progression of cancer by increasing proliferation , invasion , and/or metastasis . </S>", "<S> however , the association between and clinicopathological significance of cdh1 promoter methylation and lung cancer remains unclear . in this study </S>", "<S> , we systematically reviewed the studies of cdh1 promoter methylation and lung cancer , and evaluated the association between cdh1 promoter methylation and lung cancer using meta - analysis methods.methodsa comprehensive search of the pubmed and embase databases was performed up to july 2014 . </S>", "<S> the methodological quality of the studies was also evaluated . </S>", "<S> the data were extracted and assessed by two reviewers independently . </S>", "<S> analyses of pooled data were performed . </S>", "<S> odds ratios ( ors ) were calculated and summarized.resultsfinally , an analysis of 866 patients with non - small cell lung cancer from 13 eligible studies was performed . </S>", "<S> the cdh1 methylation level in the cancer group was significantly higher than in the controls ( or 3.89 , 95% confidence interval [ ci ] 2.875.27 , p<0.00001 ) . </S>", "<S> however , there were no correlations between cdh1 promoter methylation and clinicopathological characteristics ( sex status , or 0.78 , 95% ci 0.411.50 , p=0.46 ; smoking history , or 0.97 , 95% ci 0.531.79 , p=0.93 ; pathological type , or 0.97 , 95% ci 0.591.60 , p=0.91 ; clinical staging , or 1.48 , 95% ci 0.812.68 , p=0.2 ; lymph node metastasis , or 0.68 , 95% ci 0.133.63 , p=0.65 ; or differentiation degree , or 1.01 , 95% ci 0.343.02 , p=0.99).conclusionthe results of this meta - analysis suggest that cdh1 methylation is associated with an increased risk of lung cancer . </S>", "<S> cdh1 hypermethylation , which induces inactivation of the cdh1 gene , plays an important role in carcinogenesis and may serve as a potential drug target in lung cancer . </S>", "<S> however , cdh1 methylation does not correlate with other factors , such as smoking history , clinical stage , pathological type , sex status , lymph node metastasis , or degree of differentiation . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Publication selection", "Inclusion and exclusion criteria", "Data extraction and quality assessment", "Data analysis", "Results", "Discussion", "Conclusion"], "sections": [["lung cancer is a leading cause of cancer mortality in the usa and other developed countries.1 the two major forms of the disease are non - small cell lung cancer and small cell lung cancer , which account for 85% and 15% of all lung cancers , respectively.2 despite advances in preliminary detection and standard treatment with combination chemotherapy and radiotherapy , the prognosis for patients with lung cancer has not improved significantly in the last 20 years,3,4 and survival rates have changed little over the past 2 decades .", "currently , an enormous amount of research is aimed at understanding the molecular and cellular biology of lung cancer ; however , much more work is needed to better understand the correlation between gene regulation and lung cancer .", "growing evidence shows that aberrant hypermethylation in 5-cpg islands in the promoter regions is a major mechanism for silencing tumor suppressor or other cancer - associated genes in many kinds of human cancer.58 loss of function in cancer suppressor genes may hinder inhibition of growth of cancer cells , which leads to malignant transcription and translation during replication of dna . a number of genes , including the cyclin - dependent kinase inhibitor ( p16 ) , the tumor suppressor gene ras association domain family protein 1a , kelch - like ech - associating protein 1 , the dna repair gene mgmt , and the cystic fibrosis transmembrane conductance regulator are demonstratively methylated in lung cancer.912 cdh1", ", a member of the transmembrane glycoprotein family , also known as cadherin-1 , cam 120/80 , epithelial cadherin ( e - cadherin ) , or uvomorulin , is encoded by the cdh1 gene ( 16q22.1).13 cdh1 is a calcium - dependent cell - cell adhesion glycoprotein containing three domains , ie , five extracellular cadherin repeats , a transmembrane region , and a highly conserved cytoplasmic tail.14 \n cdh1 as a tumor suppressor gene plays an essential role in maintaining cell adhesion and adherent junctions in normal tissues .", "cdh1 expression is frequently absent in a variety of epithelial tumors , and loss of normal intercellular junctions results in promotion of cancer invasion and metastasis and is correlated with several types of cancers.1517 however , the association between and clinicopathological significance of cdh1 promoter hypermethylation and lung cancer remains unclear . in this study , we systematically investigate studies of cdh1 promoter hypermethylation and lung cancer , and validate the association between cdh1 promoter hypermethylation and lung cancer using meta - analysis methods .", "in addition , we summarize these findings and discuss tumor suppressor function , as well as the clinicopathological significance of cdh1 in lung cancer ."], ["a systematic literature searching was performed using pubmed , embase , and the web of science up to august 13 , 2014 without any language restrictions .", "the following keywords and terms were used : [ methylation or dna methylation or hypermethylation or de - methylation ] and [ cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin ( e - cadherin ) or uvomorulin ] and [ lung cancer or lung carcinoma or lung tumor ] .", "the published scientific articles were restricted to english language , and conference abstracts were excluded due to lack of sufficient data .", "titles , abstracts , and key words in the articles were initially evaluated for inclusion criteria .", "details and additional information were identified and collected from the full text of these articles .", "a study included for meta - analysis needed to have : evaluated the correlation between cdh1 methylation and lung cancer ; included a clinical cohort and controls ; included at least three patients and controls ; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression ; and used tissue data rather than blood data .", "studies that did not meet our inclusion criteria were excluded . when the same groups of patients were reported in multiple papers ,", "two researchers independently collected the information and extracted the data regarding authorship , year , source of publication , inclusion criteria , cdh1 methylation frequency , sex status , smoking history , pathological type , clinical staging , degree of differentiation , lymph node metastasis , epidermal growth factor receptor status , and prognosis in patients and controls .", "methodological evaluation was assessed by the researchers according to the remark ( reporting recommendations for tumor marker prognostic studies ) guidelines and elcwp ( european lung cancer working party ) score.18,19 the meta - analysis was performed using reviewer manager 5 ( cochrane collaboration , oxford , uk ) . pooled odds ratios ( ors ) and confidence intervals ( cis ) were calculated to assess the correlation between cdh1 methylation and lung cancer .", "cochran s q test and i \n 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i \n 2 test > 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters .", "otherwise , a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 ( statacorp , college station , tx , usa ) ."], ["a systematic literature searching was performed using pubmed , embase , and the web of science up to august 13 , 2014 without any language restrictions .", "the following keywords and terms were used : [ methylation or dna methylation or hypermethylation or de - methylation ] and [ cdh1 or cadherin-1 or cam 120/80 or epithelial cadherin ( e - cadherin ) or uvomorulin ] and [ lung cancer or lung carcinoma or lung tumor ] .", "the published scientific articles were restricted to english language , and conference abstracts were excluded due to lack of sufficient data .", "titles , abstracts , and key words in the articles were initially evaluated for inclusion criteria .", "details and additional information were identified and collected from the full text of these articles ."], ["a study included for meta - analysis needed to have : evaluated the correlation between cdh1 methylation and lung cancer ; included a clinical cohort and controls ; included at least three patients and controls ; used methylation - specific polymerase chain reaction or quantitative methylation - specific polymerase chain reaction to examine cdh1 methylation and expression ; and used tissue data rather than blood data .", "studies that did not meet our inclusion criteria were excluded . when the same groups of patients were reported in multiple papers , only the most recent and complete paper was selected to avoid overlap ."], ["two researchers independently collected the information and extracted the data regarding authorship , year , source of publication , inclusion criteria , cdh1 methylation frequency , sex status , smoking history , pathological type , clinical staging , degree of differentiation , lymph node metastasis , epidermal growth factor receptor status , and prognosis in patients and controls .", "methodological evaluation was assessed by the researchers according to the remark ( reporting recommendations for tumor marker prognostic studies ) guidelines and elcwp ( european lung cancer working party ) score.18,19"], ["the meta - analysis was performed using reviewer manager 5 ( cochrane collaboration , oxford , uk ) .", "pooled odds ratios ( ors ) and confidence intervals ( cis ) were calculated to assess the correlation between cdh1 methylation and lung cancer .", "cochran s q test and i \n 2 were used to assess heterogeneity among the studies.20 a q test showing p<0.05 or an i \n 2 test > 50% indicated significant heterogeneity and a fixed - effects model was used to calculate the parameters .", "otherwise , a random - effects model was used to pool data and attempt to identify potential sources of heterogeneity based on subgroup analyses.21,22 publication bias was assessed by begg s test and use of funnel plots.23 the analysis of meta - regression and publication bias was performed using stata version 10.0 ( statacorp , college station , tx , usa ) ."], ["the process followed to select the papers used in this report is shown in figure 1 .", "ninety papers were identified by electronic database searching and 20 further papers by manual searching .", "eighty - five papers were excluded for being duplicate publications , being an irrelevant title or abstract , or being a relevant title and abstract but not published in the english language .", "after retrieval of the full - text articles , eleven papers were excluded for not having a large enough study population or for being reviews .", "thirteen further studies were screened out ( involving 866 cases and 757 controls ) based on the inclusion and exclusion criteria in the pooled analysis .", "the characteristics of the studies are shown in table 1.2436 six papers were used to study the correlation between cdh1 methylation and sex status .", "five papers provided smoking history data , allowing an investigation of the influence of smoking on cdh1 methylation .", "three studies investigated the effect of clinical stage , ie , stage i , ii , iii , or iv . one paper discussed lymph node metastasis and another discussed degree of differentiation . analyzing tissue samples from 657 patients and 593 controls , the mean frequency of cdh1 methylation was 32% ( range 8.33% to 66.32% ) in tumor tissue and 9% ( range 0.00%27.78% ) in tissue from controls .", "this result indicates that the occurrence of cdh1 methylation is higher in tumor tissue than in normal tissue . using the fixed model , meta - analysis showed that 657 cases and 593 controls from 12 studies were pooled or as shown in figure 2 ( or 3.89 , 95% ci 2.875.27 , p<0.00001 ) .", "these findings indicate that cdh1 methylation is a key molecular event in tumor tissue but not in normal tissue .", "the results also show heterogeneity across the included studies ( i \n 2 is 61% , ie , more than 50% ) . given this significant heterogeneity , a subgroup analysis was performed to investigate sex status , smoking history , pathological type , clinical stage , differentiation degree , and lymph node metastasis to observe the relationship between cdh1 methylation and clinical characteristics ( see figure 3 ) .", "however , there was no correlation between cdh1 promoter methylation and any of these factors ( sex status , or 0.78 , 95% ci 0.411.50 , p=0.46 ; smoking history , or 0.97 , 95% ci 0.531.79 , p=0.93 ; pathological type , or 0.97 , 95% ci 0.591.60 , p=0.91 ; clinical stage , or 1.48 , 95% ci 0.812.68 , p=0.2 ; lymph node metastasis , or 0.68 , 95% ci 0.133.63 , p=0.65 ; and differentiation degree , or 1.01 , 95% ci 0.343.02 , p=0.99 ) .", "the or ranged from 0.78 to 3.89 , which was not a significant change , suggesting that the results of our meta - analysis were not significantly unstable .", "the funnel plot shown in figure 4 is partially symmetric , indicating low publication bias regarding cdh1 methylation in lung cancer ."], ["hypermethylation of tumor suppressor genes and hypomethylation of oncogenes are two essential components of the molecular mechanism involved in the epigenomic regulation of initiation and progression of cancer . as a tumor suppressor gene ,", "cdh1 maintains cell - cell adhesion and keeps epithelial cells arranged in normal arrangement and layer . in vitro studies", "demonstrate that loss of expression or function of cdh1 can activate transcription factors associated with epithelial - mesenchymal transition , leading to metastasis of cancer cells.37 \n cdh1 methylation has been detected in several types of carcinoma , including breast cancer , gastric cancer , and lung cancer.3840 a comprehensive evaluation of markers of methylation in lung cancer is needed to better understand the relationship between cdh1 methylation and lung cancer .", "although a large number of studies have demonstrated a possible relationship between cdh1 methylation and lung cancer , a meta - analysis can summarize the relevant studies and compare different subgroup characteristics . in this meta - analysis", "we analyzed data from 13 studies that included 657 tumor tissue samples and 593 control samples .", "the results show that the cdh1 methylation level in the cancer group was significantly higher than in the control group . the pooled or using the fixed - effect model was 3.89 ( 95% ci 2.875.27 versus the control group ) .", "cdh1 methylation plays a key role in the induction of lung cancer due to silencing of the tumor suppressor gene cdh1 .", "this conclusion is consistent with that of a previous study.36 since changes in cdh1 promoter hypermethylation are reversible , drug treatment promoting demethylation may be useful for delaying carcinogenesis and progression . treatment with 5-aza-2-deoxycytidine showed that migration of a549 cells decreased markedly upon restoration of cdh1.41 these preclinical studies show the therapeutic potential of restoration of tumor suppressor expression via epigenetic modulation .", "we further determined the clinicopathological significance of cdh1 promoter hypermethylation in patients with lung cancer . for smoking history ,", "the summary or was 0.97 ( 95% ci 0.531.79 ) in the 65 cases and 27 controls .", "smoking may target other specific genes for methylation or mutation , for example , methylenetetrahydrofolate reductase.33 other subgroup meta - analysis was performed , including for sex status ( or 0.78 , 95% ci 0.411.50 ) , pathological type ( or 0.97 , 95% ci 0.591.60 ) , clinical stage ( or 1.48 , 95% ci 0.812.68 ) , lymph node metastasis ( or 0.68 , 95% ci 0.133.63 ) , and degree of differentiation ( or 1.01 , 95% ci 0.343.02 ) .", "cdh1 methylation determined by clinical staging shows a slightly more significant association than the other subgroups .", "interestingly , cdh1 methylation was detected much more frequently in stages iii and iv than in stages i and ii .", "however , cdh1 methylation itself does not correlate with pathological type , sex status , lymph node metastasis , or degree of differentiation .", "possible reasons for this finding might be the widely heterogeneous results for the subgroups or the lack of cases and controls in the subgroups .", "other potentially significant factors may need to be investigated , such as patient age , tumor size , and biopsy sample control.42"], ["cdh1 methylation might be a biomarker of lung cancer , with potential value in predicting the prognosis of the disease , and warrants further studies involving more clinical cases for meta - analysis in the future .", "in addition , the potential variables on cdh1 methylation from different group database are still not clear due to the limitation of the statistical power of meta - analysis ."]]}
{"article_id": "PMC2780662", "article_text": ["nerve conduction studies ( ncs ) are an objective , quantitative , and reproducible measure of peripheral nerve function and are widely used in the diagnosis of neuropathies [ 1 , 2 ] .", "they can be used to monitor neuropathic disease progression   and the efficacy of interventions in clinical trials . however , non - uniform electrophysiologic test procedures degrade reproducibility .", "potential sources of variation include the use of different emg instruments at different test sessions or sites , inconsistent placement of recording and stimulating electrodes , use of non - standardized distance measurements , use of sub - maximal electrical stimuli , poor skin preparation resulting in high skin impedance , and failure to maintain limb temperature within an acceptable range or to compensate for temperature .", "all these factors may compromise the repeatability of ncs measurement and lead to erroneous diagnostic conclusions . aside from true physiological changes , factors that influence repeatability of ncs measurements", "are broadly grouped into two categories : inter - tester variability and intra - tester variability .", "inter - tester variability refers to variability of a test parameter measured on a single individual when repeat test measurements are made by two or more examiners .", "intra - tester variability refers to variability of a test parameter when repeat test measurements are made by a single examiner . in the absence of physiological changes ,", "both inter - tester and intra - tester variability are influenced by electrodiagnostic examination technique .", "automated ncs instruments may improve ncs repeatability by utilizing prefabricated electrode arrays and automating evoked waveform analysis .", "the objective of this study is to quantify ncs repeatability utilizing the nc - stat ( neurometrix , inc . , waltham , massachusetts ) , an automated ncs system [ 810 ] .", "we hypothesized that use of the nc - stat system would yield highly reproducible ncs results .", "all were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy .", "the study was approved and monitored by an independent review board ( copernicus group , cary , nc , usa ) .", "each upper and lower extremity nerve was tested twice ( 7 days apart ) by the same technician , utilizing the nc - stat ( neurometrix , inc . ,", "waltham , ma , usa ) , an automated ncs instrument . shown in figure 1 is a photograph of the nc - stat system components : pre - fabricated electrode arrays specific to peroneal nerve motor testing ; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis ; a communication port to transmit data for report generation .", "the nc - stat system is fda 510(k ) cleared for the performance of motor studies of the median , ulnar , peroneal and tibial nerves , and sensory studies of the median , ulnar and sural nerves .", "tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used .", "each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . a technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks .", "the electrode arrays incorporated stimulating , recording , and ground electrodes , as well as a temperature sensor .", "the device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . the evoked compound muscle ( cmap ) or compound sensory nerve ( snap ) action potentials were recorded following a series of supramaximal stimuli .", "supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities ( step size varies between 2.5 and 20 ma depending on nerve and stimulus duration ) .", "snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count .", "time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . \n", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "the median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . a volume - conducted median motor response generated by abductor pollicis brevis", "was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease .", "concurrently , an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal ( pip ) joint ( active electrode ) , with the inactive electrode 3 cm distal .", "the ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease .", "the ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist .", "the ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal .", "the stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line .", "responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe , over the vicinity of the extensor digitorum brevis muscle ( see figure 1 ) . for tibial nerve testing , stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus .", "a ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases . at the conclusion of ncs for a given subject ,", "motor responses ( cmap and f - wave ) were recorded with filter settings of 15 hz high pass and 3 khz low pass ; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters .", "supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz .", "for recording snaps , 615 individual waveforms were averaged depending upon the signal - to - noise ratio . up to 10 f - wave responses ( with 12 traces as a maximum )", "up to 20 peroneal and tibial f - wave responses ( with 40 traces as a maximum for personal , and 24 traces as a maximum for tibial ) were acquired for lower extremity tests . \n", "2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set", ". sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set .", "all ncs parameters were determined by automated computer algorithms [ 7 , 9 , 10 ] . for motor studies ,", "distal motor latency ( dml ) was the time difference between stimulus onset and initial negative deflection ( marked as  +  in the lower left panel of figure 2 ) .", "dml values from four cmap waveforms were averaged and reported ( after temperature correction ) .", "cmap amplitude was measured baseline to negative peak ( upward deflection , identified by the upper triangle ) based on the averaged cmap .", "f - wave onset latency was identified for each trace with an identified f - wave response ( traces with ", "+  , right panel of figure 2 ) , and their average was reported as the mean f - wave latency ( vertical dotted line ) .", "the distal sensory latency ( dsl ) was measured from stimulus onset to the initial negative peak ( upward deflection ) of the snap ( open circle in the upper left panel of figure 2 ) .", "the snap amplitude was measured peak to peak ( negative - positive , or vertical distance between open and closed circles ) .", "all motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values ( 32 c upper extremity , 30 c lower extremity ) with a linear correction formula : latency(corrected ) = latency ( raw))corrcoef(temperature - reference ) .", "the temperature correction factor corrcoef was previously determined in an independent study population ( 150 subjects , data on file ) , which also found dependence of cmap and snap amplitude on temperature to be not statistically significant .", "statistical measures used to quantify ncs repeatability mirrored those used in prior studies [ 2 , 4 , 5 ] .", "the pearson product - moment correlation ( cc ) was used to assess the association between ncs parameters obtained 7 days apart .", "intraclass correlation coefficient ( icc ) was used to determine the agreement between the two tests .", "coefficient of variation ( cov ) of test - retest ncs parameters was calculated and the average over all nerves was reported . relative intertrial variation ( riv )", "the riv is the difference between two tests as a percentage of the average of the two tests . because of the small sample size", ", 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . to facilitate comparison , the 5th and 95th percentile values , as reported by others ,", "were also obtained for selected parameters . the mean and standard deviation of the difference between the test and retest results", "paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean .", "all were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy .", "the study was approved and monitored by an independent review board ( copernicus group , cary , nc , usa ) .", "each upper and lower extremity nerve was tested twice ( 7 days apart ) by the same technician , utilizing the nc - stat ( neurometrix , inc . ,", "waltham , ma , usa ) , an automated ncs instrument . shown in figure 1 is a photograph of the nc - stat system components : pre - fabricated electrode arrays specific to peroneal nerve motor testing ; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis ; a communication port to transmit data for report generation .", "the nc - stat system is fda 510(k ) cleared for the performance of motor studies of the median , ulnar , peroneal and tibial nerves , and sensory studies of the median , ulnar and sural nerves .", "tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used .", "each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . a technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks .", "the electrode arrays incorporated stimulating , recording , and ground electrodes , as well as a temperature sensor .", "the device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . the evoked compound muscle ( cmap ) or compound sensory nerve ( snap ) action potentials were recorded following a series of supramaximal stimuli .", "supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities ( step size varies between 2.5 and 20 ma depending on nerve and stimulus duration ) .", "snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count .", "time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . \n", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "the median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . a volume - conducted median motor response generated by abductor pollicis brevis", "was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease .", "concurrently , an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal ( pip ) joint ( active electrode ) , with the inactive electrode 3 cm distal .", "the ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease .", "the ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist .", "the ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal .", "the stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line .", "responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe , over the vicinity of the extensor digitorum brevis muscle ( see figure 1 ) . for tibial nerve testing , stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus . both the active and inactive electrodes were located just distal to the medial malleolus .", "a ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases .", "at the conclusion of ncs for a given subject , test data were uploaded to a central database .", "motor responses ( cmap and f - wave ) were recorded with filter settings of 15 hz high pass and 3 khz low pass ; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters .", "supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz .", "615 individual waveforms were averaged depending upon the signal - to - noise ratio . up", "to 10 f - wave responses ( with 12 traces as a maximum ) were recorded for the median and ulnar nerves .", "up to 20 peroneal and tibial f - wave responses ( with 40 traces as a maximum for personal , and 24 traces as a maximum for tibial ) were acquired for lower extremity tests . \n", "2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set .", "sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set .", "all ncs parameters were determined by automated computer algorithms [ 7 , 9 , 10 ] . for motor studies , distal motor latency ( dml ) was the time difference between stimulus onset and initial negative deflection ( marked as  +  in the lower left panel of figure 2 ) .", "dml values from four cmap waveforms were averaged and reported ( after temperature correction ) .", "cmap amplitude was measured baseline to negative peak ( upward deflection , identified by the upper triangle ) based on the averaged cmap .", "f - wave onset latency was identified for each trace with an identified f - wave response ( traces with ", "+  , right panel of figure 2 ) , and their average was reported as the mean f - wave latency ( vertical dotted line ) .", "the distal sensory latency ( dsl ) was measured from stimulus onset to the initial negative peak ( upward deflection ) of the snap ( open circle in the upper left panel of figure 2 ) .", "the snap amplitude was measured peak to peak ( negative - positive , or vertical distance between open and closed circles ) .", "all motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values ( 32 c upper extremity , 30 c lower extremity ) with a linear correction formula : latency(corrected ) = latency ( raw))corrcoef(temperature - reference ) .", "the temperature correction factor corrcoef was previously determined in an independent study population ( 150 subjects , data on file ) , which also found dependence of cmap and snap amplitude on temperature to be not statistically significant .", "statistical measures used to quantify ncs repeatability mirrored those used in prior studies [ 2 , 4 , 5 ] . the pearson product - moment correlation ( cc )", "intraclass correlation coefficient ( icc ) was used to determine the agreement between the two tests .", "coefficient of variation ( cov ) of test - retest ncs parameters was calculated and the average over all nerves was reported . relative intertrial variation ( riv )", "the riv is the difference between two tests as a percentage of the average of the two tests . because of the small sample size", ", 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . to facilitate comparison , the 5th and 95th percentile values , as reported by others ,", "the mean and standard deviation of the difference between the test and retest results were also reported .", "paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean .", "their ages ranged from 24 to 52 years ( mean 37.1 , sd 8.6 years ) .", "height varied from 157 to 180 cm ( mean 173 , sd 7.9 cm ) .", "total of 15 subjects ( 5 females ) volunteered for the lower extremity repeatability study and nine of them had also enrolled in the upper extremity study .", "their age range was 2247 years ( mean 32.6 , sd 8.2 years ) and height range was 157180 cm ( mean 172 , sd 8.2 cm ) .", "the rivs for mean fwl at 5th and 95th percentiles were [ 11.4% , 9.4% ] , [ 5.1% , 3.3% ] , [ 4.9% , 4.5% ] , and [ 2.8% , 4.0% ] , respectively , for median , ulnar , peroneal , and tibial nerves . at p", "= 0.05 level , paired t - tests indicated that the means of all test and retest ncs parameters were the same .", "pearson ccs for all latency parameters were greater than 0.80 with the exception of median nerve mean fwl ( cc = 0.69 ) .", "all upper extremity amplitude parameters had ccs greater than 0.85 . for lower extremity nerves ,", "all latency results were based on temperature corrected latencies . without temperature compensation , latency parameters exhibited lower repeatability .", "for example , the cc values for ulnar nerve dml , mean fwl , and dsl without temperature correction were 0.72 , 0.93 , and 0.72 , respectively .", "standard deviation of the difference between test and retest results would have been 0.31 , 1.04 , and 0.34 .", "table 1statistical measures of nerve conduction study parameter repeatabilityriv percentiles(day 8day 1)nerveccicccov10th ( % ) 90th ( % ) meanstdevpaired t - testdistal motor latency ( dml)median0.8720.8700.0329.55.10.0550.1940.289ulnar0.8890.8320.0458.311.00.0260.1900.605peroneal0.8640.8450.06115.48.10.1240.4530.307tibial0.8500.8240.0386.88.80.0160.2390.799mean f - wave latency ( fwl)median0.6920.6790.0316.64.40.0371.9700.943ulnar0.9490.9210.0173.91.10.2860.9150.246peroneal0.8960.9010.0174.73.40.1631.5630.693tibial0.9430.9400.0131.83.50.2781.1450.363distal sensory latency ( dsl)median0.8870.8790.0287.24.00.0560.1890.270ulnar0.8150.7970.0375.79.00.0120.2070.825compound muscle action potential ( cmap ) amplitudemedian0.8790.8620.10919.219.90.0310.1980.550ulnar0.9810.9740.0455.710.70.2880.6090.088peroneal0.3920.3310.29830.899.50.8491.9370.112tibial0.7140.7280.14830.925.40.1651.6010.695sensory nerve action potential ( snap ) amplitudemedian0.9540.9540.05811.87.80.6738.4020.761ulnar0.9900.9820.0424.410.91.4855.0630.275cc : pearson product moment correlation to assess the association between test - retest results .", "riv : relative interval variation defines the difference between test - retest as a percentage of average . paired t - test results indicate the means of test and retest are not statistically different .", "dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference .", "dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference .", "fwl is the arithmetic mean of individual f - wave latencies in an f - wave set .", "statistical measures of nerve conduction study parameter repeatability cc : pearson product moment correlation to assess the association between test - retest results .", "riv : relative interval variation defines the difference between test - retest as a percentage of average . paired", "t - test results indicate the means of test and retest are not statistically different .", "dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference .", "dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference .", "fwl is the arithmetic mean of individual f - wave latencies in an f - wave set .", "several studies ( summarized in table 2 ) have examined ncs parameter repeatability [ 2 , 4 , 5 ] . in , the repeatability of ncs was evaluated for 60 sites participating in a clinical trial , with ncs oversight performed by  an experienced , insightful and knowledgeable core lab  .", "our study yielded similar results , though our study demonstrated a higher cov for peroneal cmap amplitude ( 9% vs. 29.8% ) . in", ", 132 healthy subjects were retested at a time interval of 14 weeks and icc and riv were used to measure the test - retest repeatability . in comparison with that study ,", "the riv ( 5th and 95th percentile ) for tibial nerve f - wave latency was tighter in our study ( [ 2.8% , 4.0% ] vs. [ 4.6% , 5.7% ] ) while the relationship was reversed for median nerve fwave latency results ( [ 11.4% , 9.4% ] vs. [ 6.7% , 6.7% ] ) .", "decreased repeatability of median nerve f - wave latency may be attributed to the lower amplitude signal - to - noise ratio as median nerve f - waves were acquired via a volume - conduction recording technique .", "indeed , latency repeatability was much higher for ulnar f - waves recorded directly over the muscle .", "the icc for ulnar f - wave latency was 0.92 in our study , higher than the icc of 0.59 reported in a similar study of 49 healthy adults .", "salerno and colleagues studied upper extremity sensory test - retest repeatability based on 158 active workers who were tested by a neurologist and a physiatrist .", "our results fell into the upper range of their repeatability results as measured by cc and icc . \n", "table 2repeatability results reported in the literaturesnerveccicccovdistal motor latency ( dml)median0.8260.04/0.08 > \n peroneal0.05/0.08tibial0.6170.08minimum f - wave latency ( fwl)ulnar0.59tibial0.921distal sensory latency ( dsl)median0.820.920.820.920.04ulnar0.370.640.320.63compound muscle action potential ( cmap ) amplitudemedian0.7770.07/0.11peroneal0.09tibial0.846sensory nerve action potential ( snap ) amplitudemedian0.830.880.834/0.810.880.08ulnar0.680.850.680.85 132 healthy subjects retested in 14 weeks .  ", "results for control subjects from 60 sites with core lab support in a clinical trial .  ", "33 subjects retested in 12 week , results for small electrode size ( 0.78 cm ) and all three nerves combined .  ", "repeatability results reported in the literatures   132 healthy subjects retested in 14 weeks .  ", "results for control subjects from 60 sites with core lab support in a clinical trial .  ", "33 subjects retested in 12 week , results for small electrode size ( 0.78 cm ) and all three nerves combined .  ", "it is possible that the small recording electrodes ( surface areas 2 cm ) used in our study degraded repeatability , since tjon - a - tsien and colleagues noted that when electrode size was changed from 0.78 to 7.65 cm , the cov of cmap amplitude improved from 11 to 7% ( lower cov is associated with higher repeatability ) .", "dml repeatability was less affected by electrode size in both our study and the study of .", "however , control of skin temperature is often difficult to achieve , especially in different testing environments . in the present study ,", "skin temperature adjacent to the recording site was acquired automatically using an embedded probe and latencies were adjusted to the reference temperature . as expected , temperature correction improved ncs repeatability .", "for example , the cc increased from 0.72 ( without temperature correction ) to 0.89 ( with correction ) for both motor and sensory latencies of the ulnar nerves .", "temperature correction also reduced the standard deviation of the test - retest latency difference by as much as 39% ( ulnar dml ) .", "the principal limitation of the current study was the small number of subjects evaluated relative to several prior investigations .", "the short test - retest interval of 1 week reduced the likelihood of intervening physiological changes .", "it would be valuable to quantify repeatability of the automated ncs instrument in neuropathic nerves . at the time of this study ,", "temperature correction may improve the repeatability of the amplitude parameters , especially for sensory responses .", "the reproducibility of individual ncs parameters followed the same pattern as those observed in traditional settings .", "f - wave latency had the greatest repeatability , followed by dml , dsl , and snap amplitude .", "the cmap amplitude was the least reproducible parameter , with peroneal cmap amplitude repeatability the lowest among all nerves . a larger recording electrode size should improve its repeatability .", "f - wave latency repeatability for median nerve was lower due to the lower signal - to - noise ratio inherent in volume - conduction recordings .", "as a result , over - muscle recording would be expected to enhance f - wave repeatability , which was the case for the ulnar nerve .", "further studies with longer test - retest intervals and a higher subject count would be valuable in confirming the high reproducibility of ncs parameters measured in this study . extending the study to pathologic nerves and validating the equivalent or superior performance of automated ncs instrument should increase the adoption rate of the automated ncs technology in patient management"], "abstract_text": ["<S> abstractobjectiveto quantify nerve conduction study ( ncs ) reproducibility utilizing an automated ncs system ( nc - stat , neurometrix , inc.).methodhealthy volunteers without neuropathic symptoms participated in the study . </S>", "<S> their median , ulnar , peroneal , and tibial nerves were tested twice ( 7  days apart ) by the same technician with an nc - stat instrument . </S>", "<S> pre - fabricated electrode arrays specific to each nerve were used . both motor responses </S>", "<S> ( compound motor action potential [ cmap ] and f - waves all nerves ) and sensory responses ( sensory nerve action potentials [ snap ] median and ulnar nerves only ) were recorded following supramaximal stimuli . </S>", "<S> automated algorithms determined all ncs parameters : distal motor latency ( dml ) , mean f - wave latency ( fwl ) , distal sensory latency ( dsl ) , cmap amplitude , and snap amplitude . </S>", "<S> latency was adjusted for skin temperature deviation from reference . </S>", "<S> pearson correlation coefficient ( cc ) , intraclass correlation coefficient ( icc ) , coefficient of variance ( cov ) , and relative intertrial variation ( riv ) were calculated.resultsfifteen subjects participated in either upper or lower extremity studies with nine participating in both . with the exception of cmap amplitude , all parameters had cov less than 0.06 . </S>", "<S> upper extremity amplitude parameters had ccs greater than 0.85 . </S>", "<S> ccs for latencies were greater than 0.80 except for the median nerve fwl ( cc  =  0.69 ) . </S>", "<S> for lower extremity nerves , iccs were highest for mean fwl ( > 0.90 ) , followed by dml ( > 0.82 ) and then cmap ( peroneal 0.33 , tibial 0.73 ) . </S>", "<S> the 10th to 90th riv percentiles were bounded by 7% for f - wave latencies ;  9% for all dsls ; and  11% for dml ( except peroneal at 15%).conclusionsthe reproducibility of ncs parameters obtained with an automated ncs instrument compared favorably with traditional electromyography laboratories . </S>", "<S> f - wave latencies had the highest repeatability , followed by dml , dsl , snap and cmap amplitude . given their high reproducibility , automated ncs instrument may encourage wider utilization of ncs in clinical and research applications . </S>"], "labels": null, "section_names": ["Introduction", "Methods and Materials", "Subjects", "Nerve conduction studies", "NCS Parameters", "Statistical analysis", "Results", "Discussion"], "sections": [["nerve conduction studies ( ncs ) are an objective , quantitative , and reproducible measure of peripheral nerve function and are widely used in the diagnosis of neuropathies [ 1 , 2 ] .", "they can be used to monitor neuropathic disease progression   and the efficacy of interventions in clinical trials . however , non - uniform electrophysiologic test procedures degrade reproducibility .", "potential sources of variation include the use of different emg instruments at different test sessions or sites , inconsistent placement of recording and stimulating electrodes , use of non - standardized distance measurements , use of sub - maximal electrical stimuli , poor skin preparation resulting in high skin impedance , and failure to maintain limb temperature within an acceptable range or to compensate for temperature .", "all these factors may compromise the repeatability of ncs measurement and lead to erroneous diagnostic conclusions . aside from true physiological changes , factors that influence repeatability of ncs measurements", "are broadly grouped into two categories : inter - tester variability and intra - tester variability .", "inter - tester variability refers to variability of a test parameter measured on a single individual when repeat test measurements are made by two or more examiners .", "intra - tester variability refers to variability of a test parameter when repeat test measurements are made by a single examiner . in the absence of physiological changes ,", "both inter - tester and intra - tester variability are influenced by electrodiagnostic examination technique .", "automated ncs instruments may improve ncs repeatability by utilizing prefabricated electrode arrays and automating evoked waveform analysis .", "the objective of this study is to quantify ncs repeatability utilizing the nc - stat ( neurometrix , inc . , waltham , massachusetts ) , an automated ncs system [ 810 ] .", "we hypothesized that use of the nc - stat system would yield highly reproducible ncs results ."], ["all were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy .", "the study was approved and monitored by an independent review board ( copernicus group , cary , nc , usa ) .", "each upper and lower extremity nerve was tested twice ( 7 days apart ) by the same technician , utilizing the nc - stat ( neurometrix , inc . ,", "waltham , ma , usa ) , an automated ncs instrument . shown in figure 1 is a photograph of the nc - stat system components : pre - fabricated electrode arrays specific to peroneal nerve motor testing ; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis ; a communication port to transmit data for report generation .", "the nc - stat system is fda 510(k ) cleared for the performance of motor studies of the median , ulnar , peroneal and tibial nerves , and sensory studies of the median , ulnar and sural nerves .", "tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used .", "each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . a technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks .", "the electrode arrays incorporated stimulating , recording , and ground electrodes , as well as a temperature sensor .", "the device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . the evoked compound muscle ( cmap ) or compound sensory nerve ( snap ) action potentials were recorded following a series of supramaximal stimuli .", "supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities ( step size varies between 2.5 and 20 ma depending on nerve and stimulus duration ) .", "snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count .", "time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . \n", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "the median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . a volume - conducted median motor response generated by abductor pollicis brevis", "was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease .", "concurrently , an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal ( pip ) joint ( active electrode ) , with the inactive electrode 3 cm distal .", "the ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease .", "the ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist .", "the ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal .", "the stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line .", "responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe , over the vicinity of the extensor digitorum brevis muscle ( see figure 1 ) . for tibial nerve testing , stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus .", "a ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases . at the conclusion of ncs for a given subject ,", "motor responses ( cmap and f - wave ) were recorded with filter settings of 15 hz high pass and 3 khz low pass ; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters .", "supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz .", "for recording snaps , 615 individual waveforms were averaged depending upon the signal - to - noise ratio . up to 10 f - wave responses ( with 12 traces as a maximum )", "up to 20 peroneal and tibial f - wave responses ( with 40 traces as a maximum for personal , and 24 traces as a maximum for tibial ) were acquired for lower extremity tests . \n", "2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set", ". sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set .", "all ncs parameters were determined by automated computer algorithms [ 7 , 9 , 10 ] . for motor studies ,", "distal motor latency ( dml ) was the time difference between stimulus onset and initial negative deflection ( marked as  +  in the lower left panel of figure 2 ) .", "dml values from four cmap waveforms were averaged and reported ( after temperature correction ) .", "cmap amplitude was measured baseline to negative peak ( upward deflection , identified by the upper triangle ) based on the averaged cmap .", "f - wave onset latency was identified for each trace with an identified f - wave response ( traces with ", "+  , right panel of figure 2 ) , and their average was reported as the mean f - wave latency ( vertical dotted line ) .", "the distal sensory latency ( dsl ) was measured from stimulus onset to the initial negative peak ( upward deflection ) of the snap ( open circle in the upper left panel of figure 2 ) .", "the snap amplitude was measured peak to peak ( negative - positive , or vertical distance between open and closed circles ) .", "all motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values ( 32 c upper extremity , 30 c lower extremity ) with a linear correction formula : latency(corrected ) = latency ( raw))corrcoef(temperature - reference ) .", "the temperature correction factor corrcoef was previously determined in an independent study population ( 150 subjects , data on file ) , which also found dependence of cmap and snap amplitude on temperature to be not statistically significant .", "statistical measures used to quantify ncs repeatability mirrored those used in prior studies [ 2 , 4 , 5 ] .", "the pearson product - moment correlation ( cc ) was used to assess the association between ncs parameters obtained 7 days apart .", "intraclass correlation coefficient ( icc ) was used to determine the agreement between the two tests .", "coefficient of variation ( cov ) of test - retest ncs parameters was calculated and the average over all nerves was reported . relative intertrial variation ( riv )", "the riv is the difference between two tests as a percentage of the average of the two tests . because of the small sample size", ", 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . to facilitate comparison , the 5th and 95th percentile values , as reported by others ,", "were also obtained for selected parameters . the mean and standard deviation of the difference between the test and retest results", "paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean ."], ["all were healthy office workers who lacked neurological complaints or known causes of peripheral neuropathy .", "the study was approved and monitored by an independent review board ( copernicus group , cary , nc , usa ) ."], ["each upper and lower extremity nerve was tested twice ( 7 days apart ) by the same technician , utilizing the nc - stat ( neurometrix , inc . ,", "waltham , ma , usa ) , an automated ncs instrument . shown in figure 1 is a photograph of the nc - stat system components : pre - fabricated electrode arrays specific to peroneal nerve motor testing ; an electronic monitor to be connected to the electrode arrays for nerve stimulation and waveform acquisition and analysis ; a communication port to transmit data for report generation .", "the nc - stat system is fda 510(k ) cleared for the performance of motor studies of the median , ulnar , peroneal and tibial nerves , and sensory studies of the median , ulnar and sural nerves .", "tests were performed in a commercial office setting similar to a physician s office where nc - stat systems are typically used .", "each study lasted for about 2 weeks to complete the 7-day interval test - retest protocol for 15 subjects . a technician applied pre - fabricated electrode arrays specific to each nerve based on readily identifiable anatomic landmarks .", "the electrode arrays incorporated stimulating , recording , and ground electrodes , as well as a temperature sensor .", "the device automatically checked skin impedance and determined the minimum stimulator current needed to deliver a supramaximal stimulus with amplitude ranging from 10 to 100 ma and duration between 100 and 500 s . the evoked compound muscle ( cmap ) or compound sensory nerve ( snap ) action potentials were recorded following a series of supramaximal stimuli .", "supramaximal is defined as cmap amplitudes having less than 10% variation from their mean for three stimuli of increasing intensities ( step size varies between 2.5 and 20 ma depending on nerve and stimulus duration ) .", "snaps are acquired at the motor supramaximal stimulation level since nc - stat recorded both motor and sensory responses simultaneously to minimize overall stimulus count .", "time interval between stimuli is about 23 s. the technician was trained according to the manufacturer s instructions and was blinded to the prior test results during the retest . \n", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "it consists of three components : pre - fabricated electrode arrays ( biosensor , left panel ) for a peroneal motor nerve conduction study , an electronic monitor ( connected to a biosensor during testing ) , a report generation system ( not shown ) based on electrophysiological data collected and processed by the monitor and transmitted via  docking station  ( a telecommunication port for secure data transfer via telephone line ) .", "the median stimulator cathode was placed over the midline volar wrist 3 cm proximal to the distal wrist crease . a volume - conducted median motor response generated by abductor pollicis brevis", "was recorded using paired electrodes placed over the lateral and medial aspects of the distal wrist crease .", "concurrently , an antidromic snap was recorded from the middle finger using self - adhering ring electrodes placed around the proximal interphalangeal ( pip ) joint ( active electrode ) , with the inactive electrode 3 cm distal .", "the ulnar stimulating cathode was placed over the medial volar wrist 3 cm proximal to the distal wrist crease .", "the ulnar motor response was recorded using an active electrode placed over abuductor digiti minimi and an inactive electrode placed over the lateral volar wrist .", "the ulnar snap was recorded from the small finger with the active electrode over the pip and inactive electrode 2 cm distal .", "the stimulator cathode for peroneal testing was placed lateral to the tibia at the intermalleolar line .", "responses were recorded using detector pairs placed along a line between the lateral malleolus and the 3rd toe , over the vicinity of the extensor digitorum brevis muscle ( see figure 1 ) . for tibial nerve testing , stimulating cathode was placed over the posterior tibial nerve just posterior to the medial malleolus . both the active and inactive electrodes were located just distal to the medial malleolus .", "a ground electrode and temperature sensor were interposed between stimulating and recording electrodes in all cases .", "at the conclusion of ncs for a given subject , test data were uploaded to a central database .", "motor responses ( cmap and f - wave ) were recorded with filter settings of 15 hz high pass and 3 khz low pass ; sensory responses were recorded with 175 hz high pass and 3 khz low pass filters .", "supramaximal cmap and snap were sampled at 10 khz and f - waves were acquired at 2.5 khz .", "615 individual waveforms were averaged depending upon the signal - to - noise ratio . up", "to 10 f - wave responses ( with 12 traces as a maximum ) were recorded for the median and ulnar nerves .", "up to 20 peroneal and tibial f - wave responses ( with 40 traces as a maximum for personal , and 24 traces as a maximum for tibial ) were acquired for lower extremity tests . \n", "2sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set .", "sample motor and sensory responses acquired by the nc - stat nerve conduction instrument during the repeatability study for one subject .", "top left panel shows the ulnar sensory nerve action potential ( snap ) with distal sensory latency ( dsl ) marked by an open circle .", "bottom left panel shows the ulnar compound muscle action potential ( cmap ) with distal motor latency ( dml ) marked with a plus and negative peak marked with a triangle for peak - to - base amplitude calculation .", "pluses indicate the nc - stat assigned f - wave latencies for individual traces with f - wave response .", "dotted line is the calculated mean f - wave latency for the f - wave set ."], ["all ncs parameters were determined by automated computer algorithms [ 7 , 9 , 10 ] . for motor studies , distal motor latency ( dml ) was the time difference between stimulus onset and initial negative deflection ( marked as  +  in the lower left panel of figure 2 ) .", "dml values from four cmap waveforms were averaged and reported ( after temperature correction ) .", "cmap amplitude was measured baseline to negative peak ( upward deflection , identified by the upper triangle ) based on the averaged cmap .", "f - wave onset latency was identified for each trace with an identified f - wave response ( traces with ", "+  , right panel of figure 2 ) , and their average was reported as the mean f - wave latency ( vertical dotted line ) .", "the distal sensory latency ( dsl ) was measured from stimulus onset to the initial negative peak ( upward deflection ) of the snap ( open circle in the upper left panel of figure 2 ) .", "the snap amplitude was measured peak to peak ( negative - positive , or vertical distance between open and closed circles ) .", "all motor and sensory latencies were adjusted for deviation of skin surface temperature from reference values ( 32 c upper extremity , 30 c lower extremity ) with a linear correction formula : latency(corrected ) = latency ( raw))corrcoef(temperature - reference ) .", "the temperature correction factor corrcoef was previously determined in an independent study population ( 150 subjects , data on file ) , which also found dependence of cmap and snap amplitude on temperature to be not statistically significant ."], ["statistical measures used to quantify ncs repeatability mirrored those used in prior studies [ 2 , 4 , 5 ] . the pearson product - moment correlation ( cc )", "intraclass correlation coefficient ( icc ) was used to determine the agreement between the two tests .", "coefficient of variation ( cov ) of test - retest ncs parameters was calculated and the average over all nerves was reported . relative intertrial variation ( riv )", "the riv is the difference between two tests as a percentage of the average of the two tests . because of the small sample size", ", 10th and 90th percentiles of riv were calculated for all parameters to minimize the impact of outliers . to facilitate comparison , the 5th and 95th percentile values , as reported by others ,", "the mean and standard deviation of the difference between the test and retest results were also reported .", "paired t - tests were carried out to ensure that two tests yielded ncs parameters with the same mean ."], ["their ages ranged from 24 to 52 years ( mean 37.1 , sd 8.6 years ) .", "height varied from 157 to 180 cm ( mean 173 , sd 7.9 cm ) .", "total of 15 subjects ( 5 females ) volunteered for the lower extremity repeatability study and nine of them had also enrolled in the upper extremity study .", "their age range was 2247 years ( mean 32.6 , sd 8.2 years ) and height range was 157180 cm ( mean 172 , sd 8.2 cm ) .", "the rivs for mean fwl at 5th and 95th percentiles were [ 11.4% , 9.4% ] , [ 5.1% , 3.3% ] , [ 4.9% , 4.5% ] , and [ 2.8% , 4.0% ] , respectively , for median , ulnar , peroneal , and tibial nerves . at p", "= 0.05 level , paired t - tests indicated that the means of all test and retest ncs parameters were the same .", "pearson ccs for all latency parameters were greater than 0.80 with the exception of median nerve mean fwl ( cc = 0.69 ) .", "all upper extremity amplitude parameters had ccs greater than 0.85 . for lower extremity nerves ,", "all latency results were based on temperature corrected latencies . without temperature compensation , latency parameters exhibited lower repeatability .", "for example , the cc values for ulnar nerve dml , mean fwl , and dsl without temperature correction were 0.72 , 0.93 , and 0.72 , respectively .", "standard deviation of the difference between test and retest results would have been 0.31 , 1.04 , and 0.34 .", "table 1statistical measures of nerve conduction study parameter repeatabilityriv percentiles(day 8day 1)nerveccicccov10th ( % ) 90th ( % ) meanstdevpaired t - testdistal motor latency ( dml)median0.8720.8700.0329.55.10.0550.1940.289ulnar0.8890.8320.0458.311.00.0260.1900.605peroneal0.8640.8450.06115.48.10.1240.4530.307tibial0.8500.8240.0386.88.80.0160.2390.799mean f - wave latency ( fwl)median0.6920.6790.0316.64.40.0371.9700.943ulnar0.9490.9210.0173.91.10.2860.9150.246peroneal0.8960.9010.0174.73.40.1631.5630.693tibial0.9430.9400.0131.83.50.2781.1450.363distal sensory latency ( dsl)median0.8870.8790.0287.24.00.0560.1890.270ulnar0.8150.7970.0375.79.00.0120.2070.825compound muscle action potential ( cmap ) amplitudemedian0.8790.8620.10919.219.90.0310.1980.550ulnar0.9810.9740.0455.710.70.2880.6090.088peroneal0.3920.3310.29830.899.50.8491.9370.112tibial0.7140.7280.14830.925.40.1651.6010.695sensory nerve action potential ( snap ) amplitudemedian0.9540.9540.05811.87.80.6738.4020.761ulnar0.9900.9820.0424.410.91.4855.0630.275cc : pearson product moment correlation to assess the association between test - retest results .", "riv : relative interval variation defines the difference between test - retest as a percentage of average . paired t - test results indicate the means of test and retest are not statistically different .", "dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference .", "dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference .", "fwl is the arithmetic mean of individual f - wave latencies in an f - wave set .", "statistical measures of nerve conduction study parameter repeatability cc : pearson product moment correlation to assess the association between test - retest results .", "riv : relative interval variation defines the difference between test - retest as a percentage of average . paired", "t - test results indicate the means of test and retest are not statistically different .", "dsl is the negative peak latency and snap amplitude is the peak - to - peak amplitude difference .", "dml is the onset of initial deflection and cmap is negative peak to baseline amplitude difference .", "fwl is the arithmetic mean of individual f - wave latencies in an f - wave set ."], ["several studies ( summarized in table 2 ) have examined ncs parameter repeatability [ 2 , 4 , 5 ] . in , the repeatability of ncs was evaluated for 60 sites participating in a clinical trial , with ncs oversight performed by  an experienced , insightful and knowledgeable core lab  .", "our study yielded similar results , though our study demonstrated a higher cov for peroneal cmap amplitude ( 9% vs. 29.8% ) . in", ", 132 healthy subjects were retested at a time interval of 14 weeks and icc and riv were used to measure the test - retest repeatability . in comparison with that study ,", "the riv ( 5th and 95th percentile ) for tibial nerve f - wave latency was tighter in our study ( [ 2.8% , 4.0% ] vs. [ 4.6% , 5.7% ] ) while the relationship was reversed for median nerve fwave latency results ( [ 11.4% , 9.4% ] vs. [ 6.7% , 6.7% ] ) .", "decreased repeatability of median nerve f - wave latency may be attributed to the lower amplitude signal - to - noise ratio as median nerve f - waves were acquired via a volume - conduction recording technique .", "indeed , latency repeatability was much higher for ulnar f - waves recorded directly over the muscle .", "the icc for ulnar f - wave latency was 0.92 in our study , higher than the icc of 0.59 reported in a similar study of 49 healthy adults .", "salerno and colleagues studied upper extremity sensory test - retest repeatability based on 158 active workers who were tested by a neurologist and a physiatrist .", "our results fell into the upper range of their repeatability results as measured by cc and icc . \n", "table 2repeatability results reported in the literaturesnerveccicccovdistal motor latency ( dml)median0.8260.04/0.08 > \n peroneal0.05/0.08tibial0.6170.08minimum f - wave latency ( fwl)ulnar0.59tibial0.921distal sensory latency ( dsl)median0.820.920.820.920.04ulnar0.370.640.320.63compound muscle action potential ( cmap ) amplitudemedian0.7770.07/0.11peroneal0.09tibial0.846sensory nerve action potential ( snap ) amplitudemedian0.830.880.834/0.810.880.08ulnar0.680.850.680.85 132 healthy subjects retested in 14 weeks .  ", "results for control subjects from 60 sites with core lab support in a clinical trial .  ", "33 subjects retested in 12 week , results for small electrode size ( 0.78 cm ) and all three nerves combined .  ", "repeatability results reported in the literatures   132 healthy subjects retested in 14 weeks .  ", "results for control subjects from 60 sites with core lab support in a clinical trial .  ", "33 subjects retested in 12 week , results for small electrode size ( 0.78 cm ) and all three nerves combined .  ", "it is possible that the small recording electrodes ( surface areas 2 cm ) used in our study degraded repeatability , since tjon - a - tsien and colleagues noted that when electrode size was changed from 0.78 to 7.65 cm , the cov of cmap amplitude improved from 11 to 7% ( lower cov is associated with higher repeatability ) .", "dml repeatability was less affected by electrode size in both our study and the study of .", "however , control of skin temperature is often difficult to achieve , especially in different testing environments . in the present study ,", "skin temperature adjacent to the recording site was acquired automatically using an embedded probe and latencies were adjusted to the reference temperature . as expected , temperature correction improved ncs repeatability .", "for example , the cc increased from 0.72 ( without temperature correction ) to 0.89 ( with correction ) for both motor and sensory latencies of the ulnar nerves .", "temperature correction also reduced the standard deviation of the test - retest latency difference by as much as 39% ( ulnar dml ) .", "the principal limitation of the current study was the small number of subjects evaluated relative to several prior investigations .", "the short test - retest interval of 1 week reduced the likelihood of intervening physiological changes .", "it would be valuable to quantify repeatability of the automated ncs instrument in neuropathic nerves . at the time of this study ,", "temperature correction may improve the repeatability of the amplitude parameters , especially for sensory responses .", "the reproducibility of individual ncs parameters followed the same pattern as those observed in traditional settings .", "f - wave latency had the greatest repeatability , followed by dml , dsl , and snap amplitude .", "the cmap amplitude was the least reproducible parameter , with peroneal cmap amplitude repeatability the lowest among all nerves . a larger recording electrode size should improve its repeatability .", "f - wave latency repeatability for median nerve was lower due to the lower signal - to - noise ratio inherent in volume - conduction recordings .", "as a result , over - muscle recording would be expected to enhance f - wave repeatability , which was the case for the ulnar nerve .", "further studies with longer test - retest intervals and a higher subject count would be valuable in confirming the high reproducibility of ncs parameters measured in this study . extending the study to pathologic nerves and validating the equivalent or superior performance of automated ncs instrument should increase the adoption rate of the automated ncs technology in patient management"]]}
{"article_id": "PMC3004667", "article_text": ["this study was conducted within the north central cancer treatment group , a national cancer cooperative group that is funded in part by the national cancer institute . questionnaire and clinical trial data from two separate cancer therapeutic trials , n9921 and", "these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006 , respectively . the present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials .", "eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents , gefitinib , paclitaxel , and carboplatin , which were used in one or both of the trials mentioned above .", "these criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following : 1 ) patient aged 65 years or older ; 2 ) eastern cooperative group performance score ( an often - utilized score that describes the level of function of cancer patients ) of 0 , 1 , or 2 ; 3 ) histologic or cytologic confirmation of non - small cell lung cancer ; 4 ) unresectable cancer ; 5 ) no prior chemotherapy ; 6 ) physician - anticipated life expectancy of 12 weeks or longer ; and 7 ) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial .", "in addition , all patients had to have had adequate organ function , as demonstrated by an acceptable hemogram , liver function tests , and serum creatinine at the time of study enrollment .", "other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive , such as , for example , an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel .", "all eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy .", "this 12-item instrument has been previously validated , has been tested in a variety of settings , and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens : 1 ) weekly carboplatin and paclitaxel ( intravenous ) ; 2 ) weekly carboplatin and paclitaxel ( intravenous ) potentially for four months followed by daily gefitinib ( oral ) ; or 3 ) daily gefitinib ( oral ) .", "patients were to continue cancer therapy until evidence of cancer progression , until unacceptable side effects occurred , or , for a maximum of four months , if they were receiving conventional chemotherapy .", "they were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria , version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed .", "the sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials , not for the social support aims .", "nonetheless , for the current study , we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender , performance score , and severe adverse events .", "the sample size of 113 ( 56 in each group ) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points ( 60 versus 72 ) using a two - sided alpha of 0.007 .", "scores from the lubben social support network ( items 110 on table 2 ) were summed for analyses and then transformed to a 100% scale .", "a score of 0 represents the lowest possible level of social support ; whereas , a score of 100 represents the highest possible level of social support . to transform the scores to a 100% scale ,", "a patient s individual score was divided by the patient s possible range accounting for any missed items ( maximum minus minimum ) and multiplied by 100 .", "a rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender , age ( > = 70 ) , and performance status .", "similarly , a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events ( grade 3 or worse ) versus those who did not .", "time - to - cancer progression was defined as the time from registration to documentation of cancer progression .", "survival time was defined as the time from study registration to death due to any cause .", "a cox proportional hazards model , which adjusted for cancer treatment , performance score , age , gender , and sum of target lesions at baseline , was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . in order to illustrate the influence of social support on overall survival , we divided the cohort by social support score quartiles .", "no widely accepted cut - points exist to separate social support into categories , so quartiles were used to distribute evenly the 113 patients into four groups .", "a logistic regression model , which adjusted for the same baseline factors mentioned above , was used to assess relationships between social support and adverse events .", "because of the exploratory nature of these analyses and because of the number of statistical tests performed , a p - value of < 0.01 was considered statistically significant . this decision to utilize this p - value", "was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment .", "all statistical analyses were conducted with sas software ( sas institute , cary , nc , usa ) .", "this study was conducted within the north central cancer treatment group , a national cancer cooperative group that is funded in part by the national cancer institute . questionnaire and clinical trial data from two separate cancer therapeutic trials , n9921 and", "these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006 , respectively . the present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials .", "eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents , gefitinib , paclitaxel , and carboplatin , which were used in one or both of the trials mentioned above .", "these criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following : 1 ) patient aged 65 years or older ; 2 ) eastern cooperative group performance score ( an often - utilized score that describes the level of function of cancer patients ) of 0 , 1 , or 2 ; 3 ) histologic or cytologic confirmation of non - small cell lung cancer ; 4 ) unresectable cancer ; 5 ) no prior chemotherapy ; 6 ) physician - anticipated life expectancy of 12 weeks or longer ; and 7 ) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial .", "in addition , all patients had to have had adequate organ function , as demonstrated by an acceptable hemogram , liver function tests , and serum creatinine at the time of study enrollment .", "other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive , such as , for example , an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel .", "all eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy .", "this 12-item instrument has been previously validated , has been tested in a variety of settings , and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens : 1 ) weekly carboplatin and paclitaxel ( intravenous ) ; 2 ) weekly carboplatin and paclitaxel ( intravenous ) potentially for four months followed by daily gefitinib ( oral ) ; or 3 ) daily gefitinib ( oral ) .", "patients were to continue cancer therapy until evidence of cancer progression , until unacceptable side effects occurred , or , for a maximum of four months , if they were receiving conventional chemotherapy .", "they were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria , version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed .", "the sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials , not for the social support aims .", "nonetheless , for the current study , we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender , performance score , and severe adverse events .", "the sample size of 113 ( 56 in each group ) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points ( 60 versus 72 ) using a two - sided alpha of 0.007 .", "scores from the lubben social support network ( items 110 on table 2 ) were summed for analyses and then transformed to a 100% scale .", "a score of 0 represents the lowest possible level of social support ; whereas , a score of 100 represents the highest possible level of social support . to transform the scores to a 100% scale ,", "a patient s individual score was divided by the patient s possible range accounting for any missed items ( maximum minus minimum ) and multiplied by 100 .", "a rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender , age ( > = 70 ) , and performance status .", "similarly , a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events ( grade 3 or worse ) versus those who did not .", "time - to - cancer progression was defined as the time from registration to documentation of cancer progression .", "survival time was defined as the time from study registration to death due to any cause .", "a cox proportional hazards model , which adjusted for cancer treatment , performance score , age , gender , and sum of target lesions at baseline , was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . in order to illustrate the influence of social support on overall survival , we divided the cohort by social support score quartiles .", "no widely accepted cut - points exist to separate social support into categories , so quartiles were used to distribute evenly the 113 patients into four groups .", "a logistic regression model , which adjusted for the same baseline factors mentioned above , was used to assess relationships between social support and adverse events .", "because of the exploratory nature of these analyses and because of the number of statistical tests performed , a p - value of < 0.01 was considered statistically significant . this decision to utilize this p - value", "was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment .", "all statistical analyses were conducted with sas software ( sas institute , cary , nc , usa ) .", "a total of 113 patients met the eligibility criteria , received chemotherapy on a clinical trial , and therefore participated in the current study .", "one patient did not receive chemotherapy , and two did not meet the eligibility criteria described earlier ; thus , these three patients are not included in the analyses .", "forty - two percent of the cohort consisted of women , and the median age ( range ) was 74 years ( 65 , 91 ) . at the time of study entry , a performance score of 0 , 1 , or 2 was observed in 29% , 55% , and 16% of the cohort , respectively .", "lubben social network scale data are summarized in table 2 . of note , 44% of patients reported that they see nine or more relatives at least once a month , and 27% reported that they see five to eight .", "in addition , 73% reported that when they have an important decision to make , they ", "always  have someone who can take them to their medical appointments , and 71% reported that they  always  have someone from whom they can seek help if they have medication side effects .", "the median transformed social support score for the entire cohort ( range ) was 72 ( 30100 ) .", "it should be noted that , although normative reference data are not readily available , a community - based study among a group of 7,524 older individuals observed a similarly - derived , average social support score of 63.19 among women and men in the present study , the average score was not statistically different : 72 in both ( rank sum , p = 0.87 ) ( figure 1 ) .", "additionally , there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2 : median score 74 and 70 ( rank sum , p = 0.06 ) .", "although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression ( p = 0.02 ; hazard ratio [ hr ] = 1.02 ; 95% confidence interval [ ci ] : 1.001.03 ) , these scores did not predict overall survival ( p = 0.34 ; hr = 1.01 ; 95% ci : 0.991.02 ) ( figure 2 ) .", "there was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not : median score 74 and 70 ( rank sum , p = 0.28 ) ( adjusted logistic regression p = 0.70 ; odds ratio [ or ] = 1.00 ; 95% ci : 0.971.02 ) ( table 3 ) .", "a total of 113 patients met the eligibility criteria , received chemotherapy on a clinical trial , and therefore participated in the current study .", "one patient did not receive chemotherapy , and two did not meet the eligibility criteria described earlier ; thus , these three patients are not included in the analyses .", "forty - two percent of the cohort consisted of women , and the median age ( range ) was 74 years ( 65 , 91 ) . at the time of study entry , a performance score of 0 , 1 , or 2 was observed in 29% , 55% , and 16% of the cohort , respectively .", "lubben social network scale data are summarized in table 2 . of note , 44% of patients reported that they see nine or more relatives at least once a month , and 27% reported that they see five to eight .", "in addition , 73% reported that when they have an important decision to make , they  always  have someone to talk with .", "always  have someone who can take them to their medical appointments , and 71% reported that they  always  have someone from whom they can seek help if they have medication side effects .", "the median transformed social support score for the entire cohort ( range ) was 72 ( 30100 ) .", "it should be noted that , although normative reference data are not readily available , a community - based study among a group of 7,524 older individuals observed a similarly - derived , average social support score of 63.19 among women and men in the present study , the average score was not statistically different : 72 in both ( rank sum , p = 0.87 ) ( figure 1 ) .", "additionally , there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2 : median score 74 and 70 ( rank sum , p = 0.06 ) .", "although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression ( p = 0.02 ; hazard ratio [ hr ] = 1.02 ; 95% confidence interval [ ci ] : 1.001.03 ) , these scores did not predict overall survival ( p = 0.34 ; hr = 1.01 ; 95% ci : 0.991.02 ) ( figure 2 ) .", "there was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not : median score 74 and 70 ( rank sum , p = 0.28 ) ( adjusted logistic regression p = 0.70 ; odds ratio [ or ] = 1.00 ; 95% ci : 0.971.02 ) ( table 3 ) .", "the present study was designed to assess and evaluate the role of social support in a group of geriatric non - small cell lung cancer patients who were enrolled in two different cancer therapeutic clinical trials .", "this study found that this cohort of geriatric patients had high levels of social support , as suggested by the fact that 81% reported that they ", "always  had someone who could take them to medical appointments , and 71% reported that they  always  had someone from whom they could seek help if they had medication side effects .", "however , social support was not associated with improved survival or diminished side effects from chemotherapy . moreover , no differences were observed between men and women in terms of baseline social support .", "thus , although previous studies have reported that social support carries a positive impact on clinical outcomes and although gender differences in extent of social support have been hypothesized , the present study did not observe such findings .", "why might the results of this study be divergent from the others that preceded it ?", "first , relying on a clinical trial to answer such questions provides a solid infrastructure that ensures reliable outcome data .", "the fact that all clinical trial participants were followed prospectively in such a thorough and systematic fashion makes it unlikely that patients  particularly those who have poor social support and who are thereby especially vulnerable to being lost to follow up  would , in fact , be lost to follow up .", "such artificially compromised outcomes were side - stepped by building the study around two clinical trials , as was done here .", "thus , the present study s meticulous follow up may have allowed for a more definitive and accurate assessment of the predictive capabilities  or lack thereof  of social support .", "second , and more importantly , one might argue that patients willingness to participate in a clinical trial is associated with a high level of social support and that this selection bias may have contributed to the lack of differences between groups .", "the fact that 82% of patients reported that they  always  had someone to bring them to medical appointments speaks to the possibility that this group as a whole has a high level of social support .", "similarly , the median social support score of 72 in this study appears greater than the 63 score derived from a community - based study , an observation that also suggests cancer clinical trial participants have high levels of social support.19 in short , although the findings from this study suggest that social support does not influence clinical outcomes , these findings might pertain to only a culled group of geriatric clinical trial participants .", "future studies may choose to focus further on the extent of social support among geriatric patients who participate in clinical trials , and further studies may also choose to study further whether social support is a predictive factor for the patients who can and do enroll in cancer clinical trials . two final points merit further discussion .", "first , although the current study did not find that social support was predictive of outcomes , there are a variety of other potentially clinically relevant factors that had not been captured and adjusted for .", "these include socioeconomic status , ongoing tobacco use , use of nutritional supplements , as well as several other factors .", "other investigators may choose to further study the issue of social support in the context of these other factors .", "second , although this study found that social support is not associated with clinical outcomes among elderly lung cancer patients , there remains no question that these patients have major needs that are likely exacerbated by a lack of social support .", "patients with metastatic non - small cell lung cancer suffer from a poor prognosis , as seen from both studies presented here where the prognosis was poor , and previous studies have demonstrated that elderly patients suffer from a wide constellation of cancer - related symptoms , including depression , guilt , debility , pain , and dyspnea.1013 although the present study did not observe an association between social support and clinical outcomes , there nonetheless remains a strong need for maximizing social support to help with some of the cancer - related challenges these patients face ."], "abstract_text": ["<S> background : social support is defined as a network of family / friends who provide practical and emotional help . </S>", "<S> a sizable literature describes a direct relationship between social support and improved cancer clinical outcomes . </S>", "<S> this study explored the extent of social support and its potential association with survival and adverse events in geriatric lung cancer patients.methods:one hundred thirteen patients , who were aged 65 years or older , had incurable cancer , and were enrolled in one of two chemotherapy trials , completed the lubben social network scale , a validated instrument that measures social support . </S>", "<S> all were followed for survival and chemotherapy - related adverse events.results:the median age ( range ) of the cohort was 74 years ( 6591 ) , and performance scores of 0 , 1 , or 2 were observed in 29% , 55% , and 16% , respectively . </S>", "<S> forty - two percent were women . </S>", "<S> this cohort had a high level of social support : 81% reported they  always  had someone to take them to medical appointments . however , there were no gender - based differences in social support and no associations between social support and either survival or adverse events.conclusion:in this cohort of geriatric lung cancer patients  all of whom were treated during a clinical trial  there was a high level of social support . </S>", "<S> however , there were no gender - based differences in extent of social support , and the latter did not predict clinical outcomes . </S>"], "labels": null, "section_names": ["Materials and methods", "Overview", "Patient eligibility", "Patient enrolment and follow-up", "Statistical analyses", "Results", "Demographics", "Social support scores, gender, and clinical prediction", "Discussion"], "sections": [["this study was conducted within the north central cancer treatment group , a national cancer cooperative group that is funded in part by the national cancer institute . questionnaire and clinical trial data from two separate cancer therapeutic trials , n9921 and", "these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006 , respectively . the present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials .", "eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents , gefitinib , paclitaxel , and carboplatin , which were used in one or both of the trials mentioned above .", "these criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following : 1 ) patient aged 65 years or older ; 2 ) eastern cooperative group performance score ( an often - utilized score that describes the level of function of cancer patients ) of 0 , 1 , or 2 ; 3 ) histologic or cytologic confirmation of non - small cell lung cancer ; 4 ) unresectable cancer ; 5 ) no prior chemotherapy ; 6 ) physician - anticipated life expectancy of 12 weeks or longer ; and 7 ) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial .", "in addition , all patients had to have had adequate organ function , as demonstrated by an acceptable hemogram , liver function tests , and serum creatinine at the time of study enrollment .", "other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive , such as , for example , an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel .", "all eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy .", "this 12-item instrument has been previously validated , has been tested in a variety of settings , and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens : 1 ) weekly carboplatin and paclitaxel ( intravenous ) ; 2 ) weekly carboplatin and paclitaxel ( intravenous ) potentially for four months followed by daily gefitinib ( oral ) ; or 3 ) daily gefitinib ( oral ) .", "patients were to continue cancer therapy until evidence of cancer progression , until unacceptable side effects occurred , or , for a maximum of four months , if they were receiving conventional chemotherapy .", "they were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria , version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed .", "the sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials , not for the social support aims .", "nonetheless , for the current study , we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender , performance score , and severe adverse events .", "the sample size of 113 ( 56 in each group ) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points ( 60 versus 72 ) using a two - sided alpha of 0.007 .", "scores from the lubben social support network ( items 110 on table 2 ) were summed for analyses and then transformed to a 100% scale .", "a score of 0 represents the lowest possible level of social support ; whereas , a score of 100 represents the highest possible level of social support . to transform the scores to a 100% scale ,", "a patient s individual score was divided by the patient s possible range accounting for any missed items ( maximum minus minimum ) and multiplied by 100 .", "a rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender , age ( > = 70 ) , and performance status .", "similarly , a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events ( grade 3 or worse ) versus those who did not .", "time - to - cancer progression was defined as the time from registration to documentation of cancer progression .", "survival time was defined as the time from study registration to death due to any cause .", "a cox proportional hazards model , which adjusted for cancer treatment , performance score , age , gender , and sum of target lesions at baseline , was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . in order to illustrate the influence of social support on overall survival , we divided the cohort by social support score quartiles .", "no widely accepted cut - points exist to separate social support into categories , so quartiles were used to distribute evenly the 113 patients into four groups .", "a logistic regression model , which adjusted for the same baseline factors mentioned above , was used to assess relationships between social support and adverse events .", "because of the exploratory nature of these analyses and because of the number of statistical tests performed , a p - value of < 0.01 was considered statistically significant . this decision to utilize this p - value", "was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment .", "all statistical analyses were conducted with sas software ( sas institute , cary , nc , usa ) ."], ["this study was conducted within the north central cancer treatment group , a national cancer cooperative group that is funded in part by the national cancer institute . questionnaire and clinical trial data from two separate cancer therapeutic trials , n9921 and", "these two trials recruited patients between february 2000 and february 2001 and between december 2004 and february 2006 , respectively . the present study s design was formulated prior to the initiation of the first trial and entailed merging and analyzing data from both of these prospectively conducted clinical trials ."], ["eligibility criteria for these two trials focused on selecting patients who could tolerate the chemotherapy agents , gefitinib , paclitaxel , and carboplatin , which were used in one or both of the trials mentioned above .", "these criteria are described in greater detail elsewhere,14,15 but relevant factors consistent between both trials included the following : 1 ) patient aged 65 years or older ; 2 ) eastern cooperative group performance score ( an often - utilized score that describes the level of function of cancer patients ) of 0 , 1 , or 2 ; 3 ) histologic or cytologic confirmation of non - small cell lung cancer ; 4 ) unresectable cancer ; 5 ) no prior chemotherapy ; 6 ) physician - anticipated life expectancy of 12 weeks or longer ; and 7 ) absence of a major medical illness that would preclude participation in a therapeutic cancer clinical trial .", "in addition , all patients had to have had adequate organ function , as demonstrated by an acceptable hemogram , liver function tests , and serum creatinine at the time of study enrollment .", "other eligibility criteria were more specific to each chemotherapy regimen patients were about to receive , such as , for example , an exclusion of patients with severe neuropathy in the event they were to receive paclitaxel ."], ["all eligible patients completed the lubben social network scale within 14 days of trial registration and prior to receiving any chemotherapy .", "this 12-item instrument has been previously validated , has been tested in a variety of settings , and includes questionnaire items that are highly germane to defining extent of social support.16 thereafter patients were treated with one of the following chemotherapy regimens : 1 ) weekly carboplatin and paclitaxel ( intravenous ) ; 2 ) weekly carboplatin and paclitaxel ( intravenous ) potentially for four months followed by daily gefitinib ( oral ) ; or 3 ) daily gefitinib ( oral ) .", "patients were to continue cancer therapy until evidence of cancer progression , until unacceptable side effects occurred , or , for a maximum of four months , if they were receiving conventional chemotherapy .", "they were monitored indefinitely for cancer therapy - related side effects by means of the common terminology criteria , version 2 from the national cancer institute.17 tumor response as assessed by the recist criteria,18 and overall survival was assessed ."], ["the sample sizes for the two individual trials were calculated to obtain adequate statistical power for the primary aims of these clinical trials , not for the social support aims .", "nonetheless , for the current study , we conducted a retrospective power calculation for the comparisons of two independent means to evaluate the power available for comparing the lubben social support score by the dichotomous factors of gender , performance score , and severe adverse events .", "the sample size of 113 ( 56 in each group ) with an observed standard score deviation of 17 and an observed median social support score of 72 yields 84% power to detect a difference of 12 points ( 60 versus 72 ) using a two - sided alpha of 0.007 .", "scores from the lubben social support network ( items 110 on table 2 ) were summed for analyses and then transformed to a 100% scale .", "a score of 0 represents the lowest possible level of social support ; whereas , a score of 100 represents the highest possible level of social support . to transform the scores to a 100% scale ,", "a patient s individual score was divided by the patient s possible range accounting for any missed items ( maximum minus minimum ) and multiplied by 100 .", "a rank sum test was used to evaluate whether social support scores differed based on the characteristics of gender , age ( > = 70 ) , and performance status .", "similarly , a rank sum test was also used to evaluate whether social support scores differed between patients who suffered more severe adverse events ( grade 3 or worse ) versus those who did not .", "time - to - cancer progression was defined as the time from registration to documentation of cancer progression .", "survival time was defined as the time from study registration to death due to any cause .", "a cox proportional hazards model , which adjusted for cancer treatment , performance score , age , gender , and sum of target lesions at baseline , was used to evaluate whether social support scores were related to time - to - cancer progression or overall survival . in order to illustrate the influence of social support on overall survival , we divided the cohort by social support score quartiles .", "no widely accepted cut - points exist to separate social support into categories , so quartiles were used to distribute evenly the 113 patients into four groups .", "a logistic regression model , which adjusted for the same baseline factors mentioned above , was used to assess relationships between social support and adverse events .", "because of the exploratory nature of these analyses and because of the number of statistical tests performed , a p - value of < 0.01 was considered statistically significant . this decision to utilize this p - value", "was based on the computation that the overall conventional alpha level is 0.05 and that seven multiple comparisons were made with a bonferroni adjustment .", "all statistical analyses were conducted with sas software ( sas institute , cary , nc , usa ) ."], ["a total of 113 patients met the eligibility criteria , received chemotherapy on a clinical trial , and therefore participated in the current study .", "one patient did not receive chemotherapy , and two did not meet the eligibility criteria described earlier ; thus , these three patients are not included in the analyses .", "forty - two percent of the cohort consisted of women , and the median age ( range ) was 74 years ( 65 , 91 ) . at the time of study entry , a performance score of 0 , 1 , or 2 was observed in 29% , 55% , and 16% of the cohort , respectively .", "lubben social network scale data are summarized in table 2 . of note , 44% of patients reported that they see nine or more relatives at least once a month , and 27% reported that they see five to eight .", "in addition , 73% reported that when they have an important decision to make , they ", "always  have someone who can take them to their medical appointments , and 71% reported that they  always  have someone from whom they can seek help if they have medication side effects .", "the median transformed social support score for the entire cohort ( range ) was 72 ( 30100 ) .", "it should be noted that , although normative reference data are not readily available , a community - based study among a group of 7,524 older individuals observed a similarly - derived , average social support score of 63.19 among women and men in the present study , the average score was not statistically different : 72 in both ( rank sum , p = 0.87 ) ( figure 1 ) .", "additionally , there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2 : median score 74 and 70 ( rank sum , p = 0.06 ) .", "although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression ( p = 0.02 ; hazard ratio [ hr ] = 1.02 ; 95% confidence interval [ ci ] : 1.001.03 ) , these scores did not predict overall survival ( p = 0.34 ; hr = 1.01 ; 95% ci : 0.991.02 ) ( figure 2 ) .", "there was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not : median score 74 and 70 ( rank sum , p = 0.28 ) ( adjusted logistic regression p = 0.70 ; odds ratio [ or ] = 1.00 ; 95% ci : 0.971.02 ) ( table 3 ) ."], ["a total of 113 patients met the eligibility criteria , received chemotherapy on a clinical trial , and therefore participated in the current study .", "one patient did not receive chemotherapy , and two did not meet the eligibility criteria described earlier ; thus , these three patients are not included in the analyses .", "forty - two percent of the cohort consisted of women , and the median age ( range ) was 74 years ( 65 , 91 ) . at the time of study entry , a performance score of 0 , 1 , or 2 was observed in 29% , 55% , and 16% of the cohort , respectively ."], ["lubben social network scale data are summarized in table 2 . of note , 44% of patients reported that they see nine or more relatives at least once a month , and 27% reported that they see five to eight .", "in addition , 73% reported that when they have an important decision to make , they  always  have someone to talk with .", "always  have someone who can take them to their medical appointments , and 71% reported that they  always  have someone from whom they can seek help if they have medication side effects .", "the median transformed social support score for the entire cohort ( range ) was 72 ( 30100 ) .", "it should be noted that , although normative reference data are not readily available , a community - based study among a group of 7,524 older individuals observed a similarly - derived , average social support score of 63.19 among women and men in the present study , the average score was not statistically different : 72 in both ( rank sum , p = 0.87 ) ( figure 1 ) .", "additionally , there was no statistically significant difference in social support scores among patients with an eastern cooperative oncology group performance score of 0 versus 1 and 2 : median score 74 and 70 ( rank sum , p = 0.06 ) .", "although there was a trend to suggest that higher social support scores ironically predicted a shorter time - to - cancer - progression ( p = 0.02 ; hazard ratio [ hr ] = 1.02 ; 95% confidence interval [ ci ] : 1.001.03 ) , these scores did not predict overall survival ( p = 0.34 ; hr = 1.01 ; 95% ci : 0.991.02 ) ( figure 2 ) .", "there was also no statistically significant difference in scores between patients who suffered grade 3 or worse adverse events and those who did not : median score 74 and 70 ( rank sum , p = 0.28 ) ( adjusted logistic regression p = 0.70 ; odds ratio [ or ] = 1.00 ; 95% ci : 0.971.02 ) ( table 3 ) ."], ["the present study was designed to assess and evaluate the role of social support in a group of geriatric non - small cell lung cancer patients who were enrolled in two different cancer therapeutic clinical trials .", "this study found that this cohort of geriatric patients had high levels of social support , as suggested by the fact that 81% reported that they ", "always  had someone who could take them to medical appointments , and 71% reported that they  always  had someone from whom they could seek help if they had medication side effects .", "however , social support was not associated with improved survival or diminished side effects from chemotherapy . moreover , no differences were observed between men and women in terms of baseline social support .", "thus , although previous studies have reported that social support carries a positive impact on clinical outcomes and although gender differences in extent of social support have been hypothesized , the present study did not observe such findings .", "why might the results of this study be divergent from the others that preceded it ?", "first , relying on a clinical trial to answer such questions provides a solid infrastructure that ensures reliable outcome data .", "the fact that all clinical trial participants were followed prospectively in such a thorough and systematic fashion makes it unlikely that patients  particularly those who have poor social support and who are thereby especially vulnerable to being lost to follow up  would , in fact , be lost to follow up .", "such artificially compromised outcomes were side - stepped by building the study around two clinical trials , as was done here .", "thus , the present study s meticulous follow up may have allowed for a more definitive and accurate assessment of the predictive capabilities  or lack thereof  of social support .", "second , and more importantly , one might argue that patients willingness to participate in a clinical trial is associated with a high level of social support and that this selection bias may have contributed to the lack of differences between groups .", "the fact that 82% of patients reported that they  always  had someone to bring them to medical appointments speaks to the possibility that this group as a whole has a high level of social support .", "similarly , the median social support score of 72 in this study appears greater than the 63 score derived from a community - based study , an observation that also suggests cancer clinical trial participants have high levels of social support.19 in short , although the findings from this study suggest that social support does not influence clinical outcomes , these findings might pertain to only a culled group of geriatric clinical trial participants .", "future studies may choose to focus further on the extent of social support among geriatric patients who participate in clinical trials , and further studies may also choose to study further whether social support is a predictive factor for the patients who can and do enroll in cancer clinical trials . two final points merit further discussion .", "first , although the current study did not find that social support was predictive of outcomes , there are a variety of other potentially clinically relevant factors that had not been captured and adjusted for .", "these include socioeconomic status , ongoing tobacco use , use of nutritional supplements , as well as several other factors .", "other investigators may choose to further study the issue of social support in the context of these other factors .", "second , although this study found that social support is not associated with clinical outcomes among elderly lung cancer patients , there remains no question that these patients have major needs that are likely exacerbated by a lack of social support .", "patients with metastatic non - small cell lung cancer suffer from a poor prognosis , as seen from both studies presented here where the prognosis was poor , and previous studies have demonstrated that elderly patients suffer from a wide constellation of cancer - related symptoms , including depression , guilt , debility , pain , and dyspnea.1013 although the present study did not observe an association between social support and clinical outcomes , there nonetheless remains a strong need for maximizing social support to help with some of the cancer - related challenges these patients face ."]]}
{"article_id": "PMC4318034", "article_text": ["port - wine stains ( pwss ) are congenital vascular malformations comprised of ectatic dermal capillaries and affecting 0.3 - 1% of newborn infants .", "acquired pwss are an uncommon entity developing later in life , but morphologically and histologically similar to the congenital lesions .", "we hereby report a case of 41-year - old male patient who acquired pws about a year ago .", "a 41-year - old male presented to the outpatient department of dermatology of our tertiary care hospital with complaint of asymptomatic reddish patches involving the lower part of right leg for 10 months .", "the patch started from near the ankle and gradually spread to involve whole of the medial aspect of right leg over a period of 10 months .", "we could not elicit any history of antecedent mechanical or thermal trauma , drug intake , topical application or excessive ultraviolet exposure .", "dermatological examination revealed a diffuse partially blanchable erythema extending from the right ankle and involving chiefly the medial aspect of right leg up to the knee [ figure 1 ] .", "there were also a few discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg [ figure 2 ] .", "a diffuse partially blanchable erythema extending from the right ankle and involving the medial aspect of right leg up to the knee discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg on the basis of the clinical examination , a provisional diagnosis of acquired pws was made and the patient investigated .", "the histopathological examination revealed multiple dilated , variably sized capillaries lined by a single layer of endothelial cells throughout the dermis showing lobular arrangement at places [ figure 3 ]", ". color doppler of the limb did not reveal any abnormality of the underlying arteries and veins .", "( a ) multiple dilated variably sized capillaries in the dermis ( h and e , 40 , arrows ) .", "( b ) high power showing groups of capillaries lined by a single layer of endothelial cells ( h and e , 250 , arrow ) laser therapy was discussed with the patient , but he declined treatment for the lesion .", "pwss are congenital vascular malformations usually presenting at birth in the form of pink - red to purple macules , which become darker , raised , and nodular as the person ages .", "the most accepted hypothesis is a defective embryological maturation of the sympathetic fibers , resulting in a loss of normal sympathetic control of the cutaneous vessels which leads to ectasia .", "various factors have been proposed for the occurrence of acquired pwss . of these , trauma has been found to be the most important causative factor in majority of the cases .", "trauma - induced pws was first described by fegeler in 1949 and hence called fegeler syndrome . in a review of 59 patients with acquired pws , trauma was found to be a causative factor in 17 ( 29% ) cases .", "it has been proposed that injury may result in loss of a previously effective sympathetic regulation of the cutaneous blood flow , leading to development of the lesions .", "reports of acquired port - wine stain few cases of acquired pws have also been proposed secondary to oral medications like isotretinoin , oral contraceptive pills , simvastatin , and metformin .", "isotretinoin causes skin fragility and frictional trauma , while simvastain and metformin have been shown to promote angiogenesis by upregulation of vascular endothelial growth factor .", "isolated cases of acquired pws following frostbite injury , obstruction of the peritoneovenous shunt , herpes zoster infection , cerebral arteriovenous malformation , spinal root compression , and solid brain tumor have also been reported .", "no definite cause was found for the development of lesion in our patient . in such cases , a likely possibility of having a latent vascular anomaly which manifests clinically after unnoticed trauma", "an unusual finding in our patient was the presence of a lobular arrangement of dilated vessels in the dermis which has not been observed in previous cases of congenital or acquired pws .", "interestingly , there are multiple reports of development of pyogenic granulomas ( pg ) within pws which also shows a lobular pattern of dilated vessels .", "it has been proposed that the pg probably results from an underlying arteriovenous anastamosis frequently associated with the pws .", "though , there was no clinically appreciable pyogenic granuloma like lesion , the distinct histological features seen in our patient might be attributed to a similar pathogenetic mechanism .", "as regards therapy , it includes treatment with krypton , argon , argon - pumped , tunable dye , neodymium : yttrium - aluminum garnet , copper vapor , and pulsed dye lasers .", "acquired pws tends to have a quicker and better response to pulsed dye therapy than congenital pws .", "the case report demonstrates a rare occurrence of a vascular malformation in an adult patient .", "although no definite cause could be appreciated for the development of lesion in our patient , further reports and future insights into the pathogenesis of such cases would broaden our knowledge regarding this infrequently reported phenomenon .", "this is the first report of an acquired port - wine stain from india and previous reports of such cases have also been reviewed ."], "abstract_text": ["<S> port - wine stains ( pwss ) are congenital vascular lesions caused by progressive ectasia of blood vessels located in the vascular plexus of the dermis . </S>", "<S> acquired pwss develop later in life but are identical in morphology and histology to the congenital pwss . </S>", "<S> less than 75 cases of acquired pwss have been reported in the published literature , of which there has not been a single report from india so far . </S>", "<S> various factors have been proposed for its pathogenesis like trauma , actinic exposure , drugs , tumors , and herpes zoster infection . </S>", "<S> we report an acquired port - wine stain in a 41-year - old male . </S>", "<S> the causative factors , treatment , and previous reports of this uncommon entity have also been reviewed . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["port - wine stains ( pwss ) are congenital vascular malformations comprised of ectatic dermal capillaries and affecting 0.3 - 1% of newborn infants .", "acquired pwss are an uncommon entity developing later in life , but morphologically and histologically similar to the congenital lesions .", "we hereby report a case of 41-year - old male patient who acquired pws about a year ago ."], ["a 41-year - old male presented to the outpatient department of dermatology of our tertiary care hospital with complaint of asymptomatic reddish patches involving the lower part of right leg for 10 months .", "the patch started from near the ankle and gradually spread to involve whole of the medial aspect of right leg over a period of 10 months .", "we could not elicit any history of antecedent mechanical or thermal trauma , drug intake , topical application or excessive ultraviolet exposure .", "dermatological examination revealed a diffuse partially blanchable erythema extending from the right ankle and involving chiefly the medial aspect of right leg up to the knee [ figure 1 ] .", "there were also a few discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg [ figure 2 ] .", "a diffuse partially blanchable erythema extending from the right ankle and involving the medial aspect of right leg up to the knee discrete irregularly shaped partially blanchable reddish - purple macules present on the anterior aspect of right leg on the basis of the clinical examination , a provisional diagnosis of acquired pws was made and the patient investigated .", "the histopathological examination revealed multiple dilated , variably sized capillaries lined by a single layer of endothelial cells throughout the dermis showing lobular arrangement at places [ figure 3 ]", ". color doppler of the limb did not reveal any abnormality of the underlying arteries and veins .", "( a ) multiple dilated variably sized capillaries in the dermis ( h and e , 40 , arrows ) .", "( b ) high power showing groups of capillaries lined by a single layer of endothelial cells ( h and e , 250 , arrow ) laser therapy was discussed with the patient , but he declined treatment for the lesion ."], ["pwss are congenital vascular malformations usually presenting at birth in the form of pink - red to purple macules , which become darker , raised , and nodular as the person ages .", "the most accepted hypothesis is a defective embryological maturation of the sympathetic fibers , resulting in a loss of normal sympathetic control of the cutaneous vessels which leads to ectasia .", "various factors have been proposed for the occurrence of acquired pwss . of these , trauma has been found to be the most important causative factor in majority of the cases .", "trauma - induced pws was first described by fegeler in 1949 and hence called fegeler syndrome . in a review of 59 patients with acquired pws , trauma was found to be a causative factor in 17 ( 29% ) cases .", "it has been proposed that injury may result in loss of a previously effective sympathetic regulation of the cutaneous blood flow , leading to development of the lesions .", "reports of acquired port - wine stain few cases of acquired pws have also been proposed secondary to oral medications like isotretinoin , oral contraceptive pills , simvastatin , and metformin .", "isotretinoin causes skin fragility and frictional trauma , while simvastain and metformin have been shown to promote angiogenesis by upregulation of vascular endothelial growth factor .", "isolated cases of acquired pws following frostbite injury , obstruction of the peritoneovenous shunt , herpes zoster infection , cerebral arteriovenous malformation , spinal root compression , and solid brain tumor have also been reported .", "no definite cause was found for the development of lesion in our patient . in such cases , a likely possibility of having a latent vascular anomaly which manifests clinically after unnoticed trauma", "an unusual finding in our patient was the presence of a lobular arrangement of dilated vessels in the dermis which has not been observed in previous cases of congenital or acquired pws .", "interestingly , there are multiple reports of development of pyogenic granulomas ( pg ) within pws which also shows a lobular pattern of dilated vessels .", "it has been proposed that the pg probably results from an underlying arteriovenous anastamosis frequently associated with the pws .", "though , there was no clinically appreciable pyogenic granuloma like lesion , the distinct histological features seen in our patient might be attributed to a similar pathogenetic mechanism .", "as regards therapy , it includes treatment with krypton , argon , argon - pumped , tunable dye , neodymium : yttrium - aluminum garnet , copper vapor , and pulsed dye lasers .", "acquired pws tends to have a quicker and better response to pulsed dye therapy than congenital pws .", "the case report demonstrates a rare occurrence of a vascular malformation in an adult patient .", "although no definite cause could be appreciated for the development of lesion in our patient , further reports and future insights into the pathogenesis of such cases would broaden our knowledge regarding this infrequently reported phenomenon .", "this is the first report of an acquired port - wine stain from india and previous reports of such cases have also been reviewed ."]]}
{"article_id": "PMC4353441", "article_text": ["ionizing radiation is a well - known carcinogen in humans , and breast is one of the most sensitive organs to radiogenic cancer .", "the rate of breast cancer in postwar japan was among the lowest in the world , but breast cancer contributed a disproportionately large fraction of the radiation - related cancer burden in atomic bomb survivors [ 2 , 3 ] .", "the data from the hiroshima and nagasaki survivors provides strong evidence for increased breast cancer following single acute doses of 20  cgy and linearity with increasing dose [ 36 ] .", "also , an increase in the incidence of breast cancer has been observed in areas affected by the chernobyl accident , which resulted in radioactive contamination of large areas of belarus and ukraine . a twofold increase in risk", "was observed when comparing the most ( > 40  msv cumulative dose ) and least contaminated regions .", "interestingly , the increase appeared 10 years after exposure and was most prominent in women exposed at younger age .", "more than 50,000 women in the united states have been treated with chest radiation ( 20  gy ) for a pediatric or young adult cancer .", "children treated from cancer with radiotherapy have a 2.9 relative risk of subsequent malignancy compared to those who were not [ 8 , 9 ] .", "a systematic review of 14 studies concluded that risk of breast cancer increased as early as 8 years following chest radiation and did not plateau with increasing length of follow - up .", "studies estimating low - dose radiation - induced cancer risk from diagnostic x - rays and ct scans have found a small but significant increased lifetime risk [ 11 , 12 ] . while the benefits of diagnostic x - ray and ct scans outweigh potential individual lifetime risk , their use should be justified and alternatives considered .", "we know remarkably little of molecular mechanisms that may be protective or risky for breast cancer after exposure to low - dose ionizing radiation ( ldir ) .", "identification of transcriptomic changes induced by ldir in mammary tissue will be valuable to elucidate the molecular mechanisms associated with radiation - induced breast cancer .", "long noncoding rnas ( lncrnas ) , which initially were thought of as transcriptional noise , are emerging as key regulators of a multitude of cellular processes by taking part in epigenetic , transcriptional , and posttranscriptional regulation of gene expression [ 13 , 14 ] .", "the lncrnas have a weaker evolutionary constraint and lower levels of expression compared to the protein - coding transcripts [ 15 , 16 ] but exhibit more tissue specific expression than the protein - coding genes .", "recently , a number of studies have shown that lncrna expression can be deregulated in human cancers [ 17 , 18 ] . as the functions of individual lncrnas in cancer are beginning to be elucidated ,", "they are being categorized and referred to as either tumor suppressor or oncogenic lncrnas , in the same way as traditional protein - coding cancer genes .", "however , the relevance of lncrnas in the cell and tissue response to ionizing radiation has not yet been characterized . in this study , we used agilent sureprint g3 microarrays to profile lncrna and mrna from mammary glands of balb / c mice 2 , 4 , and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10  cgy of x - radiation .", "we identified lncrna and mrna expression signatures for each time point after irradiation in comparison to sham .", "of the total 1338 lncrnas identified to be differentially expressed after ldir in either balb / c or spret / eij , 1337 had a significantly correlated expression pattern with at least one mrna that was also differentially expressed after ldir .", "our results indicate lncrnas may exert a partial or key role in the regulation of coding rna expression induced by radiation .", "balb / c and spret / eij mice were purchased from jackson laboratory , housed four per cage under a 12  hr light and 12  hr dark cycle , and fed with lab diet 5008 chow and water ad libitum .", "the mice were irradiated whole body at 8 - 9 weeks of age to a single dose of 10  cgy using a precision x - ray inc rad320 320  kvp x - ray machine , operated at 300  kv , 2  ma .", "mammary tissues were collected for gene expression profile at 2 , 4 , and 8 weeks after irradiation .", "all animal experiments were performed at lawrence berkeley national laboratory and the study was carried out in strict accordance with the guide for the care and use of laboratory animals of the national institutes of health .", "the animal use protocol was approved by the animal welfare and research committee of the lawrence berkeley national laboratory .", "agilent sureprint g3 mouse ge 8x60k microarrays were used according to the manufacturer 's protocol ( arrays contained 39,430 entrez gene rnas and 16,251 lncrnas ) .", "genes that were differentially expressed between sham and irradiated were identified by the unpaired student 's t - test with a p value cut - off of 0.05 ( p value < 0.001 for baseline strain comparison ) and a fold - change criteria of more than 1.5 . for each of the 8 experimental treatment groups , the average expression values of the 3 biological replicates", "significantly correlated pairs of mrna and lncrna were calculated using a standard permutation test . in brief , for each potential mrna and lncrna pair , the 8 mrna values were randomly rearranged and a correlation coefficient was calculated between the 8 mrnas and 8 lncrnas values .", "the p value reported in this work represents the percentage of permutations leading to a higher correlation than the original correlation between the 8 mrnas and 8 lncrnas values . in other words ,", "the lower the p value is , the more likely the mrna and lncrna pair is not randomly associated .", "gene lists were annotated with biological functions using ingenuity pathway analysis ( ipa ) , kegg pathway analysis ( http://bioinfo.vanderbilt.edu/webgestalt/ ) and david go gene ontology ( http://david.abcc.ncifcrf.gov/ ; p  0.05 ) .", "annotations for the various shapes used in the ipa networks in figures 13 are shown in figure s3 in supplementary material available online at http://dx.doi.org/10.1155/2015/461038 .", "to identify potential lncrnas and mrnas that may determine susceptibility to radiation - induced breast cancer , we profiled two inbred strains of mice with differing genetic susceptibilities : balb / c mice as more sensitive and spret / eij as more resistant .", "balb / c mice carry two dna - pkcs polymorphisms with reduced catalytic subunit activity and defective nonhomologous - end - joining of double strand breaks .", "we identified 195 lncrnas as upregulated and 95 lncrnas as downregulated in balb / c in comparison to spret / eij ( fold - change 1.5 ; p value < 0.001 ) ( figure 1(a ) ; table s1 ) .", "additionally , 582 mrnas were upregulated and 402 mrnas were downregulated in balb / c in comparison to spret / eij ( fold - change 1.5 ; p value < 0.001 ) ( figure 1(b ) ; table s1 ) .", "gene ontology analyses of differentially expressed genes between balb / c and spret / eij showed significant enrichment for metabolic processes ( p = 5.9e  06 ) , ion binding ( p = 3.00e  08 ) , and chemokine signaling ( p = 0.02 ) ( figure 1(c ) ; table s2 ) .", "our analyses identified significant strain differences in gene expression between balb / c and spret / eij mammary tissues .", "we found significant differences in the expression of a number of chemokines including cxcl10 , ccl6 , and ccl25 , which were expressed at higher levels in mammary tissues of the more sensitive and susceptible balb / c mice and which have previously been associated with breast cancer progression when over expressed . to identify lncrna and mrna expression changes induced by low - dose ionizing radiation ( ldir ) , we profiled lncrna and mrna expression from mammary glands of balb / c mice 2 , 4 , and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10  cgy of x - radiation . for balb / c mice ,", "a total of 357 , 480 , and 335 lncrnas and 550 , 911 , and 389 coding rnas were identified to be differentially expressed at weeks 2 , 4 , and 8 after ir in comparison to sham ( fold - change 1.5 ; p value < 0.05 ) , respectively ( figure 2(a ) ; table s1 ) . for spret", "/ eij , a total of 327 lncrnas and 424 mrnas were identified as differentially expressed at week 4 after irradiation in comparison to sham ( fold - change 1.5 ; p value < 0.05 ) ( figure 3(a ) ; table s1 ) .", "few coding - rnas and lncrnas were found to be differentially expressed at different time points ( figure s1(a ) and s1(b ) ) and between balb / c and spret / eij ( figure s1(c ) ) . to determine the biological functions associated with the ldir response , we excluded genes whose levels fluctuate in the mouse mammary gland across the estrous cycle .", "we recently mapped transcript - level changes across the estrous cycle in the murine mammary gland using rna sequencing and defined a comprehensive estrous variable gene signature of 3893 genes whose levels fluctuate in mammary glands of balb / c mice . comparison of our mapped ldir genes in mammary glands of balb / c and spret / eij mice with the estrous signature revealed an approximate 20% overlap in balb / c ( figure 2(b ) ) and 9% overlap in spret / eij mice ( figure 3(b ) ) .", "nonoverlapping and differentially expressed ldir genes for each of the time points were then computationally mapped to biological functions , pathways , upstream regulators , and networks .", "these analyses suggested that the ldir response signatures in mammary glands of balb / c mice transitions between time points and is distinct from the ldir response in spret / eij mice .", "two weeks after ldir exposure pathways and biological functions significantly enriched in mammary glands of balb / c mice compared to sham irradiated mice included chemokine signaling ( p = 0.01 ) , ccr3 signaling in eosinophils ( p = 0.05 ) , cellular movement ( 3.92e  04", "< p < 3.41e  02 ) , and cell death and survival ( 1.29e  03", "gene interaction networks were enriched for tissue and endocrine system injury ( figure 2(c ) top panel ) and significant predicted upstream regulators ( table s3 ) include gli2 ( p = 5.29e  04 ) and satb1 ( p = 1.98e  03 ) . similar to the two - week ldir response , gli2 was predicted to be an upstream regulator ( p = 4.52e  03 ; table s3 ) .", "gata3 and stat6 were among other significant upstream regulators associated with the four - week low - dose response ( table s3 ) .", "we furthermore observed that the mammary gland of balb / c mice four weeks after ldir was enriched for inflammatory response genes ( 1.84e  05", "< p < 1.45e  02 ) , cell - cell signaling ( 2.79e  05", "< p < 1.60e  02 ) , while gene interaction networks were enriched for lipid metabolism ( figure 2(c ) middle panel ) .", "interestingly , similar responses were observed in mammary glands of spret mice at 4  weeks after ldir including inflammatory response functions ( 1.69e  03", "< p < 4.49e  02 ) , cell - cell signaling ( 5.54e  04", "< p < 4.49e  02 ) , suggesting that the functional response is similar across strains and is independent of the gene transcript response . at 8 weeks after ldir we observed downregulation of genes involved in mammary gland development including progesterone receptor , prolactin , amphiregulin , and wnt4 ( figure 2(c ) bottom panel ; table s3 ) . to identify lncrnas potentially regulating the expression of coding rnas in response to radiation ,", "correlation coefficients on expression data were calculated for each coding rna and lncrna that were identified as differentially expressed after ldir .", "a permutation - based algorithm was then used to determine which correlations were statistically significant ( p < 0.05 ; table s4 ) .", "we observed that nearly all ldir modulated lncrnas were correlated with at least one of the ldir modulated coding mrnas ( figure 4(a ) ) . to determine whether these correlations were driven by estrous variations , we only considered genes whose expression levels were not overlapping with our previously determined estrous signature ( figure 2(b ) ) . again , we observed that nearly all differentially expressed lncrnas were correlated with at least one differentially expressed mrna suggesting that estrous cycling does not affect the strong correlation between lncrna and mrna expression after ldir . to test the robustness of these correlations , we compared the number of lncrnas associated with at least one mrna at three different p values ( p < 5e  02 , p < 5e  03 , and p < 5e  04 ) . at p", "< 5e  02 or p < 5e  03 , nearly all ( 97100% ) differentially expressed lncrnas were found to be correlated with at least one mrna ( table s5 ) . at p", "< 5e  04 , corresponding to a correlation coefficient > 0.9 , we still observed a significant fraction ( 6381% ; table s5 ) of lncrnas correlated with mrnas .", "representative correlation networks of lncrnas are shown in figure 4(b ) ( p < 5e  03 ) for each of the timepoints .", "we furthermore observed that the same lncrna correlates with different gene sets across different time points ( figure s2 ) .", "taken together these data show that ldir induces coordinated changes in lncrna and mrnas and suggests a critical role for lncrnas in mediating the low - dose radiation response .", "in this study , we demonstrate that genetic background strongly influences the expression of lncrnas and their response to low - dose radiation by transcriptomic analysis of mouse mammary glands using microarrays that contain both lncrnas and coding rnas .", "we have identified a number of lncrnas that are significantly changed after exposure to ldir at three different timepoints after radiation exposure .", "moreover , the changes in the expression of lncrnas are significantly correlated with the expression of coding rnas , suggesting that lncrnas may coordinate the tissue response to radiation via regulation of coding mrnas .", "however , the specific regulatory mechanism of this control requires further investigation , and knock - out and overexpression of the lncrna genes in mice and other model systems should be performed to increase our understanding of the regulatory mechanisms in response to ldir ."], "abstract_text": ["<S> long noncoding rnas ( lncrnas ) are emerging as key regulators of diverse cell functions and processes . </S>", "<S> however , the relevance of lncrnas in the cell and tissue response to ionizing radiation has not yet been characterized . </S>", "<S> here we used microarray profiling to determine lncrna and mrna expression in mammary glands of balb / c and spret / eij mice after low - dose ionizing radiation ( ldir ) exposure . </S>", "<S> we found that unirradiated mammary tissues of these strains differed significantly in baseline expressions of 290 lncrnas . </S>", "<S> ldir exposure ( 10  cgy ) induced a significant change in the expression of many lncrnas . </S>", "<S> the vast majority of lncrnas identified to be differentially expressed after ldir in either balb / c or spret / eij had a significantly correlated expression pattern with at least one ldir responsive mrna . </S>", "<S> functional analysis revealed that the response to ldir in balb / c mice is highly dynamic with enrichment for genes involved in tissue injury , inflammatory responses , and mammary gland development at 2 , 4 , and 8 weeks after ldir , respectively . </S>", "<S> our study demonstrates that genetic background strongly influences the expression of lncrnas and their response to radiation and that lncrnas may coordinate the tissue response to ldir exposure via regulation of coding mrnas . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusions"], "sections": [["ionizing radiation is a well - known carcinogen in humans , and breast is one of the most sensitive organs to radiogenic cancer .", "the rate of breast cancer in postwar japan was among the lowest in the world , but breast cancer contributed a disproportionately large fraction of the radiation - related cancer burden in atomic bomb survivors [ 2 , 3 ] .", "the data from the hiroshima and nagasaki survivors provides strong evidence for increased breast cancer following single acute doses of 20  cgy and linearity with increasing dose [ 36 ] .", "also , an increase in the incidence of breast cancer has been observed in areas affected by the chernobyl accident , which resulted in radioactive contamination of large areas of belarus and ukraine . a twofold increase in risk", "was observed when comparing the most ( > 40  msv cumulative dose ) and least contaminated regions .", "interestingly , the increase appeared 10 years after exposure and was most prominent in women exposed at younger age .", "more than 50,000 women in the united states have been treated with chest radiation ( 20  gy ) for a pediatric or young adult cancer .", "children treated from cancer with radiotherapy have a 2.9 relative risk of subsequent malignancy compared to those who were not [ 8 , 9 ] .", "a systematic review of 14 studies concluded that risk of breast cancer increased as early as 8 years following chest radiation and did not plateau with increasing length of follow - up .", "studies estimating low - dose radiation - induced cancer risk from diagnostic x - rays and ct scans have found a small but significant increased lifetime risk [ 11 , 12 ] . while the benefits of diagnostic x - ray and ct scans outweigh potential individual lifetime risk , their use should be justified and alternatives considered .", "we know remarkably little of molecular mechanisms that may be protective or risky for breast cancer after exposure to low - dose ionizing radiation ( ldir ) .", "identification of transcriptomic changes induced by ldir in mammary tissue will be valuable to elucidate the molecular mechanisms associated with radiation - induced breast cancer .", "long noncoding rnas ( lncrnas ) , which initially were thought of as transcriptional noise , are emerging as key regulators of a multitude of cellular processes by taking part in epigenetic , transcriptional , and posttranscriptional regulation of gene expression [ 13 , 14 ] .", "the lncrnas have a weaker evolutionary constraint and lower levels of expression compared to the protein - coding transcripts [ 15 , 16 ] but exhibit more tissue specific expression than the protein - coding genes .", "recently , a number of studies have shown that lncrna expression can be deregulated in human cancers [ 17 , 18 ] . as the functions of individual lncrnas in cancer are beginning to be elucidated ,", "they are being categorized and referred to as either tumor suppressor or oncogenic lncrnas , in the same way as traditional protein - coding cancer genes .", "however , the relevance of lncrnas in the cell and tissue response to ionizing radiation has not yet been characterized . in this study , we used agilent sureprint g3 microarrays to profile lncrna and mrna from mammary glands of balb / c mice 2 , 4 , and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10  cgy of x - radiation .", "we identified lncrna and mrna expression signatures for each time point after irradiation in comparison to sham .", "of the total 1338 lncrnas identified to be differentially expressed after ldir in either balb / c or spret / eij , 1337 had a significantly correlated expression pattern with at least one mrna that was also differentially expressed after ldir .", "our results indicate lncrnas may exert a partial or key role in the regulation of coding rna expression induced by radiation ."], ["balb / c and spret / eij mice were purchased from jackson laboratory , housed four per cage under a 12  hr light and 12  hr dark cycle , and fed with lab diet 5008 chow and water ad libitum .", "the mice were irradiated whole body at 8 - 9 weeks of age to a single dose of 10  cgy using a precision x - ray inc rad320 320  kvp x - ray machine , operated at 300  kv , 2  ma .", "mammary tissues were collected for gene expression profile at 2 , 4 , and 8 weeks after irradiation .", "all animal experiments were performed at lawrence berkeley national laboratory and the study was carried out in strict accordance with the guide for the care and use of laboratory animals of the national institutes of health .", "the animal use protocol was approved by the animal welfare and research committee of the lawrence berkeley national laboratory .", "agilent sureprint g3 mouse ge 8x60k microarrays were used according to the manufacturer 's protocol ( arrays contained 39,430 entrez gene rnas and 16,251 lncrnas ) .", "genes that were differentially expressed between sham and irradiated were identified by the unpaired student 's t - test with a p value cut - off of 0.05 ( p value < 0.001 for baseline strain comparison ) and a fold - change criteria of more than 1.5 . for each of the 8 experimental treatment groups , the average expression values of the 3 biological replicates", "significantly correlated pairs of mrna and lncrna were calculated using a standard permutation test . in brief , for each potential mrna and lncrna pair , the 8 mrna values were randomly rearranged and a correlation coefficient was calculated between the 8 mrnas and 8 lncrnas values .", "the p value reported in this work represents the percentage of permutations leading to a higher correlation than the original correlation between the 8 mrnas and 8 lncrnas values . in other words ,", "the lower the p value is , the more likely the mrna and lncrna pair is not randomly associated .", "gene lists were annotated with biological functions using ingenuity pathway analysis ( ipa ) , kegg pathway analysis ( http://bioinfo.vanderbilt.edu/webgestalt/ ) and david go gene ontology ( http://david.abcc.ncifcrf.gov/ ; p  0.05 ) .", "annotations for the various shapes used in the ipa networks in figures 13 are shown in figure s3 in supplementary material available online at http://dx.doi.org/10.1155/2015/461038 ."], ["to identify potential lncrnas and mrnas that may determine susceptibility to radiation - induced breast cancer , we profiled two inbred strains of mice with differing genetic susceptibilities : balb / c mice as more sensitive and spret / eij as more resistant .", "balb / c mice carry two dna - pkcs polymorphisms with reduced catalytic subunit activity and defective nonhomologous - end - joining of double strand breaks .", "we identified 195 lncrnas as upregulated and 95 lncrnas as downregulated in balb / c in comparison to spret / eij ( fold - change 1.5 ; p value < 0.001 ) ( figure 1(a ) ; table s1 ) .", "additionally , 582 mrnas were upregulated and 402 mrnas were downregulated in balb / c in comparison to spret / eij ( fold - change 1.5 ; p value < 0.001 ) ( figure 1(b ) ; table s1 ) .", "gene ontology analyses of differentially expressed genes between balb / c and spret / eij showed significant enrichment for metabolic processes ( p = 5.9e  06 ) , ion binding ( p = 3.00e  08 ) , and chemokine signaling ( p = 0.02 ) ( figure 1(c ) ; table s2 ) .", "our analyses identified significant strain differences in gene expression between balb / c and spret / eij mammary tissues .", "we found significant differences in the expression of a number of chemokines including cxcl10 , ccl6 , and ccl25 , which were expressed at higher levels in mammary tissues of the more sensitive and susceptible balb / c mice and which have previously been associated with breast cancer progression when over expressed . to identify lncrna and mrna expression changes induced by low - dose ionizing radiation ( ldir ) , we profiled lncrna and mrna expression from mammary glands of balb / c mice 2 , 4 , and 8 weeks after irradiation and of spret / eij mice 4 weeks after irradiation with 10  cgy of x - radiation . for balb / c mice ,", "a total of 357 , 480 , and 335 lncrnas and 550 , 911 , and 389 coding rnas were identified to be differentially expressed at weeks 2 , 4 , and 8 after ir in comparison to sham ( fold - change 1.5 ; p value < 0.05 ) , respectively ( figure 2(a ) ; table s1 ) . for spret", "/ eij , a total of 327 lncrnas and 424 mrnas were identified as differentially expressed at week 4 after irradiation in comparison to sham ( fold - change 1.5 ; p value < 0.05 ) ( figure 3(a ) ; table s1 ) .", "few coding - rnas and lncrnas were found to be differentially expressed at different time points ( figure s1(a ) and s1(b ) ) and between balb / c and spret / eij ( figure s1(c ) ) . to determine the biological functions associated with the ldir response , we excluded genes whose levels fluctuate in the mouse mammary gland across the estrous cycle .", "we recently mapped transcript - level changes across the estrous cycle in the murine mammary gland using rna sequencing and defined a comprehensive estrous variable gene signature of 3893 genes whose levels fluctuate in mammary glands of balb / c mice . comparison of our mapped ldir genes in mammary glands of balb / c and spret / eij mice with the estrous signature revealed an approximate 20% overlap in balb / c ( figure 2(b ) ) and 9% overlap in spret / eij mice ( figure 3(b ) ) .", "nonoverlapping and differentially expressed ldir genes for each of the time points were then computationally mapped to biological functions , pathways , upstream regulators , and networks .", "these analyses suggested that the ldir response signatures in mammary glands of balb / c mice transitions between time points and is distinct from the ldir response in spret / eij mice .", "two weeks after ldir exposure pathways and biological functions significantly enriched in mammary glands of balb / c mice compared to sham irradiated mice included chemokine signaling ( p = 0.01 ) , ccr3 signaling in eosinophils ( p = 0.05 ) , cellular movement ( 3.92e  04", "< p < 3.41e  02 ) , and cell death and survival ( 1.29e  03", "gene interaction networks were enriched for tissue and endocrine system injury ( figure 2(c ) top panel ) and significant predicted upstream regulators ( table s3 ) include gli2 ( p = 5.29e  04 ) and satb1 ( p = 1.98e  03 ) . similar to the two - week ldir response , gli2 was predicted to be an upstream regulator ( p = 4.52e  03 ; table s3 ) .", "gata3 and stat6 were among other significant upstream regulators associated with the four - week low - dose response ( table s3 ) .", "we furthermore observed that the mammary gland of balb / c mice four weeks after ldir was enriched for inflammatory response genes ( 1.84e  05", "< p < 1.45e  02 ) , cell - cell signaling ( 2.79e  05", "< p < 1.60e  02 ) , while gene interaction networks were enriched for lipid metabolism ( figure 2(c ) middle panel ) .", "interestingly , similar responses were observed in mammary glands of spret mice at 4  weeks after ldir including inflammatory response functions ( 1.69e  03", "< p < 4.49e  02 ) , cell - cell signaling ( 5.54e  04", "< p < 4.49e  02 ) , suggesting that the functional response is similar across strains and is independent of the gene transcript response . at 8 weeks after ldir we observed downregulation of genes involved in mammary gland development including progesterone receptor , prolactin , amphiregulin , and wnt4 ( figure 2(c ) bottom panel ; table s3 ) . to identify lncrnas potentially regulating the expression of coding rnas in response to radiation ,", "correlation coefficients on expression data were calculated for each coding rna and lncrna that were identified as differentially expressed after ldir .", "a permutation - based algorithm was then used to determine which correlations were statistically significant ( p < 0.05 ; table s4 ) .", "we observed that nearly all ldir modulated lncrnas were correlated with at least one of the ldir modulated coding mrnas ( figure 4(a ) ) . to determine whether these correlations were driven by estrous variations , we only considered genes whose expression levels were not overlapping with our previously determined estrous signature ( figure 2(b ) ) . again , we observed that nearly all differentially expressed lncrnas were correlated with at least one differentially expressed mrna suggesting that estrous cycling does not affect the strong correlation between lncrna and mrna expression after ldir . to test the robustness of these correlations , we compared the number of lncrnas associated with at least one mrna at three different p values ( p < 5e  02 , p < 5e  03 , and p < 5e  04 ) . at p", "< 5e  02 or p < 5e  03 , nearly all ( 97100% ) differentially expressed lncrnas were found to be correlated with at least one mrna ( table s5 ) . at p", "< 5e  04 , corresponding to a correlation coefficient > 0.9 , we still observed a significant fraction ( 6381% ; table s5 ) of lncrnas correlated with mrnas .", "representative correlation networks of lncrnas are shown in figure 4(b ) ( p < 5e  03 ) for each of the timepoints .", "we furthermore observed that the same lncrna correlates with different gene sets across different time points ( figure s2 ) .", "taken together these data show that ldir induces coordinated changes in lncrna and mrnas and suggests a critical role for lncrnas in mediating the low - dose radiation response ."], ["in this study , we demonstrate that genetic background strongly influences the expression of lncrnas and their response to low - dose radiation by transcriptomic analysis of mouse mammary glands using microarrays that contain both lncrnas and coding rnas .", "we have identified a number of lncrnas that are significantly changed after exposure to ldir at three different timepoints after radiation exposure .", "moreover , the changes in the expression of lncrnas are significantly correlated with the expression of coding rnas , suggesting that lncrnas may coordinate the tissue response to radiation via regulation of coding mrnas .", "however , the specific regulatory mechanism of this control requires further investigation , and knock - out and overexpression of the lncrna genes in mice and other model systems should be performed to increase our understanding of the regulatory mechanisms in response to ldir ."]]}
{"article_id": "PMC5218954", "article_text": ["\n women with psychiatric diseases are treated with antipsychotic medication(s ) with limited evidence to support the safety of their use during pregnancy.olanzapine , a second - generation antipsychotic , is a us food and drug administration pregnancy category  c drug with no unequivocal evidence of harm to the fetus.we report the first case of tracheo - esophageal fistula as a possible teratogenic effect of olanzapine exposure . \n", "women with psychiatric diseases may become pregnant and are treated with antipsychotics without any proven evidence of safety . due to ethical issues , pregnant women", "are rarely included in clinical trials , leading to a dearth of data available on the safety of antipsychotic drugs in this population .", "there have been reports of congenital anomalies in newborns of mothers exposed to antipsychotic medications during pregnancy [ 14 ] .", "olanzapine , a second - generation antipsychotic , is a us food and drug administration ( fda ) pregnancy category  c drug [ 3 , 5 ] with no unequivocal evidence of harm to the fetus .", "a literature search revealed that maternal exposure to olanzapine appears to be associated with lumbar meningomyelocele , dysplastic kidney , hip dysplasia , atrioventricular canal defect , club foot , microcephaly , ventricular septal defect , absent fingers , craniosynostosis , cleft lip , encephalocele , aqueductal stenosis , etc .", "the possibility of further teratogenic effects of olanzapine can not be ruled out . in this case report , we report the first case of tracheo - esophageal fistula ( tef ) as a possible teratogenic effect of olanzapine exposure .", "we report the case of a 29-year - old married woman with bipolar disorder with mixed episodes who had been treated with olanzapine in the psychiatric outpatient department of our institute ( lady hardinge medical college , new delhi , india ) for the last 7  years who delivered a full - term female baby with tef in september 2013 .", "one week after her marriage in february 2008 , the patient developed psychotic symptoms such as excessive talking , episodes of crying and laughing , and forgetting things frequently .", "following initial unsuccessful local treatment , she was taken to the psychiatry department of a tertiary care hospital .", "after initial management of acute symptoms there , she continued her treatment from our institute , where she was prescribed olanzapine ( oleanz 10  mg tablet orally once daily ) . in december 2008 , she had a miscarriage at 4  months gestation , following which she stopped taking the medication of her own choice . a careful history / enquiry from the parents failed to reveal any data available regarding congenital malformation of the aborted fetus . in 2009", ", she conceived again ( it is noteworthy that the patient was not taking olanzapine during this pregnancy ) and delivered a full - term healthy female child in january 2010 .", "eight months after delivery of this baby the patient had a recurrence of psychotic symptoms for which she was prescribed the same treatment . in december 2012 , she conceived a third time but due to persistent psychiatric symptoms , olanzapine was continued throughout the pregnancy by the treating psychiatrist , who considered there to be a favorable benefit : risk ratio for the patient at that time .", "she received regular antenatal care and was compliant with all prescriptions and instructions , including prophylactic folic acid and two doses of tetanus toxoid .", "there was no history of maternal alcohol intake , smoking , or exposure to other potential teratogenic drugs or exogenous hormones throughout the pregnancy .", "all baseline hematological ( hemoglobin 13.8 g / dl , total leukocyte count 6000/l , platelets 2.3 lac/l , etc . ) and biochemical investigations ( random blood sugar 98 mg / dl , blood urea 27 mg / dl , etc . )", "tests for trisomy 21 and 18 ( nuchal translucency scan and dual marker tests ) and rubella were negative .", ", she delivered a full - term female baby by vaginal delivery . immediately after delivery ,", "the baby experienced respiratory distress and excessive salivation , for which she was admitted to the neonatal intensive care unit . on examination ,", "a red rubber catheter could not be negotiated beyond 10  cm in the esophagus .", "other concomitant anomalies including vertebral , anal , renal , limb ( components of vacterl [ vertebral defects , anal atresia , cardiac defects , tracheo - esophageal fistula , renal anomalies , and limb abnormalities ] ) , eye , ear , and nasal defects were ruled out .", "the tef was repaired using esophageal  esophageal anastomosis with a drainage tube in the fifth intercostal space . on the second post - operative day", "for this reason , cervical esophagotomy was performed and a gastrostomy was inserted for feeding purposes .", "the baby was discharged in a stable condition with advice for full breast milk feeding through gastrostomy , some required medications ( multivitamin syrup , calcium , anti - emetic and antibiotics ) for an appropriate duration , and regular vaccination . however , although the baby was well until this point , she was re - hospitalized at 11  months of age because of diarrhea and unfortunately died on 19 august 2014 , probably because of widespread infection ( no definitive record of the last hospitalization is available and a post - mortem was not performed).fig . ", "1chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula \n  chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula", "a number of drugs are clearly known to be associated with teratogenicity , such as thalidomide and isotretinoin , but the majority of drugs do not have conclusive data concerning their effect on fetal development and growth . due to strict ethical constraints , at - risk populations ( including pregnant women ) are not included in most clinical trials , limiting the availability of data on the use of drugs ( including antipsychotics ) in these populations , and thus spontaneous reporting of suspected adverse drug effects constitutes an essential tool for generating data on the safety of drugs , especially in the population of pregnant women . according to a retrospective analysis of data from the lilly safety database , of the 610 prospectively identified pregnancies exposed to olanzapine with an available outcome", ", there were 401 ( 66% ) normal births , 60 ( 9.8% ) premature births , 57 ( 9.3% ) spontaneous abortions , 49 ( 8% ) perinatal complications , 27 ( 4.4% ) congenital anomalies ( cleft lip , encephalocele , and aqueductal stenosis ) , and 16 ( 2.6% ) other outcomes ( post - perinatal condition , ectopic pregnancy , post - term birth , and still birth ) .", "other reported congenital anomalies with olanzapine included meningocele , ankyloblepharon , hip dysplasia , acheiria , atrioventricular canal defect , and unilateral club foot [ 1 , 68 ] . a link between teratogenicity and antipsychotics", "is also supported by the fact that these drugs readily cross the placental barrier [ 1 , 4 , 9 ] , with 23.8% placental passage for quetiapine and 72.1% for olanzapine [ 2 , 4 , 9 ] .", "newport  et al . measured placental passage of medication from mother to fetus by measuring levels in the umbilical cord serum and found that olanzapine has the highest rate of placental passage , compared with haloperidol , risperidone , and quetiapine [ 3 , 9 ] .", "the overall incidence of esophageal atresia / tef ranges from one in every 2500 to 4500 live births .", "however , a careful literature search revealed a paucity of data on the incidence of tef in children born to women with psychiatric disease and the problem of confounding by indication can not be ruled out .", "it has been proposed that antipsychotics interfere with the action of calmodulin , which has an important role in organogenesis .", "esophageal atresia and abnormalities of pharyngeal glands have also been proposed as being related to neural crest cells .", "the drugs ( olanzapine in this case ) associated with such anomalies may be hypothesized to cause neural crest cell damage during early fetal development as a causative factor . although the neural crest cell damage - related esophageal abnormalities in many cases are also associated with cardiovascular anomalies , these were absent in the present case .", "we tried to establish the causality between the adverse outcome ( tef ) and use of olanzapine during pregnancy according to the who - umc ( world health organization ", "uppsala monitoring centre ) adverse drug reaction causality assessment scale and the naranjo causality assessment scale .", "causality is  probable ( score + 5 ) as per the naranjo scale , whereas it is ", "one of the important reasons for lower scores of causality is the non - applicability of many questions in these scales .", "the current case report of tef associated with antenatal olanzapine exposure demonstrates the need for large clinical studies to generate more conclusive data concerning use of this drug during pregnancy .", "on the basis of the present case and the literature , it is suggested that antenatal olanzapine exposure may be related to the development of tef in newborns .", "however , the present case report can only be considered as preliminary evidence and further observational prospective studies of women with antenatal exposure to olanzapine should be conducted to explore the strength of association between tef and olanzapine .", "knowledge of the teratogenic potential of a drug is essential to prevent / reduce the morbidity and mortality associated with a congenital anomaly ( tef in this case ) .", "such post - marketing reports are effective tools to disclose the adverse effects of drugs , which are difficult to detect in clinical trials and may factually change the safety status of a drug .", "written informed consent was obtained from the infant s father for publication of this case report ."], "abstract_text": ["<S> there is a dearth of evidence on the safety of the use of antipsychotics during pregnancy . </S>", "<S> olanzapine , a pregnancy category  c drug , has no unequivocal evidence of harm to the fetus . against this backdrop </S>", "<S> , we report the first case of a tracheo - esophageal fistula ( tef ) in a newborn following maternal antenatal exposure to olanzapine . </S>", "<S> a 29-year - old woman with acute psychotic disorder had been treated with olanzapine for the last 7  years . her first pregnancy , while taking olanzapine , resulted in a miscarriage at 4  months gestation , following which she discontinued olanzapine . </S>", "<S> she reconceived after a few months and delivered a full - term normal child . </S>", "<S> however , due to the recurrence of psychiatric illness after her second pregnancy , she was prescribed olanzapine again , which was continued throughout her third pregnancy . </S>", "<S> the outcome of the third pregnancy was a full - term female baby with a tef . </S>", "<S> the baby was managed surgically and discharged with satisfactory vital signs . </S>", "<S> unfortunately , however , the baby did not survive beyond 11  months of age . </S>", "<S> causality between antenatal maternal olanzapine exposure and tef in the newborn was determined to be  </S>", "<S> probable ( score + 5 ) as per the naranjo causality assessment scale . </S>", "<S> greater knowledge of this potential teratogenicity caused by olanzapine is needed to reduce morbidity and mortality in newborns . </S>"], "labels": null, "section_names": ["Key Points", "Introduction", "Case Narrative", "Discussion", "Conclusion", "Conflict of interest", "Funding", "Informed consent"], "sections": [["\n women with psychiatric diseases are treated with antipsychotic medication(s ) with limited evidence to support the safety of their use during pregnancy.olanzapine , a second - generation antipsychotic , is a us food and drug administration pregnancy category  c drug with no unequivocal evidence of harm to the fetus.we report the first case of tracheo - esophageal fistula as a possible teratogenic effect of olanzapine exposure . \n"], ["women with psychiatric diseases may become pregnant and are treated with antipsychotics without any proven evidence of safety . due to ethical issues , pregnant women", "are rarely included in clinical trials , leading to a dearth of data available on the safety of antipsychotic drugs in this population .", "there have been reports of congenital anomalies in newborns of mothers exposed to antipsychotic medications during pregnancy [ 14 ] .", "olanzapine , a second - generation antipsychotic , is a us food and drug administration ( fda ) pregnancy category  c drug [ 3 , 5 ] with no unequivocal evidence of harm to the fetus .", "a literature search revealed that maternal exposure to olanzapine appears to be associated with lumbar meningomyelocele , dysplastic kidney , hip dysplasia , atrioventricular canal defect , club foot , microcephaly , ventricular septal defect , absent fingers , craniosynostosis , cleft lip , encephalocele , aqueductal stenosis , etc .", "the possibility of further teratogenic effects of olanzapine can not be ruled out . in this case report , we report the first case of tracheo - esophageal fistula ( tef ) as a possible teratogenic effect of olanzapine exposure ."], ["we report the case of a 29-year - old married woman with bipolar disorder with mixed episodes who had been treated with olanzapine in the psychiatric outpatient department of our institute ( lady hardinge medical college , new delhi , india ) for the last 7  years who delivered a full - term female baby with tef in september 2013 .", "one week after her marriage in february 2008 , the patient developed psychotic symptoms such as excessive talking , episodes of crying and laughing , and forgetting things frequently .", "following initial unsuccessful local treatment , she was taken to the psychiatry department of a tertiary care hospital .", "after initial management of acute symptoms there , she continued her treatment from our institute , where she was prescribed olanzapine ( oleanz 10  mg tablet orally once daily ) . in december 2008 , she had a miscarriage at 4  months gestation , following which she stopped taking the medication of her own choice . a careful history / enquiry from the parents failed to reveal any data available regarding congenital malformation of the aborted fetus . in 2009", ", she conceived again ( it is noteworthy that the patient was not taking olanzapine during this pregnancy ) and delivered a full - term healthy female child in january 2010 .", "eight months after delivery of this baby the patient had a recurrence of psychotic symptoms for which she was prescribed the same treatment . in december 2012 , she conceived a third time but due to persistent psychiatric symptoms , olanzapine was continued throughout the pregnancy by the treating psychiatrist , who considered there to be a favorable benefit : risk ratio for the patient at that time .", "she received regular antenatal care and was compliant with all prescriptions and instructions , including prophylactic folic acid and two doses of tetanus toxoid .", "there was no history of maternal alcohol intake , smoking , or exposure to other potential teratogenic drugs or exogenous hormones throughout the pregnancy .", "all baseline hematological ( hemoglobin 13.8 g / dl , total leukocyte count 6000/l , platelets 2.3 lac/l , etc . ) and biochemical investigations ( random blood sugar 98 mg / dl , blood urea 27 mg / dl , etc . )", "tests for trisomy 21 and 18 ( nuchal translucency scan and dual marker tests ) and rubella were negative .", ", she delivered a full - term female baby by vaginal delivery . immediately after delivery ,", "the baby experienced respiratory distress and excessive salivation , for which she was admitted to the neonatal intensive care unit . on examination ,", "a red rubber catheter could not be negotiated beyond 10  cm in the esophagus .", "other concomitant anomalies including vertebral , anal , renal , limb ( components of vacterl [ vertebral defects , anal atresia , cardiac defects , tracheo - esophageal fistula , renal anomalies , and limb abnormalities ] ) , eye , ear , and nasal defects were ruled out .", "the tef was repaired using esophageal  esophageal anastomosis with a drainage tube in the fifth intercostal space . on the second post - operative day", "for this reason , cervical esophagotomy was performed and a gastrostomy was inserted for feeding purposes .", "the baby was discharged in a stable condition with advice for full breast milk feeding through gastrostomy , some required medications ( multivitamin syrup , calcium , anti - emetic and antibiotics ) for an appropriate duration , and regular vaccination . however , although the baby was well until this point , she was re - hospitalized at 11  months of age because of diarrhea and unfortunately died on 19 august 2014 , probably because of widespread infection ( no definitive record of the last hospitalization is available and a post - mortem was not performed).fig . ", "1chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula \n  chest x - ray posteroanterior view of the newborn baby , who was born to a mother with antenatal exposure to olanzapine , suggestive of tracheo - esophageal fistula"], ["a number of drugs are clearly known to be associated with teratogenicity , such as thalidomide and isotretinoin , but the majority of drugs do not have conclusive data concerning their effect on fetal development and growth . due to strict ethical constraints , at - risk populations ( including pregnant women ) are not included in most clinical trials , limiting the availability of data on the use of drugs ( including antipsychotics ) in these populations , and thus spontaneous reporting of suspected adverse drug effects constitutes an essential tool for generating data on the safety of drugs , especially in the population of pregnant women . according to a retrospective analysis of data from the lilly safety database , of the 610 prospectively identified pregnancies exposed to olanzapine with an available outcome", ", there were 401 ( 66% ) normal births , 60 ( 9.8% ) premature births , 57 ( 9.3% ) spontaneous abortions , 49 ( 8% ) perinatal complications , 27 ( 4.4% ) congenital anomalies ( cleft lip , encephalocele , and aqueductal stenosis ) , and 16 ( 2.6% ) other outcomes ( post - perinatal condition , ectopic pregnancy , post - term birth , and still birth ) .", "other reported congenital anomalies with olanzapine included meningocele , ankyloblepharon , hip dysplasia , acheiria , atrioventricular canal defect , and unilateral club foot [ 1 , 68 ] . a link between teratogenicity and antipsychotics", "is also supported by the fact that these drugs readily cross the placental barrier [ 1 , 4 , 9 ] , with 23.8% placental passage for quetiapine and 72.1% for olanzapine [ 2 , 4 , 9 ] .", "newport  et al . measured placental passage of medication from mother to fetus by measuring levels in the umbilical cord serum and found that olanzapine has the highest rate of placental passage , compared with haloperidol , risperidone , and quetiapine [ 3 , 9 ] .", "the overall incidence of esophageal atresia / tef ranges from one in every 2500 to 4500 live births .", "however , a careful literature search revealed a paucity of data on the incidence of tef in children born to women with psychiatric disease and the problem of confounding by indication can not be ruled out .", "it has been proposed that antipsychotics interfere with the action of calmodulin , which has an important role in organogenesis .", "esophageal atresia and abnormalities of pharyngeal glands have also been proposed as being related to neural crest cells .", "the drugs ( olanzapine in this case ) associated with such anomalies may be hypothesized to cause neural crest cell damage during early fetal development as a causative factor . although the neural crest cell damage - related esophageal abnormalities in many cases are also associated with cardiovascular anomalies , these were absent in the present case .", "we tried to establish the causality between the adverse outcome ( tef ) and use of olanzapine during pregnancy according to the who - umc ( world health organization ", "uppsala monitoring centre ) adverse drug reaction causality assessment scale and the naranjo causality assessment scale .", "causality is  probable ( score + 5 ) as per the naranjo scale , whereas it is ", "one of the important reasons for lower scores of causality is the non - applicability of many questions in these scales .", "the current case report of tef associated with antenatal olanzapine exposure demonstrates the need for large clinical studies to generate more conclusive data concerning use of this drug during pregnancy ."], ["on the basis of the present case and the literature , it is suggested that antenatal olanzapine exposure may be related to the development of tef in newborns .", "however , the present case report can only be considered as preliminary evidence and further observational prospective studies of women with antenatal exposure to olanzapine should be conducted to explore the strength of association between tef and olanzapine .", "knowledge of the teratogenic potential of a drug is essential to prevent / reduce the morbidity and mortality associated with a congenital anomaly ( tef in this case ) .", "such post - marketing reports are effective tools to disclose the adverse effects of drugs , which are difficult to detect in clinical trials and may factually change the safety status of a drug ."], [""], [""], ["written informed consent was obtained from the infant s father for publication of this case report ."]]}
{"article_id": "PMC3197260", "article_text": ["\n\t\t\t\t olfactory neuroblastoma ( onb ) is a rare malignant tumor arising from the olfactory epithelium of the nasal cavity and the paranasal sinuses .", "the anatomic origin in the superior nasal cavity leads to nonspecific symptoms that make early diagnosis difficult .", "direct extension of the tumor into the anterior cranial fossa , either at presentation or at disease recurrence , is common .", "onb is mostly a locally aggressive tumor that can spread to lymph nodes but metastatic involvement of bone , bone marrow , and other organs was also reported .", "the most commonly used staging system proposed by kadish is currently based on computer tomography and magnetic resonance imaging . in stage", "a , the tumor is limited to the nasal cavity , in stage b the tumor extends to paranasal sinus , and in stage c the tumor is extending over the nasal cavity and sinus and/or metastasized distantly .", "a histological grading system based on microscopic findings proposed by hyams at 1988 showed a clear correlation of survival with histological differentiation of disease .  ", "however most authors believe that craniofacial resection followed by rt offers the gold standard treatment in this disease . despite aggressive therapies , local , regional relapse , and distant metastasis occur frequently and often after extended periods of follow - up .", "data for this case series were acquired retrospectively from the files of division of medical oncology , cerrahpasa medical faculty , istanbul university .", "additional information regarding the clinical course and outcome was collected from the patients ' charts and phone calls to the patients , their relatives , and their general practitioners . in this study , we have reviewed 19 cases of olfactory neuroblastomas treated over 24-year period and analyzed the clinical features , treatment outcomes , and prognostic factors .", "the data of nineteen patients of olfactory neuroblastomas treated and followed up between 1986 and 2010 at division of medical oncology , cerrahpasa medical faculty , istanbul university , were analyzed retrospectively .", "patient characteristics , initial symptoms , tumor extent , histologic features , primary therapy , tumor recurrence , and treatments of recurrent disease were determined .", "patients were staged according to the kadish staging system . a single pathologist with expertise in head and neck neoplasms reviewed the histological specimens .", "local ethics committee 's approval and patients ' or their next of kin 's informed consent were taken before the study .", "treatment response was assessed by clinical examinations and computed tomography ( ct ) or magnetic resonance imaging ( mri ) .", "survival rates were calculated from diagnosis to the death of the patient using the kaplan - meier life table method .", "the madian age at diagnosis was 46 ( range : 19 to 68 years ) .", "tumor staging was kadish stage a in 3 , stage b in 5 , and stage c in 11 patients .", "four patients had metastasis of cervical lymph nodes , two brains , one bone marrow , one bone and lung metastases , and one bone , mediastinal lymph node , and liver metastases at the time of diagnosis .", "tumor localization , clinical symptoms , and presentations at the time of diagnosis are listed in table 1 .", "median time from onset of first symptoms until diagnosis was 8.8 months ( range : 1.829 months ) .", "the common presenting symptoms were nasal obstruction ( 53% ) , epistaxis ( 31% ) , rhinorrhea , and facial pain ( 26% ) .", "patient treatments consisted of surgical resection , radiation therapy , and chemotherapy ( ctx ) ( table 3 ) .", "initial treatments included surgery alone in three patients , radiotherapy ( rt ) with or without ctx in five , surgery plus postoperative rt in seven and multimodality therapy ( surgery plus postoperative ctx plus postoperative rt ) in three , and finally ctx alone in one . in 14 patients ,", "four of these patients had also radical neck dissection due to cervical lymph nodes involvement .", "nasal tumor resection in 2 patients , maxillectomy plus orbital exenteration in 1 , and external ethmoidectomy ( ee ) in 1 were also used as surgical treatments . of these 14 patients who underwent surgical tumor resection ,", "4 were treated with surgery alone , 5 with adjuvant radiation therapy after surgery , 2 with adjuvant ctx after surgery , and the remaining 3 with radiation therapy and ctx after surgery . of the remaining 5 patients , four in advanced stage and one in early stage refused surgical treatment .", "radiation therapy was administered to two of the patients , radiation therapy followed by ctx to the other two , and ctx alone to one patient .", "the average radiation dose was 57.9 gy and the ctx was chosen from cisplatin - based regimens . as a part of the primary treatment modality , 12 patients underwent radiation therapy , so did the five relapsed patients .", "as a part of primary treatment modality 8 patients received ctx , so did the three relapsed patients .   ", "fifteen patients died of tumor progression during the follow - up , of whom 9 were at an advanced stage ( stage c ) by the time of diagnosis and 6 at an early stage ( stage a / b ) .", "kadish stage c patients had a significantly poorer outcome compared to the stage a / b patients ( 2-year survival rate : 71 versus 25% , resp .", "histopathologically high - grade patients ( grade 3/4 ) had a significantly poorer outcome when compared to the lower grade ( grade 1/2 ) patients ( 2-year survival rate : 16 versus 50% , resp .", "primary tumor extensions to orbital area had a significantly poorer outcome compared to the tumors with no extension to orbital area ( 2-year survival rate : 22 versus 60% , resp .", "there was no statistically significant difference in survival rate between male and female patients ( 2-year survival rate : 40 versus 44% , resp .", "patients with age < 45 years had no poorer outcome compared to patients with age > 45 years ( 2-year survival rate : 41 versus 43% ; p = .059 ) .", "brain and bone marrow involvements showed poorer outcome compared to those without such involvement ( median survival rate : 9.1 versus 28.8 months , resp . ;", "survival rate was not significantly different among pateints of kadish c when stratified according to initial treatment ( p = .8 ) .  ", "locoregional recurrence developed in 6 and distant metastasis in 6 patients . during the follow - up period , these groups exhibited distant metastasis to the brain , spinal cord , bone , bone marrow , meninges , and lungs .", "as a treatment for recurrence and metastasis , reoperation of primary site plus radiation therapy in 4 patients and radiation therapy alone in 4 patients were offered .", "onb is a rare malignant tumor that comprises 4,65% of malignant nasal and paranasal tumors .", "some 1000 cases have been reported in the english literature since it was first introduced in 1924 .", "the incidence rate is bimodal , with peaks in the second and third decades of life and in the sixth and seventh decades of life and equal among females and males . as for our study ,", "the age at diagnosis was most common in the fourth decade and female - to - male ratio was equal .", "the average time between the appearance of the first symptom and the diagnosis was reported as 6 months and this period was similar also in our group .", "the most common symptoms at the initial diagnosis were defined as unilateral nasal obstruction and epistaxis in the literature .", "this tumor may be misdiagnosed as an undifferentiated small cell carcinoma , melanoma , rhabdomyosarcoma , or other small blue cell neoplasms .", "hyam proposed a grading system with grades 14 based on the presence or absence of seven different histopathological parameters .", "these are growth , architecture , mitotic activity , necrosis , nuclear pleomorphism , rosette formation , and fibrillary stroma .  ", ". staining paterns for neuron - spesific enolase , chromogranin , synaptophysin , s-100 , and epithelial markers can be helpful .  ", "there is evidence that histological grade of onb influences biologic behavior , particularly because it relates to disease progression , local recurrence , and metastasis . for the well - differentiated tumors a slower disease progression and less tendency for local recurrence have been described . in our study , the 2-year survival rate of this grade i / ii - iii / iv disease was 16 versus 50% , respectively .", "more aggressive treatment , such as surgery , radiation , and intensive ctx , may be useful for patients with grade iii / iv disease .", "a meta - analysis showed that the median overall survival at 5 years is 45% in 390 patients .", "the distribution of onb from 21 studies according to kadish staging system was 12% at stage a , 27% at stage b , and 61% at stage c. the mean 5-year survival is 75% for stage a , 68% for stage b , and 41% for stage c .", "the conclusions of the university of virginia stated that kadish stage is predictive of disease - related mortality .", "however , in the series of the mayo clinic it was found that kadish stage did not affect the outcome .", "danish clinicopathological study found that the staging system of kadish was able to stratify into prognostically significant groups . in our study , the 5-year survival rate of these tumors was 26% and median survival time was 23 months .", "the distribution of onb according to kadish staging system was 16% at stage a , 26% stage b , and 58% stage c. however , in our study , the 5-year survival rates of these stage groups were inferior compared to those reported in other studies .", "the cause of this poor result was presentation of 5 patients with distant metastases ( 26% ; 2 brain , 1 bone marrow , 2 other sites ) , 4 patients ( 21% ) with cervical lymph node metastasis , 12 patients ( 63% ) with high - grade histology at the time of diagnosis , and inability to evaluate the surgical margins of the resected tumors .", "the survival rate of node positive patients is 29% compared with 64% of node negative patients .", "distant metastases have been described in the pancreas , the liver , mediastinum , bone marrow , lungs , leptomeninges , skin , and breasts [ 11 , 12 ] .", "cervical nodal metastases can develop in 17 to 33% of patients and distant metastases in 10 to 40% of patients over the course of disease", "metastatic disease at presentation occurs in 10 to 50% of patients , depending on the study [ 6 , 13 , 14 ] .", "we found the local recurrence rate to be 31.5% and distant metastasis 31.5% after initial surgery . during the follow - up periods these groups exhibited distant metastases to the brain , spinal cord , bone , bone marrow , meninges , and lungs .", "patients who had brain and bone marrow metastases had poorer prognosis than others having metastatic deposits of other sites .", "involvement of the orbit is a serious prognostic factor , but when the orbital periosteum is affected without penetration into the orbital structures themselves , the eye may be preserved by resection and grafting of the periosteum without an adverse effect on survival as compared with orbital clearance .", "irrespective to the most sophisticated imaging , involvement of the dura and orbital periosteum can accurately be determined only at surgery . in our study ,", "this group 's ( 8 patients ) survival rate is poorer than those with no involvement of the orbita ( p = .042 ) .", "the institute of laryngology and otology , university college london , published a study of 42 patients treated over 23 years .", "overall and disease - free survival rates were 77 and 61% at 5 years , and 53 and 42% at 10 years , respectively .", "late recurrences were seen . based on this experience , combined therapy with craniofacial resection and radiation therapy is recommended by this group . in a retrospective study of 47 patients treated at multiple centers in germany from 1979 to 2001 ,", "the 5-year overall survival and event - free survival rate were 64% and 50% , respectively .", "patients who received multimodality therapy had a significantly better event - free survival rate compared with those who did not receive multimodality therapy ( 74 versus 41% ) .", "the authors recommend a combination therapy including ctx , surgery , and postoperative radiation therapy for kadish stage c patients . in our study ,", "initial treatment included surgery alone in three patients , rt with or without ctx in five , surgery plus postoperative rt in seven , and multimodality therapy ( surgery plus postoperative ctx plus postoperative rt ) in three , only ctx in one patient .", "however , in our study choice of treatment modality did not show a statistically significant difference in survival rate .", "the reasons for that might be including low number of patients in the study , being 5 distant site metastasis , 4 cervical lymph node metastasis at the time of diagnosis , only one orbital exentration ( although there were 8 orbital involvement ) , and 12 patients exhibiting high grade histology and inability to evaluate surgical margins in resected tumors . in conclusion , advanced tumor stage ,", "the initial histopathologic grade of iii / iv , and involvement of orbita , brain , or bone marrow metastasis were the statistically significant poor prognostic factors , whereas age , sex , and treatment modality were not prognostic factors ."], "abstract_text": ["<S> \n objective . </S>", "<S> the aim of this study was to evaluate clinicopathological findings and the efficacy of the treatment modalities used in patients with olfactory neuroblastomas . study design . </S>", "<S> retrospective record review . setting . </S>", "<S> istanbul university , cerrahpasa medical faculty , medical oncology outpatient clinic . </S>", "<S> subjects and methods . </S>", "<S> there were 3 stage a tumors , 5 stage b and 11 stage c according to the kadish staging system . </S>", "<S> there were 5 grade i / ii and 12 grade </S>", "<S> iii / iv according to the hyams ' histopathologic system . </S>", "<S> involvement to orbita was detected in eight patients at the time of diagnosis . </S>", "<S> results . </S>", "<S> the median follow - up period was 23.7   months . </S>", "<S> the 5-year survival rate for the whole group was 26% . </S>", "<S> the stage a / b groups exhibited a better survival rate than the c group with 2-year survival rates being   25 versus 71% respectively ( p = .008 ) . </S>", "<S> the grade i / ii groups exhibited a better survival rate than the grade iii / iv groups with 2-year survival rates being 50 versus 16% respectively ( p = .001 ) . </S>", "<S> the group who had orbital involvement exhibited a poor survival rate than the group of patients who had no involvement of the orbital . </S>", "<S> conclusion . in our study , </S>", "<S> tumor stage , histopathologic grading , involvement of the orbita , brain and bone marow metastases were the statistically significant prognostic factors . </S>"], "labels": null, "section_names": ["1. lntroduction", "2. Patients and Methods", "3. Statistical Analysis", "4. Results", "5. Discussion"], "sections": [["\n\t\t\t\t olfactory neuroblastoma ( onb ) is a rare malignant tumor arising from the olfactory epithelium of the nasal cavity and the paranasal sinuses .", "the anatomic origin in the superior nasal cavity leads to nonspecific symptoms that make early diagnosis difficult .", "direct extension of the tumor into the anterior cranial fossa , either at presentation or at disease recurrence , is common .", "onb is mostly a locally aggressive tumor that can spread to lymph nodes but metastatic involvement of bone , bone marrow , and other organs was also reported .", "the most commonly used staging system proposed by kadish is currently based on computer tomography and magnetic resonance imaging . in stage", "a , the tumor is limited to the nasal cavity , in stage b the tumor extends to paranasal sinus , and in stage c the tumor is extending over the nasal cavity and sinus and/or metastasized distantly .", "a histological grading system based on microscopic findings proposed by hyams at 1988 showed a clear correlation of survival with histological differentiation of disease .  ", "however most authors believe that craniofacial resection followed by rt offers the gold standard treatment in this disease . despite aggressive therapies , local , regional relapse , and distant metastasis occur frequently and often after extended periods of follow - up .", "data for this case series were acquired retrospectively from the files of division of medical oncology , cerrahpasa medical faculty , istanbul university .", "additional information regarding the clinical course and outcome was collected from the patients ' charts and phone calls to the patients , their relatives , and their general practitioners . in this study , we have reviewed 19 cases of olfactory neuroblastomas treated over 24-year period and analyzed the clinical features , treatment outcomes , and prognostic factors ."], ["the data of nineteen patients of olfactory neuroblastomas treated and followed up between 1986 and 2010 at division of medical oncology , cerrahpasa medical faculty , istanbul university , were analyzed retrospectively .", "patient characteristics , initial symptoms , tumor extent , histologic features , primary therapy , tumor recurrence , and treatments of recurrent disease were determined .", "patients were staged according to the kadish staging system . a single pathologist with expertise in head and neck neoplasms reviewed the histological specimens .", "local ethics committee 's approval and patients ' or their next of kin 's informed consent were taken before the study ."], ["treatment response was assessed by clinical examinations and computed tomography ( ct ) or magnetic resonance imaging ( mri ) .", "survival rates were calculated from diagnosis to the death of the patient using the kaplan - meier life table method ."], ["the madian age at diagnosis was 46 ( range : 19 to 68 years ) .", "tumor staging was kadish stage a in 3 , stage b in 5 , and stage c in 11 patients .", "four patients had metastasis of cervical lymph nodes , two brains , one bone marrow , one bone and lung metastases , and one bone , mediastinal lymph node , and liver metastases at the time of diagnosis .", "tumor localization , clinical symptoms , and presentations at the time of diagnosis are listed in table 1 .", "median time from onset of first symptoms until diagnosis was 8.8 months ( range : 1.829 months ) .", "the common presenting symptoms were nasal obstruction ( 53% ) , epistaxis ( 31% ) , rhinorrhea , and facial pain ( 26% ) .", "patient treatments consisted of surgical resection , radiation therapy , and chemotherapy ( ctx ) ( table 3 ) .", "initial treatments included surgery alone in three patients , radiotherapy ( rt ) with or without ctx in five , surgery plus postoperative rt in seven and multimodality therapy ( surgery plus postoperative ctx plus postoperative rt ) in three , and finally ctx alone in one . in 14 patients ,", "four of these patients had also radical neck dissection due to cervical lymph nodes involvement .", "nasal tumor resection in 2 patients , maxillectomy plus orbital exenteration in 1 , and external ethmoidectomy ( ee ) in 1 were also used as surgical treatments . of these 14 patients who underwent surgical tumor resection ,", "4 were treated with surgery alone , 5 with adjuvant radiation therapy after surgery , 2 with adjuvant ctx after surgery , and the remaining 3 with radiation therapy and ctx after surgery . of the remaining 5 patients , four in advanced stage and one in early stage refused surgical treatment .", "radiation therapy was administered to two of the patients , radiation therapy followed by ctx to the other two , and ctx alone to one patient .", "the average radiation dose was 57.9 gy and the ctx was chosen from cisplatin - based regimens . as a part of the primary treatment modality , 12 patients underwent radiation therapy , so did the five relapsed patients .", "as a part of primary treatment modality 8 patients received ctx , so did the three relapsed patients .   ", "fifteen patients died of tumor progression during the follow - up , of whom 9 were at an advanced stage ( stage c ) by the time of diagnosis and 6 at an early stage ( stage a / b ) .", "kadish stage c patients had a significantly poorer outcome compared to the stage a / b patients ( 2-year survival rate : 71 versus 25% , resp .", "histopathologically high - grade patients ( grade 3/4 ) had a significantly poorer outcome when compared to the lower grade ( grade 1/2 ) patients ( 2-year survival rate : 16 versus 50% , resp .", "primary tumor extensions to orbital area had a significantly poorer outcome compared to the tumors with no extension to orbital area ( 2-year survival rate : 22 versus 60% , resp .", "there was no statistically significant difference in survival rate between male and female patients ( 2-year survival rate : 40 versus 44% , resp .", "patients with age < 45 years had no poorer outcome compared to patients with age > 45 years ( 2-year survival rate : 41 versus 43% ; p = .059 ) .", "brain and bone marrow involvements showed poorer outcome compared to those without such involvement ( median survival rate : 9.1 versus 28.8 months , resp . ;", "survival rate was not significantly different among pateints of kadish c when stratified according to initial treatment ( p = .8 ) .  ", "locoregional recurrence developed in 6 and distant metastasis in 6 patients . during the follow - up period , these groups exhibited distant metastasis to the brain , spinal cord , bone , bone marrow , meninges , and lungs .", "as a treatment for recurrence and metastasis , reoperation of primary site plus radiation therapy in 4 patients and radiation therapy alone in 4 patients were offered ."], ["onb is a rare malignant tumor that comprises 4,65% of malignant nasal and paranasal tumors .", "some 1000 cases have been reported in the english literature since it was first introduced in 1924 .", "the incidence rate is bimodal , with peaks in the second and third decades of life and in the sixth and seventh decades of life and equal among females and males . as for our study ,", "the age at diagnosis was most common in the fourth decade and female - to - male ratio was equal .", "the average time between the appearance of the first symptom and the diagnosis was reported as 6 months and this period was similar also in our group .", "the most common symptoms at the initial diagnosis were defined as unilateral nasal obstruction and epistaxis in the literature .", "this tumor may be misdiagnosed as an undifferentiated small cell carcinoma , melanoma , rhabdomyosarcoma , or other small blue cell neoplasms .", "hyam proposed a grading system with grades 14 based on the presence or absence of seven different histopathological parameters .", "these are growth , architecture , mitotic activity , necrosis , nuclear pleomorphism , rosette formation , and fibrillary stroma .  ", ". staining paterns for neuron - spesific enolase , chromogranin , synaptophysin , s-100 , and epithelial markers can be helpful .  ", "there is evidence that histological grade of onb influences biologic behavior , particularly because it relates to disease progression , local recurrence , and metastasis . for the well - differentiated tumors a slower disease progression and less tendency for local recurrence have been described . in our study , the 2-year survival rate of this grade i / ii - iii / iv disease was 16 versus 50% , respectively .", "more aggressive treatment , such as surgery , radiation , and intensive ctx , may be useful for patients with grade iii / iv disease .", "a meta - analysis showed that the median overall survival at 5 years is 45% in 390 patients .", "the distribution of onb from 21 studies according to kadish staging system was 12% at stage a , 27% at stage b , and 61% at stage c. the mean 5-year survival is 75% for stage a , 68% for stage b , and 41% for stage c .", "the conclusions of the university of virginia stated that kadish stage is predictive of disease - related mortality .", "however , in the series of the mayo clinic it was found that kadish stage did not affect the outcome .", "danish clinicopathological study found that the staging system of kadish was able to stratify into prognostically significant groups . in our study , the 5-year survival rate of these tumors was 26% and median survival time was 23 months .", "the distribution of onb according to kadish staging system was 16% at stage a , 26% stage b , and 58% stage c. however , in our study , the 5-year survival rates of these stage groups were inferior compared to those reported in other studies .", "the cause of this poor result was presentation of 5 patients with distant metastases ( 26% ; 2 brain , 1 bone marrow , 2 other sites ) , 4 patients ( 21% ) with cervical lymph node metastasis , 12 patients ( 63% ) with high - grade histology at the time of diagnosis , and inability to evaluate the surgical margins of the resected tumors .", "the survival rate of node positive patients is 29% compared with 64% of node negative patients .", "distant metastases have been described in the pancreas , the liver , mediastinum , bone marrow , lungs , leptomeninges , skin , and breasts [ 11 , 12 ] .", "cervical nodal metastases can develop in 17 to 33% of patients and distant metastases in 10 to 40% of patients over the course of disease", "metastatic disease at presentation occurs in 10 to 50% of patients , depending on the study [ 6 , 13 , 14 ] .", "we found the local recurrence rate to be 31.5% and distant metastasis 31.5% after initial surgery . during the follow - up periods these groups exhibited distant metastases to the brain , spinal cord , bone , bone marrow , meninges , and lungs .", "patients who had brain and bone marrow metastases had poorer prognosis than others having metastatic deposits of other sites .", "involvement of the orbit is a serious prognostic factor , but when the orbital periosteum is affected without penetration into the orbital structures themselves , the eye may be preserved by resection and grafting of the periosteum without an adverse effect on survival as compared with orbital clearance .", "irrespective to the most sophisticated imaging , involvement of the dura and orbital periosteum can accurately be determined only at surgery . in our study ,", "this group 's ( 8 patients ) survival rate is poorer than those with no involvement of the orbita ( p = .042 ) .", "the institute of laryngology and otology , university college london , published a study of 42 patients treated over 23 years .", "overall and disease - free survival rates were 77 and 61% at 5 years , and 53 and 42% at 10 years , respectively .", "late recurrences were seen . based on this experience , combined therapy with craniofacial resection and radiation therapy is recommended by this group . in a retrospective study of 47 patients treated at multiple centers in germany from 1979 to 2001 ,", "the 5-year overall survival and event - free survival rate were 64% and 50% , respectively .", "patients who received multimodality therapy had a significantly better event - free survival rate compared with those who did not receive multimodality therapy ( 74 versus 41% ) .", "the authors recommend a combination therapy including ctx , surgery , and postoperative radiation therapy for kadish stage c patients . in our study ,", "initial treatment included surgery alone in three patients , rt with or without ctx in five , surgery plus postoperative rt in seven , and multimodality therapy ( surgery plus postoperative ctx plus postoperative rt ) in three , only ctx in one patient .", "however , in our study choice of treatment modality did not show a statistically significant difference in survival rate .", "the reasons for that might be including low number of patients in the study , being 5 distant site metastasis , 4 cervical lymph node metastasis at the time of diagnosis , only one orbital exentration ( although there were 8 orbital involvement ) , and 12 patients exhibiting high grade histology and inability to evaluate surgical margins in resected tumors . in conclusion , advanced tumor stage ,", "the initial histopathologic grade of iii / iv , and involvement of orbita , brain , or bone marrow metastasis were the statistically significant poor prognostic factors , whereas age , sex , and treatment modality were not prognostic factors ."]]}
{"article_id": "PMC3355710", "article_text": ["unrepaired dsbs are usually lethal for dividing cells due to the subsequent loss of the acentric fragment and the unstable break - end of the centric fragment .", "dsbs can be caused enzymatically , by ionizing irradiation , or by other s phase - independent clastogens .", "dsbs with only one break - end may appear when a replication fork meets a single - strand break or a repair - mediated parental single - strand discontinuity .", "mostly however , the break is extended by a 5-specific exonuclease activity generating 3-overhanging ends .", "as soon as overhanging ends meet at short stretches of complementary bases ( single - strand annealing , ssa ) , the distal free ends are resected , and the break becomes ligated , resulting in a ( micro-)deletion ( figure 1a ) .", "alternatively , the break - ends may invade undamaged homologous template strands and a limited dna synthesis across the break region is followed by re - ligation of both strands ( synthesis - dependent strand annealing , sdsa ) .", "the result is genetically not detectable when the template was the undamaged sister chromatid ; it appears as a gene conversion if an allele of the homologous chromosome served as a template ( figure 1c ) .", "if for break - end elongation an ectopic or an extra - chromosomal ( partially ) homologous sequence is used , the result may be linked with an insertion into the break ( figure 1b and puchta , 2005 ) . when the holiday junction ( caused by break - end invasion into a template double strand ) is resolved in connection with an exchange of the flanking region ( figure 1c ) , then , depending on the template double helix involved , a sister chromatid exchange , a crossing over , or a reciprocal translocation results .", "pathways of dsb misrepair via single - strand annealing(ssa ) or via synthesis - dependent strand annealing ( sdsa ) .", "( a ) deletion via exonucleolytic 5-end resection , ssa at complementary overhang sequences , resection of the non - aligned ends , and ligation of break - ends .", "( b ) insertion into a dsb by break - end invasion and elongationalong an ectopic and partially homologous ( vertical bars ) template.(c ) re - synthesis of break - ends after invasion into a homologous template double - strand without ( gene conversion ) or with exchange of flanking regions due to appropriate resolution of holiday junctions ( greenarrow heads ) . based on genetic experiments on budding yeast , a further mechanism ,  break - induced replication  ( bir ;", ", 1996 , 2005 ; morrow et al . , 1997 ; haber , 1999 ; for review haber , 2006 ; mceachern and haber , 2006 ; llorente et al . ,", "2008 ) has been postulated . with the exception of a direct ligation of  clean  break - ends ,", "all dsb repair events are linked with a limited replication step , bir is claimed to extend replication from the proximal break - end up to the end of the template chromatid , using a ( homologous ) undamaged double helix as template .", "mechanistically , bir appears as a non - reciprocal translocation event ( bosco and haber , 1998 ) which can be considered as a type of gene conversion extending from the breakpoint up to the telomere .", "simultaneously , the original distal fragment ( if occurring ) of the broken double helix is lost ( figure 2 ) .", "schematic models of replication and chromosome labeling patterns after bir at proximal dsb ends in s and g2 . ( a ) bir through conservative replication of a one ended dsb during s phase . the dsb appears when the replication fork arrives at a single - strand break ( arrow head ) .", "conservative replication occurs via recurrent strand invasion ( or via unidirectional fork migration ) without resolution of the holiday junction(s ) using the parental double strand as a template .", "( b ) bir during g2 phase , through conservative replication at the proximal end of a dsb ( arrow head ) via recurrent strand invasion and/or via unidirectional fork migration without resolution of the holiday junction(s ) using the undamaged sister double helix as a template .", "( c ) bir during g2 phase through semiconservative replication achieved by resolution of the holiday junction ( green arrow head ) after invasion of the elongating break - end into the template double strand .", "full lines unlabeled ; broken lines labeled by edu . the distal fragment of the broken double helix in ( b , c ) gets lost . when a homologous or a heterologous chromatid instead of the sister chromatid is involved in bir , a non - reciprocal translocation between homologous or heterologous chromosomes is mimicked .", "microhomology - mediated bir involving heterologous chromosomes leads to duplication of the template region and deletion of the region distal the original dsb .", "the problem of bir before or after regular replication in species with large chromosomes is that breakage occurring distantly to the arm end requires replication of the involved region ( larger than a chromatin unit of jointly regulated replication comprising 1  mb , zink et al . , 1998 ) more than once between two nuclear divisions , thus overriding the licensing mechanism which allows only one round of replication between two divisions .", "bir occurring during s phase ( figure 2a and hastings et al . , 2009 ) requires a recurrent 3-end invasion into a still unreplicated template chromatid ( most likely the sister chromatid ) , or a continuous migration of the replication fork toward the arm end .", "resolution of the holiday junction results in a reciprocal exchange of the distal regions between the chromatids involved .", "bir during s phase without resolution of the holiday junction circumvents the dsb , yielding a conservative replication pattern with regard to the broken chromatid distal to the breakpoint . to test whether bir may occur within chromosomes of higher plants after dsb induction", ", we treated field bean root tip meristems with the s phase - independent clastogen bleomycin during s or g2 phase and checked for the expected chromatid labeling by incorporation of the base analog ethynyldeoxyuridine ( edu , see kotogany et al . , 2010 )", "also semiconservative bir at terminal chromatid regions ( figure 2c ) was not observed after breakage in the presence of the base analog during g2 .", "our results suggest that bir either does not occur or is so infrequent that its role within the concert of dsb repair mechanisms can at best be a minor one in organisms with a large dna content ( > 1  mb ) per chromosome arm .", "the field bean , vicia faba l. , karyotype acb with six large and individually distinguishable chromosome pairs ( dbel et al . , 1978 ) is much more comfortable for aberration scoring than for instance a. thaliana with smaller chromosomes and less mitoses per root meristem .", "seeds were germinated for 3 days on wet filter paper at room temperature in the dark .", "about 12  cm long primary roots were incubated for 18  h in aerated hoagland solution , ph 5.5 , containing 1.25  mm hydroxyurea which synchronizes cells by blocking the cell cycle in early s phase . for mutagen treatment during s phase , the roots were incubated for 1.5  h in hoagland solution containing 10  g / ml bleomycin with or without 20  m edu ( invitrogen ) followed by incubation in hoagland solution for 4.5  h and then 0.05% colchicine for 2.5  h ( to arrest metaphases ) before fixation in ethanol : acetic acid ( 3:1 ) overnight .", "for scoring of chromatid aberrations , roots were washed for 10  min in distilled water , hydrolyzed for 11  min in 1 ", "n hcl at 60c , stained for 3040  min in feulgen solution and squashed in a drop of 45% acetic acid .", "only complete metaphase cells were scored for chromatid aberrations : chromatid and isochromatid breaks ( one or both sister chromatids with terminal deletion ) , interstitial deletions , duplication deletion ( the deleted part of one chromatid is inserted into a break of the sister chromatid ) , and reciprocal chromatid translocations .", "alternatively , the root tips were fixed in 4% formaldehyde for 20  min for isolation of metaphase chromosomes ( schubert et al . , 1993 ) and subsequent detection of edu incorporation using the click.it edu imaging kit from invitrogen according to manufacturer 's instruction .", "microscopic analysis of chromosomes was performed with an epifluorescence microscope ( zeiss axiophot ) using a 100/1.45 zeiss alpha - plan - fluar objective and a sony ( dxc-950p ) camera .", "images of fluorescent chromosomes were captured using filter sets f36 - 513 for dapi and f36 - 750 for alexa fluor 594 ( ahf analysentechnik , germany ) .", "dapi and alexa fluor 594 azide images were merged using adobe photoshop 6.0 software ( adobe systems , san jose , ca , usa ) . for mutagen treatment during g2 phase , primary roots were incubated in hoagland solution with hydroxyurea as above , then in hoagland solution for 4.5  h and subsequently in hoagland solution with bleomycin , with edu , or both for another 1.5  h followed by 2.5  h in colchicine before fixation , staining , and evaluation .", "the s phase - independent clastogen bleomycin induces dna breaks directly . a dose of 10  g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems ( heindorff et al . , 1987 ) .", "the same bleomycin concentration applied for 1.5  h to ( mainly ) g2 cells of root tip meristems , in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations .", "treatment with edu ( 20  m , 1.5  h ) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase .", "bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells ( table 1 ) .", "although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells , no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin .", "chromatid - type aberrations induced by edu , bleomycin , or both during g2 or s phase in field bean root tip meristems ( summary of two independent experiments ) .", "t  =  reciprocal translocation , i  =  isochromatid break , d  =  interstitial deletion , b  =  chromatid break , see figure 3a .", "when root tips were incubated in 20  m edu 8.57  h before fixation , most metaphase chromosomes ( 87.4% ) were completely labeled and some ( 12.5% ) were labeled along their euchromatin regions ( table 2 ) , indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems ( schubert and meister , 1977 ) .", "no chromosome showed asymmetric labeling up to the chromosome end , i.e. , an unlabeled terminal region of one of the sister chromatids .", "only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid .", "type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment ( two independent experiments summarized ) . *", "( see figures 2a and 3b ) , * * ( see figures 2b and 3c ) , * * * in ( ) maximum percentage of cells harboring an asymmetrically labeled chromosome . when s phase cells were treated simultaneously with edu and bleomycin , all 2130 observed chromosomes were labeled ( 81.4% completely and 18% along their euchromatin ) .", "none of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix , or if it replicated till the end of the template by unidirectional fork migration ( figure 2a and hastings et al . , 2009 ) .", "thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b ) . since 2130 chromosomes correspond to 178 cells , in about 7.3% of cells such an asymmetrically unlabeled spot occurred , representing an eightfold increase compared to the variant with edu treatment alone .", "( a ) chromatid - type aberrations after bleomycin treatment . left cell : isochromatid break ( arrow head ) , the centric , and the acentric chromatid fragments are surrounded by black dots , the homologous undamaged chromosome is surrounded by white dots .", "middle cell : symmetric reciprocal chromatid translocation ( arrow ) and two terminal chromatid breaks ( arrow heads ) .", "the latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid ( left ) or being at least 90 apart from the other break - end as in case of the broken secondary constriction ( right ) .", "right cell : interstitial deletion ( arrow ) , the deleted fragment remains attached to the undamaged sister chromatid , the chromosome involved is surrounded by black dots . (", "b ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during s phase .", "( c ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during g2 .", "the asymmetric signals appear on chromosomes ii , iv , v , and vi , respectively , at interstitial heterochromatic regions composed of homologous tandem repeats ( fuchs et al . , 1994 ) .", "when metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5  h before fixation , the corresponding cells should have been in g2 during edu treatment . indeed , most of the chromosomes ( 81.3% ) were unlabeled while 18.3% revealed late replication pattern ( symmetric labeling of heterochromatic regions ) thus indicating they were in late s phase during edu treatment .", "no chromosome showed asymmetric labeling of one chromatid up to the arm end , as expected in the case of a conservative bir ( figure 2b ) .", "also symmetric terminal labeling of sister chromatids , as expected in the case of semiconservative bir ( figure 2c ) was not observed .", "single transversal interstitial fluorescence signals on one of the sister chromatids ( figure 3c ) were observed in 10 ( 0.4% ) chromosomes .", "after simultaneous exposure to edu and to bleomycin 42.5  h before fixation , again most of the chromosomes ( 85.5% ) were completely unlabeled and thus were in g2 during treatment ; 13.2% of chromosomes showed late replication patterns , indicating that the corresponding cells were in late s during treatment .", "however , there was a > 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal ( 20 signals per 130 cells ) , usually in heterochromatic regions .", "the s phase - independent clastogen bleomycin induces dna breaks directly . a dose of 10  g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems ( heindorff et al . , 1987 ) .", "the same bleomycin concentration applied for 1.5  h to ( mainly ) g2 cells of root tip meristems , in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations .", "treatment with edu ( 20  m , 1.5  h ) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase .", "bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells ( table 1 ) .", "although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells , no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin .", "chromatid - type aberrations induced by edu , bleomycin , or both during g2 or s phase in field bean root tip meristems ( summary of two independent experiments ) .", "t  =  reciprocal translocation , i  =  isochromatid break , d  =  interstitial deletion , b  =  chromatid break , see figure 3a .", "when root tips were incubated in 20  m edu 8.57  h before fixation , most metaphase chromosomes ( 87.4% ) were completely labeled and some ( 12.5% ) were labeled along their euchromatin regions ( table 2 ) , indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems ( schubert and meister , 1977 ) .", "no chromosome showed asymmetric labeling up to the chromosome end , i.e. , an unlabeled terminal region of one of the sister chromatids .", "only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid .", "type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment ( two independent experiments summarized ) . * ( see figures 2a and 3b ) , * * ( see figures 2b and 3c ) , * * * in ( ) maximum percentage of cells harboring an asymmetrically labeled chromosome . when s phase cells were treated simultaneously with edu and bleomycin , all 2130 observed chromosomes were labeled ( 81.4% completely and 18% along their euchromatin ) .", "none of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix , or if it replicated till the end of the template by unidirectional fork migration ( figure 2a and hastings et al . , 2009 ) .", "thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b ) .", "since 2130 chromosomes correspond to 178 cells , in about 7.3% of cells such an asymmetrically unlabeled spot occurred , representing an eightfold increase compared to the variant with edu treatment alone .", "isochromatid break ( arrow head ) , the centric , and the acentric chromatid fragments are surrounded by black dots , the homologous undamaged chromosome is surrounded by white dots . middle cell : symmetric reciprocal chromatid translocation ( arrow ) and two terminal chromatid breaks ( arrow heads ) .", "the latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid ( left ) or being at least 90 apart from the other break - end as in case of the broken secondary constriction ( right ) .", "right cell : interstitial deletion ( arrow ) , the deleted fragment remains attached to the undamaged sister chromatid , the chromosome involved is surrounded by black dots . ( b ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during s phase .", "( c ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during g2 .", "the asymmetric signals appear on chromosomes ii , iv , v , and vi , respectively , at interstitial heterochromatic regions composed of homologous tandem repeats ( fuchs et al . , 1994 ) .", "when metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5  h before fixation , the corresponding cells should have been in g2 during edu treatment . indeed , most of the chromosomes ( 81.3% ) were unlabeled while 18.3% revealed late replication pattern ( symmetric labeling of heterochromatic regions ) thus indicating they were in late s phase during edu treatment .", "no chromosome showed asymmetric labeling of one chromatid up to the arm end , as expected in the case of a conservative bir ( figure 2b ) .", "also symmetric terminal labeling of sister chromatids , as expected in the case of semiconservative bir ( figure 2c ) was not observed .", "single transversal interstitial fluorescence signals on one of the sister chromatids ( figure 3c ) were observed in 10 ( 0.4% ) chromosomes .", "after simultaneous exposure to edu and to bleomycin 42.5  h before fixation , again most of the chromosomes ( 85.5% ) were completely unlabeled and thus were in g2 during treatment ; 13.2% of chromosomes showed late replication patterns , indicating that the corresponding cells were in late s during treatment .", "however , there was a > 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal ( 20 signals per 130 cells ) , usually in heterochromatic regions .", "if bir occurs in higher plants in the same way as postulated for yeast ( i.e. , as replication extending from the break position up to the corresponding chromosome arm end ) conservative replication in s or g2 ( figures 2a , b ) or semiconservative replication in g2 ( figure 2c ) should be detectable .", "of the chromatid aberrations observed after bleomycin and bleomycin plus edu treatment during s and g2 , reciprocal translocations ( 25 ) , isochromatid breaks ( 36 ) , and interstitial deletions ( 7 ) require two dsbs each , while simple chromatid breaks ( 87 ) representing terminal deletions , go back to a single dsb .", "therefore , within 600 g2 and 750 s phase cells , bleomycin caused 223 dsbs which underwent misrepair .", "thus , on average at least one dsb per six cells was induced . considering that a fraction of dsbs might have been repaired via pathways not resulting in microscopically detectable chromatid - type aberrations ( for instance by sister chromatid exchange , intrachromosomal ssa , or gene conversion ) , the actual number of dsbs may be higher .", "however , in a number of chromosomes corresponding to 308  s and g2 cells , treated simultaneously with the chromosome - breaking agent bleomycin and the base analog edu , not a single chromosome showed a labeling pattern indicative of bir . in budding yeast , more than half of diploid cells repair ho - endonuclease - induced dsbs by bir using the homologous chromosome as template ( bosco and haber , 1998 ) ; meaning more than half of the dsbs", "are repaired via bir if the endonuclease ho causes one dsb per diploid yeast cell .", "had bir occurred with a similar efficiency in the field bean , we should have observed > 223 instead of no chromosomes with asymmetric terminal edu signal .", "our result does not exclude the occurrence of bir in plants , however , the frequency of such events is below 0.45% ( less than 1 out of 223 ) of the dsbs causing chromosome structural aberrations , i.e. , more than 100-times lower than in yeast .", "unidirectional replication forks that override distal replication origins have to migrate up to the arm ends , or , alternatively , the requirement of recurrent invasion of 3-ends into other double helices , are presumptions for which experimental evidence is lacking so far .", "long range replication in addition to the regular round of replication between two nuclear divisions , represent a further unproven assumptions . to our knowledge no experimental evidence as to the occurrence of bir in animals bas been shown ( zhang et al . , 2009 ) .", "taken together these arguments imply that the existence of a bir pathway in higher eukaryotes is unlikely .", "even when such events occur , their frequency is much lower than in yeast arguing against their importance as a pathway for dsb repair at least in higher plants such as the field bean . because the 12 megabase pair ( mb ) genome size of saccharomyces cerevisiae is distributed over 32 chromosome arms , the risk that bir interferes with replication licensing at the replicon level is much smaller than for species with orders of magnitude larger genomes with several mb per arm .", "also complex chromosome rearrangements ( e.g. , difilippantonio et al . , 2002 ; zhang et al . , 2009 ) can more easily be explained by other well - documented pathways ( zhang et al . , 2009 ; schubert and lysak , 2011 ) rather than by bir via recurrent invasion of a dsb - induced 3-end into different template double helices .", "the more so as experimental approaches even in yeast can not differentiate  between bir and gene conversion events involving g2 cross over , followed by appropriate chromatid segregation ", "nevertheless , the increased frequency of interstitial asymmetric labeling patterns of chromosomes exposed to bleomycin and edu during s or g2 might indicate that conserved replication , although not reaching arm ends , could be linked with dsb repair to result in microscopically detectable stretches of gene conversion extending hundreds of kilobases ( possibly , up to the junction with the next replication unit , but not covering dna stretches of > 1  mb up to the arm ends ) . such extended gene conversion - like repair", "might be considered as bir , if the postulation  up to the chromosome terminus  is excluded .", "similar events could potentially have been responsible for the  zebra  structure of the long arm of chromosome z5a observed in a backcross progeny between elymus trachycaulus gould ex shinners and triticum aestivum l. ( zhang et al . , 2008 ) .", "the preferential appearance of local asymmetric labeling within heterochromatic regions of the field bean suggests that for late replicating repetitive dna the units of replication regulation are larger or more variable than for euchromatin .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S>  break - induced replication  ( bir ) is considered as one way to repair dna double - strand breaks ( dsbs ) . </S>", "<S> bir is defined as replication of the proximal break - ends up to the end of the broken chromosome using an undamaged ( homologous ) double - stranded template and mimicking a non - reciprocal translocation . </S>", "<S> this phenomenon was detected by genetic experiments in yeast . </S>", "<S> bir is assumed to occur also in mammals , but experimental evidence is not yet at hand . </S>", "<S> we have studied chromosomes of the field bean , vicia faba l. , with respect to the occurrence of bir after dsb induction during s and g2 phase . </S>", "<S> simultaneous incorporation of the base analog ethynyldeoxyuridine ( edu ) revealed no chromosomal replication pattern indicative of bir . </S>", "<S> thus , if occurring at all , bir does not play a major role in dsb repair in higher plants with large chromosome arms . </S>", "<S> however , the frequency of interstitial asymmetric edu incorporation within heterochromatic regions , visible on metaphase chromosomes , increased after chromosome breakage during s and g2 phase . </S>", "<S> such asymmetric labeling could be interpreted as conservative replication up to the next replicon , circumventing a dsb , and yielding an interstitial conversion - like event . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Bleomycin induces chromatid aberrations in s and G2 phase; simultaneous treatment with the base analog EdU does not further increase the aberration yield", "Chromosomal EdU incorporation patterns do not indicate break-induced replication up to an arm end", "Discussion", "Conflict of Interest Statement"], "sections": [["unrepaired dsbs are usually lethal for dividing cells due to the subsequent loss of the acentric fragment and the unstable break - end of the centric fragment .", "dsbs can be caused enzymatically , by ionizing irradiation , or by other s phase - independent clastogens .", "dsbs with only one break - end may appear when a replication fork meets a single - strand break or a repair - mediated parental single - strand discontinuity .", "mostly however , the break is extended by a 5-specific exonuclease activity generating 3-overhanging ends .", "as soon as overhanging ends meet at short stretches of complementary bases ( single - strand annealing , ssa ) , the distal free ends are resected , and the break becomes ligated , resulting in a ( micro-)deletion ( figure 1a ) .", "alternatively , the break - ends may invade undamaged homologous template strands and a limited dna synthesis across the break region is followed by re - ligation of both strands ( synthesis - dependent strand annealing , sdsa ) .", "the result is genetically not detectable when the template was the undamaged sister chromatid ; it appears as a gene conversion if an allele of the homologous chromosome served as a template ( figure 1c ) .", "if for break - end elongation an ectopic or an extra - chromosomal ( partially ) homologous sequence is used , the result may be linked with an insertion into the break ( figure 1b and puchta , 2005 ) . when the holiday junction ( caused by break - end invasion into a template double strand ) is resolved in connection with an exchange of the flanking region ( figure 1c ) , then , depending on the template double helix involved , a sister chromatid exchange , a crossing over , or a reciprocal translocation results .", "pathways of dsb misrepair via single - strand annealing(ssa ) or via synthesis - dependent strand annealing ( sdsa ) .", "( a ) deletion via exonucleolytic 5-end resection , ssa at complementary overhang sequences , resection of the non - aligned ends , and ligation of break - ends .", "( b ) insertion into a dsb by break - end invasion and elongationalong an ectopic and partially homologous ( vertical bars ) template.(c ) re - synthesis of break - ends after invasion into a homologous template double - strand without ( gene conversion ) or with exchange of flanking regions due to appropriate resolution of holiday junctions ( greenarrow heads ) . based on genetic experiments on budding yeast , a further mechanism ,  break - induced replication  ( bir ;", ", 1996 , 2005 ; morrow et al . , 1997 ; haber , 1999 ; for review haber , 2006 ; mceachern and haber , 2006 ; llorente et al . ,", "2008 ) has been postulated . with the exception of a direct ligation of  clean  break - ends ,", "all dsb repair events are linked with a limited replication step , bir is claimed to extend replication from the proximal break - end up to the end of the template chromatid , using a ( homologous ) undamaged double helix as template .", "mechanistically , bir appears as a non - reciprocal translocation event ( bosco and haber , 1998 ) which can be considered as a type of gene conversion extending from the breakpoint up to the telomere .", "simultaneously , the original distal fragment ( if occurring ) of the broken double helix is lost ( figure 2 ) .", "schematic models of replication and chromosome labeling patterns after bir at proximal dsb ends in s and g2 . ( a ) bir through conservative replication of a one ended dsb during s phase . the dsb appears when the replication fork arrives at a single - strand break ( arrow head ) .", "conservative replication occurs via recurrent strand invasion ( or via unidirectional fork migration ) without resolution of the holiday junction(s ) using the parental double strand as a template .", "( b ) bir during g2 phase , through conservative replication at the proximal end of a dsb ( arrow head ) via recurrent strand invasion and/or via unidirectional fork migration without resolution of the holiday junction(s ) using the undamaged sister double helix as a template .", "( c ) bir during g2 phase through semiconservative replication achieved by resolution of the holiday junction ( green arrow head ) after invasion of the elongating break - end into the template double strand .", "full lines unlabeled ; broken lines labeled by edu . the distal fragment of the broken double helix in ( b , c ) gets lost . when a homologous or a heterologous chromatid instead of the sister chromatid is involved in bir , a non - reciprocal translocation between homologous or heterologous chromosomes is mimicked .", "microhomology - mediated bir involving heterologous chromosomes leads to duplication of the template region and deletion of the region distal the original dsb .", "the problem of bir before or after regular replication in species with large chromosomes is that breakage occurring distantly to the arm end requires replication of the involved region ( larger than a chromatin unit of jointly regulated replication comprising 1  mb , zink et al . , 1998 ) more than once between two nuclear divisions , thus overriding the licensing mechanism which allows only one round of replication between two divisions .", "bir occurring during s phase ( figure 2a and hastings et al . , 2009 ) requires a recurrent 3-end invasion into a still unreplicated template chromatid ( most likely the sister chromatid ) , or a continuous migration of the replication fork toward the arm end .", "resolution of the holiday junction results in a reciprocal exchange of the distal regions between the chromatids involved .", "bir during s phase without resolution of the holiday junction circumvents the dsb , yielding a conservative replication pattern with regard to the broken chromatid distal to the breakpoint . to test whether bir may occur within chromosomes of higher plants after dsb induction", ", we treated field bean root tip meristems with the s phase - independent clastogen bleomycin during s or g2 phase and checked for the expected chromatid labeling by incorporation of the base analog ethynyldeoxyuridine ( edu , see kotogany et al . , 2010 )", "also semiconservative bir at terminal chromatid regions ( figure 2c ) was not observed after breakage in the presence of the base analog during g2 .", "our results suggest that bir either does not occur or is so infrequent that its role within the concert of dsb repair mechanisms can at best be a minor one in organisms with a large dna content ( > 1  mb ) per chromosome arm ."], ["the field bean , vicia faba l. , karyotype acb with six large and individually distinguishable chromosome pairs ( dbel et al . , 1978 ) is much more comfortable for aberration scoring than for instance a. thaliana with smaller chromosomes and less mitoses per root meristem .", "seeds were germinated for 3 days on wet filter paper at room temperature in the dark .", "about 12  cm long primary roots were incubated for 18  h in aerated hoagland solution , ph 5.5 , containing 1.25  mm hydroxyurea which synchronizes cells by blocking the cell cycle in early s phase . for mutagen treatment during s phase , the roots were incubated for 1.5  h in hoagland solution containing 10  g / ml bleomycin with or without 20  m edu ( invitrogen ) followed by incubation in hoagland solution for 4.5  h and then 0.05% colchicine for 2.5  h ( to arrest metaphases ) before fixation in ethanol : acetic acid ( 3:1 ) overnight .", "for scoring of chromatid aberrations , roots were washed for 10  min in distilled water , hydrolyzed for 11  min in 1 ", "n hcl at 60c , stained for 3040  min in feulgen solution and squashed in a drop of 45% acetic acid .", "only complete metaphase cells were scored for chromatid aberrations : chromatid and isochromatid breaks ( one or both sister chromatids with terminal deletion ) , interstitial deletions , duplication deletion ( the deleted part of one chromatid is inserted into a break of the sister chromatid ) , and reciprocal chromatid translocations .", "alternatively , the root tips were fixed in 4% formaldehyde for 20  min for isolation of metaphase chromosomes ( schubert et al . , 1993 ) and subsequent detection of edu incorporation using the click.it edu imaging kit from invitrogen according to manufacturer 's instruction .", "microscopic analysis of chromosomes was performed with an epifluorescence microscope ( zeiss axiophot ) using a 100/1.45 zeiss alpha - plan - fluar objective and a sony ( dxc-950p ) camera .", "images of fluorescent chromosomes were captured using filter sets f36 - 513 for dapi and f36 - 750 for alexa fluor 594 ( ahf analysentechnik , germany ) .", "dapi and alexa fluor 594 azide images were merged using adobe photoshop 6.0 software ( adobe systems , san jose , ca , usa ) . for mutagen treatment during g2 phase , primary roots were incubated in hoagland solution with hydroxyurea as above , then in hoagland solution for 4.5  h and subsequently in hoagland solution with bleomycin , with edu , or both for another 1.5  h followed by 2.5  h in colchicine before fixation , staining , and evaluation ."], ["the s phase - independent clastogen bleomycin induces dna breaks directly . a dose of 10  g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems ( heindorff et al . , 1987 ) .", "the same bleomycin concentration applied for 1.5  h to ( mainly ) g2 cells of root tip meristems , in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations .", "treatment with edu ( 20  m , 1.5  h ) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase .", "bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells ( table 1 ) .", "although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells , no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin .", "chromatid - type aberrations induced by edu , bleomycin , or both during g2 or s phase in field bean root tip meristems ( summary of two independent experiments ) .", "t  =  reciprocal translocation , i  =  isochromatid break , d  =  interstitial deletion , b  =  chromatid break , see figure 3a .", "when root tips were incubated in 20  m edu 8.57  h before fixation , most metaphase chromosomes ( 87.4% ) were completely labeled and some ( 12.5% ) were labeled along their euchromatin regions ( table 2 ) , indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems ( schubert and meister , 1977 ) .", "no chromosome showed asymmetric labeling up to the chromosome end , i.e. , an unlabeled terminal region of one of the sister chromatids .", "only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid .", "type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment ( two independent experiments summarized ) . *", "( see figures 2a and 3b ) , * * ( see figures 2b and 3c ) , * * * in ( ) maximum percentage of cells harboring an asymmetrically labeled chromosome . when s phase cells were treated simultaneously with edu and bleomycin , all 2130 observed chromosomes were labeled ( 81.4% completely and 18% along their euchromatin ) .", "none of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix , or if it replicated till the end of the template by unidirectional fork migration ( figure 2a and hastings et al . , 2009 ) .", "thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b ) . since 2130 chromosomes correspond to 178 cells , in about 7.3% of cells such an asymmetrically unlabeled spot occurred , representing an eightfold increase compared to the variant with edu treatment alone .", "( a ) chromatid - type aberrations after bleomycin treatment . left cell : isochromatid break ( arrow head ) , the centric , and the acentric chromatid fragments are surrounded by black dots , the homologous undamaged chromosome is surrounded by white dots .", "middle cell : symmetric reciprocal chromatid translocation ( arrow ) and two terminal chromatid breaks ( arrow heads ) .", "the latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid ( left ) or being at least 90 apart from the other break - end as in case of the broken secondary constriction ( right ) .", "right cell : interstitial deletion ( arrow ) , the deleted fragment remains attached to the undamaged sister chromatid , the chromosome involved is surrounded by black dots . (", "b ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during s phase .", "( c ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during g2 .", "the asymmetric signals appear on chromosomes ii , iv , v , and vi , respectively , at interstitial heterochromatic regions composed of homologous tandem repeats ( fuchs et al . , 1994 ) .", "when metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5  h before fixation , the corresponding cells should have been in g2 during edu treatment . indeed , most of the chromosomes ( 81.3% ) were unlabeled while 18.3% revealed late replication pattern ( symmetric labeling of heterochromatic regions ) thus indicating they were in late s phase during edu treatment .", "no chromosome showed asymmetric labeling of one chromatid up to the arm end , as expected in the case of a conservative bir ( figure 2b ) .", "also symmetric terminal labeling of sister chromatids , as expected in the case of semiconservative bir ( figure 2c ) was not observed .", "single transversal interstitial fluorescence signals on one of the sister chromatids ( figure 3c ) were observed in 10 ( 0.4% ) chromosomes .", "after simultaneous exposure to edu and to bleomycin 42.5  h before fixation , again most of the chromosomes ( 85.5% ) were completely unlabeled and thus were in g2 during treatment ; 13.2% of chromosomes showed late replication patterns , indicating that the corresponding cells were in late s during treatment .", "however , there was a > 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal ( 20 signals per 130 cells ) , usually in heterochromatic regions ."], ["the s phase - independent clastogen bleomycin induces dna breaks directly . a dose of 10  g / ml was previously shown to yield chromatid aberrations in up to 30% of the first post - treatment metaphases of field bean root tip meristems ( heindorff et al . , 1987 ) .", "the same bleomycin concentration applied for 1.5  h to ( mainly ) g2 cells of root tip meristems , in two independent experiments yielded on an average of 14.8% of metaphases with chromatid aberrations .", "treatment with edu ( 20  m , 1.5  h ) resulted in 0.8% metaphases with aberrations when applied during g2 and in 3.1% when applied during s phase .", "bleomycin and edu together yielded 12% of metaphases with aberration after exposure in g2 and 6.6% after exposure to s phase cells ( table 1 ) .", "although during s phase the base analog edu caused slightly more aberrations than appear in untreated control cells , no obvious synergistic effect on clastogenicity was observed when applied together with bleomycin .", "chromatid - type aberrations induced by edu , bleomycin , or both during g2 or s phase in field bean root tip meristems ( summary of two independent experiments ) .", "t  =  reciprocal translocation , i  =  isochromatid break , d  =  interstitial deletion , b  =  chromatid break , see figure 3a ."], ["when root tips were incubated in 20  m edu 8.57  h before fixation , most metaphase chromosomes ( 87.4% ) were completely labeled and some ( 12.5% ) were labeled along their euchromatin regions ( table 2 ) , indicating that the corresponding cells were in s phase during treatment as expected according to the experimental schedule based of the cell cycle duration of field bean meristems ( schubert and meister , 1977 ) .", "no chromosome showed asymmetric labeling up to the chromosome end , i.e. , an unlabeled terminal region of one of the sister chromatids .", "only one out of 1404 chromosomes displayed an unlabeled transversal region within a heterochromatic area of one sister chromatid .", "type and frequency of chromosomal labeling patterns after edu incorporation during s or g2 phase with or without bleomycin treatment ( two independent experiments summarized ) . * ( see figures 2a and 3b ) , * * ( see figures 2b and 3c ) , * * * in ( ) maximum percentage of cells harboring an asymmetrically labeled chromosome . when s phase cells were treated simultaneously with edu and bleomycin , all 2130 observed chromosomes were labeled ( 81.4% completely and 18% along their euchromatin ) .", "none of these chromosomes showed asymmetric labeling up to an arm end as to be expected if the replication fork is stalled at a single - strand break and the 3-break - end recurrently invaded the unreplicated double helix , or if it replicated till the end of the template by unidirectional fork migration ( figure 2a and hastings et al . , 2009 ) .", "thirteen chromosomes showed unlabeled transversal spots within the heterochromatic part of one sister chromatid figure 3b ) .", "since 2130 chromosomes correspond to 178 cells , in about 7.3% of cells such an asymmetrically unlabeled spot occurred , representing an eightfold increase compared to the variant with edu treatment alone .", "isochromatid break ( arrow head ) , the centric , and the acentric chromatid fragments are surrounded by black dots , the homologous undamaged chromosome is surrounded by white dots . middle cell : symmetric reciprocal chromatid translocation ( arrow ) and two terminal chromatid breaks ( arrow heads ) .", "the latter with the broken fragment either switched to the opposite site of the undamaged sister chromatid ( left ) or being at least 90 apart from the other break - end as in case of the broken secondary constriction ( right ) .", "right cell : interstitial deletion ( arrow ) , the deleted fragment remains attached to the undamaged sister chromatid , the chromosome involved is surrounded by black dots . ( b ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during s phase .", "( c ) interstitial asymmetric chromatid labeling ( arrows ) after bleomycin treatment in the presence of edu during g2 .", "the asymmetric signals appear on chromosomes ii , iv , v , and vi , respectively , at interstitial heterochromatic regions composed of homologous tandem repeats ( fuchs et al . , 1994 ) .", "when metaphase chromosomes were tested for edu incorporation after incubation in edu 42.5  h before fixation , the corresponding cells should have been in g2 during edu treatment . indeed , most of the chromosomes ( 81.3% ) were unlabeled while 18.3% revealed late replication pattern ( symmetric labeling of heterochromatic regions ) thus indicating they were in late s phase during edu treatment .", "no chromosome showed asymmetric labeling of one chromatid up to the arm end , as expected in the case of a conservative bir ( figure 2b ) .", "also symmetric terminal labeling of sister chromatids , as expected in the case of semiconservative bir ( figure 2c ) was not observed .", "single transversal interstitial fluorescence signals on one of the sister chromatids ( figure 3c ) were observed in 10 ( 0.4% ) chromosomes .", "after simultaneous exposure to edu and to bleomycin 42.5  h before fixation , again most of the chromosomes ( 85.5% ) were completely unlabeled and thus were in g2 during treatment ; 13.2% of chromosomes showed late replication patterns , indicating that the corresponding cells were in late s during treatment .", "however , there was a > 3-fold increase in chromosomes showing an interstitial transversal and asymmetric fluorescence signal ( 20 signals per 130 cells ) , usually in heterochromatic regions ."], ["if bir occurs in higher plants in the same way as postulated for yeast ( i.e. , as replication extending from the break position up to the corresponding chromosome arm end ) conservative replication in s or g2 ( figures 2a , b ) or semiconservative replication in g2 ( figure 2c ) should be detectable .", "of the chromatid aberrations observed after bleomycin and bleomycin plus edu treatment during s and g2 , reciprocal translocations ( 25 ) , isochromatid breaks ( 36 ) , and interstitial deletions ( 7 ) require two dsbs each , while simple chromatid breaks ( 87 ) representing terminal deletions , go back to a single dsb .", "therefore , within 600 g2 and 750 s phase cells , bleomycin caused 223 dsbs which underwent misrepair .", "thus , on average at least one dsb per six cells was induced . considering that a fraction of dsbs might have been repaired via pathways not resulting in microscopically detectable chromatid - type aberrations ( for instance by sister chromatid exchange , intrachromosomal ssa , or gene conversion ) , the actual number of dsbs may be higher .", "however , in a number of chromosomes corresponding to 308  s and g2 cells , treated simultaneously with the chromosome - breaking agent bleomycin and the base analog edu , not a single chromosome showed a labeling pattern indicative of bir . in budding yeast , more than half of diploid cells repair ho - endonuclease - induced dsbs by bir using the homologous chromosome as template ( bosco and haber , 1998 ) ; meaning more than half of the dsbs", "are repaired via bir if the endonuclease ho causes one dsb per diploid yeast cell .", "had bir occurred with a similar efficiency in the field bean , we should have observed > 223 instead of no chromosomes with asymmetric terminal edu signal .", "our result does not exclude the occurrence of bir in plants , however , the frequency of such events is below 0.45% ( less than 1 out of 223 ) of the dsbs causing chromosome structural aberrations , i.e. , more than 100-times lower than in yeast .", "unidirectional replication forks that override distal replication origins have to migrate up to the arm ends , or , alternatively , the requirement of recurrent invasion of 3-ends into other double helices , are presumptions for which experimental evidence is lacking so far .", "long range replication in addition to the regular round of replication between two nuclear divisions , represent a further unproven assumptions . to our knowledge no experimental evidence as to the occurrence of bir in animals bas been shown ( zhang et al . , 2009 ) .", "taken together these arguments imply that the existence of a bir pathway in higher eukaryotes is unlikely .", "even when such events occur , their frequency is much lower than in yeast arguing against their importance as a pathway for dsb repair at least in higher plants such as the field bean . because the 12 megabase pair ( mb ) genome size of saccharomyces cerevisiae is distributed over 32 chromosome arms , the risk that bir interferes with replication licensing at the replicon level is much smaller than for species with orders of magnitude larger genomes with several mb per arm .", "also complex chromosome rearrangements ( e.g. , difilippantonio et al . , 2002 ; zhang et al . , 2009 ) can more easily be explained by other well - documented pathways ( zhang et al . , 2009 ; schubert and lysak , 2011 ) rather than by bir via recurrent invasion of a dsb - induced 3-end into different template double helices .", "the more so as experimental approaches even in yeast can not differentiate  between bir and gene conversion events involving g2 cross over , followed by appropriate chromatid segregation ", "nevertheless , the increased frequency of interstitial asymmetric labeling patterns of chromosomes exposed to bleomycin and edu during s or g2 might indicate that conserved replication , although not reaching arm ends , could be linked with dsb repair to result in microscopically detectable stretches of gene conversion extending hundreds of kilobases ( possibly , up to the junction with the next replication unit , but not covering dna stretches of > 1  mb up to the arm ends ) . such extended gene conversion - like repair", "might be considered as bir , if the postulation  up to the chromosome terminus  is excluded .", "similar events could potentially have been responsible for the  zebra  structure of the long arm of chromosome z5a observed in a backcross progeny between elymus trachycaulus gould ex shinners and triticum aestivum l. ( zhang et al . , 2008 ) .", "the preferential appearance of local asymmetric labeling within heterochromatic regions of the field bean suggests that for late replicating repetitive dna the units of replication regulation are larger or more variable than for euchromatin ."], ["the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC2447884", "article_text": ["is an effort to manipulate the metabolism of an organism worthy and reasonable , \t knowing \n that this cellular process has been continuously modified and refined through \n evolution and natural selection for adapting , in the most convenient manner , to \n the ongoing environmental conditions ?", "the \n answer to this question seems obvious when three broad areas of research and \n development are identified in which manipulation of metabolic pathways is \n relevant : ( a ) drug design to treat diseases , ( b ) genetic engineering of \n organisms of biotechnological interest , and ( c ) genetic syndromes therapy .", "historically , drug design was \n the first area in which modification of metabolism was tried : the primary goal \n of drug administration is the inhibition of essential metabolic pathways , for \n example , in a parasite or a tumor cell .", "thus , any metabolic pathway can be a potential \n therapeutic target . in the absence of a solid \n theoretical background that may build a strategy for the rational design of \n drugs ,", "the pharmaceutical industry has applied the knowledge of inorganic and \n organic chemistry for the arbitrary and rather randomized modification of \n metabolic intermediaries by replacing hydrogen atoms in a model molecule with \n any other element or compound .", "the era of rational drug \n design probably started in the 50s when hans krebs proposed that , after having \n an exact description of a metabolic pathway , the  pacemaker  enzyme or \n  rate - limiting step  had to be identified .", "this approach certainly decreased the amount \n of intermediaries to be chemically modified , focusing only on the substrates , \n products , and allosteric effectors of the  rate - limiting step ,  instead of dispersing \n efforts on all the metabolic pathway intermediates .", "the experimental approaches used in the \n identification of the pacemaker , key enzymes ,  bottlenecks .", " limiting steps , or \n regulatory enzymes [ 1 , 2 ] were \n inspection of the metabolic pathway architecture : due to cell \n  economy and for reaching the highest efficiency , pathway control must \n  reside in the enzymes localized at the beginning of a pathway or after a \n  branch ( teleological approach);determination of nonequilibrium reactions : those reactions in which \n  the quotient between the mass action ratio (  ) and its equilibrium constant ( keq ) is low , /keq 1 ( thermodynamic \n  approach);identification of the steps with the lowest maximal rates ( vmax ) in cellular extracts : the key \n  enzyme of the pathway is the one that has the lowest rate ( kinetic \n  approach);enzymes with sigmoidal kinetics : steps that are susceptible to \n  alteration in their kinetic properties by compounds different from \n  substrates and products and which may coordinate the entire metabolism \n  ( nadh / nad ; nadph / nadp , atp / adp ; acetyl coa / coa ; ca / mg ; \n  high ph / low ph ) or at least two metabolic pathways ( citrate , pi , amp , \n  malonyl - coa);crossover theorem . comparing the intermediary concentrations \n  between a basal and an active steady - state pathway flux , the rate - limiting \n  step in the basal condition will be that for which its substrate \n  concentration diminishes and its product concentration increases when the \n  system changes from the basal to the active state or vice versa ( crossover \n  point on a histogram of each intermediary versus its normalized variation in concentration);the shape of the metabolic flux inhibition curve : a sigmoidal curve \n  on a plot of inhibitor concentration versus flux shows that the sensitive \n  step to the inhibitor exerts no control , that is , there is not proportionality between enzyme \n  activity inhibition and pathway flux inhibition because there is an \n   excess  of enzyme .", "on the other \n  hand , a hyperbolic curve indicates that the enzyme susceptible to the \n  inhibitor controls the flux . \n ", "inspection of the metabolic pathway architecture : due to cell \n  economy and for reaching the highest efficiency , pathway control must \n  reside in the enzymes localized at the beginning of a pathway or after a \n  branch ( teleological approach ) ; determination of nonequilibrium reactions : those reactions in which \n  the quotient between the mass action ratio (  ) and its equilibrium constant ( keq ) is low , /keq 1 ( thermodynamic \n  approach ) ; identification of the steps with the lowest maximal rates ( vmax ) in cellular extracts : the key \n  enzyme of the pathway is the one that has the lowest rate ( kinetic \n  approach ) ; enzymes with sigmoidal kinetics : steps that are susceptible to \n  alteration in their kinetic properties by compounds different from \n  substrates and products and which may coordinate the entire metabolism \n  ( nadh / nad ; nadph / nadp , atp / adp ; acetyl coa / coa ; ca / mg ; \n  high ph / low ph ) or at least two metabolic pathways ( citrate , pi , amp , \n  malonyl - coa ) ; crossover theorem . comparing the intermediary concentrations \n  between a basal and an active steady - state pathway flux", ", the rate - limiting \n  step in the basal condition will be that for which its substrate \n  concentration diminishes and its product concentration increases when the \n  system changes from the basal to the active state or vice versa ( crossover \n  point on a histogram of each intermediary versus its normalized variation in concentration ) ; the shape of the metabolic flux inhibition curve : a sigmoidal curve \n  on a plot of inhibitor concentration versus flux shows that the sensitive \n  step to the inhibitor exerts no control , that is , there is not proportionality between enzyme \n  activity inhibition and pathway flux inhibition because there is an \n   excess  of enzyme . on the other \n  hand ,", "a hyperbolic curve indicates that the enzyme susceptible to the \n  inhibitor controls the flux .", "once a site in a metabolic \n pathway has been identified with at least one of the criteria described above \n as  the rate - limiting step , ", "researchers have frequently concluded that such \n enzyme or transporter is the only limiting step of the metabolic flux and \n extend this conclusion to all cell types and to all conditions .", "for example , inspection of the glycolytic pathway ( teleological approach ) suggests that \n hexokinase ( hk ) and phosphofructokinase-1 ( pfk-1 ) ( which are at the beginning \n and after a branch of the pathway ) are the key steps of glycolysis .", "however , all studies on glycolysis in the 60s , \n 70s , and 80s were performed by taking into account only the intracellular \n reactions from hk to ldh ( i.e. , without including the glucose transport \n reaction through the plasma membrane ) and by considering glycolysis as a linear \n pathway without branches . to this", "regard , \n it is recalled that the glucose transporter ( glut ) includes a family of \n proteins and genes that are susceptible of regulation .", "thus , if the extracellular glucose is \n considered as the initial glycolytic substrate , then another potential key step \n would be glut .", "hence , if all the \n branches of the pathway are considered ( figure 1 ) , then according to the \n teleological approach there will be additional potential rate - limiting sites .", "application of the thermodynamic and kinetic approaches to glycolysis reveals that hk , \n pfk-1 , and pyruvate kinase ( pyk ) are the rate - limiting steps because in the \n living cell they catalyze reactions that are far away from equilibrium ( /keq = 1010 ) , and they are also the slowest \n enzymes in the pathway by at least one order of magnitude ( they have the lowest vmax values ) .", "the use of the enzyme cooperativity approach has established that the regulatory \n steps of glycolysis are ( i ) pfk-1 and pyk because they are allosteric enzymes \n and ( ii ) hk because it is inhibited by its products ( g6p and adp , or amp as an adp - analogue ) . \n  the application of the crossover theorem \n ( approach no .", "v ) to glycolysis has shown a consistent variation in the pfk-1 substrate \n ( f6p ) and product ( f1,6bp ) .", "up to now , \n there are few studies on control of glycolysis using the shape of the inhibitor \n titrating curve ( approach no .", "vi ) , due to the lack of specific inhibitors for \n any of the three presumed key steps .", "an exception is iodoacetate which is indeed \n a potent inhibitor of gapdh , but also of other highly reactive \n cysteine - containing enzymes [ 35 ] . by \n using iodoacetate as specific inhibitor ,", "both gapdh activity and flux showed \n identical titration curves , leading to the conclusion that gapdh was the \n rate - limiting step of glycolysis in streptococcus lactis and s. cremoris   ( see , \n however , section 3.2 ; glycolysis in lactobacteria below ) . all together , these results constitute \n the main reason why many intermediary metabolism researchers , including the \n authors of biochemistry text books , have proposed hk , pfk-1 , and pyk as the \n rate - limiting steps of glycolysis . in \n consequence , to vary the glycolytic flux , one of these enzymes has to be \n modified .", "although the above - described experimental approaches are qualitative , full control has \n been automatically assigned to the  key  steps because the concept of the rate - limiting \n step assumes that there is only one single enzyme controlling the metabolic \n pathway flux ( and the concentration of the final product of the pathway ) and , \n in consequence , assigns values of zero to the control exerted by the other \n enzymes and transporters .", "however , as analyzed for glycolysis , researchers have commonly  identified  more than one \n limiting step . in the case of oxidative \n phosphorylation ( oxphos ) , in the 70s and 80s some researchers considered \n cytochrome c oxidase as the rate - limiting step , whereas others preferred the \n atp / adp translocator or the krebs cycle", "ca - sensitive \n dehydrogenases ( for a review , see ) . rephrasing the initial question , which could be the aim of manipulating a metabolic \n pathway such as glycolysis , knowing its universal distribution in the living \n organisms ? from a clinical standpoint , the inhibition of glycolysis is relevant for the treatment of human parasitic \n or pathological diseases such as cancer .", "the glycolytic reactions are almost identical in all organisms ; in \n addition , the enzymes catalyzing these reactions are highly conserved throughout \n the evolutionary scale ( their amino acid sequences are highly similar ) . in mammals ,", "the genes of the 12 glycolytic \n enzymes are scattered throughout the genome , generally in different chromosomes , \n whereas in bacteria many of the glycolytic enzymes are clustered in operons . \n  however , there are organisms ( like some \n human parasites ) that contain enzymes with remarkable differences in their \n biochemical properties ( substrate selectivity , catalytic capacity , stability , \n and oligomeric structure ) , or in genetic expression regulation in comparison to \n the human enzymes , which could be considered as drug targets . furthermore , some glycolytic products are of commercial interest such as ethanol for wine , \n beer , and other alcoholic beverages ; co2 for bread manufacturing ; and \n lactic acid and other organic acids for cheese production .", "thus , from a biotechnological standpoint , it \n is convenient to accelerate the pathway flux to diminish the processing time and \n it is also desirable to increase the concentration of the metabolite to obtain \n robust commercial products . here", ", it is important to emphasize that the metabolic pathways are designed to attain \n changes in flux with minimal disturbances in the intermediary concentrations . for example", ", the glycolytic flux in skeletal \n muscle can increase from rest to an active state by 100 fold , without large \n changes in metabolites .", "then , it is physiologically more common to change a metabolic flux and the production of \n the final metabolite in the pathway than varying the intermediary concentrations \n .", "however , we will see that , by \n using a suitable approach of metabolic control analysis , it is possible to \n design strategies to manipulate not only fluxes but also metabolic intermediary \n concentrations .", "when the yeast saccharomyces \n cerevisiae is exposed to high glucose ( > 2% ; 0.11 m ) , the genes of all \n glycolytic enzymes are induced ( pdc and eno increase their expression by 20 fold ; \n pgk , pyk , and adh , 310 times ; and the \n others , 2 fold in average ) [ 811 ]", ". however , \n when the methodological development of genetic engineering allowed modulating the \n expression of enzymes within cells , researchers turned to the rate - limiting \n step concept to manipulate a metabolic pathway to increase flux and/or its \n intermediates , hypothesizing that the overexpression of only one , or of a few \n key glycolytic genes , should increase the flux .", "historically , heinisch   in germany was the first author to \n obtain a 3.5 fold overexpression of pfk-1 in   s. cerevisiae , but \n surprisingly he observed that the rate of ethanol production was not modified .", "subsequent experiments for increasing the \n ethanol production rate by overexpressing either each of the presumed limiting \n steps , or in combination with other glycolytic enzymes ( table 1 ) , have been \n unsuccessful and , even in some cases , a slight decrease in flux has been \n attained .", "for instance , the simultaneous \n overexpression of seven enzymes of the final section of glycolysis induced only \n a 21% increase in ethanol production after 2 hours of culture ( table 1 ) .", "this was accompanied by a 1020% decrease in pfk-1 \n expression , which might have attenuated the flux increase . in yeasts , hk is not product inhibited \n by g6p or adp ; instead , it is strongly feedback inhibited by trehalose-6-phosphate \n ( tre6p ) .", "deletion \n of the tre6p synthase gene does not \n bring about an increased ethanol production , but it rather induces a defective cellular \n growth on glucose and fructose and a lowered ethanol production , as a result of \n a highly active hk that leads to hyperaccumulation of hexose phosphate \n metabolites ( particularly f1,6bp ) and fast depletion of atp , pi , and downstream \n metabolites .", "the explanation for this event is that , in the tre6p synthase \n mutants , the rate of glucose phosphorylation exceeds the rate of glycolytic atp \n synthesis ( named  turbo effect  ) .", "heterologous expression of a tre6p - insensitive hk does not recover \n completely the wild - type phenotype .", "furthermore , deletion of the tre6p synthase gene in the \n tre6p - insensitive hk strain did affect growth , suggesting other interactions \n and functions of tre6p synthase in the control of sugar metabolism , at \n least in schizosaccharomyces pombe .", "davies and brindle   obtained a 5-fold overexpression of \n pfk-1 in s. cerevisiae , but the increase in ethanol production was not attained \n under anaerobic conditions .", "there was a slight \n increase in ethanol production in resting cells in aerobic conditions , under \n which the mitochondrial metabolism contributes to the atp supply .", "in all these works , it may be noted that \n enzyme overexpression indeed affects the concentration of several intermediaries , \n but this effect has not been further examined .", "it is worth noting that the \n experiments described in table 1 do not rigorously reproduce the physiological \n situation , in which overexpression of all the enzymes should be carried out in \n the proportions found in the organisms .", "the \n rationale behind this observation is that overexpression of only one  limiting  \n", "step leads to a flux control redistribution , a condition at which other steps \n now become rate limiting .", "thus , the \n concept of  rate - limiting step  offers no simple answer to the question of \n increasing the yeast glycolytic flux , and it rather makes this problem to \n appear as a difficult task to solve .", "in contrast , it seems that all relevant controlling steps \n have to be overexpressed , thus reproducing what natural \n selection has already successfully accomplished .", "in addition to s. cerevisiae , overexpression of glycolytic enzymes in other \n organisms such as e. coli [ 15 , 16 ] , \n lactobacteria , tomato , potato , and \n hamster ovary cells   has been accomplished , although without increasing \n flux ( table 1 ) .", "it is somewhat surprising to note that in the glycolytic enzyme \n overexpression experiments , the strong inhibitory effect of g6p ( or tre6p in s. cerevisiae ) , and citrate on hk and pfk-1 , respectively , have been neglected .", "this \n regulatory mechanism does not disappear in the cells overexpressing the enzymes \n but , on the contrary , it is exacerbated .", "then , what would be the aim of overexpressing hk , pfk-1 or any other \n allosteric , or strongly product - inhibited enzyme if they will be more \n inhibited ?", "a successful experiment of increasing \n the glycolytic flux was performed in primary cultures of rat hepatocytes .", "the transformed \n hepatocytes showed higher activity of 18.7- and 7.1-times for hk and gk , \n respectively , at 3 mm glucose , and of 6.3- and 7.1-times at 20 mm glucose .", "however , at 20 mm glucose , the flux to \n lactate was not modified in hk - transformed cells , just like the experiments \n described above ( table 1 ) .", "in contrast , \n with gk overexpression , a 3-fold increase in flux was achieved .", "the mechanistic difference is the hk \n inhibition by g6p ( 10 mm g6p inhibits hk activity by 90% ) , whereas gk is not product inhibited . \n", "the end products , lactate and h , are \n expelled and acidify the external medium which contributes to cheese flavor and \n texture and inhibits the growth of other bacteria .", "similarly to yeast , the lack of carbon source \n in lactobacteria promotes a metabolic change that leads to the production of \n formic and acetic acids , ethanol , and , in a lower proportion , l - lactic acid , \n altering the product quality .", "thus , from a commercial point of view , it does \n not seem important to know what controls the flux to lactate ( because its rate \n of production is adequate ) , but what controls the branching flux . to understand the process , and to eventually inhibit the production of secondary acids , andersen et al .  ", "constructed ldh mutants , \n using a synthetic promoter library for tuning the gene expression . in mutants \n lacking this enzyme ,", "most of the pyruvate was transformed into acetic and \n formic acids ( figure 1 ) . in turn", ", flux \n to lactate was affected in mutants expressing only 10% or less of wild - type ldh \n levels , which indicated that ldh exerts no control of the glycolytic flux in \n wild - type bacteria . only with a normal content of this enzyme ( 100% ) ,", "therefore , the flux to formic and acetic \n acids is negatively controlled by ldh , and positively by pyk [ 17 , 25 ] . as in s. cerevisiae , overexpression of \n pfk-1 , pyk , or gapdh in lactobacteria", "did not increase the flux to l - lactic \n acid [ 17 , 25 ] . similarly to e. coli glycolysis , glycolysis in l. lactis was controlled by the atp \n demand when working below its maximum capacity [ 27 , 28 ] , whereas , under high - rate \n conditions , the glucose and lactate transporters exerted the main flux control .", "furthermore , this kind of observations indicates \n that the flux control may reside outside the pathway [ 2729 ] , and it also supports the proposal \n by hofmeyr and cornish - bowden   that the end - product demand ( which is \n usually overlooked in studies of metabolism because these metabolites are \n frequently not considered as part of the pathway ) might be essential in flux \n control .", "glutathione ( -glu - cys - gly ; gsh ) is the most abundant nonproteinaceous \n thiol compound ( 110 mm ) \n in almost all living cells .", "gsh is \n involved in the oxidative stress processing , xenobiotic detoxification , and , in \n some plants and yeasts , in the inactivation of toxic heavy metals ( for a recent \n revision see ) .", "gsh is synthesized by two enzymes : -glutamylcysteine synthetase \n ( -ecs ) and glutathione synthetase ( gs ) ( figure 2 ) , \n which catalyze reactions with high - equilibrium constants ( keq > 1000 ) . under a low gsh demand ( unstressed \n conditions )", ", the producing block of enzymes has to receive information from the \n last part of the pathway to ( i ) avoid the excessive and toxic accumulation of \n the intermediary -ec and ( ii ) reach a stable steady state .", "this information transfer is mediated by gsh , \n which exerts strong competitive inhibition of -ecs   ( figure 2 ) .", "gsh and cys also exert inhibition on the \n atp - sulfurylase ( atps ) and on sulfate transporters ( figure 2 ) ( for a review , \n see ) .", "the feedback inhibition of -ecs has led several researchers to propose \n that this enzyme is the rate - limiting step of gsh synthesis [ 3335 ] .", "although there are no studies about the \n pathway 's behavior under stressed conditions , which means under a high gsh \n demand , the proposal that -ecs is the key enzyme has been automatically \n extended to any environmental condition such as heavy metal exposure . by assuming that -ecs is the rate - limiting step , many research groups \n have tried to increase , in plants and yeasts , the rate of synthesis and the \n concentration of gsh and phytochelatins ( pcs ) with the aim of fortifying their \n heavy metal resistance and storage capacity , mainly toward cd .", "the development of organisms able to grow in \n soils and water systems contaminated with heavy metals , which may have the \n ability of accumulating toxic metal ions , is of biotechnological interest for \n bioremediation strategies . with this goal in mind", ", \n researchers have then overexpressed -ecs and other pathway enzymes , including \n phytochelatin synthase ( pcs ) ( table 2 ) .", "some of these experiments have been \n partially successful in increasing gsh levels , although this has been rather \n marginal with no correlation between enzyme levels and gsh concentration .", "unfortunately , these overexpression experiments \n have not been accompanied by determinations of fluxes or other relevant metabolite \n concentrations such as pcs or cys .", "on \n the other hand , the overexpression of pcs has surprisingly induced oxidative stress \n and necrosis instead of increasing cd accumulation and resistance \n .", "this result suggests that , under high \n gsh demand ( i.e. , for pcs synthesis and for direct heavy metal \n sequestration by gsh ) , the gsh concentration does not suffice for maintaining \n the other essential gsh functions such as oxidative stress management and \n xenobiotic detoxification .", "another problem in the study \n of gsh biosynthesis for its eventual manipulation is that the pathway has been \n analyzed considering only the gsh - synthetic reactions without taking into \n account the gsh - consuming reactions ( figure 2 ) , .", "the analysis of an incomplete pathway leads to \n misleading conclusions about the control of flux .", "metabolic modeling has shown that only with \n the incorporation of the consuming reactions of the pathway end products , a true \n steady state can be established . in \n conclusion , without a solid theoretical framework , the overexpression of only \n one enzyme ( the  rate - limiting step  ) , or of many arbitrarily selected enzymes \n ( tables 1 and 2 ) , the problem of increasing the flux or metabolite \n concentrations can not be solved .", "there are some successful examples of the genetic engineering approach to manipulate \n metabolism : overexpression \n ( approx .", "23 fold ) of the five genes of the tryptophan synthesis pathway in s. \n cerevisiae , to increase ( 9-fold ) flux ; increase \n in amino acids ( trp , ile , lys , val , thr ) and \n trehalose production in corynebacterium glutamicum , in which some proteins of \n each metabolic pathway are simultaneously overexpressed , but some of them with \n mutations that confer insensitivity to feedback inhibition [ 4853 ] . in these transformed bacteria ,", "the end products \n are indeed overproduced and their excretion is accelerated;overexpression \n of pfk and pyk to increase ethanol production by 35% in e. coli , although lactic acid formation was not modified ; mannitol \n 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase \n mannitol production by 2750% in ldh - deficient lactococcus lactis ; increase \n in sorbitol production ( 5 fold ) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol \n 6-phosphate dehydrogenase ( activity up to 250 fold in mutants versus wild type ) ; overexpression \n of pfk ( 14 fold ) or ldh ( 3.5 times ) to increase 2 - 3 times the homolactic \n fermentation flux in lactococcus lactis growing on maltose , and in parallel decrease fluxes toward secondary acids and \n ethanol . \n  overexpression \n ( approx", ". 23 fold ) of the five genes of the tryptophan synthesis pathway in s. \n cerevisiae , to increase ( 9-fold ) flux ; increase \n in amino acids ( trp , ile , lys , val , thr ) and \n trehalose production in corynebacterium glutamicum , in which some proteins of \n each metabolic pathway are simultaneously overexpressed , but some of them with \n mutations that confer insensitivity to feedback inhibition [ 4853 ] .", "in these transformed bacteria , the end products \n are indeed overproduced and their excretion is accelerated ; overexpression \n of pfk and pyk to increase ethanol production by 35% in e. coli , although lactic acid formation was not modified ; mannitol \n 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase \n mannitol production by 2750% in ldh - deficient lactococcus lactis ; increase \n in sorbitol production ( 5 fold ) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol \n 6-phosphate dehydrogenase ( activity up to 250 fold in mutants versus wild type ) ; overexpression \n of pfk ( 14 fold ) or ldh ( 3.5 times ) to increase 2 - 3 times the homolactic \n fermentation flux in lactococcus lactis growing on maltose , and in parallel decrease fluxes toward secondary acids and \n ethanol .", "several glycolytic enzymes are overexpressed in \n at least 70% of human cancers . except \n for glucose transporter 1 ( glut-1 ) , the other 11 glycolytic enzymes ( hk to ldh ) \n are overexpressed in brain and nervous system cancers .", "prostate and lymphatic nodule cancers ( hodgkin \n and non - hodgkin lymphomas ; myelomas ) overexpress 10 glycolytic enzymes ( except \n for hk ; in prostate cancer glut1 is also overexpressed ) .", "there is a second group of cancers that overexpresses 68 glycolytic \n genes ( skin , kidney , stomach , testicles , lung , liver , placenta , pancreas , \n uterus , ovary , eye , head and neck , and mammary gland ) .", "a third group includes those cancers overexpressing \n 1 or 2 glycolytic genes ( bone , bone marrow , cervix , and cartilage ) . in animals ,", "gene expression of glycolytic enzymes is regulated ( both coordinately \n and individually ) under hypoxic conditions by hypoxia - responsive transcription \n factors such as hif-1 ( hypoxia - inducible factor 1 ) , sp family factors , ap-1 , and possibly mre \n ( metal response elements ) [ 8 , 5961 ] .", ". there are binding sites ( consensus sequence acgt ) for hif-1 in the promoters of genes for hk , pfk-1 , \n aldo , gapdh , pgk , eno , pyk , and ldh ( reviewed in ) .", "tpi and perhaps hpi and pgam are also induced \n by hypoxia , but it is not clear whether hif-1 mediates this induction , and whether this \n factor regulates other metabolic pathways associated with glucose \n catabolism .", "for example , although \n glycogen phosphorylase is overexpressed under hypoxia in human tissues , \n the role of hif-1 has not been demonstrated .", "if direct manipulation of \n pathway genes becomes difficult , then the overexpression or repression of \n transcription factors such as hif-1 , ap1 , and mres might solve the problem of changing \n flux , although overexpression of transcription factors may also be difficult \n due to the numerous upstream and downstream factors involved .", "the kinetoplastid parasites trypanosoma \n cruzi , trypanosoma brucei , and leishmania are the causative agents of \n chagas disease , african trypanosomiasis , and leishmaniasis , respectively . the \n available drugs to treat these diseases are highly toxic for humans .", "moreover , the \n parasites may become resistant , and hence the search for new drugs and drug \n targets is relevant for solving these public health problems . in these parasites ,", "the \n metabolism is organized in a peculiar way ; they have a subcellular structure \n called glycosome in which several metabolic pathways take place : \n gluconeogenesis , reactions of the pentose phosphate pathway , purine salvage and \n pyrimidine biosynthesis , -oxidation \n of fatty acids , fatty acid elongation , biosynthesis of ether lipids , and the \n first seven steps of glycolysis .", "glycosomal glycolytic enzymes have unique structural , kinetic , and \n regulatory features not found in their human counterparts , and therefore have \n been the subject of extensive biochemical studies to use them as drug targets .", "the rationale behind this is to synthesize \n inhibitors that affect mainly the parasitic enzymes with relatively low effect \n on the human enzymes since the infective parasite stages rely mostly on \n glycolysis for atp supply .", "there are reports on the \n design of presumed specific inhibitors for some of the t. brucei glycolytic \n enzymes : glut ( bromoacetyl-2-glucose ) , hk , hpi , pfk , aldo , tpi , gapdh , \n pgk , pyk , and glycerol-3-phosphate dehydrogenase .", "although the purified enzymes display \n very low ki values for these \n inhibitors and some of them inhibit \n parasite growth or infective capabilities , their effect on inhibiting the \n glycolytic flux has not been explored .", "therefore , it is not yet possible to \n directly ascribe the effects seen in parasite culture with the in vitro effects \n on the isolated enzymes . to identify the \n best drug targets ,", "determination of the flux control steps of glycolysis \n in t. brucei has been recently initiated .", "trypanothione ( tsh2 ) \n is a reducing agent present in trypanosomatids that is synthesized from one \n spermidine and two gsh molecules by tsh2 synthetase ( trys ) \n ( figure 3 ) .", "this metabolite and its reducing enzyme , \n tsh2 reductase ( tryr ) , replace the antioxidant and metabolic \n functions of the more common gsh / gsh reductase system present in mammals .", "in \n fact , most of the antioxidant metabolism of these parasites depend on tsh2 ( figure 3 ) [ 69 , 70 ] . thus , the enzymes of this metabolic pathway have been \n proposed as drug targets for killing the parasites .", "diminution in its gene transcription yields a \n loss of activity between 5690% , depending on \n the genetic technique [ 7275 ] . in \n knockdown t. brucei cells", "( i.e. , when tryr activity has diminished \n to less than 10% of the wild - type level ) , the parasites show growth diminution \n and higher sensitivity to h2o2 in culture and loss of \n infectiveness in mice . however , tsh2 and thiol compound contents \n were not affected .", "tryr \n downregulation by > 85% in leishmania species causes inability to survive under \n oxidative stress inside macrophages [ 7274 ] .", "in contrast , when tryr is 14- and 10 fold \n overexpressed in leishmania and t. cruzi , respectively , there are no \n significant differences in h2o2 susceptibility between \n control and transfected cells ; both types of cells are also equally resistant \n to the oxidative stress - inducers gentian violet , and nitrofurans .", "intriguingly , the cellular levels of tsh2 , \n gsh , and glutathionyl - spermidine , determined in both types of experiments ( tryr \n suppression and overexpression ) were similar in control and transformed cells .  ", "knockdown \n of trys by sirna in procyclic t. brucei causes ( i ) viability impairment and arrest of proliferation when tsh2 levels decrease to 15% of the wild - type level , ( ii ) increased sensitivity to h2o2 and alkyl hydroperoxides , ( iii ) damage to the plasma membrane , and ( iv ) \n diminution of the tsh2 content and accumulation of gsh and \n glutathionyl - spermidine .", "a similar metabolite variation ( lower tsh2 ; \n higher gsh ) was attained with a trys knockdown induced by sirna in the \n bloodstream form of t. brucei .", "this \n trys knockdown also induced an increased sensitivity to different compounds \n that affect tsh2 metabolism such as arsenicals , melarsen oxide , \n trivalent antimonials , and nifurtimox .", "indeed , western blot analysis showed , \n in addition to the expected ( 10-fold ) decrease in trys protein , a 2 - 3-folds increase in -ecs and tryr .", "the changes in expression of \n other enzymes suggest unveiled compensatory or pleiotropic effects on tsh2 metabolism .  ", "other researchers have selected -glutamylcysteine synthetase ( -ecs ) , the presumed rate - limiting step of gsh \n synthesis , as an alternative drug target of tsh2 synthesis in t. brucei ( figure 3 ) .", "knockdown of -ecs gene in the parasite induces cell death \n and depletion of gsh and tsh2 only after 80% decrease in the enzyme \n content .", "the -ecs knockdown cells are rescued from death by \n adding external gsh , which elevates the cellular gsh and tsh2 levels \n .", "glutathione synthetase ( gs ) has not been manipulated in trypanosomatids , or in any other organism , perhaps \n because it has been considered as a nonrate - limiting step of gsh and tsh2 biosynthesis . however , dna microarray \n analysis of antimonite - resistant leishmania tarentolae shows increased \n transcription of -ecs , gs , and p - glycoprotein a rnas . \n although it was not evaluated whether increase in gene transcription correlated \n with an increase in enzyme activity , it may be possible that under high gsh \n demand ( i.e. , under oxidative stress conditions ) gs might exert control of tsh2 synthesis . on the other hand , ornithine \n decarboxylase", "( odc ) overexpression in t. brucei ( the presumed limiting \n step of spermidine synthesis ) causes no change in tsh2 levels \n .", "therefore , odc does not seem to be \n a controlling step of tsh2 synthesis .", "although almost full inhibition ( > 80% ) of gene transcription or activity of any of \n these enzymes results in parasite death , the question remains of how tsh2 metabolism is affected when the enzymes are less inhibited .", "for example , in the therapeutic treatment of \n patients it is certain that drugs have to be administered for long periods of \n time .", "if the parasites are not \n completely cleared from the patient , disease recurrence and generation of drug - resistant \n parasites are possible .", "the results described \n above indicate that each enzyme by itself has low control on tsh2 synthesis and concentration ; therefore , highly specific and very potent \n inhibitors have to be designed in order to attain the required full activity blockade \n to affect tsh2 metabolism in these parasites .", "the metabolic \n control analysis ( mca ) was initially developed by kacser and burns in scotland [ 82 , 83 ] and by heinrich and rapoport \n in east germany \n  [ 84 , 85 ] .", "this analysis establishes a \n theoretical framework that explains the results observed with the enzyme \n overexpression and downregulation experiments . in addition , it helps to identify and design \n experimental strategies for the manipulation of a given process in an organism \n ( heavy metal hyperaccumulation ; increased production of ethanol , co2 , \n lactate or acetate ; or inhibition of a metabolic pathway flux with therapeutic \n purposes ) .", "mca rationalizes the quantitative \n determination of the degree of control that a given enzyme exerts on flux and \n on the concentration of metabolites .", "different \n experimental approaches have been developed to detect and direct what has to be \n done and measured , in order to identify and understand why an enzyme exerts a \n significant or a negligible control on flux and metabolite concentration in a \n metabolic pathway .", "experiments for identifying and manipulating the conceptually wrong  rate - limiting \n step . ", "to \n understand how a metabolic pathway is controlled and could be manipulated , its \n control structure has to be evaluated . the \n control structure of a pathway is constituted by the flux control coefficient ( cvi ) , which is the degree of control that the rate ( v ) of a given enzyme i exerts \n on flux j ; the concentration control coefficient ( cvi ) , which is the degree of control that a given enzyme i exerts on the concentration of a metabolite \n ( x ) ; and the elasticity coefficients .", "the control coefficients are systemic \n properties of the pathway that are mechanistically determined by the elasticity \n coefficients ( x ) , which are defined as the degree of sensitivity \n of a given enzyme vi \n  ( i.e. , the enzyme 's ability to change \n its rate ) when any of its ligands ( x : substrate , products or allosteric \n modulators ) is varied .", "viojo , in which the expression dj / dvi describes \n the variation in flux ( j ) when an infinitesimal change is done in the enzyme i concentration or activity . in practice , \n", "the infinitesimal changes in vi \n  are undetectable , and hence measurable noninfinitesimal changes are undertaken . \n ", "if a small change in vi \n  promotes a significant \n variation in j , then this enzyme exerts an elevated flux control ( figure 4 , \n position 1 ) .", "in contrast , if a rather small \n or negligible change in flux is observed when vi \n  is greatly varied , then the enzyme does not exert \n significant flux control ( figure 4 , position 2 ) . to obtain dimensionless and normalized values \n of cvi the scaling factor", "vio / jo is applied , which represents the ratio between the initial values from which \n the slope dj / dvi \n  is \n calculated .", "if all cvi of the pathway enzymes and transporters are added up , the sum comes to \n one ( summation theorem ) .", "the mca clearly distinguishes \n between the control exerted by a given enzyme on flux ( flux control \n coefficient ) and on the metabolite concentration ( concentration control \n coefficient ) .", "thus , an enzyme can have significant control on a metabolite \n concentration but not on the pathway flux .", "on one hand , the use of the rate - limiting step concept \n for manipulating metabolic pathways does not make such differentiation , which \n probably has contributed to the many unsuccessful experiments reported in the \n literature ; on the other hand , it should be clearly defined whether the aim of the \n project is to increase flux and/or a metabolite concentration since mca \n establishes for each aim a different experimental design . to determine the flux control \n coefficient of a given enzyme , small variations in the enzyme content , or \n preferentially , in activity are required , without altering the rest of the \n pathway , and then the changes in flux are determined .", "the experimental points are plotted as shown \n in figure 4 to calculate the slope at the reference point vio / jo .", "this experiment , apparently easy to perform , \n has demanded great intellectual and experimental effort .", "several experimental strategies have been \n developed to determine cvi : \n formation of heterokarionts and heterocygots ( classical genetics),titration of flux with specific inhibitors , elasticity analysis , mathematical modeling ( in \n  silico biology),in vitro reconstitution of metabolic pathways , genetic engineering to manipulate in vivo protein levels . \n ", "formation of heterokarionts and heterocygots ( classical genetics ) , titration of flux with specific inhibitors , mathematical modeling ( in \n  silico biology ) , in vitro reconstitution of metabolic pathways , genetic engineering to manipulate in vivo protein levels .", "the arginine biosynthesis in neurospora crassa was the first metabolic \n pathway in which flux control coefficients were experimentally determined by \n kacser 's laboratory .", "this fungus \n forms multinucleated mycelia that facilitate the generation of polyploid cells . \n  by mixing different ratios of spores containing \n genes encoding wild ( active ) and mutant ( inactive ) enzymes of this pathway , it \n was possible to generate heterokaryont mycelia with different content , and \n activity , of four pathway enzymes .", "the authors built plots of enzyme activity versus \n flux ( see figure 4 ) for acetyl - ornithine aminotransferase , ornithine \n transcarbamoylase , arginine - succinate synthetase , and arginine - succinate lyase . \n ", "all the experimental points of these \n heterokaryonts localized near to position 2 of figure 4 with cvi =   0.020.2 ( flux control by these enzymes was \n only 220% ) , which indicated that none of these enzymes exerted significant \n control on arginine synthesis .", "the authors did not determine the remaining flux \n control ( 75% ) , which might reside in carbamoyl - phosphate synthetase i ( this mitochondrial \n ammonium - dependent isoform can be bound to the mitochondrial inner membrane or \n form complexes with ornithine transcarbamoylase [ 87 , 88 ] ) and in mitochondrial citruline / ornithine \n transporter , both of which have been proposed as limiting steps , or might be in \n the arginine demand for protein synthesis .", "organisms \n with many alleles of one enzyme may form homo - and heterozygotes expressing \n different activity levels .", "drosophila \n melanogaster has three adh alleles encoding for isoforms with different vmax . when three natural homozygotes , a null mutant , and \n some heterozygotes were generated , different adh activities were attained but \n the ethanol consuming rate did not change ( figure 4 , position 2 ) .", "oxidative phosphorylation ( oxphos ) is \n the only pathway for which specific and potent inhibitors for many enzymes and \n transporters are available .", "oxphos is divided in two segments ( figure 5 ) : the \n oxidative system ( os ) formed by substrate transporters ( pyruvate , \n 2-oxoglutarate , glutamate , glutamate / aspartate , dicarboxylates ) , krebs cycle \n enzymes , and the respiratory chain complexes ; and the phosphorylating system \n ( ps ) constituted by the atp / adp ( ant ) and pi ( pit ) transporters , and atp \n synthase .", "when the flux ( atp synthesis ) \n is titrated by adding increasing concentrations of each specific inhibitor , \n plots are generated in which the enzyme activity is progressively diminished by \n increasing inhibitor concentration .", "hence , the cvi value depends on the type of \n inhibitor usedfor irreversible inhibition,(2)cvij  =  ( imaxjo)(djdi)[i]0 , \n for simple noncompetitive inhibition,(3)cvij  =  ( kijo)(djdi)[i]0 , \n for simple competitive inhibition,(4)cvij  =  ( -ki[(1+s)/km]jo)(djdi)[i]0 , \n  where jo is the pathway flux in the absence \n of inhibitor ; imax , minimal \n inhibitor concentration to reach maximal flux inhibition ; k \n i , inhibition constant ; s , \n substrate concentration ; km , \n michaelis - menten constant ; and d \n j / d \n i , \n initial slope ( [ i ] = 0 ) of inhibition titration curve . for irreversible inhibition,(2)cvij ", "=  ( imaxjo)(djdi)[i]0 , \n  for simple noncompetitive inhibition,(3)cvij  =  ( kijo)(djdi)[i]0 , \n  for simple competitive inhibition,(4)cvij  =  ( -ki[(1+s)/km]jo)(djdi)[i]0 , \n  to estimate flux control \n coefficients from inhibitor titration of adp - stimulated ( state 3 ) respiratory \n rates ( i.e. , mitochondrial o2 consumption coupled to atp synthesis ) , ( 2 ) for irreversible inhibitors was used \n because researchers assumed that mitochondrial inhibitors such as rotenone , \n antimycin , carboxyatractyloside , and oligomycin were ", "however , under this assumption flux control coefficients were usually \n overestimated [ 90 , 91 ] . to solve this \n problem , gellerich et al .  ", "developed ( 5 ) for noncompetitive \n tightly - bound inhibitors and , by using nonlinear regression analysis , it was \n possible to include all experimental points from the titration curve thus \n increasing accuracy in calculating cvi:(5)j  =  [ n(joji)2encojoeon[(n    co)jo    ( nji)en ] ]  +  ji  e2+(kd+ieo)ekdeo=0 , in which jo and ji are the \n respiration fluxes in the noninhibited ( e = eo ) and inhibited ( e = 0 ) states ; k \n d is the dissociation constant of \n the enzyme - inhibitor complex , and n is an empirical component that expresses the relationship between substrate \n concentration and the reaction catalyzed by the enzyme e. the analysis of data in table 3 shows that oxphos is not controlled by only one \n limiting step , but the flux control is rather distributed among several enzymes \n and transporters .", "it is worth noting \n that the value of the flux control coefficient depends on the content of enzyme \n or transporter , which varies from tissue to tissue .", "perhaps the atp / adp translocase in as-30d hepatoma mitochondria \n might reach the status of being the  oxphos limiting step  with a ca nt = 0.70 , or the pi transporter in kidney mitochondria , or the \n atp / adp translocase and the respiratory chain complex 3 in liver mitochondria , but \n", "it should be noted that other steps also exert significant control ( table 3 ) .", "although the distribution of control varies \n between tissues , the flux control mainly resides in the ps of organs with high atp \n  demand such as the heart   ( cpt+ant+atpsynthase = cps = 0.73 ) , kidney ( cps = 0.75 ; cos = 0.31 ) , and   fast - growing tumors ( cps = 0.98 ) .", "in contrast , in the \n liver   ( cos = 0.80 ; cps = 0.65 ) and brain ( cos = 0.35 ; cps = 0.41 ) , the control is shared by both systems .", "determined that the oxphos \n control was shared by the ps ( cps = 0.62 ) and the atp demand ( purified atpase ) . in turn ,", "rossignol et al .   concluded that \n the os exerted the main control ( cos = 0.68 ) , but these authors apparently used low - quality mitochondria \n ( low respiratory control values that lead to low rates of atp synthesis \n associated with high rates of respiration ) that were not incubated under near physiological \n conditions ( 10 mm pyruvate , 10 mm malate , 10 mm pi , ph 7.4 in tris buffer ) , and the authors incorrectly assumed that rotenone and antimycin \n were irreversible inhibitors .", "it is notorious \n that in all works shown in table 3 at least one of these mistakes is evident .", "there are some inhibitors for enzymes and transporters from other pathways , but they \n are not quite specific and may affect other sites . due to the fact that there are no inhibitors \n for every step in these pathways ,", "examples \n of these inhibitors are 6-chloro-6-deoxyglucose for glucose transporters in \n bacteria , 2-deoxyglucose for hpi , iodoacetate for gapdh , 1,4-dideoxy-1,4-imino - d - arabinitol \n for glycogen phosphorylase , oxalate and oxamate for ldh , 6-amino \n nicotinamide for the phosphate pentose pathway , amino - oxyacetate for \n aminotransferases and kirureninase ( tryptophan synthesis ) , norvaline for \n ornithine transcarbamylase , mercaptopycolinate for pep carboxykinase , acetazolamide \n for carbonic anhydrase , and isobutyramide for adh ( compiled by \n fell ) .", "potential uses of the experimental approachmitochondrial \n pathologies are a heterogeneous group of metabolic perturbations characterized \n by morphological abnormalities and/or oxphos dysfunction .", "mitochondrial dna analysis has revealed specific \n mutations for some mitochondriopathies . although the specific oxphos mutations causing \n the disease may appear in all tissues , the functioning of only some of them is \n altered .", "the organ 's sensitivity might be related to the different flux control \n coefficients of the mutated enzyme in the different tissues ( table 3 ) and to \n their atp supply dependence from oxphos versus \n glycolysis.mca allows for the analysis of a metabolic flux or intermediate concentration by focusing \n either on one step or by grouping enzymes in blocks or in pathways .", "thus , a comparative analysis of oxphos control \n distribution reveals that heart , kidney , some fast growing tumors ( rat as-30d \n hepatoma , mouse fibrosarcoma , human breast , lung , thyroid carcinoma , melanoma ) \n , and perhaps skeletal muscle are more susceptible to mitochondrial mutations \n in atp synthase , which is the only ps site with subunits encoded in the mitochondrial \n genome . on the other side , liver and \n brain", "might be more susceptible to mitochondrial mutations of the respiratory \n chain enzymes ( see table 3 ) .", "considering that the brain is a fully aerobic \n organ , whereas the liver depends on both oxphos ( 7080% ) and glycolysis \n ( 2030% ) for atp supply , then it can be postulated that the brain is more \n sensitive to mutations in the mitochondrial genome than the liver because subunits \n of complexes i , iii , and iv are encoded by the mitochondrial genome.titration of flux with \n specific inhibitors to determine the flux control coefficients of oxphos has been \n applied to intact tumor cells .", "the results showed that the flux control \n resided mainly in site 1 of the respiratory chain ( csitel = 0.30 ) , whereas the other evaluated sites exerted a marginal \n control .", "this observation could \n have therapeutic application if site 1 does not exert control in healthy cells , \n leading to less severe side effects.the use of inhibitors in \n intact cells to determine control coefficients might pose two problems : hydrophilic \n inhibitors such as carboxyatractyloside ( for ant ) and -cyano-4-hydroxy - cinammate ( for pyruvate transporter ) \n can not readily enter the cell due to the presence of the plasma membrane \n barrier ; the other problem is that hydrophobic but slow inhibitors , such as \n oligomycin , require long incubation times to ensure the interaction with the \n specific sites .", "these problems can be \n solved by incubating the cells for long periods of time and taking care of cell \n viability , for instance , as-30d hepatoma cells are fairly resistant to this \n mechanical manipulation as they maintain high viability after a lengthy \n incubation under smooth orbital agitation of 1 h at 37c \n .", "mitochondrial \n pathologies are a heterogeneous group of metabolic perturbations characterized \n by morphological abnormalities and/or oxphos dysfunction .", "mitochondrial dna analysis has revealed specific \n mutations for some mitochondriopathies . although the specific oxphos mutations causing \n the disease may appear in all tissues , the functioning of only some of them is \n altered .", "the organ 's sensitivity might be related to the different flux control \n coefficients of the mutated enzyme in the different tissues ( table 3 ) and to \n their atp supply dependence from oxphos versus \n glycolysis .", "mca allows for the analysis of a metabolic flux or intermediate concentration by focusing \n either on one step or by grouping enzymes in blocks or in pathways .", "thus , a comparative analysis of oxphos control \n distribution reveals that heart , kidney , some fast growing tumors ( rat as-30d \n hepatoma , mouse fibrosarcoma , human breast , lung , thyroid carcinoma , melanoma ) \n , and perhaps skeletal muscle are more susceptible to mitochondrial mutations \n in atp synthase , which is the only ps site with subunits encoded in the mitochondrial \n genome . on the other side , liver and \n brain might be more susceptible to mitochondrial mutations of the respiratory \n chain enzymes ( see table 3 ) . considering that the brain is a fully aerobic \n organ ,", "whereas the liver depends on both oxphos ( 7080% ) and glycolysis \n ( 2030% ) for atp supply , then it can be postulated that the brain is more \n sensitive to mutations in the mitochondrial genome than the liver because subunits \n of complexes i , iii , and iv are encoded by the mitochondrial genome .", "titration of flux with \n specific inhibitors to determine the flux control coefficients of oxphos has been \n applied to intact tumor cells .", "the results showed that the flux control \n resided mainly in site 1 of the respiratory chain ( csitel = 0.30 ) , whereas the other evaluated sites exerted a marginal \n control .", "this observation could \n have therapeutic application if site 1 does not exert control in healthy cells , \n leading to less severe side effects .", "the use of inhibitors in \n intact cells to determine control coefficients might pose two problems : hydrophilic \n inhibitors such as carboxyatractyloside ( for ant ) and -cyano-4-hydroxy - cinammate ( for pyruvate transporter ) \n can not readily enter the cell due to the presence of the plasma membrane \n barrier ; the other problem is that hydrophobic but slow inhibitors , such as \n oligomycin , require long incubation times to ensure the interaction with the \n specific sites .", "these problems can be \n solved by incubating the cells for long periods of time and taking care of cell \n viability , for instance , as-30d hepatoma cells are fairly resistant to this \n mechanical manipulation as they maintain high viability after a lengthy \n incubation under smooth orbital agitation of 1 h at 37c \n .", "xovio , which is a dimensionless number that show the rate variation v of a given enzyme or transporter i when the concentration of a ligand x ( substrate s , product p or allosteric modulator ) is varied in infinitesimal \n proportions .", "the elasticity coefficients are positive for those metabolites \n that increase the enzyme or transporter rate ( substrate or activator ) , and they \n are negative for the metabolites that decrease the enzyme or transporter rates \n ( product or inhibitor ) .", "an enzyme working , under a steady - state metabolic flux , at saturating conditions of s or p , \n is no longer sensitive to changes in these metabolites .", "thus , its elasticity is close to zero ( figure 6 , x = 0 ) . \n  in turn", ", an enzyme working at s or p \n concentrations well below the michaelis constant ( kms or kmp ) \n is expected to be highly sensitive to small variations in these metabolites ( figure \n 6 , x = 1 ) .", "if the kinetic parameters of an enzyme are \n known ( vmf , vmr ,   kms , and kmp ) , then the enzyme elasticity for any given metabolite \n concentration may be calculated as shown in the following equations . for substrate,(7)svi  =  s / kms1  +  s / kms  +  p / kmp  +  11    /keq , and \n for product,(8)pvi  =  p / kmp1  +  s / kms  +  p / kmp    /keq1    /keq , in which  is", "the mass \n action ratio , and keq is the equilibrium constant preferentially determined \n under physiological conditions .", "an enzyme with low elasticity can not increase ( or decrease ) its rate despite large \n variations in s ( or p ) concentration ; in consequence , such enzyme exerts a high \n flux control . in turn", ", an enzyme with a \n high elasticity can adjust its rate to the variation in s or p concentrations , \n and thus it does not interfere with the metabolic flux , exerting a low flux control .", "this inverse relationship between the \n elasticity and the flux control coefficients is expressed in a formal equation \n denominated connectivity theorem .", "a \n metabolic pathway can be divided in two blocks around an intermediary x : the \n producing ( synthetic , supply ) and the consuming ( demand ) enzyme blocks of x are i1 and i2 , respectively .", "thus , the connectivity theorem for this two - block system is(9)cv1jcv2j  =  xv2xv1 .", "the negative sign of the right part of the equation \n cancels with x , which is negative because x \n is a product of enzyme block i1 ( figure 6 ) . to obtain the flux control coefficients", "many strategies have been designed [ 90 , 103108 ] , but the most \n used and probably more trustworthy is that in which the initial pathway metabolite \n ( so ) concentration is varied to increase the x concentration ( any intermediary \n in the pathway ) , and measuring in parallel the variation in flux . under steady - state conditions , the flux rate \n is equal to the rate of end - product formation ( i.e. , lactate or alcohol for \n glycolysis ; oxygen consumption for oxphos ) and to the rate of any partial \n reaction .", "the slope , calculated at the reference \n coordinate ( xo , jo ) that is \n equivalent to ( so , vio ) , yields \n the elasticity coefficient of the consuming block of x. in another set of experiments , an inhibitor is \n added to block one or more enzymes after x. the x concentration and flux are determined and \n plotted as shown in figure 7 , from which the elasticity coefficient of the \n producing block is calculated .", "the flux \n control coefficients are determined by using the connectivity theorem and \n considering that the sum of the control coefficients comes to 1 , c1 + c2 = 1 ( summation theorem):(10)cv1j  =  xv2xv2    xv1,cv2j  =    xv1xv2    xv1 . \n ", "this method for determining cvi using the elasticities of the two blocks was called double \n modulation by kacser and burns .", "years \n later , brand and his group [ 103 , 104 ] renamed this method as top - down approach . by applying the procedure shown in figure 7 \n and using ( 10 ) for different metabolites along the metabolic pathway , it \n is possible to identify those sites that exert a higher control ( which may be \n the sites for therapeutic use or biotechnological manipulation ) and those that \n exert a negligible control under a given physiological or pathological \n situation .", "elasticity analysis has been used to evaluate the oxphos control distribution \n in tumor cells .", "almost all studies on \n this subject have been carried out with isolated mitochondria incubated in \n sucrose - based medium at 25 or 30c or with the more physiological kcl - based medium but \n still at 30c ( table 3 ) .", "furthermore , \n these studies did not consider that the product , atp , never accumulates in the \n living cells , which does occur in experiments with isolated mitochondria .", "under such a condition , a steady state in atp production can \n never be reached as in living cells . in \n other words , the distribution of control in mitochondria ( table 3 ) has been \n determined in the absence of an atp - consuming system . a remarkable exception to", "this incomplete \n experimental design was the work done by wanders et al . , in which isolated liver mitochondria were incubated \n with two different atp - consuming systems ( or adp - regenerating systems ) : hk + \n glucose and creatine kinase ( ck ) + creatine . under this", "more physiological setting , the oxphos \n flux control distributed between ant and the atp - consuming system ; however , flux \n control by the other pathway components was not examined .", "therefore , to accurately evaluate oxphos control \n distribution , mitochondria should be incubated in the presence of an \n atp - consuming system or in their natural environment ( i.e. , inside the cell ) .", "the rate of oxphos in intact \n cells is determined from the rate of oligomycin - sensitive respiration : in the \n steady state , the enzyme rates are the same and constant ; in branched pathways \n the sum of the branched fluxes equals the flux that supplies the branches .", "the global elasticity of the atp - consuming processes \n ( e.g. , synthesis of protein , \n nucleic acid , and other biomolecules , as well as ion atpases to maintain the \n ionic gradients , mechanical activity such as muscular contraction or flagellum \n and cilium movement , and secretion of hormones , digestive enzymes and \n neurotransmitters ) is estimated by inhibiting flux with low concentrations of \n oligomycin or a respiratory chain inhibitor . to determine the elasticity of the \n atp - producing block , flux , and [ atp ]", "are varied with streptomycin , an inhibitor \n of protein synthesis ( figure 7 ) .", "the \n elasticity coefficients are calculated from the initial coordinate slopes \n ( without inhibitors ) of each titration . with \n this procedure", ", it has been determined that the atp - consuming block exerts a \n significant flux control of 34% .", "remarkably , \n this flux control value obtained in cells is quite similar to the flux control \n coefficients of the atp - consuming system ( hk or ck ) reported by wanders \n et al .   with isolated \n mitochondria .", "elasticity analysis by enzyme \n blocks allows the inclusion of the end - product demand as another pathway block . \n ", "the conclusions obtained from this \n analysis have formulated the supply - demand theory , which proposes that when \n flux is controlled by one block ( demand ) , the concentration of the end - product \n is determined by the other block ( supply ) . the ratio of elasticities determines the distribution of flux control \n between supply and demand blocks . for \n instance , if x > x ( i.e. , demand becomes saturated by the end - product x , \n and hence its elasticity is near zero ) , then the demand block exerts the main \n  flux control . for concentration control , \n at larger x  x ,   smaller absolute values of both csupply and cdemand are attained ; hence , under demand saturation , \n the supply elasticity fully governs the magnitude of the variation in the \n end - product concentration . on the other \n hand ,", "when demand increases , it loses flux control and induces a diminution in \n the end - product concentration . in turn , supply gains flux control and loses \n concentration control . in the presence of feed - back inhibition ,", "the system can \n maintain the end - product concentration orders of magnitude away from \n equilibrium ( at a concentration around the k0.5 of the allosteric enzyme ) . as mentioned before ,", "the \n demand is not usually included in the pathway because it is erroneously thought \n that it is not part of it .", "but then , is \n it valid to analyze the control of a metabolite synthesis if its demand is not \n considered ?", "when the demand block is not \n included , it is assumed that the metabolic pathway produces a metabolite at the \n same rate regardless whether the metabolite demand is high or low .", "this reasoning is incorrect because a \n metabolic pathway indeed responds to changes in the metabolite demand and , more \n importantly , a pathway without end - products consumption reactions is unable to \n reach a steady state .", "the intermediary x linking the two blocks is one \n of the end - products of the producing block ( e.g. , \n pyruvate or lactate or ethanol , and atp for glycolysis ) .", "the variation in rate of the two blocks in \n response to a variation in x can be theoretical or experimentally determined ( figure 8(a ) ) .", "it is worth noting that , for this \n supply - demand approach , it is not necessary to know the kinetics of each \n pathway enzyme because the rate response of each block reflects the global kinetics \n of all participating enzymes", ". when the x \n concentration is increased , the rate of the supply block decreases ( i ) because x \n is its product and ( ii ) because usually an enzyme within this block receives \n information from the final part of the pathway , decreasing its rate through \n feedback inhibition . in turn", "to better \n visualize the effect of large rate changes , the kinetics of both blocks are \n plotted in a logarithmic scale .", "figure 8(b ) shows the kinetics described in figure 8(a ) converted to natural \n logarithm . the intersection point \n between kinetic curves , at which the supply and demand rates are identical , \n represents the pathway steady - state flux ( in the y axis ) and end - product \n concentration ( in the x axis ) .", "since the \n elasticity is also defined as x = dlnvi / dlnx , the slope at the intersection point represents the elasticity of each \n block towards the intermediary x. here , \n the use of the scalar factor is not necessary because it is included in the \n logarithmic equation . with the elasticity \n coefficients calculated from plots like those shown in figure 8 , and the \n connectivity theorem ,", "the example in figure 8(b ) shows that \n the demand exerts a high flux control ( and has low elasticity ) and the supply \n block exerts low control ( and has high elasticity ) . the \n fact that the demand may exert a high flux control in metabolite pathways has \n at least three important implications : ( a ) the supply block responds to variations \n in the demand ( high elasticity ) ; ( b ) the demand block has information transfer \n mechanisms towards the supply block that avoid the unrestricted intermediary accumulation under a low demand , \n particularly when the supply block has reactions with large keq ( > 100 ; g > 3 kcal mol at 37c ) ; and ( c ) \n if the main flux control resides in the demand block , then the supply block may \n only exert control on the intermediary concentration but not on the flux [ 30 , 32 ] .", "this last conclusion explains why it \n is incorrect to consider that an enzyme that controls flux must also control \n the intermediary concentration", ". regulatory mechanisms of \n enzyme activity are modulation of protein concentration by synthesis and \n degradation , as well as covalent modification and variation in the substrate or \n product concentrations ( which are components of the pathway ) .", "in addition , another regulatory mechanism is the \n modulation by molecules that are not part of the pathway , that is , through allosteric interaction with cooperative ( sigmoidal \n kinetics ) or noncooperative enzymes ( hyperbolic kinetics ) ( e.g. , ca activates some krebs cycle dehydrogenases ; citrate inhibits pfk-1 ; malonyl - coa inhibits \n the mitochondrial transporter of acyl - carnitine / carnitine ; or the initial \n substrate of a pathway that has not entered the system ) . for these last cases , kacser and burns \n", "proposed the use of the response coefficient r which is defined by the \n following expression:(11)rm  j  =  cvijmvi , where m is the external modulator of the i enzyme .", "the response coefficient is dj / dmmo / jo . \n  if the elasticity of the sensitive \n enzyme toward the external effector is also determined , then it is possible to \n calculate cvi by using ( 11 ) .", "unfortunately , due to the experimental complexity for determining the \n elasticity coefficient , this coefficient is often calculated in a theoretical \n way by using the respective rate equation ( michaelis - menten or hill equations ) \n and the kinetic parameters km and vmax determined by someone else under optimal \n assay conditions , which are commonly far away from the physiological ones .", "therefore , for this theoretical determination \n of elasticity only the value of the external modulator concentration is required .", "it is convenient to emphasize that the determination \n of the flux control coefficients becomes more reliable when they are calculated \n from several experimental points ( figure 7 ) , instead of only one , as occurs \n with the theoretical elasticity analysis .", "determined the flux control distribution of gluconeogenesis \n from lactate in hepatocytes by using both theoretical and experimental elasticity \n analysis and the response coefficient .", "these authors concluded that gluconeogenesis \n stimulated by glucagon was controlled by the pyruvate carboxylase ( cpc = 0.83 ) ; in the absence of this hormone , the \n control was shared by pc , pyk , eno - pgk segment , and tpi - fructose-1,6-biphosphatase \n segment .", "elasticity \n analysis has been applied to elucidate the flux control of atp - producing \n pathways in fast - growing tumor cells . for oxphos", ", this approach showed that respiratory chain complex i and \n the atp - consuming pathways were the enzymes with higher control ( cvi = 0.7 ) .", "for glycolysis , the main flux control ( cvi = 0.71 ) resided in glut + hk reactions because \n hk is strongly inhibited by its product g6p despite extensive enzyme overexpression \n .", "examples of elasticity analysis on \n other pathways are photosynthesis , ketogenesis , serine   and \n threonine synthesis in e. coli , glycolysis in yeast , glucose \n transport in yeast , dna supercoiling , glycogen synthesis in muscle , \n and galactose synthesis in yeast . in conclusion", ", the elasticity \n analysis is the most frequently used method for determining flux control \n coefficients because it does not need a group of specific inhibitors for all \n the enzymes and transporters of the pathway , neither does it require knowledge \n of the inhibitory mechanisms or kinetic constants .", "it is only necessary to produce a variation in \n the intermediary concentration x by using an inhibitor of either block or by \n directly varying the x concentration . in agreement with fell", ", it seems impossible for a researcher to analyze one by \n one the rate equation of each enzyme in a metabolic pathway to predict and \n explain the system behavior as a whole . to deal with this problem , in the last three \n decades", "some scientists have constructed mathematical models for some metabolic \n pathways using several software programs .", "thus , the specific variation of a single enzyme \n activity without altering the rest of the pathway ( figure 4 ) , which has been an \n experimentally difficult task for applying mca , becomes easier to achieve with \n reliable computing models .", "there are two basic types of modeling : ( a ) structural modeling and ( b ) kinetic \n modeling .", "the former is related to the pathway chemical reaction structure and \n does not involve kinetic information .", "the information obtained with structural modeling is the \n description of the following : the exact determination of which reactions and \n metabolites interact among them;the conservation reactions .", "there are metabolites for \n which their sum is always constant or conserved ( e.g. , nadh + nad ; nadph + nadp ; \n ubiquinol + ubiquinone ; atp + adp + amp ; coa + acetyl - coa ) .", "the identification of \n conserved metabolites might not be obvious;enzyme groups catalyzing reactions in a given relationship \n with another group of enzymes;elemental modules , which are defined as the minimal \n number of enzymes required to reach a steady state , which can be isolated from the \n system ( for a review about structural modeling ; see ) . \n ", "the exact determination of which reactions and \n metabolites interact among them ; the conservation reactions .", "there are metabolites for \n which their sum is always constant or conserved ( e.g. , nadh + nad ; nadph + nadp ; \n ubiquinol + ubiquinone ; atp + adp + amp ; coa + acetyl - coa ) .", "the identification of \n conserved metabolites might not be obvious ; enzyme groups catalyzing reactions in a given relationship \n with another group of enzymes ; elemental modules , which are defined as the minimal \n number of enzymes required to reach a steady state , which can be isolated from the \n system ( for a review about structural modeling ; see ) .", "in addition to an appropriate computing \n program , this approach requires the knowledge of the stoichiometries , rate \n equations , and keq values of each reaction in the pathway ( or the vmax in the forward and reverse \n reactions ) , as well as the intermediary concentrations reached under a given \n steady state .", "some currently used \n softwares are copasi ( http://www.copasi.org/tiki-index.php ) \n based on gepasi ( http://www.gepasi.org/ ; ) ; metamodel ; winscamp \n and jarnac   ( both available at http://www.sys-bio.org/ ) ; and pysces \n ( http://pysces.sourcesforge.net/ ; ) . for", "other programs and links , go to http://sbml.org/index.psp . to reach a steady - state flux ,", "it is necessary \n to fix the initial metabolite concentration to a constant value and the \n irreversible and constant removal of the end products . except for the final reactions in which their \n products have to be removed from the system ,", "all pathway reactions have to be \n considered as reversible , notwithstanding whether they have large keq ( if there \n is an irreversible reaction under physiological conditions , then a reversible rate \n equation that includes the keq suffices to maintain the reaction as practically \n irreversible ) .", "care should be taken to include the enzyme 's sensitivity toward \n its products because this property is related with the enzyme elasticity and hence \n with its flux control ; omission of this parameter may very likely lead to \n erroneous conclusions .", "it \n should be pointed out that the purpose of kinetic modeling is not merely to \n replicate experimental data but also to explain them .", "thus , pathway modeling is a powerful tool \n that allows for ( i ) the detection of those properties of the pathway that are \n not so obvious to visualize when the individual kinetic characteristics of the \n participating enzymes are examined ; and ( ii ) the understanding of the biochemical \n mechanisms involved in flux and intermediary concentration control .", "modeling requires the consideration of all \n reported experimental data and interactions that have been described for the \n components of a specific pathway , thus allowing for the integration of disperse \n data , discarding irrelevant facts . although all models are oversimplifications of complex cellular \n processes , they are useful for the deduction of essential relationships , for \n the design of experimental strategies that evaluate the control of a metabolic \n pathway , and for the detection of incompatibilities in the kinetic parameters \n of the participating enzymes and transporters , which may prompt the \n experimental revision of the most critical uncertainties . with \n", "the model initially constructed , the simulation results do not usually concur \n with the experimental results ; in consequence , the model normally requires \n refinement , a point at which the researcher 's thinking and knowledge of biology \n plays a fundamental role in modifying the structure and parameters of the model . \n ", "the discrepancies observed between modeling \n and experimentation unequivocally pinpoint what elements or factors have to be re - evaluated \n or incorporated so that the model approximates more closely reality ( i.e. , experimental data ) .", "the comparison of the experimentally obtained \n intermediary concentrations and fluxes with those obtained by simulation is an \n appropriate validating index of the model ; this index indicates whether the \n model approximation to the physiological situation is acceptable or whether \n re - evaluation of the kinetic properties of some enzymes and transporters and/or \n incorporation of other reactions or factors is required .", "a reason to why the results obtained by modeling may substantially differ from the \n experimental results is that the kinetic parameters of the pathway enzyme and \n transporters and the keq values used \n were determined by different research groups , under different experimental \n conditions and in different cell types .", "moreover , \n enzyme kinetic assays are carried out at low , diluted enzyme \n concentrations ( thus discarding or ignoring relevant protein - protein \n interactions ) , and at optimal ( but not physiological ) ph and  room temperature  \n ( which may be far away from the physiological values ) . in addition , no experimental information is \n usually available regarding the reactions reversibility and the product \n inhibition of the enzymes and transporters ( particularly for physiological \n irreversible reactions , i.e. , reactions with large keq ) . with worrisome \n frequency ,", "the researcher has to adjust the experimentally determined vm and km values to achieve a model behavior that acceptably resembles \n that observed in the biological system .", "apparently , \n this type of limitations as well as the sometimes overwhelming amount of \n kinetic data necessary for the construction of a kinetic model has restricted \n the number of reliable models that can be used for the prediction of the \n pathway control structure . once the kinetic model stability , robustness ,", "structural and dynamic properties have \n been evaluated , and experimentally validated , the model may become a virtual \n laboratory in which any parameter or component can be modified or replaced and \n any aspect of the pathway behavior can be explored within a wide diversity of \n circumstances or limits . at this \n stage ,", "glycolysis \n in s. bayanus , s. cerevisiae [ 113 , 123 , 124 ] , and trypanosoma brucei [ 125 , 126 ] is the \n metabolic pathway that has been more extensively modeled .", "both cell types \n have a very active glycolysis and are fully dependent on this metabolic pathway \n for atp supply , under anaerobiosis and aerobiosis , respectively .", "one advantage \n of modeling glycolysis in these cell types is that most of the kinetic \n parameters used have been experimentally determined by the same groups under \n the same experimental conditions .", "however , \n the kinetics of the reverse reactions has not been determined and thus these \n authors used kmp and keq \n values reported by others and obtained in other cell types under rather different \n experimental conditions , or they were adjusted to improve model fitting .", "nevertheless , the simulation \n results yielded relevant information on the control of the glycolytic flux . in both cases , the enzymes traditionally considered \n the rate - limiting steps , hk , atp - pfk-1 , and pyk did not contribute to the flux \n control , whereas the main control resided in glut ( 54% in the parasite and 85100% in yeast ) . under some conditions", ", hk may exert some \n control ( 15% ) in s. cerevisiae and some nonallosteric enzymes such as \n aldo , gapdh , and pgk may also exert some flux control in t. brucei .", "mca \n through kinetic modeling has been applied to several pathways : glycolysis in \n erythrocytes   in which flux control distributes between hk ( 71% ) and pfk-1 \n ( 29%);carbohydrate \n metabolism during differentiation in dictyostelium discoideum   with \n cellulose synthase ( 86% ) as the main controlling step;sucrose accumulation \n in sugar cane with hk , invertase , fructose uptake , glucose uptake , and vacuolar \n sucrose transporter having the most significant flux control ; glycerol \n synthesis in s. cerevisiae with gapdh \n ( 85% ) as the main control step ; penicillin \n synthesis in penicillium chrysogenum controlled \n ( 7598% ) either by d-(a - aminoadipyl ) cysteinylvaline synthetase ( short \n incubation times < 30 hour ) or isopenicillin n. synthetase ( long incubation \n times > 100 h ) ; calvin cycle \n  controlled by gapdh ( 50% ) and sedoheptulose-1,7-bisphosphatase ( 50%);threonine \n synthesis in e. coli controlled by homoserine dehydrogenase ( 46% ) , \n aspartate kinase ( 28% ) , and aspartate semialdehyde dehydrogenase ( 25% ) ; lysine \n production in corynebacterium glutamicum mainly \n controlled by aspartate kinase and permease ; nonoxidative \n pentose pathway in erythrocytes mainly controlled by transketolase ( 74% ) ; egf - induced mapk \n signaling in tumor cells controlled by ras - activation by egf ( 21% ) , ras \n dephosphorylation ( 43% ) , erk phosphorylation by mek ( 44% ) , and mek \n phosphorylation by ras ( 143% ) ; \n aspergillus niger  \n  arabinose utilization with \n flux control shared by arabinose reductase ( 68% ) , arabitol dehydrogenase ( 17% ) , \n and xylulose reductase ( 14% ) ; glycolysis in l. lactis in which several end products are generated ( lactate , \n organic acids , ethanol , acetoin ) .", "model predictions indicated that flux toward diacetyl and acetoin \n ( important flavor compounds ) was mainly controlled by ldh but not by \n acetolactate synthetase , the first enzyme of this branch . \n ", "glycolysis in \n erythrocytes   in which flux control distributes between hk ( 71% ) and pfk-1 \n ( 29% ) ; carbohydrate \n metabolism during differentiation in dictyostelium discoideum   with \n cellulose synthase ( 86% ) as the main controlling step ; sucrose accumulation \n in sugar cane with hk , invertase , fructose uptake , glucose uptake , and vacuolar \n sucrose transporter having the most significant flux control ; glycerol \n synthesis in s. cerevisiae with gapdh \n ( 85% ) as the main control step ; penicillin \n synthesis in penicillium chrysogenum controlled \n ( 7598% ) either by d-(a - aminoadipyl ) cysteinylvaline synthetase ( short \n incubation times < 30 hour ) or isopenicillin n. synthetase ( long incubation \n times > 100 h ) ; calvin cycle \n  controlled by gapdh ( 50% ) and sedoheptulose-1,7-bisphosphatase ( 50% ) ; threonine \n synthesis in e. coli controlled by homoserine dehydrogenase ( 46% ) , \n aspartate kinase ( 28% ) , and aspartate semialdehyde dehydrogenase ( 25% ) ; lysine \n production in corynebacterium glutamicum mainly \n controlled by aspartate kinase and permease ; nonoxidative \n pentose pathway in erythrocytes mainly controlled by transketolase ( 74% ) ; egf - induced mapk \n signaling in tumor cells controlled by ras - activation by egf ( 21% ) , ras \n dephosphorylation ( 43% ) , erk phosphorylation by mek ( 44% ) , and mek \n phosphorylation by ras ( 143% ) ; \n aspergillus niger  \n  arabinose utilization with \n flux control shared by arabinose reductase ( 68% ) , arabitol dehydrogenase ( 17% ) , \n and xylulose reductase ( 14% ) ; glycolysis in l. lactis in which several end products are generated ( lactate , \n organic acids , ethanol , acetoin ) .", "model predictions indicated that flux toward diacetyl and acetoin \n ( important flavor compounds ) was mainly controlled by ldh but not by \n acetolactate synthetase , the first enzyme of this branch .", "we modeled \n the gsh and pcs biosynthesis ( figure 2 ) to determine and understand the control \n structure of the pathway and thus be able to identify potential sites for genetic \n engineering manipulation that might lead to the generation of improved species \n in heavy metal resistance and accumulation .", "two models were constructed , one \n for higher plants and the other for yeast , both exposed to high concentrations \n of cd . due to the \n similarity in the results , only the plant results are analyzed below .", "an interesting conclusion from the gsh - pcs synthesis modeling is that control of flux \n ( and gsh concentration ) is shared between the gsh supply and demand under both unstressed \n and cd exposure \n conditions ( table 4 ) .", "this observation strongly differs from the \n idea that -ecs is the rate - limiting step [ 3335 ] . for many researchers , the concept of -ecs being the key controlling step has seemed to be \n correct because ( a ) -ecs receives information from the final part \n of the pathway , as it is potently inhibited by gsh , the pathway end - product ; \n and ( b ) -ecs is localized in the first part of the \n pathway ( figure 2 ) . in addition", "however , in most of the \n studies on the control of gsh synthesis , the gsh demand has not been \n considered .", "the gsh synthesis modeling shows that under a physiological feedback \n inhibition of -ecs by gsh a small increase in demand increases \n flux because the gsh concentration decreases and the -ecs inhibition attenuates .", "in contrast , if the demand remains constant , then \n an increase in -ecs activity or content ( by overexpression ) does \n not increase flux because the gsh inhibition is still there and operates on \n both new and old enzymes .", "the same pattern is also observed when hk is \n overexpressed to increase \n glycolytic flux since it is still inhibited by g6p ( see section 3 ) . on the other hand ,", "-ecs indeed exerts significant concentration control \n on gsh , which means that a -ecs increase results in higher gsh \n concentration ( table 4 ) .", "this last \n observation demonstrates that an enzyme controlling a metabolite concentration does \n not necessarily control the flux .", "cd exposure promotes a high gsh demand because significant oxidative stress surges , \n thus causing oxidation of gsh through gsh peroxidases , and because gsh and pcs \n are used for sequestering the toxic metal ion ; hence , a higher gsh consuming \n rate sets up . under this condition , \n modeling predicted that control was almost equally shared between the supply \n and demand blocks , but particularly between -ecs and pcs ( see figure 2 ) .", "modeling was also able to explain why pcs overexpression \n can have toxic effects on the cell .", "an increase in the gsh demand ( pcs \n overexpression ) under high - demand conditions ( cd stress ) leads to \n gsh depletion that severely compromises other processes such as the oxidative \n stress control and xenobiotic detoxification .", "the \n conclusions drawn by this model led us to propose that , to significantly increase \n the cd resistance and accumulation , -ecs and pcs should be simultaneously overexpressed \n ( table 4 ; figure 9 ) .", "this particular \n manipulation promotes an increase in the rate of gsh and pcs synthesis \n ( determined by the high - to - low transition of their flux control coefficients ) \n and in the gsh and pcs concentrations \n ( determined by their high concentration control coefficients ) .", "the model predicts that a 2-fold increase in \n the simultaneous overexpression of -ecs and pcs brings about a 1.92.4-fold \n increase in flux to gsh ( jgs ) \n and pcs ( jpcs ) and in pcs \n concentration ( figure 9 ) ; a 5-fold overexpression further increases by 4.58.1 \n times the fluxes and pcs concentration .", "this proposed enzyme overexpression \n should not exceed the gs and the complex pc - cd ( or gs - cd - gs ) vacuolar \n transporters ' maximal activities , in order to keep the cell away from a severe \n oxidative stress caused by gsh depletion or -ec accumulation .", "indeed , the concentration of gsh was maintained high and constant although \n -ec accumulated with the simultaneous overexpression \n ( figure 9 ) .", "furthermore , this enzyme \n manipulation should avoid the increase of the pc - cd and gs - cd - gs complexes in \n cytosol to toxic levels .", "in other words , \n excessive enzyme overexpression should be avoided , unless this is accompanied \n by compensating overexpression of consuming enzymes ( gs for -ecs overexpression and pcs vacuolar transporters for \n that of pcs ) . in yeasts and plants ,", "cd is ultimately inactivated by the additional interaction with s and \n the subsequent formation of stable high molecular weight complexes with pcs , cd , \n s , and gsh [ 138 , 139 ] . in \n", "parallel to the -ecs and pcs overexpression , moderate repression \n of gsh - s - transferases , which compete for the available gsh ( figure 2 ) , may also \n promote an increase in gsh concentration and pcs formation flux .", "in contrast , gene overexpression induces large \n changes in activity ; hence , further theoretical background has been developed for \n predicting the effect on flux and metabolite concentrations induced by large \n enzyme changes .", "such a theoretical background was initially developed by small \n and kacser , who depicted ( 12 ) based on the flux control coefficients to \n predict the effect promoted by large changes in enzyme activity:(12)fejmrj  =  11    i = jm(cvi0jo    ( ri    1)/ri ) , in which f is the amplification factor ( the flux increase ) , and r represents how many times the enzyme is overexpressed .", "to predict the flux changes , promoted by identical \n overexpression of two enzymes ( same r value ) with different cvi , \n the equation is \n ( 13)fejmrj  =  11    ( cij  +  cjj )    ( ( r    1)/r ) . \n \n", "figure 10 shows the effect on flux when one or more enzymes with different cvi are changed by the same r factor .", "if the sum of cvi \n of one or more enzymes is less than 0.25 , the \n impact on flux is discrete when the expression increases 5 folds ( which is the \n most common variation in the overexpression experiments analyzed in section \n 2 ) .", "but for a 3-fold overexpression of \n a group of enzymes , for which their sum of cvi is more than 0.5 , then a significant flux \n change is achieved .", "if the sum of cvi is 1 , the flux varies in a linear proportion \n with the degree of overexpression .", "it \n has to be remarked , however , that the predicted change in flux ( figure 10 ) will \n be valid until certain degree , the limits of which being determined by the \n other pathway enzymes that should stay as noncontrolling steps . \n", "figure 10 also shows the effect on flux of decreasing an enzyme activity ( third \n quadrant ) .", "this segment plot is useful when inhibition of pathway flux is being \n pursued for therapeutic purposes or for understanding the molecular basis of the \n genetic dominance and recessivity . like \n in the enzyme overexpression experiment , only a significant effect on flux is \n achieved when the enzymes with high cvi values are inhibited . for an enzyme or group of enzymes with cvi \n of 0.25 , greater than 80%", "inhibition has to be \n attained to decrease 50% the pathway flux . in this context", ", it seems feasible \n to explain why knockdown of enzymes involved in tsh2 synthesis has \n to be almost total to detect an effect on tsh2 content or to alter \n functional or pathogenic properties of the parasites ( section 4.3 ) .", "the knockdown or knockout experiments in \n trypanosomatids suggest that -ecs , trys , and tryr most probably have low flux control \n and concentration - control coefficients since their contents or activities have \n to be reduced > 80% of the normal levels to reach changes in intermediary \n levels or in oxidative stress handling .", "contrary \n to the several unsuccessful overexpression experiments carried out to increase \n the flux or metabolites of a metabolic pathway , modeling may allow for a more \n focused and appropriate design of experimental strategies of genetic \n engineering to increase flux or a given metabolite , and for selecting drug \n targets to decrease flux or metabolite concentration . for these predictions , modeling considers \n that overexpression of a controlling enzyme or transporter may promote flux or \n metabolite control redistributions . \n ", "thus , a low - control step may become a \n controlling point when overexpressing another step and , in consequence , the \n prediction shown in figure 10 based on ( 11 ) and ( 12 ) may be inaccurate . by considering the whole pathway components , \n", "modeling is also a powerful tool for predicting the effects on flux and \n metabolite concentration of varying an enzyme activity ( by overexpression or \n drug inhibition ) .", "model predictions to inhibit a pathway fluxkinetic modeling has been used to identify the flux controlling steps in trypanosoma \n brucei glycolysis for drug targeting purposes .", "interestingly , modeling has predicted \n controlling steps for the parasite pathway different from those described for \n glycolysis in human host cells [ 125 , 126 ] . \n", "the parasite lacks functional \n mitochondria and has neither krebs cycle nor oxphos enzyme activities .", "therefore , \n substrate level phosphorylation by glycolysis is the only way to generate atp \n for cellular work .", "an important \n difference in amebal glycolysis in comparison to glycolysis in human cells is \n that it contains the pyrophosphate ( ppi)-dependent enzymes phosphofructokinase \n ( ppi - pfk ) and pyruvate phosphate dikinase ( ppdk ) , which replace the highly \n modulated atp - pfk and pyk present in human cells .", "moreover , both have been \n proposed as drug targets by using ppi analogues ( bisphosphonates ) .we recently described the \n construction of a kinetic model of e. histolytica glycolysis to \n determine the control distribution of this energetically important pathway in \n the parasite .", "the model was \n constructed using the gepasi software and was based on the kinetic parameters \n determined in the purified recombinant enzymes , as well as the enzyme \n activities , fluxes , and metabolite concentrations found in the parasite .", "the results of the \n metabolic control analysis \n indicated that hk and pgam are the main flux control steps of the pathway ( 73 \n and 65% , resp . ) and perhaps glut . in \n contrast", ", the ppi - pfk and ppdk displayed low flux control ( 13 and 0.1% , resp . ) \n because they have overcapacity over the glycolytic flux .", "the amebal model allowed evaluating the \n effect on flux of  inhibiting  the pathway enzymes .", "the model predicted that in order to diminish \n by 50% the glycolytic flux ( and the atp concentration ; data not shown ) , hk and \n pgam should be inhibited by 24 and 55% , respectively , or both enzymes by 18% \n ( figure 11 ) .", "in contrast , to attain the \n same reduction in flux by inhibiting ppi - pfk and ppdk , they should be decreased \n > 70% ( figure 11 ) .", "therefore , the \n kinetic model results indicate that hk can be an appropriate drug target \n because its specific inhibition can compromise the energy levels in the \n parasite .", "they also indicate that although \n ppi - pfk and ppdk remain as promising drug targets because of their divergence \n from the human glycolytic enzymes , highly potent and very specific inhibitors \n should be designed for these enzymes in order to affect the parasite 's energy \n metabolism .", "kinetic modeling has been used to identify the flux controlling steps in trypanosoma \n brucei glycolysis for drug targeting purposes .", "interestingly , modeling has predicted \n controlling steps for the parasite pathway different from those described for \n glycolysis in human host cells [ 125 , 126 ] . \n", "the parasite lacks functional \n mitochondria and has neither krebs cycle nor oxphos enzyme activities .", "therefore , \n substrate level phosphorylation by glycolysis is the only way to generate atp \n for cellular work .", "an important \n difference in amebal glycolysis in comparison to glycolysis in human cells is \n that it contains the pyrophosphate ( ppi)-dependent enzymes phosphofructokinase \n ( ppi - pfk ) and pyruvate phosphate dikinase ( ppdk ) , which replace the highly \n modulated atp - pfk and pyk present in human cells .", "moreover , both have been \n proposed as drug targets by using ppi analogues ( bisphosphonates ) .", "we recently described the \n construction of a kinetic model of e. histolytica glycolysis to \n determine the control distribution of this energetically important pathway in \n the parasite .", "the model was \n constructed using the gepasi software and was based on the kinetic parameters \n determined in the purified recombinant enzymes , as well as the enzyme \n activities , fluxes , and metabolite concentrations found in the parasite .", "the results of the \n metabolic control analysis \n indicated that hk and pgam are the main flux control steps of the pathway ( 73 \n and 65% , resp . ) and perhaps glut . in \n contrast", ", the ppi - pfk and ppdk displayed low flux control ( 13 and 0.1% , resp . ) \n because they have overcapacity over the glycolytic flux .", "the amebal model allowed evaluating the \n effect on flux of  inhibiting  the pathway enzymes .", "the model predicted that in order to diminish \n by 50% the glycolytic flux ( and the atp concentration ; data not shown ) , hk and \n pgam should be inhibited by 24 and 55% , respectively , or both enzymes by 18% \n ( figure 11 ) .", "in contrast , to attain the \n same reduction in flux by inhibiting ppi - pfk and ppdk , they should be decreased \n > 70% ( figure 11 ) .", "therefore , the \n kinetic model results indicate that hk can be an appropriate drug target \n because its specific inhibition can compromise the energy levels in the \n parasite .", "they also indicate that although \n ppi - pfk and ppdk remain as promising drug targets because of their divergence \n from the human glycolytic enzymes , highly potent and very specific inhibitors \n should be designed for these enzymes in order to affect the parasite 's energy \n metabolism .", "another experimental approach \n for determining the enzyme control coefficients is the in vitro reconstitution \n of segments of metabolic pathways .", "it is \n recalled that for determining the flux control coefficient exerted by a given \n step on a metabolic pathway the enzyme activity has to be varied , without \n altering the other components in the system , and the flux variations are to be \n measured ( figure 4 ) .", "such an experiment can be readily made if a pathway is \n reconstituted with purified enzymes .", "some advantages of this approach are that the \n pathway structure is known , in which the components concentration may be \n manipulated and analyzed separately , and the enzyme effectors can be assayed .", "as \n the system composition is strictly controlled , the results may be highly \n reproducible .", "the main disadvantage is \n that the enzyme concentrations in the assays are diluted and thus the enzyme \n interactions are not favored . if this interaction \n is important for activity , the in vitro reconstitution may limit the \n extrapolation to the metabolic pathway inside the cell .", "there are not many studies \n describing this type of experiments , most probably due to the fact that for \n applying mca the pathway must be working under steady - state conditions . in a reconstituted system", ", only a quasi steady \n state may be reached because there is net substrate , and cofactors consumption , \n as well as product accumulation , since it is difficult to attain a constant \n substrate supply and release of products .", "one of the first experimental \n reports on control coefficient determination in a reconstituted system was \n carried out for the upper glycolytic segment with the commercially available \n rabbit muscle hk , hpi , pfk-1 , aldo , and tpi .", "each enzyme was separately titrated and the \n flux variation to glycerol-3-phosphate ( by coupling the reconstituted system to \n an excess of -gpdh ) was measured in the presence of ck to maintain \n the atp concentration constant .", "the results showed that pfk-1 and hk exerted the \n main flux control ( 65% and 20% , resp . ) , whereas the remaining 15% resided in \n the other enzymes .", "these authors observed that the addition of f1,6bp , a pfk-1 \n activator slightly diminished the flux control exerted by pfk-1 and increases \n that of hk .", "the lower glycolytic segment \n has also been reconstituted with commercial enzymes for determining the flux \n control coefficients .", "the results \n showed that flux was mainly controlled by pyk ( 60100% ) , although under some \n conditions control was shared with pgam ; eno did not contribute to the flux \n control .", "another important limitation \n of the reconstitution experiments is that the commercial availability of the \n purified enzymes from the same organism is restricted or inexistent .", "however , by using the information from the \n genome sequence projects and the recombinant dna technology , it is now possible \n to access all the enzyme genes from a metabolic pathway in the same organism , thus \n facilitating their cloning , overexpression , and purification . with this strategy ,", "we cloned , overexpressed , \n and purified the 10 glycolytic enzymes of entamoeba histolytica   for \n studying the flux control distribution in this organism by using kinetic \n modeling   and pathway reconstitution .", "the reconstitution \n experiments of the lower amebal glycolytic segment , under near physiological \n conditions of ph , temperature , and enzyme activity ( figure 12 ) showed that pgam \n and , to a lesser extent , ppdk exert the main flux control ( these amebal enzymes \n are genetically and kinetically different from their human counterparts ) with \n eno exhibiting negligible control . in turn , reconstitution of the upper amebal \n glycolytic segment has revealed that hk and , to a much lesser extent hpi , \n ppi - pfk , and ald , exerted the main flux control , with tpi having negligible \n control .", "these results strongly \n correlate with the enzyme catalytic efficiencies previously reported , in \n which hk is highly sensitive to amp inhibition , ald , and pgam have the lowest \n catalytic efficiencies among the glycolytic enzymes , leading to high flux control \n coefficients and thus becoming suitable candidates for therapeutic \n intervention .", "the reconstitution results \n also agree with the pathway modeling predictions previously analyzed ( section \n 5.4 ) , in which hk and pgam are two of the main controlling steps .", "the in vitro reconstitution \n experiments are also useful for studying the effect on control redistribution of \n an enzyme modulation that is particularly difficult to manage in vivo ; the main controlling steps \n identified with the reconstitution experiments should be further analyzed with \n other experimental strategies such as elasticity analysis in the in vivo \n systems .", "this experimental approach for determining the control coefficients could be part of \n the genetic approach analyzed in section 5.1 , but it was separated due to its \n recent methodological development and because it actually belongs to the \n molecular genetics rather than to the mendelian genetics .", "this approach is based on the in vivo modulation of the enzyme levels using the rna \n antisense technology .", "there are at least \n three strategies to \n inhibit gene expression : ( a ) the use of single stranded antisense \n oligonucleotides , which form a double stranded rna that might be degraded by rnase \n h ; ( b ) target rna degradation with catalytically active oligonucleotides , known \n as ribozymes that bind to their specific rna ; and ( c ) rna degradation using sirnas \n ( 2123 nucleotides ) .", "the rna antisense technology was applied for control coefficient determination of the \n ribulose - bisphosphate - carboxylase ( rubisco ) that fixes co2 in the \n plant calvin cycle .", "this enzyme considered \n the rate - limiting step of the calvin cycle and of the whole photosynthetic \n process , despite its high concentration ( 4 mm ) in the chloroplasts stroma that compensates \n its low catalytic efficiency .", "attempts to make rubisco a nonlimiting step , either by modifying its catalytic efficiency \n or by overexpressing it , have been unsuccessful .", "the plants were transformed with dna antisense against the \n mrna of the enzyme 's small subunit , thus promoting its degradation . for calvin cycle enzymes , the pleiotropic \n effects were minimal .", "the results showed \n that rubisco may indeed be the photosynthesis limiting step with a crubisco = 0.690.83 when plants are exposed to high \n illumination ( 1050 mol quanta ms ) , high \n humidity ( 85% ) , and low co2 concentrations ( 25 pa ) .", "however , this flux control decreases to \n 0.050.12 under moderate illumination or high co2 levels .", "unfortunately , the authors did not determine \n the control coefficients of the other pathway enzymes or the branches fluxes which \n may be significant .", "as described in section 5.4 , the results of the t. brucei glycolysis \n modeling indicated that glut was the main flux control step ( cglut  50% ) , [ 125 , 126 ] .", "this model predicted a large overcapacity for \n hk , pfk-1 , aldo , gapdh , pgam , eno , and pyk over the glycolytic flux leading to \n low flux control coefficients [ 125 , 126 ] . to validate the modeling results , the concentrations of hk , pfk-1 , pgam , \n eno , and pyk", "these knockdown expression experiments showed \n overcapacity of hk and pyk over the flux , although at lower levels than predicted \n by the model . a good correlation for \n pgam and eno", "was obtained between model predictions and experimental results . however , a large difference ( 9 folds ) was \n obtained for pfk-1 .", "this discrepancy is \n perhaps related to pleiotropic effects of pfk-1 downregulation , as these mutants \n also displayed diminution in the activities of other enzymes ( hk , eno , and pyk ) . \n ", "the combination of these two approaches , \n in silico modeling and in vivo experimentation , is complementary : on one \n hand , modeling identifies the enzymes ( out of 19 that contain the model ) that \n display the highest flux control coefficients , whereas in vivo experimentation validates the \n accuracy of the model to establish predictions about the pathway 's behavior .", "the knockdown experiments described above usually yield only two experimental points \n of the plot shown in figure 4 : the \n wild - type and the knockdown strain protein levels or enzyme activities .", "thus , with \n such an approach high levels of inhibition ( > 80% ) are mostly analyzed , \n whereas intermediate levels of downregulation ( if obtained ) are generally \n overlooked .", "therefore , knockdown \n experiments are not very useful to obtain the complete set of experimental data \n ( above and below the wild - type levels of enzyme activity with the corresponding \n flux ) for determining reliable control coefficients . a strategy to determine flux \n control coefficients from several protein levels", "has been developed by using \n adenovirus - mediated glucose-6-phosphatase ( g6pase ) overexpression under the \n control of the cytomegalovirus promoter in rat hepatocytes .", "a 2-fold g6pase overexpression did not alter cg6pase or cgk ( gk , glucokinase ) .", "however , if g6pase is overexpressed by 4 folds , then cgk \n diminished from 2.8 to 1.8 and there was a 35% lowering in glycogen synthesis .", "however , this approach allows titration of flux only above the basal \n enzyme activities found in the cell , but not below .", "these experimental inconveniences have been circumvented by using inducible gene \n expression systems based in the lac , \n lambda , nisin , gal , tetracycline , and other inducible promoters , in bacteria \n and yeast [ 151 , 152 ] . however , a problem \n frequently encountered with inducible promoters is that a steady - state of \n protein expression is difficult to attain [ 151 , 152 ] .  ", "recently , \n jensen and hammer described the design of synthetic promoter libraries ( spl ) , in \n particular for l. lactis metabolic \n optimization .", "these promoters \n maintain constant the array of the known consensus sequences for l. lactis gene transcription ( 10 and \n 35 boxes ) , while the nucleotide sequence between these boxes ( a spacer \n sequence of 17  1 bp ) is randomized , thus producing a set of promoters with \n different transcriptional strength .", "these promoter libraries allow the transcription and protein expression \n several folds above and below the wild - type levels of enzyme activity , \n thus enhancing the usefulness of this approach for mca studies .", "the \n control distribution of glycolysis in e. \n coli and l. lactis , as discussed \n in section 3.2 [ 17 , 24 , 27 , 151 ] , has been determined by using the spl \n technology . spl for yeast , mammalian and \n plant cells are also under development [ 151 , 152 ] .", "certainly , the advances in genetic \n engineering in combination with mca allow better experimental designs for \n metabolic optimization of micro - organisms of biotechnological interest .", "concluding remarks \n the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect .", "as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step .", "mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control .", "a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments ,", "it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway .", "mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) .", "for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them .", "if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes.if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients .", "these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks .", "it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis .", "however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest . \n \n", "the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect . as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step .", "mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control .", "a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments ,", "it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway .", "mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) .", "for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them .", "if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes.if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients .", "these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks .", "it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis .", "however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest . \n ", "the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect . as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step .", "mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control .  ", "a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments ,", "it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway .", "mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) .", "for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them .", "if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes .", "if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients .", "these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks .", "it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis .", "however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest ."], "abstract_text": ["<S>   the traditional experimental approaches used for changing the flux or the concentration of a particular metabolite of a metabolic pathway have been mostly based on the inhibition or over - expression of the presumed rate - limiting step .   </S>", "<S> however , the attempts to manipulate a metabolic pathway by following such approach have proved to be unsuccessful .   </S>", "<S> metabolic control analysis ( mca ) establishes how to determine , quantitatively , the degree of control that a given enzyme exerts on flux and on the concentration of metabolites , thus substituting the intuitive , qualitative concept of rate limiting step .   </S>", "<S> moreover , mca helps to understand ( i ) the underlying mechanisms by which a given enzyme exerts high or low control and ( ii ) why the control of the pathway is shared by several pathway enzymes and transporters .   by applying mca </S>", "<S> it is possible to identify the steps that should be modified to achieve a successful alteration of flux or metabolite concentration in pathways of biotechnological ( e.g. , large scale metabolite production ) or clinical relevance ( e.g. , drug therapy ) .   </S>", "<S> the different mca experimental approaches developed for the determination of the flux - control distribution in several pathways are described .   </S>", "<S> full understanding of the pathway properties when is working under a variety of conditions can help to attain a successful manipulation of flux and metabolite concentration . </S>"], "labels": null, "section_names": ["1. INTRODUCTION", "2. CONTROLLING SITES IN A METABOLIC PATHWAY", "3. IN VIVO OVEREXPRESSION EXPERIMENTS  OF ENZYMES", "4. DOWNREGULATION OF ENZYMES TO  MANIPULATE METABOLISM", "5. THEORY OF METABOLIC CONTROL ANALYSIS"], "sections": [["is an effort to manipulate the metabolism of an organism worthy and reasonable , \t knowing \n that this cellular process has been continuously modified and refined through \n evolution and natural selection for adapting , in the most convenient manner , to \n the ongoing environmental conditions ?", "the \n answer to this question seems obvious when three broad areas of research and \n development are identified in which manipulation of metabolic pathways is \n relevant : ( a ) drug design to treat diseases , ( b ) genetic engineering of \n organisms of biotechnological interest , and ( c ) genetic syndromes therapy .", "historically , drug design was \n the first area in which modification of metabolism was tried : the primary goal \n of drug administration is the inhibition of essential metabolic pathways , for \n example , in a parasite or a tumor cell .", "thus , any metabolic pathway can be a potential \n therapeutic target . in the absence of a solid \n theoretical background that may build a strategy for the rational design of \n drugs ,", "the pharmaceutical industry has applied the knowledge of inorganic and \n organic chemistry for the arbitrary and rather randomized modification of \n metabolic intermediaries by replacing hydrogen atoms in a model molecule with \n any other element or compound .", "the era of rational drug \n design probably started in the 50s when hans krebs proposed that , after having \n an exact description of a metabolic pathway , the  pacemaker  enzyme or \n  rate - limiting step  had to be identified .", "this approach certainly decreased the amount \n of intermediaries to be chemically modified , focusing only on the substrates , \n products , and allosteric effectors of the  rate - limiting step ,  instead of dispersing \n efforts on all the metabolic pathway intermediates .", "the experimental approaches used in the \n identification of the pacemaker , key enzymes ,  bottlenecks .", " limiting steps , or \n regulatory enzymes [ 1 , 2 ] were \n inspection of the metabolic pathway architecture : due to cell \n  economy and for reaching the highest efficiency , pathway control must \n  reside in the enzymes localized at the beginning of a pathway or after a \n  branch ( teleological approach);determination of nonequilibrium reactions : those reactions in which \n  the quotient between the mass action ratio (  ) and its equilibrium constant ( keq ) is low , /keq 1 ( thermodynamic \n  approach);identification of the steps with the lowest maximal rates ( vmax ) in cellular extracts : the key \n  enzyme of the pathway is the one that has the lowest rate ( kinetic \n  approach);enzymes with sigmoidal kinetics : steps that are susceptible to \n  alteration in their kinetic properties by compounds different from \n  substrates and products and which may coordinate the entire metabolism \n  ( nadh / nad ; nadph / nadp , atp / adp ; acetyl coa / coa ; ca / mg ; \n  high ph / low ph ) or at least two metabolic pathways ( citrate , pi , amp , \n  malonyl - coa);crossover theorem . comparing the intermediary concentrations \n  between a basal and an active steady - state pathway flux , the rate - limiting \n  step in the basal condition will be that for which its substrate \n  concentration diminishes and its product concentration increases when the \n  system changes from the basal to the active state or vice versa ( crossover \n  point on a histogram of each intermediary versus its normalized variation in concentration);the shape of the metabolic flux inhibition curve : a sigmoidal curve \n  on a plot of inhibitor concentration versus flux shows that the sensitive \n  step to the inhibitor exerts no control , that is , there is not proportionality between enzyme \n  activity inhibition and pathway flux inhibition because there is an \n   excess  of enzyme .", "on the other \n  hand , a hyperbolic curve indicates that the enzyme susceptible to the \n  inhibitor controls the flux . \n ", "inspection of the metabolic pathway architecture : due to cell \n  economy and for reaching the highest efficiency , pathway control must \n  reside in the enzymes localized at the beginning of a pathway or after a \n  branch ( teleological approach ) ; determination of nonequilibrium reactions : those reactions in which \n  the quotient between the mass action ratio (  ) and its equilibrium constant ( keq ) is low , /keq 1 ( thermodynamic \n  approach ) ; identification of the steps with the lowest maximal rates ( vmax ) in cellular extracts : the key \n  enzyme of the pathway is the one that has the lowest rate ( kinetic \n  approach ) ; enzymes with sigmoidal kinetics : steps that are susceptible to \n  alteration in their kinetic properties by compounds different from \n  substrates and products and which may coordinate the entire metabolism \n  ( nadh / nad ; nadph / nadp , atp / adp ; acetyl coa / coa ; ca / mg ; \n  high ph / low ph ) or at least two metabolic pathways ( citrate , pi , amp , \n  malonyl - coa ) ; crossover theorem . comparing the intermediary concentrations \n  between a basal and an active steady - state pathway flux", ", the rate - limiting \n  step in the basal condition will be that for which its substrate \n  concentration diminishes and its product concentration increases when the \n  system changes from the basal to the active state or vice versa ( crossover \n  point on a histogram of each intermediary versus its normalized variation in concentration ) ; the shape of the metabolic flux inhibition curve : a sigmoidal curve \n  on a plot of inhibitor concentration versus flux shows that the sensitive \n  step to the inhibitor exerts no control , that is , there is not proportionality between enzyme \n  activity inhibition and pathway flux inhibition because there is an \n   excess  of enzyme . on the other \n  hand ,", "a hyperbolic curve indicates that the enzyme susceptible to the \n  inhibitor controls the flux ."], ["once a site in a metabolic \n pathway has been identified with at least one of the criteria described above \n as  the rate - limiting step , ", "researchers have frequently concluded that such \n enzyme or transporter is the only limiting step of the metabolic flux and \n extend this conclusion to all cell types and to all conditions .", "for example , inspection of the glycolytic pathway ( teleological approach ) suggests that \n hexokinase ( hk ) and phosphofructokinase-1 ( pfk-1 ) ( which are at the beginning \n and after a branch of the pathway ) are the key steps of glycolysis .", "however , all studies on glycolysis in the 60s , \n 70s , and 80s were performed by taking into account only the intracellular \n reactions from hk to ldh ( i.e. , without including the glucose transport \n reaction through the plasma membrane ) and by considering glycolysis as a linear \n pathway without branches . to this", "regard , \n it is recalled that the glucose transporter ( glut ) includes a family of \n proteins and genes that are susceptible of regulation .", "thus , if the extracellular glucose is \n considered as the initial glycolytic substrate , then another potential key step \n would be glut .", "hence , if all the \n branches of the pathway are considered ( figure 1 ) , then according to the \n teleological approach there will be additional potential rate - limiting sites .", "application of the thermodynamic and kinetic approaches to glycolysis reveals that hk , \n pfk-1 , and pyruvate kinase ( pyk ) are the rate - limiting steps because in the \n living cell they catalyze reactions that are far away from equilibrium ( /keq = 1010 ) , and they are also the slowest \n enzymes in the pathway by at least one order of magnitude ( they have the lowest vmax values ) .", "the use of the enzyme cooperativity approach has established that the regulatory \n steps of glycolysis are ( i ) pfk-1 and pyk because they are allosteric enzymes \n and ( ii ) hk because it is inhibited by its products ( g6p and adp , or amp as an adp - analogue ) . \n  the application of the crossover theorem \n ( approach no .", "v ) to glycolysis has shown a consistent variation in the pfk-1 substrate \n ( f6p ) and product ( f1,6bp ) .", "up to now , \n there are few studies on control of glycolysis using the shape of the inhibitor \n titrating curve ( approach no .", "vi ) , due to the lack of specific inhibitors for \n any of the three presumed key steps .", "an exception is iodoacetate which is indeed \n a potent inhibitor of gapdh , but also of other highly reactive \n cysteine - containing enzymes [ 35 ] . by \n using iodoacetate as specific inhibitor ,", "both gapdh activity and flux showed \n identical titration curves , leading to the conclusion that gapdh was the \n rate - limiting step of glycolysis in streptococcus lactis and s. cremoris   ( see , \n however , section 3.2 ; glycolysis in lactobacteria below ) . all together , these results constitute \n the main reason why many intermediary metabolism researchers , including the \n authors of biochemistry text books , have proposed hk , pfk-1 , and pyk as the \n rate - limiting steps of glycolysis . in \n consequence , to vary the glycolytic flux , one of these enzymes has to be \n modified .", "although the above - described experimental approaches are qualitative , full control has \n been automatically assigned to the  key  steps because the concept of the rate - limiting \n step assumes that there is only one single enzyme controlling the metabolic \n pathway flux ( and the concentration of the final product of the pathway ) and , \n in consequence , assigns values of zero to the control exerted by the other \n enzymes and transporters .", "however , as analyzed for glycolysis , researchers have commonly  identified  more than one \n limiting step . in the case of oxidative \n phosphorylation ( oxphos ) , in the 70s and 80s some researchers considered \n cytochrome c oxidase as the rate - limiting step , whereas others preferred the \n atp / adp translocator or the krebs cycle", "ca - sensitive \n dehydrogenases ( for a review , see ) . rephrasing the initial question , which could be the aim of manipulating a metabolic \n pathway such as glycolysis , knowing its universal distribution in the living \n organisms ? from a clinical standpoint , the inhibition of glycolysis is relevant for the treatment of human parasitic \n or pathological diseases such as cancer .", "the glycolytic reactions are almost identical in all organisms ; in \n addition , the enzymes catalyzing these reactions are highly conserved throughout \n the evolutionary scale ( their amino acid sequences are highly similar ) . in mammals ,", "the genes of the 12 glycolytic \n enzymes are scattered throughout the genome , generally in different chromosomes , \n whereas in bacteria many of the glycolytic enzymes are clustered in operons . \n  however , there are organisms ( like some \n human parasites ) that contain enzymes with remarkable differences in their \n biochemical properties ( substrate selectivity , catalytic capacity , stability , \n and oligomeric structure ) , or in genetic expression regulation in comparison to \n the human enzymes , which could be considered as drug targets . furthermore , some glycolytic products are of commercial interest such as ethanol for wine , \n beer , and other alcoholic beverages ; co2 for bread manufacturing ; and \n lactic acid and other organic acids for cheese production .", "thus , from a biotechnological standpoint , it \n is convenient to accelerate the pathway flux to diminish the processing time and \n it is also desirable to increase the concentration of the metabolite to obtain \n robust commercial products . here", ", it is important to emphasize that the metabolic pathways are designed to attain \n changes in flux with minimal disturbances in the intermediary concentrations . for example", ", the glycolytic flux in skeletal \n muscle can increase from rest to an active state by 100 fold , without large \n changes in metabolites .", "then , it is physiologically more common to change a metabolic flux and the production of \n the final metabolite in the pathway than varying the intermediary concentrations \n .", "however , we will see that , by \n using a suitable approach of metabolic control analysis , it is possible to \n design strategies to manipulate not only fluxes but also metabolic intermediary \n concentrations ."], ["when the yeast saccharomyces \n cerevisiae is exposed to high glucose ( > 2% ; 0.11 m ) , the genes of all \n glycolytic enzymes are induced ( pdc and eno increase their expression by 20 fold ; \n pgk , pyk , and adh , 310 times ; and the \n others , 2 fold in average ) [ 811 ]", ". however , \n when the methodological development of genetic engineering allowed modulating the \n expression of enzymes within cells , researchers turned to the rate - limiting \n step concept to manipulate a metabolic pathway to increase flux and/or its \n intermediates , hypothesizing that the overexpression of only one , or of a few \n key glycolytic genes , should increase the flux .", "historically , heinisch   in germany was the first author to \n obtain a 3.5 fold overexpression of pfk-1 in   s. cerevisiae , but \n surprisingly he observed that the rate of ethanol production was not modified .", "subsequent experiments for increasing the \n ethanol production rate by overexpressing either each of the presumed limiting \n steps , or in combination with other glycolytic enzymes ( table 1 ) , have been \n unsuccessful and , even in some cases , a slight decrease in flux has been \n attained .", "for instance , the simultaneous \n overexpression of seven enzymes of the final section of glycolysis induced only \n a 21% increase in ethanol production after 2 hours of culture ( table 1 ) .", "this was accompanied by a 1020% decrease in pfk-1 \n expression , which might have attenuated the flux increase . in yeasts , hk is not product inhibited \n by g6p or adp ; instead , it is strongly feedback inhibited by trehalose-6-phosphate \n ( tre6p ) .", "deletion \n of the tre6p synthase gene does not \n bring about an increased ethanol production , but it rather induces a defective cellular \n growth on glucose and fructose and a lowered ethanol production , as a result of \n a highly active hk that leads to hyperaccumulation of hexose phosphate \n metabolites ( particularly f1,6bp ) and fast depletion of atp , pi , and downstream \n metabolites .", "the explanation for this event is that , in the tre6p synthase \n mutants , the rate of glucose phosphorylation exceeds the rate of glycolytic atp \n synthesis ( named  turbo effect  ) .", "heterologous expression of a tre6p - insensitive hk does not recover \n completely the wild - type phenotype .", "furthermore , deletion of the tre6p synthase gene in the \n tre6p - insensitive hk strain did affect growth , suggesting other interactions \n and functions of tre6p synthase in the control of sugar metabolism , at \n least in schizosaccharomyces pombe .", "davies and brindle   obtained a 5-fold overexpression of \n pfk-1 in s. cerevisiae , but the increase in ethanol production was not attained \n under anaerobic conditions .", "there was a slight \n increase in ethanol production in resting cells in aerobic conditions , under \n which the mitochondrial metabolism contributes to the atp supply .", "in all these works , it may be noted that \n enzyme overexpression indeed affects the concentration of several intermediaries , \n but this effect has not been further examined .", "it is worth noting that the \n experiments described in table 1 do not rigorously reproduce the physiological \n situation , in which overexpression of all the enzymes should be carried out in \n the proportions found in the organisms .", "the \n rationale behind this observation is that overexpression of only one  limiting  \n", "step leads to a flux control redistribution , a condition at which other steps \n now become rate limiting .", "thus , the \n concept of  rate - limiting step  offers no simple answer to the question of \n increasing the yeast glycolytic flux , and it rather makes this problem to \n appear as a difficult task to solve .", "in contrast , it seems that all relevant controlling steps \n have to be overexpressed , thus reproducing what natural \n selection has already successfully accomplished .", "in addition to s. cerevisiae , overexpression of glycolytic enzymes in other \n organisms such as e. coli [ 15 , 16 ] , \n lactobacteria , tomato , potato , and \n hamster ovary cells   has been accomplished , although without increasing \n flux ( table 1 ) .", "it is somewhat surprising to note that in the glycolytic enzyme \n overexpression experiments , the strong inhibitory effect of g6p ( or tre6p in s. cerevisiae ) , and citrate on hk and pfk-1 , respectively , have been neglected .", "this \n regulatory mechanism does not disappear in the cells overexpressing the enzymes \n but , on the contrary , it is exacerbated .", "then , what would be the aim of overexpressing hk , pfk-1 or any other \n allosteric , or strongly product - inhibited enzyme if they will be more \n inhibited ?", "a successful experiment of increasing \n the glycolytic flux was performed in primary cultures of rat hepatocytes .", "the transformed \n hepatocytes showed higher activity of 18.7- and 7.1-times for hk and gk , \n respectively , at 3 mm glucose , and of 6.3- and 7.1-times at 20 mm glucose .", "however , at 20 mm glucose , the flux to \n lactate was not modified in hk - transformed cells , just like the experiments \n described above ( table 1 ) .", "in contrast , \n with gk overexpression , a 3-fold increase in flux was achieved .", "the mechanistic difference is the hk \n inhibition by g6p ( 10 mm g6p inhibits hk activity by 90% ) , whereas gk is not product inhibited . \n", "the end products , lactate and h , are \n expelled and acidify the external medium which contributes to cheese flavor and \n texture and inhibits the growth of other bacteria .", "similarly to yeast , the lack of carbon source \n in lactobacteria promotes a metabolic change that leads to the production of \n formic and acetic acids , ethanol , and , in a lower proportion , l - lactic acid , \n altering the product quality .", "thus , from a commercial point of view , it does \n not seem important to know what controls the flux to lactate ( because its rate \n of production is adequate ) , but what controls the branching flux . to understand the process , and to eventually inhibit the production of secondary acids , andersen et al .  ", "constructed ldh mutants , \n using a synthetic promoter library for tuning the gene expression . in mutants \n lacking this enzyme ,", "most of the pyruvate was transformed into acetic and \n formic acids ( figure 1 ) . in turn", ", flux \n to lactate was affected in mutants expressing only 10% or less of wild - type ldh \n levels , which indicated that ldh exerts no control of the glycolytic flux in \n wild - type bacteria . only with a normal content of this enzyme ( 100% ) ,", "therefore , the flux to formic and acetic \n acids is negatively controlled by ldh , and positively by pyk [ 17 , 25 ] . as in s. cerevisiae , overexpression of \n pfk-1 , pyk , or gapdh in lactobacteria", "did not increase the flux to l - lactic \n acid [ 17 , 25 ] . similarly to e. coli glycolysis , glycolysis in l. lactis was controlled by the atp \n demand when working below its maximum capacity [ 27 , 28 ] , whereas , under high - rate \n conditions , the glucose and lactate transporters exerted the main flux control .", "furthermore , this kind of observations indicates \n that the flux control may reside outside the pathway [ 2729 ] , and it also supports the proposal \n by hofmeyr and cornish - bowden   that the end - product demand ( which is \n usually overlooked in studies of metabolism because these metabolites are \n frequently not considered as part of the pathway ) might be essential in flux \n control .", "glutathione ( -glu - cys - gly ; gsh ) is the most abundant nonproteinaceous \n thiol compound ( 110 mm ) \n in almost all living cells .", "gsh is \n involved in the oxidative stress processing , xenobiotic detoxification , and , in \n some plants and yeasts , in the inactivation of toxic heavy metals ( for a recent \n revision see ) .", "gsh is synthesized by two enzymes : -glutamylcysteine synthetase \n ( -ecs ) and glutathione synthetase ( gs ) ( figure 2 ) , \n which catalyze reactions with high - equilibrium constants ( keq > 1000 ) . under a low gsh demand ( unstressed \n conditions )", ", the producing block of enzymes has to receive information from the \n last part of the pathway to ( i ) avoid the excessive and toxic accumulation of \n the intermediary -ec and ( ii ) reach a stable steady state .", "this information transfer is mediated by gsh , \n which exerts strong competitive inhibition of -ecs   ( figure 2 ) .", "gsh and cys also exert inhibition on the \n atp - sulfurylase ( atps ) and on sulfate transporters ( figure 2 ) ( for a review , \n see ) .", "the feedback inhibition of -ecs has led several researchers to propose \n that this enzyme is the rate - limiting step of gsh synthesis [ 3335 ] .", "although there are no studies about the \n pathway 's behavior under stressed conditions , which means under a high gsh \n demand , the proposal that -ecs is the key enzyme has been automatically \n extended to any environmental condition such as heavy metal exposure . by assuming that -ecs is the rate - limiting step , many research groups \n have tried to increase , in plants and yeasts , the rate of synthesis and the \n concentration of gsh and phytochelatins ( pcs ) with the aim of fortifying their \n heavy metal resistance and storage capacity , mainly toward cd .", "the development of organisms able to grow in \n soils and water systems contaminated with heavy metals , which may have the \n ability of accumulating toxic metal ions , is of biotechnological interest for \n bioremediation strategies . with this goal in mind", ", \n researchers have then overexpressed -ecs and other pathway enzymes , including \n phytochelatin synthase ( pcs ) ( table 2 ) .", "some of these experiments have been \n partially successful in increasing gsh levels , although this has been rather \n marginal with no correlation between enzyme levels and gsh concentration .", "unfortunately , these overexpression experiments \n have not been accompanied by determinations of fluxes or other relevant metabolite \n concentrations such as pcs or cys .", "on \n the other hand , the overexpression of pcs has surprisingly induced oxidative stress \n and necrosis instead of increasing cd accumulation and resistance \n .", "this result suggests that , under high \n gsh demand ( i.e. , for pcs synthesis and for direct heavy metal \n sequestration by gsh ) , the gsh concentration does not suffice for maintaining \n the other essential gsh functions such as oxidative stress management and \n xenobiotic detoxification .", "another problem in the study \n of gsh biosynthesis for its eventual manipulation is that the pathway has been \n analyzed considering only the gsh - synthetic reactions without taking into \n account the gsh - consuming reactions ( figure 2 ) , .", "the analysis of an incomplete pathway leads to \n misleading conclusions about the control of flux .", "metabolic modeling has shown that only with \n the incorporation of the consuming reactions of the pathway end products , a true \n steady state can be established . in \n conclusion , without a solid theoretical framework , the overexpression of only \n one enzyme ( the  rate - limiting step  ) , or of many arbitrarily selected enzymes \n ( tables 1 and 2 ) , the problem of increasing the flux or metabolite \n concentrations can not be solved .", "there are some successful examples of the genetic engineering approach to manipulate \n metabolism : overexpression \n ( approx .", "23 fold ) of the five genes of the tryptophan synthesis pathway in s. \n cerevisiae , to increase ( 9-fold ) flux ; increase \n in amino acids ( trp , ile , lys , val , thr ) and \n trehalose production in corynebacterium glutamicum , in which some proteins of \n each metabolic pathway are simultaneously overexpressed , but some of them with \n mutations that confer insensitivity to feedback inhibition [ 4853 ] . in these transformed bacteria ,", "the end products \n are indeed overproduced and their excretion is accelerated;overexpression \n of pfk and pyk to increase ethanol production by 35% in e. coli , although lactic acid formation was not modified ; mannitol \n 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase \n mannitol production by 2750% in ldh - deficient lactococcus lactis ; increase \n in sorbitol production ( 5 fold ) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol \n 6-phosphate dehydrogenase ( activity up to 250 fold in mutants versus wild type ) ; overexpression \n of pfk ( 14 fold ) or ldh ( 3.5 times ) to increase 2 - 3 times the homolactic \n fermentation flux in lactococcus lactis growing on maltose , and in parallel decrease fluxes toward secondary acids and \n ethanol . \n  overexpression \n ( approx", ". 23 fold ) of the five genes of the tryptophan synthesis pathway in s. \n cerevisiae , to increase ( 9-fold ) flux ; increase \n in amino acids ( trp , ile , lys , val , thr ) and \n trehalose production in corynebacterium glutamicum , in which some proteins of \n each metabolic pathway are simultaneously overexpressed , but some of them with \n mutations that confer insensitivity to feedback inhibition [ 4853 ] .", "in these transformed bacteria , the end products \n are indeed overproduced and their excretion is accelerated ; overexpression \n of pfk and pyk to increase ethanol production by 35% in e. coli , although lactic acid formation was not modified ; mannitol \n 1-phosphate dehydrogenase and mannitol 1-phosphatase overexpression to increase \n mannitol production by 2750% in ldh - deficient lactococcus lactis ; increase \n in sorbitol production ( 5 fold ) in ldh - deficient lactobacillus plantarum through the overexpression of sorbitol \n 6-phosphate dehydrogenase ( activity up to 250 fold in mutants versus wild type ) ; overexpression \n of pfk ( 14 fold ) or ldh ( 3.5 times ) to increase 2 - 3 times the homolactic \n fermentation flux in lactococcus lactis growing on maltose , and in parallel decrease fluxes toward secondary acids and \n ethanol ."], ["several glycolytic enzymes are overexpressed in \n at least 70% of human cancers . except \n for glucose transporter 1 ( glut-1 ) , the other 11 glycolytic enzymes ( hk to ldh ) \n are overexpressed in brain and nervous system cancers .", "prostate and lymphatic nodule cancers ( hodgkin \n and non - hodgkin lymphomas ; myelomas ) overexpress 10 glycolytic enzymes ( except \n for hk ; in prostate cancer glut1 is also overexpressed ) .", "there is a second group of cancers that overexpresses 68 glycolytic \n genes ( skin , kidney , stomach , testicles , lung , liver , placenta , pancreas , \n uterus , ovary , eye , head and neck , and mammary gland ) .", "a third group includes those cancers overexpressing \n 1 or 2 glycolytic genes ( bone , bone marrow , cervix , and cartilage ) . in animals ,", "gene expression of glycolytic enzymes is regulated ( both coordinately \n and individually ) under hypoxic conditions by hypoxia - responsive transcription \n factors such as hif-1 ( hypoxia - inducible factor 1 ) , sp family factors , ap-1 , and possibly mre \n ( metal response elements ) [ 8 , 5961 ] .", ". there are binding sites ( consensus sequence acgt ) for hif-1 in the promoters of genes for hk , pfk-1 , \n aldo , gapdh , pgk , eno , pyk , and ldh ( reviewed in ) .", "tpi and perhaps hpi and pgam are also induced \n by hypoxia , but it is not clear whether hif-1 mediates this induction , and whether this \n factor regulates other metabolic pathways associated with glucose \n catabolism .", "for example , although \n glycogen phosphorylase is overexpressed under hypoxia in human tissues , \n the role of hif-1 has not been demonstrated .", "if direct manipulation of \n pathway genes becomes difficult , then the overexpression or repression of \n transcription factors such as hif-1 , ap1 , and mres might solve the problem of changing \n flux , although overexpression of transcription factors may also be difficult \n due to the numerous upstream and downstream factors involved .", "the kinetoplastid parasites trypanosoma \n cruzi , trypanosoma brucei , and leishmania are the causative agents of \n chagas disease , african trypanosomiasis , and leishmaniasis , respectively . the \n available drugs to treat these diseases are highly toxic for humans .", "moreover , the \n parasites may become resistant , and hence the search for new drugs and drug \n targets is relevant for solving these public health problems . in these parasites ,", "the \n metabolism is organized in a peculiar way ; they have a subcellular structure \n called glycosome in which several metabolic pathways take place : \n gluconeogenesis , reactions of the pentose phosphate pathway , purine salvage and \n pyrimidine biosynthesis , -oxidation \n of fatty acids , fatty acid elongation , biosynthesis of ether lipids , and the \n first seven steps of glycolysis .", "glycosomal glycolytic enzymes have unique structural , kinetic , and \n regulatory features not found in their human counterparts , and therefore have \n been the subject of extensive biochemical studies to use them as drug targets .", "the rationale behind this is to synthesize \n inhibitors that affect mainly the parasitic enzymes with relatively low effect \n on the human enzymes since the infective parasite stages rely mostly on \n glycolysis for atp supply .", "there are reports on the \n design of presumed specific inhibitors for some of the t. brucei glycolytic \n enzymes : glut ( bromoacetyl-2-glucose ) , hk , hpi , pfk , aldo , tpi , gapdh , \n pgk , pyk , and glycerol-3-phosphate dehydrogenase .", "although the purified enzymes display \n very low ki values for these \n inhibitors and some of them inhibit \n parasite growth or infective capabilities , their effect on inhibiting the \n glycolytic flux has not been explored .", "therefore , it is not yet possible to \n directly ascribe the effects seen in parasite culture with the in vitro effects \n on the isolated enzymes . to identify the \n best drug targets ,", "determination of the flux control steps of glycolysis \n in t. brucei has been recently initiated .", "trypanothione ( tsh2 ) \n is a reducing agent present in trypanosomatids that is synthesized from one \n spermidine and two gsh molecules by tsh2 synthetase ( trys ) \n ( figure 3 ) .", "this metabolite and its reducing enzyme , \n tsh2 reductase ( tryr ) , replace the antioxidant and metabolic \n functions of the more common gsh / gsh reductase system present in mammals .", "in \n fact , most of the antioxidant metabolism of these parasites depend on tsh2 ( figure 3 ) [ 69 , 70 ] . thus , the enzymes of this metabolic pathway have been \n proposed as drug targets for killing the parasites .", "diminution in its gene transcription yields a \n loss of activity between 5690% , depending on \n the genetic technique [ 7275 ] . in \n knockdown t. brucei cells", "( i.e. , when tryr activity has diminished \n to less than 10% of the wild - type level ) , the parasites show growth diminution \n and higher sensitivity to h2o2 in culture and loss of \n infectiveness in mice . however , tsh2 and thiol compound contents \n were not affected .", "tryr \n downregulation by > 85% in leishmania species causes inability to survive under \n oxidative stress inside macrophages [ 7274 ] .", "in contrast , when tryr is 14- and 10 fold \n overexpressed in leishmania and t. cruzi , respectively , there are no \n significant differences in h2o2 susceptibility between \n control and transfected cells ; both types of cells are also equally resistant \n to the oxidative stress - inducers gentian violet , and nitrofurans .", "intriguingly , the cellular levels of tsh2 , \n gsh , and glutathionyl - spermidine , determined in both types of experiments ( tryr \n suppression and overexpression ) were similar in control and transformed cells .  ", "knockdown \n of trys by sirna in procyclic t. brucei causes ( i ) viability impairment and arrest of proliferation when tsh2 levels decrease to 15% of the wild - type level , ( ii ) increased sensitivity to h2o2 and alkyl hydroperoxides , ( iii ) damage to the plasma membrane , and ( iv ) \n diminution of the tsh2 content and accumulation of gsh and \n glutathionyl - spermidine .", "a similar metabolite variation ( lower tsh2 ; \n higher gsh ) was attained with a trys knockdown induced by sirna in the \n bloodstream form of t. brucei .", "this \n trys knockdown also induced an increased sensitivity to different compounds \n that affect tsh2 metabolism such as arsenicals , melarsen oxide , \n trivalent antimonials , and nifurtimox .", "indeed , western blot analysis showed , \n in addition to the expected ( 10-fold ) decrease in trys protein , a 2 - 3-folds increase in -ecs and tryr .", "the changes in expression of \n other enzymes suggest unveiled compensatory or pleiotropic effects on tsh2 metabolism .  ", "other researchers have selected -glutamylcysteine synthetase ( -ecs ) , the presumed rate - limiting step of gsh \n synthesis , as an alternative drug target of tsh2 synthesis in t. brucei ( figure 3 ) .", "knockdown of -ecs gene in the parasite induces cell death \n and depletion of gsh and tsh2 only after 80% decrease in the enzyme \n content .", "the -ecs knockdown cells are rescued from death by \n adding external gsh , which elevates the cellular gsh and tsh2 levels \n .", "glutathione synthetase ( gs ) has not been manipulated in trypanosomatids , or in any other organism , perhaps \n because it has been considered as a nonrate - limiting step of gsh and tsh2 biosynthesis . however , dna microarray \n analysis of antimonite - resistant leishmania tarentolae shows increased \n transcription of -ecs , gs , and p - glycoprotein a rnas . \n although it was not evaluated whether increase in gene transcription correlated \n with an increase in enzyme activity , it may be possible that under high gsh \n demand ( i.e. , under oxidative stress conditions ) gs might exert control of tsh2 synthesis . on the other hand , ornithine \n decarboxylase", "( odc ) overexpression in t. brucei ( the presumed limiting \n step of spermidine synthesis ) causes no change in tsh2 levels \n .", "therefore , odc does not seem to be \n a controlling step of tsh2 synthesis .", "although almost full inhibition ( > 80% ) of gene transcription or activity of any of \n these enzymes results in parasite death , the question remains of how tsh2 metabolism is affected when the enzymes are less inhibited .", "for example , in the therapeutic treatment of \n patients it is certain that drugs have to be administered for long periods of \n time .", "if the parasites are not \n completely cleared from the patient , disease recurrence and generation of drug - resistant \n parasites are possible .", "the results described \n above indicate that each enzyme by itself has low control on tsh2 synthesis and concentration ; therefore , highly specific and very potent \n inhibitors have to be designed in order to attain the required full activity blockade \n to affect tsh2 metabolism in these parasites ."], ["the metabolic \n control analysis ( mca ) was initially developed by kacser and burns in scotland [ 82 , 83 ] and by heinrich and rapoport \n in east germany \n  [ 84 , 85 ] .", "this analysis establishes a \n theoretical framework that explains the results observed with the enzyme \n overexpression and downregulation experiments . in addition , it helps to identify and design \n experimental strategies for the manipulation of a given process in an organism \n ( heavy metal hyperaccumulation ; increased production of ethanol , co2 , \n lactate or acetate ; or inhibition of a metabolic pathway flux with therapeutic \n purposes ) .", "mca rationalizes the quantitative \n determination of the degree of control that a given enzyme exerts on flux and \n on the concentration of metabolites .", "different \n experimental approaches have been developed to detect and direct what has to be \n done and measured , in order to identify and understand why an enzyme exerts a \n significant or a negligible control on flux and metabolite concentration in a \n metabolic pathway .", "experiments for identifying and manipulating the conceptually wrong  rate - limiting \n step . ", "to \n understand how a metabolic pathway is controlled and could be manipulated , its \n control structure has to be evaluated . the \n control structure of a pathway is constituted by the flux control coefficient ( cvi ) , which is the degree of control that the rate ( v ) of a given enzyme i exerts \n on flux j ; the concentration control coefficient ( cvi ) , which is the degree of control that a given enzyme i exerts on the concentration of a metabolite \n ( x ) ; and the elasticity coefficients .", "the control coefficients are systemic \n properties of the pathway that are mechanistically determined by the elasticity \n coefficients ( x ) , which are defined as the degree of sensitivity \n of a given enzyme vi \n  ( i.e. , the enzyme 's ability to change \n its rate ) when any of its ligands ( x : substrate , products or allosteric \n modulators ) is varied .", "viojo , in which the expression dj / dvi describes \n the variation in flux ( j ) when an infinitesimal change is done in the enzyme i concentration or activity . in practice , \n", "the infinitesimal changes in vi \n  are undetectable , and hence measurable noninfinitesimal changes are undertaken . \n ", "if a small change in vi \n  promotes a significant \n variation in j , then this enzyme exerts an elevated flux control ( figure 4 , \n position 1 ) .", "in contrast , if a rather small \n or negligible change in flux is observed when vi \n  is greatly varied , then the enzyme does not exert \n significant flux control ( figure 4 , position 2 ) . to obtain dimensionless and normalized values \n of cvi the scaling factor", "vio / jo is applied , which represents the ratio between the initial values from which \n the slope dj / dvi \n  is \n calculated .", "if all cvi of the pathway enzymes and transporters are added up , the sum comes to \n one ( summation theorem ) .", "the mca clearly distinguishes \n between the control exerted by a given enzyme on flux ( flux control \n coefficient ) and on the metabolite concentration ( concentration control \n coefficient ) .", "thus , an enzyme can have significant control on a metabolite \n concentration but not on the pathway flux .", "on one hand , the use of the rate - limiting step concept \n for manipulating metabolic pathways does not make such differentiation , which \n probably has contributed to the many unsuccessful experiments reported in the \n literature ; on the other hand , it should be clearly defined whether the aim of the \n project is to increase flux and/or a metabolite concentration since mca \n establishes for each aim a different experimental design . to determine the flux control \n coefficient of a given enzyme , small variations in the enzyme content , or \n preferentially , in activity are required , without altering the rest of the \n pathway , and then the changes in flux are determined .", "the experimental points are plotted as shown \n in figure 4 to calculate the slope at the reference point vio / jo .", "this experiment , apparently easy to perform , \n has demanded great intellectual and experimental effort .", "several experimental strategies have been \n developed to determine cvi : \n formation of heterokarionts and heterocygots ( classical genetics),titration of flux with specific inhibitors , elasticity analysis , mathematical modeling ( in \n  silico biology),in vitro reconstitution of metabolic pathways , genetic engineering to manipulate in vivo protein levels . \n ", "formation of heterokarionts and heterocygots ( classical genetics ) , titration of flux with specific inhibitors , mathematical modeling ( in \n  silico biology ) , in vitro reconstitution of metabolic pathways , genetic engineering to manipulate in vivo protein levels .", "the arginine biosynthesis in neurospora crassa was the first metabolic \n pathway in which flux control coefficients were experimentally determined by \n kacser 's laboratory .", "this fungus \n forms multinucleated mycelia that facilitate the generation of polyploid cells . \n  by mixing different ratios of spores containing \n genes encoding wild ( active ) and mutant ( inactive ) enzymes of this pathway , it \n was possible to generate heterokaryont mycelia with different content , and \n activity , of four pathway enzymes .", "the authors built plots of enzyme activity versus \n flux ( see figure 4 ) for acetyl - ornithine aminotransferase , ornithine \n transcarbamoylase , arginine - succinate synthetase , and arginine - succinate lyase . \n ", "all the experimental points of these \n heterokaryonts localized near to position 2 of figure 4 with cvi =   0.020.2 ( flux control by these enzymes was \n only 220% ) , which indicated that none of these enzymes exerted significant \n control on arginine synthesis .", "the authors did not determine the remaining flux \n control ( 75% ) , which might reside in carbamoyl - phosphate synthetase i ( this mitochondrial \n ammonium - dependent isoform can be bound to the mitochondrial inner membrane or \n form complexes with ornithine transcarbamoylase [ 87 , 88 ] ) and in mitochondrial citruline / ornithine \n transporter , both of which have been proposed as limiting steps , or might be in \n the arginine demand for protein synthesis .", "organisms \n with many alleles of one enzyme may form homo - and heterozygotes expressing \n different activity levels .", "drosophila \n melanogaster has three adh alleles encoding for isoforms with different vmax . when three natural homozygotes , a null mutant , and \n some heterozygotes were generated , different adh activities were attained but \n the ethanol consuming rate did not change ( figure 4 , position 2 ) .", "oxidative phosphorylation ( oxphos ) is \n the only pathway for which specific and potent inhibitors for many enzymes and \n transporters are available .", "oxphos is divided in two segments ( figure 5 ) : the \n oxidative system ( os ) formed by substrate transporters ( pyruvate , \n 2-oxoglutarate , glutamate , glutamate / aspartate , dicarboxylates ) , krebs cycle \n enzymes , and the respiratory chain complexes ; and the phosphorylating system \n ( ps ) constituted by the atp / adp ( ant ) and pi ( pit ) transporters , and atp \n synthase .", "when the flux ( atp synthesis ) \n is titrated by adding increasing concentrations of each specific inhibitor , \n plots are generated in which the enzyme activity is progressively diminished by \n increasing inhibitor concentration .", "hence , the cvi value depends on the type of \n inhibitor usedfor irreversible inhibition,(2)cvij  =  ( imaxjo)(djdi)[i]0 , \n for simple noncompetitive inhibition,(3)cvij  =  ( kijo)(djdi)[i]0 , \n for simple competitive inhibition,(4)cvij  =  ( -ki[(1+s)/km]jo)(djdi)[i]0 , \n  where jo is the pathway flux in the absence \n of inhibitor ; imax , minimal \n inhibitor concentration to reach maximal flux inhibition ; k \n i , inhibition constant ; s , \n substrate concentration ; km , \n michaelis - menten constant ; and d \n j / d \n i , \n initial slope ( [ i ] = 0 ) of inhibition titration curve . for irreversible inhibition,(2)cvij ", "=  ( imaxjo)(djdi)[i]0 , \n  for simple noncompetitive inhibition,(3)cvij  =  ( kijo)(djdi)[i]0 , \n  for simple competitive inhibition,(4)cvij  =  ( -ki[(1+s)/km]jo)(djdi)[i]0 , \n  to estimate flux control \n coefficients from inhibitor titration of adp - stimulated ( state 3 ) respiratory \n rates ( i.e. , mitochondrial o2 consumption coupled to atp synthesis ) , ( 2 ) for irreversible inhibitors was used \n because researchers assumed that mitochondrial inhibitors such as rotenone , \n antimycin , carboxyatractyloside , and oligomycin were ", "however , under this assumption flux control coefficients were usually \n overestimated [ 90 , 91 ] . to solve this \n problem , gellerich et al .  ", "developed ( 5 ) for noncompetitive \n tightly - bound inhibitors and , by using nonlinear regression analysis , it was \n possible to include all experimental points from the titration curve thus \n increasing accuracy in calculating cvi:(5)j  =  [ n(joji)2encojoeon[(n    co)jo    ( nji)en ] ]  +  ji  e2+(kd+ieo)ekdeo=0 , in which jo and ji are the \n respiration fluxes in the noninhibited ( e = eo ) and inhibited ( e = 0 ) states ; k \n d is the dissociation constant of \n the enzyme - inhibitor complex , and n is an empirical component that expresses the relationship between substrate \n concentration and the reaction catalyzed by the enzyme e. the analysis of data in table 3 shows that oxphos is not controlled by only one \n limiting step , but the flux control is rather distributed among several enzymes \n and transporters .", "it is worth noting \n that the value of the flux control coefficient depends on the content of enzyme \n or transporter , which varies from tissue to tissue .", "perhaps the atp / adp translocase in as-30d hepatoma mitochondria \n might reach the status of being the  oxphos limiting step  with a ca nt = 0.70 , or the pi transporter in kidney mitochondria , or the \n atp / adp translocase and the respiratory chain complex 3 in liver mitochondria , but \n", "it should be noted that other steps also exert significant control ( table 3 ) .", "although the distribution of control varies \n between tissues , the flux control mainly resides in the ps of organs with high atp \n  demand such as the heart   ( cpt+ant+atpsynthase = cps = 0.73 ) , kidney ( cps = 0.75 ; cos = 0.31 ) , and   fast - growing tumors ( cps = 0.98 ) .", "in contrast , in the \n liver   ( cos = 0.80 ; cps = 0.65 ) and brain ( cos = 0.35 ; cps = 0.41 ) , the control is shared by both systems .", "determined that the oxphos \n control was shared by the ps ( cps = 0.62 ) and the atp demand ( purified atpase ) . in turn ,", "rossignol et al .   concluded that \n the os exerted the main control ( cos = 0.68 ) , but these authors apparently used low - quality mitochondria \n ( low respiratory control values that lead to low rates of atp synthesis \n associated with high rates of respiration ) that were not incubated under near physiological \n conditions ( 10 mm pyruvate , 10 mm malate , 10 mm pi , ph 7.4 in tris buffer ) , and the authors incorrectly assumed that rotenone and antimycin \n were irreversible inhibitors .", "it is notorious \n that in all works shown in table 3 at least one of these mistakes is evident .", "there are some inhibitors for enzymes and transporters from other pathways , but they \n are not quite specific and may affect other sites . due to the fact that there are no inhibitors \n for every step in these pathways ,", "examples \n of these inhibitors are 6-chloro-6-deoxyglucose for glucose transporters in \n bacteria , 2-deoxyglucose for hpi , iodoacetate for gapdh , 1,4-dideoxy-1,4-imino - d - arabinitol \n for glycogen phosphorylase , oxalate and oxamate for ldh , 6-amino \n nicotinamide for the phosphate pentose pathway , amino - oxyacetate for \n aminotransferases and kirureninase ( tryptophan synthesis ) , norvaline for \n ornithine transcarbamylase , mercaptopycolinate for pep carboxykinase , acetazolamide \n for carbonic anhydrase , and isobutyramide for adh ( compiled by \n fell ) .", "potential uses of the experimental approachmitochondrial \n pathologies are a heterogeneous group of metabolic perturbations characterized \n by morphological abnormalities and/or oxphos dysfunction .", "mitochondrial dna analysis has revealed specific \n mutations for some mitochondriopathies . although the specific oxphos mutations causing \n the disease may appear in all tissues , the functioning of only some of them is \n altered .", "the organ 's sensitivity might be related to the different flux control \n coefficients of the mutated enzyme in the different tissues ( table 3 ) and to \n their atp supply dependence from oxphos versus \n glycolysis.mca allows for the analysis of a metabolic flux or intermediate concentration by focusing \n either on one step or by grouping enzymes in blocks or in pathways .", "thus , a comparative analysis of oxphos control \n distribution reveals that heart , kidney , some fast growing tumors ( rat as-30d \n hepatoma , mouse fibrosarcoma , human breast , lung , thyroid carcinoma , melanoma ) \n , and perhaps skeletal muscle are more susceptible to mitochondrial mutations \n in atp synthase , which is the only ps site with subunits encoded in the mitochondrial \n genome . on the other side , liver and \n brain", "might be more susceptible to mitochondrial mutations of the respiratory \n chain enzymes ( see table 3 ) .", "considering that the brain is a fully aerobic \n organ , whereas the liver depends on both oxphos ( 7080% ) and glycolysis \n ( 2030% ) for atp supply , then it can be postulated that the brain is more \n sensitive to mutations in the mitochondrial genome than the liver because subunits \n of complexes i , iii , and iv are encoded by the mitochondrial genome.titration of flux with \n specific inhibitors to determine the flux control coefficients of oxphos has been \n applied to intact tumor cells .", "the results showed that the flux control \n resided mainly in site 1 of the respiratory chain ( csitel = 0.30 ) , whereas the other evaluated sites exerted a marginal \n control .", "this observation could \n have therapeutic application if site 1 does not exert control in healthy cells , \n leading to less severe side effects.the use of inhibitors in \n intact cells to determine control coefficients might pose two problems : hydrophilic \n inhibitors such as carboxyatractyloside ( for ant ) and -cyano-4-hydroxy - cinammate ( for pyruvate transporter ) \n can not readily enter the cell due to the presence of the plasma membrane \n barrier ; the other problem is that hydrophobic but slow inhibitors , such as \n oligomycin , require long incubation times to ensure the interaction with the \n specific sites .", "these problems can be \n solved by incubating the cells for long periods of time and taking care of cell \n viability , for instance , as-30d hepatoma cells are fairly resistant to this \n mechanical manipulation as they maintain high viability after a lengthy \n incubation under smooth orbital agitation of 1 h at 37c \n .", "mitochondrial \n pathologies are a heterogeneous group of metabolic perturbations characterized \n by morphological abnormalities and/or oxphos dysfunction .", "mitochondrial dna analysis has revealed specific \n mutations for some mitochondriopathies . although the specific oxphos mutations causing \n the disease may appear in all tissues , the functioning of only some of them is \n altered .", "the organ 's sensitivity might be related to the different flux control \n coefficients of the mutated enzyme in the different tissues ( table 3 ) and to \n their atp supply dependence from oxphos versus \n glycolysis .", "mca allows for the analysis of a metabolic flux or intermediate concentration by focusing \n either on one step or by grouping enzymes in blocks or in pathways .", "thus , a comparative analysis of oxphos control \n distribution reveals that heart , kidney , some fast growing tumors ( rat as-30d \n hepatoma , mouse fibrosarcoma , human breast , lung , thyroid carcinoma , melanoma ) \n , and perhaps skeletal muscle are more susceptible to mitochondrial mutations \n in atp synthase , which is the only ps site with subunits encoded in the mitochondrial \n genome . on the other side , liver and \n brain might be more susceptible to mitochondrial mutations of the respiratory \n chain enzymes ( see table 3 ) . considering that the brain is a fully aerobic \n organ ,", "whereas the liver depends on both oxphos ( 7080% ) and glycolysis \n ( 2030% ) for atp supply , then it can be postulated that the brain is more \n sensitive to mutations in the mitochondrial genome than the liver because subunits \n of complexes i , iii , and iv are encoded by the mitochondrial genome .", "titration of flux with \n specific inhibitors to determine the flux control coefficients of oxphos has been \n applied to intact tumor cells .", "the results showed that the flux control \n resided mainly in site 1 of the respiratory chain ( csitel = 0.30 ) , whereas the other evaluated sites exerted a marginal \n control .", "this observation could \n have therapeutic application if site 1 does not exert control in healthy cells , \n leading to less severe side effects .", "the use of inhibitors in \n intact cells to determine control coefficients might pose two problems : hydrophilic \n inhibitors such as carboxyatractyloside ( for ant ) and -cyano-4-hydroxy - cinammate ( for pyruvate transporter ) \n can not readily enter the cell due to the presence of the plasma membrane \n barrier ; the other problem is that hydrophobic but slow inhibitors , such as \n oligomycin , require long incubation times to ensure the interaction with the \n specific sites .", "these problems can be \n solved by incubating the cells for long periods of time and taking care of cell \n viability , for instance , as-30d hepatoma cells are fairly resistant to this \n mechanical manipulation as they maintain high viability after a lengthy \n incubation under smooth orbital agitation of 1 h at 37c \n .", "xovio , which is a dimensionless number that show the rate variation v of a given enzyme or transporter i when the concentration of a ligand x ( substrate s , product p or allosteric modulator ) is varied in infinitesimal \n proportions .", "the elasticity coefficients are positive for those metabolites \n that increase the enzyme or transporter rate ( substrate or activator ) , and they \n are negative for the metabolites that decrease the enzyme or transporter rates \n ( product or inhibitor ) .", "an enzyme working , under a steady - state metabolic flux , at saturating conditions of s or p , \n is no longer sensitive to changes in these metabolites .", "thus , its elasticity is close to zero ( figure 6 , x = 0 ) . \n  in turn", ", an enzyme working at s or p \n concentrations well below the michaelis constant ( kms or kmp ) \n is expected to be highly sensitive to small variations in these metabolites ( figure \n 6 , x = 1 ) .", "if the kinetic parameters of an enzyme are \n known ( vmf , vmr ,   kms , and kmp ) , then the enzyme elasticity for any given metabolite \n concentration may be calculated as shown in the following equations . for substrate,(7)svi  =  s / kms1  +  s / kms  +  p / kmp  +  11    /keq , and \n for product,(8)pvi  =  p / kmp1  +  s / kms  +  p / kmp    /keq1    /keq , in which  is", "the mass \n action ratio , and keq is the equilibrium constant preferentially determined \n under physiological conditions .", "an enzyme with low elasticity can not increase ( or decrease ) its rate despite large \n variations in s ( or p ) concentration ; in consequence , such enzyme exerts a high \n flux control . in turn", ", an enzyme with a \n high elasticity can adjust its rate to the variation in s or p concentrations , \n and thus it does not interfere with the metabolic flux , exerting a low flux control .", "this inverse relationship between the \n elasticity and the flux control coefficients is expressed in a formal equation \n denominated connectivity theorem .", "a \n metabolic pathway can be divided in two blocks around an intermediary x : the \n producing ( synthetic , supply ) and the consuming ( demand ) enzyme blocks of x are i1 and i2 , respectively .", "thus , the connectivity theorem for this two - block system is(9)cv1jcv2j  =  xv2xv1 .", "the negative sign of the right part of the equation \n cancels with x , which is negative because x \n is a product of enzyme block i1 ( figure 6 ) . to obtain the flux control coefficients", "many strategies have been designed [ 90 , 103108 ] , but the most \n used and probably more trustworthy is that in which the initial pathway metabolite \n ( so ) concentration is varied to increase the x concentration ( any intermediary \n in the pathway ) , and measuring in parallel the variation in flux . under steady - state conditions , the flux rate \n is equal to the rate of end - product formation ( i.e. , lactate or alcohol for \n glycolysis ; oxygen consumption for oxphos ) and to the rate of any partial \n reaction .", "the slope , calculated at the reference \n coordinate ( xo , jo ) that is \n equivalent to ( so , vio ) , yields \n the elasticity coefficient of the consuming block of x. in another set of experiments , an inhibitor is \n added to block one or more enzymes after x. the x concentration and flux are determined and \n plotted as shown in figure 7 , from which the elasticity coefficient of the \n producing block is calculated .", "the flux \n control coefficients are determined by using the connectivity theorem and \n considering that the sum of the control coefficients comes to 1 , c1 + c2 = 1 ( summation theorem):(10)cv1j  =  xv2xv2    xv1,cv2j  =    xv1xv2    xv1 . \n ", "this method for determining cvi using the elasticities of the two blocks was called double \n modulation by kacser and burns .", "years \n later , brand and his group [ 103 , 104 ] renamed this method as top - down approach . by applying the procedure shown in figure 7 \n and using ( 10 ) for different metabolites along the metabolic pathway , it \n is possible to identify those sites that exert a higher control ( which may be \n the sites for therapeutic use or biotechnological manipulation ) and those that \n exert a negligible control under a given physiological or pathological \n situation .", "elasticity analysis has been used to evaluate the oxphos control distribution \n in tumor cells .", "almost all studies on \n this subject have been carried out with isolated mitochondria incubated in \n sucrose - based medium at 25 or 30c or with the more physiological kcl - based medium but \n still at 30c ( table 3 ) .", "furthermore , \n these studies did not consider that the product , atp , never accumulates in the \n living cells , which does occur in experiments with isolated mitochondria .", "under such a condition , a steady state in atp production can \n never be reached as in living cells . in \n other words , the distribution of control in mitochondria ( table 3 ) has been \n determined in the absence of an atp - consuming system . a remarkable exception to", "this incomplete \n experimental design was the work done by wanders et al . , in which isolated liver mitochondria were incubated \n with two different atp - consuming systems ( or adp - regenerating systems ) : hk + \n glucose and creatine kinase ( ck ) + creatine . under this", "more physiological setting , the oxphos \n flux control distributed between ant and the atp - consuming system ; however , flux \n control by the other pathway components was not examined .", "therefore , to accurately evaluate oxphos control \n distribution , mitochondria should be incubated in the presence of an \n atp - consuming system or in their natural environment ( i.e. , inside the cell ) .", "the rate of oxphos in intact \n cells is determined from the rate of oligomycin - sensitive respiration : in the \n steady state , the enzyme rates are the same and constant ; in branched pathways \n the sum of the branched fluxes equals the flux that supplies the branches .", "the global elasticity of the atp - consuming processes \n ( e.g. , synthesis of protein , \n nucleic acid , and other biomolecules , as well as ion atpases to maintain the \n ionic gradients , mechanical activity such as muscular contraction or flagellum \n and cilium movement , and secretion of hormones , digestive enzymes and \n neurotransmitters ) is estimated by inhibiting flux with low concentrations of \n oligomycin or a respiratory chain inhibitor . to determine the elasticity of the \n atp - producing block , flux , and [ atp ]", "are varied with streptomycin , an inhibitor \n of protein synthesis ( figure 7 ) .", "the \n elasticity coefficients are calculated from the initial coordinate slopes \n ( without inhibitors ) of each titration . with \n this procedure", ", it has been determined that the atp - consuming block exerts a \n significant flux control of 34% .", "remarkably , \n this flux control value obtained in cells is quite similar to the flux control \n coefficients of the atp - consuming system ( hk or ck ) reported by wanders \n et al .   with isolated \n mitochondria .", "elasticity analysis by enzyme \n blocks allows the inclusion of the end - product demand as another pathway block . \n ", "the conclusions obtained from this \n analysis have formulated the supply - demand theory , which proposes that when \n flux is controlled by one block ( demand ) , the concentration of the end - product \n is determined by the other block ( supply ) . the ratio of elasticities determines the distribution of flux control \n between supply and demand blocks . for \n instance , if x > x ( i.e. , demand becomes saturated by the end - product x , \n and hence its elasticity is near zero ) , then the demand block exerts the main \n  flux control . for concentration control , \n at larger x  x ,   smaller absolute values of both csupply and cdemand are attained ; hence , under demand saturation , \n the supply elasticity fully governs the magnitude of the variation in the \n end - product concentration . on the other \n hand ,", "when demand increases , it loses flux control and induces a diminution in \n the end - product concentration . in turn , supply gains flux control and loses \n concentration control . in the presence of feed - back inhibition ,", "the system can \n maintain the end - product concentration orders of magnitude away from \n equilibrium ( at a concentration around the k0.5 of the allosteric enzyme ) . as mentioned before ,", "the \n demand is not usually included in the pathway because it is erroneously thought \n that it is not part of it .", "but then , is \n it valid to analyze the control of a metabolite synthesis if its demand is not \n considered ?", "when the demand block is not \n included , it is assumed that the metabolic pathway produces a metabolite at the \n same rate regardless whether the metabolite demand is high or low .", "this reasoning is incorrect because a \n metabolic pathway indeed responds to changes in the metabolite demand and , more \n importantly , a pathway without end - products consumption reactions is unable to \n reach a steady state .", "the intermediary x linking the two blocks is one \n of the end - products of the producing block ( e.g. , \n pyruvate or lactate or ethanol , and atp for glycolysis ) .", "the variation in rate of the two blocks in \n response to a variation in x can be theoretical or experimentally determined ( figure 8(a ) ) .", "it is worth noting that , for this \n supply - demand approach , it is not necessary to know the kinetics of each \n pathway enzyme because the rate response of each block reflects the global kinetics \n of all participating enzymes", ". when the x \n concentration is increased , the rate of the supply block decreases ( i ) because x \n is its product and ( ii ) because usually an enzyme within this block receives \n information from the final part of the pathway , decreasing its rate through \n feedback inhibition . in turn", "to better \n visualize the effect of large rate changes , the kinetics of both blocks are \n plotted in a logarithmic scale .", "figure 8(b ) shows the kinetics described in figure 8(a ) converted to natural \n logarithm . the intersection point \n between kinetic curves , at which the supply and demand rates are identical , \n represents the pathway steady - state flux ( in the y axis ) and end - product \n concentration ( in the x axis ) .", "since the \n elasticity is also defined as x = dlnvi / dlnx , the slope at the intersection point represents the elasticity of each \n block towards the intermediary x. here , \n the use of the scalar factor is not necessary because it is included in the \n logarithmic equation . with the elasticity \n coefficients calculated from plots like those shown in figure 8 , and the \n connectivity theorem ,", "the example in figure 8(b ) shows that \n the demand exerts a high flux control ( and has low elasticity ) and the supply \n block exerts low control ( and has high elasticity ) . the \n fact that the demand may exert a high flux control in metabolite pathways has \n at least three important implications : ( a ) the supply block responds to variations \n in the demand ( high elasticity ) ; ( b ) the demand block has information transfer \n mechanisms towards the supply block that avoid the unrestricted intermediary accumulation under a low demand , \n particularly when the supply block has reactions with large keq ( > 100 ; g > 3 kcal mol at 37c ) ; and ( c ) \n if the main flux control resides in the demand block , then the supply block may \n only exert control on the intermediary concentration but not on the flux [ 30 , 32 ] .", "this last conclusion explains why it \n is incorrect to consider that an enzyme that controls flux must also control \n the intermediary concentration", ". regulatory mechanisms of \n enzyme activity are modulation of protein concentration by synthesis and \n degradation , as well as covalent modification and variation in the substrate or \n product concentrations ( which are components of the pathway ) .", "in addition , another regulatory mechanism is the \n modulation by molecules that are not part of the pathway , that is , through allosteric interaction with cooperative ( sigmoidal \n kinetics ) or noncooperative enzymes ( hyperbolic kinetics ) ( e.g. , ca activates some krebs cycle dehydrogenases ; citrate inhibits pfk-1 ; malonyl - coa inhibits \n the mitochondrial transporter of acyl - carnitine / carnitine ; or the initial \n substrate of a pathway that has not entered the system ) . for these last cases , kacser and burns \n", "proposed the use of the response coefficient r which is defined by the \n following expression:(11)rm  j  =  cvijmvi , where m is the external modulator of the i enzyme .", "the response coefficient is dj / dmmo / jo . \n  if the elasticity of the sensitive \n enzyme toward the external effector is also determined , then it is possible to \n calculate cvi by using ( 11 ) .", "unfortunately , due to the experimental complexity for determining the \n elasticity coefficient , this coefficient is often calculated in a theoretical \n way by using the respective rate equation ( michaelis - menten or hill equations ) \n and the kinetic parameters km and vmax determined by someone else under optimal \n assay conditions , which are commonly far away from the physiological ones .", "therefore , for this theoretical determination \n of elasticity only the value of the external modulator concentration is required .", "it is convenient to emphasize that the determination \n of the flux control coefficients becomes more reliable when they are calculated \n from several experimental points ( figure 7 ) , instead of only one , as occurs \n with the theoretical elasticity analysis .", "determined the flux control distribution of gluconeogenesis \n from lactate in hepatocytes by using both theoretical and experimental elasticity \n analysis and the response coefficient .", "these authors concluded that gluconeogenesis \n stimulated by glucagon was controlled by the pyruvate carboxylase ( cpc = 0.83 ) ; in the absence of this hormone , the \n control was shared by pc , pyk , eno - pgk segment , and tpi - fructose-1,6-biphosphatase \n segment .", "elasticity \n analysis has been applied to elucidate the flux control of atp - producing \n pathways in fast - growing tumor cells . for oxphos", ", this approach showed that respiratory chain complex i and \n the atp - consuming pathways were the enzymes with higher control ( cvi = 0.7 ) .", "for glycolysis , the main flux control ( cvi = 0.71 ) resided in glut + hk reactions because \n hk is strongly inhibited by its product g6p despite extensive enzyme overexpression \n .", "examples of elasticity analysis on \n other pathways are photosynthesis , ketogenesis , serine   and \n threonine synthesis in e. coli , glycolysis in yeast , glucose \n transport in yeast , dna supercoiling , glycogen synthesis in muscle , \n and galactose synthesis in yeast . in conclusion", ", the elasticity \n analysis is the most frequently used method for determining flux control \n coefficients because it does not need a group of specific inhibitors for all \n the enzymes and transporters of the pathway , neither does it require knowledge \n of the inhibitory mechanisms or kinetic constants .", "it is only necessary to produce a variation in \n the intermediary concentration x by using an inhibitor of either block or by \n directly varying the x concentration . in agreement with fell", ", it seems impossible for a researcher to analyze one by \n one the rate equation of each enzyme in a metabolic pathway to predict and \n explain the system behavior as a whole . to deal with this problem , in the last three \n decades", "some scientists have constructed mathematical models for some metabolic \n pathways using several software programs .", "thus , the specific variation of a single enzyme \n activity without altering the rest of the pathway ( figure 4 ) , which has been an \n experimentally difficult task for applying mca , becomes easier to achieve with \n reliable computing models .", "there are two basic types of modeling : ( a ) structural modeling and ( b ) kinetic \n modeling .", "the former is related to the pathway chemical reaction structure and \n does not involve kinetic information .", "the information obtained with structural modeling is the \n description of the following : the exact determination of which reactions and \n metabolites interact among them;the conservation reactions .", "there are metabolites for \n which their sum is always constant or conserved ( e.g. , nadh + nad ; nadph + nadp ; \n ubiquinol + ubiquinone ; atp + adp + amp ; coa + acetyl - coa ) .", "the identification of \n conserved metabolites might not be obvious;enzyme groups catalyzing reactions in a given relationship \n with another group of enzymes;elemental modules , which are defined as the minimal \n number of enzymes required to reach a steady state , which can be isolated from the \n system ( for a review about structural modeling ; see ) . \n ", "the exact determination of which reactions and \n metabolites interact among them ; the conservation reactions .", "there are metabolites for \n which their sum is always constant or conserved ( e.g. , nadh + nad ; nadph + nadp ; \n ubiquinol + ubiquinone ; atp + adp + amp ; coa + acetyl - coa ) .", "the identification of \n conserved metabolites might not be obvious ; enzyme groups catalyzing reactions in a given relationship \n with another group of enzymes ; elemental modules , which are defined as the minimal \n number of enzymes required to reach a steady state , which can be isolated from the \n system ( for a review about structural modeling ; see ) .", "in addition to an appropriate computing \n program , this approach requires the knowledge of the stoichiometries , rate \n equations , and keq values of each reaction in the pathway ( or the vmax in the forward and reverse \n reactions ) , as well as the intermediary concentrations reached under a given \n steady state .", "some currently used \n softwares are copasi ( http://www.copasi.org/tiki-index.php ) \n based on gepasi ( http://www.gepasi.org/ ; ) ; metamodel ; winscamp \n and jarnac   ( both available at http://www.sys-bio.org/ ) ; and pysces \n ( http://pysces.sourcesforge.net/ ; ) . for", "other programs and links , go to http://sbml.org/index.psp . to reach a steady - state flux ,", "it is necessary \n to fix the initial metabolite concentration to a constant value and the \n irreversible and constant removal of the end products . except for the final reactions in which their \n products have to be removed from the system ,", "all pathway reactions have to be \n considered as reversible , notwithstanding whether they have large keq ( if there \n is an irreversible reaction under physiological conditions , then a reversible rate \n equation that includes the keq suffices to maintain the reaction as practically \n irreversible ) .", "care should be taken to include the enzyme 's sensitivity toward \n its products because this property is related with the enzyme elasticity and hence \n with its flux control ; omission of this parameter may very likely lead to \n erroneous conclusions .", "it \n should be pointed out that the purpose of kinetic modeling is not merely to \n replicate experimental data but also to explain them .", "thus , pathway modeling is a powerful tool \n that allows for ( i ) the detection of those properties of the pathway that are \n not so obvious to visualize when the individual kinetic characteristics of the \n participating enzymes are examined ; and ( ii ) the understanding of the biochemical \n mechanisms involved in flux and intermediary concentration control .", "modeling requires the consideration of all \n reported experimental data and interactions that have been described for the \n components of a specific pathway , thus allowing for the integration of disperse \n data , discarding irrelevant facts . although all models are oversimplifications of complex cellular \n processes , they are useful for the deduction of essential relationships , for \n the design of experimental strategies that evaluate the control of a metabolic \n pathway , and for the detection of incompatibilities in the kinetic parameters \n of the participating enzymes and transporters , which may prompt the \n experimental revision of the most critical uncertainties . with \n", "the model initially constructed , the simulation results do not usually concur \n with the experimental results ; in consequence , the model normally requires \n refinement , a point at which the researcher 's thinking and knowledge of biology \n plays a fundamental role in modifying the structure and parameters of the model . \n ", "the discrepancies observed between modeling \n and experimentation unequivocally pinpoint what elements or factors have to be re - evaluated \n or incorporated so that the model approximates more closely reality ( i.e. , experimental data ) .", "the comparison of the experimentally obtained \n intermediary concentrations and fluxes with those obtained by simulation is an \n appropriate validating index of the model ; this index indicates whether the \n model approximation to the physiological situation is acceptable or whether \n re - evaluation of the kinetic properties of some enzymes and transporters and/or \n incorporation of other reactions or factors is required .", "a reason to why the results obtained by modeling may substantially differ from the \n experimental results is that the kinetic parameters of the pathway enzyme and \n transporters and the keq values used \n were determined by different research groups , under different experimental \n conditions and in different cell types .", "moreover , \n enzyme kinetic assays are carried out at low , diluted enzyme \n concentrations ( thus discarding or ignoring relevant protein - protein \n interactions ) , and at optimal ( but not physiological ) ph and  room temperature  \n ( which may be far away from the physiological values ) . in addition , no experimental information is \n usually available regarding the reactions reversibility and the product \n inhibition of the enzymes and transporters ( particularly for physiological \n irreversible reactions , i.e. , reactions with large keq ) . with worrisome \n frequency ,", "the researcher has to adjust the experimentally determined vm and km values to achieve a model behavior that acceptably resembles \n that observed in the biological system .", "apparently , \n this type of limitations as well as the sometimes overwhelming amount of \n kinetic data necessary for the construction of a kinetic model has restricted \n the number of reliable models that can be used for the prediction of the \n pathway control structure . once the kinetic model stability , robustness ,", "structural and dynamic properties have \n been evaluated , and experimentally validated , the model may become a virtual \n laboratory in which any parameter or component can be modified or replaced and \n any aspect of the pathway behavior can be explored within a wide diversity of \n circumstances or limits . at this \n stage ,", "glycolysis \n in s. bayanus , s. cerevisiae [ 113 , 123 , 124 ] , and trypanosoma brucei [ 125 , 126 ] is the \n metabolic pathway that has been more extensively modeled .", "both cell types \n have a very active glycolysis and are fully dependent on this metabolic pathway \n for atp supply , under anaerobiosis and aerobiosis , respectively .", "one advantage \n of modeling glycolysis in these cell types is that most of the kinetic \n parameters used have been experimentally determined by the same groups under \n the same experimental conditions .", "however , \n the kinetics of the reverse reactions has not been determined and thus these \n authors used kmp and keq \n values reported by others and obtained in other cell types under rather different \n experimental conditions , or they were adjusted to improve model fitting .", "nevertheless , the simulation \n results yielded relevant information on the control of the glycolytic flux . in both cases , the enzymes traditionally considered \n the rate - limiting steps , hk , atp - pfk-1 , and pyk did not contribute to the flux \n control , whereas the main control resided in glut ( 54% in the parasite and 85100% in yeast ) . under some conditions", ", hk may exert some \n control ( 15% ) in s. cerevisiae and some nonallosteric enzymes such as \n aldo , gapdh , and pgk may also exert some flux control in t. brucei .", "mca \n through kinetic modeling has been applied to several pathways : glycolysis in \n erythrocytes   in which flux control distributes between hk ( 71% ) and pfk-1 \n ( 29%);carbohydrate \n metabolism during differentiation in dictyostelium discoideum   with \n cellulose synthase ( 86% ) as the main controlling step;sucrose accumulation \n in sugar cane with hk , invertase , fructose uptake , glucose uptake , and vacuolar \n sucrose transporter having the most significant flux control ; glycerol \n synthesis in s. cerevisiae with gapdh \n ( 85% ) as the main control step ; penicillin \n synthesis in penicillium chrysogenum controlled \n ( 7598% ) either by d-(a - aminoadipyl ) cysteinylvaline synthetase ( short \n incubation times < 30 hour ) or isopenicillin n. synthetase ( long incubation \n times > 100 h ) ; calvin cycle \n  controlled by gapdh ( 50% ) and sedoheptulose-1,7-bisphosphatase ( 50%);threonine \n synthesis in e. coli controlled by homoserine dehydrogenase ( 46% ) , \n aspartate kinase ( 28% ) , and aspartate semialdehyde dehydrogenase ( 25% ) ; lysine \n production in corynebacterium glutamicum mainly \n controlled by aspartate kinase and permease ; nonoxidative \n pentose pathway in erythrocytes mainly controlled by transketolase ( 74% ) ; egf - induced mapk \n signaling in tumor cells controlled by ras - activation by egf ( 21% ) , ras \n dephosphorylation ( 43% ) , erk phosphorylation by mek ( 44% ) , and mek \n phosphorylation by ras ( 143% ) ; \n aspergillus niger  \n  arabinose utilization with \n flux control shared by arabinose reductase ( 68% ) , arabitol dehydrogenase ( 17% ) , \n and xylulose reductase ( 14% ) ; glycolysis in l. lactis in which several end products are generated ( lactate , \n organic acids , ethanol , acetoin ) .", "model predictions indicated that flux toward diacetyl and acetoin \n ( important flavor compounds ) was mainly controlled by ldh but not by \n acetolactate synthetase , the first enzyme of this branch . \n ", "glycolysis in \n erythrocytes   in which flux control distributes between hk ( 71% ) and pfk-1 \n ( 29% ) ; carbohydrate \n metabolism during differentiation in dictyostelium discoideum   with \n cellulose synthase ( 86% ) as the main controlling step ; sucrose accumulation \n in sugar cane with hk , invertase , fructose uptake , glucose uptake , and vacuolar \n sucrose transporter having the most significant flux control ; glycerol \n synthesis in s. cerevisiae with gapdh \n ( 85% ) as the main control step ; penicillin \n synthesis in penicillium chrysogenum controlled \n ( 7598% ) either by d-(a - aminoadipyl ) cysteinylvaline synthetase ( short \n incubation times < 30 hour ) or isopenicillin n. synthetase ( long incubation \n times > 100 h ) ; calvin cycle \n  controlled by gapdh ( 50% ) and sedoheptulose-1,7-bisphosphatase ( 50% ) ; threonine \n synthesis in e. coli controlled by homoserine dehydrogenase ( 46% ) , \n aspartate kinase ( 28% ) , and aspartate semialdehyde dehydrogenase ( 25% ) ; lysine \n production in corynebacterium glutamicum mainly \n controlled by aspartate kinase and permease ; nonoxidative \n pentose pathway in erythrocytes mainly controlled by transketolase ( 74% ) ; egf - induced mapk \n signaling in tumor cells controlled by ras - activation by egf ( 21% ) , ras \n dephosphorylation ( 43% ) , erk phosphorylation by mek ( 44% ) , and mek \n phosphorylation by ras ( 143% ) ; \n aspergillus niger  \n  arabinose utilization with \n flux control shared by arabinose reductase ( 68% ) , arabitol dehydrogenase ( 17% ) , \n and xylulose reductase ( 14% ) ; glycolysis in l. lactis in which several end products are generated ( lactate , \n organic acids , ethanol , acetoin ) .", "model predictions indicated that flux toward diacetyl and acetoin \n ( important flavor compounds ) was mainly controlled by ldh but not by \n acetolactate synthetase , the first enzyme of this branch .", "we modeled \n the gsh and pcs biosynthesis ( figure 2 ) to determine and understand the control \n structure of the pathway and thus be able to identify potential sites for genetic \n engineering manipulation that might lead to the generation of improved species \n in heavy metal resistance and accumulation .", "two models were constructed , one \n for higher plants and the other for yeast , both exposed to high concentrations \n of cd . due to the \n similarity in the results , only the plant results are analyzed below .", "an interesting conclusion from the gsh - pcs synthesis modeling is that control of flux \n ( and gsh concentration ) is shared between the gsh supply and demand under both unstressed \n and cd exposure \n conditions ( table 4 ) .", "this observation strongly differs from the \n idea that -ecs is the rate - limiting step [ 3335 ] . for many researchers , the concept of -ecs being the key controlling step has seemed to be \n correct because ( a ) -ecs receives information from the final part \n of the pathway , as it is potently inhibited by gsh , the pathway end - product ; \n and ( b ) -ecs is localized in the first part of the \n pathway ( figure 2 ) . in addition", "however , in most of the \n studies on the control of gsh synthesis , the gsh demand has not been \n considered .", "the gsh synthesis modeling shows that under a physiological feedback \n inhibition of -ecs by gsh a small increase in demand increases \n flux because the gsh concentration decreases and the -ecs inhibition attenuates .", "in contrast , if the demand remains constant , then \n an increase in -ecs activity or content ( by overexpression ) does \n not increase flux because the gsh inhibition is still there and operates on \n both new and old enzymes .", "the same pattern is also observed when hk is \n overexpressed to increase \n glycolytic flux since it is still inhibited by g6p ( see section 3 ) . on the other hand ,", "-ecs indeed exerts significant concentration control \n on gsh , which means that a -ecs increase results in higher gsh \n concentration ( table 4 ) .", "this last \n observation demonstrates that an enzyme controlling a metabolite concentration does \n not necessarily control the flux .", "cd exposure promotes a high gsh demand because significant oxidative stress surges , \n thus causing oxidation of gsh through gsh peroxidases , and because gsh and pcs \n are used for sequestering the toxic metal ion ; hence , a higher gsh consuming \n rate sets up . under this condition , \n modeling predicted that control was almost equally shared between the supply \n and demand blocks , but particularly between -ecs and pcs ( see figure 2 ) .", "modeling was also able to explain why pcs overexpression \n can have toxic effects on the cell .", "an increase in the gsh demand ( pcs \n overexpression ) under high - demand conditions ( cd stress ) leads to \n gsh depletion that severely compromises other processes such as the oxidative \n stress control and xenobiotic detoxification .", "the \n conclusions drawn by this model led us to propose that , to significantly increase \n the cd resistance and accumulation , -ecs and pcs should be simultaneously overexpressed \n ( table 4 ; figure 9 ) .", "this particular \n manipulation promotes an increase in the rate of gsh and pcs synthesis \n ( determined by the high - to - low transition of their flux control coefficients ) \n and in the gsh and pcs concentrations \n ( determined by their high concentration control coefficients ) .", "the model predicts that a 2-fold increase in \n the simultaneous overexpression of -ecs and pcs brings about a 1.92.4-fold \n increase in flux to gsh ( jgs ) \n and pcs ( jpcs ) and in pcs \n concentration ( figure 9 ) ; a 5-fold overexpression further increases by 4.58.1 \n times the fluxes and pcs concentration .", "this proposed enzyme overexpression \n should not exceed the gs and the complex pc - cd ( or gs - cd - gs ) vacuolar \n transporters ' maximal activities , in order to keep the cell away from a severe \n oxidative stress caused by gsh depletion or -ec accumulation .", "indeed , the concentration of gsh was maintained high and constant although \n -ec accumulated with the simultaneous overexpression \n ( figure 9 ) .", "furthermore , this enzyme \n manipulation should avoid the increase of the pc - cd and gs - cd - gs complexes in \n cytosol to toxic levels .", "in other words , \n excessive enzyme overexpression should be avoided , unless this is accompanied \n by compensating overexpression of consuming enzymes ( gs for -ecs overexpression and pcs vacuolar transporters for \n that of pcs ) . in yeasts and plants ,", "cd is ultimately inactivated by the additional interaction with s and \n the subsequent formation of stable high molecular weight complexes with pcs , cd , \n s , and gsh [ 138 , 139 ] . in \n", "parallel to the -ecs and pcs overexpression , moderate repression \n of gsh - s - transferases , which compete for the available gsh ( figure 2 ) , may also \n promote an increase in gsh concentration and pcs formation flux .", "in contrast , gene overexpression induces large \n changes in activity ; hence , further theoretical background has been developed for \n predicting the effect on flux and metabolite concentrations induced by large \n enzyme changes .", "such a theoretical background was initially developed by small \n and kacser , who depicted ( 12 ) based on the flux control coefficients to \n predict the effect promoted by large changes in enzyme activity:(12)fejmrj  =  11    i = jm(cvi0jo    ( ri    1)/ri ) , in which f is the amplification factor ( the flux increase ) , and r represents how many times the enzyme is overexpressed .", "to predict the flux changes , promoted by identical \n overexpression of two enzymes ( same r value ) with different cvi , \n the equation is \n ( 13)fejmrj  =  11    ( cij  +  cjj )    ( ( r    1)/r ) . \n \n", "figure 10 shows the effect on flux when one or more enzymes with different cvi are changed by the same r factor .", "if the sum of cvi \n of one or more enzymes is less than 0.25 , the \n impact on flux is discrete when the expression increases 5 folds ( which is the \n most common variation in the overexpression experiments analyzed in section \n 2 ) .", "but for a 3-fold overexpression of \n a group of enzymes , for which their sum of cvi is more than 0.5 , then a significant flux \n change is achieved .", "if the sum of cvi is 1 , the flux varies in a linear proportion \n with the degree of overexpression .", "it \n has to be remarked , however , that the predicted change in flux ( figure 10 ) will \n be valid until certain degree , the limits of which being determined by the \n other pathway enzymes that should stay as noncontrolling steps . \n", "figure 10 also shows the effect on flux of decreasing an enzyme activity ( third \n quadrant ) .", "this segment plot is useful when inhibition of pathway flux is being \n pursued for therapeutic purposes or for understanding the molecular basis of the \n genetic dominance and recessivity . like \n in the enzyme overexpression experiment , only a significant effect on flux is \n achieved when the enzymes with high cvi values are inhibited . for an enzyme or group of enzymes with cvi \n of 0.25 , greater than 80%", "inhibition has to be \n attained to decrease 50% the pathway flux . in this context", ", it seems feasible \n to explain why knockdown of enzymes involved in tsh2 synthesis has \n to be almost total to detect an effect on tsh2 content or to alter \n functional or pathogenic properties of the parasites ( section 4.3 ) .", "the knockdown or knockout experiments in \n trypanosomatids suggest that -ecs , trys , and tryr most probably have low flux control \n and concentration - control coefficients since their contents or activities have \n to be reduced > 80% of the normal levels to reach changes in intermediary \n levels or in oxidative stress handling .", "contrary \n to the several unsuccessful overexpression experiments carried out to increase \n the flux or metabolites of a metabolic pathway , modeling may allow for a more \n focused and appropriate design of experimental strategies of genetic \n engineering to increase flux or a given metabolite , and for selecting drug \n targets to decrease flux or metabolite concentration . for these predictions , modeling considers \n that overexpression of a controlling enzyme or transporter may promote flux or \n metabolite control redistributions . \n ", "thus , a low - control step may become a \n controlling point when overexpressing another step and , in consequence , the \n prediction shown in figure 10 based on ( 11 ) and ( 12 ) may be inaccurate . by considering the whole pathway components , \n", "modeling is also a powerful tool for predicting the effects on flux and \n metabolite concentration of varying an enzyme activity ( by overexpression or \n drug inhibition ) .", "model predictions to inhibit a pathway fluxkinetic modeling has been used to identify the flux controlling steps in trypanosoma \n brucei glycolysis for drug targeting purposes .", "interestingly , modeling has predicted \n controlling steps for the parasite pathway different from those described for \n glycolysis in human host cells [ 125 , 126 ] . \n", "the parasite lacks functional \n mitochondria and has neither krebs cycle nor oxphos enzyme activities .", "therefore , \n substrate level phosphorylation by glycolysis is the only way to generate atp \n for cellular work .", "an important \n difference in amebal glycolysis in comparison to glycolysis in human cells is \n that it contains the pyrophosphate ( ppi)-dependent enzymes phosphofructokinase \n ( ppi - pfk ) and pyruvate phosphate dikinase ( ppdk ) , which replace the highly \n modulated atp - pfk and pyk present in human cells .", "moreover , both have been \n proposed as drug targets by using ppi analogues ( bisphosphonates ) .we recently described the \n construction of a kinetic model of e. histolytica glycolysis to \n determine the control distribution of this energetically important pathway in \n the parasite .", "the model was \n constructed using the gepasi software and was based on the kinetic parameters \n determined in the purified recombinant enzymes , as well as the enzyme \n activities , fluxes , and metabolite concentrations found in the parasite .", "the results of the \n metabolic control analysis \n indicated that hk and pgam are the main flux control steps of the pathway ( 73 \n and 65% , resp . ) and perhaps glut . in \n contrast", ", the ppi - pfk and ppdk displayed low flux control ( 13 and 0.1% , resp . ) \n because they have overcapacity over the glycolytic flux .", "the amebal model allowed evaluating the \n effect on flux of  inhibiting  the pathway enzymes .", "the model predicted that in order to diminish \n by 50% the glycolytic flux ( and the atp concentration ; data not shown ) , hk and \n pgam should be inhibited by 24 and 55% , respectively , or both enzymes by 18% \n ( figure 11 ) .", "in contrast , to attain the \n same reduction in flux by inhibiting ppi - pfk and ppdk , they should be decreased \n > 70% ( figure 11 ) .", "therefore , the \n kinetic model results indicate that hk can be an appropriate drug target \n because its specific inhibition can compromise the energy levels in the \n parasite .", "they also indicate that although \n ppi - pfk and ppdk remain as promising drug targets because of their divergence \n from the human glycolytic enzymes , highly potent and very specific inhibitors \n should be designed for these enzymes in order to affect the parasite 's energy \n metabolism .", "kinetic modeling has been used to identify the flux controlling steps in trypanosoma \n brucei glycolysis for drug targeting purposes .", "interestingly , modeling has predicted \n controlling steps for the parasite pathway different from those described for \n glycolysis in human host cells [ 125 , 126 ] . \n", "the parasite lacks functional \n mitochondria and has neither krebs cycle nor oxphos enzyme activities .", "therefore , \n substrate level phosphorylation by glycolysis is the only way to generate atp \n for cellular work .", "an important \n difference in amebal glycolysis in comparison to glycolysis in human cells is \n that it contains the pyrophosphate ( ppi)-dependent enzymes phosphofructokinase \n ( ppi - pfk ) and pyruvate phosphate dikinase ( ppdk ) , which replace the highly \n modulated atp - pfk and pyk present in human cells .", "moreover , both have been \n proposed as drug targets by using ppi analogues ( bisphosphonates ) .", "we recently described the \n construction of a kinetic model of e. histolytica glycolysis to \n determine the control distribution of this energetically important pathway in \n the parasite .", "the model was \n constructed using the gepasi software and was based on the kinetic parameters \n determined in the purified recombinant enzymes , as well as the enzyme \n activities , fluxes , and metabolite concentrations found in the parasite .", "the results of the \n metabolic control analysis \n indicated that hk and pgam are the main flux control steps of the pathway ( 73 \n and 65% , resp . ) and perhaps glut . in \n contrast", ", the ppi - pfk and ppdk displayed low flux control ( 13 and 0.1% , resp . ) \n because they have overcapacity over the glycolytic flux .", "the amebal model allowed evaluating the \n effect on flux of  inhibiting  the pathway enzymes .", "the model predicted that in order to diminish \n by 50% the glycolytic flux ( and the atp concentration ; data not shown ) , hk and \n pgam should be inhibited by 24 and 55% , respectively , or both enzymes by 18% \n ( figure 11 ) .", "in contrast , to attain the \n same reduction in flux by inhibiting ppi - pfk and ppdk , they should be decreased \n > 70% ( figure 11 ) .", "therefore , the \n kinetic model results indicate that hk can be an appropriate drug target \n because its specific inhibition can compromise the energy levels in the \n parasite .", "they also indicate that although \n ppi - pfk and ppdk remain as promising drug targets because of their divergence \n from the human glycolytic enzymes , highly potent and very specific inhibitors \n should be designed for these enzymes in order to affect the parasite 's energy \n metabolism .", "another experimental approach \n for determining the enzyme control coefficients is the in vitro reconstitution \n of segments of metabolic pathways .", "it is \n recalled that for determining the flux control coefficient exerted by a given \n step on a metabolic pathway the enzyme activity has to be varied , without \n altering the other components in the system , and the flux variations are to be \n measured ( figure 4 ) .", "such an experiment can be readily made if a pathway is \n reconstituted with purified enzymes .", "some advantages of this approach are that the \n pathway structure is known , in which the components concentration may be \n manipulated and analyzed separately , and the enzyme effectors can be assayed .", "as \n the system composition is strictly controlled , the results may be highly \n reproducible .", "the main disadvantage is \n that the enzyme concentrations in the assays are diluted and thus the enzyme \n interactions are not favored . if this interaction \n is important for activity , the in vitro reconstitution may limit the \n extrapolation to the metabolic pathway inside the cell .", "there are not many studies \n describing this type of experiments , most probably due to the fact that for \n applying mca the pathway must be working under steady - state conditions . in a reconstituted system", ", only a quasi steady \n state may be reached because there is net substrate , and cofactors consumption , \n as well as product accumulation , since it is difficult to attain a constant \n substrate supply and release of products .", "one of the first experimental \n reports on control coefficient determination in a reconstituted system was \n carried out for the upper glycolytic segment with the commercially available \n rabbit muscle hk , hpi , pfk-1 , aldo , and tpi .", "each enzyme was separately titrated and the \n flux variation to glycerol-3-phosphate ( by coupling the reconstituted system to \n an excess of -gpdh ) was measured in the presence of ck to maintain \n the atp concentration constant .", "the results showed that pfk-1 and hk exerted the \n main flux control ( 65% and 20% , resp . ) , whereas the remaining 15% resided in \n the other enzymes .", "these authors observed that the addition of f1,6bp , a pfk-1 \n activator slightly diminished the flux control exerted by pfk-1 and increases \n that of hk .", "the lower glycolytic segment \n has also been reconstituted with commercial enzymes for determining the flux \n control coefficients .", "the results \n showed that flux was mainly controlled by pyk ( 60100% ) , although under some \n conditions control was shared with pgam ; eno did not contribute to the flux \n control .", "another important limitation \n of the reconstitution experiments is that the commercial availability of the \n purified enzymes from the same organism is restricted or inexistent .", "however , by using the information from the \n genome sequence projects and the recombinant dna technology , it is now possible \n to access all the enzyme genes from a metabolic pathway in the same organism , thus \n facilitating their cloning , overexpression , and purification . with this strategy ,", "we cloned , overexpressed , \n and purified the 10 glycolytic enzymes of entamoeba histolytica   for \n studying the flux control distribution in this organism by using kinetic \n modeling   and pathway reconstitution .", "the reconstitution \n experiments of the lower amebal glycolytic segment , under near physiological \n conditions of ph , temperature , and enzyme activity ( figure 12 ) showed that pgam \n and , to a lesser extent , ppdk exert the main flux control ( these amebal enzymes \n are genetically and kinetically different from their human counterparts ) with \n eno exhibiting negligible control . in turn , reconstitution of the upper amebal \n glycolytic segment has revealed that hk and , to a much lesser extent hpi , \n ppi - pfk , and ald , exerted the main flux control , with tpi having negligible \n control .", "these results strongly \n correlate with the enzyme catalytic efficiencies previously reported , in \n which hk is highly sensitive to amp inhibition , ald , and pgam have the lowest \n catalytic efficiencies among the glycolytic enzymes , leading to high flux control \n coefficients and thus becoming suitable candidates for therapeutic \n intervention .", "the reconstitution results \n also agree with the pathway modeling predictions previously analyzed ( section \n 5.4 ) , in which hk and pgam are two of the main controlling steps .", "the in vitro reconstitution \n experiments are also useful for studying the effect on control redistribution of \n an enzyme modulation that is particularly difficult to manage in vivo ; the main controlling steps \n identified with the reconstitution experiments should be further analyzed with \n other experimental strategies such as elasticity analysis in the in vivo \n systems .", "this experimental approach for determining the control coefficients could be part of \n the genetic approach analyzed in section 5.1 , but it was separated due to its \n recent methodological development and because it actually belongs to the \n molecular genetics rather than to the mendelian genetics .", "this approach is based on the in vivo modulation of the enzyme levels using the rna \n antisense technology .", "there are at least \n three strategies to \n inhibit gene expression : ( a ) the use of single stranded antisense \n oligonucleotides , which form a double stranded rna that might be degraded by rnase \n h ; ( b ) target rna degradation with catalytically active oligonucleotides , known \n as ribozymes that bind to their specific rna ; and ( c ) rna degradation using sirnas \n ( 2123 nucleotides ) .", "the rna antisense technology was applied for control coefficient determination of the \n ribulose - bisphosphate - carboxylase ( rubisco ) that fixes co2 in the \n plant calvin cycle .", "this enzyme considered \n the rate - limiting step of the calvin cycle and of the whole photosynthetic \n process , despite its high concentration ( 4 mm ) in the chloroplasts stroma that compensates \n its low catalytic efficiency .", "attempts to make rubisco a nonlimiting step , either by modifying its catalytic efficiency \n or by overexpressing it , have been unsuccessful .", "the plants were transformed with dna antisense against the \n mrna of the enzyme 's small subunit , thus promoting its degradation . for calvin cycle enzymes , the pleiotropic \n effects were minimal .", "the results showed \n that rubisco may indeed be the photosynthesis limiting step with a crubisco = 0.690.83 when plants are exposed to high \n illumination ( 1050 mol quanta ms ) , high \n humidity ( 85% ) , and low co2 concentrations ( 25 pa ) .", "however , this flux control decreases to \n 0.050.12 under moderate illumination or high co2 levels .", "unfortunately , the authors did not determine \n the control coefficients of the other pathway enzymes or the branches fluxes which \n may be significant .", "as described in section 5.4 , the results of the t. brucei glycolysis \n modeling indicated that glut was the main flux control step ( cglut  50% ) , [ 125 , 126 ] .", "this model predicted a large overcapacity for \n hk , pfk-1 , aldo , gapdh , pgam , eno , and pyk over the glycolytic flux leading to \n low flux control coefficients [ 125 , 126 ] . to validate the modeling results , the concentrations of hk , pfk-1 , pgam , \n eno , and pyk", "these knockdown expression experiments showed \n overcapacity of hk and pyk over the flux , although at lower levels than predicted \n by the model . a good correlation for \n pgam and eno", "was obtained between model predictions and experimental results . however , a large difference ( 9 folds ) was \n obtained for pfk-1 .", "this discrepancy is \n perhaps related to pleiotropic effects of pfk-1 downregulation , as these mutants \n also displayed diminution in the activities of other enzymes ( hk , eno , and pyk ) . \n ", "the combination of these two approaches , \n in silico modeling and in vivo experimentation , is complementary : on one \n hand , modeling identifies the enzymes ( out of 19 that contain the model ) that \n display the highest flux control coefficients , whereas in vivo experimentation validates the \n accuracy of the model to establish predictions about the pathway 's behavior .", "the knockdown experiments described above usually yield only two experimental points \n of the plot shown in figure 4 : the \n wild - type and the knockdown strain protein levels or enzyme activities .", "thus , with \n such an approach high levels of inhibition ( > 80% ) are mostly analyzed , \n whereas intermediate levels of downregulation ( if obtained ) are generally \n overlooked .", "therefore , knockdown \n experiments are not very useful to obtain the complete set of experimental data \n ( above and below the wild - type levels of enzyme activity with the corresponding \n flux ) for determining reliable control coefficients . a strategy to determine flux \n control coefficients from several protein levels", "has been developed by using \n adenovirus - mediated glucose-6-phosphatase ( g6pase ) overexpression under the \n control of the cytomegalovirus promoter in rat hepatocytes .", "a 2-fold g6pase overexpression did not alter cg6pase or cgk ( gk , glucokinase ) .", "however , if g6pase is overexpressed by 4 folds , then cgk \n diminished from 2.8 to 1.8 and there was a 35% lowering in glycogen synthesis .", "however , this approach allows titration of flux only above the basal \n enzyme activities found in the cell , but not below .", "these experimental inconveniences have been circumvented by using inducible gene \n expression systems based in the lac , \n lambda , nisin , gal , tetracycline , and other inducible promoters , in bacteria \n and yeast [ 151 , 152 ] . however , a problem \n frequently encountered with inducible promoters is that a steady - state of \n protein expression is difficult to attain [ 151 , 152 ] .  ", "recently , \n jensen and hammer described the design of synthetic promoter libraries ( spl ) , in \n particular for l. lactis metabolic \n optimization .", "these promoters \n maintain constant the array of the known consensus sequences for l. lactis gene transcription ( 10 and \n 35 boxes ) , while the nucleotide sequence between these boxes ( a spacer \n sequence of 17  1 bp ) is randomized , thus producing a set of promoters with \n different transcriptional strength .", "these promoter libraries allow the transcription and protein expression \n several folds above and below the wild - type levels of enzyme activity , \n thus enhancing the usefulness of this approach for mca studies .", "the \n control distribution of glycolysis in e. \n coli and l. lactis , as discussed \n in section 3.2 [ 17 , 24 , 27 , 151 ] , has been determined by using the spl \n technology . spl for yeast , mammalian and \n plant cells are also under development [ 151 , 152 ] .", "certainly , the advances in genetic \n engineering in combination with mca allow better experimental designs for \n metabolic optimization of micro - organisms of biotechnological interest .", "concluding remarks \n the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect .", "as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step .", "mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control .", "a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments ,", "it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway .", "mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) .", "for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them .", "if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes.if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients .", "these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks .", "it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis .", "however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest . \n \n", "the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect . as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step .", "mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control .", "a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments ,", "it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway .", "mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) .", "for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them .", "if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes.if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients .", "these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks .", "it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis .", "however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest . \n ", "the frequently recurred idea of manipulating the key \n enzyme or rate - limiting step ( a concept based on a qualitative and rather \n intuitive background ) to change metabolism is incorrect . as mca has demonstrated , flux control is shared \n by multiple steps and it is not usually localized in only one step .", "mca determines quantitatively the control that \n a given enzyme exerts on the flux and on intermediary concentration and helps \n to explain why an enzyme does or does not exert control .  ", "a metabolic pathway is manipulated to change the rate \n of the end - product formation ( i.e. , \n the flux ) or the concentration of a relevant intermediary . as it is demonstrated in many unsuccessful \n experiments ,", "it is not enough to overexpress one enzyme ( the rate - limiting step ) \n or many arbitrarily selected sites of the pathway .", "mca proposes an initial experimental analysis \n that determines the structural control of the pathway and identifies the sites \n ( enzymes and transporters ) with higher control coefficients values ( i.e. , targets to be manipulated ) .", "for example , if there is a system composed of \n six enzymes and three of them have flux control coefficients with values of 0.2 \n or higher and the other three with values of 0.1 or lower , the three enzymes \n with high control coefficients must be overexpressed ( if a flux increase is \n desired ) or repressed ( if flux inhibition is the objective ) and not only one of \n them .", "if one of the selected enzymes is \n strongly inhibited by its product or has allosteric inhibition , the overexpression \n of this enzyme might not be enough to increase the flux , as it may also be \n necessary to moderately vary the product and allosteric modulator consuming \n enzymes .", "if the aim of the researcher is a metabolite \n concentration increase , which is not the end product of the pathway , mca \n suggests the overexpression of those enzymes or transporters in the supply \n block with the highest control coefficients and/or the repression of those \n enzymes in the demand block with the highest control coefficients .", "these manipulations may become complicated if \n the metabolite of interest has allosteric interactions with enzymes and \n transporters ( inhibition and activation ) of both the supply and demand blocks .", "it is recalled that ethanol production in \n yeast and lactate and acetate production in lactobacteria do not increase by \n overexpressing pfk-1 , an allosteric enzyme and the presumed rate - limiting step \n of glycolysis .", "however , the analysis of these results reveals \n that the f1,6bp concentration is indeed increased many times over the control \n level . another strategy for eliminating \n the feedback inhibition might be the introduction of mutations on the enzymes \n that are closer to the metabolite of interest ."]]}
{"article_id": "PMC4806652", "article_text": ["network - driven spindle - like oscillations are a functional hallmark of the developing cerebral cortex . during late prenatal and early postnatal stages of development ,", "spontaneous spindle - like oscillations have been identified as physiological activity patterns in various neocortical areas of different mammalian species [ 14 ] . in humans ,", "so - called  delta brushes  can be observed in eeg recordings from preterm babies already at gestational week 28 , that is ~12 weeks before normal birth of a full - term neonate ( for review see ) . in ac - filtered eeg recordings ,", "delta brushes are brief rhythmic delta waves ( 0.31.5  hz ) of 50300  v amplitude with a superimposed burst of fast rhythm ( > 8  hz , the  brush  ) .", "it has been suggested that delta brushes in human preterm infants correlate with so - called spindle bursts recorded in rodents during the early postnatal period . from a developmental point of view , these early spontaneous activity patterns in developing rodent and human cerebral cortex are probably of similar or even identical origin .", "rats and mice are altricial - born in a far less mature condition than humans . in rodents", "the degree of neocortical development at the day of birth ( postnatal day [ p ] 0 ) can be compared to the stage of human cortex between gestational weeks 28 and 32   and the cerebral cortex of a p12p14 rat is comparable to that of the full - term newborn human baby [ 6 , 8 ] . in preterm infants born between gestational weeks 28 and 32 , milh et al .   and colonnese et al", ".   recorded eeg signals containing spontaneous and stimulus - evoked delta brushes with oscillatory activity in the frequency range of 8 to 25  hz , suggesting that spontaneous delta brushes may represent a physiological neocortical activity pattern of the human fetus in utero . in the cerebral cortex of rodents , spindle bursts ( beside short gamma oscillations )", "constitute the majority of spontaneous activity during the first postnatal week ( figure 1 ) .", "these spindle bursts resemble in their appearance spindles recorded in the adult brain during sleep .", "these sleep spindles are one of the hallmarks of human stage 2 sleep  for comprehensive overviews the reader is referred to recent reviews by lthi   and by mccormick et al . .", "however , in humans sleep spindles appear 4 to 9 weeks after birth , which is much later than the disappearance of delta brushes around the end of the first postnatal week , thus excluding the hypothesis that delta brushes or spindle bursts gradually develop into sleep spindles .", "in addition , the frequency profile of spindle bursts and delta brushes displays a rather broad frequency distribution up to 25  hz , whereas sleep spindles present oscillations in a narrower band of ~1015  hz .", "the present review focuses on spindle burst activity in the cerebral cortex of the developing rat during the first postnatal week and summarizes our current understanding ( i ) on the functional properties of spindle bursts , ( ii ) the mechanisms underlying their generation , ( iii ) the synchronous patterns and cerebral networks associated with spindle bursts , and ( iv ) the physiological and pathophysiological role of spindle bursts during early cortical development .", "two distinct activity patterns can be observed in the neonatal rat cerebral cortex in vivo : gamma oscillations and spindles bursts ( figures 1 and 2 ) [ 1419 ] .", "gamma oscillations have a duration of 100 to 300  ms , a frequency of 30 to 40  hz and appear spontaneously every 10 to 30  s ( figures 1 and 2(b ) ) . the properties and functional role of gamma oscillations as well as the mechanisms underlying their generation", "spindle bursts are characterized by their spindle - like oscillatory appearance , have a duration of 0.5 to 3  s and a frequency in the range of 8 to 30  hz , and occur spontaneously every ~10  s ( figures 1 and 2(a ) ) . as recognized in full - band direct - current ( dc ) coupled recordings ,", "spindle bursts are nested in slow ( delta ) waves , which in infant rats , and preterm human babies [ 22 , 23 ] have been termed  spontaneous activity transients  ( sats ) .", "spindle bursts have been described in primary somatosensory cortex ( s1 ) including barrel cortex [ 14 , 1619 ] , in primary visual cortex ( v1 ) [ 10 , 15 ] , in primary motor cortex ( m1 ) , and in prefrontal cortex   of anesthetized and awake rats during the first postnatal week . in s1 , spontaneous and stimulus - evoked", "spindle bursts can be observed as early as p0 [ 17 , 18 ] . in rats ,", "the incidence of spindle bursts declines during the second postnatal week and sporadic spindle bursts are obscured by the ongoing neocortical activity .", "likewise in humans , delta brushes are replaced by more continuous eeg activity at birth of a full - term infant .", "this gradual developmental shift from a highly synchronized state of spontaneous activity to a desynchronized state seems to be a fundamental network property of the cerebral cortex during early neonatal stages .", "spindle bursts synchronize the activity of a local neuronal network of 200 to 400  m in diameter , which resembles the dimension of a whisker - related neocortical column in the immature barrel cortex ( figure 3 ) [ 18 , 25 ] . using a combination of voltage - sensitive dye imaging and high - density multielectrode recordings in p0-p1 rat barrel cortex in vivo , yang et al .", "could demonstrate that this early spontaneous activity constitutes the later emergence of the whisker - related barrel field map .", "these data indicate that spontaneous activity patterns at this early age form  functional precolumns ,  supporting the concept of the existence of  ontogenetic columns  in the radial unit hypothesis .", "further support for this hypothesis comes from in vivo two - photon calcium imaging in the barrel cortex of both anesthetized and nonanesthetized newborn mice , demonstrating highly synchronous spontaneous burst activity , reminiscent of spindle bursts , in local networks of 100 to 200  m in diameter .", "these data strongly indicate that early spindle bursts , and probably also gamma oscillations ( for review ) , synchronize early neocortical networks into functional columns at a developmental stage when the upper layers 2/3 have not even been formed ( i.e. , in rats at p0 ) . at this developmental stage", ", thalamocortical afferents have not reached layer 4 and instead transiently innervate the subplate ( for review [ 28 , 29 ] ) .", "thalamic afferents form transient glutamatergic synapses with surprisingly mature properties including ampa and nmda receptors [ 3032 ] . in vitro studies in acute brain slice preparations and in intact whole cortical hemisphere preparations   have demonstrated that oscillatory network activity in the frequency range of spindle bursts depend on an intact subplate [ 34 , 35 ] .", "selective removal of the subplate in s1 in vivo causes a significant decline in the occurrence of spontaneous spindle bursts and disturbances in the development of the cortical architecture in the barrel cortex .", "these data further support the hypothesis that spindle bursts in developing cerebral cortex fulfill an important role in the maturation of the neocortical architecture . in the next section we will discuss the network and molecular mechanisms underlying the generation of spindle bursts in neonatal cerebral cortex .", "the rodent cerebral cortex develops rapidly during late prenatal and early postnatal stages and at least four different activity patterns may occur sequentially between birth and the end of the first postnatal week ( for review ) .", "furthermore , the cortex shows a mediolateral and anterior - posterior gradient in development and within the same neocortical area neurons in upper layers 2/3 are ~2 days younger compared to lower cortical layers .", "many inconsistencies in the literature on the properties of spontaneous activity patterns in newborn rodents and their underlying mechanisms can be explained by the fact that these important developmental differences are often ignored .", "in addition , 2 days in early rodent cortical development make a large difference , so that the neocortex of a p0 rat ( without layer 2/3 ) can not be compared to that of a p2 rat ( with almost complete lamination ) . already in newborn ( p0-p1 ) rat barrel cortex in vivo , mechanical stimulation of a single whisker", "elicits in field potential recordings and to some extent detectable also in voltage - sensitive dye imaging ( vsdi ) a sequence of an early gamma oscillation followed by a spindle burst ( figure 3(b ) ) . at this age ,", "the thalamocortical activity reaches the developing cortical network largely via the subplate [ 17 , 31 , 32 ] and is amplified by an intrinsic gap - junction coupled network within the subplate and cortical plate [ 28 , 34 ] .", "spontaneous and evoked delta brushes can be observed in premature human neonates of 2832 weeks postconceptional age , a developmental stage when the human cerebral cortex resembles that of a newborn rat .", "impressive examples of large delta brushes are provided in the supplementary eeg videos of milh et al . , demonstrating that a single touch elicits a large oscillatory response in the somatosensory evoked potential ( sep ) recorded above the contralateral parietal cortex . in mature human cortex", ", seps with smaller amplitudes and shorter durations can be only obtained after averaging of at least 100 epochs .", "thus , in both species , rats and humans , at a comparable stage of cortical development mechanical stimulation of the sensory periphery elicits in s1 spindle bursts and delta brushes , respectively .", "simultaneous multielectrode recordings in the barrel cortex and in the ventral posteromedial nucleus ( vpm ) of the somatosensory thalamus of p0-p1 rats in vivo have demonstrated that the majority of spontaneous cortical spindle bursts and also gamma oscillations are not generated within s1 , but rather in subcortical structures or outside of s1 ( figures 4(a ) and 4(b ) ) . at this age a local , functionally defined lesion in the vpm blocks the whisker stimulation - induced cortical responses and also profoundly reduces the spontaneously occurring cortical burst activity , further demonstrating that the majority of the spontaneous spindle bursts in p0-p1 rat barrel cortex are generated in subcortical structures of the whisker - to - barrel cortex pathway ( for further information see ) . silencing the sensory periphery by injection of lidocaine into the whisker pad causes a significant reduction in the occurrence of spontaneous spindle bursts and gamma oscillations by ~50% ( figure 4(c ) ) , indicating that during this developmental period at least half of the spontaneous burst activity in s1 is related to activity in the sensory periphery .", "a similar peripheral generation of spontaneous burst activity may occur in human preterms and fetuses between gestational weeks 28 and 32 [ 9 , 22 ] .", "similar to the spindle bursts in s1 , spontaneous spindle bursts in v1 are also largely generated in the sensory periphery .", "( for review ) , provide the primary drive for spindle bursts in newborn rat v1 , as demonstrated by simultaneous recordings from the retina and v1 .", "intraocular injection of forskolin , which augmented retinal waves , increased the occurrence of v1 spindle bursts , and removal of the retina reduced the spindle bursts frequency .", "as in the somatosensory system , spindle bursts in v1 can be also evoked by stimulation of the sensory periphery .", "however , since rod- and cone - mediated visual signaling is not functional in rats during the first postnatal week , spindle bursts can not be evoked by light flashes before p8 . at that age , the neocortical response in v1 consists of an early visual evoked response followed by an evoked spindle burst .", "similar responses could be observed in v1 of preterm infants once photoreceptor mediated light responses occur in the retina   ( for review ) .", "whereas a large amount of experimental data has shown that retinal waves provide the main trigger for the cortical v1 spindles bursts , it is not completely understood which pacemaker drives the spontaneous activity in s1 and how spontaneous activity is generated in the somatosensory periphery . in p3p6 rats spontaneous whisker movements occur during active sleep and are correlated with activation of whisker - related cortical columns in the barrel cortex . in newborn rats", "the proprioceptive feedback from self - generated myoclonic movements trigger spindle bursts in s1 ( for review ) .", "spontaneous limb movements of the human fetus during the third trimester of gestation , or those of the preterm infant associated with delta brushes in s1 , are similar to these twitching movements of the neonatal rat and can be also triggered by sensory feedback .", "kreider and blumberg have demonstrated in 1-week - old rats that the mesopontine region plays a central role in the generation of myoclonic twitching .", "have shown in newborn rats that spatially confined spindle bursts in s1 are triggered in a somatotopic manner by sensory feedback signals from spontaneous muscle twitches .", "these spontaneous movements are generated by neuronal networks in the spinal cord , but spindle bursts persisted at a reduced frequency after sensory deafferentation ( spinal cord transection ) in s1 , indicating that spindle burst activity can be also generated in neocortical or thalamocortical circuits during this early period of development .", "this assumption is also supported by the observation that silencing of the sensory periphery causes only a ~50% reduction in the occurrence of spontaneous spindle bursts ( figure 4(c ) ) . however , since it can not be excluded that the remaining portion of spindle bursts actually conveys activity from adjacent or distant sensory areas ( transmitted via inter- and intrahemispheric connections , see next chapter ) , the outcome of this experiment may underestimate the contribution of the sensory periphery .", "simultaneous monitoring of forepaw movements , vsdi , and extracellular multielectrode recordings in s1 and m1 of p3p5 rats under light urethane anesthesia have demonstrated that tactile forepaw stimulation triggers spindle bursts in s1 , followed by gamma and spindle bursts in m1 ( figure 5 ) .", "focal electrical stimulation of corticospinal tract neurons in layer 5 of m1 mimicking physiologically relevant 40  hz gamma or 10  hz spindle burst activity reliably elicited forepaw movements , indicating that m1 cortical spindle bursts are capable of triggering muscle twitches at this age .", "however , only 23.7% of the spontaneous bursts in m1 triggered forepaw movements and were followed by spindle bursts in s1 ( figures 5(b)(a ) and 5(c ) ) , indicating that only a fraction of m1 activity transients triggers motor responses directly . in 40.7% of the cases , spontaneous movements preceded the burst activity in m1 and s1 ( figures 5(b)(b ) and 5(c ) ) , suggesting that this activity may arise from subcortical regions in the brainstem or spinal cord .", "the remaining 35.6% of the m1 bursts were unrelated to any movements ( figure 5(c ) ) .", "the finding that 23.7% of the movements were triggered by m1 bursts as observed by an et al .  ", "is in contrast to previous observations , which demonstrated that dissection of neocortical inputs fails to suppress muscle twitches in rat pups . in summary , these data indicate that neocortical spindle bursts in newborn rodents ( and delta brushes in human fetus during the third trimester or in preterms ) are generated by central pattern generator ( cpg ) circuits in spinal cord , brainstem , and motor cortex ( for cpg circuits in mature brain see [ 4244 ] ) .", "neuropharmacological studies provided insights into the molecular mechanisms underlying the generation and modulation of neocortical spindle bursts . in vivo   and in vitro data   suggest that gabaergic synapses are not crucial for the generation of spindle bursts or spindle burst - like activity , respectively , but are essential for their spatial confinement to a cortical ( pre- ) column .", "in contrast , spindle bursts depend on intact glutamatergic synapses including alpha - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) and n - methyl - d - aspartate ( nmda ) receptors [ 34 , 45 ] .", "carbachol - induced spindle - like oscillations in p0p3 mouse neocortex in vitro   and spindle bursts in p0p2 rat in vivo   are blocked or significantly reduced by different gap junction blockers , indicating that electrical synapses are critically involved in the generation of spindle bursts at this neonatal period", ". however , minlebaev et al .   reported for p1p3 rats a significant increase in the occurrence of spontaneous spindle bursts in s1 following application of the gap - junction blocker mefloquine .", "single - cell recordings revealed additional insights into the mechanisms underlying the generation of spindle bursts .", "spindle bursts in v1   and s1   are accompanied by a barrage of glutamatergic and gabaergic postsynaptic currents ( pscs ) that are phase - locked to the spindle oscillations ( figure 6 ) . in prefrontal cortex , glutamatergic and gabaergic synaptic inputs to excitatory pyramidal neurons are phase locked to the theta - band component of the spindle , while pscs of inhibitory interneurons are phase locked to the higher beta and gamma frequencies , suggesting that excitatory and inhibitory neurons differentially modulate the distinct components of the spindle bursts .", "further , the application of cnqx eliminates the glutamatergic pscs and completely blocks the occurrence of spindle bursts , indicating a major causal role of ampa - receptor mediated glutamatergic inputs in the generation of spindle bursts .", "additional insights have been obtained by experiments in which subplate cells were selectively ablated in s1 shortly after birth . in these animals", "these in vivo results support previous in vitro studies , in which spindle bursts elicited by cholinergic stimulation were suppressed after removal of the subplate ( see below ) [ 34 , 35 ] . taken together", ", these data suggest that thalamocortical inputs  relayed and amplified by the subplate [ 28 , 29]play an important role in the generation of spindle bursts ( e.g. , [ 17 , 45 ] ) .", "spindle bursts in the cerebral cortex of newborn rats can be elicited [ 34 , 35 ] and modulated by cholinergic mechanisms . in vitro , spindle burst - like oscillations can be reliably induced by activation of muscarinic acetylcholine receptors , predominantly of the m1 and m5 type . in vivo ,", "spindle bursts in v1 are decreased by ~50% following the application of the muscarinic receptor antagonist atropine .", "furthermore , blockade of acetylcholine esterase with physostigmine or direct stimulation of the cholinergic basal forebrain nuclei augmented the occurrence v1 spindle bursts , indicating that the cholinergic system facilitates spindle burst activity in developing cerebral cortex .", "as discussed above , spatially confined spindle bursts in newborn rat cortex synchronize a local neuronal network resembling a neocortical ( pre- ) column ( figure 3 ) . beside these intra - areal synchronization", ", spindle bursts are also synchronized between different cortical regions within one hemisphere ( intrahemispheric ) . as described above , a tight functional correlation in spontaneous and stimulus - evoked spindle burst activity exists between s1 and m1 cortex ( figure 5 ) .", "spindle bursts with similar properties as those in v1 and s1 have been also recorded in vivo in the prefrontal cortex of urethane - anesthetized rats older than p2 .", "the same authors demonstrate that the hippocampus drives this early activity in the prefrontal cortex . beside this intrahemispheric synchronization between different cortical regions , spindle bursts", "also interact between both hemispheres ( interhemispheric ) . in vivo simultaneous recordings of spontaneous activity in homotopic cortical areas in both hemispheres at the same stereotaxic", "coordinates and depth have demonstrated that the amount of interhemispheric synchronization in newborn rats is initially rather low and increases during the first postnatal week .", "this interhemispheric communication of spindle burst activity depends on an intact corpus callosum . in this regard ,", "in unanesthetized newborn rats callosotomy doubled the occurrence of spontaneous spindle bursts , suggesting that the corpus callosum modulates functionally inhibitory interactions between homotopic regions in both hemispheres during the occurrence of spindle burst activity .", "experiments in p2p15 rats demonstrated that this callosotomy - induced disinhibition is a transient feature of early development that disappears abruptly after p6 .", "it is not surprising that intra- and interhemispheric interactions of spontaneous activity in the spindle burst frequency range can be also observed in developing human cerebral cortex at early stages . using eeg and functional magnetic resonance imaging ( fmri ) omidvarnia et al .", "electric resting - state network  that shows functional intra- and interhemispheric interactions in the 815  hz frequency range . in summary , several in vitro and in vivo studies have demonstrated that spindle bursts represent elementary states of intra- and interhemispheric synchronization in the very immature cerebral cortex .", "an increasing amount of experimental and clinical data strongly indicate that spindle bursts play an important role in the physiological development of the cerebral cortex .", "experimental evidence indicates that spindle bursts may be particularly suited to interfere with early neurodevelopmental processes , and thus disturbances in spindle burst activity may cause long - term structural and functional disorders . at early stages of development spontaneous and sensory - evoked activity patterns", "influence a variety of developmental processes , such as neurogenesis , apoptosis , neuronal migration , cellular differentiation , network formation , and myelination   ( for review , see ) .", "it is not completely understood how electrical activity controls these different developmental processes and whether distinct activity patterns , such as spindle bursts , play a specific role . however , for the control of apoptotic cell death of immature neurons in vitro and in vivo the essential role of spontaneous network bursts to provide antiapoptotic signals has been demonstrated [ 52 , 56 , 57 ] . for this activity - dependent control of neuronal survival", "the phosphatidylinositol 3-kinase pathway plays an important role , while the mapk / extracellular signal - regulated kinase or the calcium / calmodulin - dependent protein kinase pathway is not directly involved .", "since one spontaneous spindle burst is associated at the single neuron level with 510 action potentials   and the frequency of spontaneous spindle bursts is ~5 per minute [ 16 , 17 ] , a single neuron discharges with 2550 action potentials per minute . under in vitro conditions this discharge frequency supports neuronal survival of developing neocortical neurons , suggesting that spontaneous spindle bursts in vivo provide an important physiological signal for the control of neuronal survival versus apoptosis in the neonatal cerebral cortex .", "notably , spindle burst and gamma activity provides an ideal physiological stimulus for the activity - dependent release of bdnf , an important antiapoptotic signal .", "balkowiec and katz   demonstrated for neuronal cultures that 3060  min of electrical burst stimulation ( 50 pulses at 2050  hz at intervals of 20  s ) increased extracellular bdnf levels by 20-fold , whereas stimulation patterns at lower frequency ( albeit producing the same number of extracellular electric shocks ) were ineffective ( for review , see ) .", "these data indicate that spontaneous spindle bursts represent a physiological trigger for the release of bdnf , which plays an important role in several aspects of development ( for review , see ) . using in vitro and in vivo models", "it has been recently shown that an experimentally induced inflammation by application of the endotoxin lipopolysaccharide induces rapid ( < 2  h ) alterations in the pattern of spontaneous spindle bursts and gamma oscillations in neonatal rodent cerebral cortex , which subsequently leads to increased apoptotic cell death .", "these inflammatory effects are specifically initiated by the microglia - derived proinflammatory cytokine tumor necrosis factor alpha and to a lesser extent by the chemokine macrophage inflammatory protein 2 .", "thus , inflammation causes a fast dysfunction in the pattern of spontaneous burst activity , which subsequently leads to increased apoptotic cell death , most likely by disturbances in the release of survival factors such as brain - derived neurotrophic factor ( bdnf ) acting on neurotrophin tropomyosin - related kinase b / c receptor .", "furthermore , removal of the subplate massively reduced spindle burst activity and led to a persistent loss of the typical barrel - like whisker representation within layer 4 , indicating that spindle bursts play a role in the development of the neocortical architecture . in summary", ", these experimental data suggest that any disturbances in the spontaneous activity of the developing cerebral cortex ( including spontaneous spindle bursts ) induce acute dysfunctions , which may cause long - term disorders .", "however , it remains to be elucidated whether spindle burst in particular can be causally related to neurodevelopmental disturbances .", "it has been recently shown in extremely preterm infants that the properties of neocortical bursts recorded with eeg and their scaling relationships correlate significantly with later cognitive development .", "these clinical data suggest that analyses of burst shapes obtained in eeg recordings from preterm and full - term newborn babies may have diagnostic use in neonatal intensive care units and predict the clinical outcome .", "while it is generally accepted that early electrical activity shapes the maturation of neocortical circuits , it remains an open question whether the specific properties of spindle burst are required or fulfill a distinct role in development . in this regard , it is possible that the spatiotemporal patterns of spindle bursts translate into a local molecular signal which fulfills an important developmental role . in particular", "as gaba does not seem to be necessary for the generation or maintenance of spindle bursts , and since gaba is essential for neuronal differentiation [ 64 , 65 ] , it is tempting to speculate that spindle bursts control the spatially confined release of gaba in developing local networks .", "another unresolved issue is that both spindle bursts and gamma oscillations can be observed during the same period of early development .", "they can be observed in early postnatal rodent brain from the day of birth (; also see ) and occur spontaneously as well as after sensory stimulation . moreover ,", "both can be observed mainly during the critical periods of the primary sensory areas , although spindle bursts probably persist for slightly longer periods .", "in addition , spindle bursts and early gamma oscillations in newborn cortex are proposed to rely on thalamocortical inputs , in contrast to gamma oscillations in adult cortex which depend on perisomatic gabaergic inhibition . beside their differences in duration , occurrence , and frequency ( see above ) , spindle bursts and gamma oscillations in newborn cortex reveal a number of additional distinctions . in the immature barrel cortex the vast majority ( > 90% ) of spontaneously occurring spindle burst spans several barrel - related columns , whereas the majority ( ~70% ) of spontaneous gamma oscillations", "are restricted to a single or two barrel related columns . in line with these observations , gamma oscillations evoked by tactile stimulation are also closely related to a single functional column , while evoked spindle bursts span over more than one column .", "thus , one reason for the coexistence of spindle bursts and gamma oscillations during early postnatal development might be a distinct role in neocortical maturation .", "gamma bursts may reflect local information processing mostly within a single functional column , thus supporting the maturation of a column related network .", "in contrast , spindle bursts probably reflect larger local network events and may thus serve to promote the connectivity between neighboring columns .", "although spindle bursts as well as gamma oscillations are spatially confined to a small network in one neocortical area , it is unclear to what extent and how this activity connects to other cortical and subcortical regions ( e.g. , see [ 18 , 19 ] ) .", "further , it remains to be studied whether the immature brain shows a spindle burst related  resting state  and how this network state is altered by sensory activation or by pathophysiological events .", "finally , it would be most interesting and important to correlate specific patterns of spontaneous activity ( e.g. , delta brush ) recorded by means of full - band direct - current eeg in preterm und full - term human neonates with the acute functional state and with the further development of the child , as impressively done by vanhatalo and colleagues [ 62 , 67 ] .", "spatially confined spindle bursts and delta brushes represent the most prominent physiological activity patterns in the developing cerebral cortex of newborn rodents and preterm human infants , respectively .", "spontaneous and stimulus - evoked spindle bursts can be observed in various neocortical areas of different mammalian species and play important roles in the early development of cortical networks .", "however , it remains to be studied in more detail how exactly spindle bursts influence the maturation of the cortex and how a potential long - term dysfunction due to disturbances in spindle burst activity can be prevented by early intervention .", "since this important type of brain activity is already present in the human fetus in utero ( either spontaneously occurring or related to sensory inputs from the uterine environment ) , a better understanding of the physiological relevance of spindle burst oscillations is of major clinical relevance ."], "abstract_text": ["<S> spontaneous and sensory evoked spindle bursts represent a functional hallmark of the developing cerebral cortex in vitro and in vivo . </S>", "<S> they have been observed in various neocortical areas of numerous species , including newborn rodents and preterm human infants . </S>", "<S> spindle bursts are generated in complex neocortical - subcortical circuits involving in many cases the participation of motor brain regions . </S>", "<S> together with early gamma oscillations , spindle bursts synchronize the activity of a local neuronal network organized in a cortical column . </S>", "<S> disturbances in spindle burst activity during corticogenesis may contribute to disorders in cortical architecture and in the activity - dependent control of programmed cell death . in this review </S>", "<S> we discuss ( i ) the functional properties of spindle bursts , ( ii ) the mechanisms underlying their generation , ( iii ) the synchronous patterns and cortical networks associated with spindle bursts , and ( iv ) the physiological and pathophysiological role of spindle bursts during early cortical development . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Functional Properties of Spindle Bursts in Neonatal Rat Cerebral Cortex ", "3. Generation of Spindle Burst Activity", "4. Synchrony and Cerebral Networks Associated with Spindles", "5. Physiological and Pathophysiological Role of Spindle-Like Oscillations in Early Brain Development", "6. Open Questions", "7. Conclusions"], "sections": [["network - driven spindle - like oscillations are a functional hallmark of the developing cerebral cortex . during late prenatal and early postnatal stages of development ,", "spontaneous spindle - like oscillations have been identified as physiological activity patterns in various neocortical areas of different mammalian species [ 14 ] . in humans ,", "so - called  delta brushes  can be observed in eeg recordings from preterm babies already at gestational week 28 , that is ~12 weeks before normal birth of a full - term neonate ( for review see ) . in ac - filtered eeg recordings ,", "delta brushes are brief rhythmic delta waves ( 0.31.5  hz ) of 50300  v amplitude with a superimposed burst of fast rhythm ( > 8  hz , the  brush  ) .", "it has been suggested that delta brushes in human preterm infants correlate with so - called spindle bursts recorded in rodents during the early postnatal period . from a developmental point of view , these early spontaneous activity patterns in developing rodent and human cerebral cortex are probably of similar or even identical origin .", "rats and mice are altricial - born in a far less mature condition than humans . in rodents", "the degree of neocortical development at the day of birth ( postnatal day [ p ] 0 ) can be compared to the stage of human cortex between gestational weeks 28 and 32   and the cerebral cortex of a p12p14 rat is comparable to that of the full - term newborn human baby [ 6 , 8 ] . in preterm infants born between gestational weeks 28 and 32 , milh et al .   and colonnese et al", ".   recorded eeg signals containing spontaneous and stimulus - evoked delta brushes with oscillatory activity in the frequency range of 8 to 25  hz , suggesting that spontaneous delta brushes may represent a physiological neocortical activity pattern of the human fetus in utero . in the cerebral cortex of rodents , spindle bursts ( beside short gamma oscillations )", "constitute the majority of spontaneous activity during the first postnatal week ( figure 1 ) .", "these spindle bursts resemble in their appearance spindles recorded in the adult brain during sleep .", "these sleep spindles are one of the hallmarks of human stage 2 sleep  for comprehensive overviews the reader is referred to recent reviews by lthi   and by mccormick et al . .", "however , in humans sleep spindles appear 4 to 9 weeks after birth , which is much later than the disappearance of delta brushes around the end of the first postnatal week , thus excluding the hypothesis that delta brushes or spindle bursts gradually develop into sleep spindles .", "in addition , the frequency profile of spindle bursts and delta brushes displays a rather broad frequency distribution up to 25  hz , whereas sleep spindles present oscillations in a narrower band of ~1015  hz .", "the present review focuses on spindle burst activity in the cerebral cortex of the developing rat during the first postnatal week and summarizes our current understanding ( i ) on the functional properties of spindle bursts , ( ii ) the mechanisms underlying their generation , ( iii ) the synchronous patterns and cerebral networks associated with spindle bursts , and ( iv ) the physiological and pathophysiological role of spindle bursts during early cortical development ."], ["two distinct activity patterns can be observed in the neonatal rat cerebral cortex in vivo : gamma oscillations and spindles bursts ( figures 1 and 2 ) [ 1419 ] .", "gamma oscillations have a duration of 100 to 300  ms , a frequency of 30 to 40  hz and appear spontaneously every 10 to 30  s ( figures 1 and 2(b ) ) . the properties and functional role of gamma oscillations as well as the mechanisms underlying their generation", "spindle bursts are characterized by their spindle - like oscillatory appearance , have a duration of 0.5 to 3  s and a frequency in the range of 8 to 30  hz , and occur spontaneously every ~10  s ( figures 1 and 2(a ) ) . as recognized in full - band direct - current ( dc ) coupled recordings ,", "spindle bursts are nested in slow ( delta ) waves , which in infant rats , and preterm human babies [ 22 , 23 ] have been termed  spontaneous activity transients  ( sats ) .", "spindle bursts have been described in primary somatosensory cortex ( s1 ) including barrel cortex [ 14 , 1619 ] , in primary visual cortex ( v1 ) [ 10 , 15 ] , in primary motor cortex ( m1 ) , and in prefrontal cortex   of anesthetized and awake rats during the first postnatal week . in s1 , spontaneous and stimulus - evoked", "spindle bursts can be observed as early as p0 [ 17 , 18 ] . in rats ,", "the incidence of spindle bursts declines during the second postnatal week and sporadic spindle bursts are obscured by the ongoing neocortical activity .", "likewise in humans , delta brushes are replaced by more continuous eeg activity at birth of a full - term infant .", "this gradual developmental shift from a highly synchronized state of spontaneous activity to a desynchronized state seems to be a fundamental network property of the cerebral cortex during early neonatal stages .", "spindle bursts synchronize the activity of a local neuronal network of 200 to 400  m in diameter , which resembles the dimension of a whisker - related neocortical column in the immature barrel cortex ( figure 3 ) [ 18 , 25 ] . using a combination of voltage - sensitive dye imaging and high - density multielectrode recordings in p0-p1 rat barrel cortex in vivo , yang et al .", "could demonstrate that this early spontaneous activity constitutes the later emergence of the whisker - related barrel field map .", "these data indicate that spontaneous activity patterns at this early age form  functional precolumns ,  supporting the concept of the existence of  ontogenetic columns  in the radial unit hypothesis .", "further support for this hypothesis comes from in vivo two - photon calcium imaging in the barrel cortex of both anesthetized and nonanesthetized newborn mice , demonstrating highly synchronous spontaneous burst activity , reminiscent of spindle bursts , in local networks of 100 to 200  m in diameter .", "these data strongly indicate that early spindle bursts , and probably also gamma oscillations ( for review ) , synchronize early neocortical networks into functional columns at a developmental stage when the upper layers 2/3 have not even been formed ( i.e. , in rats at p0 ) . at this developmental stage", ", thalamocortical afferents have not reached layer 4 and instead transiently innervate the subplate ( for review [ 28 , 29 ] ) .", "thalamic afferents form transient glutamatergic synapses with surprisingly mature properties including ampa and nmda receptors [ 3032 ] . in vitro studies in acute brain slice preparations and in intact whole cortical hemisphere preparations   have demonstrated that oscillatory network activity in the frequency range of spindle bursts depend on an intact subplate [ 34 , 35 ] .", "selective removal of the subplate in s1 in vivo causes a significant decline in the occurrence of spontaneous spindle bursts and disturbances in the development of the cortical architecture in the barrel cortex .", "these data further support the hypothesis that spindle bursts in developing cerebral cortex fulfill an important role in the maturation of the neocortical architecture . in the next section we will discuss the network and molecular mechanisms underlying the generation of spindle bursts in neonatal cerebral cortex ."], ["the rodent cerebral cortex develops rapidly during late prenatal and early postnatal stages and at least four different activity patterns may occur sequentially between birth and the end of the first postnatal week ( for review ) .", "furthermore , the cortex shows a mediolateral and anterior - posterior gradient in development and within the same neocortical area neurons in upper layers 2/3 are ~2 days younger compared to lower cortical layers .", "many inconsistencies in the literature on the properties of spontaneous activity patterns in newborn rodents and their underlying mechanisms can be explained by the fact that these important developmental differences are often ignored .", "in addition , 2 days in early rodent cortical development make a large difference , so that the neocortex of a p0 rat ( without layer 2/3 ) can not be compared to that of a p2 rat ( with almost complete lamination ) . already in newborn ( p0-p1 ) rat barrel cortex in vivo , mechanical stimulation of a single whisker", "elicits in field potential recordings and to some extent detectable also in voltage - sensitive dye imaging ( vsdi ) a sequence of an early gamma oscillation followed by a spindle burst ( figure 3(b ) ) . at this age ,", "the thalamocortical activity reaches the developing cortical network largely via the subplate [ 17 , 31 , 32 ] and is amplified by an intrinsic gap - junction coupled network within the subplate and cortical plate [ 28 , 34 ] .", "spontaneous and evoked delta brushes can be observed in premature human neonates of 2832 weeks postconceptional age , a developmental stage when the human cerebral cortex resembles that of a newborn rat .", "impressive examples of large delta brushes are provided in the supplementary eeg videos of milh et al . , demonstrating that a single touch elicits a large oscillatory response in the somatosensory evoked potential ( sep ) recorded above the contralateral parietal cortex . in mature human cortex", ", seps with smaller amplitudes and shorter durations can be only obtained after averaging of at least 100 epochs .", "thus , in both species , rats and humans , at a comparable stage of cortical development mechanical stimulation of the sensory periphery elicits in s1 spindle bursts and delta brushes , respectively .", "simultaneous multielectrode recordings in the barrel cortex and in the ventral posteromedial nucleus ( vpm ) of the somatosensory thalamus of p0-p1 rats in vivo have demonstrated that the majority of spontaneous cortical spindle bursts and also gamma oscillations are not generated within s1 , but rather in subcortical structures or outside of s1 ( figures 4(a ) and 4(b ) ) . at this age a local , functionally defined lesion in the vpm blocks the whisker stimulation - induced cortical responses and also profoundly reduces the spontaneously occurring cortical burst activity , further demonstrating that the majority of the spontaneous spindle bursts in p0-p1 rat barrel cortex are generated in subcortical structures of the whisker - to - barrel cortex pathway ( for further information see ) . silencing the sensory periphery by injection of lidocaine into the whisker pad causes a significant reduction in the occurrence of spontaneous spindle bursts and gamma oscillations by ~50% ( figure 4(c ) ) , indicating that during this developmental period at least half of the spontaneous burst activity in s1 is related to activity in the sensory periphery .", "a similar peripheral generation of spontaneous burst activity may occur in human preterms and fetuses between gestational weeks 28 and 32 [ 9 , 22 ] .", "similar to the spindle bursts in s1 , spontaneous spindle bursts in v1 are also largely generated in the sensory periphery .", "( for review ) , provide the primary drive for spindle bursts in newborn rat v1 , as demonstrated by simultaneous recordings from the retina and v1 .", "intraocular injection of forskolin , which augmented retinal waves , increased the occurrence of v1 spindle bursts , and removal of the retina reduced the spindle bursts frequency .", "as in the somatosensory system , spindle bursts in v1 can be also evoked by stimulation of the sensory periphery .", "however , since rod- and cone - mediated visual signaling is not functional in rats during the first postnatal week , spindle bursts can not be evoked by light flashes before p8 . at that age , the neocortical response in v1 consists of an early visual evoked response followed by an evoked spindle burst .", "similar responses could be observed in v1 of preterm infants once photoreceptor mediated light responses occur in the retina   ( for review ) .", "whereas a large amount of experimental data has shown that retinal waves provide the main trigger for the cortical v1 spindles bursts , it is not completely understood which pacemaker drives the spontaneous activity in s1 and how spontaneous activity is generated in the somatosensory periphery . in p3p6 rats spontaneous whisker movements occur during active sleep and are correlated with activation of whisker - related cortical columns in the barrel cortex . in newborn rats", "the proprioceptive feedback from self - generated myoclonic movements trigger spindle bursts in s1 ( for review ) .", "spontaneous limb movements of the human fetus during the third trimester of gestation , or those of the preterm infant associated with delta brushes in s1 , are similar to these twitching movements of the neonatal rat and can be also triggered by sensory feedback .", "kreider and blumberg have demonstrated in 1-week - old rats that the mesopontine region plays a central role in the generation of myoclonic twitching .", "have shown in newborn rats that spatially confined spindle bursts in s1 are triggered in a somatotopic manner by sensory feedback signals from spontaneous muscle twitches .", "these spontaneous movements are generated by neuronal networks in the spinal cord , but spindle bursts persisted at a reduced frequency after sensory deafferentation ( spinal cord transection ) in s1 , indicating that spindle burst activity can be also generated in neocortical or thalamocortical circuits during this early period of development .", "this assumption is also supported by the observation that silencing of the sensory periphery causes only a ~50% reduction in the occurrence of spontaneous spindle bursts ( figure 4(c ) ) . however , since it can not be excluded that the remaining portion of spindle bursts actually conveys activity from adjacent or distant sensory areas ( transmitted via inter- and intrahemispheric connections , see next chapter ) , the outcome of this experiment may underestimate the contribution of the sensory periphery .", "simultaneous monitoring of forepaw movements , vsdi , and extracellular multielectrode recordings in s1 and m1 of p3p5 rats under light urethane anesthesia have demonstrated that tactile forepaw stimulation triggers spindle bursts in s1 , followed by gamma and spindle bursts in m1 ( figure 5 ) .", "focal electrical stimulation of corticospinal tract neurons in layer 5 of m1 mimicking physiologically relevant 40  hz gamma or 10  hz spindle burst activity reliably elicited forepaw movements , indicating that m1 cortical spindle bursts are capable of triggering muscle twitches at this age .", "however , only 23.7% of the spontaneous bursts in m1 triggered forepaw movements and were followed by spindle bursts in s1 ( figures 5(b)(a ) and 5(c ) ) , indicating that only a fraction of m1 activity transients triggers motor responses directly . in 40.7% of the cases , spontaneous movements preceded the burst activity in m1 and s1 ( figures 5(b)(b ) and 5(c ) ) , suggesting that this activity may arise from subcortical regions in the brainstem or spinal cord .", "the remaining 35.6% of the m1 bursts were unrelated to any movements ( figure 5(c ) ) .", "the finding that 23.7% of the movements were triggered by m1 bursts as observed by an et al .  ", "is in contrast to previous observations , which demonstrated that dissection of neocortical inputs fails to suppress muscle twitches in rat pups . in summary , these data indicate that neocortical spindle bursts in newborn rodents ( and delta brushes in human fetus during the third trimester or in preterms ) are generated by central pattern generator ( cpg ) circuits in spinal cord , brainstem , and motor cortex ( for cpg circuits in mature brain see [ 4244 ] ) .", "neuropharmacological studies provided insights into the molecular mechanisms underlying the generation and modulation of neocortical spindle bursts . in vivo   and in vitro data   suggest that gabaergic synapses are not crucial for the generation of spindle bursts or spindle burst - like activity , respectively , but are essential for their spatial confinement to a cortical ( pre- ) column .", "in contrast , spindle bursts depend on intact glutamatergic synapses including alpha - amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) and n - methyl - d - aspartate ( nmda ) receptors [ 34 , 45 ] .", "carbachol - induced spindle - like oscillations in p0p3 mouse neocortex in vitro   and spindle bursts in p0p2 rat in vivo   are blocked or significantly reduced by different gap junction blockers , indicating that electrical synapses are critically involved in the generation of spindle bursts at this neonatal period", ". however , minlebaev et al .   reported for p1p3 rats a significant increase in the occurrence of spontaneous spindle bursts in s1 following application of the gap - junction blocker mefloquine .", "single - cell recordings revealed additional insights into the mechanisms underlying the generation of spindle bursts .", "spindle bursts in v1   and s1   are accompanied by a barrage of glutamatergic and gabaergic postsynaptic currents ( pscs ) that are phase - locked to the spindle oscillations ( figure 6 ) . in prefrontal cortex , glutamatergic and gabaergic synaptic inputs to excitatory pyramidal neurons are phase locked to the theta - band component of the spindle , while pscs of inhibitory interneurons are phase locked to the higher beta and gamma frequencies , suggesting that excitatory and inhibitory neurons differentially modulate the distinct components of the spindle bursts .", "further , the application of cnqx eliminates the glutamatergic pscs and completely blocks the occurrence of spindle bursts , indicating a major causal role of ampa - receptor mediated glutamatergic inputs in the generation of spindle bursts .", "additional insights have been obtained by experiments in which subplate cells were selectively ablated in s1 shortly after birth . in these animals", "these in vivo results support previous in vitro studies , in which spindle bursts elicited by cholinergic stimulation were suppressed after removal of the subplate ( see below ) [ 34 , 35 ] . taken together", ", these data suggest that thalamocortical inputs  relayed and amplified by the subplate [ 28 , 29]play an important role in the generation of spindle bursts ( e.g. , [ 17 , 45 ] ) .", "spindle bursts in the cerebral cortex of newborn rats can be elicited [ 34 , 35 ] and modulated by cholinergic mechanisms . in vitro , spindle burst - like oscillations can be reliably induced by activation of muscarinic acetylcholine receptors , predominantly of the m1 and m5 type . in vivo ,", "spindle bursts in v1 are decreased by ~50% following the application of the muscarinic receptor antagonist atropine .", "furthermore , blockade of acetylcholine esterase with physostigmine or direct stimulation of the cholinergic basal forebrain nuclei augmented the occurrence v1 spindle bursts , indicating that the cholinergic system facilitates spindle burst activity in developing cerebral cortex ."], ["as discussed above , spatially confined spindle bursts in newborn rat cortex synchronize a local neuronal network resembling a neocortical ( pre- ) column ( figure 3 ) . beside these intra - areal synchronization", ", spindle bursts are also synchronized between different cortical regions within one hemisphere ( intrahemispheric ) . as described above , a tight functional correlation in spontaneous and stimulus - evoked spindle burst activity exists between s1 and m1 cortex ( figure 5 ) .", "spindle bursts with similar properties as those in v1 and s1 have been also recorded in vivo in the prefrontal cortex of urethane - anesthetized rats older than p2 .", "the same authors demonstrate that the hippocampus drives this early activity in the prefrontal cortex . beside this intrahemispheric synchronization between different cortical regions , spindle bursts", "also interact between both hemispheres ( interhemispheric ) . in vivo simultaneous recordings of spontaneous activity in homotopic cortical areas in both hemispheres at the same stereotaxic", "coordinates and depth have demonstrated that the amount of interhemispheric synchronization in newborn rats is initially rather low and increases during the first postnatal week .", "this interhemispheric communication of spindle burst activity depends on an intact corpus callosum . in this regard ,", "in unanesthetized newborn rats callosotomy doubled the occurrence of spontaneous spindle bursts , suggesting that the corpus callosum modulates functionally inhibitory interactions between homotopic regions in both hemispheres during the occurrence of spindle burst activity .", "experiments in p2p15 rats demonstrated that this callosotomy - induced disinhibition is a transient feature of early development that disappears abruptly after p6 .", "it is not surprising that intra- and interhemispheric interactions of spontaneous activity in the spindle burst frequency range can be also observed in developing human cerebral cortex at early stages . using eeg and functional magnetic resonance imaging ( fmri ) omidvarnia et al .", "electric resting - state network  that shows functional intra- and interhemispheric interactions in the 815  hz frequency range . in summary , several in vitro and in vivo studies have demonstrated that spindle bursts represent elementary states of intra- and interhemispheric synchronization in the very immature cerebral cortex ."], ["an increasing amount of experimental and clinical data strongly indicate that spindle bursts play an important role in the physiological development of the cerebral cortex .", "experimental evidence indicates that spindle bursts may be particularly suited to interfere with early neurodevelopmental processes , and thus disturbances in spindle burst activity may cause long - term structural and functional disorders . at early stages of development spontaneous and sensory - evoked activity patterns", "influence a variety of developmental processes , such as neurogenesis , apoptosis , neuronal migration , cellular differentiation , network formation , and myelination   ( for review , see ) .", "it is not completely understood how electrical activity controls these different developmental processes and whether distinct activity patterns , such as spindle bursts , play a specific role . however , for the control of apoptotic cell death of immature neurons in vitro and in vivo the essential role of spontaneous network bursts to provide antiapoptotic signals has been demonstrated [ 52 , 56 , 57 ] . for this activity - dependent control of neuronal survival", "the phosphatidylinositol 3-kinase pathway plays an important role , while the mapk / extracellular signal - regulated kinase or the calcium / calmodulin - dependent protein kinase pathway is not directly involved .", "since one spontaneous spindle burst is associated at the single neuron level with 510 action potentials   and the frequency of spontaneous spindle bursts is ~5 per minute [ 16 , 17 ] , a single neuron discharges with 2550 action potentials per minute . under in vitro conditions this discharge frequency supports neuronal survival of developing neocortical neurons , suggesting that spontaneous spindle bursts in vivo provide an important physiological signal for the control of neuronal survival versus apoptosis in the neonatal cerebral cortex .", "notably , spindle burst and gamma activity provides an ideal physiological stimulus for the activity - dependent release of bdnf , an important antiapoptotic signal .", "balkowiec and katz   demonstrated for neuronal cultures that 3060  min of electrical burst stimulation ( 50 pulses at 2050  hz at intervals of 20  s ) increased extracellular bdnf levels by 20-fold , whereas stimulation patterns at lower frequency ( albeit producing the same number of extracellular electric shocks ) were ineffective ( for review , see ) .", "these data indicate that spontaneous spindle bursts represent a physiological trigger for the release of bdnf , which plays an important role in several aspects of development ( for review , see ) . using in vitro and in vivo models", "it has been recently shown that an experimentally induced inflammation by application of the endotoxin lipopolysaccharide induces rapid ( < 2  h ) alterations in the pattern of spontaneous spindle bursts and gamma oscillations in neonatal rodent cerebral cortex , which subsequently leads to increased apoptotic cell death .", "these inflammatory effects are specifically initiated by the microglia - derived proinflammatory cytokine tumor necrosis factor alpha and to a lesser extent by the chemokine macrophage inflammatory protein 2 .", "thus , inflammation causes a fast dysfunction in the pattern of spontaneous burst activity , which subsequently leads to increased apoptotic cell death , most likely by disturbances in the release of survival factors such as brain - derived neurotrophic factor ( bdnf ) acting on neurotrophin tropomyosin - related kinase b / c receptor .", "furthermore , removal of the subplate massively reduced spindle burst activity and led to a persistent loss of the typical barrel - like whisker representation within layer 4 , indicating that spindle bursts play a role in the development of the neocortical architecture . in summary", ", these experimental data suggest that any disturbances in the spontaneous activity of the developing cerebral cortex ( including spontaneous spindle bursts ) induce acute dysfunctions , which may cause long - term disorders .", "however , it remains to be elucidated whether spindle burst in particular can be causally related to neurodevelopmental disturbances .", "it has been recently shown in extremely preterm infants that the properties of neocortical bursts recorded with eeg and their scaling relationships correlate significantly with later cognitive development .", "these clinical data suggest that analyses of burst shapes obtained in eeg recordings from preterm and full - term newborn babies may have diagnostic use in neonatal intensive care units and predict the clinical outcome ."], ["while it is generally accepted that early electrical activity shapes the maturation of neocortical circuits , it remains an open question whether the specific properties of spindle burst are required or fulfill a distinct role in development . in this regard , it is possible that the spatiotemporal patterns of spindle bursts translate into a local molecular signal which fulfills an important developmental role . in particular", "as gaba does not seem to be necessary for the generation or maintenance of spindle bursts , and since gaba is essential for neuronal differentiation [ 64 , 65 ] , it is tempting to speculate that spindle bursts control the spatially confined release of gaba in developing local networks .", "another unresolved issue is that both spindle bursts and gamma oscillations can be observed during the same period of early development .", "they can be observed in early postnatal rodent brain from the day of birth (; also see ) and occur spontaneously as well as after sensory stimulation . moreover ,", "both can be observed mainly during the critical periods of the primary sensory areas , although spindle bursts probably persist for slightly longer periods .", "in addition , spindle bursts and early gamma oscillations in newborn cortex are proposed to rely on thalamocortical inputs , in contrast to gamma oscillations in adult cortex which depend on perisomatic gabaergic inhibition . beside their differences in duration , occurrence , and frequency ( see above ) , spindle bursts and gamma oscillations in newborn cortex reveal a number of additional distinctions . in the immature barrel cortex the vast majority ( > 90% ) of spontaneously occurring spindle burst spans several barrel - related columns , whereas the majority ( ~70% ) of spontaneous gamma oscillations", "are restricted to a single or two barrel related columns . in line with these observations , gamma oscillations evoked by tactile stimulation are also closely related to a single functional column , while evoked spindle bursts span over more than one column .", "thus , one reason for the coexistence of spindle bursts and gamma oscillations during early postnatal development might be a distinct role in neocortical maturation .", "gamma bursts may reflect local information processing mostly within a single functional column , thus supporting the maturation of a column related network .", "in contrast , spindle bursts probably reflect larger local network events and may thus serve to promote the connectivity between neighboring columns .", "although spindle bursts as well as gamma oscillations are spatially confined to a small network in one neocortical area , it is unclear to what extent and how this activity connects to other cortical and subcortical regions ( e.g. , see [ 18 , 19 ] ) .", "further , it remains to be studied whether the immature brain shows a spindle burst related  resting state  and how this network state is altered by sensory activation or by pathophysiological events .", "finally , it would be most interesting and important to correlate specific patterns of spontaneous activity ( e.g. , delta brush ) recorded by means of full - band direct - current eeg in preterm und full - term human neonates with the acute functional state and with the further development of the child , as impressively done by vanhatalo and colleagues [ 62 , 67 ] ."], ["spatially confined spindle bursts and delta brushes represent the most prominent physiological activity patterns in the developing cerebral cortex of newborn rodents and preterm human infants , respectively .", "spontaneous and stimulus - evoked spindle bursts can be observed in various neocortical areas of different mammalian species and play important roles in the early development of cortical networks .", "however , it remains to be studied in more detail how exactly spindle bursts influence the maturation of the cortex and how a potential long - term dysfunction due to disturbances in spindle burst activity can be prevented by early intervention .", "since this important type of brain activity is already present in the human fetus in utero ( either spontaneously occurring or related to sensory inputs from the uterine environment ) , a better understanding of the physiological relevance of spindle burst oscillations is of major clinical relevance ."]]}
{"article_id": "PMC3603203", "article_text": ["atrazine , 6-chloro - n2-ethyl - n4-isopropyl-1 , 3 , 5 atrazine , 4-diamine , is a selective herbicide that has been extensively used in corn production to control many broad - leaf and some grassy weeds .", "atrazine has long - term reproductive and endocrine - disrupting effects and a probable human carcinogen .", "international agency for research on cancer ( iarc ) has concluded atrazine as a group 2b carcinogen .", "the maximum contaminant level ( mcl ) for atrazine in drinking water established by the usepa is 3.0 gl .  ", "atrazine is moderately persistent in the environment and despite its low solubility , water resources contamination , it has become an international issue .", "the major dissipation route for atrazine is biodegradation , runoff , and leaching [ 4 , 5 ] .", "the rate of biodegradation of atrazine is reduced due to the adsorption , and desorption , and its bioavailability is the rate - limiting step in biodegradation .", "bioaugmentation is the addition of acclimated indigenous bacteria that can degrade the contaminant at accelerated rates .", "the bacteria are isolated from the heavily polluted soil in the laboratory through the enrichment process . the enrichment culture technique developed a mixed consortium of bacteria that are able to degrade the herbicide .", "a research was done by dehghani and colleague confirmed that atrazine biodegradation was enhanced in kavar corn field soil compared to the other soils that had not been exposed to the herbicide .", "therefore , several applications of atrazine on soil resulted in an enhancement of atrazine degradation .", "the addition of nutrient causes an increase of microbial populations , thereby , increasing the number of indigenous microorganisms capable of degrading the pollutant .", "n - containing compounds such as atrazine have been shown to serve as sources of nitrogen . in theory ,", "addition of high c / n should induce nitrogen limitation and increase selective pressure for utilization of recalcitrant n sources like s - triazines , which contain n that can be used by certain bacteria and microbial consortia .", "cometabolic biotransformation can be enhanced by an increase in microbial activity which is stimulated by addition of organic matter . in the past few decades ,", "intensive use of agricultural fertilization and herbicides has contributed to increasing concentration of n and herbicides .  ", "mixed microbial consortia [ 8 , 11 ] and strains have been isolated from soils and with a capability to complete the mineralization of the ring .", "the agrobacterium strain j14 , rhodococcus erythropolis , pseudaminobacter , and nocardioides can use atrazine as sole carbon and nitrogen source [ 1214 ] .", "laboratory experiments indicated that addition of carbon to soil inhibited atrazine biodegradation , but inorganic phosphate stimulates atrazine biodegradation .", "atrazine degradation rate was increased by addition of carbon sources to pure and mixed bacterial culture .", "ostrofsky and his coworkers ( 2001 ) found that cyanuric acid amendment increased atrazine mineralization by stimulation of general microbial population and activity . in liquid culture and in the presence of simple carbon sources ,", "( 1996 ) showed that the effect of n addition varies with the form and amount of added n .", "however , another study showed that the utilization of atrazine by pseudomonas adp in the presence of exogenous nitrogen ( nh4 , no3 , urea and glycine ) was not affected in a great amount by the presence of exogenous nitrogen .", "degradation of atrazine ( about 87% ) by p. adp and a. radiobacter was unaffected by the presence of n source , whereas no degradation occurred with bacterium m91 - 3 in media containing urea or nh4-n .", "another study reported that mineralization of atrazine by indigenous soil bacteria was inhibited by the addition of inorganic nitrogen .", "a study showed that addition of other organic amendments containing nitrogen suppressed atrazine . however , there are some limitations to the use of acclimated microbial cultures to degrade organic compound in a real field .", "physiochemical conditions of soils , and competition with native microorganisms , may destroy or reduce the inoculums and limit its degradative capacity .", "brandon and his coworkers ( 1997 ) showed that atrazine biodegradation was higher in liquid cultures than soil .", "he found that bacterial consortium in soil culture degraded 78% and 21% of atrazine at initial concentrations of 0.046 and 0.23 moles in 100 days .", "however , in liquid cultures , 90% and 56% of atrazine degraded in 80 days , respectively . in liquid culture ,", "since fars is an agricultural province and enjoys the top rank in wheat and corn production in the country in recent years , atrazine has been widely used as a selective herbicide to control broad - leaf and grassy weeds in agricultural corn fields .", "the high incidence of atrazine to contaminate water resources and the increasing concern about the toxicological properties of atrazine has made researches directed toward bioremediation of atrazine in polluted sites . therefore , the main objectives of this research are evaluating the effects of carbon and nitrogen sources on atrazine biodegradation using mixed bacterial consortium isolated from corn field soil located in south of shiraz ( kavar ) and determining the efficiency of atrazine biodegradation process in soil culture .", "kavar corn field soil with a long history of atrazine application in fars province of iran has been explored for their potential of atrazine biodegradation .", "soil samples for liquid cultures were taken from south of shiraz ( kavar ) corn field with a long history of atrazine application ( more than 10 years ) in fars province .", "soil samples for soil cultures were also taken from a field in bajgah which has been under alfalfa cultivation for 3 years and has not received atrazine in the past 10 years . disturbed soil samples were collected from 0 to 20  cm of soil depth with a hand - driven soil auger and stored at 4c until they were used .", "the soil samples were air - dried and passed through 2  mm sieve to be prepared for further microbiological examinations .", "other soil characteristics such as soil solution ph   and organic matter content   were determined .", "the soil texture in kavar corn field was loam and the amount of sand , silt , and clay distribution were in order of 47.44% , 31.5% and 17.06% .", "the native soil characteristics at kavar site was fine loamy , mixed , thermic typic haploxerepts . at bajgah field , the soil texture was clay - loam and the amount of sand , silt and clay distribution were in order of 28.7% , 33.3% and 32.0% .", "the native soil characteristics at bajgah site was fine loamy , mixed , and thermic , typic calcixerepts .", "the soil samples were transported to the laboratory in zipped plastic bags and were kept frozen at 20c until they were ready for chemical analysis .", "the soil samples were thawed and air - dried at dark in room temperature and screened through a 2.0  mm sieve for maintaining homogeneity of soil in order to reduce the variability of adsorption data .", "30  ml of dichloromethane was added to 10  g of the soil sample and shaken in a reciprocal shaker for 20 minutes .", "after filtration , the organic phase was transferred to a separating funnel and then atrazine was back extracted with 20  ml hcl ( 0.01  n ) .", "afterwards , the liquid phase was collected and transferred to a 15  ml glass vial and stored in a refrigerator prior to electrochemistry analysis .", "square wave voltammetry with the hanging mercury drop electrode ( auto lab type analyzer equipped with metrohm va stand 663 and gpes 4.9 software ) was used in this study to determine atrazine residual concentration in soil samples .", "atrazine recovery percent from soil with this method of extraction was 98% . in order to isolate mixed bacterial consortiums capable of growth on atrazine as a carbon source ,", "the selective enrichment culture and basal salt medium were prepared as described in rousseaus , .", "ten grams of wet soil was inoculated into 90  ml of atrazine medium and supplemented with sodium citrate and delvocid ( 25  mg  l ) after autoclaving .", "delvocid was used to prevent the growth of fungi and ph was also adjusted to 7.5 .", "cultures were incubated aerobically on a reciprocal shaker ( 150  rpm ) at room temperature in dark to preclude photolysis reactions .", "all enrichment cultures were subcultured on the same medium at one - week interval . from a one - week - old culture , 10  ml", "after culture was subculture for 30 and 300 days under conditions of nitrogen limitation , the remained atrazine after inoculation of the media for 10 days was quantified by electrochemistry .", "the bacterial consortium was harvested by centrifugation ( 6000  g at 40c for 20 minutes ) washed twice with 0.1  ml phosphate buffer ( ph = 7.3 ) .  ", "different carbon compounds such as glucose ( g ) , sodium citrate ( sc ) , sucrose ( su ) , starch ( st ) with three replications each at a concentration of 2  g l , and also the combination of these mentioned carbon sources such as g + sc , st + su , su + sc , st + sc , su + g , and st + g each at a total concentration of 2 ", "after inoculation of the media supported with carbon sources , they were incubated at room temperature and placed in dark for 10 days .", "control and blank without bacteria inoculation and no carbon sources ( ncs ) were also used for this study .", "after 10 days , the remained atrazine was measured . to measure the influence of nitrogen sources on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture , nitrogen sources as routine fertilizers were added to atrazine mineral salt broth containing sucrose and sodium citrate .", "ammonium nitrate and urea as fertilizers were applied to corn field at a concentration of 600825 and 200400  kg  ha , the nitrogen percent for these fertilizers were 34% and 46% , respectively .", "urea was added to atrazine minimal salt media at a concentration of 138690  mg ", "after inoculation of the media supported with nitrogen sources , they were incubated at room temperature in dark for 10 days .", "a laboratory experiment was arranged to measure the influence of ph on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture .", "a different ph from 5.5 to 8.5 with three replications was used in atrazine mineral salt broth containing sucrose and sodium citrate and no nitrogen sources .", "atrazine degradation rate by the bacterial consortium was measured in 100  ml capped erlenmeyer flask containing soil samples .", "ten grams of soil sample was brought to the desired soil moisture ( 7% and 25% ) by the addition of sterile deionized water .", "the experimental design consisted of 36 flasks with 12 treatment and three replications for each treatment .", "the initial atrazine concentrations of 1.3 and 6.7  mg  g soil are corresponded to 0.5 and 2.5  kg  ha , respectively .", "however , atrazine concentration of 20  mg  g soil is related to 7.5  kg  ha(4.43  kg a. i. ha ) which is considered a relatively high atrazine concentration and might have occurred due to an accidental spillage .", "after one day of incubation to allow herbicide sorption to the soil , inoculation with 300  l phosphate buffer containing the selected bacterial consortium was added to soil to yield 7.5  10 bacterial cell g soils as determined by plating on soil extract agar .", "the soil samples were mixed until they were homogeneously wet and then incubated at room temperature in dark until the end of the experiment .", "soil moisture was maintained constant through the incubation by weighing and correcting for any weight loss by adding sterile deionized water . to maintaining high population of atrazine degraders ,", "the inoculation with the consortium was made every 5 days over a 30-day period for a total of 6 inoculations .", "soil samples at the initial atrazine concentration of 6.7  mg  g soil were extracted at the incubation time of 2 , 10 , 20 , and 30-day and analyzed to determine the remained atrazine concentration at each time interval .", "by using a mixed bacterial consortium with a high capability of atrazine degradation isolated from kavar corn field soil , the effect of different carbon sources on atrazine biodegradation was studied ( figure 1 ) . according to this figure ,", "the percentage of atrazine biodegradation rate for different carbon sources was in the range of 9.47% to 87.72% .", "a blank sample did not adequately support the growth of consortium bacteria ( due to low turbidity ) , and the rate of atrazine degradation is lass than 3% .", "linear regression showed that there was a significant difference between different carbon sources and atrazine biodegradation rate ( p < 0.001 ) .  ", "the effect of different nitrogen sources on atrazine biodegradation was shown in tables 1 and 2 . according to table 1 ,", "the percent of atrazine biodegradation rate decreased from 87.72% to 29.58% as the ammonium nitrate concentration increased from 0.0 to 900  kg  ha soil .", "the rate of atrazine biodegradation for ammonium nitrate decreased quickly when the concentration of ammonium nitrate increased from zero to 600  kg  ha soil , after that the reduction rate of atrazine degradation was getting slower .", "the same trend has been observed for atrazine biodegradation in the presence of urea . according to table 2 ,", "the percentage of atrazine biodegradation rate decreased from 87.72% to 26.76% as the urea concentration increased from 0.0 to 500  kg  ha soil . for urea ,", "atrazine biodegradation rate under nitrogen amendment showed an initial sharp decreasing slope and then reaching constant with relative slower rate .", "variations of ph on atrazine degradation rate were examined when sucrose and sodium citrate were used as a carbon source , while there was no nitrogen source available ( figure 2 ) . according to regression analysis , it can be concluded that there was a significant difference between ph and atrazine biodegradation ( p < 0.05 ) .  \n", "table 3 showed the effect of initial atrazine concentration and soil moisture on the biodegradation of the inoculated soil . according to data on this table ,", "after 30 days of incubations atrazine degradation rate for noninoculated soil samples was low and atrazine reductions were only 7% to 19% .", "however , for inoculated soil samples , degradation rate was higher and its reductions were 19.572% . the inoculated soil samples at 25% soil moisture at initial concentration of 1.3 , 6.7 , and 20  mg  g soil , atrazine reductions were 69.5% , 60.5% and 30.5% within 30 days of incubation , respectively .  ", "figure 3 showed that atrazine biodegradation rate during the different time intervals in soil culture . \n", "figure 4 depicted the plot of the semilogarithm of initialized atrazine concentration ( c / c0 ) versus time . during the 30 days of incubation period , atrazine concentration decreased from an initial concentration of 6.7  mg  gsoil to a final concentration of 2.67  mg  gsoil . only a 5% decrease in atrazine concentration", "however , figure 3 showed that at 20 days 40% of the atrazine had degraded .", "after that a very slow increase in atrazine degradation was observed and remained constant until the end of the incubation period . assuming a pseudo - first - order reaction for the disappearance of atrazine , a plot of the natural logarithm of initialized atrazine concentration ( c / c0 ) versus time resulted in a rate constant equal to 0.0328 d ( figure 4 ) .", "the t1/2 for atrazine calculated from the plot was 21.13  d. the initial slow degradation rate of atrazine was followed by a much faster degradation rate that lasted about 5 day and then the degradation rate became slower and finally remained constant until the end of incubation period .", "carbon sources of sodium citrate and sucrose had the highest atrazine biodegradation rate . according to data ,", "mandelbaum and his coworkers ( 1993a ) used sodium citrate and sucrose as the carbon source . according to data in this research , the percentage of atrazine biodegradation rate with no carbon sources was only 5.5% .", "past studies showed that carbon in atrazine ring can not be used by many bacteria as energy sources ; however , carbon in alkylated group provided carbon for bacterial growth .", "the low amount of atrazine can not support bacterial growth , and therefore , a supplemental carbon was needed .   according to data on tables 1 and 2 ,", "atrazine utilization is repressed under nitrogen - sufficient growth condition and activated under nitrogen limitation .", "many investigations also showed a negative effect of nitrogen amendment on atrazine biodegradation by indigenous populations in soils [ 34 , 35 ] .", "however , other study reported that organic nitrogen supplied in dairy manure increased atrazine mineralization .", "data regarding the effect of ph shows that as ph increased from 5.0 to 7.0 , the rate of atrazine biodegradation increased .", "however , as ph increased from 7.0 to 8.5 this caused a reduction rate in atrazine biodegradation .", "atrazine biodegradation rates were significantly enhanced in the inoculated soils as compared to uninoculated control soils .", "after 30 days , the percent of atrazine reduction was only 12% for uninoculated soils at initial atrazine concentration of 6.7  mg  g soil and the soil moisture of 25% .", "however , soil that was inoculated every 5 days with the bacterial consortium had reduced atrazine up to 60.5% at the same initial atrazine concentration and the soil moisture by day 30 ( table 3 ) . as initial atrazine concentration increased from 1.30 to 20  mg  g soil , the percentage of atrazine reduction decreased from 69.5% to 30.5% ( inoculated soils and soil moisture 25% ) .", "the decrease in atrazine reduction at 20  g  g soil was possibly due to the result of complex interaction between microbial activity and nutrient availability .", "therefore , unbalanced nutrient supply and not atrazine toxicity was probably responsible for the decrease .  ", "figure 3 showed that atrazine degradation rate increased as time passed . according to ( table 3 ) ,", "enhanced atrazine biodegradation rates occurred with an increase in soil moisture from 7% to 25% .", "the percent of atrazine reduction increased from 32.5% to 69.5% as the soil moisture increased from 7% to 25% ( at initial atrazine concentration of 1.30  mg  g soil ) .", "many researchers found that higher atrazine biodegradation rate occurred with an increase in soil moisture .", "soil moisture influences microbial processes through direct effect ( e.g. , water availability ) or indirect effects , for example , solute diffusion , chemical availability , and aeration .", "therefore , the positive effect of increasing soil moisture was probably due to increased microbial mobility , solute diffusion , and chemical availability which all had an indirect effect on atrazine degradation . in this study , more than 60% of atrazine was degraded in 30 days of incubation periods ( figure 3 ) .", "atrazine degradation exhibited a half - life of approximately 21.13 d. in conclusion , our results confirmed that atrazine biodegradation was higher in liquid cultures than soil .", "the mixed bacterial consortium in soil culture degraded 69.5% of atrazine at initial concentrations of 1.3  mg  g soil in 30 days . however , in liquid cultures , 87.72% of all atrazine degraded in 10 days .", "results of this study suggested that atrazine bioremediation in soil utilizing atrazine - degrading bacterial consortium could be accomplished across a wide range of atrazine concentration if the soil moistures swere enough and also nutrient availability was balanced , too .", "the bacterial consortium with the ability to degrade atrazine provided a good opportunity for increasing the degradation rate of this herbicide through bioaugmentation .", "it is very important to note that these bacteria in the controlled laboratory conditions ( with the addition of carbon and nitrogen sources and without the interaction of environmental factors ) had been able to use atrazine .", "however , there are some limitations to the use of acclimated microbial cultures to degrade the herbicide in a real field .", "therefore , it is highly recommended that the feasibility of atrazine degradation to be studied by the bioaugmentation of mixed bacterial isolates to the real contaminated soil .", "the mixed bacterial consortium successful in laboratory studies may fail in the real field because of their sensitivity to high concentrations of other compounds ."], "abstract_text": ["<S> atrazine herbicide that is widely used in corn production is frequently detected in water resources . </S>", "<S> the main objectives of this research were focused on assessing the effects of carbon and nitrogen sources on atrazine biodegradation by mixed bacterial consortium and by evaluating the feasibility of using mixed bacterial consortium in soil culture . </S>", "<S> shiraz corn field soil with a long history of atrazine application has been explored for their potential of atrazine biodegradation . </S>", "<S> the influence of different carbon compounds and the effect of nitrogen sources and a different ph ( 5.58.5 ) on atrazine removal efficiency by mixed bacterial consortium in liquid culture were investigated . </S>", "<S> sodium citrate and sucrose had the highest atrazine biodegradation rate ( 87.22% ) among different carbon sources . </S>", "<S> atrazine biodegradation rate decreased more quickly by the addition of urea ( 26.76% ) compared to ammonium nitrate . </S>", "<S> based on the data obtained in this study , ph of 7.0 is optimum for atrazine biodegradation . </S>", "<S> after 30 days of incubation , the percent of atrazine reduction rates were significantly enhanced in the inoculated soils ( 60.5% ) as compared to uninoculated control soils ( 12% ) at the soil moisture content of 25% . in conclusion , </S>", "<S> bioaugmentation of soil with mixed bacterial consortium may enhance the rate of atrazine degradation in a highly polluted soil . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["atrazine , 6-chloro - n2-ethyl - n4-isopropyl-1 , 3 , 5 atrazine , 4-diamine , is a selective herbicide that has been extensively used in corn production to control many broad - leaf and some grassy weeds .", "atrazine has long - term reproductive and endocrine - disrupting effects and a probable human carcinogen .", "international agency for research on cancer ( iarc ) has concluded atrazine as a group 2b carcinogen .", "the maximum contaminant level ( mcl ) for atrazine in drinking water established by the usepa is 3.0 gl .  ", "atrazine is moderately persistent in the environment and despite its low solubility , water resources contamination , it has become an international issue .", "the major dissipation route for atrazine is biodegradation , runoff , and leaching [ 4 , 5 ] .", "the rate of biodegradation of atrazine is reduced due to the adsorption , and desorption , and its bioavailability is the rate - limiting step in biodegradation .", "bioaugmentation is the addition of acclimated indigenous bacteria that can degrade the contaminant at accelerated rates .", "the bacteria are isolated from the heavily polluted soil in the laboratory through the enrichment process . the enrichment culture technique developed a mixed consortium of bacteria that are able to degrade the herbicide .", "a research was done by dehghani and colleague confirmed that atrazine biodegradation was enhanced in kavar corn field soil compared to the other soils that had not been exposed to the herbicide .", "therefore , several applications of atrazine on soil resulted in an enhancement of atrazine degradation .", "the addition of nutrient causes an increase of microbial populations , thereby , increasing the number of indigenous microorganisms capable of degrading the pollutant .", "n - containing compounds such as atrazine have been shown to serve as sources of nitrogen . in theory ,", "addition of high c / n should induce nitrogen limitation and increase selective pressure for utilization of recalcitrant n sources like s - triazines , which contain n that can be used by certain bacteria and microbial consortia .", "cometabolic biotransformation can be enhanced by an increase in microbial activity which is stimulated by addition of organic matter . in the past few decades ,", "intensive use of agricultural fertilization and herbicides has contributed to increasing concentration of n and herbicides .  ", "mixed microbial consortia [ 8 , 11 ] and strains have been isolated from soils and with a capability to complete the mineralization of the ring .", "the agrobacterium strain j14 , rhodococcus erythropolis , pseudaminobacter , and nocardioides can use atrazine as sole carbon and nitrogen source [ 1214 ] .", "laboratory experiments indicated that addition of carbon to soil inhibited atrazine biodegradation , but inorganic phosphate stimulates atrazine biodegradation .", "atrazine degradation rate was increased by addition of carbon sources to pure and mixed bacterial culture .", "ostrofsky and his coworkers ( 2001 ) found that cyanuric acid amendment increased atrazine mineralization by stimulation of general microbial population and activity . in liquid culture and in the presence of simple carbon sources ,", "( 1996 ) showed that the effect of n addition varies with the form and amount of added n .", "however , another study showed that the utilization of atrazine by pseudomonas adp in the presence of exogenous nitrogen ( nh4 , no3 , urea and glycine ) was not affected in a great amount by the presence of exogenous nitrogen .", "degradation of atrazine ( about 87% ) by p. adp and a. radiobacter was unaffected by the presence of n source , whereas no degradation occurred with bacterium m91 - 3 in media containing urea or nh4-n .", "another study reported that mineralization of atrazine by indigenous soil bacteria was inhibited by the addition of inorganic nitrogen .", "a study showed that addition of other organic amendments containing nitrogen suppressed atrazine . however , there are some limitations to the use of acclimated microbial cultures to degrade organic compound in a real field .", "physiochemical conditions of soils , and competition with native microorganisms , may destroy or reduce the inoculums and limit its degradative capacity .", "brandon and his coworkers ( 1997 ) showed that atrazine biodegradation was higher in liquid cultures than soil .", "he found that bacterial consortium in soil culture degraded 78% and 21% of atrazine at initial concentrations of 0.046 and 0.23 moles in 100 days .", "however , in liquid cultures , 90% and 56% of atrazine degraded in 80 days , respectively . in liquid culture ,", "since fars is an agricultural province and enjoys the top rank in wheat and corn production in the country in recent years , atrazine has been widely used as a selective herbicide to control broad - leaf and grassy weeds in agricultural corn fields .", "the high incidence of atrazine to contaminate water resources and the increasing concern about the toxicological properties of atrazine has made researches directed toward bioremediation of atrazine in polluted sites . therefore , the main objectives of this research are evaluating the effects of carbon and nitrogen sources on atrazine biodegradation using mixed bacterial consortium isolated from corn field soil located in south of shiraz ( kavar ) and determining the efficiency of atrazine biodegradation process in soil culture .", "kavar corn field soil with a long history of atrazine application in fars province of iran has been explored for their potential of atrazine biodegradation ."], ["soil samples for liquid cultures were taken from south of shiraz ( kavar ) corn field with a long history of atrazine application ( more than 10 years ) in fars province .", "soil samples for soil cultures were also taken from a field in bajgah which has been under alfalfa cultivation for 3 years and has not received atrazine in the past 10 years . disturbed soil samples were collected from 0 to 20  cm of soil depth with a hand - driven soil auger and stored at 4c until they were used .", "the soil samples were air - dried and passed through 2  mm sieve to be prepared for further microbiological examinations .", "other soil characteristics such as soil solution ph   and organic matter content   were determined .", "the soil texture in kavar corn field was loam and the amount of sand , silt , and clay distribution were in order of 47.44% , 31.5% and 17.06% .", "the native soil characteristics at kavar site was fine loamy , mixed , thermic typic haploxerepts . at bajgah field , the soil texture was clay - loam and the amount of sand , silt and clay distribution were in order of 28.7% , 33.3% and 32.0% .", "the native soil characteristics at bajgah site was fine loamy , mixed , and thermic , typic calcixerepts .", "the soil samples were transported to the laboratory in zipped plastic bags and were kept frozen at 20c until they were ready for chemical analysis .", "the soil samples were thawed and air - dried at dark in room temperature and screened through a 2.0  mm sieve for maintaining homogeneity of soil in order to reduce the variability of adsorption data .", "30  ml of dichloromethane was added to 10  g of the soil sample and shaken in a reciprocal shaker for 20 minutes .", "after filtration , the organic phase was transferred to a separating funnel and then atrazine was back extracted with 20  ml hcl ( 0.01  n ) .", "afterwards , the liquid phase was collected and transferred to a 15  ml glass vial and stored in a refrigerator prior to electrochemistry analysis .", "square wave voltammetry with the hanging mercury drop electrode ( auto lab type analyzer equipped with metrohm va stand 663 and gpes 4.9 software ) was used in this study to determine atrazine residual concentration in soil samples .", "atrazine recovery percent from soil with this method of extraction was 98% . in order to isolate mixed bacterial consortiums capable of growth on atrazine as a carbon source ,", "the selective enrichment culture and basal salt medium were prepared as described in rousseaus , .", "ten grams of wet soil was inoculated into 90  ml of atrazine medium and supplemented with sodium citrate and delvocid ( 25  mg  l ) after autoclaving .", "delvocid was used to prevent the growth of fungi and ph was also adjusted to 7.5 .", "cultures were incubated aerobically on a reciprocal shaker ( 150  rpm ) at room temperature in dark to preclude photolysis reactions .", "all enrichment cultures were subcultured on the same medium at one - week interval . from a one - week - old culture , 10  ml", "after culture was subculture for 30 and 300 days under conditions of nitrogen limitation , the remained atrazine after inoculation of the media for 10 days was quantified by electrochemistry .", "the bacterial consortium was harvested by centrifugation ( 6000  g at 40c for 20 minutes ) washed twice with 0.1  ml phosphate buffer ( ph = 7.3 ) .  ", "different carbon compounds such as glucose ( g ) , sodium citrate ( sc ) , sucrose ( su ) , starch ( st ) with three replications each at a concentration of 2  g l , and also the combination of these mentioned carbon sources such as g + sc , st + su , su + sc , st + sc , su + g , and st + g each at a total concentration of 2 ", "after inoculation of the media supported with carbon sources , they were incubated at room temperature and placed in dark for 10 days .", "control and blank without bacteria inoculation and no carbon sources ( ncs ) were also used for this study .", "after 10 days , the remained atrazine was measured . to measure the influence of nitrogen sources on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture , nitrogen sources as routine fertilizers were added to atrazine mineral salt broth containing sucrose and sodium citrate .", "ammonium nitrate and urea as fertilizers were applied to corn field at a concentration of 600825 and 200400  kg  ha , the nitrogen percent for these fertilizers were 34% and 46% , respectively .", "urea was added to atrazine minimal salt media at a concentration of 138690  mg ", "after inoculation of the media supported with nitrogen sources , they were incubated at room temperature in dark for 10 days .", "a laboratory experiment was arranged to measure the influence of ph on the efficiency of atrazine biodegradation by the bacterial consortium in liquid culture .", "a different ph from 5.5 to 8.5 with three replications was used in atrazine mineral salt broth containing sucrose and sodium citrate and no nitrogen sources .", "atrazine degradation rate by the bacterial consortium was measured in 100  ml capped erlenmeyer flask containing soil samples .", "ten grams of soil sample was brought to the desired soil moisture ( 7% and 25% ) by the addition of sterile deionized water .", "the experimental design consisted of 36 flasks with 12 treatment and three replications for each treatment .", "the initial atrazine concentrations of 1.3 and 6.7  mg  g soil are corresponded to 0.5 and 2.5  kg  ha , respectively .", "however , atrazine concentration of 20  mg  g soil is related to 7.5  kg  ha(4.43  kg a. i. ha ) which is considered a relatively high atrazine concentration and might have occurred due to an accidental spillage .", "after one day of incubation to allow herbicide sorption to the soil , inoculation with 300  l phosphate buffer containing the selected bacterial consortium was added to soil to yield 7.5  10 bacterial cell g soils as determined by plating on soil extract agar .", "the soil samples were mixed until they were homogeneously wet and then incubated at room temperature in dark until the end of the experiment .", "soil moisture was maintained constant through the incubation by weighing and correcting for any weight loss by adding sterile deionized water . to maintaining high population of atrazine degraders ,", "the inoculation with the consortium was made every 5 days over a 30-day period for a total of 6 inoculations .", "soil samples at the initial atrazine concentration of 6.7  mg  g soil were extracted at the incubation time of 2 , 10 , 20 , and 30-day and analyzed to determine the remained atrazine concentration at each time interval ."], ["by using a mixed bacterial consortium with a high capability of atrazine degradation isolated from kavar corn field soil , the effect of different carbon sources on atrazine biodegradation was studied ( figure 1 ) . according to this figure ,", "the percentage of atrazine biodegradation rate for different carbon sources was in the range of 9.47% to 87.72% .", "a blank sample did not adequately support the growth of consortium bacteria ( due to low turbidity ) , and the rate of atrazine degradation is lass than 3% .", "linear regression showed that there was a significant difference between different carbon sources and atrazine biodegradation rate ( p < 0.001 ) .  ", "the effect of different nitrogen sources on atrazine biodegradation was shown in tables 1 and 2 . according to table 1 ,", "the percent of atrazine biodegradation rate decreased from 87.72% to 29.58% as the ammonium nitrate concentration increased from 0.0 to 900  kg  ha soil .", "the rate of atrazine biodegradation for ammonium nitrate decreased quickly when the concentration of ammonium nitrate increased from zero to 600  kg  ha soil , after that the reduction rate of atrazine degradation was getting slower .", "the same trend has been observed for atrazine biodegradation in the presence of urea . according to table 2 ,", "the percentage of atrazine biodegradation rate decreased from 87.72% to 26.76% as the urea concentration increased from 0.0 to 500  kg  ha soil . for urea ,", "atrazine biodegradation rate under nitrogen amendment showed an initial sharp decreasing slope and then reaching constant with relative slower rate .", "variations of ph on atrazine degradation rate were examined when sucrose and sodium citrate were used as a carbon source , while there was no nitrogen source available ( figure 2 ) . according to regression analysis , it can be concluded that there was a significant difference between ph and atrazine biodegradation ( p < 0.05 ) .  \n", "table 3 showed the effect of initial atrazine concentration and soil moisture on the biodegradation of the inoculated soil . according to data on this table ,", "after 30 days of incubations atrazine degradation rate for noninoculated soil samples was low and atrazine reductions were only 7% to 19% .", "however , for inoculated soil samples , degradation rate was higher and its reductions were 19.572% . the inoculated soil samples at 25% soil moisture at initial concentration of 1.3 , 6.7 , and 20  mg  g soil , atrazine reductions were 69.5% , 60.5% and 30.5% within 30 days of incubation , respectively .  ", "figure 3 showed that atrazine biodegradation rate during the different time intervals in soil culture . \n", "figure 4 depicted the plot of the semilogarithm of initialized atrazine concentration ( c / c0 ) versus time . during the 30 days of incubation period , atrazine concentration decreased from an initial concentration of 6.7  mg  gsoil to a final concentration of 2.67  mg  gsoil . only a 5% decrease in atrazine concentration", "however , figure 3 showed that at 20 days 40% of the atrazine had degraded .", "after that a very slow increase in atrazine degradation was observed and remained constant until the end of the incubation period . assuming a pseudo - first - order reaction for the disappearance of atrazine , a plot of the natural logarithm of initialized atrazine concentration ( c / c0 ) versus time resulted in a rate constant equal to 0.0328 d ( figure 4 ) .", "the t1/2 for atrazine calculated from the plot was 21.13  d. the initial slow degradation rate of atrazine was followed by a much faster degradation rate that lasted about 5 day and then the degradation rate became slower and finally remained constant until the end of incubation period ."], ["carbon sources of sodium citrate and sucrose had the highest atrazine biodegradation rate . according to data ,", "mandelbaum and his coworkers ( 1993a ) used sodium citrate and sucrose as the carbon source . according to data in this research , the percentage of atrazine biodegradation rate with no carbon sources was only 5.5% .", "past studies showed that carbon in atrazine ring can not be used by many bacteria as energy sources ; however , carbon in alkylated group provided carbon for bacterial growth .", "the low amount of atrazine can not support bacterial growth , and therefore , a supplemental carbon was needed .   according to data on tables 1 and 2 ,", "atrazine utilization is repressed under nitrogen - sufficient growth condition and activated under nitrogen limitation .", "many investigations also showed a negative effect of nitrogen amendment on atrazine biodegradation by indigenous populations in soils [ 34 , 35 ] .", "however , other study reported that organic nitrogen supplied in dairy manure increased atrazine mineralization .", "data regarding the effect of ph shows that as ph increased from 5.0 to 7.0 , the rate of atrazine biodegradation increased .", "however , as ph increased from 7.0 to 8.5 this caused a reduction rate in atrazine biodegradation .", "atrazine biodegradation rates were significantly enhanced in the inoculated soils as compared to uninoculated control soils .", "after 30 days , the percent of atrazine reduction was only 12% for uninoculated soils at initial atrazine concentration of 6.7  mg  g soil and the soil moisture of 25% .", "however , soil that was inoculated every 5 days with the bacterial consortium had reduced atrazine up to 60.5% at the same initial atrazine concentration and the soil moisture by day 30 ( table 3 ) . as initial atrazine concentration increased from 1.30 to 20  mg  g soil , the percentage of atrazine reduction decreased from 69.5% to 30.5% ( inoculated soils and soil moisture 25% ) .", "the decrease in atrazine reduction at 20  g  g soil was possibly due to the result of complex interaction between microbial activity and nutrient availability .", "therefore , unbalanced nutrient supply and not atrazine toxicity was probably responsible for the decrease .  ", "figure 3 showed that atrazine degradation rate increased as time passed . according to ( table 3 ) ,", "enhanced atrazine biodegradation rates occurred with an increase in soil moisture from 7% to 25% .", "the percent of atrazine reduction increased from 32.5% to 69.5% as the soil moisture increased from 7% to 25% ( at initial atrazine concentration of 1.30  mg  g soil ) .", "many researchers found that higher atrazine biodegradation rate occurred with an increase in soil moisture .", "soil moisture influences microbial processes through direct effect ( e.g. , water availability ) or indirect effects , for example , solute diffusion , chemical availability , and aeration .", "therefore , the positive effect of increasing soil moisture was probably due to increased microbial mobility , solute diffusion , and chemical availability which all had an indirect effect on atrazine degradation . in this study , more than 60% of atrazine was degraded in 30 days of incubation periods ( figure 3 ) .", "atrazine degradation exhibited a half - life of approximately 21.13 d. in conclusion , our results confirmed that atrazine biodegradation was higher in liquid cultures than soil .", "the mixed bacterial consortium in soil culture degraded 69.5% of atrazine at initial concentrations of 1.3  mg  g soil in 30 days . however , in liquid cultures , 87.72% of all atrazine degraded in 10 days .", "results of this study suggested that atrazine bioremediation in soil utilizing atrazine - degrading bacterial consortium could be accomplished across a wide range of atrazine concentration if the soil moistures swere enough and also nutrient availability was balanced , too .", "the bacterial consortium with the ability to degrade atrazine provided a good opportunity for increasing the degradation rate of this herbicide through bioaugmentation .", "it is very important to note that these bacteria in the controlled laboratory conditions ( with the addition of carbon and nitrogen sources and without the interaction of environmental factors ) had been able to use atrazine .", "however , there are some limitations to the use of acclimated microbial cultures to degrade the herbicide in a real field .", "therefore , it is highly recommended that the feasibility of atrazine degradation to be studied by the bioaugmentation of mixed bacterial isolates to the real contaminated soil .", "the mixed bacterial consortium successful in laboratory studies may fail in the real field because of their sensitivity to high concentrations of other compounds ."]]}
{"article_id": "PMC4841409", "article_text": ["neurons , oligodendrocytes ( ols ) , and astrocytes are unique cell types in the central nervous system ( cns ) of vertebrates.1 neurons process and transmit information electrochemically.2 ols generate myelin sheaths around most axons of the vertebrate cns , enabling a faster conduction of the nerve impulses .", "astrocytes provide support and nutrients for the nerve tissue.3 the different cell types in the nervous system are regulated by the precise spatiotemporal expression of specific genes.4,5 transcriptional regulation plays an important role in the determination of neural cell differentiation.6,7 \n olig genes belong to the basic helix  loop  helix transcription factor family , which encode ol lineage transcription factors 1 , 2 , and 3 ( olig1 , olig2 , and olig3 ) . with the exception of olig3 ,", "olig genes are specifically expressed in the cns , and play a critical role in cns development by controlling differentiation and maturation of ols , motor neurons ( mns ) , and astrocytes.8,9 olig2 null mice die at birth from a lack of mns.10 both gain- and loss - of - function studies were performed in an olig1 null mouse with normal myelin during development , but which were unable to remyelinate on experimental challenge.11 a second olig1 null mouse with less compensatory effect by olig2 had a more severe phenotype and died around postnatal day 14 from a complete lack of myelin .", "this mutant had mature ols , but failed to wrap myelin or even deposit lipid around axons.12 knocking out olig1 and olig2 individually or together affected differentiation and maturation of ols , suggesting functional overlap in the cns.10,13,14 until now , the role of olig2 during development of spinal cord attracted more attention .", "however , studies investigating the expression and function of olig1 in development and disease are limited .", "although it is widely known that olig1 promotes the differentiation and maturation of ols , it is unclear how these occur during development .", "exploring temporal and spatial expression and distribution of olig1 will contribute to our understanding of the role of olig1 in specialization of neural cells during development .", "therefore , in this study , we determined the expression pattern of olig1 in neural cells during rat spinal cord development .", "sprague  dawley rats were obtained from the laboratory animal center , bengbu medical college ( bengbu , people s republic of china ) .", "all experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . to produce embryonic and newborn rats , one female was cohabited with two males , and gestational age ( embryo , e )", "was designated as day 0.5 ( when vaginal plugs in female rat were observed ) .", "eighty rats were randomized to eight groups and subgroups : embryonic day 14.5 ( e14.5 ) ( n=10 ) , e18.5 ( n=10 ) , postnatal day 0 ( p0 ) ( n=10 ) , p3 ( n=10 ) , p7 ( n=10 ) , postnatal 2 weeks ( p2w ) ( n=10 ) , p4w ( n=10 ) , and adults ( n=10 ) .", "each group was randomized into two subgroups equally . in the first subgroup ( n=5 ) ,", "the spinal cords were immunohistochemically stained , and in the second subgroup ( n=5 ) , the spinal cords were subjected to western blot .", "spinal cords from embryos ( e14.5 and e18.5 ) were dissected following cervical dislocation of the pregnant rats .", "spinal cords were dissected from postnatal rats ( p0 , p3 , p7 , p2w , p4w , and adults ) and perfused intracardially with phosphate - buffered saline ( pbs ) , followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . for immunohistochemistry , spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution ( sigma , st louis , mo , usa ) at 4c for another 57 days . using a cryostat , transverse 15 m thick sections were obtained , mounted onto gelatin - coated slides ( fisher scientific , fairlawn , nj , usa ) , and stored at 80c until use . for western blot , spinal cord tissues were stored immediately at 80c until use . for immunohistochemistry staining ,", "the frozen slides were air - dried at room temperature ( rt ) for 10 minutes and rinsed with pbs for 10 minutes .", "they were blocked with 10% donkey serum in tris - buffered saline ( tbs ) containing 0.3% triton x-100 ( tbst ) for 1 hour at rt .", "the following primary antibodies were used : monoclonal mouse anti - olig1 ( igg , 1:100 , millipore , billerica , ma , usa ) , monoclonal rabbit anti - myelin basic protein ( anti - mbp ; 1:300 , millipore ) , rabbit anti - glial fibrillary acidic protein ( anti - gfap ; 1:100 , millipore ) , monoclonal mouse anti - o4 ( igm , 1:100 , millipore ) , and rabbit anti--tubulin ( 1:100 , millipore ) . the next day , the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm , igg or goat anti - rabbit igm , igg ( all 1:200 ; jackson immunoresearch lab , west grove , pa , usa ) for 1 hour at 37c .", "the slides were washed three times with pbs and coverslipped with gel aqueous mounting media ( biomeda , foster city , ca , usa ) containing hoechst 33342 ( sigma - aldrich ) to counterstain the nuclei .", "at least five randomly selected fields with a total of more than 500 cells were counted . in all experiments ,", "western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages .", "briefly , tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride ( pmsf ) ( 10 mg / ml ) and thoroughly homogenized for 5 minutes on ice , and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant .", "the protein concentrations of the supernatant were determined using a bca protein assay kit ( pierce , rockford , il , usa ) .", "the protein concentration was determined using the bio - rad dc protein assay ( every 10 l sample contain 120 g protein ) .", "protein samples containing an equal amount of protein ( 20 g ) were electrophoresed on sodium dodecyl sulfate ( sds)-polyacrylamide gels , and transferred to polyvinylidene difluoride filters ( millipore ) .", "the filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies ( in tbst-5% bovine serum albumin [ bsa ] ) including olig1 ( 1:200 , millipore ) and -actin ( 1:400 , sigma ) as markers of differentiated neural cells . after rinsing with tbst", ", the membranes were incubated with the appropriate horseradish peroxidase ( hrp)-conjugated secondary antibody ( all from kpl , gaithersburg , md , usa ) for 1 hour at rt . to visualize", "the immunoreactive proteins , the enhanced chemiluminescence ( ecl ) kit ( pierce , rockford , il , usa ) was used , following the manufacturer s instructions .", "films were digitized , and densitometry was performed using gel - pro analyzer ( media cybernetics , silver spring , md , usa ) .", "the intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin .", "the data were analyzed using spss16.0 ( ibm , armonk , ny , usa ) software .", "the data with three or more groups were analyzed by one - way analysis of variance ( anova ) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups .", "sprague  dawley rats were obtained from the laboratory animal center , bengbu medical college ( bengbu , people s republic of china ) .", "all experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . to produce embryonic and newborn rats , one female was cohabited with two males , and gestational age ( embryo , e )", "was designated as day 0.5 ( when vaginal plugs in female rat were observed ) .", "eighty rats were randomized to eight groups and subgroups : embryonic day 14.5 ( e14.5 ) ( n=10 ) , e18.5 ( n=10 ) , postnatal day 0 ( p0 ) ( n=10 ) , p3 ( n=10 ) , p7 ( n=10 ) , postnatal 2 weeks ( p2w ) ( n=10 ) , p4w ( n=10 ) , and adults ( n=10 ) .", "each group was randomized into two subgroups equally . in the first subgroup ( n=5 ) ,", "the spinal cords were immunohistochemically stained , and in the second subgroup ( n=5 ) , the spinal cords were subjected to western blot .", "spinal cords from embryos ( e14.5 and e18.5 ) were dissected following cervical dislocation of the pregnant rats .", "spinal cords were dissected from postnatal rats ( p0 , p3 , p7 , p2w , p4w , and adults ) and perfused intracardially with phosphate - buffered saline ( pbs ) , followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . for immunohistochemistry , spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution ( sigma , st louis , mo , usa ) at 4c for another 57 days . using a cryostat , transverse 15 m thick sections were obtained , mounted onto gelatin - coated slides ( fisher scientific , fairlawn , nj , usa ) , and stored at 80c until use . for western blot , spinal cord tissues were stored immediately at 80c until use .", "for immunohistochemistry staining , the frozen slides were air - dried at room temperature ( rt ) for 10 minutes and rinsed with pbs for 10 minutes .", "they were blocked with 10% donkey serum in tris - buffered saline ( tbs ) containing 0.3% triton x-100 ( tbst ) for 1 hour at rt .", "the following primary antibodies were used : monoclonal mouse anti - olig1 ( igg , 1:100 , millipore , billerica , ma , usa ) , monoclonal rabbit anti - myelin basic protein ( anti - mbp ; 1:300 , millipore ) , rabbit anti - glial fibrillary acidic protein ( anti - gfap ; 1:100 , millipore ) , monoclonal mouse anti - o4 ( igm , 1:100 , millipore ) , and rabbit anti--tubulin ( 1:100 , millipore ) .", "the next day , the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm , igg or goat anti - rabbit igm , igg ( all 1:200 ; jackson immunoresearch lab , west grove , pa , usa ) for 1 hour at 37c .", "the slides were washed three times with pbs and coverslipped with gel aqueous mounting media ( biomeda , foster city , ca , usa ) containing hoechst 33342 ( sigma - aldrich ) to counterstain the nuclei .", "at least five randomly selected fields with a total of more than 500 cells were counted . in all experiments ,", "western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages . briefly , tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride ( pmsf ) ( 10 mg / ml ) and thoroughly homogenized for 5 minutes on ice , and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant .", "the protein concentrations of the supernatant were determined using a bca protein assay kit ( pierce , rockford , il , usa ) .", "the protein concentration was determined using the bio - rad dc protein assay ( every 10 l sample contain 120 g protein ) .", "protein samples containing an equal amount of protein ( 20 g ) were electrophoresed on sodium dodecyl sulfate ( sds)-polyacrylamide gels , and transferred to polyvinylidene difluoride filters ( millipore ) .", "the filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies ( in tbst-5% bovine serum albumin [ bsa ] ) including olig1 ( 1:200 , millipore ) and -actin ( 1:400 , sigma ) as markers of differentiated neural cells . after rinsing with tbst", ", the membranes were incubated with the appropriate horseradish peroxidase ( hrp)-conjugated secondary antibody ( all from kpl , gaithersburg , md , usa ) for 1 hour at rt . to visualize the immunoreactive proteins , the enhanced chemiluminescence ( ecl ) kit ( pierce , rockford , il , usa ) was used , following the manufacturer s instructions .", "films were digitized , and densitometry was performed using gel - pro analyzer ( media cybernetics , silver spring , md , usa ) .", "the intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin .", "the data were analyzed using spss16.0 ( ibm , armonk , ny , usa ) software .", "the data with three or more groups were analyzed by one - way analysis of variance ( anova ) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups .", "western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood .", "our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points .", "however , interestingly , the expression of olig1 increased again from p2w to a high level until adulthood ( figure 1 ) .", "to determine the expression and localization of olig1 in different types of neural cells , we performed double immunostaining using the antibody against olig1 and another antibody against o4 , mbp , -tubulin , and gfap , respectively .", "our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells ( opcs ; figure 2 ) and -tubulin - positive neurons ( figure 3 ) at all time points .", "furthermore , we failed to observe expression of mbp in the spinal cord during the embryonic period ( data not shown ) .", "mbp expression was not observed prior to p3 , and no cellular coexpression of mbp and olig1 was observed ( figure 4 ) .", "the coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood .", "we also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development .", "western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood .", "our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points .", "however , interestingly , the expression of olig1 increased again from p2w to a high level until adulthood ( figure 1 ) .", "to determine the expression and localization of olig1 in different types of neural cells , we performed double immunostaining using the antibody against olig1 and another antibody against o4 , mbp , -tubulin , and gfap , respectively .", "our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells ( opcs ; figure 2 ) and -tubulin - positive neurons ( figure 3 ) at all time points .", "furthermore , we failed to observe expression of mbp in the spinal cord during the embryonic period ( data not shown ) .", "mbp expression was not observed prior to p3 , and no cellular coexpression of mbp and olig1 was observed ( figure 4 ) .", "the coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood .", "we also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development .", "the cns originates in the neuroepithelial stem cells ( nscs ) , which ultimately generate the three major cell types : neurons , ols , and astrocytes.1 in this process , transcriptional regulation plays an important role.6,7 in mice , genetic studies revealed that olig transcription factors play key roles in the genesis of mns and ols .", "gain- and loss - of - function analysis confirmed the fundamental role of olig1 in the survival and maturation of opcs.12,1517 regulation of olig1 gene expression in spinal cord of demyelinating disease promotes myelin repair after spinal cord injury .", "however , the distribution and expression of olig1 in neural cells during development is unclear . in this study", ", we systematically investigated the expression pattern of olig1 in neural cells during rat spinal cord development .", "first , using the western blot , we demonstrated the differential expression of olig1 in rat spinal cord development .", "olig1 expression increased and peaked at e18.5 , which may contribute to the differentiation of a large number of neural cells into opcs and mn precursors at this stage .", "recently , it has been reported that olig1 protein expression was significantly decreased during mouse ol progenitor differentiation.18 however , the expression of olig1 increased again from p2w , which was consistent with a recent report that olig1 was expressed in human ols during process outgrowth and maintained at a high level prior to myelination.19 next , we identified the expression of olig1 in neural cells at several time points during spinal cord development .", "we confirmed that olig1 was expressed not only in opcs but also in neurons from e14.5 to adulthood .", "cns development starts from nscs , which generate the three major cell types ( neurons , ols , and astrocytes ) of the cns.20,21 however , the intermediate steps in the differentiation of nscs in vivo have not been clarified . in the embryonic ventral spinal cord ,", "mns and ols are derived from a common progenitor pool , suggesting that these lineages share intrinsic and extrinsic regulatory mechanisms.10,13,21,22 ol progenitors were more closely aligned with neuronal subtype progenitors than with astrocytes , at least in the developing cns.23 a study demonstrated that olig2 was more likely an early - stage factor for ol precursor cells , while olig1 played a critical role in late - stage ol maturation and myelin formation.19 however , jakovcevski and zecevic24 found olig1 protein expressed in primitive neuroepithelia that can give rise to interneurons in the human fetal brain .", "a recent study suggests that olig1 regulates neuron - glial fate choice in the embryonic telencephalon.25 in conclusion , olig family members do not act independently but interact with other transcription factors to tightly control cns development.17,26 our findings raise the possibility that olig1 has some function in the formation of mns during cns development .", "mbp is a major myelin protein , which is expressed in mature ols.27,28 mbp is the major protein component of myelin at 2 weeks after birth in the rat brain , expressed a week later than in spinal cord.28 in this study , we observed that mbp expression commenced from postnatal period , and olig1 was not coexpressed with mbp during rat spinal cord development .", "opcs undergo two types of differentiation , resulting in ols in the absence of serum or type ii astrocytes in the presence of serum in vitro.2931 fate - mapping studies have shown that opcs generate a subset of astrocytes during perinatal development.32 most neurogenic bhlh transcription factors , for example , mammalian achaete - scute homologue 1 , mammalian atonal homologue , and neurogenins 1 and 2 , are only expressed transiently in progenitor cells as needed.23 in this study , we observed the transient expression of olig1 in gfap - positive cells at e14.5 , suggesting that ols and astrocytes may derive from the same progenitor cell .", "both olig1 and olig2 occur in the nucleus of ols and their progenitors occur on p1 in mouse brain.33 olig2 continues to be nuclear at all developmental stages , whereas olig1 is almost completely cytoplasmic in the adult mouse brain .", "changes in nuclear or cytoplasmic olig1 location correspond with altered morphology of opcs.11 recently , othman et al19 reported that o1-positive multipolar opcs with nuclear expression of olig1 exhibited fewer processes , while opcs with cytoplasmic olig1 manifested more extensive membrane expansion .", "nuclear olig1 facilitated mbp expression , but with greatly diminished membrane expansion.19 however , these experiments were performed in mouse and human .", "our results demonstrated that olig1 was expressed in o4-positive opcs and -tubulin - positive neurons , but was localized in the cytoplasm during the period from e14.5 to adulthood .", "we speculate that the cause for this inconsistency may be attributed to differences between animal species or other unknown factors .", "in conclusion , we systematically explored the expression pattern of olig1 in neural cells during rat spinal cord development .", "our results showed that olig1 was coexpressed with o4-positive opcs and -tubulin - positive neurons at all time points during development .", "olig1 was localized in the cytoplasm of o4- or -tubulin - positive cells during the transition from e14.5 to adult .", "our results contribute to understanding the mechanism of developmental regulation of neural cells by olig1 ."], "abstract_text": ["<S> purposeour purpose was to systematically investigate the expression pattern and role of olig1 in neural cells during rat spinal cord development.animals and methodsspinal cord tissues were dissected from sprague  dawley rats at embryonic day 14.5 ( e14.5 ) and e18.5 , postnatal day 0 ( p0 ) , p3 , p7 , postnatal 2 weeks ( p2w ) , p4w , and adults ( more than 2 months after birth ) , respectively . </S>", "<S> the expression of olig1 was determined by western blot and immunostaining . to observe expression of olig1 in different neural cell types , a double immunohistochemical staining </S>", "<S> was performed using antibodies against olig1 with o4 , -tubulin , glial fibrillary acidic protein ( gfap ) , and myelin basic protein , respectively.resultsthe expression of olig1 protein shows a significant level change in rat spinal cord at different developmental time points . starting with e14.5 , </S>", "<S> the expression gradually increased and peaked at e18.5 . </S>", "<S> olig1 decreased gradually from p3 and reached its lowest level on p7 . </S>", "<S> however , interestingly , the olig1 expression increased again from p2w , until adulthood . </S>", "<S> olig1 was coexpressed with o4-positive oligodendrocyte progenitor cells ( opcs ) and -tubulin - positive neurons at all time points during development . </S>", "<S> olig1 was also coexpressed transiently with gfap - positive astrocytes at only e14.5 . </S>", "<S> olig1 was localized in the cytoplasm of o4- and -tubulin - positive cells during the period from e14.5 to adult.conclusionthe expression of olig1 in opcs and neurons at all time points during development and in astrocytes at e14.5 suggests that olig1 may play an important role in the generation and maturation of specific neural cells during development of spinal cord . </S>", "<S> our results contribute to understanding the mechanism underlying developmental regulation of neural cells by olig1 . </S>"], "labels": null, "section_names": ["Introduction", "Animals and methods", "Animals and tissue preparation", "Immunohistochemistry", "Western blot", "Statistical analyses", "Results", "Olig1 expression in rat spinal cord development", "Olig1 expression and localization in neural cells", "Discussion", "Conclusion"], "sections": [["neurons , oligodendrocytes ( ols ) , and astrocytes are unique cell types in the central nervous system ( cns ) of vertebrates.1 neurons process and transmit information electrochemically.2 ols generate myelin sheaths around most axons of the vertebrate cns , enabling a faster conduction of the nerve impulses .", "astrocytes provide support and nutrients for the nerve tissue.3 the different cell types in the nervous system are regulated by the precise spatiotemporal expression of specific genes.4,5 transcriptional regulation plays an important role in the determination of neural cell differentiation.6,7 \n olig genes belong to the basic helix  loop  helix transcription factor family , which encode ol lineage transcription factors 1 , 2 , and 3 ( olig1 , olig2 , and olig3 ) . with the exception of olig3 ,", "olig genes are specifically expressed in the cns , and play a critical role in cns development by controlling differentiation and maturation of ols , motor neurons ( mns ) , and astrocytes.8,9 olig2 null mice die at birth from a lack of mns.10 both gain- and loss - of - function studies were performed in an olig1 null mouse with normal myelin during development , but which were unable to remyelinate on experimental challenge.11 a second olig1 null mouse with less compensatory effect by olig2 had a more severe phenotype and died around postnatal day 14 from a complete lack of myelin .", "this mutant had mature ols , but failed to wrap myelin or even deposit lipid around axons.12 knocking out olig1 and olig2 individually or together affected differentiation and maturation of ols , suggesting functional overlap in the cns.10,13,14 until now , the role of olig2 during development of spinal cord attracted more attention .", "however , studies investigating the expression and function of olig1 in development and disease are limited .", "although it is widely known that olig1 promotes the differentiation and maturation of ols , it is unclear how these occur during development .", "exploring temporal and spatial expression and distribution of olig1 will contribute to our understanding of the role of olig1 in specialization of neural cells during development .", "therefore , in this study , we determined the expression pattern of olig1 in neural cells during rat spinal cord development ."], ["sprague  dawley rats were obtained from the laboratory animal center , bengbu medical college ( bengbu , people s republic of china ) .", "all experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . to produce embryonic and newborn rats , one female was cohabited with two males , and gestational age ( embryo , e )", "was designated as day 0.5 ( when vaginal plugs in female rat were observed ) .", "eighty rats were randomized to eight groups and subgroups : embryonic day 14.5 ( e14.5 ) ( n=10 ) , e18.5 ( n=10 ) , postnatal day 0 ( p0 ) ( n=10 ) , p3 ( n=10 ) , p7 ( n=10 ) , postnatal 2 weeks ( p2w ) ( n=10 ) , p4w ( n=10 ) , and adults ( n=10 ) .", "each group was randomized into two subgroups equally . in the first subgroup ( n=5 ) ,", "the spinal cords were immunohistochemically stained , and in the second subgroup ( n=5 ) , the spinal cords were subjected to western blot .", "spinal cords from embryos ( e14.5 and e18.5 ) were dissected following cervical dislocation of the pregnant rats .", "spinal cords were dissected from postnatal rats ( p0 , p3 , p7 , p2w , p4w , and adults ) and perfused intracardially with phosphate - buffered saline ( pbs ) , followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . for immunohistochemistry , spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution ( sigma , st louis , mo , usa ) at 4c for another 57 days . using a cryostat , transverse 15 m thick sections were obtained , mounted onto gelatin - coated slides ( fisher scientific , fairlawn , nj , usa ) , and stored at 80c until use . for western blot , spinal cord tissues were stored immediately at 80c until use . for immunohistochemistry staining ,", "the frozen slides were air - dried at room temperature ( rt ) for 10 minutes and rinsed with pbs for 10 minutes .", "they were blocked with 10% donkey serum in tris - buffered saline ( tbs ) containing 0.3% triton x-100 ( tbst ) for 1 hour at rt .", "the following primary antibodies were used : monoclonal mouse anti - olig1 ( igg , 1:100 , millipore , billerica , ma , usa ) , monoclonal rabbit anti - myelin basic protein ( anti - mbp ; 1:300 , millipore ) , rabbit anti - glial fibrillary acidic protein ( anti - gfap ; 1:100 , millipore ) , monoclonal mouse anti - o4 ( igm , 1:100 , millipore ) , and rabbit anti--tubulin ( 1:100 , millipore ) . the next day , the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm , igg or goat anti - rabbit igm , igg ( all 1:200 ; jackson immunoresearch lab , west grove , pa , usa ) for 1 hour at 37c .", "the slides were washed three times with pbs and coverslipped with gel aqueous mounting media ( biomeda , foster city , ca , usa ) containing hoechst 33342 ( sigma - aldrich ) to counterstain the nuclei .", "at least five randomly selected fields with a total of more than 500 cells were counted . in all experiments ,", "western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages .", "briefly , tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride ( pmsf ) ( 10 mg / ml ) and thoroughly homogenized for 5 minutes on ice , and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant .", "the protein concentrations of the supernatant were determined using a bca protein assay kit ( pierce , rockford , il , usa ) .", "the protein concentration was determined using the bio - rad dc protein assay ( every 10 l sample contain 120 g protein ) .", "protein samples containing an equal amount of protein ( 20 g ) were electrophoresed on sodium dodecyl sulfate ( sds)-polyacrylamide gels , and transferred to polyvinylidene difluoride filters ( millipore ) .", "the filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies ( in tbst-5% bovine serum albumin [ bsa ] ) including olig1 ( 1:200 , millipore ) and -actin ( 1:400 , sigma ) as markers of differentiated neural cells . after rinsing with tbst", ", the membranes were incubated with the appropriate horseradish peroxidase ( hrp)-conjugated secondary antibody ( all from kpl , gaithersburg , md , usa ) for 1 hour at rt . to visualize", "the immunoreactive proteins , the enhanced chemiluminescence ( ecl ) kit ( pierce , rockford , il , usa ) was used , following the manufacturer s instructions .", "films were digitized , and densitometry was performed using gel - pro analyzer ( media cybernetics , silver spring , md , usa ) .", "the intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin .", "the data were analyzed using spss16.0 ( ibm , armonk , ny , usa ) software .", "the data with three or more groups were analyzed by one - way analysis of variance ( anova ) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups ."], ["sprague  dawley rats were obtained from the laboratory animal center , bengbu medical college ( bengbu , people s republic of china ) .", "all experimental protocols involving animals and their care were approved by the ethics committee of laboratory animal services center of bengbu medical college . to produce embryonic and newborn rats , one female was cohabited with two males , and gestational age ( embryo , e )", "was designated as day 0.5 ( when vaginal plugs in female rat were observed ) .", "eighty rats were randomized to eight groups and subgroups : embryonic day 14.5 ( e14.5 ) ( n=10 ) , e18.5 ( n=10 ) , postnatal day 0 ( p0 ) ( n=10 ) , p3 ( n=10 ) , p7 ( n=10 ) , postnatal 2 weeks ( p2w ) ( n=10 ) , p4w ( n=10 ) , and adults ( n=10 ) .", "each group was randomized into two subgroups equally . in the first subgroup ( n=5 ) ,", "the spinal cords were immunohistochemically stained , and in the second subgroup ( n=5 ) , the spinal cords were subjected to western blot .", "spinal cords from embryos ( e14.5 and e18.5 ) were dissected following cervical dislocation of the pregnant rats .", "spinal cords were dissected from postnatal rats ( p0 , p3 , p7 , p2w , p4w , and adults ) and perfused intracardially with phosphate - buffered saline ( pbs ) , followed by 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . for immunohistochemistry , spinal cord tissues were postfixed for 2 hours and immersed into a graded series of sucrose solution ( sigma , st louis , mo , usa ) at 4c for another 57 days . using a cryostat , transverse 15 m thick sections were obtained , mounted onto gelatin - coated slides ( fisher scientific , fairlawn , nj , usa ) , and stored at 80c until use . for western blot , spinal cord tissues were stored immediately at 80c until use ."], ["for immunohistochemistry staining , the frozen slides were air - dried at room temperature ( rt ) for 10 minutes and rinsed with pbs for 10 minutes .", "they were blocked with 10% donkey serum in tris - buffered saline ( tbs ) containing 0.3% triton x-100 ( tbst ) for 1 hour at rt .", "the following primary antibodies were used : monoclonal mouse anti - olig1 ( igg , 1:100 , millipore , billerica , ma , usa ) , monoclonal rabbit anti - myelin basic protein ( anti - mbp ; 1:300 , millipore ) , rabbit anti - glial fibrillary acidic protein ( anti - gfap ; 1:100 , millipore ) , monoclonal mouse anti - o4 ( igm , 1:100 , millipore ) , and rabbit anti--tubulin ( 1:100 , millipore ) .", "the next day , the slides were washed in pbs three times and incubated with rhodamine - conjugated goat anti - mouse igm , igg or goat anti - rabbit igm , igg ( all 1:200 ; jackson immunoresearch lab , west grove , pa , usa ) for 1 hour at 37c .", "the slides were washed three times with pbs and coverslipped with gel aqueous mounting media ( biomeda , foster city , ca , usa ) containing hoechst 33342 ( sigma - aldrich ) to counterstain the nuclei .", "at least five randomly selected fields with a total of more than 500 cells were counted . in all experiments ,"], ["western blot was used to detect the expression of olig1 in rat spinal cord tissues at different developmental stages . briefly , tissue selected from the same segment of spinal cord was immediately transferred to a homogenizer containing 1 ml lysis buffer and 10 l phenylmethanesulfonyl fluoride ( pmsf ) ( 10 mg / ml ) and thoroughly homogenized for 5 minutes on ice , and then centrifuged at 12,000 g for 30 minutes at 4c to collect the supernatant .", "the protein concentrations of the supernatant were determined using a bca protein assay kit ( pierce , rockford , il , usa ) .", "the protein concentration was determined using the bio - rad dc protein assay ( every 10 l sample contain 120 g protein ) .", "protein samples containing an equal amount of protein ( 20 g ) were electrophoresed on sodium dodecyl sulfate ( sds)-polyacrylamide gels , and transferred to polyvinylidene difluoride filters ( millipore ) .", "the filters were blocked with 5% nonfat dry milk in tbs for 1 hour at rt and then incubated overnight at 4c with primary antibodies ( in tbst-5% bovine serum albumin [ bsa ] ) including olig1 ( 1:200 , millipore ) and -actin ( 1:400 , sigma ) as markers of differentiated neural cells . after rinsing with tbst", ", the membranes were incubated with the appropriate horseradish peroxidase ( hrp)-conjugated secondary antibody ( all from kpl , gaithersburg , md , usa ) for 1 hour at rt . to visualize the immunoreactive proteins , the enhanced chemiluminescence ( ecl ) kit ( pierce , rockford , il , usa ) was used , following the manufacturer s instructions .", "films were digitized , and densitometry was performed using gel - pro analyzer ( media cybernetics , silver spring , md , usa ) .", "the intensity of immunoreactive bands for olig1 protein was normalized by the intensity of -actin ."], ["the data were analyzed using spss16.0 ( ibm , armonk , ny , usa ) software .", "the data with three or more groups were analyzed by one - way analysis of variance ( anova ) followed by post hoc tukey s t - test to determine whether there were significant differences between individual groups ."], ["western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood .", "our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points .", "however , interestingly , the expression of olig1 increased again from p2w to a high level until adulthood ( figure 1 ) .", "to determine the expression and localization of olig1 in different types of neural cells , we performed double immunostaining using the antibody against olig1 and another antibody against o4 , mbp , -tubulin , and gfap , respectively .", "our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells ( opcs ; figure 2 ) and -tubulin - positive neurons ( figure 3 ) at all time points .", "furthermore , we failed to observe expression of mbp in the spinal cord during the embryonic period ( data not shown ) .", "mbp expression was not observed prior to p3 , and no cellular coexpression of mbp and olig1 was observed ( figure 4 ) .", "the coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood .", "we also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development ."], ["western blot was used to determine olig1 expression in the rat spinal cord from e14.5 to adulthood .", "our results showed a significant change in the expression of olig1 protein in rat spinal cord at different developmental time points .", "however , interestingly , the expression of olig1 increased again from p2w to a high level until adulthood ( figure 1 ) ."], ["to determine the expression and localization of olig1 in different types of neural cells , we performed double immunostaining using the antibody against olig1 and another antibody against o4 , mbp , -tubulin , and gfap , respectively .", "our results showed that olig1 was expressed in both o4-positive oligodendrocyte progenitor cells ( opcs ; figure 2 ) and -tubulin - positive neurons ( figure 3 ) at all time points .", "furthermore , we failed to observe expression of mbp in the spinal cord during the embryonic period ( data not shown ) .", "mbp expression was not observed prior to p3 , and no cellular coexpression of mbp and olig1 was observed ( figure 4 ) .", "the coexpression of gfap and olig1 was observed at e14.5 but absent from day e18.5 to adulthood .", "we also analyzed subcellular localization of olig1 in neural cells and found that olig1 was localized in the cytoplasm of coexpressed cells at all time points during rat spinal cord development ."], ["the cns originates in the neuroepithelial stem cells ( nscs ) , which ultimately generate the three major cell types : neurons , ols , and astrocytes.1 in this process , transcriptional regulation plays an important role.6,7 in mice , genetic studies revealed that olig transcription factors play key roles in the genesis of mns and ols .", "gain- and loss - of - function analysis confirmed the fundamental role of olig1 in the survival and maturation of opcs.12,1517 regulation of olig1 gene expression in spinal cord of demyelinating disease promotes myelin repair after spinal cord injury .", "however , the distribution and expression of olig1 in neural cells during development is unclear . in this study", ", we systematically investigated the expression pattern of olig1 in neural cells during rat spinal cord development .", "first , using the western blot , we demonstrated the differential expression of olig1 in rat spinal cord development .", "olig1 expression increased and peaked at e18.5 , which may contribute to the differentiation of a large number of neural cells into opcs and mn precursors at this stage .", "recently , it has been reported that olig1 protein expression was significantly decreased during mouse ol progenitor differentiation.18 however , the expression of olig1 increased again from p2w , which was consistent with a recent report that olig1 was expressed in human ols during process outgrowth and maintained at a high level prior to myelination.19 next , we identified the expression of olig1 in neural cells at several time points during spinal cord development .", "we confirmed that olig1 was expressed not only in opcs but also in neurons from e14.5 to adulthood .", "cns development starts from nscs , which generate the three major cell types ( neurons , ols , and astrocytes ) of the cns.20,21 however , the intermediate steps in the differentiation of nscs in vivo have not been clarified . in the embryonic ventral spinal cord ,", "mns and ols are derived from a common progenitor pool , suggesting that these lineages share intrinsic and extrinsic regulatory mechanisms.10,13,21,22 ol progenitors were more closely aligned with neuronal subtype progenitors than with astrocytes , at least in the developing cns.23 a study demonstrated that olig2 was more likely an early - stage factor for ol precursor cells , while olig1 played a critical role in late - stage ol maturation and myelin formation.19 however , jakovcevski and zecevic24 found olig1 protein expressed in primitive neuroepithelia that can give rise to interneurons in the human fetal brain .", "a recent study suggests that olig1 regulates neuron - glial fate choice in the embryonic telencephalon.25 in conclusion , olig family members do not act independently but interact with other transcription factors to tightly control cns development.17,26 our findings raise the possibility that olig1 has some function in the formation of mns during cns development .", "mbp is a major myelin protein , which is expressed in mature ols.27,28 mbp is the major protein component of myelin at 2 weeks after birth in the rat brain , expressed a week later than in spinal cord.28 in this study , we observed that mbp expression commenced from postnatal period , and olig1 was not coexpressed with mbp during rat spinal cord development .", "opcs undergo two types of differentiation , resulting in ols in the absence of serum or type ii astrocytes in the presence of serum in vitro.2931 fate - mapping studies have shown that opcs generate a subset of astrocytes during perinatal development.32 most neurogenic bhlh transcription factors , for example , mammalian achaete - scute homologue 1 , mammalian atonal homologue , and neurogenins 1 and 2 , are only expressed transiently in progenitor cells as needed.23 in this study , we observed the transient expression of olig1 in gfap - positive cells at e14.5 , suggesting that ols and astrocytes may derive from the same progenitor cell .", "both olig1 and olig2 occur in the nucleus of ols and their progenitors occur on p1 in mouse brain.33 olig2 continues to be nuclear at all developmental stages , whereas olig1 is almost completely cytoplasmic in the adult mouse brain .", "changes in nuclear or cytoplasmic olig1 location correspond with altered morphology of opcs.11 recently , othman et al19 reported that o1-positive multipolar opcs with nuclear expression of olig1 exhibited fewer processes , while opcs with cytoplasmic olig1 manifested more extensive membrane expansion .", "nuclear olig1 facilitated mbp expression , but with greatly diminished membrane expansion.19 however , these experiments were performed in mouse and human .", "our results demonstrated that olig1 was expressed in o4-positive opcs and -tubulin - positive neurons , but was localized in the cytoplasm during the period from e14.5 to adulthood .", "we speculate that the cause for this inconsistency may be attributed to differences between animal species or other unknown factors ."], ["in conclusion , we systematically explored the expression pattern of olig1 in neural cells during rat spinal cord development .", "our results showed that olig1 was coexpressed with o4-positive opcs and -tubulin - positive neurons at all time points during development .", "olig1 was localized in the cytoplasm of o4- or -tubulin - positive cells during the transition from e14.5 to adult .", "our results contribute to understanding the mechanism of developmental regulation of neural cells by olig1 ."]]}
{"article_id": "PMC5361027", "article_text": ["neck pain is a common musculoskeletal disorder in modern society that can produce severe \n pain .", "67% of the population suffers from it at least once in a lifetime and the prevalence \n is about 23%1 .", "the pain exacerbates and \n fades periodically , and many patients do not fully recover from the symptoms2 .", "neck pain occurs in the upper thoracic \n spine area including the shoulder , and it is mechanical pain caused by bad postures and \n habits in most cases3 . according to \n sharon , the upper thoracic spine is involved in the physiologic motion of the neck4 .", "the decreased movement of the upper \n cervical spine can cause excessive movement of the lower cervical spine , increase fatigue in \n the sternocleidomastoid , anterior scalenus , and upper trapezius , cause changes of neck \n postures and breathing patterns , and a decrease in the range of motion5 .", "patients with chronic neck pain experience functional \n impairments including weakening of deep bending neck muscles due to the activation of neck \n surface muscles6 , increased deformity of \n the forward head posture7 , proprioception \n impairment8 , and poor balance9 .", "in addition , decreased movement of the \n cervical spine restricts the range of motion of the spine and decreases breathing \n function10 , 11 .", "slightly bent positions like the forward head posture may cause \n mechanical neck pain , cause a greater load by affecting the mobilization order of the \n muscles operating when the arm is raised , and restrict the range of motion12 .", "these changes reduce the ability to \n maintain balance and increase the risk of falls and injury of the musculoskeletal \n system9 .", "physiotherapists are using methods such as electrotherapy , therapeutic exercise , and manual \n therapy to intervene in the neck pain13 . \n in a cochrane review study of neck pain , stretching exercises for the neck and upper limbs , \n strengthening exercises , static and dynamic stabilization exercises", "maitland mobilization \n grade 3 and 4 treatment for the cervical spine and upper spine significantly decreases neck \n disability index ( ndi ) , the pain index15 , \n and increases the range of motion16 . in \n addition , an the intervention combining therapeutic exercise and manual therapy was \n significantly more effective compared to manual therapy alone17 .", "recent evidence - based studies report that treatment combining \n therapeutic exercise and manual therapy is more effective18 , but research of combined interventions is sparse .", "the purpose of \n this study was to apply joint mobilization and therapeutic exercise to the cervical spine \n and upper spine , investigate the effects on functional impairments caused by the neck pain , \n and examine differences between groups by comparing the intervention group with the group to \n which only therapeutic exercises were applied .", "this study was implemented with non - specific neck pain patients with no medical findings \n who had visited the cheongju st .", "all participants received verbal and written information about the study and signed a \n consent form .", "the patients who had undergone or would have surgery in the spine , and those who \n had neurological damage , a cervical spine fracture , osteoporosis , arthritis , a malignant \n neoplasm , a vascular disease , or a psychiatric problem were excluded .", "the subjects were \n randomly assigned to one of two groups of nine people each .", "group i was the therapeutic \n exercise group , and group ii was the group to which joint mobilization was applied in \n combination with therapeutic exercise ( table \n 1table 1.general characteristics of the subjects ( n=18)group igroup iigender ( female)99age ( years)58.0  1.659.0  2.4height ( cm)158.0  3.9157.0  4.7weight ( kg)58.0  4.659.0  6.6values are expressed as mean  sd . * significant difference between pre - post test ( p<0.05 ) .", "group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic \n exercise ) ) .", "the pretest included the visual analog scale ( vas ) , neck disability index \n ( ndi ) , active cervical range of motion ( acrom ) , static balance ability , muscle tone of the \n upper trapezius , and respiratory function .", "after the pretest , group i performed therapeutic \n exercise and group ii performed both therapeutic exercise and joint mobilization for 60 \n minutes a day , three times a week for two weeks under the guidance of a physical therapist . \n", "the participants did not receive any other interventions associated with the neck pain while \n this research was being conducted .", "the post - test was carried out with the same protocol as \n the pretest after two weeks .", "values are expressed as mean  sd . * significant difference between pre - post test ( p<0.05 ) .", "group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic \n exercise ) therapeutic exercises involved enhancing mobility , stability and muscular strength of the \n neck , improving proprioception , and performing reeducation of movement , and the intensity of \n exercises was adjusted according to the physical abilities of the individuals . for the joint \n mobilization ,", "after \n the painful sites were located by examination , the active movement test was conducted to \n find where joint mobilization would be applied , and the amount and quality of motion were \n examined .", "afterwards , passive physiological intervertebral movement ( ppivm ) testing and \n passive intervertebral accessory movement ( paivm ) test were conducted to find which joints \n had restricted or excessive movement , and determine where joint mobilization would be \n applied . the vas was employed to measure the intensity of pain19 , the ndi was used to measure neck pain disability20 , and crom instrument to assess the \n cervical range of motion21 . in order to \n check static balance ,", "myoton pro ( myoton as , estonia ) measured \n the muscle tone of the upper trapezius22 , \n and a spirometer ( pony fx spirometer ; cosmed , rome , italy ) measured the respiratory \n function23 .", ", an ibm company , \n chicago , il , usa ) . for the normality test ,", "the shapiro wilk test was conducted , confirming \n that the data are normally distributed .", "assuming homogeneity between groups , the independent \n samples t - test was performed , and the paired sample t - test was conducted for the \n within - group comparison of the measurements before and after the interventions . in addition , \n the independent samples t - test was conducted to compare the differences between groups .", "null \n hypotheses of no difference were rejected if p - values were less than 0.05 .", "vas and ndi led to significant changes in both groups , and group ii improved significantly \n more than group i ( tables 2table 2.visual analog scale of the subjectsgroup igroup iipre4.8  0.44.9  0.3post2.7  0.5 * 1.4  05**significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) , 3table 3.neck disability index of the subjectsgroup igroup iipre15.1  2.317.2  3.1post8.9  1.5 * 8.6  1.9**significant difference between pre - post test ( p<0.05 ) .", "the acrom increased significantly in both groups , and group ii \n improved significantly more on the right lateral flexion and rightward rotation than group i \n ( table 4table 4.active cervical range of motion of the subjects ( )group igroup iiflexionpre49.2  8.748.9  4.7post54.2  4.5 56.1  6.8*extensionpre54.9  4.356.4  4.9post 64.3  3.2 * 67.6  3.9*rt .", "rotationpre60.8  3.259.7  5.1post 67.6  3.6 * 67.7  4.2**significant difference between pre - post test ( p<0.05 ) .", "both groups improved significantly in static balance , with no difference \n between groups ( tables 5table 5.center of gravity sway velocities of each group under different conditions \n ( /s)group igroup iifirm - eyes openpre0.04  0.00.05  0.0 post0.03  0.00.04  0.0firm - eyes closedpre0.15  0.00.15  0.0post0.14  0.00.14 ", " 0.10.37  0.1post 0.30  0.1 * 0.32  0.1**significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) , 6table 6.center of gravity total sway distances of each group under different conditions \n ( /s)group igroup iifirm - eyes openpre294.6  64.1304.6  97.9 post270.4  66.4281.9 ", "109.2foam - eyes closedpre618.2  82.7687.3  182.8post 529.1  88.2 * 548.4  172.5**significant difference between pre - post test ( p<0.05)significant difference between groups ( p<0.05 ) ) .", "muscle tone in the left and right upper trapezius muscle improved \n significantly in both groups , and there was no difference between groups ( table 7table 7.muscle tone of the subjectsgroup igroup iirt .", "upper trapeziuspre16.0  1.5 16.0  1.2post13.7  1.3 * 14.2  1.4**significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "chronic neck pain patients suffer diverse functional impairments , including a deterioration \n of balance ability , respiratory function , range of joint motion , and proprioception11 .", "research related to neck pain suggests \n that there are more efficient interventions based on evidence through systematic reviews , \n and the interventions combining manual therapy and therapeutic exercise are described as \n more effective in recent literature18 . in \n this study , therapeutic exercises were conducted that are reported to be effective for \n patients with mechanical neck pain , namely , stretching exercise for the neck and upper \n limbs , static and dynamic stability exercise , strengthening exercise , and exercise \n reeducation14 .", "in addition , as manual \n therapy , mobilization to reduce pain and improve the movements of the soft tissue exhibiting \n contracture and restricted motions of joints ( maitland grade iii and iv)13 , 24 \n were applied to group ii .", "the effect of the two kinds of interventions , and group \n differences based on those interventions were examined . in this research , a significant \n reduction in the vas and ndi was observed in both groups , and the comparison between the \n groups showed that the intervention was more effective in group ii .", "this result is different \n from studies that found no difference between groups based on a short - term intervention17 .", "these differences may result from our \n smaller number of subjects and differences of individual characteristics .", "for the acrom , \n both groups improved significantly overall , and group ii improved more on right lateral \n flexion and rightward rotation .", "these results are consistent with studies that reported that \n mobilization applied to both the neck and spine at the same time improved cranial vertical \n angle and cranial rotation angle25 and \n research that showed that endurance exercise of the neck , stabilization exercise , and \n strength training improved the joint range of motion26 . in this study ,", "static balance was measured under a variety of conditions , including hard or \n soft bearing surfaces and open or closed eyes . a significant decrease in the sway velocity \n of the center of gravity and in", "the sway distance occurred only under eyes closed , and there \n was no difference between groups . although it was reported that patients with neck pain and \n forward head posture may exhibit reduced balance ability9 , it was possible to measure the static balance ability most \n accurately when the soft bearing surface was used and eyes were closed to block the visual \n feedback .", "muscle tone of the upper trapezius decreased significantly in both groups , and \n there was no difference between the groups .", "this may indicate that the tension and fatigue \n of the upper trapezius were reduced due to pain reduction , enhancement of mobility of the \n neck joints , and change in the mobilization sequence of movements caused by afferent \n information activation of proprioception5 . \n", "deterioration of mobility of the spine and thoracic cage was reported in patients of chronic \n neck pain , and respiratory function was also affected27 . however , in this research , no significant change in the \n respiratory function was observed .", "this may be because the patients were not given direct \n training for functional improvement of breathing muscles , although the range of motion of \n the cervical and upper thoracic spine was improved through the study interventions .", "the number of subjects was small , and since the \n period of intervention was not long , it was difficult to produce a mechanical change of \n muscles . therefore , future research is required to investigate the effect of joint \n mobilization and therapeutic exercise on the functional impairments caused by chronic neck \n pain using diverse subjects and intervention periods , and research on the persistence of the \n effect also needs to be conducted . in conclusion ,", "joint mobilization and therapeutic exercise for functional impairments \n caused by chronic neck pain significantly improved several types of functional impairment . \n", "in addition , in the group to which both joint mobilization and therapeutic exercise were \n applied , significantly more improvement in the pain index , neck disability levels , and acrom \n was seen than in the group that received only therapeutic exercise ."], "abstract_text": ["<S> [ purpose ] this study evaluated joint mobilization and therapeutic exercise applied to the \n cervical spine and upper thoracic spine for functional impairment caused by chronic neck \n pain . </S>", "<S> [ subjects and methods ] eighteen study subjects were randomly assigned to two groups \n of nine people each . </S>", "<S> therapeutic exercise only was applied to the cervical and upper \n thoracic spine for group i , while both therapeutic exercise and joint mobilization were \n applied to group ii . the visual analog scale , neck disability index , active cervical range \n of motion , static balance capacity , and muscle tone were assessed with a pre - test . </S>", "<S> the \n intervention was carried out for 60 minutes a day , three times a week , for two weeks for \n each group , followed by a post - test using the same protocol as the pre - test . </S>", "<S> [ results ] the \n visual analog scale , neck disability index , and active cervical range of motion improved \n significantly in both groups . </S>", "<S> group ii improved significantly more on right lateral \n flexion and rightward rotation . </S>", "<S> muscle tone improved significantly in the upper trapezius \n in both groups . </S>", "<S> [ conclusion ] the joint mobilization and therapeutic exercise for \n functional impairments caused by chronic neck pain had a significant effect on several \n types of functional impairment . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["neck pain is a common musculoskeletal disorder in modern society that can produce severe \n pain .", "67% of the population suffers from it at least once in a lifetime and the prevalence \n is about 23%1 .", "the pain exacerbates and \n fades periodically , and many patients do not fully recover from the symptoms2 .", "neck pain occurs in the upper thoracic \n spine area including the shoulder , and it is mechanical pain caused by bad postures and \n habits in most cases3 . according to \n sharon , the upper thoracic spine is involved in the physiologic motion of the neck4 .", "the decreased movement of the upper \n cervical spine can cause excessive movement of the lower cervical spine , increase fatigue in \n the sternocleidomastoid , anterior scalenus , and upper trapezius , cause changes of neck \n postures and breathing patterns , and a decrease in the range of motion5 .", "patients with chronic neck pain experience functional \n impairments including weakening of deep bending neck muscles due to the activation of neck \n surface muscles6 , increased deformity of \n the forward head posture7 , proprioception \n impairment8 , and poor balance9 .", "in addition , decreased movement of the \n cervical spine restricts the range of motion of the spine and decreases breathing \n function10 , 11 .", "slightly bent positions like the forward head posture may cause \n mechanical neck pain , cause a greater load by affecting the mobilization order of the \n muscles operating when the arm is raised , and restrict the range of motion12 .", "these changes reduce the ability to \n maintain balance and increase the risk of falls and injury of the musculoskeletal \n system9 .", "physiotherapists are using methods such as electrotherapy , therapeutic exercise , and manual \n therapy to intervene in the neck pain13 . \n in a cochrane review study of neck pain , stretching exercises for the neck and upper limbs , \n strengthening exercises , static and dynamic stabilization exercises", "maitland mobilization \n grade 3 and 4 treatment for the cervical spine and upper spine significantly decreases neck \n disability index ( ndi ) , the pain index15 , \n and increases the range of motion16 . in \n addition , an the intervention combining therapeutic exercise and manual therapy was \n significantly more effective compared to manual therapy alone17 .", "recent evidence - based studies report that treatment combining \n therapeutic exercise and manual therapy is more effective18 , but research of combined interventions is sparse .", "the purpose of \n this study was to apply joint mobilization and therapeutic exercise to the cervical spine \n and upper spine , investigate the effects on functional impairments caused by the neck pain , \n and examine differences between groups by comparing the intervention group with the group to \n which only therapeutic exercises were applied ."], ["this study was implemented with non - specific neck pain patients with no medical findings \n who had visited the cheongju st .", "all participants received verbal and written information about the study and signed a \n consent form .", "the patients who had undergone or would have surgery in the spine , and those who \n had neurological damage , a cervical spine fracture , osteoporosis , arthritis , a malignant \n neoplasm , a vascular disease , or a psychiatric problem were excluded .", "the subjects were \n randomly assigned to one of two groups of nine people each .", "group i was the therapeutic \n exercise group , and group ii was the group to which joint mobilization was applied in \n combination with therapeutic exercise ( table \n 1table 1.general characteristics of the subjects ( n=18)group igroup iigender ( female)99age ( years)58.0  1.659.0  2.4height ( cm)158.0  3.9157.0  4.7weight ( kg)58.0  4.659.0  6.6values are expressed as mean  sd . * significant difference between pre - post test ( p<0.05 ) .", "group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic \n exercise ) ) .", "the pretest included the visual analog scale ( vas ) , neck disability index \n ( ndi ) , active cervical range of motion ( acrom ) , static balance ability , muscle tone of the \n upper trapezius , and respiratory function .", "after the pretest , group i performed therapeutic \n exercise and group ii performed both therapeutic exercise and joint mobilization for 60 \n minutes a day , three times a week for two weeks under the guidance of a physical therapist . \n", "the participants did not receive any other interventions associated with the neck pain while \n this research was being conducted .", "the post - test was carried out with the same protocol as \n the pretest after two weeks .", "values are expressed as mean  sd . * significant difference between pre - post test ( p<0.05 ) .", "group i ( therapeautic exercise ) , group ii ( mobilization & therapeautic \n exercise ) therapeutic exercises involved enhancing mobility , stability and muscular strength of the \n neck , improving proprioception , and performing reeducation of movement , and the intensity of \n exercises was adjusted according to the physical abilities of the individuals . for the joint \n mobilization ,", "after \n the painful sites were located by examination , the active movement test was conducted to \n find where joint mobilization would be applied , and the amount and quality of motion were \n examined .", "afterwards , passive physiological intervertebral movement ( ppivm ) testing and \n passive intervertebral accessory movement ( paivm ) test were conducted to find which joints \n had restricted or excessive movement , and determine where joint mobilization would be \n applied . the vas was employed to measure the intensity of pain19 , the ndi was used to measure neck pain disability20 , and crom instrument to assess the \n cervical range of motion21 . in order to \n check static balance ,", "myoton pro ( myoton as , estonia ) measured \n the muscle tone of the upper trapezius22 , \n and a spirometer ( pony fx spirometer ; cosmed , rome , italy ) measured the respiratory \n function23 .", ", an ibm company , \n chicago , il , usa ) . for the normality test ,", "the shapiro wilk test was conducted , confirming \n that the data are normally distributed .", "assuming homogeneity between groups , the independent \n samples t - test was performed , and the paired sample t - test was conducted for the \n within - group comparison of the measurements before and after the interventions . in addition , \n the independent samples t - test was conducted to compare the differences between groups .", "null \n hypotheses of no difference were rejected if p - values were less than 0.05 ."], ["vas and ndi led to significant changes in both groups , and group ii improved significantly \n more than group i ( tables 2table 2.visual analog scale of the subjectsgroup igroup iipre4.8  0.44.9  0.3post2.7  0.5 * 1.4  05**significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) , 3table 3.neck disability index of the subjectsgroup igroup iipre15.1  2.317.2  3.1post8.9  1.5 * 8.6  1.9**significant difference between pre - post test ( p<0.05 ) .", "the acrom increased significantly in both groups , and group ii \n improved significantly more on the right lateral flexion and rightward rotation than group i \n ( table 4table 4.active cervical range of motion of the subjects ( )group igroup iiflexionpre49.2  8.748.9  4.7post54.2  4.5 56.1  6.8*extensionpre54.9  4.356.4  4.9post 64.3  3.2 * 67.6  3.9*rt .", "rotationpre60.8  3.259.7  5.1post 67.6  3.6 * 67.7  4.2**significant difference between pre - post test ( p<0.05 ) .", "both groups improved significantly in static balance , with no difference \n between groups ( tables 5table 5.center of gravity sway velocities of each group under different conditions \n ( /s)group igroup iifirm - eyes openpre0.04  0.00.05  0.0 post0.03  0.00.04  0.0firm - eyes closedpre0.15  0.00.15  0.0post0.14  0.00.14 ", " 0.10.37  0.1post 0.30  0.1 * 0.32  0.1**significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) , 6table 6.center of gravity total sway distances of each group under different conditions \n ( /s)group igroup iifirm - eyes openpre294.6  64.1304.6  97.9 post270.4  66.4281.9 ", "109.2foam - eyes closedpre618.2  82.7687.3  182.8post 529.1  88.2 * 548.4  172.5**significant difference between pre - post test ( p<0.05)significant difference between groups ( p<0.05 ) ) .", "muscle tone in the left and right upper trapezius muscle improved \n significantly in both groups , and there was no difference between groups ( table 7table 7.muscle tone of the subjectsgroup igroup iirt .", "upper trapeziuspre16.0  1.5 16.0  1.2post13.7  1.3 * 14.2  1.4**significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) .", "significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) significant difference between groups ( p<0.05 ) * significant difference between pre - post test ( p<0.05 ) ."], ["chronic neck pain patients suffer diverse functional impairments , including a deterioration \n of balance ability , respiratory function , range of joint motion , and proprioception11 .", "research related to neck pain suggests \n that there are more efficient interventions based on evidence through systematic reviews , \n and the interventions combining manual therapy and therapeutic exercise are described as \n more effective in recent literature18 . in \n this study , therapeutic exercises were conducted that are reported to be effective for \n patients with mechanical neck pain , namely , stretching exercise for the neck and upper \n limbs , static and dynamic stability exercise , strengthening exercise , and exercise \n reeducation14 .", "in addition , as manual \n therapy , mobilization to reduce pain and improve the movements of the soft tissue exhibiting \n contracture and restricted motions of joints ( maitland grade iii and iv)13 , 24 \n were applied to group ii .", "the effect of the two kinds of interventions , and group \n differences based on those interventions were examined . in this research , a significant \n reduction in the vas and ndi was observed in both groups , and the comparison between the \n groups showed that the intervention was more effective in group ii .", "this result is different \n from studies that found no difference between groups based on a short - term intervention17 .", "these differences may result from our \n smaller number of subjects and differences of individual characteristics .", "for the acrom , \n both groups improved significantly overall , and group ii improved more on right lateral \n flexion and rightward rotation .", "these results are consistent with studies that reported that \n mobilization applied to both the neck and spine at the same time improved cranial vertical \n angle and cranial rotation angle25 and \n research that showed that endurance exercise of the neck , stabilization exercise , and \n strength training improved the joint range of motion26 . in this study ,", "static balance was measured under a variety of conditions , including hard or \n soft bearing surfaces and open or closed eyes . a significant decrease in the sway velocity \n of the center of gravity and in", "the sway distance occurred only under eyes closed , and there \n was no difference between groups . although it was reported that patients with neck pain and \n forward head posture may exhibit reduced balance ability9 , it was possible to measure the static balance ability most \n accurately when the soft bearing surface was used and eyes were closed to block the visual \n feedback .", "muscle tone of the upper trapezius decreased significantly in both groups , and \n there was no difference between the groups .", "this may indicate that the tension and fatigue \n of the upper trapezius were reduced due to pain reduction , enhancement of mobility of the \n neck joints , and change in the mobilization sequence of movements caused by afferent \n information activation of proprioception5 . \n", "deterioration of mobility of the spine and thoracic cage was reported in patients of chronic \n neck pain , and respiratory function was also affected27 . however , in this research , no significant change in the \n respiratory function was observed .", "this may be because the patients were not given direct \n training for functional improvement of breathing muscles , although the range of motion of \n the cervical and upper thoracic spine was improved through the study interventions .", "the number of subjects was small , and since the \n period of intervention was not long , it was difficult to produce a mechanical change of \n muscles . therefore , future research is required to investigate the effect of joint \n mobilization and therapeutic exercise on the functional impairments caused by chronic neck \n pain using diverse subjects and intervention periods , and research on the persistence of the \n effect also needs to be conducted . in conclusion ,", "joint mobilization and therapeutic exercise for functional impairments \n caused by chronic neck pain significantly improved several types of functional impairment . \n", "in addition , in the group to which both joint mobilization and therapeutic exercise were \n applied , significantly more improvement in the pain index , neck disability levels , and acrom \n was seen than in the group that received only therapeutic exercise ."]]}
{"article_id": "PMC3417716", "article_text": ["data on the adult ( over 20 years ) population has been reported elsewhere ( 5 ) . the present analysis focused on albertan youth < 20 years of age .", "flagged in the databases and were defined as any alberta inhabitant registered under the federal indian act and entitled to treaty status with the canadian government .", "the status aboriginal identifier captures first nations and inuit peoples both on- and off - reserve with treaty status , but not mtis individuals or aboriginal individuals without treaty status who are included in the general population comparison group .", "alberta law requires that all residents and dependants register with the alberta health care insurance plan , thus the alberta central stakeholder registry functioned as a denominator .", "diabetes cases were identified by applying the national diabetes surveillance system ( ndss ) algorithm ( 13 ) , which has recently been validated for the determination of pediatric diabetes rates ( 14 ) .", "the ndss methodology of identifying has been validated in non - aboriginal populations that include persons of varying geographical locations ( 15,16 ) , and also within an aboriginal community in alberta ( 17 ) .", "the algorithm requires an individual to have either 2 physician visits or 1 hospitalization for diabetes ( icd-9 codes starting with 250 and icd-10 codes e10e14 ) within 2 years to be labelled as case of diabetes ( 13 ) .", "in contrast to the ndss case definition , pregnant women that may have had gestational diabetes were not excluded in the current analysis due to the elevated risk of subsequent diabetes ( 18 ) . also , since the numbers of diabetes cases are small among this population , only crude , unadjusted rates are provided . for both populations ,", "the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year .", "the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years ) was considered an incident year , and considered a prevalent case in subsequent years .", "prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year .", "incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) .", "odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville , md ) for crude values .", "the aapc provides a relative summary measure of the trend over a pre - specified fixed interval .", "tests of parallelism were then performed to determine if trends over time differed by group or sex .", "the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year . the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years )", "prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year .", "incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) .", "odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville ,", "the aapc provides a relative summary measure of the trend over a pre - specified fixed interval .", "tests of parallelism were then performed to determine if trends over time differed by group or sex .", "based on alberta health and wellness administrative databases , 853,733 youth ( 51.2% male ) were living in the province as of 2007 , of which 50,930 were status aboriginal ( 51.2% male ) . among these youth", "a total of 2,589 prevalent diabetes cases were apparent in 2007 , with 140 cases occurring amongst status aboriginal youth .", "the 2007 crude prevalence rate of diabetes was 0.27 and 0.31% in the status aboriginal and general population youth populations respectively ( fig . 1 , table i ) .", "incidence was 0.59 per 1,000 for status aboriginal and 0.49 per 1,000 for general population .", "no discernible differences were seen in diabetes prevalence or incidence with respect to group or sex in 2007 ( fig . 1 ,", "crude rates of diabetes among status aboriginal and general population youth in alberta , 19952007 ( adss ) . a = prevalence ; b = incidence .", "crude diabetes prevalence and incidence among status aboriginal and general population youth , 2007 crude rates of both diabetes prevalence and incidence increased in status aboriginal and general population youth over the observation period ( fig . 1 , table ii ) .", "however , prevalence grew to a greater extent among status aboriginal youth ( aapc 6.98 ) compared to those in the general population ( aapc 3.93 ; p < 0.01 ) , with status aboriginal males experiencing the largest increase in diabetes prevalence ( aapc 9.18 ) .", "ethnic differences in diabetes incidence trends were only observed among the male population , which saw a significantly larger rise in incidence for status aboriginal ( aapc 11.65 ) compared to general population males ( aapc 4.62 ; p < 0.01 ) .", "male youth , regardless of group , experienced greater increases in both prevalence and incidence over time than that of respective female youth ( table ii ) .", "age - specific crude prevalence and incidence of diabetes by group are shown in fig .", "2 . age specific crude rates of diabetes among status aboriginal and general population youth , 1995 and 2007 .", "ethnicity comparisons of aapc ( average annual percent change ) in diabetes prevalence and incidence among status aboriginal and general population youth , 19952007 p < 0.05 for aapc ethnicity comparison .", "paralleling adult populations , diabetes among youth is increasing worldwide ( 20 ) . while type 1 diabetes remains", "the majority of cases in youth , type 2 diabetes ( a disease typically regarded as an adult phenomenon ) is becoming progressively more common in children and adolescents ( 20 ) .", "we report that youth - onset diabetes is an increasing problem in alberta , especially among status aboriginals .", "though our results are in line with international data showing increasing trends ( 20,21 ) , they contradict the recent ndss reports ( 12,22 ) , which found diabetes prevalence and incidence rates remained stable at 0.3 and 0.4 ( per 1,000 ) respectfully among general population youth between 20012006 .", "it is possible that differences in ethnicities and/or socioeconomic parameters contribute to these differences . among indigenous populations", "especially , including american indian / alaskan natives , australian aborigines , the maori of new zealand , and canadian aboriginals , youth - onset diabetes is increasingly being identified ( 14,2327 ) . in canada", ", longitudinal trends of diabetes in aboriginal youth had previously only been explored in manitoba .", "dean and colleagues ( 10 ) showed the crude type 2 diabetes incidence rate rose to 0.55/1,000 ( for ages 019 ) in 2001 . similarly ,", "crude diabetes incidence increased from 0.22 per 1,000 to 0.59 per 1,000 over a 12 year time span in the current analysis .", "the observed accelerated increase in diabetes among status aboriginal youth is consistent with reports of a decrease in the age of diagnosis of diabetes among aboriginal adults . in saskatchewan", "type 2 diabetes incidence was highest among status aboriginal adults aged 4049 , while most new diabetes cases among the general population were in those > 70 years of age ( 4 ) .", "also , as indicated by the first nations and inuit regional health survey , the majority ( 53% ) of those with diabetes in aboriginal communities were < 40 years of age ( 28 ) . compared to general population , diabetes grew approximately twice as much among status aboriginal youth .", "ethnic comparisons have remained largely unexplored in youth , despite numerous studies documenting the divergence in diabetes epidemiology in adult populations . in the beginning of adss observation 1995 , incidence and prevalence of diabetes were distinctly higher among general population . by 2007", "however , status aboriginal youth have  caught up to the point where no detectable differences were apparent , likely ( as mentioned above ) due to type 2 diabetes . reinforcing this notion ,", "a recent national surveillance study amongst practicing physicians found the incidence of type 2 diabetes among aboriginal children ( 0.23/1,000 ) was strikingly 46-fold higher than caucasian children ( 0.005/1,000 ) over a 24 month period ( 29 ) .", "we observed an increased rise of diabetes among young status aboriginal males , confirming earlier studies .", "sex ratios of prevalence and incidence ( female : male ) of approximately 4:1 and 2:1 in 1995 decreased to 1:1 and 1:1 respectively in 2007 .", "additionally , the aapc in prevalence was highest amongst status aboriginal males ( aapc 9.18 ) compared to females ( aapc 5.70 ) . over a 15 year time period in manitoba , the early predominance of youth diabetes cases among aboriginal females ( 8-fold compared to males ) disappeared by 2001 ( 10 ) . from 1990 to 1998", ", american indian and alaskan native young males also experienced relative increases in diabetes prevalence almost double that of their female counterparts ( 30 ) .", "similarly , the gap in diabetes prevalence between adult status aboriginal females and males is also diminishing ( 4,5 ) .", "reasons for the accelerated rise of diabetes among status aboriginal males have not been explored , but may be due in - part to escalating childhood overweight and obesity ( 31 ) with a possible gender - based lag in the obesity epidemic where it may have occurred earlier in time amongst females . in countries with universal healthcare , administrative data have become common and invaluable sources for the population - based surveillance of many chronic diseases .", "ndss methodology has been validated in adults and youth , and is a standard for diabetes monitoring at the population level ( 16,18,32,33 ) .", "for instance , guttmann et al . ( 33 ) recently found the ndss algorithm in youth ( 019 years of age ) in ontario was associated with 100% sensitivity and 94% specificity . using a similar algorithm ( 4", "physician claims over a 2-year period ) , diabetes prevalence rose from 0.18 to 0.24 and incidence ( per 1,000 ) increased from 0.25 to 0.32 among ontarian youth between 1994 and 2003 ( 32 ) . in support , general population youth in the current study experienced nearly identical increases in prevalence and incidence between 1995 and 2003 : 0.19 to 0.25 and 0.22 to 0.32 ( per 1,000 ) respectively , with rates progressively increasing until the end of the study period in 2007 .", "we are unable to distinguish the type of diabetes in our administrative data . however , our clinical experience suggest that the majority of the diabetes in aboriginal youth is type 2 diabetes , whereas in the non - aboriginal population it is heavily dominated by type 1 diabetes .", "moreover , rates of type 1 diabetes are much lower among american indian and canadian aboriginal youth compared to the general population historically ( 34 ) .", "future research aimed at describing the contribution of both type 1 and type 2 diabetes to the observed trends is warranted .", "possible gestational diabetes cases were included and may have inflated rates in status aboriginal as gestational diabetes has been shown to be more common in adult aboriginal populations ( 35 ) .", "it is possible that increased awareness and screening in youth may have contributed to the observed increases , however for various projects ( http//:www.braiddm.ca ) we have screened 799 youth at risk ( mostly aboriginal ) in rural alberta since 2001 and have found only 3 ( 0.4% ) of those had undiagnosed diabetes ( unpublished ) . by contrast , the rates of undiagnosed diabetes are approximately 4% in at risk adults .", "thus , it appears that diabetes in youth is not often asymptomatic , and therefore is less likely to be detected by increased awareness and screening .", "our results can not be generalized to non - registered aboriginal or mtis youth , whom could not be identified and were included in the general population group .", "also , due to the small number of diabetes cases in youth , age - standardized prevalence and incidence rates were not calculated and only crude rates were reported .", "lastly , amendments to the indian act in 1985 are possibly increasing the status aboriginal denominator ( bill c-31 ) and likewise some descendants of status aboriginal people are losing their status through the  three generations  rule ( 36 ) . taken together , prudence is needed when interpreting the observed epidemiological findings . in conclusion", ", diabetes prevalence and incidence has increased among all alberta youth from 1995 to 2007 .", "status aboriginal youth , males in particular , experienced a disproportionate growth in diabetes . if unabated , increasing diabetes in youth will likely only further perpetuate the diabetes epidemic in the status aboriginal population .", "type 2 diabetes is typically the  tip of the iceberg as it is often preceded by several co - morbidities ( obesity , hypertension , metabolic syndrome , pre - diabetes , etc ) , rendering its increased diagnosis in youth a potential public health crisis .", "this work was supported in part by a team grant to the alliance for canadian health outcomes research in diabetes ( achord ; reference # : otg-88588 ) , sponsored by the canadian institutes for health research institute of nutrition , metabolism and diabetes .", "this study is based in part on de - identified data provided by the alberta health and wellness ."], "abstract_text": ["<S> objectivesto track and compare trends in diabetes rates from 1995 to 2007 for status aboriginal and general population youth.study designlongitudinal observational research study ( quantitative ) using provincial administrative data.methodsde-identified data was obtained from alberta health and wellness administrative databases for status aboriginal ( first nations and inuit people with treaty status ) and general population youth ( < 20 years ) . </S>", "<S> diabetes cases were identified using the national diabetes surveillance system algorithm . </S>", "<S> crude annual diabetes prevalence and incidence rates were calculated . </S>", "<S> the likelihood of being a prevalent case and incident case of diabetes for the 2 populations was compared for the year 2007 . </S>", "<S> average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared between the 2 groups to examine trends over time.resultswhile the prevalence of diabetes was higher in the general population in 1995 , by 2007 there were no between group differences , reflected in the significantly higher aapc of 6.98 for status aboriginal youth . status aboriginal males had a lower diabetes risk in 1995 compared with females , and experienced a greater increase in prevalence over the 13 years ( aapc 9.18 ) so that by 2007 their rates were equivalent to those of the females . </S>", "<S> differences in diabetes incidence trends were only observed among male youth , where increases in incidence were greater for status aboriginal ( aapc 11.65 ) compared to general population males ( aapc 4.62 ) ( p = 0.03).conclusionyouth - onset diabetes is an increasing problem in alberta , especially among young status aboriginal males . </S>"], "labels": null, "section_names": ["Methods", "Statistical analysis", "Results", "Discussion", "Conflict of interest and funding"], "sections": [["adss obtained de - identified data for the entire population of alberta from alberta health and wellness administrative databases ( discharge abstract database , alberta physician claims data , ambulatory care classification system and vital statistics ) .", "data on the adult ( over 20 years ) population has been reported elsewhere ( 5 ) . the present analysis focused on albertan youth < 20 years of age .", "flagged in the databases and were defined as any alberta inhabitant registered under the federal indian act and entitled to treaty status with the canadian government .", "the status aboriginal identifier captures first nations and inuit peoples both on- and off - reserve with treaty status , but not mtis individuals or aboriginal individuals without treaty status who are included in the general population comparison group .", "alberta law requires that all residents and dependants register with the alberta health care insurance plan , thus the alberta central stakeholder registry functioned as a denominator .", "diabetes cases were identified by applying the national diabetes surveillance system ( ndss ) algorithm ( 13 ) , which has recently been validated for the determination of pediatric diabetes rates ( 14 ) .", "the ndss methodology of identifying has been validated in non - aboriginal populations that include persons of varying geographical locations ( 15,16 ) , and also within an aboriginal community in alberta ( 17 ) .", "the algorithm requires an individual to have either 2 physician visits or 1 hospitalization for diabetes ( icd-9 codes starting with 250 and icd-10 codes e10e14 ) within 2 years to be labelled as case of diabetes ( 13 ) .", "in contrast to the ndss case definition , pregnant women that may have had gestational diabetes were not excluded in the current analysis due to the elevated risk of subsequent diabetes ( 18 ) . also , since the numbers of diabetes cases are small among this population , only crude , unadjusted rates are provided . for both populations ,", "the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year .", "the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years ) was considered an incident year , and considered a prevalent case in subsequent years .", "prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year .", "incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) .", "odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville , md ) for crude values .", "the aapc provides a relative summary measure of the trend over a pre - specified fixed interval .", "tests of parallelism were then performed to determine if trends over time differed by group or sex ."], ["the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year . the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years )", "prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year .", "incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) .", "odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville ,", "the aapc provides a relative summary measure of the trend over a pre - specified fixed interval .", "tests of parallelism were then performed to determine if trends over time differed by group or sex ."], ["based on alberta health and wellness administrative databases , 853,733 youth ( 51.2% male ) were living in the province as of 2007 , of which 50,930 were status aboriginal ( 51.2% male ) . among these youth", "a total of 2,589 prevalent diabetes cases were apparent in 2007 , with 140 cases occurring amongst status aboriginal youth .", "the 2007 crude prevalence rate of diabetes was 0.27 and 0.31% in the status aboriginal and general population youth populations respectively ( fig . 1 , table i ) .", "incidence was 0.59 per 1,000 for status aboriginal and 0.49 per 1,000 for general population .", "no discernible differences were seen in diabetes prevalence or incidence with respect to group or sex in 2007 ( fig . 1 ,", "crude rates of diabetes among status aboriginal and general population youth in alberta , 19952007 ( adss ) . a = prevalence ; b = incidence .", "crude diabetes prevalence and incidence among status aboriginal and general population youth , 2007 crude rates of both diabetes prevalence and incidence increased in status aboriginal and general population youth over the observation period ( fig . 1 , table ii ) .", "however , prevalence grew to a greater extent among status aboriginal youth ( aapc 6.98 ) compared to those in the general population ( aapc 3.93 ; p < 0.01 ) , with status aboriginal males experiencing the largest increase in diabetes prevalence ( aapc 9.18 ) .", "ethnic differences in diabetes incidence trends were only observed among the male population , which saw a significantly larger rise in incidence for status aboriginal ( aapc 11.65 ) compared to general population males ( aapc 4.62 ; p < 0.01 ) .", "male youth , regardless of group , experienced greater increases in both prevalence and incidence over time than that of respective female youth ( table ii ) .", "age - specific crude prevalence and incidence of diabetes by group are shown in fig .", "2 . age specific crude rates of diabetes among status aboriginal and general population youth , 1995 and 2007 .", "ethnicity comparisons of aapc ( average annual percent change ) in diabetes prevalence and incidence among status aboriginal and general population youth , 19952007 p < 0.05 for aapc ethnicity comparison ."], ["paralleling adult populations , diabetes among youth is increasing worldwide ( 20 ) . while type 1 diabetes remains", "the majority of cases in youth , type 2 diabetes ( a disease typically regarded as an adult phenomenon ) is becoming progressively more common in children and adolescents ( 20 ) .", "we report that youth - onset diabetes is an increasing problem in alberta , especially among status aboriginals .", "though our results are in line with international data showing increasing trends ( 20,21 ) , they contradict the recent ndss reports ( 12,22 ) , which found diabetes prevalence and incidence rates remained stable at 0.3 and 0.4 ( per 1,000 ) respectfully among general population youth between 20012006 .", "it is possible that differences in ethnicities and/or socioeconomic parameters contribute to these differences . among indigenous populations", "especially , including american indian / alaskan natives , australian aborigines , the maori of new zealand , and canadian aboriginals , youth - onset diabetes is increasingly being identified ( 14,2327 ) . in canada", ", longitudinal trends of diabetes in aboriginal youth had previously only been explored in manitoba .", "dean and colleagues ( 10 ) showed the crude type 2 diabetes incidence rate rose to 0.55/1,000 ( for ages 019 ) in 2001 . similarly ,", "crude diabetes incidence increased from 0.22 per 1,000 to 0.59 per 1,000 over a 12 year time span in the current analysis .", "the observed accelerated increase in diabetes among status aboriginal youth is consistent with reports of a decrease in the age of diagnosis of diabetes among aboriginal adults . in saskatchewan", "type 2 diabetes incidence was highest among status aboriginal adults aged 4049 , while most new diabetes cases among the general population were in those > 70 years of age ( 4 ) .", "also , as indicated by the first nations and inuit regional health survey , the majority ( 53% ) of those with diabetes in aboriginal communities were < 40 years of age ( 28 ) . compared to general population , diabetes grew approximately twice as much among status aboriginal youth .", "ethnic comparisons have remained largely unexplored in youth , despite numerous studies documenting the divergence in diabetes epidemiology in adult populations . in the beginning of adss observation 1995 , incidence and prevalence of diabetes were distinctly higher among general population . by 2007", "however , status aboriginal youth have  caught up to the point where no detectable differences were apparent , likely ( as mentioned above ) due to type 2 diabetes . reinforcing this notion ,", "a recent national surveillance study amongst practicing physicians found the incidence of type 2 diabetes among aboriginal children ( 0.23/1,000 ) was strikingly 46-fold higher than caucasian children ( 0.005/1,000 ) over a 24 month period ( 29 ) .", "we observed an increased rise of diabetes among young status aboriginal males , confirming earlier studies .", "sex ratios of prevalence and incidence ( female : male ) of approximately 4:1 and 2:1 in 1995 decreased to 1:1 and 1:1 respectively in 2007 .", "additionally , the aapc in prevalence was highest amongst status aboriginal males ( aapc 9.18 ) compared to females ( aapc 5.70 ) . over a 15 year time period in manitoba , the early predominance of youth diabetes cases among aboriginal females ( 8-fold compared to males ) disappeared by 2001 ( 10 ) . from 1990 to 1998", ", american indian and alaskan native young males also experienced relative increases in diabetes prevalence almost double that of their female counterparts ( 30 ) .", "similarly , the gap in diabetes prevalence between adult status aboriginal females and males is also diminishing ( 4,5 ) .", "reasons for the accelerated rise of diabetes among status aboriginal males have not been explored , but may be due in - part to escalating childhood overweight and obesity ( 31 ) with a possible gender - based lag in the obesity epidemic where it may have occurred earlier in time amongst females . in countries with universal healthcare , administrative data have become common and invaluable sources for the population - based surveillance of many chronic diseases .", "ndss methodology has been validated in adults and youth , and is a standard for diabetes monitoring at the population level ( 16,18,32,33 ) .", "for instance , guttmann et al . ( 33 ) recently found the ndss algorithm in youth ( 019 years of age ) in ontario was associated with 100% sensitivity and 94% specificity . using a similar algorithm ( 4", "physician claims over a 2-year period ) , diabetes prevalence rose from 0.18 to 0.24 and incidence ( per 1,000 ) increased from 0.25 to 0.32 among ontarian youth between 1994 and 2003 ( 32 ) . in support , general population youth in the current study experienced nearly identical increases in prevalence and incidence between 1995 and 2003 : 0.19 to 0.25 and 0.22 to 0.32 ( per 1,000 ) respectively , with rates progressively increasing until the end of the study period in 2007 .", "we are unable to distinguish the type of diabetes in our administrative data . however , our clinical experience suggest that the majority of the diabetes in aboriginal youth is type 2 diabetes , whereas in the non - aboriginal population it is heavily dominated by type 1 diabetes .", "moreover , rates of type 1 diabetes are much lower among american indian and canadian aboriginal youth compared to the general population historically ( 34 ) .", "future research aimed at describing the contribution of both type 1 and type 2 diabetes to the observed trends is warranted .", "possible gestational diabetes cases were included and may have inflated rates in status aboriginal as gestational diabetes has been shown to be more common in adult aboriginal populations ( 35 ) .", "it is possible that increased awareness and screening in youth may have contributed to the observed increases , however for various projects ( http//:www.braiddm.ca ) we have screened 799 youth at risk ( mostly aboriginal ) in rural alberta since 2001 and have found only 3 ( 0.4% ) of those had undiagnosed diabetes ( unpublished ) . by contrast , the rates of undiagnosed diabetes are approximately 4% in at risk adults .", "thus , it appears that diabetes in youth is not often asymptomatic , and therefore is less likely to be detected by increased awareness and screening .", "our results can not be generalized to non - registered aboriginal or mtis youth , whom could not be identified and were included in the general population group .", "also , due to the small number of diabetes cases in youth , age - standardized prevalence and incidence rates were not calculated and only crude rates were reported .", "lastly , amendments to the indian act in 1985 are possibly increasing the status aboriginal denominator ( bill c-31 ) and likewise some descendants of status aboriginal people are losing their status through the  three generations  rule ( 36 ) . taken together , prudence is needed when interpreting the observed epidemiological findings . in conclusion", ", diabetes prevalence and incidence has increased among all alberta youth from 1995 to 2007 .", "status aboriginal youth , males in particular , experienced a disproportionate growth in diabetes . if unabated , increasing diabetes in youth will likely only further perpetuate the diabetes epidemic in the status aboriginal population .", "type 2 diabetes is typically the  tip of the iceberg as it is often preceded by several co - morbidities ( obesity , hypertension , metabolic syndrome , pre - diabetes , etc ) , rendering its increased diagnosis in youth a potential public health crisis ."], ["this work was supported in part by a team grant to the alliance for canadian health outcomes research in diabetes ( achord ; reference # : otg-88588 ) , sponsored by the canadian institutes for health research institute of nutrition , metabolism and diabetes .", "this study is based in part on de - identified data provided by the alberta health and wellness ."]]}
{"article_id": "PMC4884626", "article_text": ["one disability that can sufficient to interfere with activities of daily living is non - syndromic hearing loss ( nshl ) ( 1 , 2 ) . people with even mild nshl have problems hearing speech when there is background noise and identifying the sounds sources ( 3 , 4 ) .", "nshl is the most common sensory deficit in humans , with an incidence of about 1 in 1,000 newborns .", "the prevalence increases during childhood , reaching a rate of 2.7 per 1,000 children before the age of 5 years and 3.5 per 1,000 adolescents .", "two thirds of people with nshl worldwide live in developing countries ( 5 , 6 ) . the transmembrane channel - like 1 ( tmc1 ) gene is considered a member of a gene family predicted to encode transmembrane proteins ( 7 , 8) .", "mutations in the tmc1 gene have been associated with profound prelingual deafness ( dfnb7/b11 ) and progressive postlingual hearing loss ( dfna36 ) ; thy have been reported in different populations ( 9 ) .", "the dfna36 and dfnb7/b11 loci are located on chromosome 9q13 - q21 ( 7 ) .", "tmc1 and tmc2 are members of a gene family predicted to encode transmembrane proteins and are located on p13 of chromosome 20 .", "the tmc1 gene encodes a sodium sensor and may function as ion transport channel or pump .", "tmc1 mrna is specifically expressed in neurosensory hair cells of the inner ear , and it is required for normal function of cochlear hair cells , although the molecular and cellular functions of the tmc1 protein are unknown ( 8) .", "since no report has yet determined the frequency of tmc1 gene mutations in the iranian population , the present study was performed to screen and identify the mutations of this gene associated with nshl using polymerase chain reaction ", "single - stranded conformation polymorphism ( pcr - sscp ) and heteroduplex analysis ( ha ) .", "this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran .", "the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan .", "finally , 100 patients ( one proband from each family ) were selected ( table 1 ) .", "nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) .", "known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research .", "total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280", "nm according to the method described by sambrook and russell ( 12 ) for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 .", "mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) .", "site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples .", "standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) .", "the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna .", "pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes .", "the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method . for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel .", "the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours .", "first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products .", "then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5 , the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) . finally , the heteroduplex fragments were visualized using standard silver nitrate staining . the samples containing shift bands on the sscp gel and", "after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc .", "for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms .", "informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) .", "all data were collected in the statistical program for the social sciences software ( spss , inc . ,", "the mean difference between groups was calculated using a t test . in this study , a p value of  0.05 was considered statistically significant .", "this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran .", "the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan .", "finally , 100 patients ( one proband from each family ) were selected ( table 1 ) .", "nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) .", "known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research .", "total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280 nm according to the method described by sambrook and russell ( 12 )", "for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 .", "mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) .", "site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples .", "standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) .", "the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna .", "pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes . the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method .", "for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel .", "the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours .", "first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products", ". then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5", ", the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) .", "the samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc .", "for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms .", "informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) .", "all data were collected in the statistical program for the social sciences software ( spss , inc . ,", "the mean difference between groups was calculated using a t test . in this study ,", "in this study , blood samples of 100 patients with hearing loss ( mean age 16.5  2.01 years , 49.2% men and 50.8% women , 74.15% married ) were collected from 10 provinces in iran .", "the connexin 26 gene is responsible for a large proportion of deafness ( about 14.6% ) , and these patients had no mutations in this gene ( 10 , 13 ) .", "extracted dna with a 260/280 nm absorbance ratio of 1.8 - 2 was subjected to gene amplification .", "exons 7 and 13 of the tmc1 gene were amplified using the pcr technique with specific and mutated oligonucleotide primers .", "after page , fragments with a length of 187 and 250 bp were revealed for exons 7 and 13 , respectively , of the tmc1 gene .", "pcr products were used for sscp and ha for investigation of mutations in the sequences .", "neither pcr - sscp nor ha showed mutations in exons 7 and 13 of the tmc1 gene ( figures 1 and 2 ) .", "line m is a 100 bp dna ladder ( fermentas , germany ) , line 5 is a sample amplified by mutant primers with typical shift ( positive control ) , and lines 2 - 4 and 6 - 13 are deafness patients samples .", "all specimens have the same template bands without shifts in the sscp bands and after ha .", "line m is a 100 bp dna ladder ( fermentas , germany ) , line 1 is positive control , line 3 is a suspected sample containing different bands compared to other samples , and lines 2 and 4 - 15 are samples from deaf patients . in the present study , to increase the accuracy of the sscp reaction , ha and mutant control ( positive control created by sdm ) were performed .", "only a number of suspected fragments in exon 7 showed a different banding pattern , but after sequencing , the mutations were not confirmed ( figure 3 ) .", "in various human populations , different dominant ( e.g. , coch , dfna5 , and pou4f3 ) and recessive genes ( e.g. , gjb2 and slc26a4 ) have been reported as the cause of hearing loss worldwide ( 7 , 9 , 14 - 16 ) .", "the tmc1 gene is an autosomal recessive gene and a common cause of hearing loss in many countries , such as india , pakistan , algeria , iraq , lebanon , sudan , tunisia , and turkey ( 15 , 17 - 19 ) .", "the prevalence of non - syndromic hearing impairment due to tmc1 in the pakistani population is 4.4% , and one study has indicated that the tmc1 protein might have an important function in the k channels of the inner hair cells ( 16 ) .", "2002 ) detected a 1.6 kb genomic deletion encompassing exon 14 of tmc1 in a recessive deafness mouse mutant , which lacks auditory responses and has hair - cell degeneration ( 7 ) . in the present study , the association of exons 7 and 13 mutations of tmc1 gene locus dfnb7/11 in deaf patients", "after gene amplification , neither sscp nor ha showed mutations in exons 7 and 13 of this gene related to hearing loss in these patients . only a suspected sample in exon 7 had a different banding pattern , but after sequencing , mutations in this exon were not identified .", "the strength of this study was the number of samples , as 890 blood samples were provided ; however , due to a lack of financial resources , we chose two exons of this gene . via the molecular analysis of the tmc1 gene in korean patients ,", "one study showed that this gene was not the cause of nshl in the korean population ( 20 ) . in our study , as in the research in korea , a relation between the tmc1 gene and nshl in the iranian population was not observed .", "( 2005 ) investigated four novel tmc1 ( dfnb7/dfnb11 ) mutations in turkish patients with congenital autosomal recessive nshl ( arnshl ) , and they indicated that tmc1 mutations account for at least 6% ( 4/65 ) of arnshl cases in gjb2-negative turkish families from the northeast and east of turkey ; however , in our study , mutation in exons 7 and 13 of tmc1 gene was not observed ( 14 ) .", "in another study in sudan , it was shown that tmc1 mutations contribute to deafness ; this confirmed and extended previous reports on the role of tmc1 in recessive non - syndromic deafness ( 20 , 21 ) .", "meanwhile , in the present study on the iranian population , no significant relation between exon 7 and 13 mutations of the tmc1 gene were observed . in pakistan ,", "2007 ) identified 10 new families segregating dfnb7/b11 deafness and tmc1 mutations , including three novel alleles ; moreover , they identified a c.100c > t mutation in exon 7 ( 16 ) .", "2008 ) identified four new mutations in the tmc1 gene and suggested an additional deafness gene at loci dfna36 and dfnb7/11 in 51 familial of turkish patients with autosomal recessive hearing loss ; these results implied the presence of mutations outside the coding region of this gene , or alternatively , at least one additional deafness - causing gene in this region ( 9 ) .", "another study in turkey reported five novel mutations in the tmc1 gene related to arnshl ( 19 ) . in comparison with", "the other populations discussed , in a review study in iran by mahdieh et al .", "the frequencies and distributions of nshl included gjb2 , gjb6 ( large deletion ) , tecta , slc26a4 , and pejvk mutations .", "the researchers indicated that mutations in gjb2 , slc26 , tecta , and pjvk genes have an important role in deafness in iran , and a screening test should be generated for better intervention and diagnostic programs ( 22 ) .", "the study of hildebrand et al . in iranian families showed that two families are related to locus dfnb7/11 , and one of them had a c.776 + 1 g > a mutation in exon 7 ( 23 ) . in 2014 , lin et al . identified novel compound heterozygous mutant alleles of tmc1 responsible for arnshl in a tibetan chinese family ( 24 ) ; meanwhile , in the present work , we did not observe a relation with mutations of exons 7 and 13 of the tmc1 gene in iranian nshl .", "the findings of the current study indicate that mutations in exons 7 and 13 of tmc1 gene are not related to hearing loss in the iranian population .", "therefore , the tmc1 gene may not related to nshl , but further studies investigating related mutations in other parts of this gene in iranian population are necessary and could help in the genetic counseling of patients and design of practical strategies for the management of auditory disorder ."], "abstract_text": ["<S> background : non - syndromic hearing loss ( nshl ) is the most common birth defect and occurs in approximately 1/1,000 newborns . </S>", "<S> nshl is a heterogeneous trait and can arise due to both genetic and environmental factors . </S>", "<S> mutations of the transmembrane channel - like 1 ( tmc1 ) gene cause non - syndromic deafness in humans and mice.objectives:the aim of the present study was to investigate the association of tmc1 gene mutations of the locus dfnb7/11 in exons 7 and 13 in a cohort of 100 patients with hearing loss in iran using polymerase chain reaction  single - stranded conformation polymorphism ( pcr - sscp ) , heteroduplex analysis ( ha ) , and dna sequencing.patients and methods : in this experimental study , the blood samples of 100 nshl patients were collected from 10 provinces in iran . </S>", "<S> these patients had a mean age of 16.5  2.01 years and 74.15% of their parents had consanguinity . </S>", "<S> dna was extracted from specimens and mutations of exons 7 and 13 of the tmc1 gene were investigated using pcr - sscp . </S>", "<S> all samples were checked via ha reaction and suspected specimens with shift bands were subjected to dna sequencing for investigation of any gene variation.results:in this study , no mutation was found in the two exons of tmc1 gene . </S>", "<S> it was concluded from these results that mutations of the tmc1 gene s special exons 7 and 13 have a low contribution in patients and are not great of clinical importance in these iranian provinces.conclusions:more studies are needed to investigate the relationship between other parts of this gene with hearing loss in different populations through the country . </S>", "<S> more research could clarify the role of this gene and its relation with deafness and provide essential information for the prevention and management of auditory disorders caused by genetic factors in the iranian population . </S>"], "labels": null, "section_names": ["1. Background", "2. Objectives", "3. Patients and Methods", "3.1. Sampling", "3.2. DNA Extraction", "3.3. Gene Amplification", "3.4. SSCP Analysis", "3.5. Heteroduplex Analysis (HA)", "3.6. Sequencing of Amplified Products", "3.7. Ethical Approval", "3.8. Statistical Analysis", "4. Results", "5. Discussion"], "sections": [["one disability that can sufficient to interfere with activities of daily living is non - syndromic hearing loss ( nshl ) ( 1 , 2 ) . people with even mild nshl have problems hearing speech when there is background noise and identifying the sounds sources ( 3 , 4 ) .", "nshl is the most common sensory deficit in humans , with an incidence of about 1 in 1,000 newborns .", "the prevalence increases during childhood , reaching a rate of 2.7 per 1,000 children before the age of 5 years and 3.5 per 1,000 adolescents .", "two thirds of people with nshl worldwide live in developing countries ( 5 , 6 ) . the transmembrane channel - like 1 ( tmc1 ) gene is considered a member of a gene family predicted to encode transmembrane proteins ( 7 , 8) .", "mutations in the tmc1 gene have been associated with profound prelingual deafness ( dfnb7/b11 ) and progressive postlingual hearing loss ( dfna36 ) ; thy have been reported in different populations ( 9 ) .", "the dfna36 and dfnb7/b11 loci are located on chromosome 9q13 - q21 ( 7 ) .", "tmc1 and tmc2 are members of a gene family predicted to encode transmembrane proteins and are located on p13 of chromosome 20 .", "the tmc1 gene encodes a sodium sensor and may function as ion transport channel or pump .", "tmc1 mrna is specifically expressed in neurosensory hair cells of the inner ear , and it is required for normal function of cochlear hair cells , although the molecular and cellular functions of the tmc1 protein are unknown ( 8) ."], ["since no report has yet determined the frequency of tmc1 gene mutations in the iranian population , the present study was performed to screen and identify the mutations of this gene associated with nshl using polymerase chain reaction ", "single - stranded conformation polymorphism ( pcr - sscp ) and heteroduplex analysis ( ha ) ."], ["this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran .", "the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan .", "finally , 100 patients ( one proband from each family ) were selected ( table 1 ) .", "nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) .", "known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research .", "total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280", "nm according to the method described by sambrook and russell ( 12 ) for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 .", "mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) .", "site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples .", "standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) .", "the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna .", "pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes .", "the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method . for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel .", "the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours .", "first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products .", "then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5 , the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) . finally , the heteroduplex fragments were visualized using standard silver nitrate staining . the samples containing shift bands on the sscp gel and", "after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc .", "for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms .", "informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) .", "all data were collected in the statistical program for the social sciences software ( spss , inc . ,", "the mean difference between groups was calculated using a t test . in this study , a p value of  0.05 was considered statistically significant ."], ["this experimental study was conducted at the cellular and molecular research center of shahrekord university of medical sciences from february 2011 to january 2012 . in the present study , we investigated the mutations of the tmc1 gene , locus dfnb7/11 , in a cohort of 100 patients with nshl in iran .", "the 890 blood samples of families with iranian origin was obtained in ethylene diamine tetra - acetic acid ( edta)-containing tubes ( sarstedt ) from 10 provinces of iran , namely semnan , sistan & baloochestan , fars , khozestan , kohgilooye va boyer ahmad , kordestan , chaharmahal & bakhtiari , booshehr , golestan , and gilan .", "finally , 100 patients ( one proband from each family ) were selected ( table 1 ) .", "nshl informational questionnaires were filled out for all families . in previous work , these patients had no mutations in the cx26 gene ( 10 ) .", "known environmental risk factors such as head trauma and use of ototoxic drugs could affect the study , so families with the possibility of exposure to these factors were excluded from the research ."], ["total genomic dna was extracted from peripheral blood samples of patients using the phenol and chloroform standard procedure ( 11 ) . the quality of extracted genomic dna was quantified by nano - drop 1000 spectrophotometer ( thermo scientific inc . , wilmington , de , usa ) at a wavelength of 260/280 nm according to the method described by sambrook and russell ( 12 )"], ["for gene amplification of exons 7 and 13 of the tmc1 gene by pcr , two sets of overlapping primers were designed due to their length using the gene runner software version 3.0 ( hastings software , inc . , hastings , ny ) , and primer sequences were blasted in the national center for biotechnology information s ( ncbi s ) genbank . the details of the designed primers are shown in table 2 .", "mutant primers created by site - directed mutagenesis ( sdm ) as positive control , tmc1-m ( tmc1 mutant primer ) .", "site - directed mutagenesis ( sdm ) after gene amplification using the designed specific primers with changes in one nucleotide was used to generate positive control samples .", "standard pcr optimization was carried out in a total volume of 50 l reaction in 0.5 ml tubes for each amplicon in a gradient palm cycler ( corbett research , australia ) .", "the pcr reaction consisted of 0.2 pm of each primer , 2.5 mm mgcl2 , 200 m dntps mix , 5 m of 10 x pcr buffer ( 200 mm tris - hcl [ ph 8.4 ] , 500 mm kcl ) , 1 unit of taq dna polymerase ( all fermentas , germany ) , and 100 ng of template dna .", "pcr temperature programs involved an initial denaturation at 94c for 5 minutes followed by 32 cycles consisting of 50 seconds of denaturation at 94c , 50 seconds of annealing at 57.5c ( exon 7 ) or 60c ( exon 13 ) , 40 seconds of extension at 72c , and a final extension at 72c for 5 minutes . the pcr amplification products ( 2 l / lane ) were loaded on 8% polyacrylamide gel ( 29:1 acrylamide : bis - acrylamide ) electrophoresis ( page ) in 1 x tbe buffer ( 10.8 g of tris - base 89 mm , 5.5 g of boric acid 2 mm , edta ( ph = 8.0 ) 4 ml of 0.5 m edta ( ph = 8.0 ) , combined all components in sufficient h2o and were stirred to dissolve ) at 85 v for 30 minutes , and the gels were stained using the silver nitrate staining method ."], ["for sscp , microtubes containing pcr products were mixed with an equal volume of formamide loading dye , heated to 96c for 15 minutes , and chilled on ice for 5 minutes before loading on the polyacrylamide gel .", "the electrophoresis tank was filled by tbe buffer 0.6 x , and tbe 1 x was used in the gel ; electrophoresis was performed with 2.5 - 5% glycerol at 20 w at 4c for 6 - 8 hours ."], ["first , 3 l of edta ( 0.5 m ) was added to 2 l of pcr products", ". then , the pcr products were heated at 95c for 3 minutes and slowly cooled to 37c over 40 minutes . after mixing with 6 x triple dye loading buffer at a volume ratio of 1:5", ", the pcr products were loaded on page with 10% urea and electrophoresis were performed at 320 v at 10 - 12c for 6 - 8 hours ( table 3 ) ."], ["the samples containing shift bands on the sscp gel and after ha were subjected to direct dna sequencing of exons 7 and 13 of the tmc1 gene in an abi 3730xl automated sequencer ( applied biosystems ) by macrogen inc ."], ["for this study , the regional research ethical committee of shahrekord university of medical sciences ( grant number 91 - 3 - 2 , january 2011 ) approved the protocol and informed consent forms .", "informed consent was obtained from all hearing loss patients before enrollment in the study based on the declaration of helsinki ( doh ) ."], ["all data were collected in the statistical program for the social sciences software ( spss , inc . ,", "the mean difference between groups was calculated using a t test . in this study ,"], ["in this study , blood samples of 100 patients with hearing loss ( mean age 16.5  2.01 years , 49.2% men and 50.8% women , 74.15% married ) were collected from 10 provinces in iran .", "the connexin 26 gene is responsible for a large proportion of deafness ( about 14.6% ) , and these patients had no mutations in this gene ( 10 , 13 ) .", "extracted dna with a 260/280 nm absorbance ratio of 1.8 - 2 was subjected to gene amplification .", "exons 7 and 13 of the tmc1 gene were amplified using the pcr technique with specific and mutated oligonucleotide primers .", "after page , fragments with a length of 187 and 250 bp were revealed for exons 7 and 13 , respectively , of the tmc1 gene .", "pcr products were used for sscp and ha for investigation of mutations in the sequences .", "neither pcr - sscp nor ha showed mutations in exons 7 and 13 of the tmc1 gene ( figures 1 and 2 ) .", "line m is a 100 bp dna ladder ( fermentas , germany ) , line 5 is a sample amplified by mutant primers with typical shift ( positive control ) , and lines 2 - 4 and 6 - 13 are deafness patients samples .", "all specimens have the same template bands without shifts in the sscp bands and after ha .", "line m is a 100 bp dna ladder ( fermentas , germany ) , line 1 is positive control , line 3 is a suspected sample containing different bands compared to other samples , and lines 2 and 4 - 15 are samples from deaf patients . in the present study , to increase the accuracy of the sscp reaction , ha and mutant control ( positive control created by sdm ) were performed .", "only a number of suspected fragments in exon 7 showed a different banding pattern , but after sequencing , the mutations were not confirmed ( figure 3 ) ."], ["in various human populations , different dominant ( e.g. , coch , dfna5 , and pou4f3 ) and recessive genes ( e.g. , gjb2 and slc26a4 ) have been reported as the cause of hearing loss worldwide ( 7 , 9 , 14 - 16 ) .", "the tmc1 gene is an autosomal recessive gene and a common cause of hearing loss in many countries , such as india , pakistan , algeria , iraq , lebanon , sudan , tunisia , and turkey ( 15 , 17 - 19 ) .", "the prevalence of non - syndromic hearing impairment due to tmc1 in the pakistani population is 4.4% , and one study has indicated that the tmc1 protein might have an important function in the k channels of the inner hair cells ( 16 ) .", "2002 ) detected a 1.6 kb genomic deletion encompassing exon 14 of tmc1 in a recessive deafness mouse mutant , which lacks auditory responses and has hair - cell degeneration ( 7 ) . in the present study , the association of exons 7 and 13 mutations of tmc1 gene locus dfnb7/11 in deaf patients", "after gene amplification , neither sscp nor ha showed mutations in exons 7 and 13 of this gene related to hearing loss in these patients . only a suspected sample in exon 7 had a different banding pattern , but after sequencing , mutations in this exon were not identified .", "the strength of this study was the number of samples , as 890 blood samples were provided ; however , due to a lack of financial resources , we chose two exons of this gene . via the molecular analysis of the tmc1 gene in korean patients ,", "one study showed that this gene was not the cause of nshl in the korean population ( 20 ) . in our study , as in the research in korea , a relation between the tmc1 gene and nshl in the iranian population was not observed .", "( 2005 ) investigated four novel tmc1 ( dfnb7/dfnb11 ) mutations in turkish patients with congenital autosomal recessive nshl ( arnshl ) , and they indicated that tmc1 mutations account for at least 6% ( 4/65 ) of arnshl cases in gjb2-negative turkish families from the northeast and east of turkey ; however , in our study , mutation in exons 7 and 13 of tmc1 gene was not observed ( 14 ) .", "in another study in sudan , it was shown that tmc1 mutations contribute to deafness ; this confirmed and extended previous reports on the role of tmc1 in recessive non - syndromic deafness ( 20 , 21 ) .", "meanwhile , in the present study on the iranian population , no significant relation between exon 7 and 13 mutations of the tmc1 gene were observed . in pakistan ,", "2007 ) identified 10 new families segregating dfnb7/b11 deafness and tmc1 mutations , including three novel alleles ; moreover , they identified a c.100c > t mutation in exon 7 ( 16 ) .", "2008 ) identified four new mutations in the tmc1 gene and suggested an additional deafness gene at loci dfna36 and dfnb7/11 in 51 familial of turkish patients with autosomal recessive hearing loss ; these results implied the presence of mutations outside the coding region of this gene , or alternatively , at least one additional deafness - causing gene in this region ( 9 ) .", "another study in turkey reported five novel mutations in the tmc1 gene related to arnshl ( 19 ) . in comparison with", "the other populations discussed , in a review study in iran by mahdieh et al .", "the frequencies and distributions of nshl included gjb2 , gjb6 ( large deletion ) , tecta , slc26a4 , and pejvk mutations .", "the researchers indicated that mutations in gjb2 , slc26 , tecta , and pjvk genes have an important role in deafness in iran , and a screening test should be generated for better intervention and diagnostic programs ( 22 ) .", "the study of hildebrand et al . in iranian families showed that two families are related to locus dfnb7/11 , and one of them had a c.776 + 1 g > a mutation in exon 7 ( 23 ) . in 2014 , lin et al . identified novel compound heterozygous mutant alleles of tmc1 responsible for arnshl in a tibetan chinese family ( 24 ) ; meanwhile , in the present work , we did not observe a relation with mutations of exons 7 and 13 of the tmc1 gene in iranian nshl .", "the findings of the current study indicate that mutations in exons 7 and 13 of tmc1 gene are not related to hearing loss in the iranian population .", "therefore , the tmc1 gene may not related to nshl , but further studies investigating related mutations in other parts of this gene in iranian population are necessary and could help in the genetic counseling of patients and design of practical strategies for the management of auditory disorder ."]]}
{"article_id": "PMC4377145", "article_text": ["microleakage between the root canal filling and root - canal walls may adversely affects the results of root - canal treatment.1 apical leakage is considered to be common cause of endodontic failure.2 hence , different endodontic filling materials , sealers and techniques have been introduced to the dental community in an attempt to improve apical seal.1 various materials have been used in root canal treatment in an attempt to achieve success .", "but , a combination of gutta - percha and a sealer are used most commonly .", "gutta - percha is considered an impermeable core material ; therefore , leakage through an obturated root canal is expected to take place at the interfaces between the sealer and dentin or the sealer and gutta - percha , or through voids within the sealer.3 apical sealing is desirable to prevent passage of bacteria and their endodotoxin apically . in vitro evaluation of apical dye penetration", "is used to estimate the sealing ability which is corresponding to in vivo amount of micro leakage with particular sealer.2 many techniques were used to evaluate the leakage of sealers such as ; colored dye penetration radio labeled tracer penetration dissolution of hard tissue clearing of teeth , spectrometry of radioisotopes electrochemical and gas chromatography .", "however , many studies showed no significant difference between these techniques.1 the aim of this in vitro study was to quantitatively evaluate the sealing properties of three different root - canal sealers ; tubliseal , sealapex and ah26 using a spectrophotometric method .", "thirty - six extracted sound mandibular molar natural teeth specimens with complete root and free from caries or cracks were collected , stored , disinfected and handled as per the recommendations and guidelines laid down by occupational safety and health administration and centers for disease control and prevention.4 the teeth were then placed in 0.9% physiologic saline solution for ten days prior to access cavity preparation .", "the samples were divided into three experimental groups ; group i : sealapex , group ii : tubliseal and group iii : ah26 with 12 samples in each group .", "the samples were then coated with nail varnish all over the root surface except 2 mm around the apical foramen . 2 ml of freshly prepared 2% methylene blue dye was taken in each vial and the apical third of the root was suspended in the dye for 72 h. samples were washed with distilled water , nail varnish removed and then placed in 20 ml of 35% nitric acid for 72 h. standard solutions of 1% , 0.5% , 0.2% , 0.05% , 0.02% and 0.01% of methylene blue in 35% nitric acid were prepared and stored for 72 h. the standard solutions and the nitric acid solutions were filtered and centrifuged for 1 min after 72 h. the supernatant was subjected to spectrophotometric analysis using a filter of 670 nm .", "the amount of leakage was extrapolated from a standard linear regression curve constructed from stock standard methylene blue dye solutions .", "obtained data were statistical analyzed with kruskal  wallis and mann  whitney u - tests using spss software version 20 ( ibm ) .", "the results of the quantitative evaluation of the sealing properties of the three root - canal sealers are shown in table 1 .", ", tubliseal showed a significant difference ( p > 0.005 ) . in comparison to sealapex", "comparisons made between tubliseal and ah26 showed no significant difference ( table 2 , chart 1 ) . mean and standard deviation values of volumetric dye penetration in groups with reference to transmission .", "three - dimensional obturation of the root canal system with a fluid impervious seal is an important factor for successful endodontic therapy .", "the root canal filling should seal the canal both apically and coronally to prevent the passage of microorganism to apex or vice versa.5 most reliable method is the use of gutta - percha cones with sealer cement .", "sealers based on zinc oxide - eugenol ( tubliseal ) , calcium hydroxide ( sealapex ) , epoxy resins ( ah26 ) were included in the present study . in present quantitative dye leakage study ,", "tubliseal demonstrated least dye leakage in comparison with other experimental groups ( table 1 ) .", "our results were in contrary to study by masoud and saleh where they found more microleakage in tubliseal group than other groups.6 sealapex is a calcium hydroxide type sealer .", "calcium hydroxide used as root canal sealer since it stimulates periapical tissues in order to maintain health or promote healing and secondly for its antimicrobial effects.7 it has been observed in some studies that , calcium hydroxide sealers showed a significant volumetric expansion during setting because of water absorption , which increases its solubility .", "the present in vitro investigation indicated maximum leakage value with sealapex among the experimental groups . in a contradictory to our results , cobankara et al .", "( 2006 ) observed sealapex with better apical sealing than the other sealers ( ah plus and rc sealer ) at 7 , 14 , and 21 days.3 ah26 is an epoxy resin based sealer that provides easy handling characteristics , good flow , good sealing to dentin and prominent antimicrobial activity.6 kumar et al . observed more micro leakage with zinc oxide - based sealer and least with resin based sealers , this is contradictory to our study.8 in our study , there was no statistical significant difference between tubliseal and ah26 in micro leakage .", "observed least amount of dye penetration for ah plus and endorez group.9 even though , the current study did not indicate any statistically significant difference between ah26 and sealapex , it is time to question the overall efficacy of calcium hydroxide sealers on the grounds that the reparative and the calcification capabilities attributed to calcium hydroxide are generally desirable before completing the obturation .", "ah26 , resin based sealer provided a better apical seal when compared with sealapex even though the results were statistically not significant .", "it mixes easily , flows well , and has ample working time , good radio - opacity , comparable solubility , good adhesion and good biocompatibility.3 in the present study , there was no significant difference between group ii and iii and group i and iii .", "did nt find any significant difference between the tested three groups ; sealer 26 , enfoflas and resin group .", "but they observed higher microleakage in sealer 26 group compared with control.11 joseph and sing evaluated the apical sealing with four root canal sealers ; ah26 , sealapex , endoflas fs and ah plus and observed no significant differences between all groups except between ah plus and endoflas.5 nagas et al .", "observed significantly lower overall leakage with ah plus group , whereas no difference was found between master cone points.12 kopper et al . observed significant dye penetration for ah plus , endofill and sealer 26.13 cobankara et al .", "observed better sealing values for roekoseal after 21 days when compared to ketac - endo and ah plus , and there was no statistically significant difference.1 dultra et al . found no statistical difference between groups for apical leakage ( endofill , ah plus , endorez and epiphany).14 in addition , before accepting a new material for routine clinical use further experiments", "should also be performed to evaluate the other aspects of the materials physical and biological properties such as biocompatibility , solubility , disintegration , radio - opacity and dimensional stability . however", ", these in vitro studies do provide comparative information of the relative performance of sealers tested under the same conditions in each particular study and clinicians can use this information to possibly choose a better sealer .", "in the present study , tubliseal sealer showed least microleage compared with sealapex and ah26 sealer .", "it is important to remember before declaring any root canal sealer as most acceptable that the results of the dye penetration studies indicate only the relative sealing ability of root canal fillings in vitro and they do not indicate their ability to prevent the penetration of bacteria into filled root canals in vivo ."], "abstract_text": ["<S> background : the aim of this study was to quantitatively analyze the amount of dye leakage with ah26 , sealapex and tubliseal sealers in endodonticaly treated teeth.materials and methods : a total of 36 extracted mandibular molar specimens were divided into three groups ; group i : sealapex , group ii : tubliseal , group ii : ah26 with 12 samples in each group . </S>", "<S> standard access cavity and biomechanical preparation was done with step back flare technique . </S>", "<S> obturations were done using respective sealers in the three different groups . </S>", "<S> then , samples were subjected to spectro photometric analysis using a filter of 670 nm . </S>", "<S> spectrophotometric analysis was performed to quantitatively analyze the amount of dye leakage with all three sealers.results:tubliseal exhibited the least microleakage . in comparison to sealapex </S>", "<S> , tubliseal showed a significant difference . compared to sealapex </S>", "<S> , ah26 showed no significant difference . </S>", "<S> comparisons made between tubliseal and ah26 showed no significant difference.conclusion:in the present study , tubliseal sealer showed least microleage compared with sealapex and ah26 sealer . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion"], "sections": [["microleakage between the root canal filling and root - canal walls may adversely affects the results of root - canal treatment.1 apical leakage is considered to be common cause of endodontic failure.2 hence , different endodontic filling materials , sealers and techniques have been introduced to the dental community in an attempt to improve apical seal.1 various materials have been used in root canal treatment in an attempt to achieve success .", "but , a combination of gutta - percha and a sealer are used most commonly .", "gutta - percha is considered an impermeable core material ; therefore , leakage through an obturated root canal is expected to take place at the interfaces between the sealer and dentin or the sealer and gutta - percha , or through voids within the sealer.3 apical sealing is desirable to prevent passage of bacteria and their endodotoxin apically . in vitro evaluation of apical dye penetration", "is used to estimate the sealing ability which is corresponding to in vivo amount of micro leakage with particular sealer.2 many techniques were used to evaluate the leakage of sealers such as ; colored dye penetration radio labeled tracer penetration dissolution of hard tissue clearing of teeth , spectrometry of radioisotopes electrochemical and gas chromatography .", "however , many studies showed no significant difference between these techniques.1 the aim of this in vitro study was to quantitatively evaluate the sealing properties of three different root - canal sealers ; tubliseal , sealapex and ah26 using a spectrophotometric method ."], ["thirty - six extracted sound mandibular molar natural teeth specimens with complete root and free from caries or cracks were collected , stored , disinfected and handled as per the recommendations and guidelines laid down by occupational safety and health administration and centers for disease control and prevention.4 the teeth were then placed in 0.9% physiologic saline solution for ten days prior to access cavity preparation .", "the samples were divided into three experimental groups ; group i : sealapex , group ii : tubliseal and group iii : ah26 with 12 samples in each group .", "the samples were then coated with nail varnish all over the root surface except 2 mm around the apical foramen . 2 ml of freshly prepared 2% methylene blue dye was taken in each vial and the apical third of the root was suspended in the dye for 72 h. samples were washed with distilled water , nail varnish removed and then placed in 20 ml of 35% nitric acid for 72 h. standard solutions of 1% , 0.5% , 0.2% , 0.05% , 0.02% and 0.01% of methylene blue in 35% nitric acid were prepared and stored for 72 h. the standard solutions and the nitric acid solutions were filtered and centrifuged for 1 min after 72 h. the supernatant was subjected to spectrophotometric analysis using a filter of 670 nm .", "the amount of leakage was extrapolated from a standard linear regression curve constructed from stock standard methylene blue dye solutions .", "obtained data were statistical analyzed with kruskal  wallis and mann  whitney u - tests using spss software version 20 ( ibm ) ."], ["the results of the quantitative evaluation of the sealing properties of the three root - canal sealers are shown in table 1 .", ", tubliseal showed a significant difference ( p > 0.005 ) . in comparison to sealapex", "comparisons made between tubliseal and ah26 showed no significant difference ( table 2 , chart 1 ) . mean and standard deviation values of volumetric dye penetration in groups with reference to transmission ."], ["three - dimensional obturation of the root canal system with a fluid impervious seal is an important factor for successful endodontic therapy .", "the root canal filling should seal the canal both apically and coronally to prevent the passage of microorganism to apex or vice versa.5 most reliable method is the use of gutta - percha cones with sealer cement .", "sealers based on zinc oxide - eugenol ( tubliseal ) , calcium hydroxide ( sealapex ) , epoxy resins ( ah26 ) were included in the present study . in present quantitative dye leakage study ,", "tubliseal demonstrated least dye leakage in comparison with other experimental groups ( table 1 ) .", "our results were in contrary to study by masoud and saleh where they found more microleakage in tubliseal group than other groups.6 sealapex is a calcium hydroxide type sealer .", "calcium hydroxide used as root canal sealer since it stimulates periapical tissues in order to maintain health or promote healing and secondly for its antimicrobial effects.7 it has been observed in some studies that , calcium hydroxide sealers showed a significant volumetric expansion during setting because of water absorption , which increases its solubility .", "the present in vitro investigation indicated maximum leakage value with sealapex among the experimental groups . in a contradictory to our results , cobankara et al .", "( 2006 ) observed sealapex with better apical sealing than the other sealers ( ah plus and rc sealer ) at 7 , 14 , and 21 days.3 ah26 is an epoxy resin based sealer that provides easy handling characteristics , good flow , good sealing to dentin and prominent antimicrobial activity.6 kumar et al . observed more micro leakage with zinc oxide - based sealer and least with resin based sealers , this is contradictory to our study.8 in our study , there was no statistical significant difference between tubliseal and ah26 in micro leakage .", "observed least amount of dye penetration for ah plus and endorez group.9 even though , the current study did not indicate any statistically significant difference between ah26 and sealapex , it is time to question the overall efficacy of calcium hydroxide sealers on the grounds that the reparative and the calcification capabilities attributed to calcium hydroxide are generally desirable before completing the obturation .", "ah26 , resin based sealer provided a better apical seal when compared with sealapex even though the results were statistically not significant .", "it mixes easily , flows well , and has ample working time , good radio - opacity , comparable solubility , good adhesion and good biocompatibility.3 in the present study , there was no significant difference between group ii and iii and group i and iii .", "did nt find any significant difference between the tested three groups ; sealer 26 , enfoflas and resin group .", "but they observed higher microleakage in sealer 26 group compared with control.11 joseph and sing evaluated the apical sealing with four root canal sealers ; ah26 , sealapex , endoflas fs and ah plus and observed no significant differences between all groups except between ah plus and endoflas.5 nagas et al .", "observed significantly lower overall leakage with ah plus group , whereas no difference was found between master cone points.12 kopper et al . observed significant dye penetration for ah plus , endofill and sealer 26.13 cobankara et al .", "observed better sealing values for roekoseal after 21 days when compared to ketac - endo and ah plus , and there was no statistically significant difference.1 dultra et al . found no statistical difference between groups for apical leakage ( endofill , ah plus , endorez and epiphany).14 in addition , before accepting a new material for routine clinical use further experiments", "should also be performed to evaluate the other aspects of the materials physical and biological properties such as biocompatibility , solubility , disintegration , radio - opacity and dimensional stability . however", ", these in vitro studies do provide comparative information of the relative performance of sealers tested under the same conditions in each particular study and clinicians can use this information to possibly choose a better sealer ."], ["in the present study , tubliseal sealer showed least microleage compared with sealapex and ah26 sealer .", "it is important to remember before declaring any root canal sealer as most acceptable that the results of the dye penetration studies indicate only the relative sealing ability of root canal fillings in vitro and they do not indicate their ability to prevent the penetration of bacteria into filled root canals in vivo ."]]}
{"article_id": "PMC2707810", "article_text": ["targeting type 4 phosphodiesterase ( pde4 ) has been recognized as a promising approach to managing copd by relieving the symptoms , slowing the progress of the disease , increasing exercise tolerance , reducing exacerbation rate , and improving quality of life ( giembycz 2001 , 2005 ; mehats et al 2003 ; spina 2003 , 2004 ; lagente et al 2005 ; lipworth 2005 ; soto and hanania 2005 ) the pressing need to develop drugs that control symptoms and reduce mortality ( pauwels et al 2001 ; gold 2005 ) and the billion - dollar marketing potential for management of copd have pushed the r&d of pde4 inhibitors into the product development pipelines of major pharmaceutical companies in the recent years .", "the early clinical trial data for the second - generation pde4 inhibitors cilomilast ( ariflo , glaxosmithkline , usa ) and roflumilast ( daxas , altana , germany ) all pointed to a successful introduction of a novel non - steroid anti - inflammatory therapy to clinicians in combating severe copd ( gamble et al 2003 ; rabe et al 2005 ) nevertheless , while the progression of developing cilomilast has idled at the approvable stage for more than two years , the announcement of the termination of the agreement to develop roflumilast between altana and pfizer has raised concerns about the therapeutic efficacy of selectively inhibiting one or two isoenzymes in the pde4 family for copd management ( pharmiweb 2005 ) . in the early six - month", " record  phase iii trial , roflumilast ( 500 mg daily ) clearly improved lung function ( ie , increased fev1 by + 97 ml ) and significantly reduced exacerbations ( acute worsening of symptoms ) compared with placebo ( rabe et al 2005 ) .", "however , in the follow - up one - year phase iii trials using exacerbations as one of the key endpoints , the results from the european copd ratio study that included 1513 patients with severe and very severe copd have failed to repeat the previously claimed efficacy .", "in addition , the new trial data confirmed that the pde4 inhibitor roflumilast s efficacy was considerably lower than the approved therapies such as fluticasone / salmeterol ( a combination therapy of glucocorticosteroid and long - acting 2-agonist ) and tiotropium bromide ( long - acting anticholinergic ) .", "the unexpectedly low long - term efficacy on exacerbation rate from roflumilast therapy made the r&d community re - examine the role of targeting pde4 in copd because one of the highest unmet needs in treating the disease is to reduce or eliminate exacerbations ( pharmiweb 2005 ) . in november of 2005", ", altana announced the withdrawal of the european marketing authorization application ( maa ) for roflumilast and decided to wait for more clinical trial data for submission of a future maa ( altana 2005a ) .", "this holdup no doubt sets back the r&d of the most promising pde4 inhibitor in development for copd .", "copd is a complex disease with pathophysiological features including inflammation ( neutrophils , macrophages , cd8 + lymphocytes infiltration , and inflammatory mediator tnf- and il-8 release ) , airway obstruction ( smooth muscle contraction , elevated cholinergic tone ) , respiratory bronchiolar ", "alveolar  vasculature remodeling ( loss of elastic recoil , alveolar destruction , and fibrosis ) , pulmonary hyperinflation , gas - exchange abnormalities , and pulmonary hypertension .", "the progressive loss of lung function leads to reductions in patients quality of life and results in exacerbations , cor pulmonale , and death .", "it is believed that the chronic non - infectious inflammation underlies the pathogenesis and the steady progression of the disease ( pauwels 2001 ; gold 2005 ) .", "the pathological changes in the patients with copd are not fully reversible and it often takes many years for a patient at risk ( cough , sputum production ) to progress into suffering from mild airflow limitation , to moderate , severe , and very severe copd ( with chronic respiratory failure ) . in the absence of a magical therapy that can stop the disease progression and reverse the abnormalities of pulmonary function , the management , including drug therapy , for copd is long - term care .", "inhibition of pde4 has been established as an effective and reliable approach to increasing intracellular camp ( conti et al 2003 ) that underlines the signaling mechanisms for the treatment of copd . in recent years", ", numerous in vitro , in vivo , and clinical trial studies demonstrated that pde4 inhibitors ( eg , rolipram , cilomilast , and roflumilast ) relax airway smooth muscles to increase air flow ( holbrook et al 1996 ; bundschuh et al 2001 ) and improve pulmonary circulation ( schermuly 2000 ; de witt 2000 ) , inhibit bronchiolar  alveolar  vasculature remodeling , and fibrosis ( kumar et al 2003 ) , reduce neutrophils ", "macrophages / cd8 + t cells infiltration and pro - inflammatory mediator release ( kumar et al 2003 ; profita et al 2003 ; wollin et al 2005 ) , improve patients exercise capacity and quality of life , and prevent the progressive loss of pulmonary function ( rabe et al 2005 ; gamble et al 2005 ) .", "with all these preferred outcomes , it seems that the pde4 inhibitors in development ( cilomilast and roflumilast ) would be an ideal armory for the healthcare community to combat copd .", "why , then , has the long - term trial with roflumilast failed to produce the expected results ?", "it could be due to a dose regimen ( 500 mg daily ) that was effective for patients with moderate to severe copd ( rabe et al 2005 ) but not adequate for those patients suffering from severe and very severe copd ( altana 2005b ) or the intrinsic low efficacy of the narrow - spectrum pde4 inhibitors .", "developing pde4 inhibitor as a therapy for copd is based on the fact that theophylline dilates airway smooth muscles and improves pulmonary function by inhibition of pde activity ( barnes 2003 ; spina 2004 ) the dose - limiting adverse reactions ( nausea , emesis , cardiac arrhythmias ) with the non - selective pde inhibitor theophylline and the first - generation pde4 inhibitor rolipram ( huang et al 2001 ; lagente et al 2005 ) directed the r&d of pde inhibitors to discover the second - generation of pde4 inhibitors cilomilast and roflumilast that have been successfully brought to the final stage for administration approval ( spina 2003 , 2004 ; lipworth 2005 ) based on the fact that the emetogenic reaction to pde4 inhibition is due to reticence of the pde4d isoenzyme ( lamontagne et al 2001 ; robichaud et al 2002 ) , several researchers in the field proposed to develop isoform - specific pde4 inhibitors that reduce or completely avoid disturbing pde4d activity and therefore do not trigger the emetic responses in the nervous system ( giembycz 2002 ; robichaud et al 2002 ; card et al 2004 ) .", "structural studies have provided evidence that the folding of catalytic domains of pde4 has a conformation involved in binding of selective inhibitors with a common scheme : ( i ) a hydrophobic sub - pocket sandwiching an inhibitor in the active site ; ( ii ) hydrogen bond(s ) to an invariant glutamine controlling the orientation inhibitor binding ( therefore the affinity or potency ) ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) ( figure 1 ) .", "the scaffold of individual pde4 isoenzyme and the structure of a given selective inhibitor govern isoenzyme - selective inhibition , depict the binding affinity , and determine the therapeutic window and rank order of potency in clinical use for the treatment of copd .", "enhancement of isoenzyme selectivity is critical for reducing side - effects of the pde4 inhibitors .", "the strength of the interaction ( hydrogen bond ) between the oxygen group(s ) of an inhibitor and the amide nitrogen group of glutamine ( gln ) 369 for pde4d and gln 443 for pde4b plays a pivotal role in determination of the potency and isoenzyme selectivity of an inhibitor ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) .", "in addition , selective pde4 inhibitors such as cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space of the pocket to yield extra binding energy ( increasing potency ) and result in greater isoenzyme selectivity ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) for example , while cilomilast s additional functional groups interact with 10 almost identical residues that form the hydrophobic sub - pocket in pde4d and pde4b , the oxygen atoms of the cyclopentyloxy and methoxy groups of cilomilast form two hydrogen bonds with the gln369 of pde4d , whereas there is only one hydrogen bond being formed between the methoxy group of cilomilast and the gln 443 of pde4b ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1 ) .", "this difference may partially explain the fact that cilomilast is approximately 10-fold more selective for pde4d than for pde4b , despite over 90% identity between the pde4b and pde4d catalytic domains .", "( giembycz 2001 ; odingo 2005 ) roflumilast shows a better fitting to the hydrophobic sub - pocket in the pde4d catalytic site than cilomilast ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xoq&template=ligands.html&l=2.1 ) , which depicts the experimental finding that roflumilast inhibits pde4d 338-fold more potently than cilomilast ( table 1 ) . as for pde4b inhibition , roflumilast s cyclopropylmethoxy and difluoromethoxy oxygen groups form two hydrogen bonds with gln 443 of pde4b ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xmu&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1 ) , which may partially explain its 585-fold greater inhibition potency than cilomilast toward pde4b ( table 1 ) .", "the dichloropyridyl substitution of roflumilast also increases its potency compared with cilomilast in terms of pde4b inhibition ( card et al 2004 ) .", "the rank order of potency for inhibition of pde4 activity and lipopolysaccharide - stimulated tnfa release , for relaxing bronchoconstriction in guinea pigs , and the daily doses for treatment of copd by roflumilast , cilomilast , rolipram , and theophylline are summarized in table 2 . by improving the inhibition ratio on pde4b / pde4d", ", roflumilast indeed lowers the emetic profile without compromising its therapeutic efficacy in comparison to cilomilast ( huang et al 2001 ; spina 2003 ) the pde4b - specific single - molecule targeting can undoubtedly reduce or eliminate an inhibitor s unwanted effects .", "however , this approach may compromise the efficacy of a pde4 inhibitor because airway and vascular smooth muscles express multiple pde4d isoforms ( eg , d1 , d2 , and d5 ) and pde4d plays a crucial role in bronchoconstriction and vascular smooth muscle contraction ( liu et al 2000 ; baillie et al 2001 ; mehats et al 2003 ) .", "an agent without 4d inhibition may end up lacking sufficient therapeutic efficacy for controlling copd ( spina 2004 ) .", "the beneficial effects on copd via relaxing airway smooth muscles and anti - inflammation mediated by pde4 inhibition with cilomilast and roflumilast have been emphasized and reviewed extensively ( spina 2004 ; lagente et al 2005 ; lipworth 2005 ; soto 2005 ) . improving pulmonary circulation has not been considered as an important therapeutic approach for managing patients with copd .", "however , the facts are 1 ) during copd exacerbations , pulmonary hypertension ( ph ) is increased , 2 ) the presence of ph is recognized as the single strongest indicator of prognosis in copd patients among the numerous clinically used lung function parameters ( doi et al 2003 ; alp et al 2006 ) , and 3 ) clinically , the higher the pulmonary arterial pressure , the shorter the life expectancy of copd patients ( barbera et al 2003 ) .", "although inhaling nitric oxide vasodilator can worsen gas exchange because of altered hypoxic regulation of ventilation - balance ( barbera 1996 ) in patients with stable copd , and vasodilators are considered as contraindications for copd patients , in their preliminary clinical trial , alp et al ( 2006 ) demonstrated that reduction of pulmonary vasculature resistance using the pde5 inhibitor sildenafil was able to significantly improve the exercise capacity ( 6-minute walk test ) of patients suffering from severe copd . a double - blinded , placebo - controlled , crossover clinical trial of sildenafil in patients ( n=10 ) with copd is going on to evaluate the effect of pde5 inhibition on patients exercise function , pulmonary function , and quality of life ( national institutes of health ) .", "although there is a lack of reports about cilomilast- or roflumilast - caused pde4 inhibition on the improvement of pulmonary circulation , in isolated perfused lung preparations , intravascular or transbronchial administration of subthreshold doses of the wide - spectrum pde4 inhibitor rolipram synergistically amplified the pulmonary vasodilatory response to inhaled pgi2 and concomitantly improved ventilation ", "more interestingly , in anesthetized cats , de witt et al ( 2000 ) found that rolipram was more potent than either siguazodan ( pde3 inhibitor ) or zaprinast ( pde5 inhibitor ) in reducing pulmonary lobar arterial pressure . when the tone in the pulmonary vascular bed was raised to a high steady level with a constant infusion of the thromboxane mimic u46619 ( 9,11-dideoxy-11 , alpha9alpha - epoxymethano prostaglandin f2 ) , intralobar injections of rolipram caused dose - related decreases in systemic arterial pressure and pulmonary arterial pressure .", "the preference reduction in pulmonary arterial pressure by inhibition of pde4 suggests that administration of pde4 inhibitors may benefit copd patients by increasing ventilation ( airway relaxation ) and improving alveolar perfusion ( vasodilation ) , therefore improving blood  gas exchange capacity and patients pulmonary function .", "thus , it is highly likely that the maximal therapeutic efficacy of targeting pde4 in the treatment of severe copd depends upon three effectors of downstream intracellular camp elevation : 1 ) anti - inflammation ( lipworth 2005 ) , 2 ) airway relaxation ( spina 2004 ) , and 3 ) vasodilation . sacrificing any one of these effectors with an isoenzyme - specific narrow - spectrum pde4 inhibitor will compromise the effectiveness of the therapy .", "how , then , is the dose - limiting dilemma ( nausea , vomiting , diarrhea , and arteritis ) ( giembycz 2005 ; spina 2004 ; lipworth 2005 ) associated with the wide - spectrum pde4 inhibition overcome ?", "the fact that there are over 60 pde isoenzymes encoded by 21 human pde genes and at least 16 pde4 isoenzymes from 4 pde4 genes ( soderling and beavo 2000 ; conti et al 2003 ; houslay and adams 2003 ; huai et al 2004 ) may render it highly possible that searching for isoenzyme - specific pde4 inhibitors will yield low - efficacy agents . in addition , mechanisms for upregulation of pde4 activity by camp - induced pde4 gene expression and pka - catalyzed phosphorylation  activation of pde4 isoforms ( conti et al 2003 ;", "wallace et al 2005 ) may very likely reverse an isoenzyme - specific , pde4 inhibitor - produced elevation of intracellular camp level , and therefore the associated biological beneficial effects .", "noticing the low therapeutic ratio and insufficient clinical efficacy of the current generation of pde4 inhibitors ( cilomilast and roflumilast ) , giembycz ( 2005 ) assumed that one potential means of improving the therapeutic ratio and safety of pde4 inhibitors may lie in the development of compounds that have broader phosphodiesterase specificity and suggested dually targeting pde4 and pde1 , pde3 , or pde7 to enhance clinical efficacy .", "this approach seems to revisit the previously well - described pde inhibitors such as theophylline or zardvarine ( schmidt et al 2000 ; barnes 2003 ) .", "it may lead to the dose - limiting drawback cycle again , because it is known that targeting camp - specific pde3 is associated with an increase in morbidity and mortality in heart failure patients ( packer et al 1991 ) . in a news feature on pharmiweb.com", ", there is a remark about pfizer s development of an inhaled dual - action pde4/spiriva ( long - lasting muscarinic antagonist tiotropium ) combination product for copd ( pharmiweb 2005 ) .", "this combined dual action modality is a favorable approach for managing patients with severe copd considering the presence of bronchoconstricting and inflammatory pathologies in the disease . in general , moderately targeting two mechanisms to reach the therapeutic goal should be more effective and safer than exploiting a single mechanism to its extended degree .", "we have proposed to co - administer ca channel antagonist ( cca ) to overcome pde4 inhibitor - caused adverse effects , especially emetic responses ( wang and wang 2005 ) because 1 ) stimuli that upregulate the camp pathway can increase the excitability of the neurons in the locus coeruleus ( lc ) that plays an important role in mediating neuronal emesis ( nestler et al 1999 ; takeda et al 2001 ) ; 2 ) the pde4d isoform is localized to neurons in the structures of the medulla including lc ( lamontagne et al 2001 ) , which are consistent with a role for pde4d in the emetic response ; and 3 ) the lc neurons fire action potentials spontaneously , resulting from endogenous properties of the membrane conductance to a persistent inward ca current , which can be blocked by diltiazem ( williams et al 1984 ; tokuyama and ho 1996 ; filosa and putnam 2003 ) .", "thus , in the presence of cca , even when a complete inhibition of pde4d leads to an elevation of camp in the lc neurons , the lc cells will not be able to fire action potentials due to a blockade of the depolarizing l - type ca currents , therefore eliminating the intrinsic dose - limiting emetic pharmacological profile of the wide - spectrum pde4 inhibition .", "in addition , ccas also relax airway smooth muscles and exhibit anti - inflammatory effects , which may synergistically augment a pde4 inhibitor s therapeutic effects on copd ( worley and kotlikoff 1990 ; szabo et al 1997 ; brown et al 2004 ) . the clinical use of ccas in treatment of pulmonary hypertension in patients with copd further support the combination therapy of a pde4 inhibitor and a cca ( sajkov et al 1997 ) .", "one concern regarding the combined therapy is the possible difference between the pharmacokinetics of the two drugs that may compromise the expected outcomes .", "this pitfall can be eliminated by developing an agent with two pharmacophores in one chemical structure , therefore being able to simultaneously target two therapeutic mechanisms such as l - type ca channels and pde4s ( wang and wang 2005 ) .", "we believe that it is worthwhile to carry out a randomized clinical trial to evaluate the safety and efficacy of dually targeting pde4 and ca channels in managing the patients with severe copd .", "the unsatisfied efficacy of using pde4 inhibitor roflumilast in treatment of severe and very severe copd has raised concerns in the r&d community about the administrative approvability for the highly expected novel therapeutic modality in combating copd .", "the extensive in vitro , in vivo , and clinical trial study data and the established clinical beneficial effects ( anti - inflammation , bronchorelaxation , pulmonary vasodilation ) associated with pde4 inhibition strongly validate targeting pde4 for controlling copd .", "development of an inhaled dual - action therapy such as pde4 inhibitor and muscarinic antagonist may be a correct approach to bringing a pde4 inhibitor to the demanding market .", "the other approach is using cca to overcome pde4 inhibitor - caused adverse effects , especially emetic responses and simultaneously enhance pde4 inhibitor s anti - inflammatory and bronchorelaxation , pulmonary vasodilation effects . of course , development of a dual agent that has with two pharmacophores in one chemical structure , therefore being able to target pde4 and l - type ca channels should also be able to improve the therapeutic window of pde4 inhibition and may make available a new therapeutic approach to managing copd ."], "abstract_text": ["<S> targeting type 4 phosphodiesterase ( pde4 ) for treatment of copd has multilevel benefits to patients by reducing inflammation , relieving bronchoconstriction , and improving pulmonary circulation . </S>", "<S> the isoenzyme - specific narrow spectrum pde4 inhibitors such as cilomilast and roflumilast may have limited clinical efficacy in managing severe and very severe copd . </S>", "<S> development of dual therapy by combining pde4 inhibition with ca2 + channel antagonism may introduce an effective novel armory for physicians to manage patients with severe copd . </S>"], "labels": null, "section_names": ["Introduction", "PDE4 inhibition and COPD", "Subtype specific PDE4 inhibition and COPD", "PDE4 inhibition and pulmonary circulation", "Approaches for improving the therapeutic efficacy of PDE4 inhibitors", "Conclusion"], "sections": [["targeting type 4 phosphodiesterase ( pde4 ) has been recognized as a promising approach to managing copd by relieving the symptoms , slowing the progress of the disease , increasing exercise tolerance , reducing exacerbation rate , and improving quality of life ( giembycz 2001 , 2005 ; mehats et al 2003 ; spina 2003 , 2004 ; lagente et al 2005 ; lipworth 2005 ; soto and hanania 2005 ) the pressing need to develop drugs that control symptoms and reduce mortality ( pauwels et al 2001 ; gold 2005 ) and the billion - dollar marketing potential for management of copd have pushed the r&d of pde4 inhibitors into the product development pipelines of major pharmaceutical companies in the recent years .", "the early clinical trial data for the second - generation pde4 inhibitors cilomilast ( ariflo , glaxosmithkline , usa ) and roflumilast ( daxas , altana , germany ) all pointed to a successful introduction of a novel non - steroid anti - inflammatory therapy to clinicians in combating severe copd ( gamble et al 2003 ; rabe et al 2005 ) nevertheless , while the progression of developing cilomilast has idled at the approvable stage for more than two years , the announcement of the termination of the agreement to develop roflumilast between altana and pfizer has raised concerns about the therapeutic efficacy of selectively inhibiting one or two isoenzymes in the pde4 family for copd management ( pharmiweb 2005 ) . in the early six - month", " record  phase iii trial , roflumilast ( 500 mg daily ) clearly improved lung function ( ie , increased fev1 by + 97 ml ) and significantly reduced exacerbations ( acute worsening of symptoms ) compared with placebo ( rabe et al 2005 ) .", "however , in the follow - up one - year phase iii trials using exacerbations as one of the key endpoints , the results from the european copd ratio study that included 1513 patients with severe and very severe copd have failed to repeat the previously claimed efficacy .", "in addition , the new trial data confirmed that the pde4 inhibitor roflumilast s efficacy was considerably lower than the approved therapies such as fluticasone / salmeterol ( a combination therapy of glucocorticosteroid and long - acting 2-agonist ) and tiotropium bromide ( long - acting anticholinergic ) .", "the unexpectedly low long - term efficacy on exacerbation rate from roflumilast therapy made the r&d community re - examine the role of targeting pde4 in copd because one of the highest unmet needs in treating the disease is to reduce or eliminate exacerbations ( pharmiweb 2005 ) . in november of 2005", ", altana announced the withdrawal of the european marketing authorization application ( maa ) for roflumilast and decided to wait for more clinical trial data for submission of a future maa ( altana 2005a ) .", "this holdup no doubt sets back the r&d of the most promising pde4 inhibitor in development for copd ."], ["copd is a complex disease with pathophysiological features including inflammation ( neutrophils , macrophages , cd8 + lymphocytes infiltration , and inflammatory mediator tnf- and il-8 release ) , airway obstruction ( smooth muscle contraction , elevated cholinergic tone ) , respiratory bronchiolar ", "alveolar  vasculature remodeling ( loss of elastic recoil , alveolar destruction , and fibrosis ) , pulmonary hyperinflation , gas - exchange abnormalities , and pulmonary hypertension .", "the progressive loss of lung function leads to reductions in patients quality of life and results in exacerbations , cor pulmonale , and death .", "it is believed that the chronic non - infectious inflammation underlies the pathogenesis and the steady progression of the disease ( pauwels 2001 ; gold 2005 ) .", "the pathological changes in the patients with copd are not fully reversible and it often takes many years for a patient at risk ( cough , sputum production ) to progress into suffering from mild airflow limitation , to moderate , severe , and very severe copd ( with chronic respiratory failure ) . in the absence of a magical therapy that can stop the disease progression and reverse the abnormalities of pulmonary function , the management , including drug therapy , for copd is long - term care .", "inhibition of pde4 has been established as an effective and reliable approach to increasing intracellular camp ( conti et al 2003 ) that underlines the signaling mechanisms for the treatment of copd . in recent years", ", numerous in vitro , in vivo , and clinical trial studies demonstrated that pde4 inhibitors ( eg , rolipram , cilomilast , and roflumilast ) relax airway smooth muscles to increase air flow ( holbrook et al 1996 ; bundschuh et al 2001 ) and improve pulmonary circulation ( schermuly 2000 ; de witt 2000 ) , inhibit bronchiolar  alveolar  vasculature remodeling , and fibrosis ( kumar et al 2003 ) , reduce neutrophils ", "macrophages / cd8 + t cells infiltration and pro - inflammatory mediator release ( kumar et al 2003 ; profita et al 2003 ; wollin et al 2005 ) , improve patients exercise capacity and quality of life , and prevent the progressive loss of pulmonary function ( rabe et al 2005 ; gamble et al 2005 ) .", "with all these preferred outcomes , it seems that the pde4 inhibitors in development ( cilomilast and roflumilast ) would be an ideal armory for the healthcare community to combat copd .", "why , then , has the long - term trial with roflumilast failed to produce the expected results ?", "it could be due to a dose regimen ( 500 mg daily ) that was effective for patients with moderate to severe copd ( rabe et al 2005 ) but not adequate for those patients suffering from severe and very severe copd ( altana 2005b ) or the intrinsic low efficacy of the narrow - spectrum pde4 inhibitors ."], ["developing pde4 inhibitor as a therapy for copd is based on the fact that theophylline dilates airway smooth muscles and improves pulmonary function by inhibition of pde activity ( barnes 2003 ; spina 2004 ) the dose - limiting adverse reactions ( nausea , emesis , cardiac arrhythmias ) with the non - selective pde inhibitor theophylline and the first - generation pde4 inhibitor rolipram ( huang et al 2001 ; lagente et al 2005 ) directed the r&d of pde inhibitors to discover the second - generation of pde4 inhibitors cilomilast and roflumilast that have been successfully brought to the final stage for administration approval ( spina 2003 , 2004 ; lipworth 2005 ) based on the fact that the emetogenic reaction to pde4 inhibition is due to reticence of the pde4d isoenzyme ( lamontagne et al 2001 ; robichaud et al 2002 ) , several researchers in the field proposed to develop isoform - specific pde4 inhibitors that reduce or completely avoid disturbing pde4d activity and therefore do not trigger the emetic responses in the nervous system ( giembycz 2002 ; robichaud et al 2002 ; card et al 2004 ) .", "structural studies have provided evidence that the folding of catalytic domains of pde4 has a conformation involved in binding of selective inhibitors with a common scheme : ( i ) a hydrophobic sub - pocket sandwiching an inhibitor in the active site ; ( ii ) hydrogen bond(s ) to an invariant glutamine controlling the orientation inhibitor binding ( therefore the affinity or potency ) ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) ( figure 1 ) .", "the scaffold of individual pde4 isoenzyme and the structure of a given selective inhibitor govern isoenzyme - selective inhibition , depict the binding affinity , and determine the therapeutic window and rank order of potency in clinical use for the treatment of copd .", "enhancement of isoenzyme selectivity is critical for reducing side - effects of the pde4 inhibitors .", "the strength of the interaction ( hydrogen bond ) between the oxygen group(s ) of an inhibitor and the amide nitrogen group of glutamine ( gln ) 369 for pde4d and gln 443 for pde4b plays a pivotal role in determination of the potency and isoenzyme selectivity of an inhibitor ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) .", "in addition , selective pde4 inhibitors such as cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space of the pocket to yield extra binding energy ( increasing potency ) and result in greater isoenzyme selectivity ( lee et al 2002 ; huai et al 2003 ; card et al 2004 ) for example , while cilomilast s additional functional groups interact with 10 almost identical residues that form the hydrophobic sub - pocket in pde4d and pde4b , the oxygen atoms of the cyclopentyloxy and methoxy groups of cilomilast form two hydrogen bonds with the gln369 of pde4d , whereas there is only one hydrogen bond being formed between the methoxy group of cilomilast and the gln 443 of pde4b ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1 ) .", "this difference may partially explain the fact that cilomilast is approximately 10-fold more selective for pde4d than for pde4b , despite over 90% identity between the pde4b and pde4d catalytic domains .", "( giembycz 2001 ; odingo 2005 ) roflumilast shows a better fitting to the hydrophobic sub - pocket in the pde4d catalytic site than cilomilast ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xom&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xoq&template=ligands.html&l=2.1 ) , which depicts the experimental finding that roflumilast inhibits pde4d 338-fold more potently than cilomilast ( table 1 ) . as for pde4b inhibition , roflumilast s cyclopropylmethoxy and difluoromethoxy oxygen groups form two hydrogen bonds with gln 443 of pde4b ( card et al 2004 ; comparing http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xmu&template=ligands.html&l=1.1 with http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/getpage.pl?pdbcode=1xlx&template=ligands.html&l=1.1 ) , which may partially explain its 585-fold greater inhibition potency than cilomilast toward pde4b ( table 1 ) .", "the dichloropyridyl substitution of roflumilast also increases its potency compared with cilomilast in terms of pde4b inhibition ( card et al 2004 ) .", "the rank order of potency for inhibition of pde4 activity and lipopolysaccharide - stimulated tnfa release , for relaxing bronchoconstriction in guinea pigs , and the daily doses for treatment of copd by roflumilast , cilomilast , rolipram , and theophylline are summarized in table 2 . by improving the inhibition ratio on pde4b / pde4d", ", roflumilast indeed lowers the emetic profile without compromising its therapeutic efficacy in comparison to cilomilast ( huang et al 2001 ; spina 2003 ) the pde4b - specific single - molecule targeting can undoubtedly reduce or eliminate an inhibitor s unwanted effects .", "however , this approach may compromise the efficacy of a pde4 inhibitor because airway and vascular smooth muscles express multiple pde4d isoforms ( eg , d1 , d2 , and d5 ) and pde4d plays a crucial role in bronchoconstriction and vascular smooth muscle contraction ( liu et al 2000 ; baillie et al 2001 ; mehats et al 2003 ) .", "an agent without 4d inhibition may end up lacking sufficient therapeutic efficacy for controlling copd ( spina 2004 ) ."], ["the beneficial effects on copd via relaxing airway smooth muscles and anti - inflammation mediated by pde4 inhibition with cilomilast and roflumilast have been emphasized and reviewed extensively ( spina 2004 ; lagente et al 2005 ; lipworth 2005 ; soto 2005 ) . improving pulmonary circulation has not been considered as an important therapeutic approach for managing patients with copd .", "however , the facts are 1 ) during copd exacerbations , pulmonary hypertension ( ph ) is increased , 2 ) the presence of ph is recognized as the single strongest indicator of prognosis in copd patients among the numerous clinically used lung function parameters ( doi et al 2003 ; alp et al 2006 ) , and 3 ) clinically , the higher the pulmonary arterial pressure , the shorter the life expectancy of copd patients ( barbera et al 2003 ) .", "although inhaling nitric oxide vasodilator can worsen gas exchange because of altered hypoxic regulation of ventilation - balance ( barbera 1996 ) in patients with stable copd , and vasodilators are considered as contraindications for copd patients , in their preliminary clinical trial , alp et al ( 2006 ) demonstrated that reduction of pulmonary vasculature resistance using the pde5 inhibitor sildenafil was able to significantly improve the exercise capacity ( 6-minute walk test ) of patients suffering from severe copd . a double - blinded , placebo - controlled , crossover clinical trial of sildenafil in patients ( n=10 ) with copd is going on to evaluate the effect of pde5 inhibition on patients exercise function , pulmonary function , and quality of life ( national institutes of health ) .", "although there is a lack of reports about cilomilast- or roflumilast - caused pde4 inhibition on the improvement of pulmonary circulation , in isolated perfused lung preparations , intravascular or transbronchial administration of subthreshold doses of the wide - spectrum pde4 inhibitor rolipram synergistically amplified the pulmonary vasodilatory response to inhaled pgi2 and concomitantly improved ventilation ", "more interestingly , in anesthetized cats , de witt et al ( 2000 ) found that rolipram was more potent than either siguazodan ( pde3 inhibitor ) or zaprinast ( pde5 inhibitor ) in reducing pulmonary lobar arterial pressure . when the tone in the pulmonary vascular bed was raised to a high steady level with a constant infusion of the thromboxane mimic u46619 ( 9,11-dideoxy-11 , alpha9alpha - epoxymethano prostaglandin f2 ) , intralobar injections of rolipram caused dose - related decreases in systemic arterial pressure and pulmonary arterial pressure .", "the preference reduction in pulmonary arterial pressure by inhibition of pde4 suggests that administration of pde4 inhibitors may benefit copd patients by increasing ventilation ( airway relaxation ) and improving alveolar perfusion ( vasodilation ) , therefore improving blood  gas exchange capacity and patients pulmonary function .", "thus , it is highly likely that the maximal therapeutic efficacy of targeting pde4 in the treatment of severe copd depends upon three effectors of downstream intracellular camp elevation : 1 ) anti - inflammation ( lipworth 2005 ) , 2 ) airway relaxation ( spina 2004 ) , and 3 ) vasodilation . sacrificing any one of these effectors with an isoenzyme - specific narrow - spectrum pde4 inhibitor will compromise the effectiveness of the therapy .", "how , then , is the dose - limiting dilemma ( nausea , vomiting , diarrhea , and arteritis ) ( giembycz 2005 ; spina 2004 ; lipworth 2005 ) associated with the wide - spectrum pde4 inhibition overcome ?"], ["the fact that there are over 60 pde isoenzymes encoded by 21 human pde genes and at least 16 pde4 isoenzymes from 4 pde4 genes ( soderling and beavo 2000 ; conti et al 2003 ; houslay and adams 2003 ; huai et al 2004 ) may render it highly possible that searching for isoenzyme - specific pde4 inhibitors will yield low - efficacy agents . in addition , mechanisms for upregulation of pde4 activity by camp - induced pde4 gene expression and pka - catalyzed phosphorylation  activation of pde4 isoforms ( conti et al 2003 ;", "wallace et al 2005 ) may very likely reverse an isoenzyme - specific , pde4 inhibitor - produced elevation of intracellular camp level , and therefore the associated biological beneficial effects .", "noticing the low therapeutic ratio and insufficient clinical efficacy of the current generation of pde4 inhibitors ( cilomilast and roflumilast ) , giembycz ( 2005 ) assumed that one potential means of improving the therapeutic ratio and safety of pde4 inhibitors may lie in the development of compounds that have broader phosphodiesterase specificity and suggested dually targeting pde4 and pde1 , pde3 , or pde7 to enhance clinical efficacy .", "this approach seems to revisit the previously well - described pde inhibitors such as theophylline or zardvarine ( schmidt et al 2000 ; barnes 2003 ) .", "it may lead to the dose - limiting drawback cycle again , because it is known that targeting camp - specific pde3 is associated with an increase in morbidity and mortality in heart failure patients ( packer et al 1991 ) . in a news feature on pharmiweb.com", ", there is a remark about pfizer s development of an inhaled dual - action pde4/spiriva ( long - lasting muscarinic antagonist tiotropium ) combination product for copd ( pharmiweb 2005 ) .", "this combined dual action modality is a favorable approach for managing patients with severe copd considering the presence of bronchoconstricting and inflammatory pathologies in the disease . in general , moderately targeting two mechanisms to reach the therapeutic goal should be more effective and safer than exploiting a single mechanism to its extended degree .", "we have proposed to co - administer ca channel antagonist ( cca ) to overcome pde4 inhibitor - caused adverse effects , especially emetic responses ( wang and wang 2005 ) because 1 ) stimuli that upregulate the camp pathway can increase the excitability of the neurons in the locus coeruleus ( lc ) that plays an important role in mediating neuronal emesis ( nestler et al 1999 ; takeda et al 2001 ) ; 2 ) the pde4d isoform is localized to neurons in the structures of the medulla including lc ( lamontagne et al 2001 ) , which are consistent with a role for pde4d in the emetic response ; and 3 ) the lc neurons fire action potentials spontaneously , resulting from endogenous properties of the membrane conductance to a persistent inward ca current , which can be blocked by diltiazem ( williams et al 1984 ; tokuyama and ho 1996 ; filosa and putnam 2003 ) .", "thus , in the presence of cca , even when a complete inhibition of pde4d leads to an elevation of camp in the lc neurons , the lc cells will not be able to fire action potentials due to a blockade of the depolarizing l - type ca currents , therefore eliminating the intrinsic dose - limiting emetic pharmacological profile of the wide - spectrum pde4 inhibition .", "in addition , ccas also relax airway smooth muscles and exhibit anti - inflammatory effects , which may synergistically augment a pde4 inhibitor s therapeutic effects on copd ( worley and kotlikoff 1990 ; szabo et al 1997 ; brown et al 2004 ) . the clinical use of ccas in treatment of pulmonary hypertension in patients with copd further support the combination therapy of a pde4 inhibitor and a cca ( sajkov et al 1997 ) .", "one concern regarding the combined therapy is the possible difference between the pharmacokinetics of the two drugs that may compromise the expected outcomes .", "this pitfall can be eliminated by developing an agent with two pharmacophores in one chemical structure , therefore being able to simultaneously target two therapeutic mechanisms such as l - type ca channels and pde4s ( wang and wang 2005 ) .", "we believe that it is worthwhile to carry out a randomized clinical trial to evaluate the safety and efficacy of dually targeting pde4 and ca channels in managing the patients with severe copd ."], ["the unsatisfied efficacy of using pde4 inhibitor roflumilast in treatment of severe and very severe copd has raised concerns in the r&d community about the administrative approvability for the highly expected novel therapeutic modality in combating copd .", "the extensive in vitro , in vivo , and clinical trial study data and the established clinical beneficial effects ( anti - inflammation , bronchorelaxation , pulmonary vasodilation ) associated with pde4 inhibition strongly validate targeting pde4 for controlling copd .", "development of an inhaled dual - action therapy such as pde4 inhibitor and muscarinic antagonist may be a correct approach to bringing a pde4 inhibitor to the demanding market .", "the other approach is using cca to overcome pde4 inhibitor - caused adverse effects , especially emetic responses and simultaneously enhance pde4 inhibitor s anti - inflammatory and bronchorelaxation , pulmonary vasodilation effects . of course , development of a dual agent that has with two pharmacophores in one chemical structure , therefore being able to target pde4 and l - type ca channels should also be able to improve the therapeutic window of pde4 inhibition and may make available a new therapeutic approach to managing copd ."]]}
{"article_id": "PMC3153715", "article_text": ["long - acting beta-2 agonists such as salmeterol and formoterol are important controller medications in the maintenance therapy of chronic stable asthma , used as an add - on drug to inhaled corticosteroid . however , their usefulness in the management of acute attacks of asthma has recently been recognized , and they are approved for use as reliever medication in europe .", "short - acting 2 agonists are the preferred drugs as the initial bronchodilator for acute asthma because of their rapid onset of action . however , due to short duration of action they require frequent administration .", "formoterol is a unique bronchodilator having rapid onset and long duration of action with a favorable safety profile .", "so it can be an ideal alternative to short - acting 2 agonists in the management of acute asthma exacerbation by providing rapid bronchodilatation and reducing the need for frequent administration .", "onset of action of formoterol is similar to salbutamol ( 13 min ) , and 8090% of brochodilatation occurs by 510 min of inhalation .", "duration of action is up to 12 h. however , duration of systemic effects with formoterol is found to be as short as salbutamol .", "arformoterol ( [ r , r ] formoterol ) is a single isomer form of formoterol , which is available in india recently .", "this study compares the efficacy and safety of arformoterol and salbutamol delivered by nebulization in the management of acute non - severe asthma .", "a total of 50 patients attending the emergency room or chest opd of a tertiary care hospital with acute non - severe asthma over a 6-month period from january 2010 to june 2010 were selected for this study .", "the inclusion criteria were as follows : ( a ) age > 18 years ; ( b ) british thoracic society definition of acute non - severe asthma ; and ( c ) ability to perform forced expiratory maneuver .", "the following patients were excluded from the study : ( a ) patients presenting with acute severe / acute life - threatening / near - fatal asthma ; ( b ) patients with a previous diagnosis of chronic obstructive pulmonary disease ( copd ) ; ( c ) history of hypersensitivity to 2 agonists ; and ( d ) pregnant or lactating women .", "the study was approved by the institutional ethics committee , and informed consent was obtained from each patient .", "the age , sex , duration of asthma symptoms , and present controller medications were recorded .", "the pulse rate , respiratory rate , and spo2 ( measured with fingertip pulse oximeter , model 6500 , nidek medical , kolkata , india ) were also recorded at the time of initial assessment .", "baseline peak expiratory flow rate ( pefr ) was measured with a peak flow meter ( peak flow master , cipla ltd . ,", "patients were then assigned by random number allocation to either of the two groups , that is , salbutamol or arformoterol group according to the medication given to them . in the salbutamol group , 5-mg salbutamol respules were administered through oxygen driven ( 6 l / min ) nebulizer ( pulmo mist ii nebuliser , nidek medical , kolkata , india ) at 20-min intervals for 1 h amounting to a total of 15 mg . in the arformoterol group ,", "total 45 g of the drug was administered as 15 g respules every 20 min through the same nebulizer .", "the drugs were administered in a double - blind manner . to eliminate the effects of other drugs on the treatment outcome", "pefr was also measured 5 min after each dose . at each time , best of the three pefr measurements was recorded .", "differences in mean pefr between the two groups were analyzed by unpaired t - test .", "one - sample test was used to compare pefr before and after each dose of the drug to determine whether there was significant improvement in either group .", "among 50 patients satisfying the inclusion criteria , 25 patients received salbutamol and 25 patients received arformoterol therapy .", "baseline characteristics of the patients ( n = 50 ) receiving salbutamol and arformoterol therapy from the table , it is evident that the demographic profile and baseline pefr in the two groups were comparable .", "in both the groups , pefr showed significant increase over the baseline values and the increase was evident after each dose of the drug .", "the increases in pefr after the first and the second dose were significantly more with arformoterol than with salbutamol , but the increase in pefr after the third dose was similar in these two groups . the comparison of improvement following the therapy with salbutamol and arformoterol is shown in table 2 .", "improvement in pefr after each dose of bronchodilator when compared with the baseline both drugs were well tolerated ; no major adverse effect was noted in either group .", "as arformoterol is the only long - acting 2 agonist available in india as a nebulizing solution , we have used this drug considering the fact that the clinical pharmacology will be similar with formoterol .", "our study has shown that both salbutamol and arformoterol are equally effective as a reliever medication as nebulizing solution in acute non - severe asthma .", "improvement in pefr was demonstrated in both the groups and following each dose of the drugs .", "the absolute increase in the pefr after the first and the second dose were more with arformoterol than with salbutamol , but the increase in the pefr after the third dose was similar with these two drugs . relatively , hydrophilic drugs such as salbutamol have a rapid onset of action due to their ability to reach the 2 receptor from the aqueous phase .", "the aqueous portion rapidly activates the 2 receptor , whereas the lipophilic portion is taken up into the cell membrane from which it diffuses slowly to stimulate 2 receptor over a prolonged period .", "several studies have shown the efficacy of formoterol in acute non - severe asthma , acute severe asthma , exercise induced bronchospasm , childhood asthma , and copd.[1317 ] most of the studies have used formoterol with terbuhaler , aerolizer , or metered dose inhaler . however , studies using formoterol nebulization are lacking .", "although few studies have shown clinically significant improvement in lung function ( fev1 ) with formoterol when compared with salbutamol , most studies have shown a comparable efficacy .", "safety of formoterol is also well documented even at high doses in patients with asthma and copd .", "although it is a long - acting drug , systemic effects with formoterol are as short as salbutamol . in few studies , the impact of high - dose formoterol on heart rate , blood pressure , serum potassium , electrocardiogram changes , and arterial blood gas were assessed which showed identical changes comparable with salbutamol", "we did not study these parameters but evaluated only the side effects reported by the patients .", "the side effects were very few , non - severe , and comparable between these two drugs .", "dryness of mouth was the most reported side effect of formoterol inhalation in one study , but we found oral irritation and headache as common side effects .", "the sample size is relatively small which limits our ability to detect small but potentially significant differences .", "this study is restricted to patients of acute non - severe asthma only ; efficacy and tolerability of arformoterol in other acute settings such as acute severe asthma and acute exacerbation of copd are yet to be explored .", "the side effect profile studied by us was only on the basis of the self - reported symptoms of the patients , and objective assessment and laboratory abnormalities were not assessed .", "we also did not follow the patients after discharge ; so long - term efficacy and safety were not studied .", "further studies are thus needed to clarify the efficacy , actual dose , dose frequency , cost - effectiveness , and long - term safety of arformoterol in acute exacerbations of asthma and copd with different levels of severity ."], "abstract_text": ["<S> arformoterol , a long - acting beta-2 agonist , has a rapid onset and long duration of action . </S>", "<S> its role as rescue medication in acute asthma attack is undetermined . to compare the efficacy and tolerability of arformoterol with salbutamol nebulization , </S>", "<S> a study was conducted among 50 patients with acute non - severe asthma . </S>", "<S> patients were randomly assigned to group 1 ( n = 25 ) and group 2 ( n = 25 ) who received three doses of salbutamol and arformoterol nebulization , respectively , at 20-min intervals . </S>", "<S> the peak expiratory flow rate ( pefr ) was measured at the baseline and 5 min after each dose . </S>", "<S> the demographics and baseline characteristics were comparable between the two groups . </S>", "<S> the mean pefr significantly increased in both these groups when compared with the baseline . </S>", "<S> the increases in the pefr in two groups were similar after the third dose . </S>", "<S> the adverse effects in both these groups were minor . </S>", "<S> arformoterol was as effective and safe as salbutamol in acute non - severe asthma . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion"], "sections": [["long - acting beta-2 agonists such as salmeterol and formoterol are important controller medications in the maintenance therapy of chronic stable asthma , used as an add - on drug to inhaled corticosteroid . however , their usefulness in the management of acute attacks of asthma has recently been recognized , and they are approved for use as reliever medication in europe .", "short - acting 2 agonists are the preferred drugs as the initial bronchodilator for acute asthma because of their rapid onset of action . however , due to short duration of action they require frequent administration .", "formoterol is a unique bronchodilator having rapid onset and long duration of action with a favorable safety profile .", "so it can be an ideal alternative to short - acting 2 agonists in the management of acute asthma exacerbation by providing rapid bronchodilatation and reducing the need for frequent administration .", "onset of action of formoterol is similar to salbutamol ( 13 min ) , and 8090% of brochodilatation occurs by 510 min of inhalation .", "duration of action is up to 12 h. however , duration of systemic effects with formoterol is found to be as short as salbutamol .", "arformoterol ( [ r , r ] formoterol ) is a single isomer form of formoterol , which is available in india recently .", "this study compares the efficacy and safety of arformoterol and salbutamol delivered by nebulization in the management of acute non - severe asthma ."], ["a total of 50 patients attending the emergency room or chest opd of a tertiary care hospital with acute non - severe asthma over a 6-month period from january 2010 to june 2010 were selected for this study .", "the inclusion criteria were as follows : ( a ) age > 18 years ; ( b ) british thoracic society definition of acute non - severe asthma ; and ( c ) ability to perform forced expiratory maneuver .", "the following patients were excluded from the study : ( a ) patients presenting with acute severe / acute life - threatening / near - fatal asthma ; ( b ) patients with a previous diagnosis of chronic obstructive pulmonary disease ( copd ) ; ( c ) history of hypersensitivity to 2 agonists ; and ( d ) pregnant or lactating women .", "the study was approved by the institutional ethics committee , and informed consent was obtained from each patient .", "the age , sex , duration of asthma symptoms , and present controller medications were recorded .", "the pulse rate , respiratory rate , and spo2 ( measured with fingertip pulse oximeter , model 6500 , nidek medical , kolkata , india ) were also recorded at the time of initial assessment .", "baseline peak expiratory flow rate ( pefr ) was measured with a peak flow meter ( peak flow master , cipla ltd . ,", "patients were then assigned by random number allocation to either of the two groups , that is , salbutamol or arformoterol group according to the medication given to them . in the salbutamol group , 5-mg salbutamol respules were administered through oxygen driven ( 6 l / min ) nebulizer ( pulmo mist ii nebuliser , nidek medical , kolkata , india ) at 20-min intervals for 1 h amounting to a total of 15 mg . in the arformoterol group ,", "total 45 g of the drug was administered as 15 g respules every 20 min through the same nebulizer .", "the drugs were administered in a double - blind manner . to eliminate the effects of other drugs on the treatment outcome", "pefr was also measured 5 min after each dose . at each time , best of the three pefr measurements was recorded .", "differences in mean pefr between the two groups were analyzed by unpaired t - test .", "one - sample test was used to compare pefr before and after each dose of the drug to determine whether there was significant improvement in either group ."], ["among 50 patients satisfying the inclusion criteria , 25 patients received salbutamol and 25 patients received arformoterol therapy .", "baseline characteristics of the patients ( n = 50 ) receiving salbutamol and arformoterol therapy from the table , it is evident that the demographic profile and baseline pefr in the two groups were comparable .", "in both the groups , pefr showed significant increase over the baseline values and the increase was evident after each dose of the drug .", "the increases in pefr after the first and the second dose were significantly more with arformoterol than with salbutamol , but the increase in pefr after the third dose was similar in these two groups . the comparison of improvement following the therapy with salbutamol and arformoterol is shown in table 2 .", "improvement in pefr after each dose of bronchodilator when compared with the baseline both drugs were well tolerated ; no major adverse effect was noted in either group ."], ["as arformoterol is the only long - acting 2 agonist available in india as a nebulizing solution , we have used this drug considering the fact that the clinical pharmacology will be similar with formoterol .", "our study has shown that both salbutamol and arformoterol are equally effective as a reliever medication as nebulizing solution in acute non - severe asthma .", "improvement in pefr was demonstrated in both the groups and following each dose of the drugs .", "the absolute increase in the pefr after the first and the second dose were more with arformoterol than with salbutamol , but the increase in the pefr after the third dose was similar with these two drugs . relatively , hydrophilic drugs such as salbutamol have a rapid onset of action due to their ability to reach the 2 receptor from the aqueous phase .", "the aqueous portion rapidly activates the 2 receptor , whereas the lipophilic portion is taken up into the cell membrane from which it diffuses slowly to stimulate 2 receptor over a prolonged period .", "several studies have shown the efficacy of formoterol in acute non - severe asthma , acute severe asthma , exercise induced bronchospasm , childhood asthma , and copd.[1317 ] most of the studies have used formoterol with terbuhaler , aerolizer , or metered dose inhaler . however , studies using formoterol nebulization are lacking .", "although few studies have shown clinically significant improvement in lung function ( fev1 ) with formoterol when compared with salbutamol , most studies have shown a comparable efficacy .", "safety of formoterol is also well documented even at high doses in patients with asthma and copd .", "although it is a long - acting drug , systemic effects with formoterol are as short as salbutamol . in few studies , the impact of high - dose formoterol on heart rate , blood pressure , serum potassium , electrocardiogram changes , and arterial blood gas were assessed which showed identical changes comparable with salbutamol", "we did not study these parameters but evaluated only the side effects reported by the patients .", "the side effects were very few , non - severe , and comparable between these two drugs .", "dryness of mouth was the most reported side effect of formoterol inhalation in one study , but we found oral irritation and headache as common side effects .", "the sample size is relatively small which limits our ability to detect small but potentially significant differences .", "this study is restricted to patients of acute non - severe asthma only ; efficacy and tolerability of arformoterol in other acute settings such as acute severe asthma and acute exacerbation of copd are yet to be explored .", "the side effect profile studied by us was only on the basis of the self - reported symptoms of the patients , and objective assessment and laboratory abnormalities were not assessed .", "we also did not follow the patients after discharge ; so long - term efficacy and safety were not studied .", "further studies are thus needed to clarify the efficacy , actual dose , dose frequency , cost - effectiveness , and long - term safety of arformoterol in acute exacerbations of asthma and copd with different levels of severity ."]]}
{"article_id": "PMC3540709", "article_text": ["hepatic angiomyolipoma ( haml ) is a rare benign mesenchymal liver tumor first described by ishak   in 1976 ; it belongs to a group of perivascular epithelioid cell tumors called pecoma . until date ,", "approximately 300 cases have been reported [ 310 ] ; however , its natural history has not been clarified .", "the tumor composed of blood vessels , smooth muscle , and adipose cells and due to the variety of predominance of these tissues , its patterns in imaging studies have resulted in a difficulty in diagnosis and misdiagnosis of the tumor as hepatocellular carcinoma ( hcc ) in some cases [ 6 , 11 ] .", "therefore , the preoperative correct diagnosis has been difficult ; however , recent advances in imaging diagnosis through a combination of ultrasonography ( us ) , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and angiography and specific immunohistochemical analysis of this tumor using human melanoma black-45 antigen ( hmb-45 ) staining have resulted in accurate diagnosis and it is reported that the current accurate preoperative diagnosis was made in 25%52% of cases [ 8 , 9 ] .", "the majority of these tumors are believed to be clinically benign during a mean follow - up period of 6.8 years ; however , an increasing number of cases and aggressive changes including growth in size , recurrence after surgical resection , metastasis , and invasive growth pattern into the parenchyma and along the vessels have been reported . in this paper", ", we have focused on the characteristic features of this tumor shown in imaging studies and by histological analysis , summarized these cases showing aggressive patterns , and discussed management of the patients and indications for surgical treatment .", "most cases were found as incidental liver tumors upon health screening or imaging examinations for other diseases .", "it usually follows a benign clinical course while some patients visited hospitals with unspecific symptoms of abdominal discomfort , fullness , and other such complaints .", "more than half of the renal aml are considered to be associated with tuberous sclerosis which features the loss of heterozygosity at tsc1 ( 9q34 ) and tsc2 ( 16p13 ) , while it is estimated to be 515% in the liver .", "thus the etiology of most of these tumors in the liver is unknown ; most cases have no history of liver diseases or specific symptoms , and no changes in laboratory data are seen .", "moreover , serum levels of the tumor markers alpha - fetoprotein , protein induced by vitamin k absence or antagonist ii , carcinoembryonic antigen , and carbohydrate antigen 19 - 9 were normal .", "since this tumor is composed of various tissues , such as lipomatous , myomatous , and angiomatous tissue in various proportions , the imaging studies show a wide array of features depending on the predominance of each tissue . the tumor showing the predominance of lipomatous tissue is likely to be correctly diagnosed ; however , myomatous and angiomatous variant poses diagnostic problems since it is difficult to be distinguished from malignant tumors .   us images may vary depending on the tissue components affected by the tumor .", "high echogenic lesions can be observed because of lipomatous and myomatous tissue , and angiomatous tissue may result in low echoic lesions in tumor images ( figure 1(a ) ) .", "if the tumor has predominance of lipomatous tissue , the differential diagnosis with hepatic hemangioma is difficult by sonography alone .", "color doppler sonography shows punctiform or filiform vascular distribution pattern if the tumor has predominance of angiomatous tissue .", "recent reports showed the diagnostic effectiveness of contrast - enhanced us ( ceus ) [ 10 , 13 ] .", "reported that ceus revealed the typical imaging characteristics of haml , that is , an inhomogeneous hyperenhancing pattern in the arterial phase and prolonged enhancement during the portal and kupffer phases .  ", "plain ct showed homogeneous or heterogeneous low - density lesions , and contrast - enhanced dynamic ct showed highly enhanced lesions in the arterial phase , prolonged enhancement in the portal phase , and , occasionally , defective lesions in the late venous phase depending on the component of the tumor tissue .", "a density of less than 20 hounsfield units in plain ct is useful to determine the involvement of lipomatous tissue .", "the difficulty is , however , to diagnose the tumor that is myomatous tissue and angiomatous variant .", "for this point , modification of size of region of interest in ct was reported to be effective for accurately diagnose renal angiomyolipoma from renal cell carcinoma .", "abdominal angiography showed marked tumor staining in the arterial phase , which remained in the portal phase ( figure 1(b ) ) . in some tumors , drainage of the hepatic veins can be observed in the late vascular phase . the first phase of ct during hepatic arteriography showed significant hypervascular lesions in the tumor ( figure 1(c ) ) , and the second phase showed the remains of staining and defective lesions in other areas ( figure 1(d ) ) .", "ct during arterial portography showed areas of defective tumors ( figure 1(d ) ) .  ", "mri is considered to be the best modality to determine the components of the tumor .", "hyperintensity on the t2-weighted image and hyper- or hypointensity on the t1-weighted image are observed depending on the component of tumor tissue [ 810 ] .", "lipomatous lesions may be determined as hyperintensity on the t1-weighted image ; they may also be determined by the chemical shift imaging technique .", "contrast - enhanced dynamic mri using gadolinium or the hepatocyte - specific contrast agent gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid showed early enhancement in the arterial phase followed by the prolonged enhancement in the portal phase and defective lesions in the hepatobiliary phase .", "macroscopic and magnifying glass view of the tumor showed a soft , white to yellow , and well - circumscribed tumor and range in size from 0.1  cm to greater than 36  cm .", "no signs of chronic inflammation or fibrosis were seen in the surrounding liver tissue . because haml comprises lipomatous , myomatous , and angiomatous tissues , microscopic examination showed a mixture of blood vessels , specialized smooth muscle cells , and adipose cells with atypical changes in classic haml ( figures 2(a ) and 2(b ) ) .", "this variation of mixture levels in one tumor reflected the differences in imaging studies and made accurate diagnosis difficult .", "it is believed that majority of the hamls behave in a benign fashion and even if some cases showed invasive growth pattern into the adjacent hepatic parenchyma , portal triad ( figure 2(c ) ) , and hepatic vein .", "tumor cells , especially myomatous cells , were stained positive for hmb-45 in most tumors ( figure 2(d ) ) , for cd34 in the endothelial cells of the blood vessels and for smooth muscle actin in spindle smooth muscle cells .", "hmb-45 is an antibody that reacts with an oncofetal premelanosome - associated glycoprotein 2 , found in neoplastic melanocytes .", "also cd63 , cd67 , desmin , s100 , and ema may also be positive but not specific .", "these cells were negative for cytokeratin 18 , cytokeratin 19 , cam 5.2 , hepatocyte paraffin-1 .", "therefore , the identification of lipomatous , myomatous , and angiomatous tissue by a positive reaction to hmb-45 currently provides the only evidence of haml [ 46 , 18 ] and can be useful for defining from the other liver tumors such as hcc after the tumor biopsy and surgical resection followed by these immunohistochemical stainings .", "although favorable prognosis can be expected for this tumor since the majority of the tumors are benign , however recently , the number of reports of haml showing malignant potential has increased [ 3 , 5 , 8 , 9 , 12 , 1624 ] which revealed significant growth , recurrence , metastasis , and poor prognosis summarized in table 1 . among those cases ,", "deng et al . reported in their case that haml showed atypical angiomatous , epithelioid components with pleomorphic and frequent mitosis in the center of the large tumor displayed p53 immunoreactivity , and mutation at exon 7 for p53 and resulted in vascular invasion , distant metastasis , and fatal outcomes .", "these results indicate that the hmb-45 staining , mitotic analysis by mib-1 ( ki67 ) , and p53 reactivity following the fine - needle biopsy of the tumor might be useful for the diagnosis of the tumor and its malignant potential .", "since haml usually follows benign clinical courses , the majority of the cases can be conservatively treated .", "however , as not a few cases showed aggressive pattern in their courses [ 3 , 5 , 8 , 9 , 12 , 1624 ] and because of the low level of accurate diagnoses by imaging studies , and because of the possibility of dissemination of tumor cells into the peritoneal cavity if a tumor is malignant , tumor biopsy has been avoided , and many hamls have been surgically resected [ 3 , 8 , 9 ] . with the increasing number of resected samples , careful comparisons of imaging studies and pathological findings", "therefore , once haml has been diagnosed by imaging studies , a fine - needle biopsy should be performed to make an accurate diagnosis   and to confirm the predominance of the tissue components , the existence of pleomorphic nuclei with high proliferation activity , p53 immunoreactivity , and mutation in p53 if possible . if the aggressive patterns such as vascular invasion , high proliferation of the tumor cells , p53 immunoreactivity were marked , or when the imaging findings and biopsy can not provide a definitive diagnosis , or if the patients have abdominal symptoms , surgical resection should be considered .", "haml is considered to be a benign mesenchymal tumor , and nonsurgical treatment with conservative management involving close followup is therefore suggested for patients with asymptomatic tumors smaller than 5  cm , which have been proved to be a typical haml by fine - needle aspiration biopsy . as a fact the renal angiomyolipoma often showed perirenal invasion , involvement of the renal vein , and the inferior vena cava , that are not considered as signs of malignant behavior and the angiomatous", ", epithelioid monophasic or pleomorphic variant might be associated with the aggressive behavior and cellular atypia , mitotic activity , and metastatic lesions are the criteria for the malignant renal angiomyolipoma . however , due to the difficulties in diagnosis by imaging studies , many hamls have been surgically resected and have been followed closely for a long period .", "in addition , by careful analysis of these recent cases [ 3 , 5 , 8 , 9 , 12 , 1624 ] summarized in table 1 , it was revealed that , although rare , hamls may have malignant potential , which may be distinguished by aggressive patterns characterized by ( 1 ) significant changes in size in short period ; ( 2 ) a change of tumor composition ( cases 1 , 2 , 5 , and 6 ) [ 9 , 16 , 17 , 19 ] ; ( 3 ) metastases to the other organs ( cases 1 , 9 , and 10 ) [ 17 , 22 , 23 ] ; ( 4 ) recurrence after curative surgical resection ( cases 3 , 7 , 9 , 10 , 11 , 12 , and 13 ) [ 3 , 8 , 18 , 20 , 2224 ] ; and ( 5 ) invasive growth into the vessels ( cases 2 , 4 , 8 , 9 , and 10 ) [ 12 , 17 , 2123 ] .  ", "ohmori et al . reported the first possibly malignant case , which showed that a significant increase in the tumor size resulted in liver dysfunction .", "fatal progression was observed in nine cases listed by multiple recurrences [ 3 , 8 , 18 , 20 , 2224 ] and metastases to the liver , peritoneum , retroperitoneal region , gastrohepatic omentum , pancreas , and lung [ 17 , 22 , 23 ] .", "the cases reported by chang , rouquie , and us   underwent surgical resection , with a suspicious of aggressive patterns and no recurrence has occurred .", "we also reported that portal thrombosis , that is , high - grade portal vein invasion , found in five cases in table 1 ( cases 2 , 8 , 9 , 10 , and 11 ) [ 3 , 17 , 2123 ] may be a clinical marker of the malignant potential and transformation of haml   as it resulted in a significantly aggressive disease and fatal course in 4 cases with multiple recurrences and metastases .", "this finding might be able to be detected by imaging studies as similar to hcc .", "in addition , pathological findings of atypical epithelioid component with high proliferation activity , p53 immunoreactivity , and mutation in p53 might be predictive markers for malignant transformation . based on these reports , as recommended by previous papers [ 3 , 8 , 9 ] ,", "long - term follow - up of haml is necessary after its diagnosis by imaging studies and biopsy specimens and curative surgical treatment .", "the majority of hamls behave as a benign tumors and conservative follow - up may be recommended ; however , with increasing number of the reports showing potentially malignant behavior , prompt surgical resection is essential for better prognosis of this tumor .", "we have reviewed noninvasive imaging studies and the role of histological diagnosis showing distinctive characteristics of haml to increase the rate of accurate diagnoses .", "in addition , we summarized the cases that showed progressive patterns of the tumor and concluded that a careful followup of the tumor even after the final diagnosis is necessary .", "we propose that tumor resection is indicated in the following scenarios : ( 1 ) the patients show symptoms ; ( 2 ) the tumor shows an aggressive growth ; ( 3 ) the tumor shows invasive growth into the vessels evidenced by fine - needle biopsy or imaging studies ; ( 4 ) the component of the tumor shows atypical epithelioid pattern , high proliferation activity , and/or p53 immunoreactivity ; and ( 5 ) a definitive diagnosis can not be made by imaging and pathological studies from malignant tumors ."], "abstract_text": ["<S> angiomyolipoma ( aml ) is a benign mesenchymal tumor that is frequently found in the kidney and , rarely , in the liver . </S>", "<S> the natural history of hepatic aml has not been clarified , and , because of the similar patterns in imaging studies , such as ultrasonography , computed tomography , and magnetic resonance imaging , some of these tumors have been overdiagnosed as hepatocellular carcinoma in the past . with an increase in the number of case reports showing detailed imaging studies and immunohistochemical staining of the tumor with human melanoma black-45 </S>", "<S> , the diagnostic accuracy is also increasing . in this paper , we focused on the role of noninvasive imaging studies and histological diagnosis showing distinctive characteristics of this tumor . </S>", "<S> in addition , because several reports have described tumor progression in terms of size , recurrence after surgical resection , metastasis to other organs , and portal thrombosis , we summarized these cases for the management and discussed the indications for the surgical treatment of this tumor . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Natural History and Laboratory Findings", "3. Imaging Studies", "4. Pathological Findings", "5. Patient Management", "6. Discussion", "7. Conclusion"], "sections": [["hepatic angiomyolipoma ( haml ) is a rare benign mesenchymal liver tumor first described by ishak   in 1976 ; it belongs to a group of perivascular epithelioid cell tumors called pecoma . until date ,", "approximately 300 cases have been reported [ 310 ] ; however , its natural history has not been clarified .", "the tumor composed of blood vessels , smooth muscle , and adipose cells and due to the variety of predominance of these tissues , its patterns in imaging studies have resulted in a difficulty in diagnosis and misdiagnosis of the tumor as hepatocellular carcinoma ( hcc ) in some cases [ 6 , 11 ] .", "therefore , the preoperative correct diagnosis has been difficult ; however , recent advances in imaging diagnosis through a combination of ultrasonography ( us ) , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and angiography and specific immunohistochemical analysis of this tumor using human melanoma black-45 antigen ( hmb-45 ) staining have resulted in accurate diagnosis and it is reported that the current accurate preoperative diagnosis was made in 25%52% of cases [ 8 , 9 ] .", "the majority of these tumors are believed to be clinically benign during a mean follow - up period of 6.8 years ; however , an increasing number of cases and aggressive changes including growth in size , recurrence after surgical resection , metastasis , and invasive growth pattern into the parenchyma and along the vessels have been reported . in this paper", ", we have focused on the characteristic features of this tumor shown in imaging studies and by histological analysis , summarized these cases showing aggressive patterns , and discussed management of the patients and indications for surgical treatment ."], ["most cases were found as incidental liver tumors upon health screening or imaging examinations for other diseases .", "it usually follows a benign clinical course while some patients visited hospitals with unspecific symptoms of abdominal discomfort , fullness , and other such complaints .", "more than half of the renal aml are considered to be associated with tuberous sclerosis which features the loss of heterozygosity at tsc1 ( 9q34 ) and tsc2 ( 16p13 ) , while it is estimated to be 515% in the liver .", "thus the etiology of most of these tumors in the liver is unknown ; most cases have no history of liver diseases or specific symptoms , and no changes in laboratory data are seen .", "moreover , serum levels of the tumor markers alpha - fetoprotein , protein induced by vitamin k absence or antagonist ii , carcinoembryonic antigen , and carbohydrate antigen 19 - 9 were normal ."], ["since this tumor is composed of various tissues , such as lipomatous , myomatous , and angiomatous tissue in various proportions , the imaging studies show a wide array of features depending on the predominance of each tissue . the tumor showing the predominance of lipomatous tissue is likely to be correctly diagnosed ; however , myomatous and angiomatous variant poses diagnostic problems since it is difficult to be distinguished from malignant tumors .   us images may vary depending on the tissue components affected by the tumor .", "high echogenic lesions can be observed because of lipomatous and myomatous tissue , and angiomatous tissue may result in low echoic lesions in tumor images ( figure 1(a ) ) .", "if the tumor has predominance of lipomatous tissue , the differential diagnosis with hepatic hemangioma is difficult by sonography alone .", "color doppler sonography shows punctiform or filiform vascular distribution pattern if the tumor has predominance of angiomatous tissue .", "recent reports showed the diagnostic effectiveness of contrast - enhanced us ( ceus ) [ 10 , 13 ] .", "reported that ceus revealed the typical imaging characteristics of haml , that is , an inhomogeneous hyperenhancing pattern in the arterial phase and prolonged enhancement during the portal and kupffer phases .  ", "plain ct showed homogeneous or heterogeneous low - density lesions , and contrast - enhanced dynamic ct showed highly enhanced lesions in the arterial phase , prolonged enhancement in the portal phase , and , occasionally , defective lesions in the late venous phase depending on the component of the tumor tissue .", "a density of less than 20 hounsfield units in plain ct is useful to determine the involvement of lipomatous tissue .", "the difficulty is , however , to diagnose the tumor that is myomatous tissue and angiomatous variant .", "for this point , modification of size of region of interest in ct was reported to be effective for accurately diagnose renal angiomyolipoma from renal cell carcinoma .", "abdominal angiography showed marked tumor staining in the arterial phase , which remained in the portal phase ( figure 1(b ) ) . in some tumors , drainage of the hepatic veins can be observed in the late vascular phase . the first phase of ct during hepatic arteriography showed significant hypervascular lesions in the tumor ( figure 1(c ) ) , and the second phase showed the remains of staining and defective lesions in other areas ( figure 1(d ) ) .", "ct during arterial portography showed areas of defective tumors ( figure 1(d ) ) .  ", "mri is considered to be the best modality to determine the components of the tumor .", "hyperintensity on the t2-weighted image and hyper- or hypointensity on the t1-weighted image are observed depending on the component of tumor tissue [ 810 ] .", "lipomatous lesions may be determined as hyperintensity on the t1-weighted image ; they may also be determined by the chemical shift imaging technique .", "contrast - enhanced dynamic mri using gadolinium or the hepatocyte - specific contrast agent gadolinium - ethoxybenzyl - diethylenetriamine pentaacetic acid showed early enhancement in the arterial phase followed by the prolonged enhancement in the portal phase and defective lesions in the hepatobiliary phase ."], ["macroscopic and magnifying glass view of the tumor showed a soft , white to yellow , and well - circumscribed tumor and range in size from 0.1  cm to greater than 36  cm .", "no signs of chronic inflammation or fibrosis were seen in the surrounding liver tissue . because haml comprises lipomatous , myomatous , and angiomatous tissues , microscopic examination showed a mixture of blood vessels , specialized smooth muscle cells , and adipose cells with atypical changes in classic haml ( figures 2(a ) and 2(b ) ) .", "this variation of mixture levels in one tumor reflected the differences in imaging studies and made accurate diagnosis difficult .", "it is believed that majority of the hamls behave in a benign fashion and even if some cases showed invasive growth pattern into the adjacent hepatic parenchyma , portal triad ( figure 2(c ) ) , and hepatic vein .", "tumor cells , especially myomatous cells , were stained positive for hmb-45 in most tumors ( figure 2(d ) ) , for cd34 in the endothelial cells of the blood vessels and for smooth muscle actin in spindle smooth muscle cells .", "hmb-45 is an antibody that reacts with an oncofetal premelanosome - associated glycoprotein 2 , found in neoplastic melanocytes .", "also cd63 , cd67 , desmin , s100 , and ema may also be positive but not specific .", "these cells were negative for cytokeratin 18 , cytokeratin 19 , cam 5.2 , hepatocyte paraffin-1 .", "therefore , the identification of lipomatous , myomatous , and angiomatous tissue by a positive reaction to hmb-45 currently provides the only evidence of haml [ 46 , 18 ] and can be useful for defining from the other liver tumors such as hcc after the tumor biopsy and surgical resection followed by these immunohistochemical stainings .", "although favorable prognosis can be expected for this tumor since the majority of the tumors are benign , however recently , the number of reports of haml showing malignant potential has increased [ 3 , 5 , 8 , 9 , 12 , 1624 ] which revealed significant growth , recurrence , metastasis , and poor prognosis summarized in table 1 . among those cases ,", "deng et al . reported in their case that haml showed atypical angiomatous , epithelioid components with pleomorphic and frequent mitosis in the center of the large tumor displayed p53 immunoreactivity , and mutation at exon 7 for p53 and resulted in vascular invasion , distant metastasis , and fatal outcomes .", "these results indicate that the hmb-45 staining , mitotic analysis by mib-1 ( ki67 ) , and p53 reactivity following the fine - needle biopsy of the tumor might be useful for the diagnosis of the tumor and its malignant potential ."], ["since haml usually follows benign clinical courses , the majority of the cases can be conservatively treated .", "however , as not a few cases showed aggressive pattern in their courses [ 3 , 5 , 8 , 9 , 12 , 1624 ] and because of the low level of accurate diagnoses by imaging studies , and because of the possibility of dissemination of tumor cells into the peritoneal cavity if a tumor is malignant , tumor biopsy has been avoided , and many hamls have been surgically resected [ 3 , 8 , 9 ] . with the increasing number of resected samples , careful comparisons of imaging studies and pathological findings", "therefore , once haml has been diagnosed by imaging studies , a fine - needle biopsy should be performed to make an accurate diagnosis   and to confirm the predominance of the tissue components , the existence of pleomorphic nuclei with high proliferation activity , p53 immunoreactivity , and mutation in p53 if possible . if the aggressive patterns such as vascular invasion , high proliferation of the tumor cells , p53 immunoreactivity were marked , or when the imaging findings and biopsy can not provide a definitive diagnosis , or if the patients have abdominal symptoms , surgical resection should be considered ."], ["haml is considered to be a benign mesenchymal tumor , and nonsurgical treatment with conservative management involving close followup is therefore suggested for patients with asymptomatic tumors smaller than 5  cm , which have been proved to be a typical haml by fine - needle aspiration biopsy . as a fact the renal angiomyolipoma often showed perirenal invasion , involvement of the renal vein , and the inferior vena cava , that are not considered as signs of malignant behavior and the angiomatous", ", epithelioid monophasic or pleomorphic variant might be associated with the aggressive behavior and cellular atypia , mitotic activity , and metastatic lesions are the criteria for the malignant renal angiomyolipoma . however , due to the difficulties in diagnosis by imaging studies , many hamls have been surgically resected and have been followed closely for a long period .", "in addition , by careful analysis of these recent cases [ 3 , 5 , 8 , 9 , 12 , 1624 ] summarized in table 1 , it was revealed that , although rare , hamls may have malignant potential , which may be distinguished by aggressive patterns characterized by ( 1 ) significant changes in size in short period ; ( 2 ) a change of tumor composition ( cases 1 , 2 , 5 , and 6 ) [ 9 , 16 , 17 , 19 ] ; ( 3 ) metastases to the other organs ( cases 1 , 9 , and 10 ) [ 17 , 22 , 23 ] ; ( 4 ) recurrence after curative surgical resection ( cases 3 , 7 , 9 , 10 , 11 , 12 , and 13 ) [ 3 , 8 , 18 , 20 , 2224 ] ; and ( 5 ) invasive growth into the vessels ( cases 2 , 4 , 8 , 9 , and 10 ) [ 12 , 17 , 2123 ] .  ", "ohmori et al . reported the first possibly malignant case , which showed that a significant increase in the tumor size resulted in liver dysfunction .", "fatal progression was observed in nine cases listed by multiple recurrences [ 3 , 8 , 18 , 20 , 2224 ] and metastases to the liver , peritoneum , retroperitoneal region , gastrohepatic omentum , pancreas , and lung [ 17 , 22 , 23 ] .", "the cases reported by chang , rouquie , and us   underwent surgical resection , with a suspicious of aggressive patterns and no recurrence has occurred .", "we also reported that portal thrombosis , that is , high - grade portal vein invasion , found in five cases in table 1 ( cases 2 , 8 , 9 , 10 , and 11 ) [ 3 , 17 , 2123 ] may be a clinical marker of the malignant potential and transformation of haml   as it resulted in a significantly aggressive disease and fatal course in 4 cases with multiple recurrences and metastases .", "this finding might be able to be detected by imaging studies as similar to hcc .", "in addition , pathological findings of atypical epithelioid component with high proliferation activity , p53 immunoreactivity , and mutation in p53 might be predictive markers for malignant transformation . based on these reports , as recommended by previous papers [ 3 , 8 , 9 ] ,", "long - term follow - up of haml is necessary after its diagnosis by imaging studies and biopsy specimens and curative surgical treatment .", "the majority of hamls behave as a benign tumors and conservative follow - up may be recommended ; however , with increasing number of the reports showing potentially malignant behavior , prompt surgical resection is essential for better prognosis of this tumor ."], ["we have reviewed noninvasive imaging studies and the role of histological diagnosis showing distinctive characteristics of haml to increase the rate of accurate diagnoses .", "in addition , we summarized the cases that showed progressive patterns of the tumor and concluded that a careful followup of the tumor even after the final diagnosis is necessary .", "we propose that tumor resection is indicated in the following scenarios : ( 1 ) the patients show symptoms ; ( 2 ) the tumor shows an aggressive growth ; ( 3 ) the tumor shows invasive growth into the vessels evidenced by fine - needle biopsy or imaging studies ; ( 4 ) the component of the tumor shows atypical epithelioid pattern , high proliferation activity , and/or p53 immunoreactivity ; and ( 5 ) a definitive diagnosis can not be made by imaging and pathological studies from malignant tumors ."]]}
{"article_id": "PMC3560653", "article_text": ["considering the therapeutic consequences , involvement of the sentinel node by metastatic disease in breast cancer patients is of great interest to the breast surgeon in order to plan the surgical approach .", "unfortunately , the physical examination of the axillae can be inaccurate in identifying pathologic lymph nodes .", "the same is also true for the pre - treatment imaging ( with or without interventional procedures on the axillary node ) , characterized by a poor negative predictive value [ 48 ] .", "using gamma probe combined with ultrasound- ( us ) guided percutaneous core needle biopsy ( cnb ) could increase the accuracy of identifying the sentinel node and allow a proper histopathologic diagnosis in order to avoid further slnb .", "the purpose of this pilot study was to assess this new pre - operative diagnostic approach .", "main inclusion criteria were : women 18 years old , invasive breast carcinoma , with no clinically positive axillary node and no history of any other malignancy .", "multicentric cancer and previous excisional biopsy were exclusion criteria . the sentinel node occult lesion localization ( snoll )", "was performed in all patients the day before surgery . a dose of 510 ( generally 7 ) mbq of technetium 99-labelled human albumin nanocolloid particles in 0.3 ml saline were administered by us - guided percutaneous injection in the area immediately surrounding the breast lesion , followed by injection of 0.2 ml saline using a 22 g needle .", "we used nanocolloid particles with a size range of 5100 nm ( nanocoll , ge healthcare ) .", "after injection , antero - posterior and lateral lymphoscintigraphic projections were obtained to identify the presence of a sentinel node and to define whether the radiocolloid has shifted to other possible sites of drainage , such as the internal mammary , intramammary , contralateral axillary or supraclavicular nodes .", "the hot spot was identified over the skin by the handheld gamma probe ( navigatog gps rmd ) and was examined using ultrasound logos hi vision gold ( hitachi ) using a breast - dedicated linear array transducer .", "the radiologist performing the us - guided biopsy established which lymph node corresponds to the hot spot under gamma probe guidance .", "we evaluated suspect us findings such as cortical thickening , especially if focal , markedly hypoechoic cortex , absence of the fatty hilum , large expansion , irregular shape , round shape , extracapsular tumoral extension or increased peripheral blood flow at power doppler .", "lymph nodes with at least 2 of these criteria were considered pathologic , and we excluded suspicious lymph nodes .", "patients were placed in a supine or contralateral - side - down oblique position on the table , with the ipsilateral hand placed behind the head .", "biopsies were performed in all patients by a single radiologist with more than 10 years experience in ultrasonography .", "cnb was performed with an automatic 18 g needle ( bard core biopsy instrument , tempe , usa ) after local anesthesia with 2% mepivacaine .", "all biopsies with a free - hand technique were performed under us guidance with direct visualization of the needle entering into the cortex of the node to confirm position of the needle tip in the appropriate location . only 1 pass", "the percutaneous biopsy was performed only if the lymph node corresponded to the hot spot .", "we considered the biopsy procedure to be successful when the obtained sample contained a large portion of solid non - fatty tissue and/or sank in formalin to be stained with haematoxylin and eosin .", "the skin above the first radioactive node was marked by the radiologist with an indelible ink tattoo to assist the surgeon .", "the puncture zone was compressed and the presence of complications such as local pain or hematoma was evaluated .", "the next day , in the operating room , radioactive lymph nodes were detected in all cases using the same gamma probe that was used to detect the hot spot for the us - guided pre - operative imaging .", "surgical slnb was performed in all cases , followed by alnd in cases of positive percutaneous biopsy .", "the surgeon was able to recognize the sentinel lymph node biopsy marked by the radiologist with the skin mark and correlate with the results of the standard slnb ( validate the ability of the preoperative diagnostic method to correctly identify the sentinel node ) . because the most frequent tumor site is the upper - outer quadrant of the breast , the surgeon generally used the same type of incision of the breast to identify the sentinel node using the gamma probe guide when the conservative surgery was performed .", "in different cases , the surgeon used a separate incision guided by the skin marker performed by the radiologist .", "the final histopathological diagnosis of slnb was compared with the results of the cnb of the sentinel node .", "the accuracy of percutaneous biopsy guided by us and gamma probe over the skin in the pre - operative staging was correlated with final pathologic reports of slnb .", "information on histological type and grade and biological characteristics ( eg , receptor status or peritumoral vascular invasion of the primary carcinoma ) were obtained from the pathology report of the breast specimen . for the pathological staging of the axillae", ", this study used the guidelines and terminology proposed by the seventh edition of the american joint committee on cancer staging manual .", "main patient characteristics are shown in table 1 and include age , type of surgery , location of primary breast cancer , nodal stage at percutaneous biopsy , pathological nodal stage , and biological features of the breast tumor .", "sentinel node identification was successful in all patients , although there were no suspicious nodes at us examination .", "the hot spot was detected over the skin by gamma probe in all 10 patients , and was examined using us - guided cnb in all patients .", "multiple sentinel nodes were not detected and no patient had the sentinel node draining to different possible sites .", "there were no major complications ( eg , clinically important bleeding , nerve injury , or infection ) related to the cnb procedure . one patient experienced a small amount of bleeding , which was stopped by simple local compression ; this was considered a minor complication . in all patients ,", "nodal metastases were found at final diagnosis in 2 of 10 patients ( table 1 ) .", "the definitive histopathological report of slnb expressed the presence of 1 micrometastasis that was not observed in the frozen section at the extemporary analysis . in the second patient , micrometastases were observed in the para - sentinel node at slnb , but no macrometastasis in the sentinel node were found .", "in all the surgically excised sentinel lymph nodes , the pathologist documented the sign of the previous core needle biopsy , verifying that the biopsied lymph node was really the sentinel node .", "in women with early breast cancer , slnb has proved to be a safe and accurate method for evaluating axillary disease , and is associated with less morbidity than complete alnd . nevertheless , slnb is not a problem - free procedure .", "patients with documented lymph node involvement at the slnb usually require further alnd as a second step during the same surgical procedure , favoring scar tissue formation and edema , and increasing the rate of complications and the surgery time .", "hospital stays are also increased in case of slnb + alnd compared with alnd alone as suggested by goyal et al . .", "pre - operative knowledge of an axillary metastatic sentinel lymph node could avoid the intraoperative slnb .", "in fact , in cases with positive preoperative lymph node cnb , a complete alnd can be performed directly as a primary procedure .", "the obvious advantages of this approach are suggested by several authors that used different methods to localize the abnormal lymph node before surgery .", "had avoided slnb in 30% of patients with a final pathology of metastatic lymph node by performing fine needle aspiration cytology ( fnac ) of the abnormal sonographic axillary node .", "sever et al10 proposed the use of intradermal peritumoral microbubble us contrast agents injection and lymphatic imaging to identify and localize the sentinel node in the preoperative stage . many studies   report a moderate diagnostic sensitivity of the percutaneous biopsy of a morphologically abnormal axillary lymph node and an even lower sensitivity in cases where the biopsied lymph node has an unaltered appearance and the procedure is performed randomly . but how can you differentiate the sentinel node from a regular lymph node especially when the pathological sentinel node is not always the largest one or the morphologically abnormal one ?", ".   found metastatic involvement in 12% of the lymph nodes with normal sonographic appearance , and declared that us - guided biopsy would miss the sentinel node in 36%   of the cases , resulting in false - negative axillary cnb , partly due to failure to identify the lymph node or the abnormal region of the lymph node to biopsy .", "furthermore , there is little information in the literature regarding the accuracy criteria for performing an axillary us . adding gamma probe over the skin could increase the sensitivity of percutaneous us - guided biopsy , providing a good approximation of detection of  true  sentinel lymph node .", "theoretically , it is well established that a negative sentinel lymph node is currently equivalent to disease - free axillae .", "the majority ( about 70% ) of women with clinically negative axillae will prove to be microscopically negative as well .", "on the other hand , it is also documented that a certain percentage of the false - negative sentinel node cnbs are for micrometastases or isolated tumor s cells .", "there is no current evidence showing that submicroscopic metastases predict an adverse outcome or that they require treatment .", "several authors [ 1820 ] demonstrated the absence of axillary recurrence during long - term follow - up in patients with sentinel node micrometastasis in which alnd was not performed .", "several studies were done , and one of the most recent addressing this question participated in an ibcsg trial that compared complete alnd ( used in cases with positive slnb ) to follow - up ( in patients with node - free macrometastasis disease ) .", "similarly , we speculate that patients with micrometastases and false - negative sentinel node proved by cnb , gamma probe and us - guided biopsy , may not need intraoperative slnb , particularly since follow - up with ultrasonography , and eventually pet , could detected early axillary disease that can be adequately treated later by therapeutic axillary dissection .", "nevertheless , avoiding the morbidity of slnb must be weighed against the risk of harboring axillary micrometastases that may potentially seed occult metastatic disease after a percutaneous biopsy . in the clinical context , considering a patient s expected life span and associated health problems , this situation might be defined as a  minimal acceptable risk  .", "slnb has also a false - negative rate ( 5% according to veronesi et al . ) and this is the reason why these patients are generally subject to regular follow - up with clinical and us examination of the axilla .", "we also suggest that cases with negative sentinel node percutaneous gamma probe and us - guided biopsy should be similarly monitored .", "several studies reported that the sensitivity of us in identifying axillary adenopathy has been increased by cytology sampling of the suspicious lymph node ; the approach is limited by the high rate of false - negatives results of the aspiration tecnique . in other words ,", "the unacceptably high rate of false - negatives results makes nodal fnac an unreliable method that can not be used to avoid intraoperative slnb .", "as known , there are a number of possible reasons for a false - negative result at fnac : inadequate specimen sampling , a low number of metastatic lymph nodes , small - sized metastasis , and failure to visualize  true  sentinel lymph node during the us examination of the axilla .", "the aim of this study was to verify the feasibility of this innovative method to detect and to biopsy the sentinel node in order to improve confidence in identification of the metastatic lymph node in the preoperative phase . in our opinion", "the decision to use large cnb rather than fnac was made because the amount of tissue taken is greater than with the latter , providing a high negative predictive value .", "it is reasonable that increasing the volume of tissue obtained from 1 step by cnb can be more effective than multiple samples of fnac to obtain a final diagnosis .", "the sensitivity of the method could be increased by performing several cnbs ( 23 or 35 samples according to garcia - ortega ) from the cortex of the presumed sentinel node .", "do not recommend the use of cnb because of the potential complications of this procedure ( eg , bleeding and nerve damage ) ; previous reports have shown that such interventional procedures in all breast cancer patients are not cost - effective .", "on the other hand , complications can be reduced if the procedures are performed by an experienced , dedicated breast radiologist ( within the breast unit ) .", "this feature is of major importance because , in early the stage , the lymph nodes usually have normal findings at us , and they are frequently small in size .", "consequently , the cutting needle must be sampling only the cortical layer if performed in a technically adequate way .", "the second element is identification of the axillary sentinel lymph node preoperatively using the gamma probe over the skin . combining the use of the gamma probe ,", "with which the  true  sentinel node can be identified with reasonable confidence , and the us - guided cnb that can avoid the sampling error dictated by the reasons mentioned above ( in case of fnac ) , the diagnostic sensitivity should , theoretically , increase . as a minimally invasive staging procedure ,", "us - guided lymph node biopsy under percutaneous gamma probe surveillance seems attractive because it provides information during the preoperative period that could allow avoidance of intraoperative slnb if the sentinel node is negative , and hence , avoid alnd in case of micrometastasis or isolated tumor s cells .", "the most relevant predictive factor for sentinel node metastases is the primary carcinoma peritumoral vascular invasion .", "it could be suggested that whenever this pathology ( or other findings ) finding is found in association with a negative percutaneous sentinel node biopsy performed under us gamma probe guidance , intraoperative slnb should be mandatory .", " have reported the pre - operative use of gamma probe over the skin to detect axillary sentinel node under us guidance .", "the latter had evaluated the sentinel node s location using the gamma probe and then marked it with a hook wire , while motomura   used the gamma probe combined with fnac to obtain the sample .", "to our knowledge , no other published study evaluated gamma probe - assisted sonographic localization combined with cnb of the presumed sentinel node as a  one -step  procedure in the pre - treatment phase of breast cancer patients .", "first , this is a pilot study that analyzed the feasibility of a new pre - treatment method proposed for detecting the sentinel node .", "second , the patient population is small because this is a pilot study , hence the results do not have a statistical impact .", "however , the concepts presented could improve the quality of lymph node sampling , particularly in early breast cancer patients", ". this could be particularly useful in cases that need slnb before neoadjuvant chemotherapy . the pre - treatment sentinel node evaluation technique described in this pilot study", "had shown a perfect concordance with the histological findings , even if the number of subjects enrolled in this study was relatively low . the accuracy and the clinical implications of the method", "in conclusion , gamma probe - assisted sonographic localization associated with cnb of the sentinel node in early breast cancer patients could be a feasible and accurate new method .", "further studies should investigate the definitive role of this method in pre - treatment breast cancer staging .", "particularly , it is necessary to investigate the clinical impact of this method in avoiding slnb , especially in cases when percutaneous sentinel node cnb is negative , without primary carcinoma peritumoral vascular invasion , in clinically node - negative breast disease patients ."], "abstract_text": ["<S> summarybackgroundthe aim of this pilot trial was to study the feasibility of sentinel node percutaneous preoperative gamma probe - guided biopsy as a valid preoperative method of assessment of nodal status compared to surgical sentinel lymph node biopsy.material/methodsthis prospective study enrolled 10 consecutive patients without evidence of axillary lymph node metastases at preoperative imaging . </S>", "<S> all patients underwent sentinel node occult lesion localization ( snoll ) using radiotracer intradermic injection that detected a  </S>", "<S> hot spot  corresponding to the sentinel node in all cases . </S>", "<S> gamma probe over the skin detection with subsequent ultrasonographically guided needle biopsy of the sentinel node were performed . </S>", "<S> the percutaneous needle core histopathological diagnosis was compared to the results of the surgical biopsy.resultspreoperative sentinel node identification was successful in all patients.conclusionsthe combination of preoperative gamma probe sentinel node detection and ultrasound - guided biopsy could represent a valid alternative to intraoperative sentinel node biopsy in clinically and ultrasonographically negative axillary nodes , resulting in shorter duration of surgery and lower intraoperative risks . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Results", "Discussion", "Conclusions"], "sections": [["the sentinel node is defined as the first tumour - draining lymphatic filter , and its involvement by metastatic disease should predict the global nodal status of the specific patient . skip metastasis in the context of a normal sentinel node is described as a very rare event . in patients with clinically node - negative disease , sentinel lymph node biopsy ( slnb ) had become the gold standard staging procedure , followed by axillary lymph node dissection ( alnd ) performed selectively in patients with previously proven ( by slnb ) sentinel node metastases .", "considering the therapeutic consequences , involvement of the sentinel node by metastatic disease in breast cancer patients is of great interest to the breast surgeon in order to plan the surgical approach .", "unfortunately , the physical examination of the axillae can be inaccurate in identifying pathologic lymph nodes .", "the same is also true for the pre - treatment imaging ( with or without interventional procedures on the axillary node ) , characterized by a poor negative predictive value [ 48 ] .", "using gamma probe combined with ultrasound- ( us ) guided percutaneous core needle biopsy ( cnb ) could increase the accuracy of identifying the sentinel node and allow a proper histopathologic diagnosis in order to avoid further slnb .", "the purpose of this pilot study was to assess this new pre - operative diagnostic approach ."], ["main inclusion criteria were : women 18 years old , invasive breast carcinoma , with no clinically positive axillary node and no history of any other malignancy .", "multicentric cancer and previous excisional biopsy were exclusion criteria . the sentinel node occult lesion localization ( snoll )", "was performed in all patients the day before surgery . a dose of 510 ( generally 7 ) mbq of technetium 99-labelled human albumin nanocolloid particles in 0.3 ml saline were administered by us - guided percutaneous injection in the area immediately surrounding the breast lesion , followed by injection of 0.2 ml saline using a 22 g needle .", "we used nanocolloid particles with a size range of 5100 nm ( nanocoll , ge healthcare ) .", "after injection , antero - posterior and lateral lymphoscintigraphic projections were obtained to identify the presence of a sentinel node and to define whether the radiocolloid has shifted to other possible sites of drainage , such as the internal mammary , intramammary , contralateral axillary or supraclavicular nodes .", "the hot spot was identified over the skin by the handheld gamma probe ( navigatog gps rmd ) and was examined using ultrasound logos hi vision gold ( hitachi ) using a breast - dedicated linear array transducer .", "the radiologist performing the us - guided biopsy established which lymph node corresponds to the hot spot under gamma probe guidance .", "we evaluated suspect us findings such as cortical thickening , especially if focal , markedly hypoechoic cortex , absence of the fatty hilum , large expansion , irregular shape , round shape , extracapsular tumoral extension or increased peripheral blood flow at power doppler .", "lymph nodes with at least 2 of these criteria were considered pathologic , and we excluded suspicious lymph nodes .", "patients were placed in a supine or contralateral - side - down oblique position on the table , with the ipsilateral hand placed behind the head .", "biopsies were performed in all patients by a single radiologist with more than 10 years experience in ultrasonography .", "cnb was performed with an automatic 18 g needle ( bard core biopsy instrument , tempe , usa ) after local anesthesia with 2% mepivacaine .", "all biopsies with a free - hand technique were performed under us guidance with direct visualization of the needle entering into the cortex of the node to confirm position of the needle tip in the appropriate location . only 1 pass", "the percutaneous biopsy was performed only if the lymph node corresponded to the hot spot .", "we considered the biopsy procedure to be successful when the obtained sample contained a large portion of solid non - fatty tissue and/or sank in formalin to be stained with haematoxylin and eosin .", "the skin above the first radioactive node was marked by the radiologist with an indelible ink tattoo to assist the surgeon .", "the puncture zone was compressed and the presence of complications such as local pain or hematoma was evaluated .", "the next day , in the operating room , radioactive lymph nodes were detected in all cases using the same gamma probe that was used to detect the hot spot for the us - guided pre - operative imaging .", "surgical slnb was performed in all cases , followed by alnd in cases of positive percutaneous biopsy .", "the surgeon was able to recognize the sentinel lymph node biopsy marked by the radiologist with the skin mark and correlate with the results of the standard slnb ( validate the ability of the preoperative diagnostic method to correctly identify the sentinel node ) . because the most frequent tumor site is the upper - outer quadrant of the breast , the surgeon generally used the same type of incision of the breast to identify the sentinel node using the gamma probe guide when the conservative surgery was performed .", "in different cases , the surgeon used a separate incision guided by the skin marker performed by the radiologist .", "the final histopathological diagnosis of slnb was compared with the results of the cnb of the sentinel node .", "the accuracy of percutaneous biopsy guided by us and gamma probe over the skin in the pre - operative staging was correlated with final pathologic reports of slnb .", "information on histological type and grade and biological characteristics ( eg , receptor status or peritumoral vascular invasion of the primary carcinoma ) were obtained from the pathology report of the breast specimen . for the pathological staging of the axillae", ", this study used the guidelines and terminology proposed by the seventh edition of the american joint committee on cancer staging manual ."], ["main patient characteristics are shown in table 1 and include age , type of surgery , location of primary breast cancer , nodal stage at percutaneous biopsy , pathological nodal stage , and biological features of the breast tumor .", "sentinel node identification was successful in all patients , although there were no suspicious nodes at us examination .", "the hot spot was detected over the skin by gamma probe in all 10 patients , and was examined using us - guided cnb in all patients .", "multiple sentinel nodes were not detected and no patient had the sentinel node draining to different possible sites .", "there were no major complications ( eg , clinically important bleeding , nerve injury , or infection ) related to the cnb procedure . one patient experienced a small amount of bleeding , which was stopped by simple local compression ; this was considered a minor complication . in all patients ,", "nodal metastases were found at final diagnosis in 2 of 10 patients ( table 1 ) .", "the definitive histopathological report of slnb expressed the presence of 1 micrometastasis that was not observed in the frozen section at the extemporary analysis . in the second patient , micrometastases were observed in the para - sentinel node at slnb , but no macrometastasis in the sentinel node were found .", "in all the surgically excised sentinel lymph nodes , the pathologist documented the sign of the previous core needle biopsy , verifying that the biopsied lymph node was really the sentinel node ."], ["in women with early breast cancer , slnb has proved to be a safe and accurate method for evaluating axillary disease , and is associated with less morbidity than complete alnd . nevertheless , slnb is not a problem - free procedure .", "patients with documented lymph node involvement at the slnb usually require further alnd as a second step during the same surgical procedure , favoring scar tissue formation and edema , and increasing the rate of complications and the surgery time .", "hospital stays are also increased in case of slnb + alnd compared with alnd alone as suggested by goyal et al . .", "pre - operative knowledge of an axillary metastatic sentinel lymph node could avoid the intraoperative slnb .", "in fact , in cases with positive preoperative lymph node cnb , a complete alnd can be performed directly as a primary procedure .", "the obvious advantages of this approach are suggested by several authors that used different methods to localize the abnormal lymph node before surgery .", "had avoided slnb in 30% of patients with a final pathology of metastatic lymph node by performing fine needle aspiration cytology ( fnac ) of the abnormal sonographic axillary node .", "sever et al10 proposed the use of intradermal peritumoral microbubble us contrast agents injection and lymphatic imaging to identify and localize the sentinel node in the preoperative stage . many studies   report a moderate diagnostic sensitivity of the percutaneous biopsy of a morphologically abnormal axillary lymph node and an even lower sensitivity in cases where the biopsied lymph node has an unaltered appearance and the procedure is performed randomly . but how can you differentiate the sentinel node from a regular lymph node especially when the pathological sentinel node is not always the largest one or the morphologically abnormal one ?", ".   found metastatic involvement in 12% of the lymph nodes with normal sonographic appearance , and declared that us - guided biopsy would miss the sentinel node in 36%   of the cases , resulting in false - negative axillary cnb , partly due to failure to identify the lymph node or the abnormal region of the lymph node to biopsy .", "furthermore , there is little information in the literature regarding the accuracy criteria for performing an axillary us . adding gamma probe over the skin could increase the sensitivity of percutaneous us - guided biopsy , providing a good approximation of detection of  true  sentinel lymph node .", "theoretically , it is well established that a negative sentinel lymph node is currently equivalent to disease - free axillae .", "the majority ( about 70% ) of women with clinically negative axillae will prove to be microscopically negative as well .", "on the other hand , it is also documented that a certain percentage of the false - negative sentinel node cnbs are for micrometastases or isolated tumor s cells .", "there is no current evidence showing that submicroscopic metastases predict an adverse outcome or that they require treatment .", "several authors [ 1820 ] demonstrated the absence of axillary recurrence during long - term follow - up in patients with sentinel node micrometastasis in which alnd was not performed .", "several studies were done , and one of the most recent addressing this question participated in an ibcsg trial that compared complete alnd ( used in cases with positive slnb ) to follow - up ( in patients with node - free macrometastasis disease ) .", "similarly , we speculate that patients with micrometastases and false - negative sentinel node proved by cnb , gamma probe and us - guided biopsy , may not need intraoperative slnb , particularly since follow - up with ultrasonography , and eventually pet , could detected early axillary disease that can be adequately treated later by therapeutic axillary dissection .", "nevertheless , avoiding the morbidity of slnb must be weighed against the risk of harboring axillary micrometastases that may potentially seed occult metastatic disease after a percutaneous biopsy . in the clinical context , considering a patient s expected life span and associated health problems , this situation might be defined as a  minimal acceptable risk  .", "slnb has also a false - negative rate ( 5% according to veronesi et al . ) and this is the reason why these patients are generally subject to regular follow - up with clinical and us examination of the axilla .", "we also suggest that cases with negative sentinel node percutaneous gamma probe and us - guided biopsy should be similarly monitored .", "several studies reported that the sensitivity of us in identifying axillary adenopathy has been increased by cytology sampling of the suspicious lymph node ; the approach is limited by the high rate of false - negatives results of the aspiration tecnique . in other words ,", "the unacceptably high rate of false - negatives results makes nodal fnac an unreliable method that can not be used to avoid intraoperative slnb .", "as known , there are a number of possible reasons for a false - negative result at fnac : inadequate specimen sampling , a low number of metastatic lymph nodes , small - sized metastasis , and failure to visualize  true  sentinel lymph node during the us examination of the axilla .", "the aim of this study was to verify the feasibility of this innovative method to detect and to biopsy the sentinel node in order to improve confidence in identification of the metastatic lymph node in the preoperative phase . in our opinion", "the decision to use large cnb rather than fnac was made because the amount of tissue taken is greater than with the latter , providing a high negative predictive value .", "it is reasonable that increasing the volume of tissue obtained from 1 step by cnb can be more effective than multiple samples of fnac to obtain a final diagnosis .", "the sensitivity of the method could be increased by performing several cnbs ( 23 or 35 samples according to garcia - ortega ) from the cortex of the presumed sentinel node .", "do not recommend the use of cnb because of the potential complications of this procedure ( eg , bleeding and nerve damage ) ; previous reports have shown that such interventional procedures in all breast cancer patients are not cost - effective .", "on the other hand , complications can be reduced if the procedures are performed by an experienced , dedicated breast radiologist ( within the breast unit ) .", "this feature is of major importance because , in early the stage , the lymph nodes usually have normal findings at us , and they are frequently small in size .", "consequently , the cutting needle must be sampling only the cortical layer if performed in a technically adequate way .", "the second element is identification of the axillary sentinel lymph node preoperatively using the gamma probe over the skin . combining the use of the gamma probe ,", "with which the  true  sentinel node can be identified with reasonable confidence , and the us - guided cnb that can avoid the sampling error dictated by the reasons mentioned above ( in case of fnac ) , the diagnostic sensitivity should , theoretically , increase . as a minimally invasive staging procedure ,", "us - guided lymph node biopsy under percutaneous gamma probe surveillance seems attractive because it provides information during the preoperative period that could allow avoidance of intraoperative slnb if the sentinel node is negative , and hence , avoid alnd in case of micrometastasis or isolated tumor s cells .", "the most relevant predictive factor for sentinel node metastases is the primary carcinoma peritumoral vascular invasion .", "it could be suggested that whenever this pathology ( or other findings ) finding is found in association with a negative percutaneous sentinel node biopsy performed under us gamma probe guidance , intraoperative slnb should be mandatory .", " have reported the pre - operative use of gamma probe over the skin to detect axillary sentinel node under us guidance .", "the latter had evaluated the sentinel node s location using the gamma probe and then marked it with a hook wire , while motomura   used the gamma probe combined with fnac to obtain the sample .", "to our knowledge , no other published study evaluated gamma probe - assisted sonographic localization combined with cnb of the presumed sentinel node as a  one -step  procedure in the pre - treatment phase of breast cancer patients .", "first , this is a pilot study that analyzed the feasibility of a new pre - treatment method proposed for detecting the sentinel node .", "second , the patient population is small because this is a pilot study , hence the results do not have a statistical impact .", "however , the concepts presented could improve the quality of lymph node sampling , particularly in early breast cancer patients", ". this could be particularly useful in cases that need slnb before neoadjuvant chemotherapy . the pre - treatment sentinel node evaluation technique described in this pilot study", "had shown a perfect concordance with the histological findings , even if the number of subjects enrolled in this study was relatively low . the accuracy and the clinical implications of the method"], ["in conclusion , gamma probe - assisted sonographic localization associated with cnb of the sentinel node in early breast cancer patients could be a feasible and accurate new method .", "further studies should investigate the definitive role of this method in pre - treatment breast cancer staging .", "particularly , it is necessary to investigate the clinical impact of this method in avoiding slnb , especially in cases when percutaneous sentinel node cnb is negative , without primary carcinoma peritumoral vascular invasion , in clinically node - negative breast disease patients ."]]}
{"article_id": "PMC4219950", "article_text": ["since the first fontan procedure was introduced by fontan in 1971 , the surgical technique has evolved and the survival rate has shown dramatic improvement . recently ,", "fontan surgery has been performed as an operation of the final stage for congenital heart disease with single ventricle physiology1 ) . in fontan circulation", "therefore , the pulmonary vascular resistance ( pvr ) must be lower than the antegrade transpulmonary flow resistance2 ) .", "thus , even a mild increase in the pvr may interfere with pulmonary circulation which in turn may result in decreased preload of ventricle , diminished cardiac output , and declined exercise tolerance3,4 ) .", "loss of pulsatility of pulmonary flow , reduced growth of the pulmonary vasculature , micro and macro thromboembolism in pulmonary circulation and reduced ventricular function are underlying mechanisms that induce the high pvr in fontan circulation5,6,7 ) .", "several studies8,9,10,11,12 ) reported the pulmonary vasodilating therapy had an effective influence on fontan patients so we want to determine whether inhaled iloprost as a pulmonary vasodilator might have a role in the clinical symptoms , hemodynamics and exercise capacity of fontan patients .", "ethical approval for this study and permission for acquiring , analyzing and reporting of patient 's data were obtained in accordance with the guidelines of the institutional review board of gachon university gil medical center ( no .", "an 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion ( doe ) .", "a physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area , liver and spleen were not palpable .", "recently , she felt more difficult to climb the stairs due to shortness of breath .", "the inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks .", "pre and post medication , laboratory test - including pro brain natriuretic peptide , cardiac catheterization , bicycle ergometer test , nyha class , and 6-minute walking test - were performed .", "after 12 weeks , the patient 's systemic blood pressure and o2 saturation were increased .", "six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii .", ", the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr .", "the results are shown in table 1 . a 22-year - old male with double outlet right ventricle", "after 12 weeks , the patient 's systemic blood pressure , o2 saturation , and nyha class were not changed significantly .", ", the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr .", "an 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion ( doe ) .", "a physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area , liver and spleen were not palpable .", "recently , she felt more difficult to climb the stairs due to shortness of breath .", "the inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks .", "pre and post medication , laboratory test - including pro brain natriuretic peptide , cardiac catheterization , bicycle ergometer test , nyha class , and 6-minute walking test - were performed .", "after 12 weeks , the patient 's systemic blood pressure and o2 saturation were increased .", "six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii .", ", the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr .", "a 22-year - old male with double outlet right ventricle status post fontan surgery at age of five years complained of mild doe only .", "after 12 weeks , the patient 's systemic blood pressure , o2 saturation , and nyha class were not changed significantly .", ", the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr .", "despite the increased survival rate of patients who has congenital heart disease with single ventricular physiology due to advanced surgical technique , the fontan surgery is still a palliative procedure .", "development of the fontan failure state might occur gradually over time ; ventricular dysfunction , aggravated cyanosis , hepatic dysfunction , coagulopathy , protein loosing enteropathy , plastic bronchitis , arrhythmia etc.1 ) .", "progressive decreasing cardiac output , and increasing central venous pressure can be also observed in fontan physiology with age1 ) .", "most patients who underwent cardiac transplant long after fontan surgery had elevated pvr before and immediately after the operation13 ) .", "indeed , pvr is an important factor in the progressive decline in efficiency of the fontan physiology .", "the loss of pulsatile blood flow of pulmonary circulation of fontan patients is positively correlated with high pvr .", "the lowering of pvr may increase pulmonary blood flow and preload reserve in fontan physiology , and for this reason the selective pulmonary vasodilating therapy is interesting as a means to improve the fontan circulation8 ) .", "in addition , nonpulsatile pulmonary blood flow may reduce capillary recruitment and endothelial function . as a result of endothelial dysfunction , impairment in production of nitric oxide , prostacyclin and prolonged overexpression of endothelin-1 level can be observed5,14 ) .", "most studies were based on high level of endothelin-1 in fontan patients9,10 ) , the endothelin receptor antagonist induced improvement of clinical , hemodynamic parameters and exercise capacity .", "experiences with sildenafil therapy on fontan patients have also been reported11,12 ) . in a recent study , rhodes", "et al.15 ) reported on the effect of inhaled iloprost on exercise function of fontan patients . in this article", "the authors administered a single treatment with iloprost nebulizer just before exercise and concluded that iloprost improves the peak oxygen pulse and peak vo2 of patients with fontan physiology . in our two case studies", ", we found that 12 weeks of iloprost therapy showed an association with improvement of clinical , hemodynamic parameters and exercise capacity in patients with fontan physiology .", "first case was considered fontan failure because of aggravated doe , functional class and pitting edema .", "unlike previous study using iloprost once , we used iloprost for three month period and positive results were achieved without major side effects . in addition , we found that iloprost was effective for fontan patients with or without symptoms .", "although more advanced curable treatments of fontan physiology have not been developed , pulmonary vasodilator therapy on fontan circulation is one of good suggestion for symptomatic treatment of fontan failure and improvement of the cardiac output ."], "abstract_text": ["<S> decreased exercise capacity after fontan surgery is relatively common and the failure of the fontan state gradually increases with age . </S>", "<S> however , there is no further treatment for patients with fontan circulation . </S>", "<S> pulmonary vasodilation therapy is an effective method to solve this problem because pulmonary vascular resistance is a major factor of the fontan problem . </S>", "<S> inhaled iloprost is a chemically stable prostacyclin analogue and a potent pulmonary vasodilator . </S>", "<S> we experienced two cases of fontan patients treated with inhaled iloprost for 12 weeks . </S>", "<S> the first patient was an 18-year - old female with pulmonary atresia with an intact ventricular septum , and the second patient was a 22-year - old male with a double outlet right ventricle . </S>", "<S> fifteen years have passed since both patients received fontan surgery . </S>", "<S> while the pulmonary pressure was not decreased significantly , improved exercise capacity and cardiac output were observed without any major side effects in both patients . the iloprost inhalation therapy was well tolerated and effective for the symptomatic treatment of fontan patients . </S>"], "labels": null, "section_names": ["Introduction", "Case reports", "1. Case 1", "2. Case 2", "Discussion"], "sections": [["since the first fontan procedure was introduced by fontan in 1971 , the surgical technique has evolved and the survival rate has shown dramatic improvement . recently ,", "fontan surgery has been performed as an operation of the final stage for congenital heart disease with single ventricle physiology1 ) . in fontan circulation", "therefore , the pulmonary vascular resistance ( pvr ) must be lower than the antegrade transpulmonary flow resistance2 ) .", "thus , even a mild increase in the pvr may interfere with pulmonary circulation which in turn may result in decreased preload of ventricle , diminished cardiac output , and declined exercise tolerance3,4 ) .", "loss of pulsatility of pulmonary flow , reduced growth of the pulmonary vasculature , micro and macro thromboembolism in pulmonary circulation and reduced ventricular function are underlying mechanisms that induce the high pvr in fontan circulation5,6,7 ) .", "several studies8,9,10,11,12 ) reported the pulmonary vasodilating therapy had an effective influence on fontan patients so we want to determine whether inhaled iloprost as a pulmonary vasodilator might have a role in the clinical symptoms , hemodynamics and exercise capacity of fontan patients .", "ethical approval for this study and permission for acquiring , analyzing and reporting of patient 's data were obtained in accordance with the guidelines of the institutional review board of gachon university gil medical center ( no ."], ["an 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion ( doe ) .", "a physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area , liver and spleen were not palpable .", "recently , she felt more difficult to climb the stairs due to shortness of breath .", "the inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks .", "pre and post medication , laboratory test - including pro brain natriuretic peptide , cardiac catheterization , bicycle ergometer test , nyha class , and 6-minute walking test - were performed .", "after 12 weeks , the patient 's systemic blood pressure and o2 saturation were increased .", "six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii .", ", the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr .", "the results are shown in table 1 . a 22-year - old male with double outlet right ventricle", "after 12 weeks , the patient 's systemic blood pressure , o2 saturation , and nyha class were not changed significantly .", ", the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr ."], ["an 18-year - old female with pulmonary atresia with intact ventricular septum status post fontan surgery at age of three years complained of aggravated dyspnea on exertion ( doe ) .", "a physical examination showed regular heart beat without murmur and mild pitting edema on the pretibial area , liver and spleen were not palpable .", "recently , she felt more difficult to climb the stairs due to shortness of breath .", "the inhaled iloprost was administered at 60 microgram / day divided by six times for 12 weeks .", "pre and post medication , laboratory test - including pro brain natriuretic peptide , cardiac catheterization , bicycle ergometer test , nyha class , and 6-minute walking test - were performed .", "after 12 weeks , the patient 's systemic blood pressure and o2 saturation were increased .", "six - minute walking distance was increased from 380 to 395 m and nyha class changed from iii to ii .", ", the cardiac index and stroke volume were increased without significant change of pulmonary pressure and pvr ."], ["a 22-year - old male with double outlet right ventricle status post fontan surgery at age of five years complained of mild doe only .", "after 12 weeks , the patient 's systemic blood pressure , o2 saturation , and nyha class were not changed significantly .", ", the cardiac index and stroke volume were also increased without significant change of pulmonary pressure and pvr ."], ["despite the increased survival rate of patients who has congenital heart disease with single ventricular physiology due to advanced surgical technique , the fontan surgery is still a palliative procedure .", "development of the fontan failure state might occur gradually over time ; ventricular dysfunction , aggravated cyanosis , hepatic dysfunction , coagulopathy , protein loosing enteropathy , plastic bronchitis , arrhythmia etc.1 ) .", "progressive decreasing cardiac output , and increasing central venous pressure can be also observed in fontan physiology with age1 ) .", "most patients who underwent cardiac transplant long after fontan surgery had elevated pvr before and immediately after the operation13 ) .", "indeed , pvr is an important factor in the progressive decline in efficiency of the fontan physiology .", "the loss of pulsatile blood flow of pulmonary circulation of fontan patients is positively correlated with high pvr .", "the lowering of pvr may increase pulmonary blood flow and preload reserve in fontan physiology , and for this reason the selective pulmonary vasodilating therapy is interesting as a means to improve the fontan circulation8 ) .", "in addition , nonpulsatile pulmonary blood flow may reduce capillary recruitment and endothelial function . as a result of endothelial dysfunction , impairment in production of nitric oxide , prostacyclin and prolonged overexpression of endothelin-1 level can be observed5,14 ) .", "most studies were based on high level of endothelin-1 in fontan patients9,10 ) , the endothelin receptor antagonist induced improvement of clinical , hemodynamic parameters and exercise capacity .", "experiences with sildenafil therapy on fontan patients have also been reported11,12 ) . in a recent study , rhodes", "et al.15 ) reported on the effect of inhaled iloprost on exercise function of fontan patients . in this article", "the authors administered a single treatment with iloprost nebulizer just before exercise and concluded that iloprost improves the peak oxygen pulse and peak vo2 of patients with fontan physiology . in our two case studies", ", we found that 12 weeks of iloprost therapy showed an association with improvement of clinical , hemodynamic parameters and exercise capacity in patients with fontan physiology .", "first case was considered fontan failure because of aggravated doe , functional class and pitting edema .", "unlike previous study using iloprost once , we used iloprost for three month period and positive results were achieved without major side effects . in addition , we found that iloprost was effective for fontan patients with or without symptoms .", "although more advanced curable treatments of fontan physiology have not been developed , pulmonary vasodilator therapy on fontan circulation is one of good suggestion for symptomatic treatment of fontan failure and improvement of the cardiac output ."]]}
{"article_id": "PMC3420559", "article_text": ["angiomyolipoma is a benign tumor that is histologically composed of groups of mature adipose tissue intermixed with convoluted thick - walled blood vessels , interlacing bundles , and irregularly arranged sheets of smooth muscle , with the kidney as the most frequent site of involvement .", "extrarenal angiomyolipoma can occur in organs such as the liver , lung , uterus , and skin .", "only two instances of this tumor arising in the tongue have previously been reported [ 1 , 2 ] .", "a 61-year - old woman was referred to our hospital with a mass in the left proglossis that was painless , but had enlarged slowly for 5 years .", "the mass measured 20  mm  20  mm and was soft and dome shaped with a dark violet - colored surface ( figure 1 ) .", "the mass was well - demarcated and easily dissected with an ultrasonic surgical aspirator ( figure 2 ) .", "histopathological examination showed an encapsulated lesion composed of a proliferation of an intricate mixture of mature adipose tissue , blood vessels , and smooth muscle ( figure 3(a ) ) .", "the patient 's postoperative course was uneventful , and there were no signs of recurrence at followup at 18 months ( figure 4 ) .", "koizumi et al .   reported a case of angiomyolipoma in the centre of the tongue .", "described a case of a angiomyolipomatous hamartoma arising in the left lateral border of the tongue . in the present case ,", "angiomyolipoma arose in the proglossis and it had a diagnosis of hemangioma because of its hardness and color .", "because the three principal components in angiomyolipoma , regardless of its location , vary greatly in proportion and distribution , its heterogeneity may cause diagnostic confusion .", "the differential diagnosis includes lipomatous or myolipomatous tumours , angiomyoma , angiolipoma , hemangioma , fibroma , and fibrolipomatous hyperplasia .", "angiomyolipoma could be considered a hamartoma , but there is no consensus that these lesions are a single entity .", "ide et al .   reported that the present lesion may not be a classic oral angiomyolipoma because it was poorly circumscribed and not encapsulated .", "they suggested the term angiomyolipomatous hamartoma to designate this lesion . in agreement with ide et al . , we diagnosed the present case as an angiomyolipoma because the mass had enlarged gradually with active proliferation and was well - encapsulated .", "in particular , tongue cancer and hemangioma are tumors with many blood vessels in the proposed area of surgical excision . for preservation of the nervous system and avoidance of bleeding , an ultrasonic surgical aspirator is effective in the resection of these tongue tumors [ 35 ] . in our case ,"], "abstract_text": ["<S> a 61-year - old woman was referred to our hospital with a mass in the left proglossis . </S>", "<S> the lesion was excised with a provisional diagnosis of hemangioma . </S>", "<S> the mass was well - demarcated and easily dissected with an ultrasonic surgical aspirator . </S>", "<S> the histopathologic diagnosis was angiomyolipoma arising in the tongue . </S>", "<S> there were no signs of recurrence at followup at 18 months . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Case Report", "3. Discussion"], "sections": [["angiomyolipoma is a benign tumor that is histologically composed of groups of mature adipose tissue intermixed with convoluted thick - walled blood vessels , interlacing bundles , and irregularly arranged sheets of smooth muscle , with the kidney as the most frequent site of involvement .", "extrarenal angiomyolipoma can occur in organs such as the liver , lung , uterus , and skin .", "only two instances of this tumor arising in the tongue have previously been reported [ 1 , 2 ] ."], ["a 61-year - old woman was referred to our hospital with a mass in the left proglossis that was painless , but had enlarged slowly for 5 years .", "the mass measured 20  mm  20  mm and was soft and dome shaped with a dark violet - colored surface ( figure 1 ) .", "the mass was well - demarcated and easily dissected with an ultrasonic surgical aspirator ( figure 2 ) .", "histopathological examination showed an encapsulated lesion composed of a proliferation of an intricate mixture of mature adipose tissue , blood vessels , and smooth muscle ( figure 3(a ) ) .", "the patient 's postoperative course was uneventful , and there were no signs of recurrence at followup at 18 months ( figure 4 ) ."], ["koizumi et al .   reported a case of angiomyolipoma in the centre of the tongue .", "described a case of a angiomyolipomatous hamartoma arising in the left lateral border of the tongue . in the present case ,", "angiomyolipoma arose in the proglossis and it had a diagnosis of hemangioma because of its hardness and color .", "because the three principal components in angiomyolipoma , regardless of its location , vary greatly in proportion and distribution , its heterogeneity may cause diagnostic confusion .", "the differential diagnosis includes lipomatous or myolipomatous tumours , angiomyoma , angiolipoma , hemangioma , fibroma , and fibrolipomatous hyperplasia .", "angiomyolipoma could be considered a hamartoma , but there is no consensus that these lesions are a single entity .", "ide et al .   reported that the present lesion may not be a classic oral angiomyolipoma because it was poorly circumscribed and not encapsulated .", "they suggested the term angiomyolipomatous hamartoma to designate this lesion . in agreement with ide et al . , we diagnosed the present case as an angiomyolipoma because the mass had enlarged gradually with active proliferation and was well - encapsulated .", "in particular , tongue cancer and hemangioma are tumors with many blood vessels in the proposed area of surgical excision . for preservation of the nervous system and avoidance of bleeding , an ultrasonic surgical aspirator is effective in the resection of these tongue tumors [ 35 ] . in our case ,"]]}
{"article_id": "PMC5204071", "article_text": ["in particular , osteoimmunology investigates how the immune system impacts on bone turnover in physiological and pathological conditions through the immunoskeletal interface .", "researchers recently hav - egained better understanding of the dialogue between immune and bone cells and a new reading register of bone remodeling is emerging , in which the various phases of bone formation and resorption , that coexist in a dynamic equilibrium , are under strict immunological control .", "bone and immune system are functionally integrated through complex homeostatic networks and , in all respects , osteoporosis could be considered a chronic immune mediated inflammatory disease which shares clinical and biological features with many other inflammatory conditions , as well as of other immune mediated diseases . in this context immune and bone systems appear to be integrated and sharing signaling pathways and regulatory mechanisms whose understanding provides a framework for obtaining new insights for the discovery of novel treatments for diseases related to both systems .", "thus osteoimmunology appears definitely as an interdisciplinary research and clinical field which also allowed new pathogenetic and clinical interpretations of well - known and common diseases , such as osteoporosis .", "its fields of interest are constantly expanding , thus enriching with an increasing number of translational implications , even in clinical practice and in various branches of medicine . here , the most important concepts in osteoimmunology are addressed in the context of physiopatological states bridging these two organ systems , including osteoporosis , ageing , menopause , inflammatory arthritis , cancer , dysmetabolism and neurological disorders .", "purpose of this review is to outline a new panorama of osteoimmunology that is not limited to immune mediated bone turnover but also consider , in the light of the latest findings in this field , interesting connections with other systems and regulatory functions over bone remodeling .", "bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals .", "antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment .", "bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis .", "bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc .", "ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes .", "moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone .", "ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function .", "the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes .", "there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] .", "the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes .", "they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) .", "the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential .", "the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation .", "oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients .", "they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages .", "ob , ocy and oc continuously communicate with each other to optimize the quality of the bone .", "for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors .", "the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption .", "the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal .", "osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption .", "rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways .", "simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation .", "many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 .", "ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade .", "a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time .", "therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption .", "the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis .", "it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation .", "wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis .", "wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy .", "the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors .", "the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression .", "cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis .", "an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis .", "other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage .", "bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals .", "antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment .", "bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis .", "bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc .", "ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes .", "moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone .", "ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function .", "the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes .", "there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] .", "the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes .", "they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) .", "the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential .", "the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation .", "oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients .", "they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages .", "ob , ocy and oc continuously communicate with each other to optimize the quality of the bone .", "for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors .", "the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption .", "the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal .", "osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption .", "rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways .", "simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation .", "many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 .", "ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade .", "a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time .", "therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption . the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis .", "it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation .", "wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis .", "wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy .", "the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors .", "the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression .", "cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis .", "an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis .", "other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage .", "both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling .", "inflammation and bone turnover share the same mediators , such as cytokines and transcription factors .", "various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk .", "t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes .", "however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells .", "two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage .", "the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype .", "the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures .", "t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease .", "th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc .", "treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- .", "for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl .", "moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation .", "rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells .", "the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus .", "thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions .", "other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal .", "the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling .", "tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation .", "their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases .", "the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc   and is also involved in the regulation of both the adaptive and innate immunity .", "it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system .", "also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function .", "therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis .", "tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells .", "for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface .", "cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen .", "in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover", ", cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption .", "eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization .", "osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator .", "the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn", "is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis .", "rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively .", "cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory .", "cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp", "boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation .", "pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages .", "the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells .", "the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin .", "cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss .", "tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production .", "moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg .", "bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth .", "therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 .", "inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis .", "the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption .", "an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis .", "all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network .", "each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines .", "for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl .", "however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression .", "in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] .", "a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated .", "the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production .", "ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) .", "treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity .", "endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 .", "these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc .", "resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob .", "osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered .", "a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis .", "finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system .", "cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis .", "oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption .", "osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors .", "they secrete a variety of proinflammatory and immunosuppressive factors . a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production .", "skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif .", "they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells .", "msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions .", "msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals . the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules .", "both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling .", "inflammation and bone turnover share the same mediators , such as cytokines and transcription factors .", "various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk .", "t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes .", "however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells .", "two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage .", "the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype .", "the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures .", "t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease .", "th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc .", "treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- .", "for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl .", "moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation .", "rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells .", "the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus .", "thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions .", "other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal .", "the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling .", "tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation .", "their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases .", "the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc   and is also involved in the regulation of both the adaptive and innate immunity .", "it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system .", "also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function .", "therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis .", "tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells .", "for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface .", "cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen .", "in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover", ", cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption .", "eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization .", "osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator .", "the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn", "is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis .", "rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively .", "cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory .", "cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp", "boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation .", "pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages .", "the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells .", "the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin .", "cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss .", "tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production .", "moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg .", "bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth .", "therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 .", "inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis .", "the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption .", "an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis .", "all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network .", "each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines .", "for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl .", "however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression .", "in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] .", "a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated .", "the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production .", "ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) .", "treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity .", "endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 .", "these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc .", "resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob .", "osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered .", "a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis .", "finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system .", "cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis .", "oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption .", "osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors .", "a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production .", "skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif .", "they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells .", "msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions .", "msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals .", "the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules .", "osteoporosis is a systemic disease of the skeleton , whose main features are loss of bone mass , bone mineral density ( bmd ) decrease and disruption of bone microarchitecture , so the skeleton becomes fragile , exposing patients to increased risk of fractures . aside from senile and postmenopausal osteoporosis , the first recognized types of primary osteoporosis , many other causes of secondary osteoporosis", "have been subsequently recognized , for example vitamin d and calcium deficits , lack of sun exposure , immobility , drugs such as cortisone , malabsorption syndromes , endocrine and dismetabolic diseases such as diabetes , disthyroidisms , hypercortisolism , and so on . only later , clinical and experimental findings evidenced a close connection between osteoporosis and immune mediated inflammatory conditions , for example , rheumatoid arthritis ( ra )   and acquired immune deficiency syndrome ( aids ) , and a common inflammatory background has been finally discovered as pathogenetic factor even in conditions of major osteoporotic risk , such as old age   and estrogen deficiency [ 57 - 59 ] .", "more recently , other unpredictable pathological conditions , such as obesity , are coming out as potential osteoporotic risk factors . even in these cases ,", "the main pathogenetic mechanism leading to bone tissue alteration seems to be inflammation . from this point of view osteoporosis", "could be therefore regarded as an immune mediated disease in which immune activation , through the induction of cytokine production and inflammation , leads to a remodeling of oc and ob activity and dysregulation of bone turnover with consequent increased bone resorption and osteoporosis .", "paradigmatic examples of the link between inflammation and osteoporosis are inflammatory arthritis , mainly ra ( fig .", "1 ) . ra is an autoimmune disease that is characterized by inflammation of the synovial joint , leading to severe structural damage and bone destruction .", "an increased bone resorption is the main pathogenetic mechanism in both disease progression leading to juxta - articular bone erosions and irreversible joint damage and systemic osteoporosis .", "bisphosphonates , drugs used for some time in the therapy of osteoporosis , are potent inhibitors of oc activity both in the primitive and secondary osteoporosis , such as that associated with autoimmune diseases . a decreased bmd in the spine and hip and higher prevalence of osteoporosis have been described in ra patients . in early untreated ra ,", "bmd is related to longer symptom duration , the presence of rheumatoid factor ( rf ) and cyclic citrulinated peptide antibodies ( anti - ccp ) , disease activity score , and the presence and progression of joint damage [ 1 , 19 ] .", "monoclonal antibodies ( moab ) against various proinflammatory cytokines and their receptors , such as tnf- , are useful in preventing and/or reversing bone erosions as well as systemic osteoporosis .", "autoimmune reactions induce rankl expression and subsequent osteoclastogenesis . that the rank/ rankl / opg pathway is central to the osteoporosis pathogenesis", "is confirmed by the elevated antiresorptive capacity of denosumab , an anti - rankl moab , utilized in osteoporosis therapy .", "activated immune cells at sites of inflammation produce a wide spectrum of proinflammatory and osteoclastogenic cytokines , resulting in bone erosions , osteitis , and peri - inflammatory and systemic bone loss .", "local peri - inflammatory bone loss and osteitis occur early and precede and predict erosive bone destruction in ra .", "moreover , peri - inflammatory bone formation is impaired , resulting in non - healing of erosions , and this allows a local vicious circle of inflammation between synovitis , osteitis , and local bone loss .", "rankl is highly expressed in the ra synovium , and inflammation - mediated bone damage is largely attributable to its abnormally high expression .", "in addition to activated t lymphocytes and macrophages , a pivotal role in inducing bone erosions is also played by rankl - expressing b cells , as highlighted by the observation of the therapeutic effect of anti - cd20 antibody in ra .", "cd4+t - cells , especially th17 cells , play a prominent role , particularly in the initiation of systemic immune response in ra .", "the interaction between immune and mesenchymal cells in joints , including synovial fibroblasts , which are characterized by hyperproliferative and hyperactive properties in response to an inflammatory environment , is of paramount importance in rheumatoid inflammation . in fig .", "thus , in the affected joints , hyperplasia of the synovial membrane is characterized by both hyperproliferation of synovial fibroblasts and massive infiltration of inflammatory immune cells , including cd4 + t cells and innate immune cells .", "autoimmune diseases , including arthritis , often result from an imbalance between treg cells and th17 cells .", "the th17 cells derived from foxp3 + t cells in ra comprise a novel th17 cell subset with a distinct pattern of gene expression and arthritogenic properties .", "the fate of plastic foxp3 + t cells may be a key determinant of the treg / th17 balance that is critically involved in the self - tolerance and autoimmunity .", "mesenchymal cells are a determinant of the development of ra that links the systemic immune response and the local disorder in the joints .", "mesenchymal cells contribute to the th17 mediated chronic inflammation by promoting the migration of th17 cells to the inflamed joint and concomitant increase in il-17 production .", "thus , the interaction of immune and mesenchymal cells plays a key role in both the chronic inflammation and bone destruction in ra .", "in particular , pathogenetic autoreactive immune cells migrate into joints and activate the mesenchymal cells resident in joint , such as synovial fibroblasts . moreover , since soluble inhibitors of the wnt pathway , such as dkk-1 , produced by synovial fibroblasts , are upregulated by tnf- , antibodies against dkk1 could be able to both promote bone formation and prevent bone erosions in ra .", "osteoporosis has long been regarded as simply the consequence of the menopausal estrogen decline . os- the progression of articular erosions is clearly driven by proinflammatory and osteoclastogenic cytokines produced by immune cells in the inflamed joint .", "both activated lymphocytes and macrophages stimulate osteoclast differentiation by producing proinflammatory mediators . activated lymphocytes of the synovial pannus overexpress rankl and tnf- that stimulate bone marrow cd11b+ ocp to proliferate and enter the bloodstream where they themselves produce inflammatory factors amplifying inflammation .", "activated macrophages in the inflamed joints produce various chemokines which drive the localization of periarticular bone ocp .", "the stimulation of osteoclastogenesis induced by the high concentrations of rankl and tnf- results in bone resorption .", "these cells do not produce antiosteoclastogenic cytokines , such as ifn- or il4 , while expressing high levels of rankl and secrete il-17 , that in turn stimulates ob , synoviocytes and fibroblasts to express rankl , and induces macrophages to produce proinflammatory cytokines , such as tnf- and il-6 in the synovium amplifying local inflammation .", "il-17 also induces the synthesis of enzymes capable of degrading the bone matrix such as matrix metalloproteinases ( mmp ) .", "these effects are balanced by treg that inhibit bone destruction by suppressing oc formation through both cell - cell contact and the secretion of inhibitory cytokines such as tgf- , il-4 and il-10 .", "teoimmunology , suggesting that immune cells take part in the bone changes typical of menopause , has led to a shift in the concept of osteoporosis , that is currently considered an inflammatory condition .", "post - menopausal osteoporosis is a clear example of the mutual influences between immune system , bone and endocrine system . like many other hormones , in addition to specific target organs ( breast and reproductive system )", ", estrogens have their receptors also on immune cells and bone , as well as on bone marrow precursors .", "( 3 ) shows the effects of estrogen deficiency on cells and molecules involved in bone metabolism .", "menopausal estrogen decline leads to proliferation and activation of t cells by increasing mhcii expression on monocytes and apc function and by inhibiting t cell apoptosis .", "these effects leads to the expansion of activated t lymphocytes resulting in hyperproduction of inflammatory cytokines and chronic oc stimulation which is responsible for bone loss and increased fracture risk .", "maintenance of inflammatory reactions leading to bone resorption and skeletal fragility is also present during senescence and inflammaging ( fig .", "4 ) , that is the condition of chronic inflammation characterizing aging , as the result of the immune system s ability to cope with stressors .", "inflammaging is now considered the background of a broad range of age - related diseases with an inflammatory pathogenesis .", "many of the biological mechanisms implicated in the aging process , such as cell senescence , proinflammatory immune profile , apoptosis and metabolism imbalance , are also implicated in bone remodeling .", "also in the absence of overt inflammatory diseases , the heightened catabolic signals induced by inflammation enhance apoptosis of ob and muscle cells , causing both osteoporosis and sarcopenia . during aging", ", the lifelong exposure to oxidative stress and chronic antigenic load leads to the loss of the regulatory process which counteracts t cell activation induced bone resorption . in aged people , lipid oxidation mediated by ros", "oxidized polyunsaturated fatty acids induce the association of ppar with -catenin and promote its degradation .", "oxidized lipids also potentiate oxidative stress , enhance ob apoptosis and inhibit bmp-2 induced ob differentiation .", "antioxidant agents seem to have some action on bone remodeling : resveratrol decreases nf - k activation induced by rankl and oc differentiation and also promotes osteogenesis in msc via the sirt1/foxo3 axis stimulation . during senescence , besides the impaired treg function , the number of effector memory cells is increased .", "these are senescent cells with proinflammatory properties , secreting several inflammatory cytokines able to influence bone remodeling .", "interestingly , this immunological pattern ( accumulation of activated cells and memory / effector lymphocytes secreting proinflammatory cytokines ) characterizing immunosenescence , is also peculiar of other immunological conditions associated with osteoporosis , such as ra , aids , chronic viral infections , etc . .", "the already complex cross - talk between bone and immune system is further expanding as they emerge new mediators involved .", "interestingly , the scenario of osteoimmunolgy is increasingly including interconnections with other organs and systems , therefore influencing several homeostatic functions in addition to bone turnover and immunity , such as hemopoiesis , metabolic and neuro - endocrine functions .", "the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis .", "hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation .", "hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells .", "interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system .", "primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors .", "bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis .", "the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis .", "of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines .", "the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women .", "the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action .", "the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone .", "the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function .", "a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 .", "these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle .", "both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development .", "cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment .", "hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis .", "ob , together with msc , are crucial components of the niche of growth of hsc .", "ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential .", "the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling .", "the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells .", "osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation .", "also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support .", "even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression .", "being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells .", "moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region .", "macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling ,", "a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation .", "the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement", ", neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob .", "in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand ,", "the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement .", "a promising new field of interest in this regard is the osteo - immune - oncology .", "there is in fact a close relationship between immune regulation of bone turnover and tumor growth .", "often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig .", "not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors .", "then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation .", "osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer .", "osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth .", "tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors . as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors .", "adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc .", "shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative", "activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob .", "in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger ", "mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal .", "not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures .", "leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain .", "leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells .", "adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance .", "it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes .", "adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob .", "fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway .", "leptin and adiponectin act on the bone through different signaling pathways with contrasting effects   ( table 2 ) .", "it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling .", "therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction .", "white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin .", "brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated .", "beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding .", "visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity .", "conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines .", "notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone .", "obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity , in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile .", "therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion", ", it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals .", "both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) .", "osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism .", "fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling .", "ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway .", "interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome .", "the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) .", "age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage .", "age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . in particular ,", "age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis .", "the binding of age to organic bone matrix may also increase the fragility of bones .", "autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions .", "however , excessive autophagy is harmful to cells and leads to damage or massive death of cells .", "autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion .", "it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn", "sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings .", "hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system .", "adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment .", "an association between major depression and osteoporosis has been recognized [ 96 - 99 ] .", "the prevalence of low bmd is higher in people with depression than the general population .", "interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] .", "neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) .", "leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner .", "it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis . the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions ,", "in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand ,", "5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) .", "there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) .", "the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal .", "the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption .", "the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism   ( fig .", "a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects .", "in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life .", "small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid  peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders .", "amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production .", "rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis .", "the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner . shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth .", "the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation .", "in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis .", "the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context", ", bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia .", "for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well .", "the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia .", "its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity .", "inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production .", "serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions .", "it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors .", "the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis .", "hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation .", "hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells .", "interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system .", "primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors .", "bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis .", "the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis .", "of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines .", "the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women .", "the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action .", "the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone .", "the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function .", "a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 .", "these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle .", "both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development .", "cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment .", "hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis .", "ob , together with msc , are crucial components of the niche of growth of hsc .", "ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential .", "the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling .", "the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells .", "osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation .", "also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support .", "even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression .", "being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells .", "moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region .", "macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling ,", "a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation .", "the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement", ", neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob .", "in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand ,", "the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement .", "a promising new field of interest in this regard is the osteo - immune - oncology .", "there is in fact a close relationship between immune regulation of bone turnover and tumor growth .", "often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis .", "tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig . 5 ) . in skeletal metastases ,", "not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors .", "then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation .", "osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer .", "osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth .", "tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors .", "as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors .", "adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc .", "shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative", "activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob .", "in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger  containing transcription factor , shift the equilibrium towards the osteoblastogenesis .", "mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal .", "not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures .", "leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain .", "leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells .", "adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance .", "it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes .", "adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob .", "fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway .", "leptin and adiponectin act on the bone through different signaling pathways with contrasting effects   ( table 2 ) .", "it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling .", "therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis .", "various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction .", "white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin .", "brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated .", "beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding .", "visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity .", "conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines .", "notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone .", "obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity ,", "in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile .", "therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion , it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals .", "both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) .", "osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism .", "fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling .", "ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway .", "interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome .", "the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) .", "age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage .", "age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases .", "in particular , age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis .", "the binding of age to organic bone matrix may also increase the fragility of bones .", "autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions .", "however , excessive autophagy is harmful to cells and leads to damage or massive death of cells .", "autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion .", "it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn", "sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings .", "hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system .", "adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment .", "an association between major depression and osteoporosis has been recognized [ 96 - 99 ] .", "the prevalence of low bmd is higher in people with depression than the general population .", "interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] .", "neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) .", "leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner .", "it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis .", "the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions , is also likely involved in interactions between the central nervous and immune systems .", "in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand ,", "5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) .", "there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) .", "the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal .", "the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption .", "the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism   ( fig .", "a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects .", "in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life .", "small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid  peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders .", "amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production .", "rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis .", "the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner .", "shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth .", "the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation .", "in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context", ", bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia .", "for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well .", "the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia .", "its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity .", "inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production .", "serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions .", "it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors .", "immune system and skeletal system interact with each other both in physiological and pathological conditions .", "considerable progress has been made in clarifying the crosstalk between bone and immune system that occurs in a complex and dynamic bone microenvironment . a central role in this crosstalk", "osteoimmunology therefore represents a new approach to studying osteoporosis that in the past was not considered an inflammatory condition .", "moreover , the discoveries of the existence of the immunoskeletal interface has also highlighted interesting repercussions for a wide range of pathological conditions beyond osteoporosis , including infections , autoimmune diseases , and neoplasia , in which same pathogenetic pathways are shared .", "osteoporosis could be therefore considered as a systemic model of integrated signaling pathways and cytokines working in a cooperative fashion .", "further important research horizons are opened with the extension of the discoveries of osteoimmunology and the disclosure of the immunoskeletal interface , whose practical implications may provide novel therapies for diseases other than osteoporosis , by targeting specific transcription factors , cytokines and their receptors . the correct understanding and decoding of the complex language", "through which immune system and bone communicate , although still in its dawn , is the essential requirement for the identification of such potentially useful therapeutic targets for both osteoporosis and other correlated inflammatory conditions , which share same mediators and signaling pathways ."], "abstract_text": ["<S> abstract : objectiveosteoimmunology investigates interactions between skeleton and immune system . in the light of recent discoveries in this field , a new reading register of osteoporosis is actually emerging , in which bone and immune cells are strictly interconnected . </S>", "<S> osteoporosis could therefore be considered a chronic immune mediated disease which shares with other age related disorders a common inflammatory background . here </S>", "<S> , we highlight these recent discoveries and the new landscape that is emerging.methodextensive literature search in pubmed central.resultswhile the inflammatory nature of osteoporosis has been clearly recognized , other interesting aspects of osteoimmunology are currently emerging . </S>", "<S> in addition , mounting evidence indicates that the immunoskeletal interface is involved in the regulation of important body functions beyond bone remodeling . </S>", "<S> bone cells take part with cells of the immune system in various immunological functions , configuring a real expanded immune system , and are therefore variously involved not only as target but also as main actors in various pathological conditions affecting primarily the immune system , such as autoimmunity and immune deficiencies , as well as in aging , menopause and other diseases sharing an inflammatory background.conclusionthe review highlights the complexity of interwoven pathways and shared mechanisms of the crosstalk between the immune and bone systems . </S>", "<S> more interestingly , the interdisciplinary field of osteoimmunology is now expanding beyond bone and immune cells , defining new homeostatic networks in which other organs and systems are functionally interconnected . </S>", "<S> therefore , the correct skeletal integrity maintenance may be also relevant to other functions outside its involvement in bone mineral homeostasis , hemopoiesis and immunity . </S>"], "labels": null, "section_names": ["Introduction", "Cellular and molecular mechanisms of bone remodeling", "The Basic Multicellular Unit", "The Receptor Network", "Crosstalk between the immune and bone system: the immunoskeletal interface", "Shared Signaling Pathways", "Parathyroid Hormone, Lymphocytes and Bone", "Cytokine and Cell Network", "Osteoporosis and inflammation: the lesson of rheumatoid arthritis", "Menopausal and senile osteoporosis", "Correlations with other systems and functions", "Hemopoiesis and Tumour Growth", "Fat Tissue and Metabolic Syndrome", "Neuroendocrine System and Major Depression", "Conclusion and perspectives", "CONFLICT OF INTEREST"], "sections": [["recently , extensive reciprocal interactions between the immune and skeletal systems have been demonstrated , resulting in the establishment of a new interdisciplinary research field , named  osteoimmunology  , which is focused on the understanding of the crosstalk between the immune and bone systems [ 1 - 3 ] .", "in particular , osteoimmunology investigates how the immune system impacts on bone turnover in physiological and pathological conditions through the immunoskeletal interface .", "researchers recently hav - egained better understanding of the dialogue between immune and bone cells and a new reading register of bone remodeling is emerging , in which the various phases of bone formation and resorption , that coexist in a dynamic equilibrium , are under strict immunological control .", "bone and immune system are functionally integrated through complex homeostatic networks and , in all respects , osteoporosis could be considered a chronic immune mediated inflammatory disease which shares clinical and biological features with many other inflammatory conditions , as well as of other immune mediated diseases . in this context immune and bone systems appear to be integrated and sharing signaling pathways and regulatory mechanisms whose understanding provides a framework for obtaining new insights for the discovery of novel treatments for diseases related to both systems .", "thus osteoimmunology appears definitely as an interdisciplinary research and clinical field which also allowed new pathogenetic and clinical interpretations of well - known and common diseases , such as osteoporosis .", "its fields of interest are constantly expanding , thus enriching with an increasing number of translational implications , even in clinical practice and in various branches of medicine . here , the most important concepts in osteoimmunology are addressed in the context of physiopatological states bridging these two organ systems , including osteoporosis , ageing , menopause , inflammatory arthritis , cancer , dysmetabolism and neurological disorders .", "purpose of this review is to outline a new panorama of osteoimmunology that is not limited to immune mediated bone turnover but also consider , in the light of the latest findings in this field , interesting connections with other systems and regulatory functions over bone remodeling ."], ["bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals .", "antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment .", "bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis .", "bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc .", "ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes .", "moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone .", "ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function .", "the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes .", "there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] .", "the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes .", "they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) .", "the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential .", "the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation .", "oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients .", "they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages .", "ob , ocy and oc continuously communicate with each other to optimize the quality of the bone .", "for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors .", "the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption .", "the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal .", "osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption .", "rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways .", "simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation .", "many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 .", "ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade .", "a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time .", "therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption .", "the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis .", "it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation .", "wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis .", "wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy .", "the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors .", "the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression .", "cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis .", "an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis .", "other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage ."], ["bone is a dynamic tissue formed by a protein and mineral salt matrix in which are embedded the bone cells , osteocytes ( ocy ) , osteoblasts ( ob ) and osteoclasts ( oc ) . in addition , many other types of cells take part in bone composition , including cartilage , stromal , hematopoietic and mesenchimal stem cells , all linked by a dense network of signals .", "antagonistic signaling between skeletal stem cell - derived subsets is a key mechanism in skeletal subset lineage commitment .", "bone tissue undergoes continuous adaptation during lifetime to preserve the structure of the skeleton and to control the mineral homeostasis .", "bone turnover requires two coordinated processes : bone formation , driven by ob and bone resorption , mediated by oc .", "ocy , through a complex network of tiny channels , transmit mechanical and microtraumatic signals leading to the activation of repair processes .", "moreover , ocy synthesize the bone matrix proteins which , along with the mineral component , determine the quality of the bone .", "ob are the precursors of ocy , i.e , the structural cells in the bone , and interact with oc to drive their differentiation and function .", "the mesenchymal stem cell ( msc ) from which the ob originate , can also give rise to chondrocytes , marrow stromal cells , and adipocytes .", "there are multiple subpopulations of perisinusoidal mesenchymal stem / progenitor cells ( mspcs ) , that have specific relationships with the different kinds of niche , i.e. the surrounding microenvironment in which the self - renewal and multilineage stem cells proliferate and differentiate [ 7 - 9 ] .", "the stem cells that maintain and repair the postnatal skeleton is an osteochondroreticular ( ocr ) stem cell that generate ob , chondrocytes , and reticular marrow stromal cells , but not adipocytes .", "they are characterized by the expression of the bone morphogenetic protein ( bmp ) antagonist gremlin 1 ( grem 1 ) .", "the perisinusoidal msc population also contains nes - gfp , leptin receptor ( lepr)-cre and cd146 expressing cells with osteogenic and adipogenic potential .", "the osteoblast precursor cells ( obp ) after increasing the osteopontin receptor ( cd44 ) and the receptor for stromal cell - derived factor 1 - sdf1 ( cxcr4 ) expression , migrate and become mature ob , attracted by vascular endothelial cells expressing sdf1 along chemotactic gradients into regions of bone formation .", "oc are multinucleated myeloid cells , specialized to remove mineralized bone matrix through the production of lysosomal enzymes , such as tartrate - resistant acid phosphatase ( trap ) and catepsin k , against which a selective inhibitor ( odanacatib ) has been recently synthesized to be employed in osteoporotic patients .", "they derive from a bone marrow precursor which gives rise also to professional antigen presenting cells ( apc ) , i.e. dendritic cells and macrophages .", "ob , ocy and oc continuously communicate with each other to optimize the quality of the bone .", "for example , ob provide essential signals for the differentiation of the myeloid lineage precursors of oc by producing macrophage colony - stimulating factor ( m - csf ) , receptor activator of nuclear factor - kb ( nf - kb ) ligand ( rankl ) and other co - stimulatory factors ."], ["the binding of rank receptor on oc and their precursors by its ligand rankl , expressed by ob and stromal cells , is the main activation signal for bone resorption .", "the ob derived m - csf links to its receptor c - fms on the surface of osteoclast cell precursors ( ocp ) , enabling the rank / rankl signal .", "osteoprotegerin ( opg ) inhibits osteoclastogenesis by acting as a decoy receptor of rankl , thus preventing bone resorption .", "rank receptor on oc , through the adapter protein tumor - necrosis - factor - receptor - associated factor 6 ( tr - af6 ) , bound to its cytoplasmic tail , activates nf - kb and other transcription factors , such as mapks , c - fos , activator protein 1 ( ap1 ) , up to nuclear factor of activated t cells ( nfatc1 ) , the hub of various signaling pathways .", "simultaneously , the activation of rank induces the phosphorylation of ig - like receptor associated adaptor proteins , such as the immunoreceptor tyrosine - based activation motif ( itam ) and fc - receptor common gamma ( fcr ) subunit . in the nucleus nfatc1 , together with other transcription factors , such as ap1 , pu.1 , microphthalmia - associated transcription factor ( mitf ) and cyclic amp responsive - element - binding protein ( creb ) , induces oc specific genes , including those codifying for calcitonin receptor , cathepsin k and trap , leading to oc differentiation and proliferation .", "many other receptor pathways interact with rank , some costimulators and amplificators , others inhibitors and modulators , and many of these are shared by immune cells . an inhibitor receptor system for rank signal is ephrin ( eph ) b2/b4 .", "ephb2 receptor on oc , stimulated by ephb4 ligand on ob , inhibits the oc differentiation blocking c - fos and the nfatc1 transcriptional cascade .", "a peculiar property of this membrane receptor complex is its capacity to control bone turnover through bidirectional signals : the cell expressing the receptor and the one that expresses the ligand influence each other at the same time .", "therefore , ephb4 activation on ob , through the induction of osteogenetic regulatory genes , contemporaneously favours the coupling of bone formation and resorption . the canonical wingless ( wnt)/ catenin pathway , involved mainly in the response to mechanical load , promotes differentiation , proliferation and mineralization activity of ob and also inhibits their apoptosis .", "it encompasses a family of proteins that bind to complex transmembrane receptors , formed by the association of frizzled ( fz ) proteins and low density lipoprotein related receptors ( lrp-5 , lrp-6 ) which stabilize the -catenin substrate that concentrates in the cytosol and migrates into the obp nucleus to regulate the transcription of target genes to induce ob differentiation and bone formation .", "wnt signaling inhibits msc commitment towards adipogenic and chondrogenic lineages , stimulating the differentiation towards osteoblastogenesis .", "wnt--catenin signaling also indirectly inhibits osteoclastogenesis and bone resorption by increasing opg secretion in ob and ocy .", "the bmp pathway acts as a wnt associative stimulator signal in skeletogenesis by expanding primitive mesenchymal cells and thus laying the foundation for subsequent endochondral ossification . at the same time , various natural inhibitors , produced both by ob and ocy , exert a negative feed - back control on wnt system , such as the wnt/-catenin signaling pathway inhibitor dickkopf homolog-1 ( dkk-1 ) , the secreted frizzled related protein ( sfrp ) and the sclerostin , synthesized by the sost gene , which binds to lrp5/6 receptors .", "the inflammatory cytokine tumor necrosis factor- ( tnf- ) induces ob apoptosis and reduces osteoblastogenesis by stimulating dkk-1 and sost expression .", "cortison augments dkk-1 and sfrp expression thus suppressing the wnt/-catenin signal on ob inducing osteoporosis .", "an anti - sclerostin moab has recently been produced as a new osteoanabolic drug useful in the therapy of osteoporosis .", "other potential osteoanabolic drugs acting on the wnt pathway are also dkk inhibitor molecules , now in the experimental stage ."], ["both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling .", "inflammation and bone turnover share the same mediators , such as cytokines and transcription factors .", "various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk .", "t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes .", "however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells .", "two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage .", "the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype .", "the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures .", "t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease .", "th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc .", "treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- .", "for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl .", "moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation .", "rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells .", "the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus .", "thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions .", "other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal .", "the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling .", "tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation .", "their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases .", "the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc   and is also involved in the regulation of both the adaptive and innate immunity .", "it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system .", "also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function .", "therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis .", "tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells .", "for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface .", "cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen .", "in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover", ", cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption .", "eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization .", "osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator .", "the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn", "is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis .", "rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively .", "cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory .", "cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp", "boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation .", "pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages .", "the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells .", "the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin .", "cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss .", "tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production .", "moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg .", "bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth .", "therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 .", "inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis .", "the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption .", "an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis .", "all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network .", "each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines .", "for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl .", "however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression .", "in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] .", "a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated .", "the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production .", "ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) .", "treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity .", "endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 .", "these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc .", "resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob .", "osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered .", "a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis .", "finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system .", "cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis .", "oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption .", "osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors .", "they secrete a variety of proinflammatory and immunosuppressive factors . a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production .", "skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif .", "they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells .", "msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions .", "msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals . the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules ."], ["both clinical observations and basic research demonstrated that mediators driving inflammatory processes are also closely involved in bone remodeling .", "inflammation and bone turnover share the same mediators , such as cytokines and transcription factors .", "various molecules mediating communication between bone cells have been identified and several immunological mediators are involved in this crosstalk .", "t lymphocytes resident in the bone marrow are the key immune cells that regulate bone remodeling and responsiveness of bone cells to parathyroid hormone ( pth ) , in physiological and pathological conditions . during inflammatory diseases or in conditions characterized by low - grade systemic inflammation , such as menopause and aging , oc bone resorption is driven by inflammatory cytokines produced by activated t lymphocytes .", "however , bone marrow t cells also support bone homeostasis by inducing bone formation via direct interactions with bone cells .", "two mechanisms are involved : the binding of t cell costimulatory molecules to their counter receptors on bone cells and their precursors , and the release of cytokines and wnt ligands that activate wnt signaling in osteoblastic cell lineage .", "the final effect of t lymphocytes on bone depends on their activation state and their specific phenotype .", "the prevailing bone marrow t cells are activated central memory cd8 + lymphocytes , secreting relatively high levels of effector cytokines , mainy tnf-. these cells are abundant in postmenopausal women with osteoporotic fractures .", "t helper ( th17 ) cells are capable of stimulating bone resorption and play a pivotal role in the bone loss of inflammatory conditions such as psoriasis , rheumatoid arthritis , periodontal disease , and inflammatory bowel disease .", "th17 cells induce osteoclastogenesis by secreting interleukin ( il)-17 , rankl , tnf- , il-1 , and il-6 , along with low levels of ifn-. moreover , il-17 stimulates the release of rankl by ob and ocy and upregulate rank expression on oc .", "treg exert anti - osteo - clastogenic activity by producing suppressor cytokines , including il-4 , il-10 , and tgf- .", "for example it is interesting to note how even the cells of the immune sytem , mainly activated t and b lymphocytes and dendritic cells , express rankl .", "moreover , ran - kl , the principal osteoclastogenic mediator , stimulates bone resorption through the nfatc1 , which is also a crucial factor in the immune system regulation .", "rankl , initially regarded as activator of apc by t lymphocytes , also plays an important role in the generation of treg , which suppress the development of cd8 + lymphocytes into cytotoxic cells .", "the expression of rankl on t lymphocytes is also central for the differentiation of medullary epithelial cells which are responsible for self - reactive t lymphocyte negative selection in the thymus .", "thus , depending on the context in which it acts , rankl can stimulate or suppress immune reactions .", "other examples of shared receptor signals are the immunoglobulin ( ig)-like receptors which amplify the nfatc1 signal .", "the toll like receptors ( tlr ) , stimulated by pathogen associated molecular patterns ( pamp ) , utilize traf6 in their cascade signaling .", "tlr are able to activate both the synthesis and release of proinflammatory and osteoclastogenic cytokines from immune cells , leading to bone resorption stimulation .", "their involvement in the bone remodeling process provides a further key in the comprehension of the osteoporosis of infectious diseases .", "the osteoclast associated receptor ( oscar ) , which belongs to the ig - like receptor family , mediates interactions between ob and oc   and is also involved in the regulation of both the adaptive and innate immunity .", "it associates with the adaptor molecule fcr subunit , which harbors an immunoreceptor tyrosine - based activation motif ( itam ) critical for calcium signaling activation in the immune system .", "also , the itam - harboring adaptor dap12 plays a role in oc differentiation and function .", "therefore , receptors such as dap12 and ig - like receptors associated with fcr , initially characterized in myeloid cells and in natural killer lymphocytes , are also involved in rank induced osteoclastogenesis .", "tnf induces the expression of oscar and other receptors important for oc differentiation on the surface of monocytoid peripheral blood cells .", "for example , cd80/cd86 blocks oc generation by binding to ctla4 , an inhibitory molecule of the monocyte induced t lymphocyte costimulation , which is highly expressed on treg surface .", "cathepsin k is expressed in oc and plays a central role in the degradation of bone matrix components , such as type i collagen .", "in addition to osteoclastic bone resorption , cathepsin k is also implicated in dendritic cell activation through tlr 9 . moreover", ", cathepsin k supports the secretion of il-6 and il-23 , inflammatory cytokines involved in the production of th17 cells , which in turn promote osteoclastic bone resorption .", "eph receptors and their associated ligands , expressed by cells found within the bone marrow microenvironment , including ob and oc , are implicated in the regulation of physiological and pathological bone remodeling , but also are central in many other different cellular processes including , in addition to immune regulation , angiogenesis , neuronal development and neoplastic metastatization .", "osteoclast semaphorin 4d sustains bone resorption by inhibiting osteoblastogenesis . since sema4d also regulates a variety of immune functions , such as antigen presentation , b lymphocyte activation and chemotaxis of monocytes , it could be regarded as an osteoimmunological mediator .", "the matrix glycoprotein osteopontin ( opn ) , produced by different types of cells , including immune cells , oc , endothelial and epithelial cells , increases bone resorption by inducing the expression of the osteoclastic immune receptor cd44 , essential for cell migration , and by directly enhancing oc attachment to bone extracellular matrix ( ecm ) , required for ocp activation . as a consequence of bone resorption , more opn", "is further released from the ecm into the bone microenvironment and into the blood , thus amplifying local and systemic osteoclastogenesis .", "rankl and cd40l expressed on t cells , apc , stromal cells and ob , activate the cognate receptors rank and cd40 in ocp and ob , respectively .", "cd40/cd40l signaling promotes macrophage activation and differentiation , antibody isotype switching , and the development of b cell memory .", "cd40l also increases the commitment of msc to the osteoblastic lineage . through cd80/86 signaling in ocp"], ["boh these conditions are mimicked by continuous pth infusion ( cpth ) , whereas daily or intermittent pth injection ( ipth ) , therapeutically employed in several osteoporotic conditions , stimulates bone formation .", "pth binds its receptors ( ppr ) on stromal cells , ob , and ocy but also on t cells and macrophages .", "the catabolic effect of cpth is mostly mediated by enhanced production of rankl and decreased production of opg by ob and stromal cells .", "the pth anabolic effect is mediated by wnt signaling activation : pth increases -catenin levels in ob , promotes lrp6 signaling and decreases the production of sclerostin .", "cpth stimulates bone cells and immune cells to release growth factors and cytokines , including il-6 and tnf- , which induce th17 cell differentiation and the production of il-17 , that plays a pivotal role in the pth induced bone loss .", "tnf- in turn stimulates oc formation and activity via multiple mechanisms , including increased rankl production .", "moreover , tnf- upregulates the expression of cd40 in ob and stromal cells , increasing their sensitivity to cpth and suppressing opg .", "bone marrow t cells provide cell surface signals and secrete cytokines that direct the differentiation of mesenchimal progenitors towards ob characterized by a high sensitivity to pth .", "therapy with teriparatide , a form of ipth treatment , increases the bone marrow levels of wnt10. bone marrow cd8 + t cells potentiate the anabolic activity of pth by providing wnt10 ."], ["inflammation results in disturbances in the immunoskeletal interface , i.e. the convergence of cells and cytokines that regulate both the immunity that the bone , causing osteoporosis .", "the inflammatory cytokines tnf- , il-1 , il-6 and il-17 are crucial in acute and chronic inflammation and strong inducers of bone resorption .", "an excessive or abnormal immune activation can induce osteoporosis , as for example in autoimmune diseases , infections and also in senile and postmenopausal osteoporosis .", "all these conditions go along with an increased inflammatory background and the presence of rankl producing activated t cells . however , in addition to osteoclastogenic cytokines , there are also cytokines which counteract bone resorption and exert osteoblastogenic properties , resulting in a complex bone remodeling cytokine network .", "each cytokine has also pleiotropic functions and it is therefore not surprising that same cytokines can exert different and often contrasting effects depending on the specific context in which they act , the maturation stage of target cells and/or the influence of other cytokines .", "for example , the pleiotropic cytokine ifn- exerts anti - osteoclastogenic effects in physiological bone remodeling , by binding to specific oc receptors and inducing traf6 proteosomal degradation with consequent inhibition of the transduction signal mediated by rankl .", "however , in postmenopausal osteoporosis , inflammation or infections , the final effect of ifn- is skewed towards bone resorption through t lymphocyte activation and rankl expression .", "in fact , ifn- is a powerful stimulator of class ii major histocompatibility complex ( mhcii ) antigen expression on apc , with consequent increased stimulation of t cells through their antigen specific receptor ( tcr ) , inducing further immune activation and production of osteoclastogenic proinflammatory cytokines [ 35 - 40 ] .", "a cascade of cytokines drives ocp homing , differentiation and activation . circulating bone marrow produced ocp function as a tank of progenitor cells for several effector cells , in relation to the different cytokines implicated .", "the activation of cd8 t cells by oc induces il-2 , il-6 , il-10 and ifn- production .", "ocp can also enhance the expression of the suppressor of cytokine signaling ( socs ) .", "treg , whose main marker is the transcription factor foxp3 , balance il-17 induced bone resorption closely interacting with oc and expressing ctla-4 , which in turn inhibits oc activity .", "endothelial cells , activated by il-1 and tnf- , attract circulating ocp at sites of inflammation where they migrate through high endothelial venules driven by the expression of cell adhesion molecules ( cam ) , such as icam-1 and cd44 .", "these cd14 + monocytoid cells , under the stimulation of rankl , become activated bone resorbing oc .", "resident tissue macrophages of bone , termed osteal macrophages , are predominantly located adjacent to ob .", "osteal macrophages play diverse roles in skeletal homeostasis , their specific functions depending on the macrophage subset considered .", "a central function of macrophages is their phagocytic ability . in particular , efferocytosis ( phagocytosis of apoptotic cells ) is a critical process in both clearing dead cells and replacement of progenitor cells to maintain bone homeostasis .", "finally , not only the immune system regulates bone remodeling , but also the bone is able to influence the immune system , actively interacting with immune cells . the same bone cells would then be able to influence or even also perform many immune functions , such as cytokine production and antigen presentation [ 46 - 48 ] . in this sense , the bone would be regarded as a sort of expanded immune system .", "cytokines secreted by bone cells drive naive t cell differentiation into several lineages , leading to expansion of mature t cell populations that further regulate bone homeostasis .", "oc selectively recruit and activate cd8 + t cells expressing cd25 and foxp3 ( oc - induced regulatory cd8 t cells ) . in turn , these cd8 + treg cells suppress bone resorption , decrease inflammatory / osteoclastogenic cytokine production , and stimulate bone formation , creating a regulatory loop : oc and rankl induce treg , and then treg blunt osteoclastic bone resorption .", "osteocalcin , expressed on mature bone cells , regulates the production of thymic - seeding t lymphoid progenitors .", "a subset of mesenchimal cells expressing osterix , a marker of bone precursors , regulate the maturation of early b lymphoid precursors by promoting pro - b to pre - b cell transition through insulin - like growth factor 1 ( igf-1 ) production .", "skeletal stem cells are also able to recruit and activate neutrophils via the release of il-6 and il-8 , ifn- , gm - csf and mif .", "they inhibit b cell proliferation , differentiation , and antibody production , and can also directly inhibit t cell function , rendering them anergic or shifting their phenotype to that of functional regulatory cells .", "msc induce macrophages to switch from classically activated proinflammatory ( m1 ) to alternatively activated anti - inflam - matory ( m2 ) phenotype , and inhibit mast cell degranulation attenuating allergic reactions .", "msc express active tlr , through which they sense bone microenvironment , recognizing exogenous ( bacterial products ) and endogenous ( heat shock proteins , rna ) danger signals .", "the common tlr signaling feature is the activation of the nf- b transcription factors implicated in controlling the expression of inflammatory cytokines and cell maturation molecules ."], ["osteoporosis is a systemic disease of the skeleton , whose main features are loss of bone mass , bone mineral density ( bmd ) decrease and disruption of bone microarchitecture , so the skeleton becomes fragile , exposing patients to increased risk of fractures . aside from senile and postmenopausal osteoporosis , the first recognized types of primary osteoporosis , many other causes of secondary osteoporosis", "have been subsequently recognized , for example vitamin d and calcium deficits , lack of sun exposure , immobility , drugs such as cortisone , malabsorption syndromes , endocrine and dismetabolic diseases such as diabetes , disthyroidisms , hypercortisolism , and so on . only later , clinical and experimental findings evidenced a close connection between osteoporosis and immune mediated inflammatory conditions , for example , rheumatoid arthritis ( ra )   and acquired immune deficiency syndrome ( aids ) , and a common inflammatory background has been finally discovered as pathogenetic factor even in conditions of major osteoporotic risk , such as old age   and estrogen deficiency [ 57 - 59 ] .", "more recently , other unpredictable pathological conditions , such as obesity , are coming out as potential osteoporotic risk factors . even in these cases ,", "the main pathogenetic mechanism leading to bone tissue alteration seems to be inflammation . from this point of view osteoporosis", "could be therefore regarded as an immune mediated disease in which immune activation , through the induction of cytokine production and inflammation , leads to a remodeling of oc and ob activity and dysregulation of bone turnover with consequent increased bone resorption and osteoporosis .", "paradigmatic examples of the link between inflammation and osteoporosis are inflammatory arthritis , mainly ra ( fig .", "1 ) . ra is an autoimmune disease that is characterized by inflammation of the synovial joint , leading to severe structural damage and bone destruction .", "an increased bone resorption is the main pathogenetic mechanism in both disease progression leading to juxta - articular bone erosions and irreversible joint damage and systemic osteoporosis .", "bisphosphonates , drugs used for some time in the therapy of osteoporosis , are potent inhibitors of oc activity both in the primitive and secondary osteoporosis , such as that associated with autoimmune diseases . a decreased bmd in the spine and hip and higher prevalence of osteoporosis have been described in ra patients . in early untreated ra ,", "bmd is related to longer symptom duration , the presence of rheumatoid factor ( rf ) and cyclic citrulinated peptide antibodies ( anti - ccp ) , disease activity score , and the presence and progression of joint damage [ 1 , 19 ] .", "monoclonal antibodies ( moab ) against various proinflammatory cytokines and their receptors , such as tnf- , are useful in preventing and/or reversing bone erosions as well as systemic osteoporosis .", "autoimmune reactions induce rankl expression and subsequent osteoclastogenesis . that the rank/ rankl / opg pathway is central to the osteoporosis pathogenesis", "is confirmed by the elevated antiresorptive capacity of denosumab , an anti - rankl moab , utilized in osteoporosis therapy .", "activated immune cells at sites of inflammation produce a wide spectrum of proinflammatory and osteoclastogenic cytokines , resulting in bone erosions , osteitis , and peri - inflammatory and systemic bone loss .", "local peri - inflammatory bone loss and osteitis occur early and precede and predict erosive bone destruction in ra .", "moreover , peri - inflammatory bone formation is impaired , resulting in non - healing of erosions , and this allows a local vicious circle of inflammation between synovitis , osteitis , and local bone loss .", "rankl is highly expressed in the ra synovium , and inflammation - mediated bone damage is largely attributable to its abnormally high expression .", "in addition to activated t lymphocytes and macrophages , a pivotal role in inducing bone erosions is also played by rankl - expressing b cells , as highlighted by the observation of the therapeutic effect of anti - cd20 antibody in ra .", "cd4+t - cells , especially th17 cells , play a prominent role , particularly in the initiation of systemic immune response in ra .", "the interaction between immune and mesenchymal cells in joints , including synovial fibroblasts , which are characterized by hyperproliferative and hyperactive properties in response to an inflammatory environment , is of paramount importance in rheumatoid inflammation . in fig .", "thus , in the affected joints , hyperplasia of the synovial membrane is characterized by both hyperproliferation of synovial fibroblasts and massive infiltration of inflammatory immune cells , including cd4 + t cells and innate immune cells .", "autoimmune diseases , including arthritis , often result from an imbalance between treg cells and th17 cells .", "the th17 cells derived from foxp3 + t cells in ra comprise a novel th17 cell subset with a distinct pattern of gene expression and arthritogenic properties .", "the fate of plastic foxp3 + t cells may be a key determinant of the treg / th17 balance that is critically involved in the self - tolerance and autoimmunity .", "mesenchymal cells are a determinant of the development of ra that links the systemic immune response and the local disorder in the joints .", "mesenchymal cells contribute to the th17 mediated chronic inflammation by promoting the migration of th17 cells to the inflamed joint and concomitant increase in il-17 production .", "thus , the interaction of immune and mesenchymal cells plays a key role in both the chronic inflammation and bone destruction in ra .", "in particular , pathogenetic autoreactive immune cells migrate into joints and activate the mesenchymal cells resident in joint , such as synovial fibroblasts . moreover , since soluble inhibitors of the wnt pathway , such as dkk-1 , produced by synovial fibroblasts , are upregulated by tnf- , antibodies against dkk1 could be able to both promote bone formation and prevent bone erosions in ra ."], ["osteoporosis has long been regarded as simply the consequence of the menopausal estrogen decline . os- the progression of articular erosions is clearly driven by proinflammatory and osteoclastogenic cytokines produced by immune cells in the inflamed joint .", "both activated lymphocytes and macrophages stimulate osteoclast differentiation by producing proinflammatory mediators . activated lymphocytes of the synovial pannus overexpress rankl and tnf- that stimulate bone marrow cd11b+ ocp to proliferate and enter the bloodstream where they themselves produce inflammatory factors amplifying inflammation .", "activated macrophages in the inflamed joints produce various chemokines which drive the localization of periarticular bone ocp .", "the stimulation of osteoclastogenesis induced by the high concentrations of rankl and tnf- results in bone resorption .", "these cells do not produce antiosteoclastogenic cytokines , such as ifn- or il4 , while expressing high levels of rankl and secrete il-17 , that in turn stimulates ob , synoviocytes and fibroblasts to express rankl , and induces macrophages to produce proinflammatory cytokines , such as tnf- and il-6 in the synovium amplifying local inflammation .", "il-17 also induces the synthesis of enzymes capable of degrading the bone matrix such as matrix metalloproteinases ( mmp ) .", "these effects are balanced by treg that inhibit bone destruction by suppressing oc formation through both cell - cell contact and the secretion of inhibitory cytokines such as tgf- , il-4 and il-10 .", "teoimmunology , suggesting that immune cells take part in the bone changes typical of menopause , has led to a shift in the concept of osteoporosis , that is currently considered an inflammatory condition .", "post - menopausal osteoporosis is a clear example of the mutual influences between immune system , bone and endocrine system . like many other hormones , in addition to specific target organs ( breast and reproductive system )", ", estrogens have their receptors also on immune cells and bone , as well as on bone marrow precursors .", "( 3 ) shows the effects of estrogen deficiency on cells and molecules involved in bone metabolism .", "menopausal estrogen decline leads to proliferation and activation of t cells by increasing mhcii expression on monocytes and apc function and by inhibiting t cell apoptosis .", "these effects leads to the expansion of activated t lymphocytes resulting in hyperproduction of inflammatory cytokines and chronic oc stimulation which is responsible for bone loss and increased fracture risk .", "maintenance of inflammatory reactions leading to bone resorption and skeletal fragility is also present during senescence and inflammaging ( fig .", "4 ) , that is the condition of chronic inflammation characterizing aging , as the result of the immune system s ability to cope with stressors .", "inflammaging is now considered the background of a broad range of age - related diseases with an inflammatory pathogenesis .", "many of the biological mechanisms implicated in the aging process , such as cell senescence , proinflammatory immune profile , apoptosis and metabolism imbalance , are also implicated in bone remodeling .", "also in the absence of overt inflammatory diseases , the heightened catabolic signals induced by inflammation enhance apoptosis of ob and muscle cells , causing both osteoporosis and sarcopenia . during aging", ", the lifelong exposure to oxidative stress and chronic antigenic load leads to the loss of the regulatory process which counteracts t cell activation induced bone resorption . in aged people , lipid oxidation mediated by ros", "oxidized polyunsaturated fatty acids induce the association of ppar with -catenin and promote its degradation .", "oxidized lipids also potentiate oxidative stress , enhance ob apoptosis and inhibit bmp-2 induced ob differentiation .", "antioxidant agents seem to have some action on bone remodeling : resveratrol decreases nf - k activation induced by rankl and oc differentiation and also promotes osteogenesis in msc via the sirt1/foxo3 axis stimulation . during senescence , besides the impaired treg function , the number of effector memory cells is increased .", "these are senescent cells with proinflammatory properties , secreting several inflammatory cytokines able to influence bone remodeling .", "interestingly , this immunological pattern ( accumulation of activated cells and memory / effector lymphocytes secreting proinflammatory cytokines ) characterizing immunosenescence , is also peculiar of other immunological conditions associated with osteoporosis , such as ra , aids , chronic viral infections , etc . ."], ["the already complex cross - talk between bone and immune system is further expanding as they emerge new mediators involved .", "interestingly , the scenario of osteoimmunolgy is increasingly including interconnections with other organs and systems , therefore influencing several homeostatic functions in addition to bone turnover and immunity , such as hemopoiesis , metabolic and neuro - endocrine functions .", "the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis .", "hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation .", "hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells .", "interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system .", "primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors .", "bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis .", "the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis .", "of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines .", "the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women .", "the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action .", "the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone .", "the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function .", "a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 .", "these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle .", "both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development .", "cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment .", "hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis .", "ob , together with msc , are crucial components of the niche of growth of hsc .", "ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential .", "the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling .", "the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells .", "osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation .", "also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support .", "even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression .", "being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells .", "moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region .", "macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling ,", "a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation .", "the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement", ", neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob .", "in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand ,", "the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement .", "a promising new field of interest in this regard is the osteo - immune - oncology .", "there is in fact a close relationship between immune regulation of bone turnover and tumor growth .", "often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis . tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig .", "not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors .", "then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation .", "osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer .", "osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth .", "tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors . as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors .", "adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc .", "shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative", "activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob .", "in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger ", "mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal .", "not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures .", "leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain .", "leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells .", "adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance .", "it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes .", "adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob .", "fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway .", "leptin and adiponectin act on the bone through different signaling pathways with contrasting effects   ( table 2 ) .", "it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling .", "therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis . various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction .", "white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin .", "brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated .", "beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding .", "visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity .", "conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines .", "notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone .", "obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity , in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile .", "therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion", ", it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals .", "both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) .", "osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism .", "fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling .", "ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway .", "interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome .", "the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) .", "age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage .", "age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases . in particular ,", "age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis .", "the binding of age to organic bone matrix may also increase the fragility of bones .", "autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions .", "however , excessive autophagy is harmful to cells and leads to damage or massive death of cells .", "autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion .", "it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn", "sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings .", "hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system .", "adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment .", "an association between major depression and osteoporosis has been recognized [ 96 - 99 ] .", "the prevalence of low bmd is higher in people with depression than the general population .", "interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] .", "neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) .", "leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner .", "it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis . the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions ,", "in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand ,", "5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) .", "there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) .", "the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal .", "the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption .", "the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism   ( fig .", "a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects .", "in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life .", "small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid  peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders .", "amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production .", "rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis .", "the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner . shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth .", "the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation .", "in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis .", "the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context", ", bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia .", "for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well .", "the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia .", "its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity .", "inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production .", "serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions .", "it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors ."], ["the crosstalk between skeletal and bone marrow is crucial to hematopoietic stem cell ( hsc ) function and throughout the hemopoiesis .", "hsc occupy specific locations in the bone marrow microenvironment , commonly referred to as niches , comprising multiple cell types able to regulate hsc proliferation and differentiation .", "hematopoietic and skeletal stem cell homeostasis are closely related : msc progeny express numerous cytokines necessary for hsc maintenance and hematopoiesis , including kit ligand and stromal - derived factor , but also stem and progenitor cells of the hematopoietic system may reciprocally regulate skeletal progenitors by expressing myriad factors associated with skeletogenesis , whose cognate receptors are highly expressed by skeletal stem cells .", "interestingly , these latter exhibit receptors to circulating hormones , such as leptin and thyroid - stymulating hormone , suggesting that skeletal stem cells and their progeny may link systemic exocrine regulation to both skeletal and hematopoietic system .", "primitive mesenchymal progenitors are more critical for hsc function , whereas lineage - restricted mesenchymal cells control more committed hematopoietic progenitors .", "bone microenvironment is therefore a composite of specialized niches providing distinctive functional units to regulate hematopoiesis .", "the estrogen deficiency induces a marked increase of inflammatory cytokines and mediators of inflammation , in particular ifn- , m - csf , tnf- , il-1 , il-6 and il-7 , in addition to prostaglandins ( pge ) and reactive oxygen species ( ros ) , which play a driving role in the development of osteoporosis .", "of particular interest is the expansion in the peripheral blood from postmenopausal women of two particular lymphocyte subsets , cd3 + cd56 + t lymphocytes , major producers of tnf- , and b220 + igm - b lymphocyte precursors , that under certain stimuli can differentiate into ocp and are strong producers of inflammatory cytokines .", "the enhanced tnf- production by activated t cells has a central role in bone loss due to estrogen decline in menopausal women .", "the same ifn- , which in some situations could be protective against osteoporosis , during estrogen deficiency has a prevailing osteoclastogenic stimulating action .", "the estrogen decline also results in the decrease of opg and tgf- that normally contrast the effects of inflammatory mediators on bone .", "the age - related increase of inflammatory cytokines results from a chronic activation of macrophages and memory / effector t cells , in addition to an impaired treg function .", "a peculiar finding of immunosenescence is the increased number of senescent memory cells expressing rankl and secreting osteoclastogenic cytokines , mainly tnf- , il-1 , il-6 and il-17 .", "these cytokines are able to facilitate ocp expansion which amplify the systemic inflammation by producing further proinflammatory factors able to recruit other inflammatory cells and perpetuating the flogistic vicious cycle .", "both the increased transcriptional activity of nf - kb due to genotoxic , inflammatory , and oxidative stresses in aging , and the chronic p53 activation induced by the age - related progressive loss of telomere length , result in impaired ob proliferation and osteoporosis development .", "cells of the immune system and bone cells derive from bone marrow precursors and develop in the same bone marrow microenvironment .", "hematopoietic and immune cells share same signaling pathways with cells of the bone and their precursors , which surprisingly do not take part in the regulation of immunity and hemopoiesis .", "ob , together with msc , are crucial components of the niche of growth of hsc .", "ob overexpression of the wnt antagonist dkk1 reduces wnt signaling in hsc and disrupts hsc self - renewal potential .", "the wnt antagonist secreted frizzled - related protein 1 ( sfrp1 ) is involved in osteoblastic mediated hsc regulation ; through its regulation of ob , pth controls the hematopoietic niche function , involving crosstalk with wnt signaling .", "the upregulation of notch ligand protein jagged-1 ( jag1 ) expression in ob and the increased notch signaling are involved in increasing hsc numbers ; angiopoietin-1 receptor activation on hsc by ob produced angiopoietin-1 promotes strict adhesion of hsc to the niche , inducing quiescence of these cells .", "osteopontin , an ob - secreted protein , participates in hsc location and is a negative regulator of their proliferation .", "also , ocy derived from ob that become embedded within the bone matrix , are involved in the complex regulation of hemopoiesis through the osteoimmune interface : mainly by the production of sclerostin , they appear to have an inhibitory effect on hsc support .", "even oc , as well as activated endothelial cells , are likely involved in the mobilization of hsc by cytokine induced cam expression .", "being the only cells capable of bone resorption , in addition to allowing the renewal of the skeleton , they also open the space in the bone marrow for hematopoietic cells .", "moreover , osteoclastic bone resorption releases calcium , which attracts and retains calcium sensing receptor expressing hsc at the endosteal region .", "macrophages play diverse roles in the bone and marrow . at the sites of bone remodeling ,", "a subset of hsc is located in close proximity to megakaryocytes in the bone marrow , where ob undergo rapid expansion in response to the secretion of megakaryocyte - derived mesenchymal growth factors , such as platelet derived growth factor ( pdgf)- , to promote hsc proliferation .", "the bone marrow microenvironment can also act as a niche for the onset and progression of neoplastic diseases , including both hematologic malignancies and metastases of solid tumors , mainly breast and prostatic cancer . in multiple myeloma , an osteolytic hematological malignancy characterized by an important skeletal involvement", ", neoplastic plasma cells produce a large amount of mediators that induce osteoclastic bone resorption and block the activity of ob .", "in addition to osteolytic factors , myeloma cells produce dkk1 , which inhibits obp differentiation by binding to the lrp5/6 coreceptors expressed on their surface . on the other hand ,", "the same bone cells produce growth factors and angiogenetic cytokines able to support the development and progression of myeloma , perpetuating a vicious circle of mutual reinforcement .", "a promising new field of interest in this regard is the osteo - immune - oncology .", "there is in fact a close relationship between immune regulation of bone turnover and tumor growth .", "often , cancer cells express rank and in proneoplastic inflammation various cell types express rankl that acts as chemotactic factor favoring the skeletal metastasis .", "tumor cells in the bone cause activation of osteoclasts that mediate bone resorption and additional growth factor release from the bone matrix , fueling a vicious circle of impaired bone turnover and tumor proliferation ( fig . 5 ) . in skeletal metastases ,", "not always the tumor associated inflammation is the expression of an immune system that successfully opposes the neoplastic growth , as previously believed , but sometimes some patterns of immune activation can be facilitators of the development of tumors .", "then the block of rankl , in addition to inhibiting bone resorption , also decreases tumor growth , enhances apoptosis of malignant cells and diminishes proneoplastic inflammation .", "osteal macrophages , as well as macrophages in other tissues , have a role in tumor progression , supporting cancers which preferentially metastasize to the skeleton , in particular breast and prostate cancer .", "osteal macrophages expressing cd68 , a phagocytic capacity marker of cells infiltrating metastatic lesions , could facilitate tumor establishment and growth .", "tumor - derived pthrp drives myeloid cell recruitment via ob produced ccl2 , which is high in the bone microenvironment and whose levels are associated with poor prognoses in primary breast tumors ."], ["as for bone , adipose tissue is a kind of immune tissue and produces immunoregulatory factors .", "adipocytes and ob derive from the same msc , as well as visceral adipose tissue macrophages and oc derive from the same hsc .", "shared transcription factors regulate the shift between the different cell lines and the subsequent differentiation cascade . by action of peroxisome proliferative", "activated receptor gamma ( ppar ) , the principal regulator of adipogenesis , the msc differentiates into adipocytes rather than into ob .", "in contrast , the expression of runt - related transcription factor 2 ( runx2 ) , associated with ob differentiation , and osterix , an ob zinc finger  containing transcription factor , shift the equilibrium towards the osteoblastogenesis .", "mechanical loading promotes ob differentiation and inhibits adipogenesis by down - regulating ppar or by stimulating a durable -catenin signal .", "not surprisingly , ppar agonists such as thiazolidinediones used to increase insulin sensitivity in type 2 diabetes , also increase the risk of osteoporotic fractures .", "leptin is a pro - inflammatory adipokine that exerts its actions via central nervous system regulation of feeding behavior , where it promotes satiety and prevents weight gain .", "leptin can also directly signal through its receptor expressed on immunocytes , where it induces tnf- and il-6 production by monocytes , chemokines by macrophages , and th1 cytokines from polarized cd4 + t cells .", "adiponectin is an anti - inflammatory adipokine whose plasma levels are strongly correlated with insulin sensitivity and glucose tolerance .", "it can directly interfere with inflammatory cytokine production in macrophages and can induce expression of the anti - inflammatory cytokine il-10 . tnf- and il-6 inhibit adiponectin production in adipocytes .", "adipose tissue produced pro - inflammatory cytokines and adipokines further modulate the activity of oc and ob .", "fat tissue , mainly visceral fat tissue , may increase bone resorption through the production of inflammatory cytokines such as il- 6 and tnf- , which stimulate oc activity through the regulation of the rankl / rank / opg pathway .", "leptin and adiponectin act on the bone through different signaling pathways with contrasting effects   ( table 2 ) .", "it promotes adipogenesis and inhibits osteogenesis in response to diet and adiposity by activating jak2/stat3 signaling .", "therefore , leptin receptors on skeletal msc function as a sensor of systemic energy homeostasis .", "various cell populations within the fat tissue can exacerbate the development of the chronic , low - grade inflammation associated with obesity and metabolic dysfunction .", "white adipocytes store lipid as triglycerides within unilocular droplets , and are responsive to various stimuli , such as insulin .", "brown adipocytes store lipid in multilocular droplets that are quickly catabolized for fuel when the tissue is stimulated .", "beige adipocytes , dispersed within white adipose tissue , can dissipate heat , similarly to classical brown adipocytes , when exposed to cold temperatures or after prolonged highfat diet feeding .", "visceral fat tissue infiltrating macrophages in the setting of diet - induced obesity , have a pro - inflammatory phenotype which initiates and/or exacerbates the chronic inflammation that contributes to adipocyte dysfunction in obesity .", "conversely , in lean humans adipose tissue macrophages may promote tissue remodeling and temper inflammation by secreting anti - inflammatory cytokines .", "notwithstanding epidemiological evidence indicates that an increase in body mass index ( bmi ) is related to increased bone mass , probably due to the effects of the mechanical load of the body weight on the skeleton , not always obesity , and mainly the increase in visceral fat mass , has a positive effect on bone .", "obesity is associated with increased leptin and decreased adiponectin serum levels . moreover , in the visceral adipose tissue there are activated macrophages producing cytokines . in obese subjects , especially with central obesity ,", "in which the visceral fat is increased , there is a significant increase of several markers of inflammation such as c reactive protein ( crp ) , il-1 , il-6 and tnf- , that can alter the quality of the bone , making it more fragile .", "therefore , these new clinical and experimental evidences definitively connect obesity and other related pathological conditions , such as metabolic syndrome , nonalcoholic fatty liver disease ( nafld ) and diabetes , to impaired bone health and fragility fractures [ 90 - 93 ] . in conclusion , it is currently emerging that adipose tissue , liver , bone and immune system modulate each other through a complex network of interconnected signals .", "both adipocytes and ob express opg and rankl and their modulation is influenced by adipokines , sex hormones , redox balance , ppar and liver x receptors ( lxr ) .", "osteocalcin , an ob secreted bone matrix noncollagen protein , takes part in calcium metabolism and in bone deposition , as well as in energy homeostasis and glucose metabolism .", "fetuin - a , produced by the liver , is involved in the regulation of bone metabolism and insulin action , vascular calcification , neurodegenerative diseases and cancer cell proliferative signaling .", "ob lineage cells express receptors for adiponectin , leptin , angiotensin ii , insulin and insulin - like growth factor-1 , able to influence rank / rankl / opg signaling pathway .", "interestingly , these hormones are implicated in the pathogenesis of type - ii diabetes , obesity and hypertension , all of which are risk factors for metabolic syndrome .", "the development and progression of diabetes - associated osteoporosis are mediated by the interaction between advanced glycation end products ( age ) and receptor of age ( rage ) .", "age are the end products of glucose , as well as other sugars , proteins , lipids , and nucleic acids via a non - enzymatic glycosylation reaction , able to bind to multiple membrane receptor proteins , including rage .", "age / rage interaction is involved in the pathophysiological processes of many inflammatory and dysmetabolic diseases .", "in particular , age are involved in the development and progression of osteoporosis by inhibiting proliferation and inducing ob apoptosis .", "the binding of age to organic bone matrix may also increase the fragility of bones .", "autophagy is a metabolic process by which eukaryotic cells degrade and recover damaged macromolecules and organelles into autophagosomes autophagy is upregulated in stressful conditions .", "however , excessive autophagy is harmful to cells and leads to damage or massive death of cells .", "autophagy deficiency increases oxidative stress levels in ob , decreases bone mineralization and induces ran - kl secretion ."], ["it is well eatablished that several neuroendocrine pathways modulate both immune responses and bone remodeling . in turn", "sympathetic nerves produce catecholamines , which are delivered to the bone microenvironment by the blood circulation or by secretion from the nerve endings .", "hsc express catecholaminergic receptors , suggesting that they are able to directly respond to signals from the sympathetic nervous system .", "adrenergic signaling reduces cxcl12 expression in the bm . affecting maintenance of hsc and the differential lineage commitment .", "an association between major depression and osteoporosis has been recognized [ 96 - 99 ] .", "the prevalence of low bmd is higher in people with depression than the general population .", "interestingly , patients on antidepressant therapy with selective serotonin reuptake inhibitors develop decreased bmd and increased risk of fracture compared to those treated with tricyclic antidepressants such as amitriptyline or patients with untreated depression , who also have a lower bmd compared with healthy controls [ 100 , 101 ] .", "neuroendocrine abnormalities of the hypothalamo - pituitary - adrenal ( hpa ) and sympathoadrenal axes are a common finding in depressed patients leading to increased catecholamine turnover and hypersecretion of corticotropin - releasing hormone ( crh ) .", "leptin is expressed in the hypothalamus and pituitary gland , where it modulates corticotropin - releasing hormone and acth secretion , probably acting in an autocrine - paracrine manner .", "it inhibits steroid - hormone secretion from the adrenal cortex but enhances catecolamine release from the adrenal medulla , activating the sympathoadrenal axis .", "the neuromodulator serotonin or 5-hydroxy - tryptamine ( 5ht ) , which is an important vasoactive mediator of allergic reactions , is also likely involved in interactions between the central nervous and immune systems .", "in addition , it has recently been emerged that proinflammatory cytokines can modulate the central nervous system 5ht signaling , resulting in the conceptualization that 5ht participates in an integrated behavioral response to pathogens and inflammatory events . on the other hand ,", "5ht receptor expression on ocy and their precursors is involved in bone metabolism and its mechanoregulation . moreover , serotonin blocks the proliferation of ocp through the suppression of intracellular transcription factor creb , which regulates many genes involved in circadian rhythms in different tissues ( period 1,2,3 and clock genes ) .", "there are two anatomically and functionally distinct pools of serotonin : the first one , synthesized through the activity of the enzyme tryptophane - hydroxylase type 2 in the central nervous system , where it functions as neurotransmitter ; the second one is synthesized in the periphery through the activity of the tryptophane - hydroxylase 1 , regulated by the low - density lipoprotein receptor ( ldlr)-related protein-5 ( lrp5 ) .", "the circulating serotonin does not exceed the blood brain barrier and is for 95% produced by intestinal enterochromaffin cells in the intestine , where it stimulates peristalsis in response to the meal .", "the peripherally produced serotonin acts as a hormone inhibiting bone formation , whereas serotonin produced in the brain functions as a neurotransmitter , enhancing bone formation and limiting bone resorption .", "the neurological mechanism of action of serotonin on bone implicates also the interaction with the adipokine leptin , in an integrated homeostatic network with fat tissue and metabolism   ( fig .", "a portion of 5ht is also produced by the mammary gland , where it acts as an autocrine - paracrine regulator of the epithelial homeostasis exerting antiproliferative and proapoptotic effects .", "in the course of pregnancy , lactation and menopause , the local serotonin production increases , contributing to the increased bone resorption typical of these phases of the woman 's life .", "small molecules able to specifically inhibit the intestinal tryptophane - hydroxylase and do not cross the blood brain barrier , have recently been proposed for the treatment of osteoporosis . in alzheimer s disease ( ad ) , a neurodegenerative disorder characterized by cortical and cerebrovascular amyloid  peptide ( a ) deposits , neurofibrillary tangles , chronic inflammation , and neuronal loss , increased bone fracture rates and reduced bmd are commonly observed , suggesting common denominators between both disorders .", "amyloid precursor protein ( app ) is transmembrane protein involved in ad pathogenesis : app gene mutations characterize early - onset ad , and many risk factors or genes associated with late - onset ad appear to affect app trafficking and a production .", "rage , acting as an app / a binding partner , is therefore implicated in the pathogenesis of both ad and osteoporosis .", "the role of rage in oc maturation and activation is also mediated by its interaction with proinflammatory associated mac-1/2 integrin , the s100 family , and the high mobility group box 1(hmgb1 ) . in particular , hmgb1 is a proin - flammatory cytokine released from activated macro - phages , that promotes rankl - induced oc differentiation in a rage - dependent manner .", "shared signaling pathways among the complex immunological machineries involved in bone remodeling activate vicious cycles underlying both bone destruction and cancer growth .", "the release of growth factors in the skeletal microenvironment as a result of osteolysis induced by oc mediated bone resorption enhances metastases and cancer cell proliferation .", "in addition to the hyperproduction of proinflammatory cytokines , rank /rankl signal alterations are central in the pathogenesis of neoplastic osteolysis . the upregulation of rankl , particularly in myeloma and breast cancer , promotes the growth of neoplastic cells which express rank and protects them from dna damage induced programmed cell death . in this context", ", bone cells may represent potential therapeutic targets in the treatment of both secondary neoplastic osteoporosis and the underlying neoplasia .", "for example , bisphosphonate treatment of individuals with multiple myeloma reduces osteolytic events and tumor burden as well .", "the block of rankl by the monoclonal antibody denosumab , in addition to inhibiting bone resorption , is also useful in reducing tumor growth , in increasing apoptosis of malignant cells and in decreasing the inflammation that supports the neoplasia .", "its predominant effect on bone is through the central nervous system : by stimulating specific receptors ( lepr ) on both serotonergic brainstem neurons and the hypothalamic ventromedial nucleus , which interact each other , it increases central sympathetic activity .", "inhibitory signals are transmitted through sympathetic fibers from the hypothalamic ventromedial nucleus to 2-adrenergic receptors ( 2-ar ) expressed on ob , suppressing their differentiation and osteocalcin production .", "serotonin is synthesized in serotonergic neurons of the central nervous system , exerting various functions in the brain ; it is also synthesized in the gut , mediating different peripheral functions .", "it acts on bone cells using three different receptors : through 5ht1b receptors , it negatively regulates bone mass , while it enhances bone formation through 5ht2b and 5ht2c receptors ."], ["immune system and skeletal system interact with each other both in physiological and pathological conditions .", "considerable progress has been made in clarifying the crosstalk between bone and immune system that occurs in a complex and dynamic bone microenvironment . a central role in this crosstalk", "osteoimmunology therefore represents a new approach to studying osteoporosis that in the past was not considered an inflammatory condition .", "moreover , the discoveries of the existence of the immunoskeletal interface has also highlighted interesting repercussions for a wide range of pathological conditions beyond osteoporosis , including infections , autoimmune diseases , and neoplasia , in which same pathogenetic pathways are shared .", "osteoporosis could be therefore considered as a systemic model of integrated signaling pathways and cytokines working in a cooperative fashion .", "further important research horizons are opened with the extension of the discoveries of osteoimmunology and the disclosure of the immunoskeletal interface , whose practical implications may provide novel therapies for diseases other than osteoporosis , by targeting specific transcription factors , cytokines and their receptors . the correct understanding and decoding of the complex language", "through which immune system and bone communicate , although still in its dawn , is the essential requirement for the identification of such potentially useful therapeutic targets for both osteoporosis and other correlated inflammatory conditions , which share same mediators and signaling pathways ."], [""]]}
{"article_id": "PMC3052620", "article_text": ["lymphangioleiomyomatosis ( lam ) is a rare idiopathic disease that exclusively occurs in women of childbearing age , and this disease is characterized by the proliferation of abnormal smooth muscle cells in the lungs and along the thoracic and abdominal lymphatics ( 1 - 3 ) .", "the disease primarily affects the lungs in the majority of cases , but extrapulmonary lam occasionally occurs with or without subsequent involvement of the lungs .", "lam involvement of the uterus is extremely rare and there have been only a few such reported cases ( 2 - 7 ) , and the imaging findings of uterine lam have only been briefly mentioned in two reports ( 2 , 5 ) . in this article , we report the cross - sectional imaging findings of extensive lam involving the uterus and pelvic cavity in a 29-year - old woman with tuberous sclerosis complex ( tsc ) and we propose that certain imaging features and the clinical history may suggest the diagnosis . to the best of our knowledge ,", "this is the first report of the imaging findings of lam involvement of female pelvic organs .", "when she was ten years old , she was diagnosed with tsc based on her history of seizure and mental retardation .", "five years previously , she underwent a pelvic magnetic resonance imaging ( mri ) study for the evaluation of menometrorrhagia and dysmenorrhea .", "the mr images showed several small ( less than 3 cm ) intramural and subserosal hemorrhagic lesions in the uterus and hematometra ( fig .", "adenomyotic cysts and uterine leiomyomas with red degeneration were considered in the differential diagnosis based on the imaging at that time .", "endometrial drainage was performed for the hematometra , but no further treatment such as hormonal therapy was done .", "for the current visit , her serum ca-125 level was 204.01 u / ml , the ca 19 - 9 level was 50.11 u / ml and the hemoglobin level was 8.7 g / dl .", "1c ) showed multiple , large , lobulated , thick - walled cystic masses involving the uterus and the entire pelvic cavity , and these masses extended to the lower abdomen .", "the attenuation of the cystic masses was higher than that of simple fluid , which suggested hemorrhagic contents .", "both kidneys were enlarged by multiple , various sized masses with attenuations matching fat and soft tissue , which are findings compatible with angiomyolipomas .", "pelvic mr imaging showed huge , irregularly - shaped , cystic masses involving the uterus and parauterine pelvic cavity , and these masses were predominantly hyperintense on the fat - saturated t1-weighted image ( fig .", "1d ) and they were heterogeneously hypointense and hyperintense on the t2-weighted image ( fig .", "these cystic masses were thought to originate from the uterine myometrium on the sagittal t2-weighted image ( fig .", "multiple loculated fluid collections with fluid - fluid levels were seen in the cul - de - sac . on the contrast - enhanced t1-weighted image", "markedly enlarged lam involving the uterus with extension to the pelvic cavity was suggested as the most probable diagnosis based on the follow - up imaging and clinical findings .", "the chest ct showed numerous , well - defined , thin - walled cysts that were diffusely distributed throughout the lungs ( fig .", "1 g ) . because the cysts were regularly round in shape and there was no associated nodular lesion , pulmonary lam was the suggested diagnosis , but she had no pulmonary symptoms .", "brain mr imaging showed multiple cortical and subcortical tubers as well as several subependymal nodules in the bilateral lateral ventricles , and this represented tuberous sclerosis ( fig .", "the intraoperative findings revealed that the uterus was enlarged and distorted by multiple subserosal and intramural hemorrhagic cystic masses .", "the cystic masses also involved both adnexae , the pelvic side wall and the omentum .", "there were severe adhesive changes between the cystic lesions and the sigmoid colon and bladder .", "microscopic examination revealed that the tumor was composed of atypical smooth muscle cells ( lam cells ) arranged in short fascicles around dilated lymphatics and a ramifying network of endothelium - lined spaces ( fig .", "immunohistochemical staining showed that the tumor cells were diffusely positive for smooth muscle actin and they were strongly multifocally positive for human melanin black-45 ( hmb-45 ) ( fig .", "the patient 's hospital course was uneventful and she was discharged on the tenth postoperative day .", "lam results from the proliferation of abnormal - appearing smooth muscle cells in the lymph vessels , which causes dilatation and obstruction in the lymph vessels and this results in cystic collections of chylous material ( 1 ) .", "these smooth muscle cells are classified in the family of perivascular epithelioid cells ( pec ) , which is a cell type that is constantly present in a group of tumors , including lam , sugar tumors of the lung and pancreas , renal angiomyolipomas and clear cell myomelanocytic tumor of the falciform ligament ( 8 , 9 ) .", "these so - called \" pecoma \" all express hmb-45 ( 8) . lam occurs in about 30% of the women with tuberous sclerosis complex ( tsc ) , which is an autosomal dominant multisystem neurocutaneous disorder of highly variable penetrance , and it is characterized by hamartomas , seizure and mental retardation ( 1 ) . due to the striking similarities in the pathological processes between the lam and tsc , lam has been considered a forme fruste of tsc , and lam is classified as a tsc - associated or sporadic form ( 5 , 10 ) .", "lymphangioleiomyomatosis predominantly affects the lung parenchyma and this is characterized by pulmonary cysts seen on ct .", "the extrapulmonary manifestation of lam is uncommon and this is mainly located in the retroperitoneum , pelvic cavity and the posterior mediastinum along the lymphatic channels ( 1 - 7 ) . in a large study of 80 patients with pulmonary lam ( 11 ) ,", "the ct imaging findings of retroperitoneal lam were described as a low - attenuating ( 3 - 25 hounsfield unit [ hu ] ) , multilobulated mass , and the ultrasound findings were reported as a cystic mass with a thick echogenic rind .", "uterine involvement of lam is extremely rare , and to the best of our knowledge , only eight cases of pathologically proven uterine lam have currently been reported ( 2 - 7 ) .", "six cases of uterine lam occurred in patients with the stigmata of tsc , and two patients did not have stigmata of tsc .", "most uterine lams were microscopic and they are incidentally found in patients undergoing evaluation for extrauterine disease ( 2 ) .", "menorrhagia and/or pelvic pain have been reported in half of the cases , the same as in our case ( 2 - 7 ) .", "the imaging findings of uterine lam were briefly mentioned in two gynaecological reports ( 2 , 5 ) .", "( 2 ) reported that uterine lam simulated high - stage endometrial stromal sarcoma , and the ct finding of uterine lam was described as a large uterine mass , which was thought to be a fibroid .", "however , the surgery revealed multiple subserosal and intramural hemorrhagic nodules ranging in size from 2.5 to 4.0 cm and bloody ascites due to tumor perforation .", "( 5 ) recently reported on a 5.5-cm hypervascular tumor between the uterus and the right ovary that showed low signal intensity on the t1-weighted mr image and intermediate signal intensity on the t2-weighted mr image .", "this mri finding is quite different from that of retroperitoneal lam and it is rather similar to that of uterine leiomyoma .", "the pathological examination in that case revealed multiple intramural leiomyomas and several fragments of irregular soft masses composed of hmb 45 positive lam cells .", "the most remarkable aspects of our case are the initial involvement of lam in the uterus that gradually grew into the pelvic cavity and the intratumoral bleeding .", "intratumoral bleeding may be caused by overdistention and rupture of the cysts . although the ct findings showed highly attenuating cystic masses involving the uterus and pelvic cavity , the mr imaging confirmed the hemorrhagic cystic nature of the masses by their signal intensity and the mr imaging exactly localized the masses .", "these imaging findings are consistent with the known imaging findings of retroperitoneal lam . however , if a patient is without a clinical history of tsc , then uterine intramural and subserosal leiomyomas with hemorrhagic and cystic degeneration , adenomyotic cysts and/or endometrial cysts , and malignant uterine and/or ovarian tumors with hemorrhage should be considered in the differential diagnosis . in summary", ", we report here on the extraordinary radiological findings of lam involving the uterus and pelvic cavity in a young woman with tsc .", "although uterine lam is rare , radiologists should consider the possibility of this disease when they see multiple cystic uterine or parauterine masses with or without internal hemorrhage in a patient with a history of tsc or pulmonary lam ."], "abstract_text": ["<S> lymphangioleiomyomatosis ( lam ) is a rare idiopathic disease and this is characterized by a proliferation of abnormal smooth muscle cells in the lungs and in the lymphatic system of the thorax and retroperitoneum . </S>", "<S> the female genital tract is rarely affected by lam . </S>", "<S> we report here on the ct and mr imaging findings of extensive lam involving the uterus and pelvic cavity , and this was seen as multiple cystic uterine and parauterine masses with internal hemorrhage in a young female with tuberous sclerosis complex . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["lymphangioleiomyomatosis ( lam ) is a rare idiopathic disease that exclusively occurs in women of childbearing age , and this disease is characterized by the proliferation of abnormal smooth muscle cells in the lungs and along the thoracic and abdominal lymphatics ( 1 - 3 ) .", "the disease primarily affects the lungs in the majority of cases , but extrapulmonary lam occasionally occurs with or without subsequent involvement of the lungs .", "lam involvement of the uterus is extremely rare and there have been only a few such reported cases ( 2 - 7 ) , and the imaging findings of uterine lam have only been briefly mentioned in two reports ( 2 , 5 ) . in this article , we report the cross - sectional imaging findings of extensive lam involving the uterus and pelvic cavity in a 29-year - old woman with tuberous sclerosis complex ( tsc ) and we propose that certain imaging features and the clinical history may suggest the diagnosis . to the best of our knowledge ,", "this is the first report of the imaging findings of lam involvement of female pelvic organs ."], ["when she was ten years old , she was diagnosed with tsc based on her history of seizure and mental retardation .", "five years previously , she underwent a pelvic magnetic resonance imaging ( mri ) study for the evaluation of menometrorrhagia and dysmenorrhea .", "the mr images showed several small ( less than 3 cm ) intramural and subserosal hemorrhagic lesions in the uterus and hematometra ( fig .", "adenomyotic cysts and uterine leiomyomas with red degeneration were considered in the differential diagnosis based on the imaging at that time .", "endometrial drainage was performed for the hematometra , but no further treatment such as hormonal therapy was done .", "for the current visit , her serum ca-125 level was 204.01 u / ml , the ca 19 - 9 level was 50.11 u / ml and the hemoglobin level was 8.7 g / dl .", "1c ) showed multiple , large , lobulated , thick - walled cystic masses involving the uterus and the entire pelvic cavity , and these masses extended to the lower abdomen .", "the attenuation of the cystic masses was higher than that of simple fluid , which suggested hemorrhagic contents .", "both kidneys were enlarged by multiple , various sized masses with attenuations matching fat and soft tissue , which are findings compatible with angiomyolipomas .", "pelvic mr imaging showed huge , irregularly - shaped , cystic masses involving the uterus and parauterine pelvic cavity , and these masses were predominantly hyperintense on the fat - saturated t1-weighted image ( fig .", "1d ) and they were heterogeneously hypointense and hyperintense on the t2-weighted image ( fig .", "these cystic masses were thought to originate from the uterine myometrium on the sagittal t2-weighted image ( fig .", "multiple loculated fluid collections with fluid - fluid levels were seen in the cul - de - sac . on the contrast - enhanced t1-weighted image", "markedly enlarged lam involving the uterus with extension to the pelvic cavity was suggested as the most probable diagnosis based on the follow - up imaging and clinical findings .", "the chest ct showed numerous , well - defined , thin - walled cysts that were diffusely distributed throughout the lungs ( fig .", "1 g ) . because the cysts were regularly round in shape and there was no associated nodular lesion , pulmonary lam was the suggested diagnosis , but she had no pulmonary symptoms .", "brain mr imaging showed multiple cortical and subcortical tubers as well as several subependymal nodules in the bilateral lateral ventricles , and this represented tuberous sclerosis ( fig .", "the intraoperative findings revealed that the uterus was enlarged and distorted by multiple subserosal and intramural hemorrhagic cystic masses .", "the cystic masses also involved both adnexae , the pelvic side wall and the omentum .", "there were severe adhesive changes between the cystic lesions and the sigmoid colon and bladder .", "microscopic examination revealed that the tumor was composed of atypical smooth muscle cells ( lam cells ) arranged in short fascicles around dilated lymphatics and a ramifying network of endothelium - lined spaces ( fig .", "immunohistochemical staining showed that the tumor cells were diffusely positive for smooth muscle actin and they were strongly multifocally positive for human melanin black-45 ( hmb-45 ) ( fig .", "the patient 's hospital course was uneventful and she was discharged on the tenth postoperative day ."], ["lam results from the proliferation of abnormal - appearing smooth muscle cells in the lymph vessels , which causes dilatation and obstruction in the lymph vessels and this results in cystic collections of chylous material ( 1 ) .", "these smooth muscle cells are classified in the family of perivascular epithelioid cells ( pec ) , which is a cell type that is constantly present in a group of tumors , including lam , sugar tumors of the lung and pancreas , renal angiomyolipomas and clear cell myomelanocytic tumor of the falciform ligament ( 8 , 9 ) .", "these so - called \" pecoma \" all express hmb-45 ( 8) . lam occurs in about 30% of the women with tuberous sclerosis complex ( tsc ) , which is an autosomal dominant multisystem neurocutaneous disorder of highly variable penetrance , and it is characterized by hamartomas , seizure and mental retardation ( 1 ) . due to the striking similarities in the pathological processes between the lam and tsc , lam has been considered a forme fruste of tsc , and lam is classified as a tsc - associated or sporadic form ( 5 , 10 ) .", "lymphangioleiomyomatosis predominantly affects the lung parenchyma and this is characterized by pulmonary cysts seen on ct .", "the extrapulmonary manifestation of lam is uncommon and this is mainly located in the retroperitoneum , pelvic cavity and the posterior mediastinum along the lymphatic channels ( 1 - 7 ) . in a large study of 80 patients with pulmonary lam ( 11 ) ,", "the ct imaging findings of retroperitoneal lam were described as a low - attenuating ( 3 - 25 hounsfield unit [ hu ] ) , multilobulated mass , and the ultrasound findings were reported as a cystic mass with a thick echogenic rind .", "uterine involvement of lam is extremely rare , and to the best of our knowledge , only eight cases of pathologically proven uterine lam have currently been reported ( 2 - 7 ) .", "six cases of uterine lam occurred in patients with the stigmata of tsc , and two patients did not have stigmata of tsc .", "most uterine lams were microscopic and they are incidentally found in patients undergoing evaluation for extrauterine disease ( 2 ) .", "menorrhagia and/or pelvic pain have been reported in half of the cases , the same as in our case ( 2 - 7 ) .", "the imaging findings of uterine lam were briefly mentioned in two gynaecological reports ( 2 , 5 ) .", "( 2 ) reported that uterine lam simulated high - stage endometrial stromal sarcoma , and the ct finding of uterine lam was described as a large uterine mass , which was thought to be a fibroid .", "however , the surgery revealed multiple subserosal and intramural hemorrhagic nodules ranging in size from 2.5 to 4.0 cm and bloody ascites due to tumor perforation .", "( 5 ) recently reported on a 5.5-cm hypervascular tumor between the uterus and the right ovary that showed low signal intensity on the t1-weighted mr image and intermediate signal intensity on the t2-weighted mr image .", "this mri finding is quite different from that of retroperitoneal lam and it is rather similar to that of uterine leiomyoma .", "the pathological examination in that case revealed multiple intramural leiomyomas and several fragments of irregular soft masses composed of hmb 45 positive lam cells .", "the most remarkable aspects of our case are the initial involvement of lam in the uterus that gradually grew into the pelvic cavity and the intratumoral bleeding .", "intratumoral bleeding may be caused by overdistention and rupture of the cysts . although the ct findings showed highly attenuating cystic masses involving the uterus and pelvic cavity , the mr imaging confirmed the hemorrhagic cystic nature of the masses by their signal intensity and the mr imaging exactly localized the masses .", "these imaging findings are consistent with the known imaging findings of retroperitoneal lam . however , if a patient is without a clinical history of tsc , then uterine intramural and subserosal leiomyomas with hemorrhagic and cystic degeneration , adenomyotic cysts and/or endometrial cysts , and malignant uterine and/or ovarian tumors with hemorrhage should be considered in the differential diagnosis . in summary", ", we report here on the extraordinary radiological findings of lam involving the uterus and pelvic cavity in a young woman with tsc .", "although uterine lam is rare , radiologists should consider the possibility of this disease when they see multiple cystic uterine or parauterine masses with or without internal hemorrhage in a patient with a history of tsc or pulmonary lam ."]]}
{"article_id": "PMC2915790", "article_text": ["ts is a key enzyme in the de novo  synthesis of dtmp , an essential precursor of dna , which catalyses the methylation of dump to dtmp .      ", "the critical role of ts in the nucleotide metabolism has made it an important target of a variety of chemotherapeutic agents including 5-fu , 5-fu prodrugs as capecitabine , and novel folate - based ts inhibitor such as raltitrexed and pemetrexed , used in the treatment of colorectal and other solid tumours .", "resistance to fluoropyrimidines and other ts inhibitors may occur through a variety of mechanisms including elevated intracellular ts levels resulting from increases in ts transcription   and translation .       in crc patients", "the ts intratumoural expression may predict for the sensitivity to 5-fu and other ts inhibitor - based chemotherapies [ 5 , 6 ]   and may also be an important prognostic marker .", "high   ts expression in early stage crc patients seems to predict for poorer overall survival in both chemotherapy - treated and untreated patients following surgery .", "also , metastatic crc patients with high ts levels are unlikely to respond to infusional treatment with 5-fu , whereas patients with low ts levels have higher than average response rate [ 8 , 9 ] . in a previous study", ", however , we demonstrated that higher ts levels are favourable prognostic factors for disease free and overall survival .", "to date the real value of the ts level as prognostic factor is doubtful and some authors are in disagreement about it .", "the ts gene , containing 7 coding exons ,   is located in chromosome 18q , for which a high percentage of monosomic loss has been reported to be cell - cycle dependent , although some recent evidence points are more oriented towards proliferation - dependence .", "it can be predicted that mutations in the ts gene might result in a modification of its structure and then in its ability to interact with the fluoropyrimidines .", "this prediction has been born out in a variety of studies [ 1215 ] .  ", "s. h. berger and f. g. berger   reported that the human colon tumour cell line hct116 produces a variant structural form of ts , in addition to the common form found in all colon cell lines tested . among the ts overproduction derivatives of hct116 , cells overexposing the novel forms are more resistant to fdurd , compared with cells overexpressing the normal form", "so , an association between drug response and altered ts structure could suggest that the novel ts form , which is encoded by a variant structural ts gene , confers relative resistance to fdurd   ; this was supported by preliminary kinetic data indicating that this novel form has a reduced affinity for fdump .", "this variant form presents a replacement of an evolutionary conserved tyrosine by a histidine at residue 33 of the ts polypeptide [ 17 , 18 ] ; this mutation represents the only difference between the two ts forms and must account for the structural and functional differences between them . in an our previous paper we performed the analysis of the ts structure in patients bearing crc   to try to demonstrate the presence of that specific mutation , but we did not find it in any patient . here , we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer and previously untreated and try to find relationship between any hypothetical ts variant form with the 5-fu treatment .        ", "we performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes ' stages ( a , b , and c ) and histological grade but we did not find any change in the ts - dna structure .", "ts structure was assessed in a series of 68 patients who underwent surgery for primary colorectal carcinoma confirmed histologically and previously untreated .", "the following exclusion criteria were applied : history of other neoplasias apart of crc and death occurring within 30 days following surgery and due to postoperative complications .", "the tumours were staged and graded according dukes ' system and their clinicopathological features were summarised in tables 1 , 2 , and 3 .", "the samples at surgery were divided in two parts : one   was frozen at 80 until analysis , and the other portion was embedded in paraffin to confirm histologically the absence of contamination by normal and necrotic tissue and lymphocytes .", "genomic dna was extracted from tumour and mucosa samples with a commercial kit purchased from qiagen ( kjvenlo , the netherlands ) and stored at 20.      the ts gene   of tumour and mucosa samples was amplified in seven different pcr reactions using dna primers of 1620 bases in length placed in the adjacent intronic regions of exons 1 , 2 , 3 , 4 , 5 , 6 , and 7 and listed in table 4 .", "amplification was performed using a perkin elmer   model 2400 thermal cycler ( boston usa ) .", "reaction mixture included 5  l of 10% dimethylsulfoxide , 5  l of 10  buffer , 1.5  l of 1.5  mm mgcl2 , 1  l of 10  mm dntps , 200500  ng of genomic dna , 200  pmol of both upstream and downstream primers , and 1 units of taq dna polymerase , in a final volume of 50  l .", "amplification was run for 35 cycles with each cycle consisting in a denaturation step at 95c for 1 minutes , a primer annealing step at 51c for 1 minute , and an extention step at 72c for 2 minutes .", "pcr was terminated by incubation at 72c for 10 minutes . the length of the amplification products ranged from 180 to 310 bases .", "dna samples , generated with independently repeated pcr products ,   were sequenced with the sequitherm excel ii dna sequencing kit ( epicenter , madison , wi ) on a li - cor 4000 ( mwg - biotech , ebersberg , germany ) sequencer .", "the enzyme structure obtained by sequencing the ts genomic dna of each sample was performed in 68 patients with operable crc , untreated with previous chemotherapy .", "primary tumours and corresponding colonic mucosa were obtained from each patient and the ts genomic structure was performed on each of them , after histological control .", "7 patients had g1 histological grade , 36 had g2 , and 25 had g3 histological grade ; 10 patients had dukes ' a tumours , 27 patients had dukes ' b , and 31 patients had dukes c cancers .", "the median age of patients was 67 years ; 30 patients were male and 38 female .", "in all the tumour samples evaluated we did not find any ts - dna variant structure in tumour : all a , b , and c dukes ' patients showed stable ts - dna , and also in the germline genome no ts - dna variants have been observed .", "the structure of the macromolecular target of a cytotoxic drug is a critical determinant of cellular sensitivity to that drug .", "recently , ts has become the subject of several studies aimed to elucidate a possible  clinical  role of ts detection either as determinant of drug resistance or as prognostic marker of the disease and predictive factor of the treatment .       ", "many studies are currently examining the real value of ts expression levels as a prognostic factor , but in the mean time significant uncertainty prevails ; whatever its prognostic influence , to date no studies have been able to establish   a  cut - off   ", "all of these in vitro studies have promising implications in the study of the role of ts in clinical practice .      ", "moreover , the unclear meaning of  levels  not only   as a prognostic indicator but also as marker predictive of resistance could be due to mechanisms different from over expression , as mutation .      ", "in fact some authors focused their attention on the ts structure with a double aim : development of a new classes of ts - inhibitors with a different mechanism of action and generation of ts mutants to develop gene - therapy strategies .", "berger and coworkers showed   that a single naturally occurring change , a tyr to his replacement , in the primary structure of the ts molecule confers relative resistance to the ts - directed antimetabolite   fdurd in hct 116 cells .", "the tyr to his mutation is the only difference between the altered form of ts and the normal form and , therefore , must be responsible for the diminished effectiveness of the enzyme as a drug target .       the data in those   studies , together with previously published experiments , strongly favour the notion that the tyr to his ' mutation is responsible for the relative fdurd resistance of cell line hct 116 [ 15 , 16 ] .  ", "however , as noted earlier ,   it is quite possible that factors in addition to this mutation contribute to the phenotype of hct116 and other colon cell lines .", "the frequency of the tyr33 to his33 mutation in the normal and in the pathological human populations is unknown .", "the altered ts may exist as a polymorphism in humans ; alternatively , it may have spontaneously arisen during tumorigenesis or during establishment of the cell lines in culture .     ", "distinguishing among these possibilities will be of great utility in assessing whether variant forms of ts identified in cultured cell lines are segregating in the human population and have an impact upon clinical response to 5-fluorouracil therapy in cancer patients .", "on the basis of this knowledge we hoped to see if in human crc samples it could be possible to find the same tyr33 to his33 substitution in the ts structure and eventually to correlate this finding with some clinicopathological parameters such as age and sex , tumour size and location , histological grade ,   dukes ' stage , 5-fu and raltitrexed treatment , and disease free and overall survivals , to see if that point mutation could be considered a reliable marker of drug resistance and of prognosis . in those samples", "we did not find that point mutation at the codon 33 .        in this study  ", "we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer ( crc ) and previously untreated and try to find a relationship between any hypothetical ts variant form with the 5-fu treatment and prognosis .        ", "we performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes ' stages ( a , b , and c ) and histological grade , but we did not find any mutation in the ts - dna structure .", "the conclusions that could be drown   are that in dukes ' a ,   b , and c crcs there are no changes in the ts - dna gene structure and that the evaluation of the ts expression is the main crc prognostic   and drug response marker .", "what remains is to evaluate the ts gene structure of the d metastatic dukes ' crcs : in these tumours it might be possible to find ts - dna structural changes related with their higher genomic instability and this fact could give an explanation of the 5-fu drug resistance and worse prognosis ."], "abstract_text": ["<S> thymidylate synthase ( ts ) catalyzes methylation \n               of dump to dtmp and it is the target for the \n               5-fluorouracil ( 5-fu ) activity . </S>", "<S> barbour et al . \n               showed that variant structural forms of ts in tumour \n               cell lines confer resistance to fluoropyrimidines . \n               </S>", "<S> we planned to perform the whole ts gene structure by \n               means of sequencing techniques in human colorectal \n               cancer ( crc ) samples to try to identify the presence \n               of any possible ts variant form that could be \n               responsible of fluoropyrimidines drug resistance and \n               of the worse prognosis . </S>", "<S> we performed the ts - dna gene \n               sequence in 68 crc from patients of a , b , and c \n               dukes ' stages and different histological grade , \n               but we did not find any mutation in the ts - dna \n               structure . in the future we intend to widen the ts \n               structure analysis to the metastatic crcs , \n               because due to their higher genomic \n               instability , they could present a ts variant form \n               responsible of the fluoropyrimidines drug resistance \n               and the worse prognosis . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["ts is a key enzyme in the de novo  synthesis of dtmp , an essential precursor of dna , which catalyses the methylation of dump to dtmp .      ", "the critical role of ts in the nucleotide metabolism has made it an important target of a variety of chemotherapeutic agents including 5-fu , 5-fu prodrugs as capecitabine , and novel folate - based ts inhibitor such as raltitrexed and pemetrexed , used in the treatment of colorectal and other solid tumours .", "resistance to fluoropyrimidines and other ts inhibitors may occur through a variety of mechanisms including elevated intracellular ts levels resulting from increases in ts transcription   and translation .       in crc patients", "the ts intratumoural expression may predict for the sensitivity to 5-fu and other ts inhibitor - based chemotherapies [ 5 , 6 ]   and may also be an important prognostic marker .", "high   ts expression in early stage crc patients seems to predict for poorer overall survival in both chemotherapy - treated and untreated patients following surgery .", "also , metastatic crc patients with high ts levels are unlikely to respond to infusional treatment with 5-fu , whereas patients with low ts levels have higher than average response rate [ 8 , 9 ] . in a previous study", ", however , we demonstrated that higher ts levels are favourable prognostic factors for disease free and overall survival .", "to date the real value of the ts level as prognostic factor is doubtful and some authors are in disagreement about it .", "the ts gene , containing 7 coding exons ,   is located in chromosome 18q , for which a high percentage of monosomic loss has been reported to be cell - cycle dependent , although some recent evidence points are more oriented towards proliferation - dependence .", "it can be predicted that mutations in the ts gene might result in a modification of its structure and then in its ability to interact with the fluoropyrimidines .", "this prediction has been born out in a variety of studies [ 1215 ] .  ", "s. h. berger and f. g. berger   reported that the human colon tumour cell line hct116 produces a variant structural form of ts , in addition to the common form found in all colon cell lines tested . among the ts overproduction derivatives of hct116 , cells overexposing the novel forms are more resistant to fdurd , compared with cells overexpressing the normal form", "so , an association between drug response and altered ts structure could suggest that the novel ts form , which is encoded by a variant structural ts gene , confers relative resistance to fdurd   ; this was supported by preliminary kinetic data indicating that this novel form has a reduced affinity for fdump .", "this variant form presents a replacement of an evolutionary conserved tyrosine by a histidine at residue 33 of the ts polypeptide [ 17 , 18 ] ; this mutation represents the only difference between the two ts forms and must account for the structural and functional differences between them . in an our previous paper we performed the analysis of the ts structure in patients bearing crc   to try to demonstrate the presence of that specific mutation , but we did not find it in any patient . here , we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer and previously untreated and try to find relationship between any hypothetical ts variant form with the 5-fu treatment .        ", "we performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes ' stages ( a , b , and c ) and histological grade but we did not find any change in the ts - dna structure ."], ["ts structure was assessed in a series of 68 patients who underwent surgery for primary colorectal carcinoma confirmed histologically and previously untreated .", "the following exclusion criteria were applied : history of other neoplasias apart of crc and death occurring within 30 days following surgery and due to postoperative complications .", "the tumours were staged and graded according dukes ' system and their clinicopathological features were summarised in tables 1 , 2 , and 3 .", "the samples at surgery were divided in two parts : one   was frozen at 80 until analysis , and the other portion was embedded in paraffin to confirm histologically the absence of contamination by normal and necrotic tissue and lymphocytes .", "genomic dna was extracted from tumour and mucosa samples with a commercial kit purchased from qiagen ( kjvenlo , the netherlands ) and stored at 20.      the ts gene   of tumour and mucosa samples was amplified in seven different pcr reactions using dna primers of 1620 bases in length placed in the adjacent intronic regions of exons 1 , 2 , 3 , 4 , 5 , 6 , and 7 and listed in table 4 .", "amplification was performed using a perkin elmer   model 2400 thermal cycler ( boston usa ) .", "reaction mixture included 5  l of 10% dimethylsulfoxide , 5  l of 10  buffer , 1.5  l of 1.5  mm mgcl2 , 1  l of 10  mm dntps , 200500  ng of genomic dna , 200  pmol of both upstream and downstream primers , and 1 units of taq dna polymerase , in a final volume of 50  l .", "amplification was run for 35 cycles with each cycle consisting in a denaturation step at 95c for 1 minutes , a primer annealing step at 51c for 1 minute , and an extention step at 72c for 2 minutes .", "pcr was terminated by incubation at 72c for 10 minutes . the length of the amplification products ranged from 180 to 310 bases .", "dna samples , generated with independently repeated pcr products ,   were sequenced with the sequitherm excel ii dna sequencing kit ( epicenter , madison , wi ) on a li - cor 4000 ( mwg - biotech , ebersberg , germany ) sequencer ."], ["the enzyme structure obtained by sequencing the ts genomic dna of each sample was performed in 68 patients with operable crc , untreated with previous chemotherapy .", "primary tumours and corresponding colonic mucosa were obtained from each patient and the ts genomic structure was performed on each of them , after histological control .", "7 patients had g1 histological grade , 36 had g2 , and 25 had g3 histological grade ; 10 patients had dukes ' a tumours , 27 patients had dukes ' b , and 31 patients had dukes c cancers .", "the median age of patients was 67 years ; 30 patients were male and 38 female .", "in all the tumour samples evaluated we did not find any ts - dna variant structure in tumour : all a , b , and c dukes ' patients showed stable ts - dna , and also in the germline genome no ts - dna variants have been observed ."], ["the structure of the macromolecular target of a cytotoxic drug is a critical determinant of cellular sensitivity to that drug .", "recently , ts has become the subject of several studies aimed to elucidate a possible  clinical  role of ts detection either as determinant of drug resistance or as prognostic marker of the disease and predictive factor of the treatment .       ", "many studies are currently examining the real value of ts expression levels as a prognostic factor , but in the mean time significant uncertainty prevails ; whatever its prognostic influence , to date no studies have been able to establish   a  cut - off   ", "all of these in vitro studies have promising implications in the study of the role of ts in clinical practice .      ", "moreover , the unclear meaning of  levels  not only   as a prognostic indicator but also as marker predictive of resistance could be due to mechanisms different from over expression , as mutation .      ", "in fact some authors focused their attention on the ts structure with a double aim : development of a new classes of ts - inhibitors with a different mechanism of action and generation of ts mutants to develop gene - therapy strategies .", "berger and coworkers showed   that a single naturally occurring change , a tyr to his replacement , in the primary structure of the ts molecule confers relative resistance to the ts - directed antimetabolite   fdurd in hct 116 cells .", "the tyr to his mutation is the only difference between the altered form of ts and the normal form and , therefore , must be responsible for the diminished effectiveness of the enzyme as a drug target .       the data in those   studies , together with previously published experiments , strongly favour the notion that the tyr to his ' mutation is responsible for the relative fdurd resistance of cell line hct 116 [ 15 , 16 ] .  ", "however , as noted earlier ,   it is quite possible that factors in addition to this mutation contribute to the phenotype of hct116 and other colon cell lines .", "the frequency of the tyr33 to his33 mutation in the normal and in the pathological human populations is unknown .", "the altered ts may exist as a polymorphism in humans ; alternatively , it may have spontaneously arisen during tumorigenesis or during establishment of the cell lines in culture .     ", "distinguishing among these possibilities will be of great utility in assessing whether variant forms of ts identified in cultured cell lines are segregating in the human population and have an impact upon clinical response to 5-fluorouracil therapy in cancer patients .", "on the basis of this knowledge we hoped to see if in human crc samples it could be possible to find the same tyr33 to his33 substitution in the ts structure and eventually to correlate this finding with some clinicopathological parameters such as age and sex , tumour size and location , histological grade ,   dukes ' stage , 5-fu and raltitrexed treatment , and disease free and overall survivals , to see if that point mutation could be considered a reliable marker of drug resistance and of prognosis . in those samples", "we did not find that point mutation at the codon 33 .        in this study  ", "we intend to proceed on the use of sequencing techniques to see if any ts variant form could be present in human cancer samples from patients who underwent surgery for primary colorectal cancer ( crc ) and previously untreated and try to find a relationship between any hypothetical ts variant form with the 5-fu treatment and prognosis .        ", "we performed the ts - dna gene sequence in 68 cancer samples from patients of different dukes ' stages ( a , b , and c ) and histological grade , but we did not find any mutation in the ts - dna structure .", "the conclusions that could be drown   are that in dukes ' a ,   b , and c crcs there are no changes in the ts - dna gene structure and that the evaluation of the ts expression is the main crc prognostic   and drug response marker .", "what remains is to evaluate the ts gene structure of the d metastatic dukes ' crcs : in these tumours it might be possible to find ts - dna structural changes related with their higher genomic instability and this fact could give an explanation of the 5-fu drug resistance and worse prognosis ."]]}
{"article_id": "PMC3941645", "article_text": ["colorectal cancer is one of the most common cancers , representing the second ( males ; 9% ) and third ( females ; 8% ) most prevalent cancer in the usa ( 1 ) , and the third overall leading cause of cancer - related death in the western world ( 2 ) .", "the past several decades have seen a dramatic increase in the incidence of colorectal cancer also in japan ( 3 ) ; in 2008 , age - standardized incidence rates in males and females in japan were 41.7 and 22.8 cases per 100 000 , with corresponding death rates of 15.2 and 8.9 per 100 000 , respectively ( 4 ) .", "the introduction of monoclonal antibody - based therapy targeting epidermal growth factor receptor ( egfr ) has provided new alternative treatment options for various types of malignancy , including colorectal cancer ( 58 ) .", "panitumumab is a high - affinity , fully human igg2 monoclonal antibody specific to the extracellular domain of egfr ( 5,9 ) .", "the clinical efficacy and tolerability of panitumumab have been well established in large , randomized , controlled clinical trials as monotherapy ( 1013 ) or in combination with cytotoxic agents ( 1417 ) , in the first- , second- and third - line , or later , settings for patients with unresectable , advanced or recurrent colorectal cancer with wild - type kirsten rat sarcoma-2 virus oncogene ( kras ) .", "panitumumab was first approved in the usa ( september 2006 ) and european union ( eu ) ( december 2007 ) as monotherapy and for use in combination therapy in the eu ( november 2011 ) . in japan ,", "panitumumab was approved ( april 2010 ) for the treatment of wild - type kras gene unresectable , advanced and recurrent colorectal cancer as monotherapy , and for use in combination therapy in the first- , second- and third - line , or later , settings based on global clinical trials ( 1012,1417 ) .", "the approval of panitumumab in japan for use in combination therapy represents the first such approval in the world .", "however , because the number of japanese individuals enrolled in the global clinical trials was relatively limited ( 13 ) , the japanese ministry of health , labour and welfare required the market authorization holders to conduct a post - marketing surveillance study , called all cases surveillance ( 18 ) , as a condition of its approval to evaluate the safety and effectiveness of all japanese patients treated with panitumumab . the main purpose of this study was to collect detailed information , particularly with regard to safety issues , on all panitumumab - treated patients ; additionally , by evaluating a large number of patients , the study aimed to identify rare adverse drug reactions ( adrs ) , which are often undetectable in smaller clinical trials .", "this post - marketing surveillance study was planned to include all patients treated with panitumumab from the start date ( 15 june 2010 ) of its launch in japan . to promote the appropriate use and evaluate safety information , a vectibix appropriate use committee , a vectibix safety evaluation committee and a vectibix interstitial lung disease ( ild ) review subcommittee", "all reported ild - like events were assessed individually by an ild review subcommittee , comprising external experts in the field of radiology , pulmonology and medical oncology .", "evaluation of ild - like events was performed using computed tomography ( ct ) and x - ray imaging , and by assessment of clinical information .", "completion of a specific registration form ( fig .  1 ) , which reported patient characteristics and treatment information , was mandatory before panitumumab treatment was initiated . the registration period of this post - marketing surveillance was june 2010 to november 2010", ". patients were carefully observed , and information during their clinical courses for 10 months ( 42 weeks ) , or until discontinuation of panitumumab for any reason , was collected through the pre - specified case report forms ( crfs ) . at the time of registration , a recommendation letter to stop initiating treatment with panitumumab", "was sent from the vectibix appropriate use committee to each attending doctor if at least one item on the dark gray zone of the registration form was checked ( fig .  1 ) .", "subsequently , the following information relating to clinical course was collected using crfs : ( 1 ) patient background including prior chemotherapy and reason for its discontinuation / termination ; ( 2 ) administration of panitumumab including its dose and date , premedication to prevent infusion reaction , concurrent medications and combined therapy ; ( 3 ) laboratory tests including serum magnesium , calcium and potassium ; ( 4 ) adverse events including time to onset , grade according to national cancer institute common terminology criteria for adverse events ( ctcae ) ver . 4.0 , treatment , causality of panitumumab and recovery and ( 5 ) outcomes including disease progression , survival or death . \n figure  1.patient registration form  excerpt .", "an adr was defined as an adverse event for which a causal relationship with panitumumab could not be ruled out .", "adrs were coded using medical dictionary for regulatory activities ( meddra ) version 15.0 . with regard to toxicity ,", "skin disorders , ild , infusion reaction , electrolyte abnormalities and cardiac disorders were monitored with special interest .", "the effectiveness of panitumumab was assessed by overall survival , calculated using the kaplan  meier method .", "overall survival time was defined as the time from the date of first administration of panitumumab to the date of death ( regardless of the cause of death ) or censored on the last date of survival ( date of confirmation of survival as recorded in the crf or , if there was no information regarding the date of confirmation of survival , the last date of panitumumab administration was used for calculation purposes ) .", "this survey was initiated in june 2010 and the data lock point of the final analysis was set at 12 september 2012 .", "during the registration period from june to november 2010 , a total of 3091 patients were registered ( completion of mandatory patient registration forms ) from 1031 clinical institutes and departments in japan . of the 3091 registered patients ,", "the crfs of five registered patients were not obtained despite repeated requests to physicians . of these , 3085 patients were eligible for safety analysis ; 1 patient was excluded from the safety analysis as no information was available regarding drug administration ( fig .  2 )", ". \n figure  2.patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "while a kras test was attempted for all registered patients , it was not determined for 79 patients ( 2.6% ) mainly due to the condition of the tissues . despite a recommendation letter to stop ,", "panitumumab was administered to three patients ( 0.1% ) with mutant kras at each physician 's discretion .", "the majority of patients ( 91.4% ) had an eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 - 1 .", "although the ecog ps of 3080 patients was reported to be 02 at registration , 5 patients with ps 3 were registered despite requests for reassessment of administration .", "the general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab .", "therefore , patients with ps 3 ( 0.7% ) or 4 ( 0.1% ) were included in this surveillance .", "there were no other registered patients who met at least one item in the dark gray zone of the registration form ( fig .  1 ) . \n", "table  1.patient demographics and baseline characteristicsall patients ( n = 3085)baseline characteristicnumber%gender  male196563.7  female112036.3age ( years )  < 65152449.4  6574105834.3  7550316.3  median ( range ) ( years)65.0 ( 1890)kras status  wild300397.3  mutant30.1  not determinable792.6primary tumor type ( duplicate counting )  colon186060.3  resected162152.5  unresected2397.8  rectal124440.3  resected105434.2  unresected1896.1  colorectal3085100.0  resected266186.3  unresected42313.7treatment line  first - line31010.1  second - line54317.6  third - line or later223272.4ecog performance status  0187760.9  194230.5  22417.8 ", "3220.7  430.1past treatment regimens  no1735.6  yes ( duplicate counting)291194.4  folfox243979.1  folfiri190761.8  bevacizumab211368.5  cetuximab91729.7kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan.mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "patient demographics and baseline characteristics kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan . mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients .", "treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines . specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "panitumumab was used in the third - line , or later , setting for metastatic disease in 72.4% of patients , and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients , such as folfox ( 79.1% ) , folfiri ( 61.8% ) and bevacizumab ( monoclonal antibody , vascular endothelial growth factor - specific angiogenesis inhibitor ; 68.5% ) ; 29.7% of patients had received cetuximab ( monoclonal antibody , egfr antagonist ) ( duplicate counting ) .", "figure  3 shows treatment regimens administered and the status of administration ( safety analysis set ) . at the start of panitumumab administration ,", "1254 ( 40.7% ) patients received monotherapy and 1831 ( 59.4% ) patients received combination chemotherapy .", "combination therapy consisted ( duplicate counting ) of folfox4 ( 3.6% ) , mfolfox6 ( 15.1% ) , folfiri ( 33.9% ) , 5fu / l - lv ( 2.0% ) , cpt-11 ( 9.0% ) , s-1 + cpt-11 ( 1.3% ) or other chemotherapy ( 3.1% ) . of the 310 patients receiving first - line treatment ,", "the majority ( 86.5% ) received combination therapy , of which 193 patients received concomitant folfox ( 62.3% ) . with regard to the use of panitumumab monotherapy in the first - line setting , primary physicians did not select combination chemotherapy considering various patient factors , such as age and general condition , because there is no limitation in the indication of panitumumab for combination or monotherapy in japan .", "combination therapy was administered in 394 of 543 ( 72.6% ) patients as second - line treatment , of whom 276 ( 50.8% ) received folfiri . almost half", "( 47.6% ) of the 2232 patients receiving third - line , or later , treatment were in the monotherapy group . \n", "the median period of treatment with panitumumab was 113.0 days ( range : 1559 days ) , with 14.6% of patients receiving panitumumab for > 10 months .", "panitumumab was discontinued in 2592 ( 84.0% ) patients in the safety analysis set ( n = 3085 ) .", "the main reasons for discontinuation ( with some duplicate counting ) were as follows : disease progression in 1903 ( 61.7% ) patients , of which 1484 patients ( 78.0% ) had confirmed disease progression by diagnostic imaging ; occurrence of adverse events in 431 ( 14.0% ) patients ; patient refusal ( 190 patients ; 6.2% ) ; lack of patient hospital visits ( 30 patients ; 1.0% ) and other reasons ( 185 patients ; 6.0% ) .", "the overall incidence of adrs in all grades was 84.1% ( 2595/3085 patients ) ( table  2 ) .", "the most common classification , according to the meddra system organ class ( soc ) , was  skin and subcutaneous tissue disorders ' ( 76.6% ; 2364/3085 ) ( table  2 ) , followed by  infections and infestations ' [ ( 25.0% ; 771/3085 ) , which included paronychia ( 23.7% ; 731/3085 ) ] ,  gastrointestinal disorders ' ( 20.8% ; 642/3085 ) and  metabolism and nutrition disorders ' ( 17.9% ; 552/3085 ) . \n", "table  2overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc)any gradegrade 3n%n%all adrs259584.179725.8 ", "skin and subcutaneous tissue disorders236476.639212.7  infections and infestations77125.01605.2  gastrointestinal disorders64220.81264.1  metabolism and nutrition disorders55217.91494.8  investigations2046.6742.4  general disorders and administration site conditions1113.6290.9  eye disorders832.780.3  nervous system disorders682.2180.6  respiratory , thoracic and mediastinal disorders632.0280.9  injury , poisoning and procedural complications521.750.2  blood and lymphatic system disorders331.1240.8  hepatobiliary disorders190.640.1  renal and urinary disorders120.470.2  musculoskeletal and connective tissue disorders100.320.1  vascular disorders90.320.1  cardiac disorders70.210.03 overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc ) the incidence of adrs in the monotherapy group ( n = 1254 ) was 80.1% ( 1004/1254 ) ; 19.7% of these were grade 3 and 5.4% were classified as serious cases . in patients receiving combination therapy ( n = 1831 ) , 86.9% reported adrs ; 30.0% of these were grade 3 and 7.9% were serious cases .", "there were no major differences in the incidence of adrs by combined regimens in the combination therapy group : folfox ( overall : 86.7% , grade 3 : 29.3% and serious cases : 9.1% ) and folfiri ( 87.4 , 29.8 and 8.1% , respectively ) .", "a summary of safety data from the post - marketing surveillance study and panitumumab clinical trials ( 19 ) is presented in table  3 . \n", "table  3.summary of safety datapost - marketing survey in japanp - mab monotherapy ( n = 1254)combination therapy ( n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders ( soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders ( soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy ( n = 1052 : kras wt)p - mab + folfox4 ( n = 322 : kras wt)p - mab + folfiri ( n = 302 : kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders ( soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders ( soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab , panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375.trial no : 2005203.trial no : 2005181 .", "summary of safety data based on the evaluation of the ild review subcommittee . including grade-2 serious cases .", "trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375 .", "the overall incidence rate of  major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) .", "dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) .", "the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus .", "the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) .", "serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively .", "the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) .", "the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) .", "the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) .", "the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not .", "the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration .", "of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively .", "of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) .", "infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) .", "infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) .", "panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases .", "two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration .", "overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) .", "the time to onset of ild varied ( table  4 ) . as determined by ct imaging ,", "diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal .", "the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n", "table  4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild ) the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient ,", "treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) . in total ,", "1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these ,", "the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) .", "meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line , or later , setting . \n", "figure  4.overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "the median overall survival time ( 95% confidence interval ) was 10.3 months ( 9.011.8 months ) , with 56.6% censored cases .", "during the registration period from june to november 2010 , a total of 3091 patients were registered ( completion of mandatory patient registration forms ) from 1031 clinical institutes and departments in japan . of the 3091 registered patients ,", "the crfs of five registered patients were not obtained despite repeated requests to physicians . of these , 3085 patients were eligible for safety analysis ; 1 patient was excluded from the safety analysis as no information was available regarding drug administration ( fig .  2 )", ". \n figure  2.patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "while a kras test was attempted for all registered patients , it was not determined for 79 patients ( 2.6% ) mainly due to the condition of the tissues . despite a recommendation letter to stop ,", "panitumumab was administered to three patients ( 0.1% ) with mutant kras at each physician 's discretion .", "the majority of patients ( 91.4% ) had an eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 - 1 .", "although the ecog ps of 3080 patients was reported to be 02 at registration , 5 patients with ps 3 were registered despite requests for reassessment of administration .", "the general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab .", "therefore , patients with ps 3 ( 0.7% ) or 4 ( 0.1% ) were included in this surveillance .", "there were no other registered patients who met at least one item in the dark gray zone of the registration form ( fig .  1 ) . \n", "table  1.patient demographics and baseline characteristicsall patients ( n = 3085)baseline characteristicnumber%gender  male196563.7  female112036.3age ( years )  < 65152449.4  6574105834.3  7550316.3  median ( range ) ( years)65.0 ( 1890)kras status  wild300397.3  mutant30.1  not determinable792.6primary tumor type ( duplicate counting )  colon186060.3  resected162152.5  unresected2397.8  rectal124440.3  resected105434.2  unresected1896.1  colorectal3085100.0  resected266186.3  unresected42313.7treatment line  first - line31010.1  second - line54317.6  third - line or later223272.4ecog performance status  0187760.9  194230.5  22417.8 ", "3220.7  430.1past treatment regimens  no1735.6  yes ( duplicate counting)291194.4  folfox243979.1  folfiri190761.8  bevacizumab211368.5  cetuximab91729.7kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan.mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "patient demographics and baseline characteristics kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan . mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients .", "treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines . specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "panitumumab was used in the third - line , or later , setting for metastatic disease in 72.4% of patients , and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients , such as folfox ( 79.1% ) , folfiri ( 61.8% ) and bevacizumab ( monoclonal antibody , vascular endothelial growth factor - specific angiogenesis inhibitor ; 68.5% ) ; 29.7% of patients had received cetuximab ( monoclonal antibody , egfr antagonist ) ( duplicate counting ) .", "figure  3 shows treatment regimens administered and the status of administration ( safety analysis set ) . at the start of panitumumab administration ,", "1254 ( 40.7% ) patients received monotherapy and 1831 ( 59.4% ) patients received combination chemotherapy .", "combination therapy consisted ( duplicate counting ) of folfox4 ( 3.6% ) , mfolfox6 ( 15.1% ) , folfiri ( 33.9% ) , 5fu / l - lv ( 2.0% ) , cpt-11 ( 9.0% ) , s-1 + cpt-11 ( 1.3% ) or other chemotherapy ( 3.1% ) . of the 310 patients receiving first - line treatment ,", "the majority ( 86.5% ) received combination therapy , of which 193 patients received concomitant folfox ( 62.3% ) . with regard to the use of panitumumab monotherapy in the first - line setting , primary physicians did not select combination chemotherapy considering various patient factors , such as age and general condition , because there is no limitation in the indication of panitumumab for combination or monotherapy in japan .", "combination therapy was administered in 394 of 543 ( 72.6% ) patients as second - line treatment , of whom 276 ( 50.8% ) received folfiri . almost half", "( 47.6% ) of the 2232 patients receiving third - line , or later , treatment were in the monotherapy group . \n", "the median period of treatment with panitumumab was 113.0 days ( range : 1559 days ) , with 14.6% of patients receiving panitumumab for > 10 months .", "panitumumab was discontinued in 2592 ( 84.0% ) patients in the safety analysis set ( n = 3085 ) .", "the main reasons for discontinuation ( with some duplicate counting ) were as follows : disease progression in 1903 ( 61.7% ) patients , of which 1484 patients ( 78.0% ) had confirmed disease progression by diagnostic imaging ; occurrence of adverse events in 431 ( 14.0% ) patients ; patient refusal ( 190 patients ; 6.2% ) ; lack of patient hospital visits ( 30 patients ; 1.0% ) and other reasons ( 185 patients ; 6.0% ) .", "the overall incidence of adrs in all grades was 84.1% ( 2595/3085 patients ) ( table  2 ) .", "the most common classification , according to the meddra system organ class ( soc ) , was  skin and subcutaneous tissue disorders ' ( 76.6% ; 2364/3085 ) ( table  2 ) , followed by  infections and infestations ' [ ( 25.0% ; 771/3085 ) , which included paronychia ( 23.7% ; 731/3085 ) ] ,  gastrointestinal disorders ' ( 20.8% ; 642/3085 ) and  metabolism and nutrition disorders ' ( 17.9% ; 552/3085 ) . \n", "table  2overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc)any gradegrade 3n%n%all adrs259584.179725.8  skin and subcutaneous tissue disorders236476.639212.7  infections and infestations77125.01605.2  gastrointestinal disorders64220.81264.1  metabolism and nutrition disorders55217.91494.8  investigations2046.6742.4  general disorders and administration site conditions1113.6290.9  eye disorders832.780.3  nervous system disorders682.2180.6  respiratory , thoracic and mediastinal disorders632.0280.9  injury , poisoning and procedural complications521.750.2  blood and lymphatic system disorders331.1240.8  hepatobiliary disorders190.640.1  renal and urinary disorders120.470.2  musculoskeletal and connective tissue disorders100.320.1  vascular disorders90.320.1  cardiac disorders70.210.03 overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc ) the incidence of adrs in the monotherapy group ( n = 1254 ) was 80.1% ( 1004/1254 ) ; 19.7% of these were grade 3 and 5.4% were classified as serious cases . in patients receiving combination therapy ( n = 1831 ) ,", "86.9% reported adrs ; 30.0% of these were grade 3 and 7.9% were serious cases .", "there were no major differences in the incidence of adrs by combined regimens in the combination therapy group : folfox ( overall : 86.7% , grade 3 : 29.3% and serious cases : 9.1% ) and folfiri ( 87.4 , 29.8 and 8.1% , respectively ) .", "a summary of safety data from the post - marketing surveillance study and panitumumab clinical trials ( 19 ) is presented in table  3 . \n", "table  3.summary of safety datapost - marketing survey in japanp - mab monotherapy ( n = 1254)combination therapy ( n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders ( soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders ( soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy ( n = 1052 : kras wt)p - mab + folfox4", "( n = 322 : kras wt)p - mab + folfiri ( n = 302 : kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders ( soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders ( soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab , panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375.trial no : 2005203.trial", "summary of safety data based on the evaluation of the ild review subcommittee . including grade-2 serious cases .", "trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375 .", "the overall incidence rate of  major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) .", "dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) .", "the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus .", "the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) .", "serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively .", "the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) .", "the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) .", "the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) .", "the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not .", "the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration .", "of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively .", "of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) .", "infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) .", "infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) .", "panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases .", "two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration .", "overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) .", "the time to onset of ild varied ( table  4 ) . as determined by ct imaging ,", "diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal .", "the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n", "table  4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild ) the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient ,", "treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) . in total ,", "1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these ,", "the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) .", "the overall incidence rate of  major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) .", "dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) .", "the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus .", "the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) .", "serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively .", "the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) .", "the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) .", "the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) .", "the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not .", "the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration .", "of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively .", "of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) .", "infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) . no grade 4 cases , or deaths due to infusion reaction ,", "infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) .", "panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases .", "two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration .", "overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) . of the total ild cases , 51.3% ( 20/39 )", "the time to onset of ild varied ( table  4 ) . as determined by ct imaging ,", "diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal .", "the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n", "table  4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild )", "the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient ,", "treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) .", "in total , 1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these ,", "the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) .", "meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line , or later , setting . \n", "figure  4.overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "the median overall survival time ( 95% confidence interval ) was 10.3 months ( 9.011.8 months ) , with 56.6% censored cases .", "the main objective of the current post - marketing surveillance study was to evaluate the safety of panitumumab in japanese patients with unresectable colorectal cancer ( and with the majority of patients possessing wild - type kras tumors ) in clinical practice .", "the overall incidence of adrs was 84.1% , with skin disorders representing the most common classification ( 78.4% ) .", "skin toxicity associated with panitumumab ( and other egfr antibodies ) is well known ( 20 ) .", "indeed , the development of skin disorders , particularly rash , is considered to be a predictive factor for the efficacy of egfr inhibitors ( 21,22 ) . as reported previously with egfr inhibitors ( 22,23 ) ,", "dermatitis acneiform ( 69.9% ) was the most frequent skin disorder reported in the current study , occurring about 2 weeks after the initiation of panitumumab . in the current study , although major skin disorders such as dermatitis acneiform , paronychia , dry skin and pruritus occurred in 78.4% of patients , 93% of the patients who experienced skin disorders were able to continue panitumumab therapy .", "the egfr - signaling pathway potentially plays a pivotal role in regulating magnesium homeostasis , and , indeed , hypomagnesemia / magnesium has been shown in patients with colorectal cancer receiving egfr - targeting antibodies ( 24 ) . in the current study ,", "46.2% of patients were evaluated for serum magnesium , and grade 3 or higher hypomagnesemia occurred in 4.0% of patients , with a shorter median time to onset in patients treated with cetuximab immediately prior to panitumumab ( 39.5 and 71 days , respectively ) , indicating that the total duration of exposure to an egfr inhibitor is an important factor . from these findings , it is considered that the longer - term administration of anti - egfr treatment worsens the severity of hypomagnesemia ( 24 ) . however , in the present study , the incidence of hypomagnesemia and hypocalcemia peaked 3 and 4 months after the start of panitumumab administration , respectively .", "moreover , the incidence of hypomagnesemia observed in the current post - marketing surveillance study is similar to that reported in a phase ii clinical trial of panitumumab in japanese patients with metastatic colorectal cancer ( 13 ) .", ", almost all patients with prior experience of infusion reaction to cetuximab could receive panitumumab safely following premedication and with a reduced speed of infusion .", "some reports have shown that panitumumab can be administered successfully in patients after a severe infusion reaction to cetuximab ( 2529 ) .", "on the other hand , post - marketing fatalities due to infusion reaction with panitumumab have been reported in patients with a history of severe cetuximab - related infusion reaction ( 30 ) . therefore , administration of panitumumab to patients with a history of infusion reaction to cetuximab is not currently recommended . pulmonary toxicity including drug - induced ild represents an important adverse reaction that is associated with egfr - targeted cancer therapy ( 31 ) , with the potential to be fatal in some patients ( 32 ) .", "we prospectively established an ild subcommittee consisting of experts to accurately evaluate the incidence of ild .", "furthermore , in order to avoid panitumumab treatment , at the time of registration , a recommendation letter was issued to reconsider treatment with panitumumab if a patient had a history or complication of ild ( fig .  1 ) .", "in the current post - marketing surveillance study of panitumumab , the overall incidence rate of ild was 1.3% , with a mortality rate of 0.6%.the incidence of ild was similar to that reported in a post - marketing survey of cetuximab in japan ( 1.2% ) ( 23 ) .", "the ild subcommittee carried out a full evaluation of ild with panitumumab , concluding that the onset of ild can occur at any time after the start of panitumumab administration and that there were no findings specific to panitumumab , either clinically or in ct imaging .", "the dad type of ild occurred in some cases , as with cetuximab and the egfr tyrosine kinase inhibitors gefitinib and erlotinib , and may be fatal . at present , no major differences in the incidence of ild or in the rate of death due to ild were found in comparison with cetuximab ( 23 ) .", "thus , ild is one of the particular concerns with the use of panitumumab as well as other anti - egfr drugs . with regard to all panitumumab - related adrs", ", there were no differences between its use as monotherapy or in combination therapy with cytotoxic agents , and the profile , incidences , onset and outcomes of these adrs were very similar to those reported in previous clinical trials .", "in contrast to the combination setting with chemotherapy regimens , the monotherapy setting represents the efficacy of panitumumab in clinical practice more accurately ; therefore , patients treated with panitumumab monotherapy as a third - line or later therapy were selected for analysis of the effectiveness of panitumumab .", "the effectiveness of panitumumab , reported previously in clinical trials , was also confirmed in the current post - marketing surveillance study . in a pivotal , phase iii , randomized ,", "controlled study ( 15 ) , the median overall survival time in the panitumumab group ( wild - type kras ) was 8.1 months ( 6.39.4 months ) ( 11 ) ; data obtained from the present surveillance study exhibit a median overall survival of 10.3 months ( 56.6% censored cases ) .", "whilst acknowledging that post - marketing surveillance studies have some inevitable limitations ( e.g. the lack of a control group ) that differ from those in well - controlled , prospective clinical trials , it is recognized that post - marketing surveillance studies provide a information based on the general population ( 33 ) .", "these factors result in greater external validity for post - marketing surveillance studies compared with well - controlled , randomized clinical trials ( 33 ) . despite these limitations , this large - scale , non - interventional study has some advantages .", "this study also provides health - care professionals with valuable information on the safety and effectiveness of panitumumab based on real - world data . in conclusion ,", "the current post - marketing surveillance study in japanese patients with unresectable colorectal cancer confirmed the safety profile and effectiveness of panitumumab that has been reported previously in clinical trials .", "it is considered that the benefit / risk balance for the use of panitumumab in patients with unresectable colorectal cancer remains favorable also in japan .", "this work was supported by takeda pharmaceutical company limited . funding to pay the open access publication charges for this article was provided by takeda pharmaceutical company .", "has received a consultancy fee from takeda , speaker honoraria from takeda , taiho and yakult , and research funding from takeda , bristol - myers squibb , merck serono and chugai .", "has received consultancy fees from taiho , chugai , takeda and merck serono , speaker honoraria from taiho , chugai , yakult , bristol - myers squibb , merck serono , kyowa hakko kirin , takeda , kaken , johnson & johnson , toray , aska and bayer , a manuscript fee from chugai , and a donation from taiho , chugai , yakult , bristol - myers squibb , merck serono , kyowa hakko kirin , takeda , daiichi - sankyo , ono , eisai , kaken and tori .", "y.k . has received a consultancy fee , speaker honoraria and non - specific research support from takeda .", "k.m . has received a consultancy fee from takeda , speaker honoraria from takeda , taiho , yakult , chugai , merck serono and bristol - myers squibb , and research funding from taiho , pfizer and merck serono .", "t.h . has received a consultancy fee from takeda and speaker honoraria from takeda , chugai , taiho , pfizer , yakult and daiichi - sankyo .", "t.y . has received a consulting fee from takeda , speaker honoraria from chugai , takeda and merck serono , and research grants from taiho , daiichi - sankyo , lily , pfizer and yakult ."], "abstract_text": ["<S> objectivepanitumumab was approved in japan in april 2010 for the treatment of kirsten rat sarcoma-2 virus oncogene wild - type unresectable and recurrent colorectal cancer . </S>", "<S> we conducted a post - marketing surveillance study to evaluate the safety and effectiveness of panitumumab.methodsafter panitumumab was commercially available in japan , all patients to be treated with panitumumab were enrolled . </S>", "<S> data on baseline characteristics , treatment outcome , and incidence and severity of adverse drug reactions were collected.resultsin total , 3091 patients were registered . in the safety analysis set ( n = 3085 ) </S>", "<S> , panitumumab was administered as monotherapy ( 40.7% ) or combination therapy ( 59.4% ) . </S>", "<S> the median treatment duration was 113 days ( range : 1559 days ) , and 451 ( 14.6% ) patients received panitumumab for 10 months . </S>", "<S> the overall incidence rate of adverse drug reactions was 84.1% , and the most common adverse drug reaction was skin disorders ( 78.4% ) . </S>", "<S> the incidence rates ( all grades ) of interstitial lung disease , infusion reaction , electrolyte abnormalities and cardiac disorders were 1.3% ( mortality rate : 0.6% ) , 1.5 , 19.3 and 0.2% , respectively . </S>", "<S> the median survival time of patients treated with panitumumab monotherapy as the third - line , or later , therapy was 10.3 months.conclusionthis post - marketing survey in clinical practice confirmed the safety and effectiveness of panitumumab . </S>", "<S> the benefit / risk balance for panitumumab in japanese patients with unresectable colorectal cancer remains favorable . </S>"], "labels": null, "section_names": ["INTRODUCTION", "PATIENTS AND METHODS", "RESULTS", "Patient Characteristics", "Treatment Course", "Safety Analysis", "Skin Disorders", "Electrolyte Abnormalities", "Infusion Reaction", "Interstitial Lung Disease", "Cardiac Disorders", "Cases of Death", "Effectiveness", "DISCUSSION", "Funding", "Conflict of interest statement"], "sections": [["colorectal cancer is one of the most common cancers , representing the second ( males ; 9% ) and third ( females ; 8% ) most prevalent cancer in the usa ( 1 ) , and the third overall leading cause of cancer - related death in the western world ( 2 ) .", "the past several decades have seen a dramatic increase in the incidence of colorectal cancer also in japan ( 3 ) ; in 2008 , age - standardized incidence rates in males and females in japan were 41.7 and 22.8 cases per 100 000 , with corresponding death rates of 15.2 and 8.9 per 100 000 , respectively ( 4 ) .", "the introduction of monoclonal antibody - based therapy targeting epidermal growth factor receptor ( egfr ) has provided new alternative treatment options for various types of malignancy , including colorectal cancer ( 58 ) .", "panitumumab is a high - affinity , fully human igg2 monoclonal antibody specific to the extracellular domain of egfr ( 5,9 ) .", "the clinical efficacy and tolerability of panitumumab have been well established in large , randomized , controlled clinical trials as monotherapy ( 1013 ) or in combination with cytotoxic agents ( 1417 ) , in the first- , second- and third - line , or later , settings for patients with unresectable , advanced or recurrent colorectal cancer with wild - type kirsten rat sarcoma-2 virus oncogene ( kras ) .", "panitumumab was first approved in the usa ( september 2006 ) and european union ( eu ) ( december 2007 ) as monotherapy and for use in combination therapy in the eu ( november 2011 ) . in japan ,", "panitumumab was approved ( april 2010 ) for the treatment of wild - type kras gene unresectable , advanced and recurrent colorectal cancer as monotherapy , and for use in combination therapy in the first- , second- and third - line , or later , settings based on global clinical trials ( 1012,1417 ) .", "the approval of panitumumab in japan for use in combination therapy represents the first such approval in the world .", "however , because the number of japanese individuals enrolled in the global clinical trials was relatively limited ( 13 ) , the japanese ministry of health , labour and welfare required the market authorization holders to conduct a post - marketing surveillance study , called all cases surveillance ( 18 ) , as a condition of its approval to evaluate the safety and effectiveness of all japanese patients treated with panitumumab . the main purpose of this study was to collect detailed information , particularly with regard to safety issues , on all panitumumab - treated patients ; additionally , by evaluating a large number of patients , the study aimed to identify rare adverse drug reactions ( adrs ) , which are often undetectable in smaller clinical trials ."], ["this post - marketing surveillance study was planned to include all patients treated with panitumumab from the start date ( 15 june 2010 ) of its launch in japan . to promote the appropriate use and evaluate safety information , a vectibix appropriate use committee , a vectibix safety evaluation committee and a vectibix interstitial lung disease ( ild ) review subcommittee", "all reported ild - like events were assessed individually by an ild review subcommittee , comprising external experts in the field of radiology , pulmonology and medical oncology .", "evaluation of ild - like events was performed using computed tomography ( ct ) and x - ray imaging , and by assessment of clinical information .", "completion of a specific registration form ( fig .  1 ) , which reported patient characteristics and treatment information , was mandatory before panitumumab treatment was initiated . the registration period of this post - marketing surveillance was june 2010 to november 2010", ". patients were carefully observed , and information during their clinical courses for 10 months ( 42 weeks ) , or until discontinuation of panitumumab for any reason , was collected through the pre - specified case report forms ( crfs ) . at the time of registration , a recommendation letter to stop initiating treatment with panitumumab", "was sent from the vectibix appropriate use committee to each attending doctor if at least one item on the dark gray zone of the registration form was checked ( fig .  1 ) .", "subsequently , the following information relating to clinical course was collected using crfs : ( 1 ) patient background including prior chemotherapy and reason for its discontinuation / termination ; ( 2 ) administration of panitumumab including its dose and date , premedication to prevent infusion reaction , concurrent medications and combined therapy ; ( 3 ) laboratory tests including serum magnesium , calcium and potassium ; ( 4 ) adverse events including time to onset , grade according to national cancer institute common terminology criteria for adverse events ( ctcae ) ver . 4.0 , treatment , causality of panitumumab and recovery and ( 5 ) outcomes including disease progression , survival or death . \n figure  1.patient registration form  excerpt .", "an adr was defined as an adverse event for which a causal relationship with panitumumab could not be ruled out .", "adrs were coded using medical dictionary for regulatory activities ( meddra ) version 15.0 . with regard to toxicity ,", "skin disorders , ild , infusion reaction , electrolyte abnormalities and cardiac disorders were monitored with special interest .", "the effectiveness of panitumumab was assessed by overall survival , calculated using the kaplan  meier method .", "overall survival time was defined as the time from the date of first administration of panitumumab to the date of death ( regardless of the cause of death ) or censored on the last date of survival ( date of confirmation of survival as recorded in the crf or , if there was no information regarding the date of confirmation of survival , the last date of panitumumab administration was used for calculation purposes ) .", "this survey was initiated in june 2010 and the data lock point of the final analysis was set at 12 september 2012 ."], ["during the registration period from june to november 2010 , a total of 3091 patients were registered ( completion of mandatory patient registration forms ) from 1031 clinical institutes and departments in japan . of the 3091 registered patients ,", "the crfs of five registered patients were not obtained despite repeated requests to physicians . of these , 3085 patients were eligible for safety analysis ; 1 patient was excluded from the safety analysis as no information was available regarding drug administration ( fig .  2 )", ". \n figure  2.patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "while a kras test was attempted for all registered patients , it was not determined for 79 patients ( 2.6% ) mainly due to the condition of the tissues . despite a recommendation letter to stop ,", "panitumumab was administered to three patients ( 0.1% ) with mutant kras at each physician 's discretion .", "the majority of patients ( 91.4% ) had an eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 - 1 .", "although the ecog ps of 3080 patients was reported to be 02 at registration , 5 patients with ps 3 were registered despite requests for reassessment of administration .", "the general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab .", "therefore , patients with ps 3 ( 0.7% ) or 4 ( 0.1% ) were included in this surveillance .", "there were no other registered patients who met at least one item in the dark gray zone of the registration form ( fig .  1 ) . \n", "table  1.patient demographics and baseline characteristicsall patients ( n = 3085)baseline characteristicnumber%gender  male196563.7  female112036.3age ( years )  < 65152449.4  6574105834.3  7550316.3  median ( range ) ( years)65.0 ( 1890)kras status  wild300397.3  mutant30.1  not determinable792.6primary tumor type ( duplicate counting )  colon186060.3  resected162152.5  unresected2397.8  rectal124440.3  resected105434.2  unresected1896.1  colorectal3085100.0  resected266186.3  unresected42313.7treatment line  first - line31010.1  second - line54317.6  third - line or later223272.4ecog performance status  0187760.9  194230.5  22417.8 ", "3220.7  430.1past treatment regimens  no1735.6  yes ( duplicate counting)291194.4  folfox243979.1  folfiri190761.8  bevacizumab211368.5  cetuximab91729.7kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan.mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "patient demographics and baseline characteristics kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan . mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients .", "treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines . specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "panitumumab was used in the third - line , or later , setting for metastatic disease in 72.4% of patients , and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients , such as folfox ( 79.1% ) , folfiri ( 61.8% ) and bevacizumab ( monoclonal antibody , vascular endothelial growth factor - specific angiogenesis inhibitor ; 68.5% ) ; 29.7% of patients had received cetuximab ( monoclonal antibody , egfr antagonist ) ( duplicate counting ) .", "figure  3 shows treatment regimens administered and the status of administration ( safety analysis set ) . at the start of panitumumab administration ,", "1254 ( 40.7% ) patients received monotherapy and 1831 ( 59.4% ) patients received combination chemotherapy .", "combination therapy consisted ( duplicate counting ) of folfox4 ( 3.6% ) , mfolfox6 ( 15.1% ) , folfiri ( 33.9% ) , 5fu / l - lv ( 2.0% ) , cpt-11 ( 9.0% ) , s-1 + cpt-11 ( 1.3% ) or other chemotherapy ( 3.1% ) . of the 310 patients receiving first - line treatment ,", "the majority ( 86.5% ) received combination therapy , of which 193 patients received concomitant folfox ( 62.3% ) . with regard to the use of panitumumab monotherapy in the first - line setting , primary physicians did not select combination chemotherapy considering various patient factors , such as age and general condition , because there is no limitation in the indication of panitumumab for combination or monotherapy in japan .", "combination therapy was administered in 394 of 543 ( 72.6% ) patients as second - line treatment , of whom 276 ( 50.8% ) received folfiri . almost half", "( 47.6% ) of the 2232 patients receiving third - line , or later , treatment were in the monotherapy group . \n", "the median period of treatment with panitumumab was 113.0 days ( range : 1559 days ) , with 14.6% of patients receiving panitumumab for > 10 months .", "panitumumab was discontinued in 2592 ( 84.0% ) patients in the safety analysis set ( n = 3085 ) .", "the main reasons for discontinuation ( with some duplicate counting ) were as follows : disease progression in 1903 ( 61.7% ) patients , of which 1484 patients ( 78.0% ) had confirmed disease progression by diagnostic imaging ; occurrence of adverse events in 431 ( 14.0% ) patients ; patient refusal ( 190 patients ; 6.2% ) ; lack of patient hospital visits ( 30 patients ; 1.0% ) and other reasons ( 185 patients ; 6.0% ) .", "the overall incidence of adrs in all grades was 84.1% ( 2595/3085 patients ) ( table  2 ) .", "the most common classification , according to the meddra system organ class ( soc ) , was  skin and subcutaneous tissue disorders ' ( 76.6% ; 2364/3085 ) ( table  2 ) , followed by  infections and infestations ' [ ( 25.0% ; 771/3085 ) , which included paronychia ( 23.7% ; 731/3085 ) ] ,  gastrointestinal disorders ' ( 20.8% ; 642/3085 ) and  metabolism and nutrition disorders ' ( 17.9% ; 552/3085 ) . \n", "table  2overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc)any gradegrade 3n%n%all adrs259584.179725.8 ", "skin and subcutaneous tissue disorders236476.639212.7  infections and infestations77125.01605.2  gastrointestinal disorders64220.81264.1  metabolism and nutrition disorders55217.91494.8  investigations2046.6742.4  general disorders and administration site conditions1113.6290.9  eye disorders832.780.3  nervous system disorders682.2180.6  respiratory , thoracic and mediastinal disorders632.0280.9  injury , poisoning and procedural complications521.750.2  blood and lymphatic system disorders331.1240.8  hepatobiliary disorders190.640.1  renal and urinary disorders120.470.2  musculoskeletal and connective tissue disorders100.320.1  vascular disorders90.320.1  cardiac disorders70.210.03 overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc ) the incidence of adrs in the monotherapy group ( n = 1254 ) was 80.1% ( 1004/1254 ) ; 19.7% of these were grade 3 and 5.4% were classified as serious cases . in patients receiving combination therapy ( n = 1831 ) , 86.9% reported adrs ; 30.0% of these were grade 3 and 7.9% were serious cases .", "there were no major differences in the incidence of adrs by combined regimens in the combination therapy group : folfox ( overall : 86.7% , grade 3 : 29.3% and serious cases : 9.1% ) and folfiri ( 87.4 , 29.8 and 8.1% , respectively ) .", "a summary of safety data from the post - marketing surveillance study and panitumumab clinical trials ( 19 ) is presented in table  3 . \n", "table  3.summary of safety datapost - marketing survey in japanp - mab monotherapy ( n = 1254)combination therapy ( n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders ( soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders ( soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy ( n = 1052 : kras wt)p - mab + folfox4 ( n = 322 : kras wt)p - mab + folfiri ( n = 302 : kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders ( soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders ( soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab , panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375.trial no : 2005203.trial no : 2005181 .", "summary of safety data based on the evaluation of the ild review subcommittee . including grade-2 serious cases .", "trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375 .", "the overall incidence rate of  major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) .", "dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) .", "the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus .", "the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) .", "serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively .", "the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) .", "the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) .", "the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) .", "the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not .", "the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration .", "of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively .", "of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) .", "infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) .", "infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) .", "panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases .", "two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration .", "overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) .", "the time to onset of ild varied ( table  4 ) . as determined by ct imaging ,", "diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal .", "the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n", "table  4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild ) the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient ,", "treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) . in total ,", "1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these ,", "the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) .", "meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line , or later , setting . \n", "figure  4.overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "the median overall survival time ( 95% confidence interval ) was 10.3 months ( 9.011.8 months ) , with 56.6% censored cases ."], ["during the registration period from june to november 2010 , a total of 3091 patients were registered ( completion of mandatory patient registration forms ) from 1031 clinical institutes and departments in japan . of the 3091 registered patients ,", "the crfs of five registered patients were not obtained despite repeated requests to physicians . of these , 3085 patients were eligible for safety analysis ; 1 patient was excluded from the safety analysis as no information was available regarding drug administration ( fig .  2 )", ". \n figure  2.patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "patient registration in the panitumumab japanese post - marketing surveillance study ( safety analysis set ) .", "while a kras test was attempted for all registered patients , it was not determined for 79 patients ( 2.6% ) mainly due to the condition of the tissues . despite a recommendation letter to stop ,", "panitumumab was administered to three patients ( 0.1% ) with mutant kras at each physician 's discretion .", "the majority of patients ( 91.4% ) had an eastern cooperative oncology group ( ecog ) performance status ( ps ) of 0 - 1 .", "although the ecog ps of 3080 patients was reported to be 02 at registration , 5 patients with ps 3 were registered despite requests for reassessment of administration .", "the general condition of 20 patients deteriorated to ps 3 or 4 before the administration of panitumumab .", "therefore , patients with ps 3 ( 0.7% ) or 4 ( 0.1% ) were included in this surveillance .", "there were no other registered patients who met at least one item in the dark gray zone of the registration form ( fig .  1 ) . \n", "table  1.patient demographics and baseline characteristicsall patients ( n = 3085)baseline characteristicnumber%gender  male196563.7  female112036.3age ( years )  < 65152449.4  6574105834.3  7550316.3  median ( range ) ( years)65.0 ( 1890)kras status  wild300397.3  mutant30.1  not determinable792.6primary tumor type ( duplicate counting )  colon186060.3  resected162152.5  unresected2397.8  rectal124440.3  resected105434.2  unresected1896.1  colorectal3085100.0  resected266186.3  unresected42313.7treatment line  first - line31010.1  second - line54317.6  third - line or later223272.4ecog performance status  0187760.9  194230.5  22417.8 ", "3220.7  430.1past treatment regimens  no1735.6  yes ( duplicate counting)291194.4  folfox243979.1  folfiri190761.8  bevacizumab211368.5  cetuximab91729.7kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan.mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients.treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines.specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "patient demographics and baseline characteristics kras , kirsten rat sarcoma-2 virus oncogene ; ecog , eastern cooperative oncology group ; folfox , 5-fluorouracil , leucovorin , oxaliplatin ; folfiri , 5-fluorouracil , leucovorin , irinotecan . mainly due to the condition of tissue samples ; reasonable attempts were made to determine the kras status for all patients .", "treatment for metastatic or recurrent disease , excluding postoperative adjuvant chemotherapy from counting treatment lines . specified ps immediately before panitumumab administration .", "although 3080 patients were reported to be ps 02 , the general condition worsened to ps 3 in 17 patients and to ps 4 in 3 patients .", "panitumumab was used in the third - line , or later , setting for metastatic disease in 72.4% of patients , and previous anticancer treatment including adjuvant chemotherapy had been administered to 94.4% of patients , such as folfox ( 79.1% ) , folfiri ( 61.8% ) and bevacizumab ( monoclonal antibody , vascular endothelial growth factor - specific angiogenesis inhibitor ; 68.5% ) ; 29.7% of patients had received cetuximab ( monoclonal antibody , egfr antagonist ) ( duplicate counting ) .", "figure  3 shows treatment regimens administered and the status of administration ( safety analysis set ) . at the start of panitumumab administration ,", "1254 ( 40.7% ) patients received monotherapy and 1831 ( 59.4% ) patients received combination chemotherapy .", "combination therapy consisted ( duplicate counting ) of folfox4 ( 3.6% ) , mfolfox6 ( 15.1% ) , folfiri ( 33.9% ) , 5fu / l - lv ( 2.0% ) , cpt-11 ( 9.0% ) , s-1 + cpt-11 ( 1.3% ) or other chemotherapy ( 3.1% ) . of the 310 patients receiving first - line treatment ,", "the majority ( 86.5% ) received combination therapy , of which 193 patients received concomitant folfox ( 62.3% ) . with regard to the use of panitumumab monotherapy in the first - line setting , primary physicians did not select combination chemotherapy considering various patient factors , such as age and general condition , because there is no limitation in the indication of panitumumab for combination or monotherapy in japan .", "combination therapy was administered in 394 of 543 ( 72.6% ) patients as second - line treatment , of whom 276 ( 50.8% ) received folfiri . almost half", "( 47.6% ) of the 2232 patients receiving third - line , or later , treatment were in the monotherapy group . \n"], ["the median period of treatment with panitumumab was 113.0 days ( range : 1559 days ) , with 14.6% of patients receiving panitumumab for > 10 months .", "panitumumab was discontinued in 2592 ( 84.0% ) patients in the safety analysis set ( n = 3085 ) .", "the main reasons for discontinuation ( with some duplicate counting ) were as follows : disease progression in 1903 ( 61.7% ) patients , of which 1484 patients ( 78.0% ) had confirmed disease progression by diagnostic imaging ; occurrence of adverse events in 431 ( 14.0% ) patients ; patient refusal ( 190 patients ; 6.2% ) ; lack of patient hospital visits ( 30 patients ; 1.0% ) and other reasons ( 185 patients ; 6.0% ) ."], ["the overall incidence of adrs in all grades was 84.1% ( 2595/3085 patients ) ( table  2 ) .", "the most common classification , according to the meddra system organ class ( soc ) , was  skin and subcutaneous tissue disorders ' ( 76.6% ; 2364/3085 ) ( table  2 ) , followed by  infections and infestations ' [ ( 25.0% ; 771/3085 ) , which included paronychia ( 23.7% ; 731/3085 ) ] ,  gastrointestinal disorders ' ( 20.8% ; 642/3085 ) and  metabolism and nutrition disorders ' ( 17.9% ; 552/3085 ) . \n", "table  2overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc)any gradegrade 3n%n%all adrs259584.179725.8  skin and subcutaneous tissue disorders236476.639212.7  infections and infestations77125.01605.2  gastrointestinal disorders64220.81264.1  metabolism and nutrition disorders55217.91494.8  investigations2046.6742.4  general disorders and administration site conditions1113.6290.9  eye disorders832.780.3  nervous system disorders682.2180.6  respiratory , thoracic and mediastinal disorders632.0280.9  injury , poisoning and procedural complications521.750.2  blood and lymphatic system disorders331.1240.8  hepatobiliary disorders190.640.1  renal and urinary disorders120.470.2  musculoskeletal and connective tissue disorders100.320.1  vascular disorders90.320.1  cardiac disorders70.210.03 overall incidence of adverse drug reactions ( adrs ) by system organ class ( soc ) the incidence of adrs in the monotherapy group ( n = 1254 ) was 80.1% ( 1004/1254 ) ; 19.7% of these were grade 3 and 5.4% were classified as serious cases . in patients receiving combination therapy ( n = 1831 ) ,", "86.9% reported adrs ; 30.0% of these were grade 3 and 7.9% were serious cases .", "there were no major differences in the incidence of adrs by combined regimens in the combination therapy group : folfox ( overall : 86.7% , grade 3 : 29.3% and serious cases : 9.1% ) and folfiri ( 87.4 , 29.8 and 8.1% , respectively ) .", "a summary of safety data from the post - marketing surveillance study and panitumumab clinical trials ( 19 ) is presented in table  3 . \n", "table  3.summary of safety datapost - marketing survey in japanp - mab monotherapy ( n = 1254)combination therapy ( n = 1831)adrs of special interestallgrade 3allgrade 3skin disorders ( soc)91873.2%1189.4%144679.0%27415.0%paronychia27221.7%332.6%45925.1%995.4%interstitial lung disease161.3%231.3%infusion reaction171.4%10.1%301.6%50.3%hypomagnesemia25720.5%614.9%26314.4%623.4%hypocalcemia594.7%161.3%774.2%261.4%cardiac disorders ( soc)20.2%00.0%50.3%10.1%clinical trialsp - mab monotherapy ( n = 1052 : kras wt)p - mab + folfox4", "( n = 322 : kras wt)p - mab + folfiri ( n = 302 : kras wt)adrs of special interestallgrade 3allgrade 3allgrade 3skin disorders ( soc)96992.1%13012.4%30895.7%11034.2%27992.4%10133.4%paronychia21420.3%121.1%6319.6%113.4%5819.2%93.0%interstitial lung disease00.0%00.0%20.6%20.6%20.7%20.7%infusion reaction353.3%50.5%247.5%82.5%62.0%10.3%hypomagnesemia797.5%222.1%8927.6%195.9%7725.5%82.6%hypocalcemia111.0%50.5%144.3%30.9%144.6%10.3%cardiac disorders ( soc)101.0%30.3%72.2%30.9%62.0%00.0%p - mab , panitumumab.based on the evaluation of the ild review subcommittee.including grade-2 serious cases.trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375.trial no : 2005203.trial", "summary of safety data based on the evaluation of the ild review subcommittee . including grade-2 serious cases .", "trial nos : 20050216 , 20040192 , 20020408 , 20030194 , 20030167 , 20030250 , 20025405 , 20030138 , 20040116 , 20030251 , 20020375 .", "the overall incidence rate of  major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) .", "dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) .", "the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus .", "the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) .", "serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively .", "the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) .", "the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) .", "the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) .", "the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not .", "the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration .", "of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively .", "of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) .", "infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) .", "infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) .", "panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases .", "two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration .", "overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) .", "the time to onset of ild varied ( table  4 ) . as determined by ct imaging ,", "diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal .", "the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n", "table  4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild ) the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient ,", "treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) . in total ,", "1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these ,", "the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) ."], ["the overall incidence rate of  major skin disorders ' ( which includes all related events ) was 78.4% ( grade 3 , 14.7% ) .", "dermatitis acneiform occurred in 69.9% of patients ( grade 3 , 10.5% ) , paronychia in 24.2% ( grade 3 , 4.3% ) , dry skin in 21.7% ( grade 3 , 2.1% ) and pruritus in 4.8% ( grade 3 , 0.4% ) .", "the median time from the first day of treatment to the onset of each of the major skin disorders was as follows : 15 days for dermatitis acneiform , 43 days for paronychia , 29 days for dry skin and 21 days for pruritus .", "the prevalence of skin disorders was similar with panitumumab monotherapy ( 74.7% ; 937/1254 ) and panitumumab plus chemotherapy ( 80.9% ; 1482/1831 ) ."], ["serum magnesium , calcium and potassium were checked at least once during treatment in 46.2% ( 1425/3085 ) , 65.4% ( 2016/3085 ) and 92.5% ( 2855/3085 ) of the patients , respectively .", "the incidence rate of electrolyte abnormalities was 19.3% ( 596/3085 ) , with that of grade 3 cases being 4.8% ( 149/3085 ) .", "the respective incidence rates of hypomagnesemia , hypocalcemia and hypokalemia in patients for whom laboratory data for these three electrolytes were available were 36.5% ( 520/1425 ) , 6.7% ( 136/2016 ) and 2.3% ( 67/2855 ) .", "the incidence rate of hypomagnesemia in the overall population was 16.9% ( 520/3085 ) , with that of grade 3 hypomagnesemia being 4.0% ( 123/3085 ) .", "the median time to hypomagnesemia onset after panitumumab treatment initiation was 63 days in all cases : 39.5 days in patients who were treated with cetuximab immediately before panitumumab and 71 days in patients who were not .", "the incidence of hypomagnesemia and hypocalcemia peaked at 34 months after the start of panitumumab administration .", "of 105 patients who experienced both hypomagnesemia and hypocalcemia , 42 patients had the same grade of hypomagnesemia and hypocalcemia ; 42 and 21 patients compared to each other had higher grades of hypomagnesemia and hypocalcemia , respectively .", "of 30 patients with grade 3 hypomagnesemia and hypocalcemia , 5 patients experienced relevant clinical symptoms accompanying electrolyte abnormalities : qtc prolongation ( 3 cases ) , rhabdomyolysis , paralysis , tetany and convulsion ( 1 case of each ) ."], ["infusion reaction occurred in 1.5% ( 47/3085 ) of patients , with an incidence rate of grade 3 or serious cases of 0.2% ( 6/3085 ) . no grade 4 cases , or deaths due to infusion reaction ,", "infusion reaction occurred at initial administration in 61.7% ( 29/47 ) of patients and in 14.9% ( 7/47 ) of patients at second administration , while the first infusion reaction occurred at the time of the third or later administration in the remaining 11 patients ( with the latest infusion reaction occurring at the 21st administration ) .", "panitumumab was administered to 70 patients who had past histories of infusion reaction due to cetuximab , following premedication and at reduced speed of infusion in almost all cases .", "two of these patients ( 2.9% ) experienced grade 1 infusion reaction , while infusion reaction occurred in 9/847 ( 1.1% ) patients without a history of infusion reaction to cetuximab administration ."], ["overall , the incidence rate of ild was 1.3% ( 39/3085 ) : 1.3% in monotherapy and 1.3% in combination therapy , resulting in a mortality rate of 0.6% ( 20/3085 ) . of the total ild cases , 51.3% ( 20/39 )", "the time to onset of ild varied ( table  4 ) . as determined by ct imaging ,", "diffuse alveolar damage ( dad ) was observed in 46.2% ( 18/39 ) of patients and 15 of the 18 dad cases were fatal .", "the remaining ild cases were diagnosed as hypersensitivity pneumonia ( 9 cases ) or organizing pneumonia ( 8 cases ) , with 4 unevaluable cases . \n", "table  4.time to onset of interstitial lung disease ( ild)time ( months)all12345678910>10ild cases84111450320139fatal cases(6)(2)(4)(0)(2)(2)(0)(1)(2)(0)(1)(20 ) time to onset of interstitial lung disease ( ild )"], ["the incidence of cardiac disorders was 0.2% ( 7/3085 ) , with death due to heart failure ( grade 5 ) reported in one patient . in this patient ,", "treated with panitumumab and folfiri , pancytopenia ( not related to panitumumab ) appeared on day 23 , followed by pneumonia ( day 28 ) and heart failure ( day 32 ) ."], ["in total , 1135 deaths were recorded , and most of them were related to progression of colorectal cancer . however , in 25 ( 2.2% ) patients , a relationship to panitumumab treatment could not be ruled out . among these ,", "the most common cause of death was ild ( 20 patients , according to the evaluation of the ild subcommittee ) ."], ["meier curve for overall survival based on 1062 patients in the safety analysis set who were receiving panitumumab monotherapy in the third - line , or later , setting . \n", "figure  4.overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "overall survival ( n= 1062 ) in patients receiving third - line , or later , therapy with panitumumab monotherapy .", "the median overall survival time ( 95% confidence interval ) was 10.3 months ( 9.011.8 months ) , with 56.6% censored cases ."], ["the main objective of the current post - marketing surveillance study was to evaluate the safety of panitumumab in japanese patients with unresectable colorectal cancer ( and with the majority of patients possessing wild - type kras tumors ) in clinical practice .", "the overall incidence of adrs was 84.1% , with skin disorders representing the most common classification ( 78.4% ) .", "skin toxicity associated with panitumumab ( and other egfr antibodies ) is well known ( 20 ) .", "indeed , the development of skin disorders , particularly rash , is considered to be a predictive factor for the efficacy of egfr inhibitors ( 21,22 ) . as reported previously with egfr inhibitors ( 22,23 ) ,", "dermatitis acneiform ( 69.9% ) was the most frequent skin disorder reported in the current study , occurring about 2 weeks after the initiation of panitumumab . in the current study , although major skin disorders such as dermatitis acneiform , paronychia , dry skin and pruritus occurred in 78.4% of patients , 93% of the patients who experienced skin disorders were able to continue panitumumab therapy .", "the egfr - signaling pathway potentially plays a pivotal role in regulating magnesium homeostasis , and , indeed , hypomagnesemia / magnesium has been shown in patients with colorectal cancer receiving egfr - targeting antibodies ( 24 ) . in the current study ,", "46.2% of patients were evaluated for serum magnesium , and grade 3 or higher hypomagnesemia occurred in 4.0% of patients , with a shorter median time to onset in patients treated with cetuximab immediately prior to panitumumab ( 39.5 and 71 days , respectively ) , indicating that the total duration of exposure to an egfr inhibitor is an important factor . from these findings , it is considered that the longer - term administration of anti - egfr treatment worsens the severity of hypomagnesemia ( 24 ) . however , in the present study , the incidence of hypomagnesemia and hypocalcemia peaked 3 and 4 months after the start of panitumumab administration , respectively .", "moreover , the incidence of hypomagnesemia observed in the current post - marketing surveillance study is similar to that reported in a phase ii clinical trial of panitumumab in japanese patients with metastatic colorectal cancer ( 13 ) .", ", almost all patients with prior experience of infusion reaction to cetuximab could receive panitumumab safely following premedication and with a reduced speed of infusion .", "some reports have shown that panitumumab can be administered successfully in patients after a severe infusion reaction to cetuximab ( 2529 ) .", "on the other hand , post - marketing fatalities due to infusion reaction with panitumumab have been reported in patients with a history of severe cetuximab - related infusion reaction ( 30 ) . therefore , administration of panitumumab to patients with a history of infusion reaction to cetuximab is not currently recommended . pulmonary toxicity including drug - induced ild represents an important adverse reaction that is associated with egfr - targeted cancer therapy ( 31 ) , with the potential to be fatal in some patients ( 32 ) .", "we prospectively established an ild subcommittee consisting of experts to accurately evaluate the incidence of ild .", "furthermore , in order to avoid panitumumab treatment , at the time of registration , a recommendation letter was issued to reconsider treatment with panitumumab if a patient had a history or complication of ild ( fig .  1 ) .", "in the current post - marketing surveillance study of panitumumab , the overall incidence rate of ild was 1.3% , with a mortality rate of 0.6%.the incidence of ild was similar to that reported in a post - marketing survey of cetuximab in japan ( 1.2% ) ( 23 ) .", "the ild subcommittee carried out a full evaluation of ild with panitumumab , concluding that the onset of ild can occur at any time after the start of panitumumab administration and that there were no findings specific to panitumumab , either clinically or in ct imaging .", "the dad type of ild occurred in some cases , as with cetuximab and the egfr tyrosine kinase inhibitors gefitinib and erlotinib , and may be fatal . at present , no major differences in the incidence of ild or in the rate of death due to ild were found in comparison with cetuximab ( 23 ) .", "thus , ild is one of the particular concerns with the use of panitumumab as well as other anti - egfr drugs . with regard to all panitumumab - related adrs", ", there were no differences between its use as monotherapy or in combination therapy with cytotoxic agents , and the profile , incidences , onset and outcomes of these adrs were very similar to those reported in previous clinical trials .", "in contrast to the combination setting with chemotherapy regimens , the monotherapy setting represents the efficacy of panitumumab in clinical practice more accurately ; therefore , patients treated with panitumumab monotherapy as a third - line or later therapy were selected for analysis of the effectiveness of panitumumab .", "the effectiveness of panitumumab , reported previously in clinical trials , was also confirmed in the current post - marketing surveillance study . in a pivotal , phase iii , randomized ,", "controlled study ( 15 ) , the median overall survival time in the panitumumab group ( wild - type kras ) was 8.1 months ( 6.39.4 months ) ( 11 ) ; data obtained from the present surveillance study exhibit a median overall survival of 10.3 months ( 56.6% censored cases ) .", "whilst acknowledging that post - marketing surveillance studies have some inevitable limitations ( e.g. the lack of a control group ) that differ from those in well - controlled , prospective clinical trials , it is recognized that post - marketing surveillance studies provide a information based on the general population ( 33 ) .", "these factors result in greater external validity for post - marketing surveillance studies compared with well - controlled , randomized clinical trials ( 33 ) . despite these limitations , this large - scale , non - interventional study has some advantages .", "this study also provides health - care professionals with valuable information on the safety and effectiveness of panitumumab based on real - world data . in conclusion ,", "the current post - marketing surveillance study in japanese patients with unresectable colorectal cancer confirmed the safety profile and effectiveness of panitumumab that has been reported previously in clinical trials .", "it is considered that the benefit / risk balance for the use of panitumumab in patients with unresectable colorectal cancer remains favorable also in japan ."], ["this work was supported by takeda pharmaceutical company limited . funding to pay the open access publication charges for this article was provided by takeda pharmaceutical company ."], ["has received a consultancy fee from takeda , speaker honoraria from takeda , taiho and yakult , and research funding from takeda , bristol - myers squibb , merck serono and chugai .", "has received consultancy fees from taiho , chugai , takeda and merck serono , speaker honoraria from taiho , chugai , yakult , bristol - myers squibb , merck serono , kyowa hakko kirin , takeda , kaken , johnson & johnson , toray , aska and bayer , a manuscript fee from chugai , and a donation from taiho , chugai , yakult , bristol - myers squibb , merck serono , kyowa hakko kirin , takeda , daiichi - sankyo , ono , eisai , kaken and tori .", "y.k . has received a consultancy fee , speaker honoraria and non - specific research support from takeda .", "k.m . has received a consultancy fee from takeda , speaker honoraria from takeda , taiho , yakult , chugai , merck serono and bristol - myers squibb , and research funding from taiho , pfizer and merck serono .", "t.h . has received a consultancy fee from takeda and speaker honoraria from takeda , chugai , taiho , pfizer , yakult and daiichi - sankyo .", "t.y . has received a consulting fee from takeda , speaker honoraria from chugai , takeda and merck serono , and research grants from taiho , daiichi - sankyo , lily , pfizer and yakult ."]]}
{"article_id": "PMC3437641", "article_text": ["nevus lipomatosus cutaneous superficialis ( nlcs ) was first described by hoffman and zuhrelle in 1921.1 it s a rare idiopathic hamartomatous benign condition characterized by the presence of an ectopic mature adipose tissue within the dermis .", "it is classified into two clinical variants : the classical form , usually composed of multiple and grouped skin - colored , pedunculated and cerebriform nodules that often coalesce to form a plaque and a second more rare form , presenting as a solitary dome - shaped sessile papule or nodule.24 we report here eight cases of nlcs in order to assess the epidemiologic , clinical , and pathological features , as well as the management of this uncommon neoplasm .", "this study was a retrospective case series including all patients with histopathologically documented nlcs , who attended the dermatology department of charles nicolle hospital in tunisia during the last 14 years ; between january 1997 and december 2010 . for each patient , we recorded the following data : age , sex , duration of the lesions , their localizations , number , and size as well as their management and evolution . ethical approval and informed consent", "eight patients with nlcs were observed , three males and five females , aged between 7 and 41 years . in patients number 4 , 5 , 7 , and 8 , lesions had begun during childhood ( at 1 , 4 , 5 , and 6 years respectively ) . for the other four patients ( number 1 , 2 , 3 , and 6 ) lesions had begun in the third to fourth decade of life .", "clinical presentation was that of the classical form in seven patients and of the solitary form in one patient ( patient number 3 ) . in four patients it was one of the limbs which had the lesion , and in the other four patients it was the trunk ( figure 1 : patient 8 ; figure 2 : patient 5 ) .", "soft , skin - colored voluminous tumor ( 10 cm x 7 cm ) of the left buttock composed of multiple nodules with a cerebriform surface .", "histopathological features were typical of nlcs showing ectopic mature adipose tissue in the dermis ( figure 3 ) .", "table 1 summarizes the main epidemiologic , clinical and pathological features , and management of nlcs in all eight patients . \n", "table 1epidemiologic , clinical and pathological features , and management of nevus lipomatosus cutaneous superficialis ( nlcs ) in all eight patients.patientsexage ( years)durationlocationclinical aspecthistopathologytreatment1m381 yearleft buttockplaque of several soft skin - colored nodules of 1 cm to 2 cmmature adipose tissue in reticular dermis without connection to the hypodermissurgical resection2f383 monthsrigth thighmultiple soft yellowish papules coalescing into plaquesectopic mature adipocytes in reticular dermisabstention3f391 yearleft shoulderone soft skin - colored nodule of 1 cmlobular proliferation of fat tissue in the dermissurgical resection4m76 yearsright shoulderlarge ( 6 cm x 2 cm ) soft skin - colored verrucous plaque composed of nodules and papulesectopic mature adipocytes in reticular dermissurgical resection5m2420 yearslumbar area1 cm to 3 cm - grouped soft skin - colored papulesepidermal acanthosis , ectopic mature adipocytes in papillary dermissurgical resection6f3210 yearsthighmultiple soft skin - colored papules coalescing into a 5 cm plaqueepidermal acanthosis ; hyperkeratosis ; dermal proliferation of mature adipocytessurgical resection7f149 yearsflanklarge ( 6 cm x 2 cm ) soft skin - colored plaque composed of nodulesthinned epidermis , ectopic mature adipose tissue in reticular dermissurgical resection8f4135 yearsleft buttocksoft , skin - colored voluminous tumor ( 10 cm x 7 cm ) composed of multiple nodules with a cerebriform surfacenormal epidermis , mature fat cells in reticular dermis without hypodermis connectionsurgical resectionf = female ; m = male .", "epidemiologic , clinical and pathological features , and management of nevus lipomatosus cutaneous superficialis ( nlcs ) in all eight patients .", "nlcs is more commonly present from birth , but can appear later in life ; there is neither sex predilection nor familial trend in this disorder.1 in this study , there were five cases in which the lesion appeared in the first three decades , and in three cases nlcs appeared after 35 years .", "two clinical patterns of nlcs exist ; a multiple form or classical type , and the solitary form . in the classical type ,", "lesions are congenital or they develop usually during the first two to three decades of life.5,6,7 it consists of multiple papules , skin colored or yellowish , coalescing into plaques with zosteriform , linear or segmental distribution .", "the lesions are slow - growing , with a smooth or cerebriform surface , and can reach a large size if left untreated .", "the largest size reported so far has been 40 cm x 28 cm.8 the most common sites are the pelvic girdle , the lower trunk , the gluteal region , and the thigh .", "the second clinical pattern of nlcs is a solitary papule or nodule mimicking skin tag,8 usually appearing later than the classical form , during the third to sixth decades of life.3,6,9 it can occur at any site , including unusual ones like the scalp , the eyelid , the nose , and the clitoris.1,1013 solitary form was also called pedonculated lipofibroma by mehregan et al . because of its distinctive clinicopathologic features in comparison with the multiple form.9,14 among the presented cases , there were seven cases with the classical form and one case with the solitary form ( case 3 ) , whilst in another tunisian study the solitary type was predominant : 11 cases out of 13.15 nlcs have an indolent and asymptomatic course . recently , a case of ulnar nerve entrapment accompanied by numbness and straining in the forearm , caused by nlcs , was reported and was treated by partial excision for symptomatic recovery.16 occasionally , nlcs may ulcerate after external trauma or ischemia . in two previously reported cases , surface of nevus", "was studded with multiple open comedons,2,17 and foul - smelling discharge may be associated.2 nlcs has been reported in association with other cutaneous disorders ; follicular papules and hypertrophic pilo - sebaceous units,18 angiokeratoma of fordyce,4 scattered leukoderma , caf - au - lait macules,2 and hemangioma.4 in our study , no associated skin abnormalities were present . to our knowledge , there are no systemic abnormalities or malignant transformation described with nlcs .", "nlcs should be clinically differenciated from nevus sebaceous , neurofibroma , lymphangioma , focal dermal hypoplasia , cylindroma , trichoepithelioma , and angiolipoma .", "histopathological evaluation is required for diagnosis showing ectopic mature adipocytes , intermingled with collagen bundles , and proliferating around the periadnexial adventitial dermis and the perivascular area .", "mature fat cells proliferate in reticular dermis and may extend to the papillary dermis.2,6,7 in the most characteristic feature of nlcs , there is usually no connection to the subcutaneous fat tissue ; for some authors this condition is necessary to establish diagnosis of nlcs.6 several studies documented increased vascularity in the subpapillary and papillary dermis ; epidermal changes have been reported , like mild to moderate acanthosis , basket weave hyperkeratosis , increased basal pigmentation , and focal elongation of rete pegs.3 adnexal structures may be reduced,3 and abnormal folliculosebaceous structures were reported in some cases such as sebaceous trichofolliculoma , folliculosebaceous cystic hamartoma , and dermoid cysts.7,19 in our study , diagnosis of nlcs was based on characteristic clinical aspects and confirmed by typical histopathological features .", "the genetic background of nlcs is still unknown , only one cytogenetic study was reported recently showing a 2p24 deletion.20 the pathogenesis of nlcs remains unknown .", "several theories have been proposed : hoffman and zuhrelle postulated that fat deposition in the dermis is secondary to degenerative changes ( metaplasia ) in the connective tissue.3 other authors hypothesized that adipocytes originate from the pericytes of dermal vessels.2,21 for others , fat cells represented a true nevus that resulted from the focal heterotopic development of adipose tissue.22 treatment is usually not necessary unless for cosmetic reasons .", "the treatment of choice is surgical excision , which is curative , and post surgical recurrence is rare .", "patients unwilling for surgery may undergo cryotherapy which yields partial but satisfactory results.8 recently , a case of classic nlcs successfully treated with co2 laser was reported with no recurrence during a follow - up period of 12 months.23 in this retrospective study , we did nt investigate chromosomal abnormalities to identify the genetic biomarkers of this congenital pathology , since cytogenetic analysis was reported in only one previous study.20", "physicians should be aware of this rare condition because early recognition enables more conservative resection of the tumor and less invasive reconstruction of the defect .", "nlcs is a rare skin malformation ; we report herein a large case series of eight patients where the classical form is predominant", ". our study may give additional data about epidemiological and clinical pattern of this hamartoma .", "further studies are needed , especially prospective studies , by using chromosomal analysis to reach conclusions about the role of the genetic abnormalities in the development of this hamartoma ."], "abstract_text": ["<S> introductionnevus lipomatosus cutaneous superficialis ( nlcs ) is a rare benign hamartomatous skin tumor characterized by dermal deposition of mature adipose tissue . </S>", "<S> it s classified in two types : the classical form with multiple soft , pedunculated , cerebriform papules and nodules that coalesce into plaques , and the solitary form that consists of a solitary papule or nodule . in this study , </S>", "<S> eight cases of nlcs are reported.methodsthe study was a retrospective case series including all patients with histopathologically documented nlcs who attended the dermatology department of charles nicolle hospital between january 1997 and december 2010 . </S>", "<S> the objective of our study was to determine the epidemioclinical characteristics , the histopathologic features , and the treatment of this hamartoma . </S>", "<S> patients included three males and five females aged between 7 and 41 years.resultsin four cases hamartoma was present since childhood , and in the other four cases it appeared in the third and fourth decades . </S>", "<S> classical form was noted in seven cases and the solitary form in one case . </S>", "<S> lesions involved limbs in four patients and trunk in four patients . </S>", "<S> seven patients underwent surgical excision , and for one case no treatment was proposed.conclusionthe multiple or classical form is largely predominant in our study . </S>", "<S> habitually , nlcs has an asymptomatic course . </S>", "<S> treatment is usually not necessary unless for cosmetic reasons ; surgical excision is curative and recurrence after is rare . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["nevus lipomatosus cutaneous superficialis ( nlcs ) was first described by hoffman and zuhrelle in 1921.1 it s a rare idiopathic hamartomatous benign condition characterized by the presence of an ectopic mature adipose tissue within the dermis .", "it is classified into two clinical variants : the classical form , usually composed of multiple and grouped skin - colored , pedunculated and cerebriform nodules that often coalesce to form a plaque and a second more rare form , presenting as a solitary dome - shaped sessile papule or nodule.24 we report here eight cases of nlcs in order to assess the epidemiologic , clinical , and pathological features , as well as the management of this uncommon neoplasm ."], ["this study was a retrospective case series including all patients with histopathologically documented nlcs , who attended the dermatology department of charles nicolle hospital in tunisia during the last 14 years ; between january 1997 and december 2010 . for each patient , we recorded the following data : age , sex , duration of the lesions , their localizations , number , and size as well as their management and evolution . ethical approval and informed consent"], ["eight patients with nlcs were observed , three males and five females , aged between 7 and 41 years . in patients number 4 , 5 , 7 , and 8 , lesions had begun during childhood ( at 1 , 4 , 5 , and 6 years respectively ) . for the other four patients ( number 1 , 2 , 3 , and 6 ) lesions had begun in the third to fourth decade of life .", "clinical presentation was that of the classical form in seven patients and of the solitary form in one patient ( patient number 3 ) . in four patients it was one of the limbs which had the lesion , and in the other four patients it was the trunk ( figure 1 : patient 8 ; figure 2 : patient 5 ) .", "soft , skin - colored voluminous tumor ( 10 cm x 7 cm ) of the left buttock composed of multiple nodules with a cerebriform surface .", "histopathological features were typical of nlcs showing ectopic mature adipose tissue in the dermis ( figure 3 ) .", "table 1 summarizes the main epidemiologic , clinical and pathological features , and management of nlcs in all eight patients . \n", "table 1epidemiologic , clinical and pathological features , and management of nevus lipomatosus cutaneous superficialis ( nlcs ) in all eight patients.patientsexage ( years)durationlocationclinical aspecthistopathologytreatment1m381 yearleft buttockplaque of several soft skin - colored nodules of 1 cm to 2 cmmature adipose tissue in reticular dermis without connection to the hypodermissurgical resection2f383 monthsrigth thighmultiple soft yellowish papules coalescing into plaquesectopic mature adipocytes in reticular dermisabstention3f391 yearleft shoulderone soft skin - colored nodule of 1 cmlobular proliferation of fat tissue in the dermissurgical resection4m76 yearsright shoulderlarge ( 6 cm x 2 cm ) soft skin - colored verrucous plaque composed of nodules and papulesectopic mature adipocytes in reticular dermissurgical resection5m2420 yearslumbar area1 cm to 3 cm - grouped soft skin - colored papulesepidermal acanthosis , ectopic mature adipocytes in papillary dermissurgical resection6f3210 yearsthighmultiple soft skin - colored papules coalescing into a 5 cm plaqueepidermal acanthosis ; hyperkeratosis ; dermal proliferation of mature adipocytessurgical resection7f149 yearsflanklarge ( 6 cm x 2 cm ) soft skin - colored plaque composed of nodulesthinned epidermis , ectopic mature adipose tissue in reticular dermissurgical resection8f4135 yearsleft buttocksoft , skin - colored voluminous tumor ( 10 cm x 7 cm ) composed of multiple nodules with a cerebriform surfacenormal epidermis , mature fat cells in reticular dermis without hypodermis connectionsurgical resectionf = female ; m = male .", "epidemiologic , clinical and pathological features , and management of nevus lipomatosus cutaneous superficialis ( nlcs ) in all eight patients ."], ["nlcs is more commonly present from birth , but can appear later in life ; there is neither sex predilection nor familial trend in this disorder.1 in this study , there were five cases in which the lesion appeared in the first three decades , and in three cases nlcs appeared after 35 years .", "two clinical patterns of nlcs exist ; a multiple form or classical type , and the solitary form . in the classical type ,", "lesions are congenital or they develop usually during the first two to three decades of life.5,6,7 it consists of multiple papules , skin colored or yellowish , coalescing into plaques with zosteriform , linear or segmental distribution .", "the lesions are slow - growing , with a smooth or cerebriform surface , and can reach a large size if left untreated .", "the largest size reported so far has been 40 cm x 28 cm.8 the most common sites are the pelvic girdle , the lower trunk , the gluteal region , and the thigh .", "the second clinical pattern of nlcs is a solitary papule or nodule mimicking skin tag,8 usually appearing later than the classical form , during the third to sixth decades of life.3,6,9 it can occur at any site , including unusual ones like the scalp , the eyelid , the nose , and the clitoris.1,1013 solitary form was also called pedonculated lipofibroma by mehregan et al . because of its distinctive clinicopathologic features in comparison with the multiple form.9,14 among the presented cases , there were seven cases with the classical form and one case with the solitary form ( case 3 ) , whilst in another tunisian study the solitary type was predominant : 11 cases out of 13.15 nlcs have an indolent and asymptomatic course . recently , a case of ulnar nerve entrapment accompanied by numbness and straining in the forearm , caused by nlcs , was reported and was treated by partial excision for symptomatic recovery.16 occasionally , nlcs may ulcerate after external trauma or ischemia . in two previously reported cases , surface of nevus", "was studded with multiple open comedons,2,17 and foul - smelling discharge may be associated.2 nlcs has been reported in association with other cutaneous disorders ; follicular papules and hypertrophic pilo - sebaceous units,18 angiokeratoma of fordyce,4 scattered leukoderma , caf - au - lait macules,2 and hemangioma.4 in our study , no associated skin abnormalities were present . to our knowledge , there are no systemic abnormalities or malignant transformation described with nlcs .", "nlcs should be clinically differenciated from nevus sebaceous , neurofibroma , lymphangioma , focal dermal hypoplasia , cylindroma , trichoepithelioma , and angiolipoma .", "histopathological evaluation is required for diagnosis showing ectopic mature adipocytes , intermingled with collagen bundles , and proliferating around the periadnexial adventitial dermis and the perivascular area .", "mature fat cells proliferate in reticular dermis and may extend to the papillary dermis.2,6,7 in the most characteristic feature of nlcs , there is usually no connection to the subcutaneous fat tissue ; for some authors this condition is necessary to establish diagnosis of nlcs.6 several studies documented increased vascularity in the subpapillary and papillary dermis ; epidermal changes have been reported , like mild to moderate acanthosis , basket weave hyperkeratosis , increased basal pigmentation , and focal elongation of rete pegs.3 adnexal structures may be reduced,3 and abnormal folliculosebaceous structures were reported in some cases such as sebaceous trichofolliculoma , folliculosebaceous cystic hamartoma , and dermoid cysts.7,19 in our study , diagnosis of nlcs was based on characteristic clinical aspects and confirmed by typical histopathological features .", "the genetic background of nlcs is still unknown , only one cytogenetic study was reported recently showing a 2p24 deletion.20 the pathogenesis of nlcs remains unknown .", "several theories have been proposed : hoffman and zuhrelle postulated that fat deposition in the dermis is secondary to degenerative changes ( metaplasia ) in the connective tissue.3 other authors hypothesized that adipocytes originate from the pericytes of dermal vessels.2,21 for others , fat cells represented a true nevus that resulted from the focal heterotopic development of adipose tissue.22 treatment is usually not necessary unless for cosmetic reasons .", "the treatment of choice is surgical excision , which is curative , and post surgical recurrence is rare .", "patients unwilling for surgery may undergo cryotherapy which yields partial but satisfactory results.8 recently , a case of classic nlcs successfully treated with co2 laser was reported with no recurrence during a follow - up period of 12 months.23 in this retrospective study , we did nt investigate chromosomal abnormalities to identify the genetic biomarkers of this congenital pathology , since cytogenetic analysis was reported in only one previous study.20"], ["physicians should be aware of this rare condition because early recognition enables more conservative resection of the tumor and less invasive reconstruction of the defect .", "nlcs is a rare skin malformation ; we report herein a large case series of eight patients where the classical form is predominant", ". our study may give additional data about epidemiological and clinical pattern of this hamartoma .", "further studies are needed , especially prospective studies , by using chromosomal analysis to reach conclusions about the role of the genetic abnormalities in the development of this hamartoma ."]]}
{"article_id": "PMC4897306", "article_text": ["the use of chemotherapy in the treatment of cancer has opened new possibilities for improvement of the quality of life of cancer patients . despite its success , treatment with some of the most effective anticancer drugs", ", the mechanism of cisplatin ( cis - diamminedichloroplatinum ( ii ) or cddp ) induced nephrotoxicity has gradually been elucidated .", "studies have shown an increase in lipid peroxides in the renal tissue of cddp - administered animals , a decrease in reduced glutathione levels , and the induction of metallothionein , an antioxidant .", "these changes have been considered to result from the generation of reactive oxygen species ( ros ) .", "studies using chemiluminescence or electron spin resonance ( esr ) have shown that cddp generates oh radical [ 5 , 6 ] .", "nephrotoxicity involves kidney damage or dysfunction arising from direct or indirect exposure to drugs and industrial or environmental chemicals .", "cisplatin ( ( cis - diamminedichloroplatinum ( ii ) or cddp ) ) , an anti - neoplastic drug have been reported to induce nephrotoxicity .", "the kidney which is the major route of cisplatin excretion also accumulates it to a greater degree than other organs [ 6 , 8 ] .", "oxidative stress , inflammation , and apoptosis are some of the mechanisms already established to explain cisplatin induced acute kidney injury .", "a number of strategies have been proposed for the prevention / management of cisplatin induced nephrotoxicity , since there is no specific treatment , with the use of some synthetic drugs which have been popular . however , these drugs have some associated risks and side - effects , hence , the need for natural alternatives of plant origin ( plant foods / extracts ) with little or no side - effect .", "plants have limitless ability to synthesize aromatic substances such as polyphenols , mainly flavonoids , and phenolic acids , which exhibit antioxidant properties due to their hydrogen - donating and metal - chelating capacities .", "polyphenols are secondary metabolites of plants and are widely distributed in plant - derived foods , such as cereals , legumes , nuts , vegetables , and fruits , and in beverages such as green or black tea , and fruit juice .", "tannic and gallic acids are two commonly phenolic acids that are structurally related . tannic acid , a naturally occurring plant polyphenol , is composed of a central glucose molecule derivatized at its hydroxyl groups with one or more galloyl residues , whereas gallic acid is a trihydroxybenzoic acid , also known as 3,4,5-trihydroxybenzoic acid , which is widely distributed in green tea , red wine and grapes , witch hazel , sumac , oak bark , and other plants .", "considerable amounts of experimental data on the antioxidant activity of both tannic acid and gallic acids with emphasis on structure - function antioxidant activity have been reported .", "also , several authors have demonstrated that tannic acid and other polyphenols have antimutagenic and anticarcinogenic activities [ 1316 ] .", "extensive studies have been carried out on the protective effect of cotreatment and posttreatment of phenolic acids against cisplatin induced nephrotoxicity .", "hence , this study was carried out to investigate and compare the protective effect of administration of both tannic and gallic acids on normal and cisplatin induced nephrotoxicity in rats .", "male albino rats weighing 110185  g used for this experiment were purchased from a private animal colony , ikere - ekiti metropolis .", "the rats were maintained at 25c on a 12 hour light / dark cycle with free access to food and water .", "they were acclimatized under these conditions for two weeks prior to the commencement of the experiments .", "the experimental study was approved by the institutional animal ethical committee of the university of ado - ekiti , nigeria .", "chemicals such as tannic acid , gallic acid , oxidized and reduced glutathione , hydrogen peroxide ( h2o2 ) , dithionitrobenzene ( dtnb ) , thiobarbituric acid ( tba ) , and adrenaline were purchased from sigma chemical co. ( st .", "ethanol , acetic acid , h2so4 , sodium carbonate , sodium citrate , sodium azide ( nan3 ) , sodium chloride , potassium dichromate , tris - hcl buffer , sodium dodecyl sulphate ( sds ) , and ascorbic acid were sourced from bdh chemicals ltd .", "may - grnwald , giemsa , and hematoxylin and eosin ( h&e ) stains were purchased from hi - media labs , mumbai .", "all the kits used for bioassay were sourced from randox laboratories ltd . , crumlin , county antrim , uk . except stated otherwise ,", "all other chemicals and reagents were of analytical grades and the water was glass - distilled .", "after two ( 2 ) weeks of acclimatization , 80 male rats were randomly divided into eight ( 8) groups of ten animals each : group i served as a normal control and received saline ( 0.85  w / v% ) orally for 7 consecutive days and on the 7th day , 1  hr after receiving the oral saline dose , the rats received a single i.p .", "group ii served as toxicant group and received saline ( 0.85% ) orally for 7 consecutive days .", "ga was orally administered at two doses , 20 and 40  mg / kg body weight ( bwt ) , to groups iii and iv , respectively , for 7 consecutive days .", "also , ta was orally administered at two doses , 20 and 40  mg / kg body weight ( bwt ) , to groups vi and vii , respectively , for 7 consecutive days . on the 7th day of pretreatment , a single i.p .", "dose of cisplatin ( 7.5  mg / kg  bwt ) after oral treatment of ga and ta was given to the animals in groups ii , iii , iv , vi , and vii .", "groups v and viii received only higher dose ( 40  mg / kg ) of ga and ta orally for 7 consecutive days , respectively , and on the 7th day 1  hr ga and ta treatment received a single i.p .", "injection of saline ( 0.85% ) to ensure that groups v and viii received only the higher dose of ga and ta ; this was done in order to test that the higher dose did not produce any kind of toxic effects .", "the doses of ta and ga used were actually selected on the basis of preliminary dose escalation studies to determine the minimum dose of ta and ga required to produce an observable effect ( data not shown ) .", "uric acid , urea , and creatinine were determined using commercially available kits ( randox laboratories uk ) .", "tissue malondialdehyde ( mda ) content was determined as described by ohkawa et al . .", "tissue antioxidant parameters were also determined ; superoxide dismutase ( sod ) by the method of alia et al .", ", catalase ( cat ) by the method of sinha , reduced glutathione ( gsh ) by the method of ellman , and glutathione peroxidase ( gpx ) by the method of rotruck et al . .", "at the end of the experiment , whole blood of the sacrificed rats were collected into edta bottles and centrifuged at 800  g for 10  min to separate the plasma .", "the plasma was then decanted into plain sample bottle and stored in a refrigerator prior to analysis .", "the rat 's tissues ( kidney ) were rapidly isolated , placed on ice , and weighed . the tissue was rinsed in cold ( 0.9%  w / v ) normal saline ( 1  :  3 , w / v ) and subsequently homogenized in sodium phosphate buffer ( ph 7.4 ) with ( 1  :  5  bt  w / v ) using mortar and pestle as homogenizer and the homogenates were centrifuged at 4,000  g .", "the uric acid concentration was determined using colorimetric method as described by collin and diehl   and morin and prox .", "briefly , 20  l of distilled water was added to 20  l of the sample which was mixed with 1  ml of hepes reagent ( 50  mm phosphate buffer , 4  mm 3,5-chloro-2-hydroxybenzenesulfonic acid ) and enzyme reagent ( 0.25  mm 4-aminophenazone , peroxidise , and uricase ) .", "thereafter , the mixture was incubated for 5  min at 37c and the absorbance at 520  nm was taken against reagent blank within 30  min .", "the urea concentration was determined using colorimetric method as described by searcy et al . .", "briefly , 10  l of sample was added to 0.1  ml of sodium nitroprusside ", "6  mm sodium nitroprusside , 1  g / l urease ) after which the mixture was incubated for 10  min at 37c .", "2.5  ml of 120  mm diluted phenol and 2.5  ml of 27  mm sodium hypochlorite solution containing 0.14  n sodium hydroxide which was then added to the reaction mixture . thereafter , the mixture was incubated for 15  min at 37c and the absorbance at 546  nm was taken against reagent blank within 8 hours .", "the creatinine concentration was determined using colorimetric alkaline picrate method as described by jaffe method .", "briefly , 50  l of distilled water was added to 2  ml of working reagent ( 35  mm picric acid and 0.32  m sodium hydroxide ) before 50  l of sample was added .", "the absorbance at 492  nm was taken twice , firstly after 30  sec and secondly after 2  min .", "the creatinine concentration was subsequently calculated against the standard , using change in the sample absorbance ( absorbance ) .", "arginase activity was determined by the measurement of urea produced by the reaction of ehrlich 's reagent according to the modified method of kaysen and strecker .", "the reaction mixture contained 1.0  mm tris - hcl buffer , 1.0  mm mncl2 ( ph 9.5 ) , 0.1  m arginase solution and 500  ml of the enzyme preparation .", "the mixture was incubated for 10  mins at 37c . the reaction was terminated by the addition of 2.5  ml ehrlich reagent ( 2.0  g of p - dimethylaminobenzaldehyde in 20.0  ml of concentrated hydrochloric acid and made up to 100  ml with distilled water ) .", "superoxide dismutase ( sod ) was determined by the method of alia et al . .", "50  l of supernatant was treated with 1000  l of 50  mm carbonate buffer ( ph 10.2 ) and 17  l of adrenaline ( 0.06  mg / ml ) .", "the absorbance was read at 480  nm in spectrophotometer for 2 minutes at 15-second intervals .", "sod activity was expressed as ui per 100  g protein ( 480 = 4.02  mmcm ) .", "the reaction mixture ( 1.5  ml ) contained 1.0  ml of 0.01  m phosphate buffer ( ph 7.0 ) , 0.1  ml of tissue homogenate , and 0.4  ml of 2  m h2o2 .", "the reaction was stopped by the addition of 2.0  ml of dichromate - acetic acid reagent ( 5% potassium dichromate and glacial acetic acid were mixed in 1  :  3 ratio ) .", "then , the absorbance was read at 620  nm : cat activity was expressed as moles of h2o2 consumed / min / g protein .", ". 1  ml of supernatant was treated with 500  l of ellman 's reagent ( 19.8  mg of 5,5dithiobisnitrobenzoic acid in 100  ml of 0.1% sodium citrate ) and 3.0  ml of 0.2  m phosphate buffer ( ph 8.0 ) .", "the activity of glutathione peroxidase ( gpx ) was assayed by the method of rotruck et al . .", "the reaction mixture containing 0.2  ml of edta ( 0.8  mm , ph 7.0 ) , 0.4  ml of phosphate buffer ( 10  mm ) , and 0.2  ml of tissue homogenate was incubated with 0.1  m of h2o2 and 0.2  ml of glutathione for 10  min .", "the activity of gpx was expressed as l  mol glutathione oxidized / min / g protein .", "the lipid peroxidation assay was carried out using the modified method of ohkawa et al . .", "briefly , 300  l of tissue homogenate , 300  l of 8.1% sds ( sodium dodecyl sulphate ) , 500  l of acetic acid / hcl ( ph = 3.4 ) , and tba ( thiobarbituric acid ) were added , and the mixture was incubated at 100c for 1 ", "thereafter , the thiobarbituric acid reactive species ( tbars ) produced was measured at 532  nm and calculated as malondialdehyde ( mda ) equivalent .", "one - way analysis of variance was used to analyze the results and duncan multiple tests were used for the post hoc analysis . statistical package for social science ( spss ) 10.0 for windows was used for the analysis .", "as evident from table 1 , administration of a single dose of cisplatin ( 7.5  mg / kg  bwt ) caused a significant increase in the biomarkers of renal function ( creatinine , urea , and uric acid ) when compared with the control group that received only saline ( 0.85%  w / v ) .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) improvement of renal function due to a decrease in the creatinine , urea , and uric acid levels when compared with the induced group ( group 2 ) . also , administration of a single i.p dose of cisplatin ( 7.5  mg / kg bwt ) caused a significant decrease in the biomarkers of oxidative stress [ superoxide dismutase ( sod ) , catalase ( cat ) , glutathione peroxidase ( gpx ) , and reduced glutathione ( gsh ) ] when compared with the control group that received only saline ( 0.85%  w / v ) ( table 2 ) .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) improvement in the body 's antioxidant status by an increase in the activities of superoxide dismutase ( sod ) , catalase ( cat ) , glutathione peroxidase ( gpx ) , and reduced glutathione ( gsh ) when compared with the induced group ( table 2 ) . likewise , there was a significant ( p < 0.05 ) increase in the malondialdehyde ( mda ) content in rat kidney administered a single i.p dose of cisplatin ( 7.5  mg / kg  bwt ) .", "however , both pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) reduction of mda content in rat kidney when compared with the induced group ( figure 1 ) . \n", "figure 2 revealed that administration of a single i.p dose of cisplatin ( 7.5  mg / kg bwt ) caused a significant ( p < 0.05 ) increase in arginase activity when compared with the control group that received only saline ( 0.85%  w / v ) .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) decrease in arginase activity when compared with the induced group ( group 2 ) .", "photomicrographs of kidney sections from various treatment groups are shown in figures 3 , 4 , and 5 .", "histopathological examination of sections from rat kidney administered a single i.p dose of cisplatin ( 7.5  mg / kg bwt ) shows severe and generalized tubular epithelial cell necrosis associated with diffuse tubular lumina when compared with the control without cisplatin .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a marked improvement on kidney damage .", "about 25% of most commonly used drugs in intensive care units ( icus ) are potentially nephrotoxic and are recognized as considerable health and economic burden worldwide . among them , cisplatin , when used in cancer chemotherapy , induces renal impairment and acute renal failure by induction of reactive oxygen species , tubulointerstitial inflammation , and apoptosis .", "although various studies have been reported on the benefits of several agents in cisplatin induced renal toxicity , the basis of nephroprotection remains elusive .", "this makes the search for strategies to prevent nephrotoxicity constitute an active area of investigation .", "hence , it is reasonable to assume that a reinforcement of antioxidant defense of renal tissue by exogenous antioxidant such as phenolic acids should be a strategy to protect the kidney from the oxidative damage . in the present study", ", we compare the protective effect of administration of gallic and tannic acids ( two commonly phenolic acids that are structurally related ) of the same dosage against normal and cisplatin induced renal injury in rats for the first time by attenuating renal oxidative stress .", "the elevations of key kidney function biomarkers such as creatinine , uric acid , and urea have been suggested to be indicative of reduced renal functions [ 30 , 31 ] .", "thus , estimation of plasma creatinine and uric acid has been employed as key test to assess kidney function [ 30 , 31 ] . in the present study , the observed elevation in plasma creatinine , urea , and uric acid levels in induced rats", "this finding is consistent with that reported by earlier studies [ 32 , 33 ] .", "however , the restoration of the plasma creatinine , urea , and uric acid level in rats treated with both gallic and tannic acids ( table 1 ) suggests that both phenolic acids have the ability to prevent kidney damage and protect the kidney against nephrotoxicity .", "this , however , may be a function of their antioxidant properties and ability to inhibit arginase activity ( figure 2 ) .", "cisplatin increased arginase activity in the rat 's kidney , while the pretreatment with both gallic and tannic acids at two doses ( 20 and 40  mg / kg bwt ) , respectively , for 7 days resulted in a decrease in kidney arginase activity ( figure 2 ) .", "arginase is a hydrolytic enzyme responsible for the conversion of l - arginine to l - ornithine and urea .", "ornithine is an important biosynthetic precursor of polyamines which have been implicated to facilitate cell proliferation in certain cancer cells .", "another important enzyme , endothelium nitric oxide synthase ( enos ) , competes with arginase for the same substrate , arginine .", "the no produced plays an important role in both regulating renal hemodynamics and modulating inflammatory and proliferating response to various stimuli .", "therefore , inhibition of arginase activity slows the progression of renal failure in renal ablation .", "the increase in the kidney and plasma mda content ( figure 1 ) in the induced rats suggests lipid peroxidation .", "this agreed with earlier studies where administration of cisplatin caused inflammation and lipid peroxidation [ 6 , 7 ] .", "this could be as a result of increased hydrogen peroxide concentration produced in the kidney due to the depletion of antioxidant enzymes : superoxide dismutase ( sod ) , reduced glutathione ( gsh ) , and catalase ( cat ) activity ( table 2 ) .", "this also is consistent with earlier study where depletion of sod , cat , and gpx in rats resulted in increased mda concentration due to lipid peroxidation .", "the depletion of these antioxidants suggests that cisplatin induced nephrotoxicity could be a result of oxidative stress or suppression of the antioxidant enzymes , as previously reported by earlier studies [ 3537 ] .", "however , the reduced kidney and plasma mda content ( figure 1 ) and restoration of sod , gsh , and cat activities ( table 2 ) in pretreated rats suggest an improvement in the in vivo antioxidant status , which may be a function of the antioxidant properties of the phenolic acids ( gallic and tannic acids )", "therefore , the observed decrease in the kidney gsh level ( table 2 ) in the induced rats suggests cisplatin induced nephrotoxicity , which is associated with a drastic reduction in kidney gsh content .", "this finding is consistent with earlier studies where gsh depletion was suggested to be due to the interaction of cisplatin with the molecules contain sulfhydryl groups [ 39 , 40 ] .", "however , restoration of gsh levels in the pretreated rats suggests the antioxidant and nephroprotective properties of the phenolic acids ( table 2 ) .", "furthermore , histopathology study revealed normal glomerulus and tubules with intact renal architecture in normal ( figure 3(a ) ) , gallic ( figure 5(a ) ) , and tannic acid ( figure 5(b ) ) group without cisplatin injection .", "degenerated tubular structures with vacuolization and loss of architecture were seen in cisplatin induced group ( figure 3(b ) ) .", "pretreatment with both gallic and tannic acids at two doses ( 20 and 40  mg / kg bwt ) , respectively , for 7 days resulted in excellent protection against nephrotoxicity induced by cisplatin and showed predominant normal kidney morphology ( figures 4(a ) , 4(b ) , 4(c ) , and 4(d ) ) .", "the results of the present study revealed that oxidative stress and apoptosis / necrosis play an important role in pathogenesis of cisplatin nephrotoxicity .", "gallic and tannic acids , two important pharmacologically active phenolic compounds , reduced cisplatin induced functional and histological renal damage .", "furthermore , they suppressed the generation of ros , lipid peroxidation , and oxidative stress in kidney tissues .", "these results indicated that both gallic and tannic acids exhibit nephroprotective effect and the possible mechanism of action by which they exert this effect could be due to their antioxidant properties and inhibition of arginase activity .", "however , tannic acid exhibited better nephroprotective potential than gallic acid which may be due to the glycosidation with a glucose moiety ."], "abstract_text": ["<S> cisplatin ( cis - diamminedichloroplatinum ( ii ) or cddp ) , used in the treatment of many solid - tissue cancers , has its chief side - effect in nephrotoxicity . </S>", "<S> hence , this study sought to investigate and compare the protective effect of gallic acid ( ga ) and tannic acid ( ta ) against cisplatin induced nephrotoxicity in rats . </S>", "<S> the rats were given a prophylactic treatment of ga and ta orally at a dose of 20 and 40  mg / kg body weight for 7 consecutive days before the administration of a single intraperitoneal ( i.p . ) injection of cisplatin ( cp ) at 7.5  mg / kg bwt . </S>", "<S> the protective effects of both ga and ta on cp induced nephrotoxicity were investigated by assaying renal function , oxidative stress biomarkers , and histopathological examination of kidney architecture . </S>", "<S> a single dose of cisplatin ( 7.5  mg / kg bwt ) injected i.p . caused a significant increase in some biomarkers of renal function ( creatinine , uric acid , and urea levels ) , with a marked elevation in malondialdehyde ( mda ) content accompanied by a significant ( p < 0.05 ) decrease in reduced glutathione ( gsh ) content ( 103.27% ) of kidney tissue as compared to control group . </S>", "<S> furthermore , a significant ( p < 0.05 ) reduction in kidney antioxidant enzymes ( sod , catalase , gpx , and gst ) activity was observed . however , pretreatment with oral administration of tannic acid and gallic acid at a dose of 20 and 40  mg / kg body weight , respectively , for 7 days prior to cisplatin administration reduced histological renal damage and suppressed the generation of ros , lipid peroxidation , and oxidative stress in kidney tissues . </S>", "<S> these results indicate that both gallic and tannic acids could serve as a preventive strategy against cisplatin induced nephrotoxicity . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["the use of chemotherapy in the treatment of cancer has opened new possibilities for improvement of the quality of life of cancer patients . despite its success , treatment with some of the most effective anticancer drugs", ", the mechanism of cisplatin ( cis - diamminedichloroplatinum ( ii ) or cddp ) induced nephrotoxicity has gradually been elucidated .", "studies have shown an increase in lipid peroxides in the renal tissue of cddp - administered animals , a decrease in reduced glutathione levels , and the induction of metallothionein , an antioxidant .", "these changes have been considered to result from the generation of reactive oxygen species ( ros ) .", "studies using chemiluminescence or electron spin resonance ( esr ) have shown that cddp generates oh radical [ 5 , 6 ] .", "nephrotoxicity involves kidney damage or dysfunction arising from direct or indirect exposure to drugs and industrial or environmental chemicals .", "cisplatin ( ( cis - diamminedichloroplatinum ( ii ) or cddp ) ) , an anti - neoplastic drug have been reported to induce nephrotoxicity .", "the kidney which is the major route of cisplatin excretion also accumulates it to a greater degree than other organs [ 6 , 8 ] .", "oxidative stress , inflammation , and apoptosis are some of the mechanisms already established to explain cisplatin induced acute kidney injury .", "a number of strategies have been proposed for the prevention / management of cisplatin induced nephrotoxicity , since there is no specific treatment , with the use of some synthetic drugs which have been popular . however , these drugs have some associated risks and side - effects , hence , the need for natural alternatives of plant origin ( plant foods / extracts ) with little or no side - effect .", "plants have limitless ability to synthesize aromatic substances such as polyphenols , mainly flavonoids , and phenolic acids , which exhibit antioxidant properties due to their hydrogen - donating and metal - chelating capacities .", "polyphenols are secondary metabolites of plants and are widely distributed in plant - derived foods , such as cereals , legumes , nuts , vegetables , and fruits , and in beverages such as green or black tea , and fruit juice .", "tannic and gallic acids are two commonly phenolic acids that are structurally related . tannic acid , a naturally occurring plant polyphenol , is composed of a central glucose molecule derivatized at its hydroxyl groups with one or more galloyl residues , whereas gallic acid is a trihydroxybenzoic acid , also known as 3,4,5-trihydroxybenzoic acid , which is widely distributed in green tea , red wine and grapes , witch hazel , sumac , oak bark , and other plants .", "considerable amounts of experimental data on the antioxidant activity of both tannic acid and gallic acids with emphasis on structure - function antioxidant activity have been reported .", "also , several authors have demonstrated that tannic acid and other polyphenols have antimutagenic and anticarcinogenic activities [ 1316 ] .", "extensive studies have been carried out on the protective effect of cotreatment and posttreatment of phenolic acids against cisplatin induced nephrotoxicity .", "hence , this study was carried out to investigate and compare the protective effect of administration of both tannic and gallic acids on normal and cisplatin induced nephrotoxicity in rats ."], ["male albino rats weighing 110185  g used for this experiment were purchased from a private animal colony , ikere - ekiti metropolis .", "the rats were maintained at 25c on a 12 hour light / dark cycle with free access to food and water .", "they were acclimatized under these conditions for two weeks prior to the commencement of the experiments .", "the experimental study was approved by the institutional animal ethical committee of the university of ado - ekiti , nigeria .", "chemicals such as tannic acid , gallic acid , oxidized and reduced glutathione , hydrogen peroxide ( h2o2 ) , dithionitrobenzene ( dtnb ) , thiobarbituric acid ( tba ) , and adrenaline were purchased from sigma chemical co. ( st .", "ethanol , acetic acid , h2so4 , sodium carbonate , sodium citrate , sodium azide ( nan3 ) , sodium chloride , potassium dichromate , tris - hcl buffer , sodium dodecyl sulphate ( sds ) , and ascorbic acid were sourced from bdh chemicals ltd .", "may - grnwald , giemsa , and hematoxylin and eosin ( h&e ) stains were purchased from hi - media labs , mumbai .", "all the kits used for bioassay were sourced from randox laboratories ltd . , crumlin , county antrim , uk . except stated otherwise ,", "all other chemicals and reagents were of analytical grades and the water was glass - distilled .", "after two ( 2 ) weeks of acclimatization , 80 male rats were randomly divided into eight ( 8) groups of ten animals each : group i served as a normal control and received saline ( 0.85  w / v% ) orally for 7 consecutive days and on the 7th day , 1  hr after receiving the oral saline dose , the rats received a single i.p .", "group ii served as toxicant group and received saline ( 0.85% ) orally for 7 consecutive days .", "ga was orally administered at two doses , 20 and 40  mg / kg body weight ( bwt ) , to groups iii and iv , respectively , for 7 consecutive days .", "also , ta was orally administered at two doses , 20 and 40  mg / kg body weight ( bwt ) , to groups vi and vii , respectively , for 7 consecutive days . on the 7th day of pretreatment , a single i.p .", "dose of cisplatin ( 7.5  mg / kg  bwt ) after oral treatment of ga and ta was given to the animals in groups ii , iii , iv , vi , and vii .", "groups v and viii received only higher dose ( 40  mg / kg ) of ga and ta orally for 7 consecutive days , respectively , and on the 7th day 1  hr ga and ta treatment received a single i.p .", "injection of saline ( 0.85% ) to ensure that groups v and viii received only the higher dose of ga and ta ; this was done in order to test that the higher dose did not produce any kind of toxic effects .", "the doses of ta and ga used were actually selected on the basis of preliminary dose escalation studies to determine the minimum dose of ta and ga required to produce an observable effect ( data not shown ) .", "uric acid , urea , and creatinine were determined using commercially available kits ( randox laboratories uk ) .", "tissue malondialdehyde ( mda ) content was determined as described by ohkawa et al . .", "tissue antioxidant parameters were also determined ; superoxide dismutase ( sod ) by the method of alia et al .", ", catalase ( cat ) by the method of sinha , reduced glutathione ( gsh ) by the method of ellman , and glutathione peroxidase ( gpx ) by the method of rotruck et al . .", "at the end of the experiment , whole blood of the sacrificed rats were collected into edta bottles and centrifuged at 800  g for 10  min to separate the plasma .", "the plasma was then decanted into plain sample bottle and stored in a refrigerator prior to analysis .", "the rat 's tissues ( kidney ) were rapidly isolated , placed on ice , and weighed . the tissue was rinsed in cold ( 0.9%  w / v ) normal saline ( 1  :  3 , w / v ) and subsequently homogenized in sodium phosphate buffer ( ph 7.4 ) with ( 1  :  5  bt  w / v ) using mortar and pestle as homogenizer and the homogenates were centrifuged at 4,000  g .", "the uric acid concentration was determined using colorimetric method as described by collin and diehl   and morin and prox .", "briefly , 20  l of distilled water was added to 20  l of the sample which was mixed with 1  ml of hepes reagent ( 50  mm phosphate buffer , 4  mm 3,5-chloro-2-hydroxybenzenesulfonic acid ) and enzyme reagent ( 0.25  mm 4-aminophenazone , peroxidise , and uricase ) .", "thereafter , the mixture was incubated for 5  min at 37c and the absorbance at 520  nm was taken against reagent blank within 30  min .", "the urea concentration was determined using colorimetric method as described by searcy et al . .", "briefly , 10  l of sample was added to 0.1  ml of sodium nitroprusside ", "6  mm sodium nitroprusside , 1  g / l urease ) after which the mixture was incubated for 10  min at 37c .", "2.5  ml of 120  mm diluted phenol and 2.5  ml of 27  mm sodium hypochlorite solution containing 0.14  n sodium hydroxide which was then added to the reaction mixture . thereafter , the mixture was incubated for 15  min at 37c and the absorbance at 546  nm was taken against reagent blank within 8 hours .", "the creatinine concentration was determined using colorimetric alkaline picrate method as described by jaffe method .", "briefly , 50  l of distilled water was added to 2  ml of working reagent ( 35  mm picric acid and 0.32  m sodium hydroxide ) before 50  l of sample was added .", "the absorbance at 492  nm was taken twice , firstly after 30  sec and secondly after 2  min .", "the creatinine concentration was subsequently calculated against the standard , using change in the sample absorbance ( absorbance ) .", "arginase activity was determined by the measurement of urea produced by the reaction of ehrlich 's reagent according to the modified method of kaysen and strecker .", "the reaction mixture contained 1.0  mm tris - hcl buffer , 1.0  mm mncl2 ( ph 9.5 ) , 0.1  m arginase solution and 500  ml of the enzyme preparation .", "the mixture was incubated for 10  mins at 37c . the reaction was terminated by the addition of 2.5  ml ehrlich reagent ( 2.0  g of p - dimethylaminobenzaldehyde in 20.0  ml of concentrated hydrochloric acid and made up to 100  ml with distilled water ) .", "superoxide dismutase ( sod ) was determined by the method of alia et al . .", "50  l of supernatant was treated with 1000  l of 50  mm carbonate buffer ( ph 10.2 ) and 17  l of adrenaline ( 0.06  mg / ml ) .", "the absorbance was read at 480  nm in spectrophotometer for 2 minutes at 15-second intervals .", "sod activity was expressed as ui per 100  g protein ( 480 = 4.02  mmcm ) .", "the reaction mixture ( 1.5  ml ) contained 1.0  ml of 0.01  m phosphate buffer ( ph 7.0 ) , 0.1  ml of tissue homogenate , and 0.4  ml of 2  m h2o2 .", "the reaction was stopped by the addition of 2.0  ml of dichromate - acetic acid reagent ( 5% potassium dichromate and glacial acetic acid were mixed in 1  :  3 ratio ) .", "then , the absorbance was read at 620  nm : cat activity was expressed as moles of h2o2 consumed / min / g protein .", ". 1  ml of supernatant was treated with 500  l of ellman 's reagent ( 19.8  mg of 5,5dithiobisnitrobenzoic acid in 100  ml of 0.1% sodium citrate ) and 3.0  ml of 0.2  m phosphate buffer ( ph 8.0 ) .", "the activity of glutathione peroxidase ( gpx ) was assayed by the method of rotruck et al . .", "the reaction mixture containing 0.2  ml of edta ( 0.8  mm , ph 7.0 ) , 0.4  ml of phosphate buffer ( 10  mm ) , and 0.2  ml of tissue homogenate was incubated with 0.1  m of h2o2 and 0.2  ml of glutathione for 10  min .", "the activity of gpx was expressed as l  mol glutathione oxidized / min / g protein .", "the lipid peroxidation assay was carried out using the modified method of ohkawa et al . .", "briefly , 300  l of tissue homogenate , 300  l of 8.1% sds ( sodium dodecyl sulphate ) , 500  l of acetic acid / hcl ( ph = 3.4 ) , and tba ( thiobarbituric acid ) were added , and the mixture was incubated at 100c for 1 ", "thereafter , the thiobarbituric acid reactive species ( tbars ) produced was measured at 532  nm and calculated as malondialdehyde ( mda ) equivalent .", "one - way analysis of variance was used to analyze the results and duncan multiple tests were used for the post hoc analysis . statistical package for social science ( spss ) 10.0 for windows was used for the analysis ."], ["as evident from table 1 , administration of a single dose of cisplatin ( 7.5  mg / kg  bwt ) caused a significant increase in the biomarkers of renal function ( creatinine , urea , and uric acid ) when compared with the control group that received only saline ( 0.85%  w / v ) .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) improvement of renal function due to a decrease in the creatinine , urea , and uric acid levels when compared with the induced group ( group 2 ) . also , administration of a single i.p dose of cisplatin ( 7.5  mg / kg bwt ) caused a significant decrease in the biomarkers of oxidative stress [ superoxide dismutase ( sod ) , catalase ( cat ) , glutathione peroxidase ( gpx ) , and reduced glutathione ( gsh ) ] when compared with the control group that received only saline ( 0.85%  w / v ) ( table 2 ) .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) improvement in the body 's antioxidant status by an increase in the activities of superoxide dismutase ( sod ) , catalase ( cat ) , glutathione peroxidase ( gpx ) , and reduced glutathione ( gsh ) when compared with the induced group ( table 2 ) . likewise , there was a significant ( p < 0.05 ) increase in the malondialdehyde ( mda ) content in rat kidney administered a single i.p dose of cisplatin ( 7.5  mg / kg  bwt ) .", "however , both pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) reduction of mda content in rat kidney when compared with the induced group ( figure 1 ) . \n", "figure 2 revealed that administration of a single i.p dose of cisplatin ( 7.5  mg / kg bwt ) caused a significant ( p < 0.05 ) increase in arginase activity when compared with the control group that received only saline ( 0.85%  w / v ) .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a significant ( p < 0.05 ) decrease in arginase activity when compared with the induced group ( group 2 ) .", "photomicrographs of kidney sections from various treatment groups are shown in figures 3 , 4 , and 5 .", "histopathological examination of sections from rat kidney administered a single i.p dose of cisplatin ( 7.5  mg / kg bwt ) shows severe and generalized tubular epithelial cell necrosis associated with diffuse tubular lumina when compared with the control without cisplatin .", "however , pretreatment with gallic and tannic acids orally at two doses , 20 and 40  mg / kg body weight , respectively , shows a marked improvement on kidney damage ."], ["about 25% of most commonly used drugs in intensive care units ( icus ) are potentially nephrotoxic and are recognized as considerable health and economic burden worldwide . among them , cisplatin , when used in cancer chemotherapy , induces renal impairment and acute renal failure by induction of reactive oxygen species , tubulointerstitial inflammation , and apoptosis .", "although various studies have been reported on the benefits of several agents in cisplatin induced renal toxicity , the basis of nephroprotection remains elusive .", "this makes the search for strategies to prevent nephrotoxicity constitute an active area of investigation .", "hence , it is reasonable to assume that a reinforcement of antioxidant defense of renal tissue by exogenous antioxidant such as phenolic acids should be a strategy to protect the kidney from the oxidative damage . in the present study", ", we compare the protective effect of administration of gallic and tannic acids ( two commonly phenolic acids that are structurally related ) of the same dosage against normal and cisplatin induced renal injury in rats for the first time by attenuating renal oxidative stress .", "the elevations of key kidney function biomarkers such as creatinine , uric acid , and urea have been suggested to be indicative of reduced renal functions [ 30 , 31 ] .", "thus , estimation of plasma creatinine and uric acid has been employed as key test to assess kidney function [ 30 , 31 ] . in the present study , the observed elevation in plasma creatinine , urea , and uric acid levels in induced rats", "this finding is consistent with that reported by earlier studies [ 32 , 33 ] .", "however , the restoration of the plasma creatinine , urea , and uric acid level in rats treated with both gallic and tannic acids ( table 1 ) suggests that both phenolic acids have the ability to prevent kidney damage and protect the kidney against nephrotoxicity .", "this , however , may be a function of their antioxidant properties and ability to inhibit arginase activity ( figure 2 ) .", "cisplatin increased arginase activity in the rat 's kidney , while the pretreatment with both gallic and tannic acids at two doses ( 20 and 40  mg / kg bwt ) , respectively , for 7 days resulted in a decrease in kidney arginase activity ( figure 2 ) .", "arginase is a hydrolytic enzyme responsible for the conversion of l - arginine to l - ornithine and urea .", "ornithine is an important biosynthetic precursor of polyamines which have been implicated to facilitate cell proliferation in certain cancer cells .", "another important enzyme , endothelium nitric oxide synthase ( enos ) , competes with arginase for the same substrate , arginine .", "the no produced plays an important role in both regulating renal hemodynamics and modulating inflammatory and proliferating response to various stimuli .", "therefore , inhibition of arginase activity slows the progression of renal failure in renal ablation .", "the increase in the kidney and plasma mda content ( figure 1 ) in the induced rats suggests lipid peroxidation .", "this agreed with earlier studies where administration of cisplatin caused inflammation and lipid peroxidation [ 6 , 7 ] .", "this could be as a result of increased hydrogen peroxide concentration produced in the kidney due to the depletion of antioxidant enzymes : superoxide dismutase ( sod ) , reduced glutathione ( gsh ) , and catalase ( cat ) activity ( table 2 ) .", "this also is consistent with earlier study where depletion of sod , cat , and gpx in rats resulted in increased mda concentration due to lipid peroxidation .", "the depletion of these antioxidants suggests that cisplatin induced nephrotoxicity could be a result of oxidative stress or suppression of the antioxidant enzymes , as previously reported by earlier studies [ 3537 ] .", "however , the reduced kidney and plasma mda content ( figure 1 ) and restoration of sod , gsh , and cat activities ( table 2 ) in pretreated rats suggest an improvement in the in vivo antioxidant status , which may be a function of the antioxidant properties of the phenolic acids ( gallic and tannic acids )", "therefore , the observed decrease in the kidney gsh level ( table 2 ) in the induced rats suggests cisplatin induced nephrotoxicity , which is associated with a drastic reduction in kidney gsh content .", "this finding is consistent with earlier studies where gsh depletion was suggested to be due to the interaction of cisplatin with the molecules contain sulfhydryl groups [ 39 , 40 ] .", "however , restoration of gsh levels in the pretreated rats suggests the antioxidant and nephroprotective properties of the phenolic acids ( table 2 ) .", "furthermore , histopathology study revealed normal glomerulus and tubules with intact renal architecture in normal ( figure 3(a ) ) , gallic ( figure 5(a ) ) , and tannic acid ( figure 5(b ) ) group without cisplatin injection .", "degenerated tubular structures with vacuolization and loss of architecture were seen in cisplatin induced group ( figure 3(b ) ) .", "pretreatment with both gallic and tannic acids at two doses ( 20 and 40  mg / kg bwt ) , respectively , for 7 days resulted in excellent protection against nephrotoxicity induced by cisplatin and showed predominant normal kidney morphology ( figures 4(a ) , 4(b ) , 4(c ) , and 4(d ) ) ."], ["the results of the present study revealed that oxidative stress and apoptosis / necrosis play an important role in pathogenesis of cisplatin nephrotoxicity .", "gallic and tannic acids , two important pharmacologically active phenolic compounds , reduced cisplatin induced functional and histological renal damage .", "furthermore , they suppressed the generation of ros , lipid peroxidation , and oxidative stress in kidney tissues .", "these results indicated that both gallic and tannic acids exhibit nephroprotective effect and the possible mechanism of action by which they exert this effect could be due to their antioxidant properties and inhibition of arginase activity .", "however , tannic acid exhibited better nephroprotective potential than gallic acid which may be due to the glycosidation with a glucose moiety ."]]}
{"article_id": "PMC2914262", "article_text": ["cavernous haemangioma is the most common primary orbital tumour in adults , accounting for 6% of all orbital lesions [ 13 ] .", "orbital schwannomas are benign peripheral nerve sheath tumours and constitute 1% of all orbital tumours [ 1 , 3 ] .", "orbital cavernous haemangiomas and schwannomas present as slow progressive enlargement and the most common presenting sign and symptom is painless proptosis [ 48 ] .", "although both orbital cavernous haemangiomas and schwannomas share many similar clinical and radiological characteristics [ 58 ] , it is important to distinguish between the tumours because they demonstrate important different features and the treatments of choice for the two are different [ 3 , 79 ] .", "magnetic resonance imaging , especially dynamic mr imaging , plays a crucial role in the differentiation of orbital cavernous haemangiomas and schwannomas [ 7 , 8 , 1014 ] .", "the spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is considered the most important finding distinguishing the two tumours [ 1014 ] . however , the literature on mr imaging findings in the differentiation of these two tumours is based on small patient populations .", "consequently , this study aims to assess mr imaging findings distinguishing orbital cavernous haemangiomas from schwannomas and to elaborate on specific differences between the two tumours .", "between june 2004 and february 2009 , 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol .", "mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients .", "most patients presented with painless insidious proptosis . some patients demonstrated a limitation of extraocular motion , diplopia and strabismus .", "informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data .", "magnetic resonance imaging was performed on a general electric ( ge ) signa 1.5-t mr imaging system ( ge healthcare , milwaukee , wi , usa ) with an eight - channel head coil . mr imaging was performed with t1-weighted ( repetition time / echo time , 400/15  ms ) and t2-weighted ( 3,000/120 ) fast spin - echo images .", "a 16-cm field of view , a 3-mm - thick section at 0 interval , and a 288    256 matrix with two signals acquired were used .", "dynamic contrast - enhanced ( dce ) mr imaging was performed by using a fast spoiled gradient recalled ( fspgr ) sequence with 8.4/4  ms , a flip angle of 15 , one excitation , a matrix of 256    160 , a field of view of 220    220  mm , and a section thickness of 3.2  mm at 0 interval . a power injector ( medrad , indianola , pa , usa ) with an injection flow rate of 2  ml /", "the acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1  mmol of gadopentetate dimeglumine ( magnevist , schering , berlin , germany ) per kilogram .", "each mr sequence yielded 12 anatomical slices in 13  s ; this was repeated 12 times with an interval between each sequence of 12  s. total imaging time was 288  s ( 4  min 48  s ) .", "dce mr imaging was evaluated by using a ge aw 4.2 workstation ( ge healthcare ) .", "a region of interest ( roi ) was drawn manually on the dynamic images for assessment of the enhancement kinetics .", "we chose the area that demonstrated the greatest degree of early enhancement so that the time  intensity curve ( tic ) could be generated .", "the area of the roi typically was 8  mm . the contrast index ( ci )", "was calculated from : ci  =  [ ( signal intensity post - contrast )    ( signal intensity pre - contrast)]/(signal intensity pre - contrast ) .", "the tics were analysed qualitatively as wash - out , persistent or plateau - shaped curves .", "magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist ( 15  years experience ) .", "location , configuration , margins , signal intensity , homogeneity and enhancement pattern of the tumour were assessed .", "the location was defined as anterior orbit ( anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery ) , posterior orbit ( posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery ) , or both anterior and posterior orbit ; extraconal or intraconal space ; superior or inferior orbit ; and nasal or temporal orbit .", "on contrast - enhanced mr imaging including dce mr imaging , the spread pattern of the contrast enhancement , enhancement pattern , ci and type of tic were evaluated .", "the spread pattern of the contrast enhancement was divided into two groups : one starting from a small point or portion and the other starting from a wide area . ", "progressive  enhancement means that on early imaging after the injection , one small point or portion of enhancement was initially noted , then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement ( fig .  1 ) .", "other patterns of enhancement included gradually heterogeneous enhancement and gradually homogeneous enhancement . \n fig . ", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) .", "f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) a 54-year - old woman with a cavernous haemangioma .", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) . f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests .", "fisher s exact test was performed when the sample size in the subgroups was deemed too small .", "between june 2004 and february 2009 , 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol .", "mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients .", "most patients presented with painless insidious proptosis . some patients demonstrated a limitation of extraocular motion , diplopia and strabismus .", "informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data .", "magnetic resonance imaging was performed on a general electric ( ge ) signa 1.5-t mr imaging system ( ge healthcare , milwaukee , wi , usa ) with an eight - channel head coil . mr imaging was performed with t1-weighted ( repetition time / echo time , 400/15  ms ) and t2-weighted ( 3,000/120 ) fast spin - echo images .", "a 16-cm field of view , a 3-mm - thick section at 0 interval , and a 288    256 matrix with two signals acquired were used .", "dynamic contrast - enhanced ( dce ) mr imaging was performed by using a fast spoiled gradient recalled ( fspgr ) sequence with 8.4/4  ms , a flip angle of 15 , one excitation , a matrix of 256    160 , a field of view of 220    220  mm , and a section thickness of 3.2  mm at 0 interval .", "a power injector ( medrad , indianola , pa , usa ) with an injection flow rate of 2  ml / s was used .", "the acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1  mmol of gadopentetate dimeglumine ( magnevist , schering , berlin , germany ) per kilogram .", "each mr sequence yielded 12 anatomical slices in 13  s ; this was repeated 12 times with an interval between each sequence of 12  s. total imaging time was 288  s ( 4  min 48  s ) .", "dce mr imaging was evaluated by using a ge aw 4.2 workstation ( ge healthcare ) .", "a region of interest ( roi ) was drawn manually on the dynamic images for assessment of the enhancement kinetics .", "we chose the area that demonstrated the greatest degree of early enhancement so that the time  intensity curve ( tic ) could be generated .", "the contrast index ( ci ) was calculated from : ci  =  [ ( signal intensity post - contrast )    ( signal intensity pre - contrast)]/(signal intensity pre - contrast ) .", "the tics were analysed qualitatively as wash - out , persistent or plateau - shaped curves .", "magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist ( 15  years experience ) .", "location , configuration , margins , signal intensity , homogeneity and enhancement pattern of the tumour were assessed .", "the location was defined as anterior orbit ( anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery ) , posterior orbit ( posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery ) , or both anterior and posterior orbit ; extraconal or intraconal space ; superior or inferior orbit ; and nasal or temporal orbit .", "on contrast - enhanced mr imaging including dce mr imaging , the spread pattern of the contrast enhancement , enhancement pattern , ci and type of tic were evaluated .", "the spread pattern of the contrast enhancement was divided into two groups : one starting from a small point or portion and the other starting from a wide area . ", "progressive  enhancement means that on early imaging after the injection , one small point or portion of enhancement was initially noted , then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement ( fig .  1 ) .", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) .", "f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) a 54-year - old woman with a cavernous haemangioma .", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) .", "f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead )", "frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests .", "fisher s exact test was performed when the sample size in the subgroups was deemed too small .", "tables  1 and 2 describe the frequency distribution of non - enhanced mr imaging features ( table  1 ) and contrast - enhanced mr imaging features ( table  2 ) .", "there was a significant difference between cavernous haemangiomas and schwannomas regarding the location ( including anterior orbit , posterior orbit , or both anterior and posterior orbit ; extraconal or intraconal space ) , configuration and margins of the mass , signal intensity and homogeneity on t1- and t2-weighted imaging , the spread pattern of the contrast enhancement , enhancement pattern and type of time ", "intensity curve ( tic ) ( p  <  0.05 ) ( figs .  1 , 2 , 3 ) . \n", "b axial t2-weighted imaging revealed that the tumour was isointense relative to muscle ( arrowhead ) .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour ( arrowhead ) .", "f axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour ( arrowhead)fig . ", "a obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour ( arrow ) and slight hypointensity of the posterior portion ( arrowhead ) relative to muscle .", "b axial t2-weighted imaging showed isointensity of the anterior portion of the tumour ( arrow ) and hyperintensity of the posterior portion ( arrowhead ) relative to muscle .", "c obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour ( arrow ) and no enhancement of the posterior portion ( arrowhead)table  1frequency distribution of non - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no . of patients594316anterior or posterior orbit0.004  anterior4779.63990.8850  posterior610.224.6425  both610.224.6425extraconal or intraconal space0.049  intraconal4474.63581.4956.2  extraconal1525.4818.6743.8nasal or temporal orbit0.250  nasal2644.11739.5956.2  temporal3355.92660.5743.8superior or inferior orbit0.937  superior2949.22148.8850  inferior3050.82251.2850configuration0.015  round610.249.3212.6  ovoid5084.73990.71168.7  irregular35.100318.7margin0.531  smooth4474.63376.71168.7  lobulated1525.41023.3531.3t1 signal intensity<0.001  hypointense711.900743.7  isointense5288.143100956.3t1 homogeneity<0.001  homogeneous5084.743100743.7  heterogeneous915.300956.3t2 signal intensity0.001  isointense58.500531.3  hyperintense5491.5431001168.7t2 homogeneity<0.001  homogeneous467843100318.7  heterogeneous1322001381.3signal intensity was compared with that of extraocular muscletable  2frequency distribution of contrast - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no .", "of patients594316spread pattern of contrast enhancement<0.001  starting from a small point or portion4372.94310000  starting from a wide area1627.10016100enhancement pattern<0.001  homogeneous or heterogeneous1627.10016100  progressive4372.94310000ci1.8    0.61.4    0.30.003tic type<0.001  persistent1220.337956.2  plateau - shaped2440.71944.2531.3  wash - out23392148.8212.50 a 42-year - old man with a schwannoma .", "b axial t2-weighted imaging revealed that the tumour was isointense relative to muscle ( arrowhead ) .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour ( arrowhead ) . f axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour ( arrowhead ) a 47-year - old woman with schwannoma .", "a obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour ( arrow ) and slight hypointensity of the posterior portion ( arrowhead ) relative to muscle .", "b axial t2-weighted imaging showed isointensity of the anterior portion of the tumour ( arrow ) and hyperintensity of the posterior portion ( arrowhead ) relative to muscle .", "c obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour ( arrow ) and no enhancement of the posterior portion ( arrowhead ) frequency distribution of non - enhanced mr imaging features signal intensity was compared with that of extraocular muscle frequency distribution of contrast - enhanced mr imaging features orbital cavernous haemangiomas were always a well - defined , ovoid , intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging .", "schwannomas often demonstrated a heterogeneously isointense or mildly hyperintense mass in the intraconal or extraconal space .", "after gadolinium administration , all 43 cavernous haemangiomas showed enhancement starting from a small point or portion and contrast media filling up the tumour later , i.e.  progressive  enhancement ( fig .  1 ) .", "compared with the cavernous haemangiomas , all 16 schwannomas showed enhancement starting from a wide area and heterogeneous or homogeneous enhancement later ( fig .  2 ) .", "orbital cavernous haemangiomas , the most common primary orbital tumour in adults , demonstrate a predilection to affect middle - aged women ( 6070% ) , with a mean age of 43 to 48  years and range of 18 to 72  years based on several large series [ 4 , 7 , 9 ] .", "the most common presenting sign and symptom of the orbital cavernous haemangioma is painless proptosis . in the past", ", orbital cavernous haemangiomas were invariably resected because they could not be conclusively discerned from other tumours clinically or radiologically .", "however , orbital cavernous haemangiomas have recently been classified as type 3 low - flow arteriovenous malformations with a direct arterial in - flow and venous out - flow mechanism ( arterial low - flow type ) rather than as true neoplasms or proliferating hamartomas , and , to our knowledge , there is no evidence of malignant transformation in the literature .", "moreover , ct and mr imaging , especially mr imaging , have evolved to deliver high sensitivity and specificity in diagnosing orbital cavernous haemangiomas and distinguishing cavernous haemangiomas from other tumours , which have played a vital role in accurate diagnosis and correct treatment planning [ 5 , 7 , 12 , 14 , 16 ] .", "therefore , recent literature supports observation as an alternative to surgical excision for asymptomatic orbital cavernous haemangiomas and surgery can be withheld until vision is threatened or cosmetic appearance resulting from proptosis is unacceptable [ 3 , 9 ] .", "orbital schwannomas , constituting 1% of all orbital tumours , are benign peripheral nerve sheath tumours originating purely from schwann cells and usually seen in young to middle - aged adults and rarely in children .", "clinical findings of orbital schwannomas are generally similar to those of cavernous haemangiomas , but they are somewhat more likely to exhibit progressive growth and symptoms .", "thus , surgical excision is the treatment of choice for orbital schwannomas , different from orbital cavernous haemangiomas [ 3 , 79 ] .", "our results demonstrate that , in presence of a well - defined , ovoid , intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging , one small point or portion of enhancement initially noted after administration of contrast medium and  progressive  enhancement on later enhancement images , favours the diagnosis of cavernous haemangioma instead of schwannoma .", "most orbital cavernous haemangiomas are identified in the intraconal space , predominantly in the lateral aspect of the intraconal space [ 7 , 9 ] . in our series ,", "only 8 of the 43 orbital cavernous haemangiomas were in the extraconal space . however , orbital schwannomas more commonly arise from sensory nerves in the orbit , specifically the supraorbital , supratrochlear and lacrimal nerves , and thus are usually outside the orbital muscle cone .", "the nasociliary nerve is the only division of the ophthalmic nerve within the annular tendon .", "therefore , orbital schwannomas can be seen as an intraconal or extraconal mass . in our series ,", "the frequency of the schwannoma being located in the extraconal or intraconal space is almost the same .", "orbital cavernous haemangiomas appear isointense to muscle on t1-weighted imaging and hyperintense to muscle on t2-weighted imaging [ 5 , 7 ] .", "although non - contrast mr imaging findings are non - specific , they may offer some advantage in the differentiation of orbital cavernous haemangiomas by demonstrating marked hyperintensity signal to muscle with internal septations on t2-weighted imaging , a hypointense circumferential rim corresponding to the fibrous pseudocapsule , and chemical shift artefact in the frequency - encoded direction secondary to high water content within the lesion surrounded by orbital fat [ 5 , 7 , 17 ] .", "orbital schwannoma is isointense with respect to the extraocular muscle on t1-weighted images and mildly hyperintense on t2-weighted images [ 6 , 18 ] .", "orbital schwannomas may undergo cavitary change , which appears as a cystic mass with straw - coloured fluid on gross pathological examination and looks homogeneously hypointense to muscle on t1-weighted images and hyperintense on t2-weighted images [ 8 , 19 ] .", "consequently , markedly homogeneous hyperintensity signal on t2-weighted imaging is one of the reliable findings distinguishing the two tumours .", "variable degrees of contrast enhancement observed in orbital cavernous haemangiomas were reported , probably related to variable imaging times after contrast medium infusion .", "however , multiphase dynamic contrast ct and mri demonstrated virtually pathognomonic imaging features of orbital cavernous haemangiomas [ 5 , 7 , 17 ] . on early imaging after the injection , one small point or portion of enhancement is initially noted because of its low - flow arterial supply , and the slow progressive accumulation of contrast medium continues within the dilated vascular spaces filling in centrally during the late venous phase .", "subsequently , homogeneous contrast enhancement occurs and persists in the delayed equilibrium phases [ 7 , 1216 ] .", "this enhancement pattern is referred to as a  progressive  enhancement pattern in the literature [ 10 , 11 , 13 ] .", "several multiphase dynamic contrast mr imaging protocols have been reported for studying orbital cavernous haemangiomas [ 7 , 11 , 12 , 14 ] .", "compared with orbital cavernous haemangiomas , no  progressive  enhancement pattern was found in orbital schwannomas [ 12 , 18 ] . therefore , our results and the literature showed that the  progressive  enhancement pattern will be the most reliable finding in distinguishing orbital cavernous haemangiomas from schwannomas .", "some reports in the literature indicated that orbital cavernous haemangiomas showed an initial central point or portion enhancement after administration of gadopentetate dimeglumine [ 7 , 1214 ] .", "however , our results show that enhancement starts from one peripheral point or portion of the tumour in most orbital cavernous haemangiomas .", "the initial enhancement point represents the connecting point of feeding vessels to the lesion , which is useful for estimating the connecting point of the feeding vessels .", "an unexpected result is that nearly half of orbital cavernous haemangiomas demonstrate a wash - out tic type frequently seen in the malignant tumour rather than a plateau - shaped or persistent tic type .", "this may be because contrast medium fills one portion of the tumour during an early phase after administration of contrast medium and contrast medium diffuses into adjacent spaces through the foramen of the fibrous interstitium between spaces at a later phase [ 1214 ] .", "thus it results in a decrease in concentration of contrast media in the portion of the tumour filled by contrast media during the early phase .", "there is a significant difference in tic type between orbital cavernous haemangiomas and schwannomas in contrast to a previous report that there is no significant difference in tic type between the two tumours .", "this is probably because we chose an roi covering the greatest degree of early enhancement rather than the whole tumour .", "recent literature on dynamic mr imaging in the breast , soft tissue and orbital masses indicates that an roi covering the greatest degree of early enhancement of the mass reflects haemodynamic alterations of the tumour better than an roi covering the whole tumour [ 10 , 21 , 22 ] .", "the multiphase dynamic contrast mr imaging findings of orbital cavernous haemangiomas have been described in the literature [ 1014 ] . however , our results provided additional information on dynamic contrast - enhanced mr imaging findings of orbital cavernous haemangiomas .", "first , short imaging time and interval of every phase on dynamic contrast mr imaging may accurately show one point or small portion of enhancement of the mass on early phase images after the injection of contrast medium and allow characterization of specific spread pattern of the contrast enhancement in small orbital cavernous haemangiomas , which can differentiate small orbital cavernous haemangiomas from small schwannomas and other tumours .", "second , tic type of orbital cavernous haemangioma is significantly different from that of schwannoma .", "previous literature showed small haemangiomas could not be differentiated from haemangiopericytomas as their enhancement appears early and strongly and calcification could be noticed in cavernous haemangiomas , contributing to the differential diagnosis [ 5 , 7 ] .", "however , calcification is occasionally seen in cavernous haemangiomas and plays a limited role in the differential diagnosis .", "our results revealed that strong enhancement starting from a wide area and rapid wash - out on dce mr imaging were seen in haemangiopericytomas that might be helpful in the differential diagnosis .", "capillary haemangiomas are a different sort of haemangiomatous tumour that are more common in children , often regress and have a different imaging appearance [ 5 , 7 ] .", "cavernous haemangiomas and schwannomas have different mr imaging features including the location , configuration and margins of the mass , signal intensity and homogeneity on t1- and t2-weighted imaging , the spread pattern of contrast enhancement , the enhancement pattern and tic type , which could be helpful in the differentiation of the two tumours .", "the spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is the most reliable finding distinguishing cavernous haemangiomas and schwannomas .", "markedly homogeneous hyperintensity signal on t2-weighted imaging is another reliable finding distinguishing the two tumours .", "mr imaging , especially dynamic contrast - enhanced mr imaging , plays a vital role in the differential diagnosis of cavernous haemangiomas and schwannomas ."], "abstract_text": ["<S> objectiveit is important to distinguish between orbital cavernous haemangioma and schwannoma because the treatments of choice for the two tumours are different . </S>", "<S> the aim was to evaluate mr imaging findings distinguishing the two tumours.methodsmagnetic resonance imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathology . </S>", "<S> location , configuration , margins , signal intensity , homogeneity and enhancement pattern of the tumour were retrospectively evaluated.resultsthere was a significant difference between cavernous haemangiomas and schwannomas regarding the location , configuration and margins of the mass , signal intensity and homogeneity on t1- and t2-weighted imaging , the spread pattern of contrast enhancement , the enhancement pattern and the type of time  </S>", "<S> intensity curve ( p  <  0.05 ) . </S>", "<S> markedly homogeneous hyperintensity signal on t2-weighted imaging and the spread pattern of the contrast enhancement favoured cavernous haemangioma rather than schwannoma ( p  <  0.01).conclusioncavernous haemangiomas and schwannomas have different mr imaging features that could be helpful in the differentiation between the tumours . </S>", "<S> the spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is the most reliable finding distinguishing cavernous haemangiomas from schwannomas . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Patient population", "MR imaging", "Image interpretation", "Statistical analysis", "Results", "Discussion", "Conclusion"], "sections": [["cavernous haemangioma is the most common primary orbital tumour in adults , accounting for 6% of all orbital lesions [ 13 ] .", "orbital schwannomas are benign peripheral nerve sheath tumours and constitute 1% of all orbital tumours [ 1 , 3 ] .", "orbital cavernous haemangiomas and schwannomas present as slow progressive enlargement and the most common presenting sign and symptom is painless proptosis [ 48 ] .", "although both orbital cavernous haemangiomas and schwannomas share many similar clinical and radiological characteristics [ 58 ] , it is important to distinguish between the tumours because they demonstrate important different features and the treatments of choice for the two are different [ 3 , 79 ] .", "magnetic resonance imaging , especially dynamic mr imaging , plays a crucial role in the differentiation of orbital cavernous haemangiomas and schwannomas [ 7 , 8 , 1014 ] .", "the spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is considered the most important finding distinguishing the two tumours [ 1014 ] . however , the literature on mr imaging findings in the differentiation of these two tumours is based on small patient populations .", "consequently , this study aims to assess mr imaging findings distinguishing orbital cavernous haemangiomas from schwannomas and to elaborate on specific differences between the two tumours ."], ["between june 2004 and february 2009 , 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol .", "mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients .", "most patients presented with painless insidious proptosis . some patients demonstrated a limitation of extraocular motion , diplopia and strabismus .", "informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data .", "magnetic resonance imaging was performed on a general electric ( ge ) signa 1.5-t mr imaging system ( ge healthcare , milwaukee , wi , usa ) with an eight - channel head coil . mr imaging was performed with t1-weighted ( repetition time / echo time , 400/15  ms ) and t2-weighted ( 3,000/120 ) fast spin - echo images .", "a 16-cm field of view , a 3-mm - thick section at 0 interval , and a 288    256 matrix with two signals acquired were used .", "dynamic contrast - enhanced ( dce ) mr imaging was performed by using a fast spoiled gradient recalled ( fspgr ) sequence with 8.4/4  ms , a flip angle of 15 , one excitation , a matrix of 256    160 , a field of view of 220    220  mm , and a section thickness of 3.2  mm at 0 interval . a power injector ( medrad , indianola , pa , usa ) with an injection flow rate of 2  ml /", "the acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1  mmol of gadopentetate dimeglumine ( magnevist , schering , berlin , germany ) per kilogram .", "each mr sequence yielded 12 anatomical slices in 13  s ; this was repeated 12 times with an interval between each sequence of 12  s. total imaging time was 288  s ( 4  min 48  s ) .", "dce mr imaging was evaluated by using a ge aw 4.2 workstation ( ge healthcare ) .", "a region of interest ( roi ) was drawn manually on the dynamic images for assessment of the enhancement kinetics .", "we chose the area that demonstrated the greatest degree of early enhancement so that the time  intensity curve ( tic ) could be generated .", "the area of the roi typically was 8  mm . the contrast index ( ci )", "was calculated from : ci  =  [ ( signal intensity post - contrast )    ( signal intensity pre - contrast)]/(signal intensity pre - contrast ) .", "the tics were analysed qualitatively as wash - out , persistent or plateau - shaped curves .", "magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist ( 15  years experience ) .", "location , configuration , margins , signal intensity , homogeneity and enhancement pattern of the tumour were assessed .", "the location was defined as anterior orbit ( anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery ) , posterior orbit ( posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery ) , or both anterior and posterior orbit ; extraconal or intraconal space ; superior or inferior orbit ; and nasal or temporal orbit .", "on contrast - enhanced mr imaging including dce mr imaging , the spread pattern of the contrast enhancement , enhancement pattern , ci and type of tic were evaluated .", "the spread pattern of the contrast enhancement was divided into two groups : one starting from a small point or portion and the other starting from a wide area . ", "progressive  enhancement means that on early imaging after the injection , one small point or portion of enhancement was initially noted , then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement ( fig .  1 ) .", "other patterns of enhancement included gradually heterogeneous enhancement and gradually homogeneous enhancement . \n fig . ", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) .", "f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) a 54-year - old woman with a cavernous haemangioma .", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) . f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests .", "fisher s exact test was performed when the sample size in the subgroups was deemed too small ."], ["between june 2004 and february 2009 , 43 patients with cavernous haemangiomas and 16 patients with schwannomas confirmed by pathological results were enrolled in the protocol .", "mr imaging including t1- and t2-weighted imaging and contrast - enhanced mr imaging was performed in all 59 patients .", "most patients presented with painless insidious proptosis . some patients demonstrated a limitation of extraocular motion , diplopia and strabismus .", "informed consent was obtained from all patients for anonymous performance of radiological studies and analysis of clinical data ."], ["magnetic resonance imaging was performed on a general electric ( ge ) signa 1.5-t mr imaging system ( ge healthcare , milwaukee , wi , usa ) with an eight - channel head coil . mr imaging was performed with t1-weighted ( repetition time / echo time , 400/15  ms ) and t2-weighted ( 3,000/120 ) fast spin - echo images .", "a 16-cm field of view , a 3-mm - thick section at 0 interval , and a 288    256 matrix with two signals acquired were used .", "dynamic contrast - enhanced ( dce ) mr imaging was performed by using a fast spoiled gradient recalled ( fspgr ) sequence with 8.4/4  ms , a flip angle of 15 , one excitation , a matrix of 256    160 , a field of view of 220    220  mm , and a section thickness of 3.2  mm at 0 interval .", "a power injector ( medrad , indianola , pa , usa ) with an injection flow rate of 2  ml / s was used .", "the acquisition of the dynamic images began concurrently with the initiation of the injection of 0.1  mmol of gadopentetate dimeglumine ( magnevist , schering , berlin , germany ) per kilogram .", "each mr sequence yielded 12 anatomical slices in 13  s ; this was repeated 12 times with an interval between each sequence of 12  s. total imaging time was 288  s ( 4  min 48  s ) .", "dce mr imaging was evaluated by using a ge aw 4.2 workstation ( ge healthcare ) .", "a region of interest ( roi ) was drawn manually on the dynamic images for assessment of the enhancement kinetics .", "we chose the area that demonstrated the greatest degree of early enhancement so that the time  intensity curve ( tic ) could be generated .", "the contrast index ( ci ) was calculated from : ci  =  [ ( signal intensity post - contrast )    ( signal intensity pre - contrast)]/(signal intensity pre - contrast ) .", "the tics were analysed qualitatively as wash - out , persistent or plateau - shaped curves ."], ["magnetic resonance imaging features including dce mr imaging were retrospectively evaluated by an experienced head and neck radiologist ( 15  years experience ) .", "location , configuration , margins , signal intensity , homogeneity and enhancement pattern of the tumour were assessed .", "the location was defined as anterior orbit ( anterior orbit is defined as the orbit anterior to the posterior ethmoidal artery ) , posterior orbit ( posterior orbit is defined as the orbit posterior to the posterior ethmoidal artery ) , or both anterior and posterior orbit ; extraconal or intraconal space ; superior or inferior orbit ; and nasal or temporal orbit .", "on contrast - enhanced mr imaging including dce mr imaging , the spread pattern of the contrast enhancement , enhancement pattern , ci and type of tic were evaluated .", "the spread pattern of the contrast enhancement was divided into two groups : one starting from a small point or portion and the other starting from a wide area . ", "progressive  enhancement means that on early imaging after the injection , one small point or portion of enhancement was initially noted , then contrast media progressively filled up the tumour and eventually the mass showed total and homogeneous enhancement ( fig .  1 ) .", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) .", "f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead ) a 54-year - old woman with a cavernous haemangioma .", "b the tumour appeared hyperintense ( arrowhead ) relative to muscle on axial t2-weighted imaging .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine , showed enhancement starting from one point of peripheral portion of the tumour ( arrowhead ) .", "f post - contrast t1-weighted imaging with fat saturation showed further enlargement of enhancement regions ( arrowhead )"], ["frequency distribution of individual mr imaging features in cavernous haemangiomas was compared with that in schwannomas with chi - squared tests .", "fisher s exact test was performed when the sample size in the subgroups was deemed too small ."], ["tables  1 and 2 describe the frequency distribution of non - enhanced mr imaging features ( table  1 ) and contrast - enhanced mr imaging features ( table  2 ) .", "there was a significant difference between cavernous haemangiomas and schwannomas regarding the location ( including anterior orbit , posterior orbit , or both anterior and posterior orbit ; extraconal or intraconal space ) , configuration and margins of the mass , signal intensity and homogeneity on t1- and t2-weighted imaging , the spread pattern of the contrast enhancement , enhancement pattern and type of time ", "intensity curve ( tic ) ( p  <  0.05 ) ( figs .  1 , 2 , 3 ) . \n", "b axial t2-weighted imaging revealed that the tumour was isointense relative to muscle ( arrowhead ) .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour ( arrowhead ) .", "f axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour ( arrowhead)fig . ", "a obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour ( arrow ) and slight hypointensity of the posterior portion ( arrowhead ) relative to muscle .", "b axial t2-weighted imaging showed isointensity of the anterior portion of the tumour ( arrow ) and hyperintensity of the posterior portion ( arrowhead ) relative to muscle .", "c obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour ( arrow ) and no enhancement of the posterior portion ( arrowhead)table  1frequency distribution of non - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no . of patients594316anterior or posterior orbit0.004  anterior4779.63990.8850  posterior610.224.6425  both610.224.6425extraconal or intraconal space0.049  intraconal4474.63581.4956.2  extraconal1525.4818.6743.8nasal or temporal orbit0.250  nasal2644.11739.5956.2  temporal3355.92660.5743.8superior or inferior orbit0.937  superior2949.22148.8850  inferior3050.82251.2850configuration0.015  round610.249.3212.6  ovoid5084.73990.71168.7  irregular35.100318.7margin0.531  smooth4474.63376.71168.7  lobulated1525.41023.3531.3t1 signal intensity<0.001  hypointense711.900743.7  isointense5288.143100956.3t1 homogeneity<0.001  homogeneous5084.743100743.7  heterogeneous915.300956.3t2 signal intensity0.001  isointense58.500531.3  hyperintense5491.5431001168.7t2 homogeneity<0.001  homogeneous467843100318.7  heterogeneous1322001381.3signal intensity was compared with that of extraocular muscletable  2frequency distribution of contrast - enhanced mr imaging featuresmr imaging featuretype of tumourp valueoverallhaemangiomaschwannoman%n%n%no .", "of patients594316spread pattern of contrast enhancement<0.001  starting from a small point or portion4372.94310000  starting from a wide area1627.10016100enhancement pattern<0.001  homogeneous or heterogeneous1627.10016100  progressive4372.94310000ci1.8    0.61.4    0.30.003tic type<0.001  persistent1220.337956.2  plateau - shaped2440.71944.2531.3  wash - out23392148.8212.50 a 42-year - old man with a schwannoma .", "b axial t2-weighted imaging revealed that the tumour was isointense relative to muscle ( arrowhead ) .", "c  e dynamic mr images obtained at 25 , 75 and 225  s , respectively , after administration of gadopentetate dimeglumine showed enhancement starting from wide areas of tumour ( arrowhead ) . f axial post - contrast t1-weighted imaging with fat saturation showed homogeneous enhancement of the tumour ( arrowhead ) a 47-year - old woman with schwannoma .", "a obliquely sagittal t1-weighted imaging identified an irregular extraconal tumour with isointensity of the anterior portion of the tumour ( arrow ) and slight hypointensity of the posterior portion ( arrowhead ) relative to muscle .", "b axial t2-weighted imaging showed isointensity of the anterior portion of the tumour ( arrow ) and hyperintensity of the posterior portion ( arrowhead ) relative to muscle .", "c obliquely sagittal post - contrast t1-weighted imaging demonstrated enhancement of the anterior portion of the tumour ( arrow ) and no enhancement of the posterior portion ( arrowhead ) frequency distribution of non - enhanced mr imaging features signal intensity was compared with that of extraocular muscle frequency distribution of contrast - enhanced mr imaging features orbital cavernous haemangiomas were always a well - defined , ovoid , intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging .", "schwannomas often demonstrated a heterogeneously isointense or mildly hyperintense mass in the intraconal or extraconal space .", "after gadolinium administration , all 43 cavernous haemangiomas showed enhancement starting from a small point or portion and contrast media filling up the tumour later , i.e.  progressive  enhancement ( fig .  1 ) .", "compared with the cavernous haemangiomas , all 16 schwannomas showed enhancement starting from a wide area and heterogeneous or homogeneous enhancement later ( fig .  2 ) ."], ["orbital cavernous haemangiomas , the most common primary orbital tumour in adults , demonstrate a predilection to affect middle - aged women ( 6070% ) , with a mean age of 43 to 48  years and range of 18 to 72  years based on several large series [ 4 , 7 , 9 ] .", "the most common presenting sign and symptom of the orbital cavernous haemangioma is painless proptosis . in the past", ", orbital cavernous haemangiomas were invariably resected because they could not be conclusively discerned from other tumours clinically or radiologically .", "however , orbital cavernous haemangiomas have recently been classified as type 3 low - flow arteriovenous malformations with a direct arterial in - flow and venous out - flow mechanism ( arterial low - flow type ) rather than as true neoplasms or proliferating hamartomas , and , to our knowledge , there is no evidence of malignant transformation in the literature .", "moreover , ct and mr imaging , especially mr imaging , have evolved to deliver high sensitivity and specificity in diagnosing orbital cavernous haemangiomas and distinguishing cavernous haemangiomas from other tumours , which have played a vital role in accurate diagnosis and correct treatment planning [ 5 , 7 , 12 , 14 , 16 ] .", "therefore , recent literature supports observation as an alternative to surgical excision for asymptomatic orbital cavernous haemangiomas and surgery can be withheld until vision is threatened or cosmetic appearance resulting from proptosis is unacceptable [ 3 , 9 ] .", "orbital schwannomas , constituting 1% of all orbital tumours , are benign peripheral nerve sheath tumours originating purely from schwann cells and usually seen in young to middle - aged adults and rarely in children .", "clinical findings of orbital schwannomas are generally similar to those of cavernous haemangiomas , but they are somewhat more likely to exhibit progressive growth and symptoms .", "thus , surgical excision is the treatment of choice for orbital schwannomas , different from orbital cavernous haemangiomas [ 3 , 79 ] .", "our results demonstrate that , in presence of a well - defined , ovoid , intraconal mass with markedly homogeneous hyperintensity signal on t2-weighted imaging , one small point or portion of enhancement initially noted after administration of contrast medium and  progressive  enhancement on later enhancement images , favours the diagnosis of cavernous haemangioma instead of schwannoma .", "most orbital cavernous haemangiomas are identified in the intraconal space , predominantly in the lateral aspect of the intraconal space [ 7 , 9 ] . in our series ,", "only 8 of the 43 orbital cavernous haemangiomas were in the extraconal space . however , orbital schwannomas more commonly arise from sensory nerves in the orbit , specifically the supraorbital , supratrochlear and lacrimal nerves , and thus are usually outside the orbital muscle cone .", "the nasociliary nerve is the only division of the ophthalmic nerve within the annular tendon .", "therefore , orbital schwannomas can be seen as an intraconal or extraconal mass . in our series ,", "the frequency of the schwannoma being located in the extraconal or intraconal space is almost the same .", "orbital cavernous haemangiomas appear isointense to muscle on t1-weighted imaging and hyperintense to muscle on t2-weighted imaging [ 5 , 7 ] .", "although non - contrast mr imaging findings are non - specific , they may offer some advantage in the differentiation of orbital cavernous haemangiomas by demonstrating marked hyperintensity signal to muscle with internal septations on t2-weighted imaging , a hypointense circumferential rim corresponding to the fibrous pseudocapsule , and chemical shift artefact in the frequency - encoded direction secondary to high water content within the lesion surrounded by orbital fat [ 5 , 7 , 17 ] .", "orbital schwannoma is isointense with respect to the extraocular muscle on t1-weighted images and mildly hyperintense on t2-weighted images [ 6 , 18 ] .", "orbital schwannomas may undergo cavitary change , which appears as a cystic mass with straw - coloured fluid on gross pathological examination and looks homogeneously hypointense to muscle on t1-weighted images and hyperintense on t2-weighted images [ 8 , 19 ] .", "consequently , markedly homogeneous hyperintensity signal on t2-weighted imaging is one of the reliable findings distinguishing the two tumours .", "variable degrees of contrast enhancement observed in orbital cavernous haemangiomas were reported , probably related to variable imaging times after contrast medium infusion .", "however , multiphase dynamic contrast ct and mri demonstrated virtually pathognomonic imaging features of orbital cavernous haemangiomas [ 5 , 7 , 17 ] . on early imaging after the injection , one small point or portion of enhancement is initially noted because of its low - flow arterial supply , and the slow progressive accumulation of contrast medium continues within the dilated vascular spaces filling in centrally during the late venous phase .", "subsequently , homogeneous contrast enhancement occurs and persists in the delayed equilibrium phases [ 7 , 1216 ] .", "this enhancement pattern is referred to as a  progressive  enhancement pattern in the literature [ 10 , 11 , 13 ] .", "several multiphase dynamic contrast mr imaging protocols have been reported for studying orbital cavernous haemangiomas [ 7 , 11 , 12 , 14 ] .", "compared with orbital cavernous haemangiomas , no  progressive  enhancement pattern was found in orbital schwannomas [ 12 , 18 ] . therefore , our results and the literature showed that the  progressive  enhancement pattern will be the most reliable finding in distinguishing orbital cavernous haemangiomas from schwannomas .", "some reports in the literature indicated that orbital cavernous haemangiomas showed an initial central point or portion enhancement after administration of gadopentetate dimeglumine [ 7 , 1214 ] .", "however , our results show that enhancement starts from one peripheral point or portion of the tumour in most orbital cavernous haemangiomas .", "the initial enhancement point represents the connecting point of feeding vessels to the lesion , which is useful for estimating the connecting point of the feeding vessels .", "an unexpected result is that nearly half of orbital cavernous haemangiomas demonstrate a wash - out tic type frequently seen in the malignant tumour rather than a plateau - shaped or persistent tic type .", "this may be because contrast medium fills one portion of the tumour during an early phase after administration of contrast medium and contrast medium diffuses into adjacent spaces through the foramen of the fibrous interstitium between spaces at a later phase [ 1214 ] .", "thus it results in a decrease in concentration of contrast media in the portion of the tumour filled by contrast media during the early phase .", "there is a significant difference in tic type between orbital cavernous haemangiomas and schwannomas in contrast to a previous report that there is no significant difference in tic type between the two tumours .", "this is probably because we chose an roi covering the greatest degree of early enhancement rather than the whole tumour .", "recent literature on dynamic mr imaging in the breast , soft tissue and orbital masses indicates that an roi covering the greatest degree of early enhancement of the mass reflects haemodynamic alterations of the tumour better than an roi covering the whole tumour [ 10 , 21 , 22 ] .", "the multiphase dynamic contrast mr imaging findings of orbital cavernous haemangiomas have been described in the literature [ 1014 ] . however , our results provided additional information on dynamic contrast - enhanced mr imaging findings of orbital cavernous haemangiomas .", "first , short imaging time and interval of every phase on dynamic contrast mr imaging may accurately show one point or small portion of enhancement of the mass on early phase images after the injection of contrast medium and allow characterization of specific spread pattern of the contrast enhancement in small orbital cavernous haemangiomas , which can differentiate small orbital cavernous haemangiomas from small schwannomas and other tumours .", "second , tic type of orbital cavernous haemangioma is significantly different from that of schwannoma .", "previous literature showed small haemangiomas could not be differentiated from haemangiopericytomas as their enhancement appears early and strongly and calcification could be noticed in cavernous haemangiomas , contributing to the differential diagnosis [ 5 , 7 ] .", "however , calcification is occasionally seen in cavernous haemangiomas and plays a limited role in the differential diagnosis .", "our results revealed that strong enhancement starting from a wide area and rapid wash - out on dce mr imaging were seen in haemangiopericytomas that might be helpful in the differential diagnosis .", "capillary haemangiomas are a different sort of haemangiomatous tumour that are more common in children , often regress and have a different imaging appearance [ 5 , 7 ] ."], ["cavernous haemangiomas and schwannomas have different mr imaging features including the location , configuration and margins of the mass , signal intensity and homogeneity on t1- and t2-weighted imaging , the spread pattern of contrast enhancement , the enhancement pattern and tic type , which could be helpful in the differentiation of the two tumours .", "the spread pattern of the contrast enhancement on dynamic contrast - enhanced mr imaging is the most reliable finding distinguishing cavernous haemangiomas and schwannomas .", "markedly homogeneous hyperintensity signal on t2-weighted imaging is another reliable finding distinguishing the two tumours .", "mr imaging , especially dynamic contrast - enhanced mr imaging , plays a vital role in the differential diagnosis of cavernous haemangiomas and schwannomas ."]]}
{"article_id": "PMC4906879", "article_text": ["gingival enlargement is a common observation in clinical practice that may occur as a result of a response to varied stimuli and/or interactions with the host and the environment .", "these lesions could be either localized to certain aspect of the oral cavity or generalized affecting larger areas .", "the possible reasons for this condition may be related to plaque , hormonal imbalances , or systemic - induced manifestation .", "these excessive gingival distensions may adversely affect speech , mastication , tooth eruption , esthetics along with major hindrance to the maintenance of routine oral hygiene .", "such cases should be treated in a methodical manner , involving a detailed medical history followed by conventional nonsurgical therapy .", "however , in order to maintain successful therapeutic outcome , it is crucial to create patient awareness and motivation , along with a timely recall visits .", "this case series describes three different conditions of localized gingival overgrowths and its management with prime emphasis on the importance of patient compliance .", "neoplasms are characterized by progressive autonomous growth that can be either a benign or a malignant course , whereas nonneoplastic lesions are usually inflammatory or represent a reaction to some kind of irritation or low - grade injury .", "most of these lesions have similar clinical findings such as sessile or pedunculated nodule located at the interdental papilla with color variations from pale pink to erythematous .", "pyogenic granuloma ( pg ) may occur in all ages and is predominant in the second decade of life in females .", "it is a common nonneoplastic , exophytic vascular growth often associated with the history of trauma or chronic irritation .", "the present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year .", "on detailed conversation with the patient , he revealed an occasional habit of smoking since 2 years with no contributory medical history .", "clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine , measuring 12 mm  15 mm with an interproximal lingual extension [ figure 1a and b ] .", "the color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft . in general ,", "early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent , whereas mature lesions tend to become firm due to the presence of more collagen network .", "there was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity .", "an intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 .", "hence , based on the location and clinical findings , a provisional diagnosis of pg was assigned .", "a treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation .", "oral hygiene instructions were given and the use of chlorhexidine mouthwash ( 0.2% clohex , dr .", "hyderabad , telangana , india ) twice a day for 1 week was advised . at the follow - up interval ,", ". a differential diagnosis of pg , peripheral fibroma , and hyperplastic gingival overgrowth was suggested .", "hence , an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence .", "the surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days .", "case 1 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43 , ( b ) lingual extension of overgrowth , ( c ) histopathological picture in confirmation with pyogenic granuloma , and ( d ) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification .", "the stained section showed parakeratinized stratified squamous epithelium , containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion .", "the underlying connective tissue stroma was delicate to dense and composed of fibroblasts , fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells , which is considered a hallmark of pg .", "there was moderate , focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [ figure 1c ] .", "the patient was recalled after a week and healing was found to be satisfactory [ figure 1d ] . correlating the clinical findings with the histological features , a diagnosis of pg", "the patient is still on follow - up since 6 months with no evidence of recurrence .", "however , literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias .", "peripheral ossifying fibroma ( pof ) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . a common gingival growth , it is typically seen on the interdental papilla and is believed to comprise about", "females are more commonly affected , and anterior maxilla is the most prevalent location of involvement .", "the etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque , calculus , microorganisms , masticatory forces , ill - fitting dentures , and poor - quality restorations .", "following the elimination of causative factors , excision of the lesion is the choice of treatment .", "the present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back .", "intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [ figure 2a and b ] .", "a tentative diagnosis of fibroma was made and patient was informed about the treatment plan .", "the patient was advised on the importance of oral care , which included brushing technique and prescription of chlorhexidine mouthwash ( 0.2% clohex , dr .", "india ) was advised twice daily so as to prevent further disease progression and was recalled after a week .", "the results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . at some areas", ", stroma appeared to be dense with ossifying areas assembled as bony trabeculae [ figure 2c ] .", "thus , based on clinical and histopathological findings , the final diagnosis was determined as pof .", "case 2 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27 , ( b ) palatal extension of overgrowth , ( c ) histopathological picture in confirmation with peripheral ossifying fibroma , and ( d ) follow - up at 2 years as reported in literature , the average time interval for recurrence is within a 12 months period .", "however , in this present case , no recurrence was seen even at 2 years follow - up [ figure 2d ] .", "gingival enlargement is a known adverse effect of calcium channel blockers , especially the dihydropyridine group .", "it is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . among the calcium channel blockers ,", "gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine ; however , the incidence with amlodipine is rare . in the present case , a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums , which had been progressively increasing in size over the previous 6 months .", "the disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene .", "the patient 's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years .", "intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [ figure 3a ] .", "the enlargement was pink in color , nodular , and slightly edematous with no surface ulcerations .", "a prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm  15 mm , soft on palpation , and bleeding on provocation present [ figure 3b ] .", "case 3 ( a ) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors , ( b ) isolated overgrowth wrt 42 and 43 , ( c ) 4 months post nonsurgical therapy , ( d ) residual isolated overgrowth wrt 42 and 43 , and ( e ) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor .", "patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits .", "she was referred to a physician for drug substitution who replaced amlodipine with atenolol , a beta - blocker .", "a full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling .", "the patient had revealed satisfactory results and the gingival tissues had shown marked regression [ figure 3c ] .", "the sessile growth revealed a reduction in size to approximately 7 mm  9 mm [ figure 3d ] .", "differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement , combined gingival enlargement , peripheral fibroma , peripheral giant cell granuloma , pg , and peripheral fibroma .", "hence , an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis .", "the nodular lesion along with surrounding tissues was excised for a complete histologic analysis [ figure 3e ] .", "the lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [ figure 3e ( i ) ] , elongated rete pegs extending deep into connective tissue [ figure 3e ( ii ) ] that exhibited densely arranged collagen fibers and new blood vessels [ figure 3e ( iii ) ] suggestive of drug - induced gingival enlargement .", "the other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [ figure 3e ( iv ) ] .", "connective tissue stroma was edematous , composed of delicate to dense collagen fibers , proliferating blood vessels , and markedly infiltrated by acute and chronic inflammatory cells [ figure 3e ( v ) ] that are seen in pg .", "thus , the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg .", "previously , a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported ; however , there has not been any report of amlodipine - induced gingival enlargement associated with pg , to the author 's knowledge .", "pyogenic granuloma ( pg ) may occur in all ages and is predominant in the second decade of life in females .", "it is a common nonneoplastic , exophytic vascular growth often associated with the history of trauma or chronic irritation .", "the present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year .", "on detailed conversation with the patient , he revealed an occasional habit of smoking since 2 years with no contributory medical history .", "clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine , measuring 12 mm  15 mm with an interproximal lingual extension [ figure 1a and b ] .", "the color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft .", "in general , early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent , whereas mature lesions tend to become firm due to the presence of more collagen network .", "there was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity .", "an intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 .", "hence , based on the location and clinical findings , a provisional diagnosis of pg was assigned .", "a treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation .", "oral hygiene instructions were given and the use of chlorhexidine mouthwash ( 0.2% clohex , dr .", "hyderabad , telangana , india ) twice a day for 1 week was advised . at the follow - up interval ,", ". a differential diagnosis of pg , peripheral fibroma , and hyperplastic gingival overgrowth was suggested .", "hence , an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence .", "the surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days .", "case 1 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43 , ( b ) lingual extension of overgrowth , ( c ) histopathological picture in confirmation with pyogenic granuloma , and ( d ) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification .", "the stained section showed parakeratinized stratified squamous epithelium , containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion .", "the underlying connective tissue stroma was delicate to dense and composed of fibroblasts , fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells , which is considered a hallmark of pg .", "there was moderate , focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [ figure 1c ] .", "the patient was recalled after a week and healing was found to be satisfactory [ figure 1d ] . correlating the clinical findings with the histological features , a diagnosis of pg", "the patient is still on follow - up since 6 months with no evidence of recurrence .", "however , literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias .", "peripheral ossifying fibroma ( pof ) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . a common gingival growth , it is typically seen on the interdental papilla and is believed to comprise about", "females are more commonly affected , and anterior maxilla is the most prevalent location of involvement .", "the etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque , calculus , microorganisms , masticatory forces , ill - fitting dentures , and poor - quality restorations . following", "the elimination of causative factors , excision of the lesion is the choice of treatment .", "the present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back .", "intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [ figure 2a and b ] .", "a tentative diagnosis of fibroma was made and patient was informed about the treatment plan .", "the patient was advised on the importance of oral care , which included brushing technique and prescription of chlorhexidine mouthwash ( 0.2% clohex , dr .", "india ) was advised twice daily so as to prevent further disease progression and was recalled after a week .", "the results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . at some areas", ", stroma appeared to be dense with ossifying areas assembled as bony trabeculae [ figure 2c ] .", "thus , based on clinical and histopathological findings , the final diagnosis was determined as pof .", "case 2 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27 , ( b ) palatal extension of overgrowth , ( c ) histopathological picture in confirmation with peripheral ossifying fibroma , and ( d ) follow - up at 2 years as reported in literature , the average time interval for recurrence is within a 12 months period .", "however , in this present case , no recurrence was seen even at 2 years follow - up [ figure 2d ] .", "gingival enlargement is a known adverse effect of calcium channel blockers , especially the dihydropyridine group .", "it is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . among the calcium channel blockers ,", "gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine ; however , the incidence with amlodipine is rare . in the present case , a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums , which had been progressively increasing in size over the previous 6 months .", "the disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene .", "the patient 's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years .", "intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [ figure 3a ] .", "the enlargement was pink in color , nodular , and slightly edematous with no surface ulcerations .", "a prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm  15 mm , soft on palpation , and bleeding on provocation present [ figure 3b ] .", "case 3 ( a ) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors , ( b ) isolated overgrowth wrt 42 and 43 , ( c ) 4 months post nonsurgical therapy , ( d ) residual isolated overgrowth wrt 42 and 43 , and ( e ) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor . a provisional diagnosis of combined gingival enlargement", "patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits .", "she was referred to a physician for drug substitution who replaced amlodipine with atenolol , a beta - blocker .", "a full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling .", "the patient had revealed satisfactory results and the gingival tissues had shown marked regression [ figure 3c ] .", "the sessile growth revealed a reduction in size to approximately 7 mm  9 mm [ figure 3d ] .", "differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement , combined gingival enlargement , peripheral fibroma , peripheral giant cell granuloma , pg , and peripheral fibroma .", "hence , an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis .", "the nodular lesion along with surrounding tissues was excised for a complete histologic analysis [ figure 3e ] .", "the lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [ figure 3e ( i ) ] , elongated rete pegs extending deep into connective tissue [ figure 3e ( ii ) ] that exhibited densely arranged collagen fibers and new blood vessels [ figure 3e ( iii ) ] suggestive of drug - induced gingival enlargement .", "the other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [ figure 3e ( iv ) ] .", "connective tissue stroma was edematous , composed of delicate to dense collagen fibers , proliferating blood vessels , and markedly infiltrated by acute and chronic inflammatory cells [ figure 3e ( v ) ] that are seen in pg .", "thus , the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg .", "previously , a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported ; however , there has not been any report of amlodipine - induced gingival enlargement associated with pg , to the author 's knowledge .", "it is possible to misdiagnose reactive lesions arising from the gingiva . a detailed case history to determine the etiology and histopathological examination is essential for an accurate diagnosis and for proper management .", "clinical differential diagnosis for localized gingival overgrowths includes fibroma , peripheral giant cell granuloma , pg , peripheral odontogenic fibroma , and peripheral ossifying fibroma . a relatively healthy oral environment provided by the dentist and maintained by", "the patient will reduce local microflora that will help in eliminating the major focus of infection . a critical factor that assists successful therapeutic outcome", "the patient should be placed on a regular dental visit , in which a complete description of the existing dental condition should be informed . creating awareness and educating patients regarding the influence of poor oral hygiene and habits to these gingival lesions are essential to avoid further recurrence ."], "abstract_text": ["<S> clinicians are often intrigued by the varied manifestations of the gingival tissue . </S>", "<S> gingival overgrowth is a common clinical finding and most of them represent a reactive hyperplasia as a direct result of plaque - related inflammatory gingival disease . </S>", "<S> these types of growth generally respond to good plaque control , removal of the causative irritants , and conservative tissue management . </S>", "<S> this case series highlights three different cases of localized gingival overgrowth and its management with emphasis on the importance of patient awareness and motivation . </S>"], "labels": null, "section_names": ["Introduction", "Case Reports", "Case 1: Pyogenic granuloma", "Case 2: Peripheral ossifying fibroma", "Case 3: Pyogenic granuloma in combination with drug-induced gingival enlargement", "Conclusion", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["gingival enlargement is a common observation in clinical practice that may occur as a result of a response to varied stimuli and/or interactions with the host and the environment .", "these lesions could be either localized to certain aspect of the oral cavity or generalized affecting larger areas .", "the possible reasons for this condition may be related to plaque , hormonal imbalances , or systemic - induced manifestation .", "these excessive gingival distensions may adversely affect speech , mastication , tooth eruption , esthetics along with major hindrance to the maintenance of routine oral hygiene .", "such cases should be treated in a methodical manner , involving a detailed medical history followed by conventional nonsurgical therapy .", "however , in order to maintain successful therapeutic outcome , it is crucial to create patient awareness and motivation , along with a timely recall visits .", "this case series describes three different conditions of localized gingival overgrowths and its management with prime emphasis on the importance of patient compliance ."], ["neoplasms are characterized by progressive autonomous growth that can be either a benign or a malignant course , whereas nonneoplastic lesions are usually inflammatory or represent a reaction to some kind of irritation or low - grade injury .", "most of these lesions have similar clinical findings such as sessile or pedunculated nodule located at the interdental papilla with color variations from pale pink to erythematous .", "pyogenic granuloma ( pg ) may occur in all ages and is predominant in the second decade of life in females .", "it is a common nonneoplastic , exophytic vascular growth often associated with the history of trauma or chronic irritation .", "the present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year .", "on detailed conversation with the patient , he revealed an occasional habit of smoking since 2 years with no contributory medical history .", "clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine , measuring 12 mm  15 mm with an interproximal lingual extension [ figure 1a and b ] .", "the color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft . in general ,", "early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent , whereas mature lesions tend to become firm due to the presence of more collagen network .", "there was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity .", "an intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 .", "hence , based on the location and clinical findings , a provisional diagnosis of pg was assigned .", "a treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation .", "oral hygiene instructions were given and the use of chlorhexidine mouthwash ( 0.2% clohex , dr .", "hyderabad , telangana , india ) twice a day for 1 week was advised . at the follow - up interval ,", ". a differential diagnosis of pg , peripheral fibroma , and hyperplastic gingival overgrowth was suggested .", "hence , an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence .", "the surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days .", "case 1 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43 , ( b ) lingual extension of overgrowth , ( c ) histopathological picture in confirmation with pyogenic granuloma , and ( d ) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification .", "the stained section showed parakeratinized stratified squamous epithelium , containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion .", "the underlying connective tissue stroma was delicate to dense and composed of fibroblasts , fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells , which is considered a hallmark of pg .", "there was moderate , focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [ figure 1c ] .", "the patient was recalled after a week and healing was found to be satisfactory [ figure 1d ] . correlating the clinical findings with the histological features , a diagnosis of pg", "the patient is still on follow - up since 6 months with no evidence of recurrence .", "however , literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias .", "peripheral ossifying fibroma ( pof ) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . a common gingival growth , it is typically seen on the interdental papilla and is believed to comprise about", "females are more commonly affected , and anterior maxilla is the most prevalent location of involvement .", "the etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque , calculus , microorganisms , masticatory forces , ill - fitting dentures , and poor - quality restorations .", "following the elimination of causative factors , excision of the lesion is the choice of treatment .", "the present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back .", "intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [ figure 2a and b ] .", "a tentative diagnosis of fibroma was made and patient was informed about the treatment plan .", "the patient was advised on the importance of oral care , which included brushing technique and prescription of chlorhexidine mouthwash ( 0.2% clohex , dr .", "india ) was advised twice daily so as to prevent further disease progression and was recalled after a week .", "the results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . at some areas", ", stroma appeared to be dense with ossifying areas assembled as bony trabeculae [ figure 2c ] .", "thus , based on clinical and histopathological findings , the final diagnosis was determined as pof .", "case 2 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27 , ( b ) palatal extension of overgrowth , ( c ) histopathological picture in confirmation with peripheral ossifying fibroma , and ( d ) follow - up at 2 years as reported in literature , the average time interval for recurrence is within a 12 months period .", "however , in this present case , no recurrence was seen even at 2 years follow - up [ figure 2d ] .", "gingival enlargement is a known adverse effect of calcium channel blockers , especially the dihydropyridine group .", "it is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . among the calcium channel blockers ,", "gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine ; however , the incidence with amlodipine is rare . in the present case , a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums , which had been progressively increasing in size over the previous 6 months .", "the disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene .", "the patient 's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years .", "intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [ figure 3a ] .", "the enlargement was pink in color , nodular , and slightly edematous with no surface ulcerations .", "a prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm  15 mm , soft on palpation , and bleeding on provocation present [ figure 3b ] .", "case 3 ( a ) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors , ( b ) isolated overgrowth wrt 42 and 43 , ( c ) 4 months post nonsurgical therapy , ( d ) residual isolated overgrowth wrt 42 and 43 , and ( e ) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor .", "patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits .", "she was referred to a physician for drug substitution who replaced amlodipine with atenolol , a beta - blocker .", "a full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling .", "the patient had revealed satisfactory results and the gingival tissues had shown marked regression [ figure 3c ] .", "the sessile growth revealed a reduction in size to approximately 7 mm  9 mm [ figure 3d ] .", "differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement , combined gingival enlargement , peripheral fibroma , peripheral giant cell granuloma , pg , and peripheral fibroma .", "hence , an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis .", "the nodular lesion along with surrounding tissues was excised for a complete histologic analysis [ figure 3e ] .", "the lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [ figure 3e ( i ) ] , elongated rete pegs extending deep into connective tissue [ figure 3e ( ii ) ] that exhibited densely arranged collagen fibers and new blood vessels [ figure 3e ( iii ) ] suggestive of drug - induced gingival enlargement .", "the other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [ figure 3e ( iv ) ] .", "connective tissue stroma was edematous , composed of delicate to dense collagen fibers , proliferating blood vessels , and markedly infiltrated by acute and chronic inflammatory cells [ figure 3e ( v ) ] that are seen in pg .", "thus , the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg .", "previously , a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported ; however , there has not been any report of amlodipine - induced gingival enlargement associated with pg , to the author 's knowledge ."], ["pyogenic granuloma ( pg ) may occur in all ages and is predominant in the second decade of life in females .", "it is a common nonneoplastic , exophytic vascular growth often associated with the history of trauma or chronic irritation .", "the present case reports a 23-year - old male patient who reported to the department of periodontology with the chief complaint of growth in the lower right front region of mouth since 1 year .", "on detailed conversation with the patient , he revealed an occasional habit of smoking since 2 years with no contributory medical history .", "clinical exploration revealed a pedunculated growth at the gingival margin with respect to lower right lateral incisor and canine , measuring 12 mm  15 mm with an interproximal lingual extension [ figure 1a and b ] .", "the color of the lesion was pale pink with the consistency being firm toward the coronal two - third whereas the lower one - third was erythematous and soft .", "in general , early stage pgs are highly vascular in appearance because they are composed predominantly of hyperplastic granulation tissue in which capillaries are prominent , whereas mature lesions tend to become firm due to the presence of more collagen network .", "there was a moderate amount of calculus present along coronal and subgingival areas of the oral cavity .", "an intraoral periapical radiograph revealed mild crestal bone loss with respect to 42 and 43 .", "hence , based on the location and clinical findings , a provisional diagnosis of pg was assigned .", "a treatment strategy was planned which included oral prophylaxis followed by review after 2 weeks for resolution of existing inflammation .", "oral hygiene instructions were given and the use of chlorhexidine mouthwash ( 0.2% clohex , dr .", "hyderabad , telangana , india ) twice a day for 1 week was advised . at the follow - up interval ,", ". a differential diagnosis of pg , peripheral fibroma , and hyperplastic gingival overgrowth was suggested .", "hence , an excisional biopsy was performed with attention to surrounding tissues so as to prevent recurrence .", "the surgical site was covered with periodontal dressing and patient was prescribed anti - inflammatory for 3 days .", "case 1 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 42 and 43 , ( b ) lingual extension of overgrowth , ( c ) histopathological picture in confirmation with pyogenic granuloma , and ( d ) follow - up at 2 weeks postexcision the sections were stained using h and e stains and a detailed histopathological picture was obtained under 10 magnification .", "the stained section showed parakeratinized stratified squamous epithelium , containing hyperplastic and edematous connective tissue dispersed at various sites within the lesion .", "the underlying connective tissue stroma was delicate to dense and composed of fibroblasts , fibrocytes with collagen fibers admixed with abundant blood vessels engorged with red blood cells , which is considered a hallmark of pg .", "there was moderate , focal aggregation of chronic inflammatory cells chiefly comprising lymphocytes and plasma cells in few areas [ figure 1c ] .", "the patient was recalled after a week and healing was found to be satisfactory [ figure 1d ] . correlating the clinical findings with the histological features , a diagnosis of pg", "the patient is still on follow - up since 6 months with no evidence of recurrence .", "however , literature has reported about 16% of pg recur after excision in spite of being reactive hyperplasias ."], ["peripheral ossifying fibroma ( pof ) is usually an isolated nonneoplastic growth on gingiva that is categorized as a hyperplastic inflammatory lesion . a common gingival growth , it is typically seen on the interdental papilla and is believed to comprise about", "females are more commonly affected , and anterior maxilla is the most prevalent location of involvement .", "the etiology of pof is uncertain and have been attributed to trauma or local irritants such as plaque , calculus , microorganisms , masticatory forces , ill - fitting dentures , and poor - quality restorations . following", "the elimination of causative factors , excision of the lesion is the choice of treatment .", "the present case was observed in a 28-year - old male who arrived at the department of periodontology with a complaint of swelling of gums in the upper left back teeth which he noticed 1 month back .", "intraoral examination revealed a sessile firm lesion interproximally between 26 and 27 [ figure 2a and b ] .", "a tentative diagnosis of fibroma was made and patient was informed about the treatment plan .", "the patient was advised on the importance of oral care , which included brushing technique and prescription of chlorhexidine mouthwash ( 0.2% clohex , dr .", "india ) was advised twice daily so as to prevent further disease progression and was recalled after a week .", "the results presented a lesion comprised of highly cellular connective tissue stroma comprising of delicate collagen and plump fibroblasts . at some areas", ", stroma appeared to be dense with ossifying areas assembled as bony trabeculae [ figure 2c ] .", "thus , based on clinical and histopathological findings , the final diagnosis was determined as pof .", "case 2 ( a ) preoperative clinical picture depicting gingival overgrowth wrt 26 and 27 , ( b ) palatal extension of overgrowth , ( c ) histopathological picture in confirmation with peripheral ossifying fibroma , and ( d ) follow - up at 2 years as reported in literature , the average time interval for recurrence is within a 12 months period .", "however , in this present case , no recurrence was seen even at 2 years follow - up [ figure 2d ] ."], ["gingival enlargement is a known adverse effect of calcium channel blockers , especially the dihydropyridine group .", "it is a serious concern for both the patient and the clinician due to its unesthetic appearance and formation of new niches for periodontopathogenic bacteria . among the calcium channel blockers ,", "gingival enlargement has most frequently been described as an adverse effect following administration of nifedipine ; however , the incidence with amlodipine is rare . in the present case , a 40-year - old female patient reported with the chief complaint of swelling in the lower front region of gums , which had been progressively increasing in size over the previous 6 months .", "the disfiguring gingival overgrowth was not only esthetically displeasing but also impaired access for oral hygiene .", "the patient 's medical history revealed that she is hypertensive since 8 years and was receiving a single dose of amlodipine 5 mg / day orally since the past 6 years .", "intraoral examination revealed severe gingival enlargement with relation to lower labial anterior gingiva involving marginal and papillary gingival [ figure 3a ] .", "the enlargement was pink in color , nodular , and slightly edematous with no surface ulcerations .", "a prominent nodular growth with a sessile base was also seen with respect to the lower right lateral incisor - canine area on buccal aspect measuring 10 mm  15 mm , soft on palpation , and bleeding on provocation present [ figure 3b ] .", "case 3 ( a ) preoperative clinical picture depicting gingival overgrowth wrt lower anteriors , ( b ) isolated overgrowth wrt 42 and 43 , ( c ) 4 months post nonsurgical therapy , ( d ) residual isolated overgrowth wrt 42 and 43 , and ( e ) histopathological picture in confirmation with drug - induced gingival enlargement associated with pyogenic granuloma the oral hygiene status of the patient was poor . a provisional diagnosis of combined gingival enlargement", "patient was advised on cessation of habit and maintenance of oral hygiene with regular follow - up visits .", "she was referred to a physician for drug substitution who replaced amlodipine with atenolol , a beta - blocker .", "a full mouth oral prophylaxis was carried out and the patient was placed on a regular recall for 3 months for reinforcement of oral hygiene practice and habit counseling .", "the patient had revealed satisfactory results and the gingival tissues had shown marked regression [ figure 3c ] .", "the sessile growth revealed a reduction in size to approximately 7 mm  9 mm [ figure 3d ] .", "differential diagnosis for the nodular overgrowth included chronic inflammatory gingival enlargement , combined gingival enlargement , peripheral fibroma , peripheral giant cell granuloma , pg , and peripheral fibroma .", "hence , an excision was necessary to be performed for maintenance of oral hygiene and to arrive at a definite diagnosis .", "the nodular lesion along with surrounding tissues was excised for a complete histologic analysis [ figure 3e ] .", "the lesion presented interesting results with one - half of the histological picture showing parakeratotic stratified squamous epithelium with acanthosis and intra- and inter - cellular edema of superficial spinous cells in some areas [ figure 3e ( i ) ] , elongated rete pegs extending deep into connective tissue [ figure 3e ( ii ) ] that exhibited densely arranged collagen fibers and new blood vessels [ figure 3e ( iii ) ] suggestive of drug - induced gingival enlargement .", "the other half of the histological picture revealed ulcerations in the epithelium at places that were covered by fibrinous exudate [ figure 3e ( iv ) ] .", "connective tissue stroma was edematous , composed of delicate to dense collagen fibers , proliferating blood vessels , and markedly infiltrated by acute and chronic inflammatory cells [ figure 3e ( v ) ] that are seen in pg .", "thus , the biopsy picture revealed a combination of drug - induced gingival overgrowth associated with pg .", "previously , a case of amlodipine - induced gingival enlargement associated with fibroepithelial hyperplasia has been reported ; however , there has not been any report of amlodipine - induced gingival enlargement associated with pg , to the author 's knowledge ."], ["it is possible to misdiagnose reactive lesions arising from the gingiva . a detailed case history to determine the etiology and histopathological examination is essential for an accurate diagnosis and for proper management .", "clinical differential diagnosis for localized gingival overgrowths includes fibroma , peripheral giant cell granuloma , pg , peripheral odontogenic fibroma , and peripheral ossifying fibroma . a relatively healthy oral environment provided by the dentist and maintained by", "the patient will reduce local microflora that will help in eliminating the major focus of infection . a critical factor that assists successful therapeutic outcome", "the patient should be placed on a regular dental visit , in which a complete description of the existing dental condition should be informed . creating awareness and educating patients regarding the influence of poor oral hygiene and habits to these gingival lesions are essential to avoid further recurrence ."], [""], [""]]}
{"article_id": "PMC3808016", "article_text": ["successful management and treatment of periodontal disease depends upon simple medical procedures such as health education , prophylaxis , scaling and root - planing , gingivectomy , gingival grafts , and different types of gingival flaps .", "periodontal pocket refers to the pathological increase in the depth of gingival sulcus , and is one of the main clinical features of periodontal disease .", "elimination of pathological changes of the pocket wall is among the objectives of surgical treatment for periodontal pockets , so that a stable and sustainable situation can be achieved .", "currently , several flap techniques have been applied for the treatment of pocket , among which modified widman flap is worth mentioning .", "this technique facilitates the use of equipment , in which pocket lining is removed with no effect on reducing the pocket depth .", "dental treatments are accompanied by patients hemodynamic changes ; for instance , increase in blood pressure and heart rate during dental treatment are influenced by various factors such as physical and physiological stress , painful stimuli , and activity of catecholamines present in local anesthetic solutions .", "heart rate , which is indicative of ventricular contraction , changes in different conditions , including pain , anxiety , stress , heart diseases , metabolic and endocrine disorders and heart muscle diseases , and is evaluated by peripheral pulse measurement or the electrocardiogram and pulse oximetry . for the measurement of blood pressure , different methods are applied , from which riva - rocci , korotokoff , dinamap , and finometer techniques can be enumerated . due to the risk of hypoxia in oral surgeries , pulse oximetry", "pulse oximeter is a reliable and sensitive apparatus in detecting small amounts of blood oxygen saturation , and in all patients , spo2 ( spo2 ) normal percentage is not less than 95% .", "a dental patient is exposed to stressors , such as physiological responses to emotional factors and/or pain .", "pain and anxiety , are important stimuli for the secretion of endogenous adrenaline , which plays a significant role in cardiovascular responses during dental treatment .", "local anesthetics mainly affect a limited area ; however , they will be absorbed from the injection site and exert general effects , especially on cardiovascular or central nervous system .", "it is obvious that more the concentration of epinephrine , the better the control of bleeding , but there will be more cardiovascular changes . in spite of the effect of local anesthetics on vital signs , their accurate application is able to dramatically reduce stress . given the above bodies of evidence and the importance of monitoring patients hemodynamic changes during oral surgery , the present study has been conducted to investigate these alterations during periodontal surgery outside the operating room with local anesthesia .", "this practical clinical trial study was carried out on patients with gingival disease referred to the clinic of dental faculty of babol university of medical sciences , and underwent modified widman flap surgery on anterior maxillary region in periodontology department .", "the equipments used in the study included ( 1 ) finger pulse oximeter ( soor afarinesh company ) , and ( 2 ) automatic sphygmomanometer ( omron company ) according to the following inclusion criteria , 50 patients ranging from 30 to 50 years participated in this study . in all patients , changes in heart rate , blood pressure , and also pulse oximetry", "were examined during five steps ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation .", "it should be noted that all the operations were performed by one surgeon , and all patients received two anesthetic cartridges containing lidocaine ( 2% ) and epinephrine ( 1:80000 ) ( darou pakhsh company ) , using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa , in anterior 1/6 region and incisive canal ; no additional anesthetic cartridge was used during surgery , and those in need of re - injection were excluded from the study .", "spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient 's finger during surgery . \n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . \n ", "having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period .", "data were analyzed by spss statistical software using anova repeated measure test , and p < 0.05 was considered as statistically significant level .", "according to the following inclusion criteria , 50 patients ranging from 30 to 50 years participated in this study . in all patients , changes in heart rate , blood pressure , and also pulse oximetry", "were examined during five steps ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation .", "it should be noted that all the operations were performed by one surgeon , and all patients received two anesthetic cartridges containing lidocaine ( 2% ) and epinephrine ( 1:80000 ) ( darou pakhsh company ) , using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa , in anterior 1/6 region and incisive canal ; no additional anesthetic cartridge was used during surgery , and those in need of re - injection were excluded from the study .", "spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient 's finger during surgery .", "\n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . \n  having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period .", "data were analyzed by spss statistical software using anova repeated measure test , and p < 0.05 was considered as statistically significant level .", "the present research has been implemented on 50 patients referring to periodontology department of dental faculty for periodontal surgery . among the study participants with the mean age of 42.58", " 7.14 years , ranging from 30 to 55 years old , there were 22 men and 28 women . in all cases , systolic and diastolic blood pressure , heart rate , and", "spo2 were evaluated during five stages ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation . the mean systolic blood pressure was highest in the second stage and lowest in the first one . in the five steps studied ,", "there was a significant difference between the first step and the second ( p < 0.0001 ) and the third ( p = 0.001 ) , the second step and the first ( p = 0.001 ) and the fourth and the fifth ( p < 0.0001 ) , the third step and the first ( p = 0.001 ) and the fourth ( p = 0.010 ) , the fourth step and the second ( p < 0.0001 ) and the third ( p = 0.010 ) , and also the fifth step and the second ( p < 0.0001 ) [ table 1 and figure 1 ] .", "comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery the maximum and the minimum diastolic blood pressures were , respectively , obtained in the second and the first stage .", "statistical analysis showed significant difference between the first stage and the second ( p = 0.001 ) , the second stage and the first ( p = 0.001 ) and the third ( p < 0.0001 ) and the fourth ( p = 0.003 ) , the third stage and the second ( p < 0.0001 ) , and the fourth step and the second ( p = 0.003 ) ; however , no meaningful relationship was found between the fifth step and the other stages [ table 2 and figure 2 ] .", "comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery according to statistical analysis , the maximum and the minimum heart rate was respectively observed in the second and the fifth stage .", "significant differences were found between the first step and the second and the third ( p < 0.0001 ) , the second and the third steps and the other stages , the fourth step and the second and the third ( p < 0.0001 ) and the fifth ( p = 0.001 ) , and also the fifth step with the second and the third ( p < 0.0001 ) and the fourth ( p = 0.001 ) steps [ table 3 and figure 3 ] .", "comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery similarly , based on statistical analysis , no significant difference has been achieved in the mean spo2 during the five stages studied ( p > 0.05 ) [ table 4 and figure 4 ] .", "comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery", "the mean systolic blood pressure was highest in the second stage and lowest in the first one . in the five steps studied ,", "there was a significant difference between the first step and the second ( p < 0.0001 ) and the third ( p = 0.001 ) , the second step and the first ( p = 0.001 ) and the fourth and the fifth ( p < 0.0001 ) , the third step and the first ( p = 0.001 ) and the fourth ( p = 0.010 ) , the fourth step and the second ( p < 0.0001 ) and the third ( p = 0.010 ) , and also the fifth step and the second ( p < 0.0001 ) [ table 1 and figure 1 ] .", "comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery", "the maximum and the minimum diastolic blood pressures were , respectively , obtained in the second and the first stage .", "statistical analysis showed significant difference between the first stage and the second ( p = 0.001 ) , the second stage and the first ( p = 0.001 ) and the third ( p < 0.0001 ) and the fourth ( p = 0.003 ) , the third stage and the second ( p < 0.0001 ) , and the fourth step and the second ( p = 0.003 ) ; however , no meaningful relationship was found between the fifth step and the other stages [ table 2 and figure 2 ] .", "comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery", "according to statistical analysis , the maximum and the minimum heart rate was respectively observed in the second and the fifth stage .", "significant differences were found between the first step and the second and the third ( p < 0.0001 ) , the second and the third steps and the other stages , the fourth step and the second and the third ( p < 0.0001 ) and the fifth ( p = 0.001 ) , and also the fifth step with the second and the third ( p < 0.0001 ) and the fourth ( p = 0.001 ) steps [ table 3 and figure 3 ] .", "comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery", "similarly , based on statistical analysis , no significant difference has been achieved in the mean spo2 during the five stages studied ( p > 0.05 ) [ table 4 and figure 4 ] .", "comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery", "hemodynamic changes during dental treatment have long been one of the main concerns among practitioners and researchers .", "increase in blood pressure and heart rate during surgery is inevitable ; however , the effect of sympathetic nervous system is not well - identified on this augmentation .", "since the use of local anesthesia does not merely contribute to increased concentration of plasma noradrenaline without dental treatment , blood pressure response appears to be depending on dental treatment process itself . as mentioned earlier , elevated blood pressure during oral surgery", "is attributed to the sympathetic nervous system . due to possessing vasoconstrictors and anesthetic components ,", "adrenaline is used as a vasoconstrictor along with lidocaine to increase the amount and duration of anesthesia .", "moreover , many researchers have pointed to changes probably effective on normal patients physiological stability .", "sedative drugs and local anesthetics can be used to control the autonomic nervous system responses and help to reduce the cardiovascular alterations .", "in addition , different people react differently to anxiety and pain , which could be significantly influential on their hemodynamic alterations . in the present study ,", "blood pressure and heart rate were remarkably increased after injection of anesthesia ( p < 0.05 ) . according to figures 1 , 2 , and 3 ,", "a meaningful difference has been found in the second step and following the anesthetic injection containing lidocaine and epinephrine ( 1:80000 ) ( p < 0.0001 ) in systolic and diastolic blood pressure and heart rate , on which the result was almost predictable .", "this can be associated with hemodynamic effects of adrenaline , as well as alpha - adrenergic effects to peripheral vasoconstriction and beta - adrenergic effects to increased cardiac contraction .", "furthermore , patient 's physiological stress before surgery and also painful stimulus following injection might lead to increased sympathetic activity and subsequently blood pressure and heart rate .", "thus , application of anxiety - reducing protocols such as psychological sedatives during treatment and minimizing patient 's waiting time can greatly control endogenous secretion of adrenaline .", "the study figures present a reduction in systolic and diastolic blood pressure following the second step and during the third , fourth , and fifth steps , indicating the elimination of stimulatory effect of adrenaline on blood pressure and heart rate and showing insignificant impact of other surgical steps on vital signs in cases of no anesthetics injection .", "furthermore , over time and acceptance of treatment processes by patient , the initial anxiety will be diminished and the person will reach to a relatively stable cardiovascular balance . since the peak effects of local anesthetics and alpha and beta epinephrine impact elimination occur in the third and the fourth steps , decrement in heart rate and blood pressure seems to be obvious in these stages . according to figure 2 ,", "diastolic blood pressure has been slightly elevated in the fifth step , after stitching at the end of the operation , which is probably due to feeling of pain by patient and decrease in the effect of anesthetic . in a study by matsumura et al .", "investigating changes in blood pressure and heart rate during dental surgery , the results revealed an increase in both parameters after anesthesia with lidocaine ( 2% ) and epinephrine ( 1:80000 ) , which is compatible to findings of the present research . in faraco", "survey on the effect of anesthetics containing lidocaine and epinephrine on cardiovascular changes during dental implant surgery , blood pressure was evaluated using the automated noninvasive oscillometric technique during ten steps ; the results showed no significant difference in blood pressure and heart rate variability , which is not in accordance to the findings achieved in this research . in another study by gedik et al . on blood pressure ,", "heart rate and temperature variability during periodontal surgery , patients were divided into four groups according to types of surgery , and blood pressure and heart rate were measured after anesthetic injection and the end of the operation ; blood pressure and heart rate were decreased in patients undergoing gingivectomy and periodontal flap , which is in consistence with the results obtained in the present research . however , no changes in heart rate and blood pressure have been observed following frenectomy and curettage , indicating that the difficulty in treatment process is an effective factor in blood pressure .", "as shown in figure 4 , no statistically significant difference has been found in the mean spo2 , measured by pulse oximetry , in different stages ( p > 0.05 ) . in other words , conventional periodontal surgery with technical and safety observations", "in an investigation by kaviani and birang , nausea , vomiting , decreased or increased blood pressure and other hypoxia complications following the use of nitrous oxide inhalation sedation during periodontal treatment have only been observed in 1 out of 32 cases , which was probably due to patient restlessness and movement during treatment .", "have addressed to pulse oximetry during oral surgery with and without intravenous sedation and found out a significant reduction in arterial blood oxygen levels in the group receiving midazolam .", "this study illustrates remarkable sedative effect of midazolam on anxious patient prior to the surgery ; nonetheless , this effect does not sustain during the operation .", "as mentioned , findings of various studies in terms of hemodynamic changes following dental treatments are contradictory , and such discrepancies could be related to different research approaches and different local anesthetics applied .", "different surgical stages will be accompanied by diverse effects on hemodynamic variability and pulse oximetry . according to the present study , blood pressure and heart rate are significantly increased after anesthetic injection and are decreased in the following stages by the elimination of adrenaline stimulatory effect . moreover", ", no significant alteration has been observed in spo2 during the operational steps in this research ."], "abstract_text": ["<S> background : changes in pulse rate and blood pressure are common consequences during oral surgeries . </S>", "<S> hypoxia during surgical process is another side effect . </S>", "<S> the objective of the present study was evaluation of blood hemoglobin oxygenation and hemodynamic changes during periodontal surgery.materials and methods : this clinical trial study was conducted upon 50 subjects aged 30 - 55 years who referred to the clinic of dental faculty of babol university and needed periodontal surgery with modified widman flaps in the anterior section of the maxilla . pulse rate , blood pressure , and pulse oximetric evaluations were recorded in five stages during surgery.results:the average of systolic and diastolic blood pressure had been in their maximum amount in the second stage of evaluation and minimum amounts were in the first one ; while pulse rate changes were greatest in the second stage and lowest in the fifth stage . analyzing the data revealed no significant difference in blood oxygen saturation ( spo2 ) measurements in none of the stages evaluated.conclusions:blood pressure and heart rate increased significantly after the injection of anesthetic drug and in further phases they were decreased after the elimination of stimulating effect of adrenaline . </S>", "<S> spo2 changes were not significantly prominent . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Study design", "Inclusion criteria", "Statistical analysis", "RESULTS", "The MeanSD systolic blood pressure", "The MeanSD diastolic blood pressure", "The MeanSD heart rate", "The MeanSD SpO", "DISCUSSION", "CONCLUSION"], "sections": [["successful management and treatment of periodontal disease depends upon simple medical procedures such as health education , prophylaxis , scaling and root - planing , gingivectomy , gingival grafts , and different types of gingival flaps .", "periodontal pocket refers to the pathological increase in the depth of gingival sulcus , and is one of the main clinical features of periodontal disease .", "elimination of pathological changes of the pocket wall is among the objectives of surgical treatment for periodontal pockets , so that a stable and sustainable situation can be achieved .", "currently , several flap techniques have been applied for the treatment of pocket , among which modified widman flap is worth mentioning .", "this technique facilitates the use of equipment , in which pocket lining is removed with no effect on reducing the pocket depth .", "dental treatments are accompanied by patients hemodynamic changes ; for instance , increase in blood pressure and heart rate during dental treatment are influenced by various factors such as physical and physiological stress , painful stimuli , and activity of catecholamines present in local anesthetic solutions .", "heart rate , which is indicative of ventricular contraction , changes in different conditions , including pain , anxiety , stress , heart diseases , metabolic and endocrine disorders and heart muscle diseases , and is evaluated by peripheral pulse measurement or the electrocardiogram and pulse oximetry . for the measurement of blood pressure , different methods are applied , from which riva - rocci , korotokoff , dinamap , and finometer techniques can be enumerated . due to the risk of hypoxia in oral surgeries , pulse oximetry", "pulse oximeter is a reliable and sensitive apparatus in detecting small amounts of blood oxygen saturation , and in all patients , spo2 ( spo2 ) normal percentage is not less than 95% .", "a dental patient is exposed to stressors , such as physiological responses to emotional factors and/or pain .", "pain and anxiety , are important stimuli for the secretion of endogenous adrenaline , which plays a significant role in cardiovascular responses during dental treatment .", "local anesthetics mainly affect a limited area ; however , they will be absorbed from the injection site and exert general effects , especially on cardiovascular or central nervous system .", "it is obvious that more the concentration of epinephrine , the better the control of bleeding , but there will be more cardiovascular changes . in spite of the effect of local anesthetics on vital signs , their accurate application is able to dramatically reduce stress . given the above bodies of evidence and the importance of monitoring patients hemodynamic changes during oral surgery , the present study has been conducted to investigate these alterations during periodontal surgery outside the operating room with local anesthesia ."], ["this practical clinical trial study was carried out on patients with gingival disease referred to the clinic of dental faculty of babol university of medical sciences , and underwent modified widman flap surgery on anterior maxillary region in periodontology department .", "the equipments used in the study included ( 1 ) finger pulse oximeter ( soor afarinesh company ) , and ( 2 ) automatic sphygmomanometer ( omron company ) according to the following inclusion criteria , 50 patients ranging from 30 to 50 years participated in this study . in all patients , changes in heart rate , blood pressure , and also pulse oximetry", "were examined during five steps ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation .", "it should be noted that all the operations were performed by one surgeon , and all patients received two anesthetic cartridges containing lidocaine ( 2% ) and epinephrine ( 1:80000 ) ( darou pakhsh company ) , using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa , in anterior 1/6 region and incisive canal ; no additional anesthetic cartridge was used during surgery , and those in need of re - injection were excluded from the study .", "spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient 's finger during surgery . \n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . \n ", "having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period .", "data were analyzed by spss statistical software using anova repeated measure test , and p < 0.05 was considered as statistically significant level ."], ["according to the following inclusion criteria , 50 patients ranging from 30 to 50 years participated in this study . in all patients , changes in heart rate , blood pressure , and also pulse oximetry", "were examined during five steps ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation .", "it should be noted that all the operations were performed by one surgeon , and all patients received two anesthetic cartridges containing lidocaine ( 2% ) and epinephrine ( 1:80000 ) ( darou pakhsh company ) , using the infiltration technique into the vestibular depth and interdental papilla and also hard palate mucosa , in anterior 1/6 region and incisive canal ; no additional anesthetic cartridge was used during surgery , and those in need of re - injection were excluded from the study .", "spo2 and hemodynamic alterations were assessed using the automatic sphygmomanometer and pulse oximeter attached to the patient 's finger during surgery ."], ["\n having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressurehaving no sensitivity to anestheticsnot being in pregnancy or lactation period . \n  having no systemic disease to be considered as contraindication of periodontal surgery or affecting the heart rate and blood pressure having no sensitivity to anesthetics not being in pregnancy or lactation period ."], ["data were analyzed by spss statistical software using anova repeated measure test , and p < 0.05 was considered as statistically significant level ."], ["the present research has been implemented on 50 patients referring to periodontology department of dental faculty for periodontal surgery . among the study participants with the mean age of 42.58", " 7.14 years , ranging from 30 to 55 years old , there were 22 men and 28 women . in all cases , systolic and diastolic blood pressure , heart rate , and", "spo2 were evaluated during five stages ; the first step before injecting the anesthetic , the second step after the anesthetic injection and before the incision , the third step after the incision , the forth step after debridement , and the fifth step following stitching and the end of the operation . the mean systolic blood pressure was highest in the second stage and lowest in the first one . in the five steps studied ,", "there was a significant difference between the first step and the second ( p < 0.0001 ) and the third ( p = 0.001 ) , the second step and the first ( p = 0.001 ) and the fourth and the fifth ( p < 0.0001 ) , the third step and the first ( p = 0.001 ) and the fourth ( p = 0.010 ) , the fourth step and the second ( p < 0.0001 ) and the third ( p = 0.010 ) , and also the fifth step and the second ( p < 0.0001 ) [ table 1 and figure 1 ] .", "comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery the maximum and the minimum diastolic blood pressures were , respectively , obtained in the second and the first stage .", "statistical analysis showed significant difference between the first stage and the second ( p = 0.001 ) , the second stage and the first ( p = 0.001 ) and the third ( p < 0.0001 ) and the fourth ( p = 0.003 ) , the third stage and the second ( p < 0.0001 ) , and the fourth step and the second ( p = 0.003 ) ; however , no meaningful relationship was found between the fifth step and the other stages [ table 2 and figure 2 ] .", "comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery according to statistical analysis , the maximum and the minimum heart rate was respectively observed in the second and the fifth stage .", "significant differences were found between the first step and the second and the third ( p < 0.0001 ) , the second and the third steps and the other stages , the fourth step and the second and the third ( p < 0.0001 ) and the fifth ( p = 0.001 ) , and also the fifth step with the second and the third ( p < 0.0001 ) and the fourth ( p = 0.001 ) steps [ table 3 and figure 3 ] .", "comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery similarly , based on statistical analysis , no significant difference has been achieved in the mean spo2 during the five stages studied ( p > 0.05 ) [ table 4 and figure 4 ] .", "comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery"], ["the mean systolic blood pressure was highest in the second stage and lowest in the first one . in the five steps studied ,", "there was a significant difference between the first step and the second ( p < 0.0001 ) and the third ( p = 0.001 ) , the second step and the first ( p = 0.001 ) and the fourth and the fifth ( p < 0.0001 ) , the third step and the first ( p = 0.001 ) and the fourth ( p = 0.010 ) , the fourth step and the second ( p < 0.0001 ) and the third ( p = 0.010 ) , and also the fifth step and the second ( p < 0.0001 ) [ table 1 and figure 1 ] .", "comparison of the meansd sb * during the five stages of modified widman flap surgery sbp changes during the five stages of modified widman flap surgery"], ["the maximum and the minimum diastolic blood pressures were , respectively , obtained in the second and the first stage .", "statistical analysis showed significant difference between the first stage and the second ( p = 0.001 ) , the second stage and the first ( p = 0.001 ) and the third ( p < 0.0001 ) and the fourth ( p = 0.003 ) , the third stage and the second ( p < 0.0001 ) , and the fourth step and the second ( p = 0.003 ) ; however , no meaningful relationship was found between the fifth step and the other stages [ table 2 and figure 2 ] .", "comparison of the meansd dbp * during the five stages of modified widman flap surgery dbp changes during the five stages of modified widman flap surgery"], ["according to statistical analysis , the maximum and the minimum heart rate was respectively observed in the second and the fifth stage .", "significant differences were found between the first step and the second and the third ( p < 0.0001 ) , the second and the third steps and the other stages , the fourth step and the second and the third ( p < 0.0001 ) and the fifth ( p = 0.001 ) , and also the fifth step with the second and the third ( p < 0.0001 ) and the fourth ( p = 0.001 ) steps [ table 3 and figure 3 ] .", "comparison of the meansd hr * during the five stages of modified widman flap surgery changes in hr during the five stages of modified widman flap surgery"], ["similarly , based on statistical analysis , no significant difference has been achieved in the mean spo2 during the five stages studied ( p > 0.05 ) [ table 4 and figure 4 ] .", "comparison of the meansd spo2 during the five stages of modified widman flap surgery spo2 variability during the five stages of modified widman flap surgery"], ["hemodynamic changes during dental treatment have long been one of the main concerns among practitioners and researchers .", "increase in blood pressure and heart rate during surgery is inevitable ; however , the effect of sympathetic nervous system is not well - identified on this augmentation .", "since the use of local anesthesia does not merely contribute to increased concentration of plasma noradrenaline without dental treatment , blood pressure response appears to be depending on dental treatment process itself . as mentioned earlier , elevated blood pressure during oral surgery", "is attributed to the sympathetic nervous system . due to possessing vasoconstrictors and anesthetic components ,", "adrenaline is used as a vasoconstrictor along with lidocaine to increase the amount and duration of anesthesia .", "moreover , many researchers have pointed to changes probably effective on normal patients physiological stability .", "sedative drugs and local anesthetics can be used to control the autonomic nervous system responses and help to reduce the cardiovascular alterations .", "in addition , different people react differently to anxiety and pain , which could be significantly influential on their hemodynamic alterations . in the present study ,", "blood pressure and heart rate were remarkably increased after injection of anesthesia ( p < 0.05 ) . according to figures 1 , 2 , and 3 ,", "a meaningful difference has been found in the second step and following the anesthetic injection containing lidocaine and epinephrine ( 1:80000 ) ( p < 0.0001 ) in systolic and diastolic blood pressure and heart rate , on which the result was almost predictable .", "this can be associated with hemodynamic effects of adrenaline , as well as alpha - adrenergic effects to peripheral vasoconstriction and beta - adrenergic effects to increased cardiac contraction .", "furthermore , patient 's physiological stress before surgery and also painful stimulus following injection might lead to increased sympathetic activity and subsequently blood pressure and heart rate .", "thus , application of anxiety - reducing protocols such as psychological sedatives during treatment and minimizing patient 's waiting time can greatly control endogenous secretion of adrenaline .", "the study figures present a reduction in systolic and diastolic blood pressure following the second step and during the third , fourth , and fifth steps , indicating the elimination of stimulatory effect of adrenaline on blood pressure and heart rate and showing insignificant impact of other surgical steps on vital signs in cases of no anesthetics injection .", "furthermore , over time and acceptance of treatment processes by patient , the initial anxiety will be diminished and the person will reach to a relatively stable cardiovascular balance . since the peak effects of local anesthetics and alpha and beta epinephrine impact elimination occur in the third and the fourth steps , decrement in heart rate and blood pressure seems to be obvious in these stages . according to figure 2 ,", "diastolic blood pressure has been slightly elevated in the fifth step , after stitching at the end of the operation , which is probably due to feeling of pain by patient and decrease in the effect of anesthetic . in a study by matsumura et al .", "investigating changes in blood pressure and heart rate during dental surgery , the results revealed an increase in both parameters after anesthesia with lidocaine ( 2% ) and epinephrine ( 1:80000 ) , which is compatible to findings of the present research . in faraco", "survey on the effect of anesthetics containing lidocaine and epinephrine on cardiovascular changes during dental implant surgery , blood pressure was evaluated using the automated noninvasive oscillometric technique during ten steps ; the results showed no significant difference in blood pressure and heart rate variability , which is not in accordance to the findings achieved in this research . in another study by gedik et al . on blood pressure ,", "heart rate and temperature variability during periodontal surgery , patients were divided into four groups according to types of surgery , and blood pressure and heart rate were measured after anesthetic injection and the end of the operation ; blood pressure and heart rate were decreased in patients undergoing gingivectomy and periodontal flap , which is in consistence with the results obtained in the present research . however , no changes in heart rate and blood pressure have been observed following frenectomy and curettage , indicating that the difficulty in treatment process is an effective factor in blood pressure .", "as shown in figure 4 , no statistically significant difference has been found in the mean spo2 , measured by pulse oximetry , in different stages ( p > 0.05 ) . in other words , conventional periodontal surgery with technical and safety observations", "in an investigation by kaviani and birang , nausea , vomiting , decreased or increased blood pressure and other hypoxia complications following the use of nitrous oxide inhalation sedation during periodontal treatment have only been observed in 1 out of 32 cases , which was probably due to patient restlessness and movement during treatment .", "have addressed to pulse oximetry during oral surgery with and without intravenous sedation and found out a significant reduction in arterial blood oxygen levels in the group receiving midazolam .", "this study illustrates remarkable sedative effect of midazolam on anxious patient prior to the surgery ; nonetheless , this effect does not sustain during the operation .", "as mentioned , findings of various studies in terms of hemodynamic changes following dental treatments are contradictory , and such discrepancies could be related to different research approaches and different local anesthetics applied ."], ["different surgical stages will be accompanied by diverse effects on hemodynamic variability and pulse oximetry . according to the present study , blood pressure and heart rate are significantly increased after anesthetic injection and are decreased in the following stages by the elimination of adrenaline stimulatory effect . moreover", ", no significant alteration has been observed in spo2 during the operational steps in this research ."]]}
{"article_id": "PMC5094925", "article_text": ["chronic kidney disease ( ckd ) is defined as a structural or functional kidney abnormality lasting for 3 or more months .", "the global prevalence of ckd is estimated to be more than 10% , and ckd has emerged as a public health problem .", "adverse outcomes of ckd such as kidney failure , cardiovascular disease ( cvd ) , and premature death can be prevented or delayed when treatment is initiated in the early stages of disease .", "as the earlier stages are often asymptomatic , ckd is usually detected during laboratory evaluation of comorbid conditions .", "glomerular filtration rate ( gfr ) is considered the index of kidney function , and for convenience", "is usually estimated using equations instead of by direct measurement . the modification of diet in renal disease ( mdrd ) study equation , which was replaced by the chronic kidney disease epidemiology collaboration 2009 equations ( ckd - epi equations ) , has been used to estimate gfr .", "however , this tended to underestimate the measured gfr and had less precision , especially at a gfr of 60 ml / min/1.73 m or more .", "the ckd - epi equations were reported as having less bias , improved precision , and greater accuracy than the mdrd study equation at a higher gfr of 60 ml / min/1.73 m or more .", "the ckd - epi equations led to a lower estimation of ckd prevalence , primarily due to a lower prevalence of gfr stage 3 , compared to the mdrd study equation .", "persistently increased protein excretion has usually been considered to be a marker of kidney damage , and screening with urine dipsticks has been acceptable for detecting proteinuria .", "recently , urinary albumin measurement was recommended as a marker of kidney damage , both for its standardization and also because albumin is the most important protein lost in the urine in most ckd", ". the albumin - to - creatinine ratio ( acr ) is the most preferred value among urinary albumin measurements .", "kidney disease : improving global outcomes ( kdigo ) has updated guidelines for defining , diagnosing , staging , and managing ckd since the establishment of the first uniform definitions and staging system for ckd in 2002 .", "more objective criteria , such as the ckd - epi equations for estimating gfr and classification of albuminuria , have been incorporated . moreover ,", "the etiology of ckd was included in the definition ; hypertension and diabetes mellitus ( dm ) are systemic diseases known to cause ckd .", "consequently , kdigo guidelines provide relatively simple and precise methods for early diagnosis of ckd , and tend to yield fewer false positives , thereby avoiding incorrect diagnosis .", "the number of patients treated with renal replacement therapy ( rrt ) including dialysis and transplantation has increased dramatically in korea , from 28,046 in 2000 to 75,042 in 2013 .", "the prevalence of rrt was 1,446 patients per million population ( pmp ) , with an incidence of 234 pmp in 2013 .", "the cause of kidney failure was estimated to be dm in 48.0% , hypertension in 19.7% , and chronic glomerulonephritis in 8.3% of cases . the previously reported ckd prevalence calculated from the mdrd study equation", "the ckd prevalence calculated from the mdrd study equation alone was 8.8% in 2005 and 7.2% in 2007 in korea national health and nutrition examination survey ( knhanes ) subjects aged  20 ; the prevalence calculated using the mdrd study equation with the dipstick proteinuria method has tended to decrease since 2001 in men and since 2005 in women among knhanes i to iv ( 1998 to 2009 ) subjects aged  20 . since there are no reports on ckd prevalence using the ckd - epi equations to estimate gfr and acr to measure albuminuria for koreans , a new analysis of ckd prevalence is necessary .", "this study was therefore designed to investigate the prevalence of ckd in the korean population using knhanes samples , based on the definition and classification by kdigo 2012 guidelines , as well as the impact of changes through a comparison of the ckd - epi and mdrd study equations for the estimation of gfr , and the associations between ckd and major risk factors , including dm , hypertension , age , and gender .", "this retrospective , population - based study was performed using data acquired by the knhanes v from 2010 to 2012 .", "knhanes v was a cross - sectional , nationally representative survey with a multistage , stratified , and probability sampling design based on sex , age , and geographical area using household registries .", "knhanes , composed of a health questionnaire survey , physical examination , and nutrition survey , has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention ( kcdc ) .", "the study protocol was approved by the institutional review board of kcdc ( 2010 - 02con-21-c ) , and written informed consents were obtained from all subjects or their guardians .", "knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 .", "the inclusion criteria were ( 1 ) adults aged  19 , and ( 2 ) available serum creatinine ( scr ) , urine albumin , and urine creatinine test results .", "individuals aged  19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 . the health examination was performed in a mobile health examination center .", "was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association .", "three readings were obtained via mercury sphygmomanometer ( baumanometer , baum , copiague , ny , usa ) , and the mean of the second and third reading was reported as the final bp for individual participants .", "body mass index ( bmi ) was calculated as body weight divided by the square of the height , and expressed in units of kg / m .", "blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast .", "a random urine sample ( first urine in the morning , if possible ) was obtained in a conical container .", "the urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day ( neodin medical institute , seoul , korea ) .", "serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako ( wako , osaka , japan ) following the jaffe method ; fasting glucose concentration ( fasting blood glucose , fbg ) by an enzymatic method using pureauto s glu ( sekisui , osaka , japan ) ; total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , and triglycerides by an enzymatic method ; and urine albumin by turbidimetric assay using albumin ( roche , mannheim , germany ) , all performed using a hitachi automatic analyzer 7600 ( hitachi high - technologies co. , tokyo , japan ) , which was calibrated using calibrator for automated systems ( roche diagnostics , indianapolis , in , usa ) .", "glycated hemoglobin ( a1c ) was assayed by high performance liquid chromatography ( hlc-723g7 a1c , tosoh , tokyo , japan ) .", "urine chemistry was tested by test strip ( urysis 2400 cassette , roche ) using urisys 2400 ( roche ) .", "low density lipoprotein cholesterol ( ldl - c ) was computed using the friedewald formula when triglycerides were less than 400 mg / dl .", "ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr .", "gfr was estimated using ckd - epi equations , taking into account gender and serum creatinine .", "individuals with gfr < 60 ml / min/1.73 m or acr  30 mg / g were classified as ckd patients . hypertension was defined as having a systolic blood pressure ( sbp ) of  140 mmhg or a diastolic blood pressure ( dbp ) of  90 mmhg , or having received a physician s diagnosis .", "dm was defined as having a1c  6.5% , fbg of  126 mg / dl , or having received a physician s diagnosis .", "dyslipidemia was defined as having total cholesterol  240 mg / dl , ldl - c  160 mg / dl , hdl - c < 40 mg / dl , triglycerides  200 mg / dl , or having received a physician s diagnosis .", "analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations .", "the study population was separated as a subpopulation , and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias .", "in addition , weight adjustments were applied to the user - missing values to prevent skews in sampling design .", "the demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables .", "the mean and the 95% confidence interval ( ci ) were presented for data on continuous variables , whereas unweighted count with weighted percentage was presented for data on categorical variables .", "the complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories , and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage .", "a subpopulation was added to strata when performing the complex samples chi - square test .", "associations of ckd , gfr , and acr categories with various risk factors , including gender , age , bmi , dm , hypertension , and dyslipidemia were expressed as an odds ratio ( or ) , which was calculated using complex samples multivariate logistic regression analyses .", "this retrospective , population - based study was performed using data acquired by the knhanes v from 2010 to 2012 .", "knhanes v was a cross - sectional , nationally representative survey with a multistage , stratified , and probability sampling design based on sex , age , and geographical area using household registries .", "knhanes , composed of a health questionnaire survey , physical examination , and nutrition survey , has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention ( kcdc ) .", "the study protocol was approved by the institutional review board of kcdc ( 2010 - 02con-21-c ) , and written informed consents were obtained from all subjects or their guardians .", "knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 .", "the inclusion criteria were ( 1 ) adults aged  19 , and ( 2 ) available serum creatinine ( scr ) , urine albumin , and urine creatinine test results .", "individuals aged  19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 .", "blood pressure ( bp ) was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association .", "three readings were obtained via mercury sphygmomanometer ( baumanometer , baum , copiague , ny , usa ) , and the mean of the second and third reading was reported as the final bp for individual participants .", "body mass index ( bmi ) was calculated as body weight divided by the square of the height , and expressed in units of kg / m .", "blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast . a random urine sample ( first urine in the morning , if possible )", "the urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day ( neodin medical institute , seoul , korea ) .", "serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako ( wako , osaka , japan ) following the jaffe method ; fasting glucose concentration ( fasting blood glucose , fbg ) by an enzymatic method using pureauto s glu ( sekisui , osaka , japan ) ; total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , and triglycerides by an enzymatic method ; and urine albumin by turbidimetric assay using albumin ( roche , mannheim , germany ) , all performed using a hitachi automatic analyzer 7600 ( hitachi high - technologies co. , tokyo , japan ) , which was calibrated using calibrator for automated systems ( roche diagnostics , indianapolis , in , usa ) .", "glycated hemoglobin ( a1c ) was assayed by high performance liquid chromatography ( hlc-723g7 a1c , tosoh , tokyo , japan ) .", "urine chemistry was tested by test strip ( urysis 2400 cassette , roche ) using urisys 2400 ( roche ) .", "low density lipoprotein cholesterol ( ldl - c ) was computed using the friedewald formula when triglycerides were less than 400 mg / dl .", "ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr .", "gfr was estimated using ckd - epi equations , taking into account gender and serum creatinine .", "individuals with gfr < 60 ml / min/1.73 m or acr  30 mg / g were classified as ckd patients .", "hypertension was defined as having a systolic blood pressure ( sbp ) of  140 mmhg or a diastolic blood pressure ( dbp ) of  90 mmhg , or having received a physician s diagnosis .", "dm was defined as having a1c  6.5% , fbg of  126 mg / dl , or having received a physician s diagnosis .", "dyslipidemia was defined as having total cholesterol  240 mg / dl , ldl - c  160 mg / dl , hdl - c < 40 mg / dl , triglycerides  200 mg / dl , or having received a physician s diagnosis .", "analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations .", "the study population was separated as a subpopulation , and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias .", "in addition , weight adjustments were applied to the user - missing values to prevent skews in sampling design .", "the demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables .", "the mean and the 95% confidence interval ( ci ) were presented for data on continuous variables , whereas unweighted count with weighted percentage was presented for data on categorical variables .", "the complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories , and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage .", "a subpopulation was added to strata when performing the complex samples chi - square test .", "associations of ckd , gfr , and acr categories with various risk factors , including gender , age , bmi , dm , hypertension , and dyslipidemia were expressed as an odds ratio ( or ) , which was calculated using complex samples multivariate logistic regression analyses .", "all statistical analyses were performed using ibm spss version 21.0 ( ibm corp . , armonk , ny , usa ) . the statistical significance level was set at p < 0.05 .", "among the total sample size of 16,576 from 2011 and 2012 , 64.7% satisfied the inclusion criteria ( 10,636 individuals : 5,388 and 5,248 from 2011 and 2012 , respectively ) as shown in fig .", "the composition of neither the total adults nor the study population was statistically different between 2011 and 2012 ( p = 0.232 and p = 0.087 , respectively ) . the study population comprised 81.8% of total adults ( 81.0% and 82.7% from 2011 and 2012 , respectively ,", "the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable , except for hdl - c , ldl - c , and prevalence of dyslipidemia ( table 1 ) .", "the percentage of men was 52.6% , and the mean age was 45.8 years ( 95% ci , 45.2 to 46.3 ) .", "mean serum creatinine was 0.86 mg / dl ( 95% ci , 0.85 to 0.86 ) , with an estimated gfr of 96 ml / min/1.73 m ( 95% ci , 96 to 97 ) .", "mean acr was 18 mg / g ( 95% ci , 15 to 21 ) , with urine albumin 22 g / ml ( 95% ci , 19 to 26 ) and urine creatinine 165 mg / dl ( 95% ci , 163 to 168 ) .", "mean sbp was 118 mmhg ( 95% ci , 117 to 119 ) , and dbp 76 mmhg ( 95% ci , 76 to 77 ) .", "mean fbg was 97 mg / dl ( 95% ci , 97 to 98 ) , and a1c 5.7% ( 95% ci , 5.67 to 5.71 ) .", "mean total cholesterol , hdl - c , ldl - c , and triglycerides were 190 mg / dl ( 95% ci , 188 to 191 ) , 49 mg / dl ( 95% ci , 49 to 50 ) , 114 mg / dl ( 95% ci , 113 to 115 ) , and 136 mg / dl ( 95% ci , 133 to 139 ) , respectively .", "the ckd prevalence in korean adults was estimated as 7.9% ( 7.8% in 2011 and 8.0% in 2012 , p = 0.770 ) ( table 1 ) .", "the prevalence of hypertension , dm , and dyslipidemia was 27.8% ( 27.2% in 2011 and 28.4% in 2012 , p = 0.380 ) , 9.3% ( 9.7% in 2011 and 9.0% in 2012 , p = 0.306 ) , and 32.8% ( 34.6% in 2011 and 31.0% in 2012 , p = 0.004 ) , respectively .", "the distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 .", "the prevalence of low , moderately increased , high , and very high ckd risk prognosis was 92.0% , 6.3% , 1.1% , and 0.6% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m was 2.2% , whereas acr  30 mg / g was 6.5% ( a2 of 5.6% and a3 of 0.9% ) .", "the prevalence of acr  30 mg / g in individuals with gfr  60 ml / min/1.73", "the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig .", "the prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation , and g2 was 29.8% and 38.7% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m by the mdrd study equation was 2.0% , which was significantly less than the 2.2% by ckd - epi equations ( p < 0.001 ) .", "the prevalence of at least 1 + proteinuria in a1 , expected to be fully negative , was 0.1% whereas  or  proteinuria in a2 and a3 , expected to be fully positive , were 5.3% and 0.3% , respectively .", "some interactions between demographic covariates , such as gender , age , and bmi , were present .", "the risk of bmi  25 for men was higher than for women ( or , 1.296 ; 95% ci , 1.164 to 1.443 ; p < 0.001 ) .", "the study population was stratified into six groups by age in 10-year increments : 19 to 28 , 29 to 38 , 39 to 48 , 49 to 58 , 59 to 68 , and  68 .", "the ors of increasing age ( using the age group of 19 to 28 as a reference ) to bmi  25 were 1.731 ( 95% ci , 1.400 to 2.139 ) , 2.150 ( 95% ci , 1.757 to 2.632 ) , 1.689 ( 95% ci , 1.376 to 2.074 ) , 2.092 ( 95% ci , 1.690 to 2.590 ) , and 1.554 ( 95% ci , 1.255 to 1.924 ; p = 0.001 ) , respectively .", "in addition , the composition of men significantly decreased in the  49 age groups ; ors of men to increasing age ( using the age group of 19 to 28 as a reference ) were 0.931 ( 95% ci , 0.784 to 1.105 ) , 0.894 ( 95% ci , 0.750 to 1.065 ) , 0.735 ( 95% ci , 0.625 to 0.864 ) , 0.691 ( 95% ci , 0.578 to 0.825 ) , and 0.505 ( 95% ci , 0.421 to 0.607 ; p = 0.001 ) , respectively . thus , the association analysis was performed separately for each gender . because ckd is defined by both gfr and albuminuria , the associations between the risk factors and either decreased gfr or increased acr were also studied .", "the frequency of women among ckd patients was 54.3% , which was significantly higher than that for men ( p < 0.001 ) . among individuals with acr  30 mg / g", ", women accounted for 55.4% ( p < 0.001 ) ; however , women constituted 48.7% of gfr < 60 ml / min/1.73 m ( p = 0.683 ) .", "the ors of gender to ckd , acr  30 mg / g , and gfr < 60 ml / min/1.73 m for men were 1.354 ( 95% ci , 1.162 to 1.577 ; p", "< 0.001 ) , and 1.057 ( 95% ci , 0.811 to 1.376 ; p = 0.683 ) , respectively .", "the prevalence of bmi  25 , ckd , gfr < 60 ml / min/1.73 m , acr  30 mg / g , dm , hypertension , and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 .", "the ors of covariates to the prevalence of ckd , gfr < 60 ml / min/1.73 m , and acr  30 mg / g , after adjusting for age , bmi , hypertension , dm , and dyslipidemia in men and women are listed in table 6 .", "hypertensive patients showed a higher risk of ckd than the normotensive population ( or , 3.394 for men ; or , 2.935 for women ; both p < 0.0001 ) .", "the ors of hypertension to cause albuminuria were 3.351 ( p < 0.001 ) for men and", "3.067 ( p < 0.001 ) for women , and to decrease gfr , 3.436 ( p < 0.001 ) for men and 1.904 ( p = 0.006 ) for women .", "diabetic patients showed a higher risk of ckd than the nondiabetic population ( or , 3.145 for men ; or , 1.983 for women ; both p < 0.001 ) .", "the ors of dm to cause albuminuria were 3.144 ( p < 0.001 ) for men and", "2.022 ( p < 0.001 ) for women , and to decrease gfr 1.965 ( p = 0.004 ) for men and 1.764 ( p = 0.013 ) for women .", "dyslipidemia showed a significant association with ckd in women ( or , 1.324 ; p = 0.023 ) , but not in men ( or , 1.175 ; p = 0.243 ) .", "increasing age and obesity ( bmi  25 ) showed a positive correlation with ckd .", "among the total sample size of 16,576 from 2011 and 2012 , 64.7% satisfied the inclusion criteria ( 10,636 individuals : 5,388 and 5,248 from 2011 and 2012 , respectively ) as shown in fig .", "the composition of neither the total adults nor the study population was statistically different between 2011 and 2012 ( p = 0.232 and p = 0.087 , respectively ) . the study population comprised 81.8% of total adults ( 81.0% and 82.7% from 2011 and 2012 , respectively ,", "the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable , except for hdl - c , ldl - c , and prevalence of dyslipidemia ( table 1 ) .", "the percentage of men was 52.6% , and the mean age was 45.8 years ( 95% ci , 45.2 to 46.3 ) .", "mean serum creatinine was 0.86 mg / dl ( 95% ci , 0.85 to 0.86 ) , with an estimated gfr of 96 ml / min/1.73 m ( 95% ci , 96 to 97 ) .", "mean acr was 18 mg / g ( 95% ci , 15 to 21 ) , with urine albumin 22 g / ml ( 95% ci , 19 to 26 ) and urine creatinine 165 mg / dl ( 95% ci , 163 to 168 ) .", "mean sbp was 118 mmhg ( 95% ci , 117 to 119 ) , and dbp 76 mmhg ( 95% ci , 76 to 77 ) .", "mean fbg was 97 mg / dl ( 95% ci , 97 to 98 ) , and a1c 5.7% ( 95% ci , 5.67 to 5.71 ) .", "mean total cholesterol , hdl - c , ldl - c , and triglycerides were 190 mg / dl ( 95% ci , 188 to 191 ) , 49 mg / dl ( 95% ci , 49 to 50 ) , 114 mg / dl ( 95% ci , 113 to 115 ) , and 136 mg / dl ( 95% ci , 133 to 139 ) , respectively .", "the ckd prevalence in korean adults was estimated as 7.9% ( 7.8% in 2011 and 8.0% in 2012 , p = 0.770 ) ( table 1 ) .", "the prevalence of hypertension , dm , and dyslipidemia was 27.8% ( 27.2% in 2011 and 28.4% in 2012 , p = 0.380 ) , 9.3% ( 9.7% in 2011 and 9.0% in 2012 , p = 0.306 ) , and 32.8% ( 34.6% in 2011 and 31.0% in 2012 , p = 0.004 ) , respectively .", "the distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 .", "the prevalence of low , moderately increased , high , and very high ckd risk prognosis was 92.0% , 6.3% , 1.1% , and 0.6% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m was 2.2% , whereas acr  30 mg / g was 6.5% ( a2 of 5.6% and a3 of 0.9% ) .", "the prevalence of acr  30 mg / g in individuals with gfr  60 ml / min/1.73", "the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig .", "2 . the prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation , and g2 was 29.8% and 38.7% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m by the mdrd study equation was 2.0% , which was significantly less than the 2.2% by ckd - epi equations ( p < 0.001 ) .", "the prevalence of at least 1 + proteinuria in a1 , expected to be fully negative , was 0.1% whereas  or  proteinuria in a2 and a3 , expected to be fully positive , were 5.3% and 0.3% , respectively .", "some interactions between demographic covariates , such as gender , age , and bmi , were present .", "the risk of bmi  25 for men was higher than for women ( or , 1.296 ; 95% ci , 1.164 to 1.443 ; p < 0.001 ) .", "the study population was stratified into six groups by age in 10-year increments : 19 to 28 , 29 to 38 , 39 to 48 , 49 to 58 , 59 to 68 , and  68 .", "the ors of increasing age ( using the age group of 19 to 28 as a reference ) to bmi  25 were 1.731 ( 95% ci , 1.400 to 2.139 ) , 2.150 ( 95% ci , 1.757 to 2.632 ) , 1.689 ( 95% ci , 1.376 to 2.074 ) , 2.092 ( 95% ci , 1.690 to 2.590 ) , and 1.554 ( 95% ci , 1.255 to 1.924 ; p = 0.001 ) , respectively .", "in addition , the composition of men significantly decreased in the  49 age groups ; ors of men to increasing age ( using the age group of 19 to 28 as a reference ) were 0.931 ( 95% ci , 0.784 to 1.105 ) , 0.894 ( 95% ci , 0.750 to 1.065 ) , 0.735 ( 95% ci , 0.625 to 0.864 ) , 0.691 ( 95% ci , 0.578 to 0.825 ) , and 0.505 ( 95% ci , 0.421 to 0.607 ; p = 0.001 ) , respectively . thus , the association analysis was performed separately for each gender . because ckd is defined by both gfr and albuminuria , the associations between the risk factors and either decreased gfr or increased acr were also studied .", "the frequency of women among ckd patients was 54.3% , which was significantly higher than that for men ( p < 0.001 ) . among individuals with acr  30 mg / g ,", "women accounted for 55.4% ( p < 0.001 ) ; however , women constituted 48.7% of gfr < 60 ml / min/1.73 m ( p = 0.683 ) .", "the ors of gender to ckd , acr  30 mg / g , and gfr < 60 ml / min/1.73 m for men were 1.354 ( 95% ci , 1.162 to 1.577 ; p", "< 0.001 ) , 1.416 ( 95% ci , 1.193 to 1.682 ; p < 0.001 ) , and 1.057 ( 95% ci , 0.811 to 1.376 ; p = 0.683 ) , respectively .", "the prevalence of bmi  25 , ckd , gfr < 60 ml / min/1.73 m , acr  30 mg / g , dm , hypertension , and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 .", "the ors of covariates to the prevalence of ckd , gfr < 60 ml / min/1.73 m , and acr  30 mg / g , after adjusting for age , bmi , hypertension , dm , and dyslipidemia in men and women are listed in table 6 .", "hypertensive patients showed a higher risk of ckd than the normotensive population ( or , 3.394 for men ; or , 2.935 for women ; both p < 0.0001 ) .", "the ors of hypertension to cause albuminuria were 3.351 ( p < 0.001 ) for men and", "3.067 ( p < 0.001 ) for women , and to decrease gfr , 3.436 ( p < 0.001 ) for men and 1.904 ( p = 0.006 ) for women .", "diabetic patients showed a higher risk of ckd than the nondiabetic population ( or , 3.145 for men ; or , 1.983 for women ; both p < 0.001 ) .", "the ors of dm to cause albuminuria were 3.144 ( p < 0.001 ) for men and", "2.022 ( p < 0.001 ) for women , and to decrease gfr 1.965 ( p = 0.004 ) for men and 1.764 ( p = 0.013 ) for women .", "dyslipidemia showed a significant association with ckd in women ( or , 1.324 ; p = 0.023 ) , but not in men ( or , 1.175 ; p = 0.243 ) . increasing age and obesity ( bmi  25 ) showed a positive correlation with ckd .", "this study is the first to report ckd prevalence using gfr estimated by using ckd - epi equations together with acr for albuminuria in the korean population .", "the prevalence of ckd was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged  19 , and the ckd risk prognosis was reported in table 2 .", "the ckd risk prognosis from moderately increased to very high reported in this study was lower than that in the usa .", "gfr and albuminuria are independent and complementary predictors of important clinical outcomes , including ckd progression , end - stage renal disease , acute kidney injury , cardiovascular mortality , and all - cause mortality [ 16 - 19 ] .", "the frequencies of gfr < 60 ml / min/1.73 m showed sharp increases starting from around age 50 for men and 60 for women , whereas those of acr  30 mg / g increased relatively gradually , starting from earlier ages , as shown in table 4 and 5 .", "the rate of decline of measured gfr was reported to double in kidney transplantation donors aged > 45 years .", "the prevalence of estimated gfr < 60 ml / min/1.73 m using ckd - epi equations and the mdrd study equation was 2.2% and 2.0% in this study , but 2.6% and 3.2% , respectively in the previous study .", "the statistical method used in the current study was complex sample analysis incorporating sampling weight , in contrast to the previous study which did not consider weight in the analysis .", "the frequencies of gfr stage g1 and g2 by ckd - epi equations were 67.7% and 29.8% , whereas those by the mdrd study equation were 59.4% and 38.7% , respectively , in this study .", "these numbers are comparable to the previous study , considering the different analytical methods ; the frequencies of gfr stage g1 and g2 by ckd - epi equations were 64.5% and 28.9% , whereas those by the mdrd study equation were 47.6% and 49.2% , respectively .", "these results confirm the tendency to underestimate gfr by the mdrd study equation when gfr is 60 ml / min/1.73 m or greater .", "the previous report of lower prevalence of gfr stage g3 using ckd - epi equations than with the mdrd study equation was not observed in this study . among the population of the previous study , the frequencies of g3 by ckd - epi equations and the mdrd study equation ( 33.2% and 37.2% , respectively ) were far greater than those among the korean population ( 2.1% and 1.9% , respectively ) .", "the prevalence of acr  30 mg / g in the entire study population , and acr  30 mg / g in individuals with gfr  60 ml / min/1.73", "the prevalence of acr  30 mg / g in the entire study population and acr  30 mg / g in individuals with gfr  60 ml / min/1.73", "m aged  35 years was 10.2% and 8.7% , respectively , in the previous korean population .", "besides the different age criteria , the populations of the previous and current study are quite different ; the previous study was designed by the researcher group considering age , gender , and city factors not using the knhanes sample data .", "although the absolute values are different , the important point in common is that a large number of people have albuminuria before gfr decreases to < 60 ml / min/1.73 m ( 85.3% and 87.7% of the albuminuria cases did not have decreased gfr in the previous and current study , respectively ) . as shown in table 3", ", test strips did not detect albuminuria in 81.5% of cases . at present , laboratory tests for albuminuria are not routinely performed during regular physical examinations in korea .", "the prevalence of ckd in men and women was 6.9% and 9.1% , respectively ( p < 0.001 ) . besides gender itself , age distribution , frequency of obesity", "( bmi  25 ) , and concurrent chronic diseases , including dm , hypertension , and dyslipidemia , were different according to gender .", "thus , the risk of ckd is higher in women than men ( or , 1.354 ; p < 0.001 ) , and the difference seems to be caused by the susceptibility to albuminuria ( or , 1.416 ; p < 0.001 ) rather than decreased gfr ( or , 1.057 ; p = 0.683 ) .", "when gfr was estimated using the mdrd study equation , the increased risks of gfr < 60 ml / min/1.73", "m in women were observed in previous studies : or , 3.16 ( p < 0.001 ) using knhanes iii and iv-1 ; frequencies of 3.4% , 9.7% , 10.2% , and 4.6% in women , and 1.0% , 5.4% , 3.1% , and 2.6% were seen in men through knhanes i - iv .", "there seemed to be no difference in acr according to gender in the previous study ( 10.1% and 10.3% in women and men , respectively ) .", "a similar observation was reported for proteinuria by gender ( frequencies of 3.3% , 2.3% , 1.4% , and 1.9% in women , and 3.1% , 3.0% , 2.8% , and 2.2% in men through knhanes", "direct comparison is difficult due to the difference in the designs of study populations   and laboratory test methods using dipstick proteinuria and acr .", "increasing age and obesity ( bmi  25 ) correlated significantly with ckd , as shown in table 6 , which agreed with previous studies [ 11 - 13 ] .", "hypertension and dm are both major risk factors and consequences of ckd [ 21 - 23 ] . concurrent chronic diseases such as dm and hypertension increased the risk of ckd consistently [ 11 - 13 ]", ". diabetic patients are susceptible to glomerular disease and hypertensive patients to renal vascular disease .", "since the prevalence of dm was higher in men than women , with the opposite for prevalence of ckd , the or of dm to ckd in men was higher than women , as shown in table 6 .", "moreover , dm was correlated with increased acr rather than decreased gfr in this study . since increased acr appeared at an earlier age in women than men , and decreased gfr", "was preceded by increased acr , the difference between ors to gfr < 60 ml / min/1.73 m and acr  30 mg / g in hypertensive women was greater than hypertensive men . on the other hand ,", "limited availability of direct ldl - c measurement , unstandardized laboratory assay methods for hdl - c , and lack of a uniform definition of dyslipidemia between the studies made it difficult to evaluate their associations .", "the major limitation of this study is the lack of the evaluation for chronicity assessment and use of single measurements for laboratory tests due to the cross - sectional data characteristics . according to the definition of ckd by the kdigo guideline", "the rationale for defining chronicity is to differentiate ckd from acute kidney diseases , which may require different interventions and have different etiologies and outcomes .", "in addition , there is still the discrepancy between measured and estimated gfr using ckd - epi equations .", "gfr estimation using equations is convenient , but measurement of gfr should be considered to diagnose a specific patient .", "finally , association analysis could not provide conclusive causal relationships due to the study characteristics .", "however , the representative features of the knhanes sample data can not be overlooked . in conclusion ,", "the prevalence of ckd using estimated gfr by the ckd - epi equation and acr for albuminuria measurement was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged  19 .", "the prevalence of albuminuria ( acr  30 mg / g ) before decline in gfr ( gfr  60 ml / min/1.73 m ) was 5.7% . despite kdigo guidelines to prevent or delay progression of ckd in its early stages , implementation remains difficult due to practical limitations .", "the failure to recognize ckd in its early stage may result in complications such as premature rrt .", "thus , urinalysis to detect albumin should be strongly recommended for patients with higher risk of ckd and cvd . as there is a consensus about the risk of ckd with dm and hypertension , regular albuminuria testing for at least these patients to detect kidney disease at an early stage", "1 . the prevalence of chronic kidney disease ( ckd ) was estimated to be 7.9% in the korea national health and nutrition examination survey samples of 2011 and 2012 in korean adults aged  19 .", "early identification of ckd paired with appropriate management and earlier referral to specialty kidney services results in economic and clinical benefits .", "the majority of ckd patients have albuminuria prior to a decrease in glomerular filtration rate .", "4 . regular laboratory tests for albuminuria in the high - risk group , especially for hypertensive or diabetes mellitus patients , should contribute to early detection of ckd ."], "abstract_text": ["<S> background / aimsan updated chronic kidney disease ( ckd ) definition and classification were proposed by kidney disease : improving global outcomes ( kdigo ) , with adoption of a new equation to estimate glomerular filtration rate ( gfr ) and albuminuria to evaluate kidney structural damage . </S>", "<S> this study was performed to estimate the prevalence of ckd in the korean adult population as defined and classified by the kdigo guidelines.methodscross-sectional samples of the fifth korean national health and nutrition examination survey for 2011 to 2012 were examined for adults aged  19 years . </S>", "<S> ckd prevalence was determined based on decreased gfr and albuminuria . </S>", "<S> the gfr was estimated using the ckd epidemiology collaboration creatinine equation , and albuminuria was evaluated using the albumin - to - creatinine ratio ( acr ) in spot urine.resultsof the 16,576 subjects participating in the survey , 10,636 ( 4,758 men , 5,878 women ) were included in the present study . </S>", "<S> the prevalence of ckd was estimated as 7.9% ( 7.8% in 2011 and 8.0% in 2012 , p = 0.770 ) . </S>", "<S> the prevalence of low , moderately increased , high , and very high ckd risk prognosis was 92.0% , 6.3% , 1.1% , and 0.6% , respectively . </S>", "<S> the prevalence of albuminuria ( acr  30 mg / g ) in individuals with gfr  60 ml </S>", "<S> / min/1.73 m2 has reached 5.7% . </S>", "<S> the odds ratios of hypertension and diabetes to ckd were 3.4 and 3.1 in men , and 2.9 and 2.0 in women ( all p < 0.001 ) , respectively.conclusionsa large percentage of ckd patients had albuminuria prior to a decrease in gfr . </S>", "<S> regular laboratory tests for albuminuria for the high - risk group , and especially for hypertensive or diabetic patients , might improve detection of ckd at an early stage . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Study population", "Anthropometry and laboratory data", "Definitions of CKD and other chronic diseases", "Statistical analysis", "RESULTS", "Characteristics of the study population", "Prevalence of CKD and other chronic diseases", "Comparison of GFR using CKD-EPI equations and the MDRD Study equation", "Comparison of ACR with proteinuria", "Associations of risk factors with CKD", "DISCUSSION", "KEY MESSAGE"], "sections": [["chronic kidney disease ( ckd ) is defined as a structural or functional kidney abnormality lasting for 3 or more months .", "the global prevalence of ckd is estimated to be more than 10% , and ckd has emerged as a public health problem .", "adverse outcomes of ckd such as kidney failure , cardiovascular disease ( cvd ) , and premature death can be prevented or delayed when treatment is initiated in the early stages of disease .", "as the earlier stages are often asymptomatic , ckd is usually detected during laboratory evaluation of comorbid conditions .", "glomerular filtration rate ( gfr ) is considered the index of kidney function , and for convenience", "is usually estimated using equations instead of by direct measurement . the modification of diet in renal disease ( mdrd ) study equation , which was replaced by the chronic kidney disease epidemiology collaboration 2009 equations ( ckd - epi equations ) , has been used to estimate gfr .", "however , this tended to underestimate the measured gfr and had less precision , especially at a gfr of 60 ml / min/1.73 m or more .", "the ckd - epi equations were reported as having less bias , improved precision , and greater accuracy than the mdrd study equation at a higher gfr of 60 ml / min/1.73 m or more .", "the ckd - epi equations led to a lower estimation of ckd prevalence , primarily due to a lower prevalence of gfr stage 3 , compared to the mdrd study equation .", "persistently increased protein excretion has usually been considered to be a marker of kidney damage , and screening with urine dipsticks has been acceptable for detecting proteinuria .", "recently , urinary albumin measurement was recommended as a marker of kidney damage , both for its standardization and also because albumin is the most important protein lost in the urine in most ckd", ". the albumin - to - creatinine ratio ( acr ) is the most preferred value among urinary albumin measurements .", "kidney disease : improving global outcomes ( kdigo ) has updated guidelines for defining , diagnosing , staging , and managing ckd since the establishment of the first uniform definitions and staging system for ckd in 2002 .", "more objective criteria , such as the ckd - epi equations for estimating gfr and classification of albuminuria , have been incorporated . moreover ,", "the etiology of ckd was included in the definition ; hypertension and diabetes mellitus ( dm ) are systemic diseases known to cause ckd .", "consequently , kdigo guidelines provide relatively simple and precise methods for early diagnosis of ckd , and tend to yield fewer false positives , thereby avoiding incorrect diagnosis .", "the number of patients treated with renal replacement therapy ( rrt ) including dialysis and transplantation has increased dramatically in korea , from 28,046 in 2000 to 75,042 in 2013 .", "the prevalence of rrt was 1,446 patients per million population ( pmp ) , with an incidence of 234 pmp in 2013 .", "the cause of kidney failure was estimated to be dm in 48.0% , hypertension in 19.7% , and chronic glomerulonephritis in 8.3% of cases . the previously reported ckd prevalence calculated from the mdrd study equation", "the ckd prevalence calculated from the mdrd study equation alone was 8.8% in 2005 and 7.2% in 2007 in korea national health and nutrition examination survey ( knhanes ) subjects aged  20 ; the prevalence calculated using the mdrd study equation with the dipstick proteinuria method has tended to decrease since 2001 in men and since 2005 in women among knhanes i to iv ( 1998 to 2009 ) subjects aged  20 . since there are no reports on ckd prevalence using the ckd - epi equations to estimate gfr and acr to measure albuminuria for koreans , a new analysis of ckd prevalence is necessary .", "this study was therefore designed to investigate the prevalence of ckd in the korean population using knhanes samples , based on the definition and classification by kdigo 2012 guidelines , as well as the impact of changes through a comparison of the ckd - epi and mdrd study equations for the estimation of gfr , and the associations between ckd and major risk factors , including dm , hypertension , age , and gender ."], ["this retrospective , population - based study was performed using data acquired by the knhanes v from 2010 to 2012 .", "knhanes v was a cross - sectional , nationally representative survey with a multistage , stratified , and probability sampling design based on sex , age , and geographical area using household registries .", "knhanes , composed of a health questionnaire survey , physical examination , and nutrition survey , has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention ( kcdc ) .", "the study protocol was approved by the institutional review board of kcdc ( 2010 - 02con-21-c ) , and written informed consents were obtained from all subjects or their guardians .", "knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 .", "the inclusion criteria were ( 1 ) adults aged  19 , and ( 2 ) available serum creatinine ( scr ) , urine albumin , and urine creatinine test results .", "individuals aged  19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 . the health examination was performed in a mobile health examination center .", "was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association .", "three readings were obtained via mercury sphygmomanometer ( baumanometer , baum , copiague , ny , usa ) , and the mean of the second and third reading was reported as the final bp for individual participants .", "body mass index ( bmi ) was calculated as body weight divided by the square of the height , and expressed in units of kg / m .", "blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast .", "a random urine sample ( first urine in the morning , if possible ) was obtained in a conical container .", "the urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day ( neodin medical institute , seoul , korea ) .", "serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako ( wako , osaka , japan ) following the jaffe method ; fasting glucose concentration ( fasting blood glucose , fbg ) by an enzymatic method using pureauto s glu ( sekisui , osaka , japan ) ; total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , and triglycerides by an enzymatic method ; and urine albumin by turbidimetric assay using albumin ( roche , mannheim , germany ) , all performed using a hitachi automatic analyzer 7600 ( hitachi high - technologies co. , tokyo , japan ) , which was calibrated using calibrator for automated systems ( roche diagnostics , indianapolis , in , usa ) .", "glycated hemoglobin ( a1c ) was assayed by high performance liquid chromatography ( hlc-723g7 a1c , tosoh , tokyo , japan ) .", "urine chemistry was tested by test strip ( urysis 2400 cassette , roche ) using urisys 2400 ( roche ) .", "low density lipoprotein cholesterol ( ldl - c ) was computed using the friedewald formula when triglycerides were less than 400 mg / dl .", "ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr .", "gfr was estimated using ckd - epi equations , taking into account gender and serum creatinine .", "individuals with gfr < 60 ml / min/1.73 m or acr  30 mg / g were classified as ckd patients . hypertension was defined as having a systolic blood pressure ( sbp ) of  140 mmhg or a diastolic blood pressure ( dbp ) of  90 mmhg , or having received a physician s diagnosis .", "dm was defined as having a1c  6.5% , fbg of  126 mg / dl , or having received a physician s diagnosis .", "dyslipidemia was defined as having total cholesterol  240 mg / dl , ldl - c  160 mg / dl , hdl - c < 40 mg / dl , triglycerides  200 mg / dl , or having received a physician s diagnosis .", "analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations .", "the study population was separated as a subpopulation , and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias .", "in addition , weight adjustments were applied to the user - missing values to prevent skews in sampling design .", "the demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables .", "the mean and the 95% confidence interval ( ci ) were presented for data on continuous variables , whereas unweighted count with weighted percentage was presented for data on categorical variables .", "the complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories , and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage .", "a subpopulation was added to strata when performing the complex samples chi - square test .", "associations of ckd , gfr , and acr categories with various risk factors , including gender , age , bmi , dm , hypertension , and dyslipidemia were expressed as an odds ratio ( or ) , which was calculated using complex samples multivariate logistic regression analyses ."], ["this retrospective , population - based study was performed using data acquired by the knhanes v from 2010 to 2012 .", "knhanes v was a cross - sectional , nationally representative survey with a multistage , stratified , and probability sampling design based on sex , age , and geographical area using household registries .", "knhanes , composed of a health questionnaire survey , physical examination , and nutrition survey , has been conducted periodically since 1998 to assess the health and nutritional status of the non - institutionalized korean population by the korea centers for disease control and prevention ( kcdc ) .", "the study protocol was approved by the institutional review board of kcdc ( 2010 - 02con-21-c ) , and written informed consents were obtained from all subjects or their guardians .", "knhanes v data of 2010 were excluded as urine albumin was not measured until 2011 .", "the inclusion criteria were ( 1 ) adults aged  19 , and ( 2 ) available serum creatinine ( scr ) , urine albumin , and urine creatinine test results .", "individuals aged  19 were classified as adults in the study due to the kdigo guideline s classification of children as birth to 18 ."], ["blood pressure ( bp ) was measured by a trained nurse following a standard protocol in accordance with the recommendations of the american heart association .", "three readings were obtained via mercury sphygmomanometer ( baumanometer , baum , copiague , ny , usa ) , and the mean of the second and third reading was reported as the final bp for individual participants .", "body mass index ( bmi ) was calculated as body weight divided by the square of the height , and expressed in units of kg / m .", "blood samples were collected from the median cubital or cephalic veins by a skilled nurse after an overnight fast . a random urine sample ( first urine in the morning , if possible )", "the urine and pretreated blood samples were refrigerated and transported to the central laboratory within the day ( neodin medical institute , seoul , korea ) .", "serum creatinine and urine creatinine were assayed by colorimetry using creatinine - hr l - type wako ( wako , osaka , japan ) following the jaffe method ; fasting glucose concentration ( fasting blood glucose , fbg ) by an enzymatic method using pureauto s glu ( sekisui , osaka , japan ) ; total cholesterol , high density lipoprotein cholesterol ( hdl - c ) , and triglycerides by an enzymatic method ; and urine albumin by turbidimetric assay using albumin ( roche , mannheim , germany ) , all performed using a hitachi automatic analyzer 7600 ( hitachi high - technologies co. , tokyo , japan ) , which was calibrated using calibrator for automated systems ( roche diagnostics , indianapolis , in , usa ) .", "glycated hemoglobin ( a1c ) was assayed by high performance liquid chromatography ( hlc-723g7 a1c , tosoh , tokyo , japan ) .", "urine chemistry was tested by test strip ( urysis 2400 cassette , roche ) using urisys 2400 ( roche ) .", "low density lipoprotein cholesterol ( ldl - c ) was computed using the friedewald formula when triglycerides were less than 400 mg / dl ."], ["ckd was defined and classified based on kdigo guidelines using both gfr and urinary acr .", "gfr was estimated using ckd - epi equations , taking into account gender and serum creatinine .", "individuals with gfr < 60 ml / min/1.73 m or acr  30 mg / g were classified as ckd patients .", "hypertension was defined as having a systolic blood pressure ( sbp ) of  140 mmhg or a diastolic blood pressure ( dbp ) of  90 mmhg , or having received a physician s diagnosis .", "dm was defined as having a1c  6.5% , fbg of  126 mg / dl , or having received a physician s diagnosis .", "dyslipidemia was defined as having total cholesterol  240 mg / dl , ldl - c  160 mg / dl , hdl - c < 40 mg / dl , triglycerides  200 mg / dl , or having received a physician s diagnosis ."], ["analyses were performed incorporating the sampling weights to obtain unbiased estimates from the complex knhanes sampling design following the knhanes recommendations .", "the study population was separated as a subpopulation , and the analysis was carried out on the entire knhanes samples for 2011 and 2012 to represent all korean adults without bias .", "in addition , weight adjustments were applied to the user - missing values to prevent skews in sampling design .", "the demographic and biochemical characteristics of the study population were summarized and compared using the complex samples general linear model for continuous variables and the complex samples chi - square test for categorical variables .", "the mean and the 95% confidence interval ( ci ) were presented for data on continuous variables , whereas unweighted count with weighted percentage was presented for data on categorical variables .", "the complex samples chi - square test was used to analyze prevalence of ckd by gfr with acr categories , and gfr comparison between mdrd and ckd - epi equations was expressed as a weighted percentage .", "a subpopulation was added to strata when performing the complex samples chi - square test .", "associations of ckd , gfr , and acr categories with various risk factors , including gender , age , bmi , dm , hypertension , and dyslipidemia were expressed as an odds ratio ( or ) , which was calculated using complex samples multivariate logistic regression analyses .", "all statistical analyses were performed using ibm spss version 21.0 ( ibm corp . , armonk , ny , usa ) . the statistical significance level was set at p < 0.05 ."], ["among the total sample size of 16,576 from 2011 and 2012 , 64.7% satisfied the inclusion criteria ( 10,636 individuals : 5,388 and 5,248 from 2011 and 2012 , respectively ) as shown in fig .", "the composition of neither the total adults nor the study population was statistically different between 2011 and 2012 ( p = 0.232 and p = 0.087 , respectively ) . the study population comprised 81.8% of total adults ( 81.0% and 82.7% from 2011 and 2012 , respectively ,", "the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable , except for hdl - c , ldl - c , and prevalence of dyslipidemia ( table 1 ) .", "the percentage of men was 52.6% , and the mean age was 45.8 years ( 95% ci , 45.2 to 46.3 ) .", "mean serum creatinine was 0.86 mg / dl ( 95% ci , 0.85 to 0.86 ) , with an estimated gfr of 96 ml / min/1.73 m ( 95% ci , 96 to 97 ) .", "mean acr was 18 mg / g ( 95% ci , 15 to 21 ) , with urine albumin 22 g / ml ( 95% ci , 19 to 26 ) and urine creatinine 165 mg / dl ( 95% ci , 163 to 168 ) .", "mean sbp was 118 mmhg ( 95% ci , 117 to 119 ) , and dbp 76 mmhg ( 95% ci , 76 to 77 ) .", "mean fbg was 97 mg / dl ( 95% ci , 97 to 98 ) , and a1c 5.7% ( 95% ci , 5.67 to 5.71 ) .", "mean total cholesterol , hdl - c , ldl - c , and triglycerides were 190 mg / dl ( 95% ci , 188 to 191 ) , 49 mg / dl ( 95% ci , 49 to 50 ) , 114 mg / dl ( 95% ci , 113 to 115 ) , and 136 mg / dl ( 95% ci , 133 to 139 ) , respectively .", "the ckd prevalence in korean adults was estimated as 7.9% ( 7.8% in 2011 and 8.0% in 2012 , p = 0.770 ) ( table 1 ) .", "the prevalence of hypertension , dm , and dyslipidemia was 27.8% ( 27.2% in 2011 and 28.4% in 2012 , p = 0.380 ) , 9.3% ( 9.7% in 2011 and 9.0% in 2012 , p = 0.306 ) , and 32.8% ( 34.6% in 2011 and 31.0% in 2012 , p = 0.004 ) , respectively .", "the distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 .", "the prevalence of low , moderately increased , high , and very high ckd risk prognosis was 92.0% , 6.3% , 1.1% , and 0.6% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m was 2.2% , whereas acr  30 mg / g was 6.5% ( a2 of 5.6% and a3 of 0.9% ) .", "the prevalence of acr  30 mg / g in individuals with gfr  60 ml / min/1.73", "the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig .", "the prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation , and g2 was 29.8% and 38.7% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m by the mdrd study equation was 2.0% , which was significantly less than the 2.2% by ckd - epi equations ( p < 0.001 ) .", "the prevalence of at least 1 + proteinuria in a1 , expected to be fully negative , was 0.1% whereas  or  proteinuria in a2 and a3 , expected to be fully positive , were 5.3% and 0.3% , respectively .", "some interactions between demographic covariates , such as gender , age , and bmi , were present .", "the risk of bmi  25 for men was higher than for women ( or , 1.296 ; 95% ci , 1.164 to 1.443 ; p < 0.001 ) .", "the study population was stratified into six groups by age in 10-year increments : 19 to 28 , 29 to 38 , 39 to 48 , 49 to 58 , 59 to 68 , and  68 .", "the ors of increasing age ( using the age group of 19 to 28 as a reference ) to bmi  25 were 1.731 ( 95% ci , 1.400 to 2.139 ) , 2.150 ( 95% ci , 1.757 to 2.632 ) , 1.689 ( 95% ci , 1.376 to 2.074 ) , 2.092 ( 95% ci , 1.690 to 2.590 ) , and 1.554 ( 95% ci , 1.255 to 1.924 ; p = 0.001 ) , respectively .", "in addition , the composition of men significantly decreased in the  49 age groups ; ors of men to increasing age ( using the age group of 19 to 28 as a reference ) were 0.931 ( 95% ci , 0.784 to 1.105 ) , 0.894 ( 95% ci , 0.750 to 1.065 ) , 0.735 ( 95% ci , 0.625 to 0.864 ) , 0.691 ( 95% ci , 0.578 to 0.825 ) , and 0.505 ( 95% ci , 0.421 to 0.607 ; p = 0.001 ) , respectively . thus , the association analysis was performed separately for each gender . because ckd is defined by both gfr and albuminuria , the associations between the risk factors and either decreased gfr or increased acr were also studied .", "the frequency of women among ckd patients was 54.3% , which was significantly higher than that for men ( p < 0.001 ) . among individuals with acr  30 mg / g", ", women accounted for 55.4% ( p < 0.001 ) ; however , women constituted 48.7% of gfr < 60 ml / min/1.73 m ( p = 0.683 ) .", "the ors of gender to ckd , acr  30 mg / g , and gfr < 60 ml / min/1.73 m for men were 1.354 ( 95% ci , 1.162 to 1.577 ; p", "< 0.001 ) , and 1.057 ( 95% ci , 0.811 to 1.376 ; p = 0.683 ) , respectively .", "the prevalence of bmi  25 , ckd , gfr < 60 ml / min/1.73 m , acr  30 mg / g , dm , hypertension , and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 .", "the ors of covariates to the prevalence of ckd , gfr < 60 ml / min/1.73 m , and acr  30 mg / g , after adjusting for age , bmi , hypertension , dm , and dyslipidemia in men and women are listed in table 6 .", "hypertensive patients showed a higher risk of ckd than the normotensive population ( or , 3.394 for men ; or , 2.935 for women ; both p < 0.0001 ) .", "the ors of hypertension to cause albuminuria were 3.351 ( p < 0.001 ) for men and", "3.067 ( p < 0.001 ) for women , and to decrease gfr , 3.436 ( p < 0.001 ) for men and 1.904 ( p = 0.006 ) for women .", "diabetic patients showed a higher risk of ckd than the nondiabetic population ( or , 3.145 for men ; or , 1.983 for women ; both p < 0.001 ) .", "the ors of dm to cause albuminuria were 3.144 ( p < 0.001 ) for men and", "2.022 ( p < 0.001 ) for women , and to decrease gfr 1.965 ( p = 0.004 ) for men and 1.764 ( p = 0.013 ) for women .", "dyslipidemia showed a significant association with ckd in women ( or , 1.324 ; p = 0.023 ) , but not in men ( or , 1.175 ; p = 0.243 ) .", "increasing age and obesity ( bmi  25 ) showed a positive correlation with ckd ."], ["among the total sample size of 16,576 from 2011 and 2012 , 64.7% satisfied the inclusion criteria ( 10,636 individuals : 5,388 and 5,248 from 2011 and 2012 , respectively ) as shown in fig .", "the composition of neither the total adults nor the study population was statistically different between 2011 and 2012 ( p = 0.232 and p = 0.087 , respectively ) . the study population comprised 81.8% of total adults ( 81.0% and 82.7% from 2011 and 2012 , respectively ,", "the anthropometric and biochemical characteristics of the study population in 2011 and 2012 were comparable , except for hdl - c , ldl - c , and prevalence of dyslipidemia ( table 1 ) .", "the percentage of men was 52.6% , and the mean age was 45.8 years ( 95% ci , 45.2 to 46.3 ) .", "mean serum creatinine was 0.86 mg / dl ( 95% ci , 0.85 to 0.86 ) , with an estimated gfr of 96 ml / min/1.73 m ( 95% ci , 96 to 97 ) .", "mean acr was 18 mg / g ( 95% ci , 15 to 21 ) , with urine albumin 22 g / ml ( 95% ci , 19 to 26 ) and urine creatinine 165 mg / dl ( 95% ci , 163 to 168 ) .", "mean sbp was 118 mmhg ( 95% ci , 117 to 119 ) , and dbp 76 mmhg ( 95% ci , 76 to 77 ) .", "mean fbg was 97 mg / dl ( 95% ci , 97 to 98 ) , and a1c 5.7% ( 95% ci , 5.67 to 5.71 ) .", "mean total cholesterol , hdl - c , ldl - c , and triglycerides were 190 mg / dl ( 95% ci , 188 to 191 ) , 49 mg / dl ( 95% ci , 49 to 50 ) , 114 mg / dl ( 95% ci , 113 to 115 ) , and 136 mg / dl ( 95% ci , 133 to 139 ) , respectively ."], ["the ckd prevalence in korean adults was estimated as 7.9% ( 7.8% in 2011 and 8.0% in 2012 , p = 0.770 ) ( table 1 ) .", "the prevalence of hypertension , dm , and dyslipidemia was 27.8% ( 27.2% in 2011 and 28.4% in 2012 , p = 0.380 ) , 9.3% ( 9.7% in 2011 and 9.0% in 2012 , p = 0.306 ) , and 32.8% ( 34.6% in 2011 and 31.0% in 2012 , p = 0.004 ) , respectively .", "the distribution of ckd prevalence classified by the gfr and acr categories is shown in table 2 .", "the prevalence of low , moderately increased , high , and very high ckd risk prognosis was 92.0% , 6.3% , 1.1% , and 0.6% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m was 2.2% , whereas acr  30 mg / g was 6.5% ( a2 of 5.6% and a3 of 0.9% ) .", "the prevalence of acr  30 mg / g in individuals with gfr  60 ml / min/1.73"], ["the prevalence of each estimated gfr category using ckd - epi equations and the mdrd study equation is shown in fig .", "2 . the prevalence of g1 was 67.9% and 59.4% by ckd - epi equations and the mdrd study equation , and g2 was 29.8% and 38.7% , respectively .", "the prevalence of gfr < 60 ml / min/1.73 m by the mdrd study equation was 2.0% , which was significantly less than the 2.2% by ckd - epi equations ( p < 0.001 ) ."], ["the prevalence of at least 1 + proteinuria in a1 , expected to be fully negative , was 0.1% whereas  or  proteinuria in a2 and a3 , expected to be fully positive , were 5.3% and 0.3% , respectively ."], ["some interactions between demographic covariates , such as gender , age , and bmi , were present .", "the risk of bmi  25 for men was higher than for women ( or , 1.296 ; 95% ci , 1.164 to 1.443 ; p < 0.001 ) .", "the study population was stratified into six groups by age in 10-year increments : 19 to 28 , 29 to 38 , 39 to 48 , 49 to 58 , 59 to 68 , and  68 .", "the ors of increasing age ( using the age group of 19 to 28 as a reference ) to bmi  25 were 1.731 ( 95% ci , 1.400 to 2.139 ) , 2.150 ( 95% ci , 1.757 to 2.632 ) , 1.689 ( 95% ci , 1.376 to 2.074 ) , 2.092 ( 95% ci , 1.690 to 2.590 ) , and 1.554 ( 95% ci , 1.255 to 1.924 ; p = 0.001 ) , respectively .", "in addition , the composition of men significantly decreased in the  49 age groups ; ors of men to increasing age ( using the age group of 19 to 28 as a reference ) were 0.931 ( 95% ci , 0.784 to 1.105 ) , 0.894 ( 95% ci , 0.750 to 1.065 ) , 0.735 ( 95% ci , 0.625 to 0.864 ) , 0.691 ( 95% ci , 0.578 to 0.825 ) , and 0.505 ( 95% ci , 0.421 to 0.607 ; p = 0.001 ) , respectively . thus , the association analysis was performed separately for each gender . because ckd is defined by both gfr and albuminuria , the associations between the risk factors and either decreased gfr or increased acr were also studied .", "the frequency of women among ckd patients was 54.3% , which was significantly higher than that for men ( p < 0.001 ) . among individuals with acr  30 mg / g ,", "women accounted for 55.4% ( p < 0.001 ) ; however , women constituted 48.7% of gfr < 60 ml / min/1.73 m ( p = 0.683 ) .", "the ors of gender to ckd , acr  30 mg / g , and gfr < 60 ml / min/1.73 m for men were 1.354 ( 95% ci , 1.162 to 1.577 ; p", "< 0.001 ) , 1.416 ( 95% ci , 1.193 to 1.682 ; p < 0.001 ) , and 1.057 ( 95% ci , 0.811 to 1.376 ; p = 0.683 ) , respectively .", "the prevalence of bmi  25 , ckd , gfr < 60 ml / min/1.73 m , acr  30 mg / g , dm , hypertension , and dyslipidemia stratified by age categories in men and women are shown in tables 4 and 5 .", "the ors of covariates to the prevalence of ckd , gfr < 60 ml / min/1.73 m , and acr  30 mg / g , after adjusting for age , bmi , hypertension , dm , and dyslipidemia in men and women are listed in table 6 .", "hypertensive patients showed a higher risk of ckd than the normotensive population ( or , 3.394 for men ; or , 2.935 for women ; both p < 0.0001 ) .", "the ors of hypertension to cause albuminuria were 3.351 ( p < 0.001 ) for men and", "3.067 ( p < 0.001 ) for women , and to decrease gfr , 3.436 ( p < 0.001 ) for men and 1.904 ( p = 0.006 ) for women .", "diabetic patients showed a higher risk of ckd than the nondiabetic population ( or , 3.145 for men ; or , 1.983 for women ; both p < 0.001 ) .", "the ors of dm to cause albuminuria were 3.144 ( p < 0.001 ) for men and", "2.022 ( p < 0.001 ) for women , and to decrease gfr 1.965 ( p = 0.004 ) for men and 1.764 ( p = 0.013 ) for women .", "dyslipidemia showed a significant association with ckd in women ( or , 1.324 ; p = 0.023 ) , but not in men ( or , 1.175 ; p = 0.243 ) . increasing age and obesity ( bmi  25 ) showed a positive correlation with ckd ."], ["this study is the first to report ckd prevalence using gfr estimated by using ckd - epi equations together with acr for albuminuria in the korean population .", "the prevalence of ckd was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged  19 , and the ckd risk prognosis was reported in table 2 .", "the ckd risk prognosis from moderately increased to very high reported in this study was lower than that in the usa .", "gfr and albuminuria are independent and complementary predictors of important clinical outcomes , including ckd progression , end - stage renal disease , acute kidney injury , cardiovascular mortality , and all - cause mortality [ 16 - 19 ] .", "the frequencies of gfr < 60 ml / min/1.73 m showed sharp increases starting from around age 50 for men and 60 for women , whereas those of acr  30 mg / g increased relatively gradually , starting from earlier ages , as shown in table 4 and 5 .", "the rate of decline of measured gfr was reported to double in kidney transplantation donors aged > 45 years .", "the prevalence of estimated gfr < 60 ml / min/1.73 m using ckd - epi equations and the mdrd study equation was 2.2% and 2.0% in this study , but 2.6% and 3.2% , respectively in the previous study .", "the statistical method used in the current study was complex sample analysis incorporating sampling weight , in contrast to the previous study which did not consider weight in the analysis .", "the frequencies of gfr stage g1 and g2 by ckd - epi equations were 67.7% and 29.8% , whereas those by the mdrd study equation were 59.4% and 38.7% , respectively , in this study .", "these numbers are comparable to the previous study , considering the different analytical methods ; the frequencies of gfr stage g1 and g2 by ckd - epi equations were 64.5% and 28.9% , whereas those by the mdrd study equation were 47.6% and 49.2% , respectively .", "these results confirm the tendency to underestimate gfr by the mdrd study equation when gfr is 60 ml / min/1.73 m or greater .", "the previous report of lower prevalence of gfr stage g3 using ckd - epi equations than with the mdrd study equation was not observed in this study . among the population of the previous study , the frequencies of g3 by ckd - epi equations and the mdrd study equation ( 33.2% and 37.2% , respectively ) were far greater than those among the korean population ( 2.1% and 1.9% , respectively ) .", "the prevalence of acr  30 mg / g in the entire study population , and acr  30 mg / g in individuals with gfr  60 ml / min/1.73", "the prevalence of acr  30 mg / g in the entire study population and acr  30 mg / g in individuals with gfr  60 ml / min/1.73", "m aged  35 years was 10.2% and 8.7% , respectively , in the previous korean population .", "besides the different age criteria , the populations of the previous and current study are quite different ; the previous study was designed by the researcher group considering age , gender , and city factors not using the knhanes sample data .", "although the absolute values are different , the important point in common is that a large number of people have albuminuria before gfr decreases to < 60 ml / min/1.73 m ( 85.3% and 87.7% of the albuminuria cases did not have decreased gfr in the previous and current study , respectively ) . as shown in table 3", ", test strips did not detect albuminuria in 81.5% of cases . at present , laboratory tests for albuminuria are not routinely performed during regular physical examinations in korea .", "the prevalence of ckd in men and women was 6.9% and 9.1% , respectively ( p < 0.001 ) . besides gender itself , age distribution , frequency of obesity", "( bmi  25 ) , and concurrent chronic diseases , including dm , hypertension , and dyslipidemia , were different according to gender .", "thus , the risk of ckd is higher in women than men ( or , 1.354 ; p < 0.001 ) , and the difference seems to be caused by the susceptibility to albuminuria ( or , 1.416 ; p < 0.001 ) rather than decreased gfr ( or , 1.057 ; p = 0.683 ) .", "when gfr was estimated using the mdrd study equation , the increased risks of gfr < 60 ml / min/1.73", "m in women were observed in previous studies : or , 3.16 ( p < 0.001 ) using knhanes iii and iv-1 ; frequencies of 3.4% , 9.7% , 10.2% , and 4.6% in women , and 1.0% , 5.4% , 3.1% , and 2.6% were seen in men through knhanes i - iv .", "there seemed to be no difference in acr according to gender in the previous study ( 10.1% and 10.3% in women and men , respectively ) .", "a similar observation was reported for proteinuria by gender ( frequencies of 3.3% , 2.3% , 1.4% , and 1.9% in women , and 3.1% , 3.0% , 2.8% , and 2.2% in men through knhanes", "direct comparison is difficult due to the difference in the designs of study populations   and laboratory test methods using dipstick proteinuria and acr .", "increasing age and obesity ( bmi  25 ) correlated significantly with ckd , as shown in table 6 , which agreed with previous studies [ 11 - 13 ] .", "hypertension and dm are both major risk factors and consequences of ckd [ 21 - 23 ] . concurrent chronic diseases such as dm and hypertension increased the risk of ckd consistently [ 11 - 13 ]", ". diabetic patients are susceptible to glomerular disease and hypertensive patients to renal vascular disease .", "since the prevalence of dm was higher in men than women , with the opposite for prevalence of ckd , the or of dm to ckd in men was higher than women , as shown in table 6 .", "moreover , dm was correlated with increased acr rather than decreased gfr in this study . since increased acr appeared at an earlier age in women than men , and decreased gfr", "was preceded by increased acr , the difference between ors to gfr < 60 ml / min/1.73 m and acr  30 mg / g in hypertensive women was greater than hypertensive men . on the other hand ,", "limited availability of direct ldl - c measurement , unstandardized laboratory assay methods for hdl - c , and lack of a uniform definition of dyslipidemia between the studies made it difficult to evaluate their associations .", "the major limitation of this study is the lack of the evaluation for chronicity assessment and use of single measurements for laboratory tests due to the cross - sectional data characteristics . according to the definition of ckd by the kdigo guideline", "the rationale for defining chronicity is to differentiate ckd from acute kidney diseases , which may require different interventions and have different etiologies and outcomes .", "in addition , there is still the discrepancy between measured and estimated gfr using ckd - epi equations .", "gfr estimation using equations is convenient , but measurement of gfr should be considered to diagnose a specific patient .", "finally , association analysis could not provide conclusive causal relationships due to the study characteristics .", "however , the representative features of the knhanes sample data can not be overlooked . in conclusion ,", "the prevalence of ckd using estimated gfr by the ckd - epi equation and acr for albuminuria measurement was 7.9% in the knhanes samples of 2011 and 2012 in korean adults aged  19 .", "the prevalence of albuminuria ( acr  30 mg / g ) before decline in gfr ( gfr  60 ml / min/1.73 m ) was 5.7% . despite kdigo guidelines to prevent or delay progression of ckd in its early stages , implementation remains difficult due to practical limitations .", "the failure to recognize ckd in its early stage may result in complications such as premature rrt .", "thus , urinalysis to detect albumin should be strongly recommended for patients with higher risk of ckd and cvd . as there is a consensus about the risk of ckd with dm and hypertension , regular albuminuria testing for at least these patients to detect kidney disease at an early stage"], ["1 . the prevalence of chronic kidney disease ( ckd ) was estimated to be 7.9% in the korea national health and nutrition examination survey samples of 2011 and 2012 in korean adults aged  19 .", "early identification of ckd paired with appropriate management and earlier referral to specialty kidney services results in economic and clinical benefits .", "the majority of ckd patients have albuminuria prior to a decrease in glomerular filtration rate .", "4 . regular laboratory tests for albuminuria in the high - risk group , especially for hypertensive or diabetes mellitus patients , should contribute to early detection of ckd ."]]}
{"article_id": "PMC4421456", "article_text": ["while it is true that not all patients are able to dialyse themselves at home , it is a sad fact that many are never given the chance , despite the fact that  patient choice has never been more discussed as an answer to almost any health care problem . although studies suggest that renal physicians and nurses regard home dialysis as the best treatment , it is in decline in most european countries , yet  in - centre hospital - based dialysis is the most expensive and most disempowering option available .", "the practical barriers to home therapy vary from unit to unit but need to be identified and overcome if patients are to benefit from self - management", ". however , it can be difficult to convince both staff and patients if they have no direct experience or training in this aspect of renal replacement therapy .", "patient pathway to ( relative ) freedom  a step which can be made much easier if staff are familiar with the  stages of change model   and have a basic understanding of motivational interviewing .", "pre - emptive living donor transplantation should always be promoted as the first - line treatment for kidney failure . however , where that is not possible , patients must receive timely , adequate and unbiased information and advice regarding the complete array of dialysis options available , including home - based peritoneal dialysis ( pd ) and haemodialysis ( hd ) .", "interestingly , a comparison of survival in canadian patients treated with nocturnal hd or deceased donor kidney transplantation showed no difference between the two treatments , suggesting that this intensive dialysis modality may be a bridge to transplantation , or even a suitable alternative , in the absence of an available living donor .", "traditionally around the world , medical and nursing staff , sometimes in conjunction with a social worker , will ", ". a clear example of this was evident in the 1990s , when the uk had a large pd population simply because space in hospital hd facilities was limited at that time , and home hd had shrunk to the extent that most uk units no longer had viable programmes . since that time ,", "in - centre hd facilities in the uk have expanded enormously , such that in some areas supply exceeds demand and the pd population has approximately halved in number . therefore , many patients currently are not able to choose home hd and patient selection presumably does not generally occur .", "staff are unlikely to be able to advocate a therapy of which they have no experience and are even less likely to advocate one which they perceive to be unavailable .", "therefore , health service managers and commissioners also have an enabling role and must embrace the value of self - care by providing the practical resources required .", "understandably , a patient may never be adequately informed about home hd or assisted automated peritoneal dialysis if these modalities are not operating in the unit concerned .", "many patients will assume that they are not suitable for a home therapy if it is not offered to them , without realizing why it has not been offered .", "ideally , every patient should learn about every dialysis modality , accepting that it is probably pointless to discuss pd with a patient who has previously had major abdominal surgery .", "if this were done , then some patients would inevitably demand the creation of home services where none currently exists , and in one or two uk units , this has already happened .", "experience within our own service suggests that where a dialysis unit enables and actively encourages self - management , patients will tend to select themselves , and if well motivated may overcome significant difficulties , such as needle phobia , housing problems and literacy issues , to exceed the expectations or predictions of even quite experienced dialysis staff .", "patients then become advocates themselves and can provide other patients with the necessary motivation to consider a home treatment , such that they approach staff , rather than vice versa .", "interestingly , the general consensus among nephrologists in canada , usa and uk in 2006 was that 45% of patients are suitable for a home therapy , and if you question the staff in your local hd unit , very few would wish themselves to be dialysed  in - centre if the need arose . despite this", ", the reality is that pd numbers are in decline and home hd is virtually non - existent in many areas .", "selection to medical and nursing staff simply does not work and that many capable patients are not finding their way through ", "there is much in our health care culture that expects care and treatment to be administered by caring and expert professionals , and this expectation is present in the providers of health care as much as it is in the recipients .", "there are of course individual differences in both staff and patients willingness to embrace self - care , but if we assume that cultures and individual preferences are not fixed , then we may find ways to move people along the self - care continuum .", "often , patients contemplating the need for renal replacement therapy are understandably and predictably hoping that dialysis will be  done for them and will probably shy away from the thought of  doing it at home. clearly , it is easier and quicker to point them in the direction of in - centre hd than to embark on a difficult and time - consuming discussion around self - care home dialysis and training . to undertake this task , the staff must first be themselves totally convinced that home treatment is in the patient s best interest  if not , the patient will rapidly detect any hint of uncertainty and further discussion is probably futile .", "this also means that all members of staff with whom the patient comes into contact must be equally capable of espousing some or all of the benefits of home therapy , from nurse , to doctor , to social worker , to dietician and others . in other words ,", "ethos must be that home therapy is not only viable but preferable and beneficial for those who are able to pursue it . for a unit s staff to be able to talk to patients with confidence", "requires direct experience of home dialysis , but obviously in many units which do not have a full range of home therapies , this may initially be impossible . under these circumstances ,", "collaboration with another unit may enable staff to gain the training and experience required to be able to talk with authority and instill confidence in the patient .", "visiting patients in their home environment is an essential part of training for both medical and nursing staff .", "it is known that patients who start dialysis after adequate preparation tend to select a home therapy [ 5 , 6 ] .", "this is encouraging , but in some instances , education can default to telling the patient a series of facts , and once the full list of facts is ticked off , the patient is expected to make a choice .", "yet , we all know from our own educational experiences that this approach alone is insufficient . on the face of it , it seems plausible that suitable patients would accept the benefits of a home therapy , once they have been  educated as to the reasons for the recommendation , and staff may be bemused when they do not do so .", "this aspect of everyday human behaviour has been most extensively studied in the field of smoking cessation , where it is recognized that someone who has accepted the need to ", "quit is much more likely to be successful than someone who has not , despite both being aware of the future health benefits .", "the stages of change model   suggests that , for most people , a change in behaviour occurs gradually , with the patient moving from being uninterested , unaware or unwilling to make a change ( pre - contemplation ) , to considering a change ( contemplation ) , to deciding and preparing to make a change ( preparation then action ) .", "therefore , before a patient is able to begin to engage in discussion about any dialysis therapy , they must have reached a point of acceptance that dialysis is necessary . if they are not at this point , then any attempt at ", "education will be largely futile , especially if it is simply repeated when it may be counter productive and alienating . even once they have accepted , the need to change a patient may still not be able to engage in the process of change due to real and significant hindrances , such as needle phobia , general anxiety , low mood or fatigue .", "5the goal for chronic kidney disease patients at the pre - contemplation stage is to begin to think about the likely 5need for dialysis in the future and where the dialysis should take place .", "the task for physicians is to empathetically engage the patient in contemplating this change to their life . during this stage , patients may appear argumentative , hopeless or in  denial , and the natural tendency is for physicians to try to  convince them with more facts , which usually engenders resistance . \n", "figure 1 describes the stages of change which anyone confronting a life changing event , such as the need to start dialysis , must pass through in order to come to terms with their new situation .", "asking the patient to indicate which rung of they ladder they feel they are on can be a helpful first step . bringing a patient to the stage of taking action in a defined timescale", "is of great importance since progressive renal disease will not wait , and it is clear that those who start dialysis in an unplanned fashion fare less well . on the other hand , a patient who has reached", "the action stage may be able to select themselves for either home or hospital therapy as they begin to understand what is , and is not , desirable and possible given their specific circumstances .", "the uk national institute for health and clinical excellence ( nice ) produced guidance on home compared to hospital hd for patients with end - stage renal failure in 2002 .", "suitable for home hd should be offered the choice of having hd in the home or in a renal unit . the definition of ", "practical characteristics of patients suitable for home hd according to nice guidance 2002 although these guidelines are entirely clear and sensible , the manchester experience is that the definition of ", "enthusiastic and determined patients who might initially have been deemed unsuitable are found to be not only capable but successful home dialysers .", "indeed , the current feeling is that very few physical disabilities preclude home treatment if the patient is keen to proceed , and level of enthusiasm is a more reliable guide .", "once a patient has arrived at the point of choosing a home therapy , the pathway to their first dialysis at home must be as smooth and pot - hole free as possible .", "this requires some resources and infrastructure to be already in place , and if they are not , then all but the most determined patient is likely to be put off .", "many patients commitment at this stage is fragile and if the path is too rough or steep , or not well signposted , they may understandably decide to turn back .", "therefore , from this point on , communication , planning and action must be slick and efficient in order to build the patient s confidence .", "the experience and confidence of the staff is crucial so that even if the patient is unsure of the process , they feel fully able to trust the team around them and to know who to turn to for reassurance or answers .", "it requires staff to engage with patients in conversations about what is important to them and what they value in more general terms ( e.g. freedom , independence , safety , quality and quantity of life ) and about what the complex hindrances to self - care may be  so that an individualized response to these sorts of questions can move patients along the self - care continuum .", "when faced with the spectre of dialysis treatment , most of us would not naturally opt for a home therapy .", "traditional methods of patient selection have not prevented a steady decline in home therapies over the past two decades , yet most dialysis unit staff say they would opt for a home therapy for themselves .", "the stages of change model coupled with basic motivational interviewing techniques can enable staff and patients to engage with each other in a more meaningful way such that many patients will be better able to understand the benefits of self - management and treatment at home .", "practical infrastructure barriers must also be tackled in order to smooth the pathway to home dialysis ."], "abstract_text": ["<S> pre - emptive living donor transplantation should always be promoted as the first - line treatment for kidney failure . </S>", "<S> where that is not possible , patients must receive timely information and advice regarding all dialysis options available , including home - based peritoneal dialysis and haemodialysis . where a dialysis unit enables and actively encourages self - management , patients will tend to select themselves , and if well motivated may overcome significant difficulties to exceed the expectations or predictions of dialysis staff . </S>", "<S> patients then become advocates themselves and can provide other patients with the necessary motivation to consider a home treatment , such that they approach staff , rather than vice versa . for staff to be able to talk to patients with confidence requires direct experience of home dialysis , but in units which do not have a full range of home therapies , this may initially be difficult . </S>", "<S> visiting patients in their home environment is an essential part of training for both medical and nursing staff . before a patient is able to begin to engage in discussion about any dialysis therapy </S>", "<S> , they must have reached a point of acceptance that dialysis is necessary . </S>", "<S> if they are not at this point , then any attempt at  education will be largely futile . </S>", "<S> once a patient has arrived at the point of choosing a home therapy , the pathway to their first dialysis at home must be as smooth and problem - free as possible . </S>"], "labels": null, "section_names": ["Introduction", "Patient selection and patient choice", "Staff education", "Patient education", "NICE guidance", "Enabling training", "Conclusions"], "sections": [["while it is true that not all patients are able to dialyse themselves at home , it is a sad fact that many are never given the chance , despite the fact that  patient choice has never been more discussed as an answer to almost any health care problem . although studies suggest that renal physicians and nurses regard home dialysis as the best treatment , it is in decline in most european countries , yet  in - centre hospital - based dialysis is the most expensive and most disempowering option available .", "the practical barriers to home therapy vary from unit to unit but need to be identified and overcome if patients are to benefit from self - management", ". however , it can be difficult to convince both staff and patients if they have no direct experience or training in this aspect of renal replacement therapy .", "patient pathway to ( relative ) freedom  a step which can be made much easier if staff are familiar with the  stages of change model   and have a basic understanding of motivational interviewing ."], ["pre - emptive living donor transplantation should always be promoted as the first - line treatment for kidney failure . however , where that is not possible , patients must receive timely , adequate and unbiased information and advice regarding the complete array of dialysis options available , including home - based peritoneal dialysis ( pd ) and haemodialysis ( hd ) .", "interestingly , a comparison of survival in canadian patients treated with nocturnal hd or deceased donor kidney transplantation showed no difference between the two treatments , suggesting that this intensive dialysis modality may be a bridge to transplantation , or even a suitable alternative , in the absence of an available living donor .", "traditionally around the world , medical and nursing staff , sometimes in conjunction with a social worker , will ", ". a clear example of this was evident in the 1990s , when the uk had a large pd population simply because space in hospital hd facilities was limited at that time , and home hd had shrunk to the extent that most uk units no longer had viable programmes . since that time ,", "in - centre hd facilities in the uk have expanded enormously , such that in some areas supply exceeds demand and the pd population has approximately halved in number . therefore , many patients currently are not able to choose home hd and patient selection presumably does not generally occur .", "staff are unlikely to be able to advocate a therapy of which they have no experience and are even less likely to advocate one which they perceive to be unavailable .", "therefore , health service managers and commissioners also have an enabling role and must embrace the value of self - care by providing the practical resources required .", "understandably , a patient may never be adequately informed about home hd or assisted automated peritoneal dialysis if these modalities are not operating in the unit concerned .", "many patients will assume that they are not suitable for a home therapy if it is not offered to them , without realizing why it has not been offered .", "ideally , every patient should learn about every dialysis modality , accepting that it is probably pointless to discuss pd with a patient who has previously had major abdominal surgery .", "if this were done , then some patients would inevitably demand the creation of home services where none currently exists , and in one or two uk units , this has already happened .", "experience within our own service suggests that where a dialysis unit enables and actively encourages self - management , patients will tend to select themselves , and if well motivated may overcome significant difficulties , such as needle phobia , housing problems and literacy issues , to exceed the expectations or predictions of even quite experienced dialysis staff .", "patients then become advocates themselves and can provide other patients with the necessary motivation to consider a home treatment , such that they approach staff , rather than vice versa ."], ["interestingly , the general consensus among nephrologists in canada , usa and uk in 2006 was that 45% of patients are suitable for a home therapy , and if you question the staff in your local hd unit , very few would wish themselves to be dialysed  in - centre if the need arose . despite this", ", the reality is that pd numbers are in decline and home hd is virtually non - existent in many areas .", "selection to medical and nursing staff simply does not work and that many capable patients are not finding their way through ", "there is much in our health care culture that expects care and treatment to be administered by caring and expert professionals , and this expectation is present in the providers of health care as much as it is in the recipients .", "there are of course individual differences in both staff and patients willingness to embrace self - care , but if we assume that cultures and individual preferences are not fixed , then we may find ways to move people along the self - care continuum .", "often , patients contemplating the need for renal replacement therapy are understandably and predictably hoping that dialysis will be  done for them and will probably shy away from the thought of  doing it at home. clearly , it is easier and quicker to point them in the direction of in - centre hd than to embark on a difficult and time - consuming discussion around self - care home dialysis and training . to undertake this task , the staff must first be themselves totally convinced that home treatment is in the patient s best interest  if not , the patient will rapidly detect any hint of uncertainty and further discussion is probably futile .", "this also means that all members of staff with whom the patient comes into contact must be equally capable of espousing some or all of the benefits of home therapy , from nurse , to doctor , to social worker , to dietician and others . in other words ,", "ethos must be that home therapy is not only viable but preferable and beneficial for those who are able to pursue it . for a unit s staff to be able to talk to patients with confidence", "requires direct experience of home dialysis , but obviously in many units which do not have a full range of home therapies , this may initially be impossible . under these circumstances ,", "collaboration with another unit may enable staff to gain the training and experience required to be able to talk with authority and instill confidence in the patient .", "visiting patients in their home environment is an essential part of training for both medical and nursing staff ."], ["it is known that patients who start dialysis after adequate preparation tend to select a home therapy [ 5 , 6 ] .", "this is encouraging , but in some instances , education can default to telling the patient a series of facts , and once the full list of facts is ticked off , the patient is expected to make a choice .", "yet , we all know from our own educational experiences that this approach alone is insufficient . on the face of it , it seems plausible that suitable patients would accept the benefits of a home therapy , once they have been  educated as to the reasons for the recommendation , and staff may be bemused when they do not do so .", "this aspect of everyday human behaviour has been most extensively studied in the field of smoking cessation , where it is recognized that someone who has accepted the need to ", "quit is much more likely to be successful than someone who has not , despite both being aware of the future health benefits .", "the stages of change model   suggests that , for most people , a change in behaviour occurs gradually , with the patient moving from being uninterested , unaware or unwilling to make a change ( pre - contemplation ) , to considering a change ( contemplation ) , to deciding and preparing to make a change ( preparation then action ) .", "therefore , before a patient is able to begin to engage in discussion about any dialysis therapy , they must have reached a point of acceptance that dialysis is necessary . if they are not at this point , then any attempt at ", "education will be largely futile , especially if it is simply repeated when it may be counter productive and alienating . even once they have accepted , the need to change a patient may still not be able to engage in the process of change due to real and significant hindrances , such as needle phobia , general anxiety , low mood or fatigue .", "5the goal for chronic kidney disease patients at the pre - contemplation stage is to begin to think about the likely 5need for dialysis in the future and where the dialysis should take place .", "the task for physicians is to empathetically engage the patient in contemplating this change to their life . during this stage , patients may appear argumentative , hopeless or in  denial , and the natural tendency is for physicians to try to  convince them with more facts , which usually engenders resistance . \n", "figure 1 describes the stages of change which anyone confronting a life changing event , such as the need to start dialysis , must pass through in order to come to terms with their new situation .", "asking the patient to indicate which rung of they ladder they feel they are on can be a helpful first step . bringing a patient to the stage of taking action in a defined timescale", "is of great importance since progressive renal disease will not wait , and it is clear that those who start dialysis in an unplanned fashion fare less well . on the other hand , a patient who has reached", "the action stage may be able to select themselves for either home or hospital therapy as they begin to understand what is , and is not , desirable and possible given their specific circumstances ."], ["the uk national institute for health and clinical excellence ( nice ) produced guidance on home compared to hospital hd for patients with end - stage renal failure in 2002 .", "suitable for home hd should be offered the choice of having hd in the home or in a renal unit . the definition of ", "practical characteristics of patients suitable for home hd according to nice guidance 2002 although these guidelines are entirely clear and sensible , the manchester experience is that the definition of ", "enthusiastic and determined patients who might initially have been deemed unsuitable are found to be not only capable but successful home dialysers .", "indeed , the current feeling is that very few physical disabilities preclude home treatment if the patient is keen to proceed , and level of enthusiasm is a more reliable guide ."], ["once a patient has arrived at the point of choosing a home therapy , the pathway to their first dialysis at home must be as smooth and pot - hole free as possible .", "this requires some resources and infrastructure to be already in place , and if they are not , then all but the most determined patient is likely to be put off .", "many patients commitment at this stage is fragile and if the path is too rough or steep , or not well signposted , they may understandably decide to turn back .", "therefore , from this point on , communication , planning and action must be slick and efficient in order to build the patient s confidence .", "the experience and confidence of the staff is crucial so that even if the patient is unsure of the process , they feel fully able to trust the team around them and to know who to turn to for reassurance or answers .", "it requires staff to engage with patients in conversations about what is important to them and what they value in more general terms ( e.g. freedom , independence , safety , quality and quantity of life ) and about what the complex hindrances to self - care may be  so that an individualized response to these sorts of questions can move patients along the self - care continuum ."], ["when faced with the spectre of dialysis treatment , most of us would not naturally opt for a home therapy .", "traditional methods of patient selection have not prevented a steady decline in home therapies over the past two decades , yet most dialysis unit staff say they would opt for a home therapy for themselves .", "the stages of change model coupled with basic motivational interviewing techniques can enable staff and patients to engage with each other in a more meaningful way such that many patients will be better able to understand the benefits of self - management and treatment at home .", "practical infrastructure barriers must also be tackled in order to smooth the pathway to home dialysis ."]]}
{"article_id": "PMC4243011", "article_text": ["the epstein  barr virus ( ebv ) was described by michael anthony epstein and yvonne barr , who isolated ebv virus particles from endemic burkitt lymphoma specimens .", "ebv is a double stranded dna virus of the herpes virus family and is the most common virus affecting humans .", "the infection occurs through oral transmission via saliva . by the age of 40 , over 90  % of the world population has experienced an ebv infection .", "ebv also plays a pathogenic role , mainly in the development of several t cell and b cell lymphoproliferations ( see below ) but is also associated with different types of rare epithelial and mesenchymal neoplasms .", "ebv is mainly a b - lymphotropic and epitheliotropic virus and rarely infects t cells and nk cells .", "the life cycle of ebv is composed of a lytic state and a latency state , the latter allowing lifelong persistence of the virus in the host .", "the replication cycle of ebv is composed of three phases : ( i ) entry into either epithelial cells or naive b cells after oropharyngeal transmission , ( ii ) lytic replication , and ( iii ) latency .", "once a cell has become infected , the viral capsid dissolves and the dna is transported into the cell nucleus . in epithelial cells ,", "the entry of ebv into the cell is usually directly followed by the lytic replication of the virus , which leads to production of infectious virions using the virus s own replication machinery .", "ebv persists in b cells throughout the differentiation from naive b cells to germinal center cells to long - lived memory b cells . in memory b cells ,", "dependent on the expression of specific ebv - associated proteins and rnas , three different viral latency types have been described , which can be reproduced to a certain extent by immunohistochemical markers   ( table  1).table 1ebv - latency types and expressed antigenslatency typeebv - associated antigens0/ieber , ebna1ii ( default program)eber , ebna1 , lmp1 , lmp2biii ( growth program)eber , ebna1 - 6 , lmp1 , lmp2a  +  blyticall lytic antigens ( e.g. , zebra ) ebv - latency types and expressed antigens", "most humans become infected with ebv during childhood , when the infection usually proceeds asymptomatically or with mild symptoms that are undistinguishable from other viral infections .", "if the infection occurs in young adults and adolescents , it can manifest as mononucleosis ( glandular fever ) .", "ebv tonsillitis differs from unspecific tonsillitis related to other causes in its histologically detectable polymorphic lymphoid infiltration with abundant plasmablasts and occasional hodgkin - like cells expanding in the interfollicular area of the tonsils . in affected lymph nodes , an extension of the interfollicular areas with a similarly polymorphic infiltration to that in tonsils", "can be observed . within the peripheral blood and bone marrow , cd8 positive lymphoblasts ( pfeiffer cells )", "might be observed . the histomorphological picture of primary ebv infection shares histomorphological features with other ebv - driven lymphoproliferative diseases ( lpd , table  2 ) .", "therefore , the definite diagnosis of infectious mononucleosis is based on the following features : ( i ) young patient age , ( ii ) sole or main manifestation in the tonsils , ( iii ) absence of immunosuppression in the patient , and most importantly ( iv ) a serological test indicating ebv primary infection ( positive igm titer).table 2spectrum of polymorphic ebv driven lpd with variable neoplastic potential spectrum of polymorphic ebv driven lpd with variable neoplastic potential following the primary infection , patients become asymptomatic virus carriers with a very limited number of ebv - positive b cells .", "a tissue correlate of this phenomenon is the occasional presence of single small ebv - encoded rna ( eber)-positive b cells , so called bystander cells , within lymph nodes , mucosa - associated lymphatic tissue , or lungs .", "rare complications of primary ebv infection , such as the chronic active ebv ( caebv ) disease and/or ebv - associated hemophagocytic syndrome have recently been reviewed in this journal .", "since the vast majority of humans have been infected by ebv in childhood and adolescence , ebv - associated lpds in adults usually arise from reactivation of the virus from ebv - infected memory cells .", "such reactivation is generally considered to consist in an escape from the normal immunosurveillance and preferentially occurs at sites representing the physiological reservoir of ebv - infected b cells , such as the lymphatic tissues and mucosa .", "in these patients , age - related decline in immunocompetence is assumed to be the cause of ebv - lpd [ 4 , 5 ] .", "however , the diagnostic pathologist should stress that a clinical work - up of the immune status is warranted once an ebv reactivation is detected .", "ebv reactivation in adults occurs primarily in the form of ebv - lpd presenting with a polymorphic histological and clinical picture .", "dependent on the clinical scenario and the preferential site of involvement , different types of polymorphic ebv - lpd can be distinguished .", "they are outlined in table  2 and include polymorphic posttransplantation lymphoproliferations ( ptld ) , ebv - positive mucocutaneous ulceration ( ebvmcu ) , and lymphomatoid granulomatosis . depending on the clinical scenario and the extent of tissue involvement , these conditions cause variable clinical symptoms .", "polymorphic ebv - lpd regress in most patients if immunocompetence can be reestablished , e.g. , by withholding immunosuppressive therapy . nevertheless , a subgroup of polymorphic ebv - lpd progress to more aggressive diseases , usually accompanied by a transformation to the morphology of a fully developed lymphoma ( designated as  monomorphic ", "polymorphic ebv - associated lymphoproliferations share histomorphological and immunophenotypical features , which are certainly not specific but should alert the histopathologist to an ebv - driven disease when analyzing lymphatic proliferations .", "these features include the following : tissue necrosisepithelial ulcerationangiotropic vasculitis - like lymphoid infiltratespolymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cellsintermingled large t cells ( often cd8 positive)b blasts , with weak cd20 expression and expression of cd30 epithelial ulceration angiotropic vasculitis - like lymphoid infiltrates polymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cells intermingled large t cells ( often cd8 positive ) b blasts , with weak cd20 expression and expression of cd30 if the clinical scenario ( immunosuppression ) or the histological features are suggestive of an ebv - lpd , testing by eber in situ hybridization should be initiated ( see below ) .", "when the diagnosis of an ebv - positive polymorphic lpd has been confirmed by detection of ebv in the tissue , the nomenclature ( classification ) of the disease is dependent on ( i ) the clinical context and ( ii ) the involved site ( table  2 ) .", "ebv - lpd histologically resembling a lymphomatoid granulomatosis might occur after organ transplantation , in which case the disorder is named according to both the clinical scenario and the histopathological picture as  ebv - positive polymorphic ptld under the histopathological picture of a lymphomatoid granulomatosis  .", "the most important diagnostic message from the pathologist to the treating clinican should be ( i ) the ebv association of the lesion and ( ii ) the clear distinction of polymorphic ebv - lpd from fully developed lymphomas.fig .", "a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown . as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head )", "f 400 histomorphology of ebv - associated lymphoproliferative diseases . a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown .", "as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head ) are indicated .", "ebv - positive lymphomas occur in immunocompetent patients , but are much more frequent in immunocompromised patients . in the latter patient group ,", "the latency type of ebv detectable in the lymphoma cells frequently but not exclusively indicates higher viral activity with expression of more ebv encoded proteins ( latency type 3 ) and sometimes active viral replication .", "lymphomas occurring in immunocompetent patients , such as hodgkin lymphoma , usually display latency types 0/1 or 2 with a more restricted pattern of ebv - associated proteins and no viral replication ( table  3 ) .", "the diagnostic criteria for these lymphomas have been described elsewhere and do not necessarily require the detection of ebv because of the presence of ebv in the lymphoma cells . in rare cases , detection of ebv is a mandatory diagnostic feature ( e.g. , in ebv - positive lpd of childhood ) .", "nevertheless , in certain lymphoma entities , e.g. , nasal and extranasal nk t cell lymphoma , angioimmunoblastic t cell lymphoma , primary effusion lymphoma , and plasmablastic lymphoma , ebv association remains an important clue towards the correct diagnosis .", "of further relevance might be the identification of ebv - positive diffuse large b cell lymphoma ( dlbcl ) of the elderly .", "histological features that are useful for identifying dlbcl candidates for screening for ebv have been described as angiotropic growth , necrosis , and polymorphic , plasmablastic or hodgkin - like differentiation and expression of cd30 .table 3ebv - positive lymphomas and latency types and their association with immunocompromised statelineageimmunocompetence of patientsentitylatency typeb cellcompetentclassical hodgkin lymphoma2endemic burkitt lymphoma0/1sporadic burkitt lymphoma0/1ebv  +  dlbcl of the elderlyvariableebv  +  dlbcl associated with chronic inflammation ( pyothorax lymphoma)predominantly 3compromisedprimary effusion lymphoma0/1plasmablastic lymphoma0/1lymphomatoid granulomatosis grade 3 and dlbcl arising from the former3monomorphic ptld \n variablelymphomas associated with hiv infection \n variablelymphoproliferative disease associated with primary immune disorders \n variableother iatrogenic immunodeficiency - associated lymphoproliferative disorders \n 3 t cellcompetentangioimmunoblastic t cell lymphoma0/1 or 2extranodal nk / t cell lymphoma2compromisedebv - positive t cell lymphoproliferative disease of childhood and young adults \n variable \n diseases under this category are further specified according to histopathology \n the immunodeficiency in many of these young patients is postulated but not always objectifiable \n dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease ebv - positive lymphomas and latency types and their association with immunocompromised state \n diseases under this category are further specified according to histopathology \n the immunodeficiency in many of these young patients is postulated but not always objectifiable \n dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease", "the diagnostic criteria for ebv - positive lymphomas are described in the current who classification . however , distinguishing polymorphic ebv - lpd and a fully developed ebv - positive lymphoma can be challenging .", "the most important feature , large sheets of ebv - positive blasts and absence of a polymorphic cellular picture , should raise suspicion of an ebv - positive lymphoma , especially a dlbcl .", "a specifically difficult task is the differentiation of a polymorphic ebv - lpd from a fully developed classical hodgkin lymphoma , because the latter may be composed of a similarly polymorphic cellular infiltrate to that of the polymorphic ebv - lpd .", "pathologists should be aware of the fact that proteins expressed in hodgkin lymphoma , such as cd15 , can also be observed in polymorphic ebv - lpd , like ebvmcu , and therefore the final diagnosis will depend not only on the histopathological features but also on the clinical context , including the site of involvement .", "for example , hodgkin lymphomas rarely involve epithelial barrier organs , but ebvmcu by definition always do ( table  2 ) . in our experience ,", "clonality testing is not helpful in differentiating ebv - positive lymphoma from polymorphic lymphoproliferations , as viral - driven lpd can also result in a clonal expansion of t and b cells .", "testing for ebna2 expression is useful in some cases , since expression of ebna2 strongly argues against hodgkin lymphoma ( table  2).table 4differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphomafeaturehodgkin lymphomapolymorphic ebv  +  lpdhistomorphologymixed cellularity infiltration with hodgkin ( like ) cells , necrosispredominant epitheloid cells and eosinophilspredominant plasmablasts , plasmacells , angiotropismimmunophenotype of large cellscd30  >  cd20cd20  >  cd30expression of ebv antigens eber  +  cells equal to lmp1  +  cells eber  +  large cells only", "ebna2 negative eber  +  cells more than lmp1  +  cells eber  +  small and large cells ebna2 + /clinical scenariopredominant immunocompetentpredominant immunocompromisedsite mediastinum nodal >  >  extranodal extranodal >  >  nodal", "mucosa differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphoma", "the question as to which specimen should be tested for ebv depends on the histopathological findings on the one hand and on the clinical context on the other hand .", "table  3 summarizes our criteria and favored method of testing for ebv , fig .", "if pathologists are confronted with lymphoid tissue histologically resembling inflammatory ( reactive ) tissue in immunocompetent patients , the decision whether or not to test for ebv is primarily based on histological features ( table  3 ) . in immunocompromised patients ,", "the method of testing should be eber in situ hybridization , which is most sensitive for detecting ebv , especially in lesions that can not necessarily be expected to express other viral antigens such as lmp1 ( table  3 ) .", "since indolent ( low grade ) b cell lymphomas are virtually never associated with ebv , testing can not be recommended . for other entities , which are listed in tables  3 and 5 , testing should be part of the standard diagnostic work - up .", "immunohistochemical staining for lmp1 is sufficient for hodgkin lymphoma , since these lymphomas express the latency type 2 .", "however , other lymphomas listed in table  5 , such as diffuse large b cell lymphomas , require testing by eber because lmp1 is frequently not detectable.fig .", "2an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) .", "eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells .", "d 400table 5recommendations for selecting tissue specimen and methods for ebv testinglymphomanodal and extranodal lymphatic tissue not presenting overt lymphoma ( polymorphic lpd)histopathological featuresclinical contexttesting by ebertesting by ebertesting by eberb cell lymphomas   endemic burkitt lymhoma hodgkin - like large cells and hodgkin or reed - sternberg cells inherited immunodeficiency   plasmablastic lymphoma   primary effusion lymphoma posttransplantation   pyothorax associated lymphoma   dlbcl with histological features suspicious for ebv association necrosis drug induced immunocompromised state angiotropism of blastic cells   any monomorphic ptld   lymphomatoid granulomatosis plasmablastic infiltrate hiv infectiont cell lymphomas   ailt   extranodal nk / t cell lymphoma   ebv - positive t cell lymphoproliferative disease of childhood and young adultstesting by eber or lmp   hodgkin lymphomatesting by eber , lmp1 and ebna2   differential diagnosis between hodgkin lymphoma and ptld \n ptld  posttransplantation lymphoproliferative disease , aitl  angioimmunoblastic t cell lymphoma an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) .", "eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells .", "original magnification a  d 400 recommendations for selecting tissue specimen and methods for ebv testing \n ptld  posttransplantation lymphoproliferative disease , aitl  angioimmunoblastic t cell lymphoma", "although epidemiologic data are lacking , ebv - lpd distinct from overt lymphoma will be increasingly recognized .", "this is due on the one hand to the aging of many populations , with an increase in age - associated ebv reactivation . on the other hand , widespread access to commercial assays and", "automatization leads to an increasing use of in situ hybridization for eber by diagnostic pathologists .", "additionally , newly recognized subgroups of lymphomas , such as the ebv - positive dlbcl of the elderly , require the detection of ebv in more lymphoma entities as part of the diagnostic process ."], "abstract_text": ["<S> lymphoproliferations associated with epstein  barr virus ( ebv ) in adult patients pose a diagnostic challenge for pathologists for several reasons . </S>", "<S> first , the ebv lymphoproliferations represent a clinically and histologically very broad spectrum ranging from self - limiting lymphoproliferations to manifest malignant lymphomas . </S>", "<S> second , the classification of these diseases is not solely based on histopathology but rather requires a synopsis of clinical as well as pathological features . and third , a resource - efficient diagnostic procedure demands a deliberate strategy for selecting the tissue specimens that are to be tested for ebv . </S>", "<S> we describe how the clinical context and histological features may indicate to histopathologists which lymphatic tissues should be tested for the presence of ebv and how these features guide the classification . </S>", "<S> we provide recommendations as to which biopsy specimens should be investigated for ebv and which methods for detecting viral association are appropriate . </S>"], "labels": null, "section_names": ["Introduction", "Primary infection by EBVclinical and histological features", "Reactivation of EBV in adults", "EBV-positive lymphomas", "Distinguishing polymorphic EBV-LPD and EBV-positive lymphoma", "Recommendations for EBV testing", "Perspective"], "sections": [["the epstein  barr virus ( ebv ) was described by michael anthony epstein and yvonne barr , who isolated ebv virus particles from endemic burkitt lymphoma specimens .", "ebv is a double stranded dna virus of the herpes virus family and is the most common virus affecting humans .", "the infection occurs through oral transmission via saliva . by the age of 40 , over 90  % of the world population has experienced an ebv infection .", "ebv also plays a pathogenic role , mainly in the development of several t cell and b cell lymphoproliferations ( see below ) but is also associated with different types of rare epithelial and mesenchymal neoplasms .", "ebv is mainly a b - lymphotropic and epitheliotropic virus and rarely infects t cells and nk cells .", "the life cycle of ebv is composed of a lytic state and a latency state , the latter allowing lifelong persistence of the virus in the host .", "the replication cycle of ebv is composed of three phases : ( i ) entry into either epithelial cells or naive b cells after oropharyngeal transmission , ( ii ) lytic replication , and ( iii ) latency .", "once a cell has become infected , the viral capsid dissolves and the dna is transported into the cell nucleus . in epithelial cells ,", "the entry of ebv into the cell is usually directly followed by the lytic replication of the virus , which leads to production of infectious virions using the virus s own replication machinery .", "ebv persists in b cells throughout the differentiation from naive b cells to germinal center cells to long - lived memory b cells . in memory b cells ,", "dependent on the expression of specific ebv - associated proteins and rnas , three different viral latency types have been described , which can be reproduced to a certain extent by immunohistochemical markers   ( table  1).table 1ebv - latency types and expressed antigenslatency typeebv - associated antigens0/ieber , ebna1ii ( default program)eber , ebna1 , lmp1 , lmp2biii ( growth program)eber , ebna1 - 6 , lmp1 , lmp2a  +  blyticall lytic antigens ( e.g. , zebra ) ebv - latency types and expressed antigens"], ["most humans become infected with ebv during childhood , when the infection usually proceeds asymptomatically or with mild symptoms that are undistinguishable from other viral infections .", "if the infection occurs in young adults and adolescents , it can manifest as mononucleosis ( glandular fever ) .", "ebv tonsillitis differs from unspecific tonsillitis related to other causes in its histologically detectable polymorphic lymphoid infiltration with abundant plasmablasts and occasional hodgkin - like cells expanding in the interfollicular area of the tonsils . in affected lymph nodes , an extension of the interfollicular areas with a similarly polymorphic infiltration to that in tonsils", "can be observed . within the peripheral blood and bone marrow , cd8 positive lymphoblasts ( pfeiffer cells )", "might be observed . the histomorphological picture of primary ebv infection shares histomorphological features with other ebv - driven lymphoproliferative diseases ( lpd , table  2 ) .", "therefore , the definite diagnosis of infectious mononucleosis is based on the following features : ( i ) young patient age , ( ii ) sole or main manifestation in the tonsils , ( iii ) absence of immunosuppression in the patient , and most importantly ( iv ) a serological test indicating ebv primary infection ( positive igm titer).table 2spectrum of polymorphic ebv driven lpd with variable neoplastic potential spectrum of polymorphic ebv driven lpd with variable neoplastic potential following the primary infection , patients become asymptomatic virus carriers with a very limited number of ebv - positive b cells .", "a tissue correlate of this phenomenon is the occasional presence of single small ebv - encoded rna ( eber)-positive b cells , so called bystander cells , within lymph nodes , mucosa - associated lymphatic tissue , or lungs .", "rare complications of primary ebv infection , such as the chronic active ebv ( caebv ) disease and/or ebv - associated hemophagocytic syndrome have recently been reviewed in this journal ."], ["since the vast majority of humans have been infected by ebv in childhood and adolescence , ebv - associated lpds in adults usually arise from reactivation of the virus from ebv - infected memory cells .", "such reactivation is generally considered to consist in an escape from the normal immunosurveillance and preferentially occurs at sites representing the physiological reservoir of ebv - infected b cells , such as the lymphatic tissues and mucosa .", "in these patients , age - related decline in immunocompetence is assumed to be the cause of ebv - lpd [ 4 , 5 ] .", "however , the diagnostic pathologist should stress that a clinical work - up of the immune status is warranted once an ebv reactivation is detected .", "ebv reactivation in adults occurs primarily in the form of ebv - lpd presenting with a polymorphic histological and clinical picture .", "dependent on the clinical scenario and the preferential site of involvement , different types of polymorphic ebv - lpd can be distinguished .", "they are outlined in table  2 and include polymorphic posttransplantation lymphoproliferations ( ptld ) , ebv - positive mucocutaneous ulceration ( ebvmcu ) , and lymphomatoid granulomatosis . depending on the clinical scenario and the extent of tissue involvement , these conditions cause variable clinical symptoms .", "polymorphic ebv - lpd regress in most patients if immunocompetence can be reestablished , e.g. , by withholding immunosuppressive therapy . nevertheless , a subgroup of polymorphic ebv - lpd progress to more aggressive diseases , usually accompanied by a transformation to the morphology of a fully developed lymphoma ( designated as  monomorphic ", "polymorphic ebv - associated lymphoproliferations share histomorphological and immunophenotypical features , which are certainly not specific but should alert the histopathologist to an ebv - driven disease when analyzing lymphatic proliferations .", "these features include the following : tissue necrosisepithelial ulcerationangiotropic vasculitis - like lymphoid infiltratespolymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cellsintermingled large t cells ( often cd8 positive)b blasts , with weak cd20 expression and expression of cd30 epithelial ulceration angiotropic vasculitis - like lymphoid infiltrates polymorphic picture rich in large cells , hodgkin - like cells , plasmablasts and plasma cells intermingled large t cells ( often cd8 positive ) b blasts , with weak cd20 expression and expression of cd30 if the clinical scenario ( immunosuppression ) or the histological features are suggestive of an ebv - lpd , testing by eber in situ hybridization should be initiated ( see below ) .", "when the diagnosis of an ebv - positive polymorphic lpd has been confirmed by detection of ebv in the tissue , the nomenclature ( classification ) of the disease is dependent on ( i ) the clinical context and ( ii ) the involved site ( table  2 ) .", "ebv - lpd histologically resembling a lymphomatoid granulomatosis might occur after organ transplantation , in which case the disorder is named according to both the clinical scenario and the histopathological picture as  ebv - positive polymorphic ptld under the histopathological picture of a lymphomatoid granulomatosis  .", "the most important diagnostic message from the pathologist to the treating clinican should be ( i ) the ebv association of the lesion and ( ii ) the clear distinction of polymorphic ebv - lpd from fully developed lymphomas.fig .", "a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown . as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head )", "f 400 histomorphology of ebv - associated lymphoproliferative diseases . a and d illustrates the features of a primary ebv infection in the oral mucosa of a child . as examples of ebv reactivation , an ebv - associated mucocutaneous ulcer in the anal mucosa in a patient treated with azathioprin for myastenia ( b and e ) and a lymphomatoid granulomatosis in the lung of a patient ( c and f ) are shown .", "as shared features in all lesions necrosis ( arrow ) and angiotropism ( arrow head ) are indicated ."], ["ebv - positive lymphomas occur in immunocompetent patients , but are much more frequent in immunocompromised patients . in the latter patient group ,", "the latency type of ebv detectable in the lymphoma cells frequently but not exclusively indicates higher viral activity with expression of more ebv encoded proteins ( latency type 3 ) and sometimes active viral replication .", "lymphomas occurring in immunocompetent patients , such as hodgkin lymphoma , usually display latency types 0/1 or 2 with a more restricted pattern of ebv - associated proteins and no viral replication ( table  3 ) .", "the diagnostic criteria for these lymphomas have been described elsewhere and do not necessarily require the detection of ebv because of the presence of ebv in the lymphoma cells . in rare cases , detection of ebv is a mandatory diagnostic feature ( e.g. , in ebv - positive lpd of childhood ) .", "nevertheless , in certain lymphoma entities , e.g. , nasal and extranasal nk t cell lymphoma , angioimmunoblastic t cell lymphoma , primary effusion lymphoma , and plasmablastic lymphoma , ebv association remains an important clue towards the correct diagnosis .", "of further relevance might be the identification of ebv - positive diffuse large b cell lymphoma ( dlbcl ) of the elderly .", "histological features that are useful for identifying dlbcl candidates for screening for ebv have been described as angiotropic growth , necrosis , and polymorphic , plasmablastic or hodgkin - like differentiation and expression of cd30 .table 3ebv - positive lymphomas and latency types and their association with immunocompromised statelineageimmunocompetence of patientsentitylatency typeb cellcompetentclassical hodgkin lymphoma2endemic burkitt lymphoma0/1sporadic burkitt lymphoma0/1ebv  +  dlbcl of the elderlyvariableebv  +  dlbcl associated with chronic inflammation ( pyothorax lymphoma)predominantly 3compromisedprimary effusion lymphoma0/1plasmablastic lymphoma0/1lymphomatoid granulomatosis grade 3 and dlbcl arising from the former3monomorphic ptld \n variablelymphomas associated with hiv infection \n variablelymphoproliferative disease associated with primary immune disorders \n variableother iatrogenic immunodeficiency - associated lymphoproliferative disorders \n 3 t cellcompetentangioimmunoblastic t cell lymphoma0/1 or 2extranodal nk / t cell lymphoma2compromisedebv - positive t cell lymphoproliferative disease of childhood and young adults \n variable \n diseases under this category are further specified according to histopathology \n the immunodeficiency in many of these young patients is postulated but not always objectifiable \n dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease ebv - positive lymphomas and latency types and their association with immunocompromised state \n diseases under this category are further specified according to histopathology \n the immunodeficiency in many of these young patients is postulated but not always objectifiable \n dlbcl diffuse large b cell lymphoma , ptld posttransplant lymphoproliferative disease"], ["the diagnostic criteria for ebv - positive lymphomas are described in the current who classification . however , distinguishing polymorphic ebv - lpd and a fully developed ebv - positive lymphoma can be challenging .", "the most important feature , large sheets of ebv - positive blasts and absence of a polymorphic cellular picture , should raise suspicion of an ebv - positive lymphoma , especially a dlbcl .", "a specifically difficult task is the differentiation of a polymorphic ebv - lpd from a fully developed classical hodgkin lymphoma , because the latter may be composed of a similarly polymorphic cellular infiltrate to that of the polymorphic ebv - lpd .", "pathologists should be aware of the fact that proteins expressed in hodgkin lymphoma , such as cd15 , can also be observed in polymorphic ebv - lpd , like ebvmcu , and therefore the final diagnosis will depend not only on the histopathological features but also on the clinical context , including the site of involvement .", "for example , hodgkin lymphomas rarely involve epithelial barrier organs , but ebvmcu by definition always do ( table  2 ) . in our experience ,", "clonality testing is not helpful in differentiating ebv - positive lymphoma from polymorphic lymphoproliferations , as viral - driven lpd can also result in a clonal expansion of t and b cells .", "testing for ebna2 expression is useful in some cases , since expression of ebna2 strongly argues against hodgkin lymphoma ( table  2).table 4differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphomafeaturehodgkin lymphomapolymorphic ebv  +  lpdhistomorphologymixed cellularity infiltration with hodgkin ( like ) cells , necrosispredominant epitheloid cells and eosinophilspredominant plasmablasts , plasmacells , angiotropismimmunophenotype of large cellscd30  >  cd20cd20  >  cd30expression of ebv antigens eber  +  cells equal to lmp1  +  cells eber  +  large cells only", "ebna2 negative eber  +  cells more than lmp1  +  cells eber  +  small and large cells ebna2 + /clinical scenariopredominant immunocompetentpredominant immunocompromisedsite mediastinum nodal >  >  extranodal extranodal >  >  nodal", "mucosa differential diagnostic of polymorphic ebv - positive lymphoproliferative disease ( lpd ) compared to hodgkin lymphoma"], ["the question as to which specimen should be tested for ebv depends on the histopathological findings on the one hand and on the clinical context on the other hand .", "table  3 summarizes our criteria and favored method of testing for ebv , fig .", "if pathologists are confronted with lymphoid tissue histologically resembling inflammatory ( reactive ) tissue in immunocompetent patients , the decision whether or not to test for ebv is primarily based on histological features ( table  3 ) . in immunocompromised patients ,", "the method of testing should be eber in situ hybridization , which is most sensitive for detecting ebv , especially in lesions that can not necessarily be expected to express other viral antigens such as lmp1 ( table  3 ) .", "since indolent ( low grade ) b cell lymphomas are virtually never associated with ebv , testing can not be recommended . for other entities , which are listed in tables  3 and 5 , testing should be part of the standard diagnostic work - up .", "immunohistochemical staining for lmp1 is sufficient for hodgkin lymphoma , since these lymphomas express the latency type 2 .", "however , other lymphomas listed in table  5 , such as diffuse large b cell lymphomas , require testing by eber because lmp1 is frequently not detectable.fig .", "2an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) .", "eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells .", "d 400table 5recommendations for selecting tissue specimen and methods for ebv testinglymphomanodal and extranodal lymphatic tissue not presenting overt lymphoma ( polymorphic lpd)histopathological featuresclinical contexttesting by ebertesting by ebertesting by eberb cell lymphomas   endemic burkitt lymhoma hodgkin - like large cells and hodgkin or reed - sternberg cells inherited immunodeficiency   plasmablastic lymphoma   primary effusion lymphoma posttransplantation   pyothorax associated lymphoma   dlbcl with histological features suspicious for ebv association necrosis drug induced immunocompromised state angiotropism of blastic cells   any monomorphic ptld   lymphomatoid granulomatosis plasmablastic infiltrate hiv infectiont cell lymphomas   ailt   extranodal nk / t cell lymphoma   ebv - positive t cell lymphoproliferative disease of childhood and young adultstesting by eber or lmp   hodgkin lymphomatesting by eber , lmp1 and ebna2   differential diagnosis between hodgkin lymphoma and ptld \n ptld  posttransplantation lymphoproliferative disease , aitl  angioimmunoblastic t cell lymphoma an ebv - associated mucocutaneous ulcer in the colonic mucosa ( a hematoxilin and eosin ) .", "eber usually labels the majority of ebv - infected cells , whereas lmp1 and ebna2 if positive in the lesion stain only a subset of all ebe - positive cells .", "original magnification a  d 400 recommendations for selecting tissue specimen and methods for ebv testing \n ptld  posttransplantation lymphoproliferative disease , aitl  angioimmunoblastic t cell lymphoma"], ["although epidemiologic data are lacking , ebv - lpd distinct from overt lymphoma will be increasingly recognized .", "this is due on the one hand to the aging of many populations , with an increase in age - associated ebv reactivation . on the other hand , widespread access to commercial assays and", "automatization leads to an increasing use of in situ hybridization for eber by diagnostic pathologists .", "additionally , newly recognized subgroups of lymphomas , such as the ebv - positive dlbcl of the elderly , require the detection of ebv in more lymphoma entities as part of the diagnostic process ."]]}
{"article_id": "PMC4841877", "article_text": ["echinococcosis or cystic hydatid disease is an endemic parasitic disease in human populations in iran and some parts of the world .", "it is caused by larvae stage of echinococcus granulosus ( 1 ) . in the life cycle of e. granulosus ,", "clinical treatment of cysts includes albendazole ( abz ) or mebendazole ( mbz ) therapy in combination with either surgical resection ( 2 ) .", "the role of enzymes in living organism is clear and remarkable and the worms are seriously dependent on these activities .", "parasite components such as enzymes have specific biological functions , which are necessary for parasite survival and are supposed to have an important role in host - parasite interactions and disease progress ( 3 ) .", "parasite s enzymes are attractive purposes that are be explored for the development of diagnostic method and vaccines .", "they mediate processes like tissue invasion , feeding , evasion ( escape the immune system ) of host immune response etc .", "glutathione - s - transferase ( gst ) is an enzyme , which has a significant role in the detoxification of parasite metabolites ( endogeous ) , host metabolites ( xenobiotics ) and drugs through their conjugation to glutathione ( 5 ) .", "gst activity in e. granulosus has been described in the cytosolic portion of protoscoleces obtained from sheep cysts and activated by pre - treatment of protoscoleces with gst inducers ( 6 ) .", "alkaline phosphates ( alp ) is an enzyme that plays an important role in dampening host immune responses and also plays a role in feeding parasites ( 7 ) .", "most phosphatases have been found in the absorption system of cestodes , excretory system in trematodes and intestinal cells of the nematode ( 8) .", "proteolytic enzymes of parasites have been given more attention than other enzymes , because they play a vital role in parasite survival and are involved in many fundamental physiologic processes ( 3 ) .", "the activities of protease described in e. granulosus ( 9 ) , has been detected in hydatid cyst fluid , cyst wall and in protoscoleces .", "this enzyme is responsible for breakdown of proteins in all living tissues in order to be used by the cells ( 10 ) .", "in addition to their known role in the catabolism , they have a part in protein processing in evasion from immune system , leaving the cyst , molting of the parasites and in diagnosis , especially cysteine proteases as serological markers .", "proteases have generally been identified as potential drug targets in parasites ( 11 , 12 ) .", "the purpose of this study was to determine the effects of abz and mbz on the activity of the gst , alp and proteases in the protoscoleces of hydatid cyst and to evaluate their inhibitory effects on enzyme activity .", "protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran ( center of iran ) .", "protoscoleces were allowed to settle in a 50 ml falcon tube , and then washed several times in phosphate - buffered saline ( pbs ph , 7.2 ) .", "viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays ( 13 ) .", "mbz used in this study was obtained from rouzdarou pharmaceutical company ( iran ) and abz was purchased from tolide daruhai dami iran company .", "no : k4111 - 500 ) , 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each ] containing 500l protoscoleces and 1", "g / ml abz and/ mbz [ stock solution 1 mg / ml of dimethyl sulphoxide ( dmso ) ] were considered as test groups and 10 culture medium [ five culture containing 500 l protoscoleces with 0.6 l dmso , and five culture medium without dmso ] regarded as control groups and were incubated at 37 c in 5% co2 ( 14 ) .", "dmso , and five culture medium the culture supernatants containing the parasite e / s products were collected at time interval , every 12 h. at the end of each time interval , the entire 1 ml of culture medium ( culture supernatants ) was removed and replaced with the same volume of fresh medium ( 13 ) .", "the precipitates were discarded and the total protein and enzyme activities were measured as follows : the concentrations of total proteins of e / s samples were measured by bradford method , which involves reacting the e / s samples with a dye that binds to protein . to measure", "the protein concentration , standard solutions ( bovine serum albumin ) and e / s products were prepared and the bradford reagent ( 100 mg coomassie brilliant blue g-250 , 50 ml 95% ethanol , 100 ml 85% phosphoric acid ) was added .", "the absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature .", "a standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated ( 15 , 16 ) . in order to measure the activity of gst in e / s samples , reagent stock including potassium phosphate buffer 0.1 m , 100 mm reduced glutathione ( gsh ) and 100 mm 1-chloro-2,4- dinitrobenzene ( cdnb ) substrates were prepared in a microtube .", "to each test , from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well .", "the same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min .", "finally , total gst activity ( u / ml ) , of samples was calculated . to calculate the specific activity of gst enzyme , the rate of enzyme activity was divided by the mg protein concentration ( 17 ) .", "eight hundred l of buffer reagent ( r1 ) and 200 l of substrate reagent ( r2 ) were poured into cuvette and mixed .", "then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure .", "sasein solution 0.65% ( 6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm , ph7.5 ) prepared and incubated at 37 c for 5 min , and then e / s samples were added to test tubes and were incubated 37 c for 10 min . the reaction was stopped using trichloroacetic acid ( 110 mm tca , prepared by diluting a 6.1n stock 1:55 with purified water ) .", "e / s products were added to control tubes simultaneously and incubated for 30 min at 37 c , and then centrifuged for 5 min at 14000 g at 25 c .", "the supernatant was poured into the test tube ; 1cc sodium carbonate solution ( 500 mm sodium carbonate solution , prepared using 53 mg / ml of anhydrous sodium carbonate in purified water ) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c .", "finally , tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm .", "solution l - tyrosin 1.1 mm , dw , na2co3 , phenol ) and reported as units /ml enzyme ( 16 ) .", "folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color . in order to determine the statistically significant difference between protein concentrations , gst ,", "alp and protease activities of e / s samples of treated and control groups , t - test was used .", "a duplicate set of samples were taken for each test and at the end , their average was taken in to account .", "protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran ( center of iran ) .", "protoscoleces were allowed to settle in a 50 ml falcon tube , and then washed several times in phosphate - buffered saline ( pbs ph , 7.2 ) .", "viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays ( 13 ) .", "mbz used in this study was obtained from rouzdarou pharmaceutical company ( iran ) and abz was purchased from tolide daruhai dami iran company .", "no : k4111 - 500 ) , 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each ] containing 500l protoscoleces and 1 g / ml abz and/ mbz [ stock solution 1 mg / ml of dimethyl sulphoxide ( dmso ) ] were considered as test groups and 10 culture medium [ five culture containing 500 l protoscoleces with 0.6 l dmso , and five culture medium without dmso ] regarded as control groups and were incubated at 37 c in 5% co2 ( 14 ) .", "the culture supernatants containing the parasite e / s products were collected at time interval , every 12 h. at the end of each time interval , the entire 1 ml of culture medium ( culture supernatants ) was removed and replaced with the same volume of fresh medium ( 13 ) .", "the precipitates were discarded and the total protein and enzyme activities were measured as follows :", "the concentrations of total proteins of e / s samples were measured by bradford method , which involves reacting the e / s samples with a dye that binds to protein . to measure the protein concentration , standard solutions ( bovine serum albumin ) and e / s products", "were prepared and the bradford reagent ( 100 mg coomassie brilliant blue g-250 , 50 ml 95% ethanol , 100 ml 85% phosphoric acid ) was added .", "the absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature .", "a standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated ( 15 , 16 ) .", "in order to measure the activity of gst in e / s samples , reagent stock including potassium phosphate buffer 0.1 m , 100 mm reduced glutathione ( gsh ) and 100 mm 1-chloro-2,4- dinitrobenzene ( cdnb ) substrates were prepared in a microtube . to each test , from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well .", "the same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min . finally , total gst activity ( u / ml ) , of samples was calculated . to calculate the specific activity of gst enzyme , the rate of enzyme activity was divided by the mg protein concentration ( 17 ) .", "eight hundred l of buffer reagent ( r1 ) and 200 l of substrate reagent ( r2 ) were poured into cuvette and mixed .", "then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure .", "sasein solution 0.65% ( 6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm , ph7.5 ) prepared and incubated at 37 c for 5 min , and then e / s samples were added to test tubes and were incubated 37 c for 10 min .", "the reaction was stopped using trichloroacetic acid ( 110 mm tca , prepared by diluting a 6.1n stock 1:55 with purified water ) .", "e / s products were added to control tubes simultaneously and incubated for 30 min at 37 c , and then centrifuged for 5 min at 14000 g at 25 c .", "the supernatant was poured into the test tube ; 1cc sodium carbonate solution ( 500 mm sodium carbonate solution , prepared using 53 mg / ml of anhydrous sodium carbonate in purified water ) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c .", "finally , tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm .", "solution l - tyrosin 1.1 mm , dw , na2co3 , phenol ) and reported as units /ml enzyme ( 16 ) .", "folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color .", "in order to determine the statistically significant difference between protein concentrations , gst , alp and protease activities of e / s samples of treated and control groups , t - test was used .", "a duplicate set of samples were taken for each test and at the end , their average was taken in to account .", "the mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml , respectively .", "the mean protein concentration in the control group was 6.06 g / ml . protein concentration of e / s products of protoscoleces treated and control groups \n p<0.05 ; between control and treated groups \n \n p>0.05 ; between treated groups \n  the results of gst , alp and protease activity are shown in table 2 .", "gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml .", "alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml , receptively .", "alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml .", "protease specific activity in abz and mbz treated protoscoleces e / s products was not detected , while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml", ". gst , alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005 , t = test or sample n / d = not detected \n gst  activity  =  od / min0.0096total  voluesample  voluedilution  factor \n  alp activity =  od  2764 \n protease  activity=(umole  tyrosine  equivalents ", "released)(total  volume  of  assay)volume  of  enzymetime  of  assayvolume  of  filtratespecific  activity  =  units / ml  enzymemg  solid \n  statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group ( p<0.05 ) .", "a significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz ( p<0.05 ) .", "meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz ( p>0.05 )", "the mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml , respectively .", "the mean protein concentration in the control group was 6.06 g / ml . protein concentration of e / s products of protoscoleces treated and control groups \n p<0.05 ; between control and treated groups \n \n p>0.05 ; between treated groups \n", "gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml .", "alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml , receptively .", "alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml .", "protease specific activity in abz and mbz treated protoscoleces e / s products was not detected , while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml .", "gst , alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005 , t = test or sample n / d = not detected \n gst  activity  =  od / min0.0096total  voluesample  voluedilution  factor \n  alp activity =  od  2764 \n protease  activity=(umole  tyrosine  equivalents  released)(total  volume  of  assay)volume  of  enzymetime  of  assayvolume  of  filtratespecific  activity  =  units / ml  enzymemg  solid \n", "statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group ( p<0.05 ) .", "a significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz ( p<0.05 ) .", "meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz ( p>0.05 )", "glutathione transferases ( gst ) are multifunctional enzyme present in both animal and plant kingdoms .", "the enzymes are regarded as parts of the phase ii detoxification system that catalyse glutathione ( gsh ) conjugation of a multitude of exogenous and endogenous toxic compounds ( 18 ) .", "gst is one of the major detoxification system component found in helminthes , particularly at high levels in cestodes and digeneas . in the tissue extracts of parasites gst activity", "gst activity exists in e / s products and in the surface of digenea worms such as schistosoma , fasciola and hookworm necator .", "gst in e / s products act as an anti - inflammatory agent and neutral lipid peroxidation products in the mucous membranes ( 20 , 21 ) .", "researches related to chemotherapy and immunotherapy , have identified glutathione of parasitic worms independently as a potential target for treatment ( 22 ) .", "these acts include the digestion of host s tissue in order to provide food for parasites , preventing blood coagulation , facilitating entry into the host s immune system , and disrupting it ( 23 ) .", "alp is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules , including nucleotides and proteins ( 24 ) .", "the phosphatases have been found frequently in the structure of absorption like tegument of cestodes , intestinal cell of the nematodes and excretory system of trematodes .", "while alkaline phosphatases have been found in adult cestode , the larval forms have a predominance of acid phosphates . this change in these enzymes", "may be associated with the growth and development of the reproductive system in adult worms ( 8) . alp as protein plays a role in immune evasion by generating the potent immunosuppressant adenosine .", "since adenosine is also an important nutrient , alp may also play a role in parasite feeding ( 7 ) .", "several studies have been carried out on characterization of hydatid cyst protoscoleces e / s products ( 25 , 26 ) . in the present study ,", "the effect of abz and mbz on protein content and some enzymatic activities of e / s products were examined .", "protein concentration in treated groups e / s products were less than that of the control group e / s sample .", "abz and mbz cause degenerative changes in intestinal cells of the worm by binding to the colchicine - sensitive site of tubulin , therefore blocks its polymerization into microtubules and inhibits the cell proliferation in metaphase stage ( 27 , 28 ) .", "based on these findings we can expect the decreasing of total protein in treated parasite culture media , which correlates our study that shows an average protein of test group is less than control group . in this study , a significant decrease observed in the activity of gst , alp and protease enzymes of the e / s products of treated groups in comparison with the control group .", "the inhibition of enzymes activity induced by mbz and abz might damage the defensive system , metabolism and nutrition of the parasite .", "the inhibition of gst activity induced by anti - helmintics may contribute to passive the detoxification mechanism ( 29 ) .", "the abz and mbz cause reduction in protease enzymes activity , and effect on the parasite activities including assault , migration , feeding , and particularly survival in the host .", "since protease enzymes are proteins , the reduction of their activity could be attributed to interference of benzimidazole with protein synthesis ( 30 ) .", "alp is an important enzyme of helminthes parasites associated with absorption and/or digestion of food materials .", "it has a role in modulating the host immune response ( 7 , 31 ) .", "higher concentrations of alp found in the some areas of intestine and sub - cuticular layers of the worm associated with protein transport processes ( 32 ) .", "various commercial drugs ( anthelmintic ) and chemical compounds are shown to bring about changes in the activity of alp in various soft - bodied helminth parasites like cestodes ( 33 ) . when taenia solium cysts was treated with praziquantel and abz , alp secretion in the culture medium", "based on the results obtained in this study , the reduction or inhibition of the enzymes ( gst and alp ) caused by abz and mbz could lead to alteration of metabolism in helminthes parasites . therefore", ", the study indicates that both anti hydatid benzimidazoles ( abz and mbz ) have an inhibitory effect on the activity of gst , alp and protease enzymes in e / s products of e. granulosus protoscoleces in vitro .", "protein concentration in e / s products of mbz groups was slightly higher than that of the abz groups but this difference was not significant .", "the results showed that the effect of abz on the activity of gst and alp is more than mbz and this may be due to the different structures of these drugs .", "in addition , the above - mentioned results might account for at least in part , difference in antihydatid mechanism of the both benzimidazole ( 5 ) .", "both drugs have an inhibitory effect on the activity of gst , alp and protease enzymes .", "the results of the two drugs indicated that abz is more effective on the enzymes activities ( gst and alp ) as compared with mbz .", "this may by attrirbuted tothe different structures of the two drug and might account for at least in part , differencein antihydatid mechanism of these benzimidazole derivation ."], "abstract_text": ["<S> background : hydatid cysts are formed in human body can be treated clinically by surgery or drugs such as albendazole ( abz ) and mebendazole ( mbz ) . </S>", "<S> the purpose of this study was comparing the effects of abz and mbz on glutathione - s - transferase , alkaline phosphatase and protease enzymes activities in protoscoleces of hydatid cyst.methods:the culture supernatants containing the parasite excretory / secretory ( e / s ) products were collected every 12 h for 72 h. the e / s products of treated samples with 1g / ml abz and mbz and the control one were collected and after centrifugation then protein concentrations were measured according to bradford method . </S>", "<S> gst , alp and protease activities of e / s products were assessed photometrically.results:the mean of gst specific activity level in treated protoscoleces with abz and mbz and in control group were obtained 69.44 , 132.83 and 225.47u / mg / protein / ml respectively . the mean alp activity level in treated protoscoleces with abz and mbz and in control group were detected 19.22 , 22.27 and 27.85 u / mg / protein / ml respectively . </S>", "<S> the protease activity level in treated protoscoleces with abz and mbz were not detected . while the mean of protease activity level in control group was 7.61u / mg / proteins . </S>", "<S> statistical analysis showed the significant difference between protein concentrations , the specific activities of gst , alp and protease enzymes in treated protoscoleces in comparison with control group ( p<0.05 ) . </S>", "<S> also , the significant difference were seen between specific activities of gst and alp enzymes in treated protoscoleces with abz in comparison with treated group with mbz ( p<0.05).conclusion : abz is more effective on the enzymes activities ( gst and alp ) as compared with mbz . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Collection of Protoscoleces", "Culture protoscoleces", "Excretory / Secretory products collection", "Protein concentration measurement of E/S products", "Glutathione-S- Transferase assay", "Alkaline phosphates assay", "Protease assay", "Statistical analysis", "Results", "Protein concentration in treated and control groups of E/S products", "GST, ALP and Protease activity in treated and control groups of E/S products", "Statistical analysis", "Discussion", "Conclusion"], "sections": [["echinococcosis or cystic hydatid disease is an endemic parasitic disease in human populations in iran and some parts of the world .", "it is caused by larvae stage of echinococcus granulosus ( 1 ) . in the life cycle of e. granulosus ,", "clinical treatment of cysts includes albendazole ( abz ) or mebendazole ( mbz ) therapy in combination with either surgical resection ( 2 ) .", "the role of enzymes in living organism is clear and remarkable and the worms are seriously dependent on these activities .", "parasite components such as enzymes have specific biological functions , which are necessary for parasite survival and are supposed to have an important role in host - parasite interactions and disease progress ( 3 ) .", "parasite s enzymes are attractive purposes that are be explored for the development of diagnostic method and vaccines .", "they mediate processes like tissue invasion , feeding , evasion ( escape the immune system ) of host immune response etc .", "glutathione - s - transferase ( gst ) is an enzyme , which has a significant role in the detoxification of parasite metabolites ( endogeous ) , host metabolites ( xenobiotics ) and drugs through their conjugation to glutathione ( 5 ) .", "gst activity in e. granulosus has been described in the cytosolic portion of protoscoleces obtained from sheep cysts and activated by pre - treatment of protoscoleces with gst inducers ( 6 ) .", "alkaline phosphates ( alp ) is an enzyme that plays an important role in dampening host immune responses and also plays a role in feeding parasites ( 7 ) .", "most phosphatases have been found in the absorption system of cestodes , excretory system in trematodes and intestinal cells of the nematode ( 8) .", "proteolytic enzymes of parasites have been given more attention than other enzymes , because they play a vital role in parasite survival and are involved in many fundamental physiologic processes ( 3 ) .", "the activities of protease described in e. granulosus ( 9 ) , has been detected in hydatid cyst fluid , cyst wall and in protoscoleces .", "this enzyme is responsible for breakdown of proteins in all living tissues in order to be used by the cells ( 10 ) .", "in addition to their known role in the catabolism , they have a part in protein processing in evasion from immune system , leaving the cyst , molting of the parasites and in diagnosis , especially cysteine proteases as serological markers .", "proteases have generally been identified as potential drug targets in parasites ( 11 , 12 ) .", "the purpose of this study was to determine the effects of abz and mbz on the activity of the gst , alp and proteases in the protoscoleces of hydatid cyst and to evaluate their inhibitory effects on enzyme activity ."], ["protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran ( center of iran ) .", "protoscoleces were allowed to settle in a 50 ml falcon tube , and then washed several times in phosphate - buffered saline ( pbs ph , 7.2 ) .", "viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays ( 13 ) .", "mbz used in this study was obtained from rouzdarou pharmaceutical company ( iran ) and abz was purchased from tolide daruhai dami iran company .", "no : k4111 - 500 ) , 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each ] containing 500l protoscoleces and 1", "g / ml abz and/ mbz [ stock solution 1 mg / ml of dimethyl sulphoxide ( dmso ) ] were considered as test groups and 10 culture medium [ five culture containing 500 l protoscoleces with 0.6 l dmso , and five culture medium without dmso ] regarded as control groups and were incubated at 37 c in 5% co2 ( 14 ) .", "dmso , and five culture medium the culture supernatants containing the parasite e / s products were collected at time interval , every 12 h. at the end of each time interval , the entire 1 ml of culture medium ( culture supernatants ) was removed and replaced with the same volume of fresh medium ( 13 ) .", "the precipitates were discarded and the total protein and enzyme activities were measured as follows : the concentrations of total proteins of e / s samples were measured by bradford method , which involves reacting the e / s samples with a dye that binds to protein . to measure", "the protein concentration , standard solutions ( bovine serum albumin ) and e / s products were prepared and the bradford reagent ( 100 mg coomassie brilliant blue g-250 , 50 ml 95% ethanol , 100 ml 85% phosphoric acid ) was added .", "the absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature .", "a standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated ( 15 , 16 ) . in order to measure the activity of gst in e / s samples , reagent stock including potassium phosphate buffer 0.1 m , 100 mm reduced glutathione ( gsh ) and 100 mm 1-chloro-2,4- dinitrobenzene ( cdnb ) substrates were prepared in a microtube .", "to each test , from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well .", "the same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min .", "finally , total gst activity ( u / ml ) , of samples was calculated . to calculate the specific activity of gst enzyme , the rate of enzyme activity was divided by the mg protein concentration ( 17 ) .", "eight hundred l of buffer reagent ( r1 ) and 200 l of substrate reagent ( r2 ) were poured into cuvette and mixed .", "then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure .", "sasein solution 0.65% ( 6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm , ph7.5 ) prepared and incubated at 37 c for 5 min , and then e / s samples were added to test tubes and were incubated 37 c for 10 min . the reaction was stopped using trichloroacetic acid ( 110 mm tca , prepared by diluting a 6.1n stock 1:55 with purified water ) .", "e / s products were added to control tubes simultaneously and incubated for 30 min at 37 c , and then centrifuged for 5 min at 14000 g at 25 c .", "the supernatant was poured into the test tube ; 1cc sodium carbonate solution ( 500 mm sodium carbonate solution , prepared using 53 mg / ml of anhydrous sodium carbonate in purified water ) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c .", "finally , tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm .", "solution l - tyrosin 1.1 mm , dw , na2co3 , phenol ) and reported as units /ml enzyme ( 16 ) .", "folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color . in order to determine the statistically significant difference between protein concentrations , gst ,", "alp and protease activities of e / s samples of treated and control groups , t - test was used .", "a duplicate set of samples were taken for each test and at the end , their average was taken in to account ."], ["protoscoleces were obtained by aseptic puncture from fertile liver hydatid cysts of ovine origin collected from an abattoir in rey city in tehran ( center of iran ) .", "protoscoleces were allowed to settle in a 50 ml falcon tube , and then washed several times in phosphate - buffered saline ( pbs ph , 7.2 ) .", "viability was determined by eosin 0.01 exclusion analysis and only protoscoleces samples with viability higher than 95% were selected for the assays ( 13 ) .", "mbz used in this study was obtained from rouzdarou pharmaceutical company ( iran ) and abz was purchased from tolide daruhai dami iran company ."], ["no : k4111 - 500 ) , 100u / ml of penicillin and 100g / ml of streptomycin as 1 ml for each ] containing 500l protoscoleces and 1 g / ml abz and/ mbz [ stock solution 1 mg / ml of dimethyl sulphoxide ( dmso ) ] were considered as test groups and 10 culture medium [ five culture containing 500 l protoscoleces with 0.6 l dmso , and five culture medium without dmso ] regarded as control groups and were incubated at 37 c in 5% co2 ( 14 ) ."], ["the culture supernatants containing the parasite e / s products were collected at time interval , every 12 h. at the end of each time interval , the entire 1 ml of culture medium ( culture supernatants ) was removed and replaced with the same volume of fresh medium ( 13 ) .", "the precipitates were discarded and the total protein and enzyme activities were measured as follows :"], ["the concentrations of total proteins of e / s samples were measured by bradford method , which involves reacting the e / s samples with a dye that binds to protein . to measure the protein concentration , standard solutions ( bovine serum albumin ) and e / s products", "were prepared and the bradford reagent ( 100 mg coomassie brilliant blue g-250 , 50 ml 95% ethanol , 100 ml 85% phosphoric acid ) was added .", "the absorbance of e / s products and standard solutions were measured at 595 nm after 5 min incubation at room temperature .", "a standard curve was prepared by using the standard solutions absorbance and the protein concentration of the samples were estimated ( 15 , 16 ) ."], ["in order to measure the activity of gst in e / s samples , reagent stock including potassium phosphate buffer 0.1 m , 100 mm reduced glutathione ( gsh ) and 100 mm 1-chloro-2,4- dinitrobenzene ( cdnb ) substrates were prepared in a microtube . to each test , from the mentioned mixture 1.8 ml and 200ul of abz and mbz treated protoscoleces e / s sample were added and mixed well .", "the same method was performed for the control groups and absorbances of gst activities were measured at 340 nm for 5 min . finally , total gst activity ( u / ml ) , of samples was calculated . to calculate the specific activity of gst enzyme , the rate of enzyme activity was divided by the mg protein concentration ( 17 ) ."], ["eight hundred l of buffer reagent ( r1 ) and 200 l of substrate reagent ( r2 ) were poured into cuvette and mixed .", "then 20 l of treated or control samples were added and mixed well and measured absorbance of sample for 5 min by every 1 min at 405 nm and enzyme activity were calculated according to the kit procedure ."], ["sasein solution 0.65% ( 6.5 mg / ml of casein in the 50 mm potassium phosphate buffer 50 mm , ph7.5 ) prepared and incubated at 37 c for 5 min , and then e / s samples were added to test tubes and were incubated 37 c for 10 min .", "the reaction was stopped using trichloroacetic acid ( 110 mm tca , prepared by diluting a 6.1n stock 1:55 with purified water ) .", "e / s products were added to control tubes simultaneously and incubated for 30 min at 37 c , and then centrifuged for 5 min at 14000 g at 25 c .", "the supernatant was poured into the test tube ; 1cc sodium carbonate solution ( 500 mm sodium carbonate solution , prepared using 53 mg / ml of anhydrous sodium carbonate in purified water ) and 200 l folin & ciocalteus phenol reagent were added and incubated for 30 min at 37 c .", "finally , tubes were centrifuged for 5 min at 14000 g at 25 c and absorbances were measured spectrophotometerically at 660 nm .", "solution l - tyrosin 1.1 mm , dw , na2co3 , phenol ) and reported as units /ml enzyme ( 16 ) .", "folin & ciocalteus phenol reagent primarily react with free tyrosine and produce a blue color ."], ["in order to determine the statistically significant difference between protein concentrations , gst , alp and protease activities of e / s samples of treated and control groups , t - test was used .", "a duplicate set of samples were taken for each test and at the end , their average was taken in to account ."], ["the mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml , respectively .", "the mean protein concentration in the control group was 6.06 g / ml . protein concentration of e / s products of protoscoleces treated and control groups \n p<0.05 ; between control and treated groups \n \n p>0.05 ; between treated groups \n  the results of gst , alp and protease activity are shown in table 2 .", "gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml .", "alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml , receptively .", "alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml .", "protease specific activity in abz and mbz treated protoscoleces e / s products was not detected , while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml", ". gst , alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005 , t = test or sample n / d = not detected \n gst  activity  =  od / min0.0096total  voluesample  voluedilution  factor \n  alp activity =  od  2764 \n protease  activity=(umole  tyrosine  equivalents ", "released)(total  volume  of  assay)volume  of  enzymetime  of  assayvolume  of  filtratespecific  activity  =  units / ml  enzymemg  solid \n  statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group ( p<0.05 ) .", "a significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz ( p<0.05 ) .", "meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz ( p>0.05 )"], ["the mean protein concentrations in e / s samples of protoseoleces exposed to abz and mb were measured 3.55 and 4.58 g/ ml , respectively .", "the mean protein concentration in the control group was 6.06 g / ml . protein concentration of e / s products of protoscoleces treated and control groups \n p<0.05 ; between control and treated groups \n \n p>0.05 ; between treated groups \n"], ["gst specific activity level of protoscoleces in abz group was 69.44 and in mbz treated group was 132.82 u / mg protein / ml .", "alp specific activity in abz and mbz treated protoscoleces were estimated as 19.22 and 22.27 u / mg protein / ml , receptively .", "alp specific activity of control group of e / s products was 27.85 u / mg protein/ ml .", "protease specific activity in abz and mbz treated protoscoleces e / s products was not detected , while protease specific activity of control group e / s products calculated 7.61 u / mg protein / ml .", "gst , alp and protease activity in e / s products of protoscoleces treated and control groups p<0/005 , t = test or sample n / d = not detected \n gst  activity  =  od / min0.0096total  voluesample  voluedilution  factor \n  alp activity =  od  2764 \n protease  activity=(umole  tyrosine  equivalents  released)(total  volume  of  assay)volume  of  enzymetime  of  assayvolume  of  filtratespecific  activity  =  units / ml  enzymemg  solid \n"], ["statistical analysis using t - test showed the significant difference between protein concentrations and specific activities of the enzymes in e / s products of protoscoleces treated with abz and mbz in comparison with control group ( p<0.05 ) .", "a significant difference was observed between specific activity of gst and alp enzymes in e / s products of protoscoleces treated with abz in comparison with the group treated with mbz ( p<0.05 ) .", "meanwhile there is no significant difference between protein concentration in e / s products of protoscoleces treated with abz compared with the group treated with mbz ( p>0.05 )"], ["glutathione transferases ( gst ) are multifunctional enzyme present in both animal and plant kingdoms .", "the enzymes are regarded as parts of the phase ii detoxification system that catalyse glutathione ( gsh ) conjugation of a multitude of exogenous and endogenous toxic compounds ( 18 ) .", "gst is one of the major detoxification system component found in helminthes , particularly at high levels in cestodes and digeneas . in the tissue extracts of parasites gst activity", "gst activity exists in e / s products and in the surface of digenea worms such as schistosoma , fasciola and hookworm necator .", "gst in e / s products act as an anti - inflammatory agent and neutral lipid peroxidation products in the mucous membranes ( 20 , 21 ) .", "researches related to chemotherapy and immunotherapy , have identified glutathione of parasitic worms independently as a potential target for treatment ( 22 ) .", "these acts include the digestion of host s tissue in order to provide food for parasites , preventing blood coagulation , facilitating entry into the host s immune system , and disrupting it ( 23 ) .", "alp is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules , including nucleotides and proteins ( 24 ) .", "the phosphatases have been found frequently in the structure of absorption like tegument of cestodes , intestinal cell of the nematodes and excretory system of trematodes .", "while alkaline phosphatases have been found in adult cestode , the larval forms have a predominance of acid phosphates . this change in these enzymes", "may be associated with the growth and development of the reproductive system in adult worms ( 8) . alp as protein plays a role in immune evasion by generating the potent immunosuppressant adenosine .", "since adenosine is also an important nutrient , alp may also play a role in parasite feeding ( 7 ) .", "several studies have been carried out on characterization of hydatid cyst protoscoleces e / s products ( 25 , 26 ) . in the present study ,", "the effect of abz and mbz on protein content and some enzymatic activities of e / s products were examined .", "protein concentration in treated groups e / s products were less than that of the control group e / s sample .", "abz and mbz cause degenerative changes in intestinal cells of the worm by binding to the colchicine - sensitive site of tubulin , therefore blocks its polymerization into microtubules and inhibits the cell proliferation in metaphase stage ( 27 , 28 ) .", "based on these findings we can expect the decreasing of total protein in treated parasite culture media , which correlates our study that shows an average protein of test group is less than control group . in this study , a significant decrease observed in the activity of gst , alp and protease enzymes of the e / s products of treated groups in comparison with the control group .", "the inhibition of enzymes activity induced by mbz and abz might damage the defensive system , metabolism and nutrition of the parasite .", "the inhibition of gst activity induced by anti - helmintics may contribute to passive the detoxification mechanism ( 29 ) .", "the abz and mbz cause reduction in protease enzymes activity , and effect on the parasite activities including assault , migration , feeding , and particularly survival in the host .", "since protease enzymes are proteins , the reduction of their activity could be attributed to interference of benzimidazole with protein synthesis ( 30 ) .", "alp is an important enzyme of helminthes parasites associated with absorption and/or digestion of food materials .", "it has a role in modulating the host immune response ( 7 , 31 ) .", "higher concentrations of alp found in the some areas of intestine and sub - cuticular layers of the worm associated with protein transport processes ( 32 ) .", "various commercial drugs ( anthelmintic ) and chemical compounds are shown to bring about changes in the activity of alp in various soft - bodied helminth parasites like cestodes ( 33 ) . when taenia solium cysts was treated with praziquantel and abz , alp secretion in the culture medium", "based on the results obtained in this study , the reduction or inhibition of the enzymes ( gst and alp ) caused by abz and mbz could lead to alteration of metabolism in helminthes parasites . therefore", ", the study indicates that both anti hydatid benzimidazoles ( abz and mbz ) have an inhibitory effect on the activity of gst , alp and protease enzymes in e / s products of e. granulosus protoscoleces in vitro .", "protein concentration in e / s products of mbz groups was slightly higher than that of the abz groups but this difference was not significant .", "the results showed that the effect of abz on the activity of gst and alp is more than mbz and this may be due to the different structures of these drugs .", "in addition , the above - mentioned results might account for at least in part , difference in antihydatid mechanism of the both benzimidazole ( 5 ) ."], ["both drugs have an inhibitory effect on the activity of gst , alp and protease enzymes .", "the results of the two drugs indicated that abz is more effective on the enzymes activities ( gst and alp ) as compared with mbz .", "this may by attrirbuted tothe different structures of the two drug and might account for at least in part , differencein antihydatid mechanism of these benzimidazole derivation ."]]}
{"article_id": "PMC3579995", "article_text": [" anal carcinoma is increasingly diagnosed in patients with human immunodeficiency virus ( hiv ) , anoreceptive intercourse , chronic inflammatory bowel disease . ", "an established association exists with human papillomavirus ( hpv ) infection and premalignant intra - epithelial dysplasia . ", "phased - array mri is recommended as the preferred imaging modality for regional staging . ", "an uncommon malignancy in the general population , squamocellular anal carcinoma ( scac ) accounts for approximately 1 % of all gastrointestinal neoplasms and less than 5 % of anorectal tumours . in past decades", ", scac was usually diagnosed at a relatively advanced age with a significant female predominance , and believed to be an indolent disease secondary to chronic irritation . in recent years , similarly to uterine cervix dysplastic changes , oncogenic human papillomavirus ( hpv ) has been detected in the vast majority ( up to 90 % ) of invasive scacs , and linked to the development of low- and high - grade premalignant anal intra - epithelial neoplasms ( ain ) , particularly with high - risk or multiple hpv serotypes infection [ 1 , 2 ] .", "furthermore , the incidence of scac is steadily increasing , particularly in patients with risk factors such as human immunodeficiency virus ( hiv ) infection , history of anoreceptive intercourse , coexistent cervical dysplasia or cancer , immunosuppression , inflammatory bowel diseases ( ibd ) and cigarette smoking .", "currently , at least half of scacs occur in relatively young ( 4060  years ) hiv - positive individuals , most often men who have sex with men ( msm ) [ 35 ] .", "the surgical anus is about 4  cm long from the anorectal junction to the perianal skin on the external anal margin ( verge ) .", "the internal anal sphincter consisting of smooth muscle is separated from the external , striated muscle sphincter by the fatty intersphincteric space . along with the puborectalis and levator ani muscles , the external anal sphincter forms the sphincter complex .", "located approximately halfway along the anus , the dentate line marks the transition from the squamous epithelium to the intestinal mucosa .", "thus , histologically scacs can be either keratinising or non - keratinising according to their origin below or above the dentate line , although with similar biological behaviour [ 6 , 7 ] .", "anal margin and anal canal scac originating distal to the dentate line drain to the inguinal and femoral lymph nodes .", "when the primary tumour arises above the dentate line , regional lymph nodes include the inguinal , internal iliac and perirectal nodes , whereas the external , common iliac and para - aortic nodes are considered non - regional [ 68 ] .", "because of its anatomical location , in most cases scac is diagnosed clinically in patients with rectal bleeding , pain , discharge or palpable masses .", "alternatively , lesions may be detected during follow - up of high - risk individuals .", "following physical examination including digital rectal and vaginal examination , ano - proctoscopy and biopsy , imaging is required to evaluate the local extent of the lesion , lymph node involvement , possible invasion of adjacent organs and distant metastases [ 6 , 9 ] .", "imaging the anal canal and perianal structures may prove technically challenging to perform and interpret .", "in past years , trans - anal ultrasound ( trus ) and magnetic resonance imaging ( mri ) techniques allowed an accurate assessment of tumour size and depth of mural invasion [ 7 , 10 , 11 ] .", "unfortunately , in patients with anal lesions , positioning of endoanal sonography probes and mri coils is hampered by pain and stricture .", "trans - anal imaging combines an excellent spatial detail with a limited field - of - view that prevents panoramic assessment of entire ischiorectal spaces and of regional lymph nodes .", "furthermore , trus has limited specificity for differentiation of residual tumour versus post - treatment fibrosis [ 1113 ] . currently , mri performed using external phased - array coils on high - magnetic - field scanners is the imaging modality of choice to investigate the anal region .", "significant advantages of mri include its native multiplanar capability , superior soft - tissue differentiation , biological non - invasiveness and optimal safety profile of gadolinium - based contrast agents .", "acquisition protocols heavily rely on high - resolution t2-weighted sequences along three planes , with coronal and axial scans planned slightly oblique , respectively parallel and perpendicular to the long axis of the anal canal . despite the increased tumour conspicuity provided by background fat suppression , short - tau inversion recovery ( stir ) sequences are less useful because of limited spatial detail and difficulty to delineate anatomic landmarks . at our centre ,", "t1-weighted sequences including fat suppression in at least one plane are routinely acquired following standard - dose intravenous gadolinium contrast , to allow detection of lesion enhancement .", "conversely , other authors discourage post - contrast mri acquisitions by stating that enhanced images do not offer additional information to the high soft tissue contrast intrinsic to t2-weighted imaging [ 7 , 10 , 1315 ] .", "although with limited contrast resolution compared with mri , volumetric multidetector computed tomography ( mdct ) acquisitions including image reformations along arbitrary planes allow visualisation of anorectal abnormalities in their cranio - caudal extent with relationship to key anatomical landmarks such as the sphincter complex [ 1618 ] .", "although the dentate line is not directly recognisable , its position can be inferred as it corresponds approximately to the upper portion of external sphincter muscles .", "sensitivity of trus and mri for the identification of scac has been reported to approach 90100 % , with high concordance regarding tumour size , although on a limited number of patients , and more precise results with ultrasound for smaller , superficial tumours .", "neoplastic tissue in the anal canal has low - to - intermediate t1 signal intensity and positive enhancement after intravenous gadolinium contrast .", "on t2-weighted and stir sequences , untreated neoplasms display intermediate signal intensity , lower to that of normal ischioanal fat and almost always superior to the internal reference standard represented by uninvolved anal sphincters and gluteal muscles ( figs .  1 , 2 ) .", "although with limited sensitivity compared with mri , on ct images scac may be detected as solid , enhancing nodules or masses within the anus ( figs .  2 , 3 ) .", "axial ( a ) and sagittal ( b ) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle , intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image ( c ) , consistent with t1 tumour ( arrowheads)fig", "axial t2-weighted ( a ) , post - contrast fat - suppressed axial ( b ) and coronal ( c ) t1-weighted images , and corresponding enhanced image from body ct ( d ) show a 5.5-cm long ( t3 ) enhancing tumour with infiltration of the left ischioanal fatty space ( arrowheads)fig .", "post - contrast axial ( a ) and coronal reformatted ( b ) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule . subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scacfig .", "4an elderly , 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri .", "sagittal t2- ( a ) and axial t1-weighted ( b ) images show 5-cm long solid , inhomogeneous neoplastic tissue ( * ) extending from the anus to encase the proximal urethra ( note catheter in place ) a 40-year - old msm with bioptic diagnosis of scac .", "axial ( a ) and sagittal ( b ) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle , intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image ( c ) , consistent with t1 tumour ( arrowheads ) a 62-year - old female with biopsy - proven scac .", "axial t2-weighted ( a ) , post - contrast fat - suppressed axial ( b ) and coronal ( c ) t1-weighted images , and corresponding enhanced image from body ct ( d ) show a 5.5-cm long ( t3 ) enhancing tumour with infiltration of the left ischioanal fatty space ( arrowheads ) a 57-year - old woman undergoing abdomino - pelvic mdct for unrelated reasons .", "post - contrast axial ( a ) and coronal reformatted ( b ) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule .", "subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scac an elderly , 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri .", "sagittal t2- ( a ) and axial t1-weighted ( b ) images show 5-cm long solid , inhomogeneous neoplastic tissue ( * ) extending from the anus to encase the proximal urethra ( note catheter in place ) staging is essential for both prognostic information and correct therapeutic planning , and is performed according to the uicc / ajcc tumour - node - metastasis ( tnm ) system ( table  1 ) , including local lesion extent , lymph node status and distant metastatic spread . in 2010", "the european society for medical oncology ( esmo ) recommended mri as the primary imaging modality to accurately stage scac , taking into account the maximum tumour diameter , possible invasion of adjacent organs and nodal involvement [ 6 , 13].table 1tumour - node - metastasis ( tnm ) staging of anal carcinoma according to lesion site of originanal canalanal marginprimary tumour ( t)txprimary tumour can not be assessedt0no evidence of primary tumourt1tumour 2  cm in greatest dimensiont2tumour 25  cm in greatest dimensiont3tumour > 5  cm in greatest dimensiont4tumour of any size invading adjacent organ(s ) e.g. vagina , urethra , bladdertumour invading deeper structures ( skeletal muscle , cartilage)regional lymph nodes ( n)nxregional lymph nodes can not be assessedn0no regional lymph node metastasisn1metastasis in perirectal lymph node(s)regional lymph node metastasisn2metastasis in unilateral internal iliac and/or inguinal lymph node(s)n3metastasis in internal iliac and perirectal lymph nodes and/or bilateral internal iliac and/or bilateral inguinal lymph nodesdistant metastasis ( m)m0no distant metastasism1distant metastasis tumour - node - metastasis ( tnm ) staging of anal carcinoma according to lesion site of origin particular care should be applied to choosing the longest lesion diameter on t2-weighted images , since correct t parameter staging relies on this measure being below 2  cm , over 5  cm or intermediate ( fig .  1 )", "mri demonstrates good correlation with physical findings concerning t stage , whereas infiltration of adjacent organs and sometimes tumour size are clinically underestimated .", "extramural neoplastic spread may involve the sphincter complex muscles ( external sphincter , levator ani and puborectalis ) and most commonly occurs towards the anterior urogenital triangle with possible vaginal , urethral or bladder involvement .", "sometimes , the tumour may also extend laterally with invasion of the ischioanal fossa ( fig . ", "2 ) , superiorly to the rectum and mesorectal compartment , or inferiorly to the skin and subcutaneous planes of the perianal region . in such instances , t2-hyperintense solid tissue is seen infiltrating the even more hyperintense fat in the ischiorectal ( fig .  2 ) and subcutaneous spaces , encasing the lower signal intensity skeletal muscles , or isointense structures such as the vagina ( fig .  5 ) , prostate and urethra ( fig . ", "notably , radiologists should remember that anal canal carcinoma directly invading the rectal wall , perianal skin , subcutaneous or the sphincter muscle does not imply assessing the tumour stage as t4 . for anal margin scac", ", a t4 lesion is defined by invasion of deeper structures such as the skeletal muscle or cartilage [ 9 , 10 , 13].fig .", "axial t2-weighted ( a ) and post - contrast fat - suppressed axial t1-weighted ( b ) images show inhomogeneous anal tissue invading the left aspect of the vagina ( arrowheads ) , with internal non - enhancing necrosis and peripheral enhancement .", "biopsy diagnosed scac with superimposed infection a 32-year - old hiv - positive woman with clinical diagnosis of anovaginal fistula .", "axial t2-weighted ( a ) and post - contrast fat - suppressed axial t1-weighted ( b ) images show inhomogeneous anal tissue invading the left aspect of the vagina ( arrowheads ) , with internal non - enhancing necrosis and peripheral enhancement .", "biopsy diagnosed scac with superimposed infection the incidence of regional nodal involvement increases with primary tumour size .", "lymph node metastases may be present ( in 25 % of cases ) even with superficial ( up to t2 ) scacs , and are unreliably assessed clinically .", "nodal staging evaluation relies on the distance from the primary tumour rather than on the number of involved nodes .", "mri is highly helpful to assess lymph node metastatic involvement , although the mere size criterion is far from accurate and associated with both false - positive and false - negative results .", "short - axis threshold values of 8  mm , 5  mm and 10  mm have been suggested for pelvic , perirectal and inguinal lymph nodes , respectively .", "additional helpful features to increase specificity include loss of the normal bean - shaped morphology and fatty hilum , internal t1 and t2 signal heterogeneity with central necrosis , and inhomogeneous enhancement ( figs . ", "6a 65-year - old woman with history of previously treated small scac 3  years earlier .", "axial t2- ( a ) and post - contrast t1-weighted ( b ) images show roundish 1-cm left inguinal node ( arrowheads ) with internal fluid - like necrosis and inhomogeneous enhancement , confirmed by ultrasound ( c ) as hypoechoic with loss of normal nodal structure .", "surgical exeresis ( postoperative status as seen in d , follow - up mri ) confirmed metastatic node from scacfig .", "axial t2-weighted ( a , b ) and post - contrast fat - suppressed t1-weighted ( c ) images show inguinal nodal metastases , larger on left side ( arrowheads ) plus bilateral enhancing perirectal adenopathies ( arrows ) a 65-year - old woman with history of previously treated small scac 3  years earlier .", "axial t2- ( a ) and post - contrast t1-weighted ( b ) images show roundish 1-cm left inguinal node ( arrowheads ) with internal fluid - like necrosis and inhomogeneous enhancement , confirmed by ultrasound ( c ) as hypoechoic with loss of normal nodal structure .", "surgical exeresis ( postoperative status as seen in d , follow - up mri ) confirmed metastatic node from scac the same patient as in fig .  2 .", "axial t2-weighted ( a , b ) and post - contrast fat - suppressed t1-weighted ( c ) images show inguinal nodal metastases , larger on left side ( arrowheads ) plus bilateral enhancing perirectal adenopathies ( arrows ) as suggested by ecco guidelines , search for distant spread is usually performed by means of contrast - enhanced body mdct , with conventional imaging appearances of liver and lung metastases . dissemination is very uncommon ( less than 5 % of patients at initial diagnosis , and is usually encountered in association with post - treatment recurrence . alternatively , in immunocompetent patients f - fluorodeoxygluocose positron emission tomography ( fdg - pet / ct ) has high specificity for nodal and visceral dissemination . at diagnosis , fdg - pet /", "ct may alter staging of anal scac in 20 % of patients , leading to inclusion of involved pelvic or inguinal lymph nodes in the radiation field [ 6 , 7 ] .", "in our experience , not infrequently anal tumours coexist with inflammatory conditions such as proctitis and abscesses . in such instances , mdct and mri provide confident detection of perirectal inflammatory changes and purulent collections that are differentiated from solid neoplastic tissue , thus allowing a correct therapeutic choice including surgical drainage as necessary ( figs .  5 , 8 , 9 , 10 ) .", "resolution of associated inflammatory changes during treatment is easily monitored by cross - sectional imaging ( figs . ", "8a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass .", "axial ( a ) and coronal reformatted ( b ) ct images show moderately heterogeneous tissue ( * ) in its entire longitudinal extent from the anorectal junction to below the anal verge , associated with large necrotic iliac adenopathies ( * in c ) .", "axial images ( b , c ) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal ( arrowheads ) associated with abscess collections with mixed gas - fluid content ( * ) and fistulas crossing the ischioanal space .", "surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . after surgical drainage and subsequent chemo - radiotherapy , mri ( c ) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracksfig .", "staging mri confirms left - sided anal thickening ( arrowheads ) with abnormal t2 signal intensity ( a ) and strong contrast enhancement ( b ) .", "incidentally , two abscess collections with necrotic content and peripheral enhancement are seen ventrally , connected to the anal canal by a fistulous track ( arrow in c ) .", "subtotal regression of changes after treatment is seen on follow - up mri ( d ) a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass .", "axial ( a ) and coronal reformatted ( b ) ct images show moderately heterogeneous tissue ( * ) in its entire longitudinal extent from the anorectal junction to below the anal verge , associated with large necrotic iliac adenopathies ( * in c ) .", "biopsy confirmed superinfected scac a 53-year - old man with purulent drainage and clinical diagnosis of perianal inflammation .", "axial images ( b , c ) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal ( arrowheads ) associated with abscess collections with mixed gas - fluid content ( * ) and fistulas crossing the ischioanal space .", "surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . after surgical drainage and subsequent chemo - radiotherapy , mri ( c ) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracks a 39-year - old hiv - infected man with biopsy - proven scac .", "staging mri confirms left - sided anal thickening ( arrowheads ) with abnormal t2 signal intensity ( a ) and strong contrast enhancement ( b ) .", "incidentally , two abscess collections with necrotic content and peripheral enhancement are seen ventrally , connected to the anal canal by a fistulous track ( arrow in c ) .", "subtotal regression of changes after treatment is seen on follow - up mri ( d ) furthermore , cross - sectional imaging particularly with mri also proves useful to differentiate anal carcinoma from other causes of local pain and perineal masses , such as pilonidal sinus diseases , gartner duct or bartolini gland cysts , tailgut cysts , uncommon soft - tissue neoplasms , urethral cancer , lymphoma or metastases [ 14 , 22 ] .", "in the past , scac was treated with abdomino - perineal resection and permanent colostomy . currently , anal margin and small canal tumours without evidence of nodal spread may be successfully excised . in all other cases ,", "the standard treatment includes radiation combined with mitomycin - c plus infusional 5-fu chemotherapy , yielding an 80 % 5-year survival rate with preservation of sphincter function .", "advanced t3/t4 tumours have worse outcomes with a 4068 % 3-year disease - free survival .", "salvage surgery with abdomino - perineal resection is reserved for persistent or recurrent tumours [ 3 , 6 , 2325 ] .", "after radio - chemotherapy , imaging follow - up with mri represents a useful complement to clinical evaluation in the assessment of therapeutic response . shortly after treatment completion , interpretation of mri is usually challenging due to the superimposition of inflammatory changes resulting from radiotherapy . performed at least 68  weeks after treatment completion , mri provides confident , reproducible assessment of post - treatment modifications .", "findings indicative of a positive response include size reduction and diminished t2 signal intensity of the treated tumour and associated adenopathies ( figs . ", "the appearance of t2-hypointense signal is consistent with fibrosis , although it does not allow excluding minor residual neoplastic foci for sure ( fig . ", "stability in size and signal intensity of any residual abnormality visible at mri in the site of the treated lesion 1 year after therapy has been reported to be strongly associated with a favourable outcome .", "locoregional and/or distant recurrence occurs in up to 35 % of treated patients , and is strongly associated with advanced ( t3t4 ) stage and nodal involvement at presentation [ 7 , 20 ] .", "persistent and locally recurrent tumours often display an aggressive behaviour , with possible extensive invasion of the adjacent organs and pelvic bony structures , and a tendency for lymphatic dissemination ( figs . ", "initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal ( a ) and contrast enhancement ( b ) ( arrowheads ) .", "complete disappearance of the lesion is observed on axial t2 ( c ) and post - contrast fat - suppressed t1-weighted ( d ) images following chemo - radiotherapyfig .", "mri staging including axial ( a ) and coronal ( b ) t2-weighted images detect a solid , 3-cm eccentric anal mass ( arrowhead ) consistent with t2 tumour , associated with right inguinal adenopathies with analogous signal features .", "after chemo - radiotherapy , mri follow - up ( c , d ) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathiesfig .", "initially , mdct ( a ) shows left - sided thickening with involvement of the external sphincter ( arrowheads ) .", "after chemo - radiotherapy , lesion regression is observed on mri with appearance of t2-hypointense fibrosis ( * ) and hyperintense nodule ( arrow ) consistent with residual tumourfig .", "14an elderly , 85-year - old lady with biopsy - proven scac and multiple comorbidities .", "initial mri ( a , b ) shows showed circumferential anal wall thickening with abnormal solid signal ( arrowheads ) measuring 6  cm in length , plus a suspicious centimetric left inguinal lymph node ( arrow in a ) , findings consistent with t3n2 lesion . after reduced chemo - radiotherapy , follow - up mri ( c , d ) 4 months later disclosed progression and partial necrosis of both primary tumour ( arrowhead ) and inguinal adenopathy ( arrow ) , plus appearance of an exophytic tissue mass protruding from the external anal orifice ( * in d ) an 83-year - old man with known scac .", "initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal ( a ) and contrast enhancement ( b ) ( arrowheads ) .", "complete disappearance of the lesion is observed on axial t2 ( c ) and post - contrast fat - suppressed t1-weighted ( d ) images following chemo - radiotherapy a 46-year - old female patient with biopsy - proven scac . mri staging including axial ( a ) and coronal ( b ) t2-weighted images detect a solid , 3-cm eccentric anal mass ( arrowhead ) consistent with t2 tumour , associated with right inguinal adenopathies with analogous signal features .", "after chemo - radiotherapy , mri follow - up ( c , d ) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathies a 46-year - old hiv - infected man with biopsy - proven scac .", "initially , mdct ( a ) shows left - sided thickening with involvement of the external sphincter ( arrowheads ) .", "after chemo - radiotherapy , lesion regression is observed on mri with appearance of t2-hypointense fibrosis ( * ) and hyperintense nodule ( arrow ) consistent with residual tumour an elderly , 85-year - old lady with biopsy - proven scac and multiple comorbidities . initial mri ( a , b ) shows showed circumferential anal wall thickening with abnormal solid signal ( arrowheads ) measuring 6  cm in length , plus a suspicious centimetric left inguinal lymph node ( arrow in a ) , findings consistent with t3n2 lesion .", "after reduced chemo - radiotherapy , follow - up mri ( c , d ) 4 months later disclosed progression and partial necrosis of both primary tumour ( arrowhead ) and inguinal adenopathy ( arrow ) , plus appearance of an exophytic tissue mass protruding from the external anal orifice ( * in d )", "after the introduction of highly active anti - retroviral treatment ( haart ) , people with hiv infection or acquired immunodeficiency syndrome ( aids ) gained a greatly improved life expectancy with better immune conditions , at the price of an increased tendency to develop tumours .", "although it is considered a non - aids - defining malignancy , currently scac ranks third ( 8.2 % ) among neoplasms observed in hiv / aids populations , with a substantially higher incidence in msms and in long - standing infected people [ 4 , 26 ] .", "dysplastic intraepithelial lesions are highly prevalent in hiv - infected people , and hiv represents a marker for coinfection with other sexually transmitted diseases such as hpv . since the risk of developing scac increases with the total time elapsed with cd4 + count below 200  cells/l , it has been hypothesised that hiv - related immune suppression acts as a cofactor to hpv in the development of anal dysplasia and progression to overt carcinoma [ 1 , 3 , 4 ] .", "screening procedures including high - resolution anoscopy and cytology smears are increasingly adopted at hiv care centres , to allow detection of scac precursors and early - stage tumours amenable to limited excision and topical therapies .", "prevention should limit the occurrence of advanced stages at diagnosis in the future [ 4 , 2629 ] .", "a high prevalence ( at least 30 % ) of anorectal complaints is characteristic of hiv - positive patients , particularly those practicing anoreceptive intercourse .", "differential diagnosis encompasses a wide spectrum of abnormalities , including non - specific anal diseases such as haemorrhoids , fissures , fistulas and abscesses , along with venereal infections , viral ulcers , and a non - negligible ( 7 % ) rate of neoplasms such as scac , lymphoma and kaposi s sarcoma .", "therefore , when performing cross - sectional imaging procedures on hiv - infected patients , even for unrelated complaints , special attention should be paid to the anal region , with a focus on the possible identification of solid , enhancing tissue consistent with tumour that indicates need for biopsy ( figs .  5 , 8 , 10 , 13 ) [ 21 , 30 , 31 ] .", "despite initial discouraging reports , in the haart era hiv - positive patients are likewise treated with standard chemo - radiation regimens , reaching satisfactory results in terms of local control and survival rates , although with increased toxicity and frequent local recurrences [ 4 , 5 , 26 , 27 , 32 ] .", "some literature reports have highlighted the increased risk of anus and lower rectum carcinomas associated with long - standing , severe perianal fistulising crohn s disease ( cd ) ( fig . ", ", scac reaches a 14 % proportion among all colorectal cancers , which is ten - times higher than the usual figure .", "furthermore , cd patients develop anorectal carcinomas at a younger age ( 20  years earlier ) than the general population . according to the hypothesised pathogenesis", ", fistulas probably allow hpv an easier access to the epithelial layers , and chronic mucosal regeneration may ultimately lead to neoplastic changes .", "however , anal tumours may occasionally develop in patients with ulcerative colitis ( uc)-related ( fig . ", "15a 40-year - old male patient with long - standing perianal crohn s disease , being treated with seton .", "contrast - enhanced mdct images ( a , b , c in cranio - caudal order ) show right - sided levator ani abscess ( * ) , extensive perianal fistulisation occupying the ischioanal space ( arrow ) , and marked solid - appearing circumferential anorectal thickening ( arrowheads ) .", "initial multiplanar mdct ( a , b ) identified enhancing anal thickening ( arrowheads ) with right - sided vaginal infiltration and solid tissue ( * ) extending to reach the internal obturator muscle . after biopsy confirmation of scac and surgical debulking with colostomy , repeat mdct ( c , d ) detected enlarging neoplastic residue ( * ) .", "shortly after chemo - radiotherapy , mri ( e ) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour ( thin arrows ) .", "mri follow - up ( e ) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence ( arrow ) .", "after negative clinical reassessment and pet findings , this post - treatment finding remained stable on further mri studies ( not shown ) a 40-year - old male patient with long - standing perianal crohn s disease , being treated with seton .", "contrast - enhanced mdct images ( a , b , c in cranio - caudal order ) show right - sided levator ani abscess ( * ) , extensive perianal fistulisation occupying the ischioanal space ( arrow ) , and marked solid - appearing circumferential anorectal thickening ( arrowheads ) .", "abdomino - perineal resection for scac was performed a 39-year - old woman with history of ulcerative colitis and perianal inflammation .", "initial multiplanar mdct ( a , b ) identified enhancing anal thickening ( arrowheads ) with right - sided vaginal infiltration and solid tissue ( * ) extending to reach the internal obturator muscle . after biopsy confirmation of scac and surgical debulking with colostomy , repeat mdct ( c , d ) detected enlarging neoplastic residue ( * ) .", "shortly after chemo - radiotherapy , mri ( e ) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour ( thin arrows ) .", "mri follow - up ( e ) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence ( arrow ) .", "after negative clinical reassessment and pet findings , this post - treatment finding remained stable on further mri studies ( not shown ) diagnosis is often unsuspected or delayed because of pre - existent , unspecific complaints and clinical assessment is hampered by complex inflammation with stricture and local pain . as a result ,", "ibd - associated anal cancers are often advanced at presentation , may require extensive surgery plus chemotherapy and radiotherapy , and are associated with a severe prognosis .", "therefore , patients with early - onset or long - standing perianal cd should undergo clinical and imaging surveillance , particularly when new or changed symptoms develop .", "radiologists should be aware of the increased risk for anorectal cancer in middle - aged ibd patients , and clearly report any solid tissue as suspicious for neoplasm and suggest biopsy ( figs . ", "the established association with hpv infection and premalignant intra - epithelial dysplastic changes provides insight into the pathogenesis of hiv- and ibd - related anal cancers , and the possibility of prevention or early diagnosis through screening of high - risk individuals [ 1 , 2 ] .", "state - of - the - art cross - sectional imaging with high - resolution mri using external phased - array coils and multiplanar mdct allow detailed , comprehensive visualisation of abnormalities involving the anus and perineal region .", "currently , mri represents the modality of choice for primary regional staging of scac , assessment of complications , of therapeutic response following chemo - radiotherapy , and of possible recurrences [ 9 , 10 , 13 ] ."], "abstract_text": ["<S> backgroundsquamocellular anal carcinoma is increasingly diagnosed in patients with risk factors.methodsstate-of-the-art imaging with magnetic resonance imaging ( mri ) using phased - array coils and volumetric multidetector computed tomography ( ct ) provides detailed visualisation of anal disorders , identification and extent assessment of neoplastic tissue , detection and characterisation of nodal and visceral metastases . </S>", "<S> mri has been recommended by the european society for medical oncology ( esmo ) as the preferred modality of choice to stage anal cancer , taking into account the maximum tumour diameter , invasion of adjacent structures and regional lymph node involvement.resultscross-sectional imaging techniques allow the identification of coexisting complications , and differentiation from other perineal abnormalities.conclusioncross-sectional imaging is useful for planning radiotherapy , surgical drainage or salvage abdomino - perineal resection . </S>", "<S> after chemo - radiotherapy , mri follow - up provides confident reassessment of therapeutic response , persistent or recurrent disease.teaching points anal carcinoma is increasingly diagnosed in patients with human immunodeficiency virus ( hiv ) , anoreceptive intercourse , chronic inflammatory bowel disease. an established association exists with human papillomavirus ( hpv ) infection and premalignant intra - epithelial dysplasia. phased - array mri is recommended as the preferred imaging modality for regional staging. imaging allows detection of infectious complications , planning of radiotherapy or salvage surgery. follow - up mri allows reliable assessment of therapeutic response after chemo - radiotherapy . </S>"], "labels": null, "section_names": ["None", "Introduction", "Regional anatomy and imaging techniques", "Imaging features and tumour staging", "Complications", "Treatment", "HIV-associated anal carcinoma", "Anal carcinoma in inflammatory bowel diseases", "Conclusion"], "sections": [[" anal carcinoma is increasingly diagnosed in patients with human immunodeficiency virus ( hiv ) , anoreceptive intercourse , chronic inflammatory bowel disease. an established association exists with human papillomavirus ( hpv ) infection and premalignant intra - epithelial dysplasia. phased - array mri is recommended as the preferred imaging modality for regional staging. imaging allows detection of infectious complications , planning of radiotherapy or salvage surgery. follow - up mri allows reliable assessment of therapeutic response after chemo - radiotherapy .", " anal carcinoma is increasingly diagnosed in patients with human immunodeficiency virus ( hiv ) , anoreceptive intercourse , chronic inflammatory bowel disease . ", "an established association exists with human papillomavirus ( hpv ) infection and premalignant intra - epithelial dysplasia . ", "phased - array mri is recommended as the preferred imaging modality for regional staging . "], ["an uncommon malignancy in the general population , squamocellular anal carcinoma ( scac ) accounts for approximately 1 % of all gastrointestinal neoplasms and less than 5 % of anorectal tumours . in past decades", ", scac was usually diagnosed at a relatively advanced age with a significant female predominance , and believed to be an indolent disease secondary to chronic irritation . in recent years , similarly to uterine cervix dysplastic changes , oncogenic human papillomavirus ( hpv ) has been detected in the vast majority ( up to 90 % ) of invasive scacs , and linked to the development of low- and high - grade premalignant anal intra - epithelial neoplasms ( ain ) , particularly with high - risk or multiple hpv serotypes infection [ 1 , 2 ] .", "furthermore , the incidence of scac is steadily increasing , particularly in patients with risk factors such as human immunodeficiency virus ( hiv ) infection , history of anoreceptive intercourse , coexistent cervical dysplasia or cancer , immunosuppression , inflammatory bowel diseases ( ibd ) and cigarette smoking .", "currently , at least half of scacs occur in relatively young ( 4060  years ) hiv - positive individuals , most often men who have sex with men ( msm ) [ 35 ] ."], ["the surgical anus is about 4  cm long from the anorectal junction to the perianal skin on the external anal margin ( verge ) .", "the internal anal sphincter consisting of smooth muscle is separated from the external , striated muscle sphincter by the fatty intersphincteric space . along with the puborectalis and levator ani muscles , the external anal sphincter forms the sphincter complex .", "located approximately halfway along the anus , the dentate line marks the transition from the squamous epithelium to the intestinal mucosa .", "thus , histologically scacs can be either keratinising or non - keratinising according to their origin below or above the dentate line , although with similar biological behaviour [ 6 , 7 ] .", "anal margin and anal canal scac originating distal to the dentate line drain to the inguinal and femoral lymph nodes .", "when the primary tumour arises above the dentate line , regional lymph nodes include the inguinal , internal iliac and perirectal nodes , whereas the external , common iliac and para - aortic nodes are considered non - regional [ 68 ] .", "because of its anatomical location , in most cases scac is diagnosed clinically in patients with rectal bleeding , pain , discharge or palpable masses .", "alternatively , lesions may be detected during follow - up of high - risk individuals .", "following physical examination including digital rectal and vaginal examination , ano - proctoscopy and biopsy , imaging is required to evaluate the local extent of the lesion , lymph node involvement , possible invasion of adjacent organs and distant metastases [ 6 , 9 ] .", "imaging the anal canal and perianal structures may prove technically challenging to perform and interpret .", "in past years , trans - anal ultrasound ( trus ) and magnetic resonance imaging ( mri ) techniques allowed an accurate assessment of tumour size and depth of mural invasion [ 7 , 10 , 11 ] .", "unfortunately , in patients with anal lesions , positioning of endoanal sonography probes and mri coils is hampered by pain and stricture .", "trans - anal imaging combines an excellent spatial detail with a limited field - of - view that prevents panoramic assessment of entire ischiorectal spaces and of regional lymph nodes .", "furthermore , trus has limited specificity for differentiation of residual tumour versus post - treatment fibrosis [ 1113 ] . currently , mri performed using external phased - array coils on high - magnetic - field scanners is the imaging modality of choice to investigate the anal region .", "significant advantages of mri include its native multiplanar capability , superior soft - tissue differentiation , biological non - invasiveness and optimal safety profile of gadolinium - based contrast agents .", "acquisition protocols heavily rely on high - resolution t2-weighted sequences along three planes , with coronal and axial scans planned slightly oblique , respectively parallel and perpendicular to the long axis of the anal canal . despite the increased tumour conspicuity provided by background fat suppression , short - tau inversion recovery ( stir ) sequences are less useful because of limited spatial detail and difficulty to delineate anatomic landmarks . at our centre ,", "t1-weighted sequences including fat suppression in at least one plane are routinely acquired following standard - dose intravenous gadolinium contrast , to allow detection of lesion enhancement .", "conversely , other authors discourage post - contrast mri acquisitions by stating that enhanced images do not offer additional information to the high soft tissue contrast intrinsic to t2-weighted imaging [ 7 , 10 , 1315 ] .", "although with limited contrast resolution compared with mri , volumetric multidetector computed tomography ( mdct ) acquisitions including image reformations along arbitrary planes allow visualisation of anorectal abnormalities in their cranio - caudal extent with relationship to key anatomical landmarks such as the sphincter complex [ 1618 ] ."], ["although the dentate line is not directly recognisable , its position can be inferred as it corresponds approximately to the upper portion of external sphincter muscles .", "sensitivity of trus and mri for the identification of scac has been reported to approach 90100 % , with high concordance regarding tumour size , although on a limited number of patients , and more precise results with ultrasound for smaller , superficial tumours .", "neoplastic tissue in the anal canal has low - to - intermediate t1 signal intensity and positive enhancement after intravenous gadolinium contrast .", "on t2-weighted and stir sequences , untreated neoplasms display intermediate signal intensity , lower to that of normal ischioanal fat and almost always superior to the internal reference standard represented by uninvolved anal sphincters and gluteal muscles ( figs .  1 , 2 ) .", "although with limited sensitivity compared with mri , on ct images scac may be detected as solid , enhancing nodules or masses within the anus ( figs .  2 , 3 ) .", "axial ( a ) and sagittal ( b ) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle , intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image ( c ) , consistent with t1 tumour ( arrowheads)fig", "axial t2-weighted ( a ) , post - contrast fat - suppressed axial ( b ) and coronal ( c ) t1-weighted images , and corresponding enhanced image from body ct ( d ) show a 5.5-cm long ( t3 ) enhancing tumour with infiltration of the left ischioanal fatty space ( arrowheads)fig .", "post - contrast axial ( a ) and coronal reformatted ( b ) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule . subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scacfig .", "4an elderly , 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri .", "sagittal t2- ( a ) and axial t1-weighted ( b ) images show 5-cm long solid , inhomogeneous neoplastic tissue ( * ) extending from the anus to encase the proximal urethra ( note catheter in place ) a 40-year - old msm with bioptic diagnosis of scac .", "axial ( a ) and sagittal ( b ) t2-weighted images show 2-cm hyperintense nodule contained within the internal sphincter muscle , intensely enhancing as seen on post - contrast fat - suppressed coronal t1-weighted image ( c ) , consistent with t1 tumour ( arrowheads ) a 62-year - old female with biopsy - proven scac .", "axial t2-weighted ( a ) , post - contrast fat - suppressed axial ( b ) and coronal ( c ) t1-weighted images , and corresponding enhanced image from body ct ( d ) show a 5.5-cm long ( t3 ) enhancing tumour with infiltration of the left ischioanal fatty space ( arrowheads ) a 57-year - old woman undergoing abdomino - pelvic mdct for unrelated reasons .", "post - contrast axial ( a ) and coronal reformatted ( b ) detailed images of the anorectal region identify an unexpected 2-cm right - sided enhancing anal nodule .", "subsequent clinical and bioptic assessment confirmed poorly symptomatic ulcerated scac an elderly , 92-year - old man with previous prostatectomy and kidney failure has unenhanced mri .", "sagittal t2- ( a ) and axial t1-weighted ( b ) images show 5-cm long solid , inhomogeneous neoplastic tissue ( * ) extending from the anus to encase the proximal urethra ( note catheter in place ) staging is essential for both prognostic information and correct therapeutic planning , and is performed according to the uicc / ajcc tumour - node - metastasis ( tnm ) system ( table  1 ) , including local lesion extent , lymph node status and distant metastatic spread . in 2010", "the european society for medical oncology ( esmo ) recommended mri as the primary imaging modality to accurately stage scac , taking into account the maximum tumour diameter , possible invasion of adjacent organs and nodal involvement [ 6 , 13].table 1tumour - node - metastasis ( tnm ) staging of anal carcinoma according to lesion site of originanal canalanal marginprimary tumour ( t)txprimary tumour can not be assessedt0no evidence of primary tumourt1tumour 2  cm in greatest dimensiont2tumour 25  cm in greatest dimensiont3tumour > 5  cm in greatest dimensiont4tumour of any size invading adjacent organ(s ) e.g. vagina , urethra , bladdertumour invading deeper structures ( skeletal muscle , cartilage)regional lymph nodes ( n)nxregional lymph nodes can not be assessedn0no regional lymph node metastasisn1metastasis in perirectal lymph node(s)regional lymph node metastasisn2metastasis in unilateral internal iliac and/or inguinal lymph node(s)n3metastasis in internal iliac and perirectal lymph nodes and/or bilateral internal iliac and/or bilateral inguinal lymph nodesdistant metastasis ( m)m0no distant metastasism1distant metastasis tumour - node - metastasis ( tnm ) staging of anal carcinoma according to lesion site of origin particular care should be applied to choosing the longest lesion diameter on t2-weighted images , since correct t parameter staging relies on this measure being below 2  cm , over 5  cm or intermediate ( fig .  1 )", "mri demonstrates good correlation with physical findings concerning t stage , whereas infiltration of adjacent organs and sometimes tumour size are clinically underestimated .", "extramural neoplastic spread may involve the sphincter complex muscles ( external sphincter , levator ani and puborectalis ) and most commonly occurs towards the anterior urogenital triangle with possible vaginal , urethral or bladder involvement .", "sometimes , the tumour may also extend laterally with invasion of the ischioanal fossa ( fig . ", "2 ) , superiorly to the rectum and mesorectal compartment , or inferiorly to the skin and subcutaneous planes of the perianal region . in such instances , t2-hyperintense solid tissue is seen infiltrating the even more hyperintense fat in the ischiorectal ( fig .  2 ) and subcutaneous spaces , encasing the lower signal intensity skeletal muscles , or isointense structures such as the vagina ( fig .  5 ) , prostate and urethra ( fig . ", "notably , radiologists should remember that anal canal carcinoma directly invading the rectal wall , perianal skin , subcutaneous or the sphincter muscle does not imply assessing the tumour stage as t4 . for anal margin scac", ", a t4 lesion is defined by invasion of deeper structures such as the skeletal muscle or cartilage [ 9 , 10 , 13].fig .", "axial t2-weighted ( a ) and post - contrast fat - suppressed axial t1-weighted ( b ) images show inhomogeneous anal tissue invading the left aspect of the vagina ( arrowheads ) , with internal non - enhancing necrosis and peripheral enhancement .", "biopsy diagnosed scac with superimposed infection a 32-year - old hiv - positive woman with clinical diagnosis of anovaginal fistula .", "axial t2-weighted ( a ) and post - contrast fat - suppressed axial t1-weighted ( b ) images show inhomogeneous anal tissue invading the left aspect of the vagina ( arrowheads ) , with internal non - enhancing necrosis and peripheral enhancement .", "biopsy diagnosed scac with superimposed infection the incidence of regional nodal involvement increases with primary tumour size .", "lymph node metastases may be present ( in 25 % of cases ) even with superficial ( up to t2 ) scacs , and are unreliably assessed clinically .", "nodal staging evaluation relies on the distance from the primary tumour rather than on the number of involved nodes .", "mri is highly helpful to assess lymph node metastatic involvement , although the mere size criterion is far from accurate and associated with both false - positive and false - negative results .", "short - axis threshold values of 8  mm , 5  mm and 10  mm have been suggested for pelvic , perirectal and inguinal lymph nodes , respectively .", "additional helpful features to increase specificity include loss of the normal bean - shaped morphology and fatty hilum , internal t1 and t2 signal heterogeneity with central necrosis , and inhomogeneous enhancement ( figs . ", "6a 65-year - old woman with history of previously treated small scac 3  years earlier .", "axial t2- ( a ) and post - contrast t1-weighted ( b ) images show roundish 1-cm left inguinal node ( arrowheads ) with internal fluid - like necrosis and inhomogeneous enhancement , confirmed by ultrasound ( c ) as hypoechoic with loss of normal nodal structure .", "surgical exeresis ( postoperative status as seen in d , follow - up mri ) confirmed metastatic node from scacfig .", "axial t2-weighted ( a , b ) and post - contrast fat - suppressed t1-weighted ( c ) images show inguinal nodal metastases , larger on left side ( arrowheads ) plus bilateral enhancing perirectal adenopathies ( arrows ) a 65-year - old woman with history of previously treated small scac 3  years earlier .", "axial t2- ( a ) and post - contrast t1-weighted ( b ) images show roundish 1-cm left inguinal node ( arrowheads ) with internal fluid - like necrosis and inhomogeneous enhancement , confirmed by ultrasound ( c ) as hypoechoic with loss of normal nodal structure .", "surgical exeresis ( postoperative status as seen in d , follow - up mri ) confirmed metastatic node from scac the same patient as in fig .  2 .", "axial t2-weighted ( a , b ) and post - contrast fat - suppressed t1-weighted ( c ) images show inguinal nodal metastases , larger on left side ( arrowheads ) plus bilateral enhancing perirectal adenopathies ( arrows ) as suggested by ecco guidelines , search for distant spread is usually performed by means of contrast - enhanced body mdct , with conventional imaging appearances of liver and lung metastases . dissemination is very uncommon ( less than 5 % of patients at initial diagnosis , and is usually encountered in association with post - treatment recurrence . alternatively , in immunocompetent patients f - fluorodeoxygluocose positron emission tomography ( fdg - pet / ct ) has high specificity for nodal and visceral dissemination . at diagnosis , fdg - pet /", "ct may alter staging of anal scac in 20 % of patients , leading to inclusion of involved pelvic or inguinal lymph nodes in the radiation field [ 6 , 7 ] ."], ["in our experience , not infrequently anal tumours coexist with inflammatory conditions such as proctitis and abscesses . in such instances , mdct and mri provide confident detection of perirectal inflammatory changes and purulent collections that are differentiated from solid neoplastic tissue , thus allowing a correct therapeutic choice including surgical drainage as necessary ( figs .  5 , 8 , 9 , 10 ) .", "resolution of associated inflammatory changes during treatment is easily monitored by cross - sectional imaging ( figs . ", "8a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass .", "axial ( a ) and coronal reformatted ( b ) ct images show moderately heterogeneous tissue ( * ) in its entire longitudinal extent from the anorectal junction to below the anal verge , associated with large necrotic iliac adenopathies ( * in c ) .", "axial images ( b , c ) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal ( arrowheads ) associated with abscess collections with mixed gas - fluid content ( * ) and fistulas crossing the ischioanal space .", "surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . after surgical drainage and subsequent chemo - radiotherapy , mri ( c ) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracksfig .", "staging mri confirms left - sided anal thickening ( arrowheads ) with abnormal t2 signal intensity ( a ) and strong contrast enhancement ( b ) .", "incidentally , two abscess collections with necrotic content and peripheral enhancement are seen ventrally , connected to the anal canal by a fistulous track ( arrow in c ) .", "subtotal regression of changes after treatment is seen on follow - up mri ( d ) a 60-year - old man with aids and clinical finding of ulcero - fungating anal mass .", "axial ( a ) and coronal reformatted ( b ) ct images show moderately heterogeneous tissue ( * ) in its entire longitudinal extent from the anorectal junction to below the anal verge , associated with large necrotic iliac adenopathies ( * in c ) .", "biopsy confirmed superinfected scac a 53-year - old man with purulent drainage and clinical diagnosis of perianal inflammation .", "axial images ( b , c ) from urgent contrast - enhanced mdct show solid circumferential thickening of the anal canal ( arrowheads ) associated with abscess collections with mixed gas - fluid content ( * ) and fistulas crossing the ischioanal space .", "surgical examination under anaesthesia including biopsies revealed ulcerated scac with superinfection . after surgical drainage and subsequent chemo - radiotherapy , mri ( c ) shows complete resolution of both inflammatory and neoplastic changes with residual t2-hypointense fibrotic tracks a 39-year - old hiv - infected man with biopsy - proven scac .", "staging mri confirms left - sided anal thickening ( arrowheads ) with abnormal t2 signal intensity ( a ) and strong contrast enhancement ( b ) .", "incidentally , two abscess collections with necrotic content and peripheral enhancement are seen ventrally , connected to the anal canal by a fistulous track ( arrow in c ) .", "subtotal regression of changes after treatment is seen on follow - up mri ( d ) furthermore , cross - sectional imaging particularly with mri also proves useful to differentiate anal carcinoma from other causes of local pain and perineal masses , such as pilonidal sinus diseases , gartner duct or bartolini gland cysts , tailgut cysts , uncommon soft - tissue neoplasms , urethral cancer , lymphoma or metastases [ 14 , 22 ] ."], ["in the past , scac was treated with abdomino - perineal resection and permanent colostomy . currently , anal margin and small canal tumours without evidence of nodal spread may be successfully excised . in all other cases ,", "the standard treatment includes radiation combined with mitomycin - c plus infusional 5-fu chemotherapy , yielding an 80 % 5-year survival rate with preservation of sphincter function .", "advanced t3/t4 tumours have worse outcomes with a 4068 % 3-year disease - free survival .", "salvage surgery with abdomino - perineal resection is reserved for persistent or recurrent tumours [ 3 , 6 , 2325 ] .", "after radio - chemotherapy , imaging follow - up with mri represents a useful complement to clinical evaluation in the assessment of therapeutic response . shortly after treatment completion , interpretation of mri is usually challenging due to the superimposition of inflammatory changes resulting from radiotherapy . performed at least 68  weeks after treatment completion , mri provides confident , reproducible assessment of post - treatment modifications .", "findings indicative of a positive response include size reduction and diminished t2 signal intensity of the treated tumour and associated adenopathies ( figs . ", "the appearance of t2-hypointense signal is consistent with fibrosis , although it does not allow excluding minor residual neoplastic foci for sure ( fig . ", "stability in size and signal intensity of any residual abnormality visible at mri in the site of the treated lesion 1 year after therapy has been reported to be strongly associated with a favourable outcome .", "locoregional and/or distant recurrence occurs in up to 35 % of treated patients , and is strongly associated with advanced ( t3t4 ) stage and nodal involvement at presentation [ 7 , 20 ] .", "persistent and locally recurrent tumours often display an aggressive behaviour , with possible extensive invasion of the adjacent organs and pelvic bony structures , and a tendency for lymphatic dissemination ( figs . ", "initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal ( a ) and contrast enhancement ( b ) ( arrowheads ) .", "complete disappearance of the lesion is observed on axial t2 ( c ) and post - contrast fat - suppressed t1-weighted ( d ) images following chemo - radiotherapyfig .", "mri staging including axial ( a ) and coronal ( b ) t2-weighted images detect a solid , 3-cm eccentric anal mass ( arrowhead ) consistent with t2 tumour , associated with right inguinal adenopathies with analogous signal features .", "after chemo - radiotherapy , mri follow - up ( c , d ) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathiesfig .", "initially , mdct ( a ) shows left - sided thickening with involvement of the external sphincter ( arrowheads ) .", "after chemo - radiotherapy , lesion regression is observed on mri with appearance of t2-hypointense fibrosis ( * ) and hyperintense nodule ( arrow ) consistent with residual tumourfig .", "14an elderly , 85-year - old lady with biopsy - proven scac and multiple comorbidities .", "initial mri ( a , b ) shows showed circumferential anal wall thickening with abnormal solid signal ( arrowheads ) measuring 6  cm in length , plus a suspicious centimetric left inguinal lymph node ( arrow in a ) , findings consistent with t3n2 lesion . after reduced chemo - radiotherapy , follow - up mri ( c , d ) 4 months later disclosed progression and partial necrosis of both primary tumour ( arrowhead ) and inguinal adenopathy ( arrow ) , plus appearance of an exophytic tissue mass protruding from the external anal orifice ( * in d ) an 83-year - old man with known scac .", "initial mri shows moderate circumferential thickening of the anus with t2-hyperintense signal ( a ) and contrast enhancement ( b ) ( arrowheads ) .", "complete disappearance of the lesion is observed on axial t2 ( c ) and post - contrast fat - suppressed t1-weighted ( d ) images following chemo - radiotherapy a 46-year - old female patient with biopsy - proven scac . mri staging including axial ( a ) and coronal ( b ) t2-weighted images detect a solid , 3-cm eccentric anal mass ( arrowhead ) consistent with t2 tumour , associated with right inguinal adenopathies with analogous signal features .", "after chemo - radiotherapy , mri follow - up ( c , d ) shows complete disappearance of both anal neoplastic solid tissue and lymphadenopathies a 46-year - old hiv - infected man with biopsy - proven scac .", "initially , mdct ( a ) shows left - sided thickening with involvement of the external sphincter ( arrowheads ) .", "after chemo - radiotherapy , lesion regression is observed on mri with appearance of t2-hypointense fibrosis ( * ) and hyperintense nodule ( arrow ) consistent with residual tumour an elderly , 85-year - old lady with biopsy - proven scac and multiple comorbidities . initial mri ( a , b ) shows showed circumferential anal wall thickening with abnormal solid signal ( arrowheads ) measuring 6  cm in length , plus a suspicious centimetric left inguinal lymph node ( arrow in a ) , findings consistent with t3n2 lesion .", "after reduced chemo - radiotherapy , follow - up mri ( c , d ) 4 months later disclosed progression and partial necrosis of both primary tumour ( arrowhead ) and inguinal adenopathy ( arrow ) , plus appearance of an exophytic tissue mass protruding from the external anal orifice ( * in d )"], ["after the introduction of highly active anti - retroviral treatment ( haart ) , people with hiv infection or acquired immunodeficiency syndrome ( aids ) gained a greatly improved life expectancy with better immune conditions , at the price of an increased tendency to develop tumours .", "although it is considered a non - aids - defining malignancy , currently scac ranks third ( 8.2 % ) among neoplasms observed in hiv / aids populations , with a substantially higher incidence in msms and in long - standing infected people [ 4 , 26 ] .", "dysplastic intraepithelial lesions are highly prevalent in hiv - infected people , and hiv represents a marker for coinfection with other sexually transmitted diseases such as hpv . since the risk of developing scac increases with the total time elapsed with cd4 + count below 200  cells/l , it has been hypothesised that hiv - related immune suppression acts as a cofactor to hpv in the development of anal dysplasia and progression to overt carcinoma [ 1 , 3 , 4 ] .", "screening procedures including high - resolution anoscopy and cytology smears are increasingly adopted at hiv care centres , to allow detection of scac precursors and early - stage tumours amenable to limited excision and topical therapies .", "prevention should limit the occurrence of advanced stages at diagnosis in the future [ 4 , 2629 ] .", "a high prevalence ( at least 30 % ) of anorectal complaints is characteristic of hiv - positive patients , particularly those practicing anoreceptive intercourse .", "differential diagnosis encompasses a wide spectrum of abnormalities , including non - specific anal diseases such as haemorrhoids , fissures , fistulas and abscesses , along with venereal infections , viral ulcers , and a non - negligible ( 7 % ) rate of neoplasms such as scac , lymphoma and kaposi s sarcoma .", "therefore , when performing cross - sectional imaging procedures on hiv - infected patients , even for unrelated complaints , special attention should be paid to the anal region , with a focus on the possible identification of solid , enhancing tissue consistent with tumour that indicates need for biopsy ( figs .  5 , 8 , 10 , 13 ) [ 21 , 30 , 31 ] .", "despite initial discouraging reports , in the haart era hiv - positive patients are likewise treated with standard chemo - radiation regimens , reaching satisfactory results in terms of local control and survival rates , although with increased toxicity and frequent local recurrences [ 4 , 5 , 26 , 27 , 32 ] ."], ["some literature reports have highlighted the increased risk of anus and lower rectum carcinomas associated with long - standing , severe perianal fistulising crohn s disease ( cd ) ( fig . ", ", scac reaches a 14 % proportion among all colorectal cancers , which is ten - times higher than the usual figure .", "furthermore , cd patients develop anorectal carcinomas at a younger age ( 20  years earlier ) than the general population . according to the hypothesised pathogenesis", ", fistulas probably allow hpv an easier access to the epithelial layers , and chronic mucosal regeneration may ultimately lead to neoplastic changes .", "however , anal tumours may occasionally develop in patients with ulcerative colitis ( uc)-related ( fig . ", "15a 40-year - old male patient with long - standing perianal crohn s disease , being treated with seton .", "contrast - enhanced mdct images ( a , b , c in cranio - caudal order ) show right - sided levator ani abscess ( * ) , extensive perianal fistulisation occupying the ischioanal space ( arrow ) , and marked solid - appearing circumferential anorectal thickening ( arrowheads ) .", "initial multiplanar mdct ( a , b ) identified enhancing anal thickening ( arrowheads ) with right - sided vaginal infiltration and solid tissue ( * ) extending to reach the internal obturator muscle . after biopsy confirmation of scac and surgical debulking with colostomy , repeat mdct ( c , d ) detected enlarging neoplastic residue ( * ) .", "shortly after chemo - radiotherapy , mri ( e ) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour ( thin arrows ) .", "mri follow - up ( e ) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence ( arrow ) .", "after negative clinical reassessment and pet findings , this post - treatment finding remained stable on further mri studies ( not shown ) a 40-year - old male patient with long - standing perianal crohn s disease , being treated with seton .", "contrast - enhanced mdct images ( a , b , c in cranio - caudal order ) show right - sided levator ani abscess ( * ) , extensive perianal fistulisation occupying the ischioanal space ( arrow ) , and marked solid - appearing circumferential anorectal thickening ( arrowheads ) .", "abdomino - perineal resection for scac was performed a 39-year - old woman with history of ulcerative colitis and perianal inflammation .", "initial multiplanar mdct ( a , b ) identified enhancing anal thickening ( arrowheads ) with right - sided vaginal infiltration and solid tissue ( * ) extending to reach the internal obturator muscle . after biopsy confirmation of scac and surgical debulking with colostomy , repeat mdct ( c , d ) detected enlarging neoplastic residue ( * ) .", "shortly after chemo - radiotherapy , mri ( e ) detected the formation of a thick hypointense fibrotic band in the site of the regressed tumour ( thin arrows ) .", "mri follow - up ( e ) identified appearance of a contralateral enhancing tissue band interpreted as suspicious for local recurrence ( arrow ) .", "after negative clinical reassessment and pet findings , this post - treatment finding remained stable on further mri studies ( not shown ) diagnosis is often unsuspected or delayed because of pre - existent , unspecific complaints and clinical assessment is hampered by complex inflammation with stricture and local pain . as a result ,", "ibd - associated anal cancers are often advanced at presentation , may require extensive surgery plus chemotherapy and radiotherapy , and are associated with a severe prognosis .", "therefore , patients with early - onset or long - standing perianal cd should undergo clinical and imaging surveillance , particularly when new or changed symptoms develop .", "radiologists should be aware of the increased risk for anorectal cancer in middle - aged ibd patients , and clearly report any solid tissue as suspicious for neoplasm and suggest biopsy ( figs . "], ["the established association with hpv infection and premalignant intra - epithelial dysplastic changes provides insight into the pathogenesis of hiv- and ibd - related anal cancers , and the possibility of prevention or early diagnosis through screening of high - risk individuals [ 1 , 2 ] .", "state - of - the - art cross - sectional imaging with high - resolution mri using external phased - array coils and multiplanar mdct allow detailed , comprehensive visualisation of abnormalities involving the anus and perineal region .", "currently , mri represents the modality of choice for primary regional staging of scac , assessment of complications , of therapeutic response following chemo - radiotherapy , and of possible recurrences [ 9 , 10 , 13 ] ."]]}
{"article_id": "PMC2917551", "article_text": ["black yeasts from the family herpotrichiellaceae ( order chaetothyriales ) are fungi with a remarkable dual ecology . on the one hand , they have a unique ability to adapt to extreme environments ( exposure to toxic chemicals , high temperature , scarcity of nutrients , acidic , and/or dry conditions ) , while on the other hand , they exhibit a significant human pathogenic potential . unlike common opportunistic fungi , herpotrichiellaceous black yeasts frequently cause infections in individuals without known underlying disease   and only occasionally in immunocompromised patients . also , more often than any other fungal group , these organisms have been reported from environments that are rich in aromatic compounds .", "the first evidence on such phenomenon arose from the frequent isolation of black yeasts from wood treated with creosote , while their occurrence on untreated wood was comparatively low , indicating that accumulation of aromatic compounds might promote the growth of these fungi .", "the concurrence of these two ecological traits in a single species has been observed for exophiala dermatitidis , isolated abundantly not only on tropical creosoted railway ties but also from clinical cases of severe mycoses [ 26 , 27 ] .", "it has been hypothesized that wild berries constitute a natural niche of e. dermatitidis , which might then be ingested by birds and humans and sporadically resulting in mycoses .", "deposition of feces on creosoted railway ties subsequently leads to the massive enrichment of this fungus .", "the related species exophiala bergeri , exophiala heteromorpha , exophiala oligosperma , and exophiala xenobiotica have also been reported as opportunistic fungi causing infections that are generally less serious and from creosoted wood [ 57 , 11 , 27 ] . however , the connection between route of infection and natural occurrence is less evident with these species .", "the tendency of herpotrichiellaceous fungi toward aromatic metabolism has been confirmed by their recurrent isolation from biofilters treating vapors of volatile aromatic hydrocarbons . besides hydrocarbon exposure , environmental conditions in biofilters", "are characterized by a relative low water activity and acidification of the filter bed , which might lead to biomass inhibition problems . in this", "respect , fungal colonization in air biofilters has generally been related to an improved bioreactor performance .", "detailed metabolic studies on fungi from gas biofilters have demonstrated their capacity to assimilate alkylbenzene hydrocarbons as the sole source of carbon and energy [ 3 , 20 , 29 ] , which appears to be quite an uncommon metabolic feature in the eukaryotes .", "molecular phylogenetic characterization of these  biofilter fungi  has shown a predominant affiliation to the genera exophiala and cladophialophora ( fam .", "herpotrichiellaceae ) , particularly to the species exophiala lecanii - corni , e. oligosperma , and the recently described e. xenobiotica , cladophialophora saturnica , and cladophialophora inmunda [ 1 , 6 ] .", "despite the medical and environmental relevance of herpotrichiellaceous black yeasts , little is understood on their biodiversity and natural occurrence . in order to address questions on niche shifts and environmental prevalence in relation to virulence factors and routes of transmission ,", "the aim of the present work is to apply a specific isolation method based on the enrichment of black yeasts by simulating in batch solid state - like cultures the environmental conditions that are found in gas biofilters for the treatment of volatile aromatic hydrocarbons .", "different environmental samples related to the life cycle of e. dermatitidis were used as inocula .", "the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table  1 .", "these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons .", "environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14  days .", "the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . \n", "table  1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95  n , 51045.96  ea2oak railway tie , between railsnear station , hollandsche rading521041.95  n , 51045.96  ea3oak railway tie , outside railsforest area , hilversum521220.68  n , 5119.66  ea4oak railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ea5concrete railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ec1berry , sorbus aucupariaroadside , de bilt52 717.18  n , 5 948.13  ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19  n , 5 931.42  ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec4berry , sorbus aucuparialapersveld park , hilversum521254.74  n , 51110.25  ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52  n , 51051.99  ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed4fresh goose feceslapersveld park , hilversum521254.74  n , 51110.25  ed5old goose feceslapersveld park , hilversum521254.74  n , 51110.25  e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source .", "serum flasks of 100  ml were filled with approximately 25  ml of perlite granules , saturated with mineral medium . each environmental sample ( 13  g )", "was washed with phosphate - buffered saline , and the suspension ( 40  ml ) was used to inoculate four different batches ( 10  ml each batch ) . as a control", ", these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth .", "the inoculated flasks were then closed with a cotton  wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene .", "this gas phase was generated by placing 10  ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate .", "a solution of 140  g  l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3  months . after this time", ", the perlite granules in each flask were washed with 5060  ml sterile water .", "one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c .", "colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters .", "a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi .", "dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions .", "the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) .", "pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50  l volumes containing 25  ng of template dna , 5  l reaction buffer ( 0.1 m tris  hcl , ph  8.0 , 0.5  m kcl , 15  mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2  mm of each dntp and 2.0  u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .", "amplification was performed with cycles of 2  min at 94c for primary denaturation , followed by 35  cycles at 94c ( 45  s ) , 52c ( 30  s ) and 72c ( 120  s ) , with a final 7-min extension step at 72c .", "amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) .", "sequence pcr was performed as follows : 95c for 1  min , followed by 30  cycles consisting of 95c for 10  s , 50c for 5  s , and 60c for 2  min .", "reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) .", "sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) .", "for the phylogenetic assignment , the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) .", "phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) .", "the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table  1 .", "these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons .", "environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14  days .", "the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . \n", "table  1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95  n , 51045.96  ea2oak railway tie , between railsnear station , hollandsche rading521041.95  n , 51045.96  ea3oak railway tie , outside railsforest area , hilversum521220.68  n , 5119.66  ea4oak railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ea5concrete railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ec1berry , sorbus aucupariaroadside , de bilt52 717.18  n , 5 948.13  ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19  n , 5 931.42  ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec4berry , sorbus aucuparialapersveld park , hilversum521254.74  n , 51110.25  ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52  n , 51051.99  ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed4fresh goose feceslapersveld park , hilversum521254.74  n , 51110.25  ed5old goose feceslapersveld park , hilversum521254.74  n , 51110.25  e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons", "the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source .", "serum flasks of 100  ml were filled with approximately 25  ml of perlite granules , saturated with mineral medium . each environmental sample ( 13  g ) was washed with phosphate - buffered saline , and the suspension ( 40  ml ) was used to inoculate four different batches ( 10  ml each batch ) . as a control", ", these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth .", "the inoculated flasks were then closed with a cotton  wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene .", "this gas phase was generated by placing 10  ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate .", "a solution of 140  g  l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3  months . after this time ,", "one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c .", "colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters .", "a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi .", "dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions .", "the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) .", "pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50  l volumes containing 25  ng of template dna , 5  l reaction buffer ( 0.1 m tris  hcl , ph  8.0 , 0.5  m kcl , 15  mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2  mm of each dntp and 2.0  u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .", "amplification was performed with cycles of 2  min at 94c for primary denaturation , followed by 35  cycles at 94c ( 45  s ) , 52c ( 30  s ) and 72c ( 120  s ) , with a final 7-min extension step at 72c .", "amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) .", "sequence pcr was performed as follows : 95c for 1  min , followed by 30  cycles consisting of 95c for 10  s , 50c for 5  s , and 60c for 2  min .", "reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) .", "sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) . for the phylogenetic assignment ,", "the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) .", "phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) .", "a total of 71 fungal strains were isolated upon enrichment of environmental samples on atmospheres of different volatile aromatic hydrocarbons ( table  2 ) . of those ,", "a single mold ( aspergillus fumigatus ) other than black yeasts was isolated from berries of sorbus aucuparia ( sample c1 ) .", "the highest isolation rates were achieved with samples taken from creosoted wooden railway ties , particularly from those arising from the tie section located outside the rails and using toluene as the incubation substrate ( 39 isolates , samples a1 and a3 ) .", "these samples were the only ones that yielded isolates when xylene or benzene was applied as enrichment substrates ( 17 and 10 strains , respectively ) , but not a single fungus was isolated when naphthalene was supplied as the sole carbon source .", "though in lower numbers , black yeasts were also isolated from the tie section located between the rails ( 14 strains , samples a2 and a4 ) . besides creosote , these samples were likely to contain fecal and mineral oil contamination as well .", "table  2number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbonssample codebenzenetoluenexylenenaphthalenetotal strainsa10411015a203003a31086024a40110011c105005c207007d206006total strains104317071one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbons one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection from the natural sampled environments , black yeasts were isolated in two out of four berries from s. aucuparia ( 12 isolates , samples c1 and c2 ) , but no fungi were obtained from berry samples of viburnum opulus and crataegus monogyna ( samples c5 and c6 , respectively ) . soil mixed with bird feces ( guano ) also yielded melanized fungi in one out of five tested samples ( six isolates , sample d2 ) .", "no black yeasts were isolated when suspensions of the samples described above ( table  1 ) were directly plated onto sga plates , without the enrichment step on volatile aromatic hydrocarbons , while several common and heavily sporulating fungal species were encountered .", "phylogenetic analysis of aligned its1 - 5.8s - its2 rrna sequences from the obtained strain collection showed that the isolates were affiliated to five major groups ( fig .  1 ) .", "sequences from reference strains deposited at the cbs collection ( utrecht , the netherlands ) that were related to the isolates in terms of their sequence homology and/or ecophysiology were also included in the analysis .", "those encompassed the type strains of e. xenobiotica , e. bergeri , exophiala spinifera , e. oligosperma , e. heteromorpha , e. dermatitidis , e. lecanii - corni , and phialophora sessilis , as well as from those of the cladophialophora species that have been so far related to the metabolism of aromatic hydrocarbons : cladophialophora immunda , c. saturnica , and cladophialophora psammophila sp . nov . , the latter species being in the process of description ( badali et al . , submitted )", ". strains of e. xenobiotica , e. bergeri , and e. dermatitidis that have previously been isolated from creosoted railway ties were also used . \n", "figure  1neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate .", "sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches", "were also included in the analysis ( genbank sequence codes are given between square brackets ) neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate .", "sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches", "were also included in the analysis ( genbank sequence codes are given between square brackets ) sequence comparisons against those from reference strains revealed that most of the isolates were similar to exophiala xenobiotica ( 32 strains ) .", "the majority was highly homologous ( 99% ) to the ex - type strain , and some were slightly deviating ( 97% homology ) .", "a second group of isolates ( nine strains ) was identified as belonging to e. bergeri on the basis of a 99% sequence homology to the ex - type strain .", "the cluster encompassing e. bergeri also included a major group ( 24 strains ) , the sequence of which deviated significantly from the ex - type strain ( 94% sequence homology ) , so that its affiliation to e. bergeri might be put into question .", "blast searches on this latter group into the genbank genomic database revealed a close match ( 98% homology ) with an unidentified exophiala strain isolated from a rock surface .", "a minor e. bergeri sibling group was composed of two strains for which no known sequence match was found .", "a third group ( three strains ) did not match any known reference type strain , but genbank searches revealed a high sequence homology ( 98% ) to the sequence of an uncultivated fungus from a municipal composting plant . except for a single isolate ( dh18150 )", "obtained from a s. aucuparia berry ( sample c1 ) , the e. xenobiotica - related strains were isolated exclusively from creosoted oak tie sections located outside the rails ( samples a1 and a3 ) .", "within this group , three strains were obtained under benzene enrichment , while the remaining 29 strains were isolated with toluene or xylene as enrichment substrates .", "besides e. xenobiotica , sample a3 also yielded five isolates upon benzene enrichment , which were identified as e. bergeri .", "the related between - the - rails section from that same tie ( sample a4 ) and the guano - rich soil ( sample d2 ) yielded 1 and 3 strains of e. bergeri , respectively , using toluene as the enrichment substrate .", "the occurrence of black yeasts in the human - dominated environment has previously been underestimated due to the application of routine microbial isolation methods . under common laboratory culture conditions ,", "numerous protocols for the selective isolation of black yeasts have been developed during the last decades ( an overview is presented in table  3 ) , which have yielded new data on the presence of these fungi in a wide range of artificial environments , sometimes with no obvious counterpart in nature .", "several methods are based on osmotolerant and oligotrophic abilities of target fungi . with these regimens ,", "the isolated black yeasts are mainly osmotolerant members of the saprophytic order dothideales , although the rock - inhabiting fungi appear to be an area of overlap between the dothideales and the chaetothyriales .", "applying enrichment methods based on the inoculation of rodents , exposure to aromatic hydrocarbons , assimilation of rare sugars , and/or incubation at high temperature , most isolated black yeast - like fungi are members of the chaetothyriales .", "it should be noted though that neither of these enrichment conditions might represent the natural niche for these fungi , and they are therefore classified with difficulty in any of the known ecological categories . \n table  3overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate resultsmethodprevalent genusorderecologyreferenceanimal baitexophiala , fonsecaea , cladophialophorachaetothyrialesopportunists[2 , 8]erythritolexophialachaetothyrialesopportunistsmineral oilexophiala , fonsecaeachaetothyrialesopportunistsraulin 40cexophialachaetothyrialesopportunistsneedleconiosporiumchaetothyrialesrock fungialkylbenzene vaporscladophialophora , exophialachaetothyrialesxenobioticsacidicexophialachaetothyrialesacidophileshortaeadothidealescrushconiosporiumchaetothyrialesrock fungihormonemadothidealeslow strengthexophialachaetothyrialesoligotrophssarcinomycesdothidealesrock fungicadophoraleotialesoligotrophshigh saltaureobasidium , hortaeadothidealeshalophiles[31 , 32]suspendcryomyces , friedmanniomycesdothidealespsychrophilesethanolbaudoiniadothidealesethanophiles overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate results the present study demonstrates that enrichment on inert solid media incubated under a controlled atmosphere rich on volatile aromatic hydrocarbons compounds displays an extraordinary selectiveness toward chaetothyrialian fungi from the herpotrichiellaceae family .", "for example , under hydrocarbon atmospheres , the berry samples were free from aureobasidium pullulans , which is a member of the dothideales , otherwise an extremely common component of honeydew mycobiota and slightly osmotic surfaces of fruits and berries .", "this species was encountered abundantly along with many contaminants on sga control plates without alkylbenzene enrichment .", "the presence of heavily sporulating airborne fungi , frequently found in soil and litter , was strongly reduced by alkylbenzenes and limited to a single strain of a. fumigatus .", "interestingly , the dothidiaceous species amorphoteca resinae , known as  creosote fungus  for its common isolation from creosoted wood , was found neither in this study nor in previous fungal isolation surveys from creosoted wood [ 26 , 27 ] .", "consistent and relatively abundant isolation of herpotrichiellaceous black yeasts from unpolluted environmental samples , such as wild berries , only after addition of volatile aromatic hydrocarbons strongly suggests that these substances play an essential role in the ecology and competitive ability of those fungi . in particular , the utilization of aromatic compounds as carbon and energy source might explain the key factor determining their success in anthropized environments .", "with advanced analytical techniques , it has been shown that volatile aromatic hydrocarbons that traditionally were associated with environmental pollution are in fact ubiquitously present in nature , though at very low concentrations .", "toluene , for example , is produced biologically in different natural environments including maturing berries .", "on the other hand , as extremophilic and slow - growing microorganisms , incubation on a support that is relatively dry and poor in nutrients is also a determinant factor for the enrichment of black yeasts , when compared to traditional liquid enrichment cultures . regarding both", "the presence of aromatic compounds and exposure to fluctuating and/or extreme environmental conditions , wood treated with creosote can be regarded as a paradigmatic environmental niche for black yeasts .", "creosote is a distillate derived entirely from tar , which is rich in a wide variety of polycyclic aromatic hydrocarbons , phenols , and cresols , and has widely been used as wood preservative against microbial decay . due to its carcinogenic character", ", the use of creosote has been banned in the european union . yet , creosoted wood utilities are still widely present in the open environment , such as telephone poles , railway cross ties , switch ties , and bridge timbers .", "also , creosote is an important soil contaminant in former wood creosoting plants . in a previous study ,", "e. dermatitidis was isolated from wild berries and massively from creosoted railway ties in thailand when using the pre - incubation in raulin s solution at low ph and incubated at 40c , this protocol being quite selective for e. dermatitidis .", "it was hypothesized that the life cycle of this species involved the occurrence on wild berries , ingestion and passage through the human intestinal tract , and feces deposition on railway ties .", "subsequent enrichment on creosoted wood might then be related not only to the oligotrophy , thermotolerance ( particularly under tropical conditions ) , acidotolerance , and moderate osmotolerance of e. dermatitidis but also to the presence of aromatic compounds .", "the temperate wild berry sampling locations that were the subject of our study ( table  1 ) had previously been analyzed for the occurrence of black yeasts [ 18 , 26 ] .", "collected berries were homogenized , diluted , and used for spread plates or were subjected to the raulin s pre - incubation protocol . in these earlier studies involving several hundreds of samples , only a single strain of e. dermatitidis", "the lack of this fungus in temperate climate conditions suggests that e. dermatitidis has an origin in the tropics .", "this hypothesis is further supported by the isolation of e. dermatitidis from creosoted railway ties in brazil , using the mineral oil - flotation protocol [ 15 , 24 ] . from the present enrichment program on volatile alkylbenzenes", ", it appears that exophiala species , though other than the thermophilic e. dermatitidis , are regularly present on substrates that are characteristic from the latter species ( berries , bird feces , and creosoted railway ties ) .", "it is known to be associated with mild cutaneous infections in humans   and is also commonly found in habitats rich in xenobiotics such as hydrocarbon - polluted soil and gas biofilters for the biodegradation of btex compounds [ 6 , 24 , 26 ] .", ", it was regarded to be an extremely rare opportunist , although a recent study showed that it was underdiagnosed due to a general lack of application of sequencing for identification . using the selective oil - flotation method ,", "e. bergeri was isolated from samples of wood treated or non - treated with creosote preservatives .", "a distinct cluster was identified within the e. bergeri group , which might belong to a yet - undescribed species .", "strains from this group were predominantly isolated from the section between the rails of sampled wooden ties , which were partly contaminated by machine oil and human feces .", "the remaining strains of e. xenobiotica and e. bergeri senso stricto were obtained from the section outside the rails , where this contaminating effect was less marked .", "three identical strains were isolated from guano - rich soil covered with hedera by enrichment under a toluene atmosphere .", "no close sequence homology against reference strains was found for these fungi , indicating that a yet - undescribed species is concerned .", "the phylogenetic affiliation of the undescribed strains will be reported in detail in a subsequent study .", "the extremophilic nature of black yeast , in combination with their capacity to metabolize aromatic pollutants , makes them ideal candidates for specific bioremediation purposes .", "prospects on the biotechnological application of these fungi until recently have been hampered by potential biohazard , but , as phylogenetic data on these organisms accumulates , it seems that severe pathogens and hydrocarbon - associated species are consistently well - separated siblings ( de hoog , unpublished ) .", "indeed , enrichment on volatile aromatic hydrocarbons in gas biofilters and similar systems thus far has never yielded pathogenic species of the higher fungal biohazard risk categories , such as cladophialophora bantiana or e. dermatitidis .", "this suggests that a certain degree of speciation between pathogenic and aromatic hydrocarbon metabolizing species has occurred in evolutionary terms . yet", ", more studies are needed to emphasize the importance of selective methods to isolate black yeasts and acquire more understanding of their ecological niches .", "the application of the enrichment method used in the present paper contributes to the isolation of fungi that have a potential for bioremediation of sites polluted with monoaromatic hydrocarbons ."], "abstract_text": ["<S> black yeast members of the herpotrichiellaceae present a complex ecological behavior : they are often isolated from rather extreme environments polluted with aromatic hydrocarbons , while they are also regularly involved in human opportunistic infections . </S>", "<S> a selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology . </S>", "<S> samples from natural ecological niches and man - made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons . </S>", "<S> benzene , toluene , and xylene were provided separately as the sole carbon and energy source via the gas phase . </S>", "<S> the assayed isolation protocol was highly specific toward mesophilic exophiala species ( 70 strains of this genus out of 71 isolates ) . </S>", "<S> those were obtained predominantly from creosote - treated railway ties ( 53 strains ) , but isolates were also found on wild berries ( 11 strains ) and in guano - rich soil samples ( six strains ) . </S>", "<S> most of the isolates were obtained on toluene ( 43 strains ) , but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi ( 17 and 10 isolates , respectively ) . based upon morphological characterizations and dna sequences of the full internal transcriber spacers ( its ) and the 8.5s rrna genes , the majority of the obtained isolates were affiliated to the recently described species exophiala xenobiotica ( 32 strains ) and exophiala bergeri ( nine strains ) . </S>", "<S> members of two other phylogenetic groups ( 24 and two strains , respectively ) somewhat related to e. bergeri were also found , and a last group ( three strains ) corresponded to an undescribed exophiala species . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Sampling", "Isolation", "Molecular Identification", "Results", "Discussion"], "sections": [["black yeasts from the family herpotrichiellaceae ( order chaetothyriales ) are fungi with a remarkable dual ecology . on the one hand , they have a unique ability to adapt to extreme environments ( exposure to toxic chemicals , high temperature , scarcity of nutrients , acidic , and/or dry conditions ) , while on the other hand , they exhibit a significant human pathogenic potential . unlike common opportunistic fungi , herpotrichiellaceous black yeasts frequently cause infections in individuals without known underlying disease   and only occasionally in immunocompromised patients . also , more often than any other fungal group , these organisms have been reported from environments that are rich in aromatic compounds .", "the first evidence on such phenomenon arose from the frequent isolation of black yeasts from wood treated with creosote , while their occurrence on untreated wood was comparatively low , indicating that accumulation of aromatic compounds might promote the growth of these fungi .", "the concurrence of these two ecological traits in a single species has been observed for exophiala dermatitidis , isolated abundantly not only on tropical creosoted railway ties but also from clinical cases of severe mycoses [ 26 , 27 ] .", "it has been hypothesized that wild berries constitute a natural niche of e. dermatitidis , which might then be ingested by birds and humans and sporadically resulting in mycoses .", "deposition of feces on creosoted railway ties subsequently leads to the massive enrichment of this fungus .", "the related species exophiala bergeri , exophiala heteromorpha , exophiala oligosperma , and exophiala xenobiotica have also been reported as opportunistic fungi causing infections that are generally less serious and from creosoted wood [ 57 , 11 , 27 ] . however , the connection between route of infection and natural occurrence is less evident with these species .", "the tendency of herpotrichiellaceous fungi toward aromatic metabolism has been confirmed by their recurrent isolation from biofilters treating vapors of volatile aromatic hydrocarbons . besides hydrocarbon exposure , environmental conditions in biofilters", "are characterized by a relative low water activity and acidification of the filter bed , which might lead to biomass inhibition problems . in this", "respect , fungal colonization in air biofilters has generally been related to an improved bioreactor performance .", "detailed metabolic studies on fungi from gas biofilters have demonstrated their capacity to assimilate alkylbenzene hydrocarbons as the sole source of carbon and energy [ 3 , 20 , 29 ] , which appears to be quite an uncommon metabolic feature in the eukaryotes .", "molecular phylogenetic characterization of these  biofilter fungi  has shown a predominant affiliation to the genera exophiala and cladophialophora ( fam .", "herpotrichiellaceae ) , particularly to the species exophiala lecanii - corni , e. oligosperma , and the recently described e. xenobiotica , cladophialophora saturnica , and cladophialophora inmunda [ 1 , 6 ] .", "despite the medical and environmental relevance of herpotrichiellaceous black yeasts , little is understood on their biodiversity and natural occurrence . in order to address questions on niche shifts and environmental prevalence in relation to virulence factors and routes of transmission ,", "the aim of the present work is to apply a specific isolation method based on the enrichment of black yeasts by simulating in batch solid state - like cultures the environmental conditions that are found in gas biofilters for the treatment of volatile aromatic hydrocarbons .", "different environmental samples related to the life cycle of e. dermatitidis were used as inocula ."], ["the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table  1 .", "these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons .", "environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14  days .", "the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . \n", "table  1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95  n , 51045.96  ea2oak railway tie , between railsnear station , hollandsche rading521041.95  n , 51045.96  ea3oak railway tie , outside railsforest area , hilversum521220.68  n , 5119.66  ea4oak railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ea5concrete railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ec1berry , sorbus aucupariaroadside , de bilt52 717.18  n , 5 948.13  ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19  n , 5 931.42  ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec4berry , sorbus aucuparialapersveld park , hilversum521254.74  n , 51110.25  ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52  n , 51051.99  ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed4fresh goose feceslapersveld park , hilversum521254.74  n , 51110.25  ed5old goose feceslapersveld park , hilversum521254.74  n , 51110.25  e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source .", "serum flasks of 100  ml were filled with approximately 25  ml of perlite granules , saturated with mineral medium . each environmental sample ( 13  g )", "was washed with phosphate - buffered saline , and the suspension ( 40  ml ) was used to inoculate four different batches ( 10  ml each batch ) . as a control", ", these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth .", "the inoculated flasks were then closed with a cotton  wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene .", "this gas phase was generated by placing 10  ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate .", "a solution of 140  g  l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3  months . after this time", ", the perlite granules in each flask were washed with 5060  ml sterile water .", "one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c .", "colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters .", "a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi .", "dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions .", "the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) .", "pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50  l volumes containing 25  ng of template dna , 5  l reaction buffer ( 0.1 m tris  hcl , ph  8.0 , 0.5  m kcl , 15  mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2  mm of each dntp and 2.0  u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .", "amplification was performed with cycles of 2  min at 94c for primary denaturation , followed by 35  cycles at 94c ( 45  s ) , 52c ( 30  s ) and 72c ( 120  s ) , with a final 7-min extension step at 72c .", "amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) .", "sequence pcr was performed as follows : 95c for 1  min , followed by 30  cycles consisting of 95c for 10  s , 50c for 5  s , and 60c for 2  min .", "reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) .", "sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) .", "for the phylogenetic assignment , the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) .", "phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) ."], ["the list of sampled sites that were used as source of inoculum for the subsequent enrichment cultures is presented in table  1 .", "these samples were collected around utrecht ( the netherlands ) and concerned wild berries from different plants , guano - rich soil , as well as samples from creosote - treated oak railway ties , which might contain both fecal pollution and contamination with aromatic hydrocarbons .", "environmental samples were collected with sterile lab tools , placed in plastic bags and plates , then stored at 4c , and processed in the laboratory within 7 to 14  days .", "the same locations had previously been sampled for black yeasts , but without enrichment on volatile aromatic hydrocarbons . \n", "table  1samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbonscodesamplesampling locationgeographic coordinates ( wgs84)a1oak railway tie , outside railsnear station , hollandsche rading521041.95  n , 51045.96  ea2oak railway tie , between railsnear station , hollandsche rading521041.95  n , 51045.96  ea3oak railway tie , outside railsforest area , hilversum521220.68  n , 5119.66  ea4oak railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ea5concrete railway tie , between railsforest area , hilversum521220.68  n , 5119.66  ec1berry , sorbus aucupariaroadside , de bilt52 717.18  n , 5 948.13  ec2berry , sorbus aucuparialight forest , voordaansepad , groenekan52 749.19  n , 5 931.42  ec3berry , sorbus aucuparialight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec4berry , sorbus aucuparialapersveld park , hilversum521254.74  n , 51110.25  ec5berry , viburnum opuluslight hedge , oostveensepad , maartensdijk52 833.70  n , 5 951.98  ec6berry , crataegus monogynahedge , vuursche pad , hollandsche rading521010.52  n , 51051.99  ed1guano - rich soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed2guano - rich soil covered with hedera sp.lapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed3guano - tic soil of jackdaw and starlinglapersveld park , hilversum , roosting under thuja521254.74  n , 51110.25  ed4fresh goose feceslapersveld park , hilversum521254.74  n , 51110.25  ed5old goose feceslapersveld park , hilversum521254.74  n , 51110.25  e samples and sampling sites in the utrecht area ( the netherlands ) used as source of inocula for the enrichment with volatile aromatic hydrocarbons"], ["the solid state - like batch culture technique on a hydrocarbon atmosphere was employed to select for fungi that are able to grow on volatile aromatic hydrocarbons as the sole carbon and energy source .", "serum flasks of 100  ml were filled with approximately 25  ml of perlite granules , saturated with mineral medium . each environmental sample ( 13  g ) was washed with phosphate - buffered saline , and the suspension ( 40  ml ) was used to inoculate four different batches ( 10  ml each batch ) . as a control", ", these suspensions were also plated on sabouraud s glucose agar ( sga ) amended with antibiotics in order to prevent bacterial growth .", "the inoculated flasks were then closed with a cotton  wool plug covered with aluminum foil and placed inside four desiccators where they were exposed , respectively , to a gaseous phase of benzene , toluene , xylene , or naphthalene .", "this gas phase was generated by placing 10  ml of a 5% ( v / v ) solution of the aromatic substrate in dibutyl - phthalate .", "a solution of 140  g  l of nacl was also added at the bottom of the desiccators to maintain an internal water activity value of 0.9 , and the whole set was incubated at 30c for at least 3  months . after this time ,", "one milliliter of 1- , 10- , or 100-fold dilutions from each soil suspension were plated in duplicate on 2% malt extract agar containing penicillin and streptomycin and incubated at 30c .", "colony growth was observed daily , and black yeast - like colonies were transferred to fresh potato dextrose agar plates for purification and provisional identification upon morphological characters ."], ["a sterile blade was used to scrape off the mycelium from the surface of agar plate cultures of previously isolated fungi .", "dna was extracted using an ultra clean microbial dna isolation kit ( mobio , carlsbad , ca 92010 , usa ) according to the manufacturer s instructions .", "the internal transcribed spacer ( its ) regions and the small subunit ( its1 - 5.8s - its2 ) of the rrna genes were amplified by using the primer set its1 ( 5-tcc gta ggt gaa cct gcg g-3 ) and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) .", "pcr reactions were performed on a gene amp pcr system 9700 ( applied biosystems , foster city , ca , usa ) in 50  l volumes containing 25  ng of template dna , 5  l reaction buffer ( 0.1 m tris  hcl , ph  8.0 , 0.5  m kcl , 15  mm mgcl2 , 0.1% gelatine , 1% triton x-100 ) , 0.2  mm of each dntp and 2.0  u taq dna polymerase ( itk diagnostics , leiden , the netherlands ) .", "amplification was performed with cycles of 2  min at 94c for primary denaturation , followed by 35  cycles at 94c ( 45  s ) , 52c ( 30  s ) and 72c ( 120  s ) , with a final 7-min extension step at 72c .", "amplicons were purified using gfx pcr dna and gel band purification kit ( ge healthcare , ltd . , buckinghamshire , uk ) .", "sequence pcr was performed as follows : 95c for 1  min , followed by 30  cycles consisting of 95c for 10  s , 50c for 5  s , and 60c for 2  min .", "reactions were purified with sephadex g-50 fine ( ge healthcare bio - sciences ab , uppsala , sweden ) , and sequencing was done on an abi 3730xl automatic sequencer ( applied biosystems ) .", "sequence data obtained in this study were adjusted using the seqman of lasergene software ( dnastar inc . , madison , wisconsin , usa ) . for the phylogenetic assignment ,", "the obtained dna sequences were compared against reference sequences by using the blastn algorithm on local cbs - knaw and public genbank databases ( ncbi , usa ) .", "phylogenetic analyses were conducted using mega version 4 ( center for evolutionary functional genomics , the biodesign institute , usa ) ."], ["a total of 71 fungal strains were isolated upon enrichment of environmental samples on atmospheres of different volatile aromatic hydrocarbons ( table  2 ) . of those ,", "a single mold ( aspergillus fumigatus ) other than black yeasts was isolated from berries of sorbus aucuparia ( sample c1 ) .", "the highest isolation rates were achieved with samples taken from creosoted wooden railway ties , particularly from those arising from the tie section located outside the rails and using toluene as the incubation substrate ( 39 isolates , samples a1 and a3 ) .", "these samples were the only ones that yielded isolates when xylene or benzene was applied as enrichment substrates ( 17 and 10 strains , respectively ) , but not a single fungus was isolated when naphthalene was supplied as the sole carbon source .", "though in lower numbers , black yeasts were also isolated from the tie section located between the rails ( 14 strains , samples a2 and a4 ) . besides creosote , these samples were likely to contain fecal and mineral oil contamination as well .", "table  2number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbonssample codebenzenetoluenexylenenaphthalenetotal strainsa10411015a203003a31086024a40110011c105005c207007d206006total strains104317071one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection number of fungal isolates obtained from solid state - like enrichment cultures incubated at 30c and a water activity of 0.9 , under an atmosphere rich in specific volatile aromatic hydrocarbons one isolated strain within this treatment was identified as aspergillus fumigatus ; this was the only non - black yeast strain from the whole obtained strain collection from the natural sampled environments , black yeasts were isolated in two out of four berries from s. aucuparia ( 12 isolates , samples c1 and c2 ) , but no fungi were obtained from berry samples of viburnum opulus and crataegus monogyna ( samples c5 and c6 , respectively ) . soil mixed with bird feces ( guano ) also yielded melanized fungi in one out of five tested samples ( six isolates , sample d2 ) .", "no black yeasts were isolated when suspensions of the samples described above ( table  1 ) were directly plated onto sga plates , without the enrichment step on volatile aromatic hydrocarbons , while several common and heavily sporulating fungal species were encountered .", "phylogenetic analysis of aligned its1 - 5.8s - its2 rrna sequences from the obtained strain collection showed that the isolates were affiliated to five major groups ( fig .  1 ) .", "sequences from reference strains deposited at the cbs collection ( utrecht , the netherlands ) that were related to the isolates in terms of their sequence homology and/or ecophysiology were also included in the analysis .", "those encompassed the type strains of e. xenobiotica , e. bergeri , exophiala spinifera , e. oligosperma , e. heteromorpha , e. dermatitidis , e. lecanii - corni , and phialophora sessilis , as well as from those of the cladophialophora species that have been so far related to the metabolism of aromatic hydrocarbons : cladophialophora immunda , c. saturnica , and cladophialophora psammophila sp . nov . , the latter species being in the process of description ( badali et al . , submitted )", ". strains of e. xenobiotica , e. bergeri , and e. dermatitidis that have previously been isolated from creosoted railway ties were also used . \n", "figure  1neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate .", "sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches", "were also included in the analysis ( genbank sequence codes are given between square brackets ) neighbor joining phylogenetic tree ( kimura 2-parameter model ) on aligned its1 - 5.8s - its2 rrna gene sequences from the fungi isolated in this study , in relation to the isolation sample and enrichment substrate .", "sequences from relevant reference type strains ( bold characters ) and from other isolates obtained previously from creosoted wood and related environments ( underlined characters ) were also added [ 26 , 27 ] . for phylogenetically unassigned groups , sequences from close genbank matches", "were also included in the analysis ( genbank sequence codes are given between square brackets ) sequence comparisons against those from reference strains revealed that most of the isolates were similar to exophiala xenobiotica ( 32 strains ) .", "the majority was highly homologous ( 99% ) to the ex - type strain , and some were slightly deviating ( 97% homology ) .", "a second group of isolates ( nine strains ) was identified as belonging to e. bergeri on the basis of a 99% sequence homology to the ex - type strain .", "the cluster encompassing e. bergeri also included a major group ( 24 strains ) , the sequence of which deviated significantly from the ex - type strain ( 94% sequence homology ) , so that its affiliation to e. bergeri might be put into question .", "blast searches on this latter group into the genbank genomic database revealed a close match ( 98% homology ) with an unidentified exophiala strain isolated from a rock surface .", "a minor e. bergeri sibling group was composed of two strains for which no known sequence match was found .", "a third group ( three strains ) did not match any known reference type strain , but genbank searches revealed a high sequence homology ( 98% ) to the sequence of an uncultivated fungus from a municipal composting plant . except for a single isolate ( dh18150 )", "obtained from a s. aucuparia berry ( sample c1 ) , the e. xenobiotica - related strains were isolated exclusively from creosoted oak tie sections located outside the rails ( samples a1 and a3 ) .", "within this group , three strains were obtained under benzene enrichment , while the remaining 29 strains were isolated with toluene or xylene as enrichment substrates .", "besides e. xenobiotica , sample a3 also yielded five isolates upon benzene enrichment , which were identified as e. bergeri .", "the related between - the - rails section from that same tie ( sample a4 ) and the guano - rich soil ( sample d2 ) yielded 1 and 3 strains of e. bergeri , respectively , using toluene as the enrichment substrate ."], ["the occurrence of black yeasts in the human - dominated environment has previously been underestimated due to the application of routine microbial isolation methods . under common laboratory culture conditions ,", "numerous protocols for the selective isolation of black yeasts have been developed during the last decades ( an overview is presented in table  3 ) , which have yielded new data on the presence of these fungi in a wide range of artificial environments , sometimes with no obvious counterpart in nature .", "several methods are based on osmotolerant and oligotrophic abilities of target fungi . with these regimens ,", "the isolated black yeasts are mainly osmotolerant members of the saprophytic order dothideales , although the rock - inhabiting fungi appear to be an area of overlap between the dothideales and the chaetothyriales .", "applying enrichment methods based on the inoculation of rodents , exposure to aromatic hydrocarbons , assimilation of rare sugars , and/or incubation at high temperature , most isolated black yeast - like fungi are members of the chaetothyriales .", "it should be noted though that neither of these enrichment conditions might represent the natural niche for these fungi , and they are therefore classified with difficulty in any of the known ecological categories . \n table  3overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate resultsmethodprevalent genusorderecologyreferenceanimal baitexophiala , fonsecaea , cladophialophorachaetothyrialesopportunists[2 , 8]erythritolexophialachaetothyrialesopportunistsmineral oilexophiala , fonsecaeachaetothyrialesopportunistsraulin 40cexophialachaetothyrialesopportunistsneedleconiosporiumchaetothyrialesrock fungialkylbenzene vaporscladophialophora , exophialachaetothyrialesxenobioticsacidicexophialachaetothyrialesacidophileshortaeadothidealescrushconiosporiumchaetothyrialesrock fungihormonemadothidealeslow strengthexophialachaetothyrialesoligotrophssarcinomycesdothidealesrock fungicadophoraleotialesoligotrophshigh saltaureobasidium , hortaeadothidealeshalophiles[31 , 32]suspendcryomyces , friedmanniomycesdothidealespsychrophilesethanolbaudoiniadothidealesethanophiles overview of selective methods used for environmental isolation of black yeasts and related melanized fungi , with approximate results the present study demonstrates that enrichment on inert solid media incubated under a controlled atmosphere rich on volatile aromatic hydrocarbons compounds displays an extraordinary selectiveness toward chaetothyrialian fungi from the herpotrichiellaceae family .", "for example , under hydrocarbon atmospheres , the berry samples were free from aureobasidium pullulans , which is a member of the dothideales , otherwise an extremely common component of honeydew mycobiota and slightly osmotic surfaces of fruits and berries .", "this species was encountered abundantly along with many contaminants on sga control plates without alkylbenzene enrichment .", "the presence of heavily sporulating airborne fungi , frequently found in soil and litter , was strongly reduced by alkylbenzenes and limited to a single strain of a. fumigatus .", "interestingly , the dothidiaceous species amorphoteca resinae , known as  creosote fungus  for its common isolation from creosoted wood , was found neither in this study nor in previous fungal isolation surveys from creosoted wood [ 26 , 27 ] .", "consistent and relatively abundant isolation of herpotrichiellaceous black yeasts from unpolluted environmental samples , such as wild berries , only after addition of volatile aromatic hydrocarbons strongly suggests that these substances play an essential role in the ecology and competitive ability of those fungi . in particular , the utilization of aromatic compounds as carbon and energy source might explain the key factor determining their success in anthropized environments .", "with advanced analytical techniques , it has been shown that volatile aromatic hydrocarbons that traditionally were associated with environmental pollution are in fact ubiquitously present in nature , though at very low concentrations .", "toluene , for example , is produced biologically in different natural environments including maturing berries .", "on the other hand , as extremophilic and slow - growing microorganisms , incubation on a support that is relatively dry and poor in nutrients is also a determinant factor for the enrichment of black yeasts , when compared to traditional liquid enrichment cultures . regarding both", "the presence of aromatic compounds and exposure to fluctuating and/or extreme environmental conditions , wood treated with creosote can be regarded as a paradigmatic environmental niche for black yeasts .", "creosote is a distillate derived entirely from tar , which is rich in a wide variety of polycyclic aromatic hydrocarbons , phenols , and cresols , and has widely been used as wood preservative against microbial decay . due to its carcinogenic character", ", the use of creosote has been banned in the european union . yet , creosoted wood utilities are still widely present in the open environment , such as telephone poles , railway cross ties , switch ties , and bridge timbers .", "also , creosote is an important soil contaminant in former wood creosoting plants . in a previous study ,", "e. dermatitidis was isolated from wild berries and massively from creosoted railway ties in thailand when using the pre - incubation in raulin s solution at low ph and incubated at 40c , this protocol being quite selective for e. dermatitidis .", "it was hypothesized that the life cycle of this species involved the occurrence on wild berries , ingestion and passage through the human intestinal tract , and feces deposition on railway ties .", "subsequent enrichment on creosoted wood might then be related not only to the oligotrophy , thermotolerance ( particularly under tropical conditions ) , acidotolerance , and moderate osmotolerance of e. dermatitidis but also to the presence of aromatic compounds .", "the temperate wild berry sampling locations that were the subject of our study ( table  1 ) had previously been analyzed for the occurrence of black yeasts [ 18 , 26 ] .", "collected berries were homogenized , diluted , and used for spread plates or were subjected to the raulin s pre - incubation protocol . in these earlier studies involving several hundreds of samples , only a single strain of e. dermatitidis", "the lack of this fungus in temperate climate conditions suggests that e. dermatitidis has an origin in the tropics .", "this hypothesis is further supported by the isolation of e. dermatitidis from creosoted railway ties in brazil , using the mineral oil - flotation protocol [ 15 , 24 ] . from the present enrichment program on volatile alkylbenzenes", ", it appears that exophiala species , though other than the thermophilic e. dermatitidis , are regularly present on substrates that are characteristic from the latter species ( berries , bird feces , and creosoted railway ties ) .", "it is known to be associated with mild cutaneous infections in humans   and is also commonly found in habitats rich in xenobiotics such as hydrocarbon - polluted soil and gas biofilters for the biodegradation of btex compounds [ 6 , 24 , 26 ] .", ", it was regarded to be an extremely rare opportunist , although a recent study showed that it was underdiagnosed due to a general lack of application of sequencing for identification . using the selective oil - flotation method ,", "e. bergeri was isolated from samples of wood treated or non - treated with creosote preservatives .", "a distinct cluster was identified within the e. bergeri group , which might belong to a yet - undescribed species .", "strains from this group were predominantly isolated from the section between the rails of sampled wooden ties , which were partly contaminated by machine oil and human feces .", "the remaining strains of e. xenobiotica and e. bergeri senso stricto were obtained from the section outside the rails , where this contaminating effect was less marked .", "three identical strains were isolated from guano - rich soil covered with hedera by enrichment under a toluene atmosphere .", "no close sequence homology against reference strains was found for these fungi , indicating that a yet - undescribed species is concerned .", "the phylogenetic affiliation of the undescribed strains will be reported in detail in a subsequent study .", "the extremophilic nature of black yeast , in combination with their capacity to metabolize aromatic pollutants , makes them ideal candidates for specific bioremediation purposes .", "prospects on the biotechnological application of these fungi until recently have been hampered by potential biohazard , but , as phylogenetic data on these organisms accumulates , it seems that severe pathogens and hydrocarbon - associated species are consistently well - separated siblings ( de hoog , unpublished ) .", "indeed , enrichment on volatile aromatic hydrocarbons in gas biofilters and similar systems thus far has never yielded pathogenic species of the higher fungal biohazard risk categories , such as cladophialophora bantiana or e. dermatitidis .", "this suggests that a certain degree of speciation between pathogenic and aromatic hydrocarbon metabolizing species has occurred in evolutionary terms . yet", ", more studies are needed to emphasize the importance of selective methods to isolate black yeasts and acquire more understanding of their ecological niches .", "the application of the enrichment method used in the present paper contributes to the isolation of fungi that have a potential for bioremediation of sites polluted with monoaromatic hydrocarbons ."]]}
{"article_id": "PMC4056141", "article_text": ["the metabolic syndrome ( mets ) is a clustering of metabolic and cardiovascular risk factors that have been widely discussed for at least 20 years .", "although some have questioned the clinical utility of metabolic syndrome , there are many reasons to believe that this entity is useful . identification of metabolic syndrome is a simple measure of finding people with a clustering of risk factors that put them at increased risk of diabetes and cardiovascular disease ( cvd ) .", "furthermore , such individuals need more intensive lifestyle interventions at an early stage to delay the disease progression to a still higher - risk category .", "also , the identification of metabolic syndrome will attract attention to various other related conditions such as fatty liver , polycystic ovary syndrome , and obstructive sleep apnea .", "since the first official definition of the metabolic syndrome put forward by a working group of the world health organization ( who ) in 1998 , a number of different definitions have been proposed .", "the latest definition given by international diabetes federation ( idf ) takes into account the evidence that abdominal obesity is an important component of the metabolic syndrome and proposes gender- and race - specific cut - offs for waist circumference ( wc ) . although the need for different wc is attributed to ethnic variation , it was observed that even within the same population , people with identical wc but different heights have different risks for metabolic syndrome .", "several studies from asia indicate that waist - to - height ratio ( whtr ) is more strongly associated with cvd risk factors than other anthropometric measures such as wc , body mass index ( bmi ) , and waist hip ratio ( whr ) .", "the aim of our study was to perform a comparative validation of wc , bmi , whr , and whtr for defining the metabolic syndrome in indian population living in the urban and rural areas of rohtak district , haryana , india .", "we used the idf definition of metabolic syndrome , excluding the measure of obesity , to determine which obesity measure among wc , bmi , whr , and whtr , and what appropriate cut - off value are most closely predictive of the non - adipose components of the idf 's definition of metabolic syndrome .", "informed consent was obtained after explaining the details of the procedure to all subjects . in rural population", "of these two rural blocks , 85 anganwadi centers ( awc ) were selected by the random selection method . from the population survey", "register of awc , male and female patients were randomly selected and called at the awc on a specified date and time after an overnight fast . in urban areas ,", "two out of total six urban health centers were selected randomly , and from the survey registers of these two health centers , male and female patients were selected randomly . a day before the study ,", "all subjects were advised to observe overnight fasting ( at least 8 hours ) and called at the nearest health center / awc in the morning .", "initial evaluation included detailed history and clinical examination of the subjects to exclude any systemic diseases .", "anthropometric indices including height ( without shoes and socks ) , weight , wc , and hip circumference were recorded for the subjects .", "height was measured to the nearest 0.1 cm using a portable stadiometer and weight measured to the nearest 0.1 kg using calibrated platform scales .", "waist circumference was measured to the nearest 0.1 cm at the midpoint between the subcostal margin and the margin of the supracristal plane according to the idf diagnostic criteria .", "hip circumference was measured to the nearest 0.1 cm around the thighs , at the height of the greater trochanter , with the patients in the standing position .", "blood pressure was recorded after patients were made to sit and rest for at least half an hour .", "blood pressure was recorded thrice at 5-minute intervals in a sitting position in the non - dominant arm , with the value to the nearest 2 mm hg , using a standard adult mercury sphygmomanometer .", "patients suffering from chronic renal , pancreatic or other severe illness , pregnant women , and women who delivered 2 months or less preceding the study , patients on lipid lowering agents , steroids , nicotinic acid , or other medications likely to cause dysglycemia , were excluded from the study . in the morning after an overnight fasting period , blood samples were obtained from the antecubital vein and transfused into vacuum tubes containing edta .", "all patients were allowed to sit and rest for at least half an hour before the blood samples were taken .", "plasma glucose , total cholesterol , triglycerides , and hdl - cholesterol were measured . according to the idf definition for the indian population , for an individual to be defined as having the metabolic syndrome ,", "he / she must be diagnosed as having central obesity defined as waist circumference 90 cm in males or 80 cm in females plus any two of the following four factors : ( 1 ) triglycerides 150 mg / dl or specific treatment for this lipid abnormality ; ( 2 ) hdl - cholesterol < 40 mg / dl in males or < 50 mg / dl in females or specific treatment for this lipid abnormality ; ( 3 ) sbp 130 mmhg , or dbp 85 mmhg or treatment for previously diagnosed hypertension ; ( 4 ) fasting plasma glucose 100 mg / dl or previously diagnosed type 2 diabetes .", "all analyses were done separately for men and women and according to their place of residence .", "continuous data were expressed as mean  sd . the optimal cut - off points for bmi , wc , whr , and whtr were obtained by selecting a point on the roc curve , which represented the largest sum of sensitivity and specificity .", "the area under the roc curve ( aurc ) was used as a measure of discrimination of a predictor .", "it measures the effectiveness of a diagnostic marker and enables the selection of an optimal threshold value ( cut - off point ) for the marker .", "according to the idf definition for the indian population , for an individual to be defined as having the metabolic syndrome , he / she must be diagnosed as having central obesity defined as waist circumference 90 cm in males or 80 cm in females plus any two of the following four factors : ( 1 ) triglycerides 150 mg / dl or specific treatment for this lipid abnormality ; ( 2 ) hdl - cholesterol < 40 mg / dl in males or < 50 mg / dl in females or specific treatment for this lipid abnormality ; ( 3 ) sbp 130 mmhg , or dbp 85 mmhg or treatment for previously diagnosed hypertension ; ( 4 ) fasting plasma glucose 100 mg / dl or previously diagnosed type 2 diabetes .", "all analyses were done separately for men and women and according to their place of residence .", "continuous data were expressed as mean  sd . the optimal cut - off points for bmi , wc , whr , and whtr were obtained by selecting a point on the roc curve , which represented the largest sum of sensitivity and specificity .", "the area under the roc curve ( aurc ) was used as a measure of discrimination of a predictor .", "it measures the effectiveness of a diagnostic marker and enables the selection of an optimal threshold value ( cut - off point ) for the marker .", "the statistical program for social sciences , version 17.0 ( spss inc . , chicago il ) , was used for all statistical analyses .", "in the present study , 3,042 individuals were screened for the prevalence of metabolic syndrome and their baseline characteristics , as shown in table 1 . out of the 3,042 individuals", "selected , 1,693 were from rural areas ( male , 814 ; and female , 879 ) and 1,349 from urban areas ( male , 704 ; and female , 645 ) . in our study participants , the prevalence of metabolic syndrome according to the idf criteria was 23.8% and 42.6% in urban men and women , respectively , while it was 14.9% and 36.3% in rural men and women , respectively . in males , high blood pressure was the most common abnormality , followed by low hdl and hypertriglyceridemia . in females ,", "impaired fasting glucose and diabetes mellitus were seen in 23.2% and 10.7% of the men , respectively , while these were seen in 22.7% and 8.7% of the women , respectively .", "baseline characteristics of study subjects stratified by gender the roc curve analysis was performed to find out optimal cut - off points for bmi , wc , whr , and whtr .", "the point on the roc , which represented the largest sum of sensitivity and specificity , was chosen to obtain the optimal cut - off point for each of these four measurements in predicting metabolic syndrome .", "figure 1 shows the roc curves of bmi , wc , whr , and whtr for predicting the presence of two or more non - adipose components of metabolic syndrome as defined for both men and women .", "the optimal cut - off value of wc in urban and rural males was > 89 cm , which was higher than that in urban and rural females , i.e. 83 cm and 79 cm , respectively , while the optimal cut - off for whtr was > 0.51 in rural females , 0.52 in rural males , and 0.53 in both urban males and females .", "whr cut - off for the prediction of metabolic syndrome was 0.87 and 0.93 , respectively , in rural females and males and 0.92 and 0.95 , respectively , in urban females and males .", "kg / m in both urban and rural males as well as urban females , while it was > 21 kg / m in rural females .", "table 2 shows aurc , optimal cut - off values , and the associated measure of each cut - off value . in both men and women , irrespective of their place of residence , wc was found to be a better predictor of metabolic syndrome than whtr [ table 2 ] .", "( a ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in urban females .", "( b ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in urban males .", "( c ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in rural females .", "( d ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in rural males . ( e )", "roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in the entire study population irrespective of gender and place of living areas under the roc curve , cut offs , sensitivity , specificity of wc , bmi , whr and whtr when we compared these anthropometric variables as a predictor of metabolic syndrome irrespective of the place of living and gender , whtr was found to have the best aurc as a predictor of metabolic syndrome at a cut - off value of 0.52 [ table 2 ] , and it scored over wc as a predictor of metabolic syndrome ( p = 0.001 ) .", "varying degree of prevalence of metabolic syndrome is reported from various regions of the world and the indian subcontinent , but very few data is available regarding the prevalence of metabolic syndrome separately in the urban and rural population of haryana and also regarding which anthropometric parameter of adiposity is best suited for urban and rural population .", "we found a prevalence of 23.8% ( 168/704 ) , 14.9% ( 122/814 ) , 42.6% ( 275/645 ) , and 36.3% ( 319/879 ) in urban men , rural men , urban women , and rural women , respectively , using the idf diagnostic criteria .", "moreover , the prevalence is more common in urban population than in rural population , which is likely due to the higher educational status and sedentary lifestyle of urban population .", "we evaluated and compared the extent to which four different anthropometric variables of adiposity ( bmi , wc , whr , and whtr ) are able to predict two or more non - adipose components of metabolic syndrome using the idf criteria .", "it was observed that in both urban and rural men and women , wc is a better predictor of metabolic syndrome than the other three anthropometric variables ( bmi , whr , and whtr ) .", "however , when the entire population was clubbed together and analyzed irrespective of their place of residence and gender , it was found that whtr scored over wc as a predictor of metabolic syndrome .", "the optimal cut - off of whtr for this prediction was 0.52 , which is in accordance with several reports from other asian countries where a cut - off value of whtr > 0.5 appears to offer a simple and reliable index of identifying individuals who face increased future risk of metabolic complications . traditionally , bmi is a widely popular index of obesity used .", "wang et al . , in their study of chinese population , found that bmi and wc are more useful than whr for predicting two or more non - adipose components of metabolic syndrome .", "however , the majority of asian population is obese and at risk of developing metabolic complications even at bmi cut - off level of 25 kg / m , which is not in accordance with the who bmi cut - off level used to define obesity .", "this has prompted experts across the globe to redefine bmi standards for asian population , and which is now set at 23 kg / m or above for asian population . in the present study ,", "the bmi cut - off of > 23 kg / m was also predictive of metabolic syndrome , except in rural females where optimal cut - off for bmi was > 21 kg / m . despite this change in bmi criteria , its use", "a large number of studies clearly suggest that the degree of central fat distribution is more clearly related to metabolic risk than bmi . in the present study ,", "the comparison of roc of bmi with that of wc showed that bmi is inferior to wc in predicting metabolic syndrome ( p = 0.0001 ) .", "wc and whr have been used as measures of central adiposity and evidences suggest a greater association of these anthropometric variables with a future metabolic risk than bmi , which is a measure of general obesity . between wc and whr ,", "several studies have shown that that wc is a better predictor of metabolic syndrome because of variations in the level of hip measurements , differences in cut - off values between men and women and among different ethnic groups , and the possibility of embarrassment to both examiner and examinee when measuring hip circumference . however , the ability of wc to be used as a universal predictor of central adiposity is limited by the use of different methods for the measurement of wc and different cut - offs used for men and women and for different ethnic groups . in the present study ,", "the optimal cut - off value of wc is > 89 cm each in urban and rural males and > 83 cm and > 79 cm in urban and rural females , respectively , and is clearly superior to whr in the prediction of metabolic syndrome ( p = 0.0001 ) .", "whtr is another anthropometric variable that has been used and found to be a better predictor of metabolic complications in various studies .", "this is because the height of an individual influences the distribution of body fat , and this factor should be taken into consideration before adopting any anthropometric variable as a measure of adiposity . on average , men are taller than women and have larger waist circumferences .", "this means that average whtr values are closer for men and women than average wc values because of adjustments for height , and the same value can be used for both genders to indicate increased risk .", "earlier reported that whtr is a better parameter of central obesity and obviates the need for numerous wc cut - offs ; it may be useful in children where existing parameters are not useful .", "they also reported that using the average height in various countries and their respective wc cut - offs as defined by the idf consensus definition , the range of whtr varies from 0.51 to 0.58 among males and from 0.47 to 0.54 among females . in the present study", ", we also observed that a single value of whtr at more than 0.52 is a better predictor of metabolic syndrome in both genders .", "moreover , for the first time in the present study , we compared both urban and rural population and found that irrespective of the place of living , a single value of whtr can be used as a marker to identify individuals with a high likelihood of contracting central adiposity and metabolic syndrome . a study conducted on adult females from singapore", "demonstrated that whtr can act as the best screening tool for cardiovascular risk . in japanese men and women ,", "whtr was found to be a better predictor of metabolic risk compared to other anthropometric indices .", "other studies also reported that the whtr is a simple and effective screening tool for cardiovascular risk factors in both men and women .", "in contrast , a dutch study revealed that height did not significantly influence the differences in measures of adiposity or intra - abdominal fat volume in women , or intra - abdominal fat areas in both genders .", "nakamura et al . reported that whtr did not confer an improved discriminatory performance compared to wc .", "kato et al . concluded that the predictive power of wc was not inferior to whtr , and wc is practically the most convenient measure for predicting metabolic syndrome because of its simplicity . in the present study ,", "the roc and aurc values for wc are better than that for whtr in case of men and women in both urban and rural areas ( p = 0.0054 ) , but if one has to use a single value for the ease and simplicity of prediction of metabolic syndrome , irrespective of gender and place of residence , then at a value of 0.52 , whtr is a good predictor of metabolic syndrome in comparison to wc where one has to use different values for different gender and place of residence . also , for disease with a high prevalence in population , such as metabolic syndrome , there is the need for a test with high specificity to ensure all true negative cases are picked up and the test will have a high positive predictive value .", "the same is true for whtr , when used irrespective of gender and place of residence , which has high specificity as well as youden 's index .", "to conclude , although the predictive value of different gender - specific wc values is clearly superior to other anthropometric measures for the diagnosis of metabolic syndrome in both urban and rural population of both genders , a single value of whtr > 0.52 irrespective of gender and area of residence can be used as a universal screening tool for the identification of individuals at high risk to the development of metabolic complications ."], "abstract_text": ["<S> aims : to compare waist circumference ( wc ) , body mass index ( bmi ) , waist hip ratio ( whr ) , and waist - to - height ratio ( whtr ) and define an appropriate cut - off , which is most closely predictive of the non - adipose components of the idf metabolic syndrome ( mets ) definition.methods and results : a total of 3,042 adults ( 1,693 in rural area and 1,349 in urban area ) were screened for the presence of mets according to the idf definition . among 3,042 adults selected as subjects , </S>", "<S> 1,518 were male and 1,524 were female . </S>", "<S> the receiver operating curve ( roc ) analysis was done to determine the optimal cut - off value and the best discriminatory value of each of these anthropometric parameters to predict two or more non - obese components of metabolic syndrome . </S>", "<S> the area under roc ( aurc ) for wc was superior to that for other anthropometric variables . </S>", "<S> the optimal cut - off value of wc in urban and rural males was > 89 cm , which is higher than that in urban and rural females at 83 cm and 79 cm , respectively ; the optimal cut - off for whtr was > 0.51 in rural females , 0.52 in rural males , and 0.53 in both urban males and females . </S>", "<S> both parameters were found to be better than bmi and whr . </S>", "<S> roc and aurc values for wc were better than those for whtr in men and women in both urban and rural areas ( p = 0.0054 ) ; however , when the entire study cohort was analyzed together , irrespective of gender and place of residence , then at a value of 0.52 , whtr scored over wc as a predictor of metabolic syndrome ( p = 0.001).conclusion : although the predictive value of different gender - specific wc values is clearly superior to other anthropometric measures for predicting two or more non - adipose components of mets , a single value of whtr irrespective of gender and the area of residence can be used as a universal screening tool for the identification of individuals at high risk of development of metabolic complications . </S>"], "labels": null, "section_names": ["I", "M", "Criteria for metabolic syndrome", "R", "D", "C"], "sections": [["the metabolic syndrome ( mets ) is a clustering of metabolic and cardiovascular risk factors that have been widely discussed for at least 20 years .", "although some have questioned the clinical utility of metabolic syndrome , there are many reasons to believe that this entity is useful . identification of metabolic syndrome is a simple measure of finding people with a clustering of risk factors that put them at increased risk of diabetes and cardiovascular disease ( cvd ) .", "furthermore , such individuals need more intensive lifestyle interventions at an early stage to delay the disease progression to a still higher - risk category .", "also , the identification of metabolic syndrome will attract attention to various other related conditions such as fatty liver , polycystic ovary syndrome , and obstructive sleep apnea .", "since the first official definition of the metabolic syndrome put forward by a working group of the world health organization ( who ) in 1998 , a number of different definitions have been proposed .", "the latest definition given by international diabetes federation ( idf ) takes into account the evidence that abdominal obesity is an important component of the metabolic syndrome and proposes gender- and race - specific cut - offs for waist circumference ( wc ) . although the need for different wc is attributed to ethnic variation , it was observed that even within the same population , people with identical wc but different heights have different risks for metabolic syndrome .", "several studies from asia indicate that waist - to - height ratio ( whtr ) is more strongly associated with cvd risk factors than other anthropometric measures such as wc , body mass index ( bmi ) , and waist hip ratio ( whr ) .", "the aim of our study was to perform a comparative validation of wc , bmi , whr , and whtr for defining the metabolic syndrome in indian population living in the urban and rural areas of rohtak district , haryana , india .", "we used the idf definition of metabolic syndrome , excluding the measure of obesity , to determine which obesity measure among wc , bmi , whr , and whtr , and what appropriate cut - off value are most closely predictive of the non - adipose components of the idf 's definition of metabolic syndrome ."], ["informed consent was obtained after explaining the details of the procedure to all subjects . in rural population", "of these two rural blocks , 85 anganwadi centers ( awc ) were selected by the random selection method . from the population survey", "register of awc , male and female patients were randomly selected and called at the awc on a specified date and time after an overnight fast . in urban areas ,", "two out of total six urban health centers were selected randomly , and from the survey registers of these two health centers , male and female patients were selected randomly . a day before the study ,", "all subjects were advised to observe overnight fasting ( at least 8 hours ) and called at the nearest health center / awc in the morning .", "initial evaluation included detailed history and clinical examination of the subjects to exclude any systemic diseases .", "anthropometric indices including height ( without shoes and socks ) , weight , wc , and hip circumference were recorded for the subjects .", "height was measured to the nearest 0.1 cm using a portable stadiometer and weight measured to the nearest 0.1 kg using calibrated platform scales .", "waist circumference was measured to the nearest 0.1 cm at the midpoint between the subcostal margin and the margin of the supracristal plane according to the idf diagnostic criteria .", "hip circumference was measured to the nearest 0.1 cm around the thighs , at the height of the greater trochanter , with the patients in the standing position .", "blood pressure was recorded after patients were made to sit and rest for at least half an hour .", "blood pressure was recorded thrice at 5-minute intervals in a sitting position in the non - dominant arm , with the value to the nearest 2 mm hg , using a standard adult mercury sphygmomanometer .", "patients suffering from chronic renal , pancreatic or other severe illness , pregnant women , and women who delivered 2 months or less preceding the study , patients on lipid lowering agents , steroids , nicotinic acid , or other medications likely to cause dysglycemia , were excluded from the study . in the morning after an overnight fasting period , blood samples were obtained from the antecubital vein and transfused into vacuum tubes containing edta .", "all patients were allowed to sit and rest for at least half an hour before the blood samples were taken .", "plasma glucose , total cholesterol , triglycerides , and hdl - cholesterol were measured . according to the idf definition for the indian population , for an individual to be defined as having the metabolic syndrome ,", "he / she must be diagnosed as having central obesity defined as waist circumference 90 cm in males or 80 cm in females plus any two of the following four factors : ( 1 ) triglycerides 150 mg / dl or specific treatment for this lipid abnormality ; ( 2 ) hdl - cholesterol < 40 mg / dl in males or < 50 mg / dl in females or specific treatment for this lipid abnormality ; ( 3 ) sbp 130 mmhg , or dbp 85 mmhg or treatment for previously diagnosed hypertension ; ( 4 ) fasting plasma glucose 100 mg / dl or previously diagnosed type 2 diabetes .", "all analyses were done separately for men and women and according to their place of residence .", "continuous data were expressed as mean  sd . the optimal cut - off points for bmi , wc , whr , and whtr were obtained by selecting a point on the roc curve , which represented the largest sum of sensitivity and specificity .", "the area under the roc curve ( aurc ) was used as a measure of discrimination of a predictor .", "it measures the effectiveness of a diagnostic marker and enables the selection of an optimal threshold value ( cut - off point ) for the marker ."], ["according to the idf definition for the indian population , for an individual to be defined as having the metabolic syndrome , he / she must be diagnosed as having central obesity defined as waist circumference 90 cm in males or 80 cm in females plus any two of the following four factors : ( 1 ) triglycerides 150 mg / dl or specific treatment for this lipid abnormality ; ( 2 ) hdl - cholesterol < 40 mg / dl in males or < 50 mg / dl in females or specific treatment for this lipid abnormality ; ( 3 ) sbp 130 mmhg , or dbp 85 mmhg or treatment for previously diagnosed hypertension ; ( 4 ) fasting plasma glucose 100 mg / dl or previously diagnosed type 2 diabetes .", "all analyses were done separately for men and women and according to their place of residence .", "continuous data were expressed as mean  sd . the optimal cut - off points for bmi , wc , whr , and whtr were obtained by selecting a point on the roc curve , which represented the largest sum of sensitivity and specificity .", "the area under the roc curve ( aurc ) was used as a measure of discrimination of a predictor .", "it measures the effectiveness of a diagnostic marker and enables the selection of an optimal threshold value ( cut - off point ) for the marker .", "the statistical program for social sciences , version 17.0 ( spss inc . , chicago il ) , was used for all statistical analyses ."], ["in the present study , 3,042 individuals were screened for the prevalence of metabolic syndrome and their baseline characteristics , as shown in table 1 . out of the 3,042 individuals", "selected , 1,693 were from rural areas ( male , 814 ; and female , 879 ) and 1,349 from urban areas ( male , 704 ; and female , 645 ) . in our study participants , the prevalence of metabolic syndrome according to the idf criteria was 23.8% and 42.6% in urban men and women , respectively , while it was 14.9% and 36.3% in rural men and women , respectively . in males , high blood pressure was the most common abnormality , followed by low hdl and hypertriglyceridemia . in females ,", "impaired fasting glucose and diabetes mellitus were seen in 23.2% and 10.7% of the men , respectively , while these were seen in 22.7% and 8.7% of the women , respectively .", "baseline characteristics of study subjects stratified by gender the roc curve analysis was performed to find out optimal cut - off points for bmi , wc , whr , and whtr .", "the point on the roc , which represented the largest sum of sensitivity and specificity , was chosen to obtain the optimal cut - off point for each of these four measurements in predicting metabolic syndrome .", "figure 1 shows the roc curves of bmi , wc , whr , and whtr for predicting the presence of two or more non - adipose components of metabolic syndrome as defined for both men and women .", "the optimal cut - off value of wc in urban and rural males was > 89 cm , which was higher than that in urban and rural females , i.e. 83 cm and 79 cm , respectively , while the optimal cut - off for whtr was > 0.51 in rural females , 0.52 in rural males , and 0.53 in both urban males and females .", "whr cut - off for the prediction of metabolic syndrome was 0.87 and 0.93 , respectively , in rural females and males and 0.92 and 0.95 , respectively , in urban females and males .", "kg / m in both urban and rural males as well as urban females , while it was > 21 kg / m in rural females .", "table 2 shows aurc , optimal cut - off values , and the associated measure of each cut - off value . in both men and women , irrespective of their place of residence , wc was found to be a better predictor of metabolic syndrome than whtr [ table 2 ] .", "( a ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in urban females .", "( b ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in urban males .", "( c ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in rural females .", "( d ) roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in rural males . ( e )", "roc curves of bmi , wc , whr , and whtr to predict the presence of two or more non - adipose components of metabolic syndrome in the entire study population irrespective of gender and place of living areas under the roc curve , cut offs , sensitivity , specificity of wc , bmi , whr and whtr when we compared these anthropometric variables as a predictor of metabolic syndrome irrespective of the place of living and gender , whtr was found to have the best aurc as a predictor of metabolic syndrome at a cut - off value of 0.52 [ table 2 ] , and it scored over wc as a predictor of metabolic syndrome ( p = 0.001 ) ."], ["varying degree of prevalence of metabolic syndrome is reported from various regions of the world and the indian subcontinent , but very few data is available regarding the prevalence of metabolic syndrome separately in the urban and rural population of haryana and also regarding which anthropometric parameter of adiposity is best suited for urban and rural population .", "we found a prevalence of 23.8% ( 168/704 ) , 14.9% ( 122/814 ) , 42.6% ( 275/645 ) , and 36.3% ( 319/879 ) in urban men , rural men , urban women , and rural women , respectively , using the idf diagnostic criteria .", "moreover , the prevalence is more common in urban population than in rural population , which is likely due to the higher educational status and sedentary lifestyle of urban population .", "we evaluated and compared the extent to which four different anthropometric variables of adiposity ( bmi , wc , whr , and whtr ) are able to predict two or more non - adipose components of metabolic syndrome using the idf criteria .", "it was observed that in both urban and rural men and women , wc is a better predictor of metabolic syndrome than the other three anthropometric variables ( bmi , whr , and whtr ) .", "however , when the entire population was clubbed together and analyzed irrespective of their place of residence and gender , it was found that whtr scored over wc as a predictor of metabolic syndrome .", "the optimal cut - off of whtr for this prediction was 0.52 , which is in accordance with several reports from other asian countries where a cut - off value of whtr > 0.5 appears to offer a simple and reliable index of identifying individuals who face increased future risk of metabolic complications . traditionally , bmi is a widely popular index of obesity used .", "wang et al . , in their study of chinese population , found that bmi and wc are more useful than whr for predicting two or more non - adipose components of metabolic syndrome .", "however , the majority of asian population is obese and at risk of developing metabolic complications even at bmi cut - off level of 25 kg / m , which is not in accordance with the who bmi cut - off level used to define obesity .", "this has prompted experts across the globe to redefine bmi standards for asian population , and which is now set at 23 kg / m or above for asian population . in the present study ,", "the bmi cut - off of > 23 kg / m was also predictive of metabolic syndrome , except in rural females where optimal cut - off for bmi was > 21 kg / m . despite this change in bmi criteria , its use", "a large number of studies clearly suggest that the degree of central fat distribution is more clearly related to metabolic risk than bmi . in the present study ,", "the comparison of roc of bmi with that of wc showed that bmi is inferior to wc in predicting metabolic syndrome ( p = 0.0001 ) .", "wc and whr have been used as measures of central adiposity and evidences suggest a greater association of these anthropometric variables with a future metabolic risk than bmi , which is a measure of general obesity . between wc and whr ,", "several studies have shown that that wc is a better predictor of metabolic syndrome because of variations in the level of hip measurements , differences in cut - off values between men and women and among different ethnic groups , and the possibility of embarrassment to both examiner and examinee when measuring hip circumference . however , the ability of wc to be used as a universal predictor of central adiposity is limited by the use of different methods for the measurement of wc and different cut - offs used for men and women and for different ethnic groups . in the present study ,", "the optimal cut - off value of wc is > 89 cm each in urban and rural males and > 83 cm and > 79 cm in urban and rural females , respectively , and is clearly superior to whr in the prediction of metabolic syndrome ( p = 0.0001 ) .", "whtr is another anthropometric variable that has been used and found to be a better predictor of metabolic complications in various studies .", "this is because the height of an individual influences the distribution of body fat , and this factor should be taken into consideration before adopting any anthropometric variable as a measure of adiposity . on average , men are taller than women and have larger waist circumferences .", "this means that average whtr values are closer for men and women than average wc values because of adjustments for height , and the same value can be used for both genders to indicate increased risk .", "earlier reported that whtr is a better parameter of central obesity and obviates the need for numerous wc cut - offs ; it may be useful in children where existing parameters are not useful .", "they also reported that using the average height in various countries and their respective wc cut - offs as defined by the idf consensus definition , the range of whtr varies from 0.51 to 0.58 among males and from 0.47 to 0.54 among females . in the present study", ", we also observed that a single value of whtr at more than 0.52 is a better predictor of metabolic syndrome in both genders .", "moreover , for the first time in the present study , we compared both urban and rural population and found that irrespective of the place of living , a single value of whtr can be used as a marker to identify individuals with a high likelihood of contracting central adiposity and metabolic syndrome . a study conducted on adult females from singapore", "demonstrated that whtr can act as the best screening tool for cardiovascular risk . in japanese men and women ,", "whtr was found to be a better predictor of metabolic risk compared to other anthropometric indices .", "other studies also reported that the whtr is a simple and effective screening tool for cardiovascular risk factors in both men and women .", "in contrast , a dutch study revealed that height did not significantly influence the differences in measures of adiposity or intra - abdominal fat volume in women , or intra - abdominal fat areas in both genders .", "nakamura et al . reported that whtr did not confer an improved discriminatory performance compared to wc .", "kato et al . concluded that the predictive power of wc was not inferior to whtr , and wc is practically the most convenient measure for predicting metabolic syndrome because of its simplicity . in the present study ,", "the roc and aurc values for wc are better than that for whtr in case of men and women in both urban and rural areas ( p = 0.0054 ) , but if one has to use a single value for the ease and simplicity of prediction of metabolic syndrome , irrespective of gender and place of residence , then at a value of 0.52 , whtr is a good predictor of metabolic syndrome in comparison to wc where one has to use different values for different gender and place of residence . also , for disease with a high prevalence in population , such as metabolic syndrome , there is the need for a test with high specificity to ensure all true negative cases are picked up and the test will have a high positive predictive value .", "the same is true for whtr , when used irrespective of gender and place of residence , which has high specificity as well as youden 's index ."], ["to conclude , although the predictive value of different gender - specific wc values is clearly superior to other anthropometric measures for the diagnosis of metabolic syndrome in both urban and rural population of both genders , a single value of whtr > 0.52 irrespective of gender and area of residence can be used as a universal screening tool for the identification of individuals at high risk to the development of metabolic complications ."]]}
{"article_id": "PMC5352288", "article_text": ["the constructive , or synthetic , biology approach is a fascinating avenue for exploring the boundary between living and nonliving matter . because the cell is the minimum unit required for life as we currently understand it , various researchers have attempted to construct artificial cells from simple , well - understood chemicals so that phenomena that occur within such artificial cells can be studied , with the ultimate goal of elucidating the origins of life and studying the fundamental functions of living cells123 . in particular , vesicles4 , which are spherical microcompartments made of amphiphilic molecules and", "can encapsulate biological molecules such as proteins56 and dna78910 , have often been used as models of biological membranes .", "vesicles can be classified as small ( defined as having a diameter of < 100 nm ) , large ( diameter < 1 m ) , or giant ( diameter > 1 m )", ". giant vesicles ( gvs ) have been studied extensively because they are similar to living cells in size , shape , and structure . owing to the size of gvs , morphological changes in gv membranes", "several methods for preparing gvs have been reported11 , including the hydration method1213 , the freeze - thaw method14 , the electroformation method1516 , and the fluidic device method1718 .", "however , encapsulating proteins and other macromolecules in gvs at high concentrations by means of these methods is difficult .", "in particular , it is extremely challenging to encapsulate biological materials in sufficient quantity ( 20 - 30 vol% ) to mimic the crowded environment inside cells1920 . to form gvs instantly , weitz and", "coworkers established a water - in - oil ( w / o ) emulsion centrifugation method2122 .", "first , because gvs prepared by this method have low lamellarity2324 , their membranes are so thin that they can be deformed easily .", "gv membrane deformation induced by ftsz ( a bacterial cell division protein ) , tubulin , and other macromolecules has been studied25262728 , and we observed polyhedron - like deformation of gv membranes induced by encapsulation of microspheres2930 .", "second , membrane proteins can be inserted into the vesicular membrane by this method , albeit with difficulty31 .", "for example , the yomo group used this method to study the in vitro synthesis and pore - forming activity of the membrane protein -hemolysin32 .", "third , it is possible to generate asymmetric gvs in which the lipid components of the inner and outer leaflets are different22 .", "generated asymmetric gvs with cationic lipids in the inner leaflet to encapsulate negatively charged polynucleotides , and with neutral lipids on the outer leaflet to decrease toxicity and nonspecific cellular uptake33 .", "fourth , the concentration and volume fraction of substances inside the gvs can be relatively high2834 .", "for example , nishimura et al . encapsulated an in vitro transcription - translation system into gvs and used the system to express green fluorescent protein ( gfp ) within the gvs36 .", "these five features make w / o emulsion centrifugation an indispensable method for generating cell - mimicking gvs . in previous work ,", "gvs generated by centrifugation were collected by means of a syringe equipped with a long 16 g stainless steel needle containing some of the final aqueous solution22 . in the hands of inexperienced technicians ,", "this collection method could easily result in contamination of the gv with some of the oil . in this study", ", we used the w / o emulsion centrifugation protocol developed by the yomo group2337 , in which precipitated gvs are collected through a hole opened at the bottom of the centrifuge tube in which they are prepared .", "we prepared gvs encapsulating 1.0  m microspheres , which are similar in size to intracellular organelles .", "the use of microspheres allowed us to estimate their concentration by calculating their volume fraction .", "establishment of a method for preparation of gvs in which materials are densely packed is an important step for creating artificial cells . to confirm the utility of our protocol for various types of inner materials", ", we also demonstrated that gfp and a small water - soluble fluorescent molecule ( uranine ) could be encapsulated in the gvs .", "prepare a stock solution of 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , 25 mm ) and a stock solution of texas red 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( texas red dhpe , 0.20 mm ) in chloroform and store the stock solutions at -20 c . \n", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas.incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n ", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas .", "incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial .", "wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres.add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively.vortex the microtube for 30 sec and then sonicate it for 10 min . \n  in a 1.5  ml", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres .", "add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "vortex the microtube for 30 sec and then sonicate it for 10 min . \n prepare a w / o emulsion containing microspheres . \n", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube.emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n ", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube .", "emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "collect the gvs . \n immediately after chilling the microtube , centrifuge it at 18,000 x g for 30 min .", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube.dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n  immediately after chilling the microtube ,", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube .", "dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass.using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n ", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass . using a micropipette ,", "deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n ", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n conduct fluorescence microscopy observations . \n", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively . \n ", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively .", "prepare a stock solution of 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , 25 mm ) and a stock solution of texas red 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( texas red dhpe , 0.20 mm ) in chloroform and store the stock solutions at -20 c . \n", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas.incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n ", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas .", "incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial .", "wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres.add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively.vortex the microtube for 30 sec and then sonicate it for 10 min . \n  in a 1.5  ml", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres .", "add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "vortex the microtube for 30 sec and then sonicate it for 10 min . \n prepare a w / o emulsion containing microspheres . \n", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube.emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n ", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube .", "emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "collect the gvs . \n immediately after chilling the microtube , centrifuge it at 18,000 x g for 30 min .", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube.dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n  immediately after chilling the microtube ,", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube .", "dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult .", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass.using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n ", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass . using a micropipette , deposit 25 l of the diluted", "precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n ", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n conduct fluorescence microscopy observations . \n", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively . \n ", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively .", "the w / o emulsion centrifugation method is illustrated photographically and schematically in figure 1 .", "the schematic image in figure 1  suggests that the most important determinant of the success of this method is that the specific gravity of the inner aqueous solution must be larger than that of the outer aqueous solution , so that the gvs will precipitate during centrifugation .", "in addition , the formation of a lipid monolayer at the w / o interface requires that the system be chilled for 10 min after the emulsion is layered on the outer aqueous solution .", "because the gvs form by transfer of emulsion droplets across the w / o interface , the osmotic pressures in the inner and outer aqueous layers must be the same . as a control experiment , we also prepared gvs containing no microspheres by means of the process shown in figure 1 and step 1.3 , except that the inner aqueous solution was prepared without microspheres . collection of the precipitated gvs after centrifugation is shown in figure 2 .", "in addition to the semitransparent phase at the very bottom of the microtube , we also observed a white , turbid intermediate phase in the outer aqueous solution ( figure 2a ) .", "this intermediate phase was rich in aggregations of microspheres and oil , whereas the bottom phase contained the gvs .", "therefore , after piercing the bottom of the microtube with a pushpin ( figure 2b ) , we collected only the first drop ( figure 2c ) , which contained massive amounts of gvs .", "it is important to make sure that no more than two drops are collected ; any additional drops may contain aggregations of microspheres and lipids , which will result in a lower density of gvs .", "the obtained vesicular dispersion often contained encapsulated materials outside of the gvs because the gvs often rupture during centrifugation . to obtain only gvs , a sorting method such as dialysis , gel filtration , or fluorescence - activated cell sorting", "if necessary for the purpose for which the precipitated gvs are to be used , they can be diluted with the outer aqueous solution . in some cases , the intermediate phase extended to the bottom of the tube , suggesting that any vesicles that formed were held together by the oil .", "we obtained differential interference microscopy and fluorescence microscopy images of the gvs without microspheres ( figure 3a , 3b ) and with microspheres ( figure 3c-3f ) .", "lipids conjugated with texas red dhpe , which emits red fluorescence , were used so that vesicle formation could be directly confirmed by visualization of the thin membrane . of the 160 gvs that we obtained , 55 encapsulated microspheres and 105 were empty , giving a ratio of encapsulation of 34% .", "we determined the volume fraction (  , vol % ) of microspheres in the gvs by means of the following method . because each gv contains dozens to several hundred microspheres , counting all the microspheres under an optical microscope", "therefore , we mixed the nonfluorescent 1.0 m microspheres with a small amount of fluorescent microspheres , which were manually counted under the fluorescence microscope . the total number ( n ) of encapsulated microspheres", "was calculated by multiplying the number ( n ) of manually counted fluorescent microspheres by 20 ( based on the original 95:5 [ v / v ] ratio of nonfluorescent to fluorescent microspheres ) .", "the value of  was then estimated as nv100/v , where v is the volume of the microspheres and v is the volume of the individual gv . note that estimation of", "n from n gives rise to counting errors , and these errors must be taken into account when calculating . the value of  was estimated from n , which was directly calculated as 20n , and this in turn resulted in the probability that accurately represents the true value is < 50% .", "in fact , n fluctuates to some extent around 20n , so we need to consider it as 20(n i ) , where i is the error in n. we estimated i in order that a probability of n i could be more than 50% obtained on the basis of a poisson distribution .", "we estimated i , which in turn allowed us to calculate 20(n  i ) and values of  that included counting errors for gvs with diameters of 10 and 15 m ( table 1 ) . according to this procedure", ", the volume fraction of microspheres in the gv shown in figure 3c was estimated to be 11 3 vol % .", "our results indicate that we successfully encapsulated 1  m microspheres in gvs at a high volume fraction .", "we were also able to encapsulate other materials into 100 mol % dopc gvs using the same outer aqueous solution and the same protocol ( figure 4 ) .", "specifically , gvs containing 0.1  m microspheres were prepared from a tris - buffered solution containing 0.1 m tris - hcl ( ph 7.5 ) , 0.15 m sucrose , and 0.5 vol % fluorescent microspheres by means of the protocol described for gvs containing 1.0-m microspheres ( figure 4a ) .", "following the protocol described above , dopc gvs containing gfp ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m sucrose , and 100 g / ml gfp ; figure 4b ) and gvs containing uranine ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m sucrose , and 30 m uranine ; figure 4c ) were also prepared .", "table 1 : numbers and volume fractions (  , vol% ) of microspheres .  ", "errors were determined as described in the text ; n = number of manually counted microspheres ; n = total number of encapsulated microspheres . figure 1 : flow chart and schematic depiction of w / o emulsion centrifugation method .", "( a ) oil solution consisting of dopc and texas red dhpe ( 100:0.3 molar ratio ) in liquid paraffin .", "( b ) inner aqueous dispersion consisting of sucrose and microspheres in tris - hcl buffer .", "( d ) mixture of 1 ml of oil solution and 300 l of inner aqueous dispersion .", "( g ) layering of 300 l of the emulsion on 1 ml of the outer aqueous solution .", "( i ) schematic depiction of the principle of the w / o emulsion centrifugation method .", "( a ) precipitated gvs just after centrifugation ( also depicted in figure 1h ) .", "the pellet containing the gvs was obtained by piercing the microtube near the bottom with a pushpin and collecting droplets from the hole .", "( c ) droplet of the precipitated gvs ( indicated by the yellow arrow ) dispersion diluted with the outer aqueous solution .", "( d ) fluorescence microscopy image of 1.0  m microspheres ( yg carboxylate microspheres ) inside a gv .", "then we estimated total number of inner microspheres ( n = 20 ( n i ) ) was 120 40 .", "it was calculated that the gv contained 1.0  m microspheres at a volume fraction (  = nv100/v ) of approximately 11  3 vol% , where v is the volume of the microspheres and v is the volume of the gvs .", "( f ) merged image of the images in panels d and e. please click here to view a larger version of this figure .", "differential interference contrast microscopy ( top ) and fluorescence microscopy ( bottom ) images of gvs containing ( a ) 0.1  m microspheres , ( b ) gfp , and ( c ) uranine .", "the specific gravities of the inner aqueous dispersion medium and the outer aqueous solution must be chosen carefully . for the w / o emulsion to precipitate into the outer aqueous solution during centrifugation", ", the specific gravity of the inner aqueous dispersion medium must be larger than that of the outer aqueous solution .", "we tried to prepare gvs using inner and outer solutions without sugars , but we obtained no gvs under these conditions , because the inner aqueous solution did not have enough mass to cross the interference between the two phases . if there is a large osmotic pressure difference between the two solutions , gvs that precipitate into the outer aqueous solution may shrink or rupture .", "therefore , the osmotic pressure inside and outside the gvs must be equal . to accomplish this , we used sucrose as a solute in the inner aqueous dispersion and glucose as a solute in the outer aqueous solution ; both sugars were at the same concentration .", "both salt22 and sugar233538 have been used for such purposes , but sugar is usually employed because it is less toxic and more soluble than salt . however ,", "if too much sugar is added , the gvs may come into contact with the bottom of the cover glass and collapse .", "one strategy is to reduce the specific gravity difference between the inner and outer aqueous solutions so that the gvs precipitate under mild conditions ; specifically , the supernatant of the precipitated gv dispersion can be exchanged with a solution that is identical to the inner aqueous solution to reduce the possibility of vesicular rupture by adhesion to the cover glass as a result of buoyancy .", "because these materials vary in specific gravity , viscosity , and surface tension , different numbers of vesicles form even when the same centrifugation conditions are used33 . to obtain the vesicles with the target properties ,", "it is essential to optimize the specific gravities and viscosities of the inner and outer aqueous solutions as well as the centrifugal acceleration.for preparation of the oil phase , incubation must occur at high temperature and in a dry environment such as an incubator or a dehydrator . in this study", ", we heated the liquid paraffin to 80 c to completely dissolve the lipid molecules.in addition , the emulsion should be prepared only as needed and should be immediately subjected to centrifugation because it is unstable just after it is prepared and the w / o droplets readily fuse to one another .", "the emulsion can be prepared in large quantities by sonication , vortexing , or tapping .", "however , using a homogenizer allows for rapid preparation of large amounts of emulsion and easier emulsification in oil with a high viscosity .", "it is also important that the emulsion be layered on the outer aqueous solution gently and rapidly and then chilled at 4 c . to shorten the time between emulsification and centrifugation , the oil - outer aqueous solution system can be prechilled", "before the emulsion is layered on it , and the whole system can then be centrifuged immediately.if the white turbidity appears faster or slower than usual , the mechanical homogenizer must be thoroughly rinsed to remove cleaning detergents .", "in addition , before emulsification , the oil solution , inner aqueous solution , and emulsifier must be returned to room temperature .", ", we were able to prepare gvs with a single interior material encapsulated at a high concentration . when the encapsulation of multiple materials is required , it is better to reduce the centrifugal acceleration .", "for example , an actin assembly system encapsulating seven compounds was achieved with centrifugation at less than 350 x g35 . in cases in which centrifugation is undesirable ,", "gvs can be obtained by adjusting the sugar concentration or by precipitating the emulsion under the influence of its own mass384041 .", "one is that oil molecules ( paraffin , in this case ) can be solubilized in the gv membrane , as has been pointed out by the weitz group21 . when insertion of a membrane protein into the gv membrane is desired , the effects of co - existing oil molecules on the protein must be considered .", ", we estimated that the volume fractions of microspheres in the gvs ranged from 4 - 30 vol% ; the volume fractions were not identical to the volume fraction of the inner aqueous solution used for gv preparation .", "although we were able to encapsulate microspheres in the gvs at a volume fraction high enough for microscopy observation , this method is not suitable for the preparation of gvs with a uniform volume fraction distribution .", "the w / o emulsion centrifugation method is commonly used for the formation of gvs containing encapsulated materials .", "recently , molecular robots containing an encapsulated dna device or a molecular device have been constructed4344 .", "gvs with compartments are the first choice for these kinds of applications ; therefore , techniques , such as ours , that could be used for encapsulating magnetic microspheres and microspheres with diverse surface functionalization can be expected to be useful44 ."], "abstract_text": ["<S> the constructive biology and the synthetic biology approach to creating artificial life involve the bottom - up assembly of biological or nonbiological materials . </S>", "<S> such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades . in particular , giant vesicles ( gvs ) </S>", "<S> have often been used as artificial cell membranes . in this paper </S>", "<S> , we describe the preparation of gvs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules . </S>", "<S> the gvs were prepared by means of a simple water - in - oil emulsion centrifugation method . </S>", "<S> specifically , a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids , and the resulting emulsion was layered carefully on the surface of another aqueous solution . </S>", "<S> the layered system was then centrifuged to generate the gvs . </S>", "<S> this powerful method was used to encapsulate materials ranging from small molecules to microspheres . </S>"], "labels": null, "section_names": ["Introduction", "Protocol", "1. Preparation of GVs by the W/OEmulsion Centrifugation Method", "2. Microscopy Observation of GVs", "Representative Results", "Discussion", "Disclosures"], "sections": [["the constructive , or synthetic , biology approach is a fascinating avenue for exploring the boundary between living and nonliving matter . because the cell is the minimum unit required for life as we currently understand it , various researchers have attempted to construct artificial cells from simple , well - understood chemicals so that phenomena that occur within such artificial cells can be studied , with the ultimate goal of elucidating the origins of life and studying the fundamental functions of living cells123 . in particular , vesicles4 , which are spherical microcompartments made of amphiphilic molecules and", "can encapsulate biological molecules such as proteins56 and dna78910 , have often been used as models of biological membranes .", "vesicles can be classified as small ( defined as having a diameter of < 100 nm ) , large ( diameter < 1 m ) , or giant ( diameter > 1 m )", ". giant vesicles ( gvs ) have been studied extensively because they are similar to living cells in size , shape , and structure . owing to the size of gvs , morphological changes in gv membranes", "several methods for preparing gvs have been reported11 , including the hydration method1213 , the freeze - thaw method14 , the electroformation method1516 , and the fluidic device method1718 .", "however , encapsulating proteins and other macromolecules in gvs at high concentrations by means of these methods is difficult .", "in particular , it is extremely challenging to encapsulate biological materials in sufficient quantity ( 20 - 30 vol% ) to mimic the crowded environment inside cells1920 . to form gvs instantly , weitz and", "coworkers established a water - in - oil ( w / o ) emulsion centrifugation method2122 .", "first , because gvs prepared by this method have low lamellarity2324 , their membranes are so thin that they can be deformed easily .", "gv membrane deformation induced by ftsz ( a bacterial cell division protein ) , tubulin , and other macromolecules has been studied25262728 , and we observed polyhedron - like deformation of gv membranes induced by encapsulation of microspheres2930 .", "second , membrane proteins can be inserted into the vesicular membrane by this method , albeit with difficulty31 .", "for example , the yomo group used this method to study the in vitro synthesis and pore - forming activity of the membrane protein -hemolysin32 .", "third , it is possible to generate asymmetric gvs in which the lipid components of the inner and outer leaflets are different22 .", "generated asymmetric gvs with cationic lipids in the inner leaflet to encapsulate negatively charged polynucleotides , and with neutral lipids on the outer leaflet to decrease toxicity and nonspecific cellular uptake33 .", "fourth , the concentration and volume fraction of substances inside the gvs can be relatively high2834 .", "for example , nishimura et al . encapsulated an in vitro transcription - translation system into gvs and used the system to express green fluorescent protein ( gfp ) within the gvs36 .", "these five features make w / o emulsion centrifugation an indispensable method for generating cell - mimicking gvs . in previous work ,", "gvs generated by centrifugation were collected by means of a syringe equipped with a long 16 g stainless steel needle containing some of the final aqueous solution22 . in the hands of inexperienced technicians ,", "this collection method could easily result in contamination of the gv with some of the oil . in this study", ", we used the w / o emulsion centrifugation protocol developed by the yomo group2337 , in which precipitated gvs are collected through a hole opened at the bottom of the centrifuge tube in which they are prepared .", "we prepared gvs encapsulating 1.0  m microspheres , which are similar in size to intracellular organelles .", "the use of microspheres allowed us to estimate their concentration by calculating their volume fraction .", "establishment of a method for preparation of gvs in which materials are densely packed is an important step for creating artificial cells . to confirm the utility of our protocol for various types of inner materials", ", we also demonstrated that gfp and a small water - soluble fluorescent molecule ( uranine ) could be encapsulated in the gvs ."], ["prepare a stock solution of 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , 25 mm ) and a stock solution of texas red 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( texas red dhpe , 0.20 mm ) in chloroform and store the stock solutions at -20 c . \n", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas.incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n ", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas .", "incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial .", "wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres.add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively.vortex the microtube for 30 sec and then sonicate it for 10 min . \n  in a 1.5  ml", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres .", "add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "vortex the microtube for 30 sec and then sonicate it for 10 min . \n prepare a w / o emulsion containing microspheres . \n", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube.emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n ", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube .", "emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "collect the gvs . \n immediately after chilling the microtube , centrifuge it at 18,000 x g for 30 min .", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube.dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n  immediately after chilling the microtube ,", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube .", "dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass.using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n ", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass . using a micropipette ,", "deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n ", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n conduct fluorescence microscopy observations . \n", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively . \n ", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively ."], ["prepare a stock solution of 1,2-dioleoyl - sn - glycero-3-phosphocholine ( dopc , 25 mm ) and a stock solution of texas red 1,2-dihexadecanoyl - sn - glycero-3-phosphoethanolamine , triethylammonium salt ( texas red dhpe , 0.20 mm ) in chloroform and store the stock solutions at -20 c . \n", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas.incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial . wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n ", "form a lipid film on the inside surface of a 5  ml glass vial by evaporating a mixture of the dopc stock solution ( 51.0 l and the texas red dhpe stock solution ( 19.2 l ) under flowing nitrogen gas .", "incubate the film under reduced pressure overnight and then add 1.0 ml of liquid paraffin ( 0.86 - 0.89 g / cm ) to the vial .", "wrap the vial in aluminum foil and incubate it at 80 c o / n at rest .", "the final concentrations of dopc and texas red dhpe are 1.3 and 3.8 x 10 mm , respectively , and the dopc : texas red dhpe molar ratio is 100:0.3 . \n", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres.add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively.vortex the microtube for 30 sec and then sonicate it for 10 min . \n  in a 1.5  ml", "lidded microtube , mix 237.5 l of a dispersion of 1.0  m nonfluorescent microspheres ( 2.5 vol% ) and 12.5 l of a dispersion of 1.0  m fluorescent microspheres ( 2.5 vol% ) ; this corresponds to a 95:5 ( v / v ) ratio of nonfluorescent to fluorescent microspheres .", "add 64 mg of sucrose followed by 125 l of tris - buffered solution ( tbs , 1 m ) and 875 l of deionized water .", "the final volume fraction of microspheres is 0.5 vol% , and the final concentrations of tris - hcl ( ph 7.5 ) and sucrose are 0.1 and 0.15 m , respectively .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "after preparation of 10 ml of a tris - buffered solution ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m glucose ) in the same procedure as inner aqueous media , place 1 ml of the solution in a 1.5  ml lidded microtube .", "vortex the microtube for 30 sec and then sonicate it for 10 min . \n prepare a w / o emulsion containing microspheres . \n", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube.emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n ", "mix 1 ml of the oil solution ( liquid paraffin containing dopc and texas red dhpe ) with 300 l of the inner aqueous solution in a 1.5  ml microtube .", "emulsify the two components in the microtube by using a mechanical homogenizer ( an agitator with blades that rotate at high speed ) operated at 10,000 rpm for 2 min at rt . \n", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "gently layer 300 l of the w / o emulsion on the upper surface of 1 ml of the outer aqueous solution at 4 c in a 1.5  ml lidded microtube .", "collect the gvs . \n immediately after chilling the microtube , centrifuge it at 18,000 x g for 30 min .", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube.dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult . \n  immediately after chilling the microtube ,", "obtain the precipitated gvs by piercing the bottom of the microtube with a pushpin and collecting one droplet in a sterilized 1.5 ml microtube .", "dilute the precipitated gv droplet 10 - 100 fold by volume with the outer aqueous solution if the obtained gvs are obtained in quantities large enough to make observation difficult ."], ["place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass.using a micropipette , deposit 25 l of the diluted precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n ", "place an adhesive incubation chamber for in situ polymerase chain reaction and hybridization ( chamber size 9 mm x 9 mm x 0.3 mm thick ) on top of a microscope cover glass . using a micropipette , deposit 25 l of the diluted", "precipitated gvs on the specimen area and immediately place another cover glass ( approximately 0.15 mm thick ) on top of the incubation chamber . \n", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n ", "record microscopy images of the vesicles with a microscope ( 10x , 20x , and 40x  objectives ) equipped with a 12v100whal - l halogen lamp . \n conduct fluorescence microscopy observations . \n", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively . \n ", "fit the units with 470 - 495  nm and 565 - 585  nm excitation filters , respectively , and with emission filters that transmit 510 - 550  nm and 600 - 690  nm light , respectively ."], ["the w / o emulsion centrifugation method is illustrated photographically and schematically in figure 1 .", "the schematic image in figure 1  suggests that the most important determinant of the success of this method is that the specific gravity of the inner aqueous solution must be larger than that of the outer aqueous solution , so that the gvs will precipitate during centrifugation .", "in addition , the formation of a lipid monolayer at the w / o interface requires that the system be chilled for 10 min after the emulsion is layered on the outer aqueous solution .", "because the gvs form by transfer of emulsion droplets across the w / o interface , the osmotic pressures in the inner and outer aqueous layers must be the same . as a control experiment , we also prepared gvs containing no microspheres by means of the process shown in figure 1 and step 1.3 , except that the inner aqueous solution was prepared without microspheres . collection of the precipitated gvs after centrifugation is shown in figure 2 .", "in addition to the semitransparent phase at the very bottom of the microtube , we also observed a white , turbid intermediate phase in the outer aqueous solution ( figure 2a ) .", "this intermediate phase was rich in aggregations of microspheres and oil , whereas the bottom phase contained the gvs .", "therefore , after piercing the bottom of the microtube with a pushpin ( figure 2b ) , we collected only the first drop ( figure 2c ) , which contained massive amounts of gvs .", "it is important to make sure that no more than two drops are collected ; any additional drops may contain aggregations of microspheres and lipids , which will result in a lower density of gvs .", "the obtained vesicular dispersion often contained encapsulated materials outside of the gvs because the gvs often rupture during centrifugation . to obtain only gvs , a sorting method such as dialysis , gel filtration , or fluorescence - activated cell sorting", "if necessary for the purpose for which the precipitated gvs are to be used , they can be diluted with the outer aqueous solution . in some cases , the intermediate phase extended to the bottom of the tube , suggesting that any vesicles that formed were held together by the oil .", "we obtained differential interference microscopy and fluorescence microscopy images of the gvs without microspheres ( figure 3a , 3b ) and with microspheres ( figure 3c-3f ) .", "lipids conjugated with texas red dhpe , which emits red fluorescence , were used so that vesicle formation could be directly confirmed by visualization of the thin membrane . of the 160 gvs that we obtained , 55 encapsulated microspheres and 105 were empty , giving a ratio of encapsulation of 34% .", "we determined the volume fraction (  , vol % ) of microspheres in the gvs by means of the following method . because each gv contains dozens to several hundred microspheres , counting all the microspheres under an optical microscope", "therefore , we mixed the nonfluorescent 1.0 m microspheres with a small amount of fluorescent microspheres , which were manually counted under the fluorescence microscope . the total number ( n ) of encapsulated microspheres", "was calculated by multiplying the number ( n ) of manually counted fluorescent microspheres by 20 ( based on the original 95:5 [ v / v ] ratio of nonfluorescent to fluorescent microspheres ) .", "the value of  was then estimated as nv100/v , where v is the volume of the microspheres and v is the volume of the individual gv . note that estimation of", "n from n gives rise to counting errors , and these errors must be taken into account when calculating . the value of  was estimated from n , which was directly calculated as 20n , and this in turn resulted in the probability that accurately represents the true value is < 50% .", "in fact , n fluctuates to some extent around 20n , so we need to consider it as 20(n i ) , where i is the error in n. we estimated i in order that a probability of n i could be more than 50% obtained on the basis of a poisson distribution .", "we estimated i , which in turn allowed us to calculate 20(n  i ) and values of  that included counting errors for gvs with diameters of 10 and 15 m ( table 1 ) . according to this procedure", ", the volume fraction of microspheres in the gv shown in figure 3c was estimated to be 11 3 vol % .", "our results indicate that we successfully encapsulated 1  m microspheres in gvs at a high volume fraction .", "we were also able to encapsulate other materials into 100 mol % dopc gvs using the same outer aqueous solution and the same protocol ( figure 4 ) .", "specifically , gvs containing 0.1  m microspheres were prepared from a tris - buffered solution containing 0.1 m tris - hcl ( ph 7.5 ) , 0.15 m sucrose , and 0.5 vol % fluorescent microspheres by means of the protocol described for gvs containing 1.0-m microspheres ( figure 4a ) .", "following the protocol described above , dopc gvs containing gfp ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m sucrose , and 100 g / ml gfp ; figure 4b ) and gvs containing uranine ( 0.1 m tris - hcl [ ph 7.5 ] , 0.15 m sucrose , and 30 m uranine ; figure 4c ) were also prepared .", "table 1 : numbers and volume fractions (  , vol% ) of microspheres .  ", "errors were determined as described in the text ; n = number of manually counted microspheres ; n = total number of encapsulated microspheres . figure 1 : flow chart and schematic depiction of w / o emulsion centrifugation method .", "( a ) oil solution consisting of dopc and texas red dhpe ( 100:0.3 molar ratio ) in liquid paraffin .", "( b ) inner aqueous dispersion consisting of sucrose and microspheres in tris - hcl buffer .", "( d ) mixture of 1 ml of oil solution and 300 l of inner aqueous dispersion .", "( g ) layering of 300 l of the emulsion on 1 ml of the outer aqueous solution .", "( i ) schematic depiction of the principle of the w / o emulsion centrifugation method .", "( a ) precipitated gvs just after centrifugation ( also depicted in figure 1h ) .", "the pellet containing the gvs was obtained by piercing the microtube near the bottom with a pushpin and collecting droplets from the hole .", "( c ) droplet of the precipitated gvs ( indicated by the yellow arrow ) dispersion diluted with the outer aqueous solution .", "( d ) fluorescence microscopy image of 1.0  m microspheres ( yg carboxylate microspheres ) inside a gv .", "then we estimated total number of inner microspheres ( n = 20 ( n i ) ) was 120 40 .", "it was calculated that the gv contained 1.0  m microspheres at a volume fraction (  = nv100/v ) of approximately 11  3 vol% , where v is the volume of the microspheres and v is the volume of the gvs .", "( f ) merged image of the images in panels d and e. please click here to view a larger version of this figure .", "differential interference contrast microscopy ( top ) and fluorescence microscopy ( bottom ) images of gvs containing ( a ) 0.1  m microspheres , ( b ) gfp , and ( c ) uranine ."], ["the specific gravities of the inner aqueous dispersion medium and the outer aqueous solution must be chosen carefully . for the w / o emulsion to precipitate into the outer aqueous solution during centrifugation", ", the specific gravity of the inner aqueous dispersion medium must be larger than that of the outer aqueous solution .", "we tried to prepare gvs using inner and outer solutions without sugars , but we obtained no gvs under these conditions , because the inner aqueous solution did not have enough mass to cross the interference between the two phases . if there is a large osmotic pressure difference between the two solutions , gvs that precipitate into the outer aqueous solution may shrink or rupture .", "therefore , the osmotic pressure inside and outside the gvs must be equal . to accomplish this , we used sucrose as a solute in the inner aqueous dispersion and glucose as a solute in the outer aqueous solution ; both sugars were at the same concentration .", "both salt22 and sugar233538 have been used for such purposes , but sugar is usually employed because it is less toxic and more soluble than salt . however ,", "if too much sugar is added , the gvs may come into contact with the bottom of the cover glass and collapse .", "one strategy is to reduce the specific gravity difference between the inner and outer aqueous solutions so that the gvs precipitate under mild conditions ; specifically , the supernatant of the precipitated gv dispersion can be exchanged with a solution that is identical to the inner aqueous solution to reduce the possibility of vesicular rupture by adhesion to the cover glass as a result of buoyancy .", "because these materials vary in specific gravity , viscosity , and surface tension , different numbers of vesicles form even when the same centrifugation conditions are used33 . to obtain the vesicles with the target properties ,", "it is essential to optimize the specific gravities and viscosities of the inner and outer aqueous solutions as well as the centrifugal acceleration.for preparation of the oil phase , incubation must occur at high temperature and in a dry environment such as an incubator or a dehydrator . in this study", ", we heated the liquid paraffin to 80 c to completely dissolve the lipid molecules.in addition , the emulsion should be prepared only as needed and should be immediately subjected to centrifugation because it is unstable just after it is prepared and the w / o droplets readily fuse to one another .", "the emulsion can be prepared in large quantities by sonication , vortexing , or tapping .", "however , using a homogenizer allows for rapid preparation of large amounts of emulsion and easier emulsification in oil with a high viscosity .", "it is also important that the emulsion be layered on the outer aqueous solution gently and rapidly and then chilled at 4 c . to shorten the time between emulsification and centrifugation , the oil - outer aqueous solution system can be prechilled", "before the emulsion is layered on it , and the whole system can then be centrifuged immediately.if the white turbidity appears faster or slower than usual , the mechanical homogenizer must be thoroughly rinsed to remove cleaning detergents .", "in addition , before emulsification , the oil solution , inner aqueous solution , and emulsifier must be returned to room temperature .", ", we were able to prepare gvs with a single interior material encapsulated at a high concentration . when the encapsulation of multiple materials is required , it is better to reduce the centrifugal acceleration .", "for example , an actin assembly system encapsulating seven compounds was achieved with centrifugation at less than 350 x g35 . in cases in which centrifugation is undesirable ,", "gvs can be obtained by adjusting the sugar concentration or by precipitating the emulsion under the influence of its own mass384041 .", "one is that oil molecules ( paraffin , in this case ) can be solubilized in the gv membrane , as has been pointed out by the weitz group21 . when insertion of a membrane protein into the gv membrane is desired , the effects of co - existing oil molecules on the protein must be considered .", ", we estimated that the volume fractions of microspheres in the gvs ranged from 4 - 30 vol% ; the volume fractions were not identical to the volume fraction of the inner aqueous solution used for gv preparation .", "although we were able to encapsulate microspheres in the gvs at a volume fraction high enough for microscopy observation , this method is not suitable for the preparation of gvs with a uniform volume fraction distribution .", "the w / o emulsion centrifugation method is commonly used for the formation of gvs containing encapsulated materials .", "recently , molecular robots containing an encapsulated dna device or a molecular device have been constructed4344 .", "gvs with compartments are the first choice for these kinds of applications ; therefore , techniques , such as ours , that could be used for encapsulating magnetic microspheres and microspheres with diverse surface functionalization can be expected to be useful44 ."], [""]]}
{"article_id": "PMC3195198", "article_text": ["right sided valvular involvement in infective endocarditis has been well - described , but lesions affecting eustachian valve are distinctly rare . in 1986 , edwards et al .", "first described the entity of eustachian valve endocarditis ( eve ) in an autopsy study of a patient with overwhelming streptococcal sepsis . in the literature only few cases have been reported so far .", "herein , we review the literature and describe 2 new cases in which the causative organisms were determined to be vancomycin resistant staphylococcus aureus ( vrsa ) , which is reported for the first time , and staphylococcus hominis .", "a 33 year old woman with a history of intravenous drug use was admitted with a history of fever , chills and cough for the last 4 days . on presentation , she was noted to have a temperature of 102.4 f ( reference range 96.8 - 100.4 f ) , an elevated pulse rate ( 110 bpm ) ( reference range 55 - 90 bpm ) , respiration rate of 26 breaths / min ( reference range 12 - 20 breaths / min ) and low blood pressure ( 90/50 mm hg ) ( reference range 100 - 139/55 - 84 mm hg ) .", "blood investigation showed an elevated white blood cell count ( 22,600 cells/l ) ( reference range 4000 - 11000 cells/l ) .", "suboptimal images by transthoracic echocardiography ( tte ) showed mobile vegetation on the tricuspid valve , along with severe tricuspid regurgitation .", "transesophageal echocardiography ( tee ) revealed a 6-cm , echogenic mass attached to the eustachian valve , [ figures 1 and 2 ] along with pulmonary valve and right ventricular wall . during the next 2 days , the patient 's condition was complicated by hemoptysis , renal failure and hypoxic respiratory failure requiring mechanical ventilation .", "transesophageal echocardiography image demonstrating a large vegetation ( 6 cm ) attached to the eustachian valve ( case 1 ) vege : vegetation , ivc : inferior vena cava .", "transesophageal echocardiographic image of vegetation attached to the right ventricular wall ( case 1 ) pulmonary ary : pulmonary artery .", "she was treated with intravenous daptomycin initially with 6 mg / kg every 24 hours , and was discharged after 4 weeks of antibiotics . a 86-year - old nursing home female with a history of dementia , severe aortic stenosis , congestive heart failure , chronic anemia secondary to gastrointestinal bleeding requiring implantable venous catheter insertion , presented with high grade temperature of 103f ( reference range 96.8 - 100.4 f ) rectally , pulse rate of 124bpm ( reference range 55 - 90 bpm ) , respiration rate of 23 breaths / min ( reference range 12 - 20 breaths / min ) and blood pressure of 128/69 mm hg ( reference range 100 - 139/55 - 84 mm hg ) .", "laboratory data showed an elevated white blood count 30,000 cells/l ( 86% polymorphs ) ( reference range 4000 - 11000 cells/l ) , low hemoglobin of 7.1 g / dl ( reference range 14.0 - 18.0 g / dl ) and normal platelets ( 323,000/l ) ( reference range 150,000 - 450,000/l ) physical examination revealed a thin , cachectic female with a late peaking systolic murmur at the left sternal border , a soft s2 and few scattered rhonchi all over the lungs .", "tte revealed moderate concentric left ventricular hypertrophy with ejection fraction of 70% , a heavily calcified aortic valve with severe aortic stenosis and moderate pulmonary hypertension ; however , no vegetations were identified .", "the patient received 4 weeks of vancomycin , 30 mg / kg in divided doses every 24 hours and became afebrile with second set of blood culture turning negative .", "a 33 year old woman with a history of intravenous drug use was admitted with a history of fever , chills and cough for the last 4 days . on presentation , she was noted to have a temperature of 102.4 f ( reference range 96.8 - 100.4 f ) , an elevated pulse rate ( 110 bpm ) ( reference range 55 - 90 bpm ) , respiration rate of 26 breaths / min ( reference range 12 - 20 breaths / min ) and low blood pressure ( 90/50 mm hg ) ( reference range 100 - 139/55 - 84 mm hg ) .", "blood investigation showed an elevated white blood cell count ( 22,600 cells/l ) ( reference range 4000 - 11000 cells/l ) .", "suboptimal images by transthoracic echocardiography ( tte ) showed mobile vegetation on the tricuspid valve , along with severe tricuspid regurgitation .", "transesophageal echocardiography ( tee ) revealed a 6-cm , echogenic mass attached to the eustachian valve , [ figures 1 and 2 ] along with pulmonary valve and right ventricular wall . during the next 2 days , the patient 's condition was complicated by hemoptysis , renal failure and hypoxic respiratory failure requiring mechanical ventilation .", "transesophageal echocardiography image demonstrating a large vegetation ( 6 cm ) attached to the eustachian valve ( case 1 ) vege : vegetation , ivc : inferior vena cava .", "transesophageal echocardiographic image of vegetation attached to the right ventricular wall ( case 1 ) pulmonary ary : pulmonary artery .", "she was treated with intravenous daptomycin initially with 6 mg / kg every 24 hours , and was discharged after 4 weeks of antibiotics .", "a 86-year - old nursing home female with a history of dementia , severe aortic stenosis , congestive heart failure , chronic anemia secondary to gastrointestinal bleeding requiring implantable venous catheter insertion , presented with high grade temperature of 103f ( reference range 96.8 - 100.4 f ) rectally , pulse rate of 124bpm ( reference range 55 - 90 bpm ) , respiration rate of 23 breaths / min ( reference range 12 - 20 breaths / min ) and blood pressure of 128/69 mm hg ( reference range 100 - 139/55 - 84 mm hg ) .", "laboratory data showed an elevated white blood count 30,000 cells/l ( 86% polymorphs ) ( reference range 4000 - 11000 cells/l ) , low hemoglobin of 7.1 g / dl ( reference range 14.0 - 18.0 g / dl ) and normal platelets ( 323,000/l ) ( reference range 150,000 - 450,000/l ) physical examination revealed a thin , cachectic female with a late peaking systolic murmur at the left sternal border , a soft s2 and few scattered rhonchi all over the lungs .", "tte revealed moderate concentric left ventricular hypertrophy with ejection fraction of 70% , a heavily calcified aortic valve with severe aortic stenosis and moderate pulmonary hypertension ; however , no vegetations were identified .", "the patient received 4 weeks of vancomycin , 30 mg / kg in divided doses every 24 hours and became afebrile with second set of blood culture turning negative .", "eustachian valve is an embryological remnant of the sinus venosus , directing oxygenated fetal blood from inferior vena cava across foramen ovale , and into the left atrium . in adults , it is non - functional and is considered a benign rudimentary structure .", "we present 2 new cases of eve with a review of literature [ table 1 ] . in the previously reported cases , the age of patients ranged from 22 years to 76 years with a median age of 44.2 years , with a male to female ratio of 5:3 .", "there seems to be an increasing trend in elderly population . in the present study ,", "the incidence of eve is not well documented ; however , a retrospective review by san roman et al . reported an incidence of 3.3% in patients with right sided endocarditis .", "summary of reported cases of eustachian value endocarditis , their location , microbial agents involved , and prognosis a predisposing factor was present in all but three cases,[35 ] with intravenous drug use ( 40% of the cases ) being the most common .", "other predisposing factors were presence of indwelling catheters , insertion of pacemaker wires , a history of rheumatic heart disease , and immunologic compromise ( chronic alcoholism , human immunodeficiency virus  [ hiv ] ) status", ". the increasing prevalence of indwelling catheters / devices may be the reason for increasing incidence of eve in the elderly population . in the present study ,", "a history of intravenous drug use was obtained in one case , and insertion of implantable venous catheter for repeated blood transfusions in the other case .", "the blood culture revealed staphylococcus aureus to be the most common pathogenic organism in 53% of the cases , which is consistent with previous reviews sawhney et al .", "the association of staphylococcus aureus positivity on blood cultures with intravenous drug abuse ( ivda ) and indwelling catheters was found to be in 100% of the cases .", "other organisms that have been reported so far include staphylococcus hominis , enterococcus cloacae , escherichia coli , proteus vulgaris , streptococcus viridans , klebsiella pneumonia   and actinomyces israeli . in our present report ,", "this is the first case of vrsa eve reported in the literature . in the second case , staphylococcus hominis ,", "a harmless commensal on human skin was the causative organism , likely related to the implantable venous access catheter insertion for chemotherapy ( which is reported for the first time also ) .", "like many other coagulase - negative staphylococci , staphylococcus hominis may occasionally cause infection in patients with compromised immune system . both cases in our series", "the finding was in concordance with previous studies in which the vegetations were seen more frequently on tee than tte .", "as the eustachian valve is situated posteriorly , the superiority of tee over tte is seen frequently . only in reports by punzo et al . and", "in 2 other cases reported by georgeson et al . , tte was frankly misleading , suggesting a ruptured chordae tendinae in one patient , and chiari 's network in the other .", "tee allows not only better visualization of the eustachian valve and chiari network , but also easily differentiates pathological masses from these normal structures .", "nevertheless , tte remains the first imaging investigation of choice ; and , tee should be performed if clinical picture strongly suggests endocarditis and no lesions are identified on tte . though eve was first described in a post - mortem autopsy case , eve seems to follow a benign clinical course presenting as right sided endocarditis , resolving with a 4 - 6 week course of culture sensitive antibiotics , as seen in 78% of patients described till date .", "few patients had a transient worsening of symptoms requiring intubation , open heart surgery with removal of eustachian valve .", "it should be strongly considered when a patient has the clinical syndrome of right - sided endocarditis , but no vegetations are identified on tte .", "we found the incidence of eve to be highest in young intra - venous drug users , and staphylococcus aureus to be the most common microbial agent .", "however , over the past 6 years eve has been seen more frequently in the elderly population with culture positivity for diverse microbial agents .", "although eve is rare , it may be unwise to rule out this diagnosis based solely on tte , especially in the setting of persistent bacteremia or pulmonary emboli ."], "abstract_text": ["<S> eustachian valve endocarditis ( eve ) is a distinctly rare and underdiagnosed entity . </S>", "<S> we report 2 new cases caused by vancomycin resistant staphylococcus aureus and staphylococcus hominis diagnosed on transesophageal echocardiography ( tee ) . although , 63% of eustachian valve endocarditis is caused by staphylococcus aureus , we report the first case of vancomycin resistant staphylococcus aureus and first case related to implantable venous access systems . </S>", "<S> eve is now seen more commonly in elderly population with diverse microbial cultures and antibiotic sensitivities . </S>", "<S> tte is the first modality for investigation of eve , however a negative tte does not preclude the diagnosis , as only 88% of cases were diagnosed on tee . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORTS", "Case 1", "Case 2", "DISCUSSION", "CONCLUSION"], "sections": [["right sided valvular involvement in infective endocarditis has been well - described , but lesions affecting eustachian valve are distinctly rare . in 1986 , edwards et al .", "first described the entity of eustachian valve endocarditis ( eve ) in an autopsy study of a patient with overwhelming streptococcal sepsis . in the literature only few cases have been reported so far .", "herein , we review the literature and describe 2 new cases in which the causative organisms were determined to be vancomycin resistant staphylococcus aureus ( vrsa ) , which is reported for the first time , and staphylococcus hominis ."], ["a 33 year old woman with a history of intravenous drug use was admitted with a history of fever , chills and cough for the last 4 days . on presentation , she was noted to have a temperature of 102.4 f ( reference range 96.8 - 100.4 f ) , an elevated pulse rate ( 110 bpm ) ( reference range 55 - 90 bpm ) , respiration rate of 26 breaths / min ( reference range 12 - 20 breaths / min ) and low blood pressure ( 90/50 mm hg ) ( reference range 100 - 139/55 - 84 mm hg ) .", "blood investigation showed an elevated white blood cell count ( 22,600 cells/l ) ( reference range 4000 - 11000 cells/l ) .", "suboptimal images by transthoracic echocardiography ( tte ) showed mobile vegetation on the tricuspid valve , along with severe tricuspid regurgitation .", "transesophageal echocardiography ( tee ) revealed a 6-cm , echogenic mass attached to the eustachian valve , [ figures 1 and 2 ] along with pulmonary valve and right ventricular wall . during the next 2 days , the patient 's condition was complicated by hemoptysis , renal failure and hypoxic respiratory failure requiring mechanical ventilation .", "transesophageal echocardiography image demonstrating a large vegetation ( 6 cm ) attached to the eustachian valve ( case 1 ) vege : vegetation , ivc : inferior vena cava .", "transesophageal echocardiographic image of vegetation attached to the right ventricular wall ( case 1 ) pulmonary ary : pulmonary artery .", "she was treated with intravenous daptomycin initially with 6 mg / kg every 24 hours , and was discharged after 4 weeks of antibiotics . a 86-year - old nursing home female with a history of dementia , severe aortic stenosis , congestive heart failure , chronic anemia secondary to gastrointestinal bleeding requiring implantable venous catheter insertion , presented with high grade temperature of 103f ( reference range 96.8 - 100.4 f ) rectally , pulse rate of 124bpm ( reference range 55 - 90 bpm ) , respiration rate of 23 breaths / min ( reference range 12 - 20 breaths / min ) and blood pressure of 128/69 mm hg ( reference range 100 - 139/55 - 84 mm hg ) .", "laboratory data showed an elevated white blood count 30,000 cells/l ( 86% polymorphs ) ( reference range 4000 - 11000 cells/l ) , low hemoglobin of 7.1 g / dl ( reference range 14.0 - 18.0 g / dl ) and normal platelets ( 323,000/l ) ( reference range 150,000 - 450,000/l ) physical examination revealed a thin , cachectic female with a late peaking systolic murmur at the left sternal border , a soft s2 and few scattered rhonchi all over the lungs .", "tte revealed moderate concentric left ventricular hypertrophy with ejection fraction of 70% , a heavily calcified aortic valve with severe aortic stenosis and moderate pulmonary hypertension ; however , no vegetations were identified .", "the patient received 4 weeks of vancomycin , 30 mg / kg in divided doses every 24 hours and became afebrile with second set of blood culture turning negative ."], ["a 33 year old woman with a history of intravenous drug use was admitted with a history of fever , chills and cough for the last 4 days . on presentation , she was noted to have a temperature of 102.4 f ( reference range 96.8 - 100.4 f ) , an elevated pulse rate ( 110 bpm ) ( reference range 55 - 90 bpm ) , respiration rate of 26 breaths / min ( reference range 12 - 20 breaths / min ) and low blood pressure ( 90/50 mm hg ) ( reference range 100 - 139/55 - 84 mm hg ) .", "blood investigation showed an elevated white blood cell count ( 22,600 cells/l ) ( reference range 4000 - 11000 cells/l ) .", "suboptimal images by transthoracic echocardiography ( tte ) showed mobile vegetation on the tricuspid valve , along with severe tricuspid regurgitation .", "transesophageal echocardiography ( tee ) revealed a 6-cm , echogenic mass attached to the eustachian valve , [ figures 1 and 2 ] along with pulmonary valve and right ventricular wall . during the next 2 days , the patient 's condition was complicated by hemoptysis , renal failure and hypoxic respiratory failure requiring mechanical ventilation .", "transesophageal echocardiography image demonstrating a large vegetation ( 6 cm ) attached to the eustachian valve ( case 1 ) vege : vegetation , ivc : inferior vena cava .", "transesophageal echocardiographic image of vegetation attached to the right ventricular wall ( case 1 ) pulmonary ary : pulmonary artery .", "she was treated with intravenous daptomycin initially with 6 mg / kg every 24 hours , and was discharged after 4 weeks of antibiotics ."], ["a 86-year - old nursing home female with a history of dementia , severe aortic stenosis , congestive heart failure , chronic anemia secondary to gastrointestinal bleeding requiring implantable venous catheter insertion , presented with high grade temperature of 103f ( reference range 96.8 - 100.4 f ) rectally , pulse rate of 124bpm ( reference range 55 - 90 bpm ) , respiration rate of 23 breaths / min ( reference range 12 - 20 breaths / min ) and blood pressure of 128/69 mm hg ( reference range 100 - 139/55 - 84 mm hg ) .", "laboratory data showed an elevated white blood count 30,000 cells/l ( 86% polymorphs ) ( reference range 4000 - 11000 cells/l ) , low hemoglobin of 7.1 g / dl ( reference range 14.0 - 18.0 g / dl ) and normal platelets ( 323,000/l ) ( reference range 150,000 - 450,000/l ) physical examination revealed a thin , cachectic female with a late peaking systolic murmur at the left sternal border , a soft s2 and few scattered rhonchi all over the lungs .", "tte revealed moderate concentric left ventricular hypertrophy with ejection fraction of 70% , a heavily calcified aortic valve with severe aortic stenosis and moderate pulmonary hypertension ; however , no vegetations were identified .", "the patient received 4 weeks of vancomycin , 30 mg / kg in divided doses every 24 hours and became afebrile with second set of blood culture turning negative ."], ["eustachian valve is an embryological remnant of the sinus venosus , directing oxygenated fetal blood from inferior vena cava across foramen ovale , and into the left atrium . in adults , it is non - functional and is considered a benign rudimentary structure .", "we present 2 new cases of eve with a review of literature [ table 1 ] . in the previously reported cases , the age of patients ranged from 22 years to 76 years with a median age of 44.2 years , with a male to female ratio of 5:3 .", "there seems to be an increasing trend in elderly population . in the present study ,", "the incidence of eve is not well documented ; however , a retrospective review by san roman et al . reported an incidence of 3.3% in patients with right sided endocarditis .", "summary of reported cases of eustachian value endocarditis , their location , microbial agents involved , and prognosis a predisposing factor was present in all but three cases,[35 ] with intravenous drug use ( 40% of the cases ) being the most common .", "other predisposing factors were presence of indwelling catheters , insertion of pacemaker wires , a history of rheumatic heart disease , and immunologic compromise ( chronic alcoholism , human immunodeficiency virus  [ hiv ] ) status", ". the increasing prevalence of indwelling catheters / devices may be the reason for increasing incidence of eve in the elderly population . in the present study ,", "a history of intravenous drug use was obtained in one case , and insertion of implantable venous catheter for repeated blood transfusions in the other case .", "the blood culture revealed staphylococcus aureus to be the most common pathogenic organism in 53% of the cases , which is consistent with previous reviews sawhney et al .", "the association of staphylococcus aureus positivity on blood cultures with intravenous drug abuse ( ivda ) and indwelling catheters was found to be in 100% of the cases .", "other organisms that have been reported so far include staphylococcus hominis , enterococcus cloacae , escherichia coli , proteus vulgaris , streptococcus viridans , klebsiella pneumonia   and actinomyces israeli . in our present report ,", "this is the first case of vrsa eve reported in the literature . in the second case , staphylococcus hominis ,", "a harmless commensal on human skin was the causative organism , likely related to the implantable venous access catheter insertion for chemotherapy ( which is reported for the first time also ) .", "like many other coagulase - negative staphylococci , staphylococcus hominis may occasionally cause infection in patients with compromised immune system . both cases in our series", "the finding was in concordance with previous studies in which the vegetations were seen more frequently on tee than tte .", "as the eustachian valve is situated posteriorly , the superiority of tee over tte is seen frequently . only in reports by punzo et al . and", "in 2 other cases reported by georgeson et al . , tte was frankly misleading , suggesting a ruptured chordae tendinae in one patient , and chiari 's network in the other .", "tee allows not only better visualization of the eustachian valve and chiari network , but also easily differentiates pathological masses from these normal structures .", "nevertheless , tte remains the first imaging investigation of choice ; and , tee should be performed if clinical picture strongly suggests endocarditis and no lesions are identified on tte . though eve was first described in a post - mortem autopsy case , eve seems to follow a benign clinical course presenting as right sided endocarditis , resolving with a 4 - 6 week course of culture sensitive antibiotics , as seen in 78% of patients described till date .", "few patients had a transient worsening of symptoms requiring intubation , open heart surgery with removal of eustachian valve ."], ["it should be strongly considered when a patient has the clinical syndrome of right - sided endocarditis , but no vegetations are identified on tte .", "we found the incidence of eve to be highest in young intra - venous drug users , and staphylococcus aureus to be the most common microbial agent .", "however , over the past 6 years eve has been seen more frequently in the elderly population with culture positivity for diverse microbial agents .", "although eve is rare , it may be unwise to rule out this diagnosis based solely on tte , especially in the setting of persistent bacteremia or pulmonary emboli ."]]}
{"article_id": "PMC3881853", "article_text": ["x - ray micro computed tomography ( mxct ) has many applications in various fields including \n medicine , geology , materials science , dentistry , art and archaeology , among \n others .", "the methodology combines a non - destructive and \n noninvasive analysis with the ability to quantify the geometrical characteristics of \n irregular specimens such as bones , archaeological remains , teeth , etc .", "the technique is \n constantly being improved through hardware and software developments and the subsequent increase in \n resolution provides researchers with new information and approaches for the \n investigation of the material properties . in the dental field , mxct has been extensively used to quantify the geometrical changes \n in the root canal anatomy during endodontic therapy , comparing pre and post \n instrumentation analyses .", "root canals receive a chemo - mechanical treatment with \n successively larger endodontic files ( figure 1 ) in \n order to prepare the anatomy and surface of the root canal to support the dental posts . \n", "this is a very demanding task as endodontic files must cut the dentin and remove the \n dental debris , preserving the curvature of the root canals without fracture .", "root \n fracture is the biggest complication during instrumentation , jeopardizing the outcome of \n endodontic therapy . despite the evolution of endodontic files over the last \n four decades and", "the large number of available endodontic systems , no system is free of \n problems and drawbacks .  ", "b : depth marking \n facilitates determination of the exact position of the file on x - ray .", "c : silicon \n rubber for working length marking . color coding indicated according to iso \n requirements ( yellow : n 20 , red : n 25 , blue : n 30 . ) .", "e : \n the number of engraved rings on the shaft determines the taper of the instrument . \n", "one ring represents taper .02 , 2 rings for .04 and 3 rings for taper .06 .", "f : \n specially designed shaft to be fitted in the handle of the low torque motor . the \n scale on the left is in mm endodontic files are made from different grades of austenitic stainless steel or ni - ti alloys characterized by a variety of geometrical \n features such as taper , cross - section , helix and rake angle , and the distance between \n the successive cutting blades .", "these \n features are critical factors in the mechanical resistance of endodontic files to load \n forces ( i.e. , bending and torsion ) , their cutting capacity , and the clearance of dental \n chips .", "the introduction of the ni - ti alloy in file \n manufacturing was followed by the development of a variety of new cross - sectional \n designs with an increased taper compared to the 0.02 employed in k- and h - type files \n made of stainless steel as dictated by the international standard organization ( iso ) \n specification 3630 .", "nevertheless , the determination of other important \n geometrical features such as the surface area and volume of the endodontic files remain \n unknown , limiting the assessment of other parameters derived from these basic features . \n", "precise estimation of the basic geometrical features might provide an additional \n approach to explain the differences in the clinical performance among various file \n designs and establish a valuable tool for development of new and more efficient \n geometrical designs .   apart from the k - files , where the surface area and volume can be calculated based on the \n untwisted tapered blank values employing mathematical formulas , the calculation of these \n features was impossible before the introduction of mxct .", "this technique has been \n extensively used to determine the geometrical features , such as shape , surface area and \n the volume of the root canals before and after treatment with endodontic \n instruments but has never been utilized to determine the geometrical features of \n endodontic files .  ", "the aim of this study was to determine , for the first time , the surface area , volume and \n specific surface area of successive file sizes of a commercially available ni - ti system . \n additionally , we tested the hypothesis that mxct is capable of discriminating the \n quantitative differences in the aforementioned geometrical properties between the \n successive sizes of endodontic files .", "these differences exist by definition due to the \n progressively increased size of the successive files used in root canal therapy .", "the \n hypothesis tested was that there are significant differences in the surface area , volume \n and specific surface area among the different sizes of endodontic files .", "three sets of flex - master ni - ti files ( vdw ; munich , germany ) , 6 files each ( iso sizes \n n 20/lot 0411310306 , n 25/lot 0410310302 and n 30/lot \n 0406310290 ) all of 0.04 taper , were utilized in this study . to avoid any inclination \n from the vertical axis ,", "the files were placed on the stage , employing a custom made \n attachment and then scanned by a mxct scanner ( skyscan model 1072 ; aartselaar , belgium ) \n operated under the following conditions : wo ka source ( 100 kv , 98 a ) , 2.36 \n m pixel size , 180 rotation , 0.9 rotation step , 1.9 sec exposure time \n averaging by two frames , and 1 mm al filter .", "the surface area and volume of all files \n were determined from the tip up to the 16 mm level , taking 6,782 horizontal slices .", "the \n files were successively scanned using the automated routine of oversize mode , for 16 h \n each .", "a fixed threshold was applied to discriminate the crosssection from the air , \n providing high contrast images of the horizontal cross - sections .", "the surface area ( sa ) and \n volume ( v ) of each file were determined by the embedded software ( ctan , skyscan , \n aartselaar , belgium ) employing the following formulas :   with n = number of slices , s = slice thickness , pi = measured periphery in slice i \n and ai = measure area in slice i the specific surface area ( the surface to volume ratio ) was also calculated .", "the \n percentage differences between the successive file sizes for the aforementioned \n properties were calculated based on the formula : horizontal slices of all scanned files were also inspected for the presence of internal \n defects such as cracks or pores .", "all data were statistically analyzed using oneway anova and snk multiple comparison \n tests at a 95% significance level , employing the file size as the discriminating \n variable .", "linearity of the surface area and volume data versus iso size was analyzed \n employing linear regression analysis .", "all data were statistically analyzed using oneway anova and snk multiple comparison \n tests at a 95% significance level , employing the file size as the discriminating \n variable .", "linearity of the surface area and volume data versus iso size was analyzed \n employing linear regression analysis .", "figure 2 demonstrates two representative \n horizontal sections ( perpendicular to the longitudinal file axis ) from the mxct . the \n left image has been taken from a region near the handle and the right from a region near \n the cutting tip of a ni - ti file , demonstrating the difference in the cross - sectional \n surface area .", "the cross - section was found to be free of internal defects such as pores \n or cracks . left and right images show two horizontal cross sections of the same file .", "the right image has a smaller cross- \n sectional surface , as it is closer to the cutting tip the quantitative results for the surface area , volume and specific surface area of \n successive file sizes are presented in table 1 . \n", "the surface area of the n 30 files showed the highest value followed by \n n 25 and n 20 .", "the increase in total surface area was 8.9% between \n n 20/n 25 and 16.6% between n 25/n 30 files , \n demonstrating close to a 100% increase for the latter .", "similar results were found for \n the volume of the ni - ti files tested . however , the increase in volume declined towards \n the larger file sizes .", "the percentage increase was estimated as 21.1% between n \n 20/n 25 and 17.2% between n 25/n 30 ) .", "the file n 20 \n showed a significantly higher specific surface area followed by n 25 and \n n 30 .", "the specific surface area showed a 10.1% decrease between n 20/n 25 , but \n only 0.4% between n 25/n 30 .", "regression analysis revealed a strong \n linearity between the surface area ( figure 3a ) and \n volume ( figure 3b ) versus file sizes .", "the raw \n data , the fitted line , the 95% confidence intervals , the yielded analytical formula , and \n the regression coefficient ( r ) are all presented in figures 3a and 3b for the \n surface area and volume , respectively .  ", "mean values and standard deviations for the surface area ( sa ) , volume ( v ) , and \n specific surface area ( ssa ) of flexmaster ni - ti files .", "the percentage differences \n ( dif ) between successive file sizes are also presented   the same superscripts indicate mean values without statistical significant \n differences ( p>0.05 ) raw data , fitted line with the 95% confidence intervals , mathematical equations \n and liner regression coefficients ( r ) , for the surface area ( a ) and \n volume ( b ) of the flexmaster ni - ti system", "the results of the current study showed significant differences for the surface area and \n volume between the files tested , thus the original hypothesis is accepted .", "it is \n noteworthy that this analysis provided significant differences for the endodontic files \n with different iso sizes , demonstrating the ability of this analysis to discriminate the \n quantitative differences of the geometrical characteristics among the different \n endodontic file sizes .", "this discrimination has also been facilitated due to the \n increased resolution of 2.36 m obtained in this study .", "the transition to scanner \n technology has a beneficial effect on the resolution of mxct from 1000 m for \n conventional tomography , which was insufficient for endodontic applications , to roughly 30 m , providing the ability \n for more extensive research , especially in the root canal anatomy after chemo - mechanical \n treatments .", "however , beyond the technological advancement , the \n resolution of a specific analysis with a given mxct scanner is limited by the size of \n the object , as it is magnification dependent .", "this is a disadvantage in addition to the \n restriction that all horizontal projections produced during rotation must be fitted to \n the maximum horizontal width of the ccd camera .", "the mxct device used in \n this study has a maximum isotropic resolution of 1.8 m but the maximum \n achievable isotropic resolution for endodontic file scanning remained at 2.36 m , \n due to dimensional constraints .", "the scanning of an alloy with a homogeneous elemental \n atomic ratio and a uniform x - ray \n absorption through its bulk facilitates image contrast and thus the selection of the \n proper threshold level to discriminate the edges of the cross - sections .", "all tested files \n were found free of internal defects such as pores or cracks , a finding which is in \n agreement with previous cross - sectional and mxct analyses .  ", "despite the increased isotropic resolution available through technological advancements , \n the accuracy of the quantitative analysis remains unknown , at least for endodontic \n applications dealing with the quantification of the geometrical features of the root \n canals and endodontic files .", "it is logical to assume that this accuracy is dependent on \n the isotropic resolution and might increase towards smaller isotropic resolutions . \n", "however , such an approach requires the development of specific standards made of the \n same material as the unknown sample , in order to preserve the same contrast .", "this could \n be a milestone for the application of mxct in the quantification of the geometrical \n features and could be an interesting subject for future research .  ", "the surface area and volume of the endodontic files , as presented for the first time by \n the present study , was found to increase towards higher file sizes with significant \n differences between the successive file sizes ( table \n 1 ) .", "linear regression analysis showed a linear correlation of both the surface \n area and volume with the nominal file sizes .", "the surface area and volume are not \n physically dependent on the nominal iso sizes and the equations presented in figure 3 are simply an engineering relationship \n between two different physical magnitudes .", "finally , the aforementioned equations are not \n universal and should be used exclusively for the tested ni - ti endodontic system . for the \n other ni - ti files , linear models with different coefficient values are anticipated .  ", "although the geometrical configuration and design of the ni - ti files are important \n factors in controlling the clinical performance of these micro - instruments , the \n assessment of their clinical performance with experimental testing remains \n questionable .", "some studies provide results with great differences , \n not only among the different brands but also within one brand and type .", "the main \n weakness of the currently available experimental protocols is that the clinical \n performance of the endodontic files has been biased as to the influence of various \n factors and has been determined separately . on the other hand , the \n results of the current study do not have any immediate clinical implications , as they \n must be combined with further experimental results of the clinical properties ( such as \n cutting efficiency , loading of dental chips , etc . ) in order to determine any possible \n correlation .", "for instance , it is logical to assume that a file with a greater surface \n area has the increased ability of loading dental chips but this requires experimental \n documentation .  ", "the surface area and volume between the successive files showed significant differences \n and thus there is no overlap between the successive file numbers , as occurs with the \n first diameter ( d1 ) below the tip . as expected , both the surface area and \n volume show an increase towards the higher file sizes .", "the percentage difference between \n n 25/n 30 ( 16.6% ) is almost double the value of n 20/n \n 25 ( 8.9% ) files sizes , whereas the percentage increase in volume was similar ( 21.1% and \n 17.2% for n 20/n 25 and n 25/n 30 , respectively ) showing \n a completely different increasing pattern .", "it should be mentioned that the percentage \n differences in the surface area show an abrupt increase towards the larger file sizes , \n while volumetric changes exhibit a declining trend .", "this implies that the surface area \n and volume progress towards larger files with a completely different pattern , a finding \n reflected by the values of the specific surface area ( figure 3 ) .", "the latter is a property that defines the total surface area \n per unit of bulk volume and has significant importance in cases \n where the surface area has a significant effect on the system studied .", "the n 20 \n files have the highest specific surface area , indicating that more surface area is \n exposed per volume unit than in n 25 and n 30 sizes . \n", "this value is characteristic of the geometrical design and it is expected to vary \n significantly among different file designs .", "files with a non - standard taper ( i.e. , \n protaper ) are anticipated to demonstrate increased specific surface area due to the \n significant reduction in file volume .  ", "it is noteworthy that beyond the estimation of the surface area and volume of the \n scanned files , the 2d and 3d analysis of the scanned files provides tremendous \n opportunities .", "firstly , all the geometrical features ( i.e. , cutting angle , depth of \n flutes , crosssectional diameter ) measured in longitudinal and cross - section analyses , \n with optical and electron microscopy can be readily measured with mxct methodology .", "this \n analysis is advantageous over traditional techniques , as it is non - destructive and thus \n there is no need for specimen preparation ( embedding , cutting , metallographic polishing , \n conductive coating ) .", "computational analysis of 3d models can be used to quantify any \n selected geometrical feature for any region of interest .", "for instance , the real surface \n area of a file working in a root canal can be easily estimated , selecting the relative \n region of interest .", "secondly , 3d modeling of scanned files can provide more accurate \n models for subsequent finite \n element analyses . on the other hand , \n", "the drawbacks of mxct methodology are the inferior resolution compared to optical and \n electron microscopy and prolonged scanning times for the model reconstruction in high \n isotropic resolution .", "another limitation is that the image quality might be affected by the beam \n hardening phenomena .", "normally , an x - ray beam contains photons with a vast range of \n energies . in physical metallurgy", ", the beam hardening phenomenon refers to the absorption \n of lower energy photons during the passing of a beam through a metal object and once \n this happens , \" beam hardening \" occurs as the mean energy of a beam increases . in this \n study , to minimize the beam hardening effect an al filter was utilized and a beam \n hardening correction routine was applied .", "the former was used to pre - harden the beam by \n cutting the lower energy photons , while the latter is a correction algorithm which was \n applied during the reconstruction process .", "the estimation of the surface area and volume \n of endodontic files might provide a new approach for the characterization of \n clinically - related properties such as cutting efficiency , loading of dentin debris etc . \n", "this study demonstrates that mxct is a powerful tool with interesting applications for \n endodontic research .", "continuing technological advancements allowing development of more \n powerful mxct scanners should accelerate the applications of this methodology in the \n near future .", "the surface area and volume demonstrated an almost linear increase , while the specific \n surface area demonstrated an abrupt decrease towards the higher file sizes .", "for the \n first time , we have shown that mxct is capable of discriminating the quantitative \n differences in the geometrical properties of the successive sizes of endodontic files . \n", "this type of analysis should be used to investigate the unexplored correlations of the \n various clinical properties of the geometrical characteristics of the endodontic files , \n opening new avenues for research in the field of endodontics .", "the authors would like to thank the research group program for funding this research \n project .", "this study has been funded by a research grant ( # rgp - vpp-206 ) from the \n research group program , deanship of scientific research , king saud university , \n riyadh , saudi arabia ."], "abstract_text": ["<S> objectivethe aim of this study was to quantify the surface area , volume and specific \n surface area of endodontic files employing quantitative x - ray micro computed \n tomography ( mxct ) . </S>", "<S> material and methodsthree sets ( six files each ) of the flex - master ni - ti system ( n 20 , 25 and \n 30 , taper .04 ) were utilized in this study . </S>", "<S> the files were scanned by mxct . </S>", "<S> the \n surface area and volume of all files were determined from the cutting tip up to 16 \n mm . </S>", "<S> the data from the surface area , volume and specific area were statistically \n evaluated using the one - way anova and snk multiple comparison tests at \n =0.05 , employing the file size as a discriminating variable . </S>", "<S> the \n correlation between the surface area and volume with nominal iso sizes were tested \n employing linear regression analysis . </S>", "<S> resultsthe surface area and volume of n 30 files showed the highest value \n followed by n 25 and n 20 and the differences were statistically \n significant . </S>", "<S> the n 20 files showed a significantly higher specific surface \n area compared to n 25 and n 30 . </S>", "<S> the increase in surface and volume \n towards higher file sizes follows a linear relationship with the nominal iso sizes \n ( r2=0.930 for surface area and r2=0.974 for volume \n respectively ) . </S>", "<S> results indicated that the surface area and volume demonstrated an \n almost linear increase while the specific surface area exhibited an abrupt \n decrease towards higher sizes . </S>", "<S> conclusionsthis study demonstrates that mxct can be effectively applied to discriminate very \n small differences in the geometrical features of endodontic micro - instruments , \n while providing quantitative information for their geometrical properties . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSIONS", "ACKNOWLEDGMENT"], "sections": [["x - ray micro computed tomography ( mxct ) has many applications in various fields including \n medicine , geology , materials science , dentistry , art and archaeology , among \n others .", "the methodology combines a non - destructive and \n noninvasive analysis with the ability to quantify the geometrical characteristics of \n irregular specimens such as bones , archaeological remains , teeth , etc .", "the technique is \n constantly being improved through hardware and software developments and the subsequent increase in \n resolution provides researchers with new information and approaches for the \n investigation of the material properties . in the dental field , mxct has been extensively used to quantify the geometrical changes \n in the root canal anatomy during endodontic therapy , comparing pre and post \n instrumentation analyses .", "root canals receive a chemo - mechanical treatment with \n successively larger endodontic files ( figure 1 ) in \n order to prepare the anatomy and surface of the root canal to support the dental posts . \n", "this is a very demanding task as endodontic files must cut the dentin and remove the \n dental debris , preserving the curvature of the root canals without fracture .", "root \n fracture is the biggest complication during instrumentation , jeopardizing the outcome of \n endodontic therapy . despite the evolution of endodontic files over the last \n four decades and", "the large number of available endodontic systems , no system is free of \n problems and drawbacks .  ", "b : depth marking \n facilitates determination of the exact position of the file on x - ray .", "c : silicon \n rubber for working length marking . color coding indicated according to iso \n requirements ( yellow : n 20 , red : n 25 , blue : n 30 . ) .", "e : \n the number of engraved rings on the shaft determines the taper of the instrument . \n", "one ring represents taper .02 , 2 rings for .04 and 3 rings for taper .06 .", "f : \n specially designed shaft to be fitted in the handle of the low torque motor . the \n scale on the left is in mm endodontic files are made from different grades of austenitic stainless steel or ni - ti alloys characterized by a variety of geometrical \n features such as taper , cross - section , helix and rake angle , and the distance between \n the successive cutting blades .", "these \n features are critical factors in the mechanical resistance of endodontic files to load \n forces ( i.e. , bending and torsion ) , their cutting capacity , and the clearance of dental \n chips .", "the introduction of the ni - ti alloy in file \n manufacturing was followed by the development of a variety of new cross - sectional \n designs with an increased taper compared to the 0.02 employed in k- and h - type files \n made of stainless steel as dictated by the international standard organization ( iso ) \n specification 3630 .", "nevertheless , the determination of other important \n geometrical features such as the surface area and volume of the endodontic files remain \n unknown , limiting the assessment of other parameters derived from these basic features . \n", "precise estimation of the basic geometrical features might provide an additional \n approach to explain the differences in the clinical performance among various file \n designs and establish a valuable tool for development of new and more efficient \n geometrical designs .   apart from the k - files , where the surface area and volume can be calculated based on the \n untwisted tapered blank values employing mathematical formulas , the calculation of these \n features was impossible before the introduction of mxct .", "this technique has been \n extensively used to determine the geometrical features , such as shape , surface area and \n the volume of the root canals before and after treatment with endodontic \n instruments but has never been utilized to determine the geometrical features of \n endodontic files .  ", "the aim of this study was to determine , for the first time , the surface area , volume and \n specific surface area of successive file sizes of a commercially available ni - ti system . \n additionally , we tested the hypothesis that mxct is capable of discriminating the \n quantitative differences in the aforementioned geometrical properties between the \n successive sizes of endodontic files .", "these differences exist by definition due to the \n progressively increased size of the successive files used in root canal therapy .", "the \n hypothesis tested was that there are significant differences in the surface area , volume \n and specific surface area among the different sizes of endodontic files ."], ["three sets of flex - master ni - ti files ( vdw ; munich , germany ) , 6 files each ( iso sizes \n n 20/lot 0411310306 , n 25/lot 0410310302 and n 30/lot \n 0406310290 ) all of 0.04 taper , were utilized in this study . to avoid any inclination \n from the vertical axis ,", "the files were placed on the stage , employing a custom made \n attachment and then scanned by a mxct scanner ( skyscan model 1072 ; aartselaar , belgium ) \n operated under the following conditions : wo ka source ( 100 kv , 98 a ) , 2.36 \n m pixel size , 180 rotation , 0.9 rotation step , 1.9 sec exposure time \n averaging by two frames , and 1 mm al filter .", "the surface area and volume of all files \n were determined from the tip up to the 16 mm level , taking 6,782 horizontal slices .", "the \n files were successively scanned using the automated routine of oversize mode , for 16 h \n each .", "a fixed threshold was applied to discriminate the crosssection from the air , \n providing high contrast images of the horizontal cross - sections .", "the surface area ( sa ) and \n volume ( v ) of each file were determined by the embedded software ( ctan , skyscan , \n aartselaar , belgium ) employing the following formulas :   with n = number of slices , s = slice thickness , pi = measured periphery in slice i \n and ai = measure area in slice i the specific surface area ( the surface to volume ratio ) was also calculated .", "the \n percentage differences between the successive file sizes for the aforementioned \n properties were calculated based on the formula : horizontal slices of all scanned files were also inspected for the presence of internal \n defects such as cracks or pores .", "all data were statistically analyzed using oneway anova and snk multiple comparison \n tests at a 95% significance level , employing the file size as the discriminating \n variable .", "linearity of the surface area and volume data versus iso size was analyzed \n employing linear regression analysis ."], ["all data were statistically analyzed using oneway anova and snk multiple comparison \n tests at a 95% significance level , employing the file size as the discriminating \n variable .", "linearity of the surface area and volume data versus iso size was analyzed \n employing linear regression analysis ."], ["figure 2 demonstrates two representative \n horizontal sections ( perpendicular to the longitudinal file axis ) from the mxct . the \n left image has been taken from a region near the handle and the right from a region near \n the cutting tip of a ni - ti file , demonstrating the difference in the cross - sectional \n surface area .", "the cross - section was found to be free of internal defects such as pores \n or cracks . left and right images show two horizontal cross sections of the same file .", "the right image has a smaller cross- \n sectional surface , as it is closer to the cutting tip the quantitative results for the surface area , volume and specific surface area of \n successive file sizes are presented in table 1 . \n", "the surface area of the n 30 files showed the highest value followed by \n n 25 and n 20 .", "the increase in total surface area was 8.9% between \n n 20/n 25 and 16.6% between n 25/n 30 files , \n demonstrating close to a 100% increase for the latter .", "similar results were found for \n the volume of the ni - ti files tested . however , the increase in volume declined towards \n the larger file sizes .", "the percentage increase was estimated as 21.1% between n \n 20/n 25 and 17.2% between n 25/n 30 ) .", "the file n 20 \n showed a significantly higher specific surface area followed by n 25 and \n n 30 .", "the specific surface area showed a 10.1% decrease between n 20/n 25 , but \n only 0.4% between n 25/n 30 .", "regression analysis revealed a strong \n linearity between the surface area ( figure 3a ) and \n volume ( figure 3b ) versus file sizes .", "the raw \n data , the fitted line , the 95% confidence intervals , the yielded analytical formula , and \n the regression coefficient ( r ) are all presented in figures 3a and 3b for the \n surface area and volume , respectively .  ", "mean values and standard deviations for the surface area ( sa ) , volume ( v ) , and \n specific surface area ( ssa ) of flexmaster ni - ti files .", "the percentage differences \n ( dif ) between successive file sizes are also presented   the same superscripts indicate mean values without statistical significant \n differences ( p>0.05 ) raw data , fitted line with the 95% confidence intervals , mathematical equations \n and liner regression coefficients ( r ) , for the surface area ( a ) and \n volume ( b ) of the flexmaster ni - ti system"], ["the results of the current study showed significant differences for the surface area and \n volume between the files tested , thus the original hypothesis is accepted .", "it is \n noteworthy that this analysis provided significant differences for the endodontic files \n with different iso sizes , demonstrating the ability of this analysis to discriminate the \n quantitative differences of the geometrical characteristics among the different \n endodontic file sizes .", "this discrimination has also been facilitated due to the \n increased resolution of 2.36 m obtained in this study .", "the transition to scanner \n technology has a beneficial effect on the resolution of mxct from 1000 m for \n conventional tomography , which was insufficient for endodontic applications , to roughly 30 m , providing the ability \n for more extensive research , especially in the root canal anatomy after chemo - mechanical \n treatments .", "however , beyond the technological advancement , the \n resolution of a specific analysis with a given mxct scanner is limited by the size of \n the object , as it is magnification dependent .", "this is a disadvantage in addition to the \n restriction that all horizontal projections produced during rotation must be fitted to \n the maximum horizontal width of the ccd camera .", "the mxct device used in \n this study has a maximum isotropic resolution of 1.8 m but the maximum \n achievable isotropic resolution for endodontic file scanning remained at 2.36 m , \n due to dimensional constraints .", "the scanning of an alloy with a homogeneous elemental \n atomic ratio and a uniform x - ray \n absorption through its bulk facilitates image contrast and thus the selection of the \n proper threshold level to discriminate the edges of the cross - sections .", "all tested files \n were found free of internal defects such as pores or cracks , a finding which is in \n agreement with previous cross - sectional and mxct analyses .  ", "despite the increased isotropic resolution available through technological advancements , \n the accuracy of the quantitative analysis remains unknown , at least for endodontic \n applications dealing with the quantification of the geometrical features of the root \n canals and endodontic files .", "it is logical to assume that this accuracy is dependent on \n the isotropic resolution and might increase towards smaller isotropic resolutions . \n", "however , such an approach requires the development of specific standards made of the \n same material as the unknown sample , in order to preserve the same contrast .", "this could \n be a milestone for the application of mxct in the quantification of the geometrical \n features and could be an interesting subject for future research .  ", "the surface area and volume of the endodontic files , as presented for the first time by \n the present study , was found to increase towards higher file sizes with significant \n differences between the successive file sizes ( table \n 1 ) .", "linear regression analysis showed a linear correlation of both the surface \n area and volume with the nominal file sizes .", "the surface area and volume are not \n physically dependent on the nominal iso sizes and the equations presented in figure 3 are simply an engineering relationship \n between two different physical magnitudes .", "finally , the aforementioned equations are not \n universal and should be used exclusively for the tested ni - ti endodontic system . for the \n other ni - ti files , linear models with different coefficient values are anticipated .  ", "although the geometrical configuration and design of the ni - ti files are important \n factors in controlling the clinical performance of these micro - instruments , the \n assessment of their clinical performance with experimental testing remains \n questionable .", "some studies provide results with great differences , \n not only among the different brands but also within one brand and type .", "the main \n weakness of the currently available experimental protocols is that the clinical \n performance of the endodontic files has been biased as to the influence of various \n factors and has been determined separately . on the other hand , the \n results of the current study do not have any immediate clinical implications , as they \n must be combined with further experimental results of the clinical properties ( such as \n cutting efficiency , loading of dental chips , etc . ) in order to determine any possible \n correlation .", "for instance , it is logical to assume that a file with a greater surface \n area has the increased ability of loading dental chips but this requires experimental \n documentation .  ", "the surface area and volume between the successive files showed significant differences \n and thus there is no overlap between the successive file numbers , as occurs with the \n first diameter ( d1 ) below the tip . as expected , both the surface area and \n volume show an increase towards the higher file sizes .", "the percentage difference between \n n 25/n 30 ( 16.6% ) is almost double the value of n 20/n \n 25 ( 8.9% ) files sizes , whereas the percentage increase in volume was similar ( 21.1% and \n 17.2% for n 20/n 25 and n 25/n 30 , respectively ) showing \n a completely different increasing pattern .", "it should be mentioned that the percentage \n differences in the surface area show an abrupt increase towards the larger file sizes , \n while volumetric changes exhibit a declining trend .", "this implies that the surface area \n and volume progress towards larger files with a completely different pattern , a finding \n reflected by the values of the specific surface area ( figure 3 ) .", "the latter is a property that defines the total surface area \n per unit of bulk volume and has significant importance in cases \n where the surface area has a significant effect on the system studied .", "the n 20 \n files have the highest specific surface area , indicating that more surface area is \n exposed per volume unit than in n 25 and n 30 sizes . \n", "this value is characteristic of the geometrical design and it is expected to vary \n significantly among different file designs .", "files with a non - standard taper ( i.e. , \n protaper ) are anticipated to demonstrate increased specific surface area due to the \n significant reduction in file volume .  ", "it is noteworthy that beyond the estimation of the surface area and volume of the \n scanned files , the 2d and 3d analysis of the scanned files provides tremendous \n opportunities .", "firstly , all the geometrical features ( i.e. , cutting angle , depth of \n flutes , crosssectional diameter ) measured in longitudinal and cross - section analyses , \n with optical and electron microscopy can be readily measured with mxct methodology .", "this \n analysis is advantageous over traditional techniques , as it is non - destructive and thus \n there is no need for specimen preparation ( embedding , cutting , metallographic polishing , \n conductive coating ) .", "computational analysis of 3d models can be used to quantify any \n selected geometrical feature for any region of interest .", "for instance , the real surface \n area of a file working in a root canal can be easily estimated , selecting the relative \n region of interest .", "secondly , 3d modeling of scanned files can provide more accurate \n models for subsequent finite \n element analyses . on the other hand , \n", "the drawbacks of mxct methodology are the inferior resolution compared to optical and \n electron microscopy and prolonged scanning times for the model reconstruction in high \n isotropic resolution .", "another limitation is that the image quality might be affected by the beam \n hardening phenomena .", "normally , an x - ray beam contains photons with a vast range of \n energies . in physical metallurgy", ", the beam hardening phenomenon refers to the absorption \n of lower energy photons during the passing of a beam through a metal object and once \n this happens , \" beam hardening \" occurs as the mean energy of a beam increases . in this \n study , to minimize the beam hardening effect an al filter was utilized and a beam \n hardening correction routine was applied .", "the former was used to pre - harden the beam by \n cutting the lower energy photons , while the latter is a correction algorithm which was \n applied during the reconstruction process .", "the estimation of the surface area and volume \n of endodontic files might provide a new approach for the characterization of \n clinically - related properties such as cutting efficiency , loading of dentin debris etc . \n", "this study demonstrates that mxct is a powerful tool with interesting applications for \n endodontic research .", "continuing technological advancements allowing development of more \n powerful mxct scanners should accelerate the applications of this methodology in the \n near future ."], ["the surface area and volume demonstrated an almost linear increase , while the specific \n surface area demonstrated an abrupt decrease towards the higher file sizes .", "for the \n first time , we have shown that mxct is capable of discriminating the quantitative \n differences in the geometrical properties of the successive sizes of endodontic files . \n", "this type of analysis should be used to investigate the unexplored correlations of the \n various clinical properties of the geometrical characteristics of the endodontic files , \n opening new avenues for research in the field of endodontics ."], ["the authors would like to thank the research group program for funding this research \n project .", "this study has been funded by a research grant ( # rgp - vpp-206 ) from the \n research group program , deanship of scientific research , king saud university , \n riyadh , saudi arabia ."]]}
{"article_id": "PMC3046833", "article_text": ["this study was approved by the institutional ethics committee review boards of chang gung memorial hospital linkou medical center and chang gung university .", "we recruited 123 patients , and all patients gave written informed consent to participate in the study .", "the screening glucose challenge test for gestational diabetes was performed as previously described ( 24 ) .", "genomic dna samples of participants were extracted and purified from anticoagulated blood with the dneasy blood & tissue kit ( qiagen , venlo , netherlands ) .", "genotyping of snps was performed using the taqman validated snp genotyping assays ( applied biosystems , foster city , ca ) .", "blood samples were collected from patients for hormone measurement 1 h after administration of the oral glucose tolerance test .", "total gip , insulin , and c - peptide levels in human serum were measured using sandwich elisa kits ( millipore , billerica , ma ; mercodia , uppsala , sweden ; and calbiotech , spring valley , ca ) .", "a 2.15-kb fragment of human gip promoter with the a allele at rs3895874 , rs3809770 , rs3848460 , and rs937301 ( 2073 bp to + 77 bp ) was chemically synthesized ( genescript inc . ,", "piscataway , nj ) and subcloned into the pgl4.2 luciferase reporter vector ( promega corp .", "promoter fragments with ancestral haplotypes were obtained using the site - directed mutagenesis . in the promoter reporter study , each experiment was conducted at least three times with three or four replicates for each treatment .", "patients glucose challenge responses and serum hormone profiles were compared with the  test or the student t test with welch correction .", "ratios of patients with glucose levels exceeding the threshold were analyzed with the  test .", "all data were presented as mean  sem , and the statistical significance cutoff value was 0.05 .", "this study was approved by the institutional ethics committee review boards of chang gung memorial hospital linkou medical center and chang gung university .", "we recruited 123 patients , and all patients gave written informed consent to participate in the study .", "the screening glucose challenge test for gestational diabetes was performed as previously described ( 24 ) .", "genomic dna samples of participants were extracted and purified from anticoagulated blood with the dneasy blood & tissue kit ( qiagen , venlo , netherlands ) .", "genotyping of snps was performed using the taqman validated snp genotyping assays ( applied biosystems , foster city , ca ) .", "blood samples were collected from patients for hormone measurement 1 h after administration of the oral glucose tolerance test .", "total gip , insulin , and c - peptide levels in human serum were measured using sandwich elisa kits ( millipore , billerica , ma ; mercodia , uppsala , sweden ; and calbiotech , spring valley , ca ) .", "a 2.15-kb fragment of human gip promoter with the a allele at rs3895874 , rs3809770 , rs3848460 , and rs937301 ( 2073 bp to + 77 bp ) was chemically synthesized ( genescript inc . ,", "piscataway , nj ) and subcloned into the pgl4.2 luciferase reporter vector ( promega corp . , madison , wi ) .", "promoter fragments with ancestral haplotypes were obtained using the site - directed mutagenesis . in the promoter reporter study , each experiment was conducted at least three times with three or four replicates for each treatment .", "patients glucose challenge responses and serum hormone profiles were compared with the  test or the student t test with welch correction . ratios of patients with glucose levels exceeding the threshold were analyzed with the  test .", "all data were presented as mean  sem , and the statistical significance cutoff value was 0.05 .", "to systematically identify putative metabolic modifiers , we curated and screened snps in 207 gene loci that have previously been implicated in the regulation of diabetes - related and/or obesity - related traits using fst ( supplementary table 1 ) .", "the empiric distribution of the fst statistic has been used to detect genomic regions that have rapidly increased in frequency as a result of local selective pressures ( 27 ) . of these 207 genes ,", "59 carried genic variants with fst values in the top 5% bracket in comparisons between the corresponding hapmap ii populations yri ( yoruba from ibadan ) , ceu ( u.s .", "residents with northern and western european ancestry ) , and asn ( pooled samples of chinese from beijing [ chb ] and japanese from tokyo [ jpt ] ; supplementary table 1 ) .", "the genic region in 29 of these 59 genes also contained another indication of local selection : long haplotypes in one of the hapmap populations .", "on the basis of the presence of highly divergent allele frequencies between eurasians and africans , the presence of ld and extended haplotypes in eurasians , and a > 30% minor allele frequency in the overall eurasian population , we identified seven snps in the 5 gene region of cdkal1 , cyb5r4 , gad2 , gip , and pparg as potential common metabolic modifiers ( table 1 ) . in earlier gwa studies ,", "select variants in cdkal1 , cyb5r4 , gad2 , and pparg were associated with type 2 diabetes - related or obesity - related traits ( 2831 ) . by contrast , there has been no report of linkage of gip variants in gwa studies .", "because the three gip variants ( rs3895874 , rs3848460 , and rs937301 ) are highly linked compared with those that appeared alone in other candidate genes ( 32 ) , and because these gip variants are partially linked with a nonsynonymous gip snp ( rs2291725 ) ( 22 ) , these gip variants have a low likelihood of being false positive ( table 1 ) .", "snps from the 5 gene region that exhibited signals of partial selection asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry ; gd , gestational diabetes ; ld , linkage - disequilibrium ; ob , obesity ; snp , single nucleotide polymorphism ; t2d , type 2 diabetes ; yri , yoruba in ibadan , nigeria . *", "the allele frequency is significantly different among hapmap ii populations ( p < 0.01 ) .", "fine mapping of the gip locus showed that these variants are linked with more than three dozen neighboring snps ( from rs9904761 to rs3895874 on chr17 : 44,31144,402 kb ) , and these linked snps exhibited fst values in the top 210% bracket in comparisons between yri and asn ( supplementary fig .", "a plot of a 250-kb region of genotypes around gip in the three hapmap ii populations showed that whereas genotypes surrounding gip in yri exhibited a high degree of homozygosity for ancestral alleles ( fig .", "1a , upper panel ) , in the same region , genotypes of asn and ceu exhibited a high degree of homozygosity for the derived alleles ( fig .", "analysis of genic snps at the gip locus between 11 populations from the hapmap iii project ( 34 ) showed that fst values were the highest for comparisons between east asian and african populations ( fig .", "this result is consistent with our recent finding that a nonsynonymous variant in the exon iv of gip ( rs2291725 ) and a 71-kb haplotype block surrounding this variant were positively selected in eurasians in the last 2,000 to 11,800 years ( 22 ) .", "differential distribution of polymorphic alleles at the gip locus in human populations . a : visual depiction of genotypes within a 250-kb region around gip in yri , ceu , and asn populations .", "polymorphic sites are color coded according to their allelic state . individuals with a homozygous genotype with ancestral allele ( aa ) and a homozygous genotype with derived allele ( aa )", "the location of snps in the gip gene region is indicated by a green rectangle .", "the position of snps within the genomic region is indicated by blue vertical lines in the bottom panel .", "b : average fst for genic snps at the gip locus between 11 populations of the hapmap phase iii dataset .", "population descriptors : asw , african ancestry in southwest u.s . ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; chb , han - chinese in beijing , china ; chd , chinese in metropolitan denver , co ; gih , gujarati indians in houston , tx ; jpt , japanese in tokyo , japan ; lwk , luhya in webuye , kenya ; mex , mexican ancestry in los angeles , ca ; mkk , maasai in kinyawa , kenya ; tsi , toscans in italy ; yri , yoruba in ibadan , nigeria .", "the highest population differentiation was found between asian ( han - chinese and japanese ) and african ( luhya , maasai , or yoruba ) populations .", "( a high - quality color representation of this figure is available in the online issue . )", "interestingly , analyses of ld and haplotype block diversity of the genomic region that encompassed the three gip variants at the 5 gene region showed that a 91-kb ld block was represented by five inferred haplotypes in ceu and four in and asn chromosomes , respectively ( fig . 2 ; supplementary fig .", "in addition , these analyses showed that the high fst values and the major shift in allele frequencies of gip variants at the 5 gene region between eurasian and african populations could be attributed to the increase of a derived haplotype ( haplotype 50 in fig .", "because the three 5 gene region variants are completely linked with the positively selected rs2291725 in asn ( 22 ) , we inferred that rs3895874 , rs3848460 , and rs937301 at the human gip promoter region were positively selected in east asian populations as well .", "frequencies of the 58 inferred haplotypes found in the 91-kb ld region ( from rs9904761 to rs3895874 ) surrounding gip in yri , ceu , and asn .", "most of the ceu and asn chromosomes contained derived haplotypes ( with aaaa residues at positions 7679 ) , whereas yri chromosomes exhibited significant divergence in the haplotype composition .", "population descriptors : asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "( a high - quality color representation of this figure is available in the online issue . )", "importantly , we found that the 5 gene region variants ( i.e. , rs3895874 , rs3809770 , rs3848460 , and rs937301 at positions 1920 , 1650 , 1158 , and 320 of gip , respectively ) are located in a haplotype block that is separated from the one containing the nonsynonymous variant rs2291725 by a hotspot for recombination in ceu and yri ( supplementary fig .", "a tetranucleotide polymorphism was represented by three inferred haplotypes in all three populations ( referred to as derived gip , ancestral gip , and ancestral gip haplotypes in the following text ; table 2 ) .", "the derived gip haplotype , which was found in 18.3% of yri chromosomes , has become the dominant haplotype and has a frequency exceeding 50 and 75% in ceu and asn , respectively ( table 2 ) .", "by contrast , the dominant ancestral haplotype in yri chromosomes ( gip , 50.8% ) was only found in 1.1% of asn chromosomes . therefore", ", these 5 gene region variants and the nonsynonymous variant rs2291725 could be selected differentially in the overall hapmap populations and represent causal mutations independent of the nonsynonymous variant rs2291725 . frequency of inferred gip haplotypes and rs3895874 genotypes at the 5 gene region in hapmap ii populations asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "* frequencies of gip haplotypes were significantly different among hapmap ii populations ( p < 0.0001 ) .", "because only functional investigations can convincingly demonstrate causal mutations , we sought to obtain direct evidence that these 5 gene region snps represent causal mutations for the population genetics observation .", "we constructed and tested gip promoter reporters with each of the three major haplotypes in transfected human embryonic kidney ( hek ) 293 t cells ( fig .", "measurement of promoter reporter activities showed that constructs with an ancestral haplotype ( gip or gip ) exhibited luciferase reporter activity 2545% higher than that of a derived haplotype ( gip , p < 0.01 ; fig .", "because the gip promoter region contains elements that are important for regulation by transcriptional factors , including pax6 and gata4 ( 23 ) , we also determined whether the gip promoter reporter activity was haplotype - dependent in the presence of these transcription factors . as expected ,", "coexpression of pax6 or gata4 increased the basal reporter activities by 1.52.5-fold and 0.70.8-fold , respectively ( fig .", "importantly , we found that reporters with an ancestral haplotype consistently exhibited significantly higher activities than those containing the derived haplotype in the presence of pax6 or gata4 ( p < 0.01 ) .", "together , these results suggested that derived alleles at rs3895874 , rs3848460 , and rs937301but not rs3809770represent functional mutations .", "the gip promoter reporter activity is haplotype - dependent . a : schematic representation of luciferase reporters containing one of the three inferred haplotypes ( derived : gip ; ancestral : gip and gip ) found in a 2.15-kb fragment of the gip promoter .", "the genomic fragment from position 2073 to + 77 bp of gip contains four snps ( from rs3895874 to rs937301 ) , and three of them are linked .", "genomic fragments that contained each of the three haplotypes were subcloned in the pgl4.2 luciferase reporter .", "b : the luciferase reporter activity is shown in hek293 t cells transfected with combinations of reporter and expression vectors for pax6 and gata4 .", "the reporter activity is haplotype - dependent in the absence or presence of select transcription factors . to compare gip promoter reporter activities , hek293 t cells in 6-well", "culture dishes were transfected with different combinations of transcription factor expression vector and a select gip promoter reporter using lipofectamine 2000 ( life technology inc . ,", ", equal amounts of pcmv and pgl4.2 expression vectors and a one - tenth aliquot of a -galactosidase expression vector were transfected . at 48 h after transfection", ", cells were free - thawed once in lysis buffer , and luciferase activity in supernatant was assayed using the steady - glo luciferase assay system ( promega corp . ) and a lumimark microplate reader ( bio - rad corp .", "the reporter activity has been expressed as the ratio of relative luciferase unit/-galactosidase activity in transfected cells .", "( a high - quality color representation of this figure is available in the online issue . ) given that earlier genome studies have not reported an association between gip variants and any trait , we speculated that the partial selection of gip variants in eurasian populations could be associated with adaptation at a life stage that is vulnerable to environmental changes and that has not been specifically studied . because pregnancy represents a critical life stage that subjects individuals to excessive metabolic load , and", "because its success has a major impact on reproductive fitness , we hypothesized that studies of phenotypic variation during pregnancy may provide a sensitive model to investigate the role of gip haplotypes . to test for an allelic effect of gip variants , we studied east asian patients for proof - of - concept testing because selection pressures are most likely ongoing in populations that exhibit the most significant evidence .", "we genotyped a panel of 123 unrelated han - chinese women who underwent a screening glucose challenge test for gestational diabetes during the 23rd to the 29th weeks of pregnancy , and these patients were assigned to three genotype groups ( gip , gip , or gip ) based on alleles at rs3895874 , rs3848460 , and rs937301 .", "the frequency of rs3895874 in these patients was similar to that of the asn population and was in the hardy  weinberg equilibrium ; frequencies of gip , gip , and gip genotypes were 0.089 , 0.480 , and 0.431 , respectively .", "in addition , alleles at rs3895874 in these patients were in absolute ld with those at rs3848460 and rs937301 . measurements of serum gip and glucose levels showed that circulating gip and glucose at 1 h after the challenge test were 20.6219.9 pg / ml , and 72230 mg / dl , respectively .", "consistent with in vitro promoter reporter assays , gip levels in patients carrying the ancestral gip haplotype ( gip heterozygote and gip homozygote ) were significantly higher than those with a homozygous gip genotype ( fig .", "by contrast , serum levels of glucose , insulin , and c - peptide , as well as age and bmi , were not significantly different among patients ( table 3 ) .", "gip haplotypes impart a difference in gip response and glucose metabolism after glucose challenge tests .", "a : measurements of serum gip and glucose levels in 123 patients during the 23rd to the 29th weeks of pregnancy at 1 h after the 50-g glucose challenge test .", "the gip level was significantly higher in patients carrying a gip haplotype compared with those homozygous for the gip haplotype .", "b : in 72 patients with the dominant gipr genotype , both serum gip and glucose levels were both significantly different between gip homozygotes and those carrying a gip haplotype .", "association between gip haplotypes and gip response after glucose challenge tests in 123 pregnant han - chinese women gip , glucose - dependent insulinotropic polypeptide ; or , odds ratio .", "significantly different from patients with a gip haplotype (  test , p < 0.05 ) . because two linked gip receptor ( gipr ) variants rs10423928 and rs1800437 ( referred to as the gipr mutation in table 3 ) have recently been shown to be associated with glucose and insulin levels after oral glucose challenge tests as well as the incretin effect in nondiabetic individuals in gwa studies ( 35,36 ) , we also genotyped these variants and sought to isolate the potential confounding effect of gipr variants .", "we found no association between gipr snps and serum glucose or hormone levels , but levels of serum glucose , in addition to gip , were significantly different between gip homozygotes , and heterozygotes and the gip homozygotes combined within the pool of patients with the dominant gipr genotype ( fig .", "moreover , we noticed that after controlling for the variation at gipr , the derived gip genotype was associated with increased risk of having a glucose level that exceeded the 140 mg / dl threshold ( odds ratio 3.53 [ 95% ci 1.259.92 ] ; p = 0.015 ; table 3 ) . among patients with a gipr genotype , 48.3% of patients with gip homozygotes exhibited glucose levels that exceeded the threshold , whereas only 20.9% of the remaining patients did", "therefore , the homozygous gip genotype could be associated with a reduced gip response and reduced capability of maintaining glucose homeostasis .", "to systematically identify putative metabolic modifiers , we curated and screened snps in 207 gene loci that have previously been implicated in the regulation of diabetes - related and/or obesity - related traits using fst ( supplementary table 1 ) .", "the empiric distribution of the fst statistic has been used to detect genomic regions that have rapidly increased in frequency as a result of local selective pressures ( 27 ) . of these 207 genes ,", "59 carried genic variants with fst values in the top 5% bracket in comparisons between the corresponding hapmap ii populations yri ( yoruba from ibadan ) , ceu ( u.s .", "residents with northern and western european ancestry ) , and asn ( pooled samples of chinese from beijing [ chb ] and japanese from tokyo [ jpt ] ; supplementary table 1 ) .", "the genic region in 29 of these 59 genes also contained another indication of local selection : long haplotypes in one of the hapmap populations .", "on the basis of the presence of highly divergent allele frequencies between eurasians and africans , the presence of ld and extended haplotypes in eurasians , and a > 30% minor allele frequency in the overall eurasian population , we identified seven snps in the 5 gene region of cdkal1 , cyb5r4 , gad2 , gip , and pparg as potential common metabolic modifiers ( table 1 ) . in earlier gwa studies ,", "select variants in cdkal1 , cyb5r4 , gad2 , and pparg were associated with type 2 diabetes - related or obesity - related traits ( 2831 ) . by contrast , there has been no report of linkage of gip variants in gwa studies .", "because the three gip variants ( rs3895874 , rs3848460 , and rs937301 ) are highly linked compared with those that appeared alone in other candidate genes ( 32 ) , and because these gip variants are partially linked with a nonsynonymous gip snp ( rs2291725 ) ( 22 ) , these gip variants have a low likelihood of being false positive ( table 1 ) .", "snps from the 5 gene region that exhibited signals of partial selection asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry ; gd , gestational diabetes ; ld , linkage - disequilibrium ; ob , obesity ; snp , single nucleotide polymorphism ; t2d , type 2 diabetes ; yri , yoruba in ibadan , nigeria . *", "the allele frequency is significantly different among hapmap ii populations ( p < 0.01 ) .", "fine mapping of the gip locus showed that these variants are linked with more than three dozen neighboring snps ( from rs9904761 to rs3895874 on chr17 : 44,31144,402 kb ) , and these linked snps exhibited fst values in the top 210% bracket in comparisons between yri and asn ( supplementary fig .", "a plot of a 250-kb region of genotypes around gip in the three hapmap ii populations showed that whereas genotypes surrounding gip in yri exhibited a high degree of homozygosity for ancestral alleles ( fig .", "1a , upper panel ) , in the same region , genotypes of asn and ceu exhibited a high degree of homozygosity for the derived alleles ( fig .", "analysis of genic snps at the gip locus between 11 populations from the hapmap iii project ( 34 ) showed that fst values were the highest for comparisons between east asian and african populations ( fig .", "this result is consistent with our recent finding that a nonsynonymous variant in the exon iv of gip ( rs2291725 ) and a 71-kb haplotype block surrounding this variant were positively selected in eurasians in the last 2,000 to 11,800 years ( 22 ) .", "differential distribution of polymorphic alleles at the gip locus in human populations . a : visual depiction of genotypes within a 250-kb region around gip in yri , ceu , and asn populations .", "polymorphic sites are color coded according to their allelic state . individuals with a homozygous genotype with ancestral allele ( aa ) and a homozygous genotype with derived allele ( aa )", "the location of snps in the gip gene region is indicated by a green rectangle .", "the position of snps within the genomic region is indicated by blue vertical lines in the bottom panel .", "b : average fst for genic snps at the gip locus between 11 populations of the hapmap phase iii dataset .", "population descriptors : asw , african ancestry in southwest u.s . ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; chb , han - chinese in beijing , china ; chd , chinese in metropolitan denver , co ; gih , gujarati indians in houston , tx ; jpt , japanese in tokyo , japan ; lwk , luhya in webuye , kenya ; mex , mexican ancestry in los angeles , ca ; mkk , maasai in kinyawa , kenya ; tsi , toscans in italy ; yri , yoruba in ibadan , nigeria .", "the highest population differentiation was found between asian ( han - chinese and japanese ) and african ( luhya , maasai , or yoruba ) populations .", "( a high - quality color representation of this figure is available in the online issue . )", "interestingly , analyses of ld and haplotype block diversity of the genomic region that encompassed the three gip variants at the 5 gene region showed that a 91-kb ld block was represented by five inferred haplotypes in ceu and four in and asn chromosomes , respectively ( fig . 2 ; supplementary fig .", "in addition , these analyses showed that the high fst values and the major shift in allele frequencies of gip variants at the 5 gene region between eurasian and african populations could be attributed to the increase of a derived haplotype ( haplotype 50 in fig .", "because the three 5 gene region variants are completely linked with the positively selected rs2291725 in asn ( 22 ) , we inferred that rs3895874 , rs3848460 , and rs937301 at the human gip promoter region were positively selected in east asian populations as well .", "frequencies of the 58 inferred haplotypes found in the 91-kb ld region ( from rs9904761 to rs3895874 ) surrounding gip in yri , ceu , and asn .", "most of the ceu and asn chromosomes contained derived haplotypes ( with aaaa residues at positions 7679 ) , whereas yri chromosomes exhibited significant divergence in the haplotype composition .", "population descriptors : asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "( a high - quality color representation of this figure is available in the online issue . )", "importantly , we found that the 5 gene region variants ( i.e. , rs3895874 , rs3809770 , rs3848460 , and rs937301 at positions 1920 , 1650 , 1158 , and 320 of gip , respectively ) are located in a haplotype block that is separated from the one containing the nonsynonymous variant rs2291725 by a hotspot for recombination in ceu and yri ( supplementary fig .", "a tetranucleotide polymorphism was represented by three inferred haplotypes in all three populations ( referred to as derived gip , ancestral gip , and ancestral gip haplotypes in the following text ; table 2 ) .", "the derived gip haplotype , which was found in 18.3% of yri chromosomes , has become the dominant haplotype and has a frequency exceeding 50 and 75% in ceu and asn , respectively ( table 2 ) .", "by contrast , the dominant ancestral haplotype in yri chromosomes ( gip , 50.8% ) was only found in 1.1% of asn chromosomes . therefore", ", these 5 gene region variants and the nonsynonymous variant rs2291725 could be selected differentially in the overall hapmap populations and represent causal mutations independent of the nonsynonymous variant rs2291725 . frequency of inferred gip haplotypes and rs3895874 genotypes at the 5 gene region in hapmap ii populations asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "* frequencies of gip haplotypes were significantly different among hapmap ii populations ( p < 0.0001 ) .", "because only functional investigations can convincingly demonstrate causal mutations , we sought to obtain direct evidence that these 5 gene region snps represent causal mutations for the population genetics observation .", "we constructed and tested gip promoter reporters with each of the three major haplotypes in transfected human embryonic kidney ( hek ) 293 t cells ( fig .", "measurement of promoter reporter activities showed that constructs with an ancestral haplotype ( gip or gip ) exhibited luciferase reporter activity 2545% higher than that of a derived haplotype ( gip , p < 0.01 ; fig .", "because the gip promoter region contains elements that are important for regulation by transcriptional factors , including pax6 and gata4 ( 23 ) , we also determined whether the gip promoter reporter activity was haplotype - dependent in the presence of these transcription factors . as expected ,", "coexpression of pax6 or gata4 increased the basal reporter activities by 1.52.5-fold and 0.70.8-fold , respectively ( fig .", "importantly , we found that reporters with an ancestral haplotype consistently exhibited significantly higher activities than those containing the derived haplotype in the presence of pax6 or gata4 ( p < 0.01 ) .", "together , these results suggested that derived alleles at rs3895874 , rs3848460 , and rs937301but not rs3809770represent functional mutations .", ". a : schematic representation of luciferase reporters containing one of the three inferred haplotypes ( derived : gip ; ancestral : gip and gip ) found in a 2.15-kb fragment of the gip promoter .", "the genomic fragment from position 2073 to + 77 bp of gip contains four snps ( from rs3895874 to rs937301 ) , and three of them are linked .", "genomic fragments that contained each of the three haplotypes were subcloned in the pgl4.2 luciferase reporter .", "b : the luciferase reporter activity is shown in hek293 t cells transfected with combinations of reporter and expression vectors for pax6 and gata4 .", "the reporter activity is haplotype - dependent in the absence or presence of select transcription factors . to compare gip promoter reporter activities , hek293 t cells in 6-well", "culture dishes were transfected with different combinations of transcription factor expression vector and a select gip promoter reporter using lipofectamine 2000 ( life technology inc . , carlsbad , ca ) .", "in each well , equal amounts of pcmv and pgl4.2 expression vectors and a one - tenth aliquot of a -galactosidase expression vector were transfected . at 48 h after transfection", ", cells were free - thawed once in lysis buffer , and luciferase activity in supernatant was assayed using the steady - glo luciferase assay system ( promega corp . ) and a lumimark microplate reader ( bio - rad corp . , hercules , ca ) .", "the reporter activity has been expressed as the ratio of relative luciferase unit/-galactosidase activity in transfected cells .", "( a high - quality color representation of this figure is available in the online issue . )", "given that earlier genome studies have not reported an association between gip variants and any trait , we speculated that the partial selection of gip variants in eurasian populations could be associated with adaptation at a life stage that is vulnerable to environmental changes and that has not been specifically studied . because pregnancy represents a critical life stage that subjects individuals to excessive metabolic load , and because its success has a major impact on reproductive fitness , we hypothesized that studies of phenotypic variation during pregnancy may provide a sensitive model to investigate the role of gip haplotypes . to test for an allelic effect of gip variants , we studied east asian patients for proof - of - concept testing because selection pressures are most likely ongoing in populations that exhibit the most significant evidence .", "we genotyped a panel of 123 unrelated han - chinese women who underwent a screening glucose challenge test for gestational diabetes during the 23rd to the 29th weeks of pregnancy , and these patients were assigned to three genotype groups ( gip , gip , or gip ) based on alleles at rs3895874 , rs3848460 , and rs937301 .", "the frequency of rs3895874 in these patients was similar to that of the asn population and was in the hardy  weinberg equilibrium ; frequencies of gip , gip , and gip genotypes were 0.089 , 0.480 , and 0.431 , respectively .", "in addition , alleles at rs3895874 in these patients were in absolute ld with those at rs3848460 and rs937301 .", "measurements of serum gip and glucose levels showed that circulating gip and glucose at 1 h after the challenge test were 20.6219.9 pg / ml , and 72230 mg / dl , respectively . consistent with in vitro promoter reporter assays , gip levels in patients carrying the ancestral gip haplotype ( gip heterozygote and gip homozygote ) were significantly higher than those with a homozygous gip genotype ( fig .", "by contrast , serum levels of glucose , insulin , and c - peptide , as well as age and bmi , were not significantly different among patients ( table 3 ) .", "gip haplotypes impart a difference in gip response and glucose metabolism after glucose challenge tests . a : measurements of serum gip and glucose levels in 123 patients during the 23rd to the 29th weeks of pregnancy at 1 h after the 50-g glucose challenge test .", "the gip level was significantly higher in patients carrying a gip haplotype compared with those homozygous for the gip haplotype .", "b : in 72 patients with the dominant gipr genotype , both serum gip and glucose levels were both significantly different between gip homozygotes and those carrying a gip haplotype .", "association between gip haplotypes and gip response after glucose challenge tests in 123 pregnant han - chinese women gip , glucose - dependent insulinotropic polypeptide ; or , odds ratio . *", "significantly different from patients with a gip haplotype (  test , p < 0.05 ) . because two linked gip receptor ( gipr ) variants rs10423928 and rs1800437 ( referred to as the gipr mutation in table 3 ) have recently been shown to be associated with glucose and insulin levels after oral glucose challenge tests as well as the incretin effect in nondiabetic individuals in gwa studies ( 35,36 ) , we also genotyped these variants and sought to isolate the potential confounding effect of gipr variants .", "we found no association between gipr snps and serum glucose or hormone levels , but levels of serum glucose , in addition to gip , were significantly different between gip homozygotes , and heterozygotes and the gip homozygotes combined within the pool of patients with the dominant gipr genotype ( fig .", "moreover , we noticed that after controlling for the variation at gipr , the derived gip genotype was associated with increased risk of having a glucose level that exceeded the 140 mg / dl threshold ( odds ratio 3.53 [ 95% ci 1.259.92 ] ; p = 0.015 ; table 3 ) . among patients with a gipr genotype , 48.3% of patients with gip homozygotes exhibited glucose levels that exceeded the threshold , whereas only 20.9% of the remaining patients did .", "therefore , the homozygous gip genotype could be associated with a reduced gip response and reduced capability of maintaining glucose homeostasis .", "on the basis of studies of signatures of selection , in vitro promoter assays , and glucose challenge tests in humans , we show that it is possible to identify causal variants related to energy - balance regulation by focusing on genic snps that were subject to environmental selection in a subset of candidate genes .", "specifically , we demonstrated that gip variants at the 5 gene region represent metabolic modifiers that contribute to phenotypic variation in gip response and glucose metabolism .", "further characterization of these causal variants would open a new venue for understanding the molecular mechanisms that underlie phenotypic variations in energy - balance regulation and improve our ability to stratify and interpret clinical outcomes associated with the gip signaling pathway . for decades", ", adaptive selection was assumed to be rare ; however , recent studies have demonstrated that adaptive substitution is pervasive in human genomes ( 1,7,37 ) . despite this progress , it is obvious that population differentiation characteristics of human genomes have yet to be fully explored because physiologic consequences of almost all of these past gene ", "environmental interactions remain to be verified experimentally ( 1,2 ) . on the other hand , because many selection pressures could be heterogeneous or reversible in a short time , the signature of selection may have eroded in genes that were responsive to cultural selection pressures ( 12,38 ) compared with those shielded from heterogeneous selection ( e.g. , the adaptation to environmental oxygen levels and altitude ) ( 5,39 ) .", "we therefore reasoned that important metabolic modifiers could be hidden in the trove of snps that showed limited evidence of positive selection and that this limitation could be particularly pertinent to modifiers associated with adaptations in response to shifts of subsistence cultures .", "consistent with this hypothesis , a survey of earlier studies of genome - wide or chromosome - wide positive selection using the so - called outlier approaches  in which candidate loci are identified in the extreme tails of empiric distributions ( 40)showed that gip variants have not been reported as positively selected ( 1,7 ) .", "the selection of gip variants was inferred after we focused the analysis on local genomic regions and assessed the significance of integrated haplotype score using coalescent simulations ( 22 ) .", "therefore , our investigation provided a proof - of - concept study for identifying causal mutations that underlie phenotypic variation of complex disease - related traits .", "this approach could open new venues for improving the translation of common variant association signals into biologic mechanisms that underlie physiologic variability or disease risk .", "neel hypothesized that mismatches between prior adaptations and new environments , or a  conflict of adaptations ,  could lead to changes in fitness or health risks ( 11 ) . because ancient variants could have been selected for the organism s reproductive success but not for its health or longevity , the ancient alleles could confer disease risks as selection pressures change .", "therefore , studies of positively selected variants that are associated with adaptations in energy - balance regulation could point not only to novel genotype ", "phenotype relationships but also to novel molecular mechanisms that mediate the potential phenotypic variation , thereby providing much - needed insight into how and which phenotypic variations in energy - balance regulation can be attributed to the selected variants . in support of the thrifty genotype hypothesis ,", "human capn10 and house - mouse insulin genes have been shown to exhibit characteristics of adaptive evolution after the emergence of agricultural societies ( 41,42 ) .", "conceptually , the high gip response associated with the ancestral gip haplotype could have been a beneficial energy - conserving mechanism when the food supply was irregular", ". the ancestral haplotype could become deleterious in the last 10 millenniums as agricultural practice became widespread .", "one possible deleterious effect of the ancestral gip haplotype in an environment that supplies abundant high - starch food resources is the hypersecretion of insulin and insulin resistance ( 43,44 ) . on the other hand , a reduced gip response associated with the derived gip haplotype", "could be protective by decreasing the extent of insulin secretion in the face of oversupply of energy inputs ( 45,46 ) . in support of this speculation , it has been well documented that in the absence of modern medicine , diabetes - associated complications and , possibly , obesity posed detrimental effects on survival when human culture shifted ( 47 ) , even though type 2 diabetes is generally considered a chronic disease in modern society .", "alternatively , an elevated glucose level associated with the derived haplotype may have improved the survival of fetuses if the population faced serious famine ", "an event frequently experienced by agricultural societies ( 48)despite the reality that an impaired glucose - tolerance response represents a risk to both the mother and fetus under normal circumstances .", "moreover , the derived haplotype could have been beneficial by reducing the obesity - promoting effect of gip ( 16 ) . in vitro and in vivo studies", "have shown that gip promotes fatty liver and other obesity - associated metabolic disorders , whereas gip antagonists suppress lipid accumulation induced by a high - fat diet ( 23 ) .", "therefore , the derived gip haplotype could be selected for its effects on the enteroadipocyte or the enteroinsular axis , or both .", "although we speculated that the derived gip haplotype may have provided protective effects in famine - plagued agricultural societies , the observation that the derived haplotype has not been fixed in any population suggests that the selection of the derived gip haplotype(s ) ( e.g. , cycles of famine ) could be opposed ( balance selection ) as populations experienced temporal changes in selection pressure ( e.g. , resumption of population growth with stable food supply ) . alternatively", ", the derived gip haplotype could simply be too young to become fixed , or the spread could be limited by the transgenerational effects associated with abnormal gestational glucose metabolism , which raise the risk of macrosomia and diabetes in the offspring ( 11 ) .", "although an association between gip variants and glucose - metabolism regulation has not been reported , gipr variants were associated with glucose and insulin levels after challenge tests as well as with bmi in gwa studies that evaluated > 29,000 individuals ( 35,36 ) .", "the finding that patients with a homozygous gip genotype have significantly higher glucose levels compared with those carrying an ancestral gip haplotype within the pool of gipr homozygotes suggested that there is a confounding effect stemming from interactions of gip and gipr variants , and that gip and gipr variants represent novel markers for the stratification of the capability to maintain glucose homeostasis during pregnancy .", "we also speculate that the significant results observed in pregnant women could be related to the fact that the success of pregnancy has a significant impact on reproductive fitness and that a major fraction of gene  environmental selections probably occurred before birth ( 49 ) .", "recent studies have corroborated this idea by showing that associations between many risk alleles and type 2 diabetes can be replicated with smaller sample sizes in patients with gestational diabetes mellitus ( 50,51 ) .", "furthermore , given the evolutionary signatures at the gip locus , the plausible molecular mechanism , and the significant results in east asian women , we speculate that the gip variant  mediated phenotypic divergence could also exist in most human populations .", "it is also important to note that the selection of gip variants represents a unique example in which the selection process involves regulatory variants that alter the glucose - induced gip response as well as a nonsynonymous variant that affects peptide bioactivity ( 22 ) .", "thus , the gip signaling pathway could represent a hotspot for selection in recent human history and play an important role in the manifestation of phenotypic variation in energy - balance regulation among individuals .", "in addition to gip variants , our study identified several cdkal1 , cyb5r4 , gad2 , and pparg variants as potential metabolic modifiers .", "recent studies have shown that variants in cdkal1 , pparg , and more than two dozen genes are associated with glycemic traits in diabetic patients ( 10 ) . surprisingly", ", none of the cdkal1 , cyb5r4 , gad2 , and pparg variants identified here have been implicated in earlier gwa studies , which suggests that these variants could be related to novel energy - balance regulatory mechanisms that operate at certain life stages or under specific physiologic conditions that have not been specifically investigated .", "future investigations of these variants could reveal additional metabolic modifiers that have arisen recently and their contributions to phenotypic variation in normal human physiology and metabolic syndrome  related traits . in conclusion", ", our data demonstrated a strong association between regulatory gip variants , and gip response and glucose metabolism , reinforcing the indication of an important role of gip signaling in diabetes - related traits from earlier gwa studies of gipr .", "importantly , our study also provided a novel approach to reveal metabolic modifiers by studying consequences of previous mismatches of physiologic capabilities and environments ."], "abstract_text": ["<S> objectivealthough recent studies have shown that human genomes contain hundreds of loci that exhibit signatures of positive selection , variants that are associated with adaptation in energy - balance regulation remain elusive . </S>", "<S> we reasoned that the difficulty in identifying such variants could be due to heterogeneity in selection pressure and that an integrative approach that incorporated experiment - based evidence and population genetics - based statistical judgments would be needed to reveal important metabolic modifiers in humans.research design and methodsto identify common metabolic modifiers that underlie phenotypic variation in diabetes - associated or obesity - associated traits in humans , or both , we screened 207 candidate loci for regulatory single nucleotide polymorphisms ( snps ) that exhibited evidence of gene  environmental interactions.resultsthree snps ( </S>", "<S> rs3895874 , rs3848460 , and rs937301 ) at the 5 gene region of human gip were identified as prime metabolic - modifier candidates at the enteroinsular axis . </S>", "<S> functional studies have shown that gip promoter reporters carrying derived alleles of these three snps ( haplotype gip1920a ) have significantly lower transcriptional activities than those with ancestral alleles at corresponding positions ( haplotype gip1920 g ) . </S>", "<S> consistently , studies of pregnant women who have undergone a screening test for gestational diabetes have shown that patients with a homozygous gip1920a / a genotype have significantly lower serum concentrations of glucose - dependent insulinotropic polypeptide ( gip ) than those carrying an ancestral gip1920 g haplotype . </S>", "<S> after controlling for a gipr variation , we showed that serum glucose concentrations of patients carrying gip1920a / a homozygotes are significantly higher than that of those carrying an ancestral gip1920 g haplotype ( odds ratio 3.53).conclusionsour proof - of - concept study indicates that common regulatory gip variants impart a difference in gip and glucose metabolism . </S>", "<S> the study also provides a rare example that identified the common variant - common phenotypic variation pattern based on evidence of moderate gene  environmental interactions . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Patients.", "Genotyping.", "GIP, insulin, and C-peptide enzyme-linked immunosorbent assay.", "In vitro ", "F", "Statistical analysis.", "RESULTS", "SNPs at the ", "The ", "The ", "DISCUSSION", "Supplementary Material"], "sections": [["this study was approved by the institutional ethics committee review boards of chang gung memorial hospital linkou medical center and chang gung university .", "we recruited 123 patients , and all patients gave written informed consent to participate in the study .", "the screening glucose challenge test for gestational diabetes was performed as previously described ( 24 ) .", "genomic dna samples of participants were extracted and purified from anticoagulated blood with the dneasy blood & tissue kit ( qiagen , venlo , netherlands ) .", "genotyping of snps was performed using the taqman validated snp genotyping assays ( applied biosystems , foster city , ca ) .", "blood samples were collected from patients for hormone measurement 1 h after administration of the oral glucose tolerance test .", "total gip , insulin , and c - peptide levels in human serum were measured using sandwich elisa kits ( millipore , billerica , ma ; mercodia , uppsala , sweden ; and calbiotech , spring valley , ca ) .", "a 2.15-kb fragment of human gip promoter with the a allele at rs3895874 , rs3809770 , rs3848460 , and rs937301 ( 2073 bp to + 77 bp ) was chemically synthesized ( genescript inc . ,", "piscataway , nj ) and subcloned into the pgl4.2 luciferase reporter vector ( promega corp .", "promoter fragments with ancestral haplotypes were obtained using the site - directed mutagenesis . in the promoter reporter study , each experiment was conducted at least three times with three or four replicates for each treatment .", "patients glucose challenge responses and serum hormone profiles were compared with the  test or the student t test with welch correction .", "ratios of patients with glucose levels exceeding the threshold were analyzed with the  test .", "all data were presented as mean  sem , and the statistical significance cutoff value was 0.05 ."], ["this study was approved by the institutional ethics committee review boards of chang gung memorial hospital linkou medical center and chang gung university .", "we recruited 123 patients , and all patients gave written informed consent to participate in the study .", "the screening glucose challenge test for gestational diabetes was performed as previously described ( 24 ) ."], ["genomic dna samples of participants were extracted and purified from anticoagulated blood with the dneasy blood & tissue kit ( qiagen , venlo , netherlands ) .", "genotyping of snps was performed using the taqman validated snp genotyping assays ( applied biosystems , foster city , ca ) ."], ["blood samples were collected from patients for hormone measurement 1 h after administration of the oral glucose tolerance test .", "total gip , insulin , and c - peptide levels in human serum were measured using sandwich elisa kits ( millipore , billerica , ma ; mercodia , uppsala , sweden ; and calbiotech , spring valley , ca ) ."], ["a 2.15-kb fragment of human gip promoter with the a allele at rs3895874 , rs3809770 , rs3848460 , and rs937301 ( 2073 bp to + 77 bp ) was chemically synthesized ( genescript inc . ,", "piscataway , nj ) and subcloned into the pgl4.2 luciferase reporter vector ( promega corp . , madison , wi ) .", "promoter fragments with ancestral haplotypes were obtained using the site - directed mutagenesis . in the promoter reporter study , each experiment was conducted at least three times with three or four replicates for each treatment ."], [""], ["patients glucose challenge responses and serum hormone profiles were compared with the  test or the student t test with welch correction . ratios of patients with glucose levels exceeding the threshold were analyzed with the  test .", "all data were presented as mean  sem , and the statistical significance cutoff value was 0.05 ."], ["to systematically identify putative metabolic modifiers , we curated and screened snps in 207 gene loci that have previously been implicated in the regulation of diabetes - related and/or obesity - related traits using fst ( supplementary table 1 ) .", "the empiric distribution of the fst statistic has been used to detect genomic regions that have rapidly increased in frequency as a result of local selective pressures ( 27 ) . of these 207 genes ,", "59 carried genic variants with fst values in the top 5% bracket in comparisons between the corresponding hapmap ii populations yri ( yoruba from ibadan ) , ceu ( u.s .", "residents with northern and western european ancestry ) , and asn ( pooled samples of chinese from beijing [ chb ] and japanese from tokyo [ jpt ] ; supplementary table 1 ) .", "the genic region in 29 of these 59 genes also contained another indication of local selection : long haplotypes in one of the hapmap populations .", "on the basis of the presence of highly divergent allele frequencies between eurasians and africans , the presence of ld and extended haplotypes in eurasians , and a > 30% minor allele frequency in the overall eurasian population , we identified seven snps in the 5 gene region of cdkal1 , cyb5r4 , gad2 , gip , and pparg as potential common metabolic modifiers ( table 1 ) . in earlier gwa studies ,", "select variants in cdkal1 , cyb5r4 , gad2 , and pparg were associated with type 2 diabetes - related or obesity - related traits ( 2831 ) . by contrast , there has been no report of linkage of gip variants in gwa studies .", "because the three gip variants ( rs3895874 , rs3848460 , and rs937301 ) are highly linked compared with those that appeared alone in other candidate genes ( 32 ) , and because these gip variants are partially linked with a nonsynonymous gip snp ( rs2291725 ) ( 22 ) , these gip variants have a low likelihood of being false positive ( table 1 ) .", "snps from the 5 gene region that exhibited signals of partial selection asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry ; gd , gestational diabetes ; ld , linkage - disequilibrium ; ob , obesity ; snp , single nucleotide polymorphism ; t2d , type 2 diabetes ; yri , yoruba in ibadan , nigeria . *", "the allele frequency is significantly different among hapmap ii populations ( p < 0.01 ) .", "fine mapping of the gip locus showed that these variants are linked with more than three dozen neighboring snps ( from rs9904761 to rs3895874 on chr17 : 44,31144,402 kb ) , and these linked snps exhibited fst values in the top 210% bracket in comparisons between yri and asn ( supplementary fig .", "a plot of a 250-kb region of genotypes around gip in the three hapmap ii populations showed that whereas genotypes surrounding gip in yri exhibited a high degree of homozygosity for ancestral alleles ( fig .", "1a , upper panel ) , in the same region , genotypes of asn and ceu exhibited a high degree of homozygosity for the derived alleles ( fig .", "analysis of genic snps at the gip locus between 11 populations from the hapmap iii project ( 34 ) showed that fst values were the highest for comparisons between east asian and african populations ( fig .", "this result is consistent with our recent finding that a nonsynonymous variant in the exon iv of gip ( rs2291725 ) and a 71-kb haplotype block surrounding this variant were positively selected in eurasians in the last 2,000 to 11,800 years ( 22 ) .", "differential distribution of polymorphic alleles at the gip locus in human populations . a : visual depiction of genotypes within a 250-kb region around gip in yri , ceu , and asn populations .", "polymorphic sites are color coded according to their allelic state . individuals with a homozygous genotype with ancestral allele ( aa ) and a homozygous genotype with derived allele ( aa )", "the location of snps in the gip gene region is indicated by a green rectangle .", "the position of snps within the genomic region is indicated by blue vertical lines in the bottom panel .", "b : average fst for genic snps at the gip locus between 11 populations of the hapmap phase iii dataset .", "population descriptors : asw , african ancestry in southwest u.s . ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; chb , han - chinese in beijing , china ; chd , chinese in metropolitan denver , co ; gih , gujarati indians in houston , tx ; jpt , japanese in tokyo , japan ; lwk , luhya in webuye , kenya ; mex , mexican ancestry in los angeles , ca ; mkk , maasai in kinyawa , kenya ; tsi , toscans in italy ; yri , yoruba in ibadan , nigeria .", "the highest population differentiation was found between asian ( han - chinese and japanese ) and african ( luhya , maasai , or yoruba ) populations .", "( a high - quality color representation of this figure is available in the online issue . )", "interestingly , analyses of ld and haplotype block diversity of the genomic region that encompassed the three gip variants at the 5 gene region showed that a 91-kb ld block was represented by five inferred haplotypes in ceu and four in and asn chromosomes , respectively ( fig . 2 ; supplementary fig .", "in addition , these analyses showed that the high fst values and the major shift in allele frequencies of gip variants at the 5 gene region between eurasian and african populations could be attributed to the increase of a derived haplotype ( haplotype 50 in fig .", "because the three 5 gene region variants are completely linked with the positively selected rs2291725 in asn ( 22 ) , we inferred that rs3895874 , rs3848460 , and rs937301 at the human gip promoter region were positively selected in east asian populations as well .", "frequencies of the 58 inferred haplotypes found in the 91-kb ld region ( from rs9904761 to rs3895874 ) surrounding gip in yri , ceu , and asn .", "most of the ceu and asn chromosomes contained derived haplotypes ( with aaaa residues at positions 7679 ) , whereas yri chromosomes exhibited significant divergence in the haplotype composition .", "population descriptors : asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "( a high - quality color representation of this figure is available in the online issue . )", "importantly , we found that the 5 gene region variants ( i.e. , rs3895874 , rs3809770 , rs3848460 , and rs937301 at positions 1920 , 1650 , 1158 , and 320 of gip , respectively ) are located in a haplotype block that is separated from the one containing the nonsynonymous variant rs2291725 by a hotspot for recombination in ceu and yri ( supplementary fig .", "a tetranucleotide polymorphism was represented by three inferred haplotypes in all three populations ( referred to as derived gip , ancestral gip , and ancestral gip haplotypes in the following text ; table 2 ) .", "the derived gip haplotype , which was found in 18.3% of yri chromosomes , has become the dominant haplotype and has a frequency exceeding 50 and 75% in ceu and asn , respectively ( table 2 ) .", "by contrast , the dominant ancestral haplotype in yri chromosomes ( gip , 50.8% ) was only found in 1.1% of asn chromosomes . therefore", ", these 5 gene region variants and the nonsynonymous variant rs2291725 could be selected differentially in the overall hapmap populations and represent causal mutations independent of the nonsynonymous variant rs2291725 . frequency of inferred gip haplotypes and rs3895874 genotypes at the 5 gene region in hapmap ii populations asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "* frequencies of gip haplotypes were significantly different among hapmap ii populations ( p < 0.0001 ) .", "because only functional investigations can convincingly demonstrate causal mutations , we sought to obtain direct evidence that these 5 gene region snps represent causal mutations for the population genetics observation .", "we constructed and tested gip promoter reporters with each of the three major haplotypes in transfected human embryonic kidney ( hek ) 293 t cells ( fig .", "measurement of promoter reporter activities showed that constructs with an ancestral haplotype ( gip or gip ) exhibited luciferase reporter activity 2545% higher than that of a derived haplotype ( gip , p < 0.01 ; fig .", "because the gip promoter region contains elements that are important for regulation by transcriptional factors , including pax6 and gata4 ( 23 ) , we also determined whether the gip promoter reporter activity was haplotype - dependent in the presence of these transcription factors . as expected ,", "coexpression of pax6 or gata4 increased the basal reporter activities by 1.52.5-fold and 0.70.8-fold , respectively ( fig .", "importantly , we found that reporters with an ancestral haplotype consistently exhibited significantly higher activities than those containing the derived haplotype in the presence of pax6 or gata4 ( p < 0.01 ) .", "together , these results suggested that derived alleles at rs3895874 , rs3848460 , and rs937301but not rs3809770represent functional mutations .", "the gip promoter reporter activity is haplotype - dependent . a : schematic representation of luciferase reporters containing one of the three inferred haplotypes ( derived : gip ; ancestral : gip and gip ) found in a 2.15-kb fragment of the gip promoter .", "the genomic fragment from position 2073 to + 77 bp of gip contains four snps ( from rs3895874 to rs937301 ) , and three of them are linked .", "genomic fragments that contained each of the three haplotypes were subcloned in the pgl4.2 luciferase reporter .", "b : the luciferase reporter activity is shown in hek293 t cells transfected with combinations of reporter and expression vectors for pax6 and gata4 .", "the reporter activity is haplotype - dependent in the absence or presence of select transcription factors . to compare gip promoter reporter activities , hek293 t cells in 6-well", "culture dishes were transfected with different combinations of transcription factor expression vector and a select gip promoter reporter using lipofectamine 2000 ( life technology inc . ,", ", equal amounts of pcmv and pgl4.2 expression vectors and a one - tenth aliquot of a -galactosidase expression vector were transfected . at 48 h after transfection", ", cells were free - thawed once in lysis buffer , and luciferase activity in supernatant was assayed using the steady - glo luciferase assay system ( promega corp . ) and a lumimark microplate reader ( bio - rad corp .", "the reporter activity has been expressed as the ratio of relative luciferase unit/-galactosidase activity in transfected cells .", "( a high - quality color representation of this figure is available in the online issue . ) given that earlier genome studies have not reported an association between gip variants and any trait , we speculated that the partial selection of gip variants in eurasian populations could be associated with adaptation at a life stage that is vulnerable to environmental changes and that has not been specifically studied . because pregnancy represents a critical life stage that subjects individuals to excessive metabolic load , and", "because its success has a major impact on reproductive fitness , we hypothesized that studies of phenotypic variation during pregnancy may provide a sensitive model to investigate the role of gip haplotypes . to test for an allelic effect of gip variants , we studied east asian patients for proof - of - concept testing because selection pressures are most likely ongoing in populations that exhibit the most significant evidence .", "we genotyped a panel of 123 unrelated han - chinese women who underwent a screening glucose challenge test for gestational diabetes during the 23rd to the 29th weeks of pregnancy , and these patients were assigned to three genotype groups ( gip , gip , or gip ) based on alleles at rs3895874 , rs3848460 , and rs937301 .", "the frequency of rs3895874 in these patients was similar to that of the asn population and was in the hardy  weinberg equilibrium ; frequencies of gip , gip , and gip genotypes were 0.089 , 0.480 , and 0.431 , respectively .", "in addition , alleles at rs3895874 in these patients were in absolute ld with those at rs3848460 and rs937301 . measurements of serum gip and glucose levels showed that circulating gip and glucose at 1 h after the challenge test were 20.6219.9 pg / ml , and 72230 mg / dl , respectively .", "consistent with in vitro promoter reporter assays , gip levels in patients carrying the ancestral gip haplotype ( gip heterozygote and gip homozygote ) were significantly higher than those with a homozygous gip genotype ( fig .", "by contrast , serum levels of glucose , insulin , and c - peptide , as well as age and bmi , were not significantly different among patients ( table 3 ) .", "gip haplotypes impart a difference in gip response and glucose metabolism after glucose challenge tests .", "a : measurements of serum gip and glucose levels in 123 patients during the 23rd to the 29th weeks of pregnancy at 1 h after the 50-g glucose challenge test .", "the gip level was significantly higher in patients carrying a gip haplotype compared with those homozygous for the gip haplotype .", "b : in 72 patients with the dominant gipr genotype , both serum gip and glucose levels were both significantly different between gip homozygotes and those carrying a gip haplotype .", "association between gip haplotypes and gip response after glucose challenge tests in 123 pregnant han - chinese women gip , glucose - dependent insulinotropic polypeptide ; or , odds ratio .", "significantly different from patients with a gip haplotype (  test , p < 0.05 ) . because two linked gip receptor ( gipr ) variants rs10423928 and rs1800437 ( referred to as the gipr mutation in table 3 ) have recently been shown to be associated with glucose and insulin levels after oral glucose challenge tests as well as the incretin effect in nondiabetic individuals in gwa studies ( 35,36 ) , we also genotyped these variants and sought to isolate the potential confounding effect of gipr variants .", "we found no association between gipr snps and serum glucose or hormone levels , but levels of serum glucose , in addition to gip , were significantly different between gip homozygotes , and heterozygotes and the gip homozygotes combined within the pool of patients with the dominant gipr genotype ( fig .", "moreover , we noticed that after controlling for the variation at gipr , the derived gip genotype was associated with increased risk of having a glucose level that exceeded the 140 mg / dl threshold ( odds ratio 3.53 [ 95% ci 1.259.92 ] ; p = 0.015 ; table 3 ) . among patients with a gipr genotype , 48.3% of patients with gip homozygotes exhibited glucose levels that exceeded the threshold , whereas only 20.9% of the remaining patients did", "therefore , the homozygous gip genotype could be associated with a reduced gip response and reduced capability of maintaining glucose homeostasis ."], ["to systematically identify putative metabolic modifiers , we curated and screened snps in 207 gene loci that have previously been implicated in the regulation of diabetes - related and/or obesity - related traits using fst ( supplementary table 1 ) .", "the empiric distribution of the fst statistic has been used to detect genomic regions that have rapidly increased in frequency as a result of local selective pressures ( 27 ) . of these 207 genes ,", "59 carried genic variants with fst values in the top 5% bracket in comparisons between the corresponding hapmap ii populations yri ( yoruba from ibadan ) , ceu ( u.s .", "residents with northern and western european ancestry ) , and asn ( pooled samples of chinese from beijing [ chb ] and japanese from tokyo [ jpt ] ; supplementary table 1 ) .", "the genic region in 29 of these 59 genes also contained another indication of local selection : long haplotypes in one of the hapmap populations .", "on the basis of the presence of highly divergent allele frequencies between eurasians and africans , the presence of ld and extended haplotypes in eurasians , and a > 30% minor allele frequency in the overall eurasian population , we identified seven snps in the 5 gene region of cdkal1 , cyb5r4 , gad2 , gip , and pparg as potential common metabolic modifiers ( table 1 ) . in earlier gwa studies ,", "select variants in cdkal1 , cyb5r4 , gad2 , and pparg were associated with type 2 diabetes - related or obesity - related traits ( 2831 ) . by contrast , there has been no report of linkage of gip variants in gwa studies .", "because the three gip variants ( rs3895874 , rs3848460 , and rs937301 ) are highly linked compared with those that appeared alone in other candidate genes ( 32 ) , and because these gip variants are partially linked with a nonsynonymous gip snp ( rs2291725 ) ( 22 ) , these gip variants have a low likelihood of being false positive ( table 1 ) .", "snps from the 5 gene region that exhibited signals of partial selection asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry ; gd , gestational diabetes ; ld , linkage - disequilibrium ; ob , obesity ; snp , single nucleotide polymorphism ; t2d , type 2 diabetes ; yri , yoruba in ibadan , nigeria . *", "the allele frequency is significantly different among hapmap ii populations ( p < 0.01 ) .", "fine mapping of the gip locus showed that these variants are linked with more than three dozen neighboring snps ( from rs9904761 to rs3895874 on chr17 : 44,31144,402 kb ) , and these linked snps exhibited fst values in the top 210% bracket in comparisons between yri and asn ( supplementary fig .", "a plot of a 250-kb region of genotypes around gip in the three hapmap ii populations showed that whereas genotypes surrounding gip in yri exhibited a high degree of homozygosity for ancestral alleles ( fig .", "1a , upper panel ) , in the same region , genotypes of asn and ceu exhibited a high degree of homozygosity for the derived alleles ( fig .", "analysis of genic snps at the gip locus between 11 populations from the hapmap iii project ( 34 ) showed that fst values were the highest for comparisons between east asian and african populations ( fig .", "this result is consistent with our recent finding that a nonsynonymous variant in the exon iv of gip ( rs2291725 ) and a 71-kb haplotype block surrounding this variant were positively selected in eurasians in the last 2,000 to 11,800 years ( 22 ) .", "differential distribution of polymorphic alleles at the gip locus in human populations . a : visual depiction of genotypes within a 250-kb region around gip in yri , ceu , and asn populations .", "polymorphic sites are color coded according to their allelic state . individuals with a homozygous genotype with ancestral allele ( aa ) and a homozygous genotype with derived allele ( aa )", "the location of snps in the gip gene region is indicated by a green rectangle .", "the position of snps within the genomic region is indicated by blue vertical lines in the bottom panel .", "b : average fst for genic snps at the gip locus between 11 populations of the hapmap phase iii dataset .", "population descriptors : asw , african ancestry in southwest u.s . ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; chb , han - chinese in beijing , china ; chd , chinese in metropolitan denver , co ; gih , gujarati indians in houston , tx ; jpt , japanese in tokyo , japan ; lwk , luhya in webuye , kenya ; mex , mexican ancestry in los angeles , ca ; mkk , maasai in kinyawa , kenya ; tsi , toscans in italy ; yri , yoruba in ibadan , nigeria .", "the highest population differentiation was found between asian ( han - chinese and japanese ) and african ( luhya , maasai , or yoruba ) populations .", "( a high - quality color representation of this figure is available in the online issue . )", "interestingly , analyses of ld and haplotype block diversity of the genomic region that encompassed the three gip variants at the 5 gene region showed that a 91-kb ld block was represented by five inferred haplotypes in ceu and four in and asn chromosomes , respectively ( fig . 2 ; supplementary fig .", "in addition , these analyses showed that the high fst values and the major shift in allele frequencies of gip variants at the 5 gene region between eurasian and african populations could be attributed to the increase of a derived haplotype ( haplotype 50 in fig .", "because the three 5 gene region variants are completely linked with the positively selected rs2291725 in asn ( 22 ) , we inferred that rs3895874 , rs3848460 , and rs937301 at the human gip promoter region were positively selected in east asian populations as well .", "frequencies of the 58 inferred haplotypes found in the 91-kb ld region ( from rs9904761 to rs3895874 ) surrounding gip in yri , ceu , and asn .", "most of the ceu and asn chromosomes contained derived haplotypes ( with aaaa residues at positions 7679 ) , whereas yri chromosomes exhibited significant divergence in the haplotype composition .", "population descriptors : asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "( a high - quality color representation of this figure is available in the online issue . )", "importantly , we found that the 5 gene region variants ( i.e. , rs3895874 , rs3809770 , rs3848460 , and rs937301 at positions 1920 , 1650 , 1158 , and 320 of gip , respectively ) are located in a haplotype block that is separated from the one containing the nonsynonymous variant rs2291725 by a hotspot for recombination in ceu and yri ( supplementary fig .", "a tetranucleotide polymorphism was represented by three inferred haplotypes in all three populations ( referred to as derived gip , ancestral gip , and ancestral gip haplotypes in the following text ; table 2 ) .", "the derived gip haplotype , which was found in 18.3% of yri chromosomes , has become the dominant haplotype and has a frequency exceeding 50 and 75% in ceu and asn , respectively ( table 2 ) .", "by contrast , the dominant ancestral haplotype in yri chromosomes ( gip , 50.8% ) was only found in 1.1% of asn chromosomes . therefore", ", these 5 gene region variants and the nonsynonymous variant rs2291725 could be selected differentially in the overall hapmap populations and represent causal mutations independent of the nonsynonymous variant rs2291725 . frequency of inferred gip haplotypes and rs3895874 genotypes at the 5 gene region in hapmap ii populations asn , pooled samples of chinese from beijing and japanese from tokyo ; ceu , utah residents with northern and western european ancestry from the centre de'etude du polymorphism humain ( ceph ) collection ; yri , yoruba in ibadan , nigeria .", "* frequencies of gip haplotypes were significantly different among hapmap ii populations ( p < 0.0001 ) ."], ["because only functional investigations can convincingly demonstrate causal mutations , we sought to obtain direct evidence that these 5 gene region snps represent causal mutations for the population genetics observation .", "we constructed and tested gip promoter reporters with each of the three major haplotypes in transfected human embryonic kidney ( hek ) 293 t cells ( fig .", "measurement of promoter reporter activities showed that constructs with an ancestral haplotype ( gip or gip ) exhibited luciferase reporter activity 2545% higher than that of a derived haplotype ( gip , p < 0.01 ; fig .", "because the gip promoter region contains elements that are important for regulation by transcriptional factors , including pax6 and gata4 ( 23 ) , we also determined whether the gip promoter reporter activity was haplotype - dependent in the presence of these transcription factors . as expected ,", "coexpression of pax6 or gata4 increased the basal reporter activities by 1.52.5-fold and 0.70.8-fold , respectively ( fig .", "importantly , we found that reporters with an ancestral haplotype consistently exhibited significantly higher activities than those containing the derived haplotype in the presence of pax6 or gata4 ( p < 0.01 ) .", "together , these results suggested that derived alleles at rs3895874 , rs3848460 , and rs937301but not rs3809770represent functional mutations .", ". a : schematic representation of luciferase reporters containing one of the three inferred haplotypes ( derived : gip ; ancestral : gip and gip ) found in a 2.15-kb fragment of the gip promoter .", "the genomic fragment from position 2073 to + 77 bp of gip contains four snps ( from rs3895874 to rs937301 ) , and three of them are linked .", "genomic fragments that contained each of the three haplotypes were subcloned in the pgl4.2 luciferase reporter .", "b : the luciferase reporter activity is shown in hek293 t cells transfected with combinations of reporter and expression vectors for pax6 and gata4 .", "the reporter activity is haplotype - dependent in the absence or presence of select transcription factors . to compare gip promoter reporter activities , hek293 t cells in 6-well", "culture dishes were transfected with different combinations of transcription factor expression vector and a select gip promoter reporter using lipofectamine 2000 ( life technology inc . , carlsbad , ca ) .", "in each well , equal amounts of pcmv and pgl4.2 expression vectors and a one - tenth aliquot of a -galactosidase expression vector were transfected . at 48 h after transfection", ", cells were free - thawed once in lysis buffer , and luciferase activity in supernatant was assayed using the steady - glo luciferase assay system ( promega corp . ) and a lumimark microplate reader ( bio - rad corp . , hercules , ca ) .", "the reporter activity has been expressed as the ratio of relative luciferase unit/-galactosidase activity in transfected cells .", "( a high - quality color representation of this figure is available in the online issue . )"], ["given that earlier genome studies have not reported an association between gip variants and any trait , we speculated that the partial selection of gip variants in eurasian populations could be associated with adaptation at a life stage that is vulnerable to environmental changes and that has not been specifically studied . because pregnancy represents a critical life stage that subjects individuals to excessive metabolic load , and because its success has a major impact on reproductive fitness , we hypothesized that studies of phenotypic variation during pregnancy may provide a sensitive model to investigate the role of gip haplotypes . to test for an allelic effect of gip variants , we studied east asian patients for proof - of - concept testing because selection pressures are most likely ongoing in populations that exhibit the most significant evidence .", "we genotyped a panel of 123 unrelated han - chinese women who underwent a screening glucose challenge test for gestational diabetes during the 23rd to the 29th weeks of pregnancy , and these patients were assigned to three genotype groups ( gip , gip , or gip ) based on alleles at rs3895874 , rs3848460 , and rs937301 .", "the frequency of rs3895874 in these patients was similar to that of the asn population and was in the hardy  weinberg equilibrium ; frequencies of gip , gip , and gip genotypes were 0.089 , 0.480 , and 0.431 , respectively .", "in addition , alleles at rs3895874 in these patients were in absolute ld with those at rs3848460 and rs937301 .", "measurements of serum gip and glucose levels showed that circulating gip and glucose at 1 h after the challenge test were 20.6219.9 pg / ml , and 72230 mg / dl , respectively . consistent with in vitro promoter reporter assays , gip levels in patients carrying the ancestral gip haplotype ( gip heterozygote and gip homozygote ) were significantly higher than those with a homozygous gip genotype ( fig .", "by contrast , serum levels of glucose , insulin , and c - peptide , as well as age and bmi , were not significantly different among patients ( table 3 ) .", "gip haplotypes impart a difference in gip response and glucose metabolism after glucose challenge tests . a : measurements of serum gip and glucose levels in 123 patients during the 23rd to the 29th weeks of pregnancy at 1 h after the 50-g glucose challenge test .", "the gip level was significantly higher in patients carrying a gip haplotype compared with those homozygous for the gip haplotype .", "b : in 72 patients with the dominant gipr genotype , both serum gip and glucose levels were both significantly different between gip homozygotes and those carrying a gip haplotype .", "association between gip haplotypes and gip response after glucose challenge tests in 123 pregnant han - chinese women gip , glucose - dependent insulinotropic polypeptide ; or , odds ratio . *", "significantly different from patients with a gip haplotype (  test , p < 0.05 ) . because two linked gip receptor ( gipr ) variants rs10423928 and rs1800437 ( referred to as the gipr mutation in table 3 ) have recently been shown to be associated with glucose and insulin levels after oral glucose challenge tests as well as the incretin effect in nondiabetic individuals in gwa studies ( 35,36 ) , we also genotyped these variants and sought to isolate the potential confounding effect of gipr variants .", "we found no association between gipr snps and serum glucose or hormone levels , but levels of serum glucose , in addition to gip , were significantly different between gip homozygotes , and heterozygotes and the gip homozygotes combined within the pool of patients with the dominant gipr genotype ( fig .", "moreover , we noticed that after controlling for the variation at gipr , the derived gip genotype was associated with increased risk of having a glucose level that exceeded the 140 mg / dl threshold ( odds ratio 3.53 [ 95% ci 1.259.92 ] ; p = 0.015 ; table 3 ) . among patients with a gipr genotype , 48.3% of patients with gip homozygotes exhibited glucose levels that exceeded the threshold , whereas only 20.9% of the remaining patients did .", "therefore , the homozygous gip genotype could be associated with a reduced gip response and reduced capability of maintaining glucose homeostasis ."], ["on the basis of studies of signatures of selection , in vitro promoter assays , and glucose challenge tests in humans , we show that it is possible to identify causal variants related to energy - balance regulation by focusing on genic snps that were subject to environmental selection in a subset of candidate genes .", "specifically , we demonstrated that gip variants at the 5 gene region represent metabolic modifiers that contribute to phenotypic variation in gip response and glucose metabolism .", "further characterization of these causal variants would open a new venue for understanding the molecular mechanisms that underlie phenotypic variations in energy - balance regulation and improve our ability to stratify and interpret clinical outcomes associated with the gip signaling pathway . for decades", ", adaptive selection was assumed to be rare ; however , recent studies have demonstrated that adaptive substitution is pervasive in human genomes ( 1,7,37 ) . despite this progress , it is obvious that population differentiation characteristics of human genomes have yet to be fully explored because physiologic consequences of almost all of these past gene ", "environmental interactions remain to be verified experimentally ( 1,2 ) . on the other hand , because many selection pressures could be heterogeneous or reversible in a short time , the signature of selection may have eroded in genes that were responsive to cultural selection pressures ( 12,38 ) compared with those shielded from heterogeneous selection ( e.g. , the adaptation to environmental oxygen levels and altitude ) ( 5,39 ) .", "we therefore reasoned that important metabolic modifiers could be hidden in the trove of snps that showed limited evidence of positive selection and that this limitation could be particularly pertinent to modifiers associated with adaptations in response to shifts of subsistence cultures .", "consistent with this hypothesis , a survey of earlier studies of genome - wide or chromosome - wide positive selection using the so - called outlier approaches  in which candidate loci are identified in the extreme tails of empiric distributions ( 40)showed that gip variants have not been reported as positively selected ( 1,7 ) .", "the selection of gip variants was inferred after we focused the analysis on local genomic regions and assessed the significance of integrated haplotype score using coalescent simulations ( 22 ) .", "therefore , our investigation provided a proof - of - concept study for identifying causal mutations that underlie phenotypic variation of complex disease - related traits .", "this approach could open new venues for improving the translation of common variant association signals into biologic mechanisms that underlie physiologic variability or disease risk .", "neel hypothesized that mismatches between prior adaptations and new environments , or a  conflict of adaptations ,  could lead to changes in fitness or health risks ( 11 ) . because ancient variants could have been selected for the organism s reproductive success but not for its health or longevity , the ancient alleles could confer disease risks as selection pressures change .", "therefore , studies of positively selected variants that are associated with adaptations in energy - balance regulation could point not only to novel genotype ", "phenotype relationships but also to novel molecular mechanisms that mediate the potential phenotypic variation , thereby providing much - needed insight into how and which phenotypic variations in energy - balance regulation can be attributed to the selected variants . in support of the thrifty genotype hypothesis ,", "human capn10 and house - mouse insulin genes have been shown to exhibit characteristics of adaptive evolution after the emergence of agricultural societies ( 41,42 ) .", "conceptually , the high gip response associated with the ancestral gip haplotype could have been a beneficial energy - conserving mechanism when the food supply was irregular", ". the ancestral haplotype could become deleterious in the last 10 millenniums as agricultural practice became widespread .", "one possible deleterious effect of the ancestral gip haplotype in an environment that supplies abundant high - starch food resources is the hypersecretion of insulin and insulin resistance ( 43,44 ) . on the other hand , a reduced gip response associated with the derived gip haplotype", "could be protective by decreasing the extent of insulin secretion in the face of oversupply of energy inputs ( 45,46 ) . in support of this speculation , it has been well documented that in the absence of modern medicine , diabetes - associated complications and , possibly , obesity posed detrimental effects on survival when human culture shifted ( 47 ) , even though type 2 diabetes is generally considered a chronic disease in modern society .", "alternatively , an elevated glucose level associated with the derived haplotype may have improved the survival of fetuses if the population faced serious famine ", "an event frequently experienced by agricultural societies ( 48)despite the reality that an impaired glucose - tolerance response represents a risk to both the mother and fetus under normal circumstances .", "moreover , the derived haplotype could have been beneficial by reducing the obesity - promoting effect of gip ( 16 ) . in vitro and in vivo studies", "have shown that gip promotes fatty liver and other obesity - associated metabolic disorders , whereas gip antagonists suppress lipid accumulation induced by a high - fat diet ( 23 ) .", "therefore , the derived gip haplotype could be selected for its effects on the enteroadipocyte or the enteroinsular axis , or both .", "although we speculated that the derived gip haplotype may have provided protective effects in famine - plagued agricultural societies , the observation that the derived haplotype has not been fixed in any population suggests that the selection of the derived gip haplotype(s ) ( e.g. , cycles of famine ) could be opposed ( balance selection ) as populations experienced temporal changes in selection pressure ( e.g. , resumption of population growth with stable food supply ) . alternatively", ", the derived gip haplotype could simply be too young to become fixed , or the spread could be limited by the transgenerational effects associated with abnormal gestational glucose metabolism , which raise the risk of macrosomia and diabetes in the offspring ( 11 ) .", "although an association between gip variants and glucose - metabolism regulation has not been reported , gipr variants were associated with glucose and insulin levels after challenge tests as well as with bmi in gwa studies that evaluated > 29,000 individuals ( 35,36 ) .", "the finding that patients with a homozygous gip genotype have significantly higher glucose levels compared with those carrying an ancestral gip haplotype within the pool of gipr homozygotes suggested that there is a confounding effect stemming from interactions of gip and gipr variants , and that gip and gipr variants represent novel markers for the stratification of the capability to maintain glucose homeostasis during pregnancy .", "we also speculate that the significant results observed in pregnant women could be related to the fact that the success of pregnancy has a significant impact on reproductive fitness and that a major fraction of gene  environmental selections probably occurred before birth ( 49 ) .", "recent studies have corroborated this idea by showing that associations between many risk alleles and type 2 diabetes can be replicated with smaller sample sizes in patients with gestational diabetes mellitus ( 50,51 ) .", "furthermore , given the evolutionary signatures at the gip locus , the plausible molecular mechanism , and the significant results in east asian women , we speculate that the gip variant  mediated phenotypic divergence could also exist in most human populations .", "it is also important to note that the selection of gip variants represents a unique example in which the selection process involves regulatory variants that alter the glucose - induced gip response as well as a nonsynonymous variant that affects peptide bioactivity ( 22 ) .", "thus , the gip signaling pathway could represent a hotspot for selection in recent human history and play an important role in the manifestation of phenotypic variation in energy - balance regulation among individuals .", "in addition to gip variants , our study identified several cdkal1 , cyb5r4 , gad2 , and pparg variants as potential metabolic modifiers .", "recent studies have shown that variants in cdkal1 , pparg , and more than two dozen genes are associated with glycemic traits in diabetic patients ( 10 ) . surprisingly", ", none of the cdkal1 , cyb5r4 , gad2 , and pparg variants identified here have been implicated in earlier gwa studies , which suggests that these variants could be related to novel energy - balance regulatory mechanisms that operate at certain life stages or under specific physiologic conditions that have not been specifically investigated .", "future investigations of these variants could reveal additional metabolic modifiers that have arisen recently and their contributions to phenotypic variation in normal human physiology and metabolic syndrome  related traits . in conclusion", ", our data demonstrated a strong association between regulatory gip variants , and gip response and glucose metabolism , reinforcing the indication of an important role of gip signaling in diabetes - related traits from earlier gwa studies of gipr .", "importantly , our study also provided a novel approach to reveal metabolic modifiers by studying consequences of previous mismatches of physiologic capabilities and environments ."], [""]]}
{"article_id": "PMC4123559", "article_text": ["the conventional definitions of pediatric adiposity depend on a measured body mass index ( bmi , kg / m ) interpreted relative to a reference distribution ( bmi normative growth charts ) for sex and age [ 13 ] .", "because it has body weight in its numerator , bmi reflects generalized ( total - body ) enlargement with a simplified correction ( as height ) for skeletal size .", "pediatric adiposity has been defined alternatively by abdominal size , most commonly a waist circumference , because increased truncal adipose tissue is correlated better than generalized adiposity with cardiometabolic dysfunction [ 4 , 5 ] . since", "waist circumference is a regional measurement , it reflects specifically only abdominal or central enlargement .", "thus , waist circumference also requires interpretation relative to its own reference distribution for sex and age .", "comparisons of risk assessments in youth have generally found little difference between bmi and waist circumference in the ability of these indicators to identify cardiometabolic risk [ 6 , 7 ] . the waist - to - height ratio ( whtr ) is an adiposity indicator with waist circumference in the numerator and a simplified correction ( as height ) for skeletal size .", "whtr does not depend on sex- or age - specific reference criteria [ 810 ] . in a large sample of us sixth - graders", ", we examined the performance of bmi z - score ( bmiz , referenced to cdc 2000 growth charts for the united states ) and whtr for the purpose of cardiometabolic risk assessment .", "participants came from the baseline enrollment ( sixth - grade students in 2006 ) of the healthy study , a cluster - randomized , controlled , primary prevention trial designed to improve indicators of adiposity and glycemic dysregulation among us middle - school children [ 12 , 13 ] .", "seven research centers recruited 42 middle schools with at least 50% of students eligible to participate in the federally subsidized national school lunch program or belonging to an ancestral minority group at increased risk of type 2 diabetes ( hispanic , non - hispanic black , or native american ) .", "a detailed protocol and background details about the healthy study are available for download at http://www.healthystudy.org/. we restricted a priori our analytic sample to students with integer ages of 11 or 12 years who were able to participate in physical education and did not have known diabetes . from 5950 eligible enrollees , we excluded 13 students due to missing or invalid data for the adiposity indicators ( height , weight , and waist circumference ) .", "additional 239 students were excluded for lack of outcome cardiometabolic risk variables , and 216 were excluded for lack of information on pubarche ( an adjustment variable associated with adrenarche , growth pattern , and insulin resistance ) [ 1416 ] .", "participant ancestry ( hispanic , non - hispanic black , non - hispanic white , other ) and sex were self - reported .", "the study was approved by each research center 's institutional review board , and informed consent from parents and assent from students were obtained prior to data collection . from measured height and weight , we calculated the sex- and age - specific bmiz based on the centers for disease control and prevention 2000 growth charts [ 11 , 17 ] .", "waist circumference was measured to the nearest 0.1  cm on bare skin just above the iliac crest following procedures of the national health and nutrition examination survey , and the whtr was calculated .", "blood pressure was recorded three times using an omron automated blood pressure monitor ( with appropriate - size cuff ) after the participant sat quietly for 5 minutes .", "pubarche was identified dichotomously from the participants ' response to a standardized question on the appearance of underarm and pubic hair .", "fasting blood samples were processed onsite and shipped to the healthy central blood laboratory ( northwest lipid research laboratories , university of washington ) .", "insulin was measured by an immunoenzymometric assay using a tosoh 1800 autoanalyzer ; the interassay and intra - assay precision analysis consistently showed a coefficient of variation ( cv ) < 10% .", "the assay had low cross - reactivity with human c - peptide ( 0% ) and proinsulin ( 2% ) .", "the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated from glucose ( mg / dl ) and insulin ( u / ml ) concentrations using the following formula : \n ( 1)homa - ir=(glucose0.05551)(insulin)22.5 .", "measurements of total plasma cholesterol , cholesterol in the lipoprotein fractions , and triglycerides were performed enzymatically on the roche modular - p autoanalyzer using well - standardized methods .", "the interassay cvs were consistently < 1.5% for total cholesterol and triglycerides and < 2% for hdl cholesterol .", "we calculated the total - to - hdl cholesterol ratio ( tc / hdlc ) , a variable that strongly predicts cardiovascular disease in adults   and may be more strongly associated than ldl - cholesterol or hdl - cholesterol concentrations with pediatric adiposity [ 22 , 23 ] .", "in addition to stratification by sex , we chose a priori to stratify our analyses by  high fatness  or  lower fatness  because the ability of adiposity indicators to identify adipose tissue mass , ectopic fat , and cardiometabolic risk variables may be stronger among children in a higher fatness category [ 2326 ] . as defined for this report , the high - fatness level included students who were above the sex - specific median value for both bmiz and whtr ; any student below the median for either adiposity indicator was designated lower fatness . for each fatness level , we prepared sex - specific , linear - regression models adjusted for ancestry ( 4 categories ) and pubarche ( yes / no ) to estimate the associations of continuous adiposity indicators with the continuous cardiometabolic risk factors ( outcomes ) . since blood pressure varies with height in children [ 27 , 28 ] our models for blood pressure outcomes included an additional adjustment for height which was entered as a continuous variable .", "for all variables except bmiz , we calculated descriptive statistics without using reference - based corrections for sex or age . for indicators or outcomes that departed markedly from a normal distribution ( whtr , homa - ir , tc / hdlc , hdl cholesterol , triglycerides , and ( only for lower - fatness students ) diastolic blood pressure ) , we transformed the variable by loge or inverse square root to approach normality prior to their use in regression models .", "our adjusted models estimated standardized beta coefficients ( change in the outcome variable ( in standard deviations ) associated with change of one standard deviation in the adiposity indicator ) for each cardiometabolic risk factor .", "cary , nc ) was used to account for variability both within and between the school clusters . in these mixed models ,", "the proportion of variation explained ( r ) by each adiposity indicator was calculated as the full model r minus r for a model omitting the adiposity indicator . to compare linear slopes among ancestral groups", ", mixed - regression models estimated nonstandardized , beta - regression coefficients ; interactions were tested between each adiposity indicator and the three ancestries represented prominently in our sample ( hispanic , black , and white ) .", "characteristics of the analytic sample are presented by sex in table 1 . as expected for sixth - grade students ,", "following further stratification by fatness level , the distributions of adiposity indicators and ancestry are summarized in table 2 .", "compared to the high - fatness groups in our sample , the lower - fatness groups had bmi and whtr distributions that resembled more closely the general population of us youth in the same age range [ 31 , 32 ] . within the high - fatness subpopulations of either sex ( table 3 ) ,", "adiposity indicators explained 19%28% of the variation in homa - ir , 4%9% of the variation in circulating lipids ( tc / hdlc , hdl cholesterol , triglycerides ) , and 5%9% of the variation in diastolic blood pressure .", "adiposity indicators explained < 3% of the variations in systolic blood pressure and fasting glucose . for each outcome variable in these high - fatness subpopulations , the effect sizes ( standardized beta coefficients ) associated with bmiz were similar to those associated with whtr . within the lower - fatness subpopulations of either sex ( table 4 ) ,", "adiposity indicators explained 8%13% of the variation in homa - ir and 2%7% of the variation in circulating lipids . for tc / hdlc and triglycerides ,", "the standardized beta coefficients tended to be weaker for bmiz ( 0.130.20 ) than for whtr ( 0.170.28 ) .", "adiposity indicators in these lower - fatness subpopulations explained < 1% of the variations in systolic blood pressure , diastolic blood pressure , and fasting glucose .", "a comparison between the two levels of fatness ( table 3 versus table 4 ) demonstrates that for either adiposity indicator the associations with homa - ir were stronger among the high - fatness students ( beta coefficients 0.430.52 ) than among the lower - fatness students ( 0.300.37 ) .", "similarly , both adiposity indicators were associated with diastolic blood pressure more strongly among high - fatness students ( 0.230.32 ; p < 0.001 for each of 4 beta coefficients ) than among lower - fatness students ( 0.030.05 ; p > 0.05 for each of four beta coefficients ) . for identification of lipid outcomes , however , we found steeper beta coefficients only among high - fatness boys ( compared to lower - fatness boys ) whose adiposity was assessed by bmiz . for both sexes assessed by whtr ,", "the associations between adiposity indicators and risk variables were not notably different between the hispanics , non - hispanic blacks , and non - hispanic whites except when related to blood pressure outcomes .", "although adiposity explained 5%7% of variation in diastolic blood pressure in the complete sample of high - fatness girls ( table 3 ) , this relationship was extremely weak for high - fatness girls who were black , as indicated by slope point estimates close to zero ( figure 1 ) .", "however , for high - fatness girls who were hispanic or white , bmiz and whtr had significant associations ( p < 0.05 ) with diastolic pressure . among high - fatness boys", "systolic blood pressure was not significantly associated with bmiz or whtr for high - fatness blacks of either sex , but the association was present for high - fatness students who were hispanic or white .", "an ancestral contrast ( blacks compared to whites ) related to systolic blood pressure was significant , however , only among the high - fatness girls assessed by bmiz ( figure 1 ; p < 0.01 ) .", "in this large sample of middle - school students at increased risk of obesity and type 2 diabetes , we found that adiposity indicators bmiz ( with reference to cdc 2000 growth charts ) and whtr ( without reference to sex and age ) had similar utility for identifying adverse levels of cardiometabolic variables .", "our findings are generally consistent with previous published reports , most of which were based on populations that had a wider age range or included less ancestral diversity .", "earlier cross - sectional studies that compared continuous whtr against bmi either without a normative growth reference [ 3335 ] or with reference - based bmi z - scores / percentile ranks [ 23 , 36 ] generally found that whtr provided stronger associations with lipid outcomes , but bmi was superior for blood pressure outcomes .", "a recent report on sixth - grade students from switzerland found that bmiz ( referenced to cdc 2000 growth charts ) and whtr provided associations with blood pressures that were weak but nearly identical . in studies of youth from the southern us , bmiz provided a slightly stronger association than whtr with homa - ir   and fasting insulin , a relationship that was complicated by nonlinearity .", "nationally representative , cross - sectional data from the us demonstrated that whtr at ages 417 years was more strongly associated than bmiz with resting heart rate . a longitudinal analysis from the same survey of adolescent and young - adult participants found that categorical whtr predicted all - cause mortality before age 55 better than categorical bmiz ( baseline ages 1218 years ) or bmi ( ages 1939 ) . from the united kingdom ,", "a large study recently reported that whtr and bmiz obtained at ages 79 years had similar associations with cross - sectional and prospective cardiometabolic risk factors in adolescence . given an approximately equal utility of bmiz and whtr for pediatric health assessments , we should consider how these adiposity indicators might perform in different settings or in the future .", "bmiz values reported in the research literature depend on standardized protocols for measuring height and weight using calibrated , high - quality scales . in nonresearch settings , however , staff training and time pressures might not be so favorable to careful measurements .", "the dependence of bmiz on normative growth references can be problematic because bmi - for - age reference values can yield discrepant inferences between populations , time periods , and ethnicities within a single country .", "worldwide  bmi growth reference based on large datasets from six countries , but subsequent reviews found that this international growth reference provided no advantage over national bmi growth references for the definition of excessive fat mass in youth   or prediction of subsequent cardiovascular risk in adulthood .", "the world health organization ( who ) more recently developed a bmi - based growth reference , the utilization of which has been described as a cumbersome task in need of simplification .", "surveys from various clinical settings have found generally that the use of bmi - for - age reference values is suboptimal [ 4750 ] .", "advocates of the whtr must address problems associated with the available protocols for measuring waist size .", "while tape measures are inexpensive and generally need little calibration , protocols for circumference measurement are still unfamiliar to many pediatric practitioners or clinic assistants .", "our study carefully measured the waist circumference just above the iliac crest , an anatomic location endorsed by prominent researchers in the united states [ 18 , 51 ] and canada . the who , however , recommends measuring waist circumference at the approximate midpoint between the lower margin of the last palpable rib and the top of the iliac crest .", "minimal waist ,  and other sites [ 54 , 55 ] . in an anthropometric study of diabetic youth , the iliac - crest protocol and", "who protocol demonstrated comparable reproducibility , but these alternative protocols yielded notable differences in the absolute value of a waist circumference obtained from the same participants .", "a study of overweight youth found that the who waist - circumference protocol had a stronger association than the iliac - crest protocol with cardiometabolic risk , and studies of adult waist circumference have likewise suggested an advantage for the who protocol [ 5860 ] .", "it follows that the whtr values calculated from the iliac - crest and who protocols should not be casually substituted for each other .", "it is possible that if our healthy study had adopted the who instead of the iliac - crest protocol for its baseline anthropometry , the re - calculated whtr indicator might have demonstrated stronger associations with cardiometabolic risk variables than those we report in this paper .", "standardization of a single waist - circumference protocol would probably advance the widespread adoption of the whtr as a low - cost adiposity indicator [ 55 , 61 ] . as an alternative to the circumference ,", "some pediatric investigators have described waist size in selected participants by measuring the external diameter sagittally ( back - to - front ) in the supine position [ 6264 ] .", "protocol might further enhance studies that are cross - sectional or involve short - term follow - up of central adiposity , but this anthropometric method needs to be tested in larger datasets that represent general youth populations . the physiological importance of tissues accumulated in the waist may help to explain why whtr was more closely associated than bmiz with variations in the levels of circulating lipid markers among our lower - fatness participants ( table 4 ) .", "an increase in waist size primarily marks expanding amounts of adipose tissue , including notably the visceral depot which is most strongly associated with an adverse metabolic phenotype [ 5 , 65 ] .", "variation in the waist circumference can explain more than 64% of the variance in the area   or volume   of visceral adipose tissue .", "an increase in bmi , by contrast , may substantially mark also the variations in gain of muscular weight or subcutaneous fat patterning that precede adulthood . along with the changes in fat mass ,", "these variations in lean mass or superficial adipose tissue contribute to the bmi calculation while contributing relatively little to metabolic risk .", "although high - fatness hispanic and white girls in our study demonstrated the expected associations between adiposity and blood pressure , we found among the high - fatness black girls ( but not black boys ) that neither bmiz nor whtr had a significant association with blood pressure outcomes ( figure 1 ) .  in comparison to young hispanics and whites ,", "young blacks tend to have abdominal adipose tissue relatively less in the visceral depot and more in the abdominal subcutaneous regions .", "another study has also reported that black girls ' waist circumference around the same age was unrelated to their diastolic blood pressure .", "since both the bmiz and whtr indicators demonstrated similar patterns of nonassociation with black girls ' blood pressure , it may be that this absence of a correlation with blood pressure is due to factors operating primarily outside the abdominal region .", "perhaps black girls benefit from an increased capacity to expand their lower - body ( gluteofemoral ) , subcutaneous , adipose - tissue stores in a manner that would increase their total body weight yet protect them from cardiometabolic risk [ 7073 ] .", "if this protective characteristic of black girls extends into their later years , it could help explain why adult black women in the us experience no increased cardiometabolic risk   or mortality   until their bmi reaches approximately 33  kg / m .", "if a well - standardized waist measurement comes into widespread use for clinical assessments of pediatric adiposity , patients and their families may improve their intuitive understanding of how excess adiposity contributes to adverse health risk .", "pediatric health care providers , too , may find it more useful to recognize risk associated with a waist increment ( corrected for height ) than with a weight increment ( corrected for height squared ) .", "adoption of the whtr could optimize both patient education and the tracking of risk . compared to bmi ,", "the whtr allows a simpler calculation without the necessity of squaring the child 's height . of interest to those concerned with child adiposity and", "cardiometabolic risk observed in different cultures or distinct time periods , the whtr will facilitate comparisons based directly on anthropometric observations without using normative reference tables that may not be suitable to all populations [ 7779 ] ."], "abstract_text": ["<S> convention defines pediatric adiposity by the body mass index z - score ( bmiz ) referenced to normative growth charts . </S>", "<S> waist - to - height ratio ( whtr ) does not depend on sex - and - age references . in the healthy study enrollment sample , </S>", "<S> we compared bmiz with whtr for ability to identify adverse cardiometabolic risk . among 5,482 sixth - grade students from 42 middle schools , we estimated explanatory variations ( r2 ) and standardized beta coefficients of bmiz or whtr for cardiometabolic risk factors : insulin resistance ( homa - ir ) , lipids , blood pressures , and glucose . </S>", "<S> for each risk outcome variable , we prepared adjusted regression models for four subpopulations stratified by sex and high versus lower fatness . for homa - ir , </S>", "<S> r2 attributed to bmiz or whtr was 19%28% among high - fatness and 8%13% among lower - fatness students . </S>", "<S> r2 for lipid variables was 4%9% among high - fatness and 2%7% among lower - fatness students . in the lower - fatness subpopulations , </S>", "<S> the standardized coefficients for total cholesterol / hdl cholesterol and triglycerides tended to be weaker for bmiz ( 0.130.20 ) than for whtr ( 0.170.28 ) . among high - fatness students , bmiz and whtr correlated with blood pressures for hispanics and whites , but not black boys ( systolic ) or girls ( systolic and diastolic ) . in 11 - 12 year olds , assessments by whtr </S>", "<S> can provide cardiometabolic risk estimates similar to conventional bmiz without requiring reference to a normative growth chart . </S>"], "labels": null, "section_names": ["1. Introduction ", "2. Methods ", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["the conventional definitions of pediatric adiposity depend on a measured body mass index ( bmi , kg / m ) interpreted relative to a reference distribution ( bmi normative growth charts ) for sex and age [ 13 ] .", "because it has body weight in its numerator , bmi reflects generalized ( total - body ) enlargement with a simplified correction ( as height ) for skeletal size .", "pediatric adiposity has been defined alternatively by abdominal size , most commonly a waist circumference , because increased truncal adipose tissue is correlated better than generalized adiposity with cardiometabolic dysfunction [ 4 , 5 ] . since", "waist circumference is a regional measurement , it reflects specifically only abdominal or central enlargement .", "thus , waist circumference also requires interpretation relative to its own reference distribution for sex and age .", "comparisons of risk assessments in youth have generally found little difference between bmi and waist circumference in the ability of these indicators to identify cardiometabolic risk [ 6 , 7 ] . the waist - to - height ratio ( whtr ) is an adiposity indicator with waist circumference in the numerator and a simplified correction ( as height ) for skeletal size .", "whtr does not depend on sex- or age - specific reference criteria [ 810 ] . in a large sample of us sixth - graders", ", we examined the performance of bmi z - score ( bmiz , referenced to cdc 2000 growth charts for the united states ) and whtr for the purpose of cardiometabolic risk assessment ."], ["participants came from the baseline enrollment ( sixth - grade students in 2006 ) of the healthy study , a cluster - randomized , controlled , primary prevention trial designed to improve indicators of adiposity and glycemic dysregulation among us middle - school children [ 12 , 13 ] .", "seven research centers recruited 42 middle schools with at least 50% of students eligible to participate in the federally subsidized national school lunch program or belonging to an ancestral minority group at increased risk of type 2 diabetes ( hispanic , non - hispanic black , or native american ) .", "a detailed protocol and background details about the healthy study are available for download at http://www.healthystudy.org/. we restricted a priori our analytic sample to students with integer ages of 11 or 12 years who were able to participate in physical education and did not have known diabetes . from 5950 eligible enrollees , we excluded 13 students due to missing or invalid data for the adiposity indicators ( height , weight , and waist circumference ) .", "additional 239 students were excluded for lack of outcome cardiometabolic risk variables , and 216 were excluded for lack of information on pubarche ( an adjustment variable associated with adrenarche , growth pattern , and insulin resistance ) [ 1416 ] .", "participant ancestry ( hispanic , non - hispanic black , non - hispanic white , other ) and sex were self - reported .", "the study was approved by each research center 's institutional review board , and informed consent from parents and assent from students were obtained prior to data collection . from measured height and weight , we calculated the sex- and age - specific bmiz based on the centers for disease control and prevention 2000 growth charts [ 11 , 17 ] .", "waist circumference was measured to the nearest 0.1  cm on bare skin just above the iliac crest following procedures of the national health and nutrition examination survey , and the whtr was calculated .", "blood pressure was recorded three times using an omron automated blood pressure monitor ( with appropriate - size cuff ) after the participant sat quietly for 5 minutes .", "pubarche was identified dichotomously from the participants ' response to a standardized question on the appearance of underarm and pubic hair .", "fasting blood samples were processed onsite and shipped to the healthy central blood laboratory ( northwest lipid research laboratories , university of washington ) .", "insulin was measured by an immunoenzymometric assay using a tosoh 1800 autoanalyzer ; the interassay and intra - assay precision analysis consistently showed a coefficient of variation ( cv ) < 10% .", "the assay had low cross - reactivity with human c - peptide ( 0% ) and proinsulin ( 2% ) .", "the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated from glucose ( mg / dl ) and insulin ( u / ml ) concentrations using the following formula : \n ( 1)homa - ir=(glucose0.05551)(insulin)22.5 .", "measurements of total plasma cholesterol , cholesterol in the lipoprotein fractions , and triglycerides were performed enzymatically on the roche modular - p autoanalyzer using well - standardized methods .", "the interassay cvs were consistently < 1.5% for total cholesterol and triglycerides and < 2% for hdl cholesterol .", "we calculated the total - to - hdl cholesterol ratio ( tc / hdlc ) , a variable that strongly predicts cardiovascular disease in adults   and may be more strongly associated than ldl - cholesterol or hdl - cholesterol concentrations with pediatric adiposity [ 22 , 23 ] .", "in addition to stratification by sex , we chose a priori to stratify our analyses by  high fatness  or  lower fatness  because the ability of adiposity indicators to identify adipose tissue mass , ectopic fat , and cardiometabolic risk variables may be stronger among children in a higher fatness category [ 2326 ] . as defined for this report , the high - fatness level included students who were above the sex - specific median value for both bmiz and whtr ; any student below the median for either adiposity indicator was designated lower fatness . for each fatness level , we prepared sex - specific , linear - regression models adjusted for ancestry ( 4 categories ) and pubarche ( yes / no ) to estimate the associations of continuous adiposity indicators with the continuous cardiometabolic risk factors ( outcomes ) . since blood pressure varies with height in children [ 27 , 28 ] our models for blood pressure outcomes included an additional adjustment for height which was entered as a continuous variable .", "for all variables except bmiz , we calculated descriptive statistics without using reference - based corrections for sex or age . for indicators or outcomes that departed markedly from a normal distribution ( whtr , homa - ir , tc / hdlc , hdl cholesterol , triglycerides , and ( only for lower - fatness students ) diastolic blood pressure ) , we transformed the variable by loge or inverse square root to approach normality prior to their use in regression models .", "our adjusted models estimated standardized beta coefficients ( change in the outcome variable ( in standard deviations ) associated with change of one standard deviation in the adiposity indicator ) for each cardiometabolic risk factor .", "cary , nc ) was used to account for variability both within and between the school clusters . in these mixed models ,", "the proportion of variation explained ( r ) by each adiposity indicator was calculated as the full model r minus r for a model omitting the adiposity indicator . to compare linear slopes among ancestral groups", ", mixed - regression models estimated nonstandardized , beta - regression coefficients ; interactions were tested between each adiposity indicator and the three ancestries represented prominently in our sample ( hispanic , black , and white ) ."], ["characteristics of the analytic sample are presented by sex in table 1 . as expected for sixth - grade students ,", "following further stratification by fatness level , the distributions of adiposity indicators and ancestry are summarized in table 2 .", "compared to the high - fatness groups in our sample , the lower - fatness groups had bmi and whtr distributions that resembled more closely the general population of us youth in the same age range [ 31 , 32 ] . within the high - fatness subpopulations of either sex ( table 3 ) ,", "adiposity indicators explained 19%28% of the variation in homa - ir , 4%9% of the variation in circulating lipids ( tc / hdlc , hdl cholesterol , triglycerides ) , and 5%9% of the variation in diastolic blood pressure .", "adiposity indicators explained < 3% of the variations in systolic blood pressure and fasting glucose . for each outcome variable in these high - fatness subpopulations , the effect sizes ( standardized beta coefficients ) associated with bmiz were similar to those associated with whtr . within the lower - fatness subpopulations of either sex ( table 4 ) ,", "adiposity indicators explained 8%13% of the variation in homa - ir and 2%7% of the variation in circulating lipids . for tc / hdlc and triglycerides ,", "the standardized beta coefficients tended to be weaker for bmiz ( 0.130.20 ) than for whtr ( 0.170.28 ) .", "adiposity indicators in these lower - fatness subpopulations explained < 1% of the variations in systolic blood pressure , diastolic blood pressure , and fasting glucose .", "a comparison between the two levels of fatness ( table 3 versus table 4 ) demonstrates that for either adiposity indicator the associations with homa - ir were stronger among the high - fatness students ( beta coefficients 0.430.52 ) than among the lower - fatness students ( 0.300.37 ) .", "similarly , both adiposity indicators were associated with diastolic blood pressure more strongly among high - fatness students ( 0.230.32 ; p < 0.001 for each of 4 beta coefficients ) than among lower - fatness students ( 0.030.05 ; p > 0.05 for each of four beta coefficients ) . for identification of lipid outcomes , however , we found steeper beta coefficients only among high - fatness boys ( compared to lower - fatness boys ) whose adiposity was assessed by bmiz . for both sexes assessed by whtr ,", "the associations between adiposity indicators and risk variables were not notably different between the hispanics , non - hispanic blacks , and non - hispanic whites except when related to blood pressure outcomes .", "although adiposity explained 5%7% of variation in diastolic blood pressure in the complete sample of high - fatness girls ( table 3 ) , this relationship was extremely weak for high - fatness girls who were black , as indicated by slope point estimates close to zero ( figure 1 ) .", "however , for high - fatness girls who were hispanic or white , bmiz and whtr had significant associations ( p < 0.05 ) with diastolic pressure . among high - fatness boys", "systolic blood pressure was not significantly associated with bmiz or whtr for high - fatness blacks of either sex , but the association was present for high - fatness students who were hispanic or white .", "an ancestral contrast ( blacks compared to whites ) related to systolic blood pressure was significant , however , only among the high - fatness girls assessed by bmiz ( figure 1 ; p < 0.01 ) ."], ["in this large sample of middle - school students at increased risk of obesity and type 2 diabetes , we found that adiposity indicators bmiz ( with reference to cdc 2000 growth charts ) and whtr ( without reference to sex and age ) had similar utility for identifying adverse levels of cardiometabolic variables .", "our findings are generally consistent with previous published reports , most of which were based on populations that had a wider age range or included less ancestral diversity .", "earlier cross - sectional studies that compared continuous whtr against bmi either without a normative growth reference [ 3335 ] or with reference - based bmi z - scores / percentile ranks [ 23 , 36 ] generally found that whtr provided stronger associations with lipid outcomes , but bmi was superior for blood pressure outcomes .", "a recent report on sixth - grade students from switzerland found that bmiz ( referenced to cdc 2000 growth charts ) and whtr provided associations with blood pressures that were weak but nearly identical . in studies of youth from the southern us , bmiz provided a slightly stronger association than whtr with homa - ir   and fasting insulin , a relationship that was complicated by nonlinearity .", "nationally representative , cross - sectional data from the us demonstrated that whtr at ages 417 years was more strongly associated than bmiz with resting heart rate . a longitudinal analysis from the same survey of adolescent and young - adult participants found that categorical whtr predicted all - cause mortality before age 55 better than categorical bmiz ( baseline ages 1218 years ) or bmi ( ages 1939 ) . from the united kingdom ,", "a large study recently reported that whtr and bmiz obtained at ages 79 years had similar associations with cross - sectional and prospective cardiometabolic risk factors in adolescence . given an approximately equal utility of bmiz and whtr for pediatric health assessments , we should consider how these adiposity indicators might perform in different settings or in the future .", "bmiz values reported in the research literature depend on standardized protocols for measuring height and weight using calibrated , high - quality scales . in nonresearch settings , however , staff training and time pressures might not be so favorable to careful measurements .", "the dependence of bmiz on normative growth references can be problematic because bmi - for - age reference values can yield discrepant inferences between populations , time periods , and ethnicities within a single country .", "worldwide  bmi growth reference based on large datasets from six countries , but subsequent reviews found that this international growth reference provided no advantage over national bmi growth references for the definition of excessive fat mass in youth   or prediction of subsequent cardiovascular risk in adulthood .", "the world health organization ( who ) more recently developed a bmi - based growth reference , the utilization of which has been described as a cumbersome task in need of simplification .", "surveys from various clinical settings have found generally that the use of bmi - for - age reference values is suboptimal [ 4750 ] .", "advocates of the whtr must address problems associated with the available protocols for measuring waist size .", "while tape measures are inexpensive and generally need little calibration , protocols for circumference measurement are still unfamiliar to many pediatric practitioners or clinic assistants .", "our study carefully measured the waist circumference just above the iliac crest , an anatomic location endorsed by prominent researchers in the united states [ 18 , 51 ] and canada . the who , however , recommends measuring waist circumference at the approximate midpoint between the lower margin of the last palpable rib and the top of the iliac crest .", "minimal waist ,  and other sites [ 54 , 55 ] . in an anthropometric study of diabetic youth , the iliac - crest protocol and", "who protocol demonstrated comparable reproducibility , but these alternative protocols yielded notable differences in the absolute value of a waist circumference obtained from the same participants .", "a study of overweight youth found that the who waist - circumference protocol had a stronger association than the iliac - crest protocol with cardiometabolic risk , and studies of adult waist circumference have likewise suggested an advantage for the who protocol [ 5860 ] .", "it follows that the whtr values calculated from the iliac - crest and who protocols should not be casually substituted for each other .", "it is possible that if our healthy study had adopted the who instead of the iliac - crest protocol for its baseline anthropometry , the re - calculated whtr indicator might have demonstrated stronger associations with cardiometabolic risk variables than those we report in this paper .", "standardization of a single waist - circumference protocol would probably advance the widespread adoption of the whtr as a low - cost adiposity indicator [ 55 , 61 ] . as an alternative to the circumference ,", "some pediatric investigators have described waist size in selected participants by measuring the external diameter sagittally ( back - to - front ) in the supine position [ 6264 ] .", "protocol might further enhance studies that are cross - sectional or involve short - term follow - up of central adiposity , but this anthropometric method needs to be tested in larger datasets that represent general youth populations . the physiological importance of tissues accumulated in the waist may help to explain why whtr was more closely associated than bmiz with variations in the levels of circulating lipid markers among our lower - fatness participants ( table 4 ) .", "an increase in waist size primarily marks expanding amounts of adipose tissue , including notably the visceral depot which is most strongly associated with an adverse metabolic phenotype [ 5 , 65 ] .", "variation in the waist circumference can explain more than 64% of the variance in the area   or volume   of visceral adipose tissue .", "an increase in bmi , by contrast , may substantially mark also the variations in gain of muscular weight or subcutaneous fat patterning that precede adulthood . along with the changes in fat mass ,", "these variations in lean mass or superficial adipose tissue contribute to the bmi calculation while contributing relatively little to metabolic risk .", "although high - fatness hispanic and white girls in our study demonstrated the expected associations between adiposity and blood pressure , we found among the high - fatness black girls ( but not black boys ) that neither bmiz nor whtr had a significant association with blood pressure outcomes ( figure 1 ) .  in comparison to young hispanics and whites ,", "young blacks tend to have abdominal adipose tissue relatively less in the visceral depot and more in the abdominal subcutaneous regions .", "another study has also reported that black girls ' waist circumference around the same age was unrelated to their diastolic blood pressure .", "since both the bmiz and whtr indicators demonstrated similar patterns of nonassociation with black girls ' blood pressure , it may be that this absence of a correlation with blood pressure is due to factors operating primarily outside the abdominal region .", "perhaps black girls benefit from an increased capacity to expand their lower - body ( gluteofemoral ) , subcutaneous , adipose - tissue stores in a manner that would increase their total body weight yet protect them from cardiometabolic risk [ 7073 ] .", "if this protective characteristic of black girls extends into their later years , it could help explain why adult black women in the us experience no increased cardiometabolic risk   or mortality   until their bmi reaches approximately 33  kg / m ."], ["if a well - standardized waist measurement comes into widespread use for clinical assessments of pediatric adiposity , patients and their families may improve their intuitive understanding of how excess adiposity contributes to adverse health risk .", "pediatric health care providers , too , may find it more useful to recognize risk associated with a waist increment ( corrected for height ) than with a weight increment ( corrected for height squared ) .", "adoption of the whtr could optimize both patient education and the tracking of risk . compared to bmi ,", "the whtr allows a simpler calculation without the necessity of squaring the child 's height . of interest to those concerned with child adiposity and", "cardiometabolic risk observed in different cultures or distinct time periods , the whtr will facilitate comparisons based directly on anthropometric observations without using normative reference tables that may not be suitable to all populations [ 7779 ] ."]]}
{"article_id": "PMC2894144", "article_text": ["the bucky shuttle   being the combination of nanosize carbon structures  fullerene   and nanotube , has many possible applications : nanoscale storage cells , devices for directed medicine transfer   and containers for effective and safe gas storage [ 7 - 13 ] .", "nanosize containers and capsules of various shapes that allow reaching a higher safety level and mass content of gas stored have been investigated for a number of years [ 11 - 13 ] .", "the engineering of nanostructured carbon opens the ways for the production of nanocapsules of complex structural shapes [ 14 - 16 ] . in this work", ", the processes of methane molecule adsorption , storage and desorption by the nanocapsule are investigated with molecular - dynamic modeling method .", "the nanocapsule - specific structure defines its adsorption qualities : at the storage stage under normal conditions , the nanocapsule contains the amount of methane that was adsorbed at normal temperature and under 40 mpa .", "the nanocapsule desorption takes place at the temperature elevation up to 350 k. there is no need to apply electric field during storage and desorption .", "methane adsorption , storage and desorption processes were modeled with the method of molecular dynamics .", "the values of hydrogen and carbon atom charges in methane molecule were obtained   using the combination of hartree  fock and becke exchange with lee  yang ", "the following atom charge values in methane molecule were obtained : carbon atom 0.628204 mulliken and hydrogen atom + 0.157051 mulliken .", "1 . nanocapsule for natural gas storage , consisting of storage chamber , junction and blocking chamber the nanocapsule consists of three parts : storage chamber , junction and blocking chamber .", "the junction consists of the nanotube ( 10,10 ) and nanotube ( 8,8 ) , moreover , the nanotube ( 8,8 ) is connected with the storage chamber , and nanotube ( 10,10 ) with the blocking chamber .", "the blocking chamber is opened and closed by the transfer of the k@c60 endohedral complex under electrostatic field action .", "the charge of + 1|e| of the k@c60 endohedral complex is uniformly distributed over the c60 shell .", "the nanotube ( 8,8 ) in the junction prevents the k@c60 from entering the storage chamber .", "the nanotube ( 8,8 ) diameter is rather large for the penetration of methane molecules , but small for the transition of the k@c60 .", "each hole in the blocking chamber is formed as a result of removing 24 carbon atoms . dangling", "the holes obtained are large enough for free penetration of methane molecules into the nanotube internal space .", "the experiment on obtaining similar holes with the application of electron beams is described in .", "it is shown that the beams can be focused on the area 1  in diameter .", "the holes in the nanotube can exist at the temperatures up to 400 k ; when the temperature elevates , the hole diameter in the nanotubes considerably decreases due to the motion and fusion of single vacancies [ 8 - 10 ] . during modeling", ", it is imitated that the nanocapsule is placed on the substrate , i.e. , the nanotube base is fixed  the nanocapsule left end", "the change in the nanotube diameter is also possible with the methods of nanostructural engineering .", "the charged endohedral complex k@c60 moves in the blocking chamber and junction under the action of external electric field .", "the electric field direction defines the nanocapsule state in the operation cycle : methane adsorption , its storage and desorption .", "the value of external electric field intensity , required for the k@c60 to move , equals 3.045  10 v / m .", "the motion of charged fullerene in the nanotube with the help of electric field is described in detail in .", "the nanocapsule operation can be split into several stages : methane adsorption , storage and desorption . at the adsorption stage ( fig .", "2 ) , the k@c60 endohedral complex is retained at the end of the blocking chamber under the action of van der waals forces . the methane molecules from the environment", "freely penetrate through the three holes into the blocking chamber and adsorb into the external space of the storage chamber .", "adsorption stage ( t= 300 k , p = 40 mpa ) the calculations are based on the following formula ( 1 ) : where  number of methane molecules , nc  number of carbon atoms in the nanotube , mass of one methane molecule and mc  mass of one carbon atom .", "figure 3 demonstrates the nanocapsule filling dynamics with methane at 300 k and under 40 mpa .", "it is clearly seen that 4 ps is enough to complete the filling of the nanocapsule storage area .", "thermodynamic conditions are p = 40 mpa and t = 300 k to block methane molecules in the storage chamber , it is necessary to move the k@c60 ion into the junction with the help of electric field as shown in fig .", "4 . external thermodynamic conditions are t = 300 k and p = 40 mpa .", "further motion of the k@c60 is blocked by the junction narrow part  nanotube ( 8,8 ) .", "nanocapsule closing stage ( t = 300 k , p = 40 mpa ) figure 5 demonstrates the dependencies of the change in the k@c60 position and its kinetic energy under the electric field action in the blocking chamber upon time . a sharp increase in the k@c60 kinetic energy equaled to 0.254 ev", "is observed at 0.5 ps , at the same time the velocity reaches 327 m / s .", "= 1 ps , the k@c60 kinetic energy decreases significantly , reaching 0.000255 ev and further up to 15 ps its value does not considerably increase .", "this is explained by minor oscillations of the k@c60 near the right end of the blocking chamber .", "the k@c60 is retained due to van der waals forces . under the constant action of electric field ,", "the k@c60 kinetic energy increases again and reaches 0.61 ev at t = 16.5 ps , i.e. , the k@c60 breaks off the blocking chamber and moves to the storage chamber to block its entrance .", "results of molecular - dynamic modeling of the k@c60 motion in the blocking chamber under the electric field action at the adsorption stage .", "the k@c60 ion position with respect to the initial ( time = 0 ps ) ion position as a function of time .", "the change in the k@c60 ion kinetic energy as a function of time in the time period from t = 16.5 ps to t = 26.5 ps , the considerable attenuating oscillations of the kinetic energy conditioned by the k@c60 motion along the blocking chamber walls adjacent to the junction entrance are observed . in this time period , each peak of the k@c60 kinetic energy corresponds to the time moments after passing the pentagonal rings in the structure of the blocking chamber . under the electric field action", ", the k@c60 penetrates into the right end of the junction  nanotube ( 10,10)and blocks the outlet of methane molecules from the storage chamber . during the penetration , a considerable increase in the kinetic energy is observed , its maximum value reaches 0.63 ev at t = 32 ps .", "after the k@c60 passes the nanotube ( 10,10 ) , the kinetic energy sharply decreases conditioned by the k@c60 deceleration in the portion of the nanotube ( 8,8 ) . in the interval from t = 41.5 ps to t = 48.5 ps , the insignificant fluctuations of the k@c60 position connected with the compressed gas pressure from one side and electric field action from another", "the value of the k@c60 kinetic energy does not exceed 0.033 ev . in the process of methane molecules adsorption ,", "the maximum velocity of the k@c60 motion is 515.5 m / s ( t = 32 ps ) .", "when transferring to the storage stage , the electric field switches off , and external thermodynamic conditions are brought to normal .", "the k@c60 , which is located in the junction , moves to the blocking chamber under the methane pressure .", "however , its transfer is insignificant ( d~5 a ) that is conditioned by the necessity to overcome the considerable energy barrier ( e = 90 kcal / mol ) to move to the blocking chamber .", "the calculations made show that there are no abnormal extensions of bonds between carbon atoms in the nanocapsule .", "methane storage stage ( t = 300 k , p = 0.1 mpa ) at the desorption stage when the temperature elevates from 300 k up to 350 k and the external pressure is normal , the k@c60 endohedral complex in the junction is pushed into the blocking chamber under the expanding methane pressure , as shown in fig .", "methane under pressure in the storage chamber freely desorbs into the external space through the holes in the blocking chamber .", "the availability of three holes in the blocking chamber and their configuration allow forcing out methane molecules and preserving the required rigidity of the construction .", "the holes are located in such a way that the k@c60 , moving under the electric field action in the blocking chamber , is unable to block all its holes at once .", "the k@c60 in the blocking chamber does not prevent methane desorption as the gas molecules flowing out do not touch it , and it is retained near the wall of the blocking chamber adjacent to the junction inlet  nanotube ( 10,10 ) due to the action of van der waals forces without the external electric field involved .", "methane desorption stage ( t = 350 k , p = 0.1 mpa ) figure 8 demonstrates the dependencies of the k@c60 location stage and its kinetic energy upon time at the temperature elevation from t = 300 k up to t = 350 k at the stage of the k@c60 desorption into the blocking chamber . at 3 ps ,", "the significant increase in the k@c60 kinetic energy equaled to 2.05 ev ( the k@c60 velocity reaches 930 m / s ) is observed .", "the k@c60 kinetic energy is consumed for overcoming the considerable energy barrier ( e = 90 kcal / mol ) formed under the action of capillary forces . when the k@c60 passes the energy barrier , the kinetic energy decreases considerably to 0.41 ev ( t = 5 ps ) . after passing the junction at 7 ps the k@c60 , kinetic energy increases again and", "when the k@c60 gets into the blocking chamber , the oscillations between its walls are observed .", "each zero value of the k@c60 kinetic energy in the interval from 7 to 23 ps corresponds to the k@c60 impact against the blocking chamber wall .", "then , the insignificant impacts of the k@c60 against the blocking chamber wall with further state stabilization near one of the pentagonal rings of the blocking chamber are observed ( fig . 7 )", "the maximum kinetic energy is 2.05 ev , which is considerably smaller than the known value ( 200 ev ) required for carbon nanotube destruction .", "the availability of even insignificant number of methane molecules in the blocking chamber considerably decreases the velocity of the k@c60 motion , and , respectively , the kinetic energy of the k@c60 impact against the blocking chamber wall .", "results of molecular - dynamic modeling of the k@c60 transfer at the temperature elevation up to 350 k. k@c60 ion position with respect to the initial ( time = 0 ps . )", "the change in the k@c60 ion kinetic energy as a function of time the methane molecules desorption process is shown in fig .", "the desorption stops at 25.4 ps , there are 53 methane molecules in the storage chamber , which is 1.38 mass%", "we demonstrated the functioning of the nanocapsule of complex structural shape for methane storage using the method of molecular dynamics .", "an obvious advantage of the nanocapsule is its operation cycle : methane is adsorbed under the elevated pressure ( 40 mpa ) and at normal temperature with further blocking of methane molecules by the k@c60 endohedral complex in the nanocapsule with the external electric field , the storage is performed in normal external conditions , and methane desorption is performed at temperature elevation up to 350 k , at which methane molecules push out the k@c60 and are desorbed from the nanocapsule .", "methane content in the nanocapsule at the storage stage is ~11.09 mass% . at the storage and desorption stages ,", "the electric field is not used , this significantly simplifies the use of nanocapsules in automobile applications .", "the multiple techniques of nanostructural engineering developed are the prerequisites for the creation of similar nanocapsules .", "calculations are made in interdepartmental supercomputer center of the russian academy of science ( moscow ) ."], "abstract_text": ["<S> the processes of methane adsorption , storage and desorption by the nanocapsule are investigated with molecular - dynamic modeling method . </S>", "<S> the specific nanocapsule shape defines its functioning uniqueness : methane is adsorbed under 40 mpa and at normal temperature with further blocking of methane molecules the k@c601 + endohedral complex in the nanocapsule by external electric field , the storage is performed under normal external conditions , and methane desorption is performed at 350 k. the methane content in the nanocapsule during storage reaches 11.09 mass% . </S>", "<S> the nanocapsule consists of tree parts : storage chamber , junction and blocking chamber . </S>", "<S> the storage chamber comprises the nanotube ( 20,20 ) . </S>", "<S> the blocking chamber is a short nanotube ( 20,20 ) with three holes . </S>", "<S> the junction consists of the nanotube ( 10,10 ) and nanotube ( 8,8 ) ; moreover , the nanotube ( 8,8 ) is connected with the storage chamber and nanotube ( 10,10 ) with the blocking chamber . </S>", "<S> the blocking chamber is opened and closed by the transfer of the k@c601 + endohedral complex under electrostatic field action . </S>"], "labels": null, "section_names": ["Introduction", "Computational Model and Details", "Results and Discussion", "Conclusion", "Acknowledgments"], "sections": [["the bucky shuttle   being the combination of nanosize carbon structures  fullerene   and nanotube , has many possible applications : nanoscale storage cells , devices for directed medicine transfer   and containers for effective and safe gas storage [ 7 - 13 ] .", "nanosize containers and capsules of various shapes that allow reaching a higher safety level and mass content of gas stored have been investigated for a number of years [ 11 - 13 ] .", "the engineering of nanostructured carbon opens the ways for the production of nanocapsules of complex structural shapes [ 14 - 16 ] . in this work", ", the processes of methane molecule adsorption , storage and desorption by the nanocapsule are investigated with molecular - dynamic modeling method .", "the nanocapsule - specific structure defines its adsorption qualities : at the storage stage under normal conditions , the nanocapsule contains the amount of methane that was adsorbed at normal temperature and under 40 mpa .", "the nanocapsule desorption takes place at the temperature elevation up to 350 k. there is no need to apply electric field during storage and desorption ."], ["methane adsorption , storage and desorption processes were modeled with the method of molecular dynamics .", "the values of hydrogen and carbon atom charges in methane molecule were obtained   using the combination of hartree  fock and becke exchange with lee  yang ", "the following atom charge values in methane molecule were obtained : carbon atom 0.628204 mulliken and hydrogen atom + 0.157051 mulliken .", "1 . nanocapsule for natural gas storage , consisting of storage chamber , junction and blocking chamber the nanocapsule consists of three parts : storage chamber , junction and blocking chamber .", "the junction consists of the nanotube ( 10,10 ) and nanotube ( 8,8 ) , moreover , the nanotube ( 8,8 ) is connected with the storage chamber , and nanotube ( 10,10 ) with the blocking chamber .", "the blocking chamber is opened and closed by the transfer of the k@c60 endohedral complex under electrostatic field action .", "the charge of + 1|e| of the k@c60 endohedral complex is uniformly distributed over the c60 shell .", "the nanotube ( 8,8 ) in the junction prevents the k@c60 from entering the storage chamber .", "the nanotube ( 8,8 ) diameter is rather large for the penetration of methane molecules , but small for the transition of the k@c60 .", "each hole in the blocking chamber is formed as a result of removing 24 carbon atoms . dangling", "the holes obtained are large enough for free penetration of methane molecules into the nanotube internal space .", "the experiment on obtaining similar holes with the application of electron beams is described in .", "it is shown that the beams can be focused on the area 1  in diameter .", "the holes in the nanotube can exist at the temperatures up to 400 k ; when the temperature elevates , the hole diameter in the nanotubes considerably decreases due to the motion and fusion of single vacancies [ 8 - 10 ] . during modeling", ", it is imitated that the nanocapsule is placed on the substrate , i.e. , the nanotube base is fixed  the nanocapsule left end", "the change in the nanotube diameter is also possible with the methods of nanostructural engineering .", "the charged endohedral complex k@c60 moves in the blocking chamber and junction under the action of external electric field .", "the electric field direction defines the nanocapsule state in the operation cycle : methane adsorption , its storage and desorption .", "the value of external electric field intensity , required for the k@c60 to move , equals 3.045  10 v / m .", "the motion of charged fullerene in the nanotube with the help of electric field is described in detail in ."], ["the nanocapsule operation can be split into several stages : methane adsorption , storage and desorption . at the adsorption stage ( fig .", "2 ) , the k@c60 endohedral complex is retained at the end of the blocking chamber under the action of van der waals forces . the methane molecules from the environment", "freely penetrate through the three holes into the blocking chamber and adsorb into the external space of the storage chamber .", "adsorption stage ( t= 300 k , p = 40 mpa ) the calculations are based on the following formula ( 1 ) : where  number of methane molecules , nc  number of carbon atoms in the nanotube , mass of one methane molecule and mc  mass of one carbon atom .", "figure 3 demonstrates the nanocapsule filling dynamics with methane at 300 k and under 40 mpa .", "it is clearly seen that 4 ps is enough to complete the filling of the nanocapsule storage area .", "thermodynamic conditions are p = 40 mpa and t = 300 k to block methane molecules in the storage chamber , it is necessary to move the k@c60 ion into the junction with the help of electric field as shown in fig .", "4 . external thermodynamic conditions are t = 300 k and p = 40 mpa .", "further motion of the k@c60 is blocked by the junction narrow part  nanotube ( 8,8 ) .", "nanocapsule closing stage ( t = 300 k , p = 40 mpa ) figure 5 demonstrates the dependencies of the change in the k@c60 position and its kinetic energy under the electric field action in the blocking chamber upon time . a sharp increase in the k@c60 kinetic energy equaled to 0.254 ev", "is observed at 0.5 ps , at the same time the velocity reaches 327 m / s .", "= 1 ps , the k@c60 kinetic energy decreases significantly , reaching 0.000255 ev and further up to 15 ps its value does not considerably increase .", "this is explained by minor oscillations of the k@c60 near the right end of the blocking chamber .", "the k@c60 is retained due to van der waals forces . under the constant action of electric field ,", "the k@c60 kinetic energy increases again and reaches 0.61 ev at t = 16.5 ps , i.e. , the k@c60 breaks off the blocking chamber and moves to the storage chamber to block its entrance .", "results of molecular - dynamic modeling of the k@c60 motion in the blocking chamber under the electric field action at the adsorption stage .", "the k@c60 ion position with respect to the initial ( time = 0 ps ) ion position as a function of time .", "the change in the k@c60 ion kinetic energy as a function of time in the time period from t = 16.5 ps to t = 26.5 ps , the considerable attenuating oscillations of the kinetic energy conditioned by the k@c60 motion along the blocking chamber walls adjacent to the junction entrance are observed . in this time period , each peak of the k@c60 kinetic energy corresponds to the time moments after passing the pentagonal rings in the structure of the blocking chamber . under the electric field action", ", the k@c60 penetrates into the right end of the junction  nanotube ( 10,10)and blocks the outlet of methane molecules from the storage chamber . during the penetration , a considerable increase in the kinetic energy is observed , its maximum value reaches 0.63 ev at t = 32 ps .", "after the k@c60 passes the nanotube ( 10,10 ) , the kinetic energy sharply decreases conditioned by the k@c60 deceleration in the portion of the nanotube ( 8,8 ) . in the interval from t = 41.5 ps to t = 48.5 ps , the insignificant fluctuations of the k@c60 position connected with the compressed gas pressure from one side and electric field action from another", "the value of the k@c60 kinetic energy does not exceed 0.033 ev . in the process of methane molecules adsorption ,", "the maximum velocity of the k@c60 motion is 515.5 m / s ( t = 32 ps ) .", "when transferring to the storage stage , the electric field switches off , and external thermodynamic conditions are brought to normal .", "the k@c60 , which is located in the junction , moves to the blocking chamber under the methane pressure .", "however , its transfer is insignificant ( d~5 a ) that is conditioned by the necessity to overcome the considerable energy barrier ( e = 90 kcal / mol ) to move to the blocking chamber .", "the calculations made show that there are no abnormal extensions of bonds between carbon atoms in the nanocapsule .", "methane storage stage ( t = 300 k , p = 0.1 mpa ) at the desorption stage when the temperature elevates from 300 k up to 350 k and the external pressure is normal , the k@c60 endohedral complex in the junction is pushed into the blocking chamber under the expanding methane pressure , as shown in fig .", "methane under pressure in the storage chamber freely desorbs into the external space through the holes in the blocking chamber .", "the availability of three holes in the blocking chamber and their configuration allow forcing out methane molecules and preserving the required rigidity of the construction .", "the holes are located in such a way that the k@c60 , moving under the electric field action in the blocking chamber , is unable to block all its holes at once .", "the k@c60 in the blocking chamber does not prevent methane desorption as the gas molecules flowing out do not touch it , and it is retained near the wall of the blocking chamber adjacent to the junction inlet  nanotube ( 10,10 ) due to the action of van der waals forces without the external electric field involved .", "methane desorption stage ( t = 350 k , p = 0.1 mpa ) figure 8 demonstrates the dependencies of the k@c60 location stage and its kinetic energy upon time at the temperature elevation from t = 300 k up to t = 350 k at the stage of the k@c60 desorption into the blocking chamber . at 3 ps ,", "the significant increase in the k@c60 kinetic energy equaled to 2.05 ev ( the k@c60 velocity reaches 930 m / s ) is observed .", "the k@c60 kinetic energy is consumed for overcoming the considerable energy barrier ( e = 90 kcal / mol ) formed under the action of capillary forces . when the k@c60 passes the energy barrier , the kinetic energy decreases considerably to 0.41 ev ( t = 5 ps ) . after passing the junction at 7 ps the k@c60 , kinetic energy increases again and", "when the k@c60 gets into the blocking chamber , the oscillations between its walls are observed .", "each zero value of the k@c60 kinetic energy in the interval from 7 to 23 ps corresponds to the k@c60 impact against the blocking chamber wall .", "then , the insignificant impacts of the k@c60 against the blocking chamber wall with further state stabilization near one of the pentagonal rings of the blocking chamber are observed ( fig . 7 )", "the maximum kinetic energy is 2.05 ev , which is considerably smaller than the known value ( 200 ev ) required for carbon nanotube destruction .", "the availability of even insignificant number of methane molecules in the blocking chamber considerably decreases the velocity of the k@c60 motion , and , respectively , the kinetic energy of the k@c60 impact against the blocking chamber wall .", "results of molecular - dynamic modeling of the k@c60 transfer at the temperature elevation up to 350 k. k@c60 ion position with respect to the initial ( time = 0 ps . )", "the change in the k@c60 ion kinetic energy as a function of time the methane molecules desorption process is shown in fig .", "the desorption stops at 25.4 ps , there are 53 methane molecules in the storage chamber , which is 1.38 mass%"], ["we demonstrated the functioning of the nanocapsule of complex structural shape for methane storage using the method of molecular dynamics .", "an obvious advantage of the nanocapsule is its operation cycle : methane is adsorbed under the elevated pressure ( 40 mpa ) and at normal temperature with further blocking of methane molecules by the k@c60 endohedral complex in the nanocapsule with the external electric field , the storage is performed in normal external conditions , and methane desorption is performed at temperature elevation up to 350 k , at which methane molecules push out the k@c60 and are desorbed from the nanocapsule .", "methane content in the nanocapsule at the storage stage is ~11.09 mass% . at the storage and desorption stages ,", "the electric field is not used , this significantly simplifies the use of nanocapsules in automobile applications .", "the multiple techniques of nanostructural engineering developed are the prerequisites for the creation of similar nanocapsules ."], ["calculations are made in interdepartmental supercomputer center of the russian academy of science ( moscow ) ."]]}
{"article_id": "PMC2907009", "article_text": ["it is an unusual \" atypical \" antipsychotic , as the \" atypical \" profile of the new antipsychotics clozapine , olanzapine , quetiapine , and risperidone has been linked to combined antagonism of serotonin 2 ( 5-ht2 ) and dopamine 2 ( d2 ) receptors , whereas amisulpride has negligible affinity for 5 ht2 receptors and is specific for dopamine d2 and d3 receptors in the limbic rather than striatal structures .", "there are reports of amisulpride being used for the management of bipolar disorders . however , there are very few reports of mania induced by amisulpride . here", "we describe a young male who developed mania while on amisulpride , a newer antipsychotic drug introduced in india .", "a 18-year - old unmarried hindu male presented with a history suggestive of schizophrenia for the last 2 years .", "the illness had an acute onset with complaints of suspiciousness , hearing voices not heard by others , violent abusive behavior , disturbed biological functions , and decreased self care .", "he was started on risperidone 4 mg along with trihexyphenidyl 2 mg and lorazepam 6 mg per day .", "gradually , over a period of 1 month , he showed improvement in the symptoms .", "subsequently , trihexyphenidyl and lorazepam were tapered and he was maintained on risperidone 4 mg per day for the next 1 and a half year .", "however , he would still be lethargic , would prefer to remain alone and would be inattentive at class .", "he would not interact with others and not take active part in various household functions .", "he was prescribed amisulpride for the negative symptoms along with risperidone 4 mg that was continued .", "it was initiated at 50 mg per day and was increased to 100 mg per day after 4 days of initiation it .", "after 10 days of 100 mg dose , the patient developed a manic episode , characterized by decreased need for sleep , over talkativeness , hyperactivity , persistent elevated mood , disinhibited behavior , over - grooming , and distractibility .", "amisulpride was stopped and he was prescribed lorazepam 4 mg on as and when required basis .", "the patient was followed up after 1 week and during this time there was substantial improvement in his manic symptoms with ymrs score of 17 .", "to the best of our knowledge of mania due to first amisulpride was reported by murphy in 2003 . however , in the case described by murphy , the patient was also on citalopram , an antidepressant , though its discontinuation did not led to improvement in manic symptoms .", "also , the patient was initiated on olanzapine immediately which would also probably have antimanic effects . in our case ,", "use of the naranjo adverse drug reaction probability scale and edward 's criteria both indicate a probable relationship between the manic episode and short - term exposure to amisulpride therapy . the manic symptoms with amisulpride can be postulated to be due to the fact that amisulpride in low doses ( < 10 mg / kg ) , preferentially blocks presynaptic d2/d3 receptors , resulting in enhanced dopamine transmission . increased dopamine metabolites and increased dopamine transmission", "this case report highlights the fact that close monitoring of patients on amisulpiride should be done for manic symptoms .", "this is also important because of the fact that atypical antipsychotic drugs ( including amisulpride ) are used for the treatment for bipolar disorder .", "open label studies have shown that amisulpride may be useful for patients with bipolar disorder .", "also , as low doses enhance dopamine transmission , and high doses reduce dopamine transmission , it might be prudent to start amisulpride at the recommended target dose at the initiation of therapy ."], "abstract_text": ["<S> amisulpride is an atypical antipsychotic used for the management of schizophrenia and other conditions like dysthymia . </S>", "<S> it has also been used for the management of bipolar disorders as an add on therapy . here , </S>", "<S> we report a patient of schizophrenia who developed a manic episode while on amisulpride . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["it is an unusual \" atypical \" antipsychotic , as the \" atypical \" profile of the new antipsychotics clozapine , olanzapine , quetiapine , and risperidone has been linked to combined antagonism of serotonin 2 ( 5-ht2 ) and dopamine 2 ( d2 ) receptors , whereas amisulpride has negligible affinity for 5 ht2 receptors and is specific for dopamine d2 and d3 receptors in the limbic rather than striatal structures .", "there are reports of amisulpride being used for the management of bipolar disorders . however , there are very few reports of mania induced by amisulpride . here", "we describe a young male who developed mania while on amisulpride , a newer antipsychotic drug introduced in india ."], ["a 18-year - old unmarried hindu male presented with a history suggestive of schizophrenia for the last 2 years .", "the illness had an acute onset with complaints of suspiciousness , hearing voices not heard by others , violent abusive behavior , disturbed biological functions , and decreased self care .", "he was started on risperidone 4 mg along with trihexyphenidyl 2 mg and lorazepam 6 mg per day .", "gradually , over a period of 1 month , he showed improvement in the symptoms .", "subsequently , trihexyphenidyl and lorazepam were tapered and he was maintained on risperidone 4 mg per day for the next 1 and a half year .", "however , he would still be lethargic , would prefer to remain alone and would be inattentive at class .", "he would not interact with others and not take active part in various household functions .", "he was prescribed amisulpride for the negative symptoms along with risperidone 4 mg that was continued .", "it was initiated at 50 mg per day and was increased to 100 mg per day after 4 days of initiation it .", "after 10 days of 100 mg dose , the patient developed a manic episode , characterized by decreased need for sleep , over talkativeness , hyperactivity , persistent elevated mood , disinhibited behavior , over - grooming , and distractibility .", "amisulpride was stopped and he was prescribed lorazepam 4 mg on as and when required basis .", "the patient was followed up after 1 week and during this time there was substantial improvement in his manic symptoms with ymrs score of 17 ."], ["to the best of our knowledge of mania due to first amisulpride was reported by murphy in 2003 . however , in the case described by murphy , the patient was also on citalopram , an antidepressant , though its discontinuation did not led to improvement in manic symptoms .", "also , the patient was initiated on olanzapine immediately which would also probably have antimanic effects . in our case ,", "use of the naranjo adverse drug reaction probability scale and edward 's criteria both indicate a probable relationship between the manic episode and short - term exposure to amisulpride therapy . the manic symptoms with amisulpride can be postulated to be due to the fact that amisulpride in low doses ( < 10 mg / kg ) , preferentially blocks presynaptic d2/d3 receptors , resulting in enhanced dopamine transmission . increased dopamine metabolites and increased dopamine transmission", "this case report highlights the fact that close monitoring of patients on amisulpiride should be done for manic symptoms .", "this is also important because of the fact that atypical antipsychotic drugs ( including amisulpride ) are used for the treatment for bipolar disorder .", "open label studies have shown that amisulpride may be useful for patients with bipolar disorder .", "also , as low doses enhance dopamine transmission , and high doses reduce dopamine transmission , it might be prudent to start amisulpride at the recommended target dose at the initiation of therapy ."]]}
{"article_id": "PMC3628128", "article_text": ["aging leads to senile diseases , including alzheimer disease ( ad ) , which has become a serious medical and socioeconomic problem .", "therefore , progress in early diagnosis and effective treatment of ad is necessary , especially in the preliminary or early stages of the disease .", "most elderly people experience physiological memory disorders ; these disorders require a careful clinical assessment , as it is extremely difficult to delineate the line between physiology and pathology .", "thus , when disorders of memory and other cognitive functions go beyond the standards of age and educational level , but have not yet reached the standards of dementia , we diagnose mild cognitive impairment ( mci ) , which includes several states that either match the standard in its wide meaning , for example age - associated memory impairment ( aami ) , age - related cognitive decline ( arcd ) , or we diagnose pathology like mci , which , although clinically similar , has a different prognosis than aami and arcd [ 14 ] . currently , base on criteria established by the mayo clinic group , mci is define as a transitional state between normal aging and alzheimer s disease , in which memory impairment is greater than expected for age , but general cognitive function and daily living activities are preserved .", "people suffering from mci , especially amnestic form ( amci ) , are at risk for developing dementia , particularly alzheimer disease ( ad ) [ 35 ] . according to various reports", ", the percentage of conversion from amci into ad ranges from 1% to 25% per year , while the mean time of dementia progression from the mci diagnosis is 4.4 years . obviously , the early identification of patients at risk for alzheimer disease from among those with mci would facilitate proper treatment help to delay the symptoms of dementia .", "therefore , searching for methods that would help us detect a higher risk of dementia in those patients , as well as looking for objective methods of early diagnosis of patients with mci , seems reasonable .", "many reports confirm a typical repeatable topographical location of neuropathological lesions at various stages of ad , as described by braak et al and pantel et al . , while emphasizing early involvement of the limbic system , including the hippocampus and entorhinal cortex .", "earlier pathology in these strategic regions in patients with a higher risk of alzheimer - type dementia , as well as in certain patients with amnestic mci , seems likely .", "atrophy of the medial temporal lobe in mci patients with a positive correlation of atrophy level and a risk of conversion to ad , inadequate for age and observed in imaging examinations ( ct , mri ) , has been clearly demonstrated [ 813 ] .", "the level of atrophy is judged by descriptive , volumetric and planimetric methods , or indirectly with the use of linear measurements of particular fluid spaces , such as fissures or certain parts of the ventricular system . despite many years of experience in structural diagnosis of the dementia diseases", ", the use of functional methods seems to be very helpful . in spect and pet examinations", "we observe the lowering of perfusion and metabolism of glucose and oxygen in temporal and parietal areas in patients with possible ad , as well as in those with mci .", "however , these methods have certain limitations such as relatively low linear resolution , which hinders precise determination of the topography of the measuring ( this problem relates to the middle temporal structures and not to the hemispheres cortex ) . a wider clinical application of magnetic resonance spectroscopy ( mrs ) , described by bootomley as  a window for metabolism", "   enabled an intravital assessment of regional metabolic disorders in certain structures of the brain .", "there have been several reports on such dysfunctions in the limbic system in patients with alzheimer disease .", "a relatively small number of spectroscopic examinations have been carried out on patients with mci .", "some of these were presented by catani et al . , who located voxels in the white matter at the ventricular triangle level   and kantarci et al .   who examined the posterior part of the cingulate gyrus in patients with mci and ad . however , these are the locations where anatomopathologic changes , typical for ad , appear later than in the medial temporal lobe ( including hippocampal formation ) .", "many authors give up on spectroscopic examinations of hippocampal structures due to their technical limitations , such as the size of examined structures and topographical relations of the surrounding area .", "the problems mentioned most often are : difficulties with field homogeneity , artifacts from cranial base area , a disqualifying weakening of the signal - to - noise ratio , and the need to reduce the volume of interest ( voi ) .", "there have been reports on the technical capacities of such examinations in various brain diseases [ 1923 ] . according to available the available literature , in the majority of papers h1mrs in mci and ad metabolites were measured in the posterior cingulated gyrus , but parts of papers evaluated the concentration of metabolites in the bilateral hippocampi of mci patients .", "this study attempted to evaluate the regional metabolic disorders using h mrs within the frontal , external and medial temporal lobes in patients with mci , as a predictor of clinical deterioration to dementia based on clinical follow - up .", "this study attempted to evaluate the regional metabolic disorders using h mrs within the frontal , external and medial temporal lobes in patients with mci , as a predictor of clinical deterioration to dementia based on clinical follow - up .", "the examination was performed on a group of 31 randomly selected subjects ( 19 females and 12 males ) with mci diagnosed under the care of the department of neurodegenerative disorders , medical research centre , polish academy of science .", "all subjects underwent neurological and psychiatric tests , routine laboratory investigations and standard neuropsychological examinations .", "patients with serious cns injuries , alcoholic abuse , diabetes , and serious hepatic and renal dysfunctions were excluded .", "mci diagnosis was established by a team of specialists after analyzing all available information and test results .", "mri and h mrs examination was performed on a 1.5 t eclipse ( marconi medical systems , usa ) scanner .", "morphological mri examination of the brain was carried out in transverse planes , parallel to the longitudinal axis of the temporal lobe in se , fse and flair sequences , in t1- and t2-weighted images , and in the frontal plane perpendicular to the longitudinal axis of the temporal lobe in flair sequence .", "we investigated the extent of cerebral atrophy , especially in medial and external temporal lobes and in frontal structures , as well as the presence of cortical - subcortical hyperintensive focuses ( which equal angiogenic lesions ) in t2-weighted images and flair sequence .", "morphological examination enabled us to exclude other pathologies such as extensive ischemic lesions , tumors , paracerebral hematomas , and hydrocephalus , which might lead to cognitive disorders .", "changes like leukoaraiosis and/or small single angiogenic focuses of gliosis were not exclusion criteria for the study .", "the voi ( volume of interest ) was located in the frontal , medial and external temporal lobe regions , separately on each side . in case of medial temporal lobe covering the hippocampal formation ,", "it was divided into 2 areas  the anterior ( the topographical point of reference in the coronal plane / layer was the level of dens ) and the posterior ( the point of reference was the output of the middle cerebellar peduncles from the pons ) . both areas were then added , and final average data sets were used in further analysis . in cases of reduced volume of the hippocampal formation , the adjacent fluid spaces ( eg , the temporal horn of the lateral ventricle and the lateral part of the transverse fissure ) were also included into the voi .", "necessary corrections of voi location were applied to particular frontal cross - sections , as well as to the axial plane , and care was taken not to cover the osseous structures of the temporal bone pyramids . for every localization , the size of voi was 8 cm . additionally , each subject had frontal measurement taken .", "h mrs examination was carried out with a single - voxel method using press sequence .", "routine 3-impulse sequences of 90 , 180 , 180 degrees and double crusher impulse were used .", "the examination was performed with an automated standardization of the field in the entire encephalon / brain ( total shimming ) and in the examined sample ( local shimming ) . for water suppression ,", "spectra were recorded within the following parameters : te=35 ms , tr=1500 ms , thickness = 15 mm , signal averages = 192 .", "assignment of resonance lines of particular metabolites was based on n - acetylaspartate signal with chemical shift set to 2.0 ppm .", "the spectra were analyzed using the manufacturer - supplied software package for the mrs ( marconi ) . in some cases ,", "it was necessary to correct the phase manually in order to obtain a maximum of symmetrical signal of residual water and to maintain a proper baseline . relative concentration ratios of particular metabolites ", "n - acetylaspartate ( naa ) , choline ( cho ) , myoinositol ( mi ) , glutamine and glutamate ( glx )  were analyzed in reference to the signal of unsuppressed water signal , and also to the signal of creatine , considering its level as an inner standard .", "results analysis of clinical and biochemical data  age , mini - mental score ( mm - score ) , folic acid , vitamin b12 and homocysteine level  was performed including initial data of all subjects enrolled into the study .", "evaluation of relative concentration ratios of metabolites from all vois , localized within frontal , medial and external temporal lobes , symmetrical on both sides , were performed in each case .", "a comparison of some metabolic ratios of mrs in patients with mci , who on follow - up has stable disease ( sd ) , disease progression ( dp ) and conversion to ad , was performed .", "the statistical analysis was carried out using the statistica for windows 7.0 ( statsoft , ok , usa ) software package .", "the examination was performed on a group of 31 randomly selected subjects ( 19 females and 12 males ) with mci diagnosed under the care of the department of neurodegenerative disorders , medical research centre , polish academy of science .", "all subjects underwent neurological and psychiatric tests , routine laboratory investigations and standard neuropsychological examinations .", "patients with serious cns injuries , alcoholic abuse , diabetes , and serious hepatic and renal dysfunctions were excluded .", "mci diagnosis was established by a team of specialists after analyzing all available information and test results .", "mri and h mrs examination was performed on a 1.5 t eclipse ( marconi medical systems , usa ) scanner .", "morphological mri examination of the brain was carried out in transverse planes , parallel to the longitudinal axis of the temporal lobe in se , fse and flair sequences , in t1- and t2-weighted images , and in the frontal plane perpendicular to the longitudinal axis of the temporal lobe in flair sequence .", "we investigated the extent of cerebral atrophy , especially in medial and external temporal lobes and in frontal structures , as well as the presence of cortical - subcortical hyperintensive focuses ( which equal angiogenic lesions ) in t2-weighted images and flair sequence .", "morphological examination enabled us to exclude other pathologies such as extensive ischemic lesions , tumors , paracerebral hematomas , and hydrocephalus , which might lead to cognitive disorders .", "changes like leukoaraiosis and/or small single angiogenic focuses of gliosis were not exclusion criteria for the study .", "the voi ( volume of interest ) was located in the frontal , medial and external temporal lobe regions , separately on each side . in case of medial temporal lobe covering the hippocampal formation ,", "it was divided into 2 areas  the anterior ( the topographical point of reference in the coronal plane / layer was the level of dens ) and the posterior ( the point of reference was the output of the middle cerebellar peduncles from the pons ) . both areas were then added , and final average data sets were used in further analysis . in cases of reduced volume of the hippocampal formation , the adjacent fluid spaces ( eg , the temporal horn of the lateral ventricle and the lateral part of the transverse fissure ) were also included into the voi .", "necessary corrections of voi location were applied to particular frontal cross - sections , as well as to the axial plane , and care was taken not to cover the osseous structures of the temporal bone pyramids . for every localization , the size of voi was 8 cm .", "h mrs examination was carried out with a single - voxel method using press sequence .", "routine 3-impulse sequences of 90 , 180 , 180 degrees and double crusher impulse were used .", "the examination was performed with an automated standardization of the field in the entire encephalon / brain ( total shimming ) and in the examined sample ( local shimming ) . for water suppression ,", "spectra were recorded within the following parameters : te=35 ms , tr=1500 ms , thickness = 15 mm , signal averages = 192 .", "assignment of resonance lines of particular metabolites was based on n - acetylaspartate signal with chemical shift set to 2.0 ppm .", "the spectra were analyzed using the manufacturer - supplied software package for the mrs ( marconi ) . in some cases ,", "it was necessary to correct the phase manually in order to obtain a maximum of symmetrical signal of residual water and to maintain a proper baseline . relative concentration ratios of particular metabolites ", "n - acetylaspartate ( naa ) , choline ( cho ) , myoinositol ( mi ) , glutamine and glutamate ( glx )  were analyzed in reference to the signal of unsuppressed water signal , and also to the signal of creatine , considering its level as an inner standard .", "results analysis of clinical and biochemical data  age , mini - mental score ( mm - score ) , folic acid , vitamin b12 and homocysteine level  was performed including initial data of all subjects enrolled into the study .", "evaluation of relative concentration ratios of metabolites from all vois , localized within frontal , medial and external temporal lobes , symmetrical on both sides , were performed in each case .", "a comparison of some metabolic ratios of mrs in patients with mci , who on follow - up has stable disease ( sd ) , disease progression ( dp ) and conversion to ad , was performed .", "the statistical analysis was carried out using the statistica for windows 7.0 ( statsoft , ok , usa ) software package .", "all patients had annual clinical follow - up at least twice . at the beginning of the study , subjects were divided into 2 groups  9 subjects who had amnestic mci , and the others who had multidomain mci .", "there were no statistical differences between groups of patients with multidomain mci and amnestic mci , including age , m - m score , level of vitamin b12 , folic acid and homocysteine ( p>0.05 ) .", "comparison of mean value in some selected parameters including both groups is presented in table 1 . during clinical follow - up", "( median 3 years ) 8 subjects had stable disease ( sd ) , 13 had progression of disease ( dp ) and 10 developed ad .", "there was no significant difference between age , m - m score , vitamin b12 , folic acid and homocysteine level among these 3 groups of subjects ( p>0.05 ) .", "statistical analyses of baseline metabolic ratios measurement using h mrs between the final 3 groups of patients , ( sd , dp and ad ) found significant difference in frontal lobes in mi / h20 ratio on left , between patients with stable disease ( 0.27 ) and those with progression ( 0.22 ) ( p=0.03 ) ( figure 1 ) . in the groups of patients with dp and those with conversion to ad", ", there was a significant difference on the left side in ratio naa / cr ( 1.77 vs. 1.43 ) , ( p=0.02 ) ( figure 2 ) .", "significant difference within temporal external lobes were found between patients with sd and dp in naa / h2o ratio on the left side ( 0.55 vs. 0.51 ) , ( p=0.04 ) ( figure 3 ) .", "additionally , there were significant differences between patients with sd and ad in chol / cr ratio ( 0.99 vs. 0.88 ) on the right side ( p=0.04 ) and in mi / cr ( 1.09 vs. 0.62 ) on the left side ( p=0.03 ) .", "a significant difference within the medial temporal lobe was found between patients with dp and ad in glx / h2o ratio ( 0.44 vs. 0.34 ) on the right side ( m - w u - test , p=0.02 ) . there were no other significant differences within h mrs metabolic ratios .", "an example of h mrs study in patient with initial diagnosis of mci and clinical deterioration during 2 years of follow - up with final diagnosis of ad is presented in ( figure 4 ) ( a  initial h mrs , and after final diagnosis , control h mrs study ", "b ) . summary of initial h mrs ratios of some metabolites in frontal , external and medial temporal lobes both sides are presented in table 3 .", "proton magnetic resonance spectroscopy  h mrs  enables an in vivo noninvasive assessment of the degree of biochemical disorders in a specified voi of the examined tissue , as well as monitoring the progress of those changes in the course of the disease and its treatment [ 5,1725 ] .", "the method we used , along with the parameters mentioned above , resulted in satisfactory quality of spectra in examined patients .", "recent studies have demonstrated the value of h1mrs in many types of cognitive disorders , not only in degenerative diseases . in mrs diagnosis ,", "normal tissues present a constant proton spectrum , while ratio changes of the metabolites can be considered as reflections of certain biochemical transformations .", "several cytological and histochemical investigations have contributed to the study of the relationship between certain chemical substances and precisely defined intracellular structures or biochemical processes  physiological as well as pathological .", "n - acetylaspartate is commonly considered as a neuronal marker due to its presence only in mature nerve cells .", "there are several reports of age - dependent lowering of naa level in certain parts of the encephalon , which is being interpreted not only as an effect of a progressive decrease in number of neurons , but also as their dysfunction .", "it also seems clear that there is a lowering of the naa concentration in atrophic structures of the limbic system in patients with ad , and a reverse correlation of the level of this metabolite with the level of dementia .", "however , the h mrs examinations of the posterior part of the cingulate gyrus ( frontal lobe ) in patients with mci , carried out by the rochester group , did not prove statistically significant differences of the naa ratios as compared to the control group .", "in our study , we found a significant difference between the ratio of naa / cr between patients with ds vs. dp in the left frontal lobe .", "we also found significant difference in naa / h2o between patients with ds and dp in the left external temporal lobe .", "we could not find any significant difference between naa / cr and naa / h2o within the medial temporal lobe on any sides .", "this could due to the frequent technical difficulties mentioned before , as measurement of the medial temporal lobe ( hippocampus area ) is often inconvenient .", "the neuronal integrity marker naa / cr or naa / h2o ratio ( independent of creatine level ) is currently declined in patients with progressive mci and those with conversion to ad compared to cognitively normal elderly subjects .", "our findings generally agree with others reports that found lower levels of naa in patients with mci and deterioration of brain function and who convert to ad .", "others reports indicated the left side more often has a drop in naa - rich neurons compare to the right side [ 2026 ] .", "medial temporal lobes ( hippocampus structures ) are widely considered as the region in which the earliest ad pathologies occur .", "therefore it seems that the lowering of naa concentration in this localization in patients with mci proves the theory of  successive  metabolic disorders in progression of dementia , according to which a lower concentration of myoinositol precedes the decline in naa concentration [ 2124 ] . in many reports , naa depletion seems to be good marker , as in metastasio et al .", "single reports indicated that the significant reduction of naa has great predictive value within the occipital area in those based on roc with threshold level < 1.61 of naa / cr ratio ; other regions and others ratios were not significant in this study . despite this inhomogeneous data", ", numerous studies have shown that naa plays an important role in neuronal integrity , with the left side more often affected in patients with progressive mci and in those with conversion to ad [ 1826 ] .", "some studies have found an elevated level of mi / cr , with good correlation of disease progression and increase of neurofibrillary tangles in patients with conversion into ad . in our study", "we did not find any of these findings ; on the contrary , we detected some depletion of mi / cr in patients with conversion to ad compared to those with stable disease , with a significant difference on the left external temporal lobe ( 1.09 vs. 0.62 ) .", "we found the same tendency in mi / h2o ratio on the left frontal lobe between patients with disease stability and disease progression .", "others reported differences between patients with mci and ad , with significantly higher signal ratio of mi / cr in ad .", "others publications did not report any difference in this metabolite ratio in the group of patients with mci .", "the differences in results of various groups of researchers could be explained by selection of the study population .", "the same research group indicated different results , dependent on study group  for instance , no difference was found in selected population of patients with amnestic mci   compare to a heterogenous population with significant difference in mi signal .", "some reports indicated that an elevated level of this metabolite in structures of the limbic system in patients with mci or dementia is connected with regional gliosis .", "this theory is supported by the presence of visibly higher mi concentrations in glia cells than in neurocytes . however , thus far the exact role of mi in mci and ad is unclear .", "there are many other disorders of the brain with mi disturbance , so specificity of this marker and ratio of mi / cr or mi / h2o seems to be low .", "recent data suggests good discrimination using mi / cr ratio in different types of dementia using roc curve .", "additional theory posits an extensive cellular capture of mi in patients at risk of dementia as the result of na / mi osmoregulator dysfunction .", "the study of adults with down syndrome , who constitute a clinically exceptional group of subjects with 100% risk of dementia similar to the alzheimer s disease , demonstrated a greater that 50% rise of the myoinositol level with age compared to the healthy control group .", "the authors associated this with an elevated activity of sodium - myoinositol transporter/ transmitter , which results from the existence of the additional 21 chromosome in down syndrome adults , which includes the gene - encoding protein of the transmitter . in our study , significant differences in mi / cr and mi / h2o were found in frontal and external temporal lobes , but not in the medial temporal lobe .", "our h mrs study shows a significant difference between chol / cr ratio only in the external temporal lobe on the left side , with a significant drop in the chol / cr ratio between patients with stable disease and ad .", "this result agrees with metastasio et al . , who showed a decreased , but not significant , depletion of chol / cr in mci patients with disease progression compare to those with stable disease in both hemispheres .", "this could be related to cholinergic neuronal damage , which is indirectly confirmed by increased activity of choline acetyltransferase ( chat ) , and which probably represents a compensatory ( but insufficient ) response in ad .", "impairment of mitochondrial activity seen in elderly patients , and more obviously in patients with ad , could be related to increase of mitochondrial membrane damage and evaluated level of membrane phospholipid   and decrease in phosphatidyl - choline and phosphatidylethanolamine described previously . in our study , significant difference in glutamine and glutamate ( glx ) ratio was noted only on the medial temporal lobe on the right side , which indicates disturbance of glx .", "this finding agrees with data presented by kantarci et al . , who found a trend toward decreased glu + gln / cr ratios from normal to mci to ad , but there were no statistically significant differences .", "detailed analysis of glutamine and glutamate in mci and ad could be explored using the new 3 t system , which currently is commercially available . in the medial temporal lobe h mrs", "has several limitations that could influence our results and our interpretation . in view of the complicated spatial shape of the medial temporal - hippocampal formation ( which is considered to be the region first changed by the progression of dementia pathology ) and its relatively small size , our volume of interest also covered other tissue structures outside the target structure , which could have caused partial falsification of the results as to its volume effect   on the other hand , these structures belong to the limbic system , and show pathological changes in the subsequent stages of the disease .", "according to reports in the literature , including the adjacent fluid spaces ( temporal horn of the lateral ventricle , lateral part of the transverse fissure ) into the voi should not affect the results .", "some researchers claim that chemical shift imaging ( csi ) , also known as single voxel spectroscopy ( svs ) , which allows recording spectra from many neighboring voxels within the examined area , is better for assessment of metabolic disorders within the hippocampus because it enables a more precise measurement .", "this theory was not corroborated in comparative studies of both of the methods used on a group of patients with temporal lobe epilepsy .", "n - acetylaspartate is commonly considered as a neuronal marker due to its presence only in mature nerve cells .", "there are several reports of age - dependent lowering of naa level in certain parts of the encephalon , which is being interpreted not only as an effect of a progressive decrease in number of neurons , but also as their dysfunction .", "it also seems clear that there is a lowering of the naa concentration in atrophic structures of the limbic system in patients with ad , and a reverse correlation of the level of this metabolite with the level of dementia .", "however , the h mrs examinations of the posterior part of the cingulate gyrus ( frontal lobe ) in patients with mci , carried out by the rochester group , did not prove statistically significant differences of the naa ratios as compared to the control group . in our study", ", we found a significant difference between the ratio of naa / cr between patients with ds vs. dp in the left frontal lobe .", "we also found significant difference in naa / h2o between patients with ds and dp in the left external temporal lobe .", "we could not find any significant difference between naa / cr and naa / h2o within the medial temporal lobe on any sides .", "this could due to the frequent technical difficulties mentioned before , as measurement of the medial temporal lobe ( hippocampus area ) is often inconvenient .", "the neuronal integrity marker naa / cr or naa / h2o ratio ( independent of creatine level ) is currently declined in patients with progressive mci and those with conversion to ad compared to cognitively normal elderly subjects .", "our findings generally agree with others reports that found lower levels of naa in patients with mci and deterioration of brain function and who convert to ad .", "others reports indicated the left side more often has a drop in naa - rich neurons compare to the right side [ 2026 ] .", "medial temporal lobes ( hippocampus structures ) are widely considered as the region in which the earliest ad pathologies occur .", "therefore it seems that the lowering of naa concentration in this localization in patients with mci proves the theory of  successive  metabolic disorders in progression of dementia , according to which a lower concentration of myoinositol precedes the decline in naa concentration [ 2124 ] . in many reports , naa depletion seems to be good marker , as in metastasio et al .", "single reports indicated that the significant reduction of naa has great predictive value within the occipital area in those based on roc with threshold level < 1.61 of naa / cr ratio ; other regions and others ratios were not significant in this study . despite this inhomogeneous data", ", numerous studies have shown that naa plays an important role in neuronal integrity , with the left side more often affected in patients with progressive mci and in those with conversion to ad [ 1826 ] .", "some studies have found an elevated level of mi / cr , with good correlation of disease progression and increase of neurofibrillary tangles in patients with conversion into ad . in our study", "we did not find any of these findings ; on the contrary , we detected some depletion of mi / cr in patients with conversion to ad compared to those with stable disease , with a significant difference on the left external temporal lobe ( 1.09 vs. 0.62 ) .", "we found the same tendency in mi / h2o ratio on the left frontal lobe between patients with disease stability and disease progression .", "others reported differences between patients with mci and ad , with significantly higher signal ratio of mi / cr in ad .", "others publications did not report any difference in this metabolite ratio in the group of patients with mci .", "the differences in results of various groups of researchers could be explained by selection of the study population .", "the same research group indicated different results , dependent on study group  for instance , no difference was found in selected population of patients with amnestic mci   compare to a heterogenous population with significant difference in mi signal .", "some reports indicated that an elevated level of this metabolite in structures of the limbic system in patients with mci or dementia is connected with regional gliosis .", "this theory is supported by the presence of visibly higher mi concentrations in glia cells than in neurocytes . however , thus far the exact role of mi in mci and ad is unclear .", "there are many other disorders of the brain with mi disturbance , so specificity of this marker and ratio of mi / cr or mi / h2o seems to be low .", "recent data suggests good discrimination using mi / cr ratio in different types of dementia using roc curve .", "additional theory posits an extensive cellular capture of mi in patients at risk of dementia as the result of na / mi osmoregulator dysfunction .", "the study of adults with down syndrome , who constitute a clinically exceptional group of subjects with 100% risk of dementia similar to the alzheimer s disease , demonstrated a greater that 50% rise of the myoinositol level with age compared to the healthy control group .", "the authors associated this with an elevated activity of sodium - myoinositol transporter/ transmitter , which results from the existence of the additional 21 chromosome in down syndrome adults , which includes the gene - encoding protein of the transmitter . in our study , significant differences in mi / cr and mi / h2o were found in frontal and external temporal lobes , but not in the medial temporal lobe .", "our h mrs study shows a significant difference between chol / cr ratio only in the external temporal lobe on the left side , with a significant drop in the chol / cr ratio between patients with stable disease and ad .", "this result agrees with metastasio et al . , who showed a decreased , but not significant , depletion of chol / cr in mci patients with disease progression compare to those with stable disease in both hemispheres .", "this could be related to cholinergic neuronal damage , which is indirectly confirmed by increased activity of choline acetyltransferase ( chat ) , and which probably represents a compensatory ( but insufficient ) response in ad .", "impairment of mitochondrial activity seen in elderly patients , and more obviously in patients with ad , could be related to increase of mitochondrial membrane damage and evaluated level of membrane phospholipid   and decrease in phosphatidyl - choline and phosphatidylethanolamine described previously .", "in our study , significant difference in glutamine and glutamate ( glx ) ratio was noted only on the medial temporal lobe on the right side , which indicates disturbance of glx .", "this finding agrees with data presented by kantarci et al . , who found a trend toward decreased glu + gln / cr ratios from normal to mci to ad , but there were no statistically significant differences .", "detailed analysis of glutamine and glutamate in mci and ad could be explored using the new 3 t system , which currently is commercially available . in the medial temporal lobe h mrs", "has several limitations that could influence our results and our interpretation . in view of the complicated spatial shape of the medial temporal - hippocampal formation ( which is considered to be the region first changed by the progression of dementia pathology ) and its relatively small size , our volume of interest also covered other tissue structures outside the target structure , which could have caused partial falsification of the results as to its volume effect   on the other hand , these structures belong to the limbic system , and show pathological changes in the subsequent stages of the disease .", "according to reports in the literature , including the adjacent fluid spaces ( temporal horn of the lateral ventricle , lateral part of the transverse fissure ) into the voi should not affect the results .", "some researchers claim that chemical shift imaging ( csi ) , also known as single voxel spectroscopy ( svs ) , which allows recording spectra from many neighboring voxels within the examined area , is better for assessment of metabolic disorders within the hippocampus because it enables a more precise measurement .", "this theory was not corroborated in comparative studies of both of the methods used on a group of patients with temporal lobe epilepsy .", "h mrs seems to be a very sensitive method that provides biochemical information using an in vivo approach in patients with initial mci , who in significant numbers developed disease progression and/or converted to ad .", "a potential advantage could be achieved by using 3 t systems , which can better discriminate quantization of glu + gln / cr and gln / cr ratios ."], "abstract_text": ["<S> summarybackgroundpurpose of study was evaluation of regional metabolic disorders using 1h mrs in patients with mci , as a predictor of clinical conversion to dementia based on clinical follow-up.material/methodsthe study group consisted of 31 subjects with diagnosis of mci based on criteria the mayo clinic group . </S>", "<S> 1h mrs was performed with a single - voxel method using press sequence . </S>", "<S> the volume of interest ( voi ) was located in the hippocampal formation and posterior part of the cingulated gyrus.resultspatients had annual clinical control at least twice . at the beginning , 9 had amnestic mci and the others had multidomain mci . during follow - up ( median 3 yrs ) 8 subjects had stable disease ( sd ) , 13 had disease progression ( dp ) and 10 develop alzheimer disease ( ad ) . </S>", "<S> baseline metabolic ratios ( 1h mrs ) between 3 groups indicated significant difference ( p<0.05 ) in left frontal lobe in mi / h20 ratio , between patients with sd ( 0.27 ) and dp . in comparing the groups with dp and ad , a significant difference in naa / cr ( 1.77 vs. 1.43 ) was found . </S>", "<S> a significant difference within left temporal external lobes was found between sd and dp in naa / h2o ratio ( 0.55 vs. 0.51 ) . </S>", "<S> an additional significant difference within medial temporal lobe was found between dp and ad in glx / h2o ratio ( 0.44 vs. 0.34 ) on the right side.conclusions1h mrs seems to be sensitive method allows prediction of which patients are liable to progress from mci to ad . </S>", "<S> combined with other biomarkers of disease staging , it is an important approach in the preclinical ad diagnosis , as well as the assessment of dementia progression . </S>"], "labels": null, "section_names": ["Background", "Aim of the study", "Material and Methods", "Inclusion criteria for patients and demographic characteristics of examined groups", "MR and ", "Statistical analysis", "Results", "Discussion", "Changes of average NAA/Cr and NAA/H", "Changes of average mI/Cr and mI/H", "Changes of average Chol/Cr and Chol/H", "Changes of average Chol/Cr and Chol/H", "Conclusions"], "sections": [["aging leads to senile diseases , including alzheimer disease ( ad ) , which has become a serious medical and socioeconomic problem .", "therefore , progress in early diagnosis and effective treatment of ad is necessary , especially in the preliminary or early stages of the disease .", "most elderly people experience physiological memory disorders ; these disorders require a careful clinical assessment , as it is extremely difficult to delineate the line between physiology and pathology .", "thus , when disorders of memory and other cognitive functions go beyond the standards of age and educational level , but have not yet reached the standards of dementia , we diagnose mild cognitive impairment ( mci ) , which includes several states that either match the standard in its wide meaning , for example age - associated memory impairment ( aami ) , age - related cognitive decline ( arcd ) , or we diagnose pathology like mci , which , although clinically similar , has a different prognosis than aami and arcd [ 14 ] . currently , base on criteria established by the mayo clinic group , mci is define as a transitional state between normal aging and alzheimer s disease , in which memory impairment is greater than expected for age , but general cognitive function and daily living activities are preserved .", "people suffering from mci , especially amnestic form ( amci ) , are at risk for developing dementia , particularly alzheimer disease ( ad ) [ 35 ] . according to various reports", ", the percentage of conversion from amci into ad ranges from 1% to 25% per year , while the mean time of dementia progression from the mci diagnosis is 4.4 years . obviously , the early identification of patients at risk for alzheimer disease from among those with mci would facilitate proper treatment help to delay the symptoms of dementia .", "therefore , searching for methods that would help us detect a higher risk of dementia in those patients , as well as looking for objective methods of early diagnosis of patients with mci , seems reasonable .", "many reports confirm a typical repeatable topographical location of neuropathological lesions at various stages of ad , as described by braak et al and pantel et al . , while emphasizing early involvement of the limbic system , including the hippocampus and entorhinal cortex .", "earlier pathology in these strategic regions in patients with a higher risk of alzheimer - type dementia , as well as in certain patients with amnestic mci , seems likely .", "atrophy of the medial temporal lobe in mci patients with a positive correlation of atrophy level and a risk of conversion to ad , inadequate for age and observed in imaging examinations ( ct , mri ) , has been clearly demonstrated [ 813 ] .", "the level of atrophy is judged by descriptive , volumetric and planimetric methods , or indirectly with the use of linear measurements of particular fluid spaces , such as fissures or certain parts of the ventricular system . despite many years of experience in structural diagnosis of the dementia diseases", ", the use of functional methods seems to be very helpful . in spect and pet examinations", "we observe the lowering of perfusion and metabolism of glucose and oxygen in temporal and parietal areas in patients with possible ad , as well as in those with mci .", "however , these methods have certain limitations such as relatively low linear resolution , which hinders precise determination of the topography of the measuring ( this problem relates to the middle temporal structures and not to the hemispheres cortex ) . a wider clinical application of magnetic resonance spectroscopy ( mrs ) , described by bootomley as  a window for metabolism", "   enabled an intravital assessment of regional metabolic disorders in certain structures of the brain .", "there have been several reports on such dysfunctions in the limbic system in patients with alzheimer disease .", "a relatively small number of spectroscopic examinations have been carried out on patients with mci .", "some of these were presented by catani et al . , who located voxels in the white matter at the ventricular triangle level   and kantarci et al .   who examined the posterior part of the cingulate gyrus in patients with mci and ad . however , these are the locations where anatomopathologic changes , typical for ad , appear later than in the medial temporal lobe ( including hippocampal formation ) .", "many authors give up on spectroscopic examinations of hippocampal structures due to their technical limitations , such as the size of examined structures and topographical relations of the surrounding area .", "the problems mentioned most often are : difficulties with field homogeneity , artifacts from cranial base area , a disqualifying weakening of the signal - to - noise ratio , and the need to reduce the volume of interest ( voi ) .", "there have been reports on the technical capacities of such examinations in various brain diseases [ 1923 ] . according to available the available literature , in the majority of papers h1mrs in mci and ad metabolites were measured in the posterior cingulated gyrus , but parts of papers evaluated the concentration of metabolites in the bilateral hippocampi of mci patients .", "this study attempted to evaluate the regional metabolic disorders using h mrs within the frontal , external and medial temporal lobes in patients with mci , as a predictor of clinical deterioration to dementia based on clinical follow - up ."], ["this study attempted to evaluate the regional metabolic disorders using h mrs within the frontal , external and medial temporal lobes in patients with mci , as a predictor of clinical deterioration to dementia based on clinical follow - up ."], ["the examination was performed on a group of 31 randomly selected subjects ( 19 females and 12 males ) with mci diagnosed under the care of the department of neurodegenerative disorders , medical research centre , polish academy of science .", "all subjects underwent neurological and psychiatric tests , routine laboratory investigations and standard neuropsychological examinations .", "patients with serious cns injuries , alcoholic abuse , diabetes , and serious hepatic and renal dysfunctions were excluded .", "mci diagnosis was established by a team of specialists after analyzing all available information and test results .", "mri and h mrs examination was performed on a 1.5 t eclipse ( marconi medical systems , usa ) scanner .", "morphological mri examination of the brain was carried out in transverse planes , parallel to the longitudinal axis of the temporal lobe in se , fse and flair sequences , in t1- and t2-weighted images , and in the frontal plane perpendicular to the longitudinal axis of the temporal lobe in flair sequence .", "we investigated the extent of cerebral atrophy , especially in medial and external temporal lobes and in frontal structures , as well as the presence of cortical - subcortical hyperintensive focuses ( which equal angiogenic lesions ) in t2-weighted images and flair sequence .", "morphological examination enabled us to exclude other pathologies such as extensive ischemic lesions , tumors , paracerebral hematomas , and hydrocephalus , which might lead to cognitive disorders .", "changes like leukoaraiosis and/or small single angiogenic focuses of gliosis were not exclusion criteria for the study .", "the voi ( volume of interest ) was located in the frontal , medial and external temporal lobe regions , separately on each side . in case of medial temporal lobe covering the hippocampal formation ,", "it was divided into 2 areas  the anterior ( the topographical point of reference in the coronal plane / layer was the level of dens ) and the posterior ( the point of reference was the output of the middle cerebellar peduncles from the pons ) . both areas were then added , and final average data sets were used in further analysis . in cases of reduced volume of the hippocampal formation , the adjacent fluid spaces ( eg , the temporal horn of the lateral ventricle and the lateral part of the transverse fissure ) were also included into the voi .", "necessary corrections of voi location were applied to particular frontal cross - sections , as well as to the axial plane , and care was taken not to cover the osseous structures of the temporal bone pyramids . for every localization , the size of voi was 8 cm . additionally , each subject had frontal measurement taken .", "h mrs examination was carried out with a single - voxel method using press sequence .", "routine 3-impulse sequences of 90 , 180 , 180 degrees and double crusher impulse were used .", "the examination was performed with an automated standardization of the field in the entire encephalon / brain ( total shimming ) and in the examined sample ( local shimming ) . for water suppression ,", "spectra were recorded within the following parameters : te=35 ms , tr=1500 ms , thickness = 15 mm , signal averages = 192 .", "assignment of resonance lines of particular metabolites was based on n - acetylaspartate signal with chemical shift set to 2.0 ppm .", "the spectra were analyzed using the manufacturer - supplied software package for the mrs ( marconi ) . in some cases ,", "it was necessary to correct the phase manually in order to obtain a maximum of symmetrical signal of residual water and to maintain a proper baseline . relative concentration ratios of particular metabolites ", "n - acetylaspartate ( naa ) , choline ( cho ) , myoinositol ( mi ) , glutamine and glutamate ( glx )  were analyzed in reference to the signal of unsuppressed water signal , and also to the signal of creatine , considering its level as an inner standard .", "results analysis of clinical and biochemical data  age , mini - mental score ( mm - score ) , folic acid , vitamin b12 and homocysteine level  was performed including initial data of all subjects enrolled into the study .", "evaluation of relative concentration ratios of metabolites from all vois , localized within frontal , medial and external temporal lobes , symmetrical on both sides , were performed in each case .", "a comparison of some metabolic ratios of mrs in patients with mci , who on follow - up has stable disease ( sd ) , disease progression ( dp ) and conversion to ad , was performed .", "the statistical analysis was carried out using the statistica for windows 7.0 ( statsoft , ok , usa ) software package ."], ["the examination was performed on a group of 31 randomly selected subjects ( 19 females and 12 males ) with mci diagnosed under the care of the department of neurodegenerative disorders , medical research centre , polish academy of science .", "all subjects underwent neurological and psychiatric tests , routine laboratory investigations and standard neuropsychological examinations .", "patients with serious cns injuries , alcoholic abuse , diabetes , and serious hepatic and renal dysfunctions were excluded .", "mci diagnosis was established by a team of specialists after analyzing all available information and test results ."], ["mri and h mrs examination was performed on a 1.5 t eclipse ( marconi medical systems , usa ) scanner .", "morphological mri examination of the brain was carried out in transverse planes , parallel to the longitudinal axis of the temporal lobe in se , fse and flair sequences , in t1- and t2-weighted images , and in the frontal plane perpendicular to the longitudinal axis of the temporal lobe in flair sequence .", "we investigated the extent of cerebral atrophy , especially in medial and external temporal lobes and in frontal structures , as well as the presence of cortical - subcortical hyperintensive focuses ( which equal angiogenic lesions ) in t2-weighted images and flair sequence .", "morphological examination enabled us to exclude other pathologies such as extensive ischemic lesions , tumors , paracerebral hematomas , and hydrocephalus , which might lead to cognitive disorders .", "changes like leukoaraiosis and/or small single angiogenic focuses of gliosis were not exclusion criteria for the study .", "the voi ( volume of interest ) was located in the frontal , medial and external temporal lobe regions , separately on each side . in case of medial temporal lobe covering the hippocampal formation ,", "it was divided into 2 areas  the anterior ( the topographical point of reference in the coronal plane / layer was the level of dens ) and the posterior ( the point of reference was the output of the middle cerebellar peduncles from the pons ) . both areas were then added , and final average data sets were used in further analysis . in cases of reduced volume of the hippocampal formation , the adjacent fluid spaces ( eg , the temporal horn of the lateral ventricle and the lateral part of the transverse fissure ) were also included into the voi .", "necessary corrections of voi location were applied to particular frontal cross - sections , as well as to the axial plane , and care was taken not to cover the osseous structures of the temporal bone pyramids . for every localization , the size of voi was 8 cm .", "h mrs examination was carried out with a single - voxel method using press sequence .", "routine 3-impulse sequences of 90 , 180 , 180 degrees and double crusher impulse were used .", "the examination was performed with an automated standardization of the field in the entire encephalon / brain ( total shimming ) and in the examined sample ( local shimming ) . for water suppression ,", "spectra were recorded within the following parameters : te=35 ms , tr=1500 ms , thickness = 15 mm , signal averages = 192 .", "assignment of resonance lines of particular metabolites was based on n - acetylaspartate signal with chemical shift set to 2.0 ppm .", "the spectra were analyzed using the manufacturer - supplied software package for the mrs ( marconi ) . in some cases ,", "it was necessary to correct the phase manually in order to obtain a maximum of symmetrical signal of residual water and to maintain a proper baseline . relative concentration ratios of particular metabolites ", "n - acetylaspartate ( naa ) , choline ( cho ) , myoinositol ( mi ) , glutamine and glutamate ( glx )  were analyzed in reference to the signal of unsuppressed water signal , and also to the signal of creatine , considering its level as an inner standard ."], ["results analysis of clinical and biochemical data  age , mini - mental score ( mm - score ) , folic acid , vitamin b12 and homocysteine level  was performed including initial data of all subjects enrolled into the study .", "evaluation of relative concentration ratios of metabolites from all vois , localized within frontal , medial and external temporal lobes , symmetrical on both sides , were performed in each case .", "a comparison of some metabolic ratios of mrs in patients with mci , who on follow - up has stable disease ( sd ) , disease progression ( dp ) and conversion to ad , was performed .", "the statistical analysis was carried out using the statistica for windows 7.0 ( statsoft , ok , usa ) software package ."], ["all patients had annual clinical follow - up at least twice . at the beginning of the study , subjects were divided into 2 groups  9 subjects who had amnestic mci , and the others who had multidomain mci .", "there were no statistical differences between groups of patients with multidomain mci and amnestic mci , including age , m - m score , level of vitamin b12 , folic acid and homocysteine ( p>0.05 ) .", "comparison of mean value in some selected parameters including both groups is presented in table 1 . during clinical follow - up", "( median 3 years ) 8 subjects had stable disease ( sd ) , 13 had progression of disease ( dp ) and 10 developed ad .", "there was no significant difference between age , m - m score , vitamin b12 , folic acid and homocysteine level among these 3 groups of subjects ( p>0.05 ) .", "statistical analyses of baseline metabolic ratios measurement using h mrs between the final 3 groups of patients , ( sd , dp and ad ) found significant difference in frontal lobes in mi / h20 ratio on left , between patients with stable disease ( 0.27 ) and those with progression ( 0.22 ) ( p=0.03 ) ( figure 1 ) . in the groups of patients with dp and those with conversion to ad", ", there was a significant difference on the left side in ratio naa / cr ( 1.77 vs. 1.43 ) , ( p=0.02 ) ( figure 2 ) .", "significant difference within temporal external lobes were found between patients with sd and dp in naa / h2o ratio on the left side ( 0.55 vs. 0.51 ) , ( p=0.04 ) ( figure 3 ) .", "additionally , there were significant differences between patients with sd and ad in chol / cr ratio ( 0.99 vs. 0.88 ) on the right side ( p=0.04 ) and in mi / cr ( 1.09 vs. 0.62 ) on the left side ( p=0.03 ) .", "a significant difference within the medial temporal lobe was found between patients with dp and ad in glx / h2o ratio ( 0.44 vs. 0.34 ) on the right side ( m - w u - test , p=0.02 ) . there were no other significant differences within h mrs metabolic ratios .", "an example of h mrs study in patient with initial diagnosis of mci and clinical deterioration during 2 years of follow - up with final diagnosis of ad is presented in ( figure 4 ) ( a  initial h mrs , and after final diagnosis , control h mrs study ", "b ) . summary of initial h mrs ratios of some metabolites in frontal , external and medial temporal lobes both sides are presented in table 3 ."], ["proton magnetic resonance spectroscopy  h mrs  enables an in vivo noninvasive assessment of the degree of biochemical disorders in a specified voi of the examined tissue , as well as monitoring the progress of those changes in the course of the disease and its treatment [ 5,1725 ] .", "the method we used , along with the parameters mentioned above , resulted in satisfactory quality of spectra in examined patients .", "recent studies have demonstrated the value of h1mrs in many types of cognitive disorders , not only in degenerative diseases . in mrs diagnosis ,", "normal tissues present a constant proton spectrum , while ratio changes of the metabolites can be considered as reflections of certain biochemical transformations .", "several cytological and histochemical investigations have contributed to the study of the relationship between certain chemical substances and precisely defined intracellular structures or biochemical processes  physiological as well as pathological .", "n - acetylaspartate is commonly considered as a neuronal marker due to its presence only in mature nerve cells .", "there are several reports of age - dependent lowering of naa level in certain parts of the encephalon , which is being interpreted not only as an effect of a progressive decrease in number of neurons , but also as their dysfunction .", "it also seems clear that there is a lowering of the naa concentration in atrophic structures of the limbic system in patients with ad , and a reverse correlation of the level of this metabolite with the level of dementia .", "however , the h mrs examinations of the posterior part of the cingulate gyrus ( frontal lobe ) in patients with mci , carried out by the rochester group , did not prove statistically significant differences of the naa ratios as compared to the control group .", "in our study , we found a significant difference between the ratio of naa / cr between patients with ds vs. dp in the left frontal lobe .", "we also found significant difference in naa / h2o between patients with ds and dp in the left external temporal lobe .", "we could not find any significant difference between naa / cr and naa / h2o within the medial temporal lobe on any sides .", "this could due to the frequent technical difficulties mentioned before , as measurement of the medial temporal lobe ( hippocampus area ) is often inconvenient .", "the neuronal integrity marker naa / cr or naa / h2o ratio ( independent of creatine level ) is currently declined in patients with progressive mci and those with conversion to ad compared to cognitively normal elderly subjects .", "our findings generally agree with others reports that found lower levels of naa in patients with mci and deterioration of brain function and who convert to ad .", "others reports indicated the left side more often has a drop in naa - rich neurons compare to the right side [ 2026 ] .", "medial temporal lobes ( hippocampus structures ) are widely considered as the region in which the earliest ad pathologies occur .", "therefore it seems that the lowering of naa concentration in this localization in patients with mci proves the theory of  successive  metabolic disorders in progression of dementia , according to which a lower concentration of myoinositol precedes the decline in naa concentration [ 2124 ] . in many reports , naa depletion seems to be good marker , as in metastasio et al .", "single reports indicated that the significant reduction of naa has great predictive value within the occipital area in those based on roc with threshold level < 1.61 of naa / cr ratio ; other regions and others ratios were not significant in this study . despite this inhomogeneous data", ", numerous studies have shown that naa plays an important role in neuronal integrity , with the left side more often affected in patients with progressive mci and in those with conversion to ad [ 1826 ] .", "some studies have found an elevated level of mi / cr , with good correlation of disease progression and increase of neurofibrillary tangles in patients with conversion into ad . in our study", "we did not find any of these findings ; on the contrary , we detected some depletion of mi / cr in patients with conversion to ad compared to those with stable disease , with a significant difference on the left external temporal lobe ( 1.09 vs. 0.62 ) .", "we found the same tendency in mi / h2o ratio on the left frontal lobe between patients with disease stability and disease progression .", "others reported differences between patients with mci and ad , with significantly higher signal ratio of mi / cr in ad .", "others publications did not report any difference in this metabolite ratio in the group of patients with mci .", "the differences in results of various groups of researchers could be explained by selection of the study population .", "the same research group indicated different results , dependent on study group  for instance , no difference was found in selected population of patients with amnestic mci   compare to a heterogenous population with significant difference in mi signal .", "some reports indicated that an elevated level of this metabolite in structures of the limbic system in patients with mci or dementia is connected with regional gliosis .", "this theory is supported by the presence of visibly higher mi concentrations in glia cells than in neurocytes . however , thus far the exact role of mi in mci and ad is unclear .", "there are many other disorders of the brain with mi disturbance , so specificity of this marker and ratio of mi / cr or mi / h2o seems to be low .", "recent data suggests good discrimination using mi / cr ratio in different types of dementia using roc curve .", "additional theory posits an extensive cellular capture of mi in patients at risk of dementia as the result of na / mi osmoregulator dysfunction .", "the study of adults with down syndrome , who constitute a clinically exceptional group of subjects with 100% risk of dementia similar to the alzheimer s disease , demonstrated a greater that 50% rise of the myoinositol level with age compared to the healthy control group .", "the authors associated this with an elevated activity of sodium - myoinositol transporter/ transmitter , which results from the existence of the additional 21 chromosome in down syndrome adults , which includes the gene - encoding protein of the transmitter . in our study , significant differences in mi / cr and mi / h2o were found in frontal and external temporal lobes , but not in the medial temporal lobe .", "our h mrs study shows a significant difference between chol / cr ratio only in the external temporal lobe on the left side , with a significant drop in the chol / cr ratio between patients with stable disease and ad .", "this result agrees with metastasio et al . , who showed a decreased , but not significant , depletion of chol / cr in mci patients with disease progression compare to those with stable disease in both hemispheres .", "this could be related to cholinergic neuronal damage , which is indirectly confirmed by increased activity of choline acetyltransferase ( chat ) , and which probably represents a compensatory ( but insufficient ) response in ad .", "impairment of mitochondrial activity seen in elderly patients , and more obviously in patients with ad , could be related to increase of mitochondrial membrane damage and evaluated level of membrane phospholipid   and decrease in phosphatidyl - choline and phosphatidylethanolamine described previously . in our study , significant difference in glutamine and glutamate ( glx ) ratio was noted only on the medial temporal lobe on the right side , which indicates disturbance of glx .", "this finding agrees with data presented by kantarci et al . , who found a trend toward decreased glu + gln / cr ratios from normal to mci to ad , but there were no statistically significant differences .", "detailed analysis of glutamine and glutamate in mci and ad could be explored using the new 3 t system , which currently is commercially available . in the medial temporal lobe h mrs", "has several limitations that could influence our results and our interpretation . in view of the complicated spatial shape of the medial temporal - hippocampal formation ( which is considered to be the region first changed by the progression of dementia pathology ) and its relatively small size , our volume of interest also covered other tissue structures outside the target structure , which could have caused partial falsification of the results as to its volume effect   on the other hand , these structures belong to the limbic system , and show pathological changes in the subsequent stages of the disease .", "according to reports in the literature , including the adjacent fluid spaces ( temporal horn of the lateral ventricle , lateral part of the transverse fissure ) into the voi should not affect the results .", "some researchers claim that chemical shift imaging ( csi ) , also known as single voxel spectroscopy ( svs ) , which allows recording spectra from many neighboring voxels within the examined area , is better for assessment of metabolic disorders within the hippocampus because it enables a more precise measurement .", "this theory was not corroborated in comparative studies of both of the methods used on a group of patients with temporal lobe epilepsy ."], ["n - acetylaspartate is commonly considered as a neuronal marker due to its presence only in mature nerve cells .", "there are several reports of age - dependent lowering of naa level in certain parts of the encephalon , which is being interpreted not only as an effect of a progressive decrease in number of neurons , but also as their dysfunction .", "it also seems clear that there is a lowering of the naa concentration in atrophic structures of the limbic system in patients with ad , and a reverse correlation of the level of this metabolite with the level of dementia .", "however , the h mrs examinations of the posterior part of the cingulate gyrus ( frontal lobe ) in patients with mci , carried out by the rochester group , did not prove statistically significant differences of the naa ratios as compared to the control group . in our study", ", we found a significant difference between the ratio of naa / cr between patients with ds vs. dp in the left frontal lobe .", "we also found significant difference in naa / h2o between patients with ds and dp in the left external temporal lobe .", "we could not find any significant difference between naa / cr and naa / h2o within the medial temporal lobe on any sides .", "this could due to the frequent technical difficulties mentioned before , as measurement of the medial temporal lobe ( hippocampus area ) is often inconvenient .", "the neuronal integrity marker naa / cr or naa / h2o ratio ( independent of creatine level ) is currently declined in patients with progressive mci and those with conversion to ad compared to cognitively normal elderly subjects .", "our findings generally agree with others reports that found lower levels of naa in patients with mci and deterioration of brain function and who convert to ad .", "others reports indicated the left side more often has a drop in naa - rich neurons compare to the right side [ 2026 ] .", "medial temporal lobes ( hippocampus structures ) are widely considered as the region in which the earliest ad pathologies occur .", "therefore it seems that the lowering of naa concentration in this localization in patients with mci proves the theory of  successive  metabolic disorders in progression of dementia , according to which a lower concentration of myoinositol precedes the decline in naa concentration [ 2124 ] . in many reports , naa depletion seems to be good marker , as in metastasio et al .", "single reports indicated that the significant reduction of naa has great predictive value within the occipital area in those based on roc with threshold level < 1.61 of naa / cr ratio ; other regions and others ratios were not significant in this study . despite this inhomogeneous data", ", numerous studies have shown that naa plays an important role in neuronal integrity , with the left side more often affected in patients with progressive mci and in those with conversion to ad [ 1826 ] ."], ["some studies have found an elevated level of mi / cr , with good correlation of disease progression and increase of neurofibrillary tangles in patients with conversion into ad . in our study", "we did not find any of these findings ; on the contrary , we detected some depletion of mi / cr in patients with conversion to ad compared to those with stable disease , with a significant difference on the left external temporal lobe ( 1.09 vs. 0.62 ) .", "we found the same tendency in mi / h2o ratio on the left frontal lobe between patients with disease stability and disease progression .", "others reported differences between patients with mci and ad , with significantly higher signal ratio of mi / cr in ad .", "others publications did not report any difference in this metabolite ratio in the group of patients with mci .", "the differences in results of various groups of researchers could be explained by selection of the study population .", "the same research group indicated different results , dependent on study group  for instance , no difference was found in selected population of patients with amnestic mci   compare to a heterogenous population with significant difference in mi signal .", "some reports indicated that an elevated level of this metabolite in structures of the limbic system in patients with mci or dementia is connected with regional gliosis .", "this theory is supported by the presence of visibly higher mi concentrations in glia cells than in neurocytes . however , thus far the exact role of mi in mci and ad is unclear .", "there are many other disorders of the brain with mi disturbance , so specificity of this marker and ratio of mi / cr or mi / h2o seems to be low .", "recent data suggests good discrimination using mi / cr ratio in different types of dementia using roc curve .", "additional theory posits an extensive cellular capture of mi in patients at risk of dementia as the result of na / mi osmoregulator dysfunction .", "the study of adults with down syndrome , who constitute a clinically exceptional group of subjects with 100% risk of dementia similar to the alzheimer s disease , demonstrated a greater that 50% rise of the myoinositol level with age compared to the healthy control group .", "the authors associated this with an elevated activity of sodium - myoinositol transporter/ transmitter , which results from the existence of the additional 21 chromosome in down syndrome adults , which includes the gene - encoding protein of the transmitter . in our study , significant differences in mi / cr and mi / h2o were found in frontal and external temporal lobes , but not in the medial temporal lobe ."], ["our h mrs study shows a significant difference between chol / cr ratio only in the external temporal lobe on the left side , with a significant drop in the chol / cr ratio between patients with stable disease and ad .", "this result agrees with metastasio et al . , who showed a decreased , but not significant , depletion of chol / cr in mci patients with disease progression compare to those with stable disease in both hemispheres .", "this could be related to cholinergic neuronal damage , which is indirectly confirmed by increased activity of choline acetyltransferase ( chat ) , and which probably represents a compensatory ( but insufficient ) response in ad .", "impairment of mitochondrial activity seen in elderly patients , and more obviously in patients with ad , could be related to increase of mitochondrial membrane damage and evaluated level of membrane phospholipid   and decrease in phosphatidyl - choline and phosphatidylethanolamine described previously ."], ["in our study , significant difference in glutamine and glutamate ( glx ) ratio was noted only on the medial temporal lobe on the right side , which indicates disturbance of glx .", "this finding agrees with data presented by kantarci et al . , who found a trend toward decreased glu + gln / cr ratios from normal to mci to ad , but there were no statistically significant differences .", "detailed analysis of glutamine and glutamate in mci and ad could be explored using the new 3 t system , which currently is commercially available . in the medial temporal lobe h mrs", "has several limitations that could influence our results and our interpretation . in view of the complicated spatial shape of the medial temporal - hippocampal formation ( which is considered to be the region first changed by the progression of dementia pathology ) and its relatively small size , our volume of interest also covered other tissue structures outside the target structure , which could have caused partial falsification of the results as to its volume effect   on the other hand , these structures belong to the limbic system , and show pathological changes in the subsequent stages of the disease .", "according to reports in the literature , including the adjacent fluid spaces ( temporal horn of the lateral ventricle , lateral part of the transverse fissure ) into the voi should not affect the results .", "some researchers claim that chemical shift imaging ( csi ) , also known as single voxel spectroscopy ( svs ) , which allows recording spectra from many neighboring voxels within the examined area , is better for assessment of metabolic disorders within the hippocampus because it enables a more precise measurement .", "this theory was not corroborated in comparative studies of both of the methods used on a group of patients with temporal lobe epilepsy ."], ["h mrs seems to be a very sensitive method that provides biochemical information using an in vivo approach in patients with initial mci , who in significant numbers developed disease progression and/or converted to ad .", "a potential advantage could be achieved by using 3 t systems , which can better discriminate quantization of glu + gln / cr and gln / cr ratios ."]]}
{"article_id": "PMC4322878", "article_text": ["intraocular pressure ( iop ) is one the most important modifiable risk factors for glaucoma progression.13 iop measurements in the ophthalmology clinic are often limited to office hours , which do not fully represent the nychthemeral iop peaks and fluctuations.4,5 previously , 24-hour iop monitoring studies have reported fluctuation as high as 8.21.4 mmhg , but many of these studies measured iop in the sitting position , overnight in a hospital environment , and over a few sampling periods , which does not truly reflect the physiological and normal setting of one s daily living , especially when the subjects are awakened at night for iop measurements.610 various studies have reported the significance of iop fluctuation on glaucoma progression.11,12 while there are studies that report otherwise,13,14 much of the existing literature has only documented inter - visit or daytime iop variability over a limited number of hours .", "this is at least partly due to limitations of how frequently repeated tonometric iop measurements can be taken . a contact lens - based sensor ( cls )", "is nowadays available for the recording of ocular dimensional profiles for up to 24 hours.15 this device has been shown to be safe and tolerable in healthy subjects and glaucoma patients as well as to provide reproducible recording of 24-hour profiles.1618 the aim of this study was to analyze the 24-hour ocular dimensional profile in normal - tension glaucoma ( ntg ) patients on medical treatment .", "informed patient consent and approval by the institutional review board of the hospital authority of hong kong were obtained prior to study commencement .", "this was a prospective cohort study from july 2012 to june 2013 , conducted at a university hospital in hong kong .", "the study recruited consenting adults ( age > 18 years old ) with unilateral or bilateral ntg who were currently on topical antiglaucoma medications .", "ntg was defined as open angle on gonioscopy ; progressive thinning of the retinal nerve fiber layer ( rnfl ) on optical coherence tomography , with corresponding glaucomatous visual field changes on the humphrey visual field analyzer ; and an iop 21 mmhg on all clinical visits based on previous medical records .", "cases with previous glaucoma surgery or laser treatment , active or previous corneal disease , and subjects with only one functional eye were excluded .", "the sensimed triggerfish ( sensimed ag , lausanne , switzerland ) is a soft silicone cls that enables recording of the ocular dimensional profile over a 24-hour period with minimal disturbance to one s daily routines and sleep cycles .", "such 24-hour profiles are related to the 24-hour iop profiles.18 dimensional changes are recorded in the corneoscleral area for 30 seconds every 5 minutes over 24 hours and each recording  burst  represents 300 data points , the medians of which are plotted as a single graph which makes up the 24-hour ocular dimensional profile measured in sensory output units of millivolt equivalents ( mveq ) .", "the device was ce - marked and thus approved for clinical use in 2009 . in healthy volunteers and glaucoma patients , the cls was found to be tolerable in normal activities of daily living and during sleep . a contact lens with base curve 8.7 mm was also found to be well adapted in most eyes , although the device also exists in steeper ( 8.4 mm ) and flatter ( 9.0 mm ) base curves.1618 the cls was placed on the subject s eye by an ophthalmologist in the outpatient clinic after a slit - lamp examination of the anterior segment and goldmann applanation tonometry ( gat ) by a single investigator . for those with unilateral disease , the cls was placed on the eye with ntg . for those with bilateral disease , a random eye assignment by card shuffling was used to determine the eye for the cls placement .", "the subject then returned home with lubricating eyedrops and carried on his or her daily activities ( both indoor and outdoor ) , apart from showering or swimming ( as the device can not be in contact with water ) .", "subjects continued their same regimen of antiglaucoma eyedrops and slept in their habitual position at night .", "after 24 hours , the subject returned to the clinic to have the cls removed followed by a slit - lamp examination and gat .", "cls profile parameters were extracted following smoothing of the profile using locally weighted polynomial regression .", "cls variability from mean : this variable reflects variability around the mean value of all raw ( not smoothed ) cls measurements in the respective period ( 24-hour , diurnal , or nocturnal ) .", "diurnal / nocturnal variability : same as above but only during the awake and asleep periods , respectively , where the sleep cycle is determined from recorded sleep times in each individual s logbook and verified by the eye - blinking frequency ( present during waking ) and the presence of rhythmic ocular pulsation waves ( present during sleep ) .", "sleep - to - wake and wake - to - sleep slopes : calculated with a generalized linear model on raw measurements in units of mveq / hour from 1 hour before sleep or wake time to 1 hour after sleep or wake time , respectively .", "number of peaks : a peak is defined as a local maximum point in the smoothed curve .", "the calculation of the number of peaks occurs as follows : each trough is noted as the start of a peak .", "the pre- and post - cls gat iops were compared using the wilcoxon signed - rank test .", "whitney u - test : \n nocturnal versus diurnal mean variability from mean;wake - to - sleep versus sleep - to - wake slopes;number of nocturnal versus diurnal peaks ; and24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "nocturnal versus diurnal mean variability from mean ; wake - to - sleep versus sleep - to - wake slopes ; number of nocturnal versus diurnal peaks ; and 24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "fisher s exact test was used to compare the differences in the number of nocturnal peaks between those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications .", "cls variability from mean : this variable reflects variability around the mean value of all raw ( not smoothed ) cls measurements in the respective period ( 24-hour , diurnal , or nocturnal ) .", "diurnal / nocturnal variability : same as above but only during the awake and asleep periods , respectively , where the sleep cycle is determined from recorded sleep times in each individual s logbook and verified by the eye - blinking frequency ( present during waking ) and the presence of rhythmic ocular pulsation waves ( present during sleep ) .", "sleep - to - wake and wake - to - sleep slopes : calculated with a generalized linear model on raw measurements in units of mveq / hour from 1 hour before sleep or wake time to 1 hour after sleep or wake time , respectively .", "number of peaks : a peak is defined as a local maximum point in the smoothed curve .", "the calculation of the number of peaks occurs as follows : each trough is noted as the start of a peak .", "the pre- and post - cls gat iops were compared using the wilcoxon signed - rank test .", "whitney u - test : \n nocturnal versus diurnal mean variability from mean;wake - to - sleep versus sleep - to - wake slopes;number of nocturnal versus diurnal peaks ; and24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "nocturnal versus diurnal mean variability from mean ; wake - to - sleep versus sleep - to - wake slopes ; number of nocturnal versus diurnal peaks ; and 24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "fisher s exact test was used to compare the differences in the number of nocturnal peaks between those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications .", "in the 18 subjects enrolled in the study , there were seven males and eleven females .", "gat iops before and after the cls wear were 15.32.2 and 13.81.7 mmhg , respectively , while on the same antiglaucoma medications ( p=0.05 ) .", "the mean rnfl thickness was 72.99.8 m and the mean deviation and pattern standard deviation on humphrey visual field analysis were 6.34.5 decibels ( db ) and 6.14.1 db , respectively .", "in the 18 subjects , 38.9% ( 7/18 ) used a single prostaglandin eyedrop administered in the evening , 44.4% ( 8/18 ) used a twice - daily non - prostaglandin eyedrop , and 17.6% ( 3/17 ) used both a nocturnal prostaglandin eyedrop plus a twice - daily antiglaucoma eyedrop .", "the mean 24-hour variability from the mean cls signal was 75.921.5 ( range : 35.7120.9 ; 95% confidence interval [ ci ] : 62.886.7 ) mveq .", "the mean nocturnal variability from the mean was 52.120.3 ( range : 23.9105.1 ; 95% ci : 41.662.5 ) mveq , which was 48.9% less than the mean diurnal variability from the mean ( 77.620.6 ) ( range : 43.9118.4 ; 95% ci : 66.988.2 ) mveq ( p=0.002 ) .", "there were no statistically significant differences in the mean diurnal , nocturnal , or 24-hour variability between those with and without prostaglandin analog treatment ( all p>0.1 ) ( table 1 ) .", "the mean number of peaks during sleep and daytime was 6.42.3 and 9.32.4 , respectively .", "the number of peaks was 54.7% less during the nocturnal period than the diurnal period ( p=0.001 ) .", "there was no significant difference between the frequencies of nocturnal peaks among those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications ( table 2 ) .", "the mean sleep time of subjects was from 8.53 pm 12.6 minutes to 7.06 am 3.6 minutes .", "the study population had a mean positive linear slope on their cls profile from wake - to - sleep with a mean of 53.242.9 mveq / hour , signifying an increase in the ocular dimensional profile when going to sleep .", "similarly , the population had a mean negative linear slope from sleep - to - wake with a mean of 42.547.6 mveq / hour , signifying a decrease in the ocular dimensional profile upon waking . on comparing the two slopes , the rate of increase when going to sleep was significantly greater than the rate of decrease upon waking ( p<0.001 ) ( table 3 ) .", "the ocular dimensional profiles of all 18 ntg subjects were unique , with individual peaks occurring at different time intervals throughout the day and night ; no two tracings were identical .", "the mean ocular dimensional profile of the study population increased during sleep ( positive wake - to - sleep slope ) and decreased upon waking ( negative sleep - to - wake slope ) , with the mean rate of increase during sleep being significantly greater than the rate of decrease upon waking ( p<0.001 ) .", "we are inclined to agree with previous postulations , wherein the increase in nocturnal iop was thought to be related to the gravitational pull of fluids to the eye and an increase in episcleral venous pressure during the supine sleep posture.19 through the use of the 24-hour cls , we affirm that the phenomenon of increased iop with supine posturing was consistent among ntg subjects under medical treatment .", "other authors have also observed the persistence of posture - induced iop changes despite treatment of ntg patients with iop - lowering medication.20 similarly , a previous japanese study reported , in a population of ntg subjects , that the iop spikes recorded from the habitual positions ( sitting up during the day and supine at night ) were positively correlated to the spikes induced from a postural - change test and after a water - drinking test.21 in contrast , renard et al reported that , in 27 subjects with suspected ntg , 24-hour iop monitoring revealed that 54.5% exhibited a diurnal acrophase and 36.4% exhibited a nocturnal acrophase , while 9.1% had no nychthemeral rhythm in the absence of medical treatment.22 the existence of concomitant obstructive sleep apnea syndrome in 80% of assessed patients could have influenced the observed iop rhythm in their cohort.22 before the availability of continuous 24-hour ocular dimensional profile recording , it has been reported that , in many glaucoma patients , the iop peaks occurred outside of office hours23,24 and that two - thirds of iop peaks in untreated glaucoma patients occurred during the nocturnal period.25 in our ntg population , we noted that there were 55% less peaks occurring nocturnally than diurnally ( p=0.001 ) .", "the reduction in the number of peaks at night in our ntg population could be related to the use of prostaglandin antiglaucoma eyedrops , since 57% of our subjects were at least on prostaglandin antiglaucoma eyedrops nocturnally .", "however , it may also be simply due to the absence of physical activity , eye blinking , and saccadic movements at night , as these parameters are known to cause iop spikes and fluctuations.26 other authors have suggested that the nocturnal iop and iop spikes may be related to visual field progression in ntg subjects.21 study on the effect of medical treatment on iop fluctuations has shown that prostaglandin antiglaucoma eyedrops offer the best sustained 24-hour iop reduction , while brimonidine offers the least.27 in our study , the mean nocturnal variability was significantly lower than the mean diurnal variability by 49% ( p=0.002 ) .", "our findings are in agreement with a previous study by pajic et al28 reporting higher coefficients of variation in cls data during the daytime than at nighttime in ntg patients .", "pajic et al reported reduced variability in nocturnal cls data independent of whether or not patients were on antiglaucoma medication.28 we have not recorded cls profiles in our patients without medical treatment and therefore do not know whether the same is valid for this patient cohort ; however , it seems plausible that the lower level of activity during sleep provides a natural reduction in variability , which iop - lowering medical treatment may further influence .", "holl et al recorded cls profiles in primary open - angle glaucoma and ocular hypertension patients and found no difference in 24-hour , diurnal , or nocturnal standard deviation of cls values in the presence of prostaglandin analog treatment as compared to without treatment.29 the variability of diurnal versus nocturnal cls values was not evaluated .", "thus , in this patient cohort , it seems that , while the overall ocular dimensional profile was higher at night , the number of peaks and variability from the mean were significantly reduced at night for ntg subjects on medical treatment .", "it was interesting to note that the gat iop after cls wear ( 13.81.7 mmhg ) was lower ( p=0.05 ) than the iop before the cls wear ( 15.32.2 mmhg ) , although the readings were measured exactly 24 hours apart .", "this seemingly lower iop after the cls use was short of statistical significance and can be explained by day - to - day variations in iop .", "there was no sample size calculation , and the relatively small sample size of the study did not allow for evaluation of differences in iop - related pattern nor stratification by the type of treatment .", "furthermore , only one 24-hour measurement with the cls was performed , therefore we could not ascertain the reproducibility of the 24-hour iop profile . at present , the ocular dimensional profile is something that can not be translated into iop and therefore has no validation for use in clinical practice .", "additional clinical research needs to be done before we can state ( based on evidence ) that the cls can be useful in optimizing the timing of medical therapy for individual patients .", "nevertheless , this is one of the few publications in the literature analyzing the 24-hour ocular dimensional profile on a continuous basis in ntg subjects receiving medical treatment .", "continuous 24-hour ocular dimensional profiles recorded in ntg patients on medical treatment revealed that the profile increased during sleep and decreased upon waking .", "there was a 50% reduction in both the variability from the mean and number of peaks during sleep as compared to daytime ."], "abstract_text": ["<S> purposeto analyze the 24-hour ocular dimensional profile in normal - tension glaucoma ( ntg ) patients on medical treatment.methodsconsecutive , medically treated ntg subjects were recruited from a university eye center . </S>", "<S> subjects were on a mean of 1.70.7 types of antiglaucoma medications and 56.6% were on a prostaglandin analog . </S>", "<S> a contact lens - based sensor device was worn in one eye of ntg patients to record the intraocular pressure ( iop)-related profile for 24 hours , recording the following : variability from mean over 24 hours , nocturnally and diurnally , as well as the number of peaks and troughs diurnally and nocturnally.resultsin 18 ntg subjects , the nocturnal variability around the mean contact lens - based sensor device signal was 48.9% less than the diurnal variability around the mean . </S>", "<S> the number of peaks was 54.7% less during the nocturnal period than during the diurnal period . </S>", "<S> the rate of increase in the ocular dimensional profile when going to sleep was significantly greater than the rate of decrease upon waking ( p<0.001).conclusionin medically treated ntg subjects , there was more variability in the iop - related pattern during the daytime and there were fewer peaks during sleep . </S>"], "labels": null, "section_names": ["Background", "Methods", "Definitions", "Statistical analysis", "Results", "Discussion", "Conclusion"], "sections": [["intraocular pressure ( iop ) is one the most important modifiable risk factors for glaucoma progression.13 iop measurements in the ophthalmology clinic are often limited to office hours , which do not fully represent the nychthemeral iop peaks and fluctuations.4,5 previously , 24-hour iop monitoring studies have reported fluctuation as high as 8.21.4 mmhg , but many of these studies measured iop in the sitting position , overnight in a hospital environment , and over a few sampling periods , which does not truly reflect the physiological and normal setting of one s daily living , especially when the subjects are awakened at night for iop measurements.610 various studies have reported the significance of iop fluctuation on glaucoma progression.11,12 while there are studies that report otherwise,13,14 much of the existing literature has only documented inter - visit or daytime iop variability over a limited number of hours .", "this is at least partly due to limitations of how frequently repeated tonometric iop measurements can be taken . a contact lens - based sensor ( cls )", "is nowadays available for the recording of ocular dimensional profiles for up to 24 hours.15 this device has been shown to be safe and tolerable in healthy subjects and glaucoma patients as well as to provide reproducible recording of 24-hour profiles.1618 the aim of this study was to analyze the 24-hour ocular dimensional profile in normal - tension glaucoma ( ntg ) patients on medical treatment ."], ["informed patient consent and approval by the institutional review board of the hospital authority of hong kong were obtained prior to study commencement .", "this was a prospective cohort study from july 2012 to june 2013 , conducted at a university hospital in hong kong .", "the study recruited consenting adults ( age > 18 years old ) with unilateral or bilateral ntg who were currently on topical antiglaucoma medications .", "ntg was defined as open angle on gonioscopy ; progressive thinning of the retinal nerve fiber layer ( rnfl ) on optical coherence tomography , with corresponding glaucomatous visual field changes on the humphrey visual field analyzer ; and an iop 21 mmhg on all clinical visits based on previous medical records .", "cases with previous glaucoma surgery or laser treatment , active or previous corneal disease , and subjects with only one functional eye were excluded .", "the sensimed triggerfish ( sensimed ag , lausanne , switzerland ) is a soft silicone cls that enables recording of the ocular dimensional profile over a 24-hour period with minimal disturbance to one s daily routines and sleep cycles .", "such 24-hour profiles are related to the 24-hour iop profiles.18 dimensional changes are recorded in the corneoscleral area for 30 seconds every 5 minutes over 24 hours and each recording  burst  represents 300 data points , the medians of which are plotted as a single graph which makes up the 24-hour ocular dimensional profile measured in sensory output units of millivolt equivalents ( mveq ) .", "the device was ce - marked and thus approved for clinical use in 2009 . in healthy volunteers and glaucoma patients , the cls was found to be tolerable in normal activities of daily living and during sleep . a contact lens with base curve 8.7 mm was also found to be well adapted in most eyes , although the device also exists in steeper ( 8.4 mm ) and flatter ( 9.0 mm ) base curves.1618 the cls was placed on the subject s eye by an ophthalmologist in the outpatient clinic after a slit - lamp examination of the anterior segment and goldmann applanation tonometry ( gat ) by a single investigator . for those with unilateral disease , the cls was placed on the eye with ntg . for those with bilateral disease , a random eye assignment by card shuffling was used to determine the eye for the cls placement .", "the subject then returned home with lubricating eyedrops and carried on his or her daily activities ( both indoor and outdoor ) , apart from showering or swimming ( as the device can not be in contact with water ) .", "subjects continued their same regimen of antiglaucoma eyedrops and slept in their habitual position at night .", "after 24 hours , the subject returned to the clinic to have the cls removed followed by a slit - lamp examination and gat .", "cls profile parameters were extracted following smoothing of the profile using locally weighted polynomial regression .", "cls variability from mean : this variable reflects variability around the mean value of all raw ( not smoothed ) cls measurements in the respective period ( 24-hour , diurnal , or nocturnal ) .", "diurnal / nocturnal variability : same as above but only during the awake and asleep periods , respectively , where the sleep cycle is determined from recorded sleep times in each individual s logbook and verified by the eye - blinking frequency ( present during waking ) and the presence of rhythmic ocular pulsation waves ( present during sleep ) .", "sleep - to - wake and wake - to - sleep slopes : calculated with a generalized linear model on raw measurements in units of mveq / hour from 1 hour before sleep or wake time to 1 hour after sleep or wake time , respectively .", "number of peaks : a peak is defined as a local maximum point in the smoothed curve .", "the calculation of the number of peaks occurs as follows : each trough is noted as the start of a peak .", "the pre- and post - cls gat iops were compared using the wilcoxon signed - rank test .", "whitney u - test : \n nocturnal versus diurnal mean variability from mean;wake - to - sleep versus sleep - to - wake slopes;number of nocturnal versus diurnal peaks ; and24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "nocturnal versus diurnal mean variability from mean ; wake - to - sleep versus sleep - to - wake slopes ; number of nocturnal versus diurnal peaks ; and 24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "fisher s exact test was used to compare the differences in the number of nocturnal peaks between those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications ."], ["cls variability from mean : this variable reflects variability around the mean value of all raw ( not smoothed ) cls measurements in the respective period ( 24-hour , diurnal , or nocturnal ) .", "diurnal / nocturnal variability : same as above but only during the awake and asleep periods , respectively , where the sleep cycle is determined from recorded sleep times in each individual s logbook and verified by the eye - blinking frequency ( present during waking ) and the presence of rhythmic ocular pulsation waves ( present during sleep ) .", "sleep - to - wake and wake - to - sleep slopes : calculated with a generalized linear model on raw measurements in units of mveq / hour from 1 hour before sleep or wake time to 1 hour after sleep or wake time , respectively .", "number of peaks : a peak is defined as a local maximum point in the smoothed curve .", "the calculation of the number of peaks occurs as follows : each trough is noted as the start of a peak ."], ["the pre- and post - cls gat iops were compared using the wilcoxon signed - rank test .", "whitney u - test : \n nocturnal versus diurnal mean variability from mean;wake - to - sleep versus sleep - to - wake slopes;number of nocturnal versus diurnal peaks ; and24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "nocturnal versus diurnal mean variability from mean ; wake - to - sleep versus sleep - to - wake slopes ; number of nocturnal versus diurnal peaks ; and 24-hour , diurnal , and nocturnal variability among prostaglandin users and nonusers .", "fisher s exact test was used to compare the differences in the number of nocturnal peaks between those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications ."], ["in the 18 subjects enrolled in the study , there were seven males and eleven females .", "gat iops before and after the cls wear were 15.32.2 and 13.81.7 mmhg , respectively , while on the same antiglaucoma medications ( p=0.05 ) .", "the mean rnfl thickness was 72.99.8 m and the mean deviation and pattern standard deviation on humphrey visual field analysis were 6.34.5 decibels ( db ) and 6.14.1 db , respectively .", "in the 18 subjects , 38.9% ( 7/18 ) used a single prostaglandin eyedrop administered in the evening , 44.4% ( 8/18 ) used a twice - daily non - prostaglandin eyedrop , and 17.6% ( 3/17 ) used both a nocturnal prostaglandin eyedrop plus a twice - daily antiglaucoma eyedrop .", "the mean 24-hour variability from the mean cls signal was 75.921.5 ( range : 35.7120.9 ; 95% confidence interval [ ci ] : 62.886.7 ) mveq .", "the mean nocturnal variability from the mean was 52.120.3 ( range : 23.9105.1 ; 95% ci : 41.662.5 ) mveq , which was 48.9% less than the mean diurnal variability from the mean ( 77.620.6 ) ( range : 43.9118.4 ; 95% ci : 66.988.2 ) mveq ( p=0.002 ) .", "there were no statistically significant differences in the mean diurnal , nocturnal , or 24-hour variability between those with and without prostaglandin analog treatment ( all p>0.1 ) ( table 1 ) .", "the mean number of peaks during sleep and daytime was 6.42.3 and 9.32.4 , respectively .", "the number of peaks was 54.7% less during the nocturnal period than the diurnal period ( p=0.001 ) .", "there was no significant difference between the frequencies of nocturnal peaks among those using a prostaglandin antiglaucoma medication versus those using other , non - prostaglandin antiglaucoma medications ( table 2 ) .", "the mean sleep time of subjects was from 8.53 pm 12.6 minutes to 7.06 am 3.6 minutes .", "the study population had a mean positive linear slope on their cls profile from wake - to - sleep with a mean of 53.242.9 mveq / hour , signifying an increase in the ocular dimensional profile when going to sleep .", "similarly , the population had a mean negative linear slope from sleep - to - wake with a mean of 42.547.6 mveq / hour , signifying a decrease in the ocular dimensional profile upon waking . on comparing the two slopes , the rate of increase when going to sleep was significantly greater than the rate of decrease upon waking ( p<0.001 ) ( table 3 ) ."], ["the ocular dimensional profiles of all 18 ntg subjects were unique , with individual peaks occurring at different time intervals throughout the day and night ; no two tracings were identical .", "the mean ocular dimensional profile of the study population increased during sleep ( positive wake - to - sleep slope ) and decreased upon waking ( negative sleep - to - wake slope ) , with the mean rate of increase during sleep being significantly greater than the rate of decrease upon waking ( p<0.001 ) .", "we are inclined to agree with previous postulations , wherein the increase in nocturnal iop was thought to be related to the gravitational pull of fluids to the eye and an increase in episcleral venous pressure during the supine sleep posture.19 through the use of the 24-hour cls , we affirm that the phenomenon of increased iop with supine posturing was consistent among ntg subjects under medical treatment .", "other authors have also observed the persistence of posture - induced iop changes despite treatment of ntg patients with iop - lowering medication.20 similarly , a previous japanese study reported , in a population of ntg subjects , that the iop spikes recorded from the habitual positions ( sitting up during the day and supine at night ) were positively correlated to the spikes induced from a postural - change test and after a water - drinking test.21 in contrast , renard et al reported that , in 27 subjects with suspected ntg , 24-hour iop monitoring revealed that 54.5% exhibited a diurnal acrophase and 36.4% exhibited a nocturnal acrophase , while 9.1% had no nychthemeral rhythm in the absence of medical treatment.22 the existence of concomitant obstructive sleep apnea syndrome in 80% of assessed patients could have influenced the observed iop rhythm in their cohort.22 before the availability of continuous 24-hour ocular dimensional profile recording , it has been reported that , in many glaucoma patients , the iop peaks occurred outside of office hours23,24 and that two - thirds of iop peaks in untreated glaucoma patients occurred during the nocturnal period.25 in our ntg population , we noted that there were 55% less peaks occurring nocturnally than diurnally ( p=0.001 ) .", "the reduction in the number of peaks at night in our ntg population could be related to the use of prostaglandin antiglaucoma eyedrops , since 57% of our subjects were at least on prostaglandin antiglaucoma eyedrops nocturnally .", "however , it may also be simply due to the absence of physical activity , eye blinking , and saccadic movements at night , as these parameters are known to cause iop spikes and fluctuations.26 other authors have suggested that the nocturnal iop and iop spikes may be related to visual field progression in ntg subjects.21 study on the effect of medical treatment on iop fluctuations has shown that prostaglandin antiglaucoma eyedrops offer the best sustained 24-hour iop reduction , while brimonidine offers the least.27 in our study , the mean nocturnal variability was significantly lower than the mean diurnal variability by 49% ( p=0.002 ) .", "our findings are in agreement with a previous study by pajic et al28 reporting higher coefficients of variation in cls data during the daytime than at nighttime in ntg patients .", "pajic et al reported reduced variability in nocturnal cls data independent of whether or not patients were on antiglaucoma medication.28 we have not recorded cls profiles in our patients without medical treatment and therefore do not know whether the same is valid for this patient cohort ; however , it seems plausible that the lower level of activity during sleep provides a natural reduction in variability , which iop - lowering medical treatment may further influence .", "holl et al recorded cls profiles in primary open - angle glaucoma and ocular hypertension patients and found no difference in 24-hour , diurnal , or nocturnal standard deviation of cls values in the presence of prostaglandin analog treatment as compared to without treatment.29 the variability of diurnal versus nocturnal cls values was not evaluated .", "thus , in this patient cohort , it seems that , while the overall ocular dimensional profile was higher at night , the number of peaks and variability from the mean were significantly reduced at night for ntg subjects on medical treatment .", "it was interesting to note that the gat iop after cls wear ( 13.81.7 mmhg ) was lower ( p=0.05 ) than the iop before the cls wear ( 15.32.2 mmhg ) , although the readings were measured exactly 24 hours apart .", "this seemingly lower iop after the cls use was short of statistical significance and can be explained by day - to - day variations in iop .", "there was no sample size calculation , and the relatively small sample size of the study did not allow for evaluation of differences in iop - related pattern nor stratification by the type of treatment .", "furthermore , only one 24-hour measurement with the cls was performed , therefore we could not ascertain the reproducibility of the 24-hour iop profile . at present , the ocular dimensional profile is something that can not be translated into iop and therefore has no validation for use in clinical practice .", "additional clinical research needs to be done before we can state ( based on evidence ) that the cls can be useful in optimizing the timing of medical therapy for individual patients .", "nevertheless , this is one of the few publications in the literature analyzing the 24-hour ocular dimensional profile on a continuous basis in ntg subjects receiving medical treatment ."], ["continuous 24-hour ocular dimensional profiles recorded in ntg patients on medical treatment revealed that the profile increased during sleep and decreased upon waking .", "there was a 50% reduction in both the variability from the mean and number of peaks during sleep as compared to daytime ."]]}
{"article_id": "PMC3969343", "article_text": ["cyclodialysis cleft is a rare clinical finding , but one that can have significant ocular complications , including decreased vision , shallowing of the anterior chamber , corneal edema , hypotony , choroidal effusions , maculopathy , vascular tortuosity , and optic disc edema .", "while it can be caused by surgical manipulation of the eye , it is more frequently a consequence of trauma.1 traumatic injury to the eye results in separation of the ciliary body from the scleral spur , which creates an aberrant outflow pathway for aqueous humor through the suprachoroidal space and often leads to hypotony .", "cyclodialysis cleft has been reported to occur in 3.4% of cases of ocular injury or globe rupture.2 additionally , one large review of 291 cases involving cyclodialysis suggested that hypotony occurs 9% of the time.3 medical treatment with atropine ophthalmic drops and laser therapy to promote reattachment of the ciliary body to the scleral spur are options for repair of this entity , but larger and more chronic cyclodialyses often require surgical repair.4,5 because of their rarity , reports on surgical repair techniques in the literature are limited .", "we share a novel , simple surgical approach to management of a case of chronic traumatic cyclodialysis cleft with a successful outcome .", "a 70-year - old caucasian male followed at the martinsburg veterans affair hospital ophthalmology clinic , martinsburg , west virginia , usa , since 2002 was found consistently to have intraocular pressure ( iop ) asymmetry , with the right eye iop ranging from 615 mmhg and usually having an iop 04 mmhg lower than the left eye .", "he was monitored for primary open - angle glaucoma and was on latanoprost eye drops in the left eye for field defects in that eye . in april 2008 ,", "the patient s history was significant for trauma to the right eye with a piece of wood over 30 years ago , which was thought to be the cause of the cleft .", "while the patient had hypotony , he maintained good visual acuity , ranging from 20/25 to 20/40 , and was therefore observed for approximately 3 years until december 2011 . at this visit ,", "his visual acuity dropped to 20/150 and he clinically developed descemet s folds with a shallow anterior chamber and an iop of 02 mmhg .", "on dilated fundus exam , he had a shallow choroidal hemorrhage and effusion in the far nasal periphery .", "the patient was initially treated medically with atropine and prednisolone acetate 1% ophthalmic drops for approximately 2 months , without improvement in iop and with persistence of choroidal hemorrhage and effusion .", "postoperatively , the choroidal effusion resolved , but the patient s iop did not improve . on repeat gonioscopy", "therefore , the patient was taken for additional surgical repair of the persistent cleft by the same technique , this time with successful closure .", "on postoperative day 1 following the second surgery , the patient complained of severe eye pain and nausea , and was found to have an lop of 58 mmhg .", "the patient was treated with topical anti - glaucoma drops and oral acetazolamide , with reduction in the iop to the mid - teens and resolution of his pain . on exam", ", there was no distortion of the pupil by sutures and the anterior chamber appeared deep ( figure 1 ) .", "the patient was subsequently tapered off the oral acetazolamide and topical drops , with stabilization of the pressure in the mid - teens and maintenance of 20/70 visual acuity at 1 year postoperatively .", "final visual acuity was limited by subsequent progression of ocular comorbidities in that eye , including cataract and proliferative diabetic retinopathy .", "a nasal conjunctival peritomy was performed , and dissection down to bare sclera was carried out and continued superotemporally to encompass the region of the cyclodialysis cleft .", "a 10 - 0 nylon suture on cs175 - 6 needle ( ethicon ) , which is a 7.0 mm -circle needle , was selected for cyclopexy .", "interrupted 10 - 0 nylon sutures were radially passed through the corneal side of the limbus , then through the iris root and ciliary body , and carried out through the sclera on the opposite side of the limbus ( figure 3 ) . to ensure that ciliary body was incorporated into each pass ,", "the sutures were tugged on intraoperatively and the iris root was evaluated for movement with mechanical pull", ". additionally , as more sutures were placed , the eye appeared to become more firm .", "we found the needle type to have a curvature and size that was very helpful for the angle and length of the pass required .", "a nasal conjunctival peritomy was performed , and dissection down to bare sclera was carried out and continued superotemporally to encompass the region of the cyclodialysis cleft .", "a 10 - 0 nylon suture on cs175 - 6 needle ( ethicon ) , which is a 7.0 mm -circle needle , was selected for cyclopexy .", "interrupted 10 - 0 nylon sutures were radially passed through the corneal side of the limbus , then through the iris root and ciliary body , and carried out through the sclera on the opposite side of the limbus ( figure 3 ) . to ensure that ciliary body was incorporated into each pass ,", "the sutures were tugged on intraoperatively and the iris root was evaluated for movement with mechanical pull .", "additionally , as more sutures were placed , the eye appeared to become more firm .", "we found the needle type to have a curvature and size that was very helpful for the angle and length of the pass required .", "multiple approaches to cleft closure have been described in the literature , including medical management , laser photocoagulation , cryotherapy , trans - scleral diathermy , gas tamponade , and anterior scleral buckle , along with various surgical repair techniques.6 while different surgical approaches have been described , many are technically challenging , particularly in the hypotonous eye . in this case report", ", we discuss the repair of a chronic traumatic cyclodialysis with indirect surgical cyclopexy , which we found to be technically easier and less invasive than other reported techniques , but equally successful .", "compared to the direct cyclopexy described in a review by ioannidis and barton,6 our indirect technique eliminated the need for direct visualization of the ciliary body , thereby reducing technical difficulty along with bleeding and wound dehiscence risk . additionally , compared to cyclopexy described by mccannel,7 our approach eliminated the additional need for clear corneal stab incision with retrieval of suture , which can also be difficult in a soft , hypotonous eye and may prolong surgical time . while our technique was successful , it required two operations to achieve successful cleft closure with improvement in iop .", "our first attempt failed , likely due to the spacing of our sutures in one particular quadrant , which we determined were too far apart .", "additionally , we used a needle with less curvature and length initially . we found that spacing the sutures approximately 1 mm apart achieved good apposition of ciliary body to scleral spur and , ultimately , closure of the cleft with restoration of physiologic iop .", "the length and curvature of the cs175 - 6 ( ethicon , cincinnati , ohio , usa ) needle also assisted with passage of each suture through the targeted tissues .", "the patient s postoperative iop spike after the second surgery was suggestive of successful closure of the cleft.1 additionally , successful closure was later confirmed by gonioscopy .", "another challenge to our case was the chronicity of the injury which was sustained 30 years prior to presentation as per the patient s self - reported history .", "there was concern that , even with successful anatomic closure , iop and visual function may not be restored due to chronic ischemic changes to the ciliary body . however , our repair achieved both anatomic and functional success , suggesting that even chronic cyclodialysis clefts can be effectively repaired .", "delgado et al reported a similar finding in a case of hypotony maculopathy secondary to cyclodialysis that was resolved with cleft closure 7 years later.8 cyclodialysis is a vision - threatening ocular injury that can often be difficult to treat medically .", "surgical repair is often necessary but can be technically difficult and challenging , particularly because of how rarely it is encountered in clinical practice .", "we present a simple , less - invasive approach to surgical cleft closure that was successful anatomically and functionally .", "additionally , we demonstrate clinical success with cleft closure in an eye with chronic hypotony ."], "abstract_text": ["<S> cyclodialysis cleft is a rare clinical finding and therefore , reports on surgical repair techniques in the literature are limited . </S>", "<S> additionally , hypotony can make repair technically challenging . </S>", "<S> we share a novel , simple surgical approach to management of a case of chronic traumatic cyclodialysis cleft with a successful outcome . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Surgical technique", "Discussion"], "sections": [["cyclodialysis cleft is a rare clinical finding , but one that can have significant ocular complications , including decreased vision , shallowing of the anterior chamber , corneal edema , hypotony , choroidal effusions , maculopathy , vascular tortuosity , and optic disc edema .", "while it can be caused by surgical manipulation of the eye , it is more frequently a consequence of trauma.1 traumatic injury to the eye results in separation of the ciliary body from the scleral spur , which creates an aberrant outflow pathway for aqueous humor through the suprachoroidal space and often leads to hypotony .", "cyclodialysis cleft has been reported to occur in 3.4% of cases of ocular injury or globe rupture.2 additionally , one large review of 291 cases involving cyclodialysis suggested that hypotony occurs 9% of the time.3 medical treatment with atropine ophthalmic drops and laser therapy to promote reattachment of the ciliary body to the scleral spur are options for repair of this entity , but larger and more chronic cyclodialyses often require surgical repair.4,5 because of their rarity , reports on surgical repair techniques in the literature are limited .", "we share a novel , simple surgical approach to management of a case of chronic traumatic cyclodialysis cleft with a successful outcome ."], ["a 70-year - old caucasian male followed at the martinsburg veterans affair hospital ophthalmology clinic , martinsburg , west virginia , usa , since 2002 was found consistently to have intraocular pressure ( iop ) asymmetry , with the right eye iop ranging from 615 mmhg and usually having an iop 04 mmhg lower than the left eye .", "he was monitored for primary open - angle glaucoma and was on latanoprost eye drops in the left eye for field defects in that eye . in april 2008 ,", "the patient s history was significant for trauma to the right eye with a piece of wood over 30 years ago , which was thought to be the cause of the cleft .", "while the patient had hypotony , he maintained good visual acuity , ranging from 20/25 to 20/40 , and was therefore observed for approximately 3 years until december 2011 . at this visit ,", "his visual acuity dropped to 20/150 and he clinically developed descemet s folds with a shallow anterior chamber and an iop of 02 mmhg .", "on dilated fundus exam , he had a shallow choroidal hemorrhage and effusion in the far nasal periphery .", "the patient was initially treated medically with atropine and prednisolone acetate 1% ophthalmic drops for approximately 2 months , without improvement in iop and with persistence of choroidal hemorrhage and effusion .", "postoperatively , the choroidal effusion resolved , but the patient s iop did not improve . on repeat gonioscopy", "therefore , the patient was taken for additional surgical repair of the persistent cleft by the same technique , this time with successful closure .", "on postoperative day 1 following the second surgery , the patient complained of severe eye pain and nausea , and was found to have an lop of 58 mmhg .", "the patient was treated with topical anti - glaucoma drops and oral acetazolamide , with reduction in the iop to the mid - teens and resolution of his pain . on exam", ", there was no distortion of the pupil by sutures and the anterior chamber appeared deep ( figure 1 ) .", "the patient was subsequently tapered off the oral acetazolamide and topical drops , with stabilization of the pressure in the mid - teens and maintenance of 20/70 visual acuity at 1 year postoperatively .", "final visual acuity was limited by subsequent progression of ocular comorbidities in that eye , including cataract and proliferative diabetic retinopathy .", "a nasal conjunctival peritomy was performed , and dissection down to bare sclera was carried out and continued superotemporally to encompass the region of the cyclodialysis cleft .", "a 10 - 0 nylon suture on cs175 - 6 needle ( ethicon ) , which is a 7.0 mm -circle needle , was selected for cyclopexy .", "interrupted 10 - 0 nylon sutures were radially passed through the corneal side of the limbus , then through the iris root and ciliary body , and carried out through the sclera on the opposite side of the limbus ( figure 3 ) . to ensure that ciliary body was incorporated into each pass ,", "the sutures were tugged on intraoperatively and the iris root was evaluated for movement with mechanical pull", ". additionally , as more sutures were placed , the eye appeared to become more firm .", "we found the needle type to have a curvature and size that was very helpful for the angle and length of the pass required ."], ["a nasal conjunctival peritomy was performed , and dissection down to bare sclera was carried out and continued superotemporally to encompass the region of the cyclodialysis cleft .", "a 10 - 0 nylon suture on cs175 - 6 needle ( ethicon ) , which is a 7.0 mm -circle needle , was selected for cyclopexy .", "interrupted 10 - 0 nylon sutures were radially passed through the corneal side of the limbus , then through the iris root and ciliary body , and carried out through the sclera on the opposite side of the limbus ( figure 3 ) . to ensure that ciliary body was incorporated into each pass ,", "the sutures were tugged on intraoperatively and the iris root was evaluated for movement with mechanical pull .", "additionally , as more sutures were placed , the eye appeared to become more firm .", "we found the needle type to have a curvature and size that was very helpful for the angle and length of the pass required ."], ["multiple approaches to cleft closure have been described in the literature , including medical management , laser photocoagulation , cryotherapy , trans - scleral diathermy , gas tamponade , and anterior scleral buckle , along with various surgical repair techniques.6 while different surgical approaches have been described , many are technically challenging , particularly in the hypotonous eye . in this case report", ", we discuss the repair of a chronic traumatic cyclodialysis with indirect surgical cyclopexy , which we found to be technically easier and less invasive than other reported techniques , but equally successful .", "compared to the direct cyclopexy described in a review by ioannidis and barton,6 our indirect technique eliminated the need for direct visualization of the ciliary body , thereby reducing technical difficulty along with bleeding and wound dehiscence risk . additionally , compared to cyclopexy described by mccannel,7 our approach eliminated the additional need for clear corneal stab incision with retrieval of suture , which can also be difficult in a soft , hypotonous eye and may prolong surgical time . while our technique was successful , it required two operations to achieve successful cleft closure with improvement in iop .", "our first attempt failed , likely due to the spacing of our sutures in one particular quadrant , which we determined were too far apart .", "additionally , we used a needle with less curvature and length initially . we found that spacing the sutures approximately 1 mm apart achieved good apposition of ciliary body to scleral spur and , ultimately , closure of the cleft with restoration of physiologic iop .", "the length and curvature of the cs175 - 6 ( ethicon , cincinnati , ohio , usa ) needle also assisted with passage of each suture through the targeted tissues .", "the patient s postoperative iop spike after the second surgery was suggestive of successful closure of the cleft.1 additionally , successful closure was later confirmed by gonioscopy .", "another challenge to our case was the chronicity of the injury which was sustained 30 years prior to presentation as per the patient s self - reported history .", "there was concern that , even with successful anatomic closure , iop and visual function may not be restored due to chronic ischemic changes to the ciliary body . however , our repair achieved both anatomic and functional success , suggesting that even chronic cyclodialysis clefts can be effectively repaired .", "delgado et al reported a similar finding in a case of hypotony maculopathy secondary to cyclodialysis that was resolved with cleft closure 7 years later.8 cyclodialysis is a vision - threatening ocular injury that can often be difficult to treat medically .", "surgical repair is often necessary but can be technically difficult and challenging , particularly because of how rarely it is encountered in clinical practice .", "we present a simple , less - invasive approach to surgical cleft closure that was successful anatomically and functionally .", "additionally , we demonstrate clinical success with cleft closure in an eye with chronic hypotony ."]]}
{"article_id": "PMC3018865", "article_text": ["acoh ( 3.00 mmol ) was added to a solution of galactoside 20 or 13 ( 1.57 mmol ) in acetone ( 3.5 ml ) and meoh ( 4.7 ml ) at 0 c .", "after 8 h , the reaction was quenched by the addition of nahco3 ( 5.95 mmol ) and then filtered .", "after concentration of the filtrate under reduced pressure , the resulting oil was purified by flash column chromatography ( etoac in hexane ) to afford alcohol 21 ( from 20 ) or 26 ( from 13 ) as a colorless oil .", "pph3 ( 1.86 mmol ) , diad ( 1.86 mmol ) , and dppa ( 1.86 mmol ) were added sequentially to a cooled solution of alcohol 21 or 26 ( 0.90 mmol ) in thf ( 20 ml ) .", "concentration under reduced pressure followed by purification of the residue by flash column chromatography ( etoac in hexane ) afforded azide 22 ( from 21 ) or 25 ( from 26 ) as a colorless oil .", "tfa ( 0.50 ml , 6.6 mmol ) was added dropwise over 5 min to a solution of azide 22 ( 200 mg , 0.19 mmol ) in ch2cl2 ( 5 ml ) at rt .", "after 30 min , the reaction mixture was concentrated under reduced pressure to afford pentaol 9 as a colorless oil ( 115 mg , quant . ) : [ ]d + 12.4 ( c 0.5 , cdcl3:cd3od , 2:1 ) ; max(film ) ( cm ) 3282s br ( oh ) , 2114s ( n3 ) , 1696 m ( c = o ) ; h nmr ( 300 mhz , cdcl3:cd3od , 2:1 ) 0.85 ( t , j = 6.0 hz , 3h ) , 1.181.39 ( stack , 22h ) , 1.401.71 ( stack , 4h ) , 1.44 ( s , 9h ) , 3.26 ( a of abx , jab = 12.6 hz , jax = 4.9 hz , 1h ) , 3.513.64 ( stack , 3h ) , 3.653.98 ( stack , 7h ) , 4.89 ( d , j = 3.3 hz , 1h ) ; c nmr ( 100 mhz , cdcl3:cd3od , 2:1 ) 14.3 ( ch3 ) , 23.2 ( ch2 ) , 26.4 ( ch2 ) , 28.6 ( ch3 ) , [ 29.9 , 30.2 , 32.4 , 32.8 ( ch2 , resonance overlap ) ] , 51.7 ( ch ) , 51.8 ( ch2 ) , 68.3 ( ch2 ) , 69.3 ( ch ) , 70.4 ( ch ) , 70.6 ( 2  ch , resonance overlap ) , 72.5 ( ch ) , 75.3 ( ch ) , 80.1 ( c ) , 156.8 ( c ) ; ms ( tof es+ ) m / z 627.3 ( [ m + na ] , 100% ) ; hrms ( tof es+ ) calcd for c29h56n4o9na [ m + na ] 627.3945 , found 627.3956 .", "tfa ( 1.0 ml , 13.2 mmol ) was added dropwise over 5 min to azide 22 ( 400 mg , 0.38 mmol ) at rt .", "the resulting colorless oil was used in the next step without further purification ( 192 mg , quant . ) .", "tfa ( 0.50 ml , 6.6 mmol ) was added dropwise over 5 min to azide 9 ( 114 mg , 0.19 mmol ) at rt .", "the resulting colorless oil was used in the next step without further purification ( 96 mg , quant . ) ."], "abstract_text": ["<S> a concise route to the 6-azido-6-deoxy--galactosyl - phytosphingosine derivative 9 is reported . </S>", "<S> orthogonal protection of the two amino groups allows elaboration of 9 into a range of 6-n - derivatized -galactosyl ceramides by late - stage introduction of the acyl chain of the ceramide and the 6-n - group in the sugar headgroup . </S>", "<S> biologically active glycolipids 6 and 8 have been synthesized to illustrate the applicability of the approach . </S>"], "labels": null, "section_names": ["Experimental Section"], "sections": [["acoh ( 3.00 mmol ) was added to a solution of galactoside 20 or 13 ( 1.57 mmol ) in acetone ( 3.5 ml ) and meoh ( 4.7 ml ) at 0 c .", "after 8 h , the reaction was quenched by the addition of nahco3 ( 5.95 mmol ) and then filtered .", "after concentration of the filtrate under reduced pressure , the resulting oil was purified by flash column chromatography ( etoac in hexane ) to afford alcohol 21 ( from 20 ) or 26 ( from 13 ) as a colorless oil .", "pph3 ( 1.86 mmol ) , diad ( 1.86 mmol ) , and dppa ( 1.86 mmol ) were added sequentially to a cooled solution of alcohol 21 or 26 ( 0.90 mmol ) in thf ( 20 ml ) .", "concentration under reduced pressure followed by purification of the residue by flash column chromatography ( etoac in hexane ) afforded azide 22 ( from 21 ) or 25 ( from 26 ) as a colorless oil .", "tfa ( 0.50 ml , 6.6 mmol ) was added dropwise over 5 min to a solution of azide 22 ( 200 mg , 0.19 mmol ) in ch2cl2 ( 5 ml ) at rt .", "after 30 min , the reaction mixture was concentrated under reduced pressure to afford pentaol 9 as a colorless oil ( 115 mg , quant . ) : [ ]d + 12.4 ( c 0.5 , cdcl3:cd3od , 2:1 ) ; max(film ) ( cm ) 3282s br ( oh ) , 2114s ( n3 ) , 1696 m ( c = o ) ; h nmr ( 300 mhz , cdcl3:cd3od , 2:1 ) 0.85 ( t , j = 6.0 hz , 3h ) , 1.181.39 ( stack , 22h ) , 1.401.71 ( stack , 4h ) , 1.44 ( s , 9h ) , 3.26 ( a of abx , jab = 12.6 hz , jax = 4.9 hz , 1h ) , 3.513.64 ( stack , 3h ) , 3.653.98 ( stack , 7h ) , 4.89 ( d , j = 3.3 hz , 1h ) ; c nmr ( 100 mhz , cdcl3:cd3od , 2:1 ) 14.3 ( ch3 ) , 23.2 ( ch2 ) , 26.4 ( ch2 ) , 28.6 ( ch3 ) , [ 29.9 , 30.2 , 32.4 , 32.8 ( ch2 , resonance overlap ) ] , 51.7 ( ch ) , 51.8 ( ch2 ) , 68.3 ( ch2 ) , 69.3 ( ch ) , 70.4 ( ch ) , 70.6 ( 2  ch , resonance overlap ) , 72.5 ( ch ) , 75.3 ( ch ) , 80.1 ( c ) , 156.8 ( c ) ; ms ( tof es+ ) m / z 627.3 ( [ m + na ] , 100% ) ; hrms ( tof es+ ) calcd for c29h56n4o9na [ m + na ] 627.3945 , found 627.3956 .", "tfa ( 1.0 ml , 13.2 mmol ) was added dropwise over 5 min to azide 22 ( 400 mg , 0.38 mmol ) at rt .", "the resulting colorless oil was used in the next step without further purification ( 192 mg , quant . ) .", "tfa ( 0.50 ml , 6.6 mmol ) was added dropwise over 5 min to azide 9 ( 114 mg , 0.19 mmol ) at rt .", "the resulting colorless oil was used in the next step without further purification ( 96 mg , quant . ) ."]]}
{"article_id": "PMC3813587", "article_text": ["ventriculoperitoneal and cystoperitoneal ( cp ) shunts are commonly used for the treatment of hydrocephalus and symptomatic intracranial cysts .", "abdominal complications , including intestinal volvulus , pseudocyst formation and migration into the gastrointestinal tract have been reported , accounting for 25% of shunt - related complications .", "bowel perforation by the shunt catheter is uncommon and accounts for 0.010.07% of all abdominal complications [ 13 ] .", "breast - related complications represent a class of thoracic shunt complication and are characterized by breast cerebrospinal fluid ( csf ) pseudocyst formation , csf galactorrhoea and shunt obstruction .", "we report a rare case of acute mastitis caused by enteric organisms passing through a cp shunt catheter , which had penetrated the transverse colon , and was related to a previous failed attempt to remove the shunt resulting in a divided catheter .", "the patient is a 56-year - old woman who underwent a surgical treatment of a lateral ventricle meningioma at the age of 29 .", "the postoperative course was uneventful and she was well without shunt - related problems for 26 years .", "she developed a brain abscess related to the shunt catheter at the age of 55 .", "a surgical removal of the catheter was attempted , but was not technically possible , and only the segment from the clavicle to the breast was removed with ligation of the two remaining ends .", "a year later , she was taken to a local hospital with continuous right - breast pain .", "the diagnosis of acute mastitis was made and she was referred to our institution for further examination . on presentation ,", "her temperature was 36.7c , and laboratory tests revealed a decreased platelet count of 103 000/l , elevated ast of 97 u / l , alt of 72 u / l and c - reactive protein level of 1.4 mg / dl .", "cultures taken from the abscess showed enteric organisms including enterococcus avium , klebsiella oxytoca and bacillus .", "abdominal ct showed that the shunt catheter was in the splenic flexure of the transverse colon with no other abnormalities ( fig .  2 ) .", "colonoscopy was performed to survey the colon and the shunt catheter was found inside the lumen at the splenic flexure ( fig . ", "acute mastitis caused by enteric organisms which had migrated through the shunt catheter after penetration into the transverse colon was the presumptive diagnosis , and the catheter removed under general anesthesia .", "the breast to the abdomen segment of the catheter was exteriorized through the right - anterior chest wall without laparotomy .", "the shunt catheter ( white arrow ) extended through the right - rectus sheath ( a ) , crossed the midline to the left side of the abdomen ( a , b ) and was inside the splenic flexure of the colon ( b , c ) .", "figure  3:colonoscopy showed the distal catheter within the transverse colon , 40 cm proximal to the anal verge ( a ) .", "an inflammatory polyp was present at the penetration site in the colon wall ( b ) .", "the shunt catheter ( white arrow ) extended through the right - rectus sheath ( a ) , crossed the midline to the left side of the abdomen ( a , b ) and was inside the splenic flexure of the colon ( b , c ) .", "colonoscopy showed the distal catheter within the transverse colon , 40 cm proximal to the anal verge ( a ) .", "an inflammatory polyp was present at the penetration site in the colon wall ( b ) .", "the penetration of shunt catheters into the intestine at the distal end is rare with a reported incidence between 0.01 and 0.07% .", "the mortality rate may be high , up to 15% , due to intracranial or intra - abdominal infections .", "several factors have been found to be responsible for this rare complication , including chronic irritation of the gastrointestinal tract , prior abdominal surgery or silicone allergy [ 2 , 3 ] .", "csf leakage and retrograde flow from the peritoneal cavity to the breast may cause breast - related complications .", "this is the first report of case with acute mastitis caused by enteric organisms migrating through a cp shunt catheter that had penetrated the transverse colon .", "the diagnosis of bowel perforation by a shunt may not be easy to make , unless the shunt protrudes from the anus [ 5 , 6 ] .", "prolonged diarrhea of unknown etiology , as well as abdominal symptoms , serves as warning signs of possible bowel perforation .", "ct can play a crucial role in the diagnosis of bowel perforation . in the present case ,", "these findings suggested that the infection was due to the retrograde flow from the colon .", "the development of mastitis was facilitated by previous division of the catheter , leaving a ligated end .", "presumably , the ligature became dislodged , leaving a direct connection from the breast tissue to the distal end of the catheter .", "previous study reports that the shunt catheter can be directly removed percutaneously in 69% ( 31/45 ) , whereas 17% ( 8/45 ) of patients require a laparotomy and repair of the bowel . if there is evidence of significant abdominal infection , such as an abscess or life - threatening peritonitis , the fistulous opening may not close spontaneously , warranting laparotomy . in the present case ,", "previous insufficient surgical treatment such as a partial removal of the catheter contributed to this shunt - related complication .", "however , it is common surgical practice to permit the distal end of a shunt catheter to remain in situ in order to simplify the surgical procedure .", "although this practice is most commonly uncomplicated , it is not unreasonable to suspect that the tube may irritate the serosal surface of the colon leading to chronic inflammation .", "it is speculated that local inflammation owing to repeated irritation of the bowel wall by the catheter tip might have contributed to the subsequent perforation . in conclusion ,", "a patient who presents with acute mastitis and has previously undergone shunt placement should undergo assessment of the abdominal segment of the catheter ."], "abstract_text": ["<S> we report a rare case of acute mastitis caused by enteric organisms passing through a cystoperitoneal shunt catheter , which had penetrated into the colon . </S>", "<S> the patient is a 56-year - old woman who underwent shunt placement for cyst formation after surgery for meningioma at the age of 29 . after 26 years , she suffered from a brain abscess and an attempt was made to surgically remove the indwelling catheter . </S>", "<S> only part of the catheter could be removed , leaving a divided and ligated catheter in situ . </S>", "<S> a year later , she described right - breast pain . </S>", "<S> ct showed that the catheter had migrated into the colon , followed by colonoscopy confirming that the catheter had indeed penetrated the colon . </S>", "<S> the breast to the abdomen segment of the catheter was exteriorized through the right - anterior chest wall without laparotomy . </S>", "<S> a patient who presents with acute mastitis and has previously undergone shunt surgery should have a careful assessment of the entire catheter . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["ventriculoperitoneal and cystoperitoneal ( cp ) shunts are commonly used for the treatment of hydrocephalus and symptomatic intracranial cysts .", "abdominal complications , including intestinal volvulus , pseudocyst formation and migration into the gastrointestinal tract have been reported , accounting for 25% of shunt - related complications .", "bowel perforation by the shunt catheter is uncommon and accounts for 0.010.07% of all abdominal complications [ 13 ] .", "breast - related complications represent a class of thoracic shunt complication and are characterized by breast cerebrospinal fluid ( csf ) pseudocyst formation , csf galactorrhoea and shunt obstruction .", "we report a rare case of acute mastitis caused by enteric organisms passing through a cp shunt catheter , which had penetrated the transverse colon , and was related to a previous failed attempt to remove the shunt resulting in a divided catheter ."], ["the patient is a 56-year - old woman who underwent a surgical treatment of a lateral ventricle meningioma at the age of 29 .", "the postoperative course was uneventful and she was well without shunt - related problems for 26 years .", "she developed a brain abscess related to the shunt catheter at the age of 55 .", "a surgical removal of the catheter was attempted , but was not technically possible , and only the segment from the clavicle to the breast was removed with ligation of the two remaining ends .", "a year later , she was taken to a local hospital with continuous right - breast pain .", "the diagnosis of acute mastitis was made and she was referred to our institution for further examination . on presentation ,", "her temperature was 36.7c , and laboratory tests revealed a decreased platelet count of 103 000/l , elevated ast of 97 u / l , alt of 72 u / l and c - reactive protein level of 1.4 mg / dl .", "cultures taken from the abscess showed enteric organisms including enterococcus avium , klebsiella oxytoca and bacillus .", "abdominal ct showed that the shunt catheter was in the splenic flexure of the transverse colon with no other abnormalities ( fig .  2 ) .", "colonoscopy was performed to survey the colon and the shunt catheter was found inside the lumen at the splenic flexure ( fig . ", "acute mastitis caused by enteric organisms which had migrated through the shunt catheter after penetration into the transverse colon was the presumptive diagnosis , and the catheter removed under general anesthesia .", "the breast to the abdomen segment of the catheter was exteriorized through the right - anterior chest wall without laparotomy .", "the shunt catheter ( white arrow ) extended through the right - rectus sheath ( a ) , crossed the midline to the left side of the abdomen ( a , b ) and was inside the splenic flexure of the colon ( b , c ) .", "figure  3:colonoscopy showed the distal catheter within the transverse colon , 40 cm proximal to the anal verge ( a ) .", "an inflammatory polyp was present at the penetration site in the colon wall ( b ) .", "the shunt catheter ( white arrow ) extended through the right - rectus sheath ( a ) , crossed the midline to the left side of the abdomen ( a , b ) and was inside the splenic flexure of the colon ( b , c ) .", "colonoscopy showed the distal catheter within the transverse colon , 40 cm proximal to the anal verge ( a ) .", "an inflammatory polyp was present at the penetration site in the colon wall ( b ) ."], ["the penetration of shunt catheters into the intestine at the distal end is rare with a reported incidence between 0.01 and 0.07% .", "the mortality rate may be high , up to 15% , due to intracranial or intra - abdominal infections .", "several factors have been found to be responsible for this rare complication , including chronic irritation of the gastrointestinal tract , prior abdominal surgery or silicone allergy [ 2 , 3 ] .", "csf leakage and retrograde flow from the peritoneal cavity to the breast may cause breast - related complications .", "this is the first report of case with acute mastitis caused by enteric organisms migrating through a cp shunt catheter that had penetrated the transverse colon .", "the diagnosis of bowel perforation by a shunt may not be easy to make , unless the shunt protrudes from the anus [ 5 , 6 ] .", "prolonged diarrhea of unknown etiology , as well as abdominal symptoms , serves as warning signs of possible bowel perforation .", "ct can play a crucial role in the diagnosis of bowel perforation . in the present case ,", "these findings suggested that the infection was due to the retrograde flow from the colon .", "the development of mastitis was facilitated by previous division of the catheter , leaving a ligated end .", "presumably , the ligature became dislodged , leaving a direct connection from the breast tissue to the distal end of the catheter .", "previous study reports that the shunt catheter can be directly removed percutaneously in 69% ( 31/45 ) , whereas 17% ( 8/45 ) of patients require a laparotomy and repair of the bowel . if there is evidence of significant abdominal infection , such as an abscess or life - threatening peritonitis , the fistulous opening may not close spontaneously , warranting laparotomy . in the present case ,", "previous insufficient surgical treatment such as a partial removal of the catheter contributed to this shunt - related complication .", "however , it is common surgical practice to permit the distal end of a shunt catheter to remain in situ in order to simplify the surgical procedure .", "although this practice is most commonly uncomplicated , it is not unreasonable to suspect that the tube may irritate the serosal surface of the colon leading to chronic inflammation .", "it is speculated that local inflammation owing to repeated irritation of the bowel wall by the catheter tip might have contributed to the subsequent perforation . in conclusion ,", "a patient who presents with acute mastitis and has previously undergone shunt placement should undergo assessment of the abdominal segment of the catheter ."]]}
{"article_id": "PMC4437384", "article_text": ["we explore , in mice , the possibility of diagnosing an early - melanoma tumor and following its growth by analyzing the changes that occur throughout the pathological process in vocs excreted in urine and feces .", "we report here total of 29 potential biomarker candidates of melanoma ; 25 of these are novel , and 4 have been previously reported by other research groups in relation to vocs detected from cultures of human melanoma cells .", "the chemical identity of only 24 of the 29 possible biomarker candidates was revealed with high confidence .", "this study will contribute to the development of a noninvasive and reliable diagnostic procedure to detect melanoma in the urine of patients .", "melanoma accounts for less than 2% of all skin cancers , but for the vast majority of deaths from skin cancer . in the united states", "alone , it is estimated that 76,100 individuals will be diagnosed with melanoma during 2014 and that 9,710 patients will die from it .", "early detection of malignant melanoma is the key factor in reducing mortality from this cancer .", "although histopathological examination is the gold standard for diagnosis of melanoma , additional , noninvasive approaches should be developed . here", ", we explore the possibility of exploiting volatile organic compounds ( vocs ) to develop such an approach ; hundreds of vocs are emitted from the human body and usually reflect the metabolic condition of the individual .", "therefore , pathological processes , such as cancer , are expected to influence the voc fingerprint of patients either by changing the ratio between different vocs or by producing new vocs .", "it has been reported that dog scan identifies , by using their olfaction sense , melanoma on the skin of patients   or melanoma samples hidden on healthy subjects .", "used gas sensor array ( also known as an  electronic nose  ) to differentiate between melanomas and nevus lesions .", "those authors compared the lesion with the adjacent skin region to reduce the skin headspace variability .", "collected biopsy samples and compared the vocs released from melanoma tissue to those released from nevus and normal tissue , using matching skin as a control .", "those authors found 32 potential biomarker candidates ; the concentration of 9 vocs increased in the presence of melanoma , and 23 vocs were detected only when melanoma cells existed and were not detected in normal cells .", "the authors used the nist 2.0 mass spectral database , with a 60% quality factor for chemical identification of the possible biomarker candidates ; however , they did not report the quality factor obtained for the different compounds by mass spectrometry ( ms ) . in a recent study ,", "kwak et al .   compared human melanoma cells to normal melanocytes cultured in vitro . by using gas chromatography- ( gc- ) ms , those authors found increased levels of both isoamyl alcohol and isovaleric acid in the headspace over melanoma cells .", "the authors also found that the melanoma cells produced some unique compounds , such as dimethyldisulfide and dimethyltrisulfide . in the present study", ", we analyzed the vocs emitted from the urine and feces of mice before ( healthy group ) and after ( cancer group ) subcutaneous injection of b16 melanoma cells .", "the present work will serve as a basis to analyze possible vocs in human patients .", "we expect some voc to be shared between the species and new and different vocs to be detected .", "the sample classification used in the present study is summarized in table 1 . the analysis of the urine samples of healthy and tumor - bearing mice revealed some differences in the vocs composition .", "there was no significant difference between the first two samples ( samples a and b ) , which were taken before the injection of the b16 melanoma cells , and the first sample taken after injection ( sample c ) . in the second sample taken after the injection ( sample d )", ", a significant difference was found in the observed vocs , although tumors were not yet palpable . in the last two samples ( samples e and f ) , tumors were palpable in all mice , and the chromatographic spectra were significantly different from those obtained in earlier samples .", "therefore , the samples were classified into three groups : healthy , early - melanoma , and late melanoma .", "typical urine chromatograms obtained from one mouse before ( sample b ) and after injection ( sample e ) are shown in figure 1 .", "some peaks in the chromatograms were present only in the tumor - bearing mice , and the area under some peaks was markedly higher or lower in the tumor - bearing mice compared to the healthy mice . by using the nist'08 and wiley mass spectral libraries , total of 120 vocs were identified in urine and 139 vocs in feces were identified in all mice samples , with an 80% quality factor ( qf ) . after subtracting the vocs that were found in the background samples ,", "a statistical analysis reduced the numbers of these peaks to 16 vocs in urine and 13 vocs in feces , which could serve as possible biomarker candidates .", "in addition , three vocs in urine and two vocs in feces , with a qf in the range of 6079% , were also included as possible biomarker candidates . of the 19 possible biomarker candidates in urine ,", "three compounds were detected only in the tumor - bearing mice ( table 2 ) , three vocs were detected only in the healthy and early - melanoma - bearing mice ( table 3 ) , and 13 vocs were detected in markedly higher concentrations in the tumor - bearing mice as compared with the healthy group ( table 4 ) .", "similarly , from the 15 possible biomarker candidates found in the headspace of feces , two vocs were detected only in the tumor - bearing mice ( table 5 ) and 13 vocs were found with a markedly higher ( 12 vocs ) or lower ( 1 voc ) concentration in the tumor - bearing mice than in the healthy group ( table 6 ) . to examine the significance of the changes in the concentrations of compounds during the different melanoma stages ( see tables 4 and 6 ) , a two - tailed paired t - test was carried out . as can be seen in table s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/841245 , some of the vocs that were identified as possible biomarker candidates in the current study have already been reported previously by other research groups as being predictive for melanoma and other cancers .", "the four potential melanoma - related biomarkers that have already been reported are isopropyl palmitate ( rt = 18.41 , qf = 72% ) , 1-hexadecanol ( rt = 14.54 , qf = 93% ) , benzaldehyde ( rt = 6.94 , qf = 97% ) , and dimethyl sulfone ( rt = 6.21 , qf = 94% ) .", "1-hexadecanol and isopropyl palmitate , a derivate of palmitic acid , were detected only in the urine of tumor - bearing mice .", "these findings are consistent with a previous study , which reported a 35-fold increase in the level of 1-hexadecanol in the case of melanoma ( as compared with a matching skin sample ) and that isopropyl palmitate was found only in the melanoma sample .", "the authors suggested that these compounds may reflect an increased de novo synthesis of fatty acids , which is a crucial metabolic alteration that cancer cells require for synthesis of a new plasma membrane .", "de novo synthesis of fatty acids could be caused by the hyperactivity of the oncogenic fatty acid synthase ( fasn ) , a common phenotype in cancer pathogenesis .", "benzaldehyde levels in feces were 132% higher in the tumor - bearing mice than in the healthy group ( p = 0.004 ) , but it is important to notice that the increases were from the early - melanoma stage to the late melanoma stage ( p = 0.002 ) , and not from the healthy group to the early - melanoma stage . in other words , benzaldehyde could be a useful possible biomarker candidate for predicting melanoma , but not in its early stage .", "the levels of dimethyl sulfone in urine showed a 199% increase from the healthy to the tumor - bearing mice groups ( p = 0.026 ) .", "as shown earlier , significantly higher amounts of this compound were found in metastatic melanoma cells , as compared with normal cells .", "this finding suggests that the metabolism of sulfur - containing amino acids by melanoma cells differs from that in normal cells .", "the gc - ms analysis of headspace in b16 melanoma cell cultures yields 12 vocs that were not detected in the headspace of the materials in the phosphate buffered saline ( pbs ) and fresh growth medium without cells ( table 7 ) .", "the compounds that were identified were isopropyl myristate ( rt = 16.76 , qf = 93% ) , decane ( rt = 7.43 , qf = 93% ) , 2,4-dimethyl-1-heptene ( rt = 4.67 , qf = 93% ) , and hexadecane ( rt = 14.7 , qf = 95% ) .", "as described above , the concentrations of isopropyl myristate were higher in both urine and feces of tumor - bearing mice .", "decane and two chemically similar compounds , 1-hexadecene and 2,4-dimethyl - heptane , were previously reported   to be present only in melanoma samples .", "the difference between 1-hexadecene and 2,4-dimethyl - heptane to hexadecane and 2,4-dimethyl-1-heptene ( resp . ) is in the presence or absence of a single double bond .", "hakim et al .   studied two hydrocarbons , decane and 2,4-dimethyl-1-heptene , that were reported as potential biomarkers of lung cancer and suggested that these compounds are probably the outcome of oxidative stress .", "as mentioned above , the tumor was palpable only in the last two samples ( late melanoma ) .", "an average lesion volume of 1664  mm ( n = 2 ) was measured three days after the last sample was taken ( see figure s1 ) . among all possible biomarker candidates that were found in the urine and feces , three compounds in urine ( figures 2(a)2(c ) ) and two compounds in feces ( figures 2(d)-2(e ) )", "three of these compounds were all ketones , that is , 5-methyl-2-heptanone ( rt = 6.87 , qf = 91% ) , 6-methyl-2-heptanone ( rt = 6.71 , qf = 94% ) , and 6-methyl-3-heptanone ( rt = 6.67 , qf = 97% ) .", "the quality factor of the two compounds found in the feces headspace was smaller than 80% ; hence they are referred to only by their retention time : rt = 8.9 and rt = 18.41 .", "6-methyl-2-heptanone has been found in the blood of patients with liver cancer   and 6-methyl-3-heptanone was found in the urine of mice with lung cancer .", "in addition , other chemically similar ketones , such as 2-heptanone and 6-hydroxy-6-methyl-3-heptanone , were also detected in urine samples from mice with lung cancer tumors .", "partial least square discriminate analysis ( pls - da ) models of urine and feces were built with the simca p+ software .", "each model included three latent variables that were calculated by the software and are , in fact , weighted linear combinations of the 12 possible biomarker candidates that most contributed to the separation between the three groups ( all the compounds in tables 26 , except compound 2 in table 2 , compound 3 in table 3 , compounds 3 and 912 in table 4 , compound 1 in table 5 , and compounds 5 and 6 in table 6 ) .", "the models are shown in figures 3 and 4 for the urine and feces models , respectively .", "the urine model exhibited good predictability of the mouse condition ( healthy or sick ) with a 92.3% sensitivity , 100% specificity , a 93.1% negative predictive value ( npv ) , and a 100% positive predictive value ( ppv ) .", "the model for feces gave less significant results , with an 80.8% sensitivity , a 96.3% specificity , an 83.9% npv , and a 95.5% ppv .", "it is important to note that eight out of nine mice were correctly predicted with an early - melanoma by using the urine model , whereas the feces model did not clearly discriminate between  healthy  and  early - melanoma  mice .", "the present study constitutes a novel proof - of - concept for the detection and monitoring of melanoma in urine and feces samples of mice at an early stage .", "our urine statistical model correctly predicted eight out of the 9 mice bearing an  early - melanoma ,  indicating that mice at that stage can be detected and distinguished from healthy mice and from mice bearing a  late melanoma .  while other research groups [ 6 , 7 ] focused on comparing human melanoma to normal and nevus skin biopsies , we attempted to distinguish between mice with an early - melanoma and healthy mice .", "our model was able to distinguish between melanoma bearing mice and healthy mice with sensitivity of 92% and specificity of 100% , as compared to studies of other groups on human samples ( e.g. , 70% sensitivity and 90% specificity reported by   and 89% sensitivity and 90% specificity reported by ) .", "these differences might be related to the low variability in the vocs of mice all having similar genetics and environment , as compared to those of humans .", "the results of this study also indicate a marked difference between healthy mice and mice bearing a  late melanoma ,  whereas the difference between healthy mice and mice bearing an  early - melanoma  is less clear .", "one possible explanation for this finding is that the concentration of the  new  compounds , which are products of the altered metabolism of the melanoma cells or of the normal cells that it affects , is below the detection threshold of our gc - ms system .", "in addition , the angiogenesis in mice bearing an  early - melanoma  is limited and , therefore , metabolites induced by melanoma cell do not disperse efficiently and are thus hard to detect . to overcome these concentration - dependent limitations", ", one should concentrate on detecting the compounds ( potential biomarker candidates ) that are responsible for the differences between healthy and  late melanoma ", "one should also develop methodologies to detect vocs directly from the regions of skin showing suspicious lesions ; a step in this direction has been taken by detecting differences in human melanoma cell line signatures by ftir spectroscopy [ 15 , 16 ] .", "some of the potential biomarker candidates that were identified in the present study were previously reported to be predictive for melanoma and other malignancies ( table s1 ) .", "those reports strengthen the validity of our findings and imply the existence of  cancer type-specific biomarkers and of common cancer biomarkers .", "some of the potential biomarker candidates are cross species compounds ; namely , they are found in both mice and human melanoma cells ; therefore , similar experiments with human melanoma cell lines as xenografts should be conducted to confirm these data .", "two additional possible biomarker candidates that may be good candidates to identify melanoma are dehydroabietic acid ( rt = 21.7 ) and 2-hexanone ( rt = 3.78 ) .", "it could be a pollutant from the sawdust , although it was not detected in the background samples .", "another possibility could be wrong identification of this compound by the code used ms database search ( qf = 81% ) .", "importantly , isopropyl myristate , the concentration of which increased in the urine and feces of tumor - bearing mice , was also found in the vapor phase of the b16 melanoma cells culture .", "this finding suggests that isopropyl myristate is a valid direct product of melanoma cell metabolism for further study .", "other compounds that were found in the headspace of the melanoma cell cultures were not detected in the urine or feces samples , and vice versa .", "this is attributed to the differences between the tumor cells microenvironment in a culture as compared to a tumor in the animal .", "we assume that there may be also contribution from the tumor microenvironment since tumor cells affect other cells in the tissue . at this point", ", the origin of the specific molecules we detected can not be determined . the observed increase in the concentration of ketones as a function of tumor growth suggests that ketogenesis pathways , incorporating various ketone formations , may be involved in the model of melanoma cancer .", "therefore , monitoring these ketones could help in tumor follow - up in melanoma patients . finally , to substantiate the findings in this study", ", we plan to perform an extended study with larger number of mice focusing on the urine vocs .", "in addition , to correlate more precisely between tumor size and voc concentration one should collect samples more often ( every 24 days ) and correlate the biomarkers to tumor volume as measured with a caliper . a different way of measuring is by expressing gfp or luciferase in the b16 cells and following their volume with an imaging device such as mri . despite the differences between mouse and human metabolism , possible biomarker candidates found in mice urine and feces", "if the possible biomarker candidates were formed in biochemical processes related to the activity of cancer cells , we can expect to find some overlap between biomarkers found in human and mice .", "urine and feces samples from nine mice were collected , twice before ( healthy group , n = 18 ) and four times after ( cancer group ,", "n = 35 , one mouse died before the last sample was obtained ) subcutaneous injection of b16 melanoma cells .", "compounds from these samples were separated and identified by using headspace solid phase microextraction ( hs - spme ) and gc / ms .", "in addition , vocs above a culture of b16 melanoma cells were also collected and analyzed .", "comparison between vocs content of the two first samples of each mouse , collected before b16 melanoma cell injection , showed high repetition of peaks in the chromatograms of each mouse .", "comparison of peak repetition in the chromatograms of different mouse in the two preinjection samples shows a markedly reduced degree of repetition ( see figure s2 in the supplementary material ) .", "these findings clearly indicate that the metabolism characteristics of each mouse have large influence on the observed chromatograms .", "consequently , we did not use a control group but compared the chromatograms of each mouse after b16 melanoma cell injection to those obtained before injection .", "female c57bl/6j inbred mice , 12 weeks old and weighing 1820  g , were obtained from harlan laboratories ( jerusalem , israel ) .", "all mice were maintained at the animal resource center of ben - gurion university of the negev , in a controlled environment ( illumination , temperature , and humidity ) free of specific pathogens .", "all animal experiments described in this work were approved by the ben - gurion university committee for the ethical care and use of animals in experiments .", "each individual mouse was kept in a separate cage ( 9 cages total ) under the same housekeeping conditions .", "the first samples were taken after a week of acclimation ( at the age of 13 weeks ) .", "b16f10 cells were cultured in an rpmi 1640 medium supplemented with 10% heat - inactivated fetal calf serum ( fcs ) , 100  u / ml penicillin , 100  mg / ml streptomycin , and 2  mm l - glutamine ( biological industries , israel ) .", "b16f10 cells ( 10 cells ) were seeded in a 10  cm diameter tissue culture dish .", "after 48  h , the medium was collected and centrifuged to eliminate cells and cell debris , and the cells were harvested by 0.23% trypsin and washed once in medium and two more times in phosphate buffered saline ( pbs ) .", "b16f10 cells were harvested by 0.23% trypsin and washed once in medium and two more times in pbs .", "one hundred microliters , containing a total of 2  10 b16f10 cells in pbs , was injected subcutaneously to the left flank of each of nine c57bl/6 mice .", "mice with tumors that reached the size of 1.5  cm diameter were sacrificed by co2 .", "urine and feces samples were collected into vials with a pipette from the new disposable plastic table cover . at the end of the sample collection", "we could not find in the literature a component in the urine or feces that allow the prediction of the hydration state of mouse ( similar to creatinine in human urine ) .", "we found 8 vocs that showed up in all the chromatograms ; however , their peak areas did not show any correlation to the total chromatogram area and they could not be used to monitor hydration state of the mice .", "thus , it was assumed that there should not be significant variations in mice hydration state since they were kept in identical conditions ( temperature and humidity ) with unlimited access to food and water .", "moreover , urine and feces samples were collected from all mice at the same time .", "it should be noted that the amount of urine and feces that were collected from the different mice varied , not enabling a direct comparison between amounts of different vocs of different mouse .", "static headspace sample extraction was achieved by exposing a 65-m polydimethylsiloxane / divinylbenzene ( pdms / dvb ) spme fiber ( supelco , bellefonte , pa , usa ) to the headspace for 10  min at 60c . following the extraction , the fiber assembly", "was transferred to the gc injection port for desorption at 250c for 5  min , with the split valve closed for 2  min .", "gc - ms analyses were performed by using an agilent 6890 series gc system ( agilent , usa ) connected to an agilent 5973 network mass selective detector ( agilent , usa ) .", "the bench top system was fitted with an spme injection sleeve 0.75  mm i d quartz liner ( supelco , bellefonte , pa , usa ) .", "the analytical column was a zebron zb-5msi fused - silica capillary column , 30  m  0.25  mm i d  0.25  m film thickness ( phenomenex , torrance , ca , usa ) .", "the carrier gas was 99.9995% pure helium ( maxima , ashdod , israel ) passed through a moisture trap ( model mt-200 - 2s ) and an agilent hydrocarbon / moisture trap ( model hmt200 - 2 ) ( agilent , china ) at flow rate of 1.0  ml  min .", "the gc was operated under the following temperature program : 50c for 3  min , ramp of 12c  min to 240c , held at 240c for 5  min , ramp of 50c  min to 260c , then held at 260c for 1  min , giving a total run of 25.23  min . for gc - ms , following electron ionization ,", "ions were scanned as the total ion current ( range : 10500 m / z at 2.97 scans  s ) . at both the beginning and end of each gc / ms analysis session the headspace over a calibration mixture ( containing equal amounts of chloroform , toluene , and dodecanethiol )", "the test is based on examination of retention time and peak shape of the solvents used in the calibration mixture .", "compounds were assigned a chemical identification by means of spectral library matching , using the nist'08 and wiley libraries .", "the databases search was performed using the chemstation software ( by agilent ) that was used to control and operate the gc / ms system .", "the assignment of chemical identity by the software is accompanied by a quality factor ( qf ) .", "the qf is also termed in some cases as matching factor . a library search procedure that identifies structural features of an unknown compound", "this procedure first retrieves library compounds whose spectra are most similar to the spectrum of the unknown compound .", "the algorithm then deduces structural features of the unknown compound from the chemical structures of the retrievals .", "the significance and reliability of each retrieved spectrum are weighted according to its similarity to the spectrum of the unknown compound .", "there are different procedures to estimate the reliability of the chemical identification of a gc peak ( see , e.g. , stein ) .", "the exact algorithm used to obtain the qf in the chemstation software is not described in the documentation ( it is considered as a commercial secret ) ; however , the manual states that qf larger than 80% is considered to be reliable .", "below this value of qf the reliability of the assignment is too low and the peaks are designated by their retention time only .", "a gc - ms analysis produces a complex chromatogram , where each peak represents a different volatile compound and the area below the peak is proportional to the amount of the compound in the sample .", "this threshold was used to ensure that all volatiles with low concentration are detected but  noise  is eliminated . to obtain estimates of the concentration of each compound , the peak area", "this normalization is performed by the chemstation software ( by agilent ) used to operate the gc / ms .", "the total chromatogram area used in the normalization is available in the output file ; hence , the actual peak area of any individual feature can be calculated .", "examination of the variations in total chromatogram area shows that the standard deviation is 17% of the mean value , a quite narrow distribution . the data analysis described in the present study", "we repeated some of the analysis using the actual area of the peaks in the chromatograms and found almost identical results to those obtained by the normalized data .", "some of the volatiles in the chromatogram were eluted at multiple rts and , therefore , we used only the rt with the best match .", "in addition , to overcome peak shifting across different chromatograms , we first grouped peaks in the same time window ( 0.3  s ) and then grouped adjacent peaks in adjacent time windows that hit the same match .", "each identified volatile was compared to all the background volatiles ( room air , plastic table cover , vial , vial septum , spme fiber , sawdust , and mouse food ) to ensure that differences were not due to a background alternation .", "a partial least squares discriminant analysis ( pls - da ) was conducted with the simca p+ code ( version 12.0.1.0 , by umetrics ab , ume , sweden ) to test whether mice with melanoma could be diagnosed by using the potential urine or feces biomarkers .", "each chromatogram vector was defined as a member in one of three classes ,  healthy ,   early - melanoma ,  and  late melanoma  in urine samples , and in one of two classes ,  healthy  and  melanoma  in the feces samples .", "two mice chosen arbitrarily were scanned three days after the last sample was taken to confirm the existence of the tumor and to estimate its volume .", "t1-weighted scanning images were acquired at 1 t by means of an aspect m2 high performance mri system ( aspect imaging technologies ltd . , israel ) , by using a gadolinium - dtpa contrast agent ( dotarem , 1  ml / kg body weight plus 0.05  ml for catheter ) ."], "abstract_text": ["<S> melanoma is the most malignant type of skin cancer . </S>", "<S> early detection of melanoma is thus critical for patient prognosis and survival . at present , </S>", "<S> examination by a skilled dermatologist followed by biopsy of suspicious lesions is the diagnostic gold standard . </S>", "<S> the aim of the present study was to examine an alternative and noninvasive method for the diagnosis of melanoma at an early stage . </S>", "<S> we identified and compared the volatile organic compounds ( vocs ) in mouse urine and feces , before and after a subcutaneous injection of b16 melanoma cells . </S>", "<S> we identified a total of 16 vocs in urine and 13 vocs in feces that could serve as potential biomarkers . </S>", "<S> statistical analysis significantly discriminated between the cancer and control groups . </S>", "<S> these results should be validated in a larger - scale animal study , after which a study could be designed in patients to develop a melanoma biomarker . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Results", "3. Discussion", "4. Methods"], "sections": [["we explore , in mice , the possibility of diagnosing an early - melanoma tumor and following its growth by analyzing the changes that occur throughout the pathological process in vocs excreted in urine and feces .", "we report here total of 29 potential biomarker candidates of melanoma ; 25 of these are novel , and 4 have been previously reported by other research groups in relation to vocs detected from cultures of human melanoma cells .", "the chemical identity of only 24 of the 29 possible biomarker candidates was revealed with high confidence .", "this study will contribute to the development of a noninvasive and reliable diagnostic procedure to detect melanoma in the urine of patients .", "melanoma accounts for less than 2% of all skin cancers , but for the vast majority of deaths from skin cancer . in the united states", "alone , it is estimated that 76,100 individuals will be diagnosed with melanoma during 2014 and that 9,710 patients will die from it .", "early detection of malignant melanoma is the key factor in reducing mortality from this cancer .", "although histopathological examination is the gold standard for diagnosis of melanoma , additional , noninvasive approaches should be developed . here", ", we explore the possibility of exploiting volatile organic compounds ( vocs ) to develop such an approach ; hundreds of vocs are emitted from the human body and usually reflect the metabolic condition of the individual .", "therefore , pathological processes , such as cancer , are expected to influence the voc fingerprint of patients either by changing the ratio between different vocs or by producing new vocs .", "it has been reported that dog scan identifies , by using their olfaction sense , melanoma on the skin of patients   or melanoma samples hidden on healthy subjects .", "used gas sensor array ( also known as an  electronic nose  ) to differentiate between melanomas and nevus lesions .", "those authors compared the lesion with the adjacent skin region to reduce the skin headspace variability .", "collected biopsy samples and compared the vocs released from melanoma tissue to those released from nevus and normal tissue , using matching skin as a control .", "those authors found 32 potential biomarker candidates ; the concentration of 9 vocs increased in the presence of melanoma , and 23 vocs were detected only when melanoma cells existed and were not detected in normal cells .", "the authors used the nist 2.0 mass spectral database , with a 60% quality factor for chemical identification of the possible biomarker candidates ; however , they did not report the quality factor obtained for the different compounds by mass spectrometry ( ms ) . in a recent study ,", "kwak et al .   compared human melanoma cells to normal melanocytes cultured in vitro . by using gas chromatography- ( gc- ) ms , those authors found increased levels of both isoamyl alcohol and isovaleric acid in the headspace over melanoma cells .", "the authors also found that the melanoma cells produced some unique compounds , such as dimethyldisulfide and dimethyltrisulfide . in the present study", ", we analyzed the vocs emitted from the urine and feces of mice before ( healthy group ) and after ( cancer group ) subcutaneous injection of b16 melanoma cells .", "the present work will serve as a basis to analyze possible vocs in human patients .", "we expect some voc to be shared between the species and new and different vocs to be detected ."], ["the sample classification used in the present study is summarized in table 1 . the analysis of the urine samples of healthy and tumor - bearing mice revealed some differences in the vocs composition .", "there was no significant difference between the first two samples ( samples a and b ) , which were taken before the injection of the b16 melanoma cells , and the first sample taken after injection ( sample c ) . in the second sample taken after the injection ( sample d )", ", a significant difference was found in the observed vocs , although tumors were not yet palpable . in the last two samples ( samples e and f ) , tumors were palpable in all mice , and the chromatographic spectra were significantly different from those obtained in earlier samples .", "therefore , the samples were classified into three groups : healthy , early - melanoma , and late melanoma .", "typical urine chromatograms obtained from one mouse before ( sample b ) and after injection ( sample e ) are shown in figure 1 .", "some peaks in the chromatograms were present only in the tumor - bearing mice , and the area under some peaks was markedly higher or lower in the tumor - bearing mice compared to the healthy mice . by using the nist'08 and wiley mass spectral libraries , total of 120 vocs were identified in urine and 139 vocs in feces were identified in all mice samples , with an 80% quality factor ( qf ) . after subtracting the vocs that were found in the background samples ,", "a statistical analysis reduced the numbers of these peaks to 16 vocs in urine and 13 vocs in feces , which could serve as possible biomarker candidates .", "in addition , three vocs in urine and two vocs in feces , with a qf in the range of 6079% , were also included as possible biomarker candidates . of the 19 possible biomarker candidates in urine ,", "three compounds were detected only in the tumor - bearing mice ( table 2 ) , three vocs were detected only in the healthy and early - melanoma - bearing mice ( table 3 ) , and 13 vocs were detected in markedly higher concentrations in the tumor - bearing mice as compared with the healthy group ( table 4 ) .", "similarly , from the 15 possible biomarker candidates found in the headspace of feces , two vocs were detected only in the tumor - bearing mice ( table 5 ) and 13 vocs were found with a markedly higher ( 12 vocs ) or lower ( 1 voc ) concentration in the tumor - bearing mice than in the healthy group ( table 6 ) . to examine the significance of the changes in the concentrations of compounds during the different melanoma stages ( see tables 4 and 6 ) , a two - tailed paired t - test was carried out . as can be seen in table s1 in supplementary material available online at http://dx.doi.org/10.1155/2015/841245 , some of the vocs that were identified as possible biomarker candidates in the current study have already been reported previously by other research groups as being predictive for melanoma and other cancers .", "the four potential melanoma - related biomarkers that have already been reported are isopropyl palmitate ( rt = 18.41 , qf = 72% ) , 1-hexadecanol ( rt = 14.54 , qf = 93% ) , benzaldehyde ( rt = 6.94 , qf = 97% ) , and dimethyl sulfone ( rt = 6.21 , qf = 94% ) .", "1-hexadecanol and isopropyl palmitate , a derivate of palmitic acid , were detected only in the urine of tumor - bearing mice .", "these findings are consistent with a previous study , which reported a 35-fold increase in the level of 1-hexadecanol in the case of melanoma ( as compared with a matching skin sample ) and that isopropyl palmitate was found only in the melanoma sample .", "the authors suggested that these compounds may reflect an increased de novo synthesis of fatty acids , which is a crucial metabolic alteration that cancer cells require for synthesis of a new plasma membrane .", "de novo synthesis of fatty acids could be caused by the hyperactivity of the oncogenic fatty acid synthase ( fasn ) , a common phenotype in cancer pathogenesis .", "benzaldehyde levels in feces were 132% higher in the tumor - bearing mice than in the healthy group ( p = 0.004 ) , but it is important to notice that the increases were from the early - melanoma stage to the late melanoma stage ( p = 0.002 ) , and not from the healthy group to the early - melanoma stage . in other words , benzaldehyde could be a useful possible biomarker candidate for predicting melanoma , but not in its early stage .", "the levels of dimethyl sulfone in urine showed a 199% increase from the healthy to the tumor - bearing mice groups ( p = 0.026 ) .", "as shown earlier , significantly higher amounts of this compound were found in metastatic melanoma cells , as compared with normal cells .", "this finding suggests that the metabolism of sulfur - containing amino acids by melanoma cells differs from that in normal cells .", "the gc - ms analysis of headspace in b16 melanoma cell cultures yields 12 vocs that were not detected in the headspace of the materials in the phosphate buffered saline ( pbs ) and fresh growth medium without cells ( table 7 ) .", "the compounds that were identified were isopropyl myristate ( rt = 16.76 , qf = 93% ) , decane ( rt = 7.43 , qf = 93% ) , 2,4-dimethyl-1-heptene ( rt = 4.67 , qf = 93% ) , and hexadecane ( rt = 14.7 , qf = 95% ) .", "as described above , the concentrations of isopropyl myristate were higher in both urine and feces of tumor - bearing mice .", "decane and two chemically similar compounds , 1-hexadecene and 2,4-dimethyl - heptane , were previously reported   to be present only in melanoma samples .", "the difference between 1-hexadecene and 2,4-dimethyl - heptane to hexadecane and 2,4-dimethyl-1-heptene ( resp . ) is in the presence or absence of a single double bond .", "hakim et al .   studied two hydrocarbons , decane and 2,4-dimethyl-1-heptene , that were reported as potential biomarkers of lung cancer and suggested that these compounds are probably the outcome of oxidative stress .", "as mentioned above , the tumor was palpable only in the last two samples ( late melanoma ) .", "an average lesion volume of 1664  mm ( n = 2 ) was measured three days after the last sample was taken ( see figure s1 ) . among all possible biomarker candidates that were found in the urine and feces , three compounds in urine ( figures 2(a)2(c ) ) and two compounds in feces ( figures 2(d)-2(e ) )", "three of these compounds were all ketones , that is , 5-methyl-2-heptanone ( rt = 6.87 , qf = 91% ) , 6-methyl-2-heptanone ( rt = 6.71 , qf = 94% ) , and 6-methyl-3-heptanone ( rt = 6.67 , qf = 97% ) .", "the quality factor of the two compounds found in the feces headspace was smaller than 80% ; hence they are referred to only by their retention time : rt = 8.9 and rt = 18.41 .", "6-methyl-2-heptanone has been found in the blood of patients with liver cancer   and 6-methyl-3-heptanone was found in the urine of mice with lung cancer .", "in addition , other chemically similar ketones , such as 2-heptanone and 6-hydroxy-6-methyl-3-heptanone , were also detected in urine samples from mice with lung cancer tumors .", "partial least square discriminate analysis ( pls - da ) models of urine and feces were built with the simca p+ software .", "each model included three latent variables that were calculated by the software and are , in fact , weighted linear combinations of the 12 possible biomarker candidates that most contributed to the separation between the three groups ( all the compounds in tables 26 , except compound 2 in table 2 , compound 3 in table 3 , compounds 3 and 912 in table 4 , compound 1 in table 5 , and compounds 5 and 6 in table 6 ) .", "the models are shown in figures 3 and 4 for the urine and feces models , respectively .", "the urine model exhibited good predictability of the mouse condition ( healthy or sick ) with a 92.3% sensitivity , 100% specificity , a 93.1% negative predictive value ( npv ) , and a 100% positive predictive value ( ppv ) .", "the model for feces gave less significant results , with an 80.8% sensitivity , a 96.3% specificity , an 83.9% npv , and a 95.5% ppv .", "it is important to note that eight out of nine mice were correctly predicted with an early - melanoma by using the urine model , whereas the feces model did not clearly discriminate between  healthy  and  early - melanoma  mice ."], ["the present study constitutes a novel proof - of - concept for the detection and monitoring of melanoma in urine and feces samples of mice at an early stage .", "our urine statistical model correctly predicted eight out of the 9 mice bearing an  early - melanoma ,  indicating that mice at that stage can be detected and distinguished from healthy mice and from mice bearing a  late melanoma .  while other research groups [ 6 , 7 ] focused on comparing human melanoma to normal and nevus skin biopsies , we attempted to distinguish between mice with an early - melanoma and healthy mice .", "our model was able to distinguish between melanoma bearing mice and healthy mice with sensitivity of 92% and specificity of 100% , as compared to studies of other groups on human samples ( e.g. , 70% sensitivity and 90% specificity reported by   and 89% sensitivity and 90% specificity reported by ) .", "these differences might be related to the low variability in the vocs of mice all having similar genetics and environment , as compared to those of humans .", "the results of this study also indicate a marked difference between healthy mice and mice bearing a  late melanoma ,  whereas the difference between healthy mice and mice bearing an  early - melanoma  is less clear .", "one possible explanation for this finding is that the concentration of the  new  compounds , which are products of the altered metabolism of the melanoma cells or of the normal cells that it affects , is below the detection threshold of our gc - ms system .", "in addition , the angiogenesis in mice bearing an  early - melanoma  is limited and , therefore , metabolites induced by melanoma cell do not disperse efficiently and are thus hard to detect . to overcome these concentration - dependent limitations", ", one should concentrate on detecting the compounds ( potential biomarker candidates ) that are responsible for the differences between healthy and  late melanoma ", "one should also develop methodologies to detect vocs directly from the regions of skin showing suspicious lesions ; a step in this direction has been taken by detecting differences in human melanoma cell line signatures by ftir spectroscopy [ 15 , 16 ] .", "some of the potential biomarker candidates that were identified in the present study were previously reported to be predictive for melanoma and other malignancies ( table s1 ) .", "those reports strengthen the validity of our findings and imply the existence of  cancer type-specific biomarkers and of common cancer biomarkers .", "some of the potential biomarker candidates are cross species compounds ; namely , they are found in both mice and human melanoma cells ; therefore , similar experiments with human melanoma cell lines as xenografts should be conducted to confirm these data .", "two additional possible biomarker candidates that may be good candidates to identify melanoma are dehydroabietic acid ( rt = 21.7 ) and 2-hexanone ( rt = 3.78 ) .", "it could be a pollutant from the sawdust , although it was not detected in the background samples .", "another possibility could be wrong identification of this compound by the code used ms database search ( qf = 81% ) .", "importantly , isopropyl myristate , the concentration of which increased in the urine and feces of tumor - bearing mice , was also found in the vapor phase of the b16 melanoma cells culture .", "this finding suggests that isopropyl myristate is a valid direct product of melanoma cell metabolism for further study .", "other compounds that were found in the headspace of the melanoma cell cultures were not detected in the urine or feces samples , and vice versa .", "this is attributed to the differences between the tumor cells microenvironment in a culture as compared to a tumor in the animal .", "we assume that there may be also contribution from the tumor microenvironment since tumor cells affect other cells in the tissue . at this point", ", the origin of the specific molecules we detected can not be determined . the observed increase in the concentration of ketones as a function of tumor growth suggests that ketogenesis pathways , incorporating various ketone formations , may be involved in the model of melanoma cancer .", "therefore , monitoring these ketones could help in tumor follow - up in melanoma patients . finally , to substantiate the findings in this study", ", we plan to perform an extended study with larger number of mice focusing on the urine vocs .", "in addition , to correlate more precisely between tumor size and voc concentration one should collect samples more often ( every 24 days ) and correlate the biomarkers to tumor volume as measured with a caliper . a different way of measuring is by expressing gfp or luciferase in the b16 cells and following their volume with an imaging device such as mri . despite the differences between mouse and human metabolism , possible biomarker candidates found in mice urine and feces", "if the possible biomarker candidates were formed in biochemical processes related to the activity of cancer cells , we can expect to find some overlap between biomarkers found in human and mice ."], ["urine and feces samples from nine mice were collected , twice before ( healthy group , n = 18 ) and four times after ( cancer group ,", "n = 35 , one mouse died before the last sample was obtained ) subcutaneous injection of b16 melanoma cells .", "compounds from these samples were separated and identified by using headspace solid phase microextraction ( hs - spme ) and gc / ms .", "in addition , vocs above a culture of b16 melanoma cells were also collected and analyzed .", "comparison between vocs content of the two first samples of each mouse , collected before b16 melanoma cell injection , showed high repetition of peaks in the chromatograms of each mouse .", "comparison of peak repetition in the chromatograms of different mouse in the two preinjection samples shows a markedly reduced degree of repetition ( see figure s2 in the supplementary material ) .", "these findings clearly indicate that the metabolism characteristics of each mouse have large influence on the observed chromatograms .", "consequently , we did not use a control group but compared the chromatograms of each mouse after b16 melanoma cell injection to those obtained before injection .", "female c57bl/6j inbred mice , 12 weeks old and weighing 1820  g , were obtained from harlan laboratories ( jerusalem , israel ) .", "all mice were maintained at the animal resource center of ben - gurion university of the negev , in a controlled environment ( illumination , temperature , and humidity ) free of specific pathogens .", "all animal experiments described in this work were approved by the ben - gurion university committee for the ethical care and use of animals in experiments .", "each individual mouse was kept in a separate cage ( 9 cages total ) under the same housekeeping conditions .", "the first samples were taken after a week of acclimation ( at the age of 13 weeks ) .", "b16f10 cells were cultured in an rpmi 1640 medium supplemented with 10% heat - inactivated fetal calf serum ( fcs ) , 100  u / ml penicillin , 100  mg / ml streptomycin , and 2  mm l - glutamine ( biological industries , israel ) .", "b16f10 cells ( 10 cells ) were seeded in a 10  cm diameter tissue culture dish .", "after 48  h , the medium was collected and centrifuged to eliminate cells and cell debris , and the cells were harvested by 0.23% trypsin and washed once in medium and two more times in phosphate buffered saline ( pbs ) .", "b16f10 cells were harvested by 0.23% trypsin and washed once in medium and two more times in pbs .", "one hundred microliters , containing a total of 2  10 b16f10 cells in pbs , was injected subcutaneously to the left flank of each of nine c57bl/6 mice .", "mice with tumors that reached the size of 1.5  cm diameter were sacrificed by co2 .", "urine and feces samples were collected into vials with a pipette from the new disposable plastic table cover . at the end of the sample collection", "we could not find in the literature a component in the urine or feces that allow the prediction of the hydration state of mouse ( similar to creatinine in human urine ) .", "we found 8 vocs that showed up in all the chromatograms ; however , their peak areas did not show any correlation to the total chromatogram area and they could not be used to monitor hydration state of the mice .", "thus , it was assumed that there should not be significant variations in mice hydration state since they were kept in identical conditions ( temperature and humidity ) with unlimited access to food and water .", "moreover , urine and feces samples were collected from all mice at the same time .", "it should be noted that the amount of urine and feces that were collected from the different mice varied , not enabling a direct comparison between amounts of different vocs of different mouse .", "static headspace sample extraction was achieved by exposing a 65-m polydimethylsiloxane / divinylbenzene ( pdms / dvb ) spme fiber ( supelco , bellefonte , pa , usa ) to the headspace for 10  min at 60c . following the extraction , the fiber assembly", "was transferred to the gc injection port for desorption at 250c for 5  min , with the split valve closed for 2  min .", "gc - ms analyses were performed by using an agilent 6890 series gc system ( agilent , usa ) connected to an agilent 5973 network mass selective detector ( agilent , usa ) .", "the bench top system was fitted with an spme injection sleeve 0.75  mm i d quartz liner ( supelco , bellefonte , pa , usa ) .", "the analytical column was a zebron zb-5msi fused - silica capillary column , 30  m  0.25  mm i d  0.25  m film thickness ( phenomenex , torrance , ca , usa ) .", "the carrier gas was 99.9995% pure helium ( maxima , ashdod , israel ) passed through a moisture trap ( model mt-200 - 2s ) and an agilent hydrocarbon / moisture trap ( model hmt200 - 2 ) ( agilent , china ) at flow rate of 1.0  ml  min .", "the gc was operated under the following temperature program : 50c for 3  min , ramp of 12c  min to 240c , held at 240c for 5  min , ramp of 50c  min to 260c , then held at 260c for 1  min , giving a total run of 25.23  min . for gc - ms , following electron ionization ,", "ions were scanned as the total ion current ( range : 10500 m / z at 2.97 scans  s ) . at both the beginning and end of each gc / ms analysis session the headspace over a calibration mixture ( containing equal amounts of chloroform , toluene , and dodecanethiol )", "the test is based on examination of retention time and peak shape of the solvents used in the calibration mixture .", "compounds were assigned a chemical identification by means of spectral library matching , using the nist'08 and wiley libraries .", "the databases search was performed using the chemstation software ( by agilent ) that was used to control and operate the gc / ms system .", "the assignment of chemical identity by the software is accompanied by a quality factor ( qf ) .", "the qf is also termed in some cases as matching factor . a library search procedure that identifies structural features of an unknown compound", "this procedure first retrieves library compounds whose spectra are most similar to the spectrum of the unknown compound .", "the algorithm then deduces structural features of the unknown compound from the chemical structures of the retrievals .", "the significance and reliability of each retrieved spectrum are weighted according to its similarity to the spectrum of the unknown compound .", "there are different procedures to estimate the reliability of the chemical identification of a gc peak ( see , e.g. , stein ) .", "the exact algorithm used to obtain the qf in the chemstation software is not described in the documentation ( it is considered as a commercial secret ) ; however , the manual states that qf larger than 80% is considered to be reliable .", "below this value of qf the reliability of the assignment is too low and the peaks are designated by their retention time only .", "a gc - ms analysis produces a complex chromatogram , where each peak represents a different volatile compound and the area below the peak is proportional to the amount of the compound in the sample .", "this threshold was used to ensure that all volatiles with low concentration are detected but  noise  is eliminated . to obtain estimates of the concentration of each compound , the peak area", "this normalization is performed by the chemstation software ( by agilent ) used to operate the gc / ms .", "the total chromatogram area used in the normalization is available in the output file ; hence , the actual peak area of any individual feature can be calculated .", "examination of the variations in total chromatogram area shows that the standard deviation is 17% of the mean value , a quite narrow distribution . the data analysis described in the present study", "we repeated some of the analysis using the actual area of the peaks in the chromatograms and found almost identical results to those obtained by the normalized data .", "some of the volatiles in the chromatogram were eluted at multiple rts and , therefore , we used only the rt with the best match .", "in addition , to overcome peak shifting across different chromatograms , we first grouped peaks in the same time window ( 0.3  s ) and then grouped adjacent peaks in adjacent time windows that hit the same match .", "each identified volatile was compared to all the background volatiles ( room air , plastic table cover , vial , vial septum , spme fiber , sawdust , and mouse food ) to ensure that differences were not due to a background alternation .", "a partial least squares discriminant analysis ( pls - da ) was conducted with the simca p+ code ( version 12.0.1.0 , by umetrics ab , ume , sweden ) to test whether mice with melanoma could be diagnosed by using the potential urine or feces biomarkers .", "each chromatogram vector was defined as a member in one of three classes ,  healthy ,   early - melanoma ,  and  late melanoma  in urine samples , and in one of two classes ,  healthy  and  melanoma  in the feces samples .", "two mice chosen arbitrarily were scanned three days after the last sample was taken to confirm the existence of the tumor and to estimate its volume .", "t1-weighted scanning images were acquired at 1 t by means of an aspect m2 high performance mri system ( aspect imaging technologies ltd . , israel ) , by using a gadolinium - dtpa contrast agent ( dotarem , 1  ml / kg body weight plus 0.05  ml for catheter ) ."]]}
{"article_id": "PMC4507440", "article_text": ["genome - wide association studies ( gwass ) have emerged as a powerful non - biased technique for identifying pathways related to human diseases with complex genetic architecture .", "understanding such complex diseases requires a broad understanding of the relevant functional nodes regulating disease - relevant pathways .", "genes do not function in isolation , and it is clear that both  gene - gene and gene - environment interactions contribute to disease susceptibility ( barreiro et  al . , 2012 ; cadwell et  al . , 2010 ; silver et  al . , 2013 ) .", "more than 140 genetic risk loci have been identified for crohn s disease , a chronic inflammatory condition affecting the gastrointestinal tract ( franke et  al . , 2010 ;", "these studies have highlighted the contribution of multiple immune and cellular pathways to crohn s disease pathogenesis , particularly those involved in microbial defense ( jostins et  al .", ", 2012 ; lassen et  al . , 2014 ; murthy et  al . , 2014 ; sadaghian sadabad et  al . , 2014 ) .", "atg16l1 ( autophagy - related protein 16-like 1 ) is a component of the core autophagy machinery , and the atg16l1 t300a polymorphism confers a modestly increased susceptibility to crohn s disease despite its relatively high prevalence in the population ( franke et  al . , 2010 ) .", "autophagy is a pro - survival intracellular degradation pathway that functions as a key mediator of a number of processes including central metabolism , cell signaling , cell death , and carcinogenesis , as well as both innate and adaptive immunity ( deretic et  al . , 2013 ; yang and klionsky , 2010 ) .", "the atg16l1 t300a polymorphism has been associated with both impaired antibacterial autophagy and altered production of cytokines by peripheral blood mononuclear cells ( pbmcs ) in response to immune stimuli ( lassen et  al . , 2014 ;", "recent studies have demonstrated that the mechanism underlying these alterations is linked to increased susceptibility of atg16l1 t300a to caspase - mediated cleavage and thus lower levels of functional atg16l1 ( lassen et  al .", "the autophagy pathway is important for defense against a number of intracellular pathogens and pathobionts including salmonella enterica serovar typhimurium , shigella flexneri , listeria monocytogenes , enterococcus faecalis , and mycobacterium tuberculosis ( benjamin et  al . , 2013 ; castillo et  al . , 2012 ;", "gutierrez et  al . , 2004 ; huett et  al . , 2012 ;", ", 2013 ; ogawa et  al . , 2005 ; tattoli et  al . , 2012 ;", ", cytoplasmic bacteria are first marked for degradation primarily by ubiquitination ( deretic et  al . , 2013 ) .", "recent studies have identified key roles for the e3 ligases lrsam1 and parkin in recognition and ubiquitination of cytoplasmic bacteria ; however , there are likely additional cellular e3 ligases that provide specificity to this pathway ( huett et  al . , 2012 ;", "ubiquitinated bacteria are then recognized by the adaptor proteins p62 , optineurin , and ndp52 , leading to the recruitment of lc3 and the autophagy machinery ( cemma et  al . , 2011 ; thurston et  al . , 2009 ; wild et  al . ,", "the autophagy pathway targets only a subpopulation of bacteria within a cell , and little is known about the molecules involved in the selective recognition and targeting of bacteria for autophagosomal degradation , despite the important pathological consequences of this process . in this study , we approached the question of the impact of the atg16l1 t300a risk variant by searching for associated responsiveness quantitative trait loci ( reqtls ) , which are defined as the effects of genetic variation on transcriptional responses of cells ( barreiro et  al . , 2012 ) .", "given that functionally similar genes are often transcriptionally coregulated , using multiple immune stimuli in genetically defined cell populations along with reqtls has enabled the identification of differential effects on molecular circuits ( gat - viks et  al . , 2013 ) . in the current study", ", we perform stimulus - specific perturbational profiling in pbmcs , quantitative mass - spectrometry - based ( ms ) proteomics , and genome - wide rna expression analysis to identify antibacterial genes that are functionally related to atg16l1 .", "using these approaches , we demonstrate a role for clec12a in antibacterial autophagy and pinpoint a functionally relevant interaction node of clec12a with an e3 ligase complex that is important for this function .", "to identify molecules involved in the atg16l1-t300a - dependent antibacterial autophagy pathway , we used perturbational profiling to measure differential gene regulation in the setting of stimulation with pathogen - associated molecular patterns and bacterial challenge .", "given that mounting evidence indicates an important role for the involvement of host - microbiota interactions and bacterial defense pathways in crohn s disease ( knights et  al . , 2013 ;", "we selected a panel of three stimuli to perturb pbmcs from healthy individuals homozygous for the  ancestral atg16l1300 t allele or the 300a risk allele ( rs2241880 ) .", "the nod2 ligand muramyl dipeptide ( mdp ) was selected based on the previous association of this stimulus with crohns - disease - specific pathways ( hugot et  al . , 2001 ;", "mdp can also modulate signals mediated through tlr2 activation , a pathway that has been implicated in crohn s disease and experimental colitis ( netea et  al . , 2004 ;", "we therefore also used the tlr2 ligand pam3cys , as well as the bacterium borrelia burgdorferi , from which these lipopeptides were initially isolated , to elicit both nod2-dependent and -independent responses . using microarrays to assess genome - wide rna expression profiles", ", we found that baseline gene expression was similar between individuals harboring the non - risk and risk alleles .", "furthermore , upon exposure to innate immune ligands , we observed strong genotype - independent induction of proinflammatory cytokines and chemokines after hierarchical clustering ( figures 1a and s1a ) .", "2012 ) , we next used factorial design analysis to examine whether transcriptional responses to pam3cys , borrelia , or mdp differed between groups stratified by the 300 t non - risk and 300a risk alleles . using multiple immune stimuli in genetically defined pbmcs along with reqtls", "has enabled differential effects on molecular circuits to be determined ( gat - viks et  al . , 2013 ) .", "our analysis identified 20 atg16l1300a - dependent reqtls under at least one stimulation condition ( figures 1b and s1b ) .", "thus , perturbational profiling of human immune cells revealed genotype - specific responses to stimulation .", "we focused additional analysis on the autophagy pathway , the cellular degradation system that has been implicated in risk of crohn s disease by gwass , which identified predisposing alleles in atg16l1 and irgm ( immunity - related gtpase family m ) ( rioux et  al . ,", "we and others have reported that the atg16l1 t300a polymorphism is associated with impaired antibacterial autophagy as well as altered production of cytokines by pbmcs in response to immune stimuli ( lassen et  al . , 2014 ; murthy et  al . , 2014 ) .", "additionally , tlr simulation , particularly through tlr2 , has been shown to induce antibacterial autophagy ( anand et  al . , 2011 ;", "2010 ) . to determine whether any of the identified genes might be associated with autophagy , we generated protein interaction networks anchored on core autophagy proteins as well as autophagy - associated components previously identified from a high - confidence interaction network derived from a systems - wide autophagy proteomics study ( behrends et  al . , 2010 ) . of the genes", "identified , rab24 was previously reported to have a direct effect on autophagy ( behrends et  al .", ", 2010 ; munaf and colombo , 2002 ) , and  three additional gene products ( hck , fez1 , and fyn ) interacted with the autophagy network via known protein - protein interactions ( figure  s1c ) .", "these observations demonstrate that the atg16l1 t300a polymorphism is associated with transcriptional modulation reflecting known protein - protein interactions with the autophagy network .", "given the previously described role for atg16l1 and irgm in antibacterial autophagy ( singh et  al .", ", 2006 ) , we next used sirna to evaluate whether any of the identified atg16l1300a - dependent reqtls affected antibacterial autophagy .", "of the 20 genes identified , 13 had detectable expression in hela cells and were suppressed by single sirnas ( figure  s1d ) .", "antibacterial autophagy was assessed in sirna - treated cells using infection with s. typhimurium , a model pathogen that is degraded by autophagy ( birmingham et  al . , 2006 ) .", "knockdown of three of these genes ( clec12a , rab24 , and evi2b ) resulted in significantly decreased salmonella - autophagosome colocalization ( figures 1c , 1d , and s1e ) .", "we next tested whether rab24 , evi2b , or clec12a play a role in classical autophagy using an lc3 flux assay , in which levels of lipidated lc3-ii are compared to lc3-i by western blot . in this assay ,", "an increase in the ratio of lc3-ii to lc3-i corresponds to an increase in autophagic flux .", "knockdown of rab24 and evi2 resulted in decreased lc3-ii accumulation when cells were treated with torin 1 , an mtor inhibitor and inducer of bulk autophagy , suggesting that these genes are involved in classical autophagy ; knockdown of atg16l1 served as a control in these experiments .", "knockdown of clec12a did not impair lc3-ii accumulation under these conditions , suggesting that clec12a functions specifically in antibacterial autophagy ( figure  1e ) ( thoreen et  al . , 2009 ) .", "additionally , to confirm that the antibacterial autophagy phenotype seen with clec12a knockdown was not due to off - target effects , we employed a knockdown rescue approach ( huett et  al . , 2012 ) by generating two sirna - resistant constructs that express full - length clec12a during knockdown of endogenous gene expression ( figure  1f ) . the autophagy defect caused by sirna against clec12a", "was rescued by overexpression of the appropriate clec12a construct ( figures 1 g and 1h ) , demonstrating that the decrease in antibacterial autophagy was specific to a reduction in clec12a expression and not due to off - target effects of sirna .", "additionally , sirna targeting clec12a did not alter bacterial entry , suggesting the observed effect was specific to the autophagy pathway ( figure  s1f ) .", "given our findings that immune cells harboring the atg16l1300a allele exhibited reduced induction of clec12a under immune stimulation conditions and that clec12a is required for efficient lc3-salmonella colocalization ( figure  1 ) , we next investigated whether a genetic interaction exists between the atg16l1 t300a snp and clec12a with respect to antibacterial autophagy . to investigate this hypothesis , we generated an atg16l1 knockout ( ko ) hela cell line using the crispr - cas9 system ( ran et  al .", "loss of atg16l1 expression was confirmed in single hela clones ( figure  s2a ) . as expected ,", "atg16l1 ko cells were unable to convert lc3-i to lc3-ii under basal or stimulatory conditions ( figure  s2b ) and had negligible lc3-bacteria colocalization ( figure  2 ) . to determine the effect of the t300a coding polymorphism ,", "we stably transduced atg16l1 ko cells with 300 t ( atg16l1 ko  + atg16l1300 t ) or 300a ( atg16l1 ko  + atg16l1300a ) alleles , achieving equivalent expression levels of atg16l1 in these lines ( figure  s2a ) . as expected , we found that basal autophagy was restored in both atg16l1 ko  + atg16l1300 t and atg16l1 ko  + atg16l1300a cell lines , with modest impairment of antibacterial autophagy in the atg16l1 ko  + atg16l1300a cells , consistent with published findings ( figure  2 ) ( conway et  al . , 2013 ; lassen et  al . , 2014 ; murthy et  al . , 2014 ) .", "targeting clec12a with sirna resulted in impaired antibacterial autophagy in the atg16l1 ko  + atg16l1300 t cell line similar to wt cells .", "we observed a more - pronounced impairment in atg16l1 ko  + atg16l1300a cells , suggesting functional interaction of the t300a polymorphism and reduced clec12a expression .", "clec12a is a c - type lectin receptor ( clr ) belonging to a family of transmembrane proteins that recognize pathogen - associated molecular patterns and engage downstream immune signal transduction pathways ( dambuza and brown , 2015 ; osorio and reis e sousa , 2011 ) .", "clec12a is highly expressed in human myeloid cells ( figure  s3a ) , and targeting antigen to clec12a results in enhanced antigen presentation on dendritic cells in mice ( lahoud et  al . , 2009 ;", "recent studies have demonstrated that clec12a binds to uric acid crystals and helps to dampen inflammation through its itim motifs ; however , it remains unclear whether this is the only ligand for clec12a ( neumann et  al .", "to determine whether clec12a plays a role in pathogen defense in  vivo , we used clec12a mice .", "bone - marrow - derived macrophages ( bmdms ) from wt or clec12a mice were infected with a strain of listeria ( egde ) known to be susceptible to autophagy in primary macrophages ( anand et  al . , 2011 ; birmingham et  al . , 2007 ; huett et  al . ,", "consistent with the salmonella infection results in sirna - treated cells , clec12a bmdms displayed lower levels of bacterial colocalization with lc3 compared with wt bmdms ( figures 3a and 3b ) .", "additionally , clec12a bmdms did not show differences in torin-1-induced autophagy , suggesting that bulk autophagy is normal in these cells ( figure  s3b ) . taken together", ", these data suggest that clec12a functions selectively in the antibacterial autophagy pathway for multiple pathogens in both mouse and human cells .", "we next investigated whether clec12a plays a role during infection with intracellular pathogens in  vivo .", "wt or clec12a mice were pre - treated with streptomycin and then infected with salmonella ( conway et  al .", "four days post - infection , stool was collected from infected mice and colony - forming units ( cfus ) were measured .", "clec12a mice had significantly higher cfu levels compared to wt mice ( figure  3c ) .", "clec12a mice also exhibited increased systemic bacterial dissemination as assessed by measuring cfus in spleens ( figure  3d ) .", "this increase in cfu was associated with a more - severe clinical disease score as assessed by overall appearance , piloerection , mobility , and posture ( figure  3e ) , as well as decreased survival ( figure  3f ) .", "taken together , these data suggest that clec12a plays a role in intracellular microbial defense both in  vitro and in  vivo . to further demonstrate the precise role for clec12a in antibacterial autophagy , we next tested whether knockdown of clec12a altered intracellular bacterial replication . using knockdown of atg16l1 as a positive control", ", we found that knockdown of clec12a in hela cells resulted in significantly increased intracellular replication of salmonella ( figure  4a ) , demonstrating that clec12a is required for restriction of intracellular bacterial replication .", "s4a ) were predominantly membrane associated and could be found surrounding intracellular dsred - labeled salmonella at both early and late time points after  infection .", "association of clec12a with salmonella peaked at 30  min post - infection and declined gradually , whereas lc3-salmonella colocalization peaked 60  min after infection , consistent with previous data ( figure  4b ) ( huett et  al . , 2012 ) .", "live cell imaging confirmed that gfp - clec12a surrounds bacteria and then dissociates at approximately 1  hr post - infection ( figure  s4b ) , consistent with the time course of peak lc3 colocalization .", "given the early association of clec12a with bacteria , we next determined whether clec12a was present at sites of bacterial entry into the host cell .", "we found that gfp - clec12a colocalized with bacteria and ruffled actin , an indicator of recent bacterial entry , suggesting that clec12a associates with bacteria concomitant with bacterial cellular entry ( figure  s4c ) ( lhocine et  al .", "we confirmed that clec12a colocalizes with salmonella and rab5 , a marker of early endosomes ( figure  s5a ) .", "however , clec12a recruitment was also  readily detected around salmonella without colocalization of rab5 , suggesting that clec12a is not exclusively associated with endosomes ( figure  s5a ) .", "next , we evaluated the colocalization of clec12a with galectin 3 and galectin 8 , which are both known to bind to sites of bacterial - induced intracellular membrane damage as well as sites of sterile membrane damage ( thurston et  al . , 2012 ) .", "clec12a colocalized with both galectin 3- and galectin 8-salmonella complexes ( figure  5a ) .", "these data confirm that clec12a associates with bacteria early after infection concomitant with membrane damage .", "clec12a also colocalized with the antibacterial autophagy adaptor ndp52 ( figure  5b ) as well as the autophagosome proteins gfp - lc3 , the atg8 homolog gabarapl2 , and atg16l1 ( figures 5c and 5d ) . in all cases ,", "these proteins surrounded intracellular bacteria in conjunction with either endogenous clec12a or gfp - clec12a ( figures 5b5d ) .", "of note , expression of gfp - clec12a gave a more - intense fluorescent signal and demonstrated increased levels of clec12a puncta within the cells , regardless of stimulation or bacterial infection .", "physical interactions between clec12a and lc3 , gabarapl2 , and atg16l1 were also confirmed by co - immunoprecipitation ( figure  s5b ) .", "we first determined whether knockdown of clec12a altered recruitment of ubiquitin to bacteria . at 1  hr post - infection , sirna knockdown of clec12a significantly reduced salmonella - ubiquitin colocalization ( figures 5e and 5f ) .", "consistent with this observation , recruitment of ndp52 to salmonella was also decreased upon sirna knockdown of clec12a ( figures 5 g and 5h ) .", "colocalization of salmonella with galectin 3 , galectin , 8 , and p62 were unchanged in cells treated with clec12a sirna ( figures s5c  s5e ) .", "these data reveal clec12a as an early bacteria - associated factor that binds to the autophagy machinery and helps initiate antibacterial autophagy through the ubiquitin - ndp52 pathway .", "recently , clec12a has been shown to be a receptor for dead cells ( neumann et  al . , 2014 ) .", "we hypothesized that clec12a could also recognize sites of membrane damage triggered by vacuolar disruption after bacterial entry ( tattoli et  al .", "galectin 3 served as a marker of damaged endosomes ( thurston et  al . , 2012 ) .", "gfp - clec12a colocalized with galectin 3 puncta after hypertonic shock and treatment with polyethylene glycol ( peg ) ( figure  5i ) .", "these results demonstrate that clec12a is recruited to sites of membrane damage and could explain the high proportion of clec12a recruitment early after bacterial entry . to develop a more - integrated model of clec12a function in pathogen response , we utilized time course rna - seq data and targeted quantitative proteomics to pinpoint clec12a - dependent protein interactions that may be important for pathogen response .", "first , we employed 3 rna - seq to quantify the transcriptome of listeria - infected wt or clec12a bmdms at various times post - infection . using this approach , we identified genes with statistically significant differences ( fdr - adjusted q value  0.05 ) in fold - change response to listeria infection between wt and clec12a bmdms at any time point post - infection ( figure  6a ) . of note ,", "expression of lamp1 , a lysosomal membrane protein and marker of salmonella - containing vacuoles , was significantly downregulated in clec12a bmdms , suggesting endomembrane dynamics might be perturbed in these cells .", "we next performed clustering and pathway analysis using an expanded list of 674 nominally significant genes that were differentially regulated in clec12a bmdms at baseline as well as in response to listeria .", "genes were grouped into ten clusters using short time - series expression miner ( stem ) , where each cluster represents one of the predefined model profiles in stem that capture potential distinct patterns of infection response ( figure  6b ; table s1 ) ( ernst and bar - joseph , 2006 ) . differences in these model profiles represent distinct regulation patterns of gene expression .", "gene ontology enrichment analysis was applied to assess the functional significance of each cluster of genes .", "the top scoring pathway that was differentially regulated in clec12a bmdms was the negative regulation of the intracellular signal transduction pathway that includes key signaling components such as ndufs3 , tmem161a , and mapkapk5 , among others ( cluster 2 ; table s1 ) .", "additionally , antifungal response genes ( s100a9 , myd88 , and ptx3 ; cluster 6 ) were also differentially regulated in the clec12a bmdms .", "selective upregulation of glucose transport ( ppbp and sorbs1 ; cluster 7 ) but downregulation of lipid transport ( scarb1 , slmo1 , and soat2 ; cluster 5 ) in clec12a cells suggested preferential regulation of metabolism by clec12a in response to listeria infection .", "these results highlight the central role of clec12a in pathogen - induced defense pathways and suggest how deficiencies in clec12a could result in impaired bacterial defense .", "next , we sought to identify important transcriptional nodes that control these responses by integrating known transcription factor - dna interaction data with our temporal gene expression profiles of infected wt and clec12a bmdms into a unified model . to identify important regulation points in the clec12a pathway , we used dynamic regulatory events miner ( drem ) , which searches for clec12a - dependent bifurcation events , defined by a transition in which a set of genes that were previously coregulated instead show divergent response / regulation profiles ( figure  s6a ) ( schulz et  al . , 2012 ) .", "based on an extension of a hidden markov model , the analysis identified egr1 , a transcription factor involved in transcriptional response to pathogens ( de grado et  al . , 2001 ; mcdermott et  al . , 2011 ) , as the earliest stage regulator that induces clec12a - dependent downregulation of specific genes ( figure  s6a ) .", "interestingly , egr1 is also known to be induced by either amino acid deprivation or er stress , which are both stimuli known to trigger autophagy ( shan et  al . , 2014 ) .", "these data suggest that reduced expression of egr1 target genes could contribute to the reduced pathogen response and autophagic targeting of bacteria in clec12a bmdms .", "other notable transcriptional regulators identified in our analysis that are known to be important for pathogen response include rela , nfe2 , hif1a , and arnt2 . taken together , this analysis suggests that deletion of clec12a results in an alteration of the response to pathogens at the transcriptional level and identifies key regulators involved in this response . to further understand the role of clec12a in antibacterial autophagy , we next applied an integrated systems approach using quantitative ms - based proteomics and coupled these results with our transcriptomic profiling analysis to uncover functional clec12a interactions relevant to bacterial defense ( figure  6c ) .", "proteomic interactors were defined as those that demonstrated significantly increased binding to flag - clec12a relative to flag vector alone after immunoprecipitation ( table s2 ) .", "interactome analysis identified highly connected nodes within the clec12a interactors ( figure  s6b ) . a specific interaction between clec12a and lc3 or atg16l1", "was not identified by ms - based proteomics ; however , this may reflect the fact that a meaningful interaction between these proteins occurs only under stimulation and represents a fraction of the total cellular pool of these proteins . to identify clec12a protein interactions that may lead to the measured transcriptomic changes in response to bacterial infection", ", we used a network optimization approach to identify high - probability sub - networks , composed of signaling and transcriptional factor interaction cascades ( figure  6c ) ( basha et  al . , 2013 ) .", "we then used a hypergeometric test to rank order clec12a - interacting proteins with the most significantly enriched set of target genes within the network to identify relevant functional nodes ( table s3 ) .", "some of the key transcriptional regulators identified in these sub - networks overlapped with those identified by drem analysis , including rela , tp53 , hif1a , and egr1 .", "this analysis pinpointed the ubiquitin - like protein , nedd8 ( neural precursor cell expressed , developmentally downregulated 8) , as both a clec12a interactor and a regulator of egr1 .", "these results suggest that the interaction between clec12a and nedd8 is a functional interaction and contributes to an early clec12a - dependent transcriptional response .", "this integrated approach also linked an e3 ubiquitin ligase complex that includes cul3 ( cullin 3 ) and the substrate adapters klhl9 ( kelch - like family member 9 ) and klhl13 ( kelch - like family member 13 ) to clec12a - dependent transcriptional responses controlled by the transcription factors cebpb and cebpd ( table s3 ) ( chen et  al . , 2014 ) .", "importantly , proteomic interactome analysis demonstrated a physical interaction between nedd8 , klhl13 , cul3 , and klhl9 , indicating that this is a functional e3 ligase complex that interacts with clec12a ( figure  6d ) .", "nedd8 is a target of some cycle - inhibiting factors ( cifs ) , which are virulence factors produced by bacterial pathogens that alter the host cell cycle ( crow et  al . , 2012 ; jubelin et  al . ,", "cif can associate directly with nedd8-modified cullin - ring complexes and inhibit their activity ( crow et  al .", "we therefore investigated whether cul3 , nedd8 , klhl9 , and klhl13 had roles in antibacterial autophagy similar to clec12a ( huett et  al .", "2013 ) . upon sirna knockdown in hela cells , klhl9 , klhl13 , and nedd8 had a dramatic effect on both lc3-salmonella colocalization and bacterial replication , consistent with a role for these proteins in antibacterial autophagy ( figures 6e and 6f ) .", "human genetics provides a powerful tool to identify and understand physiologically relevant host responses to pathogens given that host - pathogen interactions are known to have a role in many complex diseases .", "gene - gene interactions and environment - gene interactions add to the complexity of identifying causal genes and variants from gwas data , as well as determining how these variants affect pathogenesis .", "pathway - specific perturbational profiling and integrated systems approaches have previously been used in genetically defined organisms to identify genetic nodes of regulation underlying disease ( gagneur et  al . , 2013 ;", "gat - viks et  al . , 2013 ; smith and kruglyak , 2008 ; zhang et  al . , 2013 ) .", "using this strategy , we uncovered bacteria- and bacterial - ligand - specific programs affected in healthy individuals homozygous for the t300a polymorphism in the autophagy gene atg16l1 .", "interestingly , 15% of the t300a - dependent response genes were found to play a role in autophagy pathways , highlighting important snp - pathway interactions .", "our network analysis identified the clr family member clec12a as a gene that is less responsive upon tlr2 or bacterial stimulation in individuals carrying the atg16l1 \n 300a snp . pursuing the functional implication of this snp - environment interaction", ", we identified a role for clec12a in antibacterial autophagy and demonstrated that clec12a controls microbial replication in  vitro and in  vivo . using cells engineered to express the crohn s disease non - risk or risk allele of atg16l1", ", we interrogated the snp - gene interaction with clec12a to demonstrate an exacerbated effect on antibacterial autophagy in the setting of  the disease - associated snp .", "these findings are consistent with clec12a functioning at an early step in the antibacterial autophagy pathway at the level of pathogen recognition caused by membrane damage , with atg16l1 functioning downstream at the level of autophagosome formation .", "these data suggest a model in which individuals who carry the 300a risk allele have reduced clec12a responsiveness that serves to compound defects in antibacterial autophagy .", "these results highlight how non - redundant genes in the same pathway can be identified through ligand - specific transcriptional regulation .", "clrs are a large family of pattern recognition receptors , many of which are important signaling mediators in antimicrobial defense ( drummond and brown , 2013 ) .", "clec12a was recently shown to be a receptor for uric acid crystals and dead cells ( neumann et  al . , 2014 ) , and we extend this finding to demonstrate that clec12a also recognizes sites of intracellular membrane damage triggered by bacterial entry ( tattoli et  al . , 2012 ;", "additionally , we show that clec12a likely serves as an adaptor to help recruit ubiquitin and ndp52 to sites of vacuolar damage caused by bacteria and thus induce antibacterial autophagy .", "these data suggest parallels between the functions of galectins and clrs as early sensors of damage in selective autophagy pathways . additionally , a dendritic cell clr , dngr-1 , regulates endocytic handling of necrotic cell antigens to modulate cross - priming , suggesting that clrs could function broadly in the selection of endocytic cargo ( zelenay et  al . , 2012 ) .", "recent data also indicate a role for another clr , clec16a , in mitophagy , indicating that these lectins can function broadly to control target selectivity in autophagy ( soleimanpour et  al . , 2014 ) . here , we integrated time course rna - seq data and quantitative proteomics to identify relevant clec12a - dependent protein interactions that are involved in antibacterial autophagy .", "we identified an e3 ligase complex including klhl9 , nedd8 , and klhl13 that interacts with clec12a and is required for antibacterial autophagy .", "transcriptomic data suggested that the association of clec12a with this e3 ligase complex is important for the clec12a - dependent response to bacterial infection .", "virulence factors produced by some bacteria are known to directly target neddylated cullin - associated ubiquitin ligase activity , suggesting that these proteins might be broadly involved in inhibiting bacterial pathogenesis ( jubelin et  al . , 2010 ) .", "additionally , klhl9 has been previously associated with early onset autosomal dominant distal myopathy , a disease in which alterations to the autophagy pathway are thought to contribute to pathophysiology ( cirak et  al . , 2010 ) . taken together", ", these data identify a role for the recently evolved substrate adapters klhl9 and klhl13 as well as nedd8 in the restriction of bacterial infection .", "previous studies have suggested roles for the e3 ligases smurf1 , lrsam1 , and parkin in pathogen - specific autophagy ( huett et  al . , 2012 ; manzanillo et  al . , 2013 ; orvedahl et  al . , 2011 ) .", "recent studies have also highlighted roles for other e3 ubiquitin ligases in the control of bulk autophagy .", "these include a role for rnf5 in regulating the levels of atg4b ( kuang et  al . , 2012 ) , a role for cullin-5 and cullin-4 in autophagy regulation through ambra1 ( antonioli et  al . , 2014 ) , and a role for rnf216 in autophagy regulation through beclin1 ( xu et  al . , 2014 )", "additionally , several members of the trim family of proteins can act as a platform for assembly of the autophagy machinery ( mandell et  al . ,", "expression of rnf5 and rnf216 were also shown to increase pathogen susceptibility through autophagy ( kuang et  al . , 2012 ; xu et  al . , 2014 ) . taken together with our data , these findings demonstrate that e3 ubiquitin ligases play an important role in regulating autophagy at different stages of the pathway .", "specifically , e3 ubiquitin ligases create selectivity in autophagy regulation by recognizing and integrating specific signals from pathogens and cellular states .", "utilizing e3 ligases to tightly control the response to pathogens is likely a highly conserved mechanism of innate defense ( pollier et  al . , 2013 ) . here , we use integrated genomics to demonstrate a role for clec12a in antibacterial autophagy and identify an e3 ligase complex that provides insight into the selectivity of pathogen degradation .", "our in  vivo results highlight a strong role for clec12a in the restriction of microbial replication .", "it is possible given this dramatic effect that clec12a functions not only in antibacterial autophagy but in other pathogen defense pathways as well .", "this study highlights how perturbational profiling can be used to study pathways underlying immunity and pathogen defense and illustrates the potential of combining whole - genome experimental data sets to understand functional gene interactions within a relevant pathway .", "for perturbational profiling , blood was collected after written informed consent ( or waiver as approved by the institutional review board ) from healthy volunteers at radboud university nijmegen medical centre ( runmc ) .", "the study was approved by the institutional review boards and was performed in accordance with the declaration of helsinki . separation and stimulation of pbmcs from healthy individuals was performed as described previously ( netea et  al . , 2004 ) .", "microarray hybridization and genotyping of volunteers was performed as previously described ( smeekens et  al . , 2013 ) .", "factorial design analysis was performed as described previously ( cadwell et  al . , 2010 ) .", "s. typhimurium infections of hela cells and gentamicin protection assays were performed as previously described ( huett et  al . , 2012 ) .", "for entry assays , cells were infected for 20  min , washed , fixed in 4% paraformaldehyde for 15  min at room temperature , and stained as described below . for antibacterial autophagy assays in sirna - treated cells , rnai knockdown for 48  hr was performed as described above on hela cells plated on glass coverslips and infected as above .", "autophagy was induced in hela cells by treatment for 4  hr with 100  nm torin-1 , 10  g ml of e64d - pepstatin a ( sigma - aldrich ) , or mock treatment with dmso .", "cells were treated with sirna for 48  hr as described above and then autophagy was induced followed by cell lysis ( 25  mm tris [ ph 7.5 ] , 0.5% np-40 , 150  mm nacl , and protease inhibitors [ roche ] ) .", "western blotting to demonstrate lc3 lipidation was performed after equalization of protein amounts and sds - page on an anykd polyacrylamide gel ( bio - rad ) . following transfer to immobilon - p membranes ( millipore )", ", detection was performed using rabbit anti - lc3 primary ( sigma - aldrich ) , mouse anti - actin ( sigma - aldrich ) ,  and appropriate fluorescent secondary antibodies ( li - cor biosciences ) as previously described .", "the second exon of atg16l1 was targeted in hela cells using the px330 plasmid crispr system as described ( ran et  al . , 2013 ) .", "all animal studies were conducted under protocols approved by the subcommittee on research animal care ( srac ) at massachusetts general hospital .", "clec12a mice were obtained from the laboratory of g.d.b . at the university of aberdeen .", "clec12a mice were produced by conventional gene targeting of exons 14 on a c57bl/6 background ( p. redelinghuys , a. augello , r.a .", "protocols for bacterial growth and infection were performed as previously described , with slight modification ( barthel et  al . , 2003 ; conway et  al . , 2013 ) .", "quantitative proteomics using itraq labeling of peptides was performed as described previously ( lassen et  al . , 2014 ; mertins et  al . , 2014 ) .", "hela cells were reverse - transfected with plasmid expressing gfp - clec12a on glass coverslips in 12-well plates the night before the assay .", "osmotic lysis of endosomes was accomplished by exposing cells to hypertonic medium ( 0.5  m sucrose in pbs with 10% peg100 ) for 10  min .", "cells were washed twice with 1 pbs and then incubated in 60% pbs for 3  min as described previously ( thurston et  al . , 2012 ) .", "cells were returned to complete medium for 20  min and then fixed with ice - cold methanol ."], "abstract_text": ["<S> summarythe polymorphism atg16l1 t300a , associated with increased risk of crohn s disease , impairs pathogen defense mechanisms including selective autophagy , but specific pathway interactions altered by the risk allele remain unknown . here , we use perturbational profiling of human peripheral blood cells to reveal that clec12a is regulated in an atg16l1-t300a - dependent manner . </S>", "<S> antibacterial autophagy is impaired in clec12a - deficient cells , and this effect is exacerbated in the presence of the atg16l1300a risk allele . </S>", "<S> clec12a/ mice are more susceptible to salmonella infection , supporting a role for clec12a in antibacterial defense pathways in  vivo . </S>", "<S> clec12a is recruited to sites of bacterial entry , bacteria - autophagosome complexes , and sites of sterile membrane damage . </S>", "<S> integrated genomics identified a functional interaction between clec12a and an e3-ubiquitin ligase complex that functions in antibacterial autophagy . </S>", "<S> these data identify clec12a as early adaptor molecule for antibacterial autophagy and highlight perturbational profiling as a method to elucidate defense pathways in complex genetic disease . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Discussion", "Experimental Procedures", "Author Contributions"], "sections": [["genome - wide association studies ( gwass ) have emerged as a powerful non - biased technique for identifying pathways related to human diseases with complex genetic architecture .", "understanding such complex diseases requires a broad understanding of the relevant functional nodes regulating disease - relevant pathways .", "genes do not function in isolation , and it is clear that both  gene - gene and gene - environment interactions contribute to disease susceptibility ( barreiro et  al . , 2012 ; cadwell et  al . , 2010 ; silver et  al . , 2013 ) .", "more than 140 genetic risk loci have been identified for crohn s disease , a chronic inflammatory condition affecting the gastrointestinal tract ( franke et  al . , 2010 ;", "these studies have highlighted the contribution of multiple immune and cellular pathways to crohn s disease pathogenesis , particularly those involved in microbial defense ( jostins et  al .", ", 2012 ; lassen et  al . , 2014 ; murthy et  al . , 2014 ; sadaghian sadabad et  al . , 2014 ) .", "atg16l1 ( autophagy - related protein 16-like 1 ) is a component of the core autophagy machinery , and the atg16l1 t300a polymorphism confers a modestly increased susceptibility to crohn s disease despite its relatively high prevalence in the population ( franke et  al . , 2010 ) .", "autophagy is a pro - survival intracellular degradation pathway that functions as a key mediator of a number of processes including central metabolism , cell signaling , cell death , and carcinogenesis , as well as both innate and adaptive immunity ( deretic et  al . , 2013 ; yang and klionsky , 2010 ) .", "the atg16l1 t300a polymorphism has been associated with both impaired antibacterial autophagy and altered production of cytokines by peripheral blood mononuclear cells ( pbmcs ) in response to immune stimuli ( lassen et  al . , 2014 ;", "recent studies have demonstrated that the mechanism underlying these alterations is linked to increased susceptibility of atg16l1 t300a to caspase - mediated cleavage and thus lower levels of functional atg16l1 ( lassen et  al .", "the autophagy pathway is important for defense against a number of intracellular pathogens and pathobionts including salmonella enterica serovar typhimurium , shigella flexneri , listeria monocytogenes , enterococcus faecalis , and mycobacterium tuberculosis ( benjamin et  al . , 2013 ; castillo et  al . , 2012 ;", "gutierrez et  al . , 2004 ; huett et  al . , 2012 ;", ", 2013 ; ogawa et  al . , 2005 ; tattoli et  al . , 2012 ;", ", cytoplasmic bacteria are first marked for degradation primarily by ubiquitination ( deretic et  al . , 2013 ) .", "recent studies have identified key roles for the e3 ligases lrsam1 and parkin in recognition and ubiquitination of cytoplasmic bacteria ; however , there are likely additional cellular e3 ligases that provide specificity to this pathway ( huett et  al . , 2012 ;", "ubiquitinated bacteria are then recognized by the adaptor proteins p62 , optineurin , and ndp52 , leading to the recruitment of lc3 and the autophagy machinery ( cemma et  al . , 2011 ; thurston et  al . , 2009 ; wild et  al . ,", "the autophagy pathway targets only a subpopulation of bacteria within a cell , and little is known about the molecules involved in the selective recognition and targeting of bacteria for autophagosomal degradation , despite the important pathological consequences of this process . in this study , we approached the question of the impact of the atg16l1 t300a risk variant by searching for associated responsiveness quantitative trait loci ( reqtls ) , which are defined as the effects of genetic variation on transcriptional responses of cells ( barreiro et  al . , 2012 ) .", "given that functionally similar genes are often transcriptionally coregulated , using multiple immune stimuli in genetically defined cell populations along with reqtls has enabled the identification of differential effects on molecular circuits ( gat - viks et  al . , 2013 ) . in the current study", ", we perform stimulus - specific perturbational profiling in pbmcs , quantitative mass - spectrometry - based ( ms ) proteomics , and genome - wide rna expression analysis to identify antibacterial genes that are functionally related to atg16l1 .", "using these approaches , we demonstrate a role for clec12a in antibacterial autophagy and pinpoint a functionally relevant interaction node of clec12a with an e3 ligase complex that is important for this function ."], ["to identify molecules involved in the atg16l1-t300a - dependent antibacterial autophagy pathway , we used perturbational profiling to measure differential gene regulation in the setting of stimulation with pathogen - associated molecular patterns and bacterial challenge .", "given that mounting evidence indicates an important role for the involvement of host - microbiota interactions and bacterial defense pathways in crohn s disease ( knights et  al . , 2013 ;", "we selected a panel of three stimuli to perturb pbmcs from healthy individuals homozygous for the  ancestral atg16l1300 t allele or the 300a risk allele ( rs2241880 ) .", "the nod2 ligand muramyl dipeptide ( mdp ) was selected based on the previous association of this stimulus with crohns - disease - specific pathways ( hugot et  al . , 2001 ;", "mdp can also modulate signals mediated through tlr2 activation , a pathway that has been implicated in crohn s disease and experimental colitis ( netea et  al . , 2004 ;", "we therefore also used the tlr2 ligand pam3cys , as well as the bacterium borrelia burgdorferi , from which these lipopeptides were initially isolated , to elicit both nod2-dependent and -independent responses . using microarrays to assess genome - wide rna expression profiles", ", we found that baseline gene expression was similar between individuals harboring the non - risk and risk alleles .", "furthermore , upon exposure to innate immune ligands , we observed strong genotype - independent induction of proinflammatory cytokines and chemokines after hierarchical clustering ( figures 1a and s1a ) .", "2012 ) , we next used factorial design analysis to examine whether transcriptional responses to pam3cys , borrelia , or mdp differed between groups stratified by the 300 t non - risk and 300a risk alleles . using multiple immune stimuli in genetically defined pbmcs along with reqtls", "has enabled differential effects on molecular circuits to be determined ( gat - viks et  al . , 2013 ) .", "our analysis identified 20 atg16l1300a - dependent reqtls under at least one stimulation condition ( figures 1b and s1b ) .", "thus , perturbational profiling of human immune cells revealed genotype - specific responses to stimulation .", "we focused additional analysis on the autophagy pathway , the cellular degradation system that has been implicated in risk of crohn s disease by gwass , which identified predisposing alleles in atg16l1 and irgm ( immunity - related gtpase family m ) ( rioux et  al . ,", "we and others have reported that the atg16l1 t300a polymorphism is associated with impaired antibacterial autophagy as well as altered production of cytokines by pbmcs in response to immune stimuli ( lassen et  al . , 2014 ; murthy et  al . , 2014 ) .", "additionally , tlr simulation , particularly through tlr2 , has been shown to induce antibacterial autophagy ( anand et  al . , 2011 ;", "2010 ) . to determine whether any of the identified genes might be associated with autophagy , we generated protein interaction networks anchored on core autophagy proteins as well as autophagy - associated components previously identified from a high - confidence interaction network derived from a systems - wide autophagy proteomics study ( behrends et  al . , 2010 ) . of the genes", "identified , rab24 was previously reported to have a direct effect on autophagy ( behrends et  al .", ", 2010 ; munaf and colombo , 2002 ) , and  three additional gene products ( hck , fez1 , and fyn ) interacted with the autophagy network via known protein - protein interactions ( figure  s1c ) .", "these observations demonstrate that the atg16l1 t300a polymorphism is associated with transcriptional modulation reflecting known protein - protein interactions with the autophagy network .", "given the previously described role for atg16l1 and irgm in antibacterial autophagy ( singh et  al .", ", 2006 ) , we next used sirna to evaluate whether any of the identified atg16l1300a - dependent reqtls affected antibacterial autophagy .", "of the 20 genes identified , 13 had detectable expression in hela cells and were suppressed by single sirnas ( figure  s1d ) .", "antibacterial autophagy was assessed in sirna - treated cells using infection with s. typhimurium , a model pathogen that is degraded by autophagy ( birmingham et  al . , 2006 ) .", "knockdown of three of these genes ( clec12a , rab24 , and evi2b ) resulted in significantly decreased salmonella - autophagosome colocalization ( figures 1c , 1d , and s1e ) .", "we next tested whether rab24 , evi2b , or clec12a play a role in classical autophagy using an lc3 flux assay , in which levels of lipidated lc3-ii are compared to lc3-i by western blot . in this assay ,", "an increase in the ratio of lc3-ii to lc3-i corresponds to an increase in autophagic flux .", "knockdown of rab24 and evi2 resulted in decreased lc3-ii accumulation when cells were treated with torin 1 , an mtor inhibitor and inducer of bulk autophagy , suggesting that these genes are involved in classical autophagy ; knockdown of atg16l1 served as a control in these experiments .", "knockdown of clec12a did not impair lc3-ii accumulation under these conditions , suggesting that clec12a functions specifically in antibacterial autophagy ( figure  1e ) ( thoreen et  al . , 2009 ) .", "additionally , to confirm that the antibacterial autophagy phenotype seen with clec12a knockdown was not due to off - target effects , we employed a knockdown rescue approach ( huett et  al . , 2012 ) by generating two sirna - resistant constructs that express full - length clec12a during knockdown of endogenous gene expression ( figure  1f ) . the autophagy defect caused by sirna against clec12a", "was rescued by overexpression of the appropriate clec12a construct ( figures 1 g and 1h ) , demonstrating that the decrease in antibacterial autophagy was specific to a reduction in clec12a expression and not due to off - target effects of sirna .", "additionally , sirna targeting clec12a did not alter bacterial entry , suggesting the observed effect was specific to the autophagy pathway ( figure  s1f ) .", "given our findings that immune cells harboring the atg16l1300a allele exhibited reduced induction of clec12a under immune stimulation conditions and that clec12a is required for efficient lc3-salmonella colocalization ( figure  1 ) , we next investigated whether a genetic interaction exists between the atg16l1 t300a snp and clec12a with respect to antibacterial autophagy . to investigate this hypothesis , we generated an atg16l1 knockout ( ko ) hela cell line using the crispr - cas9 system ( ran et  al .", "loss of atg16l1 expression was confirmed in single hela clones ( figure  s2a ) . as expected ,", "atg16l1 ko cells were unable to convert lc3-i to lc3-ii under basal or stimulatory conditions ( figure  s2b ) and had negligible lc3-bacteria colocalization ( figure  2 ) . to determine the effect of the t300a coding polymorphism ,", "we stably transduced atg16l1 ko cells with 300 t ( atg16l1 ko  + atg16l1300 t ) or 300a ( atg16l1 ko  + atg16l1300a ) alleles , achieving equivalent expression levels of atg16l1 in these lines ( figure  s2a ) . as expected , we found that basal autophagy was restored in both atg16l1 ko  + atg16l1300 t and atg16l1 ko  + atg16l1300a cell lines , with modest impairment of antibacterial autophagy in the atg16l1 ko  + atg16l1300a cells , consistent with published findings ( figure  2 ) ( conway et  al . , 2013 ; lassen et  al . , 2014 ; murthy et  al . , 2014 ) .", "targeting clec12a with sirna resulted in impaired antibacterial autophagy in the atg16l1 ko  + atg16l1300 t cell line similar to wt cells .", "we observed a more - pronounced impairment in atg16l1 ko  + atg16l1300a cells , suggesting functional interaction of the t300a polymorphism and reduced clec12a expression .", "clec12a is a c - type lectin receptor ( clr ) belonging to a family of transmembrane proteins that recognize pathogen - associated molecular patterns and engage downstream immune signal transduction pathways ( dambuza and brown , 2015 ; osorio and reis e sousa , 2011 ) .", "clec12a is highly expressed in human myeloid cells ( figure  s3a ) , and targeting antigen to clec12a results in enhanced antigen presentation on dendritic cells in mice ( lahoud et  al . , 2009 ;", "recent studies have demonstrated that clec12a binds to uric acid crystals and helps to dampen inflammation through its itim motifs ; however , it remains unclear whether this is the only ligand for clec12a ( neumann et  al .", "to determine whether clec12a plays a role in pathogen defense in  vivo , we used clec12a mice .", "bone - marrow - derived macrophages ( bmdms ) from wt or clec12a mice were infected with a strain of listeria ( egde ) known to be susceptible to autophagy in primary macrophages ( anand et  al . , 2011 ; birmingham et  al . , 2007 ; huett et  al . ,", "consistent with the salmonella infection results in sirna - treated cells , clec12a bmdms displayed lower levels of bacterial colocalization with lc3 compared with wt bmdms ( figures 3a and 3b ) .", "additionally , clec12a bmdms did not show differences in torin-1-induced autophagy , suggesting that bulk autophagy is normal in these cells ( figure  s3b ) . taken together", ", these data suggest that clec12a functions selectively in the antibacterial autophagy pathway for multiple pathogens in both mouse and human cells .", "we next investigated whether clec12a plays a role during infection with intracellular pathogens in  vivo .", "wt or clec12a mice were pre - treated with streptomycin and then infected with salmonella ( conway et  al .", "four days post - infection , stool was collected from infected mice and colony - forming units ( cfus ) were measured .", "clec12a mice had significantly higher cfu levels compared to wt mice ( figure  3c ) .", "clec12a mice also exhibited increased systemic bacterial dissemination as assessed by measuring cfus in spleens ( figure  3d ) .", "this increase in cfu was associated with a more - severe clinical disease score as assessed by overall appearance , piloerection , mobility , and posture ( figure  3e ) , as well as decreased survival ( figure  3f ) .", "taken together , these data suggest that clec12a plays a role in intracellular microbial defense both in  vitro and in  vivo . to further demonstrate the precise role for clec12a in antibacterial autophagy , we next tested whether knockdown of clec12a altered intracellular bacterial replication . using knockdown of atg16l1 as a positive control", ", we found that knockdown of clec12a in hela cells resulted in significantly increased intracellular replication of salmonella ( figure  4a ) , demonstrating that clec12a is required for restriction of intracellular bacterial replication .", "s4a ) were predominantly membrane associated and could be found surrounding intracellular dsred - labeled salmonella at both early and late time points after  infection .", "association of clec12a with salmonella peaked at 30  min post - infection and declined gradually , whereas lc3-salmonella colocalization peaked 60  min after infection , consistent with previous data ( figure  4b ) ( huett et  al . , 2012 ) .", "live cell imaging confirmed that gfp - clec12a surrounds bacteria and then dissociates at approximately 1  hr post - infection ( figure  s4b ) , consistent with the time course of peak lc3 colocalization .", "given the early association of clec12a with bacteria , we next determined whether clec12a was present at sites of bacterial entry into the host cell .", "we found that gfp - clec12a colocalized with bacteria and ruffled actin , an indicator of recent bacterial entry , suggesting that clec12a associates with bacteria concomitant with bacterial cellular entry ( figure  s4c ) ( lhocine et  al .", "we confirmed that clec12a colocalizes with salmonella and rab5 , a marker of early endosomes ( figure  s5a ) .", "however , clec12a recruitment was also  readily detected around salmonella without colocalization of rab5 , suggesting that clec12a is not exclusively associated with endosomes ( figure  s5a ) .", "next , we evaluated the colocalization of clec12a with galectin 3 and galectin 8 , which are both known to bind to sites of bacterial - induced intracellular membrane damage as well as sites of sterile membrane damage ( thurston et  al . , 2012 ) .", "clec12a colocalized with both galectin 3- and galectin 8-salmonella complexes ( figure  5a ) .", "these data confirm that clec12a associates with bacteria early after infection concomitant with membrane damage .", "clec12a also colocalized with the antibacterial autophagy adaptor ndp52 ( figure  5b ) as well as the autophagosome proteins gfp - lc3 , the atg8 homolog gabarapl2 , and atg16l1 ( figures 5c and 5d ) . in all cases ,", "these proteins surrounded intracellular bacteria in conjunction with either endogenous clec12a or gfp - clec12a ( figures 5b5d ) .", "of note , expression of gfp - clec12a gave a more - intense fluorescent signal and demonstrated increased levels of clec12a puncta within the cells , regardless of stimulation or bacterial infection .", "physical interactions between clec12a and lc3 , gabarapl2 , and atg16l1 were also confirmed by co - immunoprecipitation ( figure  s5b ) .", "we first determined whether knockdown of clec12a altered recruitment of ubiquitin to bacteria . at 1  hr post - infection , sirna knockdown of clec12a significantly reduced salmonella - ubiquitin colocalization ( figures 5e and 5f ) .", "consistent with this observation , recruitment of ndp52 to salmonella was also decreased upon sirna knockdown of clec12a ( figures 5 g and 5h ) .", "colocalization of salmonella with galectin 3 , galectin , 8 , and p62 were unchanged in cells treated with clec12a sirna ( figures s5c  s5e ) .", "these data reveal clec12a as an early bacteria - associated factor that binds to the autophagy machinery and helps initiate antibacterial autophagy through the ubiquitin - ndp52 pathway .", "recently , clec12a has been shown to be a receptor for dead cells ( neumann et  al . , 2014 ) .", "we hypothesized that clec12a could also recognize sites of membrane damage triggered by vacuolar disruption after bacterial entry ( tattoli et  al .", "galectin 3 served as a marker of damaged endosomes ( thurston et  al . , 2012 ) .", "gfp - clec12a colocalized with galectin 3 puncta after hypertonic shock and treatment with polyethylene glycol ( peg ) ( figure  5i ) .", "these results demonstrate that clec12a is recruited to sites of membrane damage and could explain the high proportion of clec12a recruitment early after bacterial entry . to develop a more - integrated model of clec12a function in pathogen response , we utilized time course rna - seq data and targeted quantitative proteomics to pinpoint clec12a - dependent protein interactions that may be important for pathogen response .", "first , we employed 3 rna - seq to quantify the transcriptome of listeria - infected wt or clec12a bmdms at various times post - infection . using this approach , we identified genes with statistically significant differences ( fdr - adjusted q value  0.05 ) in fold - change response to listeria infection between wt and clec12a bmdms at any time point post - infection ( figure  6a ) . of note ,", "expression of lamp1 , a lysosomal membrane protein and marker of salmonella - containing vacuoles , was significantly downregulated in clec12a bmdms , suggesting endomembrane dynamics might be perturbed in these cells .", "we next performed clustering and pathway analysis using an expanded list of 674 nominally significant genes that were differentially regulated in clec12a bmdms at baseline as well as in response to listeria .", "genes were grouped into ten clusters using short time - series expression miner ( stem ) , where each cluster represents one of the predefined model profiles in stem that capture potential distinct patterns of infection response ( figure  6b ; table s1 ) ( ernst and bar - joseph , 2006 ) . differences in these model profiles represent distinct regulation patterns of gene expression .", "gene ontology enrichment analysis was applied to assess the functional significance of each cluster of genes .", "the top scoring pathway that was differentially regulated in clec12a bmdms was the negative regulation of the intracellular signal transduction pathway that includes key signaling components such as ndufs3 , tmem161a , and mapkapk5 , among others ( cluster 2 ; table s1 ) .", "additionally , antifungal response genes ( s100a9 , myd88 , and ptx3 ; cluster 6 ) were also differentially regulated in the clec12a bmdms .", "selective upregulation of glucose transport ( ppbp and sorbs1 ; cluster 7 ) but downregulation of lipid transport ( scarb1 , slmo1 , and soat2 ; cluster 5 ) in clec12a cells suggested preferential regulation of metabolism by clec12a in response to listeria infection .", "these results highlight the central role of clec12a in pathogen - induced defense pathways and suggest how deficiencies in clec12a could result in impaired bacterial defense .", "next , we sought to identify important transcriptional nodes that control these responses by integrating known transcription factor - dna interaction data with our temporal gene expression profiles of infected wt and clec12a bmdms into a unified model . to identify important regulation points in the clec12a pathway , we used dynamic regulatory events miner ( drem ) , which searches for clec12a - dependent bifurcation events , defined by a transition in which a set of genes that were previously coregulated instead show divergent response / regulation profiles ( figure  s6a ) ( schulz et  al . , 2012 ) .", "based on an extension of a hidden markov model , the analysis identified egr1 , a transcription factor involved in transcriptional response to pathogens ( de grado et  al . , 2001 ; mcdermott et  al . , 2011 ) , as the earliest stage regulator that induces clec12a - dependent downregulation of specific genes ( figure  s6a ) .", "interestingly , egr1 is also known to be induced by either amino acid deprivation or er stress , which are both stimuli known to trigger autophagy ( shan et  al . , 2014 ) .", "these data suggest that reduced expression of egr1 target genes could contribute to the reduced pathogen response and autophagic targeting of bacteria in clec12a bmdms .", "other notable transcriptional regulators identified in our analysis that are known to be important for pathogen response include rela , nfe2 , hif1a , and arnt2 . taken together , this analysis suggests that deletion of clec12a results in an alteration of the response to pathogens at the transcriptional level and identifies key regulators involved in this response . to further understand the role of clec12a in antibacterial autophagy , we next applied an integrated systems approach using quantitative ms - based proteomics and coupled these results with our transcriptomic profiling analysis to uncover functional clec12a interactions relevant to bacterial defense ( figure  6c ) .", "proteomic interactors were defined as those that demonstrated significantly increased binding to flag - clec12a relative to flag vector alone after immunoprecipitation ( table s2 ) .", "interactome analysis identified highly connected nodes within the clec12a interactors ( figure  s6b ) . a specific interaction between clec12a and lc3 or atg16l1", "was not identified by ms - based proteomics ; however , this may reflect the fact that a meaningful interaction between these proteins occurs only under stimulation and represents a fraction of the total cellular pool of these proteins . to identify clec12a protein interactions that may lead to the measured transcriptomic changes in response to bacterial infection", ", we used a network optimization approach to identify high - probability sub - networks , composed of signaling and transcriptional factor interaction cascades ( figure  6c ) ( basha et  al . , 2013 ) .", "we then used a hypergeometric test to rank order clec12a - interacting proteins with the most significantly enriched set of target genes within the network to identify relevant functional nodes ( table s3 ) .", "some of the key transcriptional regulators identified in these sub - networks overlapped with those identified by drem analysis , including rela , tp53 , hif1a , and egr1 .", "this analysis pinpointed the ubiquitin - like protein , nedd8 ( neural precursor cell expressed , developmentally downregulated 8) , as both a clec12a interactor and a regulator of egr1 .", "these results suggest that the interaction between clec12a and nedd8 is a functional interaction and contributes to an early clec12a - dependent transcriptional response .", "this integrated approach also linked an e3 ubiquitin ligase complex that includes cul3 ( cullin 3 ) and the substrate adapters klhl9 ( kelch - like family member 9 ) and klhl13 ( kelch - like family member 13 ) to clec12a - dependent transcriptional responses controlled by the transcription factors cebpb and cebpd ( table s3 ) ( chen et  al . , 2014 ) .", "importantly , proteomic interactome analysis demonstrated a physical interaction between nedd8 , klhl13 , cul3 , and klhl9 , indicating that this is a functional e3 ligase complex that interacts with clec12a ( figure  6d ) .", "nedd8 is a target of some cycle - inhibiting factors ( cifs ) , which are virulence factors produced by bacterial pathogens that alter the host cell cycle ( crow et  al . , 2012 ; jubelin et  al . ,", "cif can associate directly with nedd8-modified cullin - ring complexes and inhibit their activity ( crow et  al .", "we therefore investigated whether cul3 , nedd8 , klhl9 , and klhl13 had roles in antibacterial autophagy similar to clec12a ( huett et  al .", "2013 ) . upon sirna knockdown in hela cells , klhl9 , klhl13 , and nedd8 had a dramatic effect on both lc3-salmonella colocalization and bacterial replication , consistent with a role for these proteins in antibacterial autophagy ( figures 6e and 6f ) ."], ["human genetics provides a powerful tool to identify and understand physiologically relevant host responses to pathogens given that host - pathogen interactions are known to have a role in many complex diseases .", "gene - gene interactions and environment - gene interactions add to the complexity of identifying causal genes and variants from gwas data , as well as determining how these variants affect pathogenesis .", "pathway - specific perturbational profiling and integrated systems approaches have previously been used in genetically defined organisms to identify genetic nodes of regulation underlying disease ( gagneur et  al . , 2013 ;", "gat - viks et  al . , 2013 ; smith and kruglyak , 2008 ; zhang et  al . , 2013 ) .", "using this strategy , we uncovered bacteria- and bacterial - ligand - specific programs affected in healthy individuals homozygous for the t300a polymorphism in the autophagy gene atg16l1 .", "interestingly , 15% of the t300a - dependent response genes were found to play a role in autophagy pathways , highlighting important snp - pathway interactions .", "our network analysis identified the clr family member clec12a as a gene that is less responsive upon tlr2 or bacterial stimulation in individuals carrying the atg16l1 \n 300a snp . pursuing the functional implication of this snp - environment interaction", ", we identified a role for clec12a in antibacterial autophagy and demonstrated that clec12a controls microbial replication in  vitro and in  vivo . using cells engineered to express the crohn s disease non - risk or risk allele of atg16l1", ", we interrogated the snp - gene interaction with clec12a to demonstrate an exacerbated effect on antibacterial autophagy in the setting of  the disease - associated snp .", "these findings are consistent with clec12a functioning at an early step in the antibacterial autophagy pathway at the level of pathogen recognition caused by membrane damage , with atg16l1 functioning downstream at the level of autophagosome formation .", "these data suggest a model in which individuals who carry the 300a risk allele have reduced clec12a responsiveness that serves to compound defects in antibacterial autophagy .", "these results highlight how non - redundant genes in the same pathway can be identified through ligand - specific transcriptional regulation .", "clrs are a large family of pattern recognition receptors , many of which are important signaling mediators in antimicrobial defense ( drummond and brown , 2013 ) .", "clec12a was recently shown to be a receptor for uric acid crystals and dead cells ( neumann et  al . , 2014 ) , and we extend this finding to demonstrate that clec12a also recognizes sites of intracellular membrane damage triggered by bacterial entry ( tattoli et  al . , 2012 ;", "additionally , we show that clec12a likely serves as an adaptor to help recruit ubiquitin and ndp52 to sites of vacuolar damage caused by bacteria and thus induce antibacterial autophagy .", "these data suggest parallels between the functions of galectins and clrs as early sensors of damage in selective autophagy pathways . additionally , a dendritic cell clr , dngr-1 , regulates endocytic handling of necrotic cell antigens to modulate cross - priming , suggesting that clrs could function broadly in the selection of endocytic cargo ( zelenay et  al . , 2012 ) .", "recent data also indicate a role for another clr , clec16a , in mitophagy , indicating that these lectins can function broadly to control target selectivity in autophagy ( soleimanpour et  al . , 2014 ) . here , we integrated time course rna - seq data and quantitative proteomics to identify relevant clec12a - dependent protein interactions that are involved in antibacterial autophagy .", "we identified an e3 ligase complex including klhl9 , nedd8 , and klhl13 that interacts with clec12a and is required for antibacterial autophagy .", "transcriptomic data suggested that the association of clec12a with this e3 ligase complex is important for the clec12a - dependent response to bacterial infection .", "virulence factors produced by some bacteria are known to directly target neddylated cullin - associated ubiquitin ligase activity , suggesting that these proteins might be broadly involved in inhibiting bacterial pathogenesis ( jubelin et  al . , 2010 ) .", "additionally , klhl9 has been previously associated with early onset autosomal dominant distal myopathy , a disease in which alterations to the autophagy pathway are thought to contribute to pathophysiology ( cirak et  al . , 2010 ) . taken together", ", these data identify a role for the recently evolved substrate adapters klhl9 and klhl13 as well as nedd8 in the restriction of bacterial infection .", "previous studies have suggested roles for the e3 ligases smurf1 , lrsam1 , and parkin in pathogen - specific autophagy ( huett et  al . , 2012 ; manzanillo et  al . , 2013 ; orvedahl et  al . , 2011 ) .", "recent studies have also highlighted roles for other e3 ubiquitin ligases in the control of bulk autophagy .", "these include a role for rnf5 in regulating the levels of atg4b ( kuang et  al . , 2012 ) , a role for cullin-5 and cullin-4 in autophagy regulation through ambra1 ( antonioli et  al . , 2014 ) , and a role for rnf216 in autophagy regulation through beclin1 ( xu et  al . , 2014 )", "additionally , several members of the trim family of proteins can act as a platform for assembly of the autophagy machinery ( mandell et  al . ,", "expression of rnf5 and rnf216 were also shown to increase pathogen susceptibility through autophagy ( kuang et  al . , 2012 ; xu et  al . , 2014 ) . taken together with our data , these findings demonstrate that e3 ubiquitin ligases play an important role in regulating autophagy at different stages of the pathway .", "specifically , e3 ubiquitin ligases create selectivity in autophagy regulation by recognizing and integrating specific signals from pathogens and cellular states .", "utilizing e3 ligases to tightly control the response to pathogens is likely a highly conserved mechanism of innate defense ( pollier et  al . , 2013 ) . here , we use integrated genomics to demonstrate a role for clec12a in antibacterial autophagy and identify an e3 ligase complex that provides insight into the selectivity of pathogen degradation .", "our in  vivo results highlight a strong role for clec12a in the restriction of microbial replication .", "it is possible given this dramatic effect that clec12a functions not only in antibacterial autophagy but in other pathogen defense pathways as well .", "this study highlights how perturbational profiling can be used to study pathways underlying immunity and pathogen defense and illustrates the potential of combining whole - genome experimental data sets to understand functional gene interactions within a relevant pathway ."], ["for perturbational profiling , blood was collected after written informed consent ( or waiver as approved by the institutional review board ) from healthy volunteers at radboud university nijmegen medical centre ( runmc ) .", "the study was approved by the institutional review boards and was performed in accordance with the declaration of helsinki . separation and stimulation of pbmcs from healthy individuals was performed as described previously ( netea et  al . , 2004 ) .", "microarray hybridization and genotyping of volunteers was performed as previously described ( smeekens et  al . , 2013 ) .", "factorial design analysis was performed as described previously ( cadwell et  al . , 2010 ) .", "s. typhimurium infections of hela cells and gentamicin protection assays were performed as previously described ( huett et  al . , 2012 ) .", "for entry assays , cells were infected for 20  min , washed , fixed in 4% paraformaldehyde for 15  min at room temperature , and stained as described below . for antibacterial autophagy assays in sirna - treated cells , rnai knockdown for 48  hr was performed as described above on hela cells plated on glass coverslips and infected as above .", "autophagy was induced in hela cells by treatment for 4  hr with 100  nm torin-1 , 10  g ml of e64d - pepstatin a ( sigma - aldrich ) , or mock treatment with dmso .", "cells were treated with sirna for 48  hr as described above and then autophagy was induced followed by cell lysis ( 25  mm tris [ ph 7.5 ] , 0.5% np-40 , 150  mm nacl , and protease inhibitors [ roche ] ) .", "western blotting to demonstrate lc3 lipidation was performed after equalization of protein amounts and sds - page on an anykd polyacrylamide gel ( bio - rad ) . following transfer to immobilon - p membranes ( millipore )", ", detection was performed using rabbit anti - lc3 primary ( sigma - aldrich ) , mouse anti - actin ( sigma - aldrich ) ,  and appropriate fluorescent secondary antibodies ( li - cor biosciences ) as previously described .", "the second exon of atg16l1 was targeted in hela cells using the px330 plasmid crispr system as described ( ran et  al . , 2013 ) .", "all animal studies were conducted under protocols approved by the subcommittee on research animal care ( srac ) at massachusetts general hospital .", "clec12a mice were obtained from the laboratory of g.d.b . at the university of aberdeen .", "clec12a mice were produced by conventional gene targeting of exons 14 on a c57bl/6 background ( p. redelinghuys , a. augello , r.a .", "protocols for bacterial growth and infection were performed as previously described , with slight modification ( barthel et  al . , 2003 ; conway et  al . , 2013 ) .", "quantitative proteomics using itraq labeling of peptides was performed as described previously ( lassen et  al . , 2014 ; mertins et  al . , 2014 ) .", "hela cells were reverse - transfected with plasmid expressing gfp - clec12a on glass coverslips in 12-well plates the night before the assay .", "osmotic lysis of endosomes was accomplished by exposing cells to hypertonic medium ( 0.5  m sucrose in pbs with 10% peg100 ) for 10  min .", "cells were washed twice with 1 pbs and then incubated in 60% pbs for 3  min as described previously ( thurston et  al . , 2012 ) .", "cells were returned to complete medium for 20  min and then fixed with ice - cold methanol ."], [""]]}
{"article_id": "PMC3640597", "article_text": ["randomized controlled studies have shown that noninvasive ventilation ( niv ) reduces short - term mortality , risk of intubation and complication rate when given as an additional treatment to selected patients with an acute exacerbation in chronic obstructive pulmonary disease ( copd ) complicated by hypercapnic acute respiratory failure.1,2 plant et al2 showed a decrease of the in - hospital mortality rate from 20% to 10% when niv was given to patients with mild acidosis and in a stationary or intermediary ward in a multicenter setting .", "as with most other treatment modalities , there is likely to be a less noticeable effect when comparing real - life and clinical trial efficacy .", "this was documented in an audit performed in the uk,3 where the aim was to explore factors related to reported high mortality rates .", "niv was implemented as an add - on modality in an acute medical ward and a respiratory ward at odense university hospital , odense , denmark , in 2004,4 based on local instructions and national recommendations,5 and the modality has since been used as a standard additional treatment primarily for patients with hypercapnic acute respiratory failure and verified or presumed copd .", "specifically , patients with arterial measures of ph < 7.35 and paco2 > 6.0 kpa and mono - organic symptoms of dyspnea , respiratory rate > 25 and pao2 < 7.0 kpa without oxygen therapy are eligible for niv .", "exclusion criteria for niv treatment in the ward are untreated pneumothorax , unstable hemodynamics , and affected alertness , indicating need of intensified monitoring and intubation capability when required .", "there are few reports on long - term survival after receiving niv in a clinical setting,6 and the issue of whether there is an advantage by implementing niv in patients with severe prognosis and expected high mortality rates has not been addressed .", "the aim of this study was to analyze long - term all - cause mortality following the first years of implementing niv in a real - life acute medical ward at a university hospital in denmark , with the null hypothesis that niv has no impact on long - term survival .", "all niv treatments were registered prospectively at the acute medical and respiratory ward of the odense university hospital , with patients receiving niv for the first time in the period january 1 , 2005 to december 31 , 2007 included for this analysis .", "baseline data registered were age , sex , when registered in the records , arterial blood gas analyses initiating the indication for niv ( ph and pco2 are presented ) , diagnoses at discharge , and forced expiratory volume in 1 second ( fev1 ) .", "patients were included at the time of their first niv episode , and were followed until death or january 31 , 2012 using the danish central person register to ensure complete follow - up .", "patients were admitted to the acute medical ward from either the general practitioner or the emergency ward at the hospital .", "when patients were suspected of having an acute exacerbation of copd based on clinical presentation , prior records , arterial blood gas analyses , and chest x - ray , patients were assessed after initial standard treatment with oxygen , inhaled bronchodilator treatment , administration of systemic steroids , and , if needed , antibiotics .", "patients either continued standard medical treatment or were provided additional ventilatory support with niv in the medical acute ward .", "criteria for niv were : ( 1 ) arterial blood ph < 7.35 ; ( 2 ) pao2 < 7.0 kpa ; and ( 3 ) paco2 > 6.0 kpa in patients with mono - organic symptoms of dyspnea and respiratory rate > 25 .", "where possible , patients initiated on niv were referred to the respiratory ward the next day .", "since niv was initiated in the acute medical ward , standard niv protocols were followed ( initial inspiratory positive airway pressure 10 cm h20 and expiratory positive airway pressure 4 cm h20 ) , with oxygen supplement depending on response ( aim : peripheral oxygen saturation measure of 90%92% ) , and changes in pressure were performed depending on the clinical manifestation in accordance with the danish national guideline.5 the protocol on use of niv remained unchanged in the period of the study .", "initiation was done by internal medical staff and a respiratory physician was available for consultation if required .", "patients were monitored with repeated arterial analyses ; if the patient s status deteriorated , this patient was discussed with the attending intensive care unit ( icu ) doctor , unless the patient did not want icu treatment or the attending acute medical physician or respiratory physician had issued a  do - not - intubate ", "order , based on the severity of the underlying illness(es ) . on a few occasions ,", "niv was initiated for weaning in patients with prior longer intubation need due to respiratory failure ; these patients are reported on separately .", "patients were admitted to the acute medical ward from either the general practitioner or the emergency ward at the hospital . when patients were suspected of having an acute exacerbation of copd based on clinical presentation , prior records , arterial blood gas analyses , and chest x - ray , patients were assessed after initial standard treatment with oxygen , inhaled bronchodilator treatment , administration of systemic steroids , and , if needed , antibiotics .", "patients either continued standard medical treatment or were provided additional ventilatory support with niv in the medical acute ward .", "criteria for niv were : ( 1 ) arterial blood ph < 7.35 ; ( 2 ) pao2 < 7.0 kpa ; and ( 3 ) paco2 > 6.0 kpa in patients with mono - organic symptoms of dyspnea and respiratory rate > 25 .", "where possible , patients initiated on niv were referred to the respiratory ward the next day . since niv was initiated in the acute medical ward , standard niv protocols were followed ( initial inspiratory positive airway pressure 10 cm h20 and expiratory positive airway pressure 4 cm h20 ) , with oxygen supplement depending on response ( aim : peripheral oxygen saturation measure of 90%92% ) , and changes in pressure were performed depending on the clinical manifestation in accordance with the danish national guideline.5 the protocol on use of niv remained unchanged in the period of the study .", "initiation was done by internal medical staff and a respiratory physician was available for consultation if required .", "patients were monitored with repeated arterial analyses ; if the patient s status deteriorated , this patient was discussed with the attending intensive care unit ( icu ) doctor , unless the patient did not want icu treatment or the attending acute medical physician or respiratory physician had issued a  do - not - intubate ", "order , based on the severity of the underlying illness(es ) . on a few occasions ,", "niv was initiated for weaning in patients with prior longer intubation need due to respiratory failure ; these patients are reported on separately .", "in the study period 20052007 , niv was initiated on 390 occasions : 107 times in 2005 , 143 times in 2006 , and 140 times in 2007 . of", "the 302 patients registered , 253 patients received niv for the first time and were included in this study .", "copd was listed as the primary diagnosis in 216 patients ( 85.4% ) ; the diagnosis was confirmed by documented spirometry in 182 patients ( 8% ) and was judged as highly probable in 34 patients ( 16% ) .", "other diagnoses registered at discharge in patients presenting with acute hypercapnic respiratory failure were congestive heart failure , thoracic malignancy , and hypoventilation by other causes ( see table 1 ) .", "arterial gas analyses resulting in initiation of niv was registered in 51.4% of cases ; in these patients , the median ph was 7.24 ( 7.077.43 ) , and median pco2 ( kpa ) was 10.1 ( 6.0 , 20.3 ) .", "after 5 years , 23.7% of the patients were still alive as shown in figure 1 . for patients with documented copd ,", "the corresponding 30-day mortality was 24.3% and 5-year survival was 23.1% ( see figure 2 ) .", "there was no statistically significant difference in survival for men and women , although we did see a trend towards better long - term 5-year survival in females compared to males ( 25.7% vs 19.2% , p = 0.25 [ logrank ] ) .", "presence of copd did not affect prognosis , but the trend of better long - term 5-year survival in females compared to males was maintained ( 27.2% vs 17.9% , p = 0.083 [ logrank ] ) .", "the only statistically significant differences found between the long - term survivors and patients who died in the observation time were age and issue of a not - to - intubate order .", "survivors had a median age of 66 years compared to 74 years in nonsurvivors , and the not - to - intubate order was associated with 100% mortality .", "there was no difference in registered ph or pco2 between survivors and nonsurvivors at initiation of niv .", "descriptive data of copd patients versus non - copd patients receiving niv for the first time are shown in table 2 , and supplementary information in figure 1 .", "data on long - term survival of patients receiving niv for the first time revealed an expected high 30-day mortality rate , considerably higher than that seen in pivotal trials of niv in copd,1,2 but in line with other observational audits and studies.3,6,7 however , the 5-year survival rate was higher than earlier anticipated , which could be due to more stringent selection criteria for initiation of niv , and the fact that the study was only conducted in one center , giving patients more homogenous treatment . one significant difference between survivors and nonsurvivors was age , survivors being , as a group , younger than the group of patients who died , but the oldest patient ( 91 years ) registered in this study was alive at the end of 5 years of observation .", "we observed a trend of better long - term survival in females compared to males , in the whole niv group and especially in patients with copd , but further studies are needed to evaluate confounding variables .", "unfortunately , the registration of comorbidities was not done routinely or stringently , but , in the last couple of years , more focus has been made on screening and registration of comorbidities in copd , a benefit for future surveys .", "historically , males have had a higher exposure of cigarette smoke and occupational lung irritants8 and these factors could be contributing to the trend of worse long - term survival .", "it is well known that patients entered into clinical trials often differ significantly from patients in the usual clinical setting .", "in contrast to randomized controlled studies , this patient - based study included a much less selected group of patients having more comorbidities presenting with acute hypercapnic respiratory failure .", "patients were not limited to patients with respiratory failure due to exacerbation in copd , and , a priori , one could assume that this would affect prognosis .", "patients with other diagnoses included patients with clearly reversible disorders , such as overdoses of benzodiazepines , but several survivors were patients with other less morbid diagnoses than copd . despite the disadvantages of retrospective and therefore less complete data retrieval from records ( especially concerning arterial blood analyses ) , survivors compared to nonsurvivors were similar with regards to copd and , when registered , fev1 , ph , and pco2 . in the non - copd patients especially severe diseases such as congestive heart failure , thoracic malignancy , and end - stage fibrosis , were present among nonsurvivors but not at all present in survivors . in several of these cases ,", "niv was given as a last treatment option in patients with a not - to - intubate order .", "it seems apparent that patients labeled  not - to - intubate  have a particularly poor prognosis and , based , on our findings probably did not benefit from the treatment .", "the study included all patients initiated in an acute medical ward setting or a respiratory ward . unlike two previous prospective studies by berkius et al,6 including 93 copd patients in a multicenter icu setting , and harris et al,7 reporting use of noninvasive ventilation ( continuous positive airway pressure and bilevel positive airway pressure ) in clinical practice over 5 years , we have not included patients receiving the modality of niv initiated only in the icu setting .", "berkius et al6 reported survival data ( 5-year ) for copd patients receiving niv in the icu ( 17% ) , which was lower for patients needing intubation ( 6% ) .", "a national british copd audit from 2008 on acidosis , niv , and mortality in hospitalized patients with copd exacerbations , including 232 hospital units and 9716 patients , showed that 20% of the patients had acidotic artery blood analyses at admission.3 niv treatment in clinical practice was given to severely ill patients , many with mixed metabolic acidosis .", "further , patients fulfilling clinical criteria for niv treatment failed to receive niv , and others received it inappropriately .", "niv was also seen used as a ceiling of treatment in which efficacy was uncertain . the primary focus of this present study", "has not been evaluation of the indication for niv treatment ; since 2008 , an annual national surveillance of patients with copd ( dansk register for kol [ drkol ] ) has been launched , registration practice ( international classification of diseases [ icd]-10 ) has been reviewed , and audits performed . in denmark ,", "treatment with niv was provided to 9% of all copd patients admitted with an exacerbation in 2009 and 10% in 2010 and 2011.9", "our study confirms that patients with hypercapnic respiratory failure are a high - risk group , often with a poor prognosis .", "it casts some doubt as to what is achievable from niv in a non - icu setting and suggests that regular audits are required to ensure that the right patients are offered the right treatment when admitted with mild hypercapnic respiratory failure .", "however , the 5-year survival rate was higher than earlier anticipated , justifying the broad approach to niv treatment in this setting .", "the high 30-day mortality rate for all niv - initiated patients was higher ( 29.3% ) than for the group of patients with copd ( 24.3% ) , and this supports the notion that niv should be dedicated primarily to patients with copd in exacerbation .", "the 30-day mortality rate for copd patients receiving niv was similar in an audit from 2010 , and patients with ph < 7.25 had a worse outcome than patients presenting with higher ph at niv initiation ( unpublished data ) .", "monitoring of patient outcomes in a real - life setting is necessary to ensure optimal selection of patients for niv in a non - icu setting ."], "abstract_text": ["<S> implementation of noninvasive ventilation ( niv ) as an add - on treatment has been routinely used in a non - intensive care setting since 2004 for patients with chronic obstructive pulmonary disease ( copd ) and acute hypercapnic respiratory failure at a university hospital in denmark . </S>", "<S> although randomized controlled trials show lowered mortality rates in highly selected patients with acute exacerbation and respiratory failure , there are only few reports on long - term survival after receiving niv . </S>", "<S> we present long - term all - cause mortality data from patients receiving niv for the first time.methoddata from medical records were retrospectively retrieved from all patients receiving niv for the first time after being admitted acutely to an acute medical ward and further transfer to a respiratory ward with respiratory failure and a diagnosis of copd in the period january 1 , 2005 to december 31 , 2007 ; patients were followed until january 2012 . </S>", "<S> demographic data collected included age , sex , diagnoses at discharge , and , when present , fev1 ; a  not - to - intubate  </S>", "<S> order was also registered when listed.resultsin total , 253 patients ( 143 female , 110 male ) received niv for the first time . </S>", "<S> the median age was 72 years ( range 4691 years ) . </S>", "<S> the 30-day mortality rate was 29.3% . </S>", "<S> the 5-year survival rate was 23.7% . </S>", "<S> women showed a trend towards better survival than men ( 25.7% vs 19.2% , p = 0.25 ) , and the trend was even more pronounced for patients with copd.conclusionthe mortality rate of patients receiving niv is high , as expected in a real - life setting , but with a 5-year survival rate of 23.7% with a trend towards more female than male long - term survivors . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Organization of ward", "Results", "Discussion", "Conclusion"], "sections": [["randomized controlled studies have shown that noninvasive ventilation ( niv ) reduces short - term mortality , risk of intubation and complication rate when given as an additional treatment to selected patients with an acute exacerbation in chronic obstructive pulmonary disease ( copd ) complicated by hypercapnic acute respiratory failure.1,2 plant et al2 showed a decrease of the in - hospital mortality rate from 20% to 10% when niv was given to patients with mild acidosis and in a stationary or intermediary ward in a multicenter setting .", "as with most other treatment modalities , there is likely to be a less noticeable effect when comparing real - life and clinical trial efficacy .", "this was documented in an audit performed in the uk,3 where the aim was to explore factors related to reported high mortality rates .", "niv was implemented as an add - on modality in an acute medical ward and a respiratory ward at odense university hospital , odense , denmark , in 2004,4 based on local instructions and national recommendations,5 and the modality has since been used as a standard additional treatment primarily for patients with hypercapnic acute respiratory failure and verified or presumed copd .", "specifically , patients with arterial measures of ph < 7.35 and paco2 > 6.0 kpa and mono - organic symptoms of dyspnea , respiratory rate > 25 and pao2 < 7.0 kpa without oxygen therapy are eligible for niv .", "exclusion criteria for niv treatment in the ward are untreated pneumothorax , unstable hemodynamics , and affected alertness , indicating need of intensified monitoring and intubation capability when required .", "there are few reports on long - term survival after receiving niv in a clinical setting,6 and the issue of whether there is an advantage by implementing niv in patients with severe prognosis and expected high mortality rates has not been addressed .", "the aim of this study was to analyze long - term all - cause mortality following the first years of implementing niv in a real - life acute medical ward at a university hospital in denmark , with the null hypothesis that niv has no impact on long - term survival ."], ["all niv treatments were registered prospectively at the acute medical and respiratory ward of the odense university hospital , with patients receiving niv for the first time in the period january 1 , 2005 to december 31 , 2007 included for this analysis .", "baseline data registered were age , sex , when registered in the records , arterial blood gas analyses initiating the indication for niv ( ph and pco2 are presented ) , diagnoses at discharge , and forced expiratory volume in 1 second ( fev1 ) .", "patients were included at the time of their first niv episode , and were followed until death or january 31 , 2012 using the danish central person register to ensure complete follow - up .", "patients were admitted to the acute medical ward from either the general practitioner or the emergency ward at the hospital .", "when patients were suspected of having an acute exacerbation of copd based on clinical presentation , prior records , arterial blood gas analyses , and chest x - ray , patients were assessed after initial standard treatment with oxygen , inhaled bronchodilator treatment , administration of systemic steroids , and , if needed , antibiotics .", "patients either continued standard medical treatment or were provided additional ventilatory support with niv in the medical acute ward .", "criteria for niv were : ( 1 ) arterial blood ph < 7.35 ; ( 2 ) pao2 < 7.0 kpa ; and ( 3 ) paco2 > 6.0 kpa in patients with mono - organic symptoms of dyspnea and respiratory rate > 25 .", "where possible , patients initiated on niv were referred to the respiratory ward the next day .", "since niv was initiated in the acute medical ward , standard niv protocols were followed ( initial inspiratory positive airway pressure 10 cm h20 and expiratory positive airway pressure 4 cm h20 ) , with oxygen supplement depending on response ( aim : peripheral oxygen saturation measure of 90%92% ) , and changes in pressure were performed depending on the clinical manifestation in accordance with the danish national guideline.5 the protocol on use of niv remained unchanged in the period of the study .", "initiation was done by internal medical staff and a respiratory physician was available for consultation if required .", "patients were monitored with repeated arterial analyses ; if the patient s status deteriorated , this patient was discussed with the attending intensive care unit ( icu ) doctor , unless the patient did not want icu treatment or the attending acute medical physician or respiratory physician had issued a  do - not - intubate ", "order , based on the severity of the underlying illness(es ) . on a few occasions ,", "niv was initiated for weaning in patients with prior longer intubation need due to respiratory failure ; these patients are reported on separately ."], ["patients were admitted to the acute medical ward from either the general practitioner or the emergency ward at the hospital . when patients were suspected of having an acute exacerbation of copd based on clinical presentation , prior records , arterial blood gas analyses , and chest x - ray , patients were assessed after initial standard treatment with oxygen , inhaled bronchodilator treatment , administration of systemic steroids , and , if needed , antibiotics .", "patients either continued standard medical treatment or were provided additional ventilatory support with niv in the medical acute ward .", "criteria for niv were : ( 1 ) arterial blood ph < 7.35 ; ( 2 ) pao2 < 7.0 kpa ; and ( 3 ) paco2 > 6.0 kpa in patients with mono - organic symptoms of dyspnea and respiratory rate > 25 .", "where possible , patients initiated on niv were referred to the respiratory ward the next day . since niv was initiated in the acute medical ward , standard niv protocols were followed ( initial inspiratory positive airway pressure 10 cm h20 and expiratory positive airway pressure 4 cm h20 ) , with oxygen supplement depending on response ( aim : peripheral oxygen saturation measure of 90%92% ) , and changes in pressure were performed depending on the clinical manifestation in accordance with the danish national guideline.5 the protocol on use of niv remained unchanged in the period of the study .", "initiation was done by internal medical staff and a respiratory physician was available for consultation if required .", "patients were monitored with repeated arterial analyses ; if the patient s status deteriorated , this patient was discussed with the attending intensive care unit ( icu ) doctor , unless the patient did not want icu treatment or the attending acute medical physician or respiratory physician had issued a  do - not - intubate ", "order , based on the severity of the underlying illness(es ) . on a few occasions ,", "niv was initiated for weaning in patients with prior longer intubation need due to respiratory failure ; these patients are reported on separately ."], ["in the study period 20052007 , niv was initiated on 390 occasions : 107 times in 2005 , 143 times in 2006 , and 140 times in 2007 . of", "the 302 patients registered , 253 patients received niv for the first time and were included in this study .", "copd was listed as the primary diagnosis in 216 patients ( 85.4% ) ; the diagnosis was confirmed by documented spirometry in 182 patients ( 8% ) and was judged as highly probable in 34 patients ( 16% ) .", "other diagnoses registered at discharge in patients presenting with acute hypercapnic respiratory failure were congestive heart failure , thoracic malignancy , and hypoventilation by other causes ( see table 1 ) .", "arterial gas analyses resulting in initiation of niv was registered in 51.4% of cases ; in these patients , the median ph was 7.24 ( 7.077.43 ) , and median pco2 ( kpa ) was 10.1 ( 6.0 , 20.3 ) .", "after 5 years , 23.7% of the patients were still alive as shown in figure 1 . for patients with documented copd ,", "the corresponding 30-day mortality was 24.3% and 5-year survival was 23.1% ( see figure 2 ) .", "there was no statistically significant difference in survival for men and women , although we did see a trend towards better long - term 5-year survival in females compared to males ( 25.7% vs 19.2% , p = 0.25 [ logrank ] ) .", "presence of copd did not affect prognosis , but the trend of better long - term 5-year survival in females compared to males was maintained ( 27.2% vs 17.9% , p = 0.083 [ logrank ] ) .", "the only statistically significant differences found between the long - term survivors and patients who died in the observation time were age and issue of a not - to - intubate order .", "survivors had a median age of 66 years compared to 74 years in nonsurvivors , and the not - to - intubate order was associated with 100% mortality .", "there was no difference in registered ph or pco2 between survivors and nonsurvivors at initiation of niv .", "descriptive data of copd patients versus non - copd patients receiving niv for the first time are shown in table 2 , and supplementary information in figure 1 ."], ["data on long - term survival of patients receiving niv for the first time revealed an expected high 30-day mortality rate , considerably higher than that seen in pivotal trials of niv in copd,1,2 but in line with other observational audits and studies.3,6,7 however , the 5-year survival rate was higher than earlier anticipated , which could be due to more stringent selection criteria for initiation of niv , and the fact that the study was only conducted in one center , giving patients more homogenous treatment . one significant difference between survivors and nonsurvivors was age , survivors being , as a group , younger than the group of patients who died , but the oldest patient ( 91 years ) registered in this study was alive at the end of 5 years of observation .", "we observed a trend of better long - term survival in females compared to males , in the whole niv group and especially in patients with copd , but further studies are needed to evaluate confounding variables .", "unfortunately , the registration of comorbidities was not done routinely or stringently , but , in the last couple of years , more focus has been made on screening and registration of comorbidities in copd , a benefit for future surveys .", "historically , males have had a higher exposure of cigarette smoke and occupational lung irritants8 and these factors could be contributing to the trend of worse long - term survival .", "it is well known that patients entered into clinical trials often differ significantly from patients in the usual clinical setting .", "in contrast to randomized controlled studies , this patient - based study included a much less selected group of patients having more comorbidities presenting with acute hypercapnic respiratory failure .", "patients were not limited to patients with respiratory failure due to exacerbation in copd , and , a priori , one could assume that this would affect prognosis .", "patients with other diagnoses included patients with clearly reversible disorders , such as overdoses of benzodiazepines , but several survivors were patients with other less morbid diagnoses than copd . despite the disadvantages of retrospective and therefore less complete data retrieval from records ( especially concerning arterial blood analyses ) , survivors compared to nonsurvivors were similar with regards to copd and , when registered , fev1 , ph , and pco2 . in the non - copd patients especially severe diseases such as congestive heart failure , thoracic malignancy , and end - stage fibrosis , were present among nonsurvivors but not at all present in survivors . in several of these cases ,", "niv was given as a last treatment option in patients with a not - to - intubate order .", "it seems apparent that patients labeled  not - to - intubate  have a particularly poor prognosis and , based , on our findings probably did not benefit from the treatment .", "the study included all patients initiated in an acute medical ward setting or a respiratory ward . unlike two previous prospective studies by berkius et al,6 including 93 copd patients in a multicenter icu setting , and harris et al,7 reporting use of noninvasive ventilation ( continuous positive airway pressure and bilevel positive airway pressure ) in clinical practice over 5 years , we have not included patients receiving the modality of niv initiated only in the icu setting .", "berkius et al6 reported survival data ( 5-year ) for copd patients receiving niv in the icu ( 17% ) , which was lower for patients needing intubation ( 6% ) .", "a national british copd audit from 2008 on acidosis , niv , and mortality in hospitalized patients with copd exacerbations , including 232 hospital units and 9716 patients , showed that 20% of the patients had acidotic artery blood analyses at admission.3 niv treatment in clinical practice was given to severely ill patients , many with mixed metabolic acidosis .", "further , patients fulfilling clinical criteria for niv treatment failed to receive niv , and others received it inappropriately .", "niv was also seen used as a ceiling of treatment in which efficacy was uncertain . the primary focus of this present study", "has not been evaluation of the indication for niv treatment ; since 2008 , an annual national surveillance of patients with copd ( dansk register for kol [ drkol ] ) has been launched , registration practice ( international classification of diseases [ icd]-10 ) has been reviewed , and audits performed . in denmark ,", "treatment with niv was provided to 9% of all copd patients admitted with an exacerbation in 2009 and 10% in 2010 and 2011.9"], ["our study confirms that patients with hypercapnic respiratory failure are a high - risk group , often with a poor prognosis .", "it casts some doubt as to what is achievable from niv in a non - icu setting and suggests that regular audits are required to ensure that the right patients are offered the right treatment when admitted with mild hypercapnic respiratory failure .", "however , the 5-year survival rate was higher than earlier anticipated , justifying the broad approach to niv treatment in this setting .", "the high 30-day mortality rate for all niv - initiated patients was higher ( 29.3% ) than for the group of patients with copd ( 24.3% ) , and this supports the notion that niv should be dedicated primarily to patients with copd in exacerbation .", "the 30-day mortality rate for copd patients receiving niv was similar in an audit from 2010 , and patients with ph < 7.25 had a worse outcome than patients presenting with higher ph at niv initiation ( unpublished data ) .", "monitoring of patient outcomes in a real - life setting is necessary to ensure optimal selection of patients for niv in a non - icu setting ."]]}
{"article_id": "PMC4119343", "article_text": ["takayasu arteritis ( ta ) is a chronic inflammatory arteritis affecting the aorta and its main branches .", "the disease has a world - wide distribution , but has a high prevalence in japan .", "although there are no population based studies from india , reports have shown than the prevalence may be as high as in japan .", "most of the patients diagnosed to have this condition are women in their second and third decades of life .", "the occurrence of the disease in young children and infants is extremely rare with only a few cases reported all over the world .", "the present case report is about a 2-year - old female patient who was brought to the hospital with a history of incidentally detected hypertension of 2 months duration .", "she was born out of a non - consanguineous marriage and had an uneventful antenatal and postnatal course with normal developmental milestones and she had received vaccinations according the national immunization program , which includes bacillus calmette - guerin ( bcg ) . on examination , she was afebrile , active and well - nourished ; her weight was 16 kg and height was 80 cm .", "blood pressure was 140/90 mmhg , which was more than the 99 percentile for her height .", "her renal functions were normal ( blood urea : 8.4 mg / dl ; serum creatinine : 0.39 mg / dl )", ". serum sodium was 133.5 mmol / l , s. potassium 3.3 mmol / l , s. calcium 9.9 mg / dl and s. phosphurus 3.94 mg / dl .", "erythrocyte sedimentation rate ( esr ) was 20 mm/1 h and c - reactive protein was 6 mg / dl .", "the size of the right kidney was 7.1 cm  3.6 cm and that of the left kidney was 6.1 cm  2.9 cm .", "computed tomography aortogram showed critical stenosis of the left renal artery , at a length of 8 mm from the origin , a short segment stenosis of the right renal artery at its origin , a tight focal stenosis at the origin of the celiac artery with post - stenotic dilatation , mild stenosis at the origin of the superior mesenteric artery and narrowing of the abdominal aorta at the level of renal arteries ; there was no para - aortic lymphadenopathy [ figure 1 ] .", "the diameter of aorta at the level of diaphragm , renal arteries and just above bifurcation into iliac arteries were 6.2 mm , 5.2 mm and 6.6 mm respectively , proving that there was a narrowing at the level of renal arteries followed by post - stenotic dilatation . with these angiographic findings , in this infant with hypertension ,", "a diagnosis of ta was made according to the european league against rheumatism / pediatric rheumatology international trials organization / pediatric rheumatology european society ( eular / printo / pres ) criteria - 2008 .", "the child is being treated with nifedipine 5 g thrice daily and clonidine 50 mg thrice daily and her blood pressure is 90/55 mmhg .", "computed tomography angiogram showing bilateral renal artery stenosis and narrowing of abdominal aorta ( arrows : a : short segment stenosis of right renal artery , b : stenosis of left renal artery with small left kidney , c : narrowing of the abdominal aorta at the level of renal arteries )", "ta , also known as  pulseless disease  ,  occlusive thromboaortopathy  and  martorell syndrome  , was first systematically described by a japanese ophthalmologist mikito takayasu .", "the disease remains an enigma as the exact cause of the disease is still not elucidated .", "a human leucocyte antigen ( hla ) association and viral etiology have also been postulated .", "the clinical presentation of ta includes three phases : \n the early phase or prepulseless phase characterized by nonspecific systemic features such as malaise , arthralgia , weakness , weight loss and low grade feverthe pulseless phase characterized by claudication , amaurosis or diplopia and renovascular hypertensionthe occlusive phase characterized by transient ischemic attack , stroke , aortic regurgitation , cardiac failure , renovascular hypertension and claudication . \n ", "the early phase or prepulseless phase characterized by nonspecific systemic features such as malaise , arthralgia , weakness , weight loss and low grade fever the pulseless phase characterized by claudication , amaurosis or diplopia and renovascular hypertension the occlusive phase characterized by transient ischemic attack , stroke , aortic regurgitation , cardiac failure , renovascular hypertension and claudication . the diagnosis of ta in children is based on the eular / printo / pres criteria [ table 1 ] .", "diagnostic criteria for takayasu arteritis in children ta is certainly underdiagnosed and underreported from india , due to the fact that the disease may not present with classical features and the imaging modalities are available only in tertiary care centers .", "a study that compared the clinical manifestations of ta in india and japan reported that there was a significant variation in the manifestations of the disease in the two countries .", "in contrast to japanese patients in whom proximal aorta involvement ( takayasu conference classification - type i and ii ) was common , in indian patients descending and abdominal aorta involvement ( type iv ) was common . moreover , hypertension was the most dominant sign in indians compared with pulse lessness in the japanese .", "it was also noted that the age at diagnosis of the disease was almost a decade earlier in indians compared to japanese ( 28  10 vs. 37  14 years , p < 0.01 ) . in an indian study , it was found that aortoarteritis was the most common cause of renovasular hypertension and accounted for 59.4% of all cases .", "however , the mean age of the subjects was 27 years and the youngest child was 5 years of age . to the best of our knowledge , there are no reports from india of a case of ta in an infant .", "our patient had type iv disease , with the involvement of abdominal aorta , coeliac , superior mesenteric and both renal arteries .", "although it is suggested that glucocorticoids are the mainstay of treatment in the early or prepulseless phase , other immunosuppressive agents like methotrexate , azathioprine and anti - tumor necrosis factor agents have also been used , especially in steroid resistant cases .", "however , no evidence is available for benefit of therapy in the pediatric age group .", "however percutaneous intervention is less likely to succeed when stenosis or occlusions affect lengthy portions of an artery or the artery is heavily scarred .", "an angioplasty to the left renal artery was attempted in this case , but was unsuccessful .", "hence it was decided to continue medical management with anti - hypertensives as blood pressure could be controlled with medications and she was not given steroids as there were no clinical or laboratory indicators of an active inflammatory disease in this infant .", "although the world - wide prevalence of ta is very low , it is more prevalent in asian countries like japan and india .", "the clinical manifestations of the disease in india are different from that seen in japan .", "type iv disease involving abdominal aorta and renal arteries is more common in indians and hypertension is the most common sign .", "if the disease is active , steroids can be tried and the lesions amenable to angioplasty may be corrected by a percutaneous transluminal angioplasty ."], "abstract_text": ["<S> takayasu arteritis ( ta ) , a chronic inflammatory arteritis affecting the aorta and its main branches , is a rare condition mainly affecting young women in the second and third decades of life . </S>", "<S> occurrence of ta in infants is extremely rare , with only less than 10 cases reported all over the world until date . </S>", "<S> we report a case of a 2-year - old girl who presented with hypertension and was diagnosed to have ta with bilateral renal artery stenosis and this is probably the youngest case reported from india . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["takayasu arteritis ( ta ) is a chronic inflammatory arteritis affecting the aorta and its main branches .", "the disease has a world - wide distribution , but has a high prevalence in japan .", "although there are no population based studies from india , reports have shown than the prevalence may be as high as in japan .", "most of the patients diagnosed to have this condition are women in their second and third decades of life .", "the occurrence of the disease in young children and infants is extremely rare with only a few cases reported all over the world ."], ["the present case report is about a 2-year - old female patient who was brought to the hospital with a history of incidentally detected hypertension of 2 months duration .", "she was born out of a non - consanguineous marriage and had an uneventful antenatal and postnatal course with normal developmental milestones and she had received vaccinations according the national immunization program , which includes bacillus calmette - guerin ( bcg ) . on examination , she was afebrile , active and well - nourished ; her weight was 16 kg and height was 80 cm .", "blood pressure was 140/90 mmhg , which was more than the 99 percentile for her height .", "her renal functions were normal ( blood urea : 8.4 mg / dl ; serum creatinine : 0.39 mg / dl )", ". serum sodium was 133.5 mmol / l , s. potassium 3.3 mmol / l , s. calcium 9.9 mg / dl and s. phosphurus 3.94 mg / dl .", "erythrocyte sedimentation rate ( esr ) was 20 mm/1 h and c - reactive protein was 6 mg / dl .", "the size of the right kidney was 7.1 cm  3.6 cm and that of the left kidney was 6.1 cm  2.9 cm .", "computed tomography aortogram showed critical stenosis of the left renal artery , at a length of 8 mm from the origin , a short segment stenosis of the right renal artery at its origin , a tight focal stenosis at the origin of the celiac artery with post - stenotic dilatation , mild stenosis at the origin of the superior mesenteric artery and narrowing of the abdominal aorta at the level of renal arteries ; there was no para - aortic lymphadenopathy [ figure 1 ] .", "the diameter of aorta at the level of diaphragm , renal arteries and just above bifurcation into iliac arteries were 6.2 mm , 5.2 mm and 6.6 mm respectively , proving that there was a narrowing at the level of renal arteries followed by post - stenotic dilatation . with these angiographic findings , in this infant with hypertension ,", "a diagnosis of ta was made according to the european league against rheumatism / pediatric rheumatology international trials organization / pediatric rheumatology european society ( eular / printo / pres ) criteria - 2008 .", "the child is being treated with nifedipine 5 g thrice daily and clonidine 50 mg thrice daily and her blood pressure is 90/55 mmhg .", "computed tomography angiogram showing bilateral renal artery stenosis and narrowing of abdominal aorta ( arrows : a : short segment stenosis of right renal artery , b : stenosis of left renal artery with small left kidney , c : narrowing of the abdominal aorta at the level of renal arteries )"], ["ta , also known as  pulseless disease  ,  occlusive thromboaortopathy  and  martorell syndrome  , was first systematically described by a japanese ophthalmologist mikito takayasu .", "the disease remains an enigma as the exact cause of the disease is still not elucidated .", "a human leucocyte antigen ( hla ) association and viral etiology have also been postulated .", "the clinical presentation of ta includes three phases : \n the early phase or prepulseless phase characterized by nonspecific systemic features such as malaise , arthralgia , weakness , weight loss and low grade feverthe pulseless phase characterized by claudication , amaurosis or diplopia and renovascular hypertensionthe occlusive phase characterized by transient ischemic attack , stroke , aortic regurgitation , cardiac failure , renovascular hypertension and claudication . \n ", "the early phase or prepulseless phase characterized by nonspecific systemic features such as malaise , arthralgia , weakness , weight loss and low grade fever the pulseless phase characterized by claudication , amaurosis or diplopia and renovascular hypertension the occlusive phase characterized by transient ischemic attack , stroke , aortic regurgitation , cardiac failure , renovascular hypertension and claudication . the diagnosis of ta in children is based on the eular / printo / pres criteria [ table 1 ] .", "diagnostic criteria for takayasu arteritis in children ta is certainly underdiagnosed and underreported from india , due to the fact that the disease may not present with classical features and the imaging modalities are available only in tertiary care centers .", "a study that compared the clinical manifestations of ta in india and japan reported that there was a significant variation in the manifestations of the disease in the two countries .", "in contrast to japanese patients in whom proximal aorta involvement ( takayasu conference classification - type i and ii ) was common , in indian patients descending and abdominal aorta involvement ( type iv ) was common . moreover , hypertension was the most dominant sign in indians compared with pulse lessness in the japanese .", "it was also noted that the age at diagnosis of the disease was almost a decade earlier in indians compared to japanese ( 28  10 vs. 37  14 years , p < 0.01 ) . in an indian study , it was found that aortoarteritis was the most common cause of renovasular hypertension and accounted for 59.4% of all cases .", "however , the mean age of the subjects was 27 years and the youngest child was 5 years of age . to the best of our knowledge , there are no reports from india of a case of ta in an infant .", "our patient had type iv disease , with the involvement of abdominal aorta , coeliac , superior mesenteric and both renal arteries .", "although it is suggested that glucocorticoids are the mainstay of treatment in the early or prepulseless phase , other immunosuppressive agents like methotrexate , azathioprine and anti - tumor necrosis factor agents have also been used , especially in steroid resistant cases .", "however , no evidence is available for benefit of therapy in the pediatric age group .", "however percutaneous intervention is less likely to succeed when stenosis or occlusions affect lengthy portions of an artery or the artery is heavily scarred .", "an angioplasty to the left renal artery was attempted in this case , but was unsuccessful .", "hence it was decided to continue medical management with anti - hypertensives as blood pressure could be controlled with medications and she was not given steroids as there were no clinical or laboratory indicators of an active inflammatory disease in this infant ."], ["although the world - wide prevalence of ta is very low , it is more prevalent in asian countries like japan and india .", "the clinical manifestations of the disease in india are different from that seen in japan .", "type iv disease involving abdominal aorta and renal arteries is more common in indians and hypertension is the most common sign .", "if the disease is active , steroids can be tried and the lesions amenable to angioplasty may be corrected by a percutaneous transluminal angioplasty ."]]}
{"article_id": "PMC4249903", "article_text": ["currently , liver transplantation is the only definitive treatment for the end  stage liver diseases .", "however , its widely used was limited by the expensive costs , shortage of donor organs and invasive procedure ( 1 ) .", "hepatocyte transplantation has emerged as a feasible alternative to liver transplantation in some liver diseases ( 2 ) , but it is limited by organ donors and the low cell quality of available liver tissues ( 3 , 4 ) .", "although human embryonic stem cells ( hescs ) and umbilical cord blood stem cells ( ucbsc ) could be induced into hepatocyte - like cells in vitro ( 5 ) , their widely used were limited by the low differentiation quality , ethics , and teratoma formation ( 6 ) .", "recently , the adult stem cells ( ascs ) derived from various tissues , including bone marrow derived mesenchymal stem cells ( bmscs ) , adipose tissue derived mesenchymal stem cells ( adscs ) , and peripheral blood mononuclear cells ( pbmcs ) have been developed as new cell sources contributing to liver regeneration for their high efficiency of hepatogenic differentiation using simple procedures and no ethnic issues ( 710 ) . in the present study , we investigated whether ascs are the ideal seed cells for the liver regeneration from the followings : ascs biology , including isolation , culture , differentiation to hepatocytes , and the further role of ascs in cell therapy and tissue engineering", "in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee .", "most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) .", "side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) .", "adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally ,", "human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time ,", "g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) . then pbmcs were collected by means of aphaeresis .", "conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) .", "ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 .", "more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 .", "however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues .", "more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii .", "cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs .", "besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) .", "adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) .", "some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types .", "however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently .", "the hepatocyte - like cells from ascs were confirmed from the gene and protein expression .", "gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) .", "protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs .", "however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) .", "in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee .", "most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) .", "side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) .", "adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally , human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time ,", "pbmcs were mobilized with recombinant g - csf at 510 g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) .", "conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) .", "ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 .", "more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 .", "however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues .", "more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii .", "cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs .", "besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) .", "adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) .", "some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types .", "however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently .", "the hepatocyte - like cells from ascs were confirmed from the gene and protein expression .", "gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) .", "protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs .", "however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) .", "in the present study , we described different ascs could be induced into hepatocyte lineage cells in different culture systems in vitro .", "it is very safe and easy to acquire the enough ascs , and then induce them into functional hepatocytes in vitro .", "based on this progress , ascs transplantation might be a novel therapy for the severe liver diseases , and also will be ideal seed cells for liver tissue engineering .", "however , which ascs are better still needs us to investigate from their preparation , molecular characterization , and functional assay .", "this paper firstly provides such a concise review focused on ascs biology and differentiates potential , indicating ascs might be an ideal seed cells in cell transplant therapy and tissue engineering .", "we also believe in the future , some studies will show us the most appropriate ascs for cell transplant or tissue engineering ."], "abstract_text": ["<S> adult stem cells ( ascs ) are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues , which are the powerful sources for cell therapy and tissue engineering . </S>", "<S> bone marrow - derived mesenchymal stem cells ( bmscs ) , adipose tissue - derived mesenchymal stem cells ( adscs ) , and peripheral blood monocytes ( pbmcs ) are the common ascs , and many studies indicated that ascs isolated from various adult tissues could be induced to hepatocyte - like cells in vitro . however , the isolation , culture protocols , characterization of ascs and hepatocyte - like cells are different . </S>", "<S> this review aims to describe the isolation and culture procedures for ascs , to summarize the molecular characterization of ascs , to characterize function of hepatocyte - like cells , and to discuss the future role of ascs in cell therapy and tissue engineering . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Materials", "Isolation and expansion of BMSCs, ADSCs, and PBMCs", "Characterization of ASCs", "Protocols of ASCs transdifferentiation", "Functional analysis of hepatocyte-like cells from ASCs", "Discussion"], "sections": [["currently , liver transplantation is the only definitive treatment for the end  stage liver diseases .", "however , its widely used was limited by the expensive costs , shortage of donor organs and invasive procedure ( 1 ) .", "hepatocyte transplantation has emerged as a feasible alternative to liver transplantation in some liver diseases ( 2 ) , but it is limited by organ donors and the low cell quality of available liver tissues ( 3 , 4 ) .", "although human embryonic stem cells ( hescs ) and umbilical cord blood stem cells ( ucbsc ) could be induced into hepatocyte - like cells in vitro ( 5 ) , their widely used were limited by the low differentiation quality , ethics , and teratoma formation ( 6 ) .", "recently , the adult stem cells ( ascs ) derived from various tissues , including bone marrow derived mesenchymal stem cells ( bmscs ) , adipose tissue derived mesenchymal stem cells ( adscs ) , and peripheral blood mononuclear cells ( pbmcs ) have been developed as new cell sources contributing to liver regeneration for their high efficiency of hepatogenic differentiation using simple procedures and no ethnic issues ( 710 ) . in the present study , we investigated whether ascs are the ideal seed cells for the liver regeneration from the followings : ascs biology , including isolation , culture , differentiation to hepatocytes , and the further role of ascs in cell therapy and tissue engineering"], ["in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee .", "most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) .", "side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) .", "adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally ,", "human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time ,", "g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) . then pbmcs were collected by means of aphaeresis .", "conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) .", "ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 .", "more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 .", "however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues .", "more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii .", "cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs .", "besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) .", "adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) .", "some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types .", "however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently .", "the hepatocyte - like cells from ascs were confirmed from the gene and protein expression .", "gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) .", "protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs .", "however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) ."], ["in recent studies , samples of human bone marrow were obtained by lumbar puncture ( lp ) , human adipose tissue were obtained from abdominal subcutaneous adipose tissue of gastric cancer patients or liposuction patients , human peripheral blood were obtained from patients with hbv or healthy adult blood donors , in accordance with the local ethics committee ."], ["most laboratory data showed that bmscs could be prepared through the density degree of centrifuge , flow cytometry sorting , and sidewall sieve method ( 1114 ) .", "side - wall sieve method has become popular one for its simple operation , lower cost , less injury to cell ( 15 ) .", "adipose tissue was minced with scissors and scalpels into less than 3 mm pieces and isolation of adscs proceeded as previously described ( 16 , 17 ) . generally , human peripheral blood monocytes isolated from donors were isolated by density gradient centrifugation and further purified by adherence separation . in order to obtain more pbscs in shortly time ,", "pbmcs were mobilized with recombinant g - csf at 510 g / kg / d , administered subcutaneously daily to mobilize pbmcs from bone marrow to peripheral blood ( 18 , 19 ) ."], ["conget et al indicated that the bmscs express many surface agents including cd13 , cd44 , cd29 , cd105 , but did nt express cd1a , cd14 , cd31 , cd34 , cd56 , cd45 ( 20 ) .", "ascs derived from all three sources displayed no expression of hematopoietic markers ( cd14 , cd34 , cd45 ) , of the stem cell marker cd133 , or the marker for endothelial cells cd144 .", "more than 90% of mscs derived from the three sources expressed the typical msc marker proteins cd44 , cd73 , cd29 , and cd90 .", "however , the intensity of expression of cd90 of pbscs was significantly below that of the other tissues .", "more than 90% of the ascs derived from all three sources expressed hla i ; however , none of the mscs expressed hla ii .", "cd105 was expressed by a significantly lower percentage of pbscs compared with bm- or ad - scs , whereas , more pb- and bm - scs expressed cd106 than adscs .", "besides the fact , that they are more heterogeneous ( 23 ) , they reveal a surface antigen marker profile ( 22 , 2426 ) , and differentiation potential similar to bmscs ( 2732 ) .", "adscs are characterized as cd45 , cd34 + , cd105 + , cd31-(33 ) ( table 1 ) ."], ["some studies indicated that functional hepatocytes could be induced from ascs by some cytokines or through coculture with other cell types .", "however , the potential of the ascs transdifferentiation is generally low . therefore , researchers are keen to explore new methods to induce ascs differentiate into functional hepatocytes in vitro currently ."], ["the hepatocyte - like cells from ascs were confirmed from the gene and protein expression .", "gene expressions were identified by rt - pcr , using the common markers of hepatocytes , including alb , afp , ck18 , ck19 , and cyp3a4 ( 34 ) .", "protein expressions were usually identified by immunohistochemistry or mmunofluorescence , western blot , from the expression of albumin , ck18 , cyp3a4 , cyp1a1 , cyp2c9 and nadph - p450 ( 3538 ) . to compare the potential of hepatogenic differentiation of the different adult stem cells in vitro , researches indicated adscs have a similar differentiation potential towards the hepatic lineage , similar to bmscs .", "however , their longer culture period and proliferation capacity differ from the bmscs ( 3941 ) ."], ["in the present study , we described different ascs could be induced into hepatocyte lineage cells in different culture systems in vitro .", "it is very safe and easy to acquire the enough ascs , and then induce them into functional hepatocytes in vitro .", "based on this progress , ascs transplantation might be a novel therapy for the severe liver diseases , and also will be ideal seed cells for liver tissue engineering .", "however , which ascs are better still needs us to investigate from their preparation , molecular characterization , and functional assay .", "this paper firstly provides such a concise review focused on ascs biology and differentiates potential , indicating ascs might be an ideal seed cells in cell transplant therapy and tissue engineering .", "we also believe in the future , some studies will show us the most appropriate ascs for cell transplant or tissue engineering ."]]}
{"article_id": "PMC2694623", "article_text": ["waldenstrom 's macroglobulinemia ( wm ) is the result of a clonal proliferation of lymphocytes that produce monoclonal immunoglobulin m ( igm ) .", "it is now considered to correspond to lymphoplasmacytoid lymphoma as defined by the world health organization classification system ( 1 , 2 ) .", "many central nervous system ( cns ) complications have been described in wm patients ; the majority have been associated with blood hyperviscosity caused by igm .", "the hyperviscosity syndrome is characterized by headache , tinnitus , vertigo , blurred vision , and chronic bleeding from the nose and gums ( 3 ) .", "however , cns infiltration by plasmacytoid lymphocytes ( bing - neel syndrome ) has only rarely been reported ( 4 ) .", "a 51-yr - old woman was diagnosed with wm three years prior to this presentation .", "she received six courses of chlorambucil ( 0.3 mg / kg per day on day one to four orally ) and prednisone ( 45 mg / m per day on day one to four ) .", "next , she received fludarabine chemotherapy ( 25 mg / m per day on day one to five intravenously ) with a limited response .", "high - dose cyclophosphamide and granulocyte colony - stimulating factor were administered to induce peripheral blood progenitor cell ( pbpcs ) mobilization for autologous stem - cell transplantation ; however , the number of pbpcs collected was not sufficient .", "we then decided on a conservative treatment approach , because the patient did not have specific symptoms and the serum igm level was stable ( 3,000 - 3,500 mg / dl ) .", "the laboratory values on admission were as follows : white cell count 8,050/l with normal differential counts , hemoglobin 9.6 g / dl , platelets 296,000/l , erythrocyte sedimentation rate 144 mm / hr , total protein 9.44 g / dl , albumin 2.85 g / dl , igg 3144.4 mg / dl , iga 25.4 mg / dl , igm", "a brain computed tomography scan revealed multifocal extra - axial tumorous lesions along the dura matter .", "a brain magetic resonance imaging showed an extra - axial soft tissue tumor along the left cavernous sinus and tentorium , right frontal convexity and tentorium and falx ; the brain parenchyma appeared to be unremarkable ( fig .", "cerebrospinal fluid analysis showed the following : white cell count 43/l with neutrophils 1% , lymphocytes 52% and monocytes 47% , total protein 1.81 g / dl , glucose 104 mg / dl , igm 64.5 mg / dl and a few plasmacytoid lymphocytes on cytology .", "stereotactic biopsy of dural tissue at the falx showed a diffuse infiltration with atypical cells , which were identified immunophenotypically as plasmacytoid lymphocyte with expression of lca ( + ) , cd3 ( - ) , cd20 ( + ) , and vs38a ( + ) ( fig .", "2 ) . the patient was confirmed to have cns infiltration by atypical plasmacytoid lymphocyte infiltration ( bing - neel syndrome ) .", "the paitent received a total dose of 1,980 cgy with irradiation therapy of the whole brain that was administered in 11 fractions , and then her headache subsided , but the igm level was elevated persistently to 3,379.8 mg / dl . following radiation therapy ,", "fludarabine chemotherapy was performed ( 25 mg / m per day on 1 to 5 intravenously , two courses ) .", "a follow - up brain mri after six months revealed a marked decrease in the size of the mass in the tentorium and falx ( fig .", "the patient had no evidence of cns recurrence during the follow - up period of one year", ". however , the igm level has been increasing slowly , so further chemotherapy , including rituximab may be considered .", "in 1936 bing and neel reported the association of hyperglobulinemia , cns symptoms ( paresthesias , headache , and paralysis ) , and brain infiltration composed of plasma cells and lymphocytes in two patients ( 5 ) . in 1944 , waldenstrom described the syndrome that bears their name ( bing - neel syndrome ) ( 6 ) .", "the bing - neel syndrome , originally placed in a \" toxic - infectious \" category , appears to be the result of involvement of the cns by diffuse neoplasm infiltration .", "although they are most often peripheral , they can involve the cns . in wm , the cns may be involved by a variety of mechanisms , including hyperviscosity and direct infiltration by neoplastic cells ( 7 , 8) .", "patients with bing - neel syndrome have sometimes presented with a mass containing neoplastic cells , but the masses have been intraparenchymal rather than meningeal ( 8 , 9 ) .", "this syndrome can be subdivided into diffuse and tumoral forms . in the diffuse infiltrative form such as this case", ", malignant cells are localized mainly in leptomeningeal spaces , periventricular white - matter , pons , and medulla ( 7 , 10 - 12 ) .", "radiological findings in this syndrome have been reported , but a typical pattern has not emerged ( 13 - 15 ) . an imaging technique would be the preferable diagnostic test , although histological confirmation is necessary to establish the definitive diagnosis .", "intraventricular chemotherapy for bing - neel syndrome was reported to be effective in 1984 ( 15 ) .", "however , a review of the literature reveals that the outcome for most patients who underwent chemotherapy was poor , and the patients died within several months ( 10 , 13 , 14 ) .", "therefore , patients with bing - neel syndrome may benefit from cranial radiation therapy prior to chemotherapy ( 9 , 12 , 16 ) .", "recently , a great deal of interest has been noted by treatments with purine nucleoside analogs ( fludarabine , cladribine , and pentostatin ) because of their remarkable activity in lymphoproliferative disorders .", "it has been reported that a patient with bing - neel syndrome ( in its diffuse form ) was successfully treated with cladribine administration ( 17 ) , or radiation therapy and combination of cladribine , cyclophosphamide , and prednisone ( 18 ) . in the present case", "further treatment was not possible because of persistant bone marrow suppression . a follow - up brain mri after six months revealed a marked decrease in the size of the mass in the tentorium and falx ; the patient had no evidence of cns recurrence during the follow - up period of one year , but the igm level has been increasing slowly . we think that the major effect of deceased mass size was due to the radiation therapy in the present case .", "several retrospective and prospective studies have indicated that rituximab may induce an objective response in approximately 30 - 40% of previous treated patients with wm ( 19 ) .", "however , the effect of rituximab treatment on the cerebrospinal fluid b - cell compartment is limited in comparison with the effect on the b cells in the periphery ( 20 ) , and it has not been tried in bing - neel syndrome as yet . therefore , the effect of rituximab on cns involovement of wm need to be validated by of future studies ."], "abstract_text": ["<S> waldenstrom 's macroglobulinemia is an uncommon low - grade b - cell lymphoproliferative disorder in which monoclonal immunoglobulin m is produced . </S>", "<S> neurological symptoms due to hyperviscosity are frequent manifestations of waldenstrom 's macroglobulinemia . however , central nervous system infiltration by plasmacytoid lymphocytes ( bing - neel syndrome ) has only rarely been reported . </S>", "<S> we report a case of a 51-yr - old woman suffering from waldenstrom 's macroglobulinemia who complained of persistant headache . </S>", "<S> brain magnetic resonance imaging revealed an extra - axial soft tissue mass along the left cavernous sinus , left tentorium , right tentorium , and falx cerebri . a stereotactic biopsy of dural tissue from the falx was performed and showed plasmacytoid lymphocyte infiltration . </S>", "<S> the patient became symptom- free with irradiation of the whole brain followed by chemotherapy with fludarabine . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["waldenstrom 's macroglobulinemia ( wm ) is the result of a clonal proliferation of lymphocytes that produce monoclonal immunoglobulin m ( igm ) .", "it is now considered to correspond to lymphoplasmacytoid lymphoma as defined by the world health organization classification system ( 1 , 2 ) .", "many central nervous system ( cns ) complications have been described in wm patients ; the majority have been associated with blood hyperviscosity caused by igm .", "the hyperviscosity syndrome is characterized by headache , tinnitus , vertigo , blurred vision , and chronic bleeding from the nose and gums ( 3 ) .", "however , cns infiltration by plasmacytoid lymphocytes ( bing - neel syndrome ) has only rarely been reported ( 4 ) ."], ["a 51-yr - old woman was diagnosed with wm three years prior to this presentation .", "she received six courses of chlorambucil ( 0.3 mg / kg per day on day one to four orally ) and prednisone ( 45 mg / m per day on day one to four ) .", "next , she received fludarabine chemotherapy ( 25 mg / m per day on day one to five intravenously ) with a limited response .", "high - dose cyclophosphamide and granulocyte colony - stimulating factor were administered to induce peripheral blood progenitor cell ( pbpcs ) mobilization for autologous stem - cell transplantation ; however , the number of pbpcs collected was not sufficient .", "we then decided on a conservative treatment approach , because the patient did not have specific symptoms and the serum igm level was stable ( 3,000 - 3,500 mg / dl ) .", "the laboratory values on admission were as follows : white cell count 8,050/l with normal differential counts , hemoglobin 9.6 g / dl , platelets 296,000/l , erythrocyte sedimentation rate 144 mm / hr , total protein 9.44 g / dl , albumin 2.85 g / dl , igg 3144.4 mg / dl , iga 25.4 mg / dl , igm", "a brain computed tomography scan revealed multifocal extra - axial tumorous lesions along the dura matter .", "a brain magetic resonance imaging showed an extra - axial soft tissue tumor along the left cavernous sinus and tentorium , right frontal convexity and tentorium and falx ; the brain parenchyma appeared to be unremarkable ( fig .", "cerebrospinal fluid analysis showed the following : white cell count 43/l with neutrophils 1% , lymphocytes 52% and monocytes 47% , total protein 1.81 g / dl , glucose 104 mg / dl , igm 64.5 mg / dl and a few plasmacytoid lymphocytes on cytology .", "stereotactic biopsy of dural tissue at the falx showed a diffuse infiltration with atypical cells , which were identified immunophenotypically as plasmacytoid lymphocyte with expression of lca ( + ) , cd3 ( - ) , cd20 ( + ) , and vs38a ( + ) ( fig .", "2 ) . the patient was confirmed to have cns infiltration by atypical plasmacytoid lymphocyte infiltration ( bing - neel syndrome ) .", "the paitent received a total dose of 1,980 cgy with irradiation therapy of the whole brain that was administered in 11 fractions , and then her headache subsided , but the igm level was elevated persistently to 3,379.8 mg / dl . following radiation therapy ,", "fludarabine chemotherapy was performed ( 25 mg / m per day on 1 to 5 intravenously , two courses ) .", "a follow - up brain mri after six months revealed a marked decrease in the size of the mass in the tentorium and falx ( fig .", "the patient had no evidence of cns recurrence during the follow - up period of one year", ". however , the igm level has been increasing slowly , so further chemotherapy , including rituximab may be considered ."], ["in 1936 bing and neel reported the association of hyperglobulinemia , cns symptoms ( paresthesias , headache , and paralysis ) , and brain infiltration composed of plasma cells and lymphocytes in two patients ( 5 ) . in 1944 , waldenstrom described the syndrome that bears their name ( bing - neel syndrome ) ( 6 ) .", "the bing - neel syndrome , originally placed in a \" toxic - infectious \" category , appears to be the result of involvement of the cns by diffuse neoplasm infiltration .", "although they are most often peripheral , they can involve the cns . in wm , the cns may be involved by a variety of mechanisms , including hyperviscosity and direct infiltration by neoplastic cells ( 7 , 8) .", "patients with bing - neel syndrome have sometimes presented with a mass containing neoplastic cells , but the masses have been intraparenchymal rather than meningeal ( 8 , 9 ) .", "this syndrome can be subdivided into diffuse and tumoral forms . in the diffuse infiltrative form such as this case", ", malignant cells are localized mainly in leptomeningeal spaces , periventricular white - matter , pons , and medulla ( 7 , 10 - 12 ) .", "radiological findings in this syndrome have been reported , but a typical pattern has not emerged ( 13 - 15 ) . an imaging technique would be the preferable diagnostic test , although histological confirmation is necessary to establish the definitive diagnosis .", "intraventricular chemotherapy for bing - neel syndrome was reported to be effective in 1984 ( 15 ) .", "however , a review of the literature reveals that the outcome for most patients who underwent chemotherapy was poor , and the patients died within several months ( 10 , 13 , 14 ) .", "therefore , patients with bing - neel syndrome may benefit from cranial radiation therapy prior to chemotherapy ( 9 , 12 , 16 ) .", "recently , a great deal of interest has been noted by treatments with purine nucleoside analogs ( fludarabine , cladribine , and pentostatin ) because of their remarkable activity in lymphoproliferative disorders .", "it has been reported that a patient with bing - neel syndrome ( in its diffuse form ) was successfully treated with cladribine administration ( 17 ) , or radiation therapy and combination of cladribine , cyclophosphamide , and prednisone ( 18 ) . in the present case", "further treatment was not possible because of persistant bone marrow suppression . a follow - up brain mri after six months revealed a marked decrease in the size of the mass in the tentorium and falx ; the patient had no evidence of cns recurrence during the follow - up period of one year , but the igm level has been increasing slowly . we think that the major effect of deceased mass size was due to the radiation therapy in the present case .", "several retrospective and prospective studies have indicated that rituximab may induce an objective response in approximately 30 - 40% of previous treated patients with wm ( 19 ) .", "however , the effect of rituximab treatment on the cerebrospinal fluid b - cell compartment is limited in comparison with the effect on the b cells in the periphery ( 20 ) , and it has not been tried in bing - neel syndrome as yet . therefore , the effect of rituximab on cns involovement of wm need to be validated by of future studies ."]]}
{"article_id": "PMC3463916", "article_text": ["  the increasing prevalence of childhood obesity , especially in preschool aged children [ 1 , 2 ] , has stimulated research targeting critical periods of growth . according to freinkel 's hypothesis of  fuel - mediated teratogenesis , ", "the intrauterine environment can influence changes in gene expression and affect the development and maturation of fetal organs and tissues [ 3 , 4 ] .", "the postnatal period up to 2 years of age is also a critical period of growth . indeed ,", "rapid infant weight gain is associated with an increased risk of obesity and metabolic consequences later in life [ 58 ] .  ", "these include metabolic parameters such as in utero glucose exposure [ 911 ] , maternal prepregnancy body mass index ( bmi ) [ 1214 ] , and maternal insulin sensitivity during pregnancy . in a cohort of low - income children , maternal obesity during pregnancy more than doubled the risk of obesity in children .", "maternal diet and physical activity during pregnancy [ 1619 ] , as well as smoking may impact offspring risk of obesity and metabolic risk [ 2023 ] .", "however , there is a paucity of literature examining these risk factors in very young children .   ", "in particular , breastfeeding has a protective role against the development of obesity [ 2427 ] .", "harder et al . reported a dose - dependent association between breastfeeding and risk of obesity , where each month of prolonged breastfeeding decreased obesity risk in the child .", "beyond the first few months of life , breast - fed infants gained less weight than formula - fed infants .", "another study showed weight gain from 6 to 12 months was less in infants exclusively breastfed for 5 months compared to 2 months .", "earlier introduction to complementary foods may also contribute to increased risk of childhood overweight [ 14 , 31 ] , however the evidence is inconsistent .", "additionally , reduced physical activity and increased screen time ( television and video ) are associated with obesity risk in older children , although limited data is available in infants [ 33 , 34 ]", ".    there is little known about the impact of maternal and infant lifestyle on weight gain and adiposity at 1 year of life .", "the effect of screen time in infants on weight gain and adiposity has not been included as a potential factor in prior statistical models .", "there is also little information on the effect of maternal physical activity on weight outcomes of offspring .", "the objective of our study was to determine how maternal physical activity , maternal insulin sensitivity ( isogtt ) , prepregnancy bmi , infant feeding practices ( breastfeeding duration , age of introduction of formula , and complementary foods ) , and screen time contribute to infant weight gain and adiposity at 1 year of age .", "the study protocol was approved by the research ethics board at mount sinai hospital and the hospital for sick children .", "participants were recruited for this prospective cohort study at the time of antepartum gestational diabetes mellitus ( gdm ) screening and were consented to be followed into the postpartum .", "all consenting women then completed a 3-h oral glucose tolerance test ( ogtt ) in late 2nd or early 3rd trimester .", "venous blood samples were drawn at baseline , 60 , 120 , and 180  min after ingestion of a standard 100  g glucose load .", "the pregnant women were then classified into 3 glucose tolerance groups : ( 1 ) gdm , as defined by the national diabetes data group ( nddg ) criteria ( requires at least two of the following : fasting glucose 5.8  mmol / l , 1  h postload glucose 10.6  mmol / l , 2  h postload glucose 9.2  mmol / l , or 3  h postload glucose 8.1  mmol / l ) ; ( 2 ) gestational impaired glucose tolerance ( gigt ) , defined as meeting only one of the above criteria ; ( 3 ) normal glucose tolerance ( ngt ) , defined as not meeting any of the nddg criteria .   ", "parental demographics , medical history and anthropometrics were collected during pregnancy at the time of the ogtt .", "data collected included maternal age , maternal prepregnancy weight , maternal and paternal ethnicity , family history of diabetes , socioeconomic status , and maternal physical activity indices .", "maternal isogtt was calculated from the ogtt using the matsuda index , which is well correlated with insulin sensitivity derived from the euglycemic - hyperglycemic clamp method .", "physical activity was assessed using the baecke questionnaire , which has been validated in several populations including women of child - bearing age [ 37 , 38 ] .", "the questionnaire was completed during the ogtt , with participants reporting on their physical activity in the year preceding the pregnancy .", "the baecke questionnaire measures three domains of physical activity : ( i ) occupation - associated activity ( work index ) ; ( ii ) sport - related physical activity ( vigorous / sport index ) ; ( iii ) leisure - time physical activity not including sports ( leisure index ) .", "the work index quantifies the exertion related to occupational activities , including sitting , standing , lifting , and walking , as well as effects on the individual ( e.g. , fatigue and perspiration ) .", "the sport index characterizes vigorous / sport activity with respect to intensity ( using the updated compendium of physical activities ) .", "these indices were calculated on a scale from 15 , where 5 represented the highest level of physical activity for each category .", "for example , a score of 3 out of 5 for leisure index might indicate ", "sometimes  walking or cycling for 1530  min and  seldom  watching television .", "mean parental education score and occupation score was calculated on a scale of 17 and 19 , based on the hollingshead index , an established surrogate index of socioeconomic status . a maximum averaged parental score of 62 indicated the highest level of education and profession .  ", "infant information collected at birth included length of gestation , gender ( male or female ) , and birth weight .", "at the 1 year visit , infant weight and length were measured , and weight - for - length z - score was calculated according to the world health organization ( who ) 2006 child growth standards   to provide a surrogate measure of adiposity . the infant lifestyle questionnaire was completed by the mothers when the infants were 3 and 12 months of age and provided information on exclusive breastfeeding duration , age of introduction to formula and cereal , and screen time based on predictors found to be important for the development of obesity ( average daily exposure to tv or video viewing , meals eaten while tv is on , tv in bedroom ) [ 34 , 42 ] .", "infants born < 37 or > 42 weeks , twins , or those with medical illnesses that required prolonged hospitalization were excluded from the analysis .  ", "bivariate analysis of continuous variables with infant weight gain and weight - for - length z - score were assessed by spearman 's correlation analysis .", "independent samples t - test or one - way analysis of variance ( anova ) and post hoc analysis ( fisher 's least significant difference ) were used to test differences in the outcome variables with maternal glucose tolerance group ( gdm , gigt , or ngt ) , gdm status ( yes / no ) , family history of diabetes ( yes / no ) , infant gender ( male / female ) , ethnicity ( caucasian or non - caucasian ) , age of introduction to formula ( at birth , 15 months , 6 months or never ) , and average daily screen time divided into 3 categories ( no screen time , < 60 minutes , and 60 minutes ) .", "we converted age of introduction to formula into a categorical variable because although exclusive breastfeeding duration and age of introduction to formula are similar variables , they are not synonymous because some infants were never formula - fed ( n = 80 ) . variables determined to be significant from bivariate analyses , concurrently with variables known to influence infant weight gain and adiposity , were entered into multiple linear regression models .", "two separate models were created for weight gain from birth to 1 year and weight - for - length z - score at 1 year .", "nonmodifiable risk factors ( infant age , sex , ethnicity , birth weight , and family history of diabetes ) were forced into the model and modifiable risk factors ( maternal prepregnancy bmi , gdm status , maternal physical activity indices prior to pregnancy , socioeconomic status , maternal log isogtt , infant exclusive breastfeeding duration , age of introduction to formula , age of introduction to cereal , and daily screen time ) were entered according to the forward stepwise method .", "descriptive characteristics and lifestyle measures are presented in table 1 for mothers and infants at 1-year postpartum ( n = 246 ) . from the time of consent", "postnatally to the 1-year followup visit , 62 ( 20% ) of the original 311 infants were lost to followup .", "there were no significant differences in gdm status , prepregnancy bmi , maternal education , or maternal physical activity between mothers retained in the study versus those lost to followup .", "no significant differences were found for infant weight gain and weight - for - length z - score based on the mother 's gdm status ( yes / no ) or glucose tolerance group during pregnancy ( gdm , gigt , or ngt ) .", "independent samples t - tests also showed no significant differences in the infant outcomes for family history of diabetes ( yes / no ) and infant ethnicity ( caucasian or non - caucasian ) .", "males had greater weight gain than females ( p < 0.001 ) , however no differences between males and females for weight - for - length z - score were found ( p = 0.636 ) .", "one - way anova showed significant differences in infant weight gain for age of formula introduction ( p = 0.001 ) and screen time ( p = 0.032 ) ( figure 1 ) .", "similar trends for age of formula introduction were found for infant weight - for - length z - score at 1 year ( p = 0.012 ) . however no differences were found between 0 , < 60  min , or 60 minutes of daily screen time ( p = 0.19 ) .", "spearman 's correlation analysis ( table 2 ) showed a negative association for infant weight gain with maternal pregravid vigorous / sport index ( p = 0.031 ) , exclusive breastfeeding duration ( p < 0.001 ) , and an earlier age of introduction to cereal ( p = 0.02 ) .", "weight - for - length z - score was positively associated with maternal prepregnancy bmi ( p = 0.003 ) and birth weight ( p < 0.001 ) , and negatively associated with maternal log isogtt during pregnancy ( p = 0.031 ) and maternal pregravid vigorous / sport index ( p = 0.006 ) .", "maternal pregravid vigorous / sport index was also positively correlated with isogtt during pregnancy ( p < 0.001 ) , as expected from previous data .", "each month of prolonged exclusive breastfeeding reduced weight gain by 116.4  g , after adjustment for infant age , sex , infant ethnicity , family history of diabetes , and maternal pregravid vigorous / sport index ( p < 0.001 ) . after adjustment , each unit increase in maternal pregravid vigorous / sport index decreased weight gain by 218.6  g ( p = 0.016 ) . in total , 33% of the variance in the model for weight gain was explained by the sample .", "infant birth weight , infant ethnicity , and family history of diabetes did not significantly predict infant weight gain at 1 year . additionally , socioeconomic status , maternal prepregnancy bmi , maternal isogtt , gdm status , maternal pregravid work index , maternal pregravid leisure index , infant age of introduction to formula , and infant screen time did not emerge in the model when entered . for weight - for - length z - score , an increase in one unit of maternal prepregnancy bmi ( kg / m )", "was associated with an increase in weight - for - length z - score of 0.03 ( p = 0.016 ) , or one standard deviation change on the who child growth standards , after adjustment .", "thus , mothers with a greater prepregnancy bmi were more likely to have heavier infants normalized for length .", "in addition , each month of prolonged exclusive breastfeeding and unit increase in maternal pregravid vigorous / sport index decreased weight - for - length z - score by 0.08 ( p = 0.010 ) and 0.20 ( p = 0.031 ) , respectively .", "infant sex , infant ethnicity , and family history of diabetes were not significantly associated with infant weight - for - length z - score .", "variables that did not emerge in the model included : socioeconomic status , maternal isogtt , gdm status , maternal pregravid work index , maternal pregravid leisure index , infant age of introduction to formula , and infant screen time .", "approximately , 19% of the variance in the model for weight - for - length z - score was explained by the sample .", "  these findings demonstrate that maternal pregravid physical activity , prepregnancy bmi , and infant feeding practices have a significant influence on infant weight gain and adiposity .", "weight gain from birth to 1 year was negatively predicted by infant female sex , maternal pregravid vigorous / sport activity , and exclusive breastfeeding duration and positively predicted by infant age at time of 1 year visit .", "weight - for - length z - score at 1 year was negatively predicted by maternal pregravid vigorous / sport activity and exclusive breastfeeding duration and positively predicted by birth weight , infant age at time of 1 year visit , and maternal prepregnancy bmi .", "factors that have been previously shown to influence infant weight gain and weight - for - length z - score , such as socioeconomic status or gdm status , were not significant in our model .", "this may be due to the fact that women in this study tended to have excellent glycemic control , which was evidenced by normal birth weight in the gdm pregnancies .", "the effect of maternal prepregnancy bmi on infant adiposity is consistent with existing literature [ 1214 , 31 ] . in mothers with ngt or gdm ,", "maternal prepregnancy bmi was significantly associated with childhood overweight in both groups , even after adjustment for maternal glucose status and infant birth weight .", "furthermore , knight and colleagues reported that the impact of maternal fasting plasma glucose in nondiabetic mothers on infant growth was transient , while maternal prepregnancy bmi and paternal bmi remained correlated with offspring bmi at 2 years of age .", "thus , the authors suggested that parental obesity , shared environment , or genetic factors had a greater influence on childhood bmi than maternal fasting plasma glucose during pregnancy .  ", "after controlling for confounding factors , maternal pregravid vigorous / sport activity significantly predicted infant weight gain .", "the mean pregravid vigorous / sport index in our study was 2.4 , which suggests that 1530  min of walking or cycling per day   in mothers prior to pregnancy may be beneficial for decreasing obesity risk of offspring .", "maternal log isogtt did not predict infant growth at 1 year , after adjusting for maternal physical activity indices .", "prior analyses by our group showed pregravid vigorous / sport activity was an independent predictor of maternal isogtt and reduced glucose intolerance in pregnancy .", "taken together , pregravid physical activity may affect infant weight gain through a mechanism that partly involves maternal isogtt , however , these results suggest that maternal lifestyle may have a greater impact on infant weight gain than maternal glucose tolerance group or isogtt .", "although daily screen time was not a significant predictor of infant weight gain or adiposity in our multivariate analyses , it was unsettling to find that more than half of the infants in our study were already exposed to television at 1 year of age and 11% of infants watched more than 2 hours of television per day .", "infants with 60 minutes of screen time each day were heavier at 1 year of age than infants with 0 or < 60 minutes of daily screen time .", "one possible explanation may include decreased overall activity levels in infants exposed to television for longer periods of time .", "preschool children , more than 2 hours of television per day was associated with a higher risk of overweight and at risk for overweight and greater adiposity . despite the known effect of screen time on bmi in childhood [ 4547 ] ,", "there is currently limited information on the effect of screen time under 2 years of age .   the mechanisms to explain the effects of breastfeeding compared to formula feeding may include different physiological and behavioural factors .", "several studies [ 2427 ] , however not all [ 32 , 48 , 49 ] , suggest that breastfeeding has a protective role on later obesity risk .", "after adjusting for confounding variables , infants exclusively breast fed for a longer duration had reduced weight gain and adiposity at 1 year .", "this may be influenced by the unique metabolic factors in breast milk compared to formula , such as breast milk leptin [ 50 , 51 ] and adiponectin   that may induce earlier satiety .", "the higher protein content of formula may also contribute to increased adiposity in formula fed infants .", "koletzko and colleagues demonstrated that infants given formula with high protein content compared to infants given formula with low protein content resulted in increased weight at 2 years of age with no effect on infant length .", "it is thought that the higher protein consumption results in higher insulin secretion and stimulates the expression of insulin - like growth factor i ( igf - i ) , leading to more adipogenic activity and adipocyte differentiation [ 54 , 55 ] .", "additionally , behavioural differences between breast - fed and formula - fed infants may affect infant energy intake .", "studies have shown that there is less maternal control and a greater response to the infant 's hunger and satiety cues with breastfeeding compared to formula feeding [ 56 , 57 ] .", "there is also a lower frequency of meals and a higher uniformity of feeding volumes in formula - fed infants compared to breast - fed infants .  ", "although some studies have reported the timing of cereal introduction as a positive predictor of infant weight gain , it did not emerge in our multivariate analyses . in a recent study , huh and colleagues noted that the timing of the introduction of solid foods on the odds of obesity at 3 years of age varied by breastfeeding status .", "infants that were introduced to solid foods before 4 months and were never breast fed or stopped breastfeeding before 4 months had a sixfold increase in the odds of obesity compared to infants breast fed for more than 4 months . since our study population was introduced to cereal later than 4 months ( 5.5  1.1 months )", "altogether , our findings on infant feeding suggest that exclusive breastfeeding duration may be more predictive of infant weight gain and adiposity than age of formula or cereal introduction .  ", "this study should be viewed in context of certain strengths and limitations . due to the assessment of glucose tolerance in the late 2nd or 3rd trimester of pregnancy", ", we can not completely exclude the possibility that some individuals may have received treatments following ogtt that could affect in utero glucose exposure and isogtt on infant outcomes .", "although data was available on the type of intervention ( diet therapy or other ) , glycemic control during pregnancy could not be determined .", "other limitations included the lack of data on maternal diet and maternal physical activity during pregnancy in our study .", "proposed that maternal energy intake influenced energy intake in children more than intrauterine exposure to diabetes , which suggests maternal energy intake should be included in future studies .", "additionally , our study population had a large proportion of caucasian infants and highly educated parents , and it is unclear whether results may be extended to other ethnic backgrounds or families with lower socioeconomic status .", "loss to followup was 20% , and although there were no differences in maternal factors between those who left and remained in the study , feeding practices of the infants may have differed .", "we were also unable to report on infant physical activity due to challenges in capturing physical activity accurately and objectively in infants .", "nevertheless , we examined maternal and infant lifestyle variables that have not been previously studied together , providing further insight into the effects of the maternal and infant environment on weight gain and adiposity while controlling for important confounding variables .    in summary , increased maternal pregravid physical activity and longer exclusive breastfeeding duration may have a critical influence on reducing infant weight gain and adiposity as early as 1 year of age .", "results from this study support the recommendation for exclusive breastfeeding in infancy , and suggest maternal physical activity may also influence postnatal outcomes .", "future directions include examining the additive effects of maternal diet during and after pregnancy , infant physical activity , and sedentary time on infant weight gain and adiposity .", "the evaluation of paternal physical activity may also provide insight into the impact of family lifestyle on infant growth .", "longitudinal followup of these children will examine the effects of maternal and infant factors on bmi and adiposity in early childhood beyond 1 year of age ."], "abstract_text": ["<S> increasing evidence supports the contribution of intrauterine environmental exposures on obesity risk in offspring . </S>", "<S> few studies have included maternal and infant lifestyle factors . </S>", "<S> our objective was to study the impact of maternal physical activity , infant feeding , and screen time on offspring weight gain and adiposity . in a prospective cohort study , </S>", "<S> 246 mothers underwent testing during pregnancy to assess glucose tolerance status and insulin sensitivity . </S>", "<S> anthropometry and questionnaires on physical activity , infant feeding , and screen time were completed . </S>", "<S> multiple - linear regression was performed to examine the impact of maternal and infant factors on infant weight gain and weight - for - length z - score at 1 year . </S>", "<S> infant weight outcomes were negatively predicted by maternal pregravid vigorous / sport index and exclusive breastfeeding duration . </S>", "<S> after adjustment , each unit increase in maternal pregravid vigorous / sport index decreased infant weight gain by 218.6 g ( t = 2.44 , p = 0.016 ) and weight - for - length z - score by 0.20 ( t = 2.17 , p = 0.031 ) . </S>", "<S> each month of exclusive breastfeeding reduced infant weight gain by 116.4  g ( t = 3.97 , p < 0.001 ) and weight - for - length z - score by 0.08 ( t = 2.59 , p = 0.01 ) . </S>", "<S> maternal pregravid physical activity and exclusive breastfeeding duration are associated with weight gain and adiposity as early as 1 year of age . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["  the increasing prevalence of childhood obesity , especially in preschool aged children [ 1 , 2 ] , has stimulated research targeting critical periods of growth . according to freinkel 's hypothesis of  fuel - mediated teratogenesis , ", "the intrauterine environment can influence changes in gene expression and affect the development and maturation of fetal organs and tissues [ 3 , 4 ] .", "the postnatal period up to 2 years of age is also a critical period of growth . indeed ,", "rapid infant weight gain is associated with an increased risk of obesity and metabolic consequences later in life [ 58 ] .  ", "these include metabolic parameters such as in utero glucose exposure [ 911 ] , maternal prepregnancy body mass index ( bmi ) [ 1214 ] , and maternal insulin sensitivity during pregnancy . in a cohort of low - income children , maternal obesity during pregnancy more than doubled the risk of obesity in children .", "maternal diet and physical activity during pregnancy [ 1619 ] , as well as smoking may impact offspring risk of obesity and metabolic risk [ 2023 ] .", "however , there is a paucity of literature examining these risk factors in very young children .   ", "in particular , breastfeeding has a protective role against the development of obesity [ 2427 ] .", "harder et al . reported a dose - dependent association between breastfeeding and risk of obesity , where each month of prolonged breastfeeding decreased obesity risk in the child .", "beyond the first few months of life , breast - fed infants gained less weight than formula - fed infants .", "another study showed weight gain from 6 to 12 months was less in infants exclusively breastfed for 5 months compared to 2 months .", "earlier introduction to complementary foods may also contribute to increased risk of childhood overweight [ 14 , 31 ] , however the evidence is inconsistent .", "additionally , reduced physical activity and increased screen time ( television and video ) are associated with obesity risk in older children , although limited data is available in infants [ 33 , 34 ]", ".    there is little known about the impact of maternal and infant lifestyle on weight gain and adiposity at 1 year of life .", "the effect of screen time in infants on weight gain and adiposity has not been included as a potential factor in prior statistical models .", "there is also little information on the effect of maternal physical activity on weight outcomes of offspring .", "the objective of our study was to determine how maternal physical activity , maternal insulin sensitivity ( isogtt ) , prepregnancy bmi , infant feeding practices ( breastfeeding duration , age of introduction of formula , and complementary foods ) , and screen time contribute to infant weight gain and adiposity at 1 year of age ."], ["the study protocol was approved by the research ethics board at mount sinai hospital and the hospital for sick children .", "participants were recruited for this prospective cohort study at the time of antepartum gestational diabetes mellitus ( gdm ) screening and were consented to be followed into the postpartum .", "all consenting women then completed a 3-h oral glucose tolerance test ( ogtt ) in late 2nd or early 3rd trimester .", "venous blood samples were drawn at baseline , 60 , 120 , and 180  min after ingestion of a standard 100  g glucose load .", "the pregnant women were then classified into 3 glucose tolerance groups : ( 1 ) gdm , as defined by the national diabetes data group ( nddg ) criteria ( requires at least two of the following : fasting glucose 5.8  mmol / l , 1  h postload glucose 10.6  mmol / l , 2  h postload glucose 9.2  mmol / l , or 3  h postload glucose 8.1  mmol / l ) ; ( 2 ) gestational impaired glucose tolerance ( gigt ) , defined as meeting only one of the above criteria ; ( 3 ) normal glucose tolerance ( ngt ) , defined as not meeting any of the nddg criteria .   ", "parental demographics , medical history and anthropometrics were collected during pregnancy at the time of the ogtt .", "data collected included maternal age , maternal prepregnancy weight , maternal and paternal ethnicity , family history of diabetes , socioeconomic status , and maternal physical activity indices .", "maternal isogtt was calculated from the ogtt using the matsuda index , which is well correlated with insulin sensitivity derived from the euglycemic - hyperglycemic clamp method .", "physical activity was assessed using the baecke questionnaire , which has been validated in several populations including women of child - bearing age [ 37 , 38 ] .", "the questionnaire was completed during the ogtt , with participants reporting on their physical activity in the year preceding the pregnancy .", "the baecke questionnaire measures three domains of physical activity : ( i ) occupation - associated activity ( work index ) ; ( ii ) sport - related physical activity ( vigorous / sport index ) ; ( iii ) leisure - time physical activity not including sports ( leisure index ) .", "the work index quantifies the exertion related to occupational activities , including sitting , standing , lifting , and walking , as well as effects on the individual ( e.g. , fatigue and perspiration ) .", "the sport index characterizes vigorous / sport activity with respect to intensity ( using the updated compendium of physical activities ) .", "these indices were calculated on a scale from 15 , where 5 represented the highest level of physical activity for each category .", "for example , a score of 3 out of 5 for leisure index might indicate ", "sometimes  walking or cycling for 1530  min and  seldom  watching television .", "mean parental education score and occupation score was calculated on a scale of 17 and 19 , based on the hollingshead index , an established surrogate index of socioeconomic status . a maximum averaged parental score of 62 indicated the highest level of education and profession .  ", "infant information collected at birth included length of gestation , gender ( male or female ) , and birth weight .", "at the 1 year visit , infant weight and length were measured , and weight - for - length z - score was calculated according to the world health organization ( who ) 2006 child growth standards   to provide a surrogate measure of adiposity . the infant lifestyle questionnaire was completed by the mothers when the infants were 3 and 12 months of age and provided information on exclusive breastfeeding duration , age of introduction to formula and cereal , and screen time based on predictors found to be important for the development of obesity ( average daily exposure to tv or video viewing , meals eaten while tv is on , tv in bedroom ) [ 34 , 42 ] .", "infants born < 37 or > 42 weeks , twins , or those with medical illnesses that required prolonged hospitalization were excluded from the analysis .  ", "bivariate analysis of continuous variables with infant weight gain and weight - for - length z - score were assessed by spearman 's correlation analysis .", "independent samples t - test or one - way analysis of variance ( anova ) and post hoc analysis ( fisher 's least significant difference ) were used to test differences in the outcome variables with maternal glucose tolerance group ( gdm , gigt , or ngt ) , gdm status ( yes / no ) , family history of diabetes ( yes / no ) , infant gender ( male / female ) , ethnicity ( caucasian or non - caucasian ) , age of introduction to formula ( at birth , 15 months , 6 months or never ) , and average daily screen time divided into 3 categories ( no screen time , < 60 minutes , and 60 minutes ) .", "we converted age of introduction to formula into a categorical variable because although exclusive breastfeeding duration and age of introduction to formula are similar variables , they are not synonymous because some infants were never formula - fed ( n = 80 ) . variables determined to be significant from bivariate analyses , concurrently with variables known to influence infant weight gain and adiposity , were entered into multiple linear regression models .", "two separate models were created for weight gain from birth to 1 year and weight - for - length z - score at 1 year .", "nonmodifiable risk factors ( infant age , sex , ethnicity , birth weight , and family history of diabetes ) were forced into the model and modifiable risk factors ( maternal prepregnancy bmi , gdm status , maternal physical activity indices prior to pregnancy , socioeconomic status , maternal log isogtt , infant exclusive breastfeeding duration , age of introduction to formula , age of introduction to cereal , and daily screen time ) were entered according to the forward stepwise method ."], ["descriptive characteristics and lifestyle measures are presented in table 1 for mothers and infants at 1-year postpartum ( n = 246 ) . from the time of consent", "postnatally to the 1-year followup visit , 62 ( 20% ) of the original 311 infants were lost to followup .", "there were no significant differences in gdm status , prepregnancy bmi , maternal education , or maternal physical activity between mothers retained in the study versus those lost to followup .", "no significant differences were found for infant weight gain and weight - for - length z - score based on the mother 's gdm status ( yes / no ) or glucose tolerance group during pregnancy ( gdm , gigt , or ngt ) .", "independent samples t - tests also showed no significant differences in the infant outcomes for family history of diabetes ( yes / no ) and infant ethnicity ( caucasian or non - caucasian ) .", "males had greater weight gain than females ( p < 0.001 ) , however no differences between males and females for weight - for - length z - score were found ( p = 0.636 ) .", "one - way anova showed significant differences in infant weight gain for age of formula introduction ( p = 0.001 ) and screen time ( p = 0.032 ) ( figure 1 ) .", "similar trends for age of formula introduction were found for infant weight - for - length z - score at 1 year ( p = 0.012 ) . however no differences were found between 0 , < 60  min , or 60 minutes of daily screen time ( p = 0.19 ) .", "spearman 's correlation analysis ( table 2 ) showed a negative association for infant weight gain with maternal pregravid vigorous / sport index ( p = 0.031 ) , exclusive breastfeeding duration ( p < 0.001 ) , and an earlier age of introduction to cereal ( p = 0.02 ) .", "weight - for - length z - score was positively associated with maternal prepregnancy bmi ( p = 0.003 ) and birth weight ( p < 0.001 ) , and negatively associated with maternal log isogtt during pregnancy ( p = 0.031 ) and maternal pregravid vigorous / sport index ( p = 0.006 ) .", "maternal pregravid vigorous / sport index was also positively correlated with isogtt during pregnancy ( p < 0.001 ) , as expected from previous data .", "each month of prolonged exclusive breastfeeding reduced weight gain by 116.4  g , after adjustment for infant age , sex , infant ethnicity , family history of diabetes , and maternal pregravid vigorous / sport index ( p < 0.001 ) . after adjustment , each unit increase in maternal pregravid vigorous / sport index decreased weight gain by 218.6  g ( p = 0.016 ) . in total , 33% of the variance in the model for weight gain was explained by the sample .", "infant birth weight , infant ethnicity , and family history of diabetes did not significantly predict infant weight gain at 1 year . additionally , socioeconomic status , maternal prepregnancy bmi , maternal isogtt , gdm status , maternal pregravid work index , maternal pregravid leisure index , infant age of introduction to formula , and infant screen time did not emerge in the model when entered . for weight - for - length z - score , an increase in one unit of maternal prepregnancy bmi ( kg / m )", "was associated with an increase in weight - for - length z - score of 0.03 ( p = 0.016 ) , or one standard deviation change on the who child growth standards , after adjustment .", "thus , mothers with a greater prepregnancy bmi were more likely to have heavier infants normalized for length .", "in addition , each month of prolonged exclusive breastfeeding and unit increase in maternal pregravid vigorous / sport index decreased weight - for - length z - score by 0.08 ( p = 0.010 ) and 0.20 ( p = 0.031 ) , respectively .", "infant sex , infant ethnicity , and family history of diabetes were not significantly associated with infant weight - for - length z - score .", "variables that did not emerge in the model included : socioeconomic status , maternal isogtt , gdm status , maternal pregravid work index , maternal pregravid leisure index , infant age of introduction to formula , and infant screen time .", "approximately , 19% of the variance in the model for weight - for - length z - score was explained by the sample ."], ["  these findings demonstrate that maternal pregravid physical activity , prepregnancy bmi , and infant feeding practices have a significant influence on infant weight gain and adiposity .", "weight gain from birth to 1 year was negatively predicted by infant female sex , maternal pregravid vigorous / sport activity , and exclusive breastfeeding duration and positively predicted by infant age at time of 1 year visit .", "weight - for - length z - score at 1 year was negatively predicted by maternal pregravid vigorous / sport activity and exclusive breastfeeding duration and positively predicted by birth weight , infant age at time of 1 year visit , and maternal prepregnancy bmi .", "factors that have been previously shown to influence infant weight gain and weight - for - length z - score , such as socioeconomic status or gdm status , were not significant in our model .", "this may be due to the fact that women in this study tended to have excellent glycemic control , which was evidenced by normal birth weight in the gdm pregnancies .", "the effect of maternal prepregnancy bmi on infant adiposity is consistent with existing literature [ 1214 , 31 ] . in mothers with ngt or gdm ,", "maternal prepregnancy bmi was significantly associated with childhood overweight in both groups , even after adjustment for maternal glucose status and infant birth weight .", "furthermore , knight and colleagues reported that the impact of maternal fasting plasma glucose in nondiabetic mothers on infant growth was transient , while maternal prepregnancy bmi and paternal bmi remained correlated with offspring bmi at 2 years of age .", "thus , the authors suggested that parental obesity , shared environment , or genetic factors had a greater influence on childhood bmi than maternal fasting plasma glucose during pregnancy .  ", "after controlling for confounding factors , maternal pregravid vigorous / sport activity significantly predicted infant weight gain .", "the mean pregravid vigorous / sport index in our study was 2.4 , which suggests that 1530  min of walking or cycling per day   in mothers prior to pregnancy may be beneficial for decreasing obesity risk of offspring .", "maternal log isogtt did not predict infant growth at 1 year , after adjusting for maternal physical activity indices .", "prior analyses by our group showed pregravid vigorous / sport activity was an independent predictor of maternal isogtt and reduced glucose intolerance in pregnancy .", "taken together , pregravid physical activity may affect infant weight gain through a mechanism that partly involves maternal isogtt , however , these results suggest that maternal lifestyle may have a greater impact on infant weight gain than maternal glucose tolerance group or isogtt .", "although daily screen time was not a significant predictor of infant weight gain or adiposity in our multivariate analyses , it was unsettling to find that more than half of the infants in our study were already exposed to television at 1 year of age and 11% of infants watched more than 2 hours of television per day .", "infants with 60 minutes of screen time each day were heavier at 1 year of age than infants with 0 or < 60 minutes of daily screen time .", "one possible explanation may include decreased overall activity levels in infants exposed to television for longer periods of time .", "preschool children , more than 2 hours of television per day was associated with a higher risk of overweight and at risk for overweight and greater adiposity . despite the known effect of screen time on bmi in childhood [ 4547 ] ,", "there is currently limited information on the effect of screen time under 2 years of age .   the mechanisms to explain the effects of breastfeeding compared to formula feeding may include different physiological and behavioural factors .", "several studies [ 2427 ] , however not all [ 32 , 48 , 49 ] , suggest that breastfeeding has a protective role on later obesity risk .", "after adjusting for confounding variables , infants exclusively breast fed for a longer duration had reduced weight gain and adiposity at 1 year .", "this may be influenced by the unique metabolic factors in breast milk compared to formula , such as breast milk leptin [ 50 , 51 ] and adiponectin   that may induce earlier satiety .", "the higher protein content of formula may also contribute to increased adiposity in formula fed infants .", "koletzko and colleagues demonstrated that infants given formula with high protein content compared to infants given formula with low protein content resulted in increased weight at 2 years of age with no effect on infant length .", "it is thought that the higher protein consumption results in higher insulin secretion and stimulates the expression of insulin - like growth factor i ( igf - i ) , leading to more adipogenic activity and adipocyte differentiation [ 54 , 55 ] .", "additionally , behavioural differences between breast - fed and formula - fed infants may affect infant energy intake .", "studies have shown that there is less maternal control and a greater response to the infant 's hunger and satiety cues with breastfeeding compared to formula feeding [ 56 , 57 ] .", "there is also a lower frequency of meals and a higher uniformity of feeding volumes in formula - fed infants compared to breast - fed infants .  ", "although some studies have reported the timing of cereal introduction as a positive predictor of infant weight gain , it did not emerge in our multivariate analyses . in a recent study , huh and colleagues noted that the timing of the introduction of solid foods on the odds of obesity at 3 years of age varied by breastfeeding status .", "infants that were introduced to solid foods before 4 months and were never breast fed or stopped breastfeeding before 4 months had a sixfold increase in the odds of obesity compared to infants breast fed for more than 4 months . since our study population was introduced to cereal later than 4 months ( 5.5  1.1 months )", "altogether , our findings on infant feeding suggest that exclusive breastfeeding duration may be more predictive of infant weight gain and adiposity than age of formula or cereal introduction .  ", "this study should be viewed in context of certain strengths and limitations . due to the assessment of glucose tolerance in the late 2nd or 3rd trimester of pregnancy", ", we can not completely exclude the possibility that some individuals may have received treatments following ogtt that could affect in utero glucose exposure and isogtt on infant outcomes .", "although data was available on the type of intervention ( diet therapy or other ) , glycemic control during pregnancy could not be determined .", "other limitations included the lack of data on maternal diet and maternal physical activity during pregnancy in our study .", "proposed that maternal energy intake influenced energy intake in children more than intrauterine exposure to diabetes , which suggests maternal energy intake should be included in future studies .", "additionally , our study population had a large proportion of caucasian infants and highly educated parents , and it is unclear whether results may be extended to other ethnic backgrounds or families with lower socioeconomic status .", "loss to followup was 20% , and although there were no differences in maternal factors between those who left and remained in the study , feeding practices of the infants may have differed .", "we were also unable to report on infant physical activity due to challenges in capturing physical activity accurately and objectively in infants .", "nevertheless , we examined maternal and infant lifestyle variables that have not been previously studied together , providing further insight into the effects of the maternal and infant environment on weight gain and adiposity while controlling for important confounding variables .    in summary , increased maternal pregravid physical activity and longer exclusive breastfeeding duration may have a critical influence on reducing infant weight gain and adiposity as early as 1 year of age .", "results from this study support the recommendation for exclusive breastfeeding in infancy , and suggest maternal physical activity may also influence postnatal outcomes .", "future directions include examining the additive effects of maternal diet during and after pregnancy , infant physical activity , and sedentary time on infant weight gain and adiposity .", "the evaluation of paternal physical activity may also provide insight into the impact of family lifestyle on infant growth .", "longitudinal followup of these children will examine the effects of maternal and infant factors on bmi and adiposity in early childhood beyond 1 year of age ."]]}
{"article_id": "PMC2582979", "article_text": ["phylogenetic networks represent the evolutionary relationships of taxa , including sequences , genes , chromosomes , genomes , or species .", "there are different types of phylogenetic networks , but here , we are interested in reticulate networks , which provide an explicit representation of evolutionary history , meaning that internal  nodes  represent ancestral species , and nodes with more than two parents correspond to reticulate events such as recombination , hybridization or lateral gene transfer ( huson and bryant 2006 ) .", "reticulate networks have been extensively used in evolutionary studies , especially at the population level , where reticulate events are , in general , quite common ( posada and crandall 2001 ) .", "very few studies have tried to assess the performance of the algorithms used to reconstruct phylogenetic networks ( cassens et al .", "2007 ) , and only recently , a comprehensive computer simulation study has been completed ( woolley et al .", "one of the problems that arose during these studies was the comparison of reticulate networks .", "although several comparison metrics have been already introduced in the literature ( baroni et al .", "2008a , 2008b ; cardona , rossell , and valiente 2008b , 2008c ; cardona g , llabrs m , rossell f , valiente g , metrics for phylogenetic networks ii : nodal and triplets metrics , unpublished data ; cardona g , llabrs m , rossell f , valiente g , recent advances in metrics for phylogenetic networks , unpublished date ) , all of them have specific requirements regarding the nature of the networks to be compared in order to be ", "perfect ,  that is , to have the metric properties of nonnegativity , separation , symmetry , and triangle inequality . before continuing", "a network contains nodes ( vertices ) and  branches  ( edges ) that connect them .", "in general , we will refer to  rooted networks ,  with a direction from the past to the present ( i.e. , directed graphs ) that allows for the identification of  parent  and  child ", "the  root node  is the oldest node and has no parents .  tree nodes", "when two nodes share the same parent they are  siblings .  depending on the relationships among the nodes that occur in a network , these can be classified as  tree sibling  ( cardona , llabrs , et al .", "2008a ; cardona , rossell , and valiente 2008a ) , where every hybrid node has at least one sibling that is a tree node ;  tree child ", "2008b ; cardona , rossell , and valiente 2008a , 2008b , 2008c ; cardona g , llabrs m , rossell f , valiente g , unpublished data ) , in which every internal node has at least one child that is a tree node ;  regular  ( baroni et al .", "2004 ) , where the set of descendant leaves ( clusters ) of the nodes are all distinct ;  galled trees  ( gusfield et al .", "2004a , 2004b ) , where the paths from the most recent common ancestor ( mrca ) of the parents of a hybrid node down to the hybrid node form disjoint cycles ; and ( binary )  trees ,  which only contain tree nodes .", "these network classes are nested in this order : tree sibling  tree child  galled trees  trees , meaning a tree is also a galled tree , a tree child network , and a tree sibling network , and so on .", "regular networks , however , are not related to tree sibling , tree child , or galled tree networks ( cardona g , llabrs m , rossell f , valiente g , recent advances in metrics for phylogenetic networks , unpublished date ) . the point we want to make is that the networks resulting from reticulating evolutionary processes do not necessarily correspond with any of the idealized classes of networks described above and for which perfect metrics exist . were this true , it would imply that many phylogenetic networks could not be properly compared with the existing metrics .", "obviously , it is very important to characterize the size of this perceived gap between the algorithms and the biology , and this is precisely our goal . in order to provide a formal statistical description of this disagreement ,", "independent of particular organisms or genomic regions , we will use coalescent theory ( kingman 1982 ) to generate the type of reticulate networks that result from the evolutionary process .", "the coalescent describes the probabilities of the different genealogies for a sample of genes generally but not necessarily from the same population , and it was further extended by hudson ( 1983 ) to include recombination events .", "specifically , we will quantify the different classes of networks ( regular , tree sibling , tree child , and galled trees ) produced by the coalescent as a function of the population recombination rate .", "the standard coalescent describes the possible histories of a sample of genes back in time to their mrca ( kingman 1982 ) . in the absence of evolutionary forces like selection , migration , or recombination ,", "the only types of events that can occur are coalescent events , in which two lineages ( branches ) fuse into one .", "therefore , for a sample of n genes , there will be n  1 coalescent events until the mrca is reached .", "if recombination is included , the recombination events will result actually in the opposite pattern going backward in time , as in this case one lineage ( the recombinant ) separates into two ( the parents of the recombination event ) .", "therefore , the genealogies produced by the coalescent with recombination ( also known as arg , for  ancestral recombination graph  ) will be explicit reticulate networks as internal nodes actually represent evolutionary events . in figure 1", ", we show a typical genealogy for a sample of 10 genes . a single realization of the coalescent with recombination .", "the genealogy goes from the present ( bottom ) to the past ( up ) .", "coalescent and recombinant nodes are represented in white and gray , respectively . in our simulations , the genealogies were simulated with the program recodon ( arenas and posada 2007 ) .", "two sample sizes ( n = 10 and 50 ) and seven population recombination rates (  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 ) were explored .", "we used a continuous - time approximation where the times of the events are exponentially distributed .", "for every combination of parameters , we simulated 1,000 replicates , producing and evaluating thus a total of 14,000 genealogies .", "we varied the recombination rate that much because the recombination events actually determine the different network classes produced ( regular , tree sibling , tree child , galled trees , or binary trees ) .", "the number of raw recombination events r(n ) in the network for a sample of n genes has the expectationand thus , 0 , 2.72 , 5.44 , 10.87 , 21.74 , 43.49 , and 86.97 recombination events are expected for n = 10 , and 0 , 4.46 , 8.92 , 17.83 , 35.67 , 71.34 , and 142.68 for n = 50 , for the recombination rates enumerated above , respectively .", "the simulated genealogies were classified into five different network classes : regular , tree sibling , tree child , galled trees , and binary trees .", "first , hybrid and tree nodes in the graphs were identified as the recombinant and coalescent nodes in the genealogies , respectively , while superfluous nodes were eliminated ( fig .", "superfluous nodes have just one parent and one child and result from concatenated recombination and coalescent events .", "although they are real nodes ( and therefore count for r(n ) ) , they can not be estimated from real data and , therefore , were removed from the networks before their classification . the corresponding graph for the arg shown in figure 1 .", "the standard coalescent describes the possible histories of a sample of genes back in time to their mrca ( kingman 1982 ) . in the absence of evolutionary forces like selection , migration , or recombination ,", "the only types of events that can occur are coalescent events , in which two lineages ( branches ) fuse into one .", "therefore , for a sample of n genes , there will be n  1 coalescent events until the mrca is reached .", "if recombination is included , the recombination events will result actually in the opposite pattern going backward in time , as in this case one lineage ( the recombinant ) separates into two ( the parents of the recombination event ) .", "therefore , the genealogies produced by the coalescent with recombination ( also known as arg , for  ancestral recombination graph  ) will be explicit reticulate networks as internal nodes actually represent evolutionary events . in figure 1", ", we show a typical genealogy for a sample of 10 genes . a single realization of the coalescent with recombination .", "the genealogy goes from the present ( bottom ) to the past ( up ) .", "coalescent and recombinant nodes are represented in white and gray , respectively . in our simulations , the genealogies were simulated with the program recodon ( arenas and posada 2007 ) .", "two sample sizes ( n = 10 and 50 ) and seven population recombination rates (  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 ) were explored .", "we used a continuous - time approximation where the times of the events are exponentially distributed .", "for every combination of parameters , we simulated 1,000 replicates , producing and evaluating thus a total of 14,000 genealogies .", "we varied the recombination rate that much because the recombination events actually determine the different network classes produced ( regular , tree sibling , tree child , galled trees , or binary trees ) .", "the number of raw recombination events r(n ) in the network for a sample of n genes has the expectationand thus , 0 , 2.72 , 5.44 , 10.87 , 21.74 , 43.49 , and 86.97 recombination events are expected for n = 10 , and 0 , 4.46 , 8.92 , 17.83 , 35.67 , 71.34 , and 142.68 for n = 50 , for the recombination rates enumerated above , respectively .", "the simulated genealogies were classified into five different network classes : regular , tree sibling , tree child , galled trees , and binary trees .", "first , hybrid and tree nodes in the graphs were identified as the recombinant and coalescent nodes in the genealogies , respectively , while superfluous nodes were eliminated ( fig .", "superfluous nodes have just one parent and one child and result from concatenated recombination and coalescent events .", "although they are real nodes ( and therefore count for r(n ) ) , they can not be estimated from real data and , therefore , were removed from the networks before their classification . the corresponding graph for the arg shown in figure 1 .", "in the absence of recombination , all the simulated genealogies were binary trees , as expected .", "with recombination , different classes of reticulate networks were produced whose complexity was a function of the population recombination rate ( tables 1 and 2 ) . for n = 10 , at small recombination rates (  = 1 ) many networks were tree sibling , but half were already non - tree child . with twice as much recombination ,", "half the networks were more complex than tree sibling networks , and only a few were tree child networks or galled trees . with moderate recombination rates (  = 4 ) , only 15% of the networks were tree sibling , whereas with larger recombination rates .", "almost no network , or even none at all , could be classified into any of the standard classes .", "increasing the sample size to n = 50 just increased a little more the complexity of the networks but preserved the same trend regarding the effect of recombination . on the other hand , even at small recombination rates", "(  = 1 ) rather few networks are regular because hybrid nodes in the genealogies simulated by the coalescent with recombination have two parents and a single child , and a network containing a hybrid node with a single child is not regular unless the child is also a hybrid node .", "number of simulated networks falling in each class as a function of the recombination rate  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 for sample size n = 10 number of simulated networks falling in each class as a function of the recombination rate  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 for sample size n = 50 we will argue that the network topologies  we do not consider branch lengths  produced by the coalescent with recombination will be indistinguishable from those produced by other reticulating processes like gene conversion , hybridization , and lateral gene transfer .", "note that the coalescent with recombination results , going backward in time , in nonreciprocal exchanges of genetic material as only one of the two recombinants inherits ancestral material and will be therefore represented in the network .", "conveniently , these types of events are also typical of gene conversion , hybridization , and lateral gene transfer .", "in addition , the nodes in the arg produced by the coalescent do not have to belong to the same species ; they just represent gene copies .", "therefore , we believe that these results are likely to be very similar for models of lateral gene transfer and/or hybridization  the assumptions underlying the standard classes of networks should eventually fail given sufficient reticulation .", "we can conclude that the reticulate networks produced by the evolutionary process , at least as modeled by the coalescent with recombination , are much more convoluted than regular , tree sibling , tree child , or galled tree networks .", "these network classes  the only ones for which perfect metrics exist  are clearly insufficient to describe reticulating evolutionary processes . indeed , new network metrics need to be developed if we really want to compare reticulate phylogenetic networks estimated from real data ."], "abstract_text": ["<S> phylogenetic networks aim to represent the evolutionary history of taxa . within these , </S>", "<S> reticulate networks are explicitly able to accommodate evolutionary events like recombination , hybridization , or lateral gene transfer . </S>", "<S> although several metrics exist to compare phylogenetic networks , they make several assumptions regarding the nature of the networks that are not likely to be fulfilled by the evolutionary process . in order to characterize the potential disagreement between the algorithms and the biology , we have used the coalescent with recombination to build the type of networks produced by reticulate evolution and classified them as regular , tree sibling , tree child , or galled trees . we show that , as expected , the complexity of these reticulate networks is a function of the population recombination rate . at small recombination rates , </S>", "<S> most of the networks produced are already more complex than regular or tree sibling networks , whereas with moderate and large recombination rates , no network fit into any of the standard classes . </S>", "<S> we conclude that new metrics still need to be devised in order to properly compare two phylogenetic networks that have arisen from reticulating evolutionary process . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Simulation of Coalescent Networks", "Assignment to Network Classes", "Results"], "sections": [["phylogenetic networks represent the evolutionary relationships of taxa , including sequences , genes , chromosomes , genomes , or species .", "there are different types of phylogenetic networks , but here , we are interested in reticulate networks , which provide an explicit representation of evolutionary history , meaning that internal  nodes  represent ancestral species , and nodes with more than two parents correspond to reticulate events such as recombination , hybridization or lateral gene transfer ( huson and bryant 2006 ) .", "reticulate networks have been extensively used in evolutionary studies , especially at the population level , where reticulate events are , in general , quite common ( posada and crandall 2001 ) .", "very few studies have tried to assess the performance of the algorithms used to reconstruct phylogenetic networks ( cassens et al .", "2007 ) , and only recently , a comprehensive computer simulation study has been completed ( woolley et al .", "one of the problems that arose during these studies was the comparison of reticulate networks .", "although several comparison metrics have been already introduced in the literature ( baroni et al .", "2008a , 2008b ; cardona , rossell , and valiente 2008b , 2008c ; cardona g , llabrs m , rossell f , valiente g , metrics for phylogenetic networks ii : nodal and triplets metrics , unpublished data ; cardona g , llabrs m , rossell f , valiente g , recent advances in metrics for phylogenetic networks , unpublished date ) , all of them have specific requirements regarding the nature of the networks to be compared in order to be ", "perfect ,  that is , to have the metric properties of nonnegativity , separation , symmetry , and triangle inequality . before continuing", "a network contains nodes ( vertices ) and  branches  ( edges ) that connect them .", "in general , we will refer to  rooted networks ,  with a direction from the past to the present ( i.e. , directed graphs ) that allows for the identification of  parent  and  child ", "the  root node  is the oldest node and has no parents .  tree nodes", "when two nodes share the same parent they are  siblings .  depending on the relationships among the nodes that occur in a network , these can be classified as  tree sibling  ( cardona , llabrs , et al .", "2008a ; cardona , rossell , and valiente 2008a ) , where every hybrid node has at least one sibling that is a tree node ;  tree child ", "2008b ; cardona , rossell , and valiente 2008a , 2008b , 2008c ; cardona g , llabrs m , rossell f , valiente g , unpublished data ) , in which every internal node has at least one child that is a tree node ;  regular  ( baroni et al .", "2004 ) , where the set of descendant leaves ( clusters ) of the nodes are all distinct ;  galled trees  ( gusfield et al .", "2004a , 2004b ) , where the paths from the most recent common ancestor ( mrca ) of the parents of a hybrid node down to the hybrid node form disjoint cycles ; and ( binary )  trees ,  which only contain tree nodes .", "these network classes are nested in this order : tree sibling  tree child  galled trees  trees , meaning a tree is also a galled tree , a tree child network , and a tree sibling network , and so on .", "regular networks , however , are not related to tree sibling , tree child , or galled tree networks ( cardona g , llabrs m , rossell f , valiente g , recent advances in metrics for phylogenetic networks , unpublished date ) . the point we want to make is that the networks resulting from reticulating evolutionary processes do not necessarily correspond with any of the idealized classes of networks described above and for which perfect metrics exist . were this true , it would imply that many phylogenetic networks could not be properly compared with the existing metrics .", "obviously , it is very important to characterize the size of this perceived gap between the algorithms and the biology , and this is precisely our goal . in order to provide a formal statistical description of this disagreement ,", "independent of particular organisms or genomic regions , we will use coalescent theory ( kingman 1982 ) to generate the type of reticulate networks that result from the evolutionary process .", "the coalescent describes the probabilities of the different genealogies for a sample of genes generally but not necessarily from the same population , and it was further extended by hudson ( 1983 ) to include recombination events .", "specifically , we will quantify the different classes of networks ( regular , tree sibling , tree child , and galled trees ) produced by the coalescent as a function of the population recombination rate ."], ["the standard coalescent describes the possible histories of a sample of genes back in time to their mrca ( kingman 1982 ) . in the absence of evolutionary forces like selection , migration , or recombination ,", "the only types of events that can occur are coalescent events , in which two lineages ( branches ) fuse into one .", "therefore , for a sample of n genes , there will be n  1 coalescent events until the mrca is reached .", "if recombination is included , the recombination events will result actually in the opposite pattern going backward in time , as in this case one lineage ( the recombinant ) separates into two ( the parents of the recombination event ) .", "therefore , the genealogies produced by the coalescent with recombination ( also known as arg , for  ancestral recombination graph  ) will be explicit reticulate networks as internal nodes actually represent evolutionary events . in figure 1", ", we show a typical genealogy for a sample of 10 genes . a single realization of the coalescent with recombination .", "the genealogy goes from the present ( bottom ) to the past ( up ) .", "coalescent and recombinant nodes are represented in white and gray , respectively . in our simulations , the genealogies were simulated with the program recodon ( arenas and posada 2007 ) .", "two sample sizes ( n = 10 and 50 ) and seven population recombination rates (  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 ) were explored .", "we used a continuous - time approximation where the times of the events are exponentially distributed .", "for every combination of parameters , we simulated 1,000 replicates , producing and evaluating thus a total of 14,000 genealogies .", "we varied the recombination rate that much because the recombination events actually determine the different network classes produced ( regular , tree sibling , tree child , galled trees , or binary trees ) .", "the number of raw recombination events r(n ) in the network for a sample of n genes has the expectationand thus , 0 , 2.72 , 5.44 , 10.87 , 21.74 , 43.49 , and 86.97 recombination events are expected for n = 10 , and 0 , 4.46 , 8.92 , 17.83 , 35.67 , 71.34 , and 142.68 for n = 50 , for the recombination rates enumerated above , respectively .", "the simulated genealogies were classified into five different network classes : regular , tree sibling , tree child , galled trees , and binary trees .", "first , hybrid and tree nodes in the graphs were identified as the recombinant and coalescent nodes in the genealogies , respectively , while superfluous nodes were eliminated ( fig .", "superfluous nodes have just one parent and one child and result from concatenated recombination and coalescent events .", "although they are real nodes ( and therefore count for r(n ) ) , they can not be estimated from real data and , therefore , were removed from the networks before their classification . the corresponding graph for the arg shown in figure 1 ."], ["the standard coalescent describes the possible histories of a sample of genes back in time to their mrca ( kingman 1982 ) . in the absence of evolutionary forces like selection , migration , or recombination ,", "the only types of events that can occur are coalescent events , in which two lineages ( branches ) fuse into one .", "therefore , for a sample of n genes , there will be n  1 coalescent events until the mrca is reached .", "if recombination is included , the recombination events will result actually in the opposite pattern going backward in time , as in this case one lineage ( the recombinant ) separates into two ( the parents of the recombination event ) .", "therefore , the genealogies produced by the coalescent with recombination ( also known as arg , for  ancestral recombination graph  ) will be explicit reticulate networks as internal nodes actually represent evolutionary events . in figure 1", ", we show a typical genealogy for a sample of 10 genes . a single realization of the coalescent with recombination .", "the genealogy goes from the present ( bottom ) to the past ( up ) .", "coalescent and recombinant nodes are represented in white and gray , respectively . in our simulations , the genealogies were simulated with the program recodon ( arenas and posada 2007 ) .", "two sample sizes ( n = 10 and 50 ) and seven population recombination rates (  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 ) were explored .", "we used a continuous - time approximation where the times of the events are exponentially distributed .", "for every combination of parameters , we simulated 1,000 replicates , producing and evaluating thus a total of 14,000 genealogies .", "we varied the recombination rate that much because the recombination events actually determine the different network classes produced ( regular , tree sibling , tree child , galled trees , or binary trees ) .", "the number of raw recombination events r(n ) in the network for a sample of n genes has the expectationand thus , 0 , 2.72 , 5.44 , 10.87 , 21.74 , 43.49 , and 86.97 recombination events are expected for n = 10 , and 0 , 4.46 , 8.92 , 17.83 , 35.67 , 71.34 , and 142.68 for n = 50 , for the recombination rates enumerated above , respectively ."], ["the simulated genealogies were classified into five different network classes : regular , tree sibling , tree child , galled trees , and binary trees .", "first , hybrid and tree nodes in the graphs were identified as the recombinant and coalescent nodes in the genealogies , respectively , while superfluous nodes were eliminated ( fig .", "superfluous nodes have just one parent and one child and result from concatenated recombination and coalescent events .", "although they are real nodes ( and therefore count for r(n ) ) , they can not be estimated from real data and , therefore , were removed from the networks before their classification . the corresponding graph for the arg shown in figure 1 ."], ["in the absence of recombination , all the simulated genealogies were binary trees , as expected .", "with recombination , different classes of reticulate networks were produced whose complexity was a function of the population recombination rate ( tables 1 and 2 ) . for n = 10 , at small recombination rates (  = 1 ) many networks were tree sibling , but half were already non - tree child . with twice as much recombination ,", "half the networks were more complex than tree sibling networks , and only a few were tree child networks or galled trees . with moderate recombination rates (  = 4 ) , only 15% of the networks were tree sibling , whereas with larger recombination rates .", "almost no network , or even none at all , could be classified into any of the standard classes .", "increasing the sample size to n = 50 just increased a little more the complexity of the networks but preserved the same trend regarding the effect of recombination . on the other hand , even at small recombination rates", "(  = 1 ) rather few networks are regular because hybrid nodes in the genealogies simulated by the coalescent with recombination have two parents and a single child , and a network containing a hybrid node with a single child is not regular unless the child is also a hybrid node .", "number of simulated networks falling in each class as a function of the recombination rate  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 for sample size n = 10 number of simulated networks falling in each class as a function of the recombination rate  = 0 , 1 , 2 , 4 , 8 , 16 , and 32 for sample size n = 50 we will argue that the network topologies  we do not consider branch lengths  produced by the coalescent with recombination will be indistinguishable from those produced by other reticulating processes like gene conversion , hybridization , and lateral gene transfer .", "note that the coalescent with recombination results , going backward in time , in nonreciprocal exchanges of genetic material as only one of the two recombinants inherits ancestral material and will be therefore represented in the network .", "conveniently , these types of events are also typical of gene conversion , hybridization , and lateral gene transfer .", "in addition , the nodes in the arg produced by the coalescent do not have to belong to the same species ; they just represent gene copies .", "therefore , we believe that these results are likely to be very similar for models of lateral gene transfer and/or hybridization  the assumptions underlying the standard classes of networks should eventually fail given sufficient reticulation .", "we can conclude that the reticulate networks produced by the evolutionary process , at least as modeled by the coalescent with recombination , are much more convoluted than regular , tree sibling , tree child , or galled tree networks .", "these network classes  the only ones for which perfect metrics exist  are clearly insufficient to describe reticulating evolutionary processes . indeed , new network metrics need to be developed if we really want to compare reticulate phylogenetic networks estimated from real data ."]]}
{"article_id": "PMC3674722", "article_text": ["we recently demonstrated that diabetes directly induced dental caries in rats and mice , although a direct association between the 2 was less evident and conflicting in humans [ 14 ] .", "we demonstrated that dental caries were produced using noncariogenic diets with a low concentration of sugar in diabetic rodent models [ 57 ] , although many studies usually add large amounts of sugar to the diet to induce dental caries [ 8 , 9 ] .", "in addition , early studies on the production of experimental dental caries have reported the effects of physical properties of cariogenic diets with high concentrations of sugar on dental caries .", "these studies mainly used finely ground cariogenic diet [ 1013 ] because it is more cariogenic compared to a coarsely ground diet in rodents .", "moreover , hard and coarse foods have the ability to prevent caries [ 1417 ] .", "thus , modifying the dietary formulation may possibly enhance or reduce caries development in diabetic animals .", "however , no reports have investigated the effects of the physical properties of non - cariogenic standard diets on dental caries in diabetic animals .  ", "currently , 2 types of non - cariogenic standard diet formulations ( i.e. , powdered and pelletized diets ) are widely used in experimental rodents . in the present study ,", "we compared the effects of these diets on the development of dental caries in a diabetic rodent model focusing on the difference in hardness between the powdered and pelletized diets .", "six - week - old female f344 rats were supplied by japan slc , inc .", "the animals were housed in stainless - steel cages at a temperature of 2026c and a relative humidity of 4070% under a 12/12  h light / dark cycle , that were ventilated with filtered fresh air . to prevent infection ,", "the animals were allowed free access to tap water and fed with a widely used standard pelletized or powdered diet ( charles river formula 1 ( crf-1 ) ; oriental yeast co. , ltd . ,", "the animals were handled according to the principles for all experimental procedures outlined in the guide for the care and use of laboratory animals prepared by our institution ( setsunan university ) and the japanese association for laboratory animal science .", "the glucose levels in the fresh urine were measured semiquantitatively using urine test paper ( wako pure chemical industries , osaka , japan ) daily from day 1 to day 3 after dosing , once every week for 1 month after the first week , and once every month thereafter .", "the blood glucose levels in the tail vein samples were also measured semi - quantitatively using the glucose oxidase method ( glutest e ; sanwakagaku , aichi , japan ) once every month from the fourth week after alloxan injection .", "blood samples obtained from the tail vein and fresh urine were collected from 1:00 to 4:00  p.m. the severity of hyperglycemia was defined as follows : normal , < 200  mg / dl ; mild , > 200  mg / dl ; moderate , > 300  mg / dl ; or severe , > 400  mg / dl . the severity of glycosuria was defined as follows : normal , < 100  mg / dl ; mild , > 100  mg / dl ; moderate , > 250  mg / dl ; or severe , > 500  mg / dl . the experimental design is shown in figure 1 .", "ten rats , aged 7 weeks , were given a single dose ( 35  mg / kg body weight ) of alloxan ( sigma - aldrich japan , tokyo , japan ) via tail vein injection .", "the dose of alloxan was determined as the given dose at which a rat survives for a long period after the onset of diabetic symptoms . after the confirmation of hyperglycemia and glucosuria following the dosing of alloxan ,", "five rats were given a pelletized crf-1 diet ( diabetes - pelletized diet group ) and the remaining 5 rats were given a powdered crf-1 diet ( diabetes - powdered diet group ) .", "as a control , each of the 5 intact rats was fed a pelletized or powdered crf-1 diet ( control - pelletized diet group and control - powdered diet group ) .", "the animals were euthanized by exsanguination under deep anesthesia at the end of the observation period .", "subsequently , the mandible was removed and fixed in 10% neutral - buffered formalin ( ph 7.4 ) .", "after a 24  h fixation , the occlusal , buccolingual , and proximal surfaces of all of the molar teeth were intensively observed under a binocular stereoscope .", "soft x - ray images of the mesiodistal plane were taken under conditions of 35  kv and 2  ma for 4  min .", "the molar teeth were classified into 5 groups according to caries characteristics by observing and analyzing the radiographs : no radiolucent change ( grade 0 ) , radiolucent area only on the occlusal surface of the crown ( grade 1 ) , radiolucent areas on occlusal surface and either of the mesiodistal surfaces of the crown ( grade 2 ) , radiolucent areas over the entire surface of the crown ( grade 3 ) , and radiolucent areas over most of the surface of the dental root ( grade 4 ) .", "the mean score of the caries was used as indicator for comparing the severity of the carious lesions between the groups .", "after soft x - ray examination , a histopathological examination was performed on the mandible in all of the rats .", "after fixation with 10% neutral - buffered formalin , the sample was decalcified in a 5% solution of ethylenediaminetetraacetic acid 4 na ( edta 4 na ) for 2 weeks at 4c .", "after decalcification , the specimens were trimmed , dehydrated in a sequential ethanol series using an automated processor , and embedded in paraffin wax .", "serial 7  m thick sections on the mesiodistal plane were made through the centers of all of the molars and then stained with hematoxylin and eosin for examination using light microscopy .", "the severity of the caries lesion was graded as follows : slight , dentin caries localized in occlusal surface of dentin ; mild , dentin caries extended into the dental pulp with pulpitis and/or pulp necrosis ; moderate , dental crowns were partially decayed ; and severe , dental crowns were completely decayed ( only the molar roots remained ) .", "the wilcoxon rank - sum test was employed to compare the differences in the mean scores of the caries lesions using soft x - ray examination between the groups .", "the chi - square test was used to determine the caries incidence using soft x - ray examination and the incidence of histopathological lesions in each group of rats .", "severe hyperglycemia ( > 400  mg / dl ) and glucosuria ( > 500  mg / dl ) continued from the day after alloxan injection to the last monitoring day in all of the rats in the alloxan - treated groups .", "in addition , the blood glucose levels ranged from 78 to 120  mg / dl ( normoglycemia ) , and the urine glucose levels were less than 100  mg / dl in the control groups .  ", "typical macroscopic appearances of carious molars in diabetic rats are shown in figures 3(a ) , 3(b ) , and 3(c ) .", "macroscopically , the dental caries developed mainly in occlusal fissures and were identified as partial coronal defects of the molars in alloxan - treated diabetic rats ( figure 3(b ) ) .", "the carious lesions expanded horizontally , until the crown of the carious molar was completely invisible ( figures 3(b ) and 3(c ) ) . in contrast , control nondiabetic rats showed no changes in any of the molars ( figure 3(a ) ) .  ", "soft x - ray images of the carious lesion in diabetic rats are shown in figures 3(d ) , 3(e ) , and 3(f ) . in all of the alloxan - treated diabetic rats ,", "radiographically , the dental caries progressed both horizontally and vertically . in diabetic rats fed with a pelletized diet ,", "the carious lesion was mainly characterized as grade 2 ( figure 3(e ) ) or 4 type ( table 1 ) .", "in addition , the diabetic rats fed a powdered diet demonstrated carious lesions that were nearly of grade 4 type ( figure 3(f ) ) . in the control", "non - diabetic rats , there was no change in the molar teeth ( table 1 , figure 3(d ) ) .  ", "the histopathological characteristics of the carious lesion in diabetic rats are shown in figures 3(g ) , 3(h ) , and 3(i ) .", "histopathological carious lesions were detected in the crown as eroded dentin with bacterial colonization in alloxan - treated diabetic rats . in diabetic rats fed with the pelletized diet , many of the molars were moderately affected and the dentin caries spread over a wide area of the dental crown ( figure 3(h ) ) . in the diabetic rats fed with the powdered diet , the dental caries were markedly worsened and the crowns were nearly completely decayed ( figure 3(i ) ) .  ", "the incidence of caries teeth on the basis of each scoring in the soft x - ray examination is shown in table 1 .", "the incidence of dental caries in the diabetic rats fed on both the pelletized and powdered diets was apparently higher ( p < 0.01 ) compared to the non - diabetic control rats ( table 1 ) . in the diabetic rats fed with the pelletized diet , 96.7% of their molar teeth were affected with dental caries , and the mean caries score was 2.7 .", "in addition , in the diabetic rats fed with the powdered diet , all of their molars ( 100% ) were affected with caries , and the mean caries score was 3.7 , which was significantly higher ( p < 0.01 ) than the diabetic rats fed with the pelletized diet ( table 1 , figure 4 ) .", "no radiolucent lesions were observed in any of the molars in control non - diabetic rats .", "the incidence of histopathological carious lesions on the basis of each grade is summarized in table 2 .", "histopathologically , the severity of the carious lesions in the diabetic rats fed with a powdered diet was significantly enhanced compared to the diabetic rats fed with the pelletized diet ( table 2 ) . in control non - diabetic rats , slight carious lesions , which were macroscopically and radiographically normal ,", "were detected in a few animals ; however , there were no differences observed between the non - diabetic rats on the pelletized and powdered diets ( table 2 ) .", "the physical properties of foods such as hardness , adhesiveness , and cohesiveness are closely related with the caries - producing potential [ 19 , 20 ] . moreover ,", "the development of caries is known to be profoundly affected by food hardness , and hard and coarse foods can exert a detergent activity during mastication , which is effective in the prevention of caries [ 17 , 19 ] . furthermore , a lower degree of caries was reported in animals fed with the hardest food diet . in the present study , the severity of caries in diabetic rats was enhanced in the powdered diet group compared to the pelletized diet group .", "it was clear that food hardness affected the development of dental caries in diabetic rats fed on a non - cariogenic diet .", "hard foods are known to help flush away or neutralize undesirable material within the dental plaque and exert a cleaning effect on smooth surfaces by direct mechanical friction .", "therefore , cariogenicity will be higher in diabetic rats fed with a powdered diet than in those fed with a pelletized diet . in this study , a high incidence and severity of dental caries was confirmed in the alloxan - treated diabetic rats fed with 2 types of standard diets with a low concentration of sugar .", "furthermore , the mean caries score of the diabetic rats fed on the pelletized diet at 52 weeks after alloxan dosing in this study was almost 2-fold compared to animals at 26 weeks after alloxan dosing described in a previous report , strongly suggesting that the duration of hyperglycemia may affect caries development in diabetic animals . in this study ,", "dental caries developed and extended to all of the molars in diabetic rats fed on both the pelletized and powdered diets .", "thus , the effect of food hardness on the different diets in caries development in diabetic rats may become clearer when the dental caries mildly develop during the more early stages . in conclusion", ", food hardness could have an effect on the development of dental caries in diabetic rats , and appropriate consideration of the food formulation should be made in experimental caries studies using diabetic rodent models ."], "abstract_text": ["<S> we have previously shown that dental caries may be produced in diabetic rodent models fed with noncariogenic standard diets ; however , many studies usually add large amounts of sugar to the diet to induce dental caries . </S>", "<S> moreover , the physical properties of cariogenic diets have been reported as an important factor in the formation of caries . </S>", "<S> the aim of this study was to clarify the effect of the hardness of non - cariogenic diets on the development of dental caries in diabetic rodents . </S>", "<S> seven - week - old female f344 rats were divided into 4 groups : intact rats fed with a standard pelletized or powdered diet and alloxan - induced diabetic rats fed with a standard pelletized or powdered diet . </S>", "<S> all of the rats were sacrificed at 52 weeks of age for morphological examinations on their dental tissue . </S>", "<S> dental caries had developed and extended to all the molars in the diabetic rats that were fed with both the pelletized and powdered diets . </S>", "<S> moreover , the lesion was significantly enhanced in the powdered diet group compared to that in the pelletized diet group . in conclusion , food hardness is an important factor influencing the development of dental caries in diabetic rats . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["we recently demonstrated that diabetes directly induced dental caries in rats and mice , although a direct association between the 2 was less evident and conflicting in humans [ 14 ] .", "we demonstrated that dental caries were produced using noncariogenic diets with a low concentration of sugar in diabetic rodent models [ 57 ] , although many studies usually add large amounts of sugar to the diet to induce dental caries [ 8 , 9 ] .", "in addition , early studies on the production of experimental dental caries have reported the effects of physical properties of cariogenic diets with high concentrations of sugar on dental caries .", "these studies mainly used finely ground cariogenic diet [ 1013 ] because it is more cariogenic compared to a coarsely ground diet in rodents .", "moreover , hard and coarse foods have the ability to prevent caries [ 1417 ] .", "thus , modifying the dietary formulation may possibly enhance or reduce caries development in diabetic animals .", "however , no reports have investigated the effects of the physical properties of non - cariogenic standard diets on dental caries in diabetic animals .  ", "currently , 2 types of non - cariogenic standard diet formulations ( i.e. , powdered and pelletized diets ) are widely used in experimental rodents . in the present study ,", "we compared the effects of these diets on the development of dental caries in a diabetic rodent model focusing on the difference in hardness between the powdered and pelletized diets ."], ["six - week - old female f344 rats were supplied by japan slc , inc .", "the animals were housed in stainless - steel cages at a temperature of 2026c and a relative humidity of 4070% under a 12/12  h light / dark cycle , that were ventilated with filtered fresh air . to prevent infection ,", "the animals were allowed free access to tap water and fed with a widely used standard pelletized or powdered diet ( charles river formula 1 ( crf-1 ) ; oriental yeast co. , ltd . ,", "the animals were handled according to the principles for all experimental procedures outlined in the guide for the care and use of laboratory animals prepared by our institution ( setsunan university ) and the japanese association for laboratory animal science .", "the glucose levels in the fresh urine were measured semiquantitatively using urine test paper ( wako pure chemical industries , osaka , japan ) daily from day 1 to day 3 after dosing , once every week for 1 month after the first week , and once every month thereafter .", "the blood glucose levels in the tail vein samples were also measured semi - quantitatively using the glucose oxidase method ( glutest e ; sanwakagaku , aichi , japan ) once every month from the fourth week after alloxan injection .", "blood samples obtained from the tail vein and fresh urine were collected from 1:00 to 4:00  p.m. the severity of hyperglycemia was defined as follows : normal , < 200  mg / dl ; mild , > 200  mg / dl ; moderate , > 300  mg / dl ; or severe , > 400  mg / dl . the severity of glycosuria was defined as follows : normal , < 100  mg / dl ; mild , > 100  mg / dl ; moderate , > 250  mg / dl ; or severe , > 500  mg / dl . the experimental design is shown in figure 1 .", "ten rats , aged 7 weeks , were given a single dose ( 35  mg / kg body weight ) of alloxan ( sigma - aldrich japan , tokyo , japan ) via tail vein injection .", "the dose of alloxan was determined as the given dose at which a rat survives for a long period after the onset of diabetic symptoms . after the confirmation of hyperglycemia and glucosuria following the dosing of alloxan ,", "five rats were given a pelletized crf-1 diet ( diabetes - pelletized diet group ) and the remaining 5 rats were given a powdered crf-1 diet ( diabetes - powdered diet group ) .", "as a control , each of the 5 intact rats was fed a pelletized or powdered crf-1 diet ( control - pelletized diet group and control - powdered diet group ) .", "the animals were euthanized by exsanguination under deep anesthesia at the end of the observation period .", "subsequently , the mandible was removed and fixed in 10% neutral - buffered formalin ( ph 7.4 ) .", "after a 24  h fixation , the occlusal , buccolingual , and proximal surfaces of all of the molar teeth were intensively observed under a binocular stereoscope .", "soft x - ray images of the mesiodistal plane were taken under conditions of 35  kv and 2  ma for 4  min .", "the molar teeth were classified into 5 groups according to caries characteristics by observing and analyzing the radiographs : no radiolucent change ( grade 0 ) , radiolucent area only on the occlusal surface of the crown ( grade 1 ) , radiolucent areas on occlusal surface and either of the mesiodistal surfaces of the crown ( grade 2 ) , radiolucent areas over the entire surface of the crown ( grade 3 ) , and radiolucent areas over most of the surface of the dental root ( grade 4 ) .", "the mean score of the caries was used as indicator for comparing the severity of the carious lesions between the groups .", "after soft x - ray examination , a histopathological examination was performed on the mandible in all of the rats .", "after fixation with 10% neutral - buffered formalin , the sample was decalcified in a 5% solution of ethylenediaminetetraacetic acid 4 na ( edta 4 na ) for 2 weeks at 4c .", "after decalcification , the specimens were trimmed , dehydrated in a sequential ethanol series using an automated processor , and embedded in paraffin wax .", "serial 7  m thick sections on the mesiodistal plane were made through the centers of all of the molars and then stained with hematoxylin and eosin for examination using light microscopy .", "the severity of the caries lesion was graded as follows : slight , dentin caries localized in occlusal surface of dentin ; mild , dentin caries extended into the dental pulp with pulpitis and/or pulp necrosis ; moderate , dental crowns were partially decayed ; and severe , dental crowns were completely decayed ( only the molar roots remained ) .", "the wilcoxon rank - sum test was employed to compare the differences in the mean scores of the caries lesions using soft x - ray examination between the groups .", "the chi - square test was used to determine the caries incidence using soft x - ray examination and the incidence of histopathological lesions in each group of rats ."], ["severe hyperglycemia ( > 400  mg / dl ) and glucosuria ( > 500  mg / dl ) continued from the day after alloxan injection to the last monitoring day in all of the rats in the alloxan - treated groups .", "in addition , the blood glucose levels ranged from 78 to 120  mg / dl ( normoglycemia ) , and the urine glucose levels were less than 100  mg / dl in the control groups .  ", "typical macroscopic appearances of carious molars in diabetic rats are shown in figures 3(a ) , 3(b ) , and 3(c ) .", "macroscopically , the dental caries developed mainly in occlusal fissures and were identified as partial coronal defects of the molars in alloxan - treated diabetic rats ( figure 3(b ) ) .", "the carious lesions expanded horizontally , until the crown of the carious molar was completely invisible ( figures 3(b ) and 3(c ) ) . in contrast , control nondiabetic rats showed no changes in any of the molars ( figure 3(a ) ) .  ", "soft x - ray images of the carious lesion in diabetic rats are shown in figures 3(d ) , 3(e ) , and 3(f ) . in all of the alloxan - treated diabetic rats ,", "radiographically , the dental caries progressed both horizontally and vertically . in diabetic rats fed with a pelletized diet ,", "the carious lesion was mainly characterized as grade 2 ( figure 3(e ) ) or 4 type ( table 1 ) .", "in addition , the diabetic rats fed a powdered diet demonstrated carious lesions that were nearly of grade 4 type ( figure 3(f ) ) . in the control", "non - diabetic rats , there was no change in the molar teeth ( table 1 , figure 3(d ) ) .  ", "the histopathological characteristics of the carious lesion in diabetic rats are shown in figures 3(g ) , 3(h ) , and 3(i ) .", "histopathological carious lesions were detected in the crown as eroded dentin with bacterial colonization in alloxan - treated diabetic rats . in diabetic rats fed with the pelletized diet , many of the molars were moderately affected and the dentin caries spread over a wide area of the dental crown ( figure 3(h ) ) . in the diabetic rats fed with the powdered diet , the dental caries were markedly worsened and the crowns were nearly completely decayed ( figure 3(i ) ) .  ", "the incidence of caries teeth on the basis of each scoring in the soft x - ray examination is shown in table 1 .", "the incidence of dental caries in the diabetic rats fed on both the pelletized and powdered diets was apparently higher ( p < 0.01 ) compared to the non - diabetic control rats ( table 1 ) . in the diabetic rats fed with the pelletized diet , 96.7% of their molar teeth were affected with dental caries , and the mean caries score was 2.7 .", "in addition , in the diabetic rats fed with the powdered diet , all of their molars ( 100% ) were affected with caries , and the mean caries score was 3.7 , which was significantly higher ( p < 0.01 ) than the diabetic rats fed with the pelletized diet ( table 1 , figure 4 ) .", "no radiolucent lesions were observed in any of the molars in control non - diabetic rats .", "the incidence of histopathological carious lesions on the basis of each grade is summarized in table 2 .", "histopathologically , the severity of the carious lesions in the diabetic rats fed with a powdered diet was significantly enhanced compared to the diabetic rats fed with the pelletized diet ( table 2 ) . in control non - diabetic rats , slight carious lesions , which were macroscopically and radiographically normal ,", "were detected in a few animals ; however , there were no differences observed between the non - diabetic rats on the pelletized and powdered diets ( table 2 ) ."], ["the physical properties of foods such as hardness , adhesiveness , and cohesiveness are closely related with the caries - producing potential [ 19 , 20 ] . moreover ,", "the development of caries is known to be profoundly affected by food hardness , and hard and coarse foods can exert a detergent activity during mastication , which is effective in the prevention of caries [ 17 , 19 ] . furthermore , a lower degree of caries was reported in animals fed with the hardest food diet . in the present study , the severity of caries in diabetic rats was enhanced in the powdered diet group compared to the pelletized diet group .", "it was clear that food hardness affected the development of dental caries in diabetic rats fed on a non - cariogenic diet .", "hard foods are known to help flush away or neutralize undesirable material within the dental plaque and exert a cleaning effect on smooth surfaces by direct mechanical friction .", "therefore , cariogenicity will be higher in diabetic rats fed with a powdered diet than in those fed with a pelletized diet . in this study , a high incidence and severity of dental caries was confirmed in the alloxan - treated diabetic rats fed with 2 types of standard diets with a low concentration of sugar .", "furthermore , the mean caries score of the diabetic rats fed on the pelletized diet at 52 weeks after alloxan dosing in this study was almost 2-fold compared to animals at 26 weeks after alloxan dosing described in a previous report , strongly suggesting that the duration of hyperglycemia may affect caries development in diabetic animals . in this study ,", "dental caries developed and extended to all of the molars in diabetic rats fed on both the pelletized and powdered diets .", "thus , the effect of food hardness on the different diets in caries development in diabetic rats may become clearer when the dental caries mildly develop during the more early stages . in conclusion", ", food hardness could have an effect on the development of dental caries in diabetic rats , and appropriate consideration of the food formulation should be made in experimental caries studies using diabetic rodent models ."]]}
{"article_id": "PMC2919175", "article_text": ["encapsulation of biological macromolecules within lipid vesicles is important for biophysical research and for a variety of applications such as pharmaceuticals , foods , and cosmetics .", "although attractive interactions between solutes and the lipids used to encapsulate them can lead to high encapsulation efficiencies , such methods are only effective for certain solute / lipid combinations .", "in contrast , passive encapsulation ( i.e. , without attractive interactions ) is more general but leads to internalization of solutes at or below their concentration in the vesicle formation solution .", "solute size is a key determinant in the efficiency of passive encapsulation , with larger solutes excluded from the vesicle interior such that the internal solute concentration is lower than the external concentration during vesicle formation .", "knowledge of solute concentrations in the vesicle interior is important for understanding processes as varied as enzymatic turnover , polymerization , crystallization , or phase separation occurring in this volume .", "it is also of interest for applications in which vesicles serve as carriers for solute delivery , such as in food formulations or therapeutics .", "quantification of encapsulated solute is accomplished by different methods depending on the size of the vesicles .", "encapsulation in large unilamellar vesicles ( luvs ) and other submicrometer vesicles ( svs , which include both luvs and small unilamellar vesicles , suvs ) is quantified in terms of the bulk encapsulation efficiency ( bee ) .", "bee is the percentage of solute trapped within a batch of submicrometer vesicles as compared to the total amount of solute added .", "it is typically determined by removing all of the unencapsulated solute ( e.g. , via centrifugation or dialysis ) and then lysing the vesicles to quantify the remaining ( encapsulated ) solute .", "giant vesicles ( gvs , 1100 m),(12 ) on the other hand , are not generally studied via bulk methods due to their limited stability , but are large enough to be analyzed individually .", "for example , the concentration of a fluorescently tagged solute encapsulated within an individual giant vesicle can be quantified by comparing the internal solute fluorescence to a calibration curve.(13 ) the ability to determine encapsulation efficiency for individual gvs ( eeind ) also makes it possible to compare encapsulation between vesicles within a batch , which can vary widely .", "recently , approaches to determination of eeind for submicrometer vesicles have been reported ; these powerful new tools have not yet become routine .", "the most common gv formation techniques , electroformation(18 ) and gentle hydration,(19 ) passively encapsulate solutes , such that the expected concentration inside ( cin ) is equal to the external concentration during vesicle formation ( cout ) . for solutes with rh < 10 nm , mean cin does tend to be equal to cout ; however , we and others have measured considerable variability in cin for individual gvs in the same batch .", "for example , mean cin was found to be much less than cout for dextran polymers with hydrodynamic radius , rh > 10 nm.(14 ) solute size is also an important determinant of entrapment in submicrometer vesicles .", "for example , adrian and huang compared the encapsulation of proteins of increasing molecular weight to that of a small marker protein ( lysozyme , 14.1 kda ) in ca .", "the 97 kda -amylase was encapsulated only 50% as well as the lysozyme.(7 ) the same authors reported that proteins up to 120 kda could be encapsulated in ca .", "200 nm diameter vesicles with the same trapping efficiency as sucrose.(7 ) bee is often quite low for passive uptake because it is defined in terms of the total encapsulated volume ( capture volume , cv ) , which can be < 1% of the total volume , depending on the amount of lipid present and the morphology of the vesicles . although cv can be increased up to ca .", "50% by using more concentrated lipid,(20 ) this is not always possible for a given system .", "indeed , most papers report bees much less than 50% . to increase bee beyond what is possible by passive encapsulation ,", "for example , cationic lipids are routinely used to package nucleic acid drugs , despite concerns about their toxicity .", "solute encapsulation based on electrostatic attractions can be essentially complete , with nearly 100% of all solute molecules added to the solution being associated with liposomes . despite excellent loading , attractive interactions with encapsulating lipids can lead to more complex liposome morphologies , prevent activity of the encapsulated solute until released , and inhibit effective release of solutes upon vesicle rupture .", "while these factors are often considered acceptable in drug delivery applications , it is preferable to have free encapsulated solute of known concentration for biophysical studies , e.g. , to evaluate the effects of confinement.(30 ) encapsulation of rh > 10 nm fluorescently labeled dextran polymers in gv has been reported to improve markedly in the presence of several weight percent additional polymeric solutes.(15 ) the additional polymers acted as volume excluders , resulting in condensation of the labeled solutes and allowing them to be more easily encapsulated during vesicle formation.(15 ) macromolecular crowding is an attractive means for improving macromolecule encapsulation because of its generality.(31 ) this approach for improving encapsulation has not previously been tested for biomacromolecules such as proteins , which in many cases already exist in globular conformations and hence may not undergo as large a rh change in response to volume excluders , nor has it been tested for encapsulation in submicrometer vesicles . herein , we report the use of peg as a macromolecular crowding agent to increase the encapsulation efficiency of fluorescently labeled biomacromolecules within gvs and 200 nm diameter luvs .", "we find in general that the addition of peg increases the encapsulation efficiency of high molecular weight ( mw ) biomolecules compared to those encapsulated in di water alone .", "we conclude that volume exclusion in the presence of a crowding agent such as peg or dextran can be used as a general route to increase encapsulation efficiency of biomacromolecules in both giant and submicrometer lipid vesicles , up to levels expected for passive encapsulation of lower mw solutes ( i.e. , cin = cout ) .", "volume exclusion induced condensation is expected to have the greatest impact on large , extended molecular conformations . therefore , the macromolecules used in this work were chosen to give a range of molecular sizes and shapes .", "we first describe the effect of polymeric volume excluders on encapsulation in gvs formed by gentle hydration and then discuss encapsulation in luvs prepared by a freezethaw / extrusion protocol .", "we began by encapsulating fibrinogen , a 340 kda protein with a hydrodynamic radius ( rh ) of 11 nm , which is important in coagulation and thrombosis and is often administered to patients who have suffered severe blood loss or have lost the ability to clot blood on their own .", "fibrinogen has been reported to condense to approximately one - half of its original length during polymerization .", "fibrinogen labeled with alexa fluor 488 ( af488 ) was added during gv formation at a concentration of 33 nm , either in di water or in a 3 wt % peg 8 kda solution .", "gvs were imaged without removing them from the hydration solution , such that the external fibrinogen concentration remained 33 nm .", "figure 1 shows representative confocal fluorescence images and histograms of encapsulation efficiency for individual gvs formed in the presence of fibrinogen alone and coencapsulated with 3 wt % peg . here", ", encapsulation efficiency for the individual vesicles ( eeind ) is defined as the concentration ratio between the vesicle interior ( ci ) and the external solution ( co ) used during vesicle formation . the concentration ratio ( ci / co ) for each vesicle", "a vertical line is included at ci / co = 1 to facilitate comparison to the external fibrinogen - af488 concentration . in the absence of added peg ,", "encapsulation efficiency is quite poor ; the gv interiors appear dark , and fibrinogen - af488 concentration varied considerably from vesicle to vesicle , ranging from 1 to 54 nm ( 0.041.64  co ) with a mean of 21 nm ( 0.64  co ) .", "in contrast , vesicles formed in the presence of 3 wt % peg contained fibrinogen - af488 concentrations similar to that of the external solution , with 65% of the gvs encapsulating internal concentrations between 28 and 38 nm and a mean of 34 nm .", "( left ) confocal fluorescence images of vesicles prepared in the presence of af488-labeled fibrinogen without ( top ) and with ( bottom ) 3 wt % peg 8 kda .", "membrane composition was 3:7 egg pc : dopg with 0.05 mol % dope - rhodamine .", "( right ) histograms of the concentration ratio ( cin / cout ) for encapsulation of labeled fibrinogen in individual gvs without ( top ) and with ( bottom ) 3 wt % peg 8 kda ( co = 33 nm ) .", "we repeated this experiment with two other proteins having relatively large rh to test the generality of the method of increasing eeind via macromolecular crowding .", "thyroglobulin is a 660 kda protein with a pi of 4.6 that is important in the synthesis , storage , and secretion of thyroid hormones .", "the native structure has two slightly elongated subunits , with an rh of 10 nm.(39 ) in vivo , thyroglobulin structure is known to condense in order to increase the storage capacity of the thyroid.(40 ) encapsulation of thyroglobulin - af488 ( 18 nm ) in gvs in the absence and presence of 3 wt % peg 8 kda produced results similar to those for fibrinogen - af488 ( supporting information figure 1 ) , which had similar rh and ability to compact .", "we then repeated the experiment with catalase - af488 , a 250 kda enzyme with a pi of 5.4 and an rh of 10.4 nm .", "this protein catalyzes the decomposition of hydrogen peroxide to water and oxygen,(43 ) and has been encapsulated in submicrometer liposomes along with superoxide dismutase and injected to protect against oxygen toxicity in rats.(44 ) images and a histogram for cin are shown in supporting information figure 2 .", "eeind for was significantly increased vs control by addition of peg 8 kda during encapsulation of all three of these relatively large proteins ( student s t test , p < 0.001 ) .", "these results are consistent with the role of peg 8 kda as a volume excluder , driving the condensation of macromolecular structures . proteins , nucleic acids , and enzymes are all known to adopt condensed structures in macromolecularly crowded solutions .", "thus , we interpret the improved encapsulation of these high molecular weight proteins in 3 wt % peg 8 kda as the result of their condensation to give reduced hydrodynamic radii , which in turn caused them to be encapsulated as effectively as lower mw biomolecules .", "we next evaluated the effect of peg 8 kda on encapsulation of two smaller proteins and a dna oligomer , all of which had hydrodynamic radii less than 10 nm .", "-1-antitrypsin ( aat ) is a 52 kda protein with an rh of 3.8 nm and a pi between 4.9 and 5.1 .", "it is responsible for inhibiting elastase in vivo , which destroys elastin on connective tissues.(57 ) aat deficiency often leads to lung diseases , and clinical trials have been reported where patients are given aat infusions , which showed beneficial effects.(58 ) it has also been delivered to mice via encapsulation in submicrometer liposomes.(59 ) we began by encapsulating 1.9 m aat - af488 in gvs formed in with the presence and absence of 3 wt % peg 8 kda .", "aat - af488 was encapsulated at essentially cin = cout with or without 3% peg ( figure 2 ) .", "( left ) confocal fluorescence images of vesicles prepared in the presence of 1.9 m labeled -1-antitrypsin without ( top ) and with ( bottom ) 3 wt % peg 8 kda .", "membrane composition was 3:7 egg pc : dopg with 0.05 mol % dope - rhodamine .", "( right ) histograms of the concentration ratio ( cin / cout ) for encapsulation of labeled -1-antitrypsin in individual gvs without ( top ) and with ( bottom ) 3 wt % peg 8 kda ( co = 1.9 m ) .", "similar results were observed for encapsulation of labeled human serum albumin ( hsa ) in peg solution as compared to di water ( supporting information figure 3 ) .", "hsa has similar characteristics as aat ( i.e. , hsa has a mw of 67 kda , pi of 4.95.1 , and rh of 3.51 ) .", "we also studied the encapsulation of a single - stranded 173 base pair oligomer ( 53 kda ) labeled with fluorescein ( fam ) .", "nucleic acids are often encapsulated in submicrometer vesicles for gene delivery and in gvs to study biochemical reactions .", "dna is known to undergo condensation in the presence of divalent cations , polycations , or even neutral polymers such as peg .", "this oligonucleotide was sufficiently small to be effectively encapsulated in the gv even without addition of a crowding agent ( supporting information figure 4 ) .", "table 1 compares encapsulation for each of the biomacromolecular solutes with and without added peg . in addition to the increase in cin for the larger proteins when encapsulated in the presence of 3 wt % peg , decreased heterogeneity in cin across a population of vesicles in a batch is suggested by the standard deviations in cin ( table 1 ) and the histograms for individual solutes ( figures 1 and 2 and supporting information figures 14 ) .", "an f - test(61 ) was performed to evaluate whether differences in the variance between the control and + peg populations were statistically significant .", "we found statistically significant changes in variance for the fibrinogen ( p < 0.001 ) , catalase ( p < 0.001 ) , and aat ( p < 0.05 ) , but not for the other solutes .", "the increase in encapsulation homogeneity when fibrinogen and catalase were coencapsulated with a crowding agent arises largely from the shift to higher encapsulated concentrations .", "decreased variance for aat may also be due in part to condensation , which has been reported for aat and other small polymers , but is more likely due to peg increasing the viscosity and osmotic pressure of the solutions , as has been discussed previously .", "for example , increased viscosity could improve encapsulation heterogeneity by facilitating pore formation in the membrane , allowing more time for solutes to equilibrate between internal and external solutions.(65 ) we note that the proteins were not found to be associated with the lipid membrane of the gvs , but rather to be uniformly dispersed in the exterior and/or interior of the vesicles ( see , for example , figures 1 and 2 , left panels ) . polymerization or aggregation on microscopically visible length scales", "preparation of the vesicles in 3% peg did not inhibit their formation or change their size or morphology in any obvious way as compared to preparation in peg - free solution , consistent with our previous findings of decreased average vesicle diameter at 10 and 20 wt % peg but no noticeable difference between 0 and 3 wt % peg in terms of gv formation , size , or morphology.(15 ) the improvements in eeind observed in the presence of peg should be applicable to other crowding agents.(15 ) we therefore coencapsulated fluorescently labeled fibrinogen and 1-antitrypsin with 3 wt % dextran 500 kda for comparison .", "the 3 wt % dextran 500 kda had the same effect on encapsulation as peg 8 kda : the higher mw protein , fibrinogen , showed an increase in eeind magnitude and homogeneity with the introduction of either peg or dextran ; and the lower mw protein , aat , showed essentially no change in mean eeind , but a decreased variance ( f - test , p < 0.05 ) when dextran was added ( table 1 ) .", "theoretical eeind defined as the solute concentration in the external solution during vesicle preparation , which is given in parentheses for each solute in the first column .", "fluorescein  labeled ; hydrodynamic radius of 7.5 nm for 280 nt ssdna was reported in ref ( 66 ) under conditions where excluded volume effects can be neglected ; extrapolation of length vs rh data in ref ( 66 ) suggests 5 nm hydrodynamic radius for our 173 nt sequence .", "internal solute concentrations in sv can not be determined via simple confocal imaging as for the gv described above .", "therefore , we estimated cin and the effectiveness of solute encapsulation by comparing the percentage of total solute that has been encapsulated within a batch of vesicles ( bulk encapsulation efficiency , bee ) to the capture volume .", "capture volume was estimated based on the moles of lipid present and the size of the vesicles , assuming unilamellarity .", "bee was determined by removing the unencapsulated solute from a batch of vesicles , then lysing the vesicles and quantifying the remaining ( encapsulated ) solute . for effective passive encapsulation", ", one can expect to achieve the same concentration inside the vesicles as is present outside ( cin = cout ) .", "however , for high mw solutes , bee is often less than the capture volume ( i.e. , interior concentrations < exterior concentrations ) .", "table 2 shows the average bee for three encapsulated solutes ( carboxyfluorescein ( cf ) , 500 kda dextran - fitc , and thyroglobulin - af488 ) with and without added peg in 200 nm diameter vesicles .", "the theoretical bee for passive encapsulation in this system is 0.38% , calculated by estimating the number of vesicles formed based on the amount of lipid added to the system ( 500 m ) and then calculating the volume encapsulated by those vesicles as compared to the initial volume .", "we estimated that there were 3.4  10 lipid molecules per 200 nm diameter vesicle , for 4.5  10 vesicles that collectively encapsulated 19 l , or 0.38% of the total solution volume ( 5 ml ) , based on calculations for similar systems .", "dynamic light scattering verified that vesicle diameter remained constant whether in dilute or crowded solution , and the addition of a fluorescent lipid in the membrane verified that a negligible amount of lipid was lost to the filter during extrusion .", "errors represent the standard deviation of bee measurements taken for three separate batches of vesicles on three different days .", "encapsulation of the small fluorescent molecule , carboxyfluorescein ( cf ) , was performed as a control .", "it was expected that the effect of the coencapsulation of peg would be negligible , since the addition of peg did not significantly impact the encapsulation efficiency of cf in gvs.(15 ) the change in experimental cf bee observed between the solutions with and without peg was not statistically significant based on a student s t test ( p = 0.63 ) ( table 2 ) .", "encapsulation of fitc - dextran 500 kda and thyroglobulin - af488 did show improvement for luvs prepared in solutions containing peg 8 kda as compared to solutions without peg ( student s t test , p < 0.05 ) .", "experimentally determined bee for fitc - dextran 500 kda and thyroglobulin - af488 in water were 31% and 50% of the theoretically predicted value , respectively  these low bees are most likely due to the large size of dextran 500 kda ( rh 15 nm ) and thyroglobulin ( rh 10 nm ) .", "addition of 3 wt % peg 8 kda significantly increased the bee of fitc - dextran and thyroglobulin - af488 in these vesicles , consistent with condensation of the dextran and thyroglobulin structures via macromolecular crowding.(15 ) although the error is large , it appears that with peg 3% , the luvs are essentially encapsulating even these very large solutes at cin = cout .", "bee was increased without chemically modifying the vesicle or solute to utilize specific attractive interactions .", "in contrast to the gv encapsulation data , improvements in standard deviation were not observed in the luv experiments .", "this underscores the difference in the two measurements : the error in gv cin measurements indicates vesicle - to - vesicle variability within a batch , while variability in luv bee experiments reports on differences between entire batches prepared separately and as such may reflect variability in capture volume .", "we interpret this as the result of some cf loss from the vesicle interior during centrifugation and/or other handling , which is commonly reported in submicrometer vesicles.(69 ) these data are exciting , because while the majority of reports published on bee utilize specific interactions between liposome / solute , this method is quite general and should apply to a variety of solutes that can be condensed under conditions of macromolecular crowding . although the absolute bee in these experiments was low , we have demonstrated that addition of 3 wt % peg provides a means of improving encapsulation for large solutes such that cin = cout for luvs prepared by freezethaw / extrusion . increasing", "the amount of lipid used in a batch has been shown by others to increase cv from < 1% to as high as 50% ; which would lead to an increase in bee , as previously reported.(20 )", "our results indicate that the addition of the polymeric coencapsulants peg 8 kda and dextran 500 kda as macromolecular crowding agents during passive encapsulation of biomacromolecular solutes can substantially increase mean encapsulated concentration and in some cases homogeneity for otherwise poorly encapsulated high mw proteins in giant vesicles .", "the addition of a crowding agent also reduced vesicle - to - vesicle encapsulation variability for the lower mw aat , which was already encapsulated efficiently ( i.e. , with cin = cout ) in dilute solution .", "the mechanism for increased encapsulation is general , such that crowding agents need not be varied with the identity of the solute .", "this work is important to those using gvs as bioreactors , where knowledge of encapsulated concentration is critical to quantifying the progression of products formed during a reaction .", "measurements of bulk encapsulation efficiency ( bee ) in luvs in the presence of peg suggest that this approach works similarly in these smaller vesicles as for gvs , bringing the internal solute concentration closer to the external concentration such that the bee is determined by capture volume .", "the approach used here should be effective for a wide range of membrane / macromolecular solute combinations .", "l--phosphatidylcholine ( egg pc ) , 1,2-dioleoyl - sn - glycero-3-[phospho - rac-(1-glycerol)](sodium salt ) ( dopg ) , 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( dppc ) , cholesterol , and 1,2-dioleolyl - sn - glycero-3(phosphoethanolamine - n - lissamine rhodamine b sulfonyl ) ( ammonium salt ) ( rhodamine - dope ) were purchased from avanti polar lipids , inc .", "carboxyfluorescein , fluorescein isothiocyanate ( fitc)-labeled and unlabeled dextrans and poly(ethylene glycol ) ( peg ) were obtained from sigma .", "alexa fluor 488 and fitc labeling kits along with fibrinogen - alexa fluor 488 were purchased from molecular probes , inc .", "a fluorescein nucleic acid labeling kit was purchased from mirus bio ( madison , wi ) .", "a 173 nucleotide single - stranded oligomer with the sequence for influenza b was purchased from integrated dna technologies , inc .", "water used in these experiments was deionized to a resistance of 18 m with a nanopure diamond water system from barnstead int .", "silanized borosilicate glass culture tubes , 12  75 mm , were used to prevent biomolecules from sticking to the glass surface during formation ( kimble chase , vineland , nj ) .", "we first prepared a chloroform solution of lipids containing a 3:7 molar ratio of egg pc : dopg at a concentration of 0.26 mg / ml with 0.05 mol % rhodamine - dope .", "lipid solutions were dried under ar ( g ) to form a thin , uniform lipid film , and then , the vials were vacuum - desiccated for 2 h to remove any residual organic solvent .", "then , an aqueous solution containing a fluorescent biomolecule with or without 3 wt % unlabeled peg or dextran heated to 37 c was added along the wall of the tube , and the lipids were hydrated 48 h at this temperature .", "we chose 3 wt % peg 8 kda based on prior work with dextran 500 kda condensation , in which we observed increased encapsulation efficiency in gvs up to 10 wt % peg , with the bulk of the increase achieved by 3% and decreased average gv diameter for 10 wt % peg.(15 ) following incubation , the solutions were allowed to cool to room temperature .", "the contents of the vials , which remained undiluted , were removed with a micropipet and transferred to a microscope slide silanized with n-(triethoxysilylpropyl)-o - polyethylene oxide urethane ( gelest , inc . , morrisville , pa ) for analysis .", "coverslips were also coated with n-(triethoxysilylpropyl)-o - polyethylene oxide urethane to prevent the adhesion of fluorescently labeled biomolecules to the glass surface .", "the concentrations of crowding agents are referred to in wt % , since this is the common notation used for crowding agents in the macromolecular crowding literature ; the 3% peg 8 kda used here corresponds to 3.75 mm .", "concentrations for fluorescently labeled solutes , which are considerably lower than for the crowding agents , are given as molarities .", "a fluorescence line scan was taken across each vesicle to determine the fluorescence intensity of the alexa fluor 488 , fitc , or fluorescein inside the vesicle ( ii ) and in the undiluted bulk solution ( io , outside the vesicle ) , as reported previously .", "the concentration of biomolecule inside ( ci ) and outside ( co ) the gv was determined directly from the intensities ( ii , io ) by a calibration curve of the labeled biomolecule free in solution . from the calibration curves", ", it was determined that the bulk concentration ( co ) was equal to the initial concentration used to hydrate the vesicles .", "imaging was performed using an lsm-5 pascal laser scanning confocal microscope from carl zeiss , inc .", "( oberkochen , germany ) with a plan - apochromat 63 oil immersion objective ( 1.4 na ) , and pascal software as previously reported .", "only vesicles that were greater than 3 m in diameter and appeared to be uni- or oligolamellar were analyzed .", "dppc / cholesterol vesicles ( 80:20 mol ratio ) were prepared using a modified technique provided by the distributor to yield large unilamellar vesicles by extrusion ( luvet ) as previously reported .", "briefly , dppc and cholesterol were combined with chloroform in a round - bottom flask and evaporated for 4 h. following this , 5 ml of the solute to be encapsulated was added in either water or 3 wt % peg 8 kda to yield a final lipid concentration of 0.34 mg / ml .", "the lipid mixture was allowed to incubate in this solution at room temperature for 30 min .", "then , the flask was subjected to five freeze / thaw cycles to promote entrapment of water - soluble compounds into the vesicles.(71 ) the mixture was then extruded through a 0.2 m polycarbonate membrane for a total of 20 passes ( mini extruder kit , avanti polar lipids , inc . ,", "excess solute was removed from the vesicle suspension by washing with a 10  000 mw dialysis cassette in di water for 48 h ( slide - a - lyzer , thermo scientific , rockford , il ) and then centrifuging at 16  100  g , removing the supernatant , and replacing it with a solution free of fluorescent molecules .", "vesicle diameter was confirmed using dynamic light scattering ( zetasizer nano s , malvern instruments , worcestershire , u.k . ) .", "after centrifugation , the vesicles were lysed in 0.5% triton - x 100 , and the encapsulated concentration was determined via fluorescence measurements on a jobin yvon horiba fl3 - 21 fluorimeter ."], "abstract_text": ["<S> large solutes such as high molecular weight proteins can be difficult to encapsulate in lipid vesicles . </S>", "<S> passive trapping of these macromolecular solutes during vesicle formation typically results in concentrations inside the vesicles that are much lower than in the external solution . here , we investigated the effect of macromolecular crowding on passive encapsulation of biological macromolecules with molecular weights ranging from 52 kda to 660 kda within both individual giant lipid vesicles ( gvs , > 3 m diameter ) and populations of 200 nm diameter large unilamellar vesicles ( luvs ) . </S>", "<S> fluorescently labeled biomacromolecules were encapsulated during vesicle formation in the presence or absence of three weight percent poly(ethylene glycol ) ( peg ; 8 kda ) or dextran 500 kda , which served as crowding agents . </S>", "<S> encapsulation efficiency of the labeled biomolecules was higher for the lower molecular weight solutes , with internal concentrations essentially equal to external concentrations for labeled biomacromolecules with hydrodynamic radii ( rh ) less than 10 nm . </S>", "<S> in contrast , internal concentrations were reduced markedly for larger solutes with rh  10 nm . </S>", "<S> addition of peg or dextran during vesicle formation improved encapsulation of these larger proteins up to the same levels as observed for the smaller proteins , such that internal and external concentrations were equal . </S>", "<S> this observation is consistent with peg and dextran acting as volume excluders , reducing the hydrodynamic radius of the biomacromolecules and increasing their encapsulation . </S>", "<S> this work demonstrates a simple and general route to improved encapsulation of otherwise poorly encapsulated macromolecular solutes in both gv and luvs up to their concentration in the solution present during vesicle formation . </S>"], "labels": null, "section_names": ["Introduction", "Results and Discussion", "Conclusions", "Materials and Methods"], "sections": [["encapsulation of biological macromolecules within lipid vesicles is important for biophysical research and for a variety of applications such as pharmaceuticals , foods , and cosmetics .", "although attractive interactions between solutes and the lipids used to encapsulate them can lead to high encapsulation efficiencies , such methods are only effective for certain solute / lipid combinations .", "in contrast , passive encapsulation ( i.e. , without attractive interactions ) is more general but leads to internalization of solutes at or below their concentration in the vesicle formation solution .", "solute size is a key determinant in the efficiency of passive encapsulation , with larger solutes excluded from the vesicle interior such that the internal solute concentration is lower than the external concentration during vesicle formation .", "knowledge of solute concentrations in the vesicle interior is important for understanding processes as varied as enzymatic turnover , polymerization , crystallization , or phase separation occurring in this volume .", "it is also of interest for applications in which vesicles serve as carriers for solute delivery , such as in food formulations or therapeutics .", "quantification of encapsulated solute is accomplished by different methods depending on the size of the vesicles .", "encapsulation in large unilamellar vesicles ( luvs ) and other submicrometer vesicles ( svs , which include both luvs and small unilamellar vesicles , suvs ) is quantified in terms of the bulk encapsulation efficiency ( bee ) .", "bee is the percentage of solute trapped within a batch of submicrometer vesicles as compared to the total amount of solute added .", "it is typically determined by removing all of the unencapsulated solute ( e.g. , via centrifugation or dialysis ) and then lysing the vesicles to quantify the remaining ( encapsulated ) solute .", "giant vesicles ( gvs , 1100 m),(12 ) on the other hand , are not generally studied via bulk methods due to their limited stability , but are large enough to be analyzed individually .", "for example , the concentration of a fluorescently tagged solute encapsulated within an individual giant vesicle can be quantified by comparing the internal solute fluorescence to a calibration curve.(13 ) the ability to determine encapsulation efficiency for individual gvs ( eeind ) also makes it possible to compare encapsulation between vesicles within a batch , which can vary widely .", "recently , approaches to determination of eeind for submicrometer vesicles have been reported ; these powerful new tools have not yet become routine .", "the most common gv formation techniques , electroformation(18 ) and gentle hydration,(19 ) passively encapsulate solutes , such that the expected concentration inside ( cin ) is equal to the external concentration during vesicle formation ( cout ) . for solutes with rh < 10 nm , mean cin does tend to be equal to cout ; however , we and others have measured considerable variability in cin for individual gvs in the same batch .", "for example , mean cin was found to be much less than cout for dextran polymers with hydrodynamic radius , rh > 10 nm.(14 ) solute size is also an important determinant of entrapment in submicrometer vesicles .", "for example , adrian and huang compared the encapsulation of proteins of increasing molecular weight to that of a small marker protein ( lysozyme , 14.1 kda ) in ca .", "the 97 kda -amylase was encapsulated only 50% as well as the lysozyme.(7 ) the same authors reported that proteins up to 120 kda could be encapsulated in ca .", "200 nm diameter vesicles with the same trapping efficiency as sucrose.(7 ) bee is often quite low for passive uptake because it is defined in terms of the total encapsulated volume ( capture volume , cv ) , which can be < 1% of the total volume , depending on the amount of lipid present and the morphology of the vesicles . although cv can be increased up to ca .", "50% by using more concentrated lipid,(20 ) this is not always possible for a given system .", "indeed , most papers report bees much less than 50% . to increase bee beyond what is possible by passive encapsulation ,", "for example , cationic lipids are routinely used to package nucleic acid drugs , despite concerns about their toxicity .", "solute encapsulation based on electrostatic attractions can be essentially complete , with nearly 100% of all solute molecules added to the solution being associated with liposomes . despite excellent loading , attractive interactions with encapsulating lipids can lead to more complex liposome morphologies , prevent activity of the encapsulated solute until released , and inhibit effective release of solutes upon vesicle rupture .", "while these factors are often considered acceptable in drug delivery applications , it is preferable to have free encapsulated solute of known concentration for biophysical studies , e.g. , to evaluate the effects of confinement.(30 ) encapsulation of rh > 10 nm fluorescently labeled dextran polymers in gv has been reported to improve markedly in the presence of several weight percent additional polymeric solutes.(15 ) the additional polymers acted as volume excluders , resulting in condensation of the labeled solutes and allowing them to be more easily encapsulated during vesicle formation.(15 ) macromolecular crowding is an attractive means for improving macromolecule encapsulation because of its generality.(31 ) this approach for improving encapsulation has not previously been tested for biomacromolecules such as proteins , which in many cases already exist in globular conformations and hence may not undergo as large a rh change in response to volume excluders , nor has it been tested for encapsulation in submicrometer vesicles . herein , we report the use of peg as a macromolecular crowding agent to increase the encapsulation efficiency of fluorescently labeled biomacromolecules within gvs and 200 nm diameter luvs .", "we find in general that the addition of peg increases the encapsulation efficiency of high molecular weight ( mw ) biomolecules compared to those encapsulated in di water alone .", "we conclude that volume exclusion in the presence of a crowding agent such as peg or dextran can be used as a general route to increase encapsulation efficiency of biomacromolecules in both giant and submicrometer lipid vesicles , up to levels expected for passive encapsulation of lower mw solutes ( i.e. , cin = cout ) ."], ["volume exclusion induced condensation is expected to have the greatest impact on large , extended molecular conformations . therefore , the macromolecules used in this work were chosen to give a range of molecular sizes and shapes .", "we first describe the effect of polymeric volume excluders on encapsulation in gvs formed by gentle hydration and then discuss encapsulation in luvs prepared by a freezethaw / extrusion protocol .", "we began by encapsulating fibrinogen , a 340 kda protein with a hydrodynamic radius ( rh ) of 11 nm , which is important in coagulation and thrombosis and is often administered to patients who have suffered severe blood loss or have lost the ability to clot blood on their own .", "fibrinogen has been reported to condense to approximately one - half of its original length during polymerization .", "fibrinogen labeled with alexa fluor 488 ( af488 ) was added during gv formation at a concentration of 33 nm , either in di water or in a 3 wt % peg 8 kda solution .", "gvs were imaged without removing them from the hydration solution , such that the external fibrinogen concentration remained 33 nm .", "figure 1 shows representative confocal fluorescence images and histograms of encapsulation efficiency for individual gvs formed in the presence of fibrinogen alone and coencapsulated with 3 wt % peg . here", ", encapsulation efficiency for the individual vesicles ( eeind ) is defined as the concentration ratio between the vesicle interior ( ci ) and the external solution ( co ) used during vesicle formation . the concentration ratio ( ci / co ) for each vesicle", "a vertical line is included at ci / co = 1 to facilitate comparison to the external fibrinogen - af488 concentration . in the absence of added peg ,", "encapsulation efficiency is quite poor ; the gv interiors appear dark , and fibrinogen - af488 concentration varied considerably from vesicle to vesicle , ranging from 1 to 54 nm ( 0.041.64  co ) with a mean of 21 nm ( 0.64  co ) .", "in contrast , vesicles formed in the presence of 3 wt % peg contained fibrinogen - af488 concentrations similar to that of the external solution , with 65% of the gvs encapsulating internal concentrations between 28 and 38 nm and a mean of 34 nm .", "( left ) confocal fluorescence images of vesicles prepared in the presence of af488-labeled fibrinogen without ( top ) and with ( bottom ) 3 wt % peg 8 kda .", "membrane composition was 3:7 egg pc : dopg with 0.05 mol % dope - rhodamine .", "( right ) histograms of the concentration ratio ( cin / cout ) for encapsulation of labeled fibrinogen in individual gvs without ( top ) and with ( bottom ) 3 wt % peg 8 kda ( co = 33 nm ) .", "we repeated this experiment with two other proteins having relatively large rh to test the generality of the method of increasing eeind via macromolecular crowding .", "thyroglobulin is a 660 kda protein with a pi of 4.6 that is important in the synthesis , storage , and secretion of thyroid hormones .", "the native structure has two slightly elongated subunits , with an rh of 10 nm.(39 ) in vivo , thyroglobulin structure is known to condense in order to increase the storage capacity of the thyroid.(40 ) encapsulation of thyroglobulin - af488 ( 18 nm ) in gvs in the absence and presence of 3 wt % peg 8 kda produced results similar to those for fibrinogen - af488 ( supporting information figure 1 ) , which had similar rh and ability to compact .", "we then repeated the experiment with catalase - af488 , a 250 kda enzyme with a pi of 5.4 and an rh of 10.4 nm .", "this protein catalyzes the decomposition of hydrogen peroxide to water and oxygen,(43 ) and has been encapsulated in submicrometer liposomes along with superoxide dismutase and injected to protect against oxygen toxicity in rats.(44 ) images and a histogram for cin are shown in supporting information figure 2 .", "eeind for was significantly increased vs control by addition of peg 8 kda during encapsulation of all three of these relatively large proteins ( student s t test , p < 0.001 ) .", "these results are consistent with the role of peg 8 kda as a volume excluder , driving the condensation of macromolecular structures . proteins , nucleic acids , and enzymes are all known to adopt condensed structures in macromolecularly crowded solutions .", "thus , we interpret the improved encapsulation of these high molecular weight proteins in 3 wt % peg 8 kda as the result of their condensation to give reduced hydrodynamic radii , which in turn caused them to be encapsulated as effectively as lower mw biomolecules .", "we next evaluated the effect of peg 8 kda on encapsulation of two smaller proteins and a dna oligomer , all of which had hydrodynamic radii less than 10 nm .", "-1-antitrypsin ( aat ) is a 52 kda protein with an rh of 3.8 nm and a pi between 4.9 and 5.1 .", "it is responsible for inhibiting elastase in vivo , which destroys elastin on connective tissues.(57 ) aat deficiency often leads to lung diseases , and clinical trials have been reported where patients are given aat infusions , which showed beneficial effects.(58 ) it has also been delivered to mice via encapsulation in submicrometer liposomes.(59 ) we began by encapsulating 1.9 m aat - af488 in gvs formed in with the presence and absence of 3 wt % peg 8 kda .", "aat - af488 was encapsulated at essentially cin = cout with or without 3% peg ( figure 2 ) .", "( left ) confocal fluorescence images of vesicles prepared in the presence of 1.9 m labeled -1-antitrypsin without ( top ) and with ( bottom ) 3 wt % peg 8 kda .", "membrane composition was 3:7 egg pc : dopg with 0.05 mol % dope - rhodamine .", "( right ) histograms of the concentration ratio ( cin / cout ) for encapsulation of labeled -1-antitrypsin in individual gvs without ( top ) and with ( bottom ) 3 wt % peg 8 kda ( co = 1.9 m ) .", "similar results were observed for encapsulation of labeled human serum albumin ( hsa ) in peg solution as compared to di water ( supporting information figure 3 ) .", "hsa has similar characteristics as aat ( i.e. , hsa has a mw of 67 kda , pi of 4.95.1 , and rh of 3.51 ) .", "we also studied the encapsulation of a single - stranded 173 base pair oligomer ( 53 kda ) labeled with fluorescein ( fam ) .", "nucleic acids are often encapsulated in submicrometer vesicles for gene delivery and in gvs to study biochemical reactions .", "dna is known to undergo condensation in the presence of divalent cations , polycations , or even neutral polymers such as peg .", "this oligonucleotide was sufficiently small to be effectively encapsulated in the gv even without addition of a crowding agent ( supporting information figure 4 ) .", "table 1 compares encapsulation for each of the biomacromolecular solutes with and without added peg . in addition to the increase in cin for the larger proteins when encapsulated in the presence of 3 wt % peg , decreased heterogeneity in cin across a population of vesicles in a batch is suggested by the standard deviations in cin ( table 1 ) and the histograms for individual solutes ( figures 1 and 2 and supporting information figures 14 ) .", "an f - test(61 ) was performed to evaluate whether differences in the variance between the control and + peg populations were statistically significant .", "we found statistically significant changes in variance for the fibrinogen ( p < 0.001 ) , catalase ( p < 0.001 ) , and aat ( p < 0.05 ) , but not for the other solutes .", "the increase in encapsulation homogeneity when fibrinogen and catalase were coencapsulated with a crowding agent arises largely from the shift to higher encapsulated concentrations .", "decreased variance for aat may also be due in part to condensation , which has been reported for aat and other small polymers , but is more likely due to peg increasing the viscosity and osmotic pressure of the solutions , as has been discussed previously .", "for example , increased viscosity could improve encapsulation heterogeneity by facilitating pore formation in the membrane , allowing more time for solutes to equilibrate between internal and external solutions.(65 ) we note that the proteins were not found to be associated with the lipid membrane of the gvs , but rather to be uniformly dispersed in the exterior and/or interior of the vesicles ( see , for example , figures 1 and 2 , left panels ) . polymerization or aggregation on microscopically visible length scales", "preparation of the vesicles in 3% peg did not inhibit their formation or change their size or morphology in any obvious way as compared to preparation in peg - free solution , consistent with our previous findings of decreased average vesicle diameter at 10 and 20 wt % peg but no noticeable difference between 0 and 3 wt % peg in terms of gv formation , size , or morphology.(15 ) the improvements in eeind observed in the presence of peg should be applicable to other crowding agents.(15 ) we therefore coencapsulated fluorescently labeled fibrinogen and 1-antitrypsin with 3 wt % dextran 500 kda for comparison .", "the 3 wt % dextran 500 kda had the same effect on encapsulation as peg 8 kda : the higher mw protein , fibrinogen , showed an increase in eeind magnitude and homogeneity with the introduction of either peg or dextran ; and the lower mw protein , aat , showed essentially no change in mean eeind , but a decreased variance ( f - test , p < 0.05 ) when dextran was added ( table 1 ) .", "theoretical eeind defined as the solute concentration in the external solution during vesicle preparation , which is given in parentheses for each solute in the first column .", "fluorescein  labeled ; hydrodynamic radius of 7.5 nm for 280 nt ssdna was reported in ref ( 66 ) under conditions where excluded volume effects can be neglected ; extrapolation of length vs rh data in ref ( 66 ) suggests 5 nm hydrodynamic radius for our 173 nt sequence .", "internal solute concentrations in sv can not be determined via simple confocal imaging as for the gv described above .", "therefore , we estimated cin and the effectiveness of solute encapsulation by comparing the percentage of total solute that has been encapsulated within a batch of vesicles ( bulk encapsulation efficiency , bee ) to the capture volume .", "capture volume was estimated based on the moles of lipid present and the size of the vesicles , assuming unilamellarity .", "bee was determined by removing the unencapsulated solute from a batch of vesicles , then lysing the vesicles and quantifying the remaining ( encapsulated ) solute . for effective passive encapsulation", ", one can expect to achieve the same concentration inside the vesicles as is present outside ( cin = cout ) .", "however , for high mw solutes , bee is often less than the capture volume ( i.e. , interior concentrations < exterior concentrations ) .", "table 2 shows the average bee for three encapsulated solutes ( carboxyfluorescein ( cf ) , 500 kda dextran - fitc , and thyroglobulin - af488 ) with and without added peg in 200 nm diameter vesicles .", "the theoretical bee for passive encapsulation in this system is 0.38% , calculated by estimating the number of vesicles formed based on the amount of lipid added to the system ( 500 m ) and then calculating the volume encapsulated by those vesicles as compared to the initial volume .", "we estimated that there were 3.4  10 lipid molecules per 200 nm diameter vesicle , for 4.5  10 vesicles that collectively encapsulated 19 l , or 0.38% of the total solution volume ( 5 ml ) , based on calculations for similar systems .", "dynamic light scattering verified that vesicle diameter remained constant whether in dilute or crowded solution , and the addition of a fluorescent lipid in the membrane verified that a negligible amount of lipid was lost to the filter during extrusion .", "errors represent the standard deviation of bee measurements taken for three separate batches of vesicles on three different days .", "encapsulation of the small fluorescent molecule , carboxyfluorescein ( cf ) , was performed as a control .", "it was expected that the effect of the coencapsulation of peg would be negligible , since the addition of peg did not significantly impact the encapsulation efficiency of cf in gvs.(15 ) the change in experimental cf bee observed between the solutions with and without peg was not statistically significant based on a student s t test ( p = 0.63 ) ( table 2 ) .", "encapsulation of fitc - dextran 500 kda and thyroglobulin - af488 did show improvement for luvs prepared in solutions containing peg 8 kda as compared to solutions without peg ( student s t test , p < 0.05 ) .", "experimentally determined bee for fitc - dextran 500 kda and thyroglobulin - af488 in water were 31% and 50% of the theoretically predicted value , respectively  these low bees are most likely due to the large size of dextran 500 kda ( rh 15 nm ) and thyroglobulin ( rh 10 nm ) .", "addition of 3 wt % peg 8 kda significantly increased the bee of fitc - dextran and thyroglobulin - af488 in these vesicles , consistent with condensation of the dextran and thyroglobulin structures via macromolecular crowding.(15 ) although the error is large , it appears that with peg 3% , the luvs are essentially encapsulating even these very large solutes at cin = cout .", "bee was increased without chemically modifying the vesicle or solute to utilize specific attractive interactions .", "in contrast to the gv encapsulation data , improvements in standard deviation were not observed in the luv experiments .", "this underscores the difference in the two measurements : the error in gv cin measurements indicates vesicle - to - vesicle variability within a batch , while variability in luv bee experiments reports on differences between entire batches prepared separately and as such may reflect variability in capture volume .", "we interpret this as the result of some cf loss from the vesicle interior during centrifugation and/or other handling , which is commonly reported in submicrometer vesicles.(69 ) these data are exciting , because while the majority of reports published on bee utilize specific interactions between liposome / solute , this method is quite general and should apply to a variety of solutes that can be condensed under conditions of macromolecular crowding . although the absolute bee in these experiments was low , we have demonstrated that addition of 3 wt % peg provides a means of improving encapsulation for large solutes such that cin = cout for luvs prepared by freezethaw / extrusion . increasing", "the amount of lipid used in a batch has been shown by others to increase cv from < 1% to as high as 50% ; which would lead to an increase in bee , as previously reported.(20 )"], ["our results indicate that the addition of the polymeric coencapsulants peg 8 kda and dextran 500 kda as macromolecular crowding agents during passive encapsulation of biomacromolecular solutes can substantially increase mean encapsulated concentration and in some cases homogeneity for otherwise poorly encapsulated high mw proteins in giant vesicles .", "the addition of a crowding agent also reduced vesicle - to - vesicle encapsulation variability for the lower mw aat , which was already encapsulated efficiently ( i.e. , with cin = cout ) in dilute solution .", "the mechanism for increased encapsulation is general , such that crowding agents need not be varied with the identity of the solute .", "this work is important to those using gvs as bioreactors , where knowledge of encapsulated concentration is critical to quantifying the progression of products formed during a reaction .", "measurements of bulk encapsulation efficiency ( bee ) in luvs in the presence of peg suggest that this approach works similarly in these smaller vesicles as for gvs , bringing the internal solute concentration closer to the external concentration such that the bee is determined by capture volume .", "the approach used here should be effective for a wide range of membrane / macromolecular solute combinations ."], ["l--phosphatidylcholine ( egg pc ) , 1,2-dioleoyl - sn - glycero-3-[phospho - rac-(1-glycerol)](sodium salt ) ( dopg ) , 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( dppc ) , cholesterol , and 1,2-dioleolyl - sn - glycero-3(phosphoethanolamine - n - lissamine rhodamine b sulfonyl ) ( ammonium salt ) ( rhodamine - dope ) were purchased from avanti polar lipids , inc .", "carboxyfluorescein , fluorescein isothiocyanate ( fitc)-labeled and unlabeled dextrans and poly(ethylene glycol ) ( peg ) were obtained from sigma .", "alexa fluor 488 and fitc labeling kits along with fibrinogen - alexa fluor 488 were purchased from molecular probes , inc .", "a fluorescein nucleic acid labeling kit was purchased from mirus bio ( madison , wi ) .", "a 173 nucleotide single - stranded oligomer with the sequence for influenza b was purchased from integrated dna technologies , inc .", "water used in these experiments was deionized to a resistance of 18 m with a nanopure diamond water system from barnstead int .", "silanized borosilicate glass culture tubes , 12  75 mm , were used to prevent biomolecules from sticking to the glass surface during formation ( kimble chase , vineland , nj ) .", "we first prepared a chloroform solution of lipids containing a 3:7 molar ratio of egg pc : dopg at a concentration of 0.26 mg / ml with 0.05 mol % rhodamine - dope .", "lipid solutions were dried under ar ( g ) to form a thin , uniform lipid film , and then , the vials were vacuum - desiccated for 2 h to remove any residual organic solvent .", "then , an aqueous solution containing a fluorescent biomolecule with or without 3 wt % unlabeled peg or dextran heated to 37 c was added along the wall of the tube , and the lipids were hydrated 48 h at this temperature .", "we chose 3 wt % peg 8 kda based on prior work with dextran 500 kda condensation , in which we observed increased encapsulation efficiency in gvs up to 10 wt % peg , with the bulk of the increase achieved by 3% and decreased average gv diameter for 10 wt % peg.(15 ) following incubation , the solutions were allowed to cool to room temperature .", "the contents of the vials , which remained undiluted , were removed with a micropipet and transferred to a microscope slide silanized with n-(triethoxysilylpropyl)-o - polyethylene oxide urethane ( gelest , inc . , morrisville , pa ) for analysis .", "coverslips were also coated with n-(triethoxysilylpropyl)-o - polyethylene oxide urethane to prevent the adhesion of fluorescently labeled biomolecules to the glass surface .", "the concentrations of crowding agents are referred to in wt % , since this is the common notation used for crowding agents in the macromolecular crowding literature ; the 3% peg 8 kda used here corresponds to 3.75 mm .", "concentrations for fluorescently labeled solutes , which are considerably lower than for the crowding agents , are given as molarities .", "a fluorescence line scan was taken across each vesicle to determine the fluorescence intensity of the alexa fluor 488 , fitc , or fluorescein inside the vesicle ( ii ) and in the undiluted bulk solution ( io , outside the vesicle ) , as reported previously .", "the concentration of biomolecule inside ( ci ) and outside ( co ) the gv was determined directly from the intensities ( ii , io ) by a calibration curve of the labeled biomolecule free in solution . from the calibration curves", ", it was determined that the bulk concentration ( co ) was equal to the initial concentration used to hydrate the vesicles .", "imaging was performed using an lsm-5 pascal laser scanning confocal microscope from carl zeiss , inc .", "( oberkochen , germany ) with a plan - apochromat 63 oil immersion objective ( 1.4 na ) , and pascal software as previously reported .", "only vesicles that were greater than 3 m in diameter and appeared to be uni- or oligolamellar were analyzed .", "dppc / cholesterol vesicles ( 80:20 mol ratio ) were prepared using a modified technique provided by the distributor to yield large unilamellar vesicles by extrusion ( luvet ) as previously reported .", "briefly , dppc and cholesterol were combined with chloroform in a round - bottom flask and evaporated for 4 h. following this , 5 ml of the solute to be encapsulated was added in either water or 3 wt % peg 8 kda to yield a final lipid concentration of 0.34 mg / ml .", "the lipid mixture was allowed to incubate in this solution at room temperature for 30 min .", "then , the flask was subjected to five freeze / thaw cycles to promote entrapment of water - soluble compounds into the vesicles.(71 ) the mixture was then extruded through a 0.2 m polycarbonate membrane for a total of 20 passes ( mini extruder kit , avanti polar lipids , inc . ,", "excess solute was removed from the vesicle suspension by washing with a 10  000 mw dialysis cassette in di water for 48 h ( slide - a - lyzer , thermo scientific , rockford , il ) and then centrifuging at 16  100  g , removing the supernatant , and replacing it with a solution free of fluorescent molecules .", "vesicle diameter was confirmed using dynamic light scattering ( zetasizer nano s , malvern instruments , worcestershire , u.k . ) .", "after centrifugation , the vesicles were lysed in 0.5% triton - x 100 , and the encapsulated concentration was determined via fluorescence measurements on a jobin yvon horiba fl3 - 21 fluorimeter ."]]}
{"article_id": "PMC3399407", "article_text": ["we collected all possible literature from the year 1972 ( when kfd was first described ) to the present day conducting a medical search on kikuchi fujimoto disease ( kfd ) in english language .", "we included publications in authorized medical journals including original and review articles / theses , editorials , case reports and brief communications .", "kfd is an extremely uncommon disease but cosmopolitan with higher japanese and asiatic prevalence.1,2 however , sporadic cases have also been reported from europe . in asian woman", "kikuchi s disease is rare and benign cause of cervical lymphadenopathy.3 the disease , most often , happens to occur in young adults below 40 and seldom in children.4 at first , little female predominance was considered , but the recent literature regards it as male to female ratio 1:1.1,5 a current study on the patients with kfd conducted in taiwan reports the average age of 21.5 cardiac , hepatic and pulmonary involvement raises the morbidity .", "however , role of viruses ( epstein - barr virus and others ) in the pathogenesis of kikuchi s disease is controversial and unremarked.1 on the other hand , unger and coworkers are in favor of viral etiology as kfd manifests certain viral features ie , atypical lymphocytosis , certain histologic features , flulike respiratory prodrome and no response to antibiotic therapy.7 also , kikuchi s disease has been reported in patients with aids.8 like systemic lupus erythematosus ( sle ) , lymphocytes and histiocytes in the patients with kikuchi s disease show tubular reticular structures in their cytoplasm on electron microscopy.9 it has been opinioned that in genetically susceptible individuals , kfd may belong to exuberant t - cell mediated immune response provoked by variety of stimuli.1 even though course of cell death in kfd needs to be studied and emphasized , ohshima and his associates remarked apoptotic cell death might be involved in the pathogenesis of kfd.10 regarding to this study , proliferating cd8 t - cells may kill or be killed in the apoptotic process of this disease using fas and perforine pathways .", "kikuchi s disease begins as an acute or sub - acute condition , developing over two to three week period .", "tender cervical lymphadenopathy is the characteristic feature ( 56%98% ) of kfd , predominantly involving the posterior cervical triangle .", "size of the enlarged lymph nodes ranges from 0.5 cm to 4 cm ( occasionally 6 cm ) .", "59% patients represent painful lymphadenopathy and 1%22% patients undergo generalized lymphadenopathy.4,7,9,11 kfd , more or less , rarely involves mediastinal , peritoneal or retroperitoneal regions of the body.2 fever ( 30%50% ) associated with upper respiratory symptoms , sore throat , night sweats , weight loss , headache , rash , nausea , vomiting , and leukopenia ( about 50% ) are the other manifestations of the disease .", "12,13 atypical lymphocytes have been reported in the peripheral blood film of patients with kfd .", "extranodal involvement is rare ; however , skin , eye and bone marrow affection has been reported.1 nevertheless , kfd is linked to sle and autoimmune conditions as lymphocytes and histiocytes in the patients with kikuchi s disease show tubular reticular structures in their cytoplasm on electron microscopy.1,14 additionally , extranodal involvement in kfd is associated with frequent systemic symptoms .", "anecdotal reports bring to light the unusual features of kfd like haemophagocytic syndrome and carcinoma15 along with fatal multicentric disease .", "nervous system involvement ( septic meningitis , acute cerebellar ataxia , and encephalitis ) rarely happens to occur.16 regarding joint involvement , a case of 14 year old boy with kfd is in the record.17", "in patients with kikuchi fujimoto disease , an excisional biopsy of the involved lymph nodes is the investigation of choice .", "coagulative necrosis with ample karyorrhetic debris in paracortical areas of the involved lymph nodes is the characteristic histologic feature of kfd .", "other baseline investigations are reported unaffected . nevertheless , laboratory results of some kfd cases have reported anemia , little rise in esr and even leukopenia .", "one third individuals with kfd have shown atypical lymphocytes in their peripheral blood films.5 predominantly , t - cells ( cd8 + t - cells ) are found in kfd .", "however , neutrophils are found absent and scarce plasma cells may or may not be present . according to kuo", ", histopathologic features can be classified in three stages : proliferative , necrotizing , and xanthomatous.18 proliferative stage expresses various histiocytes , plasmacytoid monocytes and lymphoid cells containing karyorrhetic nuclear fragments , and eosinophilic apoptotic debris .", "necrotizing stage shows a degree of coagulative necrosis while xanthomatous stage is predominantly stuffed with foamy histiocytes .", "it must be born in mind that in the individuals with kfd , atypical reactive immunoblastic component is common and can be mistaken for lymphoma.4 histiocyte - associated antigens ( lysozyme , myeloperoxidase and cd68 ) are also expressed by histiocytes in kfd .", "kfd is an extremely rare disease and the differential diagnosis can be established on the basis of enlarged lymph nodes which are associated with many other disorders .", "it is necessary to born in mind the differential diagnosis of kfd as its treatment dramatically differs from other disorders .", "lymphoma ( non - hodgkin s lymphoma ) , tuberculosis , sle , plasmacytoid t - cell leukemia , kawasaki s disease , and myeloid tumor are included in the differential diagnosis of kfd.1 sometimes , because of similar clinical and histological features , it becomes problematic to differentiate kikuchi fujimoto disease from lymphadenitis associated with systemic lupus erythematosus ( sle ) .", "however , it has been reported that kfd is associated with sle . in order to exclude sle ,", "all the necessary investigations of sle ( c3 , c4 , anf , anti - sm , and le cells ) are required .", "early recognition of kikuchi fujimoto disease is of prime importance to save the patient from undergoing extensive investigations related to malignant lymphoma and other related disorders.8 histopathologic features like presence of abundant reactive histiocytes and absence of reed - sternberg cells favor kfd .", "sometimes , kfd may express histiocytes resembling with signet - ring cells and can be confused with signet - ring carcinoma .", "however , metastaic adenocarcinoma contains cells with atypical nuclei and mucin debris instead of cellular debris .", "kikuchi fujimoto disease ( histiocytic necrotizing lymhadenitis ) is a self - limiting condition that resolves spontaneously within 1 to 4 months of period .", "however , studies reveal recurrence of the disease in 3%4% of the patients.9 additionally , sle may happen to occur some years later .", "no hereditary risk has been documented in kfd.8 most of the time symptomatic relief is offered for the local and systemic complains of the disease .", "lymph node tenderness and fever is treated with analgesics , antipyretics , and nsaids . sometimes , but rarely , steroids can be used temporarily , especially in severe extranodal involvement or generalized clinical course.19 in order to run an excisional biopsy of the enlarged lymph nodes , surgical consultation may be prerequisite .", "individuals with kikuchi fujimoto disease should be examined systemically and they must be under regular follow - up in order to monitor the manifestations of sle . the course of cervical lymphadenopathy is benign and resolves spontaneously .", "very few cases have been reported as fatal . however , no standard or specific treatment of kfd has been recommended .", "kikuchi fujimoto disease is an idiopathic , extremely rare , more or less worldwide , and often under - diagnosed condition ; predominantly involving the posterior cervical lymph nodes .", "early recognition of kikuchi s disease is of prime importance to avoid extensive and expensive investigations related to malignant lymphoma other related disorders . in order to avoid misdiagnosis , awareness of this disease"], "abstract_text": ["<S> in order to determine the clinical significance of kikuchi fujimoto disease ( histiocytic necrotizing lymhadenitis ) and to review the literature available on this condition , we selected the medicine research papers in english language published between the years 1972 to 2011.kikuchi fujimoto disease ( kfd ) is an uncommon , cosmopolitan , benign and self - limiting condition with higher japanese and asian prevalence . </S>", "<S> most of the sufferers of kfd are young people who seek treatment because of having acute tender cervical lymphadenopathy , low grade fever and night sweats . </S>", "<S> coagulative necrosis with ample karyorrhetic debris in paracortical areas of the involved lymph nodes is the characteristic histologic feature of kfd . </S>", "<S> diagnosing kfd is crucial as it can be mistaken for malignant lymphoma and sle.kfd was put forth first time in 1972 by dr . </S>", "<S> masahiro kikuchi and by funimoto as lymphadenitis with reticular proliferation , histiocytes and abundant nuclear debris . </S>", "<S> it is a rare benign condition of lymph nodes and most of the clinicians and pathologists are unfamiliar with it . </S>", "<S> kfd is self - limiting disease ( within 1 to 4 months ) , however , patients should be followed up regularly as it may crop up again or progress to sle . </S>", "<S> analgesics and antipyretics help to ameliorate the symptoms . </S>"], "labels": null, "section_names": ["Literature Review", "Epidemiology", "Etiology and Pathogenesis", "Clinical Manifestations", "Diagnosis", "Differential Diagnosis", "Management", "Conclusion"], "sections": [["we collected all possible literature from the year 1972 ( when kfd was first described ) to the present day conducting a medical search on kikuchi fujimoto disease ( kfd ) in english language .", "we included publications in authorized medical journals including original and review articles / theses , editorials , case reports and brief communications ."], ["kfd is an extremely uncommon disease but cosmopolitan with higher japanese and asiatic prevalence.1,2 however , sporadic cases have also been reported from europe . in asian woman", "kikuchi s disease is rare and benign cause of cervical lymphadenopathy.3 the disease , most often , happens to occur in young adults below 40 and seldom in children.4 at first , little female predominance was considered , but the recent literature regards it as male to female ratio 1:1.1,5 a current study on the patients with kfd conducted in taiwan reports the average age of 21.5 cardiac , hepatic and pulmonary involvement raises the morbidity ."], ["however , role of viruses ( epstein - barr virus and others ) in the pathogenesis of kikuchi s disease is controversial and unremarked.1 on the other hand , unger and coworkers are in favor of viral etiology as kfd manifests certain viral features ie , atypical lymphocytosis , certain histologic features , flulike respiratory prodrome and no response to antibiotic therapy.7 also , kikuchi s disease has been reported in patients with aids.8 like systemic lupus erythematosus ( sle ) , lymphocytes and histiocytes in the patients with kikuchi s disease show tubular reticular structures in their cytoplasm on electron microscopy.9 it has been opinioned that in genetically susceptible individuals , kfd may belong to exuberant t - cell mediated immune response provoked by variety of stimuli.1 even though course of cell death in kfd needs to be studied and emphasized , ohshima and his associates remarked apoptotic cell death might be involved in the pathogenesis of kfd.10 regarding to this study , proliferating cd8 t - cells may kill or be killed in the apoptotic process of this disease using fas and perforine pathways ."], ["kikuchi s disease begins as an acute or sub - acute condition , developing over two to three week period .", "tender cervical lymphadenopathy is the characteristic feature ( 56%98% ) of kfd , predominantly involving the posterior cervical triangle .", "size of the enlarged lymph nodes ranges from 0.5 cm to 4 cm ( occasionally 6 cm ) .", "59% patients represent painful lymphadenopathy and 1%22% patients undergo generalized lymphadenopathy.4,7,9,11 kfd , more or less , rarely involves mediastinal , peritoneal or retroperitoneal regions of the body.2 fever ( 30%50% ) associated with upper respiratory symptoms , sore throat , night sweats , weight loss , headache , rash , nausea , vomiting , and leukopenia ( about 50% ) are the other manifestations of the disease .", "12,13 atypical lymphocytes have been reported in the peripheral blood film of patients with kfd .", "extranodal involvement is rare ; however , skin , eye and bone marrow affection has been reported.1 nevertheless , kfd is linked to sle and autoimmune conditions as lymphocytes and histiocytes in the patients with kikuchi s disease show tubular reticular structures in their cytoplasm on electron microscopy.1,14 additionally , extranodal involvement in kfd is associated with frequent systemic symptoms .", "anecdotal reports bring to light the unusual features of kfd like haemophagocytic syndrome and carcinoma15 along with fatal multicentric disease .", "nervous system involvement ( septic meningitis , acute cerebellar ataxia , and encephalitis ) rarely happens to occur.16 regarding joint involvement , a case of 14 year old boy with kfd is in the record.17"], ["in patients with kikuchi fujimoto disease , an excisional biopsy of the involved lymph nodes is the investigation of choice .", "coagulative necrosis with ample karyorrhetic debris in paracortical areas of the involved lymph nodes is the characteristic histologic feature of kfd .", "other baseline investigations are reported unaffected . nevertheless , laboratory results of some kfd cases have reported anemia , little rise in esr and even leukopenia .", "one third individuals with kfd have shown atypical lymphocytes in their peripheral blood films.5 predominantly , t - cells ( cd8 + t - cells ) are found in kfd .", "however , neutrophils are found absent and scarce plasma cells may or may not be present . according to kuo", ", histopathologic features can be classified in three stages : proliferative , necrotizing , and xanthomatous.18 proliferative stage expresses various histiocytes , plasmacytoid monocytes and lymphoid cells containing karyorrhetic nuclear fragments , and eosinophilic apoptotic debris .", "necrotizing stage shows a degree of coagulative necrosis while xanthomatous stage is predominantly stuffed with foamy histiocytes .", "it must be born in mind that in the individuals with kfd , atypical reactive immunoblastic component is common and can be mistaken for lymphoma.4 histiocyte - associated antigens ( lysozyme , myeloperoxidase and cd68 ) are also expressed by histiocytes in kfd ."], ["kfd is an extremely rare disease and the differential diagnosis can be established on the basis of enlarged lymph nodes which are associated with many other disorders .", "it is necessary to born in mind the differential diagnosis of kfd as its treatment dramatically differs from other disorders .", "lymphoma ( non - hodgkin s lymphoma ) , tuberculosis , sle , plasmacytoid t - cell leukemia , kawasaki s disease , and myeloid tumor are included in the differential diagnosis of kfd.1 sometimes , because of similar clinical and histological features , it becomes problematic to differentiate kikuchi fujimoto disease from lymphadenitis associated with systemic lupus erythematosus ( sle ) .", "however , it has been reported that kfd is associated with sle . in order to exclude sle ,", "all the necessary investigations of sle ( c3 , c4 , anf , anti - sm , and le cells ) are required .", "early recognition of kikuchi fujimoto disease is of prime importance to save the patient from undergoing extensive investigations related to malignant lymphoma and other related disorders.8 histopathologic features like presence of abundant reactive histiocytes and absence of reed - sternberg cells favor kfd .", "sometimes , kfd may express histiocytes resembling with signet - ring cells and can be confused with signet - ring carcinoma .", "however , metastaic adenocarcinoma contains cells with atypical nuclei and mucin debris instead of cellular debris ."], ["kikuchi fujimoto disease ( histiocytic necrotizing lymhadenitis ) is a self - limiting condition that resolves spontaneously within 1 to 4 months of period .", "however , studies reveal recurrence of the disease in 3%4% of the patients.9 additionally , sle may happen to occur some years later .", "no hereditary risk has been documented in kfd.8 most of the time symptomatic relief is offered for the local and systemic complains of the disease .", "lymph node tenderness and fever is treated with analgesics , antipyretics , and nsaids . sometimes , but rarely , steroids can be used temporarily , especially in severe extranodal involvement or generalized clinical course.19 in order to run an excisional biopsy of the enlarged lymph nodes , surgical consultation may be prerequisite .", "individuals with kikuchi fujimoto disease should be examined systemically and they must be under regular follow - up in order to monitor the manifestations of sle . the course of cervical lymphadenopathy is benign and resolves spontaneously .", "very few cases have been reported as fatal . however , no standard or specific treatment of kfd has been recommended ."], ["kikuchi fujimoto disease is an idiopathic , extremely rare , more or less worldwide , and often under - diagnosed condition ; predominantly involving the posterior cervical lymph nodes .", "early recognition of kikuchi s disease is of prime importance to avoid extensive and expensive investigations related to malignant lymphoma other related disorders . in order to avoid misdiagnosis , awareness of this disease"]]}
{"article_id": "PMC1175900", "article_text": ["does it have any bearing on outcome in the icu or during the post - icu period ?", "although not evidence based in any prospective , controlled , randomized clinical trials , it is unlikely that anyone would dispute that under - feeding eventually leads to death .", "this parallels the lack of controlled studies on the use of parachutes when jumping out of aeroplanes at high altitude .", "nevertheless , a short period of starvation appears to be a part of clinical treatment in many icus .", "this is highlighted by a report from colleagues in the netherlands presented in this issue of critical care .", "intensive care medicine is a comparatively young speciality , and within our arsenal of treatments the amount of evidence - based knowledge is often embarrassingly low .", "an illustration of this is seen in the guidelines for the surviving sepsis campaign , in which the evidence for different treatment modalities was evaluated in a formalized way .", "when we come to icu nutrition , there is little evidence at hand in terms of prospective , randomized , controlled clinical trials ; this was highlighted in recently published canadian guidelines .", ". some authors even call it ' poisonous nutrition ' and ban it from use in the icu .", "most investigators and authorities in the field would advocate enteral nutrition before parenteral , merely on the grounds that it is the natural way to feed and is cheaper .", "the evidence in favour of enteral over parenteral nutrition , or the other way around , is weak ; furthermore , it is obsolete because it was generated more than 10 years ago , with obscure indications for parenteral nutrition and with no blood sugar control . in studies comparing enteral and parenteral nutrition in patients", "whose attending physician is unsure regarding which modality will be optimal , the results demonstrate a very low feeding success rate with enteral nutrition and that complications are related to the duration of nutritional treatment , regardless of the mode of administration . in general terms it has repeatedly been shown that when patients are given only enteral nutrition , the success of feeding is below 70% .", "successful feeding may be defined in terms of the percentage of prescribed calories , number of days with at least 80% of prescribed calories delivered , or any other measure .", "the study from the netherlands presented in this issue adds further evidence to a number of publications that demonstrate that clinical practice is equivalent to delivering as little as 50% of the prescribed kilocalories .", "does this make any difference ? probably , a very large study population would be required to demonstrate that patients do worse when they are administered only 50% of the prescribed dose of antibiotics . the safety margin for bacterial kill", "when administrating antibiotics is such that reducing doses to half would require 500010,000 patients to show a difference in a prospective study . still no - one would advocate such a regimen .", "however , the side effects of antibiotics create renal insufficiency , liver insufficiency and drug fever , and predispose to development of multi - resistant bacteria .", "nevertheless , no - one would deliberately give only 50% of the prescribed dose , stating that it does not matter whether 50% or 100% of the dose is administered .", "when it comes to nutrition , however , many of us do this . in the study presented in this issue", ", the post hoc analysis shows that any action that reflects an interest in nutrition , such as placing the tube in any other position than the stomach , using some type of enhanced formula , or having a percutaneous feeding device , increases success rates with nutrition .", "these specially treated patient groups were small , and findings in these groups should perhaps not be used as a basis for clinical recommendations , but it is clear that an enhanced interest in nutrition and delivery of nutrition will increase success rates .", "so , the main reason for the systematic under - feeding that is practiced in icus is probably a lack of interest by the attending physician .", "binnekade and coworkers   do not give any information on success rates of feeding in relation to outcome .", "this is a difficult area , and studies that try to link nutritional practice to outcome must be designed carefully .", "there are examples in the literature of investigators jumping to conclusions based on insufficient information .", "nevertheless , in any patient in whom a huge energy deficit is built up , resulting in malnutrition , increased risk for complications can be predicted .", "in addition , evaluation of nutritional protocols in terms of success rate , nutrition related complications and outcome must be encouraged .", "systematic under - feeding of icu patients may be used as a marker of suboptimal care . wherever it is considered a human right not be hungry", ", the burden of producing proper evidence should be imposed on anyone who suggests that half the feed is good enough ."], "abstract_text": ["<S> systematic undernutrition of intensive care unit patients is common and neglected . is this inevitable or </S>", "<S> can better routines and protocols make a difference ? the necessity of feeding may be regarded as self - evident , but more evidence is obviously needed to strengthen this issue . in rich countries it should be a human right not to be hungry . </S>"], "labels": null, "section_names": ["None", "Abbreviations", "Competing interests"], "sections": [["does it have any bearing on outcome in the icu or during the post - icu period ?", "although not evidence based in any prospective , controlled , randomized clinical trials , it is unlikely that anyone would dispute that under - feeding eventually leads to death .", "this parallels the lack of controlled studies on the use of parachutes when jumping out of aeroplanes at high altitude .", "nevertheless , a short period of starvation appears to be a part of clinical treatment in many icus .", "this is highlighted by a report from colleagues in the netherlands presented in this issue of critical care .", "intensive care medicine is a comparatively young speciality , and within our arsenal of treatments the amount of evidence - based knowledge is often embarrassingly low .", "an illustration of this is seen in the guidelines for the surviving sepsis campaign , in which the evidence for different treatment modalities was evaluated in a formalized way .", "when we come to icu nutrition , there is little evidence at hand in terms of prospective , randomized , controlled clinical trials ; this was highlighted in recently published canadian guidelines .", ". some authors even call it ' poisonous nutrition ' and ban it from use in the icu .", "most investigators and authorities in the field would advocate enteral nutrition before parenteral , merely on the grounds that it is the natural way to feed and is cheaper .", "the evidence in favour of enteral over parenteral nutrition , or the other way around , is weak ; furthermore , it is obsolete because it was generated more than 10 years ago , with obscure indications for parenteral nutrition and with no blood sugar control . in studies comparing enteral and parenteral nutrition in patients", "whose attending physician is unsure regarding which modality will be optimal , the results demonstrate a very low feeding success rate with enteral nutrition and that complications are related to the duration of nutritional treatment , regardless of the mode of administration . in general terms it has repeatedly been shown that when patients are given only enteral nutrition , the success of feeding is below 70% .", "successful feeding may be defined in terms of the percentage of prescribed calories , number of days with at least 80% of prescribed calories delivered , or any other measure .", "the study from the netherlands presented in this issue adds further evidence to a number of publications that demonstrate that clinical practice is equivalent to delivering as little as 50% of the prescribed kilocalories .", "does this make any difference ? probably , a very large study population would be required to demonstrate that patients do worse when they are administered only 50% of the prescribed dose of antibiotics . the safety margin for bacterial kill", "when administrating antibiotics is such that reducing doses to half would require 500010,000 patients to show a difference in a prospective study . still no - one would advocate such a regimen .", "however , the side effects of antibiotics create renal insufficiency , liver insufficiency and drug fever , and predispose to development of multi - resistant bacteria .", "nevertheless , no - one would deliberately give only 50% of the prescribed dose , stating that it does not matter whether 50% or 100% of the dose is administered .", "when it comes to nutrition , however , many of us do this . in the study presented in this issue", ", the post hoc analysis shows that any action that reflects an interest in nutrition , such as placing the tube in any other position than the stomach , using some type of enhanced formula , or having a percutaneous feeding device , increases success rates with nutrition .", "these specially treated patient groups were small , and findings in these groups should perhaps not be used as a basis for clinical recommendations , but it is clear that an enhanced interest in nutrition and delivery of nutrition will increase success rates .", "so , the main reason for the systematic under - feeding that is practiced in icus is probably a lack of interest by the attending physician .", "binnekade and coworkers   do not give any information on success rates of feeding in relation to outcome .", "this is a difficult area , and studies that try to link nutritional practice to outcome must be designed carefully .", "there are examples in the literature of investigators jumping to conclusions based on insufficient information .", "nevertheless , in any patient in whom a huge energy deficit is built up , resulting in malnutrition , increased risk for complications can be predicted .", "in addition , evaluation of nutritional protocols in terms of success rate , nutrition related complications and outcome must be encouraged .", "systematic under - feeding of icu patients may be used as a marker of suboptimal care . wherever it is considered a human right not be hungry", ", the burden of producing proper evidence should be imposed on anyone who suggests that half the feed is good enough ."], [""], [""]]}
{"article_id": "PMC2137899", "article_text": ["recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) .", "because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele .", "previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) .", "the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig .", "consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) .", "this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of  contaminating  foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig .", "we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig .", "these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays .", "no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) .", "( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice .", "although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive .", "thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity .", "we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells .", "because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) .", "the resulting foxp3  foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3  cd4-cre mice .", "the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard", ", it was proposed that the autoimmunity previously reported in foxp3  cd4-cre mice ( 15 ) is due to cd4-cre  mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells .", "to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig .", "2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool .", "we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig .", "foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig .", "2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3  foxn1", "was confirmed by genomic pcr ( not depicted ) . when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) .", "foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes .", " foxn1 mice remained as healthy as foxp3  foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig .", "in contrast , foxp3  cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) . thus , foxp3 gene ablation in thymic epithelium does not result in autoimmunity .", "disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome .", "( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma .", "thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) .", "right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp  cd4-cre ( solid line ) mice .", "( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp  foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes .", "( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3  foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3  cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) .", "lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm (  ; n = 4 ) or foxp3 wild - type nude bm (  ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm (  ; n = 5 ) or foxp3 wild - type nude bm (  ; n = 6 ) .", "these results were further supported by our experiments using transfers of t cell  depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients .", "all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) .", "although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm .", "in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies", ", we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells .", "as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) .", "thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells .", "in these experiments , foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig .", "in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig .", "the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease .", "lack of autoimmunity in foxp3 nude  foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) .", "major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig .", "these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice .", "thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets .", "( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets .", "( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon", ", we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig .", "3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates .", "no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig .", "4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3  foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients .", "the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig .", "together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation .", "foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice .", "( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice .", "collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells .", "this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre  mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data", "are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology .", "thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage .", "recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) .", "because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele .", "previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) .", "the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig .", "consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) .", "this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of  contaminating  foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig .", "we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig .", "these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays .", "no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) .", "( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice .", "although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive .", "thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity .", "we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells .", "because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) .", "the resulting foxp3  foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3  cd4-cre mice .", "the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard", ", it was proposed that the autoimmunity previously reported in foxp3  cd4-cre mice ( 15 ) is due to cd4-cre  mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells .", "to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig .", "2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool .", "we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig .", "foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig .", "2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3  foxn1", "when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) .", "foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes .", " foxn1 mice remained as healthy as foxp3  foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig .", "in contrast , foxp3  cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) .", "disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome .", "( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma .", "thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) .", "right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp  cd4-cre ( solid line ) mice .", "( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp  foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes .", "( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3  foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3  cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) .", "lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm (  ; n = 4 ) or foxp3 wild - type nude bm (  ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm (  ; n = 5 ) or foxp3 wild - type nude bm (  ; n = 6 ) .", "these results were further supported by our experiments using transfers of t cell  depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients .", "all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) .", "although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm .", "in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies", ", we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells .", "as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) .", "thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells . in these experiments ,", "foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig .", "in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig .", "the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease .", "lack of autoimmunity in foxp3 nude  foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) .", "major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig .", "these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice .", "thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets .", "( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets .", "( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon", ", we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig .", "3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates .", "no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig .", "4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3  foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients .", "the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig .", "together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation .", "foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice .", "( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice .", "collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells .", "this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre  mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data", "are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology .", "thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage .", "foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background .", "b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains .", "bm chimeras were constructed using 7  10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 .", "experimental mice were age and sex matched and housed in specific pathogen - free conditions .", "disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) .", "all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington .", "510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a  pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 .", "thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) .", "after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) .", "thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti ", "s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer .", "foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background .", "b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains .", "bm chimeras were constructed using 7  10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 .", "experimental mice were age and sex matched and housed in specific pathogen - free conditions .", "disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) .", "all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington .", "510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a  pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 .", "thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) .", "after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) .", "thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti ", "s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer ."], "abstract_text": ["<S> foxp3 is essential for the commitment of differentiating thymocytes to the regulatory cd4 + t ( t reg ) cell lineage . in humans and mice with a genetic foxp3 </S>", "<S> deficiency , absence of this critical t reg cell population was suggested to be responsible for the severe autoimmune lesions . </S>", "<S> recently , it has been proposed that in addition to t reg cells , foxp3 is also expressed in thymic epithelial cells where it is involved in regulation of early thymocyte differentiation and is required to prevent autoimmunity . here , we used genetic tools to demonstrate that the thymic epithelium does not express foxp3 . furthermore , we formally showed that genetic abatement of foxp3 in the hematopoietic compartment , i.e. in t cells , is both necessary and sufficient to induce the autoimmune lesions associated with foxp3 loss . </S>", "<S> in contrast , deletion of a conditional foxp3 allele in thymic epithelial cells did not result in detectable changes in thymocyte differentiation or pathology . </S>", "<S> therefore , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage , whereas there is no role for foxp3 in thymic epithelial cells . </S>"], "labels": null, "section_names": ["RESULTS AND DISCUSSION", "No detectable Foxp3 protein expression in the thymic epithelium", "Disruption of Foxp3 in thymocytes is necessary and sufficient to cause autoimmune syndromes", "Distorted thymopoiesis associated with Foxp3 deficiency is secondary to lymphoproliferative autoimmune syndrome", "MATERIALS AND METHODS", "Mice.", "Flow cytometry and immunofluorescence.", "Online supplemental material."], "sections": [["recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) .", "because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele .", "previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) .", "the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig .", "consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) .", "this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of  contaminating  foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig .", "we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig .", "these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays .", "no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) .", "( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice .", "although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive .", "thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity .", "we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells .", "because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) .", "the resulting foxp3  foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3  cd4-cre mice .", "the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard", ", it was proposed that the autoimmunity previously reported in foxp3  cd4-cre mice ( 15 ) is due to cd4-cre  mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells .", "to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig .", "2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool .", "we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig .", "foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig .", "2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3  foxn1", "was confirmed by genomic pcr ( not depicted ) . when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) .", "foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes .", " foxn1 mice remained as healthy as foxp3  foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig .", "in contrast , foxp3  cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) . thus , foxp3 gene ablation in thymic epithelium does not result in autoimmunity .", "disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome .", "( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma .", "thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) .", "right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp  cd4-cre ( solid line ) mice .", "( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp  foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes .", "( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3  foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3  cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) .", "lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm (  ; n = 4 ) or foxp3 wild - type nude bm (  ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm (  ; n = 5 ) or foxp3 wild - type nude bm (  ; n = 6 ) .", "these results were further supported by our experiments using transfers of t cell  depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients .", "all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) .", "although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm .", "in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies", ", we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells .", "as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) .", "thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells .", "in these experiments , foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig .", "in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig .", "the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease .", "lack of autoimmunity in foxp3 nude  foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) .", "major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig .", "these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice .", "thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets .", "( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets .", "( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon", ", we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig .", "3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates .", "no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig .", "4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3  foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients .", "the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig .", "together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation .", "foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice .", "( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice .", "collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells .", "this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre  mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data", "are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology .", "thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage ."], ["recently , apparent expression of foxp3 in the majority of thymic epithelial cells was observed in experiments using flow cytometry while a subset of thymic cortical epithelial cells was found positive for foxp3 by immunofluorescence ( 16 ) .", "because epithelial cells are notorious for a high degree of nonspecific antibody binding in flow cytometric assays and are tightly associated with thymocytes in situ , potentially leading to false positive results in immunofluorescence assays , we sought to reexamine foxp3 expression in thymic epithelial cells and thymocytes by flow cytometric analysis of genetically marked foxp3-expressing cells using knock - in mice harboring a foxp3 reporter allele .", "previously , through examination of thymic tissue sections using immunofluorescence , we found foxp3 protein predominantly expressed in the medullary region in the thymus with rare foxp3 cells in the cortex ( 15 ) .", "the cortical expression of gfp was previously ascribed to expression by the few cd4cd8 double positive ( dp ) thymocytes that are gfp by flow cytometry ( 15 ) . to determine if this expression was due instead to epithelial cell expression of the foxp3 allele , we performed flow cytometric analysis of purified cd45 thymic stroma from foxp3 mice and found no expression of gfp ( fig .", "consistent with our previous studies , no expression of foxp3 was observed in thymocytes isolated from rag2 foxp3 mice ( not depicted ) .", "this finding allowed us to examine foxp3 expression in thymic stromal cells using immunofluorescence in the absence of  contaminating  foxp3-expressing thymocytes by examining gfp expression using anti - gfp antibody ( not depicted ) and polyclonal affinity - purified rabbit antibody specific for foxp3 ( fig .", "we found no sign of foxp3 expression in rag - deficient stroma above background fluorescence observed for foxp3 mice , whereas foxp3 expression was readily detectable in control rag - sufficient mice ( fig .", "these data demonstrate that the thymic epithelium did not express detectable levels of foxp3 protein within the sensitivity limit of these assays .", "no expression of foxp3 in the thymic epithelium . ( a ) expression of gfp in cd45 thymic stroma from wild - type ( shaded ) and foxp3 mice ( line ) .", "( b ) immunohistochemical analysis of foxp3 expression in the thymus of foxp3 , foxp3rag2 , and foxp3rag mice ."], ["although the aforementioned studies failed to detect foxp3 protein in thymic epithelial cells , it can be argued that our detection of foxp3 protein is not sufficiently sensitive .", "thus , the possibility remained that low level of foxp3 expression in thymic epithelial cells at a certain stage of t cell differentiation is required for prevention of autoimmunity .", "we addressed this possibility by ablation of a conditional foxp3 allele using cre recombinase expressed exclusively in thymic epithelial cells .", "because the foxn1 gene is the highly specialized regulator of thymic epithelial cell differentiation and is not expressed in bm - derived cells , we bred foxp3 mice with mice harboring cre recombinase knocked into the 3 untranslated region of the foxn1 locus ( foxn1 ; unpublished data ) .", "the resulting foxp3  foxn1 mice were examined for signs of lymphoproliferative autoimmune disease and compared with foxp3  cd4-cre mice .", "the interpretation of these experiments , however , was critically dependent on the specificity of cd4-cre and foxn1-mediated deletion . in this regard", ", it was proposed that the autoimmunity previously reported in foxp3  cd4-cre mice ( 15 ) is due to cd4-cre  mediated deletion of the foxp3 allele in a subset of cd4-expressing thymic epithelial cells .", "to directly address this issue , we first examined cd4 expression on cd45 thymic stromal cells using flow cytometry and failed to find cd4 thymic epithelial cells ( fig .", "2 a ) . next , we tested the cell type specificity of recombination mediated by cd4-cre and foxn1 using the rosa26-stop - yfp recombination reporter allele as a genetic fate - mapping tool .", "we found that cd4-cre induced recombination in the majority of thymocytes , but no recombination was detectable in the cd45 thymic stroma ( fig .", "foxn1-induced recombination occurred in the majority of cd45g8.8 thymic epithelial cells , but not cd45g8.8 stromal cells or thymocytes ( fig .", "2 b ) . a comparable extent of foxp3 deletion in purified cd45g8.8 thymic epithelial cells from foxp3  foxn1", "when the cd45g8.8 population was subdivided according to the expression of uea-1 or mhc class ii , both subsets showed similar levels of cre - mediated recombination ( not depicted ) .", "foxn1 was therefore active in the vast majority of thymic epithelial cells , but not fibroblasts or thymocytes .", " foxn1 mice remained as healthy as foxp3  foxn1 littermates and showed no signs of t cell activation ( not depicted ) , tissue pathology , or wasting disease ( fig .", "in contrast , foxp3  cd4-cre mice developed lethal autoimmune lesions indistinguishable from those in foxp3 mice in full agreement with our previously reported observations ( 15 ) .", "disruption of foxp3 in the t cell lineage is necessary and sufficient to cause autoimmune syndrome .", "( a ) no expression of cd4 in the cd45 thymic stroma . left : expression of cd4 in wild - type cd45 thymic stroma .", "thymic stromal cells were analyzed after incubation with anti - cd4 antibodies ( solid line ) or no antibody control ( shaded ) .", "right : expression of yfp in cd45 thymic stroma and cd45 thymocytes from wild - type ( shaded ) and rosa - stopyfp  cd4-cre ( solid line ) mice .", "( b ) expression of yfp from wild - type ( shaded ) and rosa - stopyfp  foxn1 ( line ) mice in cd45g8.8 epithelium , cd45g8.8 stroma , and thymocytes .", "( c ) analysis of gross clinical signs and lung and skin histopathology in foxp3  foxn1 ( n = 7 ; no histological infiltrates at 4 or 9 wk of age , no visible disease at > 16 wk of age ) , foxp3  cd4-cre ( n = 6 ; average life span 4 wk ) , foxp3 ( n = 7 ; average life span 4 wk ) , and wild - type or foxp3 mice ( n = 9 ; no histological infiltrates or visible disease at > 16 wk of age ) .", "lung and ear skin were taken from 4-wk - old male foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( d ) disease onset in neonatal rag1 mice reconstituted with foxp3 scurfy nude bm (  ; n = 4 ) or foxp3 wild - type nude bm (  ; n = 4 ) , and neonatal rag1 foxp3 scurfy mice reconstituted with foxp3 scurfy nude bm (  ; n = 5 ) or foxp3 wild - type nude bm (  ; n = 6 ) .", "these results were further supported by our experiments using transfers of t cell  depleted foxp3 and foxp3 bm , either separately or mixed at a 1:1 ratio into sublethally irradiated rag2 recipients .", "all recipients of foxp3 bm died by 67 wk of age from severe autoimmune disease , whereas recipients of foxp3 bm and mixed bm chimeras remained healthy ( not depicted ) .", "although donor bm was depleted of cd4 and cd8 t cells using magnetic bead sorting , it is impossible to formally exclude the possibility of a few pathogenic t cells remaining in the preparations of foxp3 bm .", "in addition , our experiments also differed from those previously reported by chang et al . in using mice harboring a foxp3 allele generated through targeted mutagenesis and spontaneous foxp3 mutation , respectively . to definitively exclude these possible explanations for the contradictory results from the two sets of bm transfer studies", ", we crossed the foxp3 allele to the foxn1-deficient nude mice , which are characterized by an early block in thymic epithelium differentiation and thus lack mature t cells .", "as previously reported , foxp3 nude mice were not affected by the autoimmunity ( 2 ) .", "thus , we were able to transfer bm isolated from these disease - free mice into rag1 recipients without the risk of contamination with mature pathogenic t cells . in these experiments ,", "foxp3 nude bm reconstitution of neonatal rag1 mice and foxp3rag1 mice ( serving as a positive control ) resulted in identical lethal lymphoproliferative disease ( fig .", "in contrast , control foxp3 nude bm transfers into either neonatal rag1 or foxp3rag1 did not result in disease ( fig .", "the findings above demonstrate that genetic ablation of foxp3 in the thymic epithelium is neither necessary nor sufficient for the development of autoimmune disease ."], ["lack of autoimmunity in foxp3 nude  foxp3 rag1 bm chimeras and in foxp3 foxn1 mice did not exclude the previously proposed role for expression of foxp3 in the thymic epithelium in normal thymopoiesis ( 16 ) .", "major thymic aberrations reported for foxp3 mutant mice include a decrease in thymic cellularity and in the proportion of cd4cd8 dp thymocytes ( 16 ) . to reexamine this possibility , we analyzed thymopoiesis in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice and found that both foxp3 and the foxp3 cd4-cre mice showed reduced total thymic cellularity and a decrease in the percentage of cd4cd8 dp thymocytes , whereas foxp3 foxn1 mice were identical to their wild - type littermates ( fig .", "these results demonstrate that deletion of the foxp3 gene in thymic epithelial cells does not result in detectable changes in thymic t cell maturation , and that an apparent decrease in dp thymocyte subset size and in total thymocyte numbers is due to loss of foxp3 in thymocytes rather than in the thymic epithelium and is most likely secondary to the severe autoimmunity and cytokine storm common to the foxp3 , foxp3 , and foxp3 cd4-cre mice .", "thymopoiesis was assessed by examining total thymus cellularity and by flow cytometric analysis of thymocyte subsets .", "( a ) representative cd4 and cd8 thymocyte profiles for wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "( b ) percentages of thymocytes in cd4cd8 dn , cd4cd8 dp , cd4 single positive , and cd8 single positive subsets .", "( c ) representative flow cytometric profiles of dn thymocyte subsets and ( d ) percentages of dn1 , dn2 , dn3 , and dn4 subsets in wild - type , foxp3-deficient , foxp3 cd4-cre , and foxp3 foxn1 mice .", "in addition to the aforementioned changes in thymocyte subsets , an expansion of dn1 ( cd44cd25 ) cells was reported for both foxp3 and foxp3 rag mice ( 16 ) . to examine this phenomenon", ", we first analyzed the composition of dn thymocyte subsets in foxp3 , foxp3 cd4-cre , and foxp3 foxn1 mice .", "the relative size of the dn1 - 4 thymocyte subsets was not altered in foxp3 foxn1 as compared with control mice ; however , we found relative increases in the dn1 subset in foxp3 and foxp3 cd4-cre mice ( fig .", "3 , c and d ) . to determine whether this is a cell - intrinsic defect in developing thymocytes , we analyzed dn thymocyte subsets in disease - free foxp3rag2 and control foxp3rag2 littermates .", "no differences in the relative sizes of dn1 , dn2 , and dn3 subsets were detected in these mice ( fig .", "4 , a and b ) . to exclude the possibility that very few thymic epithelial cells escaping cre - mediated deletion in foxp3  foxn1 mice are capable of supporting normal thymocyte development , we generated an additional set of bm chimeras by transferring wild - type bm into foxp3rag2 and foxp3rag2 recipients .", "the maturation of wild - type thymocytes was not different in the two sets of chimeric mice ( fig .", "together with the absence of expression of foxp3 in dn thymocytes , these experiments show that the changes in dn thymocyte maturation observed in foxp3 mutant mice are secondary effects of massive peripheral t cell activation .", "foxp3 deficiency does not affect early thymopoiesis in disease - free rag - deficient mice .", "( a ) representative flow cytometric analysis of dn1 , dn2 , dn3 , and dn4 thymocyte subsets and ( b ) their percentages in rag2 and foxp3-deficient rag2 mice .", "collectively , our data demonstrates that the thymic epithelium does not express detectable amounts of foxp3 protein , and that there are no measurable adverse effects on immunological tolerance attributable to foxp3 deficiency in thymic stromal cells or in other radiation - resistant nonhematopoietic cells .", "this conclusion is strongly supported by the lack of autoimmune manifestations and changes in thymocyte maturation upon foxn1-cre  mediated ablation of a conditional foxp3 allele in the thymic epithelium . in contrast , in control experiments , foxp3 ablation in the t cell lineage resulted in lethal autoimmune pathology typical of germline foxp3 mutation , as previously reported . in full agreement with these data", "are the results of the bm transfer experiments , which showed that foxp3 deficiency in hematopoietic cells is solely responsible for autoimmunity , and that perturbed thymocyte subsets in foxp3-deficient mice are an indirect consequence of pathology .", "thus , in mice the only known role for foxp3 remains promotion of t reg cell differentiation within the t cell lineage ."], ["foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background .", "b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains .", "bm chimeras were constructed using 7  10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 .", "experimental mice were age and sex matched and housed in specific pathogen - free conditions .", "disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) .", "all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington .", "510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a  pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 .", "thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) .", "after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) .", "thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti ", "s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer ."], ["foxp3 ( 14 ) , foxp3 ( 14 ) , foxp3 ( 15 ) , cd4-cre ( 18 ) , foxn1 ( unpublished data ; cre was inserted along with an internal ribosome entry site into the 3 untranslated region of the foxn1 gene ) , rosa - stop - yfp ( 19 ) , foxp3 , nude , rag1 , and rag2 mice have all been backcrossed to the b6 background .", "b6 foxp3 mice were backcrossed either to the b6 nude or b6 rag1 backgrounds two generations to produce foxp3 nude and foxp3 rag1 strains .", "bm chimeras were constructed using 7  10 bm cells / recipient harvested from athymic nude male mice with or without the foxp3 mutation and injected i.p . into neonatal ( 23 d ) rag1 with or without the foxp3 .", "experimental mice were age and sex matched and housed in specific pathogen - free conditions .", "disease incidence was monitored by frequent visual observation , and postmortem histological analysis of the tissues was performed using hematoxylin and eosin staining ( histology consultation services ) .", "all mice were used in accordance with guidelines from the institutional animal care committee of the university of washington ."], ["510-wk - old mice were analyzed using the following antibodies : cd45-apc , i - a  pe , cd4-pe - cy7 , cd44-pe , cd8-percp , cd25-apc , cd8-fitc , cd4-percp , and uea-1 biotin , followed by sav - percp ( all from bd biosciences ) and g8.8 supernatant conjugated to alexa 647 .", "thymic stroma preparations were enriched from three to six pooled thymi from 510-wk - old mice as described previously ( 20 ) .", "after enzymatic enrichment , cd45 thymic stromal cells were purified using cd45 microbeads ( miltenyi biotec ) and the automacs system ( miltenyi biotec ) as per the manufacturer 's recommendations before flow cytometric analysis ( 21 ) .", "thymic sections were prepared and stained as described previously ( 22 ) using rabbit polyclonal igg anti - foxp3 antibodies ( 14 ) , followed by alexa 546conjugated goat anti "], ["s1 shows early thymopoiesis for wild - type bm - derived thymocytes developing in foxp3-sufficient and foxp3-deficient hosts assessed by cd44/cd25 flow cytometric profiles of cd4cd8 thymocytes 4 wk after bm transfer ."]]}
{"article_id": "PMC4567607", "article_text": ["human parvovirus ( hpv ) b19 , being first discovered and introduced in 1975 , is a non - enveloped single - stranded dna virus from the parvoviridae family .", "the virus is transmitted by respiratory droplets and the prevalence is estimated to be high since most of the individuals are infected by the age of 15 .", "the clinical syndrome associated with hpv b19 strongly depends on the host ; for instance those suffering from hemolytic disorders , including sickle cell disease , hereditary spherocytosis ( hs ) , autoimmune hemolysis , thalassemias , and paroxysmal nocturnal hemoglobinuria ( pnh ) are susceptible to aplastic crisis .", "the virus has a predilection for infecting the erythroid progenitor cells of the bone marrow resulting in their lysis and aplastic anemia .", "thus , the bone marrow in these patients appears without erythroid precursors but with normal myeloid series .", "the hpv b19 induced aplastic crisis can unmask several hereditary hematological disorders that have been normally compensated . among these conditions ,", "when hpv b19 infects the bone marrow erythroid cells of these patients , decompensation occurs and thus the patient presents with signs and symptoms of abrupt onset severe anemia .", "several similar sporadic cases have been reported by now , but familial hpv b19 induced aplastic crisis leading to the diagnosis of hs in all family members is a very rare condition being only reported three times in the literature .", "we herein , report hpv b19 induced aplastic crisis in an asymptomatic and undiagnosed family of three with hs demonstrating pancytopenia on peripheral blood .", "a 3.5-year - old girl , the youngest child of a 4-member family , was presented with severe pallor and high fever without localizing sign with 5 days duration . on physical examination , she had jaundice in the sclera and the spleen was palpable about 3 cm below the costal margin .", "the following day , her 14-year - old brother presented with high fever of unknown origin and severe pallor . on physical examination , he was febrile ( 39 c ) and had a mild prominence of frontal and maxillary bones and jaundiced sclera .", "physical examination revealed an enlarged spleen with the tip of spleen palpated about 3 cm below the costal margins .", "the hematological indices of the three patients admitted to our department with severe pallor and fever of unknown origin according to the family history and positive findings on physical examination ( jaundice and splenomegaly ) , a work - up for hemolytic anemias , including hb electrophoresis , osmotic fragility , and autohemolysis test was performed for each patient .", "the results were consistent with the diagnosis of hs . knowing hpv b19 as the most common causative agent in the development of aplastic crisis in hemolytic anemias ;", "specifically hereditary spherocytosis polymerase chain reaction ( pcr ) for hpv b19 , dna was performed which was positive in all three patients ( figures 1 and 2 ) .", "bone marrow aspiration of the girl revealed normal marrow cellularity with mild erythroid hyperplasia and clusters of erythroid nests heralding the recovery of the erythroid series already affected by parvovirus b 19 infection . in contrary", ", cellularity was severely decreased in all three lineages in the boy implicating suppression of all hematopoietic lineages .", "both siblings received intravenous immunoglobulin ( ivig ) in a dosage of 1 gr / kg along with blood transfusion , twice for each .", "the girl recovered after 3 days with reticulocytosis of 16% , while the boy recovered 8 days later with reticulocytosis of 8% .", "their mother also had to receive blood transfusion because of having hb of 3.6 gr / dl .", "pcr for parvovirus b19 . from left to right ; the siblings , their positive and negative control , and the ladder .", "these family members were typical examples of the occurrence of aplastic crisis due to hpv b 19 complicating hs simultaneously in a family .", "only three previous reports have shown that hpv b19 can induce aplastic crisis and unmask the hs in a family . previously , green et al . in 1984 reported an adult sibling pair with hs who developed aplastic crisis after a febrile illness , which was further diagnosed to be hpv b19 infection .", "the diagnosis of hpv b19 was developed based on specific igm antibody in their sera , as pcr was not available .", "they also found that the children of one of the patients also developed hpv b19 induced aplastic crisis , which was resolved with supportive care .", "these two adult patients were treated by blood transfusion and supportive care and were discharged after 6 - 8 days of hospital care . in a similar report , mclellan and rutter in 1987 reported hpv b19 induced aplastic crisis in two teenage sisters leading to the diagnosis of hs .", "they also had a history of splenectomy in their mother at the age of 11 years .", "they both were diagnosed to have hpv b19 and underwent splenectomy and supportive care until recovery . in 1962 , chanarin et al . reported aplastic crisis in 3 members of a family ( 2 sisters and the father ) .", "the 10-year - old girl presented with 7 days history of fever of unknown origin , jaundice , and dark urine .", "the same illness was reported in 2-year - old sister and 32-year - old father who both had hs .", "the 10-year - old girl was treated with repeated transfusions , splenectomy , and supportive care for 92 days .", "based on laboratory investigations , the authors first assumed that the megaloblastic hematopoiesis accompanied by urinary excretion of large amounts of urocanic acid following oral dose of histidine hydrochloride was responsible for aplastic crisis in these patients with hs .", "however , the authors reached the conclusion that hpv b19 was responsible for the aplastic crisis in this family with hs .", "as hpv b19 was first discovered in 1975 and this family was reported in 1962 , thus the etiology was misdiagnosed . in the present study , we described a family with hs that all three members developed aplastic crisis secondary to hpv b19 infection leading to the detection of asymptomatic hs .", "those patients with hs , who remain undiagnosed , usually have mild hemolysis and the disease remains undiagnosed until the compensation is interrupted because of some environmental stressors such as infections .", "the distinguished feature of our report is the occurrence of some degrees of leukopenia , neutropenia and thrombocytopenia in two of our patients ( the mother and his son ) .", "there are few reports of mild transient pancytopenia due to hpv b19 infection and recent report on persistent severe aplastic anemia in a previously healthy adult .", "however , none of them occurred in members of a single family and one of them was persistent in a healthy subject .", "one of the theories behind the etiology of transient pancytopenia in hpv b19 infections is that the virus could be responsible for the temporary arrest of hematopoiesis that leads to aplastic crisis in persons with chronic hemolytic anemia .", "the other hypothesis is the occurrence of hpv - associated hemophagocytosis leading to pancytopenia . among our three patients ,", "two showed transient pancytopenia with more severity in the 14-year - old boy ; nevertheless , they did nt show any evidence of hemophagocytosis in bone marrow aspirate . according to available reports in the literature indicating probable efficacy of ivig in the treatment of patients with hpv b19 infections and its effect on replication on virus", "however , we can not draw any conclusion on whether the fast recovery of the patients was a positive impact of ivig or spontaneous recovery of hpv b 19 that is expected in hs patients , albeit happened very soon in our patients .", "hpv b19 induced aplastic crisis in a family leading to the diagnosis of hereditary hs is a very rare reported event in the literature .", "the distinguished feature of this report is that all affected members of a family developed some degrees of transient pancytopenia , not only anemia , all simultaneously in the course of their disease .", "however , it was transient and the recovery happened very soon in all of them within 10 days , which was attributed to the usage of ivig ."], "abstract_text": ["<S> human parvovirus ( hpv ) b19 induced aplastic crisis in a family leading to the diagnosis of hereditary spherocytosis ( hs ) is a very rare condition being barely reported in the literature . </S>", "<S> we herein report a 4-year - old girl , her brother , and their mother who all presented with progressive pallor and jaundice after a febrile illness . </S>", "<S> the hpv b19 was diagnosed using polymerase chain reaction ( pcr ) and positive serology for specific anti - hpv b19 igm . </S>", "<S> they were further diagnosed with having hs . </S>", "<S> the clinical importance of this report is that in the case of an abrupt onset of unexplained severe anemia and jaundice , one should consider underlying hemolytic anemias mostly hereditary spherocytosis complicated by hpv b19 aplastic crisis . </S>", "<S> herein , we report the occurrence of this condition , simultaneously in three members of a family . </S>", "<S> the distinguished feature of this report is that all affected family members developed some degrees of transient pancytopenia , not only anemia , all simultaneously in the course of their disease . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["human parvovirus ( hpv ) b19 , being first discovered and introduced in 1975 , is a non - enveloped single - stranded dna virus from the parvoviridae family .", "the virus is transmitted by respiratory droplets and the prevalence is estimated to be high since most of the individuals are infected by the age of 15 .", "the clinical syndrome associated with hpv b19 strongly depends on the host ; for instance those suffering from hemolytic disorders , including sickle cell disease , hereditary spherocytosis ( hs ) , autoimmune hemolysis , thalassemias , and paroxysmal nocturnal hemoglobinuria ( pnh ) are susceptible to aplastic crisis .", "the virus has a predilection for infecting the erythroid progenitor cells of the bone marrow resulting in their lysis and aplastic anemia .", "thus , the bone marrow in these patients appears without erythroid precursors but with normal myeloid series .", "the hpv b19 induced aplastic crisis can unmask several hereditary hematological disorders that have been normally compensated . among these conditions ,", "when hpv b19 infects the bone marrow erythroid cells of these patients , decompensation occurs and thus the patient presents with signs and symptoms of abrupt onset severe anemia .", "several similar sporadic cases have been reported by now , but familial hpv b19 induced aplastic crisis leading to the diagnosis of hs in all family members is a very rare condition being only reported three times in the literature .", "we herein , report hpv b19 induced aplastic crisis in an asymptomatic and undiagnosed family of three with hs demonstrating pancytopenia on peripheral blood ."], ["a 3.5-year - old girl , the youngest child of a 4-member family , was presented with severe pallor and high fever without localizing sign with 5 days duration . on physical examination , she had jaundice in the sclera and the spleen was palpable about 3 cm below the costal margin .", "the following day , her 14-year - old brother presented with high fever of unknown origin and severe pallor . on physical examination , he was febrile ( 39 c ) and had a mild prominence of frontal and maxillary bones and jaundiced sclera .", "physical examination revealed an enlarged spleen with the tip of spleen palpated about 3 cm below the costal margins .", "the hematological indices of the three patients admitted to our department with severe pallor and fever of unknown origin according to the family history and positive findings on physical examination ( jaundice and splenomegaly ) , a work - up for hemolytic anemias , including hb electrophoresis , osmotic fragility , and autohemolysis test was performed for each patient .", "the results were consistent with the diagnosis of hs . knowing hpv b19 as the most common causative agent in the development of aplastic crisis in hemolytic anemias ;", "specifically hereditary spherocytosis polymerase chain reaction ( pcr ) for hpv b19 , dna was performed which was positive in all three patients ( figures 1 and 2 ) .", "bone marrow aspiration of the girl revealed normal marrow cellularity with mild erythroid hyperplasia and clusters of erythroid nests heralding the recovery of the erythroid series already affected by parvovirus b 19 infection . in contrary", ", cellularity was severely decreased in all three lineages in the boy implicating suppression of all hematopoietic lineages .", "both siblings received intravenous immunoglobulin ( ivig ) in a dosage of 1 gr / kg along with blood transfusion , twice for each .", "the girl recovered after 3 days with reticulocytosis of 16% , while the boy recovered 8 days later with reticulocytosis of 8% .", "their mother also had to receive blood transfusion because of having hb of 3.6 gr / dl .", "pcr for parvovirus b19 . from left to right ; the siblings , their positive and negative control , and the ladder ."], ["these family members were typical examples of the occurrence of aplastic crisis due to hpv b 19 complicating hs simultaneously in a family .", "only three previous reports have shown that hpv b19 can induce aplastic crisis and unmask the hs in a family . previously , green et al . in 1984 reported an adult sibling pair with hs who developed aplastic crisis after a febrile illness , which was further diagnosed to be hpv b19 infection .", "the diagnosis of hpv b19 was developed based on specific igm antibody in their sera , as pcr was not available .", "they also found that the children of one of the patients also developed hpv b19 induced aplastic crisis , which was resolved with supportive care .", "these two adult patients were treated by blood transfusion and supportive care and were discharged after 6 - 8 days of hospital care . in a similar report , mclellan and rutter in 1987 reported hpv b19 induced aplastic crisis in two teenage sisters leading to the diagnosis of hs .", "they also had a history of splenectomy in their mother at the age of 11 years .", "they both were diagnosed to have hpv b19 and underwent splenectomy and supportive care until recovery . in 1962 , chanarin et al . reported aplastic crisis in 3 members of a family ( 2 sisters and the father ) .", "the 10-year - old girl presented with 7 days history of fever of unknown origin , jaundice , and dark urine .", "the same illness was reported in 2-year - old sister and 32-year - old father who both had hs .", "the 10-year - old girl was treated with repeated transfusions , splenectomy , and supportive care for 92 days .", "based on laboratory investigations , the authors first assumed that the megaloblastic hematopoiesis accompanied by urinary excretion of large amounts of urocanic acid following oral dose of histidine hydrochloride was responsible for aplastic crisis in these patients with hs .", "however , the authors reached the conclusion that hpv b19 was responsible for the aplastic crisis in this family with hs .", "as hpv b19 was first discovered in 1975 and this family was reported in 1962 , thus the etiology was misdiagnosed . in the present study , we described a family with hs that all three members developed aplastic crisis secondary to hpv b19 infection leading to the detection of asymptomatic hs .", "those patients with hs , who remain undiagnosed , usually have mild hemolysis and the disease remains undiagnosed until the compensation is interrupted because of some environmental stressors such as infections .", "the distinguished feature of our report is the occurrence of some degrees of leukopenia , neutropenia and thrombocytopenia in two of our patients ( the mother and his son ) .", "there are few reports of mild transient pancytopenia due to hpv b19 infection and recent report on persistent severe aplastic anemia in a previously healthy adult .", "however , none of them occurred in members of a single family and one of them was persistent in a healthy subject .", "one of the theories behind the etiology of transient pancytopenia in hpv b19 infections is that the virus could be responsible for the temporary arrest of hematopoiesis that leads to aplastic crisis in persons with chronic hemolytic anemia .", "the other hypothesis is the occurrence of hpv - associated hemophagocytosis leading to pancytopenia . among our three patients ,", "two showed transient pancytopenia with more severity in the 14-year - old boy ; nevertheless , they did nt show any evidence of hemophagocytosis in bone marrow aspirate . according to available reports in the literature indicating probable efficacy of ivig in the treatment of patients with hpv b19 infections and its effect on replication on virus", "however , we can not draw any conclusion on whether the fast recovery of the patients was a positive impact of ivig or spontaneous recovery of hpv b 19 that is expected in hs patients , albeit happened very soon in our patients ."], ["hpv b19 induced aplastic crisis in a family leading to the diagnosis of hereditary hs is a very rare reported event in the literature .", "the distinguished feature of this report is that all affected members of a family developed some degrees of transient pancytopenia , not only anemia , all simultaneously in the course of their disease .", "however , it was transient and the recovery happened very soon in all of them within 10 days , which was attributed to the usage of ivig ."]]}
{"article_id": "PMC4051987", "article_text": ["primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et  al . , 2012 ) .", "no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et  al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et  al . , 2011 ) .", "importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain .", "additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et  al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et  al . , 2000 ;", "sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus .", "sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et  al . , 2012 ) .", "the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein .", "known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et  al . , 2013 ) .", "sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor .", "for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et  al .", "recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice .", "this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et  al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et  al . , 2011 ) .", "here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et  al . , 2010 ) .", "sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et  al . , 2009 ) .", "mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et  al . , 1999 ) .", "most of the patients  carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele .", "the second knockout allele in this animal model is functionally null ( yang et  al . , 2010 ) .", "while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by  exercise intolerance and associated with ubiquitous cox deficiency .", "to test the effects of persistent increase of the nad pool on  the sco2 mouse , we first crossed it with a constitutive  parp1 mouse ( de murcia et  al . ,", "1997 ) , which shows increased levels of nad in skeletal muscle ( bai et  al . , 2011 ) .", "the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure  s1 available online ) .", "the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4  weeks starting at 2  months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure  1a ) .", "biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et  al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants ,", "these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure  1b ; table s1 ) .", "accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure  1c ) .", "the activities of complex iii and iv ( figure  1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure  1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n  = 4/group ) , as a food admix ( 400  mg / kg ) ( cant et  al . , 2012 ) for four weeks .", "metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure  s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in  vivo .", "nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure  2a ) , which showed no difference to each other .", "these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et  al . , 2012 ) .", "accordingly , the nad / nadh ratio was significantly increased ( figure  2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure  2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals .", "we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure  2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and  cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure  2d ) .", "tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment .", "as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 .", "accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure  2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure  2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals .", "the histochemical staining for cox reflected the biochemical results ( figure  2 g ) . in both c.  elegans and mammalian cells", ", nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et  al .", "we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure  2h ) .", "no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) .", "next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp  < 1  nm ; pirinen et  al . , 2014 , in this issue of cell metabolism ) at 50  mg /", "kg as a food admix for 4  weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure  s3 ) .", "weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure  3a ) .", "the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure  3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure  3c ) , indicating activation of sirt1 by mrlb-45696 .", "similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure  3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure  3f ; table s3 ) .", "histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure  3 g ) .", "again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) .", "in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure  4a ) ,  and respiratory chain activities in the brain ( figure  4b ; table  s4 ) . the intensity of cox staining was increased as well ( figure  4c ) .", "similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) .", "the nad pool is set by the balance between de novo and  salvage biosynthetic pathways and utilization by nad - consuming enzymes .", "nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et  al . , 2010 ) .", "these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) .", "nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle .", "for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et  al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt .", "parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et  al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) .", "ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis .", "these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et  al . ,", "we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr", "was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals .", "notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs .", "increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6  months ) ( pirinen et  al . , 2014 ) .", "in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4  weeks ) compared to that of  other studies ( 12  weeks ) .", "this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much  earlier than stimulation of mitochondrial proliferation and increase in mtdna content .", "likewise , prolonged nr supplementation up to 6  months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et  al . , 2013 ) .", "while we observed hardly any effect of nr in our 4  week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice .", "this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood .", "our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease .", "nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy .", "our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et  al . , 2013 ) or complex i deficiency ( karamanlidis et  al . , 2013 )", "initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et  al . , 2013 ) .", "several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et  al . , 2013 ; tutt et  al . ,", "however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects .", "mouse tissues were homogenized in 15 volumes of 10  mm potassium phosphate buffer ( ph 7.5 ) .", "mitochondrial - enriched fractions were collected after centrifugation at 800  g for 10  min in the  presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen .", "aliquots , 70  g each , were run through a 12% sds - page and electroblotted onto a  nitrocellulose membrane , which was then matched with different antibodies .", "carlo besta  neurological institute , in accordance with guidelines of the italian ministry of health .", "mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12  hr light / dark cycle and free access to water and food .", "a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et  al .", "series of 8  m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) .", "muscle quadriceps samples stored in liquid nitrogen were homogenized in 10  mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et  al . , 2004 ) .", "note that in all panels the activity of cii has been multiplied by 10 for  visualization clarity .", "nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) .", "tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) .", "mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et  al . , 2011 ) ."], "abstract_text": ["<S> summarymitochondrial disorders </S>", "<S> are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain . pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling , often fatal , disorders . </S>", "<S> pathways related to mitochondrial biogenesis are targets of sirtuin1 , a nad+-dependent protein deacetylase . as nad+ boosts the activity of sirtuin1 and other sirtuins , intracellular levels of nad+ play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell . </S>", "<S> we show here that supplementation with nicotinamide riboside , a natural nad+ precursor , or reduction of nad+ consumption by inhibiting the poly(adp - ribose ) polymerases , leads to marked improvement of the respiratory chain defect and exercise intolerance of the sco2 knockout / knockin mouse , a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis . </S>", "<S> this strategy is potentially translatable into therapy of mitochondrial disorders in humans . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Discussion", "Experimental Procedures"], "sections": [["primary mitochondrial disorders due to impaired oxidative phosphorylation ( oxphos ) are a well - established cause of severe disability and precocious death in both children and adults ( koopman et  al . , 2012 ) .", "no effective therapies are currently available for these conditions , but encouraging results have recently been obtained by stimulating mitochondrial biogenesis acting on either the ppar system ( wenz et  al . , 2008 ) or the amp - kinase ( ampk)/pgc1 axis ( viscomi et  al . , 2011 ) .", "importantly , these approaches can in principle be extended to several mitochondrial diseases with different genetic causes , as they do not point to the correction of a specific defect but are based on a more general strategy aimed at increasing the overall residual activity of the respiratory chain .", "additional targets able to activate the mitochondriogenic program and boost mitochondrial function are sirtuins 17 ( houtkooper et  al . , 2012 ) , the mammalian orthologs of the yeast silent information regulator ( sir ) 2 gene ( imai et  al . , 2000 ;", "sirtuins have different subcellular localization , sirt1 and sirt6 being mainly found in the nucleus , sirt2 in the cytosol , sirt3sirt5 in mitochondria , and sirt7 in the nucleolus .", "sirtuins are important regulators of several proteins , including key metabolic players , acting as either deacetylases or adp - ribosylases ( houtkooper et  al . , 2012 ) .", "the most - investigated member of the family is sirtuin1 ( sirt1 ) , a nad - dependent type iii nuclear deacetylase that utilizes nad as a cosubstrate to remove acetyl groups from lysine residues of a target protein .", "known targets of sirt1 are the tumor suppressor p53 , the myocyte - specific enhancer factor 2 ( mef2 ) , the forkhead box o ( foxo ) , and pgc1 , all of which regulate transcriptional programs related to increased mitochondrial function ( andreux et  al . , 2013 ) .", "sirt1 activity is directly regulated by nad availability , by substrate - dependent activation , raising the hypothesis that nad acts as a metabolic sensor .", "for instance , both nad levels and sirt1 activity increase in mammalian tissues in response to energy / nutrient stresses such as exercise ( cant and auwerx , 2009 , 2010 ) and fasting ( cant and auwerx , 2010 ; chen et  al .", "recent studies have shown that sirt1 activation can prevent diet - induced obesity in mice .", "this effect was achieved by increasing the content of nad in cells and tissues essentially through ( 1 ) dietary supplementation of suitable nad precursors , such as nicotinamide riboside , nr ( cant et  al . , 2012 ) , or ( 2 ) inhibition of nad - consuming enzymes , such as the poly(adp - ribose ) polymerase 1 , parp1 ( bai et  al . , 2011 ) .", "here , we have tested the therapeutic efficacy of these strategies on a genetic mitochondrial disease model , the sco2 knockout / knockin ( sco2 ) mouse ( yang et  al . , 2010 ) .", "sco2 encodes a metallochaperone involved in the formation of the copper redox centers into nascent complex iv ( cytochrome c oxidase , cox ) ( leary et  al . , 2009 ) .", "mutations in this gene lead to infantile fatal encephalocardiomyopathy ( papadopoulou et  al . , 1999 ) .", "most of the patients  carry at least one allele encoding the common mutation p.e140k , corresponding to the p.e129k mutation in the knockin murine sco2 allele .", "the second knockout allele in this animal model is functionally null ( yang et  al . , 2010 ) .", "while homozygous knockout individuals are embryonic lethal , the sco2 mice show a predominantly myopathic phenotype characterized by  exercise intolerance and associated with ubiquitous cox deficiency ."], ["to test the effects of persistent increase of the nad pool on  the sco2 mouse , we first crossed it with a constitutive  parp1 mouse ( de murcia et  al . ,", "1997 ) , which shows increased levels of nad in skeletal muscle ( bai et  al . , 2011 ) .", "the sco2-parp1 double mutants showed reduced fasting blood glucose levels , body weight and epididymal white adipose tissue ( wat ) compared to sco2 littermates , whereas no differences were observed between wt and parp1 littermates ( see figure  s1 available online ) .", "the endurance motor performance of sco2-parp1 double mutants , sco2 , parp1 , and wt littermates ( four animals per group ) was monitored weekly by a standard treadmill test for 4  weeks starting at 2  months of age . while sco2 mice showed markedly reduced motor performance compared to wt littermates throughout the observation time , the double mutant individuals performed as well as the wt and parp1 littermates ( figure  1a ) .", "biochemically , significant reduction of complex iv activity and , to a lesser extent , complex iii as well ( yang et  al . , 2010 ) , was measured in skeletal muscle of sco2 mice . in the muscle homogenate of sco2-parp1 double mutants ,", "these activities were comparable to that of parp1 and wt littermates , whereas complex i and ii activities were even higher ( figure  1b ; table s1 ) .", "accordingly , the intensity of the histochemical staining specific to cox was increased in skeletal muscle of the double mutants , compared to the sco2 mutants ( figure  1c ) .", "the activities of complex iii and iv ( figure  1d ; table s2 ) were also increased in the brains of double mutants relative to sco2 animals , but remained significantly lower than those measured in parp1 and wt littermates ; the histochemical reaction to cox was concordant with the biochemical data ( figure  1e ) . to test the effects of pharmacological intervention , we first administered nr to sco2 mice and wt littermates ( n  = 4/group ) , as a food admix ( 400  mg / kg ) ( cant et  al . , 2012 ) for four weeks .", "metabolic parameters in the treated groups , including reduced blood glucose , plasma fatty acids , and epididymal wat , mirrored those of parp1 mice ( figure  s2 ) , confirming that nr , or its derivative nmn , was pharmacologically active in  vivo .", "nr - treated sco2 mice significantly improved their motor performance compared to the vehicle - treated sco2 group , rapidly achieving the levels of motor endurance displayed by treated and untreated wt mice ( figure  2a ) , which showed no difference to each other .", "these results suggest that nr treatment increases mitochondrial function in the sco2 mice through nad activation of sirt1 ( cant et  al . , 2012 ) .", "accordingly , the nad / nadh ratio was significantly increased ( figure  2b ) , and the ratio between acetylated and total foxo1 , a direct target of sirt1 , was clearly reduced ( figure  2c ) in skeletal muscle of nr - treated versus vehicle - treated sco2 and wt animals .", "we did not observe differences in mtdna content ( data not shown ) and citrate synthase ( cs ) activity ( figure  2f ) , but mrna levels of several genes related to either fatty acids oxidation ( fao ) , including acox and  cd36 , or oxidative phosphorylation ( coxi , coxii , coxiv , coxva ) were significantly increased in nr - treated versus vehicle - treated sco2 but not in wt animals ( figure  2d ) .", "tfam , a key factor of mtdna transcription , was also increased , and ucp3 and pdk4 , which were downregulated in sco2 mice , returned to control levels upon nr treatment .", "as expected , we found no change in transcripts specific to pgc1 , which is activated posttranscriptionally by sirt1 , and to two pgc1 partners , nrf1 and nrf2 .", "accordingly , western blot immunovisualization demonstrated increased levels of several nuclear- and mitochondrial - dna - encoded oxphos - related proteins in nr - treated versus vehicle - treated skeletal muscle samples ( figure  2e ) . in the same specimens , mitochondrial respiratory chain activities were significantly increased ( figure  2f ; table s3 ) in nr - treated versus vehicle - treated sco2 mice , but not in wt animals .", "the histochemical staining for cox reflected the biochemical results ( figure  2 g ) . in both c.  elegans and mammalian cells", ", nr - dependent sirt1 activation can induce the mitochondrial unfolded protein response ( mtupr ) , a stress - response protective mechanism which can improve mitochondrial function ( durieux et  al .", "we found that the mtupr - specific transcripts clpp , hsp60 , and sod2 were significantly increased in muscle samples of nr - treated sco2 mice , whereas sod3 , which is unrelated to mtupr , was unchanged ( figure  2h ) .", "no effect of the nr treatment was observed on the mitochondrial respiratory chain activities in the brain of our animals ( table s4 ) .", "next , we administered a pan - parp inhibitor ( mrlb-45696 , ic50 for parp  < 1  nm ; pirinen et  al . , 2014 , in this issue of cell metabolism ) at 50  mg /", "kg as a food admix for 4  weeks . in mrlb-45696-treated sco2 mice , we observed metabolic effects similar to those of nr treatment ( figure  s3 ) .", "weekly treadmill tests showed progressive increase , up to normal , of the motor endurance in mrlb-45696-treated sco2 , whereas no change was seen in wt mice ( figure  3a ) .", "the nad / nadh ratio was significantly increased in treated versus untreated wt , but not in the sco2 , animals ( figure  3b ) , whereas the acetylated / total foxo1 ratio was reduced in both treated groups ( figure  3c ) , indicating activation of sirt1 by mrlb-45696 .", "similar to nr , mrlb-45696 increased the mrna expression levels of oxphos- and fao - related genes in both sco2 and wt mice , whereas mtdna content ( data not shown ) and cs activity ( figure  3f ) remained unchanged . in treated sco2 but not in wt animals , western blot analysis showed increased content of mitochondrial respiratory chain subunits ( figures 3d and 3e ) , which was paralleled by significantly increased mitochondrial respiratory chain activities ( figure  3f ; table s3 ) .", "histochemistry for cox showed increased staining in treated versus vehicle - treated sco2 mice ( figure  3 g ) .", "again , we found that expression of the mtupr genes hsp60 , clpp , and sod2 was significantly increased , unlike the mtupr - unrelated gene sod3 ( figures 3h and 3i ) .", "in contrast to the nr treatment , the mrlb-45696 treatment determined significant increase of cox transcripts ( figure  4a ) ,  and respiratory chain activities in the brain ( figure  4b ; table  s4 ) . the intensity of cox staining was increased as well ( figure  4c ) .", "similar results were obtained in both skeletal muscle and brain by using pj34 , a commercially available pan - parp inhibitor ( figures s3 and s4 ; table s4 ) ."], ["the nad pool is set by the balance between de novo and  salvage biosynthetic pathways and utilization by nad - consuming enzymes .", "nad is synthesized de novo from tryptophan , but the main source of nad is from salvage pathways ( houtkooper et  al . , 2010 ) .", "these require the uptake of other nad precursors from the diet , including nr . upon its entry in the cell , nr is phosphorylated by nr kinases into nicotinamide mononucleoside ( nmn ) , which is then converted to nad by nmn adenylyltransferase ( bieganowski and brenner , 2004 ) .", "nad biosynthesis and cellular levels are also controlled by a circadian clock related to the feeding / fasting cycle .", "for example , in the mitochondrial compartment nad levels regulate sirtuin 3 , a deacetylase targeting respiratory chain subunits ( peek et  al . , 2013 ) , while in the nucleus nad is a substrate of both parp1 and sirt .", "parp1 , the highest consumer of nad in mammalian tissues , is activated upon binding to damaged or abnormal dna ( durkacz et  al . , 1980 ) and catalyzes the formation of poly(adp - ribose ) polymers ( par ) using nad as a substrate , onto different acceptor proteins , including parp1 itself ( adamietz , 1987 ) .", "ablation of the parp1 gene , supplementation of nr or administration of parp inhibitors ( parpi ) can expand the nad pool and activate sirtuins , particularly sirt1 , a master regulator of mitochondrial homeostasis .", "these effects can protect mice from high - fat ( hf)-induced metabolic disease ( cant et  al . ,", "we have shown here that these treatments can correct the biochemical and clinical phenotype of the sco2 mouse , a model of genetically determined mitochondrial disease . increased transcription of genes related to both oxphos and mtupr", "was associated with activation of oxidative metabolism , increase of mitochondrial respiratory chain activities , and normalization of the endurance motor deficit , displayed by naive sco2 animals .", "notably , these effects were hardly seen in wt littermates , suggesting that mitochondrial dysfunction sensitizes muscle and possibly other tissues to activators of mitochondriogenic programs .", "increased mitochondrial function can be achieved in wt animals only by much longer - term treatments ( > 6  months ) ( pirinen et  al . , 2014 ) .", "in contrast with previous results , we found no change in mtdna copy number and cs activity in nr- or parpi - treated versus untreated animals , possibly because of the shorter timeframe of our experimental protocol ( 4  weeks ) compared to that of  other studies ( 12  weeks ) .", "this observation suggests time - dependent activation of different mitochondriogenic programs , with induction of oxphos- and fao - related genes occurring much  earlier than stimulation of mitochondrial proliferation and increase in mtdna content .", "likewise , prolonged nr supplementation up to 6  months induced mitochondrial biogenesis in the brain and improvement of cognitive dysfunction of an alzheimer disease mouse model ( gong et  al . , 2013 ) .", "while we observed hardly any effect of nr in our 4  week trial , two pan - parp inhibitors did correct the respiratory chain defect in the brains of our sco2 mice .", "this observation is particularly relevant , as the brain is an exquisite target of mitochondrial dysfunction , and progressive encephalopathy is the most frequent clinical presentation of mitochondrial disease in infancy and childhood .", "our work supports the idea that the increase of nad levels in critical tissues is an effective therapeutic option for mitochondrial disease .", "nr is a natural vitamin with no known adverse effects , which could be administered as a dietary supplement , particularly in case of isolated mitochondrial myopathy .", "our results are concordant with very recent works reporting beneficial effects of nad precursors in mouse models characterized by reduced nad / nadh ratio , such as aging ( gomes et  al . , 2013 ) or complex i deficiency ( karamanlidis et  al . , 2013 )", "initially shown to boost oxidative metabolism in diet - induced models of obesity , parp1 ablation or inhibition has recently been reported to remarkably rescue pharmacological models of liver cirrhosis , partly by correcting the associated mitochondrial impairment ( mukhopadhyay et  al . , 2013 ) .", "several parp inhibitors are currently under clinical trial as anticancer molecules , and seem to be associated with relatively mild side effects ( bundred et  al . , 2013 ; tutt et  al . ,", "however , more work is needed to evaluate their use in chronic conditions such as primary mitochondrial disorders in view of their potential genotoxic effects ."], ["mouse tissues were homogenized in 15 volumes of 10  mm potassium phosphate buffer ( ph 7.5 ) .", "mitochondrial - enriched fractions were collected after centrifugation at 800  g for 10  min in the  presence of protease inhibitors , and frozen and thawed three times in liquid nitrogen .", "aliquots , 70  g each , were run through a 12% sds - page and electroblotted onto a  nitrocellulose membrane , which was then matched with different antibodies .", "carlo besta  neurological institute , in accordance with guidelines of the italian ministry of health .", "mice were maintained in a temperature- and humidity - controlled animal - care facility , with a 12  hr light / dark cycle and free access to water and food .", "a standard treadmill apparatus ( columbus instruments , columbus , oh ) was used to measure motor exercise endurance , as described in viscomi et  al .", "series of 8  m thick sections were stained for cox and sdh , as described ( sciacco and bonilla , 1996 ) .", "muscle quadriceps samples stored in liquid nitrogen were homogenized in 10  mm phosphate buffer ( ph 7.4 ) , and the spectrophotometric activity of ci , cii , ciii , and civ , as well as cs , was measured as described ( bugiani et  al . , 2004 ) .", "note that in all panels the activity of cii has been multiplied by 10 for  visualization clarity .", "nad was extracted using acidic and alkaline extraction methods , respectively ( yang and sauve , 2006 ) .", "tissue nad was analyzed with mass spectrometry as previously described ( yang and sauve , 2006 ) .", "mtdna content and transcripts analysis was carried out by sybr green real - time pcr , as described ( viscomi et  al . , 2011 ) ."]]}
{"article_id": "PMC4376921", "article_text": ["irinotecan is commonly used in combination with oxaliplatin as a component of folfirinox chemotherapy for several gastrointestinal malignancies .", "the purpose of this case report is to describe a patient who developed acute paralysis and aphasia while receiving her initial infusion of irinotecan .", "a 67-year - old woman with newly diagnosed metastatic pancreatic adenocarcinoma presented for her first cycle of folfirinox chemotherapy . during her infusion of irinotecan", ", she developed acute onset of generalized weakness , paralysis of all extremities , and nonfluent aphasia with complete inability to communicate .", "this episode was self - limited and resolved within 2 h. prior to subsequent infusions she received intravenous repletion of potassium and had no recurrence of symptoms .", "in selected cases , coadministration of irinotecan and oxaliplatin may result in severe generalized weakness and aphasia , which may be triggered by underlying electrolyte disturbances .", "irinotecan is a topoisomerase i inhibitor derived from camptothecin , an alkaloid compound extracted from deciduous trees indigenous to eastern asia .", "its antineoplastic activity is mediated by its inhibition of double - stranded dna replication through stabilizing the cleavage complexes of topoisomerase i. its most common use is in the treatment of colorectal cancer . however , since 2010 , it has been combined with 5-fluorouracil , leucovorin , and oxaliplatin in the regimen known as folfirinox , which has been utilized as an effective therapy in patients with metastatic pancreatic cancer as well as other cancers .", "the most commonly cited adverse effects of irinotecan include late - onset diarrhea and bone marrow suppression , with clinically significant neutropenia and thrombocytopenia . a less frequent , acute cholinergic syndrome with resultant symptoms of diaphoresis , hypotension , anxiety , and abdominal cramping with diarrhea may result in severe discomfort and dehydration that can be life - threatening .", "atropine is commonly administered with the chemotherapy infusion both for the prevention and treatment of this syndrome .", "oxaliplatin is a third - generation platinum derivative that has shown to be an effective therapy in several malignancies , most commonly gastrointestinal cancers .", "side effects of oxaliplatin include a dose - limiting severe peripheral sensory neuropathy that is chronic in onset .", "less commonly , acute sensory disturbances that may be modulated by cold temperatures may occur .", "these effects are thought to be mediated by an interaction with voltage - gated sodium channels in peripheral nerves .", "here we report the case of a patient with a rare complication of combination therapy with irinotecan and oxaliplatin , i.e. severe generalized weakness , paralysis , and aphasia , and provide a synopsis of the current literature as well as a proposed therapeutic approach .", "a 67-year old asian woman with a history of poorly controlled diabetes presented with newly diagnosed metastatic pancreatic adenocarcinoma . in november 2013", ", she received her first cycle of palliative chemotherapy using folfirinox ( oxaliplatin 85 mg / m , irinotecan180 mg / m , leucovorin 400 mg / m iv , and 5-fu 2,400 mg / m iv by continuous infusion over 48 h without a bolus , with dexamethasone 10 mg and ondansetron 12 mg iv as premedication ) .", "prior to beginning therapy with folfirinox , her electrolyte levels were checked , which revealed mild hypokalemia ( 3.5 meq / l , normal values 3.55.0 ) with normal serum sodium and calcium ( na 137 mmol / l , ca 8.7 mg / dl ) .", "halfway through the irinotecan infusion , she developed acute onset of generalized weakness , paralysis of all extremities , and nonfluent aphasia with complete inability to communicate . throughout this , she was awake , alert , and aware of her surroundings , with stable vital signs . at this time", ", the infusion was stopped and she was given a second dose of atropine 1 mg as well as an iv bolus of 1 l normal saline .", "she was monitored carefully and observed to return close to her baseline status within 12 h. neither the infusion of irinotecan nor the 5-fu infusion was restarted , and she was discharged to her home with appropriate follow - up .", "she returned 2 weeks later for cycle 2 of her folfirinox therapy and felt completely well . prior to initiating the infusion ,", "her electrolyte levels were checked , which showed a low - normal potassium level of 3.7", "she received 20 meq iv kcl supplementation leading to an improvement in the potassium level to 4.4 meq / l immediately preceding the infusion .", "she was then given her second cycle of folfirinox with identical doses of the drugs as in the first cycle and was able to complete all of the therapy without recurrence of the symptoms previously experienced at her initial infusion .", "she was able to continue on therapy without event for a total of 3 cycles before she transferred her care to an institution closer to her home and was lost to follow - up .", "to date , there have been 9 reported cases of significant central nervous system toxicity during or following the administration of irinotecan , both with and without concurrent oxaliplatin administration .", "all of these cases involved the development of dysarthria , with 2 of them leading to a complete motor aphasia and 1 case with associated ataxia [ 1 , 2 , 3 , 4 , 5 ] . in each case , these symptoms developed with the initial infusion of irinotecan and completely resolved with time .", "the duration of symptoms ranged from as little as 15 min to as long as 8 h. the duration of symptoms appeared to be related to the dose of irinotecan , with doses < 200 mg / m having a quicker return to baseline ( 1545 min ) in comparison to larger doses of > 200 mg / m ( 28 h ) .", "irinotecan and its primary active metabolite , sn-38 , bind strongly to plasma proteins and tissues resulting in high plasma distribution . in animal models , irinotecan and its metabolites", "have been found to cross the blood - brain barrier into the central nervous system . in 2 patients in whom neurologic symptoms developed during irinotecan administration , hamberg et al .   examined the pharmacokinetics of irinotecan and sn-38 and found both of these values to be within the normal range .", "the acute onset of symptoms shortly after beginning irinotecan infusion also suggests that the manifestation of these symptoms is not dose or duration dependent .", "therefore , it is unlikely that altered systemic clearance of irinotecan mediates the presence or absence of neurologic symptoms in these patients .", "the degree of severe generalized weakness seen in our patient following administration of irinotecan has not previously been reported in the literature .", "though no imaging of the brain was performed in our patient , ct and mri performed in prior similar cases have failed to show any evidence of stroke or other acute cns abnormalities to explain the clinical presentation . our patient was found to have mild hypokalemia ( 3.5", "meq / l ) prior to chemotherapy ( which may have been even lower following hydration and drug administration ) , which may have contributed to her profound weakness and inability to move and speak while maintaining a normal sensorium .", "one case of acquired fanconi syndrome ( characterized by proximal tubular dysfunction resulting in electrolyte wasting of potassium , calcium , phosphate , and uric acid ) has been reported following combination therapy with capecitabine , irinotecan , and bevacizumab , but a single offending agent was not identified . correcting our patient 's electrolytes prior to the subsequent infusions and ensuring continued stability of these values throughout", "the infusion was the only change in the treatment plan and resulted in no recurrence of symptoms .", "we postulate that close monitoring and repletion of potassium was the factor that prevented further neurologic symptoms .", "as irinotecan alone has not previously been implicated in electrolyte - based neurologic complications , we also consider that the findings in our patient were not caused by irinotecan alone , but rather by combination therapy with oxaliplatin as administered in folfirinox .", "two prior case reports have noted the acute onset of severe neurologic deficits ( including generalized weakness , limb weakness , dysarthria , ophthalmoparesis , and coma ) during and shortly after the administration of oxaliplatin , which our patient received prior to initiation of irinotecan [ 8 , 9 ] . in both reported cases ,", "mmol / l ) and much more severe in the case reported by basso et al .  ", "( k 1.7 meq / l ) , which appears to correlate with the severity of symptoms . in these cases , symptoms developed during the oxaliplatin infusion or within 15 min of completion .", "our patient tolerated her oxaliplatin therapy without complications during the infusion , but it is possible that the coadministration of oxaliplatin and irinotecan may have increased the risk of developing these reversible neurologic effects", ". if this is the case , those patients receiving combination folfirinox therapy may be at significantly higher risk of developing neurologic side effects than those receiving folfox therapy alone . from our experience in this patient , and from our review of the literature , we conclude that in selected cases , the coadministration of irinotecan and oxaliplatin ( as is used in folfirinox ) may result in severe generalized weakness and aphasia .", "we believe that a careful correction of these abnormalities into the upper ranges of normal as needed , both prior to beginning and during the infusion of oxaliplatin / irinotecan , may prevent this reaction . while the symptoms may be transient and self - limited , they may equally be severe . without knowledge of their etiology and the appropriate therapy , patients may be denied further treatment or may be reexposed to a serious adverse reaction ."], "abstract_text": ["<S> backgroundirinotecan is commonly used in combination with oxaliplatin as a component of folfirinox chemotherapy for several gastrointestinal malignancies . </S>", "<S> the purpose of this case report is to describe a patient who developed acute paralysis and aphasia while receiving her initial infusion of irinotecan.case reporta 67-year - old woman with newly diagnosed metastatic pancreatic adenocarcinoma presented for her first cycle of folfirinox chemotherapy . during her infusion of irinotecan </S>", "<S> , she developed acute onset of generalized weakness , paralysis of all extremities , and nonfluent aphasia with complete inability to communicate . </S>", "<S> this episode was self - limited and resolved within 2 h. prior to subsequent infusions she received intravenous repletion of potassium and had no recurrence of symptoms.discussionin selected cases , coadministration of irinotecan and oxaliplatin may result in severe generalized weakness and aphasia , which may be triggered by underlying electrolyte disturbances . </S>", "<S> careful monitoring and correction of potassium may help prevent this reaction . </S>"], "labels": null, "section_names": ["Background", "Case Report", "Discussion", "Introduction", "Case Report", "Discussion"], "sections": [["irinotecan is commonly used in combination with oxaliplatin as a component of folfirinox chemotherapy for several gastrointestinal malignancies .", "the purpose of this case report is to describe a patient who developed acute paralysis and aphasia while receiving her initial infusion of irinotecan ."], ["a 67-year - old woman with newly diagnosed metastatic pancreatic adenocarcinoma presented for her first cycle of folfirinox chemotherapy . during her infusion of irinotecan", ", she developed acute onset of generalized weakness , paralysis of all extremities , and nonfluent aphasia with complete inability to communicate .", "this episode was self - limited and resolved within 2 h. prior to subsequent infusions she received intravenous repletion of potassium and had no recurrence of symptoms ."], ["in selected cases , coadministration of irinotecan and oxaliplatin may result in severe generalized weakness and aphasia , which may be triggered by underlying electrolyte disturbances ."], ["irinotecan is a topoisomerase i inhibitor derived from camptothecin , an alkaloid compound extracted from deciduous trees indigenous to eastern asia .", "its antineoplastic activity is mediated by its inhibition of double - stranded dna replication through stabilizing the cleavage complexes of topoisomerase i. its most common use is in the treatment of colorectal cancer . however , since 2010 , it has been combined with 5-fluorouracil , leucovorin , and oxaliplatin in the regimen known as folfirinox , which has been utilized as an effective therapy in patients with metastatic pancreatic cancer as well as other cancers .", "the most commonly cited adverse effects of irinotecan include late - onset diarrhea and bone marrow suppression , with clinically significant neutropenia and thrombocytopenia . a less frequent , acute cholinergic syndrome with resultant symptoms of diaphoresis , hypotension , anxiety , and abdominal cramping with diarrhea may result in severe discomfort and dehydration that can be life - threatening .", "atropine is commonly administered with the chemotherapy infusion both for the prevention and treatment of this syndrome .", "oxaliplatin is a third - generation platinum derivative that has shown to be an effective therapy in several malignancies , most commonly gastrointestinal cancers .", "side effects of oxaliplatin include a dose - limiting severe peripheral sensory neuropathy that is chronic in onset .", "less commonly , acute sensory disturbances that may be modulated by cold temperatures may occur .", "these effects are thought to be mediated by an interaction with voltage - gated sodium channels in peripheral nerves .", "here we report the case of a patient with a rare complication of combination therapy with irinotecan and oxaliplatin , i.e. severe generalized weakness , paralysis , and aphasia , and provide a synopsis of the current literature as well as a proposed therapeutic approach ."], ["a 67-year old asian woman with a history of poorly controlled diabetes presented with newly diagnosed metastatic pancreatic adenocarcinoma . in november 2013", ", she received her first cycle of palliative chemotherapy using folfirinox ( oxaliplatin 85 mg / m , irinotecan180 mg / m , leucovorin 400 mg / m iv , and 5-fu 2,400 mg / m iv by continuous infusion over 48 h without a bolus , with dexamethasone 10 mg and ondansetron 12 mg iv as premedication ) .", "prior to beginning therapy with folfirinox , her electrolyte levels were checked , which revealed mild hypokalemia ( 3.5 meq / l , normal values 3.55.0 ) with normal serum sodium and calcium ( na 137 mmol / l , ca 8.7 mg / dl ) .", "halfway through the irinotecan infusion , she developed acute onset of generalized weakness , paralysis of all extremities , and nonfluent aphasia with complete inability to communicate . throughout this , she was awake , alert , and aware of her surroundings , with stable vital signs . at this time", ", the infusion was stopped and she was given a second dose of atropine 1 mg as well as an iv bolus of 1 l normal saline .", "she was monitored carefully and observed to return close to her baseline status within 12 h. neither the infusion of irinotecan nor the 5-fu infusion was restarted , and she was discharged to her home with appropriate follow - up .", "she returned 2 weeks later for cycle 2 of her folfirinox therapy and felt completely well . prior to initiating the infusion ,", "her electrolyte levels were checked , which showed a low - normal potassium level of 3.7", "she received 20 meq iv kcl supplementation leading to an improvement in the potassium level to 4.4 meq / l immediately preceding the infusion .", "she was then given her second cycle of folfirinox with identical doses of the drugs as in the first cycle and was able to complete all of the therapy without recurrence of the symptoms previously experienced at her initial infusion .", "she was able to continue on therapy without event for a total of 3 cycles before she transferred her care to an institution closer to her home and was lost to follow - up ."], ["to date , there have been 9 reported cases of significant central nervous system toxicity during or following the administration of irinotecan , both with and without concurrent oxaliplatin administration .", "all of these cases involved the development of dysarthria , with 2 of them leading to a complete motor aphasia and 1 case with associated ataxia [ 1 , 2 , 3 , 4 , 5 ] . in each case , these symptoms developed with the initial infusion of irinotecan and completely resolved with time .", "the duration of symptoms ranged from as little as 15 min to as long as 8 h. the duration of symptoms appeared to be related to the dose of irinotecan , with doses < 200 mg / m having a quicker return to baseline ( 1545 min ) in comparison to larger doses of > 200 mg / m ( 28 h ) .", "irinotecan and its primary active metabolite , sn-38 , bind strongly to plasma proteins and tissues resulting in high plasma distribution . in animal models , irinotecan and its metabolites", "have been found to cross the blood - brain barrier into the central nervous system . in 2 patients in whom neurologic symptoms developed during irinotecan administration , hamberg et al .   examined the pharmacokinetics of irinotecan and sn-38 and found both of these values to be within the normal range .", "the acute onset of symptoms shortly after beginning irinotecan infusion also suggests that the manifestation of these symptoms is not dose or duration dependent .", "therefore , it is unlikely that altered systemic clearance of irinotecan mediates the presence or absence of neurologic symptoms in these patients .", "the degree of severe generalized weakness seen in our patient following administration of irinotecan has not previously been reported in the literature .", "though no imaging of the brain was performed in our patient , ct and mri performed in prior similar cases have failed to show any evidence of stroke or other acute cns abnormalities to explain the clinical presentation . our patient was found to have mild hypokalemia ( 3.5", "meq / l ) prior to chemotherapy ( which may have been even lower following hydration and drug administration ) , which may have contributed to her profound weakness and inability to move and speak while maintaining a normal sensorium .", "one case of acquired fanconi syndrome ( characterized by proximal tubular dysfunction resulting in electrolyte wasting of potassium , calcium , phosphate , and uric acid ) has been reported following combination therapy with capecitabine , irinotecan , and bevacizumab , but a single offending agent was not identified . correcting our patient 's electrolytes prior to the subsequent infusions and ensuring continued stability of these values throughout", "the infusion was the only change in the treatment plan and resulted in no recurrence of symptoms .", "we postulate that close monitoring and repletion of potassium was the factor that prevented further neurologic symptoms .", "as irinotecan alone has not previously been implicated in electrolyte - based neurologic complications , we also consider that the findings in our patient were not caused by irinotecan alone , but rather by combination therapy with oxaliplatin as administered in folfirinox .", "two prior case reports have noted the acute onset of severe neurologic deficits ( including generalized weakness , limb weakness , dysarthria , ophthalmoparesis , and coma ) during and shortly after the administration of oxaliplatin , which our patient received prior to initiation of irinotecan [ 8 , 9 ] . in both reported cases ,", "mmol / l ) and much more severe in the case reported by basso et al .  ", "( k 1.7 meq / l ) , which appears to correlate with the severity of symptoms . in these cases , symptoms developed during the oxaliplatin infusion or within 15 min of completion .", "our patient tolerated her oxaliplatin therapy without complications during the infusion , but it is possible that the coadministration of oxaliplatin and irinotecan may have increased the risk of developing these reversible neurologic effects", ". if this is the case , those patients receiving combination folfirinox therapy may be at significantly higher risk of developing neurologic side effects than those receiving folfox therapy alone . from our experience in this patient , and from our review of the literature , we conclude that in selected cases , the coadministration of irinotecan and oxaliplatin ( as is used in folfirinox ) may result in severe generalized weakness and aphasia .", "we believe that a careful correction of these abnormalities into the upper ranges of normal as needed , both prior to beginning and during the infusion of oxaliplatin / irinotecan , may prevent this reaction . while the symptoms may be transient and self - limited , they may equally be severe . without knowledge of their etiology and the appropriate therapy , patients may be denied further treatment or may be reexposed to a serious adverse reaction ."]]}
{"article_id": "PMC5195819", "article_text": ["alcohol dependence is the most prevalent substance use disorder   that can lead individuals with the disorder to serious health - related   and social problems . according to a report by nutt and colleagues ( 2007 ) , the level of potential harm and risk of alcohol use and misuse was among the top five with heroin and cocaine .", "the 12-month prevalence of alcohol dependence is reported to be approximately 2% to 6% in the general populations . in part reflecting the high potential harm and high prevalence of alcohol dependence , there have been a number of studies on how chronic alcohol use or alcohol dependence interacts with the structure of the brain of animals   and humans .", "overall brain atrophy , including the lesser volumes of gray matter and white matter with increased cerebrospinal fluid , in patients with alcohol use disorders , has consistently been reported .", "deficits in the prefrontal cortex , temporal cortex , cerebellum , striatum , hippocampus and amygdala   have also been reported in patients with alcohol dependence .", "neuropathological studies have shown that the alcohol - related neuronal and glial loss would preferentially involve the prefrontal cortex   among cortical regions .", "functional neuroimaging studies demonstrated altered metabolism or activation of the frontal cortex in association with the deteriorated neuropsychological functioning .", "cortical thickness analysis , a reliable and valid method , can capture important information on cortical structures .", "durazzo and colleagues exhibited that alcohol - dependent adults had thinner cortical regions in the left anterior cingulate cortex , bilateral frontal cortex , and bilateral insula than healthy controls .", "other researchers found that alcohol - dependent adults had reduced cortical thickness in the widespread brain regions of the superior frontal , precentral , postcentral , middle frontal , middle and superior temporal , middle temporal , and the lateral occipital cortex than healthy comparison participants . in the report by momenan and colleagues , participants with alcohol dependence exhibited thinner cortical regions that encompass the medial superior frontal cortex , insula , precentral and the precuneus of the right hemisphere as well as the superior frontal gyrus of the left hemisphere , in comparison with the healthy controls .", "previous studies have reported widespread cortical deficits without covarying out the effects associated with the global atrophy of the brain . among these widespread regions that show", "alcohol - related atrophy , we wanted to localize the cortical regions that may be particularly vulnerable to alcohol consumption .", "we therefore undertook the cortical thickness analysis adjusting for the hemispheric average cortical thickness , in 21 detoxified alcohol - dependent patients and matched 22 healthy comparison subjects .", "the objective of this study was to identify brain regions with cortical thickness alterations that exceed the level of global alterations in alcohol dependence .", "we used the whole brain - wise cortical thickness analysis , which is validated histologically   and with manual outlining method .", "we also investigated whether the magnitude of deficits are correlated with the alcohol use - related variable .", "given the studies that suggest the preferential involvement of prefrontal cortex among brain structural and functional alterations in alcohol dependence , we hypothesized that patients with alcohol dependence would have thinner prefrontal cortex in comparison with healthy comparison subjects , after correcting for the global cortical thinning associated with alcohol dependence .", "patients with alcohol dependence were enrolled from the inpatient unit of the department of neuropsychiatry in a university - affiliated hospital , seoul , south korea .", "age - matched healthy comparison subjects were recruited from the community via the local advertisement during the same study period .", "inclusion criteria for alcohol dependence group were ( 1 ) age between 20 and 70 , and ( 2 ) diagnosis of alcohol dependence according to the diagnostic and statistical manual of mental disorders - iv by 2 board - certified psychiatrists .", "exclusion criteria for both alcohol dependence and control groups were ( 1 ) any symptoms or signs of confusion , major medical disorders including kidney disease and chronic liver disease , and/or malnutrition , ( 2 ) presence or history of neurological disorders , ( 3 ) presence or history of any mental disorders other than alcohol dependence or comorbid depressive disorders , including alcohol - induced persistent dementia , alcohol - induced amnestic disorder , or alcohol withdrawal delirium ( 3 ) history of head injury , and ( 4 ) any contraindications to magnetic resonance imaging ( mri ) such as pace makers , claustrophobia , or metal implants .", "additional exclusion criteria for control group were ( 1 ) presence or history of mental disorders including alcohol abuse and ( 2 ) current alcohol consumption greater than 14 equivalent standard drinks for men , 7 for women , per time . after being detoxified for 2 weeks , all patients underwent physical examination by a physician and the routine laboratory tests to screen out any major medical disorders .", "t1 and t2 weighted mr images were obtained using 1.5 tesla siemens whole body scanner .", "repetition time [ tr]=2,050 ms , echo time [ te]=4.39 ms , inversion time [ ti]=1,100 ms , number of excitation [ nex]=2 , flip angle [ fa]=15 , slice thickness=1.3 mm , field of view [ fov]=180180 mm , acquisition matrix=256180 .", "t1 weighted images of 7 subjects were obtained with slightly different image acquisition protocol ( tr=1,960 ms , te=4.38 ms , ti=1,100 ms , nex=2 , flip angle [ fa]=15 , slice thickness=1.5 mm , fov=250 x 250 , acquisition matrix=256 x 180 ) .", "t2 weighted images were acquired in order to screen for gross brain abnormality ( tr=9,710 ms , te=120 ms , nex=2 , flip angle [ fa]=170 , slice thickness=3 mm ) .", "smoothing processes were conducted using an iterative nearest - neighbor averaging procedure with the full - width half maximum ( fwhm ) 15 mm 2-d gaussian kernel . for calculating statistical difference maps of cortical thickness between alcohol dependence and healthy comparison groups , general linear model ( glm ) with cortical thickness at each vertex as the dependent variable has been used .", "non - cortical areas of medial wall and corpus callosum were excluded from the model building .", "hemispheric average cortical thickness was included in the model as a covariate since there was a significant difference in hemispheric average cortical thickness between groups and our aim was to identify the particularly vulnerable cortical regions beyond the global brain atrophy associated with alcohol dependence .", "5,000 permutation simulations have been performed with random group - label shuffling , with a threshold for a significant vertex of p<0.05 .", "clusters with the size that would pass the family - wise error rate correction were considered significant .", "thickness values of the surface point with highest z values ( local maxima ) within the cluster , where significant group differences of cortical thickness were found , were extracted for post hoc analyses .", "pair - wise correlations were used to test whether there were associations between the magnitude of cortical thickness deficits in patients with alcohol dependence and the duration of alcohol use . considering the relatively modest sample size , sensitivity analyses to rule out the possibility that the current results may be modulated by other confounding factors such as comorbid depression , anxiety symptoms and scan parameter difference were performed .", "local maxima thickness values within the cluster extracted as described above were subjected to linear regression models that included the scan parameter difference , the presence of comorbid depression as defined by 19 or more scores on the 17-item hamilton depression rating scale ( hdrs ) , or the presence of anxiety as defined by 40 or more scores on the state - trait anxiety inventory ( stai - t )   as additional covariates .", "computations were performed using stata version 11 ( stata corp . , college station , tx , usa ) .", "patients with alcohol dependence were enrolled from the inpatient unit of the department of neuropsychiatry in a university - affiliated hospital , seoul , south korea .", "age - matched healthy comparison subjects were recruited from the community via the local advertisement during the same study period .", "inclusion criteria for alcohol dependence group were ( 1 ) age between 20 and 70 , and ( 2 ) diagnosis of alcohol dependence according to the diagnostic and statistical manual of mental disorders - iv by 2 board - certified psychiatrists .", "exclusion criteria for both alcohol dependence and control groups were ( 1 ) any symptoms or signs of confusion , major medical disorders including kidney disease and chronic liver disease , and/or malnutrition , ( 2 ) presence or history of neurological disorders , ( 3 ) presence or history of any mental disorders other than alcohol dependence or comorbid depressive disorders , including alcohol - induced persistent dementia , alcohol - induced amnestic disorder , or alcohol withdrawal delirium ( 3 ) history of head injury , and ( 4 ) any contraindications to magnetic resonance imaging ( mri ) such as pace makers , claustrophobia , or metal implants .", "additional exclusion criteria for control group were ( 1 ) presence or history of mental disorders including alcohol abuse and ( 2 ) current alcohol consumption greater than 14 equivalent standard drinks for men , 7 for women , per time . after being detoxified for 2 weeks , all patients underwent physical examination by a physician and the routine laboratory tests to screen out any major medical disorders .", "t1 and t2 weighted mr images were obtained using 1.5 tesla siemens whole body scanner .", "repetition time [ tr]=2,050 ms , echo time [ te]=4.39 ms , inversion time [ ti]=1,100 ms , number of excitation [ nex]=2 , flip angle [ fa]=15 , slice thickness=1.3 mm , field of view [ fov]=180180 mm , acquisition matrix=256180 . t1 weighted images of 7 subjects were obtained with slightly different image acquisition protocol ( tr=1,960 ms , te=4.38 ms , ti=1,100 ms , nex=2 , flip angle [ fa]=15 , slice thickness=1.5 mm , fov=250 x 250 , acquisition matrix=256 x 180 ) .", "t2 weighted images were acquired in order to screen for gross brain abnormality ( tr=9,710 ms , te=120 ms , nex=2 , flip angle [ fa]=170 , slice thickness=3 mm ) .", "smoothing processes were conducted using an iterative nearest - neighbor averaging procedure with the full - width half maximum ( fwhm ) 15 mm 2-d gaussian kernel .", "for calculating statistical difference maps of cortical thickness between alcohol dependence and healthy comparison groups , general linear model ( glm ) with cortical thickness at each vertex as the dependent variable has been used .", "non - cortical areas of medial wall and corpus callosum were excluded from the model building .", "hemispheric average cortical thickness was included in the model as a covariate since there was a significant difference in hemispheric average cortical thickness between groups and our aim was to identify the particularly vulnerable cortical regions beyond the global brain atrophy associated with alcohol dependence .", "5,000 permutation simulations have been performed with random group - label shuffling , with a threshold for a significant vertex of p<0.05 .", "clusters with the size that would pass the family - wise error rate correction were considered significant .", "thickness values of the surface point with highest z values ( local maxima ) within the cluster , where significant group differences of cortical thickness were found , were extracted for post hoc analyses .", "pair - wise correlations were used to test whether there were associations between the magnitude of cortical thickness deficits in patients with alcohol dependence and the duration of alcohol use . considering the relatively modest sample size , sensitivity analyses to rule out the possibility that the current results may be modulated by other confounding factors such as comorbid depression , anxiety symptoms and scan parameter difference were performed .", "local maxima thickness values within the cluster extracted as described above were subjected to linear regression models that included the scan parameter difference , the presence of comorbid depression as defined by 19 or more scores on the 17-item hamilton depression rating scale ( hdrs ) , or the presence of anxiety as defined by 40 or more scores on the state - trait anxiety inventory ( stai - t )   as additional covariates .", "computations were performed using stata version 11 ( stata corp . , college station , tx , usa ) .", "there were no significant differences in age and sex between diagnostic groups ( table 1 ) .", "patients with alcohol dependence drank alcohol more frequently and more heavily than healthy comparison subjects ( table 1 ) .", "patients with alcohol dependence had general cortical thinning ( left hemispheric average cortical thickness ( mm ) : 2.440.07 [ healthy comparison subjects ] vs 2.240.27 [ patients with alcohol dependence ] , t=3.35 , p=0.002 ) ( right hemispheric average cortical thickness ( mm ) : 2.450.07 [ healthy comparison subjects ] vs 2.250.23 [ patients with alcohol dependence ] , t=3.77 , p<0.001 ) . in order to identify regionally specific cortical deficits in patients with alcohol dependence compared to healthy comparison subjects , hemispheric average cortical thickness was added as a covariate in the whole brain vertex - wise analysis . in the glm model that includes age and average cortical thickness as covariates , significant cortical thickness deficits in patients with alcohol dependence , compared to healthy comparison subjects , were noted in the left superior frontal cortex , after correcting for multiple comparisons with the permutation method ( fig .", "1 ) ( cluster size=1489.5 mm ; number of vertices in the cluster=2,035 ; talairach coordinates = x [ -15.3 ] , y [ 61.8 ] , z [ 5.0 ] ; cluster p value=0.024 ) . given that different scanning parameter may influence on the cortical thickness variations , analysis was re - run with scan parameter as a covariate .", "when analysis was repeated covarying for comorbid depression that may be associated with thinner prefronto - temporal cortex , the diagnostic group effect remained significant ( p=0.001 ) . when analysis was repeated covarying for comorbid anxiety that may also be associated with thinner prefronto - temporal cortex , the diagnostic group effect remained significant in the left superior frontal cluster ( p=0.017 ) .", "post hoc correlation analysis between cortical thicknesses in the left superior frontal cluster of significant group difference and duration of alcohol use in alcohol dependence group demonstrated the significant association ( r=-0.55 ; p=0.02 ) ( fig .", "cortical thicknesses in the left superior frontal cluster was also correlated with the clinical institute withdrawal assessment for alcohol scores ( r=-0.548 , p=0.028 ) .", "otherwise we found no significant results between cortical thicknesses in the left superior frontal cluster and audit scores ( r=0.048 , p=0.859 ) ; and calculated alcohol use ( alcohol dose x frequency x duration ) ( r=-0.193 , p=0.509 ) .", "in the current study , we have identified the brain region with altered cortical thickness in patients with alcohol dependence .", "the region of cortical thickness deficits in patients with alcohol dependence encompassed primarily the superior frontal cortex , after adjusting for the effects on the global cortical atrophy induced by alcohol dependence ( fig .", "this is consistent with prior reports indicating that chronic alcohol use may have the most detrimental effects on prefrontal cortical regions .", "the level of n - acetyl aspartate , the viability marker of neurons , was decreased in prefrontal brain regions in chronic alcohol abusers .", "neuronal and glial loss has consistently been noted in the prefrontal cortical regions , particularly in the superior frontal cortex .", "alcohol use has been associated with the decreased performances in executive and attention tasks that are important function of the prefrontal cortex .", "pre - existing vulnerability in these regions may predispose individuals to alcohol dependence , since the function of these regions are to executively control over drug craving and seeking .", "these regions may be associated with the compulsive substance - related behaviors , which is also in line with our finding that shows the association between duration of alcohol use with the magnitude of the cortical deficits in this region .", "the current findings do not provide information regarding whether the pre - existing prefrontal cortical deficits have rendered individuals vulnerable to alcohol dependence , whether neurotoxicity of chronic alcohol use , including the oxidative stress , have damaged the cortex , or whether both processes have contributed to the observed findings . supporting evidence for", "the neurotoxicity as the cause of prefrontal deficits may come from the reports that show patients with longer abstinence have lesser deficits .", "it has also been suggested that subjects with alcohol dependence may have prefrontal cortex that are less recuperative from toxic effects and may undergo vicious cycle after initial exposure to substances .", "considering that alcohol dependence can be divided into two subtypes , type 1 more environmentally influenced , and type 2 more genetically induced , a comparison between type 1 and type 2 alcohol dependent patients in a study with larger sample with balanced composition of type 1 and type 2 alcohol dependence may provide an opportunity to approach this question .", "longitudinal brain imaging study that follows up patients with alcohol dependence would also provide important insights .", "this study alone does not provide direct information as to cellular level mechanisms that may underlie the observed deficits of the cortical thickness .", "however , there is a vast literature describing the impact of chronic alcohol on the brain .", "miguel - hidalgo and colleagues ( 2002 ) reported , in their study with the postmortem brain of alcohol - dependent patients without wernicke or korsakoff syndromes , that the glial pathology of reduced size and density was the most characteristic finding .", "kril and colleagues ( 1997 ) have shown selective loss of non - gabaergic pyramidal neurons .", "selective dendritic retraction , rather than cellular death , has been suggested as main pathology related to the volume loss of the gray matter .", "there is a study that examined the cortical thickness differences in subjects with fetal alcohol syndrome or prenatal alcohol exposure , compared to control subjects , which demonstrated deficits in prefronto - temporo - parietal regions .", "however , few studies have examined cortical thickness in alcohol - dependent adults . the cortical thickness analysis has been reported to provide information on an important aspect of the gray matter structure , complementary to the conventional volumetry or the vbm .", "for example , highly folded regions could have high gray matter density in vbm analysis since there would be more voxels of gray matter within a fixed radius , but thinner cortical thickness .", "this is the first study that used cortical thickness analysis in patients with alcohol dependence excluding comorbid substance use , which showed regionally specific cortical thickness deficits in alcohol dependence .", "large age range , though covaried in the statistical model , can be another weakness of the study . with the current study of a cross - sectional design , information on whether the observed cortical deficits would be progressive and", "nicotine dependence , which is highly comorbid with alcohol dependence , has also been reported to be associated with cortical atrophy , particularly that in frontal cortex .", "now that we neither were able to exclude participants with co - morbid nicotine dependence nor delineate the effects of alcohol dependence from those of nicotine dependence , the current results may have been confounded by the effects of chronic nicotine use . intellectual ability has also been reported to be associated with the prefrontal cortical development .", "the fact that the intelligence quotient was not included in the statistical model for comparisons of cortical thickness measures , is also an important limitation of the current study .", "future studies in larger samples with narrow age range and with neuropsychological assessments for the frontal lobe function , would provide valuable information . although there could be challenges in following up patients with alcohol dependence , long - term prospective longitudinal study is also warranted ."], "abstract_text": ["<S> alcohol dependence is a serious disorder that can be related with a number of potential health - related and social consequences . </S>", "<S> cortical thickness measurements would provide important information on the cortical structural alterations in patients with alcohol dependence . </S>", "<S> twenty - one patients with alcohol dependence and 22 healthy comparison subjects have been recruited and underwent high - resolution brain magnetic resonance ( mr ) imaging and clinical assessments . t1-weighted mr images were analyzed using the cortical thickness analysis program . </S>", "<S> significantly thinner cortical thickness in patients with alcohol dependence than healthy comparison subjects was noted in the left superior frontal cortical region , correcting for multiple comparisons and adjusting with age and hemispheric average cortical thickness . </S>", "<S> there was a significant association between thickness in the cluster of the left superior frontal cortex and the duration of alcohol use . </S>", "<S> the prefrontal cortical region may particularly be vulnerable to chronic alcohol exposure . </S>", "<S> it is also possible that the pre - existing deficit in this region may have rendered individuals more susceptible to alcohol dependence . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Subjects", "Brain magnetic resonance (MR) image acquisition and cortical thickness analysis", "Statistical analysis", "RESULTS", "DISCUSSION"], "sections": [["alcohol dependence is the most prevalent substance use disorder   that can lead individuals with the disorder to serious health - related   and social problems . according to a report by nutt and colleagues ( 2007 ) , the level of potential harm and risk of alcohol use and misuse was among the top five with heroin and cocaine .", "the 12-month prevalence of alcohol dependence is reported to be approximately 2% to 6% in the general populations . in part reflecting the high potential harm and high prevalence of alcohol dependence , there have been a number of studies on how chronic alcohol use or alcohol dependence interacts with the structure of the brain of animals   and humans .", "overall brain atrophy , including the lesser volumes of gray matter and white matter with increased cerebrospinal fluid , in patients with alcohol use disorders , has consistently been reported .", "deficits in the prefrontal cortex , temporal cortex , cerebellum , striatum , hippocampus and amygdala   have also been reported in patients with alcohol dependence .", "neuropathological studies have shown that the alcohol - related neuronal and glial loss would preferentially involve the prefrontal cortex   among cortical regions .", "functional neuroimaging studies demonstrated altered metabolism or activation of the frontal cortex in association with the deteriorated neuropsychological functioning .", "cortical thickness analysis , a reliable and valid method , can capture important information on cortical structures .", "durazzo and colleagues exhibited that alcohol - dependent adults had thinner cortical regions in the left anterior cingulate cortex , bilateral frontal cortex , and bilateral insula than healthy controls .", "other researchers found that alcohol - dependent adults had reduced cortical thickness in the widespread brain regions of the superior frontal , precentral , postcentral , middle frontal , middle and superior temporal , middle temporal , and the lateral occipital cortex than healthy comparison participants . in the report by momenan and colleagues , participants with alcohol dependence exhibited thinner cortical regions that encompass the medial superior frontal cortex , insula , precentral and the precuneus of the right hemisphere as well as the superior frontal gyrus of the left hemisphere , in comparison with the healthy controls .", "previous studies have reported widespread cortical deficits without covarying out the effects associated with the global atrophy of the brain . among these widespread regions that show", "alcohol - related atrophy , we wanted to localize the cortical regions that may be particularly vulnerable to alcohol consumption .", "we therefore undertook the cortical thickness analysis adjusting for the hemispheric average cortical thickness , in 21 detoxified alcohol - dependent patients and matched 22 healthy comparison subjects .", "the objective of this study was to identify brain regions with cortical thickness alterations that exceed the level of global alterations in alcohol dependence .", "we used the whole brain - wise cortical thickness analysis , which is validated histologically   and with manual outlining method .", "we also investigated whether the magnitude of deficits are correlated with the alcohol use - related variable .", "given the studies that suggest the preferential involvement of prefrontal cortex among brain structural and functional alterations in alcohol dependence , we hypothesized that patients with alcohol dependence would have thinner prefrontal cortex in comparison with healthy comparison subjects , after correcting for the global cortical thinning associated with alcohol dependence ."], ["patients with alcohol dependence were enrolled from the inpatient unit of the department of neuropsychiatry in a university - affiliated hospital , seoul , south korea .", "age - matched healthy comparison subjects were recruited from the community via the local advertisement during the same study period .", "inclusion criteria for alcohol dependence group were ( 1 ) age between 20 and 70 , and ( 2 ) diagnosis of alcohol dependence according to the diagnostic and statistical manual of mental disorders - iv by 2 board - certified psychiatrists .", "exclusion criteria for both alcohol dependence and control groups were ( 1 ) any symptoms or signs of confusion , major medical disorders including kidney disease and chronic liver disease , and/or malnutrition , ( 2 ) presence or history of neurological disorders , ( 3 ) presence or history of any mental disorders other than alcohol dependence or comorbid depressive disorders , including alcohol - induced persistent dementia , alcohol - induced amnestic disorder , or alcohol withdrawal delirium ( 3 ) history of head injury , and ( 4 ) any contraindications to magnetic resonance imaging ( mri ) such as pace makers , claustrophobia , or metal implants .", "additional exclusion criteria for control group were ( 1 ) presence or history of mental disorders including alcohol abuse and ( 2 ) current alcohol consumption greater than 14 equivalent standard drinks for men , 7 for women , per time . after being detoxified for 2 weeks , all patients underwent physical examination by a physician and the routine laboratory tests to screen out any major medical disorders .", "t1 and t2 weighted mr images were obtained using 1.5 tesla siemens whole body scanner .", "repetition time [ tr]=2,050 ms , echo time [ te]=4.39 ms , inversion time [ ti]=1,100 ms , number of excitation [ nex]=2 , flip angle [ fa]=15 , slice thickness=1.3 mm , field of view [ fov]=180180 mm , acquisition matrix=256180 .", "t1 weighted images of 7 subjects were obtained with slightly different image acquisition protocol ( tr=1,960 ms , te=4.38 ms , ti=1,100 ms , nex=2 , flip angle [ fa]=15 , slice thickness=1.5 mm , fov=250 x 250 , acquisition matrix=256 x 180 ) .", "t2 weighted images were acquired in order to screen for gross brain abnormality ( tr=9,710 ms , te=120 ms , nex=2 , flip angle [ fa]=170 , slice thickness=3 mm ) .", "smoothing processes were conducted using an iterative nearest - neighbor averaging procedure with the full - width half maximum ( fwhm ) 15 mm 2-d gaussian kernel . for calculating statistical difference maps of cortical thickness between alcohol dependence and healthy comparison groups , general linear model ( glm ) with cortical thickness at each vertex as the dependent variable has been used .", "non - cortical areas of medial wall and corpus callosum were excluded from the model building .", "hemispheric average cortical thickness was included in the model as a covariate since there was a significant difference in hemispheric average cortical thickness between groups and our aim was to identify the particularly vulnerable cortical regions beyond the global brain atrophy associated with alcohol dependence .", "5,000 permutation simulations have been performed with random group - label shuffling , with a threshold for a significant vertex of p<0.05 .", "clusters with the size that would pass the family - wise error rate correction were considered significant .", "thickness values of the surface point with highest z values ( local maxima ) within the cluster , where significant group differences of cortical thickness were found , were extracted for post hoc analyses .", "pair - wise correlations were used to test whether there were associations between the magnitude of cortical thickness deficits in patients with alcohol dependence and the duration of alcohol use . considering the relatively modest sample size , sensitivity analyses to rule out the possibility that the current results may be modulated by other confounding factors such as comorbid depression , anxiety symptoms and scan parameter difference were performed .", "local maxima thickness values within the cluster extracted as described above were subjected to linear regression models that included the scan parameter difference , the presence of comorbid depression as defined by 19 or more scores on the 17-item hamilton depression rating scale ( hdrs ) , or the presence of anxiety as defined by 40 or more scores on the state - trait anxiety inventory ( stai - t )   as additional covariates .", "computations were performed using stata version 11 ( stata corp . , college station , tx , usa ) ."], ["patients with alcohol dependence were enrolled from the inpatient unit of the department of neuropsychiatry in a university - affiliated hospital , seoul , south korea .", "age - matched healthy comparison subjects were recruited from the community via the local advertisement during the same study period .", "inclusion criteria for alcohol dependence group were ( 1 ) age between 20 and 70 , and ( 2 ) diagnosis of alcohol dependence according to the diagnostic and statistical manual of mental disorders - iv by 2 board - certified psychiatrists .", "exclusion criteria for both alcohol dependence and control groups were ( 1 ) any symptoms or signs of confusion , major medical disorders including kidney disease and chronic liver disease , and/or malnutrition , ( 2 ) presence or history of neurological disorders , ( 3 ) presence or history of any mental disorders other than alcohol dependence or comorbid depressive disorders , including alcohol - induced persistent dementia , alcohol - induced amnestic disorder , or alcohol withdrawal delirium ( 3 ) history of head injury , and ( 4 ) any contraindications to magnetic resonance imaging ( mri ) such as pace makers , claustrophobia , or metal implants .", "additional exclusion criteria for control group were ( 1 ) presence or history of mental disorders including alcohol abuse and ( 2 ) current alcohol consumption greater than 14 equivalent standard drinks for men , 7 for women , per time . after being detoxified for 2 weeks , all patients underwent physical examination by a physician and the routine laboratory tests to screen out any major medical disorders ."], ["t1 and t2 weighted mr images were obtained using 1.5 tesla siemens whole body scanner .", "repetition time [ tr]=2,050 ms , echo time [ te]=4.39 ms , inversion time [ ti]=1,100 ms , number of excitation [ nex]=2 , flip angle [ fa]=15 , slice thickness=1.3 mm , field of view [ fov]=180180 mm , acquisition matrix=256180 . t1 weighted images of 7 subjects were obtained with slightly different image acquisition protocol ( tr=1,960 ms , te=4.38 ms , ti=1,100 ms , nex=2 , flip angle [ fa]=15 , slice thickness=1.5 mm , fov=250 x 250 , acquisition matrix=256 x 180 ) .", "t2 weighted images were acquired in order to screen for gross brain abnormality ( tr=9,710 ms , te=120 ms , nex=2 , flip angle [ fa]=170 , slice thickness=3 mm ) .", "smoothing processes were conducted using an iterative nearest - neighbor averaging procedure with the full - width half maximum ( fwhm ) 15 mm 2-d gaussian kernel ."], ["for calculating statistical difference maps of cortical thickness between alcohol dependence and healthy comparison groups , general linear model ( glm ) with cortical thickness at each vertex as the dependent variable has been used .", "non - cortical areas of medial wall and corpus callosum were excluded from the model building .", "hemispheric average cortical thickness was included in the model as a covariate since there was a significant difference in hemispheric average cortical thickness between groups and our aim was to identify the particularly vulnerable cortical regions beyond the global brain atrophy associated with alcohol dependence .", "5,000 permutation simulations have been performed with random group - label shuffling , with a threshold for a significant vertex of p<0.05 .", "clusters with the size that would pass the family - wise error rate correction were considered significant .", "thickness values of the surface point with highest z values ( local maxima ) within the cluster , where significant group differences of cortical thickness were found , were extracted for post hoc analyses .", "pair - wise correlations were used to test whether there were associations between the magnitude of cortical thickness deficits in patients with alcohol dependence and the duration of alcohol use . considering the relatively modest sample size , sensitivity analyses to rule out the possibility that the current results may be modulated by other confounding factors such as comorbid depression , anxiety symptoms and scan parameter difference were performed .", "local maxima thickness values within the cluster extracted as described above were subjected to linear regression models that included the scan parameter difference , the presence of comorbid depression as defined by 19 or more scores on the 17-item hamilton depression rating scale ( hdrs ) , or the presence of anxiety as defined by 40 or more scores on the state - trait anxiety inventory ( stai - t )   as additional covariates .", "computations were performed using stata version 11 ( stata corp . , college station , tx , usa ) ."], ["there were no significant differences in age and sex between diagnostic groups ( table 1 ) .", "patients with alcohol dependence drank alcohol more frequently and more heavily than healthy comparison subjects ( table 1 ) .", "patients with alcohol dependence had general cortical thinning ( left hemispheric average cortical thickness ( mm ) : 2.440.07 [ healthy comparison subjects ] vs 2.240.27 [ patients with alcohol dependence ] , t=3.35 , p=0.002 ) ( right hemispheric average cortical thickness ( mm ) : 2.450.07 [ healthy comparison subjects ] vs 2.250.23 [ patients with alcohol dependence ] , t=3.77 , p<0.001 ) . in order to identify regionally specific cortical deficits in patients with alcohol dependence compared to healthy comparison subjects , hemispheric average cortical thickness was added as a covariate in the whole brain vertex - wise analysis . in the glm model that includes age and average cortical thickness as covariates , significant cortical thickness deficits in patients with alcohol dependence , compared to healthy comparison subjects , were noted in the left superior frontal cortex , after correcting for multiple comparisons with the permutation method ( fig .", "1 ) ( cluster size=1489.5 mm ; number of vertices in the cluster=2,035 ; talairach coordinates = x [ -15.3 ] , y [ 61.8 ] , z [ 5.0 ] ; cluster p value=0.024 ) . given that different scanning parameter may influence on the cortical thickness variations , analysis was re - run with scan parameter as a covariate .", "when analysis was repeated covarying for comorbid depression that may be associated with thinner prefronto - temporal cortex , the diagnostic group effect remained significant ( p=0.001 ) . when analysis was repeated covarying for comorbid anxiety that may also be associated with thinner prefronto - temporal cortex , the diagnostic group effect remained significant in the left superior frontal cluster ( p=0.017 ) .", "post hoc correlation analysis between cortical thicknesses in the left superior frontal cluster of significant group difference and duration of alcohol use in alcohol dependence group demonstrated the significant association ( r=-0.55 ; p=0.02 ) ( fig .", "cortical thicknesses in the left superior frontal cluster was also correlated with the clinical institute withdrawal assessment for alcohol scores ( r=-0.548 , p=0.028 ) .", "otherwise we found no significant results between cortical thicknesses in the left superior frontal cluster and audit scores ( r=0.048 , p=0.859 ) ; and calculated alcohol use ( alcohol dose x frequency x duration ) ( r=-0.193 , p=0.509 ) ."], ["in the current study , we have identified the brain region with altered cortical thickness in patients with alcohol dependence .", "the region of cortical thickness deficits in patients with alcohol dependence encompassed primarily the superior frontal cortex , after adjusting for the effects on the global cortical atrophy induced by alcohol dependence ( fig .", "this is consistent with prior reports indicating that chronic alcohol use may have the most detrimental effects on prefrontal cortical regions .", "the level of n - acetyl aspartate , the viability marker of neurons , was decreased in prefrontal brain regions in chronic alcohol abusers .", "neuronal and glial loss has consistently been noted in the prefrontal cortical regions , particularly in the superior frontal cortex .", "alcohol use has been associated with the decreased performances in executive and attention tasks that are important function of the prefrontal cortex .", "pre - existing vulnerability in these regions may predispose individuals to alcohol dependence , since the function of these regions are to executively control over drug craving and seeking .", "these regions may be associated with the compulsive substance - related behaviors , which is also in line with our finding that shows the association between duration of alcohol use with the magnitude of the cortical deficits in this region .", "the current findings do not provide information regarding whether the pre - existing prefrontal cortical deficits have rendered individuals vulnerable to alcohol dependence , whether neurotoxicity of chronic alcohol use , including the oxidative stress , have damaged the cortex , or whether both processes have contributed to the observed findings . supporting evidence for", "the neurotoxicity as the cause of prefrontal deficits may come from the reports that show patients with longer abstinence have lesser deficits .", "it has also been suggested that subjects with alcohol dependence may have prefrontal cortex that are less recuperative from toxic effects and may undergo vicious cycle after initial exposure to substances .", "considering that alcohol dependence can be divided into two subtypes , type 1 more environmentally influenced , and type 2 more genetically induced , a comparison between type 1 and type 2 alcohol dependent patients in a study with larger sample with balanced composition of type 1 and type 2 alcohol dependence may provide an opportunity to approach this question .", "longitudinal brain imaging study that follows up patients with alcohol dependence would also provide important insights .", "this study alone does not provide direct information as to cellular level mechanisms that may underlie the observed deficits of the cortical thickness .", "however , there is a vast literature describing the impact of chronic alcohol on the brain .", "miguel - hidalgo and colleagues ( 2002 ) reported , in their study with the postmortem brain of alcohol - dependent patients without wernicke or korsakoff syndromes , that the glial pathology of reduced size and density was the most characteristic finding .", "kril and colleagues ( 1997 ) have shown selective loss of non - gabaergic pyramidal neurons .", "selective dendritic retraction , rather than cellular death , has been suggested as main pathology related to the volume loss of the gray matter .", "there is a study that examined the cortical thickness differences in subjects with fetal alcohol syndrome or prenatal alcohol exposure , compared to control subjects , which demonstrated deficits in prefronto - temporo - parietal regions .", "however , few studies have examined cortical thickness in alcohol - dependent adults . the cortical thickness analysis has been reported to provide information on an important aspect of the gray matter structure , complementary to the conventional volumetry or the vbm .", "for example , highly folded regions could have high gray matter density in vbm analysis since there would be more voxels of gray matter within a fixed radius , but thinner cortical thickness .", "this is the first study that used cortical thickness analysis in patients with alcohol dependence excluding comorbid substance use , which showed regionally specific cortical thickness deficits in alcohol dependence .", "large age range , though covaried in the statistical model , can be another weakness of the study . with the current study of a cross - sectional design , information on whether the observed cortical deficits would be progressive and", "nicotine dependence , which is highly comorbid with alcohol dependence , has also been reported to be associated with cortical atrophy , particularly that in frontal cortex .", "now that we neither were able to exclude participants with co - morbid nicotine dependence nor delineate the effects of alcohol dependence from those of nicotine dependence , the current results may have been confounded by the effects of chronic nicotine use . intellectual ability has also been reported to be associated with the prefrontal cortical development .", "the fact that the intelligence quotient was not included in the statistical model for comparisons of cortical thickness measures , is also an important limitation of the current study .", "future studies in larger samples with narrow age range and with neuropsychological assessments for the frontal lobe function , would provide valuable information . although there could be challenges in following up patients with alcohol dependence , long - term prospective longitudinal study is also warranted ."]]}
{"article_id": "PMC2882589", "article_text": ["nutrient intakes , health behaviors and quality of life are interrelated and are of particular importance among rural residents . rural areas exhibit a variety of dietary habits and food - intake patterns ( lee et al . , 2002 ;", "ministry of health & welfare , 2002 ) with food intake and food selection being lower , consequently nutrient intakes being lower than urban areas .", "dietary diversity is a fundamental concept of dietary guidance internationally ( katanoda et al . , 2006 ; kennedy , 2004 ; who , 1996 ) , since increasing the diversity of foods is thought to ensure an adequate intake of essential nutrients and to promote good health ( ruel , 2003 ) .", "a low dietary diversity is a particularly severe problem among vulnerable populations of the poor ( ruel , 2003 ) and elderly ( bernstein et al . , 2002 ;", "adequate nutrient intake is essential to health promotion . hence increasing nutrient intakes as well as", "the dietary diversity of rural koreans is a potential way to improve their nutritional , and consequently , their health status and quality of life .", "( 1987 ) reported that variety among and within major food groups was associated with diet quality assessed by the mean adequacy ratio ( mar ) of 11 nutrients .", "evaluating the dietary diversity score ( dds ) and mar are , therefore , a good way to predict quality of diet and the entirety of dietary habits . in this study , we examined the dietary intakes and health behaviors of asan residents in order to improve health status and consequently the quality of life of asan residents , especially those who live in a rural area .", "asan is a medium - sized city with the unique feature with three distinctive regions since there are some areas with various kinds of industrial factories such as semi - conductor factories or automobile , air - conditioner and refrigerator manufacturing factories . additionally , other areas are mostly composed of rice farms or other kinds of rural farms in the boundaries of asan .", "some areas with a largely populated urban area ( on - yang si ) are also within the district of asan .", "therefore , asan can be easily differentiated into three distinctive regions of rural , factory and urban areas .", "the purpose of this study was to examine and compare nutrient intake , dds , mar and health behaviors of asan residents by the three distinctive regions .", "volunteers were recruited from residents of asan during january 2003 and the purposes and content of the study were explained to all participants , who provided their written , informed consent .", "a total of 930 subjects ( 351 men and 579 women ) , aged 61.4  13.8 ( mean  sd ; range 20 - 96 ) years correctly completed a 24-hour recall interview and were finally selected as participants .", "information on individual characteristics such as demography and socioeconomic status and health related behaviors was obtained using pre - coded questionnaires .", "dietary - intake data using a one - day 24-hour recall method were obtained by one - to - one interviews from the 5 to the 26 of january 2003 .", "detailed descriptions of all foods and beverages consumed and estimated food portion sizes were recorded by trained interviewers using food models , standard household measures , and full - sized colored photographs as memory aids .", "food records were converted to nutrient intakes using a computerized nutrient analysis program ( can - pro , v2.0 , the korean society of nutrition , seoul , korea ) .", "all nutrient intakes were adjusted for total energy intake in order to minimize extraneous errors in estimating dietary intake due to individual differences in total food intake .", "the energy - adjusted nutrient intake for each individual was computed by taking the residual from the regression model in which total caloric intake was the independent variable and the observed nutrient intake was the dependent variable , plus a constant equal to the expected intake of the nutrient for the mean caloric intake of the study population ( willett , 1990 ) .", "dietary diversity score ( dds ) of each individual was calculated from quantidd , which is the proportion of 17 food groups ( cereals or grains , potato and its products , simple carbohydrates or sugars , beans and its products , nuts or seeds , vegetables and their products , mushrooms , fruits and their products , meat or pork or poultry , eggs and the products , fish or shellfish and their products , seaweed and its products , milk and dairy products , fats or oils , beverages , seasonings and others ) contributing to the total amount of food intake after all the food ingredients from mixed dishes were decomposed .", "information on the 17 food groups to calculate dds from quantidd is available elsewhere ( katanoda et al . , 2006 ) .", "the mean adequate ratio ( mar ) is a mean nutrient adequate ratio ( nar ) of 12 nutrients ( protein , vitamins a , c , e and b6 , thiamin , riboflavin , niacin , folic acid , calcium , iron and zinc ) and nar of each nutrient was calculated by dividing actual nutrient intakes by recommended intakes ( ri ) of kdri ( dietary reference intakes for koreans , korean nutrition society , 2005 ) .", "nutrients without ri values defined in kdri , such as energy , fat , carbohydrate , sodium , fiber and cholesterol , were excluded in the nar calculation .", "differences among regions were assessed by anova and tukey 's post - hoc analysis for continuous variables and by the  test for categorical variables . since regions showed different social economic status ( ses ) such as income and education levels and age distributions were also different by regions , analysis of covariance ( ancova )", "was conducted to detect true differences among regions after controlling age and income levels as covariates .", "volunteers were recruited from residents of asan during january 2003 and the purposes and content of the study were explained to all participants , who provided their written , informed consent .", "a total of 930 subjects ( 351 men and 579 women ) , aged 61.4  13.8 ( mean  sd ; range 20 - 96 ) years correctly completed a 24-hour recall interview and were finally selected as participants .", "information on individual characteristics such as demography and socioeconomic status and health related behaviors was obtained using pre - coded questionnaires .", "dietary - intake data using a one - day 24-hour recall method were obtained by one - to - one interviews from the 5 to the 26 of january 2003 .", "detailed descriptions of all foods and beverages consumed and estimated food portion sizes were recorded by trained interviewers using food models , standard household measures , and full - sized colored photographs as memory aids .", "food records were converted to nutrient intakes using a computerized nutrient analysis program ( can - pro , v2.0 , the korean society of nutrition , seoul , korea ) .", "all nutrient intakes were adjusted for total energy intake in order to minimize extraneous errors in estimating dietary intake due to individual differences in total food intake .", "the energy - adjusted nutrient intake for each individual was computed by taking the residual from the regression model in which total caloric intake was the independent variable and the observed nutrient intake was the dependent variable , plus a constant equal to the expected intake of the nutrient for the mean caloric intake of the study population ( willett , 1990 ) . dietary diversity score ( dds ) of each individual was calculated from quantidd , which is the proportion of 17 food groups ( cereals or grains , potato and its products , simple carbohydrates or sugars , beans and its products , nuts or seeds , vegetables and their products , mushrooms , fruits and their products , meat or pork or poultry , eggs and the products , fish or shellfish and their products , seaweed and its products , milk and dairy products , fats or oils , beverages , seasonings and others ) contributing to the total amount of food intake after all the food ingredients from mixed dishes were decomposed .", "information on the 17 food groups to calculate dds from quantidd is available elsewhere ( katanoda et al . , 2006 ) .", "the mean adequate ratio ( mar ) is a mean nutrient adequate ratio ( nar ) of 12 nutrients ( protein , vitamins a , c , e and b6 , thiamin , riboflavin , niacin , folic acid , calcium , iron and zinc ) and nar of each nutrient was calculated by dividing actual nutrient intakes by recommended intakes ( ri ) of kdri ( dietary reference intakes for koreans , korean nutrition society , 2005 ) .", "nutrients without ri values defined in kdri , such as energy , fat , carbohydrate , sodium , fiber and cholesterol , were excluded in the nar calculation .", "differences among regions were assessed by anova and tukey 's post - hoc analysis for continuous variables and by the  test for categorical variables . since regions showed different social economic status ( ses ) such as income and education levels and age distributions were also different by regions , analysis of covariance ( ancova )", "was conducted to detect true differences among regions after controlling age and income levels as covariates .", "the fundamental characteristics of the participants and comparisons by regions are presented in table 1 . for the total participants , mean age was 61.4  13.8 years , with the men being older ( 64.8  11.7 years ;", "p<0.001 ) than the women ( 59.3  14.6 ) . for the men , factory area residents were significantly older than those living in other areas whilst for the women , urban residents were significantly younger than those living in other areas .", "the education levels were significantly different by regions ( p<0.001 ) , with more college graduates in the urban area .", "forty - five percent of the participants earned less than 500,000 won per month , which is less than half the korean average wage .", "current drinkers were significantly more in the urban area while never - drinkers were more in the rural or factory areas ( p<0.001 ) .", "however , proportions of current , past and never smokers were not significantly different by regions .", "physical activity levels were significantly higher in rural or factory areas than in the urban area ( p<0.001 ) , whereas urban residents exercised more frequently than both rural and factory area residents ( p<0.001 ) .", "there were more people who consider themselves less healthy than others with similar age in the rural and factory areas ( p<0.001 ) .", "on the other hand , more urban residents felt more stress than the rural or factory area residents ( p<0.001 ) .", "energy intakes of the participants were 1679  523 kcal for men and 1467  436 kcal for women ( table 3 ) .", "energy intake of the total participants was higher in urban area than those in the rural or factory area , but after ses variables such as age and income levels were controlled as covariates in ancova model , regional differences were no longer significant .", "meanwhile , energy intake of women in the rural area was significantly lower than that in the urban area residents and the results were not different after ses variables were controlled .", "the energy - adjusted intakes of protein , riboflavin and calcium were lower in the rural area than in factory or urban areas for the total participants and after adjustment of ses variables , the results showed same significant differences ( p<0.05 ) .", "energy - adjusted intakes of fat , vitamin c , and thiamin were significantly higher in the urban area than in the other two areas ( p<0.05 ) by anova , but ancova models did not show any significant differences . however , for vitamin e and cholesterol , both anova and ancova models showed same results of significantly higher intake in the urban area than in the other two areas ( p<0.05 ) .", "vitamin a intakes were significantly different among all three regions , being lowest in the rural area , significantly higher in the factory area and highest in the urban area by both anova and ancova ( p<0.05 ) .", "zinc intake of the factory area residents was significantly higher than that of the urban area by anova ( p<0.05 ) , but by ancova , the difference was no longer significant .", "carbohydrate , niacin , folic acid , vitamin b6 , iron , sodium and fiber intakes did not show any regional differences by both anova and ancova models .", "the result of sodium intake after adjustment of ses variables , however , showed highest intake in the factory area .", "just for the male participants , differences of fat , vitamin c , vitamin e , thiamin , riboflavin , iron and cholesterol intakes were not significant among regions while protein , vitamin a and calcium intakes were higher in the urban area and lower in the rural area .", "after ses variables were controlled in ancova model for male participants , protein , vitamin a and calcium intakes remained lowest in the rural area .", "for the women , regional differences of energy - adjusted nutrient intakes were similar to those of the total participants except for protein and niacin by both anova and ancova .", "energy - adjusted protein and niacin intakes of the women were higher in the factory area ( p<0.05 ) .", "sodium intake also showed significantly higher intake in the factory area after adjustment with ses as the case of the total participants .", "1 presents dds of the participants by regions and by sex before and after adjustment with age , income and energy intake levels .", "dds were higher in the urban area for total participants and women ( p<0.05 ) , but for men , dds was not different by regions before ses variables and energy were adjusted .", "only male showed different dds ( higher in the factory area ) while total and female participants did not show significant differences .", "2 shows mar of the participants by regions and by sex before and after adjustment with age , income and energy intake levels .", "however , mars of rural participants were lowest for total , men and women after adjustment with age , income and energy intakes ( p<0.05 ) .", "previous studies showed that selected health outcomes are dependent on adequate nutrient intakes , which rely highly on dietary quality ( foote et al .", ", 2004 ; haines et al . , 1999 ; kim et al . , 2003 ) , dietary diversity ( guthrie & scheer , 1981 ; hatloy et al . , 1998 ; kant et al . , 1991 , 1993 ; krebs - smith et al . , 1987 ) or both parameters ( bernstein et al . , 2002 ; drewnowski et al", "diet quality varies widely depending on the target population , since dietary habits are complex in nature and influenced by many factors .", "the korean rural population exhibits a distinct pattern of food choices ( ministry of health and welfare , 2002 ) and korea shows a unique pattern in dietary intake in terms of a lower proportion of energy from fat compared to both developed and developing countries ( lee et al . , 2002 ) . in this study , we described the nutrient intakes , dietary diversity ( dds ) , dietary quality ( mar ) and health related behaviors of asan residents .", "this population is unique since asan has regionally unique features so that the results of the current study may not be applicable to others residing in urban communities or geographical locations .", "our results showed that social economic status ( ses ) of asan residents was significantly different by different regions .", "as korea becomes industrialized , urbanization is more prompt and the tendency of urbanization is not different in asan .", "the younger urban resident showed higher education and income levels . about 50% of the participants of the present study were older than 65 years , and their education and income levels were lower than the korean averages .", "sixty - four percent of the total participants were educated up to the elementary school level only , with this increasing to 88% among those who are older than 65 years ( data not shown ) .", "therefore , the general ses of asan residents is lower than the korean average , with urban residents being the youngest and highest in education and income levels compared to rural or factory areas .", "rural and factory area residents did not show much difference in education and income levels although there were more elderly residents in the factory area .", "the energy intake and quantitative dietary diversity for the one - day diets was not high in this population even though all foods from mixed dishes were included after decomposition .", "only urban women showed the similar energy intake level compared to older women ( aged from 50 to 64 y ) living in big cities ( chung et al . , 2005 ) .", "however , diet quality ( mar ) of urban residents went up to 1.00 while other area residents showed mar being 0.86 for rural and 0.95 for factory residents .", "the difference of mar became significant for all participants regardless of sex after the ses factors were controlled .", "therefore , the true regional differences in quality of diet exist in the population of asan , and public health strategies to improve diet quality of rural residents should be considered .", "the proportion of carbohydrate contributing to energy intake was very high ( 69% , data not shown ) and that of fat was very low ( 15% vs. 19% korean national average ; ministry of health and welfare , 2002 ) .", "this high proportion of energy intake from carbohydrate may have limited the selection of foods from other groups in our population .", "the 1998 korean national health and nutrition survey found that the 30 most - consumed foods were similar in all areas except for rural areas , where three different kinds of kimchi were included in the top-10 most - consumed foods ( ministry of health and welfare , 2002 ) .", "many participants in the present study , especially older women from the rural area , reported that they consumed rice and kimchi almost exclusively , with few animal products and fresh fruits ( data not shown ) . the limited food choices resulting in a low dietary diversity", "may be related to the insufficient intake of essential nutrients in the korean rural population including the rural and factory area participants of this study . especially ,", "after the ses factors were controlled , significantly low dds of rural men reflect the poor quality of diet among this population .", "the current major concerns in korea as in many other countries are over nutrition and excess intakes of certain nutrients and foods rather than under nutrition ( ministry of health and welfare , 2002 ) .", "however , our results demonstrated that insufficient intakes of essential nutrients such as protein , vitamin a and e , riboflavin and calcium are still a problem in the rural area of asan even after ses was controlled .", "therefore , korean nutrition concerns should focus on dietary quality including both the concepts of nutrient deficiency and over nutrition . especially , significantly low dietary diversity of rural men and low quality measured with mar of rural men and women are of particular concern", "other health behaviors such as drinking , physical activity and perceived stress levels were much better among rural or factory residents compared to the urban residents , although exercise habits and perceived health were better in urban residents .", "( 2005 ) with korean national data also reported that self - rated usual activity showed higher in the rural area while higher exercise rate in the urban areas .", "therefore , the urban area residents of asan showed better nutrient intakes , dietary quality and exercise habits , which may result in the better perceived health compared to the rural or factory area residents of asan .", "the rural or factory area residents of asan showed much less perceived stress , however , even though nutrient intakes and dietary quality were not as good as urban residents .", "urban residents reported higher perceived stress even if they exercised more , ate better diets and showed better perceived health . in conclusion ,", "ses and quality of life measured by diet intakes and perceived health were lower in the rural area residents of asan .", "therefore , in order to achieve better quality of life and health status of the rural residents of asan , improving diet quality is essential . for the urban resident , however , despite of higher ses and diet quality , their perceived stress was higher .", "therefore , health promotion education to reduce the stress levels for urban residents of asan should be recommended ."], "abstract_text": ["<S> inadequate dietary intakes and poor health behaviors are of concern among rural residents in korea . </S>", "<S> this study is conducted to compare dietary intakes , dietary diversity score ( dds ) , mean nutrient adequacy ratio ( mar ) and health related behaviors by rural , factory and urban areas in asan . </S>", "<S> a total of 930 adults ( 351 men and 579 women ) were interviewed to assess social economic status ( ses ) , health related behaviors and food intakes by a 24-hour recall method . </S>", "<S> mean age was 61.5 years with men being older ( 64.8 years ) than women ( 59.3 years , p<0.001 ) . </S>", "<S> men in the factory area were older than rural or urban men while urban women were the youngest . </S>", "<S> education and income of urban residents were higher than other area residents . </S>", "<S> there were more current drinkers in urban area while smoking status was not different by regions . </S>", "<S> physical activity was significantly higher in rural or factory areas , whilst urban residents exercised more often ( p<0.05 ) . </S>", "<S> rural or factory area residents considered themselves less healthy than others while perceived stress was lower than urban residents . </S>", "<S> energy intakes were higher in urban residents or in men , however , after ses was controlled , energy intake did not show any differences . </S>", "<S> energy - adjusted nutrient intakes were significantly higher in the urban area ( p<0.05 ) for most nutrients except for carbohydrate , niacin , folic acid , vitamin b6 , iron and fiber . </S>", "<S> sodium intake was higher in factory area than in other areas after ses was controlled . </S>", "<S> dds of rural men and mar of both men and women in the rural area were significantly lower when ses was controlled . in conclusion , dietary intakes , diversity , adequacy and perceived health were poor in the rural area , although other health behaviors such as drinking and perceived stress were better than in the urban area . </S>", "<S> in order to improve perceived health of rural residents , good nutrition and exercise education programs are recommended . </S>"], "labels": null, "section_names": ["Introduction", "Subjects and Methods", "Subjects", "Data collection", "Dietary intakes", "Statistics", "Results", "Discussion"], "sections": [["nutrient intakes , health behaviors and quality of life are interrelated and are of particular importance among rural residents . rural areas exhibit a variety of dietary habits and food - intake patterns ( lee et al . , 2002 ;", "ministry of health & welfare , 2002 ) with food intake and food selection being lower , consequently nutrient intakes being lower than urban areas .", "dietary diversity is a fundamental concept of dietary guidance internationally ( katanoda et al . , 2006 ; kennedy , 2004 ; who , 1996 ) , since increasing the diversity of foods is thought to ensure an adequate intake of essential nutrients and to promote good health ( ruel , 2003 ) .", "a low dietary diversity is a particularly severe problem among vulnerable populations of the poor ( ruel , 2003 ) and elderly ( bernstein et al . , 2002 ;", "adequate nutrient intake is essential to health promotion . hence increasing nutrient intakes as well as", "the dietary diversity of rural koreans is a potential way to improve their nutritional , and consequently , their health status and quality of life .", "( 1987 ) reported that variety among and within major food groups was associated with diet quality assessed by the mean adequacy ratio ( mar ) of 11 nutrients .", "evaluating the dietary diversity score ( dds ) and mar are , therefore , a good way to predict quality of diet and the entirety of dietary habits . in this study , we examined the dietary intakes and health behaviors of asan residents in order to improve health status and consequently the quality of life of asan residents , especially those who live in a rural area .", "asan is a medium - sized city with the unique feature with three distinctive regions since there are some areas with various kinds of industrial factories such as semi - conductor factories or automobile , air - conditioner and refrigerator manufacturing factories . additionally , other areas are mostly composed of rice farms or other kinds of rural farms in the boundaries of asan .", "some areas with a largely populated urban area ( on - yang si ) are also within the district of asan .", "therefore , asan can be easily differentiated into three distinctive regions of rural , factory and urban areas .", "the purpose of this study was to examine and compare nutrient intake , dds , mar and health behaviors of asan residents by the three distinctive regions ."], ["volunteers were recruited from residents of asan during january 2003 and the purposes and content of the study were explained to all participants , who provided their written , informed consent .", "a total of 930 subjects ( 351 men and 579 women ) , aged 61.4  13.8 ( mean  sd ; range 20 - 96 ) years correctly completed a 24-hour recall interview and were finally selected as participants .", "information on individual characteristics such as demography and socioeconomic status and health related behaviors was obtained using pre - coded questionnaires .", "dietary - intake data using a one - day 24-hour recall method were obtained by one - to - one interviews from the 5 to the 26 of january 2003 .", "detailed descriptions of all foods and beverages consumed and estimated food portion sizes were recorded by trained interviewers using food models , standard household measures , and full - sized colored photographs as memory aids .", "food records were converted to nutrient intakes using a computerized nutrient analysis program ( can - pro , v2.0 , the korean society of nutrition , seoul , korea ) .", "all nutrient intakes were adjusted for total energy intake in order to minimize extraneous errors in estimating dietary intake due to individual differences in total food intake .", "the energy - adjusted nutrient intake for each individual was computed by taking the residual from the regression model in which total caloric intake was the independent variable and the observed nutrient intake was the dependent variable , plus a constant equal to the expected intake of the nutrient for the mean caloric intake of the study population ( willett , 1990 ) .", "dietary diversity score ( dds ) of each individual was calculated from quantidd , which is the proportion of 17 food groups ( cereals or grains , potato and its products , simple carbohydrates or sugars , beans and its products , nuts or seeds , vegetables and their products , mushrooms , fruits and their products , meat or pork or poultry , eggs and the products , fish or shellfish and their products , seaweed and its products , milk and dairy products , fats or oils , beverages , seasonings and others ) contributing to the total amount of food intake after all the food ingredients from mixed dishes were decomposed .", "information on the 17 food groups to calculate dds from quantidd is available elsewhere ( katanoda et al . , 2006 ) .", "the mean adequate ratio ( mar ) is a mean nutrient adequate ratio ( nar ) of 12 nutrients ( protein , vitamins a , c , e and b6 , thiamin , riboflavin , niacin , folic acid , calcium , iron and zinc ) and nar of each nutrient was calculated by dividing actual nutrient intakes by recommended intakes ( ri ) of kdri ( dietary reference intakes for koreans , korean nutrition society , 2005 ) .", "nutrients without ri values defined in kdri , such as energy , fat , carbohydrate , sodium , fiber and cholesterol , were excluded in the nar calculation .", "differences among regions were assessed by anova and tukey 's post - hoc analysis for continuous variables and by the  test for categorical variables . since regions showed different social economic status ( ses ) such as income and education levels and age distributions were also different by regions , analysis of covariance ( ancova )", "was conducted to detect true differences among regions after controlling age and income levels as covariates ."], ["volunteers were recruited from residents of asan during january 2003 and the purposes and content of the study were explained to all participants , who provided their written , informed consent .", "a total of 930 subjects ( 351 men and 579 women ) , aged 61.4  13.8 ( mean  sd ; range 20 - 96 ) years correctly completed a 24-hour recall interview and were finally selected as participants ."], ["information on individual characteristics such as demography and socioeconomic status and health related behaviors was obtained using pre - coded questionnaires .", "dietary - intake data using a one - day 24-hour recall method were obtained by one - to - one interviews from the 5 to the 26 of january 2003 .", "detailed descriptions of all foods and beverages consumed and estimated food portion sizes were recorded by trained interviewers using food models , standard household measures , and full - sized colored photographs as memory aids ."], ["food records were converted to nutrient intakes using a computerized nutrient analysis program ( can - pro , v2.0 , the korean society of nutrition , seoul , korea ) .", "all nutrient intakes were adjusted for total energy intake in order to minimize extraneous errors in estimating dietary intake due to individual differences in total food intake .", "the energy - adjusted nutrient intake for each individual was computed by taking the residual from the regression model in which total caloric intake was the independent variable and the observed nutrient intake was the dependent variable , plus a constant equal to the expected intake of the nutrient for the mean caloric intake of the study population ( willett , 1990 ) . dietary diversity score ( dds ) of each individual was calculated from quantidd , which is the proportion of 17 food groups ( cereals or grains , potato and its products , simple carbohydrates or sugars , beans and its products , nuts or seeds , vegetables and their products , mushrooms , fruits and their products , meat or pork or poultry , eggs and the products , fish or shellfish and their products , seaweed and its products , milk and dairy products , fats or oils , beverages , seasonings and others ) contributing to the total amount of food intake after all the food ingredients from mixed dishes were decomposed .", "information on the 17 food groups to calculate dds from quantidd is available elsewhere ( katanoda et al . , 2006 ) .", "the mean adequate ratio ( mar ) is a mean nutrient adequate ratio ( nar ) of 12 nutrients ( protein , vitamins a , c , e and b6 , thiamin , riboflavin , niacin , folic acid , calcium , iron and zinc ) and nar of each nutrient was calculated by dividing actual nutrient intakes by recommended intakes ( ri ) of kdri ( dietary reference intakes for koreans , korean nutrition society , 2005 ) .", "nutrients without ri values defined in kdri , such as energy , fat , carbohydrate , sodium , fiber and cholesterol , were excluded in the nar calculation ."], ["differences among regions were assessed by anova and tukey 's post - hoc analysis for continuous variables and by the  test for categorical variables . since regions showed different social economic status ( ses ) such as income and education levels and age distributions were also different by regions , analysis of covariance ( ancova )", "was conducted to detect true differences among regions after controlling age and income levels as covariates ."], ["the fundamental characteristics of the participants and comparisons by regions are presented in table 1 . for the total participants , mean age was 61.4  13.8 years , with the men being older ( 64.8  11.7 years ;", "p<0.001 ) than the women ( 59.3  14.6 ) . for the men , factory area residents were significantly older than those living in other areas whilst for the women , urban residents were significantly younger than those living in other areas .", "the education levels were significantly different by regions ( p<0.001 ) , with more college graduates in the urban area .", "forty - five percent of the participants earned less than 500,000 won per month , which is less than half the korean average wage .", "current drinkers were significantly more in the urban area while never - drinkers were more in the rural or factory areas ( p<0.001 ) .", "however , proportions of current , past and never smokers were not significantly different by regions .", "physical activity levels were significantly higher in rural or factory areas than in the urban area ( p<0.001 ) , whereas urban residents exercised more frequently than both rural and factory area residents ( p<0.001 ) .", "there were more people who consider themselves less healthy than others with similar age in the rural and factory areas ( p<0.001 ) .", "on the other hand , more urban residents felt more stress than the rural or factory area residents ( p<0.001 ) .", "energy intakes of the participants were 1679  523 kcal for men and 1467  436 kcal for women ( table 3 ) .", "energy intake of the total participants was higher in urban area than those in the rural or factory area , but after ses variables such as age and income levels were controlled as covariates in ancova model , regional differences were no longer significant .", "meanwhile , energy intake of women in the rural area was significantly lower than that in the urban area residents and the results were not different after ses variables were controlled .", "the energy - adjusted intakes of protein , riboflavin and calcium were lower in the rural area than in factory or urban areas for the total participants and after adjustment of ses variables , the results showed same significant differences ( p<0.05 ) .", "energy - adjusted intakes of fat , vitamin c , and thiamin were significantly higher in the urban area than in the other two areas ( p<0.05 ) by anova , but ancova models did not show any significant differences . however , for vitamin e and cholesterol , both anova and ancova models showed same results of significantly higher intake in the urban area than in the other two areas ( p<0.05 ) .", "vitamin a intakes were significantly different among all three regions , being lowest in the rural area , significantly higher in the factory area and highest in the urban area by both anova and ancova ( p<0.05 ) .", "zinc intake of the factory area residents was significantly higher than that of the urban area by anova ( p<0.05 ) , but by ancova , the difference was no longer significant .", "carbohydrate , niacin , folic acid , vitamin b6 , iron , sodium and fiber intakes did not show any regional differences by both anova and ancova models .", "the result of sodium intake after adjustment of ses variables , however , showed highest intake in the factory area .", "just for the male participants , differences of fat , vitamin c , vitamin e , thiamin , riboflavin , iron and cholesterol intakes were not significant among regions while protein , vitamin a and calcium intakes were higher in the urban area and lower in the rural area .", "after ses variables were controlled in ancova model for male participants , protein , vitamin a and calcium intakes remained lowest in the rural area .", "for the women , regional differences of energy - adjusted nutrient intakes were similar to those of the total participants except for protein and niacin by both anova and ancova .", "energy - adjusted protein and niacin intakes of the women were higher in the factory area ( p<0.05 ) .", "sodium intake also showed significantly higher intake in the factory area after adjustment with ses as the case of the total participants .", "1 presents dds of the participants by regions and by sex before and after adjustment with age , income and energy intake levels .", "dds were higher in the urban area for total participants and women ( p<0.05 ) , but for men , dds was not different by regions before ses variables and energy were adjusted .", "only male showed different dds ( higher in the factory area ) while total and female participants did not show significant differences .", "2 shows mar of the participants by regions and by sex before and after adjustment with age , income and energy intake levels .", "however , mars of rural participants were lowest for total , men and women after adjustment with age , income and energy intakes ( p<0.05 ) ."], ["previous studies showed that selected health outcomes are dependent on adequate nutrient intakes , which rely highly on dietary quality ( foote et al .", ", 2004 ; haines et al . , 1999 ; kim et al . , 2003 ) , dietary diversity ( guthrie & scheer , 1981 ; hatloy et al . , 1998 ; kant et al . , 1991 , 1993 ; krebs - smith et al . , 1987 ) or both parameters ( bernstein et al . , 2002 ; drewnowski et al", "diet quality varies widely depending on the target population , since dietary habits are complex in nature and influenced by many factors .", "the korean rural population exhibits a distinct pattern of food choices ( ministry of health and welfare , 2002 ) and korea shows a unique pattern in dietary intake in terms of a lower proportion of energy from fat compared to both developed and developing countries ( lee et al . , 2002 ) . in this study , we described the nutrient intakes , dietary diversity ( dds ) , dietary quality ( mar ) and health related behaviors of asan residents .", "this population is unique since asan has regionally unique features so that the results of the current study may not be applicable to others residing in urban communities or geographical locations .", "our results showed that social economic status ( ses ) of asan residents was significantly different by different regions .", "as korea becomes industrialized , urbanization is more prompt and the tendency of urbanization is not different in asan .", "the younger urban resident showed higher education and income levels . about 50% of the participants of the present study were older than 65 years , and their education and income levels were lower than the korean averages .", "sixty - four percent of the total participants were educated up to the elementary school level only , with this increasing to 88% among those who are older than 65 years ( data not shown ) .", "therefore , the general ses of asan residents is lower than the korean average , with urban residents being the youngest and highest in education and income levels compared to rural or factory areas .", "rural and factory area residents did not show much difference in education and income levels although there were more elderly residents in the factory area .", "the energy intake and quantitative dietary diversity for the one - day diets was not high in this population even though all foods from mixed dishes were included after decomposition .", "only urban women showed the similar energy intake level compared to older women ( aged from 50 to 64 y ) living in big cities ( chung et al . , 2005 ) .", "however , diet quality ( mar ) of urban residents went up to 1.00 while other area residents showed mar being 0.86 for rural and 0.95 for factory residents .", "the difference of mar became significant for all participants regardless of sex after the ses factors were controlled .", "therefore , the true regional differences in quality of diet exist in the population of asan , and public health strategies to improve diet quality of rural residents should be considered .", "the proportion of carbohydrate contributing to energy intake was very high ( 69% , data not shown ) and that of fat was very low ( 15% vs. 19% korean national average ; ministry of health and welfare , 2002 ) .", "this high proportion of energy intake from carbohydrate may have limited the selection of foods from other groups in our population .", "the 1998 korean national health and nutrition survey found that the 30 most - consumed foods were similar in all areas except for rural areas , where three different kinds of kimchi were included in the top-10 most - consumed foods ( ministry of health and welfare , 2002 ) .", "many participants in the present study , especially older women from the rural area , reported that they consumed rice and kimchi almost exclusively , with few animal products and fresh fruits ( data not shown ) . the limited food choices resulting in a low dietary diversity", "may be related to the insufficient intake of essential nutrients in the korean rural population including the rural and factory area participants of this study . especially ,", "after the ses factors were controlled , significantly low dds of rural men reflect the poor quality of diet among this population .", "the current major concerns in korea as in many other countries are over nutrition and excess intakes of certain nutrients and foods rather than under nutrition ( ministry of health and welfare , 2002 ) .", "however , our results demonstrated that insufficient intakes of essential nutrients such as protein , vitamin a and e , riboflavin and calcium are still a problem in the rural area of asan even after ses was controlled .", "therefore , korean nutrition concerns should focus on dietary quality including both the concepts of nutrient deficiency and over nutrition . especially , significantly low dietary diversity of rural men and low quality measured with mar of rural men and women are of particular concern", "other health behaviors such as drinking , physical activity and perceived stress levels were much better among rural or factory residents compared to the urban residents , although exercise habits and perceived health were better in urban residents .", "( 2005 ) with korean national data also reported that self - rated usual activity showed higher in the rural area while higher exercise rate in the urban areas .", "therefore , the urban area residents of asan showed better nutrient intakes , dietary quality and exercise habits , which may result in the better perceived health compared to the rural or factory area residents of asan .", "the rural or factory area residents of asan showed much less perceived stress , however , even though nutrient intakes and dietary quality were not as good as urban residents .", "urban residents reported higher perceived stress even if they exercised more , ate better diets and showed better perceived health . in conclusion ,", "ses and quality of life measured by diet intakes and perceived health were lower in the rural area residents of asan .", "therefore , in order to achieve better quality of life and health status of the rural residents of asan , improving diet quality is essential . for the urban resident , however , despite of higher ses and diet quality , their perceived stress was higher .", "therefore , health promotion education to reduce the stress levels for urban residents of asan should be recommended ."]]}
{"article_id": "PMC2988931", "article_text": ["the treatment of chronic hepatitis c ( chc ) has evolved in the last 15 years from monotherapy with interferon alpha ( ifnalpha ) to the combination treatment with pegylated ifn ( peg - ifn ) plus ribavirin for 24 - 48 weeks . up to now , several viral , host and drug - related reactions in response to ifnalpha - based therapy have been identified .", "recent studies suggest that liver inflammation in chc is controlled by several mechanisms , including host regulatory immune responses and viral polypeptides interacting with cells involved in innate and adaptive immunity .", "it is well known that cutaneous side effects of treatment with ifnalpha alone or ifnalpha plus ribavirin in patients with chc have been widely reported , beyond the fact that the virus itself can cause skin lesions . however , the cutaneous side effects during therapy of chc are of inflammatory type with local erythema , edema and , much less frequently , necrosis at the injection skin sites . by contrast ,", "skin side effects of such drugs have few data available in the literature , although the number of reports has been increasing in the last years , including cosmetic filler site injections .", "roughly one half of these patients had cutaneous findings , either alone or with systemic involvement .", "more recently there have been reported dermatological diseases in patients receiving the combination of ifn / ribavirin or ifn / ribavirin / amantadine for the treatment of chc .", "a 44-year - old man with chc genotype 1a and a viral load of hcv > 800,000 iu / ml , a2-f1 on liver biopsy according to metavir criteria , was treated for the hepatic disease .", "peg - ifnalpha-2b 1.5 g / kg weekly and 1,000 mg of ribavirin daily was introduced . in the 40th week", "the patient reported sudden intense pain , pruritus , erythema and skin hypertrophy in the left deltoid area over the seahorse tattoo and in a scar on his face ( fig .", "1 ) . a skin biopsy on the scar and tattoo showed granulomatous dermatitis ( fig .", "chest x - ray was normal and the level of angiotensin enzyme converter was 50 iu / ml ( range 18 - 55 iu / ml ) .", "we intend to call attention especially to dermatologists to the possibility of a granulomatous tattoo reaction as a side effect during chc treatment with peg - ifnalpha-2b plus ribavirin . until now", ", eight cases of sarcoidosis as a complication of ifn therapy in chc have been reported in the english language literature .", "the most relevant topic in this case is the occurrence of disease on a tattoo area .", "six months later retreatment using peg - ifnalpha-2a 180 g once a week plus ribavirin 1,000 mg daily was tried . in the 6th week , cutaneous symptoms relapsed in an unbearable way .", "the patient did not accept to continue therapy , since hydrocortisone cream was prescribed again but no satisfactory pruritus and pain improvement was noticed . a prompt regression of skin reaction occurred after stopping therapy .", "sarcoidosis is a granulomatous disorder of unknown etiology and whose epidemiology suggest a genetic tendency face to infectious agents being supposed to result of immune system deregulation leading to non - caseating granulomas as an immune reaction to an unknown persistent antigenic stimulus .", "ifn has been linked to pulmonary macrophage activation , a characteristic feature of sarcoidosis which has been assumed an exaggerated t helper 1 ( th-1 ) immune response to a variety of exogenous antigens .", "it seems very likely that a potent immunoregulatory protein for th-1 response such as ifn may induce the disease . in january 2003 the eighth case of ifn - related sarcoidosis was reported with a review of the literature .", "chest x - ray revealed hilar lymphadenopathy in three patients , with reticulonodular shadows in another three , while the patient with only cutaneous involvement had a normal chest x - ray .", "the case we report presented with cutaneous sarcoidosis during chc therapy and spontaneous regression of the lesions was noted with treatment discontinuation .", "we intend to emphasize , especially to dermatologists , the risks of a granulomatous tattoo and other cutaneous sites during chc treatment with peg - ifnalpha-2b plus ribavirin .", "thalidomide has been also shown to have specific activity to the inflammatory mediators of sarcoidosis and to be an alternative beneficial therapy ."], "abstract_text": ["<S> the treatment of chronic hepatitis c ( chc ) has evolved in the past 15 years and combination of pegylated interferon plus ribavirin is its current standard therapy . however , several side effects are commonly observed and frequently lead to transient or definitive interruption of treatment . </S>", "<S> although sarcoidosis in its systemic or cutaneous form is a very rare side effect in such circumstances , some cases have been reported even with conventional interferon . </S>", "<S> this brief review of the literature and description of a case of sarcoidosis occurring in a tattoo and a scar patient 's face , during treatment with pegylated interferon alpha-2b plus ribavirin , is an educative report directed in special to dermatologists . </S>", "<S> the lesion improved after drug interruption and recurred after retreatment with pegylated interferon alpha-2a . </S>", "<S> we conclude that this side effect must call the attention of doctors to seek for the diagnosis and therapy as soon as possible in such circumstances . </S>", "<S> no differences were noticed neither with alpha-2a nor alpha-2b pegylated interferon employment . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["the treatment of chronic hepatitis c ( chc ) has evolved in the last 15 years from monotherapy with interferon alpha ( ifnalpha ) to the combination treatment with pegylated ifn ( peg - ifn ) plus ribavirin for 24 - 48 weeks . up to now , several viral , host and drug - related reactions in response to ifnalpha - based therapy have been identified .", "recent studies suggest that liver inflammation in chc is controlled by several mechanisms , including host regulatory immune responses and viral polypeptides interacting with cells involved in innate and adaptive immunity .", "it is well known that cutaneous side effects of treatment with ifnalpha alone or ifnalpha plus ribavirin in patients with chc have been widely reported , beyond the fact that the virus itself can cause skin lesions . however , the cutaneous side effects during therapy of chc are of inflammatory type with local erythema , edema and , much less frequently , necrosis at the injection skin sites . by contrast ,", "skin side effects of such drugs have few data available in the literature , although the number of reports has been increasing in the last years , including cosmetic filler site injections .", "roughly one half of these patients had cutaneous findings , either alone or with systemic involvement .", "more recently there have been reported dermatological diseases in patients receiving the combination of ifn / ribavirin or ifn / ribavirin / amantadine for the treatment of chc ."], ["a 44-year - old man with chc genotype 1a and a viral load of hcv > 800,000 iu / ml , a2-f1 on liver biopsy according to metavir criteria , was treated for the hepatic disease .", "peg - ifnalpha-2b 1.5 g / kg weekly and 1,000 mg of ribavirin daily was introduced . in the 40th week", "the patient reported sudden intense pain , pruritus , erythema and skin hypertrophy in the left deltoid area over the seahorse tattoo and in a scar on his face ( fig .", "1 ) . a skin biopsy on the scar and tattoo showed granulomatous dermatitis ( fig .", "chest x - ray was normal and the level of angiotensin enzyme converter was 50 iu / ml ( range 18 - 55 iu / ml ) .", "we intend to call attention especially to dermatologists to the possibility of a granulomatous tattoo reaction as a side effect during chc treatment with peg - ifnalpha-2b plus ribavirin . until now", ", eight cases of sarcoidosis as a complication of ifn therapy in chc have been reported in the english language literature .", "the most relevant topic in this case is the occurrence of disease on a tattoo area .", "six months later retreatment using peg - ifnalpha-2a 180 g once a week plus ribavirin 1,000 mg daily was tried . in the 6th week , cutaneous symptoms relapsed in an unbearable way .", "the patient did not accept to continue therapy , since hydrocortisone cream was prescribed again but no satisfactory pruritus and pain improvement was noticed . a prompt regression of skin reaction occurred after stopping therapy ."], ["sarcoidosis is a granulomatous disorder of unknown etiology and whose epidemiology suggest a genetic tendency face to infectious agents being supposed to result of immune system deregulation leading to non - caseating granulomas as an immune reaction to an unknown persistent antigenic stimulus .", "ifn has been linked to pulmonary macrophage activation , a characteristic feature of sarcoidosis which has been assumed an exaggerated t helper 1 ( th-1 ) immune response to a variety of exogenous antigens .", "it seems very likely that a potent immunoregulatory protein for th-1 response such as ifn may induce the disease . in january 2003 the eighth case of ifn - related sarcoidosis was reported with a review of the literature .", "chest x - ray revealed hilar lymphadenopathy in three patients , with reticulonodular shadows in another three , while the patient with only cutaneous involvement had a normal chest x - ray .", "the case we report presented with cutaneous sarcoidosis during chc therapy and spontaneous regression of the lesions was noted with treatment discontinuation .", "we intend to emphasize , especially to dermatologists , the risks of a granulomatous tattoo and other cutaneous sites during chc treatment with peg - ifnalpha-2b plus ribavirin .", "thalidomide has been also shown to have specific activity to the inflammatory mediators of sarcoidosis and to be an alternative beneficial therapy ."]]}
{"article_id": "PMC4939675", "article_text": ["pleomorphic carcinoma is an aggressive tumour initially described in the lung by nash and stout . in the 4th edition of the world health organization", "( who ) classification of tumours of the lung , pleomorphic carcinoma is defined as a poorly differentiated ( squamous cell carcinoma or adenocarcinoma ) or undifferentiated carcinoma in which at least 10% spindle and/or giant cells are identified , or as a carcinoma constituted purely of spindle and giant cells .", "primary sites of occurrence of extrapulmonary pleomorphic carcinomas have been described in other organs such as the stomach , thyroid , gallbladder , pancreas , kidney , liver , bladder , and colon . to the best of our knowledge , only one report described a case of giant cell carcinoma of the colon resembling that observed in the lung .", "based on the recommendations of the nomenclature committee on cell death , mitotic catastrophe is defined as a tumour - suppressive mechanism occurring during or after aberrant mitosis leading to cell death or cell senescence .", "histological patterns of micro- and multinucleation have been used as morphological markers for the detection of mitotic catastrophe [ 5 , 6 ] . a high frequency of multinucleated giant cells is characteristically found in pleomorphic carcinomas . in this report , we present an additional case of primary pleomorphic carcinoma of the colon , and we underline similarities to and differences from pulmonary pleomorphic carcinoma .", "the possibility of the pleomorphic component as morphological expression of mitotic catastrophe is finally discussed .", "a 65-year - old caucasian woman with a medical history of hypercholesterolaemia , chronic obstructive pulmonary disease , and paroxysmal tachycardia was admitted to the surgery unit of the university hospital  g. martino ( messina , italy ) in 2013 for abdominal obstructive symptoms ( colicky pain to the right iliac fossa as well as borborygmi ) , change in bowel habits ( diarrhoea ) , asthenia , and severe weight loss since 6 months .", "there was no nausea or vomiting . on physical examination , a globular and tympanic abdomen , painless at superficial and deep palpation , was evident .", "she reported a history of adequate food intake with regular bowel habits , alcohol consumption ( < 20/30 g daily ) , and smoking .", "there was no family history of gastrointestinal malignancy , although familiality for bladder carcinoma was reported .", "abdominal ultrasonography highlighted an exophytic / stenosing mass at the level of the ascending colon .", "afterwards , colonoscopy also revealed an exophytic / stenosing malignant neoplastic mass in the caecum , subsequently confirmed through histological evaluation of biopsy .", "carcinoembryonic antigen ( value for smokers < 10.0 ng / ml ) and carbohydrate antigen 19 - 9 ( value < 35 iu / ml ) were within normal limits .", "a contrast - enhanced computed tomography scan showed no evidence of liver or distant metastases .", "routine haematoxylin - eosin sections were made from formalin - fixed , paraffin - embedded tissue .", "sections were also stained with periodic acid - schiff and alcian blue at ph 2.5 .", "additional sections  collected on silanized , coated slides  were used for the immunohistochemical stains , using the dako envisiontm flex , high ph detection system together with autostainer instruments .", "the commercial source , clone , and dilution of the primary antibodies are detailed in table 1 .", "macroscopic examination of the resected colon revealed an endophytic / ulcerative lesion measuring 7 cm in its greatest dimension , invading the caecum nearly to the ileocaecal valve .", "microscopically , the tumour was a poorly differentiated adenocarcinoma with a grade 4 pleomorphic component that occupied up to 10% of the whole tumour section .", "the pleomorphic component showed trabeculae and nests of neoplastic cells possessing eosinophilic cytoplasm , as well as irregularly shaped vesicular nuclei with a single large eosinophilic nucleolus .", "giant cells containing multiple nuclei , micronuclei , and prominent eosinophil nucleoli were also found ( fig . ", "the micronuclei appeared as round chromatin fragments with a diameter less than one third of the diameter of the nucleus .", "1b ) and large geographical necrotic areas with sharp boundaries with respect to the viable tumour tissue ( fig . ", "only 1 out of the 44 harvested perivisceral lymph nodes presented carcinomatous metastasis ( pt3 , pn1 , clinical stage in accordance to the 2009 ptnm system ) .", "the histochemical stains were negative for periodic acid - schiff and alcian blue in all of the pleomorphic neoplastic cells .", "on the immunohistochemical stains , these cells were diffusely and strongly positive for ceap , ckae1/ae3 , ck7 , and vimentin , but they were negative for ck20 , smooth muscle actin , desmin , synaptophysin , and -human chorionic gonadotropin .", "2b ) , whereas p53 labelling was shown in more than 90% of the tumoural nuclei ( fig . ", "we described a rare case of pleomorphic carcinoma of the right colon showing opposing clinicopathological features . although it was classified as a grade 4 tumour , it was characterized by a pushing growth pattern and presented only one lymph node with metastasis .", "these data confirm that classic grading of colorectal carcinoma is insufficient to predict a prognosis , and that further parameters such as growth patterns are needed .", "accordingly , several recent studies have shown that colorectal carcinomas with pushing margins are associated with superior disease - free survival rates when compared with tumours with infiltrative margins .", "the present tumour had some of the morphological features described in giant cell carcinoma of the lung , such as pleomorphic tumour cells , malignant giant cells , atypical mitoses , and coagulative necrosis .", "however , pleomorphic carcinoma of the lung has been reported to have a poor prognosis , whereas in our case , the patient has been well without evidence of recurrence or metastases 2 years after the operation .", "many studies suggested that pleomorphic tumours are a heterogeneous group of neoplasms arising in different anatomical sites ( e.g. lung , uterus , breast , central nervous system ) characterized by variable morphological features and prognoses [ 8 , 9 , 10 , 11 ] .", "in particular , a certain percentage of patients affected by them were found to be long - term survivors despite the anaplastic morphology of their primary neoplasms [ 8 , 10 , 11 ] .", "the differential diagnosis of pleomorphic carcinoma includes adenocarcinoma with a choriocarcinomatous component as well as mixed adenoneuroendocrine carcinoma [ 7 , 12 , 13 ] .", "a choriocarcinomatous component usually is characterized by immunoreactivity to -human chorionic gonadotropin , which was not found in our case , while the latter was excluded by the lack of expression of neuroendocrine markers such as synaptophysin and chromogranin [ 12 , 13 ] . in our case , multinucleated tumour cells contained not only abnormally large giant nuclei but also those that are abnormally small , in the form of micronuclei .", "the presence of micronuclei is usually overlooked in the reports of pleomorphic carcinoma in the current literature .", "micronuclei derive from chromosomes and/or chromosome fragments that have been irregularly distributed between daughter nuclei after abnormal mitosis . besides micronucleation and multinucleation ,", "we interpret these findings as a morphological expression of mitotic catastrophe , a particular type of cell death occurring in tumour cells after aberrant mitosis [ 5 , 6 , 14 ] .", "after mitotic catastrophe , tumour cells may continue to divide and thus develop polyploidy and/or aneuploidy .", "ki-67 is an antigen of cell proliferation frequently used in routine histopathological diagnostics as a prognostic factor in more malignancies .", "specifically , it is an epitope of a nuclear and nucleolar protein of 360 kda only expressed in nuclei of cells in active proliferation , i.e. during cell cycle phases g1 , s , g2 , and m , but not in the quiescent phase , i.e. in cell cycle phase g0 [ 17 , 18 ]", ". normally , p53 acts as a guardian of the genome , protecting cells against cancer via two main ways : by determining cell cycle arrest at g1 and g2/m and by inducing cellular apoptosis [ 19 , 20 ] .", "nuclear p53 immunohistochemical positivity in tumour cells suggests mutation in the p53 gene [ 21 , 22 ] .", "thus , immunohistochemical overexpression of p53 and ki-67 constitutes further evidence of mitotic catastrophe in the pleomorphic component of our case of colonic neoplasm .", "ionizing radiation and different classes of cytotoxic agents induce cell death through mitotic catastrophe [ 15 , 18 ] .", "histological recognition of mitotic catastrophe could be useful to predict an eventual pharmacological modulation ( induction or inhibition ) of tumour cell death .", "therefore , mitotic catastrophe has recently gained attention as a potential therapeutic target in neoplasms [ 23 , 24 , 25 ] . in summary , we reported a case of colorectal carcinoma with clinicopathological features partially similar to pleomorphic cell carcinoma of the lung , a neoplasm associated with a bad prognosis . in particular , this tumour presented only one lymph node with metastasis , and the patient is still well 2 years after the operation .", "this case presented morphological and immunohistochemical features compatible with mitotic catastrophe , a form of non - apoptotic cell death due to aberrant mitosis .", "the inclusion of mitotic catastrophe as part of a microscopic evaluation may be useful for understanding the pathogenesis of this rare entity and for new cancer treatment modalities .", "no further ethical approval was necessary to perform histology and immunohistochemistry in the case included in this report ."], "abstract_text": ["<S> pleomorphic carcinoma is an aggressive neoplasm defined by the world health organization ( who ) as a poorly differentiated ( squamous cell carcinoma or adenocarcinoma ) or undifferentiated carcinoma in which at least 10% spindle and/or giant cells are identified , or as a carcinoma constituted purely of spindle and giant cells . </S>", "<S> although this entity has initially been shown in the lung , it has been described also in extrapulmonary locations , with only one report for a colonic site . a 65-year - old woman developed a caecal tumour . </S>", "<S> gross examination revealed an endophytic / ulcerative mass 7 cm in length . </S>", "<S> microscopically , the tumour was a poorly differentiated adenocarcinoma with a pleomorphic component that occupied more than 10% of the specimen . </S>", "<S> the tumour shared these histopathological findings with pulmonary giant cell carcinoma but differed in other clinicopathological features such as a pushing growth pattern , stage pt3n1 , and an uneventful outcome 24 months after operation . </S>", "<S> the pleomorphic component showed morphological and immunohistochemical features compatible with mitotic catastrophe , a non - apoptotic cell death occurring in cycling cells after aberrant mitosis . </S>", "<S> these features included multinucleation , micronucleation , atypical mitoses , foci of geographic necrosis , as well as immunohistochemical overexpression of p53 and ki-67 . </S>", "<S> the interpretation of the pleomorphic component as morphological expression of mitotic catastrophe may be useful in comprehending the pathogenesis of this rare neoplasm , and it may have practical implications as a potential cancer therapeutic target . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Materials and Methods", "Gross Pathology and Histopathology", "Discussion", "Statement of Ethics", "Disclosure Statement"], "sections": [["pleomorphic carcinoma is an aggressive tumour initially described in the lung by nash and stout . in the 4th edition of the world health organization", "( who ) classification of tumours of the lung , pleomorphic carcinoma is defined as a poorly differentiated ( squamous cell carcinoma or adenocarcinoma ) or undifferentiated carcinoma in which at least 10% spindle and/or giant cells are identified , or as a carcinoma constituted purely of spindle and giant cells .", "primary sites of occurrence of extrapulmonary pleomorphic carcinomas have been described in other organs such as the stomach , thyroid , gallbladder , pancreas , kidney , liver , bladder , and colon . to the best of our knowledge , only one report described a case of giant cell carcinoma of the colon resembling that observed in the lung .", "based on the recommendations of the nomenclature committee on cell death , mitotic catastrophe is defined as a tumour - suppressive mechanism occurring during or after aberrant mitosis leading to cell death or cell senescence .", "histological patterns of micro- and multinucleation have been used as morphological markers for the detection of mitotic catastrophe [ 5 , 6 ] . a high frequency of multinucleated giant cells is characteristically found in pleomorphic carcinomas . in this report , we present an additional case of primary pleomorphic carcinoma of the colon , and we underline similarities to and differences from pulmonary pleomorphic carcinoma .", "the possibility of the pleomorphic component as morphological expression of mitotic catastrophe is finally discussed ."], ["a 65-year - old caucasian woman with a medical history of hypercholesterolaemia , chronic obstructive pulmonary disease , and paroxysmal tachycardia was admitted to the surgery unit of the university hospital  g. martino ( messina , italy ) in 2013 for abdominal obstructive symptoms ( colicky pain to the right iliac fossa as well as borborygmi ) , change in bowel habits ( diarrhoea ) , asthenia , and severe weight loss since 6 months .", "there was no nausea or vomiting . on physical examination , a globular and tympanic abdomen , painless at superficial and deep palpation , was evident .", "she reported a history of adequate food intake with regular bowel habits , alcohol consumption ( < 20/30 g daily ) , and smoking .", "there was no family history of gastrointestinal malignancy , although familiality for bladder carcinoma was reported .", "abdominal ultrasonography highlighted an exophytic / stenosing mass at the level of the ascending colon .", "afterwards , colonoscopy also revealed an exophytic / stenosing malignant neoplastic mass in the caecum , subsequently confirmed through histological evaluation of biopsy .", "carcinoembryonic antigen ( value for smokers < 10.0 ng / ml ) and carbohydrate antigen 19 - 9 ( value < 35 iu / ml ) were within normal limits .", "a contrast - enhanced computed tomography scan showed no evidence of liver or distant metastases ."], ["routine haematoxylin - eosin sections were made from formalin - fixed , paraffin - embedded tissue .", "sections were also stained with periodic acid - schiff and alcian blue at ph 2.5 .", "additional sections  collected on silanized , coated slides  were used for the immunohistochemical stains , using the dako envisiontm flex , high ph detection system together with autostainer instruments .", "the commercial source , clone , and dilution of the primary antibodies are detailed in table 1 ."], ["macroscopic examination of the resected colon revealed an endophytic / ulcerative lesion measuring 7 cm in its greatest dimension , invading the caecum nearly to the ileocaecal valve .", "microscopically , the tumour was a poorly differentiated adenocarcinoma with a grade 4 pleomorphic component that occupied up to 10% of the whole tumour section .", "the pleomorphic component showed trabeculae and nests of neoplastic cells possessing eosinophilic cytoplasm , as well as irregularly shaped vesicular nuclei with a single large eosinophilic nucleolus .", "giant cells containing multiple nuclei , micronuclei , and prominent eosinophil nucleoli were also found ( fig . ", "the micronuclei appeared as round chromatin fragments with a diameter less than one third of the diameter of the nucleus .", "1b ) and large geographical necrotic areas with sharp boundaries with respect to the viable tumour tissue ( fig . ", "only 1 out of the 44 harvested perivisceral lymph nodes presented carcinomatous metastasis ( pt3 , pn1 , clinical stage in accordance to the 2009 ptnm system ) .", "the histochemical stains were negative for periodic acid - schiff and alcian blue in all of the pleomorphic neoplastic cells .", "on the immunohistochemical stains , these cells were diffusely and strongly positive for ceap , ckae1/ae3 , ck7 , and vimentin , but they were negative for ck20 , smooth muscle actin , desmin , synaptophysin , and -human chorionic gonadotropin .", "2b ) , whereas p53 labelling was shown in more than 90% of the tumoural nuclei ( fig . "], ["we described a rare case of pleomorphic carcinoma of the right colon showing opposing clinicopathological features . although it was classified as a grade 4 tumour , it was characterized by a pushing growth pattern and presented only one lymph node with metastasis .", "these data confirm that classic grading of colorectal carcinoma is insufficient to predict a prognosis , and that further parameters such as growth patterns are needed .", "accordingly , several recent studies have shown that colorectal carcinomas with pushing margins are associated with superior disease - free survival rates when compared with tumours with infiltrative margins .", "the present tumour had some of the morphological features described in giant cell carcinoma of the lung , such as pleomorphic tumour cells , malignant giant cells , atypical mitoses , and coagulative necrosis .", "however , pleomorphic carcinoma of the lung has been reported to have a poor prognosis , whereas in our case , the patient has been well without evidence of recurrence or metastases 2 years after the operation .", "many studies suggested that pleomorphic tumours are a heterogeneous group of neoplasms arising in different anatomical sites ( e.g. lung , uterus , breast , central nervous system ) characterized by variable morphological features and prognoses [ 8 , 9 , 10 , 11 ] .", "in particular , a certain percentage of patients affected by them were found to be long - term survivors despite the anaplastic morphology of their primary neoplasms [ 8 , 10 , 11 ] .", "the differential diagnosis of pleomorphic carcinoma includes adenocarcinoma with a choriocarcinomatous component as well as mixed adenoneuroendocrine carcinoma [ 7 , 12 , 13 ] .", "a choriocarcinomatous component usually is characterized by immunoreactivity to -human chorionic gonadotropin , which was not found in our case , while the latter was excluded by the lack of expression of neuroendocrine markers such as synaptophysin and chromogranin [ 12 , 13 ] . in our case , multinucleated tumour cells contained not only abnormally large giant nuclei but also those that are abnormally small , in the form of micronuclei .", "the presence of micronuclei is usually overlooked in the reports of pleomorphic carcinoma in the current literature .", "micronuclei derive from chromosomes and/or chromosome fragments that have been irregularly distributed between daughter nuclei after abnormal mitosis . besides micronucleation and multinucleation ,", "we interpret these findings as a morphological expression of mitotic catastrophe , a particular type of cell death occurring in tumour cells after aberrant mitosis [ 5 , 6 , 14 ] .", "after mitotic catastrophe , tumour cells may continue to divide and thus develop polyploidy and/or aneuploidy .", "ki-67 is an antigen of cell proliferation frequently used in routine histopathological diagnostics as a prognostic factor in more malignancies .", "specifically , it is an epitope of a nuclear and nucleolar protein of 360 kda only expressed in nuclei of cells in active proliferation , i.e. during cell cycle phases g1 , s , g2 , and m , but not in the quiescent phase , i.e. in cell cycle phase g0 [ 17 , 18 ]", ". normally , p53 acts as a guardian of the genome , protecting cells against cancer via two main ways : by determining cell cycle arrest at g1 and g2/m and by inducing cellular apoptosis [ 19 , 20 ] .", "nuclear p53 immunohistochemical positivity in tumour cells suggests mutation in the p53 gene [ 21 , 22 ] .", "thus , immunohistochemical overexpression of p53 and ki-67 constitutes further evidence of mitotic catastrophe in the pleomorphic component of our case of colonic neoplasm .", "ionizing radiation and different classes of cytotoxic agents induce cell death through mitotic catastrophe [ 15 , 18 ] .", "histological recognition of mitotic catastrophe could be useful to predict an eventual pharmacological modulation ( induction or inhibition ) of tumour cell death .", "therefore , mitotic catastrophe has recently gained attention as a potential therapeutic target in neoplasms [ 23 , 24 , 25 ] . in summary , we reported a case of colorectal carcinoma with clinicopathological features partially similar to pleomorphic cell carcinoma of the lung , a neoplasm associated with a bad prognosis . in particular , this tumour presented only one lymph node with metastasis , and the patient is still well 2 years after the operation .", "this case presented morphological and immunohistochemical features compatible with mitotic catastrophe , a form of non - apoptotic cell death due to aberrant mitosis .", "the inclusion of mitotic catastrophe as part of a microscopic evaluation may be useful for understanding the pathogenesis of this rare entity and for new cancer treatment modalities ."], ["no further ethical approval was necessary to perform histology and immunohistochemistry in the case included in this report ."], [""]]}
{"article_id": "PMC2850979", "article_text": ["management of suspected scaphoid fractures with normal x - rays at emergency department ( ed ) presentation is a common clinical challenge .", "there has long been concern that failure to identify and immobilise a scaphoid fracture might result in avascular necrosis or non - union with significant functional impact for the patient and potential medico - legal risk for the practitioner .", "traditional management would see the patient immobilised in a plaster cast for 714  days followed by clinical examination and re - x - ray to identify fractures not visible on the initial films . that approach can been challenged on two fronts : whether cast immobilisation while awaiting clinical review is justified and the role of alternative imaging modalities at or near the time of initial presentation .", "the aim of this project was to characterise current management of adult patients with possible occult scaphoid fracture in australasia .", "this was an internet - based survey of directors of emergency medicine training ( demt ) throughout australasia as identified from the australasian college for emergency medicine web - site ( www.acem.org.au ) .", "demts are the designated supervisors of specialist training at hospitals accredited for training , both metropolitan and rural ( one / hospital ) . as such , they are easily identifiable , experienced specialists with a good understanding of clinical practice within the ed in which they work . participants", "were sent an invitation to participate in the confidential on - line survey by e - mail ( where one could be found ) or by post .", "data were collected using internet - based survey software ( www.surveymonkey.com ) and included the most common management strategy used for patients with possible occult scaphoid fracture ( from a range of options , see table  1 ) and whether there was a written ed guideline regarding management of such cases .", "the outcome of interest was the most common management strategy for possible occult scaphoid fractures .", "table  1management options and reported frequencymanagement strategyfrequency ( n , % ) backslab ( half - cast ) with assessment and re - x - ray at 714  days23 , 38%plaster cast ( full ) with assessment and re - x - ray at 714  days19 , 32%backslab with early ct ( within 7  days)6 , 10%backslab with early bone scan ( within 7  days)5 , 8%backslab with early mri ( within 7  days)2 , 3%support bandage with early mri ( within 7  days)1 , 1.7%support bandage with early ct ( within 7  days)1 , 1.7%plaster cast ( full ) with early ct ( within 7  days)1 , 1.7%plaster cast ( full ) with early bone scan ( within 7  days)1 , 1.7%support bandage with re - assessment at 1  week , with further tests only if symptoms or signs persist1 , 1.7%support bandage with assessment and re - x - ray at 714  days0plaster cast ( full ) with early mri ( within 7  days)0support bandage with early bone scan ( within 7  days)0othersame / next day ct1 , 1.7% management options and reported frequency this project was approved by western health under the nhrmc quality assurance guidelines .", "the most common management reported was immobilisation in a backslab ( 23 , 38% ) or full cast ( 19 , 32% ) with clinical assessment and re - x - ray in 710  days .", "ct scan within 7  days was used by 9 ( 15% ) , bone scan within 7  days by 6 ( 10% ) and mri within 7  days by 3 ( 5% ) .", "eighty - three percent of sites reported not having a written guideline / protocol for this condition .", "the rate of occult scaphoid fractures in cohorts of patients with clinical evidence suggestive of scaphoid fracture but normal initial x - rays is reported to be 016% [ 24 ] , weighted average 3.7% ( 95% ci 2.55.6% ) . in the subgroup", "who prove to have a fracture , the rate of delayed / non - union is low ( 1020% ) .", "traditional treatment for suspected occult scaphoid fracture is cast immobilisation with clinical re - assessment and re - x - ray at 710  days .", "that approach , however , has been suggested to be over - treatment , with a very low rate of significant fractures at the cost of significant time lost from work or school [ 57 ] .", "it has been suggested that symptomatic treatment with follow - up within 2  weeks for these cases is sufficient [ 6 , 8 , 9 ] .", "we found that most australasian eds continue to use plaster immobilisation for this patient group ( backslab or full cast , 70% ) and that uptake of alternative imaging modalities is only moderate ( 30% ) .", "the high use of immobilisation is at odds with reported uk practice , where only 46% of eds reported routinely using plaster immobilisation for x - ray - negative suspected scaphoid fracture .", "ct , mri and bone scan have all been shown to be effective in the diagnosis of occult scaphoid fractures , but to date no studies have shown additional benefit from early advanced imaging for hard clinical outcomes . at present , the logical role for advanced imaging is for patients with persistent symptoms and normal follow - up x - rays .", "there is a clear need for further research comparing combinations of the various immobilisation and imaging strategies and reporting clinical outcomes ( including time off work and time immobilised ) and cost - effectiveness outcomes .", "there is no evidence that lack of access to advanced imaging impacts adversely on clinical outcome .", "the weight of evidence would suggest that this is over - treatment and that a simple bandage is sufficient .", "as this was a survey , the management strategy reported may not be that actually used .", "the traditional approach to management of possible occult scaphoid fracture of immobilisation with re - x - ray at 710  days remains the most commonly used in australasia , despite evidence that this is probably over - treatment with significant consequences for patients ."], "abstract_text": ["<S> aimto characterise current management of adult patients with possible occult scaphoid fracture in australasian emergency departments.methodsinternet-based survey of directors of emergency medicine training throughout australasia . </S>", "<S> data collected included the most common management used in ed for patients with possible occult scaphoid fracture and whether there was a guideline regarding management of such cases . </S>", "<S> data are reported as descriptive statistics.results61 responses were received ( response rate 73% ) . </S>", "<S> the most common management reported was immobilisation in a backslab ( 23 , 38% ) or full cast ( 19 , 32% ) with clinical assessment and re - x - ray in 710  days . </S>", "<S> ct scan within 7  days was used by 9 ( 15% ) , bone scan within 7  days by 6 ( 10% ) and mri within 7  days by 3 ( 5% ) . </S>", "<S> very few sites were using same day / next day ct or mri . </S>", "<S> eighty - three percent of sites reported not having a guideline / protocol for this condition.conclusionthe traditional approach to management of possible occult scaphoid fracture of immobilisation with re - x - ray at 710  days remains the most commonly used in australasia , despite evidence that this is probably over - treatment with significant consequences for patients . </S>", "<S> the place of advanced imaging for investigation of potential scaphoid fractures requires further research . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusion"], "sections": [["management of suspected scaphoid fractures with normal x - rays at emergency department ( ed ) presentation is a common clinical challenge .", "there has long been concern that failure to identify and immobilise a scaphoid fracture might result in avascular necrosis or non - union with significant functional impact for the patient and potential medico - legal risk for the practitioner .", "traditional management would see the patient immobilised in a plaster cast for 714  days followed by clinical examination and re - x - ray to identify fractures not visible on the initial films . that approach can been challenged on two fronts : whether cast immobilisation while awaiting clinical review is justified and the role of alternative imaging modalities at or near the time of initial presentation .", "the aim of this project was to characterise current management of adult patients with possible occult scaphoid fracture in australasia ."], ["this was an internet - based survey of directors of emergency medicine training ( demt ) throughout australasia as identified from the australasian college for emergency medicine web - site ( www.acem.org.au ) .", "demts are the designated supervisors of specialist training at hospitals accredited for training , both metropolitan and rural ( one / hospital ) . as such , they are easily identifiable , experienced specialists with a good understanding of clinical practice within the ed in which they work . participants", "were sent an invitation to participate in the confidential on - line survey by e - mail ( where one could be found ) or by post .", "data were collected using internet - based survey software ( www.surveymonkey.com ) and included the most common management strategy used for patients with possible occult scaphoid fracture ( from a range of options , see table  1 ) and whether there was a written ed guideline regarding management of such cases .", "the outcome of interest was the most common management strategy for possible occult scaphoid fractures .", "table  1management options and reported frequencymanagement strategyfrequency ( n , % ) backslab ( half - cast ) with assessment and re - x - ray at 714  days23 , 38%plaster cast ( full ) with assessment and re - x - ray at 714  days19 , 32%backslab with early ct ( within 7  days)6 , 10%backslab with early bone scan ( within 7  days)5 , 8%backslab with early mri ( within 7  days)2 , 3%support bandage with early mri ( within 7  days)1 , 1.7%support bandage with early ct ( within 7  days)1 , 1.7%plaster cast ( full ) with early ct ( within 7  days)1 , 1.7%plaster cast ( full ) with early bone scan ( within 7  days)1 , 1.7%support bandage with re - assessment at 1  week , with further tests only if symptoms or signs persist1 , 1.7%support bandage with assessment and re - x - ray at 714  days0plaster cast ( full ) with early mri ( within 7  days)0support bandage with early bone scan ( within 7  days)0othersame / next day ct1 , 1.7% management options and reported frequency this project was approved by western health under the nhrmc quality assurance guidelines ."], ["the most common management reported was immobilisation in a backslab ( 23 , 38% ) or full cast ( 19 , 32% ) with clinical assessment and re - x - ray in 710  days .", "ct scan within 7  days was used by 9 ( 15% ) , bone scan within 7  days by 6 ( 10% ) and mri within 7  days by 3 ( 5% ) .", "eighty - three percent of sites reported not having a written guideline / protocol for this condition ."], ["the rate of occult scaphoid fractures in cohorts of patients with clinical evidence suggestive of scaphoid fracture but normal initial x - rays is reported to be 016% [ 24 ] , weighted average 3.7% ( 95% ci 2.55.6% ) . in the subgroup", "who prove to have a fracture , the rate of delayed / non - union is low ( 1020% ) .", "traditional treatment for suspected occult scaphoid fracture is cast immobilisation with clinical re - assessment and re - x - ray at 710  days .", "that approach , however , has been suggested to be over - treatment , with a very low rate of significant fractures at the cost of significant time lost from work or school [ 57 ] .", "it has been suggested that symptomatic treatment with follow - up within 2  weeks for these cases is sufficient [ 6 , 8 , 9 ] .", "we found that most australasian eds continue to use plaster immobilisation for this patient group ( backslab or full cast , 70% ) and that uptake of alternative imaging modalities is only moderate ( 30% ) .", "the high use of immobilisation is at odds with reported uk practice , where only 46% of eds reported routinely using plaster immobilisation for x - ray - negative suspected scaphoid fracture .", "ct , mri and bone scan have all been shown to be effective in the diagnosis of occult scaphoid fractures , but to date no studies have shown additional benefit from early advanced imaging for hard clinical outcomes . at present , the logical role for advanced imaging is for patients with persistent symptoms and normal follow - up x - rays .", "there is a clear need for further research comparing combinations of the various immobilisation and imaging strategies and reporting clinical outcomes ( including time off work and time immobilised ) and cost - effectiveness outcomes .", "there is no evidence that lack of access to advanced imaging impacts adversely on clinical outcome .", "the weight of evidence would suggest that this is over - treatment and that a simple bandage is sufficient .", "as this was a survey , the management strategy reported may not be that actually used ."], ["the traditional approach to management of possible occult scaphoid fracture of immobilisation with re - x - ray at 710  days remains the most commonly used in australasia , despite evidence that this is probably over - treatment with significant consequences for patients ."]]}
{"article_id": "PMC5353809", "article_text": ["women 's reproductive health is a major area of concern , especially in developing countries .", "maternal complications and poor perinatal outcome are highly associated with nonutilization of antenatal and delivery care services and poor socioeconomic conditions of the patient .", "more than half a million women die annually worldwide because of pregnancy - related complications .", "migration is the most observable and impressive fact in the growth of cities , and it is also considered as an essence of urbanization in the globe . in india , major cities have noticed an increase of around 75% population due to migration .", "migrants face numerous constraints , including lack of political representation ; inadequate housing and lack of formal residency rights ; low - paid , insecure , or hazardous work ; limited access to state - provided services such as health and education ; and discrimination based on ethnicity , religion , class , or gender .", "migrants face denial of basic entitlements including access to subsidized food , housing , drinking water , sanitation and public health facilities , and education and banking services and often work in poor conditions devoid of social security and legal protection .", "internal migrants in india constitute a large population : 307 million internal migrants or 30% of the population and by more recent estimates 326 million or 28.5% of the population . out of the total internal migrants , 70.7% are women .", "marriage is given as the prominent reason for female migration in both the rural and urban areas ", "migration of women has been acknowledged as a demographic factor which affects the reproductive health of women .", "the migrant women , who move to the urban areas , face many challenges in relation to access to health care .", "a study showed that female migrants are vulnerable to environmental , social , and institutions forces that may affect their health - seeking behavior .", "women of socially disadvantaged groups , migrant , and/or from ethnic minority groups have been recognized to be less likely to receive early prenatal care and the necessary care during pregnancy , childbirth , and postnatal period .", "empirical evidence has pointed out that migrant groups face several barriers in accessing national maternal health services .", "maternity care is classed as essential care and so can not be refused due to inability to pay .", "there is no scope for special care for such vulnerable population in india , at present .", "women suffer more because though antenatal care ( anc ) services are available , they could not reach them .", "hence , assessment of present situation of migrant women with reference to anc services utilization and institutional deliveries becomes essential .", "thus , this study was conducted with following objectives among interstate female migrants . \n to study socioeconomic and demographic profile of migrant womento estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc servicesto estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women . \n  to study socioeconomic and demographic profile of migrant women to estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc services to estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women .", "\n to study socioeconomic and demographic profile of migrant womento estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc servicesto estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women . \n  to study socioeconomic and demographic profile of migrant women to estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc services to estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women .", "a cross - sectional community - based survey was conducted among migrant women in reproductive age group in an urban slum of population ( 30,000 ) , which is the field practice area of a medical college .", "sample size was calculated using formula , where p is prevalence of the event , q is probability of nonoccurrence of event , and l is allowable error , the prevalence of anc services utilization according to the national family health survey-3 is 77% , and considering 10% allowable error , sample size comes approximately 120 .", "if there are no women fulfilling inclusion criteria in a randomly selected household , then next household was selected .", "if there is more than one woman in a household fulfilling inclusion criteria , then only one woman was selected by lottery method from that household .", "the national sample survey organization 2008 defined migrant as  a household member whose last usual place of residence ( upr ) any time in the past was different from the present place of enumeration was considered as a migrant member in a household .  in this survey , upr of a person was defined as a place ( village / town ) where the person had stayed continuously for a period of 6 months or more .", "full anc received by women : it is defined as having 3 components ( a ) at least 3 antenatal visits to health center , ( b ) at least 1 tetanus toxoid ( tt ) vaccine taken before delivery , and ( c ) consumption of 100 or more iron and folic acid ( ifa ) tablets . \n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants)married women in the age group of 1545 years who had delivered within last 2 yearsthose who consent was included in the study . \n ", "migrants ( as per the above definition ) from outside maharashtra ( interstate migrants ) married women in the age group of 1545 years who had delivered within last 2 years those who consent was included in the study .", "interviews were conducted at the randomly selected households after getting informed written consent of participants . pretested semi - structured questionnaire for face - to - face interview was prepared .", "initial visits to the above - said areas were done to build rapport with the population as they are hesitant to get interviewed .", "their household was visited with the female social workers , and meetings conducted by social workers were attended .", "while interacting with the participants , one representative from the social workers was always present .", "data were coded , entered , and analyzed using open epi software ( open source epidemiologic statistics for public health)version 3.01 .", "chi - square test was used for evaluating association between anc utilization and categorical variables .", "the national sample survey organization 2008 defined migrant as  a household member whose last usual place of residence ( upr ) any time in the past was different from the present place of enumeration was considered as a migrant member in a household .  in this survey , upr of a person was defined as a place ( village / town ) where the person had stayed continuously for a period of 6 months or more .", "full anc received by women : it is defined as having 3 components ( a ) at least 3 antenatal visits to health center , ( b ) at least 1 tetanus toxoid ( tt ) vaccine taken before delivery , and ( c ) consumption of 100 or more iron and folic acid ( ifa ) tablets .", "\n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants)married women in the age group of 1545 years who had delivered within last 2 yearsthose who consent was included in the study . \n  migrants ( as per the above definition ) from outside maharashtra ( interstate migrants ) married women in the age group of 1545 years who had delivered within last 2 years those who consent", "interviews were conducted at the randomly selected households after getting informed written consent of participants . pretested semi - structured questionnaire for face - to - face interview was prepared .", "initial visits to the above - said areas were done to build rapport with the population as they are hesitant to get interviewed .", "their household was visited with the female social workers , and meetings conducted by social workers were attended .", "while interacting with the participants , one representative from the social workers was always present .", "data were coded , entered , and analyzed using open epi software ( open source epidemiologic statistics for public health)version 3.01 .", "chi - square test was used for evaluating association between anc utilization and categorical variables .", "table 1 shows that 77% ( 93/120 ) of migrants were under the age of 29 years .", "sixty - two percent ( 74/120 ) of migrants were married before the age of 18 years though marriage before the age of 18 years is punishable by law .", "sixty - four percent ( 77/120 ) of migrants were belonged to lower socioeconomic class .", "sociodemographic profile of migrant women with their association with full antenatal care package utilization table 2 shows that almost three - fourth of migrants were hailing from uttar pradesh followed by bihar , i.e. 11.7% ( 14/120 ) .", "thirty - three percent ( 40/120 ) of migrants visited their native place at least once in 6 months .", "migration details of female migrants table 3 depicts that even after availability of reproductive and child health ( rch ) services in slum area , still 10% ( 12/120 ) of mothers did not register for anc services .", "registration percentage in the first trimester is less , i.e. , 21% ( 25/120 ) .", "these women could not be educated about various health topics such as nutrition , immunization , and family planning .", "nearly 63.4% ( 76/120 ) of mothers visited at least three times to health - care facility .", "thirty - seven percent ( 44/120 ) of mothers were not able to visit health - care facility at least three times .", "fourteen percent ( 17/120 ) of mothers did not consume a single tablet of ifa .", "ten percent ( 12/120 ) of mothers did not receive a single injection of tt .", "antenatal care services utilization by migrant women table 4 shows that 12% ( 15/120 ) of deliveries occurred in home . even after implementation of rch program with janani suraksha yojana which emphasizes on institutional delivery", "delivery practices of migrant women table 5 shows that most of the participants , i.e. , 30% ( 36/120 ) mentioned that too far location of government health facility was the most common reason for full nonutilization of anc services .", "they did not want to bear expenses toward traveling to health center , and visit to health center leads to loss of their daily wages .", "nearly 25.8% ( 31/120 ) migrant women did not think it is necessary to utilize full range of anc services .", "elder members in family did not allow 20.8% ( 25/120 ) anc mothers to go for regular anc visits as they did think it is necessary .", "they expect that their children should follow the same practice as they practiced at their time .", "poor quality service at health center 17.5% ( 21/120 ) was one of the important reasons mentioned by migrants .", "staff working at health center was not cooperative , spoke arrogantly , investigations and necessary medicines were not available are the reasons included in poor quality services .", "15.8% of migrant women said they have not utilised anc services fully as they were not having any information ( lack of knowledge ) about this services .", "a study conducted in urban slums of delhi showed that majority of migrants were hindus ( 61% ) , aged 2429 years ( 44% ) , with a literacy rate of 38% .", "majority of migrants were muslims ( 73% ) , aged 1823 years ( 39% ) .", "twenty - seven percent were illiterates , and anc care utilization was significantly associated with religion and type of family .", "a cross - sectional study conducted in tribal blocks of maharashtra showed that anc registration in the first trimester is 63.8% , 82% received tt injections , 68.5% consumed ifa tablets , and 72% paid at least three visits to health centers .", "as compared to this study , in the present study , the percentage of anc registration in the first trimester is very low , i.e. , 21% .", "most of the anc migrants registered in the second trimester . almost 91% of migrant women received at least one tt injections .", "ifa tablets are consumed by all , but 100 tablets were consumed by only 28% of migrants .", "therefore , in the present study , ifa consumption and no of anc visits to health center are very less .", "these figures clearly point out toward the inadequate utilization of anc services by migrants women .", "a cross - sectional study was carried out among recently delivered women residing in tea gardens of darjeeling district of west bengal .", "it mentioned that the major barrier toward utilization of these services was ignorance followed by distance to the health - care center .", "according to the district level household and facility survey 2007/2008 , the percentage of institutional delivery and home delivery in urban area is 87.5% and 12.5% , respectively .", "delivery at home conducted by skilled health personnel was 2.8% . in the present study ,", "a community - based cross - sectional study conducted in urban area in nainital district mentioned that high proportion of home deliveries even in urban area despite proximity to hospitals is a result of many factors including weak demand , minimal outreach services , weak community - provider linkages , and timings that often do not suit the daily - wage - earning urban poor . in the present study , out of 15 home deliveries , 11 deliveries reason mentioned was lack of transport facility to hospital .", "maternity care is an essential care which has to be made acceptable , accessible , and available at a cost affordable to women , but in case of migrant women , it is available but not acceptable and accessible due to above - mentioned reasons .", "the present study revealed low utilization of pregnancy - related health - care utilization among the study population .", "health - care workers and influential people from muslim community should be made aware and trained in explaining necessity of maternal and child health ( mch ) care .", "for this purpose , during health awareness session , elder members of the family specially mother - in - laws have to be involved on a priority basis and educated about importance of full anc care utilization and institutional delivery .", "if necessary , one - to - one counseling should be provided to convince them about correct practices . in the present study ,", "the percentage of anc registration in the first trimester is very low , i.e. , 21% .", "moreover , migrant woman feels comfortable to conduct delivery by dai if she is available .", "it means that the message of  importance of institutional delivery  is not reached or convinced to beneficiaries", ". auxiliary nurse midwife , accredited social health activist , and anganwadi workers are not reached to them .", "then , it becomes necessary to train presently working and close to population dais also so that they will conduct deliveries following proper aseptic precautions , i.e. , to follow all 7 cs - clean hand , clean blade , clean cut , clean cord , clean tie , clean towel , and clean bed .", "poor quality service by health workers is one of the reasons for nonutilization of anc services .", "health - care workers should give sufficient time to counsel patients , or one counselor should be posted for a primary health center ( phc ) area ( six subcenters ) so that he / she should visit each subcenter under the phc area at least for a day in a week and educate groups or counsel patients on various health topics with proper feedback mechanism .", "frequency of outreach activities should also be increased for migrant women staying at a far distance from health center in slums which does not come under municipal corporation .", "this study can provide new insight for policy makers to devote resources for achieving the best possible quality of mch services ."], "abstract_text": ["<S> introduction : migrant is a vulnerable population . they face several barriers in accessing health services . </S>", "<S> the immigration status presents various challenges for maternity services utilization . </S>", "<S> so this study was conducted ( 1 ) to estimate proportion of women utilizing full anc package and to find out reasons for its nonutilization ( 2 ) to estimate the proportion of institutional deliveries and reasons for home deliveries.methods:a cross - sectional community based study was conducted among migrant women in reproductive age group in an urban slum . </S>", "<S> the sample size was 120 . </S>", "<S> multistage simple random sampling was done.results:maximum numbers of migrants ( 71.7% ) were from uttar pradesh . </S>", "<S> sixty two percent married before the age of 18 years . </S>", "<S> full anc package was received by only 18% migrants . </S>", "<S> proportion of home deliveries was 12% , and all were conducted by untrained dai . </S>", "<S> lack of transport facility and availability of dai ( local birth attendant ) were the reasons mentioned for home delivery . </S>", "<S> for nonutilization of full anc package , far location of health center ( 30% ) ; it is not necessary ( 25.8% ) ; and family did not allow them to visit health center ( 21.8% ) were the reasons mentioned . </S>", "<S> religion and type of family were significantly associated with nonutilization of full anc package.conclusion:the present study revealed low utilization of anc services and high proportion of home deliveries among the migrant women even after availability of health facilities for providing anc care and to conduct deliveries in urban area . </S>"], "labels": null, "section_names": ["Introduction", "Objectives", "Materials and Methods", "Operational definition", "Inclusion criteria", "Results", "Discussion", "Conclusion", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["women 's reproductive health is a major area of concern , especially in developing countries .", "maternal complications and poor perinatal outcome are highly associated with nonutilization of antenatal and delivery care services and poor socioeconomic conditions of the patient .", "more than half a million women die annually worldwide because of pregnancy - related complications .", "migration is the most observable and impressive fact in the growth of cities , and it is also considered as an essence of urbanization in the globe . in india , major cities have noticed an increase of around 75% population due to migration .", "migrants face numerous constraints , including lack of political representation ; inadequate housing and lack of formal residency rights ; low - paid , insecure , or hazardous work ; limited access to state - provided services such as health and education ; and discrimination based on ethnicity , religion , class , or gender .", "migrants face denial of basic entitlements including access to subsidized food , housing , drinking water , sanitation and public health facilities , and education and banking services and often work in poor conditions devoid of social security and legal protection .", "internal migrants in india constitute a large population : 307 million internal migrants or 30% of the population and by more recent estimates 326 million or 28.5% of the population . out of the total internal migrants , 70.7% are women .", "marriage is given as the prominent reason for female migration in both the rural and urban areas ", "migration of women has been acknowledged as a demographic factor which affects the reproductive health of women .", "the migrant women , who move to the urban areas , face many challenges in relation to access to health care .", "a study showed that female migrants are vulnerable to environmental , social , and institutions forces that may affect their health - seeking behavior .", "women of socially disadvantaged groups , migrant , and/or from ethnic minority groups have been recognized to be less likely to receive early prenatal care and the necessary care during pregnancy , childbirth , and postnatal period .", "empirical evidence has pointed out that migrant groups face several barriers in accessing national maternal health services .", "maternity care is classed as essential care and so can not be refused due to inability to pay .", "there is no scope for special care for such vulnerable population in india , at present .", "women suffer more because though antenatal care ( anc ) services are available , they could not reach them .", "hence , assessment of present situation of migrant women with reference to anc services utilization and institutional deliveries becomes essential .", "thus , this study was conducted with following objectives among interstate female migrants . \n to study socioeconomic and demographic profile of migrant womento estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc servicesto estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women . \n  to study socioeconomic and demographic profile of migrant women to estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc services to estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women ."], ["\n to study socioeconomic and demographic profile of migrant womento estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc servicesto estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women . \n  to study socioeconomic and demographic profile of migrant women to estimate proportion of women utilizing full anc package and reasons for nonutilization of full anc services to estimate the proportion of institutional deliveries and reasons for home deliveries among the migrant women ."], ["a cross - sectional community - based survey was conducted among migrant women in reproductive age group in an urban slum of population ( 30,000 ) , which is the field practice area of a medical college .", "sample size was calculated using formula , where p is prevalence of the event , q is probability of nonoccurrence of event , and l is allowable error , the prevalence of anc services utilization according to the national family health survey-3 is 77% , and considering 10% allowable error , sample size comes approximately 120 .", "if there are no women fulfilling inclusion criteria in a randomly selected household , then next household was selected .", "if there is more than one woman in a household fulfilling inclusion criteria , then only one woman was selected by lottery method from that household .", "the national sample survey organization 2008 defined migrant as  a household member whose last usual place of residence ( upr ) any time in the past was different from the present place of enumeration was considered as a migrant member in a household .  in this survey , upr of a person was defined as a place ( village / town ) where the person had stayed continuously for a period of 6 months or more .", "full anc received by women : it is defined as having 3 components ( a ) at least 3 antenatal visits to health center , ( b ) at least 1 tetanus toxoid ( tt ) vaccine taken before delivery , and ( c ) consumption of 100 or more iron and folic acid ( ifa ) tablets . \n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants)married women in the age group of 1545 years who had delivered within last 2 yearsthose who consent was included in the study . \n ", "migrants ( as per the above definition ) from outside maharashtra ( interstate migrants ) married women in the age group of 1545 years who had delivered within last 2 years those who consent was included in the study .", "interviews were conducted at the randomly selected households after getting informed written consent of participants . pretested semi - structured questionnaire for face - to - face interview was prepared .", "initial visits to the above - said areas were done to build rapport with the population as they are hesitant to get interviewed .", "their household was visited with the female social workers , and meetings conducted by social workers were attended .", "while interacting with the participants , one representative from the social workers was always present .", "data were coded , entered , and analyzed using open epi software ( open source epidemiologic statistics for public health)version 3.01 .", "chi - square test was used for evaluating association between anc utilization and categorical variables ."], ["the national sample survey organization 2008 defined migrant as  a household member whose last usual place of residence ( upr ) any time in the past was different from the present place of enumeration was considered as a migrant member in a household .  in this survey , upr of a person was defined as a place ( village / town ) where the person had stayed continuously for a period of 6 months or more .", "full anc received by women : it is defined as having 3 components ( a ) at least 3 antenatal visits to health center , ( b ) at least 1 tetanus toxoid ( tt ) vaccine taken before delivery , and ( c ) consumption of 100 or more iron and folic acid ( ifa ) tablets ."], ["\n migrants ( as per the above definition ) from outside maharashtra ( interstate migrants)married women in the age group of 1545 years who had delivered within last 2 yearsthose who consent was included in the study . \n  migrants ( as per the above definition ) from outside maharashtra ( interstate migrants ) married women in the age group of 1545 years who had delivered within last 2 years those who consent", "interviews were conducted at the randomly selected households after getting informed written consent of participants . pretested semi - structured questionnaire for face - to - face interview was prepared .", "initial visits to the above - said areas were done to build rapport with the population as they are hesitant to get interviewed .", "their household was visited with the female social workers , and meetings conducted by social workers were attended .", "while interacting with the participants , one representative from the social workers was always present .", "data were coded , entered , and analyzed using open epi software ( open source epidemiologic statistics for public health)version 3.01 .", "chi - square test was used for evaluating association between anc utilization and categorical variables ."], ["table 1 shows that 77% ( 93/120 ) of migrants were under the age of 29 years .", "sixty - two percent ( 74/120 ) of migrants were married before the age of 18 years though marriage before the age of 18 years is punishable by law .", "sixty - four percent ( 77/120 ) of migrants were belonged to lower socioeconomic class .", "sociodemographic profile of migrant women with their association with full antenatal care package utilization table 2 shows that almost three - fourth of migrants were hailing from uttar pradesh followed by bihar , i.e. 11.7% ( 14/120 ) .", "thirty - three percent ( 40/120 ) of migrants visited their native place at least once in 6 months .", "migration details of female migrants table 3 depicts that even after availability of reproductive and child health ( rch ) services in slum area , still 10% ( 12/120 ) of mothers did not register for anc services .", "registration percentage in the first trimester is less , i.e. , 21% ( 25/120 ) .", "these women could not be educated about various health topics such as nutrition , immunization , and family planning .", "nearly 63.4% ( 76/120 ) of mothers visited at least three times to health - care facility .", "thirty - seven percent ( 44/120 ) of mothers were not able to visit health - care facility at least three times .", "fourteen percent ( 17/120 ) of mothers did not consume a single tablet of ifa .", "ten percent ( 12/120 ) of mothers did not receive a single injection of tt .", "antenatal care services utilization by migrant women table 4 shows that 12% ( 15/120 ) of deliveries occurred in home . even after implementation of rch program with janani suraksha yojana which emphasizes on institutional delivery", "delivery practices of migrant women table 5 shows that most of the participants , i.e. , 30% ( 36/120 ) mentioned that too far location of government health facility was the most common reason for full nonutilization of anc services .", "they did not want to bear expenses toward traveling to health center , and visit to health center leads to loss of their daily wages .", "nearly 25.8% ( 31/120 ) migrant women did not think it is necessary to utilize full range of anc services .", "elder members in family did not allow 20.8% ( 25/120 ) anc mothers to go for regular anc visits as they did think it is necessary .", "they expect that their children should follow the same practice as they practiced at their time .", "poor quality service at health center 17.5% ( 21/120 ) was one of the important reasons mentioned by migrants .", "staff working at health center was not cooperative , spoke arrogantly , investigations and necessary medicines were not available are the reasons included in poor quality services .", "15.8% of migrant women said they have not utilised anc services fully as they were not having any information ( lack of knowledge ) about this services ."], ["a study conducted in urban slums of delhi showed that majority of migrants were hindus ( 61% ) , aged 2429 years ( 44% ) , with a literacy rate of 38% .", "majority of migrants were muslims ( 73% ) , aged 1823 years ( 39% ) .", "twenty - seven percent were illiterates , and anc care utilization was significantly associated with religion and type of family .", "a cross - sectional study conducted in tribal blocks of maharashtra showed that anc registration in the first trimester is 63.8% , 82% received tt injections , 68.5% consumed ifa tablets , and 72% paid at least three visits to health centers .", "as compared to this study , in the present study , the percentage of anc registration in the first trimester is very low , i.e. , 21% .", "most of the anc migrants registered in the second trimester . almost 91% of migrant women received at least one tt injections .", "ifa tablets are consumed by all , but 100 tablets were consumed by only 28% of migrants .", "therefore , in the present study , ifa consumption and no of anc visits to health center are very less .", "these figures clearly point out toward the inadequate utilization of anc services by migrants women .", "a cross - sectional study was carried out among recently delivered women residing in tea gardens of darjeeling district of west bengal .", "it mentioned that the major barrier toward utilization of these services was ignorance followed by distance to the health - care center .", "according to the district level household and facility survey 2007/2008 , the percentage of institutional delivery and home delivery in urban area is 87.5% and 12.5% , respectively .", "delivery at home conducted by skilled health personnel was 2.8% . in the present study ,", "a community - based cross - sectional study conducted in urban area in nainital district mentioned that high proportion of home deliveries even in urban area despite proximity to hospitals is a result of many factors including weak demand , minimal outreach services , weak community - provider linkages , and timings that often do not suit the daily - wage - earning urban poor . in the present study , out of 15 home deliveries , 11 deliveries reason mentioned was lack of transport facility to hospital .", "maternity care is an essential care which has to be made acceptable , accessible , and available at a cost affordable to women , but in case of migrant women , it is available but not acceptable and accessible due to above - mentioned reasons ."], ["the present study revealed low utilization of pregnancy - related health - care utilization among the study population .", "health - care workers and influential people from muslim community should be made aware and trained in explaining necessity of maternal and child health ( mch ) care .", "for this purpose , during health awareness session , elder members of the family specially mother - in - laws have to be involved on a priority basis and educated about importance of full anc care utilization and institutional delivery .", "if necessary , one - to - one counseling should be provided to convince them about correct practices . in the present study ,", "the percentage of anc registration in the first trimester is very low , i.e. , 21% .", "moreover , migrant woman feels comfortable to conduct delivery by dai if she is available .", "it means that the message of  importance of institutional delivery  is not reached or convinced to beneficiaries", ". auxiliary nurse midwife , accredited social health activist , and anganwadi workers are not reached to them .", "then , it becomes necessary to train presently working and close to population dais also so that they will conduct deliveries following proper aseptic precautions , i.e. , to follow all 7 cs - clean hand , clean blade , clean cut , clean cord , clean tie , clean towel , and clean bed .", "poor quality service by health workers is one of the reasons for nonutilization of anc services .", "health - care workers should give sufficient time to counsel patients , or one counselor should be posted for a primary health center ( phc ) area ( six subcenters ) so that he / she should visit each subcenter under the phc area at least for a day in a week and educate groups or counsel patients on various health topics with proper feedback mechanism .", "frequency of outreach activities should also be increased for migrant women staying at a far distance from health center in slums which does not come under municipal corporation .", "this study can provide new insight for policy makers to devote resources for achieving the best possible quality of mch services ."], [""], [""]]}
{"article_id": "PMC3200189", "article_text": ["phytoestrogens , naturally occurring hormone - like compounds with a unique diphenolic structure found in several plants , have received much attention as dietary components as they are known to promote better health .", "phytoestrogens have been also associated with reduced incidence of various cancers , cardiovascular disease , and osteoporosis and lower cholesterol level [ 25 ] .", "some studies suggest that the dietary intake of phytoestrogens decreases the risk of breast cancer in humans [ 6 , 7 ] .", "the most extensively studied phytoestrogen in vitro and in vivo is genistein which is found richly in soybeans . however", ", biological activities of many other naturally occurring isoflavones have not been studied in detail till now .", "the phytoestrogen daidzein is an isoflavone present as a glucoside in many plants used in human diets .", "daidzein is especially concentrated in soy and soy - based products used for human consumption [ 8 , 9 ] .  ", "a higher incidence of breast cancer in western population is seen as compared to asians who consume diet containing relatively high levels of low - fat , high - fibre , high - soya content .", "asian women show protection against breast cancer in comparison to women living in usa or britain [ 11 , 12 ] which is believed to be lost upon immigration and exposure to western lifestyles within a few generations .  ", "these studies suggest that exposure to phytoestrogens at an early stage is extremely important , in order to gain their cancer - preventive effects .  ", "the female mammary gland undergoes cell division during puberty , and throughout adult life there is cyclical proliferation and involution during the estrous cycle . during early development , rising endogenous estrogen promotes mammary duct branching which ends in highly proliferative structures , terminal end buds ( tebs ) . at postnatal day ( pnd ) 21 , the number of tebs is maximal , and at puberty the onset of estrous cycles results in changing progesterone and estrogen levels that promote differentiation of tebs to the less proliferative alveolar buds or lobules [ 1517 ] .", "highest number of tumours per animal was observed when carcinogen exposure occurred in rats at postnatal day 4050 , a time when mammary gland exhibits a high density of the highly proliferative tebs .", "the incidence of carcinomas is positively correlated with the number of tebs in mammary gland of the young virgin rat at the time of carcinogen exposure [ 16 , 17 ] .", "administration of the phytoestrogen genistein to neonatal and prepubertal rats reduced tebs by promoting teb differentiation [ 18 , 19 ] .  ", "a study on sprague dawley rats ( mammary tumor model ) shows that daidzein has been an effective inhibitor of dmba - induced mammary tumor .", "chemoprevention study from our lab has also shown a decrease in tumor burden and incidence by prepubertal daidzein administration .  ", "although daidzein also has the ability to bind to the ers , the precise mechanism of action by which it shows a chemoprevention against breast cancer has not been clearly elucidated .", "considering the above literature , it could be hypothesized that daidzein could also affect the differentiation and signaling pathways as a mechanism of chemoprevention .", "hence , to explore the mechanism of actions of daidzein , its effect on cell proliferation , mammary gland differentiation , and the expression of ers in a prepubertal mammary tumour model was carried out .", "daidzein , estradiol benzoate ( eb ) , and hoechst 33258 were bought from sigma aldrich co. ( st . louis , mo , usa ) , while dimethyl sulphoxide ( dmso ) and carmine were bought from qualigens ( india ) .", ", santa cruz , calif , usa , and secondary antibodies , diaminobenzidine ( dab ) , avidin - biotin complex , and normal goat serum ( ngs ) were purchased from bangalore genei ( india ) .", "female sprague dawley rats , kept in animal facility in a 12  hr day  :  12  hr night cycle , and maintained in the animal house of the university were used for the present study .", "the studies were conducted according to the ethical guidelines of committee for control and supervision of experiments on animals , government of india , on the use of animals for scientific research .", "experiment 1a total of thirty - six animals ( female sprague dawley offsprings ) of the same age were divided into 3 groups of 12 each . on postnatal days 16 , 18 , and 20 , while group ii and iii were injected subcutaneously with 500  g / g body weight daidzein and 500  ng / g body weight of estradiol benzoate ( eb ) , respectively [ 19 , 23 ] , an equal volume of vehicle ( dmso ) was injected to the animals of group i ( control , dmso ) . on the 21st day , 18 hours after the last injection ,", "6 animals from each group were sacrificed and the rest of the 6 were maintained till the 50th day and sacrificed thereafter .", "the mammary glands obtained at both times ( pnd21 and pnd50 ) were preserved for differentiation study . \n ", "a total of thirty - six animals ( female sprague dawley offsprings ) of the same age were divided into 3 groups of 12 each . on postnatal days 16 , 18 , and 20 , while group ii and iii were injected subcutaneously with 500  g / g body weight daidzein and 500  ng / g body weight of estradiol benzoate ( eb ) , respectively [ 19 , 23 ] , an equal volume of vehicle ( dmso ) was injected to the animals of group i ( control , dmso ) . on the 21st day , 18 hours after the last injection , 6 animals from each group were sacrificed and the rest of the 6 were maintained till the 50th day and sacrificed thereafter .", "the mammary glands obtained at both times ( pnd21 and pnd50 ) were preserved for differentiation study . \n", "however , this time the mammary glands obtained at pnd21 and pnd50 were stored for immuno - histochemical experiments . \n ", "however , this time the mammary glands obtained at pnd21 and pnd50 were stored for immuno - histochemical experiments . all mounts of fourth pair of the mammary gland of the animals were prepared .", "mammary glands were dissected at the time of sacrifice , spread on the microscope slide , and then placed in neutral buffered formalin for 8  h ( 22-day - old animals ) and overnight ( 50-day - old animals ) .", "glands were defatted in acetone for 4  h or overnight , placed in 70% alcohol for 30 min , hydrated in water ( 15  min ) , and stained in alum carmine for overnight . after staining", ", glands were run through a series of graded alcohols ( 30100% ethanol ) and placed in xylene to clear the tissue .", "glands were then compressed between two glass slides for 24  h , released and allowed to expand for at least 8  h , and finally mounted using a glass coverslip and dpx .", "all mounts were then evaluated via light microscopy using the criteria established by russo and russo [ 16 , 17 ] and murrill et al . .", "all mounts were evaluated for the number of terminal end buds , terminal ducts , and lobules type i by the help of the software imagepro .  ", "the mammary glands dissected were fixed in 10% neutral buffered saline , and sectioning was done by cryostat at 20 microns .", "sections were treated with 10% normal goat serum ( ngs ) for 2  h for blocking and then incubated in the primary antibody for 3 days at 4c .", "the antibody was diluted in 0.1  m pbs containing 5% ngs and 0.5% triton - x-100 . after being washed in pbs", ", the sections were incubated in biotinylated goat antimouse igg at 1  :  200 for 18  h at 4c . following this secondary antibody step ,", "the sections were washed and placed in preformed avidin - biotin peroxidase complex ( abc ) at 1  :  400 dilutions for two hours at room temperature . for visualizations of the reaction sites , the sections were treated with the chromogen diaminobenzidine ( dab ) and hydrogen peroxide for 2 - 3  min .", "the sections were rinsed with distilled water dehydrate in ethanol and coverslipped with distyrene plasticizer xylene ( dpx ) mounting medium .", "the nuclei were stained with hoechst 33258 ( sigma ) ; 10  m sections were mounted on gelatin - coated slides and washed in pbs for 10  min ( figure 6 ) .", "they were incubated with hoechst 33258 ( 1  g / ml ) for 30  min in dark at room temperature .", "after washing twice with pbs ( 5  min each ) and once with distilled water ( 5  min ) , the slides were coverslipped with 10% glycerol and stored in dark .", "the slides were viewed in a fluorescent microscope under the blue filter ( excitation wavelength  = 343  nm , emission wavelength  = 483  nm ) . since hoechst is membrane permeant , the procedure does not require membrane permeablization .", "immunostaining was evaluated by examination of slides under a bright field microscope ( carl zeiss axioscop mot 2 ) at magnification 400x , and images were captured through a digital camera for measurement of intensity and counting of cells .  ", "intensities of immunostained cells were estimated by densitometry using morphometric software scion image . in order to count the number of er ,", "bcl2 , bax , caspase-3 positive cells and hoechst stained apoptotic nuclei , magnified views ( 400x ) of sections were captured .", "the total cells ( unstained ) and immunostained cells were counted with the help of the software imagetool ( uthscsa ) .", "values were compared using one - way anova ( sigma stat , jandel scientific ) .", "differentiation of mammary gland was studied by observing the branching of mammary gland and the terminal end buds and ducts and lobular count at two different stages ( i.e. , pnd21 and pnd50 ) after a prepubertal exposure of daidzein to the animals .", "an increase in the count of tebs , tds , and lobules has been seen in the daidzein - treated rat mammary glands at pnd21 , whereas there is a decrease in the teb and td count and further increase of lobule i count at pnd50 as compared to the control , as shown in figure 1 . a similar significant increase of tebs , tds , and lobules has also been seen in the eb - treated animals at pnd21 , and a decrease is seen in tebs and tds and an increase in mammary gland lobules at pnd50 .  ", "immunohistochemistry results at pnd21 in daidzein - and eb - treated mammary glands showed that the percentage er+ cell count decreased significantly , that is , 41.92  1.0% , p < 0.001 and 39.21  0.8% ,", "p < 0.05 , as compared to the control ( 45.7  0.5% ) , whereas intensity of daidzein - and eb - treated epithelial cells decreased up to 80.9% , p < 0.001 , and 82.8% , p < 0.001 , respectively , as compared to the control ( 100% ) . at pnd50 , daidzein - and eb - treated mammary gland epithelial cells in both the count and intensity were shown to be increased significantly .", "the percentage count of er+ cells increased to 39.2  0.7% , p < 0.001 , and 41.54  0.7% , p < 0.001 , in daidzein - and eb - treated epithelial cells respectively , from control ( 33.65  0.57% ) , and intensity increased to 121.5% , p < 0.001 , and 125.4% , p < 0.01 , in daidzein - and eb - treated epithelial cells , respectively from control ( 100% ) significantly . in 21-days - old mammary glands , the bcl2 + cell count and intensity were found to be increased significantly in daidzein - treated animals ( 40.26  0.38% , p < 0.001 ; 111% , p < 0.01 ) as well as in the eb - treated animals ( 44.16  1.02% , p <", "0.001 ; 122.4% , p < 0.001 ) from control ( 35.9  0.6% , 100% ) , whereas the cell count and intensity of bax+ cells were decreased significantly in daidzein - treated animals ( 19.37  0.22% , p < 0.05 ; 82.0% , p <", "0.001 ) as well as in eb - treated animals ( 19.7  0.68% , p < 0.05 ; 83.6% , p < 0.05 ) from control ( 23.61  0.91% , 100% ) . in 50-days - old mammary glands , bcl2 + cell count and intensity were decreased significantly in daidzein - treated animals ( 31.3  0.64% , p < 0.001 ; 82.3% , p < 0.01 ) as well as in the eb - treated animals ( 29.11  0.65% , p < 0.001 ; 79.5% , p < 0.01 ) from control ( 40.4  0.65% , 100% ) .", "simultaneously , the cell count and intensity of bax+ cells were increased significantly in daidzein - treated animals ( 40.65  0.67% , p < 0.001 ; 114% , p < 0.05 ) and in the eb - treated cells ( 39.0  0.84% ,", "p < 0.001 ; 116.3% , p < 0.01 ) from control ( 28.05  0.37% , 100% ) .", "bcl2/bax ratio was found to be enhanced in mammary gland at pnd21 but not at pnd50 .", "caspase-3 + cell count percentage was shown to be increased significantly at 50 days in mammary glands treated with daidzein ( 20.57  1.07% , p < 0.05 ) and eb ( 23.39  1.21% , p <", "also the percentage apoptotic nuclei count increased in daidzein ( 17.94  0.46% , p < 0.001 ) and eb ( 18.82  0.71% , p < 0.001 ) , compared to the control ( 13.28  0.42% ) , at 50 days , significantly ( figures 25 ) .", "our data clearly demonstrates , the prepubertal exposure to daidzein by the female sprague dawley rats produces an enhancement in the differentiation of the mammary gland as well as influences apoptosis and the er expression .", "the highest susceptibility of a rat mammary gland to a carcinogen occurs in the postpubertal virgin females around the age of 50 days , which correlates well with the higher number of tebs and the high cell proliferation , and a low incidence of mammary carcinoma is related to the loss of tebs and the low cell proliferation activity .  ", "there was an increase in the count of tebs and tds and lobules by the prepubertal administration of daidzein in pnd21 , which shows an increased proliferation in the cells , which is probably necessary for the developing of mammary gland at a prepubertal stage .", "lobules are more stable structures than the tebs and tds , hence , by the increase in the count of lobules , it could be interpreted that the undifferentiated tebs and tds are progressing gradually towards the formation of more differentiated lobules , which could be due to the influence of daidzein . in a similar kind of study with genistein , it was observed that prepubertal genistein exposure increases the count of tebs and lobules at pnd21 [ 19 , 23 ] . at pnd50 ,", "the decrease in the count of tebs and tds indicated a decreased proliferation , and coupling this with an increase in count of lobules at the same time suggests further enhancement in the differentiation of mammary glands at pubertal stage , in daidzein - treated mammary glands .", "earlier genistein had shown a similar potential to increase the mammary gland differentiation , along with reduction of teb count and increase in the lobules , at pnd50 [ 19 , 23 ] .", "some of the xenoestrogens such as diethylstilbestrol ( des ) , o , p-ddt , aroclor 1221 , aroclor 1254 , and 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) also showed an enhancement in the mammary gland differentiation when administered prepubertally .", "since the development of mammary gland is controlled by female reproductive hormones like estrogen , it is likely that exogenously the administration of the estrogen agonist estradiol benzoate ( eb ) will show an enhancement in the mammary gland development , which has also been reported earlier .", "here , the eb - treated mammary glands also showed an increase in the tebs and tds at pnd21 and a decrease in the tebs and tds count along with the increase in lobules at the old mammary gland pnd50 .", "a similarity in the results of both eb - and daidzein - treated mammary gland differentiation data suggests that daidzein might have a similar mode and magnitude of action as that of eb , when administered to rats during the prepubertal stage .", "thus , the overall results of the mammary differentiation data reveals that shortly after exposure to daidzein , there was a rapid development of the mammary gland , yielding more differentiated structures ( lobules ) and fewer undifferentiated structures ( terminal end buds ) .", "tumorigenesis experiments done earlier in our laboratory had also shown a decrease in tumour incidence and burden by treatment of daidzein to sd rats in the prepubertal stage .", "hence this chemopreventive effect of daidzein can be correlated to the increased differentiation rate of mammary gland caused by the prepubertal exposure of compound .  ", "immunohistochemical data of er- indicated that there is a decreased expression of er- in response to daidzein , seen in the mammary epithelial cells at pnd21 .", "normally , the percentage of er--positive nuclei varies according to the developmental state of the mammary gland .", "decreased expression of er- may also reflect proliferation , as proliferating epithelial cells of the mammary glands of young virgin rats do not express the receptor [ 27 , 28 ] , while its increase after 50 days suggests that er- expression might not be somewhat necessary for proliferation but may be required for the differentiation of mammary glands .", "this is consistent with our previous results which indicated that prepubertal daidzein treatment resulted in a proliferative   mammary gland at pnd21 , which led to a more differentiated structure evident at pnd50 .", "a similar result in the expression of er-  in response to daidzein and eb may suggest a common molecular mechanism of action .", "there has been a link established between the proliferating epithelial cells and the expression of er- in developing rodent mammary gland .", "the increase in the count and intensity of bcl2 at pnd21 further supported the result that there might be an increase in proliferation of cells which might have resulted in the increased count of the unstable proliferating structure , tebs , accordingly .", "later at pnd50 , there is a decrease in the bcl2 expression which indicates decreased proliferation .", "this can be related to the enhanced differentiation of the mammary glands in the daidzein - treated rats , where there was an increase in the lobule count along with a decrease in the count of teb at pnd50 . and since the differentiation of the cells has increased and proliferation has slowed down , the count of bcl2 has also decreased in 50-day - old daidzein - treated rats , significantly , as compared to control . furthermore , a decrease in the level of antiapoptotic protein bax at pnd21 and its increase in pnd50 show that apoptosis is not seen in pnd21 rather it increased in pnd50 as evaluated by the simultaneous expression levels of bcl2 and bax .", "the process of apoptosis is integral to normal mammary gland development , and the morphogenesis of ducts in the development of mammary gland is dependent on the selective death of epithelial cells to form mammary acini [ 29 , 30 ] .", "nuclear condensation and fragmentation because of degradation of dna into oligonucleosome fragments , which is a characterized feature of apoptosis , was seen to be increased in pnd50 , whereas no significant change in the apoptotic level was observed at pnd21 .", "this was further confirmed when the apoptotic marker protein , caspase-3 , which is also known to be one of the principal intracellular effectors of the apoptotic cascade , simultaneously increased in the daidzein - and eb - treated cells along with the increase in apoptotic nuclei , at pnd50 .   since daidzein and eb decrease the expression of er- in prepubertal epithelial cells along with the increase in the proliferation and differentiation of cells at the same time , it can be said that the estrogen receptor expression and the mechanism for the proliferation of cells may not be directly correlated , and both proteins might have been influenced by the test compound by separate pathways .", "both processes may be independent of each other , but both might be required for the enhancement of differentiation of the gland .", "the phytoestrogen daidzein has a potential to regulate the differentiation of mammary gland by maintaining a balance between proliferation and apoptotic death of the cells which is critical for its normal development .", "perturbations in this balance can contribute to the development of various disorders in mammary gland including cancer .", "conditions that upregulate cell proliferation or downregulate apoptosis can allow the accumulation of mutations that contribute to the subsequent development of breast cancer .", "daidzein enhances the differentiation of mammary gland in a controlled manner at all the developmental stages .  ", "although the influence of daidzein on some key proliferative and apoptotic proteins has been discussed here , investigations on other cellular pathways integral to the differentiation process can be explored to have a better understanding of the mechanism of actions of daidzein .", "thus , it can be concluded that daidzein , in a similar mechanism of action as that of eb , influences the differentiation of mammary gland by affecting cell proliferation proteins and the er- expression .", "since the more differentiated the mammary gland is , the less susceptible it is to cancer incidence , dietary intake of daidzein from an early developmental stages may be beneficial to women folk in particular , which can be further confirmed through clinical trials ."], "abstract_text": ["<S> mechanism of chemoprevention by daidzein ( 500  g / g bwt ) was examined by injecting it   subcutaneously at 16th , 18th , and 20th day postpartum , followed by counting of terminal end buds ( tebs ) , terminal ducts ( tds ) , and lobules and immunohistochemistry of er- , bcl2 , bax , and caspase-3 . </S>", "<S> dna fragmentation was also analysed to measure the apoptosis level . </S>", "<S> estradiol benzoate ( eb ) ( 500  ng / g bwt ) and dimethyl sulphoxide ( dmso ) were used as reference and vehicle , respectively . \n </S>", "<S> observations show a significant enhancement of mammary gland differentiation at postnatal day 21 ( pnd21 ) as well as pnd50 . </S>", "<S> there was a significant decrease of er- expression at pnd21 and increase in its expression   at pnd50 , in daidzein - treated animals . </S>", "<S> the ratio of expression of bcl-2 to bax proteins increased at pnd50 the same whereas , it decreased at pnd50 due to daidzein . an increased expression of caspase-3 and dna fragmentation was also seen due to daidzein at pnd50 . </S>", "<S> the mammary gland of eb - treated animals showed   response   a somewhat similar to that of daidzein - treated animals . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["phytoestrogens , naturally occurring hormone - like compounds with a unique diphenolic structure found in several plants , have received much attention as dietary components as they are known to promote better health .", "phytoestrogens have been also associated with reduced incidence of various cancers , cardiovascular disease , and osteoporosis and lower cholesterol level [ 25 ] .", "some studies suggest that the dietary intake of phytoestrogens decreases the risk of breast cancer in humans [ 6 , 7 ] .", "the most extensively studied phytoestrogen in vitro and in vivo is genistein which is found richly in soybeans . however", ", biological activities of many other naturally occurring isoflavones have not been studied in detail till now .", "the phytoestrogen daidzein is an isoflavone present as a glucoside in many plants used in human diets .", "daidzein is especially concentrated in soy and soy - based products used for human consumption [ 8 , 9 ] .  ", "a higher incidence of breast cancer in western population is seen as compared to asians who consume diet containing relatively high levels of low - fat , high - fibre , high - soya content .", "asian women show protection against breast cancer in comparison to women living in usa or britain [ 11 , 12 ] which is believed to be lost upon immigration and exposure to western lifestyles within a few generations .  ", "these studies suggest that exposure to phytoestrogens at an early stage is extremely important , in order to gain their cancer - preventive effects .  ", "the female mammary gland undergoes cell division during puberty , and throughout adult life there is cyclical proliferation and involution during the estrous cycle . during early development , rising endogenous estrogen promotes mammary duct branching which ends in highly proliferative structures , terminal end buds ( tebs ) . at postnatal day ( pnd ) 21 , the number of tebs is maximal , and at puberty the onset of estrous cycles results in changing progesterone and estrogen levels that promote differentiation of tebs to the less proliferative alveolar buds or lobules [ 1517 ] .", "highest number of tumours per animal was observed when carcinogen exposure occurred in rats at postnatal day 4050 , a time when mammary gland exhibits a high density of the highly proliferative tebs .", "the incidence of carcinomas is positively correlated with the number of tebs in mammary gland of the young virgin rat at the time of carcinogen exposure [ 16 , 17 ] .", "administration of the phytoestrogen genistein to neonatal and prepubertal rats reduced tebs by promoting teb differentiation [ 18 , 19 ] .  ", "a study on sprague dawley rats ( mammary tumor model ) shows that daidzein has been an effective inhibitor of dmba - induced mammary tumor .", "chemoprevention study from our lab has also shown a decrease in tumor burden and incidence by prepubertal daidzein administration .  ", "although daidzein also has the ability to bind to the ers , the precise mechanism of action by which it shows a chemoprevention against breast cancer has not been clearly elucidated .", "considering the above literature , it could be hypothesized that daidzein could also affect the differentiation and signaling pathways as a mechanism of chemoprevention .", "hence , to explore the mechanism of actions of daidzein , its effect on cell proliferation , mammary gland differentiation , and the expression of ers in a prepubertal mammary tumour model was carried out ."], ["daidzein , estradiol benzoate ( eb ) , and hoechst 33258 were bought from sigma aldrich co. ( st . louis , mo , usa ) , while dimethyl sulphoxide ( dmso ) and carmine were bought from qualigens ( india ) .", ", santa cruz , calif , usa , and secondary antibodies , diaminobenzidine ( dab ) , avidin - biotin complex , and normal goat serum ( ngs ) were purchased from bangalore genei ( india ) .", "female sprague dawley rats , kept in animal facility in a 12  hr day  :  12  hr night cycle , and maintained in the animal house of the university were used for the present study .", "the studies were conducted according to the ethical guidelines of committee for control and supervision of experiments on animals , government of india , on the use of animals for scientific research .", "experiment 1a total of thirty - six animals ( female sprague dawley offsprings ) of the same age were divided into 3 groups of 12 each . on postnatal days 16 , 18 , and 20 , while group ii and iii were injected subcutaneously with 500  g / g body weight daidzein and 500  ng / g body weight of estradiol benzoate ( eb ) , respectively [ 19 , 23 ] , an equal volume of vehicle ( dmso ) was injected to the animals of group i ( control , dmso ) . on the 21st day , 18 hours after the last injection ,", "6 animals from each group were sacrificed and the rest of the 6 were maintained till the 50th day and sacrificed thereafter .", "the mammary glands obtained at both times ( pnd21 and pnd50 ) were preserved for differentiation study . \n ", "a total of thirty - six animals ( female sprague dawley offsprings ) of the same age were divided into 3 groups of 12 each . on postnatal days 16 , 18 , and 20 , while group ii and iii were injected subcutaneously with 500  g / g body weight daidzein and 500  ng / g body weight of estradiol benzoate ( eb ) , respectively [ 19 , 23 ] , an equal volume of vehicle ( dmso ) was injected to the animals of group i ( control , dmso ) . on the 21st day , 18 hours after the last injection , 6 animals from each group were sacrificed and the rest of the 6 were maintained till the 50th day and sacrificed thereafter .", "the mammary glands obtained at both times ( pnd21 and pnd50 ) were preserved for differentiation study . \n", "however , this time the mammary glands obtained at pnd21 and pnd50 were stored for immuno - histochemical experiments . \n ", "however , this time the mammary glands obtained at pnd21 and pnd50 were stored for immuno - histochemical experiments . all mounts of fourth pair of the mammary gland of the animals were prepared .", "mammary glands were dissected at the time of sacrifice , spread on the microscope slide , and then placed in neutral buffered formalin for 8  h ( 22-day - old animals ) and overnight ( 50-day - old animals ) .", "glands were defatted in acetone for 4  h or overnight , placed in 70% alcohol for 30 min , hydrated in water ( 15  min ) , and stained in alum carmine for overnight . after staining", ", glands were run through a series of graded alcohols ( 30100% ethanol ) and placed in xylene to clear the tissue .", "glands were then compressed between two glass slides for 24  h , released and allowed to expand for at least 8  h , and finally mounted using a glass coverslip and dpx .", "all mounts were then evaluated via light microscopy using the criteria established by russo and russo [ 16 , 17 ] and murrill et al . .", "all mounts were evaluated for the number of terminal end buds , terminal ducts , and lobules type i by the help of the software imagepro .  ", "the mammary glands dissected were fixed in 10% neutral buffered saline , and sectioning was done by cryostat at 20 microns .", "sections were treated with 10% normal goat serum ( ngs ) for 2  h for blocking and then incubated in the primary antibody for 3 days at 4c .", "the antibody was diluted in 0.1  m pbs containing 5% ngs and 0.5% triton - x-100 . after being washed in pbs", ", the sections were incubated in biotinylated goat antimouse igg at 1  :  200 for 18  h at 4c . following this secondary antibody step ,", "the sections were washed and placed in preformed avidin - biotin peroxidase complex ( abc ) at 1  :  400 dilutions for two hours at room temperature . for visualizations of the reaction sites , the sections were treated with the chromogen diaminobenzidine ( dab ) and hydrogen peroxide for 2 - 3  min .", "the sections were rinsed with distilled water dehydrate in ethanol and coverslipped with distyrene plasticizer xylene ( dpx ) mounting medium .", "the nuclei were stained with hoechst 33258 ( sigma ) ; 10  m sections were mounted on gelatin - coated slides and washed in pbs for 10  min ( figure 6 ) .", "they were incubated with hoechst 33258 ( 1  g / ml ) for 30  min in dark at room temperature .", "after washing twice with pbs ( 5  min each ) and once with distilled water ( 5  min ) , the slides were coverslipped with 10% glycerol and stored in dark .", "the slides were viewed in a fluorescent microscope under the blue filter ( excitation wavelength  = 343  nm , emission wavelength  = 483  nm ) . since hoechst is membrane permeant , the procedure does not require membrane permeablization .", "immunostaining was evaluated by examination of slides under a bright field microscope ( carl zeiss axioscop mot 2 ) at magnification 400x , and images were captured through a digital camera for measurement of intensity and counting of cells .  ", "intensities of immunostained cells were estimated by densitometry using morphometric software scion image . in order to count the number of er ,", "bcl2 , bax , caspase-3 positive cells and hoechst stained apoptotic nuclei , magnified views ( 400x ) of sections were captured .", "the total cells ( unstained ) and immunostained cells were counted with the help of the software imagetool ( uthscsa ) .", "values were compared using one - way anova ( sigma stat , jandel scientific ) ."], ["differentiation of mammary gland was studied by observing the branching of mammary gland and the terminal end buds and ducts and lobular count at two different stages ( i.e. , pnd21 and pnd50 ) after a prepubertal exposure of daidzein to the animals .", "an increase in the count of tebs , tds , and lobules has been seen in the daidzein - treated rat mammary glands at pnd21 , whereas there is a decrease in the teb and td count and further increase of lobule i count at pnd50 as compared to the control , as shown in figure 1 . a similar significant increase of tebs , tds , and lobules has also been seen in the eb - treated animals at pnd21 , and a decrease is seen in tebs and tds and an increase in mammary gland lobules at pnd50 .  ", "immunohistochemistry results at pnd21 in daidzein - and eb - treated mammary glands showed that the percentage er+ cell count decreased significantly , that is , 41.92  1.0% , p < 0.001 and 39.21  0.8% ,", "p < 0.05 , as compared to the control ( 45.7  0.5% ) , whereas intensity of daidzein - and eb - treated epithelial cells decreased up to 80.9% , p < 0.001 , and 82.8% , p < 0.001 , respectively , as compared to the control ( 100% ) . at pnd50 , daidzein - and eb - treated mammary gland epithelial cells in both the count and intensity were shown to be increased significantly .", "the percentage count of er+ cells increased to 39.2  0.7% , p < 0.001 , and 41.54  0.7% , p < 0.001 , in daidzein - and eb - treated epithelial cells respectively , from control ( 33.65  0.57% ) , and intensity increased to 121.5% , p < 0.001 , and 125.4% , p < 0.01 , in daidzein - and eb - treated epithelial cells , respectively from control ( 100% ) significantly . in 21-days - old mammary glands , the bcl2 + cell count and intensity were found to be increased significantly in daidzein - treated animals ( 40.26  0.38% , p < 0.001 ; 111% , p < 0.01 ) as well as in the eb - treated animals ( 44.16  1.02% , p <", "0.001 ; 122.4% , p < 0.001 ) from control ( 35.9  0.6% , 100% ) , whereas the cell count and intensity of bax+ cells were decreased significantly in daidzein - treated animals ( 19.37  0.22% , p < 0.05 ; 82.0% , p <", "0.001 ) as well as in eb - treated animals ( 19.7  0.68% , p < 0.05 ; 83.6% , p < 0.05 ) from control ( 23.61  0.91% , 100% ) . in 50-days - old mammary glands , bcl2 + cell count and intensity were decreased significantly in daidzein - treated animals ( 31.3  0.64% , p < 0.001 ; 82.3% , p < 0.01 ) as well as in the eb - treated animals ( 29.11  0.65% , p < 0.001 ; 79.5% , p < 0.01 ) from control ( 40.4  0.65% , 100% ) .", "simultaneously , the cell count and intensity of bax+ cells were increased significantly in daidzein - treated animals ( 40.65  0.67% , p < 0.001 ; 114% , p < 0.05 ) and in the eb - treated cells ( 39.0  0.84% ,", "p < 0.001 ; 116.3% , p < 0.01 ) from control ( 28.05  0.37% , 100% ) .", "bcl2/bax ratio was found to be enhanced in mammary gland at pnd21 but not at pnd50 .", "caspase-3 + cell count percentage was shown to be increased significantly at 50 days in mammary glands treated with daidzein ( 20.57  1.07% , p < 0.05 ) and eb ( 23.39  1.21% , p <", "also the percentage apoptotic nuclei count increased in daidzein ( 17.94  0.46% , p < 0.001 ) and eb ( 18.82  0.71% , p < 0.001 ) , compared to the control ( 13.28  0.42% ) , at 50 days , significantly ( figures 25 ) ."], ["our data clearly demonstrates , the prepubertal exposure to daidzein by the female sprague dawley rats produces an enhancement in the differentiation of the mammary gland as well as influences apoptosis and the er expression .", "the highest susceptibility of a rat mammary gland to a carcinogen occurs in the postpubertal virgin females around the age of 50 days , which correlates well with the higher number of tebs and the high cell proliferation , and a low incidence of mammary carcinoma is related to the loss of tebs and the low cell proliferation activity .  ", "there was an increase in the count of tebs and tds and lobules by the prepubertal administration of daidzein in pnd21 , which shows an increased proliferation in the cells , which is probably necessary for the developing of mammary gland at a prepubertal stage .", "lobules are more stable structures than the tebs and tds , hence , by the increase in the count of lobules , it could be interpreted that the undifferentiated tebs and tds are progressing gradually towards the formation of more differentiated lobules , which could be due to the influence of daidzein . in a similar kind of study with genistein , it was observed that prepubertal genistein exposure increases the count of tebs and lobules at pnd21 [ 19 , 23 ] . at pnd50 ,", "the decrease in the count of tebs and tds indicated a decreased proliferation , and coupling this with an increase in count of lobules at the same time suggests further enhancement in the differentiation of mammary glands at pubertal stage , in daidzein - treated mammary glands .", "earlier genistein had shown a similar potential to increase the mammary gland differentiation , along with reduction of teb count and increase in the lobules , at pnd50 [ 19 , 23 ] .", "some of the xenoestrogens such as diethylstilbestrol ( des ) , o , p-ddt , aroclor 1221 , aroclor 1254 , and 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) also showed an enhancement in the mammary gland differentiation when administered prepubertally .", "since the development of mammary gland is controlled by female reproductive hormones like estrogen , it is likely that exogenously the administration of the estrogen agonist estradiol benzoate ( eb ) will show an enhancement in the mammary gland development , which has also been reported earlier .", "here , the eb - treated mammary glands also showed an increase in the tebs and tds at pnd21 and a decrease in the tebs and tds count along with the increase in lobules at the old mammary gland pnd50 .", "a similarity in the results of both eb - and daidzein - treated mammary gland differentiation data suggests that daidzein might have a similar mode and magnitude of action as that of eb , when administered to rats during the prepubertal stage .", "thus , the overall results of the mammary differentiation data reveals that shortly after exposure to daidzein , there was a rapid development of the mammary gland , yielding more differentiated structures ( lobules ) and fewer undifferentiated structures ( terminal end buds ) .", "tumorigenesis experiments done earlier in our laboratory had also shown a decrease in tumour incidence and burden by treatment of daidzein to sd rats in the prepubertal stage .", "hence this chemopreventive effect of daidzein can be correlated to the increased differentiation rate of mammary gland caused by the prepubertal exposure of compound .  ", "immunohistochemical data of er- indicated that there is a decreased expression of er- in response to daidzein , seen in the mammary epithelial cells at pnd21 .", "normally , the percentage of er--positive nuclei varies according to the developmental state of the mammary gland .", "decreased expression of er- may also reflect proliferation , as proliferating epithelial cells of the mammary glands of young virgin rats do not express the receptor [ 27 , 28 ] , while its increase after 50 days suggests that er- expression might not be somewhat necessary for proliferation but may be required for the differentiation of mammary glands .", "this is consistent with our previous results which indicated that prepubertal daidzein treatment resulted in a proliferative   mammary gland at pnd21 , which led to a more differentiated structure evident at pnd50 .", "a similar result in the expression of er-  in response to daidzein and eb may suggest a common molecular mechanism of action .", "there has been a link established between the proliferating epithelial cells and the expression of er- in developing rodent mammary gland .", "the increase in the count and intensity of bcl2 at pnd21 further supported the result that there might be an increase in proliferation of cells which might have resulted in the increased count of the unstable proliferating structure , tebs , accordingly .", "later at pnd50 , there is a decrease in the bcl2 expression which indicates decreased proliferation .", "this can be related to the enhanced differentiation of the mammary glands in the daidzein - treated rats , where there was an increase in the lobule count along with a decrease in the count of teb at pnd50 . and since the differentiation of the cells has increased and proliferation has slowed down , the count of bcl2 has also decreased in 50-day - old daidzein - treated rats , significantly , as compared to control . furthermore , a decrease in the level of antiapoptotic protein bax at pnd21 and its increase in pnd50 show that apoptosis is not seen in pnd21 rather it increased in pnd50 as evaluated by the simultaneous expression levels of bcl2 and bax .", "the process of apoptosis is integral to normal mammary gland development , and the morphogenesis of ducts in the development of mammary gland is dependent on the selective death of epithelial cells to form mammary acini [ 29 , 30 ] .", "nuclear condensation and fragmentation because of degradation of dna into oligonucleosome fragments , which is a characterized feature of apoptosis , was seen to be increased in pnd50 , whereas no significant change in the apoptotic level was observed at pnd21 .", "this was further confirmed when the apoptotic marker protein , caspase-3 , which is also known to be one of the principal intracellular effectors of the apoptotic cascade , simultaneously increased in the daidzein - and eb - treated cells along with the increase in apoptotic nuclei , at pnd50 .   since daidzein and eb decrease the expression of er- in prepubertal epithelial cells along with the increase in the proliferation and differentiation of cells at the same time , it can be said that the estrogen receptor expression and the mechanism for the proliferation of cells may not be directly correlated , and both proteins might have been influenced by the test compound by separate pathways .", "both processes may be independent of each other , but both might be required for the enhancement of differentiation of the gland ."], ["the phytoestrogen daidzein has a potential to regulate the differentiation of mammary gland by maintaining a balance between proliferation and apoptotic death of the cells which is critical for its normal development .", "perturbations in this balance can contribute to the development of various disorders in mammary gland including cancer .", "conditions that upregulate cell proliferation or downregulate apoptosis can allow the accumulation of mutations that contribute to the subsequent development of breast cancer .", "daidzein enhances the differentiation of mammary gland in a controlled manner at all the developmental stages .  ", "although the influence of daidzein on some key proliferative and apoptotic proteins has been discussed here , investigations on other cellular pathways integral to the differentiation process can be explored to have a better understanding of the mechanism of actions of daidzein .", "thus , it can be concluded that daidzein , in a similar mechanism of action as that of eb , influences the differentiation of mammary gland by affecting cell proliferation proteins and the er- expression .", "since the more differentiated the mammary gland is , the less susceptible it is to cancer incidence , dietary intake of daidzein from an early developmental stages may be beneficial to women folk in particular , which can be further confirmed through clinical trials ."]]}
{"article_id": "PMC4810953", "article_text": ["acute appendicitis , one of the commonest surgical emergencies , affects nearly 7% of the world 's population and accounts for about 1% of all surgical operations .", "faecoliths formed by mineral deposits layered with faecal debris and lodged in the appendix are called appendicoliths .", "the prevalence of faecoliths in the general population is 3% , and appendicoliths are seen in 10% cases of acute appendicitis .", "a 25-year - old male presented to us with pain in the right flank for 1 day .", "urinalysis was normal , and abdominal ultrasound raised a suspicion of small calculi in the right kidney .", "non - contrast computed tomography ( ncct ) abdomen revealed a few subcentimetric calculi in both kidneys without hydronephrosis or hydroureter .", "[ figure 1c and d ] ( mineral core ) , composed mainly of calcium and struvite on spectral analysis [ figure 1a and b ] .", "he underwent laparoscopic appendectomy using a 10-mm supra - umbilical camera port and two 5 mm ports at the supra - pubic region and the left iliac fossa , respectively .", "a pre - ileal appendix was found hidden in flimsy adhesions posterior to the terminal ileum .", "an incidental meckel 's diverticulum was also found [ figure 1f ] , which was not pathological and was left alone .", "the mesoappendix was cut using harmonic scalpel ( johnson and johnson make ) and the appendix was cut at the base between ligatures [ figure 1e ] .", "it was removed using a glove bag after enlarging the supra - umbilical incision because of the large stone size  2.5 cm  3.0 cm [ figure 1 g and h ] .", "ncct abdomen ( a and b ) the ct spectral analysis of the appendicolith shows the highest peak of the histogram corresponding to struvite - calcium ( c ) multiplanar reconstruction ( mpr ) coronal image showing appendicolith 2.50 cm size ( longitudinal ) ( d ) axial image showing appendicolith 2.10 cm size ( transverse ) ( e - h ) laparoscopic appendectomy , procedure , specimen and appendicolith ( e ) multiple ligations of base of appendix ( f ) meckel 's diverticulum ( g ) size of appendicolith 3 cm on longest axis ( h ) size of appendicolith 2.5 cm on the perpendicular axis", "acute appendicitis was first reported by fitz in 1886 , and wangensteen and bowers proposed the theory of an obstructive component as a causative factor in 1937 .", "other proposed aetiologies include lymphoid hyperplasia , constipation , trauma , diet , genetic predilection , hypersensitivity and mucosal ulceration .", "appendicolith on plain abdominal x - ray is a reliable sign of appendicitis ( 70% ) .", "however , computed tomography ( ct ) is more sensitive , detecting even non - calcified faecoliths .", "on ct , appendicoliths appear as laminated bodies with gas in centre or homogenous opacity . when symptomatic , they carry 90% probability of acute appendicitis and 50% higher risk of perforation and abscess formation .", "some authors have found good correlation ( 65 - 100% ) between faecoliths on ct and appendicitis , while others have not . in a retrospective review by lowe et al .", ", an appendicolith detected on ct had a sensitivity of 65% , specificity of 86% , and positive predictive value of 74% for the diagnosis of appendicitis . despite appendicoliths being common ,", "a giant appendicolith ( > 2 cm ) is extremely rare and only sporadically reported .", "others include a 2.1 cm appendicolith reported by garg and a 2.2 cm stone by kaluarachchi .", "our case is probably the largest documented stone ( 3  2.5 cm ) and the first whose chemical composition has been determined preoperatively by spectral analysis .", "the authors have not received any resources from a third party , directly or indirectly , to complete this work .", "the authors have not received any resources from a third party , directly or indirectly , to complete this work ."], "abstract_text": ["<S> acute appendicitis is one of the commonest surgical emergencies worldwide . </S>", "<S> there is considerable variation in prevalence of appendicoliths with appendicitis . </S>", "<S> most of the patients with appendicoliths are asymptomatic and they are not pathognomic for acute appendicitis . </S>", "<S> however , appendicoliths show increased association with perforation and abscess formation . </S>", "<S> appendicolith are quite common , being present in 3% of general population and in nearly 10% cases of appendicitis . however , giant appendicoliths measuring over 2 centimeters ( cms ) are extremely rare . computed tomography ( ct ) </S>", "<S> has increased their pre - operative diagnosis considerably . </S>", "<S> use of spectral analysis can give us the details of composition of the stone pre - operatively . </S>", "<S> we present a young male diagnosed pre - operatively on non - contrast computed tomography ( ncct ) to have a giant calcium struvite appendicolith . </S>", "<S> on laparoscopy he had a 3 cm stone and an incidental meckel 's diverticulum and underwent appendectomy . </S>", "<S> the case is presented for the unique size of the appendicolith alongwith review of literature . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "Financial Support and Sponsorship", "Conflicts of Interest"], "sections": [["acute appendicitis , one of the commonest surgical emergencies , affects nearly 7% of the world 's population and accounts for about 1% of all surgical operations .", "faecoliths formed by mineral deposits layered with faecal debris and lodged in the appendix are called appendicoliths .", "the prevalence of faecoliths in the general population is 3% , and appendicoliths are seen in 10% cases of acute appendicitis ."], ["a 25-year - old male presented to us with pain in the right flank for 1 day .", "urinalysis was normal , and abdominal ultrasound raised a suspicion of small calculi in the right kidney .", "non - contrast computed tomography ( ncct ) abdomen revealed a few subcentimetric calculi in both kidneys without hydronephrosis or hydroureter .", "[ figure 1c and d ] ( mineral core ) , composed mainly of calcium and struvite on spectral analysis [ figure 1a and b ] .", "he underwent laparoscopic appendectomy using a 10-mm supra - umbilical camera port and two 5 mm ports at the supra - pubic region and the left iliac fossa , respectively .", "a pre - ileal appendix was found hidden in flimsy adhesions posterior to the terminal ileum .", "an incidental meckel 's diverticulum was also found [ figure 1f ] , which was not pathological and was left alone .", "the mesoappendix was cut using harmonic scalpel ( johnson and johnson make ) and the appendix was cut at the base between ligatures [ figure 1e ] .", "it was removed using a glove bag after enlarging the supra - umbilical incision because of the large stone size  2.5 cm  3.0 cm [ figure 1 g and h ] .", "ncct abdomen ( a and b ) the ct spectral analysis of the appendicolith shows the highest peak of the histogram corresponding to struvite - calcium ( c ) multiplanar reconstruction ( mpr ) coronal image showing appendicolith 2.50 cm size ( longitudinal ) ( d ) axial image showing appendicolith 2.10 cm size ( transverse ) ( e - h ) laparoscopic appendectomy , procedure , specimen and appendicolith ( e ) multiple ligations of base of appendix ( f ) meckel 's diverticulum ( g ) size of appendicolith 3 cm on longest axis ( h ) size of appendicolith 2.5 cm on the perpendicular axis"], ["acute appendicitis was first reported by fitz in 1886 , and wangensteen and bowers proposed the theory of an obstructive component as a causative factor in 1937 .", "other proposed aetiologies include lymphoid hyperplasia , constipation , trauma , diet , genetic predilection , hypersensitivity and mucosal ulceration .", "appendicolith on plain abdominal x - ray is a reliable sign of appendicitis ( 70% ) .", "however , computed tomography ( ct ) is more sensitive , detecting even non - calcified faecoliths .", "on ct , appendicoliths appear as laminated bodies with gas in centre or homogenous opacity . when symptomatic , they carry 90% probability of acute appendicitis and 50% higher risk of perforation and abscess formation .", "some authors have found good correlation ( 65 - 100% ) between faecoliths on ct and appendicitis , while others have not . in a retrospective review by lowe et al .", ", an appendicolith detected on ct had a sensitivity of 65% , specificity of 86% , and positive predictive value of 74% for the diagnosis of appendicitis . despite appendicoliths being common ,", "a giant appendicolith ( > 2 cm ) is extremely rare and only sporadically reported .", "others include a 2.1 cm appendicolith reported by garg and a 2.2 cm stone by kaluarachchi .", "our case is probably the largest documented stone ( 3  2.5 cm ) and the first whose chemical composition has been determined preoperatively by spectral analysis .", "the authors have not received any resources from a third party , directly or indirectly , to complete this work ."], [""], ["the authors have not received any resources from a third party , directly or indirectly , to complete this work ."]]}
{"article_id": "PMC3742359", "article_text": ["it is commonly treated with conservative surgery ; prognosis is rapidly improved by performing concomitant chemotherapy after the initial surgery.1,2 gliomatosis peritonei is the metastatic implantation of mature glial tissue within the peritoneal cavity of patients with ovarian teratomas.3 as second - look or secondary debulking surgery is not generally performed for immature teratoma , as gliomatosis peritonei is not usually discovered in a patient who has completed chemotherapy and shows no evidence of disease.4 this is a rare case of a patient with ovarian teratoma recurrence in the contralateral ovary .", "the first and later occurrences were treated with conservative surgery , using local resections or cystectomies .", "gravia 0 , para 0was referred to us because of a lower abdominal mass ; magnetic resonance ( mr ) imaging revealed a tumor 20 cm in size .", "tumor marker tests revealed cancer antigen 125 = 279 u / ml , ca19 - 9 = 130.6 u / ml , squamous cell carcinoma = 3.9 ng / ml , and alpha - fet protein = 50.6 ng / ml . after providing informed consent , she selected conservative surgery .", "we performed a right salpingo - oophorectomy , partial omentectomy and peritoneal biopsy , and found lint - like scattered mass lesions 2 mm in size at the vesi - couterine pouch and the douglas pouch .", "the tumor is a mass with cystic regions of many sizes , with solid parts and adipocyte - laden parts ( fig .", "the diagnosis was stage ii c(a ) immature teratoma grade2 of the right ovary with gliomatosis peritonei .", "pathologically , the mass contained squamous epithelium , adipose tissue , bone and cartilage tissue , and immature neuroectodermal tissue , and was therefore classified as a grade 2 immature teratoma ( fig .", "peritoneal lesions , which were composed of mature glial cells , were diagnosed as gliomatosis peritonei using hematoxylin and eosin ( h&e ) stain ( fig .", "immunohistochemically , the glial cells were positive for glial fibrillary acidic protein ( gfap ) , which is expressed by numerous cell types of the central nervous system , including astrocytes , ependymal cells and glial cells ( fig .", "sixteen months after the first operation , the patient underwent cystectomy of a left ovarian tumor and a douglas pouch biopsy . pathological diagnosis was mature cystic teratoma of left ovary and gliomatosis peritonei ( fig .", "sixty months after the first operation , mr imaging revealed a left cystic ovarian tumor with solid parts 7 cm in size .", "pathologically , the mass contained immature neuroectodermal tissue , and so was classified as a grade 1 immature teratoma and gliomatosis peritonei ( fig . 2f and g ) .", "71 months after the first operation , cystectomy of a left ovarian tumor5 cm in size  and biopsies of the douglas pouch and left external iliac lymph node8 mm in size  were carried out .", "the pathological diagnosis was endometrial cyst of left ovary , gliomatosis peritonei and glial implant in lymph node ( fig .", "although this patient has never conceived a child , she has maintained a regular menstrual cycle , despite multiple surgeries for ovarian teratoma and chemotherapy .", "immature teratoma of grades 1 and 2 are considered ovarian germ cell borderline tumors , according to the world health organization s classification .", "as they occur predominantly in women in their teens and 20 s , conservative surgery is performed as a standard ; concomitant chemotherapy after the initial surgery has led to rapid improvements in prognosis.1,2 jefferys et al produced a retrospective study of 47 patients with malignant ovarian germ - cell tumors who were treated by conservative surgery and adjuvant chemotherapy . during chemotherapy , 61.7% ( 29/47 ) of patients developed amenorrhea but 91.5% of these women resumed normal menstrual function upon completion of chemotherapy .", "twenty patients ( 42.6% ) attempted conception after chemotherapy , 19 of whom ( 95% ) were successful ; 14 healthy live births were recorded , with no documented birth defects.5 in patients with ovarian germ - cell tumors , a recurrence in the contralateral ovary still could be treated by a local resection or cystectomy followed by chemotherapy if fertility was desired , thereby preserving some normal ovarian tissue , if present.5 however , there is little information about recurrence in the contralateral ovary in patients with immature teratomas . in the present case ,", "we re - performed conservative surgery for contralateral recurrence of an ovarian immature teratoma . as such cases", "are too uncommon to offer evidence for or against repeated fertility - conserving surgery , such surgery should be performed only after obtaining informed consent .", "histological grading of peritoneal implants , from grade 0 to grade 3 , is essential for therapeutic and prognostic considerations .", "grade 0 is defined by tumor consisting of only mature tissue with no mitotic activity .", "gliomatosis peritonei is the metastatic implantation of the mature glial tissue in the peritoneal cavity of patients with ovarian teratomas.3 the prognosis for gliomatosis peritonei should be very good and no further therapy should be necessary .", "peritoneal implants consisting of embryonic tissue and metastatic immature teratomas imply a poor prognosis.6 if no other teratomatous elements or malignant glial tissue can be found in the implants , the mature glial implants can be ignored and the methods of therapy should be judged only by the stage and grade of the primary ovarian teratoma.7 progression of the peritoneal implants may be as follows : ( 1 ) transforming fibroblastic and eventual disappearance ; ( 2 ) transformation to malignant tissue ( glial or teratomatous ) ; and ( 3 ) persistence without morphological changes.8 in the present case , gliomatosis peritonei was initially classified as grade 0 . because the peritoneal implants were not malignant and shrank over the 8 years , we thought them to be of the first stage ( 1 ) described above .", "although the gliomatosis peritonei shrank , a lymph node glioma was detected in the fourth surgery .", "presence of glial tissue in lymph nodes is rare , with very few reported cases .", "patients with intraperitoneal and lymph node metastases of mature glial tissue do not need therapy for such metastases .", "the prognosis for these patients is excellent , but they require long - term follow - up ."], "abstract_text": ["<S> we report a patient who has maintained a regular menstrual cycle despite undergoing cystectomy and chemotherapy for contralateral recurrence of ovarian immature teratoma with gliomatosis peritonei . </S>", "<S> we initially performed a fertility - sparing right salpingo - oophorectomy , omentectomy and peritoneal biopsy for immature teratoma with gliomatosis peritonei , with adjuvant chemotherapy ; we performed a left ovarian cystectomy and peritoneal biopsy for mature cystic teratoma with gliomatosis peritonei 16 months after the first surgery , a fertility - sparing left ovarian cystectomy and peritoneal biopsy for contralateral recurrence of ovarian immature teratoma with gliomatosis peritonei 60 months after the first surgery , and a left ovarian cystectomy and peritoneal and external iliac lymph node biopsy for endometrial cyst with gliomatosis peritonei 71 months after first surgery . </S>", "<S> the peritoneal gliomatosis lesions gradually decreased through the 4 surgeries over 8 years . </S>", "<S> the patient has maintained a regular menstrual cycle and currently shows no evidence of disease . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["it is commonly treated with conservative surgery ; prognosis is rapidly improved by performing concomitant chemotherapy after the initial surgery.1,2 gliomatosis peritonei is the metastatic implantation of mature glial tissue within the peritoneal cavity of patients with ovarian teratomas.3 as second - look or secondary debulking surgery is not generally performed for immature teratoma , as gliomatosis peritonei is not usually discovered in a patient who has completed chemotherapy and shows no evidence of disease.4 this is a rare case of a patient with ovarian teratoma recurrence in the contralateral ovary .", "the first and later occurrences were treated with conservative surgery , using local resections or cystectomies ."], ["gravia 0 , para 0was referred to us because of a lower abdominal mass ; magnetic resonance ( mr ) imaging revealed a tumor 20 cm in size .", "tumor marker tests revealed cancer antigen 125 = 279 u / ml , ca19 - 9 = 130.6 u / ml , squamous cell carcinoma = 3.9 ng / ml , and alpha - fet protein = 50.6 ng / ml . after providing informed consent , she selected conservative surgery .", "we performed a right salpingo - oophorectomy , partial omentectomy and peritoneal biopsy , and found lint - like scattered mass lesions 2 mm in size at the vesi - couterine pouch and the douglas pouch .", "the tumor is a mass with cystic regions of many sizes , with solid parts and adipocyte - laden parts ( fig .", "the diagnosis was stage ii c(a ) immature teratoma grade2 of the right ovary with gliomatosis peritonei .", "pathologically , the mass contained squamous epithelium , adipose tissue , bone and cartilage tissue , and immature neuroectodermal tissue , and was therefore classified as a grade 2 immature teratoma ( fig .", "peritoneal lesions , which were composed of mature glial cells , were diagnosed as gliomatosis peritonei using hematoxylin and eosin ( h&e ) stain ( fig .", "immunohistochemically , the glial cells were positive for glial fibrillary acidic protein ( gfap ) , which is expressed by numerous cell types of the central nervous system , including astrocytes , ependymal cells and glial cells ( fig .", "sixteen months after the first operation , the patient underwent cystectomy of a left ovarian tumor and a douglas pouch biopsy . pathological diagnosis was mature cystic teratoma of left ovary and gliomatosis peritonei ( fig .", "sixty months after the first operation , mr imaging revealed a left cystic ovarian tumor with solid parts 7 cm in size .", "pathologically , the mass contained immature neuroectodermal tissue , and so was classified as a grade 1 immature teratoma and gliomatosis peritonei ( fig . 2f and g ) .", "71 months after the first operation , cystectomy of a left ovarian tumor5 cm in size  and biopsies of the douglas pouch and left external iliac lymph node8 mm in size  were carried out .", "the pathological diagnosis was endometrial cyst of left ovary , gliomatosis peritonei and glial implant in lymph node ( fig .", "although this patient has never conceived a child , she has maintained a regular menstrual cycle , despite multiple surgeries for ovarian teratoma and chemotherapy ."], ["immature teratoma of grades 1 and 2 are considered ovarian germ cell borderline tumors , according to the world health organization s classification .", "as they occur predominantly in women in their teens and 20 s , conservative surgery is performed as a standard ; concomitant chemotherapy after the initial surgery has led to rapid improvements in prognosis.1,2 jefferys et al produced a retrospective study of 47 patients with malignant ovarian germ - cell tumors who were treated by conservative surgery and adjuvant chemotherapy . during chemotherapy , 61.7% ( 29/47 ) of patients developed amenorrhea but 91.5% of these women resumed normal menstrual function upon completion of chemotherapy .", "twenty patients ( 42.6% ) attempted conception after chemotherapy , 19 of whom ( 95% ) were successful ; 14 healthy live births were recorded , with no documented birth defects.5 in patients with ovarian germ - cell tumors , a recurrence in the contralateral ovary still could be treated by a local resection or cystectomy followed by chemotherapy if fertility was desired , thereby preserving some normal ovarian tissue , if present.5 however , there is little information about recurrence in the contralateral ovary in patients with immature teratomas . in the present case ,", "we re - performed conservative surgery for contralateral recurrence of an ovarian immature teratoma . as such cases", "are too uncommon to offer evidence for or against repeated fertility - conserving surgery , such surgery should be performed only after obtaining informed consent .", "histological grading of peritoneal implants , from grade 0 to grade 3 , is essential for therapeutic and prognostic considerations .", "grade 0 is defined by tumor consisting of only mature tissue with no mitotic activity .", "gliomatosis peritonei is the metastatic implantation of the mature glial tissue in the peritoneal cavity of patients with ovarian teratomas.3 the prognosis for gliomatosis peritonei should be very good and no further therapy should be necessary .", "peritoneal implants consisting of embryonic tissue and metastatic immature teratomas imply a poor prognosis.6 if no other teratomatous elements or malignant glial tissue can be found in the implants , the mature glial implants can be ignored and the methods of therapy should be judged only by the stage and grade of the primary ovarian teratoma.7 progression of the peritoneal implants may be as follows : ( 1 ) transforming fibroblastic and eventual disappearance ; ( 2 ) transformation to malignant tissue ( glial or teratomatous ) ; and ( 3 ) persistence without morphological changes.8 in the present case , gliomatosis peritonei was initially classified as grade 0 . because the peritoneal implants were not malignant and shrank over the 8 years , we thought them to be of the first stage ( 1 ) described above .", "although the gliomatosis peritonei shrank , a lymph node glioma was detected in the fourth surgery .", "presence of glial tissue in lymph nodes is rare , with very few reported cases .", "patients with intraperitoneal and lymph node metastases of mature glial tissue do not need therapy for such metastases .", "the prognosis for these patients is excellent , but they require long - term follow - up ."]]}
{"article_id": "PMC5026832", "article_text": ["cesarean delivery rates have been increasing in a fast manner throughout the world within past few decades ( 1 ) .", "although reasonable cesarean rates have been proposed as 510% by who , cesarean delivery rates across the world varies between 0.4% and 41% ( 2 ) .", "while cesarean birth rates had reached 28% in usa , 21% in canada , it is around 37% in brazil , 39% in mexico , 40% in china ( 2 , 3 ) .", "cesarean birth rates are also increasing in turkey in parallel to the developments in the world .", "while 6.0% of all births were realized by cesarean section in 1998 , this rate has risen to 48.1% in 2013 ( 4 , 5 ) .", "the birth and postpartum processes can imply significant risks for both mother and infant health .", "one of the most important conditions , which have effect on these risks during the birth process is the way of delivery .", "these complications may include staying at intensive care unit , postpartum depression , infection , thrombosis , hysterectomy , bleeding , blood transfusion and internal artery ligation related to maternal health , and iatrogenic prematurity , pulmonary hypertension , inability to breastfeeding , fetal respiratory syndrome in relation to newborn ( 68 ) .", "cesarean delivery rates are influenced by many non - medical factors such as cultural factors , personal characteristics of the woman and socioeconomic features ( 9 ) .", "the purpose of this study was to determine cesarean birth rates and to find out social factors affecting the cesarean birth in primiparous women .", "this study was conducted in burdur province , turkey between the dates of 1 jan 201231 dec 2012 .", "there are 78 family health units and four general hospitals including three public , and one private sector in the province of the study .", "pregnancies and pregnancy outcomes ( abortion , stillbirth and live birth ) are reported to provincial public health directorate by the hospitals and family physicians .", "a total of 737 childbirths were realized between the dates of 1 jan31 mar 2012 .", "the universe of this cross - sectional type study was formed by 223 primiparous women .", "ninety - six percent of the universe was reached ( 214/223 ) . the most prominent reasons for inability to reach the women were absence at the address given ( 2 women ) , permanent migration out of the province ( 1 woman ) and temporary visit to their parents residing in neighboring provinces ( 6 women ) .", "data collection form was made up of the questionnaire containing women s sociodemographic , biodemographic , birth characteristics and babies gender and weight .", "the dependent variable of the study is cesarean delivery , and independent variables are the factors related to women s sociodemographic , socioeconomic , health features , health behavior and violence .", "the data was collected using face - to - face interview technique after getting verbal consent from the woman , by midwives working in community health center between the dates of 15 apr31 may 2012 after necessary permissions were obtained from public health directorate .", "the midwives who would collect data were provided 3-h training , which includes the aim of the study , what each of questions targeted and the circumstances required to be considered at the stage of data collection in order to ensure standardization before data collection .", "the data was analyzed in spss 20 ( chicago , il , usa ) packaged software . in these analyses ,", "chi - square and backward lr logistic regression analyses were used , odds ratio and confidence interval was calculated .", "the independent variables ( p<0.05 ) which resulted as statistically significant in chi - square analyses , have been taken into backward logistic regression analyses .", "the universe of this cross - sectional type study was formed by 223 primiparous women .", "the most prominent reasons for inability to reach the women were absence at the address given ( 2 women ) , permanent migration out of the province ( 1 woman ) and temporary visit to their parents residing in neighboring provinces ( 6 women ) .", "data collection form was made up of the questionnaire containing women s sociodemographic , biodemographic , birth characteristics and babies gender and weight .", "the dependent variable of the study is cesarean delivery , and independent variables are the factors related to women s sociodemographic , socioeconomic , health features , health behavior and violence .", "the data was collected using face - to - face interview technique after getting verbal consent from the woman , by midwives working in community health center between the dates of 15 apr31 may 2012 after necessary permissions were obtained from public health directorate .", "the midwives who would collect data were provided 3-h training , which includes the aim of the study , what each of questions targeted and the circumstances required to be considered at the stage of data collection in order to ensure standardization before data collection .", "the data was analyzed in spss 20 ( chicago , il , usa ) packaged software . in these analyses ,", "chi - square and backward lr logistic regression analyses were used , odds ratio and confidence interval was calculated .", "the independent variables ( p<0.05 ) which resulted as statistically significant in chi - square analyses , have been taken into backward logistic regression analyses .", "when the table is monitored , it is seen that there is statistically significant relationship between the delivery mode and the place of residence , women s age , women s family type and her husband s employment status and presence of health insurance . when it is examined in terms of birth and infants characteristics", ", a significant relationship was shown between the delivery mode with doctor s influence in taking decision and the place of birth ( table 2 ) .", "sociodemographic features of women birth and infant features 3 data is missing in cesarean group the factors affecting caesarean deliveries are seen in table 3 . accordingly , when public hospitals are taken as reference , caesarean deliveries were increased by 11.2 fold in the event of the delivery in private hospital ; 6.1 fold in the event of the delivery in university hospital . again with reference to the conditions of woman s husband work on her own account , working in private sector or public sector increases 2.2 fold the caesarean birth .", "doctor s guidance increases the caesarean 4.0 times with reference to those who say doctor had no impact on the delivery mode . when the deliveries made between 17:0007:59 are taken as reference , caesarean deliveries are increased by 7.1 times between 08:0016:59 .", "women s place of residence , age , presence of health insurance , family type , husband s job , the place of birth , the birth hour and doctor s intervention to delivery mode have been identified as factors affecting the caesarean section delivery in univariate analyses performed .", "meanwhile in multivariate analyses , the place of delivery , time of the birth , doctor s effect , and husband s employment status have been identified as factors affecting the caesarean delivery among these factors .", "caesarean deliveries realized 11.2 times more in private hospitals and 6.1 times more in university hospitals in primiparous with reference to public hospitals in the study conducted .", "it was demonstrated that caesarean deliveries were increased by 12.7 times in university and top level hospitals with reference to deliveries of primiparous women in second - line hospitals in a study conducted ( 3 ) .", "caesarean deliveries are particularly among the preferable methods in terms of avoiding the complications associated with the childbirth .", "caesarean deliveries are preferred especially for pregnant women at risk due to possible complications in terms of mother and infant during delivery , cephalopelvic disproportion or high birth weight infants . in such cases ,", "if pregnant women are followed at 3rd step health institutions or are guided for delivery by 2nd step institutions , then it may increase caesarean deliveries in these institutions .", "one of the surprising aspects of the study was the 11.2 fold increase in caesarean delivery rates at private hospitals in spite of its equivalent adequacy with 2nd step health institutions in terms of the equipment with reference to public hospitals .", "caesarean delivery rates in private hospitals are significantly more than public hospitals in the studies performed ( 10 , 11 ) .", "this increment is rooted in social and economic factors as well as medical reasons . in our study , working female ratio is around 50% both in case and control groups . and a very small portion of the husbands are unemployed .", "the employed status of the husband regardless of private or public or his own account , means the woman is also under the insurance coverage . on the other hand , employed status of the husband", "caesarean deliveries are more in the women with any health insurance compared to those without insurance in all studies conducted ( 3 , 12 , 13 ) .", "this situation should be considered in conjunction with the finding of caesarean deliveries in private hospitals are , more which is another finding of the study .", "caesarean deliveries within working hours are 7.4 times more with reference to out of working hours ( 18:0008:00 ) in our study .", "caesarean operations increased on fridays and between 06:00 a.m. and 06:00 p.m. with respect to primaparous ( 14 ) . meanwhile in another study , a relationship was shown between the time of caesarean section and insurance status .", "while the time of caesarean shows dispersed within a day in kaiser type insured women , there was an increase between 4 in the morning and 6 in the evening in other type insured women having caesarean .", "the caesarean section rate is the lowest between 10 at night and 6 in the morning in all insured groups ( 15 ) .", "caesarean deliveries are also the operations performed with the patient s consent in line with physician s clinical evaluations and conviction just like in all other medical procedures .", "physicians can offer particular options about delivery mode even though a specific preference is expressed for the patient .", "if the caesarean delivery is planned , caesarean delivery will take place , however if the plan is vaginal delivery then the delivery may be realized through vaginal or caesarean section ( 16 ) .  elective caesarean ", "elective caesarean deliveries are estimated to be around 418% of all caesarean sections and 2% of all deliveries ( 17 ) .", "31.1% of the women who gave birth through caesarean section have decided caesarean delivery without doctor s influence in the study . on the other hand", ", caesarean delivery rates increase by 4.03 times with reference to those without doctor s effect for the determination of delivery mode .", "we believe that the increase with doctor s effect in caesarean deliveries occurs in two ways .", "even if the woman herself decides the delivery mode , beforehand women are informed by physicians about benefits or risks of caesarean delivery ( 16 ) or physicians directly affect the patient s decision - making process in caesarean practices due to the knowledge asymmetry between the patient and the physician ( 14 ) .", "caesarean deliveries are preferred because of physicians habits , caesarean deliveries bring more income and vaginal births take more time and defensive medicine application due to the fear of malpractice and complication during birth or pregnancy ( 18 ) .", "although the results do not show all of the factors affecting the caesarean delivery in primiparous , they also reveal that medical reasons are not the only reason in this increase trend .", "health policy makers and health professionals are required to identify the causes of this increase and to take measures .", "the strong side of the study is taking only the women who gave first birth in the study and reaching the complete universe of the study . on the other hand , indetermination and non - exclusion of caesarean sections with medical causes in study questions", "ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors ."], "abstract_text": ["<S> background : p cesarean delivery rates have been increasing throughout the world . </S>", "<S> parallel to the developments in the world the cesarean rate in turkey has risen to 48.1% in 2013 . </S>", "<S> some of the social factors were related with cesarean births . </S>", "<S> the purpose of this study was to determine cesarean birth rates and to find out social factors affecting the cesarean birth in primiparous women.methods:this study was conducted in burdur province , turkey between the dates of 1 jan 201231 dec 2012 on 223 primiparous women . </S>", "<S> the data was collected with data collection form prepared by the researchers by using face - to - face interview technique . in these analyses , </S>", "<S> chi - square and backward logistic regression analyses were used.results:in multivariate analyses , the place of delivery ( or : 11.2 [ 2.942.46 ] in private hospital and or : 6.1 [ 2.614.1 ] in university hospital ) ; time of the birth ( or : 7.1 [ 3.116.0 ] ) ; doctor s effect ( or : 4.0 [ 1.88.95 ] ) and husband s employment status ( or : 2.23 [ 1.04.7 ] ) have been identified as factors affecting the caesarean delivery in primiparous women.conclusion:although the results do not show all of the factors affecting the caesarean delivery in primiparous women , they reveal that medical reasons are not the only reason in this increase trend . </S>", "<S> health policy makers and health professionals are required to identify the causes of this increase and to take measures . </S>"], "labels": null, "section_names": ["Introduction", "Materials & Methods", "Type, universe and sampling of the research", "Variables of the research", "Collection of research data", "Statistical Analyses", "Results", "Discussion", "Conclusion", "Ethical considerations"], "sections": [["cesarean delivery rates have been increasing in a fast manner throughout the world within past few decades ( 1 ) .", "although reasonable cesarean rates have been proposed as 510% by who , cesarean delivery rates across the world varies between 0.4% and 41% ( 2 ) .", "while cesarean birth rates had reached 28% in usa , 21% in canada , it is around 37% in brazil , 39% in mexico , 40% in china ( 2 , 3 ) .", "cesarean birth rates are also increasing in turkey in parallel to the developments in the world .", "while 6.0% of all births were realized by cesarean section in 1998 , this rate has risen to 48.1% in 2013 ( 4 , 5 ) .", "the birth and postpartum processes can imply significant risks for both mother and infant health .", "one of the most important conditions , which have effect on these risks during the birth process is the way of delivery .", "these complications may include staying at intensive care unit , postpartum depression , infection , thrombosis , hysterectomy , bleeding , blood transfusion and internal artery ligation related to maternal health , and iatrogenic prematurity , pulmonary hypertension , inability to breastfeeding , fetal respiratory syndrome in relation to newborn ( 68 ) .", "cesarean delivery rates are influenced by many non - medical factors such as cultural factors , personal characteristics of the woman and socioeconomic features ( 9 ) .", "the purpose of this study was to determine cesarean birth rates and to find out social factors affecting the cesarean birth in primiparous women ."], ["this study was conducted in burdur province , turkey between the dates of 1 jan 201231 dec 2012 .", "there are 78 family health units and four general hospitals including three public , and one private sector in the province of the study .", "pregnancies and pregnancy outcomes ( abortion , stillbirth and live birth ) are reported to provincial public health directorate by the hospitals and family physicians .", "a total of 737 childbirths were realized between the dates of 1 jan31 mar 2012 .", "the universe of this cross - sectional type study was formed by 223 primiparous women .", "ninety - six percent of the universe was reached ( 214/223 ) . the most prominent reasons for inability to reach the women were absence at the address given ( 2 women ) , permanent migration out of the province ( 1 woman ) and temporary visit to their parents residing in neighboring provinces ( 6 women ) .", "data collection form was made up of the questionnaire containing women s sociodemographic , biodemographic , birth characteristics and babies gender and weight .", "the dependent variable of the study is cesarean delivery , and independent variables are the factors related to women s sociodemographic , socioeconomic , health features , health behavior and violence .", "the data was collected using face - to - face interview technique after getting verbal consent from the woman , by midwives working in community health center between the dates of 15 apr31 may 2012 after necessary permissions were obtained from public health directorate .", "the midwives who would collect data were provided 3-h training , which includes the aim of the study , what each of questions targeted and the circumstances required to be considered at the stage of data collection in order to ensure standardization before data collection .", "the data was analyzed in spss 20 ( chicago , il , usa ) packaged software . in these analyses ,", "chi - square and backward lr logistic regression analyses were used , odds ratio and confidence interval was calculated .", "the independent variables ( p<0.05 ) which resulted as statistically significant in chi - square analyses , have been taken into backward logistic regression analyses ."], ["the universe of this cross - sectional type study was formed by 223 primiparous women .", "the most prominent reasons for inability to reach the women were absence at the address given ( 2 women ) , permanent migration out of the province ( 1 woman ) and temporary visit to their parents residing in neighboring provinces ( 6 women ) ."], ["data collection form was made up of the questionnaire containing women s sociodemographic , biodemographic , birth characteristics and babies gender and weight .", "the dependent variable of the study is cesarean delivery , and independent variables are the factors related to women s sociodemographic , socioeconomic , health features , health behavior and violence ."], ["the data was collected using face - to - face interview technique after getting verbal consent from the woman , by midwives working in community health center between the dates of 15 apr31 may 2012 after necessary permissions were obtained from public health directorate .", "the midwives who would collect data were provided 3-h training , which includes the aim of the study , what each of questions targeted and the circumstances required to be considered at the stage of data collection in order to ensure standardization before data collection ."], ["the data was analyzed in spss 20 ( chicago , il , usa ) packaged software . in these analyses ,", "chi - square and backward lr logistic regression analyses were used , odds ratio and confidence interval was calculated .", "the independent variables ( p<0.05 ) which resulted as statistically significant in chi - square analyses , have been taken into backward logistic regression analyses ."], ["when the table is monitored , it is seen that there is statistically significant relationship between the delivery mode and the place of residence , women s age , women s family type and her husband s employment status and presence of health insurance . when it is examined in terms of birth and infants characteristics", ", a significant relationship was shown between the delivery mode with doctor s influence in taking decision and the place of birth ( table 2 ) .", "sociodemographic features of women birth and infant features 3 data is missing in cesarean group the factors affecting caesarean deliveries are seen in table 3 . accordingly , when public hospitals are taken as reference , caesarean deliveries were increased by 11.2 fold in the event of the delivery in private hospital ; 6.1 fold in the event of the delivery in university hospital . again with reference to the conditions of woman s husband work on her own account , working in private sector or public sector increases 2.2 fold the caesarean birth .", "doctor s guidance increases the caesarean 4.0 times with reference to those who say doctor had no impact on the delivery mode . when the deliveries made between 17:0007:59 are taken as reference , caesarean deliveries are increased by 7.1 times between 08:0016:59 ."], ["women s place of residence , age , presence of health insurance , family type , husband s job , the place of birth , the birth hour and doctor s intervention to delivery mode have been identified as factors affecting the caesarean section delivery in univariate analyses performed .", "meanwhile in multivariate analyses , the place of delivery , time of the birth , doctor s effect , and husband s employment status have been identified as factors affecting the caesarean delivery among these factors .", "caesarean deliveries realized 11.2 times more in private hospitals and 6.1 times more in university hospitals in primiparous with reference to public hospitals in the study conducted .", "it was demonstrated that caesarean deliveries were increased by 12.7 times in university and top level hospitals with reference to deliveries of primiparous women in second - line hospitals in a study conducted ( 3 ) .", "caesarean deliveries are particularly among the preferable methods in terms of avoiding the complications associated with the childbirth .", "caesarean deliveries are preferred especially for pregnant women at risk due to possible complications in terms of mother and infant during delivery , cephalopelvic disproportion or high birth weight infants . in such cases ,", "if pregnant women are followed at 3rd step health institutions or are guided for delivery by 2nd step institutions , then it may increase caesarean deliveries in these institutions .", "one of the surprising aspects of the study was the 11.2 fold increase in caesarean delivery rates at private hospitals in spite of its equivalent adequacy with 2nd step health institutions in terms of the equipment with reference to public hospitals .", "caesarean delivery rates in private hospitals are significantly more than public hospitals in the studies performed ( 10 , 11 ) .", "this increment is rooted in social and economic factors as well as medical reasons . in our study , working female ratio is around 50% both in case and control groups . and a very small portion of the husbands are unemployed .", "the employed status of the husband regardless of private or public or his own account , means the woman is also under the insurance coverage . on the other hand , employed status of the husband", "caesarean deliveries are more in the women with any health insurance compared to those without insurance in all studies conducted ( 3 , 12 , 13 ) .", "this situation should be considered in conjunction with the finding of caesarean deliveries in private hospitals are , more which is another finding of the study .", "caesarean deliveries within working hours are 7.4 times more with reference to out of working hours ( 18:0008:00 ) in our study .", "caesarean operations increased on fridays and between 06:00 a.m. and 06:00 p.m. with respect to primaparous ( 14 ) . meanwhile in another study , a relationship was shown between the time of caesarean section and insurance status .", "while the time of caesarean shows dispersed within a day in kaiser type insured women , there was an increase between 4 in the morning and 6 in the evening in other type insured women having caesarean .", "the caesarean section rate is the lowest between 10 at night and 6 in the morning in all insured groups ( 15 ) .", "caesarean deliveries are also the operations performed with the patient s consent in line with physician s clinical evaluations and conviction just like in all other medical procedures .", "physicians can offer particular options about delivery mode even though a specific preference is expressed for the patient .", "if the caesarean delivery is planned , caesarean delivery will take place , however if the plan is vaginal delivery then the delivery may be realized through vaginal or caesarean section ( 16 ) .  elective caesarean ", "elective caesarean deliveries are estimated to be around 418% of all caesarean sections and 2% of all deliveries ( 17 ) .", "31.1% of the women who gave birth through caesarean section have decided caesarean delivery without doctor s influence in the study . on the other hand", ", caesarean delivery rates increase by 4.03 times with reference to those without doctor s effect for the determination of delivery mode .", "we believe that the increase with doctor s effect in caesarean deliveries occurs in two ways .", "even if the woman herself decides the delivery mode , beforehand women are informed by physicians about benefits or risks of caesarean delivery ( 16 ) or physicians directly affect the patient s decision - making process in caesarean practices due to the knowledge asymmetry between the patient and the physician ( 14 ) .", "caesarean deliveries are preferred because of physicians habits , caesarean deliveries bring more income and vaginal births take more time and defensive medicine application due to the fear of malpractice and complication during birth or pregnancy ( 18 ) ."], ["although the results do not show all of the factors affecting the caesarean delivery in primiparous , they also reveal that medical reasons are not the only reason in this increase trend .", "health policy makers and health professionals are required to identify the causes of this increase and to take measures .", "the strong side of the study is taking only the women who gave first birth in the study and reaching the complete universe of the study . on the other hand , indetermination and non - exclusion of caesarean sections with medical causes in study questions"], ["ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors ."]]}
{"article_id": "PMC4268642", "article_text": ["las fracturas por fragilidad asociadas a la osteoporosis suponen un gran coste econmico y personal para la sociedad .", "la ingesta de calcio farmacutico o diettico es necesaria para aumentar la densidad mineral y prevenir fracturas por fragilidad .", "aunque los productos lcteos son una buena fuente de calcio , los pacientes que no pueden digerir la lactosa tienden a evitarlos y tienen un mayor riesgo de fractura que la poblacin general .", "informes anecdticos indican que las personas que no digieren bien la lactosa , al consumir leche cruda , tienen una gran reduccin de sntomas en comparacin con la leche pasteurizada", ". el mecanismo de la reduccin notificada en los sntomas , de ser verdad , se desconoce .", "el propsito del estudio actual era hacer una encuesta a las personas que beben leche cruda para determinar sus motivaciones relacionadas con la salud para consumir leche cruda , sobre todo en relacin con la mala digestin de la lactosa .", "153 de 1527 miembros de una comunidad que compra leche cruda complet una encuesta online en relacin a la leche cruda .", "el motivo principal que los encuestados citaron para beber leche cruda fue que crean que era ms sano ; el 30 % notific ciertas molestias gastrointestinales al beber leche pasteurizada , aunque casi todos ( 99 % ) notificaron consumir leche cruda sin molestias .", "a pesar de los informes de molestias gastrointestinales , un profesional mdico haba diagnosticado intolerancia a la lactosa en solo el 5 % de los encuestados , y solamente al 1 % se le haba diagnosticado intolerancia a la lactosa mediante el mtodo de referencia de prueba de hidrgeno en el aliento .", "el motivo principal para beber leche cruda es su valor saludable percibido , no su digestibilidad .", "aunque la leche cruda parece ser digerida ms fcilmente que la pasteurizada en la muestra de nuestra encuesta , se desconoce el mecanismo de digestibilidad .", "the popular media occasionally present anecdotal stories regarding the benefits of consuming raw dairy products .", "yet we found no reports in the literature supporting the anecdotal reports that lactose maldigesters are able to consume raw milk without discomfort . also unreported or under debate in the literature", "is the mechanism that allows maldigesters of pasteurized milk to consume raw milk without discomfort .", "if the enzyme is present in raw milk , it is very heat labile and would be denatured during the milk pasteurization process . naturally occurring lactobaccili and lactococci , which are killed during pasteurization ,", "another proposed mechanism of lactose maldigestion is that milk from cows with the a1 -casein variant ( commonly european breeds ) contains a peptide called -casomorphin-7 that reportedly can cause an immune response , lactose intolerance symptoms , and other health - related issues .", "these claims are not well substantiated in the literature and are controversial . further complicating the issue ,", "raw milk is often produced and sold from small family farms that typically use jersey or guernsey cows and pasture their cows on grass .", "jersey and guernsey cows typically have the a2 -casein variant , whereas holstein cows ( primarily used in large industrial dairies ) have the a1 variant .", "so what are perceived by some to be the benefits of raw milk may have as much to do with the type of cow ( a1 vs a2 ) and husbandry ( pastured and grass - fed vs confined and ration - fed ) as with the manner in which the milk is pasteurized ( or not ) .", "we intend to clinically evaluate the digestibility of raw milk vs pasteurized milk , but to guide the design of our study ,", "we need more background information . therefore , we surveyed consumers of raw milk to determine the health - related motivations for consuming raw milk , especially as they relate to lactose maldigestion .", "we developed an online survey of 16 questions , which was available from december 7 , 2012 , until january 31 , 2013 .", "the director of a raw milk  buying consortium in maryland sent an email to its 1527 members , inviting them to complete our online survey . because trafficking in raw milk intended for human consumption is against the law in maryland , survey responses were anonymous .", "this study was approved by our institutional review board , and respondents were informed that completing the survey served as informed consent .", "most of the 153 respondents ( response rate of 10% ) were females between 30 and 50 years of age ( table ) .", "of the 153 respondents , 74% reported drinking at least one glass of raw milk per day .", "results of survey of raw milk drinkers although instructed to choose only one response , 11 respondents chose one of the letter selections , then added a comment under  other ( please specify ) .", " we sought to differentiate those who consumed raw milk because they considered it more healthful than pasteurized milk from those who consumed it because they found it easier to digest .", "the concept of  easy to digest  was based on the absence of bloating , gas , diarrhea , and cramping .", "we queried on the digestibility of drinking milk under three conditions : ( 1 ) the reaction to drinking pasteurized milk before switching to raw milk ; ( 2 ) the reaction experienced currently when drinking raw milk ; and ( 3 ) the reaction of drinking pasteurized milk after having switched to raw milk .", "we also asked if there was a transition period at the onset of drinking raw milk to identify if some colonic adaptation had occurred as a result of drinking raw milk .", "we sought to identify subjects who had been diagnosed by a medical professional as lactose intolerant and if the diagnosis had been confirmed by a hydrogen breath test ( hbt ) .", "we asked about the digestibility of other dairy products , namely yogurt , cheese , and ice cream .", "it has long been known that in parts of the world where prevalence of lactose maldigestion is high , those populations nevertheless consume large quantities of yogurt .", "fermented products such as cheese and dairy products with live cultures ( yogurt ) are more easily digested than other dairy because of the presence of microbial -galactosidase . in an attempt to learn more about the potential role the -casein variant ( a1 vs a2 ) has on milk digestibility , we asked from what breed of cow the raw milk came and what type of diet the cows were fed , if the participants knew .", "lastly , we sought to assess the importance of calcium in the motivation for consuming raw milk .", "lactose intolerance is related to reduced milk consumption and therefore reduced calcium intake , which is associated with an increased risk for osteoporotic fractures .", "we analyzed differences in reported discomfort from drinking raw vs pasteurized milk and in drinking pasteurized milk before and after drinking raw milk for significance ( p<.05 ) using mcnemar 's test .", "of the 16 questions on the survey , only four were answered by all respondents , but all were answered by at least 148 ( 96.7% ) ( table ) . of the 153 respondents , most ( 86 ) cited that they consumed raw milk because they believed it to be more healthful and easier to digest .", ": 12 said raw milk tasted better than pasteurized milk ; 8 cited social / environmental concerns such as  supporting local farmers ,   it is better for the cows ,  etc ; 6 cited that they had allergies to pasteurized milk ; two claimed it cured unspecified ills ; and six cited individual reasons such as  i grew up on it  or  [ i ] do n't eat processed food .", "71 respondents reported no discomfort from drinking raw or pasteurized milk . two reported discomfort from drinking raw and pasteurized milk , and one commented that he / she does not consume cow milk but instead drinks goat milk .", "fifty - nine respondents claimed no discomfort after drinking raw milk but discomfort from drinking pasteurized milk .", "one reported no discomfort from drinking pasteurized milk but did report discomfort from drinking raw milk .", "eighteen consumed raw milk without discomfort but reported they  do no drink pasteurized milk . ", "considering the number of respondents who cited discomfort drinking milk , it was surprising that only 5 ( 3% ) respondents were diagnosed as lactose intolerant and only 1 ( 0.7% ) was confirmed lactose intolerant by means of an hbt . in terms of symptoms between before and after beginning to drink raw milk , responses included , ", "( 70 ) ;  do not have discomfort drinking pasteurized milk  ( 41 ) ; and  still get discomfort drinking pasteurized milk  ( 28 ) .", "twenty - six respondents reported no discomfort from drinking pasteurized milk , but nevertheless they do not drink pasteurized milk .", "four reported no previous discomfort but did report discomfort since beginning to drink raw milk .", "five respondents reported previously having trouble drinking pasteurized milk but no symptoms since drinking raw milk .", "two respondents reported not drinking pasteurized milk but symptoms when they did drink pasteurized milk .", "there was no significant reduction in discomfort of the respondents between the before and after drinking raw milk periods .", "in the current study , survey results suggest that the motivation for raw milk consumption is complex .", "we hypothesized that one motivation for raw milk consumption was to avoid symptoms associated with lactose maldigestion .", "yet almost half of the respondents indicated that they had no ill effects after drinking pasteurized milk . despite the number of respondents indicating some gastrointestinal ( gi ) discomfort when", "having previously consumed pasteurized milk , only 5 ( 3% ) respondents were diagnosed as lactose intolerant , and only 1 ( 0.7% ) was confirmed lactose intolerant by means of an hbt .", "these findings suggest that the symptoms of lactose intolerance were mild enough to not warrant the respondents ' seeking a medical diagnosis or that the respondents self - diagnosed and sought alternatives to pasteurized milk .", "diagnosing lactose maldigestion and the severity of the maldigestion would seem straightforward using the hbt .", "it is intriguing that a sizable percentage of those exhibiting lactose maldigestion symptoms have not been diagnosed by a medical professional as being lactose intolerant .", "it would be interesting to determine the hbt diagnosis of those who report gi discomfort .", "some indicate that what is self - diagnosed as lactose intolerance / maldigestion may be a misinterpretation of gi dysbiosis or irritable bowel syndrome .", "it may also be a self - fulfilling diagnosis because those who consider themselves to be lactose maldigesters tend to avoid dairy , which causes a change in the gut microbiota , making the bacteria less adept at decomposing lactose .", "others have found that those diagnosed as lactose maldigesters could ingest modest amounts of milk per day without incident and that symptoms associated with lactose maldigestion were misattributed .", "the underlying mechanism by which lactose in raw milk may be more readily digested , if in fact that is the case , is more difficult to determine .", "the predominant bacteria in raw milk are lactococcus lactis ( strains of which are used commercially as starter cultures in cheese making ) , but the bacteria are virtually eliminated when milk is pasteurized .", "it is likely that these native bacteria account for the alleged enhanced digestibility of raw milk over pasteurized milk , but we found no report in the literature of a direct clinical comparison . in one study ,", "yogurt with active bacterial colonies fed to maldigesters was more readily digested than pasteurized yogurt or milk with active cultures .", "therefore , yogurt maintains the stomach at a higher ph than milk , which protects the bacteria . in one study , heating yogurt and reintroducing live bacteria up to 10 bacteria", "/ ml concentrations was not as effective as standard yogurt with 10 bacteria / ml in reducing h2 in lactose maldigesters .", "furthermore , certain strains of active bacteria appear to be more efficient than others at digesting lactose .", "exposing the intestinal microflora to lactose allows the lactose - fermenting non  hydrogen - producing organisms to thrive .", "the resulting colonic adaptation results in lactose being metabolized to short - chain fatty acids and lactate instead of hydrogen , which is the main component in flatus .", "this colonic adaptation may confound the study results , which is why we included the question regarding discomfort from drinking pasteurized milk after the respondent had been consuming raw milk .", "our results did not indicate any evidence of colonic adaptation , but our results may be skewed because most respondents , after drinking raw milk , reported never drinking pasteurized milk , so it is impossible to know how they would react to pasteurized milk consumption .", "other investigators have suggested a placebo effect instead of colonic adaptation because clinical symptoms of maldigestion ( eg , bloating , diarrhea ) decreased in lactose - treated and control groups . yet", "objective analysis of h2 excretion decreased significantly in the lactose - exposed group compared with controls , suggesting some adaptation . in our survey ,", "95% of the respondents reported eating yogurt with active cultures at least once a month .", "our survey did not ask if the yogurt with active cultures consumption was similar before and after raw milk consumption , so it is unknown what role yogurt might have in colonic adaptation in our survey group .", "however , after the survey , we learned that some of the respondents made their own cheese and ice cream from raw milk .", "therefore , it is unknown what percentage of responses was associated with pasteurized commercial cheese and ice cream compared with raw - milk cheese and ice cream .", "when asked about drinking raw milk , almost 99% of respondents indicated no ill effects .", "this result suggests that , in the 20% to 30% who initially reported gi discomfort drinking pasteurized milk , the symptoms abated when consuming raw milk .", "although these findings might argue the case that raw milk is more easily digested , it is unlikely that the result would apply to all who experience gi discomfort when consuming pasteurized milk because of a selection bias in our data set .", "it is unlikely that those who would experience gi discomfort from drinking raw milk would be part of a raw dairy  buying club .", "thus , there is likely an overestimate of those lactose maldigesters who would benefit from raw milk .", "that 99% of the respondents could drink raw milk without incident is high but consistent with the published finding that 82% of patients diagnosed as lactose intolerant after ingesting pasteurized milk were capable of ingesting raw milk without incident .", "the patients in that survey were self - reporting their diagnosis by a healthcare professional , but the definition of  healthcare professional  was unclear .", "nevertheless , the information contained in that report is the best currently available for comparison .", "the current finding suggests that , at least among the group of survey respondents , milk is an important source of calcium .", "one might surmise that for those who are not able to digest milk easily , milk as a source of calcium may be eliminated from their consideration . in terms of osteoporosis prevention , it should be noted that calcium absorption from milk was not affected by lactose maldigestion status . the reduction in calcium intake in patients with lactose maldigestion", "more than 75% of the respondents reported consuming at least one glass of milk per day .", "the size of the glass was not specified , so it is unknown what magnitude of nutrients would be available for ingestion on a daily basis .", "finally , the role of a1/a2 -casein variant in cow genetics and the role of husbandry on digestibility remain elusive .", "almost 90% of the respondents reported that their milk came from grass - fed cows .", "although these results are consistent with expectations of the cows and husbandry that are associated with raw milk for human consumption , it is unknown how reliable the reporting of this information is .", "more than half of the respondents readily admitted not knowing from which breed of cow their milk came ."], "abstract_text": ["<S> background : fragility fractures associated with osteoporosis extract a large financial and personal toll on society . pharmaceutical or dietary calcium intake is needed to increase bone mineral density to prevent fragility fractures . </S>", "<S> although dairy products are a good source of calcium , patients who are unable to digest lactose tend to avoid them and are put at a greater risk for fracture than the general population . </S>", "<S> anecdotal reports suggest that lactose maldigesters , when consuming raw milk , have a dramatic reduction in symptoms relative to pasteurized milk . </S>", "<S> the mechanism of the reported reduction in symptoms , if true , is unknown . </S>", "<S> the purpose of the current study was to survey raw milk drinkers to ascertain their health - related motivations for consuming raw milk , especially as they relate to lactose maldigestion.methods:an online survey regarding raw milk was completed by 153 of 1527 members of a raw milk  buying community.results:the primary reason the respondents cited for drinking raw milk was that they believed it was more healthful ; 30% reported some gastrointestinal discomfort when drinking pasteurized milk , yet almost all ( 99% ) reported consuming raw milk without discomfort . </S>", "<S> despite the reports of gastrointestinal discomfort , only 5% of respondents had been diagnosed as lactose intolerant by a medical professional , and only 1% had been diagnosed as lactose intolerant via the gold - standard hydrogen breath test.conclusions:the primary motivation for drinking raw milk is its perceived health value , not its digestibility . </S>", "<S> although raw milk appears to be more easily digested than pasteurized milk in our survey sample , the mechanism of digestibility remains unknown . </S>"], "labels": null, "section_names": ["Antecedentes:", "Mtodos:", "Resultados:", "Conclusiones:", "INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["las fracturas por fragilidad asociadas a la osteoporosis suponen un gran coste econmico y personal para la sociedad .", "la ingesta de calcio farmacutico o diettico es necesaria para aumentar la densidad mineral y prevenir fracturas por fragilidad .", "aunque los productos lcteos son una buena fuente de calcio , los pacientes que no pueden digerir la lactosa tienden a evitarlos y tienen un mayor riesgo de fractura que la poblacin general .", "informes anecdticos indican que las personas que no digieren bien la lactosa , al consumir leche cruda , tienen una gran reduccin de sntomas en comparacin con la leche pasteurizada", ". el mecanismo de la reduccin notificada en los sntomas , de ser verdad , se desconoce .", "el propsito del estudio actual era hacer una encuesta a las personas que beben leche cruda para determinar sus motivaciones relacionadas con la salud para consumir leche cruda , sobre todo en relacin con la mala digestin de la lactosa ."], ["153 de 1527 miembros de una comunidad que compra leche cruda complet una encuesta online en relacin a la leche cruda ."], ["el motivo principal que los encuestados citaron para beber leche cruda fue que crean que era ms sano ; el 30 % notific ciertas molestias gastrointestinales al beber leche pasteurizada , aunque casi todos ( 99 % ) notificaron consumir leche cruda sin molestias .", "a pesar de los informes de molestias gastrointestinales , un profesional mdico haba diagnosticado intolerancia a la lactosa en solo el 5 % de los encuestados , y solamente al 1 % se le haba diagnosticado intolerancia a la lactosa mediante el mtodo de referencia de prueba de hidrgeno en el aliento ."], ["el motivo principal para beber leche cruda es su valor saludable percibido , no su digestibilidad .", "aunque la leche cruda parece ser digerida ms fcilmente que la pasteurizada en la muestra de nuestra encuesta , se desconoce el mecanismo de digestibilidad ."], ["the popular media occasionally present anecdotal stories regarding the benefits of consuming raw dairy products .", "yet we found no reports in the literature supporting the anecdotal reports that lactose maldigesters are able to consume raw milk without discomfort . also unreported or under debate in the literature", "is the mechanism that allows maldigesters of pasteurized milk to consume raw milk without discomfort .", "if the enzyme is present in raw milk , it is very heat labile and would be denatured during the milk pasteurization process . naturally occurring lactobaccili and lactococci , which are killed during pasteurization ,", "another proposed mechanism of lactose maldigestion is that milk from cows with the a1 -casein variant ( commonly european breeds ) contains a peptide called -casomorphin-7 that reportedly can cause an immune response , lactose intolerance symptoms , and other health - related issues .", "these claims are not well substantiated in the literature and are controversial . further complicating the issue ,", "raw milk is often produced and sold from small family farms that typically use jersey or guernsey cows and pasture their cows on grass .", "jersey and guernsey cows typically have the a2 -casein variant , whereas holstein cows ( primarily used in large industrial dairies ) have the a1 variant .", "so what are perceived by some to be the benefits of raw milk may have as much to do with the type of cow ( a1 vs a2 ) and husbandry ( pastured and grass - fed vs confined and ration - fed ) as with the manner in which the milk is pasteurized ( or not ) .", "we intend to clinically evaluate the digestibility of raw milk vs pasteurized milk , but to guide the design of our study ,", "we need more background information . therefore , we surveyed consumers of raw milk to determine the health - related motivations for consuming raw milk , especially as they relate to lactose maldigestion ."], ["we developed an online survey of 16 questions , which was available from december 7 , 2012 , until january 31 , 2013 .", "the director of a raw milk  buying consortium in maryland sent an email to its 1527 members , inviting them to complete our online survey . because trafficking in raw milk intended for human consumption is against the law in maryland , survey responses were anonymous .", "this study was approved by our institutional review board , and respondents were informed that completing the survey served as informed consent .", "most of the 153 respondents ( response rate of 10% ) were females between 30 and 50 years of age ( table ) .", "of the 153 respondents , 74% reported drinking at least one glass of raw milk per day .", "results of survey of raw milk drinkers although instructed to choose only one response , 11 respondents chose one of the letter selections , then added a comment under  other ( please specify ) .", " we sought to differentiate those who consumed raw milk because they considered it more healthful than pasteurized milk from those who consumed it because they found it easier to digest .", "the concept of  easy to digest  was based on the absence of bloating , gas , diarrhea , and cramping .", "we queried on the digestibility of drinking milk under three conditions : ( 1 ) the reaction to drinking pasteurized milk before switching to raw milk ; ( 2 ) the reaction experienced currently when drinking raw milk ; and ( 3 ) the reaction of drinking pasteurized milk after having switched to raw milk .", "we also asked if there was a transition period at the onset of drinking raw milk to identify if some colonic adaptation had occurred as a result of drinking raw milk .", "we sought to identify subjects who had been diagnosed by a medical professional as lactose intolerant and if the diagnosis had been confirmed by a hydrogen breath test ( hbt ) .", "we asked about the digestibility of other dairy products , namely yogurt , cheese , and ice cream .", "it has long been known that in parts of the world where prevalence of lactose maldigestion is high , those populations nevertheless consume large quantities of yogurt .", "fermented products such as cheese and dairy products with live cultures ( yogurt ) are more easily digested than other dairy because of the presence of microbial -galactosidase . in an attempt to learn more about the potential role the -casein variant ( a1 vs a2 ) has on milk digestibility , we asked from what breed of cow the raw milk came and what type of diet the cows were fed , if the participants knew .", "lastly , we sought to assess the importance of calcium in the motivation for consuming raw milk .", "lactose intolerance is related to reduced milk consumption and therefore reduced calcium intake , which is associated with an increased risk for osteoporotic fractures .", "we analyzed differences in reported discomfort from drinking raw vs pasteurized milk and in drinking pasteurized milk before and after drinking raw milk for significance ( p<.05 ) using mcnemar 's test ."], ["of the 16 questions on the survey , only four were answered by all respondents , but all were answered by at least 148 ( 96.7% ) ( table ) . of the 153 respondents , most ( 86 ) cited that they consumed raw milk because they believed it to be more healthful and easier to digest .", ": 12 said raw milk tasted better than pasteurized milk ; 8 cited social / environmental concerns such as  supporting local farmers ,   it is better for the cows ,  etc ; 6 cited that they had allergies to pasteurized milk ; two claimed it cured unspecified ills ; and six cited individual reasons such as  i grew up on it  or  [ i ] do n't eat processed food .", "71 respondents reported no discomfort from drinking raw or pasteurized milk . two reported discomfort from drinking raw and pasteurized milk , and one commented that he / she does not consume cow milk but instead drinks goat milk .", "fifty - nine respondents claimed no discomfort after drinking raw milk but discomfort from drinking pasteurized milk .", "one reported no discomfort from drinking pasteurized milk but did report discomfort from drinking raw milk .", "eighteen consumed raw milk without discomfort but reported they  do no drink pasteurized milk . ", "considering the number of respondents who cited discomfort drinking milk , it was surprising that only 5 ( 3% ) respondents were diagnosed as lactose intolerant and only 1 ( 0.7% ) was confirmed lactose intolerant by means of an hbt . in terms of symptoms between before and after beginning to drink raw milk , responses included , ", "( 70 ) ;  do not have discomfort drinking pasteurized milk  ( 41 ) ; and  still get discomfort drinking pasteurized milk  ( 28 ) .", "twenty - six respondents reported no discomfort from drinking pasteurized milk , but nevertheless they do not drink pasteurized milk .", "four reported no previous discomfort but did report discomfort since beginning to drink raw milk .", "five respondents reported previously having trouble drinking pasteurized milk but no symptoms since drinking raw milk .", "two respondents reported not drinking pasteurized milk but symptoms when they did drink pasteurized milk .", "there was no significant reduction in discomfort of the respondents between the before and after drinking raw milk periods ."], ["in the current study , survey results suggest that the motivation for raw milk consumption is complex .", "we hypothesized that one motivation for raw milk consumption was to avoid symptoms associated with lactose maldigestion .", "yet almost half of the respondents indicated that they had no ill effects after drinking pasteurized milk . despite the number of respondents indicating some gastrointestinal ( gi ) discomfort when", "having previously consumed pasteurized milk , only 5 ( 3% ) respondents were diagnosed as lactose intolerant , and only 1 ( 0.7% ) was confirmed lactose intolerant by means of an hbt .", "these findings suggest that the symptoms of lactose intolerance were mild enough to not warrant the respondents ' seeking a medical diagnosis or that the respondents self - diagnosed and sought alternatives to pasteurized milk .", "diagnosing lactose maldigestion and the severity of the maldigestion would seem straightforward using the hbt .", "it is intriguing that a sizable percentage of those exhibiting lactose maldigestion symptoms have not been diagnosed by a medical professional as being lactose intolerant .", "it would be interesting to determine the hbt diagnosis of those who report gi discomfort .", "some indicate that what is self - diagnosed as lactose intolerance / maldigestion may be a misinterpretation of gi dysbiosis or irritable bowel syndrome .", "it may also be a self - fulfilling diagnosis because those who consider themselves to be lactose maldigesters tend to avoid dairy , which causes a change in the gut microbiota , making the bacteria less adept at decomposing lactose .", "others have found that those diagnosed as lactose maldigesters could ingest modest amounts of milk per day without incident and that symptoms associated with lactose maldigestion were misattributed .", "the underlying mechanism by which lactose in raw milk may be more readily digested , if in fact that is the case , is more difficult to determine .", "the predominant bacteria in raw milk are lactococcus lactis ( strains of which are used commercially as starter cultures in cheese making ) , but the bacteria are virtually eliminated when milk is pasteurized .", "it is likely that these native bacteria account for the alleged enhanced digestibility of raw milk over pasteurized milk , but we found no report in the literature of a direct clinical comparison . in one study ,", "yogurt with active bacterial colonies fed to maldigesters was more readily digested than pasteurized yogurt or milk with active cultures .", "therefore , yogurt maintains the stomach at a higher ph than milk , which protects the bacteria . in one study , heating yogurt and reintroducing live bacteria up to 10 bacteria", "/ ml concentrations was not as effective as standard yogurt with 10 bacteria / ml in reducing h2 in lactose maldigesters .", "furthermore , certain strains of active bacteria appear to be more efficient than others at digesting lactose .", "exposing the intestinal microflora to lactose allows the lactose - fermenting non  hydrogen - producing organisms to thrive .", "the resulting colonic adaptation results in lactose being metabolized to short - chain fatty acids and lactate instead of hydrogen , which is the main component in flatus .", "this colonic adaptation may confound the study results , which is why we included the question regarding discomfort from drinking pasteurized milk after the respondent had been consuming raw milk .", "our results did not indicate any evidence of colonic adaptation , but our results may be skewed because most respondents , after drinking raw milk , reported never drinking pasteurized milk , so it is impossible to know how they would react to pasteurized milk consumption .", "other investigators have suggested a placebo effect instead of colonic adaptation because clinical symptoms of maldigestion ( eg , bloating , diarrhea ) decreased in lactose - treated and control groups . yet", "objective analysis of h2 excretion decreased significantly in the lactose - exposed group compared with controls , suggesting some adaptation . in our survey ,", "95% of the respondents reported eating yogurt with active cultures at least once a month .", "our survey did not ask if the yogurt with active cultures consumption was similar before and after raw milk consumption , so it is unknown what role yogurt might have in colonic adaptation in our survey group .", "however , after the survey , we learned that some of the respondents made their own cheese and ice cream from raw milk .", "therefore , it is unknown what percentage of responses was associated with pasteurized commercial cheese and ice cream compared with raw - milk cheese and ice cream .", "when asked about drinking raw milk , almost 99% of respondents indicated no ill effects .", "this result suggests that , in the 20% to 30% who initially reported gi discomfort drinking pasteurized milk , the symptoms abated when consuming raw milk .", "although these findings might argue the case that raw milk is more easily digested , it is unlikely that the result would apply to all who experience gi discomfort when consuming pasteurized milk because of a selection bias in our data set .", "it is unlikely that those who would experience gi discomfort from drinking raw milk would be part of a raw dairy  buying club .", "thus , there is likely an overestimate of those lactose maldigesters who would benefit from raw milk .", "that 99% of the respondents could drink raw milk without incident is high but consistent with the published finding that 82% of patients diagnosed as lactose intolerant after ingesting pasteurized milk were capable of ingesting raw milk without incident .", "the patients in that survey were self - reporting their diagnosis by a healthcare professional , but the definition of  healthcare professional  was unclear .", "nevertheless , the information contained in that report is the best currently available for comparison .", "the current finding suggests that , at least among the group of survey respondents , milk is an important source of calcium .", "one might surmise that for those who are not able to digest milk easily , milk as a source of calcium may be eliminated from their consideration . in terms of osteoporosis prevention , it should be noted that calcium absorption from milk was not affected by lactose maldigestion status . the reduction in calcium intake in patients with lactose maldigestion", "more than 75% of the respondents reported consuming at least one glass of milk per day .", "the size of the glass was not specified , so it is unknown what magnitude of nutrients would be available for ingestion on a daily basis .", "finally , the role of a1/a2 -casein variant in cow genetics and the role of husbandry on digestibility remain elusive .", "almost 90% of the respondents reported that their milk came from grass - fed cows .", "although these results are consistent with expectations of the cows and husbandry that are associated with raw milk for human consumption , it is unknown how reliable the reporting of this information is .", "more than half of the respondents readily admitted not knowing from which breed of cow their milk came ."]]}
{"article_id": "PMC5062637", "article_text": ["the ultrastructural localization of a molecule requires that a certain degree of electron - density , resulting in adequate electron scattering , be present in sufficient amount .", "binding of heavy atoms can be accomplished easily with regards to proteins , nucleic acids and other cellular components , in some cases with a high degree of specificity . however , the sub - cellular localization of ions is a challenge , since in many cases ions are diffusible and not easily kept in place after the procedures of sample preparation for electron microscopy ( em ) .", "many ions can offer enough scattering in spite of not being heavy metals , such as manganese , while others necessitate of blocking or precipitating techniques to be visualized .", "in particular , calcium ions are easily diffusible inside the cell , and even when protein - bound are not easily detectable . the pioneering paper by tandler et al .", "demonstrated the presence of calcium ions by em via pyroantimonate precipitation , with a good yield of the final product . the end product of the reaction , however , is probably overrated , since the technique is capable of stabilizing and precipitating calcium and other bivalent cations , such as magnesium , as proved by electron spectroscopic imaging ( esi ) analysis . since their introduction by tsien and coworkers ,", "calcium studies have used specific ca - sensitive fluorochromes , such as fura-2 , indo-1 and fluo-4 , coupled to wide - field digitized video fluorescence microfluorimetry and confocal microscopy to image acute elevations in intracellular ca concentration ( [ ca]i ) with high spatio - temporal resolution .", "cytosolic and nuclear ca signals regulate a multitude of vital cellular processes , such as gene transcription , cell cycle progression , cytoskeletal and chromatin remodelling , and migration . however , proper ca handling within the endoplasmic reticulum ( er ) , the most abundant intracellular ca reservoir , is also crucial for cell - fate decisions . the steady - state er ca level ( [ ca]er ) does not only control synthesis , folding , export and trafficking of proteins , but it also fuels mitochondrial bioenergetics thereby suppressing autophagy .", "low affinity ca indicators , such as mag - fura 2 , and genetically - encoded probes , such as the bioluminescent protein aequorin and the gfp - based fluorophores camgaroos , pericams , and cameleons , have been devised to assess er ca levels .. nevertheless , these sophisticated tools do not provide any reliable information on ca distribution within the heterogeneous structure of the er . we propose in this paper a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques .", "maranto and sandell and masland have proposed the possibility of photoconverting fluorochromes into an electrondense end product .", "the technique utilizes the photo - oxidation of diaminobenzidine ( dab ) in presence of the emission of an excited fluorochrome , and it has been shown to be efficient on different dyes and conditions .", "this method combines the specificity of available fluorophores to the high spatial resolution offered by transmission em to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles .", "the present manuscript sought to exploit dab photo - oxidation of mag - fura 2 for visualizing er ca distribution in hela cells , which are among the most widespread model cell lines used for studying intracellular ca signaling .", "hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence ,", "cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) .", "the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition .", "the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 .", "all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) .", "intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m .", "cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy .", "an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software .", "instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st .", "louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs .", "thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel", ", some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above .", "moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above . fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature .", "once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) .", "ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv .", "hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence ,", "cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) .", "the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition .", "the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 .", "all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) .", "intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m .", "cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy .", "an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software .", "instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st .", "louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs .", "thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel", ", some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above .", "moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above .", "fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature .", "once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) .", "ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv .", "calcium labeling with mag - fura 2 results in highly fluorescent staining mainly localized in the cytoplasm .", "after incubation of the cells with 20 mm ca for 1 h , the signal is present in large vesicles in the cytoplasm and around the cell nucleus , as well as in elongated structures probably related to mitochondria ( figure 1a ) .", "the faint signal visible in the nucleus is very probably due to the superposition of the cytoplasmic layer surrounding it .", "when the cells are incubated in pss , the signal is lower ( figure 1b ) and mostly present in the perinuclear vesicles , while the cytoplasmic signal far from the nucleus somehow disappears . in the last condition , without calcium and in the presence of egta , some labeling is still visible , although to a much reduced level ( figure 1c ) .", "this continuous presence of ca represents very probably the last ions the cell can not allow to lose , a sort of surviving threshold concentrations .", "supplementary figure 1 confirms that altering extracellular ca concentrations results in a consequent change in er ca homeostasis , which may have important consequences also on cytosolic ca dynamics . at em level", ", the controls did not show any electron dense precipitates , both in the absence of the fluorochrome or after aldehyde blockade ( not shown ) ; consequently , the end product obtained in the mag - fura 2 stained cells is due to the fluorochrome photoconversion .", "the dab photoconversion is an efficient way to stabilize the ions and to visualize them .", "it must be underlined here that we observe a thin section , 60 - 80 nm thick , corresponding , consequently , only to a fraction of the fluorescent signal .", "after pss incubation , the cells show a distinctive positivity ( figure 2a ) . in some cases it is possible to see the presence of precipitates without the direct association with smooth or rough er ( ser or rer , respectively ) membranes , likely related to unbound ca ions", "( figure 2b ) , and electron dense material inside the vesicles is also positive ( figure 2c ) .", "a possible suggestion of ca internalization or extrusion is shown in figure 2 d , e , where the deposits are present on the outer membrane .", "when the cells are incubated without calcium and in the presence of egta , the deposits are extremely reduced , although still present on the outer cell membrane , suggesting the presence of an extruding mechanism ( figure 2f ) . on the contrary ,", "the diffuse , low density end product is almost absent from the cytoplasm . at higher ca concentration in the medium ( figure 3a ) , the dark precipitate is detectable on the inner membrane of cytoplasmic vesicles possibly pertaining to ser , and in discrete foci with no apparent association with vesicles or membranes .", "the deposits are present within electron dense vesicles ( figure 3b ) and electron lucent structure , in some cases associated , and suggesting the idea of passage of ions from one compartment to another .", "in other cases , dab - positive material is present on the membranes of complex systems such as multivesicular bodies ( figure 3c ) or on the outer membranes of cytosolic vesicles ( figure 3d ) .", "interestingly , calcium deposits are detectable on the outer cell membrane ( figure 3e ) in spots , localized and separated , suggesting the presence of calcium channels . in figure 3f ,", "the end product of the dab photoconversion is a speckled , dense precipitate of varying size .", "however , another abundant end product is present as small sized dots , showing a markedly lower electrondensity ( figure 3f , inset ) and a widespread distribution .", "we show in this paper that mag - fura 2 photoconversion via dab oxidation is an efficient way for localizing ca ions at em level . moreover ,", "this could be considered also as a means of stabilizing their presence , thus limiting the loss of ions . as for the method ,", "photoconversion is easily carried out and reproducible , can be obtained on a good amount of cells , since the exposition in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp .", "the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary . in these conditions", ", the blackness of the product stands out from the background . in our conditions , in fact , ca ions are immediately detectable within the vesicles and the tubules of the ser , while it is much more difficult to see their presence when in the cytosol , due to their low electrondensity .", "this is a clear advantage over the conventional imaging of mag - fura 2 fluorescence that must be removed by the cytosol in order to study truly er signals .", "this is achieved by permeabilizing the plasma membrane with brief applications of antibiotics or detergents , but this procedure may destroy intracellular signaling pathways , dilute cytosolic modulators of er ca release / sequestration , and disassemble the cytoskeletal elements that maintain the contact between cell membrane and er . alternatively , the whole - cell configuration of the patch - clamp technique could be exploited to remove the dye , but this experimental approach is rather expensive and challenging , requires highly skilled electrophysiologists and has a quite low throughput .", "the final yield of photoconverted mag - fura 2 is in direct relation with the amount of ca present in the sample considered . in our condition", ", the ca - enriched medium gave a higher end product staining than the normal physiological conditions or than ca deprivation .", "it is interesting that in several cases , dense precipitates could be found in areas suggesting the local accumulation of ca nearby the mouth of plasmalemmal ca channels .", "accordingly , such precipitates are evident when extracellular ca concentration is increased , which is consistent with the notion that constitutive ca inflow from the extracellular milieu is enhanced .", "this unexpected outcome of dab photoxidation is extremely relevant in light of the master role served by local ca microdomains generated by extracellular ca influx , which regulate cell functions as diverse as cycle progression , proliferation , migration , nitric oxide production , and adenylyl cyclase activity .", "one thing that should be underlined here is that we could not visualize the ser tubule filled with the end product .", "this could be due to three different causes : i ) ca can be dispersed ( lost ) in the conditions used here ; ii ) local intraluminal ca concentration is less than expected ( which is less probable as it is unlikely to be lower than 100 m ) ; or iii ) dab photoconversion is not efficient enough for mag - fura . by comparing fluorescence images with em micrographs ,", "it is clear the difference in the signal coming from vesicles and the elongated filamentous structures , the latter being much less fluorescent .", "the relative low yield when comparing fluorescence images is definitely related to the extremely lower amount of end product which can be found within a 60 - 80 nm thin section in relation to the fluorescence originating from the whole cell . in addition , albeit [ ca]er fluctuates between 100 and 500 m , ca accumulates in specific regions of the er that may be missed in our sections . moreover , releasable ca is actually trapped by oligomers of the ca - binding proteins calnexin and calreticulin : these form a concealed ca source that is not in rapid equilibrium with the [ ca]er , but is easily accessible to ca - releasing channels upon extracellular stimulation .", "thus , the lack of ser tubules or cisternae filled with the end product of the photo - conversion , i.e. , ca , is not surprising .", "finally , the photoconversion process seems to be efficient , and gives an end product more reasonable than the precipitation technique utilized , for instance , by tandler et al . in the latter case ,", "an important amount of precipitates were found in the cell and especially in the nucleus ; our method gives a much finer end product , and this allows a superior resolution and localization . in conclusion , this method is a reliable and efficient technique for stabilizing and visualizing ca ions at em .", "it gives a high resolution end product thus allowing the fine structural ca localization . in principle", ", this approach could provide important qualitative information about the amount and sub - cellular distribution of calcium ions both in cultured cells and , more importantly , in ex vivo samples , which are far less amenable to high resolution ca imaging of er ca levels ."], "abstract_text": ["<S> we propose a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques . we have applied a new approach by photo - oxidation of diaminobenzidine in presence of the emission of an excited fluorochrome specific for calcium detection . </S>", "<S> this method combines the selectivity of available fluorophores to the high spatial resolution offered by transmission electron microscopy to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles . </S>", "<S> we show in this paper that mag - fura 2 photoconversion via diaminobenzidine oxidation is an efficient way for localizing ca2 + ions at electron microscopy level , is easily carried out and reproducible , and can be obtained on a good amount of cells , since the exposure in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp . </S>", "<S> the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Cell culture", "Intracellular Ca2", "Transmission electron microscopy", "Results", "Discussion"], "sections": [["the ultrastructural localization of a molecule requires that a certain degree of electron - density , resulting in adequate electron scattering , be present in sufficient amount .", "binding of heavy atoms can be accomplished easily with regards to proteins , nucleic acids and other cellular components , in some cases with a high degree of specificity . however , the sub - cellular localization of ions is a challenge , since in many cases ions are diffusible and not easily kept in place after the procedures of sample preparation for electron microscopy ( em ) .", "many ions can offer enough scattering in spite of not being heavy metals , such as manganese , while others necessitate of blocking or precipitating techniques to be visualized .", "in particular , calcium ions are easily diffusible inside the cell , and even when protein - bound are not easily detectable . the pioneering paper by tandler et al .", "demonstrated the presence of calcium ions by em via pyroantimonate precipitation , with a good yield of the final product . the end product of the reaction , however , is probably overrated , since the technique is capable of stabilizing and precipitating calcium and other bivalent cations , such as magnesium , as proved by electron spectroscopic imaging ( esi ) analysis . since their introduction by tsien and coworkers ,", "calcium studies have used specific ca - sensitive fluorochromes , such as fura-2 , indo-1 and fluo-4 , coupled to wide - field digitized video fluorescence microfluorimetry and confocal microscopy to image acute elevations in intracellular ca concentration ( [ ca]i ) with high spatio - temporal resolution .", "cytosolic and nuclear ca signals regulate a multitude of vital cellular processes , such as gene transcription , cell cycle progression , cytoskeletal and chromatin remodelling , and migration . however , proper ca handling within the endoplasmic reticulum ( er ) , the most abundant intracellular ca reservoir , is also crucial for cell - fate decisions . the steady - state er ca level ( [ ca]er ) does not only control synthesis , folding , export and trafficking of proteins , but it also fuels mitochondrial bioenergetics thereby suppressing autophagy .", "low affinity ca indicators , such as mag - fura 2 , and genetically - encoded probes , such as the bioluminescent protein aequorin and the gfp - based fluorophores camgaroos , pericams , and cameleons , have been devised to assess er ca levels .. nevertheless , these sophisticated tools do not provide any reliable information on ca distribution within the heterogeneous structure of the er . we propose in this paper a tool for a rapid high - resolution detection of calcium ions which can be used in parallel with other techniques .", "maranto and sandell and masland have proposed the possibility of photoconverting fluorochromes into an electrondense end product .", "the technique utilizes the photo - oxidation of diaminobenzidine ( dab ) in presence of the emission of an excited fluorochrome , and it has been shown to be efficient on different dyes and conditions .", "this method combines the specificity of available fluorophores to the high spatial resolution offered by transmission em to detect fluorescing molecules even when present in low amounts in membrane - bounded organelles .", "the present manuscript sought to exploit dab photo - oxidation of mag - fura 2 for visualizing er ca distribution in hela cells , which are among the most widespread model cell lines used for studying intracellular ca signaling ."], ["hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence ,", "cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) .", "the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition .", "the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 .", "all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) .", "intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m .", "cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy .", "an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software .", "instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st .", "louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs .", "thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel", ", some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above .", "moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above . fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature .", "once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) .", "ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv ."], ["hela cells have been cultured in iscove s modified dulbecco s medium ( imdm , lonza , basel , switzerland ) supplemented with 10% fetal bovine serum , 2% l - glutamine , 1% penicillin and streptomycin , at 37c air atmosphere with 5% co2 . after reaching 90% confluence ,", "cells have been plated in 6 wells cell culture plate ( 1x10 cells per well ) , or in chamber slides for staining and microscopic examination ( 8x10 cells per well ) .", "the day after seeding , when cells reached about 90% confluence , the medium has been removed from the wells and replaced with three solutions , one for each experimental condition .", "the solutions , different in ca concentration , are the following : physiological salt solution ( pss ) , with the following composition : 150 mm nacl , 6 mm kcl , 1.5 mm cacl2 , 1 mm mgcl2 , 10 mm glucose , and 10 mm hepes ; ca - free solution ( 0 ca ) , where ca has been substituted by 2 mm nacl , and 0.5 mm egta was added , whereas the solution with ca more concentrated is similar to pss in composition except for 120 mm nacl and 20 mm cacl2 .", "all the solutions have been brought to ph 7.4 with an osmolality of 338 mmol / kg , measured with an osmometer ( wescor 5500 ; wescor , inc . , logan , ut , usa ) ."], ["intracellular ca deposits have been marked by fluorescent dye - based calcium indicator mag - fura-2 , acetoxymethyl ( am ) ester uv light - excitable ( life tchnologies , carslbad , ca , usa ) , added to the three calcium solutions to obtain a final concentration of 5 m .", "cells have thus been incubated at 37c in the dark , for 1 h. cells cultured on chamber slides , and treated as hereto described , have been analyzed at fluorescence microscopy .", "an olympus bx51 microscope ( olympus italia srl , segrate , italy ) was used under the following conditions : 100-w hg lamp as the excitation source , 330- to 385-nm excitation filter ( excf ) , 400-nm dichroic mirror ( dm ) , and 420-nm barrier filter ( bf ) ; 100 olympus uplanfl oil - immersion objective lens ( na 1.25 ) . images were recorded with an olympus magnifier digital camera system , and stored by the olympus cellf software .", "instead , cells in plates have been fixed at room temperature for 20 minutes with a fixative solution ( 2% paraformaldehyde and 1% glutaraldehyde in pbs ) . meanwhile , a solution with dab has been freshly prepared as follows : 20 mg of dab ( sigma aldrich , st .", "louis , mo , usa ) were dissolved in 1 ml dmso , briefly vortexed and then brought to 30 ml with pbs .", "thereafter , cells have been washed twice with pbs and incubated for 3 h under a germicidal uv lamp ( sankyo denki , kanagawa , japan ; g30t8 , uv - c ) with 2 ml dab solution which is replaced with fresh solution every half an hour . in parallel", ", some cells were incubated in the absence of mag - fura 2 , treated with dab and processed as above .", "moreover , to exclude a possible interference by aldehyde groups , some slides were fixed and then treated with 0.5 m nh4cl in pbs for 30 min at 4c , then incubated with dab and processed as above ."], ["fixed and photoconverted cells have been washed twice with distilled water and postfixed with 2% osmium tetroxide in water for 1 h , at room temperature .", "once the osmium was removed and the cells rinsed several times with distilled water , cells have been scraped , centrifuged discarding supernatant , dehydrated in a graded series of ethanol and embedded in lrwhite resin ( sigma aldrich ) .", "ninety nm ultrathin sections have been cut by a reichert ultramicrotome , transferred to grids without membrane and visualized by a zeiss em900 transmission electron microscope operating at 80kv ."], ["calcium labeling with mag - fura 2 results in highly fluorescent staining mainly localized in the cytoplasm .", "after incubation of the cells with 20 mm ca for 1 h , the signal is present in large vesicles in the cytoplasm and around the cell nucleus , as well as in elongated structures probably related to mitochondria ( figure 1a ) .", "the faint signal visible in the nucleus is very probably due to the superposition of the cytoplasmic layer surrounding it .", "when the cells are incubated in pss , the signal is lower ( figure 1b ) and mostly present in the perinuclear vesicles , while the cytoplasmic signal far from the nucleus somehow disappears . in the last condition , without calcium and in the presence of egta , some labeling is still visible , although to a much reduced level ( figure 1c ) .", "this continuous presence of ca represents very probably the last ions the cell can not allow to lose , a sort of surviving threshold concentrations .", "supplementary figure 1 confirms that altering extracellular ca concentrations results in a consequent change in er ca homeostasis , which may have important consequences also on cytosolic ca dynamics . at em level", ", the controls did not show any electron dense precipitates , both in the absence of the fluorochrome or after aldehyde blockade ( not shown ) ; consequently , the end product obtained in the mag - fura 2 stained cells is due to the fluorochrome photoconversion .", "the dab photoconversion is an efficient way to stabilize the ions and to visualize them .", "it must be underlined here that we observe a thin section , 60 - 80 nm thick , corresponding , consequently , only to a fraction of the fluorescent signal .", "after pss incubation , the cells show a distinctive positivity ( figure 2a ) . in some cases it is possible to see the presence of precipitates without the direct association with smooth or rough er ( ser or rer , respectively ) membranes , likely related to unbound ca ions", "( figure 2b ) , and electron dense material inside the vesicles is also positive ( figure 2c ) .", "a possible suggestion of ca internalization or extrusion is shown in figure 2 d , e , where the deposits are present on the outer membrane .", "when the cells are incubated without calcium and in the presence of egta , the deposits are extremely reduced , although still present on the outer cell membrane , suggesting the presence of an extruding mechanism ( figure 2f ) . on the contrary ,", "the diffuse , low density end product is almost absent from the cytoplasm . at higher ca concentration in the medium ( figure 3a ) , the dark precipitate is detectable on the inner membrane of cytoplasmic vesicles possibly pertaining to ser , and in discrete foci with no apparent association with vesicles or membranes .", "the deposits are present within electron dense vesicles ( figure 3b ) and electron lucent structure , in some cases associated , and suggesting the idea of passage of ions from one compartment to another .", "in other cases , dab - positive material is present on the membranes of complex systems such as multivesicular bodies ( figure 3c ) or on the outer membranes of cytosolic vesicles ( figure 3d ) .", "interestingly , calcium deposits are detectable on the outer cell membrane ( figure 3e ) in spots , localized and separated , suggesting the presence of calcium channels . in figure 3f ,", "the end product of the dab photoconversion is a speckled , dense precipitate of varying size .", "however , another abundant end product is present as small sized dots , showing a markedly lower electrondensity ( figure 3f , inset ) and a widespread distribution ."], ["we show in this paper that mag - fura 2 photoconversion via dab oxidation is an efficient way for localizing ca ions at em level . moreover ,", "this could be considered also as a means of stabilizing their presence , thus limiting the loss of ions . as for the method ,", "photoconversion is easily carried out and reproducible , can be obtained on a good amount of cells , since the exposition in our conditions is not limited to the direct irradiation of the sample via an objective but obtained with a germicide lamp .", "the end product is sufficiently electron dense to be detected clearly when present in sufficient amount within a membrane boundary . in these conditions", ", the blackness of the product stands out from the background . in our conditions , in fact , ca ions are immediately detectable within the vesicles and the tubules of the ser , while it is much more difficult to see their presence when in the cytosol , due to their low electrondensity .", "this is a clear advantage over the conventional imaging of mag - fura 2 fluorescence that must be removed by the cytosol in order to study truly er signals .", "this is achieved by permeabilizing the plasma membrane with brief applications of antibiotics or detergents , but this procedure may destroy intracellular signaling pathways , dilute cytosolic modulators of er ca release / sequestration , and disassemble the cytoskeletal elements that maintain the contact between cell membrane and er . alternatively , the whole - cell configuration of the patch - clamp technique could be exploited to remove the dye , but this experimental approach is rather expensive and challenging , requires highly skilled electrophysiologists and has a quite low throughput .", "the final yield of photoconverted mag - fura 2 is in direct relation with the amount of ca present in the sample considered . in our condition", ", the ca - enriched medium gave a higher end product staining than the normal physiological conditions or than ca deprivation .", "it is interesting that in several cases , dense precipitates could be found in areas suggesting the local accumulation of ca nearby the mouth of plasmalemmal ca channels .", "accordingly , such precipitates are evident when extracellular ca concentration is increased , which is consistent with the notion that constitutive ca inflow from the extracellular milieu is enhanced .", "this unexpected outcome of dab photoxidation is extremely relevant in light of the master role served by local ca microdomains generated by extracellular ca influx , which regulate cell functions as diverse as cycle progression , proliferation , migration , nitric oxide production , and adenylyl cyclase activity .", "one thing that should be underlined here is that we could not visualize the ser tubule filled with the end product .", "this could be due to three different causes : i ) ca can be dispersed ( lost ) in the conditions used here ; ii ) local intraluminal ca concentration is less than expected ( which is less probable as it is unlikely to be lower than 100 m ) ; or iii ) dab photoconversion is not efficient enough for mag - fura . by comparing fluorescence images with em micrographs ,", "it is clear the difference in the signal coming from vesicles and the elongated filamentous structures , the latter being much less fluorescent .", "the relative low yield when comparing fluorescence images is definitely related to the extremely lower amount of end product which can be found within a 60 - 80 nm thin section in relation to the fluorescence originating from the whole cell . in addition , albeit [ ca]er fluctuates between 100 and 500 m , ca accumulates in specific regions of the er that may be missed in our sections . moreover , releasable ca is actually trapped by oligomers of the ca - binding proteins calnexin and calreticulin : these form a concealed ca source that is not in rapid equilibrium with the [ ca]er , but is easily accessible to ca - releasing channels upon extracellular stimulation .", "thus , the lack of ser tubules or cisternae filled with the end product of the photo - conversion , i.e. , ca , is not surprising .", "finally , the photoconversion process seems to be efficient , and gives an end product more reasonable than the precipitation technique utilized , for instance , by tandler et al . in the latter case ,", "an important amount of precipitates were found in the cell and especially in the nucleus ; our method gives a much finer end product , and this allows a superior resolution and localization . in conclusion , this method is a reliable and efficient technique for stabilizing and visualizing ca ions at em .", "it gives a high resolution end product thus allowing the fine structural ca localization . in principle", ", this approach could provide important qualitative information about the amount and sub - cellular distribution of calcium ions both in cultured cells and , more importantly , in ex vivo samples , which are far less amenable to high resolution ca imaging of er ca levels ."]]}
{"article_id": "PMC4774295", "article_text": ["nearly , 90% of metastatic tumors occur in jaw bones , especially premolar - molar region of the mandible , whereas metastases to soft tissues are extraordinarily rare and account for 0.1% of all oral malignancies .", "most metastatic tumors to the orofacial region are seen in patients aged between 40 and 70 years .", "metastasis to the soft tissues mostly involves gingiva and alveolar mucosal sites ( 54% ) followed by tongue ( 30% ) . in some cases ,", "oral metastatic tumors are found to be the first sign of metastatic spread and in some instances ; they are an indication for unidentified primary tumor of the distant site .", "the primary site differs according to oral site colonization . in men , primary tumor of the lung followed by the prostate gland and kidney", "the breast is the most common primary tumor site affecting the jawbones and soft tissues ( 41% and 24.3% , respectively ) , followed by the adrenal and female genital organs .", "clinically , these metastatic lesions appear nonaggressive , mimicking reactive or benign lesions or even simple odontogenic infections . the clinical features of these lesions include a bony swelling with tenderness , pain , ulcer , hemorrhage , paresthesia and pathological fracture .", "it is important to diagnose the origin of the tumor as the management will depend significantly on the histological type .", "although infrequent , oral metastases are rarely encountered as the first sign of occult cancer .", "hence , this article emphasizes on detailed dentoalveolar examination and early diagnosis for finding the primary focus of metastatic tumor .", "a 60-year - old male patient presented with a swelling in the right lower back teeth region of the jaw since 3 months [ figure 1 ] .", "the patient complaints of pain for 10 days , radiating to the right temporal region .", "the patient was a known hypertensive since 4 years . a well - defined solitary swelling present on the right side of mandible on extraoral examination , a well - defined , solitary swelling measuring 4 cm", " 5 cm was noted on the right side of the mandible , which was hard in consistency and fixed to the underlying skin .", "no other pathologic findings were noticed during physical examination . on intraoral examination , firm ,", "nontender swelling with ill - defined borders measuring 3 cm  3 cm was noted in the right mandibular premolar - molar region [ figure 2 ] .", "there was no ulceration , paresthesia or pathological fracture . on intraoral examination , firm ,", "nontender swelling with ill - defined borders measuring 3 cm  3 cm was noted patient was advised for oral pantamograph and computed tomography ( ct ) scan .", "ct scan of the face with three - dimension reconstruction revealed a well - defined osteolytic lesion involving the ramus and body of the right half of the mandible , measuring 54 mm  45 mm  50 mm ( anterioposterior  transverse  craniocaudal ) .", "it showed speculated periosteal lesion with multiple irregular calcifications and extended into the lingual and mandibular soft tissues [ figure 3 ] .", "based on the clinical presentation , a differential diagnosis of inflammatory swelling , odontogenic cyst , odontogenic tumor , soft tissue tumor or metastatic tumor to the jawbones was given .", "computed tomography scan of the face with three - dimensional reconstruction revealed a well - defined osteolytic lesion with multiple irregular calcifications that extended into the lingual and mandibular soft tissues incisional biopsy was done for the histopathological diagnosis .", "histopathological examination of the h and e stained tissue showed diffuse fibrous connective tissue stroma invaded by the tumor cells which were cuboidal to short columnar with eosinophilic cytoplasm arranged in alveolar / follicular pattern of varying sizes [ figure 4 ] .", "bony trabeculae were evident in some areas [ figure 5 ] . based on these features ,", "the lesion was diagnosed as a metastatic malignant tumor , probably metastasizing from the lung .", "( h&e stain , 100 ) tumor islands along with few bony trabeculae were evident in some areas .", "( h&e stain , 40 ) x - ray chest posterior - anterior view revealed consolidation of the right lower zone [ figure 6 ] .", "the ct scan of the chest with contrast revealed dense soft tissue mass lesion with speculated margins and focal pleural thickening noted at the right posterior basal segment .", "x - ray chest posterior - anterior view revealed consolidation in right lower zone of the lungs treatment for the patient was palliative external beam radiotherapy on linac given to a total dose of 3000 cgy in 10 fractions to jaw by rl / ll for 12 days which was well - tolerated by the patient .", "chemotherapy included injection cisplatin ( 75 mg)-intravenous ( iv ) ; injection etoposide ( 150 mg)-iv ; tablet vomiset ( 8 mg ) ; tablet ultracet .", "the common primary sources of tumors metastatic to the oral region are the breast , lung and kidney .", "the actual incidence of these cancers is unknown since oral metastatic disease is usually a manifestation of advanced disease .", "the mean age of occurrence is 54 years with slight male predilection . in our case ,", "also the patient was aged about the same age ( 60 years ) as supported in the literature .", "most of the patients are aware of primary tumors before the metastatic spread to the oral cavity . in our case ,", "the most common primary sites for oral metastases were the lung , kidney , liver and prostate for men ; breast , female genital organs , kidney , and colorectum for women . in our case too , lung was found to be the primary site .", "the clinical presentation of the metastatic lesions differed between the various oral sites . in the jawbones ,", "most of the patients complain of rapidly progressing swelling , pain , paresthesia , difficulty in chewing , dysphagia , disfigurement and bleeding .", "our patient presented with features such as firm , nontender swelling with ill - defined borders .", "there was no paresthesia , difficulty in chewing , dysphagia , bleeding and pathological fracture .", "pathogenesis of oral metastasis is unclear but thought to be a multistage process in which cells detach themselves from the primary tumor and get transported by lymphatic or blood vessels .", "recent studies state that cancer cells metastasizing to bone have shown to alter the physiologic balance between both bone resorption and bone formation .", "treatment of oral metastasis depends upon its presentation and the stage during which it has been identified .", "management includes chemotherapy , radiotherapy , surgical excision or a combination of the techniques under local anesthesia .", "an average survival time for lung cancer metastasis is 4 months to 1 year with a maximum survival rate of 5 years ."], "abstract_text": ["<S> oral cavity is an uncommon site for metastasis and is usually an evidence of widespread disease . </S>", "<S> the clinical presentation of the metastatic lesions differs between the various sites in the oral region . </S>", "<S> metastatic tumors account for 13% of all oral malignancies . </S>", "<S> the jawbones , particularly the mandibular molar area is more frequently affected than the oral soft tissues ( 2:1 ) . here , we report a case of a 60-year - old male patient with metastasis from lung to the mandible , where the metastasis was detected before primary tumor . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["nearly , 90% of metastatic tumors occur in jaw bones , especially premolar - molar region of the mandible , whereas metastases to soft tissues are extraordinarily rare and account for 0.1% of all oral malignancies .", "most metastatic tumors to the orofacial region are seen in patients aged between 40 and 70 years .", "metastasis to the soft tissues mostly involves gingiva and alveolar mucosal sites ( 54% ) followed by tongue ( 30% ) . in some cases ,", "oral metastatic tumors are found to be the first sign of metastatic spread and in some instances ; they are an indication for unidentified primary tumor of the distant site .", "the primary site differs according to oral site colonization . in men , primary tumor of the lung followed by the prostate gland and kidney", "the breast is the most common primary tumor site affecting the jawbones and soft tissues ( 41% and 24.3% , respectively ) , followed by the adrenal and female genital organs .", "clinically , these metastatic lesions appear nonaggressive , mimicking reactive or benign lesions or even simple odontogenic infections . the clinical features of these lesions include a bony swelling with tenderness , pain , ulcer , hemorrhage , paresthesia and pathological fracture .", "it is important to diagnose the origin of the tumor as the management will depend significantly on the histological type .", "although infrequent , oral metastases are rarely encountered as the first sign of occult cancer .", "hence , this article emphasizes on detailed dentoalveolar examination and early diagnosis for finding the primary focus of metastatic tumor ."], ["a 60-year - old male patient presented with a swelling in the right lower back teeth region of the jaw since 3 months [ figure 1 ] .", "the patient complaints of pain for 10 days , radiating to the right temporal region .", "the patient was a known hypertensive since 4 years . a well - defined solitary swelling present on the right side of mandible on extraoral examination , a well - defined , solitary swelling measuring 4 cm", " 5 cm was noted on the right side of the mandible , which was hard in consistency and fixed to the underlying skin .", "no other pathologic findings were noticed during physical examination . on intraoral examination , firm ,", "nontender swelling with ill - defined borders measuring 3 cm  3 cm was noted in the right mandibular premolar - molar region [ figure 2 ] .", "there was no ulceration , paresthesia or pathological fracture . on intraoral examination , firm ,", "nontender swelling with ill - defined borders measuring 3 cm  3 cm was noted patient was advised for oral pantamograph and computed tomography ( ct ) scan .", "ct scan of the face with three - dimension reconstruction revealed a well - defined osteolytic lesion involving the ramus and body of the right half of the mandible , measuring 54 mm  45 mm  50 mm ( anterioposterior  transverse  craniocaudal ) .", "it showed speculated periosteal lesion with multiple irregular calcifications and extended into the lingual and mandibular soft tissues [ figure 3 ] .", "based on the clinical presentation , a differential diagnosis of inflammatory swelling , odontogenic cyst , odontogenic tumor , soft tissue tumor or metastatic tumor to the jawbones was given .", "computed tomography scan of the face with three - dimensional reconstruction revealed a well - defined osteolytic lesion with multiple irregular calcifications that extended into the lingual and mandibular soft tissues incisional biopsy was done for the histopathological diagnosis .", "histopathological examination of the h and e stained tissue showed diffuse fibrous connective tissue stroma invaded by the tumor cells which were cuboidal to short columnar with eosinophilic cytoplasm arranged in alveolar / follicular pattern of varying sizes [ figure 4 ] .", "bony trabeculae were evident in some areas [ figure 5 ] . based on these features ,", "the lesion was diagnosed as a metastatic malignant tumor , probably metastasizing from the lung .", "( h&e stain , 100 ) tumor islands along with few bony trabeculae were evident in some areas .", "( h&e stain , 40 ) x - ray chest posterior - anterior view revealed consolidation of the right lower zone [ figure 6 ] .", "the ct scan of the chest with contrast revealed dense soft tissue mass lesion with speculated margins and focal pleural thickening noted at the right posterior basal segment .", "x - ray chest posterior - anterior view revealed consolidation in right lower zone of the lungs treatment for the patient was palliative external beam radiotherapy on linac given to a total dose of 3000 cgy in 10 fractions to jaw by rl / ll for 12 days which was well - tolerated by the patient .", "chemotherapy included injection cisplatin ( 75 mg)-intravenous ( iv ) ; injection etoposide ( 150 mg)-iv ; tablet vomiset ( 8 mg ) ; tablet ultracet ."], ["the common primary sources of tumors metastatic to the oral region are the breast , lung and kidney .", "the actual incidence of these cancers is unknown since oral metastatic disease is usually a manifestation of advanced disease .", "the mean age of occurrence is 54 years with slight male predilection . in our case ,", "also the patient was aged about the same age ( 60 years ) as supported in the literature .", "most of the patients are aware of primary tumors before the metastatic spread to the oral cavity . in our case ,", "the most common primary sites for oral metastases were the lung , kidney , liver and prostate for men ; breast , female genital organs , kidney , and colorectum for women . in our case too , lung was found to be the primary site .", "the clinical presentation of the metastatic lesions differed between the various oral sites . in the jawbones ,", "most of the patients complain of rapidly progressing swelling , pain , paresthesia , difficulty in chewing , dysphagia , disfigurement and bleeding .", "our patient presented with features such as firm , nontender swelling with ill - defined borders .", "there was no paresthesia , difficulty in chewing , dysphagia , bleeding and pathological fracture .", "pathogenesis of oral metastasis is unclear but thought to be a multistage process in which cells detach themselves from the primary tumor and get transported by lymphatic or blood vessels .", "recent studies state that cancer cells metastasizing to bone have shown to alter the physiologic balance between both bone resorption and bone formation .", "treatment of oral metastasis depends upon its presentation and the stage during which it has been identified .", "management includes chemotherapy , radiotherapy , surgical excision or a combination of the techniques under local anesthesia .", "an average survival time for lung cancer metastasis is 4 months to 1 year with a maximum survival rate of 5 years ."], [""], [""]]}
{"article_id": "PMC4361821", "article_text": ["the study population was all the 6 to 9 year- old students from the districts of one and two in yazd , an old and big city located in the center of iran .", "for the study group , 56 students were selected from imam hussein counseling center ( counseling center of educational organization of yazd province ) who were referred by their school staff and had received adhd diagnosis by a child psychiatrist based on dsm - iv - tr criteria and had no comorbid disorders .", "after that , the entire study group and their parents ( mother or father ) were interviewed by a clinical psychologist .", "the child symptom inventory-4 ( csi-4 ) was filed out by parents to be used for the diagnosis and differentiation between the adhd subtypes . of the participants ,", "19 were diagnosed with adhd - h , 15 with adhd - i and 22 with adhd - c . in the adhd - c group ,", "finally , 45 students ( 15 students in each adhd subtype groups ) were remained as the study group .", "the control group consisted of 30 students who were selected from the above mentioned population by stratified random sampling method . in the control group ,", "none had a history of psychiatric disorders or developmental delay based on their parents report .", "exclusion criteria was wearing glasses , color blindness , physical or psychological disorder and using psychiatric medicines .", "students who were diagnosed with adhd in the past and were under treatment with medicine were excluded from the study due to the medicines effect on attention . in the current study ,", "all the students ( from both sexes ) with all adhd subtypes were participated as the study group .", "all cases in the study and the control group were matched based on age , sex and iq level . in the current study four tools of structured interview , wechsler intelligence scale for children - revised ( wisc - r ) and child symptom inventory-4 ( csi-4 ) were used .", "child symptom inventory-4 ( csi-4 ) is a screening tool for the symptoms of behavioral and emotional disorders in children based on the diagnostic criteria of dsm - iv .", "the parent report checklist contains 97 items to screen 15 behavioral and emotional disorders including ad / hd , oppositional defiant disorder , conduct disorder , generalized anxiety disorder , social phobia , separation anxiety disorder , obsessive - compulsive disorder , specific phobia , major depressive disorder , dysthymic disorder , schizophrenia , pervasive developmental disorder , asperger s disorder and tics disorder .", "there are two methods to score csi-4 : criterion - related and norm - based . in the criterion - related method", "the score of zero was given to items which were rated as never and seldom , and the score of one was given to the items which were rated as sometimes and often .", "the final score for each subscale was obtained by adding the scores of the items related to the given subscale . in the present study ,", "this subscale consists of 18 items , in which items 1 - 9 are used for adhd - i and items 10 - 18 for adhd - h .", "the total adhd score can be obtained by adding the scores of 1 - 18 items . the cutoff point for diagnosing adhd based on the csi-4", "is 6 for both the mainly attention deficit and the mainly hyperactive - impulsive types .", "the test - retest reliability of the parent checklist in an iranian sample was reported as 0.90 .", "furthermore , in another study , cronbach s alpha of 0.92 and a sensitivity of 0.94 were reported for this inventory(11 ) .", "wechsler intelligence scale for children - revised ( wisc - r ) is a well - known scale for assessing the iq in children aged between 6 - 16 years .", "wisc - r gives two different scores for verbal and performance iq and a total iq scores .", "moderate internal consistency for total iq , verbal iq and performance iq were reported as 0.96 , 0.95 and 0.91 by wechsler , respectively(34 ) . in the present study we used total iq score to match the participants .", "wisconsin card sorting test ( wcst-64 ) is a neuropsychological test for assessing problem solving , sorting , abstract thinking , ability to maintain concepts and cognitive flexibility skills which are related to performances of the frontal lobe . in this test", ", a set of 64 cards including1 - 4shapes in forms of red triangle , green star , yellow cross and blue circle are given to the participants , and then they should place the cards based on their perception of the pattern used by the examiner .", "the pattern is a red triangle , green star , yellow cross and a blue circle . for example , if the principle is color , the true placement is that the red card regardless of the shape or number should be placed under the triangle and the examiner determines if the placement is correct or not .", "after that , the subject completes a round of ten placements correctly , and the examiner will then change the principle . the test will continue until the subject finishes the placement of ten cards for six times and place 64 cards in one category(35 ) .", "data were entered in spss-17 and analyzed by t - test and anova static tests to clarify the differences between adhd and normal group and between adhd subtypes .", "scheffe s test was also used to identify which groups were different . the mean and standard divisions ( sd ) were used for descriptive analysis .", "the study population was all the 6 to 9 year- old students from the districts of one and two in yazd , an old and big city located in the center of iran .", "for the study group , 56 students were selected from imam hussein counseling center ( counseling center of educational organization of yazd province ) who were referred by their school staff and had received adhd diagnosis by a child psychiatrist based on dsm - iv - tr criteria and had no comorbid disorders .", "after that , the entire study group and their parents ( mother or father ) were interviewed by a clinical psychologist .", "the child symptom inventory-4 ( csi-4 ) was filed out by parents to be used for the diagnosis and differentiation between the adhd subtypes . of the participants ,", "19 were diagnosed with adhd - h , 15 with adhd - i and 22 with adhd - c . in the adhd - c group ,", "finally , 45 students ( 15 students in each adhd subtype groups ) were remained as the study group .", "the control group consisted of 30 students who were selected from the above mentioned population by stratified random sampling method . in the control group ,", "none had a history of psychiatric disorders or developmental delay based on their parents report .", "exclusion criteria was wearing glasses , color blindness , physical or psychological disorder and using psychiatric medicines .", "students who were diagnosed with adhd in the past and were under treatment with medicine were excluded from the study due to the medicines effect on attention . in the current study ,", "all the students ( from both sexes ) with all adhd subtypes were participated as the study group .", "all cases in the study and the control group were matched based on age , sex and iq level .", "in the current study four tools of structured interview , wechsler intelligence scale for children - revised ( wisc - r ) and child symptom inventory-4 ( csi-4 ) were used .", "child symptom inventory-4 ( csi-4 ) is a screening tool for the symptoms of behavioral and emotional disorders in children based on the diagnostic criteria of dsm - iv .", "the parent report checklist contains 97 items to screen 15 behavioral and emotional disorders including ad / hd , oppositional defiant disorder , conduct disorder , generalized anxiety disorder , social phobia , separation anxiety disorder , obsessive - compulsive disorder , specific phobia , major depressive disorder , dysthymic disorder , schizophrenia , pervasive developmental disorder , asperger s disorder and tics disorder .", "there are two methods to score csi-4 : criterion - related and norm - based . in the criterion - related method", "the score of zero was given to items which were rated as never and seldom , and the score of one was given to the items which were rated as sometimes and often .", "the final score for each subscale was obtained by adding the scores of the items related to the given subscale . in the present study ,", "this subscale consists of 18 items , in which items 1 - 9 are used for adhd - i and items 10 - 18 for adhd - h .", "the total adhd score can be obtained by adding the scores of 1 - 18 items . the cutoff point for diagnosing adhd based on the csi-4", "is 6 for both the mainly attention deficit and the mainly hyperactive - impulsive types .", "the test - retest reliability of the parent checklist in an iranian sample was reported as 0.90 .", "furthermore , in another study , cronbach s alpha of 0.92 and a sensitivity of 0.94 were reported for this inventory(11 ) .", "wechsler intelligence scale for children - revised ( wisc - r ) is a well - known scale for assessing the iq in children aged between 6 - 16 years .", "wisc - r gives two different scores for verbal and performance iq and a total iq scores .", "moderate internal consistency for total iq , verbal iq and performance iq were reported as 0.96 , 0.95 and 0.91 by wechsler , respectively(34 ) . in the present study we used total iq score to match the participants .", "wisconsin card sorting test ( wcst-64 ) is a neuropsychological test for assessing problem solving , sorting , abstract thinking , ability to maintain concepts and cognitive flexibility skills which are related to performances of the frontal lobe . in this test", ", a set of 64 cards including1 - 4shapes in forms of red triangle , green star , yellow cross and blue circle are given to the participants , and then they should place the cards based on their perception of the pattern used by the examiner .", "the pattern is a red triangle , green star , yellow cross and a blue circle .", "for example , if the principle is color , the true placement is that the red card regardless of the shape or number should be placed under the triangle and the examiner determines if the placement is correct or not .", "after that , the subject completes a round of ten placements correctly , and the examiner will then change the principle . the test will continue until the subject finishes the placement of ten cards for six times and place 64 cards in one category(35 ) .", "data were entered in spss-17 and analyzed by t - test and anova static tests to clarify the differences between adhd and normal group and between adhd subtypes .", "scheffe s test was also used to identify which groups were different . the mean and standard divisions ( sd ) were used for descriptive analysis .", "the study group consisted of 45 school aged children ; of whom , 27 were male and 18 were female . in the control group", ", there were 30 school aged children , of whom 18were male and 12 were female .", "other characteristics of the two groups ( age , iq , adhd subtype frequency ) are presented in table 1 .", "data analysis showed that adhd and normal group were significantly different in terms of perseverative responses , perseverative errors and total errors on wsct ( tables 2 and 3 ) as shown in table 4 , adhd subtypes were significantly different in terms of perseverative responses ( p 0/01 ) and perseverative errors ( p0/001 ) . based on scheffe s test ,", "adhd - h was not different from adhd - i and adhd - c but there were significant differences between adhd - i and adhd - c in terms of perseverative responses and perseverative errors .", "the aim of the present study was to investigate the differences between adhd subtypes in terms of executive function profile .", "previous studies showed inconsistent findings in this regard . in an extended study , houghton et al .", "investigated the executive function in 94 children with adhd ( without any co - morbid disorder ) in terms of subtype and gender using five tests of the wisconsin card sorting test , the stroop color - word test , the matching familiar figures test , the trail making test , and the tower of london .", "they found that children with both types of adhd ( adhd - i and adhd - c ) differed from normal children in terms of perseveration and response inhibition , but this difference was significant in adhd - c , only ( 27 ) .", "the lack of any co - morbidity in children with adhd showed that the impairments in executive function were obviously found in adhd , particularly in the adhd - c , so providing support for barkley s theory of adhd ( 27 ) .", "consistent with this finding , in another study , klorman et al . found that children with adhd - c revealed more non - perseverative errors in wcst and solved fewer puzzles and also violated more rules on the tower of hanoi ( toh ) than adhd - i , but they were not different based on perseverative errors in wcst(28 ) . also , lawrence et al .", "their finding revealed that children with adhd were only different in set - shifting on wsct ( perseverative responses and errors ) ( 10 ) . in this respect", ", we found that children with adhd significantly differed from normal children in terms of total errors , perseverative errors and perseverative responses ( p 0.01 ) .", "also , we found that adhd subtypes operated differently in the executive function domain ( p 0.01 ) ; children with adhd predominantly combined type showed more perseverative responses and perseverative errors than children with adhd predominantly inattentive type .", "some researchers suggest that the two primary subtypes of adhd share similar neuropsychological weaknesses in inhibitory control , but there are subtype differences in response to success and failure that are contributed to a child s ultimate level of performance ( 36 ) . unlike the barkley s behavioral inhibition theory , song and hakoda found that both types of adhd ( adhd - i and adhd - c ) showed deficits in the inhabitation domain ( 24 ) .", "also , li et al . found noticeable cognitive impairments such as poor response inhibition , impaired working memory , dysfunction of planning and set - shifting in children with adhd , but there were no significant differences between the two subtypes of adhd in their study ( 12 ) .", "the first and most notable limitation of our study was that we only used one test to assess the executive function ; and the second limitation was that we did not directly evaluate the control group for psychiatric disorder and only relied on the parental report .", "our findings revealed that executive function patterns are different in children with adhd compared to normal children .", "our study also confirmed that adhd subtypes are different in terms of perseveration and response inhibition domains ; adhd - c type showed more deficits in perseveration and response inhibition .", "our findings revealed that executive function patterns are different in children with adhd compared to normal children .", "our study also confirmed that adhd subtypes are different in terms of perseveration and response inhibition domains ; adhd - c type showed more deficits in perseveration and response inhibition ."], "abstract_text": ["<S> objectivethe aim of this study was to examine the differences between adhd subtypes in executive function tasks compared to themselves and normal controls.methodin this study , 45 school aged children with attention deficit hyperactivity disorder ( adhd ) and 30 normal children who were matched based on age and iq score in wechsler intelligence scale for children - revised ( wisc - r ) were compared in terms of executive function . </S>", "<S> we used wisconsin sorting card test to assess executive function in both groups . </S>", "<S> we also used children s scores in children symptom inventory-4 ( csi-4 ) for diagnosing adhd and specifying adhd subtypes . </S>", "<S> data were entered in spss-17 and analyzed by t - test and anova static tests to clarify the differences between adhd and controls and between adhd subtypes . </S>", "<S> scheffe s test was also used to identify which groups were different from one another . </S>", "<S> the mean and standard divisions ( sd ) were used for descriptive analysis.resultsadhd subtypes are significantly different in terms of perseverative responses ( p 0/01 ) and perseverative errors ( p 0/001 ) . based on scheffe s test , </S>", "<S> attention deficit hyperactivity disorders - hyperactive type ( adhd - h ) is not that different from attention deficit hyperactivity disorders - inattention type ( adhd - i ) and attention deficit hyperactivity disorders - combined type ( adhd - c ) , but there are significant responses and perseverative differences between adhd - i and adhd - c in terms of perseverative errors . </S>", "<S> adhd - c shows more perseverative responses and perseverative errors than adhd-i.conclusionthe findings of this study revealed that executive function patterns are different in children with adhd compared to normal children . in this study </S>", "<S> it was also found that adhd subtypes are also different in terms of perseveration and response inhibition domains ; adhd - c has more deficits in these domains . </S>"], "labels": null, "section_names": ["Material and Methods", "Participants", "Instruments", "Data Analysis", "Results", "Discussion", "Limitations", "Conclusion"], "sections": [["the study population was all the 6 to 9 year- old students from the districts of one and two in yazd , an old and big city located in the center of iran .", "for the study group , 56 students were selected from imam hussein counseling center ( counseling center of educational organization of yazd province ) who were referred by their school staff and had received adhd diagnosis by a child psychiatrist based on dsm - iv - tr criteria and had no comorbid disorders .", "after that , the entire study group and their parents ( mother or father ) were interviewed by a clinical psychologist .", "the child symptom inventory-4 ( csi-4 ) was filed out by parents to be used for the diagnosis and differentiation between the adhd subtypes . of the participants ,", "19 were diagnosed with adhd - h , 15 with adhd - i and 22 with adhd - c . in the adhd - c group ,", "finally , 45 students ( 15 students in each adhd subtype groups ) were remained as the study group .", "the control group consisted of 30 students who were selected from the above mentioned population by stratified random sampling method . in the control group ,", "none had a history of psychiatric disorders or developmental delay based on their parents report .", "exclusion criteria was wearing glasses , color blindness , physical or psychological disorder and using psychiatric medicines .", "students who were diagnosed with adhd in the past and were under treatment with medicine were excluded from the study due to the medicines effect on attention . in the current study ,", "all the students ( from both sexes ) with all adhd subtypes were participated as the study group .", "all cases in the study and the control group were matched based on age , sex and iq level . in the current study four tools of structured interview , wechsler intelligence scale for children - revised ( wisc - r ) and child symptom inventory-4 ( csi-4 ) were used .", "child symptom inventory-4 ( csi-4 ) is a screening tool for the symptoms of behavioral and emotional disorders in children based on the diagnostic criteria of dsm - iv .", "the parent report checklist contains 97 items to screen 15 behavioral and emotional disorders including ad / hd , oppositional defiant disorder , conduct disorder , generalized anxiety disorder , social phobia , separation anxiety disorder , obsessive - compulsive disorder , specific phobia , major depressive disorder , dysthymic disorder , schizophrenia , pervasive developmental disorder , asperger s disorder and tics disorder .", "there are two methods to score csi-4 : criterion - related and norm - based . in the criterion - related method", "the score of zero was given to items which were rated as never and seldom , and the score of one was given to the items which were rated as sometimes and often .", "the final score for each subscale was obtained by adding the scores of the items related to the given subscale . in the present study ,", "this subscale consists of 18 items , in which items 1 - 9 are used for adhd - i and items 10 - 18 for adhd - h .", "the total adhd score can be obtained by adding the scores of 1 - 18 items . the cutoff point for diagnosing adhd based on the csi-4", "is 6 for both the mainly attention deficit and the mainly hyperactive - impulsive types .", "the test - retest reliability of the parent checklist in an iranian sample was reported as 0.90 .", "furthermore , in another study , cronbach s alpha of 0.92 and a sensitivity of 0.94 were reported for this inventory(11 ) .", "wechsler intelligence scale for children - revised ( wisc - r ) is a well - known scale for assessing the iq in children aged between 6 - 16 years .", "wisc - r gives two different scores for verbal and performance iq and a total iq scores .", "moderate internal consistency for total iq , verbal iq and performance iq were reported as 0.96 , 0.95 and 0.91 by wechsler , respectively(34 ) . in the present study we used total iq score to match the participants .", "wisconsin card sorting test ( wcst-64 ) is a neuropsychological test for assessing problem solving , sorting , abstract thinking , ability to maintain concepts and cognitive flexibility skills which are related to performances of the frontal lobe . in this test", ", a set of 64 cards including1 - 4shapes in forms of red triangle , green star , yellow cross and blue circle are given to the participants , and then they should place the cards based on their perception of the pattern used by the examiner .", "the pattern is a red triangle , green star , yellow cross and a blue circle . for example , if the principle is color , the true placement is that the red card regardless of the shape or number should be placed under the triangle and the examiner determines if the placement is correct or not .", "after that , the subject completes a round of ten placements correctly , and the examiner will then change the principle . the test will continue until the subject finishes the placement of ten cards for six times and place 64 cards in one category(35 ) .", "data were entered in spss-17 and analyzed by t - test and anova static tests to clarify the differences between adhd and normal group and between adhd subtypes .", "scheffe s test was also used to identify which groups were different . the mean and standard divisions ( sd ) were used for descriptive analysis ."], ["the study population was all the 6 to 9 year- old students from the districts of one and two in yazd , an old and big city located in the center of iran .", "for the study group , 56 students were selected from imam hussein counseling center ( counseling center of educational organization of yazd province ) who were referred by their school staff and had received adhd diagnosis by a child psychiatrist based on dsm - iv - tr criteria and had no comorbid disorders .", "after that , the entire study group and their parents ( mother or father ) were interviewed by a clinical psychologist .", "the child symptom inventory-4 ( csi-4 ) was filed out by parents to be used for the diagnosis and differentiation between the adhd subtypes . of the participants ,", "19 were diagnosed with adhd - h , 15 with adhd - i and 22 with adhd - c . in the adhd - c group ,", "finally , 45 students ( 15 students in each adhd subtype groups ) were remained as the study group .", "the control group consisted of 30 students who were selected from the above mentioned population by stratified random sampling method . in the control group ,", "none had a history of psychiatric disorders or developmental delay based on their parents report .", "exclusion criteria was wearing glasses , color blindness , physical or psychological disorder and using psychiatric medicines .", "students who were diagnosed with adhd in the past and were under treatment with medicine were excluded from the study due to the medicines effect on attention . in the current study ,", "all the students ( from both sexes ) with all adhd subtypes were participated as the study group .", "all cases in the study and the control group were matched based on age , sex and iq level ."], ["in the current study four tools of structured interview , wechsler intelligence scale for children - revised ( wisc - r ) and child symptom inventory-4 ( csi-4 ) were used .", "child symptom inventory-4 ( csi-4 ) is a screening tool for the symptoms of behavioral and emotional disorders in children based on the diagnostic criteria of dsm - iv .", "the parent report checklist contains 97 items to screen 15 behavioral and emotional disorders including ad / hd , oppositional defiant disorder , conduct disorder , generalized anxiety disorder , social phobia , separation anxiety disorder , obsessive - compulsive disorder , specific phobia , major depressive disorder , dysthymic disorder , schizophrenia , pervasive developmental disorder , asperger s disorder and tics disorder .", "there are two methods to score csi-4 : criterion - related and norm - based . in the criterion - related method", "the score of zero was given to items which were rated as never and seldom , and the score of one was given to the items which were rated as sometimes and often .", "the final score for each subscale was obtained by adding the scores of the items related to the given subscale . in the present study ,", "this subscale consists of 18 items , in which items 1 - 9 are used for adhd - i and items 10 - 18 for adhd - h .", "the total adhd score can be obtained by adding the scores of 1 - 18 items . the cutoff point for diagnosing adhd based on the csi-4", "is 6 for both the mainly attention deficit and the mainly hyperactive - impulsive types .", "the test - retest reliability of the parent checklist in an iranian sample was reported as 0.90 .", "furthermore , in another study , cronbach s alpha of 0.92 and a sensitivity of 0.94 were reported for this inventory(11 ) .", "wechsler intelligence scale for children - revised ( wisc - r ) is a well - known scale for assessing the iq in children aged between 6 - 16 years .", "wisc - r gives two different scores for verbal and performance iq and a total iq scores .", "moderate internal consistency for total iq , verbal iq and performance iq were reported as 0.96 , 0.95 and 0.91 by wechsler , respectively(34 ) . in the present study we used total iq score to match the participants .", "wisconsin card sorting test ( wcst-64 ) is a neuropsychological test for assessing problem solving , sorting , abstract thinking , ability to maintain concepts and cognitive flexibility skills which are related to performances of the frontal lobe . in this test", ", a set of 64 cards including1 - 4shapes in forms of red triangle , green star , yellow cross and blue circle are given to the participants , and then they should place the cards based on their perception of the pattern used by the examiner .", "the pattern is a red triangle , green star , yellow cross and a blue circle .", "for example , if the principle is color , the true placement is that the red card regardless of the shape or number should be placed under the triangle and the examiner determines if the placement is correct or not .", "after that , the subject completes a round of ten placements correctly , and the examiner will then change the principle . the test will continue until the subject finishes the placement of ten cards for six times and place 64 cards in one category(35 ) ."], ["data were entered in spss-17 and analyzed by t - test and anova static tests to clarify the differences between adhd and normal group and between adhd subtypes .", "scheffe s test was also used to identify which groups were different . the mean and standard divisions ( sd ) were used for descriptive analysis ."], ["the study group consisted of 45 school aged children ; of whom , 27 were male and 18 were female . in the control group", ", there were 30 school aged children , of whom 18were male and 12 were female .", "other characteristics of the two groups ( age , iq , adhd subtype frequency ) are presented in table 1 .", "data analysis showed that adhd and normal group were significantly different in terms of perseverative responses , perseverative errors and total errors on wsct ( tables 2 and 3 ) as shown in table 4 , adhd subtypes were significantly different in terms of perseverative responses ( p 0/01 ) and perseverative errors ( p0/001 ) . based on scheffe s test ,", "adhd - h was not different from adhd - i and adhd - c but there were significant differences between adhd - i and adhd - c in terms of perseverative responses and perseverative errors ."], ["the aim of the present study was to investigate the differences between adhd subtypes in terms of executive function profile .", "previous studies showed inconsistent findings in this regard . in an extended study , houghton et al .", "investigated the executive function in 94 children with adhd ( without any co - morbid disorder ) in terms of subtype and gender using five tests of the wisconsin card sorting test , the stroop color - word test , the matching familiar figures test , the trail making test , and the tower of london .", "they found that children with both types of adhd ( adhd - i and adhd - c ) differed from normal children in terms of perseveration and response inhibition , but this difference was significant in adhd - c , only ( 27 ) .", "the lack of any co - morbidity in children with adhd showed that the impairments in executive function were obviously found in adhd , particularly in the adhd - c , so providing support for barkley s theory of adhd ( 27 ) .", "consistent with this finding , in another study , klorman et al . found that children with adhd - c revealed more non - perseverative errors in wcst and solved fewer puzzles and also violated more rules on the tower of hanoi ( toh ) than adhd - i , but they were not different based on perseverative errors in wcst(28 ) . also , lawrence et al .", "their finding revealed that children with adhd were only different in set - shifting on wsct ( perseverative responses and errors ) ( 10 ) . in this respect", ", we found that children with adhd significantly differed from normal children in terms of total errors , perseverative errors and perseverative responses ( p 0.01 ) .", "also , we found that adhd subtypes operated differently in the executive function domain ( p 0.01 ) ; children with adhd predominantly combined type showed more perseverative responses and perseverative errors than children with adhd predominantly inattentive type .", "some researchers suggest that the two primary subtypes of adhd share similar neuropsychological weaknesses in inhibitory control , but there are subtype differences in response to success and failure that are contributed to a child s ultimate level of performance ( 36 ) . unlike the barkley s behavioral inhibition theory , song and hakoda found that both types of adhd ( adhd - i and adhd - c ) showed deficits in the inhabitation domain ( 24 ) .", "also , li et al . found noticeable cognitive impairments such as poor response inhibition , impaired working memory , dysfunction of planning and set - shifting in children with adhd , but there were no significant differences between the two subtypes of adhd in their study ( 12 ) ."], ["the first and most notable limitation of our study was that we only used one test to assess the executive function ; and the second limitation was that we did not directly evaluate the control group for psychiatric disorder and only relied on the parental report .", "our findings revealed that executive function patterns are different in children with adhd compared to normal children .", "our study also confirmed that adhd subtypes are different in terms of perseveration and response inhibition domains ; adhd - c type showed more deficits in perseveration and response inhibition ."], ["our findings revealed that executive function patterns are different in children with adhd compared to normal children .", "our study also confirmed that adhd subtypes are different in terms of perseveration and response inhibition domains ; adhd - c type showed more deficits in perseveration and response inhibition ."]]}
{"article_id": "PMC4848062", "article_text": ["the visual environment provides a vast assortment of information to the sighted individual during the acts of daily living . for the visually - impaired individual ,", "a need exists to compensate for the lack of this information , which has motivated the introduction of a wide variety of devices that transfer at least some visual information to another sense .", "however , the primary assistive technologies widely used today by the visually - impaired to navigate through the environment are essentially unchanged from those of twenty years ago , namely , white canes and guide dogs .", "although these two methods can facilitate the ability to travel safely in a variety of indoor and outdoor environments , neither provide the kind of assistance needed to straighten a picture frame on the wall or find a can of soup on a counter - top .", "electronic navigation aids are finding some acceptance , for example , ultrasonic canes ( ultracane from sound foresight technology , ltd . ) that provide tactile cues to objects beyond the tip of the cane , as well as portable computers with global positioning systems ( gps ) and electronic braille or speech interfaces .", "however , replacing the more general capabilities of vision to provide detailed information about objects in the environment has proven more difficult .", "there exists a need for a device that allows active interrogation and sensing of the 3d visual environment surrounding the operator while moving through everyday environments .", "there also exists a need for a device that not only allows the user to sense the environment but also provides control of specific aspects of the environment that have been detected .", "these abilities to interrogate and control the environment should not be limited to a specific , predetermined environment , such as those that already contain infrared ( ir ) transmitters .", "devices using a concept we call fingersight are intended to allow visually impaired users to remotely identify objects of interest in , and navigate through , natural environments , by acting as a sensory substitution system for the user 's sense of sight .", "there are already a number of technologies that monitor hand , eye , and body motion from a fixed or mobile camera to interpret gestures , commands , gaze direction , e.g. , .", "some of these may be used to track the operator 's interrogation of the environment , as with the commercially available eye - tracking glasses ( e.g. , tobii , tokyo ) , but more often they are used to interpret the user 's motions as commands .", "our original goal was , and largely remains , to serve the population who are visually impaired .", "most devices designed with this goal make use of sensory substitution systems , which transmit stimuli normally interpreted by one sense through another sense .", "sensory substitution systems generally consist of three components : a sensor that collects a signal , a coupling system that processes the signal , and finally an output transducer , which transmits the signal to a sense organ not normally used for that signal , .", "we specifically consider substitution for the visual system by the haptic system ( i.e. , one 's sense of touch ) .", "although hearing offers a potentially higher bandwidth than touch , it is crucial not to impede the existing use of hearing , which can be acutely well developed by those who lack normal vision , providing essential acoustic cues about the environment . as for touch , the hands offer the greatest versatility and sensitivity , but still we must not completely usurp their use , since they are essential for so many tasks in daily living .", "some employ tactile pin arrays , which feature a grid of small pins with adjustable heights .", "tactile pin arrays can be used for either passive stimulation of the fingers or during active exploration to simulate contact with surfaces and objects .", "existing systems capture an image and then display that image on a tactile screen that can be worn on a belt , for example .", "the fingers can then be used to interrogate the image depicted on the tactile screen in an effort to  visualize  the image , .", "some devices have been developed to directly convey a predetermined map from coordinates of physical space to a tactile spatial layout .", "an example is bach - y - rita 's brainport device , which uses an array of electrodes placed on the tongue to relay camera information .", "sensory substitution systems also exist that provide an audio output , such as text - to - speech devices that employ optical character recognition , canes that provide audio feedback to enhance the natural tactile forces detected at the tip itself , and the voice system , which maps image pixels into frequency and time .", "vibrotactile sensitivity is found in mechanoreceptors lying within the subcutaneous tissue , which respond maximally to frequencies of 250300 hz , as well as kinesthetic receptors . the cybertouch glove ( immersion corp .", ", san jose , ca ) uses optical tracking of each finger to determine when vibratory stimulators on that finger should be activated . some sensory aids , such as the optacon ( telesensory corp . ,", "defunct ) , aim to simulate tactile exploration of virtual objects and have been developed specifically for reading the printed page .", "our proposed device uses a vibrotactile stimulator to provide visual information gathered from a color camera system .", "the information from the camera system can be processed into a single feature whose presence or absence is communicated through the stimulator .", "for example , the camera may provide a positive signal when an edge is detected in the field of view .", "our system is similar in some ways to that of lenay ,   who attached a photocell to the tip of a finger and used it to activate a vibrator held in the other hand in an all - or - nothing manner .", "another system , developed by burch and pawluk , is mounted on the same finger as the sensor , for interacting with graphical objects on a flat screen .", "this system senses a single rgb pixel at a short distance ( i.e. , to the screen that the subject is touching ) , and vibrates based upon the color sensed .", "our system goes beyond this basic idea by detecting not just the general directional presence of light or contact with a single light source , but rather actual images ( or a series of images over time ) using a miniature camera . like burch and pawluk", "we believe that this placement promotes an intuitive coupling between the scene pointed to by the camera mounted on the fingertip and the feedback felt by that finger . in general , it is imperative for designers of any sensory substitution system to consider not only what is technologically feasible , but also what is functional in the context of the sensory and cognitive limitations of the user . in a review of sensory substitution for the blind , loomis and klatzky   pointed to the need to couple device design to the capabilities of the user for a given task .", "low bandwidth , for example , need not be a negative feature of a device if a task can competently be performed on the basis of the information provided .", "high bandwidth communication to the user is not an advantage if the target sensory channel can not process the information provided .", "furthermore , it should not be assumed that information - processing capacities of blind and sighted are equivalent ; for example , superior tactile sensory systems may be preserved in older braille readers relative to sighted populations of the same age , whereas tasks that draw on visual imagery may be impeded in a blind population without any experience of sight .", "we have developed a series of working prototypes , leading up to the one used in the experiments described in the present paper . as the progression is informative", ", we review them briefly here . our initial system permitted active interrogation of the visual surroundings with a miniature red laser attached to the fingertip ( see fig .  1 ) .", "the laser was modulated at 10 khz , allowing its reflection by objects in the environment to be detected by a non - directional phototransistor in the midst of other sources of light .", "thus , like the burch device , it sensed the light ( in this case reflected ) from a single point in space , but at a distance .", "moreover , it differed from that device by actively interrogating across the detected point for an edge .", "this was accomplished by means of a solenoid constructed from tiny magnets and a coil .", "regenerative feedback between the amplitude of the detected signal from the reflected laser and the solenoid caused the laser to vibrate vertically when the laser spot was located on any edge between light and dark objects .", "thus the system created a haptic stimulus ( vibration ) whenever a properly oriented visual edge was encountered by the laser . \n", "the laser - based system was limited to simple edges and had the disadvantage that the active visible light source could be disturbing to others .", "more importantly , limiting fingersight to what amounts to a single scanning line makes it extremely difficult to integrate more than the most rudimentary features in the visual field .", "a full video image captured at each pose of the finger offers many advantages , including immediate extraction of more complex features , identification of entire objects , and even determination of camera motion directly from the changing image .", "the recent availability of very inexpensive and small cameras , brought about largely by their use in cell phones , has led us to adopt a camera - based approach , the first version of which is shown in fig .  2 \n . a small black - and white camera ( supercircuits pc206xp 510492 ) was mounted on the subject 's finger along with a cell - phone vibrator .", "real - time analysis of the video signal from a miniature camera was used to control the cell - phone vibrator .", "the cell - phone vibrator has the advantages of low cost and size , but presents problems in that amplitude and frequency are inextricably linked together by the operating voltage .", "also , the frequency is low enough to cause noticeable vibration in the camera image , and the time for the motor to come up to speed is relatively long . \n", "two small speakers ( gui , inc . # gc0251k - nd , 1w , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 8\\omega$\\end{document } ) were converted into haptic vibratory stimulators , or tactors , by cementing a short wooden dowel onto the central cones of each speaker .", "the converted speakers were mounted on either side of the finger and held there , along with the camera , by a springed clip , leaving the palmar aspect of the fingertip bare to use for touching and grasping objects .", "we hoped , by including two such tactors , to be able to use the relative strength of the vibration from each tactor to convey a parameter such as location of an image feature along the horizontal axis . \n", "this model did not perform well for identifying the location of visual targets , primarily due to mechanical design issues .", "it proved problematic to mount the device securely on the distal phalanx of the finger . furthermore , given the influence of flexion / extension at the distal inter - phalangeal joint on the camera orientation , it was difficult for users to judge where they were aiming the device .", "in addition , there was low sensitivity to vibration the speakers mounted on the lateral aspect of the finger , as compared to the speaker on the medial aspect .", "collectively , these issues made any asymmetry in signal strength from the two tactors of little value to the user 's perception .", "the current version of fingersight uses the same speaker - based tactor shown in fig .  3 , but with only one tactor on the ( more sensitive ) medial aspect of the finger , along with a miniature color camera ( supercircuits pc208 , 510492 ) .", "the camera extended approximately 8 mm from the tip of the finger , and the speaker was attached to the side of the splint .", "the splint weighed 18.8 g and caused minimal interference with movement of the finger as a whole , though it did restrict flexion between the phalanges , thereby reducing uncertainty in camera orientation . covering the palmer aspect of the finger made it difficult to use the finger for other purposes , but we were willing to accept this for the present experiment .", "the camera and the speaker were connected to a computer system , which performed spatiotemporal edge detection ( described below ) and controlled vibrator output .", "the haptic feedback was provided by a low - frequency ( 20 hz ) audio signal sent to the speaker , the amplitude of which was adjusted to produce detectable vibrations . \n fig . ", "4.current fingersight device apparatus with camera and stimulator attached . \n  for the purposes of experimental testing", "the area of the circle was divided across its center , one semicircle containing red and one containing green pixels ( see fig .  5 ) .", "the line across the center of the circle separating red from green pixels could be set to any desired orientation .", "the circular area was surrounded by white pixels and the images were projected on a screen , where the user could point the finger - mounted camera at it . \n", "5.examples of edge stimuli , at 12 ( a ) and 4 ( b ) o'clock , respectively . \n ", "software was developed to analyze the video image from the finger - mounted camera in real time and to control the vibratory feedback to that finger .", "the goal was to generate vibrations whenever the user 's finger was pointing at , or had just passed over , the boundary between the red and green areas , irrespective of the edge 's orientation .", "optical edge detection was performed with a novel spatiotemporal kernel , designed to detect edges both in the spatial domain ( when the camera was resting on an edge ) and the temporal domain ( when the camera had moved completely past an edge between one video frame and the next ) .", "this design accommodated fast hand movements , which might cause the edge to never actually have been within the kernel in any given frame , as well as slower continual tracking along the edge .", "the spatiotemporal kernel accomplished both of these for edges of any orientation by comparing each pixel in one time frame with the corresponding pixel directly across the center of a circular kernel in the next time frame .", "if the two pixels differed , an edge was present or had just been crossed .", "it is important to note that unlike the general usage of the term  kernel  in image processing , especially for convolution , our spatiotemporal kernel was applied not at every pixel , but only at one location , the center of the image .", "just as the fovea of the eye ( the high resolution central region ) is constantly moving for human vision to function , our kernel was physically moved by the user 's finger along with the entire image , to interrogate the visual environment .", "the optimal size of the circular kernel varied depending on the scale of the edge features to be considered .", "for the particular experiment described here , the optimal radius of the kernel was determined experimentally to be 50 pixels .", "a detailed formulation of the algorithm follows : we define k as the set of pixels within the circular kernel at the center of the image . based on the particular red - green - blue ( rgb ) video image coming from the camera in real time , a color label \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n", "\\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c(x , y)$\\end{document } was created for each pixel in k , denoting whether the pixel at location \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( x , y)$\\end{document } was predominantly red , green , or ambiguous.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ c\\left(x , y\\right)=\\cases{+1/2,&$ if~predominantly~red$\\cr-1/2,&$ if~predominantly~green$\\cr 0,&$if~ambiguous$\\cr}\\eqno{\\hbox{(1)}}$$\\end{document } for our purposes ,  predominantly  indicates that the red ( r ) or blue ( b ) color value for the pixel in question was above a specific threshold , while the other two color values in the rgb pixel were both below their corresponding thresholds .", "appropriate thresholds for red , green , and blue were determined experimentally , so that red , green , and white pixels ( which registered as ambiguous ) responded appropriately .", "we also created a binary mask value \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w(x , y)$\\end{document } for each pixel that had the value of 1 wherever c was non - zero , so that boundaries including the white area surrounding the circle could be excluded from generating edges.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ w\\left(x , y\\right)=\\cases{1&$if~c\\left(x , y\\right)\\ne 0$\\cr 0&$otherwise$\\cr}\\eqno{\\hbox{(2)}}$$\\end{document } for each pixel in k , we also considered another pixel in k directly across the origin ( center of the kernel ) at location \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( -x ,- y)$\\end{document}. our measure of boundary strength was calculated by comparing the value \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c_{t}$\\end{document } for the pixel at \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( x , y)$\\end{document } in the current image at time \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t$\\end{document } to value of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c_{t-1}$\\end{document } for the pixel at \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( -x ,- y)$\\end{document } in the previous image at time \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t-1$\\end{document}. the mask values \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w_{t}$\\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w_{t-1}$\\end{document } were used to ensure that no comparisons were made using  ambiguous  pixels .", "thus a measure of edge strength \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ d$\\end{document } was computed as \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ d\\!=\\!\\!\\!\\sum_{\\left(x,\\!y\\right)\\in k}\\!\\!{\\left\\vert c_{t}\\left(x , y\\right)\\!-\\!c_{t-1}\\left(-x,\\!-y\\right)\\right\\vert w_{t}\\left(x , y\\right)w_{t-1}\\!\\left(-x,\\!-y\\right)}\\eqno{\\hbox{(3)}}$$\\end{document } if the edge measure \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ d$\\end{document } was above some threshold ( determined experimentally ) , the software reported that an edge had been detected , and triggered the haptic feedback in the form of vibration at the fingertip .", "the algorithm has the advantage that it measures equally well the conditions of sitting on an edge ( of any orientation ) for two successive timeframes and having passed completely over the edge from one timeframe to the next .", "for example , in 3d one would simply compare pixels directly across the center of a spherical rather than a circular kernel .", "twelve sighted subjects ( 10 males and 2 female ) were recruited from the university population , with an average age of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 29.4+/-{9.8}~{\\rm years}$\\end{document}. all gave informed consent .", "subjects took part in a series of trials , in which they identified the angle of an edge stimulus according to a closed set of alternatives . each edge stimulus , as described above , was projected on a display screen and consisted of a circular area 115 cm in diameter on a white background bisected with one half colored red and the other green .", "the circle was surrounded by white , so that the perimeter of the circle would not itself be interpreted as an edge ( as described above ) .", "subjects were fitted with earplugs , blindfolded , and seated with the right arm resting on a foam support 79 cm above the floor .", "the circles were projected ( epson powerlite 70c ) on a 154115 cm screen positioned 168 cm in front of the subject 's fingertip .", "the arm support was arranged so that the subject 's arm and finger were initially pointing perpendicular to the center of the projected image . during each trial ,", "the subject freely explored the display edge with the fingersight device as long as desired .", "generally , subjects maintained their arm on the rest and moved their forearm and wrist .", "angle set 1 consisted of six angles equally spaced over a 180 range in 30 steps ( from positive vertical , 90 , to just short of negative vertical , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { -}{60}^{\\circ}$\\end{document } ) , corresponding to clock - hours from 12 to 5 o'clock .", "angle set 2 consisted of seven angles equally spaced over a 90 range in 15 steps ( from positive vertical , 90 , to horizontal , 0 ) corresponding to the hours and half hours between 12 to 3 o'clock .", "subjects reported their responses using these  clock face  labels . within each set ,", "tables  1 and 2 show the confusion matrices for all subjects for angle sets 1 and 2 , respectively , plotting reported angle vs. stimulus angle .", "a confusion matrix shows correct measurements along the diagonal , with increasing error as one moves off the diagonal .", "overall , subjects had an accuracy rate ( proportion correct ) of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 0.77\\pm 0.05$\\end{document } on angle set 1 and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 0.52\\pm 0.09$\\end{document } on angle set 2 ( 95% confidence intervals ) .", "these levels are well above chance in this six - alternative choice task \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm chance}=0.16)$\\end{document}. \n table iconfusion matrix for set 1reported angle ( deg)90603003060stimulus angle ( deg)9071131011060168252003008843100101081313000068196040003260confusion matrix for angle set 1 .", "each entry is the number of occurrences of the associated stimulus / response pair . \n", "table iiconfusion matrix for set 2reported angle ( deg)0153045607590stimulus angle ( deg)0642532200151949242101301205519100450836391210600115234692750011533389900011112558confusion matrix for angle set 2 .", "each entry is the number of occurrences of the associated stimulus / response pair . \n ", "the data were also analyzed by measuring information transfer \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } , in order to determine the number of  bits  that can be transmitted by the device under the experimental conditions .\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i=\\sum_{j=1}^{k}\\sum_{i=1}^{k}{p\\left(s_{i},r_{j}\\right)\\log_{2}\\left({{p\\left(s_{i}\\vert r_{j}\\right)}\\over{p\\left(s_{i}\\right)}}\\right)}.\\eqno{\\hbox{(4)}}$$\\end{document } here , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ k$\\end{document } is the number of possible stimuli , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ s_{i}$\\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ r_{j}$\\end{document } represent a specific stimulus - response pair .", "this statistic can be estimated from the confusion matrices according to equation 6.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i_{est}=\\sum_{j=1}^{k}\\sum_{i=1}^{k}\\left({{n_{ij}}\\over{n}}\\right)\\log_{2}\\left({{n_{ij}\\times n}\\over{n_{i}\\times n_{j}}}\\right)\\!.\\eqno{\\hbox{(5)}}$$\\end{document } here , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n_{ij}$\\end{document } is the number of joint occurrences of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } and response \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ j , n_{i}$\\end{document } is the overall occurrence of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n_{j}$\\end{document } is the overall occurrence of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ j$\\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n$\\end{document } is the total number of trials .", "relative to the 2.58 bits of information in the six - alternative choice task , angle set 1 ( angles with 30 increments ) had an information transfer of 1.62 bits , while angle set 2 ( angles with 15 increments ) had an information transfer of 1.12 bits .", "we have developed a new method of providing the finger with haptic feedback about visual targets in the general environment , by permitting the finger to scan the environment directly to locate those targets .", "we have developed this idea , which we call fingersight , through a series of prototypes , exploring the particular application of detecting edges . in the process , we have developed a new spatiotemporal \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n$\\end{document}-dimensional edge detection algorithm of potentially general interest , which is simultaneously capable of detecting the presence of a stationary edge as well as having passed over an edge between successive timeframes .", "we have conducted proof - of - concept experiments showing that under active exploration the fingersight device is capable of transmitting visual spatial information through haptic channels . sighted , blindfolded subjects were able to use the device to differentiate between angles separated by as little as 15. performance with the coarser angle set , where responses were separated by 30 showed that fingersight transmitted close to 2/3 of the available information in the stimulus , with most errors being near misses .", "the information transmission of the fingersight device is intrinsically constrained by basic sensorimotor abilities of motor control , kinesthetic sensing , and spatial cognitive processing .", "was found that when blindfolded , sighted adults tried to report the orientation of a raised line that was easily tracked by touch on the plane of a tabletop , their responses pulled the true value towards the sagittal axis by about 25% ( plus some constant error ) .", "since motor control was minimized in the task in , errors can be entirely attributed to the processes of kinesthetic sensing and building a spatial representation .", "the observed level of distortion is by itself substantial enough to lead to confusion errors in the present task .", "these observations suggest that performance with fingersight could be improved by features that support motor control and augment kinesthetic feedback .", "the present experiments measure only the performance of blindfolded sighted subjects who are novices using the device ; training would be expected to considerably improve performance .", "siegle and warren have shown that distal attribution , i.e. , the direct perception of objects in external space , can occur with similar finger - mounted sensory substitution systems after a period of training .", "however the device used in their experiments had the vibrotactile stimulator mounted on the subject 's back .", "it is possible that training time may be decreased with a device such as fingersight , where the stimulus is more strongly tied to the subject 's proprioceptive knowledge of the hand 's location in space .", "it is interesting to compare the accuracy of angular measurements by fingersight in 3d space with other researchers who restricted interrogation to a fixed plane .", ", explored the ability to recognize 90- or 180-degree rotation on raised - outline drawings directly touched by the blind or sighted - blindfolded subjects , but did not explore finer angular resolution .", "postma , et al . , demonstrated the role of visual experience in such haptic spatial tasks , including the description of angles between bars on a table , showing that blindfolded sighted subjects outperformed late blind subjects , who outperformed early blind subjects .", "they found that having experienced vision , even to a limited extent , helps in the interpretation of angle by touch alone .", "the worst performance in verbally judging the angle of the bars ( demonstrated by the early blind ) was 7.2 degrees , a good deal better than our errors , possibly because they were constrained to a tabletop .", "it would also be interesting to see if sighted individuals also have a similar advantage in learning to use fingersight over blind individuals , due to previous visual experience . also relevant to the question of angle and touch", ", with haptic computer mice , which are modified to have tactile pin arrays on their upper surface .", "significant lack of accuracy in the haptic position information , which is critical for individuals to haptically piece together a 2-d graphic . ", "the inaccuracy is due to the fact that the tactile mouse ( or any normal mouse ) is a relative positioning device , dependent upon the speed of motion and orientation of the mouse to determine total displacement on the screen .", "in contrast , fingersight is inherently an absolute positioning device , given a stationary environment , and as such , fingersight may have an advantage .", "the system of burch and pawluk previously mentioned   uses a fingertip photosensor and piezoelectric stimulator to scan specially created graphical displays , in which texture is added to enhance perception of edges and orientations .", "a single photosensor thus suffices for this purpose , only because preprocessing is performed to populate regions on either side of boundaries with differing textures .", "multiple photosensors on different fingers were found to improve results with this system , because the operator 's knowledge of the spatial relationship between the fingertips could be used to integrate the inputs .", "but still the approach relies on preprocessing the image to create textures , after which individual photosensors can be effective . with fingersight , we are exploring the unadulterated 3d environment , where depending on only a few photosensors is not sufficient .", "we benefit greatly by having a multi - pixel image at each timeframe . by further processing of the camera images ,", "the fingersight device could be adapted to identify more complicated features than simple visual edges , perhaps even constituting an object recognition system for a blind operator .", "for example , it could be used to find a particular person in a crowd , or identify a door displaying a particular sign . the rapid advancement in computer vision algorithms , driven in part by the security and social networking industries , will provide ever more sophisticated capabilities .", "for example , determining camera motion and target depth from a image sequence could permit greater 3d integration of multiple perspectives and facilitate providing navigational cues to the blind operator , such as where the curbside is , whether the approaching stairwell goes up or down , or whether one is moving towards or away from the elevator .", "such analyses are not feasible for a single photosensor , but rather , they require an entire image .", "a common problem in any real - world computer vision application is the variability of lighting , and there are established techniques for solving this problem using such constructs as the  illumination cone  .", "for example , a recently developed time - of - flight ( tof ) 3d camera , the swiss ranger sr4000 ( mesa imaging , zuerich ) , can deliver a 176144 pixel image with each pixel reporting range up to 10 m with 1 cm accuracy .", "the present camera is roughly 7 \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm cm}^{3}$\\end{document } and is being integrated by at least one research group into portable devices for the blind .", "for some of these more sophisticated systems , it may become problematic to rely solely on vibrotactile feedback to the finger .", "the bandwidth of audio , especially combined with language , makes it an appealing option , though as noted above , one does not want to impede the natural use of auditory cues that are especially crucial for the vision impaired . however , intermittent use of verbal output by fingersight , and for that matter , verbal commands by the operator to the device , could prove extremely useful , while not impeding auditory cues form the environment . as noted above , it is imperative to test whether features added to the technology ultimately add to the functional capability of the user , given intrinsic limitations on human information processing .", "that said , one promising avenue is the incorporation of control capabilities into the fingersight device .", "one of our previous systems included the capability to control graphical objects on a screen . in this implementation", ", the location of a small white square on a screen is controlled by motion of the finger .", "the algorithm detects the square in the field of view of the camera , moving it on the screen to keep it constantly in the center of the camera image , while providing haptic feedback about whether the tracking system has locked onto the target .", "a variation on this system constrains the square to move along a straight line , simulating the action of a slide pot under the operator 's control .", "a further variation uses a small white triangle to simulate a knob , whose orientation is determined using standard computer vision techniques and subsequently controlled by rotation of the finger .", "clearly , such systems are not limited to actively controlling graphical objects on a screen , but could also identify inanimate objects such as a light switch or door latch .", "in such cases , remote control could still be achieved by motion of the finger once the target has been identified , using a separate control channel to turn on the light or lock the door . continued work on the control aspects of fingersight", "a final note is merited on the eventual miniaturization of a fingersight device such that it might be small enough to be worn like an artificial fingernail .", "the cameras themselves are almost small enough already , and the main considerations are power and communications .", "one can envision radio communication between a fingertip device and a pocket unit , much as wireless earphones and microphones communicate with cell phones now .", "such devices might be fully integrated into the everyday activities of the vision - impaired ."], "abstract_text": ["<S> we present a novel device mounted on the fingertip for acquiring and transmitting visual information through haptic channels . </S>", "<S> in contrast to previous systems in which the user interrogates an intermediate representation of visual information , such as a tactile display representing a camera generated image , our device uses a fingertip - mounted camera and haptic stimulator to allow the user to feel visual features directly from the environment . visual features ranging from simple intensity or oriented edges to more complex information identified automatically about objects in the environment may be translated in this manner into haptic stimulation of the finger . </S>", "<S> experiments using an initial prototype to trace a continuous straight edge have quantified the user 's ability to discriminate the angle of the edge , a potentially useful feature for higher levels analysis of the visual scene . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Conclusion"], "sections": [["the visual environment provides a vast assortment of information to the sighted individual during the acts of daily living . for the visually - impaired individual ,", "a need exists to compensate for the lack of this information , which has motivated the introduction of a wide variety of devices that transfer at least some visual information to another sense .", "however , the primary assistive technologies widely used today by the visually - impaired to navigate through the environment are essentially unchanged from those of twenty years ago , namely , white canes and guide dogs .", "although these two methods can facilitate the ability to travel safely in a variety of indoor and outdoor environments , neither provide the kind of assistance needed to straighten a picture frame on the wall or find a can of soup on a counter - top .", "electronic navigation aids are finding some acceptance , for example , ultrasonic canes ( ultracane from sound foresight technology , ltd . ) that provide tactile cues to objects beyond the tip of the cane , as well as portable computers with global positioning systems ( gps ) and electronic braille or speech interfaces .", "however , replacing the more general capabilities of vision to provide detailed information about objects in the environment has proven more difficult .", "there exists a need for a device that allows active interrogation and sensing of the 3d visual environment surrounding the operator while moving through everyday environments .", "there also exists a need for a device that not only allows the user to sense the environment but also provides control of specific aspects of the environment that have been detected .", "these abilities to interrogate and control the environment should not be limited to a specific , predetermined environment , such as those that already contain infrared ( ir ) transmitters .", "devices using a concept we call fingersight are intended to allow visually impaired users to remotely identify objects of interest in , and navigate through , natural environments , by acting as a sensory substitution system for the user 's sense of sight .", "there are already a number of technologies that monitor hand , eye , and body motion from a fixed or mobile camera to interpret gestures , commands , gaze direction , e.g. , .", "some of these may be used to track the operator 's interrogation of the environment , as with the commercially available eye - tracking glasses ( e.g. , tobii , tokyo ) , but more often they are used to interpret the user 's motions as commands .", "our original goal was , and largely remains , to serve the population who are visually impaired .", "most devices designed with this goal make use of sensory substitution systems , which transmit stimuli normally interpreted by one sense through another sense .", "sensory substitution systems generally consist of three components : a sensor that collects a signal , a coupling system that processes the signal , and finally an output transducer , which transmits the signal to a sense organ not normally used for that signal , .", "we specifically consider substitution for the visual system by the haptic system ( i.e. , one 's sense of touch ) .", "although hearing offers a potentially higher bandwidth than touch , it is crucial not to impede the existing use of hearing , which can be acutely well developed by those who lack normal vision , providing essential acoustic cues about the environment . as for touch , the hands offer the greatest versatility and sensitivity , but still we must not completely usurp their use , since they are essential for so many tasks in daily living .", "some employ tactile pin arrays , which feature a grid of small pins with adjustable heights .", "tactile pin arrays can be used for either passive stimulation of the fingers or during active exploration to simulate contact with surfaces and objects .", "existing systems capture an image and then display that image on a tactile screen that can be worn on a belt , for example .", "the fingers can then be used to interrogate the image depicted on the tactile screen in an effort to  visualize  the image , .", "some devices have been developed to directly convey a predetermined map from coordinates of physical space to a tactile spatial layout .", "an example is bach - y - rita 's brainport device , which uses an array of electrodes placed on the tongue to relay camera information .", "sensory substitution systems also exist that provide an audio output , such as text - to - speech devices that employ optical character recognition , canes that provide audio feedback to enhance the natural tactile forces detected at the tip itself , and the voice system , which maps image pixels into frequency and time .", "vibrotactile sensitivity is found in mechanoreceptors lying within the subcutaneous tissue , which respond maximally to frequencies of 250300 hz , as well as kinesthetic receptors . the cybertouch glove ( immersion corp .", ", san jose , ca ) uses optical tracking of each finger to determine when vibratory stimulators on that finger should be activated . some sensory aids , such as the optacon ( telesensory corp . ,", "defunct ) , aim to simulate tactile exploration of virtual objects and have been developed specifically for reading the printed page .", "our proposed device uses a vibrotactile stimulator to provide visual information gathered from a color camera system .", "the information from the camera system can be processed into a single feature whose presence or absence is communicated through the stimulator .", "for example , the camera may provide a positive signal when an edge is detected in the field of view .", "our system is similar in some ways to that of lenay ,   who attached a photocell to the tip of a finger and used it to activate a vibrator held in the other hand in an all - or - nothing manner .", "another system , developed by burch and pawluk , is mounted on the same finger as the sensor , for interacting with graphical objects on a flat screen .", "this system senses a single rgb pixel at a short distance ( i.e. , to the screen that the subject is touching ) , and vibrates based upon the color sensed .", "our system goes beyond this basic idea by detecting not just the general directional presence of light or contact with a single light source , but rather actual images ( or a series of images over time ) using a miniature camera . like burch and pawluk", "we believe that this placement promotes an intuitive coupling between the scene pointed to by the camera mounted on the fingertip and the feedback felt by that finger . in general , it is imperative for designers of any sensory substitution system to consider not only what is technologically feasible , but also what is functional in the context of the sensory and cognitive limitations of the user . in a review of sensory substitution for the blind , loomis and klatzky   pointed to the need to couple device design to the capabilities of the user for a given task .", "low bandwidth , for example , need not be a negative feature of a device if a task can competently be performed on the basis of the information provided .", "high bandwidth communication to the user is not an advantage if the target sensory channel can not process the information provided .", "furthermore , it should not be assumed that information - processing capacities of blind and sighted are equivalent ; for example , superior tactile sensory systems may be preserved in older braille readers relative to sighted populations of the same age , whereas tasks that draw on visual imagery may be impeded in a blind population without any experience of sight ."], ["we have developed a series of working prototypes , leading up to the one used in the experiments described in the present paper . as the progression is informative", ", we review them briefly here . our initial system permitted active interrogation of the visual surroundings with a miniature red laser attached to the fingertip ( see fig .  1 ) .", "the laser was modulated at 10 khz , allowing its reflection by objects in the environment to be detected by a non - directional phototransistor in the midst of other sources of light .", "thus , like the burch device , it sensed the light ( in this case reflected ) from a single point in space , but at a distance .", "moreover , it differed from that device by actively interrogating across the detected point for an edge .", "this was accomplished by means of a solenoid constructed from tiny magnets and a coil .", "regenerative feedback between the amplitude of the detected signal from the reflected laser and the solenoid caused the laser to vibrate vertically when the laser spot was located on any edge between light and dark objects .", "thus the system created a haptic stimulus ( vibration ) whenever a properly oriented visual edge was encountered by the laser . \n", "the laser - based system was limited to simple edges and had the disadvantage that the active visible light source could be disturbing to others .", "more importantly , limiting fingersight to what amounts to a single scanning line makes it extremely difficult to integrate more than the most rudimentary features in the visual field .", "a full video image captured at each pose of the finger offers many advantages , including immediate extraction of more complex features , identification of entire objects , and even determination of camera motion directly from the changing image .", "the recent availability of very inexpensive and small cameras , brought about largely by their use in cell phones , has led us to adopt a camera - based approach , the first version of which is shown in fig .  2 \n . a small black - and white camera ( supercircuits pc206xp 510492 ) was mounted on the subject 's finger along with a cell - phone vibrator .", "real - time analysis of the video signal from a miniature camera was used to control the cell - phone vibrator .", "the cell - phone vibrator has the advantages of low cost and size , but presents problems in that amplitude and frequency are inextricably linked together by the operating voltage .", "also , the frequency is low enough to cause noticeable vibration in the camera image , and the time for the motor to come up to speed is relatively long . \n", "two small speakers ( gui , inc . # gc0251k - nd , 1w , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 8\\omega$\\end{document } ) were converted into haptic vibratory stimulators , or tactors , by cementing a short wooden dowel onto the central cones of each speaker .", "the converted speakers were mounted on either side of the finger and held there , along with the camera , by a springed clip , leaving the palmar aspect of the fingertip bare to use for touching and grasping objects .", "we hoped , by including two such tactors , to be able to use the relative strength of the vibration from each tactor to convey a parameter such as location of an image feature along the horizontal axis . \n", "this model did not perform well for identifying the location of visual targets , primarily due to mechanical design issues .", "it proved problematic to mount the device securely on the distal phalanx of the finger . furthermore , given the influence of flexion / extension at the distal inter - phalangeal joint on the camera orientation , it was difficult for users to judge where they were aiming the device .", "in addition , there was low sensitivity to vibration the speakers mounted on the lateral aspect of the finger , as compared to the speaker on the medial aspect .", "collectively , these issues made any asymmetry in signal strength from the two tactors of little value to the user 's perception .", "the current version of fingersight uses the same speaker - based tactor shown in fig .  3 , but with only one tactor on the ( more sensitive ) medial aspect of the finger , along with a miniature color camera ( supercircuits pc208 , 510492 ) .", "the camera extended approximately 8 mm from the tip of the finger , and the speaker was attached to the side of the splint .", "the splint weighed 18.8 g and caused minimal interference with movement of the finger as a whole , though it did restrict flexion between the phalanges , thereby reducing uncertainty in camera orientation . covering the palmer aspect of the finger made it difficult to use the finger for other purposes , but we were willing to accept this for the present experiment .", "the camera and the speaker were connected to a computer system , which performed spatiotemporal edge detection ( described below ) and controlled vibrator output .", "the haptic feedback was provided by a low - frequency ( 20 hz ) audio signal sent to the speaker , the amplitude of which was adjusted to produce detectable vibrations . \n fig . ", "4.current fingersight device apparatus with camera and stimulator attached . \n  for the purposes of experimental testing", "the area of the circle was divided across its center , one semicircle containing red and one containing green pixels ( see fig .  5 ) .", "the line across the center of the circle separating red from green pixels could be set to any desired orientation .", "the circular area was surrounded by white pixels and the images were projected on a screen , where the user could point the finger - mounted camera at it . \n", "5.examples of edge stimuli , at 12 ( a ) and 4 ( b ) o'clock , respectively . \n ", "software was developed to analyze the video image from the finger - mounted camera in real time and to control the vibratory feedback to that finger .", "the goal was to generate vibrations whenever the user 's finger was pointing at , or had just passed over , the boundary between the red and green areas , irrespective of the edge 's orientation .", "optical edge detection was performed with a novel spatiotemporal kernel , designed to detect edges both in the spatial domain ( when the camera was resting on an edge ) and the temporal domain ( when the camera had moved completely past an edge between one video frame and the next ) .", "this design accommodated fast hand movements , which might cause the edge to never actually have been within the kernel in any given frame , as well as slower continual tracking along the edge .", "the spatiotemporal kernel accomplished both of these for edges of any orientation by comparing each pixel in one time frame with the corresponding pixel directly across the center of a circular kernel in the next time frame .", "if the two pixels differed , an edge was present or had just been crossed .", "it is important to note that unlike the general usage of the term  kernel  in image processing , especially for convolution , our spatiotemporal kernel was applied not at every pixel , but only at one location , the center of the image .", "just as the fovea of the eye ( the high resolution central region ) is constantly moving for human vision to function , our kernel was physically moved by the user 's finger along with the entire image , to interrogate the visual environment .", "the optimal size of the circular kernel varied depending on the scale of the edge features to be considered .", "for the particular experiment described here , the optimal radius of the kernel was determined experimentally to be 50 pixels .", "a detailed formulation of the algorithm follows : we define k as the set of pixels within the circular kernel at the center of the image . based on the particular red - green - blue ( rgb ) video image coming from the camera in real time , a color label \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n", "\\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c(x , y)$\\end{document } was created for each pixel in k , denoting whether the pixel at location \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( x , y)$\\end{document } was predominantly red , green , or ambiguous.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ c\\left(x , y\\right)=\\cases{+1/2,&$ if~predominantly~red$\\cr-1/2,&$ if~predominantly~green$\\cr 0,&$if~ambiguous$\\cr}\\eqno{\\hbox{(1)}}$$\\end{document } for our purposes ,  predominantly  indicates that the red ( r ) or blue ( b ) color value for the pixel in question was above a specific threshold , while the other two color values in the rgb pixel were both below their corresponding thresholds .", "appropriate thresholds for red , green , and blue were determined experimentally , so that red , green , and white pixels ( which registered as ambiguous ) responded appropriately .", "we also created a binary mask value \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w(x , y)$\\end{document } for each pixel that had the value of 1 wherever c was non - zero , so that boundaries including the white area surrounding the circle could be excluded from generating edges.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ w\\left(x , y\\right)=\\cases{1&$if~c\\left(x , y\\right)\\ne 0$\\cr 0&$otherwise$\\cr}\\eqno{\\hbox{(2)}}$$\\end{document } for each pixel in k , we also considered another pixel in k directly across the origin ( center of the kernel ) at location \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( -x ,- y)$\\end{document}. our measure of boundary strength was calculated by comparing the value \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c_{t}$\\end{document } for the pixel at \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( x , y)$\\end{document } in the current image at time \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t$\\end{document } to value of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c_{t-1}$\\end{document } for the pixel at \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( -x ,- y)$\\end{document } in the previous image at time \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t-1$\\end{document}. the mask values \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w_{t}$\\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ w_{t-1}$\\end{document } were used to ensure that no comparisons were made using  ambiguous  pixels .", "thus a measure of edge strength \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ d$\\end{document } was computed as \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ d\\!=\\!\\!\\!\\sum_{\\left(x,\\!y\\right)\\in k}\\!\\!{\\left\\vert c_{t}\\left(x , y\\right)\\!-\\!c_{t-1}\\left(-x,\\!-y\\right)\\right\\vert w_{t}\\left(x , y\\right)w_{t-1}\\!\\left(-x,\\!-y\\right)}\\eqno{\\hbox{(3)}}$$\\end{document } if the edge measure \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ d$\\end{document } was above some threshold ( determined experimentally ) , the software reported that an edge had been detected , and triggered the haptic feedback in the form of vibration at the fingertip .", "the algorithm has the advantage that it measures equally well the conditions of sitting on an edge ( of any orientation ) for two successive timeframes and having passed completely over the edge from one timeframe to the next .", "for example , in 3d one would simply compare pixels directly across the center of a spherical rather than a circular kernel .", "twelve sighted subjects ( 10 males and 2 female ) were recruited from the university population , with an average age of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 29.4+/-{9.8}~{\\rm years}$\\end{document}. all gave informed consent .", "subjects took part in a series of trials , in which they identified the angle of an edge stimulus according to a closed set of alternatives . each edge stimulus , as described above , was projected on a display screen and consisted of a circular area 115 cm in diameter on a white background bisected with one half colored red and the other green .", "the circle was surrounded by white , so that the perimeter of the circle would not itself be interpreted as an edge ( as described above ) .", "subjects were fitted with earplugs , blindfolded , and seated with the right arm resting on a foam support 79 cm above the floor .", "the circles were projected ( epson powerlite 70c ) on a 154115 cm screen positioned 168 cm in front of the subject 's fingertip .", "the arm support was arranged so that the subject 's arm and finger were initially pointing perpendicular to the center of the projected image . during each trial ,", "the subject freely explored the display edge with the fingersight device as long as desired .", "generally , subjects maintained their arm on the rest and moved their forearm and wrist .", "angle set 1 consisted of six angles equally spaced over a 180 range in 30 steps ( from positive vertical , 90 , to just short of negative vertical , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { -}{60}^{\\circ}$\\end{document } ) , corresponding to clock - hours from 12 to 5 o'clock .", "angle set 2 consisted of seven angles equally spaced over a 90 range in 15 steps ( from positive vertical , 90 , to horizontal , 0 ) corresponding to the hours and half hours between 12 to 3 o'clock .", "subjects reported their responses using these  clock face  labels . within each set ,"], ["tables  1 and 2 show the confusion matrices for all subjects for angle sets 1 and 2 , respectively , plotting reported angle vs. stimulus angle .", "a confusion matrix shows correct measurements along the diagonal , with increasing error as one moves off the diagonal .", "overall , subjects had an accuracy rate ( proportion correct ) of \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 0.77\\pm 0.05$\\end{document } on angle set 1 and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 0.52\\pm 0.09$\\end{document } on angle set 2 ( 95% confidence intervals ) .", "these levels are well above chance in this six - alternative choice task \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm chance}=0.16)$\\end{document}. \n table iconfusion matrix for set 1reported angle ( deg)90603003060stimulus angle ( deg)9071131011060168252003008843100101081313000068196040003260confusion matrix for angle set 1 .", "each entry is the number of occurrences of the associated stimulus / response pair . \n", "table iiconfusion matrix for set 2reported angle ( deg)0153045607590stimulus angle ( deg)0642532200151949242101301205519100450836391210600115234692750011533389900011112558confusion matrix for angle set 2 .", "each entry is the number of occurrences of the associated stimulus / response pair . \n ", "the data were also analyzed by measuring information transfer \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } , in order to determine the number of  bits  that can be transmitted by the device under the experimental conditions .\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i=\\sum_{j=1}^{k}\\sum_{i=1}^{k}{p\\left(s_{i},r_{j}\\right)\\log_{2}\\left({{p\\left(s_{i}\\vert r_{j}\\right)}\\over{p\\left(s_{i}\\right)}}\\right)}.\\eqno{\\hbox{(4)}}$$\\end{document } here , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ k$\\end{document } is the number of possible stimuli , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ s_{i}$\\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ r_{j}$\\end{document } represent a specific stimulus - response pair .", "this statistic can be estimated from the confusion matrices according to equation 6.\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i_{est}=\\sum_{j=1}^{k}\\sum_{i=1}^{k}\\left({{n_{ij}}\\over{n}}\\right)\\log_{2}\\left({{n_{ij}\\times n}\\over{n_{i}\\times n_{j}}}\\right)\\!.\\eqno{\\hbox{(5)}}$$\\end{document } here , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n_{ij}$\\end{document } is the number of joint occurrences of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } and response \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ j , n_{i}$\\end{document } is the overall occurrence of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ i$\\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n_{j}$\\end{document } is the overall occurrence of stimulus \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ j$\\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n$\\end{document } is the total number of trials .", "relative to the 2.58 bits of information in the six - alternative choice task , angle set 1 ( angles with 30 increments ) had an information transfer of 1.62 bits , while angle set 2 ( angles with 15 increments ) had an information transfer of 1.12 bits ."], ["we have developed a new method of providing the finger with haptic feedback about visual targets in the general environment , by permitting the finger to scan the environment directly to locate those targets .", "we have developed this idea , which we call fingersight , through a series of prototypes , exploring the particular application of detecting edges . in the process , we have developed a new spatiotemporal \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ n$\\end{document}-dimensional edge detection algorithm of potentially general interest , which is simultaneously capable of detecting the presence of a stationary edge as well as having passed over an edge between successive timeframes .", "we have conducted proof - of - concept experiments showing that under active exploration the fingersight device is capable of transmitting visual spatial information through haptic channels . sighted , blindfolded subjects were able to use the device to differentiate between angles separated by as little as 15. performance with the coarser angle set , where responses were separated by 30 showed that fingersight transmitted close to 2/3 of the available information in the stimulus , with most errors being near misses .", "the information transmission of the fingersight device is intrinsically constrained by basic sensorimotor abilities of motor control , kinesthetic sensing , and spatial cognitive processing .", "was found that when blindfolded , sighted adults tried to report the orientation of a raised line that was easily tracked by touch on the plane of a tabletop , their responses pulled the true value towards the sagittal axis by about 25% ( plus some constant error ) .", "since motor control was minimized in the task in , errors can be entirely attributed to the processes of kinesthetic sensing and building a spatial representation .", "the observed level of distortion is by itself substantial enough to lead to confusion errors in the present task .", "these observations suggest that performance with fingersight could be improved by features that support motor control and augment kinesthetic feedback .", "the present experiments measure only the performance of blindfolded sighted subjects who are novices using the device ; training would be expected to considerably improve performance .", "siegle and warren have shown that distal attribution , i.e. , the direct perception of objects in external space , can occur with similar finger - mounted sensory substitution systems after a period of training .", "however the device used in their experiments had the vibrotactile stimulator mounted on the subject 's back .", "it is possible that training time may be decreased with a device such as fingersight , where the stimulus is more strongly tied to the subject 's proprioceptive knowledge of the hand 's location in space .", "it is interesting to compare the accuracy of angular measurements by fingersight in 3d space with other researchers who restricted interrogation to a fixed plane .", ", explored the ability to recognize 90- or 180-degree rotation on raised - outline drawings directly touched by the blind or sighted - blindfolded subjects , but did not explore finer angular resolution .", "postma , et al . , demonstrated the role of visual experience in such haptic spatial tasks , including the description of angles between bars on a table , showing that blindfolded sighted subjects outperformed late blind subjects , who outperformed early blind subjects .", "they found that having experienced vision , even to a limited extent , helps in the interpretation of angle by touch alone .", "the worst performance in verbally judging the angle of the bars ( demonstrated by the early blind ) was 7.2 degrees , a good deal better than our errors , possibly because they were constrained to a tabletop .", "it would also be interesting to see if sighted individuals also have a similar advantage in learning to use fingersight over blind individuals , due to previous visual experience . also relevant to the question of angle and touch", ", with haptic computer mice , which are modified to have tactile pin arrays on their upper surface .", "significant lack of accuracy in the haptic position information , which is critical for individuals to haptically piece together a 2-d graphic . ", "the inaccuracy is due to the fact that the tactile mouse ( or any normal mouse ) is a relative positioning device , dependent upon the speed of motion and orientation of the mouse to determine total displacement on the screen .", "in contrast , fingersight is inherently an absolute positioning device , given a stationary environment , and as such , fingersight may have an advantage .", "the system of burch and pawluk previously mentioned   uses a fingertip photosensor and piezoelectric stimulator to scan specially created graphical displays , in which texture is added to enhance perception of edges and orientations .", "a single photosensor thus suffices for this purpose , only because preprocessing is performed to populate regions on either side of boundaries with differing textures .", "multiple photosensors on different fingers were found to improve results with this system , because the operator 's knowledge of the spatial relationship between the fingertips could be used to integrate the inputs .", "but still the approach relies on preprocessing the image to create textures , after which individual photosensors can be effective . with fingersight , we are exploring the unadulterated 3d environment , where depending on only a few photosensors is not sufficient .", "we benefit greatly by having a multi - pixel image at each timeframe . by further processing of the camera images ,", "the fingersight device could be adapted to identify more complicated features than simple visual edges , perhaps even constituting an object recognition system for a blind operator .", "for example , it could be used to find a particular person in a crowd , or identify a door displaying a particular sign . the rapid advancement in computer vision algorithms , driven in part by the security and social networking industries , will provide ever more sophisticated capabilities .", "for example , determining camera motion and target depth from a image sequence could permit greater 3d integration of multiple perspectives and facilitate providing navigational cues to the blind operator , such as where the curbside is , whether the approaching stairwell goes up or down , or whether one is moving towards or away from the elevator .", "such analyses are not feasible for a single photosensor , but rather , they require an entire image .", "a common problem in any real - world computer vision application is the variability of lighting , and there are established techniques for solving this problem using such constructs as the  illumination cone  .", "for example , a recently developed time - of - flight ( tof ) 3d camera , the swiss ranger sr4000 ( mesa imaging , zuerich ) , can deliver a 176144 pixel image with each pixel reporting range up to 10 m with 1 cm accuracy .", "the present camera is roughly 7 \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm cm}^{3}$\\end{document } and is being integrated by at least one research group into portable devices for the blind .", "for some of these more sophisticated systems , it may become problematic to rely solely on vibrotactile feedback to the finger .", "the bandwidth of audio , especially combined with language , makes it an appealing option , though as noted above , one does not want to impede the natural use of auditory cues that are especially crucial for the vision impaired . however , intermittent use of verbal output by fingersight , and for that matter , verbal commands by the operator to the device , could prove extremely useful , while not impeding auditory cues form the environment . as noted above , it is imperative to test whether features added to the technology ultimately add to the functional capability of the user , given intrinsic limitations on human information processing .", "that said , one promising avenue is the incorporation of control capabilities into the fingersight device .", "one of our previous systems included the capability to control graphical objects on a screen . in this implementation", ", the location of a small white square on a screen is controlled by motion of the finger .", "the algorithm detects the square in the field of view of the camera , moving it on the screen to keep it constantly in the center of the camera image , while providing haptic feedback about whether the tracking system has locked onto the target .", "a variation on this system constrains the square to move along a straight line , simulating the action of a slide pot under the operator 's control .", "a further variation uses a small white triangle to simulate a knob , whose orientation is determined using standard computer vision techniques and subsequently controlled by rotation of the finger .", "clearly , such systems are not limited to actively controlling graphical objects on a screen , but could also identify inanimate objects such as a light switch or door latch .", "in such cases , remote control could still be achieved by motion of the finger once the target has been identified , using a separate control channel to turn on the light or lock the door . continued work on the control aspects of fingersight", "a final note is merited on the eventual miniaturization of a fingersight device such that it might be small enough to be worn like an artificial fingernail .", "the cameras themselves are almost small enough already , and the main considerations are power and communications .", "one can envision radio communication between a fingertip device and a pocket unit , much as wireless earphones and microphones communicate with cell phones now .", "such devices might be fully integrated into the everyday activities of the vision - impaired ."]]}
{"article_id": "PMC4006502", "article_text": ["it negatively influences the child 's quality of life and is an important cause of absence from school and also a significant factor in health - related costs among children and adolescents .", "its prevalence varies greatly from 5.9 to 82% , depending on the definition criteria and the age of the patients .", "the international headache society classifies headaches into primary and secondary type in classification of headache disorders 2 edition ( ichd - ii ) .", "primary headache is not attributed to any other disorder . in the majority of patients with primary headache , general and neurological examinations are normal and", "primary headaches include migraine , tension - type headache , cluster headache , autonomic cephalalgias and other primary headache disorders .", "frequency of headache increases with age in this population . in the present study we aimed to determine epidemiologic characteristics and compare socioeconomic and some clinical factors related to primary headaches in children / adolescents ( 4 - 15 year olds ) presented to neurology clinics of tabriz university of medical sciences .", "the institutional review board at tabriz university of medical sciences approved the study protocol . after obtaining a written informed consent from parents , children with 4 - 15 years of age and with the diagnosis of primary headache ( migraine or tension - type headaches ) who presented to the neurology clinics affiliated to tabriz university of medical sciences , tabriz , iran from march 2009 to october 2011 were included in this cross - sectional study .", "a total of 190 patients fulfilled the criteria for enrollment in the study and their demographics were collected .", "we noted the type of headache , history of atopy , asphyxia during labor ( according to the history of admission in the neonatal intensive care unit after birth due to respiratory problems based on the parents report and review of the previous medical records ) and breast feeding , family history of headache , presence of headache triggers and the socioeconomic status of the family .", "diagnosis of migraine and tension - type headache was based on the international headache society diagnostic criteria for the primary headache disorders .", "socioeconomic status was determined according to the total monthly family income ( < 7,000,000 rl vs = 7,000,000 rl ) based on the index of statistical center of iran for grand cities in the years of study in addition to the highest level of education of the father ( < or = high school vs. > or = college ) .", "statistical analysis was conducted with spss , version 16.0 for window ( spss , chicago , il ) .", "data were expressed as meansd for quantitative variables and as numbers and percentages for categorical variables .", "statistical analysis was performed using the chi - square test for categorical variables and the student 's t test for numerical variables .", "eighty eight patients ( 46.3% ) had migraine and 102 patients ( 53.7% ) had tension - type headache .", "the distributions of gender and age groups in patients with migraine or tension - type headache are shown in table 1 . \n ", "the distributions of gender and age groups in patients with migraine or tension - type headache data are presented as numbers no significant differences were seen in the distribution of gender between the patients with migraine and tension - type headache .", "migraine and tension - type headache had comparable frequency in different age groups of male patients ( p=0.1 and 0.07 , respectively ) .", "in female patients the frequencies of both migraine and tension - type headache were significantly higher in the age group of 10 - 15 years as compared to the age group of 4 - 9 years ( p=0.03 and 0.04 , respectively ) .", "there was no significant differences regarding the prevalence of migraine or tension type headache between different genders in the same age group .", "the most common clinical presentations of aura in patients with migraine are demonstrated in table 2 .", "the comprehensive data regarding the epidemiologic characteristics of patients with migraine or tension - type headache in our study group are shown in table 3 . \n ", "the clinical presentations of aura in patients with migraine the characteristics compared between patients with migraine and tension - type headache as triggering factors for headache ; or : odds ratio ; ci : confidence interval history of asphyxia during labor was more prevalent among patients with migraine as compared to the tension - type headache ( p=0.007 , or = 3.24 ) . history of headache in parents was also significantly higher in patients with migraine as compared to tension - type headache ( p=0.001 , or = 3.19 ) .", "moreover , the prevalence of atopy was significantly higher in patients with migraine ( p=0.01 , or = 5.70 ) .", "prevalence of feeding with formula during infancy did not differ between the patients with either migraine or tension - type headache ( p=0.9 ) .", "the low socioeconomic status was more prevalent in patients with migraine headache ( p=0.003 , or = 2.38 ) .", "in our study primary headache was more common in older age group which is consistent with danold 's findings which showed that frequency of headache increases with age .", "tension type headache in patients was more frequent than migraine which is comparable with findings of kroner - herwig et al .", "the main finding in the present study was that gender can not predict the type of primary headache in younger children , as no gender differences were seen between the patients with migraine or tension - type headache in the age group 4 - 9 years .", "this finding is in contrast to previous report by krner - herwig et al which showed that being female was a significant predictor for three main subtypes of primary headaches", "a few other studies showed that the prevalence of migraine was higher in the males .", "our results also demonstrated that the frequencies of migraine and tension - type headache were comparable in different age groups of male patients , whereas female patients in the age group 10 - 15 years had higher frequency of both types of headache especially migraine as compared to the age group 4 - 9 years .", "this finding reveals that female adolescents had higher risk for developing primary headache specially migraine as compared to female children .", "these results agree with those reported by krner - herwig et al who showed that children with migraine were significantly older than children in the other groups .", "it could be due to hormonal effect as the trigger of migraine headache in the female adolescents .", "it was previously shown that primary headache in children is closely related to history of headache in their parents .", "our results demonstrated that when a parent showed combined headache , the risk of developing migraine in his or her child is more conspicuous as compared to the tension - type headache .", "this finding is consistent with previous reports by fallahzadeh et al and krner - herwig et al which showed a stronger association between family history of headache and being afflicted by migraine as compared to tension - type headache in children .", "our study also demonstrated that if the patients with history of peripartum asphyxia would develop primary headache it would more probably be of migraine type .", "these results partly agree with those reported by maneyapanda et al who found an association between chronic migraine headache and significant perinatal insult due to neuroplasticity and its induced hyperexcitability .", "however , it is recommended to design prospective studies to compare the frequency of primary headache ( either migraine or tension - type headache ) in patients with or without perinatal insults to elucidate the role of peripartum asphyxia as a risk factor for developing primary headaches .", "we also showed that atopy is more prevalent in patients with migraine as compared to tension - type headache .", "also ozge et al found an association between atopy and both migraine and episodic tension - type headaches .", "it has been shown that alteration in several interleukins occurs in migraine which supports an association between migraine and atopic disorders .", "although it was shown by pogliani et al that breast feeding has a protective role against primary headache disorders , our results showed that children and adolescents with history of formula feeding during infancy have comparable prevalence of developing either migraine or tension - type headache .", "our results showed that socioeconomic status of the patients could significantly influence the frequency of migraine in comparison to tension - type headache .", "ayzenberg et al in a study on 2725 adults in russia , showed that low socioeconomic status is associated with higher frequency of primary headache disorders .", "moreover , katsarava et al in another study on 1145 adult patients in the republic of georgia showed that low socioeconomic status and female gender are risk factors for development of migraine , not tension - type headache .", "moreover , the possible role of psychological factors and behavioral characteristics influencing primary headaches could have been considered .", "while some variables have been checked by the present report , further prospective studies with larger sample sizes are needed to provide more comprehensive data regarding associated factors in childhood primary headache ."], "abstract_text": ["<S> objectiveheadache is one of the most common neurologic problems in children and adolescents . </S>", "<S> primary headache including migraine and tension - type headache comprises the vast majority of headaches and are associated with marked incidence , prevalence , and individual and social cost . </S>", "<S> we aimed to assess demographic characteristics and to compare some factors related to primary headaches in children/ adolescents presented to neurology clinics of tabriz university of medical sciences.methodschildren from 4 to 15 years of age with the diagnosis of primary headache ( migraine or tension - type headaches ) who presented to the neurology clinics affiliated to tabriz university of medical sciences , tabriz , iran from march 2009 to october 2011 are included in this cross - sectional study . </S>", "<S> data regarding the type of headache , history of atopy , peripartum asphyxia , and breast feeding , family history of headache and the socioeconomic status of the family were collected . </S>", "<S> the diagnosis was based on the international headache society diagnostic criteria for the primary headache disorders.findingsone hundred ninety children ( 107 females ) with primary headache ( 88 patients with migraine and 102 patients with tension type headache ) enrolled in the study . </S>", "<S> peripartum asphyxia , history of atopy , family history of headache and low socioeconomic status ( ses ) were more common in patients with migraine ( p - values : 0.007 , 0.01 , 0.001 , 0.003 ; respectively).conclusionphysicians need to extent their knowledge regarding the primary headaches . </S>", "<S> peripartum asphyxia , history of atopy , headache in parents and low ses have been shown in the present study to be more prevalent in patients with migraine as compared to tension - type headache . </S>"], "labels": null, "section_names": ["Introduction", "Subjects and Methods", "Findings", "Discussion", "Conclusion", "Conflict of Interest"], "sections": [["it negatively influences the child 's quality of life and is an important cause of absence from school and also a significant factor in health - related costs among children and adolescents .", "its prevalence varies greatly from 5.9 to 82% , depending on the definition criteria and the age of the patients .", "the international headache society classifies headaches into primary and secondary type in classification of headache disorders 2 edition ( ichd - ii ) .", "primary headache is not attributed to any other disorder . in the majority of patients with primary headache , general and neurological examinations are normal and", "primary headaches include migraine , tension - type headache , cluster headache , autonomic cephalalgias and other primary headache disorders .", "frequency of headache increases with age in this population . in the present study we aimed to determine epidemiologic characteristics and compare socioeconomic and some clinical factors related to primary headaches in children / adolescents ( 4 - 15 year olds ) presented to neurology clinics of tabriz university of medical sciences ."], ["the institutional review board at tabriz university of medical sciences approved the study protocol . after obtaining a written informed consent from parents , children with 4 - 15 years of age and with the diagnosis of primary headache ( migraine or tension - type headaches ) who presented to the neurology clinics affiliated to tabriz university of medical sciences , tabriz , iran from march 2009 to october 2011 were included in this cross - sectional study .", "a total of 190 patients fulfilled the criteria for enrollment in the study and their demographics were collected .", "we noted the type of headache , history of atopy , asphyxia during labor ( according to the history of admission in the neonatal intensive care unit after birth due to respiratory problems based on the parents report and review of the previous medical records ) and breast feeding , family history of headache , presence of headache triggers and the socioeconomic status of the family .", "diagnosis of migraine and tension - type headache was based on the international headache society diagnostic criteria for the primary headache disorders .", "socioeconomic status was determined according to the total monthly family income ( < 7,000,000 rl vs = 7,000,000 rl ) based on the index of statistical center of iran for grand cities in the years of study in addition to the highest level of education of the father ( < or = high school vs. > or = college ) .", "statistical analysis was conducted with spss , version 16.0 for window ( spss , chicago , il ) .", "data were expressed as meansd for quantitative variables and as numbers and percentages for categorical variables .", "statistical analysis was performed using the chi - square test for categorical variables and the student 's t test for numerical variables ."], ["eighty eight patients ( 46.3% ) had migraine and 102 patients ( 53.7% ) had tension - type headache .", "the distributions of gender and age groups in patients with migraine or tension - type headache are shown in table 1 . \n ", "the distributions of gender and age groups in patients with migraine or tension - type headache data are presented as numbers no significant differences were seen in the distribution of gender between the patients with migraine and tension - type headache .", "migraine and tension - type headache had comparable frequency in different age groups of male patients ( p=0.1 and 0.07 , respectively ) .", "in female patients the frequencies of both migraine and tension - type headache were significantly higher in the age group of 10 - 15 years as compared to the age group of 4 - 9 years ( p=0.03 and 0.04 , respectively ) .", "there was no significant differences regarding the prevalence of migraine or tension type headache between different genders in the same age group .", "the most common clinical presentations of aura in patients with migraine are demonstrated in table 2 .", "the comprehensive data regarding the epidemiologic characteristics of patients with migraine or tension - type headache in our study group are shown in table 3 . \n ", "the clinical presentations of aura in patients with migraine the characteristics compared between patients with migraine and tension - type headache as triggering factors for headache ; or : odds ratio ; ci : confidence interval history of asphyxia during labor was more prevalent among patients with migraine as compared to the tension - type headache ( p=0.007 , or = 3.24 ) . history of headache in parents was also significantly higher in patients with migraine as compared to tension - type headache ( p=0.001 , or = 3.19 ) .", "moreover , the prevalence of atopy was significantly higher in patients with migraine ( p=0.01 , or = 5.70 ) .", "prevalence of feeding with formula during infancy did not differ between the patients with either migraine or tension - type headache ( p=0.9 ) .", "the low socioeconomic status was more prevalent in patients with migraine headache ( p=0.003 , or = 2.38 ) ."], ["in our study primary headache was more common in older age group which is consistent with danold 's findings which showed that frequency of headache increases with age .", "tension type headache in patients was more frequent than migraine which is comparable with findings of kroner - herwig et al .", "the main finding in the present study was that gender can not predict the type of primary headache in younger children , as no gender differences were seen between the patients with migraine or tension - type headache in the age group 4 - 9 years .", "this finding is in contrast to previous report by krner - herwig et al which showed that being female was a significant predictor for three main subtypes of primary headaches", "a few other studies showed that the prevalence of migraine was higher in the males .", "our results also demonstrated that the frequencies of migraine and tension - type headache were comparable in different age groups of male patients , whereas female patients in the age group 10 - 15 years had higher frequency of both types of headache especially migraine as compared to the age group 4 - 9 years .", "this finding reveals that female adolescents had higher risk for developing primary headache specially migraine as compared to female children .", "these results agree with those reported by krner - herwig et al who showed that children with migraine were significantly older than children in the other groups .", "it could be due to hormonal effect as the trigger of migraine headache in the female adolescents .", "it was previously shown that primary headache in children is closely related to history of headache in their parents .", "our results demonstrated that when a parent showed combined headache , the risk of developing migraine in his or her child is more conspicuous as compared to the tension - type headache .", "this finding is consistent with previous reports by fallahzadeh et al and krner - herwig et al which showed a stronger association between family history of headache and being afflicted by migraine as compared to tension - type headache in children .", "our study also demonstrated that if the patients with history of peripartum asphyxia would develop primary headache it would more probably be of migraine type .", "these results partly agree with those reported by maneyapanda et al who found an association between chronic migraine headache and significant perinatal insult due to neuroplasticity and its induced hyperexcitability .", "however , it is recommended to design prospective studies to compare the frequency of primary headache ( either migraine or tension - type headache ) in patients with or without perinatal insults to elucidate the role of peripartum asphyxia as a risk factor for developing primary headaches .", "we also showed that atopy is more prevalent in patients with migraine as compared to tension - type headache .", "also ozge et al found an association between atopy and both migraine and episodic tension - type headaches .", "it has been shown that alteration in several interleukins occurs in migraine which supports an association between migraine and atopic disorders .", "although it was shown by pogliani et al that breast feeding has a protective role against primary headache disorders , our results showed that children and adolescents with history of formula feeding during infancy have comparable prevalence of developing either migraine or tension - type headache .", "our results showed that socioeconomic status of the patients could significantly influence the frequency of migraine in comparison to tension - type headache .", "ayzenberg et al in a study on 2725 adults in russia , showed that low socioeconomic status is associated with higher frequency of primary headache disorders .", "moreover , katsarava et al in another study on 1145 adult patients in the republic of georgia showed that low socioeconomic status and female gender are risk factors for development of migraine , not tension - type headache .", "moreover , the possible role of psychological factors and behavioral characteristics influencing primary headaches could have been considered ."], ["while some variables have been checked by the present report , further prospective studies with larger sample sizes are needed to provide more comprehensive data regarding associated factors in childhood primary headache ."], [""]]}
{"article_id": "PMC3906304", "article_text": ["a total of 30 mabs that were either developed or in - licensed by companies are currently undergoing evaluation in late - stage studies ( tables 1 and 2 ) .", "five of these ( brodalumab , mabp1 , moxetumomab pasudotox , tildrakizumab , rilotumumab ) transitioned into phase 3 studies during the period from late 2012 to april 2013 .", "brodalumab ( amg827 , amgen ; khk4827 , kyowa hakko kirin ) , a human igg2 targeting the interleukin ( il)-17 receptor , is undergoing evaluation in three phase 3 studies ( amagine-1 , -2 , and -3 ) of patients with psoriasis .", "the amagine-1 study ( nct01708590 ) is evaluating the efficacy , safety , and effect of withdrawal and retreatment with brodalumab in patients with moderate - to - severe plaque psoriasis . in this study , either of two doses ( 140 mg or 210 mg ) or placebo is administered subcutaneously ( sc ) every two weeks until week 12 , when patients are rerandomized to placebo or continued treatment .", "the estimated primary completion date for the study is march 2014 . in the amagine-2 ( nct01708603 ) and amagine-3 ( nct01708603 ) studies ,", "the efficacy and safety of induction and four maintenance regimens of brodalumab compared with placebo and ustekinumab in patients with moderate - to - severe plaque psoriasis is being evaluated .", "these studies have estimated primary completion dates of august and september 2014 , respectively . note : table compiled from information available as of april 25 , 2013 .", "abbreviations : cd , cluster of differentiation ; il , interleukin ; pcsk9 , proprotein convertase subtilisin / kexin type ; sle , systemic lupus erythematosus . note : table compiled from information available as of april 25 , 2013 . * in - licensed", "; national cancer institute is sponsoring the phase 3 study in hairy cell leukemia patients .", "abbreviations : all , acute lymphoblastic leukemia ; cll , chronic lymphocytic leukemia ; hgf / sf , hepatocyte growth factor/ scatter factor ; nhl , non - hodgkin lymphoma ; nscl , non - small cell lung ; vegfr2 , vascular endothelial growth factor receptor 2 . a first phase 3 study ( nct01767857 ) of mabp1 ( xilonix , xbiotech , inc . ) , a human mab targeting il-1 , was recruiting patients as of march 2013", ". the study will evaluate overall survival of metastatic colorectal cancer patients with cachexia who are administered the mab as a monotherapy .", "the effects of mabp1 administered intravenously ( iv ) every two weeks , plus best supportive care will be compared with those of megestrol acetate administered daily plus best supportive care .", "the difference between the study arms in median overall survival from baseline to 18 mo will be compared .", "the recombinant immunotoxin moxetumomab pasudotox ( cat-8015 , astrazeneca ) is undergoing evaluation in a phase 3 study ( nct01829711 ) of patients with relapsed / refractory hairy cell leukemia .", "the study is sponsored by the national cancer institute ( nci ) ; the mab is currently licensed to astrazeneca . in 2004 , nci licensed moxetumomab pasudotox to genencor , inc .", "the mab was then acquired in 2005 by cambridge antibody technology , which was itself acquired by astrazeneca in 2006 .", "moxetumomab pasudotox comprises a mouse disulfide stabilized variable fragment with the variable heavy domain fused with a 7-mer linker and the pseudomonas aeruginosa exotoxin a pe38 fragment . in the phase 3 study", ", patients will receive 40 g / kg of the immunotoxin administered iv over 30 min on days 1 , 3 , 5 of each 28 d cycle until complete response , progressive disease , initiation of alternate therapy or unacceptable toxicity is observed .", "initiated in march 2013 , the study s estimated primary completion date is december 2014 .", "two phase 3 studies of tildrakizumab ( sch900222/mk-3222 , merck ) , a humanized mab targeting the p19 subunit of il-23 , are recruiting patients .", "initiated in december 2012 , nct01722331 is a 64-week study to evaluate the efficacy and safety / tolerability of sc administration of tildrakizumab in patients with moderate - to - severe chronic plaque psoriasis .", "patients receive 100 mg or 200 mg tildrakizumab at week 0 and week 4 , and then every 12 weeks until study end or participant discontinuation .", "patients receive the same doses of tildrakizumab , but the study includes 50 mg etanercept as well as placebo as comparators .", "the two phase 3 studies have estimated primary completion dates of june and july 2015 , respectively .", "rilotumumab ( amgen ) is a human igg2 targeting human hepatocyte growth factor / scatter factor ( hgf / sf ) that blocks binding of hgf / sf to its receptor met , thereby inhibiting the met signaling pathways .", "the mab is undergoing evaluation in a phase 3 study ( nct01697072 ) of chemotherapy ( epirubicin , cisplatin and capecitabine ) with rilotumumab or placebo for untreated advanced met - positive gastric or gastresophageal junction adenocarcinoma .", "rilotumumab or placebo is administered iv at doses of 15 mg / kg every 21 d. the primary outcome measure is overall survival in a time - frame of three years .", "initiated in october 2012 , the estimated primary completion date for the study is december 2015 .", "recently announced results for three late - stage studies of two mabs for cancer ( farletuzumab , naptumomab estafenatox ) and tabalumab , a mab treatment for autoimmune disorders , indicate that clinical endpoints were not met .", "farletuzumab ( morab-003 , morphotek / eisai ) is a humanized igg1 mab that targets human folate receptor  , which is overexpressed in most epithelial ovarian cancers , as well as some forms of endometrial , breast , renal , lung and colorectal cancers .", "the primary endpoint of progression - free survival ( pfs ) was not met in the phase 3 far-131 study ( nct00849667 ) of farletuzumab in combination with carboplatin and a taxane in patients with platinum - sensitive epithelial ovarian cancer in first relapse .", "patients received placebo or farletuzumab at either 1.25 mg / kg or 2.5 mg / kg weekly for ~six cycles .", "although the primary endpoint was not met , a trend toward improved pfs was observed in some patient subsets in a post - hoc analysis .", "the safety and efficacy of farletuzumab in combination with a platinum containing doublet in chemotherapy - nave subjects with stage iv adenocarcinoma of the lung are being evaluated in a phase 2 study ( nct01218516 ) . as announced in january 2013 by active biotech ab , initial results of a phase 2/3 study ( nct00420888 ) of naptumomab estafenatox ( abr-217620 , anyara ) in combination with interferon  as a treatment for advanced renal cell carcinoma indicated that the study did not achieve its primary clinical endpoint of overall survival in the intention - to - treat cohort .", "a confounding factor was the presence of high levels of pre - formed anti - drug antibodies found in a majority of patients in this , but not previous , studies .", "a doubling of pfs and overall survival occurred in the 25% of patients who had low or normal levels of base - line il-6 and expected levels of anti - drug antibodies .", "naptumomab estafenatox is composed of an antigen - binding fragment ( fab ) that targets metastasis - associated 5t4 fused to a mutated form of staphylococcal enterotoxin a ( sea / e-120 ) .", "phase 3 studies of tabalumab ( ly2127399 , eli lilly and co. ) as a treatment for ra have been discontinued due to lack of efficacy .", "tabalumab , a human igg4 targeting b - lymphocyte stimulator , was being evaluated in five phase 3 studies of ra patients .", "tabalumab in combination with bortezomib and dexamethasone is also being evaluated in a phase 2/3 study ( nct01602224 ) of patients with previously treated multiple myeloma .", "historically , ~50% of mabs in the commercial clinical pipeline have been studied as cancer agents ; however , ~67% of the current cohort of mabs at phase 3 is in studies for non - cancer indications ( table 1 ) .", "these 20 mabs are human or humanized , with more than half ( 55% ) targeting an interleukin or an interleukin receptor .", "the mabs are being developed as treatments for immunological disorders such as ra , sle , ms and psoriasis , but also for hypercholesterolemia , cachexia and alzheimer disease . reflecting the overall greater challenge of demonstrating safety and efficacy in cancers ,", "the molecular types of the 10 mabs in late - stage studies of cancer ( table 2 ) are more diverse than those for non - cancer indications .", "of particular note is the inclusion of a toxin in three molecules ( naptumomab estafenatox , moxetumomab pasudotox , inotuzumab ozogamicin ) and use of less than a full - length sequence for three molecules ( naptumomab estafenatox , moxetumomab pasudotox , onartuzumab ) . the majority ( 70% ) of the 10 product candidates", "are in studies of solid tumors , with the remainder undergoing evaluation as treatments for hematological cancers .", "the number of anti - cancer mabs at phase 3 is likely to increase in the next 35 y as more adcs move into and through the clinical pipeline . \n", "mabs will continue to track the progress of antibody therapeutics in clinical study throughout 2013 , and we look forward to reporting on results that may be released during the year in  which are the antibodies to watch in 2014 ?"], "abstract_text": ["<S> the transitions of antibody therapeutics to late - stage clinical development , regulatory review and the market are proceeding at a rapid pace in 2013 . since late 2012 , two monoclonal antibody ( mab ) therapeutics ( itolizumab , trastuzumab emtansine ) received their first approvals , first marketing applications for three mabs ( vedolizumab , ramucirumab , obinutuzumab ) were submitted to regulatory agencies , and five mabs ( brodalumab , mabp1 , moxetumomab pasudotox , tildrakizumab , rilotumumab ) entered their first phase 3 studies . the current total of commercially - sponsored antibody therapeutics undergoing evaluation in late - stage studies is 30 . </S>", "<S> recently announced study results for farletuzumab , naptumomab estafenatox , and tabalumab indicate that clinical endpoints were not met in some phase 3 studies of these product candidates . </S>"], "labels": null, "section_names": ["First Phase 3 studies for five mAbs", "Clinical endpoints missed in studies of three mAbs", "Late-stage pipeline update"], "sections": [["a total of 30 mabs that were either developed or in - licensed by companies are currently undergoing evaluation in late - stage studies ( tables 1 and 2 ) .", "five of these ( brodalumab , mabp1 , moxetumomab pasudotox , tildrakizumab , rilotumumab ) transitioned into phase 3 studies during the period from late 2012 to april 2013 .", "brodalumab ( amg827 , amgen ; khk4827 , kyowa hakko kirin ) , a human igg2 targeting the interleukin ( il)-17 receptor , is undergoing evaluation in three phase 3 studies ( amagine-1 , -2 , and -3 ) of patients with psoriasis .", "the amagine-1 study ( nct01708590 ) is evaluating the efficacy , safety , and effect of withdrawal and retreatment with brodalumab in patients with moderate - to - severe plaque psoriasis . in this study , either of two doses ( 140 mg or 210 mg ) or placebo is administered subcutaneously ( sc ) every two weeks until week 12 , when patients are rerandomized to placebo or continued treatment .", "the estimated primary completion date for the study is march 2014 . in the amagine-2 ( nct01708603 ) and amagine-3 ( nct01708603 ) studies ,", "the efficacy and safety of induction and four maintenance regimens of brodalumab compared with placebo and ustekinumab in patients with moderate - to - severe plaque psoriasis is being evaluated .", "these studies have estimated primary completion dates of august and september 2014 , respectively . note : table compiled from information available as of april 25 , 2013 .", "abbreviations : cd , cluster of differentiation ; il , interleukin ; pcsk9 , proprotein convertase subtilisin / kexin type ; sle , systemic lupus erythematosus . note : table compiled from information available as of april 25 , 2013 . * in - licensed", "; national cancer institute is sponsoring the phase 3 study in hairy cell leukemia patients .", "abbreviations : all , acute lymphoblastic leukemia ; cll , chronic lymphocytic leukemia ; hgf / sf , hepatocyte growth factor/ scatter factor ; nhl , non - hodgkin lymphoma ; nscl , non - small cell lung ; vegfr2 , vascular endothelial growth factor receptor 2 . a first phase 3 study ( nct01767857 ) of mabp1 ( xilonix , xbiotech , inc . ) , a human mab targeting il-1 , was recruiting patients as of march 2013", ". the study will evaluate overall survival of metastatic colorectal cancer patients with cachexia who are administered the mab as a monotherapy .", "the effects of mabp1 administered intravenously ( iv ) every two weeks , plus best supportive care will be compared with those of megestrol acetate administered daily plus best supportive care .", "the difference between the study arms in median overall survival from baseline to 18 mo will be compared .", "the recombinant immunotoxin moxetumomab pasudotox ( cat-8015 , astrazeneca ) is undergoing evaluation in a phase 3 study ( nct01829711 ) of patients with relapsed / refractory hairy cell leukemia .", "the study is sponsored by the national cancer institute ( nci ) ; the mab is currently licensed to astrazeneca . in 2004 , nci licensed moxetumomab pasudotox to genencor , inc .", "the mab was then acquired in 2005 by cambridge antibody technology , which was itself acquired by astrazeneca in 2006 .", "moxetumomab pasudotox comprises a mouse disulfide stabilized variable fragment with the variable heavy domain fused with a 7-mer linker and the pseudomonas aeruginosa exotoxin a pe38 fragment . in the phase 3 study", ", patients will receive 40 g / kg of the immunotoxin administered iv over 30 min on days 1 , 3 , 5 of each 28 d cycle until complete response , progressive disease , initiation of alternate therapy or unacceptable toxicity is observed .", "initiated in march 2013 , the study s estimated primary completion date is december 2014 .", "two phase 3 studies of tildrakizumab ( sch900222/mk-3222 , merck ) , a humanized mab targeting the p19 subunit of il-23 , are recruiting patients .", "initiated in december 2012 , nct01722331 is a 64-week study to evaluate the efficacy and safety / tolerability of sc administration of tildrakizumab in patients with moderate - to - severe chronic plaque psoriasis .", "patients receive 100 mg or 200 mg tildrakizumab at week 0 and week 4 , and then every 12 weeks until study end or participant discontinuation .", "patients receive the same doses of tildrakizumab , but the study includes 50 mg etanercept as well as placebo as comparators .", "the two phase 3 studies have estimated primary completion dates of june and july 2015 , respectively .", "rilotumumab ( amgen ) is a human igg2 targeting human hepatocyte growth factor / scatter factor ( hgf / sf ) that blocks binding of hgf / sf to its receptor met , thereby inhibiting the met signaling pathways .", "the mab is undergoing evaluation in a phase 3 study ( nct01697072 ) of chemotherapy ( epirubicin , cisplatin and capecitabine ) with rilotumumab or placebo for untreated advanced met - positive gastric or gastresophageal junction adenocarcinoma .", "rilotumumab or placebo is administered iv at doses of 15 mg / kg every 21 d. the primary outcome measure is overall survival in a time - frame of three years .", "initiated in october 2012 , the estimated primary completion date for the study is december 2015 ."], ["recently announced results for three late - stage studies of two mabs for cancer ( farletuzumab , naptumomab estafenatox ) and tabalumab , a mab treatment for autoimmune disorders , indicate that clinical endpoints were not met .", "farletuzumab ( morab-003 , morphotek / eisai ) is a humanized igg1 mab that targets human folate receptor  , which is overexpressed in most epithelial ovarian cancers , as well as some forms of endometrial , breast , renal , lung and colorectal cancers .", "the primary endpoint of progression - free survival ( pfs ) was not met in the phase 3 far-131 study ( nct00849667 ) of farletuzumab in combination with carboplatin and a taxane in patients with platinum - sensitive epithelial ovarian cancer in first relapse .", "patients received placebo or farletuzumab at either 1.25 mg / kg or 2.5 mg / kg weekly for ~six cycles .", "although the primary endpoint was not met , a trend toward improved pfs was observed in some patient subsets in a post - hoc analysis .", "the safety and efficacy of farletuzumab in combination with a platinum containing doublet in chemotherapy - nave subjects with stage iv adenocarcinoma of the lung are being evaluated in a phase 2 study ( nct01218516 ) . as announced in january 2013 by active biotech ab , initial results of a phase 2/3 study ( nct00420888 ) of naptumomab estafenatox ( abr-217620 , anyara ) in combination with interferon  as a treatment for advanced renal cell carcinoma indicated that the study did not achieve its primary clinical endpoint of overall survival in the intention - to - treat cohort .", "a confounding factor was the presence of high levels of pre - formed anti - drug antibodies found in a majority of patients in this , but not previous , studies .", "a doubling of pfs and overall survival occurred in the 25% of patients who had low or normal levels of base - line il-6 and expected levels of anti - drug antibodies .", "naptumomab estafenatox is composed of an antigen - binding fragment ( fab ) that targets metastasis - associated 5t4 fused to a mutated form of staphylococcal enterotoxin a ( sea / e-120 ) .", "phase 3 studies of tabalumab ( ly2127399 , eli lilly and co. ) as a treatment for ra have been discontinued due to lack of efficacy .", "tabalumab , a human igg4 targeting b - lymphocyte stimulator , was being evaluated in five phase 3 studies of ra patients .", "tabalumab in combination with bortezomib and dexamethasone is also being evaluated in a phase 2/3 study ( nct01602224 ) of patients with previously treated multiple myeloma ."], ["historically , ~50% of mabs in the commercial clinical pipeline have been studied as cancer agents ; however , ~67% of the current cohort of mabs at phase 3 is in studies for non - cancer indications ( table 1 ) .", "these 20 mabs are human or humanized , with more than half ( 55% ) targeting an interleukin or an interleukin receptor .", "the mabs are being developed as treatments for immunological disorders such as ra , sle , ms and psoriasis , but also for hypercholesterolemia , cachexia and alzheimer disease . reflecting the overall greater challenge of demonstrating safety and efficacy in cancers ,", "the molecular types of the 10 mabs in late - stage studies of cancer ( table 2 ) are more diverse than those for non - cancer indications .", "of particular note is the inclusion of a toxin in three molecules ( naptumomab estafenatox , moxetumomab pasudotox , inotuzumab ozogamicin ) and use of less than a full - length sequence for three molecules ( naptumomab estafenatox , moxetumomab pasudotox , onartuzumab ) . the majority ( 70% ) of the 10 product candidates", "are in studies of solid tumors , with the remainder undergoing evaluation as treatments for hematological cancers .", "the number of anti - cancer mabs at phase 3 is likely to increase in the next 35 y as more adcs move into and through the clinical pipeline . \n", "mabs will continue to track the progress of antibody therapeutics in clinical study throughout 2013 , and we look forward to reporting on results that may be released during the year in  which are the antibodies to watch in 2014 ?"]]}
{"article_id": "PMC4765276", "article_text": ["they may be seen in midline or para - axial location , from the brain to the sacral area .", "they display a particular tendency to locate in the sacrococcygeal and pre - sacral regions . reported incidence of tumors at these sites", "the oropharyngeal cavity is exceedingly rare for teratomas , 2% of all teratomas , present as large masses protruding from the oropharyngeal area .", "though oropharyngeal teratomas have a benign histopathology , they are potentially lethal as they may cause airway obstruction and respiratory compromise .", "so , principles of management in such patients comprise of the immediate postpartum establishment of a secure airway , if needed via tracheostomy and complete surgical resection of the mass .", "a 1980 g female infant born to 22-year - old mother after 38 weeks gestation presented with a growth in oral cavity associated with respiratory distress .", "the mother had undergone ultrasonography examination during pregnancy , but the report of the same was unavailable . on examination ,", "cleft palate was present and a palatal tumor of size 4 cm  3 cm was found in the cleft area [ figure 1 ] .", "it was hampering feeding . computed tomography scan  showed mass lesion in the palatal region ( attached to the palate , more on the right side ) .", "no intracranial extension [ figure 2 ] . computed tomography scan image after due preparation , the patient was operated , and the mass was excised in toto [ figure 3 ] .", "histopathologic examination  revealed a teratoma composed of mature glial tissue , choroid plexus , glands lined by respiratory type , columnar mucin secreting , and melanin pigment bearing epithelium .", "pools of myxoid matrix bearing physaliferous cells are present ( notochord  like / chondroid areas ) [ figures 4 and 5 ] .", "microscopic picture 1 microscopic picture 2 following excision , patient recovered faster and gained weight .", "now , the patient is under routine follow - up , and palatoplasty has been done .", "teratomas are the tumors which contain tissues derived from all three germ layers . these contain tissues foreign to the anatomical site of origin .", "most of the teratomas in the pediatric age group are benign , but reports of malignant teratoma do exist .", "basic histological classifications which are widely used ( arnold 's system ) are : \n dermoid tumors : these are composed of ectoderm plus mesoderm .", "this is the most common form of teratomateratoids : these are poorly differentiated tumors and contain all three germ layersteratomas : these also contain tissue from all three germ layers .", "they are histologically more identifiableepignathus : these are tumors which contain fully developed organs and appendages .", "the large cervical and nasopharyngeal teratomas which obstruct fetal swallowing of amniotic fluid may contribute to polyhydramnios .", "this is the most common form of teratoma teratoids : these are poorly differentiated tumors and contain all three germ layers teratomas : these also contain tissue from all three germ layers .", "they are histologically more identifiable epignathus : these are tumors which contain fully developed organs and appendages . the large cervical and nasopharyngeal teratomas which obstruct fetal swallowing of amniotic fluid may contribute to polyhydramnios .", "other imaging technique such as magnetic resonance imaging , is useful in finding out the anatomical relationship of these tumors .", "clinical differential diagnoses of oropharyngeal teratoma are cystic hygroma , lymphangioma , duplications and neuroblastoma .", "moreover , the histopathological differential diagnosis includes hamartoma , dermoid cyst , and a heterotopic gastrointestinal cyst . with growth , oral teratomas", "that is why , the respiratory distress may be less . the neonates with oral teratomas", "the basic principle of management of oropharyngeal teratomas is the establishment and maintenance of a secure airway .", "if an oropharyngeal teratoma is diagnosed antenatally , elective delivery or lower segment cesarean section is carried out .", "if orotracheal / nasotracheal intubation is failed / or difficult , tracheostomy must be done .", "surgical goal is to remove the complete mass in toto , as a residual tumor may cause recurrence .", "associated cleft palate can be repaired when the child grows up , that is , at the age of 1 years . during follow - up , afp should be monitored . in our case , all the afp levels were within normal range .", "early diagnosis , the establishment of the good airway , complete excision of tumor and timely follow - up should increase the survival of newborns with oral teratomas ."], "abstract_text": ["<S> for congenital teratomas , oropharyngeal cavity is one of the rarest sites ( 2% of all teratomas ) . </S>", "<S> they are rarely picked up by prenatal ultrasonography . </S>", "<S> postnatally , newborns present with respiratory distress and at this point role of pediatricians is very crucial in establishing secure airway after which they need to be carefully evaluated and surgically managed . </S>", "<S> we present a female neonate with palatal teratoma which was treated successfully with surgery . </S>"], "labels": null, "section_names": ["I", "C", "D", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["they may be seen in midline or para - axial location , from the brain to the sacral area .", "they display a particular tendency to locate in the sacrococcygeal and pre - sacral regions . reported incidence of tumors at these sites", "the oropharyngeal cavity is exceedingly rare for teratomas , 2% of all teratomas , present as large masses protruding from the oropharyngeal area .", "though oropharyngeal teratomas have a benign histopathology , they are potentially lethal as they may cause airway obstruction and respiratory compromise .", "so , principles of management in such patients comprise of the immediate postpartum establishment of a secure airway , if needed via tracheostomy and complete surgical resection of the mass ."], ["a 1980 g female infant born to 22-year - old mother after 38 weeks gestation presented with a growth in oral cavity associated with respiratory distress .", "the mother had undergone ultrasonography examination during pregnancy , but the report of the same was unavailable . on examination ,", "cleft palate was present and a palatal tumor of size 4 cm  3 cm was found in the cleft area [ figure 1 ] .", "it was hampering feeding . computed tomography scan  showed mass lesion in the palatal region ( attached to the palate , more on the right side ) .", "no intracranial extension [ figure 2 ] . computed tomography scan image after due preparation , the patient was operated , and the mass was excised in toto [ figure 3 ] .", "histopathologic examination  revealed a teratoma composed of mature glial tissue , choroid plexus , glands lined by respiratory type , columnar mucin secreting , and melanin pigment bearing epithelium .", "pools of myxoid matrix bearing physaliferous cells are present ( notochord  like / chondroid areas ) [ figures 4 and 5 ] .", "microscopic picture 1 microscopic picture 2 following excision , patient recovered faster and gained weight .", "now , the patient is under routine follow - up , and palatoplasty has been done ."], ["teratomas are the tumors which contain tissues derived from all three germ layers . these contain tissues foreign to the anatomical site of origin .", "most of the teratomas in the pediatric age group are benign , but reports of malignant teratoma do exist .", "basic histological classifications which are widely used ( arnold 's system ) are : \n dermoid tumors : these are composed of ectoderm plus mesoderm .", "this is the most common form of teratomateratoids : these are poorly differentiated tumors and contain all three germ layersteratomas : these also contain tissue from all three germ layers .", "they are histologically more identifiableepignathus : these are tumors which contain fully developed organs and appendages .", "the large cervical and nasopharyngeal teratomas which obstruct fetal swallowing of amniotic fluid may contribute to polyhydramnios .", "this is the most common form of teratoma teratoids : these are poorly differentiated tumors and contain all three germ layers teratomas : these also contain tissue from all three germ layers .", "they are histologically more identifiable epignathus : these are tumors which contain fully developed organs and appendages . the large cervical and nasopharyngeal teratomas which obstruct fetal swallowing of amniotic fluid may contribute to polyhydramnios .", "other imaging technique such as magnetic resonance imaging , is useful in finding out the anatomical relationship of these tumors .", "clinical differential diagnoses of oropharyngeal teratoma are cystic hygroma , lymphangioma , duplications and neuroblastoma .", "moreover , the histopathological differential diagnosis includes hamartoma , dermoid cyst , and a heterotopic gastrointestinal cyst . with growth , oral teratomas", "that is why , the respiratory distress may be less . the neonates with oral teratomas", "the basic principle of management of oropharyngeal teratomas is the establishment and maintenance of a secure airway .", "if an oropharyngeal teratoma is diagnosed antenatally , elective delivery or lower segment cesarean section is carried out .", "if orotracheal / nasotracheal intubation is failed / or difficult , tracheostomy must be done .", "surgical goal is to remove the complete mass in toto , as a residual tumor may cause recurrence .", "associated cleft palate can be repaired when the child grows up , that is , at the age of 1 years . during follow - up , afp should be monitored . in our case , all the afp levels were within normal range .", "early diagnosis , the establishment of the good airway , complete excision of tumor and timely follow - up should increase the survival of newborns with oral teratomas ."], [""], [""]]}
{"article_id": "PMC4691986", "article_text": ["the rationale underlying treatment recommendations based on esthetic impairment come from the social science researches , which indicate that unacceptable dental appearance may stigmatize , impede career advancement and peer group acceptance , encourage negative stereotyping , and have a negative effect on self - concept .", "orthodontic problems are usually not associated with grave mortality or morbidity ; hence , they tend to be overlooked by most health professionals as less important .", "however , studies reveal that malocclusions have a significant impact on the psychosocial health of the affected individual . for many years", ", epidemiologic studies of malocclusion suffered from considerable disagreement among investigators , especially regarding how much deviation from the ideal should be accepted within the bounds of normal .", "the orthodontic treatment need in the study group was assessed by esthetic component ( ac ) of the index of orthodontic treatment need ( iotn ) .", "it is being widely used in dental epidemiology to prioritize orthodontic treatment and seeks to quantify the likely sociopsychological effects of each patient 's malocclusion .", "the study was undertaken to assess the orthodontic treatment need in 2025-year - old patients reporting to the department of orthodontics and dentofacial orthopaedics , as assessed by orthodontists to plan future orthodontic services in the region as per priority in orthodontic triage , for worldwide comparisons , and as a powerful tool for patient counseling .", "the study was undertaken to assess the orthodontic treatment need in 2025-year - old patients reporting to the department of orthodontics and dentofacial orthopaedics , as assessed by orthodontists to plan future orthodontic services in the region as per priority in orthodontic triage , for worldwide comparisons , and as a powerful tool for patient counseling .", "the sample comprised 753 patients ( 360 females and 393 males - table 1 ) irrespective of gender , caste , creed , and socioeconomic status essentially fulfilling the following criteria : \n 2025 years old [ table 2 ] \n no history of previous / current orthodontic treatmentpresence of all permanent teeth with / without third molarsno large restorationsinformed consent from patients for participation as part of the survey . \n ", "2025 years old [ table 2 ] \n  no history of previous / current orthodontic treatment presence of all permanent teeth with / without third molars no large restorations informed consent from patients for participation as part of the survey .", "the division of sample according to gender division of the subpopulation according to the age patients were examined clinically using mouth mirror and explorer .", "ac was assessed by comparing and matching digitally clicked intraoral frontal view photograph in occlusion to the nearest resemblance on standardized photographs of iotn .", "number 1 is the most , and 10 , the least attractive arrangement of teeth .", "the anterior teeth were graded in their dental attractiveness as seen and no endeavor was made to predict the future appearance of the dentition .", "esthetic component of the index of orthodontic treatment need scale ( photographs 110 ) the ac of iotn has commonly been used to evaluate the treatment need on esthetic grounds by dentists ( operator - rated ) or patients ( self - rated ) .", "both patients and orthodontists can also assess the ac with the intent of removing the influence of parents and patients desires on the assessment of treatment need apart from considering individual variations in psychological maturity .", "esthetic component grades all the data were analyzed using the statistical package for the social sciences 13.0 software program , ibm corporation , new york , usa . results of the ac revealed that 78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and only 8.7% proved to have a definite need for orthodontic treatment .", "this states that the examiner , in general , expressed satisfaction with the esthetics of most of the screened patients , like the one shown in figure 2 unlike the one in figure 3 that without doubt required treatment . a patient categorized with slight need for orthodontic treatment a patient in severe / definite need for orthodontic treatment", "all the data were analyzed using the statistical package for the social sciences 13.0 software program , ibm corporation , new york , usa . results of the ac revealed that 78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and only 8.7% proved to have a definite need for orthodontic treatment .", "this states that the examiner , in general , expressed satisfaction with the esthetics of most of the screened patients , like the one shown in figure 2 unlike the one in figure 3 that without doubt required treatment . a patient categorized with slight need for orthodontic treatment a patient in severe / definite need for orthodontic treatment", "malocclusions may range from the severe , rather uncommon conditions such as cleft lip and palate to the more common irregularities of teeth resulting from biological variations .", "management of malocclusion is contained by a number of disciplines in dentistry , primarily orthodontics and attempts to prioritize treatment have used the measure of iotn , which relates to treatment need rather than its complexity .", "a sample of 753 patients from among those reporting to the department for a routine dental check - up was considered .", "the age group of 2025 years old was chosen because by then , the patients would be in a permanent dentition stage . at this age , there are less individual variations in dental age and occlusal development .", "the ac scores were recorded by the orthodontist unlike other studies where the patients self - assessed with the intent of removing the influence of patients desires on the assessment of treatment need apart from considering individual variations in psychological maturity .", "78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and 8.7% proved to have a definite need for orthodontic treatment [ figures 4 and 5 ] .", "esthetic component scores inference of esthetic component grading in evaluating the ac in a sample population in shiraz , 91.3% of the population was in no need for treatment , 2.44% in moderate need , and 6.21% in great need for treatment . on the contrary ,", "22.8% of the malaysian children were found to have a definite need according to ac in a study conducted by abdullah and rock in 2001 .", "analysis of registrations of the ac of the iotn should be interpreted with some caution .", "it has shortcomings , for example , the lack of photographs showing hypodontia , anterior spacing , and a class ii division 2 dentition .", "there is always some uncertainty in assessing the photographs of different types of malocclusion to identify one which corresponds to one 's own dental appearance .", "kok et al . , ( 2004 ) suggested that concern about a malocclusion is not closely related to the severity of the malocclusion in terms of esthetics as measured by the iotn - ac .", "2002 suggested that the 10 photographs should be complemented with photographs of patients from other ethnic backgrounds .", "these studies not only provide a global perspective of high orthodontic treatment need , but also confirm a persistently high orthodontic treatment need over the past decade", ". in some european countries , general dentists and orthodontists have been using indices of treatment need to prioritize state - funded orthodontic treatment for children with major irregularities .", "in addition , the demand for orthodontic treatment has increased in contemporary setting due to increasing awareness and perceptions resulting in extensive waiting rolls .", "it is essential , therefore , that health authorities should carefully prioritize and plan the provision of orthodontic treatment . the introductions of easily comprehensible , reproducible , valid , and reliable indices of therapeutic need such as the ac of iotn have allowed improved focusing of services", "\n a baseline data were obtained for planning future orthodontic services in the region as per priority in orthodontic triage .", "the data collected can help for worldwide comparisons with other populationsthe ac - iotn is , hence , being used as a powerful tool for patient counseling and planning desired orthodontic mechanotherapy in the department . \n ", "a baseline data were obtained for planning future orthodontic services in the region as per priority in orthodontic triage .", "the data collected can help for worldwide comparisons with other populations the ac - iotn is , hence , being used as a powerful tool for patient counseling and planning desired orthodontic mechanotherapy in the department ."], "abstract_text": ["<S> aim of the study : the purpose was to assess orthodontic treatment need in a subpopulation as assessed by the orthodontists.materials and methods : the study was conducted on a sample population of 753 patients aged 2025 years to assess the need for orthodontic treatment using the esthetic component ( ac ) of the index of orthodontic treatment need ( iotn).results : the ac revealed that 78.1% of the sample exhibited no or slight need for treatment , 13.2% demonstrated moderate to borderline need , and 8.7% proved to have a definite need for orthodontic treatment.conclusions:the ac - iotn can definitely be considered to be used as a powerful tool for prioritizing orthodontic triage , patient counseling , and planning desired orthodontic mechanotherapy . </S>"], "labels": null, "section_names": ["INTRODUCTION", "Aims of the study", "MATERIALS AND METHODS", "Observations", "DISCUSSION", "CONCLUSIONS", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["the rationale underlying treatment recommendations based on esthetic impairment come from the social science researches , which indicate that unacceptable dental appearance may stigmatize , impede career advancement and peer group acceptance , encourage negative stereotyping , and have a negative effect on self - concept .", "orthodontic problems are usually not associated with grave mortality or morbidity ; hence , they tend to be overlooked by most health professionals as less important .", "however , studies reveal that malocclusions have a significant impact on the psychosocial health of the affected individual . for many years", ", epidemiologic studies of malocclusion suffered from considerable disagreement among investigators , especially regarding how much deviation from the ideal should be accepted within the bounds of normal .", "the orthodontic treatment need in the study group was assessed by esthetic component ( ac ) of the index of orthodontic treatment need ( iotn ) .", "it is being widely used in dental epidemiology to prioritize orthodontic treatment and seeks to quantify the likely sociopsychological effects of each patient 's malocclusion .", "the study was undertaken to assess the orthodontic treatment need in 2025-year - old patients reporting to the department of orthodontics and dentofacial orthopaedics , as assessed by orthodontists to plan future orthodontic services in the region as per priority in orthodontic triage , for worldwide comparisons , and as a powerful tool for patient counseling ."], ["the study was undertaken to assess the orthodontic treatment need in 2025-year - old patients reporting to the department of orthodontics and dentofacial orthopaedics , as assessed by orthodontists to plan future orthodontic services in the region as per priority in orthodontic triage , for worldwide comparisons , and as a powerful tool for patient counseling ."], ["the sample comprised 753 patients ( 360 females and 393 males - table 1 ) irrespective of gender , caste , creed , and socioeconomic status essentially fulfilling the following criteria : \n 2025 years old [ table 2 ] \n no history of previous / current orthodontic treatmentpresence of all permanent teeth with / without third molarsno large restorationsinformed consent from patients for participation as part of the survey . \n ", "2025 years old [ table 2 ] \n  no history of previous / current orthodontic treatment presence of all permanent teeth with / without third molars no large restorations informed consent from patients for participation as part of the survey .", "the division of sample according to gender division of the subpopulation according to the age patients were examined clinically using mouth mirror and explorer .", "ac was assessed by comparing and matching digitally clicked intraoral frontal view photograph in occlusion to the nearest resemblance on standardized photographs of iotn .", "number 1 is the most , and 10 , the least attractive arrangement of teeth .", "the anterior teeth were graded in their dental attractiveness as seen and no endeavor was made to predict the future appearance of the dentition .", "esthetic component of the index of orthodontic treatment need scale ( photographs 110 ) the ac of iotn has commonly been used to evaluate the treatment need on esthetic grounds by dentists ( operator - rated ) or patients ( self - rated ) .", "both patients and orthodontists can also assess the ac with the intent of removing the influence of parents and patients desires on the assessment of treatment need apart from considering individual variations in psychological maturity .", "esthetic component grades all the data were analyzed using the statistical package for the social sciences 13.0 software program , ibm corporation , new york , usa . results of the ac revealed that 78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and only 8.7% proved to have a definite need for orthodontic treatment .", "this states that the examiner , in general , expressed satisfaction with the esthetics of most of the screened patients , like the one shown in figure 2 unlike the one in figure 3 that without doubt required treatment . a patient categorized with slight need for orthodontic treatment a patient in severe / definite need for orthodontic treatment"], ["all the data were analyzed using the statistical package for the social sciences 13.0 software program , ibm corporation , new york , usa . results of the ac revealed that 78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and only 8.7% proved to have a definite need for orthodontic treatment .", "this states that the examiner , in general , expressed satisfaction with the esthetics of most of the screened patients , like the one shown in figure 2 unlike the one in figure 3 that without doubt required treatment . a patient categorized with slight need for orthodontic treatment a patient in severe / definite need for orthodontic treatment"], ["malocclusions may range from the severe , rather uncommon conditions such as cleft lip and palate to the more common irregularities of teeth resulting from biological variations .", "management of malocclusion is contained by a number of disciplines in dentistry , primarily orthodontics and attempts to prioritize treatment have used the measure of iotn , which relates to treatment need rather than its complexity .", "a sample of 753 patients from among those reporting to the department for a routine dental check - up was considered .", "the age group of 2025 years old was chosen because by then , the patients would be in a permanent dentition stage . at this age , there are less individual variations in dental age and occlusal development .", "the ac scores were recorded by the orthodontist unlike other studies where the patients self - assessed with the intent of removing the influence of patients desires on the assessment of treatment need apart from considering individual variations in psychological maturity .", "78.1% exhibited no or slight need for treatment , whereas 13.2% demonstrated moderate to borderline need , and 8.7% proved to have a definite need for orthodontic treatment [ figures 4 and 5 ] .", "esthetic component scores inference of esthetic component grading in evaluating the ac in a sample population in shiraz , 91.3% of the population was in no need for treatment , 2.44% in moderate need , and 6.21% in great need for treatment . on the contrary ,", "22.8% of the malaysian children were found to have a definite need according to ac in a study conducted by abdullah and rock in 2001 .", "analysis of registrations of the ac of the iotn should be interpreted with some caution .", "it has shortcomings , for example , the lack of photographs showing hypodontia , anterior spacing , and a class ii division 2 dentition .", "there is always some uncertainty in assessing the photographs of different types of malocclusion to identify one which corresponds to one 's own dental appearance .", "kok et al . , ( 2004 ) suggested that concern about a malocclusion is not closely related to the severity of the malocclusion in terms of esthetics as measured by the iotn - ac .", "2002 suggested that the 10 photographs should be complemented with photographs of patients from other ethnic backgrounds .", "these studies not only provide a global perspective of high orthodontic treatment need , but also confirm a persistently high orthodontic treatment need over the past decade", ". in some european countries , general dentists and orthodontists have been using indices of treatment need to prioritize state - funded orthodontic treatment for children with major irregularities .", "in addition , the demand for orthodontic treatment has increased in contemporary setting due to increasing awareness and perceptions resulting in extensive waiting rolls .", "it is essential , therefore , that health authorities should carefully prioritize and plan the provision of orthodontic treatment . the introductions of easily comprehensible , reproducible , valid , and reliable indices of therapeutic need such as the ac of iotn have allowed improved focusing of services"], ["\n a baseline data were obtained for planning future orthodontic services in the region as per priority in orthodontic triage .", "the data collected can help for worldwide comparisons with other populationsthe ac - iotn is , hence , being used as a powerful tool for patient counseling and planning desired orthodontic mechanotherapy in the department . \n ", "a baseline data were obtained for planning future orthodontic services in the region as per priority in orthodontic triage .", "the data collected can help for worldwide comparisons with other populations the ac - iotn is , hence , being used as a powerful tool for patient counseling and planning desired orthodontic mechanotherapy in the department ."], [""], [""]]}
{"article_id": "PMC3810332", "article_text": ["in order to proliferate , cells must accurately transmit their chromosomes from one generation to the next . in eukaryotes ,", "the chromosomes are confined to the nucleus , the perimeter of which is defined by the nuclear envelope .", "the nuclear envelope is made of a double membrane ; the outer nuclear membrane is continuous with the endoplasmic reticulum ( er ) and contains many of the same proteins .", "the inner nuclear membrane contains a distinct set of proteins , some of which interact with chromatin .", "the outer and inner nuclear membranes are fused at sites of nuclear pore complexes ( npcs ) , macromolecular structures that allow selective passage of proteins , rna and other molecules to and from the nucleus . in most eukaryotes ,", "underlying the inner nuclear membrane is a network of proteins , called the nuclear lamina , made of lamins and lamin - associated proteins .", "this network provides rigidity to the nucleus and contributes to chromatin organization and other nuclear processes .", "interestingly , neither plants nor fungi have a lamin - based nuclear lamina , although other structures may serve a similar purpose . of note", "alterations in nuclear shape and size are characteristic of aging and certain disease states , such as various types of cancer .", "thus , understanding how nuclear shape is regulated and identifying genes that contribute to nuclear morphology are of interest from both a medical and a basic biology standpoint .", "the nuclear envelope not only serves as a diffusion barrier but also as a physical barrier separating the chromosomes from cytoplasmic structures . in most animal cells", "these cytoplasmic structures include the centrosomes , which function as microtubule organizing centers that nucleate spindle microtubules necessary for chromosome segregation .", "however , just before mitosis , the nuclear envelope stands between these microtubules and the chromosomes .", "different cell types have adopted different strategies to facilitate the access of chromosomes by microtubules : at one end of the spectrum are cells of most metazoans , in which nuclear envelope components , including the two nuclear membranes , the npcs and the nuclear lamina with their associated proteins , disperse during each and every mitosis ( fig . ", "this type of mitosis is called  open mitosis ,  and it necessitates the reassembly of the nuclear envelope around a full complement of chromosomes in each of the daughter cells once chromosome segregation is completed .", "parenthetically , the mechanism that ensures the formation of only a single nucleus at the end mitosis , as opposed to several nuclei each encompassing a subset of chromosomes , is not known . at the other end of the spectrum", "are certain fungi in which the centrosome equivalents , called spindle pole bodies , are either permanently embedded in the nuclear envelope ( such as in budding yeast ) or are embedded in the nuclear envelope prior to mitosis ( such as in fission yeast ) .", "these cells undergo what is called  closed mitosis ,  which occurs without nuclear envelope breakdown , as spindle microtubules that assemble within the nucleus can readily access the chromosomes", ". however , during closed mitosis , the nucleus must elongate in a manner that is coordinated with chromosome movement as chromosome segregation takes place ( fig . ", "the chromatin ( blue ) is contained within the nuclear envelope ( light green ) .", "as cells enter mitosis , the nuclear envelope disassembles , allowing spindle microtubules ( purple lines ) nucleated by centrosomes ( purple spheres ) to align the chromosomes on the metaphase plate .", "( b ) closed mitosis . shown is mitosis as it occurs in s. cerevisiae .", "the spindle pole body ( purple ) is embedded in the nuclear envelope throughout the cell cycle .", "after spindle pole body duplication , an intra - nuclear spindle is formed ( s. cerevisiae chromosomes do not condense enough to visualize individual chromosomes or a metaphase plate ) . during anaphase , the nucleus elongates and the nuclear envelope expands as the sister chromatids move away from each other .", "( c ) functionally open , structurally closed mitosis . a term defined by sazer to indicate a breakdown in the diffusion barrier while maintaining an intact nuclear envelope . shown is mitosis as it occurs in aspergillus nidulans . as in s. cerevisiae , during interphase the spindle pole body is embedded in the nuclear envelope .", "as cells enter mitosis the nuclear pores ( dark green ) partially disassemble , significantly reducing the diffusion barrier between the nucleus and cytoplasm . as in closed mitosis ,", "the bulge in the center of the elongated nucleus during anaphase contains the nucleolus , which is left behind during chromosome segregation .", "schizosaccharomyces japonicus cells form an intra - nuclear spindle as in cells undergoing closed or partially open mitosis .", "however , during anaphase , the nuclear envelope ruptures as the nucleus elongates without nuclear envelope expansion . the historic definition of open vs. closed mitosis was based on cytology : during open mitosis the nuclear envelope disappears ,  opening  the nucleus and exposing its content to the cytoplasm , while in closed mitosis the nuclear envelope remains intact . from a functional standpoint , the presence or absence of a nuclear envelope affects at least two processes : the free diffusion of molecules between the cytoplasm and the nucleus ( which occurs during open , but not closed , mitosis ) and the presence of a physical barrier between the chromosomes and cytoplasmic structures ( which exists in closed , but not open , mitosis ) . over the years , mitotic divisions that fall somewhere in between open and closed have been described , warranting a brief discussion on how these forms of mitosis should be defined . in aspergillus nidulans , which was originally defined as undergoing closed mitosis , npcs", "partially disassemble as cells enter mitosis , disrupting the diffusion barrier between the nucleus and the cytoplasm , while maintaining a physical barrier between the chromosomes and cytoplasmic structures ( fig . ", "a breakdown in the diffusion barrier also occurs during a brief period in anaphase of meiosis ii of the fission yeast schisosaccharomyces pombe . unlike the situation in a. nidulans", ", however , the change in permeability in s. pombe meiosis ii occurs at only a short window of nuclear division and is not accompanied by changes in either npc composition or nuclear membrane integrity .", "this led sazer to define this type of division as  functionally open but structurally closed ", ", a term that can also be applied to a. nidulans mitosis . other types of mitosis involve a partial opening in the nuclear membrane itself . in schisosaccharomyces japonicas ,", "the nuclear envelope does not expand during mitosis , nor does it disassemble . as a result , the nuclear envelope ruptures as the intra - nuclear spindle elongates ( fig . ", "interestingly , rupturing is not a result of forces exerted by the spindle , but rather a programed cell cycle event , the regulation and mechanism of which remain to be discovered . in the early embryonic divisions of the nematode caenorhabditis", "elegans the nuclear envelope is breached in pro - metaphase , allowing diffusion of cytoplasmic proteins into the nuclear space .", "a similar phenomenon is seen during drosophila syncytial divisions , where the nuclear envelope displays a partial disassembly of nuclear pore complexes and fenestrations near the centrosomes , thereby allowing microtubules to access the chromosomes .", "live cell imaging suggests that just as in closed mitosis , the nuclear envelope during drosophila syncytial divisions expands as the spindle elongates . in these three cases , however , mitosis is not structurally closed , as nuclear envelope integrity is breached and cytoplasmic structures , such as microtubules , can enter the nucleus . consequently , the term  functionally open but structurally closed  may not fully apply when describing these types of mitosis , and several studies have previously referred to these mitoses as  semi - open mitosis . ", "we propose to use  partially open mitosis  as term that encompasses all forms of mitosis that are neither fully closed nor fully open , but that exhibit a change in the diffusion barrier at some point during mitosis that may or may not be accompanied by visible structural change to the nuclear envelope . among unicellular eukaryotes", "there are many variations of nuclear envelope behavior during mitosis ( for more examples see refs .", "16 and 17 ) , and undoubtedly additional forms of mitosis still remain to be discovered .", "it is likely that the various types of mitosis have evolved to adapt to a particular environment , although it is currently not known what kinds of selective pressure shaped the different modes of mitosis .", "a possible driving force is the position of the centrosome , namely in the cytoplasm vs. embedded in the nuclear envelope , which likely coevolved with nuclear envelope behavior .", "in addition , the evolution of mitosis could have been affected by the need to eliminate rate - limiting diffusion barriers between the nucleus and the cytoplasm as occurs during open and partially open mitosis , but is either not a problem or is somehow overcome in closed mitosis .", "it is conceivable that , during syncytial divisions , partially open mitosis evolved to overcome the diffusion barrier and microtubule accessibility problems , while protecting chromosomes from aberrant attachments to microtubules from neighboring nuclei .", "the evolution of different forms of mitosis remains an unresolved , yet fascinating , question .", "closed mitosis appears at first glance to be an economical solution to the issue of microtubule - chromosome accessibility : nuclear envelope breakdown is circumvented , the chromosomes do not get  entangled  with cytoplasmic structures , and cells do not have to reassemble the nuclear envelope at the end of mitosis , risking leaving one or more chromosomes outside the nuclear perimeter .", "chief among them is how the nuclear envelope expands . in particular , how is the timing of nuclear expansion determined ?", "is it a consequence of spindle elongation or is it an independent process regulated by the cell cycle machinery ? and how are nuclear envelope components added ?", "finally , what happens to nuclear envelope expansion if cells are delayed in mitosis , for example due to checkpoint activation ?", "an insight into these questions came from a study by witkin et al . on the shape of the budding yeast nucleus . during normal growth ,", "the budding yeast nucleus is round , except during mitosis when it elongates through the bud neck to accommodate chromosome segregation ( fig . ", "witkin et al . screened for genes that when deleted lead to alterations in nuclear shape , aiming to identify genes that affect nuclear envelope dynamics .", "the authors used a collection of roughly 5000 budding yeast deletion mutants ( each deleted for a single non - essential gene ) , into which they introduced a nuclear gfp marker and a cytoplasmic rfp marker .", "this strategy then allowed them to screen visually for mutants that displayed abnormal nuclear morphology .", "one class of nuclear abnormalities was of nuclei that exhibited extensions , referred to as  flares ", ". further examination of these mutants revealed that this altered nuclear phenotype was not due to a direct involvement of the deleted genes in nuclear morphology , but rather due to the activation of a checkpoint pathway ( e.g. , the dna damage checkpoint or the spindle assembly checkpoint ) that induced a mitotic delay .", "in other words , the mutations isolated in the screen likely increased the formation of intracellular damage , which , in turn , led to a mitotic delay through checkpoint activation . indeed , other conditions that induced a mitotic delay ( e.g. , depolymerizing microtubules with nocodazole ; inhibiting the anaphase promoting complex ) also led to the formation of a nuclear flare .", "interestingly , the nuclear flare formed due to a mitotic delay was confined to the nuclear envelope region adjacent to the nucleolus ( fig .  2b , and see below ) , which normally forms a crescent - shaped structure at the nuclear periphery ( fig . ", "are images taken 20 min apart of budding yeast cells expressing a gfp - tagged nucleoplasmic marker ( pus1 ) and an rfp - tagged nucleolar marker ( nsr1 ) .", "importantly , the change in nuclear morphology was specific to a cell cycle delay in mitosis ; witkin et al . found that nuclei of cells arrested in s phase or g2 were mostly round . moreover , the formation of the nuclear flare in cells treated with nocodazole began at the same time as nuclear elongation in untreated cells and was dependent on phospholipid synthesis .", "however , phospholipid synthesis alone was not sufficient to induce flares , as g2 arrested cells accumulated just as much phospholipid as mitotic arrested cells . taken together , these results suggest that nuclear expansion during closed mitosis of budding yeast is independent of spindle elongation , but rather occurs in response to a cell cycle cue that signals mitotic entry .", "first , what regulates nuclear envelope expansion ? as noted above , the process is independent of spindle elongation , much like nuclear envelope rupture in s. japonicus .", "thus , in these yeasts there are likely targets of the cell cycle machinery that affect nuclear envelope dynamics during mitosis", "the synthesis of phospholipids , the main components of the nuclear membrane , is required for nuclear envelope expansion , as shown previously for both budding and fission yeast .", "moreover , at least one enzyme that negatively regulates membrane expansion , pah1 , is regulated by cdk1 phosphorylation ( ref .", "the observation that a nuclear flare forms when checkpoint pathways are activated suggests that phospholipid synthesis is not subjected to checkpoint regulation . however , phospholipid synthesis is unlikely to be the only cell cycle target required for nuclear expansion , because g2-arrested cells accumulate just as much phospholipid as mitotic - arrested cells , yet they do n't form a flare ( ref .", "18 ; note , however , that it is currently not known whether nuclear envelope of g2 cells also expands , but without forming a flare ) .", "it is tempting to speculate that the cell cycle target may be a protein or a process that is responsible for allocating membrane addition specifically to the nuclear envelope , either by localized phospholipid synthesis or by drawing membrane from the er preferentially during mitosis .", "spindle elongation may also play a role in nuclear envelope expansion : although the surface area of the nucleus increases during a mitotic arrest in the absence of a spindle ( i.e. , during an arrest induced by nocodazole ) , it is possible that spindle elongation contributes to the rate , and perhaps magnitude , of nuclear envelope expansion .", "observed that nuclear envelope expansion during a mitotic arrest occurs in a curious fashion : rather than expanding isometrically , thereby generating a larger sphere , the flare occurs at the nuclear envelope that is adjacent to the nucleolus ( fig . ", "whether this occurs in other organisms that undergo closed mitosis is currently not known , as cell cycle analyses typically use dna staining , rather than a nuclear envelope marker , to follow cell cycle progression .", "nonetheless , the flare induced by a mitotic arrest is similar to the nuclear expansion observed when the pah1 pathway is misregulated .", "pah1 converts phosphatidic acid to diacylglycerol . in the absence of pah1 ( or in the absence of its activators , the nem1 and spo7 phosphatase complex ) the overall levels of certain phospholipids increase ,", "the er loses its tubular shape in favor of membrane sheets , and the nucleus forms a flare at the nuclear envelope adjacent to the nucleolus .", "why , then , in both pah1 pathway mutants and in a mitotic arrest is the expansion of the nuclear envelope specific to the region adjacent to the nucleolus ?", "campbell et al . showed that in a spo7 mutant , if the nucleolus is reduced to a tiny sphere the flare still forms , with the diminished nucleolus often at the base of the flare .", "whether the nucleolus , or its remnant , affects the adjacent nuclear envelope remains unknown .", "alternatively , there may be a structure that prevents nuclear envelope expansion around the entire nuclear envelope except in the region adjacent to the nucleolus .", "there are indeed budding yeast nuclear membrane proteins that are excluded from the nucleolus ; none of the ones tested had properties consistent with an inhibitor of membrane expansion ( our lab , unpublished ) , but other such proteins may still exist .", "since the nuclear envelope is continuous with the er , it is quite possible that phospholipids are synthesized at the nuclear envelope itself .", "if this were the case , it is conceivable that the nuclear envelope adjacent to the nucleolus would have a higher capacity for phospholipid synthesis , or that membrane is added throughout the nuclear envelope , but , as mentioned above , there may be a mechanism that resists expansion everywhere except adjacent to the nucleolus .", "alternatively , the membrane could come from the er , and if this were the case then the cell would have a mechanism for allocating membrane from the er to the nuclear envelope specifically during mitosis .", "it is currently not known whether the er expands during a mitotic arrest , and if so , at what rate .", "thus , the mechanisms driving nuclear envelope dynamics , and in particular nuclear envelope expansion , remain to be uncovered .", "the study by witkin et al . highlights the dynamic nature of the nuclear envelope during the cell cycle . the observations in this study support a model whereby", "the nuclear envelope adjacent to the nucleolus is the preferential site of expansion during a mitotic delay .", "it is interesting that phospholipid synthesis is not under checkpoint control ; consequently , a cell delayed in mitosis needs to somehow deal with increased nuclear envelope surface area in the absence of chromosome segregation .", "we can envision at least three possibilities : first , sequestering the added nuclear envelope to the region adjacent to the nucleolus could preserve inter - chromosomal interactions that would otherwise be disrupted were the nucleus to expand isometrically .", "second , yeast cells are known to maintain a constant nuclear to cell volume ratio by a yet unknown mechanism .", "it is possible that by confining the extra nuclear envelope to a flare , rather than distributing it throughout the entire nuclear surface , the cell is better able to regulate its nuclear volume .", "finally , the nuclear envelope adjacent to the nucleolus may be the region of initial nuclear envelope expansion even under normal growth conditions , but in the absence of spindle elongation this expansion is not distributed throughout the entire nuclear surface .", "regardless of the mechanism , the appearance of a nuclear flare suggests that the budding yeast nuclear envelope has domains of distinct properties . how these domains differ in their ability to expand is not known .", "isolation of mutants that fail to produce a flare during a mitotic arrest will likely shed light on the mechanism that designates distinct nuclear envelope domains and on the function of flare formation . finally , can observations in closed mitosis illuminate processes related to the nuclear envelope in cells that undergo open mitosis ? as in closed mitosis , the nuclear envelope of metazoans must also expand , not during mitosis but during interphase , immediately following nuclear envelope reassembly .", "much like in yeast , the source of membrane , and both the temporal and spatial regulation of this process , are unknown .", "moreover , in certain cancer cells nuclei become larger and multi - lobed , and during aging the nuclear envelope appears ", "if so , do changes in the nuclear envelope have consequences for cell function ? despite the different modes of mitosis in budding yeast and mammalian cells , cell cycle processes have often proved to be based on conserved proteins .", "thus , studies on nuclear envelope dynamics in budding yeast will likely shed light on processes related to nuclear envelope formation , tumorigenesis and aging in higher eukaryotes ."], "abstract_text": ["<S> in eukaryotes , chromosomes are encased by a dynamic nuclear envelope . in contrast to metazoans , where the nuclear envelope disassembles during mitosis , many fungi including budding yeast undergo  closed mitosis ,  where the nuclear envelope remains intact throughout the cell cycle . </S>", "<S> consequently , during closed mitosis the nuclear envelope must expand to accommodate chromosome segregation to the two daughter cells . </S>", "<S> a recent study by witkin et al . in budding yeast showed that if progression through mitosis is delayed , for example due to checkpoint activation , the nuclear envelope continues to expand despite the block to chromosome segregation . </S>", "<S> moreover , this expansion occurs at a specific region of the nuclear envelope- adjacent to the nucleolus- forming an extension referred to as a  flare .  </S>", "<S> these observations raise questions regarding the regulation of nuclear envelope expansion both in budding yeast and in higher eukaryotes , the mechanisms confining mitotic nuclear envelope expansion to a particular region and the possible consequences of failing to regulate nuclear envelope expansion during the cell cycle . </S>"], "labels": null, "section_names": ["The Nuclear Envelope during Mitosis: Anything but Static", "Closed Mitosis: A Strategy that Generates a Complication", "Regulation of Nuclear Envelope Expansion", "The Expanding Mitotic Nuclear Envelope Uncovers Discrete Nuclear Envelope Domains", "Conclusions and Open Questions"], "sections": [["in order to proliferate , cells must accurately transmit their chromosomes from one generation to the next . in eukaryotes ,", "the chromosomes are confined to the nucleus , the perimeter of which is defined by the nuclear envelope .", "the nuclear envelope is made of a double membrane ; the outer nuclear membrane is continuous with the endoplasmic reticulum ( er ) and contains many of the same proteins .", "the inner nuclear membrane contains a distinct set of proteins , some of which interact with chromatin .", "the outer and inner nuclear membranes are fused at sites of nuclear pore complexes ( npcs ) , macromolecular structures that allow selective passage of proteins , rna and other molecules to and from the nucleus . in most eukaryotes ,", "underlying the inner nuclear membrane is a network of proteins , called the nuclear lamina , made of lamins and lamin - associated proteins .", "this network provides rigidity to the nucleus and contributes to chromatin organization and other nuclear processes .", "interestingly , neither plants nor fungi have a lamin - based nuclear lamina , although other structures may serve a similar purpose . of note", "alterations in nuclear shape and size are characteristic of aging and certain disease states , such as various types of cancer .", "thus , understanding how nuclear shape is regulated and identifying genes that contribute to nuclear morphology are of interest from both a medical and a basic biology standpoint .", "the nuclear envelope not only serves as a diffusion barrier but also as a physical barrier separating the chromosomes from cytoplasmic structures . in most animal cells", "these cytoplasmic structures include the centrosomes , which function as microtubule organizing centers that nucleate spindle microtubules necessary for chromosome segregation .", "however , just before mitosis , the nuclear envelope stands between these microtubules and the chromosomes .", "different cell types have adopted different strategies to facilitate the access of chromosomes by microtubules : at one end of the spectrum are cells of most metazoans , in which nuclear envelope components , including the two nuclear membranes , the npcs and the nuclear lamina with their associated proteins , disperse during each and every mitosis ( fig . ", "this type of mitosis is called  open mitosis ,  and it necessitates the reassembly of the nuclear envelope around a full complement of chromosomes in each of the daughter cells once chromosome segregation is completed .", "parenthetically , the mechanism that ensures the formation of only a single nucleus at the end mitosis , as opposed to several nuclei each encompassing a subset of chromosomes , is not known . at the other end of the spectrum", "are certain fungi in which the centrosome equivalents , called spindle pole bodies , are either permanently embedded in the nuclear envelope ( such as in budding yeast ) or are embedded in the nuclear envelope prior to mitosis ( such as in fission yeast ) .", "these cells undergo what is called  closed mitosis ,  which occurs without nuclear envelope breakdown , as spindle microtubules that assemble within the nucleus can readily access the chromosomes", ". however , during closed mitosis , the nucleus must elongate in a manner that is coordinated with chromosome movement as chromosome segregation takes place ( fig . ", "the chromatin ( blue ) is contained within the nuclear envelope ( light green ) .", "as cells enter mitosis , the nuclear envelope disassembles , allowing spindle microtubules ( purple lines ) nucleated by centrosomes ( purple spheres ) to align the chromosomes on the metaphase plate .", "( b ) closed mitosis . shown is mitosis as it occurs in s. cerevisiae .", "the spindle pole body ( purple ) is embedded in the nuclear envelope throughout the cell cycle .", "after spindle pole body duplication , an intra - nuclear spindle is formed ( s. cerevisiae chromosomes do not condense enough to visualize individual chromosomes or a metaphase plate ) . during anaphase , the nucleus elongates and the nuclear envelope expands as the sister chromatids move away from each other .", "( c ) functionally open , structurally closed mitosis . a term defined by sazer to indicate a breakdown in the diffusion barrier while maintaining an intact nuclear envelope . shown is mitosis as it occurs in aspergillus nidulans . as in s. cerevisiae , during interphase the spindle pole body is embedded in the nuclear envelope .", "as cells enter mitosis the nuclear pores ( dark green ) partially disassemble , significantly reducing the diffusion barrier between the nucleus and cytoplasm . as in closed mitosis ,", "the bulge in the center of the elongated nucleus during anaphase contains the nucleolus , which is left behind during chromosome segregation .", "schizosaccharomyces japonicus cells form an intra - nuclear spindle as in cells undergoing closed or partially open mitosis .", "however , during anaphase , the nuclear envelope ruptures as the nucleus elongates without nuclear envelope expansion . the historic definition of open vs. closed mitosis was based on cytology : during open mitosis the nuclear envelope disappears ,  opening  the nucleus and exposing its content to the cytoplasm , while in closed mitosis the nuclear envelope remains intact . from a functional standpoint , the presence or absence of a nuclear envelope affects at least two processes : the free diffusion of molecules between the cytoplasm and the nucleus ( which occurs during open , but not closed , mitosis ) and the presence of a physical barrier between the chromosomes and cytoplasmic structures ( which exists in closed , but not open , mitosis ) . over the years , mitotic divisions that fall somewhere in between open and closed have been described , warranting a brief discussion on how these forms of mitosis should be defined . in aspergillus nidulans , which was originally defined as undergoing closed mitosis , npcs", "partially disassemble as cells enter mitosis , disrupting the diffusion barrier between the nucleus and the cytoplasm , while maintaining a physical barrier between the chromosomes and cytoplasmic structures ( fig . ", "a breakdown in the diffusion barrier also occurs during a brief period in anaphase of meiosis ii of the fission yeast schisosaccharomyces pombe . unlike the situation in a. nidulans", ", however , the change in permeability in s. pombe meiosis ii occurs at only a short window of nuclear division and is not accompanied by changes in either npc composition or nuclear membrane integrity .", "this led sazer to define this type of division as  functionally open but structurally closed ", ", a term that can also be applied to a. nidulans mitosis . other types of mitosis involve a partial opening in the nuclear membrane itself . in schisosaccharomyces japonicas ,", "the nuclear envelope does not expand during mitosis , nor does it disassemble . as a result , the nuclear envelope ruptures as the intra - nuclear spindle elongates ( fig . ", "interestingly , rupturing is not a result of forces exerted by the spindle , but rather a programed cell cycle event , the regulation and mechanism of which remain to be discovered . in the early embryonic divisions of the nematode caenorhabditis", "elegans the nuclear envelope is breached in pro - metaphase , allowing diffusion of cytoplasmic proteins into the nuclear space .", "a similar phenomenon is seen during drosophila syncytial divisions , where the nuclear envelope displays a partial disassembly of nuclear pore complexes and fenestrations near the centrosomes , thereby allowing microtubules to access the chromosomes .", "live cell imaging suggests that just as in closed mitosis , the nuclear envelope during drosophila syncytial divisions expands as the spindle elongates . in these three cases , however , mitosis is not structurally closed , as nuclear envelope integrity is breached and cytoplasmic structures , such as microtubules , can enter the nucleus . consequently , the term  functionally open but structurally closed  may not fully apply when describing these types of mitosis , and several studies have previously referred to these mitoses as  semi - open mitosis . ", "we propose to use  partially open mitosis  as term that encompasses all forms of mitosis that are neither fully closed nor fully open , but that exhibit a change in the diffusion barrier at some point during mitosis that may or may not be accompanied by visible structural change to the nuclear envelope . among unicellular eukaryotes", "there are many variations of nuclear envelope behavior during mitosis ( for more examples see refs .", "16 and 17 ) , and undoubtedly additional forms of mitosis still remain to be discovered .", "it is likely that the various types of mitosis have evolved to adapt to a particular environment , although it is currently not known what kinds of selective pressure shaped the different modes of mitosis .", "a possible driving force is the position of the centrosome , namely in the cytoplasm vs. embedded in the nuclear envelope , which likely coevolved with nuclear envelope behavior .", "in addition , the evolution of mitosis could have been affected by the need to eliminate rate - limiting diffusion barriers between the nucleus and the cytoplasm as occurs during open and partially open mitosis , but is either not a problem or is somehow overcome in closed mitosis .", "it is conceivable that , during syncytial divisions , partially open mitosis evolved to overcome the diffusion barrier and microtubule accessibility problems , while protecting chromosomes from aberrant attachments to microtubules from neighboring nuclei .", "the evolution of different forms of mitosis remains an unresolved , yet fascinating , question ."], ["closed mitosis appears at first glance to be an economical solution to the issue of microtubule - chromosome accessibility : nuclear envelope breakdown is circumvented , the chromosomes do not get  entangled  with cytoplasmic structures , and cells do not have to reassemble the nuclear envelope at the end of mitosis , risking leaving one or more chromosomes outside the nuclear perimeter .", "chief among them is how the nuclear envelope expands . in particular , how is the timing of nuclear expansion determined ?", "is it a consequence of spindle elongation or is it an independent process regulated by the cell cycle machinery ? and how are nuclear envelope components added ?", "finally , what happens to nuclear envelope expansion if cells are delayed in mitosis , for example due to checkpoint activation ?", "an insight into these questions came from a study by witkin et al . on the shape of the budding yeast nucleus . during normal growth ,", "the budding yeast nucleus is round , except during mitosis when it elongates through the bud neck to accommodate chromosome segregation ( fig . ", "witkin et al . screened for genes that when deleted lead to alterations in nuclear shape , aiming to identify genes that affect nuclear envelope dynamics .", "the authors used a collection of roughly 5000 budding yeast deletion mutants ( each deleted for a single non - essential gene ) , into which they introduced a nuclear gfp marker and a cytoplasmic rfp marker .", "this strategy then allowed them to screen visually for mutants that displayed abnormal nuclear morphology .", "one class of nuclear abnormalities was of nuclei that exhibited extensions , referred to as  flares ", ". further examination of these mutants revealed that this altered nuclear phenotype was not due to a direct involvement of the deleted genes in nuclear morphology , but rather due to the activation of a checkpoint pathway ( e.g. , the dna damage checkpoint or the spindle assembly checkpoint ) that induced a mitotic delay .", "in other words , the mutations isolated in the screen likely increased the formation of intracellular damage , which , in turn , led to a mitotic delay through checkpoint activation . indeed , other conditions that induced a mitotic delay ( e.g. , depolymerizing microtubules with nocodazole ; inhibiting the anaphase promoting complex ) also led to the formation of a nuclear flare .", "interestingly , the nuclear flare formed due to a mitotic delay was confined to the nuclear envelope region adjacent to the nucleolus ( fig .  2b , and see below ) , which normally forms a crescent - shaped structure at the nuclear periphery ( fig . ", "are images taken 20 min apart of budding yeast cells expressing a gfp - tagged nucleoplasmic marker ( pus1 ) and an rfp - tagged nucleolar marker ( nsr1 ) .", "importantly , the change in nuclear morphology was specific to a cell cycle delay in mitosis ; witkin et al . found that nuclei of cells arrested in s phase or g2 were mostly round . moreover , the formation of the nuclear flare in cells treated with nocodazole began at the same time as nuclear elongation in untreated cells and was dependent on phospholipid synthesis .", "however , phospholipid synthesis alone was not sufficient to induce flares , as g2 arrested cells accumulated just as much phospholipid as mitotic arrested cells . taken together , these results suggest that nuclear expansion during closed mitosis of budding yeast is independent of spindle elongation , but rather occurs in response to a cell cycle cue that signals mitotic entry ."], ["first , what regulates nuclear envelope expansion ? as noted above , the process is independent of spindle elongation , much like nuclear envelope rupture in s. japonicus .", "thus , in these yeasts there are likely targets of the cell cycle machinery that affect nuclear envelope dynamics during mitosis", "the synthesis of phospholipids , the main components of the nuclear membrane , is required for nuclear envelope expansion , as shown previously for both budding and fission yeast .", "moreover , at least one enzyme that negatively regulates membrane expansion , pah1 , is regulated by cdk1 phosphorylation ( ref .", "the observation that a nuclear flare forms when checkpoint pathways are activated suggests that phospholipid synthesis is not subjected to checkpoint regulation . however , phospholipid synthesis is unlikely to be the only cell cycle target required for nuclear expansion , because g2-arrested cells accumulate just as much phospholipid as mitotic - arrested cells , yet they do n't form a flare ( ref .", "18 ; note , however , that it is currently not known whether nuclear envelope of g2 cells also expands , but without forming a flare ) .", "it is tempting to speculate that the cell cycle target may be a protein or a process that is responsible for allocating membrane addition specifically to the nuclear envelope , either by localized phospholipid synthesis or by drawing membrane from the er preferentially during mitosis .", "spindle elongation may also play a role in nuclear envelope expansion : although the surface area of the nucleus increases during a mitotic arrest in the absence of a spindle ( i.e. , during an arrest induced by nocodazole ) , it is possible that spindle elongation contributes to the rate , and perhaps magnitude , of nuclear envelope expansion ."], ["observed that nuclear envelope expansion during a mitotic arrest occurs in a curious fashion : rather than expanding isometrically , thereby generating a larger sphere , the flare occurs at the nuclear envelope that is adjacent to the nucleolus ( fig . ", "whether this occurs in other organisms that undergo closed mitosis is currently not known , as cell cycle analyses typically use dna staining , rather than a nuclear envelope marker , to follow cell cycle progression .", "nonetheless , the flare induced by a mitotic arrest is similar to the nuclear expansion observed when the pah1 pathway is misregulated .", "pah1 converts phosphatidic acid to diacylglycerol . in the absence of pah1 ( or in the absence of its activators , the nem1 and spo7 phosphatase complex ) the overall levels of certain phospholipids increase ,", "the er loses its tubular shape in favor of membrane sheets , and the nucleus forms a flare at the nuclear envelope adjacent to the nucleolus .", "why , then , in both pah1 pathway mutants and in a mitotic arrest is the expansion of the nuclear envelope specific to the region adjacent to the nucleolus ?", "campbell et al . showed that in a spo7 mutant , if the nucleolus is reduced to a tiny sphere the flare still forms , with the diminished nucleolus often at the base of the flare .", "whether the nucleolus , or its remnant , affects the adjacent nuclear envelope remains unknown .", "alternatively , there may be a structure that prevents nuclear envelope expansion around the entire nuclear envelope except in the region adjacent to the nucleolus .", "there are indeed budding yeast nuclear membrane proteins that are excluded from the nucleolus ; none of the ones tested had properties consistent with an inhibitor of membrane expansion ( our lab , unpublished ) , but other such proteins may still exist .", "since the nuclear envelope is continuous with the er , it is quite possible that phospholipids are synthesized at the nuclear envelope itself .", "if this were the case , it is conceivable that the nuclear envelope adjacent to the nucleolus would have a higher capacity for phospholipid synthesis , or that membrane is added throughout the nuclear envelope , but , as mentioned above , there may be a mechanism that resists expansion everywhere except adjacent to the nucleolus .", "alternatively , the membrane could come from the er , and if this were the case then the cell would have a mechanism for allocating membrane from the er to the nuclear envelope specifically during mitosis .", "it is currently not known whether the er expands during a mitotic arrest , and if so , at what rate .", "thus , the mechanisms driving nuclear envelope dynamics , and in particular nuclear envelope expansion , remain to be uncovered ."], ["the study by witkin et al . highlights the dynamic nature of the nuclear envelope during the cell cycle . the observations in this study support a model whereby", "the nuclear envelope adjacent to the nucleolus is the preferential site of expansion during a mitotic delay .", "it is interesting that phospholipid synthesis is not under checkpoint control ; consequently , a cell delayed in mitosis needs to somehow deal with increased nuclear envelope surface area in the absence of chromosome segregation .", "we can envision at least three possibilities : first , sequestering the added nuclear envelope to the region adjacent to the nucleolus could preserve inter - chromosomal interactions that would otherwise be disrupted were the nucleus to expand isometrically .", "second , yeast cells are known to maintain a constant nuclear to cell volume ratio by a yet unknown mechanism .", "it is possible that by confining the extra nuclear envelope to a flare , rather than distributing it throughout the entire nuclear surface , the cell is better able to regulate its nuclear volume .", "finally , the nuclear envelope adjacent to the nucleolus may be the region of initial nuclear envelope expansion even under normal growth conditions , but in the absence of spindle elongation this expansion is not distributed throughout the entire nuclear surface .", "regardless of the mechanism , the appearance of a nuclear flare suggests that the budding yeast nuclear envelope has domains of distinct properties . how these domains differ in their ability to expand is not known .", "isolation of mutants that fail to produce a flare during a mitotic arrest will likely shed light on the mechanism that designates distinct nuclear envelope domains and on the function of flare formation . finally , can observations in closed mitosis illuminate processes related to the nuclear envelope in cells that undergo open mitosis ? as in closed mitosis , the nuclear envelope of metazoans must also expand , not during mitosis but during interphase , immediately following nuclear envelope reassembly .", "much like in yeast , the source of membrane , and both the temporal and spatial regulation of this process , are unknown .", "moreover , in certain cancer cells nuclei become larger and multi - lobed , and during aging the nuclear envelope appears ", "if so , do changes in the nuclear envelope have consequences for cell function ? despite the different modes of mitosis in budding yeast and mammalian cells , cell cycle processes have often proved to be based on conserved proteins .", "thus , studies on nuclear envelope dynamics in budding yeast will likely shed light on processes related to nuclear envelope formation , tumorigenesis and aging in higher eukaryotes ."]]}
{"article_id": "PMC3687185", "article_text": ["fibro - osseous lesions ( fol ) are a poorly defined group of lesions affecting the jaws and craniofacial bones .", "all are characterized by the replacement of bone by cellular fibrous tissue containing foci of mineralization that vary in amount and appearance .", "recent world health organization classification ( 2005 ) for fibro - osseous lesions was considered while sub - dividing these into various groups [ table 1 ] .", "so proper categorization requires good correlation of the history , clinical findings , radiographic characteristics , operative findings , and histologic appearance .", "the aim of this study was to analyze various clinico - pathological and radiological features in the benign fol reported and to compare the features between fibrous dysplasia ( fd ) and cemento - ossifying fibroma ( cof ) .", "these patients were treated for fol , reported to the hospital between 1989 and 2009 .", "the clinical parameters included were age , sex , location , duration , family history , associated symptoms , and behavior of the lesion .", "the radiographic appearance , histologic features , treatment , and follow - up data were also recorded .", "basic clinical parameters such as age , sex , duration of lesion , family history , history of trauma , associated symptoms , palpatory findings , status of associated / involved teeth , associated pigmentation , site of the lesion , treatment , and recurrence details were retrospectively analyzed .", "regarding site distribution , the maxilla was divided into two anatomic regions as anterior ( midline to distal surface of canine ) and posterior ( mesial surface of first premolar distally ) .", "the mandible was divided into four anatomic regions such as anterior ( midline to distal surface of canine ) , posterior ( mesial surface of first premolar distally ) , angle ( from distal of third molar to the inferior portion of ramus ) , and ramus ( upper portion of the ramus beyond the occlusal plane ) .", "radiological features were assessed for radiolucency , radiopacity , margin of the lesion , cortical - plate expansion , involvement of antrum , displacement , and resorption of teeth .", "histopathologically , parameters such as type of bone ( mature / immature ) , cellularity , presence of cementum - like material , and nature of stroma were assessed .", "a total of 80 cases of benign fols were recorded over the 20 years . among these , cof ( all variants ) was the most frequent tumor found in 60 patients with 63 lesions ( 73.2% ) and fd was found in 20 patients ( 24.4% ) . in general for fol ,", "the age ranged from 3 years to 65 years with a mean age of 23.3 years ; the majority of cofs and fds were seen in the second decade ( 38.3% and 65% respectively ) .", "the male - female ratio for these 80 patients was 1:1 with slight female predilection ( 27 men and 33 women i.e. , 1:1.2 ) , whereas fd showed definite male predilection ( 13 men and 7 women , i.e. , 1.8:1 ) .", "however , there was no significant correlation between the lesions and the sex with p > 0.05 [ figure 1 ] .", "the sex distribution of 80 patients with fibro - osseous lesions most of the time , fol patients complained of a slow - growing swelling of the jaws and facial asymmetry ( 75 patients = 93.7% ) whereas in four patients ( 5% ) , it was an incidental finding ; all these four cases were later diagnosed to be cof . in one case of fd ,", "a biopsy was done because of a non - healing extraction socket . in case of cof ,", "the associated signs and symptoms involved pus discharge , tenderness , egg shell crackling , tender lymph nodes , ulceration of the overlying mucosa , numbness of lip and proptosis .", "none of family history and past medical history data collected were contributory to our study except few which are listed below .", "there were four cof patients recorded with history of extraction ( 2 patients ) , surgery for osteosarcoma ( 1 patient ) , and history of trauma ( 1 patient ) at the site of current presentation . when fol as a whole was taken , no specific jaw / side predilection was evident .", "the cof showed slight mandibular predilection ( 1.37:1 ) unlike that of fd which showed definitive maxillary predilection ( 2.3:1 ) .", "moreover , cof were often recorded in mandibular posterior region ( 51.2% ) , whereas at the time of presentation fd was recorded unilaterally in maxilla as a whole bone .", "there were three cases of cof noted at the midline either in maxilla ( 1 patient ) or in mandible ( 2 patients ) and three other cases of cof crossed midline with an extensive presentation in the mandible .", "the most posterior presentation of cof was noted in two of our cases in the ramus and angle of the mandible [ table 1 ]", ". extensive involvement of facial bones like ethmoidal and frontal bone was seen in fd ( 2 patients ) , in four cases of cof { juvenile ossifying fibroma ( jof ) variant } , and in two patients with aggressive form of ossifying fibroma ( of ) ( which can not be classified as jof ) .", "when the data was subjected for chi - square test , significant association was found between the fol and the site ( maxilla and mandible ) with p < 0.05 [ figure 2 ] .", "the site distribution of 80 patients with fibro - osseous lesions mobility of teeth was seen in 13 patients which included 11 cases of cof and two cases of fds . in one case of cof ,", "the lesion was associated with displacement of teeth , whereas one cof showed impacted tooth .", "radiographically , 23 ( 38.3% ) cofs showed mixed opaque and lucent areas , 19 ( 31.6% ) cases showed only radiolucent areas and 18 ( 30% ) cases showed only radiopacity . among these ,", "55 ( 91.6% ) had a well - defined border whereas 5 ( 8.3% ) cases were having diffuse outline .", "a total of eight cofs , showed involvement of antrum , displacement and resorption of the teeth . among fds", ", most of them showed mixed opaque and lucent areas ( 75% ) , diffuse borders ( 60% ) with only four cases ( 20% ) showing expansion of cortical plates , and five cases showing expansion of antrum .", "only one case showed resorption of the associated teeth . in case of cof , on 34 ( 56% )", "occasions it was provisionally diagnosed as cof and fd was considered in four cases . among 20 fds , 15 ( 75% ) times fd was considered as a provisional diagnosis and only in two occasions , it was thought in terms of cof .", "other provisional diagnosis considered were adenomatoid odontogenic tumor , ameloblastoma , aneurysmal bone cyst , odontoma , calcifying epithelial odontogenic tumor , odontogenic myxoma , central giant cell granuloma , and odontogenic keratocyst for cof and osteoma for fd .", "the cof on the post - operative histologic findings was seen as a well - demarcated lesion that was separated from the overlying cortical bone by a thin zone of fibrous tissue .", "the stroma was fibro - cellular ( 42 cases ) and fibrous ( 18 cases ) with irregular thin trabeculae of woven bone ( 43 patients ) or lamellar bone ( 11 patients ) rimmed by osteoblasts .", "basophilic , ovoid , cementum - like material was evident in 21 cofs : among these four were considered as jof  psammamatoid type . other features evident with regular histopathology", "were the presence of giant cells ( 5 cof ) , myxoid areas ( 1 jof ) , and endothelial proliferation ( 1 cof ) .", "fd showed merging of lesional bone with the normal along with highly fibrous stroma ( 8 cases ) consisting of immature trabeculae of woven bone ( 18 lesions ) giving a ", "comparison of histopathological features between cemento - ossifying fibroma and fibrous dysplasia the treatment procedures rendered in these cases has been summarized in the table 3 .", "eight patients of cof showed recurrences within a period ranging from 2 months to 4 years .", "one case of jof showed three recurrences every year . among three patients with fds , two patients had recurrences after 2 and 4 years respectively , with last one showing multiple ( 3 times ) recurrences almost after 3 years each time .", "a total of 80 cases of benign fols were recorded over the 20 years . among these , cof ( all variants ) was the most frequent tumor found in 60 patients with 63 lesions ( 73.2% ) and fd was found in 20 patients ( 24.4% ) . in general for fol ,", "the age ranged from 3 years to 65 years with a mean age of 23.3 years ; the majority of cofs and fds were seen in the second decade ( 38.3% and 65% respectively ) .", "the male - female ratio for these 80 patients was 1:1 with slight female predilection ( 27 men and 33 women i.e. , 1:1.2 ) , whereas fd showed definite male predilection ( 13 men and 7 women , i.e. , 1.8:1 ) .", "however , there was no significant correlation between the lesions and the sex with p > 0.05 [ figure 1 ] .", "the sex distribution of 80 patients with fibro - osseous lesions most of the time , fol patients complained of a slow - growing swelling of the jaws and facial asymmetry ( 75 patients = 93.7% ) whereas in four patients ( 5% ) , it was an incidental finding ; all these four cases were later diagnosed to be cof . in one case of fd ,", "a biopsy was done because of a non - healing extraction socket . in case of cof ,", "the associated signs and symptoms involved pus discharge , tenderness , egg shell crackling , tender lymph nodes , ulceration of the overlying mucosa , numbness of lip and proptosis .", "none of family history and past medical history data collected were contributory to our study except few which are listed below .", "there were four cof patients recorded with history of extraction ( 2 patients ) , surgery for osteosarcoma ( 1 patient ) , and history of trauma ( 1 patient ) at the site of current presentation . when fol as a whole was taken , no specific jaw / side predilection was evident .", "the cof showed slight mandibular predilection ( 1.37:1 ) unlike that of fd which showed definitive maxillary predilection ( 2.3:1 ) .", "moreover , cof were often recorded in mandibular posterior region ( 51.2% ) , whereas at the time of presentation fd was recorded unilaterally in maxilla as a whole bone .", "there were three cases of cof noted at the midline either in maxilla ( 1 patient ) or in mandible ( 2 patients ) and three other cases of cof crossed midline with an extensive presentation in the mandible .", "the most posterior presentation of cof was noted in two of our cases in the ramus and angle of the mandible [ table 1 ]", ". extensive involvement of facial bones like ethmoidal and frontal bone was seen in fd ( 2 patients ) , in four cases of cof { juvenile ossifying fibroma ( jof ) variant } , and in two patients with aggressive form of ossifying fibroma ( of ) ( which can not be classified as jof ) .", "when the data was subjected for chi - square test , significant association was found between the fol and the site ( maxilla and mandible ) with p < 0.05 [ figure 2 ] .", "the site distribution of 80 patients with fibro - osseous lesions mobility of teeth was seen in 13 patients which included 11 cases of cof and two cases of fds . in one case of cof ,", "the lesion was associated with displacement of teeth , whereas one cof showed impacted tooth .", "radiographically , 23 ( 38.3% ) cofs showed mixed opaque and lucent areas , 19 ( 31.6% ) cases showed only radiolucent areas and 18 ( 30% ) cases showed only radiopacity . among these ,", "55 ( 91.6% ) had a well - defined border whereas 5 ( 8.3% ) cases were having diffuse outline .", "a total of eight cofs , showed involvement of antrum , displacement and resorption of the teeth . among fds", ", most of them showed mixed opaque and lucent areas ( 75% ) , diffuse borders ( 60% ) with only four cases ( 20% ) showing expansion of cortical plates , and five cases showing expansion of antrum .", "in case of cof , on 34 ( 56% ) occasions it was provisionally diagnosed as cof and fd was considered in four cases . among 20 fds , 15 ( 75% ) times fd was considered as a provisional diagnosis and only in two occasions , it was thought in terms of cof .", "other provisional diagnosis considered were adenomatoid odontogenic tumor , ameloblastoma , aneurysmal bone cyst , odontoma , calcifying epithelial odontogenic tumor , odontogenic myxoma , central giant cell granuloma , and odontogenic keratocyst for cof and osteoma for fd .", "the cof on the post - operative histologic findings was seen as a well - demarcated lesion that was separated from the overlying cortical bone by a thin zone of fibrous tissue .", "the stroma was fibro - cellular ( 42 cases ) and fibrous ( 18 cases ) with irregular thin trabeculae of woven bone ( 43 patients ) or lamellar bone ( 11 patients ) rimmed by osteoblasts .", "basophilic , ovoid , cementum - like material was evident in 21 cofs : among these four were considered as jof  psammamatoid type .", "other features evident with regular histopathology were the presence of giant cells ( 5 cof ) , myxoid areas ( 1 jof ) , and endothelial proliferation ( 1 cof ) .", "fd showed merging of lesional bone with the normal along with highly fibrous stroma ( 8 cases ) consisting of immature trabeculae of woven bone ( 18 lesions ) giving a  chinese letter ", "eight patients of cof showed recurrences within a period ranging from 2 months to 4 years .", "one case of jof showed three recurrences every year . among three patients with fds , two patients had recurrences after 2 and 4 years respectively , with last one showing multiple ( 3 times ) recurrences almost after 3 years each time .", "the fol of the jaws comprise a diverse , interesting , and challenging group of conditions that pose difficulties in classification and treatment .", "common to all is the replacement of normal bone by a tissue composed of collagen fibers and fibroblasts that contain varying amounts of mineralized substance , which may be bony or cementum - like in appearance .", "langdon et al . , suggested that certain fols of the jaw may represent different stages in the evolution of a single disease process .", "although the first case report of fibro - osseous lesion was reported 60 years ago , there are only few retrospective studies regarding their clinic - pathological correlation . among them , the four important studies were carried out by alsharif et al . , on chinese population , ogunsalu et al . , on jamican population , langdon et al .", "comparison of clinical parameters with previous studies however , although fol are one of the common lesions occurring in india , such a retrospective study is not reported in the english language literature .", "we also tried to compare the features between cofs and fds , which are the only two groups of entities reported in this region .", "aggressive variants seen in young adults are called as jof ( who 1992 ) . according to recent who classification of fols ,", "of is a well - demarcated lesion composed of fibrocellular tissue and mineralized material of varying appearances .", "juvenile trabecular of and juvenile psammamatoid of are two histologic variants of of . due to the presence of cementum - like material of have been called as cementifying fibroma ( cf ) or cof if they have both cementum and bone - like material .", "previous studies considered cementoid lesions as separate groups which included cemento - osseous dysplasia , gigantiform cementoma , and cementoblastoma .", "however , alsharif et al . , has considered cof as cementoid lesions containing cementum - like material , since he has considered of as a separate entity . however , in 1992 the who grouped such lesions under the common denomination of cof on the grounds that they represented histologic variants of the same type of lesion . in this study , of and", "cof are combined and no cementoid lesions were reported till today to our hospital . in our data ,", "24 cases were diagnosed as of , 21 as cof , 11 as cf , and 4 as jof .", "however , we considered all 60 cases as a single entity ( cof ) in contrast to the study on chinese population where cof and of are assessed separately .", "none of these cases showed any familial history as we know there are two case reports of familial cof .", "the term fd was given by lichtenstein in 1938 , which was earlier described as osteitis fibrosa disseminate .", "it is a genetically based sporadic disease of bone that may affect single or multiple bones ( monostotic or polyostotic ) or if it is occurring in multiple adjacent craniofacial bones , it is regarded as craniofacial fd .", "fd may be part of jaffe - lichenstein 's , macunae albright 's , or mazabraud 's syndrome . in over 80% of cases", "it is monostotic , whereas all 20 cases in our data showed a solitary lesion in the jaws , although few of maxillary lesions extending up to infra - orbital margin affect vision .", ", was 27 years and 34 years , contrasting to what we got as 21 years for the present study .", "early lesions may be radiolucent , but they become increasingly radiopaque and typically show a diffuse radiopacity or  ground glass appearance  . in the present study , 75% of the lesions showed mixed opacity and lucency , whereas two cases were completely radiolucent and three cases with complete radiopaque picture .", "the key histologic features of fd are delicate trabeculae of immature bone with no osteoblastic rimming , enmeshed within a fibrous stroma giving a ", "mature bone was seen in one of our case and osteoblastic rimming was evident in few areas of five cases and stroma was mostly fibrocellular , although few showed a completely fibrous or vascular background . compared with previous studies,[468 ] both cof and fd were predominantly seen in younger population ( 25.5 years and 21 years respectively ) .", "however , in our data , there was a definite male predilection ( 1.8:1 ) for fd which was contrasting to the previous studies.[468 ] when compared with fd , many of cof presented with associated symptoms like pus discharge , egg shell crackling , tenderness , ulceration of the overlying mucosa , numbness of lip , and proptosis which is unusual for these groups of lesions ( this involvement was seen in four patients diagnosed as jof ) .", "other than jof , there are no previous reports of conventional cof showing highly aggressive behavior . only few fds presented with aggressive symptoms .", "the involvement of other facial bones ( frontal , ethmoidal , antrum ) was a frequent finding in cof when compared with fd .", "both cof and fd showed multiple recurrences . since few of our cof cases showed history of trauma and association with osteosarcoma , a careful detailing of these in the history", "is emphasized although its correlation with the occurrence of the lesion can not be established through this study . as aggressive behavior and recurrence", "was a frequent finding in few patients with cof , a long follow - up was advised .", "juvenile ossifying fibroma was of predominantly psammamatoid type and finally , not a single cementoid lesion was reported for last 20 years ."], "abstract_text": ["<S> background : fibro - osseous lesions ( fols ) are one of the commonest entities reported in the head and neck region . </S>", "<S> however , studies on these groups of lesions on indian population were not carried out before . </S>", "<S> so this motivated us to analyze the clinico - pathologic correlation of fibro - osseous lesions reported at our hospital.materials and methods : a retrospective review was made of all the lesions surgically treated in our hospital . </S>", "<S> a total of 6,175 biopsies were performed during the study period . </S>", "<S> all the cases which were histopathologically diagnosed as fols were included in the study . </S>", "<S> the demographic data , radiographic features , and histopathologic findings were analyzed and compared with similar studies on other races.results and conclusion : we could find 80 cases diagnosed as fibro - osseous lesions and information about them was documented . </S>", "<S> the most common fol reported in the region was cemento - ossifying fibroma ( cof ) ( 75% ) than fibrous dysplasia ( fd ) ( 25% ) . </S>", "<S> these were commonly occurring in 2nd decade without any sex or site predilection . </S>", "<S> however , cof was showing a slight female predominance and fd with a definite male predominance . </S>", "<S> cof was commonly seen in mandible ( posterior region ) whereas fd mainly confined to the maxilla ( as a whole bone ) . </S>", "<S> radiographically , most of cof showed well - defined mixed opaque and lucent areas whereas fd showed diffuse borders . </S>", "<S> cortical plate expansion and resorption of associated teeth was a frequent finding in cof when compared with fd . </S>", "<S> histopathologically , stroma was fibrocellular in many cases of cof , whereas most fds showed fibrous stroma , interspersed with mainly woven bone . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "Clinical findings", "Radiological findings", "Provisional diagnosis", "Pathologic findings", "Treatment and recurrence", "DISCUSSION", "CONCLUSION"], "sections": [["fibro - osseous lesions ( fol ) are a poorly defined group of lesions affecting the jaws and craniofacial bones .", "all are characterized by the replacement of bone by cellular fibrous tissue containing foci of mineralization that vary in amount and appearance .", "recent world health organization classification ( 2005 ) for fibro - osseous lesions was considered while sub - dividing these into various groups [ table 1 ] .", "so proper categorization requires good correlation of the history , clinical findings , radiographic characteristics , operative findings , and histologic appearance .", "the aim of this study was to analyze various clinico - pathological and radiological features in the benign fol reported and to compare the features between fibrous dysplasia ( fd ) and cemento - ossifying fibroma ( cof ) .", "these patients were treated for fol , reported to the hospital between 1989 and 2009 ."], ["the clinical parameters included were age , sex , location , duration , family history , associated symptoms , and behavior of the lesion .", "the radiographic appearance , histologic features , treatment , and follow - up data were also recorded .", "basic clinical parameters such as age , sex , duration of lesion , family history , history of trauma , associated symptoms , palpatory findings , status of associated / involved teeth , associated pigmentation , site of the lesion , treatment , and recurrence details were retrospectively analyzed .", "regarding site distribution , the maxilla was divided into two anatomic regions as anterior ( midline to distal surface of canine ) and posterior ( mesial surface of first premolar distally ) .", "the mandible was divided into four anatomic regions such as anterior ( midline to distal surface of canine ) , posterior ( mesial surface of first premolar distally ) , angle ( from distal of third molar to the inferior portion of ramus ) , and ramus ( upper portion of the ramus beyond the occlusal plane ) .", "radiological features were assessed for radiolucency , radiopacity , margin of the lesion , cortical - plate expansion , involvement of antrum , displacement , and resorption of teeth .", "histopathologically , parameters such as type of bone ( mature / immature ) , cellularity , presence of cementum - like material , and nature of stroma were assessed ."], ["a total of 80 cases of benign fols were recorded over the 20 years . among these , cof ( all variants ) was the most frequent tumor found in 60 patients with 63 lesions ( 73.2% ) and fd was found in 20 patients ( 24.4% ) . in general for fol ,", "the age ranged from 3 years to 65 years with a mean age of 23.3 years ; the majority of cofs and fds were seen in the second decade ( 38.3% and 65% respectively ) .", "the male - female ratio for these 80 patients was 1:1 with slight female predilection ( 27 men and 33 women i.e. , 1:1.2 ) , whereas fd showed definite male predilection ( 13 men and 7 women , i.e. , 1.8:1 ) .", "however , there was no significant correlation between the lesions and the sex with p > 0.05 [ figure 1 ] .", "the sex distribution of 80 patients with fibro - osseous lesions most of the time , fol patients complained of a slow - growing swelling of the jaws and facial asymmetry ( 75 patients = 93.7% ) whereas in four patients ( 5% ) , it was an incidental finding ; all these four cases were later diagnosed to be cof . in one case of fd ,", "a biopsy was done because of a non - healing extraction socket . in case of cof ,", "the associated signs and symptoms involved pus discharge , tenderness , egg shell crackling , tender lymph nodes , ulceration of the overlying mucosa , numbness of lip and proptosis .", "none of family history and past medical history data collected were contributory to our study except few which are listed below .", "there were four cof patients recorded with history of extraction ( 2 patients ) , surgery for osteosarcoma ( 1 patient ) , and history of trauma ( 1 patient ) at the site of current presentation . when fol as a whole was taken , no specific jaw / side predilection was evident .", "the cof showed slight mandibular predilection ( 1.37:1 ) unlike that of fd which showed definitive maxillary predilection ( 2.3:1 ) .", "moreover , cof were often recorded in mandibular posterior region ( 51.2% ) , whereas at the time of presentation fd was recorded unilaterally in maxilla as a whole bone .", "there were three cases of cof noted at the midline either in maxilla ( 1 patient ) or in mandible ( 2 patients ) and three other cases of cof crossed midline with an extensive presentation in the mandible .", "the most posterior presentation of cof was noted in two of our cases in the ramus and angle of the mandible [ table 1 ]", ". extensive involvement of facial bones like ethmoidal and frontal bone was seen in fd ( 2 patients ) , in four cases of cof { juvenile ossifying fibroma ( jof ) variant } , and in two patients with aggressive form of ossifying fibroma ( of ) ( which can not be classified as jof ) .", "when the data was subjected for chi - square test , significant association was found between the fol and the site ( maxilla and mandible ) with p < 0.05 [ figure 2 ] .", "the site distribution of 80 patients with fibro - osseous lesions mobility of teeth was seen in 13 patients which included 11 cases of cof and two cases of fds . in one case of cof ,", "the lesion was associated with displacement of teeth , whereas one cof showed impacted tooth .", "radiographically , 23 ( 38.3% ) cofs showed mixed opaque and lucent areas , 19 ( 31.6% ) cases showed only radiolucent areas and 18 ( 30% ) cases showed only radiopacity . among these ,", "55 ( 91.6% ) had a well - defined border whereas 5 ( 8.3% ) cases were having diffuse outline .", "a total of eight cofs , showed involvement of antrum , displacement and resorption of the teeth . among fds", ", most of them showed mixed opaque and lucent areas ( 75% ) , diffuse borders ( 60% ) with only four cases ( 20% ) showing expansion of cortical plates , and five cases showing expansion of antrum .", "only one case showed resorption of the associated teeth . in case of cof , on 34 ( 56% )", "occasions it was provisionally diagnosed as cof and fd was considered in four cases . among 20 fds , 15 ( 75% ) times fd was considered as a provisional diagnosis and only in two occasions , it was thought in terms of cof .", "other provisional diagnosis considered were adenomatoid odontogenic tumor , ameloblastoma , aneurysmal bone cyst , odontoma , calcifying epithelial odontogenic tumor , odontogenic myxoma , central giant cell granuloma , and odontogenic keratocyst for cof and osteoma for fd .", "the cof on the post - operative histologic findings was seen as a well - demarcated lesion that was separated from the overlying cortical bone by a thin zone of fibrous tissue .", "the stroma was fibro - cellular ( 42 cases ) and fibrous ( 18 cases ) with irregular thin trabeculae of woven bone ( 43 patients ) or lamellar bone ( 11 patients ) rimmed by osteoblasts .", "basophilic , ovoid , cementum - like material was evident in 21 cofs : among these four were considered as jof  psammamatoid type . other features evident with regular histopathology", "were the presence of giant cells ( 5 cof ) , myxoid areas ( 1 jof ) , and endothelial proliferation ( 1 cof ) .", "fd showed merging of lesional bone with the normal along with highly fibrous stroma ( 8 cases ) consisting of immature trabeculae of woven bone ( 18 lesions ) giving a ", "comparison of histopathological features between cemento - ossifying fibroma and fibrous dysplasia the treatment procedures rendered in these cases has been summarized in the table 3 .", "eight patients of cof showed recurrences within a period ranging from 2 months to 4 years .", "one case of jof showed three recurrences every year . among three patients with fds , two patients had recurrences after 2 and 4 years respectively , with last one showing multiple ( 3 times ) recurrences almost after 3 years each time ."], ["a total of 80 cases of benign fols were recorded over the 20 years . among these , cof ( all variants ) was the most frequent tumor found in 60 patients with 63 lesions ( 73.2% ) and fd was found in 20 patients ( 24.4% ) . in general for fol ,", "the age ranged from 3 years to 65 years with a mean age of 23.3 years ; the majority of cofs and fds were seen in the second decade ( 38.3% and 65% respectively ) .", "the male - female ratio for these 80 patients was 1:1 with slight female predilection ( 27 men and 33 women i.e. , 1:1.2 ) , whereas fd showed definite male predilection ( 13 men and 7 women , i.e. , 1.8:1 ) .", "however , there was no significant correlation between the lesions and the sex with p > 0.05 [ figure 1 ] .", "the sex distribution of 80 patients with fibro - osseous lesions most of the time , fol patients complained of a slow - growing swelling of the jaws and facial asymmetry ( 75 patients = 93.7% ) whereas in four patients ( 5% ) , it was an incidental finding ; all these four cases were later diagnosed to be cof . in one case of fd ,", "a biopsy was done because of a non - healing extraction socket . in case of cof ,", "the associated signs and symptoms involved pus discharge , tenderness , egg shell crackling , tender lymph nodes , ulceration of the overlying mucosa , numbness of lip and proptosis .", "none of family history and past medical history data collected were contributory to our study except few which are listed below .", "there were four cof patients recorded with history of extraction ( 2 patients ) , surgery for osteosarcoma ( 1 patient ) , and history of trauma ( 1 patient ) at the site of current presentation . when fol as a whole was taken , no specific jaw / side predilection was evident .", "the cof showed slight mandibular predilection ( 1.37:1 ) unlike that of fd which showed definitive maxillary predilection ( 2.3:1 ) .", "moreover , cof were often recorded in mandibular posterior region ( 51.2% ) , whereas at the time of presentation fd was recorded unilaterally in maxilla as a whole bone .", "there were three cases of cof noted at the midline either in maxilla ( 1 patient ) or in mandible ( 2 patients ) and three other cases of cof crossed midline with an extensive presentation in the mandible .", "the most posterior presentation of cof was noted in two of our cases in the ramus and angle of the mandible [ table 1 ]", ". extensive involvement of facial bones like ethmoidal and frontal bone was seen in fd ( 2 patients ) , in four cases of cof { juvenile ossifying fibroma ( jof ) variant } , and in two patients with aggressive form of ossifying fibroma ( of ) ( which can not be classified as jof ) .", "when the data was subjected for chi - square test , significant association was found between the fol and the site ( maxilla and mandible ) with p < 0.05 [ figure 2 ] .", "the site distribution of 80 patients with fibro - osseous lesions mobility of teeth was seen in 13 patients which included 11 cases of cof and two cases of fds . in one case of cof ,", "the lesion was associated with displacement of teeth , whereas one cof showed impacted tooth ."], ["radiographically , 23 ( 38.3% ) cofs showed mixed opaque and lucent areas , 19 ( 31.6% ) cases showed only radiolucent areas and 18 ( 30% ) cases showed only radiopacity . among these ,", "55 ( 91.6% ) had a well - defined border whereas 5 ( 8.3% ) cases were having diffuse outline .", "a total of eight cofs , showed involvement of antrum , displacement and resorption of the teeth . among fds", ", most of them showed mixed opaque and lucent areas ( 75% ) , diffuse borders ( 60% ) with only four cases ( 20% ) showing expansion of cortical plates , and five cases showing expansion of antrum ."], ["in case of cof , on 34 ( 56% ) occasions it was provisionally diagnosed as cof and fd was considered in four cases . among 20 fds , 15 ( 75% ) times fd was considered as a provisional diagnosis and only in two occasions , it was thought in terms of cof .", "other provisional diagnosis considered were adenomatoid odontogenic tumor , ameloblastoma , aneurysmal bone cyst , odontoma , calcifying epithelial odontogenic tumor , odontogenic myxoma , central giant cell granuloma , and odontogenic keratocyst for cof and osteoma for fd ."], ["the cof on the post - operative histologic findings was seen as a well - demarcated lesion that was separated from the overlying cortical bone by a thin zone of fibrous tissue .", "the stroma was fibro - cellular ( 42 cases ) and fibrous ( 18 cases ) with irregular thin trabeculae of woven bone ( 43 patients ) or lamellar bone ( 11 patients ) rimmed by osteoblasts .", "basophilic , ovoid , cementum - like material was evident in 21 cofs : among these four were considered as jof  psammamatoid type .", "other features evident with regular histopathology were the presence of giant cells ( 5 cof ) , myxoid areas ( 1 jof ) , and endothelial proliferation ( 1 cof ) .", "fd showed merging of lesional bone with the normal along with highly fibrous stroma ( 8 cases ) consisting of immature trabeculae of woven bone ( 18 lesions ) giving a  chinese letter "], ["eight patients of cof showed recurrences within a period ranging from 2 months to 4 years .", "one case of jof showed three recurrences every year . among three patients with fds , two patients had recurrences after 2 and 4 years respectively , with last one showing multiple ( 3 times ) recurrences almost after 3 years each time ."], ["the fol of the jaws comprise a diverse , interesting , and challenging group of conditions that pose difficulties in classification and treatment .", "common to all is the replacement of normal bone by a tissue composed of collagen fibers and fibroblasts that contain varying amounts of mineralized substance , which may be bony or cementum - like in appearance .", "langdon et al . , suggested that certain fols of the jaw may represent different stages in the evolution of a single disease process .", "although the first case report of fibro - osseous lesion was reported 60 years ago , there are only few retrospective studies regarding their clinic - pathological correlation . among them , the four important studies were carried out by alsharif et al . , on chinese population , ogunsalu et al . , on jamican population , langdon et al .", "comparison of clinical parameters with previous studies however , although fol are one of the common lesions occurring in india , such a retrospective study is not reported in the english language literature .", "we also tried to compare the features between cofs and fds , which are the only two groups of entities reported in this region .", "aggressive variants seen in young adults are called as jof ( who 1992 ) . according to recent who classification of fols ,", "of is a well - demarcated lesion composed of fibrocellular tissue and mineralized material of varying appearances .", "juvenile trabecular of and juvenile psammamatoid of are two histologic variants of of . due to the presence of cementum - like material of have been called as cementifying fibroma ( cf ) or cof if they have both cementum and bone - like material .", "previous studies considered cementoid lesions as separate groups which included cemento - osseous dysplasia , gigantiform cementoma , and cementoblastoma .", "however , alsharif et al . , has considered cof as cementoid lesions containing cementum - like material , since he has considered of as a separate entity . however , in 1992 the who grouped such lesions under the common denomination of cof on the grounds that they represented histologic variants of the same type of lesion . in this study , of and", "cof are combined and no cementoid lesions were reported till today to our hospital . in our data ,", "24 cases were diagnosed as of , 21 as cof , 11 as cf , and 4 as jof .", "however , we considered all 60 cases as a single entity ( cof ) in contrast to the study on chinese population where cof and of are assessed separately .", "none of these cases showed any familial history as we know there are two case reports of familial cof .", "the term fd was given by lichtenstein in 1938 , which was earlier described as osteitis fibrosa disseminate .", "it is a genetically based sporadic disease of bone that may affect single or multiple bones ( monostotic or polyostotic ) or if it is occurring in multiple adjacent craniofacial bones , it is regarded as craniofacial fd .", "fd may be part of jaffe - lichenstein 's , macunae albright 's , or mazabraud 's syndrome . in over 80% of cases", "it is monostotic , whereas all 20 cases in our data showed a solitary lesion in the jaws , although few of maxillary lesions extending up to infra - orbital margin affect vision .", ", was 27 years and 34 years , contrasting to what we got as 21 years for the present study .", "early lesions may be radiolucent , but they become increasingly radiopaque and typically show a diffuse radiopacity or  ground glass appearance  . in the present study , 75% of the lesions showed mixed opacity and lucency , whereas two cases were completely radiolucent and three cases with complete radiopaque picture .", "the key histologic features of fd are delicate trabeculae of immature bone with no osteoblastic rimming , enmeshed within a fibrous stroma giving a ", "mature bone was seen in one of our case and osteoblastic rimming was evident in few areas of five cases and stroma was mostly fibrocellular , although few showed a completely fibrous or vascular background . compared with previous studies,[468 ] both cof and fd were predominantly seen in younger population ( 25.5 years and 21 years respectively ) .", "however , in our data , there was a definite male predilection ( 1.8:1 ) for fd which was contrasting to the previous studies.[468 ] when compared with fd , many of cof presented with associated symptoms like pus discharge , egg shell crackling , tenderness , ulceration of the overlying mucosa , numbness of lip , and proptosis which is unusual for these groups of lesions ( this involvement was seen in four patients diagnosed as jof ) .", "other than jof , there are no previous reports of conventional cof showing highly aggressive behavior . only few fds presented with aggressive symptoms .", "the involvement of other facial bones ( frontal , ethmoidal , antrum ) was a frequent finding in cof when compared with fd ."], ["both cof and fd showed multiple recurrences . since few of our cof cases showed history of trauma and association with osteosarcoma , a careful detailing of these in the history", "is emphasized although its correlation with the occurrence of the lesion can not be established through this study . as aggressive behavior and recurrence", "was a frequent finding in few patients with cof , a long follow - up was advised .", "juvenile ossifying fibroma was of predominantly psammamatoid type and finally , not a single cementoid lesion was reported for last 20 years ."]]}
{"article_id": "PMC3728527", "article_text": ["green tea , unfermented and made from the leaves of the camellia sinensis plant , is one of the most popular beverages consumed across the world [ 13 ] .", "the property and chemical components of green teas are influenced by many factors , such as tea species , harvest season , climate , geographical locations , and processing . in china , among various factors , the geographical origin is recognized as an important aspect of tea . because of the similar tea species , cultivation and processing conditions in a specific tea - producing area , many teas are named after their geographical origins .", "longjing tea is a green tea produced in xihu and its surrounding areas ( hangzhou , china ) . as a famous green tea with protected designation of origin ( pdo ) ,", "longjing is recognized as one of the top green teas for its special appearance ( flat and straight leaves ) , flavor , and taste .", "however , little information has been available on the feasibility of discriminating longjing from its three specific subproducing areas , namely , xihu , qiantang , and yuezhou . as the quality and prices of longjing tea from the above three producing areas are different", ", it is necessary to develop effective analysis methods for discrimination of longjing from different subproducing areas . because of the similarity ( processing , appearance , and taste ) among different subproducing areas , the specific geographical origins of longjing are usually distinguished by sensory analysis .", "however , because it is very expensive and may take years to train a tea taster , it would be more efficient to use some nonhuman techniques .", "recent years have witnessed increased applications of electronic tongue technology to analysis of wines , milk , tea , beer , juice , and so on [ 710 ] . in these applications , a very good time stability and sensitivity", ", a good correlation between human and electronic tongue judgment has been observed , which makes it a promising alternative to human sensory analysis of teas .", "this paper was focused on developing a rapid analysis method for discriminating specific subproducing areas of longjing tea by electronic tongue and chemometrics .", "robust principal component analysis ( robpca ) [ 11 , 12 ] was used to detect outliers in each class .", "partial least squares discriminant analysis ( plsda )   was used to develop the classification model .", "155 longjing samples were collected from the local tea plantations in xihu ( 32 samples , class a ) , qiantang ( 59 samples , class b ) , and yuezhou ( 64 samples , class c ) .", "all the samples were preserved in a cool ( about 4c ) , dark , and dry place with integral packaging before preparation of tea extract .", "six gram of each sample was added with 250  ml boiling deionized water and infused for 10  min .", "the infusion was then filtered into a beaker and cooled to the room temperature ( 25c ) by water bath for electronic tongue analysis .", "the detection system consists of one reference electrode ( ag / agcl ) and 7 liquid cross selective sensors ( zz , ba , bb , ca , ga , ha , and jb ) . the cross - sensitivity and selectivity of the sensor array are listed in table 1 .", "the sensors array analyzed the solutions of tea samples with sampling interval of 1  s. each sample was measured for 150  s , which can ensure a stable response .", "all the data analysis was performed on matlab 7.0.1 ( mathworks , sherborn , ma , usa ) .", "the responses of the 7 sensors reported at 150  s were used for the subsequent data analysis .", "outliers in the data would degrade the classification models and prediction performance , so robust principal component analysis ( robpca ) was used to detect outliers in each class of samples .", "robpca can obtain robust projections of the original data points and avoid the masking effects caused by multiple outliers . with the computed robust score distance ( sd ) and", "orthogonal distance ( od ) , robpca can classify an object into one of the four groups : regular points ( with small sd and small od ) , good pca - leverage points ( with large sd and small od ) , orthogonal outliers ( with small sd and large od ) , and bad pca - leverage points ( with large sd and large od ) . with outliers", "deleted , the duplex algorithm   was used to divide the measured data into a training set and test set .", "duplex algorithm can obtain a test set of samples distributed uniformly in the range of training samples . for multiclass classification", ", plsda can be performed by regressing each column of a dummy response matrix y on the measured data x by pls . for the ith ( i = 1 , 2 , and 3 ) column in y , an element", "is set a value of 1 if the corresponding object is from class i ; otherwise , it is assigned a value of 0 . for prediction", ", a new sample is classified into class i when the ith element of its predicted response vector is nearest to 1 .", "monte carlo cross validation ( mccv )   was used to estimate the number of components in plsda by minimizing the mean percentage error of mccv ( mpemccv ) : \n ( 1)mpemccv=i=1bnii=1bmi , \n where b is the numbers of mccv data splitting , mi is the number of prediction objects , and ni is the number of wrongly predicted for the ith mccv data splitting .", "model sensitivity and specificity of prediction for each class were used to evaluate the performance of classification models : \n ( 2)sensitivity = tptp+fn , specificity = tntn+fp , \n where tp , fn , tn , and fp represent the numbers of true positives , false negatives , true negatives , and false positives , respectively . for classification ,", "objects in each class were denoted as positives and the other two classes were denoted as negatives .", "the average electronic tongue features for each class of longjing tea are shown in figure 1 . seen from figure 1 , the features of the three classes have very similar response patterns .", "the features of class b and c are very similar and different from those of class a , especially in the responses of ga and ha .", "robpca was used for outlier detection with a significance level of 0.05 . because each class of tea has a different probability distribution , robpca models were performed separately on each class .  ", "figure 2 demonstrates the robpca plots for the three classes . for each class , a robpca model with three principal components ( pcs )", "three pcs explained 89.7 , 91.5 and 92.1 percents of the total variances of each class , respectively . for outlier diagnosis ,", "bad pca - leverage points , good pca - leverage points and orthogonal outliers were excluded to obtain a representative data set distributed in the entire range of measured samples .", "the numbers of objects with large sd and od were denoted . as a result , for class", "a , two orthogonal outliers ( objects 1 and 26 ) and two good pca - leverage points ( objects 15 and 31 ) were deleted ; for class b , four orthogonal outliers ( objects 1 , 19 , 24 , and 43 ) were deleted ; for class c , three orthogonal outliers ( objects 12 , 17 , and 29 ) and one bad pca - leverage point ( object 55 ) were deleted .  ", "the duplex algorithm was then performed to divide the remaining 143 tea samples into training and test objects .", "finally , 95 samples ( class a , 18 ; class b , 37 ; and class c , 40 ) were used for training and 48 samples ( class a , 10 ; class b , 18 ; and class c , 20 ) for prediction .", "plsda model was developed and mccv was used to select the number of latent variables ( lvs ) . for mccv ,", "the original 95 training samples were randomly split into training ( 50% ) and test objects ( 50% ) for 100 times .", "the training and prediction results of a three - component plsda are demonstrated in figure 3 .", "the sensitivity / specificity of plsda for classes a , b , and c was 1.000(10/10)/1.000(38/38 ) , 1.000(18/18)/0.967(29/30 ) , and 0.950(19/20)/1.000(28/28 ) , respectively .", "the training accuracy was 1 and for prediction only one object from class c was wrongly assigned to class b , indicating the effectiveness of electronic tongue for classification of longjing tea samples .", "rapid and effective discrimination of longjing green tea from different subproducing areas was performed using electronic tongue and chemometrics .", "the sensitivity / specificity of plsda for classes a , b , and c was 1.000/1.000 , 1.000/0.967 , and 0.950/1.000 , respectively . electronic tongue and chemometrics can provide a rapid and reliable tool for discriminating the specific producing areas of longjing . compared with human sensory analysis , this method is easier to perform and the more attractive economically . in the future studies", ", the comparison of chemical methods , for example , lc / uv / ms , to the electronic tongue analysis will be performed to investigate the statistical correlation between the chemistry and the tastes of longjing tea ."], "abstract_text": ["<S> the feasibility of electronic tongue and multivariate analysis was investigated for discriminating the specific geographical origins of a chinese green tea with protected designation of origin ( pdo ) . </S>", "<S> 155 longjing tea samples from three subareas were collected and analyzed by an electronic tongue array of 7 sensors . to remove the influence of abnormal measurements and samples , robust principal component analysis ( robpca ) </S>", "<S> was used to detect outliers in each class . </S>", "<S> partial least squares discriminant analysis ( plsda ) was then used to develop a classification model . the prediction sensitivity / specificity of plsda was 1.000/1.000 , 1.000/0.967 , and 0.950/1.000 for longjing from xihu , qiantang , and yuezhou , respectively . electronic tongue and chemometrics can provide a rapid and reliable tool for discriminating the specific producing areas of longjing . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Experimental and Methods", "3. Results and Discussion", "4. Conclusions"], "sections": [["green tea , unfermented and made from the leaves of the camellia sinensis plant , is one of the most popular beverages consumed across the world [ 13 ] .", "the property and chemical components of green teas are influenced by many factors , such as tea species , harvest season , climate , geographical locations , and processing . in china , among various factors , the geographical origin is recognized as an important aspect of tea . because of the similar tea species , cultivation and processing conditions in a specific tea - producing area , many teas are named after their geographical origins .", "longjing tea is a green tea produced in xihu and its surrounding areas ( hangzhou , china ) . as a famous green tea with protected designation of origin ( pdo ) ,", "longjing is recognized as one of the top green teas for its special appearance ( flat and straight leaves ) , flavor , and taste .", "however , little information has been available on the feasibility of discriminating longjing from its three specific subproducing areas , namely , xihu , qiantang , and yuezhou . as the quality and prices of longjing tea from the above three producing areas are different", ", it is necessary to develop effective analysis methods for discrimination of longjing from different subproducing areas . because of the similarity ( processing , appearance , and taste ) among different subproducing areas , the specific geographical origins of longjing are usually distinguished by sensory analysis .", "however , because it is very expensive and may take years to train a tea taster , it would be more efficient to use some nonhuman techniques .", "recent years have witnessed increased applications of electronic tongue technology to analysis of wines , milk , tea , beer , juice , and so on [ 710 ] . in these applications , a very good time stability and sensitivity", ", a good correlation between human and electronic tongue judgment has been observed , which makes it a promising alternative to human sensory analysis of teas .", "this paper was focused on developing a rapid analysis method for discriminating specific subproducing areas of longjing tea by electronic tongue and chemometrics .", "robust principal component analysis ( robpca ) [ 11 , 12 ] was used to detect outliers in each class .", "partial least squares discriminant analysis ( plsda )   was used to develop the classification model ."], ["155 longjing samples were collected from the local tea plantations in xihu ( 32 samples , class a ) , qiantang ( 59 samples , class b ) , and yuezhou ( 64 samples , class c ) .", "all the samples were preserved in a cool ( about 4c ) , dark , and dry place with integral packaging before preparation of tea extract .", "six gram of each sample was added with 250  ml boiling deionized water and infused for 10  min .", "the infusion was then filtered into a beaker and cooled to the room temperature ( 25c ) by water bath for electronic tongue analysis .", "the detection system consists of one reference electrode ( ag / agcl ) and 7 liquid cross selective sensors ( zz , ba , bb , ca , ga , ha , and jb ) . the cross - sensitivity and selectivity of the sensor array are listed in table 1 .", "the sensors array analyzed the solutions of tea samples with sampling interval of 1  s. each sample was measured for 150  s , which can ensure a stable response .", "all the data analysis was performed on matlab 7.0.1 ( mathworks , sherborn , ma , usa ) .", "the responses of the 7 sensors reported at 150  s were used for the subsequent data analysis .", "outliers in the data would degrade the classification models and prediction performance , so robust principal component analysis ( robpca ) was used to detect outliers in each class of samples .", "robpca can obtain robust projections of the original data points and avoid the masking effects caused by multiple outliers . with the computed robust score distance ( sd ) and", "orthogonal distance ( od ) , robpca can classify an object into one of the four groups : regular points ( with small sd and small od ) , good pca - leverage points ( with large sd and small od ) , orthogonal outliers ( with small sd and large od ) , and bad pca - leverage points ( with large sd and large od ) . with outliers", "deleted , the duplex algorithm   was used to divide the measured data into a training set and test set .", "duplex algorithm can obtain a test set of samples distributed uniformly in the range of training samples . for multiclass classification", ", plsda can be performed by regressing each column of a dummy response matrix y on the measured data x by pls . for the ith ( i = 1 , 2 , and 3 ) column in y , an element", "is set a value of 1 if the corresponding object is from class i ; otherwise , it is assigned a value of 0 . for prediction", ", a new sample is classified into class i when the ith element of its predicted response vector is nearest to 1 .", "monte carlo cross validation ( mccv )   was used to estimate the number of components in plsda by minimizing the mean percentage error of mccv ( mpemccv ) : \n ( 1)mpemccv=i=1bnii=1bmi , \n where b is the numbers of mccv data splitting , mi is the number of prediction objects , and ni is the number of wrongly predicted for the ith mccv data splitting .", "model sensitivity and specificity of prediction for each class were used to evaluate the performance of classification models : \n ( 2)sensitivity = tptp+fn , specificity = tntn+fp , \n where tp , fn , tn , and fp represent the numbers of true positives , false negatives , true negatives , and false positives , respectively . for classification ,", "objects in each class were denoted as positives and the other two classes were denoted as negatives ."], ["the average electronic tongue features for each class of longjing tea are shown in figure 1 . seen from figure 1 , the features of the three classes have very similar response patterns .", "the features of class b and c are very similar and different from those of class a , especially in the responses of ga and ha .", "robpca was used for outlier detection with a significance level of 0.05 . because each class of tea has a different probability distribution , robpca models were performed separately on each class .  ", "figure 2 demonstrates the robpca plots for the three classes . for each class , a robpca model with three principal components ( pcs )", "three pcs explained 89.7 , 91.5 and 92.1 percents of the total variances of each class , respectively . for outlier diagnosis ,", "bad pca - leverage points , good pca - leverage points and orthogonal outliers were excluded to obtain a representative data set distributed in the entire range of measured samples .", "the numbers of objects with large sd and od were denoted . as a result , for class", "a , two orthogonal outliers ( objects 1 and 26 ) and two good pca - leverage points ( objects 15 and 31 ) were deleted ; for class b , four orthogonal outliers ( objects 1 , 19 , 24 , and 43 ) were deleted ; for class c , three orthogonal outliers ( objects 12 , 17 , and 29 ) and one bad pca - leverage point ( object 55 ) were deleted .  ", "the duplex algorithm was then performed to divide the remaining 143 tea samples into training and test objects .", "finally , 95 samples ( class a , 18 ; class b , 37 ; and class c , 40 ) were used for training and 48 samples ( class a , 10 ; class b , 18 ; and class c , 20 ) for prediction .", "plsda model was developed and mccv was used to select the number of latent variables ( lvs ) . for mccv ,", "the original 95 training samples were randomly split into training ( 50% ) and test objects ( 50% ) for 100 times .", "the training and prediction results of a three - component plsda are demonstrated in figure 3 .", "the sensitivity / specificity of plsda for classes a , b , and c was 1.000(10/10)/1.000(38/38 ) , 1.000(18/18)/0.967(29/30 ) , and 0.950(19/20)/1.000(28/28 ) , respectively .", "the training accuracy was 1 and for prediction only one object from class c was wrongly assigned to class b , indicating the effectiveness of electronic tongue for classification of longjing tea samples ."], ["rapid and effective discrimination of longjing green tea from different subproducing areas was performed using electronic tongue and chemometrics .", "the sensitivity / specificity of plsda for classes a , b , and c was 1.000/1.000 , 1.000/0.967 , and 0.950/1.000 , respectively . electronic tongue and chemometrics can provide a rapid and reliable tool for discriminating the specific producing areas of longjing . compared with human sensory analysis , this method is easier to perform and the more attractive economically . in the future studies", ", the comparison of chemical methods , for example , lc / uv / ms , to the electronic tongue analysis will be performed to investigate the statistical correlation between the chemistry and the tastes of longjing tea ."]]}
{"article_id": "PMC4354617", "article_text": ["aging is associated with changes in various body systems including in the neuromuscular structures , which lead to a reduction in important components of physical fitness.1 the preservation of fitness - related components such as muscular strength , endurance , and flexibility are essential to the performance of daily activities , which thus helps to maintain autonomy and quality of life in the elderly population.25 of these fitness - related components , flexibility is often less appreciated with respect to its contribution to optimal health and functional status as well as independent living for the elderly.3,6 flexibility reductions may increase the risks of injury , falling , back pain , and physical dependence in older adults.7 several mechanisms have been proposed for reductions in flexibility , among them alterations in soft - tissue structures and physical inactivity.7 when a joint is relatively inactive due to sedentary behavior , the muscles that cross it shorten , thereby reducing its range of motion . moreover ,", "independent of activity levels , the aging process plays a role in flexibility decreases.8 joint structures such cartilage , ligaments , and tendons change mechanically and biochemically with aging , increasing muscular and tendon stiffness and thus impeding mobility.911 on the other hand , maintaining a physically active lifestyle may result in improvement of functional performance with advancing age , thus enabling the execution of activities of daily living with more vigor and less fatigue.12,13 while resistance training ( rt ) is recommended for older people for the development of muscular strength,13,14 some studies have indicated that regular participation in rt programs may also contribute to increased flexibility.11,1519 in fact , there is evidence that regular rt serves as an active form of flexibility training and can improve range of motion to a similar extent as typical static stretching protocols.20 regular performance of rt may improve flexibility by reducing passive tension and stiffness of the tissues surrounding a joint.11 thus , from a time - saving standpoint , rt is a good way to develop both strength and flexibility as well as achieve improvements in body composition within a single session of training .", "the fitness - related benefits associated with rt are dependent on the manipulation of variables such as intensity , volume , exercise order , and rest intervals.12 with respect to training volume , manipulation can involve varying the number of repetitions , sets , and/or  frequency   defined here as the number of sessions performed per week .", "the american college of sports medicine recommends that older adults engage in two to three rt sessions a week for improving neuromuscular fitness.12 however , there is a current paucity of research regarding the effects of different rt weekly frequencies on flexibility adaptive responses .", "previous studies are limited to analyzing the impact of rt on flexibility without regard for manipulation of the variables that make up the training program . considering the importance of flexibility in the health and wellness of the elderly", "therefore , the main purpose of this investigation was to analyze the effect of rt performed at different weekly frequencies on flexibility in untrained older women .", "we hypothesized that higher rt frequencies would result in greater flexibility increases than lower frequencies . the rational for our hypothesis", "response relationship between training volume and muscle strength and hypertrophy,21,22 whereby greater training volumes are associated with enhanced muscular adaptations .", "a secondary aim of the study was to assess rt frequency - related changes in skeletal muscle mass .", "in accordance with the aforementioned dose  response relationship , it was hypothesized that muscle protein accretion would be greater with the higher frequency program .", "recruitment of the participants was carried out through newspaper and radio advertisements and home delivery of flyers in londrina , paran , brazil .", "a total of 350 older women responded to the advertisements , and then completed detailed health history and physical activity questionnaires .", "the women were subsequently admitted to the study if they met the following inclusion criteria : non - hypertensive ( systolic blood pressure < 140 mmhg and diastolic blood pressure < 90 mmhg ) , nondiabetic , free from cardiac or renal dysfunction , nonsmoking , not receiving hormonal replacement therapy , not performing any regular physical exercise more than once per week over the preceding 6 months , and participated in > 85% of the study training sessions .", "the remaining 64 older women ( 60 years old ) were randomly assigned to one of two groups : a group that performed rt two times per week ( n=32 ; group ", "g2x  ) or a group that performed rt three times per week ( n=32 ; group  g3x  ) . a total of 53 women ( g2x=28 ; g3x=25 ) completed the study and therefore were included in the analysis .", "participants passed a diagnostic , graded exercise stress test with twelve - lead electrocardiography reviewed by a cardiologist and were released with no restrictions for participation in this study .", "this investigation was conducted according to the declaration of helsinki , and was approved by the londrina state university ethics committee .", "the study was carried out over a period of 16 weeks , with 12 weeks dedicated to the rt program and 4 weeks used for testing .", "anthropometric , body - composition , and flexibility measurements were performed at weeks 12 and 1516 for baseline and post - training analysis , respectively , while the supervised rt program was performed during weeks 314 .", "subjects were instructed not to perform any other type of physical exercise during the entire study period .", "body mass was measured to the nearest 0.1 kg using a calibrated electronic scale ( balmak scale , model iii , so paulo , brazil ) , with the participants wearing light workout clothing and no shoes .", "height was measured with a stadiometer attached on the scale to the nearest 0.1 cm with subjects standing without shoes .", "body mass index was calculated as body mass in kilograms divided by the square root of height in meters .", "the skeletal muscle mass was estimated by the predictive equation proposed by kim et al.23 the appendicular fat - free mass used in the equation was determined by a dual energy x - ray absorptiometry scan ( lunar prodigy nrl 41990 , ge lunar , madison , wi , usa ) . before scanning , participants were instructed to remove from their person all objects containing metal .", "scans were performed with the subjects lying in the supine position along the table s longitudinal centerline axis .", "feet were taped together at the toes to immobilize the legs , while the hands were maintained in a pronated position within the scanning region .", "the software generated standard lines that set apart the limbs from the trunk and head .", "these lines were adjusted by the same technician using specific anatomical points determined by the manufacturer .", "analyses during the intervention were performed by the same technician who was blinded to intervention time point .", "retest scans resulted in a standard error of measurement of 0.29 kg and intra - class correlation coefficient of 0.997 for skeletal muscle mass . to evaluate a subject s flexibility ,", "five joint movements were adopted : cervical flexion ( cf ) , cervical extension ( ce ) , frontal hip flexion ( fhf ) , left hip flexion , and right hip flexion .", "these movements were chosen because the flexibility of the hip and cervical spine are highly important for the elderly , especially for locomotion , eye orientation , and good timeline perception.24 all measurements were obtained by a fleximeter ( code , american do brazil ltda , so paulo , brazil ) with a degree scale .", "all the procedures were made according to procedures and recommendations described elsewhere.25 briefly , in both cf and ce subjects remained lying supine on a stretching table in order to neutralize any possible compensatory movements , and the fleximeter was positioned at the side of the head , in the sagittal plane , starting with cf , where they moved the head slowly , until the chin leaned on the sternum or noticed a rigidity in the final range of motion ; for ce , the subjects moved their head back slowly . for fhf ,", "the fleximeter was positioned next to the hip just above the iliac crest , in a standing position with shoulders vertically flexed , elbows extended , fingers intertwined , legs together , then the subjects flexed frontally the hip , with knees extended throughout the movement . for lateral hip flexion , the fleximeter was placed on the medial surface of the thoracic spine , the participant remained standing , with legs together , and knees extended , but with arms crossing the trunk and hands on the contrary shoulder , and then performed the lateral trunk flexion , as a special consideration to such movement , the heel should remain supported on the ground . for all moves , after fixing the velcro attached to the fleximeter and setting the zero point , the participants executed the movements as far as they could or until tightness or discomfort in the final range of motion was felt , and at the end range of motion the evaluator recorded the measure  at this point the participants were instructed to remain in the final position until the reading was completed .", "the highest score obtained from the three measurements at each joint motion was adopted as a reference standard .", "the information obtained at baseline was not made available to the evaluator at the time of revaluation in an attempt to avoid their unduly influencing the results .", "it is worth emphasizing that the evaluator had over 2 years experience , and based on the test ", "retest , the standard error of measurement and the intra - class correlation coefficient among the movements were 2.26 degrees and 0.950 , respectively , and the maximal technical error among the movements analyzed was 1.19 degrees . during the intervention period , in each session , the instructors registered the load ( kg ) for each of the eight exercises for all the subjects .", "afterwards , training load for each subject was weekly calculated , using the sum of the load employed in the exercises as a reference for each week .", "supervised rt was carried out for 12 weeks during the morning hours , in the londrina state university facilities .", "the protocol was based on recommendations for rt in an older population to improve muscular endurance and strength.12,13 all participants were personally supervised by physical - education professionals with substantial rt experience to help ensure consistent and safe performance .", "g2x performed two rt sessions per week on tuesdays and thursdays , while g3x performed the same exercises in three sessions per week on mondays , wednesdays , and fridays .", "the rt program was a whole - body program with eight exercises comprising one exercise with free weights and seven with machines , performed exactly in the following order : chest press , horizontal leg press , seated row , knee extension , preacher curl ( free weights ) , leg curl , triceps push - down , and seated calf raise .", "participants were instructed to inhale during the eccentric phase and exhale during the concentric phase while maintaining a constant velocity of movement at a ratio of approximately 1:2 seconds ( concentric and eccentric phases , respectively ) .", "instructors adjusted the loads of each exercise according to the subject s ability and improvements in exercise capacity throughout the study in order to ensure that the subjects were exercising with as much resistance as possible while maintaining proper exercise technique .", "progression was planned so that when 15 repetitions were completed for two consecutive training sessions , weight was increased 2%5% for the upper limb exercises and 5%10% for the lower limb exercises in the next session.12 it is important to note that the participants did not perform any type of warm - up or cool - down exercises at the beginning or end of the session , respectively .", "two - way analysis of variance for repeated measures was used for within - group comparisons . in variables where sphericity was violated , as indicated by mauchly s test", ", the analyses were adjusted using a greenhouse  geisser correction . when the f - ratio was significant , bonferroni s post - hoc test was employed to identify the mean differences .", "baseline differences between groups were explored with an independent t - test . the effect size ( es )", "was calculated to verify the magnitude of the differences by cohen s d , where an es of 0.200.40 was considered small , 0.500.79 as moderate , and 0.80 as large.26 for all statistical analyses , significance was accepted at p<0.05 .", "the data were stored and analyzed using statistica software ( v 10.0 ; statsoft inc , tulsa , ok , usa ) .", "recruitment of the participants was carried out through newspaper and radio advertisements and home delivery of flyers in londrina , paran , brazil .", "a total of 350 older women responded to the advertisements , and then completed detailed health history and physical activity questionnaires .", "the women were subsequently admitted to the study if they met the following inclusion criteria : non - hypertensive ( systolic blood pressure < 140 mmhg and diastolic blood pressure < 90 mmhg ) , nondiabetic , free from cardiac or renal dysfunction , nonsmoking , not receiving hormonal replacement therapy , not performing any regular physical exercise more than once per week over the preceding 6 months , and participated in > 85% of the study training sessions .", "the remaining 64 older women ( 60 years old ) were randomly assigned to one of two groups : a group that performed rt two times per week ( n=32 ; group ", "g2x  ) or a group that performed rt three times per week ( n=32 ; group  g3x  ) . a total of 53 women ( g2x=28 ; g3x=25 ) completed the study and therefore were included in the analysis .", "participants passed a diagnostic , graded exercise stress test with twelve - lead electrocardiography reviewed by a cardiologist and were released with no restrictions for participation in this study .", "this investigation was conducted according to the declaration of helsinki , and was approved by the londrina state university ethics committee .", "the study was carried out over a period of 16 weeks , with 12 weeks dedicated to the rt program and 4 weeks used for testing .", "anthropometric , body - composition , and flexibility measurements were performed at weeks 12 and 1516 for baseline and post - training analysis , respectively , while the supervised rt program was performed during weeks 314 .", "subjects were instructed not to perform any other type of physical exercise during the entire study period .", "body mass was measured to the nearest 0.1 kg using a calibrated electronic scale ( balmak scale , model iii , so paulo , brazil ) , with the participants wearing light workout clothing and no shoes .", "height was measured with a stadiometer attached on the scale to the nearest 0.1 cm with subjects standing without shoes .", "body mass index was calculated as body mass in kilograms divided by the square root of height in meters .", "the skeletal muscle mass was estimated by the predictive equation proposed by kim et al.23 the appendicular fat - free mass used in the equation was determined by a dual energy x - ray absorptiometry scan ( lunar prodigy nrl 41990 , ge lunar , madison , wi , usa ) . before scanning , participants were instructed to remove from their person all objects containing metal .", "scans were performed with the subjects lying in the supine position along the table s longitudinal centerline axis .", "feet were taped together at the toes to immobilize the legs , while the hands were maintained in a pronated position within the scanning region .", "the software generated standard lines that set apart the limbs from the trunk and head .", "these lines were adjusted by the same technician using specific anatomical points determined by the manufacturer .", "analyses during the intervention were performed by the same technician who was blinded to intervention time point .", "retest scans resulted in a standard error of measurement of 0.29 kg and intra - class correlation coefficient of 0.997 for skeletal muscle mass .", "to evaluate a subject s flexibility , five joint movements were adopted : cervical flexion ( cf ) , cervical extension ( ce ) , frontal hip flexion ( fhf ) , left hip flexion , and right hip flexion .", "these movements were chosen because the flexibility of the hip and cervical spine are highly important for the elderly , especially for locomotion , eye orientation , and good timeline perception.24 all measurements were obtained by a fleximeter ( code , american do brazil ltda , so paulo , brazil ) with a degree scale .", "all the procedures were made according to procedures and recommendations described elsewhere.25 briefly , in both cf and ce subjects remained lying supine on a stretching table in order to neutralize any possible compensatory movements , and the fleximeter was positioned at the side of the head , in the sagittal plane , starting with cf , where they moved the head slowly , until the chin leaned on the sternum or noticed a rigidity in the final range of motion ; for ce , the subjects moved their head back slowly . for fhf", ", the fleximeter was positioned next to the hip just above the iliac crest , in a standing position with shoulders vertically flexed , elbows extended , fingers intertwined , legs together , then the subjects flexed frontally the hip , with knees extended throughout the movement . for lateral hip flexion , the fleximeter was placed on the medial surface of the thoracic spine , the participant remained standing , with legs together , and knees extended , but with arms crossing the trunk and hands on the contrary shoulder , and then performed the lateral trunk flexion , as a special consideration to such movement , the heel should remain supported on the ground .", "for all moves , after fixing the velcro attached to the fleximeter and setting the zero point , the participants executed the movements as far as they could or until tightness or discomfort in the final range of motion was felt , and at the end range of motion the evaluator recorded the measure  at this point the participants were instructed to remain in the final position until the reading was completed . the highest score obtained from the three measurements at each joint motion", "the information obtained at baseline was not made available to the evaluator at the time of revaluation in an attempt to avoid their unduly influencing the results .", "it is worth emphasizing that the evaluator had over 2 years experience , and based on the test ", "retest , the standard error of measurement and the intra - class correlation coefficient among the movements were 2.26 degrees and 0.950 , respectively , and the maximal technical error among the movements analyzed was 1.19 degrees .", "during the intervention period , in each session , the instructors registered the load ( kg ) for each of the eight exercises for all the subjects . afterwards , training load for each subject was weekly calculated , using the sum of the load employed in the exercises as a reference for each week .", "supervised rt was carried out for 12 weeks during the morning hours , in the londrina state university facilities .", "the protocol was based on recommendations for rt in an older population to improve muscular endurance and strength.12,13 all participants were personally supervised by physical - education professionals with substantial rt experience to help ensure consistent and safe performance .", "g2x performed two rt sessions per week on tuesdays and thursdays , while g3x performed the same exercises in three sessions per week on mondays , wednesdays , and fridays .", "the rt program was a whole - body program with eight exercises comprising one exercise with free weights and seven with machines , performed exactly in the following order : chest press , horizontal leg press , seated row , knee extension , preacher curl ( free weights ) , leg curl , triceps push - down , and seated calf raise .", "participants were instructed to inhale during the eccentric phase and exhale during the concentric phase while maintaining a constant velocity of movement at a ratio of approximately 1:2 seconds ( concentric and eccentric phases , respectively ) .", "instructors adjusted the loads of each exercise according to the subject s ability and improvements in exercise capacity throughout the study in order to ensure that the subjects were exercising with as much resistance as possible while maintaining proper exercise technique .", "progression was planned so that when 15 repetitions were completed for two consecutive training sessions , weight was increased 2%5% for the upper limb exercises and 5%10% for the lower limb exercises in the next session.12 it is important to note that the participants did not perform any type of warm - up or cool - down exercises at the beginning or end of the session , respectively .", "two - way analysis of variance for repeated measures was used for within - group comparisons . in variables where sphericity was violated , as indicated by mauchly s test", ", the analyses were adjusted using a greenhouse  geisser correction . when the f - ratio was significant , bonferroni s post - hoc test was employed to identify the mean differences .", "baseline differences between groups were explored with an independent t - test . the effect size ( es )", "was calculated to verify the magnitude of the differences by cohen s d , where an es of 0.200.40 was considered small , 0.500.79 as moderate , and 0.80 as large.26 for all statistical analyses , significance was accepted at p<0.05 .", "the data were stored and analyzed using statistica software ( v 10.0 ; statsoft inc , tulsa , ok , usa ) .", "a significant main effect for group - by - time interaction ( f=2.53 , p<0.05 ) was observed , in which g3x showed a higher increase than g2x ( g2x=+87.8% , es=7.31 ; g3x=+92.2% , es=10.39 ) .", "the information regarding flexibility indicators pre - and post - training according to group are presented in table 2 . a main effect of time ( p<0.01 )", "was observed for ce ( g2x=+19.1% , g3x=+20.0% ) , right hip flexion ( g2x=+14.6% , g3x=+15.9% ) , and left hip flexion ( g2x=+25.7% , g3x=+19.2% ) .", "significant group - by - time interaction ( p<0.01 ) was observed only for fhf , in which g3x showed a higher increase than g2x ( + 12.8% , and + 3.0% , respectively ) .", "figure 3 shows the absolute ( a ) and relative ( b ) variations from pre- to post - training on skeletal muscle mass indicators according to group .", "only the main effect of time reached statistical significance ( f=22.05 , p<0.001 ) , with both groups having a similar increase after the 12 weeks of the rt program .", "the ess for skeletal muscle mass changes were 0.21 and 0.43 for twice and thrice per week sessions , respectively .", "the main finding of the present study is that 12 weeks of rt was sufficient to increase or at least maintain flexibility in elderly women .", "the increases in flexibility after an rt program in older individuals have been previously reported,11,1619 and our findings are consistent with these studies .", "for example , fatouros et al18 recruited eight older men to perform 16 weeks of rt three times per week , and observed significant range - of - motion increases in knee flexion , elbow flexion , shoulder flexion , hip flexion , shoulder extension , and hip extension at study end .", "gonalves et al19 investigated the effect of 8 weeks of rt three times per week on the flexibility of elderly men and women , and noted an increase in shoulder extension , hip flexion , and hip extension after the intervention .", "barbosa et al16 investigated the effect of 10 weeks of rt three times per week in elderly women and observed an increase in flexibility as measured by the sit - and - reach test after 10 weeks .", "the exact mechanisms responsible for increased flexibility after an rt program have not yet been established in the literature and the outcomes drawn from our study do not provide mechanistic insight .", "joint movement is related to morphological elements such as muscle , bone , and connective tissues .", "in particular , muscle and fascia are responsible for ~41% of a joint s resistance to movement,27 suggesting that an rt - mediated reduction in passive tension and stiffness of these tissues translates into a greater range of motion.11 this hypothesis requires further study .", "the novel and important feature of our study was the comparison of two versus three rt sessions per week with respect to flexibility outcomes .", "results show that only fhf benefited from an increased rt frequency , while all other flexibility outcomes were similar regardless of the number of weekly sessions .", "the fact that hip - flexion range of motion showed greater improvement when training was undertaken three times per week rather than twice per week has implications for exercise prescription .", "hip flexion measures the flexibility of muscles in both the lumbar region and on the posterior thigh .", "these structures are highly relevant to the mobility of the torso and lower back , which has particular implications for functional capacity in the elderly.28 moreover , hip flexibility is a predictor of low back pain , and is strongly associated with aging.29 the muscles of the low back and the hamstrings are considered to be particularly relevant to hip mobility.24 given that es analysis showed the group with higher volume had a greater positive effect on skeletal muscle mass compared with the lower volume group , it is conceivable that the associated increased strength of muscles surrounding the hip may have led to a greater improvements in flexibility .", "an analysis of the weekly  training load  , which refers to the load used to perform a given exercise , was adopted in this study as the muscular strength indicator .", "analysis of the training load is an alternative and potentially more practical method for monitoring muscular strength changes compared with one repetition maximum test.30 our results indicate that a greater rt frequency is associated with higher increments of specific muscle strength in the sum of exercises used ( 88% and 92% , for g2x and g3x , respectively ) . to date ,", "as far as we are aware , only a few studies have investigated the effects of different training frequencies in older people,3133 with conflicting results .", "for example , both difrancisco - donoghue et al32 and taafee et al33 showed similar increases in strength between programs with high ( twice ) and low ( once ) weekly training frequencies .", "alternatively , farinatti et al31 investigated the effects of rt once , twice , and three times per week , and observed that higher frequencies promoted greater increases in strength for certain exercises ( seated dumbbell curl and knee extension ) but not for others ( bench press and standing calf raise ) .", "no statistically significant differences in changes in skeletal muscle mass were noted between training two versus three times a week in the present study .", "however , thrice - weekly training showed greater absolute increases compared with the twice - weekly condition ( 7.4% vs 4.4% , respectively ) and the ess were considerably larger when training three times a week as well ( 0.43 versus 0.21 , respectively ) .", ", g3x performed three sets per muscle per week while the g2x group performed two sets per muscle per week .", "thus , our findings suggest a potentially meaningful benefit to increasing the volume of training for a given muscle from two to three sets per week with respect to skeletal muscle mass . considering the relatively short duration of our study , future research", "is recommended to determine the extent to which such differences in skeletal muscle mass would continue or perhaps narrow over time .", "the data found in our study are limited to the joint movements analyzed and time of training applied .", "it is possible that the range of motion of these joints might continue to increase if training were to exceed 12 weeks , and the results should not be extrapolated to populations other than older women .", "moreover , the absence of monitoring the physical activity and dietary intake habits is a limitation as well . on the other hand , to our knowledge", ", the present study is the first to have investigated the effect of different rt frequencies on flexibility in older women .", "our findings indicate that rt performed at a minimum of twice per week can result in positive effects on flexibility in the elderly , reversing or slowing the aging - induced losses .", "the results of our study suggest that 12 weeks of rt improves or at least preserves the flexibility of different joint movements in older women . in addition"], "abstract_text": ["<S> objectivethe main purpose of the investigation reported here was to analyze the effect of resistance training ( rt ) performed at different weekly frequencies on flexibility in older women.participants and methodsfifty - three older women ( 60 years old ) were randomly assigned to perform rt either two ( n=28 ; group  g2x  ) , or three ( n=25 ; group  </S>", "<S> g3x  ) times per week . </S>", "<S> the rt program comprised eight exercises in which the participants performed one set of 1015 repetitions maximum for a period of 12 weeks . </S>", "<S> anthropometric , body - composition , and flexibility measurements were made at baseline and post - study . </S>", "<S> the flexibility measurements were obtained by a fleximeter.resultsa significant group - by - time interaction ( p<0.01 ) was observed for frontal hip flexion , in which g3x showed a higher increase than g2x ( + 12.8% and + 3.0% , respectively ) . </S>", "<S> both groups increased flexibility in cervical extension ( g2x=+19.1% , g3x=+20.0% ) , right hip flexion ( g2x=+14.6% , g3x=+15.9% ) , and left hip flexion ( g2x=+25.7% , g3x=+19.2% ) , with no statistical difference between groups . </S>", "<S> no statistically significant differences were noted for the increase in skeletal muscle mass between training three versus two times a week ( + 7.4% vs + 4.4% , respectively).conclusiontwelve weeks of rt improves the flexibility of different joint movements in older women , and the higher frequency induces greater increases for frontal hip flexion . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Participants", "Experimental design", "Anthropometry", "Body composition", "Flexibility measurements", "Training load", "Resistance-training program", "Statistical analyses", "Results", "Discussion", "Conclusion"], "sections": [["aging is associated with changes in various body systems including in the neuromuscular structures , which lead to a reduction in important components of physical fitness.1 the preservation of fitness - related components such as muscular strength , endurance , and flexibility are essential to the performance of daily activities , which thus helps to maintain autonomy and quality of life in the elderly population.25 of these fitness - related components , flexibility is often less appreciated with respect to its contribution to optimal health and functional status as well as independent living for the elderly.3,6 flexibility reductions may increase the risks of injury , falling , back pain , and physical dependence in older adults.7 several mechanisms have been proposed for reductions in flexibility , among them alterations in soft - tissue structures and physical inactivity.7 when a joint is relatively inactive due to sedentary behavior , the muscles that cross it shorten , thereby reducing its range of motion . moreover ,", "independent of activity levels , the aging process plays a role in flexibility decreases.8 joint structures such cartilage , ligaments , and tendons change mechanically and biochemically with aging , increasing muscular and tendon stiffness and thus impeding mobility.911 on the other hand , maintaining a physically active lifestyle may result in improvement of functional performance with advancing age , thus enabling the execution of activities of daily living with more vigor and less fatigue.12,13 while resistance training ( rt ) is recommended for older people for the development of muscular strength,13,14 some studies have indicated that regular participation in rt programs may also contribute to increased flexibility.11,1519 in fact , there is evidence that regular rt serves as an active form of flexibility training and can improve range of motion to a similar extent as typical static stretching protocols.20 regular performance of rt may improve flexibility by reducing passive tension and stiffness of the tissues surrounding a joint.11 thus , from a time - saving standpoint , rt is a good way to develop both strength and flexibility as well as achieve improvements in body composition within a single session of training .", "the fitness - related benefits associated with rt are dependent on the manipulation of variables such as intensity , volume , exercise order , and rest intervals.12 with respect to training volume , manipulation can involve varying the number of repetitions , sets , and/or  frequency   defined here as the number of sessions performed per week .", "the american college of sports medicine recommends that older adults engage in two to three rt sessions a week for improving neuromuscular fitness.12 however , there is a current paucity of research regarding the effects of different rt weekly frequencies on flexibility adaptive responses .", "previous studies are limited to analyzing the impact of rt on flexibility without regard for manipulation of the variables that make up the training program . considering the importance of flexibility in the health and wellness of the elderly", "therefore , the main purpose of this investigation was to analyze the effect of rt performed at different weekly frequencies on flexibility in untrained older women .", "we hypothesized that higher rt frequencies would result in greater flexibility increases than lower frequencies . the rational for our hypothesis", "response relationship between training volume and muscle strength and hypertrophy,21,22 whereby greater training volumes are associated with enhanced muscular adaptations .", "a secondary aim of the study was to assess rt frequency - related changes in skeletal muscle mass .", "in accordance with the aforementioned dose  response relationship , it was hypothesized that muscle protein accretion would be greater with the higher frequency program ."], ["recruitment of the participants was carried out through newspaper and radio advertisements and home delivery of flyers in londrina , paran , brazil .", "a total of 350 older women responded to the advertisements , and then completed detailed health history and physical activity questionnaires .", "the women were subsequently admitted to the study if they met the following inclusion criteria : non - hypertensive ( systolic blood pressure < 140 mmhg and diastolic blood pressure < 90 mmhg ) , nondiabetic , free from cardiac or renal dysfunction , nonsmoking , not receiving hormonal replacement therapy , not performing any regular physical exercise more than once per week over the preceding 6 months , and participated in > 85% of the study training sessions .", "the remaining 64 older women ( 60 years old ) were randomly assigned to one of two groups : a group that performed rt two times per week ( n=32 ; group ", "g2x  ) or a group that performed rt three times per week ( n=32 ; group  g3x  ) . a total of 53 women ( g2x=28 ; g3x=25 ) completed the study and therefore were included in the analysis .", "participants passed a diagnostic , graded exercise stress test with twelve - lead electrocardiography reviewed by a cardiologist and were released with no restrictions for participation in this study .", "this investigation was conducted according to the declaration of helsinki , and was approved by the londrina state university ethics committee .", "the study was carried out over a period of 16 weeks , with 12 weeks dedicated to the rt program and 4 weeks used for testing .", "anthropometric , body - composition , and flexibility measurements were performed at weeks 12 and 1516 for baseline and post - training analysis , respectively , while the supervised rt program was performed during weeks 314 .", "subjects were instructed not to perform any other type of physical exercise during the entire study period .", "body mass was measured to the nearest 0.1 kg using a calibrated electronic scale ( balmak scale , model iii , so paulo , brazil ) , with the participants wearing light workout clothing and no shoes .", "height was measured with a stadiometer attached on the scale to the nearest 0.1 cm with subjects standing without shoes .", "body mass index was calculated as body mass in kilograms divided by the square root of height in meters .", "the skeletal muscle mass was estimated by the predictive equation proposed by kim et al.23 the appendicular fat - free mass used in the equation was determined by a dual energy x - ray absorptiometry scan ( lunar prodigy nrl 41990 , ge lunar , madison , wi , usa ) . before scanning , participants were instructed to remove from their person all objects containing metal .", "scans were performed with the subjects lying in the supine position along the table s longitudinal centerline axis .", "feet were taped together at the toes to immobilize the legs , while the hands were maintained in a pronated position within the scanning region .", "the software generated standard lines that set apart the limbs from the trunk and head .", "these lines were adjusted by the same technician using specific anatomical points determined by the manufacturer .", "analyses during the intervention were performed by the same technician who was blinded to intervention time point .", "retest scans resulted in a standard error of measurement of 0.29 kg and intra - class correlation coefficient of 0.997 for skeletal muscle mass . to evaluate a subject s flexibility ,", "five joint movements were adopted : cervical flexion ( cf ) , cervical extension ( ce ) , frontal hip flexion ( fhf ) , left hip flexion , and right hip flexion .", "these movements were chosen because the flexibility of the hip and cervical spine are highly important for the elderly , especially for locomotion , eye orientation , and good timeline perception.24 all measurements were obtained by a fleximeter ( code , american do brazil ltda , so paulo , brazil ) with a degree scale .", "all the procedures were made according to procedures and recommendations described elsewhere.25 briefly , in both cf and ce subjects remained lying supine on a stretching table in order to neutralize any possible compensatory movements , and the fleximeter was positioned at the side of the head , in the sagittal plane , starting with cf , where they moved the head slowly , until the chin leaned on the sternum or noticed a rigidity in the final range of motion ; for ce , the subjects moved their head back slowly . for fhf ,", "the fleximeter was positioned next to the hip just above the iliac crest , in a standing position with shoulders vertically flexed , elbows extended , fingers intertwined , legs together , then the subjects flexed frontally the hip , with knees extended throughout the movement . for lateral hip flexion , the fleximeter was placed on the medial surface of the thoracic spine , the participant remained standing , with legs together , and knees extended , but with arms crossing the trunk and hands on the contrary shoulder , and then performed the lateral trunk flexion , as a special consideration to such movement , the heel should remain supported on the ground . for all moves , after fixing the velcro attached to the fleximeter and setting the zero point , the participants executed the movements as far as they could or until tightness or discomfort in the final range of motion was felt , and at the end range of motion the evaluator recorded the measure  at this point the participants were instructed to remain in the final position until the reading was completed .", "the highest score obtained from the three measurements at each joint motion was adopted as a reference standard .", "the information obtained at baseline was not made available to the evaluator at the time of revaluation in an attempt to avoid their unduly influencing the results .", "it is worth emphasizing that the evaluator had over 2 years experience , and based on the test ", "retest , the standard error of measurement and the intra - class correlation coefficient among the movements were 2.26 degrees and 0.950 , respectively , and the maximal technical error among the movements analyzed was 1.19 degrees . during the intervention period , in each session , the instructors registered the load ( kg ) for each of the eight exercises for all the subjects .", "afterwards , training load for each subject was weekly calculated , using the sum of the load employed in the exercises as a reference for each week .", "supervised rt was carried out for 12 weeks during the morning hours , in the londrina state university facilities .", "the protocol was based on recommendations for rt in an older population to improve muscular endurance and strength.12,13 all participants were personally supervised by physical - education professionals with substantial rt experience to help ensure consistent and safe performance .", "g2x performed two rt sessions per week on tuesdays and thursdays , while g3x performed the same exercises in three sessions per week on mondays , wednesdays , and fridays .", "the rt program was a whole - body program with eight exercises comprising one exercise with free weights and seven with machines , performed exactly in the following order : chest press , horizontal leg press , seated row , knee extension , preacher curl ( free weights ) , leg curl , triceps push - down , and seated calf raise .", "participants were instructed to inhale during the eccentric phase and exhale during the concentric phase while maintaining a constant velocity of movement at a ratio of approximately 1:2 seconds ( concentric and eccentric phases , respectively ) .", "instructors adjusted the loads of each exercise according to the subject s ability and improvements in exercise capacity throughout the study in order to ensure that the subjects were exercising with as much resistance as possible while maintaining proper exercise technique .", "progression was planned so that when 15 repetitions were completed for two consecutive training sessions , weight was increased 2%5% for the upper limb exercises and 5%10% for the lower limb exercises in the next session.12 it is important to note that the participants did not perform any type of warm - up or cool - down exercises at the beginning or end of the session , respectively .", "two - way analysis of variance for repeated measures was used for within - group comparisons . in variables where sphericity was violated , as indicated by mauchly s test", ", the analyses were adjusted using a greenhouse  geisser correction . when the f - ratio was significant , bonferroni s post - hoc test was employed to identify the mean differences .", "baseline differences between groups were explored with an independent t - test . the effect size ( es )", "was calculated to verify the magnitude of the differences by cohen s d , where an es of 0.200.40 was considered small , 0.500.79 as moderate , and 0.80 as large.26 for all statistical analyses , significance was accepted at p<0.05 .", "the data were stored and analyzed using statistica software ( v 10.0 ; statsoft inc , tulsa , ok , usa ) ."], ["recruitment of the participants was carried out through newspaper and radio advertisements and home delivery of flyers in londrina , paran , brazil .", "a total of 350 older women responded to the advertisements , and then completed detailed health history and physical activity questionnaires .", "the women were subsequently admitted to the study if they met the following inclusion criteria : non - hypertensive ( systolic blood pressure < 140 mmhg and diastolic blood pressure < 90 mmhg ) , nondiabetic , free from cardiac or renal dysfunction , nonsmoking , not receiving hormonal replacement therapy , not performing any regular physical exercise more than once per week over the preceding 6 months , and participated in > 85% of the study training sessions .", "the remaining 64 older women ( 60 years old ) were randomly assigned to one of two groups : a group that performed rt two times per week ( n=32 ; group ", "g2x  ) or a group that performed rt three times per week ( n=32 ; group  g3x  ) . a total of 53 women ( g2x=28 ; g3x=25 ) completed the study and therefore were included in the analysis .", "participants passed a diagnostic , graded exercise stress test with twelve - lead electrocardiography reviewed by a cardiologist and were released with no restrictions for participation in this study .", "this investigation was conducted according to the declaration of helsinki , and was approved by the londrina state university ethics committee ."], ["the study was carried out over a period of 16 weeks , with 12 weeks dedicated to the rt program and 4 weeks used for testing .", "anthropometric , body - composition , and flexibility measurements were performed at weeks 12 and 1516 for baseline and post - training analysis , respectively , while the supervised rt program was performed during weeks 314 .", "subjects were instructed not to perform any other type of physical exercise during the entire study period ."], ["body mass was measured to the nearest 0.1 kg using a calibrated electronic scale ( balmak scale , model iii , so paulo , brazil ) , with the participants wearing light workout clothing and no shoes .", "height was measured with a stadiometer attached on the scale to the nearest 0.1 cm with subjects standing without shoes .", "body mass index was calculated as body mass in kilograms divided by the square root of height in meters ."], ["the skeletal muscle mass was estimated by the predictive equation proposed by kim et al.23 the appendicular fat - free mass used in the equation was determined by a dual energy x - ray absorptiometry scan ( lunar prodigy nrl 41990 , ge lunar , madison , wi , usa ) . before scanning , participants were instructed to remove from their person all objects containing metal .", "scans were performed with the subjects lying in the supine position along the table s longitudinal centerline axis .", "feet were taped together at the toes to immobilize the legs , while the hands were maintained in a pronated position within the scanning region .", "the software generated standard lines that set apart the limbs from the trunk and head .", "these lines were adjusted by the same technician using specific anatomical points determined by the manufacturer .", "analyses during the intervention were performed by the same technician who was blinded to intervention time point .", "retest scans resulted in a standard error of measurement of 0.29 kg and intra - class correlation coefficient of 0.997 for skeletal muscle mass ."], ["to evaluate a subject s flexibility , five joint movements were adopted : cervical flexion ( cf ) , cervical extension ( ce ) , frontal hip flexion ( fhf ) , left hip flexion , and right hip flexion .", "these movements were chosen because the flexibility of the hip and cervical spine are highly important for the elderly , especially for locomotion , eye orientation , and good timeline perception.24 all measurements were obtained by a fleximeter ( code , american do brazil ltda , so paulo , brazil ) with a degree scale .", "all the procedures were made according to procedures and recommendations described elsewhere.25 briefly , in both cf and ce subjects remained lying supine on a stretching table in order to neutralize any possible compensatory movements , and the fleximeter was positioned at the side of the head , in the sagittal plane , starting with cf , where they moved the head slowly , until the chin leaned on the sternum or noticed a rigidity in the final range of motion ; for ce , the subjects moved their head back slowly . for fhf", ", the fleximeter was positioned next to the hip just above the iliac crest , in a standing position with shoulders vertically flexed , elbows extended , fingers intertwined , legs together , then the subjects flexed frontally the hip , with knees extended throughout the movement . for lateral hip flexion , the fleximeter was placed on the medial surface of the thoracic spine , the participant remained standing , with legs together , and knees extended , but with arms crossing the trunk and hands on the contrary shoulder , and then performed the lateral trunk flexion , as a special consideration to such movement , the heel should remain supported on the ground .", "for all moves , after fixing the velcro attached to the fleximeter and setting the zero point , the participants executed the movements as far as they could or until tightness or discomfort in the final range of motion was felt , and at the end range of motion the evaluator recorded the measure  at this point the participants were instructed to remain in the final position until the reading was completed . the highest score obtained from the three measurements at each joint motion", "the information obtained at baseline was not made available to the evaluator at the time of revaluation in an attempt to avoid their unduly influencing the results .", "it is worth emphasizing that the evaluator had over 2 years experience , and based on the test ", "retest , the standard error of measurement and the intra - class correlation coefficient among the movements were 2.26 degrees and 0.950 , respectively , and the maximal technical error among the movements analyzed was 1.19 degrees ."], ["during the intervention period , in each session , the instructors registered the load ( kg ) for each of the eight exercises for all the subjects . afterwards , training load for each subject was weekly calculated , using the sum of the load employed in the exercises as a reference for each week ."], ["supervised rt was carried out for 12 weeks during the morning hours , in the londrina state university facilities .", "the protocol was based on recommendations for rt in an older population to improve muscular endurance and strength.12,13 all participants were personally supervised by physical - education professionals with substantial rt experience to help ensure consistent and safe performance .", "g2x performed two rt sessions per week on tuesdays and thursdays , while g3x performed the same exercises in three sessions per week on mondays , wednesdays , and fridays .", "the rt program was a whole - body program with eight exercises comprising one exercise with free weights and seven with machines , performed exactly in the following order : chest press , horizontal leg press , seated row , knee extension , preacher curl ( free weights ) , leg curl , triceps push - down , and seated calf raise .", "participants were instructed to inhale during the eccentric phase and exhale during the concentric phase while maintaining a constant velocity of movement at a ratio of approximately 1:2 seconds ( concentric and eccentric phases , respectively ) .", "instructors adjusted the loads of each exercise according to the subject s ability and improvements in exercise capacity throughout the study in order to ensure that the subjects were exercising with as much resistance as possible while maintaining proper exercise technique .", "progression was planned so that when 15 repetitions were completed for two consecutive training sessions , weight was increased 2%5% for the upper limb exercises and 5%10% for the lower limb exercises in the next session.12 it is important to note that the participants did not perform any type of warm - up or cool - down exercises at the beginning or end of the session , respectively ."], ["two - way analysis of variance for repeated measures was used for within - group comparisons . in variables where sphericity was violated , as indicated by mauchly s test", ", the analyses were adjusted using a greenhouse  geisser correction . when the f - ratio was significant , bonferroni s post - hoc test was employed to identify the mean differences .", "baseline differences between groups were explored with an independent t - test . the effect size ( es )", "was calculated to verify the magnitude of the differences by cohen s d , where an es of 0.200.40 was considered small , 0.500.79 as moderate , and 0.80 as large.26 for all statistical analyses , significance was accepted at p<0.05 .", "the data were stored and analyzed using statistica software ( v 10.0 ; statsoft inc , tulsa , ok , usa ) ."], ["a significant main effect for group - by - time interaction ( f=2.53 , p<0.05 ) was observed , in which g3x showed a higher increase than g2x ( g2x=+87.8% , es=7.31 ; g3x=+92.2% , es=10.39 ) .", "the information regarding flexibility indicators pre - and post - training according to group are presented in table 2 . a main effect of time ( p<0.01 )", "was observed for ce ( g2x=+19.1% , g3x=+20.0% ) , right hip flexion ( g2x=+14.6% , g3x=+15.9% ) , and left hip flexion ( g2x=+25.7% , g3x=+19.2% ) .", "significant group - by - time interaction ( p<0.01 ) was observed only for fhf , in which g3x showed a higher increase than g2x ( + 12.8% , and + 3.0% , respectively ) .", "figure 3 shows the absolute ( a ) and relative ( b ) variations from pre- to post - training on skeletal muscle mass indicators according to group .", "only the main effect of time reached statistical significance ( f=22.05 , p<0.001 ) , with both groups having a similar increase after the 12 weeks of the rt program .", "the ess for skeletal muscle mass changes were 0.21 and 0.43 for twice and thrice per week sessions , respectively ."], ["the main finding of the present study is that 12 weeks of rt was sufficient to increase or at least maintain flexibility in elderly women .", "the increases in flexibility after an rt program in older individuals have been previously reported,11,1619 and our findings are consistent with these studies .", "for example , fatouros et al18 recruited eight older men to perform 16 weeks of rt three times per week , and observed significant range - of - motion increases in knee flexion , elbow flexion , shoulder flexion , hip flexion , shoulder extension , and hip extension at study end .", "gonalves et al19 investigated the effect of 8 weeks of rt three times per week on the flexibility of elderly men and women , and noted an increase in shoulder extension , hip flexion , and hip extension after the intervention .", "barbosa et al16 investigated the effect of 10 weeks of rt three times per week in elderly women and observed an increase in flexibility as measured by the sit - and - reach test after 10 weeks .", "the exact mechanisms responsible for increased flexibility after an rt program have not yet been established in the literature and the outcomes drawn from our study do not provide mechanistic insight .", "joint movement is related to morphological elements such as muscle , bone , and connective tissues .", "in particular , muscle and fascia are responsible for ~41% of a joint s resistance to movement,27 suggesting that an rt - mediated reduction in passive tension and stiffness of these tissues translates into a greater range of motion.11 this hypothesis requires further study .", "the novel and important feature of our study was the comparison of two versus three rt sessions per week with respect to flexibility outcomes .", "results show that only fhf benefited from an increased rt frequency , while all other flexibility outcomes were similar regardless of the number of weekly sessions .", "the fact that hip - flexion range of motion showed greater improvement when training was undertaken three times per week rather than twice per week has implications for exercise prescription .", "hip flexion measures the flexibility of muscles in both the lumbar region and on the posterior thigh .", "these structures are highly relevant to the mobility of the torso and lower back , which has particular implications for functional capacity in the elderly.28 moreover , hip flexibility is a predictor of low back pain , and is strongly associated with aging.29 the muscles of the low back and the hamstrings are considered to be particularly relevant to hip mobility.24 given that es analysis showed the group with higher volume had a greater positive effect on skeletal muscle mass compared with the lower volume group , it is conceivable that the associated increased strength of muscles surrounding the hip may have led to a greater improvements in flexibility .", "an analysis of the weekly  training load  , which refers to the load used to perform a given exercise , was adopted in this study as the muscular strength indicator .", "analysis of the training load is an alternative and potentially more practical method for monitoring muscular strength changes compared with one repetition maximum test.30 our results indicate that a greater rt frequency is associated with higher increments of specific muscle strength in the sum of exercises used ( 88% and 92% , for g2x and g3x , respectively ) . to date ,", "as far as we are aware , only a few studies have investigated the effects of different training frequencies in older people,3133 with conflicting results .", "for example , both difrancisco - donoghue et al32 and taafee et al33 showed similar increases in strength between programs with high ( twice ) and low ( once ) weekly training frequencies .", "alternatively , farinatti et al31 investigated the effects of rt once , twice , and three times per week , and observed that higher frequencies promoted greater increases in strength for certain exercises ( seated dumbbell curl and knee extension ) but not for others ( bench press and standing calf raise ) .", "no statistically significant differences in changes in skeletal muscle mass were noted between training two versus three times a week in the present study .", "however , thrice - weekly training showed greater absolute increases compared with the twice - weekly condition ( 7.4% vs 4.4% , respectively ) and the ess were considerably larger when training three times a week as well ( 0.43 versus 0.21 , respectively ) .", ", g3x performed three sets per muscle per week while the g2x group performed two sets per muscle per week .", "thus , our findings suggest a potentially meaningful benefit to increasing the volume of training for a given muscle from two to three sets per week with respect to skeletal muscle mass . considering the relatively short duration of our study , future research", "is recommended to determine the extent to which such differences in skeletal muscle mass would continue or perhaps narrow over time .", "the data found in our study are limited to the joint movements analyzed and time of training applied .", "it is possible that the range of motion of these joints might continue to increase if training were to exceed 12 weeks , and the results should not be extrapolated to populations other than older women .", "moreover , the absence of monitoring the physical activity and dietary intake habits is a limitation as well . on the other hand , to our knowledge", ", the present study is the first to have investigated the effect of different rt frequencies on flexibility in older women .", "our findings indicate that rt performed at a minimum of twice per week can result in positive effects on flexibility in the elderly , reversing or slowing the aging - induced losses ."], ["the results of our study suggest that 12 weeks of rt improves or at least preserves the flexibility of different joint movements in older women . in addition"]]}
{"article_id": "PMC5283081", "article_text": ["bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death .", "the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed .", "the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution .", "we divided the zonular fibers into two groups : the  equatorial zonule  ( blue fibers in fig .", "other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule", "were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens .", "because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues .", "once the equatorial fibers had been removed , the  hyaloid zonule  was exposed .", "the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous .", "they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face", ", it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor .", "samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor ,", "a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor .", "human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample .", "thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) .", "thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) .", "human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax .", "sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence .", "deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) .", "sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody .", "the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3  10 minutes )", ", sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) .", "sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) .", "bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each .", "samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c .", "five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard .", "five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) .", "the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al .", "briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate .", "a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c .", "peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate .", "the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation .", "bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine .", "samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark .", "an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin .", "digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction .", "peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration .", "samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed .", "the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m  2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid .", "after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m  25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c .", "peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min .", "survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2  10 .", "the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% .", "top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans .", "dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 .", ", is provided as supplementary materials s1 . the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .", "an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .", "ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .", "databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .", "common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .", "the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .", "the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .", "searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .", "2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .", "other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .", "the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .", "comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .", "target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .", "protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .", "protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles", "these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting . to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded .", "it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins .", "comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot .", "a fasta database file of all human ecm proteins was constructed . for both human and bovine samples ,", "the lists of identified proteins were used to extract separate fasta database files for identified proteins .", "an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins .", "analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins .", "common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis .", "extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample .", "the total abundances of putative zonule proteins ( using ms2 intensity  weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined .", "average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes .", "this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples", ", the ms2 intensity  weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together .", "full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 , s5 ) for each experiment are included .", "bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death .", "the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed .", "the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution .", "we divided the zonular fibers into two groups : the  equatorial zonule  ( blue fibers in fig .", "other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule", "were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens .", "because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues .", "once the equatorial fibers had been removed , the  hyaloid zonule  was exposed .", "the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous .", "they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face", ", it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor .", "samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor ,", "a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor .", "human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample .", "thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) .", "thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) .", "human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax . sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence .", "deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) .", "sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody .", "the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3  10 minutes )", ", sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) .", "sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) .", "bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each .", "samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c .", "five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard .", "five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) .", "the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al .", "briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate . a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate", "was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c .", "peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate .", "the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation .", "bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine .", "samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark .", "an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin .", "digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction .", "peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration .", "samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed .", "the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m  2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid .", "after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m  25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c .", "peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min .", "survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2  10 .", "the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% .", "top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans .", "dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 .", "the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .", "an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .", "ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .", "databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .", "common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .", "the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .", "the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .", "searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .", "2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .", "other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .", "the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .", "comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .", "target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .", "protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .", "protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles", "these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting .", "the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .", "an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .", "ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .", "databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .", "common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .", "the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .", "the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .", "searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .", "2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .", "other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .", "the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .", "comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .", "target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .", "protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .", "protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles", "these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting .", "to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded .", "it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins .", "comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot .", "a fasta database file of all human ecm proteins was constructed . for both human and bovine samples ,", "the lists of identified proteins were used to extract separate fasta database files for identified proteins .", "an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins .", "analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins . once matrisome proteins were identified", "common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis .", "extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample .", "the total abundances of putative zonule proteins ( using ms2 intensity  weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined .", "average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes .", "this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples", ", the ms2 intensity  weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together .", "relative abundances within each sample by gene were computed as described above . full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 ,", "we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) .", "for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) .", "our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins .", "liquid chromatography  ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) .", "3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species .", "two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins .", "the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans .", "the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors .", "of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs .", "however , when the data were weighted according to protein abundance , based on ms2 intensity  weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) .", "the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 .", "the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) .", "overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome .", "however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) .", "shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule .", "we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig .", ", the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig .", "the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs .", "fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) .", "emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) .", "fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) .", "all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig .", "the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome .", "fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively .", "all family members are present ( with the exception of ltbp4 in the human zonule ) .", "the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins .", "mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients .", "mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms .", "together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule .", "ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm .", "both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) .", "curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved .", "another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family .", "adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae .", "adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 .", "unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) .", "we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig .", "adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig .", "in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm .", "immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig .", "a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig .", "the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells .", "work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds .", "we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils .", "several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig .", "transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule .", "the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome .", "exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma .", "given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule .", "one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 .", "the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils .", "expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule .", "the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) .", "only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 .", "col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium .", "megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant .", "immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule .", "this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals .", "similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes .", "cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions .", "proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels .", "unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 .", "fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample .", "recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous .", "comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs .", "proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor .", "protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) .", "some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous", "three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous .", "note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule .", "the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous .", "if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values .", "although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule .", "such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample .", "the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig .", "histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 .", "we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) .", "for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) .", "our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins .", "liquid chromatography  ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) .", "3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species .", "two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins .", "the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans .", "the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors .", "of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs .", "however , when the data were weighted according to protein abundance , based on ms2 intensity  weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) .", "the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 .", "the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) .", "overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome .", "however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) .", "shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule .", "we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig .", ", the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig .", "the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs .", "fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) .", "emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) .", "fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) .", "all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig .", "the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome .", "fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively .", "all family members are present ( with the exception of ltbp4 in the human zonule ) .", "the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins .", "mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients .", "mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms .", "together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule .", "ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm .", "both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) .", "curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved .", "another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family .", "adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae .", "adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 .", "unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) .", "we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig .", "adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig .", "in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm .", "immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig .", "a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig .", "the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells .", "work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds .", "we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils .", "several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig .", "transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule .", "the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome .", "exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma .", "given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule .", "one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 .", "the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils .", "expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule .", "the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) .", "only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 .", "col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium .", "megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant .", "immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule .", "this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals .", "similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes .", "cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions .", "proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels .", "unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 .", "fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample .", "recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous . comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs .", "proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor .", "protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) .", "some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous .", "three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous .", "note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule .", "the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous .", "if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values .", "although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule .", "such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample .", "the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig .", "histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 .", "the ciliary zonule plays a key role in ocular function , centering the lens on the optical axis and transmitting the forces that mold its shape during accommodation .", "the zonule is adversely affected in several pathologic conditions , including exfoliation syndrome , homocystinuria , and marfan syndrome . to understand how and why the zonule is compromised in these diseases , a detailed analysis of its composition", "microfibrils ( the main structural elements of the zonule ) are ubiquitous components of the ecm but questions remain regarding their organization , synthesis , and role as signaling hubs .", "the ciliary zonule , a readily accessible , cell - free system composed of bundled microfibrils , represents a useful model system in which to gain insights into microfibril organization .", "the composition of the equatorial zonule was broadly similar in humans and cows , and the set of 52 proteins identified in both samples accounted for > 95% of the mass in either case .", "the fact that the human samples were from middle - aged individuals , whereas the bovine samples were from young animals , might in part explain why more proteins were identified in the bovine zonulome ( 176 vs. 144 ) .", "the bovine samples were also obtained shortly after death , whereas the human samples were obtained 2448 hours postmortem .", "it is possible that labile proteins were lost from the human samples during the postmortem period . in both humans and cows , the zonule consisted of a microfibril backbone comprised primarily of fibrillin and a relatively restricted set of glycoproteins .", "ltbp2 , a fibrillin - like protein implicated in autosomal recessive weill - marchesani syndrome type 3 ( wms3 : mim#614819 ) , was a surprisingly abundant component , comprising 10% of the total zonular protein .", "there are three fibrillin genes in humans and the presence of all three proteins was confirmed in the zonulome , consistent with earlier observations .", "fbn2 , the second most abundant fibrillin in the zonule , is synthesized early in development and is essential for normal zonule formation in mice .", "immunolocalization studies have previously failed to detect fbn2 in the adult zonule , but its presence in the human and bovine zonulome suggests that fbn2 epitopes may become inaccessible to antibody staining during postnatal development , as reported in other systems .", "several proteins implicated in tgf signaling were detected in the zonule . despite its name , latent tgf binding protein 2 ( ltbp2 ) is not a bona fide tgf-binding protein , but two other family members , ltbp1 and ltbp3 , were detected in the zonulome .", "in other systems , ltbp1 and ltbp3 facilitate the sequestration ( and release ) of tgf in the ecm through interaction with fbn1 and other components . of note", "another protein with a potential role in modulating tgf signaling was emilin-1 , a prominent component of the human and bovine zonulome .", "emilin-1 has been shown to inhibit tgf signaling by binding to pro - tgf precursor and preventing its furin - mediated maturation .", "emilins were originally identified as elastic fiber components located at the interface between microfibrils and the underlying elastin core .", "more recent studies have suggested that they associate directly with microfibrils . because zonular fibers insert into the lens capsule directly above the lens germinative zone ( the region of the lens epithelium containing the mitotically active cell population ) , tgf or other growth factors sequestered in the zonule are well placed to influence the growth of the lens directly .", "it is also possible that the cyclical stretching of the zonule during accommodation might serve to release bound factors from the surface of zonular fibers .", "one of the novel ( and abundant ) proteins detected in our study was adamtsl-6 ( thsd4 ) .", "adamts proteins and adamtsl proteins have well - documented roles in ocular biology and pathology .", "adamtsl-6 has not been identified previously in the zonule but , in other systems , is known to bind to the n terminus of fbn1 , where it may promote microfibril formation . among the proteins that we expected to detect in the zonulome , but did not , was adamts10 ; adamts10 mutations underlie weill - marchesani syndrome type 1 ( wms1 ) .", "ectopia lentis is commonly observed in wms1 , implying a mechanical defect in the ciliary zonule .", "clinically , wms1 is indistinguishable from wms2 and wms3 , syndromes caused by mutations in fbn1 and ltbp2 respectively , genes encoding two of the most abundant zonular proteins .", "immunoelectron microscopy studies have previously suggested that adamts10 is abundant in the human ciliary zonule so its absence from the proteome is perplexing . a striking feature of the zonulome in human and cows was the presence of a diverse set of protease inhibitors , the most abundant in humans being timp3 .", "the zonule is known to be exquisitely sensitive to proteolysis . before the introduction of modern cataract surgery", ", ophthalmologists removed the cataractous lens in a single piece , a procedure facilitated by brief exposure to dilute solutions of -chymotrypsin , which served to lyse the zonule and release the lens from the eye .", "the presence of timp3 and other protease inhibitors may help explain the evident durability of the zonule , despite lifelong exposure to matrix metalloproteinases and other proteases present in the ocular humors .", "the hyaloid zonule is intimately associated with the anterior face of the vitreous . during ophthalmic surgery ,", "the vitreous face behaves , physically , as if it were a distinct membrane ( the  hyaloid membrane  ) .", "ophthalmologists take great care not to damage the hyaloid membrane during surgical procedures , lest the contents of the vitreous leak .", "the current study may provide some clues as to the nature of the hyaloid membrane .", "first , it is apparent that the hyaloid membrane is reinforced by the presence of a meshwork of fibrillin - rich microfibrils , the hyaloid zonule . during dissection of the hyaloid zonule", "it is likely that proteins from the anterior - most region of the vitreous were coisolated .", "the hyaloid zonule sample was characterized by the presence of high concentrations of opticin , consistent with previous immunolocalization studies of opticin in human eyes .", "our analysis also suggested that the hyaloid sample was enriched in fibrillar collagens ( especially col2a1 , col5a2 , col5a3 , and col11a1 ) , which were detected elsewhere in the vitreous but at much lower concentrations .", "interestingly , mutations in col2a1 and col11a1 result in stickler syndrome , a condition characterized by vitreous pathology . in stickler syndrome type 1 ( caused by col2a1 mutations ) , a  membranous vitreous  is often observed ; whereas in stickler syndrome type 2 ( col11a1 ) a  beaded vitreous  is observed . in each case the anterior face of the vitreous appears to be compromised .", "thus , the phenotypes in stickler syndrome are consistent with a model in which fibrillar collagens form part of a meshwork ( along with fibrillin - rich microfibrils and opticin ) that defines and supports the vitreous face .", "one question arising from the current study is why such an unassuming structure as the ciliary zonule should require a proteome with fifty or more components ?", "in other settings , it is evident that microfibril scaffolds , decorated with latent growth factors , serve as both mechanosensors and signaling hubs .", "the pleitropic ocular effects of mutations in zonular genes ( which include congenital glaucoma , cataract , ectopia lentis , and microspherophakia ) are consistent with the notion that the role of the ciliary zonule is not solely to secure the lens in place ."], "abstract_text": ["<S> purposethe zonule of zinn ( ciliary zonule ) is a system of fibers that centers the crystalline lens on the optical axis of the eye . </S>", "<S> mutations in zonule components underlie syndromic conditions associated with a broad range of ocular pathologies , including microspherophakia and ectopia lentis . here </S>", "<S> , we used hplc  mass spectrometry to determine the molecular composition of the zonule.methodstryptic digests of human and bovine zonular samples were analyzed by hplc  mass spectrometry . </S>", "<S> the distribution of selected components was confirmed by immunofluorescence confocal microscopy . in bovine samples , </S>", "<S> the composition of the equatorial zonule was compared to that of the hyaloid zonule and vitreous humor.resultsthe 52 proteins common to the zonules of both species accounted for > 95% of the zonular protein . </S>", "<S> glycoproteins constituted the main structural components , with two proteins , fbn1 and ltbp2 , constituting 70%80% of the protein . </S>", "<S> other abundant components were mfap2 , emilin-1 , and adamtsl-6 . </S>", "<S> lysyl oxidase - like 1 , a crosslinking enzyme implicated in collagen and elastin biogenesis , was detected at significant levels . </S>", "<S> the equatorial and hyaloid zonular samples were compositionally similar to each other , although the hyaloid sample was relatively enriched in the proteoglycan opticin and the fibrillar collagens col2a1 , col11a1 , col5a2 , and col5a3.conclusionsthe zonular proteome was surprisingly complex . </S>", "<S> in addition to structural components , it contained signaling proteins , protease inhibitors , and crosslinking enzymes . </S>", "<S> the equatorial and hyaloid zonules were similar in composition , but the latter may form part of a composite structure , the hyaloid membrane , that stabilizes the vitreous face . </S>"], "labels": null, "section_names": ["Materials and Methods", "Zonule Dissection", "Histology and Immunofluorescence", "Zonule Digestion", "Vitreous Humor Digestion", "Liquid ChromatographyMass Spectroscopy Analysis of Zonule and Vitreous Humor Digests", "Bioinformatics Analysis Pipeline", "Conversion of RAW Files to MS2 Files.", "Database Searching.", "Search Results Processing.", "Determination of Contaminating Proteins", "Results", "Comparison Between Human and Bovine Zonular Fibers", "Comparison Between the Bovine Equatorial Zonule, Hyaloid Zonule, and Vitreous Humor", "Discussion", "Supplementary Material"], "sections": [["bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death .", "the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed .", "the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution .", "we divided the zonular fibers into two groups : the  equatorial zonule  ( blue fibers in fig .", "other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule", "were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens .", "because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues .", "once the equatorial fibers had been removed , the  hyaloid zonule  was exposed .", "the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous .", "they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face", ", it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor .", "samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor ,", "a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor .", "human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample .", "thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) .", "thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) .", "human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax .", "sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence .", "deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) .", "sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody .", "the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3  10 minutes )", ", sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) .", "sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) .", "bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each .", "samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c .", "five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard .", "five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) .", "the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al .", "briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate .", "a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c .", "peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate .", "the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation .", "bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine .", "samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark .", "an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin .", "digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction .", "peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration .", "samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed .", "the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m  2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid .", "after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m  25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c .", "peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min .", "survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2  10 .", "the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% .", "top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans .", "dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 .", ", is provided as supplementary materials s1 . the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .", "an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .", "ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .", "databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .", "common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .", "the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .", "the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .", "searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .", "2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .", "other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .", "the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .", "comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .", "target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .", "protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .", "protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles", "these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting . to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded .", "it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins .", "comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot .", "a fasta database file of all human ecm proteins was constructed . for both human and bovine samples ,", "the lists of identified proteins were used to extract separate fasta database files for identified proteins .", "an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins .", "analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins .", "common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis .", "extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample .", "the total abundances of putative zonule proteins ( using ms2 intensity  weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined .", "average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes .", "this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples", ", the ms2 intensity  weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together .", "full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 , s5 ) for each experiment are included ."], ["bovine eyes ( from 3-year - old animals ) were obtained from a local slaughterhouse within 3 hours of death .", "the eye globe was dissected from its anterior aspect , the cornea and iris were removed , and the front face of the ciliary processes was exposed .", "the lens , zonule , and ciliary body were released from the eye and transferred to a petri dish filled with ringer 's solution .", "we divided the zonular fibers into two groups : the  equatorial zonule  ( blue fibers in fig .", "other investigators have subdivided these fibers into anterior , equatorial , and posterior groupings , but here they were all included in a single sample , the equatorial zonule . using iridectomy scissors , fibers from the equatorial zonule", "were first transected near the ciliary body and then grasped with fine forceps and cut close to the lens .", "because the equatorial fibers are anatomically isolated within the eye , it was possible to remove them precisely , with little or no contamination from other tissues .", "once the equatorial fibers had been removed , the  hyaloid zonule  was exposed .", "the hyaloid zonule was defined as the set of fibers that were closely associated with the anterior face of the vitreous .", "they were also significantly longer than the equatorial fibers . because they were intimately connected to the vitreous face", ", it was not possible to dissect the hyaloid fibers as precisely as the equatorial fibers , and the hyaloid sample inevitably contained some elements of the anterior vitreous humor .", "samples were centrifuged for 10 minutes at maximum speed , supernatants were removed , and the zonular pellet was frozen immediately . a third sample , taken from the center of the vitreous humor ,", "a single bovine eye yielded sufficient tissue for a proteomic analysis of the equatorial zonule , hyaloid zonule , and vitreous humor .", "human eyes ( from 10 donors 2366 years of age ; table ) were obtained from a local eye bank and dissected using a similar approach to that described for the bovine eye . because the vitreous of the aged human eye was often partially liquefied , it was not possible to collect a human hyaloid zonule sample .", "thus , the human zonular sample was equivalent to the bovine equatorial zonule sample . description of human zonular samples to have sufficient material for analysis , human zonular samples were pooled ( table ) .", "thus , each pool contained combined material from four eyes of both sexes , with mean ages ranging from 38.5 ( pool d ) to 60 years ( pool c ) ."], ["human eyes were fixed for 1 week in 4% paraformaldehyde / pbs , dehydrated through graded ethanols and xylene , and embedded in paraffin wax . sections ( 4 m thick ) were cut in the midsagittal plane and processed for immunofluorescence .", "deparaffinized sections were antigen retrieved using a 60c overnight incubation in sodium citrate buffer ( 10 mm sodium citrate , 0.05% tween-20 ; ph 6.0 ) .", "sections were blocked with 3% bsa for 2 hours and incubated overnight at 4c in a 1:501:100 dilution of primary antibody .", "the following antibodies were used : adamtsl-6/thsd4 ( atlas antibodies , stockholm , sweden ) , emilin-1 ( santa cruz biotechnology , santa cruz , ca , usa ) , fbn1 ( millipore , temecula , ca , usa ) , fibulin-6/hemicentin-1 ( millipore ) , ltbp2 ( gift of tomoyuki nakamura , kansai medical university , osaka , japan ) , and mfap2 ( santa cruz biotechnology ) . after washing in pbs ( 3  10 minutes )", ", sections were incubated for 2 hours in 1:300 dilution of appropriate secondary antibody ( alexa 488conjugated goat anti - mouse or anti - rabbit ; invitrogen , carlsbad , ca , usa ) containing methyl green ( a nuclear counterstain ) .", "sections were then washed , coverslipped ( prolong gold ; invitrogen ) , and viewed using an olympus fv1000 confocal microscope ( center valley , pa , usa ) ."], ["bovine and human zonules were suspended in 50 l buffer containing 4% sds , 0.2% deoxycholic acid ( dca ) , and 100 mm ammonium bicarbonate ( ph 8) and sonicated using a fisher scientific model 60 sonic dismembranator ( thermo fischer scientific , waltham , ma , usa ) , using three treatments of 10 seconds each .", "samples were then shaken for 10 minutes at 600 rpm in a thermo - mixer at 90c .", "five microliters was removed for a pierce bicinchoninic acid ( bca ) assay ( thermo fisher scientific ) using bsa as a standard .", "five microliters of 0.5 m tris ( 2-carboxyethyl)phosphine ( tcep ) was then added , and the samples were heated at 90c for an additional 10 minutes and centrifuged at 14,000 g for 10 minutes , the supernatant was mixed with 200 l exchange buffer containing 8 m urea , 0.2% dca , and 100 mm ammonium bicarbonate , and the mixture was transferred to tween-20 passivated ultra-0.5 centrifuge filter units ( amicon ufc503008 ; emd millipore , billerica , ma , usa ) .", "the remaining sample preparation and digestion used the enhanced filter - aided sample preparation ( efasp ) method of erde et al .", "briefly , the sds containing lysis buffer was replaced with exchange buffer , proteins were alkylated with iodoacetamide , and exchange buffer was replaced with digestion buffer containing 0.2% dca and 50 mm ammonium bicarbonate . a ratio of 1:50 of sequencing grade modified trypsin ( promega , madison , wi , usa ) to substrate", "was then added , and the filter units were shaken at approximately 100 rpm for 12 hours at 37c .", "peptides were recovered by centrifugation and passage through the ultrafiltration membrane , and membranes were further washed with two 50-l portions of 50 mm ammonium bicarbonate .", "the dca in the combined filtrates was then extracted using ethyl acetate as previously described , and peptides in the aqueous phase were dried by vacuum centrifugation ."], ["bovine vitreous humor samples ( 200 l ) containing approximately 100 g protein were dried by vacuum centrifugation and dissolved in 50 l 8 m urea , 1.0 m tris ( ph 8.5 ) , 8 mm cacl2 , and 0.2 m methylamine .", "samples were reduced by addition of 4 l 0.2 m dithiothreitol and incubation at 50c for 15 minutes , followed by alkylation through addition of 4 l 0.5 m iodoacetamide and incubation at room temperature ( rt ) for 30 minutes in the dark .", "an additional 8 l 0.2 m dithiothreitol was then added , samples were incubated for an additional 15 minutes at rt , and 94 l water was added , followed by 40 l 0.1 g/l trypsin .", "digestion proceeded overnight at 37c and then 10 l formic acid was added to stop the reaction .", "peptides were solid phase extracted using sep - pak light c18 cartridges ( waters , milford , ma , usa ) , and the final eluate was dried by vacuum concentration ."], ["samples were dissolved in water containing 5% formic acid and transferred to autosampler vials , and 4 g of each zonule peptide digest ( or 2 g of each vitreous humor digest ) was analyzed .", "the samples were injected at a flow rate of 5 l / min onto an acclaim pepmap 100-m  2-cm nanoviper 5-m c18 trap ( thermo fisher scientific ) using mobile phase a containing water and 0.1% formic acid .", "after 5 minutes , the trap was switched in - line with a pepmap rslc c18 , 2 m , 75-m  25-cm easyspray column fitted in an easyspray nano electrospray source ( thermo fisher scientific ) at 40c .", "peptides were eluted with a 90-minute gradient of 7.5%30% mobile phase b containing acetonitrile and 0.1% formic acid at a flow rate of 0.3 l / min .", "survey scans were performed in the orbitrap mass analyzer at a resolution of 120,000 with a scan range of 4001500 , maximum inject time of 50 ms , and automatic gain control ( acg ) target of 2  10 .", "the quadrupole was used to isolate ions for mass spectroscopy ( ms)/ms scans with a 1.6-m / z isolation window , and fragmentation was performed by higher - energy collisional dissociation ( hcd ) with normalized collision energy of 35% .", "top speed data - dependent ms / ms spectra were collected in parallel using the ion trap , with a maximum 35-ms inject time , single scan , signal - to - noise ration ( s / n ) threshold = 2 , minimum ion intensity threshold of 5000 , and a maximum interval of 3 seconds between survey scans .", "dynamic exclusion was performed with the monoisotopic precursor selection ( mips ) filter on , exclusion of + 1 ions , 60-second exclusion time , exclusion mass tolerance of 10 ppm , and count = 1 ."], ["the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .", "an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .", "ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 .", "databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .", "common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .", "the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .", "the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .", "searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .", "2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .", "other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring .", "the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .", "comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .", "target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .", "protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .", "protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles", "these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting ."], ["the open source proteowizard toolkit was used to convert thermo raw files into compressed text files using msconvert via command line .", "an in - house python program was used to convert information in the compressed text file into appropriately formatted ms2 files .", "ms2 scans were extracted with a minimum ion count of 15 and minimum total absolute intensity of 100 ."], ["databases were downloaded from ensembl ( www.ensembl.org , in the public domain ) for bovine samples or from uniprot ( www.uniprot.org , in the public domain ) for human samples and processed with utilities available from www.proteomicanalysisworkbench.com , in the public domain .", "common contaminant sequences ( 179 ) were added before all sequences were reversed for peptide and protein error estimation using the target / decoy method .", "the ensembl bovine database ( release 79 , march 2015 ) had 22,118 protein sequences .", "the human database consisted of 20,207 uniprot reviewed canonical sequences ( swiss - prot ) downloaded july 2015 .", "searches were performed using comet version 2015.02 rev . 1 ( human samples ) or 2016.01 rev .", "2 ( bovine samples ) . monoisotopic mass parent ion and fragment ion tolerances of 1.25 and 1.0005 da were used , respectively .", "other parameters were as follows : tryptic enzyme specificity , static modifications of + 57 da on cysteine residues , variable modifications of + 16 da on methionine residues ( maximum of 2 modifications ) , and use of y- and b - ions in scoring ."], ["the paw analysis pipeline was used to control peptide sequencing errors , infer protein identities from observed peptides , and provide quantitative protein abundance estimates .", "comet search scores were transformed into discriminant scores using functions similar to those used in peptideprophet .", "target and decoy discriminant scores were displayed as histograms and overlaid for interactive setting of score thresholds to filter peptide - spectral matches at an overall false discovery rate ( fdr ) of 3% .", "protein inference used basic parsimony logic where proteins having indistinguishable peptide sets were combined into protein groups and proteins having peptide sets that were subset of other protein 's peptide sets were removed .", "protein inference was performed experiment - wide with additional requirements of a minimum of two distinct peptides per protein per biological sample applied after protein inference . in a final round of processing , straightforward extensions of parsimony principles", "these results were used for quantitative analyses using ms2 fragment ion intensity  weighted spectral counting ."], ["to more confidently determine the composition of the zonule , contaminating proteins from surrounding tissues have to be identified and excluded .", "it was assumed that the zonule would be composed predominantly of extracellular matrix ( ecm ) proteins .", "comprehensive lists of ecm proteins for human and mouse have been compiled ( http://matrisomeproject.mit.edu , in the public domain ) , and the lists of core and ecm - associated proteins for human were used to extract the corresponding protein sequence from uniprot .", "a fasta database file of all human ecm proteins was constructed . for both human and bovine samples ,", "the lists of identified proteins were used to extract separate fasta database files for identified proteins .", "an in - house python program was used to run a local installation of blast ( ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/ , in the public domain ) to determine the reciprocal best matches between the identified proteins and the human ecm proteins .", "analysis of alignment scores was used to categorize likely matches to matrisome proteins from poor alignments between matrisome proteins and contaminating proteins . once matrisome proteins were identified", "common contaminant proteins and proteins not matching to matrisome ecm proteins were excluded from quantitative analysis .", "extracellular matrix proteins constituted 90% of the bovine zonule sample and 82% of the human zonule sample .", "the total abundances of putative zonule proteins ( using ms2 intensity  weighted spectral counts ) were set to 100.00 , and the relative abundance of the proteins within each sample was determined .", "average protein abundances and their standard deviations were computed using standard excel functions . unlike the human dataset , multiple proteins from the bovine dataset mapped to individual matrisome genes .", "this was because the bovine ensemble protein database ( unlike the human swiss prot database ) contained multiple entries for protein isoforms of nearly identical sequence . to facilitate comparison to the human samples", ", the ms2 intensity  weighted spectral counts for all proteins mapping to the same matrisome gene symbol were summed together .", "relative abundances within each sample by gene were computed as described above . full , annotated protein reports ( supplementary materials s2 , s3 ) and associated detailed peptide evidence files ( supplementary materials s4 ,"], ["we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) .", "for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) .", "our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins .", "liquid chromatography  ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) .", "3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species .", "two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins .", "the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans .", "the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors .", "of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs .", "however , when the data were weighted according to protein abundance , based on ms2 intensity  weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) .", "the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 .", "the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) .", "overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome .", "however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) .", "shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule .", "we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig .", ", the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig .", "the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs .", "fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) .", "emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) .", "fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) .", "all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig .", "the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome .", "fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively .", "all family members are present ( with the exception of ltbp4 in the human zonule ) .", "the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins .", "mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients .", "mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms .", "together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule .", "ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm .", "both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) .", "curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved .", "another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family .", "adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae .", "adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 .", "unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) .", "we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig .", "adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig .", "in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm .", "immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig .", "a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig .", "the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells .", "work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds .", "we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils .", "several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig .", "transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule .", "the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome .", "exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma .", "given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule .", "one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 .", "the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils .", "expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule .", "the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) .", "only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 .", "col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium .", "megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant .", "immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule .", "this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals .", "similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes .", "cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions .", "proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels .", "unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 .", "fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample .", "recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous .", "comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs .", "proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor .", "protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) .", "some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous", "three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous .", "note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule .", "the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous .", "if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values .", "although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule .", "such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample .", "the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig .", "histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 ."], ["we began by comparing the protein composition of the human and bovine ciliary zonule ( the zonulome ) .", "for this purpose , the human zonular sample was compared with the bovine equatorial zonule sample ( see materials and methods ) .", "our initial goal was to identify those proteins common to both , reasoning that protein conservation may imply important structural or signaling roles for the set of conserved proteins .", "liquid chromatography  ms analysis of tryptic digests identified > 1000 proteins , but , following removal of common contaminants and filtering against the matrisome database , this number was reduced to 268 proteins ( 176 proteins in the bovine zonulome and 144 in the human ) .", "3 ) . in terms of mass , > 95% of the human and bovine zonules consisted of the proteins that were common to both species .", "two main classes of matrisomal proteins are defined : those constituting the core matrisome and those belonging to the group of matrisome - associated proteins .", "the core matrisome is further divided into three subcategories : ecm glycoproteins , collagens , and proteoglycans .", "the matrisome - associated protein category is also divided into three subcategories : ecm - affiliated proteins , ecm regulators , and secreted factors .", "of the six classes of matrisomal proteins , the two best represented by the number of identifications in the zonulome were glycoproteins ( 28 proteins in the human zonule and 53 in the bovine zonule ) and ecm regulators ( 23 in humans and 26 in cows ; ( figs .", "however , when the data were weighted according to protein abundance , based on ms2 intensity  weighted spectral counts , it was clear that in both cases the zonulome was dominated by glycoproteins ( figs . 4c , 4d ) .", "the nine most abundant glycoproteins in the human and bovine zonulome are ranked in figure 5 .", "the five most abundant proteins in either case were fibrillin-1 ( fbn1 ) , microfibrillar - associated protein 2 ( mfap2 ) , latent - transforming growth factor -binding protein-2 ( ltbp2 ) , emilin-1 , and thrombospondin type-1 domain - containing protein 4 ( thsd4 , also known as adamtsl-6 ) .", "overview of zonule composition in bovine ( a , c ) and human ( b , d ) samples . in terms of the number of proteins present ( a , b ) , all six categories of matrisomal proteins are well represented in the zonulome .", "however , in terms of relative protein abundance , both zonulomes are composed predominantly of glycoproteins ( c , d ) .", "shown here is a ranking of the nine most - abundant glycoproteins , which together account for 95% of the bovine zonule and 89% of the human zonule .", "we used an orthogonal technique , confocal immunofluorescence , to verify the distribution of the four most abundant glycoproteins detected in the lc - ms analysis ( fbn1 , ltbp2 , mfap2 , and emilin-1 ; fig .", ", the zonule appeared as a system of fibers projecting from the pars plana region of the nonpigmented ciliary epithelium , fanning out as they approached the lens . at their midpoint , the fibers had a relatively large diameter but near the lens surface , the large fibers divided to form smaller diameter fibers that inserted into the lens capsule ( see insets in fig .", "the three most abundant glycoproteins in the zonulome ( fbn1 , ltbp2 , and mfap2 ) labeled all zonular elements uniformly ( figs .", "fbn1 , ltbp2 , and mfap2 are distributed throughout the system of zonular fibers , including the small diameter fibers near the lens surface ( inset a in b ) and large diameter fibers at the midpoint of the zonular span ( inset b in b ) .", "emilin-1 was absent from zonular fibers near the lens surface ( arrow in d ) .", "fibrillins and ltbps comprise a superfamily of seven structurally related glycoproteins ( fbn1 - 3 and ltbp1 - 4 ) .", "all seven superfamily members were detected in the bovine zonule and only ltbp4 was absent from the human zonule ( fig .", "the fbn / ltbp superfamily constituted 83.4% of the bovine zonule and 76.0% of the human zonule and represented the most abundant protein family in either zonulome .", "fbn1 was the most abundant individual protein , accounting for 63.49% and 75.57% of the protein in the human and bovine ciliary zonule , respectively .", "all family members are present ( with the exception of ltbp4 in the human zonule ) .", "the high representation of fbn / ltbp superfamily members in the zonule is consistent with the ocular phenotypes of syndromes resulting from mutations in those proteins .", "mutations in fbn1 underlie marfan syndrome , a condition in which progressive lysis of the zonular fibers leads to ectopia lentis in most ( 71% ) patients .", "mutations in fbn1 and ltbp2 have also been shown to cause weill - marchesani syndrome ( types 2 and 3 , respectively ) , which is a disease characterized by microspherophakia and ectopia lentis , in addition to systemic symptoms .", "together , these observations support the notion that fbn1 and ltbp2 play indispensable structural roles in the ciliary zonule .", "ltbps , as their name implies , have the ability to bind and sequester latent forms of tgf in the ecm .", "both the human and the bovine zonule contained relatively high levels of tgf-2 ( lesser amounts of tgf-3 were also detected in the human sample ) .", "curiously , ltbp2 , by far the most abundant of the zonular ltbps , is the only family member incapable of binding tgf , so its role in the zonule and elsewhere is unresolved .", "another class of proteins linked with inherited ectopia lentis ( and the closely related condition ectopia lentis et pupillae , in which both the lens and the pupil are displaced ) is the adamtsl family .", "adamtsls share significant sequence similarity with the adamts family of metalloproteases but lack the protease domain found in the latter . mutations in adamtsl-4 cause isolated ectopia lentis and ectopia lentis et pupillae .", "adamtsl-4 was detected in both the bovine and human zonules ( at somewhat higher levels in the latter ) , as were adamtsl-1,-2 and -5 .", "unexpectedly , however , the most abundant member of the family was adamtsl-6 ( also known as thsd4 ) , which , in the human zonule , accounted for > 2% of the total protein ( figs . 5 , 8) .", "we used immunofluorescence to verify the expression of adamtsl-6 in the human eye ( fig .", "adamtsl-6 was strongly expressed in the large diameter fibers in a similar fashion to emilin-1 ( fig .", "in vitro studies suggest that adamtsl-6 can bind directly to fbn1 , promoting the assembly of fibrillin - rich ecm .", "immunofluorescence is largely restricted to the large diameter fibers in the center of the zonular span and absent from small fibers at the lens surface ( see fig .", "a number of protease inhibitors were detected in the zonule , including several members of the serpin family , but the most abundant inhibitor , particularly in the human sample , was timp3 ( fig .", "the only protease detected in both the human and bovine zonule was cathepsin d , a lysosomal aspartic endopeptidase that may also be secreted from cells .", "work on model systems suggest that the material properties of microfibrillar networks are shaped by both nonreducible crosslinks and disulfide bonds .", "we examined human and bovine zonules for the presence of known crosslinking enzymes that might play a role in bundling of microfibrils .", "several transglutaminases ( tgm1 - 3 ) were detected , but the most abundant crosslinker detected was lysyl oxidase - like 1 ( loxl1 ; fig .", "transglutaminase - derived crosslinks have been identified previously in fibrillin microfibrils and may be important in the zonule .", "the presence of loxl1 is intriguing because variants in the loxl1 gene have been shown to predispose to exfoliation syndrome .", "exfoliation syndrome is a condition in which disintegration of the zonular fibers and unscheduled production of matrix components results in formation of fibrillar aggregates that can clog the outflow pathways of the eye , leading to exfoliation glaucoma .", "given its demonstrated role in elastin polymerization , it is curious that loxl1 is such a prominent component of the elastin - free ciliary zonule .", "one explanation may be that fibrillin or other zonule components serve as substrates for loxl1 .", "the availability of mice deficient in loxl1 should allow us to test whether loxl1 has a role in crosslinking microfibrils .", "expression of crosslinking enzymes ( transglutaminases and lysyloxidase - like 1 ) in the ciliary zonule .", "the expression of collagens , secreted factors , and proteoglycans was compared in the human and bovine zonular samples ( supplementary tables s1 , s2 ) .", "only trace amounts of collagen were detected , of which the most abundant were col2a1 and col9a2 .", "col4 and col18 , components of basement membranes , were also detected and may represent contamination from the lens capsule or the inner limiting membrane covering the basal surface of the nonpigmented ciliary epithelium .", "megf6 , shown to interact via its egf domains with mfap5 , was one of the most abundant .", "immune components , such as cxcl14 and egfl7 , were identified in the human but not bovine zonule .", "this may reflect the fact that the human samples were largely from middle - aged individuals ( table ) , whereas the bovine samples were from young animals .", "similarly , the presence of il17d in human samples may indicate ongoing inflammatory reactions in the aged human eyes .", "cxcl14 is a chemokine implicated in the recruitment of immune cells in all types of inflammatory conditions .", "proteoglycans were not prominent zonule components in either species , although in the bovine zonule , opticin ( opt ) was detected at moderate levels ."], ["unlike the bovine zonule ( where members of a single glycoprotein family , the fbn / ltbp family , accounted for > 80% of the protein ) , the composition of the vitreous was not dominated by a restricted set of super abundant proteins ( supplementary material s3 ) . among the more abundant vitreous proteins were broad spectrum protease inhibitors ( a2ml1 , a2 m , cpmd8 , cst3 , and serping1 ) , collagens ( col9a2 , col18a1 , and col2a1 ) , fibulins ( fbln1 , fbln2 , and fbnl3 ) , the proteoglycans opt and hspg2 , and wnt signaling components sfrp2 and sfrp3 .", "fbn1 , the most abundant single component in the equatorial zonule , accounted for only 2.5% of the protein in the vitreous sample .", "recent proteomic analyses of human vitreous humor have identified many hundreds of protein components , of which a core set of 231 proteins were detected in all studies . in the present study , after filtering against the matrisome database , we identified 200 components of the bovine vitreous . comparing the bovine vitreous proteome with that of the equatorial and hyaloid zonular samples , it was evident that the zonular samples were quite distinct from the vitreous sample ( figs .", "proteins such as mfap2 and fbn2 , for example , were almost undetectable in the vitreous but relatively abundant in the zonular samples . overlapping distribution of matrisome proteins in samples from the bovine equatorial zonule , hyaloid zonule , and vitreous humor .", "protein composition of samples from the bovine vitreous humor ( gray ) , equatorial zonule ( light blue ) , and hyaloid zonule ( dark blue ) .", "some proteins are relatively abundant in all three samples ; others , such as thsd4 , are abundant in the zonular samples but barely detectable in the vitreous .", "three classes of fibrillary collagen ( type 2a , 11a , and 5a ; circled in yellow ) are significantly overrepresented in the hyaloid zonule sample compared with the equatorial zonule or vitreous .", "note that opticin ( opt ) accounts for 30% of the hyaloid zonule but was barely detectable in the equatorial zonule .", "the hyaloid zonule is associated with the vitreous face , and it is possible that during dissection , the hyaloid sample might have been contaminated with proteins emanating from the vitreous .", "if we assume that the hyaloid zonule has a similar basic composition to the equatorial zonule , then vitreous contamination should lead to values for relative protein abundance that were intermediate between the vitreous and equatorial zonule values .", "although such instances were certainly observed , there were also many examples where the abundance of a protein in the hyaloid zonule significantly exceeded ( by orders of magnitude ) that of the vitreous or the equatorial zonule .", "such proteins are likely to be unique to , or significantly enriched in , the hyaloid zonule sample .", "the set of hyaloid - enriched proteins included opt , a3i3bp , cochlin , and the collagens col2a1 , col11a1 , col5a3 , and col5a2 ( fig .", "histograms showing relative abundance of each class of matrisomal proteins in the bovine samples are included as supplementary figures s6s12 ."], ["the ciliary zonule plays a key role in ocular function , centering the lens on the optical axis and transmitting the forces that mold its shape during accommodation .", "the zonule is adversely affected in several pathologic conditions , including exfoliation syndrome , homocystinuria , and marfan syndrome . to understand how and why the zonule is compromised in these diseases , a detailed analysis of its composition", "microfibrils ( the main structural elements of the zonule ) are ubiquitous components of the ecm but questions remain regarding their organization , synthesis , and role as signaling hubs .", "the ciliary zonule , a readily accessible , cell - free system composed of bundled microfibrils , represents a useful model system in which to gain insights into microfibril organization .", "the composition of the equatorial zonule was broadly similar in humans and cows , and the set of 52 proteins identified in both samples accounted for > 95% of the mass in either case .", "the fact that the human samples were from middle - aged individuals , whereas the bovine samples were from young animals , might in part explain why more proteins were identified in the bovine zonulome ( 176 vs. 144 ) .", "the bovine samples were also obtained shortly after death , whereas the human samples were obtained 2448 hours postmortem .", "it is possible that labile proteins were lost from the human samples during the postmortem period . in both humans and cows , the zonule consisted of a microfibril backbone comprised primarily of fibrillin and a relatively restricted set of glycoproteins .", "ltbp2 , a fibrillin - like protein implicated in autosomal recessive weill - marchesani syndrome type 3 ( wms3 : mim#614819 ) , was a surprisingly abundant component , comprising 10% of the total zonular protein .", "there are three fibrillin genes in humans and the presence of all three proteins was confirmed in the zonulome , consistent with earlier observations .", "fbn2 , the second most abundant fibrillin in the zonule , is synthesized early in development and is essential for normal zonule formation in mice .", "immunolocalization studies have previously failed to detect fbn2 in the adult zonule , but its presence in the human and bovine zonulome suggests that fbn2 epitopes may become inaccessible to antibody staining during postnatal development , as reported in other systems .", "several proteins implicated in tgf signaling were detected in the zonule . despite its name , latent tgf binding protein 2 ( ltbp2 ) is not a bona fide tgf-binding protein , but two other family members , ltbp1 and ltbp3 , were detected in the zonulome .", "in other systems , ltbp1 and ltbp3 facilitate the sequestration ( and release ) of tgf in the ecm through interaction with fbn1 and other components . of note", "another protein with a potential role in modulating tgf signaling was emilin-1 , a prominent component of the human and bovine zonulome .", "emilin-1 has been shown to inhibit tgf signaling by binding to pro - tgf precursor and preventing its furin - mediated maturation .", "emilins were originally identified as elastic fiber components located at the interface between microfibrils and the underlying elastin core .", "more recent studies have suggested that they associate directly with microfibrils . because zonular fibers insert into the lens capsule directly above the lens germinative zone ( the region of the lens epithelium containing the mitotically active cell population ) , tgf or other growth factors sequestered in the zonule are well placed to influence the growth of the lens directly .", "it is also possible that the cyclical stretching of the zonule during accommodation might serve to release bound factors from the surface of zonular fibers .", "one of the novel ( and abundant ) proteins detected in our study was adamtsl-6 ( thsd4 ) .", "adamts proteins and adamtsl proteins have well - documented roles in ocular biology and pathology .", "adamtsl-6 has not been identified previously in the zonule but , in other systems , is known to bind to the n terminus of fbn1 , where it may promote microfibril formation . among the proteins that we expected to detect in the zonulome , but did not , was adamts10 ; adamts10 mutations underlie weill - marchesani syndrome type 1 ( wms1 ) .", "ectopia lentis is commonly observed in wms1 , implying a mechanical defect in the ciliary zonule .", "clinically , wms1 is indistinguishable from wms2 and wms3 , syndromes caused by mutations in fbn1 and ltbp2 respectively , genes encoding two of the most abundant zonular proteins .", "immunoelectron microscopy studies have previously suggested that adamts10 is abundant in the human ciliary zonule so its absence from the proteome is perplexing . a striking feature of the zonulome in human and cows was the presence of a diverse set of protease inhibitors , the most abundant in humans being timp3 .", "the zonule is known to be exquisitely sensitive to proteolysis . before the introduction of modern cataract surgery", ", ophthalmologists removed the cataractous lens in a single piece , a procedure facilitated by brief exposure to dilute solutions of -chymotrypsin , which served to lyse the zonule and release the lens from the eye .", "the presence of timp3 and other protease inhibitors may help explain the evident durability of the zonule , despite lifelong exposure to matrix metalloproteinases and other proteases present in the ocular humors .", "the hyaloid zonule is intimately associated with the anterior face of the vitreous . during ophthalmic surgery ,", "the vitreous face behaves , physically , as if it were a distinct membrane ( the  hyaloid membrane  ) .", "ophthalmologists take great care not to damage the hyaloid membrane during surgical procedures , lest the contents of the vitreous leak .", "the current study may provide some clues as to the nature of the hyaloid membrane .", "first , it is apparent that the hyaloid membrane is reinforced by the presence of a meshwork of fibrillin - rich microfibrils , the hyaloid zonule . during dissection of the hyaloid zonule", "it is likely that proteins from the anterior - most region of the vitreous were coisolated .", "the hyaloid zonule sample was characterized by the presence of high concentrations of opticin , consistent with previous immunolocalization studies of opticin in human eyes .", "our analysis also suggested that the hyaloid sample was enriched in fibrillar collagens ( especially col2a1 , col5a2 , col5a3 , and col11a1 ) , which were detected elsewhere in the vitreous but at much lower concentrations .", "interestingly , mutations in col2a1 and col11a1 result in stickler syndrome , a condition characterized by vitreous pathology . in stickler syndrome type 1 ( caused by col2a1 mutations ) , a  membranous vitreous  is often observed ; whereas in stickler syndrome type 2 ( col11a1 ) a  beaded vitreous  is observed . in each case the anterior face of the vitreous appears to be compromised .", "thus , the phenotypes in stickler syndrome are consistent with a model in which fibrillar collagens form part of a meshwork ( along with fibrillin - rich microfibrils and opticin ) that defines and supports the vitreous face .", "one question arising from the current study is why such an unassuming structure as the ciliary zonule should require a proteome with fifty or more components ?", "in other settings , it is evident that microfibril scaffolds , decorated with latent growth factors , serve as both mechanosensors and signaling hubs .", "the pleitropic ocular effects of mutations in zonular genes ( which include congenital glaucoma , cataract , ectopia lentis , and microspherophakia ) are consistent with the notion that the role of the ciliary zonule is not solely to secure the lens in place ."], [""]]}
{"article_id": "PMC2639293", "article_text": ["identification of specific subtypes of circulating tumor cells in the peripheral blood of cancer patients can provide important prognostic information , but to be effective , the method used must recognize all tumor cell types .", "the subtype of 19 well - characterized breast cancer cell lines was obtained by use of gene expression profiling , including normal - like , basal - like , her2-positive , and luminal a and b. cells from each line were mixed with blood from a healthy donor and subjected to the cellsearch circulating tumor cell assay .", "the cellsearch assay , which uses epithelial cell adhesion molecules on the cell surface , did not recognize normal - like breast cancer cells , although other subtypes were recognized .", "normal - like breast cancer cells have especially aggressive features , and so assays that recognize this subtype would provide valuable prognostic information .", "new assays are needed that include antibodies that specifically recognize this breast cancer subtype but not other cell types , including those of hematopoietic origin .", "identification of specific subtypes of circulating tumor cells in the peripheral blood of cancer patients can provide important prognostic information , but to be effective , the method used must recognize all tumor cell types .", "the subtype of 19 well - characterized breast cancer cell lines was obtained by use of gene expression profiling , including normal - like , basal - like , her2-positive , and luminal a and b. cells from each line were mixed with blood from a healthy donor and subjected to the cellsearch circulating tumor cell assay .", "the cellsearch assay , which uses epithelial cell adhesion molecules on the cell surface , did not recognize normal - like breast cancer cells , although other subtypes were recognized .", "normal - like breast cancer cells have especially aggressive features , and so assays that recognize this subtype would provide valuable prognostic information .", "new assays are needed that include antibodies that specifically recognize this breast cancer subtype but not other cell types , including those of hematopoietic origin .", "this study was in part financially supported by the netherlands genomic initiative / netherlands organisation for scientific research ."], "abstract_text": ["<S> identification of specific subtypes of circulating tumor cells in peripheral blood of cancer patients can provide information about the biology of metastasis and improve patient management . however , to be effective , the method used to identify circulating tumor cells must detect all tumor cell types . </S>", "<S> we investigated whether the five subtypes of human breast cancer cells that have been defined by global gene expression profiling  normal - like , basal , her2-positive , and luminal a and b  were identified by cellsearch , a us food and drug administration  approved test that uses antibodies against the cell surface  </S>", "<S> expressed epithelial cell adhesion molecule ( epcam ) to isolate circulating tumor cells . </S>", "<S> we used global gene expression profiling to determine the subtypes of a well - defined panel of 34 human breast cancer cell lines ( 15 luminal , nine normal - like , five basal - like , and five her2-positive ) . </S>", "<S> we mixed 50 - 150 cells from 10 of these cell lines with 7.5 ml of blood from a single healthy human donor , and the mixtures were subjected to the cellsearch test to isolate the breast cancer cells . </S>", "<S> we found that the cellsearch isolation method , which uses epcam on the surface of circulating tumor cells for cell isolation , did not recognize , in particular , normal - like breast cancer cells , which in general have aggressive features . </S>", "<S> new tests that include antibodies that specifically recognize normal - like breast tumor cells but not cells of hematopoietic origin are needed . </S>"], "labels": null, "section_names": ["Context and Caveats", "Prior knowledge", "Study design", "Contribution", "Implications", "Limitations", "Funding", "Supplementary Material"], "sections": [["identification of specific subtypes of circulating tumor cells in the peripheral blood of cancer patients can provide important prognostic information , but to be effective , the method used must recognize all tumor cell types .", "the subtype of 19 well - characterized breast cancer cell lines was obtained by use of gene expression profiling , including normal - like , basal - like , her2-positive , and luminal a and b. cells from each line were mixed with blood from a healthy donor and subjected to the cellsearch circulating tumor cell assay .", "the cellsearch assay , which uses epithelial cell adhesion molecules on the cell surface , did not recognize normal - like breast cancer cells , although other subtypes were recognized .", "normal - like breast cancer cells have especially aggressive features , and so assays that recognize this subtype would provide valuable prognostic information .", "new assays are needed that include antibodies that specifically recognize this breast cancer subtype but not other cell types , including those of hematopoietic origin ."], ["identification of specific subtypes of circulating tumor cells in the peripheral blood of cancer patients can provide important prognostic information , but to be effective , the method used must recognize all tumor cell types ."], ["the subtype of 19 well - characterized breast cancer cell lines was obtained by use of gene expression profiling , including normal - like , basal - like , her2-positive , and luminal a and b. cells from each line were mixed with blood from a healthy donor and subjected to the cellsearch circulating tumor cell assay ."], ["the cellsearch assay , which uses epithelial cell adhesion molecules on the cell surface , did not recognize normal - like breast cancer cells , although other subtypes were recognized ."], ["normal - like breast cancer cells have especially aggressive features , and so assays that recognize this subtype would provide valuable prognostic information .", "new assays are needed that include antibodies that specifically recognize this breast cancer subtype but not other cell types , including those of hematopoietic origin ."], [""], ["this study was in part financially supported by the netherlands genomic initiative / netherlands organisation for scientific research ."], [""]]}
{"article_id": "PMC5013237", "article_text": ["zoonotic cutaneous leishmaniasis ( zcl ) is polymorphic disease with various clinical manifestations ( 1 , 2 ) .", "leishmaniasis , a group of parasitic infections , has a worldwide distribution ( 3 ) ; the world health organization estimates a prevalence of approximately 12 million cases , with an annual mortality rate of 60,000 and an at - risk population of approximately 350 million ( 4 ) .", "approximately 90% of cl cases occur in just seven countries , including iran ( 5 ) .", "several molecular targets for diagnostic pcr testing have been evaluated in leishmania , including minicircle kinetoplast dna ( kdna ) ( 5 , 6 ) , the mini - exon ( spliced leader rna ) gene ( 7 ) , gp63 polymerase chain reaction analysis of restriction fragment length polymorphism ( pcr - rflp ) ( 8) , and the internal transcribed spacer ( its ) ( 1 , 3 , 9 , 10 ) .", "pcr is a new alternative to existing diagnostic procedures , such as direct smear of parasites from clinical specimens or by cultivation , with microscopic examination .", "in the present study , as described previously ( 1 , 11 , 12 ) , we used the rflp analysis of amplified its1 in ribosomal operons to investigate the parasite that causes zcl , and the genetic variations among l. major isolates from chabahar , a port city in southeast iran ( situated at the iran - pakistan border ) . in this region ,", "the manifestation of zcl as a new focus of disease was in doubt , but our findings showed that the species is l. major with variable genes . the disease was correlated with the clinical manifestations of zcl in chabahar , so this is a new major public health problem .", "a variety of nucleic acid detection methods that target both dna and rna have been developed ( 1 ) .", "the its1-rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples , and it enables determination of the infecting species ( 3 ) .", "the study was approved by the institutional ethics committee , and all patients signed informed consent forms .", "we evaluated the pcr - rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients .", "these patients were referred for diagnosis to the central laboratory of chabahar ( 25 17 n.70 38e ; 13,162 km ) for evaluation of skin diseases .", "the clinical samples were taken from patients suspected to have cl in different endemic areas of chabahar , including negor and other districts and villages .", "each diagnosis was confirmed by the demonstration of amastigotes on the slit smear and/or the presence of flagellated promastigotes in novy - macneal - nicolle ( nnn ) medium from skin samples of 24 patients with typical and atypical lesions . for the parasitological investigation ,", "two were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - tap tubes that contained blood agar slanted in nnn medium , then incubated at 25  1c .", "the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin .", "the diagnosis of leishmaniasis was confirmed by the presence of amastigotes in slit smears and/or the presence of flagellated promastigotes in nnn medium from typical and atypical lesions . for parasitological investigations ,", "three skin samples were collected from the edges of the lesions using sterile surgical blades .", "two of the samples were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - top tubes containing blood agar slanted in nnn medium , then incubated at 25  1c .", "parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700  g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) . for species identification ,", "the dna isolated from amastigotes in ulcers and promastigotes in cultures was extracted using a commercial extraction kit ( high pure template dna preparation kit , roche , germany ) , according to the manufacturer s instructions .", "the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l of l5.8s 5-tga tac cac tta tcg cat t-3 ( 12 ) .", "the reaction was carried out with the pcr - ready supreme mix in 47  l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix .", "amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide .", "major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls", "the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes .", "the pcr products from its1-dna pcr were digested using the haeiii restriction endonuclease enzyme and the taq1 restriction enzyme , as recommended by the manufacture ( new england biolabs , inc . ) .", "the its1 sequence was amplified from all isolates , and reference stains were digested separately with restriction enzymes , according to the manufacturer s instructions .", "a variety of nucleic acid detection methods that target both dna and rna have been developed ( 1 ) .", "the its1-rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples , and it enables determination of the infecting species ( 3 ) .", "the study was approved by the institutional ethics committee , and all patients signed informed consent forms .", "we evaluated the pcr - rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients .", "these patients were referred for diagnosis to the central laboratory of chabahar ( 25 17 n.70 38e ; 13,162 km ) for evaluation of skin diseases .", "the clinical samples were taken from patients suspected to have cl in different endemic areas of chabahar , including negor and other districts and villages .", "each diagnosis was confirmed by the demonstration of amastigotes on the slit smear and/or the presence of flagellated promastigotes in novy - macneal - nicolle ( nnn ) medium from skin samples of 24 patients with typical and atypical lesions . for the parasitological investigation ,", "two were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - tap tubes that contained blood agar slanted in nnn medium , then incubated at 25  1c .", "the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin .", "the diagnosis of leishmaniasis was confirmed by the presence of amastigotes in slit smears and/or the presence of flagellated promastigotes in nnn medium from typical and atypical lesions . for parasitological investigations ,", "three skin samples were collected from the edges of the lesions using sterile surgical blades .", "two of the samples were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - top tubes containing blood agar slanted in nnn medium , then incubated at 25  1c .", "parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700  g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) .", "for species identification , the dna isolated from amastigotes in ulcers and promastigotes in cultures was extracted using a commercial extraction kit ( high pure template dna preparation kit , roche , germany ) , according to the manufacturer s instructions .", "the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l of l5.8s 5-tga tac cac tta tcg cat t-3 ( 12 ) .", "the reaction was carried out with the pcr - ready supreme mix in 47  l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix .", "amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide .", "major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls .", "the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes .", "the pcr products from its1-dna pcr were digested using the haeiii restriction endonuclease enzyme and the taq1 restriction enzyme , as recommended by the manufacture ( new england biolabs , inc . ) .", "the its1 sequence was amplified from all isolates , and reference stains were digested separately with restriction enzymes , according to the manufacturer s instructions .", "we used the pcr - rflp assay of its1 genes for the direct identification of leishmania species in 24 out of 33 patients suspected to have cl .", "these 24 patients , who were positive on smears and cultures for l. donovani bodies demonstrated microscopically , were selected for the study .", "the patient characteristics are shown in table 1 , including gender , age , number of lesions , ulcer duration , and distribution of ulcers on the body . with the use of litsr / l5.8s primer for pcr amplification ,", "single products of the expected size of 350 bp of l. major were detected in 21 patients ( figure 1 ) . in three isolates , a nonspecific 450 bp band was seen .", "rflp was performed on the its1 region in the ribosomal operons of 24 isolates of l. major . after using the restriction enzyme haeiii , banding patterns , including fragments of 140 bp and 210 bp bands ,", "were observed in 19 cases ( lma ) . in three cases with this enzyme , 300 bp and 150 bp bands ( figure 2 )", "m , molecular size marker ( 50 bp ) ; lane 1 , standard l. major ( mrho / ir/75/er ) ; lane 2 , standard l. tropica ( mhom / ir/99/yaz1 ) ; lane 3 , pcr products ( 350 bp ) as a standard ; lane 4 , pcr products ( 450 bp ; different from standard ) ; lanes 5 - 10 , pcr products ; lane 11 , negative control .", "m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , standard l. tropica ; lanes 3 - 9 , pcr products ; lane 10 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 11 , negative control .", "digestion of its1 from ribosomal dna with taq1 enzymes enabled us to identify two different patterns . using the taq1 restriction enzyme , banding patterns including 150 bp and 200 bp", "were observed in 19 cases , same as the standard of l. major ( genotype pattern lma ) . in three isolates ,", "digestion with this enzyme showed 300 bp and 150 bp banding patterns , dissimilar to the l. major reference strain . in two cases ,", "m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , l. tropica ; lanes 3 - 9 , pcr products ; lane 10 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 11 , negative control .", "m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , l. tropica ; lane 3 , pcr products ; lane 4 , pcr products ; lane 5 , pcr products ; lane 6 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 7 , pcr products ; lanes 8 - 10 , pcr products ; lane 11 , negative control .", "leishmaniasis is a major societal health problem that mostly affects populations where essential health services are not easily accessible and the detection of cl is based on clinical characteristics ( 14 ) .", "the present study shows that cl is caused by l. major in chabahar , iran .", "parasitological methods are the gold standard for the diagnosis of cl , with a high dependence on the number of parasites in the samples and a requirement of technical skill for sampling .", "the sensitivity of these methods varies from 27% to 85% for the diagnosis of leishmaniasis , which is their main disadvantage ( 15 ) .", "in addition , contamination of the cultured medium is an occasional problem ; such material must be discarded .", "molecular techniques have proved to be sensitive and powerful tools for detecting leishmania directly in clinical samples , as well as for parasite characterization using pcr ( 1 ) .", "some studies on viannia isolates from different hosts and geographic areas have found high levels of intra- and interspecific variation .", "schonian et al . established a diagnostic its1 pcr - rflp method using the restriction enzyme haeiii for leishmaniasis , combining high sensitivity for detecting leishmania directly in clinical materials and the ability to identify all medically relevant species groups ( 12 ) . in our study , using the haeiii restriction enzyme , the restriction analysis of the amplified its1 ribosomal dna revealed two different schizodeme patterns ( lma and lmb ) in l. major . on the other hand", ", some researchers have found that haeiii was not sufficient to distinguish all species of viannia ( 16 , 17 ) .", "we found that haeiii , rsa , and hinf1 can produce two profile bandings ; alu1 produces three ; dde1 and scrf1 produce four ; and taq1 produces five ( 1 ) . in this study ,", "( 20 ) , two profile bandings ( 220 bp and 140 bp ) in haeiii digestion were detected , similar to the standard .", "taq1 digestion showed two patterns with 150 bp and 200 bp bands . in contrary , in a study in yazd ( 20 ) on molecular identification using taq1 restriction enzymes , four patterns were produced .", "it should be emphasized that leishmaniasis in chabahar is new , and genetic variation is limited .", "the results of the present study showed that cl infections were more common in males ( 48.4% ) than in females ( 24.2% ) ( table 2 ) , probably because males spend more time outdoors than females do in this region .", "some studies have shown that pcr methods achieved positive results in more than 90% of cl cases ( 13 ) .", "the crithidias are able to endosymbiotically live within some parasites related to the trypanosomatidae family and sub - families of leishmania , and move to different hosts along with them .", "some researchers believe that the only way to isolate crithidia from leishmania is through kdna and/or ribosomal rna genes .", "it has been suggested that by extracting kdna and/or rdna , then amplifying them with pcr using the pcr - rflp technique with the haeiii enzyme and isolation of bands at 350 bp for leishmania and at 450 bp for crithidia , these two parasites can be isolated from each other ( 21 ) .", "however , its1-rflp pcr showed good sensitivity , and two its1 analyses showed the same results , as in a recent study by doudi et al .", "thus , the results of the present study confirm that in the border area of sistan va baluchestan , on the peninsula of chabahar , l. major is the causative agent of cl , with genotypic variation in this area ."], "abstract_text": ["<S> backgroundzoonotic cutaneous leishmaniasis ( zcl ) is polymorphic disease that may show various clinical manifestations.objectivesthis study investigates the determination of genetic variation within the species of leishmania major isolates from new cases in chabahar , a port city in southeast iran ( situated at the iran - pakistan border ) . migration in this region indicates that leishmaniasis is spreading gradually , and a new micro - habitat focus appears each year.materials and methodsa variety of nucleic acid detection methods that target both dna and rna have been developed . </S>", "<S> the restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction ( its1-rflp pcr ) assay is a multipurpose tool for the diagnosis of leishmania from clinical samples and for enabling the determination of the infecting leishmania species . </S>", "<S> the goal of this study was the identification of species based on its1-rflp in the ribosomal operon of l. major from clinically different forms of zcl amplified by pcr , followed by the digestion of the pcr product with restriction enzymes . </S>", "<S> the profiles were observed and visualized in agarose gel under uv light . </S>", "<S> we used direct smears to identify the parasites . </S>", "<S> while taking the smear , samples were collected for culture or direct pcr . </S>", "<S> we used the pcr - rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients . </S>", "<S> pcr - its1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods.resultsof the 24 isolates , 21 had 350 bp bands ( 87.5% ) and three had 450 bp bands ( 12.5% ) . after using the restriction enzyme , </S>", "<S> banding patterns including fragments of 210 and 140 bp for l. major were detected in 19 cases.conclusionsthe l. major species causing zcl in chabahar have limited genetic variation . </S>", "<S> there seems to be little manifestation of diversity between these lesions as a new focus of disease , and new micro - habitats for the disease are appearing in parts of this region . </S>"], "labels": null, "section_names": ["1. Background", "2. Objectives", "3. Materials and Methods", "3.1. Study Area and Population", "3.2. Parasite Samples", "3.3. DNA Preparation", "3.4. Restriction Fragment Length Polymorphism", "4. Results", "5. Discussion"], "sections": [["zoonotic cutaneous leishmaniasis ( zcl ) is polymorphic disease with various clinical manifestations ( 1 , 2 ) .", "leishmaniasis , a group of parasitic infections , has a worldwide distribution ( 3 ) ; the world health organization estimates a prevalence of approximately 12 million cases , with an annual mortality rate of 60,000 and an at - risk population of approximately 350 million ( 4 ) .", "approximately 90% of cl cases occur in just seven countries , including iran ( 5 ) .", "several molecular targets for diagnostic pcr testing have been evaluated in leishmania , including minicircle kinetoplast dna ( kdna ) ( 5 , 6 ) , the mini - exon ( spliced leader rna ) gene ( 7 ) , gp63 polymerase chain reaction analysis of restriction fragment length polymorphism ( pcr - rflp ) ( 8) , and the internal transcribed spacer ( its ) ( 1 , 3 , 9 , 10 ) .", "pcr is a new alternative to existing diagnostic procedures , such as direct smear of parasites from clinical specimens or by cultivation , with microscopic examination ."], ["in the present study , as described previously ( 1 , 11 , 12 ) , we used the rflp analysis of amplified its1 in ribosomal operons to investigate the parasite that causes zcl , and the genetic variations among l. major isolates from chabahar , a port city in southeast iran ( situated at the iran - pakistan border ) . in this region ,", "the manifestation of zcl as a new focus of disease was in doubt , but our findings showed that the species is l. major with variable genes . the disease was correlated with the clinical manifestations of zcl in chabahar , so this is a new major public health problem ."], ["a variety of nucleic acid detection methods that target both dna and rna have been developed ( 1 ) .", "the its1-rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples , and it enables determination of the infecting species ( 3 ) .", "the study was approved by the institutional ethics committee , and all patients signed informed consent forms .", "we evaluated the pcr - rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients .", "these patients were referred for diagnosis to the central laboratory of chabahar ( 25 17 n.70 38e ; 13,162 km ) for evaluation of skin diseases .", "the clinical samples were taken from patients suspected to have cl in different endemic areas of chabahar , including negor and other districts and villages .", "each diagnosis was confirmed by the demonstration of amastigotes on the slit smear and/or the presence of flagellated promastigotes in novy - macneal - nicolle ( nnn ) medium from skin samples of 24 patients with typical and atypical lesions . for the parasitological investigation ,", "two were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - tap tubes that contained blood agar slanted in nnn medium , then incubated at 25  1c .", "the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin .", "the diagnosis of leishmaniasis was confirmed by the presence of amastigotes in slit smears and/or the presence of flagellated promastigotes in nnn medium from typical and atypical lesions . for parasitological investigations ,", "three skin samples were collected from the edges of the lesions using sterile surgical blades .", "two of the samples were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - top tubes containing blood agar slanted in nnn medium , then incubated at 25  1c .", "parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700  g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) . for species identification ,", "the dna isolated from amastigotes in ulcers and promastigotes in cultures was extracted using a commercial extraction kit ( high pure template dna preparation kit , roche , germany ) , according to the manufacturer s instructions .", "the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l of l5.8s 5-tga tac cac tta tcg cat t-3 ( 12 ) .", "the reaction was carried out with the pcr - ready supreme mix in 47  l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix .", "amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide .", "major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls", "the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes .", "the pcr products from its1-dna pcr were digested using the haeiii restriction endonuclease enzyme and the taq1 restriction enzyme , as recommended by the manufacture ( new england biolabs , inc . ) .", "the its1 sequence was amplified from all isolates , and reference stains were digested separately with restriction enzymes , according to the manufacturer s instructions ."], ["a variety of nucleic acid detection methods that target both dna and rna have been developed ( 1 ) .", "the its1-rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples , and it enables determination of the infecting species ( 3 ) .", "the study was approved by the institutional ethics committee , and all patients signed informed consent forms .", "we evaluated the pcr - rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients .", "these patients were referred for diagnosis to the central laboratory of chabahar ( 25 17 n.70 38e ; 13,162 km ) for evaluation of skin diseases .", "the clinical samples were taken from patients suspected to have cl in different endemic areas of chabahar , including negor and other districts and villages .", "each diagnosis was confirmed by the demonstration of amastigotes on the slit smear and/or the presence of flagellated promastigotes in novy - macneal - nicolle ( nnn ) medium from skin samples of 24 patients with typical and atypical lesions . for the parasitological investigation ,", "two were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - tap tubes that contained blood agar slanted in nnn medium , then incubated at 25  1c .", "the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin ."], ["the diagnosis of leishmaniasis was confirmed by the presence of amastigotes in slit smears and/or the presence of flagellated promastigotes in nnn medium from typical and atypical lesions . for parasitological investigations ,", "three skin samples were collected from the edges of the lesions using sterile surgical blades .", "two of the samples were used for direct smears and were stained with giemsa , and the third was inoculated into sterile screw - top tubes containing blood agar slanted in nnn medium , then incubated at 25  1c .", "parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700  g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) ."], ["for species identification , the dna isolated from amastigotes in ulcers and promastigotes in cultures was extracted using a commercial extraction kit ( high pure template dna preparation kit , roche , germany ) , according to the manufacturer s instructions .", "the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l of l5.8s 5-tga tac cac tta tcg cat t-3 ( 12 ) .", "the reaction was carried out with the pcr - ready supreme mix in 47  l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix .", "amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide .", "major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls .", "the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes ."], ["the pcr products from its1-dna pcr were digested using the haeiii restriction endonuclease enzyme and the taq1 restriction enzyme , as recommended by the manufacture ( new england biolabs , inc . ) .", "the its1 sequence was amplified from all isolates , and reference stains were digested separately with restriction enzymes , according to the manufacturer s instructions ."], ["we used the pcr - rflp assay of its1 genes for the direct identification of leishmania species in 24 out of 33 patients suspected to have cl .", "these 24 patients , who were positive on smears and cultures for l. donovani bodies demonstrated microscopically , were selected for the study .", "the patient characteristics are shown in table 1 , including gender , age , number of lesions , ulcer duration , and distribution of ulcers on the body . with the use of litsr / l5.8s primer for pcr amplification ,", "single products of the expected size of 350 bp of l. major were detected in 21 patients ( figure 1 ) . in three isolates , a nonspecific 450 bp band was seen .", "rflp was performed on the its1 region in the ribosomal operons of 24 isolates of l. major . after using the restriction enzyme haeiii , banding patterns , including fragments of 140 bp and 210 bp bands ,", "were observed in 19 cases ( lma ) . in three cases with this enzyme , 300 bp and 150 bp bands ( figure 2 )", "m , molecular size marker ( 50 bp ) ; lane 1 , standard l. major ( mrho / ir/75/er ) ; lane 2 , standard l. tropica ( mhom / ir/99/yaz1 ) ; lane 3 , pcr products ( 350 bp ) as a standard ; lane 4 , pcr products ( 450 bp ; different from standard ) ; lanes 5 - 10 , pcr products ; lane 11 , negative control .", "m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , standard l. tropica ; lanes 3 - 9 , pcr products ; lane 10 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 11 , negative control .", "digestion of its1 from ribosomal dna with taq1 enzymes enabled us to identify two different patterns . using the taq1 restriction enzyme , banding patterns including 150 bp and 200 bp", "were observed in 19 cases , same as the standard of l. major ( genotype pattern lma ) . in three isolates ,", "digestion with this enzyme showed 300 bp and 150 bp banding patterns , dissimilar to the l. major reference strain . in two cases ,", "m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , l. tropica ; lanes 3 - 9 , pcr products ; lane 10 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 11 , negative control .", "m , molecular marker ( 50 bp ) ; lane 1 , standard l. major ; lane 2 , l. tropica ; lane 3 , pcr products ; lane 4 , pcr products ; lane 5 , pcr products ; lane 6 , the only sample different from the normal samples and standard patterns , probably flagellates ; lane 7 , pcr products ; lanes 8 - 10 , pcr products ; lane 11 , negative control ."], ["leishmaniasis is a major societal health problem that mostly affects populations where essential health services are not easily accessible and the detection of cl is based on clinical characteristics ( 14 ) .", "the present study shows that cl is caused by l. major in chabahar , iran .", "parasitological methods are the gold standard for the diagnosis of cl , with a high dependence on the number of parasites in the samples and a requirement of technical skill for sampling .", "the sensitivity of these methods varies from 27% to 85% for the diagnosis of leishmaniasis , which is their main disadvantage ( 15 ) .", "in addition , contamination of the cultured medium is an occasional problem ; such material must be discarded .", "molecular techniques have proved to be sensitive and powerful tools for detecting leishmania directly in clinical samples , as well as for parasite characterization using pcr ( 1 ) .", "some studies on viannia isolates from different hosts and geographic areas have found high levels of intra- and interspecific variation .", "schonian et al . established a diagnostic its1 pcr - rflp method using the restriction enzyme haeiii for leishmaniasis , combining high sensitivity for detecting leishmania directly in clinical materials and the ability to identify all medically relevant species groups ( 12 ) . in our study , using the haeiii restriction enzyme , the restriction analysis of the amplified its1 ribosomal dna revealed two different schizodeme patterns ( lma and lmb ) in l. major . on the other hand", ", some researchers have found that haeiii was not sufficient to distinguish all species of viannia ( 16 , 17 ) .", "we found that haeiii , rsa , and hinf1 can produce two profile bandings ; alu1 produces three ; dde1 and scrf1 produce four ; and taq1 produces five ( 1 ) . in this study ,", "( 20 ) , two profile bandings ( 220 bp and 140 bp ) in haeiii digestion were detected , similar to the standard .", "taq1 digestion showed two patterns with 150 bp and 200 bp bands . in contrary , in a study in yazd ( 20 ) on molecular identification using taq1 restriction enzymes , four patterns were produced .", "it should be emphasized that leishmaniasis in chabahar is new , and genetic variation is limited .", "the results of the present study showed that cl infections were more common in males ( 48.4% ) than in females ( 24.2% ) ( table 2 ) , probably because males spend more time outdoors than females do in this region .", "some studies have shown that pcr methods achieved positive results in more than 90% of cl cases ( 13 ) .", "the crithidias are able to endosymbiotically live within some parasites related to the trypanosomatidae family and sub - families of leishmania , and move to different hosts along with them .", "some researchers believe that the only way to isolate crithidia from leishmania is through kdna and/or ribosomal rna genes .", "it has been suggested that by extracting kdna and/or rdna , then amplifying them with pcr using the pcr - rflp technique with the haeiii enzyme and isolation of bands at 350 bp for leishmania and at 450 bp for crithidia , these two parasites can be isolated from each other ( 21 ) .", "however , its1-rflp pcr showed good sensitivity , and two its1 analyses showed the same results , as in a recent study by doudi et al .", "thus , the results of the present study confirm that in the border area of sistan va baluchestan , on the peninsula of chabahar , l. major is the causative agent of cl , with genotypic variation in this area ."]]}
{"article_id": "PMC4893412", "article_text": ["it is a multisystem disorder affecting the gastrointestinal , respiratory , hepatobiliary , and reproductive systems , as well as the sweat glands ( 1 - 6 ) .", "cf is caused by a mutation in the cystictransmembrane conductance regulator ( cftr ) gene , leading to defective ion transport of the cftr protein ( 7 - 10 ) .", "although numerous mutations in the cftr gene have been reported as a primary cause in the clinical manifestations and outcome of cf ( 9 ) , recent research has focused on the role of other factors such as gender ( 11 , 12 ) , race ( 1 ) , age ( 11 ) , geographic area ( 2 ) , infection with pseudomonas aeruginosa ( 11 , 13 ) , religion ( 8) , culture ( 1 , 14 ) , and nutritional status ( 13 - 15 ) .", "recently , improvement and progress in outcomes have been observed through better understanding of the disease , finding the effective factors in the start and progression of the disease , and ultimately in better management of the disease ( 16 ) .", "these factors have led to earlier diagnosis and more timely treatment , which could affect the clinical course and change the outcome for a patient .", "therefore , the link among researchers and clinicians will be stronger due to extensive research on the disease and improved introduction of the disease ( 17 , 18 ) .", "in addition , the research results could have an effective role in changing of the culture and laws of a society .", "retrospective research , in spite of some disadvantages , plays an important role in depicting the disease ( 16 , 19 ) .", "this research has been conducted after searching the electronic databases and performing a literature review , to ensure that no similar study had been undertaken in the region .", "this study aimed to assess the association between outcomes and demographic status in azeri turkish patients with cystic fibrosis .", "this was a cross sectional study conducted at the educational and treatment children s hospital of the university of medical sciences and the medical genetic laboratory , tabriz , iran , from march 2001 to september 2014 .", "the educational and treatment children s hospital is a governmental , specialized , and referral hospital with 150 beds and six wards in northwestern iran and dr .", "m. rafeey is the top , and the most professional , physician in this field and region .", "this study was performed on azeri turks , who are members of one of the largest ethnic groups in iran ( 20 ) .", "all the medical records of the patients were reviewed using the census method , and further information was gathered from patients , or their parents , through telephone interviews .", "the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60", "meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) .", "the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors .", "patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement .", "the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end ,", "331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 .", "data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth .", "ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group .", "a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) .", "a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree", ", first cousins were classified as the first group and the others as the second group .", "demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables .", "the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test .", "logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses ,", "odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance .", "ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) .", "participants were assured of confidentiality , and consent was obtained from the patients or their parents .", "the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60 meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) .", "the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors .", "patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement .", "the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end ,", "331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 .", "data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth .", "ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group .", "a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) .", "a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree", ", first cousins were classified as the first group and the others as the second group .", "demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables .", "the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test .", "logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses ,", "odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance .", "ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) .", "participants were assured of confidentiality , and consent was obtained from the patients or their parents .", "demographic data of this study is shown in table 1.there was a significant difference between frequencies of deceased and living patients ( p < 0.001 ) . in the application of the kolmogorov - smirnov test", ", age was assumed as non - normal distribution that uses the sample median and the iqr ( minimum - maximum ) , consequently , median age for living patients was 0.4 years ( 7 days30 years ) and median age at death was 4.8 years ( 3 months  43 years ) .", "abbreviations : d , days ; iqr , minimum - maximum ; m , months ; y , years .", "all data were mentioned as frequency ( % ) . for deceased cf - patients , median age at death and living cf - patients , median age at time of the study", "have been considered , because of non - normal of age and weight at birth , median and iqr were applied .", "eighty - five patients expired during the 13 years of the study with a mean of six deaths per year and at a ratio of 1:3 . during the time of the study , 77 ( 90.6% ) of", "the 85 deceased cf patients had died under the age of four years . among the 246 living cf patients , 131 ( 53.3% ) were less than four years of age .", "( % ) . at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females .", "the  test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step ,", "there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) .", "there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) .", "there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) .", "the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages .", "finally , weight at birth was computed and compared in cf patients , both deceased and living .", "there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) .", "there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) .", "associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced .", "results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 .", "at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females .", "the  test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step ,", "there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) .", "there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) .", "there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) .", "the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages .", "finally , weight at birth was computed and compared in cf patients , both deceased and living .", "there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) .", "there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) .", "associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced .", "results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 .", "the association between non - genetic variables and mortality in individuals with cf has been established ( 2 , 6 ) . despite improved management and increased awareness , outcome remains an important issue for cf patients , because non - genetic parameters , especially demographic factors , have been ignored by researchers and clinicians .", "the current study was performed to highlight the associations between demographic parameters and outcomes in cf patients . in this study ,", "the ratio of deceased patients to living patients was 1:3 , with a significant difference .", "eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) .", "therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf .", "in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18  12 and 16 years , respectively . in this study ,", "regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients ,", "the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study ,", "also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care were risk factors for a younger median age at death in non - european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries .", "we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) . in our study", ", there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females .", "a number of studies have indicated that males have a better survival rate than females , while lannefors et al .", "in another study , accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) . cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 )", "for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males .", "expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) .", "reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study , there was a significant difference between outcome and location of residence .", "the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) .", "the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival .", "growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) .", "continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) .", "the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) .", "preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) .", "in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients .", "patients living in urban areas are usually more likely to benefit from these conditions . in this study , there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born .", "management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) .", "cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand ,", "high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families ,", "patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history .", "there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study .", "ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease .", "patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research .", "considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) .", "social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently .", "the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) .", "most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) .", "ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found .", "the mean of weight at birth in the living cf population was slightly higher than the deceased cf population .", "cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) .", "patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) .", "an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) .", "it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival .", "the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients .", "the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research .", "another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results .", "data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers .", "the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time .", "data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes .", "this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies .", "this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes .", "clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas .", "it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity .", "couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy .", "other studies were performed for evaluating the association , if any , between outcome and other demographic factors .", "in this study , the ratio of deceased patients to living patients was 1:3 , with a significant difference .", "eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) .", "therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf .", "in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18  12 and 16 years , respectively . in this study , regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients ,", "the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study ,", "also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care", "european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries .", "we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) .", "in our study , there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females .", "a number of studies have indicated that males have a better survival rate than females , while lannefors et al .", "15 ) showed 45% and 49% of males with cf expired , respectively . in another study ,", "accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) .", "cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 ) may be reasons for the differences .", "for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males .", "expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) .", "reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study", "the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) .", "the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival .", "growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) .", "continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) .", "the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) .", "preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) .", "in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients . patients living in urban areas are usually more likely to benefit from these conditions . in this study", ", there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born .", "management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) . cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand ,", "high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families ,", "patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history .", "there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study .", "ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease .", "patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research .", "considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) .", "social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently .", "the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) .", "most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) .", "ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found .", "the mean of weight at birth in the living cf population was slightly higher than the deceased cf population .", "cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) .", "patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) .", "an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) .", "it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival .", "the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients .", "the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research .", "another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results .", "data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers .", "the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time .", "data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes .", "this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies .", "this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes .", "clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas .", "it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity .", "couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy .", "other studies were performed for evaluating the association , if any , between outcome and other demographic factors ."], "abstract_text": ["<S> backgroundoutcomesforcystic fibrosis patients are improving rapidly . </S>", "<S> the demographic factors are notable variables inoutcomes , which can be evaluated and modified.objectivesthis study was designed to investigate the association between outcome and demographic factors in patients with cystic fibrosis.patients and methodsthis was a cross - sectional study and data were gathered for 331 patients using the census method , from march 2001 to september 2014 in iran . </S>", "<S> data was analyzed using logistic regression analysis , chi - square test , and independent sample t test using spss 18 . </S>", "<S> odds ratio with confidence intervals of 95% and p < 0.05 were considered significant.resultsthere were 85 ( 25.7% ) deceased patients and 246 ( 74.3% ) living patients at the time of the study . </S>", "<S> of the 246 living cf patients , 202 ( 82.2% ) were less than nine years of age , and 77 ( 90.6% ) out of the 85 deceased cf patients had died younger than four years of age . </S>", "<S> there was a significant difference between outcome and location of residence . </S>", "<S> the risk of mortality was 50% less in urban patients than in rural patients ( p = 0.03 ) . </S>", "<S> the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( p = 0.02 ) . </S>", "<S> the proportion of mortality was approximately two times , or 94% , higher for those in a consanguineous marriage than for those in a non - consanguineous marriage ( p = 0.01).conclusionsthe results demonstrated that the mortality rate was higher in cf patients with a positive family history , a consanguineous marriage , and residence in a rural area . </S>", "<S> therefore , demographic factors play an important role in the outcome of cystic fibrosis . </S>", "<S> unfortunately , these parameters , which can be managed easily and with low cost , have been overlooked . </S>"], "labels": null, "section_names": ["1. Background", "2. Objectives", "3. Patients and Methods", "3.1. Inclusion and Exclusion Criteria", "3.2. Data Extraction and Statistical Analysis", "3.3. Ethical Standards", "4. Results", "4.1. Association Between Outcome and Demographic Status", "4.2. Factors Associated With Outcome", "5. Discussion", "5.1. Demographic Characteristics", "5.2. Association Between Outcome and Demographic Status", "5.3. Strengths and Weaknesses", "5.4. Conclusions"], "sections": [["cystic fibrosis ( cf ; omim 219700 ) is the most common lethal autosomal recessive disorder ( 1 , 2 ) , with an incidence rate of 1 in 2000 - 3000 live births in europe and 1 in 10,000 births for asians living in the united kingdom ( 1 ) .", "it is a multisystem disorder affecting the gastrointestinal , respiratory , hepatobiliary , and reproductive systems , as well as the sweat glands ( 1 - 6 ) .", "cf is caused by a mutation in the cystictransmembrane conductance regulator ( cftr ) gene , leading to defective ion transport of the cftr protein ( 7 - 10 ) .", "although numerous mutations in the cftr gene have been reported as a primary cause in the clinical manifestations and outcome of cf ( 9 ) , recent research has focused on the role of other factors such as gender ( 11 , 12 ) , race ( 1 ) , age ( 11 ) , geographic area ( 2 ) , infection with pseudomonas aeruginosa ( 11 , 13 ) , religion ( 8) , culture ( 1 , 14 ) , and nutritional status ( 13 - 15 ) .", "recently , improvement and progress in outcomes have been observed through better understanding of the disease , finding the effective factors in the start and progression of the disease , and ultimately in better management of the disease ( 16 ) .", "these factors have led to earlier diagnosis and more timely treatment , which could affect the clinical course and change the outcome for a patient .", "therefore , the link among researchers and clinicians will be stronger due to extensive research on the disease and improved introduction of the disease ( 17 , 18 ) .", "in addition , the research results could have an effective role in changing of the culture and laws of a society .", "retrospective research , in spite of some disadvantages , plays an important role in depicting the disease ( 16 , 19 ) .", "this research has been conducted after searching the electronic databases and performing a literature review , to ensure that no similar study had been undertaken in the region ."], ["this study aimed to assess the association between outcomes and demographic status in azeri turkish patients with cystic fibrosis ."], ["this was a cross sectional study conducted at the educational and treatment children s hospital of the university of medical sciences and the medical genetic laboratory , tabriz , iran , from march 2001 to september 2014 .", "the educational and treatment children s hospital is a governmental , specialized , and referral hospital with 150 beds and six wards in northwestern iran and dr .", "m. rafeey is the top , and the most professional , physician in this field and region .", "this study was performed on azeri turks , who are members of one of the largest ethnic groups in iran ( 20 ) .", "all the medical records of the patients were reviewed using the census method , and further information was gathered from patients , or their parents , through telephone interviews .", "the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60", "meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) .", "the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors .", "patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement .", "the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end ,", "331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 .", "data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth .", "ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group .", "a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) .", "a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree", ", first cousins were classified as the first group and the others as the second group .", "demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables .", "the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test .", "logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses ,", "odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance .", "ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) .", "participants were assured of confidentiality , and consent was obtained from the patients or their parents ."], ["the diagnosis of cf was based on typical clinical features and confirmed by sweat chloride level > 60 meq / l , according to the method of gibson and cooke , or detection of mutations in the cftr gene known to cause cf ( 21 ) .", "the medical records of 442 patients who met the inclusion criteria were separately reviewed by two authors .", "patients with incomplete records and who were not diagnosed as having cf were excluded with the reviewer s agreement .", "the kappa agreement rate was higher than 85% , as inclusion criteria to the next phase . in the end ,", "331 cases were included in this study , which was conducted over a 13-year period from 2001 to 2014 ."], ["data included patient outcome , gender , age , residence , ranking in birth order of the family s children , family history , consanguineous marriage , degree of consanguineous marriage , and weight at birth .", "ranking in birth order of the family s children was divided into two groups , including one child as the first group , and two children or more as the second group .", "a positive family history for cf was considered as having had a first , second , or third - degree relative affected with cf ( 22 ) .", "a consanguineous marriage was defined as a union between two persons who are related as first cousins ( 3rd degree ) , first cousins once removed or double second cousins ( 4 degree ) , and second cousins ( 5 degree ) . the first degree and the second degree", ", first cousins were classified as the first group and the others as the second group .", "demographic data were analyzed using spss 18 for means and standard deviations for normally distributed continuous variables , and median and interquartile ranges were obtained for non - normally distributed continuous variables .", "the comparison between variables was analyzed with a chi - square or fisher s exact test , and independent samples t test .", "logistic regression analyses were carried out to identify variables independently associated with gender . in all analyses ,", "odds ratio ( or ) with a 95% confidence interval , and p < 0.05 indicated statistical significance ."], ["ethical aspects were considered and approval for the study was gained by the ethics committee of the university ( no = 5/4/1775 ) .", "participants were assured of confidentiality , and consent was obtained from the patients or their parents ."], ["demographic data of this study is shown in table 1.there was a significant difference between frequencies of deceased and living patients ( p < 0.001 ) . in the application of the kolmogorov - smirnov test", ", age was assumed as non - normal distribution that uses the sample median and the iqr ( minimum - maximum ) , consequently , median age for living patients was 0.4 years ( 7 days30 years ) and median age at death was 4.8 years ( 3 months  43 years ) .", "abbreviations : d , days ; iqr , minimum - maximum ; m , months ; y , years .", "all data were mentioned as frequency ( % ) . for deceased cf - patients , median age at death and living cf - patients , median age at time of the study", "have been considered , because of non - normal of age and weight at birth , median and iqr were applied .", "eighty - five patients expired during the 13 years of the study with a mean of six deaths per year and at a ratio of 1:3 . during the time of the study , 77 ( 90.6% ) of", "the 85 deceased cf patients had died under the age of four years . among the 246 living cf patients , 131 ( 53.3% ) were less than four years of age .", "( % ) . at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females .", "the  test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step ,", "there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) .", "there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) .", "there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) .", "the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages .", "finally , weight at birth was computed and compared in cf patients , both deceased and living .", "there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) .", "there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) .", "associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced .", "results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 ."], ["at first , we calculated the association between outcome and gender . at the time of the study , 51 ( 26.7% ) of the deceased patients were males and 34(24.3% ) were females .", "the  test did not show any significant difference between outcome and gender ( or = 1.13 [ 95% ci , 0.68 - 1.87 ] , p = 0.6 ) . in the next step ,", "there was a significant difference between the outcome and the geographic location ( or = 0.55 [ 95% ci , 0.32 - 0.95 ] , p = 0.03 ) .", "there was no significant difference between outcome and ranking among the family s children , while 38 ( 23.9% ) were first - born children , and 47 ( 27.3% ) were second or later born children ( or = 0.83 [ 95% ci , 0.50 - 1.37 ] , p = 0.4 ) . in this study , we analyzed family history status among these patients , and the risk of mortality was approximately two times higher in patients with a positive family history than in those with a negative family history ( or = 1.91 [ 95% ci , 1.09 - 3.36 ] , p = 0.02 ) .", "there was a statistically significant difference between the outcome and consanguineous marriage ( or = 1.94 [ 95% ci , 1.14 - 3.29 ] , p = 0.01 ) , but no significant difference in degree of positive consanguineous marriage ( or = 0.68 [ 95% ci , 0.34 - 1.37 ] , p = 0.2 ) .", "the risk of mortality was 1.94 times higher for patients in consanguineous marriages than for those in non - consanguineous marriages .", "finally , weight at birth was computed and compared in cf patients , both deceased and living .", "there was no significant difference between the outcome and weight at birth ( or = 1 [ 95% ci , 1 - 1.001 ] , p = 0.7 ) ( tables 1 and 3 ) ."], ["there was no statistically significant difference when logistic regression analysis was considered with the or for all of the variables ( p > 0.05 ) .", "associations between gender , ranking in birth order of the family s children , degree of consanguineous marriage , and weight at birth were not significant in terms of outcome , and therefore , omitted from the final model . when residential area , family history , and consanguineous marriage were entered into the model , the difference associated with outcome and or was somewhat reduced .", "results of logistic regression analysis illustrated the adjusted or for the final model and are shown in table 3 ."], ["the association between non - genetic variables and mortality in individuals with cf has been established ( 2 , 6 ) . despite improved management and increased awareness , outcome remains an important issue for cf patients , because non - genetic parameters , especially demographic factors , have been ignored by researchers and clinicians .", "the current study was performed to highlight the associations between demographic parameters and outcomes in cf patients . in this study ,", "the ratio of deceased patients to living patients was 1:3 , with a significant difference .", "eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) .", "therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf .", "in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18  12 and 16 years , respectively . in this study ,", "regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients ,", "the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study ,", "also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care were risk factors for a younger median age at death in non - european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries .", "we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) . in our study", ", there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females .", "a number of studies have indicated that males have a better survival rate than females , while lannefors et al .", "in another study , accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) . cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 )", "for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males .", "expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) .", "reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study , there was a significant difference between outcome and location of residence .", "the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) .", "the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival .", "growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) .", "continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) .", "the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) .", "preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) .", "in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients .", "patients living in urban areas are usually more likely to benefit from these conditions . in this study , there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born .", "management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) .", "cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand ,", "high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families ,", "patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history .", "there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study .", "ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease .", "patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research .", "considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) .", "social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently .", "the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) .", "most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) .", "ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found .", "the mean of weight at birth in the living cf population was slightly higher than the deceased cf population .", "cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) .", "patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) .", "an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) .", "it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival .", "the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients .", "the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research .", "another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results .", "data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers .", "the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time .", "data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes .", "this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies .", "this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes .", "clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas .", "it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity .", "couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy .", "other studies were performed for evaluating the association , if any , between outcome and other demographic factors ."], ["in this study , the ratio of deceased patients to living patients was 1:3 , with a significant difference .", "eighty - five patients expired during the period of 13 years , with a mean of six deaths per year . in a study performed by dodge et al . in the uk from 1996 to 2003 , there were 1,066 deaths , with a mean of 133 deaths per year ( 15 ) .", "therefore , based on these findings the mortality rate is lower in our study than in the uk , perhaps because this study has been performed on an ethnic group in iran with a low incidence of cf .", "in addition , it is likely that a number of deaths were not recorded or were not reported by the family in our study . in a previous study in sweden , lannefors et al . reported data from 1971 - 1999 showing that while the incidence of cf was 1/5600 live - births , the mean and median age of living cf patients were 18  12 and 16 years , respectively . in this study , regarding deceased patients , 45% were older than 18 years and 10% were older than 35 years . in deceased patients ,", "the median age at death was 26 years ( range 0 - 72 ) ( 2 ) . in another study ,", "also , this statistic had been observed in median age at death . delayed diagnosis and inappropriate health care", "european countries ( 24 ) . in our study , living cf patients had lower mean , median , and were of an older age than that reported in developed countries . also , in deceased cf patients the mean and median age at death were lower than in developed countries .", "we hypothesize that this difference may be due to the lack of proper equipment , inadequate diagnostic processes , and absence of treatment by specialists and expert staff , as well as poor nutritional status in our region compared to developed countries ( 13 , 24 ) ."], ["in our study , there was no significant difference between outcome and gender ; however , the mortality rate was somewhat higher in males than females .", "a number of studies have indicated that males have a better survival rate than females , while lannefors et al .", "15 ) showed 45% and 49% of males with cf expired , respectively . in another study ,", "accumulated data related to the mortality rate between genders reflects a higher mortality rate in cf females compared to males ( 6 ) .", "cultural issues and intrinsic ethnic differences ( 1 ) , as well as religion ( 8 , 25 ) and geographical elements , ( 9 ) may be reasons for the differences .", "for example , females are more susceptible to cf because of their body functions ; consequently , they more often report problems than males .", "expression of problems can be observed more in females than males in our region because of cultural issues ( 26 ) .", "reasons for this difference may be unclear , so it is necessary to perform additional investigations in a large population from neighboring countries to elucidate this issue . in the current study", "the mortality rate of cf was 50% higher in rural patients than urban patients , in spite of less air pollution ( 10 ) , lower stress , sufficient exercise , and organic nutritional intake in rural areas ( 13 , 27 ) .", "the availability of appropriate facilities to diagnosis and treat the disease is an important factor in patient survival .", "growth and lung health of children is influenced by proper treatment , and decreased pulmonary function is associated with morbidity and shortened survival for cf patients ( 28 ) .", "continuous care , suitable food , and daily physiotherapy are the main elements in survival of cf ( 27 ) .", "the relationship between improved nutritional support and increased survival in cf has been established , and retention of balanced and stable nutritional status is the best method of treatment for cf ( 13 , 27 ) .", "preventive treatments , such as adequate exercise , have been introduced at early ages for cf patients , and it appears to be an important factor in health maintenance in patients with cf ( 27 ) .", "in addition , a healthy lifestyle ( 27 ) , related to appropriate family functioning ( 29 ) , elevated levels of social class , education , and higher income ( 19 , 30 ) are determinates of survival for cf patients . patients living in urban areas are usually more likely to benefit from these conditions . in this study", ", there was no significant difference between outcome and ranking in birth order of the family s children , although the mortality rate was slightly higher for the second or subsequent children than for the first - born .", "management of patients with cf is one of the greatest challenges for families , and the family plays an important role in the treatment of cf ( 30 ) . cf patients need constant care such as physiotherapy , various medications , proper dietary intake , exercise , and adequate support ( 21 , 31 ) . on the other hand ,", "high socioeconomic status with adequate income , suitable occupation , and employment status were identified as factors related to a good prognosis in affected individuals ( 30 ) . in crowded families ,", "patients with a positive family history , such as a diagnostic category ( 32 , 33 ) , had a higher mortality rate than those with a negative family history .", "there was a significant difference , with the risk of mortality approximately two times , or 91% , higher with a positive family history than a negative family history , in the current study .", "ormond et al . published findings of a study stating that individuals affected by cf are segregated into four categories based on severity of disease .", "patients with a family history of cf showed a broader range of superficial severity with an unknown reason ( 34).understanding the reasons these patients have poorer prognoses than others will need additional research .", "considering consanguineous marriage as a predicating factor of outcome , children born to related parents were 94% more at risk for death than children of non - consanguineous marriage , especially in consanguineous marriage of the first - cousin type ( 3rd - degree ) .", "social conditions , religion , economic situations , and traditional beliefs are essential considerations in the preference for consanguineous marriage . in muslim populations , first - cousin unions ( 3rd - degree ) between a man and his father s or mother s brother s daughter , and the opposite , were detected frequently .", "the incidence of consanguinity has been reported to be 56% in middle east with saudi arabia having the highest prevalence at 89% in families with cf ( 35 ) .", "most studies have shown that early mortality is higher in the progeny of consanguineous unions than in non - consanguineous marriage ( 36 ) .", "ultimately , weight at birth was reviewed in deceased patients and living patients and no significant difference between them was found .", "the mean of weight at birth in the living cf population was slightly higher than the deceased cf population .", "cf is an autosomal recessive disorder affecting several organs , such as the digestive system , which is manifested in fat malabsorption , steatorrhea , and poor growth ( 2 , 21 ) .", "patients who intake adequate dietary requirements and gain proper weight have better pulmonary function ( 1 , 13 ) .", "an increased rate of mortality was associated with malnutrition , growth retardation , and declining lung function ( 1 , 13 , 37 ) .", "it is thought that in consanguineous marriage this was observed and that low weight at birth in cf patients causes shortened survival .", "the presence of ranking among the family s children , degree of consanguineous marriage , and weight at birth do not rule in the mortality of cf , although location of residence , family history , and consanguineous marriage are predictors of increased mortality in cf patients ."], ["the primary weakness of this study was that it was a retrospective study and may have limited accuracy , but it does lead the way to prospective research .", "another weak point was the investigation was limited to a specialist group , which decreases the ability to generalize the study results .", "data were limited to the children s hospital of the university of medical sciences and medical genetic laboratory in tabriz , and it was possible that some cf patients were not referred to these centers .", "the strength of this study was the adequate sample size in this population , and that it was conducted over a long period of time .", "data that was approved by two reviewers were collected from educational , referral , and therapeutic centers , and were designed to evaluate the effects of non - genetic factors on outcomes .", "this study was conducted in a population with a unique religion , ethnic origin , and culture , which could be a basis for the performance of comparative studies ."], ["this study indicates an association between outcome and the number of demographic factors in cf patients and emphasized the role of these factors in outcomes .", "clinicians need to be aware and educate patients and their families about treatments such as the correct use of medicines ( oral nutritional supplements , antibiotics , and nebulizers ) , physiotherapy , and proper nutrition , which are essential for a positive prognosis , especially in patients living in rural areas .", "it should be required that the government and relevant organizations focus on informing the public of the dangers of consanguinity .", "couples with consanguineous marriages , especially with a positive family history of the disease , require more frequent and thorough evaluations before pregnancy .", "other studies were performed for evaluating the association , if any , between outcome and other demographic factors ."]]}
{"article_id": "PMC3563348", "article_text": ["the original technique of staining the anterior capsule with trypan blue ( tb ) in eyes with no red reflex involves the injection of the dye with a cannula after filling the anterior chamber with an air bubble.1 the stability of the anterior chamber can be compromised by air escaping when the cannula is introduced and the dye is injected .", "moreover , the superficial tension of air bubble and capillary action between tb and the metal cannula can force the dye to build up within the aqueous meniscus surrounding the air bubble , making selective staining of the capsule difficult .", "several techniques using viscoelastic substances as an alternative to air have been presented to increase the stability of the anterior chamber.25 however , staining under viscoelastic material can be time - consuming and expensive , as it requires the mechanical spreading of tb onto the anterior capsule and the replacement of the viscous solution when an excessive diffusion of dye affects its transparency .", "we present an under - air staining technique of the anterior capsule using one drop of tb injected with a 30 g needle through the peripheral cornea .", "our procedure prevents the exit of air during the injection of the dye , resulting in increased stability of the anterior chamber .", "moreover , it allows selective staining of the capsule using the needle and avoids an excessive buildup of tb in the anterior chamber .", "connect a 30 g needle to a 2.25 ml luer lock syringe containing trypan blue 0.06% ( visionblue ; dutch ophthalmic research center international , zuidland , netherlands ) .", "make a standard side - port incision with a 15 blade . use a 25 g cannula connected to a 5 ml syringe to inject a big air bubble in the anterior chamber .", "introduce the 30 g needle bevel - up in the anterior chamber through the peripheral cornea between the side - port incision and the site chosen for the corneal tunnel .", "push the needle forward in the air bubble right between the anterior capsule and posterior corneal surface until the tip reaches the center of the pupil .", "unlike what occurs using a cannula , the air can not escape from the anterior chamber .", "inject gently , allowing the tb volume to grow within the needle bevel ( figure 1 ) .", "rotate the syringe 90 , letting the drop fall on the anterior capsule ( figure 2 ) .", "the needle tip allows precise control on the injected dye , similarly to a dropper . if the staining is not sufficient , it is possible to inject one more drop of tb on the unstained area .", "once the needle is removed , the perforation will close spontaneously in a few seconds , and if necessary an absorbent stick can be used to stop tiny leakages of air and/ or dye .", "inject viscoelastic substance in the anterior chamber through the side - port incision , allowing the air and the leftover dye to exit the eye .", "tb is the most used dye to perform capsulorhexis in eyes with no red reflex.6 the main staining techniques involve tb injection under air,1 under viscoelastic material,25 or directly under aqueous humor .", "an air bubble within the anterior chamber reduces the contact between the dye and the corneal endothelium and allows the delivery of undiluted tb onto the anterior capsule .", "however , the air bubble makes the anterior chamber unstable and prone to collapse when the cannula is introduced to inject the dye .", "in such an event , the anterior chamber has to be reinflated , thus increasing the duration of surgery .", "moreover , sudden reductions in volume of the anterior chamber can cause narrowing of the pupil and damage to the corneal endothelium . to prevent the air bubble from escaping the anterior chamber , it has been proposed that a small amount of high - density viscoelastic material be placed near the side - port incision.7 in our technique , we inject the dye in the anterior chamber , introducing a 30 g needle through the peripheral cornea . in this way", ", the air can not escape from the side - port incision , as often occurs when a cannula is used .", "once the needle is pulled out , the corneal perforation spontaneously closes in a few seconds .", "when the dye is injected with a blunt cannula through the side - port incision , the superficial tension of the air bubble spreads the dye into the thin liquid layer between the air and the anterior capsule.8 in this phase , a meniscus of tb forms between the metal cannula and the anterior capsule due to the capillary action and adhesive forces . to obtain the right staining ,", "the anterior capsule must be swept thoroughly with the cannula , allowing the meniscus of tb to contact the largest possible area .", "if too much dye is injected , it can migrate back between the cannula and the capsule , escaping from the paracentesis , or it could build up in the aqueous meniscus surrounding the air bubble . to avoid this ,", "toprak et al8 modified a cannula by bending it to increase the surface contact between the dye and the capsule . in our procedure", ", we use a very sharp - tipped needle , which reduces the adhesive forces with tb and allows the drop of dye to fall easily over the anterior capsule .", "many authors have described alternative staining techniques based on viscoelastic substances instead of air.25 these techniques have the advantage of making the anterior chamber more stable and of better dosing the amount of injected dye . however , when the anterior chamber is filled with viscoelastic substance , it is difficult to deliver the dye onto the capsule .", "the surgeon has to mechanically spread the dye on the largest possible surface of the capsule , and at times the dyed viscoelastic material or pockets of dye can affect the visibility of the capsule beneath . to avoid these problems , special cannulae", "have been proposed.2,4 rarely , an exchange of the viscoelastic material is needed to restore the transparency before the capsulorhexis .", "we injected tb with a 30 g needle in seven consecutive patients with white cataract and normal anterior chamber depth . in this small group of patients , we have not observed intra- or postoperative complications . after surgery , all patients showed marked improvement in their visual acuity . in six patients , we performed a regular capsulorhexis after injecting only one drop of dye . in a single patient ,", "one more drop of tb had to be injected to build up the staining of the capsule .", "an additional corneal access and a sharp needle in the anterior chamber are the major disadvantages of our technique . in eyes with shallow anterior chamber or with excessive posterior pressure", ", the use of the 30 g needle technique could be dangerous and should be avoided . in conclusion , in a small series of seven patients , the staining of the anterior capsule using tb under air with a 30 g needle allowed for enhanced stability of the anterior chamber and the use of the smallest quantity of dye ."], "abstract_text": ["<S> the original technique of staining the anterior capsule of the lens with trypan blue involves the injection of an air bubble in the anterior chamber . </S>", "<S> a drawback of this technique is the possible instability of the anterior chamber caused by the sudden exit of air when the dye is injected with the cannula through the side - port incision . </S>", "<S> other staining techniques that use viscoelastic substances to increase the stability of the anterior chamber and to dose the injected dye have been described . </S>", "<S> the authors present an under - air staining technique of the anterior capsule using one drop of trypan blue injected with a 30 g needle through the peripheral cornea . </S>", "<S> this procedure prevents the air bubble from escaping the anterior chamber and allows fast and selective staining of the capsule . </S>"], "labels": null, "section_names": ["Introduction", "Technique", "Discussion"], "sections": [["the original technique of staining the anterior capsule with trypan blue ( tb ) in eyes with no red reflex involves the injection of the dye with a cannula after filling the anterior chamber with an air bubble.1 the stability of the anterior chamber can be compromised by air escaping when the cannula is introduced and the dye is injected .", "moreover , the superficial tension of air bubble and capillary action between tb and the metal cannula can force the dye to build up within the aqueous meniscus surrounding the air bubble , making selective staining of the capsule difficult .", "several techniques using viscoelastic substances as an alternative to air have been presented to increase the stability of the anterior chamber.25 however , staining under viscoelastic material can be time - consuming and expensive , as it requires the mechanical spreading of tb onto the anterior capsule and the replacement of the viscous solution when an excessive diffusion of dye affects its transparency .", "we present an under - air staining technique of the anterior capsule using one drop of tb injected with a 30 g needle through the peripheral cornea .", "our procedure prevents the exit of air during the injection of the dye , resulting in increased stability of the anterior chamber .", "moreover , it allows selective staining of the capsule using the needle and avoids an excessive buildup of tb in the anterior chamber ."], ["connect a 30 g needle to a 2.25 ml luer lock syringe containing trypan blue 0.06% ( visionblue ; dutch ophthalmic research center international , zuidland , netherlands ) .", "make a standard side - port incision with a 15 blade . use a 25 g cannula connected to a 5 ml syringe to inject a big air bubble in the anterior chamber .", "introduce the 30 g needle bevel - up in the anterior chamber through the peripheral cornea between the side - port incision and the site chosen for the corneal tunnel .", "push the needle forward in the air bubble right between the anterior capsule and posterior corneal surface until the tip reaches the center of the pupil .", "unlike what occurs using a cannula , the air can not escape from the anterior chamber .", "inject gently , allowing the tb volume to grow within the needle bevel ( figure 1 ) .", "rotate the syringe 90 , letting the drop fall on the anterior capsule ( figure 2 ) .", "the needle tip allows precise control on the injected dye , similarly to a dropper . if the staining is not sufficient , it is possible to inject one more drop of tb on the unstained area .", "once the needle is removed , the perforation will close spontaneously in a few seconds , and if necessary an absorbent stick can be used to stop tiny leakages of air and/ or dye .", "inject viscoelastic substance in the anterior chamber through the side - port incision , allowing the air and the leftover dye to exit the eye ."], ["tb is the most used dye to perform capsulorhexis in eyes with no red reflex.6 the main staining techniques involve tb injection under air,1 under viscoelastic material,25 or directly under aqueous humor .", "an air bubble within the anterior chamber reduces the contact between the dye and the corneal endothelium and allows the delivery of undiluted tb onto the anterior capsule .", "however , the air bubble makes the anterior chamber unstable and prone to collapse when the cannula is introduced to inject the dye .", "in such an event , the anterior chamber has to be reinflated , thus increasing the duration of surgery .", "moreover , sudden reductions in volume of the anterior chamber can cause narrowing of the pupil and damage to the corneal endothelium . to prevent the air bubble from escaping the anterior chamber , it has been proposed that a small amount of high - density viscoelastic material be placed near the side - port incision.7 in our technique , we inject the dye in the anterior chamber , introducing a 30 g needle through the peripheral cornea . in this way", ", the air can not escape from the side - port incision , as often occurs when a cannula is used .", "once the needle is pulled out , the corneal perforation spontaneously closes in a few seconds .", "when the dye is injected with a blunt cannula through the side - port incision , the superficial tension of the air bubble spreads the dye into the thin liquid layer between the air and the anterior capsule.8 in this phase , a meniscus of tb forms between the metal cannula and the anterior capsule due to the capillary action and adhesive forces . to obtain the right staining ,", "the anterior capsule must be swept thoroughly with the cannula , allowing the meniscus of tb to contact the largest possible area .", "if too much dye is injected , it can migrate back between the cannula and the capsule , escaping from the paracentesis , or it could build up in the aqueous meniscus surrounding the air bubble . to avoid this ,", "toprak et al8 modified a cannula by bending it to increase the surface contact between the dye and the capsule . in our procedure", ", we use a very sharp - tipped needle , which reduces the adhesive forces with tb and allows the drop of dye to fall easily over the anterior capsule .", "many authors have described alternative staining techniques based on viscoelastic substances instead of air.25 these techniques have the advantage of making the anterior chamber more stable and of better dosing the amount of injected dye . however , when the anterior chamber is filled with viscoelastic substance , it is difficult to deliver the dye onto the capsule .", "the surgeon has to mechanically spread the dye on the largest possible surface of the capsule , and at times the dyed viscoelastic material or pockets of dye can affect the visibility of the capsule beneath . to avoid these problems , special cannulae", "have been proposed.2,4 rarely , an exchange of the viscoelastic material is needed to restore the transparency before the capsulorhexis .", "we injected tb with a 30 g needle in seven consecutive patients with white cataract and normal anterior chamber depth . in this small group of patients , we have not observed intra- or postoperative complications . after surgery , all patients showed marked improvement in their visual acuity . in six patients , we performed a regular capsulorhexis after injecting only one drop of dye . in a single patient ,", "one more drop of tb had to be injected to build up the staining of the capsule .", "an additional corneal access and a sharp needle in the anterior chamber are the major disadvantages of our technique . in eyes with shallow anterior chamber or with excessive posterior pressure", ", the use of the 30 g needle technique could be dangerous and should be avoided . in conclusion , in a small series of seven patients , the staining of the anterior capsule using tb under air with a 30 g needle allowed for enhanced stability of the anterior chamber and the use of the smallest quantity of dye ."]]}
{"article_id": "PMC3226418", "article_text": ["high resolution transmission electron microscopy and high angle annular dark - field scanning transmission electron microscopy were employed to investigate the microstructure and the epitaxial growth of the structures .", "room temperature ferroelectric and ferrimagnetic domains of the heterostructures were imaged by piezoresponse force microscopy ( pfm ) and magnetic force microscopy ( mfm ) , respectively .", "pfm and mfm investigations proved that the hybrid epitaxial nanostructures show ferroelectric and magnetic order at room temperature .", "dielectric effects occurring after repeated switching of the polarization in large planar capacitors , comprising ferrimagnetic nife2o4 dots embedded in ferroelectric pbzr0.52ti0.48o3 matrix , were studied .", "these hybrid multiferroic structures with clean and well defined epitaxial interfaces hold promise for reliable investigations of magnetoelectric coupling between the ferrimagnetic / magnetostrictive and ferroelectric / piezoelectric phases .", "there is no conflict of interest in the present study for any of the authors ."], "abstract_text": ["<S> epitaxial heterostructures combining ferroelectric ( fe ) and ferromagnetic ( fim ) oxides are a possible route to explore coupling mechanisms between the two independent order parameters , polarization and magnetization of the component phases . </S>", "<S> we report on the fabrication and properties of arrays of hybrid epitaxial nanostructures of fim nife2o4 ( nfo ) and fe pbzr0.52ti0.48o3 or pbzr0.2ti0.8o3 , with large range order and lateral dimensions from 200 nm to 1 micron.methodsthe structures were fabricated by pulsed - laser deposition . </S>", "<S> high resolution transmission electron microscopy and high angle annular dark - field scanning transmission electron microscopy were employed to investigate the microstructure and the epitaxial growth of the structures . </S>", "<S> room temperature ferroelectric and ferrimagnetic domains of the heterostructures were imaged by piezoresponse force microscopy ( pfm ) and magnetic force microscopy ( mfm ) , respectively.resultspfm and mfm investigations proved that the hybrid epitaxial nanostructures show ferroelectric and magnetic order at room temperature . </S>", "<S> dielectric effects occurring after repeated switching of the polarization in large planar capacitors , comprising ferrimagnetic nife2o4 dots embedded in ferroelectric pbzr0.52ti0.48o3 matrix , were studied.conclusionthese hybrid multiferroic structures with clean and well defined epitaxial interfaces hold promise for reliable investigations of magnetoelectric coupling between the ferrimagnetic / magnetostrictive and ferroelectric / piezoelectric phases . </S>"], "labels": null, "section_names": ["Methods", "Results", "Conclusion", "Conflict of interest and funding"], "sections": [["high resolution transmission electron microscopy and high angle annular dark - field scanning transmission electron microscopy were employed to investigate the microstructure and the epitaxial growth of the structures .", "room temperature ferroelectric and ferrimagnetic domains of the heterostructures were imaged by piezoresponse force microscopy ( pfm ) and magnetic force microscopy ( mfm ) , respectively ."], ["pfm and mfm investigations proved that the hybrid epitaxial nanostructures show ferroelectric and magnetic order at room temperature .", "dielectric effects occurring after repeated switching of the polarization in large planar capacitors , comprising ferrimagnetic nife2o4 dots embedded in ferroelectric pbzr0.52ti0.48o3 matrix , were studied ."], ["these hybrid multiferroic structures with clean and well defined epitaxial interfaces hold promise for reliable investigations of magnetoelectric coupling between the ferrimagnetic / magnetostrictive and ferroelectric / piezoelectric phases ."], ["there is no conflict of interest in the present study for any of the authors ."]]}
{"article_id": "PMC3592510", "article_text": ["malnutrition is a common and serious problem among geriatric patients , particularly for those who are diagnosed with lung cancer caused by declined functional reserve of visceral organs , decreased stability of the internal environment , and effects of acute or chronic diseases .", "malnutrition can lead to increased risk of surgery , decreased quality of life , increased incidence of postoperative complications , increased fatality rate , and decreased survival rate among elderly lung cancer inpatients.1,2 nutrition support has also been reported to improve the perioperative nutritional status and reduce the severity of postoperative complications in lung cancer.3 in the people s republic of china , the number of elderly lung cancer inpatients is increasing at a rapid rate ; thus , there is a great need to develop a tool that can accurately and effectively assess the nutritional status of these patients . the mini - nutritional assessment ( mna ) is a tool that is widely used in the usa and europe to evaluate patients nutritional status .", "this study examines the use of the mna and short - form mna ( mna - sf ) ( with a revised scoring system to better suit the elderly chinese population5 ) in assessing the nutritional status of elderly chinese lung cancer inpatients prior to scheduled surgery .", "a total of 180 elderly chinese lung cancer inpatients ( 120 men and 60 women ) aged 60 and older were randomly selected from the tianjin medical university cancer institute and hospital between june 2010 and july 2011 .", "the original mna and mna - sf scoring systems developed by guigoz4 were revised to account for differences in height , weight , and dietary behaviors between asian and western populations . for the present study , guigoz s cutoff point of < 17 for malnutrition", "was changed to < 19 , thereby modifying the overall scoring system for assessing patients nutritional status ( maximum score = 30 ) , thus : a score  24 would indicate that a patient was well nourished , a score in the range 1923 would indicate that a patient was at risk of malnutrition , and a score < 19 would indicate that a patient was malnourished.5 in addition , the body mass index ( bmi ) ( kg / m2 ) ranges were adjusted to align with the following scores : < 18.50 kg / m = 0 ( vs < 19.00 kg / m = 0 ) , 18.5021.25 kg / m = 1 ( vs 19.0021.00 kg / m = 1 ) , 21.2524.00 kg / m = 2 ( vs 21.0023.00 kg / m = 2 ) , > 24.00 kg / m = 3 ( vs > 23.00 kg / m = 3 ) .", "finally , in assessing dietary behaviors , cheese , which is infrequently consumed by the chinese population , was replaced with soy and peanut milk .", "the maximum score for the mna - sf is 14 , where a score of 12 or more reflects a well - nourished nutritional status .", "patients demographic data , including age , sex , and smoking history ( eg , date started smoking , number of cigarettes smoked per day , date ceased smoking ) , were collected by clinically trained nurses .", "anthropometric parameters and biochemical markers were determined based on previous research.5 biochemical markers were provided by clinical laboratories at the tianjin medical university cancer institute and hospital , and reference values of biochemical markers were modified to assess the nutritional status of hospitalized patients .", "all protocols used in this study were approved by the tianjin cancer institute and hospital .", "all nurses and patients participating in this study were informed about the objectives of the study and signed an informed consent document prior to participating .", "spss ( v 17.0 ; ibm , armonk , new york , ny , usa ) software was used to conduct all statistical analyses .", "means , standard deviations , cross tabulation , and frequency tables were used as descriptive statistics .", "analysis of variance for normally distributed dates and nonparametric methods , such as the wilcoxon two - sample test and the kruskal - wallis test were used to make statistical comparisons .", "a total of 180 elderly chinese lung cancer inpatients ( 120 men and 60 women ) aged 60 and older were randomly selected from the tianjin medical university cancer institute and hospital between june 2010 and july 2011 .", "the original mna and mna - sf scoring systems developed by guigoz4 were revised to account for differences in height , weight , and dietary behaviors between asian and western populations . for the present study , guigoz s cutoff point of < 17 for malnutrition", "was changed to < 19 , thereby modifying the overall scoring system for assessing patients nutritional status ( maximum score = 30 ) , thus : a score  24 would indicate that a patient was well nourished , a score in the range 1923 would indicate that a patient was at risk of malnutrition , and a score < 19 would indicate that a patient was malnourished.5 in addition , the body mass index ( bmi ) ( kg / m2 ) ranges were adjusted to align with the following scores : < 18.50 kg / m = 0 ( vs < 19.00 kg / m = 0 ) , 18.5021.25 kg / m = 1 ( vs 19.0021.00 kg / m = 1 ) , 21.2524.00 kg / m = 2 ( vs 21.0023.00 kg / m = 2 ) , > 24.00 kg / m = 3 ( vs > 23.00 kg / m = 3 ) . finally , in assessing dietary behaviors , cheese , which is infrequently consumed by the chinese population , was replaced with soy and peanut milk .", "the maximum score for the mna - sf is 14 , where a score of 12 or more reflects a well - nourished nutritional status .", "patients demographic data , including age , sex , and smoking history ( eg , date started smoking , number of cigarettes smoked per day , date ceased smoking ) , were collected by clinically trained nurses .", "anthropometric parameters and biochemical markers were determined based on previous research.5 biochemical markers were provided by clinical laboratories at the tianjin medical university cancer institute and hospital , and reference values of biochemical markers were modified to assess the nutritional status of hospitalized patients .", "all protocols used in this study were approved by the tianjin cancer institute and hospital .", "all nurses and patients participating in this study were informed about the objectives of the study and signed an informed consent document prior to participating .", "spss ( v 17.0 ; ibm , armonk , new york , ny , usa ) software was used to conduct all statistical analyses .", "means , standard deviations , cross tabulation , and frequency tables were used as descriptive statistics .", "analysis of variance for normally distributed dates and nonparametric methods , such as the wilcoxon two - sample test and the kruskal - wallis test were used to make statistical comparisons .", "average age of the 180 sample patients was 67.3  5.0 years , with a range of 60~85 years . across all patients", "assessed 9% ( n = 17 ) were malnourished ( mna score < 19 ) , 33% ( n = 59 ) were at risk of malnutrition ( mna score 1923 ) , and 58% ( n = 104 ) were well nourished ( mna score  24 ) .", "average mna - sf score was 11.5  2.3 with a range of 4 to 15 . among all patients assessed 4% ( n = 7 ) had a bmi of greater than 30", "kg / m and 7% ( n = 13 ) had a bmi of less than 20 kg / m . to measure the criterion - related validity of the mna ,", "significant correlations were found between bmi and mna scores ( p = 0.000 ) ; mna scores of well - nourished at risk of malnutrition , and malnourished patients ( p < 0.001 ) ; the bmi of well - nourished and malnourished patients ( p < 0.05 ) ; and the calf circumference ( cc ) of well - nourished and at risk of malnutrition patients with malnourished patients ( p < 0.05 ) .", "reliability was measured as homogeneity using cronbach s  coefficient and item - to - total score correlations .", "significant correlations between total mna score and mna questions were found for all questions with the exception of four related to neuropsychological problems , independence , skin problems , and number of meals eaten per day .", "for example , declined food intake was strongly correlated with weight loss during the last 3 months ( r = 0.538 ) , self - perceived nutritional status ( r = 0.485 ) , and self - perceived health status ( r = 0.482 ) .", "there was also a strong correlation between self - perceived nutritional status and self - perceived health status .", "in contrast , no significant correlations were linked to skin problems , number of meals eaten per day , and neuropsychological problems .", "mna questions that had a high number of an assigned score of 0 provided key insights : 99% ( n = 178 ) of patients could consume their food independently , 44% ( 80 ) suffered psychological stress , 26% ( 46 ) consumed low amounts of protein each day , 25% ( 45 ) had a cc < 31 cm , 15% ( 27 ) drank fewer than three cups of water per day , and 13% ( 24 ) consumed fewer than three different types of fruits and vegetables per day .", "patients who were well nourished , at risk of malnutrition , and malnourished differed significantly in their responses to anthropometrics , and global , diet , and subjective assessments ( table 3 ) .", "this suggests that all four aspects of the mna contribute to the total mna score and the nutritional status of the patients .", "average age of the 180 sample patients was 67.3  5.0 years , with a range of 60~85 years . across all patients", "assessed 9% ( n = 17 ) were malnourished ( mna score < 19 ) , 33% ( n = 59 ) were at risk of malnutrition ( mna score 1923 ) , and 58% ( n = 104 ) were well nourished ( mna score  24 ) .", "average mna - sf score was 11.5  2.3 with a range of 4 to 15 . among all patients assessed 4% ( n = 7 ) had a bmi of greater than 30", "kg / m and 7% ( n = 13 ) had a bmi of less than 20 kg / m . to measure the criterion - related validity of the mna ,", "significant correlations were found between bmi and mna scores ( p = 0.000 ) ; mna scores of well - nourished at risk of malnutrition , and malnourished patients ( p < 0.001 ) ; the bmi of well - nourished and malnourished patients ( p < 0.05 ) ; and the calf circumference ( cc ) of well - nourished and at risk of malnutrition patients with malnourished patients ( p < 0.05 ) .", "reliability was measured as homogeneity using cronbach s  coefficient and item - to - total score correlations .", "significant correlations between total mna score and mna questions were found for all questions with the exception of four related to neuropsychological problems , independence , skin problems , and number of meals eaten per day .", "for example , declined food intake was strongly correlated with weight loss during the last 3 months ( r = 0.538 ) , self - perceived nutritional status ( r = 0.485 ) , and self - perceived health status ( r = 0.482 ) .", "there was also a strong correlation between self - perceived nutritional status and self - perceived health status .", "in contrast , no significant correlations were linked to skin problems , number of meals eaten per day , and neuropsychological problems .", "mna questions that had a high number of an assigned score of 0 provided key insights : 99% ( n = 178 ) of patients could consume their food independently , 44% ( 80 ) suffered psychological stress , 26% ( 46 ) consumed low amounts of protein each day , 25% ( 45 ) had a cc < 31 cm , 15% ( 27 ) drank fewer than three cups of water per day , and 13% ( 24 ) consumed fewer than three different types of fruits and vegetables per day .", "patients who were well nourished , at risk of malnutrition , and malnourished differed significantly in their responses to anthropometrics , and global , diet , and subjective assessments ( table 3 ) .", "this suggests that all four aspects of the mna contribute to the total mna score and the nutritional status of the patients .", "published findings on the nutritional status of lung cancer inpatients vary greatly in the medical literature .", "some studies conclude that there is a high incidence of malnutrition among lung cancer inpatients .", "for example , hollaus et al6 and chauhan et al7 found that 10%20% and 30% of patients suffered from malnutrition , respectively .", "in contrast , other studies have shown that malnutrition rates are overestimated in lung cancer patients .", "jagoe et al reported that severe malnutrition was uncommon among lung cancer patients who were referred for surgery.8 however , in the present study , the incidence rate of malnutrition was found to be 42% among 180 lung cancer inpatients who were referred for surgery .", "the malnutrition or cachexia in these patients may have been caused by a slowdown in metabolism due to decreased organic functions , or absorption of the body s nutrients by malignant tumors before metastasis.9 this study also showed that there were significant correlations between mna score and bmi ( p = 0.000 ) ; the mna scores of well - nourished , at risk of malnutrition , and malnourished patients ( p < 0.001 ) ; the bmi of well - nourished and malnourished patients ( p < 0.05 ) ; and the cc of well - nourished and at - risk of malnutrition patients with malnourished patients ( p < 0.05 ) .", "secondly , all but four mna questions ( neuropsychological problems , independence , skin problems , and number of meals eaten per day ) were significantly correlated to total mna score .", "this shows that the mna is a reliable instrument ( cronbach s  coefficient = 0.698 ) for nutritional assessment .", "in addition , well - nourished , at risk of malnutrition , and malnourished patients differed significantly in their responses to mna subgroup questions related to anthropometrics , and global , diet , and subjective assessments ( table 3 ) .", "all these findings are supported by existing studies , suggesting that the mna ( with a revised scoring system to better suit the elderly chinese population ) is a valid and reliable diagnostic tool with which to assess the nutritional status of elderly chinese lung cancer inpatients.5,10 no significant correlations were found between total mna score and mna questions related to neuropsychological problems and independence ( table 2 ) .", "studies conducted by soini et al10 also found that independence was correlated with total mna score , as nurses or family members helped feed patients .", "however , neuropsychological problems were strongly correlated to total mna score in soini and murphy s studies.10,11 the differences between these findings and those of the present study may be due to the fact that , in our study , 99% ( n = 178 ) of patients could consume their food independently .", "additionally , in our study , preliminary evaluations were carried out before surgery , and some patients with neuropsychological problems may have been transferred to receive neuropsychological treatment instead of undergoing surgery . as such , the lack of cooperation from patients with neuropsychological problems may increase their risk of postoperative complications and death .", "the mna is a valid and reliable diagnostic tool that can be used to identify elderly lung cancer inpatients who are malnourished , as well as those who are at risk for malnutrition .", "the high incidence rates of preoperative malnutrition and risk of malnutrition among elderly chinese lung cancer inpatients highlights the need for further investigations to determine the relationship between nutritional status and prognosis of these patients ."], "abstract_text": ["<S> purposethis study assessed the nutritional status of elderly chinese lung cancer inpatients using a revised version of the mini - nutritional assessment ( mna ) tool.patients and methods : the revised version of the mna tool was used to assess the nutritional status of 180 elderly chinese lung cancer inpatients prior to their scheduled surgery between june 2010 and july 2011 . </S>", "<S> patients demographic data , anthropometric parameters , and biochemical markers were collected and analyzed.resultsamong the 180 inpatients who underwent the mna , 9% were malnourished ( mna score < 19 ) , 33% were at risk of malnutrition ( mna score 1923 ) , and 58% were well nourished ( mna score  24 ) . </S>", "<S> there was significant correlation between the mna scores of patients who were malnourished , at risk of malnutrition , and well nourished ( p < 0.001 ) , as well as between total mna score and most mna questions . </S>", "<S> the three patient groups with different nutritional statuses differed significantly in their responses to anthropometrics and global , diet , and subjective assessments.conclusionincidence rates of malnutrition prior to surgery are high among elderly chinese lung cancer inpatients . </S>", "<S> the revised mna is a valid and reliable tool that can be used to assess and prevent malnutrition among these inpatients . </S>"], "labels": null, "section_names": ["Introduction", "Material and methods", "Participants", "MNA/MNA-SF scoring system", "Data collection", "Ethics", "Statistical analysis", "Results", "Participants", "Correlations between total MNA score and MNA questions", "MNA questions and their intercorrelations", "MNA questions with a high frequency of zero scores", "MNA subgroup questions related to anthropometrics, and global, diet, and subjective assessments", "Discussion", "Conclusion"], "sections": [["malnutrition is a common and serious problem among geriatric patients , particularly for those who are diagnosed with lung cancer caused by declined functional reserve of visceral organs , decreased stability of the internal environment , and effects of acute or chronic diseases .", "malnutrition can lead to increased risk of surgery , decreased quality of life , increased incidence of postoperative complications , increased fatality rate , and decreased survival rate among elderly lung cancer inpatients.1,2 nutrition support has also been reported to improve the perioperative nutritional status and reduce the severity of postoperative complications in lung cancer.3 in the people s republic of china , the number of elderly lung cancer inpatients is increasing at a rapid rate ; thus , there is a great need to develop a tool that can accurately and effectively assess the nutritional status of these patients . the mini - nutritional assessment ( mna ) is a tool that is widely used in the usa and europe to evaluate patients nutritional status .", "this study examines the use of the mna and short - form mna ( mna - sf ) ( with a revised scoring system to better suit the elderly chinese population5 ) in assessing the nutritional status of elderly chinese lung cancer inpatients prior to scheduled surgery ."], ["a total of 180 elderly chinese lung cancer inpatients ( 120 men and 60 women ) aged 60 and older were randomly selected from the tianjin medical university cancer institute and hospital between june 2010 and july 2011 .", "the original mna and mna - sf scoring systems developed by guigoz4 were revised to account for differences in height , weight , and dietary behaviors between asian and western populations . for the present study , guigoz s cutoff point of < 17 for malnutrition", "was changed to < 19 , thereby modifying the overall scoring system for assessing patients nutritional status ( maximum score = 30 ) , thus : a score  24 would indicate that a patient was well nourished , a score in the range 1923 would indicate that a patient was at risk of malnutrition , and a score < 19 would indicate that a patient was malnourished.5 in addition , the body mass index ( bmi ) ( kg / m2 ) ranges were adjusted to align with the following scores : < 18.50 kg / m = 0 ( vs < 19.00 kg / m = 0 ) , 18.5021.25 kg / m = 1 ( vs 19.0021.00 kg / m = 1 ) , 21.2524.00 kg / m = 2 ( vs 21.0023.00 kg / m = 2 ) , > 24.00 kg / m = 3 ( vs > 23.00 kg / m = 3 ) .", "finally , in assessing dietary behaviors , cheese , which is infrequently consumed by the chinese population , was replaced with soy and peanut milk .", "the maximum score for the mna - sf is 14 , where a score of 12 or more reflects a well - nourished nutritional status .", "patients demographic data , including age , sex , and smoking history ( eg , date started smoking , number of cigarettes smoked per day , date ceased smoking ) , were collected by clinically trained nurses .", "anthropometric parameters and biochemical markers were determined based on previous research.5 biochemical markers were provided by clinical laboratories at the tianjin medical university cancer institute and hospital , and reference values of biochemical markers were modified to assess the nutritional status of hospitalized patients .", "all protocols used in this study were approved by the tianjin cancer institute and hospital .", "all nurses and patients participating in this study were informed about the objectives of the study and signed an informed consent document prior to participating .", "spss ( v 17.0 ; ibm , armonk , new york , ny , usa ) software was used to conduct all statistical analyses .", "means , standard deviations , cross tabulation , and frequency tables were used as descriptive statistics .", "analysis of variance for normally distributed dates and nonparametric methods , such as the wilcoxon two - sample test and the kruskal - wallis test were used to make statistical comparisons ."], ["a total of 180 elderly chinese lung cancer inpatients ( 120 men and 60 women ) aged 60 and older were randomly selected from the tianjin medical university cancer institute and hospital between june 2010 and july 2011 ."], ["the original mna and mna - sf scoring systems developed by guigoz4 were revised to account for differences in height , weight , and dietary behaviors between asian and western populations . for the present study , guigoz s cutoff point of < 17 for malnutrition", "was changed to < 19 , thereby modifying the overall scoring system for assessing patients nutritional status ( maximum score = 30 ) , thus : a score  24 would indicate that a patient was well nourished , a score in the range 1923 would indicate that a patient was at risk of malnutrition , and a score < 19 would indicate that a patient was malnourished.5 in addition , the body mass index ( bmi ) ( kg / m2 ) ranges were adjusted to align with the following scores : < 18.50 kg / m = 0 ( vs < 19.00 kg / m = 0 ) , 18.5021.25 kg / m = 1 ( vs 19.0021.00 kg / m = 1 ) , 21.2524.00 kg / m = 2 ( vs 21.0023.00 kg / m = 2 ) , > 24.00 kg / m = 3 ( vs > 23.00 kg / m = 3 ) . finally , in assessing dietary behaviors , cheese , which is infrequently consumed by the chinese population , was replaced with soy and peanut milk .", "the maximum score for the mna - sf is 14 , where a score of 12 or more reflects a well - nourished nutritional status ."], ["patients demographic data , including age , sex , and smoking history ( eg , date started smoking , number of cigarettes smoked per day , date ceased smoking ) , were collected by clinically trained nurses .", "anthropometric parameters and biochemical markers were determined based on previous research.5 biochemical markers were provided by clinical laboratories at the tianjin medical university cancer institute and hospital , and reference values of biochemical markers were modified to assess the nutritional status of hospitalized patients ."], ["all protocols used in this study were approved by the tianjin cancer institute and hospital .", "all nurses and patients participating in this study were informed about the objectives of the study and signed an informed consent document prior to participating ."], ["spss ( v 17.0 ; ibm , armonk , new york , ny , usa ) software was used to conduct all statistical analyses .", "means , standard deviations , cross tabulation , and frequency tables were used as descriptive statistics .", "analysis of variance for normally distributed dates and nonparametric methods , such as the wilcoxon two - sample test and the kruskal - wallis test were used to make statistical comparisons ."], ["average age of the 180 sample patients was 67.3  5.0 years , with a range of 60~85 years . across all patients", "assessed 9% ( n = 17 ) were malnourished ( mna score < 19 ) , 33% ( n = 59 ) were at risk of malnutrition ( mna score 1923 ) , and 58% ( n = 104 ) were well nourished ( mna score  24 ) .", "average mna - sf score was 11.5  2.3 with a range of 4 to 15 . among all patients assessed 4% ( n = 7 ) had a bmi of greater than 30", "kg / m and 7% ( n = 13 ) had a bmi of less than 20 kg / m . to measure the criterion - related validity of the mna ,", "significant correlations were found between bmi and mna scores ( p = 0.000 ) ; mna scores of well - nourished at risk of malnutrition , and malnourished patients ( p < 0.001 ) ; the bmi of well - nourished and malnourished patients ( p < 0.05 ) ; and the calf circumference ( cc ) of well - nourished and at risk of malnutrition patients with malnourished patients ( p < 0.05 ) .", "reliability was measured as homogeneity using cronbach s  coefficient and item - to - total score correlations .", "significant correlations between total mna score and mna questions were found for all questions with the exception of four related to neuropsychological problems , independence , skin problems , and number of meals eaten per day .", "for example , declined food intake was strongly correlated with weight loss during the last 3 months ( r = 0.538 ) , self - perceived nutritional status ( r = 0.485 ) , and self - perceived health status ( r = 0.482 ) .", "there was also a strong correlation between self - perceived nutritional status and self - perceived health status .", "in contrast , no significant correlations were linked to skin problems , number of meals eaten per day , and neuropsychological problems .", "mna questions that had a high number of an assigned score of 0 provided key insights : 99% ( n = 178 ) of patients could consume their food independently , 44% ( 80 ) suffered psychological stress , 26% ( 46 ) consumed low amounts of protein each day , 25% ( 45 ) had a cc < 31 cm , 15% ( 27 ) drank fewer than three cups of water per day , and 13% ( 24 ) consumed fewer than three different types of fruits and vegetables per day .", "patients who were well nourished , at risk of malnutrition , and malnourished differed significantly in their responses to anthropometrics , and global , diet , and subjective assessments ( table 3 ) .", "this suggests that all four aspects of the mna contribute to the total mna score and the nutritional status of the patients ."], ["average age of the 180 sample patients was 67.3  5.0 years , with a range of 60~85 years . across all patients", "assessed 9% ( n = 17 ) were malnourished ( mna score < 19 ) , 33% ( n = 59 ) were at risk of malnutrition ( mna score 1923 ) , and 58% ( n = 104 ) were well nourished ( mna score  24 ) .", "average mna - sf score was 11.5  2.3 with a range of 4 to 15 . among all patients assessed 4% ( n = 7 ) had a bmi of greater than 30", "kg / m and 7% ( n = 13 ) had a bmi of less than 20 kg / m . to measure the criterion - related validity of the mna ,", "significant correlations were found between bmi and mna scores ( p = 0.000 ) ; mna scores of well - nourished at risk of malnutrition , and malnourished patients ( p < 0.001 ) ; the bmi of well - nourished and malnourished patients ( p < 0.05 ) ; and the calf circumference ( cc ) of well - nourished and at risk of malnutrition patients with malnourished patients ( p < 0.05 ) ."], ["reliability was measured as homogeneity using cronbach s  coefficient and item - to - total score correlations .", "significant correlations between total mna score and mna questions were found for all questions with the exception of four related to neuropsychological problems , independence , skin problems , and number of meals eaten per day ."], ["for example , declined food intake was strongly correlated with weight loss during the last 3 months ( r = 0.538 ) , self - perceived nutritional status ( r = 0.485 ) , and self - perceived health status ( r = 0.482 ) .", "there was also a strong correlation between self - perceived nutritional status and self - perceived health status .", "in contrast , no significant correlations were linked to skin problems , number of meals eaten per day , and neuropsychological problems ."], ["mna questions that had a high number of an assigned score of 0 provided key insights : 99% ( n = 178 ) of patients could consume their food independently , 44% ( 80 ) suffered psychological stress , 26% ( 46 ) consumed low amounts of protein each day , 25% ( 45 ) had a cc < 31 cm , 15% ( 27 ) drank fewer than three cups of water per day , and 13% ( 24 ) consumed fewer than three different types of fruits and vegetables per day ."], ["patients who were well nourished , at risk of malnutrition , and malnourished differed significantly in their responses to anthropometrics , and global , diet , and subjective assessments ( table 3 ) .", "this suggests that all four aspects of the mna contribute to the total mna score and the nutritional status of the patients ."], ["published findings on the nutritional status of lung cancer inpatients vary greatly in the medical literature .", "some studies conclude that there is a high incidence of malnutrition among lung cancer inpatients .", "for example , hollaus et al6 and chauhan et al7 found that 10%20% and 30% of patients suffered from malnutrition , respectively .", "in contrast , other studies have shown that malnutrition rates are overestimated in lung cancer patients .", "jagoe et al reported that severe malnutrition was uncommon among lung cancer patients who were referred for surgery.8 however , in the present study , the incidence rate of malnutrition was found to be 42% among 180 lung cancer inpatients who were referred for surgery .", "the malnutrition or cachexia in these patients may have been caused by a slowdown in metabolism due to decreased organic functions , or absorption of the body s nutrients by malignant tumors before metastasis.9 this study also showed that there were significant correlations between mna score and bmi ( p = 0.000 ) ; the mna scores of well - nourished , at risk of malnutrition , and malnourished patients ( p < 0.001 ) ; the bmi of well - nourished and malnourished patients ( p < 0.05 ) ; and the cc of well - nourished and at - risk of malnutrition patients with malnourished patients ( p < 0.05 ) .", "secondly , all but four mna questions ( neuropsychological problems , independence , skin problems , and number of meals eaten per day ) were significantly correlated to total mna score .", "this shows that the mna is a reliable instrument ( cronbach s  coefficient = 0.698 ) for nutritional assessment .", "in addition , well - nourished , at risk of malnutrition , and malnourished patients differed significantly in their responses to mna subgroup questions related to anthropometrics , and global , diet , and subjective assessments ( table 3 ) .", "all these findings are supported by existing studies , suggesting that the mna ( with a revised scoring system to better suit the elderly chinese population ) is a valid and reliable diagnostic tool with which to assess the nutritional status of elderly chinese lung cancer inpatients.5,10 no significant correlations were found between total mna score and mna questions related to neuropsychological problems and independence ( table 2 ) .", "studies conducted by soini et al10 also found that independence was correlated with total mna score , as nurses or family members helped feed patients .", "however , neuropsychological problems were strongly correlated to total mna score in soini and murphy s studies.10,11 the differences between these findings and those of the present study may be due to the fact that , in our study , 99% ( n = 178 ) of patients could consume their food independently .", "additionally , in our study , preliminary evaluations were carried out before surgery , and some patients with neuropsychological problems may have been transferred to receive neuropsychological treatment instead of undergoing surgery . as such , the lack of cooperation from patients with neuropsychological problems may increase their risk of postoperative complications and death ."], ["the mna is a valid and reliable diagnostic tool that can be used to identify elderly lung cancer inpatients who are malnourished , as well as those who are at risk for malnutrition .", "the high incidence rates of preoperative malnutrition and risk of malnutrition among elderly chinese lung cancer inpatients highlights the need for further investigations to determine the relationship between nutritional status and prognosis of these patients ."]]}
{"article_id": "PMC4835334", "article_text": ["population aging can be considered as the most evident and dynamic aspect of modern demography , influencing the economy and public health in a significant way .", "the world has experienced a modest increase in the population above 60 years of age over the last six decades  from 8% to 10% . however", ", there were 204 million elderly in the world . in 1998 , this number more than doubled , totaling 579 million ; in 2011 , the figure reached was 893 million.1 over the next four decades , experts expect this age group to grow to 22% of the world population , which would correspond to 2.4 billion of elderly by the middle of this century.2 \n  in this sense , many elders will likely face health problems inherent to aging , as well as challenges in maintaining their independence .", "however , the elderly can stay healthy and active in society in an independent and sustainable manner,3 and the work of professionals and researchers in the multidisciplinary health field that serve this group is fundamental in achieving this state . at the same time , it is essential that the government create and apply public policies that enable and sustain educational and awareness actions regarding people qualification and the fulfillment of the population basic needs .", "as for the health of the elderly , changes in body balance ( postural instability ) have received much attention , as it is one of the symptoms that most affect their lives .", "body balance is the result of a harmonious interaction among different systems in the human body : vestibular ( inner ear  posterior labyrinth ) , visual , and somatic - sensorial ( cutaneous , muscular , and articular receptors ) systems .", "this interaction is accomplished through processes of the neural system , which organizes and executes a rapid response by means of the musculoskeletal system.4 \n  the aging process is associated with a decrease in the ability to keep balance , especially in situations where responses of the balance keeping systems to unexpected disturbances during free moving and the avoidance of obstacles are necessary .", "sensorial , motor , and adaptive balance components become vulnerable as degenerative , infectious , or damaging processes accumulate .", "although small alterations in any of the systems that comprise balance keeping do not result in significant disturbances , the involvement of multiple components may lead to severe deficits in postural stability conservation.5 \n 6 \n  the aging process may alter the dynamic posture keeping system .", "this condition is a contributing factor to falls among the elderly , albeit not the main cause.7 impairment of the vestibular system is also believed to be a factor that leads the elderly to lose balance , through the aging process of this systes organs.8 \n  alterations to postural stability are also followed by dizziness .", "this can be defined as any sensation of change in the body balance and can be rotational ( vertigo ) or non - rotational ( unbalance , oscillation , vacillation , etc . ) . both types may or may not be caused by specific vestibular system disturbances.9 \n  the presence of dizziness may cause falls , which are associated to the main cause of death in the elderly group .", "some authors also associate dizziness to partial or full inability to perform social , professional , family activities , in addition to causing physical and psychological damage , such as loss of self - confidence , depression , deficit in concentration , and performance , thus negatively interfering in the quality of life.10 \n 11 however , it is still difficult to know if psychological problems pre - exist or are a result of vestibular complications.11 \n  balance disturbances and anxiety disorders share central neural circuits involving monoaminergic components , this being the reason why anxiety is often associated with balance alterations .", "these neural circuits concentrate in a parabrachial nucleus network ( vestibular system and visceral information processing convergence point ) , also involving avoidance , anxiety , and fear symptoms.12 physical insecurity triggered by dizziness and unbalance lead to psychological insecurity , irritability , loss of self - confidence , anxiety , depression or panic , a sense of being out of reality and depersonalization.13 \n  depression  which may be caused or made worse by body imbalance  is a common condition in seniors .", "furthermore , it is associated to higher risk of morbidity and mortality , increase in health services usage , negligence in self - care , and higher risk of suicide.14 the main symptoms of depression are depressed mood and loss of interest or lack of pleasure in almost all activities , being characterized as a psychiatric syndrome . in the elderly ,", "its manifestation occurs in a heterogeneous form regarding both the presentation aspects of its etiology and its treatment.15 \n  studies relate the involvement in physical activities with a better general health condition and lesser occurrence of depression and falls in the elderly.16 \n 17 physical activity is capable of improving mental and clinical conditions of depressed elderly .", "this is because physical activity stimulates cognitive , memory , and concentration functions , in addition to raising self - esteem , improving quality of life,18 and helping decrease scores indicative of anxiety and depression,19 favoring daily activities and promoting functional independence.20 furthermore , the physically active elderly show improvement of dizziness and vertigo symptoms,21 \n 22 \n 23 decreasing , as a result , the risk of falls and associated comorbidities .", "thus , the objective of this work is to verify the correlation between physical exercise , dizziness , probability of falls , and symptoms of depression in a group of middle - aged and elderly group .", "this is a transversal and observational study with approval from the institutios ethical committee ( record n 216292011 ) .", "the sample comprised adult and elderly individuals of both sexes who took part in university extension projects .", "the inclusion factors for subjects were non - institutionalized elderly with no history of neurological or cognitive alterations , who voluntarily accepted to take part in the research and signed the informed consent form ( icf ) .", "assessment procedures included a medical history prepared for this study ; the abridged version of the geriatric depression scale ( gds ) ; and anterior functional reach test .", "the medical history assessment included questions on individuals socio - demographic and health ( age , gender , presence of diseases , treatment used ) , as well as questions about their practice of physical exercises ( frequency , time of practice , type of physical exercise ) .", "as for assessing the results of the geriatric depression scale , each answer indicating depression scored one point .", "scores below five points indicated lack of depressive symptoms ; 6 to 10 points indicated mild / moderate depressive symptoms ; and 11 to 15 points meant severe depressive symptoms.24 \n  for the functional range test was initially placed a tape measure on the wall , parallel to the floor , at the height of the acromion of each individual .", "the following were instructed to position themselves parallel to the wall , without lean on to it , with the parallels and bare feet in a comfortable position .", "the upper limb was positioned with bent shoulders at 90 , extended elbows , wrists in a neutral , flat hand position . as a result", ", it was requested that incline forward as much as possible , hold for three seconds without taking the heels off the floor .", "taking as the third metacarpal of the individual parameter was recorded in centimeters anterior displacement on the tape .", "displacements smaller than 15.2 cm indicate the risk of falls four times higher compared to those who reached more than 25.4 cm , or is highly likely to falls .", "measures between 15.2 and 25.4 cm has an average probability of falls ( twice as likely to fall than those who reach values greater than 25.4 cm ) .", "values greater than 25.4 cm indicate low probability of falls.25 \n  the authors analyzed the data using descriptive statistics through measures of central tendency ( mean and median ) and variability ( standard deviation and inner quartile ranges ) , as well as absolute and relative distribution ( n - % ) .", "the symmetric continuous distribution was evaluated by the kolmogorov - smirnov test . for the bivariate analysis between qualitative variables , the authors used the pearson qui - square test (  ) and continuity correction , which compares between observed ( real ) and expected frequencies . in contingency tables", "in which at least 25% of cell ( quads ) values presented expected frequency below five , they used fishes exact test . for continuous variables , when comparing two independent groups , the applied test was students t - test or , its non - parametric equivalent , the mann whitney test .", "multivariate analysis was accomplished through non - conditional binary logistic regression , by employing regressive procedures to test the hypotheses of association between cognitive decline and co - variables defined by the bivariate analysis ( p    0.250 ) , by obtaining the adjusted odds ratio ( or ) .", "data received statistical treatment via spss 17.0 software ( statistical package for social sciences for windows ) , adopting a significance level (  ) of 5% for the decision - making criteria .", "the sample comprised of 90 individuals , aged 69.3 (    6.8 ) in average , with minimum age of 59 years and maximum age of 84 years .", "the majority of investigated individuals was in the age group between 65 and 74 years ( 38.9% ) .", "results presented as n ( % ) , with percentage obtained as based on total valid cases ;  : nr  =  5 ( 2.5% ) . the majority of individuals interviewed ( 65.8% ) did not show depressive symptoms . regarding the anterior functional reach test ,", "the majority of evaluated individuals indeed showed low probability of fall occurrence , with an average score of 28.2 cm ( sd  =  9.2 ) points , varying from 0 to 44.5 cm ( table 2 ) .", "results presented as n ( % ) , with percentage obtained as based on total valid cases ,  : nr  =  11 ( 12.2% ) ;  : nr  =  8 ( 8.8% ) . in the analyses done on dizziness complaints ,", "the authors verified a significant association with age group ( p  <  0.05 ) , with the elderly aged 75 years or more presenting a higher probability of feeling dizziness , whereas the group aged between 65 and 74 years , this association was not verified ( table 3 ) .", "percentages obtained as based on total of each dizziness category ;  : pearson chi - square test with continuity correction ;  : t - student test for independent groups ;  : pearson chi - square test ;  : fisher 's exact test through monte carlo simulation ; * statistically significant test at 5% ; * * statistically significant test at 1% .", "moreover , it has been observed statistically significant association between the occurrence of dizziness and the presence of light / moderate depressive symptoms ( p  <  0.01 ) , as well as between the occurrence of dizziness and the average probability of falls ( p  <  0.001 ) . in terms of the association between the occurrence of dizziness and the results of the anterior functional reach test , the average in centimeters for the anterior reach ( 25.3    5.4 ) of elderly with dizziness showed to be significantly less when compared with the average of the group without dizziness complaints ( 30.1    10.6 ) ( p  <  0.01 ) .", "when comparing the occurrence of dizziness with gender , age , practice of physical exercise , and time of practice , we identified an independent relation , evidencing that dizziness is unrelated to any of these factors in the studied group . in the group of individuals that did not practice physical exercise", ", there was an association between the occurrence of dizziness and light / moderate depressive symptoms ( p  <  0.001 ) .", "however , in the same analysis done with the group that practiced some physical activity , the association between dizziness and depressive symptoms was not statistically significant ( p  >  0.05 ) ( table 4 ) .", "another evaluation confirms the importance of physical exercise . keeping the remaining variation factors not considered in the analysis ( gender , age ) constant , the group that practices physical exercise was less prone to dizziness and depressive symptoms ( table 4 ) .", "we looked for and identified the variables that could respond in a more trustworthy form to the occurrence of dizziness .", "we performed the logistic regression analysis with an adjustment to minimize the number of explanatory variables employed and maximize model precision , through the backward model , to maximize the explanatory power of the study factor . through a model defined as saturated", ", we listed all variables with minimum significance level below 0.25 through bivariate analysis ( table 3 ) as possible preceding factors for the presence of dizziness .", "they are the following : gender , age , age group , physical activity , gds score , dislocation in the anterior functional reach test , and balance . in this model , the variables for gender and age pointed out an independent relation to the occurrence of dizziness . according to the adjusted logistic regression model ,", "the following were significant predictive factors for the occurrence of dizziness : non - practice of physical exercise , classifications of light / moderate depressive symptoms , and dislocation in the anterior functional reach test .", "results described in table 5 show that the elderly who do not practice physical exercise have 2.2 times higher risk of presenting dizziness than the group that practices some physical exercise . in relation to depressive symptoms ,", "the significant risk of presenting dizziness occurred in the light / moderate degree , indicating that the elderly included in this classification had 4.5 times more chance of presenting dizziness than the group with absence of depressive symptoms .", "model parameters : pseudo - r  =  0.432 ;  -2 log likelihood  =  80.208 ; hosmer and lemeshow ( p  =  0.998 ) ; pearson 's chi - square (   =  9272 ; p ", "=  0.099 ) ; nagelkerke ( r : 0.460 ) ; correct classification : 74.4% ( dizziness : no  =  78.7% ; yes  =  67.7% ) .", "as the number of elderly with severe depressive symptoms was very low , the light / moderate and serious groups were merged , by classifying the answer for symptoms as presence or absence . over this new variable ,", "we once more calculated the risk of presence of depressive symptoms associated to dizziness , estimated at 2.1 times that of the group with absence of this symptom .", "the results verified that the group with medium probability of falls as the result for the anterior functional reach test had 4.0 times more chances of occurrence of dizziness than the group with low probability of falls . when age was kept constant", ", results indicated that the non - practice of physical exercise , the presence of depressive symptoms , and the result of the anterior functional reach test ( indicating medium probability of falls ) are factors associated to the occurrence of dizziness in the investigated sample .", "in the sample of this study , more than 60% of the individuals affirmed to not practice physical exercise , confirming other similar studies .", "the ministry of health of brazil26 made a survey , and verified that 53.7% of elderly men and 58.3% of elderly women were inactive .", "similar results were found in other study concerning the level of physical activity and its relationship with the environment ( specifically , safety ) with 1,656 elderly from florianpolis.27 by using similar parameters , pucci , reis , rech and hallal28 classified the majority of the individuals of their study as insufficiently active in leisure or walking activities . in this study ,", "current studies highlight the prevalence of dizziness reports among the elderly population.29 \n 30 \n 31 \n 32 approximately one in every five seniors currently presents dizziness or imbalance.29 in our research , 40% of the elderly reported sensation of dizziness .", "this finding is very similar to other studies done in brazil , in which the authors verified that the prevalence of dizziness in the elderly is substantial , having observed dizziness complaints from 45% of 391 elderly over 65 years of age interviewed.33 \n  another study interviewed 516 elderly people , and confirmed self - reported dizziness in 42% of them.31 thus , dizziness is a common problem for the elderly population,11 \n 34 \n 35 as these individuals show a decrease in visual acuity during aging , and loss of balance due to the reduction of ciliated cells and vestibular neurons .", "this results in loss of functions from two important components responsible for keeping balance.36 furthermore , with age , the central nervous system capabilities become impaired , affecting the processing of vestibular , visual , and proprioceptive signals responsible for maintenance of body balance .", "age also reduces capacity of modifications of the adaptive reflexes , may be responsible for the occurrence of vertigo and/or dizziness in the geriatric population.35 \n  the data from the studied sample evidenced that 33.7% of the elderly presented depressive symptoms .", "this prevalence is higher than that described by other authors,37 who report prevalence of 26.7% of depressive symptoms in a sample of elderly people residing in an urban zone . in turn , study reported depressive symptoms in 31% of the elderly evaluated , by also using the gds scale in active elderly who were part of a conviviality group , i.e. , a similar sample to the one evaluated in this study .", "additionally , the cited authors verified that 4% of the evaluated elderly presented severe depressive symptoms , which was also similar to the obtained data.38 \n  regarding anterior functional reach results , the average obtained was 28.2    9.2 cm .", "results were higher than those observed by previous studies,39 \n 40 and lower than those obtained by another study.41 values found were similar to those observed in brazil,42 which also evaluated a sample of adult and elderly people .", "the sample of elderly people in the age group between 70 and 87 years of age presented functional reach values between 29.75 ( men ) and 27.13 ( women ) .", "variations in the results of these studies can be explained through different factors , such as fall history , freight , strength , and flexibility of upper and lower limbs.42 \n  this research verified a stronger presence of dizziness in the age group above 75 years ( 41.7% ) , a significant association ( p  =  0.028 ) when compared with remaining age groups .", "such a finding is similar to that of the research with 1,273 elderly between 60 and 96 years of age , which verified reports of dizziness in 23.6% of all elderly evaluated . in this study ,", "17.8% of them were under 80 and 31% were over 80 years old.30 another longitudinal research with 620 elderly people verified that dizziness was a factor that depended on age , with prevalence of 27% in the elderly under 70 , and over 54% in the group with 90 years of age or more . among the elderly that mentioned dizziness ,", "68.2% perceived this symptom daily or weekly.43 the bigger prevalence of dizziness in the age group above 75 years of age could be due to the generalized deterioration of health in older people44 or multifactor conditions , both medical and functional.43 \n  it is worth mentioning that the presence of dizziness is directly related to the presence of falls in the elderly population;30 both conditions are considered geriatric syndromes that occur concomitantly to other harms to multiple systems of the body.5 this worsens with age .", "the elderly who suffer falls can have fractures as a consequence , which might require longer periods in bed .", "such condition is responsible for 70% of deaths resulting from accidents in individuals over 75 years in age.45 \n  further to this , there was no significant difference when comparing the presence of dizziness complaints among the elderly who practiced physical exercise whith those who did not .", "these data were different from the expectations of the authors and the findings of ruwer et al,35 because , according to these authors , the complaint of dizziness is less prevalent in the elderly that practice physical exercise .", "nevertheless , with the adjusted logistic regression , it is possible to verify that the individuals who do not practice physical exercise are 2.2 times more likely of feeling dizzy than physically active individuals are .", "this result confirms studies that compared elderly people with and without dizziness and observed that those without complaints of dizziness were engaged in physical activity.46 these authors also verified that there was a relation between dizziness and depression , which attest to the data from our study .", "the research verified a significant association between light to moderate depressive symptoms and 4.5-fold increase in the risk of feeling dizziness . regarding the anterior functional reach test , this research verified a significant association between the probability of falls and reports of dizziness .", "the elderly with medium probability of falling are four times more likely to feel dizziness than the elderly with low probability of falls , as described in another study.47 \n  the studies indicate that active elderly people are less prone to depression than the sedentary elderly.19 \n 48 \n 49 physical exercises help to reduce depression , aside from helping in its prevention .", "physical activity relates to less frequent mood disorders and may even help release some neurotransmitters , such as noradrenalin and serotonin.50 \n 51 \n  studies indicate that elderly people who practice regular physical activity mention less depressive symptoms than non - active or sedentary elderly people .", "analysis of social skills , social support , life quality and depression of elderly people in an extension project , family contexts and asylums indicated that the depressive symptom was present in 0% of practicing elderly people compare to 30% of the elderly who did not participate in the project . in the asylum , in turn , this percentage increased 40%.52 in other study about the intensity and prevalence of depressive symptoms in elderly women participating in an extension project , it was verified an absence of depressive symptoms in 80.6% of the sample.53 another study evaluated the effect of a program of aerobic physical exercise in the intensity of the ventilator threshold over depression and anxiety and life quality of healthy elderly people divided into a control and a experimental group .", "the elderly in the experimental group had a reduction in the scores of depression and anxiety and an increase in life quality , while in the control group no changes were observed.51 \n  in the research on the freight of falling and its relation with balance and mobility tests , there was a moderate correlation.40 authors state that balance and mobility enable the stability necessary to maintain a standing position , for instance . therefore , maintaining adequate levels of such capabilities reduces the risk of falls , which are directly related to level of physical activity .", "the american college of sports medicine54 mentions that the level of physical activity is inversely related to the number of limitations in the elderly .", "brazilian authors analyzed the relation between health conditions , incidence of falls , and level of physical activity in 256 elderly people .", "results indicated an association between the level of physical activity and the incidence of falls and health conditions .", "in other words , the more frequent the practice of physical activity the less the occurrence of falls .", "muscular weakness , reduction in articular mobility , degraded synergy , programming mechanisms , and motor control difficulties are the variables present in low levels of physical activity , predisposing to falls in the elderly.55 \n", "the results of this study indicated the existence of an association between the practice of physical exercise , dizziness , probability of falls , and depressive symptoms in a group of non - institutionalized middle aged adults and elderly people , evidencing that physical activity is a beneficial factor for the aging population ."], "abstract_text": ["<S> \n introduction  dizziness is a symptom that can lead to falls , which , in turn , undermine ons independence and autonomy , leading to several comorbidities . the practice of physical exercise , however , can help prevent falls . \n </S>", "<S> objective  the objective of this study is to confirm the association between physical exercise , dizziness , probability of falling , and depressive symptoms in a group of middle - aged adults and seniors . \n </S>", "<S> methods  the authors evaluated subjects based on history , the geriatric depression scale , and functional reach test . \n results  the sample consisted of 90 individuals with a mean age of 69.3    6.8 years . </S>", "<S> the authors found that 37.8% had been practicing exercise , 33.7% had depressive symptoms , and their probability of falling was above average in the functional reach test . \n </S>", "<S> conclusion  the results of this study indicated an association between dizziness , exercise practice and depressive symptoms , indicating that physical activity is a beneficial factor for the aging population . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusion"], "sections": [["population aging can be considered as the most evident and dynamic aspect of modern demography , influencing the economy and public health in a significant way .", "the world has experienced a modest increase in the population above 60 years of age over the last six decades  from 8% to 10% . however", ", there were 204 million elderly in the world . in 1998 , this number more than doubled , totaling 579 million ; in 2011 , the figure reached was 893 million.1 over the next four decades , experts expect this age group to grow to 22% of the world population , which would correspond to 2.4 billion of elderly by the middle of this century.2 \n  in this sense , many elders will likely face health problems inherent to aging , as well as challenges in maintaining their independence .", "however , the elderly can stay healthy and active in society in an independent and sustainable manner,3 and the work of professionals and researchers in the multidisciplinary health field that serve this group is fundamental in achieving this state . at the same time , it is essential that the government create and apply public policies that enable and sustain educational and awareness actions regarding people qualification and the fulfillment of the population basic needs .", "as for the health of the elderly , changes in body balance ( postural instability ) have received much attention , as it is one of the symptoms that most affect their lives .", "body balance is the result of a harmonious interaction among different systems in the human body : vestibular ( inner ear  posterior labyrinth ) , visual , and somatic - sensorial ( cutaneous , muscular , and articular receptors ) systems .", "this interaction is accomplished through processes of the neural system , which organizes and executes a rapid response by means of the musculoskeletal system.4 \n  the aging process is associated with a decrease in the ability to keep balance , especially in situations where responses of the balance keeping systems to unexpected disturbances during free moving and the avoidance of obstacles are necessary .", "sensorial , motor , and adaptive balance components become vulnerable as degenerative , infectious , or damaging processes accumulate .", "although small alterations in any of the systems that comprise balance keeping do not result in significant disturbances , the involvement of multiple components may lead to severe deficits in postural stability conservation.5 \n 6 \n  the aging process may alter the dynamic posture keeping system .", "this condition is a contributing factor to falls among the elderly , albeit not the main cause.7 impairment of the vestibular system is also believed to be a factor that leads the elderly to lose balance , through the aging process of this systes organs.8 \n  alterations to postural stability are also followed by dizziness .", "this can be defined as any sensation of change in the body balance and can be rotational ( vertigo ) or non - rotational ( unbalance , oscillation , vacillation , etc . ) . both types may or may not be caused by specific vestibular system disturbances.9 \n  the presence of dizziness may cause falls , which are associated to the main cause of death in the elderly group .", "some authors also associate dizziness to partial or full inability to perform social , professional , family activities , in addition to causing physical and psychological damage , such as loss of self - confidence , depression , deficit in concentration , and performance , thus negatively interfering in the quality of life.10 \n 11 however , it is still difficult to know if psychological problems pre - exist or are a result of vestibular complications.11 \n  balance disturbances and anxiety disorders share central neural circuits involving monoaminergic components , this being the reason why anxiety is often associated with balance alterations .", "these neural circuits concentrate in a parabrachial nucleus network ( vestibular system and visceral information processing convergence point ) , also involving avoidance , anxiety , and fear symptoms.12 physical insecurity triggered by dizziness and unbalance lead to psychological insecurity , irritability , loss of self - confidence , anxiety , depression or panic , a sense of being out of reality and depersonalization.13 \n  depression  which may be caused or made worse by body imbalance  is a common condition in seniors .", "furthermore , it is associated to higher risk of morbidity and mortality , increase in health services usage , negligence in self - care , and higher risk of suicide.14 the main symptoms of depression are depressed mood and loss of interest or lack of pleasure in almost all activities , being characterized as a psychiatric syndrome . in the elderly ,", "its manifestation occurs in a heterogeneous form regarding both the presentation aspects of its etiology and its treatment.15 \n  studies relate the involvement in physical activities with a better general health condition and lesser occurrence of depression and falls in the elderly.16 \n 17 physical activity is capable of improving mental and clinical conditions of depressed elderly .", "this is because physical activity stimulates cognitive , memory , and concentration functions , in addition to raising self - esteem , improving quality of life,18 and helping decrease scores indicative of anxiety and depression,19 favoring daily activities and promoting functional independence.20 furthermore , the physically active elderly show improvement of dizziness and vertigo symptoms,21 \n 22 \n 23 decreasing , as a result , the risk of falls and associated comorbidities .", "thus , the objective of this work is to verify the correlation between physical exercise , dizziness , probability of falls , and symptoms of depression in a group of middle - aged and elderly group ."], ["this is a transversal and observational study with approval from the institutios ethical committee ( record n 216292011 ) .", "the sample comprised adult and elderly individuals of both sexes who took part in university extension projects .", "the inclusion factors for subjects were non - institutionalized elderly with no history of neurological or cognitive alterations , who voluntarily accepted to take part in the research and signed the informed consent form ( icf ) .", "assessment procedures included a medical history prepared for this study ; the abridged version of the geriatric depression scale ( gds ) ; and anterior functional reach test .", "the medical history assessment included questions on individuals socio - demographic and health ( age , gender , presence of diseases , treatment used ) , as well as questions about their practice of physical exercises ( frequency , time of practice , type of physical exercise ) .", "as for assessing the results of the geriatric depression scale , each answer indicating depression scored one point .", "scores below five points indicated lack of depressive symptoms ; 6 to 10 points indicated mild / moderate depressive symptoms ; and 11 to 15 points meant severe depressive symptoms.24 \n  for the functional range test was initially placed a tape measure on the wall , parallel to the floor , at the height of the acromion of each individual .", "the following were instructed to position themselves parallel to the wall , without lean on to it , with the parallels and bare feet in a comfortable position .", "the upper limb was positioned with bent shoulders at 90 , extended elbows , wrists in a neutral , flat hand position . as a result", ", it was requested that incline forward as much as possible , hold for three seconds without taking the heels off the floor .", "taking as the third metacarpal of the individual parameter was recorded in centimeters anterior displacement on the tape .", "displacements smaller than 15.2 cm indicate the risk of falls four times higher compared to those who reached more than 25.4 cm , or is highly likely to falls .", "measures between 15.2 and 25.4 cm has an average probability of falls ( twice as likely to fall than those who reach values greater than 25.4 cm ) .", "values greater than 25.4 cm indicate low probability of falls.25 \n  the authors analyzed the data using descriptive statistics through measures of central tendency ( mean and median ) and variability ( standard deviation and inner quartile ranges ) , as well as absolute and relative distribution ( n - % ) .", "the symmetric continuous distribution was evaluated by the kolmogorov - smirnov test . for the bivariate analysis between qualitative variables , the authors used the pearson qui - square test (  ) and continuity correction , which compares between observed ( real ) and expected frequencies . in contingency tables", "in which at least 25% of cell ( quads ) values presented expected frequency below five , they used fishes exact test . for continuous variables , when comparing two independent groups , the applied test was students t - test or , its non - parametric equivalent , the mann whitney test .", "multivariate analysis was accomplished through non - conditional binary logistic regression , by employing regressive procedures to test the hypotheses of association between cognitive decline and co - variables defined by the bivariate analysis ( p    0.250 ) , by obtaining the adjusted odds ratio ( or ) .", "data received statistical treatment via spss 17.0 software ( statistical package for social sciences for windows ) , adopting a significance level (  ) of 5% for the decision - making criteria ."], ["the sample comprised of 90 individuals , aged 69.3 (    6.8 ) in average , with minimum age of 59 years and maximum age of 84 years .", "the majority of investigated individuals was in the age group between 65 and 74 years ( 38.9% ) .", "results presented as n ( % ) , with percentage obtained as based on total valid cases ;  : nr  =  5 ( 2.5% ) . the majority of individuals interviewed ( 65.8% ) did not show depressive symptoms . regarding the anterior functional reach test ,", "the majority of evaluated individuals indeed showed low probability of fall occurrence , with an average score of 28.2 cm ( sd  =  9.2 ) points , varying from 0 to 44.5 cm ( table 2 ) .", "results presented as n ( % ) , with percentage obtained as based on total valid cases ,  : nr  =  11 ( 12.2% ) ;  : nr  =  8 ( 8.8% ) . in the analyses done on dizziness complaints ,", "the authors verified a significant association with age group ( p  <  0.05 ) , with the elderly aged 75 years or more presenting a higher probability of feeling dizziness , whereas the group aged between 65 and 74 years , this association was not verified ( table 3 ) .", "percentages obtained as based on total of each dizziness category ;  : pearson chi - square test with continuity correction ;  : t - student test for independent groups ;  : pearson chi - square test ;  : fisher 's exact test through monte carlo simulation ; * statistically significant test at 5% ; * * statistically significant test at 1% .", "moreover , it has been observed statistically significant association between the occurrence of dizziness and the presence of light / moderate depressive symptoms ( p  <  0.01 ) , as well as between the occurrence of dizziness and the average probability of falls ( p  <  0.001 ) . in terms of the association between the occurrence of dizziness and the results of the anterior functional reach test , the average in centimeters for the anterior reach ( 25.3    5.4 ) of elderly with dizziness showed to be significantly less when compared with the average of the group without dizziness complaints ( 30.1    10.6 ) ( p  <  0.01 ) .", "when comparing the occurrence of dizziness with gender , age , practice of physical exercise , and time of practice , we identified an independent relation , evidencing that dizziness is unrelated to any of these factors in the studied group . in the group of individuals that did not practice physical exercise", ", there was an association between the occurrence of dizziness and light / moderate depressive symptoms ( p  <  0.001 ) .", "however , in the same analysis done with the group that practiced some physical activity , the association between dizziness and depressive symptoms was not statistically significant ( p  >  0.05 ) ( table 4 ) .", "another evaluation confirms the importance of physical exercise . keeping the remaining variation factors not considered in the analysis ( gender , age ) constant , the group that practices physical exercise was less prone to dizziness and depressive symptoms ( table 4 ) .", "we looked for and identified the variables that could respond in a more trustworthy form to the occurrence of dizziness .", "we performed the logistic regression analysis with an adjustment to minimize the number of explanatory variables employed and maximize model precision , through the backward model , to maximize the explanatory power of the study factor . through a model defined as saturated", ", we listed all variables with minimum significance level below 0.25 through bivariate analysis ( table 3 ) as possible preceding factors for the presence of dizziness .", "they are the following : gender , age , age group , physical activity , gds score , dislocation in the anterior functional reach test , and balance . in this model , the variables for gender and age pointed out an independent relation to the occurrence of dizziness . according to the adjusted logistic regression model ,", "the following were significant predictive factors for the occurrence of dizziness : non - practice of physical exercise , classifications of light / moderate depressive symptoms , and dislocation in the anterior functional reach test .", "results described in table 5 show that the elderly who do not practice physical exercise have 2.2 times higher risk of presenting dizziness than the group that practices some physical exercise . in relation to depressive symptoms ,", "the significant risk of presenting dizziness occurred in the light / moderate degree , indicating that the elderly included in this classification had 4.5 times more chance of presenting dizziness than the group with absence of depressive symptoms .", "model parameters : pseudo - r  =  0.432 ;  -2 log likelihood  =  80.208 ; hosmer and lemeshow ( p  =  0.998 ) ; pearson 's chi - square (   =  9272 ; p ", "=  0.099 ) ; nagelkerke ( r : 0.460 ) ; correct classification : 74.4% ( dizziness : no  =  78.7% ; yes  =  67.7% ) .", "as the number of elderly with severe depressive symptoms was very low , the light / moderate and serious groups were merged , by classifying the answer for symptoms as presence or absence . over this new variable ,", "we once more calculated the risk of presence of depressive symptoms associated to dizziness , estimated at 2.1 times that of the group with absence of this symptom .", "the results verified that the group with medium probability of falls as the result for the anterior functional reach test had 4.0 times more chances of occurrence of dizziness than the group with low probability of falls . when age was kept constant", ", results indicated that the non - practice of physical exercise , the presence of depressive symptoms , and the result of the anterior functional reach test ( indicating medium probability of falls ) are factors associated to the occurrence of dizziness in the investigated sample ."], ["in the sample of this study , more than 60% of the individuals affirmed to not practice physical exercise , confirming other similar studies .", "the ministry of health of brazil26 made a survey , and verified that 53.7% of elderly men and 58.3% of elderly women were inactive .", "similar results were found in other study concerning the level of physical activity and its relationship with the environment ( specifically , safety ) with 1,656 elderly from florianpolis.27 by using similar parameters , pucci , reis , rech and hallal28 classified the majority of the individuals of their study as insufficiently active in leisure or walking activities . in this study ,", "current studies highlight the prevalence of dizziness reports among the elderly population.29 \n 30 \n 31 \n 32 approximately one in every five seniors currently presents dizziness or imbalance.29 in our research , 40% of the elderly reported sensation of dizziness .", "this finding is very similar to other studies done in brazil , in which the authors verified that the prevalence of dizziness in the elderly is substantial , having observed dizziness complaints from 45% of 391 elderly over 65 years of age interviewed.33 \n  another study interviewed 516 elderly people , and confirmed self - reported dizziness in 42% of them.31 thus , dizziness is a common problem for the elderly population,11 \n 34 \n 35 as these individuals show a decrease in visual acuity during aging , and loss of balance due to the reduction of ciliated cells and vestibular neurons .", "this results in loss of functions from two important components responsible for keeping balance.36 furthermore , with age , the central nervous system capabilities become impaired , affecting the processing of vestibular , visual , and proprioceptive signals responsible for maintenance of body balance .", "age also reduces capacity of modifications of the adaptive reflexes , may be responsible for the occurrence of vertigo and/or dizziness in the geriatric population.35 \n  the data from the studied sample evidenced that 33.7% of the elderly presented depressive symptoms .", "this prevalence is higher than that described by other authors,37 who report prevalence of 26.7% of depressive symptoms in a sample of elderly people residing in an urban zone . in turn , study reported depressive symptoms in 31% of the elderly evaluated , by also using the gds scale in active elderly who were part of a conviviality group , i.e. , a similar sample to the one evaluated in this study .", "additionally , the cited authors verified that 4% of the evaluated elderly presented severe depressive symptoms , which was also similar to the obtained data.38 \n  regarding anterior functional reach results , the average obtained was 28.2    9.2 cm .", "results were higher than those observed by previous studies,39 \n 40 and lower than those obtained by another study.41 values found were similar to those observed in brazil,42 which also evaluated a sample of adult and elderly people .", "the sample of elderly people in the age group between 70 and 87 years of age presented functional reach values between 29.75 ( men ) and 27.13 ( women ) .", "variations in the results of these studies can be explained through different factors , such as fall history , freight , strength , and flexibility of upper and lower limbs.42 \n  this research verified a stronger presence of dizziness in the age group above 75 years ( 41.7% ) , a significant association ( p  =  0.028 ) when compared with remaining age groups .", "such a finding is similar to that of the research with 1,273 elderly between 60 and 96 years of age , which verified reports of dizziness in 23.6% of all elderly evaluated . in this study ,", "17.8% of them were under 80 and 31% were over 80 years old.30 another longitudinal research with 620 elderly people verified that dizziness was a factor that depended on age , with prevalence of 27% in the elderly under 70 , and over 54% in the group with 90 years of age or more . among the elderly that mentioned dizziness ,", "68.2% perceived this symptom daily or weekly.43 the bigger prevalence of dizziness in the age group above 75 years of age could be due to the generalized deterioration of health in older people44 or multifactor conditions , both medical and functional.43 \n  it is worth mentioning that the presence of dizziness is directly related to the presence of falls in the elderly population;30 both conditions are considered geriatric syndromes that occur concomitantly to other harms to multiple systems of the body.5 this worsens with age .", "the elderly who suffer falls can have fractures as a consequence , which might require longer periods in bed .", "such condition is responsible for 70% of deaths resulting from accidents in individuals over 75 years in age.45 \n  further to this , there was no significant difference when comparing the presence of dizziness complaints among the elderly who practiced physical exercise whith those who did not .", "these data were different from the expectations of the authors and the findings of ruwer et al,35 because , according to these authors , the complaint of dizziness is less prevalent in the elderly that practice physical exercise .", "nevertheless , with the adjusted logistic regression , it is possible to verify that the individuals who do not practice physical exercise are 2.2 times more likely of feeling dizzy than physically active individuals are .", "this result confirms studies that compared elderly people with and without dizziness and observed that those without complaints of dizziness were engaged in physical activity.46 these authors also verified that there was a relation between dizziness and depression , which attest to the data from our study .", "the research verified a significant association between light to moderate depressive symptoms and 4.5-fold increase in the risk of feeling dizziness . regarding the anterior functional reach test , this research verified a significant association between the probability of falls and reports of dizziness .", "the elderly with medium probability of falling are four times more likely to feel dizziness than the elderly with low probability of falls , as described in another study.47 \n  the studies indicate that active elderly people are less prone to depression than the sedentary elderly.19 \n 48 \n 49 physical exercises help to reduce depression , aside from helping in its prevention .", "physical activity relates to less frequent mood disorders and may even help release some neurotransmitters , such as noradrenalin and serotonin.50 \n 51 \n  studies indicate that elderly people who practice regular physical activity mention less depressive symptoms than non - active or sedentary elderly people .", "analysis of social skills , social support , life quality and depression of elderly people in an extension project , family contexts and asylums indicated that the depressive symptom was present in 0% of practicing elderly people compare to 30% of the elderly who did not participate in the project . in the asylum , in turn , this percentage increased 40%.52 in other study about the intensity and prevalence of depressive symptoms in elderly women participating in an extension project , it was verified an absence of depressive symptoms in 80.6% of the sample.53 another study evaluated the effect of a program of aerobic physical exercise in the intensity of the ventilator threshold over depression and anxiety and life quality of healthy elderly people divided into a control and a experimental group .", "the elderly in the experimental group had a reduction in the scores of depression and anxiety and an increase in life quality , while in the control group no changes were observed.51 \n  in the research on the freight of falling and its relation with balance and mobility tests , there was a moderate correlation.40 authors state that balance and mobility enable the stability necessary to maintain a standing position , for instance . therefore , maintaining adequate levels of such capabilities reduces the risk of falls , which are directly related to level of physical activity .", "the american college of sports medicine54 mentions that the level of physical activity is inversely related to the number of limitations in the elderly .", "brazilian authors analyzed the relation between health conditions , incidence of falls , and level of physical activity in 256 elderly people .", "results indicated an association between the level of physical activity and the incidence of falls and health conditions .", "in other words , the more frequent the practice of physical activity the less the occurrence of falls .", "muscular weakness , reduction in articular mobility , degraded synergy , programming mechanisms , and motor control difficulties are the variables present in low levels of physical activity , predisposing to falls in the elderly.55 \n"], ["the results of this study indicated the existence of an association between the practice of physical exercise , dizziness , probability of falls , and depressive symptoms in a group of non - institutionalized middle aged adults and elderly people , evidencing that physical activity is a beneficial factor for the aging population ."]]}
{"article_id": "PMC4951176", "article_text": ["we measured the differences in stiffness between extrafloral nectaries of v. faba and the tissue that surrounds them .", "we mounted 20 stipules from 20 , 18-day - old plants on adhesive tape that was attached ( sticky side up ) to the surface of a piece of flat styrofoam .", "we did a penetrometer test to measure the initial modulus of rigidity of the spot being tested , using a texture analyzer ( ta.xt2i , stable micro systems , surrey , uk ) fitted with a 10 stainless steel cone moving at 0.5  mm / s . in each stipule", ", one measurement was done in the center of the extrafloral nectary and 3 measurements were done in random locations on the stipule . for each stipule , an average ", "stipule stiffness  was calculated from these 3 measurements and this value was compared with the measured stiffness of the extrafloral nectary on that stipule ."], "abstract_text": ["<S> abstractextrafloral nectaries play an important role in plant defense against herbivores by providing nectar rewards that attract ants and other carnivorous insects </S>", "<S> . however , extrafloral nectaries can themselves be targets of herbivory , in addition to being exploited by nectar - robbing insects that do not provide defensive services . </S>", "<S> we recently found that the extrafloral nectaries of vicia faba plants , as well as immediately adjacent tissues , exhibit high concentrations of chemical toxins , apparently as a defense against herbivory . here </S>", "<S> we report that the nectary tissues of this plant also exhibit high levels of structural stiffness compared to surrounding tissues , likely due to cell wall lignification and the concentration of calcium oxalate crystals in nectary tissues , which may provide an additional deterrent to herbivore feeding on nectary tissues . </S>"], "labels": null, "section_names": ["Materials and methods", "Disclosure of potential conflicts of interest", "Acknowledgments"], "sections": [["we measured the differences in stiffness between extrafloral nectaries of v. faba and the tissue that surrounds them .", "we mounted 20 stipules from 20 , 18-day - old plants on adhesive tape that was attached ( sticky side up ) to the surface of a piece of flat styrofoam .", "we did a penetrometer test to measure the initial modulus of rigidity of the spot being tested , using a texture analyzer ( ta.xt2i , stable micro systems , surrey , uk ) fitted with a 10 stainless steel cone moving at 0.5  mm / s . in each stipule", ", one measurement was done in the center of the extrafloral nectary and 3 measurements were done in random locations on the stipule . for each stipule , an average ", "stipule stiffness  was calculated from these 3 measurements and this value was compared with the measured stiffness of the extrafloral nectary on that stipule ."], [""], [""]]}
{"article_id": "PMC4106404", "article_text": ["health systems based on primary health care ( phc ) distribute health care more equitably , are more cost effective and have better overall health outcomes and impact than health systems based on specialist care ( 1 ) .", "this is the foundation of the  health for all concept of alma - ata , adopted by the world health organization ( who ) in 1978 ( 2 ) and reiterated in the world health report 2008 ( 3 ) .", "worldwide , authorities have recognized that the health and well - being of a population is highly dependent on a quality phc system that is equitable , easily accessible and affordable for all members of the community ( 4 ) and that emphasizes universal coverage ( 5 ) .", "the concept of the postgraduate - trained family physician qualified to deliver equitable , high - quality phc closer to the community is now accepted in many countries around the world . however , in sub - saharan africa , family medicine is still a relatively new concept . in this article , we analyse the development of family medicine in anglophone sub - saharan africa in the recent years based on the experiences of the primafamed ( primary health care and family medicine education ) project ( 6 ) .", "lower - resource countries in sub - saharan africa face enormous health challenges and pervasive poverty . despite the work of governments and nongovernmental organizations ( ngos ) , the majority of people still do not have easy access to affordable quality health care .", "the  inverse care law ( 7 ) , noting that the fewest health - care professionals are found where they are needed the most and vice versa , is still very much applicable in most african countries .", "the poor are not only more prone to illnesses but are also unable to cope with diseases because health care is hard to access . with continuous population growth and a rather slow economic development ,", "the number of people living in poverty in sub - saharan africa has also increased , with 20.6% living on less than us$1.25 a day in 2008 ( 8) .", "it is the first level of contact of a continuing health - care process bringing health care as close as possible to where people live and work ( 9 ) .", "phc responds to the immense challenges that african countries are facing in their health systems by providing accessible , high - quality services that offer comprehensive and continuous care ( preventive , curative , rehabilitative and palliative ) at the local level , through interdisciplinary teams integrating vertical disease - oriented programmes .", "family physicians together with phc nurses ( and in some countries , mid - level care workers ) act as the clinical practitioners of the phc team . in 2009 , the 62nd world health assembly urged its member states to train and retain adequate numbers of health workers , including family physicians , and to encourage the implementation of vertical programmes in the context of integrated phc ( 10 ) . in 1968 ,", "the university of pretoria was the first university in south africa to start training specialized phc physicians , now referred to as  african family physicians ( 11 ) .", "this was followed by the other seven health science faculties in south africa . in 1997 , these eight departments of family medicine formed a network for communication and consultation , famec ( family medicine educational consortium ) , to share and exchange expertise , form a core curriculum and standardize examinations and develop appropriate assessment systems ( 12 ) . in august 2007 ,", "the concept of the african family physician in other anglophone african countries is even more recent . only in the 21st century", "did universities in anglophone countries in sub - saharan africa start family medicine training programmes , and the recently graduated african family physicians are beginning to find their place in the health systems of their respective countries .", "family medicine departments are struggling for recognition as health systems are still dominated by centralized specialist services and vertical disease - oriented approaches .", "several countries , such as namibia and botswana , did not have medical schools until very recently ( 14 ) .", "furthermore , many countries , such as the democratic republic of congo ( dr congo ) , are emerging from conflict and need to rebuild both state and infrastructure . at the wonca ( world organisation of family doctors ) africa conference in 2009 ,", "this consensus statement defined the contribution of family medicine to equity , quality and phc within an african context , as well as the role and training requirements of the african family physician ( 15 ) . due to the low number of trained doctors per capita and the high burden of disease , african family physicians work in their specific context , mainly in district hospitals with outreach to health centres , in phc teams that address the problems of the community in a comprehensive , holistic and patient - centred way whereby specific skills like surgery and district management often are essential due to the lack of other specialists .", "the consensus illustrates the ownership of the development of family medicine by african universities , one of the key points from the paris declaration ( 2005 ) and the accra agenda for action ( 2008 ) ( 16 ) .", "lower - resource countries in sub - saharan africa face enormous health challenges and pervasive poverty . despite the work of governments and nongovernmental organizations ( ngos ) , the majority of people still do not have easy access to affordable quality health care .", "the  inverse care law ( 7 ) , noting that the fewest health - care professionals are found where they are needed the most and vice versa , is still very much applicable in most african countries .", "the poor are not only more prone to illnesses but are also unable to cope with diseases because health care is hard to access . with continuous population growth and a rather slow economic development ,", "the number of people living in poverty in sub - saharan africa has also increased , with 20.6% living on less than us$1.25 a day in 2008 ( 8) .", "it is the first level of contact of a continuing health - care process bringing health care as close as possible to where people live and work ( 9 ) .", "phc responds to the immense challenges that african countries are facing in their health systems by providing accessible , high - quality services that offer comprehensive and continuous care ( preventive , curative , rehabilitative and palliative ) at the local level , through interdisciplinary teams integrating vertical disease - oriented programmes .", "family physicians together with phc nurses ( and in some countries , mid - level care workers ) act as the clinical practitioners of the phc team . in 2009 ,", "the 62nd world health assembly urged its member states to train and retain adequate numbers of health workers , including family physicians , and to encourage the implementation of vertical programmes in the context of integrated phc ( 10 ) . in 1968 ,", "the university of pretoria was the first university in south africa to start training specialized phc physicians , now referred to as  african family physicians ( 11 ) .", "this was followed by the other seven health science faculties in south africa . in 1997 , these eight departments of family medicine formed a network for communication and consultation , famec ( family medicine educational consortium ) , to share and exchange expertise , form a core curriculum and standardize examinations and develop appropriate assessment systems ( 12 ) . in august 2007 ,", "the concept of the african family physician in other anglophone african countries is even more recent . only in the 21st century", "did universities in anglophone countries in sub - saharan africa start family medicine training programmes , and the recently graduated african family physicians are beginning to find their place in the health systems of their respective countries .", "family medicine departments are struggling for recognition as health systems are still dominated by centralized specialist services and vertical disease - oriented approaches .", "several countries , such as namibia and botswana , did not have medical schools until very recently ( 14 ) . furthermore", ", many countries , such as the democratic republic of congo ( dr congo ) , are emerging from conflict and need to rebuild both state and infrastructure . at the wonca ( world organisation of family doctors ) africa conference in 2009 ,", "this consensus statement defined the contribution of family medicine to equity , quality and phc within an african context , as well as the role and training requirements of the african family physician ( 15 ) . due to the low number of trained doctors per capita and the high burden of disease , african family physicians work in their specific context , mainly in district hospitals with outreach to health centres , in phc teams that address the problems of the community in a comprehensive , holistic and patient - centred way whereby specific skills like surgery and district management often are essential due to the lack of other specialists .", "the consensus illustrates the ownership of the development of family medicine by african universities , one of the key points from the paris declaration ( 2005 ) and the accra agenda for action ( 2008 ) ( 16 ) .", "the aim of this article is to explore the extent to which the primafamed south ", "south cooperative project contributed to the development of family medicine in sub - saharan africa .", "the specific objectives are the following : the implementation of the primafamed project ; the outcomes of the primafamed project ; the development of family medicine training in sub - saharan africa from the viewpoint of the primafamed partners .", "a process analysis of the primafamed project between 2008 and 2011 is presented to discuss the implementation of the programme and the development of the network of 10 universities and associated partners in anglophone sub - saharan africa .", "finally , a swot analysis is detailed to explore the positive and negative dynamics that partners faced during the project . in 2007 ,", "a call for proposals under the name edulink was launched by the secretariat of the african , caribbean and pacific ( acp ) group of states ( 17 ) .", "together with 10 universities in eight countries in sub - saharan africa ( sudan , ghana , nigeria , dr congo , rwanda , uganda , kenya and tanzania ) , figure 1 , the department of family medicine and phc at ghent university in belgium developed the primafamed project proposal with a focus on developing family medicine training in sub - saharan africa .", "the aim of the primafamed project was to establish an institutional network between new and established departments of family medicine in universities in sub - saharan africa , within a framework of south  south cooperation ( 18 ) .", "the who world health report 2006 ,  working together for health , emphasized the need for phc training in the local community in order to deal with the brain drain from acp states ( 19 ) .", "training medical doctors in the field of family medicine , thus providing phc at the district level , responds to this call .", "the objectives of the primafamed project were formulated as follows : 1 . to contribute to the health of communities through accessible , responsive and quality health systems in sub - saharan countries by educating and training family physicians who provide interdisciplinary phc services , oriented towards the needs of individuals , their families and the communities in which they live", "develop and strengthen academic departments or units of family medicine that offer family medicine training at the undergraduate and postgraduate levels .", "the specific objectives were as follows : 1 . to develop a comprehensive vision and strategy , within the specific context of sub - saharan countries , that delineates the integral contribution of family medicine and the phc team to an equitable and quality phc system ; 2 . to establish a specific institutional network between departments and units of family medicine .", "the expected results of the primafamed project were the following : 1 . an improved institutional and administrative functioning in terms of policy , management , planning and administrative capacity building of the participating departments and units of family medicine .", "2 . improved relevance of family medicine in undergraduate and postgraduate training in the regional context .", "the partnering universities in the primafamed project were university of goma ( dr congo ) , moi university ( kenya ) , national university of rwanda , aga khan university ( tanzania ) , university of lagos ( nigeria ) , makerere university ( uganda ) , mbarara university ( uganda ) , ahfad university for women ( sudan ) , gezira university ( sudan ) and university of ghana .", "the south  south network connecting these 10 universities worked together with the eight departments of family medicine in south africa and accordingly formed a forum to share knowledge , experiences and resources .", "each of the 10 partner universities hired a local coordinator to oversee the implementation of the project , coordinate the activities and actively communicate with all partners .", "partners worked independently , integrating the primafamed objectives and the output for the project into the already existing structures and work plan at their universities . because african family physicians work mainly in district hospitals with outreach to the health centres ,", "the primafamed project consequently stimulated all partners to develop training complexes ( mainly district hospitals , several of which are located in rural areas ) for family medicine residents and to support the supply of these training complexes with the needed equipment . to further strengthen family medicine residents education , training sessions by professors from south african universities and other associated primafamed partners were held .", "a yearly conference for partners , associates and stakeholders was organized with the goal of offering trainings , sharing ideas and experiences , and strengthening the network . at the 2008 conference in kampala , the african journal of primary health care and family medicine ( 20 )", "this new open - access online journal has since published various articles related to the development of family medicine on the african continent , and researchers in the field of phc have written numerous articles on operational research and community - oriented primary care ( copc ) projects ( 21 ) . to monitor and evaluate our project ,", "this four - level scale shows the progress that took place during the 2.5-year period in which primafamed supported the partners .", "the first level corresponds to the institution being at a preparatory stage in the development of a postgraduate family medicine training programme .", "the fourth level reflects that the institution has started a postgraduate family medicine training programme with an existing curriculum by an organized department at well - organized training complexes and , most importantly , that family medicine has been accepted by the ministry of health as a specialization and that graduated family physicians are integrated into the existing health system .", "undeniably , the fourth level is not the end stage because , in our opinion , the ultimate objective would be to have an equitable and high - quality phc system with a central role for family physicians .", "department / unit of family medicine exists or is part of other departments ( community medicine )  training complexes are under development  family medicine is part of undergraduate training ", "postgraduate training has started  department / unit of family medicine exists  training complexes are in place ", "postgraduate training has started  the ministry of health has accepted family medicine as a specialization and graduated family physicians are part of the health - care system adopted from the primafamed edulink acp eu project .", "all 10 primafamed partners made progress during the 2.5 years that the project provided funding and support ( table 1 ) .", "at the start of the project , all of them had a unit or department of family medicine under development or in place , yet only three had officially started postgraduate family medicine training . at the end of the project ,", "eight had started postgraduate training , with family medicine residents located in properly equipped training hospitals .", "progress scale for development of the primafamed partners adopted from the primafamed edulink acp eu project .", "the integration of family medicine into the local health systems has been a slower process . in many countries ,", "policymakers have adopted a somewhat conservative attitude , with health systems mainly based on hospital / specialist care and on vertical programmes , which are often stimulated by donor programmes .", "a critical mass of well - trained family physicians is needed to demonstrate the effectiveness of family medicine training programmes . because all but one partner universities train only a limited number of family medicine residents simultaneously due to limited capacity , reaching that critical mass takes time . however , as the example of gezira university ( sudan ) has shown , it is possible to create a large pool of well - trained family physicians who can make a difference , in a short time . in this particular situation , because there was a vital need , policymakers and local authorities worked closely together with gezira university in development of the training ( example 1 ) . in 2012 ,", "african family physicians came together at the wonca africa conference and the primafamed network workshop in victoria falls , zimbabwe , and reaffirmed the importance of reaching that critical mass and therefore scaling up of family medicine , particularly in the african context , in which poverty is very prevalent , where rural  urban gaps remain , and where many of the millennium development goals ( mdgs ) will not be reached , including those related to child survival , maternal death and accessibility to antiretroviral drugs ( 22 ) . in 2010 , all primafamed local coordinators contributed to a swot ( strengths , weaknesses , opportunities and threats ) analysis on the development of family medicine training programmes and of family medicine as a new discipline within the health systems of their countries ( box 3 ) .", "the main findings are presented and categorized into the following levels : local / university level , national level , regional / african level and international level .", " strong partnerships and collaboration with universities and professional bodies abroad is present  the south african departments of family medicine have substantial experience in the family medicine training programmes : they can assist in training via south  south cooperation and these cooperative steps can be seen as exemplary models and  good practices", " the primafamed project and edulink acp - eu funding strengthens the development of family medicine training and is seen as the motor for the family medicine training development ", "individuals working in the family medicine departments are very motivated , proactive and willing to advocate for the cause  family physicians working as faculty are highly experienced and well qualified  the chosen district hospitals and health centres are ready for the training programmes  in several of the countries ( sudan , kenya and ghana ) , a high commitment from the ministry of health , the national council and leaders is existing  family medicine is responsive to the needs of the communities , especially in the rural areas where the majority of africans live  family medicine is not yet part of undergraduate medical training , therefore medical students are not exposed to the concept of family medicine and its principles  poor quality of intake and recruitment of family medicine residents for the family medicine training  the lack of family physicians and local teachers .", "many of the departments of family medicine depend on family physicians coming from non - african countries ( mainly , western europe and united states )  due to work overload , family medicine residents are continuously working in the hospital setting and have insufficient time to focus on the training in other aspects of family medicine , such as disease prevention and health promotion , community medicine , outpatient / continuity of care and research  the existing health systems are still weak in several countries ; plagued by poor support , weak referral systems , poor communication , poor funding and poor coordination between health centres , district hospitals and referral hospitals  dropping out of family medicine residents during the training sometimes happens in countries like dr congo because family medicine residents lose faith in their career opportunities  there is a lack of didactic materials  capacity is still too limited ", "there is a lack of awareness and knowledge of family medicine by medical graduates , specialists and the community  there is lack of adequate infrastructure in some countries , such as dr congo and ghana  there is poor communication with policymakers and little support from the government in countries like tanzania and rwanda \n   standardization of training and examinations between faculties and countries is presently not existing , although there is a strong need for accreditation and quality assurance  there is insufficient financial support for the family medicine residents in postgraduate training ", "the position of family physicians in the different health systems is unclear ( what is the career perspective of graduated family physicians ? )  there is international support for the concept of family medicine : in the world health report 2008  primary health care : now more than ever , the who advocated for the importance of moving health care out of tertiary hospitals into the community and from vertical to horizontal care , in order to respond to the needs of the community  family medicine training is growing in the whole african continent , and there is support in the surrounding countries .", "the primafamed project has been linking these different countries , in order to fight for the cause together ", "existing health systems in several countries are weak ( poor coordination , poor referral systems , poor communication and little funding )", ". family medicine can be used as a technique to strengthen these health systems  internet connection is present in several of the training centres ( and this is rapidly expanding ) ; e - learning is a very useful didactic tool for the family medicine residents  a yearly conference is organized by the primafamed network to strengthen the network and to share ideas and exchange experiences  wonca africa is organizing three yearly conferences and has been expanding in terms of number of members in the recent years  there are many existing links with organizations and universities in the north for financial and didactic support  iucea and wacp can assist in accreditation and quality assurance  several very useful books on african family medicine have been published recently and can be used as training material  the new african journal of primary health care and family medicine gives more opportunities for registrars to publish research  health - care workers and policymakers are becoming increasingly aware of the importance of the basic principles of phc and family medicine such as patient - centred care , non - communicable diseases ( ncds ) , interdisciplinary care , and continuity of care  many african ministries of health are working on health - system reforms  research in the field of family medicine is done by the registrars during the training  the primafamed network has been looking into expanding to francophone africa : mali and benin will participate in a vlir uos  funded project starting 2012 , and the african journal of primary health care and family medicine will be published in english and french from 2012 onwards  other specialities see family medicine as a threat  family medicine is a new concept and not yet part of the health system in most of the partnering countries  overload of work for doctors leads to little time for training  lack of career opportunities for family medicine physicians in countries where family medicine is not supported by the government  there is little or no funding for family medicine within the existing systems  substantial decrease in funding for further development of the family medicine training after ending of the primafamed project funding from edulink acp - eu  migration of graduates to private practice , ngos , management positions or public health ( internal brain drain ) or migration of graduates to other countries ( external brain drain ) \n   war and political instability ( e.g. dr congo , sudan and nigeria ) ; in some countries , there is a high turnover of government and with this , there often is a change of policies  increasing commercialization and privatization of health - care provision", " worldwide economic crisis , which is decreasing the funding for health care and is putting health systems under pressure  fragmentation of care through vertical disease - oriented programmes , not only for infectious diseases , but increasingly for ncds ( 23 ) .", "i. when the swot analysis was done in 2010 , the rwandan ministry of health was supportive of the new discipline and family medicine was accepted by the government as one of the specialist mmed trainings from the national university of rwanda .", "however , at the time of writing this article , the ministry of health in rwanda has changed its vision and family medicine is not seen as a priority . recently", "west african college of physicians v. especially in sudan , this is a problem as a large percentage of medical graduates move to saudi arabia for more job opportunities , better salaries or family reasons adopted from the primafamed edulink acp eu project at the local / university level , the availability of resources plays an important role . from a human resources perspective , family physicians ( in most cases from abroad ) are needed to form the faculty , train family medicine residents and develop teaching materials .", "other local professors and specialists willing to train family medicine residents in the different disciplines are important to the strengthening of both the training and the interdisciplinary network ; however , several partners noted a low level of support from other specialists because family medicine is often not well understood and is often seen as a threat .", "adequate motivation of faculty and family medicine residents is important and is influenced by many other factors such as financial backing , career opportunities and support at the local , national and international levels .", "the availability of training sites , especially at district hospitals and health centres , is crucial , including having access to physicians to train and mentor the family medicine residents and stimulating teamwork with other health - care workers such as the local nurses .", "well - written curricula ( often developed based on sample curricula from family medicine training programmes in other african universities ) and training materials are essential to create a high - quality training atmosphere .", "access to the internet and to didactic materials and equipment ( such an ultrasound machines or health education materials ) are important to strengthen the actual training", ". lack of diagnostic or therapeutic equipments or insufficient availability of essential drugs in the training hospitals influences the way the doctors learn and hinders implementation of evidence - based medicine .", "ensuring that there is a place for family medicine training in undergraduate curricula plays a crucial role in raising awareness and recruiting newly trained medical doctors . at the national level , policymakers play a pivotal role . in several countries ,", "decision - makers accept family medicine as an official specialization that is starting to take its place in the health - care system , with kenya , sudan and ghana as examples ( see examples 1 , 2 and 3 ) . several other countries have had more difficulties with the ministry of health accepting family medicine , including tanzania and rwanda .", "research on the positive outcomes of family medicine on health - care provision plays a significant role in advocating and strengthening awareness .", "a negative factor at the national level is political instability and war , as seen in eastern congo . at the regional / african level , the importance of strong partnerships and collaboration with universities and professional bodies abroad is noted .", "south african universities have excellent curricula and knowledgeable professors with extensive experience that can be leveraged via south  south cooperation .", "moreover , conferences organized by primafamed and wonca africa support the development of african family medicine .", "the establishment of the african journal of primary health care and family medicine is a great opportunity for young researchers to learn from others work and to publish their own articles . at the international level ,", "secondly , global recognition of the importance of training health - care workers in phc ( world health report 2006 and 2008 ) is vital to the development of african family medicine .", "in 2007 , a call for proposals under the name edulink was launched by the secretariat of the african , caribbean and pacific ( acp ) group of states ( 17 ) .", "together with 10 universities in eight countries in sub - saharan africa ( sudan , ghana , nigeria , dr congo , rwanda , uganda , kenya and tanzania ) , figure 1 , the department of family medicine and phc at ghent university in belgium developed the primafamed project proposal with a focus on developing family medicine training in sub - saharan africa .", "the aim of the primafamed project was to establish an institutional network between new and established departments of family medicine in universities in sub - saharan africa , within a framework of south  south cooperation ( 18 ) .", "the who world health report 2006 ,  working together for health , emphasized the need for phc training in the local community in order to deal with the brain drain from acp states ( 19 ) .", "training medical doctors in the field of family medicine , thus providing phc at the district level , responds to this call .", "the objectives of the primafamed project were formulated as follows : 1 . to contribute to the health of communities through accessible , responsive and quality health systems in sub - saharan countries by educating and training family physicians who provide interdisciplinary phc services , oriented towards the needs of individuals , their families and the communities in which they live .", "develop and strengthen academic departments or units of family medicine that offer family medicine training at the undergraduate and postgraduate levels .", "the specific objectives were as follows : 1 . to develop a comprehensive vision and strategy , within the specific context of sub - saharan countries , that delineates the integral contribution of family medicine and the phc team to an equitable and quality phc system ; 2 . to establish a specific institutional network between departments and units of family medicine .", "an improved institutional and administrative functioning in terms of policy , management , planning and administrative capacity building of the participating departments and units of family medicine .", "2 . improved relevance of family medicine in undergraduate and postgraduate training in the regional context .", "the partnering universities in the primafamed project were university of goma ( dr congo ) , moi university ( kenya ) , national university of rwanda , aga khan university ( tanzania ) , university of lagos ( nigeria ) , makerere university ( uganda ) , mbarara university ( uganda ) , ahfad university for women ( sudan ) , gezira university ( sudan ) and university of ghana .", "the south  south network connecting these 10 universities worked together with the eight departments of family medicine in south africa and accordingly formed a forum to share knowledge , experiences and resources .", "each of the 10 partner universities hired a local coordinator to oversee the implementation of the project , coordinate the activities and actively communicate with all partners .", "partners worked independently , integrating the primafamed objectives and the output for the project into the already existing structures and work plan at their universities . because african family physicians work mainly in district hospitals with outreach to the health centres ,", "the primafamed project consequently stimulated all partners to develop training complexes ( mainly district hospitals , several of which are located in rural areas ) for family medicine residents and to support the supply of these training complexes with the needed equipment .", "to further strengthen family medicine residents education , training sessions by professors from south african universities and other associated primafamed partners were held . a yearly conference for partners , associates and stakeholders", "was organized with the goal of offering trainings , sharing ideas and experiences , and strengthening the network . at the 2008 conference in kampala , the african journal of primary health care and family medicine ( 20 )", "this new open - access online journal has since published various articles related to the development of family medicine on the african continent , and researchers in the field of phc have written numerous articles on operational research and community - oriented primary care ( copc ) projects ( 21 ) . to monitor and evaluate our project ,", "this four - level scale shows the progress that took place during the 2.5-year period in which primafamed supported the partners .", "the first level corresponds to the institution being at a preparatory stage in the development of a postgraduate family medicine training programme .", "the fourth level reflects that the institution has started a postgraduate family medicine training programme with an existing curriculum by an organized department at well - organized training complexes and , most importantly , that family medicine has been accepted by the ministry of health as a specialization and that graduated family physicians are integrated into the existing health system .", "undeniably , the fourth level is not the end stage because , in our opinion , the ultimate objective would be to have an equitable and high - quality phc system with a central role for family physicians .", "department / unit of family medicine exists or is part of other departments ( community medicine )  training complexes are under development  family medicine is part of undergraduate training  department / unit of family medicine exists  training complexes are in place ", "postgraduate training has started  department / unit of family medicine exists  training complexes are in place ", "postgraduate training has started  the ministry of health has accepted family medicine as a specialization and graduated family physicians are part of the health - care system adopted from the primafamed edulink acp eu project .", "all 10 primafamed partners made progress during the 2.5 years that the project provided funding and support ( table 1 ) . at the start of the project , all of them had a unit or department of family medicine under development or in place , yet only three had officially started postgraduate family medicine training . at the end of the project ,", "eight had started postgraduate training , with family medicine residents located in properly equipped training hospitals .", "progress scale for development of the primafamed partners adopted from the primafamed edulink acp eu project .", "the integration of family medicine into the local health systems has been a slower process . in many countries ,", "policymakers have adopted a somewhat conservative attitude , with health systems mainly based on hospital / specialist care and on vertical programmes , which are often stimulated by donor programmes .", "a critical mass of well - trained family physicians is needed to demonstrate the effectiveness of family medicine training programmes . because all but one partner universities train only a limited number of family medicine residents simultaneously due to limited capacity , reaching that critical mass takes time . however , as the example of gezira university ( sudan ) has shown , it is possible to create a large pool of well - trained family physicians who can make a difference , in a short time . in this particular situation , because there was a vital need , policymakers and local authorities worked closely together with gezira university in development of the training ( example 1 ) . in 2012 ,", "african family physicians came together at the wonca africa conference and the primafamed network workshop in victoria falls , zimbabwe , and reaffirmed the importance of reaching that critical mass and therefore scaling up of family medicine , particularly in the african context , in which poverty is very prevalent , where rural  urban gaps remain , and where many of the millennium development goals ( mdgs ) will not be reached , including those related to child survival , maternal death and accessibility to antiretroviral drugs ( 22 ) .", "in 2010 , all primafamed local coordinators contributed to a swot ( strengths , weaknesses , opportunities and threats ) analysis on the development of family medicine training programmes and of family medicine as a new discipline within the health systems of their countries ( box 3 ) .", "the main findings are presented and categorized into the following levels : local / university level , national level , regional / african level and international level .", " strong partnerships and collaboration with universities and professional bodies abroad is present  the south african departments of family medicine have substantial experience in the family medicine training programmes : they can assist in training via south  south cooperation and these cooperative steps can be seen as exemplary models and  good practices  the primafamed project and edulink acp - eu funding strengthens the development of family medicine training and is seen as the motor for the family medicine training development ", "individuals working in the family medicine departments are very motivated , proactive and willing to advocate for the cause  family physicians working as faculty are highly experienced and well qualified  the chosen district hospitals and health centres are ready for the training programmes  in several of the countries ( sudan , kenya and ghana ) , a high commitment from the ministry of health , the national council and leaders is existing  family medicine is responsive to the needs of the communities , especially in the rural areas where the majority of africans live  family medicine is not yet part of undergraduate medical training , therefore medical students are not exposed to the concept of family medicine and its principles  poor quality of intake and recruitment of family medicine residents for the family medicine training  the lack of family physicians and local teachers .", "many of the departments of family medicine depend on family physicians coming from non - african countries ( mainly , western europe and united states )  due to work overload , family medicine residents are continuously working in the hospital setting and have insufficient time to focus on the training in other aspects of family medicine , such as disease prevention and health promotion , community medicine , outpatient / continuity of care and research  the existing health systems are still weak in several countries ; plagued by poor support , weak referral systems , poor communication , poor funding and poor coordination between health centres , district hospitals and referral hospitals  dropping out of family medicine residents during the training sometimes happens in countries like dr congo because family medicine residents lose faith in their career opportunities ", "there is poor guidance for the present faculty  there is a lack of awareness and knowledge of family medicine by medical graduates , specialists and the community  there is lack of adequate infrastructure in some countries , such as dr congo and ghana  there is poor communication with policymakers and little support from the government in countries like tanzania and rwanda \n   standardization of training and examinations between faculties and countries is presently not existing , although there is a strong need for accreditation and quality assurance  there is insufficient financial support for the family medicine residents in postgraduate training ", "the position of family physicians in the different health systems is unclear ( what is the career perspective of graduated family physicians ? )  there is international support for the concept of family medicine : in the world health report 2008  primary health care : now more than ever , the who advocated for the importance of moving health care out of tertiary hospitals into the community and from vertical to horizontal care , in order to respond to the needs of the community  family medicine training is growing in the whole african continent , and there is support in the surrounding countries .", "the primafamed project has been linking these different countries , in order to fight for the cause together ", "existing health systems in several countries are weak ( poor coordination , poor referral systems , poor communication and little funding ) .", "family medicine can be used as a technique to strengthen these health systems  internet connection is present in several of the training centres ( and this is rapidly expanding ) ; e - learning is a very useful didactic tool for the family medicine residents  a yearly conference is organized by the primafamed network to strengthen the network and to share ideas and exchange experiences  wonca africa is organizing three yearly conferences and has been expanding in terms of number of members in the recent years  there are many existing links with organizations and universities in the north for financial and didactic support  iucea and wacp can assist in accreditation and quality assurance  several very useful books on african family medicine have been published recently and can be used as training material  the new african journal of primary health care and family medicine gives more opportunities for registrars to publish research  health - care workers and policymakers are becoming increasingly aware of the importance of the basic principles of phc and family medicine such as patient - centred care , non - communicable diseases ( ncds ) , interdisciplinary care , and continuity of care  many african ministries of health are working on health - system reforms  research in the field of family medicine is done by the registrars during the training", " the primafamed network has been looking into expanding to francophone africa : mali and benin will participate in a vlir uos ", "funded project starting 2012 , and the african journal of primary health care and family medicine will be published in english and french from 2012 onwards  other specialities see family medicine as a threat  family medicine is a new concept and not yet part of the health system in most of the partnering countries  overload of work for doctors leads to little time for training  lack of career opportunities for family medicine physicians in countries where family medicine is not supported by the government  there is little or no funding for family medicine within the existing systems  substantial decrease in funding for further development of the family medicine training after ending of the primafamed project funding from edulink acp - eu  migration of graduates to private practice , ngos , management positions or public health ( internal brain drain ) or migration of graduates to other countries ( external brain drain ) \n   war and political instability", "( e.g. dr congo , sudan and nigeria ) ; in some countries , there is a high turnover of government and with this , there often is a change of policies  increasing commercialization and privatization of health - care provision  worldwide economic crisis , which is decreasing the funding for health care and is putting health systems under pressure  fragmentation of care through vertical disease - oriented programmes , not only for infectious diseases , but increasingly for ncds ( 23 ) .", "i. when the swot analysis was done in 2010 , the rwandan ministry of health was supportive of the new discipline and family medicine was accepted by the government as one of the specialist mmed trainings from the national university of rwanda .", "however , at the time of writing this article , the ministry of health in rwanda has changed its vision and family medicine is not seen as a priority .", "west african college of physicians v. especially in sudan , this is a problem as a large percentage of medical graduates move to saudi arabia for more job opportunities , better salaries or family reasons adopted from the primafamed edulink acp eu project at the local / university level , the availability of resources plays an important role . from a human resources perspective", ", family physicians ( in most cases from abroad ) are needed to form the faculty , train family medicine residents and develop teaching materials .", "other local professors and specialists willing to train family medicine residents in the different disciplines are important to the strengthening of both the training and the interdisciplinary network ; however , several partners noted a low level of support from other specialists because family medicine is often not well understood and is often seen as a threat .", "adequate motivation of faculty and family medicine residents is important and is influenced by many other factors such as financial backing , career opportunities and support at the local , national and international levels .", "the availability of training sites , especially at district hospitals and health centres , is crucial , including having access to physicians to train and mentor the family medicine residents and stimulating teamwork with other health - care workers such as the local nurses .", "well - written curricula ( often developed based on sample curricula from family medicine training programmes in other african universities ) and training materials are essential to create a high - quality training atmosphere .", "access to the internet and to didactic materials and equipment ( such an ultrasound machines or health education materials ) are important to strengthen the actual training .", "lack of diagnostic or therapeutic equipments or insufficient availability of essential drugs in the training hospitals influences the way the doctors learn and hinders implementation of evidence - based medicine .", "ensuring that there is a place for family medicine training in undergraduate curricula plays a crucial role in raising awareness and recruiting newly trained medical doctors . at the national level", "decision - makers accept family medicine as an official specialization that is starting to take its place in the health - care system , with kenya , sudan and ghana as examples ( see examples 1 , 2 and 3 ) . several other countries have had more difficulties with the ministry of health accepting family medicine , including tanzania and rwanda .", "research on the positive outcomes of family medicine on health - care provision plays a significant role in advocating and strengthening awareness .", "a negative factor at the national level is political instability and war , as seen in eastern congo . at the regional / african level , the importance of strong partnerships and collaboration with universities and professional bodies abroad is noted .", "south african universities have excellent curricula and knowledgeable professors with extensive experience that can be leveraged via south  south cooperation .", "moreover , conferences organized by primafamed and wonca africa support the development of african family medicine .", "the establishment of the african journal of primary health care and family medicine is a great opportunity for young researchers to learn from others work and to publish their own articles . at the international level ,", "secondly , global recognition of the importance of training health - care workers in phc ( world health report 2006 and 2008 ) is vital to the development of african family medicine .", "inverse care law through achieving universal coverage and by providing equitable and high - quality health care through well - trained health - care workers to every individual .", "training doctors who work closer to communities , where they are most needed , is an important step towards improving the health outcomes of the african population .", "however , developing a new discipline that has not yet been defined in the national health systems is a challenging task .", "the primafamed project showed that developing sustainable family medicine training programmes is a feasible but slow process with many obstacles . the south  south cooperation between the primafamed partners and the south african family medicine departments strengthened confidence in the project and reinforced the principal need for well - trained african family physicians .", "local ownership is of utmost importance , although , with no local family physicians as role models , this can be a difficult task .", "support from key figures at the level of policymakers and academicians is necessary to create this new discipline and give it a place in health systems . without the integration of family medicine into national health policy", ", it is very difficult to recruit new doctors for the training programmes because the uncertainty in career prospects negatively influences a potential candidate s decision to join these programmes .", "continuous advocacy for the discipline and for strengthening the role of the african family physician are crucial .", "exposure to family medicine and community health in the undergraduate medical curriculum is required to create awareness among new medical doctors .", "action research , such as copc ( 24 ) , in the african setting is needed to demonstrate significant outcomes and the positive influence of the discipline at the individual , community and national levels .", "recruitment , training and retention of doctors in family medicine need to be adopted in the health system of each of the african countries .", "this requires increased investment of resources in phc , both from governments and from donor organizations .", "the primafamed network that was created during the 200810 project continues to endeavour to make these action points a reality . in november 2012", ", during the wonca africa conference , representatives from many african family medicine departments came together and discussed steps forward in creating a strong family medicine and phc - oriented health - care system in various african countries .", "this led to the  statement of the primafamed network : scaling up family medicine and primary health care in africa. concrete action for scaling up is needed , including convincing ministries and leadership of medical schools to integrate family medicine and phc into the undergraduate curriculum and to train a significant proportion of medical school graduates ( between 40% and 60% ) in family medicine and phc .", "essential conditions include having accredited under- and postgraduate curricula ; well - equipped training centres for transformative learning with well - trained trainers ; national and international support networks ; a sufficient number of funded posts for family medicine residents / registrars with appropriate remuneration ; and continuous advocacy at the population and government levels ( 25 ) . continuous interaction with key players at the policymaking level and support from the government are necessary to scale up family medicine and to develop it into an essential part of the health - care systems , in order to provide equitable , high - quality health care for communities and , ultimately , to improve overall health in sub - saharan africa .", "gezira university is a public university based in central sudan , in the city of wad madani .", "in many health centres , community physicians and medical officers were in charge of patient care .", "together with policymakers , district health officers and representatives from the ministry of health identified the need for further training for medical doctors in primary health care .", "gezira university decided to develop a 1-year diploma and a 2-year in - service master of science ( msc ) in family medicine .", "both are accepted by the sudan national medical specialisation board . with the help of the primafamed project ,", "a coordinator for this development was financed , a curriculum was developed and training sites were identified and equipped with the needed material . in 200910 , the first 10 medical doctors were trained in the 1-year course . in 2010 , 120 candidates were selected for the 2-year in - service msc in family medicine . in the fall of 2012 ,", "these 120 sudanese family physicians graduated with comprehensive knowledge in district primary health care . in 2013 , 200 new candidates were selected .", "the university of gezira can be seen as a perfect example of how working together with policymakers is essential for the development of family medicine .", "in 2005 , moi university started the first family medicine training programme in anglophone eastern africa , outside south africa , nigeria and ghana , leading to the degree of master of medicine in family health .", "the development of this residency programme was a triangular effort from moi university , infa - med ( institute of family medicine , a faith - based ngo , aiming to introduce family medicine in kenya ) and the kenyan ministry of health ( moh ) .", "infa - med contributed financial support , expatriate family medicine faculty and well - established training hospitals ; and the moh provided political support to the new specialty as well as scholarships to medical doctors entering the residency programme ( 26 ) .", "a national policy ( the kenyan family medicine policy ) was developed ( 27 ) and in august 2009 , this was adopted by the kenyan government . in 2008 , the first registrars finalized the family medicine programme at moi university .", "most of these registrars are now working in connection with the department to further develop the curriculum and the training sites . a study in 2011 on the challenges of family physicians after placement showed that the ministry s posting policy needs to be improved to ensure that family physicians have a chance to perform their intended roles ( 28 ) . in 2011 , one of the first - graduated kenyan family physicians , dr patrick chege , became the head of department .", "the department of family medicine has strongly been focussing on the development of research . in 2009 ,", "a review of the present family medicine curriculum was started to improve the curriculum with a focus on the needs of the hospitals where the family physicians will be based .", "many external experts from primafamed partners and associates were consulted in this process . in january 2012 , the revised curriculum was approved by the moi university senate .", "the west african college of physicians ( wacp ) accepted family medicine in 1985 ( 29 ) and it extended to the subregion outside nigeria in 1991 ( 29 ) . in ghana , postgraduate training began in march 1999 with three family medicine residents under the auspices of the faculty of family medicine ( then general medical practice ) of the wacp .", "this was a hospital - based training programme with the initial group of trainers being private general practitioners who were elected into fellowship by the college in the early 1990s .", "it started as a 4-year programme leading to membership after the initial 2 years and fellowship after a further training for 2 years .", "that same year , the ghana college of physicians and surgeons also started another programme in family medicine to run alongside the one conducted by wacp .", "the ministry of health provides sponsorship for the programmes and accords graduates of family medicine equal status and remuneration as graduates from other specialties . in january 2008 , the undergraduate programme began in the university of ghana medical school with the first two fellowship graduates appointed as lecturers .", "these local efforts at promoting the specialty in ghana received a major boost with two significant collaborations : first with the primafamed edulink acp - eu project in 2007 , a north  south  south cooperation , and second , with the department of family medicine , university of michigan health systems in 2008 , a north  south cooperation .", "these collaborations focussed on curriculum development , faculty development , teaching of students and family medicine residents , development of training complexes and research", ". currently , there are two well - established postgraduate training complexes in accra ( korle - bu teaching hospital ) and kumasi ( komfo - anokye teaching hospital ) with 35 family medicine residents at various levels of membership and fellowship training ( 30 ) .", "funding : the primafamed project was funded by the edulink acp eu ( european commission funded programmes in the africa , caribbean and pacific group of states ) . the vlir uos ( flemish interuniversity council ) supported the funding for the yearly primafamed conferences2008 in kampala , uganda ; 2009 in rustenburg , south africa ; 2010 in swaziland and 2011 in cape town , south africa ."], "abstract_text": ["<S> background.health-care systems based on primary health care ( phc ) are more equitable and cost effective . </S>", "<S> family medicine trains medical doctors in comprehensive phc with knowledge and skills that are needed to increase quality of care . </S>", "<S> family medicine is a relatively new specialty in sub - saharan africa.objective.to explore the extent to which the primafamed south  </S>", "<S> south cooperative project contributed to the development of family medicine in sub - saharan africa.methods.the primafamed ( primary health care and family medicine education ) project worked together with 10 partner universities in sub - saharan africa to develop family medicine training programmes over a period of 2.5 years . </S>", "<S> a swot ( strengths , weaknesses , opportunities and threats ) analysis was done and the training development from 2008 to 2010 in the different partner universities was analysed.results.during the 2.5 years of the primafamed project , all partner universities made progress in the development of their family medicine training programmes . </S>", "<S> the swot analysis showed that at both national and international levels , the time is ripe to train medical doctors in family medicine and to integrate the specialty into health - care systems , although many barriers , including little awareness , lack of funding , low support from other specialists and reserved support from policymakers , are still present.conclusions.family medicine can play an important role in health - care systems in sub - saharan africa ; however , developing a new discipline is challenging . </S>", "<S> advocacy , local ownership , action research and support from governments are necessary to develop family medicine and increase its impact . </S>", "<S> the primafamed project showed that development of sustainable family medicine training programmes is a feasible but slow process . the south  south cooperation between the ten partners and the south african departments of family medicine strengthened confidence at both national and international levels . </S>"], "labels": null, "section_names": ["Introduction", "Primary health care and family medicine in Africa", "Objectives", "Methods", "The Primafamed Edulink project", "The Primafamed partners", "Outcomes of the Primafamed project", "SWOT analysis", "Conclusions", "Example 1. Sudanese family physicians", "Example 2. Family medicine training at Moi University, Eldoret, Kenya", "Example 3. Family medicine in Ghana", "Declaration"], "sections": [["health systems based on primary health care ( phc ) distribute health care more equitably , are more cost effective and have better overall health outcomes and impact than health systems based on specialist care ( 1 ) .", "this is the foundation of the  health for all concept of alma - ata , adopted by the world health organization ( who ) in 1978 ( 2 ) and reiterated in the world health report 2008 ( 3 ) .", "worldwide , authorities have recognized that the health and well - being of a population is highly dependent on a quality phc system that is equitable , easily accessible and affordable for all members of the community ( 4 ) and that emphasizes universal coverage ( 5 ) .", "the concept of the postgraduate - trained family physician qualified to deliver equitable , high - quality phc closer to the community is now accepted in many countries around the world . however , in sub - saharan africa , family medicine is still a relatively new concept . in this article , we analyse the development of family medicine in anglophone sub - saharan africa in the recent years based on the experiences of the primafamed ( primary health care and family medicine education ) project ( 6 ) .", "lower - resource countries in sub - saharan africa face enormous health challenges and pervasive poverty . despite the work of governments and nongovernmental organizations ( ngos ) , the majority of people still do not have easy access to affordable quality health care .", "the  inverse care law ( 7 ) , noting that the fewest health - care professionals are found where they are needed the most and vice versa , is still very much applicable in most african countries .", "the poor are not only more prone to illnesses but are also unable to cope with diseases because health care is hard to access . with continuous population growth and a rather slow economic development ,", "the number of people living in poverty in sub - saharan africa has also increased , with 20.6% living on less than us$1.25 a day in 2008 ( 8) .", "it is the first level of contact of a continuing health - care process bringing health care as close as possible to where people live and work ( 9 ) .", "phc responds to the immense challenges that african countries are facing in their health systems by providing accessible , high - quality services that offer comprehensive and continuous care ( preventive , curative , rehabilitative and palliative ) at the local level , through interdisciplinary teams integrating vertical disease - oriented programmes .", "family physicians together with phc nurses ( and in some countries , mid - level care workers ) act as the clinical practitioners of the phc team . in 2009 , the 62nd world health assembly urged its member states to train and retain adequate numbers of health workers , including family physicians , and to encourage the implementation of vertical programmes in the context of integrated phc ( 10 ) . in 1968 ,", "the university of pretoria was the first university in south africa to start training specialized phc physicians , now referred to as  african family physicians ( 11 ) .", "this was followed by the other seven health science faculties in south africa . in 1997 , these eight departments of family medicine formed a network for communication and consultation , famec ( family medicine educational consortium ) , to share and exchange expertise , form a core curriculum and standardize examinations and develop appropriate assessment systems ( 12 ) . in august 2007 ,", "the concept of the african family physician in other anglophone african countries is even more recent . only in the 21st century", "did universities in anglophone countries in sub - saharan africa start family medicine training programmes , and the recently graduated african family physicians are beginning to find their place in the health systems of their respective countries .", "family medicine departments are struggling for recognition as health systems are still dominated by centralized specialist services and vertical disease - oriented approaches .", "several countries , such as namibia and botswana , did not have medical schools until very recently ( 14 ) .", "furthermore , many countries , such as the democratic republic of congo ( dr congo ) , are emerging from conflict and need to rebuild both state and infrastructure . at the wonca ( world organisation of family doctors ) africa conference in 2009 ,", "this consensus statement defined the contribution of family medicine to equity , quality and phc within an african context , as well as the role and training requirements of the african family physician ( 15 ) . due to the low number of trained doctors per capita and the high burden of disease , african family physicians work in their specific context , mainly in district hospitals with outreach to health centres , in phc teams that address the problems of the community in a comprehensive , holistic and patient - centred way whereby specific skills like surgery and district management often are essential due to the lack of other specialists .", "the consensus illustrates the ownership of the development of family medicine by african universities , one of the key points from the paris declaration ( 2005 ) and the accra agenda for action ( 2008 ) ( 16 ) ."], ["lower - resource countries in sub - saharan africa face enormous health challenges and pervasive poverty . despite the work of governments and nongovernmental organizations ( ngos ) , the majority of people still do not have easy access to affordable quality health care .", "the  inverse care law ( 7 ) , noting that the fewest health - care professionals are found where they are needed the most and vice versa , is still very much applicable in most african countries .", "the poor are not only more prone to illnesses but are also unable to cope with diseases because health care is hard to access . with continuous population growth and a rather slow economic development ,", "the number of people living in poverty in sub - saharan africa has also increased , with 20.6% living on less than us$1.25 a day in 2008 ( 8) .", "it is the first level of contact of a continuing health - care process bringing health care as close as possible to where people live and work ( 9 ) .", "phc responds to the immense challenges that african countries are facing in their health systems by providing accessible , high - quality services that offer comprehensive and continuous care ( preventive , curative , rehabilitative and palliative ) at the local level , through interdisciplinary teams integrating vertical disease - oriented programmes .", "family physicians together with phc nurses ( and in some countries , mid - level care workers ) act as the clinical practitioners of the phc team . in 2009 ,", "the 62nd world health assembly urged its member states to train and retain adequate numbers of health workers , including family physicians , and to encourage the implementation of vertical programmes in the context of integrated phc ( 10 ) . in 1968 ,", "the university of pretoria was the first university in south africa to start training specialized phc physicians , now referred to as  african family physicians ( 11 ) .", "this was followed by the other seven health science faculties in south africa . in 1997 , these eight departments of family medicine formed a network for communication and consultation , famec ( family medicine educational consortium ) , to share and exchange expertise , form a core curriculum and standardize examinations and develop appropriate assessment systems ( 12 ) . in august 2007 ,", "the concept of the african family physician in other anglophone african countries is even more recent . only in the 21st century", "did universities in anglophone countries in sub - saharan africa start family medicine training programmes , and the recently graduated african family physicians are beginning to find their place in the health systems of their respective countries .", "family medicine departments are struggling for recognition as health systems are still dominated by centralized specialist services and vertical disease - oriented approaches .", "several countries , such as namibia and botswana , did not have medical schools until very recently ( 14 ) . furthermore", ", many countries , such as the democratic republic of congo ( dr congo ) , are emerging from conflict and need to rebuild both state and infrastructure . at the wonca ( world organisation of family doctors ) africa conference in 2009 ,", "this consensus statement defined the contribution of family medicine to equity , quality and phc within an african context , as well as the role and training requirements of the african family physician ( 15 ) . due to the low number of trained doctors per capita and the high burden of disease , african family physicians work in their specific context , mainly in district hospitals with outreach to health centres , in phc teams that address the problems of the community in a comprehensive , holistic and patient - centred way whereby specific skills like surgery and district management often are essential due to the lack of other specialists .", "the consensus illustrates the ownership of the development of family medicine by african universities , one of the key points from the paris declaration ( 2005 ) and the accra agenda for action ( 2008 ) ( 16 ) ."], ["the aim of this article is to explore the extent to which the primafamed south ", "south cooperative project contributed to the development of family medicine in sub - saharan africa .", "the specific objectives are the following : the implementation of the primafamed project ; the outcomes of the primafamed project ; the development of family medicine training in sub - saharan africa from the viewpoint of the primafamed partners ."], ["a process analysis of the primafamed project between 2008 and 2011 is presented to discuss the implementation of the programme and the development of the network of 10 universities and associated partners in anglophone sub - saharan africa .", "finally , a swot analysis is detailed to explore the positive and negative dynamics that partners faced during the project . in 2007 ,", "a call for proposals under the name edulink was launched by the secretariat of the african , caribbean and pacific ( acp ) group of states ( 17 ) .", "together with 10 universities in eight countries in sub - saharan africa ( sudan , ghana , nigeria , dr congo , rwanda , uganda , kenya and tanzania ) , figure 1 , the department of family medicine and phc at ghent university in belgium developed the primafamed project proposal with a focus on developing family medicine training in sub - saharan africa .", "the aim of the primafamed project was to establish an institutional network between new and established departments of family medicine in universities in sub - saharan africa , within a framework of south  south cooperation ( 18 ) .", "the who world health report 2006 ,  working together for health , emphasized the need for phc training in the local community in order to deal with the brain drain from acp states ( 19 ) .", "training medical doctors in the field of family medicine , thus providing phc at the district level , responds to this call .", "the objectives of the primafamed project were formulated as follows : 1 . to contribute to the health of communities through accessible , responsive and quality health systems in sub - saharan countries by educating and training family physicians who provide interdisciplinary phc services , oriented towards the needs of individuals , their families and the communities in which they live", "develop and strengthen academic departments or units of family medicine that offer family medicine training at the undergraduate and postgraduate levels .", "the specific objectives were as follows : 1 . to develop a comprehensive vision and strategy , within the specific context of sub - saharan countries , that delineates the integral contribution of family medicine and the phc team to an equitable and quality phc system ; 2 . to establish a specific institutional network between departments and units of family medicine .", "the expected results of the primafamed project were the following : 1 . an improved institutional and administrative functioning in terms of policy , management , planning and administrative capacity building of the participating departments and units of family medicine .", "2 . improved relevance of family medicine in undergraduate and postgraduate training in the regional context .", "the partnering universities in the primafamed project were university of goma ( dr congo ) , moi university ( kenya ) , national university of rwanda , aga khan university ( tanzania ) , university of lagos ( nigeria ) , makerere university ( uganda ) , mbarara university ( uganda ) , ahfad university for women ( sudan ) , gezira university ( sudan ) and university of ghana .", "the south  south network connecting these 10 universities worked together with the eight departments of family medicine in south africa and accordingly formed a forum to share knowledge , experiences and resources .", "each of the 10 partner universities hired a local coordinator to oversee the implementation of the project , coordinate the activities and actively communicate with all partners .", "partners worked independently , integrating the primafamed objectives and the output for the project into the already existing structures and work plan at their universities . because african family physicians work mainly in district hospitals with outreach to the health centres ,", "the primafamed project consequently stimulated all partners to develop training complexes ( mainly district hospitals , several of which are located in rural areas ) for family medicine residents and to support the supply of these training complexes with the needed equipment . to further strengthen family medicine residents education , training sessions by professors from south african universities and other associated primafamed partners were held .", "a yearly conference for partners , associates and stakeholders was organized with the goal of offering trainings , sharing ideas and experiences , and strengthening the network . at the 2008 conference in kampala , the african journal of primary health care and family medicine ( 20 )", "this new open - access online journal has since published various articles related to the development of family medicine on the african continent , and researchers in the field of phc have written numerous articles on operational research and community - oriented primary care ( copc ) projects ( 21 ) . to monitor and evaluate our project ,", "this four - level scale shows the progress that took place during the 2.5-year period in which primafamed supported the partners .", "the first level corresponds to the institution being at a preparatory stage in the development of a postgraduate family medicine training programme .", "the fourth level reflects that the institution has started a postgraduate family medicine training programme with an existing curriculum by an organized department at well - organized training complexes and , most importantly , that family medicine has been accepted by the ministry of health as a specialization and that graduated family physicians are integrated into the existing health system .", "undeniably , the fourth level is not the end stage because , in our opinion , the ultimate objective would be to have an equitable and high - quality phc system with a central role for family physicians .", "department / unit of family medicine exists or is part of other departments ( community medicine )  training complexes are under development  family medicine is part of undergraduate training ", "postgraduate training has started  department / unit of family medicine exists  training complexes are in place ", "postgraduate training has started  the ministry of health has accepted family medicine as a specialization and graduated family physicians are part of the health - care system adopted from the primafamed edulink acp eu project .", "all 10 primafamed partners made progress during the 2.5 years that the project provided funding and support ( table 1 ) .", "at the start of the project , all of them had a unit or department of family medicine under development or in place , yet only three had officially started postgraduate family medicine training . at the end of the project ,", "eight had started postgraduate training , with family medicine residents located in properly equipped training hospitals .", "progress scale for development of the primafamed partners adopted from the primafamed edulink acp eu project .", "the integration of family medicine into the local health systems has been a slower process . in many countries ,", "policymakers have adopted a somewhat conservative attitude , with health systems mainly based on hospital / specialist care and on vertical programmes , which are often stimulated by donor programmes .", "a critical mass of well - trained family physicians is needed to demonstrate the effectiveness of family medicine training programmes . because all but one partner universities train only a limited number of family medicine residents simultaneously due to limited capacity , reaching that critical mass takes time . however , as the example of gezira university ( sudan ) has shown , it is possible to create a large pool of well - trained family physicians who can make a difference , in a short time . in this particular situation , because there was a vital need , policymakers and local authorities worked closely together with gezira university in development of the training ( example 1 ) . in 2012 ,", "african family physicians came together at the wonca africa conference and the primafamed network workshop in victoria falls , zimbabwe , and reaffirmed the importance of reaching that critical mass and therefore scaling up of family medicine , particularly in the african context , in which poverty is very prevalent , where rural  urban gaps remain , and where many of the millennium development goals ( mdgs ) will not be reached , including those related to child survival , maternal death and accessibility to antiretroviral drugs ( 22 ) . in 2010 , all primafamed local coordinators contributed to a swot ( strengths , weaknesses , opportunities and threats ) analysis on the development of family medicine training programmes and of family medicine as a new discipline within the health systems of their countries ( box 3 ) .", "the main findings are presented and categorized into the following levels : local / university level , national level , regional / african level and international level .", " strong partnerships and collaboration with universities and professional bodies abroad is present  the south african departments of family medicine have substantial experience in the family medicine training programmes : they can assist in training via south  south cooperation and these cooperative steps can be seen as exemplary models and  good practices", " the primafamed project and edulink acp - eu funding strengthens the development of family medicine training and is seen as the motor for the family medicine training development ", "individuals working in the family medicine departments are very motivated , proactive and willing to advocate for the cause  family physicians working as faculty are highly experienced and well qualified  the chosen district hospitals and health centres are ready for the training programmes  in several of the countries ( sudan , kenya and ghana ) , a high commitment from the ministry of health , the national council and leaders is existing  family medicine is responsive to the needs of the communities , especially in the rural areas where the majority of africans live  family medicine is not yet part of undergraduate medical training , therefore medical students are not exposed to the concept of family medicine and its principles  poor quality of intake and recruitment of family medicine residents for the family medicine training  the lack of family physicians and local teachers .", "many of the departments of family medicine depend on family physicians coming from non - african countries ( mainly , western europe and united states )  due to work overload , family medicine residents are continuously working in the hospital setting and have insufficient time to focus on the training in other aspects of family medicine , such as disease prevention and health promotion , community medicine , outpatient / continuity of care and research  the existing health systems are still weak in several countries ; plagued by poor support , weak referral systems , poor communication , poor funding and poor coordination between health centres , district hospitals and referral hospitals  dropping out of family medicine residents during the training sometimes happens in countries like dr congo because family medicine residents lose faith in their career opportunities  there is a lack of didactic materials  capacity is still too limited ", "there is a lack of awareness and knowledge of family medicine by medical graduates , specialists and the community  there is lack of adequate infrastructure in some countries , such as dr congo and ghana  there is poor communication with policymakers and little support from the government in countries like tanzania and rwanda \n   standardization of training and examinations between faculties and countries is presently not existing , although there is a strong need for accreditation and quality assurance  there is insufficient financial support for the family medicine residents in postgraduate training ", "the position of family physicians in the different health systems is unclear ( what is the career perspective of graduated family physicians ? )  there is international support for the concept of family medicine : in the world health report 2008  primary health care : now more than ever , the who advocated for the importance of moving health care out of tertiary hospitals into the community and from vertical to horizontal care , in order to respond to the needs of the community  family medicine training is growing in the whole african continent , and there is support in the surrounding countries .", "the primafamed project has been linking these different countries , in order to fight for the cause together ", "existing health systems in several countries are weak ( poor coordination , poor referral systems , poor communication and little funding )", ". family medicine can be used as a technique to strengthen these health systems  internet connection is present in several of the training centres ( and this is rapidly expanding ) ; e - learning is a very useful didactic tool for the family medicine residents  a yearly conference is organized by the primafamed network to strengthen the network and to share ideas and exchange experiences  wonca africa is organizing three yearly conferences and has been expanding in terms of number of members in the recent years  there are many existing links with organizations and universities in the north for financial and didactic support  iucea and wacp can assist in accreditation and quality assurance  several very useful books on african family medicine have been published recently and can be used as training material  the new african journal of primary health care and family medicine gives more opportunities for registrars to publish research  health - care workers and policymakers are becoming increasingly aware of the importance of the basic principles of phc and family medicine such as patient - centred care , non - communicable diseases ( ncds ) , interdisciplinary care , and continuity of care  many african ministries of health are working on health - system reforms  research in the field of family medicine is done by the registrars during the training  the primafamed network has been looking into expanding to francophone africa : mali and benin will participate in a vlir uos  funded project starting 2012 , and the african journal of primary health care and family medicine will be published in english and french from 2012 onwards  other specialities see family medicine as a threat  family medicine is a new concept and not yet part of the health system in most of the partnering countries  overload of work for doctors leads to little time for training  lack of career opportunities for family medicine physicians in countries where family medicine is not supported by the government  there is little or no funding for family medicine within the existing systems  substantial decrease in funding for further development of the family medicine training after ending of the primafamed project funding from edulink acp - eu  migration of graduates to private practice , ngos , management positions or public health ( internal brain drain ) or migration of graduates to other countries ( external brain drain ) \n   war and political instability ( e.g. dr congo , sudan and nigeria ) ; in some countries , there is a high turnover of government and with this , there often is a change of policies  increasing commercialization and privatization of health - care provision", " worldwide economic crisis , which is decreasing the funding for health care and is putting health systems under pressure  fragmentation of care through vertical disease - oriented programmes , not only for infectious diseases , but increasingly for ncds ( 23 ) .", "i. when the swot analysis was done in 2010 , the rwandan ministry of health was supportive of the new discipline and family medicine was accepted by the government as one of the specialist mmed trainings from the national university of rwanda .", "however , at the time of writing this article , the ministry of health in rwanda has changed its vision and family medicine is not seen as a priority . recently", "west african college of physicians v. especially in sudan , this is a problem as a large percentage of medical graduates move to saudi arabia for more job opportunities , better salaries or family reasons adopted from the primafamed edulink acp eu project at the local / university level , the availability of resources plays an important role . from a human resources perspective , family physicians ( in most cases from abroad ) are needed to form the faculty , train family medicine residents and develop teaching materials .", "other local professors and specialists willing to train family medicine residents in the different disciplines are important to the strengthening of both the training and the interdisciplinary network ; however , several partners noted a low level of support from other specialists because family medicine is often not well understood and is often seen as a threat .", "adequate motivation of faculty and family medicine residents is important and is influenced by many other factors such as financial backing , career opportunities and support at the local , national and international levels .", "the availability of training sites , especially at district hospitals and health centres , is crucial , including having access to physicians to train and mentor the family medicine residents and stimulating teamwork with other health - care workers such as the local nurses .", "well - written curricula ( often developed based on sample curricula from family medicine training programmes in other african universities ) and training materials are essential to create a high - quality training atmosphere .", "access to the internet and to didactic materials and equipment ( such an ultrasound machines or health education materials ) are important to strengthen the actual training", ". lack of diagnostic or therapeutic equipments or insufficient availability of essential drugs in the training hospitals influences the way the doctors learn and hinders implementation of evidence - based medicine .", "ensuring that there is a place for family medicine training in undergraduate curricula plays a crucial role in raising awareness and recruiting newly trained medical doctors . at the national level , policymakers play a pivotal role . in several countries ,", "decision - makers accept family medicine as an official specialization that is starting to take its place in the health - care system , with kenya , sudan and ghana as examples ( see examples 1 , 2 and 3 ) . several other countries have had more difficulties with the ministry of health accepting family medicine , including tanzania and rwanda .", "research on the positive outcomes of family medicine on health - care provision plays a significant role in advocating and strengthening awareness .", "a negative factor at the national level is political instability and war , as seen in eastern congo . at the regional / african level , the importance of strong partnerships and collaboration with universities and professional bodies abroad is noted .", "south african universities have excellent curricula and knowledgeable professors with extensive experience that can be leveraged via south  south cooperation .", "moreover , conferences organized by primafamed and wonca africa support the development of african family medicine .", "the establishment of the african journal of primary health care and family medicine is a great opportunity for young researchers to learn from others work and to publish their own articles . at the international level ,", "secondly , global recognition of the importance of training health - care workers in phc ( world health report 2006 and 2008 ) is vital to the development of african family medicine ."], ["in 2007 , a call for proposals under the name edulink was launched by the secretariat of the african , caribbean and pacific ( acp ) group of states ( 17 ) .", "together with 10 universities in eight countries in sub - saharan africa ( sudan , ghana , nigeria , dr congo , rwanda , uganda , kenya and tanzania ) , figure 1 , the department of family medicine and phc at ghent university in belgium developed the primafamed project proposal with a focus on developing family medicine training in sub - saharan africa .", "the aim of the primafamed project was to establish an institutional network between new and established departments of family medicine in universities in sub - saharan africa , within a framework of south  south cooperation ( 18 ) .", "the who world health report 2006 ,  working together for health , emphasized the need for phc training in the local community in order to deal with the brain drain from acp states ( 19 ) .", "training medical doctors in the field of family medicine , thus providing phc at the district level , responds to this call .", "the objectives of the primafamed project were formulated as follows : 1 . to contribute to the health of communities through accessible , responsive and quality health systems in sub - saharan countries by educating and training family physicians who provide interdisciplinary phc services , oriented towards the needs of individuals , their families and the communities in which they live .", "develop and strengthen academic departments or units of family medicine that offer family medicine training at the undergraduate and postgraduate levels .", "the specific objectives were as follows : 1 . to develop a comprehensive vision and strategy , within the specific context of sub - saharan countries , that delineates the integral contribution of family medicine and the phc team to an equitable and quality phc system ; 2 . to establish a specific institutional network between departments and units of family medicine .", "an improved institutional and administrative functioning in terms of policy , management , planning and administrative capacity building of the participating departments and units of family medicine .", "2 . improved relevance of family medicine in undergraduate and postgraduate training in the regional context ."], ["the partnering universities in the primafamed project were university of goma ( dr congo ) , moi university ( kenya ) , national university of rwanda , aga khan university ( tanzania ) , university of lagos ( nigeria ) , makerere university ( uganda ) , mbarara university ( uganda ) , ahfad university for women ( sudan ) , gezira university ( sudan ) and university of ghana .", "the south  south network connecting these 10 universities worked together with the eight departments of family medicine in south africa and accordingly formed a forum to share knowledge , experiences and resources ."], ["each of the 10 partner universities hired a local coordinator to oversee the implementation of the project , coordinate the activities and actively communicate with all partners .", "partners worked independently , integrating the primafamed objectives and the output for the project into the already existing structures and work plan at their universities . because african family physicians work mainly in district hospitals with outreach to the health centres ,", "the primafamed project consequently stimulated all partners to develop training complexes ( mainly district hospitals , several of which are located in rural areas ) for family medicine residents and to support the supply of these training complexes with the needed equipment .", "to further strengthen family medicine residents education , training sessions by professors from south african universities and other associated primafamed partners were held . a yearly conference for partners , associates and stakeholders", "was organized with the goal of offering trainings , sharing ideas and experiences , and strengthening the network . at the 2008 conference in kampala , the african journal of primary health care and family medicine ( 20 )", "this new open - access online journal has since published various articles related to the development of family medicine on the african continent , and researchers in the field of phc have written numerous articles on operational research and community - oriented primary care ( copc ) projects ( 21 ) . to monitor and evaluate our project ,", "this four - level scale shows the progress that took place during the 2.5-year period in which primafamed supported the partners .", "the first level corresponds to the institution being at a preparatory stage in the development of a postgraduate family medicine training programme .", "the fourth level reflects that the institution has started a postgraduate family medicine training programme with an existing curriculum by an organized department at well - organized training complexes and , most importantly , that family medicine has been accepted by the ministry of health as a specialization and that graduated family physicians are integrated into the existing health system .", "undeniably , the fourth level is not the end stage because , in our opinion , the ultimate objective would be to have an equitable and high - quality phc system with a central role for family physicians .", "department / unit of family medicine exists or is part of other departments ( community medicine )  training complexes are under development  family medicine is part of undergraduate training  department / unit of family medicine exists  training complexes are in place ", "postgraduate training has started  department / unit of family medicine exists  training complexes are in place ", "postgraduate training has started  the ministry of health has accepted family medicine as a specialization and graduated family physicians are part of the health - care system adopted from the primafamed edulink acp eu project .", "all 10 primafamed partners made progress during the 2.5 years that the project provided funding and support ( table 1 ) . at the start of the project , all of them had a unit or department of family medicine under development or in place , yet only three had officially started postgraduate family medicine training . at the end of the project ,", "eight had started postgraduate training , with family medicine residents located in properly equipped training hospitals .", "progress scale for development of the primafamed partners adopted from the primafamed edulink acp eu project .", "the integration of family medicine into the local health systems has been a slower process . in many countries ,", "policymakers have adopted a somewhat conservative attitude , with health systems mainly based on hospital / specialist care and on vertical programmes , which are often stimulated by donor programmes .", "a critical mass of well - trained family physicians is needed to demonstrate the effectiveness of family medicine training programmes . because all but one partner universities train only a limited number of family medicine residents simultaneously due to limited capacity , reaching that critical mass takes time . however , as the example of gezira university ( sudan ) has shown , it is possible to create a large pool of well - trained family physicians who can make a difference , in a short time . in this particular situation , because there was a vital need , policymakers and local authorities worked closely together with gezira university in development of the training ( example 1 ) . in 2012 ,", "african family physicians came together at the wonca africa conference and the primafamed network workshop in victoria falls , zimbabwe , and reaffirmed the importance of reaching that critical mass and therefore scaling up of family medicine , particularly in the african context , in which poverty is very prevalent , where rural  urban gaps remain , and where many of the millennium development goals ( mdgs ) will not be reached , including those related to child survival , maternal death and accessibility to antiretroviral drugs ( 22 ) ."], ["in 2010 , all primafamed local coordinators contributed to a swot ( strengths , weaknesses , opportunities and threats ) analysis on the development of family medicine training programmes and of family medicine as a new discipline within the health systems of their countries ( box 3 ) .", "the main findings are presented and categorized into the following levels : local / university level , national level , regional / african level and international level .", " strong partnerships and collaboration with universities and professional bodies abroad is present  the south african departments of family medicine have substantial experience in the family medicine training programmes : they can assist in training via south  south cooperation and these cooperative steps can be seen as exemplary models and  good practices  the primafamed project and edulink acp - eu funding strengthens the development of family medicine training and is seen as the motor for the family medicine training development ", "individuals working in the family medicine departments are very motivated , proactive and willing to advocate for the cause  family physicians working as faculty are highly experienced and well qualified  the chosen district hospitals and health centres are ready for the training programmes  in several of the countries ( sudan , kenya and ghana ) , a high commitment from the ministry of health , the national council and leaders is existing  family medicine is responsive to the needs of the communities , especially in the rural areas where the majority of africans live  family medicine is not yet part of undergraduate medical training , therefore medical students are not exposed to the concept of family medicine and its principles  poor quality of intake and recruitment of family medicine residents for the family medicine training  the lack of family physicians and local teachers .", "many of the departments of family medicine depend on family physicians coming from non - african countries ( mainly , western europe and united states )  due to work overload , family medicine residents are continuously working in the hospital setting and have insufficient time to focus on the training in other aspects of family medicine , such as disease prevention and health promotion , community medicine , outpatient / continuity of care and research  the existing health systems are still weak in several countries ; plagued by poor support , weak referral systems , poor communication , poor funding and poor coordination between health centres , district hospitals and referral hospitals  dropping out of family medicine residents during the training sometimes happens in countries like dr congo because family medicine residents lose faith in their career opportunities ", "there is poor guidance for the present faculty  there is a lack of awareness and knowledge of family medicine by medical graduates , specialists and the community  there is lack of adequate infrastructure in some countries , such as dr congo and ghana  there is poor communication with policymakers and little support from the government in countries like tanzania and rwanda \n   standardization of training and examinations between faculties and countries is presently not existing , although there is a strong need for accreditation and quality assurance  there is insufficient financial support for the family medicine residents in postgraduate training ", "the position of family physicians in the different health systems is unclear ( what is the career perspective of graduated family physicians ? )  there is international support for the concept of family medicine : in the world health report 2008  primary health care : now more than ever , the who advocated for the importance of moving health care out of tertiary hospitals into the community and from vertical to horizontal care , in order to respond to the needs of the community  family medicine training is growing in the whole african continent , and there is support in the surrounding countries .", "the primafamed project has been linking these different countries , in order to fight for the cause together ", "existing health systems in several countries are weak ( poor coordination , poor referral systems , poor communication and little funding ) .", "family medicine can be used as a technique to strengthen these health systems  internet connection is present in several of the training centres ( and this is rapidly expanding ) ; e - learning is a very useful didactic tool for the family medicine residents  a yearly conference is organized by the primafamed network to strengthen the network and to share ideas and exchange experiences  wonca africa is organizing three yearly conferences and has been expanding in terms of number of members in the recent years  there are many existing links with organizations and universities in the north for financial and didactic support  iucea and wacp can assist in accreditation and quality assurance  several very useful books on african family medicine have been published recently and can be used as training material  the new african journal of primary health care and family medicine gives more opportunities for registrars to publish research  health - care workers and policymakers are becoming increasingly aware of the importance of the basic principles of phc and family medicine such as patient - centred care , non - communicable diseases ( ncds ) , interdisciplinary care , and continuity of care  many african ministries of health are working on health - system reforms  research in the field of family medicine is done by the registrars during the training", " the primafamed network has been looking into expanding to francophone africa : mali and benin will participate in a vlir uos ", "funded project starting 2012 , and the african journal of primary health care and family medicine will be published in english and french from 2012 onwards  other specialities see family medicine as a threat  family medicine is a new concept and not yet part of the health system in most of the partnering countries  overload of work for doctors leads to little time for training  lack of career opportunities for family medicine physicians in countries where family medicine is not supported by the government  there is little or no funding for family medicine within the existing systems  substantial decrease in funding for further development of the family medicine training after ending of the primafamed project funding from edulink acp - eu  migration of graduates to private practice , ngos , management positions or public health ( internal brain drain ) or migration of graduates to other countries ( external brain drain ) \n   war and political instability", "( e.g. dr congo , sudan and nigeria ) ; in some countries , there is a high turnover of government and with this , there often is a change of policies  increasing commercialization and privatization of health - care provision  worldwide economic crisis , which is decreasing the funding for health care and is putting health systems under pressure  fragmentation of care through vertical disease - oriented programmes , not only for infectious diseases , but increasingly for ncds ( 23 ) .", "i. when the swot analysis was done in 2010 , the rwandan ministry of health was supportive of the new discipline and family medicine was accepted by the government as one of the specialist mmed trainings from the national university of rwanda .", "however , at the time of writing this article , the ministry of health in rwanda has changed its vision and family medicine is not seen as a priority .", "west african college of physicians v. especially in sudan , this is a problem as a large percentage of medical graduates move to saudi arabia for more job opportunities , better salaries or family reasons adopted from the primafamed edulink acp eu project at the local / university level , the availability of resources plays an important role . from a human resources perspective", ", family physicians ( in most cases from abroad ) are needed to form the faculty , train family medicine residents and develop teaching materials .", "other local professors and specialists willing to train family medicine residents in the different disciplines are important to the strengthening of both the training and the interdisciplinary network ; however , several partners noted a low level of support from other specialists because family medicine is often not well understood and is often seen as a threat .", "adequate motivation of faculty and family medicine residents is important and is influenced by many other factors such as financial backing , career opportunities and support at the local , national and international levels .", "the availability of training sites , especially at district hospitals and health centres , is crucial , including having access to physicians to train and mentor the family medicine residents and stimulating teamwork with other health - care workers such as the local nurses .", "well - written curricula ( often developed based on sample curricula from family medicine training programmes in other african universities ) and training materials are essential to create a high - quality training atmosphere .", "access to the internet and to didactic materials and equipment ( such an ultrasound machines or health education materials ) are important to strengthen the actual training .", "lack of diagnostic or therapeutic equipments or insufficient availability of essential drugs in the training hospitals influences the way the doctors learn and hinders implementation of evidence - based medicine .", "ensuring that there is a place for family medicine training in undergraduate curricula plays a crucial role in raising awareness and recruiting newly trained medical doctors . at the national level", "decision - makers accept family medicine as an official specialization that is starting to take its place in the health - care system , with kenya , sudan and ghana as examples ( see examples 1 , 2 and 3 ) . several other countries have had more difficulties with the ministry of health accepting family medicine , including tanzania and rwanda .", "research on the positive outcomes of family medicine on health - care provision plays a significant role in advocating and strengthening awareness .", "a negative factor at the national level is political instability and war , as seen in eastern congo . at the regional / african level , the importance of strong partnerships and collaboration with universities and professional bodies abroad is noted .", "south african universities have excellent curricula and knowledgeable professors with extensive experience that can be leveraged via south  south cooperation .", "moreover , conferences organized by primafamed and wonca africa support the development of african family medicine .", "the establishment of the african journal of primary health care and family medicine is a great opportunity for young researchers to learn from others work and to publish their own articles . at the international level ,", "secondly , global recognition of the importance of training health - care workers in phc ( world health report 2006 and 2008 ) is vital to the development of african family medicine ."], ["inverse care law through achieving universal coverage and by providing equitable and high - quality health care through well - trained health - care workers to every individual .", "training doctors who work closer to communities , where they are most needed , is an important step towards improving the health outcomes of the african population .", "however , developing a new discipline that has not yet been defined in the national health systems is a challenging task .", "the primafamed project showed that developing sustainable family medicine training programmes is a feasible but slow process with many obstacles . the south  south cooperation between the primafamed partners and the south african family medicine departments strengthened confidence in the project and reinforced the principal need for well - trained african family physicians .", "local ownership is of utmost importance , although , with no local family physicians as role models , this can be a difficult task .", "support from key figures at the level of policymakers and academicians is necessary to create this new discipline and give it a place in health systems . without the integration of family medicine into national health policy", ", it is very difficult to recruit new doctors for the training programmes because the uncertainty in career prospects negatively influences a potential candidate s decision to join these programmes .", "continuous advocacy for the discipline and for strengthening the role of the african family physician are crucial .", "exposure to family medicine and community health in the undergraduate medical curriculum is required to create awareness among new medical doctors .", "action research , such as copc ( 24 ) , in the african setting is needed to demonstrate significant outcomes and the positive influence of the discipline at the individual , community and national levels .", "recruitment , training and retention of doctors in family medicine need to be adopted in the health system of each of the african countries .", "this requires increased investment of resources in phc , both from governments and from donor organizations .", "the primafamed network that was created during the 200810 project continues to endeavour to make these action points a reality . in november 2012", ", during the wonca africa conference , representatives from many african family medicine departments came together and discussed steps forward in creating a strong family medicine and phc - oriented health - care system in various african countries .", "this led to the  statement of the primafamed network : scaling up family medicine and primary health care in africa. concrete action for scaling up is needed , including convincing ministries and leadership of medical schools to integrate family medicine and phc into the undergraduate curriculum and to train a significant proportion of medical school graduates ( between 40% and 60% ) in family medicine and phc .", "essential conditions include having accredited under- and postgraduate curricula ; well - equipped training centres for transformative learning with well - trained trainers ; national and international support networks ; a sufficient number of funded posts for family medicine residents / registrars with appropriate remuneration ; and continuous advocacy at the population and government levels ( 25 ) . continuous interaction with key players at the policymaking level and support from the government are necessary to scale up family medicine and to develop it into an essential part of the health - care systems , in order to provide equitable , high - quality health care for communities and , ultimately , to improve overall health in sub - saharan africa ."], ["gezira university is a public university based in central sudan , in the city of wad madani .", "in many health centres , community physicians and medical officers were in charge of patient care .", "together with policymakers , district health officers and representatives from the ministry of health identified the need for further training for medical doctors in primary health care .", "gezira university decided to develop a 1-year diploma and a 2-year in - service master of science ( msc ) in family medicine .", "both are accepted by the sudan national medical specialisation board . with the help of the primafamed project ,", "a coordinator for this development was financed , a curriculum was developed and training sites were identified and equipped with the needed material . in 200910 , the first 10 medical doctors were trained in the 1-year course . in 2010 , 120 candidates were selected for the 2-year in - service msc in family medicine . in the fall of 2012 ,", "these 120 sudanese family physicians graduated with comprehensive knowledge in district primary health care . in 2013 , 200 new candidates were selected .", "the university of gezira can be seen as a perfect example of how working together with policymakers is essential for the development of family medicine ."], ["in 2005 , moi university started the first family medicine training programme in anglophone eastern africa , outside south africa , nigeria and ghana , leading to the degree of master of medicine in family health .", "the development of this residency programme was a triangular effort from moi university , infa - med ( institute of family medicine , a faith - based ngo , aiming to introduce family medicine in kenya ) and the kenyan ministry of health ( moh ) .", "infa - med contributed financial support , expatriate family medicine faculty and well - established training hospitals ; and the moh provided political support to the new specialty as well as scholarships to medical doctors entering the residency programme ( 26 ) .", "a national policy ( the kenyan family medicine policy ) was developed ( 27 ) and in august 2009 , this was adopted by the kenyan government . in 2008 , the first registrars finalized the family medicine programme at moi university .", "most of these registrars are now working in connection with the department to further develop the curriculum and the training sites . a study in 2011 on the challenges of family physicians after placement showed that the ministry s posting policy needs to be improved to ensure that family physicians have a chance to perform their intended roles ( 28 ) . in 2011 , one of the first - graduated kenyan family physicians , dr patrick chege , became the head of department .", "the department of family medicine has strongly been focussing on the development of research . in 2009 ,", "a review of the present family medicine curriculum was started to improve the curriculum with a focus on the needs of the hospitals where the family physicians will be based .", "many external experts from primafamed partners and associates were consulted in this process . in january 2012 , the revised curriculum was approved by the moi university senate ."], ["the west african college of physicians ( wacp ) accepted family medicine in 1985 ( 29 ) and it extended to the subregion outside nigeria in 1991 ( 29 ) . in ghana , postgraduate training began in march 1999 with three family medicine residents under the auspices of the faculty of family medicine ( then general medical practice ) of the wacp .", "this was a hospital - based training programme with the initial group of trainers being private general practitioners who were elected into fellowship by the college in the early 1990s .", "it started as a 4-year programme leading to membership after the initial 2 years and fellowship after a further training for 2 years .", "that same year , the ghana college of physicians and surgeons also started another programme in family medicine to run alongside the one conducted by wacp .", "the ministry of health provides sponsorship for the programmes and accords graduates of family medicine equal status and remuneration as graduates from other specialties . in january 2008 , the undergraduate programme began in the university of ghana medical school with the first two fellowship graduates appointed as lecturers .", "these local efforts at promoting the specialty in ghana received a major boost with two significant collaborations : first with the primafamed edulink acp - eu project in 2007 , a north  south  south cooperation , and second , with the department of family medicine , university of michigan health systems in 2008 , a north  south cooperation .", "these collaborations focussed on curriculum development , faculty development , teaching of students and family medicine residents , development of training complexes and research", ". currently , there are two well - established postgraduate training complexes in accra ( korle - bu teaching hospital ) and kumasi ( komfo - anokye teaching hospital ) with 35 family medicine residents at various levels of membership and fellowship training ( 30 ) ."], ["funding : the primafamed project was funded by the edulink acp eu ( european commission funded programmes in the africa , caribbean and pacific group of states ) . the vlir uos ( flemish interuniversity council ) supported the funding for the yearly primafamed conferences2008 in kampala , uganda ; 2009 in rustenburg , south africa ; 2010 in swaziland and 2011 in cape town , south africa ."]]}
{"article_id": "PMC3259482", "article_text": ["of those deaths , an estimated 7.3 million were due to coronary heart disease and 6.2 million due to stroke . a disproportionate amount of those deaths , over 80% , take place in low- and middle - income countries and occur at similar rates among men and women .", "not surprisingly , elevated blood pressure levels are a major cause of these diseases and are found at higher rates among low- and middle - income countries . the relationship between blood pressure levels and risk of developing cardiovascular disease is strong and well supported . in 2008 , approximately one billion adults worldwide had uncontrolled hypertension ( defined as systolic blood pressure 140  mm  hg systolic and/or diastolic blood pressure 90  mm  hg ) . given the increasing prevalence of hypertension worldwide and the associated risk for developing cardiovascular disease , public health interventions aimed at reducing blood pressure are crucial .  ", "many national and international agencies have acknowledged the role of lifestyle and diet , in particular sodium intake , on blood pressure levels .", "diets high in salt are now recognized as one of the leading risks to cardiovascular health in the world as they increase blood pressure in both children and adults .", "furthermore , a recent meta - analysis of randomized trials has demonstrated that modest reductions in dietary sodium intake are associated with significant reductions in blood pressure in both normotensives and hypertensives and a 20% reduction in cardiovascular events [ 4 , 5 ] .", "a reduction in salt intake of 6  g / day lowered blood pressure by 7/4  mm  hg diastolic in hypertensives and 4/2  mm  hg in normotensives .", "this relationship has been empirically supported and is sufficiently strong to warrant recommendations for public health interventions aimed at substantially reducing dietary sodium intake .", "furthermore , sodium reduction is noted as one of the most cost effective and most easily implemented strategies to improve population health [ 513 ] . reducing dietary salt", "is recommended by the world health organization and many national governmental and nongovernmental health organizations .", "some agencies , however , do not promote a reduction in dietary sodium , namely , nongovernmental or commercial organizations such as the salt institute , as they are sponsored by either the food or salt industries [ 9 , 11 , 1417 ] .", "regardless , it is apparent that the risks associated with an increase in salt consumption , chiefly those related to an increase in blood pressure , are linear [ 3 , 18 ] .", "most health economic models input relatively small changes in blood pressure that occur in those with normal and high blood pressure as estimated by short - term modest reductions in dietary sodium [ 7 , 8 , 19 ] .", "some models also include the gastric cancers that are positively associated with , and probably caused by , high - salt intake .", "typically , the health economic models do not include the potential impact of longer term irreversible increases in blood pressure , age - related increases in blood pressure , the epigenetic phenomena , whereby exposure to excess salt in utero may increase blood pressure in offspring , or that sodium may increase vascular and cardiac disease in the absence of changes in blood pressure [ 7 , 8 , 12 , 1921 ] .", "the burden of disease studies also do not account for diseases that have a pathophysiological basis and close association with high sodium diets ( i.e. , increased severity and frequency of asthma attacks , increased calcium containing kidney stones , osteoporosis , or obesity related to the consumption of calorie containing beverages caused by sodium - induced thirst ) . hence the burden of disease associated with excess dietary salt is not only high , but may also be underestimated .", "the objective of this commentary is to review current sodium consumption worldwide , discuss cost - effective strategies to reduce dietary sodium , as well as briefly review the role of behavioural and policy - based environmental interventions in reducing dietary sodium on a population - based scale .", "humans evolved on diets consisting of natural plant and animal foods containing small amounts of sodium , typically less than 2  g / day [ 25 , 26 ] .", "today , nearly all populations consume far greater quantities of salt than those provided in natural , unprocessed food diets .", "the world health organization currently recommends a daily consumption of less than 5 grams of salt , although some agencies recommend that no more than 1500  mg of sodium should be consumed per day [ 2729 ] , calculated as 2/3  tsp of table salt . in most populations ,", "sodium intake is 5.7  g or more / day after age 5 , with many populations consuming and average of over 10  g / day [ 3032 ] .", "furthermore , within high sodium consumption countries , only a small proportion of individuals consume the recommended levels of salt .", "for example , in canada , a country with average salt consumption of 8.5  gm / day , 85% of men and 60% of women aged 9 to 70 consume over the upper recommended limit for salt and the vast majority ( > 90% ) are above the level recommended for individuals to consume [ 33 , 34 ] .", "for example , one study found that in a population of overweight adults , a daily intake of sodium greater than 2300  mg / day was associated with a 61% increase in coronary heart disease mortality , an 89% increase in stroke mortality , and a 39% increase in all - cause mortality over a 19-year period .", "along with the other sodium - related illnesses discussed above ( i.e. , gastric cancers , kidney stones , etc . )", ", it is clear that the economic costs associated with such illnesses can be substantial .", "in countries with developed economies , salt added during the processing of foods accounts for the vast majority of dietary salt ( 7580% ) . an additional 10% of dietary salt", "is accounted for by salt that is naturally occurring in foods , while the rest is accounted for by salt added at the table or during cooking . in low- to - middle income countries where populations may have limited access to processed foods , salt added at home , in cooking , or at the table , accounts for the majority of dietary salt . reducing dietary salt is estimated to save substantial health care costs [ 7 , 10 , 16 , 30 , 3741 ] .", "for example , reducing dietary salt by 3  g / day in the united states is estimated to save 194,000 to 392,000 quality adjusted life years and reduce health care costs $ 10 to $ 24 billion us dollars a year . in canada , reducing salt consumption to recommended levels is estimated to reduce the prevalence of hypertension by 30% and to save up to $ 430 million dollars per year just in direct hypertension management costs alone . in lower - income countries , programs aimed at reducing consumption of dietary salt through an intervention largely based on education", "are estimated to cost little ( less than $ 0.40 usd per person per year ) , reduce premature deaths by close to 14 million in 10 years , and to be slightly more cost effective than strategies to reduce tobacco use ( both highly advocated interventions ) [ 8 , 40 ] .", "although there is a general lack of awareness of salt as a health issue in many countries , some countries with established salt reduction programs show increasing awareness .", "for example , in canada , 80% of people diagnosed with hypertension are attempting to reduce dietary sodium .", "in addition , many food companies have developed low - salt options to their product lines for people to choose , with some companies reducing salt additives in their full product line . in developed economies , there are substantial barriers to free choice in those who chose to eat less salt [ 16 , 44 ] . in most countries ,", "it is often the case that nutritional information is available only on the company 's website , by asking for and reviewing a binder on site , or only readily available after purchase . even in the united states and canada , countries with mandatory labelling of packaged foods , the labels are often difficult to interpret . also serving sizes may be variable and not comparable between products . in an unpublished study", ", we found that in a sample of over 100 people with diabetes who had received training on how to read a food label , not one could accurately answer how much of a processed food they could eat in a day when presented with the food label .", "in other countries , food labelling on packaged foods may not be mandatory and labelled foods may not be available .", "in remote regions and areas where populations vulnerable to the development of elevated blood pressure reside , low - salt alternative choices are typically not available .", "furthermore , these populations may not have the health literacy with which to make informed choices .", "food processors and manufacturers often use pervasive marketing techniques to create consumer demand for high - salt foods , which undermine efforts of public health and individual education interventions that attempt to reduce sodium intake .", "moreover , these marketing techniques are often directed towards children by making consumption of such foods seem  fun  .", "perhaps the greatest barrier to choosing and maintaining a low - sodium diet is that high - salt foods are ubiquitous and hence difficult for those who choose low - salt diets [ 16 , 46 ] . in canada ,", "high - salt diets are by and large perceived as unhealthy by the general population .", "however , it is often the case that the same people who recognize that canadians in general consume too much salt , believe that their personal consumption of dietary sodium is within the recommended amount .", "this suggests that even a relatively affluent , well - educated population may have difficulty identifying and avoiding high - salt foods even if they perceive it is a health issue and have chosen to follow a low - salt diet .", "some of the challenges of individual choice in selecting low salt diets is perhaps best illustrated by clinical trials where highly motivated patients are carefully and repeatedly trained how to select low - salt foods but generally can only sustain small reductions in salt intake long term [ 44 , 48 ] .", "population - based approaches to reducing dietary salt may be effective in developed economies and have shown promising results for reducing blood pressure . in the late 1950s , the japanese government implemented a campaign to reduce salt intake given the high stroke mortality rates .", "ten years later , salt intake was reduced from an average of 13.5 to 12.1  g / day overall , and from 18 to 14  g / day in the northern regions .", "the reduction resulted in a decrease in average blood pressure and an 80% reduction in stroke mortality . in the 1970s , finland 's government began a public education campaign and enforced regulations on food processing companies through a warning label on high - salt foods in order to reduce salt consumption across the country [ 50 , 51 ] .", "more than 30 years later , the overall sodium intake in finland has decreased more than 40% , with a subsequent decrease in mean diastolic blood pressure of greater than 10  mm  hg and an 80% decline in the mortality rate from heart disease and stroke .", "similar results were also seen on smaller scales in the dash ( dietary approached to stop hypertension ) trial conducted in the usa [ 3 , 53 ] .", "this trial assessed three levels of dietary sodium intake on two diets ( american diet versus dash diet ) and demonstrated that reducing sodium in either diet resulted in lower blood pressure [ 3 , 12 , 53 ] .", "more recently in the united kingdom , reductions in the amount of salt added to foods , in conjunction with a social marketing campaign , have been associated with reduction in population salt intake . in developing economies", "widespread replacement of salt with a partial salt replacement ( sodium , potassium magnesium combination ) holds great promise [ 54 , 55 ] . for example , a recent double - blind randomized controlled trial conducted in rural northern china found that replacing household salt with a reduced - sodium , high - potassium salt substitute for 1 year reduced systolic blood pressure by 5.4  mm  hg . this low cost change in diet", "has shown promising outcomes for blood pressure reduction with little to no burden on the consumer .", "clearly both a mix of population approaches and behavioural approaches targeting individuals are required to reduce sodium intake to within recommended levels [ 10 , 11 , 15 , 16 , 33 , 56 ] .", "similar methods have been successfully employed in reducing tobacco use . with respect to individually targeted efforts , for example ,", "a variety of behavioural interventions , including brief physician smoking cessation counselling , was found to meaningfully increase smoking quit rates .", "however , it has become increasingly clear that education targeted towards individuals may be necessary , but not sufficient , to motivate long - term health behaviour change [ 59 , 60 ] .", "for example , motivational interviewing , a directive patient - centred counselling approach focused on exploring and resolving ambivalence , which emerged as an effective therapeutic approach within the addictions field , has recently shown promise for other complex behaviour change problems such as weight loss in overweight and obese patients   and adherence to antihypertensive medication .", "in contrast with recommendations for behaviour change delivered through education and advice giving , motivational interviewing differs in that motivation for change is elicited from individuals , rather than imparted by a healthcare provider .", "the mix of behavioural interventions and population interventions depends on the specific circumstances of both the individual and the population . in countries with developed economies , population - based approaches , and a reduction of salt additives to food , supplemented by public education campaigns ,", "need to be the primary means of intervention to ensure that the healthy option that is low in salt is the easiest option  a basic caveat of public health interventions .", "a universal reduction in salt additives during the manufacturing process has a strong potential to reduce health disparities in vulnerable populations while improving overall population health .", "behavioural interventions may be most important to ensure the population and especially policy makers understand and are supportive of the need to reduce dietary salt .", "however , for specific individuals with strong motivation or at a greater personal risk from consuming a diet high in sodium , intensive behavioural interventions may be efficacious .", "notably sole reliance on the individual behavioural approach is likely to have a smaller impact on a population basis , to be expensive , and to increase health disparity . in developing economies , where the majority of sodium intake comes from salt added at the table and in cooking ,", "behavioural interventions are more likely to be effective in reducing overall intake than population - based means [ 8 , 32 ] . in this case", ", the individual needs to understand the consequences of excess sodium intake and change their behaviors ( i.e. , eating habits ) to reduce sodium intake . nevertheless , even in this", "partial replacement of table salt ( sodium chloride ) with various mineral salts ( mixtures of sodium , potassium , and/or magnesium and calcium ) has been shown to be highly effective to reduce overall sodium consumption and also reduce blood pressure [ 55 , 65 ] . efforts to reduce dietary salt are also likely to reduce dietary iodine , therefore monitoring dietary iodine adequacy and revising the iodine content of salt is essential to maintain population health in most settings , including developed countries .", "population interventions that ensure widespread replacement of salt with a partial salt substitute that contains iodine may be the dominant strategy to reduce sodium intake on a large - scale basis in combination with behaviour change interventions .", "given the promising outcomes observed in recent randomized controlled trials and population - based interventions , reducing dietary sodium intake to modest levels ( approximately 5  g / day ) worldwide would result in a major improvement in overall health and reduce the costs associated with diseases connected to excess sodium intake .", "however , it is apparent that relying solely on interventions that target individual behaviour is not the ideal approach for reducing sodium consumption .", "while it may contribute to behaviour change among highly motivated individuals and increase the acceptability of population - based interventions , the latter approach seems better suited for this particular health behaviour ."], "abstract_text": ["<S> excess intake of dietary salt is estimated to be one of the leading risks to health worldwide . </S>", "<S> major national and international health organizations , along with many governments around the world , have called for reductions in the consumption of dietary salt . </S>", "<S> this paper discusses behavioural and population interventions as mechanisms to reduce dietary salt . in developed countries , </S>", "<S> salt added during food processing is the dominant source of salt and largely outside of the direct control of individuals . </S>", "<S> population - based interventions have the potential to improve health and to be cost saving for these countries . in developing economies , where salt added in cooking and at the table </S>", "<S> is the dominant source , interventions based on education and behaviour change have been estimated to be highly cost effective . </S>", "<S> regardless , countries with either developed or developing economies can benefit from the integration of both population and behavioural change interventions . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Salt Consumption", "3. Cost Effectiveness of Interventions to Reduce Dietary Salt", "4. Awareness and Barriers to Change", "5. Population Interventions to Reduce Dietary Salt", "6. Individual- and Population-Based Approaches", "7. Conclusion"], "sections": [["of those deaths , an estimated 7.3 million were due to coronary heart disease and 6.2 million due to stroke . a disproportionate amount of those deaths , over 80% , take place in low- and middle - income countries and occur at similar rates among men and women .", "not surprisingly , elevated blood pressure levels are a major cause of these diseases and are found at higher rates among low- and middle - income countries . the relationship between blood pressure levels and risk of developing cardiovascular disease is strong and well supported . in 2008 , approximately one billion adults worldwide had uncontrolled hypertension ( defined as systolic blood pressure 140  mm  hg systolic and/or diastolic blood pressure 90  mm  hg ) . given the increasing prevalence of hypertension worldwide and the associated risk for developing cardiovascular disease , public health interventions aimed at reducing blood pressure are crucial .  ", "many national and international agencies have acknowledged the role of lifestyle and diet , in particular sodium intake , on blood pressure levels .", "diets high in salt are now recognized as one of the leading risks to cardiovascular health in the world as they increase blood pressure in both children and adults .", "furthermore , a recent meta - analysis of randomized trials has demonstrated that modest reductions in dietary sodium intake are associated with significant reductions in blood pressure in both normotensives and hypertensives and a 20% reduction in cardiovascular events [ 4 , 5 ] .", "a reduction in salt intake of 6  g / day lowered blood pressure by 7/4  mm  hg diastolic in hypertensives and 4/2  mm  hg in normotensives .", "this relationship has been empirically supported and is sufficiently strong to warrant recommendations for public health interventions aimed at substantially reducing dietary sodium intake .", "furthermore , sodium reduction is noted as one of the most cost effective and most easily implemented strategies to improve population health [ 513 ] . reducing dietary salt", "is recommended by the world health organization and many national governmental and nongovernmental health organizations .", "some agencies , however , do not promote a reduction in dietary sodium , namely , nongovernmental or commercial organizations such as the salt institute , as they are sponsored by either the food or salt industries [ 9 , 11 , 1417 ] .", "regardless , it is apparent that the risks associated with an increase in salt consumption , chiefly those related to an increase in blood pressure , are linear [ 3 , 18 ] .", "most health economic models input relatively small changes in blood pressure that occur in those with normal and high blood pressure as estimated by short - term modest reductions in dietary sodium [ 7 , 8 , 19 ] .", "some models also include the gastric cancers that are positively associated with , and probably caused by , high - salt intake .", "typically , the health economic models do not include the potential impact of longer term irreversible increases in blood pressure , age - related increases in blood pressure , the epigenetic phenomena , whereby exposure to excess salt in utero may increase blood pressure in offspring , or that sodium may increase vascular and cardiac disease in the absence of changes in blood pressure [ 7 , 8 , 12 , 1921 ] .", "the burden of disease studies also do not account for diseases that have a pathophysiological basis and close association with high sodium diets ( i.e. , increased severity and frequency of asthma attacks , increased calcium containing kidney stones , osteoporosis , or obesity related to the consumption of calorie containing beverages caused by sodium - induced thirst ) . hence the burden of disease associated with excess dietary salt is not only high , but may also be underestimated .", "the objective of this commentary is to review current sodium consumption worldwide , discuss cost - effective strategies to reduce dietary sodium , as well as briefly review the role of behavioural and policy - based environmental interventions in reducing dietary sodium on a population - based scale ."], ["humans evolved on diets consisting of natural plant and animal foods containing small amounts of sodium , typically less than 2  g / day [ 25 , 26 ] .", "today , nearly all populations consume far greater quantities of salt than those provided in natural , unprocessed food diets .", "the world health organization currently recommends a daily consumption of less than 5 grams of salt , although some agencies recommend that no more than 1500  mg of sodium should be consumed per day [ 2729 ] , calculated as 2/3  tsp of table salt . in most populations ,", "sodium intake is 5.7  g or more / day after age 5 , with many populations consuming and average of over 10  g / day [ 3032 ] .", "furthermore , within high sodium consumption countries , only a small proportion of individuals consume the recommended levels of salt .", "for example , in canada , a country with average salt consumption of 8.5  gm / day , 85% of men and 60% of women aged 9 to 70 consume over the upper recommended limit for salt and the vast majority ( > 90% ) are above the level recommended for individuals to consume [ 33 , 34 ] .", "for example , one study found that in a population of overweight adults , a daily intake of sodium greater than 2300  mg / day was associated with a 61% increase in coronary heart disease mortality , an 89% increase in stroke mortality , and a 39% increase in all - cause mortality over a 19-year period .", "along with the other sodium - related illnesses discussed above ( i.e. , gastric cancers , kidney stones , etc . )", ", it is clear that the economic costs associated with such illnesses can be substantial ."], ["in countries with developed economies , salt added during the processing of foods accounts for the vast majority of dietary salt ( 7580% ) . an additional 10% of dietary salt", "is accounted for by salt that is naturally occurring in foods , while the rest is accounted for by salt added at the table or during cooking . in low- to - middle income countries where populations may have limited access to processed foods , salt added at home , in cooking , or at the table , accounts for the majority of dietary salt . reducing dietary salt is estimated to save substantial health care costs [ 7 , 10 , 16 , 30 , 3741 ] .", "for example , reducing dietary salt by 3  g / day in the united states is estimated to save 194,000 to 392,000 quality adjusted life years and reduce health care costs $ 10 to $ 24 billion us dollars a year . in canada , reducing salt consumption to recommended levels is estimated to reduce the prevalence of hypertension by 30% and to save up to $ 430 million dollars per year just in direct hypertension management costs alone . in lower - income countries , programs aimed at reducing consumption of dietary salt through an intervention largely based on education", "are estimated to cost little ( less than $ 0.40 usd per person per year ) , reduce premature deaths by close to 14 million in 10 years , and to be slightly more cost effective than strategies to reduce tobacco use ( both highly advocated interventions ) [ 8 , 40 ] ."], ["although there is a general lack of awareness of salt as a health issue in many countries , some countries with established salt reduction programs show increasing awareness .", "for example , in canada , 80% of people diagnosed with hypertension are attempting to reduce dietary sodium .", "in addition , many food companies have developed low - salt options to their product lines for people to choose , with some companies reducing salt additives in their full product line . in developed economies , there are substantial barriers to free choice in those who chose to eat less salt [ 16 , 44 ] . in most countries ,", "it is often the case that nutritional information is available only on the company 's website , by asking for and reviewing a binder on site , or only readily available after purchase . even in the united states and canada , countries with mandatory labelling of packaged foods , the labels are often difficult to interpret . also serving sizes may be variable and not comparable between products . in an unpublished study", ", we found that in a sample of over 100 people with diabetes who had received training on how to read a food label , not one could accurately answer how much of a processed food they could eat in a day when presented with the food label .", "in other countries , food labelling on packaged foods may not be mandatory and labelled foods may not be available .", "in remote regions and areas where populations vulnerable to the development of elevated blood pressure reside , low - salt alternative choices are typically not available .", "furthermore , these populations may not have the health literacy with which to make informed choices .", "food processors and manufacturers often use pervasive marketing techniques to create consumer demand for high - salt foods , which undermine efforts of public health and individual education interventions that attempt to reduce sodium intake .", "moreover , these marketing techniques are often directed towards children by making consumption of such foods seem  fun  .", "perhaps the greatest barrier to choosing and maintaining a low - sodium diet is that high - salt foods are ubiquitous and hence difficult for those who choose low - salt diets [ 16 , 46 ] . in canada ,", "high - salt diets are by and large perceived as unhealthy by the general population .", "however , it is often the case that the same people who recognize that canadians in general consume too much salt , believe that their personal consumption of dietary sodium is within the recommended amount .", "this suggests that even a relatively affluent , well - educated population may have difficulty identifying and avoiding high - salt foods even if they perceive it is a health issue and have chosen to follow a low - salt diet .", "some of the challenges of individual choice in selecting low salt diets is perhaps best illustrated by clinical trials where highly motivated patients are carefully and repeatedly trained how to select low - salt foods but generally can only sustain small reductions in salt intake long term [ 44 , 48 ] ."], ["population - based approaches to reducing dietary salt may be effective in developed economies and have shown promising results for reducing blood pressure . in the late 1950s , the japanese government implemented a campaign to reduce salt intake given the high stroke mortality rates .", "ten years later , salt intake was reduced from an average of 13.5 to 12.1  g / day overall , and from 18 to 14  g / day in the northern regions .", "the reduction resulted in a decrease in average blood pressure and an 80% reduction in stroke mortality . in the 1970s , finland 's government began a public education campaign and enforced regulations on food processing companies through a warning label on high - salt foods in order to reduce salt consumption across the country [ 50 , 51 ] .", "more than 30 years later , the overall sodium intake in finland has decreased more than 40% , with a subsequent decrease in mean diastolic blood pressure of greater than 10  mm  hg and an 80% decline in the mortality rate from heart disease and stroke .", "similar results were also seen on smaller scales in the dash ( dietary approached to stop hypertension ) trial conducted in the usa [ 3 , 53 ] .", "this trial assessed three levels of dietary sodium intake on two diets ( american diet versus dash diet ) and demonstrated that reducing sodium in either diet resulted in lower blood pressure [ 3 , 12 , 53 ] .", "more recently in the united kingdom , reductions in the amount of salt added to foods , in conjunction with a social marketing campaign , have been associated with reduction in population salt intake . in developing economies", "widespread replacement of salt with a partial salt replacement ( sodium , potassium magnesium combination ) holds great promise [ 54 , 55 ] . for example , a recent double - blind randomized controlled trial conducted in rural northern china found that replacing household salt with a reduced - sodium , high - potassium salt substitute for 1 year reduced systolic blood pressure by 5.4  mm  hg . this low cost change in diet", "has shown promising outcomes for blood pressure reduction with little to no burden on the consumer ."], ["clearly both a mix of population approaches and behavioural approaches targeting individuals are required to reduce sodium intake to within recommended levels [ 10 , 11 , 15 , 16 , 33 , 56 ] .", "similar methods have been successfully employed in reducing tobacco use . with respect to individually targeted efforts , for example ,", "a variety of behavioural interventions , including brief physician smoking cessation counselling , was found to meaningfully increase smoking quit rates .", "however , it has become increasingly clear that education targeted towards individuals may be necessary , but not sufficient , to motivate long - term health behaviour change [ 59 , 60 ] .", "for example , motivational interviewing , a directive patient - centred counselling approach focused on exploring and resolving ambivalence , which emerged as an effective therapeutic approach within the addictions field , has recently shown promise for other complex behaviour change problems such as weight loss in overweight and obese patients   and adherence to antihypertensive medication .", "in contrast with recommendations for behaviour change delivered through education and advice giving , motivational interviewing differs in that motivation for change is elicited from individuals , rather than imparted by a healthcare provider .", "the mix of behavioural interventions and population interventions depends on the specific circumstances of both the individual and the population . in countries with developed economies , population - based approaches , and a reduction of salt additives to food , supplemented by public education campaigns ,", "need to be the primary means of intervention to ensure that the healthy option that is low in salt is the easiest option  a basic caveat of public health interventions .", "a universal reduction in salt additives during the manufacturing process has a strong potential to reduce health disparities in vulnerable populations while improving overall population health .", "behavioural interventions may be most important to ensure the population and especially policy makers understand and are supportive of the need to reduce dietary salt .", "however , for specific individuals with strong motivation or at a greater personal risk from consuming a diet high in sodium , intensive behavioural interventions may be efficacious .", "notably sole reliance on the individual behavioural approach is likely to have a smaller impact on a population basis , to be expensive , and to increase health disparity . in developing economies , where the majority of sodium intake comes from salt added at the table and in cooking ,", "behavioural interventions are more likely to be effective in reducing overall intake than population - based means [ 8 , 32 ] . in this case", ", the individual needs to understand the consequences of excess sodium intake and change their behaviors ( i.e. , eating habits ) to reduce sodium intake . nevertheless , even in this", "partial replacement of table salt ( sodium chloride ) with various mineral salts ( mixtures of sodium , potassium , and/or magnesium and calcium ) has been shown to be highly effective to reduce overall sodium consumption and also reduce blood pressure [ 55 , 65 ] . efforts to reduce dietary salt are also likely to reduce dietary iodine , therefore monitoring dietary iodine adequacy and revising the iodine content of salt is essential to maintain population health in most settings , including developed countries .", "population interventions that ensure widespread replacement of salt with a partial salt substitute that contains iodine may be the dominant strategy to reduce sodium intake on a large - scale basis in combination with behaviour change interventions ."], ["given the promising outcomes observed in recent randomized controlled trials and population - based interventions , reducing dietary sodium intake to modest levels ( approximately 5  g / day ) worldwide would result in a major improvement in overall health and reduce the costs associated with diseases connected to excess sodium intake .", "however , it is apparent that relying solely on interventions that target individual behaviour is not the ideal approach for reducing sodium consumption .", "while it may contribute to behaviour change among highly motivated individuals and increase the acceptability of population - based interventions , the latter approach seems better suited for this particular health behaviour ."]]}
{"article_id": "PMC3409954", "article_text": ["the associated edema which may develop within an hour is the leading cause of potential life - threatening airway compromise .", "the disease may show variation in terms of its presentation and clinical course , which needs to be addressed carefully before making any airway management plan .", "literature is convincing both for the active as well as conservative approach of airway management depending upon the patient 's clinical condition .", "the purpose of reporting this case is to highlight these issues in the management of adult patient with acute supraglottitis .", "a 52-year - old man presented in the emergency department with the complaints of sore throat for 1 day . the past medical history was unremarkable except the history of ischemic heart disease for which he was taking regular clopidogrel , losartan , and sublingual trinitrates as required .", "he was febrile with the temperature of 37.6c , respiratory rate was 22 per minute , while the oxygen saturation on room air was 95% .", "there were no signs of airway obstruction including stridor , change of voice , and dyspnea related to change in position or lying down .", "the initial management was started with humidified oxygen , intravenous dexamethasone , and adrenaline nebulization .", "the fiberoptic nasolaryngoscopy showed the significant edema and inflammation of supraglottic region more on the right side as compared to the left .", "he was quiet comfortable with the breathing and maintaining the oxygen saturation on hudson mask .", "the rest of the physical and systemic examination was normal . on the basis of initial assessment", "however , consideration was given to nasolaryngoscopy findings and the fact that we did not have overnight otorhinolaryngology cover in our district hospital .", "it was also decided that conservative airway management of patient , either in our hospital or during shifting to the specialized unit , would be very high risk .", "the three anesthetists , out of whom two had consultant grades , were present for the case .", "patient 's was kept on spontaneous ventilation with the help of nasopharyngeal airway connected to the main circuit via 15 mm outer diameter connector .", "a surgical tracheostomy was done after infiltration of local anesthesia while the patient was kept anesthetized on inhalational anesthetic on spontaneous breathing pattern .", "the patient remained stable throughout the procedure and shifted to the intensive care unit on completion of the procedure .", "the unanticipated rapidly progressive course of the disease was confirmed by the computerized tomography scan of the neck done after 24 h , which showed the marked swelling of oropharynx reducing the size of airway to not more than a pin hole [ figure 1 ] .", "patient was shifted to specialized center on the first postoperative day where he was treated medically .", "rest of the course in the hospital was uneventful and then he was discharged home .", "the reported incidence in this population group is 12.9/100,000 annually and is higher than that in the pediatric population .", "signs and symptoms at the time of presentation show variation among the patient group involved .", "sore throat , dysphagia , and change in voice are the common findings in the adults while the children usually present with the cardinal signs of airway obstruction as stridor , drooling , and dyspnea .", "the current literature supports the conservative approach for the patients in which no airway compromise is suspected .", "patients having a rapid progressive course , diabetes , or symptoms of impending airway obstruction like stridor or muffled voice are likely to be managed with the active airway intervention .", "we recommend that before taking any decision on the approach to a patient , it is very important to consider the type of health care setup and facilities available .", "the disease process may show marked variation in terms of symptoms and progression as it was happened in our patient .", "our patient presented with sore throat and he did not have any sign or symptoms of respiratory distress but still had rapid onset of massive airway edema .", "a retrospective review of nine healthy adults found that the signs of upper airway obstruction are characteristically absent in early phase of potentially fatal supraglottitis .", ". it also aids in tracking the disease progress and the response to medical treatment .", "severe swelling of the epiglottis and its extension to the arytenoids are the two factors strongly associated with the airway intervention .", "the management of our patient was based on the nasolaryngoscopy finding at the time of presentation which showed the clinically significant edema of oropharynx .", "air way manipulation in such high risk patients should be done in the operation theatre with a surgeon ready for emergency tracheostomy .", "the anesthetic options , inhalational induction using sevoflurane or awake trachesotomy , should be individualized according to the patient condition and clinical expertise available . in adult patients having acute supraglottitis ,", "clinical parameters are not the reliable indicators of the airway condition , which may deteriorate rapidly .", "a multidisciplinary approach involving the otorhinolaryngologist and anesthesiologist is of prime importance for the management of airway . either the prophylactic or conservative approach may be used depending upon the patient condition and the health care facilities available .", "it is also very important to anticipate the difficulty and all the preparations for an emergency tracheostomy should be there for securing of airway ."], "abstract_text": ["<S> airway management of adult patients with acute supraglottitis is challenging . the sign and symptoms of the disease may show marked variation in terms of severity and progression . </S>", "<S> thorough evaluation is required before selecting any particular approach . </S>", "<S> we report the case of an adult patient with acute supraglottitis , in whom active airway intervention was planned . </S>", "<S> the clinical predictors were not suggestive of any airway compromise in our patient . </S>", "<S> however , the disease was found to have an unanticipated rapidly progressive course leading to the significant edema of the oropharynx and the surrounding structures as evidenced by the laryngoscope and computerized tomography scan findings . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["the associated edema which may develop within an hour is the leading cause of potential life - threatening airway compromise .", "the disease may show variation in terms of its presentation and clinical course , which needs to be addressed carefully before making any airway management plan .", "literature is convincing both for the active as well as conservative approach of airway management depending upon the patient 's clinical condition .", "the purpose of reporting this case is to highlight these issues in the management of adult patient with acute supraglottitis ."], ["a 52-year - old man presented in the emergency department with the complaints of sore throat for 1 day . the past medical history was unremarkable except the history of ischemic heart disease for which he was taking regular clopidogrel , losartan , and sublingual trinitrates as required .", "he was febrile with the temperature of 37.6c , respiratory rate was 22 per minute , while the oxygen saturation on room air was 95% .", "there were no signs of airway obstruction including stridor , change of voice , and dyspnea related to change in position or lying down .", "the initial management was started with humidified oxygen , intravenous dexamethasone , and adrenaline nebulization .", "the fiberoptic nasolaryngoscopy showed the significant edema and inflammation of supraglottic region more on the right side as compared to the left .", "he was quiet comfortable with the breathing and maintaining the oxygen saturation on hudson mask .", "the rest of the physical and systemic examination was normal . on the basis of initial assessment", "however , consideration was given to nasolaryngoscopy findings and the fact that we did not have overnight otorhinolaryngology cover in our district hospital .", "it was also decided that conservative airway management of patient , either in our hospital or during shifting to the specialized unit , would be very high risk .", "the three anesthetists , out of whom two had consultant grades , were present for the case .", "patient 's was kept on spontaneous ventilation with the help of nasopharyngeal airway connected to the main circuit via 15 mm outer diameter connector .", "a surgical tracheostomy was done after infiltration of local anesthesia while the patient was kept anesthetized on inhalational anesthetic on spontaneous breathing pattern .", "the patient remained stable throughout the procedure and shifted to the intensive care unit on completion of the procedure .", "the unanticipated rapidly progressive course of the disease was confirmed by the computerized tomography scan of the neck done after 24 h , which showed the marked swelling of oropharynx reducing the size of airway to not more than a pin hole [ figure 1 ] .", "patient was shifted to specialized center on the first postoperative day where he was treated medically .", "rest of the course in the hospital was uneventful and then he was discharged home ."], ["the reported incidence in this population group is 12.9/100,000 annually and is higher than that in the pediatric population .", "signs and symptoms at the time of presentation show variation among the patient group involved .", "sore throat , dysphagia , and change in voice are the common findings in the adults while the children usually present with the cardinal signs of airway obstruction as stridor , drooling , and dyspnea .", "the current literature supports the conservative approach for the patients in which no airway compromise is suspected .", "patients having a rapid progressive course , diabetes , or symptoms of impending airway obstruction like stridor or muffled voice are likely to be managed with the active airway intervention .", "we recommend that before taking any decision on the approach to a patient , it is very important to consider the type of health care setup and facilities available .", "the disease process may show marked variation in terms of symptoms and progression as it was happened in our patient .", "our patient presented with sore throat and he did not have any sign or symptoms of respiratory distress but still had rapid onset of massive airway edema .", "a retrospective review of nine healthy adults found that the signs of upper airway obstruction are characteristically absent in early phase of potentially fatal supraglottitis .", ". it also aids in tracking the disease progress and the response to medical treatment .", "severe swelling of the epiglottis and its extension to the arytenoids are the two factors strongly associated with the airway intervention .", "the management of our patient was based on the nasolaryngoscopy finding at the time of presentation which showed the clinically significant edema of oropharynx .", "air way manipulation in such high risk patients should be done in the operation theatre with a surgeon ready for emergency tracheostomy .", "the anesthetic options , inhalational induction using sevoflurane or awake trachesotomy , should be individualized according to the patient condition and clinical expertise available . in adult patients having acute supraglottitis ,", "clinical parameters are not the reliable indicators of the airway condition , which may deteriorate rapidly .", "a multidisciplinary approach involving the otorhinolaryngologist and anesthesiologist is of prime importance for the management of airway . either the prophylactic or conservative approach may be used depending upon the patient condition and the health care facilities available .", "it is also very important to anticipate the difficulty and all the preparations for an emergency tracheostomy should be there for securing of airway ."]]}
{"article_id": "PMC2789965", "article_text": ["a 15-year - old boy had complained of motility limitation in the right eye since a young age .", "his visual acuity was 20/20 in each eye , although an ocular motility examination revealed markedly limited abduction and mildly limited adduction in his right eye ( fig .", "1 ) . on attempted adduction of the right eye , globe retraction ( fig .", "2 ) with narrowing of the palpebral fissure was observed , and there mild upshoot and downshoot were also observed ( fig .", "an alternate prism cover test revealed a 16 prism diopters ( pd ) right esotropia ( et ) in the primary position , 25 pd et in right gaze , 10 pd et in left gaze at distance fixation , and 4 pd et at near fixation .", "he assumed a compensatory face turn to the right and did not complain of diplopia .", "duane 's retraction syndrome was inferred as the diagnosis until we noted that the patient 's right eye had slight enophthalmos .", "hertel 's exophthalmometry revealed a 1.5 mm difference between both eyes and the margin reflex distance 1 was 0.5 mm in the right eye . computed tomography of the orbits", "was performed and demonstrated an old medial orbital wall fracture with entrapment of the medial rectus muscle and surrounding tissues ( fig .", "however , there was no history of trauma or forceps delivery according to the patient or his parents .", "the patient 's ocular motor impairment was concluded to have arose from a long - term medial orbital wall fracture and was diagnosed as ' pseudo - duane 's retraction syndrome ' .", "because open reduction surgery for an old orbital wall fracture would be very difficult , and fruitless for this patient , we decided only to perform surgery on the eye muscle .", "the 5 mm recession of the right medial rectus muscle was successfully performed in order to correct the patient 's head turn and esotropia .", "postoperatively , the patient had orthotropia in the primary position , and the anomalous head posture disappeared , though there were still some limitations in both abduction and adduction in the right eye one year after the surgery .", "duane 's retraction syndrome is a well - known congenital ocular motility disorder caused by anomalous innervation and mechanics of the lateral rectus muscle , with secondary muscle changes sometimes superimposed . in 1976 , thomas duane reported five cases of pseudo - duane 's retraction syndrome showing similar , yet different , symptoms and sign as duane 's retraction syndrome.1 other authors preferred the names of ' inverse duane , reverse duane , or mirror - image duane retraction syndrome ' owing to the presentation of opposing clinical features compared with those of classic duane 's retraction syndrome .", "the etiology is , also , completely different from that of classic duane 's retraction syndrome .", "the primary lesion in pseudo - duane 's retraction syndrome is suspected to be the medial rectus muscle .", "most cases have been reported to result from the entrapment of the medial rectus muscle within the medial orbital wall due to trauma.1,3 congenital cases,4 recurrent pterygium,5 and myocysticercosis involving the medial rectus muscle6 have also been reported to be related with inverse duane 's retraction syndrome .", "in korea , jo et al.7 reported on inverse duane 's retraction syndrome with exotropia , and lew et al.8 reported a case of congenital inverse duane 's retraction syndrome with a tight medial rectus muscle .", "recently , a case of innervational etiology , such as bilateral inverse duane 's retraction syndrome , due to simultaneous co - contraction of both horizontal rectus muscles has been reported by khan.9 to the best of our knowledge , there is no report presenting very similar clinical features as those of classic duane 's retraction syndrome arising from the entrapment of the medial rectus muscle in a medial orbital wall fracture .", "this case would most appropriately be categorized as pseudo - duane 's retraction syndrome instead of inverse or reverse duane 's retraction syndrome . there might be some possibility that the patient acquired duane 's retraction syndrome prenatally , and that the medial orbital wall then fractured after birth", ". however , several pictures of this patient around one year of age revealed that there was no conspicuous head turn , and his family recalled that the limited eye movement was not noted until 2 - 3 years of age .", "the limitation of his eye movements , therefore , could be concluded as acquired due to unnoticed orbital trauma after one year of age .", "we report a case of pseudo - duane 's retraction syndrome caused by entrapment of the medial rectus muscle in an old medial orbital wall fracture presenting very similar clinical features as those of classic duane 's retraction syndrome .", "the importance of several tests such as the forced duction test and an imaging study should be emphasized in making the diagnosis for limited eye movement ."], "abstract_text": ["<S> we report a case of pseudo - duane 's retraction syndrome with entrapment of the medial rectus muscle in an old medial orbital wall fracture presenting identical clinical symptoms as duane 's retraction syndrome . </S>", "<S> a 15-year - old boy presented with persistent limited right eye movement since a young age . </S>", "<S> examination showed marked limited abduction , mildly limited adduction , and globe retraction accompanied by narrowing of the palpebral fissure during attempted adduction in the right eye . </S>", "<S> he showed a right esotropia of 16 prism diopters and his head turned slightly to the right . </S>", "<S> a slight enophthalmos was noted in his right eye . </S>", "<S> a computed tomography scan demonstrated entrapment of the medial rectus muscle and surrounding tissues in an old medial orbital wall fracture . </S>", "<S> a forced duction test revealed a marked restriction of abduction in the right eye . </S>", "<S> a 5 mm recession of the right medial rectus muscle was performed . </S>", "<S> postoperatively , the patient 's head turn and esotropia in the primary position were successfully corrected , but there was still some limitations to his ocular movement . </S>", "<S> the importance of several tests such as the forced duction test and an imaging study should be emphasized in making a diagnosis for limitation of eye movement . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 15-year - old boy had complained of motility limitation in the right eye since a young age .", "his visual acuity was 20/20 in each eye , although an ocular motility examination revealed markedly limited abduction and mildly limited adduction in his right eye ( fig .", "1 ) . on attempted adduction of the right eye , globe retraction ( fig .", "2 ) with narrowing of the palpebral fissure was observed , and there mild upshoot and downshoot were also observed ( fig .", "an alternate prism cover test revealed a 16 prism diopters ( pd ) right esotropia ( et ) in the primary position , 25 pd et in right gaze , 10 pd et in left gaze at distance fixation , and 4 pd et at near fixation .", "he assumed a compensatory face turn to the right and did not complain of diplopia .", "duane 's retraction syndrome was inferred as the diagnosis until we noted that the patient 's right eye had slight enophthalmos .", "hertel 's exophthalmometry revealed a 1.5 mm difference between both eyes and the margin reflex distance 1 was 0.5 mm in the right eye . computed tomography of the orbits", "was performed and demonstrated an old medial orbital wall fracture with entrapment of the medial rectus muscle and surrounding tissues ( fig .", "however , there was no history of trauma or forceps delivery according to the patient or his parents .", "the patient 's ocular motor impairment was concluded to have arose from a long - term medial orbital wall fracture and was diagnosed as ' pseudo - duane 's retraction syndrome ' .", "because open reduction surgery for an old orbital wall fracture would be very difficult , and fruitless for this patient , we decided only to perform surgery on the eye muscle .", "the 5 mm recession of the right medial rectus muscle was successfully performed in order to correct the patient 's head turn and esotropia .", "postoperatively , the patient had orthotropia in the primary position , and the anomalous head posture disappeared , though there were still some limitations in both abduction and adduction in the right eye one year after the surgery ."], ["duane 's retraction syndrome is a well - known congenital ocular motility disorder caused by anomalous innervation and mechanics of the lateral rectus muscle , with secondary muscle changes sometimes superimposed . in 1976 , thomas duane reported five cases of pseudo - duane 's retraction syndrome showing similar , yet different , symptoms and sign as duane 's retraction syndrome.1 other authors preferred the names of ' inverse duane , reverse duane , or mirror - image duane retraction syndrome ' owing to the presentation of opposing clinical features compared with those of classic duane 's retraction syndrome .", "the etiology is , also , completely different from that of classic duane 's retraction syndrome .", "the primary lesion in pseudo - duane 's retraction syndrome is suspected to be the medial rectus muscle .", "most cases have been reported to result from the entrapment of the medial rectus muscle within the medial orbital wall due to trauma.1,3 congenital cases,4 recurrent pterygium,5 and myocysticercosis involving the medial rectus muscle6 have also been reported to be related with inverse duane 's retraction syndrome .", "in korea , jo et al.7 reported on inverse duane 's retraction syndrome with exotropia , and lew et al.8 reported a case of congenital inverse duane 's retraction syndrome with a tight medial rectus muscle .", "recently , a case of innervational etiology , such as bilateral inverse duane 's retraction syndrome , due to simultaneous co - contraction of both horizontal rectus muscles has been reported by khan.9 to the best of our knowledge , there is no report presenting very similar clinical features as those of classic duane 's retraction syndrome arising from the entrapment of the medial rectus muscle in a medial orbital wall fracture .", "this case would most appropriately be categorized as pseudo - duane 's retraction syndrome instead of inverse or reverse duane 's retraction syndrome . there might be some possibility that the patient acquired duane 's retraction syndrome prenatally , and that the medial orbital wall then fractured after birth", ". however , several pictures of this patient around one year of age revealed that there was no conspicuous head turn , and his family recalled that the limited eye movement was not noted until 2 - 3 years of age .", "the limitation of his eye movements , therefore , could be concluded as acquired due to unnoticed orbital trauma after one year of age .", "we report a case of pseudo - duane 's retraction syndrome caused by entrapment of the medial rectus muscle in an old medial orbital wall fracture presenting very similar clinical features as those of classic duane 's retraction syndrome .", "the importance of several tests such as the forced duction test and an imaging study should be emphasized in making the diagnosis for limited eye movement ."]]}
{"article_id": "PMC4905881", "article_text": ["lumbar spine instability has been reported to be the leading cause of chronic back \n pain1 .", "an a abnormal vertebral joint \n creates mechanical lesions and pains in the lumbar spine , decreasing stability and \n increasing the range of motion , resulting in functional degeneration2 .", "these negative patterns have a variety of forms such as \n aging , medical history , and exercise deficiency and present serious problems in the age \n group of 2040 , due to developments of modern society and recent changes in various working \n forms of work3 , 4 . herniated nucleus pulposus ( hnp ) is a representative disease of the functional vertebral \n unit that occurs when the pulposus nucleus is exposed by rupture of the annulus fibrosus and \n it is a disease that causes chronic back pains5 , 6 .", "chronic back pain patients not only have \n weak deep muscles of the lumbar region and muscle imbalance compared to normal subjects , but \n also show reduction in the re - positioning ability which leads to problem with the stability \n of the spine , due to the reduction in proprioceptive sense , which causes lumbar pain and \n recurrence7 , 8 .", "they include exercises \n for increasing muscle strength , improving functions , and maintaining posture as well as \n preventing excessive movements of the lumbar spine9 .", "exercise rehabilitation approaches for strength and stability are \n important even after surgical treatments , due to degeneration and restricted activities10 , 11 \n and conservative methods of treatment by exercise have been recommended for the purpose of \n preventing recurrence of pain and improving functions12,13,14,15,16 .", "thus , exercise therapies based on the stabilization exercises are \n considered to be necessary for preventing chronic pain and reductions in functional \n capabilities by the reduction of functional instability in various of type and the \n improvement of motor functional capabilities of the lumbar joints .", "therefore , this study aims to help adult women with lumbar disc herniation recover muscle \n strength and stability through 8-week lumber extension strength exercise programs , and to \n provide data for constructing an integrated exercise program .", "in this study , an 8-week exercise program for lumbar muscle extension strength and \n stabilization was performed by 26 females older than 20 with lumbar disc herniation .", "this \n study was approved by the hankuk university of foreign studies and the institution where it \n was performed , and it complied with the ethical principles of the declaration of helsinki . \n the purposes and process of the study were fully explained to the subjects and their consent \n was obtained before participation .", "the subjects mean  sd was 27.2  4.4  years old their mean  \n sd height was 162.3  5.1  cm and their mean  sd weight was 55.4  7.1  kg .", "the programs was conducted for a total of 8 weeks , 60 minutes a session , twice a week , to \n improve strength and stabilize lumbar extension .", "the exercise program was divided into \n warm - up exercises , main exercises and cool - down exercises .", "the warm - up exercises and \n cool - down exercises were conducted focusing on the range of joint motion ( rom ) without pain , \n utilizing cycles and steppers for 10 minutes , alternately . for the purpose of improving the \n lumbar stability and resistance muscles ,", "the main exercises were designed to be individually \n conducted in the form of a circuit training in which the number of repetitions increased \n gradually , utilizing sling and weight exercise equipment . a lumbar extension strength machine ( medx , medx inc . , ocala , fl , usa ) was utilized for the \n evaluation of lumbar extensor strength .", "the maximum static muscular strength of lumbar \n extension was measured at 7 different angles of lumbar flexion : 0 , 12 , 24 , 36 , 48 , 60 , and \n 72 degrees .", "passive tests were conducted to determine whether to limit the range of motion \n before measurement .", "the tests were conducted 12 times at 45 lbs in accordance with the medx \n exercise measurement guidelines .", "the measurement was performed after fixing the pelvis and \n thigh of the subjects on the support .", "then , the motion of the lumbar spine was limited \n during measurement by adjusting the footrest .", "the subjects were asked to increasingly extend \n the lumbar spine by sufficiently considering the limited angular range of motion of the \n joints .", "the measurements were performed in the same manner at every angle by maintaining \n maximum muscle contraction for about 2 seconds .", "for the physical stability \n measurements , the total weight distribution index ( wdi ) was calculated by assessing the \n degree of interaction and coordination of the lower body through body away while maintaining \n a standing posture . for data processing , the statistical program spss ( spss inc . ,", "the mean and standard deviation of the measurements was calculated for all metrics . to \n compare the difference between the pre - exercise and post - exercise state of the 8-week \n exercise program ,", "the paired t - test was used and statistical significance was accepted for \n values of p < 0.05 .", "significant differences were observed in lumbar extension strength ( les ) between \n pre - exercise and post - exercise at every angle of lumbar flexion , but no significant \n difference was found in wdi ( % ) ( table \n 1table 1.changes of the les and wdivariableanglepre - exercisepost - exerciseles ( ft - lbs)061.8  33.880.1  31.6**1278.7  33.898.8  33.6**2487.6  36.5116.5  32.7***3698.6  35.9129.7  32.3***48104.7  35.0137.6  30.2***60113.1  36.9146.5  30.5***72122.3  40.1158.7  34.7***wdi ( % ) 5.9  3.35.0", " 2.8values are mean  sd , les : lumbar extension strength , wdi : weight distribution \n index .", "the post - exercise les were found to be very significantly ( p<0.01 ) higher \n than pre - exercise at 0 degree and 12 degree , and extremity significantly ( p<0.001 ) higher \n than pre - exercise at 24 , 36 , 48 , and 72 degrees of lumbar flexion .", "values are mean  sd , les : lumbar extension strength , wdi : weight distribution \n index .", "this study attempted to identify the positive impacts that were exerted on extension muscle \n strength and stability by an 8-week extension strength exercise program which was performed \n by 26 females with lumbar disc herniation .", "hnp is caused by the annulus fibrosus that is squeezed into the spinal canal when the \n pulposus nucleus is torn due to a degenerative intervertebral disc .", "it has been reported \n that approximately 29% of the projected and escaped intervertebral discs are associated with \n the pulposus nucleus17 .", "chronic back \n pains resulting from hnp aggravate the instability of the lumbar spine , causing degenerative \n changes , atrophy of muscle strength , and reduce flexibility and joint range of motion due to \n trunk damage and instability18 , 19 .", "lumbar herniated nucleus pulposus does not occur due to \n lumbar pain or disability , but more frequently occurs due to deterioration of the related \n muscles or functions interacting with them adjunctively8 .", "in addition , it is said that the joint range of motion is limited \n due to the loss of muscle strength and flexibility caused by occupational factors , mode of \n action , specific changes in posture , or degenerative disease18 .", "these musculoskeletal diseases can be generally improved by utilizing conservative \n treatments such as exercise9 , 18 .", "rehabilitation exercise therapies for muscular strength \n and stability are important even after surgery10 , \n 16 .", "various types of composite \n exercises and core exercises in addition to exercises for muscular strength and stability \n are currently being utilized8 , 12 .", "these exercise therapies help to stabilize the lumbar spine through lumbar and \n trunk dynamic stabilization and exercise modulation , and increase of muscle strength20 .", "similar to the present study , a study analyzing the impacts of an 8-week functional \n exercise program for lumbar muscle strength , that was performed by 26 females with \n degenerative disc findings , found that there were significant differences between \n pre - exercise and post - exercise15 in \n lumbar muscle extension strength at all 7 angles of lumbar flexion , proving that exercise \n participation develops stability of the muscles around the lumbar spine .", "in addition , a \n study analyzing the effects of decompression therapy by 4-week joint mobilization that was \n performed for patients with lumbar herniated nucleus pulposus , reported improvements in \n joint range of motion in flexion and extension21 , proving its effectiveness . similarly , a study of13 lumbar traction for patients with lumbar \n herniated nucleus pulposus , and a study11 \n of lumbar discectomy and stabilization exercises for patients with lumbar herniated nucleus \n pulposus , both reported improvements in flexion and extension .", "the improvements in functions \n pursuant to improved muscle strength and stability were statistically significant , as were \n the results of the studies cited above .", "therefore , prevention of excessive movement of the \n lumbar spine and trunk , in the patients with chronic back pains or lumbar herniated nucleus \n pulposus can be helpful in their daily life because they secure the joint range of motion , \n thereby improving strength and stability9 .", "thus , the development of muscular strength for stabilization and integrated exercise for \n pain reduction and rehabilitation help to maintain the range of joint motion , muscle \n strength , and balance21,22,23 . for the \n improvement and rehabilitation of inpaired capacity in daily life due to back \n pain - associated injuries experienced by about 80% of the population a higher occurrence \n rate8 , 10 when recurrence is included muscle weakness , and loss15 of balance the development of an \n integrated exercise program is required .", "the above results indicate that improving muscle extension strength to enhance and improve \n the functions the patients with chronic back pain or lumbar herniated nucleus pulposus can \n help improve muscle functions and increase the range of joint motion thereby exerting a \n positive impact on physical stability ."], "abstract_text": ["<S> [ purpose ] the purpose of this study was to provide the data for constructing an \n integrated exercise program to help restore muscle strength and stability through \n extension strength exercise in adult females with lumbar disc herniation . </S>", "<S> [ subjects and \n methods ] an 8-week exercise program for lumbar muscle extension strength and stabilization \n was performed by 26 females older than 20 with lumbar disc herniation findings . [ results ] \n significant differences </S>", "<S> were found in lumbar extension muscle strength at every angle of \n lumbar flexion after participation in the 8-week stabilization exercise program ; but there \n was no significant difference in the weight distribution index . </S>", "<S> [ conclusion ] an integrated \n exercise program aiming to strengthen lumbar spine muscles , reduce pain and stabilize the \n trunk can help to maintain muscle strength and balance . in addition , improvement in \n extension strength is expected to be helpful in daily life by securing the range of joint \n motion and improving the strength and stability . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["lumbar spine instability has been reported to be the leading cause of chronic back \n pain1 .", "an a abnormal vertebral joint \n creates mechanical lesions and pains in the lumbar spine , decreasing stability and \n increasing the range of motion , resulting in functional degeneration2 .", "these negative patterns have a variety of forms such as \n aging , medical history , and exercise deficiency and present serious problems in the age \n group of 2040 , due to developments of modern society and recent changes in various working \n forms of work3 , 4 . herniated nucleus pulposus ( hnp ) is a representative disease of the functional vertebral \n unit that occurs when the pulposus nucleus is exposed by rupture of the annulus fibrosus and \n it is a disease that causes chronic back pains5 , 6 .", "chronic back pain patients not only have \n weak deep muscles of the lumbar region and muscle imbalance compared to normal subjects , but \n also show reduction in the re - positioning ability which leads to problem with the stability \n of the spine , due to the reduction in proprioceptive sense , which causes lumbar pain and \n recurrence7 , 8 .", "they include exercises \n for increasing muscle strength , improving functions , and maintaining posture as well as \n preventing excessive movements of the lumbar spine9 .", "exercise rehabilitation approaches for strength and stability are \n important even after surgical treatments , due to degeneration and restricted activities10 , 11 \n and conservative methods of treatment by exercise have been recommended for the purpose of \n preventing recurrence of pain and improving functions12,13,14,15,16 .", "thus , exercise therapies based on the stabilization exercises are \n considered to be necessary for preventing chronic pain and reductions in functional \n capabilities by the reduction of functional instability in various of type and the \n improvement of motor functional capabilities of the lumbar joints .", "therefore , this study aims to help adult women with lumbar disc herniation recover muscle \n strength and stability through 8-week lumber extension strength exercise programs , and to \n provide data for constructing an integrated exercise program ."], ["in this study , an 8-week exercise program for lumbar muscle extension strength and \n stabilization was performed by 26 females older than 20 with lumbar disc herniation .", "this \n study was approved by the hankuk university of foreign studies and the institution where it \n was performed , and it complied with the ethical principles of the declaration of helsinki . \n the purposes and process of the study were fully explained to the subjects and their consent \n was obtained before participation .", "the subjects mean  sd was 27.2  4.4  years old their mean  \n sd height was 162.3  5.1  cm and their mean  sd weight was 55.4  7.1  kg .", "the programs was conducted for a total of 8 weeks , 60 minutes a session , twice a week , to \n improve strength and stabilize lumbar extension .", "the exercise program was divided into \n warm - up exercises , main exercises and cool - down exercises .", "the warm - up exercises and \n cool - down exercises were conducted focusing on the range of joint motion ( rom ) without pain , \n utilizing cycles and steppers for 10 minutes , alternately . for the purpose of improving the \n lumbar stability and resistance muscles ,", "the main exercises were designed to be individually \n conducted in the form of a circuit training in which the number of repetitions increased \n gradually , utilizing sling and weight exercise equipment . a lumbar extension strength machine ( medx , medx inc . , ocala , fl , usa ) was utilized for the \n evaluation of lumbar extensor strength .", "the maximum static muscular strength of lumbar \n extension was measured at 7 different angles of lumbar flexion : 0 , 12 , 24 , 36 , 48 , 60 , and \n 72 degrees .", "passive tests were conducted to determine whether to limit the range of motion \n before measurement .", "the tests were conducted 12 times at 45 lbs in accordance with the medx \n exercise measurement guidelines .", "the measurement was performed after fixing the pelvis and \n thigh of the subjects on the support .", "then , the motion of the lumbar spine was limited \n during measurement by adjusting the footrest .", "the subjects were asked to increasingly extend \n the lumbar spine by sufficiently considering the limited angular range of motion of the \n joints .", "the measurements were performed in the same manner at every angle by maintaining \n maximum muscle contraction for about 2 seconds .", "for the physical stability \n measurements , the total weight distribution index ( wdi ) was calculated by assessing the \n degree of interaction and coordination of the lower body through body away while maintaining \n a standing posture . for data processing , the statistical program spss ( spss inc . ,", "the mean and standard deviation of the measurements was calculated for all metrics . to \n compare the difference between the pre - exercise and post - exercise state of the 8-week \n exercise program ,", "the paired t - test was used and statistical significance was accepted for \n values of p < 0.05 ."], ["significant differences were observed in lumbar extension strength ( les ) between \n pre - exercise and post - exercise at every angle of lumbar flexion , but no significant \n difference was found in wdi ( % ) ( table \n 1table 1.changes of the les and wdivariableanglepre - exercisepost - exerciseles ( ft - lbs)061.8  33.880.1  31.6**1278.7  33.898.8  33.6**2487.6  36.5116.5  32.7***3698.6  35.9129.7  32.3***48104.7  35.0137.6  30.2***60113.1  36.9146.5  30.5***72122.3  40.1158.7  34.7***wdi ( % ) 5.9  3.35.0", " 2.8values are mean  sd , les : lumbar extension strength , wdi : weight distribution \n index .", "the post - exercise les were found to be very significantly ( p<0.01 ) higher \n than pre - exercise at 0 degree and 12 degree , and extremity significantly ( p<0.001 ) higher \n than pre - exercise at 24 , 36 , 48 , and 72 degrees of lumbar flexion .", "values are mean  sd , les : lumbar extension strength , wdi : weight distribution \n index ."], ["this study attempted to identify the positive impacts that were exerted on extension muscle \n strength and stability by an 8-week extension strength exercise program which was performed \n by 26 females with lumbar disc herniation .", "hnp is caused by the annulus fibrosus that is squeezed into the spinal canal when the \n pulposus nucleus is torn due to a degenerative intervertebral disc .", "it has been reported \n that approximately 29% of the projected and escaped intervertebral discs are associated with \n the pulposus nucleus17 .", "chronic back \n pains resulting from hnp aggravate the instability of the lumbar spine , causing degenerative \n changes , atrophy of muscle strength , and reduce flexibility and joint range of motion due to \n trunk damage and instability18 , 19 .", "lumbar herniated nucleus pulposus does not occur due to \n lumbar pain or disability , but more frequently occurs due to deterioration of the related \n muscles or functions interacting with them adjunctively8 .", "in addition , it is said that the joint range of motion is limited \n due to the loss of muscle strength and flexibility caused by occupational factors , mode of \n action , specific changes in posture , or degenerative disease18 .", "these musculoskeletal diseases can be generally improved by utilizing conservative \n treatments such as exercise9 , 18 .", "rehabilitation exercise therapies for muscular strength \n and stability are important even after surgery10 , \n 16 .", "various types of composite \n exercises and core exercises in addition to exercises for muscular strength and stability \n are currently being utilized8 , 12 .", "these exercise therapies help to stabilize the lumbar spine through lumbar and \n trunk dynamic stabilization and exercise modulation , and increase of muscle strength20 .", "similar to the present study , a study analyzing the impacts of an 8-week functional \n exercise program for lumbar muscle strength , that was performed by 26 females with \n degenerative disc findings , found that there were significant differences between \n pre - exercise and post - exercise15 in \n lumbar muscle extension strength at all 7 angles of lumbar flexion , proving that exercise \n participation develops stability of the muscles around the lumbar spine .", "in addition , a \n study analyzing the effects of decompression therapy by 4-week joint mobilization that was \n performed for patients with lumbar herniated nucleus pulposus , reported improvements in \n joint range of motion in flexion and extension21 , proving its effectiveness . similarly , a study of13 lumbar traction for patients with lumbar \n herniated nucleus pulposus , and a study11 \n of lumbar discectomy and stabilization exercises for patients with lumbar herniated nucleus \n pulposus , both reported improvements in flexion and extension .", "the improvements in functions \n pursuant to improved muscle strength and stability were statistically significant , as were \n the results of the studies cited above .", "therefore , prevention of excessive movement of the \n lumbar spine and trunk , in the patients with chronic back pains or lumbar herniated nucleus \n pulposus can be helpful in their daily life because they secure the joint range of motion , \n thereby improving strength and stability9 .", "thus , the development of muscular strength for stabilization and integrated exercise for \n pain reduction and rehabilitation help to maintain the range of joint motion , muscle \n strength , and balance21,22,23 . for the \n improvement and rehabilitation of inpaired capacity in daily life due to back \n pain - associated injuries experienced by about 80% of the population a higher occurrence \n rate8 , 10 when recurrence is included muscle weakness , and loss15 of balance the development of an \n integrated exercise program is required .", "the above results indicate that improving muscle extension strength to enhance and improve \n the functions the patients with chronic back pain or lumbar herniated nucleus pulposus can \n help improve muscle functions and increase the range of joint motion thereby exerting a \n positive impact on physical stability ."]]}
{"article_id": "PMC4193409", "article_text": ["this article presents statistics on health care utilization , prices , expenses , employment , and work hours , as well as on national economic activity .", "some of these statistics are based on sample surveys conducted monthly or quarterly by government agencies or private organizations , and are available 1 to 3 months after the completion of the period .", "they provide the first glimpse at changes occurring within the general economy and the health care sector . the accompanying tables report selected quarterly statistics for 1990 through the first quarter of 1993 , and the calendar year aggregation of quarterly information in the past 3 years .", ", this calculation permits analysis of quarterly data to focus on the direction and magnitude of changes , without interference introduced by seasonal fluctuations . in the national health accounts , indicators such as these play an important role in the estimation of the latest historical year of health care expenditures .", "information that is more comprehensive tends to lag behind the close of a calendar year by 9 to 12 months or more .", "therefore , we rely extensively on indicators such as these to anticipate and predict changes in health care sector expenditures for the most recent year .", "other indicators help to identify specific reasons ( e.g. , increases in price inflation or declines in utilization ) for expenditure change . in the following sections", ", we will identify important indicators of health care and national economic activity and their sources .", "we then describe what these indicators tell us about general economic and health sector activity during the most recent quarter .", "since 1963 , the american hospital association ( aha ) , in cooperation with member hospitals , has been collecting data on the operation of community hospitals through its national hospital panel survey .", "community hospitals , which comprised more than 80 percent of all hospital facilities in the united states in 1991 , include all non - federal , short - term general , and other special hospitals open to the public .", "they exclude hospital units of institutions ; psychiatric facilities ; tuberculosis , other respiratory , and chronic disease hospitals ; institutions for the mentally retarded ; and alcoholism and chemical dependency hospitals .", "the sample is designed to produce estimates of community hospital indicators by bed size and region ( american hospital association , 1963 - 93 ) . in tables 1 and 2 ,", "statistics covering expenses , utilization , beds , and personnel depict trends in the operation of community hospitals annually from 1990 to 1992 and quarterly for selected quarters from 1990 through 1993 .", "figure 1 shows changes from the same quarter 1 year earlier in various measures of hospital utilization for 1984 through the first quarter of 1993 . for purposes of national health expenditures ( nhe ) ,", "survey statistics on revenues ( not shown on table 1 ) are analyzed in estimating the growth in the largest component of health care costs  community hospital expenditures .", "this one segment of nhe accounted for 33 percent of all health spending in 1991 ( letsch et al . , 1992 ) .", "the survey also identifies important factors influencing expenditure growth patterns , such as changes in the number of beds in operation , number of admissions , length of stay , use of outpatient facilities , and number of surgeries .", "the u.s . bureau of labor statistics ( bls ) collects monthly information on employment for all workers , and earnings and work hours for non - supervisory workers in a sample of 370,000 establishments .", "data are collected through cooperative agreements with state agencies that also use this information to create state and local area statistics .", "the survey is designed to collect industry - specific information on wage and salary jobs in non - agricultural industries .", "it excludes statistics on self - employed persons and on those employed in the military ( u.s . department of labor , 1993a ) .", "approximately 5 percent of the population hold more than one job at any point in time .", "( other surveys that are household - based , such as the current population survey [ cps ] , also record employment . in the cps , however , each person 's employment status is counted only once , either as employed , unemployed , or not in the labor force . ) once each year , monthly establishment - based employment statistics are adjusted to benchmarks created from annual establishment census information , resulting in revisions to previously published employment estimates .", "tables 3 and 4 and figure 2 present statistics on employment , average hourly earnings , and average weekly hours in private ( non - government ) health service establishments .", "similar statistics for the private non - agricultural sector , included on these tables , provide a basis for comparing the economy as a whole with the health sector in employment , earnings , and work hours .", "table 5 summarizes business activity in the health sector and the overall economy by measuring change in the implied non - supervisory work hours and payroll . implied work hours", "are the product of the number of non - supervisory employees and average weekly hours . implied non - supervisory payrolls", "figure 3 shows changes from the same quarter 1 year earlier in non - supervisory payroll for 1984 through the first quarter of 1993 . for purposes of nhe , changes in work hours by industry combined with changes in prices ( discussed in a later section ) can be used to gauge the direction and magnitude of expenditure change in specific industries .", "we use these composite indicators in the estimation of growth in physician and dental expenditures for the most recent period .", "we study the historical relationship of changes in this indicator to changes in expenditures , and estimate this relationship for the most recent period .", "bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services .", "tables 6 and 7 and figure 4 present information on the consumer price index ( cpi ) for all urban consumers that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states .", "( the more restrictive wage earner cpi gauges prices faced by wage earners and clerical workers", ". these workers account for 32 percent of the non - institutionalized population [ u.s .", "the index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . for most items , the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products .", "those shares or weights remain constant in all years , even though consumption patterns of the household may change over time .", "cpis for health care goods and services depict price changes for out - of - pocket expenditures made directly by consumers .", "the composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items .", "for example , the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out - of - pocket by consumers ; the remaining 97 percent of the costs of hospital care paid by private health insurers , medicare , medicaid , and other payers are not weighted into the cpi for medical care .", "in addition , some medical care sector indexes measure changes in list or charged prices , rather than in the prices actually received by providers after discounts are deducted . in several health care areas , received or transaction prices are difficult to capture , although bls is making advances in this area . in the nhe , a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by the health care financing administration ( hcfa ) to provide transaction price changes , are used as measures of inflation for the health industry .", "the indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index ( letsch , 1993 ) . in 1979", ", hcfa developed the medicare hospital input price index ( hospital market basket ) , which was designed to measure the pure price changes associated with expenditure changes for hospital services . in the early 1980s ,", "the skilled nursing facility ( snf ) and home health agency ( hha ) input price indexes , often referred to as  market baskets ,  were developed to price a consistent set of goods and services over time .", "also , in the early 1980s , the original medicare hospital input price index was revised for use in updating payment rates for the prospective payment system ( pps ) .", "all of these indexes have played an important role in helping to set medicare payment percent increases , and in understanding the contribution of input price increases to growing health expenditures .", "the input price indexes , or market baskets , are laspeyres or fixed - weight indexes that are constructed in two steps .", "for example , for the pps hospital input price index , the base period is 1987 .", "cost categories , such as food , fuel , and labor , are identified and their 1987 expenditure amounts determined . the proportion or share of total expenditures included in specific spending categories", "second , a price proxy is selected to match each expenditure category : its purpose is to measure the rate of price increase of the goods or services in that category .", "the price proxy index for each spending category is multiplied by the expenditure weight for that category .", "the sum of these products ( weights multiplied by the price index ) over all cost categories yields the composite input price index for any given time period , usually a fiscal year or a calendar year .", "the percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider .", "the input price indexes are estimated on a historical basis and forecasted over several years .", "the hcfa - chosen price proxies are forecasted under contract with data resources , inc./mcgraw - hill ( dri ) . following every calendar year quarter , in march , june , september , and december", ", dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions .", "some of the data in tables 8 through 13 are forecasted , and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received .", "the methodology and price proxy definitions used in the input price indexes are described in the federal register notices that accompany the revisions of the pps , hha , and snf cost limits .", "a description of the current structure of the pps input price index was published in the september 4 , 1990 federal register .", "the most recent pps update for payment rates was published in the september 1 , 1992 federal register .", "the latest hha regulatory input price index was published in the july 1 , 1992 federal register , and the latest snf input price index was published in the october 7 , 1992 federal register .", "periodically , the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased .", "each revision allows for new base weights , a new base year , and changes to certain price variables used for price proxies .", "each input price index is presented in two tables : the first is a percent - change table , and the second provides the actual index numbers from which the percentages were computed .", "figure 5 shows a comparison in the growth of the pps input price index with the cpi all items .", "bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services .", "tables 6 and 7 and figure 4 present information on the consumer price index ( cpi ) for all urban consumers that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states .", "( the more restrictive wage earner cpi gauges prices faced by wage earners and clerical workers", ". these workers account for 32 percent of the non - institutionalized population [ u.s .", "the index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . for most items , the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products .", "those shares or weights remain constant in all years , even though consumption patterns of the household may change over time .", "cpis for health care goods and services depict price changes for out - of - pocket expenditures made directly by consumers .", "the composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items .", "for example , the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out - of - pocket by consumers ; the remaining 97 percent of the costs of hospital care paid by private health insurers , medicare , medicaid , and other payers are not weighted into the cpi for medical care .", "in addition , some medical care sector indexes measure changes in list or charged prices , rather than in the prices actually received by providers after discounts are deducted . in several health care areas , received or transaction prices are difficult to capture , although bls is making advances in this area . in the nhe , a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by the health care financing administration ( hcfa ) to provide transaction price changes , are used as measures of inflation for the health industry .", "the indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index ( letsch , 1993 ) .", "in 1979 , hcfa developed the medicare hospital input price index ( hospital market basket ) , which was designed to measure the pure price changes associated with expenditure changes for hospital services . in the early 1980s ,", "the skilled nursing facility ( snf ) and home health agency ( hha ) input price indexes , often referred to as  market baskets ,  were developed to price a consistent set of goods and services over time .", "also , in the early 1980s , the original medicare hospital input price index was revised for use in updating payment rates for the prospective payment system ( pps ) .", "all of these indexes have played an important role in helping to set medicare payment percent increases , and in understanding the contribution of input price increases to growing health expenditures .", "the input price indexes , or market baskets , are laspeyres or fixed - weight indexes that are constructed in two steps .", "for example , for the pps hospital input price index , the base period is 1987 .", "cost categories , such as food , fuel , and labor , are identified and their 1987 expenditure amounts determined .", "second , a price proxy is selected to match each expenditure category : its purpose is to measure the rate of price increase of the goods or services in that category . the price proxy index for each spending category", "the sum of these products ( weights multiplied by the price index ) over all cost categories yields the composite input price index for any given time period , usually a fiscal year or a calendar year .", "the percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider .", "the input price indexes are estimated on a historical basis and forecasted over several years .", "the hcfa - chosen price proxies are forecasted under contract with data resources , inc./mcgraw - hill ( dri ) . following every calendar year quarter , in march , june , september , and december , dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions .", "some of the data in tables 8 through 13 are forecasted , and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received . the methodology and price proxy definitions used in the input price indexes", "are described in the federal register notices that accompany the revisions of the pps , hha , and snf cost limits .", "a description of the current structure of the pps input price index was published in the september 4 , 1990 federal register .", "the most recent pps update for payment rates was published in the september 1 , 1992 federal register .", "the latest hha regulatory input price index was published in the july 1 , 1992 federal register , and the latest snf input price index was published in the october 7 , 1992 federal register .", "periodically , the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased .", "each revision allows for new base weights , a new base year , and changes to certain price variables used for price proxies .", "each input price index is presented in two tables : the first is a percent - change table , and the second provides the actual index numbers from which the percentages were computed .", "figure 5 shows a comparison in the growth of the pps input price index with the cpi all items .", "national economic indicators provide a context for understanding health - specific indicators and how change in the health sector relates to change in the economy as a whole .", "tables 14 and 15 and figure 6 present national indicators of output , employment , and inflation .", "economy as the value of output produced within the geographic boundaries of the united states by u.s . or foreign citizens or companies .", "real  gdp removes the effects of prices from the valuation of goods and services produced , so that the growth of real gdp reflects changes in the  physical  output of the economy ( u.s . department of commerce , 1993 ) .", "growth in operating expenses for community hospitals slowed considerably in the first quarter of 1993 , increasing 7.5 percent .", "the deceleration continued steadily throughout 1992 , but appears to have leveled off in the first quarter of 1993 . all measures of utilization  admissions , inpatient days , surgical operations , and outpatient visits", " grew more slowly or decelerated faster than they had during the first quarter of 1992 at the height of the flu epidemic .", "total employment in the health service sector continued to grow at nearly the same rate as in 1992 . in the first quarter of 1993 , there were 360,900 more health services jobs than in the first quarter of 1992 , an increase of 4.3 percent . for non - supervisory workers ,", "the health service sector , maintaining steady employment growth during 1991 , appeared immune to the recent recession marked by sagging total private sector employment growth .", "in the past three quarters , growth in health services employment also appears to be unaffected by the recent upsurge in employment growth for all private , non - agricultural establishments , as health sector employment growth remains steady ( figure 2 ) .", "implied work hours ( the product of the number of non - supervisory employees and average weekly hours ) in private health service establishments grew at a rate lower than in any quarter since 1987 , 3.7 percent .", "slow growth was caused by the decline in average hours worked per week , rather than a slowing in employment growth .", "this may signal an impending slowdown in employment for the health service sector , as employers initially cut work hours rather than lay off workers when faced with falling demand for services .", "growth for implied payroll ( work hours times average hourly earnings ) in private health service establishments was also at its lowest since 1987 .", "growth in implied payroll for private hospitals has followed this trend as well , and may be one reason for the deceleration in operating - expense growth seen in the aha panel survey ( as previously noted ) .", "medical prices , as measured by the cpi for medical care , continued to increase more rapidly than prices for all other items . in the first quarter of 1993 , prices for medical care increased 6.3 percent from the first quarter of 1992 , more than two times the 2.9-percent growth for all other items .", "however , the gap between price increases for medical care and all other items has gotten smaller than what it was throughout 1992 ( figure 4 ) . although growth for nearly all components of the medical care cpi has decelerated , the most dramatic slowdown in growth occurred with the prices of prescription drugs . in calendar year 1991 , the cpi for prescription drugs increased 9.9 percent . in 1992", "first , there has been a price war among retail establishments selling prescription drugs . as the number of non - traditional types of retail outlets has grown , competition against the traditional retail pharmaceutical outlets increased .", "mass - merchandisers , such as walmart , are competing for the business of prescription drug customers .", "secondly , recent proposals on health care reform have pharmaceutical manufacturers concerned about the free - market privileges from which they have historically benefitted .", "conceivably , manufacturers and wholesalers have been voluntarily holding pricing down in an effort to avoid being faced with less desirable , mandatory price controls . evidence of the behavior of drug manufacturers can be found in the producer price index ( ppi ) for pharmaceuticals , which measures average changes in prices received by domestic producers of commodities in all stages of processing .", "growth in the ppi for pharmaceuticals decelerated at nearly the same rate as the cpi for prescription drugs . in calendar year 1991 , the ppi for pharmaceuticals increased 8.4 percent . by the first quarter of 1993 , growth in this ppi slowed to 4.6 percent .", "in the first quarter of 1993 , overall economic conditions continued to improve modestly , as shown by the eighth straight quarter of positive growth in real gdp .", "the unemployment rate ( 7.0 percent ) was lower than it had been since the fourth quarter of 1991 .", "total employment in private non - agricultural establishments increased 1.3 percent , which is the largest increase experienced in nearly 3 years .", "growth in operating expenses for community hospitals slowed considerably in the first quarter of 1993 , increasing 7.5 percent .", "the deceleration continued steadily throughout 1992 , but appears to have leveled off in the first quarter of 1993 . all measures of utilization  admissions , inpatient days , surgical operations , and outpatient visits", " grew more slowly or decelerated faster than they had during the first quarter of 1992 at the height of the flu epidemic .", "total employment in the health service sector continued to grow at nearly the same rate as in 1992 . in the first quarter of 1993 , there were 360,900 more health services jobs than in the first quarter of 1992 , an increase of 4.3 percent . for non - supervisory workers ,", "the health service sector , maintaining steady employment growth during 1991 , appeared immune to the recent recession marked by sagging total private sector employment growth . in the past three quarters , growth in health services employment also appears to be unaffected by the recent upsurge in employment growth for all private , non - agricultural establishments , as health sector employment growth remains steady ( figure 2 ) . implied work hours ( the product of the number of non - supervisory employees and average weekly hours ) in private health service establishments grew at a rate lower than in any quarter since 1987 , 3.7 percent .", "slow growth was caused by the decline in average hours worked per week , rather than a slowing in employment growth .", "this may signal an impending slowdown in employment for the health service sector , as employers initially cut work hours rather than lay off workers when faced with falling demand for services . growth for implied payroll ( work hours times average hourly earnings ) in private health service establishments was also at its lowest since 1987 .", "growth in implied payroll for private hospitals has followed this trend as well , and may be one reason for the deceleration in operating - expense growth seen in the aha panel survey ( as previously noted ) .", "medical prices , as measured by the cpi for medical care , continued to increase more rapidly than prices for all other items . in the first quarter of 1993 , prices for medical care increased 6.3 percent from the first quarter of 1992 , more than two times the 2.9-percent growth for all other items .", "however , the gap between price increases for medical care and all other items has gotten smaller than what it was throughout 1992 ( figure 4 ) . although growth for nearly all components of the medical care cpi has decelerated , the most dramatic slowdown in growth occurred with the prices of prescription drugs . in calendar year 1991 , the cpi for prescription drugs increased 9.9 percent . in 1992", "this growth decelerated to 7.6 percent . by the first quarter of 1993 , it slowed further to only 5.0 percent .", "as the number of non - traditional types of retail outlets has grown , competition against the traditional retail pharmaceutical outlets increased .", "mass - merchandisers , such as walmart , are competing for the business of prescription drug customers .", "secondly , recent proposals on health care reform have pharmaceutical manufacturers concerned about the free - market privileges from which they have historically benefitted .", "conceivably , manufacturers and wholesalers have been voluntarily holding pricing down in an effort to avoid being faced with less desirable , mandatory price controls .", "evidence of the behavior of drug manufacturers can be found in the producer price index ( ppi ) for pharmaceuticals , which measures average changes in prices received by domestic producers of commodities in all stages of processing .", "growth in the ppi for pharmaceuticals decelerated at nearly the same rate as the cpi for prescription drugs . in calendar year 1991 , the ppi for pharmaceuticals increased 8.4 percent . by the first quarter of 1993 ,", ", overall economic conditions continued to improve modestly , as shown by the eighth straight quarter of positive growth in real gdp .", "the unemployment rate ( 7.0 percent ) was lower than it had been since the fourth quarter of 1991 .", "total employment in private non - agricultural establishments increased 1.3 percent , which is the largest increase experienced in nearly 3 years ."], "abstract_text": ["<S> this regular feature of the journal includes a discussion of each of the following four topics : community hospital statistics ; employment , hours , and earnings in the private health sector ; health care prices ; and national economic indicators . </S>", "<S> these statistics are valuable in their own right for understanding the relationship between the health care sector and the overall economy . in addition </S>", "<S> , they allow us to anticipate the direction and magnitude of health care cost changes prior to the availability of more comprehensive data . </S>"], "labels": null, "section_names": ["Introduction", "Community Hospital Statistics", "Private Health Sector: Employment, Hours, and Earnings", "Prices", "Consumer Prices", "Background on Input Price Indexes", "National Economic Indicators", "First Quarter Indicators"], "sections": [["this article presents statistics on health care utilization , prices , expenses , employment , and work hours , as well as on national economic activity .", "some of these statistics are based on sample surveys conducted monthly or quarterly by government agencies or private organizations , and are available 1 to 3 months after the completion of the period .", "they provide the first glimpse at changes occurring within the general economy and the health care sector . the accompanying tables report selected quarterly statistics for 1990 through the first quarter of 1993 , and the calendar year aggregation of quarterly information in the past 3 years .", ", this calculation permits analysis of quarterly data to focus on the direction and magnitude of changes , without interference introduced by seasonal fluctuations . in the national health accounts , indicators such as these play an important role in the estimation of the latest historical year of health care expenditures .", "information that is more comprehensive tends to lag behind the close of a calendar year by 9 to 12 months or more .", "therefore , we rely extensively on indicators such as these to anticipate and predict changes in health care sector expenditures for the most recent year .", "other indicators help to identify specific reasons ( e.g. , increases in price inflation or declines in utilization ) for expenditure change . in the following sections", ", we will identify important indicators of health care and national economic activity and their sources .", "we then describe what these indicators tell us about general economic and health sector activity during the most recent quarter ."], ["since 1963 , the american hospital association ( aha ) , in cooperation with member hospitals , has been collecting data on the operation of community hospitals through its national hospital panel survey .", "community hospitals , which comprised more than 80 percent of all hospital facilities in the united states in 1991 , include all non - federal , short - term general , and other special hospitals open to the public .", "they exclude hospital units of institutions ; psychiatric facilities ; tuberculosis , other respiratory , and chronic disease hospitals ; institutions for the mentally retarded ; and alcoholism and chemical dependency hospitals .", "the sample is designed to produce estimates of community hospital indicators by bed size and region ( american hospital association , 1963 - 93 ) . in tables 1 and 2 ,", "statistics covering expenses , utilization , beds , and personnel depict trends in the operation of community hospitals annually from 1990 to 1992 and quarterly for selected quarters from 1990 through 1993 .", "figure 1 shows changes from the same quarter 1 year earlier in various measures of hospital utilization for 1984 through the first quarter of 1993 . for purposes of national health expenditures ( nhe ) ,", "survey statistics on revenues ( not shown on table 1 ) are analyzed in estimating the growth in the largest component of health care costs  community hospital expenditures .", "this one segment of nhe accounted for 33 percent of all health spending in 1991 ( letsch et al . , 1992 ) .", "the survey also identifies important factors influencing expenditure growth patterns , such as changes in the number of beds in operation , number of admissions , length of stay , use of outpatient facilities , and number of surgeries ."], ["the u.s . bureau of labor statistics ( bls ) collects monthly information on employment for all workers , and earnings and work hours for non - supervisory workers in a sample of 370,000 establishments .", "data are collected through cooperative agreements with state agencies that also use this information to create state and local area statistics .", "the survey is designed to collect industry - specific information on wage and salary jobs in non - agricultural industries .", "it excludes statistics on self - employed persons and on those employed in the military ( u.s . department of labor , 1993a ) .", "approximately 5 percent of the population hold more than one job at any point in time .", "( other surveys that are household - based , such as the current population survey [ cps ] , also record employment . in the cps , however , each person 's employment status is counted only once , either as employed , unemployed , or not in the labor force . ) once each year , monthly establishment - based employment statistics are adjusted to benchmarks created from annual establishment census information , resulting in revisions to previously published employment estimates .", "tables 3 and 4 and figure 2 present statistics on employment , average hourly earnings , and average weekly hours in private ( non - government ) health service establishments .", "similar statistics for the private non - agricultural sector , included on these tables , provide a basis for comparing the economy as a whole with the health sector in employment , earnings , and work hours .", "table 5 summarizes business activity in the health sector and the overall economy by measuring change in the implied non - supervisory work hours and payroll . implied work hours", "are the product of the number of non - supervisory employees and average weekly hours . implied non - supervisory payrolls", "figure 3 shows changes from the same quarter 1 year earlier in non - supervisory payroll for 1984 through the first quarter of 1993 . for purposes of nhe , changes in work hours by industry combined with changes in prices ( discussed in a later section ) can be used to gauge the direction and magnitude of expenditure change in specific industries .", "we use these composite indicators in the estimation of growth in physician and dental expenditures for the most recent period .", "we study the historical relationship of changes in this indicator to changes in expenditures , and estimate this relationship for the most recent period ."], ["bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services .", "tables 6 and 7 and figure 4 present information on the consumer price index ( cpi ) for all urban consumers that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states .", "( the more restrictive wage earner cpi gauges prices faced by wage earners and clerical workers", ". these workers account for 32 percent of the non - institutionalized population [ u.s .", "the index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . for most items , the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products .", "those shares or weights remain constant in all years , even though consumption patterns of the household may change over time .", "cpis for health care goods and services depict price changes for out - of - pocket expenditures made directly by consumers .", "the composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items .", "for example , the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out - of - pocket by consumers ; the remaining 97 percent of the costs of hospital care paid by private health insurers , medicare , medicaid , and other payers are not weighted into the cpi for medical care .", "in addition , some medical care sector indexes measure changes in list or charged prices , rather than in the prices actually received by providers after discounts are deducted . in several health care areas , received or transaction prices are difficult to capture , although bls is making advances in this area . in the nhe , a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by the health care financing administration ( hcfa ) to provide transaction price changes , are used as measures of inflation for the health industry .", "the indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index ( letsch , 1993 ) . in 1979", ", hcfa developed the medicare hospital input price index ( hospital market basket ) , which was designed to measure the pure price changes associated with expenditure changes for hospital services . in the early 1980s ,", "the skilled nursing facility ( snf ) and home health agency ( hha ) input price indexes , often referred to as  market baskets ,  were developed to price a consistent set of goods and services over time .", "also , in the early 1980s , the original medicare hospital input price index was revised for use in updating payment rates for the prospective payment system ( pps ) .", "all of these indexes have played an important role in helping to set medicare payment percent increases , and in understanding the contribution of input price increases to growing health expenditures .", "the input price indexes , or market baskets , are laspeyres or fixed - weight indexes that are constructed in two steps .", "for example , for the pps hospital input price index , the base period is 1987 .", "cost categories , such as food , fuel , and labor , are identified and their 1987 expenditure amounts determined . the proportion or share of total expenditures included in specific spending categories", "second , a price proxy is selected to match each expenditure category : its purpose is to measure the rate of price increase of the goods or services in that category .", "the price proxy index for each spending category is multiplied by the expenditure weight for that category .", "the sum of these products ( weights multiplied by the price index ) over all cost categories yields the composite input price index for any given time period , usually a fiscal year or a calendar year .", "the percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider .", "the input price indexes are estimated on a historical basis and forecasted over several years .", "the hcfa - chosen price proxies are forecasted under contract with data resources , inc./mcgraw - hill ( dri ) . following every calendar year quarter , in march , june , september , and december", ", dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions .", "some of the data in tables 8 through 13 are forecasted , and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received .", "the methodology and price proxy definitions used in the input price indexes are described in the federal register notices that accompany the revisions of the pps , hha , and snf cost limits .", "a description of the current structure of the pps input price index was published in the september 4 , 1990 federal register .", "the most recent pps update for payment rates was published in the september 1 , 1992 federal register .", "the latest hha regulatory input price index was published in the july 1 , 1992 federal register , and the latest snf input price index was published in the october 7 , 1992 federal register .", "periodically , the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased .", "each revision allows for new base weights , a new base year , and changes to certain price variables used for price proxies .", "each input price index is presented in two tables : the first is a percent - change table , and the second provides the actual index numbers from which the percentages were computed .", "figure 5 shows a comparison in the growth of the pps input price index with the cpi all items ."], ["bls publishes monthly information on changes in prices paid by consumers for a fixed market basket of goods and services .", "tables 6 and 7 and figure 4 present information on the consumer price index ( cpi ) for all urban consumers that measures changes in prices faced by 80 percent of the non - institutionalized population in the united states .", "( the more restrictive wage earner cpi gauges prices faced by wage earners and clerical workers", ". these workers account for 32 percent of the non - institutionalized population [ u.s .", "the index reflects changes in prices charged for the same quality and quantity of goods or services purchased in the base period . for most items , the base period of 1982 - 84 is used to define the share of consumer expenditures purchasing specific services and products .", "those shares or weights remain constant in all years , even though consumption patterns of the household may change over time .", "cpis for health care goods and services depict price changes for out - of - pocket expenditures made directly by consumers .", "the composite cpi for medical care weights together product - specific or service - specific cpis in proportion to household out - of - pocket expenditures for these items .", "for example , the composite medical care cpi measures inflation for the 3 percent of hospital expenditures that are made out - of - pocket by consumers ; the remaining 97 percent of the costs of hospital care paid by private health insurers , medicare , medicaid , and other payers are not weighted into the cpi for medical care .", "in addition , some medical care sector indexes measure changes in list or charged prices , rather than in the prices actually received by providers after discounts are deducted . in several health care areas , received or transaction prices are difficult to capture , although bls is making advances in this area . in the nhe , a combination of cpis for selected medical care items , input price indexes for nursing homes , and the cpi for hospital and related services , adjusted by the health care financing administration ( hcfa ) to provide transaction price changes , are used as measures of inflation for the health industry .", "the indexes are used to develop a fixed - weight price index for personal health care to depict price changes affecting the entire health care industry more accurately than does the overall cpi medical care index ( letsch , 1993 ) ."], ["in 1979 , hcfa developed the medicare hospital input price index ( hospital market basket ) , which was designed to measure the pure price changes associated with expenditure changes for hospital services . in the early 1980s ,", "the skilled nursing facility ( snf ) and home health agency ( hha ) input price indexes , often referred to as  market baskets ,  were developed to price a consistent set of goods and services over time .", "also , in the early 1980s , the original medicare hospital input price index was revised for use in updating payment rates for the prospective payment system ( pps ) .", "all of these indexes have played an important role in helping to set medicare payment percent increases , and in understanding the contribution of input price increases to growing health expenditures .", "the input price indexes , or market baskets , are laspeyres or fixed - weight indexes that are constructed in two steps .", "for example , for the pps hospital input price index , the base period is 1987 .", "cost categories , such as food , fuel , and labor , are identified and their 1987 expenditure amounts determined .", "second , a price proxy is selected to match each expenditure category : its purpose is to measure the rate of price increase of the goods or services in that category . the price proxy index for each spending category", "the sum of these products ( weights multiplied by the price index ) over all cost categories yields the composite input price index for any given time period , usually a fiscal year or a calendar year .", "the percent change in the input price index is an estimate of price change over time for a fixed quantity of goods and services purchased by a provider .", "the input price indexes are estimated on a historical basis and forecasted over several years .", "the hcfa - chosen price proxies are forecasted under contract with data resources , inc./mcgraw - hill ( dri ) . following every calendar year quarter , in march , june , september , and december , dri updates its macroeconomic forecasts of wages and prices based on updated historical information and revised forecast assumptions .", "some of the data in tables 8 through 13 are forecasted , and are expected to change as more recent historical data become available and subsequent quarterly forecasts are received . the methodology and price proxy definitions used in the input price indexes", "are described in the federal register notices that accompany the revisions of the pps , hha , and snf cost limits .", "a description of the current structure of the pps input price index was published in the september 4 , 1990 federal register .", "the most recent pps update for payment rates was published in the september 1 , 1992 federal register .", "the latest hha regulatory input price index was published in the july 1 , 1992 federal register , and the latest snf input price index was published in the october 7 , 1992 federal register .", "periodically , the input price indexes are revised to a new base year so that cost weights will reflect changes in the mix of goods and services that are purchased .", "each revision allows for new base weights , a new base year , and changes to certain price variables used for price proxies .", "each input price index is presented in two tables : the first is a percent - change table , and the second provides the actual index numbers from which the percentages were computed .", "figure 5 shows a comparison in the growth of the pps input price index with the cpi all items ."], ["national economic indicators provide a context for understanding health - specific indicators and how change in the health sector relates to change in the economy as a whole .", "tables 14 and 15 and figure 6 present national indicators of output , employment , and inflation .", "economy as the value of output produced within the geographic boundaries of the united states by u.s . or foreign citizens or companies .", "real  gdp removes the effects of prices from the valuation of goods and services produced , so that the growth of real gdp reflects changes in the  physical  output of the economy ( u.s . department of commerce , 1993 ) .", "growth in operating expenses for community hospitals slowed considerably in the first quarter of 1993 , increasing 7.5 percent .", "the deceleration continued steadily throughout 1992 , but appears to have leveled off in the first quarter of 1993 . all measures of utilization  admissions , inpatient days , surgical operations , and outpatient visits", " grew more slowly or decelerated faster than they had during the first quarter of 1992 at the height of the flu epidemic .", "total employment in the health service sector continued to grow at nearly the same rate as in 1992 . in the first quarter of 1993 , there were 360,900 more health services jobs than in the first quarter of 1992 , an increase of 4.3 percent . for non - supervisory workers ,", "the health service sector , maintaining steady employment growth during 1991 , appeared immune to the recent recession marked by sagging total private sector employment growth .", "in the past three quarters , growth in health services employment also appears to be unaffected by the recent upsurge in employment growth for all private , non - agricultural establishments , as health sector employment growth remains steady ( figure 2 ) .", "implied work hours ( the product of the number of non - supervisory employees and average weekly hours ) in private health service establishments grew at a rate lower than in any quarter since 1987 , 3.7 percent .", "slow growth was caused by the decline in average hours worked per week , rather than a slowing in employment growth .", "this may signal an impending slowdown in employment for the health service sector , as employers initially cut work hours rather than lay off workers when faced with falling demand for services .", "growth for implied payroll ( work hours times average hourly earnings ) in private health service establishments was also at its lowest since 1987 .", "growth in implied payroll for private hospitals has followed this trend as well , and may be one reason for the deceleration in operating - expense growth seen in the aha panel survey ( as previously noted ) .", "medical prices , as measured by the cpi for medical care , continued to increase more rapidly than prices for all other items . in the first quarter of 1993 , prices for medical care increased 6.3 percent from the first quarter of 1992 , more than two times the 2.9-percent growth for all other items .", "however , the gap between price increases for medical care and all other items has gotten smaller than what it was throughout 1992 ( figure 4 ) . although growth for nearly all components of the medical care cpi has decelerated , the most dramatic slowdown in growth occurred with the prices of prescription drugs . in calendar year 1991 , the cpi for prescription drugs increased 9.9 percent . in 1992", "first , there has been a price war among retail establishments selling prescription drugs . as the number of non - traditional types of retail outlets has grown , competition against the traditional retail pharmaceutical outlets increased .", "mass - merchandisers , such as walmart , are competing for the business of prescription drug customers .", "secondly , recent proposals on health care reform have pharmaceutical manufacturers concerned about the free - market privileges from which they have historically benefitted .", "conceivably , manufacturers and wholesalers have been voluntarily holding pricing down in an effort to avoid being faced with less desirable , mandatory price controls . evidence of the behavior of drug manufacturers can be found in the producer price index ( ppi ) for pharmaceuticals , which measures average changes in prices received by domestic producers of commodities in all stages of processing .", "growth in the ppi for pharmaceuticals decelerated at nearly the same rate as the cpi for prescription drugs . in calendar year 1991 , the ppi for pharmaceuticals increased 8.4 percent . by the first quarter of 1993 , growth in this ppi slowed to 4.6 percent .", "in the first quarter of 1993 , overall economic conditions continued to improve modestly , as shown by the eighth straight quarter of positive growth in real gdp .", "the unemployment rate ( 7.0 percent ) was lower than it had been since the fourth quarter of 1991 .", "total employment in private non - agricultural establishments increased 1.3 percent , which is the largest increase experienced in nearly 3 years ."], ["growth in operating expenses for community hospitals slowed considerably in the first quarter of 1993 , increasing 7.5 percent .", "the deceleration continued steadily throughout 1992 , but appears to have leveled off in the first quarter of 1993 . all measures of utilization  admissions , inpatient days , surgical operations , and outpatient visits", " grew more slowly or decelerated faster than they had during the first quarter of 1992 at the height of the flu epidemic .", "total employment in the health service sector continued to grow at nearly the same rate as in 1992 . in the first quarter of 1993 , there were 360,900 more health services jobs than in the first quarter of 1992 , an increase of 4.3 percent . for non - supervisory workers ,", "the health service sector , maintaining steady employment growth during 1991 , appeared immune to the recent recession marked by sagging total private sector employment growth . in the past three quarters , growth in health services employment also appears to be unaffected by the recent upsurge in employment growth for all private , non - agricultural establishments , as health sector employment growth remains steady ( figure 2 ) . implied work hours ( the product of the number of non - supervisory employees and average weekly hours ) in private health service establishments grew at a rate lower than in any quarter since 1987 , 3.7 percent .", "slow growth was caused by the decline in average hours worked per week , rather than a slowing in employment growth .", "this may signal an impending slowdown in employment for the health service sector , as employers initially cut work hours rather than lay off workers when faced with falling demand for services . growth for implied payroll ( work hours times average hourly earnings ) in private health service establishments was also at its lowest since 1987 .", "growth in implied payroll for private hospitals has followed this trend as well , and may be one reason for the deceleration in operating - expense growth seen in the aha panel survey ( as previously noted ) .", "medical prices , as measured by the cpi for medical care , continued to increase more rapidly than prices for all other items . in the first quarter of 1993 , prices for medical care increased 6.3 percent from the first quarter of 1992 , more than two times the 2.9-percent growth for all other items .", "however , the gap between price increases for medical care and all other items has gotten smaller than what it was throughout 1992 ( figure 4 ) . although growth for nearly all components of the medical care cpi has decelerated , the most dramatic slowdown in growth occurred with the prices of prescription drugs . in calendar year 1991 , the cpi for prescription drugs increased 9.9 percent . in 1992", "this growth decelerated to 7.6 percent . by the first quarter of 1993 , it slowed further to only 5.0 percent .", "as the number of non - traditional types of retail outlets has grown , competition against the traditional retail pharmaceutical outlets increased .", "mass - merchandisers , such as walmart , are competing for the business of prescription drug customers .", "secondly , recent proposals on health care reform have pharmaceutical manufacturers concerned about the free - market privileges from which they have historically benefitted .", "conceivably , manufacturers and wholesalers have been voluntarily holding pricing down in an effort to avoid being faced with less desirable , mandatory price controls .", "evidence of the behavior of drug manufacturers can be found in the producer price index ( ppi ) for pharmaceuticals , which measures average changes in prices received by domestic producers of commodities in all stages of processing .", "growth in the ppi for pharmaceuticals decelerated at nearly the same rate as the cpi for prescription drugs . in calendar year 1991 , the ppi for pharmaceuticals increased 8.4 percent . by the first quarter of 1993 ,", ", overall economic conditions continued to improve modestly , as shown by the eighth straight quarter of positive growth in real gdp .", "the unemployment rate ( 7.0 percent ) was lower than it had been since the fourth quarter of 1991 .", "total employment in private non - agricultural establishments increased 1.3 percent , which is the largest increase experienced in nearly 3 years ."]]}
{"article_id": "PMC3507402", "article_text": ["urinary retention and voiding dysfunction ( vd ) symptoms ( hesitancy , straining to void , difficulty in starting micturition , diminished stream , and sensations of incomplete emptying of the bladder ) are common and problematic features occurring after surgery for stress urinary incontinence ( sui ) , especially following a mid - urethral sling placement ( retropubic or trans - obturator procedure ) .", "they can lead to urinary infections , and can also require self - catheterization or sling section .", "the prevalence of urinary retention varies from 2.216% after surgery for sui.[13 ] preexisting voiding dysfunction is known to be a predictive factor for post - surgery urinary retention . in 2003 hong et al .", ", showed that the best predictive factor for obstructive complications was the maximum flow rate , with which it was shown to be directly correlated .", "it is thus important to screen women suffering from sui for emptying symptoms before such surgery .", "the purpose of this study was to determine whether the completion of a vd questionnaire could have a good predictive value for uroflowmetry findings , in a population of sui women showing no obvious etiologies of obstruction .", "we analyzed 415 sui women from the urodynamic database , who had undergone a filling cystometry in the department , and whose uroflowmetry indicated a total urine volume greater than 200 ml .", "women with neurological disorders , pelvic organ prolapse , urge or mixed urinary incontinence , detrusor overactivity or previous sui surgery were excluded . with these exclusion criteria ,", "these 93 women were divided into two groups : those who complained of vd symptoms ( vds group ) according to the bristol female lower urinary tract symptoms ( bfluts ) questionnaire , and those without vd symptoms ( non - vds group ) .", "we investigated the parity , age , menopausal status , and concomitant anorectal disorders of each woman .", "the women were interviewed on the presence of emptying symptoms , and completed the bfluts questionnaire [ table 1 ] , which is systematic in our department .", "we screened for five items related to voiding difficulties : hesitancy , slow or intermittent stream , straining , or sensations of incomplete emptying .", "for the purposes of data analysis , those women who answered  never  to all five items were classed in the non - vds group .", "those women for whom one or more of the answers was positive ( i.e. :  occasionally , sometimes , most of the time or all the time  ) , were classified in the vds group .", "bristol questionnarire bfluts ( urinary symptoms questionnaire ) : the five items concerning voiding dysfunction the clinical examination included a physical examination , urodynamic testing and a  free flow ", "uroflowmetry and filling cystometry at a rate of 50 ml per minute ( duet , medtronic ) .", "the filling cystometry parameters studied were : compliance , detrusor overactivity ( do ) , detrusor sensation .", "none of the patients presented with abnormal compliance , do , or abnormal detrusor sensation .", "both a quantitative and a qualitative analysis of bladder outlet obstruction ( boo ) was made by uroflowmetry .", "quantitatively , a patient was considered to have boo when a maximum flow rate of less than 15 ml / sec was observed for a urine volume greater than 200 ml , and/or the post - void residual was greater than 50 ml , as measured with a bladder scan or by catheterization .", "the urine flow was also analyzed in terms of average flow rate , urine volume , voiding time and acceleration ( defined as the maximum flow rate divided by the time taken to reach maximum flow rate ) .", "the qualitative analysis dealt with the flow patterns : a standard  bell - shaped ", "three pathologic patterns were considered : intermittent or continuous polyphasic curves , and flat curves [ figures 1a  c ] .", "we determined the sensitivity and specificity as well as the positive and negative predictive value of the vds questionnaire using the uroflowmetry data .", "( a ) intermittent polyphasic curve ( b ) continuous polyphasic curve ( c ) flat curve a comparison of the clinical characteristics and uroflowmetry of the two groups was made using a wilcoxon test for continuous data and a fischer test for categorical data .", "as the bfluts questionnaire and urodynamics were identical to those systematically used for the routine evaluation of sui women in our department , the study did not require specific ethics committee approbation .", "we analyzed 415 sui women from the urodynamic database , who had undergone a filling cystometry in the department , and whose uroflowmetry indicated a total urine volume greater than 200 ml .", "women with neurological disorders , pelvic organ prolapse , urge or mixed urinary incontinence , detrusor overactivity or previous sui surgery were excluded . with these exclusion criteria ,", "these 93 women were divided into two groups : those who complained of vd symptoms ( vds group ) according to the bristol female lower urinary tract symptoms ( bfluts ) questionnaire , and those without vd symptoms ( non - vds group ) .", "we investigated the parity , age , menopausal status , and concomitant anorectal disorders of each woman .", "the women were interviewed on the presence of emptying symptoms , and completed the bfluts questionnaire [ table 1 ] , which is systematic in our department .", "we screened for five items related to voiding difficulties : hesitancy , slow or intermittent stream , straining , or sensations of incomplete emptying . for the purposes of data analysis , those women who answered  never  to all five items were classed in the non - vds group .", "those women for whom one or more of the answers was positive ( i.e. :  occasionally , sometimes , most of the time or all the time  ) , were classified in the vds group .", "the clinical examination included a physical examination , urodynamic testing and a  free flow  uroflowmetry and filling cystometry at a rate of 50 ml per minute ( duet , medtronic ) .", "the filling cystometry parameters studied were : compliance , detrusor overactivity ( do ) , detrusor sensation .", "none of the patients presented with abnormal compliance , do , or abnormal detrusor sensation .", "both a quantitative and a qualitative analysis of bladder outlet obstruction ( boo ) was made by uroflowmetry .", "quantitatively , a patient was considered to have boo when a maximum flow rate of less than 15 ml / sec was observed for a urine volume greater than 200 ml , and/or the post - void residual was greater than 50 ml , as measured with a bladder scan or by catheterization .", "the urine flow was also analyzed in terms of average flow rate , urine volume , voiding time and acceleration ( defined as the maximum flow rate divided by the time taken to reach maximum flow rate ) .", "the qualitative analysis dealt with the flow patterns : a standard  bell - shaped ", "three pathologic patterns were considered : intermittent or continuous polyphasic curves , and flat curves [ figures 1a  c ] .", "we determined the sensitivity and specificity as well as the positive and negative predictive value of the vds questionnaire using the uroflowmetry data .", "( a ) intermittent polyphasic curve ( b ) continuous polyphasic curve ( c ) flat curve", "a comparison of the clinical characteristics and uroflowmetry of the two groups was made using a wilcoxon test for continuous data and a fischer test for categorical data .", "as the bfluts questionnaire and urodynamics were identical to those systematically used for the routine evaluation of sui women in our department , the study did not require specific ethics committee approbation .", "among the 93 women eligible for the study , 61% ( 57/93 ) complained of vd symptoms on the bfluts questionnaire ( vds group ) and 39% ( 36/93 ) had no vd symptoms ( non - vds group ) .", "patients characteristics are detailed in table 2 . no difference was observed between the two groups concerning age , parity , menopausal status , concomitant anorectal disorders and uroflowmetry parameters . in the vds group ,", "75% ( 27/36 ) of the women in the non - vds group had normal uroflowmetry , and 25% ( 9/36 ) an abnormal one without symptoms .", "the positive predictive value of the vds questionnaire was 32% and the negative predictive value was 75% [ table 3 ] .", "there was no statistical correlation between symptoms and the defined criteria of boo on uroflowmetry ( fischer test : p=0.64 ) . among the 42 women who complained of a sensation of incomplete", "emptying , only one had a real post - void residual greater than 50 ml . in this", "sui population without severe pelvic organ prolapse ( baden and walker staging system 1 ) , previous sui surgery , or neurological disorders ( the most frequent etiologies of voiding dysfunction in women ) we found a 29% ( 29/93 ) rate of abnormal uroflowmetry . characteristics of women with and without voiding difficulties symptoms in terms of age , menopausal status , parity , anorectal disorders and uroflowmetry parameters sensitivity , specificity , positive and negative predictive value of voiding dysfunction symptoms on bfluts multivariate analysis ( using multiple logistic regression method ) demonstrated no correlation between symptoms and quantitative uroflowmetry parameters ( maximum and average flow rate , acceleration , urine volume , time to maximum flow and voiding time ) , and no correlation was found between vds and age , menopausal status , parity or concomitant anorectal disorders [ table 4 ] . logistic regression method for multivariate analysis of urodynamic parameters ( maximal and average flow rate , time to void and acceleration ) , age , menopausal status , parity , concomitant anorectal disorders with respect to voiding difficulties symptoms univariate analysis of multiple factors with respect to voiding difficulties symptoms", "our results show that 25% of sui women without obstructive symptoms , i.e. without an obvious cause for bladder outlet obstruction ( no severe genital prolapse ( > grade 1 ) ) , having had no previous surgery for sui , and with no neurological disorders , have an abnormal uroflowmetry , and thus a higher risk of post - surgery obstructive disorders .", "our study clearly demonstrates the poor predictive value of the vds questionnaire , when compared with urodynamic data .", "the prevalence of voiding dysfunction symptoms among women is not easy to determine , in contrast to storage symptoms , which are more often related and analyzed .", "stress urinary incontinence and bladder outlet obstruction ( boo ) can coexist in women ; bradley found 19 cases of boo out of 104 ( 18% ) , defined on the basis of pressure flow studies among women presenting with sui . in our study , we found a high prevalence of vds ( 61% ) , probably because in the vds group we included women with any level of positive response , even those who gave one  occasionally response to the vd items ( bfluts questionnaire ) .", "urodynamic evaluation of the voiding phase in sui women before surgery is useful in order to predict the risk of vd and urinary retention , and to inform women about these potential complications .", "surgical treatments of sui using mid - urethral sling procedures are known to affect the voiding phase : the spontaneous flow curve changes to a more obstructive pattern in 40% of cases , and the maximum and mean flow rates decrease significantly after one year .", "the incidence of urinary retention after such procedures ranges from 2.216%.[13 ] postoperative vd induces a decrease in global satisfaction after surgery , and leads to an impairment of the quality of life .", "factors which correlate highly with a postoperative voiding dysfunction include a preoperative peak flow rate < 15 ml / s ( several studies ) , and an abnormal uroflow pattern or detrusor pressure < 12 cm h2o.[31114 ] therefore , screening for such factors among sui women is critical .", "one of the main limitations of the current study is the lack of a standard definition for boo in urodynamics for women .", "since voiding cystometry had not been performed , boo can not be defined urodynamically in the current study .", "farrar et al . , defined obstruction as qmax < 15 ml / s with a voided volume of 200 ml .", "we added the flow curve pattern criteria , because a pathological pattern with a normal maximal flow rate can reflect voiding difficulties .", "moreover , in pressure flow studies in women , the transurethral catheter has a significant impact on the urine flow.[1418 ] however ,  free flow ", "uroflowmetry is a composite interaction between the detrusor pressure and the resistance produced by the urethra .", "bladder outlet obstruction as well as impaired detrusor contractility can perturb uroflowmetry . in our population", ", we needed to detect voiding difficulties , whatever their origin , because among sui women both these causes of voiding disorders can promote obstructive complications after surgery .", "chassagne et al . , prospectively studied 35 clinically obstructed women and 124 control patients .", "they determined cutoff values for the pressure flow studies as : qmax<15 ml / s and pdet .", "qmax > 20 cm h2o , with a sensitivity of 74.3% and a specificity of 91.1% .", "no information was provided regarding the clinical criteria used to select the  clinically obstructed  patients .", "qmax > 20 cm h2o ) during detrusor pressure uroflow studies . among the 38 women considered as obstructed within this definition ,", "they concluded that micturition symptoms relevant to bladder outlet obstruction are non - specific , and that a complete urodynamic evaluation is essential .", "the correlation between obstructive symptoms and objective urodynamic findings is known to be poor , but has never been studied in this specific population of sui women , for which all cases with a pop - q > stage 1 , previous surgery for sui , or neurological disorders , were excluded . indeed , we excluded the most common etiologies of urodynamic assessment of bladder outlet obstruction", ". groutz et al . , reported 26% of prior anti - incontinence surgery and 24% of severe genital prolapse in a population of 38 women presenting with urodynamically assessed obstructive criteria .", "wyndaele et al . , recently confirmed the poor correlation between symptoms and urodynamic data , and a high prevalence of vds in women , whether it be considered to have a pathological origin or not .", "the absence of voiding symptoms is not a guarantee of normal micturition , since we found that almost one - third of the women not complaining of any voiding difficulty were actually shown to have an abnormal uroflowmetry .", "previous studies[1421 ] have clearly demonstrated the lack of correlation between symptoms and urodynamic diagnoses , especially for obstructive symptoms .", "we found the same results , but in a particular population of sui women , with no obvious reasons for obstruction . even in this selected population ,", "in which voiding dysfunctions should be less frequent than in the general population , we found that almost 30% of the patients had an abnormal uroflowmetry .", "no correlation was found between obstructive symptoms and boo as defined on uroflowmetry , in a specific population of sui women ."], "abstract_text": ["<S> objectives : to determine whether the completion of a voiding dysfunction ( vd ) questionnaire could have a good predictive value for uroflowmetry findings , in a population of stress urinary incontinence ( sui ) women.materials and methods : from a urodynamic database of 415 sui women , 93 with isolated sui who underwent urodynamic investigations were eligible for this study . </S>", "<S> patients with obvious etiologies of obstruction were excluded . </S>", "<S> vd symptoms were analyzed using the bristol female lower urinary tract symptoms questionnaire . </S>", "<S> bladder outlet obstruction ( boo ) was defined as a maximal flow rate under 15 ml / s for a urine volume > 200 ml , or a post - void residual volume greater than 50 ml , or an abnormal pattern of the flow curve . </S>", "<S> the sensitivity , specificity , positive and negative predictive value of questioning vd were calculated . </S>", "<S> statistical analysis was done using a wilcoxon test for continuous data and fisher exact test for categorical data , and multivariate analysis.results:reported vd had a poor specificity ( 41% ) and positive predictive value ( 32% ) of boo on uroflowmetry . </S>", "<S> no statistical correlation was found between vd symptoms and boo defined on uroflowmetry ( p=0.64 ) in this specific sui population showing no obvious etiologies of obstruction.conclusions:no correlation was found between obstructive symptoms and boo as defined on uroflowmetry , in a specific population of sui women . </S>", "<S> our results suggest that uroflowmetry may be necessary rather than multichannel urodynamics . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Population", "Questionnaire", "Urodynamics", "Statistical analysis", "Ethics", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["urinary retention and voiding dysfunction ( vd ) symptoms ( hesitancy , straining to void , difficulty in starting micturition , diminished stream , and sensations of incomplete emptying of the bladder ) are common and problematic features occurring after surgery for stress urinary incontinence ( sui ) , especially following a mid - urethral sling placement ( retropubic or trans - obturator procedure ) .", "they can lead to urinary infections , and can also require self - catheterization or sling section .", "the prevalence of urinary retention varies from 2.216% after surgery for sui.[13 ] preexisting voiding dysfunction is known to be a predictive factor for post - surgery urinary retention . in 2003 hong et al .", ", showed that the best predictive factor for obstructive complications was the maximum flow rate , with which it was shown to be directly correlated .", "it is thus important to screen women suffering from sui for emptying symptoms before such surgery .", "the purpose of this study was to determine whether the completion of a vd questionnaire could have a good predictive value for uroflowmetry findings , in a population of sui women showing no obvious etiologies of obstruction ."], ["we analyzed 415 sui women from the urodynamic database , who had undergone a filling cystometry in the department , and whose uroflowmetry indicated a total urine volume greater than 200 ml .", "women with neurological disorders , pelvic organ prolapse , urge or mixed urinary incontinence , detrusor overactivity or previous sui surgery were excluded . with these exclusion criteria ,", "these 93 women were divided into two groups : those who complained of vd symptoms ( vds group ) according to the bristol female lower urinary tract symptoms ( bfluts ) questionnaire , and those without vd symptoms ( non - vds group ) .", "we investigated the parity , age , menopausal status , and concomitant anorectal disorders of each woman .", "the women were interviewed on the presence of emptying symptoms , and completed the bfluts questionnaire [ table 1 ] , which is systematic in our department .", "we screened for five items related to voiding difficulties : hesitancy , slow or intermittent stream , straining , or sensations of incomplete emptying .", "for the purposes of data analysis , those women who answered  never  to all five items were classed in the non - vds group .", "those women for whom one or more of the answers was positive ( i.e. :  occasionally , sometimes , most of the time or all the time  ) , were classified in the vds group .", "bristol questionnarire bfluts ( urinary symptoms questionnaire ) : the five items concerning voiding dysfunction the clinical examination included a physical examination , urodynamic testing and a  free flow ", "uroflowmetry and filling cystometry at a rate of 50 ml per minute ( duet , medtronic ) .", "the filling cystometry parameters studied were : compliance , detrusor overactivity ( do ) , detrusor sensation .", "none of the patients presented with abnormal compliance , do , or abnormal detrusor sensation .", "both a quantitative and a qualitative analysis of bladder outlet obstruction ( boo ) was made by uroflowmetry .", "quantitatively , a patient was considered to have boo when a maximum flow rate of less than 15 ml / sec was observed for a urine volume greater than 200 ml , and/or the post - void residual was greater than 50 ml , as measured with a bladder scan or by catheterization .", "the urine flow was also analyzed in terms of average flow rate , urine volume , voiding time and acceleration ( defined as the maximum flow rate divided by the time taken to reach maximum flow rate ) .", "the qualitative analysis dealt with the flow patterns : a standard  bell - shaped ", "three pathologic patterns were considered : intermittent or continuous polyphasic curves , and flat curves [ figures 1a  c ] .", "we determined the sensitivity and specificity as well as the positive and negative predictive value of the vds questionnaire using the uroflowmetry data .", "( a ) intermittent polyphasic curve ( b ) continuous polyphasic curve ( c ) flat curve a comparison of the clinical characteristics and uroflowmetry of the two groups was made using a wilcoxon test for continuous data and a fischer test for categorical data .", "as the bfluts questionnaire and urodynamics were identical to those systematically used for the routine evaluation of sui women in our department , the study did not require specific ethics committee approbation ."], ["we analyzed 415 sui women from the urodynamic database , who had undergone a filling cystometry in the department , and whose uroflowmetry indicated a total urine volume greater than 200 ml .", "women with neurological disorders , pelvic organ prolapse , urge or mixed urinary incontinence , detrusor overactivity or previous sui surgery were excluded . with these exclusion criteria ,", "these 93 women were divided into two groups : those who complained of vd symptoms ( vds group ) according to the bristol female lower urinary tract symptoms ( bfluts ) questionnaire , and those without vd symptoms ( non - vds group ) .", "we investigated the parity , age , menopausal status , and concomitant anorectal disorders of each woman ."], ["the women were interviewed on the presence of emptying symptoms , and completed the bfluts questionnaire [ table 1 ] , which is systematic in our department .", "we screened for five items related to voiding difficulties : hesitancy , slow or intermittent stream , straining , or sensations of incomplete emptying . for the purposes of data analysis , those women who answered  never  to all five items were classed in the non - vds group .", "those women for whom one or more of the answers was positive ( i.e. :  occasionally , sometimes , most of the time or all the time  ) , were classified in the vds group ."], ["the clinical examination included a physical examination , urodynamic testing and a  free flow  uroflowmetry and filling cystometry at a rate of 50 ml per minute ( duet , medtronic ) .", "the filling cystometry parameters studied were : compliance , detrusor overactivity ( do ) , detrusor sensation .", "none of the patients presented with abnormal compliance , do , or abnormal detrusor sensation .", "both a quantitative and a qualitative analysis of bladder outlet obstruction ( boo ) was made by uroflowmetry .", "quantitatively , a patient was considered to have boo when a maximum flow rate of less than 15 ml / sec was observed for a urine volume greater than 200 ml , and/or the post - void residual was greater than 50 ml , as measured with a bladder scan or by catheterization .", "the urine flow was also analyzed in terms of average flow rate , urine volume , voiding time and acceleration ( defined as the maximum flow rate divided by the time taken to reach maximum flow rate ) .", "the qualitative analysis dealt with the flow patterns : a standard  bell - shaped ", "three pathologic patterns were considered : intermittent or continuous polyphasic curves , and flat curves [ figures 1a  c ] .", "we determined the sensitivity and specificity as well as the positive and negative predictive value of the vds questionnaire using the uroflowmetry data .", "( a ) intermittent polyphasic curve ( b ) continuous polyphasic curve ( c ) flat curve"], ["a comparison of the clinical characteristics and uroflowmetry of the two groups was made using a wilcoxon test for continuous data and a fischer test for categorical data ."], ["as the bfluts questionnaire and urodynamics were identical to those systematically used for the routine evaluation of sui women in our department , the study did not require specific ethics committee approbation ."], ["among the 93 women eligible for the study , 61% ( 57/93 ) complained of vd symptoms on the bfluts questionnaire ( vds group ) and 39% ( 36/93 ) had no vd symptoms ( non - vds group ) .", "patients characteristics are detailed in table 2 . no difference was observed between the two groups concerning age , parity , menopausal status , concomitant anorectal disorders and uroflowmetry parameters . in the vds group ,", "75% ( 27/36 ) of the women in the non - vds group had normal uroflowmetry , and 25% ( 9/36 ) an abnormal one without symptoms .", "the positive predictive value of the vds questionnaire was 32% and the negative predictive value was 75% [ table 3 ] .", "there was no statistical correlation between symptoms and the defined criteria of boo on uroflowmetry ( fischer test : p=0.64 ) . among the 42 women who complained of a sensation of incomplete", "emptying , only one had a real post - void residual greater than 50 ml . in this", "sui population without severe pelvic organ prolapse ( baden and walker staging system 1 ) , previous sui surgery , or neurological disorders ( the most frequent etiologies of voiding dysfunction in women ) we found a 29% ( 29/93 ) rate of abnormal uroflowmetry . characteristics of women with and without voiding difficulties symptoms in terms of age , menopausal status , parity , anorectal disorders and uroflowmetry parameters sensitivity , specificity , positive and negative predictive value of voiding dysfunction symptoms on bfluts multivariate analysis ( using multiple logistic regression method ) demonstrated no correlation between symptoms and quantitative uroflowmetry parameters ( maximum and average flow rate , acceleration , urine volume , time to maximum flow and voiding time ) , and no correlation was found between vds and age , menopausal status , parity or concomitant anorectal disorders [ table 4 ] . logistic regression method for multivariate analysis of urodynamic parameters ( maximal and average flow rate , time to void and acceleration ) , age , menopausal status , parity , concomitant anorectal disorders with respect to voiding difficulties symptoms univariate analysis of multiple factors with respect to voiding difficulties symptoms"], ["our results show that 25% of sui women without obstructive symptoms , i.e. without an obvious cause for bladder outlet obstruction ( no severe genital prolapse ( > grade 1 ) ) , having had no previous surgery for sui , and with no neurological disorders , have an abnormal uroflowmetry , and thus a higher risk of post - surgery obstructive disorders .", "our study clearly demonstrates the poor predictive value of the vds questionnaire , when compared with urodynamic data .", "the prevalence of voiding dysfunction symptoms among women is not easy to determine , in contrast to storage symptoms , which are more often related and analyzed .", "stress urinary incontinence and bladder outlet obstruction ( boo ) can coexist in women ; bradley found 19 cases of boo out of 104 ( 18% ) , defined on the basis of pressure flow studies among women presenting with sui . in our study , we found a high prevalence of vds ( 61% ) , probably because in the vds group we included women with any level of positive response , even those who gave one  occasionally response to the vd items ( bfluts questionnaire ) .", "urodynamic evaluation of the voiding phase in sui women before surgery is useful in order to predict the risk of vd and urinary retention , and to inform women about these potential complications .", "surgical treatments of sui using mid - urethral sling procedures are known to affect the voiding phase : the spontaneous flow curve changes to a more obstructive pattern in 40% of cases , and the maximum and mean flow rates decrease significantly after one year .", "the incidence of urinary retention after such procedures ranges from 2.216%.[13 ] postoperative vd induces a decrease in global satisfaction after surgery , and leads to an impairment of the quality of life .", "factors which correlate highly with a postoperative voiding dysfunction include a preoperative peak flow rate < 15 ml / s ( several studies ) , and an abnormal uroflow pattern or detrusor pressure < 12 cm h2o.[31114 ] therefore , screening for such factors among sui women is critical .", "one of the main limitations of the current study is the lack of a standard definition for boo in urodynamics for women .", "since voiding cystometry had not been performed , boo can not be defined urodynamically in the current study .", "farrar et al . , defined obstruction as qmax < 15 ml / s with a voided volume of 200 ml .", "we added the flow curve pattern criteria , because a pathological pattern with a normal maximal flow rate can reflect voiding difficulties .", "moreover , in pressure flow studies in women , the transurethral catheter has a significant impact on the urine flow.[1418 ] however ,  free flow ", "uroflowmetry is a composite interaction between the detrusor pressure and the resistance produced by the urethra .", "bladder outlet obstruction as well as impaired detrusor contractility can perturb uroflowmetry . in our population", ", we needed to detect voiding difficulties , whatever their origin , because among sui women both these causes of voiding disorders can promote obstructive complications after surgery .", "chassagne et al . , prospectively studied 35 clinically obstructed women and 124 control patients .", "they determined cutoff values for the pressure flow studies as : qmax<15 ml / s and pdet .", "qmax > 20 cm h2o , with a sensitivity of 74.3% and a specificity of 91.1% .", "no information was provided regarding the clinical criteria used to select the  clinically obstructed  patients .", "qmax > 20 cm h2o ) during detrusor pressure uroflow studies . among the 38 women considered as obstructed within this definition ,", "they concluded that micturition symptoms relevant to bladder outlet obstruction are non - specific , and that a complete urodynamic evaluation is essential .", "the correlation between obstructive symptoms and objective urodynamic findings is known to be poor , but has never been studied in this specific population of sui women , for which all cases with a pop - q > stage 1 , previous surgery for sui , or neurological disorders , were excluded . indeed , we excluded the most common etiologies of urodynamic assessment of bladder outlet obstruction", ". groutz et al . , reported 26% of prior anti - incontinence surgery and 24% of severe genital prolapse in a population of 38 women presenting with urodynamically assessed obstructive criteria .", "wyndaele et al . , recently confirmed the poor correlation between symptoms and urodynamic data , and a high prevalence of vds in women , whether it be considered to have a pathological origin or not .", "the absence of voiding symptoms is not a guarantee of normal micturition , since we found that almost one - third of the women not complaining of any voiding difficulty were actually shown to have an abnormal uroflowmetry .", "previous studies[1421 ] have clearly demonstrated the lack of correlation between symptoms and urodynamic diagnoses , especially for obstructive symptoms .", "we found the same results , but in a particular population of sui women , with no obvious reasons for obstruction . even in this selected population ,", "in which voiding dysfunctions should be less frequent than in the general population , we found that almost 30% of the patients had an abnormal uroflowmetry ."], ["no correlation was found between obstructive symptoms and boo as defined on uroflowmetry , in a specific population of sui women ."]]}
{"article_id": "PMC4707387", "article_text": ["scurvy is a disease that played an important role in history and used to be the number one killer of sailors .", "it is still diagnosed in third - world countries because of malnutrition and in epidemic form in refugee camps .", "a 35-year - old patient was seen at the emergency department of a level 1 trauma centre with spontaneous bruisings on the lower extremity .", "initially , the patient was seen by an orthopaedic surgeon at an outpatient clinic with a painful knee without any preceding trauma .", "the procedure was postponed twice . at first , the arthroscopy was cancelled because the patient had the flu .", "the second time the orthopaedic surgeon postponed the operation because of extensive bruising on the right leg ( fig .  1 )", "moreover , doctors of the internal medicine and dermatology department were consulted to explore the origin of the haematomas .", "2 ) and a biopsy was performed . at the time of the current presentation at the emergency department ,", "because of the progression of the haematomas to the patients left leg , the surgeon was consulted to rule out an active bleeding focus . \n figure  1:small bruises on the upper legs . \n", "the patient was an electrician , currently unemployed because of knee problems . since 10 weeks", "the haematomas on his right leg occurred after an episode of flu a month ago ( fig . ", "ever since , he had been exhausted , and he had spent the majority of his time in bed .", "his dentist proposed to remove the decaying teeth . according to the patient , the bad teeth", "apparently , the patients eating regimen only consisted of bread , sometimes with spread , but mostly without any additions . for this eating habit", "on physical examination , we encountered a pale patient with haematomas on his upper legs , generally green / blue or yellow coloured .", "laboratory tests revealed anaemia with a haemoglobin drop of 2.5 points in 2 weeks ( from 8.6 to 5.7 mmol / l ) .", "no surgical cause for the haematomas was found . instead , the diagnosis of a severe vitamin c deficiency (  scurvy ) by malnutrition due to his eating disorder was considered .", "after laboratory blood tests were drawn to confirm the diagnosis , vitamin c supplements were started .", "it is regrettable that the united east india company ( voc ) lost more sailors on scurvy than on the battlefield during the seventeenth century .", "scurvy was one of the limiting factors for travelling long distances on ships because of the absence of fresh food , fruit and vitamins .", "scurvy was first described by hippocrates . later in 1753 , a surgeon of the royal navy described how citrus fruits enhanced the symptoms of scurvy . in 1930 , it was discovered that depletion of vitamin c level was the cause .", "vitamin c is needed for the synthesis of collagen , which is essential for firmness of the blood vessels . in patients with scurvy ,", "the presentation of scurvy is often malaise , fatigue , stiff joints and bruising , mostly on the upper thighs and legs .", "the next stage consists of open wounds , jaundice , fever , loss of teeth and finally death .", "scurvy does not occur in most animals as they can synthesize their own vitamin c in contrast with the human , for example guinea pigs and certain monkey species .", "treatment is relatively simple with a versatile diet with citrus fruits , sweet peppers and tomatoes .", "the recommended daily quantity of vitamin c is 75 mg for women and 90 mg for man   ( http://www.who.int/en/ ) , but up to 40% more can be necessary in smokers and during infection .", "nowadays , scurvy occurs predominantly in developed countries because of malnutrition , and frequently epidemics are reported from the lesser civilized areas .", "this present case report emphasizes that scurvy may still occur in developed countries and that patients with scurvy could be encountered by physicians of multiple medical disciplines .", "scurvy should be considered in risk groups , e.g. singles , patients with eating disorder , students , elderly , alcohol abusers and patients after gastric surgery [ 5 , 6 ] .", "recently , in wales ( uk ) in 2011 , a young patient died of scurvy ( http://www.bbc.com/news/uk-wales-31039895 ) .", "however , even in these modern timeframe , groups with a high risk of vitamin deficiencies can be discerned . in our patient", ", the biopsy could have been avoided , and supplements could have been initiated more quickly in case of higher awareness of the signs and symptoms of vitamin c deficiency ."], "abstract_text": ["<S> scurvy is a disease that played an important role in ancient history and used to be a notorious cause of death in sailors . </S>", "<S> nowadays , scurvy is not a common diagnosis in the civilized world , but this case report indicates that this old - fashioned disease is not extinct at all and still exists but in a different patient category . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONFLICT OF INTEREST STATEMENT"], "sections": [["scurvy is a disease that played an important role in history and used to be the number one killer of sailors .", "it is still diagnosed in third - world countries because of malnutrition and in epidemic form in refugee camps ."], ["a 35-year - old patient was seen at the emergency department of a level 1 trauma centre with spontaneous bruisings on the lower extremity .", "initially , the patient was seen by an orthopaedic surgeon at an outpatient clinic with a painful knee without any preceding trauma .", "the procedure was postponed twice . at first , the arthroscopy was cancelled because the patient had the flu .", "the second time the orthopaedic surgeon postponed the operation because of extensive bruising on the right leg ( fig .  1 )", "moreover , doctors of the internal medicine and dermatology department were consulted to explore the origin of the haematomas .", "2 ) and a biopsy was performed . at the time of the current presentation at the emergency department ,", "because of the progression of the haematomas to the patients left leg , the surgeon was consulted to rule out an active bleeding focus . \n figure  1:small bruises on the upper legs . \n", "the patient was an electrician , currently unemployed because of knee problems . since 10 weeks", "the haematomas on his right leg occurred after an episode of flu a month ago ( fig . ", "ever since , he had been exhausted , and he had spent the majority of his time in bed .", "his dentist proposed to remove the decaying teeth . according to the patient , the bad teeth", "apparently , the patients eating regimen only consisted of bread , sometimes with spread , but mostly without any additions . for this eating habit", "on physical examination , we encountered a pale patient with haematomas on his upper legs , generally green / blue or yellow coloured .", "laboratory tests revealed anaemia with a haemoglobin drop of 2.5 points in 2 weeks ( from 8.6 to 5.7 mmol / l ) .", "no surgical cause for the haematomas was found . instead , the diagnosis of a severe vitamin c deficiency (  scurvy ) by malnutrition due to his eating disorder was considered .", "after laboratory blood tests were drawn to confirm the diagnosis , vitamin c supplements were started ."], ["it is regrettable that the united east india company ( voc ) lost more sailors on scurvy than on the battlefield during the seventeenth century .", "scurvy was one of the limiting factors for travelling long distances on ships because of the absence of fresh food , fruit and vitamins .", "scurvy was first described by hippocrates . later in 1753 , a surgeon of the royal navy described how citrus fruits enhanced the symptoms of scurvy . in 1930 , it was discovered that depletion of vitamin c level was the cause .", "vitamin c is needed for the synthesis of collagen , which is essential for firmness of the blood vessels . in patients with scurvy ,", "the presentation of scurvy is often malaise , fatigue , stiff joints and bruising , mostly on the upper thighs and legs .", "the next stage consists of open wounds , jaundice , fever , loss of teeth and finally death .", "scurvy does not occur in most animals as they can synthesize their own vitamin c in contrast with the human , for example guinea pigs and certain monkey species .", "treatment is relatively simple with a versatile diet with citrus fruits , sweet peppers and tomatoes .", "the recommended daily quantity of vitamin c is 75 mg for women and 90 mg for man   ( http://www.who.int/en/ ) , but up to 40% more can be necessary in smokers and during infection .", "nowadays , scurvy occurs predominantly in developed countries because of malnutrition , and frequently epidemics are reported from the lesser civilized areas .", "this present case report emphasizes that scurvy may still occur in developed countries and that patients with scurvy could be encountered by physicians of multiple medical disciplines .", "scurvy should be considered in risk groups , e.g. singles , patients with eating disorder , students , elderly , alcohol abusers and patients after gastric surgery [ 5 , 6 ] .", "recently , in wales ( uk ) in 2011 , a young patient died of scurvy ( http://www.bbc.com/news/uk-wales-31039895 ) .", "however , even in these modern timeframe , groups with a high risk of vitamin deficiencies can be discerned . in our patient", ", the biopsy could have been avoided , and supplements could have been initiated more quickly in case of higher awareness of the signs and symptoms of vitamin c deficiency ."], [""]]}
{"article_id": "PMC4339136", "article_text": ["human movement can be classified into walking and running , the latter of which easily \n fatigues the lower limb muscles1,2,3,4 . during running", ", the lower limbs must cope with the repeated \n transient impact of vertical ground reaction force ( grf ) , which is an abrupt collision force \n equal to about 1.5- to 3-fold the body weight5 .", "such repeated impacts are considered to cause lower limb muscle \n fatigue via chronic irritation .", "the incidence of running injury is affected by the running \n velocity and distance ran .", "investigated 17,000 patients6 and found a significantly higher incidence of osteoarthritis \n in men who were involved in running > 20 miles per week .", "koplan et al . found that the \n risk of injury increased with increasing weekly distance3 .", "on the other hand , imai et al.7 found that the number of marathon athletes who stopped running \n within four hours was significantly high .", "running \n faster generally produces greater ground reaction force ( grf ) and imposes greater stress on \n various parts of the body8 , 9 .", "running injuries occur most frequently in the knee , \n foot / ankle , and lower leg according to taunton , et al.10 , who found that the most common overuse injuries included \n patellofemoral pain syndrome , iliotibial band friction syndrome , and plantar fasciitis . running velocity is affected by various conditions including individual running ability .", "we \n postulated that the load imposed on the lower limbs would change at different running \n velocities . furthermore , because portion failure differs among individuals , the load applied \n to each muscle is probably not uniform .", "therefore , we analyzed muscle \n activity under free walking , jogging (  8.7  km / h ) , and running (  8.8  km / h ) conditions on a \n treadmill .", "ten male students without leg injuries ( age , 23.2  6.5 y ; height , 170.4  5.7  cm ; weight , \n 67.6  11.1  kg ; bmi , 23.3  3.2 ) provided written informed consent to participate after \n receiving an oral and written explanation of the study and its purpose .", "the ethics review \n board at heisei college of health sciences approved this study ( no .", "the \n participants did not routinely run but participated in recreational sports once or twice \n each week .", "muscle activity was measured by surface electromyography using a telemyo g2 and \n myoresearch xp ( noraxon usa inc . ,", "scottsdale , az , usa ) with disposable m-00-s blue sensor \n polymer electrodes ( ambu a / s , ballerup , denmark ) attached to the belly of the muscle while \n the participants moved on a gait trainer system 2 treadmill ( biodex medical systems inc .", "muscle output was recorded at 30 frames / sec using a video camera to \n synchronize the movements with the electromyographic data .", "activity was assessed at the left \n vastus medialis , vastus lateralis , hip adductors , tibialis anterior , lateral head of \n gastrocnemius , medial head gastrocnemius , and soleus .", "maximum voluntary isometric contraction ( mvc ) was determined as manual resistance against a \n maximally contracted muscle .", "the participants walked , jogged , and ran for 10 minutes in \n random order , and the myogenic potentials were measured for 30 seconds 10 minutes later . \n", "analog signals of myogenic potential were processed using a band - pass filter ( 10500  hz ) at \n a sampling frequency of 1,000  hz . the greater trochanter , lower limb joint space , and lateral malleolus of the left hip , \n knee , and ankle were marked with tape to measure the range of motion .", "one 30-second cycle of \n walking , jogging , and running recorded with the video camera was selected , and then initial \n contact , mid stance , toe off , and the range of motion of hip flexion , knee flexion , and \n ankle dorsiflexion were measured on printouts .", "in addition , using the time required for one \n cycle , stride was calculated .", "electromyographic data were analyzed using the average value of each muscle output and \n central frequency .", "the average activity was calculated from 30-second cycles of walking , \n jogging , and running , including both the swing and stance phases .", "these output values were \n divided by the mvc to determine the % mvc of each muscle .", "in addition , lower limb joint \n angles on printouts were measured using a protractor .", "the data were analyzed using a one - way \n analysis of variance , and statistical significance was accepted at p < 0.05 .", "the average walking , jogging , and running speeds were 3.6  0.4 , 6.7  0.6 , and 10.4  \n 1.3  km / h , respectively .", "the average electromyographic activities of the vastus medial , tibialis anterior , medial \n head of the gastrocnemius , and lateral head of the gastrocnemius were significantly higher \n during jogging and running compared with walking .", "the hip adductors and vastus lateralis did not significantly \n differ ( table 1table 1.average % mvc of muscles ( n=10)walkingjoggingrunningvastus medialis12.52.735.85.3 * 38.84.6*vastus lateralis17.16.136.38.137.76.8hip adductors31.26.741.07.250.17.1tibialis anterior14.31.329.33.8 * 37.63.0*lateral head of the gastrocnemius16.03.042.68.5 * 40.86.2*medial head of the gastrocnemius30.83.758.17.6 * 63.35.3*soleus18.45.039.67.844.86.2*values are meansse . * p<0.05 , vs. walking . ) .", "the maximal electromyographic activities of the vastus medialis during jogging and running \n and medial head of the gastrocnemius during running were significantly higher than during \n walking ( table 2table 2.maximum % mvc of muscles ( n=10)walkingjoggingrunningvastus medialis54.87.6105.915.2 * 135.711.5*vastus lateralis68.129.0103.117.0134.423.6hip adductors147.231.598.620.2137.320.0tibialis anterior54.16.385.820.787.87.9lateral head of the gastrocnemius69.014.4125.527.7136.515.1medial head of the gastrocnemius129.921.6190.427.8224.226.0*soleus103.351.8113.625.9179.028.6values are meansse .", "the central frequency did not significantly differ ( table 3table 3.average central frequency of muscles ( n=10)walkingjoggingrunningvastus medialis23.16.437.95.932.46.4vastus lateralis29.75.840.35.641.86.9hip adductors15.24.120.44.123.33.4tibialis anterior59.86.066.06.566.17.4lateral head of the gastrocnemius34.37.142.77.255.76.0medial head of the gastrocnemius50.07.362.59.963.19.2soleus61.27.645.18.259.711.8values are meansse ) .", "the angle of the knee at initial contact , mid stance , and toe off significantly differed , \n with the knee being more flexed during jogging and running than walking the knee being more \n flexed during running than jogging at mid stance and toe off ( table 4table 4.average left lower limb joint angles ( n=10)walkingjoggingrunninghip joint flexionic20.52.424.52.228.02.9ms10.51.623.51.5 * 28.51.7*to6.02.23.02.6 * 5.51.9*knee joint flexionic4.22.114.82.2 * 17.22.0*ms15.02.141.51.5 * 48.01.5*,**to57.11.338.92.4 * 25.42.4*,**ankle joint dorsiflexionic2.23.64.02.06.21.1ms6.51.320.01.7 * 22.01.3*to14.82.214.82.720.13.5values are meansse . * p<0.05 , vs. walking . * * p<0.05 , vs. jogging .", "ic : initial \n contact , ms : mid stance , to : toe off ) .", "ic : initial \n contact , ms : mid stance , to : toe off the average lengths of the running stride were significantly longer than those of jogging \n and walking . also , that of jogging was significantly longer than that of walking ( table 5table 5.average length of stride ( n=10)walkingjoggingrunningstride ( m)1.240.041.460.06 * 2.140.08*,**values are meansse .", "hreljac identified training , anatomy , and biomechanical factors as risk factors associated \n with running , since fast running generates considerable grf and training to run stresses the \n joints , muscles , and ligaments12 .", "the \n present study investigated differences in the amount of muscle activity during walking , \n jogging , and running .", "all participants walked freely at their own pace and then jogged at  \n 8.7  km / h and ran at  8.8  km / h .", "the central frequency of each muscle seemed to remain essentially unchanged between running \n and jogging compared with walking , and fatigue did not arise .", "the vastus lateralis and \n vastus medialis of the quadriceps not only act on the lower limb during running or walking \n but also suppress damage to the knee during weight - bearing in the stance phase .", "therefore , \n we speculated that the amount of muscle activity would increase as the grf increases .", "the \n averaged values of the vastus medialis were significantly higher during running and jogging \n compared with walking .", "brownstein et al.13 found that a knee position that is more flexed than extension is \n due to the amount of activity of the vastus medialis .", "the present study found that the knees \n were flexed significantly more during running and jogging compared with walking at initial \n contact and during mid stance .", "these findings support the notion that the vastus medialis \n works easily during jogging and running .", "in addition , kim et al.14 showed that the amount of activity in the vastus medialis \n rose in response to an elevated walking speed .", "furthermore , in a previous study15 , we compared the muscle activity during \n jogging and walking at the same velocity . in this study , the activity of the vastus medialis \n during jogging was significantly higher , and the knee joints exhibited significant flexion \n during jogging .", "load was \n especially applied in the direction of internal rotation in the stance phase . a study by ono \n", "et al.17 showed that the output of the \n vastus medialis was increased in the lower leg during internal rotation . on the other hand , \n the output at 90 of flexion between knee extension from the vicinity of the vastus \n lateralis muscle did not significantly change13 .", "in addition , the activity of the vastus lateralis muscle during \n walking was significantly higher , which could account for the small difference between \n walking and running .", "the average \n values of the adductor muscles did not significantly differ among walking , jogging , and \n running .", "along with the outer muscles of the hip joint such as the gluteus medius , the \n adductor muscles maintain the inner - outer intermediate position of the lower limb during \n running .", "the types of hip movement involved in running are primarily flexion and extension , \n with minimal adduction and abduction . in addition , because the treadmill floor is \n horizontal , the trunk is also likely to become upset .", "therefore , we considered that the \n adductor muscles do not play significant roles in walking , jogging and running .", "the averages values for the tibialis anterior muscle were significantly higher during \n running and jogging compared with walking .", "the tibialis anterior muscle contracts either \n centrifugally or isometrically before and after initial contact to control the foot to \n prevent falling18 , 19 .", "this muscle was significantly more contracted during running and \n jogging , perhaps because movement was faster .", "the average values of the lateral and medial heads of the gastrocnemius were significantly \n higher during running and jogging compared with walking .", "the gastrocnemius works from the \n loading response to the terminal stance between the stance phase18 .", "the knee joint extends in the late stance phase of the \n running stride via plantar flexion of the ankle joint .", "the force exerted on the achilles \n tendon during running exceeds 12-fold the weight of the runner20 .", "a large output by the gastrocnemius is required to \n advance the body during running .", "the average value of the soleus muscle was significantly higher during running than during \n walking .", "the soleus is a single joint muscle that runs from the proximal portion of the \n tibia and fibula to the achilles tendon .", "thus , the amount of muscle activity is not affected \n by the angle of the knee joint . because the knee flexes deeply in mid stance during running , \n more extension is required at toe off , and thus a large force is required for the triceps \n surae muscle . under such conditions", ", the soleus muscle works more easily than the \n gastrocnemius , which is a biarticular muscle . according to mann et al.21 , the angle of \n the lower limbs and the amount of muscle activity increases during running at higher \n velocities compared with walking .", "the stance phase is longer than the swing phase during \n walking but is shorter during running . according to kluitenberg et al.22 , grf on a treadmill and on the ground increases in the \n same way when", "aramptzis et al.23 reported that the mechanical power of the knee joint and \n ankle joint increases with increasing running velocity , and the present study found that , \n the stride was extended in accordance with the increase in velocity .", "we considered that \n exercise load is greater during running compared with walking due to these factors .", "the \n present study found that the amount of activity in the soleus muscle was significantly \n higher during running and jogging compared with the amounts of activity in the vastus \n medialis , tibialis anterior , and gastrocnemius muscle , which are involved in walking . \n", "therefore , it was suggested that an increase in velocity causes these muscles to carry a \n heavier load ."], "abstract_text": ["<S> [ purpose ] the present study aimed to determine changes in muscle activity while moving on \n a treadmill at various speeds . </S>", "<S> [ subjects ] the activities of the left vastus lateralis , \n vastus medialis , hip adductors , lateral head of gastrocnemius , medial head gastrocnemius , \n soleus , and tibialis anterior of 10 healthy male university students were analyzed . \n </S>", "<S> [ methods ] university students walked , jogged , and ran for 10 minutes each in random order , \n and then myogenic potentials were measured 10 minutes later for 30 seconds . </S>", "<S> the flexion \n angle of the lower limb upon initial contact , mid stance , and toe off were measured . \n </S>", "<S> [ results ] the average walking , jogging , and running speeds were 3.6  0.4 , 6.7  0.6 , and \n 10.4  1.3  km / h , respectively . </S>", "<S> the average electromyographic activities of the vastus \n medial , tibialis anterior , medial head of gastrocnemius , and lateral head of gastrocnemius \n significantly differed . </S>", "<S> all muscles were more active during jogging and running than \n walking . </S>", "<S> only the soleus was more active during running than walking , and the activities \n of the hip adductors and vastus lateralis did not significantly differ . </S>", "<S> [ conclusion ] \n velocity is faster and the angles of the lower limbs and ground reaction force ( grf ) are \n larger during running than walking . the vastus medialis and soleus worked more easily \n according to the angle of the knee joint , whereas the tibialis anterior worked more easily \n at faster velocities and the medial and lateral heads of the gastrocnemius worked more \n easily with an increased grf . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["human movement can be classified into walking and running , the latter of which easily \n fatigues the lower limb muscles1,2,3,4 . during running", ", the lower limbs must cope with the repeated \n transient impact of vertical ground reaction force ( grf ) , which is an abrupt collision force \n equal to about 1.5- to 3-fold the body weight5 .", "such repeated impacts are considered to cause lower limb muscle \n fatigue via chronic irritation .", "the incidence of running injury is affected by the running \n velocity and distance ran .", "investigated 17,000 patients6 and found a significantly higher incidence of osteoarthritis \n in men who were involved in running > 20 miles per week .", "koplan et al . found that the \n risk of injury increased with increasing weekly distance3 .", "on the other hand , imai et al.7 found that the number of marathon athletes who stopped running \n within four hours was significantly high .", "running \n faster generally produces greater ground reaction force ( grf ) and imposes greater stress on \n various parts of the body8 , 9 .", "running injuries occur most frequently in the knee , \n foot / ankle , and lower leg according to taunton , et al.10 , who found that the most common overuse injuries included \n patellofemoral pain syndrome , iliotibial band friction syndrome , and plantar fasciitis . running velocity is affected by various conditions including individual running ability .", "we \n postulated that the load imposed on the lower limbs would change at different running \n velocities . furthermore , because portion failure differs among individuals , the load applied \n to each muscle is probably not uniform .", "therefore , we analyzed muscle \n activity under free walking , jogging (  8.7  km / h ) , and running (  8.8  km / h ) conditions on a \n treadmill ."], ["ten male students without leg injuries ( age , 23.2  6.5 y ; height , 170.4  5.7  cm ; weight , \n 67.6  11.1  kg ; bmi , 23.3  3.2 ) provided written informed consent to participate after \n receiving an oral and written explanation of the study and its purpose .", "the ethics review \n board at heisei college of health sciences approved this study ( no .", "the \n participants did not routinely run but participated in recreational sports once or twice \n each week .", "muscle activity was measured by surface electromyography using a telemyo g2 and \n myoresearch xp ( noraxon usa inc . ,", "scottsdale , az , usa ) with disposable m-00-s blue sensor \n polymer electrodes ( ambu a / s , ballerup , denmark ) attached to the belly of the muscle while \n the participants moved on a gait trainer system 2 treadmill ( biodex medical systems inc .", "muscle output was recorded at 30 frames / sec using a video camera to \n synchronize the movements with the electromyographic data .", "activity was assessed at the left \n vastus medialis , vastus lateralis , hip adductors , tibialis anterior , lateral head of \n gastrocnemius , medial head gastrocnemius , and soleus .", "maximum voluntary isometric contraction ( mvc ) was determined as manual resistance against a \n maximally contracted muscle .", "the participants walked , jogged , and ran for 10 minutes in \n random order , and the myogenic potentials were measured for 30 seconds 10 minutes later . \n", "analog signals of myogenic potential were processed using a band - pass filter ( 10500  hz ) at \n a sampling frequency of 1,000  hz . the greater trochanter , lower limb joint space , and lateral malleolus of the left hip , \n knee , and ankle were marked with tape to measure the range of motion .", "one 30-second cycle of \n walking , jogging , and running recorded with the video camera was selected , and then initial \n contact , mid stance , toe off , and the range of motion of hip flexion , knee flexion , and \n ankle dorsiflexion were measured on printouts .", "in addition , using the time required for one \n cycle , stride was calculated .", "electromyographic data were analyzed using the average value of each muscle output and \n central frequency .", "the average activity was calculated from 30-second cycles of walking , \n jogging , and running , including both the swing and stance phases .", "these output values were \n divided by the mvc to determine the % mvc of each muscle .", "in addition , lower limb joint \n angles on printouts were measured using a protractor .", "the data were analyzed using a one - way \n analysis of variance , and statistical significance was accepted at p < 0.05 ."], ["the average walking , jogging , and running speeds were 3.6  0.4 , 6.7  0.6 , and 10.4  \n 1.3  km / h , respectively .", "the average electromyographic activities of the vastus medial , tibialis anterior , medial \n head of the gastrocnemius , and lateral head of the gastrocnemius were significantly higher \n during jogging and running compared with walking .", "the hip adductors and vastus lateralis did not significantly \n differ ( table 1table 1.average % mvc of muscles ( n=10)walkingjoggingrunningvastus medialis12.52.735.85.3 * 38.84.6*vastus lateralis17.16.136.38.137.76.8hip adductors31.26.741.07.250.17.1tibialis anterior14.31.329.33.8 * 37.63.0*lateral head of the gastrocnemius16.03.042.68.5 * 40.86.2*medial head of the gastrocnemius30.83.758.17.6 * 63.35.3*soleus18.45.039.67.844.86.2*values are meansse . * p<0.05 , vs. walking . ) .", "the maximal electromyographic activities of the vastus medialis during jogging and running \n and medial head of the gastrocnemius during running were significantly higher than during \n walking ( table 2table 2.maximum % mvc of muscles ( n=10)walkingjoggingrunningvastus medialis54.87.6105.915.2 * 135.711.5*vastus lateralis68.129.0103.117.0134.423.6hip adductors147.231.598.620.2137.320.0tibialis anterior54.16.385.820.787.87.9lateral head of the gastrocnemius69.014.4125.527.7136.515.1medial head of the gastrocnemius129.921.6190.427.8224.226.0*soleus103.351.8113.625.9179.028.6values are meansse .", "the central frequency did not significantly differ ( table 3table 3.average central frequency of muscles ( n=10)walkingjoggingrunningvastus medialis23.16.437.95.932.46.4vastus lateralis29.75.840.35.641.86.9hip adductors15.24.120.44.123.33.4tibialis anterior59.86.066.06.566.17.4lateral head of the gastrocnemius34.37.142.77.255.76.0medial head of the gastrocnemius50.07.362.59.963.19.2soleus61.27.645.18.259.711.8values are meansse ) .", "the angle of the knee at initial contact , mid stance , and toe off significantly differed , \n with the knee being more flexed during jogging and running than walking the knee being more \n flexed during running than jogging at mid stance and toe off ( table 4table 4.average left lower limb joint angles ( n=10)walkingjoggingrunninghip joint flexionic20.52.424.52.228.02.9ms10.51.623.51.5 * 28.51.7*to6.02.23.02.6 * 5.51.9*knee joint flexionic4.22.114.82.2 * 17.22.0*ms15.02.141.51.5 * 48.01.5*,**to57.11.338.92.4 * 25.42.4*,**ankle joint dorsiflexionic2.23.64.02.06.21.1ms6.51.320.01.7 * 22.01.3*to14.82.214.82.720.13.5values are meansse . * p<0.05 , vs. walking . * * p<0.05 , vs. jogging .", "ic : initial \n contact , ms : mid stance , to : toe off ) .", "ic : initial \n contact , ms : mid stance , to : toe off the average lengths of the running stride were significantly longer than those of jogging \n and walking . also , that of jogging was significantly longer than that of walking ( table 5table 5.average length of stride ( n=10)walkingjoggingrunningstride ( m)1.240.041.460.06 * 2.140.08*,**values are meansse ."], ["hreljac identified training , anatomy , and biomechanical factors as risk factors associated \n with running , since fast running generates considerable grf and training to run stresses the \n joints , muscles , and ligaments12 .", "the \n present study investigated differences in the amount of muscle activity during walking , \n jogging , and running .", "all participants walked freely at their own pace and then jogged at  \n 8.7  km / h and ran at  8.8  km / h .", "the central frequency of each muscle seemed to remain essentially unchanged between running \n and jogging compared with walking , and fatigue did not arise .", "the vastus lateralis and \n vastus medialis of the quadriceps not only act on the lower limb during running or walking \n but also suppress damage to the knee during weight - bearing in the stance phase .", "therefore , \n we speculated that the amount of muscle activity would increase as the grf increases .", "the \n averaged values of the vastus medialis were significantly higher during running and jogging \n compared with walking .", "brownstein et al.13 found that a knee position that is more flexed than extension is \n due to the amount of activity of the vastus medialis .", "the present study found that the knees \n were flexed significantly more during running and jogging compared with walking at initial \n contact and during mid stance .", "these findings support the notion that the vastus medialis \n works easily during jogging and running .", "in addition , kim et al.14 showed that the amount of activity in the vastus medialis \n rose in response to an elevated walking speed .", "furthermore , in a previous study15 , we compared the muscle activity during \n jogging and walking at the same velocity . in this study , the activity of the vastus medialis \n during jogging was significantly higher , and the knee joints exhibited significant flexion \n during jogging .", "load was \n especially applied in the direction of internal rotation in the stance phase . a study by ono \n", "et al.17 showed that the output of the \n vastus medialis was increased in the lower leg during internal rotation . on the other hand , \n the output at 90 of flexion between knee extension from the vicinity of the vastus \n lateralis muscle did not significantly change13 .", "in addition , the activity of the vastus lateralis muscle during \n walking was significantly higher , which could account for the small difference between \n walking and running .", "the average \n values of the adductor muscles did not significantly differ among walking , jogging , and \n running .", "along with the outer muscles of the hip joint such as the gluteus medius , the \n adductor muscles maintain the inner - outer intermediate position of the lower limb during \n running .", "the types of hip movement involved in running are primarily flexion and extension , \n with minimal adduction and abduction . in addition , because the treadmill floor is \n horizontal , the trunk is also likely to become upset .", "therefore , we considered that the \n adductor muscles do not play significant roles in walking , jogging and running .", "the averages values for the tibialis anterior muscle were significantly higher during \n running and jogging compared with walking .", "the tibialis anterior muscle contracts either \n centrifugally or isometrically before and after initial contact to control the foot to \n prevent falling18 , 19 .", "this muscle was significantly more contracted during running and \n jogging , perhaps because movement was faster .", "the average values of the lateral and medial heads of the gastrocnemius were significantly \n higher during running and jogging compared with walking .", "the gastrocnemius works from the \n loading response to the terminal stance between the stance phase18 .", "the knee joint extends in the late stance phase of the \n running stride via plantar flexion of the ankle joint .", "the force exerted on the achilles \n tendon during running exceeds 12-fold the weight of the runner20 .", "a large output by the gastrocnemius is required to \n advance the body during running .", "the average value of the soleus muscle was significantly higher during running than during \n walking .", "the soleus is a single joint muscle that runs from the proximal portion of the \n tibia and fibula to the achilles tendon .", "thus , the amount of muscle activity is not affected \n by the angle of the knee joint . because the knee flexes deeply in mid stance during running , \n more extension is required at toe off , and thus a large force is required for the triceps \n surae muscle . under such conditions", ", the soleus muscle works more easily than the \n gastrocnemius , which is a biarticular muscle . according to mann et al.21 , the angle of \n the lower limbs and the amount of muscle activity increases during running at higher \n velocities compared with walking .", "the stance phase is longer than the swing phase during \n walking but is shorter during running . according to kluitenberg et al.22 , grf on a treadmill and on the ground increases in the \n same way when", "aramptzis et al.23 reported that the mechanical power of the knee joint and \n ankle joint increases with increasing running velocity , and the present study found that , \n the stride was extended in accordance with the increase in velocity .", "we considered that \n exercise load is greater during running compared with walking due to these factors .", "the \n present study found that the amount of activity in the soleus muscle was significantly \n higher during running and jogging compared with the amounts of activity in the vastus \n medialis , tibialis anterior , and gastrocnemius muscle , which are involved in walking . \n", "therefore , it was suggested that an increase in velocity causes these muscles to carry a \n heavier load ."]]}
{"article_id": "PMC4674473", "article_text": ["the online version of this article ( doi:10.1007/s13300 - 015 - 0133-z ) contains supplementary material , which is available to authorized users .", "chronic kidney disease ( ckd ) is a common condition in patients with type 2 diabetes ( t2 dm ) .", "an estimated 2035% of patients with t2 dm have moderate to severe renal impairment [ 1 , 2 ]", ". however , many antihyperglycemic medications are contraindicated or need to be used with caution in patients with ckd , complicating t2 dm treatment choices and management .", "patients with t2 dm and ckd are particularly susceptible to safety and tolerability issues related to many classes of oral antihyperglycemic agents ( oaha ) .", "dipeptidyl peptidase-4 inhibitors ( dpp-4i ) such as sitagliptin are well tolerated in a broad range of t2 dm patient types , including those with renal disease , and may therefore be preferentially used in patients with ckd .", "prior studies have demonstrated the preferential use of sitagliptin in several populations [ 47 ] . in general , patients initiating treatment with sitagliptin were older and had more complications of diabetes and comorbidities than patients initiating other antihyperglycemic therapies [ 47 ] . if not recognized and appropriately considered in the analysis , this preferential selection of patients with specific demographic and disease characteristics for treatment with sitagliptin ( channeling bias ) could lead to inaccurate treatment effect estimates in comparative analyses that include sitagliptin .", "the objective of this study was to describe the baseline characteristics of patients with t2 dm and ckd initiating treatment with sitagliptin or non - dpp-4i oahas to ascertain whether channeling exists in this patient population .", "the truven health marketscan databases ( marketscan , truven health analytics , ann arbor , mi , usa ) contain medical claims records for more than 150  million unique patients dating from 1996 .", "the records are derived from outpatient and inpatient insurance claims for employees of over 100 employers participating in more than 12 health plans , and their beneficiaries in the united states .", "records consist of commercial claims and healthcare encounters , including information on demographics , health plan membership , international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) codes , and current procedure terminology ( cpt ) codes .", "the records of retirees with supplemental insurance are included in the database thus providing data on the elderly with continuity of care across those < 65 and 65  years of age .", "patients 25  years of age with t2 dm and ckd , with claims in the united states ( us ) between january 2006 and june 2012 , were identified in marketscan . of these patients ,", "those initiating sitagliptin or a non - dpp-4i oaha were categorized by complexity of antihyperglycemic treatment .", "patients were identified as having t2 dm if marketscan records for the patient indicated at least one inpatient or outpatient diagnosis of diabetes and at least one prescription for oaha medication .", "patients with ckd were identified by icd-9-cm diagnostic codes ( 585 , 585.3 , 585.4 , 585.5 , 585.6 , 585.9 , 403 , 403.0 , 403.00 , 403.01 , 403.1 , 403.10 , 403.11 , 403.9 , 403.90 , 403.91 , 250.4 , 404 , 404.0 , 404.00 , 404.01 , 404.02 , 404.03 , 404.1 , 404.10 , 404.11 , 404.12 , 404.13 , 404.9 , 404.90 , 404.91 , 404.92 , 404.93 , 582 , 582.0 , 582.1 , 582.2 , 582.4 , 582.8 , 582.81 , 582.89 , 582.9 ) .", "antihyperglycemic treatment was defined as : ( 1 ) initiating monotherapy ( 1 new outpatient prescription record on or after the t2 dm diagnosis ) ; ( 2 ) escalating to dual combination therapy ( 1 new prescription for a 2nd class 90  days after the 1st class , with prescription for 1st class overlapping the index date of 2nd class ) ; ( 3 ) escalating to triple combination therapy ( 1 new prescription for a 3rd class 90  days after the 2nd class , with prescriptions for 1st and 2nd classes overlapping the index date of 3rd class ) .", "patients were required to have at least 1  year of continuous enrollment in the database prior to initiation / escalation of antihyperglycemic treatment .", "patients were excluded from the analysis if they had a diagnosis of type 1 diabetes , ketoacidosis , malnutrition - associated diabetes , drug - induced diabetes or gestational diabetes without a subsequent t2 dm diagnosis code .", "patients with icd-9-cm codes explicit for mild renal disease ( stage 1 and 2 ) and patients on insulin or other injectable therapy were also excluded from the analysis .", "demographics , and clinical conditions and health care resource utilization recorded up to 5  years before therapy initiation were assessed as baseline characteristics . over 70 clinical conditions and comorbidities may have been recorded in the database , including diabetes complications , cancers , and cardiovascular ( cv ) , metabolic , gastrointestinal , hepatic , infectious , psychiatric , pulmonary , and neurological events .", "types of health care resource utilization recorded in the database included physician and emergency department visits , hospitalizations , days hospitalized , and number of medications received .", "differences between sitagliptin and non - dpp-4i oaha treatment groups were compared using absolute standardized differences ( asd ) .", "asd is the difference of two means or proportions divided by the pooled estimate of the standard deviation . unlike the traditional p value , asd is a measure of difference that is not influenced by large sample sizes and has been demonstrated to be a better measure of covariate balance [ 10 , 12 ] .", "an asd of at least 10% was used to indicate a meaningful difference between treatment groups .", "this article does not contain any new studies with human or animal subjects performed by any of the authors .", "a total of 35,922 patients with t2 dm and ckd were identified as meeting the inclusion criteria . over 45% of patients ( 46.7% ; n  =  16,742 ) initiated sitagliptin ( n  =  1234 ) or a non - dpp-4i oaha monotherapy ( n  =  15,508 ) , 40.5% ( n  =  14,540 ) initiated an escalation to dual combination therapy ( sitagliptin , n  =  2683 ; oaha , n  =  11,857 ) , and 12.9% ( n  =  4640 ) initiated an escalation to triple combination therapy ( sitagliptin , n  =  1385 ; oaha , n  =  3255 ) . roughly , 15% of patients with t2 dm and ckd ( 14.8% ; n  =  5302 ) initiated treatment with sitagliptin . in comparison , in the patients excluded from this analysis due to a lack of recorded ckd , the percentage of patients initiating sitagliptin was 7.4% .", "the greatest differences between treatment groups were observed in patients initiating monotherapy or an escalation to dual combination therapy . compared to patients initiating monotherapy with non - dpp-4i oahas ,", "patients initiating monotherapy with sitagliptin were older ( mean [ standard deviation ( sd ) ] : sitagliptin 68.8  [ 12.5 ]  years , non - dpp-4i 66.6  [ 12.8 ]  years ; asd 17% ) , were more likely to have a history of heart failure ( fig . ", "1a ; sitagliptin 23.0% , non - dpp-4i 18.6% ; asd 11% ) or arrhythmia ( fig . ", "1a ; sitagliptin 37.7% , non - dpp-4i 31.7% ; asd 13% ) , were more likely to use loop diuretics ( fig . ", "1a ; sitagliptin 44.2% , non - dpp-4i 38.0% ; asd 13% ) or beta - blockers ( fig .  1a", "; sitagliptin 66.3% , non - dpp-4i 61.3% ; asd 11% ) , and had more physician visits ( fig . ", "1b ; mean [ sd ] : sitagliptin 73.2  [ 57.6 ] physician visits , non - dpp-4i 66.3  [ 55.4 ] physician visits ; asd 12% ) .", "the differences between treatment groups ( non - dpp-4i oaha users versus sitagliptin users ) observed in patients initiating an escalation to dual therapy were similar to those observed in patients initiating monotherapy , with the exception that the between - group age difference was not as great ( mean [ sd ] : sitagliptin 71.1  [ 11.1 ]  years , non - dpp-4i 70.0  [ 11.0 ]  years ; asd 10% ) and the differences for history of arrhythmia and use of beta - blockers were not meaningful ( fig .  1c , d).fig . ", "1baseline characteristics of patients with type 2 diabetes and chronic renal disease up to 5  years before initiating treatment with sitagliptin or non - dpp-4i oral antihyperglycemic agent as monotherapy or as part of dual or triple therapy . a , c , e clinical conditions and comorbidities .", "asd of 10% indicates a meaningful difference between treatment groups . for any between - group difference of asd of at least 10%", "asd absolute standardized difference , chf congestive heart failure , dpp-4i dipeptidyl peptidase-4 inhibitor , hosp hospital , htn hypertension , meds medications , mi myocardial infarction , phys physician , tia transient ischemic attack baseline characteristics of patients with type 2 diabetes and chronic renal disease up to 5  years before initiating treatment with sitagliptin or non - dpp-4i oral antihyperglycemic agent as monotherapy or as part of dual or triple therapy . a , c , e clinical conditions and comorbidities .", "asd of 10% indicates a meaningful difference between treatment groups . for any between - group difference of asd of at least 10% ,", "asd absolute standardized difference , chf congestive heart failure , dpp-4i dipeptidyl peptidase-4 inhibitor , hosp hospital , htn hypertension , meds medications , mi myocardial infarction , phys physician , tia transient ischemic attack in patients initiating an escalation to triple combination therapy , the differences between treatment groups ( non - dpp-4i oaha users versus sitagliptin users ) were not as pronounced as those seen in patients initiating monotherapy or escalation to dual therapy , including the between - group age difference ( mean [ sd ] : sitagliptin 68.9  [ 10.9 ]  years , non - dpp-4i 68.4  [ 10.5 ]  years ; asd 5% ; fig . ", "in this study of patients with t2 dm from an employee - based insurance database , sitagliptin was initiated in a higher percentage of patients with t2 dm and ckd ( 14.8% ) compared to patients with t2 dm but no record of ckd ( 7.4% ) . unlike many other oahas , sitagliptin is approved for patients with any stage of renal disease . in light of this and its favorable renal safety profile [ 1215 ] ,", "the higher use of sitagliptin in patients with ckd observed in the current analysis is not surprising . in general ,", "patients with t2 dm and ckd who initiated treatment with sitagliptin tended to be older and were more likely to have a pre - treatment history of heart failure , arrhythmia , or use of loop diuretics or beta - blockers than patients initiating other classes of oaha . in this context , it is worth noting the results of a large , recently completed clinical trial examining the effects of adding  sitagliptin to usual care in patients with t2 dm and cv disease . in the overall study population , no difference in cv event rates compared with placebo was observed ( hazard ratio [ hr ] for the primary composite cv outcome was 0.98 ; 95%  confidence interval ( ci ) : 0.88 , 1.09 ; p  <  0.001 for noninferiority ) . additionally , in patient subgroups evaluated by renal function ,", "no difference in cv risk was noted for patients with ckd [ estimated glomerular filtration rate ( egfr ) < 60  ml / min/1.73  m ; hr  =  0.92 ; 95%  ci : 0.78 , 1.10 ) or those without ckd ( egfr 60  ml / min/1.73  m ; hr  =  1.00 ; 95%  ci : 0.89 , 1.13 ) .", "the most pronounced differences in baseline characteristics between the treatment groups were observed between patients initiating monotherapy .", "as treatment complexity increased , the differences in baseline characteristics between treatment groups persisted but were attenuated , presumably due to diminishing treatment options with increasing treatment complexity .", "these observations of channeling in patients receiving treatment with sitagliptin are similar to those previously reported in a general t2 dm population [ 47 ] .", "while the marketscan database includes insurance claims data on a large , diverse population from the us , these results may not be generalizable to the overall us population or to ex - us populations .", "in addition , the primary uses of these data are for administrative purposes , not research .", "consequently , the database has missing or limited data on a number of important disease characteristics and comorbidities .", "importantly for this study , patients with end - stage renal disease are likely underrepresented since these patients are medicare eligible .", "chronic renal disease was defined solely through icd-9-cm codes as laboratory data are not available in our dataset .", "this study further documents the presence of channeling in patients initiating treatment with sitagliptin . in this study , patients with ckd initiating treatment with sitagliptin were generally older and were more likely to have a pre - treatment history of heart failure , arrhythmia , or use of loop diuretics or beta - blockers than patients initiating other classes of oral therapies . if not recognized and analyzed appropriately , this channeling could lead to biased treatment effect estimates in comparative analyses , including those involving users of sitagliptin .", "k. g. brodovicz was an employee of merck sharp & dohme corp . , a subsidiary of merck & co. , inc . ,", "kenilworth , nj , at the time this analysis was carried out and may own stock and/or hold stock options in the company .", "kenilworth , nj , at the time this analysis was carried out and may own stock and/or hold stock options in the company .", ", a subsidiary of merck & co. , inc . , kenilworth , nj and may own stock and/or hold stock options in the company .", "this article does not contain any new studies with human or animal subjects performed by any of the authors .", "this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made ."], "abstract_text": ["<S> introductionchronic kidney disease ( ckd ) is common in patients with type 2 diabetes ( t2 dm ) and makes them particularly susceptible to safety / tolerability issues related to many classes of oral antihyperglycemic agents ( oaha ) . </S>", "<S> dipeptidyl peptidase-4 inhibitors ( dpp-4is ) like sitagliptin are generally well tolerated in patients with t2 dm and renal disease and therefore may be preferentially used in patients with ckd . to assess the extent of this preference , the characteristics of sitagliptin users with t2 dm and ckd were compared with those of other ( non - dpp-4i ) oaha users with t2 dm and ckd.methodspatients with t2 dm and ckd with claims between 2006 and 2012 were identified from a united states insurance claims database . </S>", "<S> patients starting sitagliptin or another oaha as mono , dual , or triple therapy were compared . </S>", "<S> demographic and clinical characteristics within 5  years before starting or escalating to new therapies were assessed.resultscompared to patients with ckd starting other oahas , patients with ckd starting sitagliptin as mono or dual therapy were older , had more physician visits , were more likely to have a history of heart failure and to use loop diuretics . in triple </S>", "<S> therapy patients , the differences between groups were not as pronounced , but the overall prevalences of comorbidities was higher.conclusionsimilar to prior observations in a general t2 dm population , patients with t2 dm and ckd prescribed sitagliptin tend to be older and have more comorbidities than those prescribed other classes of oaha . if not recognized and analyzed appropriately , this channeling could lead to biased treatment effect estimates in comparative analyses that include users of sitagliptin.fundingmerck & co. , inc . , </S>", "<S> kenilworth , nj , usa.electronic supplementary materialthe online version of this article ( doi:10.1007/s13300 - 015 - 0133-z ) contains supplementary material , which is available to authorized users . </S>"], "labels": null, "section_names": ["Funding", "Electronic supplementary material", "Introduction", "Methods", "Results", "Discussion", "Conclusions", "Electronic supplementary material", "Disclosures", "Compliance with ethics guidelines", "Open Access"], "sections": [[""], ["the online version of this article ( doi:10.1007/s13300 - 015 - 0133-z ) contains supplementary material , which is available to authorized users ."], ["chronic kidney disease ( ckd ) is a common condition in patients with type 2 diabetes ( t2 dm ) .", "an estimated 2035% of patients with t2 dm have moderate to severe renal impairment [ 1 , 2 ]", ". however , many antihyperglycemic medications are contraindicated or need to be used with caution in patients with ckd , complicating t2 dm treatment choices and management .", "patients with t2 dm and ckd are particularly susceptible to safety and tolerability issues related to many classes of oral antihyperglycemic agents ( oaha ) .", "dipeptidyl peptidase-4 inhibitors ( dpp-4i ) such as sitagliptin are well tolerated in a broad range of t2 dm patient types , including those with renal disease , and may therefore be preferentially used in patients with ckd .", "prior studies have demonstrated the preferential use of sitagliptin in several populations [ 47 ] . in general , patients initiating treatment with sitagliptin were older and had more complications of diabetes and comorbidities than patients initiating other antihyperglycemic therapies [ 47 ] . if not recognized and appropriately considered in the analysis , this preferential selection of patients with specific demographic and disease characteristics for treatment with sitagliptin ( channeling bias ) could lead to inaccurate treatment effect estimates in comparative analyses that include sitagliptin .", "the objective of this study was to describe the baseline characteristics of patients with t2 dm and ckd initiating treatment with sitagliptin or non - dpp-4i oahas to ascertain whether channeling exists in this patient population ."], ["the truven health marketscan databases ( marketscan , truven health analytics , ann arbor , mi , usa ) contain medical claims records for more than 150  million unique patients dating from 1996 .", "the records are derived from outpatient and inpatient insurance claims for employees of over 100 employers participating in more than 12 health plans , and their beneficiaries in the united states .", "records consist of commercial claims and healthcare encounters , including information on demographics , health plan membership , international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) codes , and current procedure terminology ( cpt ) codes .", "the records of retirees with supplemental insurance are included in the database thus providing data on the elderly with continuity of care across those < 65 and 65  years of age .", "patients 25  years of age with t2 dm and ckd , with claims in the united states ( us ) between january 2006 and june 2012 , were identified in marketscan . of these patients ,", "those initiating sitagliptin or a non - dpp-4i oaha were categorized by complexity of antihyperglycemic treatment .", "patients were identified as having t2 dm if marketscan records for the patient indicated at least one inpatient or outpatient diagnosis of diabetes and at least one prescription for oaha medication .", "patients with ckd were identified by icd-9-cm diagnostic codes ( 585 , 585.3 , 585.4 , 585.5 , 585.6 , 585.9 , 403 , 403.0 , 403.00 , 403.01 , 403.1 , 403.10 , 403.11 , 403.9 , 403.90 , 403.91 , 250.4 , 404 , 404.0 , 404.00 , 404.01 , 404.02 , 404.03 , 404.1 , 404.10 , 404.11 , 404.12 , 404.13 , 404.9 , 404.90 , 404.91 , 404.92 , 404.93 , 582 , 582.0 , 582.1 , 582.2 , 582.4 , 582.8 , 582.81 , 582.89 , 582.9 ) .", "antihyperglycemic treatment was defined as : ( 1 ) initiating monotherapy ( 1 new outpatient prescription record on or after the t2 dm diagnosis ) ; ( 2 ) escalating to dual combination therapy ( 1 new prescription for a 2nd class 90  days after the 1st class , with prescription for 1st class overlapping the index date of 2nd class ) ; ( 3 ) escalating to triple combination therapy ( 1 new prescription for a 3rd class 90  days after the 2nd class , with prescriptions for 1st and 2nd classes overlapping the index date of 3rd class ) .", "patients were required to have at least 1  year of continuous enrollment in the database prior to initiation / escalation of antihyperglycemic treatment .", "patients were excluded from the analysis if they had a diagnosis of type 1 diabetes , ketoacidosis , malnutrition - associated diabetes , drug - induced diabetes or gestational diabetes without a subsequent t2 dm diagnosis code .", "patients with icd-9-cm codes explicit for mild renal disease ( stage 1 and 2 ) and patients on insulin or other injectable therapy were also excluded from the analysis .", "demographics , and clinical conditions and health care resource utilization recorded up to 5  years before therapy initiation were assessed as baseline characteristics . over 70 clinical conditions and comorbidities may have been recorded in the database , including diabetes complications , cancers , and cardiovascular ( cv ) , metabolic , gastrointestinal , hepatic , infectious , psychiatric , pulmonary , and neurological events .", "types of health care resource utilization recorded in the database included physician and emergency department visits , hospitalizations , days hospitalized , and number of medications received .", "differences between sitagliptin and non - dpp-4i oaha treatment groups were compared using absolute standardized differences ( asd ) .", "asd is the difference of two means or proportions divided by the pooled estimate of the standard deviation . unlike the traditional p value , asd is a measure of difference that is not influenced by large sample sizes and has been demonstrated to be a better measure of covariate balance [ 10 , 12 ] .", "an asd of at least 10% was used to indicate a meaningful difference between treatment groups .", "this article does not contain any new studies with human or animal subjects performed by any of the authors ."], ["a total of 35,922 patients with t2 dm and ckd were identified as meeting the inclusion criteria . over 45% of patients ( 46.7% ; n  =  16,742 ) initiated sitagliptin ( n  =  1234 ) or a non - dpp-4i oaha monotherapy ( n  =  15,508 ) , 40.5% ( n  =  14,540 ) initiated an escalation to dual combination therapy ( sitagliptin , n  =  2683 ; oaha , n  =  11,857 ) , and 12.9% ( n  =  4640 ) initiated an escalation to triple combination therapy ( sitagliptin , n  =  1385 ; oaha , n  =  3255 ) . roughly , 15% of patients with t2 dm and ckd ( 14.8% ; n  =  5302 ) initiated treatment with sitagliptin . in comparison , in the patients excluded from this analysis due to a lack of recorded ckd , the percentage of patients initiating sitagliptin was 7.4% .", "the greatest differences between treatment groups were observed in patients initiating monotherapy or an escalation to dual combination therapy . compared to patients initiating monotherapy with non - dpp-4i oahas ,", "patients initiating monotherapy with sitagliptin were older ( mean [ standard deviation ( sd ) ] : sitagliptin 68.8  [ 12.5 ]  years , non - dpp-4i 66.6  [ 12.8 ]  years ; asd 17% ) , were more likely to have a history of heart failure ( fig . ", "1a ; sitagliptin 23.0% , non - dpp-4i 18.6% ; asd 11% ) or arrhythmia ( fig . ", "1a ; sitagliptin 37.7% , non - dpp-4i 31.7% ; asd 13% ) , were more likely to use loop diuretics ( fig . ", "1a ; sitagliptin 44.2% , non - dpp-4i 38.0% ; asd 13% ) or beta - blockers ( fig .  1a", "; sitagliptin 66.3% , non - dpp-4i 61.3% ; asd 11% ) , and had more physician visits ( fig . ", "1b ; mean [ sd ] : sitagliptin 73.2  [ 57.6 ] physician visits , non - dpp-4i 66.3  [ 55.4 ] physician visits ; asd 12% ) .", "the differences between treatment groups ( non - dpp-4i oaha users versus sitagliptin users ) observed in patients initiating an escalation to dual therapy were similar to those observed in patients initiating monotherapy , with the exception that the between - group age difference was not as great ( mean [ sd ] : sitagliptin 71.1  [ 11.1 ]  years , non - dpp-4i 70.0  [ 11.0 ]  years ; asd 10% ) and the differences for history of arrhythmia and use of beta - blockers were not meaningful ( fig .  1c , d).fig . ", "1baseline characteristics of patients with type 2 diabetes and chronic renal disease up to 5  years before initiating treatment with sitagliptin or non - dpp-4i oral antihyperglycemic agent as monotherapy or as part of dual or triple therapy . a , c , e clinical conditions and comorbidities .", "asd of 10% indicates a meaningful difference between treatment groups . for any between - group difference of asd of at least 10%", "asd absolute standardized difference , chf congestive heart failure , dpp-4i dipeptidyl peptidase-4 inhibitor , hosp hospital , htn hypertension , meds medications , mi myocardial infarction , phys physician , tia transient ischemic attack baseline characteristics of patients with type 2 diabetes and chronic renal disease up to 5  years before initiating treatment with sitagliptin or non - dpp-4i oral antihyperglycemic agent as monotherapy or as part of dual or triple therapy . a , c , e clinical conditions and comorbidities .", "asd of 10% indicates a meaningful difference between treatment groups . for any between - group difference of asd of at least 10% ,", "asd absolute standardized difference , chf congestive heart failure , dpp-4i dipeptidyl peptidase-4 inhibitor , hosp hospital , htn hypertension , meds medications , mi myocardial infarction , phys physician , tia transient ischemic attack in patients initiating an escalation to triple combination therapy , the differences between treatment groups ( non - dpp-4i oaha users versus sitagliptin users ) were not as pronounced as those seen in patients initiating monotherapy or escalation to dual therapy , including the between - group age difference ( mean [ sd ] : sitagliptin 68.9  [ 10.9 ]  years , non - dpp-4i 68.4  [ 10.5 ]  years ; asd 5% ; fig . "], ["in this study of patients with t2 dm from an employee - based insurance database , sitagliptin was initiated in a higher percentage of patients with t2 dm and ckd ( 14.8% ) compared to patients with t2 dm but no record of ckd ( 7.4% ) . unlike many other oahas , sitagliptin is approved for patients with any stage of renal disease . in light of this and its favorable renal safety profile [ 1215 ] ,", "the higher use of sitagliptin in patients with ckd observed in the current analysis is not surprising . in general ,", "patients with t2 dm and ckd who initiated treatment with sitagliptin tended to be older and were more likely to have a pre - treatment history of heart failure , arrhythmia , or use of loop diuretics or beta - blockers than patients initiating other classes of oaha . in this context , it is worth noting the results of a large , recently completed clinical trial examining the effects of adding  sitagliptin to usual care in patients with t2 dm and cv disease . in the overall study population , no difference in cv event rates compared with placebo was observed ( hazard ratio [ hr ] for the primary composite cv outcome was 0.98 ; 95%  confidence interval ( ci ) : 0.88 , 1.09 ; p  <  0.001 for noninferiority ) . additionally , in patient subgroups evaluated by renal function ,", "no difference in cv risk was noted for patients with ckd [ estimated glomerular filtration rate ( egfr ) < 60  ml / min/1.73  m ; hr  =  0.92 ; 95%  ci : 0.78 , 1.10 ) or those without ckd ( egfr 60  ml / min/1.73  m ; hr  =  1.00 ; 95%  ci : 0.89 , 1.13 ) .", "the most pronounced differences in baseline characteristics between the treatment groups were observed between patients initiating monotherapy .", "as treatment complexity increased , the differences in baseline characteristics between treatment groups persisted but were attenuated , presumably due to diminishing treatment options with increasing treatment complexity .", "these observations of channeling in patients receiving treatment with sitagliptin are similar to those previously reported in a general t2 dm population [ 47 ] .", "while the marketscan database includes insurance claims data on a large , diverse population from the us , these results may not be generalizable to the overall us population or to ex - us populations .", "in addition , the primary uses of these data are for administrative purposes , not research .", "consequently , the database has missing or limited data on a number of important disease characteristics and comorbidities .", "importantly for this study , patients with end - stage renal disease are likely underrepresented since these patients are medicare eligible .", "chronic renal disease was defined solely through icd-9-cm codes as laboratory data are not available in our dataset ."], ["this study further documents the presence of channeling in patients initiating treatment with sitagliptin . in this study , patients with ckd initiating treatment with sitagliptin were generally older and were more likely to have a pre - treatment history of heart failure , arrhythmia , or use of loop diuretics or beta - blockers than patients initiating other classes of oral therapies . if not recognized and analyzed appropriately , this channeling could lead to biased treatment effect estimates in comparative analyses , including those involving users of sitagliptin ."], [""], ["k. g. brodovicz was an employee of merck sharp & dohme corp . , a subsidiary of merck & co. , inc . ,", "kenilworth , nj , at the time this analysis was carried out and may own stock and/or hold stock options in the company .", "kenilworth , nj , at the time this analysis was carried out and may own stock and/or hold stock options in the company .", ", a subsidiary of merck & co. , inc . , kenilworth , nj and may own stock and/or hold stock options in the company ."], ["this article does not contain any new studies with human or animal subjects performed by any of the authors ."], ["this article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license ( http://creativecommons.org/licenses/by-nc/4.0/ ) , which permits any noncommercial use , distribution , and reproduction in any medium , provided you give appropriate credit to the original author(s ) and the source , provide a link to the creative commons license , and indicate if changes were made ."]]}
{"article_id": "PMC5245924", "article_text": ["the use of the kuntscher nail has been the most important advancement in trauma surgery .", "he had an acetabulum fracture and a femoral shaft fracture treated 30 years ago with a reamed kuntscher femoral nail .", "lateral hip approach was performed and after attempting to remove the nail with the specific tools being unsuccessful we decided to be more aggressive .", "firstly , we performed a simple unicortical osteotomy on the lateral side from the proximal part to below the callus in order to decompress the femoral canal without success .", "secondly , a trench in the greater trochanter around the proximal hole was performed to hit the nail from below which was still insufficient and furthermore , the hole broke when hitting the nail so we needed to drill a new hole distally .", "several cerclages closed the osteotomy and a bone graft was used to close the trench .", "with this case report , we present a new salvage technique to remove an incarcerated kuntscher nail when all the described methods have failed .", "the use of the kuntscher nailing system for the treatment of long bone fractures has been the most important advancement in trauma surgery in recent history . since its first implementation in a german hospital in the late 30 s ,", "removal of a kuntscher nail is considered a routine procedure but could be really challenging .", "bone ingrowth or overgrowth , damage to the proximal threads of the nail , and broken nails or locking screws may complicate the removal of intramedullary nails .", "the biggest challenge appears when all the described methods result in failure of removal of the nail .", "this article presents a case of an incarcerated femoral nail and describes a salvage procedure for nail extraction after all previously described methods have failed .", "the patient was involved in a plane accident 30 years before sustaining a femoral shaft fracture treated with a reamed kuntscher nail and a posterior wall acetabulum fracture treated with open reduction and internal fixation with lag screws ( fig.1 ) .", "the patient came to our outpatient clinic complaining about inguinal pain and chronic leg pain with limitation of activities of daily living , although he was able to walk for more than one hour . on physical examination ,", "the ipsilateral knee had full range of motion and the patient did not need any crutches to walk without limping .", "the postelmerldaubign scale was 14 points ( 4 for pain , 5 for walking and 5 for range of motion ) .", "as a part of the initial evaluation , a blood test with infection parameters was performed and was negative for infection .", "pre - operative radiographs showed a healed femoral fracture in all 4 cortices with an intramedullary kuntscher nail without locking screws and cortical thickening at the fracture site ( fig .", "pre - operative x - ray of the pelvic ring : intramedullary kuntscher nail in the left femur and osteosynthesis with three screws in the posterior wall of the acetabulum .", "pre - operative x - ray of the distal part of the femur : fracture site healed and distal tip of the nail .", "the patient was informed about the incipient arthritis of the hip and the possibility to perform a one - stage or two - stage surgery .", "the surgical risks were discussed with the patient , including pain after hardware removal and failure to remove the nail , specially this last point because other surgeons had tried to do the same procedure few years before and had failed after many hours of surgery .", "firstly , remove the nail at all costs and depending on the outcome of this first surgery he would assess the hip replacement surgery . the patient was put in a lateral position and a lateral hip approach was done .", "the first step was to find the proximal tip of the nail which was found seated deep in the great trochanter .", "the initial device used to remove the nail was the conical extraction tool that was unsuccessful after several attempts .", "after the overgrowth bone was removed of the proximal part of the nail a hook extraction system was engaged with difficulty .", "however , the nail did not move after multiple hits with a 1 kg hammer and finally the hook broke itself . at this point", ", we decided to use the saw to perform a simple unicortical osteotomy in the proximal third of the femur to decompress the endomedullar canal ( fig .", "3 ) . after this decompression osteotomy we tried to hit the nail from below with an impactor engaged in the proximal hole of the nail . however , the implant remained in the same position .", "clinicalintra operative image : simple unicortical osteotomy in the proximal third of the femur to decompress the endomedullar canal . after trying all these different ways to remove the nail being unsuccessful", ", we decided to continue the longitudinal osteotomy from proximal to distal in the lateral side of the femur .", "the nail was found in place with on growth and overgrowth in almost entire length of the nail .", "a new attempt to hammer the nail from below was performed and the nail started to move gradually until the proximal hole of the nail broke ( fig .", "4 ) . after so many attempts , we did not give up and proceeded to drill a new hole distally with a diamond drill ( fig .", "5 ) to finish the nail removal . clinical intra - operative image : detail of the moment when the nail started to move gradually until the proximal hole of the nail break . clinical image .", "subsequently , several cerclages and a bone allograft was used to close the longitudinal osteotomy ( fig .", "clinical intraoperative image : several cerclages closing the osteotomy with allograft bone used to close the longitudinal osteotomy .", "the patient was admitted to the hospital for pain and bleeding control . on the second day of hospitalization ,", "the patient underwent a blood transfusion as his hemoglobin was reported to be 7.1 g / dl .", "the patient was discharged on day 4 after antibiotic prophylaxis , deep vein thrombosis prophylaxis with low - weight - molecular heparin and ambulation with crutches without weight bearing . at 8 weeks of follow - up", "the patient was satisfied at 1 year of follow - up with no complications and the x - ray showed complete healing of the longitudinal osteotomy ( fig .", ", the patient is free from pain in the leg with only sporadic hip pain , so there is no indication of hip replacement surgery for the time being", "various reasons have been described in the literature for nail incarceration , including excessive callus formation closing the intramedullary canal , ingrowth of bone into the nail , bone ingrowth through the locking holes and bent or broken nails .", "there are different techniques to remove retained intramedullary nails , most of these techniques are designed for broken nails , but others are for incarcerated nails .", "there is only one case similar to our case report by lindeque et al which describes removal of tibial nail using a large longitudinal osteotomy .", "we decided to perform a two - stage procedure especially due to the incipient arthritis of the hip .", "if our patient would have had advanced stage of arthritis , we could have considered a single - staged procedure with nail removal and hip replacement or a hip replacement without removing the nail .", "goosen et al treated a similar case in which they cut the proximal end of the nail to the length needed in order to implant a hip arthroplasty at the top of the nail .", "mont et al used this technique on fifteen patients with femoral implants or extraarticular deformities .", "however , despite the aggressiveness of the procedure , the patient had a good result , and the osteotomy healed normally with no complications .", "this is the first case published in the literature using this longitudinal osteotomy to remove a femoral nail .", "it is recommended to remove it as soon as it possible to minimize later complications ."], "abstract_text": ["<S> introduction : the use of the kuntscher nail has been the most important advancement in trauma surgery . </S>", "<S> one of the problems is the difficulty to remove it . </S>", "<S> a new extraction technique is described in the present case report.case report : a 46-year - old man was referred for hip osteoarthritis . </S>", "<S> he had an acetabulum fracture and a femoral shaft fracture treated 30 years ago with a reamed kuntscher femoral nail . </S>", "<S> lateral hip approach was performed and after attempting to remove the nail with the specific tools being unsuccessful we decided to be more aggressive . </S>", "<S> firstly , we performed a simple unicortical osteotomy on the lateral side from the proximal part to below the callus in order to decompress the femoral canal without success . </S>", "<S> secondly , a trench in the greater trochanter around the proximal hole was performed to hit the nail from below which was still insufficient and furthermore , the hole broke when hitting the nail so we needed to drill a new hole distally . </S>", "<S> finally , the kuntscher nail was removed . </S>", "<S> several cerclages closed the osteotomy and a bone graft was used to close the trench . </S>", "<S> the patient had a good evolution at one year of follow-up.conclusion:with this case report , we present a new salvage technique to remove an incarcerated kuntscher nail when all the described methods have failed . </S>"], "labels": null, "section_names": ["Introduction:", "Case Report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["the use of the kuntscher nail has been the most important advancement in trauma surgery ."], ["he had an acetabulum fracture and a femoral shaft fracture treated 30 years ago with a reamed kuntscher femoral nail .", "lateral hip approach was performed and after attempting to remove the nail with the specific tools being unsuccessful we decided to be more aggressive .", "firstly , we performed a simple unicortical osteotomy on the lateral side from the proximal part to below the callus in order to decompress the femoral canal without success .", "secondly , a trench in the greater trochanter around the proximal hole was performed to hit the nail from below which was still insufficient and furthermore , the hole broke when hitting the nail so we needed to drill a new hole distally .", "several cerclages closed the osteotomy and a bone graft was used to close the trench ."], ["with this case report , we present a new salvage technique to remove an incarcerated kuntscher nail when all the described methods have failed ."], ["the use of the kuntscher nailing system for the treatment of long bone fractures has been the most important advancement in trauma surgery in recent history . since its first implementation in a german hospital in the late 30 s ,", "removal of a kuntscher nail is considered a routine procedure but could be really challenging .", "bone ingrowth or overgrowth , damage to the proximal threads of the nail , and broken nails or locking screws may complicate the removal of intramedullary nails .", "the biggest challenge appears when all the described methods result in failure of removal of the nail .", "this article presents a case of an incarcerated femoral nail and describes a salvage procedure for nail extraction after all previously described methods have failed ."], ["the patient was involved in a plane accident 30 years before sustaining a femoral shaft fracture treated with a reamed kuntscher nail and a posterior wall acetabulum fracture treated with open reduction and internal fixation with lag screws ( fig.1 ) .", "the patient came to our outpatient clinic complaining about inguinal pain and chronic leg pain with limitation of activities of daily living , although he was able to walk for more than one hour . on physical examination ,", "the ipsilateral knee had full range of motion and the patient did not need any crutches to walk without limping .", "the postelmerldaubign scale was 14 points ( 4 for pain , 5 for walking and 5 for range of motion ) .", "as a part of the initial evaluation , a blood test with infection parameters was performed and was negative for infection .", "pre - operative radiographs showed a healed femoral fracture in all 4 cortices with an intramedullary kuntscher nail without locking screws and cortical thickening at the fracture site ( fig .", "pre - operative x - ray of the pelvic ring : intramedullary kuntscher nail in the left femur and osteosynthesis with three screws in the posterior wall of the acetabulum .", "pre - operative x - ray of the distal part of the femur : fracture site healed and distal tip of the nail .", "the patient was informed about the incipient arthritis of the hip and the possibility to perform a one - stage or two - stage surgery .", "the surgical risks were discussed with the patient , including pain after hardware removal and failure to remove the nail , specially this last point because other surgeons had tried to do the same procedure few years before and had failed after many hours of surgery .", "firstly , remove the nail at all costs and depending on the outcome of this first surgery he would assess the hip replacement surgery . the patient was put in a lateral position and a lateral hip approach was done .", "the first step was to find the proximal tip of the nail which was found seated deep in the great trochanter .", "the initial device used to remove the nail was the conical extraction tool that was unsuccessful after several attempts .", "after the overgrowth bone was removed of the proximal part of the nail a hook extraction system was engaged with difficulty .", "however , the nail did not move after multiple hits with a 1 kg hammer and finally the hook broke itself . at this point", ", we decided to use the saw to perform a simple unicortical osteotomy in the proximal third of the femur to decompress the endomedullar canal ( fig .", "3 ) . after this decompression osteotomy we tried to hit the nail from below with an impactor engaged in the proximal hole of the nail . however , the implant remained in the same position .", "clinicalintra operative image : simple unicortical osteotomy in the proximal third of the femur to decompress the endomedullar canal . after trying all these different ways to remove the nail being unsuccessful", ", we decided to continue the longitudinal osteotomy from proximal to distal in the lateral side of the femur .", "the nail was found in place with on growth and overgrowth in almost entire length of the nail .", "a new attempt to hammer the nail from below was performed and the nail started to move gradually until the proximal hole of the nail broke ( fig .", "4 ) . after so many attempts , we did not give up and proceeded to drill a new hole distally with a diamond drill ( fig .", "5 ) to finish the nail removal . clinical intra - operative image : detail of the moment when the nail started to move gradually until the proximal hole of the nail break . clinical image .", "subsequently , several cerclages and a bone allograft was used to close the longitudinal osteotomy ( fig .", "clinical intraoperative image : several cerclages closing the osteotomy with allograft bone used to close the longitudinal osteotomy .", "the patient was admitted to the hospital for pain and bleeding control . on the second day of hospitalization ,", "the patient underwent a blood transfusion as his hemoglobin was reported to be 7.1 g / dl .", "the patient was discharged on day 4 after antibiotic prophylaxis , deep vein thrombosis prophylaxis with low - weight - molecular heparin and ambulation with crutches without weight bearing . at 8 weeks of follow - up", "the patient was satisfied at 1 year of follow - up with no complications and the x - ray showed complete healing of the longitudinal osteotomy ( fig .", ", the patient is free from pain in the leg with only sporadic hip pain , so there is no indication of hip replacement surgery for the time being"], ["various reasons have been described in the literature for nail incarceration , including excessive callus formation closing the intramedullary canal , ingrowth of bone into the nail , bone ingrowth through the locking holes and bent or broken nails .", "there are different techniques to remove retained intramedullary nails , most of these techniques are designed for broken nails , but others are for incarcerated nails .", "there is only one case similar to our case report by lindeque et al which describes removal of tibial nail using a large longitudinal osteotomy .", "we decided to perform a two - stage procedure especially due to the incipient arthritis of the hip .", "if our patient would have had advanced stage of arthritis , we could have considered a single - staged procedure with nail removal and hip replacement or a hip replacement without removing the nail .", "goosen et al treated a similar case in which they cut the proximal end of the nail to the length needed in order to implant a hip arthroplasty at the top of the nail .", "mont et al used this technique on fifteen patients with femoral implants or extraarticular deformities ."], ["however , despite the aggressiveness of the procedure , the patient had a good result , and the osteotomy healed normally with no complications .", "this is the first case published in the literature using this longitudinal osteotomy to remove a femoral nail .", "it is recommended to remove it as soon as it possible to minimize later complications ."]]}
{"article_id": "PMC2659690", "article_text": ["the anticancer agent 5-fluoro-2-deoxyuridine ( floxuridine ) has been shown to be clinically effective in the treatment of colon carcinoma and colorectal cancer that has metastasized to the liver . however , the adverse effects associated with chemotherapeutics are still unresolved , and many efforts have been made to minimize side - effects and maximize therapeutic efficacy .", "prodrug strategies have been increasingly utilized over the past two decades in order to overcome undesirable physicochemical properties of drugs , to improve oral bioavailability and to minimize toxic side - effects .", "a majority of the efforts have focused on antiviral and anticancer drugs and reflect the need for improved targeting , more selective action and further development of orally available alternatives .", "amino acid ester prodrugs of poorly permeant anticancer and antiviral drugs have been designed for targeted delivery via specific transporters in order to improve their oral bioavailability and metabolic disposition .", "amino acid ester prodrugs have been shown to be substrates for pept1 , pept2 , and atb transporters , and their improved oral bioavailability has been attributed to enhanced transport via carrier - mediated mechanisms .", "alkyl ester prodrugs and amino acid ester prodrugs of floxuridine , for example , have been synthesized and tested for potential improvement of oral drug delivery .", "amino acid ester prodrugs of floxuridine and an antiviral agent , acyclovir , have been shown to be substrates of the pept1 transporter .", "pept1 is stereoselective and exhibits greater affinity for l - enantiomers of amino acids than d - enantiomers.(16 ) pept1 is predominantly expressed in the small intestine , has broad substrate specificity and can transport dipeptides , tripeptides , and -lactam antibiotics .", "the valyl ester prodrug of acyclovir significantly enhances oral bioavailability of acyclovir , and several reports suggest that oligopeptide transporters are responsible for absorption enhancement .", "amino acid ester prodrugs may also facilitate enhanced delivery to pancreatic ductal cancer cells such as aspc-1 and capan-2 , since these cells express oligopeptide transporters at relatively high levels.(25 ) the activation of prodrug to the parent drug following transport is an essential step and can not be ignored . it has been shown that a specific enzyme , valacyclovirase , is primarily responsible for the conversion of valacyclovir to acyclovir .", "it has also been suggested that this enzyme might be involved in the activation of other amino acid prodrugs.(26 ) kim and colleagues reported that the substrate specificity of valacyclovirase is largely determined by the amino acid acyl - linked promoiety of the prodrug.(27 ) the metabolic conversion of floxuridine to 5-fluorouracil following systemic delivery has been shown to be detrimental to therapeutic efficacy of floxuridine .", "the mechanism of action of 5-fluorouracil ( 5-fu ) and floxuridine ( fudr ) is well understood.(30 ) 5-fu toxicity is predominantly caused by 5-fu incorporation into rna .", "however , unlike 5-fu , fudr is specifically incorporated into dna and not into rna , which leads to the minimization of adverse effects .", "several groups have reported that floxuridine is more potent than 5-fu and that the inhibition of cell proliferation is 10- to 100-fold higher than that of 5-fu .", "however , floxuridine is rapidly converted to 5-fu in many tissues , including the liver , by the enzyme thymidine phosphorylase.(34 ) as a consequence , higher doses of floxuridine are required for maintenance of clinical efficacy , leading to greater toxicity .", "therefore , protection of the glycosidic bond of floxuridine is expected to maintain the high potency of the drug and facilitate administration of low doses that can selectively kill only proliferating cells by robust inhibition of dna synthesis . improving the chemical stability of floxuridine to thymidine phosphorylase may enhance its therapeutic efficacy at low doses and obviate toxicity concerns .", "although amino acid monoester prodrugs of floxuridine have been shown to provide enhanced pept1-mediated transport as well as enzymatic activation in intestinal and liver surrogate cell systems , dipeptide analogues may exhibit even higher affinity and transport via the oligopeptide transporter .", "therefore , dipeptide prodrugs may be delivered more to a target site by carrier mediated transporters , and extra amino acid attached prodrugs could be more suitable for specific enzymatic activation at a target site than mono amino acid ester prodrugs .", "several studies on amino acid modifications to increase specific transporter affinity have been conducted . however , direct comparisons of mono amino acid and dipeptide prodrugs in terms of transporter affinity , prodrug stability and activation have not been reported . in this report", ", we describe the synthesis , characterization , and stability of dipeptide monoester prodrugs of floxuridine . various dipeptides and peptidomimetics", "have been tested to characterize the hpept1 transporter and improve its affinity , and mono amino acid ester prodrugs have been evaluated as hpept1 substrates .", "based on those reports , six amino acids were chosen to be n - terminal amino acids of the dipeptide . the smallest amino acid , glycine , and bulky amino acids like phenylalanine and tyrosine were paired with those six amino acids to form dipeptides to test the hypothesis that molecular sizes may structurally affect its ester bond stability .", "the six dipeptide promoieties , l - phenylalanalyl - l - glycine , l - leucyl - l - glycine , l - glycyl - l - leucine , l - isoleucyl - l - glycine , l - valyl - l - phenylalanine , and l - phenylalanyl - l - tyrosine , were designed for targeted delivery to an oligopeptide transporter and tested . since", "5-ester prodrugs were found to exhibit higher affinity for transporters than 3-ester prodrugs,(14 ) only 5-dipeptide monoester floxuridine prodrugs were examined in this study .", "uptake inhibition and permeability studies were conducted with caco-2 cells as well as with aspc-1 and capan-2 cells .", "the chemical stability at physiological ph and the enzymatic activation of the prodrugs in caco-2 , aspc-1 , and capan-2 cell homogenates were also evaluated to determine the effects of the amino acid / dipeptide promoiety structure on enzyme - mediated activation .", "the feasibility of selective antiproliferative action of amino acid / dipeptide floxuridine prodrugs was also explored using cancer cells that overexpress pept1 .", "finally , the stability and transport characteristics of the dipeptide monoester prodrugs of floxuridine were compared with those of the corresponding amino acid monoester prodrugs .", "the tert - butyloxycarbonyl ( boc ) protected amino acids boc - l - isoleucine , boc - l - glycine , boc - l - valine , boc - l - phenylalanine , boc - l - leucine , boc - l - glycyl - l - leucine , boc - l - phenylalanalyl - l - glycine , boc - l - leucyl - l - glycine , boc - l - isoleucyl - l - glycine , boc - l - valyl - l - phenylalanine , and boc - l - phenylalanyl - l - tyrosine were obtained from chem - impex ( wood dale , il ) .", "high - performance liquid chromatography ( hplc ) grade acetonitrile was obtained from fisher scientific ( st . louis , mo ) .", "n , n - dicyclohexylcarbodiimide ( dcc ) , n , n - dimethylaminopyridine ( dmap ) , trifluoroacetic acid ( tfa ) , and all other reagents and solvents were purchased from aldrich chemical co. ( milwaukee , wi ) .", "cell culture reagents were obtained from invitrogen ( carlsbad , ca ) , and cell culture supplies were obtained from corning ( corning , ny ) and falcon ( lincoln park , nj ) .", "the synthesis and characterization of 5-mono amino acid ester prodrugs of floxuridine have been reported previously .", "briefly , boc - protected dipeptides boc - l - glycyl - l - leucine , boc - l - phenylalanalyl - l - glycine , boc - l - leucyl - l - glycine , boc - l - isoleucyl - l - glycine , boc - l - valyl - l - phenylalanine , and boc - l - phenylalanyl - l - tyrosine , ( 1.1 mmol ) , dcc ( 1.1 mmol ) , and dmap ( 0.1 mmol ) were allowed to react with floxuridine ( 1 mmol ) in 7 ml of dry dmf for 24 h. the reaction progress was monitored by tlc ( ethyl acetate ) .", "the residue was extracted with ethyl acetate ( 30 ml ) and washed with water ( 2  20 ml ) , and saturated nacl ( 20 ml ) .", "the three spots observed on tlc were separated and purified using column chromatography ( dichloromethane ( dcm)/methanol , 20:1 ) .", "the boc group was cleaved by treating the residues with 5 ml tfa : dcm ( 1:1 ) . after 4 h", "the tfa salts of amino acid prodrugs of floxuridine were obtained as white fluffy solids . the combined yield , consisting of 3-monoester , 5-monoester , and 3,5-diesters , of each floxuridine prodrug was 60% .", "the prodrugs were determined to be 90%99% pure by reverse - phase hplc , and were easily separated from their parent compounds by reverse - phase hplc .", "the observed molecular weights of all prodrugs , determined by electrospray ionization mass spectra ( esi - ms ) obtained on a micromass lct esi - ms , were found to be consistent with those predicted by their structures .", "the structural identities of the prodrugs were then confirmed using proton nuclear magnetic resonance spectra ( h nmr ) , obtained on a 300 mhz bruker dpx-300 nmr spectrometer .", "5-l - leucyl - l - glycyl - floxuridine : yield , 15% ; percent purity , 93% ; h nmr ( dmso - d6 )  , 0.840.92 ( 6h , m , ( ch3)2 ) , 1.501.77 ( 3h , m , ch2 ) , 2.122.28 ( 2h , m , c2 ) , 3.784.35 ( 7h , m , ch of leu and ch2 of gly , c3 , c4 , c5 ) , 6.16 ( 1h , t , c1 , j = 6.0 hz ) , 7.94 ( 1h , d , chf , j = 6.9 hz ) ; esi - ms , 417.1 ( m + h ) .", "5-l - glycyl - l - leucyl - floxuridine : yield , 15% ; percent purity , 96% ; h nmr ( dmso - d6 )  , 0.85 ( 3h , d , ch3 , j = 6.5 hz ) , 0.90 ( 3h , d , ch3 , j = 6.5 hz ) , 1.511.68 ( 3h , m , ch2 ) , 2.122.26 ( 2h , m , c2 ) , 3.544.40 ( 7h , m , ch of leu and ch2 of gly , c3 , c4 ,", "c5 ) , 6.16 ( 1h , t , c1 , j = 6.0 hz ) , 7.90 ( 1h , d , chf , j = 6.8 hz ) ; esi - ms , 417.1 ( m + h ) .", "5-l - valyl - l - phenylalanyl - floxuridine : yield , 30% ; percent purity , 95% ; h nmr ( dmso - d6 )  , 0.90 ( 3h , d , ch3 , j = 6.9 hz ) , 0.95 ( 3h , d , ch3 , j = 6.9 hz ) , 2.082.20 ( 3h , m , c2 , ch of val ) , 3.02 ( 2h , m , ch2 of phe ) , 3.624.60 ( 6h , m , ch of val and phe , c3 , c4 , c5 ) , 6.14 ( 1h , t , c1 , j = 6.1 hz ) , 7.227.31 ( 5h , m , aromatic protons ) , 7.91 ( 1h , d , chf , j = 6.9 hz ) ; esi - ms , 494.1 ( m + h ) .", "5-l - isoleucyl - l - glycyl - floxuridine : yield , 15% ; percent purity , 93% ; h nmr ( dmso - d6 )  , 0.87 ( 3h , t , ch3 , j=7.3 hz ) , 0.94 ( 3h , d , ch3 , j = 6.8 hz ) , 1.17 ( 1h , m , ch2 ) , 1.52 ( 1h , m , ch2 ) , 1.81 ( 1h , m , ch ) , 2.112.28 ( 2h , m , c2 ) , 3.904.31 ( 7h , m , ch of ile and ch2 of gly , c3 , c4 ,", "c5 ) , 6.15 ( 1h , t , c1 , j = 6.5 hz ) , 7.94 ( 1h , d , chf , j = 6.9 hz ) ; esi - ms , 416.9 ( m + h ) .", "5-l - phenylalanyl - l - glycyl - floxuridine : yield , 16% ; percent purity , 98% ; h nmr ( dmso - d6 )  , 2.122.28 ( 2h , m , c2 ) , 2.923.16 ( 2h , m , ch2 of phe ) , 3.904.34 ( 7h , m , ch of phe and ch2 of gly , c3 , c4 , c5 ) , 6.15 ( 1h , t , c1 , j = 6.5 hz ) , 7.277.36 ( 5h , m , aromatic protons ) , 7.94 ( 1h , d , chf , j = 6.5 hz ) ; esi - ms , 451.1 ( m + h ) .", "5-l - phenylalanyl - l - tyrosyl - floxuridine : yield , 3.2% ; percent purity , 99% ; h nmr ( dmso - d6 )  , 2.102.32 ( 2h , m , c2 ) , 2.722.92 ( 4h , m , ch2 of phe and tyr ) , 3.904.29 ( 6h , m , ch of phe and tyr , c3 , c4 , c5 ) , 6.15 ( 1h , m , c1 ) , 6.657.32 ( 9h , m , aromatic protons ) , 7.93 ( 1h , m , chf ) ; esi - ms , 557.2 ( m + h ) .", "capan-2 cells ( passages 5054 ) , and aspc-1 cells ( passages 6365 ) from american type culture collection ( rockville , md ) were routinely maintained in rpmi-1640 containing 10% fetal bovine serum .", "caco-2 cells ( passages 3035 ) from american type culture collection ( rockville , md ) were routinely maintained in dmem containing 10% fetal bovine serum , 1% nonessential amino acids , 1 mmol / l sodium pyruvate , and 1% l - glutamine at 5% co2 and 90% relative humidity at 37 c .", "cells were grown in antibiotic - free media to avoid the possible transport interference by antibiotics .", "the cells were washed with 5 ml of ph 7.4 phosphate buffer ( 10 mmol / l ) , lysed by ultrasonication ( micro ultrasonic cell disrupter model kt40 , kontes , vineland , nj ) , and pelleted by centrifugation for 5 min at 1000 g .", "protein amount was quantified with bio - rad ( hercules , ca ) dc protein assay using bovine serum albumin as a standard .", "the protein amount was adjusted to 500 g / ml , and the hydrolysis reactions were carried out in 96-well plates ( corning ) .", "caco-2 , aspc-1 , and capan-2 cell suspensions ( 250 l ) were placed in triplicate wells , the reactions started with the addition of substrate , and cells were incubated at 37 c for 120 min . at the desired time point , sample aliquots ( 35 l )", "were removed and added to 150 l of acetonitrile ( acn ) containing 0.1% tfa .", "the mixtures were filtered with a 0.45 m filter at 1000 g for 10 min at 4 c .", "undiluted plasma ( 250 l ) was added to each well in triplicate , and substrate was added to initiate the reactions that were conducted at 37 c for 2 h. at various time points , aliquots ( 35 l ) were removed and added to 150 l of acn containing 0.1% tfa .", "the mixtures were filtered with a 0.45 m filter at 1000 g for 10 min at 4 c .", "the nonenzymatic hydrolysis of the prodrugs was determined as described above , except that each well contained ph 7.4 phosphate buffer ( 10 mmol / l ) instead of cell homogenate or human plasma .", "the stability of floxuridine and its prodrugs in the presence of thymidine phosphorylase ( tp ) was assessed by incubating the desired substrates ( 200 m ) with tp ( 2.0 ng/l ) in phosphate buffer ( ph 7.0 ) at 37 c .", "aliquots of the incubation mixture were sampled at 0 , 1 , 3 , 5 , 10 , 30 , 60 , and 120 min , and quenched with cold acetonitrile ( acn ) with 0.1% tfa , filtered through a 0.45 m membrane , and analyzed for the concentrations of prodrug , floxuridine , and 5-fu by hplc .", "caco-2 cells at nine days postseeding , and aspc-1 and capan-2 cells , both at four days postseeding , were incubated with 10 mol / l gly - sar ( 9.98 mol / l gly - sar and 0.02 mol / l [ h]gly - sar ) along with various prodrug concentrations ( 50.05 mmol / l ) for 30 min .", "the cells were washed three times with ice - cold pbs and solubilized with 10 ml of scintillation cocktail ( scintiverse , fisher scientific , st.louis , mo ) , and the amount of cell - associated radioactivity was determined by scintillation counting ( beckman ls-9000 , beckman instruments , fullerton , ca ) .", "caco-2 cell monolayers were grown on collagen - coated polytetrafluoroethylene membranes for 21 to 24 days , and capan-2 cell monolayers were grown on the same type of membrane for 14 days .", "transepithelial electrical resistance ( teer ) was monitored , and values of 240280 /cm in caco-2 and 380420 /cm in capan-2 ( total area for both cells was 4.67 cm ) were used in the study .", "apical side and basolateral sides of transwell inserts were washed with mes ( ph 6.0 ) and hepes ( ph 7.4 ) , respectively . fresh mes and hepes buffers were reapplied to transwell inserts and incubated at 37 c for 15 min . freshly prepared 0.1 mm drug solution in mes buffer ( total 1.5 ml )", "was placed in the donor chamber , and the receiver chamber was filled with hepes buffer ( total 2.5 ml ) sampling from the receiver chamber ( 200 l ) was conducted up to a period of 2 h at time intervals of 15 , 30 , 45 , 60 , 75 , 90 , and 120 min , at 37 c and replaced with an equal volume of fresh hepes buffer to maintain sink conditions in the receiver chamber .", "all samples were immediately acidified with 0.1% tfa and analyzed by reverse - phase hplc .", "the initial rates of hydrolysis were used to obtain the apparent first - order rate constants and to calculate the half - lives .", "the apparent first - order degradation rate constants of various floxuridine prodrugs at 37 c were determined by plotting the logarithm of prodrug remaining as a function of time .", "the slopes of these plots are related to the rate constant k and given by \n\n the degradation half - lives were then calculated by the equation \n\n statistical significance was evaluated with graphpad prism v. 3.0 by performing one - way analysis of variance with posthoc tukey s test to compare means . the apparent permeability ( papp ) for the prodrugs was calculated using the following equations : \n\n where jss is the steady state flux , m is the cumulative amount of prodrug , and regenerated mono amino acid prodrug , drug and 5-fu in the receiver compartment .", "the apparent permeability was calculated from steady state flux as follows : \n\n where a is the surface area of monolayer exposed to the permeant and c0 is the concentration of the prodrug in the donor solution .", "the concentrations of floxuridine and its prodrugs in the receiver and donor compartments were analyzed using hplc .", "the concentrations of prodrugs and their metabolites were determined on a waters hplc system ( waters , inc . ,", "the hplc system consisted of two waters pumps ( model 515 ) , a waters autosampler ( wisp model 712 ) , and a waters uv detector ( 996 photodiode array detector ) controlled by waters millennium 32 software ( version 3.0.1 ) .", "samples were resolved in a waters xterra c18 reverse - phase column ( 5 m , 4.6  250 mm ) equipped with a guard column . the mobile phase consisted of 1% hfba / water ( solvent a ) and", "1% hfba / acetonitrile ( solvent b ) with the solvent b gradient changing from 056% at a rate of 2%/min during a 28 min run .", "standard curves generated for each prodrug , and their parent drugs were utilized for quantitation of integrated area under peaks .", "the detection wavelength was 254 nm , and spectra were acquired in the 220380 nm range .", "the cells were seeded onto 96-well plates at 125,000 cells per well and allowed to attach / grow for 24 h before drug solutions were added .", "the culture medium ( rpmi-1640 + 10% fetal bovine serum ) was removed , and the cells were gently washed once with sterile ph 6.0 uptake buffer .", "floxuridine and floxuridine prodrugs were 2-fold serially diluted in ph 6.0 uptake buffer from 4 to 0.25 mmol / l .", "the wash buffer was removed , and 25 l of drug solution per well was added and incubated at 37 c for 2 h with aspc-1 cells and 4 h with capan-2 cells in the cell incubator . after this time period , the drug solutions were removed and the cells were gently washed twice with sterile uptake buffer .", "fresh culture medium was then added to each well after washing , and the cells were allowed to recover for 24 h before evaluating cell viability via 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2h - tetrazolium-5-carboxanilide inner salt ( xtt ) assays . a mixture ( 30 l ) containing xtt ( 1 mg / ml ) in sterile rpmi-1640 without phenol red and phenazine methosulfate ( n - methyl dibenzopyrazine methyl sulfate in sterile pbs , 0.383 mg / ml ) reagents was added to the cells and incubated at 37 c for 1 h , after which the absorbance at 450 nm was read .", "the total prodrug yield for each dipeptide was > 15% , and the purity for all prodrugs was > 90% as determined by hplc , where the impurity was the parent drug , floxuridine .", "the experiments concerning prodrug stability were performed at 37 c in ph 7.4 phosphate buffer .", "the estimated half - lives ( t1/2 ) obtained from linear regression of pseudo - first - order plots of prodrug concentration vs time for floxuridine prodrugs in ph 7.4 phosphate buffers alone and in caco-2 , aspc-1 , and capan-2 cell homogenates are listed in table 2 .", "prodrug metabolites such as floxuridine and 5-fu were monitored along with prodrug disappearance in this experiment .", "however , mass balance was not achieved because 5-fu was metabolized even further and those metabolites beyond 5-fu were not quantified ( figure 1 ) .", "dipeptide prodrugs with at least one aromatic amino acid moiety were next - best with regard to stability in enzyme - containing solutions such as plasma and cell homogenates , and those with two aromatic amino acids also exhibited chemical stability that was similar to that of 5-l - isoleucyl - floxuridine .", "all prodrugs exhibited 3- to 30-fold shorter half - lives in cell homogenates than in ph 7.4 phosphate buffer suggesting enzyme - catalyzed hydrolysis .", "with a few exceptions , the half - lives of all monoester and dipeptide prodrugs tested exhibited similar trends in the three cell homogenates and showed good linear correlations ( r = 0.870.93 ) .", "the composition of the amino acids in the dipeptide moiety exerted a profound effect on the stability of the ester bond regardless of the mode of attachment .", "thus , glycyl - containing dipeptide prodrugs were less stable in buffer alone compared to dipeptide prodrugs containing one or two aromatic amino acids .", "glycyl - containing dipeptide prodrugs were also less stable than the 5-l - glycyl ester prodrug .", "a comparison of the stability of 5-l - isoleucyl - floxuridine in various media with 5-l - isoleucyl - l - glycyl - floxuridine dramatically illustrates this effect ( table 2 ) .", "the metabolic stability of floxuridine and its amino ester prodrugs was assessed using thymidine phosphorylase .", "the results shown in table 3 indicate that floxuridine was rapidly degraded to the less active metabolite , 5-fluorouracil , by thymidine phosphorylase .", "the amino acid ester prodrugs of floxuridine were found to be quite resistant to degradation by thymidine phosphorylase .", "prodrugs containing the glycyl moiety , 5-l - glycyl , 5-l - leucyl - l - glycyl , 5-l - glycyl - l - leucyl , 5-l - phenylalanyl - l - glycyl , and 5-l - isoleucyl - l - glycyl floxuridine were 20- to 40-fold more stable than floxuridine to degradation by thymidine phosphorylase .", "the half - lives of 5-l - phenylalanyl - floxuridine , 5-l - isoleucyl - floxuridine , 5-l - valyl - l - phenylalanyl - floxuridine , and 5-l - phenylalanyl - l - tyrosyl - floxuridine were in excess of 500 min reflecting their superior resistance to metabolic degradation by thymidine phosphorylase .", "ic50 values of the amino acid / dipeptide monoester prodrugs of floxuridine for pept1 determined using inhibition of gly - sar uptake in caco-2 , aspc-1 , and capan-2 cells are summarized in table 4 .", "floxuridine showed minimal inhibition of gly - sar uptake , and ic50 values were 8.43  2.66 mm , 6.63  1.52 mm , and 16.06  6.71 mm in caco-2 , aspc-1 , and capan-2 cells , respectively .", "5-l - isoleucyl - l - glycyl - floxuridine displayed the highest affinity for oligopeptide transporters in caco-2 cells ( ic50 = 0.39  0.01 mm ) and in aspc-1 cells", "( ic50 = 0.29  0.00 mm ) and was second - best in capan-2 cells ( ic50 = 0.44  0.02 mm ) . with the exception of 5-l - leucyl and 5-l - glycyl dipeptide analogs with caco-2 cells , all dipeptide prodrugs of floxuridine exhibited 2- to 14-fold higher affinity for oligopeptide transporters than the corresponding 5-l - monoester prodrug . in particular , dipeptide prodrugs with at least one aromatic amino acid component consistently exhibited 3- to 5-fold higher affinity for the oligopeptide transporters than the corresponding monoester prodrug in all three cell lines tested .", "the apical - to - basolateral permeability of mono amino acid / dipeptide monoester prodrugs of floxuridine and parent floxuridine were determined at 37 c in caco-2 and capan-2 cell monolayers .", "all ester prodrugs of floxuridine exhibited 4- to 20-fold higher permeability than floxuridine in caco-2 cells . with the exception of 5-l - valyl - l - phenylalanyl - floxuridine ,", "the permeability of all other dipeptide prodrugs of floxuridine across caco-2 monolayers was 2- to 4-fold higher than the corresponding 5-l - mono amino acid ester floxuridine prodrug .", "the permeability trends in caco-2 monolayers are consistent with the affinity data observed in uptake inhibition studies ; however , no simple correlations between permeability and affinity were evident .", "floxuridine was impermeant with capan-2 cell monolayers ; neither floxuridine nor its metabolite 5-fu could be detected suggesting the possibility of upregulation of metabolic enzymes such as tp and dihydropyrimidine dehydrogenase ( dpd ) in capan-2 cells .", "dpd is the rate - limiting enzyme involved in the catabolism of pyrimidines and is also the main enzyme involved in the degradation of structurally related compounds like 5-fu ( figure 1 ) .", "floxuridine permeability was indeed observed in the presence of dpd inhibitors , dipyridamole and cisplatin ; however , it was 1,000- or 10,000-fold lower than that obtained with caco-2 cells ( data not shown ) .", "the permeability of 5-l - valyl - l - phenylalanyl - floxuridine across caco-2 monolayers was quite low .", "5-l - phenylalanyl - l - tyrosyl - floxuridine , however , exhibited dramatically lower permeability in capan-2 cells compared to its permeability in caco-2 cells . with the exception of 5-l - phenylalanyl - l - tyrosyl - floxuridine , the permeability of the floxuridine ester prodrugs in caco-2 cells was about 2-fold higher than the corresponding value in capan-2 cells ( r = 0.83 ) .", "unlike caco-2 monolayers , general permeability enhancement effects with dipeptide prodrugs compared to the corresponding 5-l - mono amino ester prodrug were not apparent with capan-2 cell monolayers .", "gi50 values for floxuridine and its 5-mono amino acid / dipeptide monoester prodrugs determined in cell proliferation studies with the pancreatic cancer cell lines , aspc-1 and capan-2 , are shown in table 6 .", "all prodrugs exhibit 4- to 8-fold enhanced antiproliferative activity in the two cell lines compared to parent floxuridine .", "thus , the gi50 values of all floxuridine prodrugs were in the range of 1.653.36 mm in aspc-1 and capan-2 cells as opposed to gi50 values of 22.85 mm and 17.63 mm for floxuridine in aspc-1 and capan-2 cells , respectively . these results are consistent with trends observed in gly - sar uptake inhibition studies .", "gly - sar and gly - pro did not inhibit the growth rate of capan-2 cells .", "additionally , 5-d - valyl - floxuridine exhibited antiproliferative activity similar to that of floxuridine and gly - sar , despite an affinity for pept1 that was similar to that of other mono amino acid / dipeptide monoester prodrugs of floxuridine .", "amino acid ester prodrugs have been widely employed to improve intestinal absorption of poorly permeant drugs .", "the antivirals valacyclovir and valganciclovir ( 8,38 ) are early examples of the clinical and commercial success of amino acid ester prodrug strategies .", "the enhanced oral bioavailability of these prodrugs ( 24,39 ) has been attributed to their enhanced transport by intestinal oligopeptide transporters , and to their efficient bioconversion to the parent drug by valacyclovirase .", "a variety of dipeptide and tripeptide compounds and prodrugs have also been investigated for their suitability as substrates for the pept1 transporter .", "we had previously reported the synthesis and evaluation of mono amino acid ester prodrugs of antiviral and anticancer drugs such as floxuridine , gemcitabine,(2 ) acyclovir,(15 ) and 2-bromo-5,6-dichloro-1-(-d - ribofuranosyl)benzimidazole ( bdcrb ) .", "these studies revealed that mono amino acid ester prodrugs in general provide enhanced pept1-mediated transport , a range of bioactivation rates , and enhanced glycosidic bond resistance to metabolic enzymes such as thymidine phosphorylase and cytidine deaminase . in this report", ", we describe the synthesis of dipeptide monoester prodrugs of floxuridine and their chemical stability , bioactivation and transport in caco-2 cells , a surrogate for intestinal transport , and in two pancreatic duct cell lines , aspc-1 and capan-2 , that overexpress the pept1 transporter .", "the dipeptide prodrugs appeared to be less stable in ph 7.4 buffers than the corresponding mono amino acid ester prodrugs . since no mono amino ester prodrug degradation products were detected , it is quite likely that the dipeptide monoester prodrugs degrade via parallel pathways similar to those suggested for gly - phe dipeptide alkyl ester prodrugs by larsen and colleagues.(45 ) thus , in addition to hydrolysis of the ester bond producing the dipeptide , a diketopiperazine cyclization product is also possible due to intramolecular condensation of the ester group with the free amino group of the dipeptide monoester prodrug .", "it has been reported that the rate of intramolecular aminolysis is comparable to that of ester hydrolysis and that cyclization is negligible at ph values below 6 .", "indeed , the formation of diketopiperizine was observed in a chemical stability study at ph 10 but not at lower ph values ( data not shown ) .", "the stability of the prodrugs in buffer was clearly influenced by the prodrug moiety of amino acids ; dipeptide prodrugs containing glycyl and leucyl moieties were less stable than those containing phenylalanyl dipeptide prodrugs .", "dipeptide prodrugs with two aromatic residues were the most stable in buffer as well as in cell homogenates .", "the enzymatic stabilities of 5-l - phenylalanyl - l - tyrosyl - floxuridine and 5-l - valyl - l - phenylalanyl - floxuridine were significantly enhanced compared to the other prodrugs , suggesting that bulky amino acids such as tyrosine and phenylalanine protect against enzyme - catalyzed hydrolysis of the ester linkage .", "the stability profiles of 5-l - phenylalanyl - l - tyrosyl - floxuridine and 5-l - valyl - l - phenylalanyl - floxuridine in cell homogenates , particularly caco-2 , suggest that activation to the parent drug following transport would be much slower than monoester prodrugs such as 5-l - valyl - floxuridine and reference prodrugs such as valacyclovir .", "the improved stability in biological surrogate media would facilitate prolonged systemic circulation of intact prodrugs for enhanced therapeutic action .", "the results of the affinity studies of the mono amino acid ester prodrugs for the oligopeptide transporter in caco-2 cells were generally consistent with previous findings in our laboratory,(14 ) as well as with amino acid prodrugs of acyclovir reported by beuchamp and colleagues.(48 ) no significant trends were noticeable regarding the affinity of the mono amino acid ester prodrugs in aspc-1 and capan-2 cells . with the exception of leucyl floxuridine , the affinities of the mono amino ester prodrugs were lower in aspc-1 and capan-2 compared to those observed with caco-2 cells .", "dipeptide monoester prodrugs exhibited enhanced affinity in all cell lines depending on the nature of the n - terminal amino acid moiety .", "thus , attachment of isoleucyl , phenylalanyl , or leucyl groups to glycyl floxuridine yielded 3- to 9-fold enhancement in affinity for the transporter .", "however , attachment of a glycyl promoiety to the n - terminus of leucyl floxuridine did not result in affinity enhancement .", "these findings are consistent with previous observations on the importance of the amino acid composition at the n - terminus in improving affinity for the pept1 oligopeptide transporter.(22 ) the results of apparent permeability of the floxuridine prodrugs across caco-2 monolayers are consistent with the affinity trends observed in gly - sar uptake inhibition studies . in light of previous studies with mono amino acid prodrugs of floxuridine that revealed excellent linear correlations between caco-2 permeability and pept1-mediated transport in hela / pept1 cells,(13 )", "the enhanced permeability of the dipeptide monoester prodrugs across caco-2 monolayers may indicate enhanced pept1-mediated transport of the dipeptide prodrugs .", "the extremely low permeability of 5-l - valyl - l - phenylalanyl - floxuridine in caco-2 and capan-2 cells and that of 5-l - phenylalanyl - l - tyrosyl - floxuridine in capan-2 cells are not consistent with permeability profiles of 5-l - valyl - floxuridine in caco-2 cells reported earlier(13 ) or of 5-l - phenylalanyl - floxuridine in this study .", "the low permeability of these prodrugs is similar to the low permeability across caco-2 monolayers observed for monoester prodrugs containing l - valyl - l - tyrosyl dipeptide promoieties.(17 ) the estimated clogp values ( table 1 ) of these two were indicative of their being the most lipophilic prodrugs examined , and the contribution of mdr and mrp efflux transporters in permeability studies , therefore , was tested .", "however , the permeability of 5-l - phenylalanyl - l - tyrosyl - floxuridine in capan-2 cells was not affected by 1 mm verapamil , a known efflux pump inhibitor ( data not shown ) .", "the permeabilities of the floxuridine prodrugs were consistently lower in capan-2 cells compared to their corresponding values in caco-2 monolayers .", "dipeptide monoester prodrugs did not exhibit any significant enhancement in permeability compared to the mono amino acid ester prodrugs in capan-2 cells . although the permeability across capan-2 cells for all prodrugs was significantly higher than that of floxuridine alone , meaningful trends based on structureactivity correlations between transporter affinity and membrane permeability are not evident with the limited set of promoieties examined in this study .", "the detection of only 5-fu in the basolateral receiver compartment following transport of floxuridine across caco-2 monolayers suggests the instability of the glycosidic bond of floxuridine .", "the extent of conversion of prodrugs to 5-fu following transport was substantially lower in caco-2 and capan-2 cells .", "the average percent 5-fu observed in the basolateral compartment in caco-2 monolayer studies ( 43% ; range 092% ) was higher than the corresponding average with capan-2 monolayers ( 15% ; range 035% ) . in general , conversion of dipeptide prodrugs to 5-fu", "following transport across the monolayers was about 2-fold lower than that observed with mono amino ester prodrugs .", "the results are consistent with stability profiles of floxuridine and its prodrugs in the presence of thymidine phosphorylase , an enzyme involved in the in vivo for phosphorytic cleavage of floxuridine.(49 ) floxuridine was rapidly cleaved by thymidine phosphorylase , while all amino acid ester prodrugs examined in this study were at least 20-fold more stable to glycosidic bond cleavage by thymidine phosphorylase ( table 3 ) .", "the role of esterification of the hydroxyl groups in protecting glycosidic bond cleavage by thymidine phosphorylase , the rate - determining step in deprotection and in metabolic conversion of floxuridine to 5-fu , has been discussed in a previous study.(13 ) the cell proliferation studies in the pancreatic duct cancer cell lines confirmed the enhanced potency of the amino acid ester prodrugs compared to parent floxuridine . in many cases , dipeptide prodrugs exhibited better gi50 values even though the gi50 values for dipeptide monoester prodrugs in the two cell lines were not significantly different from those obtained with mono amino acid ester prodrugs .", "the gi50 values of prodrugs did not exhibit any discernible correlations with their permeability and/or bioactivation profiles in these cells .", "the lack of potency enhancement of 5-d - valyl - floxuridine in capan-2 cells compared to floxuridine suggests that activation of the prodrugs to the parent is essential for cytotoxic action and is enzyme - specific .", "the different amino acid promoieties of prodrugs may contribute to the different rates of prodrug activation inside cancer cells by particular activation enzymes .", "therefore , it would be difficult to discern a meaningful correlation between gi50 values and prodrug permeabilities with a limited experimental time course .", "this characteristic could lead to enzyme targeted activation of prodrugs at target sites after their membrane permeation .", "intracellular anabolism of floxuridine prodrugs may illustrate that transported drugs are converted to floxuridine and 5-fu via a sequential enzymatic pathway with higher concentrations of tp present in tumor tissue ( figure 2 ) .", "taken together , our results indicate that the dipeptide monoester prodrugs exhibit significantly higher affinity for the pept1 oligopeptide transporter and 2- to 4-fold higher permeability in caco-2 and capan-2 cells than the corresponding mono amino acid ester prodrugs , suggesting their potential for improved oral absorption and uptake in cancer cells . therefore , dipeptide prodrugs might possess an advantage over amino acid monoester prodrugs for cancer target delivery .", "three dipeptide prodrugs of floxuridine , 5-l - phenylalanyl - l - tyrosyl - floxuridine , 5-l - phenylalanyl - l - glycyl - floxuridine and 5-l - isoleucyl - l - glycyl - floxuridine , displayed significantly higher affinity for the pept1 oligopeptide transporter and caco-2 permeability . the delayed enzymatic activation", ", enhanced metabolic resistance and superior affinity to oligopeptide transporters of dipeptide prodrugs may facilitate their prolonged systemic circulation and enhanced therapeutic action . with its display of respectable stability in biological surrogate media and its approximately 2- to 3-fold shorter half - life in cancer cell homogenates than ones in human plasma and caco-2 cell homogenates", ", 5-l - phenylalanyl - l - tyrosyl - floxuridine could be an optimal candidate for cancer cell targeting with enzyme - specific activation ."], "abstract_text": ["<S> dipeptide monoester prodrugs of floxuridine were synthesized , and their chemical stability in buffers , resistance to glycosidic bond metabolism , affinity for pept1 , enzymatic activation and permeability in cancer cells were determined and compared to those of mono amino acid monoester floxuridine prodrugs . </S>", "<S> prodrugs containing glycyl moieties were the least stable in ph 7.4 buffer ( t1/2 < 100 min ) . </S>", "<S> the activation of all floxuridine prodrugs was 2- to 30-fold faster in cell homogenates than their hydrolysis in buffer , suggesting enzymatic action . </S>", "<S> the enzymatic activation of dipeptide monoester prodrugs containing aromatic promoieties in cell homogenates was 5- to 20-fold slower than that of other dipeptide and most mono amino acid monoester prodrugs ( t1/2  40 to 100 min ) . </S>", "<S> all prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent floxuridine . in general , the 5-o - dipeptide monoester floxuridine prodrugs exhibited higher affinity for pept1 than the corresponding 5-o - mono amino acid ester prodrugs . </S>", "<S> the permeability of dipeptide monoester prodrugs across caco-2 and capan-2 monolayers was 2- to 4-fold higher than the corresponding mono amino acid ester prodrug . </S>", "<S> cell proliferation assays in aspc-1 and capan-2 pancreatic ductal cell lines indicated that the dipeptide monoester prodrugs were equally as potent as mono amino acid prodrugs . </S>", "<S> the transport and enzymatic profiles of 5-l - phenylalanyl - l - tyrosyl - floxuridine , 5-l - phenylalanyl - l - glycyl - floxuridine , and 5-l - isoleucyl - l - glycyl - floxuridine suggest their potential for increased oral uptake , delayed enzymatic bioconversion and enhanced resistance to metabolism to 5-fluorouracil , as well as enhanced uptake and cytotoxic activity in cancer cells , attributes that would facilitate prolonged systemic circulation for enhanced therapeutic action . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion"], "sections": [["the anticancer agent 5-fluoro-2-deoxyuridine ( floxuridine ) has been shown to be clinically effective in the treatment of colon carcinoma and colorectal cancer that has metastasized to the liver . however , the adverse effects associated with chemotherapeutics are still unresolved , and many efforts have been made to minimize side - effects and maximize therapeutic efficacy .", "prodrug strategies have been increasingly utilized over the past two decades in order to overcome undesirable physicochemical properties of drugs , to improve oral bioavailability and to minimize toxic side - effects .", "a majority of the efforts have focused on antiviral and anticancer drugs and reflect the need for improved targeting , more selective action and further development of orally available alternatives .", "amino acid ester prodrugs of poorly permeant anticancer and antiviral drugs have been designed for targeted delivery via specific transporters in order to improve their oral bioavailability and metabolic disposition .", "amino acid ester prodrugs have been shown to be substrates for pept1 , pept2 , and atb transporters , and their improved oral bioavailability has been attributed to enhanced transport via carrier - mediated mechanisms .", "alkyl ester prodrugs and amino acid ester prodrugs of floxuridine , for example , have been synthesized and tested for potential improvement of oral drug delivery .", "amino acid ester prodrugs of floxuridine and an antiviral agent , acyclovir , have been shown to be substrates of the pept1 transporter .", "pept1 is stereoselective and exhibits greater affinity for l - enantiomers of amino acids than d - enantiomers.(16 ) pept1 is predominantly expressed in the small intestine , has broad substrate specificity and can transport dipeptides , tripeptides , and -lactam antibiotics .", "the valyl ester prodrug of acyclovir significantly enhances oral bioavailability of acyclovir , and several reports suggest that oligopeptide transporters are responsible for absorption enhancement .", "amino acid ester prodrugs may also facilitate enhanced delivery to pancreatic ductal cancer cells such as aspc-1 and capan-2 , since these cells express oligopeptide transporters at relatively high levels.(25 ) the activation of prodrug to the parent drug following transport is an essential step and can not be ignored . it has been shown that a specific enzyme , valacyclovirase , is primarily responsible for the conversion of valacyclovir to acyclovir .", "it has also been suggested that this enzyme might be involved in the activation of other amino acid prodrugs.(26 ) kim and colleagues reported that the substrate specificity of valacyclovirase is largely determined by the amino acid acyl - linked promoiety of the prodrug.(27 ) the metabolic conversion of floxuridine to 5-fluorouracil following systemic delivery has been shown to be detrimental to therapeutic efficacy of floxuridine .", "the mechanism of action of 5-fluorouracil ( 5-fu ) and floxuridine ( fudr ) is well understood.(30 ) 5-fu toxicity is predominantly caused by 5-fu incorporation into rna .", "however , unlike 5-fu , fudr is specifically incorporated into dna and not into rna , which leads to the minimization of adverse effects .", "several groups have reported that floxuridine is more potent than 5-fu and that the inhibition of cell proliferation is 10- to 100-fold higher than that of 5-fu .", "however , floxuridine is rapidly converted to 5-fu in many tissues , including the liver , by the enzyme thymidine phosphorylase.(34 ) as a consequence , higher doses of floxuridine are required for maintenance of clinical efficacy , leading to greater toxicity .", "therefore , protection of the glycosidic bond of floxuridine is expected to maintain the high potency of the drug and facilitate administration of low doses that can selectively kill only proliferating cells by robust inhibition of dna synthesis . improving the chemical stability of floxuridine to thymidine phosphorylase may enhance its therapeutic efficacy at low doses and obviate toxicity concerns .", "although amino acid monoester prodrugs of floxuridine have been shown to provide enhanced pept1-mediated transport as well as enzymatic activation in intestinal and liver surrogate cell systems , dipeptide analogues may exhibit even higher affinity and transport via the oligopeptide transporter .", "therefore , dipeptide prodrugs may be delivered more to a target site by carrier mediated transporters , and extra amino acid attached prodrugs could be more suitable for specific enzymatic activation at a target site than mono amino acid ester prodrugs .", "several studies on amino acid modifications to increase specific transporter affinity have been conducted . however , direct comparisons of mono amino acid and dipeptide prodrugs in terms of transporter affinity , prodrug stability and activation have not been reported . in this report", ", we describe the synthesis , characterization , and stability of dipeptide monoester prodrugs of floxuridine . various dipeptides and peptidomimetics", "have been tested to characterize the hpept1 transporter and improve its affinity , and mono amino acid ester prodrugs have been evaluated as hpept1 substrates .", "based on those reports , six amino acids were chosen to be n - terminal amino acids of the dipeptide . the smallest amino acid , glycine , and bulky amino acids like phenylalanine and tyrosine were paired with those six amino acids to form dipeptides to test the hypothesis that molecular sizes may structurally affect its ester bond stability .", "the six dipeptide promoieties , l - phenylalanalyl - l - glycine , l - leucyl - l - glycine , l - glycyl - l - leucine , l - isoleucyl - l - glycine , l - valyl - l - phenylalanine , and l - phenylalanyl - l - tyrosine , were designed for targeted delivery to an oligopeptide transporter and tested . since", "5-ester prodrugs were found to exhibit higher affinity for transporters than 3-ester prodrugs,(14 ) only 5-dipeptide monoester floxuridine prodrugs were examined in this study .", "uptake inhibition and permeability studies were conducted with caco-2 cells as well as with aspc-1 and capan-2 cells .", "the chemical stability at physiological ph and the enzymatic activation of the prodrugs in caco-2 , aspc-1 , and capan-2 cell homogenates were also evaluated to determine the effects of the amino acid / dipeptide promoiety structure on enzyme - mediated activation .", "the feasibility of selective antiproliferative action of amino acid / dipeptide floxuridine prodrugs was also explored using cancer cells that overexpress pept1 .", "finally , the stability and transport characteristics of the dipeptide monoester prodrugs of floxuridine were compared with those of the corresponding amino acid monoester prodrugs ."], ["the tert - butyloxycarbonyl ( boc ) protected amino acids boc - l - isoleucine , boc - l - glycine , boc - l - valine , boc - l - phenylalanine , boc - l - leucine , boc - l - glycyl - l - leucine , boc - l - phenylalanalyl - l - glycine , boc - l - leucyl - l - glycine , boc - l - isoleucyl - l - glycine , boc - l - valyl - l - phenylalanine , and boc - l - phenylalanyl - l - tyrosine were obtained from chem - impex ( wood dale , il ) .", "high - performance liquid chromatography ( hplc ) grade acetonitrile was obtained from fisher scientific ( st . louis , mo ) .", "n , n - dicyclohexylcarbodiimide ( dcc ) , n , n - dimethylaminopyridine ( dmap ) , trifluoroacetic acid ( tfa ) , and all other reagents and solvents were purchased from aldrich chemical co. ( milwaukee , wi ) .", "cell culture reagents were obtained from invitrogen ( carlsbad , ca ) , and cell culture supplies were obtained from corning ( corning , ny ) and falcon ( lincoln park , nj ) .", "the synthesis and characterization of 5-mono amino acid ester prodrugs of floxuridine have been reported previously .", "briefly , boc - protected dipeptides boc - l - glycyl - l - leucine , boc - l - phenylalanalyl - l - glycine , boc - l - leucyl - l - glycine , boc - l - isoleucyl - l - glycine , boc - l - valyl - l - phenylalanine , and boc - l - phenylalanyl - l - tyrosine , ( 1.1 mmol ) , dcc ( 1.1 mmol ) , and dmap ( 0.1 mmol ) were allowed to react with floxuridine ( 1 mmol ) in 7 ml of dry dmf for 24 h. the reaction progress was monitored by tlc ( ethyl acetate ) .", "the residue was extracted with ethyl acetate ( 30 ml ) and washed with water ( 2  20 ml ) , and saturated nacl ( 20 ml ) .", "the three spots observed on tlc were separated and purified using column chromatography ( dichloromethane ( dcm)/methanol , 20:1 ) .", "the boc group was cleaved by treating the residues with 5 ml tfa : dcm ( 1:1 ) . after 4 h", "the tfa salts of amino acid prodrugs of floxuridine were obtained as white fluffy solids . the combined yield , consisting of 3-monoester , 5-monoester , and 3,5-diesters , of each floxuridine prodrug was 60% .", "the prodrugs were determined to be 90%99% pure by reverse - phase hplc , and were easily separated from their parent compounds by reverse - phase hplc .", "the observed molecular weights of all prodrugs , determined by electrospray ionization mass spectra ( esi - ms ) obtained on a micromass lct esi - ms , were found to be consistent with those predicted by their structures .", "the structural identities of the prodrugs were then confirmed using proton nuclear magnetic resonance spectra ( h nmr ) , obtained on a 300 mhz bruker dpx-300 nmr spectrometer .", "5-l - leucyl - l - glycyl - floxuridine : yield , 15% ; percent purity , 93% ; h nmr ( dmso - d6 )  , 0.840.92 ( 6h , m , ( ch3)2 ) , 1.501.77 ( 3h , m , ch2 ) , 2.122.28 ( 2h , m , c2 ) , 3.784.35 ( 7h , m , ch of leu and ch2 of gly , c3 , c4 , c5 ) , 6.16 ( 1h , t , c1 , j = 6.0 hz ) , 7.94 ( 1h , d , chf , j = 6.9 hz ) ; esi - ms , 417.1 ( m + h ) .", "5-l - glycyl - l - leucyl - floxuridine : yield , 15% ; percent purity , 96% ; h nmr ( dmso - d6 )  , 0.85 ( 3h , d , ch3 , j = 6.5 hz ) , 0.90 ( 3h , d , ch3 , j = 6.5 hz ) , 1.511.68 ( 3h , m , ch2 ) , 2.122.26 ( 2h , m , c2 ) , 3.544.40 ( 7h , m , ch of leu and ch2 of gly , c3 , c4 ,", "c5 ) , 6.16 ( 1h , t , c1 , j = 6.0 hz ) , 7.90 ( 1h , d , chf , j = 6.8 hz ) ; esi - ms , 417.1 ( m + h ) .", "5-l - valyl - l - phenylalanyl - floxuridine : yield , 30% ; percent purity , 95% ; h nmr ( dmso - d6 )  , 0.90 ( 3h , d , ch3 , j = 6.9 hz ) , 0.95 ( 3h , d , ch3 , j = 6.9 hz ) , 2.082.20 ( 3h , m , c2 , ch of val ) , 3.02 ( 2h , m , ch2 of phe ) , 3.624.60 ( 6h , m , ch of val and phe , c3 , c4 , c5 ) , 6.14 ( 1h , t , c1 , j = 6.1 hz ) , 7.227.31 ( 5h , m , aromatic protons ) , 7.91 ( 1h , d , chf , j = 6.9 hz ) ; esi - ms , 494.1 ( m + h ) .", "5-l - isoleucyl - l - glycyl - floxuridine : yield , 15% ; percent purity , 93% ; h nmr ( dmso - d6 )  , 0.87 ( 3h , t , ch3 , j=7.3 hz ) , 0.94 ( 3h , d , ch3 , j = 6.8 hz ) , 1.17 ( 1h , m , ch2 ) , 1.52 ( 1h , m , ch2 ) , 1.81 ( 1h , m , ch ) , 2.112.28 ( 2h , m , c2 ) , 3.904.31 ( 7h , m , ch of ile and ch2 of gly , c3 , c4 ,", "c5 ) , 6.15 ( 1h , t , c1 , j = 6.5 hz ) , 7.94 ( 1h , d , chf , j = 6.9 hz ) ; esi - ms , 416.9 ( m + h ) .", "5-l - phenylalanyl - l - glycyl - floxuridine : yield , 16% ; percent purity , 98% ; h nmr ( dmso - d6 )  , 2.122.28 ( 2h , m , c2 ) , 2.923.16 ( 2h , m , ch2 of phe ) , 3.904.34 ( 7h , m , ch of phe and ch2 of gly , c3 , c4 , c5 ) , 6.15 ( 1h , t , c1 , j = 6.5 hz ) , 7.277.36 ( 5h , m , aromatic protons ) , 7.94 ( 1h , d , chf , j = 6.5 hz ) ; esi - ms , 451.1 ( m + h ) .", "5-l - phenylalanyl - l - tyrosyl - floxuridine : yield , 3.2% ; percent purity , 99% ; h nmr ( dmso - d6 )  , 2.102.32 ( 2h , m , c2 ) , 2.722.92 ( 4h , m , ch2 of phe and tyr ) , 3.904.29 ( 6h , m , ch of phe and tyr , c3 , c4 , c5 ) , 6.15 ( 1h , m , c1 ) , 6.657.32 ( 9h , m , aromatic protons ) , 7.93 ( 1h , m , chf ) ; esi - ms , 557.2 ( m + h ) .", "capan-2 cells ( passages 5054 ) , and aspc-1 cells ( passages 6365 ) from american type culture collection ( rockville , md ) were routinely maintained in rpmi-1640 containing 10% fetal bovine serum .", "caco-2 cells ( passages 3035 ) from american type culture collection ( rockville , md ) were routinely maintained in dmem containing 10% fetal bovine serum , 1% nonessential amino acids , 1 mmol / l sodium pyruvate , and 1% l - glutamine at 5% co2 and 90% relative humidity at 37 c .", "cells were grown in antibiotic - free media to avoid the possible transport interference by antibiotics .", "the cells were washed with 5 ml of ph 7.4 phosphate buffer ( 10 mmol / l ) , lysed by ultrasonication ( micro ultrasonic cell disrupter model kt40 , kontes , vineland , nj ) , and pelleted by centrifugation for 5 min at 1000 g .", "protein amount was quantified with bio - rad ( hercules , ca ) dc protein assay using bovine serum albumin as a standard .", "the protein amount was adjusted to 500 g / ml , and the hydrolysis reactions were carried out in 96-well plates ( corning ) .", "caco-2 , aspc-1 , and capan-2 cell suspensions ( 250 l ) were placed in triplicate wells , the reactions started with the addition of substrate , and cells were incubated at 37 c for 120 min . at the desired time point , sample aliquots ( 35 l )", "were removed and added to 150 l of acetonitrile ( acn ) containing 0.1% tfa .", "the mixtures were filtered with a 0.45 m filter at 1000 g for 10 min at 4 c .", "undiluted plasma ( 250 l ) was added to each well in triplicate , and substrate was added to initiate the reactions that were conducted at 37 c for 2 h. at various time points , aliquots ( 35 l ) were removed and added to 150 l of acn containing 0.1% tfa .", "the mixtures were filtered with a 0.45 m filter at 1000 g for 10 min at 4 c .", "the nonenzymatic hydrolysis of the prodrugs was determined as described above , except that each well contained ph 7.4 phosphate buffer ( 10 mmol / l ) instead of cell homogenate or human plasma .", "the stability of floxuridine and its prodrugs in the presence of thymidine phosphorylase ( tp ) was assessed by incubating the desired substrates ( 200 m ) with tp ( 2.0 ng/l ) in phosphate buffer ( ph 7.0 ) at 37 c .", "aliquots of the incubation mixture were sampled at 0 , 1 , 3 , 5 , 10 , 30 , 60 , and 120 min , and quenched with cold acetonitrile ( acn ) with 0.1% tfa , filtered through a 0.45 m membrane , and analyzed for the concentrations of prodrug , floxuridine , and 5-fu by hplc .", "caco-2 cells at nine days postseeding , and aspc-1 and capan-2 cells , both at four days postseeding , were incubated with 10 mol / l gly - sar ( 9.98 mol / l gly - sar and 0.02 mol / l [ h]gly - sar ) along with various prodrug concentrations ( 50.05 mmol / l ) for 30 min .", "the cells were washed three times with ice - cold pbs and solubilized with 10 ml of scintillation cocktail ( scintiverse , fisher scientific , st.louis , mo ) , and the amount of cell - associated radioactivity was determined by scintillation counting ( beckman ls-9000 , beckman instruments , fullerton , ca ) .", "caco-2 cell monolayers were grown on collagen - coated polytetrafluoroethylene membranes for 21 to 24 days , and capan-2 cell monolayers were grown on the same type of membrane for 14 days .", "transepithelial electrical resistance ( teer ) was monitored , and values of 240280 /cm in caco-2 and 380420 /cm in capan-2 ( total area for both cells was 4.67 cm ) were used in the study .", "apical side and basolateral sides of transwell inserts were washed with mes ( ph 6.0 ) and hepes ( ph 7.4 ) , respectively . fresh mes and hepes buffers were reapplied to transwell inserts and incubated at 37 c for 15 min . freshly prepared 0.1 mm drug solution in mes buffer ( total 1.5 ml )", "was placed in the donor chamber , and the receiver chamber was filled with hepes buffer ( total 2.5 ml ) sampling from the receiver chamber ( 200 l ) was conducted up to a period of 2 h at time intervals of 15 , 30 , 45 , 60 , 75 , 90 , and 120 min , at 37 c and replaced with an equal volume of fresh hepes buffer to maintain sink conditions in the receiver chamber .", "all samples were immediately acidified with 0.1% tfa and analyzed by reverse - phase hplc .", "the initial rates of hydrolysis were used to obtain the apparent first - order rate constants and to calculate the half - lives .", "the apparent first - order degradation rate constants of various floxuridine prodrugs at 37 c were determined by plotting the logarithm of prodrug remaining as a function of time .", "the slopes of these plots are related to the rate constant k and given by \n\n the degradation half - lives were then calculated by the equation \n\n statistical significance was evaluated with graphpad prism v. 3.0 by performing one - way analysis of variance with posthoc tukey s test to compare means . the apparent permeability ( papp ) for the prodrugs was calculated using the following equations : \n\n where jss is the steady state flux , m is the cumulative amount of prodrug , and regenerated mono amino acid prodrug , drug and 5-fu in the receiver compartment .", "the apparent permeability was calculated from steady state flux as follows : \n\n where a is the surface area of monolayer exposed to the permeant and c0 is the concentration of the prodrug in the donor solution .", "the concentrations of floxuridine and its prodrugs in the receiver and donor compartments were analyzed using hplc .", "the concentrations of prodrugs and their metabolites were determined on a waters hplc system ( waters , inc . ,", "the hplc system consisted of two waters pumps ( model 515 ) , a waters autosampler ( wisp model 712 ) , and a waters uv detector ( 996 photodiode array detector ) controlled by waters millennium 32 software ( version 3.0.1 ) .", "samples were resolved in a waters xterra c18 reverse - phase column ( 5 m , 4.6  250 mm ) equipped with a guard column . the mobile phase consisted of 1% hfba / water ( solvent a ) and", "1% hfba / acetonitrile ( solvent b ) with the solvent b gradient changing from 056% at a rate of 2%/min during a 28 min run .", "standard curves generated for each prodrug , and their parent drugs were utilized for quantitation of integrated area under peaks .", "the detection wavelength was 254 nm , and spectra were acquired in the 220380 nm range .", "the cells were seeded onto 96-well plates at 125,000 cells per well and allowed to attach / grow for 24 h before drug solutions were added .", "the culture medium ( rpmi-1640 + 10% fetal bovine serum ) was removed , and the cells were gently washed once with sterile ph 6.0 uptake buffer .", "floxuridine and floxuridine prodrugs were 2-fold serially diluted in ph 6.0 uptake buffer from 4 to 0.25 mmol / l .", "the wash buffer was removed , and 25 l of drug solution per well was added and incubated at 37 c for 2 h with aspc-1 cells and 4 h with capan-2 cells in the cell incubator . after this time period , the drug solutions were removed and the cells were gently washed twice with sterile uptake buffer .", "fresh culture medium was then added to each well after washing , and the cells were allowed to recover for 24 h before evaluating cell viability via 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2h - tetrazolium-5-carboxanilide inner salt ( xtt ) assays . a mixture ( 30 l ) containing xtt ( 1 mg / ml ) in sterile rpmi-1640 without phenol red and phenazine methosulfate ( n - methyl dibenzopyrazine methyl sulfate in sterile pbs , 0.383 mg / ml ) reagents was added to the cells and incubated at 37 c for 1 h , after which the absorbance at 450 nm was read ."], ["the total prodrug yield for each dipeptide was > 15% , and the purity for all prodrugs was > 90% as determined by hplc , where the impurity was the parent drug , floxuridine .", "the experiments concerning prodrug stability were performed at 37 c in ph 7.4 phosphate buffer .", "the estimated half - lives ( t1/2 ) obtained from linear regression of pseudo - first - order plots of prodrug concentration vs time for floxuridine prodrugs in ph 7.4 phosphate buffers alone and in caco-2 , aspc-1 , and capan-2 cell homogenates are listed in table 2 .", "prodrug metabolites such as floxuridine and 5-fu were monitored along with prodrug disappearance in this experiment .", "however , mass balance was not achieved because 5-fu was metabolized even further and those metabolites beyond 5-fu were not quantified ( figure 1 ) .", "dipeptide prodrugs with at least one aromatic amino acid moiety were next - best with regard to stability in enzyme - containing solutions such as plasma and cell homogenates , and those with two aromatic amino acids also exhibited chemical stability that was similar to that of 5-l - isoleucyl - floxuridine .", "all prodrugs exhibited 3- to 30-fold shorter half - lives in cell homogenates than in ph 7.4 phosphate buffer suggesting enzyme - catalyzed hydrolysis .", "with a few exceptions , the half - lives of all monoester and dipeptide prodrugs tested exhibited similar trends in the three cell homogenates and showed good linear correlations ( r = 0.870.93 ) .", "the composition of the amino acids in the dipeptide moiety exerted a profound effect on the stability of the ester bond regardless of the mode of attachment .", "thus , glycyl - containing dipeptide prodrugs were less stable in buffer alone compared to dipeptide prodrugs containing one or two aromatic amino acids .", "glycyl - containing dipeptide prodrugs were also less stable than the 5-l - glycyl ester prodrug .", "a comparison of the stability of 5-l - isoleucyl - floxuridine in various media with 5-l - isoleucyl - l - glycyl - floxuridine dramatically illustrates this effect ( table 2 ) .", "the metabolic stability of floxuridine and its amino ester prodrugs was assessed using thymidine phosphorylase .", "the results shown in table 3 indicate that floxuridine was rapidly degraded to the less active metabolite , 5-fluorouracil , by thymidine phosphorylase .", "the amino acid ester prodrugs of floxuridine were found to be quite resistant to degradation by thymidine phosphorylase .", "prodrugs containing the glycyl moiety , 5-l - glycyl , 5-l - leucyl - l - glycyl , 5-l - glycyl - l - leucyl , 5-l - phenylalanyl - l - glycyl , and 5-l - isoleucyl - l - glycyl floxuridine were 20- to 40-fold more stable than floxuridine to degradation by thymidine phosphorylase .", "the half - lives of 5-l - phenylalanyl - floxuridine , 5-l - isoleucyl - floxuridine , 5-l - valyl - l - phenylalanyl - floxuridine , and 5-l - phenylalanyl - l - tyrosyl - floxuridine were in excess of 500 min reflecting their superior resistance to metabolic degradation by thymidine phosphorylase .", "ic50 values of the amino acid / dipeptide monoester prodrugs of floxuridine for pept1 determined using inhibition of gly - sar uptake in caco-2 , aspc-1 , and capan-2 cells are summarized in table 4 .", "floxuridine showed minimal inhibition of gly - sar uptake , and ic50 values were 8.43  2.66 mm , 6.63  1.52 mm , and 16.06  6.71 mm in caco-2 , aspc-1 , and capan-2 cells , respectively .", "5-l - isoleucyl - l - glycyl - floxuridine displayed the highest affinity for oligopeptide transporters in caco-2 cells ( ic50 = 0.39  0.01 mm ) and in aspc-1 cells", "( ic50 = 0.29  0.00 mm ) and was second - best in capan-2 cells ( ic50 = 0.44  0.02 mm ) . with the exception of 5-l - leucyl and 5-l - glycyl dipeptide analogs with caco-2 cells , all dipeptide prodrugs of floxuridine exhibited 2- to 14-fold higher affinity for oligopeptide transporters than the corresponding 5-l - monoester prodrug . in particular , dipeptide prodrugs with at least one aromatic amino acid component consistently exhibited 3- to 5-fold higher affinity for the oligopeptide transporters than the corresponding monoester prodrug in all three cell lines tested .", "the apical - to - basolateral permeability of mono amino acid / dipeptide monoester prodrugs of floxuridine and parent floxuridine were determined at 37 c in caco-2 and capan-2 cell monolayers .", "all ester prodrugs of floxuridine exhibited 4- to 20-fold higher permeability than floxuridine in caco-2 cells . with the exception of 5-l - valyl - l - phenylalanyl - floxuridine ,", "the permeability of all other dipeptide prodrugs of floxuridine across caco-2 monolayers was 2- to 4-fold higher than the corresponding 5-l - mono amino acid ester floxuridine prodrug .", "the permeability trends in caco-2 monolayers are consistent with the affinity data observed in uptake inhibition studies ; however , no simple correlations between permeability and affinity were evident .", "floxuridine was impermeant with capan-2 cell monolayers ; neither floxuridine nor its metabolite 5-fu could be detected suggesting the possibility of upregulation of metabolic enzymes such as tp and dihydropyrimidine dehydrogenase ( dpd ) in capan-2 cells .", "dpd is the rate - limiting enzyme involved in the catabolism of pyrimidines and is also the main enzyme involved in the degradation of structurally related compounds like 5-fu ( figure 1 ) .", "floxuridine permeability was indeed observed in the presence of dpd inhibitors , dipyridamole and cisplatin ; however , it was 1,000- or 10,000-fold lower than that obtained with caco-2 cells ( data not shown ) .", "the permeability of 5-l - valyl - l - phenylalanyl - floxuridine across caco-2 monolayers was quite low .", "5-l - phenylalanyl - l - tyrosyl - floxuridine , however , exhibited dramatically lower permeability in capan-2 cells compared to its permeability in caco-2 cells . with the exception of 5-l - phenylalanyl - l - tyrosyl - floxuridine , the permeability of the floxuridine ester prodrugs in caco-2 cells was about 2-fold higher than the corresponding value in capan-2 cells ( r = 0.83 ) .", "unlike caco-2 monolayers , general permeability enhancement effects with dipeptide prodrugs compared to the corresponding 5-l - mono amino ester prodrug were not apparent with capan-2 cell monolayers .", "gi50 values for floxuridine and its 5-mono amino acid / dipeptide monoester prodrugs determined in cell proliferation studies with the pancreatic cancer cell lines , aspc-1 and capan-2 , are shown in table 6 .", "all prodrugs exhibit 4- to 8-fold enhanced antiproliferative activity in the two cell lines compared to parent floxuridine .", "thus , the gi50 values of all floxuridine prodrugs were in the range of 1.653.36 mm in aspc-1 and capan-2 cells as opposed to gi50 values of 22.85 mm and 17.63 mm for floxuridine in aspc-1 and capan-2 cells , respectively . these results are consistent with trends observed in gly - sar uptake inhibition studies .", "gly - sar and gly - pro did not inhibit the growth rate of capan-2 cells .", "additionally , 5-d - valyl - floxuridine exhibited antiproliferative activity similar to that of floxuridine and gly - sar , despite an affinity for pept1 that was similar to that of other mono amino acid / dipeptide monoester prodrugs of floxuridine ."], ["amino acid ester prodrugs have been widely employed to improve intestinal absorption of poorly permeant drugs .", "the antivirals valacyclovir and valganciclovir ( 8,38 ) are early examples of the clinical and commercial success of amino acid ester prodrug strategies .", "the enhanced oral bioavailability of these prodrugs ( 24,39 ) has been attributed to their enhanced transport by intestinal oligopeptide transporters , and to their efficient bioconversion to the parent drug by valacyclovirase .", "a variety of dipeptide and tripeptide compounds and prodrugs have also been investigated for their suitability as substrates for the pept1 transporter .", "we had previously reported the synthesis and evaluation of mono amino acid ester prodrugs of antiviral and anticancer drugs such as floxuridine , gemcitabine,(2 ) acyclovir,(15 ) and 2-bromo-5,6-dichloro-1-(-d - ribofuranosyl)benzimidazole ( bdcrb ) .", "these studies revealed that mono amino acid ester prodrugs in general provide enhanced pept1-mediated transport , a range of bioactivation rates , and enhanced glycosidic bond resistance to metabolic enzymes such as thymidine phosphorylase and cytidine deaminase . in this report", ", we describe the synthesis of dipeptide monoester prodrugs of floxuridine and their chemical stability , bioactivation and transport in caco-2 cells , a surrogate for intestinal transport , and in two pancreatic duct cell lines , aspc-1 and capan-2 , that overexpress the pept1 transporter .", "the dipeptide prodrugs appeared to be less stable in ph 7.4 buffers than the corresponding mono amino acid ester prodrugs . since no mono amino ester prodrug degradation products were detected , it is quite likely that the dipeptide monoester prodrugs degrade via parallel pathways similar to those suggested for gly - phe dipeptide alkyl ester prodrugs by larsen and colleagues.(45 ) thus , in addition to hydrolysis of the ester bond producing the dipeptide , a diketopiperazine cyclization product is also possible due to intramolecular condensation of the ester group with the free amino group of the dipeptide monoester prodrug .", "it has been reported that the rate of intramolecular aminolysis is comparable to that of ester hydrolysis and that cyclization is negligible at ph values below 6 .", "indeed , the formation of diketopiperizine was observed in a chemical stability study at ph 10 but not at lower ph values ( data not shown ) .", "the stability of the prodrugs in buffer was clearly influenced by the prodrug moiety of amino acids ; dipeptide prodrugs containing glycyl and leucyl moieties were less stable than those containing phenylalanyl dipeptide prodrugs .", "dipeptide prodrugs with two aromatic residues were the most stable in buffer as well as in cell homogenates .", "the enzymatic stabilities of 5-l - phenylalanyl - l - tyrosyl - floxuridine and 5-l - valyl - l - phenylalanyl - floxuridine were significantly enhanced compared to the other prodrugs , suggesting that bulky amino acids such as tyrosine and phenylalanine protect against enzyme - catalyzed hydrolysis of the ester linkage .", "the stability profiles of 5-l - phenylalanyl - l - tyrosyl - floxuridine and 5-l - valyl - l - phenylalanyl - floxuridine in cell homogenates , particularly caco-2 , suggest that activation to the parent drug following transport would be much slower than monoester prodrugs such as 5-l - valyl - floxuridine and reference prodrugs such as valacyclovir .", "the improved stability in biological surrogate media would facilitate prolonged systemic circulation of intact prodrugs for enhanced therapeutic action .", "the results of the affinity studies of the mono amino acid ester prodrugs for the oligopeptide transporter in caco-2 cells were generally consistent with previous findings in our laboratory,(14 ) as well as with amino acid prodrugs of acyclovir reported by beuchamp and colleagues.(48 ) no significant trends were noticeable regarding the affinity of the mono amino acid ester prodrugs in aspc-1 and capan-2 cells . with the exception of leucyl floxuridine , the affinities of the mono amino ester prodrugs were lower in aspc-1 and capan-2 compared to those observed with caco-2 cells .", "dipeptide monoester prodrugs exhibited enhanced affinity in all cell lines depending on the nature of the n - terminal amino acid moiety .", "thus , attachment of isoleucyl , phenylalanyl , or leucyl groups to glycyl floxuridine yielded 3- to 9-fold enhancement in affinity for the transporter .", "however , attachment of a glycyl promoiety to the n - terminus of leucyl floxuridine did not result in affinity enhancement .", "these findings are consistent with previous observations on the importance of the amino acid composition at the n - terminus in improving affinity for the pept1 oligopeptide transporter.(22 ) the results of apparent permeability of the floxuridine prodrugs across caco-2 monolayers are consistent with the affinity trends observed in gly - sar uptake inhibition studies . in light of previous studies with mono amino acid prodrugs of floxuridine that revealed excellent linear correlations between caco-2 permeability and pept1-mediated transport in hela / pept1 cells,(13 )", "the enhanced permeability of the dipeptide monoester prodrugs across caco-2 monolayers may indicate enhanced pept1-mediated transport of the dipeptide prodrugs .", "the extremely low permeability of 5-l - valyl - l - phenylalanyl - floxuridine in caco-2 and capan-2 cells and that of 5-l - phenylalanyl - l - tyrosyl - floxuridine in capan-2 cells are not consistent with permeability profiles of 5-l - valyl - floxuridine in caco-2 cells reported earlier(13 ) or of 5-l - phenylalanyl - floxuridine in this study .", "the low permeability of these prodrugs is similar to the low permeability across caco-2 monolayers observed for monoester prodrugs containing l - valyl - l - tyrosyl dipeptide promoieties.(17 ) the estimated clogp values ( table 1 ) of these two were indicative of their being the most lipophilic prodrugs examined , and the contribution of mdr and mrp efflux transporters in permeability studies , therefore , was tested .", "however , the permeability of 5-l - phenylalanyl - l - tyrosyl - floxuridine in capan-2 cells was not affected by 1 mm verapamil , a known efflux pump inhibitor ( data not shown ) .", "the permeabilities of the floxuridine prodrugs were consistently lower in capan-2 cells compared to their corresponding values in caco-2 monolayers .", "dipeptide monoester prodrugs did not exhibit any significant enhancement in permeability compared to the mono amino acid ester prodrugs in capan-2 cells . although the permeability across capan-2 cells for all prodrugs was significantly higher than that of floxuridine alone , meaningful trends based on structureactivity correlations between transporter affinity and membrane permeability are not evident with the limited set of promoieties examined in this study .", "the detection of only 5-fu in the basolateral receiver compartment following transport of floxuridine across caco-2 monolayers suggests the instability of the glycosidic bond of floxuridine .", "the extent of conversion of prodrugs to 5-fu following transport was substantially lower in caco-2 and capan-2 cells .", "the average percent 5-fu observed in the basolateral compartment in caco-2 monolayer studies ( 43% ; range 092% ) was higher than the corresponding average with capan-2 monolayers ( 15% ; range 035% ) . in general , conversion of dipeptide prodrugs to 5-fu", "following transport across the monolayers was about 2-fold lower than that observed with mono amino ester prodrugs .", "the results are consistent with stability profiles of floxuridine and its prodrugs in the presence of thymidine phosphorylase , an enzyme involved in the in vivo for phosphorytic cleavage of floxuridine.(49 ) floxuridine was rapidly cleaved by thymidine phosphorylase , while all amino acid ester prodrugs examined in this study were at least 20-fold more stable to glycosidic bond cleavage by thymidine phosphorylase ( table 3 ) .", "the role of esterification of the hydroxyl groups in protecting glycosidic bond cleavage by thymidine phosphorylase , the rate - determining step in deprotection and in metabolic conversion of floxuridine to 5-fu , has been discussed in a previous study.(13 ) the cell proliferation studies in the pancreatic duct cancer cell lines confirmed the enhanced potency of the amino acid ester prodrugs compared to parent floxuridine . in many cases , dipeptide prodrugs exhibited better gi50 values even though the gi50 values for dipeptide monoester prodrugs in the two cell lines were not significantly different from those obtained with mono amino acid ester prodrugs .", "the gi50 values of prodrugs did not exhibit any discernible correlations with their permeability and/or bioactivation profiles in these cells .", "the lack of potency enhancement of 5-d - valyl - floxuridine in capan-2 cells compared to floxuridine suggests that activation of the prodrugs to the parent is essential for cytotoxic action and is enzyme - specific .", "the different amino acid promoieties of prodrugs may contribute to the different rates of prodrug activation inside cancer cells by particular activation enzymes .", "therefore , it would be difficult to discern a meaningful correlation between gi50 values and prodrug permeabilities with a limited experimental time course .", "this characteristic could lead to enzyme targeted activation of prodrugs at target sites after their membrane permeation .", "intracellular anabolism of floxuridine prodrugs may illustrate that transported drugs are converted to floxuridine and 5-fu via a sequential enzymatic pathway with higher concentrations of tp present in tumor tissue ( figure 2 ) .", "taken together , our results indicate that the dipeptide monoester prodrugs exhibit significantly higher affinity for the pept1 oligopeptide transporter and 2- to 4-fold higher permeability in caco-2 and capan-2 cells than the corresponding mono amino acid ester prodrugs , suggesting their potential for improved oral absorption and uptake in cancer cells . therefore , dipeptide prodrugs might possess an advantage over amino acid monoester prodrugs for cancer target delivery .", "three dipeptide prodrugs of floxuridine , 5-l - phenylalanyl - l - tyrosyl - floxuridine , 5-l - phenylalanyl - l - glycyl - floxuridine and 5-l - isoleucyl - l - glycyl - floxuridine , displayed significantly higher affinity for the pept1 oligopeptide transporter and caco-2 permeability . the delayed enzymatic activation", ", enhanced metabolic resistance and superior affinity to oligopeptide transporters of dipeptide prodrugs may facilitate their prolonged systemic circulation and enhanced therapeutic action . with its display of respectable stability in biological surrogate media and its approximately 2- to 3-fold shorter half - life in cancer cell homogenates than ones in human plasma and caco-2 cell homogenates", ", 5-l - phenylalanyl - l - tyrosyl - floxuridine could be an optimal candidate for cancer cell targeting with enzyme - specific activation ."]]}
{"article_id": "PMC3463469", "article_text": ["the mammalian order primate that includes humans , apes , and monkeys in addition to several other organisms can be traced to the late cretaceous period .", "the rhesus macaque is in many ways an ideal model organism , being closely related to humans ( sharing a common ancestor about 25 million years ago ) and also sharing similar physiology , neurobiology , and susceptibility to infectious and metabolic diseases .", "since both the m. mulatta and h. sapiens genomes have been sequenced , it is known that the evolutionary distance between them is small , with local fluctuations and low divergence , particularly in chromosome x. on average , orthologs have about 97% identity between the genomes both at the nucleotide and amino acid sequence levels .", "approximately 50% of the rhesus macaque genome consists of various repetitive sequences , similar to the human genome .", "the phenomenon of retrotransposition that occurs in eukaryotic genomes of diverse taxonomic groups is implicated in various human genetic diseases .", "insertion sites of many non - long - terminal repeat ( ltr ) retrotransposons play an important role in genome evolution and are distributed throughout the genome .", "the phenomenon behind the selection of the insertion sites of these elements has been shown to be correlated with patterns found at pre - insertion loci .", "it is well known that mobile elements insertions are capable of altering gene expression , generating genomic deletions , and they can even create new genes and gene families .", "existing repetitive elements can also cause ectopic recombinations . despite the overall similarity in retrotransposon mobilization activity in the old world monkeys and hominid lineages ,", "the retroelements that are presumed to have had the most dramatic impact in shaping primate genomes are the l1 family of line elements and the alu elements , their partner sines . besides contributing large amounts of dna to many genomes ( including at least 40% of the human genome ) they have also provided new genes , exons and other motifs involved in the physical and sequence structure of chromosomes .", "there are instances in which a previous element is now a part of the machinery that regulates gene expression .", "repetitive elements account for about 50% of the genome among all of the presently sequenced primate species ( table 1 ) . in m. mulatta ,", "two classes of mobile elements are present , class i dna transposons and class ii retrotransposons .", "the transposons can also be categorized into different families and subfamilies , based on the relationships between their sequences .", "the rhesus family consists of about 320,000 copies of many families of dna transposons and about half a million copies of endogenous retroviruses .", "the l1s and alu elements account for most of the lineage specific insertions and these have been playing an important role in shaping the complete genome .", "elan was developed earlier as a suite of tools for genome - wide retrotransposon element analysis .", "the application of modules of this bioinformatics pipeline is described as follows : ( 1 ) elefinder performs a whole genome distribution analysis of the mges through a blastn search by making the use of perl / bioperl scripts by which the output files are parsed .", "it also extracts sequence 100 bp up and downstream at each mge site identified as a preinsertion locus .", "( 2 ) dnascanner scans dna sequences such as preinsertion loci and analyses insertion hotspots of elements in detail so as to provide a set of signals or characteristics that are potentially recognized by an element for its insertion .", "the m. mulatta genome has a total of 22 chromosomes including x and y ( although the sequencing project did not sequence y ) .", "the only y chromosome that has been completely sequenced is of humans , and sequencing of the chimpanzee and mouse y chromosomes is in progress .", "the sequencing of the mammalian y chromosomes , of the organisms like rhesus macaque ( macaca mulatta ) , the white - tufted - ear marmoset ( callithrix jacchus ) , the rat ( rattus novergicus ) , the bull ( bos taurus ) and the opossum ( monodelphis domestica ) , is proposed and still under process .", "we present here a study of the primate macaca mulatta genome to identify and characterize insertion sites of the two representative retroelements present , and further , comparison with similar features of the human genome ( excluding the y chromosome ) .", "the structural and thermodynamic features as well as protein interaction measures are computed in preinsertion loci using the tool dna scanner .", "in the human genome , a full - length l1 element is around 6 kb long , and is reported to be the most successful tes in human genome by mass while alu elements , typically ~300 bp long are most successful in terms of copy number . currently , there are three macaque consensus sequences for alu : alumacya3 , alumacyb2 , and alumacyb4 and five of that for l1 : l1p4a , l1p4b , l1p4c , l1p4d , l1p4e in repbase ( version 13.5 ) .", "we first compute the nucleotide sequence divergence of l1 ( human ) with respect to the corresponding l1 ( macaca genome ) ( table 2(a ) ) .", "as can be seen , pairwise alignment shows a low percentage identity , with the major areas of dissimilarity in the 3 region .", "the macaca lineages show higher percentage similarity with each other ; with regard to the human genome , the l1 nearest to its human analog is l1p4d , while that which has diverged the most is l1p4e .", "macaca lineages show high similarity with each other and human alu seems to be most closely related to the macaca specific alumcaya3 ( table 2(b ) ) .", "tables 3 and 4 give details of the numbers of alu and l1 elements in each chromosome .", "the four groups constructed for the pre insertion loci were : intact on both ends , intact on 5 , intact on 3 and intact on neither end .", "about 1077410 copies of alus were found uniformly distributed on each chromosome . of these approximately 13.49% ( 145428 ) are truncated on the 5 end , 456752 copies ( 42.39% ) are truncated at both the ends , 30.29% are the truncated at 3 end ( 326447 ) .", "a negligible fraction ( 1.6% ) of l1 elements was intact on both sides : of the 94616 elements identified 22% ( 21044 ) were truncated at the 5 end and 1.38% ( 1314 ) were truncated at the 3 end and about 75% at both ends . as in the human genome , there are few functional alu copies in m. mulatta , and their distribution on the various chromosomes follows similar patterns ( fig . ", "the x chromosome is known to have an exceptionally high number of the l1 elements ( fig . ", "1b ) , which also contained the largest number of truncated elements ( at both the ends ) .", "the y - axis represents the frequency of elements found on the different chromosomes ( marked along the x - axis ) .", "the y - axis represents the frequency of elements found on the different chromosomes ( marked along the x - axis ) .", "the pre - insertion loci were extracted and evaluated for various physicochemical properties as described in our earlier paper .", "the positions of the extrema were similar to those seen in other cases in the physicochemical profiles generated by dnascanner .", "similar tables were calculated for each m. mulatta chromosome and for all the 14 characteristics extrema were seen in the range of -9 to -11 bp for alu element and that for the l1 element was between -2 to -19 for the majority of the cases . for l1 elements , results are similar and table 5 gives the complete information about the values and the extrema for each of the properties .", "figures  2 and 3 show the graphs obtained for 4 physicochemical properties for alu and l1 elements insertion sites in macaca genome .", "control sequences were generated by scrambling the positive data set of pre - insertion sequences ; all these above properties gave a featureless distribution ( namely no extrema ) .", "another independent set of control sequences that were obtained by randomly selecting genomic sequences of 100 bp also gave similar featureless results .", "various signals upstream of the insertion sites of alu in chromosome 1 , for ( a ) stacking energy ,", "( b ) at content , ( c ) propeller twist and ( d ) protein induced deformability .", "the y axis represents value of the property and the x - axis gives the relative position with respect to the insertion site ( taken to be 0 ) .", "various signals upstream of the insertion sites of l1 in chromosome 1 , for ( a ) a rule , ( b ) nucleosomal bending , ( c ) nucleosomal positioning,(d ) protein induced deformability .", "the y axis represents value of the property and the x - axis gives the relative position with respect to the insertion site ( taken to be 0 ) . our rationale for the choice of the various parameters", "1 ) regions with alternating purines / pyrimidines steps and at rich regions melt more readily .", "we found regions of low gc and high at content , indicating that a relatively less energy is required to melt dna near insertion sites , which in turn favors retrotransposition ( figs . ", "( 2 ) propeller twist is a property , involved in the distortion of the hydrogen bonds that hold two bases together . regions with specific dinucleotides with large propeller twist , followed by a lower propeller twist were obtained ( fig .  2c ) , which shows that latter regions may be easily distorted and are suitable for insertion .", "( 3 ) nucleosomes are involved in dna compacting and providing transcription factors access to the respective regulatory regions .", "two different nucleosomal related features , the bending energy / persistence length and the position profiles of the nucleosomes , were studied .", "regions with comparatively low energies were obtained , within the upstream areas of the insertion sites .", "( 4 ) stacking energy profiles showed a maximum near -10 , indicating that this region is unstable , leading to easy de - stacking of dna sequence , which would thereby enable an easy insertion of alu .", "( 5 ) duplex stability is a measure of the relative stability of the dna - duplex structure , which is directly dependent on sequence .", "we obtained the region around the -10 position for alu and at -19 for l1 , with a peak , representing a region which would de - stack or melt easily .", "( 6 ) dna deformability is an important property , dependent on the sequence and required for interaction with proteins .", "the dna deformability was calculated and a region ( at -10 for alu and -18 for l1 ) of low deformability was seen ; this facilitates retrotransposon insertion .", "the results of elefinder as well as the information curated in the inside database were used to find the distribution of truncation sites in the whole m. mulatta genome .", "the x - axis represents the length of element divided in bins of 25 , i.e.,125 , 2550 , and so on .", "the criteria used for the plotting was the occurrence of 5truncated and 3 intact ends and 3 truncated and 5 intact ends in the macaca genome .", "the basic aim was to identify the positions in the element sequence , where most of the times truncation occurs for the macaca genome as a whole .", "the graph plotted for the 3 truncated ends , the maximum number of hits were obtained in the last bin , i.e. , the 276300 bin has the maximum number of truncations in the macaca genome ( fig . ", "similarly , for the 5 truncated category , the maximum number of truncations was found to be in the first bin , i.e. , the 125 bin ( fig . ", "( a ) the truncation distribution of alu element in macaca mulatta for bin size 25 and the graph is plotted for 3 end of alu truncated and its 5 end being intact .", "( b ) the truncation distribution of alu element in macaca mulatta for bin size 25 and the graph is plotted for 5 end of alu truncated and its 3 end being intact .", "previously , we analyzed several genomes for distribution of mges as well as their insertion sites along with the signals facilitating their insertion . in the present study of alus ( sine ) and l1s ( line ) in the macaca genome we found that the insertion sites had physicochemical characteristics that were similar to those observed in other organisms , suggesting that these are generally important .", "the present study confirmed that the region 100 bp upstream from alu and l1 insertion sites show statistically significant distinctive properties both in the physical and structural characteristics , as well as in the energetics .", "these properties seem to play an important role in the insertion of mge . during insertion ,", "a mge causes the target site to distort in a number of ways and requires the co - operative action of a number of proteins to break bonds , unwind the dna , and to nick the target site strand .", "it is due to this series of requirements , that the insertion sites for all the chromosomes , show a characteristic set of physicochemical properties , signified by the extremum peaks in each case .", "each of the peaks for the several properties carry biological significance and may be used further for the identification of potential new insertion sites . in each case , that the nature of the extremum that has a role in defining the trend of each property was identical for both alu and l1 ( table 5 ) , explaining that the signals that are needed for the insertion of the element is same , and are probably necessary for the insertion to actually occur , although also probably are not sufficient .", "detection of the most probable truncation sites for alu elements within the complete macaca genome revealed that during insertion the truncation distribution of alu peaks toward the starting positions in the case of the trailing edge truncation and is reversed for leading edge truncations .", "we have found that lines are present in large number in all the primate genomes which includes the recently studied gorilla genome as well as callithrix jaqqus , pan troglodytes , orangutan ( fig . ", "there could be an evolutionary link in primate genomes through the spread of lines and sines ; alternately , there may have been a  master  l1 or retrotransposon copy in the genome of last common ancestor of all primates .", "the present work adds insight into primate genome architecture , showing the common structural features that promote mge insertion and genome expansion . in future work", ", we aim to compare the insertion sites across species- a task that will be facilitated as the diversity of sequenced genomes increases further .", "the genome sequence of macaca mulatta was retrieved from the ncbi ( ftp server : ftp://ftp.ncbi.nih.gov/genomes/ ) . the element sequence ( for alu and l1 )", "were obtained from repbase and pairwise alignment was performed for the l1 for the human and macaca specific l1s and its lineages using standard procedures .", "elefinder was used to find the insertion site of alu and l1 in the macaca genome .", "this tool finds the nature , distribution , genomic location , and the site of truncation for each of the insertion sites and performs comparative genome analysis and also generates several set of sequences .", "since we have previously shown that intact copies show the presence of signal as compared with the truncated groups , the analysis of the full - length elements , i.e. , of those capable of transposition was also performed .", "the tool dna scanner analyses the dna for many physico - chemical properties by using various thermodynamic , protein interactions and sequence - based features , which are beyond the t density and at density . in accordance to the choice of input parameters , the program evaluates a number of properties , using windows that move along the length of the query dna sequence .", "since downstream sequences tend to show signals that are not sufficiently statistically significant only upstream sequences were investigated for both alu and l1 elements .", "the controls were selected by scrambling sequences , randomly picking sequences from genome as well as gene sequences ."], "abstract_text": ["<S> mobile genetic elements ( mges ) are fragments of dna that can move around within the genome through retrotransposition . </S>", "<S> these are responsible for various important events such as gene inactivation , transduction , regulation of gene expression and genome expansion . </S>", "<S> the present work involves the identification and study of the distribution of alu and l1 retrotransposons in the genome of macaca mulatta , an extensively used organism in biomedical studies . </S>", "<S> we also make comparisons with mge distributions in other primate genomes and study the physicochemical properties of the local dna structure around the transposon insertion site using elan . </S>", "<S> the present work also includes computational testing of the pre - insertion loci in order to detect unique features based on dna structure , thermodynamic considerations and protein interaction measures . </S>", "<S> although there is significant sequence divergence between the elements of m. mulatta and h. sapiens , their genome wide distribution is very similar ; comparing the distribution of l1 s in all available x chromosome sequences suggests a common mechanism behind the spread of mge s in primate genomes . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Discussion", "Methods"], "sections": [["the mammalian order primate that includes humans , apes , and monkeys in addition to several other organisms can be traced to the late cretaceous period .", "the rhesus macaque is in many ways an ideal model organism , being closely related to humans ( sharing a common ancestor about 25 million years ago ) and also sharing similar physiology , neurobiology , and susceptibility to infectious and metabolic diseases .", "since both the m. mulatta and h. sapiens genomes have been sequenced , it is known that the evolutionary distance between them is small , with local fluctuations and low divergence , particularly in chromosome x. on average , orthologs have about 97% identity between the genomes both at the nucleotide and amino acid sequence levels .", "approximately 50% of the rhesus macaque genome consists of various repetitive sequences , similar to the human genome .", "the phenomenon of retrotransposition that occurs in eukaryotic genomes of diverse taxonomic groups is implicated in various human genetic diseases .", "insertion sites of many non - long - terminal repeat ( ltr ) retrotransposons play an important role in genome evolution and are distributed throughout the genome .", "the phenomenon behind the selection of the insertion sites of these elements has been shown to be correlated with patterns found at pre - insertion loci .", "it is well known that mobile elements insertions are capable of altering gene expression , generating genomic deletions , and they can even create new genes and gene families .", "existing repetitive elements can also cause ectopic recombinations . despite the overall similarity in retrotransposon mobilization activity in the old world monkeys and hominid lineages ,", "the retroelements that are presumed to have had the most dramatic impact in shaping primate genomes are the l1 family of line elements and the alu elements , their partner sines . besides contributing large amounts of dna to many genomes ( including at least 40% of the human genome ) they have also provided new genes , exons and other motifs involved in the physical and sequence structure of chromosomes .", "there are instances in which a previous element is now a part of the machinery that regulates gene expression .", "repetitive elements account for about 50% of the genome among all of the presently sequenced primate species ( table 1 ) . in m. mulatta ,", "two classes of mobile elements are present , class i dna transposons and class ii retrotransposons .", "the transposons can also be categorized into different families and subfamilies , based on the relationships between their sequences .", "the rhesus family consists of about 320,000 copies of many families of dna transposons and about half a million copies of endogenous retroviruses .", "the l1s and alu elements account for most of the lineage specific insertions and these have been playing an important role in shaping the complete genome .", "elan was developed earlier as a suite of tools for genome - wide retrotransposon element analysis .", "the application of modules of this bioinformatics pipeline is described as follows : ( 1 ) elefinder performs a whole genome distribution analysis of the mges through a blastn search by making the use of perl / bioperl scripts by which the output files are parsed .", "it also extracts sequence 100 bp up and downstream at each mge site identified as a preinsertion locus .", "( 2 ) dnascanner scans dna sequences such as preinsertion loci and analyses insertion hotspots of elements in detail so as to provide a set of signals or characteristics that are potentially recognized by an element for its insertion .", "the m. mulatta genome has a total of 22 chromosomes including x and y ( although the sequencing project did not sequence y ) .", "the only y chromosome that has been completely sequenced is of humans , and sequencing of the chimpanzee and mouse y chromosomes is in progress .", "the sequencing of the mammalian y chromosomes , of the organisms like rhesus macaque ( macaca mulatta ) , the white - tufted - ear marmoset ( callithrix jacchus ) , the rat ( rattus novergicus ) , the bull ( bos taurus ) and the opossum ( monodelphis domestica ) , is proposed and still under process .", "we present here a study of the primate macaca mulatta genome to identify and characterize insertion sites of the two representative retroelements present , and further , comparison with similar features of the human genome ( excluding the y chromosome ) .", "the structural and thermodynamic features as well as protein interaction measures are computed in preinsertion loci using the tool dna scanner ."], ["in the human genome , a full - length l1 element is around 6 kb long , and is reported to be the most successful tes in human genome by mass while alu elements , typically ~300 bp long are most successful in terms of copy number . currently , there are three macaque consensus sequences for alu : alumacya3 , alumacyb2 , and alumacyb4 and five of that for l1 : l1p4a , l1p4b , l1p4c , l1p4d , l1p4e in repbase ( version 13.5 ) .", "we first compute the nucleotide sequence divergence of l1 ( human ) with respect to the corresponding l1 ( macaca genome ) ( table 2(a ) ) .", "as can be seen , pairwise alignment shows a low percentage identity , with the major areas of dissimilarity in the 3 region .", "the macaca lineages show higher percentage similarity with each other ; with regard to the human genome , the l1 nearest to its human analog is l1p4d , while that which has diverged the most is l1p4e .", "macaca lineages show high similarity with each other and human alu seems to be most closely related to the macaca specific alumcaya3 ( table 2(b ) ) .", "tables 3 and 4 give details of the numbers of alu and l1 elements in each chromosome .", "the four groups constructed for the pre insertion loci were : intact on both ends , intact on 5 , intact on 3 and intact on neither end .", "about 1077410 copies of alus were found uniformly distributed on each chromosome . of these approximately 13.49% ( 145428 ) are truncated on the 5 end , 456752 copies ( 42.39% ) are truncated at both the ends , 30.29% are the truncated at 3 end ( 326447 ) .", "a negligible fraction ( 1.6% ) of l1 elements was intact on both sides : of the 94616 elements identified 22% ( 21044 ) were truncated at the 5 end and 1.38% ( 1314 ) were truncated at the 3 end and about 75% at both ends . as in the human genome , there are few functional alu copies in m. mulatta , and their distribution on the various chromosomes follows similar patterns ( fig . ", "the x chromosome is known to have an exceptionally high number of the l1 elements ( fig . ", "1b ) , which also contained the largest number of truncated elements ( at both the ends ) .", "the y - axis represents the frequency of elements found on the different chromosomes ( marked along the x - axis ) .", "the y - axis represents the frequency of elements found on the different chromosomes ( marked along the x - axis ) .", "the pre - insertion loci were extracted and evaluated for various physicochemical properties as described in our earlier paper .", "the positions of the extrema were similar to those seen in other cases in the physicochemical profiles generated by dnascanner .", "similar tables were calculated for each m. mulatta chromosome and for all the 14 characteristics extrema were seen in the range of -9 to -11 bp for alu element and that for the l1 element was between -2 to -19 for the majority of the cases . for l1 elements , results are similar and table 5 gives the complete information about the values and the extrema for each of the properties .", "figures  2 and 3 show the graphs obtained for 4 physicochemical properties for alu and l1 elements insertion sites in macaca genome .", "control sequences were generated by scrambling the positive data set of pre - insertion sequences ; all these above properties gave a featureless distribution ( namely no extrema ) .", "another independent set of control sequences that were obtained by randomly selecting genomic sequences of 100 bp also gave similar featureless results .", "various signals upstream of the insertion sites of alu in chromosome 1 , for ( a ) stacking energy ,", "( b ) at content , ( c ) propeller twist and ( d ) protein induced deformability .", "the y axis represents value of the property and the x - axis gives the relative position with respect to the insertion site ( taken to be 0 ) .", "various signals upstream of the insertion sites of l1 in chromosome 1 , for ( a ) a rule , ( b ) nucleosomal bending , ( c ) nucleosomal positioning,(d ) protein induced deformability .", "the y axis represents value of the property and the x - axis gives the relative position with respect to the insertion site ( taken to be 0 ) . our rationale for the choice of the various parameters", "1 ) regions with alternating purines / pyrimidines steps and at rich regions melt more readily .", "we found regions of low gc and high at content , indicating that a relatively less energy is required to melt dna near insertion sites , which in turn favors retrotransposition ( figs . ", "( 2 ) propeller twist is a property , involved in the distortion of the hydrogen bonds that hold two bases together . regions with specific dinucleotides with large propeller twist , followed by a lower propeller twist were obtained ( fig .  2c ) , which shows that latter regions may be easily distorted and are suitable for insertion .", "( 3 ) nucleosomes are involved in dna compacting and providing transcription factors access to the respective regulatory regions .", "two different nucleosomal related features , the bending energy / persistence length and the position profiles of the nucleosomes , were studied .", "regions with comparatively low energies were obtained , within the upstream areas of the insertion sites .", "( 4 ) stacking energy profiles showed a maximum near -10 , indicating that this region is unstable , leading to easy de - stacking of dna sequence , which would thereby enable an easy insertion of alu .", "( 5 ) duplex stability is a measure of the relative stability of the dna - duplex structure , which is directly dependent on sequence .", "we obtained the region around the -10 position for alu and at -19 for l1 , with a peak , representing a region which would de - stack or melt easily .", "( 6 ) dna deformability is an important property , dependent on the sequence and required for interaction with proteins .", "the dna deformability was calculated and a region ( at -10 for alu and -18 for l1 ) of low deformability was seen ; this facilitates retrotransposon insertion .", "the results of elefinder as well as the information curated in the inside database were used to find the distribution of truncation sites in the whole m. mulatta genome .", "the x - axis represents the length of element divided in bins of 25 , i.e.,125 , 2550 , and so on .", "the criteria used for the plotting was the occurrence of 5truncated and 3 intact ends and 3 truncated and 5 intact ends in the macaca genome .", "the basic aim was to identify the positions in the element sequence , where most of the times truncation occurs for the macaca genome as a whole .", "the graph plotted for the 3 truncated ends , the maximum number of hits were obtained in the last bin , i.e. , the 276300 bin has the maximum number of truncations in the macaca genome ( fig . ", "similarly , for the 5 truncated category , the maximum number of truncations was found to be in the first bin , i.e. , the 125 bin ( fig . ", "( a ) the truncation distribution of alu element in macaca mulatta for bin size 25 and the graph is plotted for 3 end of alu truncated and its 5 end being intact .", "( b ) the truncation distribution of alu element in macaca mulatta for bin size 25 and the graph is plotted for 5 end of alu truncated and its 3 end being intact ."], ["previously , we analyzed several genomes for distribution of mges as well as their insertion sites along with the signals facilitating their insertion . in the present study of alus ( sine ) and l1s ( line ) in the macaca genome we found that the insertion sites had physicochemical characteristics that were similar to those observed in other organisms , suggesting that these are generally important .", "the present study confirmed that the region 100 bp upstream from alu and l1 insertion sites show statistically significant distinctive properties both in the physical and structural characteristics , as well as in the energetics .", "these properties seem to play an important role in the insertion of mge . during insertion ,", "a mge causes the target site to distort in a number of ways and requires the co - operative action of a number of proteins to break bonds , unwind the dna , and to nick the target site strand .", "it is due to this series of requirements , that the insertion sites for all the chromosomes , show a characteristic set of physicochemical properties , signified by the extremum peaks in each case .", "each of the peaks for the several properties carry biological significance and may be used further for the identification of potential new insertion sites . in each case , that the nature of the extremum that has a role in defining the trend of each property was identical for both alu and l1 ( table 5 ) , explaining that the signals that are needed for the insertion of the element is same , and are probably necessary for the insertion to actually occur , although also probably are not sufficient .", "detection of the most probable truncation sites for alu elements within the complete macaca genome revealed that during insertion the truncation distribution of alu peaks toward the starting positions in the case of the trailing edge truncation and is reversed for leading edge truncations .", "we have found that lines are present in large number in all the primate genomes which includes the recently studied gorilla genome as well as callithrix jaqqus , pan troglodytes , orangutan ( fig . ", "there could be an evolutionary link in primate genomes through the spread of lines and sines ; alternately , there may have been a  master  l1 or retrotransposon copy in the genome of last common ancestor of all primates .", "the present work adds insight into primate genome architecture , showing the common structural features that promote mge insertion and genome expansion . in future work", ", we aim to compare the insertion sites across species- a task that will be facilitated as the diversity of sequenced genomes increases further ."], ["the genome sequence of macaca mulatta was retrieved from the ncbi ( ftp server : ftp://ftp.ncbi.nih.gov/genomes/ ) . the element sequence ( for alu and l1 )", "were obtained from repbase and pairwise alignment was performed for the l1 for the human and macaca specific l1s and its lineages using standard procedures .", "elefinder was used to find the insertion site of alu and l1 in the macaca genome .", "this tool finds the nature , distribution , genomic location , and the site of truncation for each of the insertion sites and performs comparative genome analysis and also generates several set of sequences .", "since we have previously shown that intact copies show the presence of signal as compared with the truncated groups , the analysis of the full - length elements , i.e. , of those capable of transposition was also performed .", "the tool dna scanner analyses the dna for many physico - chemical properties by using various thermodynamic , protein interactions and sequence - based features , which are beyond the t density and at density . in accordance to the choice of input parameters , the program evaluates a number of properties , using windows that move along the length of the query dna sequence .", "since downstream sequences tend to show signals that are not sufficiently statistically significant only upstream sequences were investigated for both alu and l1 elements .", "the controls were selected by scrambling sequences , randomly picking sequences from genome as well as gene sequences ."]]}
{"article_id": "PMC3088739", "article_text": ["the prevalence of ulcerative colitis ( uc ) has increased each year for the past several decades .", "a large - scale population - based cohort study shows that the flare - up rate in women is 1.2 times higher than that in men .", "accordingly , female gender appears to be a risk factor for frequent uc flare - up .", "female patients often suffer from uc symptoms during pregnancy as uc symptoms generally start to appear then , and a definitive diagnosis of uc has been confirmed in younger individuals in their teens to thirties . it has been estimated that approximately 25% of female uc patients conceive after uc diagnosis .", "in addition , the risk of flare - up and the development of associated inflammation during pregnancy is known to be increased in both the remission and the active stages of inflammatory bowel diseases ( ibd ) , including uc and crohn 's disease .", "the first trimester is considered to be a particularly high - risk time for flare - up of uc . in contrast , two european cohort studies of 634 pregnancies in 303 women showed that pregnancy improves the disease course , with a reduction in flare - ups in subsequent years [ 2 , 4 ] .", "therapeutic options are limited when flare - ups occur during pregnancy due to the concerns for the safety of fetus . according to previous reports and statements by the united states food and drug administration ( fda ) ( http://www.fda.gov/drugs/developmentapprovalprocess/developmentresources/labeling/ucm093307.htm ) , 5-aminosalicylic acid ( 5-asa ) , corticosteroids and granulocyte and monocyte adsorptive apheresis ( gma ; adacolumn )", "however , we have often observed uc that is refractory to individual treatment with these drugs or with this modality , or to intensive therapeutic approaches that combine these drugs with this modality .", "intensive gma involving two gma sessions per week has recently been shown to be more efficacious than conventional weekly gma .", "furthermore , intensive gma also safely induced rapid remission in patients with moderately active uc inflammation .", "very recently , the drug tacrolimus was approved in japan for the treatment of patients with moderate or severe uc , regardless of whether the uc is dependent on or refractory to corticosteroids .", "tacrolimus is reportedly useful for the treatment of refractory uc due to its potent immunosuppressive properties , which induce inhibition of the transcription of the early activation genes encoding interleukin-2 , tumor necrosis factor- ( tnf- ) and interferon- that are responsible for the development of inflammation .", "we report herein the case of a patient with severe uc that had developed during the first trimester of pregnancy and was refractory to combination therapy with high - dose corticosteroids and intensive gma , but was successfully treated with tacrolimus following cesarean section early in the third trimester .", "in july 2009 , a 36-year - old pregnant woman was referred to our hospital for treatment of refractory uc .", "she had been diagnosed with uc ( total colitis ) at the age of 17 .", "oral 5-asa ( 2,250 mg / day ) had maintained her in remission up to the age of 31 .", "subsequently , repeated flare - ups had occurred and she had been treated with corticosteroids or with the routine weekly gma .", "azathioprine ( aza ) administration was also tried , but had to be stopped due to aza - induced pancytopenia .", "then , oral 5-asa ( 2,250 mg / day ) plus 3 mg betamethasone enema had induced and maintained remission up to 2008 . in april 2009 , at the age of 36 years , she became intentionally pregnant for the first time , while her uc was in remission with oral 5-asa at a dose of 2,250 mg daily . however , during pregnancy , the flare - up of her total colitis required daily intravenous administration of up to 80 mg / day prednisolone , oral 5-asa 2,250 mg / day and weekly gma for 10 weeks , followed by weekly lymphocytapheresis for 3 weeks under the management of a total parenteral nutrition program at the previous hospital .", "she underwent a blood transfusion due to the development of bloody bowel movements , which was followed by severe anemia with less than 8 g / dl of hemoglobin .", "antigenemia ( c7-hrp ) positivity against cytomegalovirus was transiently detected and subsequently spontaneously converted into a negative response .", "after referral to our hospital , the patient was orally administered 20 mg prednisolone and 2,250 mg mesalamine on a daily basis .", "physical examination revealed lower abdominal tenderness with repeated bloody stools ( 810 times a day ) .", "laboratory investigations revealed a white blood cell count of 9,900/l , hemoglobin 10.1 g / dl , c - reactive protein 2.41 mg / dl , serum albumin 2.9 g / dl and an erythrocyte sedimentation rate of 50 mm / h ( table 1 ) .", "a first sigmoidoscopy showed several deep longitudinal ulcerations with erythematous and edematous changes in the mucosa from the rectum to the sigmoid colon ( fig .", "immunohistochemical findings and a polymerase chain reaction analysis of cytomegalovirus in colon biopsy specimens were also negative . upon discussion of the remaining therapeutic options ,", "she was first maintained on 20 mg prednisolone and 2,250 mg 5-asa daily ; these doses were increased to 40 mg predonisolone and 4,000 mg 5-asa daily in addition to 500 mg vancomycin daily for 1 week because of little improvement and stool culture positivity of clostridium difficile ( though cultures were negative for c. difficile toxin ) .", "the patient also underwent intensive gma therapy ( two sessions weekly ) , which was partially successful , resulting in almost complete disappearance of abdominal pain and a decrease in bowel movements to 35 times daily .", "however , her anemia was little improved and there was a gradual development of hypoalbuminemia to such an extent that transfusion of albumin was necessary every other day in order to maintain the serum level at 3.0 mg / dl , the recommended level for healthy development and safe delivery of a child .", "she was consequently unable to attain a further prolonged gestation and feared risks of blood - borne infections like hepatitis b , hepatitis c or aids viruses by repeated frequent blood transfusions .", "her first baby was prematurely delivered by cesarean section at 28 weeks and 1 day on september 9 ( birth weight 1,008 g , length 35 cm ) without any congenital malformations or neurological abnormalities .", "after cesarean section , the patient was started on oral tacrolimus ( prograf ) , aiming for serum trough levels of 1015 ng / ml for 2 weeks , followed by tapered serum trough levels of 510 ng / ml . her condition quickly improved in the following weeks and complete remission was achieved by tapering the prednisolone dose .", "total colonoscopy showed scattered regenerating areas with faintly erythematous changes in the mucosa of the transverse colon and the rectum and the appearance of mucosal healing at other sites 3 months after oral tacrolimus administration was started ( fig .", "tacrolimus is not currently approved in japan for maintenance therapy , and therefore tacrolimus administration was stopped .", "in clinical settings , therapeutic treatment for uc flare - up during pregnancy is limited because of concerns for the safety of the unborn child .", "we report herein the case of a patient with flared refractory uc during pregnancy who underwent a bridging therapy consisting of high - dose corticosteroids combined with intensive gma until it was impossible to prolong gestation any further . at that point ,", "the incidence rates of flare - up and the development of uc during pregnancy reach approximately 30% during both the remission and the active stages .", "however , it has been reported that there is no evidence to suggest that pregnancy increases the relapse risk for patients with uc [ 2 , 4 ] .", "there is also some evidence to show that pregnancy has a favorable effect on uc over time .", "a retrospective study of 111 ibd patients showed that increased parity is associated with a reduction in surgical resections .", "thus , while relapses do occur in pregnant patients , such episodes are apparently not more frequent than those in non - pregnant individuals . the key point regarding whether uc will re - emerge during pregnancy or not is the degree of activity of uc at the time of conception .", "therefore , although the present case was in remission at the time of conception , this remission may not have involved complete mucosal healing .", "currently used medications , such as corticosteroids , purine analogues , cyclosporine , tacrolimus and anti - tnf blockers , are all associated with an increased risk of opportunistic infection .", "multivariate analysis of a recent case - control study performed in ibd patients showed that the use of corticosteroids increased the likelihood of opportunistic infection with a calculated odds ratio of approximately 4 compared with the use of mesalamine .", "numerous studies have described an association between opportunistic enteric infections and exacerbation or relapse of uc .", "pathogens involved as potential triggers of uc flare - ups include c. difficile , campylobacter spp . , escherichia coli , and cytomegalovirus . in particular , the risk of c. difficile - associated diarrhea is increased , not only by antibiotic exposure , but also by immune - compromising conditions in ibd patients ,", "and there is therefore a strong focus on this risk during uc flare - ups .", "c. difficile - associated colitis in patients with ibd has a higher mortality than that in patients with c. difficile without a background of ibd . although neither toxin a nor b was detected in the present case , we eradicated c. difficle as a precautionary measure", "this treatment resulted in a partial response with 23 times fewer daily bowel movements than prior to eradication , suggesting that c. difficile infection might indeed be involved in the exacerbation of uc disease activity .", "repeated stool cultures and toxin analyses of c. difficile after eradication gave a negative result as did analysis of c7-hrp .", "pharmacologic management of uc currently relies on 5-asa , corticosteroids , immunomodulators ( aza and 6-mercaptopurine ( 6-mp ) ) , calcineurin inhibitors ( cyclosporine and tacrolimus ) , and tnf blockade .", "regarding medication during pregnancy , the most important point is to maintain remission and inactivation of disease , since the greatest risk to pregnancy is active disease .", "another key principle to disease management during uc flare - up is to remember that therapeutic treatment is limited due to concerns regarding potential fetal complications .", "5-asa remains the first - line therapy for induction and maintenance of remission for patients with mild to moderately active uc , based on an extensive history of efficacy and safety .", "prospective controlled trials and a population - based cohort study of pregnant patients who had been exposed to mesalamine did not suggest that 5-asa posed an increased risk to the fetus , despite the fact that mesalamine and its metabolite , acetyl 5-asa , are found in cord plasma .", "corticosteroid therapy is a well - established and effective treatment for patients with active uc . a large case - control study and a meta - analysis of the use of corticosteroids during the first trimester of pregnancy reported an increased risk of oral clefts in the newborns with an odds ratio of 3.35 ( 95% confidence interval 1.975.69 ) and a low overall risk of major malformations with an odds ratio of 1.45 ( 95% confidence interval 0.82.60 ) .", "intensive leukocytapheresis ( adacolumn , jimro and cellsorba ; asahi medical company , tokyo , japan ) is a relatively safe procedure that is widely used in japan for the treatment of active uc .", "a case report of a patient with uc at one center reported that gma was effective for severe uc by inducing remission with no fetal complications .", "immunosuppressive therapy using aza or 6-mp is sometimes useful for maintenance of remission in uc .", "pregnant patients treated with aza or 6-mp for ibd showed no incidence of pregnancy complications or congenital malformations . a cohort study that investigated the pregnancy outcome in female patients with", "crohn 's disease showed a higher rate of congenital anomalies following antenatal exposure to aza compared with non - treated patients .", "thus , the safety of aza and 6-mp in the treatment of pregnant patients with ibd remains controversial .", "one report that investigated 100 pregnancies among transplant recipients showed a 68% live birth rate , 12% spontaneous abortion , 3% stillbirth , and 59% premature delivery .", "in contrast , a case report of a patient with uc described a successful pregnancy and delivery of a healthy baby with rapid remission and long - term maintenance . a meta - analysis of 15 studies of pregnancy outcomes after cyclosporine therapy reported no statistical difference in the rate of fetal malformations compared to that among the general population .", "a rapid clinical response to a tnf- inhibitor ( infliximab ) is obtained when this inhibitor is used for the treatment of uc .", "infliximab does not cross the placenta in the first trimester , but feasibly is likely to do so in the second and third trimesters , thus protecting the infant from infliximab exposure during the crucial period of organogenesis .", "this result suggests that infliximab treatment does not pose a significant risk of complications during pregnancy .", "the use of aza/6-mp , tacrolimus or cyclosporine during pregnancy has never been permitted in japan .", "furthermore , until june 2010 , infliximab treatment was not approved for treatment of active uc .", "based on all of the above , we ultimately selected the following treatment regime : bridging therapy using 5-asa , corticosteroids , and intensive gma until it was impossible to further prolong gestation followed by a rescue therapy using tacrolimus after cesarean section .", "this therapy ultimately resulted in clinical remission . in conclusion , for pregnant uc patients , combined therapy consisting of high - dose corticosteroids and intensive gma is recommended until it is impossible to prolong gestation any further . when clinical remission can not be obtained , rescue therapy using tacrolimus after cesarean section may be useful .", "the authors declare that no financial or other conflict of interests exists in relation to the content of this paper ."], "abstract_text": ["<S> a 36-year - old woman who had been diagnosed with ulcerative colitis at the age of 17 years was referred to our hospital because of severe abdominal pain and repeated bloody diarrhea that persisted during pregnancy despite combination therapy with high - dose corticosteroids and weekly granulocyte and monocyte adsorptive apheresis ( gma ) . </S>", "<S> she underwent combination therapy consisting of high - dose corticosteroids , intensive gma ( two sessions per week ) and vancomycin , which was used to eradicate clostridium difficile , under total parenteral nutrition control until the estimated weight of her fetus reached 1,000 g. this combination therapy was partially successful , resulting in almost complete disappearance of abdominal pain and a marked decrease in stool frequency . </S>", "<S> however bloody diarrhea persisted and the patient developed anemia and hypoalbuminemia and was unable to prolong her gestation time . </S>", "<S> cesarean section was conducted at 28 weeks of gestation without any congenital abnormalities or neurological defects . </S>", "<S> oral administration of tacrolimus was begun 7 days after cesarean section , which was followed by rapid induction of remission . </S>", "<S> corticosteroids were then gradually tapered off . </S>", "<S> tacrolimus is one therapeutic option after cesarean section in pregnant patients who do not respond well to gma and high - dose corticosteroids for persistent active ulcerative colitis . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Disclosure Statement"], "sections": [["the prevalence of ulcerative colitis ( uc ) has increased each year for the past several decades .", "a large - scale population - based cohort study shows that the flare - up rate in women is 1.2 times higher than that in men .", "accordingly , female gender appears to be a risk factor for frequent uc flare - up .", "female patients often suffer from uc symptoms during pregnancy as uc symptoms generally start to appear then , and a definitive diagnosis of uc has been confirmed in younger individuals in their teens to thirties . it has been estimated that approximately 25% of female uc patients conceive after uc diagnosis .", "in addition , the risk of flare - up and the development of associated inflammation during pregnancy is known to be increased in both the remission and the active stages of inflammatory bowel diseases ( ibd ) , including uc and crohn 's disease .", "the first trimester is considered to be a particularly high - risk time for flare - up of uc . in contrast , two european cohort studies of 634 pregnancies in 303 women showed that pregnancy improves the disease course , with a reduction in flare - ups in subsequent years [ 2 , 4 ] .", "therapeutic options are limited when flare - ups occur during pregnancy due to the concerns for the safety of fetus . according to previous reports and statements by the united states food and drug administration ( fda ) ( http://www.fda.gov/drugs/developmentapprovalprocess/developmentresources/labeling/ucm093307.htm ) , 5-aminosalicylic acid ( 5-asa ) , corticosteroids and granulocyte and monocyte adsorptive apheresis ( gma ; adacolumn )", "however , we have often observed uc that is refractory to individual treatment with these drugs or with this modality , or to intensive therapeutic approaches that combine these drugs with this modality .", "intensive gma involving two gma sessions per week has recently been shown to be more efficacious than conventional weekly gma .", "furthermore , intensive gma also safely induced rapid remission in patients with moderately active uc inflammation .", "very recently , the drug tacrolimus was approved in japan for the treatment of patients with moderate or severe uc , regardless of whether the uc is dependent on or refractory to corticosteroids .", "tacrolimus is reportedly useful for the treatment of refractory uc due to its potent immunosuppressive properties , which induce inhibition of the transcription of the early activation genes encoding interleukin-2 , tumor necrosis factor- ( tnf- ) and interferon- that are responsible for the development of inflammation .", "we report herein the case of a patient with severe uc that had developed during the first trimester of pregnancy and was refractory to combination therapy with high - dose corticosteroids and intensive gma , but was successfully treated with tacrolimus following cesarean section early in the third trimester ."], ["in july 2009 , a 36-year - old pregnant woman was referred to our hospital for treatment of refractory uc .", "she had been diagnosed with uc ( total colitis ) at the age of 17 .", "oral 5-asa ( 2,250 mg / day ) had maintained her in remission up to the age of 31 .", "subsequently , repeated flare - ups had occurred and she had been treated with corticosteroids or with the routine weekly gma .", "azathioprine ( aza ) administration was also tried , but had to be stopped due to aza - induced pancytopenia .", "then , oral 5-asa ( 2,250 mg / day ) plus 3 mg betamethasone enema had induced and maintained remission up to 2008 . in april 2009 , at the age of 36 years , she became intentionally pregnant for the first time , while her uc was in remission with oral 5-asa at a dose of 2,250 mg daily . however , during pregnancy , the flare - up of her total colitis required daily intravenous administration of up to 80 mg / day prednisolone , oral 5-asa 2,250 mg / day and weekly gma for 10 weeks , followed by weekly lymphocytapheresis for 3 weeks under the management of a total parenteral nutrition program at the previous hospital .", "she underwent a blood transfusion due to the development of bloody bowel movements , which was followed by severe anemia with less than 8 g / dl of hemoglobin .", "antigenemia ( c7-hrp ) positivity against cytomegalovirus was transiently detected and subsequently spontaneously converted into a negative response .", "after referral to our hospital , the patient was orally administered 20 mg prednisolone and 2,250 mg mesalamine on a daily basis .", "physical examination revealed lower abdominal tenderness with repeated bloody stools ( 810 times a day ) .", "laboratory investigations revealed a white blood cell count of 9,900/l , hemoglobin 10.1 g / dl , c - reactive protein 2.41 mg / dl , serum albumin 2.9 g / dl and an erythrocyte sedimentation rate of 50 mm / h ( table 1 ) .", "a first sigmoidoscopy showed several deep longitudinal ulcerations with erythematous and edematous changes in the mucosa from the rectum to the sigmoid colon ( fig .", "immunohistochemical findings and a polymerase chain reaction analysis of cytomegalovirus in colon biopsy specimens were also negative . upon discussion of the remaining therapeutic options ,", "she was first maintained on 20 mg prednisolone and 2,250 mg 5-asa daily ; these doses were increased to 40 mg predonisolone and 4,000 mg 5-asa daily in addition to 500 mg vancomycin daily for 1 week because of little improvement and stool culture positivity of clostridium difficile ( though cultures were negative for c. difficile toxin ) .", "the patient also underwent intensive gma therapy ( two sessions weekly ) , which was partially successful , resulting in almost complete disappearance of abdominal pain and a decrease in bowel movements to 35 times daily .", "however , her anemia was little improved and there was a gradual development of hypoalbuminemia to such an extent that transfusion of albumin was necessary every other day in order to maintain the serum level at 3.0 mg / dl , the recommended level for healthy development and safe delivery of a child .", "she was consequently unable to attain a further prolonged gestation and feared risks of blood - borne infections like hepatitis b , hepatitis c or aids viruses by repeated frequent blood transfusions .", "her first baby was prematurely delivered by cesarean section at 28 weeks and 1 day on september 9 ( birth weight 1,008 g , length 35 cm ) without any congenital malformations or neurological abnormalities .", "after cesarean section , the patient was started on oral tacrolimus ( prograf ) , aiming for serum trough levels of 1015 ng / ml for 2 weeks , followed by tapered serum trough levels of 510 ng / ml . her condition quickly improved in the following weeks and complete remission was achieved by tapering the prednisolone dose .", "total colonoscopy showed scattered regenerating areas with faintly erythematous changes in the mucosa of the transverse colon and the rectum and the appearance of mucosal healing at other sites 3 months after oral tacrolimus administration was started ( fig .", "tacrolimus is not currently approved in japan for maintenance therapy , and therefore tacrolimus administration was stopped ."], ["in clinical settings , therapeutic treatment for uc flare - up during pregnancy is limited because of concerns for the safety of the unborn child .", "we report herein the case of a patient with flared refractory uc during pregnancy who underwent a bridging therapy consisting of high - dose corticosteroids combined with intensive gma until it was impossible to prolong gestation any further . at that point ,", "the incidence rates of flare - up and the development of uc during pregnancy reach approximately 30% during both the remission and the active stages .", "however , it has been reported that there is no evidence to suggest that pregnancy increases the relapse risk for patients with uc [ 2 , 4 ] .", "there is also some evidence to show that pregnancy has a favorable effect on uc over time .", "a retrospective study of 111 ibd patients showed that increased parity is associated with a reduction in surgical resections .", "thus , while relapses do occur in pregnant patients , such episodes are apparently not more frequent than those in non - pregnant individuals . the key point regarding whether uc will re - emerge during pregnancy or not is the degree of activity of uc at the time of conception .", "therefore , although the present case was in remission at the time of conception , this remission may not have involved complete mucosal healing .", "currently used medications , such as corticosteroids , purine analogues , cyclosporine , tacrolimus and anti - tnf blockers , are all associated with an increased risk of opportunistic infection .", "multivariate analysis of a recent case - control study performed in ibd patients showed that the use of corticosteroids increased the likelihood of opportunistic infection with a calculated odds ratio of approximately 4 compared with the use of mesalamine .", "numerous studies have described an association between opportunistic enteric infections and exacerbation or relapse of uc .", "pathogens involved as potential triggers of uc flare - ups include c. difficile , campylobacter spp . , escherichia coli , and cytomegalovirus . in particular , the risk of c. difficile - associated diarrhea is increased , not only by antibiotic exposure , but also by immune - compromising conditions in ibd patients ,", "and there is therefore a strong focus on this risk during uc flare - ups .", "c. difficile - associated colitis in patients with ibd has a higher mortality than that in patients with c. difficile without a background of ibd . although neither toxin a nor b was detected in the present case , we eradicated c. difficle as a precautionary measure", "this treatment resulted in a partial response with 23 times fewer daily bowel movements than prior to eradication , suggesting that c. difficile infection might indeed be involved in the exacerbation of uc disease activity .", "repeated stool cultures and toxin analyses of c. difficile after eradication gave a negative result as did analysis of c7-hrp .", "pharmacologic management of uc currently relies on 5-asa , corticosteroids , immunomodulators ( aza and 6-mercaptopurine ( 6-mp ) ) , calcineurin inhibitors ( cyclosporine and tacrolimus ) , and tnf blockade .", "regarding medication during pregnancy , the most important point is to maintain remission and inactivation of disease , since the greatest risk to pregnancy is active disease .", "another key principle to disease management during uc flare - up is to remember that therapeutic treatment is limited due to concerns regarding potential fetal complications .", "5-asa remains the first - line therapy for induction and maintenance of remission for patients with mild to moderately active uc , based on an extensive history of efficacy and safety .", "prospective controlled trials and a population - based cohort study of pregnant patients who had been exposed to mesalamine did not suggest that 5-asa posed an increased risk to the fetus , despite the fact that mesalamine and its metabolite , acetyl 5-asa , are found in cord plasma .", "corticosteroid therapy is a well - established and effective treatment for patients with active uc . a large case - control study and a meta - analysis of the use of corticosteroids during the first trimester of pregnancy reported an increased risk of oral clefts in the newborns with an odds ratio of 3.35 ( 95% confidence interval 1.975.69 ) and a low overall risk of major malformations with an odds ratio of 1.45 ( 95% confidence interval 0.82.60 ) .", "intensive leukocytapheresis ( adacolumn , jimro and cellsorba ; asahi medical company , tokyo , japan ) is a relatively safe procedure that is widely used in japan for the treatment of active uc .", "a case report of a patient with uc at one center reported that gma was effective for severe uc by inducing remission with no fetal complications .", "immunosuppressive therapy using aza or 6-mp is sometimes useful for maintenance of remission in uc .", "pregnant patients treated with aza or 6-mp for ibd showed no incidence of pregnancy complications or congenital malformations . a cohort study that investigated the pregnancy outcome in female patients with", "crohn 's disease showed a higher rate of congenital anomalies following antenatal exposure to aza compared with non - treated patients .", "thus , the safety of aza and 6-mp in the treatment of pregnant patients with ibd remains controversial .", "one report that investigated 100 pregnancies among transplant recipients showed a 68% live birth rate , 12% spontaneous abortion , 3% stillbirth , and 59% premature delivery .", "in contrast , a case report of a patient with uc described a successful pregnancy and delivery of a healthy baby with rapid remission and long - term maintenance . a meta - analysis of 15 studies of pregnancy outcomes after cyclosporine therapy reported no statistical difference in the rate of fetal malformations compared to that among the general population .", "a rapid clinical response to a tnf- inhibitor ( infliximab ) is obtained when this inhibitor is used for the treatment of uc .", "infliximab does not cross the placenta in the first trimester , but feasibly is likely to do so in the second and third trimesters , thus protecting the infant from infliximab exposure during the crucial period of organogenesis .", "this result suggests that infliximab treatment does not pose a significant risk of complications during pregnancy .", "the use of aza/6-mp , tacrolimus or cyclosporine during pregnancy has never been permitted in japan .", "furthermore , until june 2010 , infliximab treatment was not approved for treatment of active uc .", "based on all of the above , we ultimately selected the following treatment regime : bridging therapy using 5-asa , corticosteroids , and intensive gma until it was impossible to further prolong gestation followed by a rescue therapy using tacrolimus after cesarean section .", "this therapy ultimately resulted in clinical remission . in conclusion , for pregnant uc patients , combined therapy consisting of high - dose corticosteroids and intensive gma is recommended until it is impossible to prolong gestation any further . when clinical remission can not be obtained , rescue therapy using tacrolimus after cesarean section may be useful ."], ["the authors declare that no financial or other conflict of interests exists in relation to the content of this paper ."]]}
{"article_id": "PMC4247534", "article_text": ["only a few cases of pulmonary vascular tumors diagnosed using endobronchial ultrasound - guided transbronchial needle aspiration ( ebus - tbna ) have been reported in the literature .", "they frequently present as filling defects in the pulmonary vasculature on computed tomography ( ct ) making it challenging to distinguish between vascular tumor and thrombotic emboli .", "although fluorodeoxyglucose - positive emission tomography ( pet ) scan may reportedly help differentiate malignant growth from benign emboli , pet scans are not routinely performed for this purpose . increased uptake on the pet scan increases the malignant suspicion of vascular tumors but in most cases", "ebus - tbna of mediastinal and hilar lymph nodes has proven value in diagnosis and staging for lung cancer .", "in addition , ebus - tbna may provide an opportunity to identify and sample endovascular abnormalities in the mediastinal and hilar area .", "a 43-year - old white male was diagnosed with leiomyosarcoma of proximal left thigh involving femoral vessels 2 years back .", "he was found to have pulmonary embolism involving right pulmonary artery for which he was started on anticoagulation and an inferior vena cava filter was placed prior to surgery . despite being on anticoagulation postoperatively , serial surveillance imaging continued to show persistent  thrombus .  eventually , an interval increase in size of the filling defect in right main pulmonary artery extending to the right lower lobe pulmonary artery was noted on contrast - enhanced chest ct [ figure 1 ] . at this point ,", "intravascular metastatic tumor was suspected and the patient was referred to our interventional pulmonary service for diagnostic evaluation .", "the patient did not have any other imaging studies such as pet scan or magnetic resonance imaging of the chest .", "ebus ( bf - uc160f - ol8 ; olympus , tokyo , japan ) procedure was performed under general anesthesia . with the ultrasound and color flow doppler ,", "an endovascular hyper - echoic lesion was identified in right pulmonary artery [ figure 2 ] .", "only one endovascular lesion was identified , which extended to the right lower lobe branch of the pulmonary artery .", "because of the nature of the lesion 's extension in the pulmonary artery , it was not possible to accurately size it but it was estimated to be around 20 mm in the largest diameter .", "ebus - tbna of this lesion using the 22 gauge ebus needle ( olympus , na-201sx-4022 ) was performed under general anesthesia with laryngeal mask airway using propofol intravenous drip and fentanyl intravenous intermittent injections .", "a core biopsy was obtained using the 22 gauge ebus needle and a total of five passes were performed during this procedure .", "rapid - on - site evaluation of fixed slides was bloody , and no malignant cells were identified .", "the procedure was performed without any complications and cytopathological and immunohistochemical evaluation confirmed the diagnosis of metastatic leiomyosarcoma [ figure 3 ] . the patient was started on gemcitabine plus docetaxel combination chemotherapy .", "( a ) chest computed tomography ( ct ) image showing right pulmonary artery filling defect ( arrow ) diagnosed as pulmonary thrombotic emboli 2 years ago .", "( b ) chest ct 2 years later showing enlarged right pulmonary artery filling extending to the right lower arterial branch consistent with vascular tumor ( arrow ) ( a ) endobronchial ultrasound ( ebus ) image of the right hilar area showing an vascular tumor of the right lower lobe pulmonary artery ( arrow ) .", "( d ) ebus - guided transbronchial needle aspiration ( arrow ) of the right pulmonary artery tumor .", "the color doppler was used to ensure sampling the tumor area with the least blood flow ( a ) this is the tissue core biopsy of the right pulmonary mass lesion showing cells with abundant eosinophilic cytoplasm and spindled nuclei ( h and e , 200 original magnification ) .", "( b ) these cells cytologically shows fine granular chromatin without prominent nucleoli ( papanicolaou stain , 600 original magnification ) .", "( c ) immunohistochemical stains for smooth muscle actin shows strong immunoreactivity in these cells ( sma , 400 original magnification ) .", "( d ) immunohistochemical stains for desmin also shows strong positivity in these malignant cells ( desmin , 200 original magnification )", "historically , most cases of pulmonary artery tumors were diagnosed with surgery or during postmortem examination .", "successful diagnosis of primary pulmonary artery leiomyosarcoma with catheterization and biopsy has also been reported . in the last few years , several cases of primary and metastatic pulmonary artery tumors diagnosed by ebus - tbna have been reported without any procedure - related complications .", "although pulmonary artery hematoma has been described from ebus - tbna , the pulmonary artery was inadvertently punctured in that case during a routine biopsy of mediastinal lymph node .", "in addition , there have been reports of using ebus - guided trans - pulmonary vascular lymph - node sampling without any complications . here , we present the first case of metastatic leiomyosarcoma of pulmonary artery diagnosed with ebus - tbna .", "the case reported is a clear example of the challenges in diagnosis of pulmonary artery tumors due to their endovascular location and the potential risk of bleeding .", "the use of color flow doppler during ebus - tbna of pulmonary vascular tumor is essential to avoid area of higher blood flow and to perform the biopsy in the tumoral area of least blood flow . given the potential higher risk of bleeding , anticoagulation or clopidogrel therapy should probably be discontinued prior to this procedure .", "clinically , the importance of high index of suspicion is also demonstrated as the patient was initially diagnosed with pulmonary embolism .", "misdiagnosing patients with pulmonary artery tumor emboli as thrombotic emboli can result in delaying effective tumor therapy with chemotherapy or surgery .", "the diagnosis of pulmonary embolism in patients with malignant diseases can be challenging especially in ill patients where the wells score is already high .", "our patient was asymptomatic , and the chest imaging was performed as part of surveillance and staging of his sarcoma .", "intravascular metastasis was suspected as an alternate diagnosis after the presumed failure of anticoagulation therapy and increased of the pulmonary artery lesion size .", "ebus - tbna is a safe procedure and serious complications are extremely rare . until now", ", ebus - tbna of the pulmonary vascular lesions did not report any serious complications .", "ebus - tbna seems to be a safe , feasible , and minimally invasive tool for the diagnosis of vascular tumors such as metastatic pulmonary artery leiomyosarcoma ."], "abstract_text": ["<S> leiomyosarcoma of the pulmonary vasculature is an extremely rare condition that has not been previously diagnosed by endobronchial ultrasound ( ebus ) bronchoscopy . </S>", "<S> we present the case of a 43-year - old white male with a history of leiomyosarcoma who was diagnosed with pulmonary embolism 2 years ago . as the filling defects on follow - up chest computed tomography continued to worsen despite anticoagulation , </S>", "<S> ebus - guided transbronchial needle aspiration ( ebus - tbna ) of the right pulmonary artery lesion was safely and successfully performed . </S>", "<S> cytopathological examination revealed the  </S>", "<S> thrombus  to be metastatic leiomyosarcoma . in experienced hands , and carefully selected cases , ebus - tbna seems to be a safe and effective in diagnosing thoracic endovascular lesions . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["only a few cases of pulmonary vascular tumors diagnosed using endobronchial ultrasound - guided transbronchial needle aspiration ( ebus - tbna ) have been reported in the literature .", "they frequently present as filling defects in the pulmonary vasculature on computed tomography ( ct ) making it challenging to distinguish between vascular tumor and thrombotic emboli .", "although fluorodeoxyglucose - positive emission tomography ( pet ) scan may reportedly help differentiate malignant growth from benign emboli , pet scans are not routinely performed for this purpose . increased uptake on the pet scan increases the malignant suspicion of vascular tumors but in most cases", "ebus - tbna of mediastinal and hilar lymph nodes has proven value in diagnosis and staging for lung cancer .", "in addition , ebus - tbna may provide an opportunity to identify and sample endovascular abnormalities in the mediastinal and hilar area ."], ["a 43-year - old white male was diagnosed with leiomyosarcoma of proximal left thigh involving femoral vessels 2 years back .", "he was found to have pulmonary embolism involving right pulmonary artery for which he was started on anticoagulation and an inferior vena cava filter was placed prior to surgery . despite being on anticoagulation postoperatively , serial surveillance imaging continued to show persistent  thrombus .  eventually , an interval increase in size of the filling defect in right main pulmonary artery extending to the right lower lobe pulmonary artery was noted on contrast - enhanced chest ct [ figure 1 ] . at this point ,", "intravascular metastatic tumor was suspected and the patient was referred to our interventional pulmonary service for diagnostic evaluation .", "the patient did not have any other imaging studies such as pet scan or magnetic resonance imaging of the chest .", "ebus ( bf - uc160f - ol8 ; olympus , tokyo , japan ) procedure was performed under general anesthesia . with the ultrasound and color flow doppler ,", "an endovascular hyper - echoic lesion was identified in right pulmonary artery [ figure 2 ] .", "only one endovascular lesion was identified , which extended to the right lower lobe branch of the pulmonary artery .", "because of the nature of the lesion 's extension in the pulmonary artery , it was not possible to accurately size it but it was estimated to be around 20 mm in the largest diameter .", "ebus - tbna of this lesion using the 22 gauge ebus needle ( olympus , na-201sx-4022 ) was performed under general anesthesia with laryngeal mask airway using propofol intravenous drip and fentanyl intravenous intermittent injections .", "a core biopsy was obtained using the 22 gauge ebus needle and a total of five passes were performed during this procedure .", "rapid - on - site evaluation of fixed slides was bloody , and no malignant cells were identified .", "the procedure was performed without any complications and cytopathological and immunohistochemical evaluation confirmed the diagnosis of metastatic leiomyosarcoma [ figure 3 ] . the patient was started on gemcitabine plus docetaxel combination chemotherapy .", "( a ) chest computed tomography ( ct ) image showing right pulmonary artery filling defect ( arrow ) diagnosed as pulmonary thrombotic emboli 2 years ago .", "( b ) chest ct 2 years later showing enlarged right pulmonary artery filling extending to the right lower arterial branch consistent with vascular tumor ( arrow ) ( a ) endobronchial ultrasound ( ebus ) image of the right hilar area showing an vascular tumor of the right lower lobe pulmonary artery ( arrow ) .", "( d ) ebus - guided transbronchial needle aspiration ( arrow ) of the right pulmonary artery tumor .", "the color doppler was used to ensure sampling the tumor area with the least blood flow ( a ) this is the tissue core biopsy of the right pulmonary mass lesion showing cells with abundant eosinophilic cytoplasm and spindled nuclei ( h and e , 200 original magnification ) .", "( b ) these cells cytologically shows fine granular chromatin without prominent nucleoli ( papanicolaou stain , 600 original magnification ) .", "( c ) immunohistochemical stains for smooth muscle actin shows strong immunoreactivity in these cells ( sma , 400 original magnification ) .", "( d ) immunohistochemical stains for desmin also shows strong positivity in these malignant cells ( desmin , 200 original magnification )"], ["historically , most cases of pulmonary artery tumors were diagnosed with surgery or during postmortem examination .", "successful diagnosis of primary pulmonary artery leiomyosarcoma with catheterization and biopsy has also been reported . in the last few years , several cases of primary and metastatic pulmonary artery tumors diagnosed by ebus - tbna have been reported without any procedure - related complications .", "although pulmonary artery hematoma has been described from ebus - tbna , the pulmonary artery was inadvertently punctured in that case during a routine biopsy of mediastinal lymph node .", "in addition , there have been reports of using ebus - guided trans - pulmonary vascular lymph - node sampling without any complications . here , we present the first case of metastatic leiomyosarcoma of pulmonary artery diagnosed with ebus - tbna .", "the case reported is a clear example of the challenges in diagnosis of pulmonary artery tumors due to their endovascular location and the potential risk of bleeding .", "the use of color flow doppler during ebus - tbna of pulmonary vascular tumor is essential to avoid area of higher blood flow and to perform the biopsy in the tumoral area of least blood flow . given the potential higher risk of bleeding , anticoagulation or clopidogrel therapy should probably be discontinued prior to this procedure .", "clinically , the importance of high index of suspicion is also demonstrated as the patient was initially diagnosed with pulmonary embolism .", "misdiagnosing patients with pulmonary artery tumor emboli as thrombotic emboli can result in delaying effective tumor therapy with chemotherapy or surgery .", "the diagnosis of pulmonary embolism in patients with malignant diseases can be challenging especially in ill patients where the wells score is already high .", "our patient was asymptomatic , and the chest imaging was performed as part of surveillance and staging of his sarcoma .", "intravascular metastasis was suspected as an alternate diagnosis after the presumed failure of anticoagulation therapy and increased of the pulmonary artery lesion size .", "ebus - tbna is a safe procedure and serious complications are extremely rare . until now", ", ebus - tbna of the pulmonary vascular lesions did not report any serious complications ."], ["ebus - tbna seems to be a safe , feasible , and minimally invasive tool for the diagnosis of vascular tumors such as metastatic pulmonary artery leiomyosarcoma ."]]}
{"article_id": "PMC2526537", "article_text": ["transitional cell carcinoma ( tcc ) of the urinary bladder can present as a non - muscle invasive or as a muscle - invasive lesion .", "the majority of patients ( approximately 75% ) present with non - muscle invasive tumors , which are limited to the mucosa ( ta ) or the lamina propria ( t1 ) ( 1 ) .", ". however , cis tends to behave more aggressively and is often found in association with high - grade non - muscle invasive tumors .", "prognostic factors in patients with non - muscle invasive bladder cancer have been the subject of numerous publications for many years ( 2 - 18 ) .", "depending on a patient 's characteristics , the probability of recurrence after transurethral resection ( tur ) at one year ranges from about 15% to 70% ( 2 ) , and the probability of progression at five years ranges from about 7% to 40% ( 6 ) .", "non - muscle invasive tumors may be managed with tur with or without intravesical therapy .", "however , not all non - muscle invasive bladder cancer patients have the same risk for disease recurrence and progression , and the absence of risk stratification between aggressive and indolent tumors contributes to potentially excessive frequent surveillance or unnecessary intravesical therapy .", "several studies have suggested risk tables for the recurrence and progression of non - muscle invasive bladder cancer ( 5 , 7 , 9 , 10 ) , but they can not calculate the probability of a certain event , such as the probability of recurrence within five years .", "the purpose of this study was to develop and validate prognostic nomograms for disease recurrence in patients with ta , t1 transitional cell carcinomas of the bladder .", "these nonograms will facilitate patient counseling , choosing the most appropriate adjuvant treatment after tur , and determining the frequency of follow - up in individual patients .", "thirty - eight training hospitals participated in this retrospective multicenter study . between january 1998 and december 2002 , 3,060 patients with newly diagnosed", "they were treated with transurethral resection ( tur ) and diagnosed with ta or t1 tcc of the bladder based on the 2002 american joint committee on cancer ( ajcc ) tnm staging system ( 19 ) .", "patients with prior histories of bladder cancer , non - tcc histology , primary cis , or a follow - up duration of less than 12 months were excluded .", "this cohort study was also limited to tumors whose grades were determined using the 1973 world health organization system .", "the median age was 63 yr ( 21 - 98 ) , and the median follow - up duration was 44 months ( 12 - 97 ) . the clinical and pathological data ( local ) , including sex , age , tumor size ( <3 cm or 3 cm ) , multiplicity ( single or multiple ) , t category ( ta or t1 ) , tumor grade , presence of concomitant cis or squamous differentiation , and intravesical therapy were obtained from each hospital and merged .", "one - thousand and eighty - two patients were treated with intravesical therapy after tur , and drugs used for intravesical therapy were as follows : bcg in 827 patients , mitomycin - c in 108 patients , epirubicin in 135 patients , and other drugs in 12 patients .", "follow - up data were also obtained , including pathologically proven recurrence and time to first recurrence , which was defined as the time period between the date of initial diagnosis and the date of recurrence .", "patients who were still alive or who had died before a recurrence were censored at the date of the last available follow - up cystoscopy .", "we assessed the impact of various clinical and pathological features on time to first recurrence .", "the predictive accuracy of each univariate and multivariate logistic regression model was tested using the area under the receiver operating characteristics ( roc ) curve .", "all univariate and multivariate models were internally validated with 500 bootstrap re - samples as a means of calculating the most unbiased predictive accuracy . statistical significance in this study", "all statistical tests were performed using r software ( r , version 2.4.1 , the r project for statistical computing , http://www.r-project.org ) .", "three - year and five - year recurrence - free rates were 64.3% and 55.3% , respectively ( fig .", "1 ) . on univariate cox proportional hazard analysis , age , tumor size , multiplicity , tumor grade , concomitant cis , and intravesical therapy had a significant influence on recurrence - free survival ( p<0.05 ) ( table 2 ) .", "multivariate analysis revealed that age ( hazard ratio [ hr]=1.437 , p<0.001 ) , tumor size ( hr=1.328 , p=0.001 ) , multiplicity ( hr=1.505 , p<0.001 ) , tumor grade ( hr=1.347 , p=0.007 ) , concomitant cis ( hr=1.611 , p=0.007 ) , and intravesical therapy ( hr=0.681 , p<0.001 ) were independent predictors for disease - recurrence ( table 3 ) . based on this analysis , nomograms for the prediction of recurrence probabilities at three and five years were constructed ( fig .", "the scales of the nomograms reflected the coefficients from the cox model rescaled to a user - friendly ( 100 point ) range .", "the bootstrapping estimate of the predictive accuracy of the nomograms suggested that the area under the roc curve was approximately 0.599 at three years and 0.604 at five years .", "the results of bootstrap re - samples were 0.723 for the three - year estimation of the nomogram and 0.738 for the five - year estimation .", "the prognostic importance of various factors is not always consistent among various studies , and analyses of prognostic factors of the bladder tcc related to recurrence are sometimes difficult to compare ( 8) .", "this difficulty may be due to differences in the choice of the variables analyzed , their coding , and , in multivariate analyses , the correlation among the factors ( 10 ) .", "another important source of variability stems from differences in tumor status , especially between primary or recurrent tumors .", "this study showed that patient age , tumor size , multiplicity , tumor grade , cis , and intravesical therapy are significant predictors for recurrence in patients with ta , t1 tcc of the bladder .", "there was no difference in the time to the first recurrence between grade ii and grade iii tumors in the univariate analysis , so a dichotomized tumor grade ( grade i vs. grade ii / iii ) was used for the multivariate analysis ( table 2 ) . in addition , differences among intravesical instillation agents had no influence on time to first recurrence , and intravesical therapy was also simply dichotomized ( not performed vs. performed ) ( table 2 ) .", "of these , multiplicity and intravesical therapy following tur were the most influential determinants for prediction of recurrence in nomograms ( fig .", "tumor grade , tumor size , and patient age were less strong predictors than the ones mentioned previously .", "concomitant cis was a relatively weak variable , especially in nomograms for five years ( fig .", "2 ) . in addition , in this study , the study cohort was limited to tumors that were newly diagnosed between 1998 and 2002 .", "nevertheless , tumor grade , t stage , concomitant cis , tumor size , intravesical therapy , tumor status ( primary vs. recurrent ) , and recurrence rate among other factors were generally identified as independent prognostic factors for disease recurrence ( 2 - 18 ) . in some studies , age ,", "tumor location , and sex have also been regarded as independent risk factors ( 15 , 17 , 18 ) .", "notably , our nomograms showed that age is a strong determinant for prediction of recurrence .", "younger patients appear to have a more favorable prognosis because they present more frequently with non - muscle invasive , low - grade tumors .", "however , studies have shown that the risk for disease progression is the same , grade - for - grade , in younger patients as in older ones ( 20 , 21 ) .", "in contrast , our results suggest that age has a prognostic impact independent from other factors . these observations are consistent with the work by shariat et al .", "we can not explain why the age factor plays an important role in disease recurrence .", "differences in genetic and molecular aberrations in bladder tumors and accumulation of these aberrations in older patients may relate to age .", "2 may be useful for patient counseling because they predict the probability that the patient will encounter recurrent bladder cancer within the next three to five years .", "they are also effective tools for selecting patients for experimental adjuvant therapy because they are likely to be more prognostically accurate than the typical risk stratification approaches that form patient groups by placing cutoffs on variables . in the context of a randomized clinical trial", "this procedure could be accomplished by computing predicted probabilities for each patient , which would simultaneously consider all prognostic variables , and by comparing the predictions across treatment arms . in our study , we suggested two nomograms that were very similar , but there were some differences in the weight of risk score .", "the nomogram for three years would be more suitable for short - term studies , and the five - year nomogram may be helpful for long - term studies .", "additionally , nomograms may aid in determining the scheduling of follow - up visits , as patients at lower risk for relapse or a second primary tumor may require less stringent follow - up evaluations ( 23 ) .", "the prediction for the area under the roc curve was approximately 0.599 for the three - year nomogram and was 0.604 for the five - year nomogram . despite being imperfect", "another limitation is that the nomogram predicts disease recurrence only within a maximum period of five years .", "the nomogram is additionally limited because it relies on postoperative variables , making it an inadequate preoperative patient counseling tool .", "it is important to remember that adjuvant therapy performed in this data set was a mixture of various intravesical therapies , and the decision as to which instillation drug should be chosen was left to the physicians . finally , experiencing a beneficial effect on intravesical therapy in this study", "does not mean that adjuvant intravescial therapy has to be performed in all cases of non - muscle invasive bladder tumors .", "the nomograms for non - muscle invasive bladder cancer in which a point system was used are very rare .", "( 22 ) have reported that nomograms for non - muscle invasive bladder cancer can predict recurrence and progression , which included nuclear matrix protein 22 and cytology , as well as conventional predictors .", "we tested only conventional variables without ancillary markers , i.e. , nuclear matrix protein 22 .", "for this reason , the nomograms in our study may be more feasible in common clinical settings . in conclusion", ", current nomograms can be used to predict the probability of disease recurrence in patients with newly diagnosed ta , t1 transitional cell carcinoma of the bladder .", "they may be useful for patient counseling , clinical trial design , and patient follow - up planning ."], "abstract_text": ["<S> we developed nomograms to predict disease recurrence in patients with ta , t1 transitional cell carcinoma of the bladder . </S>", "<S> thirty - eight training hospitals participated in this retrospective multicenter study . between 1998 and 2002 , a total of 1,587 patients with newly diagnosed non - muscle invasive bladder cancer were enrolled in this study . </S>", "<S> patients with prior histories of bladder cancer , non - transitional cell carcinoma , or a follow - up duration of less than 12 months were excluded . with univariate and multivariate logistic regression analyses </S>", "<S> , we constructed nomograms to predict disease recurrence , and internal validation was performed using statistical techniques . </S>", "<S> three - year and five - year recurrence - free rates were 64.3% and 55.3% , respectively . </S>", "<S> multivariate analysis revealed that age ( hazard ratio [ hr]=1.437 , p<0.001 ) , tumor size ( hr=1.328 , p=0.001 ) , multiplicity ( hr=1.505 , p<0.001 ) , tumor grade ( hr=1.347 , p=0.007 ) , concomitant carcinoma in situ ( hr=1.611 , p=0.007 ) , and intravesical therapy ( hr=0.681 , </S>", "<S> p<0.001 ) were independent predictors for disease recurrence . </S>", "<S> based on these prognostic factors , nomograms for the prediction of disease recurrence were developed . </S>", "<S> these nomograms can be used to predict the probability of disease recurrence in patients with newly diagnosed ta , t1 transitional cell carcinoma of the bladder . </S>", "<S> they may be useful for patient counseling , clinical trial design , and patient follow - up planning . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["transitional cell carcinoma ( tcc ) of the urinary bladder can present as a non - muscle invasive or as a muscle - invasive lesion .", "the majority of patients ( approximately 75% ) present with non - muscle invasive tumors , which are limited to the mucosa ( ta ) or the lamina propria ( t1 ) ( 1 ) .", ". however , cis tends to behave more aggressively and is often found in association with high - grade non - muscle invasive tumors .", "prognostic factors in patients with non - muscle invasive bladder cancer have been the subject of numerous publications for many years ( 2 - 18 ) .", "depending on a patient 's characteristics , the probability of recurrence after transurethral resection ( tur ) at one year ranges from about 15% to 70% ( 2 ) , and the probability of progression at five years ranges from about 7% to 40% ( 6 ) .", "non - muscle invasive tumors may be managed with tur with or without intravesical therapy .", "however , not all non - muscle invasive bladder cancer patients have the same risk for disease recurrence and progression , and the absence of risk stratification between aggressive and indolent tumors contributes to potentially excessive frequent surveillance or unnecessary intravesical therapy .", "several studies have suggested risk tables for the recurrence and progression of non - muscle invasive bladder cancer ( 5 , 7 , 9 , 10 ) , but they can not calculate the probability of a certain event , such as the probability of recurrence within five years .", "the purpose of this study was to develop and validate prognostic nomograms for disease recurrence in patients with ta , t1 transitional cell carcinomas of the bladder .", "these nonograms will facilitate patient counseling , choosing the most appropriate adjuvant treatment after tur , and determining the frequency of follow - up in individual patients ."], ["thirty - eight training hospitals participated in this retrospective multicenter study . between january 1998 and december 2002 , 3,060 patients with newly diagnosed", "they were treated with transurethral resection ( tur ) and diagnosed with ta or t1 tcc of the bladder based on the 2002 american joint committee on cancer ( ajcc ) tnm staging system ( 19 ) .", "patients with prior histories of bladder cancer , non - tcc histology , primary cis , or a follow - up duration of less than 12 months were excluded .", "this cohort study was also limited to tumors whose grades were determined using the 1973 world health organization system .", "the median age was 63 yr ( 21 - 98 ) , and the median follow - up duration was 44 months ( 12 - 97 ) . the clinical and pathological data ( local ) , including sex , age , tumor size ( <3 cm or 3 cm ) , multiplicity ( single or multiple ) , t category ( ta or t1 ) , tumor grade , presence of concomitant cis or squamous differentiation , and intravesical therapy were obtained from each hospital and merged .", "one - thousand and eighty - two patients were treated with intravesical therapy after tur , and drugs used for intravesical therapy were as follows : bcg in 827 patients , mitomycin - c in 108 patients , epirubicin in 135 patients , and other drugs in 12 patients .", "follow - up data were also obtained , including pathologically proven recurrence and time to first recurrence , which was defined as the time period between the date of initial diagnosis and the date of recurrence .", "patients who were still alive or who had died before a recurrence were censored at the date of the last available follow - up cystoscopy .", "we assessed the impact of various clinical and pathological features on time to first recurrence .", "the predictive accuracy of each univariate and multivariate logistic regression model was tested using the area under the receiver operating characteristics ( roc ) curve .", "all univariate and multivariate models were internally validated with 500 bootstrap re - samples as a means of calculating the most unbiased predictive accuracy . statistical significance in this study", "all statistical tests were performed using r software ( r , version 2.4.1 , the r project for statistical computing , http://www.r-project.org ) ."], ["three - year and five - year recurrence - free rates were 64.3% and 55.3% , respectively ( fig .", "1 ) . on univariate cox proportional hazard analysis , age , tumor size , multiplicity , tumor grade , concomitant cis , and intravesical therapy had a significant influence on recurrence - free survival ( p<0.05 ) ( table 2 ) .", "multivariate analysis revealed that age ( hazard ratio [ hr]=1.437 , p<0.001 ) , tumor size ( hr=1.328 , p=0.001 ) , multiplicity ( hr=1.505 , p<0.001 ) , tumor grade ( hr=1.347 , p=0.007 ) , concomitant cis ( hr=1.611 , p=0.007 ) , and intravesical therapy ( hr=0.681 , p<0.001 ) were independent predictors for disease - recurrence ( table 3 ) . based on this analysis , nomograms for the prediction of recurrence probabilities at three and five years were constructed ( fig .", "the scales of the nomograms reflected the coefficients from the cox model rescaled to a user - friendly ( 100 point ) range .", "the bootstrapping estimate of the predictive accuracy of the nomograms suggested that the area under the roc curve was approximately 0.599 at three years and 0.604 at five years .", "the results of bootstrap re - samples were 0.723 for the three - year estimation of the nomogram and 0.738 for the five - year estimation ."], ["the prognostic importance of various factors is not always consistent among various studies , and analyses of prognostic factors of the bladder tcc related to recurrence are sometimes difficult to compare ( 8) .", "this difficulty may be due to differences in the choice of the variables analyzed , their coding , and , in multivariate analyses , the correlation among the factors ( 10 ) .", "another important source of variability stems from differences in tumor status , especially between primary or recurrent tumors .", "this study showed that patient age , tumor size , multiplicity , tumor grade , cis , and intravesical therapy are significant predictors for recurrence in patients with ta , t1 tcc of the bladder .", "there was no difference in the time to the first recurrence between grade ii and grade iii tumors in the univariate analysis , so a dichotomized tumor grade ( grade i vs. grade ii / iii ) was used for the multivariate analysis ( table 2 ) . in addition , differences among intravesical instillation agents had no influence on time to first recurrence , and intravesical therapy was also simply dichotomized ( not performed vs. performed ) ( table 2 ) .", "of these , multiplicity and intravesical therapy following tur were the most influential determinants for prediction of recurrence in nomograms ( fig .", "tumor grade , tumor size , and patient age were less strong predictors than the ones mentioned previously .", "concomitant cis was a relatively weak variable , especially in nomograms for five years ( fig .", "2 ) . in addition , in this study , the study cohort was limited to tumors that were newly diagnosed between 1998 and 2002 .", "nevertheless , tumor grade , t stage , concomitant cis , tumor size , intravesical therapy , tumor status ( primary vs. recurrent ) , and recurrence rate among other factors were generally identified as independent prognostic factors for disease recurrence ( 2 - 18 ) . in some studies , age ,", "tumor location , and sex have also been regarded as independent risk factors ( 15 , 17 , 18 ) .", "notably , our nomograms showed that age is a strong determinant for prediction of recurrence .", "younger patients appear to have a more favorable prognosis because they present more frequently with non - muscle invasive , low - grade tumors .", "however , studies have shown that the risk for disease progression is the same , grade - for - grade , in younger patients as in older ones ( 20 , 21 ) .", "in contrast , our results suggest that age has a prognostic impact independent from other factors . these observations are consistent with the work by shariat et al .", "we can not explain why the age factor plays an important role in disease recurrence .", "differences in genetic and molecular aberrations in bladder tumors and accumulation of these aberrations in older patients may relate to age .", "2 may be useful for patient counseling because they predict the probability that the patient will encounter recurrent bladder cancer within the next three to five years .", "they are also effective tools for selecting patients for experimental adjuvant therapy because they are likely to be more prognostically accurate than the typical risk stratification approaches that form patient groups by placing cutoffs on variables . in the context of a randomized clinical trial", "this procedure could be accomplished by computing predicted probabilities for each patient , which would simultaneously consider all prognostic variables , and by comparing the predictions across treatment arms . in our study , we suggested two nomograms that were very similar , but there were some differences in the weight of risk score .", "the nomogram for three years would be more suitable for short - term studies , and the five - year nomogram may be helpful for long - term studies .", "additionally , nomograms may aid in determining the scheduling of follow - up visits , as patients at lower risk for relapse or a second primary tumor may require less stringent follow - up evaluations ( 23 ) .", "the prediction for the area under the roc curve was approximately 0.599 for the three - year nomogram and was 0.604 for the five - year nomogram . despite being imperfect", "another limitation is that the nomogram predicts disease recurrence only within a maximum period of five years .", "the nomogram is additionally limited because it relies on postoperative variables , making it an inadequate preoperative patient counseling tool .", "it is important to remember that adjuvant therapy performed in this data set was a mixture of various intravesical therapies , and the decision as to which instillation drug should be chosen was left to the physicians . finally , experiencing a beneficial effect on intravesical therapy in this study", "does not mean that adjuvant intravescial therapy has to be performed in all cases of non - muscle invasive bladder tumors .", "the nomograms for non - muscle invasive bladder cancer in which a point system was used are very rare .", "( 22 ) have reported that nomograms for non - muscle invasive bladder cancer can predict recurrence and progression , which included nuclear matrix protein 22 and cytology , as well as conventional predictors .", "we tested only conventional variables without ancillary markers , i.e. , nuclear matrix protein 22 .", "for this reason , the nomograms in our study may be more feasible in common clinical settings . in conclusion", ", current nomograms can be used to predict the probability of disease recurrence in patients with newly diagnosed ta , t1 transitional cell carcinoma of the bladder .", "they may be useful for patient counseling , clinical trial design , and patient follow - up planning ."]]}
{"article_id": "PMC3074291", "article_text": ["endobronchial tuberculosis ( ebtb ) is a form of tuberculous infection that involves mainly the trachea and/or bronchi .", "the disease is often mistaken for more common lung diseases like pneumonia , asthma , lung cancer and other lung diseases .", "endobronchial stenosis is a common complication of ebtb despite the use of anti - tuberculous chemotherapy .", "we are presenting a case of ebtb that manifested with two endobronchial masses , which almost totally occluded the bronchial lumen and resulted in repeated episodes of post - obstructive pneumonia for more than a year prior to diagnosis .", "our patient was successfully treated with anti - tuberculous chemotherapy without the use of corticosteroids .", "repeated bronchoscopic examination after six months from initiating antituberculous treatment revealed complete resolution of the two lesions without residual bronchial stenosis .", "a 62 year old pakistani woman was admitted to hamad general hospital with history of fever for 10 days prior to admission .", "she denied any history of cough , hemoptysis , breathlessness , chest pain , nocturnal sweating or weight loss .", "prior to admission she was prescribed a seven - day course of amoxicillin that was changed to cephalexin by her family physician without improvement . for more than a year prior to this admission she had frequent visits to emergency room and a hospital admission for recurrent episodes of fever and cough that were diagnosed as community acquired pneumonia based on the radiographic finding of right lung opacity and the response to antibiotic therapy .", "three years earlier she was admitted for abdominal surgery due to a perforated duodenal ulcer and incidentally discovered to have type 2 diabetes for which she was commenced on glibenclamide .", "she had frequent visits to pakistan , the last of which was 2 years prior to her admission .", "eight months prior to her admission she was refused entry to canada because of abnormal chest radiograph .", "she was a housewife , life - long non - smoker , and never consumed alcohol .", "physical examination was remarkable for fever of 39.5 c and dull percussion note over the right lower and mid chest , without added sounds .", "laboratory investigations at admission revealed leucopenia of 1.8103/ul with neutropenia of 0.5103/ul and normal hemoglobin and platelet count .", "peripheral blood smear revealed marked leucopenia with severe neutropenia , few toxic , left - shifted neutrophils and reactive lymphocytes without malarial parasites seen ( picture of severe infection or drug induced cytopenia ) .", "bacterial culture of the sputum , 3 sputum samples for acid - fast bacilli smear and culture and human immunodeficiency virus ( hiv ) testing were all negative .", "chest radiograph revealed right lower and mid zone non - homogenous opacity ( figure 1 ) .", "computerized tomography ( ct scan ) of the chest and abdomen revealed a soft tissue enhancing mass in the lumen of the right main bronchus that was associated with distal parnechymal consolidation and multiple lymph node enlargement ( figure 2 ) .", "chest radiograph showing opacification of the right lower zone ct scan of the chest showing right bronchial mass with post - obstructive consolidation ( arrows ) bone marrow examination revealed a cellular marrow without evidence of involvement by neoplastic or granulomatous process .", "bronchoscopy was done and showed two large endobronchial masses , one in the right main bronchus and the other in the right upper lobe bronchus with glistening , whitish surface ( figure 3 ) .", "endobronchial biopsy was taken from both masses and reveled caseating granuloma with presence of acid - fast bacilli on zeihl - nelsen staining ( figure 4 ) .", "bronchoscopic picture showing a mass in the right main bronchus and another one in the right upper lobe bronchus histopathologic slides showing caseating granuloma with acid - fast bacilli ( arrow ) bronchoalveolar lavage culture for acid - fast bacilli was negative .", "she was started on intravenous meropenem for 14 days and antituberculous chemotherapy in the form of rifampicin , isoniazide , ethambutol and pyrazinamide for 2 months , and then continued on rifampicin and isoniazide for further 4 months .", "a repeated bronchoscopy 6 months later revealed complete resolution of the endobronchial masses without evidence of bronchial stenosis ( figure 5 ) .", "bronchoscopic picture of the right main bronchus and the right upper lobe bronchus after six months of treatment showing disappearance of the masses with no residual stenosis", "the disease remained infrequently reported and was mainly a postmortem pathological diagnosis until the advent of bronchoscopy in the late 1920s .", "ebtb is defined as tuberculous infection of the tracheobronchial tree with microbial and histopathological evidence [ 3 , 4 ] .", "this form of tuberculous infection continues to be an important health problem for three reasons ; firstly , its diagnosis is frequently delayed , particularly in developed countries , as the decreased incidence itself diminishes the suspicion of tuberculosis .", "secondly , bronchial stenosis is a serious complication of the disease that may develop despite efficacious antituberculous chemotherapy and thirdly , it is often misdiagnosed as bronchial asthma or lung cancer .", "the exact pathogenesis of ebtb is unknown , however , direct extension from adjacent pulmonary parenchymal lesion , implantation of the organisms from infected sputum , dissemination from the blood or erosion of a lymph node into the bronchus have all been suggested as possible mechanisms of the disease [ 3 , 8 ] .", "chung and lee identified seven subtypes of ebtb on bronchoscopic appearance ; the actively caseating type ( 43.0% ) , the edematous - hyperemic type ( 14.0% ) , the fibrostenotic type ( 10.5% ) , the tumorous type ( 10.5% ) , the granular type ( 11.4% ) , the ulcerative type ( 2.7% ) , and the non - specific bronchitic type ( 7.9% ) . among these subtypes ,", "the prognosis of tumorous ebtb was found to be most grave and unpredictable . the majority of the tumorous ebtb cases studied by chung and lee changed to fibrostenotic type within 3 months of treatment .", "tumorous ebtb is characterized by an endobronchial mass whose surface is covered by caseous material and totally or near totally occludes the bronchial lumen .", "this form of ebtb is frequently mistaken for lung cancer , adenoma or carcinoid tumors .", "the two most important goals when treating ebtb are to eradicate tubercle bacilli and to prevent bronchial stenosis .", "although anti - tuberculous chemotherapy is effective in controlling infection , it does not prevent residual bronchostenosis [ 5 , 4 ] .", "oral corticosteroids have been used empirically to prevent bronchial stenosis , nevertheless , their role remains uncertain and controversial [ 9 , 10 ] .", "many patients with ebtb will require aggressive treatment like repeated dilatation , the use of stents or even resection to treat bronchial stenosis .", "some previous studies have suggested that delay in diagnosis of ebtb is an independent predictor of the development of persistent airway stenosis .", "our patient presented with two tumorous growths that almost completely obstructed the lumens of the right main bronchus and right upper lobe bronchus and resulted in recurrent episodes of post - obstructive pneumonia for more than a year prior to diagnosis .", "considering the prognosis of tumorous type ebtb and the delay in diagnosis as a predictor for the development of bronchial stenosis , healing without stenosis in our case seems interesting .", "it may suggest that the exact mechanism behind the tendency of some patients and certain types of ebtb to develop bronchial stenosis is not yet completely understood .", "future research should focus on the pathogenesis of bronchial inflammatory reaction induced by ebtb in order to understand this mechanism ."], "abstract_text": ["<S> endobroncheal tuberculosis is defined as tuberculous infection of the tracheobronchial tree with microbial and histopathological evidence . </S>", "<S> the disease is usually mistaken for other lung diseases including lung cancer . </S>", "<S> bronchial stenosis is a common complication of this type of tuberculosis despite the use of effective anti - tuberculous chemotherapy . </S>", "<S> we are presenting a case of endobronchial tuberculosis that simulated lung cancer and healed without residual bronchial stenosis . </S>"], "labels": null, "section_names": ["Introduction", "Case presentation", "Discussion"], "sections": [["endobronchial tuberculosis ( ebtb ) is a form of tuberculous infection that involves mainly the trachea and/or bronchi .", "the disease is often mistaken for more common lung diseases like pneumonia , asthma , lung cancer and other lung diseases .", "endobronchial stenosis is a common complication of ebtb despite the use of anti - tuberculous chemotherapy .", "we are presenting a case of ebtb that manifested with two endobronchial masses , which almost totally occluded the bronchial lumen and resulted in repeated episodes of post - obstructive pneumonia for more than a year prior to diagnosis .", "our patient was successfully treated with anti - tuberculous chemotherapy without the use of corticosteroids .", "repeated bronchoscopic examination after six months from initiating antituberculous treatment revealed complete resolution of the two lesions without residual bronchial stenosis ."], ["a 62 year old pakistani woman was admitted to hamad general hospital with history of fever for 10 days prior to admission .", "she denied any history of cough , hemoptysis , breathlessness , chest pain , nocturnal sweating or weight loss .", "prior to admission she was prescribed a seven - day course of amoxicillin that was changed to cephalexin by her family physician without improvement . for more than a year prior to this admission she had frequent visits to emergency room and a hospital admission for recurrent episodes of fever and cough that were diagnosed as community acquired pneumonia based on the radiographic finding of right lung opacity and the response to antibiotic therapy .", "three years earlier she was admitted for abdominal surgery due to a perforated duodenal ulcer and incidentally discovered to have type 2 diabetes for which she was commenced on glibenclamide .", "she had frequent visits to pakistan , the last of which was 2 years prior to her admission .", "eight months prior to her admission she was refused entry to canada because of abnormal chest radiograph .", "she was a housewife , life - long non - smoker , and never consumed alcohol .", "physical examination was remarkable for fever of 39.5 c and dull percussion note over the right lower and mid chest , without added sounds .", "laboratory investigations at admission revealed leucopenia of 1.8103/ul with neutropenia of 0.5103/ul and normal hemoglobin and platelet count .", "peripheral blood smear revealed marked leucopenia with severe neutropenia , few toxic , left - shifted neutrophils and reactive lymphocytes without malarial parasites seen ( picture of severe infection or drug induced cytopenia ) .", "bacterial culture of the sputum , 3 sputum samples for acid - fast bacilli smear and culture and human immunodeficiency virus ( hiv ) testing were all negative .", "chest radiograph revealed right lower and mid zone non - homogenous opacity ( figure 1 ) .", "computerized tomography ( ct scan ) of the chest and abdomen revealed a soft tissue enhancing mass in the lumen of the right main bronchus that was associated with distal parnechymal consolidation and multiple lymph node enlargement ( figure 2 ) .", "chest radiograph showing opacification of the right lower zone ct scan of the chest showing right bronchial mass with post - obstructive consolidation ( arrows ) bone marrow examination revealed a cellular marrow without evidence of involvement by neoplastic or granulomatous process .", "bronchoscopy was done and showed two large endobronchial masses , one in the right main bronchus and the other in the right upper lobe bronchus with glistening , whitish surface ( figure 3 ) .", "endobronchial biopsy was taken from both masses and reveled caseating granuloma with presence of acid - fast bacilli on zeihl - nelsen staining ( figure 4 ) .", "bronchoscopic picture showing a mass in the right main bronchus and another one in the right upper lobe bronchus histopathologic slides showing caseating granuloma with acid - fast bacilli ( arrow ) bronchoalveolar lavage culture for acid - fast bacilli was negative .", "she was started on intravenous meropenem for 14 days and antituberculous chemotherapy in the form of rifampicin , isoniazide , ethambutol and pyrazinamide for 2 months , and then continued on rifampicin and isoniazide for further 4 months .", "a repeated bronchoscopy 6 months later revealed complete resolution of the endobronchial masses without evidence of bronchial stenosis ( figure 5 ) .", "bronchoscopic picture of the right main bronchus and the right upper lobe bronchus after six months of treatment showing disappearance of the masses with no residual stenosis"], ["the disease remained infrequently reported and was mainly a postmortem pathological diagnosis until the advent of bronchoscopy in the late 1920s .", "ebtb is defined as tuberculous infection of the tracheobronchial tree with microbial and histopathological evidence [ 3 , 4 ] .", "this form of tuberculous infection continues to be an important health problem for three reasons ; firstly , its diagnosis is frequently delayed , particularly in developed countries , as the decreased incidence itself diminishes the suspicion of tuberculosis .", "secondly , bronchial stenosis is a serious complication of the disease that may develop despite efficacious antituberculous chemotherapy and thirdly , it is often misdiagnosed as bronchial asthma or lung cancer .", "the exact pathogenesis of ebtb is unknown , however , direct extension from adjacent pulmonary parenchymal lesion , implantation of the organisms from infected sputum , dissemination from the blood or erosion of a lymph node into the bronchus have all been suggested as possible mechanisms of the disease [ 3 , 8 ] .", "chung and lee identified seven subtypes of ebtb on bronchoscopic appearance ; the actively caseating type ( 43.0% ) , the edematous - hyperemic type ( 14.0% ) , the fibrostenotic type ( 10.5% ) , the tumorous type ( 10.5% ) , the granular type ( 11.4% ) , the ulcerative type ( 2.7% ) , and the non - specific bronchitic type ( 7.9% ) . among these subtypes ,", "the prognosis of tumorous ebtb was found to be most grave and unpredictable . the majority of the tumorous ebtb cases studied by chung and lee changed to fibrostenotic type within 3 months of treatment .", "tumorous ebtb is characterized by an endobronchial mass whose surface is covered by caseous material and totally or near totally occludes the bronchial lumen .", "this form of ebtb is frequently mistaken for lung cancer , adenoma or carcinoid tumors .", "the two most important goals when treating ebtb are to eradicate tubercle bacilli and to prevent bronchial stenosis .", "although anti - tuberculous chemotherapy is effective in controlling infection , it does not prevent residual bronchostenosis [ 5 , 4 ] .", "oral corticosteroids have been used empirically to prevent bronchial stenosis , nevertheless , their role remains uncertain and controversial [ 9 , 10 ] .", "many patients with ebtb will require aggressive treatment like repeated dilatation , the use of stents or even resection to treat bronchial stenosis .", "some previous studies have suggested that delay in diagnosis of ebtb is an independent predictor of the development of persistent airway stenosis .", "our patient presented with two tumorous growths that almost completely obstructed the lumens of the right main bronchus and right upper lobe bronchus and resulted in recurrent episodes of post - obstructive pneumonia for more than a year prior to diagnosis .", "considering the prognosis of tumorous type ebtb and the delay in diagnosis as a predictor for the development of bronchial stenosis , healing without stenosis in our case seems interesting .", "it may suggest that the exact mechanism behind the tendency of some patients and certain types of ebtb to develop bronchial stenosis is not yet completely understood .", "future research should focus on the pathogenesis of bronchial inflammatory reaction induced by ebtb in order to understand this mechanism ."]]}
{"article_id": "PMC3678173", "article_text": ["the patients were subjected to specialists opinion ( e.g. , ophthalmologist , ent or medical specialist ) and relevant investigations ( e.g. , fundus , x - ray skull , c.t .", "the cases suffering from organic mental disorders , chronic psychiatric disorders , epilepsy or drug dependence were excluded from the study .", "out of 3245 patients seen during the study period , 100 ( 3.2% ) were suffering from migraine .", "maximum patients belonged to age - group of 31 - 40 years ( 40% ) followed by 21 - 30 years ( 22% ) .", "a majority of migraineurs were married ( 76% ) , illiterate or just literate ( 55% ) , hindus ( 86% ) and housewives ( 56% ) [ table 1 ] .", "socio - demographic characteristics of patients with migraine family history of migraine was present in 12% cases whereas family history of depression was present in 6% .", "maximum patients had a total duration of migraine as one to three years ( 40% ) followed by less one year duration ( 32% ) .", "the commonest frequency of migraine was 1 - 2/week ( 54% ) or 2 - 3/month ( 26% ) .", "the provoking factors were reported in 18 patients ( 18% ) [ table 2 ] .", "migraine without aura ( previously called common migraine ) was the commonest variety ( 81% ) followed by migraine with aura ( previously called classis migraine ) ( 17% ) and complications of migraine ( 2% ) ( only two cases reported complications due to migraine  both with fainting spells ) .", "the personality profile of the patients showed anankastic traits ( 25% ) , dysthymic traits ( 20% ) , histrionic traits ( 17% ) , anxious traits ( 14% ) , schizotypal traits ( 10% ) , cyclothymic traits ( 6% ) and others ( 8% ) .", "the physical comorbidity included hypertension ( 12% ) , cervical spondylosis ( 8% ) , refractive errors ( 7% ) , chronic sinusitis ( 3% ) and diabetes mellitus ( 2% ) .", "characteristic of migraine generalized anxiety disorder ( f41.1 ) was the commonest psychiatric diagnosis ( 34% ) followed by mixed anxiety and depressive disorder ( f41.2 ) ( 18% ) , 22 patients ( 22% ) did not have any psychiatric disorder [ table 3 ] .", "migraine is reported to be more common in persons between the age of 20 and 45 years . in the present study also", ", migraine was found to be more common among age group of 31 - 40 years ( 40% ) followed by 21 - 30 years of age ( 22% ) .", "this is also consistent with other studies[91416 ] and a majority of patients were married ( 76% ) .", "unilateral type of headache was much more common than the bilateral type and the headache was throbbing in nature in a majority of migraneurs .", "there is a wide range of age of onset of migraine reported in the literature ( 5 to 28 years ) with an average age at onset in mid - teens .", "they were fatigue , lack of adequate sleep , intake of alcohol , menstruation and oral contraceptives .", "the other provoking factors for migraine as reported in the literature e.g. , aged cheeses , caffeine , chocolate , concentrated sugar , dairy products , fermented , pickled food , fruits ( bananas , figs , raisins , pineapple etc .", ", ) vegetables ( onions , pea , pods , nuts , peanuts ) , yeast products , meats with nitrites , monosodium glutamate , saccharin etc . , could not be identified or reported by the patients in the present study .", "migraine without aura ( previously called common migraine ) was the commonest subtype seen ( in 81% ) .", "this is consistent with the reported findings that lifetime prevalence of migraine without aura is about 13% as compared to migraine with aura seen in less than 4% population .", "generalized anxiety disorder ( f41.1 ) was the commonest disorder followed by mixed anxiety and depressive disorder ( f41.2 ) and depressive episode ( f32 ) .", "mixed dissociative ( coversion ) disorders ( f44.7 ) ( fits and fainting ) were also seen among female migraineurs .", "previous clinical and community studies[1724 ] have also reported a strong association between migraine and depression as well as anxiety disorders and also vice - versa .", "the literature also indicates that patients with migraine are at reduced risk of suffering from anxiety , mood disorders and substance - related disorders compared with medication overuse headache sufferers .", "retrospective studies on the onset and course of these syndromes showed that the onset of anxiety generally preceded that of migraine whereas the onset of depression tended to occur after that of migraine .", "the studies on association of personality traits and migraine demonstrate increased prevalence of neuroticism - anxiety and depression as compared to healthy controls .", "but the present study , a cross sectional one , did not determine whether the onset of anxiety disorder and depression preceded or followed the migraine .", "health - related outcomes were worst in those with both migraines and a psychiatric disorder and intermediate in those with either condition alone .", "future long term follow up studies on larger sample are warranted to study this association .", "the present study confirms that majority ( 78% ) of patients with migraine had psychiatric disorders ."], "abstract_text": ["<S> background : migraine is a common disorder which has psychiatric sequelae.objective:the objective of this study was to determine the clinical pattern and psychiatric comorbidity of migraine.materials and methods:100 cases of migraine seen over a period of one year were analysed to know the sociodemographic characteristics , clinical pattern and psychiatric morbidity.results:maximum patients were between 31 - 40 years of age group ( 40% ) , females ( 78.0% ) , married ( 76% ) and housewives ( 56.0% ) . family history of migraine was present in 12% cases . </S>", "<S> average age of onset was 22 years . </S>", "<S> unilateral and throbbing type of headache was most common . </S>", "<S> the commonest frequency was one to two per week . </S>", "<S> migraine without aura was commonest sub - type ( 80% ) . </S>", "<S> generalized anxiety disorder ( f41.1 ) was the most common psychiatric disorder ( 34% ) , followed by mixed anxiety and depressive disorder ( f41.2 ) ( 18% ) and depressive episode ( f32 ) ( 14% ) . in 22% cases , </S>", "<S> no psychiatric disorder could be elicited.conclusion:the present study confirms that majority patients with migraine had psychiatric disorders . </S>", "<S> this needs timely detection and appropriate intervention to treat and control the migraine effectively . </S>"], "labels": null, "section_names": ["MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["the present study was conducted in the psychiatry outpatient department of a tertiary care hospital during one year after obtaining clearance from institutional ethical committee .", "their sociodemographic profile , characteristics of migraine , psychiatric history , detailed physical examination as well as mental status examination was recorded .", "the patients were subjected to specialists opinion ( e.g. , ophthalmologist , ent or medical specialist ) and relevant investigations ( e.g. , fundus , x - ray skull , c.t .", "the cases suffering from organic mental disorders , chronic psychiatric disorders , epilepsy or drug dependence were excluded from the study ."], ["out of 3245 patients seen during the study period , 100 ( 3.2% ) were suffering from migraine .", "maximum patients belonged to age - group of 31 - 40 years ( 40% ) followed by 21 - 30 years ( 22% ) .", "a majority of migraineurs were married ( 76% ) , illiterate or just literate ( 55% ) , hindus ( 86% ) and housewives ( 56% ) [ table 1 ] .", "socio - demographic characteristics of patients with migraine family history of migraine was present in 12% cases whereas family history of depression was present in 6% .", "maximum patients had a total duration of migraine as one to three years ( 40% ) followed by less one year duration ( 32% ) .", "the commonest frequency of migraine was 1 - 2/week ( 54% ) or 2 - 3/month ( 26% ) .", "the provoking factors were reported in 18 patients ( 18% ) [ table 2 ] .", "migraine without aura ( previously called common migraine ) was the commonest variety ( 81% ) followed by migraine with aura ( previously called classis migraine ) ( 17% ) and complications of migraine ( 2% ) ( only two cases reported complications due to migraine  both with fainting spells ) .", "the personality profile of the patients showed anankastic traits ( 25% ) , dysthymic traits ( 20% ) , histrionic traits ( 17% ) , anxious traits ( 14% ) , schizotypal traits ( 10% ) , cyclothymic traits ( 6% ) and others ( 8% ) .", "the physical comorbidity included hypertension ( 12% ) , cervical spondylosis ( 8% ) , refractive errors ( 7% ) , chronic sinusitis ( 3% ) and diabetes mellitus ( 2% ) .", "characteristic of migraine generalized anxiety disorder ( f41.1 ) was the commonest psychiatric diagnosis ( 34% ) followed by mixed anxiety and depressive disorder ( f41.2 ) ( 18% ) , 22 patients ( 22% ) did not have any psychiatric disorder [ table 3 ] ."], ["migraine is reported to be more common in persons between the age of 20 and 45 years . in the present study also", ", migraine was found to be more common among age group of 31 - 40 years ( 40% ) followed by 21 - 30 years of age ( 22% ) .", "this is also consistent with other studies[91416 ] and a majority of patients were married ( 76% ) .", "unilateral type of headache was much more common than the bilateral type and the headache was throbbing in nature in a majority of migraneurs .", "there is a wide range of age of onset of migraine reported in the literature ( 5 to 28 years ) with an average age at onset in mid - teens .", "they were fatigue , lack of adequate sleep , intake of alcohol , menstruation and oral contraceptives .", "the other provoking factors for migraine as reported in the literature e.g. , aged cheeses , caffeine , chocolate , concentrated sugar , dairy products , fermented , pickled food , fruits ( bananas , figs , raisins , pineapple etc .", ", ) vegetables ( onions , pea , pods , nuts , peanuts ) , yeast products , meats with nitrites , monosodium glutamate , saccharin etc . , could not be identified or reported by the patients in the present study .", "migraine without aura ( previously called common migraine ) was the commonest subtype seen ( in 81% ) .", "this is consistent with the reported findings that lifetime prevalence of migraine without aura is about 13% as compared to migraine with aura seen in less than 4% population .", "generalized anxiety disorder ( f41.1 ) was the commonest disorder followed by mixed anxiety and depressive disorder ( f41.2 ) and depressive episode ( f32 ) .", "mixed dissociative ( coversion ) disorders ( f44.7 ) ( fits and fainting ) were also seen among female migraineurs .", "previous clinical and community studies[1724 ] have also reported a strong association between migraine and depression as well as anxiety disorders and also vice - versa .", "the literature also indicates that patients with migraine are at reduced risk of suffering from anxiety , mood disorders and substance - related disorders compared with medication overuse headache sufferers .", "retrospective studies on the onset and course of these syndromes showed that the onset of anxiety generally preceded that of migraine whereas the onset of depression tended to occur after that of migraine .", "the studies on association of personality traits and migraine demonstrate increased prevalence of neuroticism - anxiety and depression as compared to healthy controls .", "but the present study , a cross sectional one , did not determine whether the onset of anxiety disorder and depression preceded or followed the migraine .", "health - related outcomes were worst in those with both migraines and a psychiatric disorder and intermediate in those with either condition alone .", "future long term follow up studies on larger sample are warranted to study this association ."], ["the present study confirms that majority ( 78% ) of patients with migraine had psychiatric disorders ."]]}
{"article_id": "PMC3594951", "article_text": ["medullary thyroid carcinoma ( mtc ) is a rare neuroendocrine cancer that originates from thyroid parafollicular calcitonin-(ct- ) producing cells .", "mtc accounts for approximately 4% of all thyroid malignancies ; approximately 75% of these cases occur in the sporadic form , and 25% occur in the hereditary form [ 13 ] .", "mtc usually has a favorable prognosis , with a 10-year survival rate of 70%80% , if it is diagnosed and treated at an early stage when the tumor is confined to the thyroid .", "unfortunately , most cases of mtc present at diagnosis with metastases to the local and regional lymph nodes and to distant organs , especially the lungs , liver , and bones . patients with metastatic mtc have a 10-year overall survival rate of 40% , and metastasis is the main cause of death in patients with mtc [ 4 , 6 ] .", "locally advanced and distant metastatic diseases are incurable , as surgical resection and conventional radio- and cytotoxic chemotherapies are not effective against metastatic mtc [ 7 , 8 ] . clinical trials of various combinations of chemotherapeutic drugs have yielded unsatisfactory results [ 9 , 10 ] . however , research over the last years has led to a good understanding of the genetic defects and altered molecular pathways that are associated with the development of mtc .", "thus , multiple promising therapeutic agents that target these genetic alterations have been developed to treat progressive and advanced mtc . activating mutations of the tyrosine kinase receptor ( tkr ) rearranged during transfection ( ret )", "this discovery has led to the development and introduction of targeted therapies , such as tyrosine kinase inhibitors ( tkis ) that target ret .", "several tkis directed toward ret kinase have been tested in vitro , preclinical , and clinical studies with promising results .", "unfortunately , these agents are not likely to be curative , as the longest duration of response observed was approximately 4 years , and the maintenance of agent - dependent effects may require continuous therapies , which are not without important side effects . the main reasons for the failure of these agents to cure mtc are the development of resistance to tkis that target the ret and other cell receptors and the activities of other signal transduction pathways that are involved in mtc tumorigenesis and progression but not directly targeted by tkis .", "in recent years , the discovery of mechanisms of resistance to tkis and of several other molecular events that contribute to mtc transformation and metastasis suggested that combinatory therapy may result in a more significant tumor growth inhibition .", "this has led to the development of novel compounds that have been used in several clinical trials , including tkis that can target multiple tkrs simultaneously in addition to ret and agents that can target other altered signaling pathways .", "other studies have demonstrated the potential for immunotherapy in combination with agents that target signal transduction pathways that are important for mtc growth .", "because the aim of these targeted therapies is to extend lifespan and increase the quality of life , it is very important to limit the toxicities of therapeutic agents , either alone or in combination .", "the possibility of testing these novel drugs in vitro ( in primary thyroid cancer cells ) and in vivo may help to improve the personalization of treatments .", "the role of the ret oncogene in the tumorigenesis of mtc has been characterized extensively .", "the ret gene encodes a transmembrane tyrosine kinase that binds to glial cell line - derived neurotrophic factor ( gdnf ) family ligands .", "ret signaling leads to the activation of the ras / mitogen - activated protein kinase ( mapk ) and the phosphatidylinositol 3 kinase ( pi3k)/akt pathways and has key roles in cell growth , differentiation , and survival . activating point mutations of the tkr ret", "have been reported in nearly all hereditary cases of mtc ; some of these mutations are included in the men2a , familial mtc , or men2b syndromes in which there is a genotypic / phenotypic correlation between the type of ret mutation and clinical features .", "germline mutations in the ret proto - oncogene are responsible for hereditary mtc , while somatic ret mutations are responsible for sporadic mtc .", "however , this paper will mainly focus on additional cellular signaling pathways other than ret responsible of mtc tumorigenesis and progression and potential targeted approaches for the treatment of advanced or metastatic mtc .", "although activating mutations of the tkr ret are believed to be the primary oncogenic event in the development of a majority of mtc cases , it is clear that ret cooperates with other signal transduction pathways to promote mtc tumorigenesis .", "in addition to ret , other kinase receptors may play a role in the development and progression of mtcs .  ", "similar to the ret receptor , the epidermal growth factor receptor ( egfr ) is a tkr that is associated with the regulation of cell growth , proliferation , and apoptosis .", "dimerization of the receptor following ligand binding results in transphosphorylation and the subsequent activation of several downstream signal pathways .", "egfr has been shown to be frequently overexpressed in various types of thyroid carcinomas , including mtc , and to play a role in cancer development and progression .", "in contrast , a recent report analyzing different mtc on tissue microarrays has demonstrated that only 20% of cases revealed moderate to strong reactivity for egfr , whereas the majority of the cases revealed weak and very focal positivity .", "with respect to the numbers of egfr gene copies in mtcs , the researchers did not detect amplifications but did find polysomes in 15% of the examined tumors .", "additionally , egfr was activated in a subset of mtcs , which suggests that this subset of patients might benefit from drugs that target also egfr .", "recent findings have shown that the ligand - induced activation of egfr can stimulate ret activation beyond signaling and growth stimulation .", "several egfr inhibitors have been shown to markedly inhibit the growth of the mtc tt and mz - crc-1 cell lines . because ret activation seems to be influenced by egfr , a recent study investigated whether egfr activation could be related to specific ret mutations in mtcs .", "the researchers found that tumors with the most aggressive ret mutations ( in codons 883/918 ) exhibited reduced egfr expression compared to other ret mutations .", "it could be speculated that the most aggressive ret mutations are less dependent on egfr activation .", "in fact , in the work by croyle et al . , in which cell lines with ret mutations in codon 634 and codon 918 were compared , the effect of egfr inhibition on the codon 918 mutated cell line appeared to be reduced , in agreement with the previous data . because the activation status of egfr seems to be related to ret activation", "however , no differences have been found in egfr activation between ret - positive and -negative tumors , which likely indicates that other molecular mechanisms lead to ret activation , such as increased ret gene copy numbers , altered promoter activity , or increased transcription in the ret mutation - negative tumors .", "these data suggest that egfr status determination in mtcs might be important but certainly deserves further investigations .", "the vascular endothelial growth factor receptor ( vegfr ) pathway is also important in the pathogenesis of mtc .", "there are three transmembrane receptors that mediate the angiogenic and lymphogenic effects of vegf : vegfr-1 , vegfr-2 , and vegfr-3 .", "overexpression of vegf and vegfr-2 has been found in mtc compared to normal thyroid tissue .", "the vegf proteins ( vegf - a , b , c , and d ) , which are secreted by tumor cells , act as ligands for the vegfr-2 receptors on endothelial cells and promote a signaling cascade through different pathways , such as plc--pkc - raf - mek - mapk and pi3k - akt , that stimulate cellular proliferation , migration , and survival and induce neoangiogenesis . angiogenesis is an essential alteration in cell physiology that predisposes the development of malignancy and is fundamental in tumor growth and metastasis . similarly", "to egfr , the overexpression of vegfr-2 in mtc has been shown to correlate with metastasis .  ", "several multitargeting tyrosine kinase inhibitors that block vegfr have shown promising clinical antitumor activity ; unfortunately , in most thyroid carcinomas and other solid tumors , the antiangiogenic effects are often only transitory and really often may have late paradoxically protumorigenic effects .", "additionally , it seems that a modest significant association has been observed between vegfr-2 expression and ret mutation status in primary tumors .   the met proto - oncogene codes for the tk receptor for the hepatocyte growth factor ( hgf ) .", "met hyperactivation reportedly correlates with the metastatic abilities of tumor cells . met and hgf coexpression has been observed in a subset of mtc tumors and is associated with multifocality in mtc , which makes this interaction a potentially important target . in one report", "importantly , ret can induce the overexpression of c - met in this type of thyroid tumor .  ", "the fibroblast growth factor receptor 4 ( fgfr4 ) has also been reported to be overexpressed in mtc cell lines .", "inhibition of fgfr phosphorylation with the small molecule fgfr inhibitor pd173074 resulted in an arrest of cell proliferation and tumor growth . moreover , the dual inhibition of ret and fgfr combined with tyrosine kinase inhibitors resulted in greater suppressions of cell proliferation in vitro and tumor control in vivo than that which was achieved with either agent alone .", "these data highlight ret and fgfr4 as therapeutic targets and suggest a potential role for the use of combined tyrosine kinase inhibitors in the management of inoperable medullary thyroid cancers . finally , the platelet - derived growth factor receptor ( pdgfr ) also seems to play a role in differentiated thyroid cancer , although its role and function have not been fully investigated in mtc .", "several other signal transduction pathways have been implicated as contributors to mtc tumor growth , as illustrated in two recent studies [ 5 , 30 ] .", "these pathways include ret interactions with prb , p53 , p18 , and p27 as well as the phosphatidylinositol 3-kinase / akt / mtor and ras / raf / mek / erk pathways . \n", "ret interactions with tumor suppressor genes \n \n ( a ) prb and p53 . the tumor suppressor genes rb1 ( retinoblastoma ; prb protein ) and tp53 ( p53 protein ) are frequently mutated in human cancers , and it appears that , in cancer , both pathways must be inactivated to overcome senescence or apoptosis .", "there is extensive genetic evidence that the prb and p53 pathways are involved in mtc in rodents .", "further , the loss of tp53 further increased mtc formation in rb1-deficient mice [ 32 , 33 ] .", "it has been shown that in mice , ret cooperates with the inactivation of prb / p53 to cause experimental mtc .", "prb / p53 mutant mice have been shown to acquire ret mutations that are analogous to activating germline mutations that are observed in human men2a and familial medullary thyroid carcinoma ( fmtc ) .", "this suggests that murine mtc requires mutational dysregulations within both the ret and nuclear tumor suppressor gene pathways .", "however , mouse models may not mimic human disease , and a systematic analysis of the genes in the rb1 and tp53 pathways in human samples will help to clarify their roles in human mtc formation .", "this information may be important for the development of novel targeted therapeutic approaches for mtc .  \n", "thyroid tumors show low expression of the cyclin - dependent kinase inhibitor ( cdki ) p27 ( kip1 ) , and recent evidence demonstrates that p27 is downregulated by the active ret mutant , ret / ptc1 , which is found in papillary thyroid carcinomas .", "these data implicate decreased p27 activity as an important event during thyroid tumorigenesis . however , p27 mice develop men - like tumors only in combination with the loss of p18 ( ink4c ) , another cdki .", "this suggests that p18 and p27 are functional collaborators in the suppression of tumorigenesis , that the loss of both is critical to the development of men tumors , and that both p18 and p27 are regulated by ret .  \n", "the pi3k - akt - mammalian target of the rapamycin ( mtor ) cascade is important in tumorigenesis due to its ability to promote cell growth , proliferation , and survival .", "several examples provide evidence to support a role for the activation of the pi3k / akt / mtor signaling cascade in medullary thyroid cancer [ 3638 ] .  ", "several mechanisms have been shown to be involved in the activation of pi3k signaling in medullary thyroid cancer .", "a mutation of men2a ( ret - men2a ) has been shown to activate pi3k and its downstream effector , the serine / threonine kinase akt / protein kinase b .", "previous studies have demonstrated that a mutation of tyr-1062 , which is the intracellular docking site for shc and enigma on ret , abolishes the ret - men2a transforming activity .", "these studies further revealed that the mutation of tyr-1062 abrogates the binding of the p85 regulatory subunit of pi3k to ret - men2a and subsequent stimulation of the pi3k / akt pathway .", "furthermore , retroviral transduction of rat fibroblasts with a dominant - interfering form of pi3k was shown to suppress ret - men2a - dependent transformation , whereas the overexpression of akt enhanced ret - men2a oncogenic potential . in summary , these data are consistent with the notion that ret - mediated cell - transforming effects are critically dependent on the activation of the pi3k / akt / mtor pathway . in cell lines , the pi3k - akt / mtor pathway has been shown to be important in the pathogenesis of men2b .", "ret - men2b ( ret m918 t ) is more effectively autophosphorylated at ret y1062 than is ret - men2a , which subsequently leads to increased constitutive activation of the ras / mitogen - activated protein kinase ( mapk ) and pi3k / akt / mtor signaling cascades .", "furthermore , previous data report other possible mechanisms of pi3k activation in thyroid carcinomas , such as the overexpression of rai ( shcc / n - shc ) , which is a substrate of ret oncoproteins [ 5 , 42 ] .", "finally , the loss of expression of the phosphatase and tensin homologue ( pten ) gene , a tumor suppressor gene , may have a role in the activation of pi3k / akt in thyroid tumors .", "the absence of functional pten protein expression has been observed in various cancer cells and has led to the constitutive activation of downstream components of the pi3k pathway , including the akt and mtor kinases .", "preclinical models showed that inactivation of these kinases is able to reverse the effects of pten loss .", "these data raise the possibility that drugs that target either these kinases or pi3k itself might have significant therapeutic activity against pten - null cancers .", "mutations in major nodes of this signaling cascade have been observed in human cancers ; these mutations include gain - of - function mutations and amplifications of the genes encoding pi3k and akt [ 44 , 45 ] .", "however , a recent study on a series of 49 mtcs has shown that the pi3k genes were not mutated , and that the activation of the pi3k pathway was significantly associated with the status of ret mutations .", "in fact , using protein expression analysis , the same authors confirmed that the akt / mtor pathway was highly activated in mtc , especially in cases with germline ret mutations .", "interestingly , this association was not dependent on the type of mutation ( in either the codons of the juxta - membrane or of the tyrosine kinase portion of the receptors ) but was dependent on the hereditary nature of the mutation .", "in contrast , medullary carcinomas with sporadic ret mutations or with wild - type ret were observed with heterogeneous expression of akt / mtor pathway molecules , which suggests the need for further elucidation of alternative activation mechanisms . in a recent study ,", "the pi3k / akt / mtor pathway was shown to be activated in mtc , particularly in the metastatic lymph nodes , and this pathway was shown to sustain malignant features of different mtc cell models .", "moreover , it has been shown that the selective inhibition of the mtor pathway in a germline - ret - mutated mtc cell line can effectively decrease cell viability and block the phosphorylated status of mtor signaling molecules , which confirms previously published in vitro data .", "another study used metformin , which is an antidiabetic agent that decreases the proliferation of cancer cells through the 5-amp - activated protein kinase - dependent inhibition of mtor , in mtc cell lines to show that the growth - inhibitory effects on the cells were associated with the downregulation of both the mtor/6sk and perk signaling pathways .", "altogether , these results strongly suggest that mtor might be a very efficacious target in patients with advanced or metastatic mtc .", "noteworthy , mtor inhibitors are currently being used in clinical trials for the treatment of medullary thyroid carcinoma .  \n ras - raf - mek - erk pathway .", "unlike hereditary mtc , in which ret mutations are the critical events , in sporadic mtc , the genetic or molecular biomarkers have not been fully established .", "ras is a frequently mutated oncogene in a broad spectrum of human tumors , including thyroid carcinoma , mainly in the follicular forms . in this context , investigators aimed to determine whether mutations in the ras oncogene could play a possible role in the carcinogenesis of sporadic mtc .", "the initial study analyzed 15 sporadic mtcs for mutations in known hot spots ( codons 12 , 13 , and 61 ) of the h- and k - ras oncogenes by a direct sequencing technique , although no ras mutations were detected in any of the examined tumors .", "a different study on 49 mtc confirmed that ras mutations are a rare event in this type of tumor , regardless of ret mutational status .", "by contrast , different studies have demonstrated the presence of ras mutations in mtc . a mutational screening for h- , k- , n - ras , and braf in 13 sporadic and inherited mtcs revealed one sporadic ret - negative mtc ( stage iii ) with a mutation in the h - ras codon 13 ( g13r ) .", "another study analyzed 188 hereditary and sporadic mtcs for ras mutational status , revealing a low prevalence of mutations , which were confined only to ret - negative sporadic mtc .", "furthermore , recent studies have reported a quite high prevalence of ras mutations in sporadic mtc , particularly in ret - negative mtc ; 68% ( 17 of 25 ) of the ret - negative mtcs had mutations of ras compared to only 2.5% ( 1/40 ) of the ret - positive mtcs .", "these findings were confirmed by a different study that analyzed 17 sporadic mtcs by exome sequencing and found dominant and mutually exclusive oncogenic mutations in ret and ras ( h- and k - ras ) genes in 17 sporadic mtcs . as expected ,", "most ras mutations corresponded to mutational hot spots in exons 2 and 3 , although some mutations were also detected in exon 4 .", "this study confirms that ret and ras mutations are mutually exclusive , and that they are probably 2 different oncogenic driver events in mtc .", "these results suggest that the activation of the proto - oncogenes ras and ret represents alternative genetic events in sporadic mtc tumorigenesis , and that more sensitive sequencing techniques such as next generation sequencing are necessary to detect mutations . for decades ,", "the ras and raf families of oncogenes have been known to be transforming genes . however , in many normal cultured cell types , the sustained expression of activated ras or its downstream effector , raf , can elicit cell cycle arrest or senescence . the ras / raf - mediated growth arrest has been proposed as a defense mechanism against the inappropriate activation of the ras / raf signal transduction pathway [ 56 , 57 ] . according to this hypothesis , spontaneous mutations in ras genes , which may occur stochastically in all cell types , would be innocuous for the organism because these mutations would lead to growth arrest or senescence .", "hence , for carcinogenesis to occur in response to ras / raf activation , the growth arrest response must be inactivated .", "the growth arrest response to ras / raf activation is not limited to normal cells .", "several tumor cell lines that were derived from different tumor types , including medullary thyroid carcinoma , also experienced growth arrest , usually accompanied by differentiation or senescence [ 5860 ] .", "these findings indicate that some tumors retain a capability for growth arrest in response to ras / raf activation .", "the mechanism by which ras or raf activation can induce growth arrest is not completely understood .", "some investigators have reported that ras or raf activation could induce the expression and secretion of a protein that mediated differentiation and g1 cell cycle arrest in mtc cells . by protein purification and mass spectrometry , this protein has been identified as the leukemia inhibitory factor ( lif ) .", "stat3 activation was necessary for lif - mediated growth arrest and differentiation in mtc cells .", "in addition , the ras / raf / mek / erk pathway could also mediate growth arrest and differentiation by a second mechanism that is independent of lif / jak / stat3 .", "this novel autocrine - paracrine mechanism , which mediates crosstalk between the ras / raf / mek / erk and the jak - stat pathways , defines a novel mechanism of ras / raf - induced cell growth arrest [ 61 , 62 ] .", "the inactivation of gsk-3beta in tt cells by well - known gsk-3beta inhibitors , such as lithium chloride ( licl ) and sb216763 , is associated with both growth suppression and a significant decrease in neuroendocrine markers , such as human achaete - scute complex - like 1 and chromogranin a. growth inhibition by gsk-3beta inactivation was found to be associated with cell cycle arrest due to an increase in the levels of cyclin - dependent kinase inhibitors , such as p21 , p27 , and p15 . additionally , tt xenografts mice , treated with licl , showed a significant reduction in tumor volume compared with those that were treated with a control .", "therefore , gsk-3beta is a key downstream target of the raf-1 pathway in tt cells , and the inactivation of gsk-3beta alone is sufficient to inhibit the growth of tt cells both in vitro and in vivo .  \n", "-catenin is a ubiquitously expressed multifunctional protein that plays an important role in cellular adhesion . a novel ret--catenin signaling pathway was found to be a critical contributor to enhanced cell proliferation and tumor progression in thyroid cancer .", "gujral et al . showed that ret could induce -catenin - mediated transcription , cell proliferation , and transformation in vitro and that -catenin nuclear localization and subsequent mediation of -catenin by ret are key secondary events in tumor growth and metastasis in vivo .", "this novel interaction suggests a mechanism that may underlie the broad and early metastatic potential of mtc .", "these data suggest an unrecognized role for -catenin signaling that may have implications for tyrosine kinase - mediated tumorigenesis in multiple tumor types and provide another potential target for therapeutic agents . in support , a recent study performed on tissue microarray observed that wnt pathway proteins , including wisp-1 , wisp-2 , and -catenin , were actively expressed in mtc .  \n", "the nuclear factor kappa - b ( nf-b ) proteins , a family of transcription factors that are found virtually in all cells , are known to play crucial roles in the growth of many human malignancies .", "the ability of nf-b to target a large number of genes that regulate cell proliferation , differentiation , survival , and apoptosis provides clues towards its dysregulation during the processes of tumorigenesis , metastatic progression , and therapeutic resistance of tumors .", "nf-b is constitutively active in mtc through the ret - induced phosphorylation , ubiquitination , and proteosomal degradation of inhibitors of nf-b ( ikb ) , which allows nf-b to enter the nucleus and bind to dna .", "nf-b is frequently activated in mtc , and the activation of ret by somatic or germline mutations may be responsible for nf-b activation in these tumors .", "these results suggest that the nf-b pathway may be an important target for drug development in mtc .  \n", "a recent study examined the expression of proteins involved in angiogenesis , inflammation , apoptosis , cell cycle , cell - to - cell contact , and carcinogenesis using high - throughput tissue microarrays from 23 patients with mtc .", "these authors identified several novel potentially important protein targets such as cox-1/2 , bcl-2a , gst- , gli-1 , gli-2 , gli-3 , and bmi-1 that may be therapeutically targeted in mtc .", "for example , cox-1 and cox-2 , which are two inflammation - related factors , were significantly expressed in these cases , suggesting that nonsteroidal anti - inflammatory drugs may provide benefit in some patients with mtc .", "then , the finding of antiapoptotic bcl-2a and gst- overexpression in mtc suggests that bcl-2a and gst- inhibitors might be a treatment option for patients with advanced or metastatic mtc .", "the same is applied to gli-1 , gli-2 , and gli-3 , members of sonic hedgehog homolog ( shh ) pathway , and bmi-1 , a cell - cycle marker that resulted overexpressed in these mtc samples .", "the studies of these markers , particularly the members of the shh , may improve our understanding of mechanism of resistance to current chemotherapeutic and/or tki regimens and identify novel potential therapeutic approaches .", "due to increased knowledge of the molecular pathogenesis of mtc , therapeutic agents that target specific altered pathways have been developed ( figure 1 ) . because the alterations of protein kinases and their pathways are involved in mtc development , several tyrosine kinase receptors inhibitors ( tkis ) have been tested in vitro , preclinical , and clinical studies .", "ret is certainly an attractive target for several types of tumors particularly for parafollicular c - cells - derived tumors , which are addicted to ret and its activity .", "tkis are small organic compounds that affect tyrosine kinase - dependent oncogenic pathways by competing with atp - binding sites of the tyrosine kinase catalytic domains .", "occupation of these sites inhibits autophosphorylation and activation of the tyrosine kinases and prevents the further activation of intracellular signaling pathways .", "several tkis that are directed against ret kinase have been developed for the treatment of mtc , but there is no currently available tyrosine kinase inhibitor specific to ret .", "however , several multitargeted tyrosine kinase inhibitors have demonstrated significant activity against ret ( table 1 ) , including vandetanib , sorafenib , motesanib , imatinib , and sunitinib [ 66 , 68 ] .", "the inhibition of only one kinase receptor may induce the compensatory activation of other tks and consequent resistance to treatment with tkis [ 69 , 70 ] .", "therefore , the simultaneous inhibition of multiple activated tks may be the best way to approach mtc [ 7173 ] .", "vandetanib ( zd6474 ) is an oral tki that targets ret , vegfr-2 , and -3 , and at higher concentrations , egfr .", "vandetanib selectively inhibits pathways that are critical for tumor growth and angiogenesis without leading to direct cytotoxic effects on tumor or endothelial cells .", "most of the mutant ret oncoproteins have demonstrated sensitivity to vandetanib , while mutations in which valine 804 is substituted by either leucine or methionine ( as observed in some cases of men2a ) rendered the ret kinase significantly resistant to vandetanib .", "this resistance is likely due to steric hindrance , as the val804gly mutation increased the sensitivity of ret to vandetanib .", "when ret activity was inhibited , overstimulation of egfr was able to partially compensate for ret through a partial rescue of mapk pathway activation .", "the inhibition of egfr by vandetanib has been shown to prevent this rescue of mapk pathway activation .", "these data support the idea that the dual inhibition of ret and egfr is important , as it may overcome the risk of an escape by mtc cells from a blockade of ret through the compensatory overstimulation of egfr .", "in addition , the expression of egfr has been demonstrated in tumor - associated endothelial cells . in this", "respect , anti - egfr agents have been shown to block the proliferation and migration of endothelial cells .", "therefore , the anti - egfr activity of vandetanib might result in an additional direct antiangiogenetic mechanism .", "one phase ii clinical trial showed the antitumor activity of vandetanib ( 300  mg / day ) in patients with hereditary mtc . in this study ,", "20% of the patients showed a partial response to vandetanib ( > 30% reduction in tumor diameter ) , while an additional 53% of patients presented with disease stability at 24 weeks . only 1 patient showed disease progression while receiving this agent . of interest", ", this patient was not affected by any of two ret mutations correlated to vandetanib resistance in which valine 804 is substituted by leucine or methionine , but by y791f ret germline mutation .", "another clinical trial , in which 19 patients with hereditary mtc were treated with vandetanib ( 100  mg / day ) , showed similar results .", "vandetanib is the only tki approved for the treatment of symptomatic or progressive mtc in patients with unresectable locally advanced or metastatic disease .", "the approval of vandetanib in april 2011 by the us food and drug administration ( fda ) was based on the results of the phase iii clinical trial , ", "study d4200c00058 ,  in which 331 patients with unresectable locally advanced or metastatic mtc were randomly assigned to receive vandetanib ( 231 ) or a placebo ( 100 ) .", "this study showed that the median progression - free survival duration ( pfs ) was 11 months longer in the group that received vandetanib than in the placebo control group and that 45% of the patients had an objective response rate ( orr ) .", "common adverse events ( any grade ) occurred more frequently with vandetanib compared to the placebo and included diarrhea ( 56% versus 26% ) , rash ( 45% versus 11% ) , nausea ( 33% versus 16% ) , hypertension ( 32% versus 5% ) , and headache ( 26% versus 9% ) .", "the impact of overall survival of mtc patients treated with this compound is presently unknown . \n", "xl184 ( cabozantinib ) is an oral selective inhibitor of ret , c - met , and vegfr-2 .", "the phase i trial results indicated that cabozantinib is active in patients with mtc , including those who harbor somatic ret mutations and are potentially at high risk for progression and death . a global phase iii pivotal study in mtc is ongoing ( http://www.clinicaltrials.gov/ number nct00704730 ) . \n", "sorafenib ( bay 43 - 9006)is a multikinase inhibitor that targets braf , vegfr-2 , vegfr-3 , kit ( a stem cell growth factor proto - oncogene involved in cell survival and differentiation ) , and pdgfr .", "a phase ii clinical trial , in which sorafenib ( 400  mg / twice daily ) was given to 21 patients with metastatic medullary carcinoma , reported that of the patients with sporadic mtc , 87.5% achieved disease stability and 6.3% demonstrated partial responses .", "the treatment was prematurely terminated in mtc hereditary patients due to slow accrual . in a similar trial ,", "recently , a study of sorafenib was conducted on advanced thyroid carcinoma patients , and partial responses were reported in six out of the 12 ( 50% ) patients with mtc , although the small number of patients requires further prospective studies .  \n", "sunitinib ( su011248 ) is a small molecule inhibitor that targets many of the same protein kinases as sorafenib , including vegfr , pdgfr , kit , and ret . in a phase ii clinical trial ,", "33 patients with either well - differentiated thyroid carcinoma or mtc were treated with sunitinib .", "one patient had a complete response , 28% of the patients had partial responses , and 46% of the patients exhibited disease stability .", "the intermediate results of the phase ii thysu study also showed the efficacy of sunitinib in advanced medullary thyroid carcinoma .", "motesanib ( amg 706 ) is a multikinase inhibitor that targets vegfr receptors 1 , 2 , and 3 , pdgfr , and kit and exerts antiangiogenic and direct antitumor activities .", "a phase ii study performed in 91 patients with locally advanced or metastatic , progressive or symptomatic , mtc demonstrated that although the objective response rate was low , a significant proportion of the mtc patients ( 81% ) achieved disease stability while receiving motesanib .", "a recent study investigated the effects of motesanib on wild - type and mutant ret activity in vitro and on tumor xenograft growth in a mouse model of mtc .", "the results of this study demonstrated that motesanib inhibited thyroid tumor xenograft growth , predominantly through the inhibition of angiogenesis and possibly via the direct inhibition of vegfr2 and ret , which were expressed in tumor cells .", "these data suggest that angiogenic pathways and specifically the vegf pathway are still important for mtc cells .  \n", "imatinib ( sti571 ) is a tki that inhibits ret , pdgfr , and kit .", "a phase ii trial in which imatinib was tested in metastatic mtc patients yielded disappointing results .", "the patients showed no objective responses ; however , a minority of patients achieved disease stability .", "several tk inhibitors have been tested in clinical trials , but the most effective inhibitor and whether there is any specificity for particular ret mutations remain unknown .", "a recent study compared the effects of four tkis ( axitinib , sunitinib , vandetanib , and xl184 ) on cell proliferation , ret expression and autophosphorylation , and erk activation in cell lines that express men2a ( mtc - tt ) and men2b ( mz - crc-1 ) mutation .", "the findings showed that the inhibitors were specific for different mutations , with xl184 being the most potent inhibitor against the men2a mutation and vandetanib the most effective against the men2b mutation in vitro .", "no tk inhibitor was superior for all tested cell lines , which indicates that mutation - specific therapies could be beneficial in mtc treatment .", "one of the mechanisms for the resistance of mtc to chemotherapeutic drugs is multidrug resistance ( mdr ) .", "mdr in cancer cells has been attributed to the overexpression of several plasma membrane atp - dependent efflux pumps , such as mdr-1 .", "the enzyme cyclooxygenase ( cox-2 ) has been shown to regulate mdr-1 expression in rat mesangial cells .", "furthermore , a study has shown that in vitro treatment of an mtc - derived cell line with rofecoxib , a cox-2 inhibitor , was able to sensitize mtc cells to doxorubicin . a recent study , performed in vitro ,", "has shown that celecoxib , another cox-2 inhibitor , was able to induce both mtc cell apoptosis and sensitization to vinorelbine , thus enhancing the chemotherapeutic effect of this drug .", "a clinical trial , in which the in vivo activities of celecoxib were explored in mtc patients who can not benefit from available treatments , would be desirable after accounting for the possible cardiovascular risks of this drug .", "several other therapeutic agents are being investigated for their uses in the treatment of thyroid carcinomas , including mtc .", "these agents inhibit targets that are involved in development of mtc other than the tyrosine kinase receptors .", "we previously discussed that ras operates in a complex signaling network with multiple activators and effectors , which allows them to regulate many cellular functions such as cell proliferation , differentiation , apoptosis , and senescence .", "phosphatidylinositol 3-kinase ( pi3k ) is one of the main effector pathways of ras , regulating cell growth , cell cycle entry , cell survival , cytoskeleton reorganization , and metabolism .", "the presence of ras mutations in sporadic mtc and most importantly the frequent activation of the pi3k / akt / mtor pathway in several aggressive and metastatic mtcs strongly suggest that this cellular signaling pathway is a good candidate for targeted therapies against mtc .", "in fact , several in vitro evidences have demonstrated that the indirect blocking of this pathway , by pi3k inhibitors , or direct inhibition of mtor   can mediate the induction of apoptosis and a decrease in cell viability in the mtc tt cell line .", "currently , there are several clinical trials in which patients with radioiodine - refractory differentiated and medullary thyroid carcinomas were recruited to test the efficacy of everolimus ( rad001 ) , a novel inhibitor of mtor , in combination with other drugs ( nct01625520 and nct01270321 ) .", "recently , researchers have developed a liposomal form of everolimus and have demonstrated the anticancer efficacy of this formulation against tt cells .", "studies have shown that metformin can decrease the proliferation of cancer cells through 5-amp - activated protein kinase-(ampk- ) dependent inhibition of mtor .", "some researchers have reported growth inhibitory effects of metformin against mtc cell lines ( tt and mz - crc-1 ) , which were attributable to the downregulation of both mtor/6sk and perk signaling .", "the expression of the molecular targets of metformin in human mtc cells suggests that this drug may be potentially useful in the treatment of mtc .  ", "pi3k - akt - mtor and raf - mek - erk signaling have been shown to be important in the resistance of thyroid cancer cells to apoptosis and the promotion of tumor progression .", "in this context , targeted anti - vegfr therapy or raf inhibition may be ineffective if pi3k signaling remains intact .", "therefore , two promising drugs , raf265 , a raf inhibitor that is active against vegfr2 , and bez235 , a pi3k inhibitor , were tested alone and in combination in preclinical mtc models that represented the key genotypes observed in refractory thyroid cancers .", "the study findings showed that a combination treatment with agents that inhibited both raf and pi3k pathways strongly inhibited growth both in vitro and in vivo .", "in addition , the investigators showed for the first time that raf265 potently inhibits the constitutively active ret , a form of the kinase that is observed in men2a .", "persistent ret activation , a frequent event in mtc , leads to the activation of the pi3k / akt / mtor , erk / mapk , and jak / stat3 pathways .", "recently , the efficacy of the jak1/2 inhibitor azd1480 against the growth of thyroid cancer was tested in vitro and in vivo in thyroid cancer cell lines that expressed oncogenic ret .", "the findings showed that azd1480 efficiently inhibited the growth and tumorigenesis of thyroid cancer cell lines that harbored oncogenic ret alterations , likely through the inhibition of pi3k - akt signaling ; this result supports the use of this inhibitor in patients with thyroid cancer , particularly in those with advanced mtc , for whom there are no effective therapeutic options .", "thus , somatostatin ( srif ) peptide analogues in combination with tkis may be a promising approach for the treatment of these tumors .", "the presence of different combinations of srif receptor ( sstr ) subtypes in a given patient may explain the variable clinical response to srif analogues and may promote the search for more selective drugs with different affinities to the various receptor subtypes .", "the currently available somatostatin analogues ( octreotide and lanreotide ) act preferentially through the somatostatin receptor subtype 2 ( sst2 ) . in mtc", ", these compounds have been reported to exert antisecretive effects on calcitonin but unfortunately are not reported to have antiproliferative effects .", "pasireotide ( som230 ) is a new somatostatin analogue that has a peculiar binding profile with high affinity for sst1 , sst2 , sst3 , and sst5 .", "preliminary data from a phase ii study of patients with metastatic carcinoma show that som230 is effective , and some clinical trials are exploring the efficacy of som230 alone or in combination with rad001 in patients with mtc ( nct01625520 and nct01270321 ) .  ", "the nf-b pathway is also a potential target for drug development , and a number of compounds have been developed to inhibit this pathway at different levels in cancer cells .", "studies have demonstrated that the proteosome inhibitor bortezomib exerted a promising antitumor effect in human mtc cells through the inhibition of ib degradation , which led to the inactivation of the transcriptional factor nf-b .", "patients are currently being recruited for a phase i / ii trial to study the combination of vandetanib plus bortezomib ( http://www.clinicaltrials.gov/ ) .", "immunization with tumor antigen - pulsed autologous dendritic cells ( dcs ) resulted in protective immunity and the rejection of various established human tumors .", "specifically , vaccination immunotherapy with calcitonin and/or carcinoembryonic antigen ( cea ) peptide - pulsed dcs was shown to result in the induction of a cellular , antigen - specific immune response in patients with mtc , which led to clinical responses in some patients .", "therefore , for the first time , a potential dc vaccination therapy was developed for patients with metastatic mtc .", "another study , which was performed in a transgenic mtc mouse model , has confirmed this finding .  ", "have reported on the in vitro findings of a vaccination trial in 5 mtc patients , who were treated with dcs that were generated using a new protocol , which consisted of granulocyte - macrophage colony - stimulating factor and interferon - alpha ( ifn - dcs ) .", "these investigators demonstrated that immunization with ifn - dcs led to a tumor epitope - specific th1 immune response in mtc patients .", "furthermore , a pilot trial of 10 patients has assessed the safety and efficacy , in terms of immune responses and clinical activities , of the dcs . in this study", "monitoring of the patients included serial measurements of calcitonin tumor markers , radiological imaging , and immunological in vitro tests , including t - cell interferon - gamma detection and cytotoxicity assays .", "after a median followup of 11 months ( range 726 ) , 3 ( 30% ) of the 10 patients exhibited disease stability , while 7 ( 70% ) of the patients progressed during treatment .", "a combined treatment with different tumor cell lysate - pulsed dcs increased the likelihood of a calcitonin tumor marker response and should therefore be preferred over monotherapy with dcs pulsed with a single lysate .  ", "thus , immunotherapy may be a promising alternative therapy in combination with agents that target specific signal transduction pathways for aggressive forms of mtc that are resistant to classical therapies .", "a significant antitumor effect was also observed with radioimmunotherapy using an anti - cea i - f6 monoclonal antibody in mtc - bearing nude mice . nevertheless , no complete responses were observed .", "similarly to chemotherapy , drugs that target the tumor microenvironment might improve the efficacy of radioimmunotherapy .", "this hypothesis was confirmed by a recent study in which pretreatment of mice grafted with the tt human medullary thyroid cancer cell line with antiangiogenic therapies was found to improve the efficacy of radioimmunotherapy with acceptable toxicity .", "another independent study , which was conducted in a mouse model of mtc , has found similar results with the angiogenesis inhibitor bevacizumab .", "future investigations will be performed to better understand how antiangiogenic agents enhance the efficacy of radioimmunotherapy .", "epigenetic drugs are expected to target the two main mechanisms of epigenetic alterations , dna methylation and acetylation , and are regarded with increasing interest by both endocrinologists and oncologists . concluded trials of such drugs have shown that few patients with advanced thyroid cancer responded completely , which suggests that these treatments were effective at stabilizing progressive disease .", "therefore , definitive results from clinical trials will clarify the true effectiveness of epigenetic drugs in these tumors .", "epigenetic drugs , when used in combination with other target molecules , might significantly increase response rates to treatment in advanced thyroid cancer patients , either by relaxing the chromatin structure to make dna more accessible to the effects of a dna targeting drug or by acting synergistically with antimitotic drugs . thus", ", epigenetic therapy may be a promising novel approach for the treatment of some cases of mtc .  ", "tumor cells often devise strategies to bypass the effects of antineoplastic agents , and the selection of therapy - resistant clones is frequently the reason for treatment failure .", "one characteristic of endocrine cancer is a general resistance to conventional chemotherapies or radiotherapies that would normally lead to apoptosis of the cancer cells .", "mtc can develop resistances to cytotoxic drugs due to the expression of the multidrug resistance ( mdr ) 1 gene .", "drugs that can oppose this mechanism of resistance to traditional chemotherapies may increase the sensitivity of mtc tumor cells to chemotherapy .", "for example , celecoxib , a cox-2 inhibitor , has been shown to potentiate the chemotherapeutic effect of vinorelbine in mtc .", "therefore , the synergistic actions of a cytotoxic drug and a compound that increases the sensitivity of mtc cells to such a drug could provide a treatment method for patients who can not benefit from tkis .", "however , the most promising results in patients with chemotherapy and radiotherapy - unresponsive mtc were obtained with tkis .", "multitargeted tyrosine kinase inhibitors , which inhibit multiple rtks simultaneously , including ret , have been developed to bypass this potential resistance mechanism .", "one complication is that such inhibitors may not only be more effective for targeting receptors in tumor cells but may also exhibit greater toxicity .", "thus , the challenge is to balance the increased efficacy of these inhibitors with the potential for a broader array of side effects .", "moreover , some mtc patients can not take advantage of these therapies due to specific ret mutations that confer resistance to tkis ( e.g. , ret v804 confers resistance to vandetanib ) [ 76 , 114 ] .", "finally an important study reported the existence of cancer stem - like cells in mtc , which exhibit the features of self - renewal and of multiple lineage differentiation that is dependent on ret proto - oncogene receptor activity suggesting a potential mechanism of resistance to cytotoxic or tkis agents .", "most cases of metastatic mtc are incurable due to resistance to conventional chemo- and radiotherapies . in recent decades , the identification of genetic defects and altered cellular signaling pathways involved in human mtc tumorigenesis have led to the development of targeted therapies , of which the most important are tyrosine kinase inhibitors . however , the low rates of partial responses or complete responses and the short duration of responses in mtc patients who were observed while taking these drugs prompted researchers to develop new drugs and alternative therapies to be combined with multi - tkis and to reduce potential cross - toxicity effects . to this end , a recent study has shown synergistic effects with a combination of sorafenib and the mek inhibitor azd6244 against a human mtc cell line .", "concomitant use of a raf inhibitor , raf265 , and a dual pi3k / mtor inhibitor , bez-235 , was another effective combinatorial therapy against thyroid cancer in xenograft mouse models .", "the mtor cascade is emerging probably as one of the most important deregulated pathways in advanced and metastatic mtc and certainly deserves further study . targeting mtor in combination might be efficacious in patients with this tumor types .", "however , a complete overview of all signaling pathways , including their interactions , role of the activating gene mutations that contribute to mtc tumorigenesis , and mechanisms of intrinsic and acquired resistance to treatments , is required and will permit us to identify the best therapy for each patient .", "novel combined or sequential therapies request a further step in the knowledge of the cellular signaling of this tumor . finally , larger multi - institutional clinical trials to fully understand the clinical benefits of these therapies are warranted ."], "abstract_text": ["<S> parafollicular c - cell - derived medullary thyroid cancer ( mtc ) comprises 3% to 4% of all thyroid cancers . while cytotoxic treatments have been shown to have limited efficacy , targeted molecular therapies that inhibit rearranged during transfection ( ret ) and other tyrosine kinase receptors that are mainly involved in angiogenesis have shown great promise in the treatment of metastatic or locally advanced mtc . </S>", "<S> multi - tyrosine kinase inhibitors such as vandetanib , which is already approved for the treatment of progressive mtc , and cabozantinib have shown distinct advantages with regard to rates of disease response and control . however , these types of tyrosine kinase inhibitor compounds are able to concurrently block several types of targets , which limits the understanding of ret as a specific target . moreover , </S>", "<S> important resistances to tyrosine kinase inhibitors can occur , which limit the long - term efficacy of these treatments . deregulated cellular signaling pathways and genetic alterations in mtc , particularly the activation of the ras / mammalian target of rapamycin ( mtor ) cascades and ret crosstalk signaling , are now emerging as novel and potentially promising therapeutic treatments for aggressive mtc . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Key Cellular Signaling Pathways and Alterations in MTC", "3. Potential Targeted Therapies for MTC", "4. Conclusions"], "sections": [["medullary thyroid carcinoma ( mtc ) is a rare neuroendocrine cancer that originates from thyroid parafollicular calcitonin-(ct- ) producing cells .", "mtc accounts for approximately 4% of all thyroid malignancies ; approximately 75% of these cases occur in the sporadic form , and 25% occur in the hereditary form [ 13 ] .", "mtc usually has a favorable prognosis , with a 10-year survival rate of 70%80% , if it is diagnosed and treated at an early stage when the tumor is confined to the thyroid .", "unfortunately , most cases of mtc present at diagnosis with metastases to the local and regional lymph nodes and to distant organs , especially the lungs , liver , and bones . patients with metastatic mtc have a 10-year overall survival rate of 40% , and metastasis is the main cause of death in patients with mtc [ 4 , 6 ] .", "locally advanced and distant metastatic diseases are incurable , as surgical resection and conventional radio- and cytotoxic chemotherapies are not effective against metastatic mtc [ 7 , 8 ] . clinical trials of various combinations of chemotherapeutic drugs have yielded unsatisfactory results [ 9 , 10 ] . however , research over the last years has led to a good understanding of the genetic defects and altered molecular pathways that are associated with the development of mtc .", "thus , multiple promising therapeutic agents that target these genetic alterations have been developed to treat progressive and advanced mtc . activating mutations of the tyrosine kinase receptor ( tkr ) rearranged during transfection ( ret )", "this discovery has led to the development and introduction of targeted therapies , such as tyrosine kinase inhibitors ( tkis ) that target ret .", "several tkis directed toward ret kinase have been tested in vitro , preclinical , and clinical studies with promising results .", "unfortunately , these agents are not likely to be curative , as the longest duration of response observed was approximately 4 years , and the maintenance of agent - dependent effects may require continuous therapies , which are not without important side effects . the main reasons for the failure of these agents to cure mtc are the development of resistance to tkis that target the ret and other cell receptors and the activities of other signal transduction pathways that are involved in mtc tumorigenesis and progression but not directly targeted by tkis .", "in recent years , the discovery of mechanisms of resistance to tkis and of several other molecular events that contribute to mtc transformation and metastasis suggested that combinatory therapy may result in a more significant tumor growth inhibition .", "this has led to the development of novel compounds that have been used in several clinical trials , including tkis that can target multiple tkrs simultaneously in addition to ret and agents that can target other altered signaling pathways .", "other studies have demonstrated the potential for immunotherapy in combination with agents that target signal transduction pathways that are important for mtc growth .", "because the aim of these targeted therapies is to extend lifespan and increase the quality of life , it is very important to limit the toxicities of therapeutic agents , either alone or in combination .", "the possibility of testing these novel drugs in vitro ( in primary thyroid cancer cells ) and in vivo may help to improve the personalization of treatments ."], ["the role of the ret oncogene in the tumorigenesis of mtc has been characterized extensively .", "the ret gene encodes a transmembrane tyrosine kinase that binds to glial cell line - derived neurotrophic factor ( gdnf ) family ligands .", "ret signaling leads to the activation of the ras / mitogen - activated protein kinase ( mapk ) and the phosphatidylinositol 3 kinase ( pi3k)/akt pathways and has key roles in cell growth , differentiation , and survival . activating point mutations of the tkr ret", "have been reported in nearly all hereditary cases of mtc ; some of these mutations are included in the men2a , familial mtc , or men2b syndromes in which there is a genotypic / phenotypic correlation between the type of ret mutation and clinical features .", "germline mutations in the ret proto - oncogene are responsible for hereditary mtc , while somatic ret mutations are responsible for sporadic mtc .", "however , this paper will mainly focus on additional cellular signaling pathways other than ret responsible of mtc tumorigenesis and progression and potential targeted approaches for the treatment of advanced or metastatic mtc .", "although activating mutations of the tkr ret are believed to be the primary oncogenic event in the development of a majority of mtc cases , it is clear that ret cooperates with other signal transduction pathways to promote mtc tumorigenesis .", "in addition to ret , other kinase receptors may play a role in the development and progression of mtcs .  ", "similar to the ret receptor , the epidermal growth factor receptor ( egfr ) is a tkr that is associated with the regulation of cell growth , proliferation , and apoptosis .", "dimerization of the receptor following ligand binding results in transphosphorylation and the subsequent activation of several downstream signal pathways .", "egfr has been shown to be frequently overexpressed in various types of thyroid carcinomas , including mtc , and to play a role in cancer development and progression .", "in contrast , a recent report analyzing different mtc on tissue microarrays has demonstrated that only 20% of cases revealed moderate to strong reactivity for egfr , whereas the majority of the cases revealed weak and very focal positivity .", "with respect to the numbers of egfr gene copies in mtcs , the researchers did not detect amplifications but did find polysomes in 15% of the examined tumors .", "additionally , egfr was activated in a subset of mtcs , which suggests that this subset of patients might benefit from drugs that target also egfr .", "recent findings have shown that the ligand - induced activation of egfr can stimulate ret activation beyond signaling and growth stimulation .", "several egfr inhibitors have been shown to markedly inhibit the growth of the mtc tt and mz - crc-1 cell lines . because ret activation seems to be influenced by egfr , a recent study investigated whether egfr activation could be related to specific ret mutations in mtcs .", "the researchers found that tumors with the most aggressive ret mutations ( in codons 883/918 ) exhibited reduced egfr expression compared to other ret mutations .", "it could be speculated that the most aggressive ret mutations are less dependent on egfr activation .", "in fact , in the work by croyle et al . , in which cell lines with ret mutations in codon 634 and codon 918 were compared , the effect of egfr inhibition on the codon 918 mutated cell line appeared to be reduced , in agreement with the previous data . because the activation status of egfr seems to be related to ret activation", "however , no differences have been found in egfr activation between ret - positive and -negative tumors , which likely indicates that other molecular mechanisms lead to ret activation , such as increased ret gene copy numbers , altered promoter activity , or increased transcription in the ret mutation - negative tumors .", "these data suggest that egfr status determination in mtcs might be important but certainly deserves further investigations .", "the vascular endothelial growth factor receptor ( vegfr ) pathway is also important in the pathogenesis of mtc .", "there are three transmembrane receptors that mediate the angiogenic and lymphogenic effects of vegf : vegfr-1 , vegfr-2 , and vegfr-3 .", "overexpression of vegf and vegfr-2 has been found in mtc compared to normal thyroid tissue .", "the vegf proteins ( vegf - a , b , c , and d ) , which are secreted by tumor cells , act as ligands for the vegfr-2 receptors on endothelial cells and promote a signaling cascade through different pathways , such as plc--pkc - raf - mek - mapk and pi3k - akt , that stimulate cellular proliferation , migration , and survival and induce neoangiogenesis . angiogenesis is an essential alteration in cell physiology that predisposes the development of malignancy and is fundamental in tumor growth and metastasis . similarly", "to egfr , the overexpression of vegfr-2 in mtc has been shown to correlate with metastasis .  ", "several multitargeting tyrosine kinase inhibitors that block vegfr have shown promising clinical antitumor activity ; unfortunately , in most thyroid carcinomas and other solid tumors , the antiangiogenic effects are often only transitory and really often may have late paradoxically protumorigenic effects .", "additionally , it seems that a modest significant association has been observed between vegfr-2 expression and ret mutation status in primary tumors .   the met proto - oncogene codes for the tk receptor for the hepatocyte growth factor ( hgf ) .", "met hyperactivation reportedly correlates with the metastatic abilities of tumor cells . met and hgf coexpression has been observed in a subset of mtc tumors and is associated with multifocality in mtc , which makes this interaction a potentially important target . in one report", "importantly , ret can induce the overexpression of c - met in this type of thyroid tumor .  ", "the fibroblast growth factor receptor 4 ( fgfr4 ) has also been reported to be overexpressed in mtc cell lines .", "inhibition of fgfr phosphorylation with the small molecule fgfr inhibitor pd173074 resulted in an arrest of cell proliferation and tumor growth . moreover , the dual inhibition of ret and fgfr combined with tyrosine kinase inhibitors resulted in greater suppressions of cell proliferation in vitro and tumor control in vivo than that which was achieved with either agent alone .", "these data highlight ret and fgfr4 as therapeutic targets and suggest a potential role for the use of combined tyrosine kinase inhibitors in the management of inoperable medullary thyroid cancers . finally , the platelet - derived growth factor receptor ( pdgfr ) also seems to play a role in differentiated thyroid cancer , although its role and function have not been fully investigated in mtc .", "several other signal transduction pathways have been implicated as contributors to mtc tumor growth , as illustrated in two recent studies [ 5 , 30 ] .", "these pathways include ret interactions with prb , p53 , p18 , and p27 as well as the phosphatidylinositol 3-kinase / akt / mtor and ras / raf / mek / erk pathways . \n", "ret interactions with tumor suppressor genes \n \n ( a ) prb and p53 . the tumor suppressor genes rb1 ( retinoblastoma ; prb protein ) and tp53 ( p53 protein ) are frequently mutated in human cancers , and it appears that , in cancer , both pathways must be inactivated to overcome senescence or apoptosis .", "there is extensive genetic evidence that the prb and p53 pathways are involved in mtc in rodents .", "further , the loss of tp53 further increased mtc formation in rb1-deficient mice [ 32 , 33 ] .", "it has been shown that in mice , ret cooperates with the inactivation of prb / p53 to cause experimental mtc .", "prb / p53 mutant mice have been shown to acquire ret mutations that are analogous to activating germline mutations that are observed in human men2a and familial medullary thyroid carcinoma ( fmtc ) .", "this suggests that murine mtc requires mutational dysregulations within both the ret and nuclear tumor suppressor gene pathways .", "however , mouse models may not mimic human disease , and a systematic analysis of the genes in the rb1 and tp53 pathways in human samples will help to clarify their roles in human mtc formation .", "this information may be important for the development of novel targeted therapeutic approaches for mtc .  \n", "thyroid tumors show low expression of the cyclin - dependent kinase inhibitor ( cdki ) p27 ( kip1 ) , and recent evidence demonstrates that p27 is downregulated by the active ret mutant , ret / ptc1 , which is found in papillary thyroid carcinomas .", "these data implicate decreased p27 activity as an important event during thyroid tumorigenesis . however , p27 mice develop men - like tumors only in combination with the loss of p18 ( ink4c ) , another cdki .", "this suggests that p18 and p27 are functional collaborators in the suppression of tumorigenesis , that the loss of both is critical to the development of men tumors , and that both p18 and p27 are regulated by ret .  \n", "the pi3k - akt - mammalian target of the rapamycin ( mtor ) cascade is important in tumorigenesis due to its ability to promote cell growth , proliferation , and survival .", "several examples provide evidence to support a role for the activation of the pi3k / akt / mtor signaling cascade in medullary thyroid cancer [ 3638 ] .  ", "several mechanisms have been shown to be involved in the activation of pi3k signaling in medullary thyroid cancer .", "a mutation of men2a ( ret - men2a ) has been shown to activate pi3k and its downstream effector , the serine / threonine kinase akt / protein kinase b .", "previous studies have demonstrated that a mutation of tyr-1062 , which is the intracellular docking site for shc and enigma on ret , abolishes the ret - men2a transforming activity .", "these studies further revealed that the mutation of tyr-1062 abrogates the binding of the p85 regulatory subunit of pi3k to ret - men2a and subsequent stimulation of the pi3k / akt pathway .", "furthermore , retroviral transduction of rat fibroblasts with a dominant - interfering form of pi3k was shown to suppress ret - men2a - dependent transformation , whereas the overexpression of akt enhanced ret - men2a oncogenic potential . in summary , these data are consistent with the notion that ret - mediated cell - transforming effects are critically dependent on the activation of the pi3k / akt / mtor pathway . in cell lines , the pi3k - akt / mtor pathway has been shown to be important in the pathogenesis of men2b .", "ret - men2b ( ret m918 t ) is more effectively autophosphorylated at ret y1062 than is ret - men2a , which subsequently leads to increased constitutive activation of the ras / mitogen - activated protein kinase ( mapk ) and pi3k / akt / mtor signaling cascades .", "furthermore , previous data report other possible mechanisms of pi3k activation in thyroid carcinomas , such as the overexpression of rai ( shcc / n - shc ) , which is a substrate of ret oncoproteins [ 5 , 42 ] .", "finally , the loss of expression of the phosphatase and tensin homologue ( pten ) gene , a tumor suppressor gene , may have a role in the activation of pi3k / akt in thyroid tumors .", "the absence of functional pten protein expression has been observed in various cancer cells and has led to the constitutive activation of downstream components of the pi3k pathway , including the akt and mtor kinases .", "preclinical models showed that inactivation of these kinases is able to reverse the effects of pten loss .", "these data raise the possibility that drugs that target either these kinases or pi3k itself might have significant therapeutic activity against pten - null cancers .", "mutations in major nodes of this signaling cascade have been observed in human cancers ; these mutations include gain - of - function mutations and amplifications of the genes encoding pi3k and akt [ 44 , 45 ] .", "however , a recent study on a series of 49 mtcs has shown that the pi3k genes were not mutated , and that the activation of the pi3k pathway was significantly associated with the status of ret mutations .", "in fact , using protein expression analysis , the same authors confirmed that the akt / mtor pathway was highly activated in mtc , especially in cases with germline ret mutations .", "interestingly , this association was not dependent on the type of mutation ( in either the codons of the juxta - membrane or of the tyrosine kinase portion of the receptors ) but was dependent on the hereditary nature of the mutation .", "in contrast , medullary carcinomas with sporadic ret mutations or with wild - type ret were observed with heterogeneous expression of akt / mtor pathway molecules , which suggests the need for further elucidation of alternative activation mechanisms . in a recent study ,", "the pi3k / akt / mtor pathway was shown to be activated in mtc , particularly in the metastatic lymph nodes , and this pathway was shown to sustain malignant features of different mtc cell models .", "moreover , it has been shown that the selective inhibition of the mtor pathway in a germline - ret - mutated mtc cell line can effectively decrease cell viability and block the phosphorylated status of mtor signaling molecules , which confirms previously published in vitro data .", "another study used metformin , which is an antidiabetic agent that decreases the proliferation of cancer cells through the 5-amp - activated protein kinase - dependent inhibition of mtor , in mtc cell lines to show that the growth - inhibitory effects on the cells were associated with the downregulation of both the mtor/6sk and perk signaling pathways .", "altogether , these results strongly suggest that mtor might be a very efficacious target in patients with advanced or metastatic mtc .", "noteworthy , mtor inhibitors are currently being used in clinical trials for the treatment of medullary thyroid carcinoma .  \n ras - raf - mek - erk pathway .", "unlike hereditary mtc , in which ret mutations are the critical events , in sporadic mtc , the genetic or molecular biomarkers have not been fully established .", "ras is a frequently mutated oncogene in a broad spectrum of human tumors , including thyroid carcinoma , mainly in the follicular forms . in this context , investigators aimed to determine whether mutations in the ras oncogene could play a possible role in the carcinogenesis of sporadic mtc .", "the initial study analyzed 15 sporadic mtcs for mutations in known hot spots ( codons 12 , 13 , and 61 ) of the h- and k - ras oncogenes by a direct sequencing technique , although no ras mutations were detected in any of the examined tumors .", "a different study on 49 mtc confirmed that ras mutations are a rare event in this type of tumor , regardless of ret mutational status .", "by contrast , different studies have demonstrated the presence of ras mutations in mtc . a mutational screening for h- , k- , n - ras , and braf in 13 sporadic and inherited mtcs revealed one sporadic ret - negative mtc ( stage iii ) with a mutation in the h - ras codon 13 ( g13r ) .", "another study analyzed 188 hereditary and sporadic mtcs for ras mutational status , revealing a low prevalence of mutations , which were confined only to ret - negative sporadic mtc .", "furthermore , recent studies have reported a quite high prevalence of ras mutations in sporadic mtc , particularly in ret - negative mtc ; 68% ( 17 of 25 ) of the ret - negative mtcs had mutations of ras compared to only 2.5% ( 1/40 ) of the ret - positive mtcs .", "these findings were confirmed by a different study that analyzed 17 sporadic mtcs by exome sequencing and found dominant and mutually exclusive oncogenic mutations in ret and ras ( h- and k - ras ) genes in 17 sporadic mtcs . as expected ,", "most ras mutations corresponded to mutational hot spots in exons 2 and 3 , although some mutations were also detected in exon 4 .", "this study confirms that ret and ras mutations are mutually exclusive , and that they are probably 2 different oncogenic driver events in mtc .", "these results suggest that the activation of the proto - oncogenes ras and ret represents alternative genetic events in sporadic mtc tumorigenesis , and that more sensitive sequencing techniques such as next generation sequencing are necessary to detect mutations . for decades ,", "the ras and raf families of oncogenes have been known to be transforming genes . however , in many normal cultured cell types , the sustained expression of activated ras or its downstream effector , raf , can elicit cell cycle arrest or senescence . the ras / raf - mediated growth arrest has been proposed as a defense mechanism against the inappropriate activation of the ras / raf signal transduction pathway [ 56 , 57 ] . according to this hypothesis , spontaneous mutations in ras genes , which may occur stochastically in all cell types , would be innocuous for the organism because these mutations would lead to growth arrest or senescence .", "hence , for carcinogenesis to occur in response to ras / raf activation , the growth arrest response must be inactivated .", "the growth arrest response to ras / raf activation is not limited to normal cells .", "several tumor cell lines that were derived from different tumor types , including medullary thyroid carcinoma , also experienced growth arrest , usually accompanied by differentiation or senescence [ 5860 ] .", "these findings indicate that some tumors retain a capability for growth arrest in response to ras / raf activation .", "the mechanism by which ras or raf activation can induce growth arrest is not completely understood .", "some investigators have reported that ras or raf activation could induce the expression and secretion of a protein that mediated differentiation and g1 cell cycle arrest in mtc cells . by protein purification and mass spectrometry , this protein has been identified as the leukemia inhibitory factor ( lif ) .", "stat3 activation was necessary for lif - mediated growth arrest and differentiation in mtc cells .", "in addition , the ras / raf / mek / erk pathway could also mediate growth arrest and differentiation by a second mechanism that is independent of lif / jak / stat3 .", "this novel autocrine - paracrine mechanism , which mediates crosstalk between the ras / raf / mek / erk and the jak - stat pathways , defines a novel mechanism of ras / raf - induced cell growth arrest [ 61 , 62 ] .", "the inactivation of gsk-3beta in tt cells by well - known gsk-3beta inhibitors , such as lithium chloride ( licl ) and sb216763 , is associated with both growth suppression and a significant decrease in neuroendocrine markers , such as human achaete - scute complex - like 1 and chromogranin a. growth inhibition by gsk-3beta inactivation was found to be associated with cell cycle arrest due to an increase in the levels of cyclin - dependent kinase inhibitors , such as p21 , p27 , and p15 . additionally , tt xenografts mice , treated with licl , showed a significant reduction in tumor volume compared with those that were treated with a control .", "therefore , gsk-3beta is a key downstream target of the raf-1 pathway in tt cells , and the inactivation of gsk-3beta alone is sufficient to inhibit the growth of tt cells both in vitro and in vivo .  \n", "-catenin is a ubiquitously expressed multifunctional protein that plays an important role in cellular adhesion . a novel ret--catenin signaling pathway was found to be a critical contributor to enhanced cell proliferation and tumor progression in thyroid cancer .", "gujral et al . showed that ret could induce -catenin - mediated transcription , cell proliferation , and transformation in vitro and that -catenin nuclear localization and subsequent mediation of -catenin by ret are key secondary events in tumor growth and metastasis in vivo .", "this novel interaction suggests a mechanism that may underlie the broad and early metastatic potential of mtc .", "these data suggest an unrecognized role for -catenin signaling that may have implications for tyrosine kinase - mediated tumorigenesis in multiple tumor types and provide another potential target for therapeutic agents . in support , a recent study performed on tissue microarray observed that wnt pathway proteins , including wisp-1 , wisp-2 , and -catenin , were actively expressed in mtc .  \n", "the nuclear factor kappa - b ( nf-b ) proteins , a family of transcription factors that are found virtually in all cells , are known to play crucial roles in the growth of many human malignancies .", "the ability of nf-b to target a large number of genes that regulate cell proliferation , differentiation , survival , and apoptosis provides clues towards its dysregulation during the processes of tumorigenesis , metastatic progression , and therapeutic resistance of tumors .", "nf-b is constitutively active in mtc through the ret - induced phosphorylation , ubiquitination , and proteosomal degradation of inhibitors of nf-b ( ikb ) , which allows nf-b to enter the nucleus and bind to dna .", "nf-b is frequently activated in mtc , and the activation of ret by somatic or germline mutations may be responsible for nf-b activation in these tumors .", "these results suggest that the nf-b pathway may be an important target for drug development in mtc .  \n", "a recent study examined the expression of proteins involved in angiogenesis , inflammation , apoptosis , cell cycle , cell - to - cell contact , and carcinogenesis using high - throughput tissue microarrays from 23 patients with mtc .", "these authors identified several novel potentially important protein targets such as cox-1/2 , bcl-2a , gst- , gli-1 , gli-2 , gli-3 , and bmi-1 that may be therapeutically targeted in mtc .", "for example , cox-1 and cox-2 , which are two inflammation - related factors , were significantly expressed in these cases , suggesting that nonsteroidal anti - inflammatory drugs may provide benefit in some patients with mtc .", "then , the finding of antiapoptotic bcl-2a and gst- overexpression in mtc suggests that bcl-2a and gst- inhibitors might be a treatment option for patients with advanced or metastatic mtc .", "the same is applied to gli-1 , gli-2 , and gli-3 , members of sonic hedgehog homolog ( shh ) pathway , and bmi-1 , a cell - cycle marker that resulted overexpressed in these mtc samples .", "the studies of these markers , particularly the members of the shh , may improve our understanding of mechanism of resistance to current chemotherapeutic and/or tki regimens and identify novel potential therapeutic approaches ."], ["due to increased knowledge of the molecular pathogenesis of mtc , therapeutic agents that target specific altered pathways have been developed ( figure 1 ) . because the alterations of protein kinases and their pathways are involved in mtc development , several tyrosine kinase receptors inhibitors ( tkis ) have been tested in vitro , preclinical , and clinical studies .", "ret is certainly an attractive target for several types of tumors particularly for parafollicular c - cells - derived tumors , which are addicted to ret and its activity .", "tkis are small organic compounds that affect tyrosine kinase - dependent oncogenic pathways by competing with atp - binding sites of the tyrosine kinase catalytic domains .", "occupation of these sites inhibits autophosphorylation and activation of the tyrosine kinases and prevents the further activation of intracellular signaling pathways .", "several tkis that are directed against ret kinase have been developed for the treatment of mtc , but there is no currently available tyrosine kinase inhibitor specific to ret .", "however , several multitargeted tyrosine kinase inhibitors have demonstrated significant activity against ret ( table 1 ) , including vandetanib , sorafenib , motesanib , imatinib , and sunitinib [ 66 , 68 ] .", "the inhibition of only one kinase receptor may induce the compensatory activation of other tks and consequent resistance to treatment with tkis [ 69 , 70 ] .", "therefore , the simultaneous inhibition of multiple activated tks may be the best way to approach mtc [ 7173 ] .", "vandetanib ( zd6474 ) is an oral tki that targets ret , vegfr-2 , and -3 , and at higher concentrations , egfr .", "vandetanib selectively inhibits pathways that are critical for tumor growth and angiogenesis without leading to direct cytotoxic effects on tumor or endothelial cells .", "most of the mutant ret oncoproteins have demonstrated sensitivity to vandetanib , while mutations in which valine 804 is substituted by either leucine or methionine ( as observed in some cases of men2a ) rendered the ret kinase significantly resistant to vandetanib .", "this resistance is likely due to steric hindrance , as the val804gly mutation increased the sensitivity of ret to vandetanib .", "when ret activity was inhibited , overstimulation of egfr was able to partially compensate for ret through a partial rescue of mapk pathway activation .", "the inhibition of egfr by vandetanib has been shown to prevent this rescue of mapk pathway activation .", "these data support the idea that the dual inhibition of ret and egfr is important , as it may overcome the risk of an escape by mtc cells from a blockade of ret through the compensatory overstimulation of egfr .", "in addition , the expression of egfr has been demonstrated in tumor - associated endothelial cells . in this", "respect , anti - egfr agents have been shown to block the proliferation and migration of endothelial cells .", "therefore , the anti - egfr activity of vandetanib might result in an additional direct antiangiogenetic mechanism .", "one phase ii clinical trial showed the antitumor activity of vandetanib ( 300  mg / day ) in patients with hereditary mtc . in this study ,", "20% of the patients showed a partial response to vandetanib ( > 30% reduction in tumor diameter ) , while an additional 53% of patients presented with disease stability at 24 weeks . only 1 patient showed disease progression while receiving this agent . of interest", ", this patient was not affected by any of two ret mutations correlated to vandetanib resistance in which valine 804 is substituted by leucine or methionine , but by y791f ret germline mutation .", "another clinical trial , in which 19 patients with hereditary mtc were treated with vandetanib ( 100  mg / day ) , showed similar results .", "vandetanib is the only tki approved for the treatment of symptomatic or progressive mtc in patients with unresectable locally advanced or metastatic disease .", "the approval of vandetanib in april 2011 by the us food and drug administration ( fda ) was based on the results of the phase iii clinical trial , ", "study d4200c00058 ,  in which 331 patients with unresectable locally advanced or metastatic mtc were randomly assigned to receive vandetanib ( 231 ) or a placebo ( 100 ) .", "this study showed that the median progression - free survival duration ( pfs ) was 11 months longer in the group that received vandetanib than in the placebo control group and that 45% of the patients had an objective response rate ( orr ) .", "common adverse events ( any grade ) occurred more frequently with vandetanib compared to the placebo and included diarrhea ( 56% versus 26% ) , rash ( 45% versus 11% ) , nausea ( 33% versus 16% ) , hypertension ( 32% versus 5% ) , and headache ( 26% versus 9% ) .", "the impact of overall survival of mtc patients treated with this compound is presently unknown . \n", "xl184 ( cabozantinib ) is an oral selective inhibitor of ret , c - met , and vegfr-2 .", "the phase i trial results indicated that cabozantinib is active in patients with mtc , including those who harbor somatic ret mutations and are potentially at high risk for progression and death . a global phase iii pivotal study in mtc is ongoing ( http://www.clinicaltrials.gov/ number nct00704730 ) . \n", "sorafenib ( bay 43 - 9006)is a multikinase inhibitor that targets braf , vegfr-2 , vegfr-3 , kit ( a stem cell growth factor proto - oncogene involved in cell survival and differentiation ) , and pdgfr .", "a phase ii clinical trial , in which sorafenib ( 400  mg / twice daily ) was given to 21 patients with metastatic medullary carcinoma , reported that of the patients with sporadic mtc , 87.5% achieved disease stability and 6.3% demonstrated partial responses .", "the treatment was prematurely terminated in mtc hereditary patients due to slow accrual . in a similar trial ,", "recently , a study of sorafenib was conducted on advanced thyroid carcinoma patients , and partial responses were reported in six out of the 12 ( 50% ) patients with mtc , although the small number of patients requires further prospective studies .  \n", "sunitinib ( su011248 ) is a small molecule inhibitor that targets many of the same protein kinases as sorafenib , including vegfr , pdgfr , kit , and ret . in a phase ii clinical trial ,", "33 patients with either well - differentiated thyroid carcinoma or mtc were treated with sunitinib .", "one patient had a complete response , 28% of the patients had partial responses , and 46% of the patients exhibited disease stability .", "the intermediate results of the phase ii thysu study also showed the efficacy of sunitinib in advanced medullary thyroid carcinoma .", "motesanib ( amg 706 ) is a multikinase inhibitor that targets vegfr receptors 1 , 2 , and 3 , pdgfr , and kit and exerts antiangiogenic and direct antitumor activities .", "a phase ii study performed in 91 patients with locally advanced or metastatic , progressive or symptomatic , mtc demonstrated that although the objective response rate was low , a significant proportion of the mtc patients ( 81% ) achieved disease stability while receiving motesanib .", "a recent study investigated the effects of motesanib on wild - type and mutant ret activity in vitro and on tumor xenograft growth in a mouse model of mtc .", "the results of this study demonstrated that motesanib inhibited thyroid tumor xenograft growth , predominantly through the inhibition of angiogenesis and possibly via the direct inhibition of vegfr2 and ret , which were expressed in tumor cells .", "these data suggest that angiogenic pathways and specifically the vegf pathway are still important for mtc cells .  \n", "imatinib ( sti571 ) is a tki that inhibits ret , pdgfr , and kit .", "a phase ii trial in which imatinib was tested in metastatic mtc patients yielded disappointing results .", "the patients showed no objective responses ; however , a minority of patients achieved disease stability .", "several tk inhibitors have been tested in clinical trials , but the most effective inhibitor and whether there is any specificity for particular ret mutations remain unknown .", "a recent study compared the effects of four tkis ( axitinib , sunitinib , vandetanib , and xl184 ) on cell proliferation , ret expression and autophosphorylation , and erk activation in cell lines that express men2a ( mtc - tt ) and men2b ( mz - crc-1 ) mutation .", "the findings showed that the inhibitors were specific for different mutations , with xl184 being the most potent inhibitor against the men2a mutation and vandetanib the most effective against the men2b mutation in vitro .", "no tk inhibitor was superior for all tested cell lines , which indicates that mutation - specific therapies could be beneficial in mtc treatment .", "one of the mechanisms for the resistance of mtc to chemotherapeutic drugs is multidrug resistance ( mdr ) .", "mdr in cancer cells has been attributed to the overexpression of several plasma membrane atp - dependent efflux pumps , such as mdr-1 .", "the enzyme cyclooxygenase ( cox-2 ) has been shown to regulate mdr-1 expression in rat mesangial cells .", "furthermore , a study has shown that in vitro treatment of an mtc - derived cell line with rofecoxib , a cox-2 inhibitor , was able to sensitize mtc cells to doxorubicin . a recent study , performed in vitro ,", "has shown that celecoxib , another cox-2 inhibitor , was able to induce both mtc cell apoptosis and sensitization to vinorelbine , thus enhancing the chemotherapeutic effect of this drug .", "a clinical trial , in which the in vivo activities of celecoxib were explored in mtc patients who can not benefit from available treatments , would be desirable after accounting for the possible cardiovascular risks of this drug .", "several other therapeutic agents are being investigated for their uses in the treatment of thyroid carcinomas , including mtc .", "these agents inhibit targets that are involved in development of mtc other than the tyrosine kinase receptors .", "we previously discussed that ras operates in a complex signaling network with multiple activators and effectors , which allows them to regulate many cellular functions such as cell proliferation , differentiation , apoptosis , and senescence .", "phosphatidylinositol 3-kinase ( pi3k ) is one of the main effector pathways of ras , regulating cell growth , cell cycle entry , cell survival , cytoskeleton reorganization , and metabolism .", "the presence of ras mutations in sporadic mtc and most importantly the frequent activation of the pi3k / akt / mtor pathway in several aggressive and metastatic mtcs strongly suggest that this cellular signaling pathway is a good candidate for targeted therapies against mtc .", "in fact , several in vitro evidences have demonstrated that the indirect blocking of this pathway , by pi3k inhibitors , or direct inhibition of mtor   can mediate the induction of apoptosis and a decrease in cell viability in the mtc tt cell line .", "currently , there are several clinical trials in which patients with radioiodine - refractory differentiated and medullary thyroid carcinomas were recruited to test the efficacy of everolimus ( rad001 ) , a novel inhibitor of mtor , in combination with other drugs ( nct01625520 and nct01270321 ) .", "recently , researchers have developed a liposomal form of everolimus and have demonstrated the anticancer efficacy of this formulation against tt cells .", "studies have shown that metformin can decrease the proliferation of cancer cells through 5-amp - activated protein kinase-(ampk- ) dependent inhibition of mtor .", "some researchers have reported growth inhibitory effects of metformin against mtc cell lines ( tt and mz - crc-1 ) , which were attributable to the downregulation of both mtor/6sk and perk signaling .", "the expression of the molecular targets of metformin in human mtc cells suggests that this drug may be potentially useful in the treatment of mtc .  ", "pi3k - akt - mtor and raf - mek - erk signaling have been shown to be important in the resistance of thyroid cancer cells to apoptosis and the promotion of tumor progression .", "in this context , targeted anti - vegfr therapy or raf inhibition may be ineffective if pi3k signaling remains intact .", "therefore , two promising drugs , raf265 , a raf inhibitor that is active against vegfr2 , and bez235 , a pi3k inhibitor , were tested alone and in combination in preclinical mtc models that represented the key genotypes observed in refractory thyroid cancers .", "the study findings showed that a combination treatment with agents that inhibited both raf and pi3k pathways strongly inhibited growth both in vitro and in vivo .", "in addition , the investigators showed for the first time that raf265 potently inhibits the constitutively active ret , a form of the kinase that is observed in men2a .", "persistent ret activation , a frequent event in mtc , leads to the activation of the pi3k / akt / mtor , erk / mapk , and jak / stat3 pathways .", "recently , the efficacy of the jak1/2 inhibitor azd1480 against the growth of thyroid cancer was tested in vitro and in vivo in thyroid cancer cell lines that expressed oncogenic ret .", "the findings showed that azd1480 efficiently inhibited the growth and tumorigenesis of thyroid cancer cell lines that harbored oncogenic ret alterations , likely through the inhibition of pi3k - akt signaling ; this result supports the use of this inhibitor in patients with thyroid cancer , particularly in those with advanced mtc , for whom there are no effective therapeutic options .", "thus , somatostatin ( srif ) peptide analogues in combination with tkis may be a promising approach for the treatment of these tumors .", "the presence of different combinations of srif receptor ( sstr ) subtypes in a given patient may explain the variable clinical response to srif analogues and may promote the search for more selective drugs with different affinities to the various receptor subtypes .", "the currently available somatostatin analogues ( octreotide and lanreotide ) act preferentially through the somatostatin receptor subtype 2 ( sst2 ) . in mtc", ", these compounds have been reported to exert antisecretive effects on calcitonin but unfortunately are not reported to have antiproliferative effects .", "pasireotide ( som230 ) is a new somatostatin analogue that has a peculiar binding profile with high affinity for sst1 , sst2 , sst3 , and sst5 .", "preliminary data from a phase ii study of patients with metastatic carcinoma show that som230 is effective , and some clinical trials are exploring the efficacy of som230 alone or in combination with rad001 in patients with mtc ( nct01625520 and nct01270321 ) .  ", "the nf-b pathway is also a potential target for drug development , and a number of compounds have been developed to inhibit this pathway at different levels in cancer cells .", "studies have demonstrated that the proteosome inhibitor bortezomib exerted a promising antitumor effect in human mtc cells through the inhibition of ib degradation , which led to the inactivation of the transcriptional factor nf-b .", "patients are currently being recruited for a phase i / ii trial to study the combination of vandetanib plus bortezomib ( http://www.clinicaltrials.gov/ ) .", "immunization with tumor antigen - pulsed autologous dendritic cells ( dcs ) resulted in protective immunity and the rejection of various established human tumors .", "specifically , vaccination immunotherapy with calcitonin and/or carcinoembryonic antigen ( cea ) peptide - pulsed dcs was shown to result in the induction of a cellular , antigen - specific immune response in patients with mtc , which led to clinical responses in some patients .", "therefore , for the first time , a potential dc vaccination therapy was developed for patients with metastatic mtc .", "another study , which was performed in a transgenic mtc mouse model , has confirmed this finding .  ", "have reported on the in vitro findings of a vaccination trial in 5 mtc patients , who were treated with dcs that were generated using a new protocol , which consisted of granulocyte - macrophage colony - stimulating factor and interferon - alpha ( ifn - dcs ) .", "these investigators demonstrated that immunization with ifn - dcs led to a tumor epitope - specific th1 immune response in mtc patients .", "furthermore , a pilot trial of 10 patients has assessed the safety and efficacy , in terms of immune responses and clinical activities , of the dcs . in this study", "monitoring of the patients included serial measurements of calcitonin tumor markers , radiological imaging , and immunological in vitro tests , including t - cell interferon - gamma detection and cytotoxicity assays .", "after a median followup of 11 months ( range 726 ) , 3 ( 30% ) of the 10 patients exhibited disease stability , while 7 ( 70% ) of the patients progressed during treatment .", "a combined treatment with different tumor cell lysate - pulsed dcs increased the likelihood of a calcitonin tumor marker response and should therefore be preferred over monotherapy with dcs pulsed with a single lysate .  ", "thus , immunotherapy may be a promising alternative therapy in combination with agents that target specific signal transduction pathways for aggressive forms of mtc that are resistant to classical therapies .", "a significant antitumor effect was also observed with radioimmunotherapy using an anti - cea i - f6 monoclonal antibody in mtc - bearing nude mice . nevertheless , no complete responses were observed .", "similarly to chemotherapy , drugs that target the tumor microenvironment might improve the efficacy of radioimmunotherapy .", "this hypothesis was confirmed by a recent study in which pretreatment of mice grafted with the tt human medullary thyroid cancer cell line with antiangiogenic therapies was found to improve the efficacy of radioimmunotherapy with acceptable toxicity .", "another independent study , which was conducted in a mouse model of mtc , has found similar results with the angiogenesis inhibitor bevacizumab .", "future investigations will be performed to better understand how antiangiogenic agents enhance the efficacy of radioimmunotherapy .", "epigenetic drugs are expected to target the two main mechanisms of epigenetic alterations , dna methylation and acetylation , and are regarded with increasing interest by both endocrinologists and oncologists . concluded trials of such drugs have shown that few patients with advanced thyroid cancer responded completely , which suggests that these treatments were effective at stabilizing progressive disease .", "therefore , definitive results from clinical trials will clarify the true effectiveness of epigenetic drugs in these tumors .", "epigenetic drugs , when used in combination with other target molecules , might significantly increase response rates to treatment in advanced thyroid cancer patients , either by relaxing the chromatin structure to make dna more accessible to the effects of a dna targeting drug or by acting synergistically with antimitotic drugs . thus", ", epigenetic therapy may be a promising novel approach for the treatment of some cases of mtc .  ", "tumor cells often devise strategies to bypass the effects of antineoplastic agents , and the selection of therapy - resistant clones is frequently the reason for treatment failure .", "one characteristic of endocrine cancer is a general resistance to conventional chemotherapies or radiotherapies that would normally lead to apoptosis of the cancer cells .", "mtc can develop resistances to cytotoxic drugs due to the expression of the multidrug resistance ( mdr ) 1 gene .", "drugs that can oppose this mechanism of resistance to traditional chemotherapies may increase the sensitivity of mtc tumor cells to chemotherapy .", "for example , celecoxib , a cox-2 inhibitor , has been shown to potentiate the chemotherapeutic effect of vinorelbine in mtc .", "therefore , the synergistic actions of a cytotoxic drug and a compound that increases the sensitivity of mtc cells to such a drug could provide a treatment method for patients who can not benefit from tkis .", "however , the most promising results in patients with chemotherapy and radiotherapy - unresponsive mtc were obtained with tkis .", "multitargeted tyrosine kinase inhibitors , which inhibit multiple rtks simultaneously , including ret , have been developed to bypass this potential resistance mechanism .", "one complication is that such inhibitors may not only be more effective for targeting receptors in tumor cells but may also exhibit greater toxicity .", "thus , the challenge is to balance the increased efficacy of these inhibitors with the potential for a broader array of side effects .", "moreover , some mtc patients can not take advantage of these therapies due to specific ret mutations that confer resistance to tkis ( e.g. , ret v804 confers resistance to vandetanib ) [ 76 , 114 ] .", "finally an important study reported the existence of cancer stem - like cells in mtc , which exhibit the features of self - renewal and of multiple lineage differentiation that is dependent on ret proto - oncogene receptor activity suggesting a potential mechanism of resistance to cytotoxic or tkis agents ."], ["most cases of metastatic mtc are incurable due to resistance to conventional chemo- and radiotherapies . in recent decades , the identification of genetic defects and altered cellular signaling pathways involved in human mtc tumorigenesis have led to the development of targeted therapies , of which the most important are tyrosine kinase inhibitors . however , the low rates of partial responses or complete responses and the short duration of responses in mtc patients who were observed while taking these drugs prompted researchers to develop new drugs and alternative therapies to be combined with multi - tkis and to reduce potential cross - toxicity effects . to this end , a recent study has shown synergistic effects with a combination of sorafenib and the mek inhibitor azd6244 against a human mtc cell line .", "concomitant use of a raf inhibitor , raf265 , and a dual pi3k / mtor inhibitor , bez-235 , was another effective combinatorial therapy against thyroid cancer in xenograft mouse models .", "the mtor cascade is emerging probably as one of the most important deregulated pathways in advanced and metastatic mtc and certainly deserves further study . targeting mtor in combination might be efficacious in patients with this tumor types .", "however , a complete overview of all signaling pathways , including their interactions , role of the activating gene mutations that contribute to mtc tumorigenesis , and mechanisms of intrinsic and acquired resistance to treatments , is required and will permit us to identify the best therapy for each patient .", "novel combined or sequential therapies request a further step in the knowledge of the cellular signaling of this tumor . finally , larger multi - institutional clinical trials to fully understand the clinical benefits of these therapies are warranted ."]]}
{"article_id": "PMC2873758", "article_text": ["all of the effects of antimicrobial agents against microbes , including the delineation of microbial resistance , are based upon the results of in vitro susceptibility testing .", "most of these susceptibility tests only measure bacteriostatic activity even though the agent being tested may have bactericidal activity .", "these authors point out the paucity of studies that have critically evaluated the effectiveness of antimicrobial therapy with results of in vitro susceptibility tests .", "such critical evaluations are not easily done , as susceptibility tests do not take into account the normal host defense mechanisms .", "however , the detection of resistance is somewhat predictive of poor outcome , although in the normal host this may be less clinically important due to the interaction of host defenses ( 17,18 ) .", "the ability of bactericidal activity to influence therapeutic efficacy and clinical outcome has been evaluated in infections that typically are refractory to antimicrobial therapy .", "these infections include endocarditis , meningitis , osteomyelitis , and infections in the neutropenic host .", "all are similar in that antimicrobial penetration and host defense mechanisms at the site of infection are limited .", "both experimental models of infection ( 1922 ) and clinical studies ( 2327 ) have shown that bactericidal activity predicts therapeutic efficacy and results in improved clinical outcome .", "bactericidal activity has been considered less important in respiratory tract infections , with the exception being acute infectious exacerbations in cystic fibrosis ( 28 ) .", "however , the relevance of pharmacokinetic and pharmacodynamics in the selection of antibiotics for respiratory tract infection has become increasingly recognized ( 29 ) .", "issues such as drug concentration at the site of infection , bactericidal activity , postantibiotic effect , and duration of therapy needed to achieve these effects are now being considered when antimicrobial agents are selected for the therapy of respiratory tract infections ( 29,30 ) .", "although host factors may allow a bacteriostatic agent to be used successfully in an infected patient , these factors appear to be less able to curtail the emergence of resistance .", "resistance , as a rule , occurs more rapidly with bacteriostatic agents such as tetracyclines , sulfonamides , and macrolides than it does with bactericidal agents such as beta - lactams and aminoglycosides ( 3133 ) .", "beta - lactam agents have been the antimicrobial agents of choice for the therapy of pneumococcal pneumonia since penicillin was first clinically introduced in the 1940s ( 17 ) .", "the emergence of macrolide resistance in s. pneumoniae has been rapid in comparison and has even been described during treatment of pneumococcal pneumonia ( 34 ) .", "bactericidal activity thus may be useful in the therapy of respiratory tract infections as a means to curtail , but not avoid , the emergence of resistance .", "the key to resolving the problem of antibacterial resistance lies in identifying the mechanisms that engender it ( 3133 ) . among the most important mechanisms are decreased ability of antibacterials to penetrate the bacterial cell wall , active efflux of antibacterial agents , inactivation of antibacterial agents , destruction of antibacterial agents , alteration of antibacterial target sites , development of bypass pathways around antibacterial targets , and constitutive phenotypic variation in bacterial physiology .", "subsequent exposure of the microorganism to a specific agent may then select the mutant , leading to the emergence of resistance . some resistance mechanisms , such as bacterial production of beta - lactamase , are inducible or can be derepressed ( 35 ) , requiring either upregulation or mutation of genetic material .", "thus , if resistance is to be suppressed , the opportunity for bacterial upregulation or mutation of genetic material must be minimized .", "one way to minimize upregulation , mutation , or both is by using bactericidal rather than merely bacteriostatic agents .", "microorganisms inhibited by a bacteriostatic agent or exposed to an insufficient concentration of a bactericidal agent remain alive and , ipso facto , retain the potential to become resistant or promulgate any resistance selected by the exposure to the antimicrobial agent .", "an example of this principle can be seen with the upper respiratory tract pathogen , streptococcus pyogenes , which to date has not developed resistance to penicillin , a bactericidal agent , but has developed resistance to erythromycin , a bacteriostatic agent ( 3638 ) .", "erythromycin resistance in s. pyogenes largely is due to upregulation of efflux ( 36 ) or to ribosomal mutation ( 37 ) .", "antimicrobial agents that kill this pathogen should be less likely to promulgate any strains having such resistance . another example is seen with the omp genes of gram - negative microorganisms .", "these omp genes encode porins that are sometimes flanked by insertion sequences . in the presence of the bacteriostatic agent , the mobility of insertion sequence  flanking omp genes can be attenuated and will result in disruption of the omp genes .", "the reduced expression of these porins may lead to reduced uptake of the inducer , the antibacterial agent .", "specifically , insertion sequence interruption of the ompk36 porin gene in respiratory tract pathogen klebsiella pneumoniae has been shown to interfere in the expression of this porin gene and has resulted in clinical failure ( 39 ) .", "if a bactericidal agent kills a pathogen such as klebsiella before mutation of the porin gene , resistance is less likely to develop .", "these two examples illustrate the desirability of achieving bactericidal activity to curtail the emergence of resistance .", "bactericidal activity can be achieved through the mechanism of action for a single antimicrobial agent or by the use of combination therapy , or both .", "sulfamethoxazole / trimethoprim ( smx - tmp ) is an example of a combination of two agents , each of which alone is bacteriostatic , that achieves bactericidal activity .", "sulfamethoxazole inhibits dihydropteroate synthase , the bacterial enzyme that catalyzes the incorporation of p - aminobenzoid acid into dihydropteroic acid , the immediate precursor of folic acid , while trimethoprim was specifically synthesized as an inhibitor of dihydrofolate reductase ( 40 ) .", "smx - tmp has long been used for the therapy of respiratory tract infection ( 41 ) and has proven particularly useful in the treatment of acute exacerbations of chronic bronchitis .", "in fact , the world health organization continues to be recommend smx - tmp as the first - line treatment for pneumonia in children because of its low cost and ease of dosing .", "resistance to smx - tmp has emerged more slowly than for either agent used alone ( 42 ) .", "however , emergence of resistance to s. pneumoniae has occurred ( 43 ) and now may limit the use of smx - tmp in respiratory tract infections .", "combinations of antimicrobial agents are also used in the therapy of bacterial endocarditis to achieve synergism leading to increased bactericidal activity and improved sterilization of infected valves .", "bactericidal synergy for s. epidermidis can be demonstrated in vitro for the combination of vancomycin , rifampin , and gentamicin , which correlates well with the therapeutic results in an experimental animal model ( 44 ) . in a comparable clinical study of patients with prosthetic valve endocarditis caused by staphylococcus epidermidis ,", "90% were cured with a combination of vancomycin , rifampin , and/or gentamicin , compared with only 50% cured among those receiving vancomycin alone ( 45 ) .", "combination therapy for respiratory tract infections is less well studied except for acute respiratory tract infections occurring in cystic fibrosis patients .", "for example , combination therapy for treatment of pseudomonas aeruginosa pulmonary infections in cystic fibrosis patients achieved a cure rate of 89% if peak serum bactericidal titers were > 1:128 ( 28 ) .", "in contrast , 100% of patients failed therapy if their peak serum bactericidal titers were < 1:16 .", "other examples of the importance of bactericidal activity are the fluoroquinolones . studies of the bactericidal action of the quinolones against escherichia coli demonstrate at least two independent and important mechanisms of action .", "these bactericidal agents are only effective if the bacteria are actively dividing or synthesizing proteins and mrna .", "the bactericidal activity of the quinolone nalidixic acid , for example , is minimized by chloramphenicol , which prevents protein synthesis , and by rifampin , which prevents rna synthesis . however , ciprofloxacin and ofloxacin / levofloxacin respond differently .", "although the bactericidal action of these fluoroquinolones against e. coli is reduced , bactericidal action is not entirely eliminated by chloramphenicol or rifampin .", "this lack of elimination of bactericidal action suggests that these agents possess a secondary bactericidal mechanism of action that does not depend on the synthesis of protein and rna , and that may be active when the bacteria are in a nonreplicating state ( 46 ) . to understand this secondary bactericidal effect ,", "consisting of approximately 20 genes , this system repairs structural damage to dna caused by antibacterial agents , mainly through bypass repair ( 4650 ) .", "another effect of the sos response , activated by fluoroquinolone - induced damage to the bacterial dna , is the discontinuation of cell replication .", "in addition to high concentrations of fluoroquinolone , which trigger the secondary bactericidal mechanism , higher concentrations at dna targets also play a role in the emergence of resistance because the postantibiotic effect of the fluoroquinolones is dependent upon concentration , time , and the microorganism .", "if the concentration of fluoroquinolone attained at the bacterial dna targets is high enough to activate the sos system for a duration that exceeds the capability of the particular microorganism to repair its dna damage and replicate , the microorganism dies .", "if the fluoroquinolone concentration is not adequate , however , a race occurs between cumulative damage over time and the selection of a resistant mutant .", "the concentration of fluoroquinolone required for sos - mediated discontinuation of cell replication is expressed as a peak concentration / minimum inhibitory concentration ( mic ) ratio and appears to require a ratio of approximately 10:1 ( 50,51 ) .", "a rat model for pseudomonas sepsis demonstrated that peak concentration / mic ratios > 20:1 once per day produced significantly ( p<0.5 ) better survival  which may result in the selection of a mutant with altered topoisomerase  than did regimens using the same dosage on a more fractionated schedule ( 52 ) .", "dosages that led to peak concentration / mic ratios < 10 times the mic did not result in as high a survival rate .", "indeed , when the peak concentration / mic ratio was < 10 times the mic , the best survival was predicted by the area under the curve / mic ratio , since repeated exposure to the fluoroquinolone causes damage cumulatively .", "the length of time that fluoroquinolone levels in plasma exceeded the mic had no influence on survival .", "the emergence of fluoroquinolone resistance with respect to staphylococcus aureus and p. aeruginosa has been well - documented ( 53 ) . this major problem is due to a wide variety of fluoroquinolone - resistance mechanisms ( 54,55 ) , particularly the mutation of dna gyrase ( 56 ) .", "while this type of resistance generally results in mics only four- to eight - fold higher than the susceptible isolate , recent studies have reported the development of high - level resistance ( e.g. , ciprofloxacin mic for p. aeruginosa of 1,024 mg / l ) mediated by efflux pumps targeting multiple antibacterial agents ( 57,58 ) .", "these multidrug efflux pumps could be overcome by high fluoroquinolone concentrations , some of which , however , would not be clinically achievable .", "a rabbit meningitis model further demonstrates how the inability to achieve peak concentration / mic ratios > 10:1 influences the postantibiotic effect . in an in vivo study ,", "an exposure to ciprofloxacin at the mic had minimal impact ( 59 ) , underscoring the value of bactericidal activity with respect to fluoroquinolone therapy .", "the greater the activity of the fluoroquinolone , the more likely the agent will achieve serum or tissue levels that are > 10 times the mic , which in turn determines the secondary bactericidal and postantibiotic effects .", "consequently , newer fluoroquinolones such as gemifloxacin ( 60 ) and others now under development have markedly increased activity compared with traditional agents .", "for example , ciprofloxacin has mics against streptococcus pneumoniae of approximately 0.5 mg / l , while gemifloxacin has mics of approximately 0.03 mg / l .", "an important issue associated with the use of fluoroquinolones in the therapy of respiratory tract infections is the fact that fluoroquinolones also are used to treat other infections .", "the use of these agents for other infections means that the population already had been exposed to fluoroquinolones before their widespread use in respiratory tract infections .", "exposure of normal flora in these patients to subbactericidal concentrations of fluoroquinolones may allow resistant strains to emerge .", "cross - resistance is a well - recognized problem with fluoroquinolones ( 55 ) , and the enormous prior exposure of the population to these agents may have created resistant strains in the normal flora of the mucosal surfaces , skin , gastrointestinal tract , and reproductive tract . in addition , prior exposure may result in increasing mics due to subtle mutations of topoisomerases , which then may leave the microorganism only one step from a mutation that will produce overt resistance ( 55 ) .", "an example of such subtle topoisomerase mutation is seen with fluoroquinolones such as levofloxacin , which have been recommended and widely used for the therapy of pneumococcal pneumonia when penicillin resistance to s. pneumoniae is a problem ( 61 ) . unfortunately , the population has had considerable prior exposure to earlier fluoroquinolones , which has allowed rapid emergence of fluoroquinolone resistance in s. pneumoniae ( 62 ) .", "failure of treatment of pneumococcal pneumonia due to resistance to levofloxacin recently has been described ( 63 ) .", "this example confirms the problem of cross - resistance and further mutations resulting in increased resistance and suggests that newer fluoroquinolones such as gemifloxacin may be less effective or even ineffective against s. pneumoniae .", "other examples of the importance of bactericidal activity are the fluoroquinolones . studies of the bactericidal action of the quinolones against escherichia coli demonstrate at least two independent and important mechanisms of action .", "these bactericidal agents are only effective if the bacteria are actively dividing or synthesizing proteins and mrna .", "the bactericidal activity of the quinolone nalidixic acid , for example , is minimized by chloramphenicol , which prevents protein synthesis , and by rifampin , which prevents rna synthesis . however , ciprofloxacin and ofloxacin / levofloxacin respond differently .", "although the bactericidal action of these fluoroquinolones against e. coli is reduced , bactericidal action is not entirely eliminated by chloramphenicol or rifampin .", "this lack of elimination of bactericidal action suggests that these agents possess a secondary bactericidal mechanism of action that does not depend on the synthesis of protein and rna , and that may be active when the bacteria are in a nonreplicating state ( 46 ) . to understand this secondary bactericidal effect ,", "consisting of approximately 20 genes , this system repairs structural damage to dna caused by antibacterial agents , mainly through bypass repair ( 4650 ) .", "another effect of the sos response , activated by fluoroquinolone - induced damage to the bacterial dna , is the discontinuation of cell replication .", "in addition to high concentrations of fluoroquinolone , which trigger the secondary bactericidal mechanism , higher concentrations at dna targets also play a role in the emergence of resistance because the postantibiotic effect of the fluoroquinolones is dependent upon concentration , time , and the microorganism .", "if the concentration of fluoroquinolone attained at the bacterial dna targets is high enough to activate the sos system for a duration that exceeds the capability of the particular microorganism to repair its dna damage and replicate , the microorganism dies .", "if the fluoroquinolone concentration is not adequate , however , a race occurs between cumulative damage over time and the selection of a resistant mutant .", "the concentration of fluoroquinolone required for sos - mediated discontinuation of cell replication is expressed as a peak concentration / minimum inhibitory concentration ( mic ) ratio and appears to require a ratio of approximately 10:1 ( 50,51 ) .", "a rat model for pseudomonas sepsis demonstrated that peak concentration / mic ratios > 20:1 once per day produced significantly ( p<0.5 ) better survival  which may result in the selection of a mutant with altered topoisomerase  than did regimens using the same dosage on a more fractionated schedule ( 52 ) .", "dosages that led to peak concentration / mic ratios < 10 times the mic did not result in as high a survival rate .", "indeed , when the peak concentration / mic ratio was < 10 times the mic , the best survival was predicted by the area under the curve / mic ratio , since repeated exposure to the fluoroquinolone causes damage cumulatively .", "the length of time that fluoroquinolone levels in plasma exceeded the mic had no influence on survival .", "the emergence of fluoroquinolone resistance with respect to staphylococcus aureus and p. aeruginosa has been well - documented ( 53 ) . this major problem is due to a wide variety of fluoroquinolone - resistance mechanisms ( 54,55 ) , particularly the mutation of dna gyrase ( 56 ) .", "while this type of resistance generally results in mics only four- to eight - fold higher than the susceptible isolate , recent studies have reported the development of high - level resistance ( e.g. , ciprofloxacin mic for p. aeruginosa of 1,024 mg / l ) mediated by efflux pumps targeting multiple antibacterial agents ( 57,58 ) .", "these multidrug efflux pumps could be overcome by high fluoroquinolone concentrations , some of which , however , would not be clinically achievable .", "a rabbit meningitis model further demonstrates how the inability to achieve peak concentration / mic ratios > 10:1 influences the postantibiotic effect . in an in vivo study ,", "an exposure to ciprofloxacin at the mic had minimal impact ( 59 ) , underscoring the value of bactericidal activity with respect to fluoroquinolone therapy .", "the greater the activity of the fluoroquinolone , the more likely the agent will achieve serum or tissue levels that are > 10 times the mic , which in turn determines the secondary bactericidal and postantibiotic effects .", "consequently , newer fluoroquinolones such as gemifloxacin ( 60 ) and others now under development have markedly increased activity compared with traditional agents .", "for example , ciprofloxacin has mics against streptococcus pneumoniae of approximately 0.5 mg / l , while gemifloxacin has mics of approximately 0.03 mg / l .", "an important issue associated with the use of fluoroquinolones in the therapy of respiratory tract infections is the fact that fluoroquinolones also are used to treat other infections .", "the use of these agents for other infections means that the population already had been exposed to fluoroquinolones before their widespread use in respiratory tract infections .", "exposure of normal flora in these patients to subbactericidal concentrations of fluoroquinolones may allow resistant strains to emerge .", "cross - resistance is a well - recognized problem with fluoroquinolones ( 55 ) , and the enormous prior exposure of the population to these agents may have created resistant strains in the normal flora of the mucosal surfaces , skin , gastrointestinal tract , and reproductive tract . in addition , prior exposure may result in increasing mics due to subtle mutations of topoisomerases , which then may leave the microorganism only one step from a mutation that will produce overt resistance ( 55 ) .", "an example of such subtle topoisomerase mutation is seen with fluoroquinolones such as levofloxacin , which have been recommended and widely used for the therapy of pneumococcal pneumonia when penicillin resistance to s. pneumoniae is a problem ( 61 ) . unfortunately , the population has had considerable prior exposure to earlier fluoroquinolones , which has allowed rapid emergence of fluoroquinolone resistance in s. pneumoniae ( 62 ) .", "failure of treatment of pneumococcal pneumonia due to resistance to levofloxacin recently has been described ( 63 ) .", "this example confirms the problem of cross - resistance and further mutations resulting in increased resistance and suggests that newer fluoroquinolones such as gemifloxacin may be less effective or even ineffective against s. pneumoniae .", "the term macrolide was originally applied to specific compounds produced by various streptomyces species containing , as part of their structure , a macrocyclic lactone to which various deoxy sugars are attached .", "these bacteriostatic compounds bind to bacterial 50s ribosomes , inhibiting protein synthesis without a concomitant inhibition of nucleic acid synthesis .", "the classification has since been modified to include other structurally diverse agents , such as lincosamides and streptogramins , which are also produced by streptomyces species and target the 50s ribosome .", "the term mls ( macrolide , lincosamide , and streptogramin ) has become the accepted nomenclature for this important group of antibacterial agents  except when the emphasis is on structural similarity , in which case the erythromycin congeners ( erythromycin - a , clarithromycin , and azithromycin ) are often referred to as the  true macrolides  ( 64 ) .", "although antibacterial agents in the mls class have been largely bacteriostatic , newer members demonstrate bactericidal activity .", "these new agents include quinupristin / dalfopristin and telithromycin . the bactericidal mechanism of action of quinupristin / dalfopristin , a combination of two streptogramins , is unique ( 65 ) .", "dalfopristin is an olefinic macrolactone that binds to the 50s subunit of the prokaryotic ribosome and interferes with the function of peptidyl transferase , thereby inactivating the donor and acceptor sites of the ribosome .", "in addition , dalfopristin triggers a conformational change in the ribosome that greatly increases the affinity of quinupristin , a peptidic macrolactone , which also binds to the 50s subunit of the ribosome and halts peptide chain elongation .", "consequently , protein synthesis is not only halted transiently by either component used alone but also halted permanently by the two components in combination , resulting in synergistic and concentration - independent bactericidal activity against many pathogens .", "this binding of both macrolactones distinguishes quinupristin / dalfopristin from other antibiotic classes ( 66,67 ) , with the attendant prolongation of the postantibiotic effect ( 68 ) representing a distinct advantage over older agents ( 69 ) .", "quinupristin / dalfopristin is bactericidal against staphylococci and streptococci such as s. pneumoniae , generally bacteriostatic against enterococcus faecium , and inactive against e. faecalis ( 70 ) . because quinupristin / dalfopristin is available only in an intravenous formulation , its utility for treating respiratory tract infections is limited to hospitalized patients", "moreover , clinical data on quinupristin / dalfopristin therapy of pneumococcal pneumonia caused by macrolide resistant strains of s. pneumoniae is lacking .", "telithromycin , the first of a new class of antibacterials , the ketolides , is approved for use in europe and is currently being reviewed by the u. s. food and drug administration ( 71 ) .", "the clinical use of telithromycin in germany , as well as safety data presented to the food and drug administration , suggests that the toxicity and adverse reactions are similar to those of clarithromycin .", "this similarity is not surprising , as the ketolides are novel semisynthetic erythromycin - a derivatives structurally similar to clarithromycin .", "the c6-hydroxyl of erythromycin - a has been replaced by a methoxy group , as in clarithromycin , improving acid stability .", "the main structural innovation is the lack of the neutral sugar , cladinose , in position c3 .", "the 3-l - cladinose sugar moiety is removed , and the resulting 3-hydroxy group is oxidized to a 3-keto group , which is responsible for preventing induction of macrolide resistance ( 72 ) .", "telithromycin is produced through substitution at positions 11 and 12 of the erythronolide a ring with a butyl imidazolyl pyridinyl side chain .", "the resulting c11,c12 carbamate extension facilitates a distinctly different and more effective interaction with domain ii of the 23s rrna than occurs with erythromycin - a or clarithromycin ( 73 ) and is responsible for increased activity against erythromycin - a  resistant gram - positive cocci , such as s. pneumoniae , which develop resistance due to increased efflux ( 74,75 ) .", "moreover , the bactericidal action is effective against a number of other key respiratory pathogens , such as h. influenzae and c. pneumoniae ( 7678 ) , other gram - negative bacilli , such as m. catarrhalis and bordetella pertussis , and the intracellular pathogen legionella pneumophila ( 79 ) .", "unlike erythromycin - a ( which interacts with bacterial 23s ribosomal rna by contacts limited to hairpin 35 in domain ii of the ribosomal rna and to the peptidyl transferase loop in domain v ) , telithromycin is not a true macrolide because the l - cladinose moiety at position c3 has been replaced by a keto group and by alkylaryl side chains at positions c11,c12 .", "although both erythromycin - a and telithromycin bind to the peptidyl transferase loop ( the site of methylation by resistant bacteria ) , telithromycin binds much more avidly to hairpin 35 than erythromycin - a .", "in fact , telithromycin interacts strongly with two domains of the bacterial 23s rrna ( domains ii and v ) , which fold together in the tertiary 23s rrna to form a single drug - binding pocket ( 80,81 ) .", "the lack of the l - cladinose moiety , as well as the enhanced binding at domain ii and v , may explain why ketolides are associated with a low potential for inducing resistance ( 82,83 ) and contributes to telithromycin s sustained activity against mlsb - resistant strains ( in particular those with domain v modifications ) ( 73 ) .", "these features , in addition to the mic and the amount of drug delivered to the infection site , are considered strong predictors of a positive outcome ( 29,30 ) . however , the population has had enormous exposure to earlier macrolides .", "this influence means that resistance due to efflux or methylation of the 23s ribosome ( domain v ) may have already occurred in a large number of pneumococcal isolates .", "macrolide - resistant strains of s. pneumoniae to date have had a low incidence of cross - resistance to telithromycin ( 82,83 ) . however , increased efflux or other mutations might result in resistance to ketolides . to date", ", only two ketolide - resistant strains of s. pneumoniae have been identified ( 84 ) in over 10,000 pneumococcal isolates screened by the protekt study ( 10 ) .", "clearly , careful monitoring for ketolide resistance by surveillance studies such as the protekt study will need to be continued .", "meeting the challenge presented by the increasing numbers of bacterial pathogens resistant to common antibiotic treatments will require new types of antibacterial agents .", "therapies that maximize bactericidal effects are important because they reduce the development of bacterial resistance mechanisms .", "therefore , the use of bactericidal agents such as telithromycin for therapy of respiratory tract infections may well ensure that the antibacterial era endures long into the 21st century .", "however , careful monitoring of resistance will be needed to ensure that this agent remains active against common pathogens .", "stratton is associate professor of pathology and medicine and director of the clinical microbiology laboratory at the vanderbilt university medical center in nashville , tennessee .", "his research interests include the mechanisms of antimicrobial activity , antimicrobial resistance , and the pathogenesis of chlamydia pneumoniae ."], "abstract_text": ["<S> the global emergence of antibacterial resistance among common and atypical respiratory pathogens in the last decade necessitates the strategic application of antibacterial agents . </S>", "<S> the use of bactericidal rather than bacteriostatic agents as first - line therapy is recommended because the eradication of microorganisms serves to curtail , although not avoid , the development of bacterial resistance . </S>", "<S> bactericidal activity is achieved with specific classes of antimicrobial agents as well as by combination therapy . </S>", "<S> newer classes of antibacterial agents , such as the fluoroquinolones and certain members of the macrolide / lincosamine / streptogramin class have increased bactericidal activity compared with traditional agents . </S>", "<S> more recently , the ketolides ( novel , semisynthetic , erythromycin - a derivatives ) have demonstrated potent bactericidal activity against key respiratory pathogens , including streptococcus pneumoniae , haemophilus influenzae , chlamydia pneumoniae , and moraxella catarrhalis . </S>", "<S> moreover , the ketolides are associated with a low potential for inducing resistance , making them promising first - line agents for respiratory tract infections . </S>"], "labels": null, "section_names": ["Clinical Relevance of Bacteriostatic versus Bactericidal Activity", "Antibacterial Resistance Mechanisms: Bactericidal versus Bacteriostatic Activity", "Bactericidal Activity Achieved by Combination Therapy", "Bactericidal Activity Achieved with Novel Bactericidal Agents", "Fluoroquinolones", "Macrolides, Lincosamides, and Streptogramins (MLS)", "Ketolides: Telithromycin", "Conclusion"], "sections": [["all of the effects of antimicrobial agents against microbes , including the delineation of microbial resistance , are based upon the results of in vitro susceptibility testing .", "most of these susceptibility tests only measure bacteriostatic activity even though the agent being tested may have bactericidal activity .", "these authors point out the paucity of studies that have critically evaluated the effectiveness of antimicrobial therapy with results of in vitro susceptibility tests .", "such critical evaluations are not easily done , as susceptibility tests do not take into account the normal host defense mechanisms .", "however , the detection of resistance is somewhat predictive of poor outcome , although in the normal host this may be less clinically important due to the interaction of host defenses ( 17,18 ) .", "the ability of bactericidal activity to influence therapeutic efficacy and clinical outcome has been evaluated in infections that typically are refractory to antimicrobial therapy .", "these infections include endocarditis , meningitis , osteomyelitis , and infections in the neutropenic host .", "all are similar in that antimicrobial penetration and host defense mechanisms at the site of infection are limited .", "both experimental models of infection ( 1922 ) and clinical studies ( 2327 ) have shown that bactericidal activity predicts therapeutic efficacy and results in improved clinical outcome .", "bactericidal activity has been considered less important in respiratory tract infections , with the exception being acute infectious exacerbations in cystic fibrosis ( 28 ) .", "however , the relevance of pharmacokinetic and pharmacodynamics in the selection of antibiotics for respiratory tract infection has become increasingly recognized ( 29 ) .", "issues such as drug concentration at the site of infection , bactericidal activity , postantibiotic effect , and duration of therapy needed to achieve these effects are now being considered when antimicrobial agents are selected for the therapy of respiratory tract infections ( 29,30 ) .", "although host factors may allow a bacteriostatic agent to be used successfully in an infected patient , these factors appear to be less able to curtail the emergence of resistance .", "resistance , as a rule , occurs more rapidly with bacteriostatic agents such as tetracyclines , sulfonamides , and macrolides than it does with bactericidal agents such as beta - lactams and aminoglycosides ( 3133 ) .", "beta - lactam agents have been the antimicrobial agents of choice for the therapy of pneumococcal pneumonia since penicillin was first clinically introduced in the 1940s ( 17 ) .", "the emergence of macrolide resistance in s. pneumoniae has been rapid in comparison and has even been described during treatment of pneumococcal pneumonia ( 34 ) .", "bactericidal activity thus may be useful in the therapy of respiratory tract infections as a means to curtail , but not avoid , the emergence of resistance ."], ["the key to resolving the problem of antibacterial resistance lies in identifying the mechanisms that engender it ( 3133 ) . among the most important mechanisms are decreased ability of antibacterials to penetrate the bacterial cell wall , active efflux of antibacterial agents , inactivation of antibacterial agents , destruction of antibacterial agents , alteration of antibacterial target sites , development of bypass pathways around antibacterial targets , and constitutive phenotypic variation in bacterial physiology .", "subsequent exposure of the microorganism to a specific agent may then select the mutant , leading to the emergence of resistance . some resistance mechanisms , such as bacterial production of beta - lactamase , are inducible or can be derepressed ( 35 ) , requiring either upregulation or mutation of genetic material .", "thus , if resistance is to be suppressed , the opportunity for bacterial upregulation or mutation of genetic material must be minimized .", "one way to minimize upregulation , mutation , or both is by using bactericidal rather than merely bacteriostatic agents .", "microorganisms inhibited by a bacteriostatic agent or exposed to an insufficient concentration of a bactericidal agent remain alive and , ipso facto , retain the potential to become resistant or promulgate any resistance selected by the exposure to the antimicrobial agent .", "an example of this principle can be seen with the upper respiratory tract pathogen , streptococcus pyogenes , which to date has not developed resistance to penicillin , a bactericidal agent , but has developed resistance to erythromycin , a bacteriostatic agent ( 3638 ) .", "erythromycin resistance in s. pyogenes largely is due to upregulation of efflux ( 36 ) or to ribosomal mutation ( 37 ) .", "antimicrobial agents that kill this pathogen should be less likely to promulgate any strains having such resistance . another example is seen with the omp genes of gram - negative microorganisms .", "these omp genes encode porins that are sometimes flanked by insertion sequences . in the presence of the bacteriostatic agent , the mobility of insertion sequence  flanking omp genes can be attenuated and will result in disruption of the omp genes .", "the reduced expression of these porins may lead to reduced uptake of the inducer , the antibacterial agent .", "specifically , insertion sequence interruption of the ompk36 porin gene in respiratory tract pathogen klebsiella pneumoniae has been shown to interfere in the expression of this porin gene and has resulted in clinical failure ( 39 ) .", "if a bactericidal agent kills a pathogen such as klebsiella before mutation of the porin gene , resistance is less likely to develop .", "these two examples illustrate the desirability of achieving bactericidal activity to curtail the emergence of resistance ."], ["bactericidal activity can be achieved through the mechanism of action for a single antimicrobial agent or by the use of combination therapy , or both .", "sulfamethoxazole / trimethoprim ( smx - tmp ) is an example of a combination of two agents , each of which alone is bacteriostatic , that achieves bactericidal activity .", "sulfamethoxazole inhibits dihydropteroate synthase , the bacterial enzyme that catalyzes the incorporation of p - aminobenzoid acid into dihydropteroic acid , the immediate precursor of folic acid , while trimethoprim was specifically synthesized as an inhibitor of dihydrofolate reductase ( 40 ) .", "smx - tmp has long been used for the therapy of respiratory tract infection ( 41 ) and has proven particularly useful in the treatment of acute exacerbations of chronic bronchitis .", "in fact , the world health organization continues to be recommend smx - tmp as the first - line treatment for pneumonia in children because of its low cost and ease of dosing .", "resistance to smx - tmp has emerged more slowly than for either agent used alone ( 42 ) .", "however , emergence of resistance to s. pneumoniae has occurred ( 43 ) and now may limit the use of smx - tmp in respiratory tract infections .", "combinations of antimicrobial agents are also used in the therapy of bacterial endocarditis to achieve synergism leading to increased bactericidal activity and improved sterilization of infected valves .", "bactericidal synergy for s. epidermidis can be demonstrated in vitro for the combination of vancomycin , rifampin , and gentamicin , which correlates well with the therapeutic results in an experimental animal model ( 44 ) . in a comparable clinical study of patients with prosthetic valve endocarditis caused by staphylococcus epidermidis ,", "90% were cured with a combination of vancomycin , rifampin , and/or gentamicin , compared with only 50% cured among those receiving vancomycin alone ( 45 ) .", "combination therapy for respiratory tract infections is less well studied except for acute respiratory tract infections occurring in cystic fibrosis patients .", "for example , combination therapy for treatment of pseudomonas aeruginosa pulmonary infections in cystic fibrosis patients achieved a cure rate of 89% if peak serum bactericidal titers were > 1:128 ( 28 ) .", "in contrast , 100% of patients failed therapy if their peak serum bactericidal titers were < 1:16 ."], ["other examples of the importance of bactericidal activity are the fluoroquinolones . studies of the bactericidal action of the quinolones against escherichia coli demonstrate at least two independent and important mechanisms of action .", "these bactericidal agents are only effective if the bacteria are actively dividing or synthesizing proteins and mrna .", "the bactericidal activity of the quinolone nalidixic acid , for example , is minimized by chloramphenicol , which prevents protein synthesis , and by rifampin , which prevents rna synthesis . however , ciprofloxacin and ofloxacin / levofloxacin respond differently .", "although the bactericidal action of these fluoroquinolones against e. coli is reduced , bactericidal action is not entirely eliminated by chloramphenicol or rifampin .", "this lack of elimination of bactericidal action suggests that these agents possess a secondary bactericidal mechanism of action that does not depend on the synthesis of protein and rna , and that may be active when the bacteria are in a nonreplicating state ( 46 ) . to understand this secondary bactericidal effect ,", "consisting of approximately 20 genes , this system repairs structural damage to dna caused by antibacterial agents , mainly through bypass repair ( 4650 ) .", "another effect of the sos response , activated by fluoroquinolone - induced damage to the bacterial dna , is the discontinuation of cell replication .", "in addition to high concentrations of fluoroquinolone , which trigger the secondary bactericidal mechanism , higher concentrations at dna targets also play a role in the emergence of resistance because the postantibiotic effect of the fluoroquinolones is dependent upon concentration , time , and the microorganism .", "if the concentration of fluoroquinolone attained at the bacterial dna targets is high enough to activate the sos system for a duration that exceeds the capability of the particular microorganism to repair its dna damage and replicate , the microorganism dies .", "if the fluoroquinolone concentration is not adequate , however , a race occurs between cumulative damage over time and the selection of a resistant mutant .", "the concentration of fluoroquinolone required for sos - mediated discontinuation of cell replication is expressed as a peak concentration / minimum inhibitory concentration ( mic ) ratio and appears to require a ratio of approximately 10:1 ( 50,51 ) .", "a rat model for pseudomonas sepsis demonstrated that peak concentration / mic ratios > 20:1 once per day produced significantly ( p<0.5 ) better survival  which may result in the selection of a mutant with altered topoisomerase  than did regimens using the same dosage on a more fractionated schedule ( 52 ) .", "dosages that led to peak concentration / mic ratios < 10 times the mic did not result in as high a survival rate .", "indeed , when the peak concentration / mic ratio was < 10 times the mic , the best survival was predicted by the area under the curve / mic ratio , since repeated exposure to the fluoroquinolone causes damage cumulatively .", "the length of time that fluoroquinolone levels in plasma exceeded the mic had no influence on survival .", "the emergence of fluoroquinolone resistance with respect to staphylococcus aureus and p. aeruginosa has been well - documented ( 53 ) . this major problem is due to a wide variety of fluoroquinolone - resistance mechanisms ( 54,55 ) , particularly the mutation of dna gyrase ( 56 ) .", "while this type of resistance generally results in mics only four- to eight - fold higher than the susceptible isolate , recent studies have reported the development of high - level resistance ( e.g. , ciprofloxacin mic for p. aeruginosa of 1,024 mg / l ) mediated by efflux pumps targeting multiple antibacterial agents ( 57,58 ) .", "these multidrug efflux pumps could be overcome by high fluoroquinolone concentrations , some of which , however , would not be clinically achievable .", "a rabbit meningitis model further demonstrates how the inability to achieve peak concentration / mic ratios > 10:1 influences the postantibiotic effect . in an in vivo study ,", "an exposure to ciprofloxacin at the mic had minimal impact ( 59 ) , underscoring the value of bactericidal activity with respect to fluoroquinolone therapy .", "the greater the activity of the fluoroquinolone , the more likely the agent will achieve serum or tissue levels that are > 10 times the mic , which in turn determines the secondary bactericidal and postantibiotic effects .", "consequently , newer fluoroquinolones such as gemifloxacin ( 60 ) and others now under development have markedly increased activity compared with traditional agents .", "for example , ciprofloxacin has mics against streptococcus pneumoniae of approximately 0.5 mg / l , while gemifloxacin has mics of approximately 0.03 mg / l .", "an important issue associated with the use of fluoroquinolones in the therapy of respiratory tract infections is the fact that fluoroquinolones also are used to treat other infections .", "the use of these agents for other infections means that the population already had been exposed to fluoroquinolones before their widespread use in respiratory tract infections .", "exposure of normal flora in these patients to subbactericidal concentrations of fluoroquinolones may allow resistant strains to emerge .", "cross - resistance is a well - recognized problem with fluoroquinolones ( 55 ) , and the enormous prior exposure of the population to these agents may have created resistant strains in the normal flora of the mucosal surfaces , skin , gastrointestinal tract , and reproductive tract . in addition , prior exposure may result in increasing mics due to subtle mutations of topoisomerases , which then may leave the microorganism only one step from a mutation that will produce overt resistance ( 55 ) .", "an example of such subtle topoisomerase mutation is seen with fluoroquinolones such as levofloxacin , which have been recommended and widely used for the therapy of pneumococcal pneumonia when penicillin resistance to s. pneumoniae is a problem ( 61 ) . unfortunately , the population has had considerable prior exposure to earlier fluoroquinolones , which has allowed rapid emergence of fluoroquinolone resistance in s. pneumoniae ( 62 ) .", "failure of treatment of pneumococcal pneumonia due to resistance to levofloxacin recently has been described ( 63 ) .", "this example confirms the problem of cross - resistance and further mutations resulting in increased resistance and suggests that newer fluoroquinolones such as gemifloxacin may be less effective or even ineffective against s. pneumoniae ."], ["other examples of the importance of bactericidal activity are the fluoroquinolones . studies of the bactericidal action of the quinolones against escherichia coli demonstrate at least two independent and important mechanisms of action .", "these bactericidal agents are only effective if the bacteria are actively dividing or synthesizing proteins and mrna .", "the bactericidal activity of the quinolone nalidixic acid , for example , is minimized by chloramphenicol , which prevents protein synthesis , and by rifampin , which prevents rna synthesis . however , ciprofloxacin and ofloxacin / levofloxacin respond differently .", "although the bactericidal action of these fluoroquinolones against e. coli is reduced , bactericidal action is not entirely eliminated by chloramphenicol or rifampin .", "this lack of elimination of bactericidal action suggests that these agents possess a secondary bactericidal mechanism of action that does not depend on the synthesis of protein and rna , and that may be active when the bacteria are in a nonreplicating state ( 46 ) . to understand this secondary bactericidal effect ,", "consisting of approximately 20 genes , this system repairs structural damage to dna caused by antibacterial agents , mainly through bypass repair ( 4650 ) .", "another effect of the sos response , activated by fluoroquinolone - induced damage to the bacterial dna , is the discontinuation of cell replication .", "in addition to high concentrations of fluoroquinolone , which trigger the secondary bactericidal mechanism , higher concentrations at dna targets also play a role in the emergence of resistance because the postantibiotic effect of the fluoroquinolones is dependent upon concentration , time , and the microorganism .", "if the concentration of fluoroquinolone attained at the bacterial dna targets is high enough to activate the sos system for a duration that exceeds the capability of the particular microorganism to repair its dna damage and replicate , the microorganism dies .", "if the fluoroquinolone concentration is not adequate , however , a race occurs between cumulative damage over time and the selection of a resistant mutant .", "the concentration of fluoroquinolone required for sos - mediated discontinuation of cell replication is expressed as a peak concentration / minimum inhibitory concentration ( mic ) ratio and appears to require a ratio of approximately 10:1 ( 50,51 ) .", "a rat model for pseudomonas sepsis demonstrated that peak concentration / mic ratios > 20:1 once per day produced significantly ( p<0.5 ) better survival  which may result in the selection of a mutant with altered topoisomerase  than did regimens using the same dosage on a more fractionated schedule ( 52 ) .", "dosages that led to peak concentration / mic ratios < 10 times the mic did not result in as high a survival rate .", "indeed , when the peak concentration / mic ratio was < 10 times the mic , the best survival was predicted by the area under the curve / mic ratio , since repeated exposure to the fluoroquinolone causes damage cumulatively .", "the length of time that fluoroquinolone levels in plasma exceeded the mic had no influence on survival .", "the emergence of fluoroquinolone resistance with respect to staphylococcus aureus and p. aeruginosa has been well - documented ( 53 ) . this major problem is due to a wide variety of fluoroquinolone - resistance mechanisms ( 54,55 ) , particularly the mutation of dna gyrase ( 56 ) .", "while this type of resistance generally results in mics only four- to eight - fold higher than the susceptible isolate , recent studies have reported the development of high - level resistance ( e.g. , ciprofloxacin mic for p. aeruginosa of 1,024 mg / l ) mediated by efflux pumps targeting multiple antibacterial agents ( 57,58 ) .", "these multidrug efflux pumps could be overcome by high fluoroquinolone concentrations , some of which , however , would not be clinically achievable .", "a rabbit meningitis model further demonstrates how the inability to achieve peak concentration / mic ratios > 10:1 influences the postantibiotic effect . in an in vivo study ,", "an exposure to ciprofloxacin at the mic had minimal impact ( 59 ) , underscoring the value of bactericidal activity with respect to fluoroquinolone therapy .", "the greater the activity of the fluoroquinolone , the more likely the agent will achieve serum or tissue levels that are > 10 times the mic , which in turn determines the secondary bactericidal and postantibiotic effects .", "consequently , newer fluoroquinolones such as gemifloxacin ( 60 ) and others now under development have markedly increased activity compared with traditional agents .", "for example , ciprofloxacin has mics against streptococcus pneumoniae of approximately 0.5 mg / l , while gemifloxacin has mics of approximately 0.03 mg / l .", "an important issue associated with the use of fluoroquinolones in the therapy of respiratory tract infections is the fact that fluoroquinolones also are used to treat other infections .", "the use of these agents for other infections means that the population already had been exposed to fluoroquinolones before their widespread use in respiratory tract infections .", "exposure of normal flora in these patients to subbactericidal concentrations of fluoroquinolones may allow resistant strains to emerge .", "cross - resistance is a well - recognized problem with fluoroquinolones ( 55 ) , and the enormous prior exposure of the population to these agents may have created resistant strains in the normal flora of the mucosal surfaces , skin , gastrointestinal tract , and reproductive tract . in addition , prior exposure may result in increasing mics due to subtle mutations of topoisomerases , which then may leave the microorganism only one step from a mutation that will produce overt resistance ( 55 ) .", "an example of such subtle topoisomerase mutation is seen with fluoroquinolones such as levofloxacin , which have been recommended and widely used for the therapy of pneumococcal pneumonia when penicillin resistance to s. pneumoniae is a problem ( 61 ) . unfortunately , the population has had considerable prior exposure to earlier fluoroquinolones , which has allowed rapid emergence of fluoroquinolone resistance in s. pneumoniae ( 62 ) .", "failure of treatment of pneumococcal pneumonia due to resistance to levofloxacin recently has been described ( 63 ) .", "this example confirms the problem of cross - resistance and further mutations resulting in increased resistance and suggests that newer fluoroquinolones such as gemifloxacin may be less effective or even ineffective against s. pneumoniae ."], ["the term macrolide was originally applied to specific compounds produced by various streptomyces species containing , as part of their structure , a macrocyclic lactone to which various deoxy sugars are attached .", "these bacteriostatic compounds bind to bacterial 50s ribosomes , inhibiting protein synthesis without a concomitant inhibition of nucleic acid synthesis .", "the classification has since been modified to include other structurally diverse agents , such as lincosamides and streptogramins , which are also produced by streptomyces species and target the 50s ribosome .", "the term mls ( macrolide , lincosamide , and streptogramin ) has become the accepted nomenclature for this important group of antibacterial agents  except when the emphasis is on structural similarity , in which case the erythromycin congeners ( erythromycin - a , clarithromycin , and azithromycin ) are often referred to as the  true macrolides  ( 64 ) .", "although antibacterial agents in the mls class have been largely bacteriostatic , newer members demonstrate bactericidal activity .", "these new agents include quinupristin / dalfopristin and telithromycin . the bactericidal mechanism of action of quinupristin / dalfopristin , a combination of two streptogramins , is unique ( 65 ) .", "dalfopristin is an olefinic macrolactone that binds to the 50s subunit of the prokaryotic ribosome and interferes with the function of peptidyl transferase , thereby inactivating the donor and acceptor sites of the ribosome .", "in addition , dalfopristin triggers a conformational change in the ribosome that greatly increases the affinity of quinupristin , a peptidic macrolactone , which also binds to the 50s subunit of the ribosome and halts peptide chain elongation .", "consequently , protein synthesis is not only halted transiently by either component used alone but also halted permanently by the two components in combination , resulting in synergistic and concentration - independent bactericidal activity against many pathogens .", "this binding of both macrolactones distinguishes quinupristin / dalfopristin from other antibiotic classes ( 66,67 ) , with the attendant prolongation of the postantibiotic effect ( 68 ) representing a distinct advantage over older agents ( 69 ) .", "quinupristin / dalfopristin is bactericidal against staphylococci and streptococci such as s. pneumoniae , generally bacteriostatic against enterococcus faecium , and inactive against e. faecalis ( 70 ) . because quinupristin / dalfopristin is available only in an intravenous formulation , its utility for treating respiratory tract infections is limited to hospitalized patients", "moreover , clinical data on quinupristin / dalfopristin therapy of pneumococcal pneumonia caused by macrolide resistant strains of s. pneumoniae is lacking ."], ["telithromycin , the first of a new class of antibacterials , the ketolides , is approved for use in europe and is currently being reviewed by the u. s. food and drug administration ( 71 ) .", "the clinical use of telithromycin in germany , as well as safety data presented to the food and drug administration , suggests that the toxicity and adverse reactions are similar to those of clarithromycin .", "this similarity is not surprising , as the ketolides are novel semisynthetic erythromycin - a derivatives structurally similar to clarithromycin .", "the c6-hydroxyl of erythromycin - a has been replaced by a methoxy group , as in clarithromycin , improving acid stability .", "the main structural innovation is the lack of the neutral sugar , cladinose , in position c3 .", "the 3-l - cladinose sugar moiety is removed , and the resulting 3-hydroxy group is oxidized to a 3-keto group , which is responsible for preventing induction of macrolide resistance ( 72 ) .", "telithromycin is produced through substitution at positions 11 and 12 of the erythronolide a ring with a butyl imidazolyl pyridinyl side chain .", "the resulting c11,c12 carbamate extension facilitates a distinctly different and more effective interaction with domain ii of the 23s rrna than occurs with erythromycin - a or clarithromycin ( 73 ) and is responsible for increased activity against erythromycin - a  resistant gram - positive cocci , such as s. pneumoniae , which develop resistance due to increased efflux ( 74,75 ) .", "moreover , the bactericidal action is effective against a number of other key respiratory pathogens , such as h. influenzae and c. pneumoniae ( 7678 ) , other gram - negative bacilli , such as m. catarrhalis and bordetella pertussis , and the intracellular pathogen legionella pneumophila ( 79 ) .", "unlike erythromycin - a ( which interacts with bacterial 23s ribosomal rna by contacts limited to hairpin 35 in domain ii of the ribosomal rna and to the peptidyl transferase loop in domain v ) , telithromycin is not a true macrolide because the l - cladinose moiety at position c3 has been replaced by a keto group and by alkylaryl side chains at positions c11,c12 .", "although both erythromycin - a and telithromycin bind to the peptidyl transferase loop ( the site of methylation by resistant bacteria ) , telithromycin binds much more avidly to hairpin 35 than erythromycin - a .", "in fact , telithromycin interacts strongly with two domains of the bacterial 23s rrna ( domains ii and v ) , which fold together in the tertiary 23s rrna to form a single drug - binding pocket ( 80,81 ) .", "the lack of the l - cladinose moiety , as well as the enhanced binding at domain ii and v , may explain why ketolides are associated with a low potential for inducing resistance ( 82,83 ) and contributes to telithromycin s sustained activity against mlsb - resistant strains ( in particular those with domain v modifications ) ( 73 ) .", "these features , in addition to the mic and the amount of drug delivered to the infection site , are considered strong predictors of a positive outcome ( 29,30 ) . however , the population has had enormous exposure to earlier macrolides .", "this influence means that resistance due to efflux or methylation of the 23s ribosome ( domain v ) may have already occurred in a large number of pneumococcal isolates .", "macrolide - resistant strains of s. pneumoniae to date have had a low incidence of cross - resistance to telithromycin ( 82,83 ) . however , increased efflux or other mutations might result in resistance to ketolides . to date", ", only two ketolide - resistant strains of s. pneumoniae have been identified ( 84 ) in over 10,000 pneumococcal isolates screened by the protekt study ( 10 ) .", "clearly , careful monitoring for ketolide resistance by surveillance studies such as the protekt study will need to be continued ."], ["meeting the challenge presented by the increasing numbers of bacterial pathogens resistant to common antibiotic treatments will require new types of antibacterial agents .", "therapies that maximize bactericidal effects are important because they reduce the development of bacterial resistance mechanisms .", "therefore , the use of bactericidal agents such as telithromycin for therapy of respiratory tract infections may well ensure that the antibacterial era endures long into the 21st century .", "however , careful monitoring of resistance will be needed to ensure that this agent remains active against common pathogens .", "stratton is associate professor of pathology and medicine and director of the clinical microbiology laboratory at the vanderbilt university medical center in nashville , tennessee .", "his research interests include the mechanisms of antimicrobial activity , antimicrobial resistance , and the pathogenesis of chlamydia pneumoniae ."]]}
{"article_id": "PMC3873009", "article_text": ["sponges play an important role in the benthic - pelagic coupling on coral reefs ( lesser 2006 ) .", "the sponge community sequesters and processes organic matter and as such conserve energy and nutrients for the reef ecosystem .", "it is generally assumed that sponges on the fore - reef slope mainly trap allochthonous organic matter that may compensate the losses of material from reef ecosystems ( e.g. , richter et al .", "most reef sponges are extremely efficient in filter feeding ( e.g. , reiswig 1974 ; lesser 2006 ) .", "they preferably filter the picoplankton fraction from the flowing water ( e.g. , pile 1999 ; ribes et al . 2003 , 2005 ) .", "the major food resource of coral reef sponges , however , may well be dissolved organic matter ( reiswig 1990 ; yahel et al .", "2003 ) in coral cavities are fueled mainly by dissolved organic matter ( dom ) ( de goeij et al .", "encrusting sponges cover together with coralline algae > 50% of the walls in coral cavities , caves , and undersides of corals ( vasseur 1974 ; richter and wunsch 1999 ; wunsch et al .", "this cryptic surface of 3-dimensionally well - developed reefs may exceed the open reef surface ( ginsburg 1983 ; richter et al .", "the sheer size of this reef habitat already preludes the grand role of its biota in organic matter cycling .", "dom removal by the cryptic biota on coral reefs can be huge ( up to 500  mmol  c  m cryptic surface d ) , with cavity sponges accounting for 70% of the consumed dom in coral reef cavities ( de goeij and van duyl 2007 ;", "these recent findings draw direct attention to the origin and sources of dom on coral reefs .", "phytoplankton ( including photoautotrophic bacteria ) usually is the main source of reactive dom in oligotrophic ocean water ; it releases dom as photosynthetic product or through food web processes and lysis ( carlson 2002 ) . on coral reefs", "extra sources of reactive dom include release of dom by corals through mucus production ( johannes 1967 ; richman et al .", "1975 ; crossland et al . 1980 ; naumann et al . 2010 ) and extracellular release of dom by benthic algae ( e.g. , wada et al . 2007 ;", "the main consumers of reactive dom are usually heterotrophic bacteria ( e.g. , harvey et al .", "the fate of mucus and dissolved organic matter in reef overlying waters has been ascribed to consumption and mineralization by bacteria in the reef ambient water and in sediments ( e.g. , gast et al .", "the first indication that reef - derived dom may also be assimilated by higher trophic levels came from the abundance of the dietary fatty acid biomarker 20:46 in the encrusting dom feeding cavity sponge halisarca caerulea ( de goeij et al .", "1991 ) and in coralline algae , rhodophyta ( viso and marty 1993 ) and its presence in sponges suggests that reef - derived organic matter may be an important food source for coral reef sponges .", "this study aims ( 1 ) to determine whether cavity sponges mainly rely on reef - produced or open water - derived dissolved organic matter and ( 2 ) to identify key sources of dom for cavity sponges . as putative food compounds different fractions of suspended matter", ", bacterioplankton , coral mucus , and coralline algae as proxies for dom were analyzed . through stable isotopes and fatty acid biomarkers , we characterized energy sources of cavity sponges collected from coral reefs and open water on the southwest coast of curaao , caribbean ( 1212n , 6856w ) .", "stable isotopes are a classical way to trace food sources of aquatic animals ( peterson and fry 1987 ) . the carbon and nitrogen stable isotope signatures of 12 sponge species were compared with those of putative food compounds .", "fatty acid biomarkers have been repeatedly used as source - specific indicators of dissolved and particulate organic matter both in environmental and in food web studies ( e.g. hall et al .", "the fa pattern of six sponge species and three types of reef food resources were analyzed , and the relative input of different sources to the diet of the sponges was determined .", "samples were collected from march 14 to 22 , 2006 , along four transects perpendicular to the sw coast of curaao ( fig .  1 ) .", "transects were placed 37  km apart from se to nw along the coast at lagun jan thiel ( ljt ) , at the ocean cruise terminal in otrabanda ( ct ) , at buoy 1 ( b1 ) of the carmabi reef close to piscaderabaai , and at slangenbaai ( sb ) .", "transects from the shore to open water cross the narrow fringing reefs and water masses representative for the variation in coral community composition and water quality along the sw coast of curaao ( van duyl 1985 ; gast et al .", "1999 ; van duyl and gast 2001 ) . along each transect ( ljt , ct , b1 , and sb ) , we sampled 2 stations : fig . ", "( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbeanan off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water anda coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "position of sampling transects ( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbean an off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water and a coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "water samples were taken with a 6  l niskin bottle operated by a scuba diver .", "the niskin bottle was repeatedly filled and hauled on board a small open boat and emptied in a clean and sample washed 20  l container . at each station", "( n  =  8) , we collected 20  l. on the fore - reef slope stations ( n  =  4 ) , we collected a few small pieces of the stony corals montastraea annularis and madracis mirabilis ( also siderastrea siderea and porites astreoides but only at b1 ) between 15 and 17  m depth , which were kept in seawater under natural light conditions until mucus collection on the next day . from the entrance of coral cavities at 1517  m depth on the fore - reef slope , we chiseled crusts of calcareous algae . from the same cavities ,", "various species of encrusting sponges were collected , up to eight different species per station .", "the 20-l water samples were filtered over 47-mm - diameter combusted glass microfiber filters ( whatman ) , first over a gfc filter ( nominal pore size 1.2  m ) and then over a gff filter ( nominal pore size 0.7  m ) .", "after sea water filtration , filters were shortly washed with milli - q water ( mq ) to remove salt and were dried at 50c in a stove and stored in aluminum foil in the freezer until processing .", "the remaining bacterioplankton in the gff filtrate was concentrated with a vivaspin filter cartridge driven by a master volt tube pump .", "the concentrate was subsequently filtered over a 0.2  m pore size 25-mm - diameter anopore disc ( aluminum oxide membrane filter , whatman ) .", "the discs were briefly washed with mq after seawater filtration and dried at 50c in a stove ( 12  h ) . after drying , the filter was crumbled in a clean acid - washed glass funnel , the integrated polypropylene support ring of the filter was removed and the filter fragments were transferred to combusted silver capsules , which were closed with tweezers .", "subsequently , the folded capsules were placed in coded trays and stored in the freezer until processing .", "live corals were air - exposed during mucus collection and positioned upside down connected to a stand .", "after shortly spraying the live coral tissue with mq , the mucus produced by the coral was collected via an acid - washed glass funnel in combusted glass vials .", "after collection of approximately 2  ml mucus per coral , mucus samples were dried in a stove at 50c before being stored dry in the freezer .", "the cavity sponges and coralline algae were quickly washed with mq to remove salt and the tissue was subsequently removed from the underlying substratum by knife and scalpel .", "small pieces were fixed in ethanol ( 80% ) for taxonomical identification of the different sponge species .", "the rest of the pieces were collected in combusted glass vials dried at 50c and stored dry in the freezer .", "sponges and putative food sources were subject to stable isotope analysis . both carbon and nitrogen ( except for bacteria for which only corg was measured ) isotopic composition of the samples were determined using a fisons cn analyser coupled on line via a finnigan conflo 2 interface , to a finnigan delta s mass spectrometer .", "the carbon and nitrogen isotope ratios are expressed as corg and n relative to the vienna pee dee belemnite standard and air , respectively , and the standard error of the measurements is ~0.15. all carbon isotope samples ( except bacteria that were caught on aluminum filters ) were acidified ( tissue directly with acid and glass filters in acid vapor ) prior to measurements and corrected for individual sets of blanks ( isotope mass balance corrections ) .", "seawater as source of carbonates leading to possible enrichment of corg in case of un - acidified aluminum bacteria filters was eliminated by the rinse with mq water at the end of filtration .", "the dried sponge , coralline algae , and mucus material were first homogenized in a mortar . for isotope measurements , adequate amount of material was transferred to pre - combusted silver boats for acidification within cups , followed by oven drying to remove excess acid . from the remaining grounded material , samples from the b1 reef station", "were also analyzed for their total fatty acid composition using a method of one - step procedure of fatty acid extraction and methylation ( masood et al .", "fatty acid extraction and preparation of methyl esters ( fame ) were carried out according to masood et al .", "( 2005 ) with reagent volumes adapted for use in 2.5-ml gc - vials using fame c19:0 as internal standard to calculate concentration of fas ( van gaever et al .", "these individual samples were analyzed separately for their fa compositions employing a large volume splitless injection method on a thermo finnigan trace ultra gc .", "main items of this method are the large volume liner with glass wool , pre - column deactivated silica 5  m    0.53  m , and analytical column sge bpx-70 50  m    0.32  mm    0.25  m .", "the identification of fames was based on the comparison of retention times with authentic commercially available reference material and standards .", "fatty acid source designation was achieved using data on distinctive fa for bacteria , algal carbon , and higher organisms ( e.g. , parrish et al . 2000 ; boschker and middelburg 2002 ; volkman 2006 ) .", "we ran one- and two - way anovas with non - transformed stable isotope data to determine whether variations in stable isotopes of sponges were due to sponge species and/or sponge location assuming no interaction effects ( not enough replication for testing interaction ) .", "the tukey s hsd method was used for pair wise comparisons of different sponge species ( multiple comparisons of means ) .", "anova s were also run for isotope signals of reef and open water - derived suspended matter .", "the siar stable isotope mixing model analysis ( parnell et al . 2010 ) was applied to investigate probable solutions for the diet of sponges based on dual stable isotope data of four sources ( gfc and gff fractions of suspended matter , crustose coralline algae , and coral mucus ) .", "we used the following trophic enrichment factors with standard deviations for the sponges : 3.5    0.5 for n and 1    1 for  c ( vander zanden and rasmussen 2001 ; behringer and butler iv 2006 ) .", "the siar model takes data on organism isotopes and fits a bayesian model to their dietary habits based upon a gaussian likelihood with a mixture dirichlet - distributed prior on the mean .", "model fitting is via markov chain monte carlo ( mcmc ) which produces simulations of plausible values for each dietary source consistent with the data .", "the software package siar ( stable isotope analysis in r ) is freely available from the package section of the comprehensive r archive network website  http://cran.r - project.org/ ( parnell and jackson 2010 ) .", "fatty acid composition patterns were explored with multivariate analyses of fas composition profiles ( non - transformed percentage abundance ) using the program primer version 5 ( clarke and gorley 2006 ) .", "multi - dimensional scaling ( mds plots ) of fa composition and dual isotope label patterns were compared for six sponge species and the three different reef - derived resources ( cca , mucus of m. annularis , and mucus of m. mirabilis ) .", "similarities between fa profiles were investigated using the simper ( similarity of percentages ) function , and statistical differences were determined using the anosim ( analysis of similarities ) function .", "samples were collected from march 14 to 22 , 2006 , along four transects perpendicular to the sw coast of curaao ( fig .  1 ) .", "transects were placed 37  km apart from se to nw along the coast at lagun jan thiel ( ljt ) , at the ocean cruise terminal in otrabanda ( ct ) , at buoy 1 ( b1 ) of the carmabi reef close to piscaderabaai , and at slangenbaai ( sb ) .", "transects from the shore to open water cross the narrow fringing reefs and water masses representative for the variation in coral community composition and water quality along the sw coast of curaao ( van duyl 1985 ; gast et al .", "1999 ; van duyl and gast 2001 ) . along each transect ( ljt , ct , b1 , and sb ) , we sampled 2 stations : fig . ", "( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbeanan off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water anda coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "position of sampling transects ( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbean an off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water and a coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "water samples were taken with a 6  l niskin bottle operated by a scuba diver .", "the niskin bottle was repeatedly filled and hauled on board a small open boat and emptied in a clean and sample washed 20  l container . at each station", "( n  =  8) , we collected 20  l. on the fore - reef slope stations ( n  =  4 ) , we collected a few small pieces of the stony corals montastraea annularis and madracis mirabilis ( also siderastrea siderea and porites astreoides but only at b1 ) between 15 and 17  m depth , which were kept in seawater under natural light conditions until mucus collection on the next day . from the entrance of coral cavities at 1517  m depth on the fore - reef slope , we chiseled crusts of calcareous algae . from the same cavities ,", "various species of encrusting sponges were collected , up to eight different species per station .", "the 20-l water samples were filtered over 47-mm - diameter combusted glass microfiber filters ( whatman ) , first over a gfc filter ( nominal pore size 1.2  m ) and then over a gff filter ( nominal pore size 0.7  m ) .", "after sea water filtration , filters were shortly washed with milli - q water ( mq ) to remove salt and were dried at 50c in a stove and stored in aluminum foil in the freezer until processing .", "the remaining bacterioplankton in the gff filtrate was concentrated with a vivaspin filter cartridge driven by a master volt tube pump .", "the concentrate was subsequently filtered over a 0.2  m pore size 25-mm - diameter anopore disc ( aluminum oxide membrane filter , whatman ) .", "the discs were briefly washed with mq after seawater filtration and dried at 50c in a stove ( 12  h ) . after drying , the filter was crumbled in a clean acid - washed glass funnel , the integrated polypropylene support ring of the filter was removed and the filter fragments were transferred to combusted silver capsules , which were closed with tweezers .", "subsequently , the folded capsules were placed in coded trays and stored in the freezer until processing .", "live corals were air - exposed during mucus collection and positioned upside down connected to a stand .", "after shortly spraying the live coral tissue with mq , the mucus produced by the coral was collected via an acid - washed glass funnel in combusted glass vials .", "after collection of approximately 2  ml mucus per coral , mucus samples were dried in a stove at 50c before being stored dry in the freezer .", "the cavity sponges and coralline algae were quickly washed with mq to remove salt and the tissue was subsequently removed from the underlying substratum by knife and scalpel .", "small pieces were fixed in ethanol ( 80% ) for taxonomical identification of the different sponge species .", "the rest of the pieces were collected in combusted glass vials dried at 50c and stored dry in the freezer .", "sponges and putative food sources were subject to stable isotope analysis . both carbon and nitrogen ( except for bacteria for which only corg was measured ) isotopic composition of the samples were determined using a fisons cn analyser coupled on line via a finnigan conflo 2 interface , to a finnigan delta s mass spectrometer .", "the carbon and nitrogen isotope ratios are expressed as corg and n relative to the vienna pee dee belemnite standard and air , respectively , and the standard error of the measurements is ~0.15. all carbon isotope samples ( except bacteria that were caught on aluminum filters ) were acidified ( tissue directly with acid and glass filters in acid vapor ) prior to measurements and corrected for individual sets of blanks ( isotope mass balance corrections ) .", "seawater as source of carbonates leading to possible enrichment of corg in case of un - acidified aluminum bacteria filters was eliminated by the rinse with mq water at the end of filtration .", "the dried sponge , coralline algae , and mucus material were first homogenized in a mortar . for isotope measurements , adequate amount of material was transferred to pre - combusted silver boats for acidification within cups , followed by oven drying to remove excess acid . from the remaining grounded material , samples from the b1 reef station", "were also analyzed for their total fatty acid composition using a method of one - step procedure of fatty acid extraction and methylation ( masood et al .", "fatty acid extraction and preparation of methyl esters ( fame ) were carried out according to masood et al .", "( 2005 ) with reagent volumes adapted for use in 2.5-ml gc - vials using fame c19:0 as internal standard to calculate concentration of fas ( van gaever et al .", "these individual samples were analyzed separately for their fa compositions employing a large volume splitless injection method on a thermo finnigan trace ultra gc .", "main items of this method are the large volume liner with glass wool , pre - column deactivated silica 5  m    0.53  m , and analytical column sge bpx-70 50  m    0.32  mm    0.25  m .", "the identification of fames was based on the comparison of retention times with authentic commercially available reference material and standards .", "fatty acid source designation was achieved using data on distinctive fa for bacteria , algal carbon , and higher organisms ( e.g. , parrish et al .", "we ran one- and two - way anovas with non - transformed stable isotope data to determine whether variations in stable isotopes of sponges were due to sponge species and/or sponge location assuming no interaction effects ( not enough replication for testing interaction ) .", "the tukey s hsd method was used for pair wise comparisons of different sponge species ( multiple comparisons of means ) .", "anova s were also run for isotope signals of reef and open water - derived suspended matter .", "the siar stable isotope mixing model analysis ( parnell et al . 2010 ) was applied to investigate probable solutions for the diet of sponges based on dual stable isotope data of four sources ( gfc and gff fractions of suspended matter , crustose coralline algae , and coral mucus ) .", "we used the following trophic enrichment factors with standard deviations for the sponges : 3.5    0.5 for n and 1    1 for  c ( vander zanden and rasmussen 2001 ; behringer and butler iv 2006 ) .", "the siar model takes data on organism isotopes and fits a bayesian model to their dietary habits based upon a gaussian likelihood with a mixture dirichlet - distributed prior on the mean .", "model fitting is via markov chain monte carlo ( mcmc ) which produces simulations of plausible values for each dietary source consistent with the data .", "the software package siar ( stable isotope analysis in r ) is freely available from the package section of the comprehensive r archive network website  http://cran.r - project.org/ ( parnell and jackson 2010 ) .", "fatty acid composition patterns were explored with multivariate analyses of fas composition profiles ( non - transformed percentage abundance ) using the program primer version 5 ( clarke and gorley 2006 ) .", "multi - dimensional scaling ( mds plots ) of fa composition and dual isotope label patterns were compared for six sponge species and the three different reef - derived resources ( cca , mucus of m. annularis , and mucus of m. mirabilis ) .", "similarities between fa profiles were investigated using the simper ( similarity of percentages ) function , and statistical differences were determined using the anosim ( analysis of similarities ) function .", "the sponge species collected on the reef stations were identified on basis of their tissue and skeletal characteristics .", "2007 ) to 20.7 ( scopalina ruetzleri ) and n values ranging from 2.9 ( monanchora arbuscula ) to 7.1 ( scopalina ruetzleri ) ( table  1 ) .", "variations in corg and n data were significantly related to sponge species ( anova , f(5,17 )  =  30.3 and f(5,17 )  =  15.8 , p  <  0.01 for both ) and not to reef station ( f(3,17 )  =  1.16 and f(3,17 )  =  0.83 , p  >  0.35 for both ) .", "0.05 ) were found between s. ruetzleri and h. caerulea , s. ruetzleri and niphates erecta , n. erecta and m. arbuscula , which were sampled at three to four reef sites ( table  1 ) .", "differences in dual stable isotope values between sponge species were small when compared to isotope values of sources .", "therefore , the sponge data of the different transects were combined in the fig . ", "2a , b. the stable isotope signals of particulate organic matter size fractions were not significantly different between the open water and reef water ( anova , p  >  0.05 ) , but were kept separate in fig . ", "2a , b. figure  2a shows that open water and reef water bacteria have on average slightly different c signatures .", "variations in corg values of bacterioplankton were large between stations ( ljt and sb vs. ct and b1 , table  2 ) . only the reef water bacteria of station sb ( c : 17.9 ) were in trophic reach of sponges ( < 2 difference ) .", "the sponge trophic range overlaps with the range of coralline algae ( average c of 16.9    4.2 ) .", "coral mucus of montastraea annularis and madracis mirabilis was on average heavier in corg content than the sponges ( table  2 ) .", "coral mucus and coralline algae are potential sponge food because of a trophic level n increase of 34 ( fig . ", "2b).table  1sponge species collected at four reef stations with their respective average isotope signaturescryptic biotastationljtlagun jan thielctcruise terminalb1carmabi reefsbslangenbaaicodecncncncn \n halisarca caerulea \n\n n  =  7sp118.793.6918.454.1218.333.1618.484.0218.204.0917.874.0718.734.63 \n clathria ( thalysis ) raraechelae \n\n n  =  1sp218.914.03 \n chondrilla caribensis \n\n n  =  1sp317.163.83 \n chelonaplysilla erecta \n\n n  =  1sp419.944.68 \n desmapsamma anchorata \n\n n  =  1sp518.815.61 \n scopalina ruetzleri \n\n", "n  =  6sp619.765.9020.264.8520.795.4820.026.1319.417.0720.065.86 \n eurypon laughlini \n\n n  =  218.955.6718.515.78 \n topsentia ophirhaphidites \n\n n  =  219.635.3719.275.82 \n niphates erecta \n\n n  =  617.704.0117.654.7917.934.4717.994.2117.974.7118.004.71 \n callyspongia vaginalis \n\n n  =  217.723.7917.474.37 \n monanchora arbuscula \n\n n  =  520.383.8320.403.4219.823.2819.952.8419.473.63 \n phorbas amaranthus \n\n", "n  =  118.864.40codes sp1sp6 refer to sponges collected at b1 and correspond to sponge data presented in fig .  3 \n fig . ", "2plots of carbon ( a ) and nitrogen ( b ) stable isotope signatures of material collected from the four reef and four open water stations ( average and standard error ) .", "sponges ( n  =  35 ) , bacteria ( bact n  =  8) , coral mucus ( n  =  9 ) , glass microfiber filters coarse ( gfc ) and fine ( gff ) represent values of suspended matter ( spm ) collected either from reef water or from open water offshore ( n  =  4 or 5 ) .", "crustose coralline algae from two reef stations ( n  =  3)table  2average isotope signatures of putative food sources for sponges collected from reef stations ljt , ct , b1 , and sb ( replicates , n  =  23 , standard errors range from 0 to 0.4 , not shown)substrate sourcesstationljtlagun jan thielctcruise terminalb1carmabi reefsbslangenbaaicodecncncncnspm - gfc  open water21.775.2723.873.3722.967.0021.613.94  reef water23.724.5123.385.1723.833.6722.532.66  crevice water21.402.96spm - gff  open water20.863.8123.921.9725.872.8421.524.69  reef water21.194.5324.863.0024.364.4022.093.80  crevice water24.083.20bacteria  open water21.93nd26.02nd27.31nd19.42nd  reef water15.36nd26.40nd27.41nd17.85ndmacroalgae  coralline algaecca13.373.6025.201.3012.124.05coral mucus  m. annularis \n mu115.460.5415.784.1216.462.3514.243.21  m. mirabilis \n mu217.952.1216.534.2417.641.71  s. siderea \n 17.800.55  p. astreoides \n 16.871.11suspended particulate matter ( spm ) fractions collected on coarse ( gfc ) and fine ( gff ) glass fiber filters .", "codes cca and mu1 , mu2 refer to data collected at b1 and correspond to data presented in fig . ", "3 \n\n nd not done sponge species collected at four reef stations with their respective average isotope signatures codes sp1sp6 refer to sponges collected at b1 and correspond to sponge data presented in fig . ", "3 \n  plots of carbon ( a ) and nitrogen ( b ) stable isotope signatures of material collected from the four reef and four open water stations ( average and standard error ) . sponges ( n  =  35 ) , bacteria ( bact n  =  8) , coral mucus ( n  =  9 ) , glass microfiber filters coarse ( gfc ) and fine ( gff ) represent values of suspended matter ( spm ) collected either from reef water or from open water offshore ( n  =  4 or 5 ) .", "crustose coralline algae from two reef stations ( n  =  3 ) average isotope signatures of putative food sources for sponges collected from reef stations ljt , ct , b1 , and sb ( replicates , n  =  23 , standard errors range from 0 to 0.4 , not shown ) suspended particulate matter ( spm ) fractions collected on coarse ( gfc ) and fine ( gff ) glass fiber filters . bacterioplankton ( bacteria ) was collected on aluminum oxide ( anopore ) membrane filters .", "codes cca and mu1 , mu2 refer to data collected at b1 and correspond to data presented in fig . ", "3 \n  pooled analysis with the mixing model ( no distinction between sites or sponge species ) show that mucus ( mu1 and mu2 combined ) contributed to the average cavity sponge diet between 44 and 57% ( = 95% low and high confidence limits , mode 50% ) , cca between 0 and 10% ( mode 2% ) , and the different spm fractions between 0 and 33% ( mode 7% ) for gfc and 14 and 47% ( mode 37% ) for gff ( table  3a ) . of the overall diet 51% ( mode ) was reef - derived .", "therefore , we ran the siar mixing model also separate for the stations for which cca data were available , ct and b1 ( tables  2 , 3b , c ) . pooled analysis of sponges at ct ( no distinction between sponge species ) reflected a higher cca contribution ( between 6 and 43% , mode 31% ) than mucus ( 046% , mode 17% ) . together", "suspended matter supplemented the diet between 0 and 40% for the gfc fraction ( mode 17% ) and 1155% for the gff fraction ( mode of gfc and gff fractions together 51% , table  3b ) . at b1 , where coral cover is higher than at ct ( van duyl 1985 ; pers comm ) , the diet of an average cavity sponge ( no distinction between sponge species ) was dominated by reef - derived sources ( 73% ) with coral mucus and cca contributing for 60 and 13% ( mode ) and with 95% confidence between 5068% and 030% , respectively ( table  3c ) .", "the contribution of suspended matter fractions gfc and gff was within the 028/030% range ( modes 11 and 5% ) , respectively.table  3dual isotope mixing model analyses of the diet of spongessourceslow 95%high 95%modemeanmode%mean%(a ) average diet of sponges ( four stations ) \n gfc ro00.3260.0710.138  gff ro0.1400.4740.3710.32144.245.9  cca00.0950.0150.039  mucmix0.4350.5720.4980.50251.354.1(b ) diet of sponges at ct \n gfc ro00.4020.1700.194  gff ro0.1120.5520.3350.33350.552.7  cca0.0630.4280.3080.254  mucmix00.4550.1720.2184847.2(c ) diet of sponges at b1 \n gfc ro00.2750.1060.122  gff ro00.3000.0530.13015.925.2  cca0.0010.3010.1310.155  mucmix0.5020.6820.5960.59372.774.8the sources comprise two suspended matter size fractions , gfc ro > 1.2  m and gff ro > 0.7 and < 1.2  m from reef overlying and open water ( ro ) , cca representing crustose coralline algae and mucmix representing coral mucus of two different coral species , m. annularis and m. mirabilis .", "the 95% credibility intervals are presented of the different sources as well as the mode and the mean . mode% and", "mean% show the contribution of plankton - derived ( gfc and gff ) and reef - derived organic matter ( cca and mucmix ) in the diet as %   dual isotope mixing model analyses of the diet of sponges the sources comprise two suspended matter size fractions , gfc ro > 1.2  m and gff ro > 0.7 and < 1.2  m from reef overlying and open water ( ro ) , cca representing crustose coralline algae and mucmix representing coral mucus of two different coral species , m. annularis and m. mirabilis .", "the 95% credibility intervals are presented of the different sources as well as the mode and the mean . mode% and", "mean% show the contribution of plankton - derived ( gfc and gff ) and reef - derived organic matter ( cca and mucmix ) in the diet as %   for six sponge species , cca and mucus of two coral species at b1 dual isotopes as well as fatty acid patterns were measured ( fig .  3 ; table  4 ) .", "fas in sources were also present in most sponges , but the diversity in fas was higher in sponges .", "differences between sources ( cca and mucus ) and sponges in the fatty acid compositions are illustrated in fig . ", "the euclidean distances show that the fas of sponges cluster together and are distinct from the sources .", "main fas of the sponge cluster included , in order of importance 16:0 , 20:46 , 18:0 , 20:53 , 18:17c , and branched 20:0 ( phytanic acid ) whereas in the case of the potential energy sources this included 16:0 , 18:0 , 18:19c , and 20:46 ( table  4 ) .", "the source groups were significantly different ( anosim , global r  =  0.8 , p  <  0.05 ) from the sponge group and simper analysis revealed that the differences are the relatively higher percentage abundance of 16:0 , 20:46 , 18:0 , and 18:19c in coral mucus and coralline algae but higher percentage abundance of phytanic acid , 24:0 and 20:53 in the sponges .", "these 7 fas together accounted for ~60% of the fa dissimilarity ( table  4 ; fig . ", "4 ) . among the three different sources , coralline algae , mucus of montastraea annularis , and madracis mirabilis , composition patterns were also significantly different ( anosim , global r  =  1 , p  <  0.05 ) .", "simper analysis revealed two very dominant fatty acids in the coralline algae ( 16:0 and 20:46 ) that together accounted for ~73% of the fa ; in mucus from m. annularis ( mu1 ) , four abundant fas made up ~70% ( 16:0 , 18:0 , 20:53 , and 18:19c ) ; in mucus from m. mirabilis ( mu2 ) , three abundant fas made up ~70% ( 16:0 , 18:0 , and 20:46 , table  2 ) .", "the major fas accounting for the dissimilarity between mucus from the two different coral species is the relatively higher percentage abundance of 20:46 and 18:0 in m. mirabilis ( mu2 ) but a relatively higher percentage abundance of 18:17c and 20:53 in m. annularis ( mu1 ) with these 4 fas accounting for ~60% of the dissimilarity between the coral species ( table  4 ) .", "three groups of sponges were distinguished among the 6 sponge species ( fig .  4 : h. caerulea ( sp1 ) , c. caribensis ( sp3 ) and c. erecta ( sp4 )", "make up the first group ; c. raraechelae ( sp2 ) and d. anchorata ( sp5 ) the second and s. ruetzleri ( sp6 ) the third group ) .", "simper analysis revealed different combinations of fas , in order of dominance , accounting for ~70% of sponge group similarity : for the first group ( sp1 , sp3 , and sp4 ) fas 16:0 , br 20:0 ( phytanic acid ) , 18:0 , 20:46 , i15:0 , and 16:17c are the main fas . in the second group ( sp2 and sp5 ) fas 16:0 , 20:46 , 24:0 , 18:17c , 22:0 , 20:53 , and 20:63 determined primarily the composition pattern . in the third group ( sp6 ) , these were the five fas : 16:0 , 20:46 , 18:0 , 20:53 , and 14:0 ( table  4).fig . ", "3carbon and nitrogen isotopes of six sponge species ( sp1sp6 ) and putative organic matter sources for sponges at reef site b1 .", "the average and standard error for sp1 ( n  =  3 ) and sp6 ( n  =  2 ) , gfc - spm ( n  =  3 ) , gff - spm ( n  =  3 ) is presented .", "other data points ( sp2 , 3 , 4 , and 5 , cca and mucus from two coral species , montastraea annularis ( mu1 ) and madracis mirabilis ( mu2 ) do not have replicates other than repetitive sample analysis of the same specimen ) .", "see also tables  1 , 2 \n table  4fatty acid composition of six sponge species ( sp1sp6 , see table  1 ) , one crustose coralline alga ( cca ) and mucus ( mu ) from two coral species ( see table  2 ) , all collected from reef site b1sp1sp2sp3sp4sp5sp6ccamu1mu2i14:0 \n 0 ( 0)0 ( 0)0.7 ( 0.02)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)14:06.6 ( 0.55)2.6 ( 0.05)2.3 ( 0.08)3.6 ( 0.40)3.8 ( 0.55)9.1 ( 0.24)3.5 ( 0.10)5.3 ( 0.21)2.6 ( 0.05)ai15:0 \n 0.6 ( 0.05)0 ( 0)2.6 ( 0.02)0.5 ( 0.27)0.1 ( 0.07)0.3 ( 0.06)0.04 ( 0.04)0 ( 0)0 ( 0)i15:0 \n 5.3 ( 0.24)0.3 ( 0.06)9.8 ( 0.05)5.4 ( 0.25)0.8 ( 0.04)0.5 ( 0.16)0 ( 0)3.8 ( 1.90)4.2 ( 0.08)15:01.2 ( 0.11)0.5 ( 0.01)1.1 ( 0.07)1.4 ( 0.07)1.7 ( 0.15)7.5 ( 0.23)0.3 ( 0.07)0 ( 0)0 ( 0)i16:0 \n 0.6 ( 0.08)0.4 ( 0.02)1.3 ( 0.02)0.4 ( 0.20)0.7 ( 0.05)1.9 ( 0.05)0.3 ( 0.02)0 ( 0)0 ( 0)10me-16:0 \n 0 ( 0)0 ( 0)0 ( 0)0 ( 0)0.4 ( 0.07)0 ( 0)0 ( 0)0 ( 0)0 ( 0)16:260 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0.51 ( 0.03)16:240 ( 0)0.2 ( 0.03)0 ( 0)0 ( 0)0.5 ( 0.00)0.1 ( 0.09)0.04 ( 0.04)0 ( 0)0 ( 0)16:15c0 ( 0)0.7 ( 0.03)0.9 ( 0.03)0.4 ( 0.18)0.6 ( 0.03)0.4 ( 0.02)0 ( 0)0 ( 0)0 ( 0)16:17c \n 3.3 ( 0.16)1.6 ( 0.02)12.6 ( 0.03)4.0 ( 0.11)4.2 ( 0.16)2.1 ( 0.04)0.5 ( 0.06)3.9 ( 0.50)0.6 ( 0.04)16:17 t \n 0 ( 0)0.15 ( 0.02)0.79 ( 0.01)0 ( 0)0.1 ( 0.07)0.3 ( 0.04)0 ( 0)0 ( 0)0 ( 0)16:017.1 ( 1.73)13.2 ( 0.27)18.4 ( 0.19)20.5 ( 0.39)12.1 ( 0.62)19.6 ( 0.52)40.7 ( 0.21)30.8 ( 0.84)27.8 ( 0.66)10me-17:0 \n 0 ( 0)0 ( 0)0.8 ( 0.15)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)17:17c0.9 ( 0.66)0.1 ( 0.00)1.9 ( 0.10)8.5 ( 0.42)0.5 ( 0.11)0.7 ( 0.22)0.4 ( 0.12)0 ( 0)0 ( 0)17:01.9 ( 0.08)0.5 ( 0.04)0.5 ( 0.49)2.6 ( 0.13)1.4 ( 0.00)0.5 ( 0.09)0.3 ( 0.00)0.5 ( 0.03)0 ( 0)i18:0 \n 0 ( 0)0.6 ( 0.05)0.3 ( 0.05)0 ( 0)0.6 ( 0.09)0.4 ( 0.02)0 ( 0)0 ( 0)0 ( 0)18:530.2 ( 0.22)1.1 ( 0.03)0.2 ( 0.05)0 ( 0)1.7 ( 0.23)0.1 ( 0.05)0.1 ( 0.00)1.5 ( 0.04)0 ( 0)18:50 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)2.1 ( 1.03)0 ( 0)18:360.6 ( 0.56)0.3 ( 0.02)0.1 ( 0.04)0 ( 0)0 ( 0)0 ( 0)0 ( 0)4.1 ( 0.31)0 ( 0)18:330.5 ( 0.25)0.1 ( 0.07)0 ( 0)0.8 ( 0.21)0.3 ( 0.26)0 ( 0)0 ( 0)0 ( 0)0 ( 0)18:26c1.8 ( 0.12)0.8 ( 0.07)0.3 ( 0.02)2.2 ( 0.09)0.6 ( 0.30)0.9 ( 0.06)0.8 ( 0.02)0.4 ( 0.20)0.8 ( 0.05)18:17c5.7 ( 0.12)9.7 ( 0.06)3.3 ( 0.10)5.5 ( 0.04)6.6 ( 0.15)2.0 ( 0.06)4.5 ( 0.05)8.1 ( 0.33)0.7 ( 0.03)18:19c2.5 ( 0.50)1.9 ( 0.06)1.9 ( 0.11)4.2 ( 0.14)1.4 ( 0.26)2.0 ( 0.08)11.2 ( 0.10)9.1 ( 0.32)7.3 ( 0.12)18:09.4 ( 0.25)4.0 ( 1.61)9.7 ( 0.23)10.2 ( 0.29)5.6 ( 0.46)11.5 ( 0.06)3.0 ( 0.02)15.5 ( 0.34)25.4 ( 0.74)i20:0 \n 0.2 ( 0.20)0.8 ( 0.01)0 ( 0)0 ( 0)1.1 ( 0.14)0 ( 0)0 ( 0)0 ( 0)0 ( 0)20:534.5 ( 0.26)6.3 ( 0.24)3.4 ( 0.01)3.0 ( 0.25)5.0 ( 0.36)11.7 ( 0.26)0.1 ( 0.05)11.0 ( 0.15)4.4 ( 0.44)20:430 ( 0)0.10 ( 0.05)0 ( 0)0 ( 0)0.4 ( 0.08)0.7 ( 0.35)0.3 ( 0.30)0 ( 0)0 ( 0)20:4610.6 ( 0.68)11.1 ( 0.24)9.1 ( 0.25)8.1 ( 0.11)10.7 ( 0.48)15.2 ( 0.27)32.2 ( 0.75)0 ( 0)15.0 ( 0.34)20:360.7 ( 0.13)0.7 ( 0.12)0.5 ( 0.06)0.6 ( 0.08)0.5 ( 0.03)0.5 ( 0.05)0.4 ( 0.03)0 ( 0)0 ( 0)20:330 ( 0)0.1 ( 0.07)0 ( 0)0.1 ( 0.12)0.3 ( 0.13)0.1 ( 0.13)0 ( 0)0 ( 0)0 ( 0)phytanic acid15.5 ( 1.34)2.9 ( 0.13)13.4 ( 0.26)8.3 ( 0.29)1.8 ( 0.30)1.3 ( 0.19)0.2 ( 0.03)0 ( 0)0 ( 0)20:19c0.6 ( 0.39)0.2 ( 0.05)0.1 ( 0.00)0 ( 0)0.5 ( 0.10)0.1 ( 0.05)0.2 ( 0.04)0 ( 0)0.3 ( 0.28)20:290.3 ( 0.15)0.3 ( 0.05)0 ( 0)0.8 ( 0.02)0.9 ( 0.40)2.6 ( 0.04)0.1 ( 0.04)0 ( 0)0 ( 0)20:00.9 ( 0.04)2.2 ( 0.06)0.8 ( 0.04)1.7 ( 0.05)3.5 ( 0.16)2.5 ( 0.03)0.3 ( 0.05)3.1 ( 0.14)6.4 ( 0.21)22:631.0 ( 0.01)8.4 ( 0.14)0.1 ( 0.06)2.6 ( 0.27)3.6 ( 0.28)1.9 ( 0.02)0 ( 0)0 ( 0)0 ( 0)22:530 ( 0)0 ( 0)0.1 ( 0.09)2.8 ( 0.30)0 ( 0)0 ( 0)0.1 ( 0.03)0 ( 0)0 ( 0)22:460.5 ( 0.06)1.9 ( 0.14)0 ( 0)0.6 ( 0.58)0.3 ( 0.12)0 ( 0)0 ( 0)0.9 ( 0.44)1.9 ( 0.14)22:1110.2 ( 0.20)0.1 ( 0.03)0.5 ( 0.06)0.1 ( 0.11)0 ( 0)0 ( 0)0.1 ( 0.03)0 ( 0)0 ( 0)22:19c0 ( 0)0 ( 0)0.1 ( 0.05)0 ( 0)0 ( 0)1.0 ( 0.08)0.1 ( 0.07)0 ( 0)0 ( 0)22:01.9 ( 0.18)4.2 ( 0.25)0.7 ( 0.03)0.4 ( 0.19)18.1 ( 0.89)0.8 ( 0.20)0.2 ( 0.05)0 ( 0)2.2 ( 0.09)23:01.5 ( 0.17)2.1 ( 0.34)0.9 ( 0.05)0.1 ( 0.12)2.0 ( 0.14)0.1 ( 0.06)0 ( 0)0 ( 0)0 ( 0)24:03.5 ( 0.31)20.3 ( 0.96)1.1 ( 0.10)0.6 ( 0.06)7.8 ( 0.32)1.8 ( 0.04)0.3 ( 0.05)0 ( 0)0 ( 0)% bact - spec6.6/9.92.2/3.816.2/28.76.3/10.33.7/7.83.4/5.50.3/0.83.8/7.74.2/4.8average %    se ( n  =  3 ) \n bacteria - specific fa and last row is average % bacteria specific fa ( bact - spec ) with / without inclusion of 16:17cfig . ", "4multidimensional scaling ( mds ) plot showing similarity in fatty acid composition profiles of six sponge species ( sp16 ) , crustose coralline algae ( cca ) and the mucus of two coral species , montastraea annularis and madracis mirabilis , at reef site b1 ( see table  4 for list of fatty acids ) carbon and nitrogen isotopes of six sponge species ( sp1sp6 ) and putative organic matter sources for sponges at reef site b1 .", "the average and standard error for sp1 ( n  =  3 ) and sp6 ( n  =  2 ) , gfc - spm ( n  =  3 ) , gff - spm ( n  =  3 ) is presented .", "other data points ( sp2 , 3 , 4 , and 5 , cca and mucus from two coral species , montastraea annularis ( mu1 ) and madracis mirabilis ( mu2 ) do not have replicates other than repetitive sample analysis of the same specimen ) . see also tables  1 , 2 \n  fatty acid composition of six sponge species ( sp1sp6 , see table  1 ) , one crustose coralline alga ( cca ) and mucus ( mu ) from two coral species ( see table  2 ) , all collected from reef site b1 average %    se ( n  =  3 ) \n bacteria - specific fa and last row is average % bacteria specific fa ( bact - spec ) with / without inclusion of 16:17c multidimensional scaling ( mds ) plot showing similarity in fatty acid composition profiles of six sponge species ( sp16 ) , crustose coralline algae ( cca ) and the mucus of two coral species , montastraea annularis and madracis mirabilis , at reef site b1 ( see table  4 for list of fatty acids ) for these three distinguished sponge groups at b1 , we also ran the isotope mixing model ( table  5 ) .", "the first group appears to rely mainly on reef - derived sources ( mucus and cca , mode and mean both > 75% ) .", "the second group probably relied less on mucus and more on cca and used more plankton - derived food compared with group 1 .", "ruetzleri only ) consists for about 51% ( mean ) of reef - derived and 49% ( mean ) of suspended matter ( plankton ) derived food .", "group 1 has a smaller centroid euclidean distance ( 23.64 ) to the average mucus mixture than groups 2 and 3 ( 32.43 and 24.55 ) in fig . ", "this concurs with a larger contribution of mucus ( 66% ) to the diet of group 1 than that of the other two groups ( 36 and 33% ) ( fig .  4 ; table  5 ) .", "the lower contribution of cca in the diet of group 1 ( 10% mode ) compared to group 3 ( 19% mode ) was also reflected in fig .  4 .", "we did not find this for group 2 , which had the longest euclidean distance to cca ( 41.81 vs. 33.78 of group 3 and 37.37 of group 1 ) , while cca - derived food comprised 22% in the diet of this group . to further explore the relation between the stable isotope and fa data sets", ", we compared the matrices of fas and dual isotope signatures of sponges and reef resources ( figs .  3 , 4 ) and found a significant correlation between the two matrices ( primer , rho  =  0.625 , p  =  < 0.01).table  5results of dual isotope mixing model analyses for different sponge groups at b1sourceslow 95%high 95%modemeanmode%mean%(a ) diet of group 1 \n\n h. caerulea , c. caribensis , c. erecta ( sp1 , sp3 , sp4)gfc ro00.2840.0360.117gff ro00.2440.0360.1047.222.1cca00.2860.0950.132mucmix0.5040.7960.6580.64775.377.9(b ) diet of group 2 \n\n c. raraechelae , d. anchorata ( sp2 , sp5)gfc ro00.4320.1670.216gff ro00.430.2020.20836.942.4cca00.4510.2190.219mucmix0.0770.6060.3570.35757.657.6(c ) diet of group 3 \n\n s. ruetzleri ( sp6)gfc ro0.0030.4870.2920.259gff ro00.4530.2850.23257.749.1cca00.4210.1880.207mucmix0.0230.5160.3270.30251.550.9 for further explanation , see table  3 \n  results of dual isotope mixing model analyses for different sponge groups at b1   for further explanation , see table  3 \n", "cryptic encrusting sponges living in coral cavities on the fore - reef slope of fringing reefs sequester most of their organic matter from the passing water . they efficiently filter the pico- and nanoplankton , but dissolved organic matter ( dom ) appears to be the major resource of various sponge species living on the open reef and in coral cavities ( yahel et al . 2003 ; de goeij et al .", ". it may be dom released from phytoplankton , dom supplied to the reef from outside or dom released from the reef benthos .", "gradients in dom concentrations over the reef suggest that there is a net release of dom from coral reef waters to adjacent open waters ( van duyl and gast 2001 ) .", "only 1020% of the dom in reef water appears to be available for sponges ( de goeij et al .", "2008a ) . by analyses of dual stable isotope signatures ( corg and n ) and fa patterns of sponges and their potential food sources , we obtained circumstantial evidence that the reef itself mainly feeds her cavity sponges .", "this remarkable finding implies that cryptic sponges support reef internal nutrient recycling and conservation of reef organic matter .", "it was surprising that none of the 12 sponge species examined appeared to live mainly on suspended particulate organic matter or dom derived from this source .", "the corg values of the spm fractions were in the range reported for marine suspended matter ( peterson and fry 1987 ) .", "bacterioplankton values were within 2 of spm in open water , which indicates that bacteria were the primary consumers of spm .", "this makes it highly likely that spm represents predominantly phytoplankton and that the bacterioplankton reflects the c isotope ratio of reactive dom released by phytoplankton ( e.g. , harvey et al .", "the c of spm did not change during passage over the reef , for instance by obtaining a typical c spm reef signal ( 17 to 20 : land et al .", "the n of spm did not change either and remained ~4. moreover , at two of the four stations ( ct and b1 ) comparable corg values of predominantly heterotrophic bacterioplankton ( 26 , 27 , table  2 ) between open water and reef bottom water suggest that this bacterioplankton remained dependent on phytoplankton doc . such low c values have been reported , for e.g. , synechococcus ( c 33 to 25 , brutemark et al . 2009 ) and prochlorococcus , both abundant in reef and open water along curaao ( van duyl et al . 2002 ) .", "residence time of the spm and bacterioplankton in reef overlying waters may have been too short for changes in isotopic ratios either due to rapid water exchange with the open water and/or due to spm removal by benthic suspension feeders .", "phytoplankton concentrations in reef surrounding waters in curaao are usually low ( van duyl et al . 2002 ) , and the organic matter demand of cryptic biota , cryptic sponges in particular , is high .", "cavity biota between 10 and 15  m depth on the fore - reef slope in curaao consume on average 350  mmol  c  m  d of which cavity sponges consume on average 256  mmol  c  md ( 73% ) with the doc fraction comprising > 90% of the diet ( de goeij et al .", "this organic matter supply is orders of magnitude higher than the supply of pelagic primary production to reefs ( richter et al .", "it is evident that the supply of open water - derived phytoplankton and its dom release is insufficient to satisfy the total organic carbon requirements of coral reef cavity sponges . in accordance ,", "the isotope mixing model results confirmed a spm contribution of less than < 50% in the diet of most sponges .", "this implies that the examined sponges with average corg of 18.9 and n of 4.6 mainly assimilate other sources than pelagic primary production .", "presence of distinct reactive dom in reef bottom water compared to open water was demonstrated by the increased c value of bacterioplankton at two of the four stations ( ljt and sb ) .", "bacterioplankton apparently assimilated a source of carbon not available in the open water , a source , which may also be available to the dom feeding cavity sponges .", "benthic primary producers usually have higher corg values than pelagic primary producers ( france 1995 ; fry 2006 ) .", "thus , assimilation of benthic algae - derived reactive doc may explain the elevated c values in bacterioplankton in reef bottom water at ljt and sb .", "bacteria respond quickly when exposed to changing resources in the vicinity of the reef bottom .", "enhanced bacterioplankton production in reef bottom waters , cavity waters , and coral mucus point to the reef bottom as the source of inorganic nutrients and reactive dom ( van duyl and gast 2001 ; scheffers et al .", "however , the c reef signal of bacterioplankton ( 17.9 to 15.4 ) was higher than that of most examined sponges ( 20.8 to 17.2 ) suggesting that sponges and bacterioplankton may not fully depend on the same source of reef - derived dom .", "sponges may exploit a wider size range of the operationally defined dom fraction than bacterioplankton ( de goeij et al .", "benthic primary producers such as corals and benthic macroalgae have been reported to release reactive dom ( e.g. , crossland 1983 , 1987 ; wild et al .", "the c values of the benthic substrates ( coral mucus of two dominant coral species and crustose coralline algae ) were on average higher in c than the spm and open water bacterioplankton and were more in range of the sponge stable isotope signals .", "the n values of reef - derived substrates with values 34 lower than those of sponges on average suggest a direct trophic link ( one trophic level distance ) .", "the lower n values of reef substrates versus open water substrates may be due to enhanced n2 fixation by the reef benthos ( davey et al .", "discharge from land or pollution may counteract this effect by introducing organic and inorganic n enriched in n. this may partly explain the variations in n in mucus , coralline algae , and sponges between different stations . the range of corg in crustose coralline algae ( cca ) of 25.2 to 12.1 was unexpectedly large , possibly due to the unique ontogeny of cca .", "younger portions of the cca may have lower corg values than older portions possibly because rapid photosynthesis in younger parts depends more on respired co2 than photosynthesis in older parts ( lee and carpenter 2001 ) . anyway , the lower range of corg values of cca was within 2 of our sponges .", "moreover , the n of cca ( 1.34.0 ) make it a likely food source for the majority of cavity sponges with 34 higher n values than the cca .", "little is known of exudation of organic matter by cca apart from the releases of allelochemicals ( e.g. , kim et al .", "total dom release by cca has not been determined as far as we know , but their productivity equals that of fleshy macroalgae and turf algae on reefs ( chrisholm 2003 ) . reported c values of 18.7 to 16.8 for dictyota sp.,19.3 for halimeda sp .", "( raz - guzman macbeth and de la lanza espina 1991 ; lepoint et al .", "2000 ) , and n values of 0.53 of macroalgae on coral reefs ( yamamuro et al . 1995 ; france et al .", "1998 ) fall in the same range as cca ( corg 25.2 to 12.1 , n 1.34 ) .", "therefore , dom released from these brown and green macroalgae may have also contributed to the isotope signal in sponges .", "trophic transfers of organic matter derived from benthic macroalgae and mangroves , to sponges have been reported before ( behringer and butler iv 2006 ; granek et al .", "2009 ) and appear to be important in the nutrition of sponges . unlike cca , coral mucus has already been described as an important food source on coral reefs ( wild et al .", "1980 ; wild et al . 2004 ) and may be a suitable source of dom for cavity sponges .", "the c of the mucus samples of montastraea annularis and madracis mirabilis ( range 17.9 to 14.2 ) is higher than that of most cavity sponges , implying that sponges do not feed exclusively on mucus or mucus - derived dom .", "coral - derived organic matter is a reactive substrate for bacterioplankton growth ( ferrier - pags et al .", "2000 ) and may have contributed to the c reef signal of bacterioplankton at ljt ( 15.4 ) .", "the average n of mucus ( 1.9 ) and that of cavity sponges ( 4.6 ) are consistent with mucus / mucus - derived dom as a food source for most cavity sponges .", "it is evident that individually , neither phytoplankton , bacterioplankton , cca ( benthic macroalgae ) nor coral mucus can account for the sponge dual isotope signatures .", "the isotope mixing model analysis showed that cavity sponges incorporate from half to three quarters of their organic matter from mucus and cca ( benthic macroalgae ) with mucus as the dominant source of reef - derived organic matter ( up to 66% of the diet ) .", "it appears that with decreasing coral cover the contribution of coral mucus to the diet of sponges decreases and shifts to a larger contribution of cca ( benthic macroalgae ) and phytoplankton . at station", "ct with a lower coral cover than at the other reef stations , mucus only contributed 17% ( mode ) to the diet of sponges while the bulk food was provided by cca including other benthic macroalgae ( 31% ) and small phytoplankton ( 34% , gff fraction ) .", "this was different at b1 , where coral cover on the slope is relatively high ( van duyl 1985 ; bak and nieuwland 1995 ) and the sponges reflect a diet of mainly mucus ( 60% , mode ) with 13% cca .", "this suggests that the sponge community is opportunistic and consumes om according to availability irrespective of the dom source .", "this does not imply that all examined sponge species at a particular reef site have similar diets .", "this is further corroborated with our fa profiles of sponges at b1 . the overall similarity in fa composition pattern of", "the three groups within these six sponge species ( based on significantly different fa compositions ) also had different diets according to the isotope mixing model analysis , implying differences in the proportional use of mucus , benthic algae ( including cca ) , and phytoplankton - derived dom .", "this suggests that there is trophic niche segregation among sponges which may be an important factor facilitating co - existence of different sponge species ( thurber 2007 ) .", "furthermore , the differences in diet may to some extent be reflected in the fa compositions of sponge groups suggesting that the selected food resources may indeed form part of the diet of sponges .", "the fatty acid profiles of our coral mucus and cca sources resemble those reported for coral tissue and cca ( e.g. , latyshev et al . 1991 ;", "although many dominant fas such as 14:0 , 16:0 , and 18:0 ( table  2 ) are common to many organisms , some fas have been identified as potential tracer of food source in sponges . for this study area specifically , for example , de goeij et al .", "( 2008b ) conclude from isotope tracer experiments that the dominant fa 20:46 found in the sponge halisarca caerulea has an exogenous source .", "interestingly , 20:46 is found dominant in all 6 sponge species examined and it is also dominant in cca and m. mirabilis mucus .", "the high cover of cca in cavities ( 30% of the hard bottom surface in cavities , scheffers et al .", "2004 ) , its close proximity to cavity sponges and the fact that cca was rich in 20:46 , may have contributed to the 20:46 content in sponges . in sponge groups 2 and 3 , this fa was the second most abundant ( > 10% of total fas ) and the diet of these sponge groups consisted for 1922% ( mode ) of cca ( including other macroalgae ) . in sponge group 1 , the predominantly mucus feeding group ,", "20:46 was the third most abundant fa with a lower relative abundance than in the other groups ( < 10% ) .", "this coincided with a lower contribution of cca in their diet ( 10% mode ) .", "trophic transfer of this fa may be directly linked to the consumption of cca as part of macroalgal - derived organic matter .", "phytanic acid , which can be considered as a sponge biomarker ( de goeij et al .", "2008b ) was the second most abundant fa in group 1 . for its synthesis ,", "degradation products of chlorophyll a are required , possibly delivered to the sponge by sponge associated phototrophic microbes , considering the relatively high amount of the likely bacterial specific fa 16:17c as well as the overall high % of bacteria specific fas in group 1 ( up to 29% compared with up to 10% of total fas in the other groups , table  4 ) .", "sponge species in group 1 indeed harbor - rich microbial communities ( hill et al . 2005 ; erwin and thacker 2007 ) .", "mutualism between sponges and their associated microorganisms may have influenced the diet of group 1 compared with groups 2 and 3 .", "weisz et al . ( 2007 ) show that sponges with high versus low abundances of associated microbes may have distinct diets . in line , we show that the diet of sponges ( based on stable isotopes as well as fas ) varies with abundance of bacteria specific fas .", "interestingly , sponges with a relatively high abundance of associated microbes ( group 1 ) appear to be more dependent on reef - derived food than other sponges .", "the fa 20:53 , a dominant fa in ( benthic ) diatoms and the mucus of corals , particularly in mucus of m. annularis was most abundant in s. ruetzleri ( group 3 ) .", "the siar model analysis showed that the contribution of phytoplankton - derived food was highest in this sponge ( mean 49% and mode 57% , both plankton size fractions together ) , which may suggest that it feeds on diatoms or diatom - derived om rich in 20:53 .", "the other sponge groups ( 1 and 2 ) may obtain 20:53 predominantly by consuming mucus considering the higher contribution of mucus and lower plankton - derived food in the diet of these groups than in the diet of s. ruetzleri ( group 3 ) .", "diatom - derived om as well as mucus may have contributed to the abundance of 20:53 in sponges . considering the congruencies between the fa compositions and the dual stable isotope signals in the trophic transfer and relations between sources and consumers , it was not surprising that the matrices ( based on euclidean distances ) of the reef sources and consumers of both approaches were significantly correlated .", "both methods apparently lead to comparable results with fa composition and biomarkers identifying the food items of consumers , and evidencing trophic transfer and with dual stable isotope analyses estimating the contribution of the different sources to the diet of consumers .", "results of both methods strongly support a trophic transfer from zooxanthellate corals and cca to cavity sponges .", "the quantitative contribution of cca to the diet of sponges remains unresolved , because its dual isotope signal overlaps with that of other benthic macroalgae .", "results unambiguously point to the reef benthos as the main source of organic matter for encrusting cavity sponges on coral reefs .", "the contribution of open water - derived organic matter as food for the cavity sponge community was usually less than 50% .", "food sources for sponges on the reef comprise dom derived from coral mucus and benthic macroalgae ( cca ) .", "the prominent presence of a characteristic fatty acid of coral mucus and cca , 20:46 in sponges further confirms this trophic transfer .", "( 2008b , 2009 ) found that the cavity sponge halisarca caerulea converts assimilated dom in sponge biomass and detrital material . combining this with the knowledge that ( 1 ) the cover of cavity sponges is high on fore - reef slopes ( scheffers et al .", "2004 ) , ( 2 ) dom assimilation by cavity sponges is high ( de goeij et al .", "2008a ) , and ( 3 ) most of the om assimilated by sponges is reef - derived ( this study ) , we argue that cryptic sponges play a key role in conserving reef - produced dom for the reef system by converting it into pom . whether the  cryptic ", "sponge link in the coral reef food web is favorable for the coral reef health status remains to be investigated ."], "abstract_text": ["<S> the diet of cavity sponges on the narrow fringing reefs of curaao , caribbean was studied . </S>", "<S> the origin and resources of the bulk food of these sponges , i.e. , dissolved organic matter ( dom ) , were identified using stable carbon and nitrogen isotopes and fatty acid biomarkers . </S>", "<S> we found that phytoplankton and its derived dom from the adjacent open sea and from reef overlying water is not the main source of food for most of the sponges examined nor is bacterioplankton . </S>", "<S> interestingly , dual stable isotope signatures ( 13corg , 15norg ) and fatty acid biomarkers appoint coral mucus and organic matter derived from crustose coralline algae ( cca ) as probable food sources for encrusting sponges . </S>", "<S> mucus - derived dom may contribute up to 66% to the diet of examined sponges based on results of dual isotope mixing model analysis . </S>", "<S> the contribution of cca ( as purported representative for benthic algae ) was smaller with values up to 31% . </S>", "<S> together , mucus- and cca - derived substrates contributed for 4873% to the diet of sponges . </S>", "<S> the presence of the exogenous fatty acid 20:46 in sponges , which is abundant in coral mucus of madracis mirabilis and in cca , highlights these reef - derived resources as sources of nutrition for dom feeding cavity sponges . </S>", "<S> the relatively high concentrations of exogenous 20:46 in all sponges examined supports our hypothesis that the bulk of the food of the cavity sponge community is reef - derived . </S>", "<S> our results imply that cavity sponges play an important role in conserving food and energy produced within the reef . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Sampling sites and sample collection", "Sample treatment", "Statistical analyses", "Results", "Discussion", "Conclusion"], "sections": [["sponges play an important role in the benthic - pelagic coupling on coral reefs ( lesser 2006 ) .", "the sponge community sequesters and processes organic matter and as such conserve energy and nutrients for the reef ecosystem .", "it is generally assumed that sponges on the fore - reef slope mainly trap allochthonous organic matter that may compensate the losses of material from reef ecosystems ( e.g. , richter et al .", "most reef sponges are extremely efficient in filter feeding ( e.g. , reiswig 1974 ; lesser 2006 ) .", "they preferably filter the picoplankton fraction from the flowing water ( e.g. , pile 1999 ; ribes et al . 2003 , 2005 ) .", "the major food resource of coral reef sponges , however , may well be dissolved organic matter ( reiswig 1990 ; yahel et al .", "2003 ) in coral cavities are fueled mainly by dissolved organic matter ( dom ) ( de goeij et al .", "encrusting sponges cover together with coralline algae > 50% of the walls in coral cavities , caves , and undersides of corals ( vasseur 1974 ; richter and wunsch 1999 ; wunsch et al .", "this cryptic surface of 3-dimensionally well - developed reefs may exceed the open reef surface ( ginsburg 1983 ; richter et al .", "the sheer size of this reef habitat already preludes the grand role of its biota in organic matter cycling .", "dom removal by the cryptic biota on coral reefs can be huge ( up to 500  mmol  c  m cryptic surface d ) , with cavity sponges accounting for 70% of the consumed dom in coral reef cavities ( de goeij and van duyl 2007 ;", "these recent findings draw direct attention to the origin and sources of dom on coral reefs .", "phytoplankton ( including photoautotrophic bacteria ) usually is the main source of reactive dom in oligotrophic ocean water ; it releases dom as photosynthetic product or through food web processes and lysis ( carlson 2002 ) . on coral reefs", "extra sources of reactive dom include release of dom by corals through mucus production ( johannes 1967 ; richman et al .", "1975 ; crossland et al . 1980 ; naumann et al . 2010 ) and extracellular release of dom by benthic algae ( e.g. , wada et al . 2007 ;", "the main consumers of reactive dom are usually heterotrophic bacteria ( e.g. , harvey et al .", "the fate of mucus and dissolved organic matter in reef overlying waters has been ascribed to consumption and mineralization by bacteria in the reef ambient water and in sediments ( e.g. , gast et al .", "the first indication that reef - derived dom may also be assimilated by higher trophic levels came from the abundance of the dietary fatty acid biomarker 20:46 in the encrusting dom feeding cavity sponge halisarca caerulea ( de goeij et al .", "1991 ) and in coralline algae , rhodophyta ( viso and marty 1993 ) and its presence in sponges suggests that reef - derived organic matter may be an important food source for coral reef sponges .", "this study aims ( 1 ) to determine whether cavity sponges mainly rely on reef - produced or open water - derived dissolved organic matter and ( 2 ) to identify key sources of dom for cavity sponges . as putative food compounds different fractions of suspended matter", ", bacterioplankton , coral mucus , and coralline algae as proxies for dom were analyzed . through stable isotopes and fatty acid biomarkers , we characterized energy sources of cavity sponges collected from coral reefs and open water on the southwest coast of curaao , caribbean ( 1212n , 6856w ) .", "stable isotopes are a classical way to trace food sources of aquatic animals ( peterson and fry 1987 ) . the carbon and nitrogen stable isotope signatures of 12 sponge species were compared with those of putative food compounds .", "fatty acid biomarkers have been repeatedly used as source - specific indicators of dissolved and particulate organic matter both in environmental and in food web studies ( e.g. hall et al .", "the fa pattern of six sponge species and three types of reef food resources were analyzed , and the relative input of different sources to the diet of the sponges was determined ."], ["samples were collected from march 14 to 22 , 2006 , along four transects perpendicular to the sw coast of curaao ( fig .  1 ) .", "transects were placed 37  km apart from se to nw along the coast at lagun jan thiel ( ljt ) , at the ocean cruise terminal in otrabanda ( ct ) , at buoy 1 ( b1 ) of the carmabi reef close to piscaderabaai , and at slangenbaai ( sb ) .", "transects from the shore to open water cross the narrow fringing reefs and water masses representative for the variation in coral community composition and water quality along the sw coast of curaao ( van duyl 1985 ; gast et al .", "1999 ; van duyl and gast 2001 ) . along each transect ( ljt , ct , b1 , and sb ) , we sampled 2 stations : fig . ", "( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbeanan off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water anda coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "position of sampling transects ( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbean an off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water and a coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "water samples were taken with a 6  l niskin bottle operated by a scuba diver .", "the niskin bottle was repeatedly filled and hauled on board a small open boat and emptied in a clean and sample washed 20  l container . at each station", "( n  =  8) , we collected 20  l. on the fore - reef slope stations ( n  =  4 ) , we collected a few small pieces of the stony corals montastraea annularis and madracis mirabilis ( also siderastrea siderea and porites astreoides but only at b1 ) between 15 and 17  m depth , which were kept in seawater under natural light conditions until mucus collection on the next day . from the entrance of coral cavities at 1517  m depth on the fore - reef slope , we chiseled crusts of calcareous algae . from the same cavities ,", "various species of encrusting sponges were collected , up to eight different species per station .", "the 20-l water samples were filtered over 47-mm - diameter combusted glass microfiber filters ( whatman ) , first over a gfc filter ( nominal pore size 1.2  m ) and then over a gff filter ( nominal pore size 0.7  m ) .", "after sea water filtration , filters were shortly washed with milli - q water ( mq ) to remove salt and were dried at 50c in a stove and stored in aluminum foil in the freezer until processing .", "the remaining bacterioplankton in the gff filtrate was concentrated with a vivaspin filter cartridge driven by a master volt tube pump .", "the concentrate was subsequently filtered over a 0.2  m pore size 25-mm - diameter anopore disc ( aluminum oxide membrane filter , whatman ) .", "the discs were briefly washed with mq after seawater filtration and dried at 50c in a stove ( 12  h ) . after drying , the filter was crumbled in a clean acid - washed glass funnel , the integrated polypropylene support ring of the filter was removed and the filter fragments were transferred to combusted silver capsules , which were closed with tweezers .", "subsequently , the folded capsules were placed in coded trays and stored in the freezer until processing .", "live corals were air - exposed during mucus collection and positioned upside down connected to a stand .", "after shortly spraying the live coral tissue with mq , the mucus produced by the coral was collected via an acid - washed glass funnel in combusted glass vials .", "after collection of approximately 2  ml mucus per coral , mucus samples were dried in a stove at 50c before being stored dry in the freezer .", "the cavity sponges and coralline algae were quickly washed with mq to remove salt and the tissue was subsequently removed from the underlying substratum by knife and scalpel .", "small pieces were fixed in ethanol ( 80% ) for taxonomical identification of the different sponge species .", "the rest of the pieces were collected in combusted glass vials dried at 50c and stored dry in the freezer .", "sponges and putative food sources were subject to stable isotope analysis . both carbon and nitrogen ( except for bacteria for which only corg was measured ) isotopic composition of the samples were determined using a fisons cn analyser coupled on line via a finnigan conflo 2 interface , to a finnigan delta s mass spectrometer .", "the carbon and nitrogen isotope ratios are expressed as corg and n relative to the vienna pee dee belemnite standard and air , respectively , and the standard error of the measurements is ~0.15. all carbon isotope samples ( except bacteria that were caught on aluminum filters ) were acidified ( tissue directly with acid and glass filters in acid vapor ) prior to measurements and corrected for individual sets of blanks ( isotope mass balance corrections ) .", "seawater as source of carbonates leading to possible enrichment of corg in case of un - acidified aluminum bacteria filters was eliminated by the rinse with mq water at the end of filtration .", "the dried sponge , coralline algae , and mucus material were first homogenized in a mortar . for isotope measurements , adequate amount of material was transferred to pre - combusted silver boats for acidification within cups , followed by oven drying to remove excess acid . from the remaining grounded material , samples from the b1 reef station", "were also analyzed for their total fatty acid composition using a method of one - step procedure of fatty acid extraction and methylation ( masood et al .", "fatty acid extraction and preparation of methyl esters ( fame ) were carried out according to masood et al .", "( 2005 ) with reagent volumes adapted for use in 2.5-ml gc - vials using fame c19:0 as internal standard to calculate concentration of fas ( van gaever et al .", "these individual samples were analyzed separately for their fa compositions employing a large volume splitless injection method on a thermo finnigan trace ultra gc .", "main items of this method are the large volume liner with glass wool , pre - column deactivated silica 5  m    0.53  m , and analytical column sge bpx-70 50  m    0.32  mm    0.25  m .", "the identification of fames was based on the comparison of retention times with authentic commercially available reference material and standards .", "fatty acid source designation was achieved using data on distinctive fa for bacteria , algal carbon , and higher organisms ( e.g. , parrish et al . 2000 ; boschker and middelburg 2002 ; volkman 2006 ) .", "we ran one- and two - way anovas with non - transformed stable isotope data to determine whether variations in stable isotopes of sponges were due to sponge species and/or sponge location assuming no interaction effects ( not enough replication for testing interaction ) .", "the tukey s hsd method was used for pair wise comparisons of different sponge species ( multiple comparisons of means ) .", "anova s were also run for isotope signals of reef and open water - derived suspended matter .", "the siar stable isotope mixing model analysis ( parnell et al . 2010 ) was applied to investigate probable solutions for the diet of sponges based on dual stable isotope data of four sources ( gfc and gff fractions of suspended matter , crustose coralline algae , and coral mucus ) .", "we used the following trophic enrichment factors with standard deviations for the sponges : 3.5    0.5 for n and 1    1 for  c ( vander zanden and rasmussen 2001 ; behringer and butler iv 2006 ) .", "the siar model takes data on organism isotopes and fits a bayesian model to their dietary habits based upon a gaussian likelihood with a mixture dirichlet - distributed prior on the mean .", "model fitting is via markov chain monte carlo ( mcmc ) which produces simulations of plausible values for each dietary source consistent with the data .", "the software package siar ( stable isotope analysis in r ) is freely available from the package section of the comprehensive r archive network website  http://cran.r - project.org/ ( parnell and jackson 2010 ) .", "fatty acid composition patterns were explored with multivariate analyses of fas composition profiles ( non - transformed percentage abundance ) using the program primer version 5 ( clarke and gorley 2006 ) .", "multi - dimensional scaling ( mds plots ) of fa composition and dual isotope label patterns were compared for six sponge species and the three different reef - derived resources ( cca , mucus of m. annularis , and mucus of m. mirabilis ) .", "similarities between fa profiles were investigated using the simper ( similarity of percentages ) function , and statistical differences were determined using the anosim ( analysis of similarities ) function ."], ["samples were collected from march 14 to 22 , 2006 , along four transects perpendicular to the sw coast of curaao ( fig .  1 ) .", "transects were placed 37  km apart from se to nw along the coast at lagun jan thiel ( ljt ) , at the ocean cruise terminal in otrabanda ( ct ) , at buoy 1 ( b1 ) of the carmabi reef close to piscaderabaai , and at slangenbaai ( sb ) .", "transects from the shore to open water cross the narrow fringing reefs and water masses representative for the variation in coral community composition and water quality along the sw coast of curaao ( van duyl 1985 ; gast et al .", "1999 ; van duyl and gast 2001 ) . along each transect ( ljt , ct , b1 , and sb ) , we sampled 2 stations : fig . ", "( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbeanan off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water anda coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "position of sampling transects ( ljt , ct , b1 , and sb ) along the southwest coast of curaao , with an inset of the geographic position of the island in the caribbean an off - shore open water station about 12  km distance from the reef where water was collected at 1517  m depth in the blue water and a coral reef station where we collected bottom water at 1517  m depth on the fore - reef slope , approximately 0.5  m above the coral bottom .", "water samples were taken with a 6  l niskin bottle operated by a scuba diver .", "the niskin bottle was repeatedly filled and hauled on board a small open boat and emptied in a clean and sample washed 20  l container . at each station", "( n  =  8) , we collected 20  l. on the fore - reef slope stations ( n  =  4 ) , we collected a few small pieces of the stony corals montastraea annularis and madracis mirabilis ( also siderastrea siderea and porites astreoides but only at b1 ) between 15 and 17  m depth , which were kept in seawater under natural light conditions until mucus collection on the next day . from the entrance of coral cavities at 1517  m depth on the fore - reef slope , we chiseled crusts of calcareous algae . from the same cavities ,", "various species of encrusting sponges were collected , up to eight different species per station ."], ["the 20-l water samples were filtered over 47-mm - diameter combusted glass microfiber filters ( whatman ) , first over a gfc filter ( nominal pore size 1.2  m ) and then over a gff filter ( nominal pore size 0.7  m ) .", "after sea water filtration , filters were shortly washed with milli - q water ( mq ) to remove salt and were dried at 50c in a stove and stored in aluminum foil in the freezer until processing .", "the remaining bacterioplankton in the gff filtrate was concentrated with a vivaspin filter cartridge driven by a master volt tube pump .", "the concentrate was subsequently filtered over a 0.2  m pore size 25-mm - diameter anopore disc ( aluminum oxide membrane filter , whatman ) .", "the discs were briefly washed with mq after seawater filtration and dried at 50c in a stove ( 12  h ) . after drying , the filter was crumbled in a clean acid - washed glass funnel , the integrated polypropylene support ring of the filter was removed and the filter fragments were transferred to combusted silver capsules , which were closed with tweezers .", "subsequently , the folded capsules were placed in coded trays and stored in the freezer until processing .", "live corals were air - exposed during mucus collection and positioned upside down connected to a stand .", "after shortly spraying the live coral tissue with mq , the mucus produced by the coral was collected via an acid - washed glass funnel in combusted glass vials .", "after collection of approximately 2  ml mucus per coral , mucus samples were dried in a stove at 50c before being stored dry in the freezer .", "the cavity sponges and coralline algae were quickly washed with mq to remove salt and the tissue was subsequently removed from the underlying substratum by knife and scalpel .", "small pieces were fixed in ethanol ( 80% ) for taxonomical identification of the different sponge species .", "the rest of the pieces were collected in combusted glass vials dried at 50c and stored dry in the freezer .", "sponges and putative food sources were subject to stable isotope analysis . both carbon and nitrogen ( except for bacteria for which only corg was measured ) isotopic composition of the samples were determined using a fisons cn analyser coupled on line via a finnigan conflo 2 interface , to a finnigan delta s mass spectrometer .", "the carbon and nitrogen isotope ratios are expressed as corg and n relative to the vienna pee dee belemnite standard and air , respectively , and the standard error of the measurements is ~0.15. all carbon isotope samples ( except bacteria that were caught on aluminum filters ) were acidified ( tissue directly with acid and glass filters in acid vapor ) prior to measurements and corrected for individual sets of blanks ( isotope mass balance corrections ) .", "seawater as source of carbonates leading to possible enrichment of corg in case of un - acidified aluminum bacteria filters was eliminated by the rinse with mq water at the end of filtration .", "the dried sponge , coralline algae , and mucus material were first homogenized in a mortar . for isotope measurements , adequate amount of material was transferred to pre - combusted silver boats for acidification within cups , followed by oven drying to remove excess acid . from the remaining grounded material , samples from the b1 reef station", "were also analyzed for their total fatty acid composition using a method of one - step procedure of fatty acid extraction and methylation ( masood et al .", "fatty acid extraction and preparation of methyl esters ( fame ) were carried out according to masood et al .", "( 2005 ) with reagent volumes adapted for use in 2.5-ml gc - vials using fame c19:0 as internal standard to calculate concentration of fas ( van gaever et al .", "these individual samples were analyzed separately for their fa compositions employing a large volume splitless injection method on a thermo finnigan trace ultra gc .", "main items of this method are the large volume liner with glass wool , pre - column deactivated silica 5  m    0.53  m , and analytical column sge bpx-70 50  m    0.32  mm    0.25  m .", "the identification of fames was based on the comparison of retention times with authentic commercially available reference material and standards .", "fatty acid source designation was achieved using data on distinctive fa for bacteria , algal carbon , and higher organisms ( e.g. , parrish et al ."], ["we ran one- and two - way anovas with non - transformed stable isotope data to determine whether variations in stable isotopes of sponges were due to sponge species and/or sponge location assuming no interaction effects ( not enough replication for testing interaction ) .", "the tukey s hsd method was used for pair wise comparisons of different sponge species ( multiple comparisons of means ) .", "anova s were also run for isotope signals of reef and open water - derived suspended matter .", "the siar stable isotope mixing model analysis ( parnell et al . 2010 ) was applied to investigate probable solutions for the diet of sponges based on dual stable isotope data of four sources ( gfc and gff fractions of suspended matter , crustose coralline algae , and coral mucus ) .", "we used the following trophic enrichment factors with standard deviations for the sponges : 3.5    0.5 for n and 1    1 for  c ( vander zanden and rasmussen 2001 ; behringer and butler iv 2006 ) .", "the siar model takes data on organism isotopes and fits a bayesian model to their dietary habits based upon a gaussian likelihood with a mixture dirichlet - distributed prior on the mean .", "model fitting is via markov chain monte carlo ( mcmc ) which produces simulations of plausible values for each dietary source consistent with the data .", "the software package siar ( stable isotope analysis in r ) is freely available from the package section of the comprehensive r archive network website  http://cran.r - project.org/ ( parnell and jackson 2010 ) .", "fatty acid composition patterns were explored with multivariate analyses of fas composition profiles ( non - transformed percentage abundance ) using the program primer version 5 ( clarke and gorley 2006 ) .", "multi - dimensional scaling ( mds plots ) of fa composition and dual isotope label patterns were compared for six sponge species and the three different reef - derived resources ( cca , mucus of m. annularis , and mucus of m. mirabilis ) .", "similarities between fa profiles were investigated using the simper ( similarity of percentages ) function , and statistical differences were determined using the anosim ( analysis of similarities ) function ."], ["the sponge species collected on the reef stations were identified on basis of their tissue and skeletal characteristics .", "2007 ) to 20.7 ( scopalina ruetzleri ) and n values ranging from 2.9 ( monanchora arbuscula ) to 7.1 ( scopalina ruetzleri ) ( table  1 ) .", "variations in corg and n data were significantly related to sponge species ( anova , f(5,17 )  =  30.3 and f(5,17 )  =  15.8 , p  <  0.01 for both ) and not to reef station ( f(3,17 )  =  1.16 and f(3,17 )  =  0.83 , p  >  0.35 for both ) .", "0.05 ) were found between s. ruetzleri and h. caerulea , s. ruetzleri and niphates erecta , n. erecta and m. arbuscula , which were sampled at three to four reef sites ( table  1 ) .", "differences in dual stable isotope values between sponge species were small when compared to isotope values of sources .", "therefore , the sponge data of the different transects were combined in the fig . ", "2a , b. the stable isotope signals of particulate organic matter size fractions were not significantly different between the open water and reef water ( anova , p  >  0.05 ) , but were kept separate in fig . ", "2a , b. figure  2a shows that open water and reef water bacteria have on average slightly different c signatures .", "variations in corg values of bacterioplankton were large between stations ( ljt and sb vs. ct and b1 , table  2 ) . only the reef water bacteria of station sb ( c : 17.9 ) were in trophic reach of sponges ( < 2 difference ) .", "the sponge trophic range overlaps with the range of coralline algae ( average c of 16.9    4.2 ) .", "coral mucus of montastraea annularis and madracis mirabilis was on average heavier in corg content than the sponges ( table  2 ) .", "coral mucus and coralline algae are potential sponge food because of a trophic level n increase of 34 ( fig . ", "2b).table  1sponge species collected at four reef stations with their respective average isotope signaturescryptic biotastationljtlagun jan thielctcruise terminalb1carmabi reefsbslangenbaaicodecncncncn \n halisarca caerulea \n\n n  =  7sp118.793.6918.454.1218.333.1618.484.0218.204.0917.874.0718.734.63 \n clathria ( thalysis ) raraechelae \n\n n  =  1sp218.914.03 \n chondrilla caribensis \n\n n  =  1sp317.163.83 \n chelonaplysilla erecta \n\n n  =  1sp419.944.68 \n desmapsamma anchorata \n\n n  =  1sp518.815.61 \n scopalina ruetzleri \n\n", "n  =  6sp619.765.9020.264.8520.795.4820.026.1319.417.0720.065.86 \n eurypon laughlini \n\n n  =  218.955.6718.515.78 \n topsentia ophirhaphidites \n\n n  =  219.635.3719.275.82 \n niphates erecta \n\n n  =  617.704.0117.654.7917.934.4717.994.2117.974.7118.004.71 \n callyspongia vaginalis \n\n n  =  217.723.7917.474.37 \n monanchora arbuscula \n\n n  =  520.383.8320.403.4219.823.2819.952.8419.473.63 \n phorbas amaranthus \n\n", "n  =  118.864.40codes sp1sp6 refer to sponges collected at b1 and correspond to sponge data presented in fig .  3 \n fig . ", "2plots of carbon ( a ) and nitrogen ( b ) stable isotope signatures of material collected from the four reef and four open water stations ( average and standard error ) .", "sponges ( n  =  35 ) , bacteria ( bact n  =  8) , coral mucus ( n  =  9 ) , glass microfiber filters coarse ( gfc ) and fine ( gff ) represent values of suspended matter ( spm ) collected either from reef water or from open water offshore ( n  =  4 or 5 ) .", "crustose coralline algae from two reef stations ( n  =  3)table  2average isotope signatures of putative food sources for sponges collected from reef stations ljt , ct , b1 , and sb ( replicates , n  =  23 , standard errors range from 0 to 0.4 , not shown)substrate sourcesstationljtlagun jan thielctcruise terminalb1carmabi reefsbslangenbaaicodecncncncnspm - gfc  open water21.775.2723.873.3722.967.0021.613.94  reef water23.724.5123.385.1723.833.6722.532.66  crevice water21.402.96spm - gff  open water20.863.8123.921.9725.872.8421.524.69  reef water21.194.5324.863.0024.364.4022.093.80  crevice water24.083.20bacteria  open water21.93nd26.02nd27.31nd19.42nd  reef water15.36nd26.40nd27.41nd17.85ndmacroalgae  coralline algaecca13.373.6025.201.3012.124.05coral mucus  m. annularis \n mu115.460.5415.784.1216.462.3514.243.21  m. mirabilis \n mu217.952.1216.534.2417.641.71  s. siderea \n 17.800.55  p. astreoides \n 16.871.11suspended particulate matter ( spm ) fractions collected on coarse ( gfc ) and fine ( gff ) glass fiber filters .", "codes cca and mu1 , mu2 refer to data collected at b1 and correspond to data presented in fig . ", "3 \n\n nd not done sponge species collected at four reef stations with their respective average isotope signatures codes sp1sp6 refer to sponges collected at b1 and correspond to sponge data presented in fig . ", "3 \n  plots of carbon ( a ) and nitrogen ( b ) stable isotope signatures of material collected from the four reef and four open water stations ( average and standard error ) . sponges ( n  =  35 ) , bacteria ( bact n  =  8) , coral mucus ( n  =  9 ) , glass microfiber filters coarse ( gfc ) and fine ( gff ) represent values of suspended matter ( spm ) collected either from reef water or from open water offshore ( n  =  4 or 5 ) .", "crustose coralline algae from two reef stations ( n  =  3 ) average isotope signatures of putative food sources for sponges collected from reef stations ljt , ct , b1 , and sb ( replicates , n  =  23 , standard errors range from 0 to 0.4 , not shown ) suspended particulate matter ( spm ) fractions collected on coarse ( gfc ) and fine ( gff ) glass fiber filters . bacterioplankton ( bacteria ) was collected on aluminum oxide ( anopore ) membrane filters .", "codes cca and mu1 , mu2 refer to data collected at b1 and correspond to data presented in fig . ", "3 \n  pooled analysis with the mixing model ( no distinction between sites or sponge species ) show that mucus ( mu1 and mu2 combined ) contributed to the average cavity sponge diet between 44 and 57% ( = 95% low and high confidence limits , mode 50% ) , cca between 0 and 10% ( mode 2% ) , and the different spm fractions between 0 and 33% ( mode 7% ) for gfc and 14 and 47% ( mode 37% ) for gff ( table  3a ) . of the overall diet 51% ( mode ) was reef - derived .", "therefore , we ran the siar mixing model also separate for the stations for which cca data were available , ct and b1 ( tables  2 , 3b , c ) . pooled analysis of sponges at ct ( no distinction between sponge species ) reflected a higher cca contribution ( between 6 and 43% , mode 31% ) than mucus ( 046% , mode 17% ) . together", "suspended matter supplemented the diet between 0 and 40% for the gfc fraction ( mode 17% ) and 1155% for the gff fraction ( mode of gfc and gff fractions together 51% , table  3b ) . at b1 , where coral cover is higher than at ct ( van duyl 1985 ; pers comm ) , the diet of an average cavity sponge ( no distinction between sponge species ) was dominated by reef - derived sources ( 73% ) with coral mucus and cca contributing for 60 and 13% ( mode ) and with 95% confidence between 5068% and 030% , respectively ( table  3c ) .", "the contribution of suspended matter fractions gfc and gff was within the 028/030% range ( modes 11 and 5% ) , respectively.table  3dual isotope mixing model analyses of the diet of spongessourceslow 95%high 95%modemeanmode%mean%(a ) average diet of sponges ( four stations ) \n gfc ro00.3260.0710.138  gff ro0.1400.4740.3710.32144.245.9  cca00.0950.0150.039  mucmix0.4350.5720.4980.50251.354.1(b ) diet of sponges at ct \n gfc ro00.4020.1700.194  gff ro0.1120.5520.3350.33350.552.7  cca0.0630.4280.3080.254  mucmix00.4550.1720.2184847.2(c ) diet of sponges at b1 \n gfc ro00.2750.1060.122  gff ro00.3000.0530.13015.925.2  cca0.0010.3010.1310.155  mucmix0.5020.6820.5960.59372.774.8the sources comprise two suspended matter size fractions , gfc ro > 1.2  m and gff ro > 0.7 and < 1.2  m from reef overlying and open water ( ro ) , cca representing crustose coralline algae and mucmix representing coral mucus of two different coral species , m. annularis and m. mirabilis .", "the 95% credibility intervals are presented of the different sources as well as the mode and the mean . mode% and", "mean% show the contribution of plankton - derived ( gfc and gff ) and reef - derived organic matter ( cca and mucmix ) in the diet as %   dual isotope mixing model analyses of the diet of sponges the sources comprise two suspended matter size fractions , gfc ro > 1.2  m and gff ro > 0.7 and < 1.2  m from reef overlying and open water ( ro ) , cca representing crustose coralline algae and mucmix representing coral mucus of two different coral species , m. annularis and m. mirabilis .", "the 95% credibility intervals are presented of the different sources as well as the mode and the mean . mode% and", "mean% show the contribution of plankton - derived ( gfc and gff ) and reef - derived organic matter ( cca and mucmix ) in the diet as %   for six sponge species , cca and mucus of two coral species at b1 dual isotopes as well as fatty acid patterns were measured ( fig .  3 ; table  4 ) .", "fas in sources were also present in most sponges , but the diversity in fas was higher in sponges .", "differences between sources ( cca and mucus ) and sponges in the fatty acid compositions are illustrated in fig . ", "the euclidean distances show that the fas of sponges cluster together and are distinct from the sources .", "main fas of the sponge cluster included , in order of importance 16:0 , 20:46 , 18:0 , 20:53 , 18:17c , and branched 20:0 ( phytanic acid ) whereas in the case of the potential energy sources this included 16:0 , 18:0 , 18:19c , and 20:46 ( table  4 ) .", "the source groups were significantly different ( anosim , global r  =  0.8 , p  <  0.05 ) from the sponge group and simper analysis revealed that the differences are the relatively higher percentage abundance of 16:0 , 20:46 , 18:0 , and 18:19c in coral mucus and coralline algae but higher percentage abundance of phytanic acid , 24:0 and 20:53 in the sponges .", "these 7 fas together accounted for ~60% of the fa dissimilarity ( table  4 ; fig . ", "4 ) . among the three different sources , coralline algae , mucus of montastraea annularis , and madracis mirabilis , composition patterns were also significantly different ( anosim , global r  =  1 , p  <  0.05 ) .", "simper analysis revealed two very dominant fatty acids in the coralline algae ( 16:0 and 20:46 ) that together accounted for ~73% of the fa ; in mucus from m. annularis ( mu1 ) , four abundant fas made up ~70% ( 16:0 , 18:0 , 20:53 , and 18:19c ) ; in mucus from m. mirabilis ( mu2 ) , three abundant fas made up ~70% ( 16:0 , 18:0 , and 20:46 , table  2 ) .", "the major fas accounting for the dissimilarity between mucus from the two different coral species is the relatively higher percentage abundance of 20:46 and 18:0 in m. mirabilis ( mu2 ) but a relatively higher percentage abundance of 18:17c and 20:53 in m. annularis ( mu1 ) with these 4 fas accounting for ~60% of the dissimilarity between the coral species ( table  4 ) .", "three groups of sponges were distinguished among the 6 sponge species ( fig .  4 : h. caerulea ( sp1 ) , c. caribensis ( sp3 ) and c. erecta ( sp4 )", "make up the first group ; c. raraechelae ( sp2 ) and d. anchorata ( sp5 ) the second and s. ruetzleri ( sp6 ) the third group ) .", "simper analysis revealed different combinations of fas , in order of dominance , accounting for ~70% of sponge group similarity : for the first group ( sp1 , sp3 , and sp4 ) fas 16:0 , br 20:0 ( phytanic acid ) , 18:0 , 20:46 , i15:0 , and 16:17c are the main fas . in the second group ( sp2 and sp5 ) fas 16:0 , 20:46 , 24:0 , 18:17c , 22:0 , 20:53 , and 20:63 determined primarily the composition pattern . in the third group ( sp6 ) , these were the five fas : 16:0 , 20:46 , 18:0 , 20:53 , and 14:0 ( table  4).fig . ", "3carbon and nitrogen isotopes of six sponge species ( sp1sp6 ) and putative organic matter sources for sponges at reef site b1 .", "the average and standard error for sp1 ( n  =  3 ) and sp6 ( n  =  2 ) , gfc - spm ( n  =  3 ) , gff - spm ( n  =  3 ) is presented .", "other data points ( sp2 , 3 , 4 , and 5 , cca and mucus from two coral species , montastraea annularis ( mu1 ) and madracis mirabilis ( mu2 ) do not have replicates other than repetitive sample analysis of the same specimen ) .", "see also tables  1 , 2 \n table  4fatty acid composition of six sponge species ( sp1sp6 , see table  1 ) , one crustose coralline alga ( cca ) and mucus ( mu ) from two coral species ( see table  2 ) , all collected from reef site b1sp1sp2sp3sp4sp5sp6ccamu1mu2i14:0 \n 0 ( 0)0 ( 0)0.7 ( 0.02)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)14:06.6 ( 0.55)2.6 ( 0.05)2.3 ( 0.08)3.6 ( 0.40)3.8 ( 0.55)9.1 ( 0.24)3.5 ( 0.10)5.3 ( 0.21)2.6 ( 0.05)ai15:0 \n 0.6 ( 0.05)0 ( 0)2.6 ( 0.02)0.5 ( 0.27)0.1 ( 0.07)0.3 ( 0.06)0.04 ( 0.04)0 ( 0)0 ( 0)i15:0 \n 5.3 ( 0.24)0.3 ( 0.06)9.8 ( 0.05)5.4 ( 0.25)0.8 ( 0.04)0.5 ( 0.16)0 ( 0)3.8 ( 1.90)4.2 ( 0.08)15:01.2 ( 0.11)0.5 ( 0.01)1.1 ( 0.07)1.4 ( 0.07)1.7 ( 0.15)7.5 ( 0.23)0.3 ( 0.07)0 ( 0)0 ( 0)i16:0 \n 0.6 ( 0.08)0.4 ( 0.02)1.3 ( 0.02)0.4 ( 0.20)0.7 ( 0.05)1.9 ( 0.05)0.3 ( 0.02)0 ( 0)0 ( 0)10me-16:0 \n 0 ( 0)0 ( 0)0 ( 0)0 ( 0)0.4 ( 0.07)0 ( 0)0 ( 0)0 ( 0)0 ( 0)16:260 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0.51 ( 0.03)16:240 ( 0)0.2 ( 0.03)0 ( 0)0 ( 0)0.5 ( 0.00)0.1 ( 0.09)0.04 ( 0.04)0 ( 0)0 ( 0)16:15c0 ( 0)0.7 ( 0.03)0.9 ( 0.03)0.4 ( 0.18)0.6 ( 0.03)0.4 ( 0.02)0 ( 0)0 ( 0)0 ( 0)16:17c \n 3.3 ( 0.16)1.6 ( 0.02)12.6 ( 0.03)4.0 ( 0.11)4.2 ( 0.16)2.1 ( 0.04)0.5 ( 0.06)3.9 ( 0.50)0.6 ( 0.04)16:17 t \n 0 ( 0)0.15 ( 0.02)0.79 ( 0.01)0 ( 0)0.1 ( 0.07)0.3 ( 0.04)0 ( 0)0 ( 0)0 ( 0)16:017.1 ( 1.73)13.2 ( 0.27)18.4 ( 0.19)20.5 ( 0.39)12.1 ( 0.62)19.6 ( 0.52)40.7 ( 0.21)30.8 ( 0.84)27.8 ( 0.66)10me-17:0 \n 0 ( 0)0 ( 0)0.8 ( 0.15)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)17:17c0.9 ( 0.66)0.1 ( 0.00)1.9 ( 0.10)8.5 ( 0.42)0.5 ( 0.11)0.7 ( 0.22)0.4 ( 0.12)0 ( 0)0 ( 0)17:01.9 ( 0.08)0.5 ( 0.04)0.5 ( 0.49)2.6 ( 0.13)1.4 ( 0.00)0.5 ( 0.09)0.3 ( 0.00)0.5 ( 0.03)0 ( 0)i18:0 \n 0 ( 0)0.6 ( 0.05)0.3 ( 0.05)0 ( 0)0.6 ( 0.09)0.4 ( 0.02)0 ( 0)0 ( 0)0 ( 0)18:530.2 ( 0.22)1.1 ( 0.03)0.2 ( 0.05)0 ( 0)1.7 ( 0.23)0.1 ( 0.05)0.1 ( 0.00)1.5 ( 0.04)0 ( 0)18:50 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)0 ( 0)2.1 ( 1.03)0 ( 0)18:360.6 ( 0.56)0.3 ( 0.02)0.1 ( 0.04)0 ( 0)0 ( 0)0 ( 0)0 ( 0)4.1 ( 0.31)0 ( 0)18:330.5 ( 0.25)0.1 ( 0.07)0 ( 0)0.8 ( 0.21)0.3 ( 0.26)0 ( 0)0 ( 0)0 ( 0)0 ( 0)18:26c1.8 ( 0.12)0.8 ( 0.07)0.3 ( 0.02)2.2 ( 0.09)0.6 ( 0.30)0.9 ( 0.06)0.8 ( 0.02)0.4 ( 0.20)0.8 ( 0.05)18:17c5.7 ( 0.12)9.7 ( 0.06)3.3 ( 0.10)5.5 ( 0.04)6.6 ( 0.15)2.0 ( 0.06)4.5 ( 0.05)8.1 ( 0.33)0.7 ( 0.03)18:19c2.5 ( 0.50)1.9 ( 0.06)1.9 ( 0.11)4.2 ( 0.14)1.4 ( 0.26)2.0 ( 0.08)11.2 ( 0.10)9.1 ( 0.32)7.3 ( 0.12)18:09.4 ( 0.25)4.0 ( 1.61)9.7 ( 0.23)10.2 ( 0.29)5.6 ( 0.46)11.5 ( 0.06)3.0 ( 0.02)15.5 ( 0.34)25.4 ( 0.74)i20:0 \n 0.2 ( 0.20)0.8 ( 0.01)0 ( 0)0 ( 0)1.1 ( 0.14)0 ( 0)0 ( 0)0 ( 0)0 ( 0)20:534.5 ( 0.26)6.3 ( 0.24)3.4 ( 0.01)3.0 ( 0.25)5.0 ( 0.36)11.7 ( 0.26)0.1 ( 0.05)11.0 ( 0.15)4.4 ( 0.44)20:430 ( 0)0.10 ( 0.05)0 ( 0)0 ( 0)0.4 ( 0.08)0.7 ( 0.35)0.3 ( 0.30)0 ( 0)0 ( 0)20:4610.6 ( 0.68)11.1 ( 0.24)9.1 ( 0.25)8.1 ( 0.11)10.7 ( 0.48)15.2 ( 0.27)32.2 ( 0.75)0 ( 0)15.0 ( 0.34)20:360.7 ( 0.13)0.7 ( 0.12)0.5 ( 0.06)0.6 ( 0.08)0.5 ( 0.03)0.5 ( 0.05)0.4 ( 0.03)0 ( 0)0 ( 0)20:330 ( 0)0.1 ( 0.07)0 ( 0)0.1 ( 0.12)0.3 ( 0.13)0.1 ( 0.13)0 ( 0)0 ( 0)0 ( 0)phytanic acid15.5 ( 1.34)2.9 ( 0.13)13.4 ( 0.26)8.3 ( 0.29)1.8 ( 0.30)1.3 ( 0.19)0.2 ( 0.03)0 ( 0)0 ( 0)20:19c0.6 ( 0.39)0.2 ( 0.05)0.1 ( 0.00)0 ( 0)0.5 ( 0.10)0.1 ( 0.05)0.2 ( 0.04)0 ( 0)0.3 ( 0.28)20:290.3 ( 0.15)0.3 ( 0.05)0 ( 0)0.8 ( 0.02)0.9 ( 0.40)2.6 ( 0.04)0.1 ( 0.04)0 ( 0)0 ( 0)20:00.9 ( 0.04)2.2 ( 0.06)0.8 ( 0.04)1.7 ( 0.05)3.5 ( 0.16)2.5 ( 0.03)0.3 ( 0.05)3.1 ( 0.14)6.4 ( 0.21)22:631.0 ( 0.01)8.4 ( 0.14)0.1 ( 0.06)2.6 ( 0.27)3.6 ( 0.28)1.9 ( 0.02)0 ( 0)0 ( 0)0 ( 0)22:530 ( 0)0 ( 0)0.1 ( 0.09)2.8 ( 0.30)0 ( 0)0 ( 0)0.1 ( 0.03)0 ( 0)0 ( 0)22:460.5 ( 0.06)1.9 ( 0.14)0 ( 0)0.6 ( 0.58)0.3 ( 0.12)0 ( 0)0 ( 0)0.9 ( 0.44)1.9 ( 0.14)22:1110.2 ( 0.20)0.1 ( 0.03)0.5 ( 0.06)0.1 ( 0.11)0 ( 0)0 ( 0)0.1 ( 0.03)0 ( 0)0 ( 0)22:19c0 ( 0)0 ( 0)0.1 ( 0.05)0 ( 0)0 ( 0)1.0 ( 0.08)0.1 ( 0.07)0 ( 0)0 ( 0)22:01.9 ( 0.18)4.2 ( 0.25)0.7 ( 0.03)0.4 ( 0.19)18.1 ( 0.89)0.8 ( 0.20)0.2 ( 0.05)0 ( 0)2.2 ( 0.09)23:01.5 ( 0.17)2.1 ( 0.34)0.9 ( 0.05)0.1 ( 0.12)2.0 ( 0.14)0.1 ( 0.06)0 ( 0)0 ( 0)0 ( 0)24:03.5 ( 0.31)20.3 ( 0.96)1.1 ( 0.10)0.6 ( 0.06)7.8 ( 0.32)1.8 ( 0.04)0.3 ( 0.05)0 ( 0)0 ( 0)% bact - spec6.6/9.92.2/3.816.2/28.76.3/10.33.7/7.83.4/5.50.3/0.83.8/7.74.2/4.8average %    se ( n  =  3 ) \n bacteria - specific fa and last row is average % bacteria specific fa ( bact - spec ) with / without inclusion of 16:17cfig . ", "4multidimensional scaling ( mds ) plot showing similarity in fatty acid composition profiles of six sponge species ( sp16 ) , crustose coralline algae ( cca ) and the mucus of two coral species , montastraea annularis and madracis mirabilis , at reef site b1 ( see table  4 for list of fatty acids ) carbon and nitrogen isotopes of six sponge species ( sp1sp6 ) and putative organic matter sources for sponges at reef site b1 .", "the average and standard error for sp1 ( n  =  3 ) and sp6 ( n  =  2 ) , gfc - spm ( n  =  3 ) , gff - spm ( n  =  3 ) is presented .", "other data points ( sp2 , 3 , 4 , and 5 , cca and mucus from two coral species , montastraea annularis ( mu1 ) and madracis mirabilis ( mu2 ) do not have replicates other than repetitive sample analysis of the same specimen ) . see also tables  1 , 2 \n  fatty acid composition of six sponge species ( sp1sp6 , see table  1 ) , one crustose coralline alga ( cca ) and mucus ( mu ) from two coral species ( see table  2 ) , all collected from reef site b1 average %    se ( n  =  3 ) \n bacteria - specific fa and last row is average % bacteria specific fa ( bact - spec ) with / without inclusion of 16:17c multidimensional scaling ( mds ) plot showing similarity in fatty acid composition profiles of six sponge species ( sp16 ) , crustose coralline algae ( cca ) and the mucus of two coral species , montastraea annularis and madracis mirabilis , at reef site b1 ( see table  4 for list of fatty acids ) for these three distinguished sponge groups at b1 , we also ran the isotope mixing model ( table  5 ) .", "the first group appears to rely mainly on reef - derived sources ( mucus and cca , mode and mean both > 75% ) .", "the second group probably relied less on mucus and more on cca and used more plankton - derived food compared with group 1 .", "ruetzleri only ) consists for about 51% ( mean ) of reef - derived and 49% ( mean ) of suspended matter ( plankton ) derived food .", "group 1 has a smaller centroid euclidean distance ( 23.64 ) to the average mucus mixture than groups 2 and 3 ( 32.43 and 24.55 ) in fig . ", "this concurs with a larger contribution of mucus ( 66% ) to the diet of group 1 than that of the other two groups ( 36 and 33% ) ( fig .  4 ; table  5 ) .", "the lower contribution of cca in the diet of group 1 ( 10% mode ) compared to group 3 ( 19% mode ) was also reflected in fig .  4 .", "we did not find this for group 2 , which had the longest euclidean distance to cca ( 41.81 vs. 33.78 of group 3 and 37.37 of group 1 ) , while cca - derived food comprised 22% in the diet of this group . to further explore the relation between the stable isotope and fa data sets", ", we compared the matrices of fas and dual isotope signatures of sponges and reef resources ( figs .  3 , 4 ) and found a significant correlation between the two matrices ( primer , rho  =  0.625 , p  =  < 0.01).table  5results of dual isotope mixing model analyses for different sponge groups at b1sourceslow 95%high 95%modemeanmode%mean%(a ) diet of group 1 \n\n h. caerulea , c. caribensis , c. erecta ( sp1 , sp3 , sp4)gfc ro00.2840.0360.117gff ro00.2440.0360.1047.222.1cca00.2860.0950.132mucmix0.5040.7960.6580.64775.377.9(b ) diet of group 2 \n\n c. raraechelae , d. anchorata ( sp2 , sp5)gfc ro00.4320.1670.216gff ro00.430.2020.20836.942.4cca00.4510.2190.219mucmix0.0770.6060.3570.35757.657.6(c ) diet of group 3 \n\n s. ruetzleri ( sp6)gfc ro0.0030.4870.2920.259gff ro00.4530.2850.23257.749.1cca00.4210.1880.207mucmix0.0230.5160.3270.30251.550.9 for further explanation , see table  3 \n  results of dual isotope mixing model analyses for different sponge groups at b1   for further explanation , see table  3 \n"], ["cryptic encrusting sponges living in coral cavities on the fore - reef slope of fringing reefs sequester most of their organic matter from the passing water . they efficiently filter the pico- and nanoplankton , but dissolved organic matter ( dom ) appears to be the major resource of various sponge species living on the open reef and in coral cavities ( yahel et al . 2003 ; de goeij et al .", ". it may be dom released from phytoplankton , dom supplied to the reef from outside or dom released from the reef benthos .", "gradients in dom concentrations over the reef suggest that there is a net release of dom from coral reef waters to adjacent open waters ( van duyl and gast 2001 ) .", "only 1020% of the dom in reef water appears to be available for sponges ( de goeij et al .", "2008a ) . by analyses of dual stable isotope signatures ( corg and n ) and fa patterns of sponges and their potential food sources , we obtained circumstantial evidence that the reef itself mainly feeds her cavity sponges .", "this remarkable finding implies that cryptic sponges support reef internal nutrient recycling and conservation of reef organic matter .", "it was surprising that none of the 12 sponge species examined appeared to live mainly on suspended particulate organic matter or dom derived from this source .", "the corg values of the spm fractions were in the range reported for marine suspended matter ( peterson and fry 1987 ) .", "bacterioplankton values were within 2 of spm in open water , which indicates that bacteria were the primary consumers of spm .", "this makes it highly likely that spm represents predominantly phytoplankton and that the bacterioplankton reflects the c isotope ratio of reactive dom released by phytoplankton ( e.g. , harvey et al .", "the c of spm did not change during passage over the reef , for instance by obtaining a typical c spm reef signal ( 17 to 20 : land et al .", "the n of spm did not change either and remained ~4. moreover , at two of the four stations ( ct and b1 ) comparable corg values of predominantly heterotrophic bacterioplankton ( 26 , 27 , table  2 ) between open water and reef bottom water suggest that this bacterioplankton remained dependent on phytoplankton doc . such low c values have been reported , for e.g. , synechococcus ( c 33 to 25 , brutemark et al . 2009 ) and prochlorococcus , both abundant in reef and open water along curaao ( van duyl et al . 2002 ) .", "residence time of the spm and bacterioplankton in reef overlying waters may have been too short for changes in isotopic ratios either due to rapid water exchange with the open water and/or due to spm removal by benthic suspension feeders .", "phytoplankton concentrations in reef surrounding waters in curaao are usually low ( van duyl et al . 2002 ) , and the organic matter demand of cryptic biota , cryptic sponges in particular , is high .", "cavity biota between 10 and 15  m depth on the fore - reef slope in curaao consume on average 350  mmol  c  m  d of which cavity sponges consume on average 256  mmol  c  md ( 73% ) with the doc fraction comprising > 90% of the diet ( de goeij et al .", "this organic matter supply is orders of magnitude higher than the supply of pelagic primary production to reefs ( richter et al .", "it is evident that the supply of open water - derived phytoplankton and its dom release is insufficient to satisfy the total organic carbon requirements of coral reef cavity sponges . in accordance ,", "the isotope mixing model results confirmed a spm contribution of less than < 50% in the diet of most sponges .", "this implies that the examined sponges with average corg of 18.9 and n of 4.6 mainly assimilate other sources than pelagic primary production .", "presence of distinct reactive dom in reef bottom water compared to open water was demonstrated by the increased c value of bacterioplankton at two of the four stations ( ljt and sb ) .", "bacterioplankton apparently assimilated a source of carbon not available in the open water , a source , which may also be available to the dom feeding cavity sponges .", "benthic primary producers usually have higher corg values than pelagic primary producers ( france 1995 ; fry 2006 ) .", "thus , assimilation of benthic algae - derived reactive doc may explain the elevated c values in bacterioplankton in reef bottom water at ljt and sb .", "bacteria respond quickly when exposed to changing resources in the vicinity of the reef bottom .", "enhanced bacterioplankton production in reef bottom waters , cavity waters , and coral mucus point to the reef bottom as the source of inorganic nutrients and reactive dom ( van duyl and gast 2001 ; scheffers et al .", "however , the c reef signal of bacterioplankton ( 17.9 to 15.4 ) was higher than that of most examined sponges ( 20.8 to 17.2 ) suggesting that sponges and bacterioplankton may not fully depend on the same source of reef - derived dom .", "sponges may exploit a wider size range of the operationally defined dom fraction than bacterioplankton ( de goeij et al .", "benthic primary producers such as corals and benthic macroalgae have been reported to release reactive dom ( e.g. , crossland 1983 , 1987 ; wild et al .", "the c values of the benthic substrates ( coral mucus of two dominant coral species and crustose coralline algae ) were on average higher in c than the spm and open water bacterioplankton and were more in range of the sponge stable isotope signals .", "the n values of reef - derived substrates with values 34 lower than those of sponges on average suggest a direct trophic link ( one trophic level distance ) .", "the lower n values of reef substrates versus open water substrates may be due to enhanced n2 fixation by the reef benthos ( davey et al .", "discharge from land or pollution may counteract this effect by introducing organic and inorganic n enriched in n. this may partly explain the variations in n in mucus , coralline algae , and sponges between different stations . the range of corg in crustose coralline algae ( cca ) of 25.2 to 12.1 was unexpectedly large , possibly due to the unique ontogeny of cca .", "younger portions of the cca may have lower corg values than older portions possibly because rapid photosynthesis in younger parts depends more on respired co2 than photosynthesis in older parts ( lee and carpenter 2001 ) . anyway , the lower range of corg values of cca was within 2 of our sponges .", "moreover , the n of cca ( 1.34.0 ) make it a likely food source for the majority of cavity sponges with 34 higher n values than the cca .", "little is known of exudation of organic matter by cca apart from the releases of allelochemicals ( e.g. , kim et al .", "total dom release by cca has not been determined as far as we know , but their productivity equals that of fleshy macroalgae and turf algae on reefs ( chrisholm 2003 ) . reported c values of 18.7 to 16.8 for dictyota sp.,19.3 for halimeda sp .", "( raz - guzman macbeth and de la lanza espina 1991 ; lepoint et al .", "2000 ) , and n values of 0.53 of macroalgae on coral reefs ( yamamuro et al . 1995 ; france et al .", "1998 ) fall in the same range as cca ( corg 25.2 to 12.1 , n 1.34 ) .", "therefore , dom released from these brown and green macroalgae may have also contributed to the isotope signal in sponges .", "trophic transfers of organic matter derived from benthic macroalgae and mangroves , to sponges have been reported before ( behringer and butler iv 2006 ; granek et al .", "2009 ) and appear to be important in the nutrition of sponges . unlike cca , coral mucus has already been described as an important food source on coral reefs ( wild et al .", "1980 ; wild et al . 2004 ) and may be a suitable source of dom for cavity sponges .", "the c of the mucus samples of montastraea annularis and madracis mirabilis ( range 17.9 to 14.2 ) is higher than that of most cavity sponges , implying that sponges do not feed exclusively on mucus or mucus - derived dom .", "coral - derived organic matter is a reactive substrate for bacterioplankton growth ( ferrier - pags et al .", "2000 ) and may have contributed to the c reef signal of bacterioplankton at ljt ( 15.4 ) .", "the average n of mucus ( 1.9 ) and that of cavity sponges ( 4.6 ) are consistent with mucus / mucus - derived dom as a food source for most cavity sponges .", "it is evident that individually , neither phytoplankton , bacterioplankton , cca ( benthic macroalgae ) nor coral mucus can account for the sponge dual isotope signatures .", "the isotope mixing model analysis showed that cavity sponges incorporate from half to three quarters of their organic matter from mucus and cca ( benthic macroalgae ) with mucus as the dominant source of reef - derived organic matter ( up to 66% of the diet ) .", "it appears that with decreasing coral cover the contribution of coral mucus to the diet of sponges decreases and shifts to a larger contribution of cca ( benthic macroalgae ) and phytoplankton . at station", "ct with a lower coral cover than at the other reef stations , mucus only contributed 17% ( mode ) to the diet of sponges while the bulk food was provided by cca including other benthic macroalgae ( 31% ) and small phytoplankton ( 34% , gff fraction ) .", "this was different at b1 , where coral cover on the slope is relatively high ( van duyl 1985 ; bak and nieuwland 1995 ) and the sponges reflect a diet of mainly mucus ( 60% , mode ) with 13% cca .", "this suggests that the sponge community is opportunistic and consumes om according to availability irrespective of the dom source .", "this does not imply that all examined sponge species at a particular reef site have similar diets .", "this is further corroborated with our fa profiles of sponges at b1 . the overall similarity in fa composition pattern of", "the three groups within these six sponge species ( based on significantly different fa compositions ) also had different diets according to the isotope mixing model analysis , implying differences in the proportional use of mucus , benthic algae ( including cca ) , and phytoplankton - derived dom .", "this suggests that there is trophic niche segregation among sponges which may be an important factor facilitating co - existence of different sponge species ( thurber 2007 ) .", "furthermore , the differences in diet may to some extent be reflected in the fa compositions of sponge groups suggesting that the selected food resources may indeed form part of the diet of sponges .", "the fatty acid profiles of our coral mucus and cca sources resemble those reported for coral tissue and cca ( e.g. , latyshev et al . 1991 ;", "although many dominant fas such as 14:0 , 16:0 , and 18:0 ( table  2 ) are common to many organisms , some fas have been identified as potential tracer of food source in sponges . for this study area specifically , for example , de goeij et al .", "( 2008b ) conclude from isotope tracer experiments that the dominant fa 20:46 found in the sponge halisarca caerulea has an exogenous source .", "interestingly , 20:46 is found dominant in all 6 sponge species examined and it is also dominant in cca and m. mirabilis mucus .", "the high cover of cca in cavities ( 30% of the hard bottom surface in cavities , scheffers et al .", "2004 ) , its close proximity to cavity sponges and the fact that cca was rich in 20:46 , may have contributed to the 20:46 content in sponges . in sponge groups 2 and 3 , this fa was the second most abundant ( > 10% of total fas ) and the diet of these sponge groups consisted for 1922% ( mode ) of cca ( including other macroalgae ) . in sponge group 1 , the predominantly mucus feeding group ,", "20:46 was the third most abundant fa with a lower relative abundance than in the other groups ( < 10% ) .", "this coincided with a lower contribution of cca in their diet ( 10% mode ) .", "trophic transfer of this fa may be directly linked to the consumption of cca as part of macroalgal - derived organic matter .", "phytanic acid , which can be considered as a sponge biomarker ( de goeij et al .", "2008b ) was the second most abundant fa in group 1 . for its synthesis ,", "degradation products of chlorophyll a are required , possibly delivered to the sponge by sponge associated phototrophic microbes , considering the relatively high amount of the likely bacterial specific fa 16:17c as well as the overall high % of bacteria specific fas in group 1 ( up to 29% compared with up to 10% of total fas in the other groups , table  4 ) .", "sponge species in group 1 indeed harbor - rich microbial communities ( hill et al . 2005 ; erwin and thacker 2007 ) .", "mutualism between sponges and their associated microorganisms may have influenced the diet of group 1 compared with groups 2 and 3 .", "weisz et al . ( 2007 ) show that sponges with high versus low abundances of associated microbes may have distinct diets . in line , we show that the diet of sponges ( based on stable isotopes as well as fas ) varies with abundance of bacteria specific fas .", "interestingly , sponges with a relatively high abundance of associated microbes ( group 1 ) appear to be more dependent on reef - derived food than other sponges .", "the fa 20:53 , a dominant fa in ( benthic ) diatoms and the mucus of corals , particularly in mucus of m. annularis was most abundant in s. ruetzleri ( group 3 ) .", "the siar model analysis showed that the contribution of phytoplankton - derived food was highest in this sponge ( mean 49% and mode 57% , both plankton size fractions together ) , which may suggest that it feeds on diatoms or diatom - derived om rich in 20:53 .", "the other sponge groups ( 1 and 2 ) may obtain 20:53 predominantly by consuming mucus considering the higher contribution of mucus and lower plankton - derived food in the diet of these groups than in the diet of s. ruetzleri ( group 3 ) .", "diatom - derived om as well as mucus may have contributed to the abundance of 20:53 in sponges . considering the congruencies between the fa compositions and the dual stable isotope signals in the trophic transfer and relations between sources and consumers , it was not surprising that the matrices ( based on euclidean distances ) of the reef sources and consumers of both approaches were significantly correlated .", "both methods apparently lead to comparable results with fa composition and biomarkers identifying the food items of consumers , and evidencing trophic transfer and with dual stable isotope analyses estimating the contribution of the different sources to the diet of consumers .", "results of both methods strongly support a trophic transfer from zooxanthellate corals and cca to cavity sponges .", "the quantitative contribution of cca to the diet of sponges remains unresolved , because its dual isotope signal overlaps with that of other benthic macroalgae ."], ["results unambiguously point to the reef benthos as the main source of organic matter for encrusting cavity sponges on coral reefs .", "the contribution of open water - derived organic matter as food for the cavity sponge community was usually less than 50% .", "food sources for sponges on the reef comprise dom derived from coral mucus and benthic macroalgae ( cca ) .", "the prominent presence of a characteristic fatty acid of coral mucus and cca , 20:46 in sponges further confirms this trophic transfer .", "( 2008b , 2009 ) found that the cavity sponge halisarca caerulea converts assimilated dom in sponge biomass and detrital material . combining this with the knowledge that ( 1 ) the cover of cavity sponges is high on fore - reef slopes ( scheffers et al .", "2004 ) , ( 2 ) dom assimilation by cavity sponges is high ( de goeij et al .", "2008a ) , and ( 3 ) most of the om assimilated by sponges is reef - derived ( this study ) , we argue that cryptic sponges play a key role in conserving reef - produced dom for the reef system by converting it into pom . whether the  cryptic ", "sponge link in the coral reef food web is favorable for the coral reef health status remains to be investigated ."]]}
{"article_id": "PMC3419429", "article_text": ["the asialoglycoprotein receptor ( asgpr ) ( also known as the ashwell receptor ) mediates the capture and endocytosis of galactose- ( gal ) and n - acetylgalactosamine- ( galnac ) terminating glycoproteins .", "the relevance of this function has been the subject of much debate ; the primary role may be the removal of potentially hazardous glycoconjugates arising from normal tissue turnover , tissue injury , disease , and other causes .", "studies using knock - out mice have provided evidence for direct involvement of the asgpr in removal of abnormally sialylated plasma glycoproteins .", "mice lacking the sialyltransferase st3gal4 showed prolonged bleeding which was attributed to asgpr - mediated clearance of at least one plasma hemostatic component , von willebrand factor ( vwf ) , that showed decreased sialylation .", "findings in mice lacking the asgpr demonstrated that , during sepsis , the receptor removed components of hemostasis ( vwf and platelets ) that had been desialylated by bacterial neuraminidase and thereby allowed hemostatic adaptation that moderated disseminated intravascular coagulation and improved host survival .", "the asgpr may therefore be poised for rapid clearance of plasma glycoproteins that , for whatever reason , show decreased or abnormal sialylation .", "the highest level of expression of the asgpr is in the liver , in which it is located on the sinusoidal face of hepatocytes .", "low - level expression has been shown in various other cell types , such as , peritoneal macrophages in rat , human intestinal epithelial cells , mouse testis , and the rat thyroid gland .", "the galactosyl homeostasis theory proposes that the balance of both gal and galnac glycoconjugates is important for normal tissue physiology [ 1 , 9 ] ; whether or not expression of the asgpr at these other sites represents a need for localized constitutive control of such glycoconjugates is not known .", "a major area of interest focussing on the asgpr is the targeted gene transfer / delivery of drugs to the liver [ 10 , 11 ] .", "for example , using galnac as a ligand on a sirna - containing complex , the latter was successfully targeted to hepatocytes after simple intravenous injection into the tail vein in mice .", "moreover , sirna - mediated knock - down of targeted genes was demonstrated within the hepatocytes . besides its potential importance in allowing liver - specific drug targeting", ", the asgpr is a key factor in the design and administration of glycoprotein pharmaceuticals more generally : the activity of the receptor can impact upon drug half life and thereby the window of therapeutic efficacy .", "human asgpr is composed of two types of subunit : a major subunit ( asialoglycoprotein receptor 1 , asgr1 ) and minor subunit ( asialoglycoprotein receptor 2 , asgr2 ) .", "both subunits are type ii , single pass proteins that broadly comprise a cytoplasmic domain , transmembrane domain , and extracellular carbohydrate recognition domain ( crd ) [ 14 , 15 ]", ". the subunits may exist as asgr1-asgr2 heterooligomers , asgr1homotrimers and homotetramers , and asgr2 homodimers and homotetramers .", "these different quaternary forms may allow for functional differences , such as , substrate specificity or rate of endocytosis [ 1 , 16 ] . the genes encoding asgr1 and asgr2 ( asgr1 and asgr2 , resp . )", "are located on the short arm of autosome 17 , approximately 58.6 kilobases ( kb ) apart .", "the genes are evolutionarily related but differ significantly in their structural organization : asgr1 comprises 8 exons and is approximately 6 kb long , asgr2 contains 9 exons occupying 13.5  kb of dna [ 14 , 15 ] . until recently , asgr1 was thought to yield one transcript encoding a single protein .", "however , in 2010 , liu et al . demonstrated the existence of two alternatively spliced transcripts .", "the longer transcript , t1 , contains all 8 exons and is by far the more abundant ; it encodes full - length asgr1 ( isoform a , 291 aminoacids ) .", "the shorter transcript , t2 , has an in - frame deletion of exon 3 resulting in the loss of 39 residues ( isoform b , 252 amino acids ) .", "asgr2 gives rise to five transcripts ( th2 , t1 , t2 , t3 , and t4 ) encoding four isoforms ( a to d ) that contain different in - frame deletions arising from alternative exon splicing events .", "isoforms a and c contain 5 aminoacids that serve as a proteolysis cleavage signal near the junction between the transmembrane domain and the crd .", "cleavage at this site results in secretion of the crd as a soluble protein [ 18 , 19 ] .", "isoforms b and d lack this signal therefore they are not proteolytically cleaved but rather remain membrane bound where they may oligomerize with asgr1 isoform a to form native asgpr at the cell surface .", "the secreted forms of asgr1 and asgr2 are able to associate into soluble asgpr [ 17 , 19 ] .", "there is evidence to suggest that the soluble receptor may bind free substrates in the circulation and carry them to the liver for uptake and degradation . aside from membrane attachment", "/ secretion , it is not known whether further functional variation , such as , substrate specificity , may be associated with the different isoforms of asgr1 and asgr2 . based on the observation above that the asgpr", "is expressed in rat peritoneal macrophages , it was considered possible that monocytes , the lineage precursor of tissue macrophages , may also express the receptor . to the best of our knowledge ,", "the present study investigated the expression of asgr1 and asgr2 in human peripheral blood monocytes .", "the data showed that both asgr1 and asgr2 are expressed in human monocytes in the circulation , that expression can not be detected in lymphocytes and granulocytes , that the transcripts of both genes differ between individuals and that , in a given individual , the transcription profile for asgr2 is restricted to one of two patterns .", "the findings are of potential importance for health and disease in a variety of disciplines .", "genes , transcripts , and proteins are referred to by their formal scientific names as listed in the national centre for biotechnology information ( ncbi ) , which complies with standardized international nomenclature .  ", "gene sizes , exon numbering , and so forth were obtained from the reference sequences listed in table 1 .", "edta - anticoagulated whole blood in excess of that required for routine blood tests was anonymized and handled in accordance with medical research council guidelines .", "citrate- or edta - anticoagulated whole blood ( 5  ml ) was centrifuged at 200  g for 10 minutes , ambient temperature to prepare platelet - rich plasma ( prp ) .", "two - thirds of the volume of the prp was carefully removed from above the buffy coat , centrifuged at 1800  g for 10 minutes to pellet the platelets , and then the supernatant plasma returned to the original blood sample .", "as described above , the blood sample was loaded onto a discontinuous histopaque ( sigma aldrich , dorset , uk ) gradient comprising histopaque 1119 ( 2  ml , lower layer ) and histopaque 1077 ( 3  ml , upper layer ) .", "following centrifugation at 800  g for 1  h at ambient temperature , the cells at the interface between the plasma and histopaque 1077 ( monocytes and lymphocytes ) were harvested ; the cells at the interface between histopaque 1077 and 1119 ( principally granulocytes with a small proportion of lymphocytes ) were also harvested .", "harvested cells were washed by resuspension in 10  ml phosphate buffered saline ph 7.2 ( pbs ) followed by centrifugation at 250  g for 10 minutes at ambient temperature .", "the cell pellet was resuspended in pbs ( 0.7  ml ) and an aliquot ( 0.2  ml ) was diluted 1  :  1 ( v / v ) with pbs and used for determination of cell counts on a pentra 120 ( horiba abx , montpellier , france ) . citrate- or edta - anticoagulated whole blood ( 5  ml ) was depleted of platelets as described above ( separation of white blood cells ) . the blood sample ( 3  ml )", "was then loaded onto histopaque 1077 ( 3  ml ) and centrifuged to separate the white blood cells ( wbc ) ( 400  g , 30  min , ambient temperature ) .", "the wbc interface was harvested , washed , and an aliquot used for determination of cell counts as described above ( separation of white blood cells ) .  ", "the remaining cells , suspended in pbs , were separated according to forward scatter and side scatter light characteristics using a moflo high speed cell sorter ( beckman coulter , high wycombe , bucks , uk ) .", "gating was restrictive rather than permissive : each gate was set only for the core population of cells that had the required scatter characteristics . in particular , the lymphocyte gate was set to minimise the possibility of small monocytes being harvested .", "purity checks were subsequently performed by reanalysis of aliquots of the sorted cells in the flow cytometer ; purity was typically around 98% . all reagents and consumables for cell culture were supplied by invitrogen life technologies ltd , paisley , ireland .", "( human hepatocyte carcinoma )   and thp1 ( human acute monocytic leukemia )   cells were cultured in rpmi1640 medium + l - glutamine , supplemented with 10% ( v / v ) fetal calf serum and containing penicillin ( 50  units / ml ) and streptomycin ( 50  g / ml ) .", "cells were grown at 37c in water - saturated air ( 95% , v / v ) , co2 ( 5% , v / v ) in 25  cm flasks .", "hepg2 cells ( adherent cell line ) were liberated from the flask wall mechanically using a plastic scraper and were separated by exposure to shear force .", "hepg2 has been shown by others to express high levels of the ashwell receptor   and was used as a positive control in all relevant experiments .", "rna was extracted using rneasy kits ( qiagen , west sussex , uk ) according to the manufacturer 's instructions .", "yield was determined spectrophotometrically and then 1.0  g was reverse transcribed into cdna using random hexamers and the high capacity cdna reverse transcription kit ( life technologies ltd / applied biosystems , paisley , uk ) according to the manufacturer 's instructions .", "standard polymerase chain reactions ( pcrs ) typically contained approximately 30  ng cdna in a mixture of dntps ( 100  mol / l each ) , tris - hcl ph8.0 ( 10  mmol / l ) , mgcl2 ( 1.5  mmol / l ) , taq dna polymerase ( 1u ) ( applied biosystems , warwickshire , uk ) , and primers ( figure 1 ) in a final volume of 25  l . all primers were used at 0.5 ", "pcr was done using a 2720 dna thermal cycler ( applied biosystems , warwickshire , uk ) .", "the primer sequences ( 5 to 3 ) were as follows :  asg1rtf gaaagatgaagtcgctagagt  asg1rtr aggctccgcaggtcagacac  a1cdsf1 gtagcgcgacggccagtactgaagaacctgggaatcagac  a1cdsr1 cagggcgcagcgatgacagctcctcaccttcggaacatca  a1test2  gaccaaggagtatcaagacctt  asg2rtf cacacctggtggtcatcaac  asg2rtr aattatctggctgagtgacag  asgr2rtf3 agctgagctcggaggaaaatg  asgr2rtr2 gcagctcggcttgcagctgtg  a2cdsf1 gtagcgcgacggccagtcccagccctcagagcaacctca  a2cdsr1 cagggcgcagcgatgactcaacagagaagccagagctggg  b2mrtf tccgtggccttagctgtgct  b2mrtr ccagtccttgctgaaagaca  n13f  gtagcgcgacggccagt  n13r cagggcgcagcgatgac asg1rtf gaaagatgaagtcgctagagt asg1rtr aggctccgcaggtcagacac a1cdsf1 gtagcgcgacggccagtactgaagaacctgggaatcagac a1cdsr1 cagggcgcagcgatgacagctcctcaccttcggaacatca a1test2  gaccaaggagtatcaagacctt asg2rtf cacacctggtggtcatcaac asg2rtr aattatctggctgagtgacag asgr2rtf3 agctgagctcggaggaaaatg asgr2rtr2 gcagctcggcttgcagctgtg a2cdsf1 gtagcgcgacggccagtcccagccctcagagcaacctca a2cdsr1 cagggcgcagcgatgactcaacagagaagccagagctggg b2mrtf tccgtggccttagctgtgct b2mrtr ccagtccttgctgaaagaca n13f  gtagcgcgacggccagt n13r cagggcgcagcgatgac primers were designed for rt - pcr according to the following criteria : ( 1 ) they flanked at least one intron ; ( 2 ) they were specific for asgr1 or asgr2 transcripts , cross hybridization was not possible despite the sequence homology between the coding sequences of the two genes . n13f and n13r are , respectively , modified m13 universal and reverse sequencing primers as previously described . underlined nucleotides are not part of asgr1 or asgr2 sequence but are tails corresponding to n13f or n13r to facilitate sequence analysis . for nested pcr ,", "the first - round product was then diluted 10-fold with water and 1  l of diluent was used as the substrate in the second round of pcr .", "pcr conditions were as follows :  standard pcr : 94c for 30  s ; 60c for 30  s ; 72c for 1  min . ", "nested pcr : 1st round 94c for 30  s ; 60c for 30  s ; 72c for 1  min 30  s.  nested pcr : 2nd round 94c for 30  s ; 60c for 30  s ; 72c for 1  min.routinely , 30 cycles of amplification were used ; however , for real time pcr , 55 cycles were employed . ", "standard pcr : 94c for 30  s ; 60c for 30  s ; 72c for 1  min .", "nested pcr : 1st round 94c for 30  s ; 60c for 30  s ; 72c for 1  min 30  s. nested pcr : 2nd round 94c for 30  s ; 60c for 30  s ; 72c for 1  min . \n", "asgr1 and asgr2 transcript levels were measured using lightcycler faststart dna master sybr green i ( roche products ltd , hertfordshire , uk ) according to the manufacturer 's instructions , with 3  mmol / l mgcl2 final concentration in the reaction .", "reactions contained 2  ng cdna per 20  l and relevant primers ( figure 1 ) .", "analyses were done in duplicate in a lightcycler ii using lightcycler v4.0 software ( perkin elmer - applied biosystems , warwickshire , uk ) using the following conditions : 94c for 10  min followed by 55 cycles comprising 94c for 10  s ; 64c for 5  s ; 72c for 10  s. pcr products were purified using the highpure kit ( boehringer mannheim , nottingham , uk ) and then sequenced using bigdye3.1 according the manufacturer 's instructions ( perkin elmer - applied biosystems , warrington , uk ) .", "sequencing primers were either the 5 pcr primer , 3 pcr primer , n13f or n13r according to the amplification product .", "sequencing reaction products were purified using qiaquick pcr purification columns ( qiagen , west sussex , uk ) and then electrophoresed on an applied biosystems 3130xl genetic analyser .", "agarose gel electrophoresis : the analyses used 2% ( w / v ) gels and 1x tbe buffer ( tris ( 90  mmol / l ) , boric acid ( 90  mmol / l ) , edta ( 1.25  mmol / l ) , ph 8.0 ) ; visualization was done using ethidium bromide staining and uv light .", "polyacrylamide gel electrophoresis : gels comprised polyacrylamide ( total acrylamide = 5% , w / v ; cross link = 3.3% , w / v ) and 1x tbe buffer , visualization was done using silver staining .", "the dna size standard pbr322/mspi ( new england biolabs , hertfordshire , uk ) was used for all electrophoretic analyses .", "initially , peripheral blood mononuclear cells ( pbmcs ) were harvested and screened for asgr1 and asgr2 expression without fractionation into monocytes / lymphocytes / granulocytes .", "rt - pcr of rna extracted from pbmcs demonstrated the presence of both asgr1 and asgr2 transcripts ( figure 2(a ) ) .", "these were also detected in the monocyte cell line thp1 ( figure 2(a ) ) . following these initial findings", ", pbmcs were fractionated in order to localize the cells in which asgr1 and asgr2 expression was present .", "histopaque gradients allowed separation of cells into monocytes + lymphocytes ( m + l ) and granulocytes + lymphocytes ( g + l ) .", "these cell preparations typically contained 10% monocytes/90% lymphocytes ( m + l ) and 94% granulocytes/10% lymphocytes ( g + l ) ( data not shown ) . using rt - pcr , asgr1 and asgr2 transcripts", "were detected in rna from the m + l fraction but not in rna from the g + l fraction ( figure 2(b ) ) .", "these findings were reproducible for blood samples taken from 3 different individuals ( data not shown ) .", "the g + l fraction did not yield a product for the transcripts even after 50 cycles of rt - pcr amplification ( figure 2(b ) ) .", "some nonspecific products were obtained as a result of this high number of cycles ; however , these did not interfere with the main result of the experiment ( figure 2(b ) ) . because lymphocytes were common to both cell fractions , and because the g + l fraction was reproducibly negative for asgr1 and asgr2 transcripts , the data suggested that expression of the two genes was localized to monocytes . to confirm this ,", "monocytes and lymphocytes were formally cell sorted using flow cytometry , rna extracted and screened using rt - pcr .", "monocytes gave a positive result for both asgr1 and asgr2 transcripts , whilst lymphocytes did not give a pcr product , even after 50 cycles of amplification ( figure 2(c ) ) .", "this high cycle number resulted in the burst - through of some nonspecific products ; however , these did not interfere with the interpretation ( figure 2(c ) ) .", "these data provide strong evidence for expression of asgr1 and asgr2 in monocytes but not in lymphocytes or granulocytes .", "the longer transcript ( t1 ) encodes isoform a ( full - length asgr1 ) , the shorter transcript ( t2 ) has an in - frame deletion of 117 nucleotides in the coding sequence resulting in a shorter isoform ( isoform b ) ( figure 1(a ) ) .", "the latter lacks the transmembrane domain and is secreted as a soluble protein . in the liver ,", "t1 has been shown to be the predominant isoform , with very little t2 detectable . using a nested pcr approach to screen rna from the m + l cell fraction of peripheral blood ,", "different individuals showed the presence of either t1 or of both t1 and t2 ( figure 3 ) . among the five individuals screened , none showed the presence of transcript t2 on its own . in combination with the data in figure 2 ( which showed that asgr1 transcripts are detected in monocytes and not lymphocytes ) , these data suggest that the monocyte transcription profile for asgr1 differs between individuals .", "five transcripts have been described for asgr2 , giving rise to four protein isoforms ( figure 1(b ) ) . using a rt - pcr designed to detect transcripts encoding different isoforms , the product profiles obtained for rna extracted from the histopaque m+l cell fraction differed among individuals .", "two distinct profiles were obtained , the first corresponded to transcripts encoding isoforms a , b , c , and d , the second to transcripts encoding isoforms b and d only ( figure 4 ) .", "asgr2 isoforms a and c can give rise to soluble protein via proteolysis between the transmembrane domain and the crd .", "isoforms b and d lack the proteolysis site and can not produce the soluble form .", "the data therefore indicate that in some individuals , monocytes may produce both soluble and membrane - bound asgr2 , whilst in others , the soluble form is not produced by these cells .", "real - time pcr was optimized for primer pairs asg1rtf + asg1rtr , asg2rtf + asg2rtr ( which , respectively , amplify all asgr1 and all asgr2 transcripts ) and b2mrtf + b2mrtr ( which amplify the reference target gene , b2 m ) . for each primer pair", ", a specific product was obtained that had a characteristic melting curve ( figure 5 ) . following pcr optimization", ", transcripts were measured using relative quantification , in which the target was asgr1 or asgr2 , the reference was b2 m , the calibrator was hepg2 , and the unknown was flow sorted monocytes or thp1 . b2 m is a recommended reference target for quantitative pcr , having the advantages of a single transcript , no processed pseudogene and expression at similar levels in a wide range of tissues .", "flow - sorted monocytes and thp1 cells gave similar results : relative to expression in hepg2 , asgr1 transcripts were between 1.53e-07 to 7.53e-06-fold less , whilst asgr2 transcripts were between 7.16e-05 and 3.78e-04-fold less .", "figure 5 illustrates relative quantification of asgr1 and asgr2 transcripts in cell - sorted monocytes .", "the ratio of expression of asgr1  :  asgr2 in monocytes and the monocyte cell line thp1 was approximately 1  :  100 . to ascertain whether thp1 would be a suitable cell line for future asgpr studies in monocytes , the coding sequences of the asgr1 and asgr2 transcripts produced by these cells were determined .", "a nested pcr strategy was used as follows : the first round of synthesis amplified the entire coding sequence plus some 5 and 3 flanking sequence and then second round pcrs amplified nested portions within the first round product .", "one of the nested portions contained the region that differed between transcripts and gave products of different size according to the transcript .", "these products were electrophoresed , excised from the gel , reamplified individually , and then sequenced , to give the sequence of each of the coding regions that differed between transcripts .", "thp1 asgr1 and asgr2 transcripts corresponded with previously described splice variants found in the liver ( data not shown ) .", "the findings demonstrate that thp1 cells have the capacity to transcribe and process correctly , transcripts encoding functional isoforms of the asgpr . taken together with the results in figures 3 and 4 ,", "the data provide evidence that monocytes express correctly processed transcripts from both asgr1 and asgr2 and thereby have the potential to produce functional asgpr .", "the coding sequence for asgr1 in thp1 differed from the ncbi reference sequence ( table 1 ) by a single nucleotide : c.267g > a , for which the monocyte cell line was homozygous ( data not shown ) .", "this change is silent at the protein level ( codon 89 , aag , changes to aaa , both encoding lysine ) .", "the change is a naturally occurring variant ( rs55714927 ) listed in the international snp database ( dbsnp ) .", "the thp1 asgr2 coding sequence for any transcript did not differ to the corresponding ncbi reference sequence ( table 1 ) ( data not shown ) .", "in this study , expression of asgr1 and asgr2 was demonstrated in peripheral blood monocytes . for both genes ,", "transcript profiles were obtained that corresponded with known splice variants found in the liver . to the best of our knowledge ,", "this is the first report of the expression of correctly processed transcripts of asgr1 and asgr2 by human monocytes . in rat", ", a transcript encoding an asialoglycoprotein - binding protein was isolated from a peritoneal macrophage cdna library and was highly homologous to that of the rodent hepatic asgpr .", "the data were interpreted to indicate that the rodent macrophage asialoglycoprotein - binding protein and the liver asgpr were encoded by related genes ; however , it now seems possible that alternative splicing may underlie the differences .", "the findings of the present study do not suggest expression of alternative asgpr - related genes in monocytes , rather they are entirely consistent with transcription of asgr1 and asgr2 , as occurs in the liver .", "the various transcripts of asgr1 and asgr2 encode different isoforms of the proteins ( figure 1 ) .", "it is not known whether the isoforms differ in their specificity or functionality , however it is predicted that certain isoforms have the potential to give rise to soluble forms of each protein .", "asgr1 transcript t2 has an in - frame deletion of 117  bp that encode the transmembrane domain ; the resulting isoform ", ", th2 and t3 encode isoforms  a  ( t1 and th2 ' ) and  c ", "( t3 ) which contain a proteolysis signal that , upon cleavage , gives rise to soluble protein .", "transcripts t2 and t4 , respectively , encode isoforms b and d which lack the proteolysis signal and are membrane bound .", "the data presented here indicate that monocytes are able to express transcripts encoding soluble and membrane - bound isoforms of the asgpr proteins .", "asgr2 was found to give rise to two transcript profiles , only one of which was present in the monocytes from any given individual .", "one profile corresponded with transcripts encoding both soluble and membrane - bound asgr2 isoforms ( a , b , c , and d ) , the other with membrane - bound asgr2 isoforms ( b and d ) only .", "this novel finding indicates a fundamental difference between individuals . the underlying basis for this and whether or not it has physiologic significance in health or disease merit further exploration .  ", "the level of asgr1 and asgr2 expression was considerably less in monocytes and the monocyte cell line thp1 compared with the liver cell line hepg2 .", "it should be borne in mind that hepg2 expresses high levels of the receptor   and the monocyte expression was relative to this .", "that monocyte transcripts were not rare within the cells was indicated by the fact that they were readily detected using rt - pcr at routine cycle numbers .", "low level of expression of the asgpr has been reported previously in certain nonhepatic tissues ( rat macrophages , human intestinal epithelial cells , mouse testis , and rat thyroid gland ) .", "it would be relevant to explore whether asgr1 and asgr2 transcription alters upon monocyte activation .", "the relative expression of asgr1 and asgr2 in monocytes and thp1 ( 1  :  100 ) differed notably from ratios reported in liver ( 1  :  2   and 1  :  6 ) .", "the difference may be real , or it may reflect differences in methodology between studies or possibly a difference in the amplification efficiency of the primer pair used for asgr1 compared with that used for asgr2 .", "the true relative expression of the two genes by monocytes therefore remains to be established .", "an important question arising from these results is whether monocytes translate asgr1 and asgr2 transcripts and produce functional asgpr .", "the restricted expression of the genes in monocytes , but not in lymphocytes or granulocytes , suggests the transcripts are not produced randomly but specifically .", "based on the fundamental principles of biology , this is likely to be for translation , it is difficult to think why else different transcripts encoding different isoforms may be produced by one specific cell type in the blood .", "the restricted tissue specificity for asgpr expression in the body , and the lower expression level observed at the nonhepatic locations , could signal a specific role or function at those sites . in the case of monocytes , the receptor could , at the very least , serve a scavenger function where there is infection or tissue injury .", "expression of correctly processed transcripts of asgr1 and asgr2 by circulating monocytes has potentially significant implications in several important areas .", "if monocytes express functional asgpr , they may contribute towards the normal physiological processes undertaken by the hepatocyte receptor .", "whilst hepatocyte asgpr is localized to the liver and relies upon the circulation to deliver ligands to it , the monocyte receptor would represent a mobile pool that can reach ligands in most parts of the body .", "monocyte asgpr may additionally interact with ligands in the blood , as does liver asgpr .", "thus , the monocyte receptor , if it is produced , may overlap functionally with that of the liver but may have additional physiological roles elsewhere in the body .", "hepatic asgpr has been shown to be directly involved in the normal turnover of an important protein of primary hemostasis , vwf .", "studies in knock - out mice indicated that the asgpr is engaged in the constitutive control of vwf level and can , additionally , remove hemostatic components ( vwf and platelets ) desialylated by bacterial neuraminidase , a possible survival mechanism in disseminated intravascular coagulation .", "the results in these studies were ascribed to the activity of the liver receptor ; however , our data raise the possibility that monocytes may contribute to the relevant physiological processes via the asgpr .", "the findings have implications for the design of drugs targeting the liver via the asgpr and drugs that are cleared by this receptor [ 29 , 30 ] .", "various strategies have been used , for example , conjugation of drug with galactosyl terminating molecules , as has been done with antiviral nucleoside analogues in the treatment of chronic viral hepatitis .", "besides drug targeting , strategies to deliver genes or other nucleic acids to the liver have also made use of the asgpr [ 1012 ] .", "relatively recently , successful in vivo delivery of sirnas to the liver in mice has been achieved via simple intravenous injection employing asgpr - specific conjugates containing galnac .", "pharmacological targeting of the asgpr could not be considered to be predominantly liver - specific if monocytes express the receptor .", "\n asgr1 and asgr2 are expressed in peripheral blood monocytes in humans . for both genes ,", "monocytes have the ability to produce correctly spliced transcripts encoding each of the known protein isoforms .", "however , there are interindividual differences in the transcript profiles ; of particular note , despite several possible asgr2 transcripts , just two combinations were found to occur .", "this observation indicates that , in any given individual , asgr2 mrna splicing is restricted to one of two profiles .", "quantitative studies indicate that expression of both asgr1 and asgr2 is lower in monocytes than in the liver , but none - the - less is readily detected in monocytes ( in comparison to other blood cells in which expression could not be demonstrated ) .", "the liver may represent a static site to which blood carries glycoconjugates for asgpr - mediated removal , whilst monocytes may represent a mobile pool that can reach sites where the activity of the receptor is needed .", "the data presented here open various important avenues for future research , a notable one of which is the measurement and characterization of monocyte asgpr protein .", "the results for the cell line thp1 indicated it could be used as a model system for the investigation of monocyte aspgr function and activity .", "monocytes play a pivotal role in inflammation and immunity , the finding of asgpr transcripts within these cells offers new insight into their biochemistry and functional potential ."], "abstract_text": ["<S> \n background . </S>", "<S> the asialoglycoprotein receptor ( asgpr ) is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease . </S>", "<S> the receptor comprises two proteins , asialoglycoprotein receptor 1 and 2 ( asgr1 and asgr2 ) , encoded by the genes asgr1 and asgr2 . </S>", "<S> design and methods . using reverse transcription amplification ( rt - pcr ) , expression of asgr1 and asgr2 was investigated in human peripheral blood monocytes . </S>", "<S> results . </S>", "<S> monocytes were found to express asgr1 and asgr2 transcripts . correctly spliced transcript variants encoding different isoforms of asgr1 and asgr2 were present in monocytes . </S>", "<S> the profile of transcript variants from both asgr1 and asgr2 differed among individuals . </S>", "<S> transcript expression levels were compared with the hepatocyte cell line hepg2 which produces high levels of asgpr . </S>", "<S> monocyte transcripts were 4 to 6 orders of magnitude less than in hepg2 but nonetheless readily detectable using standard rt - pcr . </S>", "<S> the monocyte cell line thp1 gave similar results to monocytes harvested from peripheral blood , indicating it may provide a suitable model system for studying asgpr function in this cell type . conclusions . </S>", "<S> monocytes transcribe and correctly process transcripts encoding the constituent proteins of the asgpr . </S>", "<S> monocytes may therefore represent a mobile pool of the receptor , capable of reaching sites remote from the liver . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Design and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["the asialoglycoprotein receptor ( asgpr ) ( also known as the ashwell receptor ) mediates the capture and endocytosis of galactose- ( gal ) and n - acetylgalactosamine- ( galnac ) terminating glycoproteins .", "the relevance of this function has been the subject of much debate ; the primary role may be the removal of potentially hazardous glycoconjugates arising from normal tissue turnover , tissue injury , disease , and other causes .", "studies using knock - out mice have provided evidence for direct involvement of the asgpr in removal of abnormally sialylated plasma glycoproteins .", "mice lacking the sialyltransferase st3gal4 showed prolonged bleeding which was attributed to asgpr - mediated clearance of at least one plasma hemostatic component , von willebrand factor ( vwf ) , that showed decreased sialylation .", "findings in mice lacking the asgpr demonstrated that , during sepsis , the receptor removed components of hemostasis ( vwf and platelets ) that had been desialylated by bacterial neuraminidase and thereby allowed hemostatic adaptation that moderated disseminated intravascular coagulation and improved host survival .", "the asgpr may therefore be poised for rapid clearance of plasma glycoproteins that , for whatever reason , show decreased or abnormal sialylation .", "the highest level of expression of the asgpr is in the liver , in which it is located on the sinusoidal face of hepatocytes .", "low - level expression has been shown in various other cell types , such as , peritoneal macrophages in rat , human intestinal epithelial cells , mouse testis , and the rat thyroid gland .", "the galactosyl homeostasis theory proposes that the balance of both gal and galnac glycoconjugates is important for normal tissue physiology [ 1 , 9 ] ; whether or not expression of the asgpr at these other sites represents a need for localized constitutive control of such glycoconjugates is not known .", "a major area of interest focussing on the asgpr is the targeted gene transfer / delivery of drugs to the liver [ 10 , 11 ] .", "for example , using galnac as a ligand on a sirna - containing complex , the latter was successfully targeted to hepatocytes after simple intravenous injection into the tail vein in mice .", "moreover , sirna - mediated knock - down of targeted genes was demonstrated within the hepatocytes . besides its potential importance in allowing liver - specific drug targeting", ", the asgpr is a key factor in the design and administration of glycoprotein pharmaceuticals more generally : the activity of the receptor can impact upon drug half life and thereby the window of therapeutic efficacy .", "human asgpr is composed of two types of subunit : a major subunit ( asialoglycoprotein receptor 1 , asgr1 ) and minor subunit ( asialoglycoprotein receptor 2 , asgr2 ) .", "both subunits are type ii , single pass proteins that broadly comprise a cytoplasmic domain , transmembrane domain , and extracellular carbohydrate recognition domain ( crd ) [ 14 , 15 ]", ". the subunits may exist as asgr1-asgr2 heterooligomers , asgr1homotrimers and homotetramers , and asgr2 homodimers and homotetramers .", "these different quaternary forms may allow for functional differences , such as , substrate specificity or rate of endocytosis [ 1 , 16 ] . the genes encoding asgr1 and asgr2 ( asgr1 and asgr2 , resp . )", "are located on the short arm of autosome 17 , approximately 58.6 kilobases ( kb ) apart .", "the genes are evolutionarily related but differ significantly in their structural organization : asgr1 comprises 8 exons and is approximately 6 kb long , asgr2 contains 9 exons occupying 13.5  kb of dna [ 14 , 15 ] . until recently , asgr1 was thought to yield one transcript encoding a single protein .", "however , in 2010 , liu et al . demonstrated the existence of two alternatively spliced transcripts .", "the longer transcript , t1 , contains all 8 exons and is by far the more abundant ; it encodes full - length asgr1 ( isoform a , 291 aminoacids ) .", "the shorter transcript , t2 , has an in - frame deletion of exon 3 resulting in the loss of 39 residues ( isoform b , 252 amino acids ) .", "asgr2 gives rise to five transcripts ( th2 , t1 , t2 , t3 , and t4 ) encoding four isoforms ( a to d ) that contain different in - frame deletions arising from alternative exon splicing events .", "isoforms a and c contain 5 aminoacids that serve as a proteolysis cleavage signal near the junction between the transmembrane domain and the crd .", "cleavage at this site results in secretion of the crd as a soluble protein [ 18 , 19 ] .", "isoforms b and d lack this signal therefore they are not proteolytically cleaved but rather remain membrane bound where they may oligomerize with asgr1 isoform a to form native asgpr at the cell surface .", "the secreted forms of asgr1 and asgr2 are able to associate into soluble asgpr [ 17 , 19 ] .", "there is evidence to suggest that the soluble receptor may bind free substrates in the circulation and carry them to the liver for uptake and degradation . aside from membrane attachment", "/ secretion , it is not known whether further functional variation , such as , substrate specificity , may be associated with the different isoforms of asgr1 and asgr2 . based on the observation above that the asgpr", "is expressed in rat peritoneal macrophages , it was considered possible that monocytes , the lineage precursor of tissue macrophages , may also express the receptor . to the best of our knowledge ,", "the present study investigated the expression of asgr1 and asgr2 in human peripheral blood monocytes .", "the data showed that both asgr1 and asgr2 are expressed in human monocytes in the circulation , that expression can not be detected in lymphocytes and granulocytes , that the transcripts of both genes differ between individuals and that , in a given individual , the transcription profile for asgr2 is restricted to one of two patterns .", "the findings are of potential importance for health and disease in a variety of disciplines ."], ["genes , transcripts , and proteins are referred to by their formal scientific names as listed in the national centre for biotechnology information ( ncbi ) , which complies with standardized international nomenclature .  ", "gene sizes , exon numbering , and so forth were obtained from the reference sequences listed in table 1 .", "edta - anticoagulated whole blood in excess of that required for routine blood tests was anonymized and handled in accordance with medical research council guidelines .", "citrate- or edta - anticoagulated whole blood ( 5  ml ) was centrifuged at 200  g for 10 minutes , ambient temperature to prepare platelet - rich plasma ( prp ) .", "two - thirds of the volume of the prp was carefully removed from above the buffy coat , centrifuged at 1800  g for 10 minutes to pellet the platelets , and then the supernatant plasma returned to the original blood sample .", "as described above , the blood sample was loaded onto a discontinuous histopaque ( sigma aldrich , dorset , uk ) gradient comprising histopaque 1119 ( 2  ml , lower layer ) and histopaque 1077 ( 3  ml , upper layer ) .", "following centrifugation at 800  g for 1  h at ambient temperature , the cells at the interface between the plasma and histopaque 1077 ( monocytes and lymphocytes ) were harvested ; the cells at the interface between histopaque 1077 and 1119 ( principally granulocytes with a small proportion of lymphocytes ) were also harvested .", "harvested cells were washed by resuspension in 10  ml phosphate buffered saline ph 7.2 ( pbs ) followed by centrifugation at 250  g for 10 minutes at ambient temperature .", "the cell pellet was resuspended in pbs ( 0.7  ml ) and an aliquot ( 0.2  ml ) was diluted 1  :  1 ( v / v ) with pbs and used for determination of cell counts on a pentra 120 ( horiba abx , montpellier , france ) . citrate- or edta - anticoagulated whole blood ( 5  ml ) was depleted of platelets as described above ( separation of white blood cells ) . the blood sample ( 3  ml )", "was then loaded onto histopaque 1077 ( 3  ml ) and centrifuged to separate the white blood cells ( wbc ) ( 400  g , 30  min , ambient temperature ) .", "the wbc interface was harvested , washed , and an aliquot used for determination of cell counts as described above ( separation of white blood cells ) .  ", "the remaining cells , suspended in pbs , were separated according to forward scatter and side scatter light characteristics using a moflo high speed cell sorter ( beckman coulter , high wycombe , bucks , uk ) .", "gating was restrictive rather than permissive : each gate was set only for the core population of cells that had the required scatter characteristics . in particular , the lymphocyte gate was set to minimise the possibility of small monocytes being harvested .", "purity checks were subsequently performed by reanalysis of aliquots of the sorted cells in the flow cytometer ; purity was typically around 98% . all reagents and consumables for cell culture were supplied by invitrogen life technologies ltd , paisley , ireland .", "( human hepatocyte carcinoma )   and thp1 ( human acute monocytic leukemia )   cells were cultured in rpmi1640 medium + l - glutamine , supplemented with 10% ( v / v ) fetal calf serum and containing penicillin ( 50  units / ml ) and streptomycin ( 50  g / ml ) .", "cells were grown at 37c in water - saturated air ( 95% , v / v ) , co2 ( 5% , v / v ) in 25  cm flasks .", "hepg2 cells ( adherent cell line ) were liberated from the flask wall mechanically using a plastic scraper and were separated by exposure to shear force .", "hepg2 has been shown by others to express high levels of the ashwell receptor   and was used as a positive control in all relevant experiments .", "rna was extracted using rneasy kits ( qiagen , west sussex , uk ) according to the manufacturer 's instructions .", "yield was determined spectrophotometrically and then 1.0  g was reverse transcribed into cdna using random hexamers and the high capacity cdna reverse transcription kit ( life technologies ltd / applied biosystems , paisley , uk ) according to the manufacturer 's instructions .", "standard polymerase chain reactions ( pcrs ) typically contained approximately 30  ng cdna in a mixture of dntps ( 100  mol / l each ) , tris - hcl ph8.0 ( 10  mmol / l ) , mgcl2 ( 1.5  mmol / l ) , taq dna polymerase ( 1u ) ( applied biosystems , warwickshire , uk ) , and primers ( figure 1 ) in a final volume of 25  l . all primers were used at 0.5 ", "pcr was done using a 2720 dna thermal cycler ( applied biosystems , warwickshire , uk ) .", "the primer sequences ( 5 to 3 ) were as follows :  asg1rtf gaaagatgaagtcgctagagt  asg1rtr aggctccgcaggtcagacac  a1cdsf1 gtagcgcgacggccagtactgaagaacctgggaatcagac  a1cdsr1 cagggcgcagcgatgacagctcctcaccttcggaacatca  a1test2  gaccaaggagtatcaagacctt  asg2rtf cacacctggtggtcatcaac  asg2rtr aattatctggctgagtgacag  asgr2rtf3 agctgagctcggaggaaaatg  asgr2rtr2 gcagctcggcttgcagctgtg  a2cdsf1 gtagcgcgacggccagtcccagccctcagagcaacctca  a2cdsr1 cagggcgcagcgatgactcaacagagaagccagagctggg  b2mrtf tccgtggccttagctgtgct  b2mrtr ccagtccttgctgaaagaca  n13f  gtagcgcgacggccagt  n13r cagggcgcagcgatgac asg1rtf gaaagatgaagtcgctagagt asg1rtr aggctccgcaggtcagacac a1cdsf1 gtagcgcgacggccagtactgaagaacctgggaatcagac a1cdsr1 cagggcgcagcgatgacagctcctcaccttcggaacatca a1test2  gaccaaggagtatcaagacctt asg2rtf cacacctggtggtcatcaac asg2rtr aattatctggctgagtgacag asgr2rtf3 agctgagctcggaggaaaatg asgr2rtr2 gcagctcggcttgcagctgtg a2cdsf1 gtagcgcgacggccagtcccagccctcagagcaacctca a2cdsr1 cagggcgcagcgatgactcaacagagaagccagagctggg b2mrtf tccgtggccttagctgtgct b2mrtr ccagtccttgctgaaagaca n13f  gtagcgcgacggccagt n13r cagggcgcagcgatgac primers were designed for rt - pcr according to the following criteria : ( 1 ) they flanked at least one intron ; ( 2 ) they were specific for asgr1 or asgr2 transcripts , cross hybridization was not possible despite the sequence homology between the coding sequences of the two genes . n13f and n13r are , respectively , modified m13 universal and reverse sequencing primers as previously described . underlined nucleotides are not part of asgr1 or asgr2 sequence but are tails corresponding to n13f or n13r to facilitate sequence analysis . for nested pcr ,", "the first - round product was then diluted 10-fold with water and 1  l of diluent was used as the substrate in the second round of pcr .", "pcr conditions were as follows :  standard pcr : 94c for 30  s ; 60c for 30  s ; 72c for 1  min . ", "nested pcr : 1st round 94c for 30  s ; 60c for 30  s ; 72c for 1  min 30  s.  nested pcr : 2nd round 94c for 30  s ; 60c for 30  s ; 72c for 1  min.routinely , 30 cycles of amplification were used ; however , for real time pcr , 55 cycles were employed . ", "standard pcr : 94c for 30  s ; 60c for 30  s ; 72c for 1  min .", "nested pcr : 1st round 94c for 30  s ; 60c for 30  s ; 72c for 1  min 30  s. nested pcr : 2nd round 94c for 30  s ; 60c for 30  s ; 72c for 1  min . \n", "asgr1 and asgr2 transcript levels were measured using lightcycler faststart dna master sybr green i ( roche products ltd , hertfordshire , uk ) according to the manufacturer 's instructions , with 3  mmol / l mgcl2 final concentration in the reaction .", "reactions contained 2  ng cdna per 20  l and relevant primers ( figure 1 ) .", "analyses were done in duplicate in a lightcycler ii using lightcycler v4.0 software ( perkin elmer - applied biosystems , warwickshire , uk ) using the following conditions : 94c for 10  min followed by 55 cycles comprising 94c for 10  s ; 64c for 5  s ; 72c for 10  s. pcr products were purified using the highpure kit ( boehringer mannheim , nottingham , uk ) and then sequenced using bigdye3.1 according the manufacturer 's instructions ( perkin elmer - applied biosystems , warrington , uk ) .", "sequencing primers were either the 5 pcr primer , 3 pcr primer , n13f or n13r according to the amplification product .", "sequencing reaction products were purified using qiaquick pcr purification columns ( qiagen , west sussex , uk ) and then electrophoresed on an applied biosystems 3130xl genetic analyser .", "agarose gel electrophoresis : the analyses used 2% ( w / v ) gels and 1x tbe buffer ( tris ( 90  mmol / l ) , boric acid ( 90  mmol / l ) , edta ( 1.25  mmol / l ) , ph 8.0 ) ; visualization was done using ethidium bromide staining and uv light .", "polyacrylamide gel electrophoresis : gels comprised polyacrylamide ( total acrylamide = 5% , w / v ; cross link = 3.3% , w / v ) and 1x tbe buffer , visualization was done using silver staining .", "the dna size standard pbr322/mspi ( new england biolabs , hertfordshire , uk ) was used for all electrophoretic analyses ."], ["initially , peripheral blood mononuclear cells ( pbmcs ) were harvested and screened for asgr1 and asgr2 expression without fractionation into monocytes / lymphocytes / granulocytes .", "rt - pcr of rna extracted from pbmcs demonstrated the presence of both asgr1 and asgr2 transcripts ( figure 2(a ) ) .", "these were also detected in the monocyte cell line thp1 ( figure 2(a ) ) . following these initial findings", ", pbmcs were fractionated in order to localize the cells in which asgr1 and asgr2 expression was present .", "histopaque gradients allowed separation of cells into monocytes + lymphocytes ( m + l ) and granulocytes + lymphocytes ( g + l ) .", "these cell preparations typically contained 10% monocytes/90% lymphocytes ( m + l ) and 94% granulocytes/10% lymphocytes ( g + l ) ( data not shown ) . using rt - pcr , asgr1 and asgr2 transcripts", "were detected in rna from the m + l fraction but not in rna from the g + l fraction ( figure 2(b ) ) .", "these findings were reproducible for blood samples taken from 3 different individuals ( data not shown ) .", "the g + l fraction did not yield a product for the transcripts even after 50 cycles of rt - pcr amplification ( figure 2(b ) ) .", "some nonspecific products were obtained as a result of this high number of cycles ; however , these did not interfere with the main result of the experiment ( figure 2(b ) ) . because lymphocytes were common to both cell fractions , and because the g + l fraction was reproducibly negative for asgr1 and asgr2 transcripts , the data suggested that expression of the two genes was localized to monocytes . to confirm this ,", "monocytes and lymphocytes were formally cell sorted using flow cytometry , rna extracted and screened using rt - pcr .", "monocytes gave a positive result for both asgr1 and asgr2 transcripts , whilst lymphocytes did not give a pcr product , even after 50 cycles of amplification ( figure 2(c ) ) .", "this high cycle number resulted in the burst - through of some nonspecific products ; however , these did not interfere with the interpretation ( figure 2(c ) ) .", "these data provide strong evidence for expression of asgr1 and asgr2 in monocytes but not in lymphocytes or granulocytes .", "the longer transcript ( t1 ) encodes isoform a ( full - length asgr1 ) , the shorter transcript ( t2 ) has an in - frame deletion of 117 nucleotides in the coding sequence resulting in a shorter isoform ( isoform b ) ( figure 1(a ) ) .", "the latter lacks the transmembrane domain and is secreted as a soluble protein . in the liver ,", "t1 has been shown to be the predominant isoform , with very little t2 detectable . using a nested pcr approach to screen rna from the m + l cell fraction of peripheral blood ,", "different individuals showed the presence of either t1 or of both t1 and t2 ( figure 3 ) . among the five individuals screened , none showed the presence of transcript t2 on its own . in combination with the data in figure 2 ( which showed that asgr1 transcripts are detected in monocytes and not lymphocytes ) , these data suggest that the monocyte transcription profile for asgr1 differs between individuals .", "five transcripts have been described for asgr2 , giving rise to four protein isoforms ( figure 1(b ) ) . using a rt - pcr designed to detect transcripts encoding different isoforms , the product profiles obtained for rna extracted from the histopaque m+l cell fraction differed among individuals .", "two distinct profiles were obtained , the first corresponded to transcripts encoding isoforms a , b , c , and d , the second to transcripts encoding isoforms b and d only ( figure 4 ) .", "asgr2 isoforms a and c can give rise to soluble protein via proteolysis between the transmembrane domain and the crd .", "isoforms b and d lack the proteolysis site and can not produce the soluble form .", "the data therefore indicate that in some individuals , monocytes may produce both soluble and membrane - bound asgr2 , whilst in others , the soluble form is not produced by these cells .", "real - time pcr was optimized for primer pairs asg1rtf + asg1rtr , asg2rtf + asg2rtr ( which , respectively , amplify all asgr1 and all asgr2 transcripts ) and b2mrtf + b2mrtr ( which amplify the reference target gene , b2 m ) . for each primer pair", ", a specific product was obtained that had a characteristic melting curve ( figure 5 ) . following pcr optimization", ", transcripts were measured using relative quantification , in which the target was asgr1 or asgr2 , the reference was b2 m , the calibrator was hepg2 , and the unknown was flow sorted monocytes or thp1 . b2 m is a recommended reference target for quantitative pcr , having the advantages of a single transcript , no processed pseudogene and expression at similar levels in a wide range of tissues .", "flow - sorted monocytes and thp1 cells gave similar results : relative to expression in hepg2 , asgr1 transcripts were between 1.53e-07 to 7.53e-06-fold less , whilst asgr2 transcripts were between 7.16e-05 and 3.78e-04-fold less .", "figure 5 illustrates relative quantification of asgr1 and asgr2 transcripts in cell - sorted monocytes .", "the ratio of expression of asgr1  :  asgr2 in monocytes and the monocyte cell line thp1 was approximately 1  :  100 . to ascertain whether thp1 would be a suitable cell line for future asgpr studies in monocytes , the coding sequences of the asgr1 and asgr2 transcripts produced by these cells were determined .", "a nested pcr strategy was used as follows : the first round of synthesis amplified the entire coding sequence plus some 5 and 3 flanking sequence and then second round pcrs amplified nested portions within the first round product .", "one of the nested portions contained the region that differed between transcripts and gave products of different size according to the transcript .", "these products were electrophoresed , excised from the gel , reamplified individually , and then sequenced , to give the sequence of each of the coding regions that differed between transcripts .", "thp1 asgr1 and asgr2 transcripts corresponded with previously described splice variants found in the liver ( data not shown ) .", "the findings demonstrate that thp1 cells have the capacity to transcribe and process correctly , transcripts encoding functional isoforms of the asgpr . taken together with the results in figures 3 and 4 ,", "the data provide evidence that monocytes express correctly processed transcripts from both asgr1 and asgr2 and thereby have the potential to produce functional asgpr .", "the coding sequence for asgr1 in thp1 differed from the ncbi reference sequence ( table 1 ) by a single nucleotide : c.267g > a , for which the monocyte cell line was homozygous ( data not shown ) .", "this change is silent at the protein level ( codon 89 , aag , changes to aaa , both encoding lysine ) .", "the change is a naturally occurring variant ( rs55714927 ) listed in the international snp database ( dbsnp ) .", "the thp1 asgr2 coding sequence for any transcript did not differ to the corresponding ncbi reference sequence ( table 1 ) ( data not shown ) ."], ["in this study , expression of asgr1 and asgr2 was demonstrated in peripheral blood monocytes . for both genes ,", "transcript profiles were obtained that corresponded with known splice variants found in the liver . to the best of our knowledge ,", "this is the first report of the expression of correctly processed transcripts of asgr1 and asgr2 by human monocytes . in rat", ", a transcript encoding an asialoglycoprotein - binding protein was isolated from a peritoneal macrophage cdna library and was highly homologous to that of the rodent hepatic asgpr .", "the data were interpreted to indicate that the rodent macrophage asialoglycoprotein - binding protein and the liver asgpr were encoded by related genes ; however , it now seems possible that alternative splicing may underlie the differences .", "the findings of the present study do not suggest expression of alternative asgpr - related genes in monocytes , rather they are entirely consistent with transcription of asgr1 and asgr2 , as occurs in the liver .", "the various transcripts of asgr1 and asgr2 encode different isoforms of the proteins ( figure 1 ) .", "it is not known whether the isoforms differ in their specificity or functionality , however it is predicted that certain isoforms have the potential to give rise to soluble forms of each protein .", "asgr1 transcript t2 has an in - frame deletion of 117  bp that encode the transmembrane domain ; the resulting isoform ", ", th2 and t3 encode isoforms  a  ( t1 and th2 ' ) and  c ", "( t3 ) which contain a proteolysis signal that , upon cleavage , gives rise to soluble protein .", "transcripts t2 and t4 , respectively , encode isoforms b and d which lack the proteolysis signal and are membrane bound .", "the data presented here indicate that monocytes are able to express transcripts encoding soluble and membrane - bound isoforms of the asgpr proteins .", "asgr2 was found to give rise to two transcript profiles , only one of which was present in the monocytes from any given individual .", "one profile corresponded with transcripts encoding both soluble and membrane - bound asgr2 isoforms ( a , b , c , and d ) , the other with membrane - bound asgr2 isoforms ( b and d ) only .", "this novel finding indicates a fundamental difference between individuals . the underlying basis for this and whether or not it has physiologic significance in health or disease merit further exploration .  ", "the level of asgr1 and asgr2 expression was considerably less in monocytes and the monocyte cell line thp1 compared with the liver cell line hepg2 .", "it should be borne in mind that hepg2 expresses high levels of the receptor   and the monocyte expression was relative to this .", "that monocyte transcripts were not rare within the cells was indicated by the fact that they were readily detected using rt - pcr at routine cycle numbers .", "low level of expression of the asgpr has been reported previously in certain nonhepatic tissues ( rat macrophages , human intestinal epithelial cells , mouse testis , and rat thyroid gland ) .", "it would be relevant to explore whether asgr1 and asgr2 transcription alters upon monocyte activation .", "the relative expression of asgr1 and asgr2 in monocytes and thp1 ( 1  :  100 ) differed notably from ratios reported in liver ( 1  :  2   and 1  :  6 ) .", "the difference may be real , or it may reflect differences in methodology between studies or possibly a difference in the amplification efficiency of the primer pair used for asgr1 compared with that used for asgr2 .", "the true relative expression of the two genes by monocytes therefore remains to be established .", "an important question arising from these results is whether monocytes translate asgr1 and asgr2 transcripts and produce functional asgpr .", "the restricted expression of the genes in monocytes , but not in lymphocytes or granulocytes , suggests the transcripts are not produced randomly but specifically .", "based on the fundamental principles of biology , this is likely to be for translation , it is difficult to think why else different transcripts encoding different isoforms may be produced by one specific cell type in the blood .", "the restricted tissue specificity for asgpr expression in the body , and the lower expression level observed at the nonhepatic locations , could signal a specific role or function at those sites . in the case of monocytes , the receptor could , at the very least , serve a scavenger function where there is infection or tissue injury .", "expression of correctly processed transcripts of asgr1 and asgr2 by circulating monocytes has potentially significant implications in several important areas .", "if monocytes express functional asgpr , they may contribute towards the normal physiological processes undertaken by the hepatocyte receptor .", "whilst hepatocyte asgpr is localized to the liver and relies upon the circulation to deliver ligands to it , the monocyte receptor would represent a mobile pool that can reach ligands in most parts of the body .", "monocyte asgpr may additionally interact with ligands in the blood , as does liver asgpr .", "thus , the monocyte receptor , if it is produced , may overlap functionally with that of the liver but may have additional physiological roles elsewhere in the body .", "hepatic asgpr has been shown to be directly involved in the normal turnover of an important protein of primary hemostasis , vwf .", "studies in knock - out mice indicated that the asgpr is engaged in the constitutive control of vwf level and can , additionally , remove hemostatic components ( vwf and platelets ) desialylated by bacterial neuraminidase , a possible survival mechanism in disseminated intravascular coagulation .", "the results in these studies were ascribed to the activity of the liver receptor ; however , our data raise the possibility that monocytes may contribute to the relevant physiological processes via the asgpr .", "the findings have implications for the design of drugs targeting the liver via the asgpr and drugs that are cleared by this receptor [ 29 , 30 ] .", "various strategies have been used , for example , conjugation of drug with galactosyl terminating molecules , as has been done with antiviral nucleoside analogues in the treatment of chronic viral hepatitis .", "besides drug targeting , strategies to deliver genes or other nucleic acids to the liver have also made use of the asgpr [ 1012 ] .", "relatively recently , successful in vivo delivery of sirnas to the liver in mice has been achieved via simple intravenous injection employing asgpr - specific conjugates containing galnac .", "pharmacological targeting of the asgpr could not be considered to be predominantly liver - specific if monocytes express the receptor ."], ["\n asgr1 and asgr2 are expressed in peripheral blood monocytes in humans . for both genes ,", "monocytes have the ability to produce correctly spliced transcripts encoding each of the known protein isoforms .", "however , there are interindividual differences in the transcript profiles ; of particular note , despite several possible asgr2 transcripts , just two combinations were found to occur .", "this observation indicates that , in any given individual , asgr2 mrna splicing is restricted to one of two profiles .", "quantitative studies indicate that expression of both asgr1 and asgr2 is lower in monocytes than in the liver , but none - the - less is readily detected in monocytes ( in comparison to other blood cells in which expression could not be demonstrated ) .", "the liver may represent a static site to which blood carries glycoconjugates for asgpr - mediated removal , whilst monocytes may represent a mobile pool that can reach sites where the activity of the receptor is needed .", "the data presented here open various important avenues for future research , a notable one of which is the measurement and characterization of monocyte asgpr protein .", "the results for the cell line thp1 indicated it could be used as a model system for the investigation of monocyte aspgr function and activity .", "monocytes play a pivotal role in inflammation and immunity , the finding of asgpr transcripts within these cells offers new insight into their biochemistry and functional potential ."]]}
{"article_id": "PMC4882466", "article_text": ["the prevalence of obesity in the united states has increased over the past several decades . according to the national health and nutrition examination survey ( nhanes ) results , between the years of 1988 and 1994", ", 22.9% of adults aged 20 years and older exceeded the criteria for adult obesity [ body mass index ( bmi )  30 kgm ] ( 17 ) . by the 20112012 survey ,", "that percentage had increased to 35.1% ( 19 ) . this represents more than a 50% increase in obesity prevalence among american adults aged 20 years and older over the course of two decades .", "perhaps more concerning is the prevalence of obesity among american youth , defined by the centers for disease control and prevention ( cdc ) as a bmi greater than or equal to the 95 percentile on the cdc growth charts ( 2 ) . among children and adolescents aged 2", " 19 years , the prevalence of obesity increased from 5.5% between the years of 1976 and 1980 to 16.9% between 2007 and 2008 ( 18 ) and remained 16.9% between 2011 and 2012 ( 19 ) .", "analyses of several large - scale data pools confirm that obesity contributes to the risk for development of many negative health outcomes including coronary heart disease , hypertriglyceridemia , hypercholesterolemia , hypertension , and type ii diabetes ( 20 , 23 ) .", "using secondary data , thompson and colleagues developed a model to analyze the relationship between bmi and several disease conditions in adults ( 23 ) .", "notably , they found that the risks for hypertension and type ii diabetes are at least 2- and 3-fold higher for obese individuals with bmi values of 32.5 kgm and 37.5 kgm , respectively , as compared to normal - weight individuals with bmi values of 22.5 kgm .", "obesity - related health risks are not limited to adults . according to an analysis of bogalusa heart study data , the odds ratios between childhood obesity and many cardiovascular disease risk factors , including hypercholesterolemia , hypertriglyceridemia , hyperinsulinemia , and hypertension are between 2.4 and 12.6 , indicating a strong , positive association between childhood obesity and these specific risk factors ( 8) .", "finkelstein and colleagues estimated that in 2006 , the annual per capita medical expenditure for an obese person was $ 1,429 ( adjusted to reflect 2008 dollars ) higher than for a person of normal weight , which represents 42% higher medical costs for obese individuals compared to individuals with normal body weights ( 5 ) .", "data from over 3,000 counties and county equivalents across the united states indicate that in 2006 , approximately 54 million individuals met the criteria for obesity ( 16 ) .", "this equates to an extra $ 77 billion ( adjusted to reflect 2008 dollars ) in medical spending attributable to obesity in 2006 alone . beginning in the 2013  2014 school year , the president s council on fitness , sports & nutrition adopted the presidential youth fitness program to replace the president s challenge physical fitness test ( 22 ) .", "this program aims to encourage healthy , active lifestyles among youth . as part of this program ,", "schools use the presidential youth fitness program s fitnessgram to assess health - related fitness and use these data to inform physical education instruction . the fitnessgram scientific advisory board has developed four categories for classification of body composition : very lean , healthy fitness zone ( hfz ) , needs improvement  some risk ( ni  sr ) , and needs improvement  high risk ( ni  hr ) .", "risk level was established based on body fat percentage , and body fat cutoff percentages were converted to corresponding bmi values . for ten - year olds , the ni ", "hr category includes boys with a bmi  20.8 kgm and girls with a bmi  21.0 kgm ( 12 , 13 , 15 ) .", "these bmi cutoff values are lower than those utilized by the cdc , which defines childhood obesity as having a bmi at or above the 95 percentile on sex - specific bmi - for - age growth charts ( 2 ) .", "the corresponding bmi values that the cdc uses to indicate obesity are 22.03 kgm for boys aged 10.00  10.49 years , 24.16 kgm for boys aged 10.50  10.99 years , 23.18 kgm for girls aged 10.00  10.49 years , and 23.35 kgm for girls aged 10.50  10.99 years ( 11 ) .", "thus , using the cdc childhood obesity estimates could result in a substantial underestimation of the number of children at risk for negative health outcomes based upon bmi .", "children identified as obese by bmi have an increased risk of developing health complications that were previously thought to be limited to adults such as hypertension and type ii diabetes mellitus ( 3 ) .", "these conditions are also well - known risk factors for the development of cardiovascular disease .", "obese children aged ten years and older are likely to remain obese into early adulthood ( 28 ) , and therefore , accrue medical costs associated with obesity - related conditions .", "knowledge of the medical costs associated with childhood obesity can provide a basis by which to justify the cost of childhood obesity prevention and treatment efforts . to this end ,", "finkelstein and colleagues analyzed the lifetime direct medical costs for an obese ten - year - old beyond the costs that a ten - year - old at a normal body weight would incur over his or her lifespan ( 7 ) .", "the six investigations included in the meta - analysis each estimated longevity using a variety of factors such as probability of survival based on weight status ( 6 ) , gender , and race ( 4 , 14 , 2325 , 27 ) . based upon this meta - analysis ,", "two low- and high - end cost estimates were provided : one that accounted for adulthood weight gain among normal - weight children and one that compared against children who remained at normal weight through adulthood .", "results suggested that an obese ten - year - old , typically in the fifth grade , will incur between $ 12,660 ( low - end ; recommended ) and $ 19,630 ( high - end ) of incremental direct medical costs when accounting for weight gain through adulthood and between $ 16,310 ( low - end ) and $ 19,350 ( high - end ) with a recommended cost estimate of $ 19,000 when contrasted with a child who remains at normal weight throughout adulthood ( 4 , 7 , 14 , 23 , 24 , 27 ) .", "using these recommended estimates along with results from the fitnessgram body composition classifications , the purpose of this analysis is to estimate the lifetime economic impact of childhood obesity for this single age cohort in the two most populous states , california and texas .", "fitnessgram data from the school years 20102011 , 20112012 , and 20122013 were obtained for california ( 1 ) and texas ( 21 ) .", "these data were school - level and included boys and girls from all public school districts in these two states .", "the total number of fifth grade students tested for bmi in california and in texas and the number of fifth grade students in each bmi category were calculated for each of the three school years .", "the fitnessgram scientific advisory board has determined that bmi values at or above 20.8 kgm for ten - year - old boys and 21.0 kgm for ten - year - old girls represent high health risk ( 15 ) .", "the california report included the total number of fifth graders tested for body composition as well as the number and percentage of students in each fitness zone .", "the texas report included data for each grade level and further separated the data by district and gender .", "the total number of fifth graders tested for body composition and the number and percentage of students in each fitness zone were calculated using excel .", "then , using the low , high , and recommended cost estimates suggested by finkelstein and colleagues ( 7 ) , the lifetime direct medical costs attributable to obesity were calculated for students in the ni  hr category for each of the three school years in california and in texas by multiplying the number of students in this category by the corresponding cost estimate per obese student", "the first set of estimates compared the direct medical costs for obese children against the direct medical costs for normal - weight children who gain weight as adults .", "the second set of estimates compared the direct medical costs for obese children against the direct medical costs for normal - weight children who remain at normal body weight as adults the results reflect the lifetime direct medical costs attributable to obesity for each fifth grade cohort over the three - year period in california and texas .", "the analysis used the costs estimated for a ten - year - old obese child ; however , it should be noted that students in the fifth grade range from ages 911 .", "the percentage of fifth grade students with bmi values in each category remained stable from the 2010  2011 school year through the 2012  2013 school year within each state ( see table 1 ) . just over half of fifth graders in each state", "were identified as having bmi values in the hfz , whereas at least one - third of fifth graders in each state were identified as having bmi values in the ni ", "hr for bmi , and in texas , between 36.5 and 36.8 percent of students were categorized as ni ", "hr for bmi during this timeframe . despite their geographical distance from one another , california and texas had comparable percentages of students in each category for each of the three school years .", "hr zone for each year in each state was multiplied by the estimated per capita lifetime direct medical costs of obesity previously established by finkelstein and colleagues ( 7 ) .", "the resulting values represent the low , high , and recommended statewide lifetime costs of obesity for the ten - year - old cohorts accounting for adulthood weight gain ( see table 2 ) and compared to children who maintain normal weight as adults ( see table 3 ) .", "using the presidential youth fitness program s fitnessgram body composition data , results indicate that in each of the two most populous states , childhood obesity for each cohort of fifth graders over the 3-year timeframe will cost between $ 1.4 and $ 3.0 billion ( compared to normal - weight children who become overweight as adults ) and $ 1.8 and $ 3.0 billion ( compared to normal - weight children who remain at normal weight as adults ) beyond the direct medical expenses for children of normal weight who remain at normal weight throughout adulthood .", "these results represent the costs solely for fifth graders in california and texas and not the additional accrued expenses for children of other ages as finkelstein and colleagues only estimated costs for the ten - year - old age group ( 7 ) . additionally , these values represent only the direct medical costs and not indirect costs , such as absenteeism , which would result in higher estimates . throughout the three years for", "which data were analyzed , the number of fifth graders who presented with bmi values in the ni ", "this underscores the need for the development of effective childhood obesity prevention and reduction efforts .", "fitnessgram results indicate that on average , 33.9% of fifth graders in california and 36.7% of fifth graders in texas have bmi values that place them at high risk for problematic health outcomes .", "the number of ten - year - olds in the us population was just over 4 million on july 1 each year from 2010 to 2012 ( 26 ) . if the percentage of fifth graders with bmi values in the ni ", "hr category in california and texas were extrapolated to the us population of ten - year - olds , this results in a potential lifetime economic burden of approximately $ 17 billion ( accounting for adulthood weight gain ) or $ 25 billion ( not accounting for adulthood weight gain ) in direct medical costs beyond that of individuals at a healthy weight for each single - year age cohort .", "in contrast to these findings , nhanes data from 2011  2012 indicate that 17.7% of children aged 6  11 were classified as obese by bmi according to the cdc standards ( 19 ) . using this obesity estimate along with recommended cost estimates , the increased lifetime economic burden of obesity would be approximately $ 9 billion ( accounting for adulthood weight gain ) or $ 13.5 billion ( not accounting for adulthood weight gain ) in direct medical costs for each single - year age cohort", ". this discrepancy might be partially due to the method used for the determination of childhood obesity .", "the cdc defines obesity for youth ages 2  19 as having a bmi greater than or equal to the 95 percentile on sex - specific bmi - for - age growth charts ( 2 ) . using this method ,", "the minimum bmi values that indicate obesity are 22.03 kgm for boys aged 10.00  10.49 years , 24.16 kgm for boys aged 10.50  10.99 years , 23.18 kgm for girls aged 10.00  10.49 years , and 23.35 kgm for girls aged 10.50  10.99 years ( 11 ) .", "the bmi values that indicate childhood obesity per the cdc method are higher than those utilized in the fitnessgram to indicate high risk of excess adiposity - associated health problems .", "the fitnessgram scientific advisory board has identified body fat percentages that correspond with increased metabolic syndrome and cardiovascular disease risk factors in children and calculated the associated bmi values ( 9 , 13 ) .", "these bmi values , 20.8 kgm for ten - year - old boys and 21.0 kgm for ten - year - old girls , were identified as the cutoff values for high - risk bmi ( 15 ) .", "meta - analysis conducted by javed and colleagues suggests that the cdc bmi standard to determine childhood obesity status compared to reference measures of adiposity defined as a high body fat percentage ( i.e. , dual - energy x - ray absorptiometry scan , hydrostatic weighing , or air - displacement plethysmography ) is highly specific ( pooled specificity = 0.93 ) but less sensitive ( pooled sensitivity = 0.73 ) and thus might fail to detect excess body fatness in over 25% of cases ( 10 ) .", "therefore , the lower bmi cutoff values used to determine high - risk bmi in the fitnessgram will increase the sensitivity of the childhood obesity estimate . while the fitnessgram is a useful tool to help identify children who are at increased risk based on bmi and researchers can use this information to estimate the economic impact of obesity , a limitation of this study is that fitnessgram results were not available for many states .", "is administered to students nationwide as part of the presidential youth fitness program , more data will become available and more rigorous analysis should be performed on nationwide data", ". however , the current analysis uses data from the two most populous states in the union and underscores the large economic impact of childhood obesity . using bmi data from the presidential youth fitness program s fitnessgram over a three - year timespan and the estimated lifetime direct medical costs associated with obesity as recommended by finkelstein and colleagues ( 7 ) ,", "results indicate that the estimated lifetime medical costs of childhood obesity in the two most populous states , california and texas , are between $ 1.4 and $ 3.0 billion for each single - year age cohort analyzed .", "this information can be used to encourage spending , resource development , and prevention program implementation to reduce obesity in these two states .", "further analyses should be conducted to estimate the economic burden associated with childhood obesity using the criterion - referenced fitnessgram bmi standards across the united states when data are available ."], "abstract_text": ["<S> the prevalence of childhood obesity in the united states increased more than three - fold from 1976  1980 to 2007  2008 . the presidential youth fitness program s fitnessgram is the current method recommended by the president s council on fitness , sports & nutrition for assessing health - related fitness factors , including body composition . </S>", "<S> fitnessgram data from california and texas , the two most populous states , over a three - year time span indicate that more than one - third of fifth grade students , typically ten - year - olds , are obese . </S>", "<S> previous studies report that an obese ten - year - old child who remains obese into adulthood will incur elevated direct medical costs beyond his or her normal - weight peers over a lifetime . </S>", "<S> the recommended elevated cost estimates are approximately $ 12,660 when comparing against a normal - weight child who gains weight as an adult and approximately $ 19,000 compared to a child who remains at normal weight as an adult . by applying these figures to fitnessgram results from california and texas </S>", "<S> , each group of fifth grade students in each of the two states will incur between $ 1.4 and $ 3.0 billion in direct medical costs over a lifetime . when the percentage of obese fifth graders is extrapolated to the rest of the united states 4 million ten - year - olds , this results in </S>", "<S> more than $ 17 billion ( accounting for adulthood weight gain ) or $ 25 billion ( not accounting for adulthood weight gain ) in added direct lifetime medical costs attributable to obesity for this single - year age cohort . </S>", "<S> this information should be used to influence spending decisions and resource allocation to obesity reduction and prevention efforts . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION"], "sections": [["the prevalence of obesity in the united states has increased over the past several decades . according to the national health and nutrition examination survey ( nhanes ) results , between the years of 1988 and 1994", ", 22.9% of adults aged 20 years and older exceeded the criteria for adult obesity [ body mass index ( bmi )  30 kgm ] ( 17 ) . by the 20112012 survey ,", "that percentage had increased to 35.1% ( 19 ) . this represents more than a 50% increase in obesity prevalence among american adults aged 20 years and older over the course of two decades .", "perhaps more concerning is the prevalence of obesity among american youth , defined by the centers for disease control and prevention ( cdc ) as a bmi greater than or equal to the 95 percentile on the cdc growth charts ( 2 ) . among children and adolescents aged 2", " 19 years , the prevalence of obesity increased from 5.5% between the years of 1976 and 1980 to 16.9% between 2007 and 2008 ( 18 ) and remained 16.9% between 2011 and 2012 ( 19 ) .", "analyses of several large - scale data pools confirm that obesity contributes to the risk for development of many negative health outcomes including coronary heart disease , hypertriglyceridemia , hypercholesterolemia , hypertension , and type ii diabetes ( 20 , 23 ) .", "using secondary data , thompson and colleagues developed a model to analyze the relationship between bmi and several disease conditions in adults ( 23 ) .", "notably , they found that the risks for hypertension and type ii diabetes are at least 2- and 3-fold higher for obese individuals with bmi values of 32.5 kgm and 37.5 kgm , respectively , as compared to normal - weight individuals with bmi values of 22.5 kgm .", "obesity - related health risks are not limited to adults . according to an analysis of bogalusa heart study data , the odds ratios between childhood obesity and many cardiovascular disease risk factors , including hypercholesterolemia , hypertriglyceridemia , hyperinsulinemia , and hypertension are between 2.4 and 12.6 , indicating a strong , positive association between childhood obesity and these specific risk factors ( 8) .", "finkelstein and colleagues estimated that in 2006 , the annual per capita medical expenditure for an obese person was $ 1,429 ( adjusted to reflect 2008 dollars ) higher than for a person of normal weight , which represents 42% higher medical costs for obese individuals compared to individuals with normal body weights ( 5 ) .", "data from over 3,000 counties and county equivalents across the united states indicate that in 2006 , approximately 54 million individuals met the criteria for obesity ( 16 ) .", "this equates to an extra $ 77 billion ( adjusted to reflect 2008 dollars ) in medical spending attributable to obesity in 2006 alone . beginning in the 2013  2014 school year , the president s council on fitness , sports & nutrition adopted the presidential youth fitness program to replace the president s challenge physical fitness test ( 22 ) .", "this program aims to encourage healthy , active lifestyles among youth . as part of this program ,", "schools use the presidential youth fitness program s fitnessgram to assess health - related fitness and use these data to inform physical education instruction . the fitnessgram scientific advisory board has developed four categories for classification of body composition : very lean , healthy fitness zone ( hfz ) , needs improvement  some risk ( ni  sr ) , and needs improvement  high risk ( ni  hr ) .", "risk level was established based on body fat percentage , and body fat cutoff percentages were converted to corresponding bmi values . for ten - year olds , the ni ", "hr category includes boys with a bmi  20.8 kgm and girls with a bmi  21.0 kgm ( 12 , 13 , 15 ) .", "these bmi cutoff values are lower than those utilized by the cdc , which defines childhood obesity as having a bmi at or above the 95 percentile on sex - specific bmi - for - age growth charts ( 2 ) .", "the corresponding bmi values that the cdc uses to indicate obesity are 22.03 kgm for boys aged 10.00  10.49 years , 24.16 kgm for boys aged 10.50  10.99 years , 23.18 kgm for girls aged 10.00  10.49 years , and 23.35 kgm for girls aged 10.50  10.99 years ( 11 ) .", "thus , using the cdc childhood obesity estimates could result in a substantial underestimation of the number of children at risk for negative health outcomes based upon bmi .", "children identified as obese by bmi have an increased risk of developing health complications that were previously thought to be limited to adults such as hypertension and type ii diabetes mellitus ( 3 ) .", "these conditions are also well - known risk factors for the development of cardiovascular disease .", "obese children aged ten years and older are likely to remain obese into early adulthood ( 28 ) , and therefore , accrue medical costs associated with obesity - related conditions .", "knowledge of the medical costs associated with childhood obesity can provide a basis by which to justify the cost of childhood obesity prevention and treatment efforts . to this end ,", "finkelstein and colleagues analyzed the lifetime direct medical costs for an obese ten - year - old beyond the costs that a ten - year - old at a normal body weight would incur over his or her lifespan ( 7 ) .", "the six investigations included in the meta - analysis each estimated longevity using a variety of factors such as probability of survival based on weight status ( 6 ) , gender , and race ( 4 , 14 , 2325 , 27 ) . based upon this meta - analysis ,", "two low- and high - end cost estimates were provided : one that accounted for adulthood weight gain among normal - weight children and one that compared against children who remained at normal weight through adulthood .", "results suggested that an obese ten - year - old , typically in the fifth grade , will incur between $ 12,660 ( low - end ; recommended ) and $ 19,630 ( high - end ) of incremental direct medical costs when accounting for weight gain through adulthood and between $ 16,310 ( low - end ) and $ 19,350 ( high - end ) with a recommended cost estimate of $ 19,000 when contrasted with a child who remains at normal weight throughout adulthood ( 4 , 7 , 14 , 23 , 24 , 27 ) .", "using these recommended estimates along with results from the fitnessgram body composition classifications , the purpose of this analysis is to estimate the lifetime economic impact of childhood obesity for this single age cohort in the two most populous states , california and texas ."], ["fitnessgram data from the school years 20102011 , 20112012 , and 20122013 were obtained for california ( 1 ) and texas ( 21 ) .", "these data were school - level and included boys and girls from all public school districts in these two states .", "the total number of fifth grade students tested for bmi in california and in texas and the number of fifth grade students in each bmi category were calculated for each of the three school years .", "the fitnessgram scientific advisory board has determined that bmi values at or above 20.8 kgm for ten - year - old boys and 21.0 kgm for ten - year - old girls represent high health risk ( 15 ) .", "the california report included the total number of fifth graders tested for body composition as well as the number and percentage of students in each fitness zone .", "the texas report included data for each grade level and further separated the data by district and gender .", "the total number of fifth graders tested for body composition and the number and percentage of students in each fitness zone were calculated using excel .", "then , using the low , high , and recommended cost estimates suggested by finkelstein and colleagues ( 7 ) , the lifetime direct medical costs attributable to obesity were calculated for students in the ni  hr category for each of the three school years in california and in texas by multiplying the number of students in this category by the corresponding cost estimate per obese student", "the first set of estimates compared the direct medical costs for obese children against the direct medical costs for normal - weight children who gain weight as adults .", "the second set of estimates compared the direct medical costs for obese children against the direct medical costs for normal - weight children who remain at normal body weight as adults the results reflect the lifetime direct medical costs attributable to obesity for each fifth grade cohort over the three - year period in california and texas .", "the analysis used the costs estimated for a ten - year - old obese child ; however , it should be noted that students in the fifth grade range from ages 911 ."], ["the percentage of fifth grade students with bmi values in each category remained stable from the 2010  2011 school year through the 2012  2013 school year within each state ( see table 1 ) . just over half of fifth graders in each state", "were identified as having bmi values in the hfz , whereas at least one - third of fifth graders in each state were identified as having bmi values in the ni ", "hr for bmi , and in texas , between 36.5 and 36.8 percent of students were categorized as ni ", "hr for bmi during this timeframe . despite their geographical distance from one another , california and texas had comparable percentages of students in each category for each of the three school years .", "hr zone for each year in each state was multiplied by the estimated per capita lifetime direct medical costs of obesity previously established by finkelstein and colleagues ( 7 ) .", "the resulting values represent the low , high , and recommended statewide lifetime costs of obesity for the ten - year - old cohorts accounting for adulthood weight gain ( see table 2 ) and compared to children who maintain normal weight as adults ( see table 3 ) ."], ["using the presidential youth fitness program s fitnessgram body composition data , results indicate that in each of the two most populous states , childhood obesity for each cohort of fifth graders over the 3-year timeframe will cost between $ 1.4 and $ 3.0 billion ( compared to normal - weight children who become overweight as adults ) and $ 1.8 and $ 3.0 billion ( compared to normal - weight children who remain at normal weight as adults ) beyond the direct medical expenses for children of normal weight who remain at normal weight throughout adulthood .", "these results represent the costs solely for fifth graders in california and texas and not the additional accrued expenses for children of other ages as finkelstein and colleagues only estimated costs for the ten - year - old age group ( 7 ) . additionally , these values represent only the direct medical costs and not indirect costs , such as absenteeism , which would result in higher estimates . throughout the three years for", "which data were analyzed , the number of fifth graders who presented with bmi values in the ni ", "this underscores the need for the development of effective childhood obesity prevention and reduction efforts .", "fitnessgram results indicate that on average , 33.9% of fifth graders in california and 36.7% of fifth graders in texas have bmi values that place them at high risk for problematic health outcomes .", "the number of ten - year - olds in the us population was just over 4 million on july 1 each year from 2010 to 2012 ( 26 ) . if the percentage of fifth graders with bmi values in the ni ", "hr category in california and texas were extrapolated to the us population of ten - year - olds , this results in a potential lifetime economic burden of approximately $ 17 billion ( accounting for adulthood weight gain ) or $ 25 billion ( not accounting for adulthood weight gain ) in direct medical costs beyond that of individuals at a healthy weight for each single - year age cohort .", "in contrast to these findings , nhanes data from 2011  2012 indicate that 17.7% of children aged 6  11 were classified as obese by bmi according to the cdc standards ( 19 ) . using this obesity estimate along with recommended cost estimates , the increased lifetime economic burden of obesity would be approximately $ 9 billion ( accounting for adulthood weight gain ) or $ 13.5 billion ( not accounting for adulthood weight gain ) in direct medical costs for each single - year age cohort", ". this discrepancy might be partially due to the method used for the determination of childhood obesity .", "the cdc defines obesity for youth ages 2  19 as having a bmi greater than or equal to the 95 percentile on sex - specific bmi - for - age growth charts ( 2 ) . using this method ,", "the minimum bmi values that indicate obesity are 22.03 kgm for boys aged 10.00  10.49 years , 24.16 kgm for boys aged 10.50  10.99 years , 23.18 kgm for girls aged 10.00  10.49 years , and 23.35 kgm for girls aged 10.50  10.99 years ( 11 ) .", "the bmi values that indicate childhood obesity per the cdc method are higher than those utilized in the fitnessgram to indicate high risk of excess adiposity - associated health problems .", "the fitnessgram scientific advisory board has identified body fat percentages that correspond with increased metabolic syndrome and cardiovascular disease risk factors in children and calculated the associated bmi values ( 9 , 13 ) .", "these bmi values , 20.8 kgm for ten - year - old boys and 21.0 kgm for ten - year - old girls , were identified as the cutoff values for high - risk bmi ( 15 ) .", "meta - analysis conducted by javed and colleagues suggests that the cdc bmi standard to determine childhood obesity status compared to reference measures of adiposity defined as a high body fat percentage ( i.e. , dual - energy x - ray absorptiometry scan , hydrostatic weighing , or air - displacement plethysmography ) is highly specific ( pooled specificity = 0.93 ) but less sensitive ( pooled sensitivity = 0.73 ) and thus might fail to detect excess body fatness in over 25% of cases ( 10 ) .", "therefore , the lower bmi cutoff values used to determine high - risk bmi in the fitnessgram will increase the sensitivity of the childhood obesity estimate . while the fitnessgram is a useful tool to help identify children who are at increased risk based on bmi and researchers can use this information to estimate the economic impact of obesity , a limitation of this study is that fitnessgram results were not available for many states .", "is administered to students nationwide as part of the presidential youth fitness program , more data will become available and more rigorous analysis should be performed on nationwide data", ". however , the current analysis uses data from the two most populous states in the union and underscores the large economic impact of childhood obesity . using bmi data from the presidential youth fitness program s fitnessgram over a three - year timespan and the estimated lifetime direct medical costs associated with obesity as recommended by finkelstein and colleagues ( 7 ) ,", "results indicate that the estimated lifetime medical costs of childhood obesity in the two most populous states , california and texas , are between $ 1.4 and $ 3.0 billion for each single - year age cohort analyzed .", "this information can be used to encourage spending , resource development , and prevention program implementation to reduce obesity in these two states .", "further analyses should be conducted to estimate the economic burden associated with childhood obesity using the criterion - referenced fitnessgram bmi standards across the united states when data are available ."]]}
{"article_id": "PMC4053678", "article_text": ["brown tumor is a non - neoplastic giant cell lesion characterized by increased circulating levels of parathyroid hormone ( pth ) .", "it is usually an uncommon lesion occurring with the frequency of 4.5% in primary hyperparathyroidism ( hpt ) and 1.5 - 1.7% in cases of secondary hpt , with overall incidence of 0.1% .", "hpt can be classified into : primary , which occurs due to hyperplasia , benign or malignant neoplasm of one or more parathyroid gland .", "secondary hpt is caused as a result of hypocalcemia , vitamin d deficiency or secondary to chronic renal insufficiency , which acts as a stimulus for pth production .", "tertiary hpt is associated with renal failure and autonomous functioning glands in long - standing secondary hpt cases .", "the fourth type of hpt has been recognized , which occurs due to increased pth levels synthesized in patients with malignant diseases .", "we report an interesting and rather unique case of a brown tumor of maxilla and mandible developing after acute exogenous thyroxine poisoning in a young female patient under treatment for hypothyroidism and ectopic intrathoracic parathyroid adenoma .", "a 23-year - old female reported to our department with the chief complaint of a swelling on the left side of the lower jaw for past 4 months producing facial asymmetry .", "patient was a known hypothyroid and was under exogenous levothyroxine ( l - thyroxine ) therapy for the past 3 years .", "she gave history of l - thyroxine poisoning ( approximately 4000 mcg stat dose ) 4 months back after which she developed rapidly growing swelling on the left side of the jaw .", "extraoral examination revealed the presence of a well - circumscribed expansile swelling in the left mandibular body region measuring 5 cm  4 cm , which was hard , non - tender and non - mobile without any surface changes .", "intraorally , obliteration of buccal vestibule on the left side with intact normal mucosa was seen .", "no mobility , loss of vitality or tenderness was elicited with any teeth in the left quadrant . biochemical assay and", "blood analysis revealed an increased value of serum alkaline phosphatase ( 420 u / l ; normal range : 100 - 172 u / l ) , serum pth ( 370.40 pg / ml ; normal range : 50 - 300 pg / ml ) and serum calcium ( 13.3 mgs / dl ; normal range : 8.5 - 11.0 mg / dl ) .", "the serum phosphorus level was decreased ( 2.4 mg / dl ; normal range : 2.7 - 4.5 mg / dl ) .", "cone beam computed tomography ( ct ) scan of the facial region revealed a well - defined soft tissue lesion within the left body of the mandible of approximately 3.5 cm  2.8 cm [ figure 1a and d ] .", "it also represented well - defined hypodense lesions in relation to the right body of the mandible measuring 2.5 cm  1.0 cm [ figure 1d ] and anterior maxilla , measuring 3.5 cm  3.6 cm [ figure 1b ] .", "there was also lytic lesion on the left side skull bone with generalized reduction in bone density [ figure 1c ] .", "a full body skeletal survey was also performed , which revealed no such lesions in long bones .", "( a ) computed tomography scan ( axial section ) showing well - defined soft tissue lesion within the left body of the mandible .", "( d ) 3d reconstruction showing bilateral lytic lesion within the body of the mandible and generalized reduction in the bone density ( arrow indicating the lesion ) ultrasound of neck failed to reveal any pathology in thyroid and parathyroid glands . to further assess the parathyroid gland status tc sestamibi - spect parathyroid scintigraphy", "was carried out , which demonstrated relatively prominent flow of activity toward left submandibular salivary gland region .", "abnormal ovoid region of intense tracer concentration in the anterior mediastinum in right paratracheal region was noted .", "the findings were suggestive of ectopic ( intrathoracic ) parathyroid gland with neoplastic changes in left submandibular salivary gland region [ figure 2a ] .", "( a ) tc sestamibi - single photon emission computed tomography parathyroid scintigraphy image showing intense tracer concentration in right paratracheal region and left submandibular region .", "( b ) computed tomography scan ( axial section ) showing well defined mass in intrathoracic prevascular space ct scan of the chest was taken , which revealed a moderate size nodular lesion showing heterogeneous enhancement in right prevascular space measuring 3.0 cm  2.4 cm [ figure 2b ] . the history , biochemical values and imaging reports corroborated with the clinical features of the brown tumor of hpt associated with pathologic ectopic parathyroid gland", ". an incisional biopsy of the mandibular lesion was performed under local anesthesia that revealed fibro collagenous tissue containing plenty of osteoclastic giant cells dispersed throughout the lesion with small fragments of reactive bone , the features consistent with reparative giant cell granuloma .", "thoracotomy was carried out and ectopic intrathoracic parathyroid gland was excised [ figure 3a ] .", "a combination of sharp and blunt dissection was used to excise the mass and it was delivered per oral in total [ figure 3b ] .", "post - operative serum calcium level was 10.6 mg / dl and pth level 11.90 pg / mg . intra - operative view of ( a ) ectopic parathyroid gland .", "( b ) left mandibular lesion the intrathoracic mass and mandibular specimen was sent for histopathological examination .", "the intrathoracic mass revealed parathyroid neoplasm suggestive of atypical parathyroid adenoma [ figure 4a ] .", "the mandibular lesion presented a giant cell proliferation in the background of a variably fibrotic stroma .", "the giant cells were arranged in sheets with little intervening spindle cell stroma . at the periphery osteopenic bone trabecule", "these features were suggestive of brown tumor of hpt [ figure 4b ] . ( a ) histopathological section of mandibular lesion showing giant cell proliferation with variable areas of haemorrhage and hemosiderin deposition ( h and e , 20 ) .", "( b ) histopathological section of parathyroid neoplasm composed of nuclear pleomorphism with cystic changes , suggestive of parathyroid adenoma ( h and e , 20 )", "pth plays a key role in calcium and phosphate balance between extracellular fluid and bones .", "brown tumor is relatively an uncommon lesion associated with hpt , which results in an abnormal osteoclastic and osteoblastic activity resulting in resorption and fibrous replacement of the bone .", "brown tumor is more commonly found in ribs , clavicles , pelvis , femur and facial bones . in craniofacial region mandible", "radiographically , this lesion appears as well - defined unilocular or multilocular radiolucencies causing cortical plate expansion and often thinning of the cortical plates .", "the density of jaw is decreased due to generalized demineralization of the medullary bones along with change in trabecule pattern giving a mixed radiopaque - radiolucent appearance .", "brown tumor mimics giant cell lesions and it can be distinguished from the latter based upon the clinical history and biochemical profile of the patient indicating hpt . due to overlapping clinical and radiological features , patients presenting in maxillofacial department with suspected giant cell lesion should undergo biochemical assay to rule out hpt .", "other differentials to be included are cherubism , aneurysmal bone cyst , paget 's disease , langerhans cell histiocytosis , osteosarcoma and osteomyelitis .", "histopathologically , brown tumor reveals multinucleated giant cells in a background of spindle cell proliferation along with a large amount of hemosiderin deposition , vascularity and hemorrhage giving brown appearance to this lesion .", "the most interesting aspect of this case report is association of brown tumor with ectopic intrathoracic parathyroid gland with normal functioning anatomic parathyroid glands .", "ectopic parathyroid glands are uncommon , mainly arising due to abnormal migration of parathyroid tissue during embryogenesis .", "it has been reported that in almost 16% cases of hpt , ectopic parathyroid glands are present , which may often go unnoticed and are a cause for failed parathyroid exploration .", "the uniqueness of the case is further defined by its occurrence at a very young age ( 23 years ) along with its bilateral presentation with simultaneous involvement of maxilla , mandible and skull bones in a brief period .", "this patient was hypothyroid , under thyroxine therapy for past 3 years with an episode of acute poisoning of l - thyroxine before the onset of swelling of the jaw .", "the routine biochemical examination of patient taken 3 months prior to the onset of swelling revealed normal serum calcium and phosphorus levels and hypothyroid state . unfortunately , consultant endocrinologist and physicians failed to observe any relation between the hyperparathyroid state of the patient and exogenous thyroxine .", "to best of our knowledge , no case of brown tumor of hpt with such aggressive behavior and wide spectrum of clinical findings associated with acute thyroxine ingestion has been reported in the past .", "hence , we assume that either bolus toxic dose of thyroxine or chronic state of hypothyroidism has caused a high pth and serum calcium levels resulting in a hyperparathyroid state causing bone remodeling events in the craniofacial region .", "few authors have reported that prolonged thyroid - stimulating hormone ( tsh ) stimulation may lead to hpt or a state of hpt in hypothyroidism and vice versa .", "this case report establishes a credible support for this hypothesis and brings out a definite correlation between tsh inhibition and onset of hpt .", "the treatment of brown tumor mainly focuses on correction of the underlying disorder and maintenance of normal pth and serum calcium levels .", "use of systemic or intralesional corticosteroid have been reported to reduce the size of the lesion .", "long - term follow - up of such lesion is mandatory as variable clinical behavior of the lesion following normalization of the pth and serum calcium levels has been reported .", "we have also adopted a similar protocol with surgical excision of the pathological ectopic parathyroid gland and mandibular tumor , which was causing severe disfigurement of the patient 's face .", "pth and serum calcium levels were found to be normal within 3 days after surgery .", "follow - up radiographs of the patient has revealed marked regression in the craniofacial lesions .", "ultra sonographic scan , ct scan and full body skeletal survey in conjunction with complete biochemical analysis can be carried out to assess the pathological parathyroid gland and extent of lesions in long bones .", "latest imaging techniques such as tc - sestamibi scan and fluorine - fluorodeoxyglucose - positron emission tomography / ct have been proved to be an effective and useful diagnostic modality in assessing location and functioning of parathyroid glands .", "our patient represents a rare case of brown tumor with a wide spectrum of associated clinical findings . in conclusion", ", the management of the brown tumor should involve early diagnosis , complete biochemical assay and full body skeletal survey followed by normalization of pth , serum calcium and phosphorus levels and parathyroidectomy , if indicated . in the absence of any pathology of anatomic parathyroid", "this case emphasizes the need for periodic biochemical investigations in the hypothyroid patients on exogenous thyroxine therapy ."], "abstract_text": ["<S> brown tumor is a giant cell lesion associated with hyperparathyroidism . </S>", "<S> it is a non - neoplastic condition and represents terminal stage of the remodeling process in hyperparathyroid state . </S>", "<S> we report a case of brown tumor with multiple lesions in craniofacial region associated with ectopic parathyroid adenoma revealed after acute l - thyroxine poisoning . </S>", "<S> this case report emphasizes on the need for routine biochemical investigations along with serum calcium , phosphorus and parathyroid hormone levels in patients on thyroxine therapy . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["brown tumor is a non - neoplastic giant cell lesion characterized by increased circulating levels of parathyroid hormone ( pth ) .", "it is usually an uncommon lesion occurring with the frequency of 4.5% in primary hyperparathyroidism ( hpt ) and 1.5 - 1.7% in cases of secondary hpt , with overall incidence of 0.1% .", "hpt can be classified into : primary , which occurs due to hyperplasia , benign or malignant neoplasm of one or more parathyroid gland .", "secondary hpt is caused as a result of hypocalcemia , vitamin d deficiency or secondary to chronic renal insufficiency , which acts as a stimulus for pth production .", "tertiary hpt is associated with renal failure and autonomous functioning glands in long - standing secondary hpt cases .", "the fourth type of hpt has been recognized , which occurs due to increased pth levels synthesized in patients with malignant diseases .", "we report an interesting and rather unique case of a brown tumor of maxilla and mandible developing after acute exogenous thyroxine poisoning in a young female patient under treatment for hypothyroidism and ectopic intrathoracic parathyroid adenoma ."], ["a 23-year - old female reported to our department with the chief complaint of a swelling on the left side of the lower jaw for past 4 months producing facial asymmetry .", "patient was a known hypothyroid and was under exogenous levothyroxine ( l - thyroxine ) therapy for the past 3 years .", "she gave history of l - thyroxine poisoning ( approximately 4000 mcg stat dose ) 4 months back after which she developed rapidly growing swelling on the left side of the jaw .", "extraoral examination revealed the presence of a well - circumscribed expansile swelling in the left mandibular body region measuring 5 cm  4 cm , which was hard , non - tender and non - mobile without any surface changes .", "intraorally , obliteration of buccal vestibule on the left side with intact normal mucosa was seen .", "no mobility , loss of vitality or tenderness was elicited with any teeth in the left quadrant . biochemical assay and", "blood analysis revealed an increased value of serum alkaline phosphatase ( 420 u / l ; normal range : 100 - 172 u / l ) , serum pth ( 370.40 pg / ml ; normal range : 50 - 300 pg / ml ) and serum calcium ( 13.3 mgs / dl ; normal range : 8.5 - 11.0 mg / dl ) .", "the serum phosphorus level was decreased ( 2.4 mg / dl ; normal range : 2.7 - 4.5 mg / dl ) .", "cone beam computed tomography ( ct ) scan of the facial region revealed a well - defined soft tissue lesion within the left body of the mandible of approximately 3.5 cm  2.8 cm [ figure 1a and d ] .", "it also represented well - defined hypodense lesions in relation to the right body of the mandible measuring 2.5 cm  1.0 cm [ figure 1d ] and anterior maxilla , measuring 3.5 cm  3.6 cm [ figure 1b ] .", "there was also lytic lesion on the left side skull bone with generalized reduction in bone density [ figure 1c ] .", "a full body skeletal survey was also performed , which revealed no such lesions in long bones .", "( a ) computed tomography scan ( axial section ) showing well - defined soft tissue lesion within the left body of the mandible .", "( d ) 3d reconstruction showing bilateral lytic lesion within the body of the mandible and generalized reduction in the bone density ( arrow indicating the lesion ) ultrasound of neck failed to reveal any pathology in thyroid and parathyroid glands . to further assess the parathyroid gland status tc sestamibi - spect parathyroid scintigraphy", "was carried out , which demonstrated relatively prominent flow of activity toward left submandibular salivary gland region .", "abnormal ovoid region of intense tracer concentration in the anterior mediastinum in right paratracheal region was noted .", "the findings were suggestive of ectopic ( intrathoracic ) parathyroid gland with neoplastic changes in left submandibular salivary gland region [ figure 2a ] .", "( a ) tc sestamibi - single photon emission computed tomography parathyroid scintigraphy image showing intense tracer concentration in right paratracheal region and left submandibular region .", "( b ) computed tomography scan ( axial section ) showing well defined mass in intrathoracic prevascular space ct scan of the chest was taken , which revealed a moderate size nodular lesion showing heterogeneous enhancement in right prevascular space measuring 3.0 cm  2.4 cm [ figure 2b ] . the history , biochemical values and imaging reports corroborated with the clinical features of the brown tumor of hpt associated with pathologic ectopic parathyroid gland", ". an incisional biopsy of the mandibular lesion was performed under local anesthesia that revealed fibro collagenous tissue containing plenty of osteoclastic giant cells dispersed throughout the lesion with small fragments of reactive bone , the features consistent with reparative giant cell granuloma .", "thoracotomy was carried out and ectopic intrathoracic parathyroid gland was excised [ figure 3a ] .", "a combination of sharp and blunt dissection was used to excise the mass and it was delivered per oral in total [ figure 3b ] .", "post - operative serum calcium level was 10.6 mg / dl and pth level 11.90 pg / mg . intra - operative view of ( a ) ectopic parathyroid gland .", "( b ) left mandibular lesion the intrathoracic mass and mandibular specimen was sent for histopathological examination .", "the intrathoracic mass revealed parathyroid neoplasm suggestive of atypical parathyroid adenoma [ figure 4a ] .", "the mandibular lesion presented a giant cell proliferation in the background of a variably fibrotic stroma .", "the giant cells were arranged in sheets with little intervening spindle cell stroma . at the periphery osteopenic bone trabecule", "these features were suggestive of brown tumor of hpt [ figure 4b ] . ( a ) histopathological section of mandibular lesion showing giant cell proliferation with variable areas of haemorrhage and hemosiderin deposition ( h and e , 20 ) .", "( b ) histopathological section of parathyroid neoplasm composed of nuclear pleomorphism with cystic changes , suggestive of parathyroid adenoma ( h and e , 20 )"], ["pth plays a key role in calcium and phosphate balance between extracellular fluid and bones .", "brown tumor is relatively an uncommon lesion associated with hpt , which results in an abnormal osteoclastic and osteoblastic activity resulting in resorption and fibrous replacement of the bone .", "brown tumor is more commonly found in ribs , clavicles , pelvis , femur and facial bones . in craniofacial region mandible", "radiographically , this lesion appears as well - defined unilocular or multilocular radiolucencies causing cortical plate expansion and often thinning of the cortical plates .", "the density of jaw is decreased due to generalized demineralization of the medullary bones along with change in trabecule pattern giving a mixed radiopaque - radiolucent appearance .", "brown tumor mimics giant cell lesions and it can be distinguished from the latter based upon the clinical history and biochemical profile of the patient indicating hpt . due to overlapping clinical and radiological features , patients presenting in maxillofacial department with suspected giant cell lesion should undergo biochemical assay to rule out hpt .", "other differentials to be included are cherubism , aneurysmal bone cyst , paget 's disease , langerhans cell histiocytosis , osteosarcoma and osteomyelitis .", "histopathologically , brown tumor reveals multinucleated giant cells in a background of spindle cell proliferation along with a large amount of hemosiderin deposition , vascularity and hemorrhage giving brown appearance to this lesion .", "the most interesting aspect of this case report is association of brown tumor with ectopic intrathoracic parathyroid gland with normal functioning anatomic parathyroid glands .", "ectopic parathyroid glands are uncommon , mainly arising due to abnormal migration of parathyroid tissue during embryogenesis .", "it has been reported that in almost 16% cases of hpt , ectopic parathyroid glands are present , which may often go unnoticed and are a cause for failed parathyroid exploration .", "the uniqueness of the case is further defined by its occurrence at a very young age ( 23 years ) along with its bilateral presentation with simultaneous involvement of maxilla , mandible and skull bones in a brief period .", "this patient was hypothyroid , under thyroxine therapy for past 3 years with an episode of acute poisoning of l - thyroxine before the onset of swelling of the jaw .", "the routine biochemical examination of patient taken 3 months prior to the onset of swelling revealed normal serum calcium and phosphorus levels and hypothyroid state . unfortunately , consultant endocrinologist and physicians failed to observe any relation between the hyperparathyroid state of the patient and exogenous thyroxine .", "to best of our knowledge , no case of brown tumor of hpt with such aggressive behavior and wide spectrum of clinical findings associated with acute thyroxine ingestion has been reported in the past .", "hence , we assume that either bolus toxic dose of thyroxine or chronic state of hypothyroidism has caused a high pth and serum calcium levels resulting in a hyperparathyroid state causing bone remodeling events in the craniofacial region .", "few authors have reported that prolonged thyroid - stimulating hormone ( tsh ) stimulation may lead to hpt or a state of hpt in hypothyroidism and vice versa .", "this case report establishes a credible support for this hypothesis and brings out a definite correlation between tsh inhibition and onset of hpt .", "the treatment of brown tumor mainly focuses on correction of the underlying disorder and maintenance of normal pth and serum calcium levels .", "use of systemic or intralesional corticosteroid have been reported to reduce the size of the lesion .", "long - term follow - up of such lesion is mandatory as variable clinical behavior of the lesion following normalization of the pth and serum calcium levels has been reported .", "we have also adopted a similar protocol with surgical excision of the pathological ectopic parathyroid gland and mandibular tumor , which was causing severe disfigurement of the patient 's face .", "pth and serum calcium levels were found to be normal within 3 days after surgery .", "follow - up radiographs of the patient has revealed marked regression in the craniofacial lesions .", "ultra sonographic scan , ct scan and full body skeletal survey in conjunction with complete biochemical analysis can be carried out to assess the pathological parathyroid gland and extent of lesions in long bones .", "latest imaging techniques such as tc - sestamibi scan and fluorine - fluorodeoxyglucose - positron emission tomography / ct have been proved to be an effective and useful diagnostic modality in assessing location and functioning of parathyroid glands .", "our patient represents a rare case of brown tumor with a wide spectrum of associated clinical findings . in conclusion", ", the management of the brown tumor should involve early diagnosis , complete biochemical assay and full body skeletal survey followed by normalization of pth , serum calcium and phosphorus levels and parathyroidectomy , if indicated . in the absence of any pathology of anatomic parathyroid", "this case emphasizes the need for periodic biochemical investigations in the hypothyroid patients on exogenous thyroxine therapy ."]]}
{"article_id": "PMC3376802", "article_text": ["during april  october 2009 , bird bandings were conducted in the protected area of finca ribavellosa in la rioja , spain ( 4214n , 254w ) .", "ticks were collected from birds and classified through taxonomic keys ( 7 ) and molecular methods ( 8) .", "dna was individually extracted by using 2 incubations of 20 minutes each with ammonium hydroxide ( 1 ml of 25% ammonia and 19 ml of milli - q water that had been autoclaved ) at 100c and 90c .", "dna extracts were used as templates for pcrs targeting fragment genes for tick classification and for bacteria detection ( table 1 ) .", "two negative controls , 1 containing water instead of template dna and the other with template dna but without primers , and a positive control ( a tick extract , a. phagocytophilum , b. burgdorferi sensu stricto , or r. slovaca ) were included in all pcrs .", "amplification products were sequenced , and nucleotide sequences were compared with those available in genbank by using a blast search ( www.ncbi.nlm.nih.gov/blast/blast.cgi ) .", "phylogenetic and molecular evolutionary analyses were conducted by using mega4 ( 16 in technical appendix ) .", ", reference ; msp , p44 major surface protein gene ; flab , flagellin gene ; ompb , 120-kda genus common antigen gene ; ompa , 190-kda protein antigen gene ; glta , citrate synthase gene . ", "r  =  a / g ; w  =  a / t .", "a total of 222 ticks belonging to the species haemaphysalis punctata ( n = 1 ) , ixodes frontalis ( n = 7 ) , i. arboricola ( n = 26 ) , i. ricinus ( n = 181 ) , and other ixodes spp .", "two nucleotide sequences for the 16s rrna fragment gene of i. arboricola ticks were recorded ( genbank accession nos . jf791812 and jf791813 ) ( table 2 ) . *", "l , larva ; msp , p44 major surface protein gene ; n , nymph ; flab , flagellin gene ; 5s-23s is , 5s-23s rrna intergenic spacer ; ompb , 120-kda genus common antigen gene ; ompa , 190-kda protein antigen gene ; glta , citrate synthase gene . ", "a. phagocytophilum was detected only in 1 larva of an i. ricinus tick ( 0.5% ) .", "the most prevalent genospecies was b. garinii ( n = 19 ) , which was detected in i. ricinus ( n = 16 ) , h. punctata ( n = 1 ) , i. frontalis ( n = 1 ) , and ixodes sp .", "b. valaisiana was amplified in 9 samples ( 8 i. ricinus and 1 ixodes sp .", "r. monacensis ( n = 1 ) , r. helvetica ( n = 1 ) , r. sibirica sibirica ( n = 1 ) , and rickettsia spp .", "furthermore , according to glta , ompa , and ompb sequence analysis , a possible new rickettsia sp . was found in 25 i. arboricola ticks and 2 i. ricinus ticks .", "for these 27 samples , highest identities with r. heilongjiangensis ( 97.1% ) and r. japonica ( 99.1% ) were found for ompa ( genbank accession no .", "jf758826 ) nucleotide sequences , respectively , whereas glta nucleotide sequences were identical to those from both rickettsia spp . according to multilocus sequence typing ( data not shown ) and genetic criteria agreed on by experts , a candidatus status could be assigned .", "we named it candidatus rickettsia vini ( 17 in technical appendix ) ( table 2 ) .", "( figure ) . the phylogenetic position of candidatus rickettsia vini based on the ompa nucleotide sequences in a study of the role of birds in dispersal of etiologic agents of tick - borne zoonoses , spain , 2009 .", "the percentage of replicate trees in which the associated taxa clustered in the bootstrap test ( 1,000 replicates ) is shown next to the branches .", "the tree is drawn to scale , with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree .", "the evolutionary distances were computed by using the kimura 2-parameter method and are in the units of the number of base substitutions per site .", "the presence of anaplasma , borrelia , and rickettsia species in ticks removed from passerine birds corroborates the role of these vertebrates in the epidemiology and dispersion of tick - borne pathogens in spain and in other zones of the planet .", "some of the parasitized birds in our study , such as the european robin ( erithacus rubecula ) or eurasian blackcap ( sylvia atricapilla ) , are considered migratory or partial migratory birds .", "in addition , these species share an ecologic niche and ectoparasites ( horizontal transmission ) with other migratory birds that cover long distances from africa to the eurasian region . except for i. arboricola , the tick species captured in this study previously had been found on birds in spain ( 18 in technical appendix ) .", "the high prevalence of i. ricinus ticks was expected because it is the most frequent tick in this area , and the immature stages of this tick frequently parasitize birds .", "i. ricinus ticks are the main vectors of a. phagocytophilum in europe , and this microorganism has been detected on vegetation in the studied area ( 1 ) .", "however , the low prevalence ( 0.5% ) of a. phagocytophilum in the ticks in our study corroborates data from other studies ( 19,20 in technical appendix ) .", "the presence of a. phagocytophilum in a larva in our study supports the role of birds as reservoirs of a. phagocytophilum .", "the prevalence ( 13.1% ) of b. burgdorferi in our samples is similar to prevalences reported in other studies in europe in which i. ricinus is the main species of tick captured from birds ( 19 in technical appendix ) . in spain , b. garinii , b. valaisiana , and b. afzelii", "have been detected in ticks from birds ( 18 in technical appendix ) . according to our data ,", "the human pathogen b. garinii was the most prevalent species , as reported in birds from europe ( 21 in technical appendix ) .", "although it has been recently detected in ticks from birds in norway ( 22 in technical appendix ) , its finding in spain was unexpected .", "regarding rickettsia species , r. monacensis and r. helvetica are among the human pathogens detected in our study .", "both species have been identified in ticks from birds in europe ( 19,20,23 in technical appendix ) . on the contrary ,", "candidatus rickettsia vini , a potential new rickettsia species , also detected in our study , has not been related to human disease ( 17 in technical appendix ) .", "several genospecies closely related to r. heilongjiangensis and r. japonica have been identified in ixodes spp .", "r. sibirica sibirica , responsible for siberian tick typhus in western people s republic of china and in siberia , was also amplified in an i. ricinus larva in this study .", "our data confirm the involvement of birds in the cycle of human tick - borne diseases ."], "abstract_text": ["<S> we amplified gene sequences from anaplasma phagocytophilum , borrelia garinii , b. valaisiana , b. turdi , rickettsia monacensis , r. helvetica , r. sibirica sibirica , and rickettsia spp . </S>", "<S> ( including candidatus rickettsia vini ) in ticks removed from birds in spain . </S>", "<S> the findings support the role of passerine birds as possible dispersers of these tick - borne pathogens . </S>"], "labels": null, "section_names": ["The Study", "Conclusions", "Supplementary Material"], "sections": [["during april  october 2009 , bird bandings were conducted in the protected area of finca ribavellosa in la rioja , spain ( 4214n , 254w ) .", "ticks were collected from birds and classified through taxonomic keys ( 7 ) and molecular methods ( 8) .", "dna was individually extracted by using 2 incubations of 20 minutes each with ammonium hydroxide ( 1 ml of 25% ammonia and 19 ml of milli - q water that had been autoclaved ) at 100c and 90c .", "dna extracts were used as templates for pcrs targeting fragment genes for tick classification and for bacteria detection ( table 1 ) .", "two negative controls , 1 containing water instead of template dna and the other with template dna but without primers , and a positive control ( a tick extract , a. phagocytophilum , b. burgdorferi sensu stricto , or r. slovaca ) were included in all pcrs .", "amplification products were sequenced , and nucleotide sequences were compared with those available in genbank by using a blast search ( www.ncbi.nlm.nih.gov/blast/blast.cgi ) .", "phylogenetic and molecular evolutionary analyses were conducted by using mega4 ( 16 in technical appendix ) .", ", reference ; msp , p44 major surface protein gene ; flab , flagellin gene ; ompb , 120-kda genus common antigen gene ; ompa , 190-kda protein antigen gene ; glta , citrate synthase gene . ", "r  =  a / g ; w  =  a / t .", "a total of 222 ticks belonging to the species haemaphysalis punctata ( n = 1 ) , ixodes frontalis ( n = 7 ) , i. arboricola ( n = 26 ) , i. ricinus ( n = 181 ) , and other ixodes spp .", "two nucleotide sequences for the 16s rrna fragment gene of i. arboricola ticks were recorded ( genbank accession nos . jf791812 and jf791813 ) ( table 2 ) . *", "l , larva ; msp , p44 major surface protein gene ; n , nymph ; flab , flagellin gene ; 5s-23s is , 5s-23s rrna intergenic spacer ; ompb , 120-kda genus common antigen gene ; ompa , 190-kda protein antigen gene ; glta , citrate synthase gene . ", "a. phagocytophilum was detected only in 1 larva of an i. ricinus tick ( 0.5% ) .", "the most prevalent genospecies was b. garinii ( n = 19 ) , which was detected in i. ricinus ( n = 16 ) , h. punctata ( n = 1 ) , i. frontalis ( n = 1 ) , and ixodes sp .", "b. valaisiana was amplified in 9 samples ( 8 i. ricinus and 1 ixodes sp .", "r. monacensis ( n = 1 ) , r. helvetica ( n = 1 ) , r. sibirica sibirica ( n = 1 ) , and rickettsia spp .", "furthermore , according to glta , ompa , and ompb sequence analysis , a possible new rickettsia sp . was found in 25 i. arboricola ticks and 2 i. ricinus ticks .", "for these 27 samples , highest identities with r. heilongjiangensis ( 97.1% ) and r. japonica ( 99.1% ) were found for ompa ( genbank accession no .", "jf758826 ) nucleotide sequences , respectively , whereas glta nucleotide sequences were identical to those from both rickettsia spp . according to multilocus sequence typing ( data not shown ) and genetic criteria agreed on by experts , a candidatus status could be assigned .", "we named it candidatus rickettsia vini ( 17 in technical appendix ) ( table 2 ) .", "( figure ) . the phylogenetic position of candidatus rickettsia vini based on the ompa nucleotide sequences in a study of the role of birds in dispersal of etiologic agents of tick - borne zoonoses , spain , 2009 .", "the percentage of replicate trees in which the associated taxa clustered in the bootstrap test ( 1,000 replicates ) is shown next to the branches .", "the tree is drawn to scale , with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree .", "the evolutionary distances were computed by using the kimura 2-parameter method and are in the units of the number of base substitutions per site ."], ["the presence of anaplasma , borrelia , and rickettsia species in ticks removed from passerine birds corroborates the role of these vertebrates in the epidemiology and dispersion of tick - borne pathogens in spain and in other zones of the planet .", "some of the parasitized birds in our study , such as the european robin ( erithacus rubecula ) or eurasian blackcap ( sylvia atricapilla ) , are considered migratory or partial migratory birds .", "in addition , these species share an ecologic niche and ectoparasites ( horizontal transmission ) with other migratory birds that cover long distances from africa to the eurasian region . except for i. arboricola , the tick species captured in this study previously had been found on birds in spain ( 18 in technical appendix ) .", "the high prevalence of i. ricinus ticks was expected because it is the most frequent tick in this area , and the immature stages of this tick frequently parasitize birds .", "i. ricinus ticks are the main vectors of a. phagocytophilum in europe , and this microorganism has been detected on vegetation in the studied area ( 1 ) .", "however , the low prevalence ( 0.5% ) of a. phagocytophilum in the ticks in our study corroborates data from other studies ( 19,20 in technical appendix ) .", "the presence of a. phagocytophilum in a larva in our study supports the role of birds as reservoirs of a. phagocytophilum .", "the prevalence ( 13.1% ) of b. burgdorferi in our samples is similar to prevalences reported in other studies in europe in which i. ricinus is the main species of tick captured from birds ( 19 in technical appendix ) . in spain , b. garinii , b. valaisiana , and b. afzelii", "have been detected in ticks from birds ( 18 in technical appendix ) . according to our data ,", "the human pathogen b. garinii was the most prevalent species , as reported in birds from europe ( 21 in technical appendix ) .", "although it has been recently detected in ticks from birds in norway ( 22 in technical appendix ) , its finding in spain was unexpected .", "regarding rickettsia species , r. monacensis and r. helvetica are among the human pathogens detected in our study .", "both species have been identified in ticks from birds in europe ( 19,20,23 in technical appendix ) . on the contrary ,", "candidatus rickettsia vini , a potential new rickettsia species , also detected in our study , has not been related to human disease ( 17 in technical appendix ) .", "several genospecies closely related to r. heilongjiangensis and r. japonica have been identified in ixodes spp .", "r. sibirica sibirica , responsible for siberian tick typhus in western people s republic of china and in siberia , was also amplified in an i. ricinus larva in this study .", "our data confirm the involvement of birds in the cycle of human tick - borne diseases ."], [""]]}
{"article_id": "PMC4674516", "article_text": ["korea became an  aging society  in 2000 , and in 2008 , the prevalence of individuals aged 65  years or older was 10.3  % . in 2018 , korea will become an  aged society  when 14.4  % of its population comprise the elderly , and a  super - aged society  by 2026 [ 1 , 2 ] .", "to overcome this situation , the korean government introduced a new social insurance scheme for long - term care ( ltc ) in july 2008 .", "the ltc insurance program provides social services for elderly people with a few functional limitations .", "the purpose of this insurance system was to meet the markedly increased demand for elderly healthcare as a result of a rapidly aging population , elevated expectations for healthcare , and a change in the pattern of medical conditions in the elderly from acute illness to chronic disability .", "this insurance uses the ltc grade for denoting how much help is required for an elderly person in their daily lives .", "after the implementation of ltc insurance , the number of nursing facilities in korea also increased sharply from 1717 locations ( 68,581 people ) in 2008 to 3751 locations ( 116,782 people ) .", "researchers have documented the widespread incidence of inappropriate medication use in elderly persons and reported an estimated prevalence from 4.8 to 45.6  % [ 511 ] .", "the proper use of medicines and monitoring for adverse effects are important factors in the treatment of elderly patients .", "aging is associated with a reduction in first - pass metabolism and therefore an increase in the bioavailability and distribution of drugs , which increases the risk of adverse effects ; these risks grow exponentially when a number of different drugs are used .", "because of the potentially serious consequences of exaggerated or incorrect prescriptions , screening tools have been created to detect inadequacies in drug prescriptions . in 2012 , the american geriatrics society ( ags ) and an interdisciplinary panel of experts in geriatric care and pharmacotherapy reached a consensus on the 2012 ags beers criteria .", "fifty - three medications or medication classes encompass the final updated criteria , which are divided into three categories : potentially inappropriate medications ( pims ) or classes to avoid in older adults ; pims or classes to avoid in older adults with certain diseases and syndromes that the listed drugs can exacerbate ; and medications to be used with caution in older adults .", "elderly nursing home residents regularly receive complex multi - drug therapy because of the presence of both acute and chronic diseases . for this reason ,", "there have been few reports dealing with the prevalence of pim prescriptions or their adverse effects on elderly patients in south korea [ 14 , 15 ] . to our knowledge", ", there have been no reports on the prevalence of pim prescriptions in south korea ltc facilities using the 2012 version of the beers criteria .", "hence , we evaluate the frequency of inappropriate medication use in elderly patients admitted to nursing homes in korea by applying the newly revised 2012 ags beers criteria .", "a retrospective cross - sectional survey study was performed in patients aged 65  years or older , which produced a sample of 824 people admitted to 20 nursing facilities in northwest korea from january 2012 to february 2012 .", "we excluded four patients who did not take any medications , 259 patients with incomplete medical records , and 32 patients who were not included in any ltci programs .", "we assessed the patients age , sex , co - medication , comorbidity , activities of daily living ( adl ) , length of stay , grade of ltc insurance for seniors , and the bed size and business type of the ltc care facilities .", "the ltc grade is based on standards of five areas of physical functions ( adl ) , cognition , behavioral changes ( behavioral problems ) , demand on nursing care , and need for rehabilitation , and judgment standards made in consideration of service necessary according to the state of functions . on a checklist with a maximum score of 100 , a score of 55 and over makes an individual eligible for insured care .", "categories of ltc grade are defined as follows : grade 1 ( most severe)the elderly person has one of the following handicaps : he or she can not go to or get out of bed unaided ; experiences behavioral difficulties , impaired judgment , and frequent memory loss as a result of severe brain injury ; or completely needs full assistance with all the activities of daily life and records a ltc acknowledgment score of at least 95 ; grade 2 ( severe)the elderly person can not eat , defecate nor dress themselves unaided ; has impaired judgment and memory loss due to dementia ; needs considerable help in moving or moving in a wheelchair ; spends most parts of daily life in bed and records an ltc acknowledgment score of at least 75 but less than 95 ; grade 3 ( moderate)the elderly person partially needs considerable ltc protection ; some degree of help for eating , dressing / undressing , using the toilet , looking after himself or herself , performing household tasks , and performing their everyday activities outside the home ; and records an ltc acknowledgement score of at least 55 but less than 75 ; no grade ( mild)the elderly person needs some help in one or two activities of daily life , including bathing and dressing / undressing and records an ltc acknowledgment score of less than 55 .", "prescriptions were assessed for the use of pims according to the 2012 ags beers criteria .", "we focused on evaluating 34 pims to avoid in older adults and one of the categories in the 2012 update to the ags s beers criteria .", "drug  disease interactions and drugs to be used with caution in geriatric patients were also assessed using the beers criteria .", "anti - cholinergics include brompheniramine , carbinoxamine , chlorpheniramine , clemastine , cyproheptadine , dexbrompheniramine , dexchlorpheniramine , diphenhydramine ( oral ) , doxylamine , hydroxyzine , promethazine , and triprolidine .", "cardiovascular medications include alpha blockers , alpha agonists , guanabenz , guanfacine , methyldopa , reserpine ( > 0.1  mg / day ) , anti - arrhythmic drugs ( class ia , ic , iii ) , and amiodarone .", "central nervous system drugs include tertiary tricyclic antidepressants , antipsychotics , barbiturates , and benzodiazepines .", "we calculated the descriptive statistics to illustrate characteristics of the study population by comparing persons on pims with those not on pims .", "the distributions of baseline descriptive statistics are expressed as proportions and means with standard deviations . in the case of categorical variables , cross - tabulations with chi - square tests", "were used in comparing the differences . for continuous variables , the statistically significant difference in means was determined by the independent t test .", "concurrent pim use was defined as the number of pim prescriptions within an individual incidence year .", "logistic regression analysis was performed to identify the risk factors for pim exposure where pim use ( 0 or 1 ) was the dependent variable and the independent variables included age , sex , adl , length of stay , number of comorbidities , number of co - medications , and grade of ltc insurance .", "use of pims has been consistently associated with female sex , higher number of medications , higher number of chronic diseases , and older age [ 1621 ] .", "all statistical analyses were performed using spss 18.0 for windows ( spss inc . , chicago , il , usa ) .", "this study recruited 529 patients ; 221 of these did not use a pim , while the remaining 308 patients were currently taking pims .", "significant differences between those prescribed and those not prescribed a pim were found for sex , age , deductible class , number of different medications used , number of chronic conditions , total healthcare costs in the previous year , and number of concurrent pims .", "the average number of medications in the pim group was 4.0    2.3 , while the non - pim group s average number of medications was 6.4    2.9 ( p  <  0.001 ) .", "in addition , the average number of comorbidities was 3.3    1.4 in the pim group and 3.8    1.5 in the non - pim group ( p  <  0.001 ) .", "the number of patients whose ltc insurance for seniors was grade 3 was relatively higher in the pim group than it was in the non - pim group .", "the six most commonly prescribed classes of medicines within the anatomical therapeutic chemical classification system were as follows : central nervous system ( n  =  930 ; 36.4  % ) , alimentary tract and metabolism ( n  =  553 ; 21.6  % ) , cardiovascular system ( n  =  388 ; 15.1  % ) , blood and blood - forming organs ( n  =  236 ; 9.2  % ) , respiratory system ( n  =  115 ; 4.5  % ) , and musculoskeletal system ( n  =  72 ; 2.8  % ) .", "table  2 presents the number of pims prescribed in ltc facilities.table  1general characteristics of the residents in long - term care facilitiesvariablesnon - pims [ n  =  221 ( % ) ] pims [ n  =  308 ( % ) ] \n p valueage ( years)81.6    8.980.2    8.40.067sex ( male)64 ( 29.0%)59 ( 19.2%)0.080number of co - medications4.0    2.36.4    2.9<0.001 \n 03105 ( 47.5%)51 ( 16.6% )  4582 ( 37.1%)112 ( 36.4% )  6732 ( 14.5%)103 ( 33.4% )  82 ( 0.9%)42 ( 13.6%)number of co - morbidities3.3    1.43.77    1.5<0.001 \n 0270 ( 31.7%)58 ( 18.8% )  352 ( 23.5%)90 ( 29.2% )  461 ( 27.6%)66 ( 21.4% )   538 ( 17.2%)94 ( 30.5%)number of adls1.2    1.21.3    1.20.405  adl  bathing8 ( 3.6%)7 ( 2.3%)0.357  dressing52 ( 23.5%)62 ( 20.1%)0.348  eating132 ( 59.7%)209 ( 67.9%)0.054  toileting58 ( 26.2%)87 ( 28.2%)0.611  walking22 ( 10.0%)42 ( 13.6%)0.200length of stay ( months)28.4    25.926.7    29.20.494grade of long - term care insurance for senior  138 ( 17.2%)31 ( 10.1%)0.008 \n 283 ( 37.6%)100 ( 32.5% )  3100 ( 45.2%)177 ( 57.5%)bed size of long - term care facilities  < 2954 ( 24.4%)74 ( 24.0%)0.950 \n 305966 ( 29.9%)96 ( 31.2% )  60101 ( 45.7%)138 ( 44.8%)business type of long - term care facilities  corporation159 ( 71.9%)223 ( 72.4%)0.908 \n private operator62 ( 28.1%)85 ( 27.6%)values are presented as number ( % ) \n pim potentially inappropriate medication , adl activities of daily living \n p value applied to row 47 inclusive \n p value applied to row 912 inclusive \n p value applied to row 2022 inclusive \n p value applied to row 2325 inclusive \n p value applied to row 2627 inclusivetable  2number of potentially inappropriate medications in the residents of long - term care facilitiesnumber of potentially inappropriate medicationsnumber ( % ) 0221 ( 41.8)1189 ( 35.7)292 ( 17.4)320 ( 3.8)46 ( 1.1)51 ( 0.2)total529 ( 100.0 ) general characteristics of the residents in long - term care facilities values are presented as number ( % ) \n pim potentially inappropriate medication , adl activities of daily living \n p value applied to row 47 inclusive \n p value applied to row 912 inclusive \n p value applied to row 2022 inclusive \n p value applied to row 2325 inclusive \n p value applied to row 2627 inclusive number of potentially inappropriate medications in the residents of long - term care facilities the six most commonly prescribed classes of medications in our patients were as follows : central nervous system ( n  =  271 ; 58.7  % ) , anticholinergics ( n  =  98 ; 21.2  % ) , cardiovascular system ( n  =  50 ; 10.8  % ) , endocrine system ( n  =  22 ; 4.8  % ) , analgesics ( n  =  9 ; 1.9  % ) , and anti - parkinsonian drugs ( n  =  9 ; 1.9  % ) ( table  3 ) . specifically , the most common pim subtype was quetiapine ( n  =  131 ; 28.4  % ) , followed by chlorpheniramine ( n  =  73 ; 15.8  % ) , risperidone ( n  =  30 ; 6.5  % ) , zolpidem ( n  =  27 ; 5.8  % ) , and spironolactone ( n  =  26 , 5.6  % ) ( table  4 ) .", "multivariate logistic regression analyses were applied , and the factors that exhibited significant associations with pim prescriptions are shown in table  5.table  3six most commonly prescribed potentially inappropriate medications classesmedications class ( atc code)number ( % ) central nervous system ( n05-n06)271 ( 58.7)anticholinergics ( n04a)98 ( 21.2)cardiovascular system ( c)50 ( 10.8)endocrine system ( g03)22 ( 4.8)analgesics ( n02)9 ( 1.9)anti - parkinsonian drugs ( n04 )  9 ( 1.9)medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classificationtable  4prevalence of potentially inappropriate medications according to individual drugsdrugs ( atc code)number ( % ) quetiapine ( n05ah04)131 ( 28.4)chlorpeniramine ( r06ab04)73 ( 15.8)risperidone ( n05ax08)30 ( 6.5)zolpidem ( n05cf02)27 ( 5.8)spironolactone ( c03da01)26 ( 5.6)olanzapine ( n05ah03 )  21 ( 4.5)lorazepam ( n05ba06)19 ( 4.1)alprazolam ( n05ba12)16 ( 3.5)hydroxyzine ( n05bb01)13 ( 2.8)cyproheptadine ( r06ax02)12 ( 2.6)doxazosin ( c02ca04)12 ( 2.6)sliding scale insulin ( a10ab01)12 ( 2.6)amitriptyline ( n06aa09)11 ( 2.4)megestrol ( g03ac05)10 ( 2.2)meloxicam ( n01ac06)9 ( 1.9)benztropine ( n04ac01)7 ( 1.5)nifedipine ( c08ca05)7 ( 1.5)diazepam ( n05ba01)6 ( 1.3)clonazepam ( n03ae01)5 ( 1.1)terazosin ( g04ca03)3 ( 0.6)total462 ( 100)medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classificationtable  5logistic regression analysis for potentially inappropriate medications used in the residents of long - term care facilitiesvariables \n  \n standard error \n p valueadjusted or ( 95  % ci)age0.0030.0120.8001.00 ( 0.981.02)sex ( female)0.1360.3160.6661.15 ( 0.622.13)number of co - medications  031.00  450.9030.243<0.0012.47 ( 1.533.97 )  671.6880.316<0.0015.41 ( 2.9110.06 )  83.6000.761<0.00136.61 ( 8.24162.68)number of co - morbidities  021.00  30.4880.2710.0731.63 ( 0.862.76 )  40.0740.2780.7910.93 ( 0.541.60 )  50.4380.2940.1371.55 ( 0.872.76)grade of long - term care insurance for seniors ", "11.00  20.2710.3120.3851.31 ( 0.712.42 )  30.7090.3440.0392.03 ( 1.043.99)adjusted for activities of daily living , length of stay , bed size and business type of long - term care facilitiesgrade of long - term care insurance for seniors made based on standards of five areas of physical functions ( activities of daily living ) , cognition , behavioral changes , demand on nursing care , and need for rehabilitation and judgment standards made in consideration of service necessary according to the state of functions . categories of ltc grade are defined as follows : grade 1 ( most severe ) ; grade 2 ( severe ) ; grade 3 ( moderate ) six most commonly prescribed potentially inappropriate medications classes medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classification prevalence of potentially inappropriate medications according to individual drugs medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classification logistic regression analysis for potentially inappropriate medications used in the residents of long - term care facilities adjusted for activities of daily living , length of stay , bed size and business type of long - term care facilities grade of long - term care insurance for seniors made based on standards of five areas of physical functions ( activities of daily living ) , cognition , behavioral changes , demand on nursing care , and need for rehabilitation and judgment standards made in consideration of service necessary according to the state of functions .", "categories of ltc grade are defined as follows : grade 1 ( most severe ) ; grade 2 ( severe ) ; grade 3 ( moderate )", "the present study focuses on a growing public health problem : inappropriate medication prescriptions in ltc facilities .", "a disturbingly high level of pims were prescribed ( 58.2  % ) , which is of concern , indicating room for improvement .", "a comparison with the results from studies conducted previously worldwide is not straightforward owing to variations in methods , such as data and duration of collection , study criteria , and characteristics of the population groups . despite these variations ,", "this prevalence was similar to those observed in the usa , with values of 49  % of elderly patients who were admitted with one or more of the seven most common medical diagnoses   and 55.3  % in those undergoing surgery during their hospitalization , and 56.1  % of those in acute care hospitals in japan .", "the results described in the multivariate analysis regarding the factors associated with a potentially inappropriate prescription showed that the likelihood was determined by the number of drugs prescribed during the patient s hospitalization .", ", the results of a logistic regression model showed that multiple diseases and a higher use of drugs during hospital stay predicted pim prescription .", "multivariate analyses also revealed that the number of drugs prescribed seemed to be one of the most important independent predictors for receiving an inappropriate medication [ 27 , 28 ] .", "furthermore , inappropriate medication use was higher with the ltc insurance grade 3 , which means less dependence and a requirement of low - level care .", "this can be explained by the fact that most of the residents in korean ltc facilities have been diagnosed with dementia or another neurodegenerative disorder .", "considering central nervous system drugs ( 58.7 % ) were the most prescribed class of inappropriate medications , such prescriptions are used to manage behavioral and psychiatric symptoms .", "when interpreting the findings of the current study , some limitations to the methodology that could bias estimates are worth mentioning .", "the first limitation lies with the fact that the study was cross sectional , which does not allow us to identify the causal relationship of pim with associated factors in nature .", "second , the beers criteria have been examined extensively in research since their development and found to be effective in identifying pim and advising close monitoring of certain medications .", "however , evidence supporting the effectiveness of the beers criteria in consistently reducing the adverse effects of drugs or decreasing costs or mortality is lacking [ 29 , 30 ] .", "the beers criteria does not take into account geriatric patients in palliative care or hospice , does not detect prescribing omissions , underuse of medications , inappropriate dosing of medications , or duplication of drug classes [ 31 , 32 ] .", "third , although the information was gathered mainly from a chart review , recall bias may have been a problem because patients reported their current medications through a structured interview that took place before admission , including the duration of treatment and the dosage ; this information may have been inaccurate .", "fourth , this sample is unlikely to be representative of all koreans because the study was conducted in one region of the country and , therefore , the ability to generalize the findings is questionable .", "central nervous system drugs ( 58.7  % ) were the most prescribed class of inappropriate medications in korean ltc facilities .", "inappropriate medication use was associated with the number of co - medications and ltc insurance grade 3 , which means less dependence and a requirement of low - level care .", "central nervous system drugs ( 58.7  % ) were the most prescribed class of inappropriate medications .", "there was a relationship between inappropriate medication use and the number of co - medications .", "the frequency of inappropriate medication prescriptions was higher among patients whose ltc insurance for seniors was grade 3 , which means less dependence and a requirement of low - level care .", "this retrospective study was carried out after obtaining the permission of the institutional review board of myoung - ji hospital in korea and has been performed in accordance with the ethical standards of the declaration of helsinki .", "kang soo lee , sang - hwan kim , and hee - jin hwang have no conflicts of interest to declare .", "kang soo lee , sang - hwan kim , and hee - jin hwang certify that no funding has been received for the conduct of this study and/or preparation of this manuscript ."], "abstract_text": ["<S> backgroundelderly residents of long - term care facilities are more vulnerable to being prescribed inappropriate medications because of the high incidence of co - medication in this population resulting from the presence of multiple chronic diseases and also age - related changes in pharmacokinetics and pharmacodynamics.objectivewe evaluated the frequency of potentially inappropriate medications and factors influencing their frequency.methodsa retrospective cross - sectional study was conducted in 20 long - term care facilities located in the northwest regions of south korea for 824 patients aged 65  years and older who were assessed between january and february of 2012 . </S>", "<S> potentially inappropriate medications were identified using the 2012 american geriatric society s beers criteria . </S>", "<S> we assessed the relationship between the frequency of potentially inappropriate medications prescribed and patient age , sex , co - medications , comorbidity , activities of daily living , length of stay , grade of long - term care insurance for seniors , and the bed size and business type of the long - term care facility.resultsof the 529 participants who satisfied our inclusion criteria , 308 ( 58.2  % ) had received at least one inappropriate medication according to the 2012 beers criteria . </S>", "<S> the most frequently prescribed classes of inappropriate medications were central nervous system drugs ( 58.7  % ) , anti - cholinergics ( 21.2  % ) , and cardiovascular medications ( 10.8  % ) . </S>", "<S> the most commonly used drugs were quetiapine ( 28.4  % ) , chlorpheniramine ( 15.8  % ) , risperidone ( 6.5  % ) , and zolpidem ( 5.8  % ) . </S>", "<S> inappropriate medication use was associated with the number of co - medications and long - term care insurance grade 3 , which means less dependence and a requirement of low - level care.conclusionscentral nervous system drugs ( 58.7  % ) were the most prescribed class of inappropriate medications . </S>", "<S> quetiapine was the drug most often given inappropriately ( 28.4  % ) . </S>", "<S> there was a relationship between inappropriate medication use and the number of co - medications . </S>", "<S> the frequency of inappropriate medication prescriptions was higher among patients whose long - term care insurance for seniors was grade 3 , which means less dependence and a requirement of low - level care . </S>"], "labels": null, "section_names": ["Key Points", "Introduction", "Methods", "Results", "Discussion", "Conclusions", "None", "None", "None"], "sections": [["central nervous system drugs ( 58.7  % ) were the most prescribed class of inappropriate medications in korean long - term care facilities.inappropriate medication use was associated with the number of co - medications and long - term care insurance grade 3 , which means less dependence and a requirement of low - level care.effective management of nursing home residents belonging to long - term care grade 3 via reducing potentially inappropriate medications is considered ."], ["korea became an  aging society  in 2000 , and in 2008 , the prevalence of individuals aged 65  years or older was 10.3  % . in 2018 , korea will become an  aged society  when 14.4  % of its population comprise the elderly , and a  super - aged society  by 2026 [ 1 , 2 ] .", "to overcome this situation , the korean government introduced a new social insurance scheme for long - term care ( ltc ) in july 2008 .", "the ltc insurance program provides social services for elderly people with a few functional limitations .", "the purpose of this insurance system was to meet the markedly increased demand for elderly healthcare as a result of a rapidly aging population , elevated expectations for healthcare , and a change in the pattern of medical conditions in the elderly from acute illness to chronic disability .", "this insurance uses the ltc grade for denoting how much help is required for an elderly person in their daily lives .", "after the implementation of ltc insurance , the number of nursing facilities in korea also increased sharply from 1717 locations ( 68,581 people ) in 2008 to 3751 locations ( 116,782 people ) .", "researchers have documented the widespread incidence of inappropriate medication use in elderly persons and reported an estimated prevalence from 4.8 to 45.6  % [ 511 ] .", "the proper use of medicines and monitoring for adverse effects are important factors in the treatment of elderly patients .", "aging is associated with a reduction in first - pass metabolism and therefore an increase in the bioavailability and distribution of drugs , which increases the risk of adverse effects ; these risks grow exponentially when a number of different drugs are used .", "because of the potentially serious consequences of exaggerated or incorrect prescriptions , screening tools have been created to detect inadequacies in drug prescriptions . in 2012 , the american geriatrics society ( ags ) and an interdisciplinary panel of experts in geriatric care and pharmacotherapy reached a consensus on the 2012 ags beers criteria .", "fifty - three medications or medication classes encompass the final updated criteria , which are divided into three categories : potentially inappropriate medications ( pims ) or classes to avoid in older adults ; pims or classes to avoid in older adults with certain diseases and syndromes that the listed drugs can exacerbate ; and medications to be used with caution in older adults .", "elderly nursing home residents regularly receive complex multi - drug therapy because of the presence of both acute and chronic diseases . for this reason ,", "there have been few reports dealing with the prevalence of pim prescriptions or their adverse effects on elderly patients in south korea [ 14 , 15 ] . to our knowledge", ", there have been no reports on the prevalence of pim prescriptions in south korea ltc facilities using the 2012 version of the beers criteria .", "hence , we evaluate the frequency of inappropriate medication use in elderly patients admitted to nursing homes in korea by applying the newly revised 2012 ags beers criteria ."], ["a retrospective cross - sectional survey study was performed in patients aged 65  years or older , which produced a sample of 824 people admitted to 20 nursing facilities in northwest korea from january 2012 to february 2012 .", "we excluded four patients who did not take any medications , 259 patients with incomplete medical records , and 32 patients who were not included in any ltci programs .", "we assessed the patients age , sex , co - medication , comorbidity , activities of daily living ( adl ) , length of stay , grade of ltc insurance for seniors , and the bed size and business type of the ltc care facilities .", "the ltc grade is based on standards of five areas of physical functions ( adl ) , cognition , behavioral changes ( behavioral problems ) , demand on nursing care , and need for rehabilitation , and judgment standards made in consideration of service necessary according to the state of functions . on a checklist with a maximum score of 100 , a score of 55 and over makes an individual eligible for insured care .", "categories of ltc grade are defined as follows : grade 1 ( most severe)the elderly person has one of the following handicaps : he or she can not go to or get out of bed unaided ; experiences behavioral difficulties , impaired judgment , and frequent memory loss as a result of severe brain injury ; or completely needs full assistance with all the activities of daily life and records a ltc acknowledgment score of at least 95 ; grade 2 ( severe)the elderly person can not eat , defecate nor dress themselves unaided ; has impaired judgment and memory loss due to dementia ; needs considerable help in moving or moving in a wheelchair ; spends most parts of daily life in bed and records an ltc acknowledgment score of at least 75 but less than 95 ; grade 3 ( moderate)the elderly person partially needs considerable ltc protection ; some degree of help for eating , dressing / undressing , using the toilet , looking after himself or herself , performing household tasks , and performing their everyday activities outside the home ; and records an ltc acknowledgement score of at least 55 but less than 75 ; no grade ( mild)the elderly person needs some help in one or two activities of daily life , including bathing and dressing / undressing and records an ltc acknowledgment score of less than 55 .", "prescriptions were assessed for the use of pims according to the 2012 ags beers criteria .", "we focused on evaluating 34 pims to avoid in older adults and one of the categories in the 2012 update to the ags s beers criteria .", "drug  disease interactions and drugs to be used with caution in geriatric patients were also assessed using the beers criteria .", "anti - cholinergics include brompheniramine , carbinoxamine , chlorpheniramine , clemastine , cyproheptadine , dexbrompheniramine , dexchlorpheniramine , diphenhydramine ( oral ) , doxylamine , hydroxyzine , promethazine , and triprolidine .", "cardiovascular medications include alpha blockers , alpha agonists , guanabenz , guanfacine , methyldopa , reserpine ( > 0.1  mg / day ) , anti - arrhythmic drugs ( class ia , ic , iii ) , and amiodarone .", "central nervous system drugs include tertiary tricyclic antidepressants , antipsychotics , barbiturates , and benzodiazepines .", "we calculated the descriptive statistics to illustrate characteristics of the study population by comparing persons on pims with those not on pims .", "the distributions of baseline descriptive statistics are expressed as proportions and means with standard deviations . in the case of categorical variables , cross - tabulations with chi - square tests", "were used in comparing the differences . for continuous variables , the statistically significant difference in means was determined by the independent t test .", "concurrent pim use was defined as the number of pim prescriptions within an individual incidence year .", "logistic regression analysis was performed to identify the risk factors for pim exposure where pim use ( 0 or 1 ) was the dependent variable and the independent variables included age , sex , adl , length of stay , number of comorbidities , number of co - medications , and grade of ltc insurance .", "use of pims has been consistently associated with female sex , higher number of medications , higher number of chronic diseases , and older age [ 1621 ] .", "all statistical analyses were performed using spss 18.0 for windows ( spss inc . , chicago , il , usa ) ."], ["this study recruited 529 patients ; 221 of these did not use a pim , while the remaining 308 patients were currently taking pims .", "significant differences between those prescribed and those not prescribed a pim were found for sex , age , deductible class , number of different medications used , number of chronic conditions , total healthcare costs in the previous year , and number of concurrent pims .", "the average number of medications in the pim group was 4.0    2.3 , while the non - pim group s average number of medications was 6.4    2.9 ( p  <  0.001 ) .", "in addition , the average number of comorbidities was 3.3    1.4 in the pim group and 3.8    1.5 in the non - pim group ( p  <  0.001 ) .", "the number of patients whose ltc insurance for seniors was grade 3 was relatively higher in the pim group than it was in the non - pim group .", "the six most commonly prescribed classes of medicines within the anatomical therapeutic chemical classification system were as follows : central nervous system ( n  =  930 ; 36.4  % ) , alimentary tract and metabolism ( n  =  553 ; 21.6  % ) , cardiovascular system ( n  =  388 ; 15.1  % ) , blood and blood - forming organs ( n  =  236 ; 9.2  % ) , respiratory system ( n  =  115 ; 4.5  % ) , and musculoskeletal system ( n  =  72 ; 2.8  % ) .", "table  2 presents the number of pims prescribed in ltc facilities.table  1general characteristics of the residents in long - term care facilitiesvariablesnon - pims [ n  =  221 ( % ) ] pims [ n  =  308 ( % ) ] \n p valueage ( years)81.6    8.980.2    8.40.067sex ( male)64 ( 29.0%)59 ( 19.2%)0.080number of co - medications4.0    2.36.4    2.9<0.001 \n 03105 ( 47.5%)51 ( 16.6% )  4582 ( 37.1%)112 ( 36.4% )  6732 ( 14.5%)103 ( 33.4% )  82 ( 0.9%)42 ( 13.6%)number of co - morbidities3.3    1.43.77    1.5<0.001 \n 0270 ( 31.7%)58 ( 18.8% )  352 ( 23.5%)90 ( 29.2% )  461 ( 27.6%)66 ( 21.4% )   538 ( 17.2%)94 ( 30.5%)number of adls1.2    1.21.3    1.20.405  adl  bathing8 ( 3.6%)7 ( 2.3%)0.357  dressing52 ( 23.5%)62 ( 20.1%)0.348  eating132 ( 59.7%)209 ( 67.9%)0.054  toileting58 ( 26.2%)87 ( 28.2%)0.611  walking22 ( 10.0%)42 ( 13.6%)0.200length of stay ( months)28.4    25.926.7    29.20.494grade of long - term care insurance for senior  138 ( 17.2%)31 ( 10.1%)0.008 \n 283 ( 37.6%)100 ( 32.5% )  3100 ( 45.2%)177 ( 57.5%)bed size of long - term care facilities  < 2954 ( 24.4%)74 ( 24.0%)0.950 \n 305966 ( 29.9%)96 ( 31.2% )  60101 ( 45.7%)138 ( 44.8%)business type of long - term care facilities  corporation159 ( 71.9%)223 ( 72.4%)0.908 \n private operator62 ( 28.1%)85 ( 27.6%)values are presented as number ( % ) \n pim potentially inappropriate medication , adl activities of daily living \n p value applied to row 47 inclusive \n p value applied to row 912 inclusive \n p value applied to row 2022 inclusive \n p value applied to row 2325 inclusive \n p value applied to row 2627 inclusivetable  2number of potentially inappropriate medications in the residents of long - term care facilitiesnumber of potentially inappropriate medicationsnumber ( % ) 0221 ( 41.8)1189 ( 35.7)292 ( 17.4)320 ( 3.8)46 ( 1.1)51 ( 0.2)total529 ( 100.0 ) general characteristics of the residents in long - term care facilities values are presented as number ( % ) \n pim potentially inappropriate medication , adl activities of daily living \n p value applied to row 47 inclusive \n p value applied to row 912 inclusive \n p value applied to row 2022 inclusive \n p value applied to row 2325 inclusive \n p value applied to row 2627 inclusive number of potentially inappropriate medications in the residents of long - term care facilities the six most commonly prescribed classes of medications in our patients were as follows : central nervous system ( n  =  271 ; 58.7  % ) , anticholinergics ( n  =  98 ; 21.2  % ) , cardiovascular system ( n  =  50 ; 10.8  % ) , endocrine system ( n  =  22 ; 4.8  % ) , analgesics ( n  =  9 ; 1.9  % ) , and anti - parkinsonian drugs ( n  =  9 ; 1.9  % ) ( table  3 ) . specifically , the most common pim subtype was quetiapine ( n  =  131 ; 28.4  % ) , followed by chlorpheniramine ( n  =  73 ; 15.8  % ) , risperidone ( n  =  30 ; 6.5  % ) , zolpidem ( n  =  27 ; 5.8  % ) , and spironolactone ( n  =  26 , 5.6  % ) ( table  4 ) .", "multivariate logistic regression analyses were applied , and the factors that exhibited significant associations with pim prescriptions are shown in table  5.table  3six most commonly prescribed potentially inappropriate medications classesmedications class ( atc code)number ( % ) central nervous system ( n05-n06)271 ( 58.7)anticholinergics ( n04a)98 ( 21.2)cardiovascular system ( c)50 ( 10.8)endocrine system ( g03)22 ( 4.8)analgesics ( n02)9 ( 1.9)anti - parkinsonian drugs ( n04 )  9 ( 1.9)medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classificationtable  4prevalence of potentially inappropriate medications according to individual drugsdrugs ( atc code)number ( % ) quetiapine ( n05ah04)131 ( 28.4)chlorpeniramine ( r06ab04)73 ( 15.8)risperidone ( n05ax08)30 ( 6.5)zolpidem ( n05cf02)27 ( 5.8)spironolactone ( c03da01)26 ( 5.6)olanzapine ( n05ah03 )  21 ( 4.5)lorazepam ( n05ba06)19 ( 4.1)alprazolam ( n05ba12)16 ( 3.5)hydroxyzine ( n05bb01)13 ( 2.8)cyproheptadine ( r06ax02)12 ( 2.6)doxazosin ( c02ca04)12 ( 2.6)sliding scale insulin ( a10ab01)12 ( 2.6)amitriptyline ( n06aa09)11 ( 2.4)megestrol ( g03ac05)10 ( 2.2)meloxicam ( n01ac06)9 ( 1.9)benztropine ( n04ac01)7 ( 1.5)nifedipine ( c08ca05)7 ( 1.5)diazepam ( n05ba01)6 ( 1.3)clonazepam ( n03ae01)5 ( 1.1)terazosin ( g04ca03)3 ( 0.6)total462 ( 100)medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classificationtable  5logistic regression analysis for potentially inappropriate medications used in the residents of long - term care facilitiesvariables \n  \n standard error \n p valueadjusted or ( 95  % ci)age0.0030.0120.8001.00 ( 0.981.02)sex ( female)0.1360.3160.6661.15 ( 0.622.13)number of co - medications  031.00  450.9030.243<0.0012.47 ( 1.533.97 )  671.6880.316<0.0015.41 ( 2.9110.06 )  83.6000.761<0.00136.61 ( 8.24162.68)number of co - morbidities  021.00  30.4880.2710.0731.63 ( 0.862.76 )  40.0740.2780.7910.93 ( 0.541.60 )  50.4380.2940.1371.55 ( 0.872.76)grade of long - term care insurance for seniors ", "11.00  20.2710.3120.3851.31 ( 0.712.42 )  30.7090.3440.0392.03 ( 1.043.99)adjusted for activities of daily living , length of stay , bed size and business type of long - term care facilitiesgrade of long - term care insurance for seniors made based on standards of five areas of physical functions ( activities of daily living ) , cognition , behavioral changes , demand on nursing care , and need for rehabilitation and judgment standards made in consideration of service necessary according to the state of functions . categories of ltc grade are defined as follows : grade 1 ( most severe ) ; grade 2 ( severe ) ; grade 3 ( moderate ) six most commonly prescribed potentially inappropriate medications classes medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classification prevalence of potentially inappropriate medications according to individual drugs medication classes are stratified according to 2012 american geriatrics society beers criteria for potentially inappropriate medication use in older adults ( ) means codes of anatomical therapeutic chemical ( atc ) classification logistic regression analysis for potentially inappropriate medications used in the residents of long - term care facilities adjusted for activities of daily living , length of stay , bed size and business type of long - term care facilities grade of long - term care insurance for seniors made based on standards of five areas of physical functions ( activities of daily living ) , cognition , behavioral changes , demand on nursing care , and need for rehabilitation and judgment standards made in consideration of service necessary according to the state of functions .", "categories of ltc grade are defined as follows : grade 1 ( most severe ) ; grade 2 ( severe ) ; grade 3 ( moderate )"], ["the present study focuses on a growing public health problem : inappropriate medication prescriptions in ltc facilities .", "a disturbingly high level of pims were prescribed ( 58.2  % ) , which is of concern , indicating room for improvement .", "a comparison with the results from studies conducted previously worldwide is not straightforward owing to variations in methods , such as data and duration of collection , study criteria , and characteristics of the population groups . despite these variations ,", "this prevalence was similar to those observed in the usa , with values of 49  % of elderly patients who were admitted with one or more of the seven most common medical diagnoses   and 55.3  % in those undergoing surgery during their hospitalization , and 56.1  % of those in acute care hospitals in japan .", "the results described in the multivariate analysis regarding the factors associated with a potentially inappropriate prescription showed that the likelihood was determined by the number of drugs prescribed during the patient s hospitalization .", ", the results of a logistic regression model showed that multiple diseases and a higher use of drugs during hospital stay predicted pim prescription .", "multivariate analyses also revealed that the number of drugs prescribed seemed to be one of the most important independent predictors for receiving an inappropriate medication [ 27 , 28 ] .", "furthermore , inappropriate medication use was higher with the ltc insurance grade 3 , which means less dependence and a requirement of low - level care .", "this can be explained by the fact that most of the residents in korean ltc facilities have been diagnosed with dementia or another neurodegenerative disorder .", "considering central nervous system drugs ( 58.7 % ) were the most prescribed class of inappropriate medications , such prescriptions are used to manage behavioral and psychiatric symptoms .", "when interpreting the findings of the current study , some limitations to the methodology that could bias estimates are worth mentioning .", "the first limitation lies with the fact that the study was cross sectional , which does not allow us to identify the causal relationship of pim with associated factors in nature .", "second , the beers criteria have been examined extensively in research since their development and found to be effective in identifying pim and advising close monitoring of certain medications .", "however , evidence supporting the effectiveness of the beers criteria in consistently reducing the adverse effects of drugs or decreasing costs or mortality is lacking [ 29 , 30 ] .", "the beers criteria does not take into account geriatric patients in palliative care or hospice , does not detect prescribing omissions , underuse of medications , inappropriate dosing of medications , or duplication of drug classes [ 31 , 32 ] .", "third , although the information was gathered mainly from a chart review , recall bias may have been a problem because patients reported their current medications through a structured interview that took place before admission , including the duration of treatment and the dosage ; this information may have been inaccurate .", "fourth , this sample is unlikely to be representative of all koreans because the study was conducted in one region of the country and , therefore , the ability to generalize the findings is questionable .", "central nervous system drugs ( 58.7  % ) were the most prescribed class of inappropriate medications in korean ltc facilities .", "inappropriate medication use was associated with the number of co - medications and ltc insurance grade 3 , which means less dependence and a requirement of low - level care ."], ["central nervous system drugs ( 58.7  % ) were the most prescribed class of inappropriate medications .", "there was a relationship between inappropriate medication use and the number of co - medications .", "the frequency of inappropriate medication prescriptions was higher among patients whose ltc insurance for seniors was grade 3 , which means less dependence and a requirement of low - level care ."], ["this retrospective study was carried out after obtaining the permission of the institutional review board of myoung - ji hospital in korea and has been performed in accordance with the ethical standards of the declaration of helsinki ."], ["kang soo lee , sang - hwan kim , and hee - jin hwang have no conflicts of interest to declare ."], ["kang soo lee , sang - hwan kim , and hee - jin hwang certify that no funding has been received for the conduct of this study and/or preparation of this manuscript ."]]}
{"article_id": "PMC4498860", "article_text": ["in patients with chronic hepatitis and in those with liver cirrhosis , detection of increasing incidences of hepatocellular carcinoma ( hcc ) requires careful monitoring every 36  months using ultrasound sonography ( us ) , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and frequent blood tests .", "once hcc is detected , patients experience repeated recurrence , despite the fact that a variety of hcc treatment methods have been developed , such as anti - viral agents , surgical treatment , percutaneous ethanol injection ( peit ) , radiofrequency ablation ( rfa ) , trans - catheter arterial chemoembolization ( tace ) , and molecular - target drugs . in the last 10  years , tumor vaccines generated from patients ' own formalin - fixed hcc tissues", "the autologous formalin - fixed tumor vaccine ( aftv ) has been reported to reduce the recurrence risk of primary resected hepatitis b virus - background hcc by 81% 1 . in one case ,", "recurrence of multiple - recurrent hepatitis c virus ( hcv)-back ground hcc was clearly suppressed long - term without any additional treatment 2 . here", ", we report a case in which aftv injection resulted in suppression of multiple - recurrent hcv - background hcc recurrence . in this case", ", we detected a significant density of glypican-3 ( gpc3)-specific cytotoxic t lymphocytes ( ctl ) in the blood at 12  months after the last intradermal injection of aftv .", "a 55-year - old female patient was diagnosed with hcv - related hepatitis in 2002 .", "liver biopsy showed active chronic hepatitis accompanied by bridging fibrosis and necrotic inflammatory reactions . to reduce the activity of chronic hepatitis , the patient was treated with consensus interferon for 24  weeks .", "results showed complete remission of the hepatitis as liver transaminases normalized , as well as conversion of seropositive hcv to negative up to the present .", "when she was 57  years old , two 20-mm hccs were observed in left lobes s2 and s3 , and a left hepatic lobectomy was performed in june 2004 . in addition , two 10-mm hccs in s5 and s7 were treated with radiofrequency ablation ( rfa ) in september 2008 .", "the 10-mm hcc in s5 was detected again in january 2011 , and was treated with trans - catheter arterial chemoembolization ( tace ) and rfa . in january 2012 , a new 10-mm hcc was found in s6 , and a resection was done after treatment with rfa .", "pathologies obtained after the two hepatectomies showed poorly differentiated hcc in the 2004 specimen and moderately differentiated hcc in the 2012 specimen ( fig.1 ) .", "the liver function has been kept good and the bilirubin levels were < 2  mg / dl .", "the patient did not hope liver transplantation but desired aftv therapy because of the rapid decline in the recurrence interval , that is , 51- , 28- , 12- , and 4-months .", "pathology and glypican-3 ( gpc3 ) expression in hepatocellular carcinomas . ( a ) h&e staining and ( b ) gpc3 immunohistochemistry of representative tissue from a 2004-resected hepatocellular carcinoma ( hcc ) specimen of the present case .", "( c ) h&e staining and ( d ) gpc3 immunohistochemistry from a 2012-resected specimen .", "gpc3 expression ( b , d ) was observed in areas with hcc ( arrows ) , but not in cirrhotic areas ( white stars ) .", "we prepared aftv as reported previously 3 using a total of 1.56  g of autologous hcc tissue recovered from formalin - fixed and paraffin - embedded tumors from the two operations .", "according to the therapeutic schedule employed 1 , a single course of aftv therapy applied from september to october 2012 consisted of the following : ( 1 ) a delayed - type hypersensitivity ( dth ) test with immunoadjuvant - free fixed hcc fragments applied to a forearm was initiated 48  h before the first aftv injection to check for allergic reaction .", "( 2 ) up to three rounds of aftv were injected intradermally into the upper arm at 2  week intervals .", "( 3 ) a second dth test , performed 2  weeks after the last aftv injection to the other forearm , showed a positive dth response . ( 4 ) ultrasound sonography and blood tests for tumor markers which were conducted every 3  months , and computerized tomography every 6  months , revealed no evidence of recurrence or generation of new hcc lesions for more than 27  months .", "immunohistochemistry to detect gpc3 4 in the hcc specimen resected in 2004 revealed strong expression of gpc in the hcc lesion ( fig.1b ) .", "we also detected gpc3 expression in the 2012 specimen resected after treatment with rfa ( fig.1d ) .", "however , we did not observe any gpc3 expression within cirrhotic lesions in either specimen ( fig.1b and d ) . to evaluate aftv - induced gpc3-reactive ctl", ", we carried out interferon--specific elispot assays as described previously 5 , using a target human hcc cell line expressing gpc3 ( sk - hep-1/gpc3 ) .", "the cell line was developed from sk - hep-1/hla - a0201/2402 cells by transfecting with human gpc3-gene . a control cell line ( sk - hep-1/vec )", "although hla subtypes of the patient and the target cell line differ ( hla - a0206/3303 and hla - a0201/2402 , respectively ) , hla - a2 ( a0201)-restricted gpc3 epitopes are cross - reactive with hla - a0206 , allowing detection of gpc3-specific ctl that are reactive to the target cell line 5 .", "glypican-3-specific ctls were clearly observed in peripheral blood drawn 12  months after the first injection of aftv ( average 21.5 spots against sk - hep-1/hgpc3 cells vs. nine spots against sk - hep-1/vec control cells ) ( fig.2 ) .", "lymphocytes were prepared from the patient 's blood drawn at 12  months after autologous formalin - fixed tumor vaccine ( aftv ) injection .", "we detected hla - a2-restricted glypican-3 ( gpc3 ) peptide - specific cytotoxic t lymphocytes in the 12-month blood .", "sk - hep-1/hgpc3 , human gpc3 gene - transfected sk - hep-1 cells ( sk - hep-1 with hla - a a0201/2402 ) , a human hepatocellular carcinoma cell line unable to express gpc3 ; sk - hep-1/vec , control vector gene - transfected sk - hep-1 cell line .", "effector cells ( cd8 t cells ) , 1    10  cells / well ; target cells ( sk - hep-1 cells ) , 5    10  cells / well .", "gpc3 , a heparan - sulfate proteoglycans linked to the outer surface of cell membrane via a glycosylphosphatidylinositol anchor 6 , is frequently overexpressed in hcc 7,8 and in precancerous lesions in the liver 9 .", "for this reason , a recombinant humanized monoclonal antibody against gpc3 10 and a gpc3-peptide vaccine 11 have been developed for treatment of hcc .", "treatments with these new agents were well tolerated , and noted measurable immune responses and antitumor efficacy . however , clinical outcome were not necessarily prominent in these phase i trials compared to the previous expectations derived from preclinical studies .", "autologous formalin - fixed tumor vaccine differs from these specified molecule - targeted agents in that the autologous tumor , containing a wide array of unidentified antigens , is the source of antigens .", "thus , ctl induced with aftv made from tumor tissue must be polyclonal by nature ; for example , polyclonal ctl induced in vitro with a formalin - fixed carcinoembryonic antigen protein 12 . at present , no one provided any evidence that the impure tumor antigenic formalin - fixed autologous hcc tissue is truly active to induce a specific cellular immune response against a specified tumor antigenic molecule .", "the present case is the first one we could identify gpc3-specific cellular immune response based on the autologous formalin - fixed hcc .", "while we detected gpc3-specific ctl in the patient 's blood 12  months after aftv treatment , few gpc3-specific ctl were detected 16  months after the treatment ( data not shown ) .", "these results may suggest that , although the conspicuous life span of gpc3-specific ctl in blood is < 16  months , the immunoprotective effect of aftv continues beyond gpc3-specific ctl lifespan in blood , since this patient presently continued to show no evidence of hcc recurrence 32  months after the second resection of hcc .", "we consider two hypotheses to explain this continued protection : ( 1 ) the gpc3-specific ctl may have differentiated into memory t cells , which migrate to lymphatic tissues and are therefore not conspicuously detectable in peripheral blood .", "( 2 ) the gpc3-specific ctl apoptosed after killing all target hcc cells in vivo , thereby eliminating all tumor antigens and preventing replicative stimulation to active ctl .", "this is the first case we could identify gpc3-specific cellular immune response based on impure tumor antigenic formalin - fixed autologous hcc tissue .", "present results suggest that aftv should be taken in consideration as one of treatment modalities in cases involving potential hcc derived from chronic hepatitis and liver cirrhosis ."], "abstract_text": ["<S> key clinical messageautologous formalin - fixed tumor vaccine ( aftv ) suppressed re - recurrence for more than 32  months of multiple - recurrent hepatocellular carcinoma based on hepatitis c virus - induced liver cirrhosis in a case with previous recurrence interval , 51- , 28- , 12- , and 4-months . </S>", "<S> we detected glypican-3-specific cytotoxic t lymphocytes in the peripheral blood at 12  months after aftv . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion", "Conflict of Interest"], "sections": [["in patients with chronic hepatitis and in those with liver cirrhosis , detection of increasing incidences of hepatocellular carcinoma ( hcc ) requires careful monitoring every 36  months using ultrasound sonography ( us ) , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and frequent blood tests .", "once hcc is detected , patients experience repeated recurrence , despite the fact that a variety of hcc treatment methods have been developed , such as anti - viral agents , surgical treatment , percutaneous ethanol injection ( peit ) , radiofrequency ablation ( rfa ) , trans - catheter arterial chemoembolization ( tace ) , and molecular - target drugs . in the last 10  years , tumor vaccines generated from patients ' own formalin - fixed hcc tissues", "the autologous formalin - fixed tumor vaccine ( aftv ) has been reported to reduce the recurrence risk of primary resected hepatitis b virus - background hcc by 81% 1 . in one case ,", "recurrence of multiple - recurrent hepatitis c virus ( hcv)-back ground hcc was clearly suppressed long - term without any additional treatment 2 . here", ", we report a case in which aftv injection resulted in suppression of multiple - recurrent hcv - background hcc recurrence . in this case", ", we detected a significant density of glypican-3 ( gpc3)-specific cytotoxic t lymphocytes ( ctl ) in the blood at 12  months after the last intradermal injection of aftv ."], ["a 55-year - old female patient was diagnosed with hcv - related hepatitis in 2002 .", "liver biopsy showed active chronic hepatitis accompanied by bridging fibrosis and necrotic inflammatory reactions . to reduce the activity of chronic hepatitis , the patient was treated with consensus interferon for 24  weeks .", "results showed complete remission of the hepatitis as liver transaminases normalized , as well as conversion of seropositive hcv to negative up to the present .", "when she was 57  years old , two 20-mm hccs were observed in left lobes s2 and s3 , and a left hepatic lobectomy was performed in june 2004 . in addition , two 10-mm hccs in s5 and s7 were treated with radiofrequency ablation ( rfa ) in september 2008 .", "the 10-mm hcc in s5 was detected again in january 2011 , and was treated with trans - catheter arterial chemoembolization ( tace ) and rfa . in january 2012 , a new 10-mm hcc was found in s6 , and a resection was done after treatment with rfa .", "pathologies obtained after the two hepatectomies showed poorly differentiated hcc in the 2004 specimen and moderately differentiated hcc in the 2012 specimen ( fig.1 ) .", "the liver function has been kept good and the bilirubin levels were < 2  mg / dl .", "the patient did not hope liver transplantation but desired aftv therapy because of the rapid decline in the recurrence interval , that is , 51- , 28- , 12- , and 4-months .", "pathology and glypican-3 ( gpc3 ) expression in hepatocellular carcinomas . ( a ) h&e staining and ( b ) gpc3 immunohistochemistry of representative tissue from a 2004-resected hepatocellular carcinoma ( hcc ) specimen of the present case .", "( c ) h&e staining and ( d ) gpc3 immunohistochemistry from a 2012-resected specimen .", "gpc3 expression ( b , d ) was observed in areas with hcc ( arrows ) , but not in cirrhotic areas ( white stars ) .", "we prepared aftv as reported previously 3 using a total of 1.56  g of autologous hcc tissue recovered from formalin - fixed and paraffin - embedded tumors from the two operations .", "according to the therapeutic schedule employed 1 , a single course of aftv therapy applied from september to october 2012 consisted of the following : ( 1 ) a delayed - type hypersensitivity ( dth ) test with immunoadjuvant - free fixed hcc fragments applied to a forearm was initiated 48  h before the first aftv injection to check for allergic reaction .", "( 2 ) up to three rounds of aftv were injected intradermally into the upper arm at 2  week intervals .", "( 3 ) a second dth test , performed 2  weeks after the last aftv injection to the other forearm , showed a positive dth response . ( 4 ) ultrasound sonography and blood tests for tumor markers which were conducted every 3  months , and computerized tomography every 6  months , revealed no evidence of recurrence or generation of new hcc lesions for more than 27  months .", "immunohistochemistry to detect gpc3 4 in the hcc specimen resected in 2004 revealed strong expression of gpc in the hcc lesion ( fig.1b ) .", "we also detected gpc3 expression in the 2012 specimen resected after treatment with rfa ( fig.1d ) .", "however , we did not observe any gpc3 expression within cirrhotic lesions in either specimen ( fig.1b and d ) . to evaluate aftv - induced gpc3-reactive ctl", ", we carried out interferon--specific elispot assays as described previously 5 , using a target human hcc cell line expressing gpc3 ( sk - hep-1/gpc3 ) .", "the cell line was developed from sk - hep-1/hla - a0201/2402 cells by transfecting with human gpc3-gene . a control cell line ( sk - hep-1/vec )", "although hla subtypes of the patient and the target cell line differ ( hla - a0206/3303 and hla - a0201/2402 , respectively ) , hla - a2 ( a0201)-restricted gpc3 epitopes are cross - reactive with hla - a0206 , allowing detection of gpc3-specific ctl that are reactive to the target cell line 5 .", "glypican-3-specific ctls were clearly observed in peripheral blood drawn 12  months after the first injection of aftv ( average 21.5 spots against sk - hep-1/hgpc3 cells vs. nine spots against sk - hep-1/vec control cells ) ( fig.2 ) .", "lymphocytes were prepared from the patient 's blood drawn at 12  months after autologous formalin - fixed tumor vaccine ( aftv ) injection .", "we detected hla - a2-restricted glypican-3 ( gpc3 ) peptide - specific cytotoxic t lymphocytes in the 12-month blood .", "sk - hep-1/hgpc3 , human gpc3 gene - transfected sk - hep-1 cells ( sk - hep-1 with hla - a a0201/2402 ) , a human hepatocellular carcinoma cell line unable to express gpc3 ; sk - hep-1/vec , control vector gene - transfected sk - hep-1 cell line .", "effector cells ( cd8 t cells ) , 1    10  cells / well ; target cells ( sk - hep-1 cells ) , 5    10  cells / well ."], ["gpc3 , a heparan - sulfate proteoglycans linked to the outer surface of cell membrane via a glycosylphosphatidylinositol anchor 6 , is frequently overexpressed in hcc 7,8 and in precancerous lesions in the liver 9 .", "for this reason , a recombinant humanized monoclonal antibody against gpc3 10 and a gpc3-peptide vaccine 11 have been developed for treatment of hcc .", "treatments with these new agents were well tolerated , and noted measurable immune responses and antitumor efficacy . however , clinical outcome were not necessarily prominent in these phase i trials compared to the previous expectations derived from preclinical studies .", "autologous formalin - fixed tumor vaccine differs from these specified molecule - targeted agents in that the autologous tumor , containing a wide array of unidentified antigens , is the source of antigens .", "thus , ctl induced with aftv made from tumor tissue must be polyclonal by nature ; for example , polyclonal ctl induced in vitro with a formalin - fixed carcinoembryonic antigen protein 12 . at present , no one provided any evidence that the impure tumor antigenic formalin - fixed autologous hcc tissue is truly active to induce a specific cellular immune response against a specified tumor antigenic molecule .", "the present case is the first one we could identify gpc3-specific cellular immune response based on the autologous formalin - fixed hcc .", "while we detected gpc3-specific ctl in the patient 's blood 12  months after aftv treatment , few gpc3-specific ctl were detected 16  months after the treatment ( data not shown ) .", "these results may suggest that , although the conspicuous life span of gpc3-specific ctl in blood is < 16  months , the immunoprotective effect of aftv continues beyond gpc3-specific ctl lifespan in blood , since this patient presently continued to show no evidence of hcc recurrence 32  months after the second resection of hcc .", "we consider two hypotheses to explain this continued protection : ( 1 ) the gpc3-specific ctl may have differentiated into memory t cells , which migrate to lymphatic tissues and are therefore not conspicuously detectable in peripheral blood .", "( 2 ) the gpc3-specific ctl apoptosed after killing all target hcc cells in vivo , thereby eliminating all tumor antigens and preventing replicative stimulation to active ctl ."], ["this is the first case we could identify gpc3-specific cellular immune response based on impure tumor antigenic formalin - fixed autologous hcc tissue .", "present results suggest that aftv should be taken in consideration as one of treatment modalities in cases involving potential hcc derived from chronic hepatitis and liver cirrhosis ."], [""]]}
{"article_id": "PMC153442", "article_text": ["in this and the next issue of critical care , john haddad   presents a comprehensive review on the contributions of transcription factors to lung injury .", "the topic is large and ever changing , and the complete coverage of nuclear factor-b ( nf-b ) and hypoxia - inducible factor ( hif)-1 is spread over two issues . in critical care", "the majority of these patients progress to the point at which they require intubation and mechanical ventilation , with an associated high mortality .", "recent studies   have demonstrated that low tidal volume ( 6 ml / kg ) ventilation strategies dramatically reduce this mortality .", "however , many other clinical trials of various treatments have failed at either reducing the lung injury or accelerating the healing to the end - point of reduced mortality . in this age of genomics and proteomics", "we continue to explore the association of gene and environment . with respect to lung injury ,", "we need to identify and understand the mechanisms that predispose patients to the excessive inflammation resulting from an overactive innate immune response that characterizes sepsis and lung injury . these include stimuli , signal transduction ( receptors , enzyme cascades , transcription factors ) , gene(s ) response and the measured clinical phenotype .", "john haddad , in his two - part review , identifies many ' clinical stimuli ' in cell culture , animal model and patient studies representing an oxidative stress that can generate a response via nf-b or hif-1 dependent signalling .", "nf-b was originally described in b lymphocytes , but it is now recognized as a member of the rel family of transcription factors and is a critical response element in many cytokine - dependent events or inflammatory conditions . as a result of this link ,", "nf-b has become a major target for new therapeutic approaches in such clinical disease states as asthma , cancer , arthritis , and cardiovascular and neurodegenerative conditions .", "haddad   discusses the roles of critical care conditions such as hyperoxia , haemorrhage and resuscitation ; the ' stress response ' to illness ( interleukin-6 , interleukin-8 , tumour necrosis factor , rantes [ regulated upon activation , normal t cell expressed and secreted ] ) ; and mechanical ventilation and ischaemia / reperfusion . in all of these conditions", "these dynamic variations in cellular redox or oxidative stress , if in disequilibrium , may regulate gene expression and lead to apoptosis ( cell death without inflammation ) , inflammation and lung injury .", "the master regulatory element of hypoxic conditions and adapting oxidative stresses to gene expression is hif-1 .", "hif-1 , a dna - binding protein , has increased stability and binding in hypoxic conditions and is degraded rapidly in normoxia .", "the accumulation of the -subunits allows for  heterodimer formation and translocation into the nucleus during hypoxia .", "this process leads to selective upregulation of genes whose products are involved in hypoxia and inflammatory lung injury .", "these include erythropoietin , vascular endothelial growth factor ( vegf ) and glucose transporter . work by haddad   has demonstrated that proinflammatory cytokines also activate hif-1 stability and dna binding .", "this effect was most profound in hypoxic conditions and was , in fact , greater than that in hypoxaemia alone .", "it is felt that hif-1 , via its action on vegf expression , is directly related to lung injury by endothelial barrier dysfunction mediated by vegf and recognized clinically as increased pulmonary vascular permeability .", "haddad   discusses in detail how hypoxia and inflammatory stimuli initiate many signalling cascades via hif-1 to generate a response phenotype to these oxidative stresses .", "understanding the molecular signalling that couples oxidative stresses via nf-b or hif-1 to acute lung injury should generate new therapeutic options .", "haddad   discusses the rationale for the use of tyloxapol to reduce proinflammatory cytokines , n - acetyl cysteine ( a glutathione precursor ) to reduce neutrophil - associated alveolitis in chronic conditions such as cystic fibrosis , and the use of pyrrolidine dithiocarbamate in transplantation to reduce neutrophil - associated oxidant lung injury .", "two new compounds , isohelenin and lisofylline , a phosphodiesterase inhibitor , are described as being able to reduce proinflammatory cytokines and ameliorate oxidant lung injury in animal models . as exciting as this emerging field is , with its predictable contribution to future ' bench to bedside ' discussions , a more complete mechanistic understanding and future clinical trials will assist in the realization of improved treatment and reduced mortality from oxidant - mediated lung injury .", "hif = hypoxia - inducible factor ; nf-b = nuclear factor-b ; vegf = vascular endothelial growth factor ."], "abstract_text": ["<S> the role of oxidative stress in regulating transcription factors and specific gene responses in critical illness is a new and emerging area . a better understanding of the proinflammatory oxidant stimuli of reactive oxygen species generation and how this generates the clinical phenotype of acute lung injury by regulating gene expression may allow the development of new therapeutic strategies . in his review john haddad describes the present data and role for transcription factors nuclear factor-b and hypoxia - inducible factor-1 in acute lung injury . </S>"], "labels": null, "section_names": ["Introduction", "Nuclear factor-B: response to stimuli", "Hypoxia-inducible factor-1: role in hypoxaemia-initiated lung injury", "Conclusion", "Competing interests", "Abbreviations"], "sections": [["in this and the next issue of critical care , john haddad   presents a comprehensive review on the contributions of transcription factors to lung injury .", "the topic is large and ever changing , and the complete coverage of nuclear factor-b ( nf-b ) and hypoxia - inducible factor ( hif)-1 is spread over two issues . in critical care", "the majority of these patients progress to the point at which they require intubation and mechanical ventilation , with an associated high mortality .", "recent studies   have demonstrated that low tidal volume ( 6 ml / kg ) ventilation strategies dramatically reduce this mortality .", "however , many other clinical trials of various treatments have failed at either reducing the lung injury or accelerating the healing to the end - point of reduced mortality . in this age of genomics and proteomics", "we continue to explore the association of gene and environment . with respect to lung injury ,", "we need to identify and understand the mechanisms that predispose patients to the excessive inflammation resulting from an overactive innate immune response that characterizes sepsis and lung injury . these include stimuli , signal transduction ( receptors , enzyme cascades , transcription factors ) , gene(s ) response and the measured clinical phenotype .", "john haddad , in his two - part review , identifies many ' clinical stimuli ' in cell culture , animal model and patient studies representing an oxidative stress that can generate a response via nf-b or hif-1 dependent signalling ."], ["nf-b was originally described in b lymphocytes , but it is now recognized as a member of the rel family of transcription factors and is a critical response element in many cytokine - dependent events or inflammatory conditions . as a result of this link ,", "nf-b has become a major target for new therapeutic approaches in such clinical disease states as asthma , cancer , arthritis , and cardiovascular and neurodegenerative conditions .", "haddad   discusses the roles of critical care conditions such as hyperoxia , haemorrhage and resuscitation ; the ' stress response ' to illness ( interleukin-6 , interleukin-8 , tumour necrosis factor , rantes [ regulated upon activation , normal t cell expressed and secreted ] ) ; and mechanical ventilation and ischaemia / reperfusion . in all of these conditions", "these dynamic variations in cellular redox or oxidative stress , if in disequilibrium , may regulate gene expression and lead to apoptosis ( cell death without inflammation ) , inflammation and lung injury ."], ["the master regulatory element of hypoxic conditions and adapting oxidative stresses to gene expression is hif-1 .", "hif-1 , a dna - binding protein , has increased stability and binding in hypoxic conditions and is degraded rapidly in normoxia .", "the accumulation of the -subunits allows for  heterodimer formation and translocation into the nucleus during hypoxia .", "this process leads to selective upregulation of genes whose products are involved in hypoxia and inflammatory lung injury .", "these include erythropoietin , vascular endothelial growth factor ( vegf ) and glucose transporter . work by haddad   has demonstrated that proinflammatory cytokines also activate hif-1 stability and dna binding .", "this effect was most profound in hypoxic conditions and was , in fact , greater than that in hypoxaemia alone .", "it is felt that hif-1 , via its action on vegf expression , is directly related to lung injury by endothelial barrier dysfunction mediated by vegf and recognized clinically as increased pulmonary vascular permeability .", "haddad   discusses in detail how hypoxia and inflammatory stimuli initiate many signalling cascades via hif-1 to generate a response phenotype to these oxidative stresses ."], ["understanding the molecular signalling that couples oxidative stresses via nf-b or hif-1 to acute lung injury should generate new therapeutic options .", "haddad   discusses the rationale for the use of tyloxapol to reduce proinflammatory cytokines , n - acetyl cysteine ( a glutathione precursor ) to reduce neutrophil - associated alveolitis in chronic conditions such as cystic fibrosis , and the use of pyrrolidine dithiocarbamate in transplantation to reduce neutrophil - associated oxidant lung injury .", "two new compounds , isohelenin and lisofylline , a phosphodiesterase inhibitor , are described as being able to reduce proinflammatory cytokines and ameliorate oxidant lung injury in animal models . as exciting as this emerging field is , with its predictable contribution to future ' bench to bedside ' discussions , a more complete mechanistic understanding and future clinical trials will assist in the realization of improved treatment and reduced mortality from oxidant - mediated lung injury ."], [""], ["hif = hypoxia - inducible factor ; nf-b = nuclear factor-b ; vegf = vascular endothelial growth factor ."]]}
{"article_id": "PMC2981782", "article_text": ["we enrolled 13 consecutive patients between march 2005 and march 2009 and who met the following inclusion criteria : 1 ) they were diagnosed with acral lentiginous melanoma by punch biopsy ; 2 ) their skin lesion confined to the foot ; 3 ) there was no skin or soft tissue disease on the ipsilateral lower leg .", "we routinely checked the preoperative ultrasonography for popliteal and inguinal lymph node metastasis , and we performed computed tomography of the chest and positron emission tomography to assess for distant metastasis .", "a distally - based island sural flap was used in 10 cases for hindfoot reconstruction , and the lateral supramalleolar fasciocutaneous flap was used in 3 cases for lateral arch reconstruction of the mid- and forefoot after wide excision of malignant melanomas ( table 1 ) .", "eight patients were women and 5 were men with mean age of 60.3 years ( range , 35 to 73 years ) .", "the mean duration of follow - up was 23.3 months ( range , 6 to 48 months ) .", "one patient had ipsilateral inguinal and popliteal lymph node metastasis , and one patient had ipsilateral inguinal lymph node metastasis at the time of diagnosis .", "we performed wide excision of the malignant melanoma with a 2 cm margin in all the patients .", "after wide excision , we confirmed that the margin of resection was free of tumor by frozen section , and then we measured the exact size of the defect .", "the distally based island sural flap was outlined at the junction of the two heads of g the astrocnemius muscle .", "a line of incision was traced over the presumed course of the sural nerve and the lesser saphenous vein .", "the pivotal point of the pedicle was three fingers breadth proximal to the tip of the lateral malleolus .", "1 ) . the lateral supramalleolar fasciocutaneous flap need an essential landmark , which is the perforating branch of the peroneal artery as it pierces the interosseous membrane at the distal tibiofibular angle about 5 cm proximal to the tip of the lateral malleolus .", "the fasciocutaneous flap contained the superficial peroneal nerve , the perforating branch from the peroneal artery and the venae comitantes ( fig .", "2 ) . we attempted neurorrhaphy between the proximally cut superficial peroneal nerve and the distally cut lateral plantar nerve in the two lateral supramalleolar flaps . at a mean time of 21 days after the first operation", ", the skin graft on the flap donor site and opening the pedicle tunnel was carried out with full thickness skin , which was obtained from the ipsilateral inguinal area .", "inguinal lymph node dissection was concomitantly performed , after harvesting the full thickness skin graft , in 10 patients who had \" thick melanomas \" ( > 1.0 mm in thickness by breslow microstaging ) and who had lymph node metastasis seen on the preoperative ultrasonography .", "three patients with \" thin melanoma \" (  1.0 mm in thickness by breslow microstaging ) had a split thickness skin graft that was harvested from the thigh without inguinal lymph node dissection.9 ) low molecular weight dextran was routinely used for 5 days postoperatively .", "partial and full weight bearing ambulation could be started after 6 weeks and 12 weeks after the first operation without concern for wound healing .", "we performed wide excision of the malignant melanoma with a 2 cm margin in all the patients .", "after wide excision , we confirmed that the margin of resection was free of tumor by frozen section , and then we measured the exact size of the defect .", "the distally based island sural flap was outlined at the junction of the two heads of g the astrocnemius muscle .", "a line of incision was traced over the presumed course of the sural nerve and the lesser saphenous vein .", "the pivotal point of the pedicle was three fingers breadth proximal to the tip of the lateral malleolus .", "1 ) . the lateral supramalleolar fasciocutaneous flap need an essential landmark , which is the perforating branch of the peroneal artery as it pierces the interosseous membrane at the distal tibiofibular angle about 5 cm proximal to the tip of the lateral malleolus .", "the fasciocutaneous flap contained the superficial peroneal nerve , the perforating branch from the peroneal artery and the venae comitantes ( fig .", "2 ) . we attempted neurorrhaphy between the proximally cut superficial peroneal nerve and the distally cut lateral plantar nerve in the two lateral supramalleolar flaps . at a mean time of 21 days after the first operation", ", the skin graft on the flap donor site and opening the pedicle tunnel was carried out with full thickness skin , which was obtained from the ipsilateral inguinal area .", "inguinal lymph node dissection was concomitantly performed , after harvesting the full thickness skin graft , in 10 patients who had \" thick melanomas \" ( > 1.0 mm in thickness by breslow microstaging ) and who had lymph node metastasis seen on the preoperative ultrasonography .", "three patients with \" thin melanoma \" (  1.0 mm in thickness by breslow microstaging ) had a split thickness skin graft that was harvested from the thigh without inguinal lymph node dissection.9 )", "partial and full weight bearing ambulation could be started after 6 weeks and 12 weeks after the first operation without concern for wound healing .", "one patient , who had a split thickness skin graft without lymph node dissection , was revealed to have pulmonary and inguinal lymph node metastasis at 4 months postoperatively , and the patient died of disease at 13 months . among those who underwent elective inguinal lymph node dissection ,", "2 patients were revealed to be alive with disease and 3 patients died of disease at 7 , 10 , 13 , 15 months later after operation respectively .", "the length of the flap varied from 7.5 to 12 cm ( mean length , 9.6 cm ) and the width varied from 6.5 to 12 cm ( mean width , 8.8 cm ) . in 4 cases , superficial necrosis developed , but this was successfully treated by debridement and suture or skin graft ( table 1 ) .", "all 13 flaps survived completely and they provided stable defect coverage , good contour and normal weight bearing ambulation .", "as for the patients who had neurorrhaphy , the recovery of sensation was not observed .", "distally based fasciocutaneous flaps on the leg were first introduced in 1983.10 ) the main advantages of these flaps are a well - defined surface that is independent of a lengthwidth ratio and the preservation of the main vascular axis . in most cases ,", "the sural nerve descends in between the two heads of the gastrocnemius muscle and it penetrates the deep fascia at the median point of the leg .", "the superficial sural artery originates from the popliteal or sural arteries , and it reaches the sural nerve at 2 cm to 3 cm after its emergence , then subdivides into the medial , median and lateral superficial sural arteries .", "the median superficial sural artery follows the course of the sural nerve , and then it emits numerous branches toward the skin at the lower half of the leg , along its superficial path .", "the peroneal artery , in turn , makes other anastomoses , notably through its descending branch , and especially with the anterior tibial artery .", "several authors have stated that the principal anastomosis occurs at above to 5 centimeters from the lateral malleolus.2,11 - 13 ) although there are some reports of a preoperative lesser saphenous vein mapping method that used doppler ultrasound4 ) or using a simple rubber tourniquet for making the superficial vessels prominent,2 ) we did not use these techniques .", "however , the incision was initiated in the proximal extreme of the flap , we identified the lesser saphenous vein , sural artery and nerve , and then we made further incision with centralizing on the presumed vessel course as much as possible .", "the distally based sural flap had a tendency of having a short flap pedicle for coverage of a forefoot defect , as compared to that of the lateral supramalleolar flap .", "the lateral supramalleolar flap is a distally based fasciocutaneous flap that is vascularized by branches of a perforating branch of the dorsal peroneal artery .", "this branch is constant and it emerges 5 cm above the lateral malleolus , with 2 or 3 ascending cutaneous branches and a deep descending branch . the ascending branches are included in the intermuscular septum between the extensor digitorum longus and the peroneus brevis , and the ascending branches perforate the fascia and then they supply the skin over the lower lateral half of the leg .", "the descending branch runs distally below the deep fascia , and it anastomoses with the anterolateral malleolar branch of the tibialis anterior artery , and then it continues into the foot to form anastomoses with the tarsi arteries and dorsal arch .", "the point of emergence of the perforating branch of the dorsal peroneal artery could be detected \" by palpating the groove just above and anterior to the lateral malleolus.\"14 ) this represents the pivot point .", "the axis of the flap is represented by a midline drawn from the anterior tibial crest to the posterior margin of the fibula .", "voche et al.5 ) distinguished the type of arterial blood supply to the lateral supramalleloar flap depending on how far distally the flap is raised , and this can include a mixed antegrade and retrograde blood supply .", "so this flap can have a longer pedicle length due to the various possible vascular networks and the flap can reach to the forefoot .", "almeida et al.2 ) reported on the complications in their 71 cases of a reverse - flow island sural flap , including partial ( 22.1% ) or total ( 4.2% ) flap necrosis , infection ( 8.5% ) and venous congestion ( 4.1% ) .", "voche et al.5 ) reported venous congestion and partial flap necrosis ( 5 to 30% ) in their 41 cases of using the lateral supramalleolar flap .", "they presented the delayed neurofasicocutaneous sural flap , which is initially completely elevated and it is then fixed again at the donor site using running sutures for 7 to 15 days . after confirming the flap 's survival , the flap was raised again and transposed into the defect .", "this delayed procedure could be an alternative to overcome these problems and to increase the reliability and viability of the flap .", "tan et al.15 ) treated three patients with a supercharged reverse flow sural flap to reduce venous congestion and edema , and the venous drainage was achieved by end - to - end anastomosis between the free end of the lesser saphenous vein and a superficial vein on the medial site of the ankle . among our 4 patients with partial necrosis , 2 occurred in the distally based sural artery flap .", "partial necrosis developed on the lateral margin of the flap that was transposed medially on the plantar surface .", "the flap sizes of these patients were not too much larger than that of the other patients , but the routes of passage of the sural vascular pedicle were more laterally located from the flap center . therefore , at the time of sural flap dissection , the skin island should be redrawn and shifted either medially or laterally as the dissection progresses , while trying to keep the pedicle centralized with regard to the flap .", "the other two cases of partial necrosis developed on the distal portion of the transposed lateral supramelleolar flap .", "these flaps were large size ( 12  9 , 10.5  9 cm ) with long pedicle lengths to reach the metatarso - phalangeal joint of the fifth toe .", "we confirmed well - blanched , transposed flap color to the lateral arch of the forefoot and then we completely sutured the pedicle tunnel . from these cases , we suggest that an opened pedicle tunnel can be beneficial for the long length pedicle and a large flap size . when the defects are larger and more anterior , free tissue transfer is often required because it can provide a large amount of tissue.6 ) however , there are some disadvantages such as donor - site morbidity , an increased operation time , the use of a major vessel of the leg and the necessity of microsurgical expertise .", "moreover , langstein et al.16 ) reported the free tissue transfer for limb salvage of soft tissue malignancies on the foot . according to their results , over 50% of the patients with local recurrence and persistent disease required below knee amputation .", "voche et al.5 ) suggested the lateral supramalleolar flap is reliable and useful for coverage of the lower leg , ankle and foot skin defects , but coverage of the weight - bearing surface of the foot should be avoided .", "however , we had satisfactory results for a the large defect of the lateral half of the midfoot and forefoot , including a weight bearing area , so there seems to be no problem if the pedicle can be placed in a non - weight bearing portion or delayed weight bearing is performed .", "one patient developed multiple inguinal lymph node and lung metastases after a late split thickness skin graft on the donor defect area and the patient had received a lateral supramalleolar flap .", "this all suggested the necessity of inguinal lymph node biopsy or dissection , and many articles have emphasized that sentinel lymph node biopsy needs to be done in a patient in the malignant melanoma.17 ) if this patient had received a full thickness skin graft instead of a split skin graft , then we would have performed inguinal lymph node dissection .", "our delayed full thickness skin graft had benefits with respect to confirming the flap 's viability in a critical period , the one - time - approach for ipsilateral lymph node dissection after harvesting the full thickness skin and the safety for the pedicle tunnel that remained open rather than performing primary closure . in conclusion ,", "the distally based sural flap and lateral supramalleolar flap provide effective coverage of variable sized soft tissue defects on the foot after wide excision of malignant melanoma , this is quick and safe surgery without the necessity of microsurgical expertise , it preserves the major arteries of the leg and the donor - site morbidity is acceptable .", "the distally based sural flap is useful for the hindfoot , and the lateral supramalleolar flap is good for lateral archs of the mid- and forefoot ."], "abstract_text": ["<S> backgroundwe report on our experience with using a distally based island flap for soft tissue reconstruction of the foot in limb salvage surgery for malignant melanoma patients.methodsa distally based sural flap was used for 10 cases for the hindfoot reconstruction , and a lateral supramalleolar flap was used for 3 cases for the lateral arch reconstruction of the mid- and forefoot after wide excision of malignant melanomas.resultsthe length of the flap varied from 7.5 cm to 12 cm ( mean , 9.6 cm ) and the width varied from 6.5 cm to 12 cm ( mean , 8.8 cm ) . superficial necrosis developed in four flaps , but </S>", "<S> this was successfully treated by debridement and suture or a skin graft . </S>", "<S> all thirteen flaps survived completely and they provided good contour , stable and durable coverage for normal weight bearing.conclusionsthe distally based sural flap is considered to be useful for reconstructing the hindfoot , and the lateral supramalleolar flap is good for reconstructing the lateral archs of the mid- and forefoot after resection of malignant melanoma of the foot . </S>"], "labels": null, "section_names": ["METHODS", "Surgical Technique", "Postoperative Care", "RESULTS", "DISCUSSION"], "sections": [["we enrolled 13 consecutive patients between march 2005 and march 2009 and who met the following inclusion criteria : 1 ) they were diagnosed with acral lentiginous melanoma by punch biopsy ; 2 ) their skin lesion confined to the foot ; 3 ) there was no skin or soft tissue disease on the ipsilateral lower leg .", "we routinely checked the preoperative ultrasonography for popliteal and inguinal lymph node metastasis , and we performed computed tomography of the chest and positron emission tomography to assess for distant metastasis .", "a distally - based island sural flap was used in 10 cases for hindfoot reconstruction , and the lateral supramalleolar fasciocutaneous flap was used in 3 cases for lateral arch reconstruction of the mid- and forefoot after wide excision of malignant melanomas ( table 1 ) .", "eight patients were women and 5 were men with mean age of 60.3 years ( range , 35 to 73 years ) .", "the mean duration of follow - up was 23.3 months ( range , 6 to 48 months ) .", "one patient had ipsilateral inguinal and popliteal lymph node metastasis , and one patient had ipsilateral inguinal lymph node metastasis at the time of diagnosis .", "we performed wide excision of the malignant melanoma with a 2 cm margin in all the patients .", "after wide excision , we confirmed that the margin of resection was free of tumor by frozen section , and then we measured the exact size of the defect .", "the distally based island sural flap was outlined at the junction of the two heads of g the astrocnemius muscle .", "a line of incision was traced over the presumed course of the sural nerve and the lesser saphenous vein .", "the pivotal point of the pedicle was three fingers breadth proximal to the tip of the lateral malleolus .", "1 ) . the lateral supramalleolar fasciocutaneous flap need an essential landmark , which is the perforating branch of the peroneal artery as it pierces the interosseous membrane at the distal tibiofibular angle about 5 cm proximal to the tip of the lateral malleolus .", "the fasciocutaneous flap contained the superficial peroneal nerve , the perforating branch from the peroneal artery and the venae comitantes ( fig .", "2 ) . we attempted neurorrhaphy between the proximally cut superficial peroneal nerve and the distally cut lateral plantar nerve in the two lateral supramalleolar flaps . at a mean time of 21 days after the first operation", ", the skin graft on the flap donor site and opening the pedicle tunnel was carried out with full thickness skin , which was obtained from the ipsilateral inguinal area .", "inguinal lymph node dissection was concomitantly performed , after harvesting the full thickness skin graft , in 10 patients who had \" thick melanomas \" ( > 1.0 mm in thickness by breslow microstaging ) and who had lymph node metastasis seen on the preoperative ultrasonography .", "three patients with \" thin melanoma \" (  1.0 mm in thickness by breslow microstaging ) had a split thickness skin graft that was harvested from the thigh without inguinal lymph node dissection.9 ) low molecular weight dextran was routinely used for 5 days postoperatively .", "partial and full weight bearing ambulation could be started after 6 weeks and 12 weeks after the first operation without concern for wound healing ."], ["we performed wide excision of the malignant melanoma with a 2 cm margin in all the patients .", "after wide excision , we confirmed that the margin of resection was free of tumor by frozen section , and then we measured the exact size of the defect .", "the distally based island sural flap was outlined at the junction of the two heads of g the astrocnemius muscle .", "a line of incision was traced over the presumed course of the sural nerve and the lesser saphenous vein .", "the pivotal point of the pedicle was three fingers breadth proximal to the tip of the lateral malleolus .", "1 ) . the lateral supramalleolar fasciocutaneous flap need an essential landmark , which is the perforating branch of the peroneal artery as it pierces the interosseous membrane at the distal tibiofibular angle about 5 cm proximal to the tip of the lateral malleolus .", "the fasciocutaneous flap contained the superficial peroneal nerve , the perforating branch from the peroneal artery and the venae comitantes ( fig .", "2 ) . we attempted neurorrhaphy between the proximally cut superficial peroneal nerve and the distally cut lateral plantar nerve in the two lateral supramalleolar flaps . at a mean time of 21 days after the first operation", ", the skin graft on the flap donor site and opening the pedicle tunnel was carried out with full thickness skin , which was obtained from the ipsilateral inguinal area .", "inguinal lymph node dissection was concomitantly performed , after harvesting the full thickness skin graft , in 10 patients who had \" thick melanomas \" ( > 1.0 mm in thickness by breslow microstaging ) and who had lymph node metastasis seen on the preoperative ultrasonography .", "three patients with \" thin melanoma \" (  1.0 mm in thickness by breslow microstaging ) had a split thickness skin graft that was harvested from the thigh without inguinal lymph node dissection.9 )"], ["partial and full weight bearing ambulation could be started after 6 weeks and 12 weeks after the first operation without concern for wound healing ."], ["one patient , who had a split thickness skin graft without lymph node dissection , was revealed to have pulmonary and inguinal lymph node metastasis at 4 months postoperatively , and the patient died of disease at 13 months . among those who underwent elective inguinal lymph node dissection ,", "2 patients were revealed to be alive with disease and 3 patients died of disease at 7 , 10 , 13 , 15 months later after operation respectively .", "the length of the flap varied from 7.5 to 12 cm ( mean length , 9.6 cm ) and the width varied from 6.5 to 12 cm ( mean width , 8.8 cm ) . in 4 cases , superficial necrosis developed , but this was successfully treated by debridement and suture or skin graft ( table 1 ) .", "all 13 flaps survived completely and they provided stable defect coverage , good contour and normal weight bearing ambulation .", "as for the patients who had neurorrhaphy , the recovery of sensation was not observed ."], ["distally based fasciocutaneous flaps on the leg were first introduced in 1983.10 ) the main advantages of these flaps are a well - defined surface that is independent of a lengthwidth ratio and the preservation of the main vascular axis . in most cases ,", "the sural nerve descends in between the two heads of the gastrocnemius muscle and it penetrates the deep fascia at the median point of the leg .", "the superficial sural artery originates from the popliteal or sural arteries , and it reaches the sural nerve at 2 cm to 3 cm after its emergence , then subdivides into the medial , median and lateral superficial sural arteries .", "the median superficial sural artery follows the course of the sural nerve , and then it emits numerous branches toward the skin at the lower half of the leg , along its superficial path .", "the peroneal artery , in turn , makes other anastomoses , notably through its descending branch , and especially with the anterior tibial artery .", "several authors have stated that the principal anastomosis occurs at above to 5 centimeters from the lateral malleolus.2,11 - 13 ) although there are some reports of a preoperative lesser saphenous vein mapping method that used doppler ultrasound4 ) or using a simple rubber tourniquet for making the superficial vessels prominent,2 ) we did not use these techniques .", "however , the incision was initiated in the proximal extreme of the flap , we identified the lesser saphenous vein , sural artery and nerve , and then we made further incision with centralizing on the presumed vessel course as much as possible .", "the distally based sural flap had a tendency of having a short flap pedicle for coverage of a forefoot defect , as compared to that of the lateral supramalleolar flap .", "the lateral supramalleolar flap is a distally based fasciocutaneous flap that is vascularized by branches of a perforating branch of the dorsal peroneal artery .", "this branch is constant and it emerges 5 cm above the lateral malleolus , with 2 or 3 ascending cutaneous branches and a deep descending branch . the ascending branches are included in the intermuscular septum between the extensor digitorum longus and the peroneus brevis , and the ascending branches perforate the fascia and then they supply the skin over the lower lateral half of the leg .", "the descending branch runs distally below the deep fascia , and it anastomoses with the anterolateral malleolar branch of the tibialis anterior artery , and then it continues into the foot to form anastomoses with the tarsi arteries and dorsal arch .", "the point of emergence of the perforating branch of the dorsal peroneal artery could be detected \" by palpating the groove just above and anterior to the lateral malleolus.\"14 ) this represents the pivot point .", "the axis of the flap is represented by a midline drawn from the anterior tibial crest to the posterior margin of the fibula .", "voche et al.5 ) distinguished the type of arterial blood supply to the lateral supramalleloar flap depending on how far distally the flap is raised , and this can include a mixed antegrade and retrograde blood supply .", "so this flap can have a longer pedicle length due to the various possible vascular networks and the flap can reach to the forefoot .", "almeida et al.2 ) reported on the complications in their 71 cases of a reverse - flow island sural flap , including partial ( 22.1% ) or total ( 4.2% ) flap necrosis , infection ( 8.5% ) and venous congestion ( 4.1% ) .", "voche et al.5 ) reported venous congestion and partial flap necrosis ( 5 to 30% ) in their 41 cases of using the lateral supramalleolar flap .", "they presented the delayed neurofasicocutaneous sural flap , which is initially completely elevated and it is then fixed again at the donor site using running sutures for 7 to 15 days . after confirming the flap 's survival , the flap was raised again and transposed into the defect .", "this delayed procedure could be an alternative to overcome these problems and to increase the reliability and viability of the flap .", "tan et al.15 ) treated three patients with a supercharged reverse flow sural flap to reduce venous congestion and edema , and the venous drainage was achieved by end - to - end anastomosis between the free end of the lesser saphenous vein and a superficial vein on the medial site of the ankle . among our 4 patients with partial necrosis , 2 occurred in the distally based sural artery flap .", "partial necrosis developed on the lateral margin of the flap that was transposed medially on the plantar surface .", "the flap sizes of these patients were not too much larger than that of the other patients , but the routes of passage of the sural vascular pedicle were more laterally located from the flap center . therefore , at the time of sural flap dissection , the skin island should be redrawn and shifted either medially or laterally as the dissection progresses , while trying to keep the pedicle centralized with regard to the flap .", "the other two cases of partial necrosis developed on the distal portion of the transposed lateral supramelleolar flap .", "these flaps were large size ( 12  9 , 10.5  9 cm ) with long pedicle lengths to reach the metatarso - phalangeal joint of the fifth toe .", "we confirmed well - blanched , transposed flap color to the lateral arch of the forefoot and then we completely sutured the pedicle tunnel . from these cases , we suggest that an opened pedicle tunnel can be beneficial for the long length pedicle and a large flap size . when the defects are larger and more anterior , free tissue transfer is often required because it can provide a large amount of tissue.6 ) however , there are some disadvantages such as donor - site morbidity , an increased operation time , the use of a major vessel of the leg and the necessity of microsurgical expertise .", "moreover , langstein et al.16 ) reported the free tissue transfer for limb salvage of soft tissue malignancies on the foot . according to their results , over 50% of the patients with local recurrence and persistent disease required below knee amputation .", "voche et al.5 ) suggested the lateral supramalleolar flap is reliable and useful for coverage of the lower leg , ankle and foot skin defects , but coverage of the weight - bearing surface of the foot should be avoided .", "however , we had satisfactory results for a the large defect of the lateral half of the midfoot and forefoot , including a weight bearing area , so there seems to be no problem if the pedicle can be placed in a non - weight bearing portion or delayed weight bearing is performed .", "one patient developed multiple inguinal lymph node and lung metastases after a late split thickness skin graft on the donor defect area and the patient had received a lateral supramalleolar flap .", "this all suggested the necessity of inguinal lymph node biopsy or dissection , and many articles have emphasized that sentinel lymph node biopsy needs to be done in a patient in the malignant melanoma.17 ) if this patient had received a full thickness skin graft instead of a split skin graft , then we would have performed inguinal lymph node dissection .", "our delayed full thickness skin graft had benefits with respect to confirming the flap 's viability in a critical period , the one - time - approach for ipsilateral lymph node dissection after harvesting the full thickness skin and the safety for the pedicle tunnel that remained open rather than performing primary closure . in conclusion ,", "the distally based sural flap and lateral supramalleolar flap provide effective coverage of variable sized soft tissue defects on the foot after wide excision of malignant melanoma , this is quick and safe surgery without the necessity of microsurgical expertise , it preserves the major arteries of the leg and the donor - site morbidity is acceptable .", "the distally based sural flap is useful for the hindfoot , and the lateral supramalleolar flap is good for lateral archs of the mid- and forefoot ."]]}
{"article_id": "PMC270725", "article_text": ["techniques and equipment to accomplish endotracheal ( et ) intubation were the precursor to modern day invasive mechanical ventilation . in recent years , however , the popularity of the et tube has waned .", "clinically , the et tube is seen as an impediment to spontaneous breathing , a transit route for bacteria to the lower airway , and  with the advent of noninvasive ventilation  a device to be avoided when possible .", "of particular interest has been the effect of the et tube on work of breathing and methods to eliminate this work .", "commonly , pressure support ventilation ( psv ) has been suggested as the technique of choice for eliminating imposed work due to the et tube .", "more recently , the technique of automatic tube compensation ( atc ) has become available to specifically address this issue . in this issue of critical care , maeda and", "colleagues   compare the technique of atc , as provided by the drager evita 4 ( dragerwerks , lubeck , germany ) and the puritan bennett 840 ( carlsbad , ca , usa ) , versus psv in reducing imposed work of breathing in a lung model . before i comment on the merits of the study ,", "clearly , before the advent of pressure support ventilation in the early 1980s , patients were successfully weaned using t - piece trials and intermittent mandatory ventilation , with no apparent untoward effects .", "in 1986 , shapiro and coworkers   presented data from three normal volunteers breathing through et tubes at a constant tidal volume of 500 ml .", "that report is widely quoted but is limited by the use of unintubated normal individuals and the requirement for a constant tidal volume during increasing respiratory rates .", "additionally , close review of the data demonstrates that with a size 8.0 et tube the work of breathing in joules per minute does not become excessive until minute ventilation exceeds 15 l / min .", "brochard and colleagues   compared work of breathing during assisted ventilation , four levels of pressure support , continuous positive airway pressure , and after extubation in an effort to determine the role of pressure support in overcoming the imposed work presented by the et tube . that trial evaluated both patients with normal lungs and those with chronic obstructive pulmonary disease ( copd ) . the authors concluded that the pressure support level that eliminated imposed work was between 3 and 14 cmh2o .", "interestingly , this was determined retrospectively by matching the work of breathing after extubation to the level of pressure support that resulted in equivalent work of breathing during mechanical ventilation .", "recent work suggests that the work of breathing postextubation may actually increase compared with work of breathing through the et tube , raising questions about the conclusion of the study by brochard and colleagues .", "weissman   evaluated flow - volume loops in 18 postoperative patients intubated with size 7.0 and 8.0 et tubes and found that only ' minimal limitation to airflow ' occurred at volumes and frequencies associated with tidal breathing .", "that study is one of the very few that contemplate the idea that et tube resistance may not be important when the appropriate size is chosen and the patients pulmonary function has improved to the point that weaning may be considered . of course", ", there are numerous other studies that suggest that et tube resistance may represent a significant impediment to spontaneous breathing .", "however , it is important to note that although there remains concern about et tube resistance , and the literature is replete with lung model studies of increased work of breathing , there is not a single clinical trial that suggests that spontaneous breathing through the et tube results in untoward outcomes .", "the report by maeda and coworkers   in this issue of critical care evaluates the two most popular methods of overcoming et tube resistance , namely psv and atc .", "the authors concluded that tube compensation could not overcome the pressure  time product associated with triggering and that pressure support is as effective as atc at 100% .", "there are , however , tremendous disadvantages to a lung model study in comparing these techniques .", "calculating the pressure  time product and work includes the work required to trigger the ventilator , which can only be overcome by direct measurement of tracheal pressure and triggering from the tracheal signal .", "this method has been proposed by many , and advanced by the group from gainesville . additionally ,", "although the lung model allows consistency , it can not react to differences in gas delivery . in several human studies comparing psv with atc", ", the slow flow and longer inspiratory time associated with psv has been associated with dysynchrony . when used in a patient with copd ,", "the patient 's high airway resistance and increased compliance allow even small amounts of psv to cause hyperinflation and auto - peep ( positive end - expiratory pressure ) .", "alterations in pulmonary mechanics , along with the preference of the copd patient for short inspiratory times and long expiratory times , result in neuromechanical dysynchrony during psv .", "the main proposed advantages of atc over psv are reduced work of breathing as patient demand varies , preservation of a normal , variable breathing pattern , and improved synchrony . in a recent trial of spontaneous breathing before extubation , haberthur and coworkers   failed to show any advantage of atc over psv or t - tube trials .", "one issue not addressed in the study by maeda and coworkers is the role of expiratory compensation .", "one distinct difference in the operation of the drager evita 4 and puritan bennett 840 is that evita 4 provides both inspiratory and expiratory compensation for et tube resistance . in these cases", ", the airway pressure may be allowed to drop below peep to facilitate expiratory flow .", "new modes and new techniques are developed in the hope of resolving clinical problems and often concentrate on a short - term physiologic end - point .", "conventional wisdom suggests that the et tube is an impediment to efficient spontaneous breathing , yet clinical evidence during spontaneous breathing trials appears to argue to the contrary", ". the real question may be whether overcoming et tube resistance is necessary , not whether atc is as good as or better than psv .", "the future of atc , like many new techniques , may not be in overcoming et tube resistance , but as a method of support during spontaneous breathing that improves patient - ventilator synchrony as compared with psv .", "additional clinical studies are required to complement the excellent laboratory work by the group from osaka reported in this issue .", "atc = automatic tube compensation ; copd = chronic obstructive pulmonary disease ; et = endotracheal ; peep = positive end - expiratory pressure ; psv = pressure support ventilation ."], "abstract_text": ["<S> concerns about the work of breathing imposed by the endotracheal tube have led clinicians to routinely use pressure support to overcome this resistive component . </S>", "<S> more recently , ventilator manufacturers have introduced systems to automatically overcome endotracheal tube resistance , regardless of tube diameter or patient demand for flow . despite the theoretical advantages </S>", "<S> , neither method appears to provide superior performance . stepping back </S>", "<S> , the real question may be , is overcoming endotracheal tube resistance really important ? </S>"], "labels": null, "section_names": ["None", "Competing interests", "Abbreviations"], "sections": [["techniques and equipment to accomplish endotracheal ( et ) intubation were the precursor to modern day invasive mechanical ventilation . in recent years , however , the popularity of the et tube has waned .", "clinically , the et tube is seen as an impediment to spontaneous breathing , a transit route for bacteria to the lower airway , and  with the advent of noninvasive ventilation  a device to be avoided when possible .", "of particular interest has been the effect of the et tube on work of breathing and methods to eliminate this work .", "commonly , pressure support ventilation ( psv ) has been suggested as the technique of choice for eliminating imposed work due to the et tube .", "more recently , the technique of automatic tube compensation ( atc ) has become available to specifically address this issue . in this issue of critical care , maeda and", "colleagues   compare the technique of atc , as provided by the drager evita 4 ( dragerwerks , lubeck , germany ) and the puritan bennett 840 ( carlsbad , ca , usa ) , versus psv in reducing imposed work of breathing in a lung model . before i comment on the merits of the study ,", "clearly , before the advent of pressure support ventilation in the early 1980s , patients were successfully weaned using t - piece trials and intermittent mandatory ventilation , with no apparent untoward effects .", "in 1986 , shapiro and coworkers   presented data from three normal volunteers breathing through et tubes at a constant tidal volume of 500 ml .", "that report is widely quoted but is limited by the use of unintubated normal individuals and the requirement for a constant tidal volume during increasing respiratory rates .", "additionally , close review of the data demonstrates that with a size 8.0 et tube the work of breathing in joules per minute does not become excessive until minute ventilation exceeds 15 l / min .", "brochard and colleagues   compared work of breathing during assisted ventilation , four levels of pressure support , continuous positive airway pressure , and after extubation in an effort to determine the role of pressure support in overcoming the imposed work presented by the et tube . that trial evaluated both patients with normal lungs and those with chronic obstructive pulmonary disease ( copd ) . the authors concluded that the pressure support level that eliminated imposed work was between 3 and 14 cmh2o .", "interestingly , this was determined retrospectively by matching the work of breathing after extubation to the level of pressure support that resulted in equivalent work of breathing during mechanical ventilation .", "recent work suggests that the work of breathing postextubation may actually increase compared with work of breathing through the et tube , raising questions about the conclusion of the study by brochard and colleagues .", "weissman   evaluated flow - volume loops in 18 postoperative patients intubated with size 7.0 and 8.0 et tubes and found that only ' minimal limitation to airflow ' occurred at volumes and frequencies associated with tidal breathing .", "that study is one of the very few that contemplate the idea that et tube resistance may not be important when the appropriate size is chosen and the patients pulmonary function has improved to the point that weaning may be considered . of course", ", there are numerous other studies that suggest that et tube resistance may represent a significant impediment to spontaneous breathing .", "however , it is important to note that although there remains concern about et tube resistance , and the literature is replete with lung model studies of increased work of breathing , there is not a single clinical trial that suggests that spontaneous breathing through the et tube results in untoward outcomes .", "the report by maeda and coworkers   in this issue of critical care evaluates the two most popular methods of overcoming et tube resistance , namely psv and atc .", "the authors concluded that tube compensation could not overcome the pressure  time product associated with triggering and that pressure support is as effective as atc at 100% .", "there are , however , tremendous disadvantages to a lung model study in comparing these techniques .", "calculating the pressure  time product and work includes the work required to trigger the ventilator , which can only be overcome by direct measurement of tracheal pressure and triggering from the tracheal signal .", "this method has been proposed by many , and advanced by the group from gainesville . additionally ,", "although the lung model allows consistency , it can not react to differences in gas delivery . in several human studies comparing psv with atc", ", the slow flow and longer inspiratory time associated with psv has been associated with dysynchrony . when used in a patient with copd ,", "the patient 's high airway resistance and increased compliance allow even small amounts of psv to cause hyperinflation and auto - peep ( positive end - expiratory pressure ) .", "alterations in pulmonary mechanics , along with the preference of the copd patient for short inspiratory times and long expiratory times , result in neuromechanical dysynchrony during psv .", "the main proposed advantages of atc over psv are reduced work of breathing as patient demand varies , preservation of a normal , variable breathing pattern , and improved synchrony . in a recent trial of spontaneous breathing before extubation , haberthur and coworkers   failed to show any advantage of atc over psv or t - tube trials .", "one issue not addressed in the study by maeda and coworkers is the role of expiratory compensation .", "one distinct difference in the operation of the drager evita 4 and puritan bennett 840 is that evita 4 provides both inspiratory and expiratory compensation for et tube resistance . in these cases", ", the airway pressure may be allowed to drop below peep to facilitate expiratory flow .", "new modes and new techniques are developed in the hope of resolving clinical problems and often concentrate on a short - term physiologic end - point .", "conventional wisdom suggests that the et tube is an impediment to efficient spontaneous breathing , yet clinical evidence during spontaneous breathing trials appears to argue to the contrary", ". the real question may be whether overcoming et tube resistance is necessary , not whether atc is as good as or better than psv .", "the future of atc , like many new techniques , may not be in overcoming et tube resistance , but as a method of support during spontaneous breathing that improves patient - ventilator synchrony as compared with psv .", "additional clinical studies are required to complement the excellent laboratory work by the group from osaka reported in this issue ."], [""], ["atc = automatic tube compensation ; copd = chronic obstructive pulmonary disease ; et = endotracheal ; peep = positive end - expiratory pressure ; psv = pressure support ventilation ."]]}
{"article_id": "PMC5384695", "article_text": ["bed rest , heart failure , depletion of intravascular volume , and aging often induce hypotension when the body or trunk is upright.15 generally , a tachycardic response occurs due to vagal withdrawal according to the downward blood shift that occurs under orthostatic stress in healthy humans,6 although orthostatic hypotension presents as an excessive drop in cardiac output ( q ) and lower tachycardic response in frail and elderly patients as a consequence of inadequate vascular contraction or autonomic dysfunction.7,8 numerous studies have suggested that advancing age is associated with decreases in vagal nerve activity.912 age - related decreases in vagal nerve activity have been suggested as contributing factors for orthostatic hypotension in elderly individuals.7,13 orthostatic hypotension has been reported in more than 60% of frail older patients in an acute geriatric ward.5 previous studies have observed orthostatic hypotension in 54% of seated older inpatients resting on a bed for more than 12 h.14 in light of these facts , methods of preventing hypovolemia and hypotension during orthostatic stress need to be investigated .", "pointed out that studies of orthostatic hypotension in frail patients , such as those with decompensated heart failure , are scarce and preventive interventions remain insufficient.2 clinically , frail patients are placed in fowler s position or a semiseated position on the bed .", "these positions are often clinically used without distinction between acute- or chronic - phase illness .", "the patient is placed in a reclined position with the backrest between 30 and 60 and with flexed or straight knees in fowler s position.15,16 breathing , eating , and other routine daily activities are considered to be facilitated in fowler s position.15,1720 some studies have suggested a relationship between trunk angle and hemodynamics during fowler s position.2124 cicolini et al . reported that blood pressure in fowler s position was intermediate between the seated and supine positions in both young healthy individuals and hypertensive patients.23,24 those studies indicated that the effects of orthostatic stress on hemodynamics in fowler s position may be substantial in frail patients .", "how posture can prevent the hypovolemia and hypotension caused by orthostatic stress during fowler s position is important and should be addressed .", "our previous study indicated that fowler s position with an upright upper trunk is effective for maintaining circulatory volume with lower tachycardia via vagal withdrawal compared with having the whole trunk upright in younger subjects.25 however , whether an upright upper trunk in fowler s position allows easy maintenance of circulatory volume in cases with autonomic hypofunction associated with aging remains unclear .", "we postulated that stroke volume ( sv ) would be higher and heart rate ( hr ) would be lower with the upper trunk upright than with the whole trunk upright in fowler s position in both older and younger subjects .", "in addition , we presumed that the lower hr would be caused by reduced sympathetic activation in older individuals but by reduced vagal withdrawal in younger individuals .", "the present study was performed as a cross - sectional study to assess our hypothesis with a fundamental study of the effects of the two fowler s positions on hemodynamics and hr variability parameters in healthy older and younger subjects .", "we assessed hemodynamics and hr variability in 11 male younger subjects ( mean age  standard error of the mean [ sem ] : 20.50.2 years ; range : 2022 years ; weight : 60.61.8 kg ; height : 170.21.8 cm ) and 11 male community - dwelling older subjects ( mean age  sem : 69.51.2 years ; range : 6479 years ; weight : 62.02.1 kg ; height : 165.41.8 cm ) .", "all subjects were interviewed regarding medical history , and blood pressure was checked after a period of rest .", "final decisions on subject selections were made by consultations between a medical doctor and researchers .", "potential subjects were excluded when they had a history of cardiovascular disease , respiratory disease or other chronic medical disease , or were not normotensive ( systolic bold pressure below 140 mmhg and diastolic blood pressure below 90 mmhg ) .", "all subjects in the final analysis were healthy and had no history of chronic or acute cardiovascular or respiratory disease or other chronic diseases .", "subjects were instructed to refrain from beverages containing caffeine or alcohol for 24 h before starting experiments and to refrain from eating and drinking after 2,200 h on the day prior to the experiments that started in the morning or to consume a light breakfast before experiments that started in the afternoon .", "all experiments were implemented between 1,100 and 1,400 h , because blood pressure fluctuates according to a circadian pattern stabilizing at approximately noon .", "the ethics commission of the international university of health and welfare approved the study , and all participants provided voluntary written consent to participate after being fully informed about the procedure , risks , and protocol .", "we collected physiological data with the patient in the supine position and two fowler s positions . prior to experiments", ", subjects rested in a thermoneutral room at 28c for 15 min and were set up for electrocardiography ( ecg ) , noninvasive arterial pressure monitoring , and impedance cardiography ( icg ) .", "measurements in the supine position were made with sufficient time between measurements in the two fowler s positions . between measurements in each fowler s position , subjects rested for more than 3 min .", "data were recorded for 5 min in each of the supine position and the two fowler s positions .", "subjects were placed in the fowler s positions on a bed at 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) and 60 of whole trunk inclination ( wt60 ) ( figure 1 ) .", "we defined the upper and lower segments at ut60 around the spinous process of the 10th thoracic vertebra .", "the height of the bottom of the upper and lower trunk was adjusted according to individual trunk size .", "all experiments were performed in our laboratory ( odawara , kanagawa , japan ) under controlled thermoneutral temperature .", "hr was estimated from type ii ecg ( ecg 100c ; biopac systems , goleta , ca , usa ) in all experiments .", "icg ( nico 100c ; biopac systems ) was performed using eight spot electrodes ( vitrode m ; nihon kohden , tokyo , japan ) attached to the neck and lower thorax of each subject for icg based on a previous study.26 sv was estimated from transthoracic bioimpedance using bernstein s method that has been validated as offering high reliability comparable to the technique of thermodilution.26 icg allows continuous noninvasive evaluation of sv .", "noninvasive continuous arterial pressure was measured in the radial artery by tonometry using an arterial pressure monitor ( bp-608ev ; omron colin , tokyo , japan ) .", "mean arterial pressure ( map ) was estimated as one third pulse pressure plus diastolic pressure .", "ecg , thoracic bioimpedance , and continuous arterial pressure signals were recorded on a personal computer using the mp150 data acquisition system ( biopac systems ) at a sampling rate of 1 khz .", "we performed frequency domain and time domain analysis of ecg for the assessment of vagal function .", "we calculated logarithm - transformed respiratory sinus arrhythmia ( ln rsa ) from the power spectrum of the rr intervals ( 0.150.4 hz ) .", "we thus calculated the square root of the mean of the sum of the squares of differences between adjacent rr intervals ( rmssd ) , number of pairs of adjacent rr intervals differing by more than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of rr intervals ( pnn50 ) for 5 min .", "all parameters of hr variability reflect vagal modulation.27,28 changes in hemodynamic variables from supine and hr variability parameters in the three positions in subjects from the two age groups were compared by two - way mixed - design analysis of variance ( anova ) .", "when three positions  two age group interactions were significant , one - way repeated - measures anova of the three positions in each subject and one - way anova of subjects from the two age groups in each position were used to evaluate simple main effects .", "subsequent post hoc analysis to determine differences between the three positions in each age group were evaluated using a paired t - test with shaffer s multiple comparison procedure in each age group.29 the level of statistical significance was set at p<0.05 .", "data were statistically analyzed using r for windows version 3.3.0 statistical software ( www.r-project.org ) and the car 2.14 package .", "we assessed hemodynamics and hr variability in 11 male younger subjects ( mean age  standard error of the mean [ sem ] : 20.50.2 years ; range : 2022 years ; weight : 60.61.8 kg ; height : 170.21.8 cm ) and 11 male community - dwelling older subjects ( mean age  sem : 69.51.2 years ; range : 6479 years ; weight : 62.02.1 kg ; height : 165.41.8 cm ) .", "all subjects were interviewed regarding medical history , and blood pressure was checked after a period of rest .", "final decisions on subject selections were made by consultations between a medical doctor and researchers .", "potential subjects were excluded when they had a history of cardiovascular disease , respiratory disease or other chronic medical disease , or were not normotensive ( systolic bold pressure below 140 mmhg and diastolic blood pressure below 90 mmhg ) .", "all subjects in the final analysis were healthy and had no history of chronic or acute cardiovascular or respiratory disease or other chronic diseases .", "subjects were instructed to refrain from beverages containing caffeine or alcohol for 24 h before starting experiments and to refrain from eating and drinking after 2,200 h on the day prior to the experiments that started in the morning or to consume a light breakfast before experiments that started in the afternoon .", "all experiments were implemented between 1,100 and 1,400 h , because blood pressure fluctuates according to a circadian pattern stabilizing at approximately noon .", "the ethics commission of the international university of health and welfare approved the study , and all participants provided voluntary written consent to participate after being fully informed about the procedure , risks , and protocol .", "we collected physiological data with the patient in the supine position and two fowler s positions . prior to experiments", ", subjects rested in a thermoneutral room at 28c for 15 min and were set up for electrocardiography ( ecg ) , noninvasive arterial pressure monitoring , and impedance cardiography ( icg ) .", "measurements in the supine position were made with sufficient time between measurements in the two fowler s positions . between measurements in each fowler s position , subjects rested for more than 3 min .", "data were recorded for 5 min in each of the supine position and the two fowler s positions .", "subjects were placed in the fowler s positions on a bed at 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) and 60 of whole trunk inclination ( wt60 ) ( figure 1 ) .", "we defined the upper and lower segments at ut60 around the spinous process of the 10th thoracic vertebra .", "the height of the bottom of the upper and lower trunk was adjusted according to individual trunk size .", "all experiments were performed in our laboratory ( odawara , kanagawa , japan ) under controlled thermoneutral temperature .", "hr was estimated from type ii ecg ( ecg 100c ; biopac systems , goleta , ca , usa ) in all experiments .", "icg ( nico 100c ; biopac systems ) was performed using eight spot electrodes ( vitrode m ; nihon kohden , tokyo , japan ) attached to the neck and lower thorax of each subject for icg based on a previous study.26 sv was estimated from transthoracic bioimpedance using bernstein s method that has been validated as offering high reliability comparable to the technique of thermodilution.26 icg allows continuous noninvasive evaluation of sv .", "noninvasive continuous arterial pressure was measured in the radial artery by tonometry using an arterial pressure monitor ( bp-608ev ; omron colin , tokyo , japan ) .", "mean arterial pressure ( map ) was estimated as one third pulse pressure plus diastolic pressure .", "ecg , thoracic bioimpedance , and continuous arterial pressure signals were recorded on a personal computer using the mp150 data acquisition system ( biopac systems ) at a sampling rate of 1 khz .", "we performed frequency domain and time domain analysis of ecg for the assessment of vagal function .", "we calculated logarithm - transformed respiratory sinus arrhythmia ( ln rsa ) from the power spectrum of the rr intervals ( 0.150.4 hz ) .", "we thus calculated the square root of the mean of the sum of the squares of differences between adjacent rr intervals ( rmssd ) , number of pairs of adjacent rr intervals differing by more than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of rr intervals ( pnn50 ) for 5 min .", "changes in hemodynamic variables from supine and hr variability parameters in the three positions in subjects from the two age groups were compared by two - way mixed - design analysis of variance ( anova ) .", "when three positions  two age group interactions were significant , one - way repeated - measures anova of the three positions in each subject and one - way anova of subjects from the two age groups in each position were used to evaluate simple main effects .", "subsequent post hoc analysis to determine differences between the three positions in each age group were evaluated using a paired t - test with shaffer s multiple comparison procedure in each age group.29 the level of statistical significance was set at p<0.05 .", "data were statistically analyzed using r for windows version 3.3.0 statistical software ( www.r-project.org ) and the car 2.14 package .", "figure 2 shows the results of changes in hemodynamic variables from supine at all positions . change in hr ( hr ) increased and change in sv ( sv ) decreased with the change from supine to an upright trunk in younger and older subjects .", "two - way mixed - design anova of hr and sv revealed significant main effects of the three positions ( hr , f = 31.73 ; sv , f = 15.73 ; p<0.05 ) , but no significant main effects of the two age groups ( p>0.05 ) and no significant three positions  two age groups interactions ( p>0.05 ) .", "multiple comparisons for each age group showed that hr ( ut60 and wt60 in younger , 2.91.2 beats / min and 6.61.4 beats / min , respectively ; ut60 and wt60 in older , 2.80.7 beats / min and 5.00.9 beats / min , respectively ) was significantly lower at ut60 than at wt60 and at supine than at ut60 and wt60 .", "in addition , sv ( ut60 and wt60 in younger , 4.42.8 ml and 13.72.9 ml , respectively ; ut60 and wt60 in older , 0.33.3 ml and 7.43.1 ml , respectively ) was significantly higher at ut60 and supine than at wt60 in older and younger subjects ( p<0.05 ) .", "anova for changes in q ( q : ut60 and wt60 in younger , 0.10.2 l and 0.30.2 l , respectively ; ut60 and wt60 in older , 0.20.2 l and 0.00.2 l , respectively ) revealed a significant main effect of the three positions ( q , f = 3.55 , p<0.05 ) but no significant main effect of the two age groups ( p>0.05 ) and no significant interaction of three positions  two age groups ( p>0.05 ) .", "multiple comparisons among each age group showed no significant differences between the three positions ( p>0.05 ) .", "anova of changes in map ( map : ut60 and wt60 in younger , 1.21.9 mmhg and 4.24.0 mmhg , respectively ; ut60 and wt60 in older , 0.52.8 mmhg and 2.72.6 mmhg , respectively ) revealed no significant main effects for the three positions ( p>0.05 ) or two age groups ( p>0.05 ) and no significant interaction of the three positions  two age groups ( p>0.05 ) .", "although all parameters decreased with the change from supine to upright trunk in younger subjects , significant differences were not seen between all positions in older subjects .", "these parameters in older subjects were lower compared with younger subjects for each position ( table 2 ) .", "two - way mixed - design anova of ln rsa , rmssd , nn50 , and pnn50 revealed significant main effects of the three positions ( p<0.05 ) and two age groups ( p<0.05 ) but significant three positions  two age groups interactions ( p<0.05 ) .", "post hoc analysis revealed significant simple main effects of the three positions in younger subjects ( ln rsa in younger , f = 8.67 ; rmssd in younger , f = 6.71 ; nn50 in younger , f = 5.95 ; pnn50 in younger , f = 7.12 ; p<0.05 ) but no significant simple main effect of the three positions in older subjects ( p>0.05 ) and significant simple main effects of the two age groups among each position ( ln rsa of supine , f = 34.6 ; rmssd of supine , f = 25.18 ; nn50 of supine , f = 27.45 ; pnn50 of supine , f = 29.77 ; ln rsa of ut60 , f = 24.72 ; rmssd of ut60 , f = 19.87 ; nn50 of ut60 , f = 20.43 ; pnn50 of ut60 , f = 19.46 ; ln rsa of wt60 , f = 14.36 ; rmssd of wt60 , f = 4.95 ; nn50 of wt60 , f = 6.59 ; pnn50 of wt60 , f = 6.12 ; p<0.05 ) .", "multiple comparisons among the three positions showed that all hr variability parameters were significantly higher at ut60 and supine position than at wt60 in younger subjects ( p<0.05 ) .", "figure 2 shows the results of changes in hemodynamic variables from supine at all positions . change in hr ( hr ) increased and change in sv ( sv ) decreased with the change from supine to an upright trunk in younger and older subjects .", "two - way mixed - design anova of hr and sv revealed significant main effects of the three positions ( hr , f = 31.73 ; sv , f = 15.73 ; p<0.05 ) , but no significant main effects of the two age groups ( p>0.05 ) and no significant three positions  two age groups interactions ( p>0.05 ) .", "multiple comparisons for each age group showed that hr ( ut60 and wt60 in younger , 2.91.2 beats / min and 6.61.4 beats / min , respectively ; ut60 and wt60 in older , 2.80.7 beats / min and 5.00.9 beats / min , respectively ) was significantly lower at ut60 than at wt60 and at supine than at ut60 and wt60 .", "in addition , sv ( ut60 and wt60 in younger , 4.42.8 ml and 13.72.9 ml , respectively ; ut60 and wt60 in older , 0.33.3 ml and 7.43.1 ml , respectively ) was significantly higher at ut60 and supine than at wt60 in older and younger subjects ( p<0.05 ) .", "anova for changes in q ( q : ut60 and wt60 in younger , 0.10.2 l and 0.30.2 l , respectively ; ut60 and wt60 in older , 0.20.2 l and 0.00.2 l , respectively ) revealed a significant main effect of the three positions ( q , f = 3.55 , p<0.05 ) but no significant main effect of the two age groups ( p>0.05 ) and no significant interaction of three positions  two age groups ( p>0.05 ) .", "multiple comparisons among each age group showed no significant differences between the three positions ( p>0.05 ) .", "anova of changes in map ( map : ut60 and wt60 in younger , 1.21.9 mmhg and 4.24.0 mmhg , respectively ; ut60 and wt60 in older , 0.52.8 mmhg and 2.72.6 mmhg , respectively ) revealed no significant main effects for the three positions ( p>0.05 ) or two age groups ( p>0.05 ) and no significant interaction of the three positions  two age groups ( p>0.05 ) .", "although all parameters decreased with the change from supine to upright trunk in younger subjects , significant differences were not seen between all positions in older subjects .", "these parameters in older subjects were lower compared with younger subjects for each position ( table 2 ) .", "two - way mixed - design anova of ln rsa , rmssd , nn50 , and pnn50 revealed significant main effects of the three positions ( p<0.05 ) and two age groups ( p<0.05 ) but significant three positions  two age groups interactions ( p<0.05 ) .", "post hoc analysis revealed significant simple main effects of the three positions in younger subjects ( ln rsa in younger , f = 8.67 ; rmssd in younger , f = 6.71 ; nn50 in younger , f = 5.95 ; pnn50 in younger , f = 7.12 ; p<0.05 ) but no significant simple main effect of the three positions in older subjects ( p>0.05 ) and significant simple main effects of the two age groups among each position ( ln rsa of supine , f = 34.6 ; rmssd of supine , f = 25.18 ; nn50 of supine , f = 27.45 ; pnn50 of supine , f = 29.77 ; ln rsa of ut60 , f = 24.72 ; rmssd of ut60 , f = 19.87 ; nn50 of ut60 , f = 20.43 ; pnn50 of ut60 , f = 19.46 ; ln rsa of wt60 , f = 14.36 ; rmssd of wt60 , f = 4.95 ; nn50 of wt60 , f = 6.59 ; pnn50 of wt60 , f = 6.12 ; p<0.05 ) .", "multiple comparisons among the three positions showed that all hr variability parameters were significantly higher at ut60 and supine position than at wt60 in younger subjects ( p<0.05 ) .", "the major findings of the present study were as follows : 1 ) the reductions in sv and tachycardic response were smaller with the upper trunk upright compared with the whole trunk upright , in healthy young and older individuals , and 2 ) the reduced tachycardic response with the upright upper trunk would be caused by decreased vagal withdrawal in younger subjects and reduced sympathetic activation in older subjects .", "orthostatic stress reportedly occurs with the shift in blood volume to the abdomen and legs in healthy individuals.30,31 at ut60 , subdivision of the trunk into upper and lower segments was defined by the spinous process of the 10th thoracic vertebra , under the presumption that since the heart is located close to the center of rotation of the bottom of the dorsal thorax , even if the upper trunk is upright , the level of the heart is lower with ut60 than with wt60 . in addition , the abdomen and pelvic segments at ut60 were not upright compared with wt60 .", "orthostatic stress might therefore be smaller at ut60 than at wt60 , suggesting differences in the distribution of the blood volume shift to lower parts of the body at ut60 compared to wt60 in both subject groups .", "moreover , these results were observed to be similar to the findings of our previous study in healthy young subjects.25 some previous studies have reported the effects of advancing age on sv under orthostatic stress induced by lower body negative pressure ( lbnp ) .", "these previous studies revealed that the reduction of sv was significantly lower among older individuals than among younger individuals under higher lbnp , although changes in sv did not differ between age groups under lower level lbnp.32,33 tsutsui et al . indicated that hypovolemia was less among older subjects than among the younger during higher level lbnp because leg compliance in the older subjects was reduced according to decreased capacitance function with age.33 with the present findings , significant differences in sv between age groups with the two fowler s positions were not observed .", "these results appear similar to previous findings during lower level lbnp but appear to differ from previous findings for higher - level lbnp.33 in fowler s position , since only the trunk and head were upright and the lower extremities were similar to the level in the supine position , orthostatic stress would be limited .", "differences in leg compliance with aging were thus indicated to have no effect on differences in sv between younger and older subjects , because the shift in blood volume to the lower extremities was regarded as too small . in this study ,", "sv was less than approximately 14 ml during the two fowler s positions in both age groups .", "positive hr occurred with the change from supine to upright trunk and hr was lower at ut60 than at wt60 in both age groups .", "these tachycardic responses were suggested to be the result of the negative sv being lower at ut60 than at wt60 .", "however , since all hr variability parameters in older subjects differed from those of younger subjects in the three positions , the mechanisms underlying the tachycardic responses reflected in the present results were suggested to differ between younger and older subjects .", "generally , tachycardic response less than 100 beats / min is attributed to vagal withdrawal.34 we therefore concluded that the tachycardic responses in the present results were caused by vagal withdrawal in younger subjects .", "in addition , vagal withdrawal would be lower at ut60 than at wt60 because ln rsa , rmssd , nn50 , and pnn50 were higher at ut60 compared with wt60 in younger subjects .", "however , no significant differences were seen among all positions and ln rsa , rmssd , nn50 , and pnn50 were lower in older subjects than in younger subjects in each position .", "vagal function is well known to reduce with age.912 these results suggested that vagal function was reduced in older subjects and tachycardic response was caused by mechanisms other than vagal withdrawal .", "nevertheless , the tachycardic response was significantly different between the two fowler s positions in older subjects .", "some previous studies have indicated that sympathetic nerve function and sympathetic baroreflex function are maintained with age.10,32,3537 the tachycardic response of older subjects with an upright trunk might thus be suggested to result from sympathetic activation in the present study , rather than vagal withdrawal . from the above , compared with wt60 , ut60 might inhibit decreases in sv and inhibit tachycardic response by reducing vagal withdrawal in younger subjects and by reducing sympathetic activation in older subjects .", "although sv was higher at ut60 than at wt60 in both age groups , q and map did not differ between the two fowler s positions in either age group or between the two age groups in either fowler s position .", "these results suggest that the tachycardic response and vasomotor contraction response maintained q and map to compensate for reduced sv under orthostatic stress regardless of age .", "such findings may be attributed to the regulation of tachycardic response and vasomotor contraction via vagal withdrawal in younger individuals and via sympathetic activation in older individuals .", "our findings indicate that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic response compared to an upright whole trunk regardless of age , although the autonomic mechanisms underlying tachycardic responses differed between younger and older adults . an upright upper trunk in fowler s position might help to reduce orthostatic stress and facilitate routine activities and conversation in frail patients .", "some studies have suggested that leg compression bandaging could prevent orthostatic hypotension in older patients.2,38 although that method might indeed be effective , that approach requires a more time - consuming procedure and the patient may feel a sense of uncomfortable restriction from the bandaging .", "our proposed method offers the advantages that the procedure to tilt the upper trunk is very simple and patients do not require compression bandaging or use of stockings to maintain circulatory volume when the patient wants to raise the body on the bed .", "the present results suggest that reduced tachycardic response with an upright upper trunk may be regulated by reduced sympathetic activation compared with having the whole trunk upright during fowler s position in older subjects .", "however , the present study did not directly assess sympathetic nerve activity in the two fowler s positions or while supine . to corroborate our findings ,", "confirmation by direct measurement of sympathetic nerve activity or venous plasma norepinephrine concentration in fowler s positions in younger and older subjects may be necessary . in consideration of using a fowler s position such as ut60 at clinical sites , validation in patients with orthostatic intolerance", "is needed because the influences of an upright upper trunk on hemodynamics and cardiovascular regulation in such patients have yet to be completely clarified .", "we demonstrated that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic responses compared to having the whole trunk upright in both younger and older subjects .", "such inhibition of tachycardic responses might be caused by reduced vagal withdrawal in younger subjects and by reduced sympathetic activation in older subjects .", "an upright upper trunk during fowler s position might be useful to maintain circulatory volume for reduced orthostatic stress and to facilitate daily activities in frail patients at clinical sites ."], "abstract_text": ["<S> backgrounddownward shifts in blood volume with changing position generally cause tachycardic responses . </S>", "<S> age - related decreases in vagal nerve activity could contribute to orthostatic hypotension in older individuals . </S>", "<S> fowler s position is a reclined position with the back between 30 and 60 , used to facilitate breathing , eating , and other routine daily activities in frail and elderly patients.objectivethis study examined whether stroke volume ( sv ) was higher and heart rate ( hr ) lower in fowler s position with an upright upper trunk than in fowler s position with the whole trunk upright in both older and younger subjects , based on the assumption that lower hr would result from reduced sympathetic activation in older individuals.methodswe assessed hemodynamics and hr variability from electrocardiography , noninvasive arterial pressure and impedance cardiography in 11 younger male subjects ( age range , 2022 years ) and 11 older male subjects ( age range , 6479 years ) , using three positions : supine , or fowler s positions with either 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) or 60 of whole trunk inclination ( wt60 ) . </S>", "<S> comparisons were then made between age groups and between positions.resultsreductions in sv and tachycardic response were smaller with ut60 than with wt60 , in both younger and older subjects . </S>", "<S> in addition , reduced tachycardic response with upright upper trunk appeared attributable to decreased vagal withdrawal in younger subjects and to reduced sympathetic activation in older subjects.conclusionour findings indicate that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic response compared to an upright whole trunk regardless of age , although the autonomic mechanisms underlying tachycardic responses differed between younger and older adults . </S>", "<S> an upright upper trunk in fowler s position might help to reduce orthostatic stress and facilitate routine activities and conversation in frail patients . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Subjects", "Procedure", "Measurements", "Statistical analysis", "Results", "Hemodynamics", "HR variability", "Discussion", "Limitations", "Conclusion"], "sections": [["bed rest , heart failure , depletion of intravascular volume , and aging often induce hypotension when the body or trunk is upright.15 generally , a tachycardic response occurs due to vagal withdrawal according to the downward blood shift that occurs under orthostatic stress in healthy humans,6 although orthostatic hypotension presents as an excessive drop in cardiac output ( q ) and lower tachycardic response in frail and elderly patients as a consequence of inadequate vascular contraction or autonomic dysfunction.7,8 numerous studies have suggested that advancing age is associated with decreases in vagal nerve activity.912 age - related decreases in vagal nerve activity have been suggested as contributing factors for orthostatic hypotension in elderly individuals.7,13 orthostatic hypotension has been reported in more than 60% of frail older patients in an acute geriatric ward.5 previous studies have observed orthostatic hypotension in 54% of seated older inpatients resting on a bed for more than 12 h.14 in light of these facts , methods of preventing hypovolemia and hypotension during orthostatic stress need to be investigated .", "pointed out that studies of orthostatic hypotension in frail patients , such as those with decompensated heart failure , are scarce and preventive interventions remain insufficient.2 clinically , frail patients are placed in fowler s position or a semiseated position on the bed .", "these positions are often clinically used without distinction between acute- or chronic - phase illness .", "the patient is placed in a reclined position with the backrest between 30 and 60 and with flexed or straight knees in fowler s position.15,16 breathing , eating , and other routine daily activities are considered to be facilitated in fowler s position.15,1720 some studies have suggested a relationship between trunk angle and hemodynamics during fowler s position.2124 cicolini et al . reported that blood pressure in fowler s position was intermediate between the seated and supine positions in both young healthy individuals and hypertensive patients.23,24 those studies indicated that the effects of orthostatic stress on hemodynamics in fowler s position may be substantial in frail patients .", "how posture can prevent the hypovolemia and hypotension caused by orthostatic stress during fowler s position is important and should be addressed .", "our previous study indicated that fowler s position with an upright upper trunk is effective for maintaining circulatory volume with lower tachycardia via vagal withdrawal compared with having the whole trunk upright in younger subjects.25 however , whether an upright upper trunk in fowler s position allows easy maintenance of circulatory volume in cases with autonomic hypofunction associated with aging remains unclear .", "we postulated that stroke volume ( sv ) would be higher and heart rate ( hr ) would be lower with the upper trunk upright than with the whole trunk upright in fowler s position in both older and younger subjects .", "in addition , we presumed that the lower hr would be caused by reduced sympathetic activation in older individuals but by reduced vagal withdrawal in younger individuals .", "the present study was performed as a cross - sectional study to assess our hypothesis with a fundamental study of the effects of the two fowler s positions on hemodynamics and hr variability parameters in healthy older and younger subjects ."], ["we assessed hemodynamics and hr variability in 11 male younger subjects ( mean age  standard error of the mean [ sem ] : 20.50.2 years ; range : 2022 years ; weight : 60.61.8 kg ; height : 170.21.8 cm ) and 11 male community - dwelling older subjects ( mean age  sem : 69.51.2 years ; range : 6479 years ; weight : 62.02.1 kg ; height : 165.41.8 cm ) .", "all subjects were interviewed regarding medical history , and blood pressure was checked after a period of rest .", "final decisions on subject selections were made by consultations between a medical doctor and researchers .", "potential subjects were excluded when they had a history of cardiovascular disease , respiratory disease or other chronic medical disease , or were not normotensive ( systolic bold pressure below 140 mmhg and diastolic blood pressure below 90 mmhg ) .", "all subjects in the final analysis were healthy and had no history of chronic or acute cardiovascular or respiratory disease or other chronic diseases .", "subjects were instructed to refrain from beverages containing caffeine or alcohol for 24 h before starting experiments and to refrain from eating and drinking after 2,200 h on the day prior to the experiments that started in the morning or to consume a light breakfast before experiments that started in the afternoon .", "all experiments were implemented between 1,100 and 1,400 h , because blood pressure fluctuates according to a circadian pattern stabilizing at approximately noon .", "the ethics commission of the international university of health and welfare approved the study , and all participants provided voluntary written consent to participate after being fully informed about the procedure , risks , and protocol .", "we collected physiological data with the patient in the supine position and two fowler s positions . prior to experiments", ", subjects rested in a thermoneutral room at 28c for 15 min and were set up for electrocardiography ( ecg ) , noninvasive arterial pressure monitoring , and impedance cardiography ( icg ) .", "measurements in the supine position were made with sufficient time between measurements in the two fowler s positions . between measurements in each fowler s position , subjects rested for more than 3 min .", "data were recorded for 5 min in each of the supine position and the two fowler s positions .", "subjects were placed in the fowler s positions on a bed at 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) and 60 of whole trunk inclination ( wt60 ) ( figure 1 ) .", "we defined the upper and lower segments at ut60 around the spinous process of the 10th thoracic vertebra .", "the height of the bottom of the upper and lower trunk was adjusted according to individual trunk size .", "all experiments were performed in our laboratory ( odawara , kanagawa , japan ) under controlled thermoneutral temperature .", "hr was estimated from type ii ecg ( ecg 100c ; biopac systems , goleta , ca , usa ) in all experiments .", "icg ( nico 100c ; biopac systems ) was performed using eight spot electrodes ( vitrode m ; nihon kohden , tokyo , japan ) attached to the neck and lower thorax of each subject for icg based on a previous study.26 sv was estimated from transthoracic bioimpedance using bernstein s method that has been validated as offering high reliability comparable to the technique of thermodilution.26 icg allows continuous noninvasive evaluation of sv .", "noninvasive continuous arterial pressure was measured in the radial artery by tonometry using an arterial pressure monitor ( bp-608ev ; omron colin , tokyo , japan ) .", "mean arterial pressure ( map ) was estimated as one third pulse pressure plus diastolic pressure .", "ecg , thoracic bioimpedance , and continuous arterial pressure signals were recorded on a personal computer using the mp150 data acquisition system ( biopac systems ) at a sampling rate of 1 khz .", "we performed frequency domain and time domain analysis of ecg for the assessment of vagal function .", "we calculated logarithm - transformed respiratory sinus arrhythmia ( ln rsa ) from the power spectrum of the rr intervals ( 0.150.4 hz ) .", "we thus calculated the square root of the mean of the sum of the squares of differences between adjacent rr intervals ( rmssd ) , number of pairs of adjacent rr intervals differing by more than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of rr intervals ( pnn50 ) for 5 min .", "all parameters of hr variability reflect vagal modulation.27,28 changes in hemodynamic variables from supine and hr variability parameters in the three positions in subjects from the two age groups were compared by two - way mixed - design analysis of variance ( anova ) .", "when three positions  two age group interactions were significant , one - way repeated - measures anova of the three positions in each subject and one - way anova of subjects from the two age groups in each position were used to evaluate simple main effects .", "subsequent post hoc analysis to determine differences between the three positions in each age group were evaluated using a paired t - test with shaffer s multiple comparison procedure in each age group.29 the level of statistical significance was set at p<0.05 .", "data were statistically analyzed using r for windows version 3.3.0 statistical software ( www.r-project.org ) and the car 2.14 package ."], ["we assessed hemodynamics and hr variability in 11 male younger subjects ( mean age  standard error of the mean [ sem ] : 20.50.2 years ; range : 2022 years ; weight : 60.61.8 kg ; height : 170.21.8 cm ) and 11 male community - dwelling older subjects ( mean age  sem : 69.51.2 years ; range : 6479 years ; weight : 62.02.1 kg ; height : 165.41.8 cm ) .", "all subjects were interviewed regarding medical history , and blood pressure was checked after a period of rest .", "final decisions on subject selections were made by consultations between a medical doctor and researchers .", "potential subjects were excluded when they had a history of cardiovascular disease , respiratory disease or other chronic medical disease , or were not normotensive ( systolic bold pressure below 140 mmhg and diastolic blood pressure below 90 mmhg ) .", "all subjects in the final analysis were healthy and had no history of chronic or acute cardiovascular or respiratory disease or other chronic diseases .", "subjects were instructed to refrain from beverages containing caffeine or alcohol for 24 h before starting experiments and to refrain from eating and drinking after 2,200 h on the day prior to the experiments that started in the morning or to consume a light breakfast before experiments that started in the afternoon .", "all experiments were implemented between 1,100 and 1,400 h , because blood pressure fluctuates according to a circadian pattern stabilizing at approximately noon .", "the ethics commission of the international university of health and welfare approved the study , and all participants provided voluntary written consent to participate after being fully informed about the procedure , risks , and protocol ."], ["we collected physiological data with the patient in the supine position and two fowler s positions . prior to experiments", ", subjects rested in a thermoneutral room at 28c for 15 min and were set up for electrocardiography ( ecg ) , noninvasive arterial pressure monitoring , and impedance cardiography ( icg ) .", "measurements in the supine position were made with sufficient time between measurements in the two fowler s positions . between measurements in each fowler s position , subjects rested for more than 3 min .", "data were recorded for 5 min in each of the supine position and the two fowler s positions .", "subjects were placed in the fowler s positions on a bed at 30 of lower trunk inclination and 60 of upper trunk inclination ( ut60 ) and 60 of whole trunk inclination ( wt60 ) ( figure 1 ) .", "we defined the upper and lower segments at ut60 around the spinous process of the 10th thoracic vertebra .", "the height of the bottom of the upper and lower trunk was adjusted according to individual trunk size .", "all experiments were performed in our laboratory ( odawara , kanagawa , japan ) under controlled thermoneutral temperature ."], ["hr was estimated from type ii ecg ( ecg 100c ; biopac systems , goleta , ca , usa ) in all experiments .", "icg ( nico 100c ; biopac systems ) was performed using eight spot electrodes ( vitrode m ; nihon kohden , tokyo , japan ) attached to the neck and lower thorax of each subject for icg based on a previous study.26 sv was estimated from transthoracic bioimpedance using bernstein s method that has been validated as offering high reliability comparable to the technique of thermodilution.26 icg allows continuous noninvasive evaluation of sv .", "noninvasive continuous arterial pressure was measured in the radial artery by tonometry using an arterial pressure monitor ( bp-608ev ; omron colin , tokyo , japan ) .", "mean arterial pressure ( map ) was estimated as one third pulse pressure plus diastolic pressure .", "ecg , thoracic bioimpedance , and continuous arterial pressure signals were recorded on a personal computer using the mp150 data acquisition system ( biopac systems ) at a sampling rate of 1 khz .", "we performed frequency domain and time domain analysis of ecg for the assessment of vagal function .", "we calculated logarithm - transformed respiratory sinus arrhythmia ( ln rsa ) from the power spectrum of the rr intervals ( 0.150.4 hz ) .", "we thus calculated the square root of the mean of the sum of the squares of differences between adjacent rr intervals ( rmssd ) , number of pairs of adjacent rr intervals differing by more than 50 ms ( nn50 ) and the proportion derived by dividing nn50 by the total number of rr intervals ( pnn50 ) for 5 min ."], ["changes in hemodynamic variables from supine and hr variability parameters in the three positions in subjects from the two age groups were compared by two - way mixed - design analysis of variance ( anova ) .", "when three positions  two age group interactions were significant , one - way repeated - measures anova of the three positions in each subject and one - way anova of subjects from the two age groups in each position were used to evaluate simple main effects .", "subsequent post hoc analysis to determine differences between the three positions in each age group were evaluated using a paired t - test with shaffer s multiple comparison procedure in each age group.29 the level of statistical significance was set at p<0.05 .", "data were statistically analyzed using r for windows version 3.3.0 statistical software ( www.r-project.org ) and the car 2.14 package ."], ["figure 2 shows the results of changes in hemodynamic variables from supine at all positions . change in hr ( hr ) increased and change in sv ( sv ) decreased with the change from supine to an upright trunk in younger and older subjects .", "two - way mixed - design anova of hr and sv revealed significant main effects of the three positions ( hr , f = 31.73 ; sv , f = 15.73 ; p<0.05 ) , but no significant main effects of the two age groups ( p>0.05 ) and no significant three positions  two age groups interactions ( p>0.05 ) .", "multiple comparisons for each age group showed that hr ( ut60 and wt60 in younger , 2.91.2 beats / min and 6.61.4 beats / min , respectively ; ut60 and wt60 in older , 2.80.7 beats / min and 5.00.9 beats / min , respectively ) was significantly lower at ut60 than at wt60 and at supine than at ut60 and wt60 .", "in addition , sv ( ut60 and wt60 in younger , 4.42.8 ml and 13.72.9 ml , respectively ; ut60 and wt60 in older , 0.33.3 ml and 7.43.1 ml , respectively ) was significantly higher at ut60 and supine than at wt60 in older and younger subjects ( p<0.05 ) .", "anova for changes in q ( q : ut60 and wt60 in younger , 0.10.2 l and 0.30.2 l , respectively ; ut60 and wt60 in older , 0.20.2 l and 0.00.2 l , respectively ) revealed a significant main effect of the three positions ( q , f = 3.55 , p<0.05 ) but no significant main effect of the two age groups ( p>0.05 ) and no significant interaction of three positions  two age groups ( p>0.05 ) .", "multiple comparisons among each age group showed no significant differences between the three positions ( p>0.05 ) .", "anova of changes in map ( map : ut60 and wt60 in younger , 1.21.9 mmhg and 4.24.0 mmhg , respectively ; ut60 and wt60 in older , 0.52.8 mmhg and 2.72.6 mmhg , respectively ) revealed no significant main effects for the three positions ( p>0.05 ) or two age groups ( p>0.05 ) and no significant interaction of the three positions  two age groups ( p>0.05 ) .", "although all parameters decreased with the change from supine to upright trunk in younger subjects , significant differences were not seen between all positions in older subjects .", "these parameters in older subjects were lower compared with younger subjects for each position ( table 2 ) .", "two - way mixed - design anova of ln rsa , rmssd , nn50 , and pnn50 revealed significant main effects of the three positions ( p<0.05 ) and two age groups ( p<0.05 ) but significant three positions  two age groups interactions ( p<0.05 ) .", "post hoc analysis revealed significant simple main effects of the three positions in younger subjects ( ln rsa in younger , f = 8.67 ; rmssd in younger , f = 6.71 ; nn50 in younger , f = 5.95 ; pnn50 in younger , f = 7.12 ; p<0.05 ) but no significant simple main effect of the three positions in older subjects ( p>0.05 ) and significant simple main effects of the two age groups among each position ( ln rsa of supine , f = 34.6 ; rmssd of supine , f = 25.18 ; nn50 of supine , f = 27.45 ; pnn50 of supine , f = 29.77 ; ln rsa of ut60 , f = 24.72 ; rmssd of ut60 , f = 19.87 ; nn50 of ut60 , f = 20.43 ; pnn50 of ut60 , f = 19.46 ; ln rsa of wt60 , f = 14.36 ; rmssd of wt60 , f = 4.95 ; nn50 of wt60 , f = 6.59 ; pnn50 of wt60 , f = 6.12 ; p<0.05 ) .", "multiple comparisons among the three positions showed that all hr variability parameters were significantly higher at ut60 and supine position than at wt60 in younger subjects ( p<0.05 ) ."], ["figure 2 shows the results of changes in hemodynamic variables from supine at all positions . change in hr ( hr ) increased and change in sv ( sv ) decreased with the change from supine to an upright trunk in younger and older subjects .", "two - way mixed - design anova of hr and sv revealed significant main effects of the three positions ( hr , f = 31.73 ; sv , f = 15.73 ; p<0.05 ) , but no significant main effects of the two age groups ( p>0.05 ) and no significant three positions  two age groups interactions ( p>0.05 ) .", "multiple comparisons for each age group showed that hr ( ut60 and wt60 in younger , 2.91.2 beats / min and 6.61.4 beats / min , respectively ; ut60 and wt60 in older , 2.80.7 beats / min and 5.00.9 beats / min , respectively ) was significantly lower at ut60 than at wt60 and at supine than at ut60 and wt60 .", "in addition , sv ( ut60 and wt60 in younger , 4.42.8 ml and 13.72.9 ml , respectively ; ut60 and wt60 in older , 0.33.3 ml and 7.43.1 ml , respectively ) was significantly higher at ut60 and supine than at wt60 in older and younger subjects ( p<0.05 ) .", "anova for changes in q ( q : ut60 and wt60 in younger , 0.10.2 l and 0.30.2 l , respectively ; ut60 and wt60 in older , 0.20.2 l and 0.00.2 l , respectively ) revealed a significant main effect of the three positions ( q , f = 3.55 , p<0.05 ) but no significant main effect of the two age groups ( p>0.05 ) and no significant interaction of three positions  two age groups ( p>0.05 ) .", "multiple comparisons among each age group showed no significant differences between the three positions ( p>0.05 ) .", "anova of changes in map ( map : ut60 and wt60 in younger , 1.21.9 mmhg and 4.24.0 mmhg , respectively ; ut60 and wt60 in older , 0.52.8 mmhg and 2.72.6 mmhg , respectively ) revealed no significant main effects for the three positions ( p>0.05 ) or two age groups ( p>0.05 ) and no significant interaction of the three positions  two age groups ( p>0.05 ) ."], ["although all parameters decreased with the change from supine to upright trunk in younger subjects , significant differences were not seen between all positions in older subjects .", "these parameters in older subjects were lower compared with younger subjects for each position ( table 2 ) .", "two - way mixed - design anova of ln rsa , rmssd , nn50 , and pnn50 revealed significant main effects of the three positions ( p<0.05 ) and two age groups ( p<0.05 ) but significant three positions  two age groups interactions ( p<0.05 ) .", "post hoc analysis revealed significant simple main effects of the three positions in younger subjects ( ln rsa in younger , f = 8.67 ; rmssd in younger , f = 6.71 ; nn50 in younger , f = 5.95 ; pnn50 in younger , f = 7.12 ; p<0.05 ) but no significant simple main effect of the three positions in older subjects ( p>0.05 ) and significant simple main effects of the two age groups among each position ( ln rsa of supine , f = 34.6 ; rmssd of supine , f = 25.18 ; nn50 of supine , f = 27.45 ; pnn50 of supine , f = 29.77 ; ln rsa of ut60 , f = 24.72 ; rmssd of ut60 , f = 19.87 ; nn50 of ut60 , f = 20.43 ; pnn50 of ut60 , f = 19.46 ; ln rsa of wt60 , f = 14.36 ; rmssd of wt60 , f = 4.95 ; nn50 of wt60 , f = 6.59 ; pnn50 of wt60 , f = 6.12 ; p<0.05 ) .", "multiple comparisons among the three positions showed that all hr variability parameters were significantly higher at ut60 and supine position than at wt60 in younger subjects ( p<0.05 ) ."], ["the major findings of the present study were as follows : 1 ) the reductions in sv and tachycardic response were smaller with the upper trunk upright compared with the whole trunk upright , in healthy young and older individuals , and 2 ) the reduced tachycardic response with the upright upper trunk would be caused by decreased vagal withdrawal in younger subjects and reduced sympathetic activation in older subjects .", "orthostatic stress reportedly occurs with the shift in blood volume to the abdomen and legs in healthy individuals.30,31 at ut60 , subdivision of the trunk into upper and lower segments was defined by the spinous process of the 10th thoracic vertebra , under the presumption that since the heart is located close to the center of rotation of the bottom of the dorsal thorax , even if the upper trunk is upright , the level of the heart is lower with ut60 than with wt60 . in addition , the abdomen and pelvic segments at ut60 were not upright compared with wt60 .", "orthostatic stress might therefore be smaller at ut60 than at wt60 , suggesting differences in the distribution of the blood volume shift to lower parts of the body at ut60 compared to wt60 in both subject groups .", "moreover , these results were observed to be similar to the findings of our previous study in healthy young subjects.25 some previous studies have reported the effects of advancing age on sv under orthostatic stress induced by lower body negative pressure ( lbnp ) .", "these previous studies revealed that the reduction of sv was significantly lower among older individuals than among younger individuals under higher lbnp , although changes in sv did not differ between age groups under lower level lbnp.32,33 tsutsui et al . indicated that hypovolemia was less among older subjects than among the younger during higher level lbnp because leg compliance in the older subjects was reduced according to decreased capacitance function with age.33 with the present findings , significant differences in sv between age groups with the two fowler s positions were not observed .", "these results appear similar to previous findings during lower level lbnp but appear to differ from previous findings for higher - level lbnp.33 in fowler s position , since only the trunk and head were upright and the lower extremities were similar to the level in the supine position , orthostatic stress would be limited .", "differences in leg compliance with aging were thus indicated to have no effect on differences in sv between younger and older subjects , because the shift in blood volume to the lower extremities was regarded as too small . in this study ,", "sv was less than approximately 14 ml during the two fowler s positions in both age groups .", "positive hr occurred with the change from supine to upright trunk and hr was lower at ut60 than at wt60 in both age groups .", "these tachycardic responses were suggested to be the result of the negative sv being lower at ut60 than at wt60 .", "however , since all hr variability parameters in older subjects differed from those of younger subjects in the three positions , the mechanisms underlying the tachycardic responses reflected in the present results were suggested to differ between younger and older subjects .", "generally , tachycardic response less than 100 beats / min is attributed to vagal withdrawal.34 we therefore concluded that the tachycardic responses in the present results were caused by vagal withdrawal in younger subjects .", "in addition , vagal withdrawal would be lower at ut60 than at wt60 because ln rsa , rmssd , nn50 , and pnn50 were higher at ut60 compared with wt60 in younger subjects .", "however , no significant differences were seen among all positions and ln rsa , rmssd , nn50 , and pnn50 were lower in older subjects than in younger subjects in each position .", "vagal function is well known to reduce with age.912 these results suggested that vagal function was reduced in older subjects and tachycardic response was caused by mechanisms other than vagal withdrawal .", "nevertheless , the tachycardic response was significantly different between the two fowler s positions in older subjects .", "some previous studies have indicated that sympathetic nerve function and sympathetic baroreflex function are maintained with age.10,32,3537 the tachycardic response of older subjects with an upright trunk might thus be suggested to result from sympathetic activation in the present study , rather than vagal withdrawal . from the above , compared with wt60 , ut60 might inhibit decreases in sv and inhibit tachycardic response by reducing vagal withdrawal in younger subjects and by reducing sympathetic activation in older subjects .", "although sv was higher at ut60 than at wt60 in both age groups , q and map did not differ between the two fowler s positions in either age group or between the two age groups in either fowler s position .", "these results suggest that the tachycardic response and vasomotor contraction response maintained q and map to compensate for reduced sv under orthostatic stress regardless of age .", "such findings may be attributed to the regulation of tachycardic response and vasomotor contraction via vagal withdrawal in younger individuals and via sympathetic activation in older individuals .", "our findings indicate that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic response compared to an upright whole trunk regardless of age , although the autonomic mechanisms underlying tachycardic responses differed between younger and older adults . an upright upper trunk in fowler s position might help to reduce orthostatic stress and facilitate routine activities and conversation in frail patients .", "some studies have suggested that leg compression bandaging could prevent orthostatic hypotension in older patients.2,38 although that method might indeed be effective , that approach requires a more time - consuming procedure and the patient may feel a sense of uncomfortable restriction from the bandaging .", "our proposed method offers the advantages that the procedure to tilt the upper trunk is very simple and patients do not require compression bandaging or use of stockings to maintain circulatory volume when the patient wants to raise the body on the bed ."], ["the present results suggest that reduced tachycardic response with an upright upper trunk may be regulated by reduced sympathetic activation compared with having the whole trunk upright during fowler s position in older subjects .", "however , the present study did not directly assess sympathetic nerve activity in the two fowler s positions or while supine . to corroborate our findings ,", "confirmation by direct measurement of sympathetic nerve activity or venous plasma norepinephrine concentration in fowler s positions in younger and older subjects may be necessary . in consideration of using a fowler s position such as ut60 at clinical sites , validation in patients with orthostatic intolerance", "is needed because the influences of an upright upper trunk on hemodynamics and cardiovascular regulation in such patients have yet to be completely clarified ."], ["we demonstrated that an upright upper trunk during fowler s position allowed maintenance of sv and inhibited tachycardic responses compared to having the whole trunk upright in both younger and older subjects .", "such inhibition of tachycardic responses might be caused by reduced vagal withdrawal in younger subjects and by reduced sympathetic activation in older subjects .", "an upright upper trunk during fowler s position might be useful to maintain circulatory volume for reduced orthostatic stress and to facilitate daily activities in frail patients at clinical sites ."]]}
{"article_id": "PMC3014710", "article_text": ["alzheimer 's disease ( ad ) is a progressive , neurodegenerative brain disorder which affects over 37 million people worldwide with an estimated global cost of over $ 600 billion in 2010     [ 1 , 2 ] .", "ad is a growing socioeconomic and financial burden due to its strong correlation with ageing ; around 1 in 3 people aged over 80 years have ad , which means that a rapid rise in ad cases is anticipated as life expectancy continues to increase .", "although several therapeutics are currently available to slow disease progression , there is currently no way to halt or prevent ad .", "ad is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles in the brain .", "the major constituents of senile plaques are the amyloid- ( a ) peptides , which are derived from the proteolytic processing of the amyloid precursor protein ( app ) within lipid rafts .", "the a peptide , notably a142 , is highly aggregation prone and self - assembles to form a heterogeneous mixture of oligomers and protofibrils , ultimately depositing as fibrils in senile plaques .", "an accumulating body of evidence indicates that soluble a oligomers , which correlate strongly with disease onset and severity , are the major neurotoxic species in ad     [ 58 ] .", "although a oligomers are neurotoxic at nanomolar concentrations and cause ad - related memory deficits , the cellular mechanisms of toxicity are poorly characterised .", "recently , several neuronal receptors which bind a oligomers have been identified , including the cellular prion protein ( prp )      and glutamate receptors     [ 10 , 11 ] among others .", "interestingly , these receptors reside primarily within , or partition into , cholesterol - rich microdomains within the plasma membrane known as lipid rafts .  ", "the three steps which underlie a oligomer - mediated neuropathology in ad , are ( 1 ) a production , ( 2 ) a assembly into oligomers and ( 3 ) a oligomers interacting with neuronal receptors .", "crucially , all three of these processes occur in lipid raft domains of the plasma membrane which are considered to play a key role in the development of ad . in this paper", ", we will outline the pivotal role that lipid rafts play in linking together the generation , self - assembly and toxicity of a oligomers , which underlie the development of the neuropathology in ad .", "a major focus will be upon the interaction between a oligomers and their putative cellular receptors .", "the multitude of different lipids and proteins within the plasma membrane were once thought to be distributed homogeneously across the entire lipid bilayer , as proposed by the fluid mosaic model in 1972 . however , the plasma membrane is now known to be more akin to a sea of disordered phospholipids , in which float microdomains with distinct lipid compositions , known as lipid rafts .", "lipid rafts are small ( 10200  nm ) , heterogeneous and highly dynamic assemblies that are enriched in specific components , namely cholesterol and sphingolipids ( figure 1 )     [ 14 , 15 ] .", "biochemically , lipid rafts are defined by their relative insolubility in nonionic detergents at low temperature , conferring upon them the alternative name , detergent - resistant membranes ( drms ) .", "lipid rafts are also known as liquid - ordered domains because the highly saturated sphingolipid acyl chains enable closer lipid packing , and therefore more restricted lateral movement , than the mainly unsaturated acyl chains of the phospholipids in the surrounding nonraft regions of the membrane .", "functionally , lipid rafts serve to compartmentalise cellular processes by concentrating certain proteins and lipids within the same microenvironment .", "lipid rafts are particularly enriched in glycosyl - phosphatidylinositol ( gpi)-anchored and acylated proteins due to the preferential intercalation of the saturated acyl chains into the liquid - ordered environment .", "other proteins can also associate with lipid rafts either directly or through binding to other cofactors or ligands     .", "the dynamic clustering and pinching off of lipid rafts regulates the spatial and temporal assembly of signalling and trafficking molecules , forming short - lived but vital signalling platforms     .", "lipid rafts are implicated in various essential cellular functions , including signal transduction , cell adhesion and protein / lipid sorting .", "of particular relevance here are cell signalling , sorting and axon guidance , as these processes are essential for neural development and synaptic plasticity     [ 19 , 20 ] .", "crucially , neuronal lipid rafts are also required for the maintenance of dendritic spines and healthy synapses , which are vital for neural communication including learning and memory ; processes which fail in ad     .", "the observation that lipid rafts are much more abundant in mature hippocampal neurons than in other cell types emphasises their physiological importance within the memory centre of the healthy brain , and may explain why hippocampal neurons are a primary target for a oligomer toxicity and destruction in ad     .", "lipid rafts are involved in the regulation of app processing and the generation of the a peptide which is the driving force in ad pathology     [ 23 , 24 ] . for comprehensive reviews detailing the involvement of membrane rafts in ad and a production ,", "the a peptide is produced by the lipid raft dependent amyloidogenic processing of its precursor protein , app ( figure 1 ) .", "the amyloidogenic cleavage of full - length app is initiated by the -site app cleaving enzyme-1 ( bace1 ) , a transmembrane aspartic metalloprotease . a large", ", soluble ectodomain ( sapp ) is released to leave behind a membrane - anchored c - terminal fragment ( c99 ) which retains the intact a sequence .", "the second amyloidogenic cleavage of app involves a -secretase complex which contains presenilin-1 or presenilin-2 ( the catalytic component ) , presenilin enhancer-2 ( pen2 ) , nicastrin and anterior pharynx defective-1 ( aph1 ) .", "the -secretase complex cleaves the remaining c99 stub to release a peptides of between 3942 residues in length , depending upon the precise cleavage site , along with the app intracellular domain ( aicd ) .", "although the majority of full - length app is localised to nonraft regions of the plasma membrane , where nonamyloidogenic cleavage by the -secretases adam 9 , 10 , and 17      precludes a formation , a subset of both app and bace1 partitions into lipid rafts along with -secretase components .", "both bace1 and the -secretase subunits undergo posttranslational s - palmitoylation which aids their targeting to lipid raft domains . in the case of app ,", "a direct interaction with cholesterol  the major component of lipid rafts  was recently identified .", "high cholesterol increases the partitioning of app , along with bace1 and -secretase components , into lipid rafts     . a large body of evidence points towards lipid rafts being the physiological site of amyloidogenic a production by bace1 and the -secretase complex .", "for example , both the copatching of app and bace1 by cross - linking antibodies      and the exclusive targeting of bace1 to lipid rafts by the addition of a gpi - anchor      significantly increased app cleavage at the -secretase site .", "furthermore , enrichments in lipid raft components , namely cholesterol and ganglioside gm1 , promote the generation of a     [ 31 , 33 ] .", "all four of the -secretase subunits are also enriched and active within lipid raft fractions derived from human brain     [ 34 , 35 ] and lipid raft - type membranes in vitro     [ 36 , 37 ] . in the brain ,", "the majority of a is found within detergent - resistant , glycolipid - enriched rafts , along with -secretase components     .  ", "the composition of lipid rafts purified from ad brains has been shown to be abnormal , with the rafts being more ordered and more viscous     , which implies that the modulation of lipid raft composition may present a therapeutic avenue for modulating ad - related neuropathology .", "this has led to a number of researchers investigating whether depleting lipid raft components could lower a production and therefore prevent ad .", "cholesterol , being a major component of lipid rafts and a risk factor for ad , was the obvious choice to target . for a recent review of the involvement of cholesterol in ad ,", "cholesterol depletion has indeed been shown to reduce app partitioning into lipid rafts which precludes its interaction with bace1 and -secretase components , thus lowering a production     .", "hypercholesterolaemia is linked to increased a production and deposition in the brain , both in humans     [ 4345 ] and in rodents     [ 4648 ] and is linked to an increased risk of developing ad .", "cholesterol depletion also lowers a production in cultured cells      and one study showed that a 70% reduction in cholesterol in living hippocampal neurons was sufficient to completely abolish a production . taking this into account , cholesterol - lowering drugs known as", "some retrospective epidemiological studies have shown that the administration of statins , which lower cholesterol levels , can reduce the incidence of dementia , including ad     [ 5153 ] .", "however , other studies have shown no correlation between statin usage and dementia      and the effect of statins upon disease progression and cognitive decline in ad patients has been challenged . intriguingly , it was revealed recently that a production actually reduces cholesterol in cultured cells of neuronal origin by increasing efflux , possibly acting as a chaperone to remove excess cholesterol from the brain to the circulation .    ", "although a reduction in cholesterol may go some way towards reducing a levels in the brain , much longer - term epidemiological studies and clinical trials initiated before significant neuronal loss and cognitive function are apparent are required in order to further elucidate the effects of lowering cholesterol levels upon ad onset and neuropathology .", "lipid rafts contain many essential components other than cholesterol , such as sphingolipids , and it is likely that the modulation of just one factor will not completely abolish a production in vivo .", "it is important to remember that cholesterol metabolism in the brain is largely isolated from the rest of the body by the blood - brain barrier . as nearly all of the cholesterol in the brain", "is synthesised in situ , the modulation of cholesterol levels within neurons represents a more difficult pharmaceutical challenge and the blood - brain barrier permeability of the drugs used needs to be considered .", "furthermore , even if cholestxerol depletion mediates a reduction in a levels , a oligomers effect neurotoxicity and memory impairments at low nanomolar concentrations     .", "therefore , residual levels of a production may be sufficient for continued a oligomer - mediated toxicity .", "the a peptide is natively unfolded and , under certain conditions , it aggregates to form a heterogeneous mixture of soluble oligomers , protofibrils and fibrils .", "it was accepted for a long time that the a fibrils that deposit in neuritic plaques , which are observed post mortem in diseased brains , were responsible for neurotoxicity in ad .", "a fibrils have been reported to induce neuronal dysfunction and cell death , although fibrils are less potent neurotoxins than soluble forms of a     [ 60 , 61 ] .", "interestingly , fibrils have been found to become more neurotoxic upon fragmentation     , raising the possibility that soluble species released from fibril ends may underlie their neurotoxicity .", "a plethora of studies have now demonstrated that levels of soluble a oligomers in the brain correlate much better than plaques or fibrils with ad onset , progression and severity     [ 5 , 6 , 8 , 63 , 64 ] . within the last fifteen years , a large number of studies from research groups worldwide have reported the existence of many different oligomeric assemblies from various sources , including ad brain and cerebrospinal fluid ( csf ) samples , secreted into the conditioned medium of cultured cells or prepared artificially from recombinant or synthetic a peptides .", "a heterogeneous range of sizes and peptide conformations have been observed among these natural and artificial a oligomers , including dimers and trimers     [ 66 , 67 ] , tetramers , hexamers and the dodecameric a*56     , globulomers     , ring - shaped annular protofibrils   and higher molecular weight a-derived diffusible ligands ( addls ) which can comprise hundreds of monomeric subunits     [ 9 , 70 ] ( figure 2 ) . however , despite the disparity in size and source , a oligomers appear to share important functional properties .", "notably , both natural and synthetic a oligomer preparations bind to hippocampal neurons and cells of neuronal lineage , causing a loss of dendritic spines , neurotoxicity , the inhibition of long - term synaptic potentiation ( ltp : an electrophysiological correlate of learning and memory ) and impairments in working memory at nanomolar concentrations     [ 64 , 67 , 68 , 7073 ] .", "the preferential binding and toxicity of a oligomers towards neurons in the hippocampus may explain why a oligomers correlate with ad severity and disease progression     [ 9 , 68 , 70 ] .", "a is a physiological peptide which is present in the brain tissue and csf of healthy subjects throughout life , without necessarily causing neurodegeneration     [ 7476 ] .", "many studies have shown that monomeric , nonaggregated a does not cause the neurotoxic effects that are mediated by a oligomers .", "in fact , monomeric a has recently been reported to have neuroprotective roles in the brain     [ 77 , 78 ] .", "the aggregation of a is necessary for its toxicity   and the emerging picture is that soluble a oligomers are the proximate neurotoxins in ad     [ 8 , 80 ] .", "the aggregation of a is therefore a critical step in the development of ad pathogenesis , and one in which lipid rafts appear to play a fundamental role .", "neuronal sensitivity to a-induced toxicity has been found to be dependent upon a binding to the cell membrane      and a has been identified in lipid rafts from cultured cells and from human and rodent brains .", "soluble a dimers accumulate rapidly , and have been found at elevated levels , in lipid raft fractions isolated from human and transgenic mouse model ad brains     .", "importantly , a has been shown to accumulate in presynaptic terminals in ad cortex where it colocalises with the lipid raft markers cholesterol and ganglioside gm1 . taken together ,", "these data suggest that a accumulation and aggregation within lipid rafts may underlie ad neuropathology .", "as cholesterol is a major component of lipid rafts , it was postulated to facilitate a oligomerisation on neuronal membranes . the brain is particularly enriched in cholesterol , harbouring over 23% of the body 's total complement but comprising only around 2% of total body mass . however , the role of cholesterol in promoting the assembly of a is controversial and conflicting evidence has been presented in recent years .", "the main difficulty is being able to distinguish between the key role of cholesterol in building the lipid raft domains necessary for a production and the suggested role of cholesterol in promoting a oligomerisation .", "as discussed previously , raised cholesterol has been linked to ad ; is this solely due to an increase in total lipid raft composition of the plasma membrane which increases amyloidogenic processing of app to yield more a peptide or due to a direct effect on a oligomerisation ?  ", "a growing body of evidence suggests that certain components of lipid raft domains may play a much more sinister role in catalysing the conversion of the aggregation - prone a peptide to its neurotoxic , oligomeric states .", "cholesterol is known to modulate the interaction of the a peptide with lipid bilayers     .", "further , a oligomers isolated from ad patients associate with drms in a cholesterol - dependent manner , and cholesterol depletion reduces the aggregation of a     .", "it is currently unknown , however , whether this latter effect is due to a direct interaction between a and cholesterol , or due to the overall depletion in lipid raft domains and/or the subsequent change in composition and properties brought on by a reduction in cholesterol .", "conversely , a recent study revealed that increasing the level of cholesterol in human neuroblastoma cells actually reduced the ability of synthetic a oligomers to bind , in spite of the colocalisation of the a oligomers with the lipid raft component ganglioside gm1 .", "these data agree with the authors ' previous finding that an increased level of membrane cholesterol exerts a protective effect against a oligomer toxicity     . in the more recent study  ", "it was proposed that a fluctuation in cholesterol levels may alter the physical properties of lipid rafts thereby modulating oligomer binding .  ", "cholesterol can also facilitate a aggregation through the structural modification of other lipid raft components .", "a recent study using reconstituted membranes revealed a structural role for cholesterol in modulating the conformation of glycosphingolipids .", "depending on the type of glycosphingolipid , cholesterol can either facilitate ( such as for ganglioside gm1 ) or inhibit the interaction of a peptides with lipid rafts through fine - tuning of the glycosphingolipid conformation .", "this reinforces the notion that a binding to , and aggregation upon , neuronal lipid raft domains can not be ascribed to a single component , but rather that multiple players are likely to be involved .", "in fact , mounting evidence suggests that gangliosides within lipid rafts appear to be the main driving force behind the oligomerisation of a on neuronal membranes .", "the development of ad within certain brain regions has been found to correlate with increased ganglioside levels     .", "gangliosides are glycosphingolipids with one or more sialic acid moieties attached to the sugar chain .", "gangliosides are found predominantly in the central nervous system , where they are enriched in lipid rafts due to the preferential packing of their saturated acyl chains within the liquid - ordered phase . a study in 1995 revealed that a population of membrane - bound a tightly bound to gangliosides exists in ad brains .", "more recently , exogenously - applied a was shown to bind to neuronal membranes and to redistribute into lipid rafts where it colocalised with ganglioside gm1 in a time - dependent manner .", "gm1 facilitated the binding and accumulation of a oligomers at lipid raft domains and appeared to be required for the a oligomer - mediated lipid peroxidation of drms .", "ganglioside gm1 contains just one sialic acid moiety and plays important physiological roles in neuronal function .", "a appears to interact with the sialic acid moiety of gangliosides such as gm1 and these bound aggregates can go on to seed further a aggregation     .", "the interaction between sialic acid and a induces a conformational rearrangement of the a peptide chain   which may potentiate a oligomerisation .", "drms derived from ganglioside - rich rat brain , but not from liver , were found to promote the oligomerisation of a .", "further , this study revealed that the removal of cholesterol or protein from these raft fractions did not prevent a aggregation , providing evidence that neither cholesterol nor protein is essential for this process .", "however , lipid raft fractions containing very low levels of gangliosides still retained some a oligomerisation ability , and therefore ganglioside - independent aggregation mechanisms can not be ruled out .", "when the first synthetic a oligomers were prepared from a142 peptide by the klein laboratory in 1998 , it was observed that their binding to hippocampal neurons and cultured nerve cells was abolished by treating the cells with trypsin     .", "this , coupled with the low oligomer concentration ( 5  nm ) required for neurotoxicity , implied that specific protein receptors were responsible for the binding of a oligomers and for the subsequent transduction and amplification of neurotoxicity .", "indeed , a recent study found that a oligomer binding to neurons was saturable with an estimated apparent kd of ~0.4  nm     .", "this finding implied that one or more high - affinity receptors are responsible for a oligomer binding and subsequent neurotoxicity .", "immunofluorescence microscopy has revealed that a oligomers bind to dendritic spines of hippocampal neurons where they colocalise with postsynaptic markers     [ 9 , 98 , 99 ] .", "interestingly , a oligomer binding to neurons has a punctate appearance     , which is reminiscent of the appearance of lipid raft localised proteins     .", "several putative neuronal receptors for a have been identified in recent years , namely proteins that are related to mechanisms of memory and neuroprotection in the brain .", "noteworthy , all of these receptors either reside primarily within , or can partition into , lipid raft domains at the surface of neurons .", "lipid rafts may therefore hold the key to understanding how the deleterious effects of a oligomers are transduced through binding to specific receptors within these microdomains . in 2009 ,", "laurn and colleagues reported that the cellular prion protein ( prp ) is a specific , high - affinity neuronal receptor for a142 oligomers     .", "prp is a gpi - anchored protein that is expressed at high levels in the brain , particularly at synapses and axons , where it resides in lipid rafts .", "the misfolded form of the prion protein ( prp ) is infamous for being the causative agent in mad cow disease ( bovine spongiform encephalopathy , bse ) and its human equivalent , creutzfeldt - jakob disease ( cjd ) .", "although the correctly - folded prp   is critical for prion disease pathogenesis , its physiological function remains enigmatic , with potential neuroprotective roles in oxidative stress defence , metal ion homeostasis and antiapoptosis     . in a search to identify neuronal receptors for a oligomers , laurn et al .    ", "screened a mouse brain expression library of 225,000 cdna constructs from which only two positive clones , both encoding full - length   prp , were isolated that were able to bind a oligomers with high affinity and specificity .", "interestingly , the   prp homologues shadoo and doppel were found not to bind a oligomers to any significant degree . a further , more focussed screen of 352 clones encoding transmembrane proteins identified amyloid- precursor - like protein 1 ( aplp1 ) and transmembrane protein 30b ( tmem30b ) as weak a receptors , although their specificity for oligomeric a was poor .", "the 7 nicotinic acetylcholine receptor ( nachr7 ) and the receptor for advanced glycation end products ( rage ) were also assayed due to their previously reported affinities for a peptides     [ 103 , 104 ] , although neither displayed high - affinity a oligomer binding .", "therefore , prp was the only identified receptor to display both high affinity and high specificity for a oligomers . a direct interaction between prp and a oligomers was confirmed and the core oligomer binding region of prp   was narrowed down to amino acids 95110 , a positively charged cluster rich in lysine residues     .  ", "prp   was also shown to mediate the inhibition of ltp that is induced when hippocampal slices were incubated with a oligomers at nanomolar concentrations     .", "a follow - up in vivo study revealed that the presence of   prp   is required for the a oligomer - mediated memory impairments in an ad model mouse .", "taken together , these data indicate a strong association between a oligomers binding to prp   within lipid rafts of hippocampal neurons and the induction of memory deficits that are characteristic of ad . nevertheless , there has been some dispute over the role of prp   in transducing the deleterious effects of a oligomers in vivo , as other studies have reported data which oppose this theory .", "first , balducci and colleagues reported that although a oligomers bind tightly to    prp    they cause impairments in long - term memory in mice independently of    prp     . in this study , the effects of synthetic a oligomers upon wild - type mice were observed , whereas gimbel et al .     ", "further , the synthetic depsipeptide and the oligomer preparation method utilised by balducci et al .      differed from those used by gimbel and coworkers     , raising the possibility that   prp   does not have the same binding affinity for all types of a oligomers .", "second , the aguzzi group crossed an ad mouse model , which suffers from a-dependent memory deficits in the form of ltp impairment , with mice expressing either wild - type   prp , a secreted form of   prp   ( lacking its gpi anchor ) or no   prp   .", "they found that the presence or absence of wild - type   prp    had no effect upon the a-mediated inhibition of ltp .", "however , expression of the secreted form of   prp   was found to suppress the impairment in ltp , which the authors proposed may be due to the potential chelation and subsequent degradation of a oligomers by soluble   prp    in the extracellular milieu .", "third , kessels and coworkers reported the influence of   prp    upon hippocampal neurons expressing a c - terminally truncated form of app in a viral expression construct .", "the same loss of dendritic spines and inhibition of ltp were observed in the presence and absence of    prp , suggesting that a-mediated synaptic defects do not require   prp .", "however , laurn and colleagues have emphasised the differences in the model system utilised by kessels and coworkers in their study which may account for the opposing data , namely the viral expression of app , a higher concentration of a oligomers and a difference in the observed suppression of synaptic plasticity .  ", "further investigation is needed to clarify the role of    prp    in modulating the a oligomer - mediated impairments in memory and ltp .", "differences in the oligomer preparations , age and genotype of the mouse models , the nature of the promoter elements driving gene expression and the particular memory tests employed by the different authors may account for the discrepancies in the data .  ", "the binding of a oligomers to prp   is not the first time that prp   has been linked to ad .", "senile plaques from a subset of ad patients were observed to contain   prp      and abundant a deposits have been observed in some cjd cases     .", "furthermore , the met / val 129 polymorphism in the prnp gene that encodes prp    is a risk factor for early - onset ad     . in 2007", ", we demonstrated that prp negatively modulates a production through inhibition of the app cleaving enzyme , bace1     .", "these data , along with the recent discovery that prp   binds to a oligomers and transduces their deleterious effects , raises the intriguing possibility of a feedback loop .", "we propose that , physiologically , prp   maintains a production at a low level through bace1 inhibition , but in ad this interaction may be disrupted by a oligomers binding to   prp   and causing its segregation from bace1 .", "therefore , a oligomers binding to prp   may also promote their own production through the ablation of bace1 inhibition by    prp .", "more recently , levels of   prp    have been shown to be reduced in ad brains     [ 115 , 116 ] possibly arguing against   prp    being involved in mediating the neurotoxic effects of a oligomers , at least in the terminal stages of the disease .", "it is important to note that laurn and colleagues reported that the removal of prp   from hippocampal neurons only reduced a oligomer binding by approximately 50%     .", "this suggests that other receptors not identified in the expression library screen due to nonpreferential binding conditions or a low affinity for the particular type of a oligomers that were used , and/or nonprotein lipid raft components , may play equally crucial roles in a oligomer binding and neurotoxicity", ". glutamate receptors , which possibly exist in a complex with prp    ,   represent a candidate interacting partner for a oligomers which could explain the deleterious effects upon hippocampal synaptic plasticity .", "synaptic failure and impairments in synaptic plasticity are hallmarks of early ad neuropathology     [ 100 , 118 , 119 ] .", "ltp and long - term depression ( ltd ) are mechanistic dimmer switches which facilitate synaptic plasticity by strengthening or weakening communication across a synapse , respectively , with ltp being essential for hippocampal - dependent learning and memory     [ 120 , 121 ] .", "numerous lines of study have confirmed that soluble a oligomers from various sources , including those isolated from ad brains , disrupt hippocampal ltp in vitro and in vivo and cause impairments in learning and memory     [ 9 , 67 , 70 , 107 , 122 , 123 ] .", "although not all studies agree , it has also been demonstrated that a oligomers can provoke ltd which opposes ltp     [ 67 , 124 , 125 ] .", "neuronal receptors which modulate ltp and/or ltd are therefore likely candidates for the specific binding of a oligomers .", "additionally , glutamate receptor dysfunction has been implicated in ad which is characterised by memory deficits caused by impaired synaptic plasticity .", "glutamate receptors consist of two classes ; ionotropic ( cation - specific ion channels ) and metabotropic ( g - protein - coupled ) .", "n - methyl - d - aspartate receptors ( nmdars ) constitute a major class of glutamate receptors in the mammalian brain which localise to the postsynaptic membrane of excitatory synapses .", "the membrane channel is usually blocked by mg ions which are displaced when synaptic transmission results in depolarisation and glutamate release and binding .", "nmdar channel opening leads to the rapid influx of ca   which triggers ltp induction .", "longer - term effects which maintain the reinforced synapse include the activation of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors ( ampars ) , altered gene expression and kinase activity and the growth of new dendritic spines .", "interestingly , nmdar activation can also stimulate ltd , having the opposing effect of synapse weakening , and this appears to depend upon the nature of the stimulus and the subtype of nmdar involved .", "nmdars localise to lipid raft domains where they interact with flotillins     [ 131 , 132 ] although they can move laterally between raft and nonraft domains in response to cues including phosphorylation .", "statins , which deplete cellular cholesterol thus reducing lipid raft formation , have been shown to reduce the localisation of nmdars to lipid raft domains , which has a neuroprotective effect     .", "mounting evidence points towards a central role for nmdars in the modulation of a oligomer toxicity .", "soluble a oligomers inhibit nmdar - dependent ltp     [ 70 , 135 ] and exhibit postsynaptic binding to hippocampal neurons which express nmdar subunits glun1 and glun2b     . a reduction in nmdar subunits glun1 and glun2b", "crucially , a recent study has confirmed that a oligomer - mediated early synaptic dysfunction depends upon the activation of glun2b - containing nmdars .", "a oligomers were found to decrease the nmdar - dependent influx of ca into dendritic spines     , and to reduce dendritic spine and synapse density   in a mechanism which involve the subsequent phosphorylation of tau     .", "nmdar antagonists , including one which is specific for glun2b subunits , were able to reverse the a-induced loss of dendritic spine density     [ 100 , 137 , 139 ] .", "these effects are consistent with a oligomers blocking the nmdar - mediated stimulation of ltp whilst promoting nmdar - mediated ltd .", "in addition , a oligomers have been shown to stimulate the excessive generation of reactive oxygen species ( ros ) through an nmdar - dependent mechanism     , suggesting a link between aberrant ros regulation and a-induced cognitive impairment .", "furthermore , evidence to confirm a direct interaction between a oligomers and nmdar subunits has recently been presented .", "partial colocalisation was observed between nmdar glun2b and a oligomers in hippocampal slices , which increased upon the addition of glutamate , although the maximum colocalisation was less than 50%     .", "however , an indirect model proposed by venkitaramani and colleagues suggests that the a oligomer - mediated decrease in glun2b - containing nmdars results from the former binding to -7 nicotinic acetylcholine receptors ( 7nachr ) , which activates striatal - enriched tyrosine phosphatase ( step ) , in turn stimulating nmdar internalisation .", "more recent data has revealed elevated levels of step in a mouse model of ad and in human ad brains , and that the removal of step abrogates the a-mediated reduction in nmdars at the cell surface     . whether or not a oligomers interact with nmdars directly", ", growing evidence suggests that nmdars play an important role in transducing the deleterious effects of a oligomers upon synaptic functionality .", "the mglur5 metabotropic glutamate receptor plays important regulatory roles in neuronal calcium mobilisation and the modulation of ltp and excitatory postsynaptic potentials in hippocampal neurons     [ 144 , 145 ] .", "recently , mglur5 was identified as a novel a oligomer receptor in a study of the behaviour of fluorescently - labelled a oligomers on hippocampal neurons and their interaction with neuronal receptors .", "the a oligomers bound to excitatory synapses where their mobility decreased as they aggregated to form larger clusters over time .", "consistent with previous data , a oligomers caused a removal in nmdars from synapses and were found to coimmunoprecipitate with nmdar subunits .", "interestingly , the a oligomers also formed complexes with mglur5 receptors , which caused their lateral redistribution into dendritic spines followed by ca dysregulation .", "renner and colleagues also observed a time - dependent increase in lipid raft - localised mglur5s which suggests that a oligomers reduce the mobility of mglurs , causing their aberrant aggregation within pathological signalling platforms . when mglur5 was removed from mouse hippocampal neurons , a oligomer binding was reduced by approximately 80% and the loss of nmdars from the cell surface was prevented .", "metabotropic glutamate receptors have been implicated previously in the pathogenesis of ad and other neurodegenerative disorders .", "impaired mglur signalling in the cortex of ad patients has been shown to correlate with ad - related neuropathological changes     .", "a recent study revealed that the a peptide upregulates the expression of mglur5s in astrocytes , protective nonneuronal cells which are implicated in ad pathogenesis and inflammation . increased levels of mglur5s", "were observed in the brains of down 's syndrome patients     ; a disease in which elevated levels of a result from the triplication of the app gene .  ", "various other lipid raft - associated proteins have been reported to effect a-mediated synaptic dysfunction .", "for instance , the removal of nerve growth factor receptors ( ngfrs ) , including trka and p75 neurotrophin receptor , from cells treated with gm1-induced a oligomers caused a significant reduction in oligomer - mediated cytoxicity     .", "ngfr dysfunction and aberrant ngf signalling is associated with ad and increased a production     [ 152 , 153 ] .", "although no direct interaction has been shown to our knowledge , it is possible that interplay between a oligomers and ngfrs may form part of a positive feedback loop which serves to reinforce a oligomer production , whilst blocking ngf signalling with deleterious effects upon neuronal survival .", "physiologically , ngf binds to trka causing the translocation and clustering of receptors within lipid rafts     .", "the binding of a oligomers to trka and other ngfrs may therefore cause aberrant lipid raft clustering which prevents or disrupts the formation of the normal signalling platforms .", "recent research proposes that impaired insulin signalling may be involved in ad , even leading to the hypothesis that ad represents a third type of diabetes .", "insulin receptors , which are robustly expressed in hippocampal neurons , were found to bind a oligomers and to undergo internalisation from dendritic spines     .", "perturbations in insulin signalling in the brain caused by a oligomers may impair memory and ltp .", "interestingly , insulin receptor subunits are also enriched in lipid raft domains in hippocampal neurons .", "the emerging picture is that lipid rafts accommodate multiple receptors for a oligomers , namely prp   along with nmdar , mglur5 and possibly other , lower affinity receptors .", "interestingly , there is evidence to suggest that these three lipid raft - associated receptors interact together .", "it has also been reported that   prp   inhibited nmdar function in hippocampal neurons and coimmunoprecipitated with nmdar subunits .", "the functional and physical links between these a oligomer receptors suggest the existence of a multi - component , a oligomer binding raft complex , comprising of prp , mglur5 and nmdar ( figure 3 ) .", "whether the formation of this complex is required for oligomer binding , or whether the interaction of a oligomers with the individual proteins induces its assembly , is a  chicken and egg  situation .", "one possible hypothesis is that a oligomers promote the clustering of   prp   and glutamate receptors into pathological mega - scaffolds which induce both toxic loss- and gain - of - function downstream effects .", "for instance , the aberrant localisation of glutamate receptors may impede neuronal signalling mechanisms including ltp , while the clustering or internalisation of nmdars may promote their ltd - inducing functionality .", "the combined effects of oligomer binding upon more than one glutamate receptor is likely to be a large disturbance in ca homeostasis which results in pathological signalling cascades .", "interestingly , the prp - mediated response to oxidative stress is thought to induce signalling cascades which can modulate ca flux and synaptic plasticity .", "furthermore , a oligomers may cause the internalisation or loss of function of components such as prp   thus reducing neuroprotection against oxidative stress at the cell surface .", "the clustering of a oligomers at lipid raft domains may also cause damage to physiologically important signalling rafts , thus impairing neuronal function .", "furthermore , the a oligomer - induced redistribution of neuronal proteins into lipid rafts may influence their nonraft interacting partners , with additional deleterious effects upon neuronal function and integrity .", "neuronal lipid rafts are crucial modulators of a production and aggregation , leading to the accumulation of neurotoxic a oligomers in the brain which drive ad pathology .", "recent evidence now incriminates lipid rafts as pathological signalling platforms in which a oligomer receptors , such as prp   and glutamate receptors , cluster .", "a oligomer binding appears to induce the aberrant localisation of these proteins with deleterious effects upon their physiological functions including hippocampal ltp , which underlies memory , and defence against oxidative stress . in this way", ", lipid rafts appear to be directly responsible for the transduction of a oligomer - mediated memory impairments and neurotoxicity which characterise ad .", "lipid rafts are not only implicated in ad but may also be the key to a range of neurodegenerative proteinopathies , including parkinson 's disease , huntington 's disease , amyotrophic lateral sclerosis and prion diseases ( reviewed in ) . indeed , lipid raft disruption protects neurons against the toxicity of other oligomers besides a      and lipid rafts may therefore represent generic platforms for oligomer - mediated neurotoxicity .", "understanding the cell biology of the downstream effects of amyloid oligomers binding to neuronal lipid raft proteins may uncover potential therapeutic targets for the prevention of ad and other neurodegenerative diseases ."], "abstract_text": ["<S> lipid rafts are membrane microdomains , enriched in cholesterol and sphingolipids , into which specific subsets of proteins and lipids partition , creating cell - signalling platforms that are vital for neuronal functions . </S>", "<S> lipid rafts play at least three crucial roles in alzheimer 's disease ( ad ) , namely , in promoting the generation of the amyloid- ( a ) peptide , facilitating its aggregation upon neuronal membranes to form toxic oligomers and hosting specific neuronal receptors through which the ad - related neurotoxicity and memory impairments of the a oligomers are transduced . </S>", "<S> recent evidence suggests that a oligomers may exert their deleterious effects through binding to , and causing the aberrant clustering of , lipid raft proteins including the cellular prion protein and glutamate receptors . </S>", "<S> the formation of these pathogenic lipid raft - based platforms may be critical for the toxic signalling mechanisms that underlie synaptic dysfunction and neuropathology in ad . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Lipid Rafts", "3. Lipid Raft Components Promote A", "4. A", "5. Conclusions"], "sections": [["alzheimer 's disease ( ad ) is a progressive , neurodegenerative brain disorder which affects over 37 million people worldwide with an estimated global cost of over $ 600 billion in 2010     [ 1 , 2 ] .", "ad is a growing socioeconomic and financial burden due to its strong correlation with ageing ; around 1 in 3 people aged over 80 years have ad , which means that a rapid rise in ad cases is anticipated as life expectancy continues to increase .", "although several therapeutics are currently available to slow disease progression , there is currently no way to halt or prevent ad .", "ad is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles in the brain .", "the major constituents of senile plaques are the amyloid- ( a ) peptides , which are derived from the proteolytic processing of the amyloid precursor protein ( app ) within lipid rafts .", "the a peptide , notably a142 , is highly aggregation prone and self - assembles to form a heterogeneous mixture of oligomers and protofibrils , ultimately depositing as fibrils in senile plaques .", "an accumulating body of evidence indicates that soluble a oligomers , which correlate strongly with disease onset and severity , are the major neurotoxic species in ad     [ 58 ] .", "although a oligomers are neurotoxic at nanomolar concentrations and cause ad - related memory deficits , the cellular mechanisms of toxicity are poorly characterised .", "recently , several neuronal receptors which bind a oligomers have been identified , including the cellular prion protein ( prp )      and glutamate receptors     [ 10 , 11 ] among others .", "interestingly , these receptors reside primarily within , or partition into , cholesterol - rich microdomains within the plasma membrane known as lipid rafts .  ", "the three steps which underlie a oligomer - mediated neuropathology in ad , are ( 1 ) a production , ( 2 ) a assembly into oligomers and ( 3 ) a oligomers interacting with neuronal receptors .", "crucially , all three of these processes occur in lipid raft domains of the plasma membrane which are considered to play a key role in the development of ad . in this paper", ", we will outline the pivotal role that lipid rafts play in linking together the generation , self - assembly and toxicity of a oligomers , which underlie the development of the neuropathology in ad .", "a major focus will be upon the interaction between a oligomers and their putative cellular receptors ."], ["the multitude of different lipids and proteins within the plasma membrane were once thought to be distributed homogeneously across the entire lipid bilayer , as proposed by the fluid mosaic model in 1972 . however , the plasma membrane is now known to be more akin to a sea of disordered phospholipids , in which float microdomains with distinct lipid compositions , known as lipid rafts .", "lipid rafts are small ( 10200  nm ) , heterogeneous and highly dynamic assemblies that are enriched in specific components , namely cholesterol and sphingolipids ( figure 1 )     [ 14 , 15 ] .", "biochemically , lipid rafts are defined by their relative insolubility in nonionic detergents at low temperature , conferring upon them the alternative name , detergent - resistant membranes ( drms ) .", "lipid rafts are also known as liquid - ordered domains because the highly saturated sphingolipid acyl chains enable closer lipid packing , and therefore more restricted lateral movement , than the mainly unsaturated acyl chains of the phospholipids in the surrounding nonraft regions of the membrane .", "functionally , lipid rafts serve to compartmentalise cellular processes by concentrating certain proteins and lipids within the same microenvironment .", "lipid rafts are particularly enriched in glycosyl - phosphatidylinositol ( gpi)-anchored and acylated proteins due to the preferential intercalation of the saturated acyl chains into the liquid - ordered environment .", "other proteins can also associate with lipid rafts either directly or through binding to other cofactors or ligands     .", "the dynamic clustering and pinching off of lipid rafts regulates the spatial and temporal assembly of signalling and trafficking molecules , forming short - lived but vital signalling platforms     .", "lipid rafts are implicated in various essential cellular functions , including signal transduction , cell adhesion and protein / lipid sorting .", "of particular relevance here are cell signalling , sorting and axon guidance , as these processes are essential for neural development and synaptic plasticity     [ 19 , 20 ] .", "crucially , neuronal lipid rafts are also required for the maintenance of dendritic spines and healthy synapses , which are vital for neural communication including learning and memory ; processes which fail in ad     .", "the observation that lipid rafts are much more abundant in mature hippocampal neurons than in other cell types emphasises their physiological importance within the memory centre of the healthy brain , and may explain why hippocampal neurons are a primary target for a oligomer toxicity and destruction in ad     .", "lipid rafts are involved in the regulation of app processing and the generation of the a peptide which is the driving force in ad pathology     [ 23 , 24 ] . for comprehensive reviews detailing the involvement of membrane rafts in ad and a production ,", "the a peptide is produced by the lipid raft dependent amyloidogenic processing of its precursor protein , app ( figure 1 ) .", "the amyloidogenic cleavage of full - length app is initiated by the -site app cleaving enzyme-1 ( bace1 ) , a transmembrane aspartic metalloprotease . a large", ", soluble ectodomain ( sapp ) is released to leave behind a membrane - anchored c - terminal fragment ( c99 ) which retains the intact a sequence .", "the second amyloidogenic cleavage of app involves a -secretase complex which contains presenilin-1 or presenilin-2 ( the catalytic component ) , presenilin enhancer-2 ( pen2 ) , nicastrin and anterior pharynx defective-1 ( aph1 ) .", "the -secretase complex cleaves the remaining c99 stub to release a peptides of between 3942 residues in length , depending upon the precise cleavage site , along with the app intracellular domain ( aicd ) .", "although the majority of full - length app is localised to nonraft regions of the plasma membrane , where nonamyloidogenic cleavage by the -secretases adam 9 , 10 , and 17      precludes a formation , a subset of both app and bace1 partitions into lipid rafts along with -secretase components .", "both bace1 and the -secretase subunits undergo posttranslational s - palmitoylation which aids their targeting to lipid raft domains . in the case of app ,", "a direct interaction with cholesterol  the major component of lipid rafts  was recently identified .", "high cholesterol increases the partitioning of app , along with bace1 and -secretase components , into lipid rafts     . a large body of evidence points towards lipid rafts being the physiological site of amyloidogenic a production by bace1 and the -secretase complex .", "for example , both the copatching of app and bace1 by cross - linking antibodies      and the exclusive targeting of bace1 to lipid rafts by the addition of a gpi - anchor      significantly increased app cleavage at the -secretase site .", "furthermore , enrichments in lipid raft components , namely cholesterol and ganglioside gm1 , promote the generation of a     [ 31 , 33 ] .", "all four of the -secretase subunits are also enriched and active within lipid raft fractions derived from human brain     [ 34 , 35 ] and lipid raft - type membranes in vitro     [ 36 , 37 ] . in the brain ,", "the majority of a is found within detergent - resistant , glycolipid - enriched rafts , along with -secretase components     .  ", "the composition of lipid rafts purified from ad brains has been shown to be abnormal , with the rafts being more ordered and more viscous     , which implies that the modulation of lipid raft composition may present a therapeutic avenue for modulating ad - related neuropathology .", "this has led to a number of researchers investigating whether depleting lipid raft components could lower a production and therefore prevent ad .", "cholesterol , being a major component of lipid rafts and a risk factor for ad , was the obvious choice to target . for a recent review of the involvement of cholesterol in ad ,", "cholesterol depletion has indeed been shown to reduce app partitioning into lipid rafts which precludes its interaction with bace1 and -secretase components , thus lowering a production     .", "hypercholesterolaemia is linked to increased a production and deposition in the brain , both in humans     [ 4345 ] and in rodents     [ 4648 ] and is linked to an increased risk of developing ad .", "cholesterol depletion also lowers a production in cultured cells      and one study showed that a 70% reduction in cholesterol in living hippocampal neurons was sufficient to completely abolish a production . taking this into account , cholesterol - lowering drugs known as", "some retrospective epidemiological studies have shown that the administration of statins , which lower cholesterol levels , can reduce the incidence of dementia , including ad     [ 5153 ] .", "however , other studies have shown no correlation between statin usage and dementia      and the effect of statins upon disease progression and cognitive decline in ad patients has been challenged . intriguingly , it was revealed recently that a production actually reduces cholesterol in cultured cells of neuronal origin by increasing efflux , possibly acting as a chaperone to remove excess cholesterol from the brain to the circulation .    ", "although a reduction in cholesterol may go some way towards reducing a levels in the brain , much longer - term epidemiological studies and clinical trials initiated before significant neuronal loss and cognitive function are apparent are required in order to further elucidate the effects of lowering cholesterol levels upon ad onset and neuropathology .", "lipid rafts contain many essential components other than cholesterol , such as sphingolipids , and it is likely that the modulation of just one factor will not completely abolish a production in vivo .", "it is important to remember that cholesterol metabolism in the brain is largely isolated from the rest of the body by the blood - brain barrier . as nearly all of the cholesterol in the brain", "is synthesised in situ , the modulation of cholesterol levels within neurons represents a more difficult pharmaceutical challenge and the blood - brain barrier permeability of the drugs used needs to be considered .", "furthermore , even if cholestxerol depletion mediates a reduction in a levels , a oligomers effect neurotoxicity and memory impairments at low nanomolar concentrations     .", "therefore , residual levels of a production may be sufficient for continued a oligomer - mediated toxicity ."], ["the a peptide is natively unfolded and , under certain conditions , it aggregates to form a heterogeneous mixture of soluble oligomers , protofibrils and fibrils .", "it was accepted for a long time that the a fibrils that deposit in neuritic plaques , which are observed post mortem in diseased brains , were responsible for neurotoxicity in ad .", "a fibrils have been reported to induce neuronal dysfunction and cell death , although fibrils are less potent neurotoxins than soluble forms of a     [ 60 , 61 ] .", "interestingly , fibrils have been found to become more neurotoxic upon fragmentation     , raising the possibility that soluble species released from fibril ends may underlie their neurotoxicity .", "a plethora of studies have now demonstrated that levels of soluble a oligomers in the brain correlate much better than plaques or fibrils with ad onset , progression and severity     [ 5 , 6 , 8 , 63 , 64 ] . within the last fifteen years , a large number of studies from research groups worldwide have reported the existence of many different oligomeric assemblies from various sources , including ad brain and cerebrospinal fluid ( csf ) samples , secreted into the conditioned medium of cultured cells or prepared artificially from recombinant or synthetic a peptides .", "a heterogeneous range of sizes and peptide conformations have been observed among these natural and artificial a oligomers , including dimers and trimers     [ 66 , 67 ] , tetramers , hexamers and the dodecameric a*56     , globulomers     , ring - shaped annular protofibrils   and higher molecular weight a-derived diffusible ligands ( addls ) which can comprise hundreds of monomeric subunits     [ 9 , 70 ] ( figure 2 ) . however , despite the disparity in size and source , a oligomers appear to share important functional properties .", "notably , both natural and synthetic a oligomer preparations bind to hippocampal neurons and cells of neuronal lineage , causing a loss of dendritic spines , neurotoxicity , the inhibition of long - term synaptic potentiation ( ltp : an electrophysiological correlate of learning and memory ) and impairments in working memory at nanomolar concentrations     [ 64 , 67 , 68 , 7073 ] .", "the preferential binding and toxicity of a oligomers towards neurons in the hippocampus may explain why a oligomers correlate with ad severity and disease progression     [ 9 , 68 , 70 ] .", "a is a physiological peptide which is present in the brain tissue and csf of healthy subjects throughout life , without necessarily causing neurodegeneration     [ 7476 ] .", "many studies have shown that monomeric , nonaggregated a does not cause the neurotoxic effects that are mediated by a oligomers .", "in fact , monomeric a has recently been reported to have neuroprotective roles in the brain     [ 77 , 78 ] .", "the aggregation of a is necessary for its toxicity   and the emerging picture is that soluble a oligomers are the proximate neurotoxins in ad     [ 8 , 80 ] .", "the aggregation of a is therefore a critical step in the development of ad pathogenesis , and one in which lipid rafts appear to play a fundamental role .", "neuronal sensitivity to a-induced toxicity has been found to be dependent upon a binding to the cell membrane      and a has been identified in lipid rafts from cultured cells and from human and rodent brains .", "soluble a dimers accumulate rapidly , and have been found at elevated levels , in lipid raft fractions isolated from human and transgenic mouse model ad brains     .", "importantly , a has been shown to accumulate in presynaptic terminals in ad cortex where it colocalises with the lipid raft markers cholesterol and ganglioside gm1 . taken together ,", "these data suggest that a accumulation and aggregation within lipid rafts may underlie ad neuropathology .", "as cholesterol is a major component of lipid rafts , it was postulated to facilitate a oligomerisation on neuronal membranes . the brain is particularly enriched in cholesterol , harbouring over 23% of the body 's total complement but comprising only around 2% of total body mass . however , the role of cholesterol in promoting the assembly of a is controversial and conflicting evidence has been presented in recent years .", "the main difficulty is being able to distinguish between the key role of cholesterol in building the lipid raft domains necessary for a production and the suggested role of cholesterol in promoting a oligomerisation .", "as discussed previously , raised cholesterol has been linked to ad ; is this solely due to an increase in total lipid raft composition of the plasma membrane which increases amyloidogenic processing of app to yield more a peptide or due to a direct effect on a oligomerisation ?  ", "a growing body of evidence suggests that certain components of lipid raft domains may play a much more sinister role in catalysing the conversion of the aggregation - prone a peptide to its neurotoxic , oligomeric states .", "cholesterol is known to modulate the interaction of the a peptide with lipid bilayers     .", "further , a oligomers isolated from ad patients associate with drms in a cholesterol - dependent manner , and cholesterol depletion reduces the aggregation of a     .", "it is currently unknown , however , whether this latter effect is due to a direct interaction between a and cholesterol , or due to the overall depletion in lipid raft domains and/or the subsequent change in composition and properties brought on by a reduction in cholesterol .", "conversely , a recent study revealed that increasing the level of cholesterol in human neuroblastoma cells actually reduced the ability of synthetic a oligomers to bind , in spite of the colocalisation of the a oligomers with the lipid raft component ganglioside gm1 .", "these data agree with the authors ' previous finding that an increased level of membrane cholesterol exerts a protective effect against a oligomer toxicity     . in the more recent study  ", "it was proposed that a fluctuation in cholesterol levels may alter the physical properties of lipid rafts thereby modulating oligomer binding .  ", "cholesterol can also facilitate a aggregation through the structural modification of other lipid raft components .", "a recent study using reconstituted membranes revealed a structural role for cholesterol in modulating the conformation of glycosphingolipids .", "depending on the type of glycosphingolipid , cholesterol can either facilitate ( such as for ganglioside gm1 ) or inhibit the interaction of a peptides with lipid rafts through fine - tuning of the glycosphingolipid conformation .", "this reinforces the notion that a binding to , and aggregation upon , neuronal lipid raft domains can not be ascribed to a single component , but rather that multiple players are likely to be involved .", "in fact , mounting evidence suggests that gangliosides within lipid rafts appear to be the main driving force behind the oligomerisation of a on neuronal membranes .", "the development of ad within certain brain regions has been found to correlate with increased ganglioside levels     .", "gangliosides are glycosphingolipids with one or more sialic acid moieties attached to the sugar chain .", "gangliosides are found predominantly in the central nervous system , where they are enriched in lipid rafts due to the preferential packing of their saturated acyl chains within the liquid - ordered phase . a study in 1995 revealed that a population of membrane - bound a tightly bound to gangliosides exists in ad brains .", "more recently , exogenously - applied a was shown to bind to neuronal membranes and to redistribute into lipid rafts where it colocalised with ganglioside gm1 in a time - dependent manner .", "gm1 facilitated the binding and accumulation of a oligomers at lipid raft domains and appeared to be required for the a oligomer - mediated lipid peroxidation of drms .", "ganglioside gm1 contains just one sialic acid moiety and plays important physiological roles in neuronal function .", "a appears to interact with the sialic acid moiety of gangliosides such as gm1 and these bound aggregates can go on to seed further a aggregation     .", "the interaction between sialic acid and a induces a conformational rearrangement of the a peptide chain   which may potentiate a oligomerisation .", "drms derived from ganglioside - rich rat brain , but not from liver , were found to promote the oligomerisation of a .", "further , this study revealed that the removal of cholesterol or protein from these raft fractions did not prevent a aggregation , providing evidence that neither cholesterol nor protein is essential for this process .", "however , lipid raft fractions containing very low levels of gangliosides still retained some a oligomerisation ability , and therefore ganglioside - independent aggregation mechanisms can not be ruled out ."], ["when the first synthetic a oligomers were prepared from a142 peptide by the klein laboratory in 1998 , it was observed that their binding to hippocampal neurons and cultured nerve cells was abolished by treating the cells with trypsin     .", "this , coupled with the low oligomer concentration ( 5  nm ) required for neurotoxicity , implied that specific protein receptors were responsible for the binding of a oligomers and for the subsequent transduction and amplification of neurotoxicity .", "indeed , a recent study found that a oligomer binding to neurons was saturable with an estimated apparent kd of ~0.4  nm     .", "this finding implied that one or more high - affinity receptors are responsible for a oligomer binding and subsequent neurotoxicity .", "immunofluorescence microscopy has revealed that a oligomers bind to dendritic spines of hippocampal neurons where they colocalise with postsynaptic markers     [ 9 , 98 , 99 ] .", "interestingly , a oligomer binding to neurons has a punctate appearance     , which is reminiscent of the appearance of lipid raft localised proteins     .", "several putative neuronal receptors for a have been identified in recent years , namely proteins that are related to mechanisms of memory and neuroprotection in the brain .", "noteworthy , all of these receptors either reside primarily within , or can partition into , lipid raft domains at the surface of neurons .", "lipid rafts may therefore hold the key to understanding how the deleterious effects of a oligomers are transduced through binding to specific receptors within these microdomains . in 2009 ,", "laurn and colleagues reported that the cellular prion protein ( prp ) is a specific , high - affinity neuronal receptor for a142 oligomers     .", "prp is a gpi - anchored protein that is expressed at high levels in the brain , particularly at synapses and axons , where it resides in lipid rafts .", "the misfolded form of the prion protein ( prp ) is infamous for being the causative agent in mad cow disease ( bovine spongiform encephalopathy , bse ) and its human equivalent , creutzfeldt - jakob disease ( cjd ) .", "although the correctly - folded prp   is critical for prion disease pathogenesis , its physiological function remains enigmatic , with potential neuroprotective roles in oxidative stress defence , metal ion homeostasis and antiapoptosis     . in a search to identify neuronal receptors for a oligomers , laurn et al .    ", "screened a mouse brain expression library of 225,000 cdna constructs from which only two positive clones , both encoding full - length   prp , were isolated that were able to bind a oligomers with high affinity and specificity .", "interestingly , the   prp homologues shadoo and doppel were found not to bind a oligomers to any significant degree . a further , more focussed screen of 352 clones encoding transmembrane proteins identified amyloid- precursor - like protein 1 ( aplp1 ) and transmembrane protein 30b ( tmem30b ) as weak a receptors , although their specificity for oligomeric a was poor .", "the 7 nicotinic acetylcholine receptor ( nachr7 ) and the receptor for advanced glycation end products ( rage ) were also assayed due to their previously reported affinities for a peptides     [ 103 , 104 ] , although neither displayed high - affinity a oligomer binding .", "therefore , prp was the only identified receptor to display both high affinity and high specificity for a oligomers . a direct interaction between prp and a oligomers was confirmed and the core oligomer binding region of prp   was narrowed down to amino acids 95110 , a positively charged cluster rich in lysine residues     .  ", "prp   was also shown to mediate the inhibition of ltp that is induced when hippocampal slices were incubated with a oligomers at nanomolar concentrations     .", "a follow - up in vivo study revealed that the presence of   prp   is required for the a oligomer - mediated memory impairments in an ad model mouse .", "taken together , these data indicate a strong association between a oligomers binding to prp   within lipid rafts of hippocampal neurons and the induction of memory deficits that are characteristic of ad . nevertheless , there has been some dispute over the role of prp   in transducing the deleterious effects of a oligomers in vivo , as other studies have reported data which oppose this theory .", "first , balducci and colleagues reported that although a oligomers bind tightly to    prp    they cause impairments in long - term memory in mice independently of    prp     . in this study , the effects of synthetic a oligomers upon wild - type mice were observed , whereas gimbel et al .     ", "further , the synthetic depsipeptide and the oligomer preparation method utilised by balducci et al .      differed from those used by gimbel and coworkers     , raising the possibility that   prp   does not have the same binding affinity for all types of a oligomers .", "second , the aguzzi group crossed an ad mouse model , which suffers from a-dependent memory deficits in the form of ltp impairment , with mice expressing either wild - type   prp , a secreted form of   prp   ( lacking its gpi anchor ) or no   prp   .", "they found that the presence or absence of wild - type   prp    had no effect upon the a-mediated inhibition of ltp .", "however , expression of the secreted form of   prp   was found to suppress the impairment in ltp , which the authors proposed may be due to the potential chelation and subsequent degradation of a oligomers by soluble   prp    in the extracellular milieu .", "third , kessels and coworkers reported the influence of   prp    upon hippocampal neurons expressing a c - terminally truncated form of app in a viral expression construct .", "the same loss of dendritic spines and inhibition of ltp were observed in the presence and absence of    prp , suggesting that a-mediated synaptic defects do not require   prp .", "however , laurn and colleagues have emphasised the differences in the model system utilised by kessels and coworkers in their study which may account for the opposing data , namely the viral expression of app , a higher concentration of a oligomers and a difference in the observed suppression of synaptic plasticity .  ", "further investigation is needed to clarify the role of    prp    in modulating the a oligomer - mediated impairments in memory and ltp .", "differences in the oligomer preparations , age and genotype of the mouse models , the nature of the promoter elements driving gene expression and the particular memory tests employed by the different authors may account for the discrepancies in the data .  ", "the binding of a oligomers to prp   is not the first time that prp   has been linked to ad .", "senile plaques from a subset of ad patients were observed to contain   prp      and abundant a deposits have been observed in some cjd cases     .", "furthermore , the met / val 129 polymorphism in the prnp gene that encodes prp    is a risk factor for early - onset ad     . in 2007", ", we demonstrated that prp negatively modulates a production through inhibition of the app cleaving enzyme , bace1     .", "these data , along with the recent discovery that prp   binds to a oligomers and transduces their deleterious effects , raises the intriguing possibility of a feedback loop .", "we propose that , physiologically , prp   maintains a production at a low level through bace1 inhibition , but in ad this interaction may be disrupted by a oligomers binding to   prp   and causing its segregation from bace1 .", "therefore , a oligomers binding to prp   may also promote their own production through the ablation of bace1 inhibition by    prp .", "more recently , levels of   prp    have been shown to be reduced in ad brains     [ 115 , 116 ] possibly arguing against   prp    being involved in mediating the neurotoxic effects of a oligomers , at least in the terminal stages of the disease .", "it is important to note that laurn and colleagues reported that the removal of prp   from hippocampal neurons only reduced a oligomer binding by approximately 50%     .", "this suggests that other receptors not identified in the expression library screen due to nonpreferential binding conditions or a low affinity for the particular type of a oligomers that were used , and/or nonprotein lipid raft components , may play equally crucial roles in a oligomer binding and neurotoxicity", ". glutamate receptors , which possibly exist in a complex with prp    ,   represent a candidate interacting partner for a oligomers which could explain the deleterious effects upon hippocampal synaptic plasticity .", "synaptic failure and impairments in synaptic plasticity are hallmarks of early ad neuropathology     [ 100 , 118 , 119 ] .", "ltp and long - term depression ( ltd ) are mechanistic dimmer switches which facilitate synaptic plasticity by strengthening or weakening communication across a synapse , respectively , with ltp being essential for hippocampal - dependent learning and memory     [ 120 , 121 ] .", "numerous lines of study have confirmed that soluble a oligomers from various sources , including those isolated from ad brains , disrupt hippocampal ltp in vitro and in vivo and cause impairments in learning and memory     [ 9 , 67 , 70 , 107 , 122 , 123 ] .", "although not all studies agree , it has also been demonstrated that a oligomers can provoke ltd which opposes ltp     [ 67 , 124 , 125 ] .", "neuronal receptors which modulate ltp and/or ltd are therefore likely candidates for the specific binding of a oligomers .", "additionally , glutamate receptor dysfunction has been implicated in ad which is characterised by memory deficits caused by impaired synaptic plasticity .", "glutamate receptors consist of two classes ; ionotropic ( cation - specific ion channels ) and metabotropic ( g - protein - coupled ) .", "n - methyl - d - aspartate receptors ( nmdars ) constitute a major class of glutamate receptors in the mammalian brain which localise to the postsynaptic membrane of excitatory synapses .", "the membrane channel is usually blocked by mg ions which are displaced when synaptic transmission results in depolarisation and glutamate release and binding .", "nmdar channel opening leads to the rapid influx of ca   which triggers ltp induction .", "longer - term effects which maintain the reinforced synapse include the activation of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors ( ampars ) , altered gene expression and kinase activity and the growth of new dendritic spines .", "interestingly , nmdar activation can also stimulate ltd , having the opposing effect of synapse weakening , and this appears to depend upon the nature of the stimulus and the subtype of nmdar involved .", "nmdars localise to lipid raft domains where they interact with flotillins     [ 131 , 132 ] although they can move laterally between raft and nonraft domains in response to cues including phosphorylation .", "statins , which deplete cellular cholesterol thus reducing lipid raft formation , have been shown to reduce the localisation of nmdars to lipid raft domains , which has a neuroprotective effect     .", "mounting evidence points towards a central role for nmdars in the modulation of a oligomer toxicity .", "soluble a oligomers inhibit nmdar - dependent ltp     [ 70 , 135 ] and exhibit postsynaptic binding to hippocampal neurons which express nmdar subunits glun1 and glun2b     . a reduction in nmdar subunits glun1 and glun2b", "crucially , a recent study has confirmed that a oligomer - mediated early synaptic dysfunction depends upon the activation of glun2b - containing nmdars .", "a oligomers were found to decrease the nmdar - dependent influx of ca into dendritic spines     , and to reduce dendritic spine and synapse density   in a mechanism which involve the subsequent phosphorylation of tau     .", "nmdar antagonists , including one which is specific for glun2b subunits , were able to reverse the a-induced loss of dendritic spine density     [ 100 , 137 , 139 ] .", "these effects are consistent with a oligomers blocking the nmdar - mediated stimulation of ltp whilst promoting nmdar - mediated ltd .", "in addition , a oligomers have been shown to stimulate the excessive generation of reactive oxygen species ( ros ) through an nmdar - dependent mechanism     , suggesting a link between aberrant ros regulation and a-induced cognitive impairment .", "furthermore , evidence to confirm a direct interaction between a oligomers and nmdar subunits has recently been presented .", "partial colocalisation was observed between nmdar glun2b and a oligomers in hippocampal slices , which increased upon the addition of glutamate , although the maximum colocalisation was less than 50%     .", "however , an indirect model proposed by venkitaramani and colleagues suggests that the a oligomer - mediated decrease in glun2b - containing nmdars results from the former binding to -7 nicotinic acetylcholine receptors ( 7nachr ) , which activates striatal - enriched tyrosine phosphatase ( step ) , in turn stimulating nmdar internalisation .", "more recent data has revealed elevated levels of step in a mouse model of ad and in human ad brains , and that the removal of step abrogates the a-mediated reduction in nmdars at the cell surface     . whether or not a oligomers interact with nmdars directly", ", growing evidence suggests that nmdars play an important role in transducing the deleterious effects of a oligomers upon synaptic functionality .", "the mglur5 metabotropic glutamate receptor plays important regulatory roles in neuronal calcium mobilisation and the modulation of ltp and excitatory postsynaptic potentials in hippocampal neurons     [ 144 , 145 ] .", "recently , mglur5 was identified as a novel a oligomer receptor in a study of the behaviour of fluorescently - labelled a oligomers on hippocampal neurons and their interaction with neuronal receptors .", "the a oligomers bound to excitatory synapses where their mobility decreased as they aggregated to form larger clusters over time .", "consistent with previous data , a oligomers caused a removal in nmdars from synapses and were found to coimmunoprecipitate with nmdar subunits .", "interestingly , the a oligomers also formed complexes with mglur5 receptors , which caused their lateral redistribution into dendritic spines followed by ca dysregulation .", "renner and colleagues also observed a time - dependent increase in lipid raft - localised mglur5s which suggests that a oligomers reduce the mobility of mglurs , causing their aberrant aggregation within pathological signalling platforms . when mglur5 was removed from mouse hippocampal neurons , a oligomer binding was reduced by approximately 80% and the loss of nmdars from the cell surface was prevented .", "metabotropic glutamate receptors have been implicated previously in the pathogenesis of ad and other neurodegenerative disorders .", "impaired mglur signalling in the cortex of ad patients has been shown to correlate with ad - related neuropathological changes     .", "a recent study revealed that the a peptide upregulates the expression of mglur5s in astrocytes , protective nonneuronal cells which are implicated in ad pathogenesis and inflammation . increased levels of mglur5s", "were observed in the brains of down 's syndrome patients     ; a disease in which elevated levels of a result from the triplication of the app gene .  ", "various other lipid raft - associated proteins have been reported to effect a-mediated synaptic dysfunction .", "for instance , the removal of nerve growth factor receptors ( ngfrs ) , including trka and p75 neurotrophin receptor , from cells treated with gm1-induced a oligomers caused a significant reduction in oligomer - mediated cytoxicity     .", "ngfr dysfunction and aberrant ngf signalling is associated with ad and increased a production     [ 152 , 153 ] .", "although no direct interaction has been shown to our knowledge , it is possible that interplay between a oligomers and ngfrs may form part of a positive feedback loop which serves to reinforce a oligomer production , whilst blocking ngf signalling with deleterious effects upon neuronal survival .", "physiologically , ngf binds to trka causing the translocation and clustering of receptors within lipid rafts     .", "the binding of a oligomers to trka and other ngfrs may therefore cause aberrant lipid raft clustering which prevents or disrupts the formation of the normal signalling platforms .", "recent research proposes that impaired insulin signalling may be involved in ad , even leading to the hypothesis that ad represents a third type of diabetes .", "insulin receptors , which are robustly expressed in hippocampal neurons , were found to bind a oligomers and to undergo internalisation from dendritic spines     .", "perturbations in insulin signalling in the brain caused by a oligomers may impair memory and ltp .", "interestingly , insulin receptor subunits are also enriched in lipid raft domains in hippocampal neurons .", "the emerging picture is that lipid rafts accommodate multiple receptors for a oligomers , namely prp   along with nmdar , mglur5 and possibly other , lower affinity receptors .", "interestingly , there is evidence to suggest that these three lipid raft - associated receptors interact together .", "it has also been reported that   prp   inhibited nmdar function in hippocampal neurons and coimmunoprecipitated with nmdar subunits .", "the functional and physical links between these a oligomer receptors suggest the existence of a multi - component , a oligomer binding raft complex , comprising of prp , mglur5 and nmdar ( figure 3 ) .", "whether the formation of this complex is required for oligomer binding , or whether the interaction of a oligomers with the individual proteins induces its assembly , is a  chicken and egg  situation .", "one possible hypothesis is that a oligomers promote the clustering of   prp   and glutamate receptors into pathological mega - scaffolds which induce both toxic loss- and gain - of - function downstream effects .", "for instance , the aberrant localisation of glutamate receptors may impede neuronal signalling mechanisms including ltp , while the clustering or internalisation of nmdars may promote their ltd - inducing functionality .", "the combined effects of oligomer binding upon more than one glutamate receptor is likely to be a large disturbance in ca homeostasis which results in pathological signalling cascades .", "interestingly , the prp - mediated response to oxidative stress is thought to induce signalling cascades which can modulate ca flux and synaptic plasticity .", "furthermore , a oligomers may cause the internalisation or loss of function of components such as prp   thus reducing neuroprotection against oxidative stress at the cell surface .", "the clustering of a oligomers at lipid raft domains may also cause damage to physiologically important signalling rafts , thus impairing neuronal function .", "furthermore , the a oligomer - induced redistribution of neuronal proteins into lipid rafts may influence their nonraft interacting partners , with additional deleterious effects upon neuronal function and integrity ."], ["neuronal lipid rafts are crucial modulators of a production and aggregation , leading to the accumulation of neurotoxic a oligomers in the brain which drive ad pathology .", "recent evidence now incriminates lipid rafts as pathological signalling platforms in which a oligomer receptors , such as prp   and glutamate receptors , cluster .", "a oligomer binding appears to induce the aberrant localisation of these proteins with deleterious effects upon their physiological functions including hippocampal ltp , which underlies memory , and defence against oxidative stress . in this way", ", lipid rafts appear to be directly responsible for the transduction of a oligomer - mediated memory impairments and neurotoxicity which characterise ad .", "lipid rafts are not only implicated in ad but may also be the key to a range of neurodegenerative proteinopathies , including parkinson 's disease , huntington 's disease , amyotrophic lateral sclerosis and prion diseases ( reviewed in ) . indeed , lipid raft disruption protects neurons against the toxicity of other oligomers besides a      and lipid rafts may therefore represent generic platforms for oligomer - mediated neurotoxicity .", "understanding the cell biology of the downstream effects of amyloid oligomers binding to neuronal lipid raft proteins may uncover potential therapeutic targets for the prevention of ad and other neurodegenerative diseases ."]]}
{"article_id": "PMC3354799", "article_text": ["in dentistry , the concept of aesthetics is extremely subjective and is related to beauty , harmony and the needs of the patient .", "the interactions between new restorative materials and techniques allow the reproduction of dental structures , restoring form and function in such a way that restorative procedures become imperceptible .", "nevertheless , during the evaluation of aesthetically compromised teeth , we encounter unfavorable clinical situations of great complexity , characterized by deep invasions of the mineralized structures .", "the reproduction of the optical characteristics of the teeth , such as translucency , opalescence and fluorescence , requires a considerable knowledge of restorative techniques and the materials currently available .", "dental enamel defects have been associated with a broad spectrum of aetiologies , including genetic and epigenetic factors such as systemic , local and environmental factors .", "a serious disturbance that occurs during the stages of enamel formation will impact the quality and/or quantity of the enamel formed , depending on the phase of amelogenesis that is affected and the duration of the stimulus on the ameloblasts .", "the consequence of this disturbance in the formation of the organic enamel matrix is enamel hypoplasia , which can be characterized as small grooves , depressions and cracks in the enamel surface that can be viewed in mild cases .", "this article presents a case report of a restorative treatment of enamel hypoplasia using hybrid composite resin to mask color alteration and enamel defects of the anterior upper teeth .", "a 22-year - old female patient was referred to the dental clinic , reporting a visual discomfort from the presence of irregularities and discoloration in the maxillary incisors . dental history and", "clinical examination revealed that she had a soft form of enamel hypoplasia [ figure 1 ] .", "clinical examination also evidenced an enamel defect in the maxillary lateral and central incisors , with rough surfaces with irregular limits that principally involve the middle third of the crown [ figure 2 ] .", "initial clinical aspect of the maxillary incisors a buccal view of the central and lateral incisors with hypoplastic alterations the clinical situation revealed that it was not possible to re - establish aesthetics and function without the use of a restorative procedure .", "the position and pattern of the enamel irregularities , the occlusion and a tooth remnant with a large substrate suggested that a composite resin restoration would be a reliable option for this case .", "the patient was systemically healthy , presented an overall plaque index and gingival index of below 10% and the restorative area was free from visible plaque .", "a slight enameloplasty , using a spherical 1015f diamond bur ( kg sorensen , so paulo , brazil ) and manual instruments , was performed on both the irregularities and the limits of the tooth defect [ figures 3 and 4 ] .", "the regularization of the defects created a good substrate that was favorable for adhesive restorations .", "delimitation of the tooth irregularities with graphite enameloplasty with a spherical diamond bur for smoothing out defects the color was recorded using the vitapan classical scale ( vita zahnfabrik , bad sckingen , germany ) , and the shade a2/a3 was considered as the initial color .", "briefly , the dental surface was acid etched ( 35% phosphoric acid ) [ figure 5 ] , rinsed for 30 s and dried with absorbent paper .", "a two - component adhesive system ( adhese , ivoclar vivadent ag , schaan , liechtenstein ) was applied on the dentin and the enamel and was light - cured for 10 s with an intensity of 1400 mw / cm ( radii led curing light , sdi , australia ) [ figure 6 ] .", "the dental surface was acid etched ( 35% phosphoric acid ) a two - component adhesive system was applied on the dentin and on the enamel a combination of the incremental and stratified layering technique was used to fill the tooth using a highly aesthetic nanohybrid composite resin , ips - empress ( ivoclar vivadent ag ) .", "the composite was added in increments of 1.52 mm and was light - cured after every layer , according to the manufacturer 's instructions .", "first , the dentin was simulated with a thin layer of a microhybrid composite ( da3 ) and a final layer with an enamel composite ( ea2 ) , which was placed with a fine # 2 brush ( cosmedent , chicago , usa ) for fine detailing / texturizing to simulate the enamel , increasing the final brightness of the restoration [ figure 7 ] . a clinical view of the central incisor immediately after the restorative procedures", "the contouring was refined using 30-blade carbide trimming burs ( 9714ff , kg sorensen ) , and the final polishing was performed with a high - luster polishing paste ( opal l , renfert gmbh , hilzingen , germany ) using goat - hair brushes and cotton buffs ( renfert gmbh ) .", "four months after the restoration , a good final aspect was observed and the lateral smile view exhibited an imperceptible restoration [ figures 8 and 9 ] .", "with the valuation of aesthetics , minimally invasive restorative techniques have provided an expansion of the current conservative philosophy .", "nevertheless , during the evaluation of aesthetically compromised teeth , we encounter adverse clinical conditions of great complexity , marked by the invasion of the mineralized structures at depth .", "enamel hypoplasia is an incomplete or defective formation in the organic matrix of the enamel and remaining certain areas susceptible to decay ; it is responsible for a major proportion of lesions .", "the irregularities present in a hypoplasia provide favorable conditions for the retention of plaque and the early development of caries lesions , which progress and reach deep into the enamel and the dentin .", "one of the signs of hypoplastic lesions is diminishing enamel luster and dental surfaces that have become eroded with cavitations and irregular wear because of the loss of the microanatomy affecting the color , morphology and texture of teeth . on some occasions , hypoplasia is mistaken for fluorosis ; however , enamel hypoplasia is an incomplete or defective formation in the organic matrix of the enamel , triggered by diseases , systemic disorders , trauma and infections in the pulp of deciduous teeth .", "it manifests with partial or complete absence of the enamel , which can be systemic ( when it affects a group of teeth ) or local ( when it has asymmetric distribution and is relegated to a single tooth ) . because it is neither fully transparent nor fully opaque ,", "some modern composites provide optical similarities consistent with natural teeth , yielding satisfactory levels of opalescence , value and chroma .", "it is important to consider that the final restoration is dependent on both the thickness and the varying degrees of translucency and opacity of the several layers of the composite . of all dental structures", "describe a minimally invasive technique performed in cases of enamel hypoplasia based on enamel microabrasion and complemented with composite resin restorations .", "the procedures employed are simple , but they require knowledge of the real causes of the staining and comprehension of restorative techniques . various treatment protocols may be performed , depending on the level of involvement and the severity of the lesions .", "composite resin restorations are fully capable of reproducing the appearance of a natural tooth with highly aesthetic outcomes . in this context", ", the main goal of the treatment of enamel hypoplasia is to re - establish the anatomical harmony between occlusion , function and aesthetics and to restore patient self - esteem , promoting social and psychological benefits .", "in conclusion , this case report demonstrates that restorative rehabilitation , in addition to promoting health , may provide a more favorable aesthetic appearance for the smile , matching the tooth polychromatic and raising the self - esteem of the patient ."], "abstract_text": ["<S> enamel hypoplasia is a developmental defect of the enamel that is produced by a disturbance in the formation of the organic enamel matrix , clinically visible as enamel defects . </S>", "<S> disorders that occur during the stages of enamel development and maturation reduce the amount or thickness of the enamel , resulting in white spots , tiny grooves , depressions and fissures in the enamel surface . </S>", "<S> the complexity and intensity of the dental deformity lesions will conduct the ideal treatment - associating conservative techniques . </S>", "<S> this article presents a case report of a restorative treatment of enamel hypoplasia using hybrid composite resin to mask color alteration and enamel defects . </S>", "<S> an aesthetic appearance that respects the tooth polychromatic and the self - esteem of the patient can be achieved with this approach . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["in dentistry , the concept of aesthetics is extremely subjective and is related to beauty , harmony and the needs of the patient .", "the interactions between new restorative materials and techniques allow the reproduction of dental structures , restoring form and function in such a way that restorative procedures become imperceptible .", "nevertheless , during the evaluation of aesthetically compromised teeth , we encounter unfavorable clinical situations of great complexity , characterized by deep invasions of the mineralized structures .", "the reproduction of the optical characteristics of the teeth , such as translucency , opalescence and fluorescence , requires a considerable knowledge of restorative techniques and the materials currently available .", "dental enamel defects have been associated with a broad spectrum of aetiologies , including genetic and epigenetic factors such as systemic , local and environmental factors .", "a serious disturbance that occurs during the stages of enamel formation will impact the quality and/or quantity of the enamel formed , depending on the phase of amelogenesis that is affected and the duration of the stimulus on the ameloblasts .", "the consequence of this disturbance in the formation of the organic enamel matrix is enamel hypoplasia , which can be characterized as small grooves , depressions and cracks in the enamel surface that can be viewed in mild cases .", "this article presents a case report of a restorative treatment of enamel hypoplasia using hybrid composite resin to mask color alteration and enamel defects of the anterior upper teeth ."], ["a 22-year - old female patient was referred to the dental clinic , reporting a visual discomfort from the presence of irregularities and discoloration in the maxillary incisors . dental history and", "clinical examination revealed that she had a soft form of enamel hypoplasia [ figure 1 ] .", "clinical examination also evidenced an enamel defect in the maxillary lateral and central incisors , with rough surfaces with irregular limits that principally involve the middle third of the crown [ figure 2 ] .", "initial clinical aspect of the maxillary incisors a buccal view of the central and lateral incisors with hypoplastic alterations the clinical situation revealed that it was not possible to re - establish aesthetics and function without the use of a restorative procedure .", "the position and pattern of the enamel irregularities , the occlusion and a tooth remnant with a large substrate suggested that a composite resin restoration would be a reliable option for this case .", "the patient was systemically healthy , presented an overall plaque index and gingival index of below 10% and the restorative area was free from visible plaque .", "a slight enameloplasty , using a spherical 1015f diamond bur ( kg sorensen , so paulo , brazil ) and manual instruments , was performed on both the irregularities and the limits of the tooth defect [ figures 3 and 4 ] .", "the regularization of the defects created a good substrate that was favorable for adhesive restorations .", "delimitation of the tooth irregularities with graphite enameloplasty with a spherical diamond bur for smoothing out defects the color was recorded using the vitapan classical scale ( vita zahnfabrik , bad sckingen , germany ) , and the shade a2/a3 was considered as the initial color .", "briefly , the dental surface was acid etched ( 35% phosphoric acid ) [ figure 5 ] , rinsed for 30 s and dried with absorbent paper .", "a two - component adhesive system ( adhese , ivoclar vivadent ag , schaan , liechtenstein ) was applied on the dentin and the enamel and was light - cured for 10 s with an intensity of 1400 mw / cm ( radii led curing light , sdi , australia ) [ figure 6 ] .", "the dental surface was acid etched ( 35% phosphoric acid ) a two - component adhesive system was applied on the dentin and on the enamel a combination of the incremental and stratified layering technique was used to fill the tooth using a highly aesthetic nanohybrid composite resin , ips - empress ( ivoclar vivadent ag ) .", "the composite was added in increments of 1.52 mm and was light - cured after every layer , according to the manufacturer 's instructions .", "first , the dentin was simulated with a thin layer of a microhybrid composite ( da3 ) and a final layer with an enamel composite ( ea2 ) , which was placed with a fine # 2 brush ( cosmedent , chicago , usa ) for fine detailing / texturizing to simulate the enamel , increasing the final brightness of the restoration [ figure 7 ] . a clinical view of the central incisor immediately after the restorative procedures", "the contouring was refined using 30-blade carbide trimming burs ( 9714ff , kg sorensen ) , and the final polishing was performed with a high - luster polishing paste ( opal l , renfert gmbh , hilzingen , germany ) using goat - hair brushes and cotton buffs ( renfert gmbh ) .", "four months after the restoration , a good final aspect was observed and the lateral smile view exhibited an imperceptible restoration [ figures 8 and 9 ] ."], ["with the valuation of aesthetics , minimally invasive restorative techniques have provided an expansion of the current conservative philosophy .", "nevertheless , during the evaluation of aesthetically compromised teeth , we encounter adverse clinical conditions of great complexity , marked by the invasion of the mineralized structures at depth .", "enamel hypoplasia is an incomplete or defective formation in the organic matrix of the enamel and remaining certain areas susceptible to decay ; it is responsible for a major proportion of lesions .", "the irregularities present in a hypoplasia provide favorable conditions for the retention of plaque and the early development of caries lesions , which progress and reach deep into the enamel and the dentin .", "one of the signs of hypoplastic lesions is diminishing enamel luster and dental surfaces that have become eroded with cavitations and irregular wear because of the loss of the microanatomy affecting the color , morphology and texture of teeth . on some occasions , hypoplasia is mistaken for fluorosis ; however , enamel hypoplasia is an incomplete or defective formation in the organic matrix of the enamel , triggered by diseases , systemic disorders , trauma and infections in the pulp of deciduous teeth .", "it manifests with partial or complete absence of the enamel , which can be systemic ( when it affects a group of teeth ) or local ( when it has asymmetric distribution and is relegated to a single tooth ) . because it is neither fully transparent nor fully opaque ,", "some modern composites provide optical similarities consistent with natural teeth , yielding satisfactory levels of opalescence , value and chroma .", "it is important to consider that the final restoration is dependent on both the thickness and the varying degrees of translucency and opacity of the several layers of the composite . of all dental structures", "describe a minimally invasive technique performed in cases of enamel hypoplasia based on enamel microabrasion and complemented with composite resin restorations .", "the procedures employed are simple , but they require knowledge of the real causes of the staining and comprehension of restorative techniques . various treatment protocols may be performed , depending on the level of involvement and the severity of the lesions .", "composite resin restorations are fully capable of reproducing the appearance of a natural tooth with highly aesthetic outcomes . in this context", ", the main goal of the treatment of enamel hypoplasia is to re - establish the anatomical harmony between occlusion , function and aesthetics and to restore patient self - esteem , promoting social and psychological benefits ."], ["in conclusion , this case report demonstrates that restorative rehabilitation , in addition to promoting health , may provide a more favorable aesthetic appearance for the smile , matching the tooth polychromatic and raising the self - esteem of the patient ."]]}
{"article_id": "PMC3649205", "article_text": ["gastrointestinal lipomas are rare , mostly small and detected incidentally during endoscopic examination ( 1 ) .", "these tumours are benign proliferations of mature fatty tissue arising in the submucosa and predominately arise in the large intestine ( 51 - 70% ) preferentially on the right side .", "the majority of symptomatic lesions occur in adults in the sixth decade of life ( 2 ) .", "lipohypertrophy of the ileo - caecal valve is classified as a variant of normal ( 1 ) . the vast majority are asymptomatic but they can manifest due to intussusceptions , ulceration leading to haemorrhage or iron deficiency anaemia , intestinal obstruction or present as an abdominal mass . most are compressible and submucosal although they can rarely develop in the serosa and appear as a fold or polyp .", "they contain mature adipose tissue and usually have a thick capsule surrounding the tumour ( 2 ) .", "these changes if extensive can mimic a liposarcoma ; however , frank liposarcoma of the colon is extremely rare and requires the presence of lipoblasts ( 2 ) .", "mucosal biopsies can be unhelpful and yield either intact mucosa , granulation / inflammatory changes or fat necrosis .", ". it can be difficult to separate the two and even endoscopic ultrasound can be non - diagnostic and adequate histology sampling may require a resection ( 3 ) . where a biopsy diagnosis has been made endoscopic resection or limited resection can be undertaken removing the need for major resectional surgery ( 3 ) .", "where this is not technically feasible a minimally invasive approach using either mini - laparotomy or laparoscopic resection has successfully been reported ( 4,5 ) .", "a 74 year old man with a background of chronic obstructive airways disease ( coad ) was referred for urgent assessment by his general practioner with a 1 month history of lower abdominal pain and altered bowel habit .", "abdominal and rectal examination was unremarkable and routine blood testing including carcinoembryonic antigen ( cea ) was normal .", "a large ulcerating polypoidal lesion was found at his hepatic flexure ( figure 1 ) .", "colonoscopy demonstrating large pedunculated ulcerating lesion found at hepatic flexure histology from biopsies taken showed no evidence of neoplasia or dysplasia but was felt to most likely be unrepresentative of the endoscopic lesion .", "a staging ct scan of chest , abdomen and pelvis was arranged as part of pre - operative staging protocol and this demonstrated a large 3.3 x 4.3 x 3.4 cm heterogeneous enhancing colonic polyp , which predominately enhanced with fatty attenuation ( figure 2 ) .", "proximal to the lesion was circumferential colonic wall thickening and surrounding fat stranding suspicious of colonic malignancy .", "there was concern about colonic malignancy and following discussion at a mdt meeting he was offered a surgical resection .", "ct scan of the abdomen showing the lesion ( marked a ) a laparoscopic extended right hemicolectomy was performed under general anaesthesia .", "there were no complications and the patient made an excellent recovery and was discharged home .", "gross and microscopic histological examination showed a submucosal lipoma with areas of fat necrosis and surface ulceration with extensive granulation tissue .", "polypoidal tumour measuring 4 cm with an area of central ulceration gross slices of the tumour show adipose tissue expanding the submucosa .", "there are areas of fat necrosis and mucosal ulceration microscopy shows lobules of mature adipocytes .", "imaging may reveal tissue with the signal and uptake of adipose tissue but a confident diagnosis may not be possible .", "endoscopists , radiologists and pathologists need to be aware of this entity . careful discussion and review of the investigations at an mdt meeting may permit a pre - operative diagnosis .", "endoscopic resection can be used where technically feasible but where doubt remains or this is not feasible then laparoscopic or open resection may be required ."], "abstract_text": ["<S> lipomas of the alimentary tract are rare tumours that can mimic malignant lesions . </S>", "<S> they are often small and asymptomatic although larger tumours can present with intusussception or as abdominal masses . </S>", "<S> we present a case of a transverse colon submucosal lipoma masquerading as a colonic adenocarcinoma leading to resection.a 74 year - old - man was referred urgently for assessment with altered bowel habits , and lower abdominal discomfort along with a positive faecal - occult - blood sample . </S>", "<S> colonoscopy demonstrated a large polypoidal lesion at the hepatic flexure with ulceration . </S>", "<S> biopsies were inconclusive . a staging ct scan confirmed a 3.3 x 4.3 x 3.4 cm polyp with colonic wall thickening suspicious of malignancy . </S>", "<S> an extended right hemi - colectomy was performed . </S>", "<S> histology showed a large submucosal lipoma with 12 reactive lymph nodes.colonic lipoma often present as incidental findings detected on either imaging or endoscopically whilst investigating other symptoms . </S>", "<S> their appearances can mimic colonic malignancy and surgical resection may be required . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["gastrointestinal lipomas are rare , mostly small and detected incidentally during endoscopic examination ( 1 ) .", "these tumours are benign proliferations of mature fatty tissue arising in the submucosa and predominately arise in the large intestine ( 51 - 70% ) preferentially on the right side .", "the majority of symptomatic lesions occur in adults in the sixth decade of life ( 2 ) .", "lipohypertrophy of the ileo - caecal valve is classified as a variant of normal ( 1 ) . the vast majority are asymptomatic but they can manifest due to intussusceptions , ulceration leading to haemorrhage or iron deficiency anaemia , intestinal obstruction or present as an abdominal mass . most are compressible and submucosal although they can rarely develop in the serosa and appear as a fold or polyp .", "they contain mature adipose tissue and usually have a thick capsule surrounding the tumour ( 2 ) .", "these changes if extensive can mimic a liposarcoma ; however , frank liposarcoma of the colon is extremely rare and requires the presence of lipoblasts ( 2 ) .", "mucosal biopsies can be unhelpful and yield either intact mucosa , granulation / inflammatory changes or fat necrosis .", ". it can be difficult to separate the two and even endoscopic ultrasound can be non - diagnostic and adequate histology sampling may require a resection ( 3 ) . where a biopsy diagnosis has been made endoscopic resection or limited resection can be undertaken removing the need for major resectional surgery ( 3 ) .", "where this is not technically feasible a minimally invasive approach using either mini - laparotomy or laparoscopic resection has successfully been reported ( 4,5 ) ."], ["a 74 year old man with a background of chronic obstructive airways disease ( coad ) was referred for urgent assessment by his general practioner with a 1 month history of lower abdominal pain and altered bowel habit .", "abdominal and rectal examination was unremarkable and routine blood testing including carcinoembryonic antigen ( cea ) was normal .", "a large ulcerating polypoidal lesion was found at his hepatic flexure ( figure 1 ) .", "colonoscopy demonstrating large pedunculated ulcerating lesion found at hepatic flexure histology from biopsies taken showed no evidence of neoplasia or dysplasia but was felt to most likely be unrepresentative of the endoscopic lesion .", "a staging ct scan of chest , abdomen and pelvis was arranged as part of pre - operative staging protocol and this demonstrated a large 3.3 x 4.3 x 3.4 cm heterogeneous enhancing colonic polyp , which predominately enhanced with fatty attenuation ( figure 2 ) .", "proximal to the lesion was circumferential colonic wall thickening and surrounding fat stranding suspicious of colonic malignancy .", "there was concern about colonic malignancy and following discussion at a mdt meeting he was offered a surgical resection .", "ct scan of the abdomen showing the lesion ( marked a ) a laparoscopic extended right hemicolectomy was performed under general anaesthesia .", "there were no complications and the patient made an excellent recovery and was discharged home .", "gross and microscopic histological examination showed a submucosal lipoma with areas of fat necrosis and surface ulceration with extensive granulation tissue .", "polypoidal tumour measuring 4 cm with an area of central ulceration gross slices of the tumour show adipose tissue expanding the submucosa .", "there are areas of fat necrosis and mucosal ulceration microscopy shows lobules of mature adipocytes ."], ["imaging may reveal tissue with the signal and uptake of adipose tissue but a confident diagnosis may not be possible .", "endoscopists , radiologists and pathologists need to be aware of this entity . careful discussion and review of the investigations at an mdt meeting may permit a pre - operative diagnosis .", "endoscopic resection can be used where technically feasible but where doubt remains or this is not feasible then laparoscopic or open resection may be required ."]]}
{"article_id": "PMC4609433", "article_text": ["pekinensis ) is a diploid ( 2n = 2x = 20 ) dicot with a genomic size of 550  mb ( http://www.brassica.info/resource/ ) .", "the species originated in china and now has become one of the most important and widely cultivated leaf vegetables in asia .", "the tight leafy head is the main edible part . after a long history of domestication", ", chinese cabbage evolves into different cultivars with a variety of characteristics , such as rosette leaf morphology , heading leaf morphology , leafy head shape , size , and structure , flowering time , nutrient composition , and resistance to biotic and abiotic .", "a better understanding of the molecular mechanism of evolution of chinese cabbage and further development of marker - assisted selection ( mas ) will accelerate the selection process of improved cultivars to meet the growing consumers and environmental needs .", "although progress has been made in underlining the molecular mechanism [ 25 ] , many aspects are still unclear .", "molecular markers have been widely used to study the genetic basis of important traits and map regulatory genes in plants .", "markers tightly linked with important agronomic traits can potentially be used for molecular breeding to develop improved cultivars .", "many molecular markers and genetic maps of chinese cabbage have been reported previously [ 625 ] .", "however , there is still a great need to develop novel molecular markers for construction of high - density linkage maps for genetics and molecular studies of important traits in chinese cabbage .", "simple sequence repeat ( ssr ) markers or microsatellite markers are among the most important markers in plants .", "ssrs have been widely used in genetic mapping and molecular breeding in plants because they are highly abundant and have significant polymorphism .", "other factors , like accessibility for detection , reliability , and codominance , also make them perfect markers for such purposes .", "ssrs found in transcribed sequences are called expressed sequence - simple sequence repeats ( est - ssrs ) . compared with genomic - ssrs detected in noncoding sequences ,", "est - ssrs are more efficient for qtl mapping , gene targeting , and mas .", "as transcribed sequences are more conserved than noncoding sequences , the transferability of est - ssrs is better than genomic - ssrs [ 2830 ] , which can be utilized for cross genome comparison and evolutionary analysis [ 27 , 31 ] .", "additionally , abundant ests were generated in recent years with the development of next - generation sequencing approaches , making identification of est - ssrs more practical and cost - efficient .", "many est - ssrs have been identified in chinese cabbage [ 16 , 20 , 25 , 3336 ] . because whole genome sequencing of chinese cabbage is still underway , new est - ssrs", "could also be identified for further studies such as high - density genetic linkage map construction , gene / qtl mapping , and cultivar identification . in our previous study , the whole transcriptomes were analyzed for the rosette leaves and folding leaves of a typical heading chinese cabbage , namely , fushanbaotou , using a solexa / illumina rna - seq platform , and a large - scale est database was generated . in this study , we further assembled those ests from the rl and fl libraries into nonredundant unigenes .", "a total of 10,420 est - ssrs were identified , among which 2744 est - ssrs are detected for the first time , according to the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .", "we characterized these identified est - ssrs and designed 7877 pcr primer pairs for 1561 est - ssrs .", "furthermore , serving as a validation purpose , we tested polymorphisms of 24 est - ssrs .", "we expect this study can pave the road for further investigation of new est - ssr markers and for construction of high - density genetic maps .", "for est - ssr identification and primer design , a typical heading chinese cabbage , namely , fushanbaotou , was used in this study . for primer assessment and ssr polymorphism analysis ,", "a panel of twenty - four cultivars of chinese cabbage was used , including nineteen morphologically diverse cultivars of brassica rapa l. ssp .", "pekinensis ( b. pekinensis l. ) and five brassica rapa l. chinensis ( b. chinensis l. ) .", "leaves were collected after they were grown for two weeks from ten seedlings of each cultivar and were pooled together for dna extraction .", "we assembled the clean read dataset presented by wang et al .   from the rl and fl libraries according to the methods described by wang et al .   using the trinity software ( http://trinityrnaseq.sourceforge.net/ ) .", "redundant sequences were removed and overlapping unigenes were assembled into continuous sequences by the tigr gene indices clustering ( tgicl ) tools .", "similarity was set at 94% and an overlap length was set at 100  bp .", "parameters were set with a minimum number of 12 , 6 , 5 , 5 , 4 , and 4 repeat units for identification of mono- , di- , tri- , tetra- , penta- , and hexanucleotide motifs , respectively .", "primer length ranged from 18 to 28  bp ( with an optimality at 23 ) .", "the physical positions of the est - ssrs identified in the study were determined by aligning the ssrs and flanking sequences ( 50  bp at each side ) to the brassica rapa ( chiifu-401 ) reference genome ( http://brassicadb.org/brad/ ) using blastn .", "new est - ssrs were identified by comparing with previously reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .", "the dna sample of the chinese cabbage fushanbaotou was used as template to detect the availability of ssr primers designed above .", "the dna samples of those aforementioned twenty - four cultivars of chinese cabbage were used as templates for ssr polymorphism analysis .", "the polymorphisms of est - ssrs were validated by 6% denaturing polyacrylamide gel electrophoresis , 12% nondenaturing polyacrylamide gel electrophoresis , and sequencing .", "high quality clean read data from the rl and fl libraries by wang et al .  ", "a total of 99,684 and 95,411 contigs were obtained , with an average length of 333 and 342  bp and a median length ( n50 ) of 531 and 536  bp , from the rl and fl libraries , respectively ( table 1 ) .", "contigs from the same transcript were detected with paired - end reads , as well as the distances between these contigs . using the trinity software package , we assembled these contigs into unigenes , in which ns were removed .", "a total of 46,294 and 48,473 unigenes from the rl and fl libraries were obtained with an average length of 707 and 680  bp and a median length ( n50 ) of 1000 and 980  bp , respectively ( table 1 ) .", "size distribution of the contigs and unigenes is consistent with the rl and fl libraries as shown in figure 1 , indicating that our illumina sequencing solution is reliable and reproducible .", "unigenes from the two samples were combined ; redundant unigenes were removed ; and the rest was assembled with tgicl   to form a single dataset , which represents 40.7  mb of sequence and contains a total of 51,694 nonredundant unigenes , with an average read length of 788  bp , and a median read length ( n50 ) of 1154  bp ( table 1 ) .", "the sequences of the unigenes are listed in table s1 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/473028 ) .", "the length of 24,271 nonredundant unigenes ( 46.95% ) is between 200 and 500  bp ; the length of 13,613 ( 26.33% ) is between 501 and 1,000  bp , and the length of 13,810 ( 26.72% ) is longer than 1,000  bp ( figure 1 ) .", "a total of 10420 est - ssrs were detected with the microsatellite software ( misa ; http://pgrc.ipk-gatersleben.de/misa/ ) in 8571 unigenes , accounting for 16.6% of total nonredundant unigenes ( tables 2 and s2 ) .", "the mean ssr density is one per 3.9  kb , corresponding to one for every 5.0 nonredundant unigenes .", "1502 unigenes ( 17.5% ) harbored more than one ssr and 666 ssrs ( 6.4% ) were present in compound formation that had more than one repeat type ( table 2 ) .", "the ssrs with tri- and dinucleotide repeat motifs were the most common ( 4,405 , 42.27% ; 4,043 , 38.80% , resp . ) , followed by mono- ( 1,644 , 15.78% ) , hexa- ( 126 , 1.21% ) , penta- ( 112 , 1.07% ) and tetra- ( 90 , 0.86% ) nucleotide repeat motifs ( figure 2 ) .", "the most common two repeat motif types accounted for 81.07% of the total ssrs detected , and the rest repeat motifs types only accounted for 18.93% .", "the iterate number of repeat units in an est - ssr ranged from 4 to 25 .", "the occurrence frequency of est - ssts with different iterate numbers was found to be unequal either .", "est - ssrs with iterate number of 5 ( 2832 , 27.18% ) were the most common ones , followed by 6 ( 2739 , 26.29% ) , 7 ( 1368 , 13.13% ) , 8 ( 703 , 6.75% ) , 12 ( 542 , 5.20% ) , and 9 ( 480 , 4.61% ) ( table s3 ) .", "a dinucleotide containing est - ssrs with a maximum of 25 repeat units was identified . for est - ssrs with more than 10 repeat units ,", "the mononucleotide repeat motifs were the most abundant , accounting for 93.46% of these est - ssrs .", "the lengths of est - ssr sequences ranged from 12 to 65  bp ( table s4 ) .", "the longest one is a pentanucleotide containing est - ssr with 65  bp in length .", "the lengths of most est - ssrs are from 12 to 20  bp , accounting for 91.47% of the total est - ssrs , followed by est - ssrs with 2130  bp in length ( 874 ssrs , 8.39% ) .", "only 13 est - ssrs were identified with over 30  bp , accounting for 0.12% of the total est - ssrs .", "a total of 124 est - ssr motifs were identified , including 2 mono- , 3 di- , 10 tri- , 13 tetra- , 33 penta- , and 63 hexanucleotide repeat units containing est - ssrs .", "the dominant motif identified in our est - ssrs was ag / ct ( 3,519 , 33.8% ) , followed by a / t ( 1,562 , 15.0% ) , aag / ctt ( 1,445 , 13.9% ) , agg / cct ( 776 , 7.4% ) , atc / atg ( 627 , 6.0% ) , aac / gtt ( 392 , 4.4% ) , acc / ggt ( 392 , 3.8% ) , ac / gt ( 349 , 3.3% ) , and agc / ctg ( 317 , 3.0% ) ( figure 3 ) .", "the other 115 motifs have low frequency , accounting only for 9.3% of total est - ssrs .", "physical locations of the est - ssrs were assigned by searching against the nonredundant ( nr ) protein database of ncbi ( http://www.ncbi.nlm.nih.gov/ ) and the brassica database ( http://brassicadb.org/brad/ ) using blastx .", "our results showed that 4329 est - ssrs ( 44.4% ) were located in coding regions ( cdss ) , 3456 ( 35.5% ) in 5-utrs , and 1297 ( 13.3% ) in 3-utrs ( figure 4 , table s4 ) .", "locations of the remaining 672 est - ssrs ( 6.9% ) were not successfully assigned ( figure 4 , table s4 ) .", "for the est - ssrs localized in the cds region , trinucleotide repeats were the most common ones , accounting for 62.72% of the total est - ssrs localized in this region , followed by dinucleotide repeats ( 897 , 20.72% ) , mononucleotide repeats ( 325 , 7.51 ) , and compound formation ( 287 , 6.63% ) ( table s4 ) .", "dinucleotide repeats ( 1909 , 55.24% ) were the dominant types in 5-utrs , followed by trinucleotide repeats ( 730 , 21.12% ) , mononucleotide repeats ( 483 , 13.98% ) , and compound formation ones ( 214 , 6.19% ) ( table s4 ) .", "mono- , di- , and trinucleotide repeat est - ssrs were the top three types found in 3-utrs , accounting for 35.08% , 30.07% , and 28.60% of the total est - ssrs localized in these regions , respectively .", "the est - ssrs and the flanking sequences ( 50  bp on each side ) were aligned to the brassica rapa ( chiifu-401 ) reference genome ( http://brassicadb.org/brad/ ) using blastn to determine their physical positions .", "new est - ssrs were identified by comparing with the earlier reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .", "a total of 2744 new est - ssrs ( 26.3% ) were identified in the study . of the 7676 known ssrs ( 73.6% ) , 2317 est - ssrs ( 22.2% ) show polymorphism with different repeat numbers , and 5359 ( 51.4% ) were exactly the same with the earlier reported ssrs based on the brassica rapa ( chiifu-401 ) genomic sequence   ( table s2 ) .", "a total of 7877 pcr primer pairs from the unique sequences flanking 1561 est - ssr loci were designed according to the criteria described in section 2 using primer 3 ( table s5 ) . for each est - ssr locus , a maximum of 5 alternative primer pairs was designed .", "the other 8859 est - ssrs , which had no appropriate pcr primer pairs designed as their flanking sequences , did not fulfill the primer design criteria mentioned above . for the 1561 est - ssrs with pcr primers designed ,", "pcr primers of those aforementioned 24 loci with n  20  bp were selected for primer synthesis and amplification evaluation in chinese cabbage fushanbaotou .", "nineteen ( 79.2% ) of these 24 est - ssr loci successfully yielded pcr amplicons in fushanbaotou .", "we sequenced these nineteen pcr amplicons and found that the amplicons in thirteen loci were exactly the same as expected ; two were longer than the expected size , and four were shorter ( table 3 ) .", "size deviation of five est - ssrs loci with the expected sizes ( br - es6 , br - es7 , br - es8 , br - es12 , and br - es18 ) was due to the variations of ssr repeat motifs ( table s6 ) .", "one amplicon ( br - es16 ) deviated from the expected sizes and had an additional 86  bp containing a ( tc)9 motif near the ssr repeat motif region ( table s6 ) .", "nineteen effective primer pairs were used for polymorphism validation for these aforementioned 24 chinese cabbage cultivars .", "the results showed that 17 loci ( 89.5% ) were polymorphic ( figure 5 ) .", "a total of 56 alleles at the 17 polymorphic loci were identified and the average number of alleles per ssr locus was 3.29 with a range between 2 and 6 .", "a maximum of 6 alleles was detected for br - es16 and br - es18 loci .", "br - es6 and br - es11 had no polymorphic allele in all 24 cultivars in this study ( figure 5 , tables 3 and s4 ) .", "of the 17 polymorphic loci , twelve loci were polymorphic in all cultivars of b. pekinensis l. and b. chinensis l. three loci ( br - es2 , br - es9 , and br - es19 ) had no polymorphism in the cultivars of b. pekinensis l. but had polymorphism in the cultivars of b. chinensis l. , while two loci ( br - es4 and br - es7 ) were polymorphic in the cultivars of b. pekinensis l. but were not polymorphic in the cultivars of b. chinensis l. ( figure 5 , table s8 ) .", "we sequenced the polymorphic alleles of the 17 polymorphic loci and found that polymorphisms of 9 loci ( br - es1 , br - es4 , br - es7 , br - es8 , br - es10 , br - es14 , br - es17 , br - es18 , and br - es19 ) were because of different iterate numbers of ssr repeat motifs . in another 6 polymorphic loci ( br - es2 , br - es3 , br - es12 , br - es13 , br - es15 , and br - es16 ) ,", "the most polymorphic alleles were found in the repeat motifs with additional changes in other regions ( table s7 ) .", "for example , compared with the allele br - es3 - 160  bp in fushanbaotou , the polymorphic alleles br - es3 - 163  bp and 145  bp had different iterate numbers of the tag / atc repeat motif , while the polymorphic allele 99  bp had not only a different number of the repeat motif , but also a deletion in another region ( table s7 ) .", "the other two polymorphic loci , br - es5 and br - es9 , had polymorphisms that are not related with the repeat numbers of ssr motifs ( table s7 ) .", "illumina paired - end rna sequencing is one of the fast immerging next - generation sequencing ( ngs ) technologies . because of its advantages in high - throughput , high accuracy , and low cost", ", illumina paired - end sequencing has been widely used for de novo transcriptome sequencing and assembly and transcriptome quality and quantity analysis in many plants [ 37 , 38 , 42 , 43 ] . in our previous study ,", "the transcriptome of rosette and folding leaves in chinese cabbage was analyzed using the illumina paired - end rna sequencing technology , and abundant clean reads and ests with high quality were obtained .", "the large quantity of clean reads would increase coverage depth of transcriptome nucleotide , enhance sequencing accuracy , and provide useful information for developing new tools for genetic mapping and molecular breeding of chinese cabbage . in this study , we further assembled the clean reads into contigs and unigenes from the rl and fl libraries , respectively .", "the parameters for both contigs and unigenes between the two libraries had no significant differences ( table 1 ) , indicating our illumina sequencing solutions have high reliability and reproducibility .", "the unigenes of the two libraries were further assembled and a total of 51,694 nonredundant unigenes were obtained from the 40.7  mb sequence data .", "we discovered more nonredundant unigenes than those in previous studies [ 35 , 36 ] , which represent a large portion of the chinese cabbage transcriptome and are important for a comprehensive understanding of est - ssrs .", "a total of 10,420 ssrs with over 12  bp were identified from the deep transcriptome sequence dataset of chinese cabbage .", "the frequency of occurrence of ssrs is slightly higher than those reported in previous studies on chinese cabbage ( about 8.415.6% ) [ 20 , 3436 ] and also higher than those of other dicotyledonous species such as peanut ( 6.8% ) , sweetpotato ( 8.2% ) , sesame ( 8.9% ) , pigeonpea ( 7.6% ) , grapes ( 2.5% ) , pepper ( 4.9% ) , and flax ( 3.5% ) , but it is lower than those of coffee ( 18.5% ) , radish ( 23.8% ) , and caster bean ( 28.4% ) .", "detection of est - ssrs depends on a number of factors such as genome structure , tools and parameters for est - ssrs detection and exploration , and size of dataset for unigene assembly .", "the frequency of ssrs with different sizes of repeat units is not evenly distributed in plants .", "previous studies showed dinucleotide ssr loci are the most abundant class in safflower , pigeonpea , and sesame , whereas trinucleotide repeats are the most frequent ones in barley , sweetpotato , jatropha curcas , iris , pepper , caster bean , flax , cucurbita pepo , and radish . in ramie   and wheat , dinucleotide and trinucleotide", "trinucleotide ( 4405 , 42.3% ) was found to be the most common repeat motif class in chinese cabbage , followed by dinucleotide ( 4043 , 38.8% ) ( figure 2 ) .", "however , on the genomic level , of chinese cabbage , dinucleotide is the most common repeat motif , followed by trinucleotide .", "we found the most dominant mononucleotide repeat motif in chinese cabbage was a / t ( 1,562 , accounting for 15.0% of the total est - ssrs ) , which is consistent with previous reports for chinese cabbage   and for other plants such as arabidopsis , rice , wheat , radish , castor bean , gossypium raimondii , oil palm , and eggplant .", "for dinucleotide motif , ag / ct was the most common repeat motif , accounting for 87.0% of the total dinucleotide est - ssrs .", "it is in close agreement with the results in previous studies for genic ssrs in chinese cabbage [ 20 , 36 ] and those in most other plants such as sweetpotato , iris , sesame , and radish .", "the ag / ct repeat motif was also the most dominant repeat among all the est - ssrs identified in this study , accounting for 33.8% of the total est - ssrs .", "however , for genomic - ssrs in chinese cabbage , at / ta is the most common dinucleotide motif .", "the aag / ctt ( 1,445 , 13.9% ) motif was the most frequent motif among trinucleotide est - ssrs in the study , which is consistent with the results in previous studies in chinese cabbage [ 25 , 36 ] and many dicot species , for example , arabidopsis , soybean , peanut , sweetpotato , radish , and sesame .", "in many monocot species such as maize , barley , and sorghum [ 66 , 67 ] , ccg / ggc is the most dominant trinucleotide repeat motif .", "it is considered a specific feature of monocot genomes due to the high gc content in monocot genomes .", "of all 10420 est - ssrs identified in this study , more than 70% have been identified and presented in the ssr marker database ( http://oilcrops.info/ssrdb ) , among which over half were exactly the same with the earlier reported ssrs based on the brassica rapa ( chiifu-401 ) genomic sequence ( table s2 ) .", "2317 est - ssrs ( 22.2% ) with polymorphism in different repeat numbers could further be used for identification of chiifu-401 and fushanbaotou and for genetic linkage map constructions using these two cultivars as parents .", "a total of 2744 new est - ssrs ( 26.3% ) were identified in the study , which , in combination with previously discovered est - ssrs , could be used for high - density genetic linkage map construction , gene / qtl mapping , cultivar identification , and so forth . in the present study ,", "79.2% of the est - ssrs primer pairs selected for primer evaluation successfully generated high quality amplicons , indicating that the ests from the high - throughput rna sequencing of chinese cabbage transcriptome are suitable for specific primer design .", "the unsuccessfully designed primer pairs may be due to splice sites , large introns , chimeric primer(s ) , or poor quality sequences .", "we found that all amplicons contained the expected ssrs and the ssrs in 13 amplicons were exactly the same as predicted ( table s6 ) .", "the deviation of est - ssr pcr amplicons from the expected size is likely due to the presence of introns , large insertions or repeat number variations , a lack of specificity , or assembly errors . in the present study", ", we found five of six amplicons with unexpected sizes had different iterate number of ssr repeat units , while the other one had a 86  bp insertion near the expected ssr repeat motif region ( table s6 ) .", "these results suggested that the unigenes assembled from the high - throughput rna sequencing of chinese cabbage transcriptome are reliable , and the est - ssrs identified in our dataset could be used for further studies , such as genetic mapping and cultivar identification .", "most of the est - ssr loci ( accounting for 89.5% of the tested loci ) were found to be polymorphic among the 24 tested cabbage cultivars .", "the mean number of alleles per ssr locus was 3.29 with a range between 2 and 6 ( table 3 ) , indicating that polymorphism of est - ssrs in chinese cabbage is relatively high .", "most of the polymorphisms of the tested est - ssr loci are due to the variations of ssr repeat motifs in this study .", "there were only two loci where the polymorphisms were not related to the ssr repeat motif variations ( table s6 ) .", "the results indicate that the est - ssrs identified and the pcr primers designed in this study could further be used for constructing high - density genetic linkage maps , mapping quantitative trait loci , assessing germplasm polymorphism and evolution , marker - assisted selection , and cloning functional gene in chinese cabbage . in summary , we assembled a large set of clean reads with high quality derived from the chinese cabbage transcriptome using high - throughput rna sequencing technology with a solexa / illumina platform .", "a total of 51,694 nonredundant unigenes were obtained from 40.7  mb sequence data , providing substantial knowledge for est - ssr identification and characterization .", "10,420 est - ssrs were identified and characterized , and pcr primer pairs for 1561 est - ssrs were designed . by comparing with previously reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) , we identified a total of 2744 new est - ssrs .", "primer pairs for 24 est - ssrs were selected for primer evaluation , and 79.2% of the 24 est - ssr loci successfully generated high quality amplicons . among the effective primers , 89.5% of them showed polymorphism in 24 cultivars of chinese cabbage .", "the est - ssrs developed in this study , in combination with previously reported est - ssrs , will provide valuable resources for constructing high - density genetic linkage maps , mapping quantitative trait loci , assessing germplasm polymorphism and evolution , marker - assisted selection , and cloning functional gene in chinese cabbage . to our knowledge , this is the first successful attempt to develop large quantity of est - ssrs with high quality based on the transcriptome of chinese cabbage using high - throughput rna sequencing technology ."], "abstract_text": ["<S> simple sequence repeats ( ssrs ) are among the most important markers for population analysis and have been widely used in plant genetic mapping and molecular breeding . expressed sequence tag - ssr ( est - ssr ) markers , located in the coding regions , are potentially more efficient for qtl mapping , gene targeting , and marker - assisted breeding . in this study </S>", "<S> , we investigated 51,694 nonredundant unigenes , assembled from clean reads from deep transcriptome sequencing with a solexa / illumina platform , for identification and development of est - ssrs in chinese cabbage . in total , 10,420 est - ssrs with over 12  bp were identified and characterized , among which 2744 est - ssrs are new and 2317 are known ones showing polymorphism with previously reported ssrs . </S>", "<S> a total of 7877 pcr primer pairs for 1561 est - ssr loci were designed , and primer pairs for twenty - four est - ssrs were selected for primer evaluation . in nineteen est - ssr loci ( 79.2% ) , amplicons were successfully generated with high quality . </S>", "<S> seventeen ( 89.5% ) showed polymorphism in twenty - four cultivars of chinese cabbage . </S>", "<S> the polymorphic alleles of each polymorphic locus were sequenced , and the results showed that most polymorphisms were due to variations of ssr repeat motifs . </S>", "<S> the est - ssrs identified and characterized in this study have important implications for developing new tools for genetics and molecular breeding in chinese cabbage . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["pekinensis ) is a diploid ( 2n = 2x = 20 ) dicot with a genomic size of 550  mb ( http://www.brassica.info/resource/ ) .", "the species originated in china and now has become one of the most important and widely cultivated leaf vegetables in asia .", "the tight leafy head is the main edible part . after a long history of domestication", ", chinese cabbage evolves into different cultivars with a variety of characteristics , such as rosette leaf morphology , heading leaf morphology , leafy head shape , size , and structure , flowering time , nutrient composition , and resistance to biotic and abiotic .", "a better understanding of the molecular mechanism of evolution of chinese cabbage and further development of marker - assisted selection ( mas ) will accelerate the selection process of improved cultivars to meet the growing consumers and environmental needs .", "although progress has been made in underlining the molecular mechanism [ 25 ] , many aspects are still unclear .", "molecular markers have been widely used to study the genetic basis of important traits and map regulatory genes in plants .", "markers tightly linked with important agronomic traits can potentially be used for molecular breeding to develop improved cultivars .", "many molecular markers and genetic maps of chinese cabbage have been reported previously [ 625 ] .", "however , there is still a great need to develop novel molecular markers for construction of high - density linkage maps for genetics and molecular studies of important traits in chinese cabbage .", "simple sequence repeat ( ssr ) markers or microsatellite markers are among the most important markers in plants .", "ssrs have been widely used in genetic mapping and molecular breeding in plants because they are highly abundant and have significant polymorphism .", "other factors , like accessibility for detection , reliability , and codominance , also make them perfect markers for such purposes .", "ssrs found in transcribed sequences are called expressed sequence - simple sequence repeats ( est - ssrs ) . compared with genomic - ssrs detected in noncoding sequences ,", "est - ssrs are more efficient for qtl mapping , gene targeting , and mas .", "as transcribed sequences are more conserved than noncoding sequences , the transferability of est - ssrs is better than genomic - ssrs [ 2830 ] , which can be utilized for cross genome comparison and evolutionary analysis [ 27 , 31 ] .", "additionally , abundant ests were generated in recent years with the development of next - generation sequencing approaches , making identification of est - ssrs more practical and cost - efficient .", "many est - ssrs have been identified in chinese cabbage [ 16 , 20 , 25 , 3336 ] . because whole genome sequencing of chinese cabbage is still underway , new est - ssrs", "could also be identified for further studies such as high - density genetic linkage map construction , gene / qtl mapping , and cultivar identification . in our previous study , the whole transcriptomes were analyzed for the rosette leaves and folding leaves of a typical heading chinese cabbage , namely , fushanbaotou , using a solexa / illumina rna - seq platform , and a large - scale est database was generated . in this study , we further assembled those ests from the rl and fl libraries into nonredundant unigenes .", "a total of 10,420 est - ssrs were identified , among which 2744 est - ssrs are detected for the first time , according to the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .", "we characterized these identified est - ssrs and designed 7877 pcr primer pairs for 1561 est - ssrs .", "furthermore , serving as a validation purpose , we tested polymorphisms of 24 est - ssrs .", "we expect this study can pave the road for further investigation of new est - ssr markers and for construction of high - density genetic maps ."], ["for est - ssr identification and primer design , a typical heading chinese cabbage , namely , fushanbaotou , was used in this study . for primer assessment and ssr polymorphism analysis ,", "a panel of twenty - four cultivars of chinese cabbage was used , including nineteen morphologically diverse cultivars of brassica rapa l. ssp .", "pekinensis ( b. pekinensis l. ) and five brassica rapa l. chinensis ( b. chinensis l. ) .", "leaves were collected after they were grown for two weeks from ten seedlings of each cultivar and were pooled together for dna extraction .", "we assembled the clean read dataset presented by wang et al .   from the rl and fl libraries according to the methods described by wang et al .   using the trinity software ( http://trinityrnaseq.sourceforge.net/ ) .", "redundant sequences were removed and overlapping unigenes were assembled into continuous sequences by the tigr gene indices clustering ( tgicl ) tools .", "similarity was set at 94% and an overlap length was set at 100  bp .", "parameters were set with a minimum number of 12 , 6 , 5 , 5 , 4 , and 4 repeat units for identification of mono- , di- , tri- , tetra- , penta- , and hexanucleotide motifs , respectively .", "primer length ranged from 18 to 28  bp ( with an optimality at 23 ) .", "the physical positions of the est - ssrs identified in the study were determined by aligning the ssrs and flanking sequences ( 50  bp at each side ) to the brassica rapa ( chiifu-401 ) reference genome ( http://brassicadb.org/brad/ ) using blastn .", "new est - ssrs were identified by comparing with previously reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .", "the dna sample of the chinese cabbage fushanbaotou was used as template to detect the availability of ssr primers designed above .", "the dna samples of those aforementioned twenty - four cultivars of chinese cabbage were used as templates for ssr polymorphism analysis .", "the polymorphisms of est - ssrs were validated by 6% denaturing polyacrylamide gel electrophoresis , 12% nondenaturing polyacrylamide gel electrophoresis , and sequencing ."], ["high quality clean read data from the rl and fl libraries by wang et al .  ", "a total of 99,684 and 95,411 contigs were obtained , with an average length of 333 and 342  bp and a median length ( n50 ) of 531 and 536  bp , from the rl and fl libraries , respectively ( table 1 ) .", "contigs from the same transcript were detected with paired - end reads , as well as the distances between these contigs . using the trinity software package , we assembled these contigs into unigenes , in which ns were removed .", "a total of 46,294 and 48,473 unigenes from the rl and fl libraries were obtained with an average length of 707 and 680  bp and a median length ( n50 ) of 1000 and 980  bp , respectively ( table 1 ) .", "size distribution of the contigs and unigenes is consistent with the rl and fl libraries as shown in figure 1 , indicating that our illumina sequencing solution is reliable and reproducible .", "unigenes from the two samples were combined ; redundant unigenes were removed ; and the rest was assembled with tgicl   to form a single dataset , which represents 40.7  mb of sequence and contains a total of 51,694 nonredundant unigenes , with an average read length of 788  bp , and a median read length ( n50 ) of 1154  bp ( table 1 ) .", "the sequences of the unigenes are listed in table s1 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/473028 ) .", "the length of 24,271 nonredundant unigenes ( 46.95% ) is between 200 and 500  bp ; the length of 13,613 ( 26.33% ) is between 501 and 1,000  bp , and the length of 13,810 ( 26.72% ) is longer than 1,000  bp ( figure 1 ) .", "a total of 10420 est - ssrs were detected with the microsatellite software ( misa ; http://pgrc.ipk-gatersleben.de/misa/ ) in 8571 unigenes , accounting for 16.6% of total nonredundant unigenes ( tables 2 and s2 ) .", "the mean ssr density is one per 3.9  kb , corresponding to one for every 5.0 nonredundant unigenes .", "1502 unigenes ( 17.5% ) harbored more than one ssr and 666 ssrs ( 6.4% ) were present in compound formation that had more than one repeat type ( table 2 ) .", "the ssrs with tri- and dinucleotide repeat motifs were the most common ( 4,405 , 42.27% ; 4,043 , 38.80% , resp . ) , followed by mono- ( 1,644 , 15.78% ) , hexa- ( 126 , 1.21% ) , penta- ( 112 , 1.07% ) and tetra- ( 90 , 0.86% ) nucleotide repeat motifs ( figure 2 ) .", "the most common two repeat motif types accounted for 81.07% of the total ssrs detected , and the rest repeat motifs types only accounted for 18.93% .", "the iterate number of repeat units in an est - ssr ranged from 4 to 25 .", "the occurrence frequency of est - ssts with different iterate numbers was found to be unequal either .", "est - ssrs with iterate number of 5 ( 2832 , 27.18% ) were the most common ones , followed by 6 ( 2739 , 26.29% ) , 7 ( 1368 , 13.13% ) , 8 ( 703 , 6.75% ) , 12 ( 542 , 5.20% ) , and 9 ( 480 , 4.61% ) ( table s3 ) .", "a dinucleotide containing est - ssrs with a maximum of 25 repeat units was identified . for est - ssrs with more than 10 repeat units ,", "the mononucleotide repeat motifs were the most abundant , accounting for 93.46% of these est - ssrs .", "the lengths of est - ssr sequences ranged from 12 to 65  bp ( table s4 ) .", "the longest one is a pentanucleotide containing est - ssr with 65  bp in length .", "the lengths of most est - ssrs are from 12 to 20  bp , accounting for 91.47% of the total est - ssrs , followed by est - ssrs with 2130  bp in length ( 874 ssrs , 8.39% ) .", "only 13 est - ssrs were identified with over 30  bp , accounting for 0.12% of the total est - ssrs .", "a total of 124 est - ssr motifs were identified , including 2 mono- , 3 di- , 10 tri- , 13 tetra- , 33 penta- , and 63 hexanucleotide repeat units containing est - ssrs .", "the dominant motif identified in our est - ssrs was ag / ct ( 3,519 , 33.8% ) , followed by a / t ( 1,562 , 15.0% ) , aag / ctt ( 1,445 , 13.9% ) , agg / cct ( 776 , 7.4% ) , atc / atg ( 627 , 6.0% ) , aac / gtt ( 392 , 4.4% ) , acc / ggt ( 392 , 3.8% ) , ac / gt ( 349 , 3.3% ) , and agc / ctg ( 317 , 3.0% ) ( figure 3 ) .", "the other 115 motifs have low frequency , accounting only for 9.3% of total est - ssrs .", "physical locations of the est - ssrs were assigned by searching against the nonredundant ( nr ) protein database of ncbi ( http://www.ncbi.nlm.nih.gov/ ) and the brassica database ( http://brassicadb.org/brad/ ) using blastx .", "our results showed that 4329 est - ssrs ( 44.4% ) were located in coding regions ( cdss ) , 3456 ( 35.5% ) in 5-utrs , and 1297 ( 13.3% ) in 3-utrs ( figure 4 , table s4 ) .", "locations of the remaining 672 est - ssrs ( 6.9% ) were not successfully assigned ( figure 4 , table s4 ) .", "for the est - ssrs localized in the cds region , trinucleotide repeats were the most common ones , accounting for 62.72% of the total est - ssrs localized in this region , followed by dinucleotide repeats ( 897 , 20.72% ) , mononucleotide repeats ( 325 , 7.51 ) , and compound formation ( 287 , 6.63% ) ( table s4 ) .", "dinucleotide repeats ( 1909 , 55.24% ) were the dominant types in 5-utrs , followed by trinucleotide repeats ( 730 , 21.12% ) , mononucleotide repeats ( 483 , 13.98% ) , and compound formation ones ( 214 , 6.19% ) ( table s4 ) .", "mono- , di- , and trinucleotide repeat est - ssrs were the top three types found in 3-utrs , accounting for 35.08% , 30.07% , and 28.60% of the total est - ssrs localized in these regions , respectively .", "the est - ssrs and the flanking sequences ( 50  bp on each side ) were aligned to the brassica rapa ( chiifu-401 ) reference genome ( http://brassicadb.org/brad/ ) using blastn to determine their physical positions .", "new est - ssrs were identified by comparing with the earlier reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) .", "a total of 2744 new est - ssrs ( 26.3% ) were identified in the study . of the 7676 known ssrs ( 73.6% ) , 2317 est - ssrs ( 22.2% ) show polymorphism with different repeat numbers , and 5359 ( 51.4% ) were exactly the same with the earlier reported ssrs based on the brassica rapa ( chiifu-401 ) genomic sequence   ( table s2 ) .", "a total of 7877 pcr primer pairs from the unique sequences flanking 1561 est - ssr loci were designed according to the criteria described in section 2 using primer 3 ( table s5 ) . for each est - ssr locus , a maximum of 5 alternative primer pairs was designed .", "the other 8859 est - ssrs , which had no appropriate pcr primer pairs designed as their flanking sequences , did not fulfill the primer design criteria mentioned above . for the 1561 est - ssrs with pcr primers designed ,", "pcr primers of those aforementioned 24 loci with n  20  bp were selected for primer synthesis and amplification evaluation in chinese cabbage fushanbaotou .", "nineteen ( 79.2% ) of these 24 est - ssr loci successfully yielded pcr amplicons in fushanbaotou .", "we sequenced these nineteen pcr amplicons and found that the amplicons in thirteen loci were exactly the same as expected ; two were longer than the expected size , and four were shorter ( table 3 ) .", "size deviation of five est - ssrs loci with the expected sizes ( br - es6 , br - es7 , br - es8 , br - es12 , and br - es18 ) was due to the variations of ssr repeat motifs ( table s6 ) .", "one amplicon ( br - es16 ) deviated from the expected sizes and had an additional 86  bp containing a ( tc)9 motif near the ssr repeat motif region ( table s6 ) .", "nineteen effective primer pairs were used for polymorphism validation for these aforementioned 24 chinese cabbage cultivars .", "the results showed that 17 loci ( 89.5% ) were polymorphic ( figure 5 ) .", "a total of 56 alleles at the 17 polymorphic loci were identified and the average number of alleles per ssr locus was 3.29 with a range between 2 and 6 .", "a maximum of 6 alleles was detected for br - es16 and br - es18 loci .", "br - es6 and br - es11 had no polymorphic allele in all 24 cultivars in this study ( figure 5 , tables 3 and s4 ) .", "of the 17 polymorphic loci , twelve loci were polymorphic in all cultivars of b. pekinensis l. and b. chinensis l. three loci ( br - es2 , br - es9 , and br - es19 ) had no polymorphism in the cultivars of b. pekinensis l. but had polymorphism in the cultivars of b. chinensis l. , while two loci ( br - es4 and br - es7 ) were polymorphic in the cultivars of b. pekinensis l. but were not polymorphic in the cultivars of b. chinensis l. ( figure 5 , table s8 ) .", "we sequenced the polymorphic alleles of the 17 polymorphic loci and found that polymorphisms of 9 loci ( br - es1 , br - es4 , br - es7 , br - es8 , br - es10 , br - es14 , br - es17 , br - es18 , and br - es19 ) were because of different iterate numbers of ssr repeat motifs . in another 6 polymorphic loci ( br - es2 , br - es3 , br - es12 , br - es13 , br - es15 , and br - es16 ) ,", "the most polymorphic alleles were found in the repeat motifs with additional changes in other regions ( table s7 ) .", "for example , compared with the allele br - es3 - 160  bp in fushanbaotou , the polymorphic alleles br - es3 - 163  bp and 145  bp had different iterate numbers of the tag / atc repeat motif , while the polymorphic allele 99  bp had not only a different number of the repeat motif , but also a deletion in another region ( table s7 ) .", "the other two polymorphic loci , br - es5 and br - es9 , had polymorphisms that are not related with the repeat numbers of ssr motifs ( table s7 ) ."], ["illumina paired - end rna sequencing is one of the fast immerging next - generation sequencing ( ngs ) technologies . because of its advantages in high - throughput , high accuracy , and low cost", ", illumina paired - end sequencing has been widely used for de novo transcriptome sequencing and assembly and transcriptome quality and quantity analysis in many plants [ 37 , 38 , 42 , 43 ] . in our previous study ,", "the transcriptome of rosette and folding leaves in chinese cabbage was analyzed using the illumina paired - end rna sequencing technology , and abundant clean reads and ests with high quality were obtained .", "the large quantity of clean reads would increase coverage depth of transcriptome nucleotide , enhance sequencing accuracy , and provide useful information for developing new tools for genetic mapping and molecular breeding of chinese cabbage . in this study , we further assembled the clean reads into contigs and unigenes from the rl and fl libraries , respectively .", "the parameters for both contigs and unigenes between the two libraries had no significant differences ( table 1 ) , indicating our illumina sequencing solutions have high reliability and reproducibility .", "the unigenes of the two libraries were further assembled and a total of 51,694 nonredundant unigenes were obtained from the 40.7  mb sequence data .", "we discovered more nonredundant unigenes than those in previous studies [ 35 , 36 ] , which represent a large portion of the chinese cabbage transcriptome and are important for a comprehensive understanding of est - ssrs .", "a total of 10,420 ssrs with over 12  bp were identified from the deep transcriptome sequence dataset of chinese cabbage .", "the frequency of occurrence of ssrs is slightly higher than those reported in previous studies on chinese cabbage ( about 8.415.6% ) [ 20 , 3436 ] and also higher than those of other dicotyledonous species such as peanut ( 6.8% ) , sweetpotato ( 8.2% ) , sesame ( 8.9% ) , pigeonpea ( 7.6% ) , grapes ( 2.5% ) , pepper ( 4.9% ) , and flax ( 3.5% ) , but it is lower than those of coffee ( 18.5% ) , radish ( 23.8% ) , and caster bean ( 28.4% ) .", "detection of est - ssrs depends on a number of factors such as genome structure , tools and parameters for est - ssrs detection and exploration , and size of dataset for unigene assembly .", "the frequency of ssrs with different sizes of repeat units is not evenly distributed in plants .", "previous studies showed dinucleotide ssr loci are the most abundant class in safflower , pigeonpea , and sesame , whereas trinucleotide repeats are the most frequent ones in barley , sweetpotato , jatropha curcas , iris , pepper , caster bean , flax , cucurbita pepo , and radish . in ramie   and wheat , dinucleotide and trinucleotide", "trinucleotide ( 4405 , 42.3% ) was found to be the most common repeat motif class in chinese cabbage , followed by dinucleotide ( 4043 , 38.8% ) ( figure 2 ) .", "however , on the genomic level , of chinese cabbage , dinucleotide is the most common repeat motif , followed by trinucleotide .", "we found the most dominant mononucleotide repeat motif in chinese cabbage was a / t ( 1,562 , accounting for 15.0% of the total est - ssrs ) , which is consistent with previous reports for chinese cabbage   and for other plants such as arabidopsis , rice , wheat , radish , castor bean , gossypium raimondii , oil palm , and eggplant .", "for dinucleotide motif , ag / ct was the most common repeat motif , accounting for 87.0% of the total dinucleotide est - ssrs .", "it is in close agreement with the results in previous studies for genic ssrs in chinese cabbage [ 20 , 36 ] and those in most other plants such as sweetpotato , iris , sesame , and radish .", "the ag / ct repeat motif was also the most dominant repeat among all the est - ssrs identified in this study , accounting for 33.8% of the total est - ssrs .", "however , for genomic - ssrs in chinese cabbage , at / ta is the most common dinucleotide motif .", "the aag / ctt ( 1,445 , 13.9% ) motif was the most frequent motif among trinucleotide est - ssrs in the study , which is consistent with the results in previous studies in chinese cabbage [ 25 , 36 ] and many dicot species , for example , arabidopsis , soybean , peanut , sweetpotato , radish , and sesame .", "in many monocot species such as maize , barley , and sorghum [ 66 , 67 ] , ccg / ggc is the most dominant trinucleotide repeat motif .", "it is considered a specific feature of monocot genomes due to the high gc content in monocot genomes .", "of all 10420 est - ssrs identified in this study , more than 70% have been identified and presented in the ssr marker database ( http://oilcrops.info/ssrdb ) , among which over half were exactly the same with the earlier reported ssrs based on the brassica rapa ( chiifu-401 ) genomic sequence ( table s2 ) .", "2317 est - ssrs ( 22.2% ) with polymorphism in different repeat numbers could further be used for identification of chiifu-401 and fushanbaotou and for genetic linkage map constructions using these two cultivars as parents .", "a total of 2744 new est - ssrs ( 26.3% ) were identified in the study , which , in combination with previously discovered est - ssrs , could be used for high - density genetic linkage map construction , gene / qtl mapping , cultivar identification , and so forth . in the present study ,", "79.2% of the est - ssrs primer pairs selected for primer evaluation successfully generated high quality amplicons , indicating that the ests from the high - throughput rna sequencing of chinese cabbage transcriptome are suitable for specific primer design .", "the unsuccessfully designed primer pairs may be due to splice sites , large introns , chimeric primer(s ) , or poor quality sequences .", "we found that all amplicons contained the expected ssrs and the ssrs in 13 amplicons were exactly the same as predicted ( table s6 ) .", "the deviation of est - ssr pcr amplicons from the expected size is likely due to the presence of introns , large insertions or repeat number variations , a lack of specificity , or assembly errors . in the present study", ", we found five of six amplicons with unexpected sizes had different iterate number of ssr repeat units , while the other one had a 86  bp insertion near the expected ssr repeat motif region ( table s6 ) .", "these results suggested that the unigenes assembled from the high - throughput rna sequencing of chinese cabbage transcriptome are reliable , and the est - ssrs identified in our dataset could be used for further studies , such as genetic mapping and cultivar identification .", "most of the est - ssr loci ( accounting for 89.5% of the tested loci ) were found to be polymorphic among the 24 tested cabbage cultivars .", "the mean number of alleles per ssr locus was 3.29 with a range between 2 and 6 ( table 3 ) , indicating that polymorphism of est - ssrs in chinese cabbage is relatively high .", "most of the polymorphisms of the tested est - ssr loci are due to the variations of ssr repeat motifs in this study .", "there were only two loci where the polymorphisms were not related to the ssr repeat motif variations ( table s6 ) .", "the results indicate that the est - ssrs identified and the pcr primers designed in this study could further be used for constructing high - density genetic linkage maps , mapping quantitative trait loci , assessing germplasm polymorphism and evolution , marker - assisted selection , and cloning functional gene in chinese cabbage . in summary , we assembled a large set of clean reads with high quality derived from the chinese cabbage transcriptome using high - throughput rna sequencing technology with a solexa / illumina platform .", "a total of 51,694 nonredundant unigenes were obtained from 40.7  mb sequence data , providing substantial knowledge for est - ssr identification and characterization .", "10,420 est - ssrs were identified and characterized , and pcr primer pairs for 1561 est - ssrs were designed . by comparing with previously reported ssrs in the ssr marker database for brassica ( http://oilcrops.info/ssrdb ) , we identified a total of 2744 new est - ssrs .", "primer pairs for 24 est - ssrs were selected for primer evaluation , and 79.2% of the 24 est - ssr loci successfully generated high quality amplicons . among the effective primers , 89.5% of them showed polymorphism in 24 cultivars of chinese cabbage .", "the est - ssrs developed in this study , in combination with previously reported est - ssrs , will provide valuable resources for constructing high - density genetic linkage maps , mapping quantitative trait loci , assessing germplasm polymorphism and evolution , marker - assisted selection , and cloning functional gene in chinese cabbage . to our knowledge , this is the first successful attempt to develop large quantity of est - ssrs with high quality based on the transcriptome of chinese cabbage using high - throughput rna sequencing technology ."]]}
{"article_id": "PMC3543581", "article_text": ["much of the current success in clinical positron emission tomography ( pet ) can be attributed to the development of 2-(f ) fluoro-2-deoxy - d - glucose ( f18-fdg ) .", "it is the most commonly used radiopharmaceutical in the pet , with a half - life of 110 min . before the release of f18-fdg for human use , the quality control tests need to be performed to check the safety of the product .", "the bet using gel clot method is a 60-min test and typically performed after the decay of the f18-fdg sample to determine the endotoxin content .", "the gel clot is technically the most simple and was the first bet approved by the food and drug administration ( fda ) .", "endotoxin is a subset of pyrogens that are strictly of gram - negative origin , a natural complex of lipopolysaccharides occurring in the outer layer of the bilayered gram - negative bacterial cell . from the circulating blood cells of limulus polyphemus , called amebocytes ,", "a clear lysate is obtained that forms an opaque gel in the presence of extremely low concentrations of bacterial endotoxins .", "pharmacopeia ( usp ) , pharmacopeia , f18-fdg should not contain more than 175/v usp endotoxin units ( eu ) per milliliter of f18-fdg injection , in which v is the maximum administered total dose in milliliters , at the expiration time .", "however , no such standards have been recommended by indian pharmacopoeia commission ( ipc ) till date . the ipc is taking measures to include radiopharmaceuticals in the indian pharmacopoeia .", "the objective of this study protocol was to set up a standard pyrogen testing facility and to establish documented evidence if the process employed for bet testing of f18-fdg by gel clot method produces the desired results consistently , when performed as per the standard operating procedures .", "random samples of the f18-fdg from september 2010 to march 2011 were subjected to the gel clot bet .", "f18-fdg was synthesized in the in - house medical cyclotron and radiochemistry lab at the department of nuclear medicine and pet centre , using the tracerlab mxfdg ( ge healthcare , mnster , germany ) .", "the assay was performed at four different maximum valid dilutions ( mvds , i.e. 1:10 , 1:100 , 1:350 and 1:700 ) .", "the ph of the samples was measured to ensure a ph within the range 6.08.0 for the validity of the gel clot procedure .", "the standard gel clot test for assessing the bacterial endotoxin content consisted of four controls : negative water control ( nwc ) , positive water control ( pwc ) , negative product control ( npc ) and positive product control ( ppc ) .", "the nwc is the endotoxin - free limulus amaebocyte lysate ( lal ) reagent water .", "the matched control standard endotoxin ( cse ) , lal reagent and lal reagent water obtained from charles river laboratories india private limited , bangalore , india , were used in this study .", "the sensitivity of the lal reagent contained in each vial was 0.125 eu / ml . the test for confirmation of lysate sensitivity was carried out with every new batch of the lysate as per the usp < 85 > recommendations .", "the cse having predetermined amount of endotoxin was prepared according to the directions for use given in the specific certificate of analysis ( coa ) .", "the cse stock solution of 20 eu / ml was prepared with endotoxin - free water and vortexed for 5 min .", "all the dilutions were made in the depyrogenated 16  125 mm glass tubes using endotoxin - free water and pyrogen - free sterile pipettes tips .", "depyrogenations were performed in the dry heat oven at 250c for 1 hour , when the endotoxin - free glass tubes were not obtained commercially .", "the bet was performed using 0.1 ml of f18-fdg sample and 0.1 ml of reconstituted lal reagent per tube .", "the lal reagent was constituted after the addition of lal reagent water ( endotoxin free ) in the freeze - dried power of lysate .", "the pwc and ppc used for these tests were at the endotoxin concentration of 0.5 eu / ml . the four dilutions of the f18-fdg , i.e. 1:10 , 1:100 , 1:350 and 1:700 , were freshly prepared from the batches and subjected to the gel clot assay .", "the ppc was separately prepared for all the dilutions at the endotoxin concentration of 0.25 eu / ml . the reaction solution was mixed thoroughly and placed immediately in the incubator at 37c1c for 602 min . at the end of the incubation period ,", "if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive .", "a positive test indicated that the concentration of endotoxin in the tube is greater than or equal to the sensitivity of the lal reagent .", "any other state of the reaction mixture constituted a negative test , which indicated an endotoxin concentration less than the lal reagent sensitivity .", "the test was considered negative when the tube is inverted and the gel breaks or collapses .", "random samples of the f18-fdg from september 2010 to march 2011 were subjected to the gel clot bet .", "f18-fdg was synthesized in the in - house medical cyclotron and radiochemistry lab at the department of nuclear medicine and pet centre , using the tracerlab mxfdg ( ge healthcare , mnster , germany ) .", "the assay was performed at four different maximum valid dilutions ( mvds , i.e. 1:10 , 1:100 , 1:350 and 1:700 ) .", "the ph of the samples was measured to ensure a ph within the range 6.08.0 for the validity of the gel clot procedure .", "the standard gel clot test for assessing the bacterial endotoxin content consisted of four controls : negative water control ( nwc ) , positive water control ( pwc ) , negative product control ( npc ) and positive product control ( ppc ) .", "the nwc is the endotoxin - free limulus amaebocyte lysate ( lal ) reagent water .", "the matched control standard endotoxin ( cse ) , lal reagent and lal reagent water obtained from charles river laboratories india private limited , bangalore , india , were used in this study .", "the sensitivity of the lal reagent contained in each vial was 0.125 eu / ml . the test for confirmation of lysate sensitivity was carried out with every new batch of the lysate as per the usp < 85 > recommendations .", "the cse having predetermined amount of endotoxin was prepared according to the directions for use given in the specific certificate of analysis ( coa ) .", "the cse stock solution of 20 eu / ml was prepared with endotoxin - free water and vortexed for 5 min .", "all the dilutions were made in the depyrogenated 16  125 mm glass tubes using endotoxin - free water and pyrogen - free sterile pipettes tips .", "depyrogenations were performed in the dry heat oven at 250c for 1 hour , when the endotoxin - free glass tubes were not obtained commercially .", "the bet was performed using 0.1 ml of f18-fdg sample and 0.1 ml of reconstituted lal reagent per tube .", "the lal reagent was constituted after the addition of lal reagent water ( endotoxin free ) in the freeze - dried power of lysate .", "the pwc and ppc used for these tests were at the endotoxin concentration of 0.5 eu / ml . the four dilutions of the f18-fdg , i.e. 1:10 , 1:100 , 1:350 and 1:700 , were freshly prepared from the batches and subjected to the gel clot assay .", "the ppc was separately prepared for all the dilutions at the endotoxin concentration of 0.25 eu / ml . the reaction solution was mixed thoroughly and placed immediately in the incubator at 37c1c for 602 min . at the end of the incubation period ,", "if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive .", "a positive test indicated that the concentration of endotoxin in the tube is greater than or equal to the sensitivity of the lal reagent .", "any other state of the reaction mixture constituted a negative test , which indicated an endotoxin concentration less than the lal reagent sensitivity .", "the test was considered negative when the tube is inverted and the gel breaks or collapses .", "the mean ph was 7.05 ( 6.57.5 ) . the total numbers of tests performed on the f18-fdg were 100 excluding nwc , pwc and ppc .", "table 1 summarizes the results of the undiluted f18-fdg on the positive control solutions at an endotoxin concentration of 0.5 eu / ml ( 4 ) . of the 10 undiluted f18-fdg batches ,", "this indicated that all the batches did not contain endotoxin greater than 0.5 eu / ml .", "all the 20 positive endotoxin control pwc vials gelled . only 18 out of 20 ppc vials gelled after incubation at 37c for 60 min .", "results for undiluted f18-fdg sample table 2 summarizes the results for the 60-min gel clot assay on the four different dilutions of the f18-fdg samples .", "all the 20 positive endotoxin control pwc vials gelled . none of the 20 1:10 f18-fdg sample vials gelled after 60-min incubation at 37c . however , 1 out of 20 ppc vials for the 1:10 dilution did not gel probably due to some interference with the gel formation at the lysate sensitivity of 0.125 eu / ml .", "all the remaining vials for the 1:100 , 1 : 350 and 1:700 dilutions did not gel .", "this indicated that all the vials did not contain endotoxin greater than 0.125 eu / ml . results for the diluted f18-fdg samples", "the gel clot method is often considered as the most accurate and sensitive procedure for testing endotoxin content in injectable radiopharmaceutical products because fewer false - positive and false - negative results are observed when this method is used . the total time taken to complete", "the lal reaction requires optimal ph range . in the undiluted samples , the measured ph ( 7.05 ) was well within the acceptable range ( i.e. 6.08.0 ) for the gel clot assay .", "the f18-fdg produced in the tracerlab mx synthesis module is neutralized in the citrate buffer which contains multivalent ions .", "nakao et al . reported the effect of the citrate salt concentration on the endotoxin test for the f18-fdg preparations .", "these authors reported that the undiluted sample showed the recovery less than 20% , beyond the acceptance of the usp .", "similar results were found in the present study with the undiluted samples as two vials of the ppc ( endotoxin concentration equal to 0.5 eu / ml ) did not gel , indicating the interference with the gel formation . the interference may be due to the presence of the citrate ions . at the expiration time , the maximum administered total dose in milliliters of fdg should contain less than 175 eu .", "therefore , the total f18-fdg preparation at any time did not contain more than 8 eu ( 0.5 eu / ml  16 ml ) .", "however , the undiluted sample did show the inhibition of the gel formation as shown in table 1 .", "therefore , the sample was tested for the various dilutions 1:10 , 1:100 , 1:350 and 1:700 .", "the 350 and 700 dilution factors were considered according to the limit of 175 eu / ml ( single patient dose supplied to the other institute or when the final elution is in 23 ml due to some technical errors ) . the rate of inhibition improved with the dilutions in table 2 .", "the gel clot assay has a major drawback which limits its use for the short - lived radiopharmaceuticals .", "in addition to this drawback , errors made by technical personnel and the misinterpretation of results are also common problems .", "the 60-min bet is described in usp<85 > ,  bacterial endotoxins test  , and is also recommended for pyrogenicity testing in the draft guidance of the fda on current good manufacturing practice ( cgmp ) for pet drug products . because the remainder of the required quality assurance ( qa ) testing for f18-fdg injection , with the exception of the sterility test , can be completed in approximately 2030 min , delaying the release of the short - lived f18-fdg injection for an additional 3040 min is not practical and is , in fact , wasteful .", "an in - process 20-min bet must be performed before release of the drug product , and a standard 60-min bet must also be completed . some other techniques to determine endotoxins", "are also known , such as the chromogenic ( color development ) and the turbidimetric ( turbidity development ) tests , both of which can provide valuable quantitative and qualitative information about the endotoxin concentration in samples . regardless of which method is used , pet laboratories and pharmacies should ensure that their technicians are well trained and educated in the endotoxin testing method of choice .", "this study suggests that the bet needs to be performed , standardized and documented in each cyclotron and pet facility .", "the dilution of the test sample is the easiest means to resolve the potential product inhibition / enhancement problem during the gel clot testing procedure .", "the final f18-fdg preparation at any time point did not contain more than 8 eu , thus making it safe for human administration ."], "abstract_text": ["<S> background : the bacterial endotoxin test ( bet ) performed using gel clot method is a 60-min test and typically performed after the decay of the 2-(18f ) fluoro-2-deoxy - d - glucose ( f18-fdg ) sample to determine the endotoxin content . </S>", "<S> the objective of this study protocol was to perform bet testing of f18-fdg by gel clot method.materials and methods : ten random decayed samples of the f18-fdg were subjected to the gel clot bet . </S>", "<S> the assay was performed with undiluted f18-fdg and at four different maximum valid dilutions of 1:10 , 1:100 , 1:350 and 1:700 ( total number of tests = 100 ) . </S>", "<S> the sensitivity of the lal reagent used was 0.125 eu / ml . endotoxin dilutions were freshly prepared from control standard endotoxin ( cse ) stock solution for each f18-fdg batch testing . if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive . </S>", "<S> any other state of the reaction mixture constituted a negative test.results:in the undiluted samples , the measured ph ( 7.05 ) was well within the acceptable range ( i.e. 6.08.0 ) for the gel clot assay . </S>", "<S> of the 10 undiluted f18-fdg batches and all the diluted samples , none gelled after 60-min incubation period at 37c . </S>", "<S> however , the undiluted f18-fdg did inhibit gel formation at the lysate sensitivity of 0.125 eu / ml.conclusion : the total volume of fdg produced was 16 ml in the synthesis module . </S>", "<S> the total f18-fdg preparation at any time did not contain more than 8 eu ( 0.5 eu / ml  16 ml ) . </S>", "<S> thus , the product is safe for human administration . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "F18-FDG sample preparation", "Preparation of endotoxin stock solution and endotoxin dilutions", "Glassware for BET", "Routine test procedure", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["much of the current success in clinical positron emission tomography ( pet ) can be attributed to the development of 2-(f ) fluoro-2-deoxy - d - glucose ( f18-fdg ) .", "it is the most commonly used radiopharmaceutical in the pet , with a half - life of 110 min . before the release of f18-fdg for human use , the quality control tests need to be performed to check the safety of the product .", "the bet using gel clot method is a 60-min test and typically performed after the decay of the f18-fdg sample to determine the endotoxin content .", "the gel clot is technically the most simple and was the first bet approved by the food and drug administration ( fda ) .", "endotoxin is a subset of pyrogens that are strictly of gram - negative origin , a natural complex of lipopolysaccharides occurring in the outer layer of the bilayered gram - negative bacterial cell . from the circulating blood cells of limulus polyphemus , called amebocytes ,", "a clear lysate is obtained that forms an opaque gel in the presence of extremely low concentrations of bacterial endotoxins .", "pharmacopeia ( usp ) , pharmacopeia , f18-fdg should not contain more than 175/v usp endotoxin units ( eu ) per milliliter of f18-fdg injection , in which v is the maximum administered total dose in milliliters , at the expiration time .", "however , no such standards have been recommended by indian pharmacopoeia commission ( ipc ) till date . the ipc is taking measures to include radiopharmaceuticals in the indian pharmacopoeia .", "the objective of this study protocol was to set up a standard pyrogen testing facility and to establish documented evidence if the process employed for bet testing of f18-fdg by gel clot method produces the desired results consistently , when performed as per the standard operating procedures ."], ["random samples of the f18-fdg from september 2010 to march 2011 were subjected to the gel clot bet .", "f18-fdg was synthesized in the in - house medical cyclotron and radiochemistry lab at the department of nuclear medicine and pet centre , using the tracerlab mxfdg ( ge healthcare , mnster , germany ) .", "the assay was performed at four different maximum valid dilutions ( mvds , i.e. 1:10 , 1:100 , 1:350 and 1:700 ) .", "the ph of the samples was measured to ensure a ph within the range 6.08.0 for the validity of the gel clot procedure .", "the standard gel clot test for assessing the bacterial endotoxin content consisted of four controls : negative water control ( nwc ) , positive water control ( pwc ) , negative product control ( npc ) and positive product control ( ppc ) .", "the nwc is the endotoxin - free limulus amaebocyte lysate ( lal ) reagent water .", "the matched control standard endotoxin ( cse ) , lal reagent and lal reagent water obtained from charles river laboratories india private limited , bangalore , india , were used in this study .", "the sensitivity of the lal reagent contained in each vial was 0.125 eu / ml . the test for confirmation of lysate sensitivity was carried out with every new batch of the lysate as per the usp < 85 > recommendations .", "the cse having predetermined amount of endotoxin was prepared according to the directions for use given in the specific certificate of analysis ( coa ) .", "the cse stock solution of 20 eu / ml was prepared with endotoxin - free water and vortexed for 5 min .", "all the dilutions were made in the depyrogenated 16  125 mm glass tubes using endotoxin - free water and pyrogen - free sterile pipettes tips .", "depyrogenations were performed in the dry heat oven at 250c for 1 hour , when the endotoxin - free glass tubes were not obtained commercially .", "the bet was performed using 0.1 ml of f18-fdg sample and 0.1 ml of reconstituted lal reagent per tube .", "the lal reagent was constituted after the addition of lal reagent water ( endotoxin free ) in the freeze - dried power of lysate .", "the pwc and ppc used for these tests were at the endotoxin concentration of 0.5 eu / ml . the four dilutions of the f18-fdg , i.e. 1:10 , 1:100 , 1:350 and 1:700 , were freshly prepared from the batches and subjected to the gel clot assay .", "the ppc was separately prepared for all the dilutions at the endotoxin concentration of 0.25 eu / ml . the reaction solution was mixed thoroughly and placed immediately in the incubator at 37c1c for 602 min . at the end of the incubation period ,", "if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive .", "a positive test indicated that the concentration of endotoxin in the tube is greater than or equal to the sensitivity of the lal reagent .", "any other state of the reaction mixture constituted a negative test , which indicated an endotoxin concentration less than the lal reagent sensitivity .", "the test was considered negative when the tube is inverted and the gel breaks or collapses ."], ["random samples of the f18-fdg from september 2010 to march 2011 were subjected to the gel clot bet .", "f18-fdg was synthesized in the in - house medical cyclotron and radiochemistry lab at the department of nuclear medicine and pet centre , using the tracerlab mxfdg ( ge healthcare , mnster , germany ) .", "the assay was performed at four different maximum valid dilutions ( mvds , i.e. 1:10 , 1:100 , 1:350 and 1:700 ) .", "the ph of the samples was measured to ensure a ph within the range 6.08.0 for the validity of the gel clot procedure ."], ["the standard gel clot test for assessing the bacterial endotoxin content consisted of four controls : negative water control ( nwc ) , positive water control ( pwc ) , negative product control ( npc ) and positive product control ( ppc ) .", "the nwc is the endotoxin - free limulus amaebocyte lysate ( lal ) reagent water .", "the matched control standard endotoxin ( cse ) , lal reagent and lal reagent water obtained from charles river laboratories india private limited , bangalore , india , were used in this study .", "the sensitivity of the lal reagent contained in each vial was 0.125 eu / ml . the test for confirmation of lysate sensitivity was carried out with every new batch of the lysate as per the usp < 85 > recommendations .", "the cse having predetermined amount of endotoxin was prepared according to the directions for use given in the specific certificate of analysis ( coa ) .", "the cse stock solution of 20 eu / ml was prepared with endotoxin - free water and vortexed for 5 min ."], ["all the dilutions were made in the depyrogenated 16  125 mm glass tubes using endotoxin - free water and pyrogen - free sterile pipettes tips .", "depyrogenations were performed in the dry heat oven at 250c for 1 hour , when the endotoxin - free glass tubes were not obtained commercially ."], ["the bet was performed using 0.1 ml of f18-fdg sample and 0.1 ml of reconstituted lal reagent per tube .", "the lal reagent was constituted after the addition of lal reagent water ( endotoxin free ) in the freeze - dried power of lysate .", "the pwc and ppc used for these tests were at the endotoxin concentration of 0.5 eu / ml . the four dilutions of the f18-fdg , i.e. 1:10 , 1:100 , 1:350 and 1:700 , were freshly prepared from the batches and subjected to the gel clot assay .", "the ppc was separately prepared for all the dilutions at the endotoxin concentration of 0.25 eu / ml . the reaction solution was mixed thoroughly and placed immediately in the incubator at 37c1c for 602 min . at the end of the incubation period ,", "if the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180 , the test was considered positive .", "a positive test indicated that the concentration of endotoxin in the tube is greater than or equal to the sensitivity of the lal reagent .", "any other state of the reaction mixture constituted a negative test , which indicated an endotoxin concentration less than the lal reagent sensitivity .", "the test was considered negative when the tube is inverted and the gel breaks or collapses ."], ["the mean ph was 7.05 ( 6.57.5 ) . the total numbers of tests performed on the f18-fdg were 100 excluding nwc , pwc and ppc .", "table 1 summarizes the results of the undiluted f18-fdg on the positive control solutions at an endotoxin concentration of 0.5 eu / ml ( 4 ) . of the 10 undiluted f18-fdg batches ,", "this indicated that all the batches did not contain endotoxin greater than 0.5 eu / ml .", "all the 20 positive endotoxin control pwc vials gelled . only 18 out of 20 ppc vials gelled after incubation at 37c for 60 min .", "results for undiluted f18-fdg sample table 2 summarizes the results for the 60-min gel clot assay on the four different dilutions of the f18-fdg samples .", "all the 20 positive endotoxin control pwc vials gelled . none of the 20 1:10 f18-fdg sample vials gelled after 60-min incubation at 37c . however , 1 out of 20 ppc vials for the 1:10 dilution did not gel probably due to some interference with the gel formation at the lysate sensitivity of 0.125 eu / ml .", "all the remaining vials for the 1:100 , 1 : 350 and 1:700 dilutions did not gel .", "this indicated that all the vials did not contain endotoxin greater than 0.125 eu / ml . results for the diluted f18-fdg samples"], ["the gel clot method is often considered as the most accurate and sensitive procedure for testing endotoxin content in injectable radiopharmaceutical products because fewer false - positive and false - negative results are observed when this method is used . the total time taken to complete", "the lal reaction requires optimal ph range . in the undiluted samples , the measured ph ( 7.05 ) was well within the acceptable range ( i.e. 6.08.0 ) for the gel clot assay .", "the f18-fdg produced in the tracerlab mx synthesis module is neutralized in the citrate buffer which contains multivalent ions .", "nakao et al . reported the effect of the citrate salt concentration on the endotoxin test for the f18-fdg preparations .", "these authors reported that the undiluted sample showed the recovery less than 20% , beyond the acceptance of the usp .", "similar results were found in the present study with the undiluted samples as two vials of the ppc ( endotoxin concentration equal to 0.5 eu / ml ) did not gel , indicating the interference with the gel formation . the interference may be due to the presence of the citrate ions . at the expiration time , the maximum administered total dose in milliliters of fdg should contain less than 175 eu .", "therefore , the total f18-fdg preparation at any time did not contain more than 8 eu ( 0.5 eu / ml  16 ml ) .", "however , the undiluted sample did show the inhibition of the gel formation as shown in table 1 .", "therefore , the sample was tested for the various dilutions 1:10 , 1:100 , 1:350 and 1:700 .", "the 350 and 700 dilution factors were considered according to the limit of 175 eu / ml ( single patient dose supplied to the other institute or when the final elution is in 23 ml due to some technical errors ) . the rate of inhibition improved with the dilutions in table 2 .", "the gel clot assay has a major drawback which limits its use for the short - lived radiopharmaceuticals .", "in addition to this drawback , errors made by technical personnel and the misinterpretation of results are also common problems .", "the 60-min bet is described in usp<85 > ,  bacterial endotoxins test  , and is also recommended for pyrogenicity testing in the draft guidance of the fda on current good manufacturing practice ( cgmp ) for pet drug products . because the remainder of the required quality assurance ( qa ) testing for f18-fdg injection , with the exception of the sterility test , can be completed in approximately 2030 min , delaying the release of the short - lived f18-fdg injection for an additional 3040 min is not practical and is , in fact , wasteful .", "an in - process 20-min bet must be performed before release of the drug product , and a standard 60-min bet must also be completed . some other techniques to determine endotoxins", "are also known , such as the chromogenic ( color development ) and the turbidimetric ( turbidity development ) tests , both of which can provide valuable quantitative and qualitative information about the endotoxin concentration in samples . regardless of which method is used , pet laboratories and pharmacies should ensure that their technicians are well trained and educated in the endotoxin testing method of choice ."], ["this study suggests that the bet needs to be performed , standardized and documented in each cyclotron and pet facility .", "the dilution of the test sample is the easiest means to resolve the potential product inhibition / enhancement problem during the gel clot testing procedure .", "the final f18-fdg preparation at any time point did not contain more than 8 eu , thus making it safe for human administration ."]]}
{"article_id": "PMC3552635", "article_text": ["prostate cancer becomes one of the most common cancers in developed and developing countries [ 13 ] .", "it is because life expectancy rised ; age is one of the prostate cancer risk factors .", "moreover there is wide availability of prostate - specific antigen ( psa ) serum test , which makes possible to diagnose clinically silent , low stage disease .", "number of such patients is growing and makes management of this disease even harder in every day oncology care ; optimal management is still controversial .", "there is a wide range of treatment methods and one of the most important outcome risk factors is local tumor control .", "there are risk categories , in which are propositions of treatment ( table 1 ) .", "very low risk category was introduced by national comprehensive cancer center ( nccn ) in 2010 . \n ", "risk categories and treatment options according to nccn   prostate specific antigen density if predicted probability of nodal involvement is  2% neoadjuvant , concomitant or adjuvant in selected patients without fixation radical prostatectomy ( rp ) remains one of basic curative therapy for prostate cancer , especially for low and inermediate risk patients .", "complications include perioperative short- term side effects as blood loss , wound infection or from anesthesia , and long - term , postoperative , as incontinence or impotence [ 58 ] .", "three - dimensional conformal radiotherapy ( 3dcrt ) , dynamic techniques like intense - modulated ( imrt ) or arc radiotherapy and extracranial stereotactic techniques allow to prepare normal tissue sparing plans with clinical target volume ( ctv ) high dose prescription . not only photon beams , but also particles , e.g. protons are used for highly conformal irradiation . every day", "image - guided radiotherapy ( igrt ) use , especially with intraprostatic seed markers , makes possible dose escalation and less toxicity with smaller margins contouring .", "brachytherapy , combined with external - beam radiotherapy ( ebrt ) or alone , remains one of best tools for absolute dose escalation inside prostate .", "due to low alpha / beta ratio for prostate cancer these protocols are favorable , in which hypofractionation is used .", "both low - dose ( ldr ) and high - dose rate ( hdr ) brachytherapy ( bt ) are used in prostate cancer patients .", "27 kev , half - life : 59 days ) or palladium-103 ( pd ; mean photon energy  21kev , half - life : 17 days )", "hdr brachytherapy is , particularly , based on iridium-192 ( ir ; mean photon energy  400 kev , half - life : 74 days ) stepping source , which is driven into temporary implant catheters by computer - controlled unit .", "it also makes high dose escalation possible , but interstitial implant is needed [ 12 , 13 ] .", "radiotherapy complications are acute and late , mostly in bladder and rectum , like incontinence , diarrhea , urinary stricture , impotence and proctitis .", "outcome is similar to surgery , however tolerance of treatment seems better for radiotherapy excluding proctitis rates , which are higher in irradiated patients .", "patient for rt are usually those , who can not undergo surgery , older than 65 years or with locally advanced disease , e.g. extracapsular spread [ 5 , 14 , 15 ] .", "patients with shorter life expectancy or inconvinced to treat their disease are recommended for psa testing at least every six months , digital rectal examination ( dre ) at least every twelve months and , especially those with longer life expectancy , for repeated prostate biopsy at least every year . if there is disease progression observed , then patient should undergo clinical evaluation to choose optimal treatment option .", "although clinical progression is still not well defined and requires physician judgement and treatment option should be chosen wisely after patient discussion with his doctor [ 5 , 16 ] .", "comparative analysis of psa free survival outcomes by prostate cancer results study group has been published , recently .", "it contains comparison of results from 848 articles selected from over 18000 published between 2000 and 2010 .", "only radical treatment was considered containing bt ( including hdr ) , ebrt ( including imrt ) , rp , proton thereapy , hifu and cryotherapy .", "pcrsg agreed unanimously for study inclusion criteria as : patient stratification into pretreatment groups ( according to d'amico , zelefsky or nccn ) , psa free time endpoint ( astro , phoenix , psa < 0.2 ng / ml for rp ) , both clinical and pathological staging , minimum of 72 gy ebrt ( imrt or 3dcrt ) , results published in peer - review journals only , minimum median follow - up of 5 years .", "morover minimum patients number for each risk group was accepted as 100 for low and intermediate and 50 for high risk group .", "results of this study suggest that bt alone , particularly seed implant , provides superior psa free survival in low risk patients .", "ebrt combined with bt seems to be equal to bt alone , and better than ebrt alone or rp , in intermediate risk group .", "combined irradiation ( ebrt with bt ) with or without androgen deprivation therapy seems to be superior for high - risk patients .", "although these results are encouraging to choose bt as an element of managment , it should be remembered that selection bias may play main role , mostly in intermediate risk group . according to lack of large , randomized studies these may help to choose ultimate treatment decision both for physician and his patient .", "bt seems to be superior option for dose escalation and psa control in prostate cancer patients .", "this is also reason to recall guidelines of european and north american societies on prostate cancer brachytherapy , both hdr and ldr .", "european association of urology states that transperineal bt alone qualification criteria are : clinical stage between t1c and t2a , without nodal involvment or metastases , six or less gleason score diagnosed with sufficient number of random biopsies , initial psa level of 10 ng / ml or less , no more than 50% of biopsy cores with cancer , prostate volume of less than 50 cm and a good ipss ( < 20 fair tolerance ; < 9 good tolerance ) . ldr - bt is recommended both by eau and gec / estro as safe and effective in low risk group of prostate cancer patients with life expectancy longer than 5 years .", "post - turp patients should avoid this procedure , however if turp was performed several years earlier some patients can undergo ldr - bt after careful evaluation .", "hdr - bt is recommended as a dose escalation technique combined with ebrt for patients with intermediate or high risk of failure with life expectancy longer than 5 years .", "the exclusion criteria are : prostate volume bigger than 60 cm ( hormonal downsizing to reduce benign prostate hyperplasia is indicated ) , transurethral resection of prostate within six months , infiltration of external sphincter of the bladder neck , significant urinary obstructive symptoms ( residual urine volume > 50 cm , ipss > 12 and qmax < 15 cm / s ) , technical issues ( pubic arch interference , trus prostate - rectum distance less then 5 mm , lack of proper positioning of patient , anaesthesia or complementary ebrt contraindications ) .", "moreover these guidelines state that hdr - bt alone should be considered as an investigational treatment with proper fractionation .", "salvage hdr - bt should be also performed in a prospective clinical trial , only .", "abs states that hdr - bt provides highly conformal treatment with less irradiation for pelvic organs - at - risk and excellent outcome .", "all patients should be considered for these aproach , particularly for intermediate and high relapse risk patients as an irradiation boost or as a monotherapy for low risk patients .", "prior rectal surgery or pelvic irradiation , i.e. rectal cancer treatment , in prostate cancer patients should be carefully evaluated and complications and outcome risk factors should be discussed with special hdr - bt center expertise .", "inflammatory bowel disease patients should be considered for hdr - bt if not suitable for rp , asymptomatic and not required treatment for at least 0.5 to 10 years .", "hdr - bt alone seems to be better than ebrt alone or combined with hdr boost , because of smaller bowel volumes irradiated .", "turp should be performed at least 90 days before hdr - bt , an aerated gel should be given into urethtra to outline its shape and special attention should be given to not exceed 110% of prescription dose .", "most feasible glands for hdr - bt are those beneath 50 cm , but abs guidelines allow to treat larger ones .", "pubic arch interference , baseline urinary function and application technique ( fixed template or freehand ) should be carefully considered .", "patients should be informed about potential risks of erectile dysfunction or acute and late urinary side effects .", "abs states that absolute contraindications include preexisting rectal fistule , impossibility of anaesthesia and no proof of malignancy .", "absolute contraindications include poor performance status and life expectancy shorter than 10 years , spinal or general anaesthesia not possible , large post turp defects and lack of rectum , which makes trus impossible .", "ipss value over 20 points can correlate with increased urinary toxicity and needs careful evaluation .", "prostate volume over 60 cm is not a contraindication and even with pubic arch interference can be considered for ldr - bt with short adt course ( 3 - 4 months ) to downsize the gland .", "also freehand implantation is acceptable , but experienced practitioners should perform ldr - bt in such cases .", "abs states that ldr - bt alone should be done in low risk patients and as option for intermediate group .", "ebrt and permanent seed combination is considered as standard of care for high risk patients .", "hdr - bt , according to gec / estro - eau recommendations , should be done under trus guidance with template , its proper fixation and treatment planning software .", "additional imaging with ct or mri after implantation may be used . clinical target volume ( ctv ) for hdr - bt can be whole gland (  prostate surface ) with homogenous needles distribution or with addition of visible tumor boost inside ctv .", "prostate surface dose prescription differs among cancer centers and is between 6 to 10 gy per fraction with total dose of 12 - 20 gy in 2 to 4 fractions combined with ebrt of 45 to 54 gy in 6 - 7 weeks .", "organs - at - risk doses should not exceed 120% of mtd for urethra and less then 6 gy per fraction for rectum . those doses should be precisely stated and valuated in clinical trials with long follow - up including penile bulb maximum dose .", "proximal tips of catheters should be visualized and images should be taken at least 9 mm above and below ctv .", "organs - at - risk should be confined including rectum , urethra , bladder and penile bulb .", "minimum of 14 catheters should be placed or more , particularly if  boost within boost is planned . especially urethra piercing should be avoided .", "at least 90% of ctv should be covered with prescribed dose with aiming 95% of ctv .", "hdr - bt boost is given in one to six and hdr - bt alone in three to six fractions .", "due to wide heterogeneity of fractionation schedules , oar constraints are based on experienced hdr centers references , e.g. from less then 105% to less then 125% for urethra .", "ldr - bt should be performed under trus guidance according to gec / estro guidelines . if possible gross tumor volume ( gtv ) should be delineated on pre - treatment scans .", "ctv should contain whole prostate with 3 mm margin in all directions except posterior ( rectal wall ) and cranial ( bladder neck ) if needed .", "dose prescription to 100% isodose should be 145 gy for i and 125 gy for pd seeds .", "at least 95% of ctv should be covered with 100% isodose with no more than 50% of ctv with isodose 150% .", "maximum dose in 2 cm for rectal wall should be no more than 145 gy and in 0.1 cm less than 200 gy .", "150% of prescribed dose should cover less than 10% of urethra volume and 130% of this dose should cover less than 30% of its volume .", "it should be done as a preplanning , either as a separate procedure or on the implanting day in operational room .", "main procedure should be performed with trus and template ; patient position and probe angle should be similar to preplanned .", "minimum is biplanar , high resolution ( 5 - 12 mhz ) trus with dedicated planning software .", "team should consist at least of trained radiation oncologist and qualified medical physicist , but presence of urologist , certified dosimetrist or other supportive staff is appropriate .", "abs does not suggest any seed deposition technique , however it should have excellent outcome , be reproducible and with optimal dosimetry . both stranded and loose seeds can be chosen for implantation .", "i and pd implants showed excellent outcomes , so abs do not favor any of them .", "ldr - bt alone prescription dose to ptv should range from 140 - 160 gy for i seeds and 110 - 125 gy for pd .", "if combination of ebrt and seed implantation is planned , i ptv dose should be between 108 - 110 gy and pd ptv dose should be between 90 - 100 gy .", "ebrt dose should be 41.4 - 50.4 gy ( 1.8 gy / day , but 2 gy / day is also acceptable ) .", "ldr - bt is usually performed 0 - 8 weeks after ebrt , but there is no evidence for optimal sequence of these .", "reverse approach is also accepted , which may theoretically be better , because of simultaneous irradiation from implant and ebrt and possibility of adjusting ebrt if needed .", "dose constrains for organs at risk should not exceed 150% of prescribed dose in 5% and 125% of prescribed dose in 30% of urethral volume in the preplan .", "prescribed dose should be in less then 1 cm of rectum on day 1 dosimetry and in less then 1.3 cm on day 30 .", "although ct performed immediately or one day after the implantation has several positives , e.g. early detection of dosimetric problems , but after implant oedema can derive dosimetric underestimations .", "it is 12 - 20 days after implantation for pd and 23 - 37 days for i. erectile function organs at risk e.g. penile bulb are still not agreed , though there are no recommendations on these .", "both ldr and hdr - bt patients should be seen 6 weeks after implantation ( last one for temporary implants ) according to gec / estro recommendations .", "should be examined every 3 months , every 6 months until 5 years after treatment and then annually .", "dre , psa test , side effects evaluation ( urinary , rectal , potency toxicity and ipss ) and trus should be done [ 18 , 20 ] .", "abs suggests to follow - up hdr - bt patients every six months for first 2 - 3 years , and then at least annually .", "it should consist of dre , psa test and toxicity evaluation ( urinary , rectal and sexual function ) .", "phoenix definition of biochemical relapse is recommended , although case individual assessment is required and patient should be informed about psa bounce .", "ldr - bt patients should be seen every six to twelve months with regular intervals .", "post bt rectal biopsies or urinary instrumentation should be avoided and all of the risks and benefits should be considered [ 21 , 22 ] .", "brachytherapy is supreme tool in prostate cancer management with a wide range of options in every risk group ( table 2 ) .", "excellent outcomes and relatively small irradiated volume seems more beneficial than any risk of intraoperative implantation , particularly in wisely selected patients .", "feasibility of seeding or catheterization is basic for choosing appropriate candidates for bt , however there are articles describing series of patient with non - fitting anatomy , i.e. prostate smaller than 20 cm or bigger then 50 cm or post turp patients [ 24 , 25 ] .", "assesment should consist of careful clinical examination including dre , patient 's history ( i.e. urologic , prior pelvic rt , surgery or trauma , inflammatory bowel disease , connective tissue dissorders ) , pretherapy psa serum level , biopsy proven cancer with gleason score and clinical staging .", "trus stays basic imaging , but ct and mri are also very useful , either before procedure and as a planning guidance .", "new abilities of mri , i.e. spectroscopy and diffusion , and 11c pet - ct should be discussed more in next recommendations [ 2628 ] .", "gec / estro and eau recommendations on prostate cancer treatment with temporary implants with stepping sources are from 2005 and on permanent seeds from 2000 with 2007 update .", "although general approach for bt , both hdr and ldr , has not particular differences across the world , european recommendations need an update . \n ", "recommendations comparison for gec/ estro and abs [ 18 , 2022 ] under investigation for high risk patients to institutional review board "], "abstract_text": ["<S> prostate cancer , due to wide availability of psa tests , is very often diagnosed in early stage , nowadays . </S>", "<S> this makes management of this disease even harder in every day oncology care . </S>", "<S> there is a wide range of treatment options including surgery , radiotherapy and active surveillance , but essential question is which treatment patient and oncologist should decide for . </S>", "<S> due to recent publication of prostate cancer results study group , in which brachytherapy is one of supreme curative options for prostate cancer , we decided to overview most present european and north american recommendations . </S>", "<S> national comprehensive cancer network , american society for radiation oncology , american brachytherapy society , european association of urology and groupe europen de curiethrapie of european society for therapeutic radiation oncology guidelines are overviewed , particularly focusing on hdr and ldr brachytherapy . </S>"], "labels": null, "section_names": ["Purpose", "Patient selection", "Application and treatment planning", "Follow-up", "Summary"], "sections": [["prostate cancer becomes one of the most common cancers in developed and developing countries [ 13 ] .", "it is because life expectancy rised ; age is one of the prostate cancer risk factors .", "moreover there is wide availability of prostate - specific antigen ( psa ) serum test , which makes possible to diagnose clinically silent , low stage disease .", "number of such patients is growing and makes management of this disease even harder in every day oncology care ; optimal management is still controversial .", "there is a wide range of treatment methods and one of the most important outcome risk factors is local tumor control .", "there are risk categories , in which are propositions of treatment ( table 1 ) .", "very low risk category was introduced by national comprehensive cancer center ( nccn ) in 2010 . \n ", "risk categories and treatment options according to nccn   prostate specific antigen density if predicted probability of nodal involvement is  2% neoadjuvant , concomitant or adjuvant in selected patients without fixation radical prostatectomy ( rp ) remains one of basic curative therapy for prostate cancer , especially for low and inermediate risk patients .", "complications include perioperative short- term side effects as blood loss , wound infection or from anesthesia , and long - term , postoperative , as incontinence or impotence [ 58 ] .", "three - dimensional conformal radiotherapy ( 3dcrt ) , dynamic techniques like intense - modulated ( imrt ) or arc radiotherapy and extracranial stereotactic techniques allow to prepare normal tissue sparing plans with clinical target volume ( ctv ) high dose prescription . not only photon beams , but also particles , e.g. protons are used for highly conformal irradiation . every day", "image - guided radiotherapy ( igrt ) use , especially with intraprostatic seed markers , makes possible dose escalation and less toxicity with smaller margins contouring .", "brachytherapy , combined with external - beam radiotherapy ( ebrt ) or alone , remains one of best tools for absolute dose escalation inside prostate .", "due to low alpha / beta ratio for prostate cancer these protocols are favorable , in which hypofractionation is used .", "both low - dose ( ldr ) and high - dose rate ( hdr ) brachytherapy ( bt ) are used in prostate cancer patients .", "27 kev , half - life : 59 days ) or palladium-103 ( pd ; mean photon energy  21kev , half - life : 17 days )", "hdr brachytherapy is , particularly , based on iridium-192 ( ir ; mean photon energy  400 kev , half - life : 74 days ) stepping source , which is driven into temporary implant catheters by computer - controlled unit .", "it also makes high dose escalation possible , but interstitial implant is needed [ 12 , 13 ] .", "radiotherapy complications are acute and late , mostly in bladder and rectum , like incontinence , diarrhea , urinary stricture , impotence and proctitis .", "outcome is similar to surgery , however tolerance of treatment seems better for radiotherapy excluding proctitis rates , which are higher in irradiated patients .", "patient for rt are usually those , who can not undergo surgery , older than 65 years or with locally advanced disease , e.g. extracapsular spread [ 5 , 14 , 15 ] .", "patients with shorter life expectancy or inconvinced to treat their disease are recommended for psa testing at least every six months , digital rectal examination ( dre ) at least every twelve months and , especially those with longer life expectancy , for repeated prostate biopsy at least every year . if there is disease progression observed , then patient should undergo clinical evaluation to choose optimal treatment option .", "although clinical progression is still not well defined and requires physician judgement and treatment option should be chosen wisely after patient discussion with his doctor [ 5 , 16 ] .", "comparative analysis of psa free survival outcomes by prostate cancer results study group has been published , recently .", "it contains comparison of results from 848 articles selected from over 18000 published between 2000 and 2010 .", "only radical treatment was considered containing bt ( including hdr ) , ebrt ( including imrt ) , rp , proton thereapy , hifu and cryotherapy .", "pcrsg agreed unanimously for study inclusion criteria as : patient stratification into pretreatment groups ( according to d'amico , zelefsky or nccn ) , psa free time endpoint ( astro , phoenix , psa < 0.2 ng / ml for rp ) , both clinical and pathological staging , minimum of 72 gy ebrt ( imrt or 3dcrt ) , results published in peer - review journals only , minimum median follow - up of 5 years .", "morover minimum patients number for each risk group was accepted as 100 for low and intermediate and 50 for high risk group .", "results of this study suggest that bt alone , particularly seed implant , provides superior psa free survival in low risk patients .", "ebrt combined with bt seems to be equal to bt alone , and better than ebrt alone or rp , in intermediate risk group .", "combined irradiation ( ebrt with bt ) with or without androgen deprivation therapy seems to be superior for high - risk patients .", "although these results are encouraging to choose bt as an element of managment , it should be remembered that selection bias may play main role , mostly in intermediate risk group . according to lack of large , randomized studies these may help to choose ultimate treatment decision both for physician and his patient .", "bt seems to be superior option for dose escalation and psa control in prostate cancer patients .", "this is also reason to recall guidelines of european and north american societies on prostate cancer brachytherapy , both hdr and ldr ."], ["european association of urology states that transperineal bt alone qualification criteria are : clinical stage between t1c and t2a , without nodal involvment or metastases , six or less gleason score diagnosed with sufficient number of random biopsies , initial psa level of 10 ng / ml or less , no more than 50% of biopsy cores with cancer , prostate volume of less than 50 cm and a good ipss ( < 20 fair tolerance ; < 9 good tolerance ) . ldr - bt is recommended both by eau and gec / estro as safe and effective in low risk group of prostate cancer patients with life expectancy longer than 5 years .", "post - turp patients should avoid this procedure , however if turp was performed several years earlier some patients can undergo ldr - bt after careful evaluation .", "hdr - bt is recommended as a dose escalation technique combined with ebrt for patients with intermediate or high risk of failure with life expectancy longer than 5 years .", "the exclusion criteria are : prostate volume bigger than 60 cm ( hormonal downsizing to reduce benign prostate hyperplasia is indicated ) , transurethral resection of prostate within six months , infiltration of external sphincter of the bladder neck , significant urinary obstructive symptoms ( residual urine volume > 50 cm , ipss > 12 and qmax < 15 cm / s ) , technical issues ( pubic arch interference , trus prostate - rectum distance less then 5 mm , lack of proper positioning of patient , anaesthesia or complementary ebrt contraindications ) .", "moreover these guidelines state that hdr - bt alone should be considered as an investigational treatment with proper fractionation .", "salvage hdr - bt should be also performed in a prospective clinical trial , only .", "abs states that hdr - bt provides highly conformal treatment with less irradiation for pelvic organs - at - risk and excellent outcome .", "all patients should be considered for these aproach , particularly for intermediate and high relapse risk patients as an irradiation boost or as a monotherapy for low risk patients .", "prior rectal surgery or pelvic irradiation , i.e. rectal cancer treatment , in prostate cancer patients should be carefully evaluated and complications and outcome risk factors should be discussed with special hdr - bt center expertise .", "inflammatory bowel disease patients should be considered for hdr - bt if not suitable for rp , asymptomatic and not required treatment for at least 0.5 to 10 years .", "hdr - bt alone seems to be better than ebrt alone or combined with hdr boost , because of smaller bowel volumes irradiated .", "turp should be performed at least 90 days before hdr - bt , an aerated gel should be given into urethtra to outline its shape and special attention should be given to not exceed 110% of prescription dose .", "most feasible glands for hdr - bt are those beneath 50 cm , but abs guidelines allow to treat larger ones .", "pubic arch interference , baseline urinary function and application technique ( fixed template or freehand ) should be carefully considered .", "patients should be informed about potential risks of erectile dysfunction or acute and late urinary side effects .", "abs states that absolute contraindications include preexisting rectal fistule , impossibility of anaesthesia and no proof of malignancy .", "absolute contraindications include poor performance status and life expectancy shorter than 10 years , spinal or general anaesthesia not possible , large post turp defects and lack of rectum , which makes trus impossible .", "ipss value over 20 points can correlate with increased urinary toxicity and needs careful evaluation .", "prostate volume over 60 cm is not a contraindication and even with pubic arch interference can be considered for ldr - bt with short adt course ( 3 - 4 months ) to downsize the gland .", "also freehand implantation is acceptable , but experienced practitioners should perform ldr - bt in such cases .", "abs states that ldr - bt alone should be done in low risk patients and as option for intermediate group .", "ebrt and permanent seed combination is considered as standard of care for high risk patients ."], ["hdr - bt , according to gec / estro - eau recommendations , should be done under trus guidance with template , its proper fixation and treatment planning software .", "additional imaging with ct or mri after implantation may be used . clinical target volume ( ctv ) for hdr - bt can be whole gland (  prostate surface ) with homogenous needles distribution or with addition of visible tumor boost inside ctv .", "prostate surface dose prescription differs among cancer centers and is between 6 to 10 gy per fraction with total dose of 12 - 20 gy in 2 to 4 fractions combined with ebrt of 45 to 54 gy in 6 - 7 weeks .", "organs - at - risk doses should not exceed 120% of mtd for urethra and less then 6 gy per fraction for rectum . those doses should be precisely stated and valuated in clinical trials with long follow - up including penile bulb maximum dose .", "proximal tips of catheters should be visualized and images should be taken at least 9 mm above and below ctv .", "organs - at - risk should be confined including rectum , urethra , bladder and penile bulb .", "minimum of 14 catheters should be placed or more , particularly if  boost within boost is planned . especially urethra piercing should be avoided .", "at least 90% of ctv should be covered with prescribed dose with aiming 95% of ctv .", "hdr - bt boost is given in one to six and hdr - bt alone in three to six fractions .", "due to wide heterogeneity of fractionation schedules , oar constraints are based on experienced hdr centers references , e.g. from less then 105% to less then 125% for urethra .", "ldr - bt should be performed under trus guidance according to gec / estro guidelines . if possible gross tumor volume ( gtv ) should be delineated on pre - treatment scans .", "ctv should contain whole prostate with 3 mm margin in all directions except posterior ( rectal wall ) and cranial ( bladder neck ) if needed .", "dose prescription to 100% isodose should be 145 gy for i and 125 gy for pd seeds .", "at least 95% of ctv should be covered with 100% isodose with no more than 50% of ctv with isodose 150% .", "maximum dose in 2 cm for rectal wall should be no more than 145 gy and in 0.1 cm less than 200 gy .", "150% of prescribed dose should cover less than 10% of urethra volume and 130% of this dose should cover less than 30% of its volume .", "it should be done as a preplanning , either as a separate procedure or on the implanting day in operational room .", "main procedure should be performed with trus and template ; patient position and probe angle should be similar to preplanned .", "minimum is biplanar , high resolution ( 5 - 12 mhz ) trus with dedicated planning software .", "team should consist at least of trained radiation oncologist and qualified medical physicist , but presence of urologist , certified dosimetrist or other supportive staff is appropriate .", "abs does not suggest any seed deposition technique , however it should have excellent outcome , be reproducible and with optimal dosimetry . both stranded and loose seeds can be chosen for implantation .", "i and pd implants showed excellent outcomes , so abs do not favor any of them .", "ldr - bt alone prescription dose to ptv should range from 140 - 160 gy for i seeds and 110 - 125 gy for pd .", "if combination of ebrt and seed implantation is planned , i ptv dose should be between 108 - 110 gy and pd ptv dose should be between 90 - 100 gy .", "ebrt dose should be 41.4 - 50.4 gy ( 1.8 gy / day , but 2 gy / day is also acceptable ) .", "ldr - bt is usually performed 0 - 8 weeks after ebrt , but there is no evidence for optimal sequence of these .", "reverse approach is also accepted , which may theoretically be better , because of simultaneous irradiation from implant and ebrt and possibility of adjusting ebrt if needed .", "dose constrains for organs at risk should not exceed 150% of prescribed dose in 5% and 125% of prescribed dose in 30% of urethral volume in the preplan .", "prescribed dose should be in less then 1 cm of rectum on day 1 dosimetry and in less then 1.3 cm on day 30 .", "although ct performed immediately or one day after the implantation has several positives , e.g. early detection of dosimetric problems , but after implant oedema can derive dosimetric underestimations .", "it is 12 - 20 days after implantation for pd and 23 - 37 days for i. erectile function organs at risk e.g. penile bulb are still not agreed , though there are no recommendations on these ."], ["both ldr and hdr - bt patients should be seen 6 weeks after implantation ( last one for temporary implants ) according to gec / estro recommendations .", "should be examined every 3 months , every 6 months until 5 years after treatment and then annually .", "dre , psa test , side effects evaluation ( urinary , rectal , potency toxicity and ipss ) and trus should be done [ 18 , 20 ] .", "abs suggests to follow - up hdr - bt patients every six months for first 2 - 3 years , and then at least annually .", "it should consist of dre , psa test and toxicity evaluation ( urinary , rectal and sexual function ) .", "phoenix definition of biochemical relapse is recommended , although case individual assessment is required and patient should be informed about psa bounce .", "ldr - bt patients should be seen every six to twelve months with regular intervals .", "post bt rectal biopsies or urinary instrumentation should be avoided and all of the risks and benefits should be considered [ 21 , 22 ] ."], ["brachytherapy is supreme tool in prostate cancer management with a wide range of options in every risk group ( table 2 ) .", "excellent outcomes and relatively small irradiated volume seems more beneficial than any risk of intraoperative implantation , particularly in wisely selected patients .", "feasibility of seeding or catheterization is basic for choosing appropriate candidates for bt , however there are articles describing series of patient with non - fitting anatomy , i.e. prostate smaller than 20 cm or bigger then 50 cm or post turp patients [ 24 , 25 ] .", "assesment should consist of careful clinical examination including dre , patient 's history ( i.e. urologic , prior pelvic rt , surgery or trauma , inflammatory bowel disease , connective tissue dissorders ) , pretherapy psa serum level , biopsy proven cancer with gleason score and clinical staging .", "trus stays basic imaging , but ct and mri are also very useful , either before procedure and as a planning guidance .", "new abilities of mri , i.e. spectroscopy and diffusion , and 11c pet - ct should be discussed more in next recommendations [ 2628 ] .", "gec / estro and eau recommendations on prostate cancer treatment with temporary implants with stepping sources are from 2005 and on permanent seeds from 2000 with 2007 update .", "although general approach for bt , both hdr and ldr , has not particular differences across the world , european recommendations need an update . \n ", "recommendations comparison for gec/ estro and abs [ 18 , 2022 ] under investigation for high risk patients to institutional review board "]]}
{"article_id": "PMC3388424", "article_text": ["only 2030% of patients with group a beta hemolytic streptococcus ( gabhs ) pharyngitis presents with classical symptoms of the disease .", "reliance on clinical judgment alone has a poor predictive value and results in 80% to 95% overestimation of disease [ 2 , 3 ] .", "diagnostic strategies for acute gabhs pharyngitis are thus based on epidemiological factors , signs , and symptoms , as well as the result of throat cultures ( tcs ) .", "several studies have shown that the use of throat culture leads to more judicious use of antibiotics [ 57 ] .", "physicians prescribe antibiotics for acute pharyngitis as they are concerned that patients with this complaint may be suffering from gabhs infection that if left untreated might develop suppurative complications , such as , tonsillar abscess or nonsuppurative complications , such as , rheumatic fever [ 6 , 8 ] . antibiotics , however ; confer only minor symptomatic benefits for gabhs sore throat .", "they shorten the duration of symptoms by merely half a day on average [ 8 , 9 ] .  ", "more recent studies have shown that antibiotic use only reduced the incidence of rheumatic fever by a mere 0.5 cases per 100,000 .", "the importance of preventing rheumatic fever has lessened as the incidence of rheumatic fever and rheumatic heart disease has declined significantly in the last 20 years , from a mean annual incidence of 13.4 per 100,000 to 5 per 100,000 .", "prevalence has decreased as well from 5.7 per 1,000 in the eighties to 0.5 per 1,000 in 2000 [ 8 , 10 , 11 ] .  ", "this failure probably stems from the fact that about 20% of children with gabhs is infected with bacteria which contain m protein , a virulence factor located on the surface of the bacterial wall that confers resistance to commonly used antibiotics .", "newer beta - lactamase - resistant antibiotics did not prevent this treatment failure [ 13 , 14 ] .  ", "review of the literature from 1945 to 1999 , which includes 10,484 cases of gabhs sore throat , found that antibiotic treatment reduced the occurrence of acute otitis media , a common complication of this disease , by a mere 25% , compared to the placebo group and sinusitis by only 50% .", "rheumatic fever , a nonsuppurative complication , was reduced by less than 33% , compared to placebo [ 8 , 10 , 11 , 15 ] .  ", "in addition to the uncertainly in the scientific literature , parents seem to be uncertain regarding the benefits of antibiotic treatment for acute gabhs pharyngitis and tend to stop treatment earlier than prescribed . in a pilot study", ", we randomly followed 75 children with gabhs pharyngitis for 6 months and have found that more than 75% of them did not complete ten days of antibiotics .", "this finding led us to conduct a multisite , prospective cohort observational study , the results of which are reported here .", "the goal of this study was to determine whether noncompliance with antibiotic treatment affects short - term or long - term complications .", "two central , primarily rural , and agricultural regions of the largest health maintenance organization ( hmo ) in israel , comprising approximately one million patients . using a standard protocol", ", we located from our computerized data base 107,840 patients , aged 6 months to 18 years , who were examined by their primary care physician for upper respiratory tract infection , tonsillitis , pharyngitis , sore throat , tonsillopharyngitis , neck pain , cervical lymphadenopathy , pta , rpa , from january 1 , 1999 until december 31 , 2000 .", "we then accessed the charts of 78,473 of these children who were diagnosed with infected throat or one of the differential variants , excluding all children diagnosed as having viral upper respiratory infections .", "47,000 of these patients were formally diagnosed with acute pharyngitis or acute tonsillitis and received a prescription for antibiotics , indicating that their physician suspected bacterial disease . in the index visit , 35,000 of these children had at least four out of five symptoms in the modified centor criteria used for this study and nadir modified breese epidemiological and clinical score card ( ecsc ) that has 91% sensitivity and 98% specificity when the score was above 15 ( score between 4 and 36 ) for the diagnosis of gabhs [ 1719 ] .", "colonies yielding beta - hemolysis were grouped for surface carbohydrate assessment by using a latex bead agglutination test ( figure 1 ) . of the 6336 children ( with positive cultures with 4 or more centor criteria and 15 or higher ecsc ) , 4,775 parents consented to enroll their children to the study ( figure 1 ) . excluded from the study were children who were diagnosed as gabhs chronic carriers or who had suffered from post - gabhs complications ; had any chronic illness , such as , renal or hepatic impairment ; had bleeding disorder ; had congenital or acquired immunodeficiency or suffered from malignancy .  ", "initial patient / parent contact was made by one of the authors ( m. sarrell ) within 3 to 5 days of the initial positive throat culture . at that time , initial information regarding the illness and whether a prescription for antibiotics was given by the primary care physician .  ", "the attending physicians of the two thousand study patients were contacted by email within 48 hours of the enrollment by one author ( m. sarrell ) .", "the physicians were requested to inform the authors of any additional cultures taken during therapy , and to request that they obtain two additional throat cultures and engage in improve adherence strategies by providing information , counseling , reminders , reinforcement , and if needed personal attention or supervision .", "while repeated cultures are not routinely recommended for asymptomatic patients who have completed a course of antimicrobial therapy , in light of the poor compliance with treatment in the pilot study , performance of such follow - up cultures was considered important for the purposes of the study .", "the first follow - up culture was performed within 10 to 14 days of the initial positive culture regardless of treatment status .", "the purpose of this culture was verification of antibiotics treatment failure or persistence of gabhs in the oropharynx of the untreated patients .", "the second additional throat culture was taken between days 21 and 30 after the initial positive culture , regardless of treatment status , to ascertain the presence of residual gabhs or recurrences . treating physicians", "were also requested to obtain blood for liver enzymes , renal function tests , and urine analysis from all the participants and to perform annual follow - up evaluation thereafter .  ", "a second patient / parent contact was made by our study coordinator within 10 to 14 days of initiation of antibiotic treatment .", "she collected information about demographic characteristics , past medical history , febrile status , need for repeat throat culture during the treatment period ( that was not part of the study protocol ) , and type of medication prescribed . during this contact she obtained information about the number of days of actual treatment , omission compliance and complications , the patients / parents perceived as deriving from the treatment ( or lack thereof ) .", "the computerized charts of the participants were searched within 2 to 4 weeks of the second patient / parent contact for additional information , including demographic characteristics , medical and environmental history , initial clinical data , such as , in - office fever evaluation , results of the physical examination , additional culture taken , type of antibiotics used , and disposition of prescription received .", "a second search of the computerized medical charts was performed by our research assistant between 30 and 90 days of initiation of medical treatment , to ascertain that the 2 requested throat cultures were obtained . relapse or recurrence of clinical or bacterial pharyngitis ,", "suppurative or nonsuppurative complication , or even whether the participants complained of any sore throat within 30 days of completion of treatment were also evaluated . in order to assure that all possible short - term complications that occurred within 90 days of the index case were obtained ,", "an additional comprehensive search of the hmo database was done within 120 days of the second computer search .", "we ascertained that findings that were either not available on the original computerized chart or were seen by other than their primary care physician , ( e.g. , emergency departments , patients that relocated ) , were not overlooked .", "the charts of the participants were then reviewed by one of the authors on a yearly basis , from january 2000 to january 2010 , noting possible late nonsuppurative complications of gabhs infection .", "no patients were lost to followup , even if they had changed physicians , due to our ability to track them through the centralized database to their new physician or another hmo .", "the children that were enlisted to the army ( and thus not members of any hmo during their military service ) were contacted either through their former attending physician or the military physician .  ", "minor treatment failure was defined as any clinical or bacterial recurrence of pharyngitis during the short - term follow - up period and its correlation to compliance with treatment .  ", "major treatment failure was defined as retropharyngeal or peritonsillar abscess or long - term complications , such as rheumatic fever .  ", "suppurative complications were chosen as a model , because the nonsuppurative complications ( rheumatic heart disease , arthritis , carditis ) have been practically eradicated in our region .", "this cohort study was designed to analyze rare events ( according to the cioms classification 110 events per 10,000 children years ) .", "the annual incidence of peritonsillar abscess ( pta ) in our region is 24 cases per 100,000 and the incidence of retropharyngeal abscess ( rpa ) is 57 cases per 100,000 .  ", "power calculations suggested 6,500 to 7,000 person - years of intervention would be needed to detect a 22% difference in pta and rpa between the fully treated ( ft ) and partially treated ( pt ) arms of the study population .", "furthermore , one - sided alpha of 0.025 , a statistical power of 95% , and the pta / rpa incidence given above showed that approximately 19,000 children - years would be needed to show the noninferiority of ft versus pt . since the primary outcome of interest is the pta / rpa hazard ratio between ft and pt .", "the null hypothesis to be tested is hr pta / rpa > 2 ( i.e. , the pta / rpa hazard ratio for ft versus pt is higher or equal to 2 ) .", "the pta / rpa hazard ratio was calculated for ft versus pt .   for purposes of analysis ,", "participants were divided into four subgroups based on length of treatment : 1st subgroup ( untreated ) , those who did not receive any treatment , 2nd subgroup ( partially treated ) , children that received antibiotics for 1 to 3 days , 3rd subgroup ( mostly - treated ) , children treated for 4 to 6 days , and 4th subgroup ( fully - treated ) children treated between 7 to 10 days .", "data are presented as proportions ( with 99% confidence intervals [ cis ] ) , means ( with sds ) , or medians ( with interquartile ranges ) , using pearson  tests , student 's tests , or fisher exact test .", "comparisons of length of treatment according to time and treatment were assessed using the repeated measures and analysis of variance and the paired t test .", "a 2-tailed p value of.05 was used to determine the statistical significance of differences observed between groups and to calculate confidence intervals around differences in sample means and odds ratios .", "we used the mcnamara test to measure the changes between the groups and their subgroups regard to the length of antibiotic treatment .", "over half of their children ( 1023 , 51% ) were between the ages of 6 months and 6.9 years , and over half 1,039 ( 52% ) were female .", "the majority of children ( 1,821 , 91% ) were prescribed penicillin or amoxicillin , allergic or intolerant to penicillin were treated with cephalosporin 25 ( 1.5% ) , erythromycin 109 ( 5.5% ) , and azithromycin 45 ( 2.5% ) , all medication were prescribed twice daily for 10 days , except azithromycin once daily for 5 days .", "no statistical correlation was found between the type of antibiotics , the children received , or the demographic characteristics and the complications found in the later medical examinations .", "only 196 children ( 9.8% ) actually completed 10 days of antibiotic treatment . despite having received a prescription from their physician , two hundred and thirteen participants ( 11% )", "did not start taking any treatment whatsoever , including those who did not even purchase antibiotics .", "as shown in table 1 a no statistical correlation was found concerning length frequency and duration , palatability , number of daily dose of treatment , but a statistically significant difference was found between all the subgroups concerning the length of antibiotics treatment ( p   <", "the majority of children ( 1192 , 59.6% ) had 3 days or less of fever , defined as any rectal temperature less than 38.5c or oral temperature less than 37.8c .", "the majority of children ( 1591 , 80% ) received medication for four to six days at the most ( partially treated subgroup ) .", "as illustrated in table 1 , the association between the duration of fever and the number of days of treatment was statistically significant ( p < .0001 ) .  ", "of the 306 ( 15.3% ) children with clinically diagnosed recurrent tonsillopharyngitis , only 236 ( 12.3% ) had positive gabhs findings on the throat culture taken within 10 to 14 days after conclusion of the primary infection .", "an additional thirty - four ( 1.7% ) had a positive second study culture ( taken 2130 days after the index positive gabhs culture ) .", "of note is the fact that the majority ( 156 , 66% ) of the positive study culture at 1014 days were found among the subgroup treated for 7 to 10 days .", "no such positive results were found in the subgroup treated for 1 up to 3 days .", "furthermore , no positive gabhs throat cultures were found on the second study culture in the untreated group .", "the majority ( 26 , 76% ) of positive gabhs cultures were in the mostly treated subgroups .", "as illustrated in table 2 , these findings were both statistically significant ( p  <  .0001 ) ( table 2 ) .  ", "cervical lymphadenitis , acute otitis media , and impetigo were the only suppurative complications noted .", "110 ( 5.5% ) children developed cervical lymphadenitis , most ( 52 , 47% ) among the 6 to 7 days treatment subgroup and 33 ( 33% ) among the 4 to 5 days treatment subgroup , a significant difference among the treatment subgroups ( p < .0001 ) .", "additionally , no children developed nonsuppurative complications during 10 years of followup nor did any of the children develop iga nephropathy during the follow - up period .", "furthermore , they were no association between the five modified centor criteria and development of complication , even when stratified by type of antibiotics or the season of the year . altogether , 304 ( 15% )", "new onset cases of acute otitis media ( aom ) were diagnosed within 30 days of the initial diagnosis .", "however , only 31 ( 10% ) of those were in the untreated subgroup , as compared to 141 ( 46% ) in the 7 to 10 days treatment subgroup , 98 ( 38% ) in the 4 to 6 days treatment subgroup and 98 ( 33% ) in the 1 to 3 days subgroup , a statistically significant difference among the all treatment subgroups ( p  <  .0001 ) .   attempting to elucidate the possible causes for the differences between the recurrence of gabhs and the length of antibiotic treatment or clinical score on enrolment or illness severity , a multivariate stepwise logistic analysis was performed .", "the duration of fever was the most significant predictor for such recurrence , age under 6 years being less significant , while treatment for 7 to 10 days had no significant influence on the recurrence of gabhs .  ", "of note is that the contribution of the duration of fever was apparent even after controlling for the concomitant influence of age , gender , medical history , single- or two - parent home , type of antibiotics or the season of the year , and concomitant illnesses , such as , conjunctivitis , otitis media , upper respiratory infection , gastroenteritis , or lymphadenitis , which may have otherwise explained the influence of the length of treatment in relation to those illnesses ( table 3 ) .", "this study found a very poor parent / child compliance to antibiotic treatment prescribed for symptomatic , culture - positive gabhs tonsillopharyngitis .", "11% of children did not start taking any treatment at all , and only 10% completed a full course of treatment .", "the reason for this low rate of compliance is unclear , but it coincides with other reported studies [ 14 , 16 ] . we speculate that a large proportion of lack of compliance is the parent 's sensation that antibiotics are potentially dangerous   and overprescribed [ 18 , 19 ] . despite this poor compliance , in our study as in others , there was a very low rate of suppurative complications .", "furthermore , despite the poor compliance , we found no increase in the incidence of acute rheumatic fever , the most dreaded complication of gabhs , in our patients .", "in fact , since the year 2000 , the incidence of rf in our region has declined from 2.2 per 100,000 to 0.2 per 100,000 in 2008 , according to the epidemiological department of the israeli ministry of health .", "this is in concordance with other developed countries , including , the united states , where the original recommendation for 10 days of antibiotic treatment of gabhs originated . in that country ,", "currently there are only 10 cases of rf per 100,000 patients with gabhs pharyngitis , and only 1 case per 10,000 patients with acute rheumatic fever develop rheumatic heart disease [ 2022 ] .", "in fact , concomitant with the increased use of antibiotics , recurrence of gabhs in the usa rose from 9% and 10.7% in the years 1975 to 1979 , respectively , to 25.9% and 37.5% in the years 1995 to 1996 , despite the decline in acute rheumatic fever .", "14% of patients in our study had a recurrent infection , proven by a gabhs - positive throat cultures .", "this percentage is similar to other studies which found that penicillin failed to eradicate gabhs from the throat in approximately 13% to 26% of the patients evaluated [ 23 , 24 ] .", "the majority of recurrences in our study were in the younger group ( mean age of 10.2 years , and 60% of them younger than 9.9 years ) .", "this is consistent with other published studies where such recurrences were more frequent among children aged 1 to 8 years than among children aged between 13 and 19 years [ 24 , 25 ] .   historically ,", "prescribing 10 days of oral penicillin began in the 1950s , substituting the intramuscular injections of long - acting parenteral penicillin , based on surrogate markers of eradication of gabhs from the tonsillopharynx . however , no study has conclusively proven that this practice unequivocally prevents acute rheumatic fever [ 26 , 27 ] . even though orally prescribed penicillin appeared to be equally effective for clinical and laboratory resolution of signs and symptoms , it is difficult to administer and expensive , considering the staggering financial burden of approximately 140 office visits per annum per 1,000 children younger than 15 years [ 28 , 29 ] . substituting azithromycin or cephalosporins for penicillin was found to produce better bacteriological and clinical results and also required a shorter course of treatment [ 30 , 31 ] .   ", "our results may not apply to adults , sick people , or chronic gabhs carriers .", "it does not address the optimal length of treatment required to achieve appropriate eradication of the microbe or whether complete eradication is required at all .", "the method of pill / doses counts is not a good measure of adherence , but due to it simplicity and empiric nature it was found adequate for this study .", "we were unable to assess the true variation of the incidence of acute rheumatic fever , due to the fact that this disease has been practically eradicated from our population .", "our data suggest that the large majority of parents / patients stop administering antibiotics to their children who suffer from gabhs prior to the completion of the recommended course .", "a more judicious use of antibiotics would promote and improve compliance , cut costs , and prove more convenient to parents and children alike ."], "abstract_text": ["<S> \n background . </S>", "<S> uncertainty exists concerning the necessity of 10-day antibiotic treatment of group a beta hemolytic streptococcus ( gabhs ) pharyngitis . objective . to assess the incidence of gabhs recurrence and suppurative and nonsuppurative complications in relation to compliance </S>", "<S> . methods . </S>", "<S> ( design ) . prospective cohort observational study . </S>", "<S> ( subjects ) . </S>", "<S> 2,000 children aged 6 months to 18 years with sore throat and positive gabhs culture . </S>", "<S> ( main outcome measures ) . </S>", "<S> recurrence of symptomatic culture positive gabhs pharyngitis , incidence of suppurative , and long - term , regional , nonsuppurative complications of gabhs pharyngitis , over a ten year period . results . </S>", "<S> 213 ( 11% ) of the children received no treatment . </S>", "<S> most children received antibiotics for only 46 days ( in correlation with the duration of fever , which in most cases lasted up to 3 days ) . </S>", "<S> three hundred and six ( 15.3% ) children had clinically diagnosed recurrent tonsillopharyngitis ; 236 ( 12.3% ) had positive gabhs findings within 10 to 14 days and thirty - four ( 1.7% ) within 2130 days after the index positive gabhs culture . </S>", "<S> the remaining 1.3% had no positive culture despite the clinical findings . </S>", "<S> almost all recurrences [ 236 ( 11.6% ) ] occurred within 14 days and 156 ( 7.6% ) in the fully treated group . </S>", "<S> the presence of fever during the first 3 days of the disease was the most significant predictor for recurrence . </S>", "<S> other predictors were the age younger than 6 years and the presence of cervical lymphadenitis . </S>", "<S> no increase in the incidence of nonsuppurative or suppurative complications was noted during the 10-year follow - up period , compared to the past incidence of those complications in israel . </S>", "<S> conclusions . </S>", "<S> our data suggests that the majority of children discontinue antibiotics for gabhs tonsillopharyngitis a day or two after the fever subsides . </S>", "<S> the incidence of complications in our study was not affected by this poor compliance . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusion "], "sections": [["only 2030% of patients with group a beta hemolytic streptococcus ( gabhs ) pharyngitis presents with classical symptoms of the disease .", "reliance on clinical judgment alone has a poor predictive value and results in 80% to 95% overestimation of disease [ 2 , 3 ] .", "diagnostic strategies for acute gabhs pharyngitis are thus based on epidemiological factors , signs , and symptoms , as well as the result of throat cultures ( tcs ) .", "several studies have shown that the use of throat culture leads to more judicious use of antibiotics [ 57 ] .", "physicians prescribe antibiotics for acute pharyngitis as they are concerned that patients with this complaint may be suffering from gabhs infection that if left untreated might develop suppurative complications , such as , tonsillar abscess or nonsuppurative complications , such as , rheumatic fever [ 6 , 8 ] . antibiotics , however ; confer only minor symptomatic benefits for gabhs sore throat .", "they shorten the duration of symptoms by merely half a day on average [ 8 , 9 ] .  ", "more recent studies have shown that antibiotic use only reduced the incidence of rheumatic fever by a mere 0.5 cases per 100,000 .", "the importance of preventing rheumatic fever has lessened as the incidence of rheumatic fever and rheumatic heart disease has declined significantly in the last 20 years , from a mean annual incidence of 13.4 per 100,000 to 5 per 100,000 .", "prevalence has decreased as well from 5.7 per 1,000 in the eighties to 0.5 per 1,000 in 2000 [ 8 , 10 , 11 ] .  ", "this failure probably stems from the fact that about 20% of children with gabhs is infected with bacteria which contain m protein , a virulence factor located on the surface of the bacterial wall that confers resistance to commonly used antibiotics .", "newer beta - lactamase - resistant antibiotics did not prevent this treatment failure [ 13 , 14 ] .  ", "review of the literature from 1945 to 1999 , which includes 10,484 cases of gabhs sore throat , found that antibiotic treatment reduced the occurrence of acute otitis media , a common complication of this disease , by a mere 25% , compared to the placebo group and sinusitis by only 50% .", "rheumatic fever , a nonsuppurative complication , was reduced by less than 33% , compared to placebo [ 8 , 10 , 11 , 15 ] .  ", "in addition to the uncertainly in the scientific literature , parents seem to be uncertain regarding the benefits of antibiotic treatment for acute gabhs pharyngitis and tend to stop treatment earlier than prescribed . in a pilot study", ", we randomly followed 75 children with gabhs pharyngitis for 6 months and have found that more than 75% of them did not complete ten days of antibiotics .", "this finding led us to conduct a multisite , prospective cohort observational study , the results of which are reported here .", "the goal of this study was to determine whether noncompliance with antibiotic treatment affects short - term or long - term complications ."], ["two central , primarily rural , and agricultural regions of the largest health maintenance organization ( hmo ) in israel , comprising approximately one million patients . using a standard protocol", ", we located from our computerized data base 107,840 patients , aged 6 months to 18 years , who were examined by their primary care physician for upper respiratory tract infection , tonsillitis , pharyngitis , sore throat , tonsillopharyngitis , neck pain , cervical lymphadenopathy , pta , rpa , from january 1 , 1999 until december 31 , 2000 .", "we then accessed the charts of 78,473 of these children who were diagnosed with infected throat or one of the differential variants , excluding all children diagnosed as having viral upper respiratory infections .", "47,000 of these patients were formally diagnosed with acute pharyngitis or acute tonsillitis and received a prescription for antibiotics , indicating that their physician suspected bacterial disease . in the index visit , 35,000 of these children had at least four out of five symptoms in the modified centor criteria used for this study and nadir modified breese epidemiological and clinical score card ( ecsc ) that has 91% sensitivity and 98% specificity when the score was above 15 ( score between 4 and 36 ) for the diagnosis of gabhs [ 1719 ] .", "colonies yielding beta - hemolysis were grouped for surface carbohydrate assessment by using a latex bead agglutination test ( figure 1 ) . of the 6336 children ( with positive cultures with 4 or more centor criteria and 15 or higher ecsc ) , 4,775 parents consented to enroll their children to the study ( figure 1 ) . excluded from the study were children who were diagnosed as gabhs chronic carriers or who had suffered from post - gabhs complications ; had any chronic illness , such as , renal or hepatic impairment ; had bleeding disorder ; had congenital or acquired immunodeficiency or suffered from malignancy .  ", "initial patient / parent contact was made by one of the authors ( m. sarrell ) within 3 to 5 days of the initial positive throat culture . at that time , initial information regarding the illness and whether a prescription for antibiotics was given by the primary care physician .  ", "the attending physicians of the two thousand study patients were contacted by email within 48 hours of the enrollment by one author ( m. sarrell ) .", "the physicians were requested to inform the authors of any additional cultures taken during therapy , and to request that they obtain two additional throat cultures and engage in improve adherence strategies by providing information , counseling , reminders , reinforcement , and if needed personal attention or supervision .", "while repeated cultures are not routinely recommended for asymptomatic patients who have completed a course of antimicrobial therapy , in light of the poor compliance with treatment in the pilot study , performance of such follow - up cultures was considered important for the purposes of the study .", "the first follow - up culture was performed within 10 to 14 days of the initial positive culture regardless of treatment status .", "the purpose of this culture was verification of antibiotics treatment failure or persistence of gabhs in the oropharynx of the untreated patients .", "the second additional throat culture was taken between days 21 and 30 after the initial positive culture , regardless of treatment status , to ascertain the presence of residual gabhs or recurrences . treating physicians", "were also requested to obtain blood for liver enzymes , renal function tests , and urine analysis from all the participants and to perform annual follow - up evaluation thereafter .  ", "a second patient / parent contact was made by our study coordinator within 10 to 14 days of initiation of antibiotic treatment .", "she collected information about demographic characteristics , past medical history , febrile status , need for repeat throat culture during the treatment period ( that was not part of the study protocol ) , and type of medication prescribed . during this contact she obtained information about the number of days of actual treatment , omission compliance and complications , the patients / parents perceived as deriving from the treatment ( or lack thereof ) .", "the computerized charts of the participants were searched within 2 to 4 weeks of the second patient / parent contact for additional information , including demographic characteristics , medical and environmental history , initial clinical data , such as , in - office fever evaluation , results of the physical examination , additional culture taken , type of antibiotics used , and disposition of prescription received .", "a second search of the computerized medical charts was performed by our research assistant between 30 and 90 days of initiation of medical treatment , to ascertain that the 2 requested throat cultures were obtained . relapse or recurrence of clinical or bacterial pharyngitis ,", "suppurative or nonsuppurative complication , or even whether the participants complained of any sore throat within 30 days of completion of treatment were also evaluated . in order to assure that all possible short - term complications that occurred within 90 days of the index case were obtained ,", "an additional comprehensive search of the hmo database was done within 120 days of the second computer search .", "we ascertained that findings that were either not available on the original computerized chart or were seen by other than their primary care physician , ( e.g. , emergency departments , patients that relocated ) , were not overlooked .", "the charts of the participants were then reviewed by one of the authors on a yearly basis , from january 2000 to january 2010 , noting possible late nonsuppurative complications of gabhs infection .", "no patients were lost to followup , even if they had changed physicians , due to our ability to track them through the centralized database to their new physician or another hmo .", "the children that were enlisted to the army ( and thus not members of any hmo during their military service ) were contacted either through their former attending physician or the military physician .  ", "minor treatment failure was defined as any clinical or bacterial recurrence of pharyngitis during the short - term follow - up period and its correlation to compliance with treatment .  ", "major treatment failure was defined as retropharyngeal or peritonsillar abscess or long - term complications , such as rheumatic fever .  ", "suppurative complications were chosen as a model , because the nonsuppurative complications ( rheumatic heart disease , arthritis , carditis ) have been practically eradicated in our region .", "this cohort study was designed to analyze rare events ( according to the cioms classification 110 events per 10,000 children years ) .", "the annual incidence of peritonsillar abscess ( pta ) in our region is 24 cases per 100,000 and the incidence of retropharyngeal abscess ( rpa ) is 57 cases per 100,000 .  ", "power calculations suggested 6,500 to 7,000 person - years of intervention would be needed to detect a 22% difference in pta and rpa between the fully treated ( ft ) and partially treated ( pt ) arms of the study population .", "furthermore , one - sided alpha of 0.025 , a statistical power of 95% , and the pta / rpa incidence given above showed that approximately 19,000 children - years would be needed to show the noninferiority of ft versus pt . since the primary outcome of interest is the pta / rpa hazard ratio between ft and pt .", "the null hypothesis to be tested is hr pta / rpa > 2 ( i.e. , the pta / rpa hazard ratio for ft versus pt is higher or equal to 2 ) .", "the pta / rpa hazard ratio was calculated for ft versus pt .   for purposes of analysis ,", "participants were divided into four subgroups based on length of treatment : 1st subgroup ( untreated ) , those who did not receive any treatment , 2nd subgroup ( partially treated ) , children that received antibiotics for 1 to 3 days , 3rd subgroup ( mostly - treated ) , children treated for 4 to 6 days , and 4th subgroup ( fully - treated ) children treated between 7 to 10 days .", "data are presented as proportions ( with 99% confidence intervals [ cis ] ) , means ( with sds ) , or medians ( with interquartile ranges ) , using pearson  tests , student 's tests , or fisher exact test .", "comparisons of length of treatment according to time and treatment were assessed using the repeated measures and analysis of variance and the paired t test .", "a 2-tailed p value of.05 was used to determine the statistical significance of differences observed between groups and to calculate confidence intervals around differences in sample means and odds ratios .", "we used the mcnamara test to measure the changes between the groups and their subgroups regard to the length of antibiotic treatment ."], ["over half of their children ( 1023 , 51% ) were between the ages of 6 months and 6.9 years , and over half 1,039 ( 52% ) were female .", "the majority of children ( 1,821 , 91% ) were prescribed penicillin or amoxicillin , allergic or intolerant to penicillin were treated with cephalosporin 25 ( 1.5% ) , erythromycin 109 ( 5.5% ) , and azithromycin 45 ( 2.5% ) , all medication were prescribed twice daily for 10 days , except azithromycin once daily for 5 days .", "no statistical correlation was found between the type of antibiotics , the children received , or the demographic characteristics and the complications found in the later medical examinations .", "only 196 children ( 9.8% ) actually completed 10 days of antibiotic treatment . despite having received a prescription from their physician , two hundred and thirteen participants ( 11% )", "did not start taking any treatment whatsoever , including those who did not even purchase antibiotics .", "as shown in table 1 a no statistical correlation was found concerning length frequency and duration , palatability , number of daily dose of treatment , but a statistically significant difference was found between all the subgroups concerning the length of antibiotics treatment ( p   <", "the majority of children ( 1192 , 59.6% ) had 3 days or less of fever , defined as any rectal temperature less than 38.5c or oral temperature less than 37.8c .", "the majority of children ( 1591 , 80% ) received medication for four to six days at the most ( partially treated subgroup ) .", "as illustrated in table 1 , the association between the duration of fever and the number of days of treatment was statistically significant ( p < .0001 ) .  ", "of the 306 ( 15.3% ) children with clinically diagnosed recurrent tonsillopharyngitis , only 236 ( 12.3% ) had positive gabhs findings on the throat culture taken within 10 to 14 days after conclusion of the primary infection .", "an additional thirty - four ( 1.7% ) had a positive second study culture ( taken 2130 days after the index positive gabhs culture ) .", "of note is the fact that the majority ( 156 , 66% ) of the positive study culture at 1014 days were found among the subgroup treated for 7 to 10 days .", "no such positive results were found in the subgroup treated for 1 up to 3 days .", "furthermore , no positive gabhs throat cultures were found on the second study culture in the untreated group .", "the majority ( 26 , 76% ) of positive gabhs cultures were in the mostly treated subgroups .", "as illustrated in table 2 , these findings were both statistically significant ( p  <  .0001 ) ( table 2 ) .  ", "cervical lymphadenitis , acute otitis media , and impetigo were the only suppurative complications noted .", "110 ( 5.5% ) children developed cervical lymphadenitis , most ( 52 , 47% ) among the 6 to 7 days treatment subgroup and 33 ( 33% ) among the 4 to 5 days treatment subgroup , a significant difference among the treatment subgroups ( p < .0001 ) .", "additionally , no children developed nonsuppurative complications during 10 years of followup nor did any of the children develop iga nephropathy during the follow - up period .", "furthermore , they were no association between the five modified centor criteria and development of complication , even when stratified by type of antibiotics or the season of the year . altogether , 304 ( 15% )", "new onset cases of acute otitis media ( aom ) were diagnosed within 30 days of the initial diagnosis .", "however , only 31 ( 10% ) of those were in the untreated subgroup , as compared to 141 ( 46% ) in the 7 to 10 days treatment subgroup , 98 ( 38% ) in the 4 to 6 days treatment subgroup and 98 ( 33% ) in the 1 to 3 days subgroup , a statistically significant difference among the all treatment subgroups ( p  <  .0001 ) .   attempting to elucidate the possible causes for the differences between the recurrence of gabhs and the length of antibiotic treatment or clinical score on enrolment or illness severity , a multivariate stepwise logistic analysis was performed .", "the duration of fever was the most significant predictor for such recurrence , age under 6 years being less significant , while treatment for 7 to 10 days had no significant influence on the recurrence of gabhs .  ", "of note is that the contribution of the duration of fever was apparent even after controlling for the concomitant influence of age , gender , medical history , single- or two - parent home , type of antibiotics or the season of the year , and concomitant illnesses , such as , conjunctivitis , otitis media , upper respiratory infection , gastroenteritis , or lymphadenitis , which may have otherwise explained the influence of the length of treatment in relation to those illnesses ( table 3 ) ."], ["this study found a very poor parent / child compliance to antibiotic treatment prescribed for symptomatic , culture - positive gabhs tonsillopharyngitis .", "11% of children did not start taking any treatment at all , and only 10% completed a full course of treatment .", "the reason for this low rate of compliance is unclear , but it coincides with other reported studies [ 14 , 16 ] . we speculate that a large proportion of lack of compliance is the parent 's sensation that antibiotics are potentially dangerous   and overprescribed [ 18 , 19 ] . despite this poor compliance , in our study as in others , there was a very low rate of suppurative complications .", "furthermore , despite the poor compliance , we found no increase in the incidence of acute rheumatic fever , the most dreaded complication of gabhs , in our patients .", "in fact , since the year 2000 , the incidence of rf in our region has declined from 2.2 per 100,000 to 0.2 per 100,000 in 2008 , according to the epidemiological department of the israeli ministry of health .", "this is in concordance with other developed countries , including , the united states , where the original recommendation for 10 days of antibiotic treatment of gabhs originated . in that country ,", "currently there are only 10 cases of rf per 100,000 patients with gabhs pharyngitis , and only 1 case per 10,000 patients with acute rheumatic fever develop rheumatic heart disease [ 2022 ] .", "in fact , concomitant with the increased use of antibiotics , recurrence of gabhs in the usa rose from 9% and 10.7% in the years 1975 to 1979 , respectively , to 25.9% and 37.5% in the years 1995 to 1996 , despite the decline in acute rheumatic fever .", "14% of patients in our study had a recurrent infection , proven by a gabhs - positive throat cultures .", "this percentage is similar to other studies which found that penicillin failed to eradicate gabhs from the throat in approximately 13% to 26% of the patients evaluated [ 23 , 24 ] .", "the majority of recurrences in our study were in the younger group ( mean age of 10.2 years , and 60% of them younger than 9.9 years ) .", "this is consistent with other published studies where such recurrences were more frequent among children aged 1 to 8 years than among children aged between 13 and 19 years [ 24 , 25 ] .   historically ,", "prescribing 10 days of oral penicillin began in the 1950s , substituting the intramuscular injections of long - acting parenteral penicillin , based on surrogate markers of eradication of gabhs from the tonsillopharynx . however , no study has conclusively proven that this practice unequivocally prevents acute rheumatic fever [ 26 , 27 ] . even though orally prescribed penicillin appeared to be equally effective for clinical and laboratory resolution of signs and symptoms , it is difficult to administer and expensive , considering the staggering financial burden of approximately 140 office visits per annum per 1,000 children younger than 15 years [ 28 , 29 ] . substituting azithromycin or cephalosporins for penicillin was found to produce better bacteriological and clinical results and also required a shorter course of treatment [ 30 , 31 ] .   ", "our results may not apply to adults , sick people , or chronic gabhs carriers .", "it does not address the optimal length of treatment required to achieve appropriate eradication of the microbe or whether complete eradication is required at all .", "the method of pill / doses counts is not a good measure of adherence , but due to it simplicity and empiric nature it was found adequate for this study .", "we were unable to assess the true variation of the incidence of acute rheumatic fever , due to the fact that this disease has been practically eradicated from our population ."], ["our data suggest that the large majority of parents / patients stop administering antibiotics to their children who suffer from gabhs prior to the completion of the recommended course .", "a more judicious use of antibiotics would promote and improve compliance , cut costs , and prove more convenient to parents and children alike ."]]}
{"article_id": "PMC4616107", "article_text": ["myofascial pain syndrome ( mps ) can be caused by frequent muscle or fascial stiffness as a \n result of prolonged tension and muscle fatigue due to repetitive stress of muscles or \n overuse of particular muscles1 , 2 .", "mps activates trigger points in 54% of women and 45% of men , and is regarded as \n the most common cause of pain in the musculoskeletal system3 . in particular , the upper trapezius requires head movement according \n to the direction of view , but it can be vulnerable to damage when the hands and arms are \n used repetitively in work that requires precise control4,5,6 . once myofascial trigger points are activated , changes in the \n structural characteristics and contraction function of the muscle will occur .", "the most \n distinctive changes are taut bands , tender nodules , referred pain , local twitch response , \n muscle weakness , and restricted range of motion3 .", "to evaluate the contraction function of skeletal muscle , various methods can be used . among \n them ,", "surface electromyography ( emg ) analyzes functional changes in muscle by measuring \n quantitative changes in motor unit action potential that is activated by muscle \n contraction7 , 8 .", "surface emg is widely used in kinetic analysis to diagnose normal \n or abnormal function in muscles and nerves according to amplitude and frequency9 .", "it has several advantages , as it is \n noninvasive and convenient , and can perform measurement even during dynamic motion10 , 11 .", "although a number of clinical reports on the pathologic mechanisms or clinical diagnosis \n and treatment of mps can be found , few studies have been conducted on the changes in \n contraction function in myofascial trigger point areas or on the electrophysiological \n characteristics and neuromuscular physiological information .", "this study aimed to compare the \n electrophysiological characteristics of normal muscles versus muscles with latent or active \n myofascial trigger points , and to identify their neuromuscular physiological \n characteristics , in order to provide a quantitative evaluation of mps and clinical \n foundational data for its diagnosis .", "this study was approved by the \n research agency , and all participants provided written informed consent .", "subjects were \n selected among those with no known neurologic disease , no regular exercise habit , and no \n drug use that could affect the experimental result .", "subjects in the active myofascial trigger point group were those diagnosed with mps \n by a physician and whose clinical characteristics were pain in the upper trapezius during \n rest , taut bands , tender nodules , referred pain , local twitch response , and muscle weakness . \n", "subjects in the latent myofascial trigger point group were those not diagnosed with mps but \n who felt tenderness when the upper trapezius was stimulated .", "mean age , height , and weight , respectively , were 23.27 \n  4.21  years , 172.32  8.44  cm , and 64.14  6.54  kg in the active trigger point group ; 23.74 \n  6.47  years , 174.76  6.22  cm , and 66.74  5.74  kg in the latent trigger point group ; and \n 24.17  3.14  years , 171.21  9.64  cm , and 63.44  7.51  kg in the control group . to measure maximum voluntary isometric contraction ( mvic ) , the shoulders and heads of the \n subjects were firmly fixed and pulled as the dynamometer as maximum power , thereby measuring \n mvic against the upper trapezius .", "endurance time was measured from the start of mvic to a \n 50% reduction in contraction force .", "median frequency ( mdf ) was measured by using emg at the \n muscle belly of the upper trapezius during mvic .", "muscle fatigue index was calculated by \n obtaining the mdf at a section where the mvic force was reduced from 100% to 50% , and remove \n the 50% mdf from 100% mdf was divided into 100% mdf .", "the sampling rate for the surface emg \n signal was set at 1,000  hz , and the frequency band filter was set at 20 to 450  hz , using one \n channel .", "storage and analysis of the emg signals was performed using acqknowledge 3.8.1 \n ( biopac systems , goleta , ca , usa ) .", "the average of three measurements was calculated and used \n in the analyses ; 10 minutes of rest was given between measurements to prevent muscle \n fatigue .", "statistical analyses were performed using spss version 18.0 for windows ( spss , inc . \n", "one - way analysis of variance was conducted to examine differences among \n groups with respect to the measured items , while the tukey test was conducted for post hoc \n analysis .", "there were no significant differences in mvic or endurance time among the three groups . \n however , mdf was significantly different among all three groups ( p < 0.05 ) ( table 1table 1.comparison of mvic , et , mdf , and fi among the three groupsgroup a ( n = 30)group b ( n = 30)group c ( n = 30)post hocmvic ,  kg19.811.5 * 22.19.423.510.2/et , s48.59.749.510.251.28.8/mdf ,  hz102.521.488.523.184.517.4a <", "cmfi0.150.070.080.040.060.04a < cdata are presented as mean  sd . * significant difference among the three groups ( p \n < 0.05 ) .", "group a : active myofascial trigger points ; group b : latent myofascial \n trigger points ; group c : control .", "et : endurance time ; mdf : median frequency ; mfi : \n muscle fatigue index ; mvic : maximal voluntary isometric contraction ) .", "tukey post hoc results showed that the active myofascial \n trigger point group was significantly different from the control group .", "muscle fatigue index \n also was significantly different among all three groups ( p < 0.05 ) ( table 1 ) .", "tukey post hoc results showed \n that the active myofascial trigger point group was significantly different from the control \n group .", "data are presented as mean  sd . * significant difference among the three groups ( p \n < 0.05 ) .", "group a : active myofascial trigger points ; group b : latent myofascial \n trigger points ; group c : control .", "et : endurance time ; mdf : median frequency ; mfi : \n muscle fatigue index ; mvic : maximal voluntary isometric contraction", "this study aimed to measure mvic , endurance , mdf , and muscle fatigue index of normal \n muscles versus muscles with latent or active myofascial trigger points , thereby identifying \n the neuromuscular physiological characteristics of muscles with active myofascial trigger \n points and providing clinical foundational data that are applicable for the quantitative \n evaluation and diagnosis of mps .", "surface emg , which was used to evaluate the functionality of trigger points in this study , \n is nonintrusive and convenient ; thus , it is widely used in studies on the functional \n characteristics of muscle , by analyzing the electrical activity of muscle .", "the results of \n mdf according to surface emg show changes in recruitment of fast - twitch muscle fibers and \n conduction velocity of motor unit action potential12 .", "in addition , increases in mdf reflect recruitment of type ii \n fibers , and muscle fatigue index can be analyzed according to the relationship between type \n ii fibers and mdf13 , 14 . in the present experiment ,", "mdf was significantly higher in the \n active trigger point muscles than in normal muscles .", "the above results indicate that as \n trigger points were activated , recruitment of motor unit action potential of type ii fibers \n also increased .", "in addition , muscle fatigue index also increased due to the increased \n distribution of type ii fibers .", "however , mvic was lower in active trigger point muscles than \n in normal muscles .", "although a high correlation between muscle strength and mdf has been \n reported15 , 16 , the same correlation was not found in this study .", "this result was \n due to increased recruitment of motor unit action potential of type ii fibers in active \n trigger point muscles , which more easily induced muscle fatigue as a physiological \n phenomenon , thereby reducing muscle strength .", "in other words , the greater the fatigue , the \n weaker the muscle strength17 ; endurance \n also was decreased due to muscle weakening .", "therefore , this study s results will be effective in complementing the physical therapy \n diagnosis of mps  which , until now , has focused on physical examination only  by understanding \n not only the usefulness of electrophysiological analysis in mps diagnosis , but also the \n neuromuscular physiological characteristics of active trigger point muscles ."], "abstract_text": ["<S> [ purpose ] this study compared the differences in electrophysiological characteristics of \n normal muscles versus muscles with latent or active myofascial trigger points , and \n identified the neuromuscular physiological characteristics of muscles with active \n myofascial trigger points , thereby providing a quantitative evaluation of myofascial pain \n syndrome and clinical foundational data for its diagnosis . [ subjects ] ninety adults in \n their 20s participated in this study . </S>", "<S> subjects were equally divided into three groups : the \n active myofascial trigger point group , the latent myofascial trigger point group , and the \n control group . [ methods ] </S>", "<S> maximum voluntary isometric contraction ( mvic ) , endurance , median \n frequency ( mdf ) , and muscle fatigue index were measured in all subjects . </S>", "<S> [ results ] no \n significant differences in mvic or endurance were revealed among the three groups . \n </S>", "<S> however , the active trigger point group had significantly different mdf and muscle fatigue \n index compared with the control group . [ conclusion ] given that muscles with active \n myofascial trigger points had an increased mdf and suffered muscle fatigue more easily , \n increased recruitment of motor unit action potential of type ii fibers was evident . \n </S>", "<S> therefore , electrophysiological analysis of these myofascial trigger points can be applied \n to evaluate the effect of physical therapy and provide a quantitative diagnosis of \n myofascial pain syndrome . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["myofascial pain syndrome ( mps ) can be caused by frequent muscle or fascial stiffness as a \n result of prolonged tension and muscle fatigue due to repetitive stress of muscles or \n overuse of particular muscles1 , 2 .", "mps activates trigger points in 54% of women and 45% of men , and is regarded as \n the most common cause of pain in the musculoskeletal system3 . in particular , the upper trapezius requires head movement according \n to the direction of view , but it can be vulnerable to damage when the hands and arms are \n used repetitively in work that requires precise control4,5,6 . once myofascial trigger points are activated , changes in the \n structural characteristics and contraction function of the muscle will occur .", "the most \n distinctive changes are taut bands , tender nodules , referred pain , local twitch response , \n muscle weakness , and restricted range of motion3 .", "to evaluate the contraction function of skeletal muscle , various methods can be used . among \n them ,", "surface electromyography ( emg ) analyzes functional changes in muscle by measuring \n quantitative changes in motor unit action potential that is activated by muscle \n contraction7 , 8 .", "surface emg is widely used in kinetic analysis to diagnose normal \n or abnormal function in muscles and nerves according to amplitude and frequency9 .", "it has several advantages , as it is \n noninvasive and convenient , and can perform measurement even during dynamic motion10 , 11 .", "although a number of clinical reports on the pathologic mechanisms or clinical diagnosis \n and treatment of mps can be found , few studies have been conducted on the changes in \n contraction function in myofascial trigger point areas or on the electrophysiological \n characteristics and neuromuscular physiological information .", "this study aimed to compare the \n electrophysiological characteristics of normal muscles versus muscles with latent or active \n myofascial trigger points , and to identify their neuromuscular physiological \n characteristics , in order to provide a quantitative evaluation of mps and clinical \n foundational data for its diagnosis ."], ["this study was approved by the \n research agency , and all participants provided written informed consent .", "subjects were \n selected among those with no known neurologic disease , no regular exercise habit , and no \n drug use that could affect the experimental result .", "subjects in the active myofascial trigger point group were those diagnosed with mps \n by a physician and whose clinical characteristics were pain in the upper trapezius during \n rest , taut bands , tender nodules , referred pain , local twitch response , and muscle weakness . \n", "subjects in the latent myofascial trigger point group were those not diagnosed with mps but \n who felt tenderness when the upper trapezius was stimulated .", "mean age , height , and weight , respectively , were 23.27 \n  4.21  years , 172.32  8.44  cm , and 64.14  6.54  kg in the active trigger point group ; 23.74 \n  6.47  years , 174.76  6.22  cm , and 66.74  5.74  kg in the latent trigger point group ; and \n 24.17  3.14  years , 171.21  9.64  cm , and 63.44  7.51  kg in the control group . to measure maximum voluntary isometric contraction ( mvic ) , the shoulders and heads of the \n subjects were firmly fixed and pulled as the dynamometer as maximum power , thereby measuring \n mvic against the upper trapezius .", "endurance time was measured from the start of mvic to a \n 50% reduction in contraction force .", "median frequency ( mdf ) was measured by using emg at the \n muscle belly of the upper trapezius during mvic .", "muscle fatigue index was calculated by \n obtaining the mdf at a section where the mvic force was reduced from 100% to 50% , and remove \n the 50% mdf from 100% mdf was divided into 100% mdf .", "the sampling rate for the surface emg \n signal was set at 1,000  hz , and the frequency band filter was set at 20 to 450  hz , using one \n channel .", "storage and analysis of the emg signals was performed using acqknowledge 3.8.1 \n ( biopac systems , goleta , ca , usa ) .", "the average of three measurements was calculated and used \n in the analyses ; 10 minutes of rest was given between measurements to prevent muscle \n fatigue .", "statistical analyses were performed using spss version 18.0 for windows ( spss , inc . \n", "one - way analysis of variance was conducted to examine differences among \n groups with respect to the measured items , while the tukey test was conducted for post hoc \n analysis ."], ["there were no significant differences in mvic or endurance time among the three groups . \n however , mdf was significantly different among all three groups ( p < 0.05 ) ( table 1table 1.comparison of mvic , et , mdf , and fi among the three groupsgroup a ( n = 30)group b ( n = 30)group c ( n = 30)post hocmvic ,  kg19.811.5 * 22.19.423.510.2/et , s48.59.749.510.251.28.8/mdf ,  hz102.521.488.523.184.517.4a <", "cmfi0.150.070.080.040.060.04a < cdata are presented as mean  sd . * significant difference among the three groups ( p \n < 0.05 ) .", "group a : active myofascial trigger points ; group b : latent myofascial \n trigger points ; group c : control .", "et : endurance time ; mdf : median frequency ; mfi : \n muscle fatigue index ; mvic : maximal voluntary isometric contraction ) .", "tukey post hoc results showed that the active myofascial \n trigger point group was significantly different from the control group .", "muscle fatigue index \n also was significantly different among all three groups ( p < 0.05 ) ( table 1 ) .", "tukey post hoc results showed \n that the active myofascial trigger point group was significantly different from the control \n group .", "data are presented as mean  sd . * significant difference among the three groups ( p \n < 0.05 ) .", "group a : active myofascial trigger points ; group b : latent myofascial \n trigger points ; group c : control .", "et : endurance time ; mdf : median frequency ; mfi : \n muscle fatigue index ; mvic : maximal voluntary isometric contraction"], ["this study aimed to measure mvic , endurance , mdf , and muscle fatigue index of normal \n muscles versus muscles with latent or active myofascial trigger points , thereby identifying \n the neuromuscular physiological characteristics of muscles with active myofascial trigger \n points and providing clinical foundational data that are applicable for the quantitative \n evaluation and diagnosis of mps .", "surface emg , which was used to evaluate the functionality of trigger points in this study , \n is nonintrusive and convenient ; thus , it is widely used in studies on the functional \n characteristics of muscle , by analyzing the electrical activity of muscle .", "the results of \n mdf according to surface emg show changes in recruitment of fast - twitch muscle fibers and \n conduction velocity of motor unit action potential12 .", "in addition , increases in mdf reflect recruitment of type ii \n fibers , and muscle fatigue index can be analyzed according to the relationship between type \n ii fibers and mdf13 , 14 . in the present experiment ,", "mdf was significantly higher in the \n active trigger point muscles than in normal muscles .", "the above results indicate that as \n trigger points were activated , recruitment of motor unit action potential of type ii fibers \n also increased .", "in addition , muscle fatigue index also increased due to the increased \n distribution of type ii fibers .", "however , mvic was lower in active trigger point muscles than \n in normal muscles .", "although a high correlation between muscle strength and mdf has been \n reported15 , 16 , the same correlation was not found in this study .", "this result was \n due to increased recruitment of motor unit action potential of type ii fibers in active \n trigger point muscles , which more easily induced muscle fatigue as a physiological \n phenomenon , thereby reducing muscle strength .", "in other words , the greater the fatigue , the \n weaker the muscle strength17 ; endurance \n also was decreased due to muscle weakening .", "therefore , this study s results will be effective in complementing the physical therapy \n diagnosis of mps  which , until now , has focused on physical examination only  by understanding \n not only the usefulness of electrophysiological analysis in mps diagnosis , but also the \n neuromuscular physiological characteristics of active trigger point muscles ."]]}
{"article_id": "PMC4330232", "article_text": [" primary or secondary neurondocrine ( ne ) tumours of the female genital tract are rare .", " cervical small cell carcinoma and ovarian carcinoids are the most common gynaecological ne tumours .", "neuroendocrine tumours comprise a set of neoplasms that arises from the diffuse neuroendocrine cell system .", "these tumours are more commonly identified in the gastrointestinal tract , pancreas , lung and thymus .", "recently , a simplified terminology has been proposed , dividing ne tumours in two groups : poorly differentiated neuroendocrine carcinomas ( necs ) and well - differentiated neuroendocrine tumours ( nets ) .", "necs include small cell carcinoma and large cell neuroendocrine carcinoma , while nets account for typical and atypical carcinoids [ 14 ] .", "the clinical features of gynaecological ne tumours generally depend on the organ of origin and are non - specific", ". however , some patients may present with paraneoplastic syndromes owing to the production of several peptides and hormones .", "the absence of endocrine clinical syndromes in most patients with ne tumours is possibly related to the release of insufficient amounts or inactive hormones .", "the purpose of this article is to review the epidemiological , clinical and imaging features with pathological correlation of primary gynaecological ne tumours , emphasising the importance of differentiating them from metastatic disease and other carcinomas with an ne component .", "for the classification of ne differentiation , tumour cells have to express at least two ne markers such as chromogranin a , synaptophysin or neuron - specific enolase .", "a great variety of other peptides and hormones may be found , including calcitonin , gastrin , serotonin , substance p , vasoactive intestinal peptide , pancreatic polypeptide , somatostatin and adrenocorticotrophic hormone [ 1 , 3 ] .", "some cancers display a combination of ne and non - ne features , usually either glandular or squamous components .", "the amount of ne component within a non - ne carcinoma may range from a single ne cell to a well - identifiable ne tumour cell population . in this set , two terms are widely accepted : mixed exocrine - endocrine carcinoma ( meec ) and ( adeno)carcinoma with ( focal ) ne differentiation .", "the finding of a focal non - ne differentiation in almost pure ne tumours is less common . according to the world health organisation ( who ) classification of ne tumours , the diagnosis of a mixed exocrine - endocrine tumour should take into account at least two major diagnostic parameters : an extension of at least 30  % for each component and the recognition of structural ne features such as well - differentiated organoid or solid / diffuse growth patterns .", "in fact , no reasonable explanation is provided for the limit of 30  % , and consequently morphological criteria should also be considered [ 7 , 8 ] .", "carcinomas with focal ne differentiation have been identified in several non - ne tumours of the gynaecological tract , such as endometrial endometrioid adenocarcinomas , endocervical adenocarcinomas and adenosquamous carcinomas , and even ovarian surface epithelial neoplasms of mucinous , endometrioid and serous types .", "these tumours display less than one - third of ne cells and a non - ne growth pattern , and the finding of these ne cells has not been shown to affect outcome [ 5 , 7 ] .", "few data are currently at our disposal regarding the prognosis of mixed exocrine - endocrine carcinoma ( meec ) in comparison to pure ne or non - ne tumours . however , the therapeutic schemes are different and the accurate histological diagnosis is mandatory", "the differentiation of small cell ne carcinomas from poorly differentiated squamous cell carcinoma ( scc ) with ne features may be especially difficult . sometimes , and particularly for cervical cancers ,", "the recognition of the ne component is only possible after the hysterectomy , since the tissue obtained on cervical biopsy may be insufficient , leading to the diagnosis of poorly differentiated cervical cancer .", "due to the rarity of these tumours , data in the literature concerning their imaging features are scarce . we know that small cell carcinoma and large cell ne carcinoma tend to behave aggressively , in contrast to carcinoids , which are typically confined to the organ .", "nevertheless , these are non - specific features and the available data regarding these extremely rare tumours are limited and discourage scientific generalisations .", "consequently , no reliable guidelines , for either diagnosis or treatment , are established . for staging purposes", ", physicians and radiologists should consider the currently used revised international federation of gynecology and obstetrics ( figo ) staging system [ 911 ] .", "the imaging approach to gynaecological ne tumours is similar to that for other histological types . for the cervix ,", "uterus and adnexa , ultrasound ( us ) using both transabdominal and transvaginal probes plays a critical role in the initial diagnosis .", "the use of computed tomography ( ct ) or positron emission tomography - computed tomography ( pet - ct ) is established in cervical and endometrial tumours for detection of nodal and distant metastases , which may be particularly useful for poorly differentiated ne carcinomas , typically invasive and metastatic . in ovarian cancers", ", ct is also indicated to evaluate the extent of disease and detect nonresectable metastases . magnetic resonance imaging ( mri )", "may be used for characterising indeterminate adnexal masses and for pre - treatment staging purposes , essentially in cervical and endometrial tumours .", "mri may replace ct in the staging of adnexal tumours in young females , pregnancy , renal insufficiency and allergy to iodinated contrast [ 10 , 12 , 13 ] ( table  1).table 1histological classification of ne tumours . table created by the authors based on the literature review [ 14 , 7]poorly differentiated neuroendocrine carcinomas ( necs)small cell carcinomalarge cell neuroendocrine carcinomawell - differentiated neuroendocrine tumours ( nets)typical carcinoidatypical carcinoidcombination of ne and non - ne featuresmixed exocrine - endocrine carcinoma ( meec ) ( adeno ) carcinoma with ( focal ) ne differentiation histological classification of ne tumours .", "table created by the authors based on the literature review [ 14 , 7 ] ne tumours of the vagina and vulva are much rarer .", "us plays a limited role in the diagnosis , but ct and mri may be used for staging purposes .", "mri is primarily used for local staging of vaginal tumours and should be performed after vaginal filling with gel . particularly for typical and atypical carcinoids ( well - differentiated neuroendocrine tumours ) ,", "patients with negative octreotide scans should not be considered for somatostatin analogue therapy and typically show better responses to chemotherapy schemes .", "primary carcinoid tumours of the ovary are rare and account for less than 5  % of all carcinoid tumours and for less than 0.1  % of all ovarian neoplasms .", "these are germ cell tumours in origin and classified as teratomas with a predominance of ne features .", "the great majority is asymptomatic and incidentally found on cross - sectional or ultrasound imaging .", "the median age of diagnosis is 55  years of age ( range from 17 to 83  years ) , with most of the patients being peri- or postmenopausal [ 1 , 1417 ] .", "these tumours are typically unilateral , slow growing and diagnosed in an early stage . in a 5-decade analysis performed by modlin et al .", ", 66  % of 113 carcinoid ovarian tumours were localised lesions , while 22  % 31  % presented with distant spread", ". they may exist in pure forms , predominantly solid with small cysts or as a nodule within a mature teratoma , mucinous cystadenoma or even brenner tumour .", "ovarian carcinoids contain well - differentiated ne cells and some subtypes are similar to the gastrointestinal carcinoids .", "they are immunoreactive to at least one neuroendocrine marker , including chromogranin , synaptophysin or leu-7 , and may also be present in peptide hormones such as pancreatic polypeptide , gastrin , vasoactive intestinal peptides and glucagon .", "insular carcinoid is the most common type of ovarian carcinoid , typically presenting with a pelvic mass .", "near 30  % of patients will have signs and symptoms of carcinoid syndrome , including facial flushing , diarrhoea , bronchospasm and oedema .", "it happens because serotonin - like substances are directly released into the systemic circulation through the ovarian venous system , bypassing hepatic deactivation .", "carcinoid syndrome is less common in the other subtypes ( 13  % in trabecular and 3.2  % in strumal carcinoids ) .", "trabecular carcinoid constitutes the second most common ovarian carcinoid tumour and 25  % of patients will present with constipation due to peptide yy production , which inhibits intestinal motility [ 15 , 17 ] .", "some cases of hirsutism are also thought to be related to peptide yy production , which is known to inhibit peripheral steroid - producing cells [ 14 , 19 ] .", "mucinous carcinoids are rare ( 1.5  % ) , may be pure or associated with mature teratomas . in these cases ,", "the differential diagnosis must include metastasis from a gastrointestinal tumour and primary mucinous appendiceal carcinoid .", "we should also consider strumal carcinoids , tumours that contain normal thyroid and carcinoid tissues .", "eight per cent of the patients may present with thyroid hormone - related symptoms and some ovarian strumal carcinoids are also reported to cause severe constipation owing to peptide yy production [ 15 , 22 ] .", "primary carcinoid tumours typically behave in a benign fashion , and the prognosis is generally favourable .", "if the tumour presents with stage i , survival may be greater than 90  % ; however , for women with advanced stage disease , the prognosis is poor and the 5-year survival is approximately 33  % . attending to the more favourable prognosis of ovarian carcinoid tumours ,", "the differentiation of such lesions from other highly malignant ovarian neoplasms is mandatory in order to choose the most appropriate therapeutic options .", "particular note should be taken in relation to mucinous carcinoid , which behaves more aggressively and may present with extra - ovarian spread and lymph node involvement . while performing the diagnosis of an ovarian carcinoid , it is important to rule out ovarian metastases , essentially from the gastrointestinal tract .", "the insular and trabecular subtypes most commonly have metastases in the ovary simulating a primary tumour .", "metastatic carcinoid is frequently bilateral , while primary ovarian carcinoids tend to be unilateral [ 1 , 23 ] .", "compared the features of primary insular carcinoids and metastatic midgut carcinoids and concluded that the presence of extra - ovarian tumour , bilaterality , multinodularity , vascular invasion and the absence of teratomatous elements are suggestive of an extra - ovarian origin .", "both small cell carcinoma and large cell ne carcinoma of the ovary are highly malignant and aggressive , regardless of the stage .", "ovarian small cell carcinoma resembles the pulmonary type and is often associated with common epithelial tumours , typically an endometrioid carcinoma , suggesting an origin from ovarian surface epithelium .", "its immune profile includes positivity for neuron - specific enolase and less commonly for chromogranin [ 1 , 5 ] .", "large cell ne carcinomas have been associated with benign and malignant surface epithelial - stromal tumours , and immunohistochemical markers for chromogranin are typically positive [ 25 , 26 ] .", "we review three cases of neuroendocrine ovarian tumours , one primary carcinoid and two secondary carcinoid tumours ( figs .  1 , 2 and 3 ) . once again , we emphasise that few data are provided in the literature regarding the imaging features of these tumours", "all the cases show well - circumscribed , predominately solid ovarian tumours . however , fig .  1 exemplifies a typical benign behaviour , while fig . ", "figure  2 represents an atypical presentation of metastatic disease , with overlap between benign and malignant features.fig .", "1 \n a  e struma carcinoid of the right ovary . a 49-year - old female complaining of pelvic and lower abdominal pain . ultrasound ( a ) and axial enhanced ct image ( b ) .", "large , heterogeneous , mixed tumour of the ovary , predominantly solid , with necrotic areas and calcifications", ". the solid portions are moderately echogenic on us and hyperdense on enhanced ct , while cystic areas are anechogenic on us and hypodense on ct .", "this tumour is composed of areas of thyroid tissue and carcinoid ( h&e ) , the latter having acinar and trabecular architecture ( c ) .", "the tumour cells show positive cytoplasmatic staining for chromogranine ( d ) and for synaptophysin ( not shown ) .", "the thyroid area with compact folicular structure presents intense immunoreactivity for thyroglobulin ( e)fig . 2 \n a  d metastatic ovarian carcinoid of unknown primary origin in a 23-year - old female complaining of pelvic pain and amenorrhoea .", "sagittal t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation , after intravenous gadolinium administration ( b ) .", "large , well - defined solid lesion of the left ovary , cranially and anteriorly located in relation to the uterus .", "on t2wi , it displays intermediate signal intensity and shows a central high signal region .", "the lesion shows peripheral , intense contrast enhancement and a large central area of necrosis .", "h&e section of the ovary occupied by a tumour with insular pattern composed by neuroendocrine cells ( c ) .", "these cells have intense cytoplasmatic staining for synaptophysin ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown)fig . 3 \n a  b metastatic , bilateral ovarian carcinoid , from a primary jejunal tumour in a 28-year - old female complaining of pelvic pain and dyspareunia .", "multiple liver metastases were also seen ( not shown ) \n a  e struma carcinoid of the right ovary .", "large , heterogeneous , mixed tumour of the ovary , predominantly solid , with necrotic areas and calcifications .", "the solid portions are moderately echogenic on us and hyperdense on enhanced ct , while cystic areas are anechogenic on us and hypodense on ct .", "this tumour is composed of areas of thyroid tissue and carcinoid ( h&e ) , the latter having acinar and trabecular architecture ( c ) .", "the tumour cells show positive cytoplasmatic staining for chromogranine ( d ) and for synaptophysin ( not shown ) .", "the thyroid area with compact folicular structure presents intense immunoreactivity for thyroglobulin ( e ) \n a  d metastatic ovarian carcinoid of unknown primary origin in a 23-year - old female complaining of pelvic pain and amenorrhoea .", "sagittal t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation , after intravenous gadolinium administration ( b ) . large , well - defined solid lesion of the left ovary , cranially and anteriorly located in relation to the uterus .", "on t2wi , it displays intermediate signal intensity and shows a central high signal region .", "the lesion shows peripheral , intense contrast enhancement and a large central area of necrosis .", "h&e section of the ovary occupied by a tumour with insular pattern composed by neuroendocrine cells ( c ) .", "these cells have intense cytoplasmatic staining for synaptophysin ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown ) \n a  b metastatic , bilateral ovarian carcinoid , from a primary jejunal tumour in a 28-year - old female complaining of pelvic pain and dyspareunia . coronal ( a ) and axial ( b ) enhanced ct images .", "large cell ne carcinoma and carcinoid types are rarer than the former and few data can be found in the literature .", "staging and treatment strategies for both small and large cell cervical carcinoma globally follow the same criteria used for common histological types of cervical carcinomas .", "small cell carcinoma accounts for 16  % of cervical carcinomas [ 6 , 27 ] .", "median age of diagnosis is in the 5th decade , ranging from 21 to 87  years .", "some patients present with a cervical mass and an abnormal pap smear may be found .", "this tumour is strongly associated with human papilloma virus 18 ( hpv-18 ) , but hpv-16-positive tumours have also been demonstrated .", "the most commonly found are hypercalcaemia ( parathormone ) , hypoglycaemia ( insulin ) , carcinoid syndrome ( serotonin ) , cushing s syndrome ( corticotropin ) , siadh ( vasopressin ) and myasthenia gravis .", "most tumours are voluminous , exceeding 6  cm , and show deep cervical infiltration and necrosis", ". often , growth may be exophytic , which appears to be associated with a better prognosis . nevertheless , it is generally a highly aggressive tumour with a worse prognosis than that of poorly differentiated squamous cell carcinomas .", "the rates of lymphovascular space invasion and extra - pelvic recurrences for bone , supraclavicular lymph nodes and lung are particularly high [ 6 , 27 , 28 , 30 ] . according to mccusker et al . , females with small cell tumours have 1.84 times greater risk of death compared to patients with squamous cell carcinomas .", "it appears to have a similar outcome to small cell carcinoma and its treatment remains challenging because of the high rate of recurrence and distant metastases even with early stage disease [ 2 , 32 ] .", "we review three cases of cervical small cell carcinoma ( figs .  4 , 5 and 6 ) .", "cervical necs resemble cervical squamous cell carcinoma , displaying the same mri features : low signal intensity on t1-weighted images ( t1wi ) and high signal intensity on t2-weighted images ( t2wi ) .", "no histological distinction can be provided regarding only imaging features . however , according to the literature , cervical necs are generally highly aggressive and expected to be diagnosed in advanced figo stages .", "concordantly , we illustrate necs in iv - a ( invasion of adjacent organs ) ( fig .  4 ) and iii - b ( invasion of the pelvic side wall ) ( fig . ", "6 ) stages.fig . 4 \n a  b cervical neuroendocrine tumour ( figo iv - a ; invasion of adjacent organs ) .", "a 32-year - old female complaining of vaginal bleeding after intercourse and fetid vaginal discharge .", "sagittal t2-weighted mr image ( a ) and axial t2-weighted mr image ( b ) .", "the parametria are circumferentially infiltrated and the tumour extends anteriorly to the bladder , with transmural invasion and fistulae ( arrow ) .", ". 5 \n a  e cervical small cell carcinoma ( figo ii - a ; invasion of the upper two - thirds of vagina with no parametrial invasion ) .", "sagittal t2-weighted mr image ( a ) , axial t2-weighted mr images ( b ) and sagittal diffusion weighted image ( dwi ) , b(c ) retroverted uterus with an anterior cervical tumour , highly restrictive on dwi .", "no parametrial invasion is noted , but huge , bilateral obturator lymph nodes are found ( arrows ) .", "the left ovary shows follicular activity and is posteriorly displaced ( arrowhead ) . in this h&e section the cervix is occupied by a solid tumour , composed of small cells , with uniform nuclei and scarce cytoplasm ( d ) .", "some tumour cells show positive cytoplasmatic staining for synaptophysin ( e ) and for chromogranin ( not shown)fig . 6 \n a  b cervical small cell carcinoma ( figo iii - b ; extension to the pelvic side wall ) . a 64  year - old female complaining of menorrhagia .", "axial t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation after intravenous gadolinium administration ( b ) .", "cervical tumour extending posteriorly to the pelvic wall ( asterisk ) and superiorly to the left adnexal area , where it forms a huge solid , heterogeneous mass with central necrotic areas ( arrow ) .", "this mass shows intense enhancement after gadolinium administration and is adherent to the uterine corpus , which may be found inferiorly .", "bilateral internal iliac lymph nodes are also noted ( arrowheads ) \n a  b cervical neuroendocrine tumour ( figo iv - a ; invasion of adjacent organs ) .", "a 32-year - old female complaining of vaginal bleeding after intercourse and fetid vaginal discharge .", "sagittal t2-weighted mr image ( a ) and axial t2-weighted mr image ( b ) . large cervical cancer with infiltration of the lower segment of the uterine corpus .", "the parametria are circumferentially infiltrated and the tumour extends anteriorly to the bladder , with transmural invasion and fistulae ( arrow ) .", "bilateral , nodal metastases of the external iliac groups are also seen ( arrowheads ) \n a  e cervical small cell carcinoma ( figo ii - a ; invasion of the upper two - thirds of vagina with no parametrial invasion ) .", "sagittal t2-weighted mr image ( a ) , axial t2-weighted mr images ( b ) and sagittal diffusion weighted image ( dwi ) , b(c ) retroverted uterus with an anterior cervical tumour , highly restrictive on dwi .", "no parametrial invasion is noted , but huge , bilateral obturator lymph nodes are found ( arrows ) .", "the left ovary shows follicular activity and is posteriorly displaced ( arrowhead ) . in this h&e section", "the cervix is occupied by a solid tumour , composed of small cells , with uniform nuclei and scarce cytoplasm ( d ) .", "some tumour cells show positive cytoplasmatic staining for synaptophysin ( e ) and for chromogranin ( not shown ) \n a  b cervical small cell carcinoma ( figo iii - b ; extension to the pelvic side wall ) .", "axial t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation after intravenous gadolinium administration ( b ) .", "cervical tumour extending posteriorly to the pelvic wall ( asterisk ) and superiorly to the left adnexal area , where it forms a huge solid , heterogeneous mass with central necrotic areas ( arrow ) .", "this mass shows intense enhancement after gadolinium administration and is adherent to the uterine corpus , which may be found inferiorly .", "well - differentiated neuroendocrine tumours ( nets ) include typical and atypical carcinoid tumours . among the uncommon sites of primary carcinoid tumours ,", "the uterine cervix constitutes one of the rarest locations , representing only 0.5  % to 5  % of cervical cancers .", "carcinoid syndrome is very rare and even in the lack of clinically evident carcinoid syndrome , many patients present with elevated urinary 5-hydroxyindoleacetic acid ( 5-hiaa ) .", "however , the diagnosis of a cervical carcinoid is frequently postoperative and laboratorial investigation is typically not helpful since it is not performed in the absence of symptoms .", "these tumours display a very aggressive , malignant behaviour , and their prognosis is poor ( 2- or 3-year survival rates , ranging from 12.5 to 33  % , according to some studies ) .", "atypical carcinoids of the cervix are also very rare and , according to a review of yoshida et al . , only 13 cases have been reported in the literature .", "the reported endometrial neoplasms with ne differentiation essentially include small cell carcinoma and endometrial adenocarcinoma with ne cells .", "small cell carcinoma of the endometrium resembles that of the lung and is uncommon , accounting for less than 1  % of all endometrial carcinomas .", "like in other endometrial carcinomas , abnormal bleeding is the primary presenting symptom , but some patients present with advanced disease , sometimes with pain from metastases .", "these tumours usually present as bulky intraluminal masses , and deep myometrial invasion ( 3350  % ) and extra - uterine spread ( near 50  % ) are common .", "pelvic lymph nodes , adnexa and the peritoneum constitute the typical sites of extra - uterine involvement .", "the overall prognosis is poor [ 5 , 35 ] . in 50 to 66  % ,", "endometrial small cell carcinoma displays an admixed component of grade 1 or 2 endometrioid adenocarcinoma , and few cases showed a malignant mixed mullerian tumour within a predominant small cell carcinoma .", "we review two cases of poorly differentiated endometrial carcinoma with ne differentiation ( figs .  7 and 8) . like in cervical carcinoma", ", imaging does not allow histological distinction , since all endometrial cancers tend to present similarly : low to moderate signal on t2wi and hypointensity relative to the hyperintense enhancing myometrium on dynamic contrast - enhanced mri sequences .", "however , when a highly aggressive endometrial tumour is found , a neuroendocrine nature may be suspected .", "there are no consistent data regarding the prognosis of non - pure ne endometrial tumours ; however , the cases we illustrate were diagnosed in advanced figo stages , iv - b ( distant metastases ) ( fig . ", "7 ) and iii - c1 ( involvement of pelvic nodes ) ( fig . ", "7 \n a  d poorly differentiated endometrial carcinoma with ne differentiation ( figo iv - b ; distant metastases ) .", "axial t2-weighted mr image ( a ) and axial enhanced ct image ( b ) .", "there is no invasion of the cervical stroma or bladder ( not shown ) , but a right obturator node ( arrow ) is found .", "ct evaluation shows that the disease has disseminated outside the pelvis , revealing para - aortic lymph nodes ( arrowhead ) as well as retroperitoneal and peritoneal metastases ( asterisks ) .", "vagina biopsy ( h&e ) infiltrated by a solid tumour composed by sheets of uniform cells , with small oval nuclei and scarse cytoplasm ( c ) .", "these cells have an intense cytoplasmatic staining for neuron - specific enolase ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown ) .", "8 \n a  d undifferentiated endometrial carcinoma with ne differentiation ( figo iii - c1 ; involvement of pelvic nodes ) .", "endometrial tumour prolapsing into the cervical canal , without invasion of the cervical stroma and myometrium .", "the tumour extends bilaterally through the lumen of the tubes to the parametrial tissues ( arrows ) .", "h&e section of the tumour with a solid pattern composed by large cells with pleomorphic nuclei , mitoses and scarce cytoplasm ( c ) .", "these cells have small chromogranine - positive granules ( d ) and small synaptophysin granules in the cytoplasm ( not shown ) and are strongly positive for pancytokeratin cam 5.2 ( not shown ) \n a  d poorly differentiated endometrial carcinoma with ne differentiation ( figo iv - b ; distant metastases ) .", "axial t2-weighted mr image ( a ) and axial enhanced ct image ( b ) .", "there is no invasion of the cervical stroma or bladder ( not shown ) , but a right obturator node ( arrow ) is found .", "ct evaluation shows that the disease has disseminated outside the pelvis , revealing para - aortic lymph nodes ( arrowhead ) as well as retroperitoneal and peritoneal metastases ( asterisks ) .", "vagina biopsy ( h&e ) infiltrated by a solid tumour composed by sheets of uniform cells , with small oval nuclei and scarse cytoplasm ( c ) .", "these cells have an intense cytoplasmatic staining for neuron - specific enolase ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown ) .", "this tumour was synaptophysin negative \n a  d undifferentiated endometrial carcinoma with ne differentiation ( figo iii - c1 ; involvement of pelvic nodes ) .", "endometrial tumour prolapsing into the cervical canal , without invasion of the cervical stroma and myometrium .", "the tumour extends bilaterally through the lumen of the tubes to the parametrial tissues ( arrows ) .", "h&e section of the tumour with a solid pattern composed by large cells with pleomorphic nuclei , mitoses and scarce cytoplasm ( c ) .", "these cells have small chromogranine - positive granules ( d ) and small synaptophysin granules in the cytoplasm ( not shown ) and are strongly positive for pancytokeratin cam 5.2 ( not shown )", "neuroendocrine tumours of the fallopian tube , vagina and vulva are very rare and only a few cases have been reported . in the fallopian tube ,", "these tumours encompass carcinoids and small cell carcinoma , and they are not well known . in the vagina ,", "almost ten cases of small cell carcinoma have been reported , occurring in females between 41 and 78  years of age .", "like in other vaginal tumours , the most frequent complaint was vaginal bleeding or discharge .", "metastatic disease was common in this small cohort , so the prognosis is thought to be poor . in the vulva , neuroendocrine differentiation may be found in small cell carcinoma of the bartholin gland and merkel cell tumours , which tend to present as vulvar masses .", "two groups are currently considered : necs , including small cell carcinoma and large cell ne carcinoma , and nets , which encompass typical and atypical carcinoids .", "the clinical features of theses tumours are non - specific and depend on the organ of origin and on the extension and aggressiveness of the disease .", "the imaging evaluation should follow the same criteria used for other tumours , and figo remains the preferred staging system .", "the diagnosis is obviously histological , and the role of imaging tools essentially includes initial detection , staging and biopsy guidance .", "however , some points should be taken into account when imaging studies are performed : cervical small cell carcinoma and ovarian carcinoid are the most common gynaecological ne tumours.cervical small cell carcinomas usually behave aggressively.ovarian carcinoids tend to behave in a benign fashion.while managing an ovarian carcinoid , extra - ovarian extension , bilaterality and multinodularity should raise the suspicion of metastatic disease.endometrial ne tumours are very rare and are usually small cell carcinoma or adenocarcinoma with ne differentiation.as for other tumours , us , ct , pet - ct and mri remain the most commonly used imaging tools , but octreotide scans may be useful for typical and atypical carcinoids .", "ovarian carcinoids tend to behave in a benign fashion . while managing an ovarian carcinoid , extra - ovarian extension , bilaterality and multinodularity should raise the suspicion of metastatic disease .", "endometrial ne tumours are very rare and are usually small cell carcinoma or adenocarcinoma with ne differentiation . as for other tumours , us , ct , pet - ct and mri remain the most commonly used imaging tools , but octreotide scans may be useful for typical and atypical carcinoids ."], "abstract_text": ["<S> backgroundboth primary and secondary gynaecological neuroendocrine ( ne ) tumours are uncommon , and the literature is scarce concerning their imaging features.methodsthis article reviews the epidemiological , clinical and imaging features with pathological correlation of gynaecological ne tumours.resultsthe clinical features of gynaecological ne tumours are non - specific and depend on the organ of origin and on the extension and aggressiveness of the disease . </S>", "<S> the imaging approach to these tumours is similar to that for other histological types and the revised international federation of gynecology and obstetrics ( figo ) staging system also applies to ne tumours . </S>", "<S> neuroendocrine tumours were recently divided into two groups : poorly differentiated neuroendocrine carcinomas ( necs ) and well - differentiated neuroendocrine tumours ( nets ) . </S>", "<S> necs include small cell carcinoma and large cell neuroendocrine carcinoma , while nets account for typical and atypical carcinoids . </S>", "<S> cervical small cell carcinoma and ovarian carcinoid are the most common gynaecological ne tumours . </S>", "<S> the former typically behaves aggressively ; the latter usually behaves in a benign fashion and tends to be confined to the organ.conclusionwhile dealing with ovarian carcinoids , extra - ovarian extension , bilaterality and multinodularity raise the suspicion of metastatic disease . </S>", "<S> ne tumours of the endometrium and other gynaecological locations are very rare.teaching points primary or secondary neurondocrine ( ne ) tumours of the female genital tract are rare. cervical small cell carcinoma and ovarian carcinoids are the most common gynaecological ne tumours. cervical small cell carcinomas usually behave aggressively. ovarian carcinoids tend to behave in a benign fashion. the imaging approach to gynaecological ne tumours and other histological types is similar . </S>"], "labels": null, "section_names": ["None", "Introduction", "General histological considerations", "General imaging considerations", "Ovary", "Well-differentiated neuroendocrine tumours (NETs)", "Poorly differentiated neuroendocrine carcinomas (NECs)", "Cervix", "Poorly differentiated neuroendocrine carcinomas (NECs)", "Well-differentiated neuroendocrine tumours (NETs)", "Endometrium", "Other locations", "Conclusion and teaching points", "Conflict of Interest"], "sections": [[" primary or secondary neurondocrine ( ne ) tumours of the female genital tract are rare .", " cervical small cell carcinoma and ovarian carcinoids are the most common gynaecological ne tumours ."], ["neuroendocrine tumours comprise a set of neoplasms that arises from the diffuse neuroendocrine cell system .", "these tumours are more commonly identified in the gastrointestinal tract , pancreas , lung and thymus .", "recently , a simplified terminology has been proposed , dividing ne tumours in two groups : poorly differentiated neuroendocrine carcinomas ( necs ) and well - differentiated neuroendocrine tumours ( nets ) .", "necs include small cell carcinoma and large cell neuroendocrine carcinoma , while nets account for typical and atypical carcinoids [ 14 ] .", "the clinical features of gynaecological ne tumours generally depend on the organ of origin and are non - specific", ". however , some patients may present with paraneoplastic syndromes owing to the production of several peptides and hormones .", "the absence of endocrine clinical syndromes in most patients with ne tumours is possibly related to the release of insufficient amounts or inactive hormones .", "the purpose of this article is to review the epidemiological , clinical and imaging features with pathological correlation of primary gynaecological ne tumours , emphasising the importance of differentiating them from metastatic disease and other carcinomas with an ne component ."], ["for the classification of ne differentiation , tumour cells have to express at least two ne markers such as chromogranin a , synaptophysin or neuron - specific enolase .", "a great variety of other peptides and hormones may be found , including calcitonin , gastrin , serotonin , substance p , vasoactive intestinal peptide , pancreatic polypeptide , somatostatin and adrenocorticotrophic hormone [ 1 , 3 ] .", "some cancers display a combination of ne and non - ne features , usually either glandular or squamous components .", "the amount of ne component within a non - ne carcinoma may range from a single ne cell to a well - identifiable ne tumour cell population . in this set , two terms are widely accepted : mixed exocrine - endocrine carcinoma ( meec ) and ( adeno)carcinoma with ( focal ) ne differentiation .", "the finding of a focal non - ne differentiation in almost pure ne tumours is less common . according to the world health organisation ( who ) classification of ne tumours , the diagnosis of a mixed exocrine - endocrine tumour should take into account at least two major diagnostic parameters : an extension of at least 30  % for each component and the recognition of structural ne features such as well - differentiated organoid or solid / diffuse growth patterns .", "in fact , no reasonable explanation is provided for the limit of 30  % , and consequently morphological criteria should also be considered [ 7 , 8 ] .", "carcinomas with focal ne differentiation have been identified in several non - ne tumours of the gynaecological tract , such as endometrial endometrioid adenocarcinomas , endocervical adenocarcinomas and adenosquamous carcinomas , and even ovarian surface epithelial neoplasms of mucinous , endometrioid and serous types .", "these tumours display less than one - third of ne cells and a non - ne growth pattern , and the finding of these ne cells has not been shown to affect outcome [ 5 , 7 ] .", "few data are currently at our disposal regarding the prognosis of mixed exocrine - endocrine carcinoma ( meec ) in comparison to pure ne or non - ne tumours . however , the therapeutic schemes are different and the accurate histological diagnosis is mandatory", "the differentiation of small cell ne carcinomas from poorly differentiated squamous cell carcinoma ( scc ) with ne features may be especially difficult . sometimes , and particularly for cervical cancers ,", "the recognition of the ne component is only possible after the hysterectomy , since the tissue obtained on cervical biopsy may be insufficient , leading to the diagnosis of poorly differentiated cervical cancer ."], ["due to the rarity of these tumours , data in the literature concerning their imaging features are scarce . we know that small cell carcinoma and large cell ne carcinoma tend to behave aggressively , in contrast to carcinoids , which are typically confined to the organ .", "nevertheless , these are non - specific features and the available data regarding these extremely rare tumours are limited and discourage scientific generalisations .", "consequently , no reliable guidelines , for either diagnosis or treatment , are established . for staging purposes", ", physicians and radiologists should consider the currently used revised international federation of gynecology and obstetrics ( figo ) staging system [ 911 ] .", "the imaging approach to gynaecological ne tumours is similar to that for other histological types . for the cervix ,", "uterus and adnexa , ultrasound ( us ) using both transabdominal and transvaginal probes plays a critical role in the initial diagnosis .", "the use of computed tomography ( ct ) or positron emission tomography - computed tomography ( pet - ct ) is established in cervical and endometrial tumours for detection of nodal and distant metastases , which may be particularly useful for poorly differentiated ne carcinomas , typically invasive and metastatic . in ovarian cancers", ", ct is also indicated to evaluate the extent of disease and detect nonresectable metastases . magnetic resonance imaging ( mri )", "may be used for characterising indeterminate adnexal masses and for pre - treatment staging purposes , essentially in cervical and endometrial tumours .", "mri may replace ct in the staging of adnexal tumours in young females , pregnancy , renal insufficiency and allergy to iodinated contrast [ 10 , 12 , 13 ] ( table  1).table 1histological classification of ne tumours . table created by the authors based on the literature review [ 14 , 7]poorly differentiated neuroendocrine carcinomas ( necs)small cell carcinomalarge cell neuroendocrine carcinomawell - differentiated neuroendocrine tumours ( nets)typical carcinoidatypical carcinoidcombination of ne and non - ne featuresmixed exocrine - endocrine carcinoma ( meec ) ( adeno ) carcinoma with ( focal ) ne differentiation histological classification of ne tumours .", "table created by the authors based on the literature review [ 14 , 7 ] ne tumours of the vagina and vulva are much rarer .", "us plays a limited role in the diagnosis , but ct and mri may be used for staging purposes .", "mri is primarily used for local staging of vaginal tumours and should be performed after vaginal filling with gel . particularly for typical and atypical carcinoids ( well - differentiated neuroendocrine tumours ) ,", "patients with negative octreotide scans should not be considered for somatostatin analogue therapy and typically show better responses to chemotherapy schemes ."], [""], ["primary carcinoid tumours of the ovary are rare and account for less than 5  % of all carcinoid tumours and for less than 0.1  % of all ovarian neoplasms .", "these are germ cell tumours in origin and classified as teratomas with a predominance of ne features .", "the great majority is asymptomatic and incidentally found on cross - sectional or ultrasound imaging .", "the median age of diagnosis is 55  years of age ( range from 17 to 83  years ) , with most of the patients being peri- or postmenopausal [ 1 , 1417 ] .", "these tumours are typically unilateral , slow growing and diagnosed in an early stage . in a 5-decade analysis performed by modlin et al .", ", 66  % of 113 carcinoid ovarian tumours were localised lesions , while 22  % 31  % presented with distant spread", ". they may exist in pure forms , predominantly solid with small cysts or as a nodule within a mature teratoma , mucinous cystadenoma or even brenner tumour .", "ovarian carcinoids contain well - differentiated ne cells and some subtypes are similar to the gastrointestinal carcinoids .", "they are immunoreactive to at least one neuroendocrine marker , including chromogranin , synaptophysin or leu-7 , and may also be present in peptide hormones such as pancreatic polypeptide , gastrin , vasoactive intestinal peptides and glucagon .", "insular carcinoid is the most common type of ovarian carcinoid , typically presenting with a pelvic mass .", "near 30  % of patients will have signs and symptoms of carcinoid syndrome , including facial flushing , diarrhoea , bronchospasm and oedema .", "it happens because serotonin - like substances are directly released into the systemic circulation through the ovarian venous system , bypassing hepatic deactivation .", "carcinoid syndrome is less common in the other subtypes ( 13  % in trabecular and 3.2  % in strumal carcinoids ) .", "trabecular carcinoid constitutes the second most common ovarian carcinoid tumour and 25  % of patients will present with constipation due to peptide yy production , which inhibits intestinal motility [ 15 , 17 ] .", "some cases of hirsutism are also thought to be related to peptide yy production , which is known to inhibit peripheral steroid - producing cells [ 14 , 19 ] .", "mucinous carcinoids are rare ( 1.5  % ) , may be pure or associated with mature teratomas . in these cases ,", "the differential diagnosis must include metastasis from a gastrointestinal tumour and primary mucinous appendiceal carcinoid .", "we should also consider strumal carcinoids , tumours that contain normal thyroid and carcinoid tissues .", "eight per cent of the patients may present with thyroid hormone - related symptoms and some ovarian strumal carcinoids are also reported to cause severe constipation owing to peptide yy production [ 15 , 22 ] .", "primary carcinoid tumours typically behave in a benign fashion , and the prognosis is generally favourable .", "if the tumour presents with stage i , survival may be greater than 90  % ; however , for women with advanced stage disease , the prognosis is poor and the 5-year survival is approximately 33  % . attending to the more favourable prognosis of ovarian carcinoid tumours ,", "the differentiation of such lesions from other highly malignant ovarian neoplasms is mandatory in order to choose the most appropriate therapeutic options .", "particular note should be taken in relation to mucinous carcinoid , which behaves more aggressively and may present with extra - ovarian spread and lymph node involvement . while performing the diagnosis of an ovarian carcinoid , it is important to rule out ovarian metastases , essentially from the gastrointestinal tract .", "the insular and trabecular subtypes most commonly have metastases in the ovary simulating a primary tumour .", "metastatic carcinoid is frequently bilateral , while primary ovarian carcinoids tend to be unilateral [ 1 , 23 ] .", "compared the features of primary insular carcinoids and metastatic midgut carcinoids and concluded that the presence of extra - ovarian tumour , bilaterality , multinodularity , vascular invasion and the absence of teratomatous elements are suggestive of an extra - ovarian origin ."], ["both small cell carcinoma and large cell ne carcinoma of the ovary are highly malignant and aggressive , regardless of the stage .", "ovarian small cell carcinoma resembles the pulmonary type and is often associated with common epithelial tumours , typically an endometrioid carcinoma , suggesting an origin from ovarian surface epithelium .", "its immune profile includes positivity for neuron - specific enolase and less commonly for chromogranin [ 1 , 5 ] .", "large cell ne carcinomas have been associated with benign and malignant surface epithelial - stromal tumours , and immunohistochemical markers for chromogranin are typically positive [ 25 , 26 ] .", "we review three cases of neuroendocrine ovarian tumours , one primary carcinoid and two secondary carcinoid tumours ( figs .  1 , 2 and 3 ) . once again , we emphasise that few data are provided in the literature regarding the imaging features of these tumours", "all the cases show well - circumscribed , predominately solid ovarian tumours . however , fig .  1 exemplifies a typical benign behaviour , while fig . ", "figure  2 represents an atypical presentation of metastatic disease , with overlap between benign and malignant features.fig .", "1 \n a  e struma carcinoid of the right ovary . a 49-year - old female complaining of pelvic and lower abdominal pain . ultrasound ( a ) and axial enhanced ct image ( b ) .", "large , heterogeneous , mixed tumour of the ovary , predominantly solid , with necrotic areas and calcifications", ". the solid portions are moderately echogenic on us and hyperdense on enhanced ct , while cystic areas are anechogenic on us and hypodense on ct .", "this tumour is composed of areas of thyroid tissue and carcinoid ( h&e ) , the latter having acinar and trabecular architecture ( c ) .", "the tumour cells show positive cytoplasmatic staining for chromogranine ( d ) and for synaptophysin ( not shown ) .", "the thyroid area with compact folicular structure presents intense immunoreactivity for thyroglobulin ( e)fig . 2 \n a  d metastatic ovarian carcinoid of unknown primary origin in a 23-year - old female complaining of pelvic pain and amenorrhoea .", "sagittal t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation , after intravenous gadolinium administration ( b ) .", "large , well - defined solid lesion of the left ovary , cranially and anteriorly located in relation to the uterus .", "on t2wi , it displays intermediate signal intensity and shows a central high signal region .", "the lesion shows peripheral , intense contrast enhancement and a large central area of necrosis .", "h&e section of the ovary occupied by a tumour with insular pattern composed by neuroendocrine cells ( c ) .", "these cells have intense cytoplasmatic staining for synaptophysin ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown)fig . 3 \n a  b metastatic , bilateral ovarian carcinoid , from a primary jejunal tumour in a 28-year - old female complaining of pelvic pain and dyspareunia .", "multiple liver metastases were also seen ( not shown ) \n a  e struma carcinoid of the right ovary .", "large , heterogeneous , mixed tumour of the ovary , predominantly solid , with necrotic areas and calcifications .", "the solid portions are moderately echogenic on us and hyperdense on enhanced ct , while cystic areas are anechogenic on us and hypodense on ct .", "this tumour is composed of areas of thyroid tissue and carcinoid ( h&e ) , the latter having acinar and trabecular architecture ( c ) .", "the tumour cells show positive cytoplasmatic staining for chromogranine ( d ) and for synaptophysin ( not shown ) .", "the thyroid area with compact folicular structure presents intense immunoreactivity for thyroglobulin ( e ) \n a  d metastatic ovarian carcinoid of unknown primary origin in a 23-year - old female complaining of pelvic pain and amenorrhoea .", "sagittal t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation , after intravenous gadolinium administration ( b ) . large , well - defined solid lesion of the left ovary , cranially and anteriorly located in relation to the uterus .", "on t2wi , it displays intermediate signal intensity and shows a central high signal region .", "the lesion shows peripheral , intense contrast enhancement and a large central area of necrosis .", "h&e section of the ovary occupied by a tumour with insular pattern composed by neuroendocrine cells ( c ) .", "these cells have intense cytoplasmatic staining for synaptophysin ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown ) \n a  b metastatic , bilateral ovarian carcinoid , from a primary jejunal tumour in a 28-year - old female complaining of pelvic pain and dyspareunia . coronal ( a ) and axial ( b ) enhanced ct images ."], ["large cell ne carcinoma and carcinoid types are rarer than the former and few data can be found in the literature ."], ["staging and treatment strategies for both small and large cell cervical carcinoma globally follow the same criteria used for common histological types of cervical carcinomas .", "small cell carcinoma accounts for 16  % of cervical carcinomas [ 6 , 27 ] .", "median age of diagnosis is in the 5th decade , ranging from 21 to 87  years .", "some patients present with a cervical mass and an abnormal pap smear may be found .", "this tumour is strongly associated with human papilloma virus 18 ( hpv-18 ) , but hpv-16-positive tumours have also been demonstrated .", "the most commonly found are hypercalcaemia ( parathormone ) , hypoglycaemia ( insulin ) , carcinoid syndrome ( serotonin ) , cushing s syndrome ( corticotropin ) , siadh ( vasopressin ) and myasthenia gravis .", "most tumours are voluminous , exceeding 6  cm , and show deep cervical infiltration and necrosis", ". often , growth may be exophytic , which appears to be associated with a better prognosis . nevertheless , it is generally a highly aggressive tumour with a worse prognosis than that of poorly differentiated squamous cell carcinomas .", "the rates of lymphovascular space invasion and extra - pelvic recurrences for bone , supraclavicular lymph nodes and lung are particularly high [ 6 , 27 , 28 , 30 ] . according to mccusker et al . , females with small cell tumours have 1.84 times greater risk of death compared to patients with squamous cell carcinomas .", "it appears to have a similar outcome to small cell carcinoma and its treatment remains challenging because of the high rate of recurrence and distant metastases even with early stage disease [ 2 , 32 ] .", "we review three cases of cervical small cell carcinoma ( figs .  4 , 5 and 6 ) .", "cervical necs resemble cervical squamous cell carcinoma , displaying the same mri features : low signal intensity on t1-weighted images ( t1wi ) and high signal intensity on t2-weighted images ( t2wi ) .", "no histological distinction can be provided regarding only imaging features . however , according to the literature , cervical necs are generally highly aggressive and expected to be diagnosed in advanced figo stages .", "concordantly , we illustrate necs in iv - a ( invasion of adjacent organs ) ( fig .  4 ) and iii - b ( invasion of the pelvic side wall ) ( fig . ", "6 ) stages.fig . 4 \n a  b cervical neuroendocrine tumour ( figo iv - a ; invasion of adjacent organs ) .", "a 32-year - old female complaining of vaginal bleeding after intercourse and fetid vaginal discharge .", "sagittal t2-weighted mr image ( a ) and axial t2-weighted mr image ( b ) .", "the parametria are circumferentially infiltrated and the tumour extends anteriorly to the bladder , with transmural invasion and fistulae ( arrow ) .", ". 5 \n a  e cervical small cell carcinoma ( figo ii - a ; invasion of the upper two - thirds of vagina with no parametrial invasion ) .", "sagittal t2-weighted mr image ( a ) , axial t2-weighted mr images ( b ) and sagittal diffusion weighted image ( dwi ) , b(c ) retroverted uterus with an anterior cervical tumour , highly restrictive on dwi .", "no parametrial invasion is noted , but huge , bilateral obturator lymph nodes are found ( arrows ) .", "the left ovary shows follicular activity and is posteriorly displaced ( arrowhead ) . in this h&e section the cervix is occupied by a solid tumour , composed of small cells , with uniform nuclei and scarce cytoplasm ( d ) .", "some tumour cells show positive cytoplasmatic staining for synaptophysin ( e ) and for chromogranin ( not shown)fig . 6 \n a  b cervical small cell carcinoma ( figo iii - b ; extension to the pelvic side wall ) . a 64  year - old female complaining of menorrhagia .", "axial t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation after intravenous gadolinium administration ( b ) .", "cervical tumour extending posteriorly to the pelvic wall ( asterisk ) and superiorly to the left adnexal area , where it forms a huge solid , heterogeneous mass with central necrotic areas ( arrow ) .", "this mass shows intense enhancement after gadolinium administration and is adherent to the uterine corpus , which may be found inferiorly .", "bilateral internal iliac lymph nodes are also noted ( arrowheads ) \n a  b cervical neuroendocrine tumour ( figo iv - a ; invasion of adjacent organs ) .", "a 32-year - old female complaining of vaginal bleeding after intercourse and fetid vaginal discharge .", "sagittal t2-weighted mr image ( a ) and axial t2-weighted mr image ( b ) . large cervical cancer with infiltration of the lower segment of the uterine corpus .", "the parametria are circumferentially infiltrated and the tumour extends anteriorly to the bladder , with transmural invasion and fistulae ( arrow ) .", "bilateral , nodal metastases of the external iliac groups are also seen ( arrowheads ) \n a  e cervical small cell carcinoma ( figo ii - a ; invasion of the upper two - thirds of vagina with no parametrial invasion ) .", "sagittal t2-weighted mr image ( a ) , axial t2-weighted mr images ( b ) and sagittal diffusion weighted image ( dwi ) , b(c ) retroverted uterus with an anterior cervical tumour , highly restrictive on dwi .", "no parametrial invasion is noted , but huge , bilateral obturator lymph nodes are found ( arrows ) .", "the left ovary shows follicular activity and is posteriorly displaced ( arrowhead ) . in this h&e section", "the cervix is occupied by a solid tumour , composed of small cells , with uniform nuclei and scarce cytoplasm ( d ) .", "some tumour cells show positive cytoplasmatic staining for synaptophysin ( e ) and for chromogranin ( not shown ) \n a  b cervical small cell carcinoma ( figo iii - b ; extension to the pelvic side wall ) .", "axial t2-weighted mr image ( a ) and axial t1-weighted mr image with fat saturation after intravenous gadolinium administration ( b ) .", "cervical tumour extending posteriorly to the pelvic wall ( asterisk ) and superiorly to the left adnexal area , where it forms a huge solid , heterogeneous mass with central necrotic areas ( arrow ) .", "this mass shows intense enhancement after gadolinium administration and is adherent to the uterine corpus , which may be found inferiorly ."], ["well - differentiated neuroendocrine tumours ( nets ) include typical and atypical carcinoid tumours . among the uncommon sites of primary carcinoid tumours ,", "the uterine cervix constitutes one of the rarest locations , representing only 0.5  % to 5  % of cervical cancers .", "carcinoid syndrome is very rare and even in the lack of clinically evident carcinoid syndrome , many patients present with elevated urinary 5-hydroxyindoleacetic acid ( 5-hiaa ) .", "however , the diagnosis of a cervical carcinoid is frequently postoperative and laboratorial investigation is typically not helpful since it is not performed in the absence of symptoms .", "these tumours display a very aggressive , malignant behaviour , and their prognosis is poor ( 2- or 3-year survival rates , ranging from 12.5 to 33  % , according to some studies ) .", "atypical carcinoids of the cervix are also very rare and , according to a review of yoshida et al . , only 13 cases have been reported in the literature ."], ["the reported endometrial neoplasms with ne differentiation essentially include small cell carcinoma and endometrial adenocarcinoma with ne cells .", "small cell carcinoma of the endometrium resembles that of the lung and is uncommon , accounting for less than 1  % of all endometrial carcinomas .", "like in other endometrial carcinomas , abnormal bleeding is the primary presenting symptom , but some patients present with advanced disease , sometimes with pain from metastases .", "these tumours usually present as bulky intraluminal masses , and deep myometrial invasion ( 3350  % ) and extra - uterine spread ( near 50  % ) are common .", "pelvic lymph nodes , adnexa and the peritoneum constitute the typical sites of extra - uterine involvement .", "the overall prognosis is poor [ 5 , 35 ] . in 50 to 66  % ,", "endometrial small cell carcinoma displays an admixed component of grade 1 or 2 endometrioid adenocarcinoma , and few cases showed a malignant mixed mullerian tumour within a predominant small cell carcinoma .", "we review two cases of poorly differentiated endometrial carcinoma with ne differentiation ( figs .  7 and 8) . like in cervical carcinoma", ", imaging does not allow histological distinction , since all endometrial cancers tend to present similarly : low to moderate signal on t2wi and hypointensity relative to the hyperintense enhancing myometrium on dynamic contrast - enhanced mri sequences .", "however , when a highly aggressive endometrial tumour is found , a neuroendocrine nature may be suspected .", "there are no consistent data regarding the prognosis of non - pure ne endometrial tumours ; however , the cases we illustrate were diagnosed in advanced figo stages , iv - b ( distant metastases ) ( fig . ", "7 ) and iii - c1 ( involvement of pelvic nodes ) ( fig . ", "7 \n a  d poorly differentiated endometrial carcinoma with ne differentiation ( figo iv - b ; distant metastases ) .", "axial t2-weighted mr image ( a ) and axial enhanced ct image ( b ) .", "there is no invasion of the cervical stroma or bladder ( not shown ) , but a right obturator node ( arrow ) is found .", "ct evaluation shows that the disease has disseminated outside the pelvis , revealing para - aortic lymph nodes ( arrowhead ) as well as retroperitoneal and peritoneal metastases ( asterisks ) .", "vagina biopsy ( h&e ) infiltrated by a solid tumour composed by sheets of uniform cells , with small oval nuclei and scarse cytoplasm ( c ) .", "these cells have an intense cytoplasmatic staining for neuron - specific enolase ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown ) .", "8 \n a  d undifferentiated endometrial carcinoma with ne differentiation ( figo iii - c1 ; involvement of pelvic nodes ) .", "endometrial tumour prolapsing into the cervical canal , without invasion of the cervical stroma and myometrium .", "the tumour extends bilaterally through the lumen of the tubes to the parametrial tissues ( arrows ) .", "h&e section of the tumour with a solid pattern composed by large cells with pleomorphic nuclei , mitoses and scarce cytoplasm ( c ) .", "these cells have small chromogranine - positive granules ( d ) and small synaptophysin granules in the cytoplasm ( not shown ) and are strongly positive for pancytokeratin cam 5.2 ( not shown ) \n a  d poorly differentiated endometrial carcinoma with ne differentiation ( figo iv - b ; distant metastases ) .", "axial t2-weighted mr image ( a ) and axial enhanced ct image ( b ) .", "there is no invasion of the cervical stroma or bladder ( not shown ) , but a right obturator node ( arrow ) is found .", "ct evaluation shows that the disease has disseminated outside the pelvis , revealing para - aortic lymph nodes ( arrowhead ) as well as retroperitoneal and peritoneal metastases ( asterisks ) .", "vagina biopsy ( h&e ) infiltrated by a solid tumour composed by sheets of uniform cells , with small oval nuclei and scarse cytoplasm ( c ) .", "these cells have an intense cytoplasmatic staining for neuron - specific enolase ( d ) and have small chromogranine - positive granules in the cytoplasm ( not shown ) .", "this tumour was synaptophysin negative \n a  d undifferentiated endometrial carcinoma with ne differentiation ( figo iii - c1 ; involvement of pelvic nodes ) .", "endometrial tumour prolapsing into the cervical canal , without invasion of the cervical stroma and myometrium .", "the tumour extends bilaterally through the lumen of the tubes to the parametrial tissues ( arrows ) .", "h&e section of the tumour with a solid pattern composed by large cells with pleomorphic nuclei , mitoses and scarce cytoplasm ( c ) .", "these cells have small chromogranine - positive granules ( d ) and small synaptophysin granules in the cytoplasm ( not shown ) and are strongly positive for pancytokeratin cam 5.2 ( not shown )"], ["neuroendocrine tumours of the fallopian tube , vagina and vulva are very rare and only a few cases have been reported . in the fallopian tube ,", "these tumours encompass carcinoids and small cell carcinoma , and they are not well known . in the vagina ,", "almost ten cases of small cell carcinoma have been reported , occurring in females between 41 and 78  years of age .", "like in other vaginal tumours , the most frequent complaint was vaginal bleeding or discharge .", "metastatic disease was common in this small cohort , so the prognosis is thought to be poor . in the vulva , neuroendocrine differentiation may be found in small cell carcinoma of the bartholin gland and merkel cell tumours , which tend to present as vulvar masses ."], ["two groups are currently considered : necs , including small cell carcinoma and large cell ne carcinoma , and nets , which encompass typical and atypical carcinoids .", "the clinical features of theses tumours are non - specific and depend on the organ of origin and on the extension and aggressiveness of the disease .", "the imaging evaluation should follow the same criteria used for other tumours , and figo remains the preferred staging system .", "the diagnosis is obviously histological , and the role of imaging tools essentially includes initial detection , staging and biopsy guidance .", "however , some points should be taken into account when imaging studies are performed : cervical small cell carcinoma and ovarian carcinoid are the most common gynaecological ne tumours.cervical small cell carcinomas usually behave aggressively.ovarian carcinoids tend to behave in a benign fashion.while managing an ovarian carcinoid , extra - ovarian extension , bilaterality and multinodularity should raise the suspicion of metastatic disease.endometrial ne tumours are very rare and are usually small cell carcinoma or adenocarcinoma with ne differentiation.as for other tumours , us , ct , pet - ct and mri remain the most commonly used imaging tools , but octreotide scans may be useful for typical and atypical carcinoids .", "ovarian carcinoids tend to behave in a benign fashion . while managing an ovarian carcinoid , extra - ovarian extension , bilaterality and multinodularity should raise the suspicion of metastatic disease .", "endometrial ne tumours are very rare and are usually small cell carcinoma or adenocarcinoma with ne differentiation . as for other tumours , us , ct , pet - ct and mri remain the most commonly used imaging tools , but octreotide scans may be useful for typical and atypical carcinoids ."], [""]]}
{"article_id": "PMC3731973", "article_text": ["adenoid cystic carcinoma ( adcc ) is a malignant salivary gland tumor that was first described by billroth in 1859 under the name cylindroma attributing to its cribriform appearance formed by the tumor cells with cylindrical pseudolumina or pseudospaces .", "the term  adenoid cystic carcinoma  was introduced by ewing ( foote and frazell ) in 1954 .", "adcc is a relatively rare malignant salivary gland tumor comprising less than 1% of all malignancies of head and neck .", "it is the 5 most common malignancy of salivary gland origin , representing 5 - 10% of all salivary gland neoplasms .", "the parotid and submandibular glands are the two most common sites for adcc accounting for 55% of the cases . among the major glands ,", "adcc accounts for 8.3% of all palatal salivary gland tumors and 17.7% of malignant palatal salivary gland tumors ( afip series ) .", "the other less common sites are lower lip , retromolar tonsillar pillar area , sublingual gland , buccal mucosa and floor of the mouth .", "the nose and paranasal sinuses represent the next most common sites for minor gland adccs .", "the tumor is most often clinically deceptive by its small size and slow growth , which actually overlies its extensive subclinical invasion and marked ability for early metastasis making the prognosis questionable .", "the three recognized histological variants of adcc are cribriform , tubular and solid although , cribriform is the most commonest and solid is the least commonest .", "it is not uncommon to have more than one histopathologic pattern in a single neoplasm and rather all three patterns can be observed in the majority of tumors .", "the main reason for histological typing is to assess the prognostic difference between histologic types .", "tubular pattern ( well differentiated ) is believed to have the best prognosis compared to the cribriform pattern ( moderately differentiated ) and solid pattern ( poorly differentiated ) .", "a 60-year - old male patient reported to the department of oral and maxillofacial surgery with a chief complaint of pain and discharge in the upper left back region of the jaw since one and half years . according to the patient one year prior to our consultation , he got the maxillary molars extracted from a local dentist due to pain and mobility of the teeth in that area . upon consultation patient revealed that the pain continued to exist even after the extraction .", "the patient is a known hypertensive and is on medication since three years for the same .", "the patient had smoking habit , which he quit only a couple of years ago .", "intraoral examination revealed a solitary erythematous swelling with diffuse borders involving the left posterior portion of hard palate and was also seen involving the soft palate on the same side .", "the swelling was seen extending from mesial aspect of first premolar to 1 cm posterior to tuberosity on left side , involving the palatal area but not crossing the midline [ figure 1 ] .", "intraorally , the lesion was seen involving the hard and soft palate but not crossing the midline the swelling was nontender .", "the clinical differential diagnosis included a benign or malignant neoplasm of minor salivary glands , a neoplasm of maxillary sinus .", "ct scan showing complete obliteration of left maxillary sinus an incisional biopsy of palate and sinus lining was performed .", "islands of uniform cells arranged in cord - like pattern with hyperchromatic nuclei were seen enclosing round to oval pseudocystic spaces in the stroma .", "the tumor cells are also seen arranged in the form of solid islands [ figure 4 ] and ductal pattern [ figure 5 ] .", "focal areas showed small cords and longitudinal tubules of isomorphic cells set in a background of densely hyalinized stroma [ figure 6 ] .", "variable - sized cribriform spaces formed by small deeply basophilic cells imparting swiss cheese pattern solid pattern composed of nests of basaloid cells and mitotic figures tubular pattern characterized by ductal structures formed by layers of isomorphic basaloid cells tubules of isomorphic tumor cells set in a densely hyalinized stroma a diagnosis of adcc ( cribriform pattern ) was established .", "the patient was treated by wide surgical excision with clear margins and hemi - maxillectomy of left maxillary region with post - radiotherapy .", "the present case was staged as t4n0m0 based on american joint committee on cancer as a guide to prognosis .", "the patient was under regular follow - up and is free of the disease at one - year follow - up .", "adenoid cystic carcinoma most often presents a diagnostic and treatment challenge owing to the rarity of the lesion .", "most findings regarding adcc are actually based upon studies with a small number of patients and there is a need for further information regarding its clinical behaviour as well as treatment modalities and their results .", "adcc may occur at any age although in most cases the patients age ranged from 24 to 78 years .", "the age of patients affected with major salivary gland tumors has been shown to be younger ( mean 44 years ) compared to the age of those who developed tumors of the minor glands ( mean 54 years ) and shows female predilection ( f : m  1.2:1 ) .", "pain is a common and important finding , occurring early in the course of the disease before there is a noticeable swelling .", "palate is more commonly involved generally the area of greater palatine foramen . among the malignant neoplasms of minor salivary glands , the most common was mucoepidermoid carcinoma ( 21.8% ) followed by polymorphous low grade adenocarcinoma ( plga ) ( 7.1% ) and", "adccs of the minor glands have been reported to have worse prognosis than those of the major salivary glands .", "tumors involving the nose , paranasal sinuses and maxillary sinus have the worst prognosis as they are usually detected with higher stages at the time of diagnosis . tumors of minor salivary glands usually have the tendency to infiltrate extra glandular soft tissues and bone thereby allowing increased dissemination of the tumor .", "lymph node involvement is uncommon ( < 5% of cases ) and is usually due to contiguous spread rather than lymphatic permeation or embolization .", "the differential diagnosis of adcc includes plga , basal cell adenoma ( bca ) , mixed tumor and basaloid squamous cell carcinoma ( bsc ) .", "the cribriform pattern so typical of adcc may also rarely be seen in bca , mixed tumor and plga , and the distinction between plga and adcc is more challenging .", "a polymorphous architecture characterizes plga whereas adcc has a more limited range of histologic patterns with no more than three patterns .", "plga shows uniform cell population with cytologically bland , round or oval vesicular nuclei and pale eosinophilic cytoplasm where as cells in adcc have clear cytoplasm , angular , hyperchromatic nuclei and may show mitotic activity .", "the ki-67 index is reported to be 10 times higher in adcc compared to plga .", "smooth muscle markers of myoepithelial differentiation are positive in adcc but negative in plga . though plga may form solid areas , they lack the overall high - grade feel associated with adcc .", "occasional foci in pleomorphic adenoma ( pa ) can resemble adcc but the presence of typical myxochondroid matrix and plasmacytoid or spindle shaped cells helps to avoid confusion .", "cribriform structures may sometimes be observed in bca and such cases can be differentiated from adcc on the basis of gradual structural alteration from areas typical of bca .", "the peripheral palisading and focal squamous differentiation with whorling pattern present in bca are not usually encountered in adcc .", "the basement membrane material secreted by bsc tends to dissect between tumor cells rather than form crisp cribriform spaces seen in adcc .", "focal keratinization , attachment to rete pegs , and presence of surface squamous dysplasia or carcinoma in situ helps to distinguish it from adcc .", "immunohistochemical studies demonstrated that the pseudocysts are positive for periodic acid schiff reagent ( pas ) and alcian blue and contain basement membrane components such as type iv collagen , heparin sulfate and laminin isoforms .", "epithelial cells are positive for carcinoembryonic antigen ( cea ) and epithelial membrane antigen ( ema ) .", "duct lining cells are positive for c - kit ( cd117 ) and myoepithelial cells are positive for s-100 protein , calponin , p63 , smooth muscle actin and myosin .", "expression of s-100 , glial fibrillary acidic protein ( gfap ) and neural cell adhesion molecule have been correlated with the presence of perineural invasion .", "molecular genetic data : alterations in chromosomes 6q , 9p and 17p12 - 13 are the most frequent cytogenetic alterations reported .", "hypermethylation of the promoter region of the p16 gene was associated with higher histologic grades of malignancy .", "possible treatment modalities available for the treatment of adcc are surgery , radiotherapy , chemotherapy and combined therapy .", "it requires excision with the widest surgical margins possible as tumor cells extend well beyond the clinical and radiographic margins .", "neutron therapy , which involves larger particles of greater energy can achieve reasonable local control as a primary therapeutic modality .", "main factors associated with patient survival are anatomic location , primary lesion size , presence or absence of metastasis at the time of diagnosis , perineural invasion and histological variant .", "distant metastasis occurs in 25%-50% of patients and lung is the most common involved site .", "the 5-year survival rate after effective treatment is 75% , but long - term survival rates are low ( 10 years-20% and 15 years-10% ) .", "the present patient was recalled at every 6-month interval in view of the sinus involvement and propensity for distant metastasis .", "lesions involving the sinus tend to have a poor prognosis due to its infiltrative growth and distant metastasis ."], "abstract_text": ["<S> adenoid cystic carcinoma is an uncommon , slow growing malignant salivary gland tumor that is characterized by wide local infiltration , perineural spread , propensity to local recurrence and distant metastasis . in this paper , the authors present a case of adenoid cystic carcinoma affecting the palate and involving the maxillary sinus in a 60-year - old male patient along with a brief review of literature . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["adenoid cystic carcinoma ( adcc ) is a malignant salivary gland tumor that was first described by billroth in 1859 under the name cylindroma attributing to its cribriform appearance formed by the tumor cells with cylindrical pseudolumina or pseudospaces .", "the term  adenoid cystic carcinoma  was introduced by ewing ( foote and frazell ) in 1954 .", "adcc is a relatively rare malignant salivary gland tumor comprising less than 1% of all malignancies of head and neck .", "it is the 5 most common malignancy of salivary gland origin , representing 5 - 10% of all salivary gland neoplasms .", "the parotid and submandibular glands are the two most common sites for adcc accounting for 55% of the cases . among the major glands ,", "adcc accounts for 8.3% of all palatal salivary gland tumors and 17.7% of malignant palatal salivary gland tumors ( afip series ) .", "the other less common sites are lower lip , retromolar tonsillar pillar area , sublingual gland , buccal mucosa and floor of the mouth .", "the nose and paranasal sinuses represent the next most common sites for minor gland adccs .", "the tumor is most often clinically deceptive by its small size and slow growth , which actually overlies its extensive subclinical invasion and marked ability for early metastasis making the prognosis questionable .", "the three recognized histological variants of adcc are cribriform , tubular and solid although , cribriform is the most commonest and solid is the least commonest .", "it is not uncommon to have more than one histopathologic pattern in a single neoplasm and rather all three patterns can be observed in the majority of tumors .", "the main reason for histological typing is to assess the prognostic difference between histologic types .", "tubular pattern ( well differentiated ) is believed to have the best prognosis compared to the cribriform pattern ( moderately differentiated ) and solid pattern ( poorly differentiated ) ."], ["a 60-year - old male patient reported to the department of oral and maxillofacial surgery with a chief complaint of pain and discharge in the upper left back region of the jaw since one and half years . according to the patient one year prior to our consultation , he got the maxillary molars extracted from a local dentist due to pain and mobility of the teeth in that area . upon consultation patient revealed that the pain continued to exist even after the extraction .", "the patient is a known hypertensive and is on medication since three years for the same .", "the patient had smoking habit , which he quit only a couple of years ago .", "intraoral examination revealed a solitary erythematous swelling with diffuse borders involving the left posterior portion of hard palate and was also seen involving the soft palate on the same side .", "the swelling was seen extending from mesial aspect of first premolar to 1 cm posterior to tuberosity on left side , involving the palatal area but not crossing the midline [ figure 1 ] .", "intraorally , the lesion was seen involving the hard and soft palate but not crossing the midline the swelling was nontender .", "the clinical differential diagnosis included a benign or malignant neoplasm of minor salivary glands , a neoplasm of maxillary sinus .", "ct scan showing complete obliteration of left maxillary sinus an incisional biopsy of palate and sinus lining was performed .", "islands of uniform cells arranged in cord - like pattern with hyperchromatic nuclei were seen enclosing round to oval pseudocystic spaces in the stroma .", "the tumor cells are also seen arranged in the form of solid islands [ figure 4 ] and ductal pattern [ figure 5 ] .", "focal areas showed small cords and longitudinal tubules of isomorphic cells set in a background of densely hyalinized stroma [ figure 6 ] .", "variable - sized cribriform spaces formed by small deeply basophilic cells imparting swiss cheese pattern solid pattern composed of nests of basaloid cells and mitotic figures tubular pattern characterized by ductal structures formed by layers of isomorphic basaloid cells tubules of isomorphic tumor cells set in a densely hyalinized stroma a diagnosis of adcc ( cribriform pattern ) was established .", "the patient was treated by wide surgical excision with clear margins and hemi - maxillectomy of left maxillary region with post - radiotherapy .", "the present case was staged as t4n0m0 based on american joint committee on cancer as a guide to prognosis .", "the patient was under regular follow - up and is free of the disease at one - year follow - up ."], ["adenoid cystic carcinoma most often presents a diagnostic and treatment challenge owing to the rarity of the lesion .", "most findings regarding adcc are actually based upon studies with a small number of patients and there is a need for further information regarding its clinical behaviour as well as treatment modalities and their results .", "adcc may occur at any age although in most cases the patients age ranged from 24 to 78 years .", "the age of patients affected with major salivary gland tumors has been shown to be younger ( mean 44 years ) compared to the age of those who developed tumors of the minor glands ( mean 54 years ) and shows female predilection ( f : m  1.2:1 ) .", "pain is a common and important finding , occurring early in the course of the disease before there is a noticeable swelling .", "palate is more commonly involved generally the area of greater palatine foramen . among the malignant neoplasms of minor salivary glands , the most common was mucoepidermoid carcinoma ( 21.8% ) followed by polymorphous low grade adenocarcinoma ( plga ) ( 7.1% ) and", "adccs of the minor glands have been reported to have worse prognosis than those of the major salivary glands .", "tumors involving the nose , paranasal sinuses and maxillary sinus have the worst prognosis as they are usually detected with higher stages at the time of diagnosis . tumors of minor salivary glands usually have the tendency to infiltrate extra glandular soft tissues and bone thereby allowing increased dissemination of the tumor .", "lymph node involvement is uncommon ( < 5% of cases ) and is usually due to contiguous spread rather than lymphatic permeation or embolization .", "the differential diagnosis of adcc includes plga , basal cell adenoma ( bca ) , mixed tumor and basaloid squamous cell carcinoma ( bsc ) .", "the cribriform pattern so typical of adcc may also rarely be seen in bca , mixed tumor and plga , and the distinction between plga and adcc is more challenging .", "a polymorphous architecture characterizes plga whereas adcc has a more limited range of histologic patterns with no more than three patterns .", "plga shows uniform cell population with cytologically bland , round or oval vesicular nuclei and pale eosinophilic cytoplasm where as cells in adcc have clear cytoplasm , angular , hyperchromatic nuclei and may show mitotic activity .", "the ki-67 index is reported to be 10 times higher in adcc compared to plga .", "smooth muscle markers of myoepithelial differentiation are positive in adcc but negative in plga . though plga may form solid areas , they lack the overall high - grade feel associated with adcc .", "occasional foci in pleomorphic adenoma ( pa ) can resemble adcc but the presence of typical myxochondroid matrix and plasmacytoid or spindle shaped cells helps to avoid confusion .", "cribriform structures may sometimes be observed in bca and such cases can be differentiated from adcc on the basis of gradual structural alteration from areas typical of bca .", "the peripheral palisading and focal squamous differentiation with whorling pattern present in bca are not usually encountered in adcc .", "the basement membrane material secreted by bsc tends to dissect between tumor cells rather than form crisp cribriform spaces seen in adcc .", "focal keratinization , attachment to rete pegs , and presence of surface squamous dysplasia or carcinoma in situ helps to distinguish it from adcc .", "immunohistochemical studies demonstrated that the pseudocysts are positive for periodic acid schiff reagent ( pas ) and alcian blue and contain basement membrane components such as type iv collagen , heparin sulfate and laminin isoforms .", "epithelial cells are positive for carcinoembryonic antigen ( cea ) and epithelial membrane antigen ( ema ) .", "duct lining cells are positive for c - kit ( cd117 ) and myoepithelial cells are positive for s-100 protein , calponin , p63 , smooth muscle actin and myosin .", "expression of s-100 , glial fibrillary acidic protein ( gfap ) and neural cell adhesion molecule have been correlated with the presence of perineural invasion .", "molecular genetic data : alterations in chromosomes 6q , 9p and 17p12 - 13 are the most frequent cytogenetic alterations reported .", "hypermethylation of the promoter region of the p16 gene was associated with higher histologic grades of malignancy .", "possible treatment modalities available for the treatment of adcc are surgery , radiotherapy , chemotherapy and combined therapy .", "it requires excision with the widest surgical margins possible as tumor cells extend well beyond the clinical and radiographic margins .", "neutron therapy , which involves larger particles of greater energy can achieve reasonable local control as a primary therapeutic modality .", "main factors associated with patient survival are anatomic location , primary lesion size , presence or absence of metastasis at the time of diagnosis , perineural invasion and histological variant .", "distant metastasis occurs in 25%-50% of patients and lung is the most common involved site .", "the 5-year survival rate after effective treatment is 75% , but long - term survival rates are low ( 10 years-20% and 15 years-10% ) .", "the present patient was recalled at every 6-month interval in view of the sinus involvement and propensity for distant metastasis ."], ["lesions involving the sinus tend to have a poor prognosis due to its infiltrative growth and distant metastasis ."]]}
{"article_id": "PMC4306322", "article_text": ["the quantity of trauma victims and the management involved in attending them leads to a great workload in most hospitals and places an enormous amount of pressure on the health care system .", "furthermore , traumatic injuries have been reported as the leading cause of death in the first 40 years of life and appear to be on the increase . during the last decade , interpersonal violence ( ipv ) was one of the main etiological factors of fractures of the maxillofacial complex .", "road traffic accidents remain the most important cause of maxillofacial trauma worldwide , although falls and assaults have become more significant in north america , brazil and europe . blunt and cutting wounds on the head and neck are also common .", "legal underreporting is common due to associations with alcohol abuse , illicit drug use , firearms and acts of violence against women , children , and the elderly , which generate fear , shame , low self - esteem and a sense of powerlessness .", "facial fractures associated to ipv have functional , aesthetic and psychological effects on the victims , as well as a socioeconomic impact on the health systems of many countries .these victims are more likely to suffer from depression , anxiety and post - traumatic stress disorder .", "epidemiological studies help to identify demands , develop prevention programs and structure an integrated care system .", "the aim of this retrospective study was to compare the peculiarities of maxillofacial injuries caused by interpersonal violence with other etiologic factors .", "this retrospective study involved data collected from clinical notes and surgical records of patients treated at seven different hospitals located in three different cities in so paulo state , brazil .", "the sample was selected based on analysis of medical records of facial trauma patients attended by the department of oral and maxillofacial surgery in the piracicaba dental school at the university of campinas from january 1999 to december 2012 .", "a standardized form was designed to investigate the epidemiologic features of maxillofacial traumas , considering aspects such as age , gender , diagnosis , soft tissue lesions , facial injuries and general injuries .", "the exclusion criterion was incomplete information about the type of trauma on the medical chart .", "the origin of the facial trauma was classified as follows : traffic accidents ( automobile and motorcycle ) ; work accidents ; sport accidents ; falls ; interpersonal violence ; cycling accidents ; pedestrian accidents and others .", "facial injury sites were classified as follows : upper ( frontal bone ) ; middle ( maxilla , nasal bones , zygomatic - orbital complex , naso - orbital ethmoidal ) and lower ( mandible ) .", "mandibular fractures were described according to the anatomic site : condylar fractures ; angle fractures ; body fractures and fractures of symphysis .", "the present study was approved by the research ethics committee of piracicaba dental school , state university of campinas ( fop / unicamp ) under protocol number 75/2013 .", "data were submitted to statistical analysis ( simple descriptive statistics and chi - squared test ) with the support of the statistical package for social sciences ( spss 18.0 for windows , spss inc , chicago , il ) . binary logistic regression and the chi - square test were performed and the results were considered relevant when p < 0.05 .", "these included absolute frequencies ( raw counts ) and relative frequencies ( proportions or percentages of the total number of observations ) .", "based on the 13-year study period , 3,724 patient referrals to the maxillofacial surgery department were enrolled in the present study .", "the median age at the time of admission was 29.2 years , with a standard deviation of 17.06 years .", "a total of 612 patients ( 16.4% ) exhibited facial injuries related to ipv . in this group ,", "male victims prevailed with 81% ( n = 496 ) and the mean age was 31.28 years , with a standard deviation of 13.33 years .", "weekly consumption of alcohol was reported by 32.4% and tobacco use was 43.8% . in total ,", "79 patients ( 12.8% ) reported using non - injecting illicit drugs and 14 ( 2.2% ) stated that they currently used injecting drugs . in 176 cases ,", "isolated alveolar - dental injuries were observed in 35 patients . with regards to maxillofacial fractures ,", "table 2 shows the distribution of diagnoses in the general sample and among patients who were victims of physical assault . \n", "distribution of patients with facial injuries according to etiological factors \n \n n = number of patients . \n \n", "distribution of facial injury diagnoses among a general sample and patients whose facial injury was associated with interpersonal violence ( ipv ) \n \n n = number of patients . \n  in the cases of mandibular fractures ,", "the anatomic regions most affected were the body and the mandibular angle , with 36 cases in each region , followed by the condyle ( 25 cases ) , symphysis ( 9 cases ) and isolated mandibular ramus ( 2 cases ) . in 41 patients , fractures involving more than one anatomic region of the mandible", "lacerations and abrasions on the face were identified in 181 and 105 patients , respectively .", "table 3 displays the distribution of body injuries in these patients , when compared to the general sample . \n", "distribution of body injuries among patients with facial trauma and patients whose facial injury was associated with interpersonal violence ( ipv ) \n \n the calculation is based on whole sample ( n = 3724 ) . \n \n", "the calculation is based on patients that involved with ipv ( n = 612 ) . \n \n", "comparison of features of facial injury of victims of interpersonal violence ( ipv ) and other etiologic factors \n \n n = number of patients ;  = chi - square", ". \n  a greater number of fractures and lacerations of the face was observed , although with fewer facial abrasions . with regards to the area of bone fractures ,", "fewer injuries occurred in other regions of the body , mainly due to fewer injuries of the upper and lower limbs .", "however , patients that were associated with ipv exhibited a greater number of injuries to the neurocranium .", "binary logistic regression was performed to determine whether fractures in facial injuries were affected by gender , age ( median 30 years ) , drug use , the presence of lacerations or the presence of abrasions .", "fractures were associated with gender : male patients are more at risk than females ( or = 1.61 , p < 0.002 , 95% ci : 1.05 to 2.46 ) .", "although motor vehicle accidents are still the main cause of maxillofacial fractures in some developing countries , recent data from developed countries have shown that ipv is the dominant cause .", "ipv is one of the leading causes of maxillofacial fractures and its prevalence in a population varies according to geographic region and appears to be associated with factors such as socioeconomic status , population density and age characteristics .", "the prevalence of male victims has been reported in most studies , ranging from 61% to 92% , as well as a mean age of approximately 30 years [ 10 - 14 ] .", "however , children are also affected and several studies have reported the patterns and severity of these injuries . this age group is susceptible to craniofacial trauma due to their greater cranial mass - to - body weight ratio . compared to adults , less", "65 patients were under 18 years of age , although most of them ( 36 patients ) were aged between 16 and 18 years .", "these patients mainly exhibited nasal bone ( 21 patients ) and mandibular fractures ( 12 patients ) , results which are in accordance with the literature .", "the prevalence of male victims is influenced by cultural models of gender , defined as the set of attributes , values , roles and behavior patterns that are expected from a man in a particular culture .", "generally , the traits valued in this socialization are aggression , domination and exploitation of danger , which could contribute to their association with ipv .", "ipv among females is mainly associated with domestic violence . in the present study , it was not possible to determine the pattern of violence suffered .", "when the victim is female , the case should be investigated regardless of the site of the injury , as there is a greater possibility that they are the victim of physical aggression due to the vulnerability of the gender .", "another relevant factor is the differences in female participation in social activities , making them more ( or less ) prone to urban violence .", "patients who were victims of physical assault , unlike other etiologies such as sports accidents , are often affected by problems such as drug use and poverty . in the present study", ", we found high consumption rates of alcohol , tobacco and non - injecting drugs .", "its consumption results in an increase in confidence , followed by the subjective feeling of increased mental and physical capacity , as well as impaired judgment and coordination .", ".   found that alcohol was involved in 87% of maxillofacial fractures caused by ipv , compared with 58% for motor vehicle accidents .", "studies have reported that fractures of the mandible are the most prevalent , followed by zygomatic complex fractures [ 17 - 19 ] .", "furthermore , we showed that the prevalence of mandibular and nasal bone fractures was higher in this group of patients .", "the literature indicates that trauma associated with ipv are less severe than those associated with other etiological factors due to the magnitude and speed of impact . despite this fact , patients in which ipv was the etiologic factor exhibited a higher prevalence of bone fractures when compared to all other patients treated .", "possibly , some patients with minor injuries do not seek medical care due to shame or fear .", "a high frequency of soft tissue lesions were found on the face , particularly lacerations . in the literature ,", "the most common orofacial injuries are lip injuries , followed by other soft tissue injuries on the face , gingival and oral mucosa injuries , and teeth and/or periodontal injuries .", "the pattern of facial fractures is related to the type , direction and force of impact , as well as the anatomical features of the area .", "ipv injuries are usually caused by punches or kicks , for which the face is the main target region .", "the face is probably the target for most acts of physical aggression as it is easily reached , due to its location at the same height as the aggressor s raised arm .", "it has also been suggested that the aggressor either consciously or unconsciously wishes to affect the victim s self - esteem .", "males appear to be injured by more dangerous mechanisms and are more frequently injured by objects than females .", "the mandible , which is mobile and prominent on the face , is more susceptible to trauma . in the present study ,", "the region of the mandibular angle has been considered the least resistant , mainly due to the presence of the mandibular third molar .", "these accidents often cause pan - facial , cranial , orthopedic , abdominal and cervical spine injuries , which may require multidisciplinary input , with significant interventions such as intubation , craniotomy and tracheostomy .", "conversely , ipv tends to be associated with isolated fractures , particularly in the mandibular region .", "the present study confirmed that most patients were affected by a fracture in only one region of the face . a lower prevalence of injuries was recorded for other parts of body .", "although the world health organization considers the response of health - care services to victims of violence to be an international priority , health professionals are generally not qualified to identify the injuries or council victims .", "the role of health professionals is to identify the etiology of the injury and , in cases of physical aggression , provide the victim with information on where to seek help .", "moreover , health professionals are not well prepared to ask sensitive questions and are unaware of victim - support institutions .", "the findings of the present study demonstrated that facial trauma caused by interpersonal violence seem to be associated with a higher rate of facial fractures and lacerations when compared to all other patients with facial injuries .", "male patients are more likely to exhibit severe injuries and exhibited a greater amount of bone fractures . prominent areas of the face and neurocranium should be carefully examined as they tend to be more prone to injuries .", "glaucia maria bovi ambrosano , associate professor , department of community dentistry , campinas state university piracicaba dental school , for statistical analysis assistance ."], "abstract_text": ["<S> abstractobjectivesthe aim of this retrospective study was to compare the peculiarities of maxillofacial injuries caused by interpersonal violence with other etiologic factors.material and methodsmedical records of 3,724 patients with maxillofacial injuries in so paulo state ( brazil ) were retrospectively analyzed . </S>", "<S> the data were submitted to statistical analysis ( simple descriptive statistics and chi - squared test ) using spss 18.0 software.resultsdata of 612 patients with facial injuries caused by violence were analyzed . </S>", "<S> the majority of the patients were male ( 81% ; n = 496 ) , with a mean age of 31.28 years ( standard deviation of 13.33 years ) . </S>", "<S> these patients were more affected by mandibular and nose fractures , when compared with all other patients ( p < 0.01 ) , although fewer injuries were recorded in other body parts ( 2 = 17.54 ; p < 0.01 ) ; victims of interpersonal violence exhibited more injuries when the neurocranium was analyzed in isolation ( 2 = 6.85 ; p < 0.01).conclusionsfacial trauma due to interpersonal violence seem to be related to a higher rate of facial fractures and lacerations when compared to all patients with facial injuries . </S>", "<S> prominent areas of the face and neurocranium were more affected by injuries . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS", "ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS"], "sections": [["the quantity of trauma victims and the management involved in attending them leads to a great workload in most hospitals and places an enormous amount of pressure on the health care system .", "furthermore , traumatic injuries have been reported as the leading cause of death in the first 40 years of life and appear to be on the increase . during the last decade , interpersonal violence ( ipv ) was one of the main etiological factors of fractures of the maxillofacial complex .", "road traffic accidents remain the most important cause of maxillofacial trauma worldwide , although falls and assaults have become more significant in north america , brazil and europe . blunt and cutting wounds on the head and neck are also common .", "legal underreporting is common due to associations with alcohol abuse , illicit drug use , firearms and acts of violence against women , children , and the elderly , which generate fear , shame , low self - esteem and a sense of powerlessness .", "facial fractures associated to ipv have functional , aesthetic and psychological effects on the victims , as well as a socioeconomic impact on the health systems of many countries .these victims are more likely to suffer from depression , anxiety and post - traumatic stress disorder .", "epidemiological studies help to identify demands , develop prevention programs and structure an integrated care system .", "the aim of this retrospective study was to compare the peculiarities of maxillofacial injuries caused by interpersonal violence with other etiologic factors ."], ["this retrospective study involved data collected from clinical notes and surgical records of patients treated at seven different hospitals located in three different cities in so paulo state , brazil .", "the sample was selected based on analysis of medical records of facial trauma patients attended by the department of oral and maxillofacial surgery in the piracicaba dental school at the university of campinas from january 1999 to december 2012 .", "a standardized form was designed to investigate the epidemiologic features of maxillofacial traumas , considering aspects such as age , gender , diagnosis , soft tissue lesions , facial injuries and general injuries .", "the exclusion criterion was incomplete information about the type of trauma on the medical chart .", "the origin of the facial trauma was classified as follows : traffic accidents ( automobile and motorcycle ) ; work accidents ; sport accidents ; falls ; interpersonal violence ; cycling accidents ; pedestrian accidents and others .", "facial injury sites were classified as follows : upper ( frontal bone ) ; middle ( maxilla , nasal bones , zygomatic - orbital complex , naso - orbital ethmoidal ) and lower ( mandible ) .", "mandibular fractures were described according to the anatomic site : condylar fractures ; angle fractures ; body fractures and fractures of symphysis .", "the present study was approved by the research ethics committee of piracicaba dental school , state university of campinas ( fop / unicamp ) under protocol number 75/2013 .", "data were submitted to statistical analysis ( simple descriptive statistics and chi - squared test ) with the support of the statistical package for social sciences ( spss 18.0 for windows , spss inc , chicago , il ) . binary logistic regression and the chi - square test were performed and the results were considered relevant when p < 0.05 .", "these included absolute frequencies ( raw counts ) and relative frequencies ( proportions or percentages of the total number of observations ) ."], ["based on the 13-year study period , 3,724 patient referrals to the maxillofacial surgery department were enrolled in the present study .", "the median age at the time of admission was 29.2 years , with a standard deviation of 17.06 years .", "a total of 612 patients ( 16.4% ) exhibited facial injuries related to ipv . in this group ,", "male victims prevailed with 81% ( n = 496 ) and the mean age was 31.28 years , with a standard deviation of 13.33 years .", "weekly consumption of alcohol was reported by 32.4% and tobacco use was 43.8% . in total ,", "79 patients ( 12.8% ) reported using non - injecting illicit drugs and 14 ( 2.2% ) stated that they currently used injecting drugs . in 176 cases ,", "isolated alveolar - dental injuries were observed in 35 patients . with regards to maxillofacial fractures ,", "table 2 shows the distribution of diagnoses in the general sample and among patients who were victims of physical assault . \n", "distribution of patients with facial injuries according to etiological factors \n \n n = number of patients . \n \n", "distribution of facial injury diagnoses among a general sample and patients whose facial injury was associated with interpersonal violence ( ipv ) \n \n n = number of patients . \n  in the cases of mandibular fractures ,", "the anatomic regions most affected were the body and the mandibular angle , with 36 cases in each region , followed by the condyle ( 25 cases ) , symphysis ( 9 cases ) and isolated mandibular ramus ( 2 cases ) . in 41 patients , fractures involving more than one anatomic region of the mandible", "lacerations and abrasions on the face were identified in 181 and 105 patients , respectively .", "table 3 displays the distribution of body injuries in these patients , when compared to the general sample . \n", "distribution of body injuries among patients with facial trauma and patients whose facial injury was associated with interpersonal violence ( ipv ) \n \n the calculation is based on whole sample ( n = 3724 ) . \n \n", "the calculation is based on patients that involved with ipv ( n = 612 ) . \n \n", "comparison of features of facial injury of victims of interpersonal violence ( ipv ) and other etiologic factors \n \n n = number of patients ;  = chi - square", ". \n  a greater number of fractures and lacerations of the face was observed , although with fewer facial abrasions . with regards to the area of bone fractures ,", "fewer injuries occurred in other regions of the body , mainly due to fewer injuries of the upper and lower limbs .", "however , patients that were associated with ipv exhibited a greater number of injuries to the neurocranium .", "binary logistic regression was performed to determine whether fractures in facial injuries were affected by gender , age ( median 30 years ) , drug use , the presence of lacerations or the presence of abrasions .", "fractures were associated with gender : male patients are more at risk than females ( or = 1.61 , p < 0.002 , 95% ci : 1.05 to 2.46 ) ."], ["although motor vehicle accidents are still the main cause of maxillofacial fractures in some developing countries , recent data from developed countries have shown that ipv is the dominant cause .", "ipv is one of the leading causes of maxillofacial fractures and its prevalence in a population varies according to geographic region and appears to be associated with factors such as socioeconomic status , population density and age characteristics .", "the prevalence of male victims has been reported in most studies , ranging from 61% to 92% , as well as a mean age of approximately 30 years [ 10 - 14 ] .", "however , children are also affected and several studies have reported the patterns and severity of these injuries . this age group is susceptible to craniofacial trauma due to their greater cranial mass - to - body weight ratio . compared to adults , less", "65 patients were under 18 years of age , although most of them ( 36 patients ) were aged between 16 and 18 years .", "these patients mainly exhibited nasal bone ( 21 patients ) and mandibular fractures ( 12 patients ) , results which are in accordance with the literature .", "the prevalence of male victims is influenced by cultural models of gender , defined as the set of attributes , values , roles and behavior patterns that are expected from a man in a particular culture .", "generally , the traits valued in this socialization are aggression , domination and exploitation of danger , which could contribute to their association with ipv .", "ipv among females is mainly associated with domestic violence . in the present study , it was not possible to determine the pattern of violence suffered .", "when the victim is female , the case should be investigated regardless of the site of the injury , as there is a greater possibility that they are the victim of physical aggression due to the vulnerability of the gender .", "another relevant factor is the differences in female participation in social activities , making them more ( or less ) prone to urban violence .", "patients who were victims of physical assault , unlike other etiologies such as sports accidents , are often affected by problems such as drug use and poverty . in the present study", ", we found high consumption rates of alcohol , tobacco and non - injecting drugs .", "its consumption results in an increase in confidence , followed by the subjective feeling of increased mental and physical capacity , as well as impaired judgment and coordination .", ".   found that alcohol was involved in 87% of maxillofacial fractures caused by ipv , compared with 58% for motor vehicle accidents .", "studies have reported that fractures of the mandible are the most prevalent , followed by zygomatic complex fractures [ 17 - 19 ] .", "furthermore , we showed that the prevalence of mandibular and nasal bone fractures was higher in this group of patients .", "the literature indicates that trauma associated with ipv are less severe than those associated with other etiological factors due to the magnitude and speed of impact . despite this fact , patients in which ipv was the etiologic factor exhibited a higher prevalence of bone fractures when compared to all other patients treated .", "possibly , some patients with minor injuries do not seek medical care due to shame or fear .", "a high frequency of soft tissue lesions were found on the face , particularly lacerations . in the literature ,", "the most common orofacial injuries are lip injuries , followed by other soft tissue injuries on the face , gingival and oral mucosa injuries , and teeth and/or periodontal injuries .", "the pattern of facial fractures is related to the type , direction and force of impact , as well as the anatomical features of the area .", "ipv injuries are usually caused by punches or kicks , for which the face is the main target region .", "the face is probably the target for most acts of physical aggression as it is easily reached , due to its location at the same height as the aggressor s raised arm .", "it has also been suggested that the aggressor either consciously or unconsciously wishes to affect the victim s self - esteem .", "males appear to be injured by more dangerous mechanisms and are more frequently injured by objects than females .", "the mandible , which is mobile and prominent on the face , is more susceptible to trauma . in the present study ,", "the region of the mandibular angle has been considered the least resistant , mainly due to the presence of the mandibular third molar .", "these accidents often cause pan - facial , cranial , orthopedic , abdominal and cervical spine injuries , which may require multidisciplinary input , with significant interventions such as intubation , craniotomy and tracheostomy .", "conversely , ipv tends to be associated with isolated fractures , particularly in the mandibular region .", "the present study confirmed that most patients were affected by a fracture in only one region of the face . a lower prevalence of injuries was recorded for other parts of body .", "although the world health organization considers the response of health - care services to victims of violence to be an international priority , health professionals are generally not qualified to identify the injuries or council victims .", "the role of health professionals is to identify the etiology of the injury and , in cases of physical aggression , provide the victim with information on where to seek help .", "moreover , health professionals are not well prepared to ask sensitive questions and are unaware of victim - support institutions ."], ["the findings of the present study demonstrated that facial trauma caused by interpersonal violence seem to be associated with a higher rate of facial fractures and lacerations when compared to all other patients with facial injuries .", "male patients are more likely to exhibit severe injuries and exhibited a greater amount of bone fractures . prominent areas of the face and neurocranium should be carefully examined as they tend to be more prone to injuries ."], ["glaucia maria bovi ambrosano , associate professor , department of community dentistry , campinas state university piracicaba dental school , for statistical analysis assistance ."]]}
{"article_id": "PMC4004215", "article_text": ["the influence of protecting \n groups on the reactivity and selectivity \n of glycosyl donors is a widely recognized and exploited phenomenon .", "the corresponding influence of protecting groups on the anomeric \n equilibrium , while evident for many years , is less widely studied and exploited .", "recent examples of the latter \n include the recognition that 2n,3o - oxazolidinones both facilitate anomerization in the 2-amino-2-deoxyhexopyranosides \n and stabilize the axial over the equatorial anomer , with similar effects being apparent in hexopyranosides carrying \n a 2,3-o - cyclic carbonate group , and the realization that cyclic 4,6-o - acetals modulate \n the anomeric effect in the hexopyranoses .", "our interest in the use of the n - acetyl-4o,5n - oxazolidinone protected \n sialyl donors , and their n - desacetyl \n counterparts , which have emerged as some of \n the most powerful and equatorially selective systems for the preparation \n of sialyl o - glycosides , as well as their c- and s - counterparts , prompted us \n to examine the effect of these systems on the anomeric equilibrium \n of sialyl glycosides .", "the magnitude of the anomeric effect is typically \n assessed either by mutarotation of anomeric hemiacetals or by the \n brnsted or lewis acid - mediated equilibration of glycosides , \n and such methods have been used to determine the position of the anomeric \n equilibrium in both n - acetyl neuraminic acid itself \n and its methyl glycoside , leading to the conclusion that the axial glycoside is significantly \n more strongly favored in the sialic acids ( figure 1 ) than in glucose and that mutarotation of the hemiacetal \n takes place via a ring - opening mechanism .", "the attempted synthesis of a n - acetyl-4o,5n - protected sialyl \n hemiacetal 2 by hydrolysis of the corresponding thioglycoside 3 resulted in the formation of a complex mixture of the two \n anomers \n of 2 and of the acyclic form 4 , and its \n hydrate 5 , as determined by nmr spectroscopy , in which \n the latter two species predominated ( figure 2 ) .", "the complexity of the spectra together with the predominance of \n the open - chain forms revealed the strain placed on the pyranose forms \n by the presence of the trans - fused oxazolidinone \n ring , even if no distortion of the pyranose ring is evident from x - ray \n structures , and precluded use of the standard \n methods in this investigation .", "compounds employed in and arising from \n the mutarotation of an oxazolidinone - protected \n sialose derivative .", "accordingly , we turned \n our attention to the use of neutral methods \n for the equilibration of sialic acid glycosides such as would not \n involve the intermediacy of the anomeric hemiacetal and would be compatible \n with the readily cleaved activated oxazolidinone \n ring .", "precedent for the equilibration of anomeric stereochemistry \n through the reversible homolytic cleavage and reformation of anomeric \n c  h , c  co , and c  te bonds , suggested \n that the answer might lie in radical reactions .", "the need to approximate \n as closely as possible the properties of o - glycosidic \n bonds suggested the synthesis and equilibration of o - glycosyl derivatives of sterically hindered n , n - disubstituted hydroxylamines by the fischer ", "albeit less substituted than \n the systems required for the radical equilibration process , glycosyl \n hydroxylamines are found in nature in the enediyne antibiotics calicheamicin \n 16 and esperamicin 7 , and simple hydroxylamines have been employed as glycosidic bond \n surrogates in neoglycoconjugates ( figure 3 ) .", "moreover , the distinct conformational preferences of the o - glycosyl hydroxylamine linkage have been advanced as a \n design element used by nature to assist in the binding of calicheamicin \n to the minor groove of dna.o - glycosyl hydroxylamines have been identified as metabolites of various \n drugs , including glucosides and glucuronides \n of the antioxidant 2,2,6,6-tetramethylpiperidin-1-ol .", "natural products featuring the o - glycosyl hydroxylamine \n moiety . in a preliminary communication \n we established the concept with \n sialyl glycosides of 2,2,4,4-tetramethylpiperidin-1-ol in a single \n solvent . in this article", "we report in \n full on the synthesis of the o - sialyl glycosides \n of two further hydroxylamines of differing steric bulk and conduct \n equilibration studies in three solvents spanning a broad range of \n polarities .", "the influence of the sialyl protecting groups , solvent \n polarity , and hydroxylamine steric bulk on the thermal equilibration \n reactions of the sialyl hydroxylamines are presented and afford insight \n into the influence of the trans - fused oxazolidinone \n group in sialic acid chemistry .", "the lack of kinetic selectivity of \n sialyl anomeric radicals is discussed from the viewpoint of their \n conformation , which is based on literature electron spin resonance \n data of cognate radicals .", "despite the presence of o - glycosyl hydroxylamines \n in various enediyne antibiotics and their use in neoglycoconjugates , \n literature methods for their synthesis are limited to the glycosylation \n of ( i ) various n - acylated hydroxylamines , e.g. , n - hydroxyphthalimide and n - carboethoxy \n hydroxylamine , ( ii ) oximes , and ( iii ) nitrones , each with subsequent manipulation of the functionality at nitrogen . \n", "these methods are , however , not suitable for the synthesis of the \n sterically hindered o - glycosyl n , n - dialkylhydroxylamines required for this investigation , \n and we therefore turned to the design of alternative routes .", "a brief \n investigation of the direct glycosylation of n - hydroxy-2,2,6,6-tetramethylpiperidine \n with efficient sialyl donors such as developed previously in our laboratory was unfruitful , perhaps for reasons of steric \n hindrance , and we turned therefore to radical reactions and their \n relative indifference to steric constraints .", "previous workers have generated sialyl anomeric radicals for the \n purposes of c - sialoside formation by the action of \n stannyl radicals on sialyl chlorides , but we have \n preferred simple photolysis of a readily available s - sialyl xanthate ester 8 .", "we note in passing that attempted photochemical equilibration of \n xanthate 8 resulted , in a process reminiscent of the \n barton ", "fortunately , 254 nm \n photolysis of 8 through pyrex \n in a rayonnet photoreactor in the presence of the stable nitroxyl \n radicals tempo ( 10 ) , tmio ( 11 ) , and sg1 ( 12 ) resulted in the formation of the desired o - glycosyl hydroxylamines 1416 as \n reported in scheme 2 .", "attempted application \n of this method to dbno 13(107 ) resulted only in the formation of the glycal 9 . that \n the photolysis of 8 can be interrupted with appropriate \n radical traps before the formation of glycal 9 indicates \n that it takes place via a two - step process giving an initial radical \n pair followed by disproportionation ( scheme 1 ) .", "this observation , which is critical to the success of the project , \n differs from the original conclusion of the barton and porter laboratories \n regarding the photoelimination of thiobenzoate esters .", "those workers \n preferred a concerted elimination from a photochemically excited state \n of the thiocarbonyl ester on the basis of ( i ) the observation of a \n short - lived transient on irradiation of the thiobenzoate , and ( ii ) \n the inability to trap radical intermediates with ( unspecified ) radical \n traps .", "we also note , however , and more \n in line with the results observed here , that the photolysis of s - acyl xanthate esters is known to proceed with homolytic \n scission of the s - acyl bond resulting in acyl radical \n formation . returning to the synthesis of the o - glycosyl hydroxylamines , \n when the photolysis of 8 was conducted in the presence \n of tempo ( 10 ) the product 14", "was obtained \n as an approximately 1:2 mixture , favoring the isomer ( - ) with \n the axial hydroxylamino residue , in 69% yield together with 10% of \n the glycal . with tmio ( 11 ) , the adduct 15 was obtained in 72% yield as an approximate 1:2.2 : \n mixture .", "photolysis in dichloroethane in the presence of racemic sg1 \n ( 12 ) gave only the glycal 9 , but the anomeric \n radical could be intercepted in 45% yield when neat sg1 ( 12 equiv ) \n was used as solvent .", "finally , photolysis in the presence of di(t - butyl)nitroxide in dichloroethane afforded only the glycal 9 .", "given the use of racemic sg1 , there are four potential \n diastereomers of 16 , but the product was isolated as \n a mixture of two predominant but inseparable isomers , the major one \n of which we tentatively assign as an equatorial ( - ) adduct \n and the minor as an axial ( - ) adduct without commenting \n on the configuration at the stereogenic \n center adjacent to the phosphoryl group .", "the anomeric stereochemistry \n in each of the adducts 1416 is assigned \n on the basis of the j heteronuclear coupling \n constant between the anomeric carboxyl carbon and the axial methylene \n proton at c3 in the pyranose ring , which is diagnostic in the sialic \n acid glycosides .", "these assignments are also supported by noe measurements for the \n axial ( - ) anomer of 14 and subsequent members \n of the same series ( vide infra ) , which reveal the spatial proximity \n of the axial h6 in the pyranose ring and one or more of the methyl \n singlets in the hydroxylamine moiety .", "resubmission of 14 to the photolysis conditions did not result in any change in the \n anomeric ratio consistent with the lack of a suitable chromophore , \n and indicating the products 1416 to be kinetic mixtures .", "rate constants for the trapping of \n alkyl radicals by nitroxyl radicals , while high ( 10 to \n 10 s ) , are nevertheless below the \n diffusion controlled limit for stabilized alkyl radicals and are dependent \n on steric bulk in both the radical and the trap .", "the stereoselectivities observed in the formation of 1416 therefore must factor in the inherent face \n selectivity of the intermediate radical 18 ( figure 4 ) and the steric bulk of the trap , which according \n to bowry and ingold , follows the trend dbno > tempo > tmio on \n the \n basis of kinetic trapping data ( sg1 not being included in the study ) .", "we conclude therefore that radical 18 shows a modest axial selectivity that can be overridden by the use \n of the presumably more bulky trap sg1 .", "this modest axial selectivity \n is consistent with the work of paulsen and matschulat , who recorded \n an axial / equatorial trapping preference of 1.8:1 for the reaction \n of 18 with allyltributylstannane at 60 c in thf , but not with the work of nagy and bednarski , \n who reported an approximate axial / equatorial ratio of 1:1 for the \n same reaction conducted at room temperature under photochemical conditions .", "we depict radical 18 as a \n close to planar -radical \n with extensive delocalization onto the carboxyl oxygen resulting in \n two distinct conformers about the exocyclic c1c2 bond ( figure 4 ) on the basis of literature esr data for simple \n model radicals .", "thus , esr studies of the tri - o - acetyl-2-deoxy-1-glucosyl \n radical 19 show it to adopt a chair conformation with \n an out of plane bend of 6 for the anomeric carbon consistent \n with a radical that is 90% sp hybridized .", "the methoxycarbonylmethyl radical 20 is a planar \n -radical with extensive delocalization onto the carboxyl oxygen \n and a barrier to rotation about the ch2co2me bond of 11 kcal mol , and the methoxy(methoxycarbonyl)methyl radical 21 displays esr parameters that closely mirror those of 20 , including the hyperfine splitting ah indicative of coupling to the ester methyl hydrogens , which is diagnostic \n of extensive delocalization . finally", ", \n the close model radicals 22 and 23 also \n show esr spectral parameters consistent with those for 20 , including hyperfine splitting by the ester methyl hydrogens and \n two distinct rotamers about the c5co2me bond .", "the two radicals 22 and 23 , isomeric at \n the -position , adopt different conformations so as in each \n case to maximize the overlap of the -acetoxy group with the \n singly filled orbital , a phenomenon known as quasi - homoanomeric stabilization .", "it follows that when the -position is unsubstituted as in \n radicals 18 and 19 , and the constraint of \n the quasi - homoanomeric interaction absent , the chair conformation \n will be retained .", "predicted structure of the intermediate \n radical 18 and literature structures of the model radicals 1923 with key esr parameters ( hyperfine \n splitting constants a in mt ) . presumably , it is the close to -type , approximately \n planar \n structure of radical 18 and the presence of the anomeric \n carboxyl group that accounts for the low kinetic stereoselectivities \n in its reactions with radical traps , which contrast with the high \n axial selectivity seen in the chemistry of simple per acetylated glycosyl \n radicals , including 19 , and their 1-alkoxy derivatives .", "thus , trapping of radical 18 along the axial direction , \n while favored on stereoelectronic grounds and leading directly to \n the chair conformation of the product , involves a 1,3-diaxial interaction \n between the axial h4 and h6 and the partially formed c2o bond , \n while equatorial trapping results in 1,3-diaxial interaction of h4 \n and h6 with the fully formed c2-co2me bond albeit while \n placing the incoming bulky hydroxylamine in the sterically more accessible \n equatorial position ( figure 5 ) .", "this latter \n mode only begins to predominate with the most bulky nitroxide trap , \n sg1 ( 12 ) and the formation of 16 ( scheme 2 ) .", "a similar rationale has been previously invoked \n for exo  endo - selectivity , \n with competing steric interactions between a full covalent bond to \n a substituent at the radical center and partially formed covalent \n bond to an incoming trap for the bicyclic radical 24 .", "competing transition states for the trapping of radical 18 showing 1,3-diaxial interactions with h4 and h6 , and structure \n of \n the radical 24 . with a series of glycosyl hydroxylamines in hand , and with \n a view \n to examining the influence of protecting groups on the anomeric equilibrium , \n", "a set of standard protecting group manipulations were applied to 14 and 15 , as illustrated in scheme 3 , giving a number of derivatives for subsequent \n equilibration studies .", "a series of equilibration \n reactions were then conducted by heating \n individually ( 0.050.1 m ) solutions of substances 14 , 15 , and 3033 to \n 90 c in each of deuteriobenzene , deuterio-1,2-dichloroethane , \n and deuterioacetonitrile , with periodic monitoring by nmr spectroscopy \n until no further change was observed ( table 1 ) .", "attempted equilibration of the sg1 derivative 16 resulted \n only in the formation of the elimination product 9 , for \n which reason , coupled with the problem of extra diastereomers arising \n from the stereogenic center in the aglycone , the sg1 series was not \n pursued further .", "a further compound , the n , n - diacetyl tempo glycoside 25 was also subjected \n to equilibration in deuterio-1,2-dichloroethane ( table 1 ) ; however , further work with this compound was not conducted \n in other solvents , nor was the tmio analogue prepared .", "the mechanism \n of the equilibration process involves reversible homolytic scission \n of the glycosyl - hydroxylamine c  o bond to an anomeric radical \n and a persistent aminoxyl radical ( scheme 4 ) .", "ingold radical effect that the recombination is intermolecular \n rather than intramolecular within the confines of the initial radical \n pair .", "the intermolecular nature of the process enables its use in \n combinatorial library generation , and \n for the present purposes it was readily demonstrated by a simple crossover \n experiment .", "thus , heating of 15 in the presence of an \n equimolar amount of tempo in 1,2-dichloroethane to 90 c for \n nine days , after which no further change was observed , resulted in \n the formation of an approximately 1:2 mixture of 14 and 15 , both as a mixture of anomers favoring the -anomers . \n", "the predominace of the tmio sialosides 15 over the tempo \n sialosides 14 in this equilibrated mixture indicates \n the greater thermodynamic stability of 15 over 14 , which is consistent with the less hindered , more tied \n back nature and therefore less persistent \n nature of the tmio radical 11 in comparison to the tempo \n radical 10", ". successful achievement of the \n equilibration process required rigorous \n exclusion of oxygen , which was achieved by copious sparring with argon , \n and possibly moisture , as with some batches of deuterioacetonitrile \n a further type of byproduct exemplified by 34 was observed \n ( scheme 5 ) . while this thermal oxidative decarboxylation \n process of a sialic acid glycoside is certainly interesting in view \n of other sialic acid decarboxylation processes , both oxidative and reductive , we have not pursed \n it further at present , especially as it could be avoided by degassing \n and use of fresh deuterioacetonitrile .", "first , as compared to sialic acid itself ( figure 1 ) and to simple methyl sialosides the o - sialyl hydroxylamines studied \n all show an inverted preference for the equatorial over the axial \n glycosides .", "second , there is a solvent effect on the position of the \n anomeric equilibrium with the equatorial ( - ) anomers generally \n being more highly favored in the higher polarity solvents .", "third , \n the magnitude of this solvent effect is on the whole greater in the \n tmio series ( table 1 , entries 2 , 5 , and 7 ) \n than in the tempo series ( table 1 , entries \n 1 , 4 , and 6 ) .", "fourth , the anomeric ratio at equilibrium is a function \n of the protecting groups employed with the same trends being seen \n for the both the tempo and tmio series of compounds .", "the general \n preference of the equatorial over the axial glycosides \n seen in table 1 , which contrasts sharply with \n the strong anomeric effect in simple sialosides , reflects the significant \n steric bulk of the hindered hydroxylamine aglycones and the attendant \n destabilization of the axial glycosides through classical 1,3-diaxial \n interactions .", "this reversal of the anomeric preference between simple \n sialosides and those of hindered hydroxylamines does not detract from \n the validity of comparisons within the later series of compounds that \n form the core of this article .", "the observed solvent effect , which \n is consistent with the well - known general \n trend whereby the anomeric effect is greatest in less polar solvents , \n simply reflects the more polar nature of equatorial versus axial glycosides . \n", "the greater solvent dependence of the tmio series as compared to the \n tempo series of compounds presumably arises from the more polar nature \n of the tmio group relative to the tempo group , which is a function \n of the presence of the electron - withdrawing arene in the tmio moiety .", "the most consistent trend , which holds for \n both the tempo and tmio series of compounds and for all three solvents \n assayed , involves the change in anomeric ratio as a function of protecting \n groups at the o4 and n5 positions .", "thus , there is generally a greater \n preference for the equatorial glycoside , or a smaller anomeric effect , \n in the 4o,5n - oxazolidinone series \n ( table 1 , entries 4 and 5 ) , than in the n - acetyl-4o,5n - oxazolidinone \n series ( table 1 , entries 6 and 7 ) , which in \n turn show a greater equatorial preference than the 4o,5n - diacetyl series ( table 1 , entries 1 and 2 ) .", "although less data is available , the 4o,5n,5n - triacetyl compounds \n ( table 1 , entry 3 ) appear to fall between the \n diacetyl and the n - acetyloxazolidinones .", "decreasing preference \n for the axial ( - ) sialoside as a function \n of protecting group . turning to the reasons underlying the general greater preference \n for the equatorial glycosides ( figure 6 ) in \n the oxazolidinone series , we discount steric arguments based on a \n smaller steric clash between the aglycone and the o4 protecting group \n in the oxazolidinones because the usual exoanomeric effect conformation about the glycosidic \n bond will orient the aglycone toward the rear of the molecule and \n away from o4 ( figure 7 ) .", "indeed , the typical \n exoanomeric effect conformation with an n  o  anomeric \n c  ring oxygen torsion angle of -60 is evident \n in the x - ray crystal structure of a calicheamicin derivative , in the x - ray structures of a simple o - glycosyl \n hydroxylamine , and of various o - glycosyl oximes and hydroxyimides , albeit a molecular mechanics - based computational \n paper appears to suggest a n  o  anomeric c  ring \n oxygen torsion angle of 170. newman \n projection about the hydroxylamine n  o bond showing \n the typical exoanomeric effect conformation of the o - glycosyl hydroxylamines .", "rather we suggest that the influence \n of the oxazolidinone on the \n anomeric effect is rooted in the highly polar nature of the oxazolidinone \n system with its strong dipole moment aligned with the axis of the \n carbonyl bond ( figure 8) .", "thus , as illustrated in figure 9 , the oxazolidinone \n moiety exhibits a powerful electron - withdrawing effect in the mean \n plane of the pyranose ring due to the alignment of the carbonyl dipole \n with the polar c4o4 and c5n5 bonds .", "this is to be \n contrasted with the 4o,5n - diacetyl \n system in which , assuming the standard minimum energy conformations \n of the ester and amide groups with the latter apparent from \n the 180 h5c5n5nh torsion angle with jnh , h5 = 10 hz , the two carbonyl \n dipoles are oriented in opposite directions and approximately perpendicular \n to the c4o4 and c5n5 bonds resulting overall in a \n smaller electron - withdrawing effect .", "the electron - withdrawing effect \n of the n - acetyloxazolidinone system , while still \n powerful because of the alignment of the oxazolidinone carbonyl with \n the c4o4 and c5n5 bonds in the mean plane of the pyranose \n ring , is moderated by the orientation of the acetyl group , which , \n as is clear from the available x - ray crystal structures and literature data with simple models , is oriented so as to minimize the overall dipole ( figure 8) at least in weakly polar environments .", "dipole moments \n ( debye units ) of open chain and cyclic carbonyl \n functionalities in nonpolar solvents .", "orientations of the key o4 and n5 protecting group carbonyl dipole \n moments in the o - sialyl hydroxylamines ( side chains \n omitted for clarity ) .", "if the anomeric effect is understood in terms of donation \n of electron \n density from a lone pair ( n ) on the ring oxygen into \n the synperiplanar c  o  * orbital of the axial glycosidic \n bond , the influence of the oxazolidinone can be \n attributed to the lowering of the energy of the lone pair due to the \n presence of the strongly electron - withdrawing group , which in turn \n reduces the n * interaction and so \n diminishes the anomeric effect . in valence bond terms", "this equates \n to the destabilization of the double bond  no bond resonance \n form owing to the increased energy of the oxocarbenium ion arising \n from the presence of the strongly electron - withdrawing protecting \n group ( figure 10 ) .", "it can also be argued in \n view of the high dipole moment of the oxazolidinone group that there \n is a dipolar component to the stabilization of the equatorial glycosides . \n", "thus , in the axial glycosides the vectors of the oxazolidinone dipole \n and the anomeric carbon oxygen bond dipole subtend an angle of approximately \n 90 , whereas in the equatorial glycoside the angle subtended \n is approximately 60 , suggesting that the equatorial glycosides \n will be more highly stabilized in less polar media , as is the case \n ( table 1 ) .", "a new method of equilibration of sialyl anomeric \n c  o bonds \n has been developed on the basis of the fischer  ingold persistent \n radical effect .", "this method enables the investigation of protecting \n groups on the anomeric equilibrium and is compatible with acid and \n base sensitive functionality .", "the trans - fused oxazolidinone \n moiety is found by this method to stabilize equatorial glycosides \n over their axial counterparts , i.e. , to reduce the magnitude of the \n anomeric effect .", "this effect , which adds to a series of recent observations \n on the influence of cyclic protecting groups on the anomeric effect , is a manifestation of the strongly electron - withdrawing nature of \n the trans - fused oxazolidinone , ( and by extrapolation trans - fused cyclic carbonates ) , which is a direct consequence \n of the highly dipolar nature of such heterocycles .", "the strongly electron - withdrawing \n nature of these groups manifest by the present study more than likely \n also plays an important role in the high kinetic selectivity observed \n in the trans - fused oxazolidinone ( and cyclic carbonate)-directed \n -sialoside synthesis methods .", "the unusually poor kinetic diastereoselectivity \n of anomeric radicals in the sialic acid series is a function of the \n -type planar nature of these radicals coupled with the existence \n of competing 1,3-diaxial interactions in the diastereomeric transition \n states for the formation of both anomers ."], "abstract_text": ["<S> a method \n for the investigation of the influence of protecting groups \n on the anomeric equilibrium in the sialic acid glycosides has been \n developed on the basis of the equilibration of o - sialyl \n hydroxylamines by reversible homolytic scission of the glycosidic \n bond following the dictates of the fischer  </S>", "<S> ingold persistent \n radical effect . </S>", "<S> it is found that a trans - fused 4o,5n - oxazolidinone group stabilizes the \n equatorial glycoside , i.e. , reduces the anomeric effect , when compared \n to the 4o,5n - diacetyl protected \n systems . </S>", "<S> this effect is discussed in terms of the powerful electron - withdrawing \n nature of the oxazolidinone system , which in turn is a function of \n its strong dipole moment in the mean plane of the pyranose ring system . \n </S>", "<S> the new equilibration method displays a small solvent effect and is \n most pronounced in less polar media consistent with the anomeric effect \n in general . </S>", "<S> the unusual ( for anomeric radicals ) poor kinetic selectivity \n of anomeric sialyl radicals is discussed in terms of the planar -type \n structure of these radicals and of competing 1,3-diaxial interactions \n in the diastereomeric transition states for trapping on the - \n and -faces of the radical . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Conclusions"], "sections": [["the influence of protecting \n groups on the reactivity and selectivity \n of glycosyl donors is a widely recognized and exploited phenomenon .", "the corresponding influence of protecting groups on the anomeric \n equilibrium , while evident for many years , is less widely studied and exploited .", "recent examples of the latter \n include the recognition that 2n,3o - oxazolidinones both facilitate anomerization in the 2-amino-2-deoxyhexopyranosides \n and stabilize the axial over the equatorial anomer , with similar effects being apparent in hexopyranosides carrying \n a 2,3-o - cyclic carbonate group , and the realization that cyclic 4,6-o - acetals modulate \n the anomeric effect in the hexopyranoses .", "our interest in the use of the n - acetyl-4o,5n - oxazolidinone protected \n sialyl donors , and their n - desacetyl \n counterparts , which have emerged as some of \n the most powerful and equatorially selective systems for the preparation \n of sialyl o - glycosides , as well as their c- and s - counterparts , prompted us \n to examine the effect of these systems on the anomeric equilibrium \n of sialyl glycosides .", "the magnitude of the anomeric effect is typically \n assessed either by mutarotation of anomeric hemiacetals or by the \n brnsted or lewis acid - mediated equilibration of glycosides , \n and such methods have been used to determine the position of the anomeric \n equilibrium in both n - acetyl neuraminic acid itself \n and its methyl glycoside , leading to the conclusion that the axial glycoside is significantly \n more strongly favored in the sialic acids ( figure 1 ) than in glucose and that mutarotation of the hemiacetal \n takes place via a ring - opening mechanism .", "the attempted synthesis of a n - acetyl-4o,5n - protected sialyl \n hemiacetal 2 by hydrolysis of the corresponding thioglycoside 3 resulted in the formation of a complex mixture of the two \n anomers \n of 2 and of the acyclic form 4 , and its \n hydrate 5 , as determined by nmr spectroscopy , in which \n the latter two species predominated ( figure 2 ) .", "the complexity of the spectra together with the predominance of \n the open - chain forms revealed the strain placed on the pyranose forms \n by the presence of the trans - fused oxazolidinone \n ring , even if no distortion of the pyranose ring is evident from x - ray \n structures , and precluded use of the standard \n methods in this investigation .", "compounds employed in and arising from \n the mutarotation of an oxazolidinone - protected \n sialose derivative .", "accordingly , we turned \n our attention to the use of neutral methods \n for the equilibration of sialic acid glycosides such as would not \n involve the intermediacy of the anomeric hemiacetal and would be compatible \n with the readily cleaved activated oxazolidinone \n ring .", "precedent for the equilibration of anomeric stereochemistry \n through the reversible homolytic cleavage and reformation of anomeric \n c  h , c  co , and c  te bonds , suggested \n that the answer might lie in radical reactions .", "the need to approximate \n as closely as possible the properties of o - glycosidic \n bonds suggested the synthesis and equilibration of o - glycosyl derivatives of sterically hindered n , n - disubstituted hydroxylamines by the fischer ", "albeit less substituted than \n the systems required for the radical equilibration process , glycosyl \n hydroxylamines are found in nature in the enediyne antibiotics calicheamicin \n 16 and esperamicin 7 , and simple hydroxylamines have been employed as glycosidic bond \n surrogates in neoglycoconjugates ( figure 3 ) .", "moreover , the distinct conformational preferences of the o - glycosyl hydroxylamine linkage have been advanced as a \n design element used by nature to assist in the binding of calicheamicin \n to the minor groove of dna.o - glycosyl hydroxylamines have been identified as metabolites of various \n drugs , including glucosides and glucuronides \n of the antioxidant 2,2,6,6-tetramethylpiperidin-1-ol .", "natural products featuring the o - glycosyl hydroxylamine \n moiety . in a preliminary communication \n we established the concept with \n sialyl glycosides of 2,2,4,4-tetramethylpiperidin-1-ol in a single \n solvent . in this article", "we report in \n full on the synthesis of the o - sialyl glycosides \n of two further hydroxylamines of differing steric bulk and conduct \n equilibration studies in three solvents spanning a broad range of \n polarities .", "the influence of the sialyl protecting groups , solvent \n polarity , and hydroxylamine steric bulk on the thermal equilibration \n reactions of the sialyl hydroxylamines are presented and afford insight \n into the influence of the trans - fused oxazolidinone \n group in sialic acid chemistry .", "the lack of kinetic selectivity of \n sialyl anomeric radicals is discussed from the viewpoint of their \n conformation , which is based on literature electron spin resonance \n data of cognate radicals ."], ["despite the presence of o - glycosyl hydroxylamines \n in various enediyne antibiotics and their use in neoglycoconjugates , \n literature methods for their synthesis are limited to the glycosylation \n of ( i ) various n - acylated hydroxylamines , e.g. , n - hydroxyphthalimide and n - carboethoxy \n hydroxylamine , ( ii ) oximes , and ( iii ) nitrones , each with subsequent manipulation of the functionality at nitrogen . \n", "these methods are , however , not suitable for the synthesis of the \n sterically hindered o - glycosyl n , n - dialkylhydroxylamines required for this investigation , \n and we therefore turned to the design of alternative routes .", "a brief \n investigation of the direct glycosylation of n - hydroxy-2,2,6,6-tetramethylpiperidine \n with efficient sialyl donors such as developed previously in our laboratory was unfruitful , perhaps for reasons of steric \n hindrance , and we turned therefore to radical reactions and their \n relative indifference to steric constraints .", "previous workers have generated sialyl anomeric radicals for the \n purposes of c - sialoside formation by the action of \n stannyl radicals on sialyl chlorides , but we have \n preferred simple photolysis of a readily available s - sialyl xanthate ester 8 .", "we note in passing that attempted photochemical equilibration of \n xanthate 8 resulted , in a process reminiscent of the \n barton ", "fortunately , 254 nm \n photolysis of 8 through pyrex \n in a rayonnet photoreactor in the presence of the stable nitroxyl \n radicals tempo ( 10 ) , tmio ( 11 ) , and sg1 ( 12 ) resulted in the formation of the desired o - glycosyl hydroxylamines 1416 as \n reported in scheme 2 .", "attempted application \n of this method to dbno 13(107 ) resulted only in the formation of the glycal 9 . that \n the photolysis of 8 can be interrupted with appropriate \n radical traps before the formation of glycal 9 indicates \n that it takes place via a two - step process giving an initial radical \n pair followed by disproportionation ( scheme 1 ) .", "this observation , which is critical to the success of the project , \n differs from the original conclusion of the barton and porter laboratories \n regarding the photoelimination of thiobenzoate esters .", "those workers \n preferred a concerted elimination from a photochemically excited state \n of the thiocarbonyl ester on the basis of ( i ) the observation of a \n short - lived transient on irradiation of the thiobenzoate , and ( ii ) \n the inability to trap radical intermediates with ( unspecified ) radical \n traps .", "we also note , however , and more \n in line with the results observed here , that the photolysis of s - acyl xanthate esters is known to proceed with homolytic \n scission of the s - acyl bond resulting in acyl radical \n formation . returning to the synthesis of the o - glycosyl hydroxylamines , \n when the photolysis of 8 was conducted in the presence \n of tempo ( 10 ) the product 14", "was obtained \n as an approximately 1:2 mixture , favoring the isomer ( - ) with \n the axial hydroxylamino residue , in 69% yield together with 10% of \n the glycal . with tmio ( 11 ) , the adduct 15 was obtained in 72% yield as an approximate 1:2.2 : \n mixture .", "photolysis in dichloroethane in the presence of racemic sg1 \n ( 12 ) gave only the glycal 9 , but the anomeric \n radical could be intercepted in 45% yield when neat sg1 ( 12 equiv ) \n was used as solvent .", "finally , photolysis in the presence of di(t - butyl)nitroxide in dichloroethane afforded only the glycal 9 .", "given the use of racemic sg1 , there are four potential \n diastereomers of 16 , but the product was isolated as \n a mixture of two predominant but inseparable isomers , the major one \n of which we tentatively assign as an equatorial ( - ) adduct \n and the minor as an axial ( - ) adduct without commenting \n on the configuration at the stereogenic \n center adjacent to the phosphoryl group .", "the anomeric stereochemistry \n in each of the adducts 1416 is assigned \n on the basis of the j heteronuclear coupling \n constant between the anomeric carboxyl carbon and the axial methylene \n proton at c3 in the pyranose ring , which is diagnostic in the sialic \n acid glycosides .", "these assignments are also supported by noe measurements for the \n axial ( - ) anomer of 14 and subsequent members \n of the same series ( vide infra ) , which reveal the spatial proximity \n of the axial h6 in the pyranose ring and one or more of the methyl \n singlets in the hydroxylamine moiety .", "resubmission of 14 to the photolysis conditions did not result in any change in the \n anomeric ratio consistent with the lack of a suitable chromophore , \n and indicating the products 1416 to be kinetic mixtures .", "rate constants for the trapping of \n alkyl radicals by nitroxyl radicals , while high ( 10 to \n 10 s ) , are nevertheless below the \n diffusion controlled limit for stabilized alkyl radicals and are dependent \n on steric bulk in both the radical and the trap .", "the stereoselectivities observed in the formation of 1416 therefore must factor in the inherent face \n selectivity of the intermediate radical 18 ( figure 4 ) and the steric bulk of the trap , which according \n to bowry and ingold , follows the trend dbno > tempo > tmio on \n the \n basis of kinetic trapping data ( sg1 not being included in the study ) .", "we conclude therefore that radical 18 shows a modest axial selectivity that can be overridden by the use \n of the presumably more bulky trap sg1 .", "this modest axial selectivity \n is consistent with the work of paulsen and matschulat , who recorded \n an axial / equatorial trapping preference of 1.8:1 for the reaction \n of 18 with allyltributylstannane at 60 c in thf , but not with the work of nagy and bednarski , \n who reported an approximate axial / equatorial ratio of 1:1 for the \n same reaction conducted at room temperature under photochemical conditions .", "we depict radical 18 as a \n close to planar -radical \n with extensive delocalization onto the carboxyl oxygen resulting in \n two distinct conformers about the exocyclic c1c2 bond ( figure 4 ) on the basis of literature esr data for simple \n model radicals .", "thus , esr studies of the tri - o - acetyl-2-deoxy-1-glucosyl \n radical 19 show it to adopt a chair conformation with \n an out of plane bend of 6 for the anomeric carbon consistent \n with a radical that is 90% sp hybridized .", "the methoxycarbonylmethyl radical 20 is a planar \n -radical with extensive delocalization onto the carboxyl oxygen \n and a barrier to rotation about the ch2co2me bond of 11 kcal mol , and the methoxy(methoxycarbonyl)methyl radical 21 displays esr parameters that closely mirror those of 20 , including the hyperfine splitting ah indicative of coupling to the ester methyl hydrogens , which is diagnostic \n of extensive delocalization . finally", ", \n the close model radicals 22 and 23 also \n show esr spectral parameters consistent with those for 20 , including hyperfine splitting by the ester methyl hydrogens and \n two distinct rotamers about the c5co2me bond .", "the two radicals 22 and 23 , isomeric at \n the -position , adopt different conformations so as in each \n case to maximize the overlap of the -acetoxy group with the \n singly filled orbital , a phenomenon known as quasi - homoanomeric stabilization .", "it follows that when the -position is unsubstituted as in \n radicals 18 and 19 , and the constraint of \n the quasi - homoanomeric interaction absent , the chair conformation \n will be retained .", "predicted structure of the intermediate \n radical 18 and literature structures of the model radicals 1923 with key esr parameters ( hyperfine \n splitting constants a in mt ) . presumably , it is the close to -type , approximately \n planar \n structure of radical 18 and the presence of the anomeric \n carboxyl group that accounts for the low kinetic stereoselectivities \n in its reactions with radical traps , which contrast with the high \n axial selectivity seen in the chemistry of simple per acetylated glycosyl \n radicals , including 19 , and their 1-alkoxy derivatives .", "thus , trapping of radical 18 along the axial direction , \n while favored on stereoelectronic grounds and leading directly to \n the chair conformation of the product , involves a 1,3-diaxial interaction \n between the axial h4 and h6 and the partially formed c2o bond , \n while equatorial trapping results in 1,3-diaxial interaction of h4 \n and h6 with the fully formed c2-co2me bond albeit while \n placing the incoming bulky hydroxylamine in the sterically more accessible \n equatorial position ( figure 5 ) .", "this latter \n mode only begins to predominate with the most bulky nitroxide trap , \n sg1 ( 12 ) and the formation of 16 ( scheme 2 ) .", "a similar rationale has been previously invoked \n for exo  endo - selectivity , \n with competing steric interactions between a full covalent bond to \n a substituent at the radical center and partially formed covalent \n bond to an incoming trap for the bicyclic radical 24 .", "competing transition states for the trapping of radical 18 showing 1,3-diaxial interactions with h4 and h6 , and structure \n of \n the radical 24 . with a series of glycosyl hydroxylamines in hand , and with \n a view \n to examining the influence of protecting groups on the anomeric equilibrium , \n", "a set of standard protecting group manipulations were applied to 14 and 15 , as illustrated in scheme 3 , giving a number of derivatives for subsequent \n equilibration studies .", "a series of equilibration \n reactions were then conducted by heating \n individually ( 0.050.1 m ) solutions of substances 14 , 15 , and 3033 to \n 90 c in each of deuteriobenzene , deuterio-1,2-dichloroethane , \n and deuterioacetonitrile , with periodic monitoring by nmr spectroscopy \n until no further change was observed ( table 1 ) .", "attempted equilibration of the sg1 derivative 16 resulted \n only in the formation of the elimination product 9 , for \n which reason , coupled with the problem of extra diastereomers arising \n from the stereogenic center in the aglycone , the sg1 series was not \n pursued further .", "a further compound , the n , n - diacetyl tempo glycoside 25 was also subjected \n to equilibration in deuterio-1,2-dichloroethane ( table 1 ) ; however , further work with this compound was not conducted \n in other solvents , nor was the tmio analogue prepared .", "the mechanism \n of the equilibration process involves reversible homolytic scission \n of the glycosyl - hydroxylamine c  o bond to an anomeric radical \n and a persistent aminoxyl radical ( scheme 4 ) .", "ingold radical effect that the recombination is intermolecular \n rather than intramolecular within the confines of the initial radical \n pair .", "the intermolecular nature of the process enables its use in \n combinatorial library generation , and \n for the present purposes it was readily demonstrated by a simple crossover \n experiment .", "thus , heating of 15 in the presence of an \n equimolar amount of tempo in 1,2-dichloroethane to 90 c for \n nine days , after which no further change was observed , resulted in \n the formation of an approximately 1:2 mixture of 14 and 15 , both as a mixture of anomers favoring the -anomers . \n", "the predominace of the tmio sialosides 15 over the tempo \n sialosides 14 in this equilibrated mixture indicates \n the greater thermodynamic stability of 15 over 14 , which is consistent with the less hindered , more tied \n back nature and therefore less persistent \n nature of the tmio radical 11 in comparison to the tempo \n radical 10", ". successful achievement of the \n equilibration process required rigorous \n exclusion of oxygen , which was achieved by copious sparring with argon , \n and possibly moisture , as with some batches of deuterioacetonitrile \n a further type of byproduct exemplified by 34 was observed \n ( scheme 5 ) . while this thermal oxidative decarboxylation \n process of a sialic acid glycoside is certainly interesting in view \n of other sialic acid decarboxylation processes , both oxidative and reductive , we have not pursed \n it further at present , especially as it could be avoided by degassing \n and use of fresh deuterioacetonitrile .", "first , as compared to sialic acid itself ( figure 1 ) and to simple methyl sialosides the o - sialyl hydroxylamines studied \n all show an inverted preference for the equatorial over the axial \n glycosides .", "second , there is a solvent effect on the position of the \n anomeric equilibrium with the equatorial ( - ) anomers generally \n being more highly favored in the higher polarity solvents .", "third , \n the magnitude of this solvent effect is on the whole greater in the \n tmio series ( table 1 , entries 2 , 5 , and 7 ) \n than in the tempo series ( table 1 , entries \n 1 , 4 , and 6 ) .", "fourth , the anomeric ratio at equilibrium is a function \n of the protecting groups employed with the same trends being seen \n for the both the tempo and tmio series of compounds .", "the general \n preference of the equatorial over the axial glycosides \n seen in table 1 , which contrasts sharply with \n the strong anomeric effect in simple sialosides , reflects the significant \n steric bulk of the hindered hydroxylamine aglycones and the attendant \n destabilization of the axial glycosides through classical 1,3-diaxial \n interactions .", "this reversal of the anomeric preference between simple \n sialosides and those of hindered hydroxylamines does not detract from \n the validity of comparisons within the later series of compounds that \n form the core of this article .", "the observed solvent effect , which \n is consistent with the well - known general \n trend whereby the anomeric effect is greatest in less polar solvents , \n simply reflects the more polar nature of equatorial versus axial glycosides . \n", "the greater solvent dependence of the tmio series as compared to the \n tempo series of compounds presumably arises from the more polar nature \n of the tmio group relative to the tempo group , which is a function \n of the presence of the electron - withdrawing arene in the tmio moiety .", "the most consistent trend , which holds for \n both the tempo and tmio series of compounds and for all three solvents \n assayed , involves the change in anomeric ratio as a function of protecting \n groups at the o4 and n5 positions .", "thus , there is generally a greater \n preference for the equatorial glycoside , or a smaller anomeric effect , \n in the 4o,5n - oxazolidinone series \n ( table 1 , entries 4 and 5 ) , than in the n - acetyl-4o,5n - oxazolidinone \n series ( table 1 , entries 6 and 7 ) , which in \n turn show a greater equatorial preference than the 4o,5n - diacetyl series ( table 1 , entries 1 and 2 ) .", "although less data is available , the 4o,5n,5n - triacetyl compounds \n ( table 1 , entry 3 ) appear to fall between the \n diacetyl and the n - acetyloxazolidinones .", "decreasing preference \n for the axial ( - ) sialoside as a function \n of protecting group . turning to the reasons underlying the general greater preference \n for the equatorial glycosides ( figure 6 ) in \n the oxazolidinone series , we discount steric arguments based on a \n smaller steric clash between the aglycone and the o4 protecting group \n in the oxazolidinones because the usual exoanomeric effect conformation about the glycosidic \n bond will orient the aglycone toward the rear of the molecule and \n away from o4 ( figure 7 ) .", "indeed , the typical \n exoanomeric effect conformation with an n  o  anomeric \n c  ring oxygen torsion angle of -60 is evident \n in the x - ray crystal structure of a calicheamicin derivative , in the x - ray structures of a simple o - glycosyl \n hydroxylamine , and of various o - glycosyl oximes and hydroxyimides , albeit a molecular mechanics - based computational \n paper appears to suggest a n  o  anomeric c  ring \n oxygen torsion angle of 170. newman \n projection about the hydroxylamine n  o bond showing \n the typical exoanomeric effect conformation of the o - glycosyl hydroxylamines .", "rather we suggest that the influence \n of the oxazolidinone on the \n anomeric effect is rooted in the highly polar nature of the oxazolidinone \n system with its strong dipole moment aligned with the axis of the \n carbonyl bond ( figure 8) .", "thus , as illustrated in figure 9 , the oxazolidinone \n moiety exhibits a powerful electron - withdrawing effect in the mean \n plane of the pyranose ring due to the alignment of the carbonyl dipole \n with the polar c4o4 and c5n5 bonds .", "this is to be \n contrasted with the 4o,5n - diacetyl \n system in which , assuming the standard minimum energy conformations \n of the ester and amide groups with the latter apparent from \n the 180 h5c5n5nh torsion angle with jnh , h5 = 10 hz , the two carbonyl \n dipoles are oriented in opposite directions and approximately perpendicular \n to the c4o4 and c5n5 bonds resulting overall in a \n smaller electron - withdrawing effect .", "the electron - withdrawing effect \n of the n - acetyloxazolidinone system , while still \n powerful because of the alignment of the oxazolidinone carbonyl with \n the c4o4 and c5n5 bonds in the mean plane of the pyranose \n ring , is moderated by the orientation of the acetyl group , which , \n as is clear from the available x - ray crystal structures and literature data with simple models , is oriented so as to minimize the overall dipole ( figure 8) at least in weakly polar environments .", "dipole moments \n ( debye units ) of open chain and cyclic carbonyl \n functionalities in nonpolar solvents .", "orientations of the key o4 and n5 protecting group carbonyl dipole \n moments in the o - sialyl hydroxylamines ( side chains \n omitted for clarity ) .", "if the anomeric effect is understood in terms of donation \n of electron \n density from a lone pair ( n ) on the ring oxygen into \n the synperiplanar c  o  * orbital of the axial glycosidic \n bond , the influence of the oxazolidinone can be \n attributed to the lowering of the energy of the lone pair due to the \n presence of the strongly electron - withdrawing group , which in turn \n reduces the n * interaction and so \n diminishes the anomeric effect . in valence bond terms", "this equates \n to the destabilization of the double bond  no bond resonance \n form owing to the increased energy of the oxocarbenium ion arising \n from the presence of the strongly electron - withdrawing protecting \n group ( figure 10 ) .", "it can also be argued in \n view of the high dipole moment of the oxazolidinone group that there \n is a dipolar component to the stabilization of the equatorial glycosides . \n", "thus , in the axial glycosides the vectors of the oxazolidinone dipole \n and the anomeric carbon oxygen bond dipole subtend an angle of approximately \n 90 , whereas in the equatorial glycoside the angle subtended \n is approximately 60 , suggesting that the equatorial glycosides \n will be more highly stabilized in less polar media , as is the case \n ( table 1 ) ."], ["a new method of equilibration of sialyl anomeric \n c  o bonds \n has been developed on the basis of the fischer  ingold persistent \n radical effect .", "this method enables the investigation of protecting \n groups on the anomeric equilibrium and is compatible with acid and \n base sensitive functionality .", "the trans - fused oxazolidinone \n moiety is found by this method to stabilize equatorial glycosides \n over their axial counterparts , i.e. , to reduce the magnitude of the \n anomeric effect .", "this effect , which adds to a series of recent observations \n on the influence of cyclic protecting groups on the anomeric effect , is a manifestation of the strongly electron - withdrawing nature of \n the trans - fused oxazolidinone , ( and by extrapolation trans - fused cyclic carbonates ) , which is a direct consequence \n of the highly dipolar nature of such heterocycles .", "the strongly electron - withdrawing \n nature of these groups manifest by the present study more than likely \n also plays an important role in the high kinetic selectivity observed \n in the trans - fused oxazolidinone ( and cyclic carbonate)-directed \n -sialoside synthesis methods .", "the unusually poor kinetic diastereoselectivity \n of anomeric radicals in the sialic acid series is a function of the \n -type planar nature of these radicals coupled with the existence \n of competing 1,3-diaxial interactions in the diastereomeric transition \n states for the formation of both anomers ."]]}
{"article_id": "PMC3452075", "article_text": ["familial hemiplegic migraine ( fhm ) is an autosomal dominant rare type of migraine with aura characterized by some degree of hemiparesis .", "fhm1 , fhm2 and fhm3 are caused by mutations in the ion - channel genes cacna1a , atp1a2 and scn1a , respectively [ 24 ] .", "the majority of fhm families have pure fhm , but cerebellar ataxia is frequent in fhm families with mutation in the cacna1a gene [ 5 , 6 ] .", "we present a norwegian family with a v628  m mutation in the atp1a2 gene ( fig .  1 ) .", "1the pedigree , the arrow indicate the proband , squares are men and circles are women , filled symbols are affected , and forward slash indicate deceased the pedigree , the arrow indicate the proband , squares are men and circles are women , filled symbols are affected , and forward slash indicate deceased", "a 16-year - old male was referred to the neurological ward on a hot summer day due to a prolonged attack of fhm .", "the patient had played soccer and shortly after heading a ball he experienced a left - sided homonymous hemianopsia , followed by left - sided sensory symptoms and motor weakness , and then a contralateral migrainous headache . from the age of 2  years he experienced several attacks of sudden drowsiness , nausea and headache .", "most attacks were preceded by head trauma and were interpreted as commotio . at the age of 6  years", "he also experienced motor weakness and reduced coordination of the hand during attacks , and at the age of 9  years this was followed by dysphasia and somnolence .", "several ct - scans of the brain were normal . at the age of 10  years he had the diagnosis fhm after a prolonged attack with visual disturbances , scotoma , sensory symptoms , motor weakness , aphasia , somnolence and contralateral headache .", "he was discharged with 75  mg acetylsalicyl acid daily , and 160  mg propranolol daily .", "after initiation of the prophylactic treatment , the attack frequency 12 times per year was unchanged , but the duration of the aura symptoms and headache was reduced to 12 and 2436  h duration , respectively . prior to hospital admission", "the patient barely drank or ate anything for 5  days due to nausea and severe migrainous headache . at first", ", he received treatment with metoclopramide , paracetamole and ketobemidon and 1,000  ml i.v .", "blood test including blood count , b-12 , creatine kinase , electrolytes , liver status , tsh , antinuclear antibodies , anticardiolipin antibodies and rheumatoid factor were all normal .", "due to fear of vasoconstriction and migraine infarction , several measures were evaluated including continuous conservative management and verapamil treatment .", "after external advice continuous infusion of intravenous nimodipine 510  ml / h ( dose ) was initiated .", "the patient s symptoms worsened within hours , and the following morning he had a generalized tonic ", "total infusion dose was then 16  mg . an acute mri showed mild swelling of the right parietal lobe ( figs .  2 , 3 ) .", "he continued to have some degree of hemiparesis along with apraxia , homonymous hemianopsia and mild cognitive failure , i.e. , 25 of 30 points on the mini mental state examination ( mmse ) , and mildly impaired memory score and moderately impaired construction score on the cognistat . at that time", "a single photon emission computed tomography ( spect ) showed reduced perfusion in the right hemisphere , predominantly in the temporal and parietal lobe ( fig .  4 ) .", "the patient was discharged after 3  weeks hospitalization and re - admitted 4  weeks later on day 55 . at that time he suffered from dizziness and memory deficits .", "clinical examination disclosed a partial homonymous hemianopsia , and the mmse score was 29 due to one point lost on the memory task .", "this provoked an attack of 1-week duration , and he was advised to refrain from intensive physical activity.fig . ", "2t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regionsfig . ", "3apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regionsfig . ", "499  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regions apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regions 99  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions grandmother .", "the proband s deceased grandmother on the mother s side was reported to have had attacks of hemiplegia , but never had epileptic seizures .", "it usually started with unilateral sensory symptoms followed by unilateral hemiparesis and then she got a contralateral headache . initially the attacks lasted up to 4  days and occurred from two to four times every month .", "the attack duration and frequency declined during adolescence , and she experienced her last attack at age 38  years .", "the attacks were often provoked by physical activity and the affected side varied between attacks .", "he experienced his first attack at the age of 13  year and had not more than 15 attacks since then .", "the attacks usually start with dizziness followed by homonymous visual disturbances , unilateral sensory symptoms , aphasia and unilateral weakness and then a contralateral headache .", "the aura symptoms usually last from 1 to 6  h , while the headache last up to 24  h. the aura symptoms change side from attack to attack .", "the nucleotide 1987 g  >  a substitution causes a change of the amino acid valine to methionine ( v628  m ) . both 16-year - old patients and mother had the mutation , while the elder brother was not tested .", "a 16-year - old male was referred to the neurological ward on a hot summer day due to a prolonged attack of fhm .", "the patient had played soccer and shortly after heading a ball he experienced a left - sided homonymous hemianopsia , followed by left - sided sensory symptoms and motor weakness , and then a contralateral migrainous headache . from the age of 2  years he experienced several attacks of sudden drowsiness , nausea and headache .", "most attacks were preceded by head trauma and were interpreted as commotio . at the age of 6  years", "he also experienced motor weakness and reduced coordination of the hand during attacks , and at the age of 9  years this was followed by dysphasia and somnolence .", "several ct - scans of the brain were normal . at the age of 10  years he had the diagnosis fhm after a prolonged attack with visual disturbances , scotoma , sensory symptoms , motor weakness , aphasia , somnolence and contralateral headache .", "he was discharged with 75  mg acetylsalicyl acid daily , and 160  mg propranolol daily .", "after initiation of the prophylactic treatment , the attack frequency 12 times per year was unchanged , but the duration of the aura symptoms and headache was reduced to 12 and 2436  h duration , respectively . prior to hospital admission", "the patient barely drank or ate anything for 5  days due to nausea and severe migrainous headache . at first", ", he received treatment with metoclopramide , paracetamole and ketobemidon and 1,000  ml i.v .", "blood test including blood count , b-12 , creatine kinase , electrolytes , liver status , tsh , antinuclear antibodies , anticardiolipin antibodies and rheumatoid factor were all normal .", "due to fear of vasoconstriction and migraine infarction , several measures were evaluated including continuous conservative management and verapamil treatment .", "after external advice continuous infusion of intravenous nimodipine 510  ml / h ( dose ) was initiated .", "the patient s symptoms worsened within hours , and the following morning he had a generalized tonic ", "total infusion dose was then 16  mg . an acute mri showed mild swelling of the right parietal lobe ( figs .  2 , 3 ) .", "he continued to have some degree of hemiparesis along with apraxia , homonymous hemianopsia and mild cognitive failure , i.e. , 25 of 30 points on the mini mental state examination ( mmse ) , and mildly impaired memory score and moderately impaired construction score on the cognistat . at that time", "a single photon emission computed tomography ( spect ) showed reduced perfusion in the right hemisphere , predominantly in the temporal and parietal lobe ( fig .  4 ) .", "the patient was discharged after 3  weeks hospitalization and re - admitted 4  weeks later on day 55 . at that time he suffered from dizziness and memory deficits .", "clinical examination disclosed a partial homonymous hemianopsia , and the mmse score was 29 due to one point lost on the memory task .", "this provoked an attack of 1-week duration , and he was advised to refrain from intensive physical activity.fig . ", "2t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regionsfig . ", "3apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regionsfig . ", "499  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regions apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regions 99  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions", "the proband s deceased grandmother on the mother s side was reported to have had attacks of hemiplegia , but never had epileptic seizures . mother .", "it usually started with unilateral sensory symptoms followed by unilateral hemiparesis and then she got a contralateral headache . initially the attacks lasted up to 4  days and occurred from two to four times every month .", "the attack duration and frequency declined during adolescence , and she experienced her last attack at age 38  years .", "the attacks were often provoked by physical activity and the affected side varied between attacks .", "he experienced his first attack at the age of 13  year and had not more than 15 attacks since then .", "the attacks usually start with dizziness followed by homonymous visual disturbances , unilateral sensory symptoms , aphasia and unilateral weakness and then a contralateral headache .", "the aura symptoms usually last from 1 to 6  h , while the headache last up to 24  h. the aura symptoms change side from attack to attack .", "the nucleotide 1987 g  >  a substitution causes a change of the amino acid valine to methionine ( v628  m ) . both 16-year - old patients and mother had the mutation , while the elder brother was not tested .", "the phenotype  genotype correlation of the identified mutation ( v628  m ) is strengthened by the fact that it has also been reported in a turkish family .", "the norwegian family had attacks of pure fhm , with pronounced intra - familial variation in frequency , severity and duration of attacks .", "physical activity provoked attacks in the affected alive , and mild head trauma provoked attacks in the proband .", "mutations in the atp1a2 gene usually cause pure fhm , but alternating hemiplegia of childhood , benign familial infantile convulsions , cerebellar symptoms , severe episodic neurological deficits and mental retardation have also been reported [ 818 ] .", "the hypovolemia caused by dehydration and arterial hypotension probably added to sustain the prolonged fhm attack , but these factors alone has not previously been sufficient to cause a generalized tonic ", "the seizure is most likely caused by nimodipine , as the proband and his mother both noticed worsening of the aura symptoms after initiation of the intravenous nimodipine infusion .", "nimodipine is a calcium - channel blocker that dilates cerebral vessels and it is used to prevent delayed cerebral ischemia following subarachnoid hemorrhage . in theory nimodipine might prevent the vasospasm of fhm by lowering ca - influx .", "however , the continuous intravenous infusion of nimodipine probably aggravated the arterial hypotension , a known side effect .", "this caused additional hypoperfusion and hypoxia in the right hemisphere , which lead to a generalized tonic ", "clonic seizure and a secondary edema as shown on mri and spect ( for details see above ) .", "we conclude that nimodipine is contraindicated in the management of prolonged fhm attack , and recommend conservative management except from supplement of sufficient intravenous fluid in nauseated patients in order to avoid hypovolemia ."], "abstract_text": ["<S> we present a norwegian family with familial hemiplegic migraine ( fhm ) with possibly four affected in three generations . </S>", "<S> the family had a point mutation in the atp1a2 gene that caused a change of the amino acid valine to methionine ( v628  m ) . </S>", "<S> the symptoms were pure fhm with intra- and interindividual variability , and epilepsy is not part of the clinical picture . </S>", "<S> attacks could be provoked by physical activity . </S>", "<S> the proband had prolonged attacks of fhm , and was hospitalized due to such an attack provoked by a minor head trauma . </S>", "<S> the initial management was conservative , but due to persistence of the hemiplegia on day  9 , a continuous nimodipine infusion was initiated in order to prevent cerebrovascular vasospasm . </S>", "<S> however , the nimodipine infusion worsened the patient s symptoms and possibly provoked a generalized tonic  </S>", "<S> clonic seizure due to vasodilatation and reduced cerebral blood flow . </S>", "<S> the mri showed cortical edema and the spect showed reduced perfusion on the contralateral side of the hemiplegia . </S>", "<S> we conclude that nimodipine is contraindicated in the management of prolonged fhm attacks , and recommend conservative management and supplement of sufficient intravenous fluid in nauseated patients in order to avoid hypovolemia . </S>"], "labels": null, "section_names": ["Introduction", "Case reports", "Proband", "Family history", "Genetic testing", "Discussion"], "sections": [["familial hemiplegic migraine ( fhm ) is an autosomal dominant rare type of migraine with aura characterized by some degree of hemiparesis .", "fhm1 , fhm2 and fhm3 are caused by mutations in the ion - channel genes cacna1a , atp1a2 and scn1a , respectively [ 24 ] .", "the majority of fhm families have pure fhm , but cerebellar ataxia is frequent in fhm families with mutation in the cacna1a gene [ 5 , 6 ] .", "we present a norwegian family with a v628  m mutation in the atp1a2 gene ( fig .  1 ) .", "1the pedigree , the arrow indicate the proband , squares are men and circles are women , filled symbols are affected , and forward slash indicate deceased the pedigree , the arrow indicate the proband , squares are men and circles are women , filled symbols are affected , and forward slash indicate deceased"], ["a 16-year - old male was referred to the neurological ward on a hot summer day due to a prolonged attack of fhm .", "the patient had played soccer and shortly after heading a ball he experienced a left - sided homonymous hemianopsia , followed by left - sided sensory symptoms and motor weakness , and then a contralateral migrainous headache . from the age of 2  years he experienced several attacks of sudden drowsiness , nausea and headache .", "most attacks were preceded by head trauma and were interpreted as commotio . at the age of 6  years", "he also experienced motor weakness and reduced coordination of the hand during attacks , and at the age of 9  years this was followed by dysphasia and somnolence .", "several ct - scans of the brain were normal . at the age of 10  years he had the diagnosis fhm after a prolonged attack with visual disturbances , scotoma , sensory symptoms , motor weakness , aphasia , somnolence and contralateral headache .", "he was discharged with 75  mg acetylsalicyl acid daily , and 160  mg propranolol daily .", "after initiation of the prophylactic treatment , the attack frequency 12 times per year was unchanged , but the duration of the aura symptoms and headache was reduced to 12 and 2436  h duration , respectively . prior to hospital admission", "the patient barely drank or ate anything for 5  days due to nausea and severe migrainous headache . at first", ", he received treatment with metoclopramide , paracetamole and ketobemidon and 1,000  ml i.v .", "blood test including blood count , b-12 , creatine kinase , electrolytes , liver status , tsh , antinuclear antibodies , anticardiolipin antibodies and rheumatoid factor were all normal .", "due to fear of vasoconstriction and migraine infarction , several measures were evaluated including continuous conservative management and verapamil treatment .", "after external advice continuous infusion of intravenous nimodipine 510  ml / h ( dose ) was initiated .", "the patient s symptoms worsened within hours , and the following morning he had a generalized tonic ", "total infusion dose was then 16  mg . an acute mri showed mild swelling of the right parietal lobe ( figs .  2 , 3 ) .", "he continued to have some degree of hemiparesis along with apraxia , homonymous hemianopsia and mild cognitive failure , i.e. , 25 of 30 points on the mini mental state examination ( mmse ) , and mildly impaired memory score and moderately impaired construction score on the cognistat . at that time", "a single photon emission computed tomography ( spect ) showed reduced perfusion in the right hemisphere , predominantly in the temporal and parietal lobe ( fig .  4 ) .", "the patient was discharged after 3  weeks hospitalization and re - admitted 4  weeks later on day 55 . at that time he suffered from dizziness and memory deficits .", "clinical examination disclosed a partial homonymous hemianopsia , and the mmse score was 29 due to one point lost on the memory task .", "this provoked an attack of 1-week duration , and he was advised to refrain from intensive physical activity.fig . ", "2t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regionsfig . ", "3apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regionsfig . ", "499  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regions apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regions 99  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions grandmother .", "the proband s deceased grandmother on the mother s side was reported to have had attacks of hemiplegia , but never had epileptic seizures .", "it usually started with unilateral sensory symptoms followed by unilateral hemiparesis and then she got a contralateral headache . initially the attacks lasted up to 4  days and occurred from two to four times every month .", "the attack duration and frequency declined during adolescence , and she experienced her last attack at age 38  years .", "the attacks were often provoked by physical activity and the affected side varied between attacks .", "he experienced his first attack at the age of 13  year and had not more than 15 attacks since then .", "the attacks usually start with dizziness followed by homonymous visual disturbances , unilateral sensory symptoms , aphasia and unilateral weakness and then a contralateral headache .", "the aura symptoms usually last from 1 to 6  h , while the headache last up to 24  h. the aura symptoms change side from attack to attack .", "the nucleotide 1987 g  >  a substitution causes a change of the amino acid valine to methionine ( v628  m ) . both 16-year - old patients and mother had the mutation , while the elder brother was not tested ."], ["a 16-year - old male was referred to the neurological ward on a hot summer day due to a prolonged attack of fhm .", "the patient had played soccer and shortly after heading a ball he experienced a left - sided homonymous hemianopsia , followed by left - sided sensory symptoms and motor weakness , and then a contralateral migrainous headache . from the age of 2  years he experienced several attacks of sudden drowsiness , nausea and headache .", "most attacks were preceded by head trauma and were interpreted as commotio . at the age of 6  years", "he also experienced motor weakness and reduced coordination of the hand during attacks , and at the age of 9  years this was followed by dysphasia and somnolence .", "several ct - scans of the brain were normal . at the age of 10  years he had the diagnosis fhm after a prolonged attack with visual disturbances , scotoma , sensory symptoms , motor weakness , aphasia , somnolence and contralateral headache .", "he was discharged with 75  mg acetylsalicyl acid daily , and 160  mg propranolol daily .", "after initiation of the prophylactic treatment , the attack frequency 12 times per year was unchanged , but the duration of the aura symptoms and headache was reduced to 12 and 2436  h duration , respectively . prior to hospital admission", "the patient barely drank or ate anything for 5  days due to nausea and severe migrainous headache . at first", ", he received treatment with metoclopramide , paracetamole and ketobemidon and 1,000  ml i.v .", "blood test including blood count , b-12 , creatine kinase , electrolytes , liver status , tsh , antinuclear antibodies , anticardiolipin antibodies and rheumatoid factor were all normal .", "due to fear of vasoconstriction and migraine infarction , several measures were evaluated including continuous conservative management and verapamil treatment .", "after external advice continuous infusion of intravenous nimodipine 510  ml / h ( dose ) was initiated .", "the patient s symptoms worsened within hours , and the following morning he had a generalized tonic ", "total infusion dose was then 16  mg . an acute mri showed mild swelling of the right parietal lobe ( figs .  2 , 3 ) .", "he continued to have some degree of hemiparesis along with apraxia , homonymous hemianopsia and mild cognitive failure , i.e. , 25 of 30 points on the mini mental state examination ( mmse ) , and mildly impaired memory score and moderately impaired construction score on the cognistat . at that time", "a single photon emission computed tomography ( spect ) showed reduced perfusion in the right hemisphere , predominantly in the temporal and parietal lobe ( fig .  4 ) .", "the patient was discharged after 3  weeks hospitalization and re - admitted 4  weeks later on day 55 . at that time he suffered from dizziness and memory deficits .", "clinical examination disclosed a partial homonymous hemianopsia , and the mmse score was 29 due to one point lost on the memory task .", "this provoked an attack of 1-week duration , and he was advised to refrain from intensive physical activity.fig . ", "2t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regionsfig . ", "3apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regionsfig . ", "499  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions t2-weighted mri on day  10 showing hyperintensity and swelling of grey matter in the right temporal and parietal regions apparent diffusion coefficient ( adc ) map on day 10 showing restricted diffusion in the right temporal and parietal regions 99  m tc - hmpao - spect on day  22 showing cerebral blood flow hypoperfusion in the right hemisphere , predominantly in the temporal and parietal regions"], ["the proband s deceased grandmother on the mother s side was reported to have had attacks of hemiplegia , but never had epileptic seizures . mother .", "it usually started with unilateral sensory symptoms followed by unilateral hemiparesis and then she got a contralateral headache . initially the attacks lasted up to 4  days and occurred from two to four times every month .", "the attack duration and frequency declined during adolescence , and she experienced her last attack at age 38  years .", "the attacks were often provoked by physical activity and the affected side varied between attacks .", "he experienced his first attack at the age of 13  year and had not more than 15 attacks since then .", "the attacks usually start with dizziness followed by homonymous visual disturbances , unilateral sensory symptoms , aphasia and unilateral weakness and then a contralateral headache .", "the aura symptoms usually last from 1 to 6  h , while the headache last up to 24  h. the aura symptoms change side from attack to attack ."], ["the nucleotide 1987 g  >  a substitution causes a change of the amino acid valine to methionine ( v628  m ) . both 16-year - old patients and mother had the mutation , while the elder brother was not tested ."], ["the phenotype  genotype correlation of the identified mutation ( v628  m ) is strengthened by the fact that it has also been reported in a turkish family .", "the norwegian family had attacks of pure fhm , with pronounced intra - familial variation in frequency , severity and duration of attacks .", "physical activity provoked attacks in the affected alive , and mild head trauma provoked attacks in the proband .", "mutations in the atp1a2 gene usually cause pure fhm , but alternating hemiplegia of childhood , benign familial infantile convulsions , cerebellar symptoms , severe episodic neurological deficits and mental retardation have also been reported [ 818 ] .", "the hypovolemia caused by dehydration and arterial hypotension probably added to sustain the prolonged fhm attack , but these factors alone has not previously been sufficient to cause a generalized tonic ", "the seizure is most likely caused by nimodipine , as the proband and his mother both noticed worsening of the aura symptoms after initiation of the intravenous nimodipine infusion .", "nimodipine is a calcium - channel blocker that dilates cerebral vessels and it is used to prevent delayed cerebral ischemia following subarachnoid hemorrhage . in theory nimodipine might prevent the vasospasm of fhm by lowering ca - influx .", "however , the continuous intravenous infusion of nimodipine probably aggravated the arterial hypotension , a known side effect .", "this caused additional hypoperfusion and hypoxia in the right hemisphere , which lead to a generalized tonic ", "clonic seizure and a secondary edema as shown on mri and spect ( for details see above ) .", "we conclude that nimodipine is contraindicated in the management of prolonged fhm attack , and recommend conservative management except from supplement of sufficient intravenous fluid in nauseated patients in order to avoid hypovolemia ."]]}
{"article_id": "PMC293473", "article_text": ["a few days later , four more cases occurred among postal workers at the brentwood mail processing facility in the washington area .  ", "the appearance of new cases in different locations around the country suggested contamination with b. anthracis of several postal facilities . in each location , the population potentially exposed was large .", "public health , criminal investigation , and postal authorities at local , state and federal levels investigated the suspected bioterrorism event . within hours of the discovery of clusters of cases ,", "the centers for disease control and prevention(cdc ) deployed teams to four different locations to support state and local investigations .", "four letters containing anthrax spores were known to have been postmarked and processed in the hamilton mail processing center ( hmpc ) located in hamilton township , a suburb of trenton , new jersey .", "the hmpc is part of a complicated network of stations and buildings designed to move millions of pieces of mail per day .", "it is not a typical post office , but a very large facility located in a 281,387 square foot building with many operations areas containing mechanized equipment .", "it was typically staffed by 250 employees per shift and visited by numerous others in the course of processing 2 million items per day .", "on october 19 , , , , , field investigation in trenton was initiated by a cdc team composed of an epidemiologist , environmental experts , occupational physicians , epidemic intelligence service ( eis ) officers , a public health informatics fellow , and support personnel .", "the team was headquartered in the new jersey department of health and senior services ( njdhss ) and had access to the local area network and computing environment of the department .", "this article describes the experience of the informatics fellow and the new jersey geographic information system(gis ) coordinator during the first two weeks of the investigation in assessing information system needs and organizing on - site computer support for the new jersey arm of the investigation .", "early in the investigation , the number of people exposed to anthrax was estimated to be large .", "the response team planned to sample for anthrax contamination in the facility ; identify the exposed population ; locate , culture , and give prophylactic antibiotics to those most likely to be exposed ; and monitor the exposed and treated persons over time for potential long - term effects .", "information on each person and sample was to be obtained , stored , analyzed , and augmented over time .", "for epidemiologic investigation and management purposes , summary reports had to be generated frequently . because of the large number of agencies involved and the rapid progress of events , a system of document management for the many drafts of guidelines and other communications was also needed .", "immediate needs for output included lists of employees , clients , visitors , and other potentially exposed persons .", "the lists were used by eis officers for individual follow - up and were linked to the map of floor plans of the postal facility and environmental sampling results to help identify persons with the greatest likelihood of exposure .", "investigators and managers needed summary information , including the percentage of exposed patients receiving medication and the distribution of exposed persons by county .", "the media relations ' officer of the team produced reports as often as twice a day for the public .", "information needs of the emergency response team in new jersey not all potential data sources were available during the investigation .", "the database system in the building was unavailable , as the computers had been turned off due to concern that fans in the computers might create air currents that would jeopardize safety or alter environmental sampling results .", "the list of employees in the facility computers was therefore unavailable to the investigators and had to be assembled from other sources .", "digital autocad drawing files of the facility were also stored on a workstation within the facility , and were likewise unavailable  a situation that complicated the production of accurate and representative floor - plan maps .", "laboratory results included cultures of both human and environmental samples performed by the new jersey state laboratory and cdc laboratories . managing reports from samples processed in different laboratories required linking of multiple unique identifiers .", "electronic documents that were produced or revised during the investigation included survey results , background documents , guidelines , antibiotic stockpile logistics , and press releases .", "frequent revisions made it necessary to identify and provide access to the latest documents and to track document revisions .", "the information system developed consisted of three main components : a database of possibly exposed persons , a gis of the physical plant and equipment , and a document repository .", "the exposed - person database required merging of files in multiple formats , manual data entry , rapid analysis , and frequent production of reports .", "gis work was done using floor plans produced in arcview gis 3.2a ( environmental systems research institute , inc . , redlands , ca ) by screen - digitizing scanned not - to - scale xerographic copies of autocad drawings of the postal building .", "documents were made available to the team by using web pages ( html ) on the new jersey department of health intranet to link to the latest copies of the files .", "building outlines , walls , work areas , equipment , furniture , the ventilation system and diffusers , and environmental sampling locations were digitized and stored as shapefiles , a format used by both the arcview gis and epi map software .", "the geographical features were then linked to environmental sampling data to specific locations in the building using epi map .", "a unique sample identification number was used as the key for linking the shapefile to the environmental sample database ( figure 1 ) .", "as the map creation process progressed over the 2-week period , additional , larger size paper drawings were provided to which a complete floor plan and heating , ventilation , and air conditioning systems components could be added .", "another version of the floor plan has been published and is available on the internet .", "( environmental.png ) preliminary sampling at the mail facility 1 to support investigations in other facilities where the letters were manipulated , digitized floor plans of the carteret and west trenton post offices were produced from rudimentary sketches . since epi map is public domain software , it was installed on a number of computers without need for licensing making a gis tool freely available to the investigators .", "the document repository was created using html on an intranet site inside the firewall of the njdhss .", "the web page included up - to - date information such as press releases and a list of activities for the day .", "it was updated twice a day or more often when important information had to be communicated to the rest of the investigation team .", "each link was checked on a daily basis to ensure that the correct document was being accessed .", "early in the investigation , the number of people exposed to anthrax was estimated to be large .", "the response team planned to sample for anthrax contamination in the facility ; identify the exposed population ; locate , culture , and give prophylactic antibiotics to those most likely to be exposed ; and monitor the exposed and treated persons over time for potential long - term effects .", "information on each person and sample was to be obtained , stored , analyzed , and augmented over time .", "for epidemiologic investigation and management purposes , summary reports had to be generated frequently . because of the large number of agencies involved and the rapid progress of events , a system of document management for the many drafts of guidelines and other communications was also needed .", "immediate needs for output included lists of employees , clients , visitors , and other potentially exposed persons .", "the lists were used by eis officers for individual follow - up and were linked to the map of floor plans of the postal facility and environmental sampling results to help identify persons with the greatest likelihood of exposure .", "investigators and managers needed summary information , including the percentage of exposed patients receiving medication and the distribution of exposed persons by county .", "the media relations ' officer of the team produced reports as often as twice a day for the public .", "information needs of the emergency response team in new jersey not all potential data sources were available during the investigation .", "the database system in the building was unavailable , as the computers had been turned off due to concern that fans in the computers might create air currents that would jeopardize safety or alter environmental sampling results .", "the list of employees in the facility computers was therefore unavailable to the investigators and had to be assembled from other sources .", "digital autocad drawing files of the facility were also stored on a workstation within the facility , and were likewise unavailable  a situation that complicated the production of accurate and representative floor - plan maps .", "laboratory results included cultures of both human and environmental samples performed by the new jersey state laboratory and cdc laboratories . managing reports from samples processed in different laboratories required linking of multiple unique identifiers .", "electronic documents that were produced or revised during the investigation included survey results , background documents , guidelines , antibiotic stockpile logistics , and press releases .", "frequent revisions made it necessary to identify and provide access to the latest documents and to track document revisions .", "the information system developed consisted of three main components : a database of possibly exposed persons , a gis of the physical plant and equipment , and a document repository .", "the exposed - person database required merging of files in multiple formats , manual data entry , rapid analysis , and frequent production of reports .", "gis work was done using floor plans produced in arcview gis 3.2a ( environmental systems research institute , inc . , redlands , ca ) by screen - digitizing scanned not - to - scale xerographic copies of autocad drawings of the postal building .", "documents were made available to the team by using web pages ( html ) on the new jersey department of health intranet to link to the latest copies of the files .", "building outlines , walls , work areas , equipment , furniture , the ventilation system and diffusers , and environmental sampling locations were digitized and stored as shapefiles , a format used by both the arcview gis and epi map software . the geographical features were then linked to environmental sampling data to specific locations in the building using epi map .", "a unique sample identification number was used as the key for linking the shapefile to the environmental sample database ( figure 1 ) .", "as the map creation process progressed over the 2-week period , additional , larger size paper drawings were provided to which a complete floor plan and heating , ventilation , and air conditioning systems components could be added .", "another version of the floor plan has been published and is available on the internet .", "( environmental.png ) preliminary sampling at the mail facility 1 to support investigations in other facilities where the letters were manipulated , digitized floor plans of the carteret and west trenton post offices were produced from rudimentary sketches . since epi map is public domain software , it was installed on a number of computers without need for licensing making a gis tool freely available to the investigators .", "the document repository was created using html on an intranet site inside the firewall of the njdhss .", "the web page included up - to - date information such as press releases and a list of activities for the day .", "it was updated twice a day or more often when important information had to be communicated to the rest of the investigation team .", "each link was checked on a daily basis to ensure that the correct document was being accessed .", "recent publications on informatics and bioterrorism have focused on detection of bioterrorist events . some have reviewed the informatics contribution to past emergencies and formulated proposals for various kinds of surveillance and/or response [ 6 - 10 ] .", "the goal of this paper is to present needs , difficulties , and solutions encountered in the first 2 weeks of a single field investigation .", "every epidemic investigation is unique , but a comprehensive review of activities carried out during an actual investigation may focus discussion on understanding and improving the informatics response in such events . at the beginning of this investigation ,", "although the njdhss provided a secure computer environment in which to work , many elements of the system had to be designed and set up during the investigation .", "much of the base information did not exist ( e.g. , gis of the hamilton facility ) or had been made unavailable by the event under investigation ( e.g. , hamilton facility autocad digital drawing files and employee lists ) . as in most field investigations , both data sources and hypotheses shifted and evolved over the course of the investigation . during an investigation , information is critical for decision - making processes and for detection of cases , identification of risk factors , and managing prevention and control measures . in the first hours or days , the emergency response team is required to design and implement an information system capable of providing reliable , timely information .", "experience in developing such \" emergency information systems \" and with the software being used plays an important role in determining the time needed to complete the task . in this event ,", "close collaboration between the epidemiologist and informaticians was supported by software designed for field investigation and for exchange of data with other commercial products .", "the presence of trained professionals from a gis center was an important element in being able to map the site of the investigation .", "use of industry standard shapefiles and microsoft access databases allowed working copies to be distributed and used in public domain software as needed .", "a database system was operational within two days of the beginning of operations , and the gis elements evolved over a two - week period as floor plans became available .", "potential problems identified initially included lags in entry of clinical data , lack of access to data sources because computer systems were disrupted or inaccessible , and the variety of formats and platforms that were designed for other purposes .", "at least one source produced data in different formats on different days . in field investigation ,", "the informatics environment is defined by the agencies , networks , and individual computers from which data are obtained and with which exchange of data occurs . unless the entire investigation is internal to a single agency or company , each of the partners  state , county , health care facility , law enforcement agency , industry , or other entity  has its own informatics environment .", "the standards most likely to be common to all the partners are those of the computer industry , such as the internet , microsoft windows , and popular gis standards .", "those conducting the investigation must be prepared to adapt to the standards used by the data source partners rather than the reverse . in this investigation ,", "existing database systems provided useful information , but the data items were available on paper , in excel files , in text files , in access files , and in arcview shapefiles .", "the informatician is responsible for merging and integrating elements stored in different database formats to make the information available to the emergency response team .", "the need for a speedy public health response is a challenge for informaticians because the steps that are typically time consuming , such as requirements generation , analysis , design , implementation , and testing of a new system , must be completed in hours rather than in weeks or months . in this experience ,", "some parts of the system were functional within two days after arrival at the investigation site .", "epi info 2000   was used to set up databases for manual data entry and updating and for importing and merging data provided by collaborating sources in a variety of microsoft windows formats .", "the system developed took advantage of several features available in epi info   through its use of commercial component software and computer industry standards :  rapid development of a relational database , in which identifying information was localized to a single table for security purposes  importation of data from several of the 20 different file formats that can be read in epi info 2000  the ability to perform data management interactively but to preserve and replay the steps through automatically generated programs or scripts  the ability to link and display microsoft access data with arcview shapefiles to create maps  rapid development , with minimal coding , simplifying the testing and debugging process  the ability to add or delete variables from a database merely by revising a form on the screen .", " the availability of analytic output and epidemiologic statistics in the same program used for data management  interaction with high end gis programs and transition of database management to a commercial program through epi", "info 's incorporation of gis and windows standards  public domain status of the program allowed downloading the latest version from the internet and distributing it to any number of partners without licensing difficulties geographical and spatial analysis has gained importance in public health during the last few years .", "the availability of maps during the investigation can be attributed to the fact that the njdhss had made resources available from its gis center .", "the gis professional was able to create the shapefiles while the rest of the emergency response team ( ert ) focused on data collection and data management .", "once created , maps were widely available for use among all members of the team , and epi map 2000 could be used to link data in microsoft access files to the shapefiles of the building floor plans .", "the main use of gis in this investigation was to create a relatively detailed base map and to locate environmental sampling and provide spatial analysis and visual support to the investigation .", "we identified and located all samples taken in the different facilities . in a matter of days we connected the database system with the map files to provide up to date information regarding sample processing and results .", "the maps created provided a new perspective of the magnitude of contamination inside the facility and also identified non - contaminated areas .", "we extended the mapping capabilities to illustrate the area of coverage of the active and passive surveillance systems implemented in surrounding communities for the detection of new cases ( figure 2 ) .", "the surveillance system was originally restricted to nine counties and was latter extended to others .", "changes in the number of centers included in the surveillance system were reflected in different versions of the same map to document changes over time .", "( surveillance.png ) surveillance of new cases in new jersey because of the diversity of the data sources during the investigation , the database system required frequent maintenance .", "we were able to automate most of the data management routines using epi info program ( scripting ) files and to produce automatic reports .", "laboratory results were available through the local area network in the new jersey state health department where the team worked .", "although we might have been able to connect to the laboratory system to obtain real - time updates , for security reasons , the laboratory results for the investigation were exported and became available to the emergency response team at the end of each day .", "there was therefore a modest , but significant delay in availability of laboratory information . during emergency field situations ,", "any system intended for use by more than one group of people should be easy to use , compatible with other software , and well documented .", "the database was documented using entity relationship ( er ) diagrams ( figure 3 ) .", "epi info has a command to display the structure of a table , making it easy to document the system for the replacement team that arrived after two weeks .", "this second team preferred to work with microsoft access rather than epi info , but the fact that epi info 2000 stores data in generic access 97 tables facilitated the transition .", "( databasemodel.eps ) example of the entity relationship diagram for the database system implemented document tracking and version control are important when geographically separated teams are working on the same set of problems . in this case", "each one of the field sites had a set of experts developing documents and recommendations that applied to the whole event and not only to the geographic area of investigation .", "it was important to keep the most recent set of recommendations available to all members of the emergency response team for decision - making and information dissemination . during the investigation", ", we were able to access the most recent documents with minimal effort , using links in a web page .", "a more sophisticated version could be developed to allow tracking changes in a document over time .", "emergency situations require rapid design and implementation of databases to provide reliable information in the field . having the ability to respond rapidly to emergency situations requires planning and preparedness .", "key elements in preparation are in paying sufficient attention to informatics support and having both staff and software that can adapt quickly to new situations and computing environments .", "the likelihood of success is greater if the same people and software provide support for more routine epidemic investigations in this field investigation , the informatics environment included excel spreadsheets , files in several other formats , paper questionnaires and computer printouts , an existing gis capability using arcview , and the need for frequent revisions in database structure , frequent summary reports , and protection of personal identifiers .", "software for field use must be flexible enough to interact with and unite numerous data sources and computing environments .", "investigators must be prepared for new situations , and be ready to adapt new techniques and software as the needs of an investigation unfold .", "the traditional steps in systems development of requirements gathering , analysis , design , implementation , and testing must be traversed in rapid sequence during the first few hours or days of an investigation and repeated again as the system evolves and capabilities are added .", "more formalized approach to systems development needs to be either extensively modified or compressed to account for the time - critical nature of the situation .", "the use of commercial software standards such as windows file formats , web pages , and mainstream gis programs facilitates the rapid implementation of systems in the field .", "the design of systems for emergencies should include methods for storing and maintaining version control of documents and images in addition to more structured data .", "a floor plan of the contaminated hamilton facility was available only as an incomplete and poor quality 8 1/2  11 inch photocopy of the original ' e ' sized autocad drawing .", "it was scanned as a tiff image , imported into arcview gis 3.2 software and used as a backdrop to create shapefiles of the building outline , walls , operational areas and equipment placements , and niosh and fbi sampling locations .", "shapefiles were developed using a scanned copy of the floor plan of each facility and digitized using arcview .", "the elements of information to be displayed were divided into several layers showing samples taken by different agencies and their location .", "after completion , the shapefiles were linked to the data using epi map 2000 , a component of epi info that is arcviewcompatible .", "elements of information stored in databases created in epi info 2000 were sent directly to epi map for displaying ( figure 1 ) .", "the process was automated by using scripts or programs . to obtain data on persons possibly exposed , a liaison between the u.s .", "he was able to obtain ms - excelfiles or computer printouts listing personnel for each of the facilities under investigation .", "additional information was added to those lists to record presence in the facility during the exposure period , sex of the employee , and post exposure prophylaxis ( pep ) .", "most of the clinical follow - up services , including pep , were provided by the local hospital .", "the informatics department of the hospital extracted relevant clinical data from the database system and made them available to the investigation team as excel tables on some days and comma delimited text files on other days .", "primary data items collected by eis officers and other staff were entered into epi info 2000 or into excel and then imported into epi info 2000 ( microsoft access 97 ) format .", "potentially exposed populations such as regular visitors and relatives of postal workers were identified and manually entered into the main database as the investigation proceeded .", "demographic characteristics of potentially exposed persons were assembled from various sources , including the local hospital where pep was administered . when pep was offered to a large group of people , tracking of different treatment protocols and their results became necessary .", "paper forms were designed not to become obsolete if protocols are modified during the follow up , and similar revisions in the database were expected .", "database management was done in epi info 2000 , the windows version of epi info .", "epi info 2000 was developed at cdc from commercial component software and visual basic and offers compatibility with microsoft access files and 20 other standard database formats .", "it includes a mapping program that uses the arcview compatible shapefile format and allows the display of data from access tables on shapefiles .", "most of the basic statistics needed for reporting and analysis are built in so that we did not have to spend time coding or debugging algorithms .", "the data collection and data management tools in epi info do not require expert database managers .", "the database system consisted of a central database designed and maintained in epi info 2000 .", "the unique key at first contained information ( was \" intelligent \" ) , but we designed a mechanism to replace intelligent keys by non - intelligent keys .", "the intelligent key contained facility code , deployment place ( nj ) exposed unique identifier , and postal facility involved .", "we included the deployment site in the key assuming that the information would be linked with other sites at a latter time . from the beginning of the operation", ", security was a concern . because of the complexity of the investigation we decided that all sensitive information  defined as information that would allow identification of specific individuals  would be handled separately from the rest of the database .", "we created a single flat table with all identifiers and related the table to the rest of the database system . with that structure", "identifiers were not sent to cdc headquarters in atlanta , for example , and sensitive data were protected by the security features of the njdhss computing environment .", "a commercial html editor ( microsoft front page 2000 ) was used for the maintenance of the document repository web page .", "web pages included in the system did not contain any special scripting that required an advanced web programming techniques .", "the level of html coding was basic , and mostly inserted automatically by front page .", "the document repository allowed coding and storing versions of documents during the progress of the investigation .", "the document check - in process included a simple computer - based form with the name of the document , the author , and a time stamp .", "the first two elements were used to create a catalog , and the timestamp was used for versioning control .", "a floor plan of the contaminated hamilton facility was available only as an incomplete and poor quality 8 1/2  11 inch photocopy of the original ' e ' sized autocad drawing .", "it was scanned as a tiff image , imported into arcview gis 3.2 software and used as a backdrop to create shapefiles of the building outline , walls , operational areas and equipment placements , and niosh and fbi sampling locations .", "shapefiles were developed using a scanned copy of the floor plan of each facility and digitized using arcview .", "the elements of information to be displayed were divided into several layers showing samples taken by different agencies and their location .", "after completion , the shapefiles were linked to the data using epi map 2000 , a component of epi info that is arcviewcompatible .", "elements of information stored in databases created in epi info 2000 were sent directly to epi map for displaying ( figure 1 ) .", "to obtain data on persons possibly exposed , a liaison between the u.s . postal service and the epidemiologic team", "he was able to obtain ms - excelfiles or computer printouts listing personnel for each of the facilities under investigation .", "additional information was added to those lists to record presence in the facility during the exposure period , sex of the employee , and post exposure prophylaxis ( pep ) .", "most of the clinical follow - up services , including pep , were provided by the local hospital .", "the informatics department of the hospital extracted relevant clinical data from the database system and made them available to the investigation team as excel tables on some days and comma delimited text files on other days .", "primary data items collected by eis officers and other staff were entered into epi info 2000 or into excel and then imported into epi info 2000 ( microsoft access 97 ) format .", "potentially exposed populations such as regular visitors and relatives of postal workers were identified and manually entered into the main database as the investigation proceeded .", "demographic characteristics of potentially exposed persons were assembled from various sources , including the local hospital where pep was administered . when pep was offered to a large group of people ,", "paper forms were designed not to become obsolete if protocols are modified during the follow up , and similar revisions in the database were expected .", "database management was done in epi info 2000 , the windows version of epi info .", "epi info 2000 was developed at cdc from commercial component software and visual basic and offers compatibility with microsoft access files and 20 other standard database formats .", "it includes a mapping program that uses the arcview compatible shapefile format and allows the display of data from access tables on shapefiles .", "most of the basic statistics needed for reporting and analysis are built in so that we did not have to spend time coding or debugging algorithms .", "the data collection and data management tools in epi info do not require expert database managers .", "the database system consisted of a central database designed and maintained in epi info 2000 .", "the unique key at first contained information ( was \" intelligent \" ) , but we designed a mechanism to replace intelligent keys by non - intelligent keys .", "the intelligent key contained facility code , deployment place ( nj ) exposed unique identifier , and postal facility involved .", "we included the deployment site in the key assuming that the information would be linked with other sites at a latter time .", "from the beginning of the operation , security was a concern . because of the complexity of the investigation we decided that all sensitive information  defined as information that would allow identification of specific individuals  would be handled separately from the rest of the database .", "we created a single flat table with all identifiers and related the table to the rest of the database system . with that structure", "identifiers were not sent to cdc headquarters in atlanta , for example , and sensitive data were protected by the security features of the njdhss computing environment .", "a commercial html editor ( microsoft front page 2000 ) was used for the maintenance of the document repository web page .", "web pages included in the system did not contain any special scripting that required an advanced web programming techniques .", "the level of html coding was basic , and mostly inserted automatically by front page .", "the document repository allowed coding and storing versions of documents during the progress of the investigation .", "members of the team were instructed to check in documents on a regular basis . the document check - in process included a simple computer - based form with the name of the document , the author , and a time stamp .", "the first two elements were used to create a catalog , and the timestamp was used for versioning control .", "jz performed the analysis of informatics needs , set up the epi info portions of the field system and the document management system , and drafted the manuscript .", "rs developed the arcview gis system and shapefiles used in the investigation , and contributed to the manuscript .", "ad provided advice on the analysis of informatics needs and the development of the epi info applications through daily telephone conversations and wrote portions of the manuscript .", "esri ( environmental systems research institute ) provided a special license for the free distribution of mapobjects software within epiinfo / epimap 2000 ."], "abstract_text": ["<S> backgroundthe investigation of potential exposure to anthrax spores in a trenton , new jersey , mail - processing facility required rapid assessment of informatics needs and adaptation of existing informatics tools to new physical and information - processing environments . because the affected building and its computers were closed down , data to list potentially exposed persons and map building floor plans were unavailable from the primary source.resultscontrolling the effects of anthrax contamination required identification and follow - up of potentially exposed persons . </S>", "<S> risk of exposure had to be estimated from the geographic relationship between work history and environmental sample sites within the contaminated facility . to assist in establishing geographic relationships , floor plan maps of the postal facility </S>", "<S> were constructed in arcview geographic information system ( gis ) software and linked to a database of personnel and visitors using epi info and epi map 2000 . </S>", "<S> a repository for maintaining the latest versions of various documents was set up using web page hyperlinks.conclusionsduring public health emergencies , such as bioterrorist attacks and disease epidemics , computerized information systems for data management , analysis , and communication may be needed within hours of beginning the investigation . </S>", "<S> available sources of data and output requirements of the system may be changed frequently during the course of the investigation . </S>", "<S> integrating data from a variety of sources may require entering or importing data from a variety of digital and paper formats . </S>", "<S> spatial representation of data is particularly valuable for assessing environmental exposure . </S>", "<S> written documents , guidelines , and memos important to the epidemic were frequently revised . in this investigation , </S>", "<S> a database was operational on the second day and the gis component during the second week of the investigation . </S>"], "labels": null, "section_names": ["Background", "Results", "Assessment of Information Needs", "System Implementation", "Discussion", "Conclusions", "Methods", "Geographic Information System", "Data Collection and Input", "Database Management", "Security", "Document Management", "Authors' contributions", "Acknowledgements"], "sections": [["a few days later , four more cases occurred among postal workers at the brentwood mail processing facility in the washington area .  ", "the appearance of new cases in different locations around the country suggested contamination with b. anthracis of several postal facilities . in each location , the population potentially exposed was large .", "public health , criminal investigation , and postal authorities at local , state and federal levels investigated the suspected bioterrorism event . within hours of the discovery of clusters of cases ,", "the centers for disease control and prevention(cdc ) deployed teams to four different locations to support state and local investigations .", "four letters containing anthrax spores were known to have been postmarked and processed in the hamilton mail processing center ( hmpc ) located in hamilton township , a suburb of trenton , new jersey .", "the hmpc is part of a complicated network of stations and buildings designed to move millions of pieces of mail per day .", "it is not a typical post office , but a very large facility located in a 281,387 square foot building with many operations areas containing mechanized equipment .", "it was typically staffed by 250 employees per shift and visited by numerous others in the course of processing 2 million items per day .", "on october 19 , , , , , field investigation in trenton was initiated by a cdc team composed of an epidemiologist , environmental experts , occupational physicians , epidemic intelligence service ( eis ) officers , a public health informatics fellow , and support personnel .", "the team was headquartered in the new jersey department of health and senior services ( njdhss ) and had access to the local area network and computing environment of the department .", "this article describes the experience of the informatics fellow and the new jersey geographic information system(gis ) coordinator during the first two weeks of the investigation in assessing information system needs and organizing on - site computer support for the new jersey arm of the investigation ."], ["early in the investigation , the number of people exposed to anthrax was estimated to be large .", "the response team planned to sample for anthrax contamination in the facility ; identify the exposed population ; locate , culture , and give prophylactic antibiotics to those most likely to be exposed ; and monitor the exposed and treated persons over time for potential long - term effects .", "information on each person and sample was to be obtained , stored , analyzed , and augmented over time .", "for epidemiologic investigation and management purposes , summary reports had to be generated frequently . because of the large number of agencies involved and the rapid progress of events , a system of document management for the many drafts of guidelines and other communications was also needed .", "immediate needs for output included lists of employees , clients , visitors , and other potentially exposed persons .", "the lists were used by eis officers for individual follow - up and were linked to the map of floor plans of the postal facility and environmental sampling results to help identify persons with the greatest likelihood of exposure .", "investigators and managers needed summary information , including the percentage of exposed patients receiving medication and the distribution of exposed persons by county .", "the media relations ' officer of the team produced reports as often as twice a day for the public .", "information needs of the emergency response team in new jersey not all potential data sources were available during the investigation .", "the database system in the building was unavailable , as the computers had been turned off due to concern that fans in the computers might create air currents that would jeopardize safety or alter environmental sampling results .", "the list of employees in the facility computers was therefore unavailable to the investigators and had to be assembled from other sources .", "digital autocad drawing files of the facility were also stored on a workstation within the facility , and were likewise unavailable  a situation that complicated the production of accurate and representative floor - plan maps .", "laboratory results included cultures of both human and environmental samples performed by the new jersey state laboratory and cdc laboratories . managing reports from samples processed in different laboratories required linking of multiple unique identifiers .", "electronic documents that were produced or revised during the investigation included survey results , background documents , guidelines , antibiotic stockpile logistics , and press releases .", "frequent revisions made it necessary to identify and provide access to the latest documents and to track document revisions .", "the information system developed consisted of three main components : a database of possibly exposed persons , a gis of the physical plant and equipment , and a document repository .", "the exposed - person database required merging of files in multiple formats , manual data entry , rapid analysis , and frequent production of reports .", "gis work was done using floor plans produced in arcview gis 3.2a ( environmental systems research institute , inc . , redlands , ca ) by screen - digitizing scanned not - to - scale xerographic copies of autocad drawings of the postal building .", "documents were made available to the team by using web pages ( html ) on the new jersey department of health intranet to link to the latest copies of the files .", "building outlines , walls , work areas , equipment , furniture , the ventilation system and diffusers , and environmental sampling locations were digitized and stored as shapefiles , a format used by both the arcview gis and epi map software .", "the geographical features were then linked to environmental sampling data to specific locations in the building using epi map .", "a unique sample identification number was used as the key for linking the shapefile to the environmental sample database ( figure 1 ) .", "as the map creation process progressed over the 2-week period , additional , larger size paper drawings were provided to which a complete floor plan and heating , ventilation , and air conditioning systems components could be added .", "another version of the floor plan has been published and is available on the internet .", "( environmental.png ) preliminary sampling at the mail facility 1 to support investigations in other facilities where the letters were manipulated , digitized floor plans of the carteret and west trenton post offices were produced from rudimentary sketches . since epi map is public domain software , it was installed on a number of computers without need for licensing making a gis tool freely available to the investigators .", "the document repository was created using html on an intranet site inside the firewall of the njdhss .", "the web page included up - to - date information such as press releases and a list of activities for the day .", "it was updated twice a day or more often when important information had to be communicated to the rest of the investigation team .", "each link was checked on a daily basis to ensure that the correct document was being accessed ."], ["early in the investigation , the number of people exposed to anthrax was estimated to be large .", "the response team planned to sample for anthrax contamination in the facility ; identify the exposed population ; locate , culture , and give prophylactic antibiotics to those most likely to be exposed ; and monitor the exposed and treated persons over time for potential long - term effects .", "information on each person and sample was to be obtained , stored , analyzed , and augmented over time .", "for epidemiologic investigation and management purposes , summary reports had to be generated frequently . because of the large number of agencies involved and the rapid progress of events , a system of document management for the many drafts of guidelines and other communications was also needed .", "immediate needs for output included lists of employees , clients , visitors , and other potentially exposed persons .", "the lists were used by eis officers for individual follow - up and were linked to the map of floor plans of the postal facility and environmental sampling results to help identify persons with the greatest likelihood of exposure .", "investigators and managers needed summary information , including the percentage of exposed patients receiving medication and the distribution of exposed persons by county .", "the media relations ' officer of the team produced reports as often as twice a day for the public .", "information needs of the emergency response team in new jersey not all potential data sources were available during the investigation .", "the database system in the building was unavailable , as the computers had been turned off due to concern that fans in the computers might create air currents that would jeopardize safety or alter environmental sampling results .", "the list of employees in the facility computers was therefore unavailable to the investigators and had to be assembled from other sources .", "digital autocad drawing files of the facility were also stored on a workstation within the facility , and were likewise unavailable  a situation that complicated the production of accurate and representative floor - plan maps .", "laboratory results included cultures of both human and environmental samples performed by the new jersey state laboratory and cdc laboratories . managing reports from samples processed in different laboratories required linking of multiple unique identifiers .", "electronic documents that were produced or revised during the investigation included survey results , background documents , guidelines , antibiotic stockpile logistics , and press releases .", "frequent revisions made it necessary to identify and provide access to the latest documents and to track document revisions ."], ["the information system developed consisted of three main components : a database of possibly exposed persons , a gis of the physical plant and equipment , and a document repository .", "the exposed - person database required merging of files in multiple formats , manual data entry , rapid analysis , and frequent production of reports .", "gis work was done using floor plans produced in arcview gis 3.2a ( environmental systems research institute , inc . , redlands , ca ) by screen - digitizing scanned not - to - scale xerographic copies of autocad drawings of the postal building .", "documents were made available to the team by using web pages ( html ) on the new jersey department of health intranet to link to the latest copies of the files .", "building outlines , walls , work areas , equipment , furniture , the ventilation system and diffusers , and environmental sampling locations were digitized and stored as shapefiles , a format used by both the arcview gis and epi map software . the geographical features were then linked to environmental sampling data to specific locations in the building using epi map .", "a unique sample identification number was used as the key for linking the shapefile to the environmental sample database ( figure 1 ) .", "as the map creation process progressed over the 2-week period , additional , larger size paper drawings were provided to which a complete floor plan and heating , ventilation , and air conditioning systems components could be added .", "another version of the floor plan has been published and is available on the internet .", "( environmental.png ) preliminary sampling at the mail facility 1 to support investigations in other facilities where the letters were manipulated , digitized floor plans of the carteret and west trenton post offices were produced from rudimentary sketches . since epi map is public domain software , it was installed on a number of computers without need for licensing making a gis tool freely available to the investigators .", "the document repository was created using html on an intranet site inside the firewall of the njdhss .", "the web page included up - to - date information such as press releases and a list of activities for the day .", "it was updated twice a day or more often when important information had to be communicated to the rest of the investigation team .", "each link was checked on a daily basis to ensure that the correct document was being accessed ."], ["recent publications on informatics and bioterrorism have focused on detection of bioterrorist events . some have reviewed the informatics contribution to past emergencies and formulated proposals for various kinds of surveillance and/or response [ 6 - 10 ] .", "the goal of this paper is to present needs , difficulties , and solutions encountered in the first 2 weeks of a single field investigation .", "every epidemic investigation is unique , but a comprehensive review of activities carried out during an actual investigation may focus discussion on understanding and improving the informatics response in such events . at the beginning of this investigation ,", "although the njdhss provided a secure computer environment in which to work , many elements of the system had to be designed and set up during the investigation .", "much of the base information did not exist ( e.g. , gis of the hamilton facility ) or had been made unavailable by the event under investigation ( e.g. , hamilton facility autocad digital drawing files and employee lists ) . as in most field investigations , both data sources and hypotheses shifted and evolved over the course of the investigation . during an investigation , information is critical for decision - making processes and for detection of cases , identification of risk factors , and managing prevention and control measures . in the first hours or days , the emergency response team is required to design and implement an information system capable of providing reliable , timely information .", "experience in developing such \" emergency information systems \" and with the software being used plays an important role in determining the time needed to complete the task . in this event ,", "close collaboration between the epidemiologist and informaticians was supported by software designed for field investigation and for exchange of data with other commercial products .", "the presence of trained professionals from a gis center was an important element in being able to map the site of the investigation .", "use of industry standard shapefiles and microsoft access databases allowed working copies to be distributed and used in public domain software as needed .", "a database system was operational within two days of the beginning of operations , and the gis elements evolved over a two - week period as floor plans became available .", "potential problems identified initially included lags in entry of clinical data , lack of access to data sources because computer systems were disrupted or inaccessible , and the variety of formats and platforms that were designed for other purposes .", "at least one source produced data in different formats on different days . in field investigation ,", "the informatics environment is defined by the agencies , networks , and individual computers from which data are obtained and with which exchange of data occurs . unless the entire investigation is internal to a single agency or company , each of the partners  state , county , health care facility , law enforcement agency , industry , or other entity  has its own informatics environment .", "the standards most likely to be common to all the partners are those of the computer industry , such as the internet , microsoft windows , and popular gis standards .", "those conducting the investigation must be prepared to adapt to the standards used by the data source partners rather than the reverse . in this investigation ,", "existing database systems provided useful information , but the data items were available on paper , in excel files , in text files , in access files , and in arcview shapefiles .", "the informatician is responsible for merging and integrating elements stored in different database formats to make the information available to the emergency response team .", "the need for a speedy public health response is a challenge for informaticians because the steps that are typically time consuming , such as requirements generation , analysis , design , implementation , and testing of a new system , must be completed in hours rather than in weeks or months . in this experience ,", "some parts of the system were functional within two days after arrival at the investigation site .", "epi info 2000   was used to set up databases for manual data entry and updating and for importing and merging data provided by collaborating sources in a variety of microsoft windows formats .", "the system developed took advantage of several features available in epi info   through its use of commercial component software and computer industry standards :  rapid development of a relational database , in which identifying information was localized to a single table for security purposes  importation of data from several of the 20 different file formats that can be read in epi info 2000  the ability to perform data management interactively but to preserve and replay the steps through automatically generated programs or scripts  the ability to link and display microsoft access data with arcview shapefiles to create maps  rapid development , with minimal coding , simplifying the testing and debugging process  the ability to add or delete variables from a database merely by revising a form on the screen .", " the availability of analytic output and epidemiologic statistics in the same program used for data management  interaction with high end gis programs and transition of database management to a commercial program through epi", "info 's incorporation of gis and windows standards  public domain status of the program allowed downloading the latest version from the internet and distributing it to any number of partners without licensing difficulties geographical and spatial analysis has gained importance in public health during the last few years .", "the availability of maps during the investigation can be attributed to the fact that the njdhss had made resources available from its gis center .", "the gis professional was able to create the shapefiles while the rest of the emergency response team ( ert ) focused on data collection and data management .", "once created , maps were widely available for use among all members of the team , and epi map 2000 could be used to link data in microsoft access files to the shapefiles of the building floor plans .", "the main use of gis in this investigation was to create a relatively detailed base map and to locate environmental sampling and provide spatial analysis and visual support to the investigation .", "we identified and located all samples taken in the different facilities . in a matter of days we connected the database system with the map files to provide up to date information regarding sample processing and results .", "the maps created provided a new perspective of the magnitude of contamination inside the facility and also identified non - contaminated areas .", "we extended the mapping capabilities to illustrate the area of coverage of the active and passive surveillance systems implemented in surrounding communities for the detection of new cases ( figure 2 ) .", "the surveillance system was originally restricted to nine counties and was latter extended to others .", "changes in the number of centers included in the surveillance system were reflected in different versions of the same map to document changes over time .", "( surveillance.png ) surveillance of new cases in new jersey because of the diversity of the data sources during the investigation , the database system required frequent maintenance .", "we were able to automate most of the data management routines using epi info program ( scripting ) files and to produce automatic reports .", "laboratory results were available through the local area network in the new jersey state health department where the team worked .", "although we might have been able to connect to the laboratory system to obtain real - time updates , for security reasons , the laboratory results for the investigation were exported and became available to the emergency response team at the end of each day .", "there was therefore a modest , but significant delay in availability of laboratory information . during emergency field situations ,", "any system intended for use by more than one group of people should be easy to use , compatible with other software , and well documented .", "the database was documented using entity relationship ( er ) diagrams ( figure 3 ) .", "epi info has a command to display the structure of a table , making it easy to document the system for the replacement team that arrived after two weeks .", "this second team preferred to work with microsoft access rather than epi info , but the fact that epi info 2000 stores data in generic access 97 tables facilitated the transition .", "( databasemodel.eps ) example of the entity relationship diagram for the database system implemented document tracking and version control are important when geographically separated teams are working on the same set of problems . in this case", "each one of the field sites had a set of experts developing documents and recommendations that applied to the whole event and not only to the geographic area of investigation .", "it was important to keep the most recent set of recommendations available to all members of the emergency response team for decision - making and information dissemination . during the investigation", ", we were able to access the most recent documents with minimal effort , using links in a web page .", "a more sophisticated version could be developed to allow tracking changes in a document over time ."], ["emergency situations require rapid design and implementation of databases to provide reliable information in the field . having the ability to respond rapidly to emergency situations requires planning and preparedness .", "key elements in preparation are in paying sufficient attention to informatics support and having both staff and software that can adapt quickly to new situations and computing environments .", "the likelihood of success is greater if the same people and software provide support for more routine epidemic investigations in this field investigation , the informatics environment included excel spreadsheets , files in several other formats , paper questionnaires and computer printouts , an existing gis capability using arcview , and the need for frequent revisions in database structure , frequent summary reports , and protection of personal identifiers .", "software for field use must be flexible enough to interact with and unite numerous data sources and computing environments .", "investigators must be prepared for new situations , and be ready to adapt new techniques and software as the needs of an investigation unfold .", "the traditional steps in systems development of requirements gathering , analysis , design , implementation , and testing must be traversed in rapid sequence during the first few hours or days of an investigation and repeated again as the system evolves and capabilities are added .", "more formalized approach to systems development needs to be either extensively modified or compressed to account for the time - critical nature of the situation .", "the use of commercial software standards such as windows file formats , web pages , and mainstream gis programs facilitates the rapid implementation of systems in the field .", "the design of systems for emergencies should include methods for storing and maintaining version control of documents and images in addition to more structured data ."], ["a floor plan of the contaminated hamilton facility was available only as an incomplete and poor quality 8 1/2  11 inch photocopy of the original ' e ' sized autocad drawing .", "it was scanned as a tiff image , imported into arcview gis 3.2 software and used as a backdrop to create shapefiles of the building outline , walls , operational areas and equipment placements , and niosh and fbi sampling locations .", "shapefiles were developed using a scanned copy of the floor plan of each facility and digitized using arcview .", "the elements of information to be displayed were divided into several layers showing samples taken by different agencies and their location .", "after completion , the shapefiles were linked to the data using epi map 2000 , a component of epi info that is arcviewcompatible .", "elements of information stored in databases created in epi info 2000 were sent directly to epi map for displaying ( figure 1 ) .", "the process was automated by using scripts or programs . to obtain data on persons possibly exposed , a liaison between the u.s .", "he was able to obtain ms - excelfiles or computer printouts listing personnel for each of the facilities under investigation .", "additional information was added to those lists to record presence in the facility during the exposure period , sex of the employee , and post exposure prophylaxis ( pep ) .", "most of the clinical follow - up services , including pep , were provided by the local hospital .", "the informatics department of the hospital extracted relevant clinical data from the database system and made them available to the investigation team as excel tables on some days and comma delimited text files on other days .", "primary data items collected by eis officers and other staff were entered into epi info 2000 or into excel and then imported into epi info 2000 ( microsoft access 97 ) format .", "potentially exposed populations such as regular visitors and relatives of postal workers were identified and manually entered into the main database as the investigation proceeded .", "demographic characteristics of potentially exposed persons were assembled from various sources , including the local hospital where pep was administered . when pep was offered to a large group of people , tracking of different treatment protocols and their results became necessary .", "paper forms were designed not to become obsolete if protocols are modified during the follow up , and similar revisions in the database were expected .", "database management was done in epi info 2000 , the windows version of epi info .", "epi info 2000 was developed at cdc from commercial component software and visual basic and offers compatibility with microsoft access files and 20 other standard database formats .", "it includes a mapping program that uses the arcview compatible shapefile format and allows the display of data from access tables on shapefiles .", "most of the basic statistics needed for reporting and analysis are built in so that we did not have to spend time coding or debugging algorithms .", "the data collection and data management tools in epi info do not require expert database managers .", "the database system consisted of a central database designed and maintained in epi info 2000 .", "the unique key at first contained information ( was \" intelligent \" ) , but we designed a mechanism to replace intelligent keys by non - intelligent keys .", "the intelligent key contained facility code , deployment place ( nj ) exposed unique identifier , and postal facility involved .", "we included the deployment site in the key assuming that the information would be linked with other sites at a latter time . from the beginning of the operation", ", security was a concern . because of the complexity of the investigation we decided that all sensitive information  defined as information that would allow identification of specific individuals  would be handled separately from the rest of the database .", "we created a single flat table with all identifiers and related the table to the rest of the database system . with that structure", "identifiers were not sent to cdc headquarters in atlanta , for example , and sensitive data were protected by the security features of the njdhss computing environment .", "a commercial html editor ( microsoft front page 2000 ) was used for the maintenance of the document repository web page .", "web pages included in the system did not contain any special scripting that required an advanced web programming techniques .", "the level of html coding was basic , and mostly inserted automatically by front page .", "the document repository allowed coding and storing versions of documents during the progress of the investigation .", "the document check - in process included a simple computer - based form with the name of the document , the author , and a time stamp .", "the first two elements were used to create a catalog , and the timestamp was used for versioning control ."], ["a floor plan of the contaminated hamilton facility was available only as an incomplete and poor quality 8 1/2  11 inch photocopy of the original ' e ' sized autocad drawing .", "it was scanned as a tiff image , imported into arcview gis 3.2 software and used as a backdrop to create shapefiles of the building outline , walls , operational areas and equipment placements , and niosh and fbi sampling locations .", "shapefiles were developed using a scanned copy of the floor plan of each facility and digitized using arcview .", "the elements of information to be displayed were divided into several layers showing samples taken by different agencies and their location .", "after completion , the shapefiles were linked to the data using epi map 2000 , a component of epi info that is arcviewcompatible .", "elements of information stored in databases created in epi info 2000 were sent directly to epi map for displaying ( figure 1 ) ."], ["to obtain data on persons possibly exposed , a liaison between the u.s . postal service and the epidemiologic team", "he was able to obtain ms - excelfiles or computer printouts listing personnel for each of the facilities under investigation .", "additional information was added to those lists to record presence in the facility during the exposure period , sex of the employee , and post exposure prophylaxis ( pep ) .", "most of the clinical follow - up services , including pep , were provided by the local hospital .", "the informatics department of the hospital extracted relevant clinical data from the database system and made them available to the investigation team as excel tables on some days and comma delimited text files on other days .", "primary data items collected by eis officers and other staff were entered into epi info 2000 or into excel and then imported into epi info 2000 ( microsoft access 97 ) format .", "potentially exposed populations such as regular visitors and relatives of postal workers were identified and manually entered into the main database as the investigation proceeded .", "demographic characteristics of potentially exposed persons were assembled from various sources , including the local hospital where pep was administered . when pep was offered to a large group of people ,", "paper forms were designed not to become obsolete if protocols are modified during the follow up , and similar revisions in the database were expected ."], ["database management was done in epi info 2000 , the windows version of epi info .", "epi info 2000 was developed at cdc from commercial component software and visual basic and offers compatibility with microsoft access files and 20 other standard database formats .", "it includes a mapping program that uses the arcview compatible shapefile format and allows the display of data from access tables on shapefiles .", "most of the basic statistics needed for reporting and analysis are built in so that we did not have to spend time coding or debugging algorithms .", "the data collection and data management tools in epi info do not require expert database managers .", "the database system consisted of a central database designed and maintained in epi info 2000 .", "the unique key at first contained information ( was \" intelligent \" ) , but we designed a mechanism to replace intelligent keys by non - intelligent keys .", "the intelligent key contained facility code , deployment place ( nj ) exposed unique identifier , and postal facility involved .", "we included the deployment site in the key assuming that the information would be linked with other sites at a latter time ."], ["from the beginning of the operation , security was a concern . because of the complexity of the investigation we decided that all sensitive information  defined as information that would allow identification of specific individuals  would be handled separately from the rest of the database .", "we created a single flat table with all identifiers and related the table to the rest of the database system . with that structure", "identifiers were not sent to cdc headquarters in atlanta , for example , and sensitive data were protected by the security features of the njdhss computing environment ."], ["a commercial html editor ( microsoft front page 2000 ) was used for the maintenance of the document repository web page .", "web pages included in the system did not contain any special scripting that required an advanced web programming techniques .", "the level of html coding was basic , and mostly inserted automatically by front page .", "the document repository allowed coding and storing versions of documents during the progress of the investigation .", "members of the team were instructed to check in documents on a regular basis . the document check - in process included a simple computer - based form with the name of the document , the author , and a time stamp .", "the first two elements were used to create a catalog , and the timestamp was used for versioning control ."], ["jz performed the analysis of informatics needs , set up the epi info portions of the field system and the document management system , and drafted the manuscript .", "rs developed the arcview gis system and shapefiles used in the investigation , and contributed to the manuscript .", "ad provided advice on the analysis of informatics needs and the development of the epi info applications through daily telephone conversations and wrote portions of the manuscript ."], ["esri ( environmental systems research institute ) provided a special license for the free distribution of mapobjects software within epiinfo / epimap 2000 ."]]}
{"article_id": "PMC3762035", "article_text": ["duodenal ulcer perforation is still a common complication of chronic peptic ulcer disease . despite the wide spread use of anti - secretory and h. pylori eradication therapy", "most of the literature pertaining to the situation in the developed world , showed that the disease is largely confined to the elderly patients taking ulcerogenic medications.[15 ] the situation in the developed nations is summed up by johnson :  the surgeon s major role in the management of peptic ulcer disease will be the performance of life - saving emergency operations in the elderly unfit patient .  in contrast to the developing world , the patients are younger and have a long life - time of potentially useful activity ahead of them[18 ] as anti - secretory and anti - h .", "pylori eradication drugs are been freely used , we had expected an improvement in this disease condition .", "hence , we set out in this review to look at the prevalence , pattern of presentation , risk factors and management outcome in the study environment and we also set out to look at the management pattern if sufficient for a semi - urban area in a developing world .", "this review was conducted at the federal medical centre ido - ekiti , southwest nigeria .", "the case records of all patients with clinically established diagnosis of pdus , managed in this center from january 2004 to december 2011 were reviewed .", "the diagnostic protocol for every patient at presentation included : the clinical findings , abdominal ultrasound , and chest x - rays which in some cases gave further credence to the diagnosis .", "full blood count , urea and electrolytes , creatinine , and urinalysis results were documented .", "the patients were optimized for surgery with intravenous fluids , antibiotics , and urethral catheterization .", "clinical and intra - operative findings , treatment outcome and follow - ups were all evaluated .", "none of the patients had a prior diagnosis of their ulcer by an upper gastro - intestinal endoscopy ( ugie ) before presentation .", "although , 20 ( 66.6% ) of them had peptic ulcer symptoms with , inadequate or no medical treatment .", "the remainder , 10 ( 33.3% ) presented for the first time with perforation , with no prior treatment for peptic ulcer disease .", "the notable risk factor was the free abuse of herbal concoction ( admixture of local gin , spices , roots and bitters ) for body pains .", "fifteen patients took alcohol and only two took non - steroidal anti - inflammatory drugs [ table 1 ] .", "a good proportion of the patients 76.6% were referred to us from private health institutions , while the rest came to our center from their homes .", "after adequate resuscitation , all the 30 patients had emergency surgery of simple closure of their perforations , re - enforced with an omental patch and thorough peritoneal larvage .", "most of the 26 managed patients had a favorable outcome ; were discharged home and followed - up at the clinic for 4 - 6 weeks but later lost to follow - up .", "pdu is still a major complication of chronic peptic ulcer disease seen quite often in the study center , as well as other centers of the world as a frequent cause of acute abdomen .", "although experience from some parts of nigeria showed that gastric outlet obstruction has overtaken pdu in incidence , it is still frequently seen in many centers in nigeria , as well as many centers in the developed world.[17 ] this study has also highlighted that the perforation affected mostly the younger age group ( in their 3 -6 decade ) [ table 2 ] , which is in keeping with studies in many centers in the developing world.[111 ] much has been written about non - operative management of pdu in the western world . since the early works of croft et al . , there has been considerable interests in the non - operative management of pdus . in a randomized trial comparing surgical and non - surgical therapy , for pdu", ", they showed a mortality rate of 5% in each group and no difference in morbidity .", ", have also shown that non - operative therapy could be effective in selected patients .", "they showed that as much as 81% of their patients were treated conservatively , but 43% later had surgery .", "some writers have claimed that virtually all patients with pdu , could be managed conservatively .", "however , conservative management has not been accepted by many workers ; whereas , most surgeons have difficulty in understanding how a patient who has wide spread peritonitis with food debris widely distributed throughout the abdominal cavity can improve without operation .", "undoubtedly , there are patients with small leaks , from a perforation and with relatively mild peritoneal contamination who may be managed conservatively ; these are in a minority .", "we did not consider this treatment option because of the delay in presentation , 2 - 7 days as well as the socio - economic implications of long hospitalization .", "all patients were operated as soon as they were resuscitated and optimized for surgery . in the pre h. pylori era , in the developed world , it has been shown that only 30% of patients with pdu treated with simple closure and thorough peritoneal larvage had good long - term result . in the present era , ( h. pylori era ) , studies have shown , that operation may achieve long - term satisfactory results , only when h. pylori infections , present in 50 - 70% of patients with pdu is totally eliminated .", "however , all have agreed that it is sometimes difficult to determine the h. pylori status of patients having emergent operation for perforated d.u . in this study ,", "none of the patients had an ugie done before presentation and surgery ; and in all of them , their h. pylori status was not known : although , 20 ( 66.6% ) of them were erratically treated at one time or the other for peptic ulcer disease symptoms .", "all patients had simple closure of their perforation with omental patch reinforcement , thorough peritoneal larvage and h. pylori eradication therapy was instituted .", "this is only palliative and is sufficient for the elderly patients in the developed world .", "patients who have suffered one perforation may suffer another one if anti - secretory drugs are not maintained ; as it were , for life .", "this life - long treatment is obviously not within the reach of an average patient in the developing world . in this study ,", "most workers , from the developing world have also shown that pdu , affects mostly the younger age group.[18 ] all have attested to the fact that this group are not likely to be anti eradication drug compliant for life .", "the costs of these drugs , re - infection by h. pylori organisms even after the eradication and dyspeptic symptoms after surgery,[18 ] shows that this palliative surgery ( omental patch ) is obviously not appropriate for a resource poor center , vis - a - vis for the developing world ; that a shift towards the free use of definitive ulcer surgery should be the goal .", "dempsey summed it all ,  using the possible h. pylori infection eradication as an excuse not to do a definitive ulcer operation in any patient with perforated d - u is irrational .", " therefore the various surgical options that have been proposed by workers in the field include : \n omental patch closure + highly selective vagotomyomental patch closure + bilateral truncal vagotomy ( btv)resectional surgery + truncal vagotomypyloroplasty + btv . \n", "dakubo et al . , writing from a west african sub - region had shown statistically that age greater than 60 years , alcohol intake and resectional surgery are key factors in predicting post - operative morbidity and mortality .", "it stands therefore to reason that since the majority of the patients in the study center vis - a - vis developing world are below 60 years , they stand to benefit from a curative definitive ulcer surgery . pyloroplasty ( inco - operating the perforation ) and btv is quite safe and highly recommended because of its short operative time , simplicity and easily reproducible by all , even trainee surgeons .", "omental patch closure + highly selective vagotomy omental patch closure + bilateral truncal vagotomy ( btv ) resectional surgery + truncal vagotomy pyloroplasty + btv .", "pdu disease is still a frequent cause of acute abdomen in many centers of the developing world where it affects mostly the youths .", "the relative high costs of triple regime therapy for h. pylori eradication , fear of re - perforation and easy loss to follow - up , call for an urgent re - appraisal of the present method of surgical treatment .", "therefore , a more liberal role for wide spread definitive ulcer surgery is suggested for most of our young patients ."], "abstract_text": ["<S> introduction : perforated duodenal ulcer ( pdu ) is still seen frequently in the study center inspite of the free use of effective medical curative therapy . </S>", "<S> we then set out to ascertain the pattern of presentation , peculiar risk factors in the study environment , re - evaluate our method of management , and to see if it is adequate for patients in a developing country.materials and methods : this is a retrospective study of patients admitted and managed for pdus , between january 2004 and december 2011 at the federal medical centre , idoekiti , southwest nigeria . </S>", "<S> the records of patients were retrieved and demographic data relating to age , sex , symptoms , duration , diagnosis , intra - operative findings , and management outcome were extracted . </S>", "<S> the results were analyzed.results:a total of 30 patients were admitted and operated during this period . </S>", "<S> twenty - eight of them were males and two were females . </S>", "<S> the mean age was 47 years and the male : female ratio was 14:1 . </S>", "<S> the duration of symptoms before presentation ranged from 2 to 7 days . </S>", "<S> none of the patients had a prior diagnosis of their ulcers , by an upper gastro intestinal endoscopy before presentation ; although most had dyspeptic symptoms , with inadequate or no medical treatment . </S>", "<S> the notable peculiar risk factor was the abuse of local herbal concoction for body pains by all the patients . </S>", "<S> seven patients smokes , 15 consumes alcohol , and only two take non - steroidal anti - inflammatory drugs for body pains . </S>", "<S> most of the managed patients ; 26 were satisfactorily discharged home and later followed - up at the surgical out - patient department . </S>", "<S> four mortality was recorded during the period of study.conclusion:pdu is still a major complication of chronic peptic ulcer disease . </S>", "<S> simple omental patch and h. pylori eradication is no longer appropriate as a mode of treatment for the youths who are mostly affected in the center . </S>", "<S> we therefore , suggest a more wide spread use of definitive ulcer surgery for most of our patients with no pre - operative risk factors . </S>"], "labels": null, "section_names": ["I", "M", "R", "D", "C"], "sections": [["duodenal ulcer perforation is still a common complication of chronic peptic ulcer disease . despite the wide spread use of anti - secretory and h. pylori eradication therapy", "most of the literature pertaining to the situation in the developed world , showed that the disease is largely confined to the elderly patients taking ulcerogenic medications.[15 ] the situation in the developed nations is summed up by johnson :  the surgeon s major role in the management of peptic ulcer disease will be the performance of life - saving emergency operations in the elderly unfit patient .  in contrast to the developing world , the patients are younger and have a long life - time of potentially useful activity ahead of them[18 ] as anti - secretory and anti - h .", "pylori eradication drugs are been freely used , we had expected an improvement in this disease condition .", "hence , we set out in this review to look at the prevalence , pattern of presentation , risk factors and management outcome in the study environment and we also set out to look at the management pattern if sufficient for a semi - urban area in a developing world ."], ["this review was conducted at the federal medical centre ido - ekiti , southwest nigeria .", "the case records of all patients with clinically established diagnosis of pdus , managed in this center from january 2004 to december 2011 were reviewed .", "the diagnostic protocol for every patient at presentation included : the clinical findings , abdominal ultrasound , and chest x - rays which in some cases gave further credence to the diagnosis .", "full blood count , urea and electrolytes , creatinine , and urinalysis results were documented .", "the patients were optimized for surgery with intravenous fluids , antibiotics , and urethral catheterization .", "clinical and intra - operative findings , treatment outcome and follow - ups were all evaluated ."], ["none of the patients had a prior diagnosis of their ulcer by an upper gastro - intestinal endoscopy ( ugie ) before presentation .", "although , 20 ( 66.6% ) of them had peptic ulcer symptoms with , inadequate or no medical treatment .", "the remainder , 10 ( 33.3% ) presented for the first time with perforation , with no prior treatment for peptic ulcer disease .", "the notable risk factor was the free abuse of herbal concoction ( admixture of local gin , spices , roots and bitters ) for body pains .", "fifteen patients took alcohol and only two took non - steroidal anti - inflammatory drugs [ table 1 ] .", "a good proportion of the patients 76.6% were referred to us from private health institutions , while the rest came to our center from their homes .", "after adequate resuscitation , all the 30 patients had emergency surgery of simple closure of their perforations , re - enforced with an omental patch and thorough peritoneal larvage .", "most of the 26 managed patients had a favorable outcome ; were discharged home and followed - up at the clinic for 4 - 6 weeks but later lost to follow - up ."], ["pdu is still a major complication of chronic peptic ulcer disease seen quite often in the study center , as well as other centers of the world as a frequent cause of acute abdomen .", "although experience from some parts of nigeria showed that gastric outlet obstruction has overtaken pdu in incidence , it is still frequently seen in many centers in nigeria , as well as many centers in the developed world.[17 ] this study has also highlighted that the perforation affected mostly the younger age group ( in their 3 -6 decade ) [ table 2 ] , which is in keeping with studies in many centers in the developing world.[111 ] much has been written about non - operative management of pdu in the western world . since the early works of croft et al . , there has been considerable interests in the non - operative management of pdus . in a randomized trial comparing surgical and non - surgical therapy , for pdu", ", they showed a mortality rate of 5% in each group and no difference in morbidity .", ", have also shown that non - operative therapy could be effective in selected patients .", "they showed that as much as 81% of their patients were treated conservatively , but 43% later had surgery .", "some writers have claimed that virtually all patients with pdu , could be managed conservatively .", "however , conservative management has not been accepted by many workers ; whereas , most surgeons have difficulty in understanding how a patient who has wide spread peritonitis with food debris widely distributed throughout the abdominal cavity can improve without operation .", "undoubtedly , there are patients with small leaks , from a perforation and with relatively mild peritoneal contamination who may be managed conservatively ; these are in a minority .", "we did not consider this treatment option because of the delay in presentation , 2 - 7 days as well as the socio - economic implications of long hospitalization .", "all patients were operated as soon as they were resuscitated and optimized for surgery . in the pre h. pylori era , in the developed world , it has been shown that only 30% of patients with pdu treated with simple closure and thorough peritoneal larvage had good long - term result . in the present era , ( h. pylori era ) , studies have shown , that operation may achieve long - term satisfactory results , only when h. pylori infections , present in 50 - 70% of patients with pdu is totally eliminated .", "however , all have agreed that it is sometimes difficult to determine the h. pylori status of patients having emergent operation for perforated d.u . in this study ,", "none of the patients had an ugie done before presentation and surgery ; and in all of them , their h. pylori status was not known : although , 20 ( 66.6% ) of them were erratically treated at one time or the other for peptic ulcer disease symptoms .", "all patients had simple closure of their perforation with omental patch reinforcement , thorough peritoneal larvage and h. pylori eradication therapy was instituted .", "this is only palliative and is sufficient for the elderly patients in the developed world .", "patients who have suffered one perforation may suffer another one if anti - secretory drugs are not maintained ; as it were , for life .", "this life - long treatment is obviously not within the reach of an average patient in the developing world . in this study ,", "most workers , from the developing world have also shown that pdu , affects mostly the younger age group.[18 ] all have attested to the fact that this group are not likely to be anti eradication drug compliant for life .", "the costs of these drugs , re - infection by h. pylori organisms even after the eradication and dyspeptic symptoms after surgery,[18 ] shows that this palliative surgery ( omental patch ) is obviously not appropriate for a resource poor center , vis - a - vis for the developing world ; that a shift towards the free use of definitive ulcer surgery should be the goal .", "dempsey summed it all ,  using the possible h. pylori infection eradication as an excuse not to do a definitive ulcer operation in any patient with perforated d - u is irrational .", " therefore the various surgical options that have been proposed by workers in the field include : \n omental patch closure + highly selective vagotomyomental patch closure + bilateral truncal vagotomy ( btv)resectional surgery + truncal vagotomypyloroplasty + btv . \n", "dakubo et al . , writing from a west african sub - region had shown statistically that age greater than 60 years , alcohol intake and resectional surgery are key factors in predicting post - operative morbidity and mortality .", "it stands therefore to reason that since the majority of the patients in the study center vis - a - vis developing world are below 60 years , they stand to benefit from a curative definitive ulcer surgery . pyloroplasty ( inco - operating the perforation ) and btv is quite safe and highly recommended because of its short operative time , simplicity and easily reproducible by all , even trainee surgeons .", "omental patch closure + highly selective vagotomy omental patch closure + bilateral truncal vagotomy ( btv ) resectional surgery + truncal vagotomy pyloroplasty + btv ."], ["pdu disease is still a frequent cause of acute abdomen in many centers of the developing world where it affects mostly the youths .", "the relative high costs of triple regime therapy for h. pylori eradication , fear of re - perforation and easy loss to follow - up , call for an urgent re - appraisal of the present method of surgical treatment .", "therefore , a more liberal role for wide spread definitive ulcer surgery is suggested for most of our young patients ."]]}
{"article_id": "PMC5138045", "article_text": ["hemophilia b is a chronic hereditary disease characterized by a deficiency in factor ix ( fix ) activity .", "it is less common than hemophilia a which is due to factor viii ( fviii ) deficiency . according to the world federation of hemophilia global survey of 2014 ,", "178,500 persons were identified with hemophilia across 106 countries , of whom only 16.1% had hemophilia b.1 hemophilia b is an x - linked disease .", "missense , frameshift , and nonsense mutations are the most frequently seen , and deletions are rarely described.2 the severity of the disease is related to clotting factor levels .", "the disease is classified as severe , moderate , or mild when the clotting factor levels are < 1 , 15 , or > 5 , respectively.3 people with mild - to - moderate hemophilia rarely bleed unless after trauma or invasive procedures .", "frequency and severity of bleeding is greatest in severe hemophilia b. data from the us show that only 36% of patients with hemophilia b have the severe form of the disease.4 recurrent and spontaneous bleeding into joints and/or soft tissues since early infancy are hallmarks of severity .", "gastrointestinal and intracranial bleeding can be life threatening.3 repeated bleeding into joints without adequate treatment can result in crippling chronic joint disease , pain , and reduced quality of life.3,58 the introduction and availability of factor concentrate has dramatically improved the treatment of hemophilia with a significant decrease in morbidity and mortality , and an increase in quality of life.9 early on - demand treatment of acute bleeding episodes decreases the number of joint deformities compared to untreated or minimally treated patient.10,11 nevertheless , long - term musculoskeletal follow - up of on - demand treatment in hemophilia showed a progressive deterioration of the joint functions in these patients . as on - demand treatment appears to be clearly suboptimal , prophylaxis  meaning preventive use of clotting factor given at regular intervals  was proposed and adopted since several decades in sweden.12 in 1994 , the medical and scientific advisory council ( masac ) of the us national hemophilia foundation issued guidelines stating that prophylaxis with twice- or thrice - weekly fix infusion , at a dose of 2540 iu / kg , should be considered the optimal treatment for hemophilia b.13 the european pediatric network for haemophilia management define primary prophylaxis as regular treatment started after first episode of bleeding and secondary prophylaxis as regular or intermittent regular treatment started after several episodes of bleeding.14,15 the world federation of hemophila ( wfh ) guidelines recommend prophylaxis to prevent bleed and joint destruction and preserve normal musculoskeletal function .", "moreover , it is advisable to give it to all patients before performing any activities associated with an increased risk of trauma .", "prophylaxis should be the state - of - art treatment.3 despite the guidelines , prophylaxis has not been universally adopted .", "medical , psychosocial , and cost controversies limit the implementation of prophylaxis.16 in the canadian 2006 survey , only 32% of patients with severe hemophilia b received prophylaxis , while 69% of patients with hemophilia a received the treatment .", "this difference was particularly notable in the 02 years age - group ( 17% in hemophilia b versus 53% in hemophilia a ) and also present in patients > 18 years of age ( 20% versus 55% ) .", "this difference could be explained by a less severe phenotype , less frequent joint bleeds , later presentation in life , paucity of clinical trials studying the use of prophylaxis , or simply a current treatment tradition.17 moderate hemophilia b is diagnosed much more frequently than the severe ( 37% versus 27% ) form .", "positivity to cross - reacting material , which corresponds to measurable level of fix antigen in plasma , is frequently observed in hemophilia b. this could be explained by the frequency of non - null mutation .", "most mutations in hemophilia b are missense mutations and these result in mild - to - moderate form of the disease and account for variable factor levels in plasma.18 studies reporting prophylactic use of fix in hemophilia b patients are limited in number , and concern mainly small populations within larger clinical trials.1922 benefits of prophylaxis remain questionable even if intuitively the tendency is in favor.23,24 there is currently no evidence to suggest or refute prophylaxis.25 following a 6-month regimen of prophylaxis with recombinant fix ( rix ) , patients achieved significantly meaningful improvements in physical health - related quality of life ( hrqol ) .", "those who switched from intermittent prophylaxis to on - demand prophylaxis experienced improvements in physical and mental hrqol.26 there is no general agreement on the optimal prophylaxis regimen , and some schemes differ from those proposed by the consensus perspectives on prophylactic therapy for hemophilia , held in london , september 2021 , 2002.14 given the paucity of controlled clinical trials regarding prophylaxis in subjects with hemophilia b , choice of treatment approach is limited to clinical judgment , clinical experience , and interpretation of currently available data based on pharmacokinetics ( pk ) , bleeding phenotype , and type of fix used . in recent years , greater", "attention has been paid to the pk of coagulation factor , dosing intervals , and trough levels .", "pk parameters had an increasing role in management choice even if there was no clear evidence on clinical efficacy .", "they are routinely measured in clinical practice to guide treatment dosing , particularly for primary prophylaxis , and in connection with surgery.27 in 2011 , the european medicines agency considered appropriate incremental recovery , half - life , area under the curve ( auc ) , and clearance to be the most important surrogate end points for efficacy of fix products.28 pk parameters of fix are not well characterized or widely investigated .", "the pk of fix are more complicated than those of fviii , and also differ between plasma - derived and recombinant forms resulting in variation between studies .", "fix has a longer half - life in the circulation than fviii.29 according to sampling time , the half - life of fix could be different .", "after administration of 75 iu / kg of nonacog alfa to patients aged 1261 years , the half - life was reported as 2224 hours when sampled at 72 hours,30 and after administration of 50 iu / g , the half - life was reported as 34 hours when sampled at 96 hours.31 a sampling schedule at 48 hours gives shorter half - life . in powell s study , the residual fix observed 1 week after administration of fix is supportive of longer half - life.31 strategy to achieve prophylaxis is to maintain the plasma level of factor activity at or above 1 u / dl.3 in current bio - assays , accuracy can be expected to be rather poor at the conventional target of 1 u / dl , which is the lower limit of the assays.32 this issue with assaying was clearly demonstrated by the discrepancies in factor levels in patients with mild - to - moderate and severe hemophilia in the uk national external quality assessment service data.33 furthermore , measuring plasma fix activity may not fully reflect the hemostatic efficacy of infused fix .", "fix is a small protein ( 55 kda ) with access to both intravascular and extravascular compartments.34 a number of clinical observations in hemophilia b patients suggest the presence of extravascular  pool  of fix .", "continuous infusion of fix results in reduced dose requirement of fix to maintain a level of 100%.35 furthermore , there are experimental data demonstrating that fix binds to collagen iv .", "this may be a source of hemostatically active fix which is not measurable by plasma assays.34,36 experiments on baboons showed that perfusion of excess bovine fix increased the circulating fix proportionally .", "reanalysis of these data suggests that the extravascular component contains at least threefold more fix than that present in the circulation.34 two experiments on mouse demonstrated the affinity of fix to collagen iv and its impact on hemostasis . in a knock - in mouse model expressing a fix variant with reduced affinity to collagen iv , hemostasis was delayed despite high levels of fix .", "bolus infusion of fix variant with enhanced collagen affinity demonstrates a prolonged hemostatic effect in hemophilia b mouse .", "this hemostatic effect persists days after plasma levels reach less than 1%.37 fix ( and fixa ) binds rapidly and reversibly to vascular endothelium and subendothelial extracellular matrix .", "this is mediated by the interaction of specific residues in the fix gla domain with collagen iv , located predominantly in the basement membrane.38,39 the clinical significance of maintaining a 1% trough level is mainly applicable in fviii deficiency .", "such evidence does not exist in fix deficiency and is widely debated.40 in a cohort of 64 patients ( 51 hemophilia a and 13 hemophilia b ) described by ahnstrom et al , some patients did not bleed with a trough level less than 1% , while others bled with a trough level higher than 3% .", "they found that there is no relationship between factor level and incidence of bleed and that the correlation was very weak .", "authors suggest that dosing in prophylactic treatment should be individualized.41 due to interindividual variations in pk parameters , targeting a particular trough level may not be appropriate for every individual.41 relationship between fix trough levels and therapeutic outcomes has not been confirmed in clinical trials.40 baseline factor levels are not the only determinants of bleeding phenotype in hemophilia . with the same factor level", ", the severity and frequency of bleeding may be different for people with hemophilia.42 in hemophilia b , where limited data exist , there is a need to strike a balance between clinical and pk end points in the evaluation of clinical efficacy.43 in hemophilia , bleeding frequency is considered a key indicator of the efficacy of the treatment regimen . the hemophilia severity score ( hss ) has been developed as a method to predict bleeding score in hemophilia.44 it includes in its assessment the annual joint bleeding rate , annual factor consumption , and wfh orthopedic score .", "this score was used by vyas et al to evaluate 178 hemophilia patients without inhibitors in a single us center ( hemophilia a [ n=139 ] , hemophilia b [ n=39 ] ) .", "they found heterogeneity of hemophilia phenotype and widespread variability in the hss values of patients with the same baseline factor activity.45 data from a single - center cohort study of 171 patients with severe hemophilia a and b in the netherlands demonstrated the importance of clinical issues in determining the phenotype .", "they found that age at first joint bleed was an indicator of bleeding pattern.46 there was higher annual clotting factor consumption in subjects who experienced a joint bleed at an early age comparing to those who experienced later in life.47 according to the united kingdom haemophilia centres doctors organisation ( ukhcdo ) annual report , a large variation in rates of clotting factor concentrate consumption in patients with same diagnosis was also widely observed.48 clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment for hemophilia b. the role of pk - guided therapy and its relationship to clinical efficacy remain open issues to be established .", "historically , effective treatment of hemophilia b started with fresh frozen plasma and prothrombin complex concentrate .", "in the early 1990s , highly purified plasma - derived factor concentrate was introduced.49,50 since then , large numbers have become commercially available.51 improvement in sourcing and purification procedure of blood components and the introduction of different steps of viral inactivation increased the safety of plasma - derived concentrates.52 despite these improvements , concerns remain and transfusion - transmitted emerging pathogens like prion and unencapsulated virus should not be overlooked.5356 in 1997 , the first purified rfix product , nonacog alfa , received us food and drug administration ( fda ) approval followed in 2014 by another rfix product , bax326 .", "these rfix products have a lower risk of pathogenic contamination and have low activated fix activity , which confers low thrombogenic potential in humans.2 traditionally , in vivo recovery ( ivr ) is the parameter used to characterize the pk properties of coagulation factors.57 following bolus injection , recombinant protein has lower ivr compared to plasma - derived fix .", "terminal half - life is similar for the two products ( 1719 hours).40,58,59 pk studies that enrolled 308 subjects with hemophilia b in three comparative studies showed that the ivr of rfix is approximately one - third to one - half that of plasma - derived fix.58,60,61 this disparity in ivr was also noted by kisker et al in a double - blind crossover study of plasma - derived fix and rfix in 15 subjects older than 5 years with severe hemophilia b.62 this disparity could be related to baseline fix levels , body weight , and age.58,63 while some individuals have identical recoveries with both types of concentrates , others have pronounced differences.64 a recent survey in italy indicated that plasma - derived fix and rfix are used in similar doses with similar outcomes.65 despite disparity in recovery , recombinant coagulant factors are nowadays the preferred treatment option for hemophiliacs and , in particular , for pediatric patients mainly in light of the wide margin of safety and efficacy .", "wfh  who and nnh propose the twice- or thrice - weekly prophylaxis dose of 50 iu / kg . this regimen was extrapolated from the fviii prophylaxis experience , early pk data , and the few studies done on fix . in the original phase 3 nonacog alfa study in previously treated subjects aged 12 years , the mean dose was 40.3 iu / kg administered 23 times weekly.22 in the phase 3 trial of reformulated nonacog alfa in previously treated subjects , the median dose was 51.7 iu / kg administered 23 times weekly.30 frequency and dose of prophylaxis were determined by the investigator in these two studies . both the studies demonstrated safety and efficacy in persons with hemophilia b. however , rigorous regimens of prophylaxis are difficult and adherence remains a problem.66 only 45% of individuals with severe hemophilia b are currently receiving prophylaxis in the us.67 cost of clotting factor is certainly a main obstacle for the implementation of such management .", "it is the largest predictor of overall cost in the care of people with hemophilia.68,69 however , the leading reasons for lack of adherence to the prescribed regimen are time consumption , convenient access to peripheral vein , forgetfulness , and the feeling of being healthy with the disappearance of symptoms with time.70,71 many patients find it very difficult to spend 1520 minutes every morning to mix and infuse intravenous factor .", "this is certainly more difficult in children where venous access could be difficult and where central venous access devices are associated with the concomitant risk of infection and thrombosis . in an european survey carried out by de moerloose between october 2005 and september 2006 , an interview was conducted with 30 patients in each of six european countries ( france , germany , italy , spain , sweden , and the uk ) , resulting in a total of 180 patients .", "the feeling of reduction , fluctuation , or disappearance of symptoms was cited in 38% of cases , forgetfulness in 36% , lack of time for the treatment in 30% , and convenience in 30% .", "forgetfulness was the reason most often cited by patients on prophylaxis ( 46%).72 therefore , new approaches of management must be proposed to achieve adherence and make patients lives easier . despite recent promising success in gene therapy for hemophilia b ,", "a cure for hemophilia is not yet available.73,74 novel clotting formulations with longer half - life represent a major advance but at a high financial cost .", "it has the potential of increasing convenience to patient , owing to fewer infusions , less preparation and infusion time , and preservation of venous access in those for whom venous access is a challenge.75 there is some evidence to support such a regimen . according to powel s study ,", "residual fix was observed 1 week after administration of fix.31 maintaining a 1% trough level is widely debated in fix deficiency.40 experimental data demonstrate that fix has access to both intravascular and extravascular compartments with potential availability of clinically significant extravascular stores of fix.34 patients with hemophilia b seem to have less severe phenotype and less frequent joint bleed than those with hemophilia a. vyas et al found heterogeneity of hemophilia phenotype and widespread variability in the hss values of 178 patients with the same baseline factor activity.45 in 1976 , morfini et al published the first randomized trial comparing once - weekly and twice - weekly prophylaxis with on - demand treatment in ten subjects with hemophilia b over 1 year.20 a total of 7.5 iu / kg was administered to the twice - weekly group and 15 iu / kg to the once - weekly group .", "the two prophylactic groups had a significant reduction in bleeding episodes when compared to the observed 1-year period preceding the trial ( p<0.005 ) .", "patients having measurable fix in plasma for a higher number of days bleed less.20 in 2014 , valentino et al published a phase 4 multicenter , randomized , open - label , four - period crossover study that evaluated the efficacy and safety of nonacog alfa as a prophylaxis regimen ( 50 iu / kg twice - weekly or 100 iu / kg once - weekly ) compared with on - demand administration.75 inclusion criteria were being male , aged 665 years , with severe - to - moderately severe hemophilia ( fix 2 ) , and 12 or more bleeding episodes , including six or more hemarthroses episodes .", "the primary end point was the annual bleeding rate ( abr ) of two prophylactic regimens compared to on - demand treatment .", "the abr was 35.1 , 2.6 , and 4.6 for the on - demand treatment , 50 iu / kg twice a week , and 100 iu / kg once a week , respectively .", "a significant difference was observed between the two prophylaxis groups and the on - demand group ( p<0.0001 ) with a reduction of bleed of 89.4% in favor of the prophylaxis group .", "no patient discontinued the prophylactic treatment because of inefficacy or less - than - expected effect .", "there was no serious adverse event , or report of thrombogenicity , or occurrence of inhibitors.75 the use of high dose of fix ( 100 iu / kg per dose ) was of concern as it resulted in thrombogenicity . in a review conducted by rendo et al of five clinical trials in patients with hemophilia b , patients who received 100 iu / kg per dose of nonacog", "alfa reported no thrombotic events based on clinical findings and laboratory markers such as thrombin ", "antithrombin ( tat ) and d - dimers.77 this finding was also confirmed in a study conducted by kavakli et al .", "this study evaluated the efficacy and safety of once - weekly prophylaxis with 100 iu / kg nonacog alfa compared with on - demand treatment in 25 adolescent and adult patients for a period of 52 weeks.76 at visits 2 and 4 of the study , assessment of thrombosis was done with monitoring of d - dimers and tat iii complex .", "none of the patients developed clinical thrombotic event and there was no inhibitor occurrence . otherwise , mean abr was significantly lower in the prophylactic group comparing to the on - demand treatment group .", "there was 3.6 bleeding events for the prophylactic group and 32.9 events for the on - demand group ( p<0.0001 ) .", "most interestingly , eight of 17 patients had a fix superior to 2 u / dl 1 week after dosing .", "no severe adverse event was observed and once - weekly prophylaxis was well tolerated.77 the studies of valentino75 and kavakli76 showed efficacy of once - weekly prophylaxis with no unexpected safety issues , no thrombotic event , no occurrence of inhibitors , and significant reduction in bleeding episodes .", "the population size of the studies was small , although this is quite frequent in hemophilia b studies .", "forty - one patients were evaluated in the twice - weekly prophylaxis group of the valantino study and 25 patients in the kavakli study . besides , the study conducted by valantino was of short duration .", "this is relatively a too short period to confirm safety and efficacy of a product .", "the kavakli study was conducted over a 52-week period and supports safety and efficacy of this regimen in a better way .", "otherwise , the two studies were conducted in previously treated patients and 92% of the patients included in the kavakli study had tertiary prophylaxis .", "the benefit of such regimen in untreated patient or in primary prophylaxis or secondary prophylaxis remains to be demonstrated .", "effect of switching from twice - weekly to once - weekly prophylaxis is not yet known .", "in 1994 , the medical and scientific advisory council ( masac ) of the us national hemophilia foundation issued guidelines stating that prophylaxis with twice- or thrice - weekly fix infusion , at a dose of 2540 iu / kg , should be considered the optimal treatment for hemophilia b.13 the european pediatric network for haemophilia management define primary prophylaxis as regular treatment started after first episode of bleeding and secondary prophylaxis as regular or intermittent regular treatment started after several episodes of bleeding.14,15 the world federation of hemophila ( wfh ) guidelines recommend prophylaxis to prevent bleed and joint destruction and preserve normal musculoskeletal function . moreover , it is advisable to give it to all patients before performing any activities associated with an increased risk of trauma .", "prophylaxis should be the state - of - art treatment.3 despite the guidelines , prophylaxis has not been universally adopted .", "medical , psychosocial , and cost controversies limit the implementation of prophylaxis.16 in the canadian 2006 survey , only 32% of patients with severe hemophilia b received prophylaxis , while 69% of patients with hemophilia a received the treatment .", "this difference was particularly notable in the 02 years age - group ( 17% in hemophilia b versus 53% in hemophilia a ) and also present in patients > 18 years of age ( 20% versus 55% ) .", "this difference could be explained by a less severe phenotype , less frequent joint bleeds , later presentation in life , paucity of clinical trials studying the use of prophylaxis , or simply a current treatment tradition.17 moderate hemophilia b is diagnosed much more frequently than the severe ( 37% versus 27% ) form .", "positivity to cross - reacting material , which corresponds to measurable level of fix antigen in plasma , is frequently observed in hemophilia b. this could be explained by the frequency of non - null mutation .", "most mutations in hemophilia b are missense mutations and these result in mild - to - moderate form of the disease and account for variable factor levels in plasma.18 studies reporting prophylactic use of fix in hemophilia b patients are limited in number , and concern mainly small populations within larger clinical trials.1922 benefits of prophylaxis remain questionable even if intuitively the tendency is in favor.23,24 there is currently no evidence to suggest or refute prophylaxis.25 following a 6-month regimen of prophylaxis with recombinant fix ( rix ) , patients achieved significantly meaningful improvements in physical health - related quality of life ( hrqol )", ". those who switched from intermittent prophylaxis to on - demand prophylaxis experienced improvements in physical and mental hrqol.26", "there is no general agreement on the optimal prophylaxis regimen , and some schemes differ from those proposed by the consensus perspectives on prophylactic therapy for hemophilia , held in london , september 2021 , 2002.14 given the paucity of controlled clinical trials regarding prophylaxis in subjects with hemophilia b , choice of treatment approach is limited to clinical judgment , clinical experience , and interpretation of currently available data based on pharmacokinetics ( pk ) , bleeding phenotype , and type of fix used .", "in recent years , greater attention has been paid to the pk of coagulation factor , dosing intervals , and trough levels .", "pk parameters had an increasing role in management choice even if there was no clear evidence on clinical efficacy .", "they are routinely measured in clinical practice to guide treatment dosing , particularly for primary prophylaxis , and in connection with surgery.27 in 2011 , the european medicines agency considered appropriate incremental recovery , half - life , area under the curve ( auc ) , and clearance to be the most important surrogate end points for efficacy of fix products.28 pk parameters of fix are not well characterized or widely investigated .", "the pk of fix are more complicated than those of fviii , and also differ between plasma - derived and recombinant forms resulting in variation between studies .", "fix has a longer half - life in the circulation than fviii.29 according to sampling time , the half - life of fix could be different .", "after administration of 75 iu / kg of nonacog alfa to patients aged 1261 years , the half - life was reported as 2224 hours when sampled at 72 hours,30 and after administration of 50 iu / g , the half - life was reported as 34 hours when sampled at 96 hours.31 a sampling schedule at 48 hours gives shorter half - life . in powell s study , the residual fix observed 1 week after administration of fix is supportive of longer half - life.31 strategy to achieve prophylaxis is to maintain the plasma level of factor activity at or above 1 u / dl.3 in current bio - assays , accuracy can be expected to be rather poor at the conventional target of 1 u / dl , which is the lower limit of the assays.32 this issue with assaying was clearly demonstrated by the discrepancies in factor levels in patients with mild - to - moderate and severe hemophilia in the uk national external quality assessment service data.33 furthermore , measuring plasma fix activity may not fully reflect the hemostatic efficacy of infused fix .", "fix is a small protein ( 55 kda ) with access to both intravascular and extravascular compartments.34 a number of clinical observations in hemophilia b patients suggest the presence of extravascular  pool  of fix .", "continuous infusion of fix results in reduced dose requirement of fix to maintain a level of 100%.35 furthermore , there are experimental data demonstrating that fix binds to collagen iv .", "this may be a source of hemostatically active fix which is not measurable by plasma assays.34,36 experiments on baboons showed that perfusion of excess bovine fix increased the circulating fix proportionally .", "reanalysis of these data suggests that the extravascular component contains at least threefold more fix than that present in the circulation.34 two experiments on mouse demonstrated the affinity of fix to collagen iv and its impact on hemostasis . in a knock - in mouse model expressing a fix variant with reduced affinity to collagen iv , hemostasis was delayed despite high levels of fix .", "bolus infusion of fix variant with enhanced collagen affinity demonstrates a prolonged hemostatic effect in hemophilia b mouse .", "this hemostatic effect persists days after plasma levels reach less than 1%.37 fix ( and fixa ) binds rapidly and reversibly to vascular endothelium and subendothelial extracellular matrix .", "this is mediated by the interaction of specific residues in the fix gla domain with collagen iv , located predominantly in the basement membrane.38,39 the clinical significance of maintaining a 1% trough level is mainly applicable in fviii deficiency .", "such evidence does not exist in fix deficiency and is widely debated.40 in a cohort of 64 patients ( 51 hemophilia a and 13 hemophilia b ) described by ahnstrom et al , some patients did not bleed with a trough level less than 1% , while others bled with a trough level higher than 3% .", "they found that there is no relationship between factor level and incidence of bleed and that the correlation was very weak .", "authors suggest that dosing in prophylactic treatment should be individualized.41 due to interindividual variations in pk parameters , targeting a particular trough level may not be appropriate for every individual.41 relationship between fix trough levels and therapeutic outcomes has not been confirmed in clinical trials.40", "baseline factor levels are not the only determinants of bleeding phenotype in hemophilia . with the same factor level", ", the severity and frequency of bleeding may be different for people with hemophilia.42 in hemophilia b , where limited data exist , there is a need to strike a balance between clinical and pk end points in the evaluation of clinical efficacy.43 in hemophilia , bleeding frequency is considered a key indicator of the efficacy of the treatment regimen . the hemophilia severity score ( hss ) has been developed as a method to predict bleeding score in hemophilia.44 it includes in its assessment the annual joint bleeding rate , annual factor consumption , and wfh orthopedic score .", "this score was used by vyas et al to evaluate 178 hemophilia patients without inhibitors in a single us center ( hemophilia a [ n=139 ] , hemophilia b [ n=39 ] ) .", "they found heterogeneity of hemophilia phenotype and widespread variability in the hss values of patients with the same baseline factor activity.45 data from a single - center cohort study of 171 patients with severe hemophilia a and b in the netherlands demonstrated the importance of clinical issues in determining the phenotype .", "they found that age at first joint bleed was an indicator of bleeding pattern.46 there was higher annual clotting factor consumption in subjects who experienced a joint bleed at an early age comparing to those who experienced later in life.47 according to the united kingdom haemophilia centres doctors organisation ( ukhcdo ) annual report , a large variation in rates of clotting factor concentrate consumption in patients with same diagnosis was also widely observed.48 clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment for hemophilia b. the role of pk - guided therapy and its relationship to clinical efficacy remain open issues to be established .", "historically , effective treatment of hemophilia b started with fresh frozen plasma and prothrombin complex concentrate . in the early 1990s ,", "highly purified plasma - derived factor concentrate was introduced.49,50 since then , large numbers have become commercially available.51 improvement in sourcing and purification procedure of blood components and the introduction of different steps of viral inactivation increased the safety of plasma - derived concentrates.52 despite these improvements , concerns remain and transfusion - transmitted emerging pathogens like prion and unencapsulated virus should not be overlooked.5356 in 1997 , the first purified rfix product , nonacog alfa , received us food and drug administration ( fda ) approval followed in 2014 by another rfix product , bax326 .", "these rfix products have a lower risk of pathogenic contamination and have low activated fix activity , which confers low thrombogenic potential in humans.2 traditionally , in vivo recovery ( ivr ) is the parameter used to characterize the pk properties of coagulation factors.57 following bolus injection , recombinant protein has lower ivr compared to plasma - derived fix .", "terminal half - life is similar for the two products ( 1719 hours).40,58,59 pk studies that enrolled 308 subjects with hemophilia b in three comparative studies showed that the ivr of rfix is approximately one - third to one - half that of plasma - derived fix.58,60,61 this disparity in ivr was also noted by kisker et al in a double - blind crossover study of plasma - derived fix and rfix in 15 subjects older than 5 years with severe hemophilia b.62 this disparity could be related to baseline fix levels , body weight , and age.58,63 while some individuals have identical recoveries with both types of concentrates , others have pronounced differences.64 a recent survey in italy indicated that plasma - derived fix and rfix are used in similar doses with similar outcomes.65 despite disparity in recovery , recombinant coagulant factors are nowadays the preferred treatment option for hemophiliacs and , in particular , for pediatric patients mainly in light of the wide margin of safety and efficacy .", "wfh  who and nnh propose the twice- or thrice - weekly prophylaxis dose of 50 iu / kg . this regimen was extrapolated from the fviii prophylaxis experience , early pk data , and the few studies done on fix . in the original phase 3 nonacog alfa study in previously treated subjects aged 12 years , the mean dose was 40.3 iu / kg administered 23 times weekly.22 in the phase 3 trial of reformulated nonacog alfa in previously treated subjects , the median dose was 51.7 iu / kg administered 23 times weekly.30 frequency and dose of prophylaxis were determined by the investigator in these two studies . both the studies demonstrated safety and efficacy in persons with hemophilia b. however , rigorous regimens of prophylaxis are difficult and adherence remains a problem.66 only 45% of individuals with severe hemophilia b are currently receiving prophylaxis in the us.67 cost of clotting factor is certainly a main obstacle for the implementation of such management .", "it is the largest predictor of overall cost in the care of people with hemophilia.68,69 however , the leading reasons for lack of adherence to the prescribed regimen are time consumption , convenient access to peripheral vein , forgetfulness , and the feeling of being healthy with the disappearance of symptoms with time.70,71 many patients find it very difficult to spend 1520 minutes every morning to mix and infuse intravenous factor .", "this is certainly more difficult in children where venous access could be difficult and where central venous access devices are associated with the concomitant risk of infection and thrombosis . in an european survey carried out by de moerloose between october 2005 and september 2006 ,", "an interview was conducted with 30 patients in each of six european countries ( france , germany , italy , spain , sweden , and the uk ) , resulting in a total of 180 patients .", "the feeling of reduction , fluctuation , or disappearance of symptoms was cited in 38% of cases , forgetfulness in 36% , lack of time for the treatment in 30% , and convenience in 30% .", "forgetfulness was the reason most often cited by patients on prophylaxis ( 46%).72 therefore , new approaches of management must be proposed to achieve adherence and make patients lives easier . despite recent promising success in gene therapy for hemophilia b ,", "a cure for hemophilia is not yet available.73,74 novel clotting formulations with longer half - life represent a major advance but at a high financial cost .", "it has the potential of increasing convenience to patient , owing to fewer infusions , less preparation and infusion time , and preservation of venous access in those for whom venous access is a challenge.75 there is some evidence to support such a regimen .", "according to powel s study , residual fix was observed 1 week after administration of fix.31 maintaining a 1% trough level is widely debated in fix deficiency.40 experimental data demonstrate that fix has access to both intravascular and extravascular compartments with potential availability of clinically significant extravascular stores of fix.34 patients with hemophilia b seem to have less severe phenotype and less frequent joint bleed than those with hemophilia a. vyas et al found heterogeneity of hemophilia phenotype and widespread variability in the hss values of 178 patients with the same baseline factor activity.45 in 1976 , morfini et al published the first randomized trial comparing once - weekly and twice - weekly prophylaxis with on - demand treatment in ten subjects with hemophilia b over 1 year.20 a total of 7.5 iu / kg was administered to the twice - weekly group and 15 iu / kg to the once - weekly group .", "the two prophylactic groups had a significant reduction in bleeding episodes when compared to the observed 1-year period preceding the trial ( p<0.005 ) .", "patients having measurable fix in plasma for a higher number of days bleed less.20 in 2014 , valentino et al published a phase 4 multicenter , randomized , open - label , four - period crossover study that evaluated the efficacy and safety of nonacog alfa as a prophylaxis regimen ( 50 iu / kg twice - weekly or 100 iu / kg once - weekly ) compared with on - demand administration.75 inclusion criteria were being male , aged 665 years , with severe - to - moderately severe hemophilia ( fix 2 ) , and 12 or more bleeding episodes , including six or more hemarthroses episodes .", "the primary end point was the annual bleeding rate ( abr ) of two prophylactic regimens compared to on - demand treatment .", "the abr was 35.1 , 2.6 , and 4.6 for the on - demand treatment , 50 iu / kg twice a week , and 100 iu / kg once a week , respectively .", "a significant difference was observed between the two prophylaxis groups and the on - demand group ( p<0.0001 ) with a reduction of bleed of 89.4% in favor of the prophylaxis group .", "no patient discontinued the prophylactic treatment because of inefficacy or less - than - expected effect .", "there was no serious adverse event , or report of thrombogenicity , or occurrence of inhibitors.75 the use of high dose of fix ( 100 iu / kg per dose ) was of concern as it resulted in thrombogenicity . in a review conducted by rendo et al of five clinical trials in patients with hemophilia b , patients who received 100", "iu / kg per dose of nonacog alfa reported no thrombotic events based on clinical findings and laboratory markers such as thrombin ", "antithrombin ( tat ) and d - dimers.77 this finding was also confirmed in a study conducted by kavakli et al .", "this study evaluated the efficacy and safety of once - weekly prophylaxis with 100 iu / kg nonacog alfa compared with on - demand treatment in 25 adolescent and adult patients for a period of 52 weeks.76 at visits 2 and 4 of the study , assessment of thrombosis was done with monitoring of d - dimers and tat iii complex .", "none of the patients developed clinical thrombotic event and there was no inhibitor occurrence . otherwise , mean abr was significantly lower in the prophylactic group comparing to the on - demand treatment group .", "there was 3.6 bleeding events for the prophylactic group and 32.9 events for the on - demand group ( p<0.0001 ) .", "most interestingly , eight of 17 patients had a fix superior to 2 u / dl 1 week after dosing .", "no severe adverse event was observed and once - weekly prophylaxis was well tolerated.77 the studies of valentino75 and kavakli76 showed efficacy of once - weekly prophylaxis with no unexpected safety issues , no thrombotic event , no occurrence of inhibitors , and significant reduction in bleeding episodes .", "the population size of the studies was small , although this is quite frequent in hemophilia b studies .", "forty - one patients were evaluated in the twice - weekly prophylaxis group of the valantino study and 25 patients in the kavakli study . besides , the study conducted by valantino was of short duration .", "this is relatively a too short period to confirm safety and efficacy of a product .", "the kavakli study was conducted over a 52-week period and supports safety and efficacy of this regimen in a better way .", "otherwise , the two studies were conducted in previously treated patients and 92% of the patients included in the kavakli study had tertiary prophylaxis .", "the benefit of such regimen in untreated patient or in primary prophylaxis or secondary prophylaxis remains to be demonstrated .", "effect of switching from twice - weekly to once - weekly prophylaxis is not yet known .", "the current unmet challenges with regard to management of hemophilia b are financial sustainability , inhibitor occurrence , and poor uptake of prophylaxis .", "prophylactic regimen must be more convenient to patients and their caregivers , while being effective and safe to improve adherence to treatment .", "further research is needed to identify ideal prophylaxis regimens for patients with varying severity of hemophilia b. once - weekly prophylaxis with rix may be a viable treatment option for patients with hemophilia b. prophylaxis regimen at 100 iu / kg may be a safe and effective alternative to twice - weekly prophylaxis at 50 iu / kg .", "however , it remains unknown how patients with a less severe bleeding phenotype or with better baseline joint status might respond to once - weekly dosing with rix . in many regions of the world ,", "fix doses less than the standard 50 iu / kg twice - weekly dose are followed .", "to minimize the cost of treatment and to tailor individual treatment to clinical response , it would be of interest to investigate the effect of once - weekly administration of lower doses of fix ."], "abstract_text": ["<S> regular prophylactic treatment in severe hemophilia should be considered an optimal treatment . </S>", "<S> there is no general agreement on the optimal prophylaxis regimen , and adherence to prophylaxis is a main challenge due to medical , psychosocial , and cost controversies . </S>", "<S> improved approaches in prophylaxis regimen of hemophilia b are needed to make patients lives easier . </S>", "<S> there is some evidence to support the efficacy of once - weekly prophylaxis . </S>", "<S> longer sampling schedules are required for the determination of pharmacokinetic ( pk ) properties of factor ix ( fix ) . </S>", "<S> the half - life of fix seems to be longer than previously described and is expected to be 34 hours . </S>", "<S> the clinical significance of maintaining a 1% trough level is widely debated in hemophilia b. the overall relationship between factor concentrate levels and incidence of joint bleeding was found to be very weak . </S>", "<S> data also indicate that the distribution of fix into an extravascular fix compartment may contribute to hemostasis independently of circulating plasma fix levels . </S>", "<S> clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment . </S>", "<S> role of pk - guided therapy remains to be established . </S>", "<S> two prospective randomized studies had evaluated the efficacy and safety of 100 iu / kg once - weekly prophylaxis with nonacog alfa , and this prophylaxis regimen was found to be associated with lower annual bleeding rate compared with on - demand treatment in adolescents and adults with moderately severe - to - severe hemophilia b. secondary prophylaxis therapy with 100 iu / kg nonacog alfa once weekly reduced annual bleeding rate by 89.4% relative to on - demand treatment . </S>", "<S> residual fix may be supportive of effectiveness . </S>", "<S> once - weekly prophylaxis was well tolerated in the two studies , with a safety profile similar to that reported during the on - demand treatment period . to individually tailor treatment to clinical response and to minimize costs of factor concentrate , </S>", "<S> it would be of interest to investigate the efficacy of lower doses of the drug administered once a week . </S>"], "labels": null, "section_names": ["Introduction", "Prophylaxis", "Prophylaxis regimen", "Impact of pharmacokinetics on FIX prophylaxis regimen", "Impact of hemophilia phenotype on prophylaxis regimen", "Impact of type of FIX used in prophylaxis regimen", "Dose and frequency of prophylaxis regimen", "Conclusion"], "sections": [["hemophilia b is a chronic hereditary disease characterized by a deficiency in factor ix ( fix ) activity .", "it is less common than hemophilia a which is due to factor viii ( fviii ) deficiency . according to the world federation of hemophilia global survey of 2014 ,", "178,500 persons were identified with hemophilia across 106 countries , of whom only 16.1% had hemophilia b.1 hemophilia b is an x - linked disease .", "missense , frameshift , and nonsense mutations are the most frequently seen , and deletions are rarely described.2 the severity of the disease is related to clotting factor levels .", "the disease is classified as severe , moderate , or mild when the clotting factor levels are < 1 , 15 , or > 5 , respectively.3 people with mild - to - moderate hemophilia rarely bleed unless after trauma or invasive procedures .", "frequency and severity of bleeding is greatest in severe hemophilia b. data from the us show that only 36% of patients with hemophilia b have the severe form of the disease.4 recurrent and spontaneous bleeding into joints and/or soft tissues since early infancy are hallmarks of severity .", "gastrointestinal and intracranial bleeding can be life threatening.3 repeated bleeding into joints without adequate treatment can result in crippling chronic joint disease , pain , and reduced quality of life.3,58 the introduction and availability of factor concentrate has dramatically improved the treatment of hemophilia with a significant decrease in morbidity and mortality , and an increase in quality of life.9 early on - demand treatment of acute bleeding episodes decreases the number of joint deformities compared to untreated or minimally treated patient.10,11 nevertheless , long - term musculoskeletal follow - up of on - demand treatment in hemophilia showed a progressive deterioration of the joint functions in these patients . as on - demand treatment appears to be clearly suboptimal , prophylaxis  meaning preventive use of clotting factor given at regular intervals  was proposed and adopted since several decades in sweden.12 in 1994 , the medical and scientific advisory council ( masac ) of the us national hemophilia foundation issued guidelines stating that prophylaxis with twice- or thrice - weekly fix infusion , at a dose of 2540 iu / kg , should be considered the optimal treatment for hemophilia b.13 the european pediatric network for haemophilia management define primary prophylaxis as regular treatment started after first episode of bleeding and secondary prophylaxis as regular or intermittent regular treatment started after several episodes of bleeding.14,15 the world federation of hemophila ( wfh ) guidelines recommend prophylaxis to prevent bleed and joint destruction and preserve normal musculoskeletal function .", "moreover , it is advisable to give it to all patients before performing any activities associated with an increased risk of trauma .", "prophylaxis should be the state - of - art treatment.3 despite the guidelines , prophylaxis has not been universally adopted .", "medical , psychosocial , and cost controversies limit the implementation of prophylaxis.16 in the canadian 2006 survey , only 32% of patients with severe hemophilia b received prophylaxis , while 69% of patients with hemophilia a received the treatment .", "this difference was particularly notable in the 02 years age - group ( 17% in hemophilia b versus 53% in hemophilia a ) and also present in patients > 18 years of age ( 20% versus 55% ) .", "this difference could be explained by a less severe phenotype , less frequent joint bleeds , later presentation in life , paucity of clinical trials studying the use of prophylaxis , or simply a current treatment tradition.17 moderate hemophilia b is diagnosed much more frequently than the severe ( 37% versus 27% ) form .", "positivity to cross - reacting material , which corresponds to measurable level of fix antigen in plasma , is frequently observed in hemophilia b. this could be explained by the frequency of non - null mutation .", "most mutations in hemophilia b are missense mutations and these result in mild - to - moderate form of the disease and account for variable factor levels in plasma.18 studies reporting prophylactic use of fix in hemophilia b patients are limited in number , and concern mainly small populations within larger clinical trials.1922 benefits of prophylaxis remain questionable even if intuitively the tendency is in favor.23,24 there is currently no evidence to suggest or refute prophylaxis.25 following a 6-month regimen of prophylaxis with recombinant fix ( rix ) , patients achieved significantly meaningful improvements in physical health - related quality of life ( hrqol ) .", "those who switched from intermittent prophylaxis to on - demand prophylaxis experienced improvements in physical and mental hrqol.26 there is no general agreement on the optimal prophylaxis regimen , and some schemes differ from those proposed by the consensus perspectives on prophylactic therapy for hemophilia , held in london , september 2021 , 2002.14 given the paucity of controlled clinical trials regarding prophylaxis in subjects with hemophilia b , choice of treatment approach is limited to clinical judgment , clinical experience , and interpretation of currently available data based on pharmacokinetics ( pk ) , bleeding phenotype , and type of fix used . in recent years , greater", "attention has been paid to the pk of coagulation factor , dosing intervals , and trough levels .", "pk parameters had an increasing role in management choice even if there was no clear evidence on clinical efficacy .", "they are routinely measured in clinical practice to guide treatment dosing , particularly for primary prophylaxis , and in connection with surgery.27 in 2011 , the european medicines agency considered appropriate incremental recovery , half - life , area under the curve ( auc ) , and clearance to be the most important surrogate end points for efficacy of fix products.28 pk parameters of fix are not well characterized or widely investigated .", "the pk of fix are more complicated than those of fviii , and also differ between plasma - derived and recombinant forms resulting in variation between studies .", "fix has a longer half - life in the circulation than fviii.29 according to sampling time , the half - life of fix could be different .", "after administration of 75 iu / kg of nonacog alfa to patients aged 1261 years , the half - life was reported as 2224 hours when sampled at 72 hours,30 and after administration of 50 iu / g , the half - life was reported as 34 hours when sampled at 96 hours.31 a sampling schedule at 48 hours gives shorter half - life . in powell s study , the residual fix observed 1 week after administration of fix is supportive of longer half - life.31 strategy to achieve prophylaxis is to maintain the plasma level of factor activity at or above 1 u / dl.3 in current bio - assays , accuracy can be expected to be rather poor at the conventional target of 1 u / dl , which is the lower limit of the assays.32 this issue with assaying was clearly demonstrated by the discrepancies in factor levels in patients with mild - to - moderate and severe hemophilia in the uk national external quality assessment service data.33 furthermore , measuring plasma fix activity may not fully reflect the hemostatic efficacy of infused fix .", "fix is a small protein ( 55 kda ) with access to both intravascular and extravascular compartments.34 a number of clinical observations in hemophilia b patients suggest the presence of extravascular  pool  of fix .", "continuous infusion of fix results in reduced dose requirement of fix to maintain a level of 100%.35 furthermore , there are experimental data demonstrating that fix binds to collagen iv .", "this may be a source of hemostatically active fix which is not measurable by plasma assays.34,36 experiments on baboons showed that perfusion of excess bovine fix increased the circulating fix proportionally .", "reanalysis of these data suggests that the extravascular component contains at least threefold more fix than that present in the circulation.34 two experiments on mouse demonstrated the affinity of fix to collagen iv and its impact on hemostasis . in a knock - in mouse model expressing a fix variant with reduced affinity to collagen iv , hemostasis was delayed despite high levels of fix .", "bolus infusion of fix variant with enhanced collagen affinity demonstrates a prolonged hemostatic effect in hemophilia b mouse .", "this hemostatic effect persists days after plasma levels reach less than 1%.37 fix ( and fixa ) binds rapidly and reversibly to vascular endothelium and subendothelial extracellular matrix .", "this is mediated by the interaction of specific residues in the fix gla domain with collagen iv , located predominantly in the basement membrane.38,39 the clinical significance of maintaining a 1% trough level is mainly applicable in fviii deficiency .", "such evidence does not exist in fix deficiency and is widely debated.40 in a cohort of 64 patients ( 51 hemophilia a and 13 hemophilia b ) described by ahnstrom et al , some patients did not bleed with a trough level less than 1% , while others bled with a trough level higher than 3% .", "they found that there is no relationship between factor level and incidence of bleed and that the correlation was very weak .", "authors suggest that dosing in prophylactic treatment should be individualized.41 due to interindividual variations in pk parameters , targeting a particular trough level may not be appropriate for every individual.41 relationship between fix trough levels and therapeutic outcomes has not been confirmed in clinical trials.40 baseline factor levels are not the only determinants of bleeding phenotype in hemophilia . with the same factor level", ", the severity and frequency of bleeding may be different for people with hemophilia.42 in hemophilia b , where limited data exist , there is a need to strike a balance between clinical and pk end points in the evaluation of clinical efficacy.43 in hemophilia , bleeding frequency is considered a key indicator of the efficacy of the treatment regimen . the hemophilia severity score ( hss ) has been developed as a method to predict bleeding score in hemophilia.44 it includes in its assessment the annual joint bleeding rate , annual factor consumption , and wfh orthopedic score .", "this score was used by vyas et al to evaluate 178 hemophilia patients without inhibitors in a single us center ( hemophilia a [ n=139 ] , hemophilia b [ n=39 ] ) .", "they found heterogeneity of hemophilia phenotype and widespread variability in the hss values of patients with the same baseline factor activity.45 data from a single - center cohort study of 171 patients with severe hemophilia a and b in the netherlands demonstrated the importance of clinical issues in determining the phenotype .", "they found that age at first joint bleed was an indicator of bleeding pattern.46 there was higher annual clotting factor consumption in subjects who experienced a joint bleed at an early age comparing to those who experienced later in life.47 according to the united kingdom haemophilia centres doctors organisation ( ukhcdo ) annual report , a large variation in rates of clotting factor concentrate consumption in patients with same diagnosis was also widely observed.48 clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment for hemophilia b. the role of pk - guided therapy and its relationship to clinical efficacy remain open issues to be established .", "historically , effective treatment of hemophilia b started with fresh frozen plasma and prothrombin complex concentrate .", "in the early 1990s , highly purified plasma - derived factor concentrate was introduced.49,50 since then , large numbers have become commercially available.51 improvement in sourcing and purification procedure of blood components and the introduction of different steps of viral inactivation increased the safety of plasma - derived concentrates.52 despite these improvements , concerns remain and transfusion - transmitted emerging pathogens like prion and unencapsulated virus should not be overlooked.5356 in 1997 , the first purified rfix product , nonacog alfa , received us food and drug administration ( fda ) approval followed in 2014 by another rfix product , bax326 .", "these rfix products have a lower risk of pathogenic contamination and have low activated fix activity , which confers low thrombogenic potential in humans.2 traditionally , in vivo recovery ( ivr ) is the parameter used to characterize the pk properties of coagulation factors.57 following bolus injection , recombinant protein has lower ivr compared to plasma - derived fix .", "terminal half - life is similar for the two products ( 1719 hours).40,58,59 pk studies that enrolled 308 subjects with hemophilia b in three comparative studies showed that the ivr of rfix is approximately one - third to one - half that of plasma - derived fix.58,60,61 this disparity in ivr was also noted by kisker et al in a double - blind crossover study of plasma - derived fix and rfix in 15 subjects older than 5 years with severe hemophilia b.62 this disparity could be related to baseline fix levels , body weight , and age.58,63 while some individuals have identical recoveries with both types of concentrates , others have pronounced differences.64 a recent survey in italy indicated that plasma - derived fix and rfix are used in similar doses with similar outcomes.65 despite disparity in recovery , recombinant coagulant factors are nowadays the preferred treatment option for hemophiliacs and , in particular , for pediatric patients mainly in light of the wide margin of safety and efficacy .", "wfh  who and nnh propose the twice- or thrice - weekly prophylaxis dose of 50 iu / kg . this regimen was extrapolated from the fviii prophylaxis experience , early pk data , and the few studies done on fix . in the original phase 3 nonacog alfa study in previously treated subjects aged 12 years , the mean dose was 40.3 iu / kg administered 23 times weekly.22 in the phase 3 trial of reformulated nonacog alfa in previously treated subjects , the median dose was 51.7 iu / kg administered 23 times weekly.30 frequency and dose of prophylaxis were determined by the investigator in these two studies . both the studies demonstrated safety and efficacy in persons with hemophilia b. however , rigorous regimens of prophylaxis are difficult and adherence remains a problem.66 only 45% of individuals with severe hemophilia b are currently receiving prophylaxis in the us.67 cost of clotting factor is certainly a main obstacle for the implementation of such management .", "it is the largest predictor of overall cost in the care of people with hemophilia.68,69 however , the leading reasons for lack of adherence to the prescribed regimen are time consumption , convenient access to peripheral vein , forgetfulness , and the feeling of being healthy with the disappearance of symptoms with time.70,71 many patients find it very difficult to spend 1520 minutes every morning to mix and infuse intravenous factor .", "this is certainly more difficult in children where venous access could be difficult and where central venous access devices are associated with the concomitant risk of infection and thrombosis . in an european survey carried out by de moerloose between october 2005 and september 2006 , an interview was conducted with 30 patients in each of six european countries ( france , germany , italy , spain , sweden , and the uk ) , resulting in a total of 180 patients .", "the feeling of reduction , fluctuation , or disappearance of symptoms was cited in 38% of cases , forgetfulness in 36% , lack of time for the treatment in 30% , and convenience in 30% .", "forgetfulness was the reason most often cited by patients on prophylaxis ( 46%).72 therefore , new approaches of management must be proposed to achieve adherence and make patients lives easier . despite recent promising success in gene therapy for hemophilia b ,", "a cure for hemophilia is not yet available.73,74 novel clotting formulations with longer half - life represent a major advance but at a high financial cost .", "it has the potential of increasing convenience to patient , owing to fewer infusions , less preparation and infusion time , and preservation of venous access in those for whom venous access is a challenge.75 there is some evidence to support such a regimen . according to powel s study ,", "residual fix was observed 1 week after administration of fix.31 maintaining a 1% trough level is widely debated in fix deficiency.40 experimental data demonstrate that fix has access to both intravascular and extravascular compartments with potential availability of clinically significant extravascular stores of fix.34 patients with hemophilia b seem to have less severe phenotype and less frequent joint bleed than those with hemophilia a. vyas et al found heterogeneity of hemophilia phenotype and widespread variability in the hss values of 178 patients with the same baseline factor activity.45 in 1976 , morfini et al published the first randomized trial comparing once - weekly and twice - weekly prophylaxis with on - demand treatment in ten subjects with hemophilia b over 1 year.20 a total of 7.5 iu / kg was administered to the twice - weekly group and 15 iu / kg to the once - weekly group .", "the two prophylactic groups had a significant reduction in bleeding episodes when compared to the observed 1-year period preceding the trial ( p<0.005 ) .", "patients having measurable fix in plasma for a higher number of days bleed less.20 in 2014 , valentino et al published a phase 4 multicenter , randomized , open - label , four - period crossover study that evaluated the efficacy and safety of nonacog alfa as a prophylaxis regimen ( 50 iu / kg twice - weekly or 100 iu / kg once - weekly ) compared with on - demand administration.75 inclusion criteria were being male , aged 665 years , with severe - to - moderately severe hemophilia ( fix 2 ) , and 12 or more bleeding episodes , including six or more hemarthroses episodes .", "the primary end point was the annual bleeding rate ( abr ) of two prophylactic regimens compared to on - demand treatment .", "the abr was 35.1 , 2.6 , and 4.6 for the on - demand treatment , 50 iu / kg twice a week , and 100 iu / kg once a week , respectively .", "a significant difference was observed between the two prophylaxis groups and the on - demand group ( p<0.0001 ) with a reduction of bleed of 89.4% in favor of the prophylaxis group .", "no patient discontinued the prophylactic treatment because of inefficacy or less - than - expected effect .", "there was no serious adverse event , or report of thrombogenicity , or occurrence of inhibitors.75 the use of high dose of fix ( 100 iu / kg per dose ) was of concern as it resulted in thrombogenicity . in a review conducted by rendo et al of five clinical trials in patients with hemophilia b , patients who received 100 iu / kg per dose of nonacog", "alfa reported no thrombotic events based on clinical findings and laboratory markers such as thrombin ", "antithrombin ( tat ) and d - dimers.77 this finding was also confirmed in a study conducted by kavakli et al .", "this study evaluated the efficacy and safety of once - weekly prophylaxis with 100 iu / kg nonacog alfa compared with on - demand treatment in 25 adolescent and adult patients for a period of 52 weeks.76 at visits 2 and 4 of the study , assessment of thrombosis was done with monitoring of d - dimers and tat iii complex .", "none of the patients developed clinical thrombotic event and there was no inhibitor occurrence . otherwise , mean abr was significantly lower in the prophylactic group comparing to the on - demand treatment group .", "there was 3.6 bleeding events for the prophylactic group and 32.9 events for the on - demand group ( p<0.0001 ) .", "most interestingly , eight of 17 patients had a fix superior to 2 u / dl 1 week after dosing .", "no severe adverse event was observed and once - weekly prophylaxis was well tolerated.77 the studies of valentino75 and kavakli76 showed efficacy of once - weekly prophylaxis with no unexpected safety issues , no thrombotic event , no occurrence of inhibitors , and significant reduction in bleeding episodes .", "the population size of the studies was small , although this is quite frequent in hemophilia b studies .", "forty - one patients were evaluated in the twice - weekly prophylaxis group of the valantino study and 25 patients in the kavakli study . besides , the study conducted by valantino was of short duration .", "this is relatively a too short period to confirm safety and efficacy of a product .", "the kavakli study was conducted over a 52-week period and supports safety and efficacy of this regimen in a better way .", "otherwise , the two studies were conducted in previously treated patients and 92% of the patients included in the kavakli study had tertiary prophylaxis .", "the benefit of such regimen in untreated patient or in primary prophylaxis or secondary prophylaxis remains to be demonstrated .", "effect of switching from twice - weekly to once - weekly prophylaxis is not yet known ."], ["in 1994 , the medical and scientific advisory council ( masac ) of the us national hemophilia foundation issued guidelines stating that prophylaxis with twice- or thrice - weekly fix infusion , at a dose of 2540 iu / kg , should be considered the optimal treatment for hemophilia b.13 the european pediatric network for haemophilia management define primary prophylaxis as regular treatment started after first episode of bleeding and secondary prophylaxis as regular or intermittent regular treatment started after several episodes of bleeding.14,15 the world federation of hemophila ( wfh ) guidelines recommend prophylaxis to prevent bleed and joint destruction and preserve normal musculoskeletal function . moreover , it is advisable to give it to all patients before performing any activities associated with an increased risk of trauma .", "prophylaxis should be the state - of - art treatment.3 despite the guidelines , prophylaxis has not been universally adopted .", "medical , psychosocial , and cost controversies limit the implementation of prophylaxis.16 in the canadian 2006 survey , only 32% of patients with severe hemophilia b received prophylaxis , while 69% of patients with hemophilia a received the treatment .", "this difference was particularly notable in the 02 years age - group ( 17% in hemophilia b versus 53% in hemophilia a ) and also present in patients > 18 years of age ( 20% versus 55% ) .", "this difference could be explained by a less severe phenotype , less frequent joint bleeds , later presentation in life , paucity of clinical trials studying the use of prophylaxis , or simply a current treatment tradition.17 moderate hemophilia b is diagnosed much more frequently than the severe ( 37% versus 27% ) form .", "positivity to cross - reacting material , which corresponds to measurable level of fix antigen in plasma , is frequently observed in hemophilia b. this could be explained by the frequency of non - null mutation .", "most mutations in hemophilia b are missense mutations and these result in mild - to - moderate form of the disease and account for variable factor levels in plasma.18 studies reporting prophylactic use of fix in hemophilia b patients are limited in number , and concern mainly small populations within larger clinical trials.1922 benefits of prophylaxis remain questionable even if intuitively the tendency is in favor.23,24 there is currently no evidence to suggest or refute prophylaxis.25 following a 6-month regimen of prophylaxis with recombinant fix ( rix ) , patients achieved significantly meaningful improvements in physical health - related quality of life ( hrqol )", ". those who switched from intermittent prophylaxis to on - demand prophylaxis experienced improvements in physical and mental hrqol.26"], ["there is no general agreement on the optimal prophylaxis regimen , and some schemes differ from those proposed by the consensus perspectives on prophylactic therapy for hemophilia , held in london , september 2021 , 2002.14 given the paucity of controlled clinical trials regarding prophylaxis in subjects with hemophilia b , choice of treatment approach is limited to clinical judgment , clinical experience , and interpretation of currently available data based on pharmacokinetics ( pk ) , bleeding phenotype , and type of fix used ."], ["in recent years , greater attention has been paid to the pk of coagulation factor , dosing intervals , and trough levels .", "pk parameters had an increasing role in management choice even if there was no clear evidence on clinical efficacy .", "they are routinely measured in clinical practice to guide treatment dosing , particularly for primary prophylaxis , and in connection with surgery.27 in 2011 , the european medicines agency considered appropriate incremental recovery , half - life , area under the curve ( auc ) , and clearance to be the most important surrogate end points for efficacy of fix products.28 pk parameters of fix are not well characterized or widely investigated .", "the pk of fix are more complicated than those of fviii , and also differ between plasma - derived and recombinant forms resulting in variation between studies .", "fix has a longer half - life in the circulation than fviii.29 according to sampling time , the half - life of fix could be different .", "after administration of 75 iu / kg of nonacog alfa to patients aged 1261 years , the half - life was reported as 2224 hours when sampled at 72 hours,30 and after administration of 50 iu / g , the half - life was reported as 34 hours when sampled at 96 hours.31 a sampling schedule at 48 hours gives shorter half - life . in powell s study , the residual fix observed 1 week after administration of fix is supportive of longer half - life.31 strategy to achieve prophylaxis is to maintain the plasma level of factor activity at or above 1 u / dl.3 in current bio - assays , accuracy can be expected to be rather poor at the conventional target of 1 u / dl , which is the lower limit of the assays.32 this issue with assaying was clearly demonstrated by the discrepancies in factor levels in patients with mild - to - moderate and severe hemophilia in the uk national external quality assessment service data.33 furthermore , measuring plasma fix activity may not fully reflect the hemostatic efficacy of infused fix .", "fix is a small protein ( 55 kda ) with access to both intravascular and extravascular compartments.34 a number of clinical observations in hemophilia b patients suggest the presence of extravascular  pool  of fix .", "continuous infusion of fix results in reduced dose requirement of fix to maintain a level of 100%.35 furthermore , there are experimental data demonstrating that fix binds to collagen iv .", "this may be a source of hemostatically active fix which is not measurable by plasma assays.34,36 experiments on baboons showed that perfusion of excess bovine fix increased the circulating fix proportionally .", "reanalysis of these data suggests that the extravascular component contains at least threefold more fix than that present in the circulation.34 two experiments on mouse demonstrated the affinity of fix to collagen iv and its impact on hemostasis . in a knock - in mouse model expressing a fix variant with reduced affinity to collagen iv , hemostasis was delayed despite high levels of fix .", "bolus infusion of fix variant with enhanced collagen affinity demonstrates a prolonged hemostatic effect in hemophilia b mouse .", "this hemostatic effect persists days after plasma levels reach less than 1%.37 fix ( and fixa ) binds rapidly and reversibly to vascular endothelium and subendothelial extracellular matrix .", "this is mediated by the interaction of specific residues in the fix gla domain with collagen iv , located predominantly in the basement membrane.38,39 the clinical significance of maintaining a 1% trough level is mainly applicable in fviii deficiency .", "such evidence does not exist in fix deficiency and is widely debated.40 in a cohort of 64 patients ( 51 hemophilia a and 13 hemophilia b ) described by ahnstrom et al , some patients did not bleed with a trough level less than 1% , while others bled with a trough level higher than 3% .", "they found that there is no relationship between factor level and incidence of bleed and that the correlation was very weak .", "authors suggest that dosing in prophylactic treatment should be individualized.41 due to interindividual variations in pk parameters , targeting a particular trough level may not be appropriate for every individual.41 relationship between fix trough levels and therapeutic outcomes has not been confirmed in clinical trials.40"], ["baseline factor levels are not the only determinants of bleeding phenotype in hemophilia . with the same factor level", ", the severity and frequency of bleeding may be different for people with hemophilia.42 in hemophilia b , where limited data exist , there is a need to strike a balance between clinical and pk end points in the evaluation of clinical efficacy.43 in hemophilia , bleeding frequency is considered a key indicator of the efficacy of the treatment regimen . the hemophilia severity score ( hss ) has been developed as a method to predict bleeding score in hemophilia.44 it includes in its assessment the annual joint bleeding rate , annual factor consumption , and wfh orthopedic score .", "this score was used by vyas et al to evaluate 178 hemophilia patients without inhibitors in a single us center ( hemophilia a [ n=139 ] , hemophilia b [ n=39 ] ) .", "they found heterogeneity of hemophilia phenotype and widespread variability in the hss values of patients with the same baseline factor activity.45 data from a single - center cohort study of 171 patients with severe hemophilia a and b in the netherlands demonstrated the importance of clinical issues in determining the phenotype .", "they found that age at first joint bleed was an indicator of bleeding pattern.46 there was higher annual clotting factor consumption in subjects who experienced a joint bleed at an early age comparing to those who experienced later in life.47 according to the united kingdom haemophilia centres doctors organisation ( ukhcdo ) annual report , a large variation in rates of clotting factor concentrate consumption in patients with same diagnosis was also widely observed.48 clinical assessment of the frequency and severity of bleeds remain an important measure of the efficacy of treatment for hemophilia b. the role of pk - guided therapy and its relationship to clinical efficacy remain open issues to be established ."], ["historically , effective treatment of hemophilia b started with fresh frozen plasma and prothrombin complex concentrate . in the early 1990s ,", "highly purified plasma - derived factor concentrate was introduced.49,50 since then , large numbers have become commercially available.51 improvement in sourcing and purification procedure of blood components and the introduction of different steps of viral inactivation increased the safety of plasma - derived concentrates.52 despite these improvements , concerns remain and transfusion - transmitted emerging pathogens like prion and unencapsulated virus should not be overlooked.5356 in 1997 , the first purified rfix product , nonacog alfa , received us food and drug administration ( fda ) approval followed in 2014 by another rfix product , bax326 .", "these rfix products have a lower risk of pathogenic contamination and have low activated fix activity , which confers low thrombogenic potential in humans.2 traditionally , in vivo recovery ( ivr ) is the parameter used to characterize the pk properties of coagulation factors.57 following bolus injection , recombinant protein has lower ivr compared to plasma - derived fix .", "terminal half - life is similar for the two products ( 1719 hours).40,58,59 pk studies that enrolled 308 subjects with hemophilia b in three comparative studies showed that the ivr of rfix is approximately one - third to one - half that of plasma - derived fix.58,60,61 this disparity in ivr was also noted by kisker et al in a double - blind crossover study of plasma - derived fix and rfix in 15 subjects older than 5 years with severe hemophilia b.62 this disparity could be related to baseline fix levels , body weight , and age.58,63 while some individuals have identical recoveries with both types of concentrates , others have pronounced differences.64 a recent survey in italy indicated that plasma - derived fix and rfix are used in similar doses with similar outcomes.65 despite disparity in recovery , recombinant coagulant factors are nowadays the preferred treatment option for hemophiliacs and , in particular , for pediatric patients mainly in light of the wide margin of safety and efficacy ."], ["wfh  who and nnh propose the twice- or thrice - weekly prophylaxis dose of 50 iu / kg . this regimen was extrapolated from the fviii prophylaxis experience , early pk data , and the few studies done on fix . in the original phase 3 nonacog alfa study in previously treated subjects aged 12 years , the mean dose was 40.3 iu / kg administered 23 times weekly.22 in the phase 3 trial of reformulated nonacog alfa in previously treated subjects , the median dose was 51.7 iu / kg administered 23 times weekly.30 frequency and dose of prophylaxis were determined by the investigator in these two studies . both the studies demonstrated safety and efficacy in persons with hemophilia b. however , rigorous regimens of prophylaxis are difficult and adherence remains a problem.66 only 45% of individuals with severe hemophilia b are currently receiving prophylaxis in the us.67 cost of clotting factor is certainly a main obstacle for the implementation of such management .", "it is the largest predictor of overall cost in the care of people with hemophilia.68,69 however , the leading reasons for lack of adherence to the prescribed regimen are time consumption , convenient access to peripheral vein , forgetfulness , and the feeling of being healthy with the disappearance of symptoms with time.70,71 many patients find it very difficult to spend 1520 minutes every morning to mix and infuse intravenous factor .", "this is certainly more difficult in children where venous access could be difficult and where central venous access devices are associated with the concomitant risk of infection and thrombosis . in an european survey carried out by de moerloose between october 2005 and september 2006 ,", "an interview was conducted with 30 patients in each of six european countries ( france , germany , italy , spain , sweden , and the uk ) , resulting in a total of 180 patients .", "the feeling of reduction , fluctuation , or disappearance of symptoms was cited in 38% of cases , forgetfulness in 36% , lack of time for the treatment in 30% , and convenience in 30% .", "forgetfulness was the reason most often cited by patients on prophylaxis ( 46%).72 therefore , new approaches of management must be proposed to achieve adherence and make patients lives easier . despite recent promising success in gene therapy for hemophilia b ,", "a cure for hemophilia is not yet available.73,74 novel clotting formulations with longer half - life represent a major advance but at a high financial cost .", "it has the potential of increasing convenience to patient , owing to fewer infusions , less preparation and infusion time , and preservation of venous access in those for whom venous access is a challenge.75 there is some evidence to support such a regimen .", "according to powel s study , residual fix was observed 1 week after administration of fix.31 maintaining a 1% trough level is widely debated in fix deficiency.40 experimental data demonstrate that fix has access to both intravascular and extravascular compartments with potential availability of clinically significant extravascular stores of fix.34 patients with hemophilia b seem to have less severe phenotype and less frequent joint bleed than those with hemophilia a. vyas et al found heterogeneity of hemophilia phenotype and widespread variability in the hss values of 178 patients with the same baseline factor activity.45 in 1976 , morfini et al published the first randomized trial comparing once - weekly and twice - weekly prophylaxis with on - demand treatment in ten subjects with hemophilia b over 1 year.20 a total of 7.5 iu / kg was administered to the twice - weekly group and 15 iu / kg to the once - weekly group .", "the two prophylactic groups had a significant reduction in bleeding episodes when compared to the observed 1-year period preceding the trial ( p<0.005 ) .", "patients having measurable fix in plasma for a higher number of days bleed less.20 in 2014 , valentino et al published a phase 4 multicenter , randomized , open - label , four - period crossover study that evaluated the efficacy and safety of nonacog alfa as a prophylaxis regimen ( 50 iu / kg twice - weekly or 100 iu / kg once - weekly ) compared with on - demand administration.75 inclusion criteria were being male , aged 665 years , with severe - to - moderately severe hemophilia ( fix 2 ) , and 12 or more bleeding episodes , including six or more hemarthroses episodes .", "the primary end point was the annual bleeding rate ( abr ) of two prophylactic regimens compared to on - demand treatment .", "the abr was 35.1 , 2.6 , and 4.6 for the on - demand treatment , 50 iu / kg twice a week , and 100 iu / kg once a week , respectively .", "a significant difference was observed between the two prophylaxis groups and the on - demand group ( p<0.0001 ) with a reduction of bleed of 89.4% in favor of the prophylaxis group .", "no patient discontinued the prophylactic treatment because of inefficacy or less - than - expected effect .", "there was no serious adverse event , or report of thrombogenicity , or occurrence of inhibitors.75 the use of high dose of fix ( 100 iu / kg per dose ) was of concern as it resulted in thrombogenicity . in a review conducted by rendo et al of five clinical trials in patients with hemophilia b , patients who received 100", "iu / kg per dose of nonacog alfa reported no thrombotic events based on clinical findings and laboratory markers such as thrombin ", "antithrombin ( tat ) and d - dimers.77 this finding was also confirmed in a study conducted by kavakli et al .", "this study evaluated the efficacy and safety of once - weekly prophylaxis with 100 iu / kg nonacog alfa compared with on - demand treatment in 25 adolescent and adult patients for a period of 52 weeks.76 at visits 2 and 4 of the study , assessment of thrombosis was done with monitoring of d - dimers and tat iii complex .", "none of the patients developed clinical thrombotic event and there was no inhibitor occurrence . otherwise , mean abr was significantly lower in the prophylactic group comparing to the on - demand treatment group .", "there was 3.6 bleeding events for the prophylactic group and 32.9 events for the on - demand group ( p<0.0001 ) .", "most interestingly , eight of 17 patients had a fix superior to 2 u / dl 1 week after dosing .", "no severe adverse event was observed and once - weekly prophylaxis was well tolerated.77 the studies of valentino75 and kavakli76 showed efficacy of once - weekly prophylaxis with no unexpected safety issues , no thrombotic event , no occurrence of inhibitors , and significant reduction in bleeding episodes .", "the population size of the studies was small , although this is quite frequent in hemophilia b studies .", "forty - one patients were evaluated in the twice - weekly prophylaxis group of the valantino study and 25 patients in the kavakli study . besides , the study conducted by valantino was of short duration .", "this is relatively a too short period to confirm safety and efficacy of a product .", "the kavakli study was conducted over a 52-week period and supports safety and efficacy of this regimen in a better way .", "otherwise , the two studies were conducted in previously treated patients and 92% of the patients included in the kavakli study had tertiary prophylaxis .", "the benefit of such regimen in untreated patient or in primary prophylaxis or secondary prophylaxis remains to be demonstrated .", "effect of switching from twice - weekly to once - weekly prophylaxis is not yet known ."], ["the current unmet challenges with regard to management of hemophilia b are financial sustainability , inhibitor occurrence , and poor uptake of prophylaxis .", "prophylactic regimen must be more convenient to patients and their caregivers , while being effective and safe to improve adherence to treatment .", "further research is needed to identify ideal prophylaxis regimens for patients with varying severity of hemophilia b. once - weekly prophylaxis with rix may be a viable treatment option for patients with hemophilia b. prophylaxis regimen at 100 iu / kg may be a safe and effective alternative to twice - weekly prophylaxis at 50 iu / kg .", "however , it remains unknown how patients with a less severe bleeding phenotype or with better baseline joint status might respond to once - weekly dosing with rix . in many regions of the world ,", "fix doses less than the standard 50 iu / kg twice - weekly dose are followed .", "to minimize the cost of treatment and to tailor individual treatment to clinical response , it would be of interest to investigate the effect of once - weekly administration of lower doses of fix ."]]}
{"article_id": "PMC2896657", "article_text": ["protease inhibitors have been purified from an array of leguminous and nonleguminous species encompassing torresea cearensis , erythrina caffra , dolichos lablab , crotalaria paulina , medicago scutellata [ 5 , 6 ] , canavalia gladiata , pisum sativum , dimorphandra mollis , swartzia pickellii , psophocarpus tetragonolobus , delonix regina , poecilanthe parviflora , adenanthera pavonina , cajanus cajan , dolichos biflorus , phaseolus acutifolius , arachis hypogaea , leucaena leucocephala , bauhinia bauhinioides , bauhinia variegata , bauhinia ungulata , vigna unguiculata , lens culinaris , glycine max , peltophorum dubium , pithecellobium dulce , glycine soja , and barley .", "protease inhibitors impair the activity of insect midgut proteases and thus adversely affect protein digestion and health in insects .", "they represent one of the multitude of entomotoxic proteins [ 30 , 31 ] .  ", "in addition to insecticidal activity [ 3235 ] , protease inhibitors demonstrate antiproliferative and antitumor activities [ 3645 ] .", "the objective of the present study was to isolate and characterize proteins with protease inhibitory activity from white cloud beans .", "an aqueous extract of the beans ( 250  g ) was produced by blending in distilled water ( 3  ml / g ) followed by centrifugation ( 14000  g for 25 minutes at 4c ) .  ", "the resulting supernatant was applied to a 5  20  cm column of deae - cellulose ( sigma ) in 10  mm tris - hcl buffer ( ph 7.4 ) .  ", "after elution of unadsorbed proteins ( fraction d1 ) , the column was eluted successively with 0.2  m nacl and 1  m nacl in the tris - hcl buffer .  ", "fraction d2 eluted with 0.2  m nacl was dialyzed to remove nacl and then subjected to affinity chromatography on a 5  15  cm of affi - gel blue gel ( bio - rad ) in 10  mm tris hcl buffer ( ph 7.4 ) .   the unadsorbed fraction ( b1 )", "was dialyzed against 10  mm nh4oac buffer ( ph 5 ) and then applied to a 2.5  20  cm column of sp - sepharose ( ge healthcare ) .  ", "after elution of unadsorbed proteins ( fraction s1 ) , the column was eluted with a 0 - 1  m nacl concentration gradient in the nh4oac buffer .  ", "the first and second adsorbed fractions ( sp2 and sp3 ) were then further purified by gel filtration on a superdex 75  hr 10/30 column ( ge healthcare ) in 0.2  m nh4hco3 buffer ( ph 8.5 ) using an akta purifier ( ge healthcare ) .  ", "the molecular mass of the isolated proteins was determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) following the procedure of laemmli and favre .  ", "gel filtration on an fplc - superdex 75 column , previously calibrated with molecular mass marker proteins ( ge healthcare ) , was employed to determine the molecular mass of the protein .  ", "the n - terminal sequence of the protein was analyzed by using a hewlett - packard hp g1000a edman degradation unit and an hp 1000 hplc system . the test sample (", "20  l ) was added to 160  l of a 1% casein solution in 0.1  m tris - hcl buffer ( ph 7.4 ) .  ", "trypsin ( 20  l of a 0.5  mg / ml solution ) was then added and the mixture was incubated at 37c for 15 minutes followed by addition of trichloroacetic acid ( 0.4  ml , 5% ) that was added to bring the reaction to an end .  ", "after centrifugation the absorbance of the resulting supernatant , which indicates the amount of casein fragments produced by trypsin , was read at 280  nm .  ", "the % inhibition of trypsin activity is equal to the % decrease in absorbance of the supernatant .     ", "the isolated trypsin inhibitor ( 2.5  m ) was treated with dithiothreitol ( dtt ) at the final concentration 2.5 , 10 and 40  mm for 5 , 20 , and 80 minutes at 37c .", "soybean trypsin inhibitor from sigma ( 2.5  m ) was similarly treated and used as a positive control . the reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each dtt concentration .", "residual trypsin inhibitor activity was measured at ph 7.4 as described above in assay for trypsin inhibitory activity .", "the highest iodoacetamide concentration used in the test was devoid of any effect on trypsin activity and the trypsin inhibitory activity of isolated trypsin inhibitor and soybean trypsin inhibitor .", "the cell lines l1210 ( human leukemia ) and mbl2 ( murine lymphoma ) were obtained from american type culture collection .  ", "the cell line was maintained in dulbecco modified eagles ' medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 100  mg / l streptomycin , and 100  iu / ml penicillin , at 37c in a humidified atmosphere of 5%  co2 .  ", "cells ( 1  10 ) in their exponential growth phase were seeded into each well of a 96-well culture plate ( nunc , denmark ) and incubated for 3 hours prior to addition of the trypsin inhibitor .  ", "incubation was performed for an additional 48 hours .   radioactive precursor , 1  ci ( [ h - methyl]-thymidine , from ge healthcare ) , was then introduced to each well and the incubation continued for 6 hours .  ", "the assay for hiv reverse transcriptase inhibitory activity was carried out in view of the report that trypsin inhibitors manifest this activity [ 50 , 51 ] .", "it was conducted according to instructions supplied with the assay kit from boehringer mannheim ( germany ) .", "the assay makes following use of the ability of reverse transcriptase to synthesize dna , commencing from the template / primer hybrid poly ( a ) oligo ( dt ) 15 .  ", "the digoxigenin- and biotin - labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule , which is freshly synthesized by the reverse transcriptase ( rt ) .  ", "the detection and quantification of synthesized dna as a parameter for rt activity are based on a sandwich elisa protocol .  ", "biotin - labeled dna binds to the surface of microtiter plate modules that have been precoated with strepatavidin .   in the following step", ", an antibody to digoxigenin , conjugated to peroxidase , binds to the digoxigenin - labeled dna .   in the last step", "the peroxidase enzyme affects the cleavage of the substrate , yielding a colored reaction product .  ", "the absorbance of the sample at 405  nm which is directly correlated to the level of rt activity can be measured using a microtiter plate ( elisa ) reader .  ", "a fixed amount ( 46  ng ) of recombinant hiv-1 reverse transcriptase was used .  ", "the inhibitory activity of the trypsin inhibitor was calculated as percent inhibition compared to a control without the trypsin inhibitor .", "the plasmid that expressed his - tagged wild - type hiv-1 integrase , pt7 - 7-his ( y | tx)-hiv-1-in , was a generous gift from professor s.a .", "a 1-liter culture of e. coli bl21 ( de3 ) cells containing the expressing plasmid was grown at 37c until it reached od600 0.7 - 0.8 .", "cells were induced by addition of 0.8  mm iptg ( isopropyl--d - thiogalactopyranoside ) and harvested , after 4 hours of incubation , by centrifugation at 6000  g for 10 minutes at 4c .", "cells were suspended at a concentration of 10  ml / g wet cell paste in 20  mm tris - hcl buffer ( ph 8.0 ) containing 0.1  mm edta , 2  mm -mercaptoethanol , 0.5  m nacl , and 5  mm imidazole .", "after incubation at 4c for 1 hour , the lysate was sonicated and centrifuged at 40  000  g at 4c for 20 minutes .", "the pellet was homogenized in 50  ml buffer a ( 20  mm tris - hcl , ph 8.0 , 2  m nacl , 2  mm -mercaptoethanol ) which contained 5  mm imidazole .", "the suspension was rotated at 4c for 1 hour and cleared by centrifugation at 40  000  g at 4c for 20 minutes .", "the supernatant was applied to a 1  ml chelating sepharose ( ge healthcare ) column charged with 50  mm imidazole .", "the column was eluted with five column volumes of buffer a containing 5  mm imidazole , and the protein was eluted with three column volumes of buffer a containing 200 and 400  mm imidazole , respectively .", "protein containing fractions were pooled , and edta was added to a final concentration of 5  mm , followed by dialysis against buffer b ( 20  mm hepes , ph 7.5 , 1  mm edta , 1  m nacl , 20% glycerol ) containing 2  mm -mercaptoethanol and then against buffer b containing 1  mm dithiothreitol .", "a nonradioactive elisa - based hiv-1 integrase assay was carried out according to the dna - coated plate method . in this study", ", 1  g of smal - linearized pbluescript sk was coated onto each well in the presence of 2  m nacl as target dna .", "the donor dna was prepared by annealing vu5br   ( 5-biotin - gtgtggaaaatctcta- gcagt-3 ) and vu5   ( 5-actgctagagattttccacac-3 ) in 10  mm tris - hc1 , ph 8.0 , 1  mm edta , and 0.1  m nacl at 80c , followed by 30 minutes at room temperature .", "integrase reaction was conducted in 20  mm hepes ( ph 7.5 ) containing 10  mm mncl2 , 30  mm nacl , 10  mm dithiothreitol , and 0.05% nonidet - p40 ( sigma ) . after the reaction ,", "biotinylated dna immobilized on the wells was incubated with streptavidin - conjugated alkaline phosphatase ( boehringer - mannheim , mannheim , germany ) , followed by colorimetric detection with 1  mg / ml p - nitrophenyl phosphate in 10% diethanolamine buffer ( ph 9.8 ) containing 0.5  mm mgcl2 .", "the activity of sars coronavirus ( cov ) protease was reflected by a cleavage of designed substrate which is composed of two proteins linked by a cleavage site for sars cov protease .", "the reaction was carried out in a mixture containing 5  m sars cov protease , 5  m sample , and 20  m substrate and buffer [ 20  mm tris - hcl ( ph 7.5 ) , 20  mm nacl and 10  mm beta - mercaptoethanol ] for 40 minutes at 37c . the reaction was then terminated by heating at 100c for 2 minutes .", "then the reaction mixture was analysed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) .", "if sars cov protease is inhibited by the test sample , there is only one band , which is the intact substrate , shown in sds - page .", "the assay for antifungal activity toward botrytis cinerea and fuserium oxysporum was executed in 100  mm  15  mm petri plates containing 10  ml of potato dextrose agar .   after the mycelial colony had grown to a sufficiently large size , sterile blank paper disks ( 0.625  cm in diameter )", "were laid at a distance of 0.5  cm away from the edge of the mycelial colony .   an aliquot ( 15  l ) of the trypsin inhibitor was added to a disk .   the plates were exposed at 23c for 72 hours until mycelial growth had enveloped the disks containing the control and had produced crescents of inhibition around disks containing samples with antifungal activity .", "anion exchange chromatography of the bean extract on deae - cellulose resolved it into three fractions , an unadsorbed fraction d1 together with two adsorbed fractions d2 and d3 .  ", "after affinity chromatography of fraction d2 on affi - gel blue gel , the activity appeared in the unadsorbed fraction b1 ( data not shown ) .  ", "ion exchange chromatography of fraction b1 on sp - sepharose resolved it into a small unadsorbed fraction sp1 and three large adsorbed fractions ( sp2 , sp3 , and sp4 ) of about the same size ( figure 1 ) .", "final purification of sp2 on superdex 75 produced two fractions , sp2su1 and sp2su2 ( figure 2(a ) ) .", "gel filtration of sp3 on superdex 75 yielded two fractions sp3su1 and sp3su2 ( figure 2(b ) ) .", "fractions sp2su2 and sp3su2 , both with a molecular mass of 16  kda as determined by gel filtration on superdex 75 , were the only fractions with trypsin inhibitory activity .", "both sp2su2 and sp3su2 displayed a molecular mass of 16  kda in sds - page ( figure 3 ) and gel filtration ( figure 2 ) .", "these two white cloud bean trypsin inhibitors inhibited trypsin with an ic50 of about 0.6  m ( figure 4 ) .", "dtt treatment curtailed the trypsin inhibiting activity in a dose- and time - dependent manner ( table 3 ) .", "the two trypsin inhibitors did not inhibit hiv-1 reverse transcriptase when tested at various concentrations up to 100  m ( table 4 ) .", "they lacked antifungal activity when tested up to 24  g / disk ( 100  m , 15  l ) ( data not shown ) .  ", "the ic50 values of the inhibitory effects of the trypsin inhibitors on l1210 cells were , respectively , 21.5  m and 28.8  m ( table 4 ) .", "however , there was no activity toward mbl2 cells when tested up to 100  m .  ", "the present study disclosed the production of two trypsin inhibitors with closely related n - terminal sequences chromatographic behavior and bioactivities by the white cloud bean variety of phaseolus vulgaris .  ", "the trypsin inhibitors demonstrate the same molecular mass ; both are adsorbed on deae - cellulose and unadsorbed on affi - gel blue gel but can be separated by ion exchange chromatography on sp - sepharose .  ", "they have approximately the same trypsin inhibitory potency , and neither of them inhibits hiv-1 reverse transcriptase inhibitory activity , hiv-1 integrase inhibitory activity , sars coronavirus proteinase inhibitory activity , or fungal growth .  ", "both inhibitors exhibit an antiproliferative activity toward l1210 cells albeit with a small difference in potency , while there is little activity toward mbl2 cells .  ", "the difference in yields of the two trypsin inhibitors from the white cloud beans is only slight .", "the two trypsin inhibitors exhibit n - terminal sequence homology with those of other leguminous trypsin inhibitors such as inhibitors mungbean ( vigna mungo ) , cowpea ( vigna unguiculata ) , and lima bean ( phaseolus lunatus ) .  ", "whereas leguminous antifungal proteins [ 48 , 49 ] , like nonleguminous antifungal proteins , are unadsorbed on deae - cellulose and adsorbed on affi - gel blue gel , white cloud bean trypsin inhibitors are adsorbed on deae - cellulose and unadsorbed on affi - gel blue gel .  ", "thus the purification procedure adopted in the present investigation can be conveniently used to separate trypsin inhibitors from antifungal proteins .  ", "the antiproliferative activity of white cloud bean trypsin inhibitors is consistent with similar observations on field bean trypsin inhibitor [ 39 , 40 , 42 , 43 , 54 ] .", "it is noteworthy that white cloud bean trypsin inhibitors do not exert a similar action on lymphoma mbl2 cells .  ", "in contrast to broad bean trypsin inhibitor [ 48 , 56 ] , those from white cloud bean do not inhibit hiv-1 reverse transcriptase .  ", "this is reminiscent of the finding that lentil and vigna mungo inhibitors have little or no hiv-1 reverse transcriptase inhibitory and antifungal activities [ 57 , 58 ] . in summary ,", "the isolation of two trypsin inhibitors with very similar biochemical and biological characteristics from white cloud beans was achieved in the present investigation .  ", "white cloud bean trypsin inhibitors demonstrate antiproliferative activity against tumor cells but do no inhibit mycelial growth or hiv-1 reverse transcriptase .  ", "previously isolated p. vulgaris trypsin inhibitors have not been so tested [ 6064 ] .  ", "they reportedly have a molecular mass of about 9  kda [ 63 , 65 ] or 13  kda , smaller than the value of 16  kda obtained for white cloud bean trypsin inhibitors ."], "abstract_text": ["<S> a purification protocol that comprised ion exchange chromatography on deae - cellulose , affinity chromatography on affi - gel blue gel , ion exchange chromatography on sp - sepharose , and gel filtration by fplc on superdex 75 was complied to isolate two trypsin inhibitors from phaseolus vulgaris cv  white cloud bean  . both trypsin inhibitors exhibited a molecular mass of 16  kda and reduced the activity of trypsin with an ic50 value of about 0.6  m . </S>", "<S> dithiothreitol attenuated the trypsin inhibitory activity , signifying that an intact disulfide bond is indispensable to the activity . </S>", "<S> [ methyl-3h ] thymidine incorporation by leukemia l1210 cells was inhibited with an ic50 value of 28.8  m and 21.5  m , respectively . </S>", "<S> they were lacking in activity toward lymphoma mbl2 cells and inhibitory effect on hiv-1 reverse transcriptase and fungal growth when tested up to 100  m . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion and Conclusions"], "sections": [["protease inhibitors have been purified from an array of leguminous and nonleguminous species encompassing torresea cearensis , erythrina caffra , dolichos lablab , crotalaria paulina , medicago scutellata [ 5 , 6 ] , canavalia gladiata , pisum sativum , dimorphandra mollis , swartzia pickellii , psophocarpus tetragonolobus , delonix regina , poecilanthe parviflora , adenanthera pavonina , cajanus cajan , dolichos biflorus , phaseolus acutifolius , arachis hypogaea , leucaena leucocephala , bauhinia bauhinioides , bauhinia variegata , bauhinia ungulata , vigna unguiculata , lens culinaris , glycine max , peltophorum dubium , pithecellobium dulce , glycine soja , and barley .", "protease inhibitors impair the activity of insect midgut proteases and thus adversely affect protein digestion and health in insects .", "they represent one of the multitude of entomotoxic proteins [ 30 , 31 ] .  ", "in addition to insecticidal activity [ 3235 ] , protease inhibitors demonstrate antiproliferative and antitumor activities [ 3645 ] .", "the objective of the present study was to isolate and characterize proteins with protease inhibitory activity from white cloud beans ."], ["an aqueous extract of the beans ( 250  g ) was produced by blending in distilled water ( 3  ml / g ) followed by centrifugation ( 14000  g for 25 minutes at 4c ) .  ", "the resulting supernatant was applied to a 5  20  cm column of deae - cellulose ( sigma ) in 10  mm tris - hcl buffer ( ph 7.4 ) .  ", "after elution of unadsorbed proteins ( fraction d1 ) , the column was eluted successively with 0.2  m nacl and 1  m nacl in the tris - hcl buffer .  ", "fraction d2 eluted with 0.2  m nacl was dialyzed to remove nacl and then subjected to affinity chromatography on a 5  15  cm of affi - gel blue gel ( bio - rad ) in 10  mm tris hcl buffer ( ph 7.4 ) .   the unadsorbed fraction ( b1 )", "was dialyzed against 10  mm nh4oac buffer ( ph 5 ) and then applied to a 2.5  20  cm column of sp - sepharose ( ge healthcare ) .  ", "after elution of unadsorbed proteins ( fraction s1 ) , the column was eluted with a 0 - 1  m nacl concentration gradient in the nh4oac buffer .  ", "the first and second adsorbed fractions ( sp2 and sp3 ) were then further purified by gel filtration on a superdex 75  hr 10/30 column ( ge healthcare ) in 0.2  m nh4hco3 buffer ( ph 8.5 ) using an akta purifier ( ge healthcare ) .  ", "the molecular mass of the isolated proteins was determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) following the procedure of laemmli and favre .  ", "gel filtration on an fplc - superdex 75 column , previously calibrated with molecular mass marker proteins ( ge healthcare ) , was employed to determine the molecular mass of the protein .  ", "the n - terminal sequence of the protein was analyzed by using a hewlett - packard hp g1000a edman degradation unit and an hp 1000 hplc system . the test sample (", "20  l ) was added to 160  l of a 1% casein solution in 0.1  m tris - hcl buffer ( ph 7.4 ) .  ", "trypsin ( 20  l of a 0.5  mg / ml solution ) was then added and the mixture was incubated at 37c for 15 minutes followed by addition of trichloroacetic acid ( 0.4  ml , 5% ) that was added to bring the reaction to an end .  ", "after centrifugation the absorbance of the resulting supernatant , which indicates the amount of casein fragments produced by trypsin , was read at 280  nm .  ", "the % inhibition of trypsin activity is equal to the % decrease in absorbance of the supernatant .     ", "the isolated trypsin inhibitor ( 2.5  m ) was treated with dithiothreitol ( dtt ) at the final concentration 2.5 , 10 and 40  mm for 5 , 20 , and 80 minutes at 37c .", "soybean trypsin inhibitor from sigma ( 2.5  m ) was similarly treated and used as a positive control . the reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each dtt concentration .", "residual trypsin inhibitor activity was measured at ph 7.4 as described above in assay for trypsin inhibitory activity .", "the highest iodoacetamide concentration used in the test was devoid of any effect on trypsin activity and the trypsin inhibitory activity of isolated trypsin inhibitor and soybean trypsin inhibitor .", "the cell lines l1210 ( human leukemia ) and mbl2 ( murine lymphoma ) were obtained from american type culture collection .  ", "the cell line was maintained in dulbecco modified eagles ' medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) , 100  mg / l streptomycin , and 100  iu / ml penicillin , at 37c in a humidified atmosphere of 5%  co2 .  ", "cells ( 1  10 ) in their exponential growth phase were seeded into each well of a 96-well culture plate ( nunc , denmark ) and incubated for 3 hours prior to addition of the trypsin inhibitor .  ", "incubation was performed for an additional 48 hours .   radioactive precursor , 1  ci ( [ h - methyl]-thymidine , from ge healthcare ) , was then introduced to each well and the incubation continued for 6 hours .  ", "the assay for hiv reverse transcriptase inhibitory activity was carried out in view of the report that trypsin inhibitors manifest this activity [ 50 , 51 ] .", "it was conducted according to instructions supplied with the assay kit from boehringer mannheim ( germany ) .", "the assay makes following use of the ability of reverse transcriptase to synthesize dna , commencing from the template / primer hybrid poly ( a ) oligo ( dt ) 15 .  ", "the digoxigenin- and biotin - labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule , which is freshly synthesized by the reverse transcriptase ( rt ) .  ", "the detection and quantification of synthesized dna as a parameter for rt activity are based on a sandwich elisa protocol .  ", "biotin - labeled dna binds to the surface of microtiter plate modules that have been precoated with strepatavidin .   in the following step", ", an antibody to digoxigenin , conjugated to peroxidase , binds to the digoxigenin - labeled dna .   in the last step", "the peroxidase enzyme affects the cleavage of the substrate , yielding a colored reaction product .  ", "the absorbance of the sample at 405  nm which is directly correlated to the level of rt activity can be measured using a microtiter plate ( elisa ) reader .  ", "a fixed amount ( 46  ng ) of recombinant hiv-1 reverse transcriptase was used .  ", "the inhibitory activity of the trypsin inhibitor was calculated as percent inhibition compared to a control without the trypsin inhibitor .", "the plasmid that expressed his - tagged wild - type hiv-1 integrase , pt7 - 7-his ( y | tx)-hiv-1-in , was a generous gift from professor s.a .", "a 1-liter culture of e. coli bl21 ( de3 ) cells containing the expressing plasmid was grown at 37c until it reached od600 0.7 - 0.8 .", "cells were induced by addition of 0.8  mm iptg ( isopropyl--d - thiogalactopyranoside ) and harvested , after 4 hours of incubation , by centrifugation at 6000  g for 10 minutes at 4c .", "cells were suspended at a concentration of 10  ml / g wet cell paste in 20  mm tris - hcl buffer ( ph 8.0 ) containing 0.1  mm edta , 2  mm -mercaptoethanol , 0.5  m nacl , and 5  mm imidazole .", "after incubation at 4c for 1 hour , the lysate was sonicated and centrifuged at 40  000  g at 4c for 20 minutes .", "the pellet was homogenized in 50  ml buffer a ( 20  mm tris - hcl , ph 8.0 , 2  m nacl , 2  mm -mercaptoethanol ) which contained 5  mm imidazole .", "the suspension was rotated at 4c for 1 hour and cleared by centrifugation at 40  000  g at 4c for 20 minutes .", "the supernatant was applied to a 1  ml chelating sepharose ( ge healthcare ) column charged with 50  mm imidazole .", "the column was eluted with five column volumes of buffer a containing 5  mm imidazole , and the protein was eluted with three column volumes of buffer a containing 200 and 400  mm imidazole , respectively .", "protein containing fractions were pooled , and edta was added to a final concentration of 5  mm , followed by dialysis against buffer b ( 20  mm hepes , ph 7.5 , 1  mm edta , 1  m nacl , 20% glycerol ) containing 2  mm -mercaptoethanol and then against buffer b containing 1  mm dithiothreitol .", "a nonradioactive elisa - based hiv-1 integrase assay was carried out according to the dna - coated plate method . in this study", ", 1  g of smal - linearized pbluescript sk was coated onto each well in the presence of 2  m nacl as target dna .", "the donor dna was prepared by annealing vu5br   ( 5-biotin - gtgtggaaaatctcta- gcagt-3 ) and vu5   ( 5-actgctagagattttccacac-3 ) in 10  mm tris - hc1 , ph 8.0 , 1  mm edta , and 0.1  m nacl at 80c , followed by 30 minutes at room temperature .", "integrase reaction was conducted in 20  mm hepes ( ph 7.5 ) containing 10  mm mncl2 , 30  mm nacl , 10  mm dithiothreitol , and 0.05% nonidet - p40 ( sigma ) . after the reaction ,", "biotinylated dna immobilized on the wells was incubated with streptavidin - conjugated alkaline phosphatase ( boehringer - mannheim , mannheim , germany ) , followed by colorimetric detection with 1  mg / ml p - nitrophenyl phosphate in 10% diethanolamine buffer ( ph 9.8 ) containing 0.5  mm mgcl2 .", "the activity of sars coronavirus ( cov ) protease was reflected by a cleavage of designed substrate which is composed of two proteins linked by a cleavage site for sars cov protease .", "the reaction was carried out in a mixture containing 5  m sars cov protease , 5  m sample , and 20  m substrate and buffer [ 20  mm tris - hcl ( ph 7.5 ) , 20  mm nacl and 10  mm beta - mercaptoethanol ] for 40 minutes at 37c . the reaction was then terminated by heating at 100c for 2 minutes .", "then the reaction mixture was analysed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) .", "if sars cov protease is inhibited by the test sample , there is only one band , which is the intact substrate , shown in sds - page .", "the assay for antifungal activity toward botrytis cinerea and fuserium oxysporum was executed in 100  mm  15  mm petri plates containing 10  ml of potato dextrose agar .   after the mycelial colony had grown to a sufficiently large size , sterile blank paper disks ( 0.625  cm in diameter )", "were laid at a distance of 0.5  cm away from the edge of the mycelial colony .   an aliquot ( 15  l ) of the trypsin inhibitor was added to a disk .   the plates were exposed at 23c for 72 hours until mycelial growth had enveloped the disks containing the control and had produced crescents of inhibition around disks containing samples with antifungal activity ."], ["anion exchange chromatography of the bean extract on deae - cellulose resolved it into three fractions , an unadsorbed fraction d1 together with two adsorbed fractions d2 and d3 .  ", "after affinity chromatography of fraction d2 on affi - gel blue gel , the activity appeared in the unadsorbed fraction b1 ( data not shown ) .  ", "ion exchange chromatography of fraction b1 on sp - sepharose resolved it into a small unadsorbed fraction sp1 and three large adsorbed fractions ( sp2 , sp3 , and sp4 ) of about the same size ( figure 1 ) .", "final purification of sp2 on superdex 75 produced two fractions , sp2su1 and sp2su2 ( figure 2(a ) ) .", "gel filtration of sp3 on superdex 75 yielded two fractions sp3su1 and sp3su2 ( figure 2(b ) ) .", "fractions sp2su2 and sp3su2 , both with a molecular mass of 16  kda as determined by gel filtration on superdex 75 , were the only fractions with trypsin inhibitory activity .", "both sp2su2 and sp3su2 displayed a molecular mass of 16  kda in sds - page ( figure 3 ) and gel filtration ( figure 2 ) .", "these two white cloud bean trypsin inhibitors inhibited trypsin with an ic50 of about 0.6  m ( figure 4 ) .", "dtt treatment curtailed the trypsin inhibiting activity in a dose- and time - dependent manner ( table 3 ) .", "the two trypsin inhibitors did not inhibit hiv-1 reverse transcriptase when tested at various concentrations up to 100  m ( table 4 ) .", "they lacked antifungal activity when tested up to 24  g / disk ( 100  m , 15  l ) ( data not shown ) .  ", "the ic50 values of the inhibitory effects of the trypsin inhibitors on l1210 cells were , respectively , 21.5  m and 28.8  m ( table 4 ) .", "however , there was no activity toward mbl2 cells when tested up to 100  m .  "], ["the present study disclosed the production of two trypsin inhibitors with closely related n - terminal sequences chromatographic behavior and bioactivities by the white cloud bean variety of phaseolus vulgaris .  ", "the trypsin inhibitors demonstrate the same molecular mass ; both are adsorbed on deae - cellulose and unadsorbed on affi - gel blue gel but can be separated by ion exchange chromatography on sp - sepharose .  ", "they have approximately the same trypsin inhibitory potency , and neither of them inhibits hiv-1 reverse transcriptase inhibitory activity , hiv-1 integrase inhibitory activity , sars coronavirus proteinase inhibitory activity , or fungal growth .  ", "both inhibitors exhibit an antiproliferative activity toward l1210 cells albeit with a small difference in potency , while there is little activity toward mbl2 cells .  ", "the difference in yields of the two trypsin inhibitors from the white cloud beans is only slight .", "the two trypsin inhibitors exhibit n - terminal sequence homology with those of other leguminous trypsin inhibitors such as inhibitors mungbean ( vigna mungo ) , cowpea ( vigna unguiculata ) , and lima bean ( phaseolus lunatus ) .  ", "whereas leguminous antifungal proteins [ 48 , 49 ] , like nonleguminous antifungal proteins , are unadsorbed on deae - cellulose and adsorbed on affi - gel blue gel , white cloud bean trypsin inhibitors are adsorbed on deae - cellulose and unadsorbed on affi - gel blue gel .  ", "thus the purification procedure adopted in the present investigation can be conveniently used to separate trypsin inhibitors from antifungal proteins .  ", "the antiproliferative activity of white cloud bean trypsin inhibitors is consistent with similar observations on field bean trypsin inhibitor [ 39 , 40 , 42 , 43 , 54 ] .", "it is noteworthy that white cloud bean trypsin inhibitors do not exert a similar action on lymphoma mbl2 cells .  ", "in contrast to broad bean trypsin inhibitor [ 48 , 56 ] , those from white cloud bean do not inhibit hiv-1 reverse transcriptase .  ", "this is reminiscent of the finding that lentil and vigna mungo inhibitors have little or no hiv-1 reverse transcriptase inhibitory and antifungal activities [ 57 , 58 ] . in summary ,", "the isolation of two trypsin inhibitors with very similar biochemical and biological characteristics from white cloud beans was achieved in the present investigation .  ", "white cloud bean trypsin inhibitors demonstrate antiproliferative activity against tumor cells but do no inhibit mycelial growth or hiv-1 reverse transcriptase .  ", "previously isolated p. vulgaris trypsin inhibitors have not been so tested [ 6064 ] .  ", "they reportedly have a molecular mass of about 9  kda [ 63 , 65 ] or 13  kda , smaller than the value of 16  kda obtained for white cloud bean trypsin inhibitors ."]]}
{"article_id": "PMC4622453", "article_text": ["although about two - thirds of patients with epilepsy ( pwe ) treated with antiepileptic drugs ( aeds ) reach seizure - freedom , about one - third remains drug - resistant to the current therapies.1 despite the introduction of new aeds with a better pharmacokinetic and safety profile compared to old generation aeds , today , one of the major causes of failure of antiepileptic treatment is poor adherence often due to occurrence of adverse drug reactions ( adrs ) , leading up to 25% of patients to discontinue treatment before the achievement of effective doses and with a consequent increase of health care costs.2,3 during the last 25 years , many efforts have been directed to the development of new aeds with different mechanisms of action able to reduce brain hyperexcitability ; recently considerable interest has been focused on synaptic vesicle protein 2a ( sv2a ) and its role as a target for aeds.4 the first drug of this new class approved for epilepsy is levetiracetam ( lev ) , and from this lead compound , several racetam analogs have been synthesized.5 based on target - drug program , brivaracetam ( brv ) ( 2s)-2-[(4r)-2-oxo-4-propylpyrro - lidinyl]butanamide , an n - propyl analog of lev has been identified and has entered clinical trials for pwe;6 it is an sv2a ligand with high selectivity and a 1030-fold higher potency , depending on the experimental conditions , when compared to lev.7,8 the purpose of this review is to present updated data available on the pharmacology , efficacy , and tolerability of brv .", "we have conducted a systematic search in the pubmed and cochrane library databases up to august 14 , 2015 summarizing all relevant data for efficacy , safety , and tolerability of brv in the treatment of partial - onset seizure .", "although the correlation between sv2a binding and anti - convulsant potency in animal models has been previously demonstrated , the role of sv2a in neurotransmission still remains unclear.9 in fact , even if it has been established that sv2a protein is involved in normal synaptic vesicle function , the exact mechanism of synaptic vesicle cycling regulation and neurotransmitter release remains unknown.5,9 several preclinical studies have demonstrated that brv is more potent and efficacious than lev in animal models of both focal and generalized epilepsy.10 in particular , in fully amygdala - kindled rats , brv induced a significant suppression in motor - seizure severity from a dose of 21.2 mg / kg , whereas lev caused a similar effect from a dose of 170 mg / kg .", "brv also significantly reduced the after - discharge duration at the highest dose tested ( 212.3 mg / kg ) , whereas lev was inactive on this parameter up to 1,700 mg / kg.10 moreover , brv protected significantly against both partial and generalized seizures in fully amygdala - kindled mice resistant to phenytoin ( effective dose 50 [ ed50 ] : 68.3 mg / kg , intraperitoneal [ ip]).10 recently , it has been demonstrated that brv has a higher brain permeability than lev , with consequently more rapid onset of action after acute dosing in audiogenic mice ; this fast onset of action might also have a potential clinical relevance for the treatment of status epilepticus or cluster seizures.11 another study confirmed the protective activity of brv against kindled seizures with focal seizure threshold and significantly severity significantly modified.12 furthermore , a recent study , using a rapid kindling model in p14 , p21 , p28 , and p60 rats , has evaluated two doses of brv 10 and 100 mg / kg , demonstrating that brv 100 mg / kg significantly increased the after - discharge threshold at all ages , whereas brv 10 mg / kg increased after - discharge threshold in p60 , p28 , and p21 rats .", "brv also reduced the after - discharge duration , achieving statistical significance with 10 and 100 mg / kg at p60 and with 100 mg / kg , at p21 .", "at p60 , brv increases the number of stimulations required to reach stage 45 seizures in a dose - dependent manner . at p28 and p21 , brv increased the number of stimulations required to develop stage 45 seizures in a dose - dependent manner , with almost complete elimination of stage 45 seizures.13 moreover , brv showed a marked synergism with diazepam to reduce seizure duration in self - sustained status epilepticus induced by stimulation of the perforant path.14 a recent study has evaluated the antiseizure and antimyoclonic activities of brv in comparison to lev in an animal model of posthypoxic myoclonus , showing higher efficacy of brv ( 0.3 mg / kg ) than lev ( 3 mg / kg ) against posthypoxic seizures.15 furthermore , recent experiments conducted in transgenic mice with alzheimer s disease supported an adjunctive and peculiar role of brv that not only revealed an efficacy against spike - wave discharges similar to ethosuximide , but it showed the ability to reverse memory impairment , thus extending the potential spectrum of action of this new aed.16 in addition to sv2a block , brv also exhibits inhibitory activity on neuronal voltage - gated sodium channels ( vgsc ) playing a role as a partial antagonist , as has been reported for other aeds.17,18 in particular , experimental studies on primary cortical cultures have demonstrated that brv is able to prolong the sodium channel time recovery from fast inactivation , and this effect could reduce the availability of sodium channel during high - frequency repetitive firing.17,19 even if this data has been refuted by another recent experimental study that has showed that this vgsc inhibition does not impair sustained repetitive firing in neurons of neuroblastoma cells,20 this is an important aspect , which deserves to be better clarified , since the lack of effect of brv to reduce neuronal excitability by blocking high repetitive firing in neurons might exclude that the modulation of vgsc contributes to antiseizure effects of brv .", "brv presents a favorable pharmacokinetic profile , linear and predictable , with low intersubject variability and almost 100% bioavailability.2123 the pharmacokinetic properties of brv have been studied in healthy adult volunteers , in the elderly , in patients with pwe , and in those with hepatic or renal impairment.2225 the pharmacokinetic differences in elderly subjects compared to healthy volunteers are not so important as to require any dose adjustment.25 absorption of brv is unaffected by the presence of food , including high fat meals ; after oral administration , brv is rapidly absorbed by the gastrointestinal tract , and displays linear and dose - proportional profile over the dose range tested.22,26 its distribution volume is close to total body water ( vz=0.5 l / kg ) , and it binds weakly to plasma proteins ( 17.5% ) .", "its terminal half - life is ~9 hours.21 saliva and plasma brv levels are highly correlated , brv crosses the mucosa by passive diffusion , therefore , the saliva concentration of brv is correlated with plasma concentration .", "it is possible to speculate that saliva might be a suitable sample for monitoring brv levels when blood sampling could be a limiting factor.23 brv is extensively metabolized through several metabolic pathways and is fully excreted by urine ( only 8%11% remains unchanged ) .", "brv is eliminated primarily by metabolism , with the major metabolic pathway involving hydrolysis of the acetamide group resulting in formation of an acid metabolite ( brv - ac ; 34.2% of a radiolabeled dose in urine).21 a secondary pathway , mainly mediated by cytochrome p450 ( cyp ) 2c19,27 forms a hydroxy metabolite ( brv - oh ; 15.9% of dose in urine).21 a combination of these two pathways leads to the formation of a hydroxyacid metabolite ( 15.2% of dose in urine);21,28 only 8.6% of the dose is recovered as the unchanged compound in urine.21 all three metabolites of brv are pharmacologically inactive ( unpublished results ) in an open - label study conducted in patients with liver disease , the plasma half - life of brv was prolonged up to 17.4 hours in correlation with the severity of hepatic impairment ; however , the exposure to brv is increased by 50%60% in patients with hepatic impairment , irrespective of severity classified by child ", "pugh score.25,26 these data suggest that the maximum daily dose of brv might be reduced by one - third in patients with hepatic impairment.25 in severe renal impairment , the exposure to a single oral dose of 200 mg brv not requiring dialysis ( creatinine clearance < 15 ml / min ) , and renal clearance of three metabolites ( acid , hydroxy , and hydroxyacid ) , was decreased 10-fold in patients with severe renal impairment.24 nevertheless , there are data showing a toxicological coverage for metabolites , without the evidence of any safety issues ( ucb data file ) . based on these observations , a dose adjustment for brv should not be required at any stage of renal dysfunction .", "the efficacy of brv as add - on therapy in patients with uncontrolled partial seizures has been assessed in six randomized placebo - controlled clinical trials ( table 1).29 in the first two studies , brv as adjunctive therapy in adult patients with partial epilepsy and poor control with 12 concomitant aeds , at different doses ( 5 , 20 , 50 , and 50150 mg / d ) has been evaluated.30,31 french et al30 reported a statistically significant reduction of seizure frequency achieved at the 50 mg / d dose , with high tolerability and infrequent adrs . in particular , the percentage reduction over placebo in focal seizure frequency / week was directly correlated to brv dose , respectively 9.8% at 5 mg / d , 14.9% at 20 mg / d , and 22.1% at 50 mg / d , with a median percent reduction from baseline in seizure frequency / week of 21.7% ( placebo ) , 29.9% ( brv5 ) , 42.6% ( brv20 ) , and 53.1% ( brv50 ) ; 50% responder rates were 16.7% ( placebo ) , 32.0% ( brv5 ) , 44.2% ( brv20 ) , and 55.8% ( brv50 ) ; seizure freedom rates during the 7-week treatment period were 1.9% ( placebo ) , 8.0% ( brv5 ) , 7.7% ( brv20 ) , and 7.7% ( brv50 ) . on the other hand , higher doses of brv would not seem to be more effective , in fact , van paesschen et al31 did not find significant differences in seizure frequency reduction between brv 50 and 150 mg / d during the 7-week maintenance period .", "in particular , the median seizure frequency / week was 1.00 , 1.96 , and 1.86 in the group treated with brv 50 mg / d , brv 150 mg / d , and placebo , respectively.31 the reduction in baseline - adjusted seizure frequency / week over placebo during the maintenance period was 14.7% in the brv 50 mg / d group and was 13.6% in the brv 150 mg / d group ; however , a significant difference over placebo was observed on several secondary efficacy outcomes ( 10 weeks combined up - titration and maintenance period).31 in fact , after the 10-week treatment period , the median seizure frequency / week was 1.10 , 2.05 , and 1.95 in the brv 50 mg / d , brv 150 mg / d , and placebo groups , respectively . in the maintenance period ,", "50% responder rates were 23.1% for placebo compared with 39.6% for brv50 and 33.3% for brv150 . during the treatment period , 50% responder rates were 17.3% for placebo compared with 35.8% for brv50 and 30.8% for brv150 .", "nine patients were seizure - free during the 10-week treatment period in the brv50 group ( three in the brv150 group , only one in the placebo group ) . in the study by ryvlin et al,32", "the efficacy and safety / tolerability of brv ( at doses of 20 , 50 , and 100 mg / d ) in patients with uncontrolled partial seizures with / without secondary generalization , despite treatment with one or two concomitant aeds , was investigated .", "the percent reduction over placebo in baseline - adjusted seizure frequency / week was 6.8% , 6.5% , and 11.7% in the brv 20 , 50 , and 100 mg / d groups , respectively .", "the percent reduction over placebo in baseline - adjusted seizure frequency/28 days was 9.2% and 20.5% in the brv 50 and 100 mg / d groups , respectively .", "median percent reductions from baseline were 30.0% for brv 20 mg / d , 26.8% for brv 50 mg / d , and 32.5% for brv 100 mg / d in comparison to 17.0% for placebo .", "responder rates ( 50% ) were 27.3% , 27.3% , and 36.0% for brv 20 , 50 , and 100 mg / d , respectively , in comparison to 20.0% for placebo .", "based on these results , only brv 100 mg / d was able to significantly reduce seizure frequency / week over placebo .", "indeed , in their randomized placebo - controlled trial , adjunctive brv at a daily dose of 50 mg significantly decreased seizure frequency , while lower dosages ( 5 and 20 mg / d ) did not achieve significant differences.33 in more detail , the percent reduction in partial - onset seizure frequency / week in comparison to placebo was 0.9% ( p=0.885 ) for brv 5 mg / d , 4.1% ( p=0.492 ) for brv 20 mg / d , and 12.8% ( p=0.025 ) for brv 50 mg / d ; in the brv 50 mg / d group , statistical significance was also observed for the 50% responder rate ( brv 32.7% vs placebo 16.7% ) and median percent reduction from baseline in focal seizure frequency / week ( brv 30.5% vs placebo 17.8% ) .", "recently a randomized , multicenter , double - blind phase iii trial was conducted by klein et al34 to evaluate the efficacy and the safety profile of brv at fixed doses of 100200 mg / d in adult patients with refractory partial onset seizures .", "responder rate was 21.6% for placebo group , 38.9% for brv 100 mg / d , and 37.8% for brv 200 mg / d ; the percent reduction of partial onset seizures in 28-day frequency was 22.8% for brv 100 mg and 23.2% for brv 200 mg .", "kwan et al35 conducted a double - blind , randomized , placebo - controlled trial investigating the safety and tolerability profile of adjunctive brv ( at individualized tailored doses ranging from 20 to 150 mg / d ) in patients with partial or generalized refractory epilepsies .", "the percent of reduction of focal seizure frequency / week in the brv group in comparison to placebo was 7.3% , while only in the generalized seizures group , the number of seizure days / week decreased from 1.42 at baseline to 0.63 during the treatment period in brv - treated patients ( n=36 ) , and from 1.47 at baseline to 1.26 during the treatment period in the placebo group ( n=13).35 the median percent reduction in baseline - adjusted seizure frequency / week was 26.9% brv vs 18.9% placebo , and the 50% responder rate was 30.3% brv vs 16.7% placebo .", "the median percent reduction from baseline in generalized seizure days / week was 42.6% vs 20.7% , and the 50% responder rate was 44.4% vs 15.4% in brv - treated and placebo - treated patients , respectively .", "similar to lev , brv might become a useful aed for the treatment of myoclonic seizures occurring in the setting of idiopathic generalized epilepsies ( eg , juvenile myoclonic epilepsy)36 or of progressive myoclonic epilepsies.37 two randomized , placebo - controlled trials evaluating efficacy and safety of adjunctive brv ( 5150 mg / d ) in unverricht ", "lundborg disease , the most common and less severe form of progressive myoclonic epilepsies,38 failed to show a significant improvement of myoclonus in these patients.39 however , sample size was small ( 106 patients randomized in the two trials ) , and the patients were allowed to receive lev.39 moreover , it is well known that myoclonus may present high inter- and intrapatients variability ( with patients experiencing  good days  and  bad days  ) in unverricht ", "currently an open - label , multicenter , follow - up study to evaluate the long - term safety and efficacy of brv is ongoing ( brite study - nct01339559 ) .", "finally , to date , in all studies performed , brv was evaluated as an oral tablet formulation , and no data are available about intravenous infusion since the study nct02088957 , aiming at a comparison of the efficacy and safety of intravenous brv vs phenytoin in adult subjects with nonconvulsive electrographic seizures , was terminated for low enrollment .", "the most commonly reported adverse effects with brv in adults were primarily related to the central nervous system and included somnolence , fatigue , and dizziness.22 these adverse effects were mild to moderate , and the tolerability profile is so excellent that it did not impair therapeutic compliance . in fact , the daily dose of brv ( 20150 mg ) was well tolerated and associated with 6.1% of discontinuation rates due to adrs compared to 5.0% of the placebo group.35 furthermore , adrs induced by brv seem to be time - related , disappearing during the course of treatment .", "the entity of sedative effects of brv measured by psychometric tests is dose - related in healthy men and appeared clearly from 600 mg upwards as a decrease in attention , motor control , and alertness.22 moreover , the type and the severity of adrs are not influenced by food.22 as demonstrated in healthy males , a twice - daily dosing regimen could be a good clinical practice to reduce blood fluctuations and peak , which might influence the appearance of adverse events.22 no effects on cardiac function were reported even at very high daily dosages ( up to 800 mg / d).40 no data about fertility and/or potential teratogenic effect of brv in humans are currently available ; however , no adverse effects were detected up to the highest tested oral dose of 400 mg / kg / d on fertility , and no effects on pregnancy or fetal development at 600 mg / d were observed in animals.26 seizure aggravation or the appearance of new generalized seizures was rare : three studies reported this adverse event , occurring in similar proportions between placebo and treated group ( 4.3% vs 5.2% , p=0.67).32,33,35 in the above - reported five randomized clinical trials , 1,639 subjects were included in an intention - to - treat analysis ( 1,214 treated with brv and 425 with placebo).3035 no differences were observed in the proportion of subjects experiencing at least one adverse event ( 65.5% with brv vs 60.5% with placebo , p=0.10 ) .", "most events were mild to moderate ; actually , comprehensive withdrawal rate due to adverse events was quite low and similar in brv and placebo arms ( 5.4% with brv vs 4.2% with placebo , p=0.37 ) .", "serious adverse events were quite rare and equally distributed ( 2.9% with brv vs 4.4% with placebo , p=0.16 ) .", "adverse events that were observed in at least 5% of subjects in either group are listed in table 2 .", "the proportion of patients reporting fatigue and somnolence was significantly higher in brv group compared to placebo ( table 2 ) .", "irritability was reported in three studies only , and it was present in a small proportion of subjects ( 3% receiving brv , 1% receiving placebo , p=0.36 ) .", "because of its advantageous pharmacokinetic profile , brv treatment does not appear to influence plasma concentrations of other aeds such as carbamazepine , lamotrigine , lev , oxcarbazepine , topiramate , or valproic acid.41 however , carbamazepine plasma levels are slightly reduced by coadministration of brv ( 400 mg / d ) , while levels of carbamazepine - epoxide are increased in a dose - dependent manner.26,42 this increase of plasma concentration of carbamazepine - epoxide is the result of inhibition by brv of epoxide hydrolase that metabolizes carbamazepine - epoxide into carbamazepine - diol.42 high doses of brv ( 400 mg / d ) cause a moderate decrease of ethinylestradiol and levonorgestrel plasma levels ( components of oral contraceptives ) but this posological range has no impact on ovulation .", "no effect on contraceptive disposition is reported at therapeutic doses of brv 100 mg / d.39 interestingly , there is a possible negative interaction between brv and lev , in fact , the concomitant use of both drugs may reduce brv efficacy ; however , this evidence is not robust because the number of patients with concomitant lev was very small , other studies may be useful to assess this apparent pharmacodynamic interaction.33", "brv is a novel aed whose efficacy in partial epilepsies has been studied and established in five randomized controlled trials;3035 furthermore , two recent meta - analyses have confirmed significant effects for brv in patients with refractory partial seizures.29,43 considering that brv shares part of its mechanism of action with lev and that its ability to inhibit vgsc ( still debated ) is in common with several other aeds , it will be very intriguing to see how this drug will behave in real - life clinical practice .", "accordingly , it could be hypothesized that brv might possess at least the same effectiveness as lev .", "based on this hypothesis , brv may be reasonably considered as a valuable add - on aed in patients with partial seizures , also considering its suggested good tolerability .", "however , specific studies are needed to confirm its efficacy in specific epileptic syndromes , for example , a decreased expression of sv2a in the hippocampus of patients with temporal lobe epilepsy with hippocampal sclerosis has been documented and might represent a pharmacoresistance mechanism in some cases ; however , lev has been reported to be effective.44,45 in addition , because of its good safety and pharmacokinetic profile , brv might be ideal for use in monotherapy , as previously demonstrated for lev.46 finally , few studies have been performed , and more randomized double - blind studies are needed to confirm these considerations and to demonstrate if brv might really confirm its promises and become a new tool for epileptologists ."], "abstract_text": ["<S> brivaracetam ( brv ) , a high - affinity synaptic vesicle protein 2a ligand , reported to be 1030-fold more potent than levetiracetam ( lev ) , is highly effective in a wide range of experimental models of focal and generalized seizures . </S>", "<S> brv and lev similarly bind to synaptic vesicle protein 2a , while differentiating for other pharmacological effects ; in fact , brv does not inhibit high voltage ca2 + channels and ampa receptors as lev . </S>", "<S> furthermore , brv apparently exhibits inhibitory activity on neuronal voltage - gated sodium channels playing a role as a partial antagonist . </S>", "<S> brv is currently waiting for approval both in the united states and the european union as adjunctive therapy for patients with partial seizures . in patients with photosensitive epilepsy , </S>", "<S> brv showed a dose - dependent effect in suppressing or attenuating the photoparoxysmal response . in well - controlled trials conducted to date , adjunctive brv demonstrated efficacy and good tolerability in patients with focal epilepsy </S>", "<S> . </S>", "<S> brv has a linear pharmacokinetic profile . </S>", "<S> brv is extensively metabolized and excreted by urine ( only 8%11% unchanged ) . </S>", "<S> the metabolites of brv are inactive , and hydrolysis of the acetamide group is the mainly involved metabolic pathway ; hepatic impairment probably requires dose adjustment . </S>", "<S> brv does not seem to influence other antiepileptic drug plasma levels . </S>", "<S> six clinical trials have so far been completed indicating that brv is effective in controlling seizures when used at doses between 50 and 200 mg / d . </S>", "<S> the drug is generally well - tolerated with only mild - to - moderate side effects ; this is confirmed by the low discontinuation rate observed in these clinical studies . </S>", "<S> the most common side effects are related to central nervous system and include fatigue , dizziness , and somnolence ; these apparently disappear during treatment . in this review , we analyzed brv , focusing on the current evidences from experimental animal models to clinical studies with particular interest on potential use in clinical practice . </S>", "<S> finally , pharmacological properties of brv are summarized with a description of its pharmacokinetics , safety , and potential / known drug  drug interactions . </S>"], "labels": null, "section_names": ["Introduction", "BRV mechanism of action and summary of its efficacy in experimental animal models of epilepsy", "Pharmacokinetic profile of BRV", "Efficacy of BRV in PWE: effect on partial seizures", "Safety profile of BRV", "Known drugdrug interactions", "Conclusion"], "sections": [["although about two - thirds of patients with epilepsy ( pwe ) treated with antiepileptic drugs ( aeds ) reach seizure - freedom , about one - third remains drug - resistant to the current therapies.1 despite the introduction of new aeds with a better pharmacokinetic and safety profile compared to old generation aeds , today , one of the major causes of failure of antiepileptic treatment is poor adherence often due to occurrence of adverse drug reactions ( adrs ) , leading up to 25% of patients to discontinue treatment before the achievement of effective doses and with a consequent increase of health care costs.2,3 during the last 25 years , many efforts have been directed to the development of new aeds with different mechanisms of action able to reduce brain hyperexcitability ; recently considerable interest has been focused on synaptic vesicle protein 2a ( sv2a ) and its role as a target for aeds.4 the first drug of this new class approved for epilepsy is levetiracetam ( lev ) , and from this lead compound , several racetam analogs have been synthesized.5 based on target - drug program , brivaracetam ( brv ) ( 2s)-2-[(4r)-2-oxo-4-propylpyrro - lidinyl]butanamide , an n - propyl analog of lev has been identified and has entered clinical trials for pwe;6 it is an sv2a ligand with high selectivity and a 1030-fold higher potency , depending on the experimental conditions , when compared to lev.7,8 the purpose of this review is to present updated data available on the pharmacology , efficacy , and tolerability of brv .", "we have conducted a systematic search in the pubmed and cochrane library databases up to august 14 , 2015 summarizing all relevant data for efficacy , safety , and tolerability of brv in the treatment of partial - onset seizure ."], ["although the correlation between sv2a binding and anti - convulsant potency in animal models has been previously demonstrated , the role of sv2a in neurotransmission still remains unclear.9 in fact , even if it has been established that sv2a protein is involved in normal synaptic vesicle function , the exact mechanism of synaptic vesicle cycling regulation and neurotransmitter release remains unknown.5,9 several preclinical studies have demonstrated that brv is more potent and efficacious than lev in animal models of both focal and generalized epilepsy.10 in particular , in fully amygdala - kindled rats , brv induced a significant suppression in motor - seizure severity from a dose of 21.2 mg / kg , whereas lev caused a similar effect from a dose of 170 mg / kg .", "brv also significantly reduced the after - discharge duration at the highest dose tested ( 212.3 mg / kg ) , whereas lev was inactive on this parameter up to 1,700 mg / kg.10 moreover , brv protected significantly against both partial and generalized seizures in fully amygdala - kindled mice resistant to phenytoin ( effective dose 50 [ ed50 ] : 68.3 mg / kg , intraperitoneal [ ip]).10 recently , it has been demonstrated that brv has a higher brain permeability than lev , with consequently more rapid onset of action after acute dosing in audiogenic mice ; this fast onset of action might also have a potential clinical relevance for the treatment of status epilepticus or cluster seizures.11 another study confirmed the protective activity of brv against kindled seizures with focal seizure threshold and significantly severity significantly modified.12 furthermore , a recent study , using a rapid kindling model in p14 , p21 , p28 , and p60 rats , has evaluated two doses of brv 10 and 100 mg / kg , demonstrating that brv 100 mg / kg significantly increased the after - discharge threshold at all ages , whereas brv 10 mg / kg increased after - discharge threshold in p60 , p28 , and p21 rats .", "brv also reduced the after - discharge duration , achieving statistical significance with 10 and 100 mg / kg at p60 and with 100 mg / kg , at p21 .", "at p60 , brv increases the number of stimulations required to reach stage 45 seizures in a dose - dependent manner . at p28 and p21 , brv increased the number of stimulations required to develop stage 45 seizures in a dose - dependent manner , with almost complete elimination of stage 45 seizures.13 moreover , brv showed a marked synergism with diazepam to reduce seizure duration in self - sustained status epilepticus induced by stimulation of the perforant path.14 a recent study has evaluated the antiseizure and antimyoclonic activities of brv in comparison to lev in an animal model of posthypoxic myoclonus , showing higher efficacy of brv ( 0.3 mg / kg ) than lev ( 3 mg / kg ) against posthypoxic seizures.15 furthermore , recent experiments conducted in transgenic mice with alzheimer s disease supported an adjunctive and peculiar role of brv that not only revealed an efficacy against spike - wave discharges similar to ethosuximide , but it showed the ability to reverse memory impairment , thus extending the potential spectrum of action of this new aed.16 in addition to sv2a block , brv also exhibits inhibitory activity on neuronal voltage - gated sodium channels ( vgsc ) playing a role as a partial antagonist , as has been reported for other aeds.17,18 in particular , experimental studies on primary cortical cultures have demonstrated that brv is able to prolong the sodium channel time recovery from fast inactivation , and this effect could reduce the availability of sodium channel during high - frequency repetitive firing.17,19 even if this data has been refuted by another recent experimental study that has showed that this vgsc inhibition does not impair sustained repetitive firing in neurons of neuroblastoma cells,20 this is an important aspect , which deserves to be better clarified , since the lack of effect of brv to reduce neuronal excitability by blocking high repetitive firing in neurons might exclude that the modulation of vgsc contributes to antiseizure effects of brv ."], ["brv presents a favorable pharmacokinetic profile , linear and predictable , with low intersubject variability and almost 100% bioavailability.2123 the pharmacokinetic properties of brv have been studied in healthy adult volunteers , in the elderly , in patients with pwe , and in those with hepatic or renal impairment.2225 the pharmacokinetic differences in elderly subjects compared to healthy volunteers are not so important as to require any dose adjustment.25 absorption of brv is unaffected by the presence of food , including high fat meals ; after oral administration , brv is rapidly absorbed by the gastrointestinal tract , and displays linear and dose - proportional profile over the dose range tested.22,26 its distribution volume is close to total body water ( vz=0.5 l / kg ) , and it binds weakly to plasma proteins ( 17.5% ) .", "its terminal half - life is ~9 hours.21 saliva and plasma brv levels are highly correlated , brv crosses the mucosa by passive diffusion , therefore , the saliva concentration of brv is correlated with plasma concentration .", "it is possible to speculate that saliva might be a suitable sample for monitoring brv levels when blood sampling could be a limiting factor.23 brv is extensively metabolized through several metabolic pathways and is fully excreted by urine ( only 8%11% remains unchanged ) .", "brv is eliminated primarily by metabolism , with the major metabolic pathway involving hydrolysis of the acetamide group resulting in formation of an acid metabolite ( brv - ac ; 34.2% of a radiolabeled dose in urine).21 a secondary pathway , mainly mediated by cytochrome p450 ( cyp ) 2c19,27 forms a hydroxy metabolite ( brv - oh ; 15.9% of dose in urine).21 a combination of these two pathways leads to the formation of a hydroxyacid metabolite ( 15.2% of dose in urine);21,28 only 8.6% of the dose is recovered as the unchanged compound in urine.21 all three metabolites of brv are pharmacologically inactive ( unpublished results ) in an open - label study conducted in patients with liver disease , the plasma half - life of brv was prolonged up to 17.4 hours in correlation with the severity of hepatic impairment ; however , the exposure to brv is increased by 50%60% in patients with hepatic impairment , irrespective of severity classified by child ", "pugh score.25,26 these data suggest that the maximum daily dose of brv might be reduced by one - third in patients with hepatic impairment.25 in severe renal impairment , the exposure to a single oral dose of 200 mg brv not requiring dialysis ( creatinine clearance < 15 ml / min ) , and renal clearance of three metabolites ( acid , hydroxy , and hydroxyacid ) , was decreased 10-fold in patients with severe renal impairment.24 nevertheless , there are data showing a toxicological coverage for metabolites , without the evidence of any safety issues ( ucb data file ) . based on these observations , a dose adjustment for brv should not be required at any stage of renal dysfunction ."], ["the efficacy of brv as add - on therapy in patients with uncontrolled partial seizures has been assessed in six randomized placebo - controlled clinical trials ( table 1).29 in the first two studies , brv as adjunctive therapy in adult patients with partial epilepsy and poor control with 12 concomitant aeds , at different doses ( 5 , 20 , 50 , and 50150 mg / d ) has been evaluated.30,31 french et al30 reported a statistically significant reduction of seizure frequency achieved at the 50 mg / d dose , with high tolerability and infrequent adrs . in particular , the percentage reduction over placebo in focal seizure frequency / week was directly correlated to brv dose , respectively 9.8% at 5 mg / d , 14.9% at 20 mg / d , and 22.1% at 50 mg / d , with a median percent reduction from baseline in seizure frequency / week of 21.7% ( placebo ) , 29.9% ( brv5 ) , 42.6% ( brv20 ) , and 53.1% ( brv50 ) ; 50% responder rates were 16.7% ( placebo ) , 32.0% ( brv5 ) , 44.2% ( brv20 ) , and 55.8% ( brv50 ) ; seizure freedom rates during the 7-week treatment period were 1.9% ( placebo ) , 8.0% ( brv5 ) , 7.7% ( brv20 ) , and 7.7% ( brv50 ) . on the other hand , higher doses of brv would not seem to be more effective , in fact , van paesschen et al31 did not find significant differences in seizure frequency reduction between brv 50 and 150 mg / d during the 7-week maintenance period .", "in particular , the median seizure frequency / week was 1.00 , 1.96 , and 1.86 in the group treated with brv 50 mg / d , brv 150 mg / d , and placebo , respectively.31 the reduction in baseline - adjusted seizure frequency / week over placebo during the maintenance period was 14.7% in the brv 50 mg / d group and was 13.6% in the brv 150 mg / d group ; however , a significant difference over placebo was observed on several secondary efficacy outcomes ( 10 weeks combined up - titration and maintenance period).31 in fact , after the 10-week treatment period , the median seizure frequency / week was 1.10 , 2.05 , and 1.95 in the brv 50 mg / d , brv 150 mg / d , and placebo groups , respectively . in the maintenance period ,", "50% responder rates were 23.1% for placebo compared with 39.6% for brv50 and 33.3% for brv150 . during the treatment period , 50% responder rates were 17.3% for placebo compared with 35.8% for brv50 and 30.8% for brv150 .", "nine patients were seizure - free during the 10-week treatment period in the brv50 group ( three in the brv150 group , only one in the placebo group ) . in the study by ryvlin et al,32", "the efficacy and safety / tolerability of brv ( at doses of 20 , 50 , and 100 mg / d ) in patients with uncontrolled partial seizures with / without secondary generalization , despite treatment with one or two concomitant aeds , was investigated .", "the percent reduction over placebo in baseline - adjusted seizure frequency / week was 6.8% , 6.5% , and 11.7% in the brv 20 , 50 , and 100 mg / d groups , respectively .", "the percent reduction over placebo in baseline - adjusted seizure frequency/28 days was 9.2% and 20.5% in the brv 50 and 100 mg / d groups , respectively .", "median percent reductions from baseline were 30.0% for brv 20 mg / d , 26.8% for brv 50 mg / d , and 32.5% for brv 100 mg / d in comparison to 17.0% for placebo .", "responder rates ( 50% ) were 27.3% , 27.3% , and 36.0% for brv 20 , 50 , and 100 mg / d , respectively , in comparison to 20.0% for placebo .", "based on these results , only brv 100 mg / d was able to significantly reduce seizure frequency / week over placebo .", "indeed , in their randomized placebo - controlled trial , adjunctive brv at a daily dose of 50 mg significantly decreased seizure frequency , while lower dosages ( 5 and 20 mg / d ) did not achieve significant differences.33 in more detail , the percent reduction in partial - onset seizure frequency / week in comparison to placebo was 0.9% ( p=0.885 ) for brv 5 mg / d , 4.1% ( p=0.492 ) for brv 20 mg / d , and 12.8% ( p=0.025 ) for brv 50 mg / d ; in the brv 50 mg / d group , statistical significance was also observed for the 50% responder rate ( brv 32.7% vs placebo 16.7% ) and median percent reduction from baseline in focal seizure frequency / week ( brv 30.5% vs placebo 17.8% ) .", "recently a randomized , multicenter , double - blind phase iii trial was conducted by klein et al34 to evaluate the efficacy and the safety profile of brv at fixed doses of 100200 mg / d in adult patients with refractory partial onset seizures .", "responder rate was 21.6% for placebo group , 38.9% for brv 100 mg / d , and 37.8% for brv 200 mg / d ; the percent reduction of partial onset seizures in 28-day frequency was 22.8% for brv 100 mg and 23.2% for brv 200 mg .", "kwan et al35 conducted a double - blind , randomized , placebo - controlled trial investigating the safety and tolerability profile of adjunctive brv ( at individualized tailored doses ranging from 20 to 150 mg / d ) in patients with partial or generalized refractory epilepsies .", "the percent of reduction of focal seizure frequency / week in the brv group in comparison to placebo was 7.3% , while only in the generalized seizures group , the number of seizure days / week decreased from 1.42 at baseline to 0.63 during the treatment period in brv - treated patients ( n=36 ) , and from 1.47 at baseline to 1.26 during the treatment period in the placebo group ( n=13).35 the median percent reduction in baseline - adjusted seizure frequency / week was 26.9% brv vs 18.9% placebo , and the 50% responder rate was 30.3% brv vs 16.7% placebo .", "the median percent reduction from baseline in generalized seizure days / week was 42.6% vs 20.7% , and the 50% responder rate was 44.4% vs 15.4% in brv - treated and placebo - treated patients , respectively .", "similar to lev , brv might become a useful aed for the treatment of myoclonic seizures occurring in the setting of idiopathic generalized epilepsies ( eg , juvenile myoclonic epilepsy)36 or of progressive myoclonic epilepsies.37 two randomized , placebo - controlled trials evaluating efficacy and safety of adjunctive brv ( 5150 mg / d ) in unverricht ", "lundborg disease , the most common and less severe form of progressive myoclonic epilepsies,38 failed to show a significant improvement of myoclonus in these patients.39 however , sample size was small ( 106 patients randomized in the two trials ) , and the patients were allowed to receive lev.39 moreover , it is well known that myoclonus may present high inter- and intrapatients variability ( with patients experiencing  good days  and  bad days  ) in unverricht ", "currently an open - label , multicenter , follow - up study to evaluate the long - term safety and efficacy of brv is ongoing ( brite study - nct01339559 ) .", "finally , to date , in all studies performed , brv was evaluated as an oral tablet formulation , and no data are available about intravenous infusion since the study nct02088957 , aiming at a comparison of the efficacy and safety of intravenous brv vs phenytoin in adult subjects with nonconvulsive electrographic seizures , was terminated for low enrollment ."], ["the most commonly reported adverse effects with brv in adults were primarily related to the central nervous system and included somnolence , fatigue , and dizziness.22 these adverse effects were mild to moderate , and the tolerability profile is so excellent that it did not impair therapeutic compliance . in fact , the daily dose of brv ( 20150 mg ) was well tolerated and associated with 6.1% of discontinuation rates due to adrs compared to 5.0% of the placebo group.35 furthermore , adrs induced by brv seem to be time - related , disappearing during the course of treatment .", "the entity of sedative effects of brv measured by psychometric tests is dose - related in healthy men and appeared clearly from 600 mg upwards as a decrease in attention , motor control , and alertness.22 moreover , the type and the severity of adrs are not influenced by food.22 as demonstrated in healthy males , a twice - daily dosing regimen could be a good clinical practice to reduce blood fluctuations and peak , which might influence the appearance of adverse events.22 no effects on cardiac function were reported even at very high daily dosages ( up to 800 mg / d).40 no data about fertility and/or potential teratogenic effect of brv in humans are currently available ; however , no adverse effects were detected up to the highest tested oral dose of 400 mg / kg / d on fertility , and no effects on pregnancy or fetal development at 600 mg / d were observed in animals.26 seizure aggravation or the appearance of new generalized seizures was rare : three studies reported this adverse event , occurring in similar proportions between placebo and treated group ( 4.3% vs 5.2% , p=0.67).32,33,35 in the above - reported five randomized clinical trials , 1,639 subjects were included in an intention - to - treat analysis ( 1,214 treated with brv and 425 with placebo).3035 no differences were observed in the proportion of subjects experiencing at least one adverse event ( 65.5% with brv vs 60.5% with placebo , p=0.10 ) .", "most events were mild to moderate ; actually , comprehensive withdrawal rate due to adverse events was quite low and similar in brv and placebo arms ( 5.4% with brv vs 4.2% with placebo , p=0.37 ) .", "serious adverse events were quite rare and equally distributed ( 2.9% with brv vs 4.4% with placebo , p=0.16 ) .", "adverse events that were observed in at least 5% of subjects in either group are listed in table 2 .", "the proportion of patients reporting fatigue and somnolence was significantly higher in brv group compared to placebo ( table 2 ) .", "irritability was reported in three studies only , and it was present in a small proportion of subjects ( 3% receiving brv , 1% receiving placebo , p=0.36 ) ."], ["because of its advantageous pharmacokinetic profile , brv treatment does not appear to influence plasma concentrations of other aeds such as carbamazepine , lamotrigine , lev , oxcarbazepine , topiramate , or valproic acid.41 however , carbamazepine plasma levels are slightly reduced by coadministration of brv ( 400 mg / d ) , while levels of carbamazepine - epoxide are increased in a dose - dependent manner.26,42 this increase of plasma concentration of carbamazepine - epoxide is the result of inhibition by brv of epoxide hydrolase that metabolizes carbamazepine - epoxide into carbamazepine - diol.42 high doses of brv ( 400 mg / d ) cause a moderate decrease of ethinylestradiol and levonorgestrel plasma levels ( components of oral contraceptives ) but this posological range has no impact on ovulation .", "no effect on contraceptive disposition is reported at therapeutic doses of brv 100 mg / d.39 interestingly , there is a possible negative interaction between brv and lev , in fact , the concomitant use of both drugs may reduce brv efficacy ; however , this evidence is not robust because the number of patients with concomitant lev was very small , other studies may be useful to assess this apparent pharmacodynamic interaction.33"], ["brv is a novel aed whose efficacy in partial epilepsies has been studied and established in five randomized controlled trials;3035 furthermore , two recent meta - analyses have confirmed significant effects for brv in patients with refractory partial seizures.29,43 considering that brv shares part of its mechanism of action with lev and that its ability to inhibit vgsc ( still debated ) is in common with several other aeds , it will be very intriguing to see how this drug will behave in real - life clinical practice .", "accordingly , it could be hypothesized that brv might possess at least the same effectiveness as lev .", "based on this hypothesis , brv may be reasonably considered as a valuable add - on aed in patients with partial seizures , also considering its suggested good tolerability .", "however , specific studies are needed to confirm its efficacy in specific epileptic syndromes , for example , a decreased expression of sv2a in the hippocampus of patients with temporal lobe epilepsy with hippocampal sclerosis has been documented and might represent a pharmacoresistance mechanism in some cases ; however , lev has been reported to be effective.44,45 in addition , because of its good safety and pharmacokinetic profile , brv might be ideal for use in monotherapy , as previously demonstrated for lev.46 finally , few studies have been performed , and more randomized double - blind studies are needed to confirm these considerations and to demonstrate if brv might really confirm its promises and become a new tool for epileptologists ."]]}
{"article_id": "PMC3990118", "article_text": ["\n acute intussusception is one of the most common causes of acute abdomen in infants .", "management includes both surgical and non - surgical methods , with gradually increasing importance recently being accorded to the latter .", "conservative measures , including hydrostatic reduction , are the methods of choice for initial management . though widely accepted , the expertise is still not widely available , especially in developing countries .", "\n a six - month - old child presented with complaints of repeated episodes of vomiting that contained only ingested milk .", "this was associated with three episodes of loose stools , initially watery and later with fresh red blood per rectum with jelly - like consistency .", "there was no history of fever , decreased urine output , lethargy , abnormal body movements , or difficulty in breathing .", "her immunizations were fully up to date with no history of rota virus immunization . \n \n on admission", "abdominal examination was remarkable for an ill - defined mass that could be felt in the right hypochondrium .", "she was initially kept nil per oral and started on conservative measures , including antibiotics .", "an urgent abdominal ultrasonography was done , which showed a round mass - like lesion giving a target appearance in the right hypochondrium ( suggestive of ileocolic intussusception ) .", "she was taken for immediate intervention , and as per surgical opinion , a hydrostatic reduction under radiological guidance was started . \n \n", "the child was first stabilized using intravenous fluids to treat dehydration , and a pre - procedure x - ray abdomen was performed to rule out intestinal perforation .", "the procedure was done in the ultrasonography room in the presence of a treating physician and a pediatric surgeon . without using any sedation ,", "a 16 f foley catheter was inserted into the rectum in the supine position and the balloon was gently inflated while maintaining a tight anal seal . \n \n", "the patient s vitals , which included abdominal girth and distension , were monitored throughout the procedure .", "lactate solution , warmed to body temperature , was then slowly introduced into the catheter through a bag hanging at a height of approximately 100 cm . during reduction ,", "the intussusceptum was observed under continuous ultrasound guidance as it proceeded to the cecum and then reduced across the ileocecal valve ( figures 1 - 4 ) . \n \n", "ileal loop ( star ) within ascending colon ( arrow ) , longitudinal view . \n \n", "this was associated with the patient s improved condition , which included less crying and irritability .", "the parents were told about the possibility of recurrence and asked to pay close attention for the onset of symptoms . \n", "\n acute intussusception is defined as the telescoping of the proximal bowel ( intussusceptum ) into the distal bowel ( intussuscipiens ) .", "the usual locations include the ileocolic region and ileocecal junction , which comprise the majority of cases .", "intussusception mostly presents between four months and two years , while the peak incidence is found around middle infancy . \n \n", "diagnosis usually needs a high suspicion on the part of the treating physician , as presentation can be quite varied , including irritability , vomiting , and poor feeding .", "characteristic manifestations include crying episodes , fresh red blood in stools ( hematochezia ) , and a mass in the abdomen .", "differential diagnoses include acute gastroenteritis , dysentery , sepsis syndrome , and volvulus , amongst others .", "the diagnosis is usually confirmed sonographically , which is highly accurate and has a reported sensitivity that exceeds 90% .", "various signs have been described in the literature , depicting intussusception as an abdominal mass with a target sign on the transverse section , a pseudokidney ( or sandwich sign ) on the longitudinal section , and a crescent - in - doughnut sign on axial images . \n \n", "the management of intussusception has seen a paradigm shift in recent years . in the past , the preferred non - surgical method involved use of a barium enema for reduction , followed by the air insufflation method . \n \n", "hydrostatic reduction using ultrasound - guided saline reduction has recently gained acceptance as the procedure of choice for initial nonsurgical management of intussusceptions in children .", "this popularity can be attributed to the avoidance of radiation exposure to the child and the treating team , as well as to the high success rates that have been achieved . \n \n", "there is still some controversy regarding the exact method of the procedure , which can be attributed to the poor training of physicians and to the general belief that intussusception is entirely a surgical problem , especially in developing countries .", "this procedure should be undertaken with a close liaison between pediatrics , pediatric surgery , and radiology teams to obtain the best results .", "the surgeon and physician should always be present at the time of reduction for clinical monitoring . \n \n", "after adequate optimization , including temperature controlled settings , the procedure should ideally be undertaken without any sedation .", "this is helpful for assessing a successful reduction using clinical criteria , as described later . \n \n", "we used ringer s lactate due to its near physiological constitution , avoiding the risk of chemical peritonitis due to a barium enema or tension pneumoperitoneum due to an air enema , as seen in cases of intestinal perforation .", "the risk of intestinal perforation following such a procedure is rarely encountered due to less hydrostatic pressure exerted and an even amount of pressure exerted on the bowel walls as compared to air insufflations . even if intestinal perforation occurs", "we chose 100 cm h2o pressure to lessen the risk of perforation , though different studies have used a wide range of pressures ( 75 to 125 cm h2o).6 , \n 9 \n \n the main ultrasonographical criteria of successful reduction is the flow of the fluid from the cecum into the terminal ileum .", "general improvement in the clinical profile of the patient including relief from crying , stable vitals , increasing abdominal distension during the procedure ( which indicates filling of the small bowel ) , and the disappearance of the abdominal mass , should also be given due consideration when deciding to terminate the procedure . \n \n", "a repeat ultrasound should always be done after the procedure as the chances of recurrence is high , especially in the immediate post - procedure period .", ", this method should be undertaken as teamwork , and not considered the work of a single specialty , which is the best way of treating the patients according to the standards of today . \n"], "abstract_text": ["<S> we present a case of infantile intussusception treated successfully using hydrostatic reduction , highlighting the importance of non - surgical techniques as the first line of management for this entity . </S>", "<S> physicians should strive for mastery over such techniques by extensive training to prevent unwanted surgical procedures in such cases . </S>"], "labels": null, "section_names": ["\nINTRODUCTION\n", "\nCASE REPORT\n", "\nDISCUSSION\n", "\nCONFLICT OF INTEREST\n"], "sections": [["\n acute intussusception is one of the most common causes of acute abdomen in infants .", "management includes both surgical and non - surgical methods , with gradually increasing importance recently being accorded to the latter .", "conservative measures , including hydrostatic reduction , are the methods of choice for initial management . though widely accepted , the expertise is still not widely available , especially in developing countries ."], ["\n a six - month - old child presented with complaints of repeated episodes of vomiting that contained only ingested milk .", "this was associated with three episodes of loose stools , initially watery and later with fresh red blood per rectum with jelly - like consistency .", "there was no history of fever , decreased urine output , lethargy , abnormal body movements , or difficulty in breathing .", "her immunizations were fully up to date with no history of rota virus immunization . \n \n on admission", "abdominal examination was remarkable for an ill - defined mass that could be felt in the right hypochondrium .", "she was initially kept nil per oral and started on conservative measures , including antibiotics .", "an urgent abdominal ultrasonography was done , which showed a round mass - like lesion giving a target appearance in the right hypochondrium ( suggestive of ileocolic intussusception ) .", "she was taken for immediate intervention , and as per surgical opinion , a hydrostatic reduction under radiological guidance was started . \n \n", "the child was first stabilized using intravenous fluids to treat dehydration , and a pre - procedure x - ray abdomen was performed to rule out intestinal perforation .", "the procedure was done in the ultrasonography room in the presence of a treating physician and a pediatric surgeon . without using any sedation ,", "a 16 f foley catheter was inserted into the rectum in the supine position and the balloon was gently inflated while maintaining a tight anal seal . \n \n", "the patient s vitals , which included abdominal girth and distension , were monitored throughout the procedure .", "lactate solution , warmed to body temperature , was then slowly introduced into the catheter through a bag hanging at a height of approximately 100 cm . during reduction ,", "the intussusceptum was observed under continuous ultrasound guidance as it proceeded to the cecum and then reduced across the ileocecal valve ( figures 1 - 4 ) . \n \n", "ileal loop ( star ) within ascending colon ( arrow ) , longitudinal view . \n \n", "this was associated with the patient s improved condition , which included less crying and irritability .", "the parents were told about the possibility of recurrence and asked to pay close attention for the onset of symptoms . \n"], ["\n acute intussusception is defined as the telescoping of the proximal bowel ( intussusceptum ) into the distal bowel ( intussuscipiens ) .", "the usual locations include the ileocolic region and ileocecal junction , which comprise the majority of cases .", "intussusception mostly presents between four months and two years , while the peak incidence is found around middle infancy . \n \n", "diagnosis usually needs a high suspicion on the part of the treating physician , as presentation can be quite varied , including irritability , vomiting , and poor feeding .", "characteristic manifestations include crying episodes , fresh red blood in stools ( hematochezia ) , and a mass in the abdomen .", "differential diagnoses include acute gastroenteritis , dysentery , sepsis syndrome , and volvulus , amongst others .", "the diagnosis is usually confirmed sonographically , which is highly accurate and has a reported sensitivity that exceeds 90% .", "various signs have been described in the literature , depicting intussusception as an abdominal mass with a target sign on the transverse section , a pseudokidney ( or sandwich sign ) on the longitudinal section , and a crescent - in - doughnut sign on axial images . \n \n", "the management of intussusception has seen a paradigm shift in recent years . in the past , the preferred non - surgical method involved use of a barium enema for reduction , followed by the air insufflation method . \n \n", "hydrostatic reduction using ultrasound - guided saline reduction has recently gained acceptance as the procedure of choice for initial nonsurgical management of intussusceptions in children .", "this popularity can be attributed to the avoidance of radiation exposure to the child and the treating team , as well as to the high success rates that have been achieved . \n \n", "there is still some controversy regarding the exact method of the procedure , which can be attributed to the poor training of physicians and to the general belief that intussusception is entirely a surgical problem , especially in developing countries .", "this procedure should be undertaken with a close liaison between pediatrics , pediatric surgery , and radiology teams to obtain the best results .", "the surgeon and physician should always be present at the time of reduction for clinical monitoring . \n \n", "after adequate optimization , including temperature controlled settings , the procedure should ideally be undertaken without any sedation .", "this is helpful for assessing a successful reduction using clinical criteria , as described later . \n \n", "we used ringer s lactate due to its near physiological constitution , avoiding the risk of chemical peritonitis due to a barium enema or tension pneumoperitoneum due to an air enema , as seen in cases of intestinal perforation .", "the risk of intestinal perforation following such a procedure is rarely encountered due to less hydrostatic pressure exerted and an even amount of pressure exerted on the bowel walls as compared to air insufflations . even if intestinal perforation occurs", "we chose 100 cm h2o pressure to lessen the risk of perforation , though different studies have used a wide range of pressures ( 75 to 125 cm h2o).6 , \n 9 \n \n the main ultrasonographical criteria of successful reduction is the flow of the fluid from the cecum into the terminal ileum .", "general improvement in the clinical profile of the patient including relief from crying , stable vitals , increasing abdominal distension during the procedure ( which indicates filling of the small bowel ) , and the disappearance of the abdominal mass , should also be given due consideration when deciding to terminate the procedure . \n \n", "a repeat ultrasound should always be done after the procedure as the chances of recurrence is high , especially in the immediate post - procedure period .", ", this method should be undertaken as teamwork , and not considered the work of a single specialty , which is the best way of treating the patients according to the standards of today . \n"], [""]]}
{"article_id": "PMC3508130", "article_text": ["aging , a multifactorial process of enormous complexity , is characterized by impairment of physiochemical and biological aspects of cellular functions ( harman 1992 ) .", "oxidative stress , an unavoidable consequence in the metabolism of oxygen in aerobic cells , is a major factor in the aging process and , in the course of many chronic diseases , associated with aging ( mattson 2002 ) .", "many predisposing conditions which increase in prevalence during aging , such as obesity , insulin resistance , inflammation , changes in the activity of the hypothalamus  hypophysis suprarenal axis , stress , and hypertension , contribute to increase prevalence of cardiovascular diseases ( veronica and esther 2012 ) .", "lipid infiltration in the myocardium is the foremost disorder encountered in the development of the aging process ( johannsen and ravussin 2010 ) .", "aging is frequently accompanied by several pathological conditions and some associated phenomena such as increased lipid peroxidation , generation of free radicals , and increased peroxidation of nitric oxide ( no ) to its toxic species , resulting from oxidative stress which significantly alters the incidence of cardiovascular diseases ( guarner et al .", "alteration in glutathione - dependent antioxidant system is expected to exert a significant impact on physiological and metabolic functions of cellular membranes .", "enhanced lipid peroxidation and deterioration of membrane structure has been well established during the aging process ( yu 2005 ) . preventing and treating cardiovascular diseases would be useful in promoting normal aging .", "the identification of natural molecule with antioxidant , antilipidemic , and membrane - stabilizing properties is , therefore , one strategy to facilitate healthy aging .", "chitosan is one of the most abundant naturally occurring polysaccharides present in shellfish , clams , krill , oysters , squid , fungi , and insects ( cardenas et al .", "it is a polymer of -(1 - 4)-d - glucosamine ( fig .  1 ) , and it is chemically similar to that of the plant fiber , cellulose . it has been reported to possess antilipidemic ( santhosh et al .", "previously , anandan et al . ( 2004 ) observed the antiulcerogenic potential of chitosan against hcl ", "the free radical quenching property of this marine polysaccharide has also been studied in detail ( xing et al .", "it has profound applications in the fields of clarification and purification , chromatography , paper and textiles , photography , food and nutrition , agriculture , pharmaceutical and medical , cosmetics , biodegradable membranes , and biotechnology ( santhosh et al .", "though the beneficial effects of chitosan have been extensively studied , the antiaging effect of chitosan has not yet been explored.fig .", "1structure of chitosan structure of chitosan in the present study , an attempt has been made to assess the salubrious effect of dietary chitosan intake on myocardial lipid peroxidation and glutathione - dependent antioxidant status in young and aged rats by virtue of its antioxidant and hypolipidemic properties .", "reduced glutathione , tetraethoxypropane , and 2-thiobarbituric acid were procured from m / s sigma chemical company , st .", "chitosan ( mw , 750,000  da ; viscosity , 8  cp ; deacetylation rate , 8587  % ; purity , 98.6  % ) used in the experiment was a kind gift from dr . t. k. thankkappan , principal scientist , central institute of fisheries technology , cochin , india .", "male wistar strain albino rats , weighing 120150  g [ 18 young rats of 23  months old ( mean age , 79.2    6.53  days ) ] and 350400  g [ 18 aged rats of 2025  months old ( mean age , 712    51.6  days ) ] , were selected for the study .", "the animals were housed individually in polypropylene cages under hygienic and standard environmental conditions ( 28    2  c ; humidity , 6070  % ; 12  h light / dark cycle ) .", "the animals were allowed a standard diet ( m / s sai foods , bangalore , india ; table  1 ) and water ad libitum .", "the experiment was carried out according to guidelines of the committee for the purpose of control and supervision of experiments on animals , new delhi , india , and approved by the institutional animal ethics committee of the central institute of fisheries technology , cochin , india.table 1composition of standard diets .", "no.ingredientscomposition ( g/100  g diet)1carbohydrate ( nitrogen free)56.22crude protein22.03ash7.54crude oil4.25crude fiber3.06glucose2.57vitamins1.88sand silica1.49calcium0.810phosphorus0.6 composition of standard diet seven days after acclimatization , the animals were divided into two major groups : group i consisted of 18 normal young rats and group ii consisted of 18 normal aged rats .", "each group was further subdivided into three groups ( six rats each ) : one control group ( group ia and group iia ) and two experimental groups based on the duration of supplementation of chitosan at 2  % level along with feed 30  days ( group ib and group iib ) and 60  days ( group ic and group iic ) . at the end of the experimental period ,", "the animals were sacrificed , and the blood was collected in a heparinized tube for the separation of plasma .", "the heart tissue was excised immediately and homogenized in ice - cold 0.1-m tris  hcl buffer in a potter ", "the homogenate was used for the estimation of lipid peroxides ( lpo ; ohkawa et al .", "1979 ) , reduced glutathione ( gsh ; ellman 1959 ) , glutathione reductase ( gr ; stall et al .", "high - density lipoprotein - cholesterol ( hdl - cholesterol ) in plasma was determined by the method of izzo et al .", "( 1981 ) , and low - density lipoprotein - cholesterol ( ldl - cholesterol ) was estimated according to the calculation of friedwald et al .", "( 1974 ) after extracting total lipids according to the method of folch et al .", "multiple comparisons of the significant anova were performed by duncan 's multiple range comparison test . a p value  < 0.05 was considered as statistically significant .", "all data were analyzed with the aid of statistical package program spss 12.0 for windows .", "reduced glutathione , tetraethoxypropane , and 2-thiobarbituric acid were procured from m / s sigma chemical company , st .", "chitosan ( mw , 750,000  da ; viscosity , 8  cp ; deacetylation rate , 8587  % ; purity , 98.6  % ) used in the experiment was a kind gift from dr . t. k. thankkappan , principal scientist , central institute of fisheries technology , cochin , india .", "male wistar strain albino rats , weighing 120150  g [ 18 young rats of 23  months old ( mean age , 79.2    6.53  days ) ] and 350400  g [ 18 aged rats of 2025  months old ( mean age , 712    51.6  days ) ] , were selected for the study .", "the animals were housed individually in polypropylene cages under hygienic and standard environmental conditions ( 28    2  c ; humidity , 6070  % ; 12  h light / dark cycle ) .", "the animals were allowed a standard diet ( m / s sai foods , bangalore , india ; table  1 ) and water ad libitum .", "the experiment was carried out according to guidelines of the committee for the purpose of control and supervision of experiments on animals , new delhi , india , and approved by the institutional animal ethics committee of the central institute of fisheries technology , cochin , india.table 1composition of standard diets .", "no.ingredientscomposition ( g/100  g diet)1carbohydrate ( nitrogen free)56.22crude protein22.03ash7.54crude oil4.25crude fiber3.06glucose2.57vitamins1.88sand silica1.49calcium0.810phosphorus0.6 composition of standard diet", "seven days after acclimatization , the animals were divided into two major groups : group i consisted of 18 normal young rats and group ii consisted of 18 normal aged rats .", "each group was further subdivided into three groups ( six rats each ) : one control group ( group ia and group iia ) and two experimental groups based on the duration of supplementation of chitosan at 2  % level along with feed 30  days ( group ib and group iib ) and 60  days ( group ic and group iic ) . at the end of the experimental period ,", "the animals were sacrificed , and the blood was collected in a heparinized tube for the separation of plasma .", "the heart tissue was excised immediately and homogenized in ice - cold 0.1-m tris  hcl buffer in a potter ", "the homogenate was used for the estimation of lipid peroxides ( lpo ; ohkawa et al .", "1979 ) , reduced glutathione ( gsh ; ellman 1959 ) , glutathione reductase ( gr ; stall et al . 1969 ) , and glutathione peroxidase ( gpx ; paglia and valentine 1967 ) .", "high - density lipoprotein - cholesterol ( hdl - cholesterol ) in plasma was determined by the method of izzo et al .", "( 1981 ) , and low - density lipoprotein - cholesterol ( ldl - cholesterol ) was estimated according to the calculation of friedwald et al .", "( 1974 ) after extracting total lipids according to the method of folch et al .", "multiple comparisons of the significant anova were performed by duncan 's multiple range comparison test . a p value  < 0.05 was considered as statistically significant .", "all data were analyzed with the aid of statistical package program spss 12.0 for windows .", "significant ( p  <  0.05 ) variation was observed in the body weight ( grams ) of young ( initial , 129    9.05 ; final , 249    18.3 ) and aged ( initial , 382    27.2 ; final , 336    23.1 ) chitosan - supplemented groups of rats .", "the significant ( p  <  0.05 ) loss observed in the body weight of chitosan - supplemented aged animals might be related to the fibrous nature of chitosan .", "interestingly , the total food intake ( grams per 60  days ) of aged chitosan - supplemented rats ( 904    78.4 ) was comparable to that of young rats ( 745    59.5 ) .", "level of total cholesterol was significantly ( p  <  0.05 ) higher in plasma and heart tissue of group iia aged rats as compared to group ia young control rats , indicating the development of mild age - associated hypercholesterolemic condition ( tables  2 and 3 ) .", "the level of ldl - cholesterol was slightly ( p  <  0.05 ) higher in group iia aged rats , whereas hdl - cholesterol levels were significantly lower compared to group ia young animals ( table  2 ) .", "this aspect might be due to the augmented mobilization of ldl - cholesterol from the blood into the cell membranes , resulting in abnormal cholesterol deposition in the myocardium .", "in the present study , the dietary supplementation with chitosan significantly reduced the total cholesterol level in plasma and myocardial tissue of group iic aged rats as compared to group iia rats .", "it also kept the levels of ldl - cholesterol and hdl - cholesterol in plasma comparable to that of group ic rats .", "2009 ) , which showed that the positively charged amino groups of chitosan possess the ability to bind negatively charged molecules such as lipids and bile acids , inducing a greater fractional excretion in the feces . also", "( 2007 ) suggested that chitosan improve lipid metabolism by regulating total cholesterol and ldl - cholesterol by upregulation of hepatic ldl receptor mrna expression , increasing the excretion of fecal bile acids .", "previous studies ( yao and chiang 2002 ) pointed out that chitosan supplementation was capable of lowering the levels of plasma total cholesterol and ldl - cholesterol in experimental animals . in the present study , a slight decline in the level of total cholesterol and ldl - cholesterol were also noted in group ic chitosan - fed young rats , ascertaining the anticholesterolemic property of chitosan ( ylitalo et al .", "2002).table 2effect of dietary chitosan supplementation on total cholesterol , hdl - cholesterol , and ldl - cholesterol in plasma of young and aged ratsparametersyoung ratsaged ratsgroup ia ( control)group ib ( 30  days)group ic ( 60  days)group iia ( control)group iib ( 30  days)group iic ( 60  days)total cholesterol79.9    5.36 a , b76.2    4.98 a , b73.1    5.07 a98.4    9.12 c91.7    8.56 c87.3    8.21 b , chdl - cholesterol42.2    3.11 a , b ,", "c44.9    3.48 b , c47.5    3.29 c33.2    2.68 d36.4    2.56 a39.7    3.74 a , bldl - cholesterol24.3    1.42 a22.7    1.31 a , b20.3    1.18 b48.4    2.72 c43.1    2.14 d38.3    2.27 eresults are mean    sd for six rats .", "values expressed : total cholesterol , hdl - cholesterol , and ldl - cholesterol , milligrams per deciliter .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test)table 3effect of dietary chitosan supplementation on the levels of total cholesterol , lipid peroxides , and reduced glutathione ( gsh ) and the activities of glutathione - dependent antioxidant enzymes [ glutathione peroxidase ( gpx ) and glutathione reductase ( gr ) ] in the heart tissue of young and aged ratsparametersyoung ratsaged ratsgroup ia ( control)group ib ( 30  days)group ic ( 60  days)group iia ( control)group iib ( 30  days)group iic ( 60  days)total cholesterol2.72    0.17 a , b2.56    0.14 a2.43    0.16 a3.68    0.32 c3.29    0.21 d3.04    0.25 b , dlipid peroxides1.08    0.07 a0.98    0.06 a0.97    0.07 a2.58    0.14 b1.96    0.09 c1.54    0.11 dgsh10.9    0.84 a12.3    1.02 a , b14.5    1.17 c7.54    0.61 d8.76    0.69 d , e9.52    0.85 a , egpx5.23    0.35 a5.51    0.41 a , b5.98    0.37 b3.12    0.18 c4.05    0.26 d4.56    0.34 dgr0.43    0.02 a0.52    0.04 b0.58    0.03 c0.22    0.01 d0.29    0.03 e0.36    0.02 fresults are mean    sd for six animals .", "values expressed : total cholesterol , milligrams per gram wet tissue ; lipid peroxides , nanomoles mda released per milligram protein ; gsh , micrograms per milligram protein ; gpx , micrograms gsh oxidized per minute per milligram protein ; gr , nanomoles nadph oxidized per minute per milligram protein .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test ) effect of dietary chitosan supplementation on total cholesterol , hdl - cholesterol , and ldl - cholesterol in plasma of young and aged rats results are mean    sd for six rats .", "values expressed : total cholesterol , hdl - cholesterol , and ldl - cholesterol , milligrams per deciliter .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test ) effect of dietary chitosan supplementation on the levels of total cholesterol , lipid peroxides , and reduced glutathione ( gsh ) and the activities of glutathione - dependent antioxidant enzymes [ glutathione peroxidase ( gpx ) and glutathione reductase ( gr ) ] in the heart tissue of young and aged rats results are mean    sd for six animals .", "values expressed : total cholesterol , milligrams per gram wet tissue ; lipid peroxides , nanomoles mda released per milligram protein ; gsh , micrograms per milligram protein ; gpx , micrograms gsh oxidized per minute per milligram protein ; gr , nanomoles nadph oxidized per minute per milligram protein .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test ) sumiyoshi and kimura ( 2006 ) suggested that the lipid - lowering effects of chitosan might be mediated by increases in fecal fat and/or bile acid excretion resulting from the binding of bile acids , and by a decrease in the absorption of dietary cholesterol from the small intestine .", "( 2009 ) have shown that normal intestinal microbiota might also positively influence immune responses and protect against the development of inflammatory diseases systemically through the formation of short - chain fatty acids by fermentation of dietary fiber in the intestine .", "chitosan , a biopolymer of glucosamine derived from chitin that is chemically similar to that of cellulose , acts as a dietary fiber in gastrointestinal tract ( gallaher et al .", "it is possible that chitosan may function through the generation of gut metabolites , such as short - chain fatty acids / short - chain oligosaccharides , in attenuating the development of inflammatory processes related to aging . in the present study", ", there was a significant ( p  <  0.05 ) increase in the level of lipid peroxidation observed with a concomitant reduction in the level of nonenzymatic ( gsh ) and enzymatic ( gpx and gr ) antioxidants in the heart tissue of group iia aged rats as compared to group ia young control animals ( table  3 ) .", "this concurs with the earlier findings ( subramanian and james 2010 ) , which indicated that the higher vulnerability of aged myocardium to peroxidative damage was mainly due to a decline in the level of free radical scavengers .", "depletion of gsh results in enhanced lipid peroxidation , and excessive lipid peroxidation can cause increased gsh consumption during aging ( denniss et al .", "the reduction in the activity of gpx and gr may be due to the reduced availability of gsh .", "gpx offers protection to the cellular and subcellular membranes from the peroxidative damage by eliminating hydrogen peroxide and lipid peroxide ( li et al .", "inhibition of gsh - dependent antioxidant enzymes makes myocardial cell membranes more susceptible to oxidative damage in aging .", "gsh and gsh - dependent enzyme systems may be directly related to the pathogenic mechanisms related to age - associated disorders ( goncharova et al .", "2007 ) . in our study , the dietary chitosan intake significantly attenuated the age - associated oxidative stress and maintained the level of the glutathione - dependent antioxidant status in the heart tissue at near normal .", "( 2003 ) have shown that chitosan has strong antioxidative effects , which decrease free radical production and increase antioxidant enzyme activities during ccl4-induced lipid peroxidation in rats .", "( 2004 ) have suggested that chitosan may eliminate various free radicals by the action of nitrogen on the c-2 position of the chitosan .", "( 2001 ) reported that the scavenging mechanism of chitosan is related to the fact that the free radicals can react with the hydrogen ion from the ammonium ions to form a stable molecule .", "the normal young rats receiving chitosan ( group ic ) did not show any significant change when compared with normal ( group ia ) rats , indicating that it does not per se have any adverse effects .", "in conclusion , the overall antiaging effect of dietary chitosan intake is probably related to its ability to inhibit the increased accumulation of lipids both in the systemic circulation and in the myocardium by its antilipidemic property , or to normal maintenance of the activities of glutathione - dependent antioxidant enzymes and the level of gsh , which protect myocardial membrane against oxidative stress by decreasing lipid peroxidation reactions ."], "abstract_text": ["<S> aging has been defined as the changes that occur in living organisms with the passage of time that lead to functional impairment and ultimately to death . </S>", "<S> free radical - induced oxidative damage has long been thought to be the most important consequence of the aging process . in the present study </S>", "<S> , an attempt has been made to study the salubrious effects of dietary supplementation of chitosan on glutathione - dependent antioxidant defense system in young and aged rats . </S>", "<S> the dietary supplementation of chitosan significantly reduced the age - associated dyslipidemic abnormalities noted in the levels of total cholesterol , hdl - cholesterol , and ldl - cholesterol in plasma and heart tissue . </S>", "<S> its administration significantly ( p  <  0.05 ) attenuated the oxidative stress in the heart tissue of aged rats through the counteraction of free radical formation by maintaining the enzymatic [ glutathione peroxidase ( gpx ) and glutathione reductase ( gr ) ] and non - enzymatic [ reduced glutathione ( gsh ) ] status at levels comparable to that of normal young rats . </S>", "<S> our results conclude that dietary intake of chitosan restores the depleted myocardial antioxidant status and suggest that it could be an effective therapeutic agent in treatment of age - associated disorders where hypercholesterolemia and oxidative stress are the major causative factors . </S>"], "labels": null, "section_names": ["Introduction", "Experimental", "Drugs and chemicals", "Animals", "Experimental protocol", "Statistical analysis", "Results and discussion"], "sections": [["aging , a multifactorial process of enormous complexity , is characterized by impairment of physiochemical and biological aspects of cellular functions ( harman 1992 ) .", "oxidative stress , an unavoidable consequence in the metabolism of oxygen in aerobic cells , is a major factor in the aging process and , in the course of many chronic diseases , associated with aging ( mattson 2002 ) .", "many predisposing conditions which increase in prevalence during aging , such as obesity , insulin resistance , inflammation , changes in the activity of the hypothalamus  hypophysis suprarenal axis , stress , and hypertension , contribute to increase prevalence of cardiovascular diseases ( veronica and esther 2012 ) .", "lipid infiltration in the myocardium is the foremost disorder encountered in the development of the aging process ( johannsen and ravussin 2010 ) .", "aging is frequently accompanied by several pathological conditions and some associated phenomena such as increased lipid peroxidation , generation of free radicals , and increased peroxidation of nitric oxide ( no ) to its toxic species , resulting from oxidative stress which significantly alters the incidence of cardiovascular diseases ( guarner et al .", "alteration in glutathione - dependent antioxidant system is expected to exert a significant impact on physiological and metabolic functions of cellular membranes .", "enhanced lipid peroxidation and deterioration of membrane structure has been well established during the aging process ( yu 2005 ) . preventing and treating cardiovascular diseases would be useful in promoting normal aging .", "the identification of natural molecule with antioxidant , antilipidemic , and membrane - stabilizing properties is , therefore , one strategy to facilitate healthy aging .", "chitosan is one of the most abundant naturally occurring polysaccharides present in shellfish , clams , krill , oysters , squid , fungi , and insects ( cardenas et al .", "it is a polymer of -(1 - 4)-d - glucosamine ( fig .  1 ) , and it is chemically similar to that of the plant fiber , cellulose . it has been reported to possess antilipidemic ( santhosh et al .", "previously , anandan et al . ( 2004 ) observed the antiulcerogenic potential of chitosan against hcl ", "the free radical quenching property of this marine polysaccharide has also been studied in detail ( xing et al .", "it has profound applications in the fields of clarification and purification , chromatography , paper and textiles , photography , food and nutrition , agriculture , pharmaceutical and medical , cosmetics , biodegradable membranes , and biotechnology ( santhosh et al .", "though the beneficial effects of chitosan have been extensively studied , the antiaging effect of chitosan has not yet been explored.fig .", "1structure of chitosan structure of chitosan in the present study , an attempt has been made to assess the salubrious effect of dietary chitosan intake on myocardial lipid peroxidation and glutathione - dependent antioxidant status in young and aged rats by virtue of its antioxidant and hypolipidemic properties ."], ["reduced glutathione , tetraethoxypropane , and 2-thiobarbituric acid were procured from m / s sigma chemical company , st .", "chitosan ( mw , 750,000  da ; viscosity , 8  cp ; deacetylation rate , 8587  % ; purity , 98.6  % ) used in the experiment was a kind gift from dr . t. k. thankkappan , principal scientist , central institute of fisheries technology , cochin , india .", "male wistar strain albino rats , weighing 120150  g [ 18 young rats of 23  months old ( mean age , 79.2    6.53  days ) ] and 350400  g [ 18 aged rats of 2025  months old ( mean age , 712    51.6  days ) ] , were selected for the study .", "the animals were housed individually in polypropylene cages under hygienic and standard environmental conditions ( 28    2  c ; humidity , 6070  % ; 12  h light / dark cycle ) .", "the animals were allowed a standard diet ( m / s sai foods , bangalore , india ; table  1 ) and water ad libitum .", "the experiment was carried out according to guidelines of the committee for the purpose of control and supervision of experiments on animals , new delhi , india , and approved by the institutional animal ethics committee of the central institute of fisheries technology , cochin , india.table 1composition of standard diets .", "no.ingredientscomposition ( g/100  g diet)1carbohydrate ( nitrogen free)56.22crude protein22.03ash7.54crude oil4.25crude fiber3.06glucose2.57vitamins1.88sand silica1.49calcium0.810phosphorus0.6 composition of standard diet seven days after acclimatization , the animals were divided into two major groups : group i consisted of 18 normal young rats and group ii consisted of 18 normal aged rats .", "each group was further subdivided into three groups ( six rats each ) : one control group ( group ia and group iia ) and two experimental groups based on the duration of supplementation of chitosan at 2  % level along with feed 30  days ( group ib and group iib ) and 60  days ( group ic and group iic ) . at the end of the experimental period ,", "the animals were sacrificed , and the blood was collected in a heparinized tube for the separation of plasma .", "the heart tissue was excised immediately and homogenized in ice - cold 0.1-m tris  hcl buffer in a potter ", "the homogenate was used for the estimation of lipid peroxides ( lpo ; ohkawa et al .", "1979 ) , reduced glutathione ( gsh ; ellman 1959 ) , glutathione reductase ( gr ; stall et al .", "high - density lipoprotein - cholesterol ( hdl - cholesterol ) in plasma was determined by the method of izzo et al .", "( 1981 ) , and low - density lipoprotein - cholesterol ( ldl - cholesterol ) was estimated according to the calculation of friedwald et al .", "( 1974 ) after extracting total lipids according to the method of folch et al .", "multiple comparisons of the significant anova were performed by duncan 's multiple range comparison test . a p value  < 0.05 was considered as statistically significant .", "all data were analyzed with the aid of statistical package program spss 12.0 for windows ."], ["reduced glutathione , tetraethoxypropane , and 2-thiobarbituric acid were procured from m / s sigma chemical company , st .", "chitosan ( mw , 750,000  da ; viscosity , 8  cp ; deacetylation rate , 8587  % ; purity , 98.6  % ) used in the experiment was a kind gift from dr . t. k. thankkappan , principal scientist , central institute of fisheries technology , cochin , india ."], ["male wistar strain albino rats , weighing 120150  g [ 18 young rats of 23  months old ( mean age , 79.2    6.53  days ) ] and 350400  g [ 18 aged rats of 2025  months old ( mean age , 712    51.6  days ) ] , were selected for the study .", "the animals were housed individually in polypropylene cages under hygienic and standard environmental conditions ( 28    2  c ; humidity , 6070  % ; 12  h light / dark cycle ) .", "the animals were allowed a standard diet ( m / s sai foods , bangalore , india ; table  1 ) and water ad libitum .", "the experiment was carried out according to guidelines of the committee for the purpose of control and supervision of experiments on animals , new delhi , india , and approved by the institutional animal ethics committee of the central institute of fisheries technology , cochin , india.table 1composition of standard diets .", "no.ingredientscomposition ( g/100  g diet)1carbohydrate ( nitrogen free)56.22crude protein22.03ash7.54crude oil4.25crude fiber3.06glucose2.57vitamins1.88sand silica1.49calcium0.810phosphorus0.6 composition of standard diet"], ["seven days after acclimatization , the animals were divided into two major groups : group i consisted of 18 normal young rats and group ii consisted of 18 normal aged rats .", "each group was further subdivided into three groups ( six rats each ) : one control group ( group ia and group iia ) and two experimental groups based on the duration of supplementation of chitosan at 2  % level along with feed 30  days ( group ib and group iib ) and 60  days ( group ic and group iic ) . at the end of the experimental period ,", "the animals were sacrificed , and the blood was collected in a heparinized tube for the separation of plasma .", "the heart tissue was excised immediately and homogenized in ice - cold 0.1-m tris  hcl buffer in a potter ", "the homogenate was used for the estimation of lipid peroxides ( lpo ; ohkawa et al .", "1979 ) , reduced glutathione ( gsh ; ellman 1959 ) , glutathione reductase ( gr ; stall et al . 1969 ) , and glutathione peroxidase ( gpx ; paglia and valentine 1967 ) .", "high - density lipoprotein - cholesterol ( hdl - cholesterol ) in plasma was determined by the method of izzo et al .", "( 1981 ) , and low - density lipoprotein - cholesterol ( ldl - cholesterol ) was estimated according to the calculation of friedwald et al .", "( 1974 ) after extracting total lipids according to the method of folch et al ."], ["multiple comparisons of the significant anova were performed by duncan 's multiple range comparison test . a p value  < 0.05 was considered as statistically significant .", "all data were analyzed with the aid of statistical package program spss 12.0 for windows ."], ["significant ( p  <  0.05 ) variation was observed in the body weight ( grams ) of young ( initial , 129    9.05 ; final , 249    18.3 ) and aged ( initial , 382    27.2 ; final , 336    23.1 ) chitosan - supplemented groups of rats .", "the significant ( p  <  0.05 ) loss observed in the body weight of chitosan - supplemented aged animals might be related to the fibrous nature of chitosan .", "interestingly , the total food intake ( grams per 60  days ) of aged chitosan - supplemented rats ( 904    78.4 ) was comparable to that of young rats ( 745    59.5 ) .", "level of total cholesterol was significantly ( p  <  0.05 ) higher in plasma and heart tissue of group iia aged rats as compared to group ia young control rats , indicating the development of mild age - associated hypercholesterolemic condition ( tables  2 and 3 ) .", "the level of ldl - cholesterol was slightly ( p  <  0.05 ) higher in group iia aged rats , whereas hdl - cholesterol levels were significantly lower compared to group ia young animals ( table  2 ) .", "this aspect might be due to the augmented mobilization of ldl - cholesterol from the blood into the cell membranes , resulting in abnormal cholesterol deposition in the myocardium .", "in the present study , the dietary supplementation with chitosan significantly reduced the total cholesterol level in plasma and myocardial tissue of group iic aged rats as compared to group iia rats .", "it also kept the levels of ldl - cholesterol and hdl - cholesterol in plasma comparable to that of group ic rats .", "2009 ) , which showed that the positively charged amino groups of chitosan possess the ability to bind negatively charged molecules such as lipids and bile acids , inducing a greater fractional excretion in the feces . also", "( 2007 ) suggested that chitosan improve lipid metabolism by regulating total cholesterol and ldl - cholesterol by upregulation of hepatic ldl receptor mrna expression , increasing the excretion of fecal bile acids .", "previous studies ( yao and chiang 2002 ) pointed out that chitosan supplementation was capable of lowering the levels of plasma total cholesterol and ldl - cholesterol in experimental animals . in the present study , a slight decline in the level of total cholesterol and ldl - cholesterol were also noted in group ic chitosan - fed young rats , ascertaining the anticholesterolemic property of chitosan ( ylitalo et al .", "2002).table 2effect of dietary chitosan supplementation on total cholesterol , hdl - cholesterol , and ldl - cholesterol in plasma of young and aged ratsparametersyoung ratsaged ratsgroup ia ( control)group ib ( 30  days)group ic ( 60  days)group iia ( control)group iib ( 30  days)group iic ( 60  days)total cholesterol79.9    5.36 a , b76.2    4.98 a , b73.1    5.07 a98.4    9.12 c91.7    8.56 c87.3    8.21 b , chdl - cholesterol42.2    3.11 a , b ,", "c44.9    3.48 b , c47.5    3.29 c33.2    2.68 d36.4    2.56 a39.7    3.74 a , bldl - cholesterol24.3    1.42 a22.7    1.31 a , b20.3    1.18 b48.4    2.72 c43.1    2.14 d38.3    2.27 eresults are mean    sd for six rats .", "values expressed : total cholesterol , hdl - cholesterol , and ldl - cholesterol , milligrams per deciliter .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test)table 3effect of dietary chitosan supplementation on the levels of total cholesterol , lipid peroxides , and reduced glutathione ( gsh ) and the activities of glutathione - dependent antioxidant enzymes [ glutathione peroxidase ( gpx ) and glutathione reductase ( gr ) ] in the heart tissue of young and aged ratsparametersyoung ratsaged ratsgroup ia ( control)group ib ( 30  days)group ic ( 60  days)group iia ( control)group iib ( 30  days)group iic ( 60  days)total cholesterol2.72    0.17 a , b2.56    0.14 a2.43    0.16 a3.68    0.32 c3.29    0.21 d3.04    0.25 b , dlipid peroxides1.08    0.07 a0.98    0.06 a0.97    0.07 a2.58    0.14 b1.96    0.09 c1.54    0.11 dgsh10.9    0.84 a12.3    1.02 a , b14.5    1.17 c7.54    0.61 d8.76    0.69 d , e9.52    0.85 a , egpx5.23    0.35 a5.51    0.41 a , b5.98    0.37 b3.12    0.18 c4.05    0.26 d4.56    0.34 dgr0.43    0.02 a0.52    0.04 b0.58    0.03 c0.22    0.01 d0.29    0.03 e0.36    0.02 fresults are mean    sd for six animals .", "values expressed : total cholesterol , milligrams per gram wet tissue ; lipid peroxides , nanomoles mda released per milligram protein ; gsh , micrograms per milligram protein ; gpx , micrograms gsh oxidized per minute per milligram protein ; gr , nanomoles nadph oxidized per minute per milligram protein .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test ) effect of dietary chitosan supplementation on total cholesterol , hdl - cholesterol , and ldl - cholesterol in plasma of young and aged rats results are mean    sd for six rats .", "values expressed : total cholesterol , hdl - cholesterol , and ldl - cholesterol , milligrams per deciliter .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test ) effect of dietary chitosan supplementation on the levels of total cholesterol , lipid peroxides , and reduced glutathione ( gsh ) and the activities of glutathione - dependent antioxidant enzymes [ glutathione peroxidase ( gpx ) and glutathione reductase ( gr ) ] in the heart tissue of young and aged rats results are mean    sd for six animals .", "values expressed : total cholesterol , milligrams per gram wet tissue ; lipid peroxides , nanomoles mda released per milligram protein ; gsh , micrograms per milligram protein ; gpx , micrograms gsh oxidized per minute per milligram protein ; gr , nanomoles nadph oxidized per minute per milligram protein .", "values that have a different letter ( a , b , c , d , e , f ) differ significantly with each other ( p  <  0.05 ; duncan 's multiple range test ) sumiyoshi and kimura ( 2006 ) suggested that the lipid - lowering effects of chitosan might be mediated by increases in fecal fat and/or bile acid excretion resulting from the binding of bile acids , and by a decrease in the absorption of dietary cholesterol from the small intestine .", "( 2009 ) have shown that normal intestinal microbiota might also positively influence immune responses and protect against the development of inflammatory diseases systemically through the formation of short - chain fatty acids by fermentation of dietary fiber in the intestine .", "chitosan , a biopolymer of glucosamine derived from chitin that is chemically similar to that of cellulose , acts as a dietary fiber in gastrointestinal tract ( gallaher et al .", "it is possible that chitosan may function through the generation of gut metabolites , such as short - chain fatty acids / short - chain oligosaccharides , in attenuating the development of inflammatory processes related to aging . in the present study", ", there was a significant ( p  <  0.05 ) increase in the level of lipid peroxidation observed with a concomitant reduction in the level of nonenzymatic ( gsh ) and enzymatic ( gpx and gr ) antioxidants in the heart tissue of group iia aged rats as compared to group ia young control animals ( table  3 ) .", "this concurs with the earlier findings ( subramanian and james 2010 ) , which indicated that the higher vulnerability of aged myocardium to peroxidative damage was mainly due to a decline in the level of free radical scavengers .", "depletion of gsh results in enhanced lipid peroxidation , and excessive lipid peroxidation can cause increased gsh consumption during aging ( denniss et al .", "the reduction in the activity of gpx and gr may be due to the reduced availability of gsh .", "gpx offers protection to the cellular and subcellular membranes from the peroxidative damage by eliminating hydrogen peroxide and lipid peroxide ( li et al .", "inhibition of gsh - dependent antioxidant enzymes makes myocardial cell membranes more susceptible to oxidative damage in aging .", "gsh and gsh - dependent enzyme systems may be directly related to the pathogenic mechanisms related to age - associated disorders ( goncharova et al .", "2007 ) . in our study , the dietary chitosan intake significantly attenuated the age - associated oxidative stress and maintained the level of the glutathione - dependent antioxidant status in the heart tissue at near normal .", "( 2003 ) have shown that chitosan has strong antioxidative effects , which decrease free radical production and increase antioxidant enzyme activities during ccl4-induced lipid peroxidation in rats .", "( 2004 ) have suggested that chitosan may eliminate various free radicals by the action of nitrogen on the c-2 position of the chitosan .", "( 2001 ) reported that the scavenging mechanism of chitosan is related to the fact that the free radicals can react with the hydrogen ion from the ammonium ions to form a stable molecule .", "the normal young rats receiving chitosan ( group ic ) did not show any significant change when compared with normal ( group ia ) rats , indicating that it does not per se have any adverse effects .", "in conclusion , the overall antiaging effect of dietary chitosan intake is probably related to its ability to inhibit the increased accumulation of lipids both in the systemic circulation and in the myocardium by its antilipidemic property , or to normal maintenance of the activities of glutathione - dependent antioxidant enzymes and the level of gsh , which protect myocardial membrane against oxidative stress by decreasing lipid peroxidation reactions ."]]}
{"article_id": "PMC3886103", "article_text": ["cleft lip and palate ( clp ) patients are going through an extensive treatment with \n orthodontic and surgical correction of the position of teeth and hard and soft \n tissues .", "the aim of the primary surgery is to close the clefts of the lip and the \n soft palate .", "the aim of the secondary surgery is to close the cleft of the alveolus \n by bone grafting .", "surgery of the clefts induces formation of scar tissue , which \n affects the soft tissue matrix that guides the development of the maxillary complex . \n", "the scar tissue as well as a reduced growth of the mid face often causes a maxillary \n retrognatism , that adversely affects the chewing function , the ability to speak , the \n facial aesthetics , social life   as well as psychological and general \n wellbeing of the clp - patients . for that \n reason ,", "between 25 to 60% of the clp - patients need a surgical treatment of the \n maxillary retrognatism", ". this \n surgical - orthodontic treatment is the last part of the rehabilitation of the \n clp - patient , which has the objective to create a good dental occlusion and function , \n more harmonic facial aesthetics and increase the quality of life .", "since the \n 1970 's the standard treatment of clp - patients with a maxillary hypoplasia has \n been a le fort i osteotomy with a bone graft .", "treating extensive sagittal discrepancies maxillary surgery has to \n be combined with mandibular set - back .", "maxillary surgery is difficult in clp - patients \n because of the scar tissue , a tendency to relapse , poor bone quality and quantity \n and a reduced blood supply in the affected area .", "these conditions are responsible for a typical relapse of 20 - 25% \n . to compensate for this an overcorrection", "is often made during the \n surgical procedure , which can lead to an \n unpredictable final result .", "polley and figueroa   described the use of \n distraction osteogenesis ( do ) as an alternative treatment of maxillary hypoplasia \n using an external bone distraction device .", "the principle of this treatment was to \n induce formation of immature bone in the gap after a le fort i osteotomy by gradual \n tensile strength separating the two segments .", "studies of the treatment have shown a significantly reduced \n tendency of relapse , favourable changes \n of the soft tissue   and changes of the \n velopharyngeal closure similar to that of conventional advancement .", "the duration of the course of treatment is up to 16 weeks longer when choosing do . in \n this period", "the appliance penetrates the buccal mucosa in the sulcus and the patient \n must take care of the daily activation and keep it clean .", "the aim of this retrospective pilot study was to examine and compare the cleft lip \n and palate patients ' satisfaction after treatment with either maxillary distraction \n or traditional advancement of the maxilla after a le fort i osteotomy .", "in the period 1996 - 2007 forty - two clp - patients with need for advancement of the \n maxilla were surgically treated at the department of oral and maxillofacial surgery , \n aarhus university hospital by either traditional advancement of the maxilla or \n distraction osteogenesis . until 2000", "the standard regime of treatment was a \n traditional advancement of the maxillary complex after le fort i osteotomy . since \n 2000 maxillary distraction has provided an alternative in treatment of clp - patients \n requiring a maxillary advancement of 10 mm or more .", "consequently , the patients in \n this study consisted of two groups of clp - patients with the need for \n surgical - orthodontic correction of maxillary hypoplasia .", "a criterion for both groups \n was that the postsurgical orthodontics had to be finished if the patient was to be \n included in the study .", "conventional group ( conv ) included patients treated with \n traditional advancement of the maxilla after a le fort i osteotomy in the period \n 1996 - 2007 .", "the group do included patients treated with maxillary distraction table 1 . patients ' characteristics according to gender , type of cleft , type \n of surgery performed , type of distractors used and age at surgery unsegmented le fort i osteotomy and internal distraction . unsegmented le fort i osteotomy and external distraction .", "m = male ; f = female ; uclp = unilateral cleft lip and palate ; bclp = \n bilateral cleft lip and palate ; cl .", "fifteen patients met the inclusion criteria in the do group and ten patients in the \n conv group .", "the composition of the two groups according to gender , type of cleft , \n type of distraction , type of distractors used , age at surgery and type of surgery \n performed is presented in table 1 .", "group do consisted of 10 males and 5 females with \n a mean age at the time of surgery of 17.5 ( sd 2.3 ) years while group conv consisted \n of 5 males and 5 females with a mean age at the time of surgery of 17.8 ( sd 2.6 ) \n years . a quantitative method , a questionnaire ,", "existing literature was reviewed and a list of \n relevant subjects was made and operationalized in a questionnaire containing 13 \n questions about the patients ' perception of aesthetics and function .", "the patients were asked to answer by making a mark on a vas - scale scaled 0 - 100 \n . the score 0 indicating the highest \n level of satisfaction with functional parameters or facial aesthetics and the score \n 100 indicating the lowest level of satisfaction .", "the end points of the scales were \n unequivocal and easy to understand , for example no pain / intolerable pain ; very \n satisfied / very unsatisfied ; no discomfort / worst imaginable discomfort .", "the \n accompanying letter contained information about the aim of the study and how to \n answer .", "the patients were asked to return the questionnaire by pre - paid mail after \n two weeks .", "the study was approved \n by the central denmark region committees on biomedical research ethics .", "all patients had presurgical orthodontic treatment and were treated surgically with a \n high le fort i osteotomy . in the conv group", "the treatment was a conventional \n combined orthodontic - surgical treatment with model surgery and splint fabrication \n for the maxillary position and completed with postsurgical orthodontics .", "the \n patients in the conv group had an average advancement of 6.98 mm ( range 5 - 11 mm ) . \n", "patients from the do group had the distraction device placed in pre - planned position \n calculated from lateral cephalograms and adjusted on a three - dimensional print of \n the skull based on a computed tomography ( ct ) or a cone beam ct ( cbct ) .", "the appliances used for distraction were synthes internal distractor ( synthes , west \n chester , pa , usa ) , kls martin internal distractor ( kls martin group , tuttlingen , \n germany ) and rigid external device ( red ) kls martin ( kls martin group , tuttlingen , \n germany ) for the external distraction procedures ( table 1 ) .", "all the patients treated with distraction were registered for model surgery using the \n same procedure as for the conventional treated patients using face bow registration , \n wax bite and articulator mounting .", "the patients had surgical splints fabricated and \n in the cases of do the splint was used as a guide for using intermaxillary elastics \n after the active distraction .", "the maxilla was thereby manipulated into the final \n planned position . in five of the do patients the palatal defect caused an unstable \n intersegmental position .", "the use of the splint was refrained from and intermaxillary \n elastics were used to guide the segments into occlusion . during the surgical procedure the internal distraction devices were adapted to the \n maxilla in the planned position .", "the screw holes were marked and the devices were \n removed in order to complete the le fort i osteotomy .", "after ensuring mobility of the \n maxilla the devices were fixed to the maxilla and tested for interference free \n activation .", "the mean latency period after device placement was 4.9 days ( range 4 - 7 days ) . \n during the active phase of distraction", "the appliances were activated on a daily \n basis either by the patient or an assistant , usually a family member .", "activation was \n done twice a day with a rate of 0.5 mm , corresponding to 1.0 mm daily .", "activation \n went on until the planned position of the maxilla was achieved and intermaxillary \n elastics were then used to adjust the final position .", "the mean periodof active \n distraction was 17.7 days ( range 13 - 28 days ) and the mean advancement of the \n maxilla was 12 mm ( range 6 - 16 mm ) .", "after an average consolidation period of 77.3 \n days ( range 35 - 213 days ) , the patients were readmitted and the devices removed in \n general anaesthesia .", "the treatment was a conventional combined orthodontic - surgical treatment with \n model surgery and splint fabrication for the maxillary position and completed with \n postsurgical orthodontics .", "unpaired t - tests were performed to analyse intergroup differences regarding duration \n of orthodontic treatment and vas - scores in spss 18.0 ( ibm , usa ) . statistical \n significance was defined as p < 0.05 .", "the duration of the orthodontic treatment was in average 10 months longer in the do \n group than the conv group and this difference was significant ( p < 0.05 ) . neither \n the age distribution nor the period of time between surgery and completion of the \n questionnaire differed significantly ( p < 0.05 ) table 2 .", "distribution of the patients ' groups according to gender , age at \n surgery and period between surgery and completion of the \n questionnaire after the primary surgery with insertion of the \n distractors . after the surgical procedure .", "unpaired t - test , states p < 0.05 between patients of do \n and conv groups .", "nineteen out of 25 patients returned the questionnaires and the total response ratio \n was 76% .", "results of the patients ' satisfaction on a continuous visual \n analog - scale ( vas ) on a vas - scale scaled 0 - 100 with the score 0 indicating the \n highest level of satisfaction with functional parameters or facial \n aesthetics and the score 100 indicating the lowest level of \n satisfaction .", "both groups felt a great deal of satisfaction with their appearance both according to \n themselves and the perceptions of relatives and other people 's reaction in \n general .", "both groups were satisfied with their general well being and felt few \n restrictions or discomfort during social activities . according to functional \n parameters the groups were alike .", "both groups were minimally affected by pain and \n sensory disturbances and reported a great deal of satisfaction with speech and \n breathing .", "the discomfort during eating and drinking and sleep were low as well in \n both groups .", "the greatest difference occurred in the parameter satisfaction with the \n duration of the treatment course .", "the do group scored higher on the vas - scale \n according to less satisfaction with the duration than the conv group .", "statistical \n analyses revealed no significant differences ( p > 0.05 ) among the groups which \n may be explained by the numbers of patients included in the study .", "distraction osteogenesis has become a widely used treatment of maxillary hypoplasia \n in clp - patients because of the reports of better stability and the possibility for \n larger advancements .", "however , it is a more complicated treatment because of the \n period with active distraction , the obligate need for good cooperation and the long \n consolidation phase with the patient still wearing the appliance .", "the present study \n is a pilot study and evaluates retrospectively patients ' satisfaction in two groups \n of clp - patients treated for maxillary hypoplasia with either maxillary distraction \n or conventional le fort i advancement .", "the number of patients was limited and \n especially the conv group was small and the number of patients responding was lower \n than in the do group .", "the results indicated a high level of satisfaction with the facial aesthetic at the \n end of treatment in both the do and the conv group .", "the parameters pain and sensory \n disturbances did not differ a lot between the groups in this study .", "it could be \n hypothesized that more extensive osteotomies and preoperative advancements in conv \n could induced more sensory disturbances and pain compared with a gradual movement \n and distraction histogenesis of soft tissues when using do .", "the groups had comparable scores according to the functional parameters eating and \n drinking , sleep , speech and breathing .", "do was significantly less satisfied with the \n duration of the treatment than conv probably according to a longer duration of the \n orthodontic treatment .", "the influence of the period with the distractors mounted on \n the level of satisfaction was not measured but it can be hypothesized that this \n period is troublesome .", "further studies of this period between mounting and removal \n is needed as the prolonged total treatment time is a disadvantage and perhaps the \n most important difference when choosing do instead of conventional treatment", ". a \n greater understanding of the cellular processes during the period of distraction and \n the period of consolidation and possibility to accelerate the genesis and maturation \n of bone by the use of pharmacological agents could reduce the duration of these \n periods . by a reduction of the duration of these aforementioned periods", "the \n probable inconveniences could be reduced and possibly of a greater patients ' \n satisfaction and lesser discomfort could be achieved . another way to reduce the \n strain on the", "do patients would be the continuous development of the distractors , \n e.g. reduction in size , partly removable appliances , continuous activation etc . \n .", "patients ' satisfaction in association with distraction is a subject sparsely \n examined in the existing literature .", "no studies describes patients ' satisfaction \n during internal distraction while only a single retrospective survey with a quite \n small population has studied patients ' satisfaction during external distraction .", "concluded that most of \n the patients receiving red treatment had an increased satisfaction with facial \n aesthetics after the treatment than before the treatment in spite of a great deal of \n dissatisfaction with the facial aesthetics during the course of treatment .", "a long term study of patients ' perception of function and satisfaction showed that \n orthognathic surgery resulted in a subjective estimation of function , appearance , \n health , and interpersonal relationships that was higher than that among pretreatment \n and no - treatment control groups .", "a newly \n prospective study of patients receiving orthognathic surgery showed that perception \n of function and general satisfaction significantly increased after orthognathic \n surgery and that the parameters were positive correlated .", "this study also showed a general high level of \n satisfaction with appearance and functional parameters in clp - patients treated with \n either do or conventional treatment .", "the retrospective design of this survey and the two years between the operation and \n the completion of the questionnaire diminished the reliability of the patients ' \n answers .", "the use of a questionnaires cause problems with the internal validity , \n because the questionnaire is designed with a limited amount of topics , that might \n not all be the most valid according to every single patient but representing a \n selection bias . further use of this questionnaire necessitates a validation .", "instead \n a validated questionnaire , for instance the oral health impact profile or \n orthognathic quality of life questionnaire can be used in further studies .", "prospective surveys should \n be based on a validated questionnaire and include the period between mounting and \n removal of the distractors .", "a comparative study of patients ' satisfaction during \n three different courses of treatment , traditional advancement , external distraction \n and internal distraction , could probably lead to a greater understanding of this \n important subject .", "1 . cleft lip and palate patients experience a high level of satisfaction with \n functional parameters and aesthetics after surgical - orthodontic treatment of \n maxillary hypoplasia .", "patients treated with distraction osteogenesis were less satisfied with the \n duration of their treatment than the conventional group .", "no external \n funding , apart from the support of the authors ' institution , was available for \n this study ."], "abstract_text": ["<S> abstractobjectivesto compare cleft lip and palate patients ' satisfaction with aesthetics and \n functional parameters after conventional advancement of the maxilla or by \n the use of distraction osteogenesis.material and methodscase series observational study . </S>", "<S> group of distraction osteogenesis ( do ) \n consisted of 15 patients treated with distraction osteogenesis while group \n conventional ( conv ) included 10 patients treated with traditional \n advancement of the maxilla . </S>", "<S> patients were asked to fill out a questionnaire \n about their subjective evaluation of satisfaction with facial aesthetics and \n functional parameters on a continuous visual analog - scale ( vas ) when the \n treatment was finished.resultsthe total response rate was 76% . </S>", "<S> preoperatively the two groups did not differ \n significantly according to group characteristics . at follow - up </S>", "<S> both groups \n were satisfied with aesthetics and functional parameters . </S>", "<S> the do group was \n less satisfied with the duration of the treatment than the conv group . </S>", "<S> there \n were no statistically significant differences among the groups regarding \n functional parameters or facial aesthetics.conclusionscleft lip and palate patients experienced a high level of satisfaction with \n functional parameters and aesthetics as a result of surgical maxillary \n advancement . </S>", "<S> the patients treated with distraction osteogenesis were less \n satisfied with the duration of the treatment . </S>", "<S> further studies are \n needed . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS", "ACKNOWLEDGEMENTS AND DISCLOSURE STATEMENTS"], "sections": [["cleft lip and palate ( clp ) patients are going through an extensive treatment with \n orthodontic and surgical correction of the position of teeth and hard and soft \n tissues .", "the aim of the primary surgery is to close the clefts of the lip and the \n soft palate .", "the aim of the secondary surgery is to close the cleft of the alveolus \n by bone grafting .", "surgery of the clefts induces formation of scar tissue , which \n affects the soft tissue matrix that guides the development of the maxillary complex . \n", "the scar tissue as well as a reduced growth of the mid face often causes a maxillary \n retrognatism , that adversely affects the chewing function , the ability to speak , the \n facial aesthetics , social life   as well as psychological and general \n wellbeing of the clp - patients . for that \n reason ,", "between 25 to 60% of the clp - patients need a surgical treatment of the \n maxillary retrognatism", ". this \n surgical - orthodontic treatment is the last part of the rehabilitation of the \n clp - patient , which has the objective to create a good dental occlusion and function , \n more harmonic facial aesthetics and increase the quality of life .", "since the \n 1970 's the standard treatment of clp - patients with a maxillary hypoplasia has \n been a le fort i osteotomy with a bone graft .", "treating extensive sagittal discrepancies maxillary surgery has to \n be combined with mandibular set - back .", "maxillary surgery is difficult in clp - patients \n because of the scar tissue , a tendency to relapse , poor bone quality and quantity \n and a reduced blood supply in the affected area .", "these conditions are responsible for a typical relapse of 20 - 25% \n . to compensate for this an overcorrection", "is often made during the \n surgical procedure , which can lead to an \n unpredictable final result .", "polley and figueroa   described the use of \n distraction osteogenesis ( do ) as an alternative treatment of maxillary hypoplasia \n using an external bone distraction device .", "the principle of this treatment was to \n induce formation of immature bone in the gap after a le fort i osteotomy by gradual \n tensile strength separating the two segments .", "studies of the treatment have shown a significantly reduced \n tendency of relapse , favourable changes \n of the soft tissue   and changes of the \n velopharyngeal closure similar to that of conventional advancement .", "the duration of the course of treatment is up to 16 weeks longer when choosing do . in \n this period", "the appliance penetrates the buccal mucosa in the sulcus and the patient \n must take care of the daily activation and keep it clean .", "the aim of this retrospective pilot study was to examine and compare the cleft lip \n and palate patients ' satisfaction after treatment with either maxillary distraction \n or traditional advancement of the maxilla after a le fort i osteotomy ."], ["in the period 1996 - 2007 forty - two clp - patients with need for advancement of the \n maxilla were surgically treated at the department of oral and maxillofacial surgery , \n aarhus university hospital by either traditional advancement of the maxilla or \n distraction osteogenesis . until 2000", "the standard regime of treatment was a \n traditional advancement of the maxillary complex after le fort i osteotomy . since \n 2000 maxillary distraction has provided an alternative in treatment of clp - patients \n requiring a maxillary advancement of 10 mm or more .", "consequently , the patients in \n this study consisted of two groups of clp - patients with the need for \n surgical - orthodontic correction of maxillary hypoplasia .", "a criterion for both groups \n was that the postsurgical orthodontics had to be finished if the patient was to be \n included in the study .", "conventional group ( conv ) included patients treated with \n traditional advancement of the maxilla after a le fort i osteotomy in the period \n 1996 - 2007 .", "the group do included patients treated with maxillary distraction table 1 . patients ' characteristics according to gender , type of cleft , type \n of surgery performed , type of distractors used and age at surgery unsegmented le fort i osteotomy and internal distraction . unsegmented le fort i osteotomy and external distraction .", "m = male ; f = female ; uclp = unilateral cleft lip and palate ; bclp = \n bilateral cleft lip and palate ; cl .", "fifteen patients met the inclusion criteria in the do group and ten patients in the \n conv group .", "the composition of the two groups according to gender , type of cleft , \n type of distraction , type of distractors used , age at surgery and type of surgery \n performed is presented in table 1 .", "group do consisted of 10 males and 5 females with \n a mean age at the time of surgery of 17.5 ( sd 2.3 ) years while group conv consisted \n of 5 males and 5 females with a mean age at the time of surgery of 17.8 ( sd 2.6 ) \n years . a quantitative method , a questionnaire ,", "existing literature was reviewed and a list of \n relevant subjects was made and operationalized in a questionnaire containing 13 \n questions about the patients ' perception of aesthetics and function .", "the patients were asked to answer by making a mark on a vas - scale scaled 0 - 100 \n . the score 0 indicating the highest \n level of satisfaction with functional parameters or facial aesthetics and the score \n 100 indicating the lowest level of satisfaction .", "the end points of the scales were \n unequivocal and easy to understand , for example no pain / intolerable pain ; very \n satisfied / very unsatisfied ; no discomfort / worst imaginable discomfort .", "the \n accompanying letter contained information about the aim of the study and how to \n answer .", "the patients were asked to return the questionnaire by pre - paid mail after \n two weeks .", "the study was approved \n by the central denmark region committees on biomedical research ethics .", "all patients had presurgical orthodontic treatment and were treated surgically with a \n high le fort i osteotomy . in the conv group", "the treatment was a conventional \n combined orthodontic - surgical treatment with model surgery and splint fabrication \n for the maxillary position and completed with postsurgical orthodontics .", "the \n patients in the conv group had an average advancement of 6.98 mm ( range 5 - 11 mm ) . \n", "patients from the do group had the distraction device placed in pre - planned position \n calculated from lateral cephalograms and adjusted on a three - dimensional print of \n the skull based on a computed tomography ( ct ) or a cone beam ct ( cbct ) .", "the appliances used for distraction were synthes internal distractor ( synthes , west \n chester , pa , usa ) , kls martin internal distractor ( kls martin group , tuttlingen , \n germany ) and rigid external device ( red ) kls martin ( kls martin group , tuttlingen , \n germany ) for the external distraction procedures ( table 1 ) .", "all the patients treated with distraction were registered for model surgery using the \n same procedure as for the conventional treated patients using face bow registration , \n wax bite and articulator mounting .", "the patients had surgical splints fabricated and \n in the cases of do the splint was used as a guide for using intermaxillary elastics \n after the active distraction .", "the maxilla was thereby manipulated into the final \n planned position . in five of the do patients the palatal defect caused an unstable \n intersegmental position .", "the use of the splint was refrained from and intermaxillary \n elastics were used to guide the segments into occlusion . during the surgical procedure the internal distraction devices were adapted to the \n maxilla in the planned position .", "the screw holes were marked and the devices were \n removed in order to complete the le fort i osteotomy .", "after ensuring mobility of the \n maxilla the devices were fixed to the maxilla and tested for interference free \n activation .", "the mean latency period after device placement was 4.9 days ( range 4 - 7 days ) . \n during the active phase of distraction", "the appliances were activated on a daily \n basis either by the patient or an assistant , usually a family member .", "activation was \n done twice a day with a rate of 0.5 mm , corresponding to 1.0 mm daily .", "activation \n went on until the planned position of the maxilla was achieved and intermaxillary \n elastics were then used to adjust the final position .", "the mean periodof active \n distraction was 17.7 days ( range 13 - 28 days ) and the mean advancement of the \n maxilla was 12 mm ( range 6 - 16 mm ) .", "after an average consolidation period of 77.3 \n days ( range 35 - 213 days ) , the patients were readmitted and the devices removed in \n general anaesthesia .", "the treatment was a conventional combined orthodontic - surgical treatment with \n model surgery and splint fabrication for the maxillary position and completed with \n postsurgical orthodontics .", "unpaired t - tests were performed to analyse intergroup differences regarding duration \n of orthodontic treatment and vas - scores in spss 18.0 ( ibm , usa ) . statistical \n significance was defined as p < 0.05 ."], ["the duration of the orthodontic treatment was in average 10 months longer in the do \n group than the conv group and this difference was significant ( p < 0.05 ) . neither \n the age distribution nor the period of time between surgery and completion of the \n questionnaire differed significantly ( p < 0.05 ) table 2 .", "distribution of the patients ' groups according to gender , age at \n surgery and period between surgery and completion of the \n questionnaire after the primary surgery with insertion of the \n distractors . after the surgical procedure .", "unpaired t - test , states p < 0.05 between patients of do \n and conv groups .", "nineteen out of 25 patients returned the questionnaires and the total response ratio \n was 76% .", "results of the patients ' satisfaction on a continuous visual \n analog - scale ( vas ) on a vas - scale scaled 0 - 100 with the score 0 indicating the \n highest level of satisfaction with functional parameters or facial \n aesthetics and the score 100 indicating the lowest level of \n satisfaction .", "both groups felt a great deal of satisfaction with their appearance both according to \n themselves and the perceptions of relatives and other people 's reaction in \n general .", "both groups were satisfied with their general well being and felt few \n restrictions or discomfort during social activities . according to functional \n parameters the groups were alike .", "both groups were minimally affected by pain and \n sensory disturbances and reported a great deal of satisfaction with speech and \n breathing .", "the discomfort during eating and drinking and sleep were low as well in \n both groups .", "the greatest difference occurred in the parameter satisfaction with the \n duration of the treatment course .", "the do group scored higher on the vas - scale \n according to less satisfaction with the duration than the conv group .", "statistical \n analyses revealed no significant differences ( p > 0.05 ) among the groups which \n may be explained by the numbers of patients included in the study ."], ["distraction osteogenesis has become a widely used treatment of maxillary hypoplasia \n in clp - patients because of the reports of better stability and the possibility for \n larger advancements .", "however , it is a more complicated treatment because of the \n period with active distraction , the obligate need for good cooperation and the long \n consolidation phase with the patient still wearing the appliance .", "the present study \n is a pilot study and evaluates retrospectively patients ' satisfaction in two groups \n of clp - patients treated for maxillary hypoplasia with either maxillary distraction \n or conventional le fort i advancement .", "the number of patients was limited and \n especially the conv group was small and the number of patients responding was lower \n than in the do group .", "the results indicated a high level of satisfaction with the facial aesthetic at the \n end of treatment in both the do and the conv group .", "the parameters pain and sensory \n disturbances did not differ a lot between the groups in this study .", "it could be \n hypothesized that more extensive osteotomies and preoperative advancements in conv \n could induced more sensory disturbances and pain compared with a gradual movement \n and distraction histogenesis of soft tissues when using do .", "the groups had comparable scores according to the functional parameters eating and \n drinking , sleep , speech and breathing .", "do was significantly less satisfied with the \n duration of the treatment than conv probably according to a longer duration of the \n orthodontic treatment .", "the influence of the period with the distractors mounted on \n the level of satisfaction was not measured but it can be hypothesized that this \n period is troublesome .", "further studies of this period between mounting and removal \n is needed as the prolonged total treatment time is a disadvantage and perhaps the \n most important difference when choosing do instead of conventional treatment", ". a \n greater understanding of the cellular processes during the period of distraction and \n the period of consolidation and possibility to accelerate the genesis and maturation \n of bone by the use of pharmacological agents could reduce the duration of these \n periods . by a reduction of the duration of these aforementioned periods", "the \n probable inconveniences could be reduced and possibly of a greater patients ' \n satisfaction and lesser discomfort could be achieved . another way to reduce the \n strain on the", "do patients would be the continuous development of the distractors , \n e.g. reduction in size , partly removable appliances , continuous activation etc . \n .", "patients ' satisfaction in association with distraction is a subject sparsely \n examined in the existing literature .", "no studies describes patients ' satisfaction \n during internal distraction while only a single retrospective survey with a quite \n small population has studied patients ' satisfaction during external distraction .", "concluded that most of \n the patients receiving red treatment had an increased satisfaction with facial \n aesthetics after the treatment than before the treatment in spite of a great deal of \n dissatisfaction with the facial aesthetics during the course of treatment .", "a long term study of patients ' perception of function and satisfaction showed that \n orthognathic surgery resulted in a subjective estimation of function , appearance , \n health , and interpersonal relationships that was higher than that among pretreatment \n and no - treatment control groups .", "a newly \n prospective study of patients receiving orthognathic surgery showed that perception \n of function and general satisfaction significantly increased after orthognathic \n surgery and that the parameters were positive correlated .", "this study also showed a general high level of \n satisfaction with appearance and functional parameters in clp - patients treated with \n either do or conventional treatment .", "the retrospective design of this survey and the two years between the operation and \n the completion of the questionnaire diminished the reliability of the patients ' \n answers .", "the use of a questionnaires cause problems with the internal validity , \n because the questionnaire is designed with a limited amount of topics , that might \n not all be the most valid according to every single patient but representing a \n selection bias . further use of this questionnaire necessitates a validation .", "instead \n a validated questionnaire , for instance the oral health impact profile or \n orthognathic quality of life questionnaire can be used in further studies .", "prospective surveys should \n be based on a validated questionnaire and include the period between mounting and \n removal of the distractors .", "a comparative study of patients ' satisfaction during \n three different courses of treatment , traditional advancement , external distraction \n and internal distraction , could probably lead to a greater understanding of this \n important subject ."], ["1 . cleft lip and palate patients experience a high level of satisfaction with \n functional parameters and aesthetics after surgical - orthodontic treatment of \n maxillary hypoplasia .", "patients treated with distraction osteogenesis were less satisfied with the \n duration of their treatment than the conventional group ."], ["no external \n funding , apart from the support of the authors ' institution , was available for \n this study ."]]}
{"article_id": "PMC3134056", "article_text": ["a rising trend in the incidence and prevalence of inflammatory bowel disease ( ibd ) in asia has been recognized for the past two decades .", "the thiopurine drugs azathioprine ( aza ) and mercaptopurine are widely used for the treatment of ibd and have proven to be effective in both inducing and maintaining remission of crohn 's disease and ulcerative colitis ; however adverse effects can occur in 928% of patients , and these adverse effects often necessitate a dose reduction or discontinuation .", "nevertheless , some reports suggested that neutropenia during aza therapy reduced the relapse rates in ibd patients [ 1 , 2 ] , and that the eradication of sensitized leukocytes , which was done by leukapheresis or bone marrow transplantation , improved ibd in some cases [ 3 , 4 ] .", "there have also been some cases of ulcerative colitis with prolonged remission following aza - induced pancytopenia [ 5 , 6 ] .", "a 22-year - old man with a known history of crohn 's colitis who had undergone fistulectomy for complex perianal fistula 2 years previously was admitted with a complaint of severe weight loss ( 25 kg during 2 years ) , diarrhea , abdominal pain and recurrent perianal tenderness .", "physical exam revealed fistular openings and subcutaneous abscess pockets with severe tenderness on the anus and buttock .", "he was not able to maintain a sitting or supine position , so his social activity was limited .", "contrast - enhanced computed tomography scan of the abdomen showed multifocal inflammatory wall thickening and thick - walled abscess pockets with the beaded appearance of subcutaneous small abscesses in the precoccygeal and perianal area ( fig .", "abscess drainage and a seton operation were performed , then aza was administered at a starting dose of 25 mg ; this was increased up to 50 mg after 2 weeks .", "he then revisited the hospital and presented with high fever ( 39.2c ) and myalgia . on general peripheral blood test , leukocytes were 180/l ( segmented cells 6% ) , hemoglobin was 6.6 g / dl and platelets were 48,000/l .", "after 2 weeks of administering human recombinant granulocyte colony - stimulating factor and broad - spectrum antibiotic therapy , his fever subsided and the cell count fully recovered .", "escherichia coli was found on blood culture , but it was sensitive to the antibiotics .", "after recovering from cytopenia , the frequency of bowel movements decreased and the perianal pain and oozing improved gradually .", "follow - up colonoscopy was performed 3 months later and demonstrated diffuse fibrotic scar instead of multiple ulcerations ( fig .", "he gained 10 kg within 6 months and is now on regular follow - up without clinical recurrence ( cdai score = 114 ) .", "therefore , a conventional immunosuppressive dose of less than 2.5 mg / kg / day causes predictable mild leukopenia .", "rarely however , sudden , severe and unexpected myelosuppression , which is possibly idiosyncratic , has also been reported when low to moderate doses ( < 2 mg / kg / day ) of the drug were used . in these cases ,", "the cytopenic patients are vulnerable to the development of sepsis that necessitates hospital admission for intensive care .", "however , the preferential suppression of neutrophils develops when leukopenia occurs , which suggests that aza 's antiinflammatory effects are mediated through its effects on neutrophils as well .", "in fact , clinical relapse of ibd is characterized by increased neutrophil migration into the intestine .", ". moreover , some reports have described cases of ulcerative colitis with prolonged remission following pancytopenia .", "burke et al . reported prolonged remission in excess of 4 years following induced pancytopenia , where 3 of the 4 patients who developed severe neutropenia remained in remission for up to 21 months following that episode .", "first , given that altered host immunity plays an important role in ibd activity , it is possible that this patient 's host immunity was reset following the pancytopenia caused by aza . according to one report that analyzed the course of patients with crohn 's disease and ulcerative colitis and who underwent allogeneic stem cell transplantation for myeloid leukemia and myelodysplastic syndrome , 10 of 11 patients showed no ibd activity after stem cell transplantation , and the colonoscopy after complete discontinuation of prophylactic posttransplant immunosuppression revealed no pathologic findings .", "second , the possible benefit of granulocyte macrophage colony - stimulating factor ( gm - csf ) can be hypothesized .", "dieckgraefe and korzenik investigated the safety and possible benefit of gm - csf for the treatment of 15 patients with moderate to severe crohn 's disease .", "third , the original bowel flora is temporarily altered after total gut decontamination and long - term antibacterial treatment .", "therefore , the most likely theory seems to be the modification of a genetically determined immune abnormality that is responsible for the chronic intestinal inflammation by the reset of the immune system or modification of an altered microbial environment .", "however , it is unclear whether this mechanism can affect the long - term clinical course .", "some studies have reported that tpmt genotype or activity does not predict the development of aza - induced myelotoxicity . on the other hand ,", "some reports have suggested a substantial correlation between low enzyme activity and the development of myelotoxicity [ 13 , 14 ] .", "reported that assessment of the tpmt genotype or activity can reduce the risk of myelotoxicity in approximately one - third of patients . in our case ,", "the tpmt was the wild type and this result raised doubts whether tpmt can be a reasonable screening test to identify patients with a high risk of severe myelotoxicity due to an inherited deficiency of tpmt activity . in conclusion , we report here a patient who had crohn 's colitis and complicated perianal fistulas that required immunosuppression , and who achieved endoscopically determined remission that showed accelerated mucosal healing as well as clinical remission after aza - induced pancytopenia ."], "abstract_text": ["<S> the immunosuppressant azathioprine ( aza ) is widely used in the treatment of inflammatory bowel disease ( ibd ) for both inducing and maintaining remission . </S>", "<S> however , the adverse effects of aza can often necessitate a dose reduction or discontinuation . </S>", "<S> bone marrow suppression is one of the most serious complications with aza treatment . on the other hand , </S>", "<S> some reports have suggested that neutropenia during aza therapy reduced the relapse rates of ibd patients , and there have been some cases where eradication of the sensitized leukocytes by leukapheresis or bone marrow transplantation improved the ibd , which may explain the relevant role of neutropenia in controlling disease activity . </S>", "<S> this report describes the case of a 22-year - old male patient who had crohn 's colitis and complicated perianal fistulas that required immunosuppression ; he achieved endoscopically determined remission and showed accelerated mucosal healing as well as clinical remission following the aza - induced pancytopenia . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["a rising trend in the incidence and prevalence of inflammatory bowel disease ( ibd ) in asia has been recognized for the past two decades .", "the thiopurine drugs azathioprine ( aza ) and mercaptopurine are widely used for the treatment of ibd and have proven to be effective in both inducing and maintaining remission of crohn 's disease and ulcerative colitis ; however adverse effects can occur in 928% of patients , and these adverse effects often necessitate a dose reduction or discontinuation .", "nevertheless , some reports suggested that neutropenia during aza therapy reduced the relapse rates in ibd patients [ 1 , 2 ] , and that the eradication of sensitized leukocytes , which was done by leukapheresis or bone marrow transplantation , improved ibd in some cases [ 3 , 4 ] .", "there have also been some cases of ulcerative colitis with prolonged remission following aza - induced pancytopenia [ 5 , 6 ] ."], ["a 22-year - old man with a known history of crohn 's colitis who had undergone fistulectomy for complex perianal fistula 2 years previously was admitted with a complaint of severe weight loss ( 25 kg during 2 years ) , diarrhea , abdominal pain and recurrent perianal tenderness .", "physical exam revealed fistular openings and subcutaneous abscess pockets with severe tenderness on the anus and buttock .", "he was not able to maintain a sitting or supine position , so his social activity was limited .", "contrast - enhanced computed tomography scan of the abdomen showed multifocal inflammatory wall thickening and thick - walled abscess pockets with the beaded appearance of subcutaneous small abscesses in the precoccygeal and perianal area ( fig .", "abscess drainage and a seton operation were performed , then aza was administered at a starting dose of 25 mg ; this was increased up to 50 mg after 2 weeks .", "he then revisited the hospital and presented with high fever ( 39.2c ) and myalgia . on general peripheral blood test , leukocytes were 180/l ( segmented cells 6% ) , hemoglobin was 6.6 g / dl and platelets were 48,000/l .", "after 2 weeks of administering human recombinant granulocyte colony - stimulating factor and broad - spectrum antibiotic therapy , his fever subsided and the cell count fully recovered .", "escherichia coli was found on blood culture , but it was sensitive to the antibiotics .", "after recovering from cytopenia , the frequency of bowel movements decreased and the perianal pain and oozing improved gradually .", "follow - up colonoscopy was performed 3 months later and demonstrated diffuse fibrotic scar instead of multiple ulcerations ( fig .", "he gained 10 kg within 6 months and is now on regular follow - up without clinical recurrence ( cdai score = 114 ) ."], ["therefore , a conventional immunosuppressive dose of less than 2.5 mg / kg / day causes predictable mild leukopenia .", "rarely however , sudden , severe and unexpected myelosuppression , which is possibly idiosyncratic , has also been reported when low to moderate doses ( < 2 mg / kg / day ) of the drug were used . in these cases ,", "the cytopenic patients are vulnerable to the development of sepsis that necessitates hospital admission for intensive care .", "however , the preferential suppression of neutrophils develops when leukopenia occurs , which suggests that aza 's antiinflammatory effects are mediated through its effects on neutrophils as well .", "in fact , clinical relapse of ibd is characterized by increased neutrophil migration into the intestine .", ". moreover , some reports have described cases of ulcerative colitis with prolonged remission following pancytopenia .", "burke et al . reported prolonged remission in excess of 4 years following induced pancytopenia , where 3 of the 4 patients who developed severe neutropenia remained in remission for up to 21 months following that episode .", "first , given that altered host immunity plays an important role in ibd activity , it is possible that this patient 's host immunity was reset following the pancytopenia caused by aza . according to one report that analyzed the course of patients with crohn 's disease and ulcerative colitis and who underwent allogeneic stem cell transplantation for myeloid leukemia and myelodysplastic syndrome , 10 of 11 patients showed no ibd activity after stem cell transplantation , and the colonoscopy after complete discontinuation of prophylactic posttransplant immunosuppression revealed no pathologic findings .", "second , the possible benefit of granulocyte macrophage colony - stimulating factor ( gm - csf ) can be hypothesized .", "dieckgraefe and korzenik investigated the safety and possible benefit of gm - csf for the treatment of 15 patients with moderate to severe crohn 's disease .", "third , the original bowel flora is temporarily altered after total gut decontamination and long - term antibacterial treatment .", "therefore , the most likely theory seems to be the modification of a genetically determined immune abnormality that is responsible for the chronic intestinal inflammation by the reset of the immune system or modification of an altered microbial environment .", "however , it is unclear whether this mechanism can affect the long - term clinical course .", "some studies have reported that tpmt genotype or activity does not predict the development of aza - induced myelotoxicity . on the other hand ,", "some reports have suggested a substantial correlation between low enzyme activity and the development of myelotoxicity [ 13 , 14 ] .", "reported that assessment of the tpmt genotype or activity can reduce the risk of myelotoxicity in approximately one - third of patients . in our case ,", "the tpmt was the wild type and this result raised doubts whether tpmt can be a reasonable screening test to identify patients with a high risk of severe myelotoxicity due to an inherited deficiency of tpmt activity . in conclusion , we report here a patient who had crohn 's colitis and complicated perianal fistulas that required immunosuppression , and who achieved endoscopically determined remission that showed accelerated mucosal healing as well as clinical remission after aza - induced pancytopenia ."]]}
{"article_id": "PMC4664812", "article_text": ["diabetic macular edema ( dme ) is a common complication of diabetic retinopathy ( dr ) and a leading cause of visual loss in this population [ 1 , 2 ] .", "major components of dme are retinal microvascular dysfunction and blood - retinal barrier ( brb ) breakdown with consequent increase in vascular permeability that allows plasma compounds to leak into the retina [ 35 ] .", "there is evidence that upregulation of angiogenic and inflammatory factors , including vascular endothelial growth factor ( vegf ) , and downregulation of antiangiogenic factors as well as redox shift contribute to the breakdown of the brb in dr [ 510 ] .", "oxidative stress and inflammation also play an important role in the pathogenesis of dr and dme .", "vegf causes conformational changes in the tight junctions of the retinal vascular endothelial cells and plays a major role in the increased vascular permeability and brb breakdown in diabetic eyes [ 5 , 7 , 11 , 12 ] .", "vitreous vegf levels correlate significantly with the severity of dr , but dme can occur in nonproliferative dr ( npdr ) as well as proliferative dr ( pdr ) .", "interventional studies on ranibizumab , a monoclonal antibody against vegf , have shown that intraocular injections of ranibizumab significantly reduce foveal thickness and improve visual acuity in patients with dme [ 14 , 15 ] .", "however , the conclusions of the studies were not based on the comparison of the real intravitreal concentration of vegf with the foveal thickness in oct ; they only asses the retinal thickness before and after therapy . in our earlier study", ", we found that also the intravitreal uric acid ( ua ) concentrations correlated significantly with degree of dr .", "we suspect that ua may play a role in the pathogenesis of dr and dme : studies of ua strongly suggest that its redox potential affects endothelial function   and might contribute to the brb breakdown .", "the correlation of intravitreal ua with vegf in npdr and dme has not been studied yet .", "optical coherence tomography ( oct ) has enabled clinicians to noninvasively evaluate the effect of dr on retinal thickness in a standard clinical setting [ 18 , 19 ] . however , there are very limited data on how oct parameters in dme correlate with vitreous levels of vegf and other biochemical parameters .", "the aim of our study was to analyse the vitreous and serum of diabetic patients with dme and severe npdr and compare them to nondiabetic controls .", "the analysis focused on vegf and ua as two possible pathogenetic factors in the development of dme .", "we compared blood and vitreous levels of vegf , ua , and protein between the two study groups and describe their correlation with the changes seen in oct .", "first group involved 16 subjects with type 2 diabetes mellitus ( dm ) with npdr and cystoid dme . in this group ,", "the mean duration of dm was 18.6  8.3 years and 15 patients ( 93.75% ) were treated with insulin and 1 patient ( 6.25% ) was treated with peroral antidiabetics .", "a group of 13 nondiabetic subjects with idiopathic epiretinal membrane and diffuse retinal thickening served as control .", "dm duration was defined as the duration from the first diagnosis of dm to the time of vitreous sampling .", "all patients underwent a standard ophthalmologic examination including measurement of best corrected visual acuity , slit - lamp biomicroscopy , indirect ophthalmoscopy , and oct .", "the retinopathy was graded according to the early treatment diabetic retinopathy study research group and patients enrolled in the study had moderate to severe nonproliferative dr ( npdr ) .", "the center involving dme was defined clinically and confirmed by retinal thickening in cross - sectional spectral domain ( sd ) oct scans .", "the indications for vitrectomy in this study were macular edema and preoperative best corrected visual acuity ( bcva ) more than 0.3 logmar ( logarithm of the minimum angle of resolution ) and in the diabetic group no or poor response to previous therapy with photocoagulation or intravitreal injection .", "exclusion criteria were as follows : ( a ) history of intraocular haemorrhage , ( b ) prior vitreoretinal surgery , ( c ) other ocular surgeries or laser coagulation less than 6 months prior to the operation , ( d ) history of ocular inflammation , ( e ) proliferative dr or other retinal conditions causing neovascularisation , ( f ) ophthalmic disorders associated with macular edema , and ( g ) treatment with intravitreal anti - vegf or steroid injections ( e.g. , triamcinolone , dexamethasone , bevacizumab , ranibizumab , and aflibercept ) less than 6 months prior to the operation . at the time of the study , all patients were in a stable clinical condition without clinical or laboratory signs of acute inflammation .", "the research was approved by the local institutional ethics committee , faculty of medicine and dentistry , palacky university olomouc , czech republic .", "data and sample collection was independent of all treatment decisions . it did not affect a patient 's access to treatment and fully complied with all ethical and legal requirements for noninterventional data collection in the czech republic .", "all patients gave written informed consent to the treatment , as well as data collection .", "the reported investigations were in accordance with the principles of the current version of the declaration of helsinki .", "oct examinations were performed one day before vitrectomy with spectral domain oct ( cirrus hd - oct , carl zeiss meditec ag , jena , germany ) using macular cube acquisition according to the manufacturer 's protocol .", "the macular cube 512  128 scan consists of 128 raster scans with 512 a - scans , within a 6  6  mm macular area . the mean central retinal thickness ( crt , i.e. , central subfield thickness ) from the internal limiting membrane to the retinal pigment epithelium at the fovea", "was defined as the mean retinal thickness in a 1  mm diameter circular zone concentred on the fovea .", "also cube volume ( cv ) and cube average thickness ( cat ) of the scanned area were calculated by cirrus hd - oct software and checked for accuracy .", "the cv is calculated from the 1  mm diameter zone and cat from the central 6  mm diameter zone concentred on the fovea .", "based on previous studies that evaluated morphological changes in dme [ 22 , 23 ] , the central scan through the fovea was assessed for the presence of intraretinal cysts and serous retinal detachment ( srd ) by an independent examiner .", "vitrectomy was performed to improve visual acuity and to decrease retinal thickness in the macula .", "each patient underwent standard three - port therapeutic pars plana vitrectomy using current surgical techniques ( the alcon constellation vision system ) . before opening the infusion port at the start of the vitrectomy ,", "undiluted vitreous samples were obtained and collected in sterile tubes ( cca . 0.3  ml ) .", "overnight fasting blood samples were drawn from the antecubital vein at the time of vitrectomy and used for biochemical assay .", "the concentration of ua was estimated using enzymatic methods ( uricase - peroxidase ) with photometric detection ( modular , roche , germany ) .", "hba1c was measured by high performance liquid chromatography and calibration was traced to the reference method of the international federation of clinical chemistry ( variant ii , bio - rad ; http://www.bio-rad.com/ ) .", "the concentration of vegf was quantified by enzyme linked immunosorbent assay ( elisa ) using a commercial human vegf kit ( r and d systems , minneapolis , mn , usa ) according to the manufacturer 's protocol .", "the limits of quantification for vegf were min = 31.2  pg / ml and max = 1000  pg / ml , respectively .", "we calculated the median with 1st and 3rd quartile ( iqr , interquartile range ) . in 16 subjects , the intravitreal vegf and in 3 subjects the intravitreal ua concentration were under the detection limit ; these subjects were included in the statistical analysis to avoid selection bias .", "hence , we used the nonparametric analysis for ordinal variables , and the concentrations under the detection limit were assigned ", "the comparison between dme group and control group was done by mann - whitney u test and fisher 's exact test .", "biochemical analysis of the vitreous showed significant differences between dm and control group in the concentration of vegf , ua , and total protein but not albumin as shown in table 2 and figures 13 . in all nondiabetic control subjects ,", "the concentration of vegf in vitreous was under the detection limit of 31.2  pg / ml . in the diabetic group , ua concentration in vitreous", "correlated significantly with vitreous vegf concentration (  = 0.559 , p = 0.03 ) . however , in dme vitreous vegf and ua did not correlate with the total vitreous protein .", "further , in the control group , no significant correlation between the biochemical analytes in vitreous was found .  ", "figure 4 shows the relationship between vitreous vegf and vitreous ua of dme and control group .", "median of serum concentration of ua in diabetic patients was significantly elevated compared with the control group ( 337.0  mol / l , iqr : 324.0407.0 ", "mol / l in dm group versus 259.5  mol / l , iqr : 220.0334.8  mol / l in control group ; p = 0.025 ) . also median concentration of vegf in serum of diabetic patients ( 414.3  pg / ml , iqr : 293.1512.0  pg / ml ) was higher than in controls ( 332.7  pg / ml , iqr : 149.4551.8  pg / ml ) , but the difference was not significant .", "there was a significant correlation between ua concentrations in serum and vitreous (  = 0.652 , p = 0.016 ) in the control group but not in dme .", "further , no significant correlation between concentrations of vegf in serum and vitreous was found in both groups .", "the median crt , cat , and cv did not differ significantly between both groups and are listed in table 3 .", "significant difference was found in presence of srd between the groups as shown in table 3 . in the diabetic group", ", there was a significant correlation between crt and cat (  = 0.589 , p = 0.016 ) .", "the crt of dm subjects also correlated significantly with the cv (  = 0.581 , p = 0.018 ) .", "however , the strongest correlation in the dm group was between cat and cv (  = 0.999 , p < 0.001 ) .", "the srd was found in the oct scans of 6 diabetic eyes , but its presence did not correlate with any of the other oct parameters .", "further , among all oct parameters , only cv correlated significantly with the concentration of vitreous vegf in the dm group (  = 0.515 , p = 0.041 ) .", "the crt , cat , cv , and srd show in dm and control subjects no significant correlation to vitreous concentrations of ua , albumin , or total protein .", "the correlation of logmar bcva with changes in oct parameters and vitreous content was also evaluated and we found it to be nonsignificant in both groups .", "there was also no correlation between oct parameters and serous concentrations of ua or vegf .", "the results demonstrate that biochemical analysis of the vitreous showed significant higher concentrations of vegf , ua , and total protein in dm and control group . moreover , in patients with dme intravitreal levels of ua correlate significantly with intravitreal levels of vegf .", "furthermore , we found that the cv measured with cirrus hd - oct correlate significantly with the concentration of vegf in the vitreous of patients with npdr and dme . in our earlier study , we showed that the levels of intravitreal ua correlated significantly with the degree of dr   and recently also serum ua concentration has been found to be associated with increase in severity of dr .", "finding significant higher ua concentration in vitreous of dm compared to controls and a correlation between ua and vegf in the vitreous of npdr patients supports our assumption that ua too may be one contributing causal factor in the pathogenesis of dr .", "ua is a degradation product of metabolism and under normal conditions ua acts as an antioxidant . in diabetics", ", hyperglycaemia induces redox stress , which leads to consumption of the naturally occurring local antioxidants protecting capillary endothelium .", "this results in urate redox shuttle , meaning that ua paradoxically becomes prooxidant and contributes to endothelial dysfunction through oxidative - redox stress .", "johnson et al . showed that local ischemia results , via enzymatic activation , in increased ua production as well as oxidant formation .", "decreased total antioxidant status was shown to contribute to the progression of pdr via induction of vegf . on the other side , high ua concentration in the vitreous of diabetic patients", "the vegf - induced production of reactive oxygen species was attenuated by urate ; however , it did not modify the vegf - induced changes in permeability of monolayers .", "this could explain the correlation of ua and vegf in the vitreous of diabetic group found in the present study .", "it has to be elucidated whether ua is originating from leakage of retinal vessels , which is increased in dr , or from local production .", "although total vitreous protein was significantly higher in the diabetic group compared to controls , its level did not correlate with both ua and vegf .", "furthermore , in the diabetic subjects we found no correlation between serum and vitreous level of ua .", "however , to be able to distinguish the origin of increased ua in the vitreous , further analyses , for example , with tagged ua , should be done . since there was a correlation between vitreous ua and vegf but no correlation between vitreous ua and oct parameters", ", we conclude that ua has a probable relation with diabetic microangiopathy and accordingly dr but not directly with the development of dme .", "recent studies have shown that vegf causes conformational changes in the tight junctions of retinal vascular endothelial cells and plays a major role in the elevated vascular permeability in diabetic eyes with dme .", "it is well known that the vitreous vegf levels correlate significantly with the severity of dr [ 1113 , 28 ] .", "few authors have also found a significant correlation between retinal thickness at the fovea measured on oct and vegf concentrations in the vitreous [ 29 , 30 ] and aqueous .", "the association of vitreous vegf levels and dme morphology was studied by sonoda et al .  ", "and these authors showed no significant differences in vegf concentrations in cystoid versus diffuse aspect of dme . in the present study", ", there was no significant correlation between vitreous vegf levels and crt of diabetic patients .", "however , the results show that in dme increase in cv correlated with increased concentration of vegf in the vitreous .", "the retinal thickness at the central fovea in funatsu et al .   was calculated as average foveal thickness from 4 manual measurements per patient .", "used the average thickness of the central area with 1000  m in diameter calculated by humphrey oct .", "defined the central macular thickness as the average thickness of the central 500  m in diameter .", "the mean crt in dme is widely accepted as the new surrogate marker for evaluating treatment efficacy .", "this is also because the changes in the fovea are deciding for the visual acuity . in our study ,", "the crt , cat , and cv in both dm and control group did not correlate with the logmar bcva .", "there are also studies evaluating the effect of the anti - vegf therapy using both the mean foveal thickness and cv . on the other hand ,", "since dme usually affects the macular area and not only the foveal region , assessment of vegf concentration in clinical practice using the cv is comprehensible . like us , sonoda et al", ". showed that there was no significant correlation between intravitreal vegf levels and the amount of subretinal fluid in dme .", "other studies have reported that eyes with serous retinal detachment often have a poor prognosis after treatment [ 22 , 36 ] .", "the strength of the study described here is that it determined the relationship between the levels of intravitreal biochemical parameters and retinal morphology at the same time .", "the limitation was the small sample size ( 29 eyes ) . this was caused by decreasing use of vitrectomy for dme and this curtailed collection of vitreous samples .", "although our vitrectomy for dme might be considered overtreatment , it was a comparatively effective method as it stabilized the intraocular condition of dme and the efficacy was maintained for a long period . in interpreting or generalizing our results , it should be remembered that the findings demonstrate association of vegf levels in the vitreous with the cube volume , but they do not prove cause and effect .", "further , the study was focused on vegf and ua ; however , the pathogenesis of the dme is complex and still not fully understood .", "vitreous concentrations of uric acid and vegf were significantly higher in dm subjects than in controls . moreover , vitreous ua concentration correlated significantly with the vitreous vegf concentrations in patients with npdr and cystoid dme . increased vegf concentrations are known to be involved in the pathogenesis of dme .", "our results suggest that , apart from vegf , the role of ua in the pathogenesis and progression of dr should also be considered . comparing oct parameters to the vitreous levels of ua and vegf", ", we found that increased concentration of intravitreal vegf in patients with npdr and cystoid dme correlated with increase of cube volume calculated by cirrus hd - oct . since dme usually affects the macular area and not only the foveal region , the assessment of the vegf concentration in clinical practice using the cube volume is comprehensible .", "this oct parameter could be used to assess the efficacy of anti - vegf therapy ."], "abstract_text": ["<S> \n purpose . we investigated two factors linked to diabetic macular edema ( dme ) , vitreous and serum levels of vascular endothelial growth factor ( vegf ) and uric acid ( ua ) in patients with dme , and compared the results with changes in optical coherence tomography ( oct ) and visual acuity ( va ) . </S>", "<S> methods . </S>", "<S> a prospective study of 29 eyes , 16 cystoid dme and nonproliferative diabetic retinopathy ( dr ) and 13 nondiabetic controls . biochemical analysis of vitreous and serum samples was performed and oct scans were graded according to central retinal thickness ( crt ) , cube volume ( cv ) , cube average thickness ( cat ) , and serous retinal detachment ( srd ) . results . in dme group , intravitreal concentrations of vegf ( p < 0.001 ) , ua ( p = 0.038 ) , and total protein ( p < 0.001 ) were significantly higher than in control group . in dme subjects , intravitreal ua correlated significantly with intravitreal vegf (  = 0.559 , p = 0.03 ) but not with total vitreous protein and serum ua . increased </S>", "<S> intravitreal vegf in dme group correlated with increase in cv (  = 0.515/p = 0.041 ) . </S>", "<S> none of the oct parameters correlated with the va . </S>", "<S> conclusions . </S>", "<S> the results suggest that the cv might be assessor of anti - vegf therapy efficacy . </S>", "<S> second , apart from vegf , the role of ua in the pathogenesis and progression of dr should be considered . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusions"], "sections": [["diabetic macular edema ( dme ) is a common complication of diabetic retinopathy ( dr ) and a leading cause of visual loss in this population [ 1 , 2 ] .", "major components of dme are retinal microvascular dysfunction and blood - retinal barrier ( brb ) breakdown with consequent increase in vascular permeability that allows plasma compounds to leak into the retina [ 35 ] .", "there is evidence that upregulation of angiogenic and inflammatory factors , including vascular endothelial growth factor ( vegf ) , and downregulation of antiangiogenic factors as well as redox shift contribute to the breakdown of the brb in dr [ 510 ] .", "oxidative stress and inflammation also play an important role in the pathogenesis of dr and dme .", "vegf causes conformational changes in the tight junctions of the retinal vascular endothelial cells and plays a major role in the increased vascular permeability and brb breakdown in diabetic eyes [ 5 , 7 , 11 , 12 ] .", "vitreous vegf levels correlate significantly with the severity of dr , but dme can occur in nonproliferative dr ( npdr ) as well as proliferative dr ( pdr ) .", "interventional studies on ranibizumab , a monoclonal antibody against vegf , have shown that intraocular injections of ranibizumab significantly reduce foveal thickness and improve visual acuity in patients with dme [ 14 , 15 ] .", "however , the conclusions of the studies were not based on the comparison of the real intravitreal concentration of vegf with the foveal thickness in oct ; they only asses the retinal thickness before and after therapy . in our earlier study", ", we found that also the intravitreal uric acid ( ua ) concentrations correlated significantly with degree of dr .", "we suspect that ua may play a role in the pathogenesis of dr and dme : studies of ua strongly suggest that its redox potential affects endothelial function   and might contribute to the brb breakdown .", "the correlation of intravitreal ua with vegf in npdr and dme has not been studied yet .", "optical coherence tomography ( oct ) has enabled clinicians to noninvasively evaluate the effect of dr on retinal thickness in a standard clinical setting [ 18 , 19 ] . however , there are very limited data on how oct parameters in dme correlate with vitreous levels of vegf and other biochemical parameters .", "the aim of our study was to analyse the vitreous and serum of diabetic patients with dme and severe npdr and compare them to nondiabetic controls .", "the analysis focused on vegf and ua as two possible pathogenetic factors in the development of dme .", "we compared blood and vitreous levels of vegf , ua , and protein between the two study groups and describe their correlation with the changes seen in oct ."], ["first group involved 16 subjects with type 2 diabetes mellitus ( dm ) with npdr and cystoid dme . in this group ,", "the mean duration of dm was 18.6  8.3 years and 15 patients ( 93.75% ) were treated with insulin and 1 patient ( 6.25% ) was treated with peroral antidiabetics .", "a group of 13 nondiabetic subjects with idiopathic epiretinal membrane and diffuse retinal thickening served as control .", "dm duration was defined as the duration from the first diagnosis of dm to the time of vitreous sampling .", "all patients underwent a standard ophthalmologic examination including measurement of best corrected visual acuity , slit - lamp biomicroscopy , indirect ophthalmoscopy , and oct .", "the retinopathy was graded according to the early treatment diabetic retinopathy study research group and patients enrolled in the study had moderate to severe nonproliferative dr ( npdr ) .", "the center involving dme was defined clinically and confirmed by retinal thickening in cross - sectional spectral domain ( sd ) oct scans .", "the indications for vitrectomy in this study were macular edema and preoperative best corrected visual acuity ( bcva ) more than 0.3 logmar ( logarithm of the minimum angle of resolution ) and in the diabetic group no or poor response to previous therapy with photocoagulation or intravitreal injection .", "exclusion criteria were as follows : ( a ) history of intraocular haemorrhage , ( b ) prior vitreoretinal surgery , ( c ) other ocular surgeries or laser coagulation less than 6 months prior to the operation , ( d ) history of ocular inflammation , ( e ) proliferative dr or other retinal conditions causing neovascularisation , ( f ) ophthalmic disorders associated with macular edema , and ( g ) treatment with intravitreal anti - vegf or steroid injections ( e.g. , triamcinolone , dexamethasone , bevacizumab , ranibizumab , and aflibercept ) less than 6 months prior to the operation . at the time of the study , all patients were in a stable clinical condition without clinical or laboratory signs of acute inflammation .", "the research was approved by the local institutional ethics committee , faculty of medicine and dentistry , palacky university olomouc , czech republic .", "data and sample collection was independent of all treatment decisions . it did not affect a patient 's access to treatment and fully complied with all ethical and legal requirements for noninterventional data collection in the czech republic .", "all patients gave written informed consent to the treatment , as well as data collection .", "the reported investigations were in accordance with the principles of the current version of the declaration of helsinki .", "oct examinations were performed one day before vitrectomy with spectral domain oct ( cirrus hd - oct , carl zeiss meditec ag , jena , germany ) using macular cube acquisition according to the manufacturer 's protocol .", "the macular cube 512  128 scan consists of 128 raster scans with 512 a - scans , within a 6  6  mm macular area . the mean central retinal thickness ( crt , i.e. , central subfield thickness ) from the internal limiting membrane to the retinal pigment epithelium at the fovea", "was defined as the mean retinal thickness in a 1  mm diameter circular zone concentred on the fovea .", "also cube volume ( cv ) and cube average thickness ( cat ) of the scanned area were calculated by cirrus hd - oct software and checked for accuracy .", "the cv is calculated from the 1  mm diameter zone and cat from the central 6  mm diameter zone concentred on the fovea .", "based on previous studies that evaluated morphological changes in dme [ 22 , 23 ] , the central scan through the fovea was assessed for the presence of intraretinal cysts and serous retinal detachment ( srd ) by an independent examiner .", "vitrectomy was performed to improve visual acuity and to decrease retinal thickness in the macula .", "each patient underwent standard three - port therapeutic pars plana vitrectomy using current surgical techniques ( the alcon constellation vision system ) . before opening the infusion port at the start of the vitrectomy ,", "undiluted vitreous samples were obtained and collected in sterile tubes ( cca . 0.3  ml ) .", "overnight fasting blood samples were drawn from the antecubital vein at the time of vitrectomy and used for biochemical assay .", "the concentration of ua was estimated using enzymatic methods ( uricase - peroxidase ) with photometric detection ( modular , roche , germany ) .", "hba1c was measured by high performance liquid chromatography and calibration was traced to the reference method of the international federation of clinical chemistry ( variant ii , bio - rad ; http://www.bio-rad.com/ ) .", "the concentration of vegf was quantified by enzyme linked immunosorbent assay ( elisa ) using a commercial human vegf kit ( r and d systems , minneapolis , mn , usa ) according to the manufacturer 's protocol .", "the limits of quantification for vegf were min = 31.2  pg / ml and max = 1000  pg / ml , respectively .", "we calculated the median with 1st and 3rd quartile ( iqr , interquartile range ) . in 16 subjects , the intravitreal vegf and in 3 subjects the intravitreal ua concentration were under the detection limit ; these subjects were included in the statistical analysis to avoid selection bias .", "hence , we used the nonparametric analysis for ordinal variables , and the concentrations under the detection limit were assigned ", "the comparison between dme group and control group was done by mann - whitney u test and fisher 's exact test ."], ["biochemical analysis of the vitreous showed significant differences between dm and control group in the concentration of vegf , ua , and total protein but not albumin as shown in table 2 and figures 13 . in all nondiabetic control subjects ,", "the concentration of vegf in vitreous was under the detection limit of 31.2  pg / ml . in the diabetic group , ua concentration in vitreous", "correlated significantly with vitreous vegf concentration (  = 0.559 , p = 0.03 ) . however , in dme vitreous vegf and ua did not correlate with the total vitreous protein .", "further , in the control group , no significant correlation between the biochemical analytes in vitreous was found .  ", "figure 4 shows the relationship between vitreous vegf and vitreous ua of dme and control group .", "median of serum concentration of ua in diabetic patients was significantly elevated compared with the control group ( 337.0  mol / l , iqr : 324.0407.0 ", "mol / l in dm group versus 259.5  mol / l , iqr : 220.0334.8  mol / l in control group ; p = 0.025 ) . also median concentration of vegf in serum of diabetic patients ( 414.3  pg / ml , iqr : 293.1512.0  pg / ml ) was higher than in controls ( 332.7  pg / ml , iqr : 149.4551.8  pg / ml ) , but the difference was not significant .", "there was a significant correlation between ua concentrations in serum and vitreous (  = 0.652 , p = 0.016 ) in the control group but not in dme .", "further , no significant correlation between concentrations of vegf in serum and vitreous was found in both groups .", "the median crt , cat , and cv did not differ significantly between both groups and are listed in table 3 .", "significant difference was found in presence of srd between the groups as shown in table 3 . in the diabetic group", ", there was a significant correlation between crt and cat (  = 0.589 , p = 0.016 ) .", "the crt of dm subjects also correlated significantly with the cv (  = 0.581 , p = 0.018 ) .", "however , the strongest correlation in the dm group was between cat and cv (  = 0.999 , p < 0.001 ) .", "the srd was found in the oct scans of 6 diabetic eyes , but its presence did not correlate with any of the other oct parameters .", "further , among all oct parameters , only cv correlated significantly with the concentration of vitreous vegf in the dm group (  = 0.515 , p = 0.041 ) .", "the crt , cat , cv , and srd show in dm and control subjects no significant correlation to vitreous concentrations of ua , albumin , or total protein .", "the correlation of logmar bcva with changes in oct parameters and vitreous content was also evaluated and we found it to be nonsignificant in both groups .", "there was also no correlation between oct parameters and serous concentrations of ua or vegf .", "the results demonstrate that biochemical analysis of the vitreous showed significant higher concentrations of vegf , ua , and total protein in dm and control group . moreover , in patients with dme intravitreal levels of ua correlate significantly with intravitreal levels of vegf .", "furthermore , we found that the cv measured with cirrus hd - oct correlate significantly with the concentration of vegf in the vitreous of patients with npdr and dme . in our earlier study , we showed that the levels of intravitreal ua correlated significantly with the degree of dr   and recently also serum ua concentration has been found to be associated with increase in severity of dr .", "finding significant higher ua concentration in vitreous of dm compared to controls and a correlation between ua and vegf in the vitreous of npdr patients supports our assumption that ua too may be one contributing causal factor in the pathogenesis of dr .", "ua is a degradation product of metabolism and under normal conditions ua acts as an antioxidant . in diabetics", ", hyperglycaemia induces redox stress , which leads to consumption of the naturally occurring local antioxidants protecting capillary endothelium .", "this results in urate redox shuttle , meaning that ua paradoxically becomes prooxidant and contributes to endothelial dysfunction through oxidative - redox stress .", "johnson et al . showed that local ischemia results , via enzymatic activation , in increased ua production as well as oxidant formation .", "decreased total antioxidant status was shown to contribute to the progression of pdr via induction of vegf . on the other side , high ua concentration in the vitreous of diabetic patients", "the vegf - induced production of reactive oxygen species was attenuated by urate ; however , it did not modify the vegf - induced changes in permeability of monolayers .", "this could explain the correlation of ua and vegf in the vitreous of diabetic group found in the present study .", "it has to be elucidated whether ua is originating from leakage of retinal vessels , which is increased in dr , or from local production .", "although total vitreous protein was significantly higher in the diabetic group compared to controls , its level did not correlate with both ua and vegf .", "furthermore , in the diabetic subjects we found no correlation between serum and vitreous level of ua .", "however , to be able to distinguish the origin of increased ua in the vitreous , further analyses , for example , with tagged ua , should be done . since there was a correlation between vitreous ua and vegf but no correlation between vitreous ua and oct parameters", ", we conclude that ua has a probable relation with diabetic microangiopathy and accordingly dr but not directly with the development of dme .", "recent studies have shown that vegf causes conformational changes in the tight junctions of retinal vascular endothelial cells and plays a major role in the elevated vascular permeability in diabetic eyes with dme .", "it is well known that the vitreous vegf levels correlate significantly with the severity of dr [ 1113 , 28 ] .", "few authors have also found a significant correlation between retinal thickness at the fovea measured on oct and vegf concentrations in the vitreous [ 29 , 30 ] and aqueous .", "the association of vitreous vegf levels and dme morphology was studied by sonoda et al .  ", "and these authors showed no significant differences in vegf concentrations in cystoid versus diffuse aspect of dme . in the present study", ", there was no significant correlation between vitreous vegf levels and crt of diabetic patients .", "however , the results show that in dme increase in cv correlated with increased concentration of vegf in the vitreous .", "the retinal thickness at the central fovea in funatsu et al .   was calculated as average foveal thickness from 4 manual measurements per patient .", "used the average thickness of the central area with 1000  m in diameter calculated by humphrey oct .", "defined the central macular thickness as the average thickness of the central 500  m in diameter .", "the mean crt in dme is widely accepted as the new surrogate marker for evaluating treatment efficacy .", "this is also because the changes in the fovea are deciding for the visual acuity . in our study ,", "the crt , cat , and cv in both dm and control group did not correlate with the logmar bcva .", "there are also studies evaluating the effect of the anti - vegf therapy using both the mean foveal thickness and cv . on the other hand ,", "since dme usually affects the macular area and not only the foveal region , assessment of vegf concentration in clinical practice using the cv is comprehensible . like us , sonoda et al", ". showed that there was no significant correlation between intravitreal vegf levels and the amount of subretinal fluid in dme .", "other studies have reported that eyes with serous retinal detachment often have a poor prognosis after treatment [ 22 , 36 ] .", "the strength of the study described here is that it determined the relationship between the levels of intravitreal biochemical parameters and retinal morphology at the same time .", "the limitation was the small sample size ( 29 eyes ) . this was caused by decreasing use of vitrectomy for dme and this curtailed collection of vitreous samples .", "although our vitrectomy for dme might be considered overtreatment , it was a comparatively effective method as it stabilized the intraocular condition of dme and the efficacy was maintained for a long period . in interpreting or generalizing our results , it should be remembered that the findings demonstrate association of vegf levels in the vitreous with the cube volume , but they do not prove cause and effect .", "further , the study was focused on vegf and ua ; however , the pathogenesis of the dme is complex and still not fully understood ."], ["vitreous concentrations of uric acid and vegf were significantly higher in dm subjects than in controls . moreover , vitreous ua concentration correlated significantly with the vitreous vegf concentrations in patients with npdr and cystoid dme . increased vegf concentrations are known to be involved in the pathogenesis of dme .", "our results suggest that , apart from vegf , the role of ua in the pathogenesis and progression of dr should also be considered . comparing oct parameters to the vitreous levels of ua and vegf", ", we found that increased concentration of intravitreal vegf in patients with npdr and cystoid dme correlated with increase of cube volume calculated by cirrus hd - oct . since dme usually affects the macular area and not only the foveal region , the assessment of the vegf concentration in clinical practice using the cube volume is comprehensible .", "this oct parameter could be used to assess the efficacy of anti - vegf therapy ."]]}
{"article_id": "PMC4539181", "article_text": ["diabetes is the most frequent cause of end - stage renal disease in industrialised countries [ 1 , 2 ] .", "clinically , diabetic nephropathy is characterized by the development of albuminuria and a subsequent decline in glomerular filtration rate .", "this severe complication significantly influences the risk of cardiovascular disease as well as mortality and quality of life [ 3 , 4 ] . at present", "the most important identified risk factors are diabetes duration , arterial blood pressure , and glycaemic regulation . as evident from an increasing incidence of affected patients ,", "however , there is still a great need for new strategies in the treatment and prevention of diabetic nephropathy .", "clear evidence indicates that the pathogenesis of diabetic nephropathy is multifactorial and triggered by a complex series of pathophysiological events .", "the inflammatory response in diabetes is highly complex involving proinflammatory cytokines and chemokines , for example , il1", "the impact of complement activation on the diabetic kidney may well , in part , be mediated through induction of cytokine response and inflammation [ 911 ] .", "several studies have linked diabetic late - complications to the complement system of the innate immune system [ 12 , 13 ] .", "the complement system plays a crucial role in recognition and clearance of infectious microbes and the system forms a link between innate and adaptive immunity .", "the activation of the complement system results in the release of multiple inflammatory signaling molecules .", "ultimately complement activation leads to the formation of pore - forming membrane attack complexes ( macs ) that are inserted in the cell membranes to mediate lyses of the cell through osmotic stress .", "however , in mammalian cells , it has been shown that sublytic amounts of mac can increase production of il-8 and monocyte chemoattractant protein 1 dependent on nfb nuclear translocation .", "furthermore , macs are shown to have a mitogenic effect and cause release of basic fibroblast growth factor and platelet - derived growth factor from endothelial cells leading to fibrosis in neighboring cells including glomerular mesangial cells [ 16 , 17 ] .", "the latter effects of mac may explain the link between complement and diabetic kidney damage .", "three activations pathways exist : the classical , the alternative , and the lectin pathway .", "the present paper focuses on the lectin pathway , in which at least five soluble pattern - recognition molecules are characterized that may activate the complement system , that is , mannan - binding lectin ( mbl ) , h - ficolin , l - ficolin , m - ficolin , and collectin - k1 .", "the three ficolins utilize a fibrinogen - like domain that binds , for example , n - acetylglucosamine , n - acetylgalactosamine , and n - acetyl - neuraminic acid , whereas mbl and cl - k1 have a carbohydrate recognition domain and through this bind specifically to patterns of monosaccharides . a very recent publication reports a close association between ficolin and diabetic nephropathy in patients with type 1 diabetes .", "the observational design of the study , however , limits its ability to study a cause - effect relationship . when ficolins bind they all initiate activation of associated serine proteases ( mbl associated serine proteases , masps ) , which subsequently cleave the complement factors , c2 and c4 , leading to further complement activation [ 20 , 21 ] .", "eventually , complement activation leads to the formation of macs causing cell lyses or induction of fibrosis . the balance between activation and inhibition of the complement cascade is tightly controlled by regulatory proteins in order to prevent damage of healthy host cells . in diabetes", ", inappropriate effects of the complement system may be present as glycation - induced dysfunction of the complement inhibitory mechanism and consequently overactivation of the system is indicated [ 2325 ] .", "it is speculated that diabetic patients are exposed to uncontrolled complement attack partly due to altered molecular patterns on the cell surfaces as a consequence of high blood glucose [ 24 , 25 ] .", "most significantly , an association is seen between diabetic nephropathy and the lectin pathway [ 2628 ] . we have previously demonstrated direct cause - effect relationship between presence of mbl and worsening of kidney injury in a mouse model of diabetic nephropathy [ 29 , 30 ] .", "we speculate that ficolins also exert detrimental effects in diabetes similar to mbl through activation of the lectin pathway as indicated in patients with type 1 diabetes .", "this study aimed to investigate the impact of ficolin b ( the orthologue to human m - ficolin ) on the development of diabetic nephropathy in a mouse model of type 1 diabetes .", "we used 11-week - old , female ficolin b knockout mice and age - matched , female c57bl/6j bomtac wild - type mice ( taconic , ry , denmark ) .", "the knockout ficolin b model was backcrossed more than 10 generations to a c57bl/6j bomtac genetic background ( own breeding ) . in each cage", "there were three to eight mice and they had free access to tap water and standard chow ( altromin number 1324 ; lage , germany ) .", "the environment was stable with a 12-hour light - dark cycle , temperature at 21  1c , and humidity of 55  5% .", "the ficolin b knockout mice and the wild - type mice were randomized into a diabetic and nondiabetic group ; thus four groups were made : ( 1 ) diabetic knockout mice ( n = 6 ) , ( 2 ) nondiabetic knockout mice ( n = 7 ) , ( 3 ) diabetic wild - type mice ( n = 11 ) , and ( 4 ) nondiabetic wild - type mice ( n = 11 ) .", "diabetes was induced by intraperitoneal injections of streptozotocin ( stz ) dissolved in a cold 10  mm citrate buffer ( doses of 55  mg / kg body weight , sigma aldrich , st louis , mo , usa ) on five consecutive days .", "the 18-week experiment was initiated when the mice were classified as diabetic ( blood glucose > 15  mm ) . animals with more than 15% sustained weight loss , signs of illness , or persistent ketonuria were excluded from the study .", "blood glucose was measured from tail vein by contour ( bayer diabetes care , kgs .", "lyngby , denmark ) . with combur test d strip ( roche diagnostics gmbh , mannheim , germany )", "two mice from each diabetic group were excluded because of insufficient increase in blood glucose levels .", "furthermore , two mice from the diabetic knockout group were excluded because of weight loss > 15% of body weight .", "the excluded mice were not included in the number of animals per group indicated above .", "spot urine was collected in eppendorf tubes on five consecutive days prior to sacrifice of the animals .", "the blood samples were drawn from under the tongue at baseline and from the retroorbital venous plexus at study end and collected in potassium edta tubes ( sarstedt , nmbrecht , germany ) .", "the animals were anesthetized by an intraperitoneal dose of ketamine at 0.5  mg / g body weight and xylazine at 0.2  mg / g body weight ( ketaminol 4 vet and narcoxyl vet , resp . ,", "urinary albumin excretion was determined by mouse albumin elisa quantification kit ( bethyl laboratories , inc . ,", "urine creatinine was measured by isocratic high - performance liquid chromatography ( hplc ) on a zorbax scx300 column ( agilent , usa ) using a slight modification of a method first reported by yuen et al . .", "in brief , 5  l urine was added to 100  l acetonitrile containing 0.5% acetic acid and vortexed for 15 seconds to extract the creatinine .", "after 15  min of 20c storage and centrifugation the supernatants were evaporated and then reconstituted with 25  l 5  mm sodium acetate , ph 4.1 .", "duplicate samples ( 10  ul each ) were fractionated on a 50  mm  2.1  mm zorbax scx300 column with an in - front scx guard column .", "isocratic hplc was performed at a flow rate of 1  ml / min , and uv absorbance was monitored at 225  nm .", "a standard curve was created by including a 2-fold dilution series of creatinine anhydrous ( sigma aldrich ) .", "this study was designed with two independent factors ; diabetes / nondiabetes and knockout / wild - type and thus analysed by two - way anova for normal distributed variable with equal variance .", "the main focus of interest was the interaction between the diabetic factor and the knockout factor ; that is , does ficolin b modify the effects of diabetes on the effect parameters ? if no interaction was found , the independent effects of diabetes and ficolin b on the kidney were estimated .", "for pairwise comparison , normal distributed data was tested with student 's t - test , whereas otherwise the wilcoxon mann - whitney rank sum test was used .", "data are given as mean ( 95% confidence interval ( ci ) ) unless else is stated .", "at baseline , the knockout mice on average weighed 20.0  g , which was slightly less than the wild type mice , 20.8  g ( p = 0.04 ) .", "no difference was found between the two diabetic groups or between the two nondiabetic groups ( table 1 ) .", "after 18 weeks an expected difference in body weight was observed between the diabetic and the nondiabetic mice independently of knockout status ( p < 0.001 ) .", "the nondiabetic mice weighed 3.2  g ( ci : 2.0  g4.3  g ) more than the diabetic mice .", "furthermore the diabetic knockout mice were significantly smaller than the diabetic wild type ( p < 0.05 ) .", "as presented in table 1 , blood glucose , estimated as area under the curve ( auc ) , did not differ between the two diabetic groups ( p = 0.69 ) or between the two nondiabetic groups ( p = 0.13 ) .", "the kidney weight was equally increased in diabetic wild - type mice , 24% ( ci : 13%36% ) , and in the diabetic knockout mice , 29% ( ci : 12%47% ) , compared to the respective control groups ( figure 2(a ) ) . no interaction between knockout and diabetes was found ( p = 0.60 ) , indicating that wild - type and knockout mice develop the same degree of diabetes - induced renal hypertrophy .", "the considerable body weight difference between the two diabetic groups at study end indicated that the kidney weight was to be normalised to the body weight .", "ficolin b did not modify the diabetes - induced increase in kidney weight when testing for interaction ( p = 0.11 ) .", "furthermore , no significant statistical difference was found in kidney weight per body weight between the diabetic wild - type , 1.95  mg / g , and the diabetic knockout , 2.89  mg / g ( p = 0.09 ) .", "the albumin - to - creatinine ration ( acr ) was higher among the diabetic wild - type mice , 76  mg / g ( ci : 50103  mg / g ) , compared to the nondiabetic wild - type mice , 44  mg / g ( ci : 2563  mg / g ) , p = 0.07 .", "similarly , the acr of diabetic knockout mice was 96  mg / g ( ci : 71122  mg / g ) compared to the nondiabetic knockout group , 34  mg / g ( ci : 2344  mg / g ) , p < 0.001 . as depicted in figure 3 no interaction was observed between diabetes and ficolin b knockout , p = 0.21 .", "in the present study we found no association between diabetes - induced kidney changes and the presence of ficolin b. we conclude that ficolin b is not responsible for , or a crucial contributory factor in , the pathophysiology of diabetic nephropathy . in our study , the kidney weight and to some extent the acr were altered by diabetes as expected .", "the diabetes - induced increase in kidney weight , measured by comparing the diabetic mice with the nondiabetic mice , was not statistically different between the wild - type and ficolin b knockout mice .", "the diabetes - induced increase in kidney weight was 24% in the wild - type mice and 29% in the ficolin b knockout mice .", "taking the lower body weight of the knockout mice into account , the difference in renal hypertrophy was still insignificant when comparing the wild - type mice and the ficolin b knockout mice .", "similarly , the diabetic change seen in acr was not altered in the absence of ficolin b. the experimental setup including four groups matched on age , body weight , and genetical background was a strength to the study , as the diabetes factor and the knockout factor were the only modulators of the outcome .", "most importantly both diabetic groups did reach and sustain blood glucose levels of above 15  mm . at study end ,", "the body weight differed among groups , which impeded the analyses of the diabetic kidney damage , because the knockout mice appear to be more vulnerable to type 1 diabetes mellitus .", "our study provides important new information on the association between the lectin pathway and diabetic kidney damage .", "we are the first to investigate the role of ficolin b ( which corresponds to ficolin m in human ) in the inflammatory response of diabetic nephropathy . in mice with deficiency of mbl ,", "the classical functional and physical renal changes normally seen in this experimental model of type 1 diabetes were modified [ 29 , 30 ] .", "the fact that ficolin b does not appear to modulate diabetic effects on the kidney emphasizes the importance of mbl compared with ficolin b. both mbl and ficolin b activate the lectin pathway of the complement system , but only deficiency of mbl has been shown to protect against diabetic kidney damage .", "this indicates that the role of the lectin pathway in the development of diabetic nephropathy is complex and may depend on the specific carbohydrate - binding properties of mbl as previously described .", "the function of other complement factors in the first parts of the lectin pathway ( e.g. , ficolin a and masps ) in the pathology of diabetic kidney disease remains unknown and must be explored in further studies .", "one study indicates that ficolin a and ficolin b exert a cooperatively defensive role in destroying streptococcus pneumoniae , suggesting a synergetic immunological effect .", "this emphasises the need for further investigations involving both mouse ficolins . in order to fully understand the involvement of the lectin pathway in the development of diabetic nephropathy , an additional parallel experiment with masps is of particular interest given that they represent the limiting downward step in the complement activation", "in conclusion , this study demonstrates that ficolin b does not modify the kidney weight and acr in a type 1 diabetes mouse model .", "this indicates that the role of the lectin pathway in the development of diabetic nephropathy is specific and that hyperglycaemia - induced glycations on renal cells may be more prone to bind mbl than ficolin b."], "abstract_text": ["<S> \n background . </S>", "<S> the innate immune system may have adverse effects in diabetes and cardiovascular disease . </S>", "<S> the complement system seems to play a key role through erroneous complement activation via hyperglycaemia - induced neoepitopes . </S>", "<S> recently mannan - binding lectin ( mbl ) was shown to worsen diabetic kidney changes . </S>", "<S> we hypothesize that mouse ficolin b exerts detrimental effects in the diabetic kidney as seen for mbl . </S>", "<S> methods . </S>", "<S> we induced diabetes with streptozotocin in female wild - type mice and ficolin b knockout mice and included two similar nondiabetic groups . </S>", "<S> renal hypertrophy and excretion of urinary albumin and creatinine were quantified to assess diabetic kidney damage . </S>", "<S> results . in the wild - type groups , </S>", "<S> the kidney weighed 24% more in the diabetic mice compared to the controls . the diabetes - induced increase in kidney weight was 29% in the ficolin b knockout mice , that is , equal to wild - type animals ( two - way anova , p = 0.60 ) . in the wild - type mice the albumin - to - creatinine ratio ( acr ) was 32.5  </S>", "<S> mg / g higher in the diabetic mice compared to the controls . </S>", "<S> the difference was 62.5  mg / g in the ficolin b knockout mice , but this was not significantly different from the wild - type animals ( two - way anova , p = 0.21 ) . </S>", "<S> conclusions . in conclusion , </S>", "<S> the diabetes - induced effects on kidney weight and acr were not modified by the presence or absence of ficolin b. </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["diabetes is the most frequent cause of end - stage renal disease in industrialised countries [ 1 , 2 ] .", "clinically , diabetic nephropathy is characterized by the development of albuminuria and a subsequent decline in glomerular filtration rate .", "this severe complication significantly influences the risk of cardiovascular disease as well as mortality and quality of life [ 3 , 4 ] . at present", "the most important identified risk factors are diabetes duration , arterial blood pressure , and glycaemic regulation . as evident from an increasing incidence of affected patients ,", "however , there is still a great need for new strategies in the treatment and prevention of diabetic nephropathy .", "clear evidence indicates that the pathogenesis of diabetic nephropathy is multifactorial and triggered by a complex series of pathophysiological events .", "the inflammatory response in diabetes is highly complex involving proinflammatory cytokines and chemokines , for example , il1", "the impact of complement activation on the diabetic kidney may well , in part , be mediated through induction of cytokine response and inflammation [ 911 ] .", "several studies have linked diabetic late - complications to the complement system of the innate immune system [ 12 , 13 ] .", "the complement system plays a crucial role in recognition and clearance of infectious microbes and the system forms a link between innate and adaptive immunity .", "the activation of the complement system results in the release of multiple inflammatory signaling molecules .", "ultimately complement activation leads to the formation of pore - forming membrane attack complexes ( macs ) that are inserted in the cell membranes to mediate lyses of the cell through osmotic stress .", "however , in mammalian cells , it has been shown that sublytic amounts of mac can increase production of il-8 and monocyte chemoattractant protein 1 dependent on nfb nuclear translocation .", "furthermore , macs are shown to have a mitogenic effect and cause release of basic fibroblast growth factor and platelet - derived growth factor from endothelial cells leading to fibrosis in neighboring cells including glomerular mesangial cells [ 16 , 17 ] .", "the latter effects of mac may explain the link between complement and diabetic kidney damage .", "three activations pathways exist : the classical , the alternative , and the lectin pathway .", "the present paper focuses on the lectin pathway , in which at least five soluble pattern - recognition molecules are characterized that may activate the complement system , that is , mannan - binding lectin ( mbl ) , h - ficolin , l - ficolin , m - ficolin , and collectin - k1 .", "the three ficolins utilize a fibrinogen - like domain that binds , for example , n - acetylglucosamine , n - acetylgalactosamine , and n - acetyl - neuraminic acid , whereas mbl and cl - k1 have a carbohydrate recognition domain and through this bind specifically to patterns of monosaccharides . a very recent publication reports a close association between ficolin and diabetic nephropathy in patients with type 1 diabetes .", "the observational design of the study , however , limits its ability to study a cause - effect relationship . when ficolins bind they all initiate activation of associated serine proteases ( mbl associated serine proteases , masps ) , which subsequently cleave the complement factors , c2 and c4 , leading to further complement activation [ 20 , 21 ] .", "eventually , complement activation leads to the formation of macs causing cell lyses or induction of fibrosis . the balance between activation and inhibition of the complement cascade is tightly controlled by regulatory proteins in order to prevent damage of healthy host cells . in diabetes", ", inappropriate effects of the complement system may be present as glycation - induced dysfunction of the complement inhibitory mechanism and consequently overactivation of the system is indicated [ 2325 ] .", "it is speculated that diabetic patients are exposed to uncontrolled complement attack partly due to altered molecular patterns on the cell surfaces as a consequence of high blood glucose [ 24 , 25 ] .", "most significantly , an association is seen between diabetic nephropathy and the lectin pathway [ 2628 ] . we have previously demonstrated direct cause - effect relationship between presence of mbl and worsening of kidney injury in a mouse model of diabetic nephropathy [ 29 , 30 ] .", "we speculate that ficolins also exert detrimental effects in diabetes similar to mbl through activation of the lectin pathway as indicated in patients with type 1 diabetes .", "this study aimed to investigate the impact of ficolin b ( the orthologue to human m - ficolin ) on the development of diabetic nephropathy in a mouse model of type 1 diabetes ."], ["we used 11-week - old , female ficolin b knockout mice and age - matched , female c57bl/6j bomtac wild - type mice ( taconic , ry , denmark ) .", "the knockout ficolin b model was backcrossed more than 10 generations to a c57bl/6j bomtac genetic background ( own breeding ) . in each cage", "there were three to eight mice and they had free access to tap water and standard chow ( altromin number 1324 ; lage , germany ) .", "the environment was stable with a 12-hour light - dark cycle , temperature at 21  1c , and humidity of 55  5% .", "the ficolin b knockout mice and the wild - type mice were randomized into a diabetic and nondiabetic group ; thus four groups were made : ( 1 ) diabetic knockout mice ( n = 6 ) , ( 2 ) nondiabetic knockout mice ( n = 7 ) , ( 3 ) diabetic wild - type mice ( n = 11 ) , and ( 4 ) nondiabetic wild - type mice ( n = 11 ) .", "diabetes was induced by intraperitoneal injections of streptozotocin ( stz ) dissolved in a cold 10  mm citrate buffer ( doses of 55  mg / kg body weight , sigma aldrich , st louis , mo , usa ) on five consecutive days .", "the 18-week experiment was initiated when the mice were classified as diabetic ( blood glucose > 15  mm ) . animals with more than 15% sustained weight loss , signs of illness , or persistent ketonuria were excluded from the study .", "blood glucose was measured from tail vein by contour ( bayer diabetes care , kgs .", "lyngby , denmark ) . with combur test d strip ( roche diagnostics gmbh , mannheim , germany )", "two mice from each diabetic group were excluded because of insufficient increase in blood glucose levels .", "furthermore , two mice from the diabetic knockout group were excluded because of weight loss > 15% of body weight .", "the excluded mice were not included in the number of animals per group indicated above .", "spot urine was collected in eppendorf tubes on five consecutive days prior to sacrifice of the animals .", "the blood samples were drawn from under the tongue at baseline and from the retroorbital venous plexus at study end and collected in potassium edta tubes ( sarstedt , nmbrecht , germany ) .", "the animals were anesthetized by an intraperitoneal dose of ketamine at 0.5  mg / g body weight and xylazine at 0.2  mg / g body weight ( ketaminol 4 vet and narcoxyl vet , resp . ,", "urinary albumin excretion was determined by mouse albumin elisa quantification kit ( bethyl laboratories , inc . ,", "urine creatinine was measured by isocratic high - performance liquid chromatography ( hplc ) on a zorbax scx300 column ( agilent , usa ) using a slight modification of a method first reported by yuen et al . .", "in brief , 5  l urine was added to 100  l acetonitrile containing 0.5% acetic acid and vortexed for 15 seconds to extract the creatinine .", "after 15  min of 20c storage and centrifugation the supernatants were evaporated and then reconstituted with 25  l 5  mm sodium acetate , ph 4.1 .", "duplicate samples ( 10  ul each ) were fractionated on a 50  mm  2.1  mm zorbax scx300 column with an in - front scx guard column .", "isocratic hplc was performed at a flow rate of 1  ml / min , and uv absorbance was monitored at 225  nm .", "a standard curve was created by including a 2-fold dilution series of creatinine anhydrous ( sigma aldrich ) .", "this study was designed with two independent factors ; diabetes / nondiabetes and knockout / wild - type and thus analysed by two - way anova for normal distributed variable with equal variance .", "the main focus of interest was the interaction between the diabetic factor and the knockout factor ; that is , does ficolin b modify the effects of diabetes on the effect parameters ? if no interaction was found , the independent effects of diabetes and ficolin b on the kidney were estimated .", "for pairwise comparison , normal distributed data was tested with student 's t - test , whereas otherwise the wilcoxon mann - whitney rank sum test was used .", "data are given as mean ( 95% confidence interval ( ci ) ) unless else is stated ."], ["at baseline , the knockout mice on average weighed 20.0  g , which was slightly less than the wild type mice , 20.8  g ( p = 0.04 ) .", "no difference was found between the two diabetic groups or between the two nondiabetic groups ( table 1 ) .", "after 18 weeks an expected difference in body weight was observed between the diabetic and the nondiabetic mice independently of knockout status ( p < 0.001 ) .", "the nondiabetic mice weighed 3.2  g ( ci : 2.0  g4.3  g ) more than the diabetic mice .", "furthermore the diabetic knockout mice were significantly smaller than the diabetic wild type ( p < 0.05 ) .", "as presented in table 1 , blood glucose , estimated as area under the curve ( auc ) , did not differ between the two diabetic groups ( p = 0.69 ) or between the two nondiabetic groups ( p = 0.13 ) .", "the kidney weight was equally increased in diabetic wild - type mice , 24% ( ci : 13%36% ) , and in the diabetic knockout mice , 29% ( ci : 12%47% ) , compared to the respective control groups ( figure 2(a ) ) . no interaction between knockout and diabetes was found ( p = 0.60 ) , indicating that wild - type and knockout mice develop the same degree of diabetes - induced renal hypertrophy .", "the considerable body weight difference between the two diabetic groups at study end indicated that the kidney weight was to be normalised to the body weight .", "ficolin b did not modify the diabetes - induced increase in kidney weight when testing for interaction ( p = 0.11 ) .", "furthermore , no significant statistical difference was found in kidney weight per body weight between the diabetic wild - type , 1.95  mg / g , and the diabetic knockout , 2.89  mg / g ( p = 0.09 ) .", "the albumin - to - creatinine ration ( acr ) was higher among the diabetic wild - type mice , 76  mg / g ( ci : 50103  mg / g ) , compared to the nondiabetic wild - type mice , 44  mg / g ( ci : 2563  mg / g ) , p = 0.07 .", "similarly , the acr of diabetic knockout mice was 96  mg / g ( ci : 71122  mg / g ) compared to the nondiabetic knockout group , 34  mg / g ( ci : 2344  mg / g ) , p < 0.001 . as depicted in figure 3 no interaction was observed between diabetes and ficolin b knockout , p = 0.21 ."], ["in the present study we found no association between diabetes - induced kidney changes and the presence of ficolin b. we conclude that ficolin b is not responsible for , or a crucial contributory factor in , the pathophysiology of diabetic nephropathy . in our study , the kidney weight and to some extent the acr were altered by diabetes as expected .", "the diabetes - induced increase in kidney weight , measured by comparing the diabetic mice with the nondiabetic mice , was not statistically different between the wild - type and ficolin b knockout mice .", "the diabetes - induced increase in kidney weight was 24% in the wild - type mice and 29% in the ficolin b knockout mice .", "taking the lower body weight of the knockout mice into account , the difference in renal hypertrophy was still insignificant when comparing the wild - type mice and the ficolin b knockout mice .", "similarly , the diabetic change seen in acr was not altered in the absence of ficolin b. the experimental setup including four groups matched on age , body weight , and genetical background was a strength to the study , as the diabetes factor and the knockout factor were the only modulators of the outcome .", "most importantly both diabetic groups did reach and sustain blood glucose levels of above 15  mm . at study end ,", "the body weight differed among groups , which impeded the analyses of the diabetic kidney damage , because the knockout mice appear to be more vulnerable to type 1 diabetes mellitus .", "our study provides important new information on the association between the lectin pathway and diabetic kidney damage .", "we are the first to investigate the role of ficolin b ( which corresponds to ficolin m in human ) in the inflammatory response of diabetic nephropathy . in mice with deficiency of mbl ,", "the classical functional and physical renal changes normally seen in this experimental model of type 1 diabetes were modified [ 29 , 30 ] .", "the fact that ficolin b does not appear to modulate diabetic effects on the kidney emphasizes the importance of mbl compared with ficolin b. both mbl and ficolin b activate the lectin pathway of the complement system , but only deficiency of mbl has been shown to protect against diabetic kidney damage .", "this indicates that the role of the lectin pathway in the development of diabetic nephropathy is complex and may depend on the specific carbohydrate - binding properties of mbl as previously described .", "the function of other complement factors in the first parts of the lectin pathway ( e.g. , ficolin a and masps ) in the pathology of diabetic kidney disease remains unknown and must be explored in further studies .", "one study indicates that ficolin a and ficolin b exert a cooperatively defensive role in destroying streptococcus pneumoniae , suggesting a synergetic immunological effect .", "this emphasises the need for further investigations involving both mouse ficolins . in order to fully understand the involvement of the lectin pathway in the development of diabetic nephropathy , an additional parallel experiment with masps is of particular interest given that they represent the limiting downward step in the complement activation"], ["in conclusion , this study demonstrates that ficolin b does not modify the kidney weight and acr in a type 1 diabetes mouse model .", "this indicates that the role of the lectin pathway in the development of diabetic nephropathy is specific and that hyperglycaemia - induced glycations on renal cells may be more prone to bind mbl than ficolin b."]]}
{"article_id": "PMC4323503", "article_text": ["how to choose surgical approach in severe multilevel cervical cord compression case is a hot debate on spine surgery .", "some surgeons think anterior surgery should be of the primary choice , because of direct decompression , restoring intervertebral height and reconstruction of stability7,12 ) .", "some surgeons prefer posterior approach , owing to easy decompression , low surgical risks and complications14 ) . whereas other surgeons suggest that combined anterior and posterior approach at one stage achieve more sufficient decompression though with increasing of complications and costs10 ) .", "multiple factors should be considered when a surgery is to be performed , especially on patients with multilevel cord compression from both ventral and dorsal parts .", "which approach is better to decompress the cord sufficiently and maintain the spinal column simultaneously , anterior or posterior ?", "however , in clinical practice , surgeon 's experience and surgical skills are usually of the primary consideration .", "although anterior discectomy and fusion ( acdf ) , or corpectomy and fusion ( accf ) has satisfactory results in treatment of multilevel lesions of cervical spondylosis and ossification of the posterior longitudinal ligament ( opll ) , some patients remain complain of persistence or recurrence of new symptoms .", "reports elucidated the causes of unsatisfactory results and the revision techniques have so far been limited .", "thus , the purpose of this study was to investigate the causes for failed anterior multilevel acdf or accf , and the outcomes of secondary laminoplasty .", "between feb 2003 and may 2011 , 23 patients who complained of persistence of primary symptoms or occurrence of new symptoms for more than 3 months after multilevel anterior cervical surgery ( acdf or accf ) in our spine surgery center were reviewed in the study .", "patients demonstrated as neck pain , cervical movement restriction , spasticity , sensory loss , weakness , or sphincter dysfunction to different extent .", "therapeutic treatments included traction , collar support , physical therapy , functional exercise and analgesics . of 23 patients ,", "the study population consisted of 12 men and 5 women , with a mean age of 59.95.6 ( range , 49 - 78 ) years old .", "main indications for the second surgery were when patients displayed signs of cord compression at least at 2 levels associated with / without canal stenosis corresponding to signs of myelopathy .", "major exclusion criteria were symptomatic treatment less than 3 months , axial neck pain as the solitary symptom , cervical instability , active systemic infection , and other contraindications in routine surgery .", "instability documented as angle difference ( cobb angle ) more than 15 degrees and/or listhesis more than 3 mm on extension and flexion lateral views .", "the patient underwent a general anesthesia and is positioned prone with the head and neck held in slight flexion on the operating table .", "a cervical midline incision is performed down to the cervical fascia , and the cervical musculature is reflected off of the involved cervical lamina .", "the decompression levels extended to one level cranial or / and caudal to compressive lesions are included .", "a 4-mm laminotomy is created just medial to the level of the pedicles on the clinically more symptomatic side , or on the most narrowed side on images , if there is no dominant symptom side .", "then the involved ligaments and flavum is removed until the pulse of dura sac is seen .", "the external cortex is thin out to a 3- to 4-mm trough on the contralateral lamina , again just medial to the level of the pedicles , and inner cortex is preserved .", "the laminae are lifted carefully to create a greenstick fracture on the hinge side , expanding the spinal canal diameter", ". then cervical titanium mini - plate ( medtronic sofamer danek , memphis , usa or depuy synthes , bettlach , switzerland ) is placed at each level secured by two 4-mm screws onto the lamina and two at the level of the lateral masses . at last", "the patient is placed in a hard cervical collar for the first 6 weeks after surgery .", "patients ' ages , gender , disease , the first surgical technique and levels , intervals between the first surgery and the onset of new symptoms or the persistence of primary symptoms , re - surgical levels , and causes for revision were examined .", "the outcomes were evaluated preoperatively , at 1 week , 2 months , 6 months , and the final visit .", "scores are based on the rating of motor function ( fingers , 0 to 4 points ; shoulder and elbows , -2 to 0 points ; and lower extremity , 0 to 4 points ) , sensory function ( upper extremity , 0 to 2 points ; lower extremity , 0 to 2 points ; and trunk , 0 to 2 points ) and urinary bladder function ( 0 to 3 points ) . a normal joa score is 17 points .", "recovery rate was calculated as : ( scores at follow up - scores preoperatively)/(17-scores preoperatively)100% .", "recovery rate more than 75% was classified as excellent , 50 - 75% good , 25 - 50% fair , and less than 25% poor ; the excellent to good rate was calculated .", "patients were required to rate their neck pain , using the visual analogue scale ( vas ; 0 mm = no pain , 100 mm = worst pain ) at different intervals , supplemented with at 1 day postoperatively .", "cervical alignment was created by a line parallel to the inferior aspect of the c2 body and a line parallel to that of the c7 body , according to cobb 's method on neutral lateral view ( fig .", "all data were analyzed by sas 8.0 ( statistical analysis system , sas institute inc . ,", "between feb 2003 and may 2011 , 23 patients who complained of persistence of primary symptoms or occurrence of new symptoms for more than 3 months after multilevel anterior cervical surgery ( acdf or accf ) in our spine surgery center were reviewed in the study .", "patients demonstrated as neck pain , cervical movement restriction , spasticity , sensory loss , weakness , or sphincter dysfunction to different extent .", "therapeutic treatments included traction , collar support , physical therapy , functional exercise and analgesics . of 23 patients ,", "the study population consisted of 12 men and 5 women , with a mean age of 59.95.6 ( range , 49 - 78 ) years old .", "main indications for the second surgery were when patients displayed signs of cord compression at least at 2 levels associated with / without canal stenosis corresponding to signs of myelopathy .", "major exclusion criteria were symptomatic treatment less than 3 months , axial neck pain as the solitary symptom , cervical instability , active systemic infection , and other contraindications in routine surgery .", "instability documented as angle difference ( cobb angle ) more than 15 degrees and/or listhesis more than 3 mm on extension and flexion lateral views .", "the patient underwent a general anesthesia and is positioned prone with the head and neck held in slight flexion on the operating table .", "a cervical midline incision is performed down to the cervical fascia , and the cervical musculature is reflected off of the involved cervical lamina .", "the decompression levels extended to one level cranial or / and caudal to compressive lesions are included .", "a 4-mm laminotomy is created just medial to the level of the pedicles on the clinically more symptomatic side , or on the most narrowed side on images , if there is no dominant symptom side .", "then the involved ligaments and flavum is removed until the pulse of dura sac is seen .", "the external cortex is thin out to a 3- to 4-mm trough on the contralateral lamina , again just medial to the level of the pedicles , and inner cortex is preserved .", "the laminae are lifted carefully to create a greenstick fracture on the hinge side , expanding the spinal canal diameter", ". then cervical titanium mini - plate ( medtronic sofamer danek , memphis , usa or depuy synthes , bettlach , switzerland ) is placed at each level secured by two 4-mm screws onto the lamina and two at the level of the lateral masses . at last ,", "the patient is placed in a hard cervical collar for the first 6 weeks after surgery .", "patients ' ages , gender , disease , the first surgical technique and levels , intervals between the first surgery and the onset of new symptoms or the persistence of primary symptoms , re - surgical levels , and causes for revision were examined .", "the outcomes were evaluated preoperatively , at 1 week , 2 months , 6 months , and the final visit .", "scores are based on the rating of motor function ( fingers , 0 to 4 points ; shoulder and elbows , -2 to 0 points ; and lower extremity , 0 to 4 points ) , sensory function ( upper extremity , 0 to 2 points ; lower extremity , 0 to 2 points ; and trunk , 0 to 2 points ) and urinary bladder function ( 0 to 3 points ) .", "recovery rate was calculated as : ( scores at follow up - scores preoperatively)/(17-scores preoperatively)100% .", "recovery rate more than 75% was classified as excellent , 50 - 75% good , 25 - 50% fair , and less than 25% poor ; the excellent to good rate was calculated .", "patients were required to rate their neck pain , using the visual analogue scale ( vas ; 0 mm = no pain , 100 mm = worst pain ) at different intervals , supplemented with at 1 day postoperatively .", "cervical alignment was created by a line parallel to the inferior aspect of the c2 body and a line parallel to that of the c7 body , according to cobb 's method on neutral lateral view ( fig .", "all data were analyzed by sas 8.0 ( statistical analysis system , sas institute inc . , cary , nc , usa ) .", "mean duration between the first surgery and the onset of new symptoms or persistence of symptoms was 14.54.2 ( range , 1 to 44 ) months .", "causes for revision surgery were inappropriate approach in 3 patients ( 17.6% ) , insufficient decompression in 4 patients ( 23.5% ) , adjacent degeneration in 2 patients ( 11.8% ) , and disease progression in 8 patients ( 47.1% ) .", "there were 8 patients of disease progression , with 4 cases of opll and 4 cases of cervical spondylotic myelopathy ( csm ) .", "the progression levels increased from 1.250.2 at the first surgery to 2.250.4 at the second surgery on opll patients , and from 1.50.2 to 2.50.4 on csm patients .", "mean follow - up duration was 29.712.1 ( range , 6 - 84 ) months .", "mean joa score was 10.54.1 preoperatively , 11.84.2 at 1 week , 13.54.4 at 2 months , 14.14.8 at 6 months and", "13.94.6 at the final visit ( table 2 ) ; significant improvement achieved at 2 months ( p<0.05 ) and maintained thereafter ( p>0.05 ) .", "mean recovery rate was 20.0% , 46.1% , 55.4% , and 52.3% after surgery , respectively ; significant improvement was detected at 2 months ( p<0.05 ) and maintained with the time ( p>0.05 ) .", "one case had excellent result , 5 good , 7 fair , and 4 poor , with an excellent to good rate of 35.3% at 1 week ; two cases had excellent results , 8 good , 5 fair , and 2 poor , with an excellent to good rate of 58.8% at 2 months ; three cases had excellent results , 9 good , 4 fair , and 1 poor , with an excellent to good rate of 70.6% at 6 months ; three cases had excellent results , 8 good , 4 fair , and 2 poor , with an excellent to good rate of 64.7% at the final visit .", "mean vas score was 29.116.7 preoperatively , 51.127.6 at 1 day , 30.717.2 at 1 week , 9.19.9 at 2 months , 8.57.9 at 6 months , and 9.08.2 at the final visit .", "vas score decreased at 1 week ( p<0.05 ) and at 2 months ( p<0.05 ) .", "mean lordotic angle was 11.710.3 preoperatively , 11.110.2 at 1 week , 11.710.3 at 2 months , 10.99.7 at 6 months and", "10.39.1 at the final visit ; no significance was observed during the follow up ( p>0.05 ) ( fig .", "one case demonstrated as axial pain and relieved 2 weeks after pain management and physical therapy .", "acdf or accf has excellent and good results in treatment of cervical spondylosis and opll .", "the anterior approach removes pathological compression to the cord directly , stabilizes spine column , and maintains cervical alignment .", "however , if the disease involved multiple levels or posterior compression , the ideal effects of anterior approach are difficult to achieve3,5 ) .", "it is not easy in performing sufficient decompression and preserving cervical range of motion on acdf or accf .", "besides , corpectomy of multilevel vertebrae in series destroys the integrity of spine column and has a risk of nonunion . especially for patients with congenital or acquired narrowed canal ,", "the compression to cord remains after initial anterior surgery . in the study , 3 patients demonstrated narrowed canal associated with posterior compression and persistent even after aggravation of symptoms after anterior surgery .", "however , after the secondary laminoplasty , the canal is enlarged significantly and the cord is given more space to breathe1 ) .", "if the compression was mainly from the posterior part associated with or without canal stenosis , the posterior approach should be performed .", "second cause was adjacent degeneration . in a study by matsumoto et al.11 ) that acdf patients had significantly higher incidence of progression of disc degeneration than control subjects .", "prasarn et al.15 ) thought biomechanics altered after fusion , with an increased adjacent - segment motion in cervical spine . after fixation and fusion , there is a change in the load of the stiffed fusion which accelerated the degeneration of adjacent disc and endplate , especially on the caudal level .", "faldini et al.6 ) thought proper lordotic alignment could prevent adjacent degeneration . in our opinion , though factors of degeneration were not systematically elucidated , maintaining lordosis and exercising cervical dorsal musculatures could help a lot .", "it was thought that factors influencing decompression included surgical approach , technique and experience of surgeon , and overall knowledge of pathologic structure .", "osteophyte , ossified nucleus pulposus , opll and abnormal curvature venous plexus increased complexity of decompression , especially in patients with multilevel lesions or on narrowed canal .", "the last but not the least was disease progression . in the study , half of patients displayed as extension of cord compression though after sufficient initial anterior decompression .", "the symptoms aggravated gradually until the patients seek for the surgeons . after the revised posterior surgery , the enlarged canal was enough to compensate for the spinal cord in a narrowed space for a long time .", "thus , in the study , symptoms of patients with disease progression alleviated immediately after revision .", "factors of progression are not clear , which may include metabolism , heredity , endocrine secretion and biomechanics .", "choi et al.4 ) also believed that age , severity of disease , irreversible changes of gray matter in cord , duration of symptoms , and diabetes mellitus took important parts .", "patients ' age , general status , extent and range of compression , and safety should be considered , when planning a revision surgery .", "pang et al.13 ) thought although anterior surgery was more effective in relieving cord compromise , inadequate decompression and disease progression might require secondary laminoplasty .", "scar tissue around anterior region after anterior surgery increased risks of neurovascular , trachea or esophagus injury .", "be - sides , once the cage and vertebrae above and below are fused , it was difficult to remove the protruded or ossified materials along posterior wall of vertebrae , especially in multilevel segments .", "it was thought that myelopathy could be treated via anterior or posterior approaches , but risks and complications in acdf increase .", "average joa score , recovery rate , and excellent to good rate did n't improve until at 2 months , and attained the maximum at 6 months , with a slight deterioration thereafter .", "however , it was reported by hyun et al.9 ) that loss of alignment was time - dependent but no further decreased after 18 months . in the study ,", "the lordosis maintained during the entire follow up . in a study by baba et al.2 ) , excellent to good rate of secondary laminoplasty was 55.6% .", "they thought that narrowed canal added to the risk of recurrence , and additional laminoplasty could prevent structural compromise occurring adjacent vertebrae .", "symptoms and signs had no obvious alleviation , with high - signal intensity remained persistent .", "yagi et al.18 ) also thought that long - term outcome was significantly worse in these patients .", "the outcome was also worse in patients with expansion of high - signal area , which the causes might be not only from necrosis secondary to ischemia of the anterior spinal artery , but also from the repeated minor traumas inflicted on the cord from an unstable spine . combining the research and related literatures , we thought that multiple factors affect outcomes of revision surgery , which included age , extent lesion of cord , surgical times , mental disease , and so on .", "edwards et al.5 ) deemed that both corpectomy and laminoplasty arrested progression in multilevel myelopathy , which resulted in significant neurologic recovery and pain reduction .", "however , the laminoplasty cohort required less pain medication than did the corpectomy cohort . given the higher prevalence of complications in anterior approach , it was believed that laminoplasty might be the preferred treatment in the absence of preoperative kyphosis . however , although it is a safe and effective technique , complications include paralysis of c5 nerve root , axial pain , and instability of spine , cerebral spinal fluid leakage and close of \" opening door\"16 ) .", "laminoplasty preserving the c7 muscle attachment is very important . in the study , except for the decompression extending to t1 , all the other cases preserve muscle insertions into the c7 spinous process .", "it was also reported that this preservation was associated with a significantly decreased frequency of postoperative axial neck pain8 ) .", "future studies will seek to research in a prospective , randomized and controlled way with a longer duration . despite limitations ,", "laminoplasty should be considered as a primary procedure or as a revision procedure versus anterior approach in multilevel lesions because of effective decompression , preservation cervical alignment and minimum morbidity .", "laminoplasty has satisfactory results in failed anterior surgery , with a low incidence of complications ."], "abstract_text": ["<S> objectiveto investigate the causes for failed anterior cervical surgery and the outcomes of secondary laminoplasty.methodsseventeen patients failed anterior multilevel cervical surgery and the following conservative treatments between feb 2003 and may 2011 underwent secondary laminoplasty . </S>", "<S> outcomes were evaluated by the japanese orthopaedic association ( joa ) scale and visual analogue scale ( vas ) before the secondary surgery , at 1 week , 2 months , 6 months , and the final visit . </S>", "<S> cervical alignment , causes for revision and complications were also assessed.resultswith a mean follow - up of 29.712.1 months , joa score , recovery rate and excellent to good rate improved significantly at 2 months ( p<0.05 ) and maintained thereafter ( p>0.05 ) . </S>", "<S> mean vas score decreased postoperatively ( p<0.05 ) . </S>", "<S> lordotic angle maintained during the entire follow up ( p>0.05 ) . </S>", "<S> the causes for secondary surgery were inappropriate approach in 3 patients , insufficient decompression in 4 patients , adjacent degeneration in 2 patients , and disease progression in 8 patients . </S>", "<S> complications included one case of c5 palsy , axial pain and cerebrospinal fluid leakage , respectively.conclusionlaminoplasty has satisfactory results in failed multilevel anterior surgery , with a low incidence of complications . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Study population", "Surgical procedures", "Outcome measures", "Statistics", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["how to choose surgical approach in severe multilevel cervical cord compression case is a hot debate on spine surgery .", "some surgeons think anterior surgery should be of the primary choice , because of direct decompression , restoring intervertebral height and reconstruction of stability7,12 ) .", "some surgeons prefer posterior approach , owing to easy decompression , low surgical risks and complications14 ) . whereas other surgeons suggest that combined anterior and posterior approach at one stage achieve more sufficient decompression though with increasing of complications and costs10 ) .", "multiple factors should be considered when a surgery is to be performed , especially on patients with multilevel cord compression from both ventral and dorsal parts .", "which approach is better to decompress the cord sufficiently and maintain the spinal column simultaneously , anterior or posterior ?", "however , in clinical practice , surgeon 's experience and surgical skills are usually of the primary consideration .", "although anterior discectomy and fusion ( acdf ) , or corpectomy and fusion ( accf ) has satisfactory results in treatment of multilevel lesions of cervical spondylosis and ossification of the posterior longitudinal ligament ( opll ) , some patients remain complain of persistence or recurrence of new symptoms .", "reports elucidated the causes of unsatisfactory results and the revision techniques have so far been limited .", "thus , the purpose of this study was to investigate the causes for failed anterior multilevel acdf or accf , and the outcomes of secondary laminoplasty ."], ["between feb 2003 and may 2011 , 23 patients who complained of persistence of primary symptoms or occurrence of new symptoms for more than 3 months after multilevel anterior cervical surgery ( acdf or accf ) in our spine surgery center were reviewed in the study .", "patients demonstrated as neck pain , cervical movement restriction , spasticity , sensory loss , weakness , or sphincter dysfunction to different extent .", "therapeutic treatments included traction , collar support , physical therapy , functional exercise and analgesics . of 23 patients ,", "the study population consisted of 12 men and 5 women , with a mean age of 59.95.6 ( range , 49 - 78 ) years old .", "main indications for the second surgery were when patients displayed signs of cord compression at least at 2 levels associated with / without canal stenosis corresponding to signs of myelopathy .", "major exclusion criteria were symptomatic treatment less than 3 months , axial neck pain as the solitary symptom , cervical instability , active systemic infection , and other contraindications in routine surgery .", "instability documented as angle difference ( cobb angle ) more than 15 degrees and/or listhesis more than 3 mm on extension and flexion lateral views .", "the patient underwent a general anesthesia and is positioned prone with the head and neck held in slight flexion on the operating table .", "a cervical midline incision is performed down to the cervical fascia , and the cervical musculature is reflected off of the involved cervical lamina .", "the decompression levels extended to one level cranial or / and caudal to compressive lesions are included .", "a 4-mm laminotomy is created just medial to the level of the pedicles on the clinically more symptomatic side , or on the most narrowed side on images , if there is no dominant symptom side .", "then the involved ligaments and flavum is removed until the pulse of dura sac is seen .", "the external cortex is thin out to a 3- to 4-mm trough on the contralateral lamina , again just medial to the level of the pedicles , and inner cortex is preserved .", "the laminae are lifted carefully to create a greenstick fracture on the hinge side , expanding the spinal canal diameter", ". then cervical titanium mini - plate ( medtronic sofamer danek , memphis , usa or depuy synthes , bettlach , switzerland ) is placed at each level secured by two 4-mm screws onto the lamina and two at the level of the lateral masses . at last", "the patient is placed in a hard cervical collar for the first 6 weeks after surgery .", "patients ' ages , gender , disease , the first surgical technique and levels , intervals between the first surgery and the onset of new symptoms or the persistence of primary symptoms , re - surgical levels , and causes for revision were examined .", "the outcomes were evaluated preoperatively , at 1 week , 2 months , 6 months , and the final visit .", "scores are based on the rating of motor function ( fingers , 0 to 4 points ; shoulder and elbows , -2 to 0 points ; and lower extremity , 0 to 4 points ) , sensory function ( upper extremity , 0 to 2 points ; lower extremity , 0 to 2 points ; and trunk , 0 to 2 points ) and urinary bladder function ( 0 to 3 points ) . a normal joa score is 17 points .", "recovery rate was calculated as : ( scores at follow up - scores preoperatively)/(17-scores preoperatively)100% .", "recovery rate more than 75% was classified as excellent , 50 - 75% good , 25 - 50% fair , and less than 25% poor ; the excellent to good rate was calculated .", "patients were required to rate their neck pain , using the visual analogue scale ( vas ; 0 mm = no pain , 100 mm = worst pain ) at different intervals , supplemented with at 1 day postoperatively .", "cervical alignment was created by a line parallel to the inferior aspect of the c2 body and a line parallel to that of the c7 body , according to cobb 's method on neutral lateral view ( fig .", "all data were analyzed by sas 8.0 ( statistical analysis system , sas institute inc . ,"], ["between feb 2003 and may 2011 , 23 patients who complained of persistence of primary symptoms or occurrence of new symptoms for more than 3 months after multilevel anterior cervical surgery ( acdf or accf ) in our spine surgery center were reviewed in the study .", "patients demonstrated as neck pain , cervical movement restriction , spasticity , sensory loss , weakness , or sphincter dysfunction to different extent .", "therapeutic treatments included traction , collar support , physical therapy , functional exercise and analgesics . of 23 patients ,", "the study population consisted of 12 men and 5 women , with a mean age of 59.95.6 ( range , 49 - 78 ) years old .", "main indications for the second surgery were when patients displayed signs of cord compression at least at 2 levels associated with / without canal stenosis corresponding to signs of myelopathy .", "major exclusion criteria were symptomatic treatment less than 3 months , axial neck pain as the solitary symptom , cervical instability , active systemic infection , and other contraindications in routine surgery .", "instability documented as angle difference ( cobb angle ) more than 15 degrees and/or listhesis more than 3 mm on extension and flexion lateral views ."], ["the patient underwent a general anesthesia and is positioned prone with the head and neck held in slight flexion on the operating table .", "a cervical midline incision is performed down to the cervical fascia , and the cervical musculature is reflected off of the involved cervical lamina .", "the decompression levels extended to one level cranial or / and caudal to compressive lesions are included .", "a 4-mm laminotomy is created just medial to the level of the pedicles on the clinically more symptomatic side , or on the most narrowed side on images , if there is no dominant symptom side .", "then the involved ligaments and flavum is removed until the pulse of dura sac is seen .", "the external cortex is thin out to a 3- to 4-mm trough on the contralateral lamina , again just medial to the level of the pedicles , and inner cortex is preserved .", "the laminae are lifted carefully to create a greenstick fracture on the hinge side , expanding the spinal canal diameter", ". then cervical titanium mini - plate ( medtronic sofamer danek , memphis , usa or depuy synthes , bettlach , switzerland ) is placed at each level secured by two 4-mm screws onto the lamina and two at the level of the lateral masses . at last ,", "the patient is placed in a hard cervical collar for the first 6 weeks after surgery ."], ["patients ' ages , gender , disease , the first surgical technique and levels , intervals between the first surgery and the onset of new symptoms or the persistence of primary symptoms , re - surgical levels , and causes for revision were examined .", "the outcomes were evaluated preoperatively , at 1 week , 2 months , 6 months , and the final visit .", "scores are based on the rating of motor function ( fingers , 0 to 4 points ; shoulder and elbows , -2 to 0 points ; and lower extremity , 0 to 4 points ) , sensory function ( upper extremity , 0 to 2 points ; lower extremity , 0 to 2 points ; and trunk , 0 to 2 points ) and urinary bladder function ( 0 to 3 points ) .", "recovery rate was calculated as : ( scores at follow up - scores preoperatively)/(17-scores preoperatively)100% .", "recovery rate more than 75% was classified as excellent , 50 - 75% good , 25 - 50% fair , and less than 25% poor ; the excellent to good rate was calculated .", "patients were required to rate their neck pain , using the visual analogue scale ( vas ; 0 mm = no pain , 100 mm = worst pain ) at different intervals , supplemented with at 1 day postoperatively .", "cervical alignment was created by a line parallel to the inferior aspect of the c2 body and a line parallel to that of the c7 body , according to cobb 's method on neutral lateral view ( fig ."], ["all data were analyzed by sas 8.0 ( statistical analysis system , sas institute inc . , cary , nc , usa ) ."], ["mean duration between the first surgery and the onset of new symptoms or persistence of symptoms was 14.54.2 ( range , 1 to 44 ) months .", "causes for revision surgery were inappropriate approach in 3 patients ( 17.6% ) , insufficient decompression in 4 patients ( 23.5% ) , adjacent degeneration in 2 patients ( 11.8% ) , and disease progression in 8 patients ( 47.1% ) .", "there were 8 patients of disease progression , with 4 cases of opll and 4 cases of cervical spondylotic myelopathy ( csm ) .", "the progression levels increased from 1.250.2 at the first surgery to 2.250.4 at the second surgery on opll patients , and from 1.50.2 to 2.50.4 on csm patients .", "mean follow - up duration was 29.712.1 ( range , 6 - 84 ) months .", "mean joa score was 10.54.1 preoperatively , 11.84.2 at 1 week , 13.54.4 at 2 months , 14.14.8 at 6 months and", "13.94.6 at the final visit ( table 2 ) ; significant improvement achieved at 2 months ( p<0.05 ) and maintained thereafter ( p>0.05 ) .", "mean recovery rate was 20.0% , 46.1% , 55.4% , and 52.3% after surgery , respectively ; significant improvement was detected at 2 months ( p<0.05 ) and maintained with the time ( p>0.05 ) .", "one case had excellent result , 5 good , 7 fair , and 4 poor , with an excellent to good rate of 35.3% at 1 week ; two cases had excellent results , 8 good , 5 fair , and 2 poor , with an excellent to good rate of 58.8% at 2 months ; three cases had excellent results , 9 good , 4 fair , and 1 poor , with an excellent to good rate of 70.6% at 6 months ; three cases had excellent results , 8 good , 4 fair , and 2 poor , with an excellent to good rate of 64.7% at the final visit .", "mean vas score was 29.116.7 preoperatively , 51.127.6 at 1 day , 30.717.2 at 1 week , 9.19.9 at 2 months , 8.57.9 at 6 months , and 9.08.2 at the final visit .", "vas score decreased at 1 week ( p<0.05 ) and at 2 months ( p<0.05 ) .", "mean lordotic angle was 11.710.3 preoperatively , 11.110.2 at 1 week , 11.710.3 at 2 months , 10.99.7 at 6 months and", "10.39.1 at the final visit ; no significance was observed during the follow up ( p>0.05 ) ( fig .", "one case demonstrated as axial pain and relieved 2 weeks after pain management and physical therapy ."], ["acdf or accf has excellent and good results in treatment of cervical spondylosis and opll .", "the anterior approach removes pathological compression to the cord directly , stabilizes spine column , and maintains cervical alignment .", "however , if the disease involved multiple levels or posterior compression , the ideal effects of anterior approach are difficult to achieve3,5 ) .", "it is not easy in performing sufficient decompression and preserving cervical range of motion on acdf or accf .", "besides , corpectomy of multilevel vertebrae in series destroys the integrity of spine column and has a risk of nonunion . especially for patients with congenital or acquired narrowed canal ,", "the compression to cord remains after initial anterior surgery . in the study , 3 patients demonstrated narrowed canal associated with posterior compression and persistent even after aggravation of symptoms after anterior surgery .", "however , after the secondary laminoplasty , the canal is enlarged significantly and the cord is given more space to breathe1 ) .", "if the compression was mainly from the posterior part associated with or without canal stenosis , the posterior approach should be performed .", "second cause was adjacent degeneration . in a study by matsumoto et al.11 ) that acdf patients had significantly higher incidence of progression of disc degeneration than control subjects .", "prasarn et al.15 ) thought biomechanics altered after fusion , with an increased adjacent - segment motion in cervical spine . after fixation and fusion , there is a change in the load of the stiffed fusion which accelerated the degeneration of adjacent disc and endplate , especially on the caudal level .", "faldini et al.6 ) thought proper lordotic alignment could prevent adjacent degeneration . in our opinion , though factors of degeneration were not systematically elucidated , maintaining lordosis and exercising cervical dorsal musculatures could help a lot .", "it was thought that factors influencing decompression included surgical approach , technique and experience of surgeon , and overall knowledge of pathologic structure .", "osteophyte , ossified nucleus pulposus , opll and abnormal curvature venous plexus increased complexity of decompression , especially in patients with multilevel lesions or on narrowed canal .", "the last but not the least was disease progression . in the study , half of patients displayed as extension of cord compression though after sufficient initial anterior decompression .", "the symptoms aggravated gradually until the patients seek for the surgeons . after the revised posterior surgery , the enlarged canal was enough to compensate for the spinal cord in a narrowed space for a long time .", "thus , in the study , symptoms of patients with disease progression alleviated immediately after revision .", "factors of progression are not clear , which may include metabolism , heredity , endocrine secretion and biomechanics .", "choi et al.4 ) also believed that age , severity of disease , irreversible changes of gray matter in cord , duration of symptoms , and diabetes mellitus took important parts .", "patients ' age , general status , extent and range of compression , and safety should be considered , when planning a revision surgery .", "pang et al.13 ) thought although anterior surgery was more effective in relieving cord compromise , inadequate decompression and disease progression might require secondary laminoplasty .", "scar tissue around anterior region after anterior surgery increased risks of neurovascular , trachea or esophagus injury .", "be - sides , once the cage and vertebrae above and below are fused , it was difficult to remove the protruded or ossified materials along posterior wall of vertebrae , especially in multilevel segments .", "it was thought that myelopathy could be treated via anterior or posterior approaches , but risks and complications in acdf increase .", "average joa score , recovery rate , and excellent to good rate did n't improve until at 2 months , and attained the maximum at 6 months , with a slight deterioration thereafter .", "however , it was reported by hyun et al.9 ) that loss of alignment was time - dependent but no further decreased after 18 months . in the study ,", "the lordosis maintained during the entire follow up . in a study by baba et al.2 ) , excellent to good rate of secondary laminoplasty was 55.6% .", "they thought that narrowed canal added to the risk of recurrence , and additional laminoplasty could prevent structural compromise occurring adjacent vertebrae .", "symptoms and signs had no obvious alleviation , with high - signal intensity remained persistent .", "yagi et al.18 ) also thought that long - term outcome was significantly worse in these patients .", "the outcome was also worse in patients with expansion of high - signal area , which the causes might be not only from necrosis secondary to ischemia of the anterior spinal artery , but also from the repeated minor traumas inflicted on the cord from an unstable spine . combining the research and related literatures , we thought that multiple factors affect outcomes of revision surgery , which included age , extent lesion of cord , surgical times , mental disease , and so on .", "edwards et al.5 ) deemed that both corpectomy and laminoplasty arrested progression in multilevel myelopathy , which resulted in significant neurologic recovery and pain reduction .", "however , the laminoplasty cohort required less pain medication than did the corpectomy cohort . given the higher prevalence of complications in anterior approach , it was believed that laminoplasty might be the preferred treatment in the absence of preoperative kyphosis . however , although it is a safe and effective technique , complications include paralysis of c5 nerve root , axial pain , and instability of spine , cerebral spinal fluid leakage and close of \" opening door\"16 ) .", "laminoplasty preserving the c7 muscle attachment is very important . in the study , except for the decompression extending to t1 , all the other cases preserve muscle insertions into the c7 spinous process .", "it was also reported that this preservation was associated with a significantly decreased frequency of postoperative axial neck pain8 ) .", "future studies will seek to research in a prospective , randomized and controlled way with a longer duration . despite limitations ,", "laminoplasty should be considered as a primary procedure or as a revision procedure versus anterior approach in multilevel lesions because of effective decompression , preservation cervical alignment and minimum morbidity ."], ["laminoplasty has satisfactory results in failed anterior surgery , with a low incidence of complications ."]]}
{"article_id": "PMC3669588", "article_text": ["population aging is the process by which older individuals become a proportionally larger share of the total population .", "it was the most distinctive global demographic event that the world witnessed in the past century and is an important event for the twenty - first century too .", "it was initially experienced by the more developed countries and has recently become apparent in the developing countries .", "population aging will be faced globally by all countries in this century , although at varying levels of intensity and time . according to the world health organization ( who )", ", world 's elderly population i.e. , people 60 years of age and older is approximately 650 million at present and by 2050 , it is forecast to reach 2 billion . in 2008 , five out of the top ten causes of mortality worldwide , other than injuries , were non - communicable diseases ( ncds ) and expected to go up to seven out of ten by the year 2030 . by then about 76% of the deaths in the world will be due to ncds .", "government and the who have already recognized the huge burden of preventable disease , disability , death and distress caused by the ncds . the present international health agenda guided by who focuses on four conditions ( cardiovascular disease , diabetes , cancer and chronic respiratory disease ) responsible for most of the premature mortality and four  lifestyle risk factors ( smoking , harmful alcohol use , lack of physical activity and high salt , high fat diets ) leading to above conditions .", "medications play crucial role in geriatric health care as they treat chronic diseases , alleviate pain and improve quality of life .", "age - related changes in drug disposition and pharmacodynamic responses have significant clinical implications , and increased use of a number of medications in elderly raises the risk of medicine - related problems that may occur . as medication use and the incidence of adverse drug outcomes increase with advancing age , it is important to ensure quality use of medicines in older people towards attaining a higher goal of healthy and active aging .", "but , use of drugs by the elderly and their clinical outcomes especially adverse drug reactions ( adrs ) have not been prominent research topic and continues to be given low priority in national and international public health arena , at least in a developing country like india . however , we feel that this probably needs more attention in india because we have not completely dealt with the scourge of communicable diseases yet .", "in addition , 70% of all older people now live in low- or middle - income countries , including india where sustainable pharmacovigilance systems has not developed as yet .", "the relationship between increased use of drugs and elderly is well established . consequently , increased use of medications in elderly increases the risk of adrs .", "studies from around the world have shown a definite correlation between increasing age and adr rate , at least for some medical conditions .", "although very few such studies are available in indian settings , harugeri et al . in a hospital setting found that the prevalence of adr - related hospital admissions was 5.9% , while in another such study in india , it was observed to be 6.7% . for india ,", "harugeri et al . predicted that 18000 bed days in a given time would be due to adrs in elderly and total cost of hospital stay due to adrs is estimated to be us $ 4350 ( inr 200100 ) , that is us $ 80.5 per patient ( 108.7% of per capita per year expenditure on health ) .", "there has been much debate on whether advancing age by itself is a cause of increased risk of adrs but merely a marker for comorbidity , altered pharmacokinetics , and polypharmacy .", "gurwitz and avorn concluded that  patient - specific physiological and functional characteristics are probably more important than any chronological measure in predicting both adverse and beneficial outcomes associated with specific drug therapies  , while studies around the world have clearly shown that the risks of adrs ( including interactions ) is related to the number of medicines taken and sometimes due to inappropriate use of medicines .", "( 1991 ) observed an exponential rather than the linear relation between the risks of adr and the number of medicines taken in 9000 elderly italian patients receiving 10 medicines .", "review of several studies by steward rb et al . found that the patients aged above 65 years use an average of two to six prescribed medications , and 1 - 3.4 non - prescribed medications .", "out of all the factors that are most consistently associated with adverse drug reactions , polypharmacy is considered to be the most important .", "thus , studies have correlated the integral association between old age and increased rate of adrs arising out of confounding association between age and polypharmacy contributed by age - related changes in pharmacodynamics and pharmacokinetics at least for some medical conditions .", "the classical pharmacological classification of adrs by rawlins and thompson divides adr into two major subtypes : type a reactions , which are dose - dependent and predictable , and unpredictable type b (  bizarre or idiosyncratic ) reactions .", "majority of adrs ( 80% ) causing admission or occurring in hospital setting are type a reactions .", "they are predictable and potentially avoidable in nature as they are related to accentuation of known pharmacological effects of the drug .", "drugs associated with type a reactions are generally with low therapeutic index and commonly used among elderly .", "the lists of medicines most likely to be used in the elderly include antibiotics , anticoagulants , digoxin , diuretics , hypoglycemic agents , antineoplastic agents and non - steroidal anti - inflammatory drugs ( nsaids ) and these are responsible for 60% of adrs leading to hospital admission and 70% of adrs occurring in hospital .", "type b adrs are usually uncommon , but rarely may sometimes cause serious toxicities . therefore , adrs in elderly are largely contributed by prescribing error e.g. , large doses of drugs without taking into account , the effect of age and frailty on drug disposition , especially renal and hepatic clearance", ". the other contributing factor may be because of not considering the increased pharmacodynamic sensitivity of the elderly to several commonly used drugs , e.g. , central nervous system and cardiovascular drugs . since only around 3000 subjects receive a medicine prior to marketing", ", it is not surprising that less frequent ( particularly type b ) adrs are often recognized only after marketing .", "the capacity of premarketing studies to recognize adrs is further reduced by the limited numbers of patients in the age group of 65 or older in trials and that even smaller numbers of the oldest old .", "in addition , long latency diseases like cancer are difficult to detect on account of the short duration of study .", "the latter has been defined in the literature in relative terms ( for example , the administration of an excessive number of drugs ) and in absolute terms , ranging from two to more than six simultaneous medications .", "in contrast to general population , the incidence of combination therapy is found to be greatest in the elderly .", "a drug combination may sometimes cause synergistic toxicity , which is greater than the sum of the risks of toxicity of either agent used alone .", "for example , the combination of corticosteroids and nsaids : the risks of development of nsaid - induced peptic ulcer in older patients may increases by 10% among elderly .", "however , concurrent use of corticosteroids and nsaids had shown a risk of peptic ulcer disease that was 15 times greater than that of non - users of either drug .", "similarly , the relative risk of hospitalization for hemorrhagic peptic ulcer disease in elderly patients ( above 65 years ) has been observed to be increased by manifolds on concurrent use of oral anticoagulants and nsaids , while the risks were lower when used alone .", "this suggests that there is certainly a need to recognize and consider the aspects of  synergism of toxicity  while prescribing medications in elderly .", "however , polypharmacy at times can effectively control certain disease condition e.g. , hypertension and epilepsy .", "respecting the individual needs of every individual patient is the preferred approach of prescribing in elderly .", "some of the steps that may be helpful in minimizing adrs in elderly may be : \n maintaining accurate record of all medications in use : may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines.number of medications ( prescribed and non - prescribed ) : monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs.individual doses : reducing the doses wherever appropriate and titrating them carefully from a low starting dose , if pharmacodynamic sensitivity is likely to be the problem.simple regimens of medication : choose the preparation suitable for the patient and minimize the dose whenever needed , but avoid advising the patient to break a single tablet into two or three equal pieces.ensuring safe management of medications by patients : involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur .", "minimize hoarding of previously used and expired drugs.therapeutic drug monitoring is encouraged , but should not replace clinical observation .", "considering an adr as a possible cause for any new problem.utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines . \n  maintaining accurate record of all medications in use", ": may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines .", "number of medications ( prescribed and non - prescribed ) : monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs .", "individual doses : reducing the doses wherever appropriate and titrating them carefully from a low starting dose , if pharmacodynamic sensitivity is likely to be the problem .", "simple regimens of medication : choose the preparation suitable for the patient and minimize the dose whenever needed , but avoid advising the patient to break a single tablet into two or three equal pieces . ensuring safe management of medications by patients : involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur .", "considering an adr as a possible cause for any new problem . utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines .", "drugs are double - edged weapons and increased use of drugs by elderly increases the risk of adverse drug reactions causing increased morbidity and mortality . in addition , economic consequences of adrs constitute a problem of considerable magnitude .", "developing countries have rapidly aging population and the governments are seeking guidance in promoting healthy and active aging", ". however , strategies to increase opportunities for identifying adrs and related problems have not been emphasized in current policy responses in india to meet the increase in elderly population and chronic conditions .", "in addition , there is definite paucity of good quality research and data on adrs in india .", "further , growing pharmacotherapy with increase in the number of diseases in the elderly will require more timely and accurate drug safety data .", "pharmacoepidemiological studies that encompass large numbers of elderly drug users are needed to obtain this information as increased knowledge of the frequency and cost of adverse drug reactions is important in enabling more rational therapeutic decisions by individual clinicians and more optimal social policy . given quantitative information on medication risks , clinicians will be able to change the use of the drug ( prescribe for fewer patients , use safer alternatives when available , and use in lower doses for shorter duration ) or can take measures to minimize side effects ( prescribe prophylactic medications , increase monitoring for side effects , and intensify patient education ) .", "policy changes that can result from better data on drug toxicities include withdrawal of drug from the market , change in drug labeling , educational programs to physician and changes in academic curriculum of pharmacology .", "this will result in promoting and ensuring a good health of older people , which can be of great benefit to their families and communities .", "in the long run , more precise estimates of the true costs associated with adrs could stimulate development of prophylaxis and of alternative therapies ."], "abstract_text": ["<S> medications probably are the single most important health care technology in preventing illness , disability , and death in the geriatric population . </S>", "<S> age - related changes in drug disposition and pharmacodynamic responses have significant clinical implications ; increased use of a number of medications raises the risk that medicine - related problems may occur . </S>", "<S> the relationship between increased use of drugs including the prescription medication and elderly is well established . </S>", "<S> majority of adrs ( 80% ) causing admission or occurring in hospital are type a reactions . </S>", "<S> although less common occurring in elderly , type b adrs may sometimes cause serious toxicity . </S>", "<S> studies have correlated the integral association between old age and increased rate of adverse drug reactions arising out of confounding association between age and polypharmacy contributed by age - related changes in pharmacodynamics and pharmacokinetics at least for some medical conditions . </S>", "<S> a drug combination may sometimes cause synergistic toxicity which is greater than the sum of the risks of toxicity of either agent used alone . </S>", "<S> but , strategies to increase opportunities for identifying adrs and related problems have not been emphasised in current international policy responses especially in india to the increase in elderly population and chronic conditions . </S>", "<S> careful epidemiological studies that encompass large numbers of elderly drug users are required to obtain this information as increased knowledge of the frequency and cost of adverse drug reactions is important in enabling both more rational therapeutic decisions by individual clinicians and more optimal social policy . </S>"], "labels": null, "section_names": ["BACKGROUND", "EXTENT OF ADVERSE DRUG REACTION PROBLEM IN ELDERLY", "NATURE OF THE PROBLEM", "POLYPHARMACY AND ELDERLY", "STEPS OF MINIMIZING ADR IN ELDERLY", "CONCLUSION"], "sections": [["population aging is the process by which older individuals become a proportionally larger share of the total population .", "it was the most distinctive global demographic event that the world witnessed in the past century and is an important event for the twenty - first century too .", "it was initially experienced by the more developed countries and has recently become apparent in the developing countries .", "population aging will be faced globally by all countries in this century , although at varying levels of intensity and time . according to the world health organization ( who )", ", world 's elderly population i.e. , people 60 years of age and older is approximately 650 million at present and by 2050 , it is forecast to reach 2 billion . in 2008 , five out of the top ten causes of mortality worldwide , other than injuries , were non - communicable diseases ( ncds ) and expected to go up to seven out of ten by the year 2030 . by then about 76% of the deaths in the world will be due to ncds .", "government and the who have already recognized the huge burden of preventable disease , disability , death and distress caused by the ncds . the present international health agenda guided by who focuses on four conditions ( cardiovascular disease , diabetes , cancer and chronic respiratory disease ) responsible for most of the premature mortality and four  lifestyle risk factors ( smoking , harmful alcohol use , lack of physical activity and high salt , high fat diets ) leading to above conditions .", "medications play crucial role in geriatric health care as they treat chronic diseases , alleviate pain and improve quality of life .", "age - related changes in drug disposition and pharmacodynamic responses have significant clinical implications , and increased use of a number of medications in elderly raises the risk of medicine - related problems that may occur . as medication use and the incidence of adverse drug outcomes increase with advancing age , it is important to ensure quality use of medicines in older people towards attaining a higher goal of healthy and active aging .", "but , use of drugs by the elderly and their clinical outcomes especially adverse drug reactions ( adrs ) have not been prominent research topic and continues to be given low priority in national and international public health arena , at least in a developing country like india . however , we feel that this probably needs more attention in india because we have not completely dealt with the scourge of communicable diseases yet .", "in addition , 70% of all older people now live in low- or middle - income countries , including india where sustainable pharmacovigilance systems has not developed as yet ."], ["the relationship between increased use of drugs and elderly is well established . consequently , increased use of medications in elderly increases the risk of adrs .", "studies from around the world have shown a definite correlation between increasing age and adr rate , at least for some medical conditions .", "although very few such studies are available in indian settings , harugeri et al . in a hospital setting found that the prevalence of adr - related hospital admissions was 5.9% , while in another such study in india , it was observed to be 6.7% . for india ,", "harugeri et al . predicted that 18000 bed days in a given time would be due to adrs in elderly and total cost of hospital stay due to adrs is estimated to be us $ 4350 ( inr 200100 ) , that is us $ 80.5 per patient ( 108.7% of per capita per year expenditure on health ) .", "there has been much debate on whether advancing age by itself is a cause of increased risk of adrs but merely a marker for comorbidity , altered pharmacokinetics , and polypharmacy .", "gurwitz and avorn concluded that  patient - specific physiological and functional characteristics are probably more important than any chronological measure in predicting both adverse and beneficial outcomes associated with specific drug therapies  , while studies around the world have clearly shown that the risks of adrs ( including interactions ) is related to the number of medicines taken and sometimes due to inappropriate use of medicines .", "( 1991 ) observed an exponential rather than the linear relation between the risks of adr and the number of medicines taken in 9000 elderly italian patients receiving 10 medicines .", "review of several studies by steward rb et al . found that the patients aged above 65 years use an average of two to six prescribed medications , and 1 - 3.4 non - prescribed medications .", "out of all the factors that are most consistently associated with adverse drug reactions , polypharmacy is considered to be the most important .", "thus , studies have correlated the integral association between old age and increased rate of adrs arising out of confounding association between age and polypharmacy contributed by age - related changes in pharmacodynamics and pharmacokinetics at least for some medical conditions ."], ["the classical pharmacological classification of adrs by rawlins and thompson divides adr into two major subtypes : type a reactions , which are dose - dependent and predictable , and unpredictable type b (  bizarre or idiosyncratic ) reactions .", "majority of adrs ( 80% ) causing admission or occurring in hospital setting are type a reactions .", "they are predictable and potentially avoidable in nature as they are related to accentuation of known pharmacological effects of the drug .", "drugs associated with type a reactions are generally with low therapeutic index and commonly used among elderly .", "the lists of medicines most likely to be used in the elderly include antibiotics , anticoagulants , digoxin , diuretics , hypoglycemic agents , antineoplastic agents and non - steroidal anti - inflammatory drugs ( nsaids ) and these are responsible for 60% of adrs leading to hospital admission and 70% of adrs occurring in hospital .", "type b adrs are usually uncommon , but rarely may sometimes cause serious toxicities . therefore , adrs in elderly are largely contributed by prescribing error e.g. , large doses of drugs without taking into account , the effect of age and frailty on drug disposition , especially renal and hepatic clearance", ". the other contributing factor may be because of not considering the increased pharmacodynamic sensitivity of the elderly to several commonly used drugs , e.g. , central nervous system and cardiovascular drugs . since only around 3000 subjects receive a medicine prior to marketing", ", it is not surprising that less frequent ( particularly type b ) adrs are often recognized only after marketing .", "the capacity of premarketing studies to recognize adrs is further reduced by the limited numbers of patients in the age group of 65 or older in trials and that even smaller numbers of the oldest old .", "in addition , long latency diseases like cancer are difficult to detect on account of the short duration of study ."], ["the latter has been defined in the literature in relative terms ( for example , the administration of an excessive number of drugs ) and in absolute terms , ranging from two to more than six simultaneous medications .", "in contrast to general population , the incidence of combination therapy is found to be greatest in the elderly .", "a drug combination may sometimes cause synergistic toxicity , which is greater than the sum of the risks of toxicity of either agent used alone .", "for example , the combination of corticosteroids and nsaids : the risks of development of nsaid - induced peptic ulcer in older patients may increases by 10% among elderly .", "however , concurrent use of corticosteroids and nsaids had shown a risk of peptic ulcer disease that was 15 times greater than that of non - users of either drug .", "similarly , the relative risk of hospitalization for hemorrhagic peptic ulcer disease in elderly patients ( above 65 years ) has been observed to be increased by manifolds on concurrent use of oral anticoagulants and nsaids , while the risks were lower when used alone .", "this suggests that there is certainly a need to recognize and consider the aspects of  synergism of toxicity  while prescribing medications in elderly .", "however , polypharmacy at times can effectively control certain disease condition e.g. , hypertension and epilepsy ."], ["respecting the individual needs of every individual patient is the preferred approach of prescribing in elderly .", "some of the steps that may be helpful in minimizing adrs in elderly may be : \n maintaining accurate record of all medications in use : may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines.number of medications ( prescribed and non - prescribed ) : monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs.individual doses : reducing the doses wherever appropriate and titrating them carefully from a low starting dose , if pharmacodynamic sensitivity is likely to be the problem.simple regimens of medication : choose the preparation suitable for the patient and minimize the dose whenever needed , but avoid advising the patient to break a single tablet into two or three equal pieces.ensuring safe management of medications by patients : involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur .", "minimize hoarding of previously used and expired drugs.therapeutic drug monitoring is encouraged , but should not replace clinical observation .", "considering an adr as a possible cause for any new problem.utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines . \n  maintaining accurate record of all medications in use", ": may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines .", "number of medications ( prescribed and non - prescribed ) : monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs .", "individual doses : reducing the doses wherever appropriate and titrating them carefully from a low starting dose , if pharmacodynamic sensitivity is likely to be the problem .", "simple regimens of medication : choose the preparation suitable for the patient and minimize the dose whenever needed , but avoid advising the patient to break a single tablet into two or three equal pieces . ensuring safe management of medications by patients : involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur .", "considering an adr as a possible cause for any new problem . utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines ."], ["drugs are double - edged weapons and increased use of drugs by elderly increases the risk of adverse drug reactions causing increased morbidity and mortality . in addition , economic consequences of adrs constitute a problem of considerable magnitude .", "developing countries have rapidly aging population and the governments are seeking guidance in promoting healthy and active aging", ". however , strategies to increase opportunities for identifying adrs and related problems have not been emphasized in current policy responses in india to meet the increase in elderly population and chronic conditions .", "in addition , there is definite paucity of good quality research and data on adrs in india .", "further , growing pharmacotherapy with increase in the number of diseases in the elderly will require more timely and accurate drug safety data .", "pharmacoepidemiological studies that encompass large numbers of elderly drug users are needed to obtain this information as increased knowledge of the frequency and cost of adverse drug reactions is important in enabling more rational therapeutic decisions by individual clinicians and more optimal social policy . given quantitative information on medication risks , clinicians will be able to change the use of the drug ( prescribe for fewer patients , use safer alternatives when available , and use in lower doses for shorter duration ) or can take measures to minimize side effects ( prescribe prophylactic medications , increase monitoring for side effects , and intensify patient education ) .", "policy changes that can result from better data on drug toxicities include withdrawal of drug from the market , change in drug labeling , educational programs to physician and changes in academic curriculum of pharmacology .", "this will result in promoting and ensuring a good health of older people , which can be of great benefit to their families and communities .", "in the long run , more precise estimates of the true costs associated with adrs could stimulate development of prophylaxis and of alternative therapies ."]]}
{"article_id": "PMC5125187", "article_text": ["day care or ambulatory anaesthesia is as old as the history of anaesthesia itself . in the last few decades , it has gained significant popularity mainly because of the enormous cost savings and has therefore been accepted by the hospitals , patients and the insurance companies alike .", "however , the quality and excellence of care needs to be defined and defining good care is a challenge .", "unanticipated admission , whatever the cause , is an indicator of the quality of care because it negates the basic goal of preventing hospitalisation and same day discharge .", "while day care surgery in india is limited to a few hospitals , there is a dearth of data on hospitalisation following such intervention .", "this study aimed to examine the incidence and factors associated with unanticipated admission following day care surgery at a tertiary level hospital in south india .", "patients belonging to american society of anesthesiologists ( asa ) physical status classes 1 and 2 , undergoing surgeries under general or regional anaesthetic techniques along with appropriate nerve blocks for post - operative pain relief were included .", "it was planned to use the median value of controls for continuous exposure variables ( such as time of anaesthesia and time of starting last case ) to define exposure status .", "we required 70 cases and 70 controls to detect an odds ratio ( or ) of 2.5 with a power of 80% and  error of 0.05% . to permit adjustment for confounders , two controls per case", "were chosen , which required the number of subjects to be 53 cases and 106 controls .", "demographic characteristics , patients weight , pre - operative medical illness , personal habits , asa status , the diagnosis and surgical procedures , time since last meal , duration of anaesthesia and surgery , experience of the surgeon and anaesthesiologist , and intraoperative management ( techniques , drugs , monitoring , etc . ) were documented .", "the number of hours fasted was divided into 68 h , 911 h and > 11 h. the outcomes measured were the admission of patients following day care surgery , and the causes of unanticipated admissions .", "discharge of the patient was decided after both the surgeon and the anaesthesiologist cleared the patient .", "the admission of the patient to the hospital was also decided by the surgeon or the anaesthetist depending on the complication the patient had .", "those who need to stay overnight from the day care centre were considered as unanticipated admissions ( the centre functions from 7 am to 9 pm with the morning session from 7:30 am to 11:30 am and the afternoon session from 12:30 pm to 4:30 pm .", "the reasons for unanticipated admission were classified into four main groups : ( i ) anaesthetic reasons included nausea vomiting , giddiness , prolonged general anaesthesia and others , ( ii ) surgical reasons included pain , bleeding , complicated surgery requiring wound dressing , wound drainage or antibiotics , ( iii ) medical reasons such as fever , drug allergies and other complications , ( iv ) social reasons included patients or surgeons request . a nested case ", "descriptive statistics in the form of frequencies , median and means , standard deviations and percentages were calculated . the chi - square test and or were calculated to determine which factors were associated with unanticipated admission . the or , their 95% confidence intervals ( cis ) and corresponding p values were calculated for all variables .", "for the statistically significant variables , the mantel haenszel or was calculated to identify confounding , if any . for analysing the different narcotics used , each narcotic was given a score using morphine as a reference ; 10 mg of morphine = 100 g of fentanyl = 75 mg of pethidine = 100 mg of tramadol .", "hence , a narcotic score of one equals 1 mg of morphine = 10 g of fentanyl = 7.5 mg of pethidine = 10 mg of tramadol .", "for example , if a patient received 100 g of fentanyl and 6 mg of morphine during the surgery , the patient would have a narcotic score of 16 .", "the equivalent narcotic dose was totalled and the narcotic score for each patient was then calculated .", "a narcotic score of 10 was used as the cut - off and the tests of significance done .", "data were analysed using the statistical package for social sciences ( spss ) version 13.0 ( spss inc .", "during the 3-month study , from the cohort of 776 patients , 63 patients with unanticipated admission formed the case group and were compared to twice the number of unhospitalised patients , which formed the control group . at the end of the day , after all the patients had been discharged or admitted from the day care centre , lots were drawn from the discharged group , which randomly selected controls .", "of these , anaesthetic complications accounted for 33.33% , surgical complications were 15.87% , medical were 6.35% and social reasons were 44.44% .", "social reasons included admission on request of the surgeon and admission on request of the patient or parents of a child .", "the reasons for anaesthetic admission were post - operative nausea and vomiting ( ponv ) , sweating and giddiness in the recovery room , prolonged general anaesthetic ( > 2 h ) , probable aspiration , urinary retention , a failed anaesthetic , which required switching to another technique and post - operative pain .", "the surgical reasons were bleeding ; complicated surgery needing wound drainage , dressings or antibiotics .", "medical causes were persistent high blood pressure needing treatment , fever , seizures and drug reactions .", "reasons for unanticipated admissions the orthopaedic surgery speciality had the maximum admissions ( 34.9% ) , followed by abdominal surgeries , 27% .", "ear nose and throat ( ent ) had 11.1% admissions ; vascular surgeries accounted for 3.17% whereas urology had none .", "the type of surgery and technique of anaesthesia among unanticipated admissions are shown in tables 2 and 3 .", "type of surgery requiring readmission technique of anaesthesia in those requiring readmission individual risk factors for admissions and or 's were calculated using univariate logistic regression [ table 4 ] .", "the possibility of confounding was investigated using stratified analysis and calculation of the mantel haenszel or .", "univariate analysis showing factors associated with unanticipated admissions the factors significantly associated with unanticipated admissions were the duration of anaesthesia , duration of surgery , experience of the surgeon , time of starting last case , hours of fasting and a high narcotics score .", "anaesthesia lasting more than 50 min and surgery lasting more than 35 min were at a higher risk of admission .", "patients whose surgery started after 3 pm were also at a significantly high risk of admission .", "the study found that patients operated on after 3 pm are 8 times at risk of being admitted than if their surgery started before 3 pm .", "it was found that those fasting shorter hours ( 68 h ) had 2.2 times greater risk of admission .", "intraoperative use of narcotics was analysed in both groups , and this was not statistically significant .", "( p = 0.06 with an or of 0.84 and a 95% ci of 0.411.71 ) the dose of narcotic used during anaesthesia was analysed using the narcotic score and patients with a score of > 10 were 2.86 times at a greater risk of admission with a 95% ci of 1.177.09 .", "it was also found that patients operated on by consultant surgeons were 3.2 times more likely to be admitted but this was not analysed further as consultant surgeons operated upon almost 90% of patients .", "the above statistically significant variables were entered into a bivariate logistic regression model [ table 5 ] .", "anaesthesia time 50 min and surgery time 35 min were highly correlated and hence only anaesthesia time 50 min was entered into the model .", "the anaesthesia time 50 min ( p = 0.002 ; or : 3.18 ; 95% ci : 1.506.72 ) and starting of last case after 3 pm ( p = 0.002 ; or : 10.10 ; 95% ci : 2.4242.15 ) remained statistically significant for unanticipated admissions .", "the day care centre is an architecturally and functionally integrated independent unit with areas for admission , surgery , recovery and discharge .", "it consists of an admission area with a lounge for relatives , two operating theatres , and a four - bedded recovery room with adequate monitoring facilities , oxygen and suction apparatus and in addition a six bedded day surgery ward .", "the surgical disciplines using the day care centre include ent , orthopaedics , general surgery , vascular surgery and urology .", "unanticipated admissions following day care surgery are a quality marker of efficiency of ambulatory surgery services .", "the incidence of unanticipated admission in this study was 4.51% that compares favourably with other published reports , which document rates between 1.04% and 5.4% .", "patients were admitted for an average of 4.5 days however 70% of patients were admitted only for 1 day .", "the factors associated with unanticipated admissions identified by the study were long duration of anaesthesia and surgery , starting of last case after 3 pm , shorter hours of fasting and greater dose of narcotics used .", ". anaesthesia lasting longer than 50 min and surgery lasting longer than 35 min were significantly associated with an increased risk of admissions .", "the study has found that longer anaesthesia is associated with a 3.85 times greater and longer surgery has four times increased risk of admissions .", "this study documented that surgeries started after 3 pm had a higher risk of unanticipated admissions and is consistent with those reported in literature .", "fortier et al . have reported that completion of surgery after 3 pm was a significant factor", ". this may be because patients having surgery later in the day had less time to recover and be fit for discharge as compared to patients whose surgery was started earlier in the day .", "the use of intraoperative narcotics was statistically significant on the univariate analysis but after adjusting for confounders using bivariate analysis , it was not significant .", "the study showed that the incidence of ponv was one of the major anaesthetic risk factors for admission , which accounted for 51.14% of anaesthetic admissions .", "therefore , a comparison between the narcotic score and ponv was made , and a significant association was found .", "patients with a narcotic score of > 10 were nine times more likely to have ponv requiring admission .", "the ability to provide adequate pain relief without exacerbating ponv remains one of the major challenges for providers of outpatient anaesthesia and surgery .", "balanced analgesia techniques involving the use of opioids and other groups of analgesic drugs such as local anaesthetics and non - steroidal anti - inflammatory drugs are now being commonly used .", "post - operative pain is the most commonly reported complication of day care anaesthesia with up to 50% of patients experiencing wound pain . however", ", this study did not show that instead it demonstrated that the use of opiates can lead to ponv requiring admission .", "it was found that shorter fasting time of < 8 h induced a greater risk of unanticipated admission .", "the univariate analysis showed that shorter fasting time was at 2.2 times the risk of admission .", "the bivariate analysis showed that shorter fasting times had 1.9 times the risk of admission though not statistically significant .", "this group of patients may be the patients who are on the afternoon list starting at 12:30 pm , are allowed a light breakfast at 6 am and therefore have a higher risk of ponv .", "the duration of fasting and time of starting last case were stratified , and the mantel haenszel or calculated .", "since both the crude or and the mantel haenszel odd ratio were almost equal it was concluded that there was no confounding factor involved .", "the uncorrected incidence of unanticipated admission in our study was 8.11% , and this is because our day care facility was relatively new and the threshold level for admission was very low .", "surgeons request accounted for 36.5% of all cases when no real discernable reason could be made out .", "it may be that the surgeon suspects that he might have done a little more extensive procedure on the patient than anticipated , but does not fall under the category of complications . a small number ( 7.9% ) also included request for admission from the patient or parents of a child .", "the idea that patients can go home on the same day as the surgery is a novel one and one which patients find difficult to reconcile to and therefore request for admission .", "therefore , if both these groups are removed , the overall incidence works out to the value of 4.51% .", "our setup is part of a large tertiary hospital and in case of any apprehension , admission from the day care centre to the ward is relatively easy and more readily facilitated .", "the variables that were not significant were weight of the patient , distance travelled , pre - existing medical disease , and asa status , type of anaesthetic and experience of the anaesthetist .", "type of surgery and gender also had no detectable effect on admission , as evidenced in other studies .", "although it was expected that pre - existing medical disease , type of anaesthetic and type of surgery would be significant , it was not so in our study . a large sample size and doing asa 3 and 4 patients in the day care facility", "the factors associated with unanticipated admissions included longer duration of anaesthesia and surgery , later time of starting last case , shorter hours of fasting and the greater dose of narcotics used .", "these factors can guide decision making to facilitate improved efficiency of the day care centre .", "the low rates of medical causes of admissions reflect a good pre - anaesthetic assessment in terms of the suitability of patients ."], "abstract_text": ["<S> background and aims : day care surgery offers respite from hospitalisation for specific surgical procedures and has many advantages . however , occasionally patients who undergo such surgery require hospitalisation for unanticipated complications . </S>", "<S> we aimed to determine their incidence and to identify factors associated with unanticipated admissions in a tertiary care hospital in south india.methods:during the 3-month study , 63 cases requiring admission and 126 randomly selected controls were taken from the 776 procedures that were performed were compared . </S>", "<S> the variables studied were patients demographic characteristics , pre - operative medical illness , personal habits , american society of anesthesiologists status , the diagnosis and surgical procedures , time since last meal , duration of anaesthesia and surgery , experience of the surgeon and anaesthetist , and intraoperative management ( techniques , drugs , monitoring , etc . ) . </S>", "<S> univariate and bivariate statistics were used to determine factors associated with unanticipated admissions.results:the incidence of unanticipated admissions following day care surgery was 8.11% . </S>", "<S> the reasons for admission were anaesthetic ( 33.33% ) , surgical ( 15.87% ) , medical ( 6.34% ) and social ( 44.44% ) . </S>", "<S> the factors significantly associated with unanticipated admissions included duration of anaesthesia more than 50 min ( odds ratio [ or ] : 3.179 ; 95% confidence interval [ ci ] : 1.5036.722 ) , and starting the last case after 3 pm ( or : 10.095 ; 95% ci : 2.41842.148).conclusion : unanticipated admissions following day care surgery occur mainly due to anaesthetic , surgical , medical and social reasons . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["day care or ambulatory anaesthesia is as old as the history of anaesthesia itself . in the last few decades , it has gained significant popularity mainly because of the enormous cost savings and has therefore been accepted by the hospitals , patients and the insurance companies alike .", "however , the quality and excellence of care needs to be defined and defining good care is a challenge .", "unanticipated admission , whatever the cause , is an indicator of the quality of care because it negates the basic goal of preventing hospitalisation and same day discharge .", "while day care surgery in india is limited to a few hospitals , there is a dearth of data on hospitalisation following such intervention .", "this study aimed to examine the incidence and factors associated with unanticipated admission following day care surgery at a tertiary level hospital in south india ."], ["patients belonging to american society of anesthesiologists ( asa ) physical status classes 1 and 2 , undergoing surgeries under general or regional anaesthetic techniques along with appropriate nerve blocks for post - operative pain relief were included .", "it was planned to use the median value of controls for continuous exposure variables ( such as time of anaesthesia and time of starting last case ) to define exposure status .", "we required 70 cases and 70 controls to detect an odds ratio ( or ) of 2.5 with a power of 80% and  error of 0.05% . to permit adjustment for confounders , two controls per case", "were chosen , which required the number of subjects to be 53 cases and 106 controls .", "demographic characteristics , patients weight , pre - operative medical illness , personal habits , asa status , the diagnosis and surgical procedures , time since last meal , duration of anaesthesia and surgery , experience of the surgeon and anaesthesiologist , and intraoperative management ( techniques , drugs , monitoring , etc . ) were documented .", "the number of hours fasted was divided into 68 h , 911 h and > 11 h. the outcomes measured were the admission of patients following day care surgery , and the causes of unanticipated admissions .", "discharge of the patient was decided after both the surgeon and the anaesthesiologist cleared the patient .", "the admission of the patient to the hospital was also decided by the surgeon or the anaesthetist depending on the complication the patient had .", "those who need to stay overnight from the day care centre were considered as unanticipated admissions ( the centre functions from 7 am to 9 pm with the morning session from 7:30 am to 11:30 am and the afternoon session from 12:30 pm to 4:30 pm .", "the reasons for unanticipated admission were classified into four main groups : ( i ) anaesthetic reasons included nausea vomiting , giddiness , prolonged general anaesthesia and others , ( ii ) surgical reasons included pain , bleeding , complicated surgery requiring wound dressing , wound drainage or antibiotics , ( iii ) medical reasons such as fever , drug allergies and other complications , ( iv ) social reasons included patients or surgeons request . a nested case ", "descriptive statistics in the form of frequencies , median and means , standard deviations and percentages were calculated . the chi - square test and or were calculated to determine which factors were associated with unanticipated admission . the or , their 95% confidence intervals ( cis ) and corresponding p values were calculated for all variables .", "for the statistically significant variables , the mantel haenszel or was calculated to identify confounding , if any . for analysing the different narcotics used , each narcotic was given a score using morphine as a reference ; 10 mg of morphine = 100 g of fentanyl = 75 mg of pethidine = 100 mg of tramadol .", "hence , a narcotic score of one equals 1 mg of morphine = 10 g of fentanyl = 7.5 mg of pethidine = 10 mg of tramadol .", "for example , if a patient received 100 g of fentanyl and 6 mg of morphine during the surgery , the patient would have a narcotic score of 16 .", "the equivalent narcotic dose was totalled and the narcotic score for each patient was then calculated .", "a narcotic score of 10 was used as the cut - off and the tests of significance done .", "data were analysed using the statistical package for social sciences ( spss ) version 13.0 ( spss inc ."], ["during the 3-month study , from the cohort of 776 patients , 63 patients with unanticipated admission formed the case group and were compared to twice the number of unhospitalised patients , which formed the control group . at the end of the day , after all the patients had been discharged or admitted from the day care centre , lots were drawn from the discharged group , which randomly selected controls .", "of these , anaesthetic complications accounted for 33.33% , surgical complications were 15.87% , medical were 6.35% and social reasons were 44.44% .", "social reasons included admission on request of the surgeon and admission on request of the patient or parents of a child .", "the reasons for anaesthetic admission were post - operative nausea and vomiting ( ponv ) , sweating and giddiness in the recovery room , prolonged general anaesthetic ( > 2 h ) , probable aspiration , urinary retention , a failed anaesthetic , which required switching to another technique and post - operative pain .", "the surgical reasons were bleeding ; complicated surgery needing wound drainage , dressings or antibiotics .", "medical causes were persistent high blood pressure needing treatment , fever , seizures and drug reactions .", "reasons for unanticipated admissions the orthopaedic surgery speciality had the maximum admissions ( 34.9% ) , followed by abdominal surgeries , 27% .", "ear nose and throat ( ent ) had 11.1% admissions ; vascular surgeries accounted for 3.17% whereas urology had none .", "the type of surgery and technique of anaesthesia among unanticipated admissions are shown in tables 2 and 3 .", "type of surgery requiring readmission technique of anaesthesia in those requiring readmission individual risk factors for admissions and or 's were calculated using univariate logistic regression [ table 4 ] .", "the possibility of confounding was investigated using stratified analysis and calculation of the mantel haenszel or .", "univariate analysis showing factors associated with unanticipated admissions the factors significantly associated with unanticipated admissions were the duration of anaesthesia , duration of surgery , experience of the surgeon , time of starting last case , hours of fasting and a high narcotics score .", "anaesthesia lasting more than 50 min and surgery lasting more than 35 min were at a higher risk of admission .", "patients whose surgery started after 3 pm were also at a significantly high risk of admission .", "the study found that patients operated on after 3 pm are 8 times at risk of being admitted than if their surgery started before 3 pm .", "it was found that those fasting shorter hours ( 68 h ) had 2.2 times greater risk of admission .", "intraoperative use of narcotics was analysed in both groups , and this was not statistically significant .", "( p = 0.06 with an or of 0.84 and a 95% ci of 0.411.71 ) the dose of narcotic used during anaesthesia was analysed using the narcotic score and patients with a score of > 10 were 2.86 times at a greater risk of admission with a 95% ci of 1.177.09 .", "it was also found that patients operated on by consultant surgeons were 3.2 times more likely to be admitted but this was not analysed further as consultant surgeons operated upon almost 90% of patients .", "the above statistically significant variables were entered into a bivariate logistic regression model [ table 5 ] .", "anaesthesia time 50 min and surgery time 35 min were highly correlated and hence only anaesthesia time 50 min was entered into the model .", "the anaesthesia time 50 min ( p = 0.002 ; or : 3.18 ; 95% ci : 1.506.72 ) and starting of last case after 3 pm ( p = 0.002 ; or : 10.10 ; 95% ci : 2.4242.15 ) remained statistically significant for unanticipated admissions ."], ["the day care centre is an architecturally and functionally integrated independent unit with areas for admission , surgery , recovery and discharge .", "it consists of an admission area with a lounge for relatives , two operating theatres , and a four - bedded recovery room with adequate monitoring facilities , oxygen and suction apparatus and in addition a six bedded day surgery ward .", "the surgical disciplines using the day care centre include ent , orthopaedics , general surgery , vascular surgery and urology .", "unanticipated admissions following day care surgery are a quality marker of efficiency of ambulatory surgery services .", "the incidence of unanticipated admission in this study was 4.51% that compares favourably with other published reports , which document rates between 1.04% and 5.4% .", "patients were admitted for an average of 4.5 days however 70% of patients were admitted only for 1 day .", "the factors associated with unanticipated admissions identified by the study were long duration of anaesthesia and surgery , starting of last case after 3 pm , shorter hours of fasting and greater dose of narcotics used .", ". anaesthesia lasting longer than 50 min and surgery lasting longer than 35 min were significantly associated with an increased risk of admissions .", "the study has found that longer anaesthesia is associated with a 3.85 times greater and longer surgery has four times increased risk of admissions .", "this study documented that surgeries started after 3 pm had a higher risk of unanticipated admissions and is consistent with those reported in literature .", "fortier et al . have reported that completion of surgery after 3 pm was a significant factor", ". this may be because patients having surgery later in the day had less time to recover and be fit for discharge as compared to patients whose surgery was started earlier in the day .", "the use of intraoperative narcotics was statistically significant on the univariate analysis but after adjusting for confounders using bivariate analysis , it was not significant .", "the study showed that the incidence of ponv was one of the major anaesthetic risk factors for admission , which accounted for 51.14% of anaesthetic admissions .", "therefore , a comparison between the narcotic score and ponv was made , and a significant association was found .", "patients with a narcotic score of > 10 were nine times more likely to have ponv requiring admission .", "the ability to provide adequate pain relief without exacerbating ponv remains one of the major challenges for providers of outpatient anaesthesia and surgery .", "balanced analgesia techniques involving the use of opioids and other groups of analgesic drugs such as local anaesthetics and non - steroidal anti - inflammatory drugs are now being commonly used .", "post - operative pain is the most commonly reported complication of day care anaesthesia with up to 50% of patients experiencing wound pain . however", ", this study did not show that instead it demonstrated that the use of opiates can lead to ponv requiring admission .", "it was found that shorter fasting time of < 8 h induced a greater risk of unanticipated admission .", "the univariate analysis showed that shorter fasting time was at 2.2 times the risk of admission .", "the bivariate analysis showed that shorter fasting times had 1.9 times the risk of admission though not statistically significant .", "this group of patients may be the patients who are on the afternoon list starting at 12:30 pm , are allowed a light breakfast at 6 am and therefore have a higher risk of ponv .", "the duration of fasting and time of starting last case were stratified , and the mantel haenszel or calculated .", "since both the crude or and the mantel haenszel odd ratio were almost equal it was concluded that there was no confounding factor involved .", "the uncorrected incidence of unanticipated admission in our study was 8.11% , and this is because our day care facility was relatively new and the threshold level for admission was very low .", "surgeons request accounted for 36.5% of all cases when no real discernable reason could be made out .", "it may be that the surgeon suspects that he might have done a little more extensive procedure on the patient than anticipated , but does not fall under the category of complications . a small number ( 7.9% ) also included request for admission from the patient or parents of a child .", "the idea that patients can go home on the same day as the surgery is a novel one and one which patients find difficult to reconcile to and therefore request for admission .", "therefore , if both these groups are removed , the overall incidence works out to the value of 4.51% .", "our setup is part of a large tertiary hospital and in case of any apprehension , admission from the day care centre to the ward is relatively easy and more readily facilitated .", "the variables that were not significant were weight of the patient , distance travelled , pre - existing medical disease , and asa status , type of anaesthetic and experience of the anaesthetist .", "type of surgery and gender also had no detectable effect on admission , as evidenced in other studies .", "although it was expected that pre - existing medical disease , type of anaesthetic and type of surgery would be significant , it was not so in our study . a large sample size and doing asa 3 and 4 patients in the day care facility"], ["the factors associated with unanticipated admissions included longer duration of anaesthesia and surgery , later time of starting last case , shorter hours of fasting and the greater dose of narcotics used .", "these factors can guide decision making to facilitate improved efficiency of the day care centre .", "the low rates of medical causes of admissions reflect a good pre - anaesthetic assessment in terms of the suitability of patients ."], [""], [""]]}
{"article_id": "PMC270606", "article_text": ["the relationship between the clotting cascade and the promotion or inhibition of the anti - inflammatory response continues to be defined through basic research .", "the potential key role of proteins that were originally believed to be exclusively involved in coagulation in the inflammatory response provides an exciting potential mechanism(s ) for modification through the application of new therapies .", "the accompanying review by riewald and ruf   outlines the available information on the possible steps involving some of the various proteins that are common to both coagulation and inflammation .", "the complexity of the multiple actions of the proteins in both inflammation and coagulation should excite the clinician with regard to potential new therapies that may be beneficial via activation of one or both of the concurrent pathways .", "however , before clinicians can administer new therapies that utilize new knowledge on the actions of coagulation cascade components , and hence improve the outcomes of their patients , several gaps in our current understanding must be addressed .", "this information is imperative if the clinician is to take basic knowledge ' to the bedside ' , where individual patients can benefit .", "administration of intrinsic or synthetic analogues of coagulation cascade components ( e.g. protein c , thrombin , and factor viia or xa ) , coagulation inhibitors ( e.g. heparin or analogues ) , or agonists or antagonists of the protease - activated receptors , in an attempt to alter the individual patient 's coagulation / inflammation balance , could theoretically produce responses ranging from beneficial to detrimental .", "knowledge of the ' most beneficial ' balance between augmenting or inhibiting the contribution of the clotting cascade components to inflammation would be required for determining the selection and dosing of potential new therapies .", "a safe assumption is that any new therapies that alter the intrinsic response to coagulation and inflammatory stimulants would also potentially produce detrimental effects on these , and potentially other , physiological responses .", "a simple example of such a potential for detrimental effects is the administration of protein c resulting in undesirable haemorrhage .", "this example illustrates that a broader knowledge of the impact of altering the coagulation/ inflammatory response will be required before clinicians can comfortably utilize new therapies in patient care .", "it is unlikely that any new therapies resulting from the new knowledge of the contribution of the clotting cascade to inflammation can be applied to all patients .", "it can be anticipated that certain patients would have contraindications to the new therapies or that dosages would need to be adjusted on the basis of pharmacokinetic or pharmacodynamic variables .", "the clinician will need to know what these contraindications and variables are before the new therapies can be used confidently .", "some , such as coagulation disorders , may be known and obvious , but any new strategies that could potentially alter the balance of a complex physiological response may be anticipated to be influenced by many , as yet unknown , factors in the individual patient .", "an example is the concurrent administration of heparin , which appears to antagonize the anti - inflammatory effect of antithrombin iii in septic patients .", "a more complete understanding of the impact of concurrent conditions , therapies and the patient 's genetic make - up on outcome will be required if we are to obtain the maximum benefit from new therapies .", "for any new therapy resulting from our expanded knowledge of the clotting cascade in the inflammatory response to be tailored to the needs of the individual patient , a method(s ) for rapidly and easily assessing the patient 's dynamic inflammatory response will be required .", "unless any new therapies have very benign toxicities , the need to assess the need , dosage and response to treatment will mandate sensitive and specific monitoring techniques .", "bedside or clinical laboratory techniques that could be applicable to most clinical environments would allow the benefit of any new therapies to be applied to the greatest number of patients .", "conversely , any new treatments that require complex , laborious monitoring techniques will have limited applicability .", "as our knowledge of the contribution of the intrinsic coagulation components increases , the development of many potential new therapies will hopefully emerge for clinical use", ". these may be totally new therapeutic entities or new strategies for using existing drugs or clotting factors .", "such developments will create the new dilemma for the clinician , namely that of trying to select between different treatment options for the individual patient .", "clinical trials outlining the potential anticipated benefit(s ) and the appropriate target patient population for each new therapeutic strategy will need to be completed", ". this will allow the clinician to ensure that the most appropriate strategy can be selected for the individual patient .", "the above - mentioned concerns aside , the clinician should be excited about the expanding knowledge of the role of coagulation cascade components in modifying the inflammatory response", ". such knowledge will hopefully result in new strategies and therapies to manage the septic patient ."], "abstract_text": ["<S> the relationship between blood coagulation factors and the promotion or inhibition of the anti - inflammatory response continues to be defined through basic research . </S>", "<S> the potential key role of blood coagulation factors in the response during sepsis provides an exciting potential mechanism(s ) for modification through the application of new therapies . </S>", "<S> the complexity of the potential multiple actions of the proteins , such as protein c , should allow for development of new therapies to minimize the detrimental inflammatory response . </S>", "<S> however , several gaps in our current understanding must be bridged before the clinician can take the basic knowledge ' to the bedside ' , where individual patients will benefit . </S>"], "labels": null, "section_names": ["Introduction", "Optimum balance between pro-/anticoagulation and pro-/anti-inflammation", "Altering the intrinsic balance of coagulation and inflammation: detrimental consequences", "Patient variables that must be considered if the balance is to be altered", "Assessing individual intrinsic response at the bedside", "What agents have the 'best' activity in altering the intrinsic coagulation/inflammation balance?", "Conclusion", "Conflicting interests"], "sections": [["the relationship between the clotting cascade and the promotion or inhibition of the anti - inflammatory response continues to be defined through basic research .", "the potential key role of proteins that were originally believed to be exclusively involved in coagulation in the inflammatory response provides an exciting potential mechanism(s ) for modification through the application of new therapies .", "the accompanying review by riewald and ruf   outlines the available information on the possible steps involving some of the various proteins that are common to both coagulation and inflammation .", "the complexity of the multiple actions of the proteins in both inflammation and coagulation should excite the clinician with regard to potential new therapies that may be beneficial via activation of one or both of the concurrent pathways .", "however , before clinicians can administer new therapies that utilize new knowledge on the actions of coagulation cascade components , and hence improve the outcomes of their patients , several gaps in our current understanding must be addressed .", "this information is imperative if the clinician is to take basic knowledge ' to the bedside ' , where individual patients can benefit ."], ["administration of intrinsic or synthetic analogues of coagulation cascade components ( e.g. protein c , thrombin , and factor viia or xa ) , coagulation inhibitors ( e.g. heparin or analogues ) , or agonists or antagonists of the protease - activated receptors , in an attempt to alter the individual patient 's coagulation / inflammation balance , could theoretically produce responses ranging from beneficial to detrimental .", "knowledge of the ' most beneficial ' balance between augmenting or inhibiting the contribution of the clotting cascade components to inflammation would be required for determining the selection and dosing of potential new therapies ."], ["a safe assumption is that any new therapies that alter the intrinsic response to coagulation and inflammatory stimulants would also potentially produce detrimental effects on these , and potentially other , physiological responses .", "a simple example of such a potential for detrimental effects is the administration of protein c resulting in undesirable haemorrhage .", "this example illustrates that a broader knowledge of the impact of altering the coagulation/ inflammatory response will be required before clinicians can comfortably utilize new therapies in patient care ."], ["it is unlikely that any new therapies resulting from the new knowledge of the contribution of the clotting cascade to inflammation can be applied to all patients .", "it can be anticipated that certain patients would have contraindications to the new therapies or that dosages would need to be adjusted on the basis of pharmacokinetic or pharmacodynamic variables .", "the clinician will need to know what these contraindications and variables are before the new therapies can be used confidently .", "some , such as coagulation disorders , may be known and obvious , but any new strategies that could potentially alter the balance of a complex physiological response may be anticipated to be influenced by many , as yet unknown , factors in the individual patient .", "an example is the concurrent administration of heparin , which appears to antagonize the anti - inflammatory effect of antithrombin iii in septic patients .", "a more complete understanding of the impact of concurrent conditions , therapies and the patient 's genetic make - up on outcome will be required if we are to obtain the maximum benefit from new therapies ."], ["for any new therapy resulting from our expanded knowledge of the clotting cascade in the inflammatory response to be tailored to the needs of the individual patient , a method(s ) for rapidly and easily assessing the patient 's dynamic inflammatory response will be required .", "unless any new therapies have very benign toxicities , the need to assess the need , dosage and response to treatment will mandate sensitive and specific monitoring techniques .", "bedside or clinical laboratory techniques that could be applicable to most clinical environments would allow the benefit of any new therapies to be applied to the greatest number of patients .", "conversely , any new treatments that require complex , laborious monitoring techniques will have limited applicability ."], ["as our knowledge of the contribution of the intrinsic coagulation components increases , the development of many potential new therapies will hopefully emerge for clinical use", ". these may be totally new therapeutic entities or new strategies for using existing drugs or clotting factors .", "such developments will create the new dilemma for the clinician , namely that of trying to select between different treatment options for the individual patient .", "clinical trials outlining the potential anticipated benefit(s ) and the appropriate target patient population for each new therapeutic strategy will need to be completed", ". this will allow the clinician to ensure that the most appropriate strategy can be selected for the individual patient ."], ["the above - mentioned concerns aside , the clinician should be excited about the expanding knowledge of the role of coagulation cascade components in modifying the inflammatory response", ". such knowledge will hopefully result in new strategies and therapies to manage the septic patient ."], [""]]}
{"article_id": "PMC5428134", "article_text": ["myelofibrosis ( mf ) belongs to the category of myeloproliferative neoplasms ( mpns ) and may present as a primary disorder ( primary myelofibrosis [ pmf ] ) or evolve from polycythaemia vera ( pv ) or essential thrombocythaemia ( et ) to post - pv or post - et mf.1 it is characterised by the clonal proliferation of a pluripotent haematopoietic stem cell,2 in which the abnormal stem cell population releases several cytokines and growth factors into the bone marrow microenvironment , thus leading to an increase in bone marrow fibrosis , stromal changes , involvement of extramedullary organs such as the spleen and liver , and consequent clinical manifestations.3 myelofibrosis has an incidence of about 0.58 new cases per 100 000 person - years , but a higher prevalence of 6 per 100 000 person - years because of its chronic and disabling course.4 median age at diagnosis is 67 years , without any significant difference in distribution between the sexes .", "the diagnosis of mf is currently based on the world health organization 2016 criteria , which include the jak2v617f mutation that is detected in 50% to 60% of all cases.58 mutations in genes other than jak2 such as mpl mutations ( frequency : 5%10%)9,10 and somatically acquired mutations in the calr gene ( frequency : 15%20%)11,12 have also been described . however , about 10% of patients with mf do not develop any known mutation and are considered to have ", "triple - negative mf.13 in addition to these 3 driver mutations , numerous other somatic mutations involving epigenetic processes ( ezh2 , tet2 , asxl1 , and dnmt3a ) , spliceosome machinery ( srsf2 , sf3b1 , and u2af1 ) , and disease evolution ( eg , tp53 , idh1/2 , and ikzf ) have been identified in mf.1416 some of these mutations , such as those in dntm3a17 or tet2,18 have not been shown to correlate with survival outcome .", "conversely , mutations in asxl1 , srsf2 , and ezh2 predicted short survival in a large cohort of patients .", "more specifically , a report by tefferi et al19 points to the calr/asxl1 + profile as the most detrimental mutation profile in pmf .", "the symptoms mainly include those associated with splenomegaly ( abdominal distension and pain , early satiety , splenic infarction , dyspnoea , and diarrhoea ) and constitutional symptoms such as fatigue , cachexia , pruritus , bone pain , weight loss , and fever ; these worsen patients role functioning and quality of life ( qol ) .", "median survival ranges from approximately 3.5 to 5.5 years,20,21 and the most frequent cause of death in patients with mf is transformation to acute myeloid leukaemia ( 20% ) , but most patients die because of other disease - related events , such as progression without transformation , infections , and thrombo - haemorrhagic complications.20 prognosis is currently based on 3 different prognostic scoring systems , which mainly refer to age , constitutional symptoms , anaemia , white blood cell counts , and percentage of peripheral blood blasts : international prognostic scoring system ( ipss ) , which is applicable at diagnosis20 ; dynamic international prognostic scoring system ( dipss)22 ; and dipss - plus , which can be applied at any time during followup . the last incorporates 3 additional independent risk factors : red blood cell ( rbc ) transfusion requirement , platelet counts of < 100  10/l , and an unfavourable karyotype ( table 1).23 until recently , mf has remained orphan of curative treatments : the only treatment that has a clearly demonstrated impact on disease progression is allogeneic haematopoietic stem cell transplantation ( allo - hsct ) , but treatment - related mortality is high and only a minority of patients are eligible for such intensive therapy.24 the previously used treatments were palliative and have only limited benefits in qol and symptom control .", "however , the discovery of jak2 mutations , which has established that dysregulation of the jak - stat signalling pathway is a major contributor to the pathogenesis of mpns , has also led to the development of small - molecule jak1/2 inhibitors , the first of which ( ruxolitinib ) has been approved for the treatment of mf in the united states and europe . in this article , we report on old and new therapeutic strategies that proved effective in early preclinical and clinical trials and subsequently in the daily clinical practice for patients with mf , particularly concerning the topics of anaemia , splenomegaly , iron overload ( io ) , and allo - hsct .", "the management of anaemia can be one of the most challenging aspects of treating patients with mf ( table 2 ) .", "blood transfusion is the standard therapy for symptomatically anaemic patients , and the transfusion target should be assessed individually .", "corticosteroids ( eg , prednisone 0.5 mg / kg / day ) may be temporarily effective in treating anaemia and constitutional symptoms and are usually used in combination with other therapies.25 erythropoiesis - stimulating agents ( esas ) are worth trying in mf patients with moderate , nontransfusion - dependent anaemia and a low serum erythropoietin level ( < 125", "iu / l ) , although rapid spleen enlargement during treatment has occasionally been reported .", "response rates vary from 23% to 60% in different studies , with no clear evidence favouring darbepoetinalfa over conventional recombinant erythropoietins .", "furthermore , responses are usually short - lived ( < 1 year ) , and as no prospective randomised study of the value of esas has yet been published , they are not indicated in anaemic subjects with established transfusion dependency.26 if there are no contraindications , androgen preparations or danazol ( a semisynthetic attenuated androgen ) can be used .", "they have been shown to stimulate erythropoiesis in patients with refractory anaemia , leading to increased haemoglobin ( hb ) levels , reticulocytosis , and a decreased need for rbc transfusions27 ; however , documentation of their efficacy as single agents is largely restricted to retrospective studies .", "one of these reported responses in 11 of 30 patients with mf , including 8 with a complete response,28 with a lack of transfusion dependence and higher pretreatment hb levels predicting response . in another retrospective study", ", responses were observed in 17 of 39 patients with mf taking danazol , including 8 ( 21% ) with an increase in hb of 1.5 g / dl , hb levels of > 10 g / dl , and transfusion independence for 8 weeks.29 however , there were no identifiable patients characteristics ( such as transfusion dependency , baseline hb level , or cytogenetic results ) that influenced outcome .", "these findings have been confirmed in a recent series of 50 patients with mf30 ; the slightly lower rate of anaemia response ( 30% ) should be attributed to the use of more stringent response criteria.31 in terms of predicting response , the only pretreatment variable showing a trend for an association with response to danazol was transfusion dependency , with only 18.5% of the responders in this subgroup of patients against 43.5% in the subgroup not requiring transfusions .", "the main limitations of using danazol are toxicities , including fluid retention , increased libido , liver function test abnormalities , headache , and virilisation .", "all patients receiving danazol should therefore be monitored using monthly liver function tests during initial therapy and periodic liver ultrasound examinations to detect any hepatic malignancy .", "the antiangiogenic and immunomodulatory properties of thalidomide , lenalidomide , and pomalidomide make them potentially effective medical therapies for mf , with some responses in patients with anaemia , thrombocytopenia , and splenomegaly ; potential modifications to the bone marrow microenvironment ; and a possible reduction in bone marrow fibrosis .", "the combination of thalidomide and prednisone has been evaluated in 21 patients with mf , 62% of whom showed an anaemia response.32 however , the high incidence of neuropathy associated with thalidomide limits its usefulness . furthermore , because of the risk of thrombosis , prophylaxis with aspirin is recommended in all patients with a platelet count of > 50  10/l .", "this combination is therefore not usually selected for the first - line management of anaemia .", "a phase ii clinical trial ( nct00227591 ) assessed the therapeutic efficacy of lenalidomide combined with prednisone in 42 patients with mf .", "clinical improvements in anaemia and splenomegaly were observed in , respectively , 19% and 10% of the subjects .", "similar to thalidomide , lenalidomide was burdened by toxicity , including cytopenia ( at least 1 grade 34 event in 88% of patients ) and nonhaematologic toxicity ( at least 1 grade 34 event in 45% of patients).33 a second study of lenalidomide plus prednisone in 40 patients with intermediate - risk or high - risk mf led to an overall response rate based on international working group criteria of 30% for anaemia and 42% for splenomegaly , with a median time to response of 12 weeks . however , grade 3 and 4 adverse events ( aes ) were reported , mainly cytopenia.34 a recently updated report of this study after a median follow - up of 9 years35 showed that treatment responses improved over time , with 14 patients ( 35% ) responding overall .", "more specifically , 39% of the patients showed a response in terms of reduction in spleen size , and the overall anaemia response rate was 32% .", "however , there was no significant difference in baseline characteristics between the patients who responded and those who did not .", "an analysis combining the results of 3 phase ii trials indicated that lenalidomide - based therapy may be more effective than thalidomide - based therapy , and fewer patients treated with lenalidomide plus prednisone discontinued therapy due to toxicity than those receiving thalidomide - based therapy .", "in addition , there was no significant difference in the response to lenalidomide alone and lenalidomide plus prednisone ; however , response duration was significantly longer in patients who received lenalidomide plus prednisone.36 pomalidomide , a more potent immunomodulatory drug , has been evaluated in a multicentre , double - blind , placebo - controlled phase iii study ( nct01178281).37 however , the study failed to meet the primary endpoint as an equal proportion of patients with mf in the pomalidomide ( n = 152 ) and placebo arm ( n = 77 ) achieved an anaemia response ( 16% vs 16% , p = 1 ) . on the contrary ,", "the platelet response was significantly better in patients who received pomalidomide ( 22% vs 0% ) .", "cytoreductive agents have been the treatment of choice for most mf patients with symptomatic splenomegaly ( table 3 ) .", "hydroxyurea ( hu ) , an s - phase cell cycle  specific nucleotide - depleting agent that inhibits ribonucleotide reductase,38 is one of the most widely used medical therapies for patients with appreciably symptomatic splenomegaly,39,40 although it induces only modest responses at higher doses ( 12 g daily ) and mainly in subjects with nonmassive splenomegaly ( < 15 cm).45 although hu is generally well tolerated , the modest improvement in symptoms is temporary , and exacerbated cytopenia frequently limits treatment . in patients who do not respond to", "hu , it has been shown that the oral alkylating agents , melphalan and busulfan , improve splenomegaly and other disease symptoms , but they may also exacerbate cytopenia and possibly increase the frequency of leukaemic transformation .", "furthermore , they are mainly used in older patients as they are relatively manageable insofar as frequent laboratory monitoring is not required , unlike in the case of hu or other cytoreductive agents.41,46 in cases of massive refractory splenomegaly , it has been found that monthly courses of intravenous cladribine ( 2-chlorodeoxyadenosine ) lead to a response in up to 50% of patients , with severe but reversible cytopenia being the main toxicity.42 interferon - alfa ( standard and pegylated versions ) has proved to have only a minimal clinical effect in reducing splenomegaly , and therefore , its use is not generally recommended.43 hypomethylating agents , such as azacitidine and decitabine , have also been studied in mf , but currently play only a limited role in its treatment.47 more recently , thomas et al48 demonstrated that methotrexate ( mtx ) may act as an inhibitor of the jak - stat pathway and that this activity is likely to be specific and not related to a general effect on protein phosphorylation : the drug s in vitro activity was observed at a concentration equivalent to that used in patients taking low - dose mtx ( 525 mg / wk ) .", "what is important is that its efficacy in controlling haematologic parameters , systemic symptoms , and splenomegaly has been confirmed in vivo in 2 recent case reports.49 splenectomy is a palliative debulking measure used in patients with mpns .", "its indications are mainly symptomatic massive splenomegaly , symptomatic portal hypertension with oesophageal varices and/or bleeding , profound cachexia , transfusion - dependent anaemia , and/or severe hypercatabolic symptoms .", "removal of the spleen improves mechanical symptoms ( ie , early satiety and pain ) in most cases and is often followed by weight gain in cachectic patients , but it is usually not effective against other constitutional symptoms .", "improvements in anaemia and thrombocytopenia after splenectomy have been reported in , respectively , 50% and < 30% of patients .", "progressive hepatomegaly sometimes follows splenectomy , probably due to the migration of haematopoiesis , and a markedly enlarged liver is a contraindication to splenectomy .", "current data concerning an increased rate of leukaemic transformation after spleen removal are still discordant.44,50 however , given the high complication rate and limited benefit of splenectomy , appropriate patient selection is crucial.51 splenic radiotherapy , on a fractioned basis , at a daily dose of 0.4 to 1 gy , with weekly evaluation of spleen size and haematologic values until therapeutic effect is achieved or haematologic toxicity develops , can be used to treat mpns with an adequate platelet count ( > 50  10/l ) , as extramedullary haematopoiesis has proved to be considerably sensitive to external beam radiotherapy in patients with mf . however , it leads to only transient benefits and may exacerbate cytopenia , particularly thrombocytopenia.50 it also has to be remembered that radiation can also cause local fibrosis with splenic adhesions to surrounding tissues that make a subsequent splenectomy technically more complicated and increase the morbidity and mortality of the procedure . in general , traditional treatment options are limited and insufficient to address the morbidity and mortality associated with mf . however , as mentioned above , the discovery of mutations leading to constitutive activation of the jak - stat signalling pathway raises hope that mf may be cured by selective jak1/2 inhibitors , as happens in the case of chronic myeloid leukaemia treated with bcr - abl1 tyrosine kinase inhibitors .", "ruxolitinib ( jakavi ; novartis , basel , switzerland ) was the first jak1/2 inhibitor to become commercially available for the treatment of mf.52 in preclinical jak2v617f - positive mpn mouse models , it induced a considerable downregulation of jak - dependent proinflammatory cytokines , reduced mouse splenomegaly , and showed antiproliferative and proapoptotic activities .", "it is the only jak inhibitor approved in the united states for the treatment of splenomegaly in subjects with intermediate / high - risk mf and in europe for the treatment of splenomegaly and/or constitutional symptoms in patients with intermediate-2/high - risk mf.53 these approvals were based on the results of 2 phase iii randomised studies : comfort - i ( ruxolitinib vs placebo ) and comfort - ii ( ruxolitinib vs best available therapy [ bat]).54,55 the primary endpoint of both studies was a > 35% reduction in spleen volume after 24 ( comfort - i ) or 48 weeks of treatment ( comfort - ii ) , which was reached by , respectively , 41.7% and 28.5% of the patients treated with ruxolitinib , as against , respectively , 0.7% and 0% of the patients receiving placebo or bat ( p < .0001).54,55 overall , more than 90% of the patients enrolled in both studies experienced some reduction in spleen volume at some time during the follow - up , and the reduction remained stable in most of the patients after a median follow - up of 3 ( comfort - i ) and 5 years ( comfort - ii).56,57 the therapeutic success of ruxolitinib is not limited to reducing spleen volume because , unlike the drugs previously used to treat mf , it is efficacious in relieving constitutional symptoms ; reducing abdominal discomfort , appetite loss , itching , fatigue , and night sweats ; and improving the qol of most treated patients . as the drug s activity is independent of jak2 mutational status and not specific for the neoplastic clone , the response rate is similar in patients with and without the jak2v617f mutation because of its anti - jak1mediated effect .", "the uk , open - label , phase ii robust study evaluated its safety and efficacy in patients with mf , including those at intermediate-1 risk .", "the treatment was successful in 50% of the population as a whole and 57% of the intermediate-1risk patients .", "reduction in spleen length and symptoms was observed in all of the risk groups , and improvements in the myelofibrosis symptom assessment form total symptom score were seen in 80% of intermediate-1 , 72.7% of intermediate-2 , and 72.2% of high - risk patients.58 similarly , the phase iiib expanded - access jak inhibitor ruxolitinib in myelofibrosis patients ( jump ) trial for patients with mf without access to ruxolitinib outside of a clinical study found that the drug s safety and efficacy profile in intermediate-1risk patients was consistent with that in the study population as a whole and with that previously reported in intermediate-2risk and high - risk patients.59 the toxicity of ruxolitinib treatment is mainly haematologic due to the drug s interference with an essential pathway for haematopoiesis , as demonstrated in the comfort studies ; in both trials , thrombocytopenia was the dose - limiting toxicity , and anaemia was the most common haematologic ae .", "a number of studies have shown that ruxolitinib affects many cytokines and interferes with the immune process necessary for the pathogenesis of mpns , but it also affects the function of various immune cells and may therefore favour an increased incidence of opportunistic and nonopportunistic infections.60,61 for example , ruxolitinib impairs natural killer cell differentiation and function and inhibits dendritic cell activation and migration , and antigen - specific t - cell responses in a dose - dependent manner in vitro and in vivo .", "however , despite warnings about this increased risk,6264 a recent update of the jump study described a low incidence of infections : the all - grade infections observed in 1% of patients included nasopharyngitis ( 6.3% ) , urinary tract infection ( 6% ) , pneumonia ( 5.3% ) , bronchitis ( 4.2% ) , herpes zoster ( 3.6% ) , influenza ( 3% ) , upper respiratory tract infection ( 2.9% ) , cystitis ( 2.5% ) , gastroenteritis ( 1.8% ) , respiratory tract infection ( 1.8% ) , and oral herpes ( 1.6% ) .", "other infections included tuberculosis in 3 patients ( 0.3% ) and legionella pneumonia in 1 patient ( 0.1% ) .", "no hepatitis b reactivation was reported , and only 6 patients ( 0.5% ) discontinued treatment because of grade 3 pneumonia.59 the patients receiving ruxolitinib in the comfort - ii study experienced higher rates of viral and bacterial infection than those receiving conventional therapy , but most of the infections were grade 1 or 2 and did not lead to any dose reductions or the discontinuation of trial medication .", "furthermore , the rates of infection tended to decrease with longer exposure to the drug .", "however , as patients with mf are already predisposed to infections65 and the long - term risks of ruxolitinib treatment are still unknown , treated patients should be carefully monitored , and prophylaxis for herpes zoster or other infections should be considered on a case - by - case basis , depending on local risk .", "given its promising results , a further indication for ruxolitinib treatment is as a therapeutic bridge to allo - hsct .", "furthermore , an increasing number of reports appeared in the literature , describing the morphologic changes in the bone marrow occurring in ruxolitinib - treated patients , mostly focusing on modifications in bone marrow fibrosis degree.6670 by the beginning of 2014 , a number of other jak2 inhibitors were being tested : fedratinib,71 pacritinib,72 ly2784544,73 and momelotinib.74 however , the clinical trials of fedratinib and pacritinib were soon discontinued because of safety problems : wernicke encephalopathy ( fedratinib ) and bleeding ( pacritinib ) . momelotinib ( formerly known as cyt387 ) is a small - molecule , adenosine triphosphate ", "its kinase profiling indicates that it has good selectivity over other jak family kinases ( jak3 , tyk2 ) and excellent selectivity over other tyrosine and serine / threonine kinases.75 the preclinical data provide a rationale for the use of momelotinib in bcr - abl1negative mpns , and a multicentre phase i / ii trial involving 166 patients with intermediate / high - risk mf showed that the drug is well tolerated at oral doses of 150 or 300 mg once daily or 250 mg twice daily and led to improvements in splenomegaly , constitutional symptoms , and transfusion requirement.74 of particular interest are the transfusion independence responses , which were observed in more than half of the rbc transfusion  dependent subjects with a maximum transfusion - free period exceeding 2 years .", "in addition , the percentage of all subjects requiring rbc transfusions substantially decreased over the treatment period .", "more precisely , the overall anaemia response rate was 54% in transfusion - dependent patients with a median time to a confirmed anaemia response of 12 weeks ( range : 84293 days ) . as has been previously reported , treatment with momelotinib led to a rapid and sustained reduction in splenomegaly in approximately 31% of all cases , with a median time to response of 15 days , and the constitutional symptoms of most of the patients disappeared within 6 months . in terms of safety , about 20% of the patients experienced a first - dose effect ( dizziness , flushing , and hypotension ) that was self - limited .", "grade 3/4 haematologic and nonhaematologic aes were infrequent with the exception of thrombocytopenia , which occurred in approximately 17% of patients .", "grade 3/4 nonhaematologic laboratory aes included hyperlipasaemia ( 4% ) and increased liver enzymes ( grade 3 and 4 increase in aspartate aminotransferase in , respectively , 1% and < 1% of the patients ; a grade 3 increase in alanine aminotransferase in 2% ) . mainly , grade 1 treatment  related sensory peripheral neuropathy was reported , but there were no treatment - related deaths . in brief , momelotinib seems to lead to a significant and lasting improvement in anaemia , splenomegaly , and constitutional symptoms at doses of 150 or 300 mg / day or 150 mg twice daily .", "the efficacy and aes of momelotinib will be further evaluated in 2 currently ongoing phase iii trials : a randomised bat - controlled study of mf patients with anaemia and thrombocytopenia previously treated with ruxolitinib and a randomised study comparing momelotinib and ruxolitinib in patients with mf ( nct02101268-nct01969838 ) .", "more recently , the efficacy and safety of 3 dose levels of a potent and selective oral jak1 inhibitor , incb039110 , have been evaluated in an open - label phase ii study , resulting in clinically meaningful symptom relief , modest spleen volume reduction , and limited myelosuppression.76 in particular , only 1 patient discontinued for grade 3 thrombocytopenia , whereas nonhaematologic aes were largely of grade 1 or 2 and most commonly represented by fatigue .", "nearly 40% of patients with mf are anaemic at the time of diagnosis , including 25% who are already transfusion dependent,77,78 and more than 60% will develop clinically significant anaemia during the course of follow - up.49 the clinical impact of io and its potential relationship to the heightened inflammatory response of patients with mf warrant consideration not only because potential liver dysfunction , cardiac disease , and other complications of io probably contribute to patient morbidity and mortality but also because the growing evidence of impaired haematopoiesis attributable to bone marrow haemosiderosis suggests a viable therapeutic target.79,80 each unit of rbc contains 200 to 250 mg of iron , and as the reticuloendothelial system can clear approximately 10 to 15 g ( corresponding to 50 rbc units ) , any excess is deposited in tissues and leads to organ damage.81 iron overload is a concern when treating patients with mf , which is why iron chelation therapy ( ict ) has been used to counteract its potentially negative effects .", "however , it has to be admitted that there is a lack of prospective , randomised , controlled trials of the use of ict in patients with mf . one small retrospective study of 10 patients with mf demonstrated an erythroid response in 40% of cases receiving oral ict with deferasirox ( dfx ) , thus allowing these patients to reduce their transfusion requirement ; it also revealed a trend towards better overall survival in the responding patients.82 other data coming from a number of reported case studies79,80,83,84 also indicate that ict improves anaemia and decreases transfusion dependence in patients with mf . finally , a recent retrospective , multicentre analysis of 28 patients with mf and io secondary to transfusion dependence found that 11 patients ( 42.3% ) achieved a stable and consistent reduction in ferritin levels ( < 1000 ng / ml ) , and 6 of 26 patients ( 23% ) showed a persistent ( > 3 months ) increase in hb levels to > 1.5 g / dl , with the disappearance of transfusion dependence in 4 cases .", "however , comparison of the baseline characteristics of the patients who achieved an erythroid response and those who did not achieve did not reveal any significant differences that could be considered predictive.85 deferoxamine ( desferal ) is a linear ligand that forms 1:1 complexes with iron that maintain a net charge , allow for membrane permeability , and provide access to intracellular iron stores86 that are then excreted primarily in urine.87,88 it is administered in the form of an intravenous or subcutaneous infusion , and because of its short plasma half - life , the efficacy of the treatment correlates with the duration of infusion , and it is only effective if administered at high doses between 5 and 7 times per week.89 when administered as a continuous 24-hour infusion for 6 to 7 days per week in patients with high - risk -thalassemia , it can reverse iron - induced cardiac dysfunction and increase long - term survival.90 however , treatment - related side effects include infusion site discomfort ( nearly 100% , with the development of local erythema or induration in some cases),91 visual changes ( 0%10% ) , generally transient auditory neurotoxicity ( 20%25%),92,93 increased serum creatinine levels ( 22% ) , vomiting ( 16% ) , abdominal discomfort ( 14% ) , constipation ( 14% ) , arthralgia ( 14% ) , nausea ( 11% ) , rash ( 5% ) , and diarrhoea ( 5%).94 deferasirox is an oral iron chelator frequently used in clinical practice in the united states and europe that has a long half - life of 8 to 16 hours and can be administered once daily.89 it forms a 2:1 complex with iron,95 which is then excreted largely in the bile and faeces ( much less in urine).96,97 unlike other iron chelators , it is thought that dfx also affects haematopoietic stem cell differentiation by means of a reactive oxygen species  mediated mechanism , which may underlie the erythropoeitic response seen in some dfx - treated patients.95 the most frequently reported adverse effects are gastrointestinal toxicity ( 21%64% ) , diarrhoea ( 46% ) , abdominal pain ( 15%28% ) , nausea ( 24% ) , vomiting ( 21% ) , and constipation ( 10%).94,98101 patients have also been reported to experience renal dysfunction ( 10%64% , usually nonprogressive at the start of treatment and improving after a dose reduction ) , skin rash ( 4%39% ) , arthralgia ( 15%),94 and transaminitis ( 4%70%),81,98101 and there have been rarer reports of auditory neurotoxicity ( 1%6% ) as a potential side effect.100,101 it is not entirely clear whether ict can reverse the ill effects of io , and there are no completed studies that provide prospective evidence of a beneficial impact in terms of the restoration of normal haematopoiesis or outcomes in patients with mf .", "consequently , treatment decisions concerning the use of ict in patients with mf continue to be extrapolated from the data of myelodysplastic syndromes .", "allogeneic haematopoietic stem cell transplantation is still the only intervention that has been shown to be a potential cure for mf or a means of prolonging the survival of these patients .", "data from the most recent studies suggest that the expected 3-year progression - free survival rate is in the range of 40% to 50%.102 the adoption of reduced intensity conditioning regimens has recently made allo - hsct applicable to a larger proportion of patients.103 however , decisions concerning allo - hsct are based on inductive reasoning and require a considerable professional experience .", "key questions include patient selection , donor selection , pre- and posttransplant management , conditioning regimen , and prevention and management of post - transplant relapses .", "international prognostic scoring systems ( ie , ipss , dipss , and dipss - plus)20,22,23 are the most comprehensive means of risk stratification currently available to guide therapeutic decision making , although the influence of driver mutations and the acquisition of additional mutations during the natural course of the disease may further refine this process .", "all patients with mf aged < 70 years with ipss , dipss , or dipss - plus intermediate-2risk or high - risk disease and a reasonable performance status , and without any significant competing comorbid conditions , should be considered potential candidates for allo - hsct .", "patients aged < 65 years with intermediate-1risk disease should only be considered candidates if they present with refractory , transfusion - dependent anaemia or > 2% of peripheral blood blasts , or adverse cytogenetics ( as defined by the dipss - plus classification ) . finally , patients with low - risk disease should not undergo allo - hsct.104 individual transplant - specific prognostic factors should be considered in every candidate for allo - hsct to be able to make individualised decisions . in this context , the transplant - specific high - risk factors include a spleen extending more than 22 cm below the costal margin , having been transfused with more than 20 rbc units , having received a transplantation from an hla nonidentical donor , a poor performance status ( an eastern cooperative oncology group status of > 2 ) , a high comorbidity index ( a haematopoietic cell transplantation comorbidity index score of > 3 ) , and the presence of portal hypertension .", "completely matched rather than mismatched donors should be selected because , as reported in the european blood and marrow transplantation registry , the cumulative incidence of nonrelapsed mortality after 1 year is , respectively , 12% and 38% and is not different between hla - identical siblings and 10/10 matched unrelated donors ( 10% vs 13%).105 however , haploidentical related donors are an attractive alternative source of haematopoietic stem cells.106 it is important to note that peripheral blood is considered the most appropriate source of haematopoietic stem cells in the case of hla - matched sibling and unrelated donors . when splenectomy is performed before allo - hsct , it may facilitate disease eradication .", "some reports have also shown faster engraftment in splenectomised patients ; however , the pretransplant use of splenectomy remains controversial as no study has yet prospectively evaluated the effect of protocol - based splenectomy before transplantation . in the case of older patients and/or those with comorbidities ,", "a less intense conditioning regimen is more appropriate , whereas patients with advanced disease and a good performance status should undergo a more intensified regimen.104 finally , in patients relapsing with constitutional symptoms or splenomegaly , jak1/2 inhibitor treatment is recommended but remains experimental . to address this question ,", "ruxolitinib is being administered to eligible patients with mf for 60 days before definitive allo - hsct in a prospective multicentre phase ii study conducted by the myeloproliferative disorders research consortium ( nct01790295 ) .", "traditional mf treatments are primarily palliative and have proved to be inadequate to address the considerable morbidity and mortality associated with this disabling disease .", "more specifically , concerning anaemia , there have been various therapeutic attempts , but rbc transfusions still remain the most frequently used approach , even though io represents an increasingly frequent clinical challenge . considering instead splenomegaly , besides hu , ruxolitinib , as well as other investigational jak1/2 inhibitors , offers new hope for these patients as they have been shown to lead not only to significant reduction in splenomegaly but also to the palliation of disease - related symptoms . however , allo - hsct is still the only intervention that has evidence indicating it is potentially curative .", "obviously , in such a context , participation into a clinical trial should be encouraged whenever possible , with the purpose of making new drugs available ."], "abstract_text": ["<S> myelofibrosis ( mf ) is a bcr - abl1negative myeloproliferative neoplasm that is mainly characterised by reactive bone marrow fibrosis , extramedullary haematopoiesis , anaemia , hepatosplenomegaly , constitutional symptoms , leukaemic progression , and shortened survival . as such , this malignancy is still orphan of curative treatments </S>", "<S> ; indeed , the only treatment that has a clearly demonstrated impact on disease progression is allogeneic haematopoietic stem cell transplantation , but only a minority of patients are eligible for such intensive therapy . </S>", "<S> however , more recently , the discovery of jak2 mutations has also led to the development of small - molecule jak1/2 inhibitors , the first of which , ruxolitinib , has been approved for the treatment of mf in the united states and europe . in this article , we report on old and new therapeutic strategies that proved effective in early preclinical and clinical trials , and subsequently in the daily clinical practice , for patients with mf , particularly concerning the topics of anaemia , splenomegaly , iron overload , and allogeneic stem cell transplantation . </S>"], "labels": null, "section_names": ["Introduction", "Anaemia", "Splenomegaly", "Iron Overload", "Stem Cell Transplantation", "Conclusions"], "sections": [["myelofibrosis ( mf ) belongs to the category of myeloproliferative neoplasms ( mpns ) and may present as a primary disorder ( primary myelofibrosis [ pmf ] ) or evolve from polycythaemia vera ( pv ) or essential thrombocythaemia ( et ) to post - pv or post - et mf.1 it is characterised by the clonal proliferation of a pluripotent haematopoietic stem cell,2 in which the abnormal stem cell population releases several cytokines and growth factors into the bone marrow microenvironment , thus leading to an increase in bone marrow fibrosis , stromal changes , involvement of extramedullary organs such as the spleen and liver , and consequent clinical manifestations.3 myelofibrosis has an incidence of about 0.58 new cases per 100 000 person - years , but a higher prevalence of 6 per 100 000 person - years because of its chronic and disabling course.4 median age at diagnosis is 67 years , without any significant difference in distribution between the sexes .", "the diagnosis of mf is currently based on the world health organization 2016 criteria , which include the jak2v617f mutation that is detected in 50% to 60% of all cases.58 mutations in genes other than jak2 such as mpl mutations ( frequency : 5%10%)9,10 and somatically acquired mutations in the calr gene ( frequency : 15%20%)11,12 have also been described . however , about 10% of patients with mf do not develop any known mutation and are considered to have ", "triple - negative mf.13 in addition to these 3 driver mutations , numerous other somatic mutations involving epigenetic processes ( ezh2 , tet2 , asxl1 , and dnmt3a ) , spliceosome machinery ( srsf2 , sf3b1 , and u2af1 ) , and disease evolution ( eg , tp53 , idh1/2 , and ikzf ) have been identified in mf.1416 some of these mutations , such as those in dntm3a17 or tet2,18 have not been shown to correlate with survival outcome .", "conversely , mutations in asxl1 , srsf2 , and ezh2 predicted short survival in a large cohort of patients .", "more specifically , a report by tefferi et al19 points to the calr/asxl1 + profile as the most detrimental mutation profile in pmf .", "the symptoms mainly include those associated with splenomegaly ( abdominal distension and pain , early satiety , splenic infarction , dyspnoea , and diarrhoea ) and constitutional symptoms such as fatigue , cachexia , pruritus , bone pain , weight loss , and fever ; these worsen patients role functioning and quality of life ( qol ) .", "median survival ranges from approximately 3.5 to 5.5 years,20,21 and the most frequent cause of death in patients with mf is transformation to acute myeloid leukaemia ( 20% ) , but most patients die because of other disease - related events , such as progression without transformation , infections , and thrombo - haemorrhagic complications.20 prognosis is currently based on 3 different prognostic scoring systems , which mainly refer to age , constitutional symptoms , anaemia , white blood cell counts , and percentage of peripheral blood blasts : international prognostic scoring system ( ipss ) , which is applicable at diagnosis20 ; dynamic international prognostic scoring system ( dipss)22 ; and dipss - plus , which can be applied at any time during followup . the last incorporates 3 additional independent risk factors : red blood cell ( rbc ) transfusion requirement , platelet counts of < 100  10/l , and an unfavourable karyotype ( table 1).23 until recently , mf has remained orphan of curative treatments : the only treatment that has a clearly demonstrated impact on disease progression is allogeneic haematopoietic stem cell transplantation ( allo - hsct ) , but treatment - related mortality is high and only a minority of patients are eligible for such intensive therapy.24 the previously used treatments were palliative and have only limited benefits in qol and symptom control .", "however , the discovery of jak2 mutations , which has established that dysregulation of the jak - stat signalling pathway is a major contributor to the pathogenesis of mpns , has also led to the development of small - molecule jak1/2 inhibitors , the first of which ( ruxolitinib ) has been approved for the treatment of mf in the united states and europe . in this article , we report on old and new therapeutic strategies that proved effective in early preclinical and clinical trials and subsequently in the daily clinical practice for patients with mf , particularly concerning the topics of anaemia , splenomegaly , iron overload ( io ) , and allo - hsct ."], ["the management of anaemia can be one of the most challenging aspects of treating patients with mf ( table 2 ) .", "blood transfusion is the standard therapy for symptomatically anaemic patients , and the transfusion target should be assessed individually .", "corticosteroids ( eg , prednisone 0.5 mg / kg / day ) may be temporarily effective in treating anaemia and constitutional symptoms and are usually used in combination with other therapies.25 erythropoiesis - stimulating agents ( esas ) are worth trying in mf patients with moderate , nontransfusion - dependent anaemia and a low serum erythropoietin level ( < 125", "iu / l ) , although rapid spleen enlargement during treatment has occasionally been reported .", "response rates vary from 23% to 60% in different studies , with no clear evidence favouring darbepoetinalfa over conventional recombinant erythropoietins .", "furthermore , responses are usually short - lived ( < 1 year ) , and as no prospective randomised study of the value of esas has yet been published , they are not indicated in anaemic subjects with established transfusion dependency.26 if there are no contraindications , androgen preparations or danazol ( a semisynthetic attenuated androgen ) can be used .", "they have been shown to stimulate erythropoiesis in patients with refractory anaemia , leading to increased haemoglobin ( hb ) levels , reticulocytosis , and a decreased need for rbc transfusions27 ; however , documentation of their efficacy as single agents is largely restricted to retrospective studies .", "one of these reported responses in 11 of 30 patients with mf , including 8 with a complete response,28 with a lack of transfusion dependence and higher pretreatment hb levels predicting response . in another retrospective study", ", responses were observed in 17 of 39 patients with mf taking danazol , including 8 ( 21% ) with an increase in hb of 1.5 g / dl , hb levels of > 10 g / dl , and transfusion independence for 8 weeks.29 however , there were no identifiable patients characteristics ( such as transfusion dependency , baseline hb level , or cytogenetic results ) that influenced outcome .", "these findings have been confirmed in a recent series of 50 patients with mf30 ; the slightly lower rate of anaemia response ( 30% ) should be attributed to the use of more stringent response criteria.31 in terms of predicting response , the only pretreatment variable showing a trend for an association with response to danazol was transfusion dependency , with only 18.5% of the responders in this subgroup of patients against 43.5% in the subgroup not requiring transfusions .", "the main limitations of using danazol are toxicities , including fluid retention , increased libido , liver function test abnormalities , headache , and virilisation .", "all patients receiving danazol should therefore be monitored using monthly liver function tests during initial therapy and periodic liver ultrasound examinations to detect any hepatic malignancy .", "the antiangiogenic and immunomodulatory properties of thalidomide , lenalidomide , and pomalidomide make them potentially effective medical therapies for mf , with some responses in patients with anaemia , thrombocytopenia , and splenomegaly ; potential modifications to the bone marrow microenvironment ; and a possible reduction in bone marrow fibrosis .", "the combination of thalidomide and prednisone has been evaluated in 21 patients with mf , 62% of whom showed an anaemia response.32 however , the high incidence of neuropathy associated with thalidomide limits its usefulness . furthermore , because of the risk of thrombosis , prophylaxis with aspirin is recommended in all patients with a platelet count of > 50  10/l .", "this combination is therefore not usually selected for the first - line management of anaemia .", "a phase ii clinical trial ( nct00227591 ) assessed the therapeutic efficacy of lenalidomide combined with prednisone in 42 patients with mf .", "clinical improvements in anaemia and splenomegaly were observed in , respectively , 19% and 10% of the subjects .", "similar to thalidomide , lenalidomide was burdened by toxicity , including cytopenia ( at least 1 grade 34 event in 88% of patients ) and nonhaematologic toxicity ( at least 1 grade 34 event in 45% of patients).33 a second study of lenalidomide plus prednisone in 40 patients with intermediate - risk or high - risk mf led to an overall response rate based on international working group criteria of 30% for anaemia and 42% for splenomegaly , with a median time to response of 12 weeks . however , grade 3 and 4 adverse events ( aes ) were reported , mainly cytopenia.34 a recently updated report of this study after a median follow - up of 9 years35 showed that treatment responses improved over time , with 14 patients ( 35% ) responding overall .", "more specifically , 39% of the patients showed a response in terms of reduction in spleen size , and the overall anaemia response rate was 32% .", "however , there was no significant difference in baseline characteristics between the patients who responded and those who did not .", "an analysis combining the results of 3 phase ii trials indicated that lenalidomide - based therapy may be more effective than thalidomide - based therapy , and fewer patients treated with lenalidomide plus prednisone discontinued therapy due to toxicity than those receiving thalidomide - based therapy .", "in addition , there was no significant difference in the response to lenalidomide alone and lenalidomide plus prednisone ; however , response duration was significantly longer in patients who received lenalidomide plus prednisone.36 pomalidomide , a more potent immunomodulatory drug , has been evaluated in a multicentre , double - blind , placebo - controlled phase iii study ( nct01178281).37 however , the study failed to meet the primary endpoint as an equal proportion of patients with mf in the pomalidomide ( n = 152 ) and placebo arm ( n = 77 ) achieved an anaemia response ( 16% vs 16% , p = 1 ) . on the contrary ,", "the platelet response was significantly better in patients who received pomalidomide ( 22% vs 0% ) ."], ["cytoreductive agents have been the treatment of choice for most mf patients with symptomatic splenomegaly ( table 3 ) .", "hydroxyurea ( hu ) , an s - phase cell cycle  specific nucleotide - depleting agent that inhibits ribonucleotide reductase,38 is one of the most widely used medical therapies for patients with appreciably symptomatic splenomegaly,39,40 although it induces only modest responses at higher doses ( 12 g daily ) and mainly in subjects with nonmassive splenomegaly ( < 15 cm).45 although hu is generally well tolerated , the modest improvement in symptoms is temporary , and exacerbated cytopenia frequently limits treatment . in patients who do not respond to", "hu , it has been shown that the oral alkylating agents , melphalan and busulfan , improve splenomegaly and other disease symptoms , but they may also exacerbate cytopenia and possibly increase the frequency of leukaemic transformation .", "furthermore , they are mainly used in older patients as they are relatively manageable insofar as frequent laboratory monitoring is not required , unlike in the case of hu or other cytoreductive agents.41,46 in cases of massive refractory splenomegaly , it has been found that monthly courses of intravenous cladribine ( 2-chlorodeoxyadenosine ) lead to a response in up to 50% of patients , with severe but reversible cytopenia being the main toxicity.42 interferon - alfa ( standard and pegylated versions ) has proved to have only a minimal clinical effect in reducing splenomegaly , and therefore , its use is not generally recommended.43 hypomethylating agents , such as azacitidine and decitabine , have also been studied in mf , but currently play only a limited role in its treatment.47 more recently , thomas et al48 demonstrated that methotrexate ( mtx ) may act as an inhibitor of the jak - stat pathway and that this activity is likely to be specific and not related to a general effect on protein phosphorylation : the drug s in vitro activity was observed at a concentration equivalent to that used in patients taking low - dose mtx ( 525 mg / wk ) .", "what is important is that its efficacy in controlling haematologic parameters , systemic symptoms , and splenomegaly has been confirmed in vivo in 2 recent case reports.49 splenectomy is a palliative debulking measure used in patients with mpns .", "its indications are mainly symptomatic massive splenomegaly , symptomatic portal hypertension with oesophageal varices and/or bleeding , profound cachexia , transfusion - dependent anaemia , and/or severe hypercatabolic symptoms .", "removal of the spleen improves mechanical symptoms ( ie , early satiety and pain ) in most cases and is often followed by weight gain in cachectic patients , but it is usually not effective against other constitutional symptoms .", "improvements in anaemia and thrombocytopenia after splenectomy have been reported in , respectively , 50% and < 30% of patients .", "progressive hepatomegaly sometimes follows splenectomy , probably due to the migration of haematopoiesis , and a markedly enlarged liver is a contraindication to splenectomy .", "current data concerning an increased rate of leukaemic transformation after spleen removal are still discordant.44,50 however , given the high complication rate and limited benefit of splenectomy , appropriate patient selection is crucial.51 splenic radiotherapy , on a fractioned basis , at a daily dose of 0.4 to 1 gy , with weekly evaluation of spleen size and haematologic values until therapeutic effect is achieved or haematologic toxicity develops , can be used to treat mpns with an adequate platelet count ( > 50  10/l ) , as extramedullary haematopoiesis has proved to be considerably sensitive to external beam radiotherapy in patients with mf . however , it leads to only transient benefits and may exacerbate cytopenia , particularly thrombocytopenia.50 it also has to be remembered that radiation can also cause local fibrosis with splenic adhesions to surrounding tissues that make a subsequent splenectomy technically more complicated and increase the morbidity and mortality of the procedure . in general , traditional treatment options are limited and insufficient to address the morbidity and mortality associated with mf . however , as mentioned above , the discovery of mutations leading to constitutive activation of the jak - stat signalling pathway raises hope that mf may be cured by selective jak1/2 inhibitors , as happens in the case of chronic myeloid leukaemia treated with bcr - abl1 tyrosine kinase inhibitors .", "ruxolitinib ( jakavi ; novartis , basel , switzerland ) was the first jak1/2 inhibitor to become commercially available for the treatment of mf.52 in preclinical jak2v617f - positive mpn mouse models , it induced a considerable downregulation of jak - dependent proinflammatory cytokines , reduced mouse splenomegaly , and showed antiproliferative and proapoptotic activities .", "it is the only jak inhibitor approved in the united states for the treatment of splenomegaly in subjects with intermediate / high - risk mf and in europe for the treatment of splenomegaly and/or constitutional symptoms in patients with intermediate-2/high - risk mf.53 these approvals were based on the results of 2 phase iii randomised studies : comfort - i ( ruxolitinib vs placebo ) and comfort - ii ( ruxolitinib vs best available therapy [ bat]).54,55 the primary endpoint of both studies was a > 35% reduction in spleen volume after 24 ( comfort - i ) or 48 weeks of treatment ( comfort - ii ) , which was reached by , respectively , 41.7% and 28.5% of the patients treated with ruxolitinib , as against , respectively , 0.7% and 0% of the patients receiving placebo or bat ( p < .0001).54,55 overall , more than 90% of the patients enrolled in both studies experienced some reduction in spleen volume at some time during the follow - up , and the reduction remained stable in most of the patients after a median follow - up of 3 ( comfort - i ) and 5 years ( comfort - ii).56,57 the therapeutic success of ruxolitinib is not limited to reducing spleen volume because , unlike the drugs previously used to treat mf , it is efficacious in relieving constitutional symptoms ; reducing abdominal discomfort , appetite loss , itching , fatigue , and night sweats ; and improving the qol of most treated patients . as the drug s activity is independent of jak2 mutational status and not specific for the neoplastic clone , the response rate is similar in patients with and without the jak2v617f mutation because of its anti - jak1mediated effect .", "the uk , open - label , phase ii robust study evaluated its safety and efficacy in patients with mf , including those at intermediate-1 risk .", "the treatment was successful in 50% of the population as a whole and 57% of the intermediate-1risk patients .", "reduction in spleen length and symptoms was observed in all of the risk groups , and improvements in the myelofibrosis symptom assessment form total symptom score were seen in 80% of intermediate-1 , 72.7% of intermediate-2 , and 72.2% of high - risk patients.58 similarly , the phase iiib expanded - access jak inhibitor ruxolitinib in myelofibrosis patients ( jump ) trial for patients with mf without access to ruxolitinib outside of a clinical study found that the drug s safety and efficacy profile in intermediate-1risk patients was consistent with that in the study population as a whole and with that previously reported in intermediate-2risk and high - risk patients.59 the toxicity of ruxolitinib treatment is mainly haematologic due to the drug s interference with an essential pathway for haematopoiesis , as demonstrated in the comfort studies ; in both trials , thrombocytopenia was the dose - limiting toxicity , and anaemia was the most common haematologic ae .", "a number of studies have shown that ruxolitinib affects many cytokines and interferes with the immune process necessary for the pathogenesis of mpns , but it also affects the function of various immune cells and may therefore favour an increased incidence of opportunistic and nonopportunistic infections.60,61 for example , ruxolitinib impairs natural killer cell differentiation and function and inhibits dendritic cell activation and migration , and antigen - specific t - cell responses in a dose - dependent manner in vitro and in vivo .", "however , despite warnings about this increased risk,6264 a recent update of the jump study described a low incidence of infections : the all - grade infections observed in 1% of patients included nasopharyngitis ( 6.3% ) , urinary tract infection ( 6% ) , pneumonia ( 5.3% ) , bronchitis ( 4.2% ) , herpes zoster ( 3.6% ) , influenza ( 3% ) , upper respiratory tract infection ( 2.9% ) , cystitis ( 2.5% ) , gastroenteritis ( 1.8% ) , respiratory tract infection ( 1.8% ) , and oral herpes ( 1.6% ) .", "other infections included tuberculosis in 3 patients ( 0.3% ) and legionella pneumonia in 1 patient ( 0.1% ) .", "no hepatitis b reactivation was reported , and only 6 patients ( 0.5% ) discontinued treatment because of grade 3 pneumonia.59 the patients receiving ruxolitinib in the comfort - ii study experienced higher rates of viral and bacterial infection than those receiving conventional therapy , but most of the infections were grade 1 or 2 and did not lead to any dose reductions or the discontinuation of trial medication .", "furthermore , the rates of infection tended to decrease with longer exposure to the drug .", "however , as patients with mf are already predisposed to infections65 and the long - term risks of ruxolitinib treatment are still unknown , treated patients should be carefully monitored , and prophylaxis for herpes zoster or other infections should be considered on a case - by - case basis , depending on local risk .", "given its promising results , a further indication for ruxolitinib treatment is as a therapeutic bridge to allo - hsct .", "furthermore , an increasing number of reports appeared in the literature , describing the morphologic changes in the bone marrow occurring in ruxolitinib - treated patients , mostly focusing on modifications in bone marrow fibrosis degree.6670 by the beginning of 2014 , a number of other jak2 inhibitors were being tested : fedratinib,71 pacritinib,72 ly2784544,73 and momelotinib.74 however , the clinical trials of fedratinib and pacritinib were soon discontinued because of safety problems : wernicke encephalopathy ( fedratinib ) and bleeding ( pacritinib ) . momelotinib ( formerly known as cyt387 ) is a small - molecule , adenosine triphosphate ", "its kinase profiling indicates that it has good selectivity over other jak family kinases ( jak3 , tyk2 ) and excellent selectivity over other tyrosine and serine / threonine kinases.75 the preclinical data provide a rationale for the use of momelotinib in bcr - abl1negative mpns , and a multicentre phase i / ii trial involving 166 patients with intermediate / high - risk mf showed that the drug is well tolerated at oral doses of 150 or 300 mg once daily or 250 mg twice daily and led to improvements in splenomegaly , constitutional symptoms , and transfusion requirement.74 of particular interest are the transfusion independence responses , which were observed in more than half of the rbc transfusion  dependent subjects with a maximum transfusion - free period exceeding 2 years .", "in addition , the percentage of all subjects requiring rbc transfusions substantially decreased over the treatment period .", "more precisely , the overall anaemia response rate was 54% in transfusion - dependent patients with a median time to a confirmed anaemia response of 12 weeks ( range : 84293 days ) . as has been previously reported , treatment with momelotinib led to a rapid and sustained reduction in splenomegaly in approximately 31% of all cases , with a median time to response of 15 days , and the constitutional symptoms of most of the patients disappeared within 6 months . in terms of safety , about 20% of the patients experienced a first - dose effect ( dizziness , flushing , and hypotension ) that was self - limited .", "grade 3/4 haematologic and nonhaematologic aes were infrequent with the exception of thrombocytopenia , which occurred in approximately 17% of patients .", "grade 3/4 nonhaematologic laboratory aes included hyperlipasaemia ( 4% ) and increased liver enzymes ( grade 3 and 4 increase in aspartate aminotransferase in , respectively , 1% and < 1% of the patients ; a grade 3 increase in alanine aminotransferase in 2% ) . mainly , grade 1 treatment  related sensory peripheral neuropathy was reported , but there were no treatment - related deaths . in brief , momelotinib seems to lead to a significant and lasting improvement in anaemia , splenomegaly , and constitutional symptoms at doses of 150 or 300 mg / day or 150 mg twice daily .", "the efficacy and aes of momelotinib will be further evaluated in 2 currently ongoing phase iii trials : a randomised bat - controlled study of mf patients with anaemia and thrombocytopenia previously treated with ruxolitinib and a randomised study comparing momelotinib and ruxolitinib in patients with mf ( nct02101268-nct01969838 ) .", "more recently , the efficacy and safety of 3 dose levels of a potent and selective oral jak1 inhibitor , incb039110 , have been evaluated in an open - label phase ii study , resulting in clinically meaningful symptom relief , modest spleen volume reduction , and limited myelosuppression.76 in particular , only 1 patient discontinued for grade 3 thrombocytopenia , whereas nonhaematologic aes were largely of grade 1 or 2 and most commonly represented by fatigue ."], ["nearly 40% of patients with mf are anaemic at the time of diagnosis , including 25% who are already transfusion dependent,77,78 and more than 60% will develop clinically significant anaemia during the course of follow - up.49 the clinical impact of io and its potential relationship to the heightened inflammatory response of patients with mf warrant consideration not only because potential liver dysfunction , cardiac disease , and other complications of io probably contribute to patient morbidity and mortality but also because the growing evidence of impaired haematopoiesis attributable to bone marrow haemosiderosis suggests a viable therapeutic target.79,80 each unit of rbc contains 200 to 250 mg of iron , and as the reticuloendothelial system can clear approximately 10 to 15 g ( corresponding to 50 rbc units ) , any excess is deposited in tissues and leads to organ damage.81 iron overload is a concern when treating patients with mf , which is why iron chelation therapy ( ict ) has been used to counteract its potentially negative effects .", "however , it has to be admitted that there is a lack of prospective , randomised , controlled trials of the use of ict in patients with mf . one small retrospective study of 10 patients with mf demonstrated an erythroid response in 40% of cases receiving oral ict with deferasirox ( dfx ) , thus allowing these patients to reduce their transfusion requirement ; it also revealed a trend towards better overall survival in the responding patients.82 other data coming from a number of reported case studies79,80,83,84 also indicate that ict improves anaemia and decreases transfusion dependence in patients with mf . finally , a recent retrospective , multicentre analysis of 28 patients with mf and io secondary to transfusion dependence found that 11 patients ( 42.3% ) achieved a stable and consistent reduction in ferritin levels ( < 1000 ng / ml ) , and 6 of 26 patients ( 23% ) showed a persistent ( > 3 months ) increase in hb levels to > 1.5 g / dl , with the disappearance of transfusion dependence in 4 cases .", "however , comparison of the baseline characteristics of the patients who achieved an erythroid response and those who did not achieve did not reveal any significant differences that could be considered predictive.85 deferoxamine ( desferal ) is a linear ligand that forms 1:1 complexes with iron that maintain a net charge , allow for membrane permeability , and provide access to intracellular iron stores86 that are then excreted primarily in urine.87,88 it is administered in the form of an intravenous or subcutaneous infusion , and because of its short plasma half - life , the efficacy of the treatment correlates with the duration of infusion , and it is only effective if administered at high doses between 5 and 7 times per week.89 when administered as a continuous 24-hour infusion for 6 to 7 days per week in patients with high - risk -thalassemia , it can reverse iron - induced cardiac dysfunction and increase long - term survival.90 however , treatment - related side effects include infusion site discomfort ( nearly 100% , with the development of local erythema or induration in some cases),91 visual changes ( 0%10% ) , generally transient auditory neurotoxicity ( 20%25%),92,93 increased serum creatinine levels ( 22% ) , vomiting ( 16% ) , abdominal discomfort ( 14% ) , constipation ( 14% ) , arthralgia ( 14% ) , nausea ( 11% ) , rash ( 5% ) , and diarrhoea ( 5%).94 deferasirox is an oral iron chelator frequently used in clinical practice in the united states and europe that has a long half - life of 8 to 16 hours and can be administered once daily.89 it forms a 2:1 complex with iron,95 which is then excreted largely in the bile and faeces ( much less in urine).96,97 unlike other iron chelators , it is thought that dfx also affects haematopoietic stem cell differentiation by means of a reactive oxygen species  mediated mechanism , which may underlie the erythropoeitic response seen in some dfx - treated patients.95 the most frequently reported adverse effects are gastrointestinal toxicity ( 21%64% ) , diarrhoea ( 46% ) , abdominal pain ( 15%28% ) , nausea ( 24% ) , vomiting ( 21% ) , and constipation ( 10%).94,98101 patients have also been reported to experience renal dysfunction ( 10%64% , usually nonprogressive at the start of treatment and improving after a dose reduction ) , skin rash ( 4%39% ) , arthralgia ( 15%),94 and transaminitis ( 4%70%),81,98101 and there have been rarer reports of auditory neurotoxicity ( 1%6% ) as a potential side effect.100,101 it is not entirely clear whether ict can reverse the ill effects of io , and there are no completed studies that provide prospective evidence of a beneficial impact in terms of the restoration of normal haematopoiesis or outcomes in patients with mf .", "consequently , treatment decisions concerning the use of ict in patients with mf continue to be extrapolated from the data of myelodysplastic syndromes ."], ["allogeneic haematopoietic stem cell transplantation is still the only intervention that has been shown to be a potential cure for mf or a means of prolonging the survival of these patients .", "data from the most recent studies suggest that the expected 3-year progression - free survival rate is in the range of 40% to 50%.102 the adoption of reduced intensity conditioning regimens has recently made allo - hsct applicable to a larger proportion of patients.103 however , decisions concerning allo - hsct are based on inductive reasoning and require a considerable professional experience .", "key questions include patient selection , donor selection , pre- and posttransplant management , conditioning regimen , and prevention and management of post - transplant relapses .", "international prognostic scoring systems ( ie , ipss , dipss , and dipss - plus)20,22,23 are the most comprehensive means of risk stratification currently available to guide therapeutic decision making , although the influence of driver mutations and the acquisition of additional mutations during the natural course of the disease may further refine this process .", "all patients with mf aged < 70 years with ipss , dipss , or dipss - plus intermediate-2risk or high - risk disease and a reasonable performance status , and without any significant competing comorbid conditions , should be considered potential candidates for allo - hsct .", "patients aged < 65 years with intermediate-1risk disease should only be considered candidates if they present with refractory , transfusion - dependent anaemia or > 2% of peripheral blood blasts , or adverse cytogenetics ( as defined by the dipss - plus classification ) . finally , patients with low - risk disease should not undergo allo - hsct.104 individual transplant - specific prognostic factors should be considered in every candidate for allo - hsct to be able to make individualised decisions . in this context , the transplant - specific high - risk factors include a spleen extending more than 22 cm below the costal margin , having been transfused with more than 20 rbc units , having received a transplantation from an hla nonidentical donor , a poor performance status ( an eastern cooperative oncology group status of > 2 ) , a high comorbidity index ( a haematopoietic cell transplantation comorbidity index score of > 3 ) , and the presence of portal hypertension .", "completely matched rather than mismatched donors should be selected because , as reported in the european blood and marrow transplantation registry , the cumulative incidence of nonrelapsed mortality after 1 year is , respectively , 12% and 38% and is not different between hla - identical siblings and 10/10 matched unrelated donors ( 10% vs 13%).105 however , haploidentical related donors are an attractive alternative source of haematopoietic stem cells.106 it is important to note that peripheral blood is considered the most appropriate source of haematopoietic stem cells in the case of hla - matched sibling and unrelated donors . when splenectomy is performed before allo - hsct , it may facilitate disease eradication .", "some reports have also shown faster engraftment in splenectomised patients ; however , the pretransplant use of splenectomy remains controversial as no study has yet prospectively evaluated the effect of protocol - based splenectomy before transplantation . in the case of older patients and/or those with comorbidities ,", "a less intense conditioning regimen is more appropriate , whereas patients with advanced disease and a good performance status should undergo a more intensified regimen.104 finally , in patients relapsing with constitutional symptoms or splenomegaly , jak1/2 inhibitor treatment is recommended but remains experimental . to address this question ,", "ruxolitinib is being administered to eligible patients with mf for 60 days before definitive allo - hsct in a prospective multicentre phase ii study conducted by the myeloproliferative disorders research consortium ( nct01790295 ) ."], ["traditional mf treatments are primarily palliative and have proved to be inadequate to address the considerable morbidity and mortality associated with this disabling disease .", "more specifically , concerning anaemia , there have been various therapeutic attempts , but rbc transfusions still remain the most frequently used approach , even though io represents an increasingly frequent clinical challenge . considering instead splenomegaly , besides hu , ruxolitinib , as well as other investigational jak1/2 inhibitors , offers new hope for these patients as they have been shown to lead not only to significant reduction in splenomegaly but also to the palliation of disease - related symptoms . however , allo - hsct is still the only intervention that has evidence indicating it is potentially curative .", "obviously , in such a context , participation into a clinical trial should be encouraged whenever possible , with the purpose of making new drugs available ."]]}
{"article_id": "PMC3784683", "article_text": ["a 45-year - old iranian woman was referred to the private clinic in the city of hamadan for implant consultation .", "her past medical history was not notable , and there was no evidence of systemic disease .", "she had no history of trauma to the mandible . in the extra - oral examination ,", "intra - oral examination revealed normal oral mucosa , the absence of soft tissue expansion , and teeth of a normal color .", "all of the teeth were asymptomatic , with no pain or tenderness on percussion or palpation .", "the involved teeth were vital in an electric stimulation test . for assessment before implant insertion , cbct had been ordered . during evaluation of the implant insertion areas on promax3d cbct ( planmeca oy , helsinki , finland ) , a radiolucent - radiopaque mixed lesion located on the apices of the lower incisors was observed . on the axial , sagittal , and coronal", "cbct images , the extension of the lesion was observed from the mesial side of the right mandibular lateral incisor to the distal side of the left mandibular lateral incisor .", "it was a multifocal lesion in which solitary lesions were reached together and made a larger lesion .", "the total dimension of the lesion was about 16.6 mm in the mesiodistal direction and 6.9 mm in the longest superior - inferior direction . on the panoramic reconstructed cbct image ,", "the lesion associated with the left mandibular lateral incisor was radiolucent , whereas the lesion on the apex of the left central incisor was mixed radiolucent - radiopaque and the lesion associated with the right central incisor was radiopaque with a radiolucent rim around the lesion of this tooth ( fig .", "the state of the lesion relative to the buccal and lingual cortical plates could be assessed , which might not be possible on the conventional radiographs . on the axial image ,", "one of them was located at the mesial side of the right canine and the other between the left central and lateral incisors ( fig .", "cross - sectional images , the discontinuity of the lingual cortex was found at the area between the two central incisors on several consecutive sectional images ( fig .", "this cortical discontinuity was more obvious on the three - dimensional ( 3d ) cbct images ( fig .", "however , it should be considered that the cortical bone could be seen to have destruction on 3d images , even though it would have been thin without discontinuity on the cross - sectional images . on the digital periapical radiograph that was taken for", "periodontal ligament space widening was found , especially around the root of the left lateral incisor .", "based on the patient clinical and radiographic findings , a diagnosis of multifocal periapical cemento - osseous dysplasia was made .", "pcod is a specific lesion within this group of conditions that usually occurs in middle - aged black women.4 a systematic review of the literature on cod indicated an ethnic distribution of 59% , 37% , and 3% for blacks , asians ( japanese , chinese , and korean ) , and caucasians including indian cases , respectively , in case reports.8 zegarelli et al reported the incidence of pcod in the general population to be 2 - 3/1000.9 periapical cemento - osseous dysplasia generally does not cause cortical bone expansion or perforation.9 - 12 alsufyani and lam reviewed the clinical and radiographic characteristics of 118 patients with cod.13 they showed that 71.6% of 118 patients had no cortical expansion , 76% had intact lamina dura , and 93% had a normal periodontal ligament space .", "modern advances in imaging help the dentist to use the cbct with the appropriate field of view and spatial resolution ; therefore , the internal mineralized structure of the pathologic lesions can be investigated with a low radiation dose.14 axial cbct images clearly demonstrate the location and extension of the lesion .", "the expansion and perforation of the cortical plates can be evaluated on cbct even if they are slight .", "this report showed the discontinuity of the lingual cortex on some axial and cross - sectional cbct images .", "this characteristic of the lesion was an unusual feature among the cases of pcod reported up to now that may be due to its large size and needed to be checked in further regular follow - ups .", "this feature also could be found in an exaggerated fashion on the 3d cbct images .", "although 3d imaging could help the radiologist in diagnosing the lesion for the first look , it should be noted that it is not a reliable viewing modality for assigning the real status of the lesion in relation to its surrounding bone structures . in many cod cases that have been misdiagnosed and/or mismanaged ,", "the lesions were identified in their early stages as a periapical rarefying osteitis such as periapical abscess , granuloma , or cyst , and unnecessary endodontic treatment was performed . therefore , vitality tests are especially important for differential diagnosis . in the radiolucent - radiopaque mixed stage and the radiopaque stage , the differential diagnosis might include chronic sclerosing osteomyelitis , cementoossifying fibroma , odontoma , cementoblastoma , and osteoblastoma.13 periapical cemento - osseous dysplasia is usually detected on routine radiological examinations . however , the differential diagnosis of our case could be calcifying cystic odontogenic tumor and cemento - ossifying fibroma . by using cbct discrimination of pcod from these lesions that exhibit similar internal calcification on conventional radiography", "high density mass in pcod which is centered in low density area is different from findings of calcifying cystic odontogenic tumor in which calcification is observed at or near the cyst wall.15 also , cemento - ossifying fibroma has more obvious concentric buccolingual expansion on multiplanar cbct image . unless pcod is symptomatic ,", "treatment is usually not needed because development and maturation of the lesion is self - limiting .", "intervention may cause secondary infection of the cementum - like radiopacities , which may in turn induce osteomyelitis in these lesions.13 however , if pcod demonstrates unusual changes or becomes symptomatic , surgical intervention would then be needed.7 this case was considered as no treatment with only periodic follow - up check ."], "abstract_text": ["<S> periapical cemento - osseous dysplasia ( pcod ) is a subtype of cemento - osseous dysplasia that usually occurs in middle - aged black women . </S>", "<S> this report described a case of a 45-year - old iranian woman who was diagnosed with pcod on the basis of cone beam computed tomographic ( cbct ) findings . </S>", "<S> cbct enabled detailed visualization of the bone changes . </S>", "<S> this report described the special radiographic characteristics of pcod , including discontinuity of the lingual cortex on the cbct sectional and three - dimensional images . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 45-year - old iranian woman was referred to the private clinic in the city of hamadan for implant consultation .", "her past medical history was not notable , and there was no evidence of systemic disease .", "she had no history of trauma to the mandible . in the extra - oral examination ,", "intra - oral examination revealed normal oral mucosa , the absence of soft tissue expansion , and teeth of a normal color .", "all of the teeth were asymptomatic , with no pain or tenderness on percussion or palpation .", "the involved teeth were vital in an electric stimulation test . for assessment before implant insertion , cbct had been ordered . during evaluation of the implant insertion areas on promax3d cbct ( planmeca oy , helsinki , finland ) , a radiolucent - radiopaque mixed lesion located on the apices of the lower incisors was observed . on the axial , sagittal , and coronal", "cbct images , the extension of the lesion was observed from the mesial side of the right mandibular lateral incisor to the distal side of the left mandibular lateral incisor .", "it was a multifocal lesion in which solitary lesions were reached together and made a larger lesion .", "the total dimension of the lesion was about 16.6 mm in the mesiodistal direction and 6.9 mm in the longest superior - inferior direction . on the panoramic reconstructed cbct image ,", "the lesion associated with the left mandibular lateral incisor was radiolucent , whereas the lesion on the apex of the left central incisor was mixed radiolucent - radiopaque and the lesion associated with the right central incisor was radiopaque with a radiolucent rim around the lesion of this tooth ( fig .", "the state of the lesion relative to the buccal and lingual cortical plates could be assessed , which might not be possible on the conventional radiographs . on the axial image ,", "one of them was located at the mesial side of the right canine and the other between the left central and lateral incisors ( fig .", "cross - sectional images , the discontinuity of the lingual cortex was found at the area between the two central incisors on several consecutive sectional images ( fig .", "this cortical discontinuity was more obvious on the three - dimensional ( 3d ) cbct images ( fig .", "however , it should be considered that the cortical bone could be seen to have destruction on 3d images , even though it would have been thin without discontinuity on the cross - sectional images . on the digital periapical radiograph that was taken for", "periodontal ligament space widening was found , especially around the root of the left lateral incisor .", "based on the patient clinical and radiographic findings , a diagnosis of multifocal periapical cemento - osseous dysplasia was made ."], ["pcod is a specific lesion within this group of conditions that usually occurs in middle - aged black women.4 a systematic review of the literature on cod indicated an ethnic distribution of 59% , 37% , and 3% for blacks , asians ( japanese , chinese , and korean ) , and caucasians including indian cases , respectively , in case reports.8 zegarelli et al reported the incidence of pcod in the general population to be 2 - 3/1000.9 periapical cemento - osseous dysplasia generally does not cause cortical bone expansion or perforation.9 - 12 alsufyani and lam reviewed the clinical and radiographic characteristics of 118 patients with cod.13 they showed that 71.6% of 118 patients had no cortical expansion , 76% had intact lamina dura , and 93% had a normal periodontal ligament space .", "modern advances in imaging help the dentist to use the cbct with the appropriate field of view and spatial resolution ; therefore , the internal mineralized structure of the pathologic lesions can be investigated with a low radiation dose.14 axial cbct images clearly demonstrate the location and extension of the lesion .", "the expansion and perforation of the cortical plates can be evaluated on cbct even if they are slight .", "this report showed the discontinuity of the lingual cortex on some axial and cross - sectional cbct images .", "this characteristic of the lesion was an unusual feature among the cases of pcod reported up to now that may be due to its large size and needed to be checked in further regular follow - ups .", "this feature also could be found in an exaggerated fashion on the 3d cbct images .", "although 3d imaging could help the radiologist in diagnosing the lesion for the first look , it should be noted that it is not a reliable viewing modality for assigning the real status of the lesion in relation to its surrounding bone structures . in many cod cases that have been misdiagnosed and/or mismanaged ,", "the lesions were identified in their early stages as a periapical rarefying osteitis such as periapical abscess , granuloma , or cyst , and unnecessary endodontic treatment was performed . therefore , vitality tests are especially important for differential diagnosis . in the radiolucent - radiopaque mixed stage and the radiopaque stage , the differential diagnosis might include chronic sclerosing osteomyelitis , cementoossifying fibroma , odontoma , cementoblastoma , and osteoblastoma.13 periapical cemento - osseous dysplasia is usually detected on routine radiological examinations . however , the differential diagnosis of our case could be calcifying cystic odontogenic tumor and cemento - ossifying fibroma . by using cbct discrimination of pcod from these lesions that exhibit similar internal calcification on conventional radiography", "high density mass in pcod which is centered in low density area is different from findings of calcifying cystic odontogenic tumor in which calcification is observed at or near the cyst wall.15 also , cemento - ossifying fibroma has more obvious concentric buccolingual expansion on multiplanar cbct image . unless pcod is symptomatic ,", "treatment is usually not needed because development and maturation of the lesion is self - limiting .", "intervention may cause secondary infection of the cementum - like radiopacities , which may in turn induce osteomyelitis in these lesions.13 however , if pcod demonstrates unusual changes or becomes symptomatic , surgical intervention would then be needed.7 this case was considered as no treatment with only periodic follow - up check ."]]}
{"article_id": "PMC4245860", "article_text": ["controversy exists as to whether endodontic procedures \n are the primary cause for loss of strength .", "panitvisai and messer ( 1995 ) reported that cuspal deflection \n increased with increasing the extension of cavity preparations and was greatest when \n endodontic access was incorporated into a preparation .", "it has been reported that \n endodontically treated teeth and their contralateral vital pairs exhibited similar \n biomechanical properties , such as punch shear strength , toughness , and load required for \n fracture .", "although the insertion of a post does not strengthen or reinforce endodontically treated \n teeth , it is basically used to provide sufficient retention of \n the core material which in turn is used to retain a fixed restoration . for the inserted post to perform its \n function , several variables must be put into consideration such as post length , \n diameter , geometric design , and surface configuration . additionally , special attention should be given to the \n material the post is made of in order to ensure adequate distribution of the absorbed \n stresses and to prevent root fracture during .", "regarding with consideration to the well - known success of \n metallic posts there are now many concerns regarding the associated inhomogeneous stress \n distribution , biological side effects due to microleakage and corrosion , and the \n influence of their dark color under an all - ceramic restorations .", "the introduction of non - metallic fiber reinforced composite ( frc ) posts helped improving \n stress distribution because their elastic modulus is similar to that of dentin as \n indicated by several clinical and laboratory studies .", "frc post systems showed more frequent favorable failure \n modes than did metal post systems .", "although fiber posts proved effective \n in withstanding compressive loads in posterior teeth , they behave differently in anterior teeth where non - axial biting \n forces prevail and where their flexural behavior becomes more effective .", "recent studies suggested that glass - fiber posts contributed to the reinforcement \n and strengthening of endodontically treated teeth under full coverage crowns .", "a ferrule or encircling band of cast metal around the remaining coronal surface can \n provide protective reinforcement to endodontically treated teeth by encapsulation of the \n remaining coronal structure and by resisting functional lever forces during mastication . \n", "a minimum 1 to 2 mm of ferrule height is necessary to achieve such protective \n effect .", "recent clinical \n studies reported that the ferrule structure have a direct influence on the clinical \n success rate of endodontically treated premolars restored with fiber posts and the \n failure events were due mainly to post debonding .", "an in \n vitro study reported that incomplete crown ferrule was associated with \n greater variation in load capacity after chewing simulation , while other studies found no effect of different \n ferrule heights in teeth restored with fiber posts and resin cores .", "the aim of this study was to evaluate the fracture resistance and failure pattern of \n endodontically treated mandibular premolars restored with different ferrule heights in \n combination with fiber posts and all - ceramic crowns .", "the null hypothesis was that \n different ferrule heights do not improve the fracture resistance or the failure pattern \n of the tested restorations .", "all \n external debris were removed with an ultrasonic scaler , and examined stereoscopically \n at 10x magnification to verify the absence of cracks , defects and dental caries . \n", "teeth were stored in a 0.5% chloramine t ( prolabo , paris , france ) in saline solution . \n", "buccolingual and mesiodistal coronal dimensions plus root length of all selected \n teeth were measured using a digital caliper ( digimatic calipers , mitsutoyo , tokyo , \n japan ) and only teeth with the following mean dimensions were selected : 14.1 mm root \n length , 7.3 mm buccolingual width , and 4.9 mm mesiodistal width .", "standardized root canal preparations were made using the following procedure : initial \n probing using no .", "10 k - files ( flexo files , maillefer , ballaigues , switzerland ) ; the \n root canal length was established through direct observation of the file extruding \n from the apical foramen .", "the specimens were then prepared endodontically with a \n stepback procedure with size 45 ( flex r file ; union broach , york pa ) .", "after \n intermittent rinsing with 2.5% sodium hypochlorite solution , the endodontic treatment \n was completed using manual lateral condensation method ( ah plus , dentsply , de tray , \n konstanz , germany ) .", "after endodontic treatment , each root was thinly covered with a silicone impression \n material ( aquasil , dentsply ) to simulate thickness of periodontal ligament .", "all specimens were embedded in \n self - polymerized acrylic resin ( orthoresin , dentsply , degudent gmbh , postfach 1364 , \n d-63403 hanau , germany ) poured into a mold while maintaining 2 mm below the cervical \n line exposed .", "the teeth were embedded along their long axis using a surveyor ( ney \n surveyor ; dentsply ) .", "a new diamond \n point ( lot - nr 1599 , dfs dental and technical products , gmbh , germany ) was attached to \n the milling machine ( k9 milling apparatus-990 , kavo , germany ) for every group .", "the \n mrd gauged diamond had a self - limiting tip , which produced a 1-mm - deep chamfer and \n the margins and the angle of convergence were standardized .", "after preparation of the finish line , the coronal dentinal extension was modified \n accordingly ( figure 1 ) : group 1 ( control ) : 1 mm \n circumferential ferrule from the gingival margin without a fiber post ; group 2 : 1 mm \n circumferential ferrule with fiber post and resin core ; group 3 : non - uniform ferrule \n height ( 2 mm buccally and 1 mm lingually ) with fiber post and resin core ; group 4 : a \n non - uniform ferrule height ( 3 mm buccally and 2 mm lingually ) with fiber and resin \n core post ; group 5 : received no ferrule preparation with fiber post and resin \n core .", "schematic representation of the different tested groups in the control group , excess gutta - percha was removed to a depth of 2 mm from the \n coronal surface of the preparation , using a carbide bur ( 171l-012 , brasseler , usa ) . \n", "the coronal walls were etched with 36% phosphoric acid ( total etch , ivoclar - vivadent , \n schaan , liechtenstein ) for 15 s , washed with water spray and then gently air - dried . \n", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied using a microbrush \n and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) .", "a hybrid composite \n resin ( tetric ceram , ivoclar - vivadent ) was applied and light polymerized for 40 \n s. for the remaining groups , post space was created using no .", "1 peeso reamer ( union \n broach co. , long island , ny , usa ) and corresponding calibrating drill ( frc postec \n plus , # 1 , ivoclar - vivadent ) leaving 4 mm of apical gutta - percha intact .", "a translucent \n glass fiber reinforced composite post ( frc postec plus # 1 ivoclar - vivadent , schaan , \n liechtenstein ) was used .", "each post was cut to a suitable length with a diamond bur so \n that it was covered with at least 2 mm of resin composite occlusally .", "post cementation was carried out with self - polymerized resin cement ( multilink , \n ivoclar vivadent ) following manufacturer s instructions .", "silane coupling agent \n ( monobond - s , ivoclar - vivadent ) was applied on the post surface for 60 s and then \n air - dried . after post cementation , the surrounding dentin surface was etched with 36% \n phosphoric acid ( total etch , ivoclar - vivadent ) for 15 s , washed with water spray and \n gently air - dried .", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied \n using a microbrush and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) . \n", "then core build - up was performed using hybrid composite resin ( tetric ceram , \n ivoclar - vivadent ) in 1-mm - thick increments and light polymerized ( astralis 10 ; \n ivocar - vivadent ) for 40 s until the core was restored to predetermined dimensions . \n", "the final layer was placed using a transparent matrix to allow for shape consistency \n between specimens .", "the dimensions of the prepared cores were confirmed with a \n measuring microscope with 30x magnification lens with precision of 5 um .", "a single - phase impression was made using polyvinylsiloxane impression material \n ( virtual , ivoclar - vivadent ) and master dies were fabricated with type 4 die stone \n ( jad stone , whip mix , louisville , kentucky , usa ) . a press ceramic ( e max , a3 , \n ivoclar - vivadent ) was selected to fabricate all - ceramic crowns of the restored \n specimens .", "crown dimensions were standardized by using a mold for the external shape \n of each specimen .", "the fitting surface of the crowns was pretreated with hydrofluoric \n acid ( ips ceramic etching gel , ivoclar - vivadent ) for 20 s , rinsed off , air dried , \n silanized for 60 s and air dried .", "dentine primer liquids were mixed and applied on \n the whole prepared tooth surface for 15 s. resin cement was dispensed from the \n automix syringe directly into the inner surface of the crowns which were seated and \n held in position under fixed load of 20 n ; excess resin was removed immediately with \n a micro brush . exposed margins were covered with glycerin gel and rinsed off after \n complete polymerization of the resin cement . the specimens were stored in distilled \n water at 37c for 7 days prior to testing .", "all specimens were subjected to cyclic loading according to the following regime : \n sinusoidal load between 50 and 200 n at a rate of 2 hertz .", "all specimens received \n 15,000 cycles and surface damage was prevented by insertion of a 0.5 mm silicon sheet \n between the occlusal surface of the ceramic crown and the loading indenter ( 3 mm \n diameter ) of the pneumatically activated loading machine .", "after completion of cyclic \n loading , a universal testing machine ( instron 8500 plus , instron , 100 royal st . \n", "canton , ma , usa ) was used to deliver a compressive load to the specimens at a \n crosshead speed of 1 mm / min at 45 degree angle to the long axis of the teeth ( root \n apex tilted lingually ) until failure .", "load - time curves were recorded using a \n universal testing machine s computer software .", "the \n failure load of the specimen was determined when the forceversus - time graph showed an \n abrupt change in load , indicating a sudden decrease in the specimen s resistance to \n compressive loading .", "after loading , the failure mode recorded for each specimen and classified as either \n favorable facture above the cement - enamel junction ( repairable ) or catastrophic \n fracture of the root below cement - enamel junction ( non repairable ) .", "these inspections \n were made using a stereomicroscope ( stereoscopic zoom microscope , smz-1000 , nikon , \n japan ) and during inspection , the teeth were trans - illuminated with a fiber optic \n cable .", "complete or partial debonding of the crown or of the post and core were also \n considered as favorable failure modes .", "data were analyzed statistically by one - way \n anova and tukey s post - hoc test was used for pair - wise comparisons \n ( =0.05 ) .", "all \n external debris were removed with an ultrasonic scaler , and examined stereoscopically \n at 10x magnification to verify the absence of cracks , defects and dental caries . \n", "teeth were stored in a 0.5% chloramine t ( prolabo , paris , france ) in saline solution . \n", "buccolingual and mesiodistal coronal dimensions plus root length of all selected \n teeth were measured using a digital caliper ( digimatic calipers , mitsutoyo , tokyo , \n japan ) and only teeth with the following mean dimensions were selected : 14.1 mm root \n length , 7.3 mm buccolingual width , and 4.9 mm mesiodistal width .", "standardized root canal preparations were made using the following procedure : initial \n probing using no .", "10 k - files ( flexo files , maillefer , ballaigues , switzerland ) ; the \n root canal length was established through direct observation of the file extruding \n from the apical foramen .", "the specimens were then prepared endodontically with a \n stepback procedure with size 45 ( flex r file ; union broach , york pa ) .", "after \n intermittent rinsing with 2.5% sodium hypochlorite solution , the endodontic treatment \n was completed using manual lateral condensation method ( ah plus , dentsply , de tray , \n konstanz , germany ) .", "after endodontic treatment , each root was thinly covered with a silicone impression \n material ( aquasil , dentsply ) to simulate thickness of periodontal ligament .", "all specimens were embedded in \n self - polymerized acrylic resin ( orthoresin , dentsply , degudent gmbh , postfach 1364 , \n d-63403 hanau , germany ) poured into a mold while maintaining 2 mm below the cervical \n line exposed .", "the teeth were embedded along their long axis using a surveyor ( ney \n surveyor ; dentsply ) .", "a new diamond \n point ( lot - nr 1599 , dfs dental and technical products , gmbh , germany ) was attached to \n the milling machine ( k9 milling apparatus-990 , kavo , germany ) for every group .", "the \n mrd gauged diamond had a self - limiting tip , which produced a 1-mm - deep chamfer and \n the margins and the angle of convergence were standardized .", "after preparation of the finish line , the coronal dentinal extension was modified \n accordingly ( figure 1 ) : group 1 ( control ) : 1 mm \n circumferential ferrule from the gingival margin without a fiber post ; group 2 : 1 mm \n circumferential ferrule with fiber post and resin core ; group 3 : non - uniform ferrule \n height ( 2 mm buccally and 1 mm lingually ) with fiber post and resin core ; group 4 : a \n non - uniform ferrule height ( 3 mm buccally and 2 mm lingually ) with fiber and resin \n core post ; group 5 : received no ferrule preparation with fiber post and resin \n core .", "in the control group , excess gutta - percha was removed to a depth of 2 mm from the \n coronal surface of the preparation , using a carbide bur ( 171l-012 , brasseler , usa ) . \n", "the coronal walls were etched with 36% phosphoric acid ( total etch , ivoclar - vivadent , \n schaan , liechtenstein ) for 15 s , washed with water spray and then gently air - dried . \n", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied using a microbrush \n and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) .", "a hybrid composite \n resin ( tetric ceram , ivoclar - vivadent ) was applied and light polymerized for 40 \n s. for the remaining groups , post space was created using no .", "1 peeso reamer ( union \n broach co. , long island , ny , usa ) and corresponding calibrating drill ( frc postec \n plus , # 1 , ivoclar - vivadent ) leaving 4 mm of apical gutta - percha intact . a translucent \n glass fiber reinforced composite post ( frc postec plus # 1 ivoclar - vivadent , schaan , \n liechtenstein ) was used .", "each post was cut to a suitable length with a diamond bur so \n that it was covered with at least 2 mm of resin composite occlusally .", "post cementation was carried out with self - polymerized resin cement ( multilink , \n ivoclar vivadent ) following manufacturer s instructions .", "silane coupling agent \n ( monobond - s , ivoclar - vivadent ) was applied on the post surface for 60 s and then \n air - dried . after post cementation , the surrounding dentin surface was etched with 36% \n phosphoric acid ( total etch , ivoclar - vivadent ) for 15 s , washed with water spray and \n gently air - dried .", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied \n using a microbrush and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) . \n", "then core build - up was performed using hybrid composite resin ( tetric ceram , \n ivoclar - vivadent ) in 1-mm - thick increments and light polymerized ( astralis 10 ; \n ivocar - vivadent ) for 40 s until the core was restored to predetermined dimensions . \n", "the final layer was placed using a transparent matrix to allow for shape consistency \n between specimens .", "the dimensions of the prepared cores were confirmed with a \n measuring microscope with 30x magnification lens with precision of 5 um .", "a single - phase impression was made using polyvinylsiloxane impression material \n ( virtual , ivoclar - vivadent ) and master dies were fabricated with type 4 die stone \n ( jad stone , whip mix , louisville , kentucky , usa ) . a press ceramic ( e max , a3 , \n ivoclar - vivadent ) was selected to fabricate all - ceramic crowns of the restored \n specimens .", "crown dimensions were standardized by using a mold for the external shape \n of each specimen .", "the fitting surface of the crowns was pretreated with hydrofluoric \n acid ( ips ceramic etching gel , ivoclar - vivadent ) for 20 s , rinsed off , air dried , \n silanized for 60 s and air dried .", "dentine primer liquids were mixed and applied on \n the whole prepared tooth surface for 15 s. resin cement was dispensed from the \n automix syringe directly into the inner surface of the crowns which were seated and \n held in position under fixed load of 20 n ; excess resin was removed immediately with \n a micro brush . exposed margins were covered with glycerin gel and rinsed off after \n complete polymerization of the resin cement .", "the specimens were stored in distilled \n water at 37c for 7 days prior to testing .", "all specimens were subjected to cyclic loading according to the following regime : \n sinusoidal load between 50 and 200 n at a rate of 2 hertz .", "all specimens received \n 15,000 cycles and surface damage was prevented by insertion of a 0.5 mm silicon sheet \n between the occlusal surface of the ceramic crown and the loading indenter ( 3 mm \n diameter ) of the pneumatically activated loading machine .", "after completion of cyclic \n loading , a universal testing machine ( instron 8500 plus , instron , 100 royal st . \n", "canton , ma , usa ) was used to deliver a compressive load to the specimens at a \n crosshead speed of 1 mm / min at 45 degree angle to the long axis of the teeth ( root \n apex tilted lingually ) until failure .", "load - time curves were recorded using a \n universal testing machine s computer software .", "the \n failure load of the specimen was determined when the forceversus - time graph showed an \n abrupt change in load , indicating a sudden decrease in the specimen s resistance to \n compressive loading .", "after loading , the failure mode recorded for each specimen and classified as either \n favorable facture above the cement - enamel junction ( repairable ) or catastrophic \n fracture of the root below cement - enamel junction ( non repairable ) .", "these inspections \n were made using a stereomicroscope ( stereoscopic zoom microscope , smz-1000 , nikon , \n japan ) and during inspection , the teeth were trans - illuminated with a fiber optic \n cable . complete or partial debonding of the crown or of the post and core were also \n considered as favorable failure modes .", "data were analyzed statistically by one - way \n anova and tukey s post - hoc test was used for pair - wise comparisons \n ( =0.05 ) .", "statistical analysis revealed no significant differences between the failure load of the \n tested groups and the control group restored without fiber post \n ( p<0.780 ) .", "although fracture resistance of the control group was \n comparable to that of the other groups that received fiber post , there was a higher \n catastrophic failure rate ( 70% ) in the control specimens in the form of vertical root \n fracture .", "the groups restored with fiber post , on the other hand , had almost complete \n favorable fracture in the form of cervical fracture above the cervical line , previous \n data are summarized in table 1 . failure load ( standard deviation ) and failure type of tested groups similar superscript letter indicates no significant difference between \n different test groups in all specimens , the cemented ceramic crown was fractured without evidence of debonding \n of either the crown or the cemented post .", "the internal surface of the fractured ceramic \n fragments demonstrated evidence of resin cement and part of the core material indicating \n cohesive fracture of the resin core .", "considering the results obtained in this study , different ferrule heights did not \n improve the fracture resistance or the failure pattern of the tested specimens . on the \n other hand , the use of fiber posts with a modulus of elasticity close to that of dentin \n changed the catastrophic failure type of the control group to almost complete favorable \n fracture for the other four groups .", "the \n dynamic cyclic loading program was intended to quickly screen any possible weakness in \n the cemented restorations .", "providing occlusal protection prevented generation of cone \n cracks in the brittle ceramic crowns which was the reason why all specimens survived \n without failure .", "longer periods of cyclic loading are required to shed light on the \n long - term performance of these restorations , but investigating this issue was not within \n the scope of this study .", "the results of the fracture resistance test showed that the amount of residual coronal \n structure ( ferrule height ) did not increase significantly the fracture resistance of \n endodontically treated teeth .", "these results are in agreement with those of previous \n studies ; while contrary \n results were reported by other authors .", "this could be \n explained by the fact that fiber posts transmitted the forces and distributed the \n loading stresses over a bigger surface area of the tooth structure similarly for all \n tested groups .", "these results may also be interpreted as the resin \n bonded fiber posts with resin composite core exerted a reinforcing effect by supporting \n the remaining tooth structure regardless of the ferrule design .", "these \n non - metallic post systems have gained widespread popularity in recent years because of \n other advantages such as their superior esthetics , ease of retrievability , and simple \n application technique , which allow the clinician to complete the procedure in a single \n short appointment . according to the results of the present study , specimens in the control group ( no fiber \n post ) revealed higher catastrophic failure rate compared to the other groups , which \n indicates that insertion of a fiber post may enhance the clinical performance of \n endodontically treated teeth even if the failure load remains relatively not \n effected .", "the presence of a uniform 1-mm - thick coronal structure \n ( group 2 ) resulted in the highest fracture resistance value , especially when compared to \n the control group , which is in agreement with the results of previous studies .", "a point of clinical relevance is that the fracture resistance of specimens with \n non - uniform coronal structure ( group 3 and 4 ) was lower than the specimens without a \n ferrule ( group 5 ) , with consideration to maintaining as much as possible of sound tooth \n structure .", "it could be advised to adjust the coronal structure evenly to provide a flat \n seat for the core build up material , which could improve the fracture resistance of \n endodontically , treated teeth .", "lithia disilicate - based all - ceramic crowns were used in this study because of its high \n flexural strength ( 400 mpa ) , easily etched using hydrofluoric acid , readily silanized \n and bonded with any suitable resin cement .", "it has also been reported that the mean \n chewing force of adults ranges between 7 to 15 kg , and the maximum biting force could \n reach up to 90 kg .", "as the fracture \n loads in all groups in the present study were found to be greater than the ordinary \n chewing force , and even greater than the maximum biting force , their mechanical strength \n could be considered satisfactory from a clinical point of view . in terms of the failure modes ,", "the obtained results are in accordance with those of \n previous studies , showing that the application of fiber posts resulted in \n more favorable fracture patternswhile \n the specimens restored without posts showed a higher incidence of catastrophic failure . \n", "it could be postulated that specimens restored with fiber - reinforced post systems \n offered more homogenous stress distribution due to their modulus of elasticity close to \n that of dentin resulting in a better stress distribution that occurs at the post - dentin \n interface .", "this could explain why all favorable fractures were \n limited to the cervical portion of the root including the core - dentin interface , since \n the stresses were concentrated in the cervical area and the outer root surface . \n supporting this opinion is the cohesive fracture of the resin core material , which \n remained attached to the fractured segments of ceramic crowns , thus indicating good bond \n strength between the core build up material and the cemented ceramic crown . the limited \n number of specimens and the difficulty related to reproducing the complexity of \n functional loads in the oral environment may be some of the shortcomings of the present \n study . further investigations including finite element analysis and long - term cyclic \n loading studies are recommended to complement the present study .", "within the limitations of this study , it may be concluded that increasing the ferrule \n length did not improve the fracture resistance of endodontically treated teeth restored \n with glass ceramic crowns .", "insertion of a fiber post could reduce the percentage of \n catastrophic failure of these restorations under functional loads ."], "abstract_text": ["<S> objectivethis study investigated the effect of different ferrule heights on endodontically \n treated premolars.materials and methodsfifty sound mandibular first premolars were endodontically treated and then \n restored with 7-mm fiber post ( frc postec plus # 1 ivoclar - vivadent ) luted with \n self - polymerized resin cement ( multilink , ivoclar vivadent ) while the coronal \n section was restored with hybrid composite core build - up material ( tetric ceram , \n ivoclar - vivadent ) , which received all - ceramic crown . </S>", "<S> different ferrule heights \n were investigated : 1-mm circumferential ferrule without post and core ( group 1 \n used as control ) , a circumferential 1-mm ferrule ( group 2 ) , non - uniform ferrule \n 2-mm buccally and 1-mm lingually ( group 3 ) , non - uniform ferrule 3-mm buccally and \n 2-mm lingually ( group 4 ) , and finally no ferrule preparation ( group 5 ) . </S>", "<S> the \n fracture load and failure pattern of the tested groups were investigated by \n applying axial load to the ceramic crowns ( n=10 ) . </S>", "<S> data were analyzed statistically \n by one - way anova and tukey s post - hoc test was used for pair - wise \n comparisons ( =0.05).resultsthere were no significant differences among the failure load of all tested groups \n ( p<0.780 ) . </S>", "<S> the control group had the lowest fracture resistance \n ( 891.43202.22 n ) and the highest catastrophic failure rate \n ( p<0.05 ) . compared to the control group , the use of fiber post reduced the \n percentage of catastrophic failure while increasing the ferrule height did not \n influence the fracture resistance of the restored specimens.conclusionswithin the limitations of this study , increasing the ferrule length did not \n influence the fracture resistance of endodontically treated teeth restored with \n glass ceramic crowns . </S>", "<S> insertion of a fiber post could reduce the percentage of \n catastrophic failure of these restorations under function . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "Preparation of specimens", "Full crown preparation", "Post-space preparation", "Fracture resistance", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["endodontically treated teeth often loose substantial tooth structure from previous \n caries , pre - existing restorations , and/or endodontic treatment , which reduce tooth bulk and thickness of healthy dentin \n resulting in increased chances of fracture under function .", "controversy exists as to whether endodontic procedures \n are the primary cause for loss of strength .", "panitvisai and messer ( 1995 ) reported that cuspal deflection \n increased with increasing the extension of cavity preparations and was greatest when \n endodontic access was incorporated into a preparation .", "it has been reported that \n endodontically treated teeth and their contralateral vital pairs exhibited similar \n biomechanical properties , such as punch shear strength , toughness , and load required for \n fracture .", "although the insertion of a post does not strengthen or reinforce endodontically treated \n teeth , it is basically used to provide sufficient retention of \n the core material which in turn is used to retain a fixed restoration . for the inserted post to perform its \n function , several variables must be put into consideration such as post length , \n diameter , geometric design , and surface configuration . additionally , special attention should be given to the \n material the post is made of in order to ensure adequate distribution of the absorbed \n stresses and to prevent root fracture during .", "regarding with consideration to the well - known success of \n metallic posts there are now many concerns regarding the associated inhomogeneous stress \n distribution , biological side effects due to microleakage and corrosion , and the \n influence of their dark color under an all - ceramic restorations .", "the introduction of non - metallic fiber reinforced composite ( frc ) posts helped improving \n stress distribution because their elastic modulus is similar to that of dentin as \n indicated by several clinical and laboratory studies .", "frc post systems showed more frequent favorable failure \n modes than did metal post systems .", "although fiber posts proved effective \n in withstanding compressive loads in posterior teeth , they behave differently in anterior teeth where non - axial biting \n forces prevail and where their flexural behavior becomes more effective .", "recent studies suggested that glass - fiber posts contributed to the reinforcement \n and strengthening of endodontically treated teeth under full coverage crowns .", "a ferrule or encircling band of cast metal around the remaining coronal surface can \n provide protective reinforcement to endodontically treated teeth by encapsulation of the \n remaining coronal structure and by resisting functional lever forces during mastication . \n", "a minimum 1 to 2 mm of ferrule height is necessary to achieve such protective \n effect .", "recent clinical \n studies reported that the ferrule structure have a direct influence on the clinical \n success rate of endodontically treated premolars restored with fiber posts and the \n failure events were due mainly to post debonding .", "an in \n vitro study reported that incomplete crown ferrule was associated with \n greater variation in load capacity after chewing simulation , while other studies found no effect of different \n ferrule heights in teeth restored with fiber posts and resin cores .", "the aim of this study was to evaluate the fracture resistance and failure pattern of \n endodontically treated mandibular premolars restored with different ferrule heights in \n combination with fiber posts and all - ceramic crowns .", "the null hypothesis was that \n different ferrule heights do not improve the fracture resistance or the failure pattern \n of the tested restorations ."], ["all \n external debris were removed with an ultrasonic scaler , and examined stereoscopically \n at 10x magnification to verify the absence of cracks , defects and dental caries . \n", "teeth were stored in a 0.5% chloramine t ( prolabo , paris , france ) in saline solution . \n", "buccolingual and mesiodistal coronal dimensions plus root length of all selected \n teeth were measured using a digital caliper ( digimatic calipers , mitsutoyo , tokyo , \n japan ) and only teeth with the following mean dimensions were selected : 14.1 mm root \n length , 7.3 mm buccolingual width , and 4.9 mm mesiodistal width .", "standardized root canal preparations were made using the following procedure : initial \n probing using no .", "10 k - files ( flexo files , maillefer , ballaigues , switzerland ) ; the \n root canal length was established through direct observation of the file extruding \n from the apical foramen .", "the specimens were then prepared endodontically with a \n stepback procedure with size 45 ( flex r file ; union broach , york pa ) .", "after \n intermittent rinsing with 2.5% sodium hypochlorite solution , the endodontic treatment \n was completed using manual lateral condensation method ( ah plus , dentsply , de tray , \n konstanz , germany ) .", "after endodontic treatment , each root was thinly covered with a silicone impression \n material ( aquasil , dentsply ) to simulate thickness of periodontal ligament .", "all specimens were embedded in \n self - polymerized acrylic resin ( orthoresin , dentsply , degudent gmbh , postfach 1364 , \n d-63403 hanau , germany ) poured into a mold while maintaining 2 mm below the cervical \n line exposed .", "the teeth were embedded along their long axis using a surveyor ( ney \n surveyor ; dentsply ) .", "a new diamond \n point ( lot - nr 1599 , dfs dental and technical products , gmbh , germany ) was attached to \n the milling machine ( k9 milling apparatus-990 , kavo , germany ) for every group .", "the \n mrd gauged diamond had a self - limiting tip , which produced a 1-mm - deep chamfer and \n the margins and the angle of convergence were standardized .", "after preparation of the finish line , the coronal dentinal extension was modified \n accordingly ( figure 1 ) : group 1 ( control ) : 1 mm \n circumferential ferrule from the gingival margin without a fiber post ; group 2 : 1 mm \n circumferential ferrule with fiber post and resin core ; group 3 : non - uniform ferrule \n height ( 2 mm buccally and 1 mm lingually ) with fiber post and resin core ; group 4 : a \n non - uniform ferrule height ( 3 mm buccally and 2 mm lingually ) with fiber and resin \n core post ; group 5 : received no ferrule preparation with fiber post and resin \n core .", "schematic representation of the different tested groups in the control group , excess gutta - percha was removed to a depth of 2 mm from the \n coronal surface of the preparation , using a carbide bur ( 171l-012 , brasseler , usa ) . \n", "the coronal walls were etched with 36% phosphoric acid ( total etch , ivoclar - vivadent , \n schaan , liechtenstein ) for 15 s , washed with water spray and then gently air - dried . \n", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied using a microbrush \n and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) .", "a hybrid composite \n resin ( tetric ceram , ivoclar - vivadent ) was applied and light polymerized for 40 \n s. for the remaining groups , post space was created using no .", "1 peeso reamer ( union \n broach co. , long island , ny , usa ) and corresponding calibrating drill ( frc postec \n plus , # 1 , ivoclar - vivadent ) leaving 4 mm of apical gutta - percha intact .", "a translucent \n glass fiber reinforced composite post ( frc postec plus # 1 ivoclar - vivadent , schaan , \n liechtenstein ) was used .", "each post was cut to a suitable length with a diamond bur so \n that it was covered with at least 2 mm of resin composite occlusally .", "post cementation was carried out with self - polymerized resin cement ( multilink , \n ivoclar vivadent ) following manufacturer s instructions .", "silane coupling agent \n ( monobond - s , ivoclar - vivadent ) was applied on the post surface for 60 s and then \n air - dried . after post cementation , the surrounding dentin surface was etched with 36% \n phosphoric acid ( total etch , ivoclar - vivadent ) for 15 s , washed with water spray and \n gently air - dried .", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied \n using a microbrush and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) . \n", "then core build - up was performed using hybrid composite resin ( tetric ceram , \n ivoclar - vivadent ) in 1-mm - thick increments and light polymerized ( astralis 10 ; \n ivocar - vivadent ) for 40 s until the core was restored to predetermined dimensions . \n", "the final layer was placed using a transparent matrix to allow for shape consistency \n between specimens .", "the dimensions of the prepared cores were confirmed with a \n measuring microscope with 30x magnification lens with precision of 5 um .", "a single - phase impression was made using polyvinylsiloxane impression material \n ( virtual , ivoclar - vivadent ) and master dies were fabricated with type 4 die stone \n ( jad stone , whip mix , louisville , kentucky , usa ) . a press ceramic ( e max , a3 , \n ivoclar - vivadent ) was selected to fabricate all - ceramic crowns of the restored \n specimens .", "crown dimensions were standardized by using a mold for the external shape \n of each specimen .", "the fitting surface of the crowns was pretreated with hydrofluoric \n acid ( ips ceramic etching gel , ivoclar - vivadent ) for 20 s , rinsed off , air dried , \n silanized for 60 s and air dried .", "dentine primer liquids were mixed and applied on \n the whole prepared tooth surface for 15 s. resin cement was dispensed from the \n automix syringe directly into the inner surface of the crowns which were seated and \n held in position under fixed load of 20 n ; excess resin was removed immediately with \n a micro brush . exposed margins were covered with glycerin gel and rinsed off after \n complete polymerization of the resin cement . the specimens were stored in distilled \n water at 37c for 7 days prior to testing .", "all specimens were subjected to cyclic loading according to the following regime : \n sinusoidal load between 50 and 200 n at a rate of 2 hertz .", "all specimens received \n 15,000 cycles and surface damage was prevented by insertion of a 0.5 mm silicon sheet \n between the occlusal surface of the ceramic crown and the loading indenter ( 3 mm \n diameter ) of the pneumatically activated loading machine .", "after completion of cyclic \n loading , a universal testing machine ( instron 8500 plus , instron , 100 royal st . \n", "canton , ma , usa ) was used to deliver a compressive load to the specimens at a \n crosshead speed of 1 mm / min at 45 degree angle to the long axis of the teeth ( root \n apex tilted lingually ) until failure .", "load - time curves were recorded using a \n universal testing machine s computer software .", "the \n failure load of the specimen was determined when the forceversus - time graph showed an \n abrupt change in load , indicating a sudden decrease in the specimen s resistance to \n compressive loading .", "after loading , the failure mode recorded for each specimen and classified as either \n favorable facture above the cement - enamel junction ( repairable ) or catastrophic \n fracture of the root below cement - enamel junction ( non repairable ) .", "these inspections \n were made using a stereomicroscope ( stereoscopic zoom microscope , smz-1000 , nikon , \n japan ) and during inspection , the teeth were trans - illuminated with a fiber optic \n cable .", "complete or partial debonding of the crown or of the post and core were also \n considered as favorable failure modes .", "data were analyzed statistically by one - way \n anova and tukey s post - hoc test was used for pair - wise comparisons \n ( =0.05 ) ."], ["all \n external debris were removed with an ultrasonic scaler , and examined stereoscopically \n at 10x magnification to verify the absence of cracks , defects and dental caries . \n", "teeth were stored in a 0.5% chloramine t ( prolabo , paris , france ) in saline solution . \n", "buccolingual and mesiodistal coronal dimensions plus root length of all selected \n teeth were measured using a digital caliper ( digimatic calipers , mitsutoyo , tokyo , \n japan ) and only teeth with the following mean dimensions were selected : 14.1 mm root \n length , 7.3 mm buccolingual width , and 4.9 mm mesiodistal width .", "standardized root canal preparations were made using the following procedure : initial \n probing using no .", "10 k - files ( flexo files , maillefer , ballaigues , switzerland ) ; the \n root canal length was established through direct observation of the file extruding \n from the apical foramen .", "the specimens were then prepared endodontically with a \n stepback procedure with size 45 ( flex r file ; union broach , york pa ) .", "after \n intermittent rinsing with 2.5% sodium hypochlorite solution , the endodontic treatment \n was completed using manual lateral condensation method ( ah plus , dentsply , de tray , \n konstanz , germany ) .", "after endodontic treatment , each root was thinly covered with a silicone impression \n material ( aquasil , dentsply ) to simulate thickness of periodontal ligament .", "all specimens were embedded in \n self - polymerized acrylic resin ( orthoresin , dentsply , degudent gmbh , postfach 1364 , \n d-63403 hanau , germany ) poured into a mold while maintaining 2 mm below the cervical \n line exposed .", "the teeth were embedded along their long axis using a surveyor ( ney \n surveyor ; dentsply ) ."], ["a new diamond \n point ( lot - nr 1599 , dfs dental and technical products , gmbh , germany ) was attached to \n the milling machine ( k9 milling apparatus-990 , kavo , germany ) for every group .", "the \n mrd gauged diamond had a self - limiting tip , which produced a 1-mm - deep chamfer and \n the margins and the angle of convergence were standardized .", "after preparation of the finish line , the coronal dentinal extension was modified \n accordingly ( figure 1 ) : group 1 ( control ) : 1 mm \n circumferential ferrule from the gingival margin without a fiber post ; group 2 : 1 mm \n circumferential ferrule with fiber post and resin core ; group 3 : non - uniform ferrule \n height ( 2 mm buccally and 1 mm lingually ) with fiber post and resin core ; group 4 : a \n non - uniform ferrule height ( 3 mm buccally and 2 mm lingually ) with fiber and resin \n core post ; group 5 : received no ferrule preparation with fiber post and resin \n core ."], ["in the control group , excess gutta - percha was removed to a depth of 2 mm from the \n coronal surface of the preparation , using a carbide bur ( 171l-012 , brasseler , usa ) . \n", "the coronal walls were etched with 36% phosphoric acid ( total etch , ivoclar - vivadent , \n schaan , liechtenstein ) for 15 s , washed with water spray and then gently air - dried . \n", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied using a microbrush \n and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) .", "a hybrid composite \n resin ( tetric ceram , ivoclar - vivadent ) was applied and light polymerized for 40 \n s. for the remaining groups , post space was created using no .", "1 peeso reamer ( union \n broach co. , long island , ny , usa ) and corresponding calibrating drill ( frc postec \n plus , # 1 , ivoclar - vivadent ) leaving 4 mm of apical gutta - percha intact . a translucent \n glass fiber reinforced composite post ( frc postec plus # 1 ivoclar - vivadent , schaan , \n liechtenstein ) was used .", "each post was cut to a suitable length with a diamond bur so \n that it was covered with at least 2 mm of resin composite occlusally .", "post cementation was carried out with self - polymerized resin cement ( multilink , \n ivoclar vivadent ) following manufacturer s instructions .", "silane coupling agent \n ( monobond - s , ivoclar - vivadent ) was applied on the post surface for 60 s and then \n air - dried . after post cementation , the surrounding dentin surface was etched with 36% \n phosphoric acid ( total etch , ivoclar - vivadent ) for 15 s , washed with water spray and \n gently air - dried .", "one coat of adhesive resin ( excite , ivoclar - vivadent ) was applied \n using a microbrush and light polymerized for 20 s ( astralis 10 , ivoclar - vivadent ) . \n", "then core build - up was performed using hybrid composite resin ( tetric ceram , \n ivoclar - vivadent ) in 1-mm - thick increments and light polymerized ( astralis 10 ; \n ivocar - vivadent ) for 40 s until the core was restored to predetermined dimensions . \n", "the final layer was placed using a transparent matrix to allow for shape consistency \n between specimens .", "the dimensions of the prepared cores were confirmed with a \n measuring microscope with 30x magnification lens with precision of 5 um .", "a single - phase impression was made using polyvinylsiloxane impression material \n ( virtual , ivoclar - vivadent ) and master dies were fabricated with type 4 die stone \n ( jad stone , whip mix , louisville , kentucky , usa ) . a press ceramic ( e max , a3 , \n ivoclar - vivadent ) was selected to fabricate all - ceramic crowns of the restored \n specimens .", "crown dimensions were standardized by using a mold for the external shape \n of each specimen .", "the fitting surface of the crowns was pretreated with hydrofluoric \n acid ( ips ceramic etching gel , ivoclar - vivadent ) for 20 s , rinsed off , air dried , \n silanized for 60 s and air dried .", "dentine primer liquids were mixed and applied on \n the whole prepared tooth surface for 15 s. resin cement was dispensed from the \n automix syringe directly into the inner surface of the crowns which were seated and \n held in position under fixed load of 20 n ; excess resin was removed immediately with \n a micro brush . exposed margins were covered with glycerin gel and rinsed off after \n complete polymerization of the resin cement .", "the specimens were stored in distilled \n water at 37c for 7 days prior to testing ."], ["all specimens were subjected to cyclic loading according to the following regime : \n sinusoidal load between 50 and 200 n at a rate of 2 hertz .", "all specimens received \n 15,000 cycles and surface damage was prevented by insertion of a 0.5 mm silicon sheet \n between the occlusal surface of the ceramic crown and the loading indenter ( 3 mm \n diameter ) of the pneumatically activated loading machine .", "after completion of cyclic \n loading , a universal testing machine ( instron 8500 plus , instron , 100 royal st . \n", "canton , ma , usa ) was used to deliver a compressive load to the specimens at a \n crosshead speed of 1 mm / min at 45 degree angle to the long axis of the teeth ( root \n apex tilted lingually ) until failure .", "load - time curves were recorded using a \n universal testing machine s computer software .", "the \n failure load of the specimen was determined when the forceversus - time graph showed an \n abrupt change in load , indicating a sudden decrease in the specimen s resistance to \n compressive loading .", "after loading , the failure mode recorded for each specimen and classified as either \n favorable facture above the cement - enamel junction ( repairable ) or catastrophic \n fracture of the root below cement - enamel junction ( non repairable ) .", "these inspections \n were made using a stereomicroscope ( stereoscopic zoom microscope , smz-1000 , nikon , \n japan ) and during inspection , the teeth were trans - illuminated with a fiber optic \n cable . complete or partial debonding of the crown or of the post and core were also \n considered as favorable failure modes .", "data were analyzed statistically by one - way \n anova and tukey s post - hoc test was used for pair - wise comparisons \n ( =0.05 ) ."], ["statistical analysis revealed no significant differences between the failure load of the \n tested groups and the control group restored without fiber post \n ( p<0.780 ) .", "although fracture resistance of the control group was \n comparable to that of the other groups that received fiber post , there was a higher \n catastrophic failure rate ( 70% ) in the control specimens in the form of vertical root \n fracture .", "the groups restored with fiber post , on the other hand , had almost complete \n favorable fracture in the form of cervical fracture above the cervical line , previous \n data are summarized in table 1 . failure load ( standard deviation ) and failure type of tested groups similar superscript letter indicates no significant difference between \n different test groups in all specimens , the cemented ceramic crown was fractured without evidence of debonding \n of either the crown or the cemented post .", "the internal surface of the fractured ceramic \n fragments demonstrated evidence of resin cement and part of the core material indicating \n cohesive fracture of the resin core ."], ["considering the results obtained in this study , different ferrule heights did not \n improve the fracture resistance or the failure pattern of the tested specimens . on the \n other hand , the use of fiber posts with a modulus of elasticity close to that of dentin \n changed the catastrophic failure type of the control group to almost complete favorable \n fracture for the other four groups .", "the \n dynamic cyclic loading program was intended to quickly screen any possible weakness in \n the cemented restorations .", "providing occlusal protection prevented generation of cone \n cracks in the brittle ceramic crowns which was the reason why all specimens survived \n without failure .", "longer periods of cyclic loading are required to shed light on the \n long - term performance of these restorations , but investigating this issue was not within \n the scope of this study .", "the results of the fracture resistance test showed that the amount of residual coronal \n structure ( ferrule height ) did not increase significantly the fracture resistance of \n endodontically treated teeth .", "these results are in agreement with those of previous \n studies ; while contrary \n results were reported by other authors .", "this could be \n explained by the fact that fiber posts transmitted the forces and distributed the \n loading stresses over a bigger surface area of the tooth structure similarly for all \n tested groups .", "these results may also be interpreted as the resin \n bonded fiber posts with resin composite core exerted a reinforcing effect by supporting \n the remaining tooth structure regardless of the ferrule design .", "these \n non - metallic post systems have gained widespread popularity in recent years because of \n other advantages such as their superior esthetics , ease of retrievability , and simple \n application technique , which allow the clinician to complete the procedure in a single \n short appointment . according to the results of the present study , specimens in the control group ( no fiber \n post ) revealed higher catastrophic failure rate compared to the other groups , which \n indicates that insertion of a fiber post may enhance the clinical performance of \n endodontically treated teeth even if the failure load remains relatively not \n effected .", "the presence of a uniform 1-mm - thick coronal structure \n ( group 2 ) resulted in the highest fracture resistance value , especially when compared to \n the control group , which is in agreement with the results of previous studies .", "a point of clinical relevance is that the fracture resistance of specimens with \n non - uniform coronal structure ( group 3 and 4 ) was lower than the specimens without a \n ferrule ( group 5 ) , with consideration to maintaining as much as possible of sound tooth \n structure .", "it could be advised to adjust the coronal structure evenly to provide a flat \n seat for the core build up material , which could improve the fracture resistance of \n endodontically , treated teeth .", "lithia disilicate - based all - ceramic crowns were used in this study because of its high \n flexural strength ( 400 mpa ) , easily etched using hydrofluoric acid , readily silanized \n and bonded with any suitable resin cement .", "it has also been reported that the mean \n chewing force of adults ranges between 7 to 15 kg , and the maximum biting force could \n reach up to 90 kg .", "as the fracture \n loads in all groups in the present study were found to be greater than the ordinary \n chewing force , and even greater than the maximum biting force , their mechanical strength \n could be considered satisfactory from a clinical point of view . in terms of the failure modes ,", "the obtained results are in accordance with those of \n previous studies , showing that the application of fiber posts resulted in \n more favorable fracture patternswhile \n the specimens restored without posts showed a higher incidence of catastrophic failure . \n", "it could be postulated that specimens restored with fiber - reinforced post systems \n offered more homogenous stress distribution due to their modulus of elasticity close to \n that of dentin resulting in a better stress distribution that occurs at the post - dentin \n interface .", "this could explain why all favorable fractures were \n limited to the cervical portion of the root including the core - dentin interface , since \n the stresses were concentrated in the cervical area and the outer root surface . \n supporting this opinion is the cohesive fracture of the resin core material , which \n remained attached to the fractured segments of ceramic crowns , thus indicating good bond \n strength between the core build up material and the cemented ceramic crown . the limited \n number of specimens and the difficulty related to reproducing the complexity of \n functional loads in the oral environment may be some of the shortcomings of the present \n study . further investigations including finite element analysis and long - term cyclic \n loading studies are recommended to complement the present study ."], ["within the limitations of this study , it may be concluded that increasing the ferrule \n length did not improve the fracture resistance of endodontically treated teeth restored \n with glass ceramic crowns .", "insertion of a fiber post could reduce the percentage of \n catastrophic failure of these restorations under functional loads ."]]}
{"article_id": "PMC4033860", "article_text": ["dichlorvos ( dimethyl-2 , 2-dichloro vinyl phosphate ) is an organophosphorous ( op ) insecticide with moderate human toxicity , used for insect control in food storage areas , greenhouses , barns and control of insects on livestock .", "parenteral dichlorvos administration with suicidal intention is rarely reported in the literature . in one case report", ", extensive muscle necrosis was reported following intramuscular injection of dichlorvos . in another case , following injection of dichlorvos subcutaneously in both wrists , a young patient developed compartment syndrome requiring fasciotomy . in both cases", ", i describe the case of a young female patient , who developed atypical signs of op poisoning following subcutaneous injection of dichlorvos .", "the present case report is about a 37-year - old , previously healthy female , who was admitted to our intensive care unit 18-h after alleged self - injection of 76% dichlorvos ( nuvos ) 3 ml in the upper left arm ( 3 times with a 1 ml insulin syringe ) . within an hour of the injections", ", she had normal body temperature with heart rate of 90/min , blood pressure 110/70 mmhg , respiratory rate 14/min and pulse oximeter showing 100% saturation on room air .", "no crackles were heard on chest auscultation . on the nervous system examination , she was jittery with slurred speech and normal cranial nerves .", "she had hypertonia , brisk tendon reflexes , bilateral sustained ankle clonus and bilateral extensor plantar reflex .", "local examination revealed a blister and a black necrotizing lesion over the anterior aspect of the left arm [ figure 1 ] .", "she was rapidly atropinized with incremental boluses of atropine ( total dose 12 mg ) in next 30 min ( targeting a dry mouth ) followed by infusion of 1.8 mg / h .", "pralidoxime infusion was started at 500 mg / h following a loading dose of 1000 mg over 20 min .", "intravenous clindamycin ( 600 mg q 8 h ) was administered empirically for presumed local wound infection .", "debridement of the wound showed predominant involvement of the subcutaneous tissue plane with minimal necrosis of the superficial fibers of biceps brachii .", "initial routine laboratory investigations were unremarkable except elevated total count ( 16200/mm ) and mild respiratory alkalosis .", "local skin necrosis with surrounding blisters her clinical course was complicated by respiratory arrest 9-h after hospital admission ( 27-h after the injection of dichlorovos ) leading to hypoxia and bradycardia .", "arterial blood gas analysis post resuscitation showed - ph 7.28 , po2 - 36 mmhg , pco2 - 54 mmhg , hco3 - 22 mmhg .", "serum choline esterase ( pseudo cholinesterase ) level of the same day showed a very low value of < 1 ku /", "she could be successfully weaned off from the ventilator on day 4 . on day 5 , while on atropine infusion she was found to be delirious .", "she was also noticed to have extrapyramidal manifestations including rigidity and left sided gaze preference .", "in this case , other than the unusual route of administration , a number of atypical manifestations were found .", "this includes relatively delayed respiratory failure and central nervous system ( cns ) manifestations with few muscarinic signs .", "two independent mechanisms are thought to be responsible for respiratory failure following op insecticide poisoning  cholinergic cns depression in the early stage ( associated with other cholinergic features ) and late progressive neuromuscular transmission defect .", "second mechanism is part of the syndrome described as  type ii paralysis  by wadia et al . or intermediate syndrome by senanayake and karalliedde . our patient developed respiratory arrest 27-h after exposure to dichlorovos .", "in her clinical course she never had neck or proximal muscle weakness , though the clinical findings were not correlated electrophysiologically .", "usually cns signs are manifested early in the course in the presence of peripheral muscarinic signs . in a large series published on neurological manifestations of op compounds , 8 of 200 patients had pyramidal tract signs in the form of only hyper - reflexia manifesting within 24 h of exposure .", "all these patients were unconscious with florid cholinergic manifestations , including constricted pupils . in their original series of intermediate syndrome senanayake and karalliedde , observed hyperreflexia in one of their patients ( with flexor plantar reflex ) and even she had typical muscarinic signs .", "extrapyramidal signs of op poisoning are often transient and may be missed in a sick patient . in a taiwanese series", "cns manifestations ( both pyramidal tract and extrapyramidal ) were delayed and were associated with relatively scanty peripheral muscarinic signs .", "possible explanation for these atypical manifestations in our patient could be route specific ( in this case subcutaneous ) effect of op compound ( dichlorovos ) causing isolated delayed cns depression , with few peripheral cholinergic signs .", "animal experiments have shown route specific ( subcutaneous or intravenous ) cardiovascular and neuromuscular effect of op compounds in rats ."], "abstract_text": ["<S> current case report describes a 37-year - old female patient who was admitted to the hospital following subcutaneous injection of dichlorvos with an insulin syringe . </S>", "<S> the only peripheral cholinergic sign observed on admission was excessive salivation with bilateral pyramidal tract signs . </S>", "<S> locally she had necrosis of skin and subcutaneous tissue with surrounding blisters . </S>", "<S> in the subsequent course of her illness , she developed respiratory arrest requiring ventilator support . </S>", "<S> she also had delayed extrapyramidal manifestations . </S>", "<S> relevant literature is reviewed . </S>", "<S> possibility of route - specific , delayed predominant central nervous system effect of dichlorvos postulated . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["dichlorvos ( dimethyl-2 , 2-dichloro vinyl phosphate ) is an organophosphorous ( op ) insecticide with moderate human toxicity , used for insect control in food storage areas , greenhouses , barns and control of insects on livestock .", "parenteral dichlorvos administration with suicidal intention is rarely reported in the literature . in one case report", ", extensive muscle necrosis was reported following intramuscular injection of dichlorvos . in another case , following injection of dichlorvos subcutaneously in both wrists , a young patient developed compartment syndrome requiring fasciotomy . in both cases", ", i describe the case of a young female patient , who developed atypical signs of op poisoning following subcutaneous injection of dichlorvos ."], ["the present case report is about a 37-year - old , previously healthy female , who was admitted to our intensive care unit 18-h after alleged self - injection of 76% dichlorvos ( nuvos ) 3 ml in the upper left arm ( 3 times with a 1 ml insulin syringe ) . within an hour of the injections", ", she had normal body temperature with heart rate of 90/min , blood pressure 110/70 mmhg , respiratory rate 14/min and pulse oximeter showing 100% saturation on room air .", "no crackles were heard on chest auscultation . on the nervous system examination , she was jittery with slurred speech and normal cranial nerves .", "she had hypertonia , brisk tendon reflexes , bilateral sustained ankle clonus and bilateral extensor plantar reflex .", "local examination revealed a blister and a black necrotizing lesion over the anterior aspect of the left arm [ figure 1 ] .", "she was rapidly atropinized with incremental boluses of atropine ( total dose 12 mg ) in next 30 min ( targeting a dry mouth ) followed by infusion of 1.8 mg / h .", "pralidoxime infusion was started at 500 mg / h following a loading dose of 1000 mg over 20 min .", "intravenous clindamycin ( 600 mg q 8 h ) was administered empirically for presumed local wound infection .", "debridement of the wound showed predominant involvement of the subcutaneous tissue plane with minimal necrosis of the superficial fibers of biceps brachii .", "initial routine laboratory investigations were unremarkable except elevated total count ( 16200/mm ) and mild respiratory alkalosis .", "local skin necrosis with surrounding blisters her clinical course was complicated by respiratory arrest 9-h after hospital admission ( 27-h after the injection of dichlorovos ) leading to hypoxia and bradycardia .", "arterial blood gas analysis post resuscitation showed - ph 7.28 , po2 - 36 mmhg , pco2 - 54 mmhg , hco3 - 22 mmhg .", "serum choline esterase ( pseudo cholinesterase ) level of the same day showed a very low value of < 1 ku /", "she could be successfully weaned off from the ventilator on day 4 . on day 5 , while on atropine infusion she was found to be delirious .", "she was also noticed to have extrapyramidal manifestations including rigidity and left sided gaze preference ."], ["in this case , other than the unusual route of administration , a number of atypical manifestations were found .", "this includes relatively delayed respiratory failure and central nervous system ( cns ) manifestations with few muscarinic signs .", "two independent mechanisms are thought to be responsible for respiratory failure following op insecticide poisoning  cholinergic cns depression in the early stage ( associated with other cholinergic features ) and late progressive neuromuscular transmission defect .", "second mechanism is part of the syndrome described as  type ii paralysis  by wadia et al . or intermediate syndrome by senanayake and karalliedde . our patient developed respiratory arrest 27-h after exposure to dichlorovos .", "in her clinical course she never had neck or proximal muscle weakness , though the clinical findings were not correlated electrophysiologically .", "usually cns signs are manifested early in the course in the presence of peripheral muscarinic signs . in a large series published on neurological manifestations of op compounds , 8 of 200 patients had pyramidal tract signs in the form of only hyper - reflexia manifesting within 24 h of exposure .", "all these patients were unconscious with florid cholinergic manifestations , including constricted pupils . in their original series of intermediate syndrome senanayake and karalliedde , observed hyperreflexia in one of their patients ( with flexor plantar reflex ) and even she had typical muscarinic signs .", "extrapyramidal signs of op poisoning are often transient and may be missed in a sick patient . in a taiwanese series", "cns manifestations ( both pyramidal tract and extrapyramidal ) were delayed and were associated with relatively scanty peripheral muscarinic signs .", "possible explanation for these atypical manifestations in our patient could be route specific ( in this case subcutaneous ) effect of op compound ( dichlorovos ) causing isolated delayed cns depression , with few peripheral cholinergic signs .", "animal experiments have shown route specific ( subcutaneous or intravenous ) cardiovascular and neuromuscular effect of op compounds in rats ."]]}
{"article_id": "PMC4595706", "article_text": ["transesophageal echocardiography ( tee ) is considered to be safe and relatively noninvasive diagnostic tool,1 ) however severe complications have been reported .", "the incidence of major tee - related complications range from 0.2% to 0.5% and mortality is reported to be < 0.01%.2345 ) among these complications , problems related to insertion of tee probe take a large proportion , including perforation of hypopharynx and esophagus . for our knowledge , there have been reported only few cases of hypopharyngeal perforation , especially not been reported in asian population to date .", "we describe the clinical presentations and secondary complications associated with oropharyngeal injury with the case of a 74-year - old man who suffered a hypopharyngeal laceration after tee .", "a 74-year - old man was referred to our cardiovascular center for the evaluation and treatment of mitral regurgitation ( mr ) .", "he had been complaining of aggravation of dyspnea ( new york heart association functional class iv ) for the preceding 3 weeks , and atrial fibrillation and severe mr were detected from other clinic .", "the patient had medical history of hypertension and chronic obstructive pulmonary disease for 15 years , and received endovascular aneurysm repair for abdominal aortic aneurysm 8 years ago .", "the patient was admitted to the intensive care unit because of uncompensated heart failure and careful control of pulmonary edema with chronic kidney disease .", "the patient presented tachypnea and orthopnea before the tee procedure , less than minimal dose of sedative agent was administrated to lessen patient 's discomfort , 1 mg of lorazepam , 25 mg of fentanyl , intravenously . as we checked for the mental status ,", "the insertion of tee probe was performed with the patient in left lateral decubitus position according to the following standardized technique : the probe was inserted through the midline and gently advanced to pass the first pharyngeal curvature corresponding to the base of the tongue .", "the probe was then extended , and the patient was asked to swallow , at which point the probe was further advanced to enter the esophageal inlet . when the probe has reached to the root of tongue ,", "the patient suddenly changed his position form left decubitus to supine position and gave force to the neck , and resisted to probe insertion .", "the probe got lodged at right - side of hypopharyngeal area so that failed to advance . at second attempt ,", "tee demonstrated severe eccentric mr with medial commissural prolapse due to chordae rupture ( a3-p3 commissure ) with left ventricular dilatation , mild tricuspid regurgitation ( fig .", "the physical examination revealed tenderness and crepitus on right anterolateral area of neck ( level iii ) 3 hours later after tee .", "iatrogenic hypopharyngeal or esophageal injury was suspected , antibiotic treatment with piperacillin / tazobactam was initiated and the patient was not allowed to eat or drink .", "computed tomography ( ct ) scan revealed subcutaneous emphysema without involvement of mediastinum , but the level of injury was not detected ( fig .", "fiberoptic nasolaryngoscopic examination identified edema of the right posterolateral wall of the hypopharynx and hypopharyngeal bruise but there was no evidence of rupture or perforation .", "laboratory analysis revealed an elevation of the white blood cell count ( 20.36  10/l ) and c - reactive protein ( crp ; 28.54 mg / dl ) .", "we tried to find any evidence of esophageal injury because treatment strategy would be different if the esophagus was involved .", "an ent specialist and a gi specialist agreed with hypopharyngeal injury without esophageal damage after multiple tests .", "we concluded the lesion was limited to the hypopharynx because the presence of hematoma at the right side of hypopharynx , consistent with the direction and the depth of the probe passage .", "further , there was no evidence of esophageal injury on serial follow - up multimodality imaging studies .", "after 5 days from the injury , the subcutaneous emphysema disappeared and the patient remained afebrile with improvement of the leukocyte count and crp ( fig .", "however painful neck mass around anterolateral area of neck was noticed ( level iii ) .", "the follow - up ct without contrast showed a right parapharyngeal and retropharyngeal abscess secondary to hypopharyngeal injury ( fig .", "yellowish fluid in the abscess was analyzed for cytology and revealed as acute inflammatory cells predominantly neutrophils . as the patient 's systemic status was stable with decrease of the leukocyte count and crp , the antibiotics ( piperacillin / tazobactam )", "was maintained for 14 days with the drainage of abscess because no organisms were identified from the abscess fluid culture and repeated blood cultures .", "on 7th day after tee , second swallowing study was performed and no leakage was demonstrated ( fig .", "4b ) , infectious parameters were continuously decreased , and follow - up fiberoptic nasolaryngoscopic examination demonstrated no evidence of perforation or rupture in hypopharyngeal cavity .", "after 14 days of antibiotic therapy , when the infection was controlled completely , the patient had successful mitral valve repair with tricuspid annuloplasty with st .", "the overall incidence of severe hypopharyngeal and esophageal complications from traditional tee is low.1)3)6 ) risk factors of hypopharyngeal and upper gastrointestinal complications have been reported in few studies .", "patient related risk factors include older age , short stature , presence of cervical spinal disease , chronic steroid use , presence of congestive heart failure.3)7)8 ) the presence of gastroesophageal pathology such as zenker 's diverticulum , esophageal stricture or obstructing mass , fibrosis secondary to prior chest radiation could be potential risk factor . anatomical variation like massive cardiomegaly1)9 ) or tracheoesophageal fistula may be correlated to increase the risk .", "procedure related risk factors include inexperience of the cardiologist , and resistance to prove insertion with poor cooperation . to avoid direct injury to the hypopharynx and upper esophagus during a blind insertion of probe , appropriate conscious sedation and local anesthetic spray", "direct visualization of the passage of the probe with direct laryngoscope could be recommended to minimize blind injury of throat .", "if there are procedural difficulties such as multiple attempts at passage of the tee probe or resistance during insertion , the examination should be stopped .", "in addition , indirect injury is also result from increased continuous pressure on the probe positioned in the upper esophagus , therefore , the exam should be stopped if the resistance is continuing .", "finally , it is very important to prevent sudden position change of the patient during the probe insertion .", "it needs pre - procedural enough education , adequate sedation , careful insertion of tee probe , and an experienced assistant . in our case ,", "we assumed that the patient 's poor cooperation and insufficient sedation might cause the wrong direction of the tee probe and advancing force of tee probe into the throat had led the probe to get lodged into the right pyriform sinus .", "treatment of hypopharyngeal perforation depends on the cause , timing of diagnosis , and type of lesion.101112 ) in a study of hinojar et al.,10 ) 3 patients out of 7 patients were managed conservatively , whereas other 4 patients got surgical treatment . mostly in the former cases , the time duration to the diagnosis was less than 1 - 2 days and involvement of infection", "was limited to pharyngeal space . in current case , our patient complained pain in his throat and we detected subcutaneous emphysema within 3 hours after injury that may prevent aggravation of infection and lead good prognosis .", "it is important not only to understand possible complications of the procedure and to recognize the risk factors of pharyngeal and esophageal injury , but also to detect the complication as soon as possible if it occurred , to get better prognosis ."], "abstract_text": ["<S> transesophageal echocardiography ( tee ) is considered relatively safe but semi - invasive . </S>", "<S> the hypopharyngeal and esophageal injury is infrequent complication of tee but could be serious , even life - threatening . </S>", "<S> we present a case of a 74-year - old man who experienced a deep neck infection secondary to hypopharyngeal injury following tee . </S>", "<S> the diagnosis was made because of the subcutaneous emphysema developed 3 hours after tee . in spite of antibiotics therapy with prolonged fasting , a right parapharyngeal and retropharyngeal abscess </S>", "<S> was developed 5 days later . with ultrasound - guided drainage of abscess and continuous antibiotic treatment , infection was controlled . </S>", "<S> the patent underwent mitral valve repair after 14 days of antibiotic therapy . </S>", "<S> the patient recovered uneventfully . for cardiologists performing tee , </S>", "<S> it is required to know complications and their risk factors to minimize hypopharyngeal and esophageal injury . </S>"], "labels": null, "section_names": ["Introduction", "Case", "Discussion"], "sections": [["transesophageal echocardiography ( tee ) is considered to be safe and relatively noninvasive diagnostic tool,1 ) however severe complications have been reported .", "the incidence of major tee - related complications range from 0.2% to 0.5% and mortality is reported to be < 0.01%.2345 ) among these complications , problems related to insertion of tee probe take a large proportion , including perforation of hypopharynx and esophagus . for our knowledge , there have been reported only few cases of hypopharyngeal perforation , especially not been reported in asian population to date .", "we describe the clinical presentations and secondary complications associated with oropharyngeal injury with the case of a 74-year - old man who suffered a hypopharyngeal laceration after tee ."], ["a 74-year - old man was referred to our cardiovascular center for the evaluation and treatment of mitral regurgitation ( mr ) .", "he had been complaining of aggravation of dyspnea ( new york heart association functional class iv ) for the preceding 3 weeks , and atrial fibrillation and severe mr were detected from other clinic .", "the patient had medical history of hypertension and chronic obstructive pulmonary disease for 15 years , and received endovascular aneurysm repair for abdominal aortic aneurysm 8 years ago .", "the patient was admitted to the intensive care unit because of uncompensated heart failure and careful control of pulmonary edema with chronic kidney disease .", "the patient presented tachypnea and orthopnea before the tee procedure , less than minimal dose of sedative agent was administrated to lessen patient 's discomfort , 1 mg of lorazepam , 25 mg of fentanyl , intravenously . as we checked for the mental status ,", "the insertion of tee probe was performed with the patient in left lateral decubitus position according to the following standardized technique : the probe was inserted through the midline and gently advanced to pass the first pharyngeal curvature corresponding to the base of the tongue .", "the probe was then extended , and the patient was asked to swallow , at which point the probe was further advanced to enter the esophageal inlet . when the probe has reached to the root of tongue ,", "the patient suddenly changed his position form left decubitus to supine position and gave force to the neck , and resisted to probe insertion .", "the probe got lodged at right - side of hypopharyngeal area so that failed to advance . at second attempt ,", "tee demonstrated severe eccentric mr with medial commissural prolapse due to chordae rupture ( a3-p3 commissure ) with left ventricular dilatation , mild tricuspid regurgitation ( fig .", "the physical examination revealed tenderness and crepitus on right anterolateral area of neck ( level iii ) 3 hours later after tee .", "iatrogenic hypopharyngeal or esophageal injury was suspected , antibiotic treatment with piperacillin / tazobactam was initiated and the patient was not allowed to eat or drink .", "computed tomography ( ct ) scan revealed subcutaneous emphysema without involvement of mediastinum , but the level of injury was not detected ( fig .", "fiberoptic nasolaryngoscopic examination identified edema of the right posterolateral wall of the hypopharynx and hypopharyngeal bruise but there was no evidence of rupture or perforation .", "laboratory analysis revealed an elevation of the white blood cell count ( 20.36  10/l ) and c - reactive protein ( crp ; 28.54 mg / dl ) .", "we tried to find any evidence of esophageal injury because treatment strategy would be different if the esophagus was involved .", "an ent specialist and a gi specialist agreed with hypopharyngeal injury without esophageal damage after multiple tests .", "we concluded the lesion was limited to the hypopharynx because the presence of hematoma at the right side of hypopharynx , consistent with the direction and the depth of the probe passage .", "further , there was no evidence of esophageal injury on serial follow - up multimodality imaging studies .", "after 5 days from the injury , the subcutaneous emphysema disappeared and the patient remained afebrile with improvement of the leukocyte count and crp ( fig .", "however painful neck mass around anterolateral area of neck was noticed ( level iii ) .", "the follow - up ct without contrast showed a right parapharyngeal and retropharyngeal abscess secondary to hypopharyngeal injury ( fig .", "yellowish fluid in the abscess was analyzed for cytology and revealed as acute inflammatory cells predominantly neutrophils . as the patient 's systemic status was stable with decrease of the leukocyte count and crp , the antibiotics ( piperacillin / tazobactam )", "was maintained for 14 days with the drainage of abscess because no organisms were identified from the abscess fluid culture and repeated blood cultures .", "on 7th day after tee , second swallowing study was performed and no leakage was demonstrated ( fig .", "4b ) , infectious parameters were continuously decreased , and follow - up fiberoptic nasolaryngoscopic examination demonstrated no evidence of perforation or rupture in hypopharyngeal cavity .", "after 14 days of antibiotic therapy , when the infection was controlled completely , the patient had successful mitral valve repair with tricuspid annuloplasty with st ."], ["the overall incidence of severe hypopharyngeal and esophageal complications from traditional tee is low.1)3)6 ) risk factors of hypopharyngeal and upper gastrointestinal complications have been reported in few studies .", "patient related risk factors include older age , short stature , presence of cervical spinal disease , chronic steroid use , presence of congestive heart failure.3)7)8 ) the presence of gastroesophageal pathology such as zenker 's diverticulum , esophageal stricture or obstructing mass , fibrosis secondary to prior chest radiation could be potential risk factor . anatomical variation like massive cardiomegaly1)9 ) or tracheoesophageal fistula may be correlated to increase the risk .", "procedure related risk factors include inexperience of the cardiologist , and resistance to prove insertion with poor cooperation . to avoid direct injury to the hypopharynx and upper esophagus during a blind insertion of probe , appropriate conscious sedation and local anesthetic spray", "direct visualization of the passage of the probe with direct laryngoscope could be recommended to minimize blind injury of throat .", "if there are procedural difficulties such as multiple attempts at passage of the tee probe or resistance during insertion , the examination should be stopped .", "in addition , indirect injury is also result from increased continuous pressure on the probe positioned in the upper esophagus , therefore , the exam should be stopped if the resistance is continuing .", "finally , it is very important to prevent sudden position change of the patient during the probe insertion .", "it needs pre - procedural enough education , adequate sedation , careful insertion of tee probe , and an experienced assistant . in our case ,", "we assumed that the patient 's poor cooperation and insufficient sedation might cause the wrong direction of the tee probe and advancing force of tee probe into the throat had led the probe to get lodged into the right pyriform sinus .", "treatment of hypopharyngeal perforation depends on the cause , timing of diagnosis , and type of lesion.101112 ) in a study of hinojar et al.,10 ) 3 patients out of 7 patients were managed conservatively , whereas other 4 patients got surgical treatment . mostly in the former cases , the time duration to the diagnosis was less than 1 - 2 days and involvement of infection", "was limited to pharyngeal space . in current case , our patient complained pain in his throat and we detected subcutaneous emphysema within 3 hours after injury that may prevent aggravation of infection and lead good prognosis .", "it is important not only to understand possible complications of the procedure and to recognize the risk factors of pharyngeal and esophageal injury , but also to detect the complication as soon as possible if it occurred , to get better prognosis ."]]}
{"article_id": "PMC3469105", "article_text": ["it has been reported that the prevalence and incidence of dementia in the united states have been either stable or even declining over the last 2 decades of the 1990s .", "this question is particularly relevant to the case of japan which is an economically advanced country like the us , but is believed to have a different level of vascular disease risk .", "studies conducted in the late 1980s and early 1990s reported that vascular dementia ( vad ) was more prevalent than alzheimer 's disease ( ad ) in japan , compared with the us and other western countries where ad is more prevalent than vad . in studies among japanese americans conducted in the early 1990s [ 4 , 5 ] ,", "the prevalence ratio of ad to vad was higher than that reported among japanese in japan and more closely resembled that found in the caucasian population .", "this suggests that there might have been environmental factors that changed the risks of developing subtypes of dementia after japanese immigrated to the us .", "on the other hand , more recent studies conducted in the late 1990s suggest that the cross - national differences found in the past may have been due to differences in the diagnostic methods used [ 6 , 7 ] .", "standardization of diagnosis is one of the challenges of cross - national comparisons of dementia prevalence .", "there is an ongoing debate as to whether : ( 1 ) the higher proportion of vad found in the past studies in japan could be due to differences in diagnostic criteria used in japan and the us , ( 2 ) the similar ad / vad ratio with that of the us found in recent japanese studies could be due to decreased cerebrovascular disease incidence over the past decades in japan , or ( 3 ) there is no systematic time trend in ad / vad ratio in japan and the observed variation is due to regional differences within japan . despite a growing interest in the influence of vascular disease and its risk factors on alzheimer 's disease ( ad ) [ 812 ] ,", "our aims in the current study are to : ( 1 ) summarize epidemiological studies of dementia in japan including relevant details of study protocols and diagnostic criteria , ( 2 ) compare age - specific prevalence of all - cause dementia among studies , and ( 3 ) assess the trends in ad versus vad over time .", "previous studies which concluded an increase in ad / vad over time in japan [ 1315 ] did not examine the diagnostic criteria used , nor did they examine age - specific ad / vad ratios .", "  we selected dementia prevalence studies in japan that were designed to be representative of specific communities or prefectures with at least 500 study participants aged 65 and older , and whose age - specific prevalence ( for either 5- or 10-year age intervals ) for ad and vad was published between 1990 and 2009 in international journals , using medline with the search words  japan ,   dementia ,  and  prevalence . ", "we used the former criterion ( n  500 ) in order to have a large enough sample size to allow meaningful between - cohort comparisons of dementia prevalence .", "eight studies met these criteria : the hisayama study [ 16 , 17 ] conducted at four time points , the okinawa study , the radiation effect research foundation adult health survey ( rerf - ahs , a.k.a hiroshima study ) , the tajiri project , and the ama - cho study .", "brief descriptions of each study cohort follow ( see also table 1 for further summary ) .  ", "hisayama is a rural community adjacent to the city of fukuoka , a major city of kyushu island .", "cross - sectional dementia prevalence was estimated 4 times , in 1985 , 1992 , 1998 , and 2005 [ 16 , 17 ] .   ", "random sampling was conducted to recruit study participants in the selected sites between 1991 and 1992 .   in 1958 ,", "the atomic bomb casualty commission began the adult health study ( ahs ) to survey the occurrence of illnesses and changes in physiological and biochemical function resulting from exposure to atomicbomb radiation .", "the original ahs cohort consisted of atomicbomb survivors and their controls , selected from residents in hiroshima and nagasaki . between 1992 and 1996 , those aged 60 and older who were residents of hiroshima and members of the ahs were examined .", "the tajiri project is a community - based study started in 1988 for the prevention of stroke , dementia , and bed - confinement in tajiri , an agricultural area in northern japan . in 1998 , all residents 65 years and older were targeted for the dementia prevalence study [ 6 , 20 ] .  ", "this study was conducted in the municipality of ama - cho , a rural island town in the northwestern part of japan .", "all residents as of the prevalence day of march 1 , 2008 were requested to participate in the dementia prevalence study .   ", "differences in overall dementia prevalence among 8 studies were examined using poisson regression models based on the number of dementia cases ( weighted numbers of cases for okinawa studies ) and the number of participants by 10 year age groups ( except the youngest age group where 5-year ages were used : 6569 , 7079 , 8089 , and 90 + ) to provide large enough sample sizes in each group for meaningful statistical comparisons , yet controlling for changing age composition over time .", "the okinawa 1992 study sample was used as a reference group because it was the largest and was conducted at the mid - point of assessment years among the 8 studies .", "\n table 1 gives a brief description of each study and the criteria used to define dementia and dementia subtypes .", "all assessments , except in the tajiri study , were based on multistage assessments , during which all participants were screened in phase 1 ( screening phase ) , and selected participants and controls from phase 1 received final diagnoses from physicians in phase 2 ( clinical assessment phase ) .", "the dementia diagnostic criteria used in japanese studies were either dsm - iii , dsm - iii - r , or dsm - iv . the diagnostic criteria used to define subtypes of dementia varied among the studies in japan .", "the following criteria were used to define vad : hachinski ischemic scores , ninds - airen , dsm - iv , and the alzheimer 's disease diagnostic and treatment centers ( addtc )   criteria . for ad , dsm - iii - r , dsm - iv , and nincds - adrda   were used .   the overall prevalence of dementia among those aged 65 and older ranged from 5.6% ( hisayama 1992 ) to 11.3% ( ama - cho 2008 ) ( table 1 ) .", "poisson regression models for dementia prevalence showed that compared with the okinawa 1992 study , hiroshima 1996 ( p = 0.0002 ) , tajiri 1998 ( p < 0.0001 ) , hisayama 2005 ( p < 0.0001 ) , and ama - cho 2008 ( p = 0.007 ) had a higher prevalence of all - cause dementia , controlling for sex and age groups ( table 2 ) .", "there were no difference between the okinawa 1992 study and hisayama studies conducted in 1985 , 1992 , and 1998 .", "no difference was found between men and women .   as a post hoc analysis , we also ran poisson regression models using only the hisayama studies ( 4 time points ) .", "the results showed that compared with the all - cause dementia prevalence in 1985 , that of 2005 was higher ( coefficient : 0.32 , 95% ci : 0.010.64 , p = 0.04 ) controlling for sex and age groups ( not shown in table 1 ) .  ", "ad / vad ratios among those aged 65 and older are listed in the last column of table 1 .", "the okinawa ( 1992 ) , hiroshima ( 1996 ) , tajiri ( 1998 ) , and ama - cho ( 2008 ) studies used the same criteria for diagnoses of ad ( nincds - adrda ) and the ad / vad ratio among those aged 65 and older was 1.85 , 1.85 , 3.33 , and 4.12 for the above studies , respectively .", "the hisayama studies which conducted 4 cross - sectional studies using the same diagnostic criteria for subtypes of dementia also showed the increasing trend in the ratio of ad / vad , ranging from 0.52 in 1985 to 1.96 in 1998 , and 1.92 in 2005 . the tajiri project , which conducted an mri substudy to identify dementia etiologies , showed that the proportion of ad among total dementia cases differed largely depending on the criteria used : 62.5% ( ad / vad = 3.3 ) using ninds - airen criteria , and 40.6% ( ad /", "vad = 1.0 ) or 56.2% ( ad / vad = 2.3 ) depending on assessors using dsm - iv criteria .   ", "even though we selected studies with relatively large sample sizes ( n  500 ) , the number of cases was too small to conduct meaningful comparisons of age - specific ad / vad ratios for the age group 6570 ( < 5 for each case ) .", "therefore , we list age - specific prevalence of dementia and ad / vad ratios for the age groups 7079 , 8089 , and 90 and older in table 3 .", "the youngest age group examined here ( age 7079 ) had a prevalence of vad that was consistently higher than that of ad in japan ( i.e. , ad / vad < 1 ) except in more recent studies conducted in 2005 ( hisayama ) and 2008 ( ama - cho ) . except for these two recent studies ( hisayama 2005 and ama - cho 2008 ) ,", "eight major prevalence studies conducted in japan were reviewed in an attempt to identify trends in prevalence of all - cause dementia and subtypes of dementia , paying careful attention to diagnostic protocols .", "we found that compared with the okinawa 1992 study , studies conducted in later years ( 1994 ( hiroshima ) , 1998 ( tajiri ) , 2005 ( hisayama ) , and 2008 ( ama - cho ) ) had a higher prevalence of all - cause dementia , after controlling for age groups and sex . within 4 studies conducted in hisayama ( 1985 , 1992 , 1998 , and 2005 ) , we also found that the prevalence in 2005 was higher than that in 1985 , after controlling for age groups and sex .", "thus , the dementia prevalence seems to be increasing in japan , in contrast to the us where decreasing or stable prevalence of all - cause dementia has been reported .   ", "two diseases that could have high impact on the prevalence of dementia at national levels are cerebrovascular disease and type 2 diabetes , as shown by their relatively high population attributable risk % ( par% ) of dementia in the united states [ 12 , 28 ] . according to the national nutrition survey in japan , those with hemoglobin a1c values  6.0% ( possible type 2 diabetes )", "were estimated to be 22.8% , 37.4% , and 40.9% among men aged 70 and older in year 1997 , 2002 and 2007 , respectively , and the comparative figures among women were 27.2% , 28.2% , and 34.6% . on the other hand ,", "the decline in stroke incidence reached plateaus around the late 1990s after a continuous sharp decline beginning in 1960 , thus further declines in dementia due to stroke would not be expected after the 1990s .", "however , it is possible that the prevalence of small vessel cerebrovascular disease with resultant microinfarcts that would not be accounted for in these vascular disease statistics could play a latent or underappreciated role in causing or contributing to dementia .", "the increase in type 2 diabetes , the metabolic syndrome and its associated vascular complications ( risk factors for ad ) , with a plateau in declining trends in major stroke incidence , could have lead to an increase in dementia prevalence in recent years in japan .", "it is also possible that increasing public awareness of dementia in recent years is resulting in enhanced recognition of functional and cognitive declines that might previously have been dismissed as", " longer survival of those who suffered from stroke / tia due to advanced medical treatment could also increase the prevalence of dementia to some extent .", "unfortunately , autopsy confirmation in large proportions of the participants in these epidemiologic studies is not available , thus limiting conclusions as to more specific underlying etiologies .  ", "we found that okinawa had a lower overall prevalence of dementia compared with other cohorts except hisayama studies in 1985 , 1992 , and 1998 .", "these two regions ( okinawa and hisayama ) have had lower incidence of cerebrovascular diseases in comparison with other regions studied here .", "for example , the rate of cerebrovascular mortality , as a proxy of cerebrovascular disease incidence , declined sharply between 1975 and 2000 by over 65% in all regions and declined further between 2000 and 2005 by over 10% in all regions ( table 4 ) , with the above two regions constantly having lower rates than other regions .", "historically , salt consumption has been high in the northern part of japan because vegetables and marine products are cured with salt to preserve them during the longer winter months and consumed with processed white rice .", "this dietary pattern is believed to be one of the reasons of the higher prevalence of hypertension and large vessel cerebrovascular disease in the northern prefectures in japan .", "the low dementia prevalence in okinawa and hisayama ( except 2008 ) , which is located in the southern part of japan , could not only be due to a lower rate of vad resulting from lower cerebrovascular disease incidence , but also due to lower ad prevalence resulting from reduced vascular injuries [ 8 , 12 , 33 , 34 ] .   vascular dementia ( vad )", "was believed to be more prevalent than alzheimer 's disease ( ad ) in japan in the 1980s , in contrast to the us or other western countries , but studies conducted in the late 1990s and after showed patterns that were more similar to the us [ 13 , 15 , 35 ] .", "westernization  of the dementia prevalence pattern could be partly due to the declining stoke incidence observed during the 1980s as described above", ". however , it could also be due to changes in diagnostic criteria used in japan .", "for example , in the tajiri study , patients received a diagnosis of  possible ad with cvd  by means of the ninds - airen criteria , provided that the vascular effect on dementia was considered to be too ambiguous to diagnose as vad , which lead to relatively high proportion of ad out of all dementia cases ( over 62% ) .", "the study also demonstrated the difficulty of obtaining consensus on the definition of vad , even for experienced neurologists using the same criteria ; two neurologists blinded to each other 's diagnosis did not agree when diagnosing vad versus ad under dsm - iv criteria : the proportion of vad out of total dementia cases ranged from 40.6% to 56.2% , depending on assessors .", "we expect that lack of imaging data may result in underdiagnosis of subcortical vascular brain injury ( e.g. , white matter hyperintensity , and silent brain infarcts , etc . ) and thus lead to underestimation of vad", ". however , more recent studies including the tajiri , hisayama ( 1998 ; 2005 ) , hiroshima and ama - cho studies used imaging data , and these tend to show higher ad / vad ratios ( i.e. , not higher vad prevalence ) compared with earlier studies .", "therefore , it is likely that changes in diagnostic criteria at least partly explain the higher proportion of diagnosed ad in recent years .    in all studies except more recent studies conducted after 2000 ,", "as age increased , the proportion of ad among the total dementia cases increased : the youngest age group examined ( age 7079 ) had a prevalence of vad that was consistently higher than that of ad in japan ( i.e. , ad / vad < 1 ) . on the other hand , among the older age groups ,", "in fact , among the oldest age group ( age  90 ) , ad / vad ratios in japan were not necessarily lower than the us figures even among studies conducted in the early 1990s , ranging from 2.5 ( hisayama 1985 ) to 6.0 ( hisayama 1992 ) . in the aging , demographics , and memory study ( adams )", "[ 36 , 37 ] , which is the first study in the us to calculate a nationally representative dementia prevalence , the age - specific ad / vad ratios were found to be 2.36 , 4.43 , and 4.79 for age group 7179 , 8089 , 90 and older , respectively , using dsm - iii - r   and dsm - iv   criteria for dementia and nincds - adrda criteria for ad , that is , the age - associated increase in ad prevalence seems to be more evident in japan than in the us .", "this could be partly due to the fact that the gender gap in life expectancy is larger in japan compared with us and the proportion of women increases more steeply as age increases in japan .", "for example , in 1990 , the life expectancy at age 65 was 18.9 years for women and 15.1 years for men in the us , that is , a gender gap of 3.8 years , while the comparative figure in japan was 20.0 years and 16.2 years , with the gender gap of 4.8 years .", "similarly , in 2008 , the life expectancy at age 65 was 19.8 years for women and 17.1 years for men in the us , with the gender gap of 2.7 years , while the comparative figure in japan was 23.6 years and 18.6 years , with the gender gap of 5.0 years .", "cerebrovascular disease is more common among men than women , and we expect a higher proportion of vad among men than women . a steeper age - associated increase in ad prevalence found in japan , therefore", ", could be due to a higher proportion of women among the older old age groups in japan .", "comparisons of age - specific ad / vad ratios between men and women could clarify this issue , but we were unable to do so due to the small sample size once we stratify prevalence by sex and age groups , especially among those aged 90 and older .", "however , at least among the younger two age groups ( ages 7079 ; 8089 ) , we see an increasing trend in the proportion of ad cases as age group goes up in both men and women ( data not shown ) .", "these results also suggest that it is important to consider the age / sex structure of samples when we compare the ad / vad ratio among different cohorts", "although incidence of dementia and its subtype would give a more accurate picture regarding the trend , there is a paucity of dementia incidence studies in japan . to our knowledge , only three incidence studies", "have been reported thus far : ( 1 ) the hisayama cohort , following their 1985 cohort for 7 years ; ( 2 ) the tajiri project , following a sub - sample of their 1998 cohort   for up to 7 years ; ( 3 ) the hiroshima study ( radiation effects research foundation adult health study ) , following their 19921996 prevalence cohort until the year 2003 .", "the incidence of all - cause dementia was 19.3 per 1000 person - years for men and 20.9 for women in the hisayama study , 33.9 for men and 44.0 for women in the tajiri study , and 12.0 for men and 16.6 for women in the hiroshima study .", "the hisayama and hisorhisma studies reported significant gender differences in subtypes of dementia incidence : hisayama reported that the incidence of vad increased with age and was consistently higher than that of ad for men , while the incidence of ad was higher than that of vad for women age 75 years or older .", "the incidence of ad markedly increased after the age of 80 in either sex , but overall , vad was more common in the hisayama study .", "the hiroshima study also reported that probable ad showed the most remarkable increase with age , and probable vad was significantly lower among women .", "overall , ad was more common in this study , possibly because the hiroshima study was conducted later than the hisayama study , after a further decline in stroke incidence had occurred . in all 3 incidence studies ,", "nincds - adrda criteria were used for ad and ninds - airen for vad . in japan ,", "ad incidence and prevalence tend to increase with age more than vad and older old women predominantly have ad rather than vad .", "therefore , when aggregated , the overall prevalence of ad may become higher as more women survive in the oldest old age group . as we mentioned earlier , it is important to consider the age / sex structure of samples when examining trends in the ad / vad ratio .    in conclusion ,", "our systematic review shows that ( 1 ) there is an increasing trend in overall dementia in japan .", "although we can not confirm definitively from the current study , the possible explanation of the increase could be a shift in health conditions among the elderly in japan including the increase in diabetes mellitus in more recent years despite the plateau in decline in stroke incidence during the late 1990s ; ( 2 ) the similar ad / vad ratio found in recent studies in japan with that of the us could be due to a combination of at least 3 factors : ( a ) shifting diagnostic criteria ( more in line with us consensus diagnosis ) , ( b ) possible shifting in health conditions among the elderly in japan ( decline in stroke incidence , but increase in metabolic disease e.g. , type 2 diabetes , hyperlipidemia , and atherosclerosis ) , ( c ) an increase in the proportion of the oldest old who had an historically higher prevalence of ad ( as opposed to vad ) in japan , and ( d ) regional variations ( i.e. , a north - to - south gradient in vad ) possibly due to large difference in dietary patterns .   ", "the study limitations include relatively small sample sizes , especially for the oldest old group .", "the okinawa study took into account the potential dementia cases among those who were screened at phase 1 ( screening phase ) , but did not participate in phase 2 ( clinical assessment phase ) , using weights generated from phase 1 .", "however , as with most other epidemiological studies of dementia , there was no way of precisely estimating the frequency of dementia among those who did not participate in the screening phase .", "four japanese prevalence studies [ 15 , 4143 ] were not included in this study because the age - specific prevalence of ad and vad were not provided in the published articles .", "although it would be interesting to deconstruct the changes in all - cause - dementia prevalence and ad / vad ratios into the potential explanatory factors , different criteria used to define subtypes of dementia would not allow this type of quantitative assessment .", "one of the strengths of our study is that we could ascertain the detailed screening procedures and diagnostic criteria by contacting the investigators of the original studies .", "future studies could aid in monitoring changing prevalence patterns and their causes by including some of the following elements .", "first , as with hisayama studies , it would be ideal for studies to examine the prevalence of dementia and its subtypes repeatedly at the same locations using the same criteria .", "second , it would be helpful if future epidemiology studies would recruit more participants aged 90 and older ( e.g. , through aggressive age - stratified sampling protocols ) to improve estimates of prevalence in this age group .", "third , more comprehensive diagnostic criteria should be used for inter - cohort comparisons : as suggested by viswanathan and colleagues , ad and vad exist in a continuum of disease .", "it might be more meaningful if we could apply , for example , more specific clinicopathological criteria for mixed dementia [ 4450 ] than those currently used .", "increased use of standardized neuroimaging such as the alzheimer 's disease neuroimaging initiative ( adni )   might aid in the development of more specific and comparable criteria for the diagnosis of vad . finally , autopsy confirmation of the underlying potential causes of dementia in epidemiological studies would go a long way to help resolve these important uncertainties in the shifting patterns over time of the dementias ."], "abstract_text": ["<S> there is a paucity of data regarding trends in dementia and its subtype prevalence in japan . </S>", "<S> our aims in the current paper are to : ( 1 ) summarize epidemiological studies of dementia in japan including relevant details of study protocol and diagnostic criteria , ( 2 ) compare the age - specific prevalence of all - cause dementia among studies , and ( 3 ) assess the trends in alzheimer 's disease ( ad ) versus vascular dementia ( vad ) over time . </S>", "<S> we reviewed diagnostic criteria , all - cause dementia prevalence , and the ad / vad ratio from 8 large population studies of dementia in japan . compared with the okinawa 1992 study , </S>", "<S> studies conducted in 1994 , 1998 , 2005 , and 2008 had a higher prevalence of all - cause dementia using poisson regression models , after controlling for age and sex . </S>", "<S> in contrast to the us and some european countries , all - cause dementia prevalence is increasing in japan . </S>", "<S> the prevalence of ad as opposed to vad seems to be increasing over time , but large variability in diagnostic criteria , possible regional variability , and differences in prevalence of subtypes of dementia between men and women make it difficult to draw a conclusion about this trend at the national level . </S>", "<S> further studies , for example , comparing the population attributable risk of vascular diseases to the prevalence and incidence of dementia could help to clarify the regional variations in etiological subtypes . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion "], "sections": [["it has been reported that the prevalence and incidence of dementia in the united states have been either stable or even declining over the last 2 decades of the 1990s .", "this question is particularly relevant to the case of japan which is an economically advanced country like the us , but is believed to have a different level of vascular disease risk .", "studies conducted in the late 1980s and early 1990s reported that vascular dementia ( vad ) was more prevalent than alzheimer 's disease ( ad ) in japan , compared with the us and other western countries where ad is more prevalent than vad . in studies among japanese americans conducted in the early 1990s [ 4 , 5 ] ,", "the prevalence ratio of ad to vad was higher than that reported among japanese in japan and more closely resembled that found in the caucasian population .", "this suggests that there might have been environmental factors that changed the risks of developing subtypes of dementia after japanese immigrated to the us .", "on the other hand , more recent studies conducted in the late 1990s suggest that the cross - national differences found in the past may have been due to differences in the diagnostic methods used [ 6 , 7 ] .", "standardization of diagnosis is one of the challenges of cross - national comparisons of dementia prevalence .", "there is an ongoing debate as to whether : ( 1 ) the higher proportion of vad found in the past studies in japan could be due to differences in diagnostic criteria used in japan and the us , ( 2 ) the similar ad / vad ratio with that of the us found in recent japanese studies could be due to decreased cerebrovascular disease incidence over the past decades in japan , or ( 3 ) there is no systematic time trend in ad / vad ratio in japan and the observed variation is due to regional differences within japan . despite a growing interest in the influence of vascular disease and its risk factors on alzheimer 's disease ( ad ) [ 812 ] ,", "our aims in the current study are to : ( 1 ) summarize epidemiological studies of dementia in japan including relevant details of study protocols and diagnostic criteria , ( 2 ) compare age - specific prevalence of all - cause dementia among studies , and ( 3 ) assess the trends in ad versus vad over time .", "previous studies which concluded an increase in ad / vad over time in japan [ 1315 ] did not examine the diagnostic criteria used , nor did they examine age - specific ad / vad ratios ."], ["  we selected dementia prevalence studies in japan that were designed to be representative of specific communities or prefectures with at least 500 study participants aged 65 and older , and whose age - specific prevalence ( for either 5- or 10-year age intervals ) for ad and vad was published between 1990 and 2009 in international journals , using medline with the search words  japan ,   dementia ,  and  prevalence . ", "we used the former criterion ( n  500 ) in order to have a large enough sample size to allow meaningful between - cohort comparisons of dementia prevalence .", "eight studies met these criteria : the hisayama study [ 16 , 17 ] conducted at four time points , the okinawa study , the radiation effect research foundation adult health survey ( rerf - ahs , a.k.a hiroshima study ) , the tajiri project , and the ama - cho study .", "brief descriptions of each study cohort follow ( see also table 1 for further summary ) .  ", "hisayama is a rural community adjacent to the city of fukuoka , a major city of kyushu island .", "cross - sectional dementia prevalence was estimated 4 times , in 1985 , 1992 , 1998 , and 2005 [ 16 , 17 ] .   ", "random sampling was conducted to recruit study participants in the selected sites between 1991 and 1992 .   in 1958 ,", "the atomic bomb casualty commission began the adult health study ( ahs ) to survey the occurrence of illnesses and changes in physiological and biochemical function resulting from exposure to atomicbomb radiation .", "the original ahs cohort consisted of atomicbomb survivors and their controls , selected from residents in hiroshima and nagasaki . between 1992 and 1996 , those aged 60 and older who were residents of hiroshima and members of the ahs were examined .", "the tajiri project is a community - based study started in 1988 for the prevention of stroke , dementia , and bed - confinement in tajiri , an agricultural area in northern japan . in 1998 , all residents 65 years and older were targeted for the dementia prevalence study [ 6 , 20 ] .  ", "this study was conducted in the municipality of ama - cho , a rural island town in the northwestern part of japan .", "all residents as of the prevalence day of march 1 , 2008 were requested to participate in the dementia prevalence study .   ", "differences in overall dementia prevalence among 8 studies were examined using poisson regression models based on the number of dementia cases ( weighted numbers of cases for okinawa studies ) and the number of participants by 10 year age groups ( except the youngest age group where 5-year ages were used : 6569 , 7079 , 8089 , and 90 + ) to provide large enough sample sizes in each group for meaningful statistical comparisons , yet controlling for changing age composition over time .", "the okinawa 1992 study sample was used as a reference group because it was the largest and was conducted at the mid - point of assessment years among the 8 studies ."], ["\n table 1 gives a brief description of each study and the criteria used to define dementia and dementia subtypes .", "all assessments , except in the tajiri study , were based on multistage assessments , during which all participants were screened in phase 1 ( screening phase ) , and selected participants and controls from phase 1 received final diagnoses from physicians in phase 2 ( clinical assessment phase ) .", "the dementia diagnostic criteria used in japanese studies were either dsm - iii , dsm - iii - r , or dsm - iv . the diagnostic criteria used to define subtypes of dementia varied among the studies in japan .", "the following criteria were used to define vad : hachinski ischemic scores , ninds - airen , dsm - iv , and the alzheimer 's disease diagnostic and treatment centers ( addtc )   criteria . for ad , dsm - iii - r , dsm - iv , and nincds - adrda   were used .   the overall prevalence of dementia among those aged 65 and older ranged from 5.6% ( hisayama 1992 ) to 11.3% ( ama - cho 2008 ) ( table 1 ) .", "poisson regression models for dementia prevalence showed that compared with the okinawa 1992 study , hiroshima 1996 ( p = 0.0002 ) , tajiri 1998 ( p < 0.0001 ) , hisayama 2005 ( p < 0.0001 ) , and ama - cho 2008 ( p = 0.007 ) had a higher prevalence of all - cause dementia , controlling for sex and age groups ( table 2 ) .", "there were no difference between the okinawa 1992 study and hisayama studies conducted in 1985 , 1992 , and 1998 .", "no difference was found between men and women .   as a post hoc analysis , we also ran poisson regression models using only the hisayama studies ( 4 time points ) .", "the results showed that compared with the all - cause dementia prevalence in 1985 , that of 2005 was higher ( coefficient : 0.32 , 95% ci : 0.010.64 , p = 0.04 ) controlling for sex and age groups ( not shown in table 1 ) .  ", "ad / vad ratios among those aged 65 and older are listed in the last column of table 1 .", "the okinawa ( 1992 ) , hiroshima ( 1996 ) , tajiri ( 1998 ) , and ama - cho ( 2008 ) studies used the same criteria for diagnoses of ad ( nincds - adrda ) and the ad / vad ratio among those aged 65 and older was 1.85 , 1.85 , 3.33 , and 4.12 for the above studies , respectively .", "the hisayama studies which conducted 4 cross - sectional studies using the same diagnostic criteria for subtypes of dementia also showed the increasing trend in the ratio of ad / vad , ranging from 0.52 in 1985 to 1.96 in 1998 , and 1.92 in 2005 . the tajiri project , which conducted an mri substudy to identify dementia etiologies , showed that the proportion of ad among total dementia cases differed largely depending on the criteria used : 62.5% ( ad / vad = 3.3 ) using ninds - airen criteria , and 40.6% ( ad /", "vad = 1.0 ) or 56.2% ( ad / vad = 2.3 ) depending on assessors using dsm - iv criteria .   ", "even though we selected studies with relatively large sample sizes ( n  500 ) , the number of cases was too small to conduct meaningful comparisons of age - specific ad / vad ratios for the age group 6570 ( < 5 for each case ) .", "therefore , we list age - specific prevalence of dementia and ad / vad ratios for the age groups 7079 , 8089 , and 90 and older in table 3 .", "the youngest age group examined here ( age 7079 ) had a prevalence of vad that was consistently higher than that of ad in japan ( i.e. , ad / vad < 1 ) except in more recent studies conducted in 2005 ( hisayama ) and 2008 ( ama - cho ) . except for these two recent studies ( hisayama 2005 and ama - cho 2008 ) ,"], ["eight major prevalence studies conducted in japan were reviewed in an attempt to identify trends in prevalence of all - cause dementia and subtypes of dementia , paying careful attention to diagnostic protocols .", "we found that compared with the okinawa 1992 study , studies conducted in later years ( 1994 ( hiroshima ) , 1998 ( tajiri ) , 2005 ( hisayama ) , and 2008 ( ama - cho ) ) had a higher prevalence of all - cause dementia , after controlling for age groups and sex . within 4 studies conducted in hisayama ( 1985 , 1992 , 1998 , and 2005 ) , we also found that the prevalence in 2005 was higher than that in 1985 , after controlling for age groups and sex .", "thus , the dementia prevalence seems to be increasing in japan , in contrast to the us where decreasing or stable prevalence of all - cause dementia has been reported .   ", "two diseases that could have high impact on the prevalence of dementia at national levels are cerebrovascular disease and type 2 diabetes , as shown by their relatively high population attributable risk % ( par% ) of dementia in the united states [ 12 , 28 ] . according to the national nutrition survey in japan , those with hemoglobin a1c values  6.0% ( possible type 2 diabetes )", "were estimated to be 22.8% , 37.4% , and 40.9% among men aged 70 and older in year 1997 , 2002 and 2007 , respectively , and the comparative figures among women were 27.2% , 28.2% , and 34.6% . on the other hand ,", "the decline in stroke incidence reached plateaus around the late 1990s after a continuous sharp decline beginning in 1960 , thus further declines in dementia due to stroke would not be expected after the 1990s .", "however , it is possible that the prevalence of small vessel cerebrovascular disease with resultant microinfarcts that would not be accounted for in these vascular disease statistics could play a latent or underappreciated role in causing or contributing to dementia .", "the increase in type 2 diabetes , the metabolic syndrome and its associated vascular complications ( risk factors for ad ) , with a plateau in declining trends in major stroke incidence , could have lead to an increase in dementia prevalence in recent years in japan .", "it is also possible that increasing public awareness of dementia in recent years is resulting in enhanced recognition of functional and cognitive declines that might previously have been dismissed as", " longer survival of those who suffered from stroke / tia due to advanced medical treatment could also increase the prevalence of dementia to some extent .", "unfortunately , autopsy confirmation in large proportions of the participants in these epidemiologic studies is not available , thus limiting conclusions as to more specific underlying etiologies .  ", "we found that okinawa had a lower overall prevalence of dementia compared with other cohorts except hisayama studies in 1985 , 1992 , and 1998 .", "these two regions ( okinawa and hisayama ) have had lower incidence of cerebrovascular diseases in comparison with other regions studied here .", "for example , the rate of cerebrovascular mortality , as a proxy of cerebrovascular disease incidence , declined sharply between 1975 and 2000 by over 65% in all regions and declined further between 2000 and 2005 by over 10% in all regions ( table 4 ) , with the above two regions constantly having lower rates than other regions .", "historically , salt consumption has been high in the northern part of japan because vegetables and marine products are cured with salt to preserve them during the longer winter months and consumed with processed white rice .", "this dietary pattern is believed to be one of the reasons of the higher prevalence of hypertension and large vessel cerebrovascular disease in the northern prefectures in japan .", "the low dementia prevalence in okinawa and hisayama ( except 2008 ) , which is located in the southern part of japan , could not only be due to a lower rate of vad resulting from lower cerebrovascular disease incidence , but also due to lower ad prevalence resulting from reduced vascular injuries [ 8 , 12 , 33 , 34 ] .   vascular dementia ( vad )", "was believed to be more prevalent than alzheimer 's disease ( ad ) in japan in the 1980s , in contrast to the us or other western countries , but studies conducted in the late 1990s and after showed patterns that were more similar to the us [ 13 , 15 , 35 ] .", "westernization  of the dementia prevalence pattern could be partly due to the declining stoke incidence observed during the 1980s as described above", ". however , it could also be due to changes in diagnostic criteria used in japan .", "for example , in the tajiri study , patients received a diagnosis of  possible ad with cvd  by means of the ninds - airen criteria , provided that the vascular effect on dementia was considered to be too ambiguous to diagnose as vad , which lead to relatively high proportion of ad out of all dementia cases ( over 62% ) .", "the study also demonstrated the difficulty of obtaining consensus on the definition of vad , even for experienced neurologists using the same criteria ; two neurologists blinded to each other 's diagnosis did not agree when diagnosing vad versus ad under dsm - iv criteria : the proportion of vad out of total dementia cases ranged from 40.6% to 56.2% , depending on assessors .", "we expect that lack of imaging data may result in underdiagnosis of subcortical vascular brain injury ( e.g. , white matter hyperintensity , and silent brain infarcts , etc . ) and thus lead to underestimation of vad", ". however , more recent studies including the tajiri , hisayama ( 1998 ; 2005 ) , hiroshima and ama - cho studies used imaging data , and these tend to show higher ad / vad ratios ( i.e. , not higher vad prevalence ) compared with earlier studies .", "therefore , it is likely that changes in diagnostic criteria at least partly explain the higher proportion of diagnosed ad in recent years .    in all studies except more recent studies conducted after 2000 ,", "as age increased , the proportion of ad among the total dementia cases increased : the youngest age group examined ( age 7079 ) had a prevalence of vad that was consistently higher than that of ad in japan ( i.e. , ad / vad < 1 ) . on the other hand , among the older age groups ,", "in fact , among the oldest age group ( age  90 ) , ad / vad ratios in japan were not necessarily lower than the us figures even among studies conducted in the early 1990s , ranging from 2.5 ( hisayama 1985 ) to 6.0 ( hisayama 1992 ) . in the aging , demographics , and memory study ( adams )", "[ 36 , 37 ] , which is the first study in the us to calculate a nationally representative dementia prevalence , the age - specific ad / vad ratios were found to be 2.36 , 4.43 , and 4.79 for age group 7179 , 8089 , 90 and older , respectively , using dsm - iii - r   and dsm - iv   criteria for dementia and nincds - adrda criteria for ad , that is , the age - associated increase in ad prevalence seems to be more evident in japan than in the us .", "this could be partly due to the fact that the gender gap in life expectancy is larger in japan compared with us and the proportion of women increases more steeply as age increases in japan .", "for example , in 1990 , the life expectancy at age 65 was 18.9 years for women and 15.1 years for men in the us , that is , a gender gap of 3.8 years , while the comparative figure in japan was 20.0 years and 16.2 years , with the gender gap of 4.8 years .", "similarly , in 2008 , the life expectancy at age 65 was 19.8 years for women and 17.1 years for men in the us , with the gender gap of 2.7 years , while the comparative figure in japan was 23.6 years and 18.6 years , with the gender gap of 5.0 years .", "cerebrovascular disease is more common among men than women , and we expect a higher proportion of vad among men than women . a steeper age - associated increase in ad prevalence found in japan , therefore", ", could be due to a higher proportion of women among the older old age groups in japan .", "comparisons of age - specific ad / vad ratios between men and women could clarify this issue , but we were unable to do so due to the small sample size once we stratify prevalence by sex and age groups , especially among those aged 90 and older .", "however , at least among the younger two age groups ( ages 7079 ; 8089 ) , we see an increasing trend in the proportion of ad cases as age group goes up in both men and women ( data not shown ) .", "these results also suggest that it is important to consider the age / sex structure of samples when we compare the ad / vad ratio among different cohorts", "although incidence of dementia and its subtype would give a more accurate picture regarding the trend , there is a paucity of dementia incidence studies in japan . to our knowledge , only three incidence studies", "have been reported thus far : ( 1 ) the hisayama cohort , following their 1985 cohort for 7 years ; ( 2 ) the tajiri project , following a sub - sample of their 1998 cohort   for up to 7 years ; ( 3 ) the hiroshima study ( radiation effects research foundation adult health study ) , following their 19921996 prevalence cohort until the year 2003 .", "the incidence of all - cause dementia was 19.3 per 1000 person - years for men and 20.9 for women in the hisayama study , 33.9 for men and 44.0 for women in the tajiri study , and 12.0 for men and 16.6 for women in the hiroshima study .", "the hisayama and hisorhisma studies reported significant gender differences in subtypes of dementia incidence : hisayama reported that the incidence of vad increased with age and was consistently higher than that of ad for men , while the incidence of ad was higher than that of vad for women age 75 years or older .", "the incidence of ad markedly increased after the age of 80 in either sex , but overall , vad was more common in the hisayama study .", "the hiroshima study also reported that probable ad showed the most remarkable increase with age , and probable vad was significantly lower among women .", "overall , ad was more common in this study , possibly because the hiroshima study was conducted later than the hisayama study , after a further decline in stroke incidence had occurred . in all 3 incidence studies ,", "nincds - adrda criteria were used for ad and ninds - airen for vad . in japan ,", "ad incidence and prevalence tend to increase with age more than vad and older old women predominantly have ad rather than vad .", "therefore , when aggregated , the overall prevalence of ad may become higher as more women survive in the oldest old age group . as we mentioned earlier , it is important to consider the age / sex structure of samples when examining trends in the ad / vad ratio .    in conclusion ,", "our systematic review shows that ( 1 ) there is an increasing trend in overall dementia in japan .", "although we can not confirm definitively from the current study , the possible explanation of the increase could be a shift in health conditions among the elderly in japan including the increase in diabetes mellitus in more recent years despite the plateau in decline in stroke incidence during the late 1990s ; ( 2 ) the similar ad / vad ratio found in recent studies in japan with that of the us could be due to a combination of at least 3 factors : ( a ) shifting diagnostic criteria ( more in line with us consensus diagnosis ) , ( b ) possible shifting in health conditions among the elderly in japan ( decline in stroke incidence , but increase in metabolic disease e.g. , type 2 diabetes , hyperlipidemia , and atherosclerosis ) , ( c ) an increase in the proportion of the oldest old who had an historically higher prevalence of ad ( as opposed to vad ) in japan , and ( d ) regional variations ( i.e. , a north - to - south gradient in vad ) possibly due to large difference in dietary patterns .   ", "the study limitations include relatively small sample sizes , especially for the oldest old group .", "the okinawa study took into account the potential dementia cases among those who were screened at phase 1 ( screening phase ) , but did not participate in phase 2 ( clinical assessment phase ) , using weights generated from phase 1 .", "however , as with most other epidemiological studies of dementia , there was no way of precisely estimating the frequency of dementia among those who did not participate in the screening phase .", "four japanese prevalence studies [ 15 , 4143 ] were not included in this study because the age - specific prevalence of ad and vad were not provided in the published articles .", "although it would be interesting to deconstruct the changes in all - cause - dementia prevalence and ad / vad ratios into the potential explanatory factors , different criteria used to define subtypes of dementia would not allow this type of quantitative assessment .", "one of the strengths of our study is that we could ascertain the detailed screening procedures and diagnostic criteria by contacting the investigators of the original studies .", "future studies could aid in monitoring changing prevalence patterns and their causes by including some of the following elements .", "first , as with hisayama studies , it would be ideal for studies to examine the prevalence of dementia and its subtypes repeatedly at the same locations using the same criteria .", "second , it would be helpful if future epidemiology studies would recruit more participants aged 90 and older ( e.g. , through aggressive age - stratified sampling protocols ) to improve estimates of prevalence in this age group .", "third , more comprehensive diagnostic criteria should be used for inter - cohort comparisons : as suggested by viswanathan and colleagues , ad and vad exist in a continuum of disease .", "it might be more meaningful if we could apply , for example , more specific clinicopathological criteria for mixed dementia [ 4450 ] than those currently used .", "increased use of standardized neuroimaging such as the alzheimer 's disease neuroimaging initiative ( adni )   might aid in the development of more specific and comparable criteria for the diagnosis of vad . finally , autopsy confirmation of the underlying potential causes of dementia in epidemiological studies would go a long way to help resolve these important uncertainties in the shifting patterns over time of the dementias ."]]}
{"article_id": "PMC4483456", "article_text": ["chronic low back pain ( clbp ) refers to chronic pain around the lumbar vertebra sustained \n for over three months without serious pathological characteristics , regardless of the \n existence of sciatalgia1 .", "clbp causes pain \n and changes in the muscles , as well as decreases in contractile force and muscle activity . \n", "studies of therapeutic approaches to clbp pain reduction , muscular strength improvement , and \n spinal movement enhancement have investigated the effects of myofacial release , osteopathy , \n massage , lumbar stabilization exercises , and proprioceptive neuromuscular facilitation \n ( pnf)5,6,7 . among these therapeutic approaches ,", "joint mobilization using pnf has a positive effect on \n pain , muscle strength , and range of motion ( rom)8 , \n 9 .", "it is applied as a passive movement by \n therapists10 , 11 . in particular , kaltenborn - evjenth orthopedic manual therapy \n ( keomt ) is an effective treatment for improving rom and pain .", "keomt is a safe and \n well - established procedure that slowly utilizes the convex - concave rule ( traction or gliding \n on the treatment side)10 , 12 .", "hindle et al . \n reported pnf is effective at improving rom and muscular strength13 .", "kim et al . reported that lumbar stabilization therapy \n using pnf for clbp significantly improved the thicknesses of the external oblique and \n multifidus muscles , and the functional level8 .", "in addition , pnf has been used to improve the muscular strength of \n athletes14 .", "this case study was a single patient with clbp and a lumbar transitional vertebra who has \n six lumbar vertebrae ( not five ) .", "a lumbar transitional vertebra is defined as a congenital \n malformation that appears as a deformation of the fifth lumbar ( l5 ) or first sacrum ( s1 ) \n vertebra15 .", "vergauwen reported that \n lumbar transitional vertebrae patients more commonly experience disc protrusion , \n degeneration , and spinal stenosis than patients without lumbar transitional vertebrae16 .", "therefore , the \n purpose of this study was to identify the effects of joint mobilization using keomt and pnf \n on a patient with clbp and a lumbar transitional vertebra .", "the subject was selected from among the patients of sarang hospital ( yongin , south korea ) . \n", "the subject \n agreed to participate in the study after receiving explanations regarding the purpose and \n procedures of the experiment , and signed an informed consent statement before participation . \n", "the protocol for this study was approved by the local ethics committee of the namseoul \n university of cheonan .", "computer \n tomography ( ct ) showed six lumbar vertebrae , which is one more lumbar vertebra than a normal \n person ( fig .", "the subject was aged \n 29 , and had a weight of 67  kg , a height of 158  cm , and a bmi of 26.90 .", "the initial symptom developed about two years earlier , and at the time of the \n study the subject was not receiving any medical or physical therapy .", "the major complaint of \n the subject was a numb feeling , especially in the hind part of the left leg .", "she also \n complained of a stinging pain during daily activities , such as lifting , lumbar twist , and \n flexion .", "the program consisted of a 40-min session , 3 days a week for 4 weeks ( october 131 , 2014 ) , \n during which joint mobilization was performed using keomt and pnf techniques .", "joint mobilization using keomt lasted 20  min , and it included lumbar segmental traction and \n lumbar segmental mobilization ( flexion , extension ) in a side - lying position .", "it was performed by a therapist who had \n completed a keomt spine advance course .", "the pnf exercise included shoulder flexion , abduction , and \n external rotation in a supine position , and dynamic reversal of the antagonist was \n performed17 , which indirectly exercised \n the abdominal muscle through irradiation of the shoulder muscle .", "it was performed by a \n therapist who had completed the pnf level i , ii courses .", "the subject was assessed for spinal motion , low back pain , and thickness of the multifidus . \n", "spinal motion was assessed using a spinal mouse ( idiag , swiss ) , which measures the spinal \n curvature , flexion , and extension ( thoracic and lumbar vertebrae ) .", "low back pain was \n assessed using a visual analogue scale ( vas ) and the oswestry disability index ( odi ) .", "the \n vas assesses subjective pain through a which has a possible range of 0 to 10 .", "the odi assesses low back pain , and has a possible range of 0 to \n 100% .", "it is composed of ten items , including pain severity , self - management , lifting , \n sitting , standing , sleeping , etc .", "the thickness of the multifidus was measured using ct ( general electric , \n korea ) .", "the multifidus muscles of the fourth lumbar vertebra ( l4)20 was scanned by a radiological thechnician at sarang \n hospital .", "the spinal curvature , flexion , and extension of thoracic and lumbar vertebrae were \n measured . in the thoracic vertebra ,", "the rom of flexion increased from 14 to 20 , and the rom of \n extension increased from 1 to 4. in the lumbar vertebra , the angle of spinal curvature \n increased from 12 to 20. the rom in flexion increased from 33 to 47 , and the rom of \n extension increased from 2 to 7 ( table \n 1table 1.change of thoracic and lumbar movementvariablecurvature ( )flexion ( )extension ( )prepostprepostprepostthoracic2632142014lumbar1220334727 ) .", "the subject mentioned she was less uncomfortable in \n daily life , e.g. during face washing , dishwashing , hair washing , etc .", "the odi percentage \n score decreased from 48.88 to 24.44 ( table \n 2table 2.change of vas and odivariablevas ( score)odi ( % ) prepostprepost7.5348.8824.44 ) .", "the thickness of the multifidus was measured at the fourth lumbar vertebra ( left and \n right ) .", "the left multifidus increased from 572.09 to 662.09 mm , and the right \n multifidus increased from 479.84 to 530.90 mm ( table 3table 3.change of multifidus thicknessvariableprepostmultifidus ( mm)l : 572.09l : 662.09r : 479.84r : 530.90l : left ; r : right ) .", "3.multifidus thickness on a ct ( post ) show the multifidus thickness on a ct .", "multifidus thickness on a ct ( pre ) multifidus thickness on a ct ( post )", "this study was a case study of a single patient with clbp and a lumbar transitional \n vertebra .", "the purpose of this study was to identify the effects of joint mobilization using \n keomt and pnf techniques in a patient with clbp and a lumbar transitional vertebra .", "the \n spinal motion , pain , and thickness of the multifidus were evaluated and compared between \n pre- and post - assessment .", "florentino et al . reported that most nurses possess at least one musculoskeletal disorder , \n among which low back pain is the most frequent ( 60.9%)21 .", "after the intervention , the angle of spinal curvature increased , and the roms of flexion \n and extension of the thoracic and lumbar vertebrae also increased , indication that joint \n mobilization using keomt and pnf had a positive effect on spinal motion .", "ko et al . reported \n that clbp patients in a thoracic joint mobilization group showed greater improvements in \n spinal motion and pain reduction than a william exercise group22 .", "our present results were similar , suggesting that joint \n mobilization has a positive effect on spinal motion .", "after the intervention , the vas score decreased \n from 7.5 to 3 , and the odi score decreased from 48.88% to 24.44% .", "orthopedic manual therapy \n has positive effects on the spinal function of clbp patients6 , especially items of self - management , such as bathing , dressing , and \n sleeping .", "reported pain reduction after joint mobilization using keomt was performed for \n osteoarthritic elderly people23 , and \n lpez et al .", "reported joint postero - anterior mobilization had an effect on the pain and rom \n of clbp patients9 .", "lee reported a pnf group demonstrated greater pain reduction and increased muscle activity \n than a ball training group , and jung reported pnf reduced the pain of clbp patients24 , 25 .", ". therefore , the results \n of previous studies agree with those of the present study .", "the spinal muscle contracts prior to \n motion and controls the movement28 . in \n particular , the multifidus muscle is important for lumbar stabilization . the superficial \n multifidus plays a role in spinal direction control , and", "after the intervention , the \n left multifidus increased from 572.09 to 662.09 mm , and the right multifidus was \n increased from 479.84 to 530.90 mm .", "kim et al . reported a pnf group showed \n greater increases in the thicknesses of the multifidus and external oblique muscles than a \n general physiotherapy group and a lumbar stabilization group8 .", "in addition , a resistance exercise using pnf not only reinforced \n muscular strength causing maximal muscular contraction , but it also stabilized the body \n position30 .", "these results indicate pnf \n has a positive effect on the thickness of the multifidus . according to our present results ,", "joint mobilization using keomt and pnf had a positive \n effect on the spinal motion , pain , and thickness of the multifidus of a patient with clbp \n and a lumbar transitional vertebra .", "this study was a single subject case study , but it is \n meaningful in that it presented spinal motion , pain , and muscle activity in a patient with a \n lumbar transitional vertebra . a larger number of patients will need to be studied in the \n future ."], "abstract_text": ["<S> [ purpose ] the purpose of this case study was to identify the effects of \n joint mobilization using kaltenborn - evjenth orthopedic manual therapy ( keomt ) and \n proprioceptive neuromuscular facilitation ( pnf ) techniques on a patient with chronic low \n back pain ( clbp ) and a lumbar transitional vertebra . </S>", "<S> [ methods ] the intervention methods \n were joint mobilization using keomt and pnf techniques . the program consisted of 40-min \n sessions 3 days a week for 4 weeks . the spinal motion ( thoracic and lumbar vertebrae ) , \n pain , and thickness of the multifidus were measured . </S>", "<S> [ results ] the angle of spinal \n curvature increased , and the range of motions ( roms ) flexion and extension increased in \n the thoracic and lumbar vertebrae . the pain score as measured on a visual analogue scale \n ( vas ) and the oswestry disability index ( odi ) score decreased . </S>", "<S> the thickness of the \n multifidus ( l4 ) increased on the left and right sides . </S>", "<S> [ conclusion ] these results suggest \n that joint mobilization using keomt and pnf techniques had a positive effect on the spinal \n motion , pain , and thickness of the multifidus of a patient with chronic low back pain and \n a lumbar transitional vertebra . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECT AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["chronic low back pain ( clbp ) refers to chronic pain around the lumbar vertebra sustained \n for over three months without serious pathological characteristics , regardless of the \n existence of sciatalgia1 .", "clbp causes pain \n and changes in the muscles , as well as decreases in contractile force and muscle activity . \n", "studies of therapeutic approaches to clbp pain reduction , muscular strength improvement , and \n spinal movement enhancement have investigated the effects of myofacial release , osteopathy , \n massage , lumbar stabilization exercises , and proprioceptive neuromuscular facilitation \n ( pnf)5,6,7 . among these therapeutic approaches ,", "joint mobilization using pnf has a positive effect on \n pain , muscle strength , and range of motion ( rom)8 , \n 9 .", "it is applied as a passive movement by \n therapists10 , 11 . in particular , kaltenborn - evjenth orthopedic manual therapy \n ( keomt ) is an effective treatment for improving rom and pain .", "keomt is a safe and \n well - established procedure that slowly utilizes the convex - concave rule ( traction or gliding \n on the treatment side)10 , 12 .", "hindle et al . \n reported pnf is effective at improving rom and muscular strength13 .", "kim et al . reported that lumbar stabilization therapy \n using pnf for clbp significantly improved the thicknesses of the external oblique and \n multifidus muscles , and the functional level8 .", "in addition , pnf has been used to improve the muscular strength of \n athletes14 .", "this case study was a single patient with clbp and a lumbar transitional vertebra who has \n six lumbar vertebrae ( not five ) .", "a lumbar transitional vertebra is defined as a congenital \n malformation that appears as a deformation of the fifth lumbar ( l5 ) or first sacrum ( s1 ) \n vertebra15 .", "vergauwen reported that \n lumbar transitional vertebrae patients more commonly experience disc protrusion , \n degeneration , and spinal stenosis than patients without lumbar transitional vertebrae16 .", "therefore , the \n purpose of this study was to identify the effects of joint mobilization using keomt and pnf \n on a patient with clbp and a lumbar transitional vertebra ."], ["the subject was selected from among the patients of sarang hospital ( yongin , south korea ) . \n", "the subject \n agreed to participate in the study after receiving explanations regarding the purpose and \n procedures of the experiment , and signed an informed consent statement before participation . \n", "the protocol for this study was approved by the local ethics committee of the namseoul \n university of cheonan .", "computer \n tomography ( ct ) showed six lumbar vertebrae , which is one more lumbar vertebra than a normal \n person ( fig .", "the subject was aged \n 29 , and had a weight of 67  kg , a height of 158  cm , and a bmi of 26.90 .", "the initial symptom developed about two years earlier , and at the time of the \n study the subject was not receiving any medical or physical therapy .", "the major complaint of \n the subject was a numb feeling , especially in the hind part of the left leg .", "she also \n complained of a stinging pain during daily activities , such as lifting , lumbar twist , and \n flexion .", "the program consisted of a 40-min session , 3 days a week for 4 weeks ( october 131 , 2014 ) , \n during which joint mobilization was performed using keomt and pnf techniques .", "joint mobilization using keomt lasted 20  min , and it included lumbar segmental traction and \n lumbar segmental mobilization ( flexion , extension ) in a side - lying position .", "it was performed by a therapist who had \n completed a keomt spine advance course .", "the pnf exercise included shoulder flexion , abduction , and \n external rotation in a supine position , and dynamic reversal of the antagonist was \n performed17 , which indirectly exercised \n the abdominal muscle through irradiation of the shoulder muscle .", "it was performed by a \n therapist who had completed the pnf level i , ii courses .", "the subject was assessed for spinal motion , low back pain , and thickness of the multifidus . \n", "spinal motion was assessed using a spinal mouse ( idiag , swiss ) , which measures the spinal \n curvature , flexion , and extension ( thoracic and lumbar vertebrae ) .", "low back pain was \n assessed using a visual analogue scale ( vas ) and the oswestry disability index ( odi ) .", "the \n vas assesses subjective pain through a which has a possible range of 0 to 10 .", "the odi assesses low back pain , and has a possible range of 0 to \n 100% .", "it is composed of ten items , including pain severity , self - management , lifting , \n sitting , standing , sleeping , etc .", "the thickness of the multifidus was measured using ct ( general electric , \n korea ) .", "the multifidus muscles of the fourth lumbar vertebra ( l4)20 was scanned by a radiological thechnician at sarang \n hospital ."], ["the spinal curvature , flexion , and extension of thoracic and lumbar vertebrae were \n measured . in the thoracic vertebra ,", "the rom of flexion increased from 14 to 20 , and the rom of \n extension increased from 1 to 4. in the lumbar vertebra , the angle of spinal curvature \n increased from 12 to 20. the rom in flexion increased from 33 to 47 , and the rom of \n extension increased from 2 to 7 ( table \n 1table 1.change of thoracic and lumbar movementvariablecurvature ( )flexion ( )extension ( )prepostprepostprepostthoracic2632142014lumbar1220334727 ) .", "the subject mentioned she was less uncomfortable in \n daily life , e.g. during face washing , dishwashing , hair washing , etc .", "the odi percentage \n score decreased from 48.88 to 24.44 ( table \n 2table 2.change of vas and odivariablevas ( score)odi ( % ) prepostprepost7.5348.8824.44 ) .", "the thickness of the multifidus was measured at the fourth lumbar vertebra ( left and \n right ) .", "the left multifidus increased from 572.09 to 662.09 mm , and the right \n multifidus increased from 479.84 to 530.90 mm ( table 3table 3.change of multifidus thicknessvariableprepostmultifidus ( mm)l : 572.09l : 662.09r : 479.84r : 530.90l : left ; r : right ) .", "3.multifidus thickness on a ct ( post ) show the multifidus thickness on a ct .", "multifidus thickness on a ct ( pre ) multifidus thickness on a ct ( post )"], ["this study was a case study of a single patient with clbp and a lumbar transitional \n vertebra .", "the purpose of this study was to identify the effects of joint mobilization using \n keomt and pnf techniques in a patient with clbp and a lumbar transitional vertebra .", "the \n spinal motion , pain , and thickness of the multifidus were evaluated and compared between \n pre- and post - assessment .", "florentino et al . reported that most nurses possess at least one musculoskeletal disorder , \n among which low back pain is the most frequent ( 60.9%)21 .", "after the intervention , the angle of spinal curvature increased , and the roms of flexion \n and extension of the thoracic and lumbar vertebrae also increased , indication that joint \n mobilization using keomt and pnf had a positive effect on spinal motion .", "ko et al . reported \n that clbp patients in a thoracic joint mobilization group showed greater improvements in \n spinal motion and pain reduction than a william exercise group22 .", "our present results were similar , suggesting that joint \n mobilization has a positive effect on spinal motion .", "after the intervention , the vas score decreased \n from 7.5 to 3 , and the odi score decreased from 48.88% to 24.44% .", "orthopedic manual therapy \n has positive effects on the spinal function of clbp patients6 , especially items of self - management , such as bathing , dressing , and \n sleeping .", "reported pain reduction after joint mobilization using keomt was performed for \n osteoarthritic elderly people23 , and \n lpez et al .", "reported joint postero - anterior mobilization had an effect on the pain and rom \n of clbp patients9 .", "lee reported a pnf group demonstrated greater pain reduction and increased muscle activity \n than a ball training group , and jung reported pnf reduced the pain of clbp patients24 , 25 .", ". therefore , the results \n of previous studies agree with those of the present study .", "the spinal muscle contracts prior to \n motion and controls the movement28 . in \n particular , the multifidus muscle is important for lumbar stabilization . the superficial \n multifidus plays a role in spinal direction control , and", "after the intervention , the \n left multifidus increased from 572.09 to 662.09 mm , and the right multifidus was \n increased from 479.84 to 530.90 mm .", "kim et al . reported a pnf group showed \n greater increases in the thicknesses of the multifidus and external oblique muscles than a \n general physiotherapy group and a lumbar stabilization group8 .", "in addition , a resistance exercise using pnf not only reinforced \n muscular strength causing maximal muscular contraction , but it also stabilized the body \n position30 .", "these results indicate pnf \n has a positive effect on the thickness of the multifidus . according to our present results ,", "joint mobilization using keomt and pnf had a positive \n effect on the spinal motion , pain , and thickness of the multifidus of a patient with clbp \n and a lumbar transitional vertebra .", "this study was a single subject case study , but it is \n meaningful in that it presented spinal motion , pain , and muscle activity in a patient with a \n lumbar transitional vertebra . a larger number of patients will need to be studied in the \n future ."]]}
{"article_id": "PMC3609962", "article_text": [" to learn about the normal anatomy of the neural tube on mri  to recognise the mr appearance of neural tube defects  to understand the value of mri in assessing ntds", "technological developments in diagnostic imaging have improved the diagnosis and treatment of congenital anomalies immensely , and this progress is especially evident in neural tube defects ( ntds ) .", "ultrasonography allows accurate assessment of the neural tube . fetal magnetic resonance imaging ( mri ) complements ultrasonography , confirming the suspected anomaly and even detecting malformations that were not detected at ultrasonography .", "the additional information provided by mri may lead to changes in the management of the pregnancy and/or of the delivery [ 25 ] . in this article", "we describe the radiological , pathological and clinical features of the most common ntds , and finally emphasise an integrated clinical and radiological approach for accurate diagnosis .", "in our institution , all fetal mri studies are performed after ultrasonography and mri never constitutes the first imaging study of the fetus . none of the women or fetuses receives any special preparation .", "although it is important to be extremely careful when undertaking any procedure on a fetus , there is no evidence of associated risk to fetal development with fetal mri [ 68 ] .", "we use a 1.5-tesla scanner with a four - element phased - array body coil .", "a complete study requires between 12 and 15 sequences , so it takes about 2530  min .", "the fetal mri images are obtained using ultrafast sequences to minimise artefacts due to fetal movements .", "it seems that slow , rhythmic maternal respiration reduces fetal movements , so it is not necessary to acquire images with maternal breath - holding .", "t2-weighted single shot fast spin - echo ( ssfse ) or half - fourier single - shot turbo spin - echo ( haste ) images provide most of the information necessary for diagnosis .", "gradient echo sequences ( echo - planar imaging [ epi ] and true fast imaging with steady state precession [ fisp ] ) have greater ferromagnetic susceptibility , so they make it possible to identify bony and vascular structures [ 1 , 9 ] . both the head and spine must be studied in all three planes . even when the only indication for fetal mri is a suspected ntd , all the fetal structures should always be studied to rule out other possible anomalies .", "the first phase , gastrulation , begins in the 2nd to 3rd weeks of gestation ; in this phase , the two layers of the embryonic plate divide into three definitive layers ( the ectoderm , mesoderm and endoderm ) . in the second phase , neurulation , which takes place in the 3rd to 4th weeks of gestation", ", the cells that will become the notochord interact with the overlying ectodermic tissue , causing this tissue to thicken , fold over and fuse , thus forming the neural tube .", "the fusion begins in the medial dorsal zone of the embryo and progresses in a zipper - like fashion cranially as the rostral neuropore and caudally as the caudal neuropore .", "the closure of the two ends does not occur simultaneously : the cranial end closes before the caudal end .", "the final phase , secondary neurulation , takes place in the 5th to 6th weeks of gestation ; in this phase a secondary neural tube forms from the cells of hensen s node , which is a mass of totipotent cells located dorsally that intervene in the formation of the notochord and later migrate caudally to the presacral region .", "this secondary neural tube is initially solid ; it undergoes a process of retrogressive differentiation called canalisation in which it cavitates to form the medullary cone and the filum terminale .", "the interruption of this process at any point , whether in the cranial end or in the caudal end , will result in an ntd [ 11 , 12 ] .", "the spinal column and spinal cord are the same length until 1215  weeks gestation , after which the spinal column grows more than the spinal cord until the medullary cone is situated approximately at the level of the second lumbar vertebra , where it will be at birth and will remain without significant changes throughout life [ 11 , 12 ] .", "mri is an excellent method for evaluating the structures of the fetal head and spine .", "images can be obtained in any plane , regardless of the position of the fetus in the uterus . the cranial vault ( fig . ", "1a , b ) is seen as a hypointense structure in t2-weighted images , even early in gestation .", "1a ) can also be identified in any plane , but their visualisation improves as gestation advances.fig .", "a sagittal and b coronal t2-weighted haste images of the fetal head show the osseous structures of the base of the skull ( arrows in a ) and bones of the cranial vault ( arrows in b ) .", "the vertebral bodies are hypointense with a band of higher intensity in the centre ( white arrows ) , the intervertebral spaces are hyperintense ( black arrows ) .", "d coronal t2-weighted haste image shows the vertebral pedicles as hypointense rounded structures on both sides of the spinal canal ( arrowheads ) . the interpedicular distance in the lumbar spine", "is slightly increased ( arrows ) ; this is a normal finding that should not be confused with spina bifida .", "e axial image at the level of l1l2 shows the medullary cone ( white arrow ) inside the spinal canal ( the asterisks mark the kidneys ) .", "f the medullary cone is no longer seen in this axial image of the spinal canal below the kidneys ; sometimes the nerve roots can be identified ( white arrows ) a , b normal anatomy of the neural tube .", "a sagittal and b coronal t2-weighted haste images of the fetal head show the osseous structures of the base of the skull ( arrows in a ) and bones of the cranial vault ( arrows in b ) .", "the vertebral bodies are hypointense with a band of higher intensity in the centre ( white arrows ) , the intervertebral spaces are hyperintense ( black arrows ) .", "d coronal t2-weighted haste image shows the vertebral pedicles as hypointense rounded structures on both sides of the spinal canal ( arrowheads ) . the interpedicular distance in the lumbar spine", "is slightly increased ( arrows ) ; this is a normal finding that should not be confused with spina bifida .", "e axial image at the level of l1l2 shows the medullary cone ( white arrow ) inside the spinal canal ( the asterisks mark the kidneys ) .", "f the medullary cone is no longer seen in this axial image of the spinal canal below the kidneys ; sometimes the nerve roots can be identified ( white arrows ) the vertebral bodies have low signal intensity on t2-weighted sequences and are clearly seen on sagittal images .", "the intervertebral spaces are hyperintense and are clearly depicted in the sagittal plane ( fig . ", "the interpedicular distance should be constant throughout the spinal column , although it does increase slightly in the lumbar region and this widening should not be confused with an ntd ( fig . ", "the posterior elements of the vertebrae are best seen in the axial plane ( fig .  1e , f ) .", "caution is warranted in the assessment of possible scoliosis or kyphosis , because the spinal column can be altered by the position of the fetus in the uterus ; these pseudoalterations are more common as gestation advances , resulting in proportionately less space within the uterine cavity ( fig . ", "the spinal canal is hyperintense in t2-weighted sequences because it is filled with cerebrospinal fluid .", "although the spinal cord is well depicted in all three planes , it can probably be evaluated best in the axial plane ( fig . ", "the position of the fetus within the uterus results in a certain degree of thoracolumbar kyphosis that displaces the spinal cord anteriorly in the thoracic spine , making it difficult to see because it is adjacent to the vertebral bodies ( fig . ", "precaution should be taken to ensure that the spinal cord disappears below the fetal kidneys ( fig .  1e , f ) ;", "sometimes , small hypointense structures that correspond to nerve roots are seen distal to the medullary cone ( fig . ", "it is important to evaluate the paravertebral soft tissues in mri studies , especially in fetuses with spina bifida or destructive processes or tumours that could affect these tissues . the skin and the paravertebral muscular structures are hypointense in t2-weighted sequences", ". the spinal canal should be closed posteriorly by soft tissues ( fig .  1c , e , f ) .", "ntds are a heterogeneous group of anomalies that result from the partial or total failure of the neural tube to close .", "ntds can affect the cranial region ( anencephaly ) or the spinal region ( spina bifida ) ; spinal ntds can be divided into open or closed defects , depending on whether they are covered by a layer of skin   ( fig . ", "2neural tube defects the overall worldwide incidence of ntds is between one and ten per 1,000 live births [ 14 , 15 ] .", "the rate of ntds has varied widely over time and has a clear geographical distribution .", "the highest incidences have been reported in mexico , ireland , india and northern china [ 16 , 17 ] .", "a family history of ntds is absent in 9095% of cases , and ntds are reported in 3% of miscarriages . some genetic factors like single gene mutations ( meckel - gruber and", "roberts syndromes ) and chromosomopathies ( trisomies 13 and 18 , triploidy ) have been identified .", "some maternal factors ( hyperthermia , diabetes , hyperinsulinaemia , obesity , psychosocial or emotional stress [ 1823 ] ) and some environmental factors ( maternal medication with anticonvulsants like valproic acid and carbamazepine ) can increase the incidence of ntds .", "prenatal screening for ntds includes the measurement of diverse markers like acetylcholinesterase and alpha - fetoprotein in amniotic fluid and in maternal serum ( alpha - fetoprotein is a glycoprotein secreted by the liver and by the fetal yolk sac ) . a recent meta - analysis   confirmed that measuring alpha - fetoprotein in maternal serum during the second trimester is useful in screening for ntds .", "nevertheless , this marker is only useful for detecting open ntds ; furthermore , alpha - fetoprotein levels are also elevated in a long list of fetal anomalies , including omphalocele , turner s syndrome , and sacrococcygeal teratoma , amongst others .", "it is the absence of a major portion of the brain , skull and scalp above the orbits .", "there are two subtypes : anencephaly , in which the cranial vault , cerebral hemispheres and diencephalic structures are replaced by amorphous neurovascular tissue , and exencephaly , in which the cranial vault is absent but relatively normal amounts of brain tissue , albeit abnormally developed , is present .", "some authors consider exencephaly an early stage of anencephaly , because the unprotected anomalous brain tissue located outside the cranial vault will eventually take on the appearance of anencephaly after being destroyed by the trauma induced by constant collision against the walls of the uterus . in both exencephaly and anencephaly ,", "the base of the skull , the facial structures , and the orbits are present and are usually normal .", "mri is usually not used to evaluate fetuses with these conditions because ultrasonography usually detects this anomaly very early and mri is not required to confirm this diagnosis .", "fetal mri studies show the absence of osseous structures above the orbits and the absence of brain tissue or alterations in brain tissue when it is present . it is considered a lethal anomaly ,", "the specific name of the anomaly depends on the structures contained in the herniated sac ; for example , a meningocele contains meninges and cerebrospinal fluid , an encephalomeningocele contains meninges and brain tissue and an encephalomeningocystocele or encephalomeningohydrocele contains meninges , brain tissue , and part of the ventricular system ( fig .  3 ) .", "most encephaloceles occur along the midline in the occipital or frontal region , but they can also involve the anterior cranial fossa ( into the nasal sinuses ) or even the base of the skull ( into the sphenoid or ethmoid sinus).fig . 3encephalocele .", "a sagittal and b axial t2-weighted haste images show the protrusion of the occipital lobe and occipital horn of the ventricular system ( black arrows in a and b ) through a defect in the bone and soft tissues ( white arrows in a ) .", "c ultrasound image also shows the protrusion of the occipital lobe ( white arrows ) .", "d , e images of the same fetus at 31  weeks gestation show a decrease in the extruded encephalic component ( arrow in e ) and an increase in the associated meningeal component ( arrow in d ) encephalocele .", "a sagittal and b axial t2-weighted haste images show the protrusion of the occipital lobe and occipital horn of the ventricular system ( black arrows in a and b ) through a defect in the bone and soft tissues ( white arrows in a ) .", "c ultrasound image also shows the protrusion of the occipital lobe ( white arrows ) .", "d , e images of the same fetus at 31  weeks gestation show a decrease in the extruded encephalic component ( arrow in e ) and an increase in the associated meningeal component ( arrow in d ) encephaloceles are usually diagnosed by ultrasonography .", "mri s role is to specify the type of lesion , which is more important in smaller lesions and in those that affect the anterior cranial fossa or base of the skull .", "it is essential to evaluate the rest of the structures in the head and the neural tube , fundamentally the posterior fossa because it can be difficult to evaluate with ultrasonography .", "3d ) as well as the presence of brain tissue inside the herniated sac in cases of encephalomeningocele ( fig .  3a , b , e ) .", "other anomalies like open spina bifida or ventriculomegaly that are often associated with encephaloceles are easy to evaluate with mri .", "the differential diagnosis for encephalocele is based on the size , content and location of the lesion .", "the main differential diagnosis for encephaloceles located in the occipital region is with cystic hygroma , whereas for those located in the frontal region , the differential diagnosis is with teratoma and haemangioma of the scalp .", "both teratomas and haemangiomas of the scalp are shown as slightly heterogeneous but well - defined solid masses with variable signal intensity ; haemangiomas may have areas of signal voiding due to their intense vascularisation .", "an encephalocele may form part of a syndrome like meckel - gruber syndrome and it can be associated with other anomalies like ventriculomegaly , spina bifida ( 715% of all encephaloceles ) , microcephaly , or cleft lip and/or cleft palate .", "the prognosis depends on the size , location and extent of the lesion as well as on whether other anomalies are present .", "iniencephaly is an ntd affecting the occipital bone combined with rachischisis of the cervicothoracic spine and retroflexion of the head ( fig .  4 ) .", "fetuses with iniencephaly have a variable defect in the occipital bone with an enlarged foramen magnum , the partial or total absence of cervicothoracic vertebrae with irregular fusion of those that are present and incomplete closure of the vertebral bodies and arches .", "these alterations result in significant shortening of the spine and of the fetus due to marked lordosis with hyperextension of the head .", "iniencephaly can be open ( associated with encephalocele ) or closed ( spinal anomaly without encephalocele ) .fig .", "the spine is short , the head is hyperextended , and there is a nuchal fold ( arrow ) .", "the spine is short , the head is hyperextended , and there is a nuchal fold ( arrow ) .", "b pathological specimen the differential diagnosis is with klippel - feil syndrome ( short neck and fusion of the cervical vertebrae ) . some authors believe that klippel - feil syndrome is a milder form of iniencephaly . the prognosis of iniencephaly in the neonatal period is dismal , so whether other anomalies are present is unimportant .", "spinal dysraphism is the result of a congenital vertebral defect due to the failure of the caudal neuropore to close that leads to the exposure of the contents of the medullary canal to the exterior .", "the defect usually involves the posterior vertebral elements , although it can also involve the vertebral body itself .", "the defect is usually located in the lumbosacral region , but it can occur at any level and it can even involve the entire spine .", "the absence of skin and muscle in these areas is due to the failure to induct the overlying ectodermal and mesodermal tissues .", "other theories postulate the existence of an unbalance in the production and reabsorption of cerebrospinal fluid during the embryonic period that leads to an excessive accumulation in the already closed neural tube ( hydromyelia ) , which in turn causes a secondary separation from the dorsal wall .", "spinal dysraphism comprises a wide spectrum of anomalies ; these can be divided into open and closed defects .", "rachischisis is the most severe form of spina bifida ; in this lethal condition , an extensive segment of the rachis is absent , resulting in extensive exposure of neural tissue .", "myelomeningocele and myeloschisis ( myelocele ) are the most common spinal dysraphism , accounting for 85% of all cases [ 12 , 32 ] . in these lesions ,", "neural contents are exposed to the exterior through midline defects in the bone and skin [ 12 , 33 ] . when the spinal canal is directly exposed , the anomaly is called myeloschisis or myelocele   ( fig .  5 ) , and when intramedullary structures lined with meninges protrude or herniate due to the expansion of the underlying subarachnoid space , it is called myelomeningocele ( fig .  6 ) .", "myelomeningocele is the most common open spinal dysraphism [ 32 , 35 ] . by definition", ", there must be a vertebral defect , the most common of which is the absence of the posterior vertebral arches ( figs . ", "the main characteristic that allows myelomeningoceles to be differentiated from myeloschisis is the position of the neural placode , a remnant of the unfolded ( non - neurulated ) neural tube , with respect to the surface of the skin . in myelomeningocele , the neural placode protrudes behind the surface of the skin ( fig . ", "6a , b , f ) , whereas in myeloschisis it is at the same level ( fig . ", "another , extremely rare type of open dysraphism is the hemimyelomeningocele or hemimyelocele : in this anomaly , diastematomyelia is associated with one of the above - mentioned anomalies in which one of the hemicords does not complete neurulation [ 32 , 36 ] .", "this finding is important because if diastematomyelia is not suspected , the surgical repair of the myelomeningocele will not include the repair of the tethered cord that is nearly always present in diastematomyelia [ 1 , 9 , 37].fig .", "a , b axial t2-weighted haste image of the fetal cranium shows the deformity of the frontal bones ( black arrows in a ) and the characteristic beaklike shape of the ventricular horns ( arrows in a ) .", "the posterior fossa deformity can also be seen : the cisterna magnum is absent and the foramen magnum is enlarged due to the descent of the structures of the posterior fossa toward the spinal canal ( arrows in b ) .", "c sagittal t2-weighted haste image shows a cutaneous defect or thinning in the lumbosacral region ( arrowhead ) with no mass protruding through the defect as well as an associated chiari malformation ( black arrow ) .", "d axial image at the level of the cutaneous defect shows the abnormally low neural placode within the spinal canal without expansion of the subarachnoid spaces", "a sagittal t2-weighted haste image shows a small cystic mass protruding at the thoracolumbar level ( black arrow )", ". associated hydrocephalus ( asterisk ) and a chiari malformation ( white arrow ) are also seen .", "b axial t2-weighted haste image at the lumbosacral level shows the neural placode protruding outside the surface of the skin due to expansion of the adjacent subarachnoid space ( black arrow ) . c coronal image at the spinal level shows increased interpedicular distance at the level of the neural defect .", "d ultrasound image of a different patient shows a cystic mass protruding at lumbar level ( white arrows ) .", "a , b axial t2-weighted haste image of the fetal cranium shows the deformity of the frontal bones ( black arrows in a ) and the characteristic beaklike shape of the ventricular horns ( arrows in a ) .", "the posterior fossa deformity can also be seen : the cisterna magnum is absent and the foramen magnum is enlarged due to the descent of the structures of the posterior fossa toward the spinal canal ( arrows in b ) . c sagittal t2-weighted haste image shows a cutaneous defect or thinning in the lumbosacral region ( arrowhead ) with no mass protruding through the defect as well as an associated chiari malformation ( black arrow ) .", "d axial image at the level of the cutaneous defect shows the abnormally low neural placode within the spinal canal without expansion of the subarachnoid spaces .", "a sagittal t2-weighted haste image shows a small cystic mass protruding at the thoracolumbar level ( black arrow ) .", "associated hydrocephalus ( asterisk ) and a chiari malformation ( white arrow ) are also seen .", "b axial t2-weighted haste image at the lumbosacral level shows the neural placode protruding outside the surface of the skin due to expansion of the adjacent subarachnoid space ( black arrow ) .", "c coronal image at the spinal level shows increased interpedicular distance at the level of the neural defect .", "d ultrasound image of a different patient shows a cystic mass protruding at lumbar level ( white arrows ) .", "e pathological specimen . f sagittal diagrams of the anomaly open spina bifida is nearly always found together with chiari type ii malformation , which is an anomaly of the posterior fossa consisting of herniation of part of the cerebellum ( vermis and fourth ventricle ) and of the brainstem through the foramen magnum ( figs . ", "this association is thought to be due to the collapse of the primitive fetal ventricular system ( brought about by the loss of cerebrospinal fluid through the spinal defect ) , which prevents the rhombencephalic vesicle ( from which the brainstem , fourth ventricle , and cerebellum are derived ) from expanding and results in deficient development of the posterior fossa and herniation .", "recent studies have reported worse prognoses for fetuses with greater cerebellar herniation , related to an increased incidence of seizures , vesical dysfunction , and dependency for ambulation .", "open spina bifida is accompanied by a collapse of the extra - axial subarachnoid spaces ( fig . ", "6a ) secondary to mechanical obstruction from the chiari malformation or to dysfunctional reabsorption of cerebrospinal fluid [ 38 , 40 ] in 75% of cases .", "this is why it is essential to evaluate the spine carefully to rule out spinal dysraphism when ventriculomegaly is detected at any gestational age .", "likewise , the shape of the ventricles is abnormal owing to the acute angle of the ventricular horns   ( fig . ", "5a ) . as in ultrasonography , in mri both direct and the indirect signs of spina bifida at the level of the fetal cranium ( the lemon sign : special shape of the cranium due to deformity of the frontal bones [ fig . ", "5a ] and the banana sign : special shape of the posterior fossa secondary to displacement of the cerebellar hemispheres toward the cervical canal , which results in the obliteration of the cisterna magnum [ fig . ", "other cranial anomalies , like the collapse of the subarachnoid space , a beak - shaped mesencephalic tectum , dysgenesis of the corpus callosum and subependymal heterotopias are all better seen on mri .", "the differential diagnosis will depend fundamentally on the contents and location of the spinal defect .", "the intracranial anomalies that accompany ntds can help in differentiating between these two entities , because teratomas are not usually accompanied by intracranial anomalies .", "distinguishing between open and closed spinal dysraphism is of the utmost importance because the long - term functional and neurological prognoses are notably better in cases of closed dysraphism than in cases of open dysraphism [ 39 , 41 ] .", "it is of the utmost importance to obtain an accurate and specific assessment of the anomaly due to the progressive incorporation of in utero treatments .", "latest trials in fetal surgery for myelomeningocele have shown a significant reduction of vp shunt placement , an improvement in the overall neuromotor function and a significantly reversed hindbrain herniation in those patients who underwent fetal repair compared with those who underwent postnatal surgery .", "this trial has also revealed that in utero therapy increases the risk of spontaneous rupture of membranes , oligohydramnios and preterm delivery .", "fetal mri provides a better overview for the neurosurgeon who is familiar with mri and not as much with ultrasound .", "specific details with respect to the level and magnitude of the defect can help the intra - uterine management of the spinal anomaly .", "the resolution of mri is less than ultrasound , but the diagnostic value of mri is less dependent on the fetal position .", "the term  closed  is more appropriate than  occult  , because cutaneous signs that raise suspicion of these anomalies are present in up to 50% of cases [ 34 , 43 ] .", "the soft tissues that cover the spine dorsally can be identified as a hypointense line on sagittal and axial t2-weighted images ( figs . ", ", this line is interrupted at the level of the lesion ; this finding is essential for differentiating between open and closed dysraphism .", "closed dysraphism is not usually associated to cranial alterations like chiari malformation or ventriculomegaly [ 1 , 9 ] and does not manifest with elevated alpha - fetoprotein .", "a sagittal t2-weighted haste image shows an interruption in the posterior vertebral arches in the lumbar region ( arrow ) . a posterior fossa anomaly with an elevated vermis and dilation of the iv ventricle is also seen ( asterisk ) .", "b axial image at the lumbar level showing the cystic mass protruding through the spinal defect ( black arrow ) .", "two nerve roots ( white arrow ) can be seen arising from the non - neurulated neural placode inside the spinal defect .", "a sagittal t2-weighted haste image shows an interruption in the posterior vertebral arches in the lumbar region ( arrow ) . a posterior fossa anomaly with an elevated vermis and dilation of the iv ventricle is also seen ( asterisk ) .", "b axial image at the lumbar level showing the cystic mass protruding through the spinal defect ( black arrow ) .", "two nerve roots ( white arrow ) can be seen arising from the non - neurulated neural placode inside the spinal defect .", "d sagittal diagram of the anomaly there are three main types of closed spinal dysraphisms with masses .", "in the first , further subdivided into lipomyelomeningocele and lipomyeloschisis ( lipomyelocele ) ( fig . ", "8) , direct contact between the mesenchymal tissue and neural tube affects the differentiation of fatty tissue .", "clinically , these lesions are characterised by the presence of a lipomatous subcutaneous mass above the gluteal cleft . in these cases , it is important to note the point where the lipoma joins the neural placode to differentiate between a lipomyelomeningocele and lipomyeloschisis .", "the point of union in lipomyelomeningoceles is outside the neural canal due to the expansion of the subarachnoid space , whereas in lipomyeloschisis the lipoma joins the neural placode inside the neural canal after entering through a defect in the bone ( fig . ", "a sagittal t2-weighted haste image shows a deformity of the spinal canal completely covered by soft tissues ( arrows ) .", "b coronal image showing the widening of the spinal canal at the lumbar level ( white arrows ) and abnormally low termination of the medullary cone ( black arrow ) below the kidneys .", "c ct study after birth shows the deformed , widened spinal canal ( arrowheads ) and soft - tissue calcification behind the bone defect ( white arrows ) .", "d sagittal t1-weighted mri image obtained after birth shows the abnormally low medullary cone , a lipomatous component within the spine ( white arrows ) , and soft - tissue calcification ( black arrow ) .", "e axial t1-weighted image showing spinal cord duplication ( black arrows ) and the lipomatous component within the spine ( asterisk ) .", "a sagittal t2-weighted haste image shows a deformity of the spinal canal completely covered by soft tissues ( arrows ) .", "b coronal image showing the widening of the spinal canal at the lumbar level ( white arrows ) and abnormally low termination of the medullary cone ( black arrow ) below the kidneys .", "c ct study after birth shows the deformed , widened spinal canal ( arrowheads ) and soft - tissue calcification behind the bone defect ( white arrows ) .", "d sagittal t1-weighted mri image obtained after birth shows the abnormally low medullary cone , a lipomatous component within the spine ( white arrows ) , and soft - tissue calcification ( black arrow ) .", "e axial t1-weighted image showing spinal cord duplication ( black arrows ) and the lipomatous component within the spine ( asterisk ) .", "g sagittal diagrams of the anomaly the second type is a meningocele with cutaneous lining .", "anterior meningoceles are much less common and tend to be located in the presacral region .", "the third type , myelocystocele ( fig .  7 ) , consists of the herniation of a segment of the spinal cord with a dilated ependymal canal within a meningocele ; these lesions can occur at any point in the spine .", "myelocystoceles are similar to myelomeningoceles ; the two entities are differentiated by the thickness of the wall of the herniated sac ( fig . ", "7b ) , the absence of the chiari malformation , and the absence of elevated alpha - fetoprotein levels [ 1 , 9 ] .", "simple lesions include intradural lipomas , filum terminale lipomas , tight filum terminale syndrome , persistent terminal ventricle , and dermal sinus .", "intradural lipomas are lipomas located within the spine that are contained within the dural sac .", "these lesions generally affect the lumbosacral region and are usually associated with the tethered cord syndrome .", "filum terminale lipomas or fibrolipomatous thickening of the filum terminale can be considered normal variants unless there is clinical evidence of a tethered cord .", "filum terminale lipomas are identified as linear thickening of the filum terminale that is hyperintense on t1-weighted sequences .", "the tight filum terminale syndrome is characterised by shortening and hypertrophy of the filum terminale and consequent cord tethering syndrome .", "persistent terminal ventricle consists of small linear cavities inside the medullary cone immediately above the filum terminale .", "a dermal sinus is an epithelial fistula that connects the neural tissue or the meninges with the surface of the skin .", "dermal sinuses are usually located in the lumbosacral region and are usually found together with an intraspinal dermoid cyst .", "clinically , they present with a small midline pit , a cutaneous nevus , an area of hyperpigmentation , or a capillary haemangioma . detecting all these findings prenatally", "is extremely difficult ; however , the fistulous tract can be identified using high resolution mri techniques .", "these patients require surgical repair after birth because the fistulous tract that communicates the skin with the neural tissue can predispose to infections of the neural contents .", "complex closed spinal dysraphisms can be divided into disorders of midline notochord integration and disorders of notochord malformation .", "disorders of midline notochord integration include enteric fistula , which is an abnormal connection between the digestive tract and the surface of the skin that may or may not be accompanied by a neurenteric cyst .", "neurenteric cysts are lined with epithelial mucosa similar to that of the gastrointestinal tract and are typically located in the anterior cervicothoracic region .", "8) , which consists of a longitudinal splitting of the spinal cord into two hemicords .", "there are two types of diastematomyelia : in type 1 , both hemicords are wrapped in independent meningeal linings and are separated by an osseous or cartilaginous septum ; whereas in type 2 , the two hemicords share a common meningeal lining [ 32 , 48 ] .", "the septum in type 1 diastematomyelia can be seen as a hypointense linear structure within the spinal canal on t2-weighted sequences .", "caution should be taken not to confuse this septum with the spinal cord , as both structures have the same signal intensity .", "the soft tissues and the skin that cover these anomalies are usually intact unless they form part of a more complex anomaly .", "diastematomyelia is normally accompanied by vertebral anomalies like hemivertebrae or butterfly vertebrae , tethered cord , or open spina bifida ( hemimyelomeningocele or hemimyelocele ) .", "disorders of notochord malformation include caudal regression syndrome , which consists of the total or partial agenesis of the lumbar and sacral spine , probably secondary to a disturbance in the formation of the secondary neural tube .", "caudal regression syndrome is divided into two types : in type 1 , the medullary cone is high and ends abruptly ; in type 2 , the spinal cord is tethered and the medullary cone is low - lying [ 32 , 46 ] .", "caudal regression syndrome comprises a wide spectrum of anomalies ranging from sacral agenesis or dysgenesis to sirenomelia ( fusion of the lower limbs ) .", "some authors believe that type 1 and type 2 caudal regression actually represent two distinct entities .", "caudal regression can be found in association with other anomalies like omphalocele , bladder exstrophy , imperforate anus and spinal defects ( oeis complex ) ; vertebral anomalies , anal anomalies , cardiac anomalies , tracheoesophageal fistula , limb malformations , hydrocephalus ( vacterl - h ) ; and sacral agenesis , anal atresia and presacral teratoma or meningocele ( currarino triad ) .", "another disorder of notochord malformation is segmental spinal dysgenesis , which includes agenesis or dysgenesis of the thoracic or lumbar spine , alterations of the spinal cord and nerve roots , congenital paraparesis or paraplegia , and deformities in the lower limbs .", "their prenatal diagnosis can predict the viability of the fetus , has implications for family counselling about the pregnancy , and orients the prognosis and management of the newborn .", "ultrasonography remains the primary imaging test for the prenatal diagnosis of ntds , but sometimes it does not enable an accurate diagnosis so other diagnostic techniques are necessary .", "fetal mri has advanced rapidly in the last 25  years , developing from an experimental technique to become a fundamental tool in normal clinical practice in many centres around the world .", "mri s ability to detect complex anomalies that involve different organs has been widely reported .", "fetal mri should be restricted to centres with experienced staff and should never be considered a replacement for ultrasonography , which continues to be the technique of choice to evaluate fetal morphology ."], "abstract_text": ["<S> objectiveto assess the role of magnetic resonance imaging ( mri ) in the prenatal diagnosis of neural tube defects ( ntds).backgroundntds comprise a heterogeneous group of congenital anomalies that derive from the failure of the neural tube to close . advances in ultrasonography and mri </S>", "<S> have considerably improved the diagnosis and treatment of ntds both before and after birth . </S>", "<S> ultrasonography is the first technique in the morphological study of the fetus , and it often makes it possible to detect or suspect ntds . </S>", "<S> fetal mri is a complementary technique that makes it possible to clear up uncertain ultrasonographic findings and to detect associated anomalies that might go undetected at ultrasonography . </S>", "<S> the progressive incorporation of intrauterine treatments makes an accurate diagnosis of ntds essential to ensure optimal perinatal management . </S>", "<S> the ability of fetal mri to detect complex anomalies that affect different organs has been widely reported , and it can be undertaken whenever ntds are suspected.conclusionwe describe the normal appearance of fetal neural tube on mri , and we discuss the most common anomalies involving the structures and the role of fetal mri in their assessment.key points to learn about the normal anatomy of the neural tube on mri to recognise the mr appearance of neural tube defects to understand the value of mri in assessing ntds </S>"], "labels": null, "section_names": ["None", "Introduction", "MRI protocol and technique", "Anatomy and embryology", "Normal anatomy of the neural tube on MRI", "Neural tube defects", "Anencephaly/exencephaly", "Encephalocele", "Iniencephaly", "Spinal dysraphism or spina bifida", "Conclusion"], "sections": [[" to learn about the normal anatomy of the neural tube on mri  to recognise the mr appearance of neural tube defects  to understand the value of mri in assessing ntds"], ["technological developments in diagnostic imaging have improved the diagnosis and treatment of congenital anomalies immensely , and this progress is especially evident in neural tube defects ( ntds ) .", "ultrasonography allows accurate assessment of the neural tube . fetal magnetic resonance imaging ( mri ) complements ultrasonography , confirming the suspected anomaly and even detecting malformations that were not detected at ultrasonography .", "the additional information provided by mri may lead to changes in the management of the pregnancy and/or of the delivery [ 25 ] . in this article", "we describe the radiological , pathological and clinical features of the most common ntds , and finally emphasise an integrated clinical and radiological approach for accurate diagnosis ."], ["in our institution , all fetal mri studies are performed after ultrasonography and mri never constitutes the first imaging study of the fetus . none of the women or fetuses receives any special preparation .", "although it is important to be extremely careful when undertaking any procedure on a fetus , there is no evidence of associated risk to fetal development with fetal mri [ 68 ] .", "we use a 1.5-tesla scanner with a four - element phased - array body coil .", "a complete study requires between 12 and 15 sequences , so it takes about 2530  min .", "the fetal mri images are obtained using ultrafast sequences to minimise artefacts due to fetal movements .", "it seems that slow , rhythmic maternal respiration reduces fetal movements , so it is not necessary to acquire images with maternal breath - holding .", "t2-weighted single shot fast spin - echo ( ssfse ) or half - fourier single - shot turbo spin - echo ( haste ) images provide most of the information necessary for diagnosis .", "gradient echo sequences ( echo - planar imaging [ epi ] and true fast imaging with steady state precession [ fisp ] ) have greater ferromagnetic susceptibility , so they make it possible to identify bony and vascular structures [ 1 , 9 ] . both the head and spine must be studied in all three planes . even when the only indication for fetal mri is a suspected ntd , all the fetal structures should always be studied to rule out other possible anomalies ."], ["the first phase , gastrulation , begins in the 2nd to 3rd weeks of gestation ; in this phase , the two layers of the embryonic plate divide into three definitive layers ( the ectoderm , mesoderm and endoderm ) . in the second phase , neurulation , which takes place in the 3rd to 4th weeks of gestation", ", the cells that will become the notochord interact with the overlying ectodermic tissue , causing this tissue to thicken , fold over and fuse , thus forming the neural tube .", "the fusion begins in the medial dorsal zone of the embryo and progresses in a zipper - like fashion cranially as the rostral neuropore and caudally as the caudal neuropore .", "the closure of the two ends does not occur simultaneously : the cranial end closes before the caudal end .", "the final phase , secondary neurulation , takes place in the 5th to 6th weeks of gestation ; in this phase a secondary neural tube forms from the cells of hensen s node , which is a mass of totipotent cells located dorsally that intervene in the formation of the notochord and later migrate caudally to the presacral region .", "this secondary neural tube is initially solid ; it undergoes a process of retrogressive differentiation called canalisation in which it cavitates to form the medullary cone and the filum terminale .", "the interruption of this process at any point , whether in the cranial end or in the caudal end , will result in an ntd [ 11 , 12 ] .", "the spinal column and spinal cord are the same length until 1215  weeks gestation , after which the spinal column grows more than the spinal cord until the medullary cone is situated approximately at the level of the second lumbar vertebra , where it will be at birth and will remain without significant changes throughout life [ 11 , 12 ] ."], ["mri is an excellent method for evaluating the structures of the fetal head and spine .", "images can be obtained in any plane , regardless of the position of the fetus in the uterus . the cranial vault ( fig . ", "1a , b ) is seen as a hypointense structure in t2-weighted images , even early in gestation .", "1a ) can also be identified in any plane , but their visualisation improves as gestation advances.fig .", "a sagittal and b coronal t2-weighted haste images of the fetal head show the osseous structures of the base of the skull ( arrows in a ) and bones of the cranial vault ( arrows in b ) .", "the vertebral bodies are hypointense with a band of higher intensity in the centre ( white arrows ) , the intervertebral spaces are hyperintense ( black arrows ) .", "d coronal t2-weighted haste image shows the vertebral pedicles as hypointense rounded structures on both sides of the spinal canal ( arrowheads ) . the interpedicular distance in the lumbar spine", "is slightly increased ( arrows ) ; this is a normal finding that should not be confused with spina bifida .", "e axial image at the level of l1l2 shows the medullary cone ( white arrow ) inside the spinal canal ( the asterisks mark the kidneys ) .", "f the medullary cone is no longer seen in this axial image of the spinal canal below the kidneys ; sometimes the nerve roots can be identified ( white arrows ) a , b normal anatomy of the neural tube .", "a sagittal and b coronal t2-weighted haste images of the fetal head show the osseous structures of the base of the skull ( arrows in a ) and bones of the cranial vault ( arrows in b ) .", "the vertebral bodies are hypointense with a band of higher intensity in the centre ( white arrows ) , the intervertebral spaces are hyperintense ( black arrows ) .", "d coronal t2-weighted haste image shows the vertebral pedicles as hypointense rounded structures on both sides of the spinal canal ( arrowheads ) . the interpedicular distance in the lumbar spine", "is slightly increased ( arrows ) ; this is a normal finding that should not be confused with spina bifida .", "e axial image at the level of l1l2 shows the medullary cone ( white arrow ) inside the spinal canal ( the asterisks mark the kidneys ) .", "f the medullary cone is no longer seen in this axial image of the spinal canal below the kidneys ; sometimes the nerve roots can be identified ( white arrows ) the vertebral bodies have low signal intensity on t2-weighted sequences and are clearly seen on sagittal images .", "the intervertebral spaces are hyperintense and are clearly depicted in the sagittal plane ( fig . ", "the interpedicular distance should be constant throughout the spinal column , although it does increase slightly in the lumbar region and this widening should not be confused with an ntd ( fig . ", "the posterior elements of the vertebrae are best seen in the axial plane ( fig .  1e , f ) .", "caution is warranted in the assessment of possible scoliosis or kyphosis , because the spinal column can be altered by the position of the fetus in the uterus ; these pseudoalterations are more common as gestation advances , resulting in proportionately less space within the uterine cavity ( fig . ", "the spinal canal is hyperintense in t2-weighted sequences because it is filled with cerebrospinal fluid .", "although the spinal cord is well depicted in all three planes , it can probably be evaluated best in the axial plane ( fig . ", "the position of the fetus within the uterus results in a certain degree of thoracolumbar kyphosis that displaces the spinal cord anteriorly in the thoracic spine , making it difficult to see because it is adjacent to the vertebral bodies ( fig . ", "precaution should be taken to ensure that the spinal cord disappears below the fetal kidneys ( fig .  1e , f ) ;", "sometimes , small hypointense structures that correspond to nerve roots are seen distal to the medullary cone ( fig . ", "it is important to evaluate the paravertebral soft tissues in mri studies , especially in fetuses with spina bifida or destructive processes or tumours that could affect these tissues . the skin and the paravertebral muscular structures are hypointense in t2-weighted sequences", ". the spinal canal should be closed posteriorly by soft tissues ( fig .  1c , e , f ) ."], ["ntds are a heterogeneous group of anomalies that result from the partial or total failure of the neural tube to close .", "ntds can affect the cranial region ( anencephaly ) or the spinal region ( spina bifida ) ; spinal ntds can be divided into open or closed defects , depending on whether they are covered by a layer of skin   ( fig . ", "2neural tube defects the overall worldwide incidence of ntds is between one and ten per 1,000 live births [ 14 , 15 ] .", "the rate of ntds has varied widely over time and has a clear geographical distribution .", "the highest incidences have been reported in mexico , ireland , india and northern china [ 16 , 17 ] .", "a family history of ntds is absent in 9095% of cases , and ntds are reported in 3% of miscarriages . some genetic factors like single gene mutations ( meckel - gruber and", "roberts syndromes ) and chromosomopathies ( trisomies 13 and 18 , triploidy ) have been identified .", "some maternal factors ( hyperthermia , diabetes , hyperinsulinaemia , obesity , psychosocial or emotional stress [ 1823 ] ) and some environmental factors ( maternal medication with anticonvulsants like valproic acid and carbamazepine ) can increase the incidence of ntds .", "prenatal screening for ntds includes the measurement of diverse markers like acetylcholinesterase and alpha - fetoprotein in amniotic fluid and in maternal serum ( alpha - fetoprotein is a glycoprotein secreted by the liver and by the fetal yolk sac ) . a recent meta - analysis   confirmed that measuring alpha - fetoprotein in maternal serum during the second trimester is useful in screening for ntds .", "nevertheless , this marker is only useful for detecting open ntds ; furthermore , alpha - fetoprotein levels are also elevated in a long list of fetal anomalies , including omphalocele , turner s syndrome , and sacrococcygeal teratoma , amongst others ."], ["it is the absence of a major portion of the brain , skull and scalp above the orbits .", "there are two subtypes : anencephaly , in which the cranial vault , cerebral hemispheres and diencephalic structures are replaced by amorphous neurovascular tissue , and exencephaly , in which the cranial vault is absent but relatively normal amounts of brain tissue , albeit abnormally developed , is present .", "some authors consider exencephaly an early stage of anencephaly , because the unprotected anomalous brain tissue located outside the cranial vault will eventually take on the appearance of anencephaly after being destroyed by the trauma induced by constant collision against the walls of the uterus . in both exencephaly and anencephaly ,", "the base of the skull , the facial structures , and the orbits are present and are usually normal .", "mri is usually not used to evaluate fetuses with these conditions because ultrasonography usually detects this anomaly very early and mri is not required to confirm this diagnosis .", "fetal mri studies show the absence of osseous structures above the orbits and the absence of brain tissue or alterations in brain tissue when it is present . it is considered a lethal anomaly ,"], ["the specific name of the anomaly depends on the structures contained in the herniated sac ; for example , a meningocele contains meninges and cerebrospinal fluid , an encephalomeningocele contains meninges and brain tissue and an encephalomeningocystocele or encephalomeningohydrocele contains meninges , brain tissue , and part of the ventricular system ( fig .  3 ) .", "most encephaloceles occur along the midline in the occipital or frontal region , but they can also involve the anterior cranial fossa ( into the nasal sinuses ) or even the base of the skull ( into the sphenoid or ethmoid sinus).fig . 3encephalocele .", "a sagittal and b axial t2-weighted haste images show the protrusion of the occipital lobe and occipital horn of the ventricular system ( black arrows in a and b ) through a defect in the bone and soft tissues ( white arrows in a ) .", "c ultrasound image also shows the protrusion of the occipital lobe ( white arrows ) .", "d , e images of the same fetus at 31  weeks gestation show a decrease in the extruded encephalic component ( arrow in e ) and an increase in the associated meningeal component ( arrow in d ) encephalocele .", "a sagittal and b axial t2-weighted haste images show the protrusion of the occipital lobe and occipital horn of the ventricular system ( black arrows in a and b ) through a defect in the bone and soft tissues ( white arrows in a ) .", "c ultrasound image also shows the protrusion of the occipital lobe ( white arrows ) .", "d , e images of the same fetus at 31  weeks gestation show a decrease in the extruded encephalic component ( arrow in e ) and an increase in the associated meningeal component ( arrow in d ) encephaloceles are usually diagnosed by ultrasonography .", "mri s role is to specify the type of lesion , which is more important in smaller lesions and in those that affect the anterior cranial fossa or base of the skull .", "it is essential to evaluate the rest of the structures in the head and the neural tube , fundamentally the posterior fossa because it can be difficult to evaluate with ultrasonography .", "3d ) as well as the presence of brain tissue inside the herniated sac in cases of encephalomeningocele ( fig .  3a , b , e ) .", "other anomalies like open spina bifida or ventriculomegaly that are often associated with encephaloceles are easy to evaluate with mri .", "the differential diagnosis for encephalocele is based on the size , content and location of the lesion .", "the main differential diagnosis for encephaloceles located in the occipital region is with cystic hygroma , whereas for those located in the frontal region , the differential diagnosis is with teratoma and haemangioma of the scalp .", "both teratomas and haemangiomas of the scalp are shown as slightly heterogeneous but well - defined solid masses with variable signal intensity ; haemangiomas may have areas of signal voiding due to their intense vascularisation .", "an encephalocele may form part of a syndrome like meckel - gruber syndrome and it can be associated with other anomalies like ventriculomegaly , spina bifida ( 715% of all encephaloceles ) , microcephaly , or cleft lip and/or cleft palate .", "the prognosis depends on the size , location and extent of the lesion as well as on whether other anomalies are present ."], ["iniencephaly is an ntd affecting the occipital bone combined with rachischisis of the cervicothoracic spine and retroflexion of the head ( fig .  4 ) .", "fetuses with iniencephaly have a variable defect in the occipital bone with an enlarged foramen magnum , the partial or total absence of cervicothoracic vertebrae with irregular fusion of those that are present and incomplete closure of the vertebral bodies and arches .", "these alterations result in significant shortening of the spine and of the fetus due to marked lordosis with hyperextension of the head .", "iniencephaly can be open ( associated with encephalocele ) or closed ( spinal anomaly without encephalocele ) .fig .", "the spine is short , the head is hyperextended , and there is a nuchal fold ( arrow ) .", "the spine is short , the head is hyperextended , and there is a nuchal fold ( arrow ) .", "b pathological specimen the differential diagnosis is with klippel - feil syndrome ( short neck and fusion of the cervical vertebrae ) . some authors believe that klippel - feil syndrome is a milder form of iniencephaly . the prognosis of iniencephaly in the neonatal period is dismal , so whether other anomalies are present is unimportant ."], ["spinal dysraphism is the result of a congenital vertebral defect due to the failure of the caudal neuropore to close that leads to the exposure of the contents of the medullary canal to the exterior .", "the defect usually involves the posterior vertebral elements , although it can also involve the vertebral body itself .", "the defect is usually located in the lumbosacral region , but it can occur at any level and it can even involve the entire spine .", "the absence of skin and muscle in these areas is due to the failure to induct the overlying ectodermal and mesodermal tissues .", "other theories postulate the existence of an unbalance in the production and reabsorption of cerebrospinal fluid during the embryonic period that leads to an excessive accumulation in the already closed neural tube ( hydromyelia ) , which in turn causes a secondary separation from the dorsal wall .", "spinal dysraphism comprises a wide spectrum of anomalies ; these can be divided into open and closed defects .", "rachischisis is the most severe form of spina bifida ; in this lethal condition , an extensive segment of the rachis is absent , resulting in extensive exposure of neural tissue .", "myelomeningocele and myeloschisis ( myelocele ) are the most common spinal dysraphism , accounting for 85% of all cases [ 12 , 32 ] . in these lesions ,", "neural contents are exposed to the exterior through midline defects in the bone and skin [ 12 , 33 ] . when the spinal canal is directly exposed , the anomaly is called myeloschisis or myelocele   ( fig .  5 ) , and when intramedullary structures lined with meninges protrude or herniate due to the expansion of the underlying subarachnoid space , it is called myelomeningocele ( fig .  6 ) .", "myelomeningocele is the most common open spinal dysraphism [ 32 , 35 ] . by definition", ", there must be a vertebral defect , the most common of which is the absence of the posterior vertebral arches ( figs . ", "the main characteristic that allows myelomeningoceles to be differentiated from myeloschisis is the position of the neural placode , a remnant of the unfolded ( non - neurulated ) neural tube , with respect to the surface of the skin . in myelomeningocele , the neural placode protrudes behind the surface of the skin ( fig . ", "6a , b , f ) , whereas in myeloschisis it is at the same level ( fig . ", "another , extremely rare type of open dysraphism is the hemimyelomeningocele or hemimyelocele : in this anomaly , diastematomyelia is associated with one of the above - mentioned anomalies in which one of the hemicords does not complete neurulation [ 32 , 36 ] .", "this finding is important because if diastematomyelia is not suspected , the surgical repair of the myelomeningocele will not include the repair of the tethered cord that is nearly always present in diastematomyelia [ 1 , 9 , 37].fig .", "a , b axial t2-weighted haste image of the fetal cranium shows the deformity of the frontal bones ( black arrows in a ) and the characteristic beaklike shape of the ventricular horns ( arrows in a ) .", "the posterior fossa deformity can also be seen : the cisterna magnum is absent and the foramen magnum is enlarged due to the descent of the structures of the posterior fossa toward the spinal canal ( arrows in b ) .", "c sagittal t2-weighted haste image shows a cutaneous defect or thinning in the lumbosacral region ( arrowhead ) with no mass protruding through the defect as well as an associated chiari malformation ( black arrow ) .", "d axial image at the level of the cutaneous defect shows the abnormally low neural placode within the spinal canal without expansion of the subarachnoid spaces", "a sagittal t2-weighted haste image shows a small cystic mass protruding at the thoracolumbar level ( black arrow )", ". associated hydrocephalus ( asterisk ) and a chiari malformation ( white arrow ) are also seen .", "b axial t2-weighted haste image at the lumbosacral level shows the neural placode protruding outside the surface of the skin due to expansion of the adjacent subarachnoid space ( black arrow ) . c coronal image at the spinal level shows increased interpedicular distance at the level of the neural defect .", "d ultrasound image of a different patient shows a cystic mass protruding at lumbar level ( white arrows ) .", "a , b axial t2-weighted haste image of the fetal cranium shows the deformity of the frontal bones ( black arrows in a ) and the characteristic beaklike shape of the ventricular horns ( arrows in a ) .", "the posterior fossa deformity can also be seen : the cisterna magnum is absent and the foramen magnum is enlarged due to the descent of the structures of the posterior fossa toward the spinal canal ( arrows in b ) . c sagittal t2-weighted haste image shows a cutaneous defect or thinning in the lumbosacral region ( arrowhead ) with no mass protruding through the defect as well as an associated chiari malformation ( black arrow ) .", "d axial image at the level of the cutaneous defect shows the abnormally low neural placode within the spinal canal without expansion of the subarachnoid spaces .", "a sagittal t2-weighted haste image shows a small cystic mass protruding at the thoracolumbar level ( black arrow ) .", "associated hydrocephalus ( asterisk ) and a chiari malformation ( white arrow ) are also seen .", "b axial t2-weighted haste image at the lumbosacral level shows the neural placode protruding outside the surface of the skin due to expansion of the adjacent subarachnoid space ( black arrow ) .", "c coronal image at the spinal level shows increased interpedicular distance at the level of the neural defect .", "d ultrasound image of a different patient shows a cystic mass protruding at lumbar level ( white arrows ) .", "e pathological specimen . f sagittal diagrams of the anomaly open spina bifida is nearly always found together with chiari type ii malformation , which is an anomaly of the posterior fossa consisting of herniation of part of the cerebellum ( vermis and fourth ventricle ) and of the brainstem through the foramen magnum ( figs . ", "this association is thought to be due to the collapse of the primitive fetal ventricular system ( brought about by the loss of cerebrospinal fluid through the spinal defect ) , which prevents the rhombencephalic vesicle ( from which the brainstem , fourth ventricle , and cerebellum are derived ) from expanding and results in deficient development of the posterior fossa and herniation .", "recent studies have reported worse prognoses for fetuses with greater cerebellar herniation , related to an increased incidence of seizures , vesical dysfunction , and dependency for ambulation .", "open spina bifida is accompanied by a collapse of the extra - axial subarachnoid spaces ( fig . ", "6a ) secondary to mechanical obstruction from the chiari malformation or to dysfunctional reabsorption of cerebrospinal fluid [ 38 , 40 ] in 75% of cases .", "this is why it is essential to evaluate the spine carefully to rule out spinal dysraphism when ventriculomegaly is detected at any gestational age .", "likewise , the shape of the ventricles is abnormal owing to the acute angle of the ventricular horns   ( fig . ", "5a ) . as in ultrasonography , in mri both direct and the indirect signs of spina bifida at the level of the fetal cranium ( the lemon sign : special shape of the cranium due to deformity of the frontal bones [ fig . ", "5a ] and the banana sign : special shape of the posterior fossa secondary to displacement of the cerebellar hemispheres toward the cervical canal , which results in the obliteration of the cisterna magnum [ fig . ", "other cranial anomalies , like the collapse of the subarachnoid space , a beak - shaped mesencephalic tectum , dysgenesis of the corpus callosum and subependymal heterotopias are all better seen on mri .", "the differential diagnosis will depend fundamentally on the contents and location of the spinal defect .", "the intracranial anomalies that accompany ntds can help in differentiating between these two entities , because teratomas are not usually accompanied by intracranial anomalies .", "distinguishing between open and closed spinal dysraphism is of the utmost importance because the long - term functional and neurological prognoses are notably better in cases of closed dysraphism than in cases of open dysraphism [ 39 , 41 ] .", "it is of the utmost importance to obtain an accurate and specific assessment of the anomaly due to the progressive incorporation of in utero treatments .", "latest trials in fetal surgery for myelomeningocele have shown a significant reduction of vp shunt placement , an improvement in the overall neuromotor function and a significantly reversed hindbrain herniation in those patients who underwent fetal repair compared with those who underwent postnatal surgery .", "this trial has also revealed that in utero therapy increases the risk of spontaneous rupture of membranes , oligohydramnios and preterm delivery .", "fetal mri provides a better overview for the neurosurgeon who is familiar with mri and not as much with ultrasound .", "specific details with respect to the level and magnitude of the defect can help the intra - uterine management of the spinal anomaly .", "the resolution of mri is less than ultrasound , but the diagnostic value of mri is less dependent on the fetal position .", "the term  closed  is more appropriate than  occult  , because cutaneous signs that raise suspicion of these anomalies are present in up to 50% of cases [ 34 , 43 ] .", "the soft tissues that cover the spine dorsally can be identified as a hypointense line on sagittal and axial t2-weighted images ( figs . ", ", this line is interrupted at the level of the lesion ; this finding is essential for differentiating between open and closed dysraphism .", "closed dysraphism is not usually associated to cranial alterations like chiari malformation or ventriculomegaly [ 1 , 9 ] and does not manifest with elevated alpha - fetoprotein .", "a sagittal t2-weighted haste image shows an interruption in the posterior vertebral arches in the lumbar region ( arrow ) . a posterior fossa anomaly with an elevated vermis and dilation of the iv ventricle is also seen ( asterisk ) .", "b axial image at the lumbar level showing the cystic mass protruding through the spinal defect ( black arrow ) .", "two nerve roots ( white arrow ) can be seen arising from the non - neurulated neural placode inside the spinal defect .", "a sagittal t2-weighted haste image shows an interruption in the posterior vertebral arches in the lumbar region ( arrow ) . a posterior fossa anomaly with an elevated vermis and dilation of the iv ventricle is also seen ( asterisk ) .", "b axial image at the lumbar level showing the cystic mass protruding through the spinal defect ( black arrow ) .", "two nerve roots ( white arrow ) can be seen arising from the non - neurulated neural placode inside the spinal defect .", "d sagittal diagram of the anomaly there are three main types of closed spinal dysraphisms with masses .", "in the first , further subdivided into lipomyelomeningocele and lipomyeloschisis ( lipomyelocele ) ( fig . ", "8) , direct contact between the mesenchymal tissue and neural tube affects the differentiation of fatty tissue .", "clinically , these lesions are characterised by the presence of a lipomatous subcutaneous mass above the gluteal cleft . in these cases , it is important to note the point where the lipoma joins the neural placode to differentiate between a lipomyelomeningocele and lipomyeloschisis .", "the point of union in lipomyelomeningoceles is outside the neural canal due to the expansion of the subarachnoid space , whereas in lipomyeloschisis the lipoma joins the neural placode inside the neural canal after entering through a defect in the bone ( fig . ", "a sagittal t2-weighted haste image shows a deformity of the spinal canal completely covered by soft tissues ( arrows ) .", "b coronal image showing the widening of the spinal canal at the lumbar level ( white arrows ) and abnormally low termination of the medullary cone ( black arrow ) below the kidneys .", "c ct study after birth shows the deformed , widened spinal canal ( arrowheads ) and soft - tissue calcification behind the bone defect ( white arrows ) .", "d sagittal t1-weighted mri image obtained after birth shows the abnormally low medullary cone , a lipomatous component within the spine ( white arrows ) , and soft - tissue calcification ( black arrow ) .", "e axial t1-weighted image showing spinal cord duplication ( black arrows ) and the lipomatous component within the spine ( asterisk ) .", "a sagittal t2-weighted haste image shows a deformity of the spinal canal completely covered by soft tissues ( arrows ) .", "b coronal image showing the widening of the spinal canal at the lumbar level ( white arrows ) and abnormally low termination of the medullary cone ( black arrow ) below the kidneys .", "c ct study after birth shows the deformed , widened spinal canal ( arrowheads ) and soft - tissue calcification behind the bone defect ( white arrows ) .", "d sagittal t1-weighted mri image obtained after birth shows the abnormally low medullary cone , a lipomatous component within the spine ( white arrows ) , and soft - tissue calcification ( black arrow ) .", "e axial t1-weighted image showing spinal cord duplication ( black arrows ) and the lipomatous component within the spine ( asterisk ) .", "g sagittal diagrams of the anomaly the second type is a meningocele with cutaneous lining .", "anterior meningoceles are much less common and tend to be located in the presacral region .", "the third type , myelocystocele ( fig .  7 ) , consists of the herniation of a segment of the spinal cord with a dilated ependymal canal within a meningocele ; these lesions can occur at any point in the spine .", "myelocystoceles are similar to myelomeningoceles ; the two entities are differentiated by the thickness of the wall of the herniated sac ( fig . ", "7b ) , the absence of the chiari malformation , and the absence of elevated alpha - fetoprotein levels [ 1 , 9 ] .", "simple lesions include intradural lipomas , filum terminale lipomas , tight filum terminale syndrome , persistent terminal ventricle , and dermal sinus .", "intradural lipomas are lipomas located within the spine that are contained within the dural sac .", "these lesions generally affect the lumbosacral region and are usually associated with the tethered cord syndrome .", "filum terminale lipomas or fibrolipomatous thickening of the filum terminale can be considered normal variants unless there is clinical evidence of a tethered cord .", "filum terminale lipomas are identified as linear thickening of the filum terminale that is hyperintense on t1-weighted sequences .", "the tight filum terminale syndrome is characterised by shortening and hypertrophy of the filum terminale and consequent cord tethering syndrome .", "persistent terminal ventricle consists of small linear cavities inside the medullary cone immediately above the filum terminale .", "a dermal sinus is an epithelial fistula that connects the neural tissue or the meninges with the surface of the skin .", "dermal sinuses are usually located in the lumbosacral region and are usually found together with an intraspinal dermoid cyst .", "clinically , they present with a small midline pit , a cutaneous nevus , an area of hyperpigmentation , or a capillary haemangioma . detecting all these findings prenatally", "is extremely difficult ; however , the fistulous tract can be identified using high resolution mri techniques .", "these patients require surgical repair after birth because the fistulous tract that communicates the skin with the neural tissue can predispose to infections of the neural contents .", "complex closed spinal dysraphisms can be divided into disorders of midline notochord integration and disorders of notochord malformation .", "disorders of midline notochord integration include enteric fistula , which is an abnormal connection between the digestive tract and the surface of the skin that may or may not be accompanied by a neurenteric cyst .", "neurenteric cysts are lined with epithelial mucosa similar to that of the gastrointestinal tract and are typically located in the anterior cervicothoracic region .", "8) , which consists of a longitudinal splitting of the spinal cord into two hemicords .", "there are two types of diastematomyelia : in type 1 , both hemicords are wrapped in independent meningeal linings and are separated by an osseous or cartilaginous septum ; whereas in type 2 , the two hemicords share a common meningeal lining [ 32 , 48 ] .", "the septum in type 1 diastematomyelia can be seen as a hypointense linear structure within the spinal canal on t2-weighted sequences .", "caution should be taken not to confuse this septum with the spinal cord , as both structures have the same signal intensity .", "the soft tissues and the skin that cover these anomalies are usually intact unless they form part of a more complex anomaly .", "diastematomyelia is normally accompanied by vertebral anomalies like hemivertebrae or butterfly vertebrae , tethered cord , or open spina bifida ( hemimyelomeningocele or hemimyelocele ) .", "disorders of notochord malformation include caudal regression syndrome , which consists of the total or partial agenesis of the lumbar and sacral spine , probably secondary to a disturbance in the formation of the secondary neural tube .", "caudal regression syndrome is divided into two types : in type 1 , the medullary cone is high and ends abruptly ; in type 2 , the spinal cord is tethered and the medullary cone is low - lying [ 32 , 46 ] .", "caudal regression syndrome comprises a wide spectrum of anomalies ranging from sacral agenesis or dysgenesis to sirenomelia ( fusion of the lower limbs ) .", "some authors believe that type 1 and type 2 caudal regression actually represent two distinct entities .", "caudal regression can be found in association with other anomalies like omphalocele , bladder exstrophy , imperforate anus and spinal defects ( oeis complex ) ; vertebral anomalies , anal anomalies , cardiac anomalies , tracheoesophageal fistula , limb malformations , hydrocephalus ( vacterl - h ) ; and sacral agenesis , anal atresia and presacral teratoma or meningocele ( currarino triad ) .", "another disorder of notochord malformation is segmental spinal dysgenesis , which includes agenesis or dysgenesis of the thoracic or lumbar spine , alterations of the spinal cord and nerve roots , congenital paraparesis or paraplegia , and deformities in the lower limbs ."], ["their prenatal diagnosis can predict the viability of the fetus , has implications for family counselling about the pregnancy , and orients the prognosis and management of the newborn .", "ultrasonography remains the primary imaging test for the prenatal diagnosis of ntds , but sometimes it does not enable an accurate diagnosis so other diagnostic techniques are necessary .", "fetal mri has advanced rapidly in the last 25  years , developing from an experimental technique to become a fundamental tool in normal clinical practice in many centres around the world .", "mri s ability to detect complex anomalies that involve different organs has been widely reported .", "fetal mri should be restricted to centres with experienced staff and should never be considered a replacement for ultrasonography , which continues to be the technique of choice to evaluate fetal morphology ."]]}
{"article_id": "PMC4197389", "article_text": ["patient acuity has been increasing , patient turnover is higher , and patients are frequently of advanced age with several comorbid disorders .", "limited resources and the pressure for higher efficiency can result in the suboptimal delivery of healthcare . during treatment within the healthcare system , a significant percentage of patients ( 316% ) suffer from adverse events through system errors- with 1565% of adverse patient events being attributed to communication failure .", "patient care can involve several health care providers with several transfers between providers . in this transition ,", "poor or incomplete relay of patient data may result in errors , near misses and adverse events .", "standardized and thorough communication of critical information is an essential component of patient safety , .", "although individual us based handover guidelines , and handover tools   have been developed , these practices are not widespread .", "a previous survey study observed that 31% of mds experienced daily clinical problems which they believed avoidable if provided with adequate hand - over processes .", "widespread us practice of verbal hand - over has been observed to be highly inefficient .", "the world health organization listed  communication during patient care handovers  as one of its high 5 patient safety initiatives .", "the joint commission international listed their 2nd national patient safety goal as to  improve the effectiveness of communication among caregivers  .", "there is a lack of studies on how anaesthesiologists hand over their patients , which is surprising given that anesthesiology is a specialty that has always aimed at placing patient safety as its top priority", ". the standards of postoperative care issued by the american society of anesthesiology does not state how handover should be performed nor what information it should include , leaving the handover process decision up to the individual clinician .", "studies analyzing anesthetic team handover in the recovery room observed that the majority of handovers were remarkably brief and insufficient- .", "in these studies , critical details such as the preoperative state of the patient , type of operation performed , amount and kinds of analgesics given and problems encountered during the case were either barely mentioned or underplayed . in some instances significant intraoperative events were completely disregarded .", "additionally , there were frequent distractions present at the time of the hand - over .", "there are no reported studies on handover practices performed by anesthesia providers when transition of care occurs intraoperatively .", "this preliminary pilot study was a qualitative rather than quantitative survey to highlight the scope of the current us intraoperative handover of patient information .", "this study had institutional approval ( wayne state university irb ) . due to the anonymity of the suvey content ,", "this study used an online questionnaire format with 8 survey questions created using a computerized online survey program ( survey monkey , palo alto , ca , usa , ) .", "an e - mail with a link to the questionnaire was sent to anesthesia providers at all anesthesia residency programs nationwide ( 120 out the 132 us programs encompassing around 4500 residents and their attending academic mdas ) and a smaller survey selection of crnas ( 10 institutions about 300 crnas in the metropolitan area of detroit , mi ) .", "all questionnaires were anonymous and were only sent to us anesthesia providers working in an academic hospital setting with an associated residency program in anesthesiology .", "the only demographics of surveyed anesthesia health care providers obtained were for medical training level ( certified registered nurse anesthetist -crna , medical doctor of anesthesiology resident mdar , or medical doctor of anesthesiology mda ) .", "from all questionnaires received ( n  =  216 ) , 87 ( 40.3% ) of respondents were mdas , 71 ( 32.9% ) were mdar , and 58 were crnas ( 26.9% ; table  1 ) .", "the response rate to this survey was low with a response from approximately 5% of the resident population in us anesthesia programs and approximately 20% of the crnas surveyed in the metropolitan detroit area .", "of all respondents , 108 ( 49.1 % ) stated that they did not have a handover protocol at the institution where they practiced ( table  1 ) . of the respondents who did have an institutional handover policy", "furrthermmore , 85.7% respondents reported not having a departmental standardized handover form ( table  2 ) .", "of all responders , 84.8% ( 173/204 ) had an experience where they had not received sufficient information during a patient handover .", "only 7.4% ( 15/203 ) of respondents had never experienced complications or mismanagement due to poor or incomplete handover . in comparison ,", "59.6% ( 121/203 ) reported rarely having complications , 30.5% ( 62/203 ) reported sometimes having complications and 2.5% ( 5/203 ) reported frequently having complications ( table  3 ) .", "respondents were asked to respond on the frequency in which they addressed the specific handover information such as allergies and medications , past medical history , intraoperative events ( anesthesia related and surgery related ) , postoperative plans ( extubation / icu , etc . ) , special concerns ( code status , jehovah 's witness , type and screen antibodies , airway information ( ie .", "h / o difficult intubation ) and significant diagnostic studies ( e.g blood lab , ekg , chest x - ray , stress tests , pulmonary function tests , etc . ) . for this specific handover information ,", "the majority of providers frequently or always relayed information regarding patient allergies ( 89.2% ) , past medical history ( 99% ) , anesthesia related intraoperative events ( 99% ) , surgery related intraoperative events ( 91.7% ) , airway information ( 97.5% ) and significant diagnostic studies ( 84.2% ; fig .  1 ) .", "however , information about medications ( 31.4% ) , postoperative plan ( 19.1% ) , code status ( 75.4% ) , information about refusal of blood ( 21.1% ) and type and screen antibodies ( 49% ) were either sometimes , rarely or never included during handover . from the specific list of patient handover information factors ,", "80.5% ( 165/204 ) of responders stated that these factors would be sufficient for an effective , complete handover . on a scale from 15 ,", "key - ( 1  =   never , 2  =   rarely , 3  =   sometimes , 4  =   frequently , 5  =   always .", "mdar - medical doctor of anesthesiology resident , crna- certified registered nurse anesthetist , mda - medical doctor of anesthesiology .", "ponv : post - operative nauseaand vommitting ; mh : malignant hyperthermia ; iv : intravenous .", "this pilot study was a qualitative rather than quantitative survey to highlight the scope of the current problem of us intraoperative handover of patient information .", "poor transition of patient care from the operating room has a high potential for adverse patient outcomes .", "our study indicates that the current intraoperatvive handover processes among anesthesia providers do not have standard uniform practices , and there may be a high proportion of insufficient patient handover practices .", "these findings are surprising given that the anesthesiology specialty places patient safety as one of its top objectives and that all survey respondents practiced in an academic institution .", "major limitations of this study were the low response rate of 216 respondents and that due to annonymous nature of this survey the number of individual academic institutitions could not be determined .", "additionally , there is the possibility that there was a bias towards an increased proportion of responses from anesthesia providers who had prior poor handover experiences and were unsatisified with current practice .", "the lack of standardized handover process has resulted in substandard patient care in us healthcare setting , .", ", we hope that our study will stimulate a discussion of us handover practices in the anesthesia field . increased awareness in handover practices", "we believe that patient handover practices should be discussed and an optimal response would be the formation of an expert consortium with the aim to develop and implement standardized protocols ."], "abstract_text": ["<S> abstractcurrently , no reported studies have evaluated intraoperative handover among anesthesia providers . </S>", "<S> studies on anesthetic handover in the us recovery room setting observed that handover processes are insufficient and , in many instances , significant intraoperative events are disregarded . </S>", "<S> an online survey tool was sent to anesthesia providers at us anesthesia residency programs nationwide ( 120 out of the 132 us programs encompassing around 4500 residents and their academic mdas ) and a smaller survey selection of crnas ( 10 institutions about 300 crnas in the metropolitan area of detroit , mi , usa ) to collect information on handover practices . </S>", "<S> the response rate to this survey ( n  =  216 ) was comprised of approximately 5% ( n  =  71 ) of the resident population in us anesthesia programs , 5% ( n  =  87 ) of mdas , and 20% ( n  =  58 ) of the crnas . </S>", "<S> out of all respondents ( n  =  212 ) , 49.1 % had no hand - over protocol at their institution and 88% of respondents who did have institutional handover protocols believed them insufficient for effective patient handover . in addiiton , </S>", "<S> 84.8% of all responders reported situations where there was insufficient information received during a patient handover . </S>", "<S> only 7% of the respondents reported never experiencing complications or mismanagement due to poor or incomplete hand - overs . </S>", "<S> in contrast , 60% reported rarely having complications , 31% reported sometimes having complications , and 3% reported frequent complications . </S>", "<S> in conclusion , handover transition of patient care is a vulnerable and potentially life - threatening event in the operating room . </S>", "<S> our preliminary study suggests that current intraoperatvive handover practices among anesthesia providers are suboptimal and that national patient handover guidelines are required to improve patient safety . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["patient acuity has been increasing , patient turnover is higher , and patients are frequently of advanced age with several comorbid disorders .", "limited resources and the pressure for higher efficiency can result in the suboptimal delivery of healthcare . during treatment within the healthcare system , a significant percentage of patients ( 316% ) suffer from adverse events through system errors- with 1565% of adverse patient events being attributed to communication failure .", "patient care can involve several health care providers with several transfers between providers . in this transition ,", "poor or incomplete relay of patient data may result in errors , near misses and adverse events .", "standardized and thorough communication of critical information is an essential component of patient safety , .", "although individual us based handover guidelines , and handover tools   have been developed , these practices are not widespread .", "a previous survey study observed that 31% of mds experienced daily clinical problems which they believed avoidable if provided with adequate hand - over processes .", "widespread us practice of verbal hand - over has been observed to be highly inefficient .", "the world health organization listed  communication during patient care handovers  as one of its high 5 patient safety initiatives .", "the joint commission international listed their 2nd national patient safety goal as to  improve the effectiveness of communication among caregivers  .", "there is a lack of studies on how anaesthesiologists hand over their patients , which is surprising given that anesthesiology is a specialty that has always aimed at placing patient safety as its top priority", ". the standards of postoperative care issued by the american society of anesthesiology does not state how handover should be performed nor what information it should include , leaving the handover process decision up to the individual clinician .", "studies analyzing anesthetic team handover in the recovery room observed that the majority of handovers were remarkably brief and insufficient- .", "in these studies , critical details such as the preoperative state of the patient , type of operation performed , amount and kinds of analgesics given and problems encountered during the case were either barely mentioned or underplayed . in some instances significant intraoperative events were completely disregarded .", "additionally , there were frequent distractions present at the time of the hand - over .", "there are no reported studies on handover practices performed by anesthesia providers when transition of care occurs intraoperatively .", "this preliminary pilot study was a qualitative rather than quantitative survey to highlight the scope of the current us intraoperative handover of patient information ."], ["this study had institutional approval ( wayne state university irb ) . due to the anonymity of the suvey content ,", "this study used an online questionnaire format with 8 survey questions created using a computerized online survey program ( survey monkey , palo alto , ca , usa , ) .", "an e - mail with a link to the questionnaire was sent to anesthesia providers at all anesthesia residency programs nationwide ( 120 out the 132 us programs encompassing around 4500 residents and their attending academic mdas ) and a smaller survey selection of crnas ( 10 institutions about 300 crnas in the metropolitan area of detroit , mi ) .", "all questionnaires were anonymous and were only sent to us anesthesia providers working in an academic hospital setting with an associated residency program in anesthesiology .", "the only demographics of surveyed anesthesia health care providers obtained were for medical training level ( certified registered nurse anesthetist -crna , medical doctor of anesthesiology resident mdar , or medical doctor of anesthesiology mda ) ."], ["from all questionnaires received ( n  =  216 ) , 87 ( 40.3% ) of respondents were mdas , 71 ( 32.9% ) were mdar , and 58 were crnas ( 26.9% ; table  1 ) .", "the response rate to this survey was low with a response from approximately 5% of the resident population in us anesthesia programs and approximately 20% of the crnas surveyed in the metropolitan detroit area .", "of all respondents , 108 ( 49.1 % ) stated that they did not have a handover protocol at the institution where they practiced ( table  1 ) . of the respondents who did have an institutional handover policy", "furrthermmore , 85.7% respondents reported not having a departmental standardized handover form ( table  2 ) .", "of all responders , 84.8% ( 173/204 ) had an experience where they had not received sufficient information during a patient handover .", "only 7.4% ( 15/203 ) of respondents had never experienced complications or mismanagement due to poor or incomplete handover . in comparison ,", "59.6% ( 121/203 ) reported rarely having complications , 30.5% ( 62/203 ) reported sometimes having complications and 2.5% ( 5/203 ) reported frequently having complications ( table  3 ) .", "respondents were asked to respond on the frequency in which they addressed the specific handover information such as allergies and medications , past medical history , intraoperative events ( anesthesia related and surgery related ) , postoperative plans ( extubation / icu , etc . ) , special concerns ( code status , jehovah 's witness , type and screen antibodies , airway information ( ie .", "h / o difficult intubation ) and significant diagnostic studies ( e.g blood lab , ekg , chest x - ray , stress tests , pulmonary function tests , etc . ) . for this specific handover information ,", "the majority of providers frequently or always relayed information regarding patient allergies ( 89.2% ) , past medical history ( 99% ) , anesthesia related intraoperative events ( 99% ) , surgery related intraoperative events ( 91.7% ) , airway information ( 97.5% ) and significant diagnostic studies ( 84.2% ; fig .  1 ) .", "however , information about medications ( 31.4% ) , postoperative plan ( 19.1% ) , code status ( 75.4% ) , information about refusal of blood ( 21.1% ) and type and screen antibodies ( 49% ) were either sometimes , rarely or never included during handover . from the specific list of patient handover information factors ,", "80.5% ( 165/204 ) of responders stated that these factors would be sufficient for an effective , complete handover . on a scale from 15 ,", "key - ( 1  =   never , 2  =   rarely , 3  =   sometimes , 4  =   frequently , 5  =   always .", "mdar - medical doctor of anesthesiology resident , crna- certified registered nurse anesthetist , mda - medical doctor of anesthesiology .", "ponv : post - operative nauseaand vommitting ; mh : malignant hyperthermia ; iv : intravenous ."], ["this pilot study was a qualitative rather than quantitative survey to highlight the scope of the current problem of us intraoperative handover of patient information .", "poor transition of patient care from the operating room has a high potential for adverse patient outcomes .", "our study indicates that the current intraoperatvive handover processes among anesthesia providers do not have standard uniform practices , and there may be a high proportion of insufficient patient handover practices .", "these findings are surprising given that the anesthesiology specialty places patient safety as one of its top objectives and that all survey respondents practiced in an academic institution .", "major limitations of this study were the low response rate of 216 respondents and that due to annonymous nature of this survey the number of individual academic institutitions could not be determined .", "additionally , there is the possibility that there was a bias towards an increased proportion of responses from anesthesia providers who had prior poor handover experiences and were unsatisified with current practice .", "the lack of standardized handover process has resulted in substandard patient care in us healthcare setting , .", ", we hope that our study will stimulate a discussion of us handover practices in the anesthesia field . increased awareness in handover practices", "we believe that patient handover practices should be discussed and an optimal response would be the formation of an expert consortium with the aim to develop and implement standardized protocols ."]]}
{"article_id": "PMC155639", "article_text": ["slc / ccl21 , normally expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts t cells and dendritic cells ( dc ) [ 1 - 8 ] .", "slc / ccl21 recruits both nave lymphocytes and antigen stimulated dc into t cell zones of secondary lymphoid organs , co - localizing these early immune response constituents that culminate in cognate t cell activation .", "the capacity to facilitate the co - localization of both dc and t cells forms a strong rationale for the use of slc / ccl21 in cancer therapy . in previous studies", "we have shown that the slc / ccl21-mediated anti - tumor responses are accompanied by the enhanced elaboration of ifn , ip-10/cxcl10 , mig / cxcl9 , gm - csf and il-12 , but decreased pge2 , vegf , il-10 , and tgf- at the tumor site .", "in addition , there was a marked increase in cd4and cd8cells secreting ifn and gm - csf as well as dc expressing cd11c infiltrating the tumors .", "mig / cxcl 9 and ip-10/cxcl10 are glutamine - leucine - arginine ( elr)-negative cxc chemokines that are potent chemoattractants for activated t cells [ 11 - 13 ]", ". these chemokines also have in common the ability to signal through a g protein - coupled receptor , cxcr3 , expressed on activated t lymphocytes .", "ip-10/cxcl10 , mig / cxcl9 and ifn are known to have potent anti - tumor activities in vivo [ 14 - 17 ] .", "both mig / cxcl9 and ip-10/cxcl10 are important for the anti - tumor activity mediated by ifn inducing cytokines such as il-12 .", "based on these findings we hypothesized that augmentation in ifn , mig / cxcl9 , and ip-10/cxcl10 would play an important role in the slc / ccl21-mediated anti - tumor response . here", "we show that the cytokines ifn , ip-10/cxcl10 and mig / cxcl9 serve as effector molecules in slc / ccl21-mediated anti - tumor responses .", "host antigen presenting cells ( apc ) are critical for the cross - presentation of tumor antigens .", "however tumors have the capacity to limit apc maturation , function and infiltration of the tumor site [ 21 - 24 ] .", "thus molecules that attract host apc and t cells could serve as potent agents for cancer immunotherapy .", "a potentially effective pathway to restore ag presentation is the establishment of a chemotactic gradient that favors localization of both activated dc and type 1 cytokine producing lymphocytes at the tumor site .", "slc / ccl21 , a cc chemokine expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts naive t cells and dcs [ 1 - 8 ] . because dcs are potent apcs that function as principal activators of t cells , the capacity to facilitate the co - localization of both dc and t cells may reverse tumor - mediated immune suppression and orchestrate effective cell - mediated immune responses [ 9,10,25 - 27 ] .", "in addition to its immunotherapeutic potential , slc / ccl21 has been found to have potent angiostatic effects , thus adding further support for its use in cancer therapy . based on these dual capacities ,", "we as well as others have begun pre - clinical evaluation of slc / ccl21 in several tumor models [ 9,10,25 - 27 ] . utilizing two transplantable murine lung cancer models", ", we have previously shown that the anti - tumor efficacy of slc / ccl21 is t cell - dependent . in both models recombinant", "slc / ccl21 administered intratumorally led to complete tumor eradication in 40 % of the treated mice .", "studies performed in cd4 and cd8 knockout mice also revealed a requirement for both cd4 and cd8 lymphocytes subsets for slc / ccl21 mediated tumor regression .", "consistent with these findings we found that slc therapy did not alter tumor growth in scid mice . in immune competent mice ,", "intratumoral injection of slc / ccl21 led to a significant increase in cd4 and cd8 t lymphocytes as well as dc infiltrating the tumor and the draining lymph nodes .", "the cellular infiltrates were accompanied by an enhanced elaboration of il-12 , ifn , ip-10/cxcl10 and mig / cxcl9 at the tumor site .", "slc / ccl21 is documented to have direct anti - angiogenic effects ; the tumor reductions observed in this model could have been due to participation by t cells secreting ifn leading to inhibition of angiogenesis via mig / cxcl9 and ip-10/cxcl10 , as well as t cell - dependent immunity .", "both mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3-expressing activated t lymphocytes that could further amplify ifn at the tumor site .", "hence we postulated that in addition to ifn , ip-10/cxcl10 and mig / cxcl9 would also be important contributors to the tumor reduction in the context of slc / ccl21 therapy .", "to determine the importance of mig / cxcl9 , ip-10/cxcl10 and ifn in the slc / ccl21 mediated antitumor response , these cytokines were depleted in slc / ccl21-treated mice .", "anti - ip-10/cxcl10 , mig / cxcl9 and ifn antibodies each partially but yet significantly inhibited the anti - tumor efficacy of slc / ccl21 ( figure 1 * p < 0.01 compared to the control antibody group ) .", "neutralization of ifn caused a significant decrease in both mig / cxcl9 and ip-10/cxcl10 indicating that these chemokines are largely ifn dependent .", "thus , an increase in ifn at the tumor site of slc / ccl21-treated mice could explain the relative increases in ip-10/cxcl10 and mig / cxcl9 .", "the converse was also observed ; ifn production at the tumor site was found to be mig / cxcl9- and ip-10/cxcl10-dependent as indicated by the fact that neutralization of these cytokines caused a significant decrease in ifn(figure 2 ) .", "neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines , thus indicating that ifn , mig / cxcl9 and ip-10/cxcl10 are interdependent in the slc / ccl21-mediated anti - tumor responses . based on these findings , we speculated that the decrease of ip-10/cxcl10 and mig / cxcl9 led to a decrease in ifn by limiting the influx of t cells producing ifn at the tumor site .", "because mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3 expressing activated t lymphocytes , we determined if neutralizing these chemokines in vivo in the slc / ccl21-treated mice would decrease the frequency of cxcr3 expressing t cells at the tumor sites .", "compared to slc / ccl21-treated mice receiving control antibody , flow cytometric evaluation of single - cell suspensions of non - necrotic tumors from slc / ccl21-treated mice receiving neutralizing antibodies to mig / cxcl9 , ip-10/cxcl10 and ifn showed 10 , 12 and 31 % decreases , respectively , in the gated cd3 cxcr3 t cell populations ( table 1 ) .", "the fact that neutralization of ifn was the most efficient at decreasing the total number of cxcr3-activated t cells may be due to the decrease in the ifn-dependent cxcr3 ligands mig / cxcl9 and ip-10/cxcl10 .", "individually , neutralizing mig / cxcl9 and/or ip-10/cxcl10 only led to a partial decrease in the total number of cxcr3-activated t cells . because mig / cxcl9 and ip-10/cxcl10 share the same receptor ( cxcr3 ) , one possible explanation of a partial decrease in cxcr3-activated t cells is that neutralization of one ligand may overexpose the receptor to the other ligand .", "furthermore , because murine slc / ccl21 binds cxcr3 receptor , slc / ccl21 may be recruiting cxcr3 t cells directly .", "an alternative explanation is that residual cytokines such as ifn - inducible t cell  chemoattractant ( itac / cxcl11 ) present after in vivo neutralization of ifn , mig , ip-10 may have remaining activity and attract cxcr3 t cells .", "our results imply that all 3 cytokines , ifn , mig and ip-10 play interrelated roles in the recruitment of cxcr3-activated t cells in slc / ccl21-mediated anti - tumor responses . while the number of cd4 and cd8 cells remained unaltered ,", "compared to slc / ccl21-treated mice receiving control antibody , mice administered neutralizing antibodies to mig / cxcl9 , ip-10/cxcl10 and ifn showed respective decreases of 38 , 28 and 16% in the number of cd11cdec205dc infiltrating the tumor ( table 1 ) . at present", "it is not clear why there is a decrease in dc following neutralization of mig / cxcl9 , ip-10/cxcl10 and ifn. a plausible explanation is that these cytokines may be involved in complex interactions with other molecules that are chemotactic for dc .", "further studies are required to delineate the mechanisms responsible for mig / cxcl9 , ip-10/cxcl10 and ifn mediated localization of dc at the tumor site .", "5 days following tumor implantation , mice were treated intratumorally with recombinant murine slc / ccl21 ( 0.5 g ) 3x / week .", "one day before slc / ccl21 administration , mice were given the respective cytokine antibody by i.p .", "antibodies to : ip-10/cxcl10 , mig / cxcl9 and ifn significantly inhibited the antitumor efficacy of slc / ccl21 ( * p < 0.01 compared to the control antibody treated group ) .", "n = 8 mice group slc / ccl21-treated mice had a significant induction in ifn , mig / cxcl9 and ip-10/cxcl10 at the tumor site compared to diluent treated control tumor bearing mice ( p < 0.001 ) .", "assessment of cytokine production at the tumor site of slc / ccl21 treated mice receiving anti - ifn , anti mig / cxcl9 and anti ip-10/cxcl10 showed an interdependence of ifn , mig / cxcl9 and ip-10/cxcl10 : neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines ( * p < 0.01 compared to the control antibody treated group ) .", "n= 8 mice per group neutralization of ip-10 , mig and ifn reduces the frequency of cd3cxcr3 t lymphocytes and cd11cdec205dc at the tumor site of slc / ccl21-treated mice single cell suspensions of non - necrotic tumor nodules from slc / ccl21 treated mice receiving neutralizing antibodies to p-10/cxcl10 , mig / cxcl9 , ifn , control ab and diluent treated tumor bearing mice were prepared .", "cell surface staining for t cell markers cd4 , cd8 , cd3 , and the chemokine receptor cxcr3 as well as dc markers cd11c and dec205 were evaluated by flow cytometry .", "cells were identified as lymphocytes or dc by gating on the forward and side scatter profiles ; 10,000 gated events were collected and analyzed using cell quest software . within the gated t lymphocyte population , intratumoral injection of slc / ccl21 led to an increase in the frequency of cd4 , cd8 , cd3cxcr3events compared to the diluent control ( * p < 0.001 ) . within the gated dc population ,", "intratumoral injection of slc / ccl21 led to an increase in the frequency of cd11cdec205events compared to the diluent control ( * p < 0.001 ) . compared to slc / ccl21-treated mice receiving control antibody , mice receiving neutralizing antibodies to ip-10/cxcl10 , mig / cxcl9 , and ifn showed decrease in the gated cd3cxcr3 t cell as well as cd11cdec205 dc events ( * * p < 0.01 ) ( n = 8 mice per group ) .", "our results suggest that the anti - tumor properties of slc / ccl21 may be due to its chemotactic capacity in co - localization of dc and t cells as well as in part to the induction of key cytokines such as ifn , ip-10/cxcl10 and mig / cxcl9 .", "abs to murine ifn , recombinant ifn were obtained from pharmingen ( san diego , ca ) .", "abs to murine mig / cxcl9 , ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d ( minneapolis , mn ) .", "polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described .", "anti - mouse ifn monoclonal ( r4 - 462 , american type culture collection , rockville , maryland ,", "md ) neutralizing antibody was purified by affinity chromatography from scid mice ascites , which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . for flow cytometry , labeled antibodies ( fitc , pe , percp ) against the t cell surface markers to cd3 , cd4 , cd8 and against the dc markers , cd11c and dec 205 and the appropriate controls were purchased from pharmingen .", "a weakly immunogenic lung cancer , lewis lung carcinoma ( 3ll , h-2 ) obtained from atcc ( crl-1642 , llc ) was utilized for assessment of cytokines important for slc / ccl21  mediated anti - tumor responses in vivo .", "the cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium ( irvine scientific , santa anna , ca ) supplemented with 10% fetal bovine serum ( fbs ) ( gemini bioproducts , calabasas , ca ) , penicillin ( 100 u / ml ) , streptomycin ( 0.1 mg / ml ) , 2 mm glutamine ( jrh biosciences , lenexa , ks ) and maintained at 37c in a humidified atmosphere containing 5% co2 in air .", "the cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . for tumorigenesis experiments ,", "injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week .", "five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 ( pepro tech , rocky hill , nj ) or murine serum albumin ( utilized as an irrelevant protein for control injections ) were administered three times per week for two weeks as previously described .", "24 h prior to slc / ccl21 treatment , and then three times a week , mice were injected i.p . with 1 ml / dose of anti - ip-10/cxcl10 , or anti - mig / cxcl9 , or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies ( goat igg and rat igg ) at equivalent doses for the duration of the experiment .", "in response to these antibodies , there was a significant reduction of the respective cytokines in vivo ( figure 2 ) .", "mig / cxcl9 , ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described .", "flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer ( becton dickinson , san jose , ca ) in the university of california , los angeles , jonsson cancer center flow cytometry core facility . cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles .", "10,000-gated events were collected and analyzed using cell quest software ( becton dickinson ) . for staining , two or three fluorochromes ( pe , fitc , percp ) ( pharmingen ) were used to gate on the cd3 , cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . for cxcr3 expression , t cells", "statistical analyses of the data in figures 1 , 2 and in table 1 were performed using the paired student t test .", "abs to murine ifn , recombinant ifn were obtained from pharmingen ( san diego , ca ) .", "abs to murine mig / cxcl9 , ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d ( minneapolis , mn ) .", "polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described .", "anti - mouse ifn monoclonal ( r4 - 462 , american type culture collection , rockville , maryland ,", "md ) neutralizing antibody was purified by affinity chromatography from scid mice ascites , which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . for flow cytometry , labeled antibodies ( fitc , pe , percp ) against the t cell surface markers to cd3 , cd4 , cd8 and against the dc markers , cd11c and dec 205 and the appropriate controls were purchased from pharmingen .", "a weakly immunogenic lung cancer , lewis lung carcinoma ( 3ll , h-2 ) obtained from atcc ( crl-1642 , llc ) was utilized for assessment of cytokines important for slc / ccl21  mediated anti - tumor responses in vivo .", "the cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium ( irvine scientific , santa anna , ca ) supplemented with 10% fetal bovine serum ( fbs ) ( gemini bioproducts , calabasas , ca ) , penicillin ( 100 u / ml ) , streptomycin ( 0.1 mg / ml ) , 2 mm glutamine ( jrh biosciences , lenexa , ks ) and maintained at 37c in a humidified atmosphere containing 5% co2 in air .", "the cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . for tumorigenesis experiments ,", "injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week .", "five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 ( pepro tech , rocky hill , nj ) or murine serum albumin ( utilized as an irrelevant protein for control injections ) were administered three times per week for two weeks as previously described .", "24 h prior to slc / ccl21 treatment , and then three times a week , mice were injected i.p . with 1 ml / dose of anti - ip-10/cxcl10 , or anti - mig / cxcl9 , or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies ( goat igg and rat igg ) at equivalent doses for the duration of the experiment .", "in response to these antibodies , there was a significant reduction of the respective cytokines in vivo ( figure 2 ) .", "mig / cxcl9 , ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described .", "flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer ( becton dickinson , san jose , ca ) in the university of california , los angeles , jonsson cancer center flow cytometry core facility .", "cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles .", "10,000-gated events were collected and analyzed using cell quest software ( becton dickinson ) . for staining , two or three fluorochromes ( pe , fitc , percp ) ( pharmingen ) were used to gate on the cd3 , cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . for cxcr3 expression ,", "statistical analyses of the data in figures 1 , 2 and in table 1 were performed using the paired student t test .", "authors sh , lxz , mh , rkb and mdb contributed to the discussion of the article .", "authors rms and smd together with ss conceived of the study , participated in its design and drafted the manuscript .", "we thank kimberly atianzar for excellent technical assistance in conducting the elisa measurements . national institutes of health grant r01 ca78654 , ca85686 , p50 ca90388 , medical research funds from the department of veterans affairs , the research enhancement award program in cancer gene medicine and the tobacco - related disease research program of the university of california supported this work ."], "abstract_text": ["<S> backgroundslc / ccl21 , normally expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts t cells and dendritic cells ( dc ) . </S>", "<S> we have previously shown that slc / ccl21-mediated anti - tumor responses are accompanied by significant induction of ifn and the cxc chemokines , monokine induced by ifn ( mig / cxcl9 ) and ifn-inducible protein-10 ( ip-10/cxcl10).resultswe assessed the importance of ifn , ip-10/cxcl10 and mig / cxcl9 in slc / ccl21 therapy . in vivo depletion of ip-10/cxcl10 , mig / cxcl9 or ifn significantly reduced the anti - tumor efficacy of slc / ccl21 . </S>", "<S> assessment of cytokine production at the tumor site showed an interdependence of ifn , mig / cxcl9 and ip-10/cxcl10 ; neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines . </S>", "<S> similarly , neutralization of any one of these cytokines led to a decrease in the frequency of cxcr3+ve t cells and cd11c+ve dc at the tumor site.conclusionthese findings indicate that the full potency of slc / ccl21-mediated anti - tumor responses require in part the induction of ifn , mig / cxcl9 and ip-10/cxcl10 . </S>"], "labels": null, "section_names": ["Background", "Results and Discussion", "Conclusions", "Materials and Methods", "Reagents", "Cell culture and tumorigenesis model", "Cytokine ELISA", "Flow Cytometry", "Statistical Analysis", "Author's contributions", "Acknowledgements"], "sections": [["slc / ccl21 , normally expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts t cells and dendritic cells ( dc ) [ 1 - 8 ] .", "slc / ccl21 recruits both nave lymphocytes and antigen stimulated dc into t cell zones of secondary lymphoid organs , co - localizing these early immune response constituents that culminate in cognate t cell activation .", "the capacity to facilitate the co - localization of both dc and t cells forms a strong rationale for the use of slc / ccl21 in cancer therapy . in previous studies", "we have shown that the slc / ccl21-mediated anti - tumor responses are accompanied by the enhanced elaboration of ifn , ip-10/cxcl10 , mig / cxcl9 , gm - csf and il-12 , but decreased pge2 , vegf , il-10 , and tgf- at the tumor site .", "in addition , there was a marked increase in cd4and cd8cells secreting ifn and gm - csf as well as dc expressing cd11c infiltrating the tumors .", "mig / cxcl 9 and ip-10/cxcl10 are glutamine - leucine - arginine ( elr)-negative cxc chemokines that are potent chemoattractants for activated t cells [ 11 - 13 ]", ". these chemokines also have in common the ability to signal through a g protein - coupled receptor , cxcr3 , expressed on activated t lymphocytes .", "ip-10/cxcl10 , mig / cxcl9 and ifn are known to have potent anti - tumor activities in vivo [ 14 - 17 ] .", "both mig / cxcl9 and ip-10/cxcl10 are important for the anti - tumor activity mediated by ifn inducing cytokines such as il-12 .", "based on these findings we hypothesized that augmentation in ifn , mig / cxcl9 , and ip-10/cxcl10 would play an important role in the slc / ccl21-mediated anti - tumor response . here", "we show that the cytokines ifn , ip-10/cxcl10 and mig / cxcl9 serve as effector molecules in slc / ccl21-mediated anti - tumor responses ."], ["host antigen presenting cells ( apc ) are critical for the cross - presentation of tumor antigens .", "however tumors have the capacity to limit apc maturation , function and infiltration of the tumor site [ 21 - 24 ] .", "thus molecules that attract host apc and t cells could serve as potent agents for cancer immunotherapy .", "a potentially effective pathway to restore ag presentation is the establishment of a chemotactic gradient that favors localization of both activated dc and type 1 cytokine producing lymphocytes at the tumor site .", "slc / ccl21 , a cc chemokine expressed in high endothelial venules and in t cell zones of spleen and lymph nodes , strongly attracts naive t cells and dcs [ 1 - 8 ] . because dcs are potent apcs that function as principal activators of t cells , the capacity to facilitate the co - localization of both dc and t cells may reverse tumor - mediated immune suppression and orchestrate effective cell - mediated immune responses [ 9,10,25 - 27 ] .", "in addition to its immunotherapeutic potential , slc / ccl21 has been found to have potent angiostatic effects , thus adding further support for its use in cancer therapy . based on these dual capacities ,", "we as well as others have begun pre - clinical evaluation of slc / ccl21 in several tumor models [ 9,10,25 - 27 ] . utilizing two transplantable murine lung cancer models", ", we have previously shown that the anti - tumor efficacy of slc / ccl21 is t cell - dependent . in both models recombinant", "slc / ccl21 administered intratumorally led to complete tumor eradication in 40 % of the treated mice .", "studies performed in cd4 and cd8 knockout mice also revealed a requirement for both cd4 and cd8 lymphocytes subsets for slc / ccl21 mediated tumor regression .", "consistent with these findings we found that slc therapy did not alter tumor growth in scid mice . in immune competent mice ,", "intratumoral injection of slc / ccl21 led to a significant increase in cd4 and cd8 t lymphocytes as well as dc infiltrating the tumor and the draining lymph nodes .", "the cellular infiltrates were accompanied by an enhanced elaboration of il-12 , ifn , ip-10/cxcl10 and mig / cxcl9 at the tumor site .", "slc / ccl21 is documented to have direct anti - angiogenic effects ; the tumor reductions observed in this model could have been due to participation by t cells secreting ifn leading to inhibition of angiogenesis via mig / cxcl9 and ip-10/cxcl10 , as well as t cell - dependent immunity .", "both mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3-expressing activated t lymphocytes that could further amplify ifn at the tumor site .", "hence we postulated that in addition to ifn , ip-10/cxcl10 and mig / cxcl9 would also be important contributors to the tumor reduction in the context of slc / ccl21 therapy .", "to determine the importance of mig / cxcl9 , ip-10/cxcl10 and ifn in the slc / ccl21 mediated antitumor response , these cytokines were depleted in slc / ccl21-treated mice .", "anti - ip-10/cxcl10 , mig / cxcl9 and ifn antibodies each partially but yet significantly inhibited the anti - tumor efficacy of slc / ccl21 ( figure 1 * p < 0.01 compared to the control antibody group ) .", "neutralization of ifn caused a significant decrease in both mig / cxcl9 and ip-10/cxcl10 indicating that these chemokines are largely ifn dependent .", "thus , an increase in ifn at the tumor site of slc / ccl21-treated mice could explain the relative increases in ip-10/cxcl10 and mig / cxcl9 .", "the converse was also observed ; ifn production at the tumor site was found to be mig / cxcl9- and ip-10/cxcl10-dependent as indicated by the fact that neutralization of these cytokines caused a significant decrease in ifn(figure 2 ) .", "neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines , thus indicating that ifn , mig / cxcl9 and ip-10/cxcl10 are interdependent in the slc / ccl21-mediated anti - tumor responses . based on these findings , we speculated that the decrease of ip-10/cxcl10 and mig / cxcl9 led to a decrease in ifn by limiting the influx of t cells producing ifn at the tumor site .", "because mig / cxcl9 and ip-10/cxcl10 are chemotactic for stimulated cxcr3 expressing activated t lymphocytes , we determined if neutralizing these chemokines in vivo in the slc / ccl21-treated mice would decrease the frequency of cxcr3 expressing t cells at the tumor sites .", "compared to slc / ccl21-treated mice receiving control antibody , flow cytometric evaluation of single - cell suspensions of non - necrotic tumors from slc / ccl21-treated mice receiving neutralizing antibodies to mig / cxcl9 , ip-10/cxcl10 and ifn showed 10 , 12 and 31 % decreases , respectively , in the gated cd3 cxcr3 t cell populations ( table 1 ) .", "the fact that neutralization of ifn was the most efficient at decreasing the total number of cxcr3-activated t cells may be due to the decrease in the ifn-dependent cxcr3 ligands mig / cxcl9 and ip-10/cxcl10 .", "individually , neutralizing mig / cxcl9 and/or ip-10/cxcl10 only led to a partial decrease in the total number of cxcr3-activated t cells . because mig / cxcl9 and ip-10/cxcl10 share the same receptor ( cxcr3 ) , one possible explanation of a partial decrease in cxcr3-activated t cells is that neutralization of one ligand may overexpose the receptor to the other ligand .", "furthermore , because murine slc / ccl21 binds cxcr3 receptor , slc / ccl21 may be recruiting cxcr3 t cells directly .", "an alternative explanation is that residual cytokines such as ifn - inducible t cell  chemoattractant ( itac / cxcl11 ) present after in vivo neutralization of ifn , mig , ip-10 may have remaining activity and attract cxcr3 t cells .", "our results imply that all 3 cytokines , ifn , mig and ip-10 play interrelated roles in the recruitment of cxcr3-activated t cells in slc / ccl21-mediated anti - tumor responses . while the number of cd4 and cd8 cells remained unaltered ,", "compared to slc / ccl21-treated mice receiving control antibody , mice administered neutralizing antibodies to mig / cxcl9 , ip-10/cxcl10 and ifn showed respective decreases of 38 , 28 and 16% in the number of cd11cdec205dc infiltrating the tumor ( table 1 ) . at present", "it is not clear why there is a decrease in dc following neutralization of mig / cxcl9 , ip-10/cxcl10 and ifn. a plausible explanation is that these cytokines may be involved in complex interactions with other molecules that are chemotactic for dc .", "further studies are required to delineate the mechanisms responsible for mig / cxcl9 , ip-10/cxcl10 and ifn mediated localization of dc at the tumor site .", "5 days following tumor implantation , mice were treated intratumorally with recombinant murine slc / ccl21 ( 0.5 g ) 3x / week .", "one day before slc / ccl21 administration , mice were given the respective cytokine antibody by i.p .", "antibodies to : ip-10/cxcl10 , mig / cxcl9 and ifn significantly inhibited the antitumor efficacy of slc / ccl21 ( * p < 0.01 compared to the control antibody treated group ) .", "n = 8 mice group slc / ccl21-treated mice had a significant induction in ifn , mig / cxcl9 and ip-10/cxcl10 at the tumor site compared to diluent treated control tumor bearing mice ( p < 0.001 ) .", "assessment of cytokine production at the tumor site of slc / ccl21 treated mice receiving anti - ifn , anti mig / cxcl9 and anti ip-10/cxcl10 showed an interdependence of ifn , mig / cxcl9 and ip-10/cxcl10 : neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines ( * p < 0.01 compared to the control antibody treated group ) .", "n= 8 mice per group neutralization of ip-10 , mig and ifn reduces the frequency of cd3cxcr3 t lymphocytes and cd11cdec205dc at the tumor site of slc / ccl21-treated mice single cell suspensions of non - necrotic tumor nodules from slc / ccl21 treated mice receiving neutralizing antibodies to p-10/cxcl10 , mig / cxcl9 , ifn , control ab and diluent treated tumor bearing mice were prepared .", "cell surface staining for t cell markers cd4 , cd8 , cd3 , and the chemokine receptor cxcr3 as well as dc markers cd11c and dec205 were evaluated by flow cytometry .", "cells were identified as lymphocytes or dc by gating on the forward and side scatter profiles ; 10,000 gated events were collected and analyzed using cell quest software . within the gated t lymphocyte population , intratumoral injection of slc / ccl21 led to an increase in the frequency of cd4 , cd8 , cd3cxcr3events compared to the diluent control ( * p < 0.001 ) . within the gated dc population ,", "intratumoral injection of slc / ccl21 led to an increase in the frequency of cd11cdec205events compared to the diluent control ( * p < 0.001 ) . compared to slc / ccl21-treated mice receiving control antibody , mice receiving neutralizing antibodies to ip-10/cxcl10 , mig / cxcl9 , and ifn showed decrease in the gated cd3cxcr3 t cell as well as cd11cdec205 dc events ( * * p < 0.01 ) ( n = 8 mice per group ) ."], ["our results suggest that the anti - tumor properties of slc / ccl21 may be due to its chemotactic capacity in co - localization of dc and t cells as well as in part to the induction of key cytokines such as ifn , ip-10/cxcl10 and mig / cxcl9 ."], ["abs to murine ifn , recombinant ifn were obtained from pharmingen ( san diego , ca ) .", "abs to murine mig / cxcl9 , ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d ( minneapolis , mn ) .", "polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described .", "anti - mouse ifn monoclonal ( r4 - 462 , american type culture collection , rockville , maryland ,", "md ) neutralizing antibody was purified by affinity chromatography from scid mice ascites , which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . for flow cytometry , labeled antibodies ( fitc , pe , percp ) against the t cell surface markers to cd3 , cd4 , cd8 and against the dc markers , cd11c and dec 205 and the appropriate controls were purchased from pharmingen .", "a weakly immunogenic lung cancer , lewis lung carcinoma ( 3ll , h-2 ) obtained from atcc ( crl-1642 , llc ) was utilized for assessment of cytokines important for slc / ccl21  mediated anti - tumor responses in vivo .", "the cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium ( irvine scientific , santa anna , ca ) supplemented with 10% fetal bovine serum ( fbs ) ( gemini bioproducts , calabasas , ca ) , penicillin ( 100 u / ml ) , streptomycin ( 0.1 mg / ml ) , 2 mm glutamine ( jrh biosciences , lenexa , ks ) and maintained at 37c in a humidified atmosphere containing 5% co2 in air .", "the cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . for tumorigenesis experiments ,", "injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week .", "five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 ( pepro tech , rocky hill , nj ) or murine serum albumin ( utilized as an irrelevant protein for control injections ) were administered three times per week for two weeks as previously described .", "24 h prior to slc / ccl21 treatment , and then three times a week , mice were injected i.p . with 1 ml / dose of anti - ip-10/cxcl10 , or anti - mig / cxcl9 , or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies ( goat igg and rat igg ) at equivalent doses for the duration of the experiment .", "in response to these antibodies , there was a significant reduction of the respective cytokines in vivo ( figure 2 ) .", "mig / cxcl9 , ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described .", "flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer ( becton dickinson , san jose , ca ) in the university of california , los angeles , jonsson cancer center flow cytometry core facility . cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles .", "10,000-gated events were collected and analyzed using cell quest software ( becton dickinson ) . for staining , two or three fluorochromes ( pe , fitc , percp ) ( pharmingen ) were used to gate on the cd3 , cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . for cxcr3 expression , t cells", "statistical analyses of the data in figures 1 , 2 and in table 1 were performed using the paired student t test ."], ["abs to murine ifn , recombinant ifn were obtained from pharmingen ( san diego , ca ) .", "abs to murine mig / cxcl9 , ip-10/cxcl10 and recombinant cytokine standards was purchased from r&d ( minneapolis , mn ) .", "polyclonal goat anti - murine mig / cxcl9 and anti - murine ip-10/cxcl10 specific anti - serum were produced and characterized as previously described .", "anti - mouse ifn monoclonal ( r4 - 462 , american type culture collection , rockville , maryland ,", "md ) neutralizing antibody was purified by affinity chromatography from scid mice ascites , which was generated 34 weeks after intraperitoneal injection of 10r4 - 462 hybridoma cells per mouse . for flow cytometry , labeled antibodies ( fitc , pe , percp ) against the t cell surface markers to cd3 , cd4 , cd8 and against the dc markers , cd11c and dec 205 and the appropriate controls were purchased from pharmingen ."], ["a weakly immunogenic lung cancer , lewis lung carcinoma ( 3ll , h-2 ) obtained from atcc ( crl-1642 , llc ) was utilized for assessment of cytokines important for slc / ccl21  mediated anti - tumor responses in vivo .", "the cells were routinely cultured as monolayers in 25 cmtissue culture flasks containing rpmi 1640 medium ( irvine scientific , santa anna , ca ) supplemented with 10% fetal bovine serum ( fbs ) ( gemini bioproducts , calabasas , ca ) , penicillin ( 100 u / ml ) , streptomycin ( 0.1 mg / ml ) , 2 mm glutamine ( jrh biosciences , lenexa , ks ) and maintained at 37c in a humidified atmosphere containing 5% co2 in air .", "the cell lines were mycoplasma free and cells were utilized up to the tenth passage before thawing frozen stock cells from liquid n2 . for tumorigenesis experiments ,", "injection in the right supra scapular area of c57bl/6 mice and tumor volumes were monitored three times each week .", "five day established tumors were treated with intratumoral injection of 0.5 g of recombinant murine slc / ccl21 ( pepro tech , rocky hill , nj ) or murine serum albumin ( utilized as an irrelevant protein for control injections ) were administered three times per week for two weeks as previously described .", "24 h prior to slc / ccl21 treatment , and then three times a week , mice were injected i.p . with 1 ml / dose of anti - ip-10/cxcl10 , or anti - mig / cxcl9 , or 100 g / dose of purified monoclonal anti - ifn or appropriate control antibodies ( goat igg and rat igg ) at equivalent doses for the duration of the experiment .", "in response to these antibodies , there was a significant reduction of the respective cytokines in vivo ( figure 2 ) ."], ["mig / cxcl9 , ip-10/cxcl10 and ifn were quantified using a modification of a double ligand method as previously described ."], ["flow cytometric analyses for t cell and dc markers were performed on a facscan flow cytometer ( becton dickinson , san jose , ca ) in the university of california , los angeles , jonsson cancer center flow cytometry core facility .", "cells were identified as lymphocytes or dc by gating based on forward and side scatter profiles .", "10,000-gated events were collected and analyzed using cell quest software ( becton dickinson ) . for staining , two or three fluorochromes ( pe , fitc , percp ) ( pharmingen ) were used to gate on the cd3 , cd4 and cd8 t lymphocyte population or cd11c dc in single cell suspensions from tumor nodule . for cxcr3 expression ,"], ["statistical analyses of the data in figures 1 , 2 and in table 1 were performed using the paired student t test ."], ["authors sh , lxz , mh , rkb and mdb contributed to the discussion of the article .", "authors rms and smd together with ss conceived of the study , participated in its design and drafted the manuscript ."], ["we thank kimberly atianzar for excellent technical assistance in conducting the elisa measurements . national institutes of health grant r01 ca78654 , ca85686 , p50 ca90388 , medical research funds from the department of veterans affairs , the research enhancement award program in cancer gene medicine and the tobacco - related disease research program of the university of california supported this work ."]]}
{"article_id": "PMC2655632", "article_text": ["neonatal bacterial meningitis continues to be \n the most common serious infection of the central nervous system ( cns ) in \n newborns with high morbidity and mortality despite the availability of \n effective bactericidal antibiotics over the last sixty years [ 1 , 2 ] .", "this high morbidity and mortality are due to inadequate \n knowledge of the pathogenesis of this disease . \n", "e. coli is the most common gram - negative \n bacterium causing neonatal sepsis and meningitis .", "bacterial meningitis in \n newborns is due to hematogenous spread of the pathogen to the meninges .", "the \n most important issue in the pathogenesis of e. \n coli meningitis is how circulating pathogens cross the blood - brain barrier \n ( bbb ) , which is mainly composed of brain microvascular endothelial cells ( bmecs )   [ 3 , 4 ] .", "our \n previous studies showed that e. coli k1 invasion of human bmec was \n significantly greater with stationary - phase ( sp ) cultures than with \n exponential - phase cultures , suggesting that expression of e. coli k1 invasion - associated virulence \n genes is strongly regulated in a growth - phase - dependent manner .", "a nonsense mutation in the sp regulatory gene rpos was identified in e. coli k1 strains e44 and ihe3034 . \n", "complementation with the wild type e. coli k12 rpos gene \n significantly enhanced ihe3034 invasion of bmec , but failed to improve the invasion activity of another e. \n coli k1 strain e44 .", "these studies suggest that the growth - phase - dependent \n invasion of bmec by ihe3034 is affected by rpos and that e44 carries a \n loss - of - function mutation in the rpos gene .", "several virulence \n factors , including ibe ( termed after   invasion of   brain endothelial \n cells ) proteins [ 6 , 7 ] , ompa   , yiijp , fimh , asla , and traj \n , have been identified in various strains of e. coli in the in vitro and in vivo models of the \n bbb as invasins .", "most of those \n invasion genes are present in the e. coli k-12 genome [ 4 , 16 ] .", "however , the ibea gene encoding a 50 kda protein has been found to be unique to some pathogenic e. coli k1 strains ( e.g. , c5 and rs218 ) , \n while laboratory strains of e. coli k-12 ( e.g. , dh5 and hb101 ) , as well \n as noninvasive e.   coli ( e.g. , e412 ) , \n lack ibea .", "recently , vimentin has \n been identified as an ibea - binding protein on the surface of human bmec . using the ibea gene \n as a probe", ", we have identified a 20.3 kb genomic locus as a genetic island of meningitic e. coli containing ibea ( gima ) .", "this \n locus is situated between yjid and yjie , adjacent to the fim operon , and \n absent in nonpathogenic e. coli k12 \n strains .", "the first three operons \n ( ptnipkc , cgldtec , gcxkrci ) may \n be involved in energy metabolism and the last operon ( iberat ) contributes to e. \n coli k1 invasion of bmec .", "our previous work showed that gima - mediated \n invasion of human endothelial cells is regulated by carbon source .", "this is \n consistent with the observations by others that carbon source modulates \n expression of virulence factors in several pathogenic bacteria .", "ibea and ibet contribute \n to e. coli k1 invasion and adhesion \n [ 18 , 19 ] .", "our previous studies suggest that iber is a novel regulatory protein that is present in pathogenic e. coli k1 .", "it belongs to the \n ntrc / nifa family of transcriptional activators , carrying a sigma 54-interaction \n domain and showing significant \n sequence homology to various regulatory proteins for glycerol metabolism operon \n in citrobacter freundii ( p45512 ) , acetoacetate metabolism in e. coli k12 , \n sigma l - dependent transcription in b. \n subtilis ( p54529 ) , nif - specific regulation in herbaspirillum seropedicae , dhar transcription in e. coli k12 , and globe signal transduction \n in clostridium beijerinckii [ 4 , 20 ] .", "however , it is unknown how \n iber contributes to the pathogenesis of meningitic infection by modulating the \n virulence of the pathogen . as e44 carries a nonsense mutation in the rpos gene and exhibits strong invasion activity in the sp , there \n", "we speculated that iber is an rpos - like regulator in sp gene expression in e44 . in order to dissect the \n regulation of sp gene expression in e44 that is associated with the \n pathogenesis of e. coli meningitis , a comparative proteomic analysis of \n an iber deletion mutant ( br2 ) and its parent strain e44", "our studies suggested that the iber gene was involved in \n regulating sp gene expression related to stress resistance and pathogenesis .", "the bacterial strain and plasmid vectors and their relevant \n characteristics are shown in table 1 .", "the mutant strains used in this study \n were derived from e44 , which is a rifampin - resistant strain derived from a \n neonatal meningitis isolate , e. coli k1 rs218 ( o18 : k1 : h7 )   [ 4 , 21 ] .", "sm10 ( pir ) , dh5   ( pir ) , and pcvd442 were used for making \n isogenic deletion mutants of iber and tnaa [ 6 , 10 , 11 ] .", "e. \n coli k12 strain mc4100   and its rpos \n\t\t\t\t\t insertion mutation tn10 mutant rh90 \n were used as positive and negative controls for rpos , respectively .", "plasmid \n pstyabb , which carries the gene for monooxygenase , was used for indole assay \n .", "e. coli strains were grown at 37c in luria broth ( lb ; 1% \n tryptone , 0.5% yeast extract , 0.5% nacl ) or brain heart infusion ( bhi , difco \n laboratories , detroit , mich , usa ) broth and were stored at 70c in lb plus 20% \n glycerol . when it was necessary , the medium was supplemented with ampicillin \n ( 100 g / ml ) and rifampin ( 100 g / ml ) for the positive selection of plasmids or \n bacterial strains ( table 1 ) . all genetic manipulations were done by using standard \n methods , as described elsewhere .", "plasmid dna was extracted by using a \n plasmid mini kit ( qiagen , calif , \n  usa ) .", "dna fragments were purified and were extracted from agarose \n gel slices , using qiaquick gel extraction kit ( qiagen ) .", "competent e. coli cells were made in 10% glycerol \n and were transformed by electroporation as described previously [ 6 , 7 ] . to determine the role of the iber gene in the \n growth - phase - dependent e. coli k1 invasion of bmec , an isogenic deletion \n mutant of iber", "two pcr dna fragments , b ( 1.2 kb ) and r ( 1.0 kb ) , flanking a 1.8-kb \n region to be deleted were produced from two pairs of primers ( iber - s1/iber - b1 \n for b and iber - b2/iber - x2 for r , see table 2 ) .", "the two fragments were ligated \n to make a 2.2 kb fragment ( br ) that carries an iber internal deletion . \n", "the br fragment was subcloned between sali and xbai sites on pcvd442 , and \n the resulting recombinant plasmid was named pcbr2 .", "the mutants named br2 were \n obtained by mating e44 with sm10 pir that carries pcbr2 as described previously \n .", "we used pcr and dna sequencing to confirm the internal deletion in the iber deletion mutant br2 and the desired \n chromosomal gene iber of the mutant with primers iber - s1 and iber - x2 \n ( table 2 ) .", "amplification was done by using the following cycle profile : 35 \n cycles at 94c for 1 minute , 58c for 1 minute , and 70c for 1.5 minutes . for the tnaa in - frame deletion", "briefly , 2 pcr dna fragments , ftn5 ( 1.0-kb ) \n and ftn3 ( 1.4-kb ) , were made to flank a 1.3 kb region containing tnaa to \n be deleted , by using 2 primer pairs ( tn - s1/tn - b1 for ftn5 and tn - b2/tn - x2 for \n ftn3 , see table 2 ) .", "then the two fragments were ligated to make a 2.4 kb \n fragment ( ftn53 ) that carried a tnaa internal deletion .", "the ftn53 \n fragment was subcloned into pcvd442 between sali and xbai sites to get the \n suicide plasmid pctna2 . to get the tnaa in - frame deletion mutant , tna44", ", \n conjugation and screening were carried out as described above . the tnaa gene deletion in the mutant tna44 \n", "was confirmed by pcr using the primers tn - s1 and tn - x2 ( table 2 ) .", "human bmecs were routinely cultured in rpmi 1640 medium \n ( mediatech , herndon , va , \n usa ) containing 10% heat inactivated fetal bovine serum , 10% nu - serum , 2 mm glutamine , 1 mm sodium pyruvate , essential amino acids , vitamins , penicillin \n g ( 50 g / ml ) , and streptomycin ( 100 g / ml ) at 37c in 5% co2 . \n ", "the \n number of intracellular bacteria was determined on blood agar plates after the \n extracellular bacteria were killed by incubation of the monolayers with \n experimental medium containing gentamicin ( 100 g / ml ) for 1 hour .", "results were \n expressed as percent invasion ( 100      ( number of intracellular bacteria \n recovered)/(number of bacteria inoculated ) ) .", "briefly , e. \n coli strain e44 and the iber deletion mutant br2 were cultured \n in bhi medium overnight without agitation .", "the bacterial cells were harvested \n at the stationary - phase ( od = 2.53.0 ) by \n centrifugation at 6000      g for 10 minutes .", "denaturing protein extraction ( phenol \n extraction procedure ) was performed according to saravanan and rose .", "the lyophilized pellets were \n dissolved in rehydration buffer ( 7 m urea , 2 m thiourea , 4% chaps , 0.2% ph 310 biolytes , 1% \n dtt ; 1 mg dry pellets for 200 l buffer ) and shaken on vortex for 1 hour at \n room temperature .", "the first and second dimensions of the page were performed at \n least in triplicate ( to reduce the likelihood of differences based solely upon \n gel - to - gel variability ) according to the standard protocols developed and \n defined by bio - rad .", "solubilized total e. \n coli protein samples ( 200 l each ) were loaded on 11 cm immobilized ph \n gradient ( ipg ) strips ( ph 47 ) . \n", "rehydration / loading was done passively ( no voltage ) for 1 hour , followed \n immediately by 14 hours of active rehydration ( 50 v ) at 20c .", "isoelectric \n focusing of the ipg strips was performed at 20c using a 50 a   current limit \n per strip to prevent damage to the strip and the instrument .", "electrophoresis \n was carried out as follows : step 1 , 250 v for 20 minutes ; step 2 , a rapid ramp \n to 8000 v ; step 3 , \n focusing at 8000 v for 55 000 vhr ; step 4 , \n hold at 400 v. due to latent ionic components in the sample the actual running \n voltage was only approximately 6500 v. after the first dimensional run was completed , the ipg \n strips were equilibrated in buffer i ( 6 m urea , 0.375 m tris - hcl ph 8.8 , 2% sds , \n 20% glycerol , and 2%dtt ) for 15 minutes and then in buffer ii ( 6 m urea , 0.375 m \n tris - hcl ph 8.8 , 2% sds , 20% glycerol , and 2%dtt ) for additional 15 minutes . \n", "the second dimension sds - page was performed with 15% resolving gels and 5% \n stacking gels ( 160      180      0.5 mm ) .", "the 2de gels were scanned at a 200 bpi resolution with \n typhoon scanner ( amersham biosciences , nj , usa ) , and analyzed with imagemaster \n 2d platinum version 6.0 ( ge healthcare bio - science , nj , usa ) .", "only those protein spots having \n differences in density of 1.5-fold or greater between the groups were chosen . \n", "moreover , all protein spots selected for analysis were shown to have \n significant difference in protein density ( mean      sem , p < .01 ) by software of spss 10.0 .", "protein bands were excised from preparative coomassie blue - stained \n gels and washed several times with destaining solutions ( 25 mm nh4hco3 for 15 minutes and then with 50% acetonitrile containing 25 mm nh4hco3 for 15 minutes ) .", "gel pieces were then dehydrated with 100% acetonitrile , dried , \n and then incubated with a reducing solution ( 25 mm nh4hco3 containing 10 mm dithiothreitol ) for 1 hour at 56c and subsequently with an \n alkylating solution ( 25 mm nh4hco3 containing 55 mm \n iodoacetamide ) for 45 minutes at room temperature .", "after reduction and \n alkylation , gels were washed several times with the destaining solutions and \n finally with pure water for 15 minutes before being treated again with 100% acetonitrile . \n", "depending on the protein content , 2 - 3 l of 0.1 g/l modified \n trypsin ( promega , wiss , usa , \n sequencing grade ) in 25 mm nh4hco3 was added over the gel \n spots and incubated for 30 minutes .", "about 710 l of 25 mm nh4hco3 was then added to cover the gel spots and incubated at 37c overnight .", "the \n in - gel digestion products were extracted with formic acid / acetonitrile \n solutions followed by evaporation .", "samples were desalted using mziptip c18 \n pipette tips ( millipore , mass , usa ) before ms / ms analysis .", "the sample was \n resuspended in 10 l of 0.1% formic acid , injected via an autosampler ( surveyor , \n thermofinnigan , calif , \n usa ) and subjected to reverse phase liquid chromatography using thermofinnigan \n surveyor ms - pump in conjunction with a biobasic 18 100      0.18 mm reverse - phase \n capillary column ( thermofinnigan , calif , usa ) .", "mass analysis was done using a \n thermofinnigan lcq deca xp plus ion trap mass spectrometer equipped with a \n nanospray ion source ( thermofinnigan , calif , usa ) employing a 4 cm metal emitter ( proxeon , \n odense , denmark ) .", "spray voltage of the mass spectrometer was set to 2.9 kv and capillary temperature was set at 190c .", "the column \n equilibrated for 5 minutes at 1.5 l / min with 95% solution a and 5% solution b \n ( a , 0.1% formic acid in water ; b , 0.1% formic acid in acetonitrile ) followed by \n a linear gradient was initiated 5 minutes after sample injection ramping to 65% \n solution a over 45 minutes .", "solution a was increased to 80% over the \n subsequent 5 minutes and held at 80% for 5 minutes , after which the column was \n reequilibrated back to 5% solution a ( aqueous ) .", "a \n data - dependent acquisition mode was used where each of the top five ions for a \n given scan was subjected to ms / ms analysis .", "the protein identification was conducted with the ms / ms \n search software mascot 1.9 with confirmatory or complementary analyses using \n turbosequest as implemented in the bioworks 3.2 .", "e. coli genome \n sequences at the national center for biotechnology information ( ncbi ) were used \n as the primary search databases and searches were complemented with the ncbi \n nonredundant protein database . for \n determination of indole production ,", "we followed the method of indole conversion \n into indigo as described previously [ 12 , 27 ] , with minor modifications .", "all \n strains e44 , br2 , tna44 , mg1655 , and rh90 were transformed with the pstyabb \n plasmid , which constitutively expresses the styab protein converting indole to \n indigo .", "the bacteria were incubated in m9 medium containing 0.4% glucose and \n 100 g / ml ampicillin overnight with shaking .", "then the bacteria were collected by \n centrifugation and resuspended to od600 = 0.2 in bhi medium supplied \n with 100 g / ml ampicillin , and incubated at 37c without shaking . to determine \n the indigo formation at different time points ,", "the samples were read at 600 nm to determine the indigo concentration \n by comparison to a standard curve .", "bacteria were grown in bhi broth at 37c overnight without \n shaking , and collected by centrifugation .", "the number of cells was measured on \n the basis of their od at 600 nm .", "bacteria were suspended and diluted to 10 cells / ml in pbs for the following assays . for heat shock , 100 l of bacteria \n", "was heated at 54c for 3 minutes . for alkali endurance , the bacterial \n suspension", "was mixed with equal volume of tris buffer ( 1 m , ph = 10.0 ) and 8 \n volumes of water ( final concentration , 100 mm , ph 10.0 ) and incubated at 37c \n for 30 minutes . for acid endurance ,", "1/10 volume of the bacterial suspension was \n mixed with lb containing acetic acid ( final concentration , 90 mm , ph 2.8 ) and \n incubated at 37c for 20 minutes . for high osmolarity challenge", ", bacteria were \n mixed with an equal volume of 4.8 m nacl ( final concentration , 2.4 m ) and \n incubated at 37c for 1 hour . for oxidative stress ,", "bacteria were harvested and \n resuspended in an equal volume of pbs containing 10 m h2o2 incubated at 37c   for 30 minutes .", "after exposure to these stresses , bacteria \n were diluted in 0.9% saline and plated in duplicate on lb agar plates .", "the surviving rate with stress was \n calculated from the ratio of the bacterial number under stress condition to the \n bacteria number under nonstress condition .", "the surviving rate without stress was \n calculated from the bacteria number grown on plates .", "the gene iber in e. coli k1 e44 is predicted as \n the only regulatory protein present in the iberat operon in gima by \n bioinformatics approaches . to determine the role of iber gene in \n the growth - phase - dependent invasion of bmec by meningitic e. coli k1 ,", "an isogenic in - frame deletion mutant of iber was \n made by chromosomal gene replacement with the recombinant suicide plasmid pcbr2 \n carrying a 2.2 kb dna fragment with iber internal deletion ( figure 1(a ) ) . \n", "the 2.2 kb dna fragment was generated by ligation of two pcr amplicons ( 1.2 and \n 1.0 kbs ) flanking the \n 1.8 kb iber coding region .", "the iber deletion mutant was obtained \n by mating e44 with sm10 pir carrying pcbr2 .", "the mutant colony morphology on lb \n agar plates and growth rate in lb broth were the same as the parent strain e44 . \n", "the deletion of iber was confirmed by colony pcr and dna sequencing \n ( figure 1(b ) ) . in order to examine the virulence phenotype of the iber deletion mutant , a comparative study of the invasiveness of e44 ( parent \n strain ) , br2 , and", "the complemented br2 was carried out . as shown in figure 1(c ) , \n the relative invasion rate of br2 was significantly reduced as compared to that \n of e44 and the plasmid pwks1030 carrying the iber gene was able to complement the noninvasive phenotype of br2 , \n suggesting that the iber gene contributes to the e. coli e44 \n invasion process . to determine the role of iber in regulating sp gene expressionof meningitic e. coli k1 , the wild type e. coli e44 and its iber deletion mutant br2 \n were cultured in bhi broth overnight .", "the total proteins of each strain were \n extracted from the cells as described in section 2 .  ", "the \n experiment was performed three times with two sets of independently grown \n cultures .", "only spots showing the same pattern in three independent runs were \n retained and quantified using the software imagemaster 2d platinum version 6.0 . \n", "figures 2(a ) and 2(b ) showed the protein patterns of e44 and br2 , respectively . \n", "all the upregulated spots and downregulated spots satisfying the criteria as \n mentioned above were marked on both the 2d maps . they were excised and \n identified by lc - ms / ms ( table 3 ) .", "eight protein spots were found to be \n differentially expressed in br2 \n as compared to its parent strain e44 . among them", ", 4 protein spots were \n significantly upregulated in br2 including elongation factor ef - tu ( tufb , spot u1 ) , \n glyceraldehyde-3-phosphate dehydrogenase a ( gapa , spot u2 ) , outer membrane protein 3a ( ompa , spot u3 ) , and alkyl hydroperoxide \n reductase ( ahpc , spot u4 ) , \n while 4 protein spots were of decreased abundance in br2 , including \n dihydrolipoamide dehydrogenase ( lpda , \n spot d1 ) , tryptophanase ( tnaa , \n spot d2 ) , and two isoforms of outer membrane protein c ( ompc , spot d3 , and d4 ) .", "figure 3(a ) showed the enlargements of \n each changed protein marked with black arrows and spot numbers .", "the relative \n ratios of each downregulated protein and upregulated protein were shown in figures \n 3(b ) and 3(c ) .", "we classified the proteins into two main categories on the \n basis of their roles in the sp growth of e. coli cells : ( a ) response to \n environmental modifications ( including tnaa , \n tufb , ompc , ompa , and ahpc ) \n and ( b ) central metabolism ( including lpda \n and gapa ) . since iber was hypothesized as an \n sp - regulator contributing to the growth regulation and virulence of e44 , its \n role in the invasion process and resistance to stress conditions should be \n further characterized .", "tnaa , a tryptophanase , degrades tryptophan , resulting in \n the formation of indole , which has been proposed to act as an extracellular \n signal in stationary phase cells of e. coli [ 28 , 29 ] .", "production of \n indole , via the enzymatic activity of tnaa , is also induced during biofilm \n formation .", "tnaa , which was \n controlled by rpos in other e. coli strains , is one of the most \n important transcriptional regulators for the gene expressions in sp cells . \n", "tufb ( ef - tu ) is responsible for binding and transporting the appropriate \n codon - specified aminoacyl - trna to the aminoacyl ( a ) site of the ribosome [ 32 , 33 ] .", "in addition to its function in translation elongation , elongation factor \n tu is implicated in protein folding and protection from stress like a chaperone \n molecule .", "ompc , as well as ompf , is a porin protein present on the \n outer membrane of e. coli , responding to the osmotic challenge .", "ompr , as \n a regulator , activates transcription of ompf and ompc , and \n changes the ratio of these two , so that the total level of porin proteins \n remains approximately constant [ 36 , 37 ] .", "ompf , which produces slightly wider \n pores ( 1.2 nm ) than does ompc , predominates at low osmotic strength , whereas \n ompc ( 1.1 nm ) predominates at high osmotic strength . in e. coli , \n the expression of ompc", "it has been proposed that the smaller pores formed by ompc could \n reduce the diffusion of larger hydrophobic and negatively charged molecules \n when bacteria encounter high osmolarity conditions as in the host compartments . \n", "presumably , this protein is very important for the stress resistance of e. \n coli in the stationary phase . in this study , the downregulation of ompc \n resulting from the iber deletion might decrease resistance to high \n osmolarity .", "in addition , it had been reported that ompc is involved in invasion \n of epithelial cells by crohn 's disease - associated e. coli strain lf82 and shigella flexneri [ 39 , 40 ] , suggesting \n that ompc might be involved in e44 invasion of the host tissue barriers .", "ompa \n is a major protein in the outer membrane of both pathogenic and nonpathogenic \n\t\t\t\t\t e. coli \n\t\t\t\t\t  .", "as shown in our previous study , the ompa - deletion mutant of e44 was \n significantly more sensitive than that of its parent strain to sds , cholate , \n acidic environment , and high osmolarity .", "ompa is downregulated upon entry \n into sp by sigmae , which plays a central role in maintaining cell envelope \n integrity both under stress conditions and during normal growth [ 42 , 43 ] .", "we \n demonstrated here that ompa was upregulated in the iber mutant br2 ( figure 3 ) , suggesting that ompa expression is suppressed \n upon entry   into sp by iber in a manner similar to sigmae .", "alkyl hydroperoxidase ( ahpc ) functions as a primary scavenger of endogenous h2o2 at a low ( 10 m ) concentration .", "all of ahpc , katg , \n and kate genes are known to participate in the antioxidant defense \n mechanism against h2o2-induced stress in e. coli . \n", "it has been reported that sp - inducible rpos regulates kate gene \n expression and oxyr regulates ahpc and katg genes [ 45 , 46 ] .", "our \n previous study has demonstrated that e. coli k1 rs218 had a nonsense \n mutation in its rpos gene , resulting in a negligible kate activity , but no obvious difference in katg . in this study ,", "the \n increase in ahpc expression indicated that the iber deletion led to an increased oxidative stress in sp compared with the wild type \n strain , suggesting that iber is involved in the resistance to oxidative \n stress upon entry into sp . \n\t\t\t\t\t", "lipoamide \n dehydrogenase ( lpda ) , which is the same as dihydrolipoamide \n dehydrogenase ( dldh ) , makes up the e3 component of pyruvate dehydrogenase \n complex , 2-oxo glutarate dehydrogenase , and branched - chain 2-oxo acid \n dehydrogenase complexes .", "dldh has been identified as virulence factors \n contributing to the pathogenesis of bacterial infections caused by mycobacterium tuberculosis and streptococcus pneumoniae because it \n enhances their survival within the host cells [ 47 , 48 ] . as shown in figure 3 , \n", "lpda was downregulated in the iber deletion mutant br2 , suggesting that this enzyme might be involved in the \n virulence of meningitic e. coli k1 via enhancing the pathogen survival \n within the host .", "recently , gapa in e. \n coli has been identified as one of a few proteins , which harbors \n functionally important thiol groups against oxidative stress .", "as gapa is \n upregulated in br2 ( figure 3 ) , iber may be involved in the negative control of gapa in sp . in summary ,", "all \n these proteins contribute to growth - related carbon source metabolism or stress \n resistance .", "they are associated with the sp regulation . among these proteins , tnaa is the most \n important one as it produces the signal molecule indole and is regulated by \n rpos . since e44 carries a loss - of - function mutation in its rpos gene , there should be alternative signaling pathway(s ) to complement the \n functional deficiency of rpos in this pathogenic e. coli strain .", "our proteomic analyses showed that the tnaa expression was significantly affected by iber , which might be functionally \n equivalent to rpos .", "therefore , our \n focus for further studies was placed on how tnaa is regulated by iber . to test our hypothesis that iber is an rpos - like regulator , \n the tnaa in - frame deletion mutant tna44 was generated with the same gene replacement approach that \n was used for the iber deletion mutant .", "tna44 was obtained by mating e44 with sm10 pir carrying the \n recombinant suicide plasmid pctna2 which contains the truncated tnaa gene .", "although overall growth rates did not \n differ between the mutants ( br2 and tna44 ) and their parent strain e44 ( figure 4(a ) ) , \n the invasive capability of tna44 ( 19% ) and br2 ( 35% ) was significantly reduced \n as compared to that of e44 ( 100% ) ( figures 4(c)-4(d ) ) .", "these data \n suggest that tnaa is an important downstream regulator that is required foriber - modulated e. coli k1 invasion .", "as our proteomics analysis had shown that tnaa expression was induced by iber in sp and the tnaa and iber deletion resulted in \n a deficiency in indole production in sp - cultures ( figure 4(b ) ) , we further tested \n whether the tnaa product indole , as an sp extracellular signal molecule , played \n a role in the process of invasion .", "indole is converted to indigo ( which is not \n further degraded in e. coli ) by several monooxygenases , thus providing \n an easy method for its determination .", "a plasmid pstyabb , carrying the gene \n for styrene monooxygenase , was used to monitor the indole production through \n its conversion to indigo .", "the plasmid was transformed into e. coli strains e44 , br2 , and tna44 , and the indole production was measured at \n different time points for these stains in bhi media ( figure 4(b ) ) .", "by contrast , \n the indole production was almost abolished in the tnaa deletion mutant \n and severely reduced in the iber deletion mutant ( figure 4(b ) ) .", "these \n results demonstrated that the indole production was controlled by iber through tnaa . to examine whether indole could compliment the noninvasive \n phenotype of tnaa and iber deletion mutants , indole was supplied in the bhi medium at 100 m to the tna44 \n and br2 sp cultures .", "the \n result showed that indole was able to significantly \n enhance the relative invasion rate of tna44 ( 19% to 69% ) and br2 ( 35% to 65% ) as compared to \n that of e44 , suggesting that indole could partly compliment the noninvasive phenotype of tna44 \n ( figure 4(c ) ) and br2 ( figure 4(d ) ) .", "lacour and landini have shown that the rpos gene in e. coli k12 strain \n mg1655 controls the production of indole , which acts as a signal molecule in sp \n cells , via regulation of tnaa , the indole - producing enzyme .", "as tnaa is \n regulated by iber in e44 , it is most likely that iber is a novel regulator to \n complement the functional deficiency of rpos in e44 .", "it has been reported that rpos is able to positively and \n negatively control expression of a large set of genes when bacteria enter into \n the sp [ 46 , 50 , 51 ] . during such \n transition ,", "bacteria undergo physiological changes that allow their sp \n organisms to survive better in such insults as heat , high - osmotic environment , \n starvation , uv radiation , h2o2 ,   and acid than their \n exponential counterpart [ 50 , 52 ] .", "the loss of rpos resulted in the decrease of \n stress resistance and cell survival in the sp [ 5 , 53 ] .", "although our study \n showed that the loss of iber did not affect the growth rate in bhi \n medium , the survival rates of the iber deletion mutant br2 in the sp \n significantly decreased as compared to that of the wild type strain e44 even \n without any stress treatment ( figure 5(a ) ) .", "our proteomics analysis also \n revealed that the most significant proteomic changes in the iber deletion mutant were related to bacterial response to environmental \n modifications .", "for example , ahpc , as a primary scavenger of endogenous h2o2 , \n  was upregulated in the iber   deletion mutant , implying that the loss of iber resulted in the decreased survival rates of bacterial cells under an oxidative \n stress in the sp .", "ompc , as a porin protein , was downregulated in br2 , perhaps \n resulting in the decreased resistance to osmolarity stress .", "these results \n suggested that iber plays a regulatory role in response to stress \n conditions in e44 that carries a nonsense mutation in rpos .", "to examine \n the function of iber in response to stress environments , we performed \n several survival assays under different stress conditions , including heat shock \n ( 54c for 3 minutes ) , alkali endurance ( tris , ph = 10 for 30 minutes ) , acid \n endurance ( acetic acid , ph = 2.8 for 20 minutes ) , high osmolarity challenge ( 2.5 m nacl for 1 hour ) , and oxidative stress ( 10 m h2o2 for \n 30 minutes ) .", "in all the survival experiments , the wild type strain e44 showed \n higher survival rates than the iber deletion mutant , indicating that the iber gene is required for all these \n stress resistances ( figure 5(b ) ) . especially in the heat shock assay , the loss \n of iber resulted in over 95% cells death , indicating that iber played a vital role in temperature sensitivity in this strain . in the other \n stress treatments ,", "the iber deletion also significantly reduced the \n survival rates ( more than 60% cell death ) of br2 as compared to that of the \n wild type strain e44 , suggesting that iber had a global regulatory role in the \n resistance to acid , alkali , high osmolarity and oxidative stress . in the \n survival assays for the e. coli control strain mg1655", ", the rpos deletion mutant rh90 also decreased the survival levels in these five stress \n conditions , showing the similar patterns like iber in response to stress \n environments ( data not shown ) . combining the proteomics analysis and the stress \n survival studies ,", "we conclude that iber is an rpos - like regulator to control \n gene expression of proteins that are critical for stress - resistance and cell \n survival in the sp in e44 , which carries a loss - of - function mutation in the rpos gene .", "currently , most e. coli meningitis studies are done with sp cultures in which the pathogen invasion of \n human bmec is significantly greater than the log phase cultures . in most \n strains of e. coli ,", "rpos plays a \n central role in regulating the sp regulatory genes for protecting cells against \n starvation and stress damage .", "rpos , the major sp regulator , has also been shown \n to regulate the expression of microbial virulence genes in various bacteria \n including e. coli k1 ( o157 : h7 ) , salmonella \n typhimurium , shigella flexneri , yersinia enterocolitica , vibrio \n cholera , and borrelia burgdorfer ,   [ 5 , 53 , 54 ] .", "surprisingly , however , rpos was found to be inactive in meningitic \n strain e44 .", "the current proteomic studies may provide an answer to the \n long - standing question regarding the sp gene regulation in e44 . combining the \n proteomics analysis , virulence determination , and the stress survival studies \n of the iber mutant br2", ", we have \n demonstrated for the first time that iber serves as an rpos - like regulator to \n control gene expression that is critical for stress - resistance and cell \n survival in the sp of e44 .", "iber is not a structural homologue of   rpos as iber \n and rpos do not share any significant sequence homology .", "rpos ( also known as  , \n  , or katf ) is a global regulator in e. coli , which is the second principal  subunit after the major  factor . in e44", ", however , iber appears not to be a master regulator on the \n basis of its genomic prevalence and functional spectrum .", "the prevalence of the \n gimalocus carrying iber is highly dependent on the origin \n of the strain and on the subgroup it belongs to ( a , b1 , b2 , and d ) ( 4 ) . in all \n the studies , where the presence of this locushas been analyzed ,", "gima was found to be restricted to the b2 \n subgroup , a subgroup that includes strains with the highest virulence in mice \n and the highest level of virulence determinants ( 4 ) .", "our proteomic studies showed that a limited number \n of genes were regulated by iber , suggesting that iber is a regulator with a \n narrow functional spectrum .", "in other e. \n coli strains , either pathogenic or probiotic strains , functional \n heterogeneity of rpos in stress tolerance was widely observed [ 5 , 53 , 55 ] . \n", "those studies have shown that some e. \n coli strains can maintain their stress tolerance capability or \n significantly modulate their stress resistance phenotype independent of their rpos genotypes .", "such adaptation processes \n compromising the rpos - dependent stress responses may have significant impact on \n bacterial survival in environments , as well as in the host 's stomach and \n intestine [ 53 , 55 ] .", "iber , a regulator in the gima regulon , may contribute to \n bacterial virulence adaptation process in e44 to complement the functional \n deficiency of rpos .", "another significant finding of our proteomic studies is that \n iber in meningitic strain e44 is able to upregulate tnaa , which is controlled \n by rpos in other e. coli strains \n .", "the virulence determination of the tnaa mutant showed that tnaa and its product indole were required for e44 invasion \n of human bmec .", "the generation of indole , via the tryptophanase activity of \n tnaa , was also observed during the formation of biofilm in e. coli and other bacteria [ 28 , 56 ] .", "in addition to the initiation \n of indole - mediated signaling , tnaa ( tryptophanase ) is able to catabolize \n tryptophan , cysteine , and serine to pyruvate [ 29 , 56 ] .", "the three proteins \n significantly upregulated by iber are tnaa , lpda , and ompc , all of which are \n directly or indirectly involved in pyruvate metabolism .", "ompc , an \n osmotically regulated porin , may facilitate nutrient uptake . on the other \n hand , the three operons ( ptnipkc , cgldtec , and gcxkrci ) in the gima regulon may also directly or indirectly \n contribute to pyruvate metabolism by converting dihydroxyacetone , glycerol , and \n glycerate to pyruvate .", "it has been shown that the capability to catabolize \n carbon source is an important parameter in the ability to persist and compete \n in stationary phase .", "this raises the possibility that signaling by indole , \n which is regulated by iber via tnaa , may play a critical role in the pathways that \n prepare the pathogens for a nutrient - poor environment ( e.g. , cns ) when the \n carbon source becomes limited for energy production ."], "abstract_text": ["<S> \n iber is a regulator present in meningitic escherichia coli strain e44 that carries a loss - of - function mutation in the stationary - phase ( sp ) regulatory gene rpos . in order to determine whether iber is an sp regulator in e44 </S>", "<S> , two - dimensional gel electrophoresis and lc - ms were used to compare the proteomes of a noninvasive iber deletion mutant br2 and its parent strain e44 in the sp . </S>", "<S> four up - regulated ( tufb , gapa , ompa , ahpc ) and three down - regulated ( lpda , tnaa , opmc ) proteins in br2 were identified when compared to e44 . </S>", "<S> all these proteins contribute to energy metabolism or stress resistance , which is related to sp regulation . </S>", "<S> one of the down - regulated proteins , tryptophanase ( tnaa ) , which is regulated by rpos in other e. coli strains , is associated with sp regulation via production of a signal molecule indole . </S>", "<S> our studies demonstrated that tnaa was required for e44 invasion , and that indole was able to restore the noninvasive phenotype of the tnaa mutant . </S>", "<S> the production of indole was significantly reduced in br2 , indicating that iber is required for the indole production via tnaa . </S>", "<S> survival studies under different stress conditions indicated that iber contributed to bacteria stress resistance in the sp . </S>", "<S> taken together , iber is a novel regulator contributing to the sp regulation . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussions", "4. Concluding Remarks"], "sections": [["neonatal bacterial meningitis continues to be \n the most common serious infection of the central nervous system ( cns ) in \n newborns with high morbidity and mortality despite the availability of \n effective bactericidal antibiotics over the last sixty years [ 1 , 2 ] .", "this high morbidity and mortality are due to inadequate \n knowledge of the pathogenesis of this disease . \n", "e. coli is the most common gram - negative \n bacterium causing neonatal sepsis and meningitis .", "bacterial meningitis in \n newborns is due to hematogenous spread of the pathogen to the meninges .", "the \n most important issue in the pathogenesis of e. \n coli meningitis is how circulating pathogens cross the blood - brain barrier \n ( bbb ) , which is mainly composed of brain microvascular endothelial cells ( bmecs )   [ 3 , 4 ] .", "our \n previous studies showed that e. coli k1 invasion of human bmec was \n significantly greater with stationary - phase ( sp ) cultures than with \n exponential - phase cultures , suggesting that expression of e. coli k1 invasion - associated virulence \n genes is strongly regulated in a growth - phase - dependent manner .", "a nonsense mutation in the sp regulatory gene rpos was identified in e. coli k1 strains e44 and ihe3034 . \n", "complementation with the wild type e. coli k12 rpos gene \n significantly enhanced ihe3034 invasion of bmec , but failed to improve the invasion activity of another e. \n coli k1 strain e44 .", "these studies suggest that the growth - phase - dependent \n invasion of bmec by ihe3034 is affected by rpos and that e44 carries a \n loss - of - function mutation in the rpos gene .", "several virulence \n factors , including ibe ( termed after   invasion of   brain endothelial \n cells ) proteins [ 6 , 7 ] , ompa   , yiijp , fimh , asla , and traj \n , have been identified in various strains of e. coli in the in vitro and in vivo models of the \n bbb as invasins .", "most of those \n invasion genes are present in the e. coli k-12 genome [ 4 , 16 ] .", "however , the ibea gene encoding a 50 kda protein has been found to be unique to some pathogenic e. coli k1 strains ( e.g. , c5 and rs218 ) , \n while laboratory strains of e. coli k-12 ( e.g. , dh5 and hb101 ) , as well \n as noninvasive e.   coli ( e.g. , e412 ) , \n lack ibea .", "recently , vimentin has \n been identified as an ibea - binding protein on the surface of human bmec . using the ibea gene \n as a probe", ", we have identified a 20.3 kb genomic locus as a genetic island of meningitic e. coli containing ibea ( gima ) .", "this \n locus is situated between yjid and yjie , adjacent to the fim operon , and \n absent in nonpathogenic e. coli k12 \n strains .", "the first three operons \n ( ptnipkc , cgldtec , gcxkrci ) may \n be involved in energy metabolism and the last operon ( iberat ) contributes to e. \n coli k1 invasion of bmec .", "our previous work showed that gima - mediated \n invasion of human endothelial cells is regulated by carbon source .", "this is \n consistent with the observations by others that carbon source modulates \n expression of virulence factors in several pathogenic bacteria .", "ibea and ibet contribute \n to e. coli k1 invasion and adhesion \n [ 18 , 19 ] .", "our previous studies suggest that iber is a novel regulatory protein that is present in pathogenic e. coli k1 .", "it belongs to the \n ntrc / nifa family of transcriptional activators , carrying a sigma 54-interaction \n domain and showing significant \n sequence homology to various regulatory proteins for glycerol metabolism operon \n in citrobacter freundii ( p45512 ) , acetoacetate metabolism in e. coli k12 , \n sigma l - dependent transcription in b. \n subtilis ( p54529 ) , nif - specific regulation in herbaspirillum seropedicae , dhar transcription in e. coli k12 , and globe signal transduction \n in clostridium beijerinckii [ 4 , 20 ] .", "however , it is unknown how \n iber contributes to the pathogenesis of meningitic infection by modulating the \n virulence of the pathogen . as e44 carries a nonsense mutation in the rpos gene and exhibits strong invasion activity in the sp , there \n", "we speculated that iber is an rpos - like regulator in sp gene expression in e44 . in order to dissect the \n regulation of sp gene expression in e44 that is associated with the \n pathogenesis of e. coli meningitis , a comparative proteomic analysis of \n an iber deletion mutant ( br2 ) and its parent strain e44", "our studies suggested that the iber gene was involved in \n regulating sp gene expression related to stress resistance and pathogenesis ."], ["the bacterial strain and plasmid vectors and their relevant \n characteristics are shown in table 1 .", "the mutant strains used in this study \n were derived from e44 , which is a rifampin - resistant strain derived from a \n neonatal meningitis isolate , e. coli k1 rs218 ( o18 : k1 : h7 )   [ 4 , 21 ] .", "sm10 ( pir ) , dh5   ( pir ) , and pcvd442 were used for making \n isogenic deletion mutants of iber and tnaa [ 6 , 10 , 11 ] .", "e. \n coli k12 strain mc4100   and its rpos \n\t\t\t\t\t insertion mutation tn10 mutant rh90 \n were used as positive and negative controls for rpos , respectively .", "plasmid \n pstyabb , which carries the gene for monooxygenase , was used for indole assay \n .", "e. coli strains were grown at 37c in luria broth ( lb ; 1% \n tryptone , 0.5% yeast extract , 0.5% nacl ) or brain heart infusion ( bhi , difco \n laboratories , detroit , mich , usa ) broth and were stored at 70c in lb plus 20% \n glycerol . when it was necessary , the medium was supplemented with ampicillin \n ( 100 g / ml ) and rifampin ( 100 g / ml ) for the positive selection of plasmids or \n bacterial strains ( table 1 ) . all genetic manipulations were done by using standard \n methods , as described elsewhere .", "plasmid dna was extracted by using a \n plasmid mini kit ( qiagen , calif , \n  usa ) .", "dna fragments were purified and were extracted from agarose \n gel slices , using qiaquick gel extraction kit ( qiagen ) .", "competent e. coli cells were made in 10% glycerol \n and were transformed by electroporation as described previously [ 6 , 7 ] . to determine the role of the iber gene in the \n growth - phase - dependent e. coli k1 invasion of bmec , an isogenic deletion \n mutant of iber", "two pcr dna fragments , b ( 1.2 kb ) and r ( 1.0 kb ) , flanking a 1.8-kb \n region to be deleted were produced from two pairs of primers ( iber - s1/iber - b1 \n for b and iber - b2/iber - x2 for r , see table 2 ) .", "the two fragments were ligated \n to make a 2.2 kb fragment ( br ) that carries an iber internal deletion . \n", "the br fragment was subcloned between sali and xbai sites on pcvd442 , and \n the resulting recombinant plasmid was named pcbr2 .", "the mutants named br2 were \n obtained by mating e44 with sm10 pir that carries pcbr2 as described previously \n .", "we used pcr and dna sequencing to confirm the internal deletion in the iber deletion mutant br2 and the desired \n chromosomal gene iber of the mutant with primers iber - s1 and iber - x2 \n ( table 2 ) .", "amplification was done by using the following cycle profile : 35 \n cycles at 94c for 1 minute , 58c for 1 minute , and 70c for 1.5 minutes . for the tnaa in - frame deletion", "briefly , 2 pcr dna fragments , ftn5 ( 1.0-kb ) \n and ftn3 ( 1.4-kb ) , were made to flank a 1.3 kb region containing tnaa to \n be deleted , by using 2 primer pairs ( tn - s1/tn - b1 for ftn5 and tn - b2/tn - x2 for \n ftn3 , see table 2 ) .", "then the two fragments were ligated to make a 2.4 kb \n fragment ( ftn53 ) that carried a tnaa internal deletion .", "the ftn53 \n fragment was subcloned into pcvd442 between sali and xbai sites to get the \n suicide plasmid pctna2 . to get the tnaa in - frame deletion mutant , tna44", ", \n conjugation and screening were carried out as described above . the tnaa gene deletion in the mutant tna44 \n", "was confirmed by pcr using the primers tn - s1 and tn - x2 ( table 2 ) .", "human bmecs were routinely cultured in rpmi 1640 medium \n ( mediatech , herndon , va , \n usa ) containing 10% heat inactivated fetal bovine serum , 10% nu - serum , 2 mm glutamine , 1 mm sodium pyruvate , essential amino acids , vitamins , penicillin \n g ( 50 g / ml ) , and streptomycin ( 100 g / ml ) at 37c in 5% co2 . \n ", "the \n number of intracellular bacteria was determined on blood agar plates after the \n extracellular bacteria were killed by incubation of the monolayers with \n experimental medium containing gentamicin ( 100 g / ml ) for 1 hour .", "results were \n expressed as percent invasion ( 100      ( number of intracellular bacteria \n recovered)/(number of bacteria inoculated ) ) .", "briefly , e. \n coli strain e44 and the iber deletion mutant br2 were cultured \n in bhi medium overnight without agitation .", "the bacterial cells were harvested \n at the stationary - phase ( od = 2.53.0 ) by \n centrifugation at 6000      g for 10 minutes .", "denaturing protein extraction ( phenol \n extraction procedure ) was performed according to saravanan and rose .", "the lyophilized pellets were \n dissolved in rehydration buffer ( 7 m urea , 2 m thiourea , 4% chaps , 0.2% ph 310 biolytes , 1% \n dtt ; 1 mg dry pellets for 200 l buffer ) and shaken on vortex for 1 hour at \n room temperature .", "the first and second dimensions of the page were performed at \n least in triplicate ( to reduce the likelihood of differences based solely upon \n gel - to - gel variability ) according to the standard protocols developed and \n defined by bio - rad .", "solubilized total e. \n coli protein samples ( 200 l each ) were loaded on 11 cm immobilized ph \n gradient ( ipg ) strips ( ph 47 ) . \n", "rehydration / loading was done passively ( no voltage ) for 1 hour , followed \n immediately by 14 hours of active rehydration ( 50 v ) at 20c .", "isoelectric \n focusing of the ipg strips was performed at 20c using a 50 a   current limit \n per strip to prevent damage to the strip and the instrument .", "electrophoresis \n was carried out as follows : step 1 , 250 v for 20 minutes ; step 2 , a rapid ramp \n to 8000 v ; step 3 , \n focusing at 8000 v for 55 000 vhr ; step 4 , \n hold at 400 v. due to latent ionic components in the sample the actual running \n voltage was only approximately 6500 v. after the first dimensional run was completed , the ipg \n strips were equilibrated in buffer i ( 6 m urea , 0.375 m tris - hcl ph 8.8 , 2% sds , \n 20% glycerol , and 2%dtt ) for 15 minutes and then in buffer ii ( 6 m urea , 0.375 m \n tris - hcl ph 8.8 , 2% sds , 20% glycerol , and 2%dtt ) for additional 15 minutes . \n", "the second dimension sds - page was performed with 15% resolving gels and 5% \n stacking gels ( 160      180      0.5 mm ) .", "the 2de gels were scanned at a 200 bpi resolution with \n typhoon scanner ( amersham biosciences , nj , usa ) , and analyzed with imagemaster \n 2d platinum version 6.0 ( ge healthcare bio - science , nj , usa ) .", "only those protein spots having \n differences in density of 1.5-fold or greater between the groups were chosen . \n", "moreover , all protein spots selected for analysis were shown to have \n significant difference in protein density ( mean      sem , p < .01 ) by software of spss 10.0 .", "protein bands were excised from preparative coomassie blue - stained \n gels and washed several times with destaining solutions ( 25 mm nh4hco3 for 15 minutes and then with 50% acetonitrile containing 25 mm nh4hco3 for 15 minutes ) .", "gel pieces were then dehydrated with 100% acetonitrile , dried , \n and then incubated with a reducing solution ( 25 mm nh4hco3 containing 10 mm dithiothreitol ) for 1 hour at 56c and subsequently with an \n alkylating solution ( 25 mm nh4hco3 containing 55 mm \n iodoacetamide ) for 45 minutes at room temperature .", "after reduction and \n alkylation , gels were washed several times with the destaining solutions and \n finally with pure water for 15 minutes before being treated again with 100% acetonitrile . \n", "depending on the protein content , 2 - 3 l of 0.1 g/l modified \n trypsin ( promega , wiss , usa , \n sequencing grade ) in 25 mm nh4hco3 was added over the gel \n spots and incubated for 30 minutes .", "about 710 l of 25 mm nh4hco3 was then added to cover the gel spots and incubated at 37c overnight .", "the \n in - gel digestion products were extracted with formic acid / acetonitrile \n solutions followed by evaporation .", "samples were desalted using mziptip c18 \n pipette tips ( millipore , mass , usa ) before ms / ms analysis .", "the sample was \n resuspended in 10 l of 0.1% formic acid , injected via an autosampler ( surveyor , \n thermofinnigan , calif , \n usa ) and subjected to reverse phase liquid chromatography using thermofinnigan \n surveyor ms - pump in conjunction with a biobasic 18 100      0.18 mm reverse - phase \n capillary column ( thermofinnigan , calif , usa ) .", "mass analysis was done using a \n thermofinnigan lcq deca xp plus ion trap mass spectrometer equipped with a \n nanospray ion source ( thermofinnigan , calif , usa ) employing a 4 cm metal emitter ( proxeon , \n odense , denmark ) .", "spray voltage of the mass spectrometer was set to 2.9 kv and capillary temperature was set at 190c .", "the column \n equilibrated for 5 minutes at 1.5 l / min with 95% solution a and 5% solution b \n ( a , 0.1% formic acid in water ; b , 0.1% formic acid in acetonitrile ) followed by \n a linear gradient was initiated 5 minutes after sample injection ramping to 65% \n solution a over 45 minutes .", "solution a was increased to 80% over the \n subsequent 5 minutes and held at 80% for 5 minutes , after which the column was \n reequilibrated back to 5% solution a ( aqueous ) .", "a \n data - dependent acquisition mode was used where each of the top five ions for a \n given scan was subjected to ms / ms analysis .", "the protein identification was conducted with the ms / ms \n search software mascot 1.9 with confirmatory or complementary analyses using \n turbosequest as implemented in the bioworks 3.2 .", "e. coli genome \n sequences at the national center for biotechnology information ( ncbi ) were used \n as the primary search databases and searches were complemented with the ncbi \n nonredundant protein database . for \n determination of indole production ,", "we followed the method of indole conversion \n into indigo as described previously [ 12 , 27 ] , with minor modifications .", "all \n strains e44 , br2 , tna44 , mg1655 , and rh90 were transformed with the pstyabb \n plasmid , which constitutively expresses the styab protein converting indole to \n indigo .", "the bacteria were incubated in m9 medium containing 0.4% glucose and \n 100 g / ml ampicillin overnight with shaking .", "then the bacteria were collected by \n centrifugation and resuspended to od600 = 0.2 in bhi medium supplied \n with 100 g / ml ampicillin , and incubated at 37c without shaking . to determine \n the indigo formation at different time points ,", "the samples were read at 600 nm to determine the indigo concentration \n by comparison to a standard curve .", "bacteria were grown in bhi broth at 37c overnight without \n shaking , and collected by centrifugation .", "the number of cells was measured on \n the basis of their od at 600 nm .", "bacteria were suspended and diluted to 10 cells / ml in pbs for the following assays . for heat shock , 100 l of bacteria \n", "was heated at 54c for 3 minutes . for alkali endurance , the bacterial \n suspension", "was mixed with equal volume of tris buffer ( 1 m , ph = 10.0 ) and 8 \n volumes of water ( final concentration , 100 mm , ph 10.0 ) and incubated at 37c \n for 30 minutes . for acid endurance ,", "1/10 volume of the bacterial suspension was \n mixed with lb containing acetic acid ( final concentration , 90 mm , ph 2.8 ) and \n incubated at 37c for 20 minutes . for high osmolarity challenge", ", bacteria were \n mixed with an equal volume of 4.8 m nacl ( final concentration , 2.4 m ) and \n incubated at 37c for 1 hour . for oxidative stress ,", "bacteria were harvested and \n resuspended in an equal volume of pbs containing 10 m h2o2 incubated at 37c   for 30 minutes .", "after exposure to these stresses , bacteria \n were diluted in 0.9% saline and plated in duplicate on lb agar plates .", "the surviving rate with stress was \n calculated from the ratio of the bacterial number under stress condition to the \n bacteria number under nonstress condition .", "the surviving rate without stress was \n calculated from the bacteria number grown on plates ."], ["the gene iber in e. coli k1 e44 is predicted as \n the only regulatory protein present in the iberat operon in gima by \n bioinformatics approaches . to determine the role of iber gene in \n the growth - phase - dependent invasion of bmec by meningitic e. coli k1 ,", "an isogenic in - frame deletion mutant of iber was \n made by chromosomal gene replacement with the recombinant suicide plasmid pcbr2 \n carrying a 2.2 kb dna fragment with iber internal deletion ( figure 1(a ) ) . \n", "the 2.2 kb dna fragment was generated by ligation of two pcr amplicons ( 1.2 and \n 1.0 kbs ) flanking the \n 1.8 kb iber coding region .", "the iber deletion mutant was obtained \n by mating e44 with sm10 pir carrying pcbr2 .", "the mutant colony morphology on lb \n agar plates and growth rate in lb broth were the same as the parent strain e44 . \n", "the deletion of iber was confirmed by colony pcr and dna sequencing \n ( figure 1(b ) ) . in order to examine the virulence phenotype of the iber deletion mutant , a comparative study of the invasiveness of e44 ( parent \n strain ) , br2 , and", "the complemented br2 was carried out . as shown in figure 1(c ) , \n the relative invasion rate of br2 was significantly reduced as compared to that \n of e44 and the plasmid pwks1030 carrying the iber gene was able to complement the noninvasive phenotype of br2 , \n suggesting that the iber gene contributes to the e. coli e44 \n invasion process . to determine the role of iber in regulating sp gene expressionof meningitic e. coli k1 , the wild type e. coli e44 and its iber deletion mutant br2 \n were cultured in bhi broth overnight .", "the total proteins of each strain were \n extracted from the cells as described in section 2 .  ", "the \n experiment was performed three times with two sets of independently grown \n cultures .", "only spots showing the same pattern in three independent runs were \n retained and quantified using the software imagemaster 2d platinum version 6.0 . \n", "figures 2(a ) and 2(b ) showed the protein patterns of e44 and br2 , respectively . \n", "all the upregulated spots and downregulated spots satisfying the criteria as \n mentioned above were marked on both the 2d maps . they were excised and \n identified by lc - ms / ms ( table 3 ) .", "eight protein spots were found to be \n differentially expressed in br2 \n as compared to its parent strain e44 . among them", ", 4 protein spots were \n significantly upregulated in br2 including elongation factor ef - tu ( tufb , spot u1 ) , \n glyceraldehyde-3-phosphate dehydrogenase a ( gapa , spot u2 ) , outer membrane protein 3a ( ompa , spot u3 ) , and alkyl hydroperoxide \n reductase ( ahpc , spot u4 ) , \n while 4 protein spots were of decreased abundance in br2 , including \n dihydrolipoamide dehydrogenase ( lpda , \n spot d1 ) , tryptophanase ( tnaa , \n spot d2 ) , and two isoforms of outer membrane protein c ( ompc , spot d3 , and d4 ) .", "figure 3(a ) showed the enlargements of \n each changed protein marked with black arrows and spot numbers .", "the relative \n ratios of each downregulated protein and upregulated protein were shown in figures \n 3(b ) and 3(c ) .", "we classified the proteins into two main categories on the \n basis of their roles in the sp growth of e. coli cells : ( a ) response to \n environmental modifications ( including tnaa , \n tufb , ompc , ompa , and ahpc ) \n and ( b ) central metabolism ( including lpda \n and gapa ) . since iber was hypothesized as an \n sp - regulator contributing to the growth regulation and virulence of e44 , its \n role in the invasion process and resistance to stress conditions should be \n further characterized .", "tnaa , a tryptophanase , degrades tryptophan , resulting in \n the formation of indole , which has been proposed to act as an extracellular \n signal in stationary phase cells of e. coli [ 28 , 29 ] .", "production of \n indole , via the enzymatic activity of tnaa , is also induced during biofilm \n formation .", "tnaa , which was \n controlled by rpos in other e. coli strains , is one of the most \n important transcriptional regulators for the gene expressions in sp cells . \n", "tufb ( ef - tu ) is responsible for binding and transporting the appropriate \n codon - specified aminoacyl - trna to the aminoacyl ( a ) site of the ribosome [ 32 , 33 ] .", "in addition to its function in translation elongation , elongation factor \n tu is implicated in protein folding and protection from stress like a chaperone \n molecule .", "ompc , as well as ompf , is a porin protein present on the \n outer membrane of e. coli , responding to the osmotic challenge .", "ompr , as \n a regulator , activates transcription of ompf and ompc , and \n changes the ratio of these two , so that the total level of porin proteins \n remains approximately constant [ 36 , 37 ] .", "ompf , which produces slightly wider \n pores ( 1.2 nm ) than does ompc , predominates at low osmotic strength , whereas \n ompc ( 1.1 nm ) predominates at high osmotic strength . in e. coli , \n the expression of ompc", "it has been proposed that the smaller pores formed by ompc could \n reduce the diffusion of larger hydrophobic and negatively charged molecules \n when bacteria encounter high osmolarity conditions as in the host compartments . \n", "presumably , this protein is very important for the stress resistance of e. \n coli in the stationary phase . in this study , the downregulation of ompc \n resulting from the iber deletion might decrease resistance to high \n osmolarity .", "in addition , it had been reported that ompc is involved in invasion \n of epithelial cells by crohn 's disease - associated e. coli strain lf82 and shigella flexneri [ 39 , 40 ] , suggesting \n that ompc might be involved in e44 invasion of the host tissue barriers .", "ompa \n is a major protein in the outer membrane of both pathogenic and nonpathogenic \n\t\t\t\t\t e. coli \n\t\t\t\t\t  .", "as shown in our previous study , the ompa - deletion mutant of e44 was \n significantly more sensitive than that of its parent strain to sds , cholate , \n acidic environment , and high osmolarity .", "ompa is downregulated upon entry \n into sp by sigmae , which plays a central role in maintaining cell envelope \n integrity both under stress conditions and during normal growth [ 42 , 43 ] .", "we \n demonstrated here that ompa was upregulated in the iber mutant br2 ( figure 3 ) , suggesting that ompa expression is suppressed \n upon entry   into sp by iber in a manner similar to sigmae .", "alkyl hydroperoxidase ( ahpc ) functions as a primary scavenger of endogenous h2o2 at a low ( 10 m ) concentration .", "all of ahpc , katg , \n and kate genes are known to participate in the antioxidant defense \n mechanism against h2o2-induced stress in e. coli . \n", "it has been reported that sp - inducible rpos regulates kate gene \n expression and oxyr regulates ahpc and katg genes [ 45 , 46 ] .", "our \n previous study has demonstrated that e. coli k1 rs218 had a nonsense \n mutation in its rpos gene , resulting in a negligible kate activity , but no obvious difference in katg . in this study ,", "the \n increase in ahpc expression indicated that the iber deletion led to an increased oxidative stress in sp compared with the wild type \n strain , suggesting that iber is involved in the resistance to oxidative \n stress upon entry into sp . \n\t\t\t\t\t", "lipoamide \n dehydrogenase ( lpda ) , which is the same as dihydrolipoamide \n dehydrogenase ( dldh ) , makes up the e3 component of pyruvate dehydrogenase \n complex , 2-oxo glutarate dehydrogenase , and branched - chain 2-oxo acid \n dehydrogenase complexes .", "dldh has been identified as virulence factors \n contributing to the pathogenesis of bacterial infections caused by mycobacterium tuberculosis and streptococcus pneumoniae because it \n enhances their survival within the host cells [ 47 , 48 ] . as shown in figure 3 , \n", "lpda was downregulated in the iber deletion mutant br2 , suggesting that this enzyme might be involved in the \n virulence of meningitic e. coli k1 via enhancing the pathogen survival \n within the host .", "recently , gapa in e. \n coli has been identified as one of a few proteins , which harbors \n functionally important thiol groups against oxidative stress .", "as gapa is \n upregulated in br2 ( figure 3 ) , iber may be involved in the negative control of gapa in sp . in summary ,", "all \n these proteins contribute to growth - related carbon source metabolism or stress \n resistance .", "they are associated with the sp regulation . among these proteins , tnaa is the most \n important one as it produces the signal molecule indole and is regulated by \n rpos . since e44 carries a loss - of - function mutation in its rpos gene , there should be alternative signaling pathway(s ) to complement the \n functional deficiency of rpos in this pathogenic e. coli strain .", "our proteomic analyses showed that the tnaa expression was significantly affected by iber , which might be functionally \n equivalent to rpos .", "therefore , our \n focus for further studies was placed on how tnaa is regulated by iber . to test our hypothesis that iber is an rpos - like regulator , \n the tnaa in - frame deletion mutant tna44 was generated with the same gene replacement approach that \n was used for the iber deletion mutant .", "tna44 was obtained by mating e44 with sm10 pir carrying the \n recombinant suicide plasmid pctna2 which contains the truncated tnaa gene .", "although overall growth rates did not \n differ between the mutants ( br2 and tna44 ) and their parent strain e44 ( figure 4(a ) ) , \n the invasive capability of tna44 ( 19% ) and br2 ( 35% ) was significantly reduced \n as compared to that of e44 ( 100% ) ( figures 4(c)-4(d ) ) .", "these data \n suggest that tnaa is an important downstream regulator that is required foriber - modulated e. coli k1 invasion .", "as our proteomics analysis had shown that tnaa expression was induced by iber in sp and the tnaa and iber deletion resulted in \n a deficiency in indole production in sp - cultures ( figure 4(b ) ) , we further tested \n whether the tnaa product indole , as an sp extracellular signal molecule , played \n a role in the process of invasion .", "indole is converted to indigo ( which is not \n further degraded in e. coli ) by several monooxygenases , thus providing \n an easy method for its determination .", "a plasmid pstyabb , carrying the gene \n for styrene monooxygenase , was used to monitor the indole production through \n its conversion to indigo .", "the plasmid was transformed into e. coli strains e44 , br2 , and tna44 , and the indole production was measured at \n different time points for these stains in bhi media ( figure 4(b ) ) .", "by contrast , \n the indole production was almost abolished in the tnaa deletion mutant \n and severely reduced in the iber deletion mutant ( figure 4(b ) ) .", "these \n results demonstrated that the indole production was controlled by iber through tnaa . to examine whether indole could compliment the noninvasive \n phenotype of tnaa and iber deletion mutants , indole was supplied in the bhi medium at 100 m to the tna44 \n and br2 sp cultures .", "the \n result showed that indole was able to significantly \n enhance the relative invasion rate of tna44 ( 19% to 69% ) and br2 ( 35% to 65% ) as compared to \n that of e44 , suggesting that indole could partly compliment the noninvasive phenotype of tna44 \n ( figure 4(c ) ) and br2 ( figure 4(d ) ) .", "lacour and landini have shown that the rpos gene in e. coli k12 strain \n mg1655 controls the production of indole , which acts as a signal molecule in sp \n cells , via regulation of tnaa , the indole - producing enzyme .", "as tnaa is \n regulated by iber in e44 , it is most likely that iber is a novel regulator to \n complement the functional deficiency of rpos in e44 .", "it has been reported that rpos is able to positively and \n negatively control expression of a large set of genes when bacteria enter into \n the sp [ 46 , 50 , 51 ] . during such \n transition ,", "bacteria undergo physiological changes that allow their sp \n organisms to survive better in such insults as heat , high - osmotic environment , \n starvation , uv radiation , h2o2 ,   and acid than their \n exponential counterpart [ 50 , 52 ] .", "the loss of rpos resulted in the decrease of \n stress resistance and cell survival in the sp [ 5 , 53 ] .", "although our study \n showed that the loss of iber did not affect the growth rate in bhi \n medium , the survival rates of the iber deletion mutant br2 in the sp \n significantly decreased as compared to that of the wild type strain e44 even \n without any stress treatment ( figure 5(a ) ) .", "our proteomics analysis also \n revealed that the most significant proteomic changes in the iber deletion mutant were related to bacterial response to environmental \n modifications .", "for example , ahpc , as a primary scavenger of endogenous h2o2 , \n  was upregulated in the iber   deletion mutant , implying that the loss of iber resulted in the decreased survival rates of bacterial cells under an oxidative \n stress in the sp .", "ompc , as a porin protein , was downregulated in br2 , perhaps \n resulting in the decreased resistance to osmolarity stress .", "these results \n suggested that iber plays a regulatory role in response to stress \n conditions in e44 that carries a nonsense mutation in rpos .", "to examine \n the function of iber in response to stress environments , we performed \n several survival assays under different stress conditions , including heat shock \n ( 54c for 3 minutes ) , alkali endurance ( tris , ph = 10 for 30 minutes ) , acid \n endurance ( acetic acid , ph = 2.8 for 20 minutes ) , high osmolarity challenge ( 2.5 m nacl for 1 hour ) , and oxidative stress ( 10 m h2o2 for \n 30 minutes ) .", "in all the survival experiments , the wild type strain e44 showed \n higher survival rates than the iber deletion mutant , indicating that the iber gene is required for all these \n stress resistances ( figure 5(b ) ) . especially in the heat shock assay , the loss \n of iber resulted in over 95% cells death , indicating that iber played a vital role in temperature sensitivity in this strain . in the other \n stress treatments ,", "the iber deletion also significantly reduced the \n survival rates ( more than 60% cell death ) of br2 as compared to that of the \n wild type strain e44 , suggesting that iber had a global regulatory role in the \n resistance to acid , alkali , high osmolarity and oxidative stress . in the \n survival assays for the e. coli control strain mg1655", ", the rpos deletion mutant rh90 also decreased the survival levels in these five stress \n conditions , showing the similar patterns like iber in response to stress \n environments ( data not shown ) . combining the proteomics analysis and the stress \n survival studies ,", "we conclude that iber is an rpos - like regulator to control \n gene expression of proteins that are critical for stress - resistance and cell \n survival in the sp in e44 , which carries a loss - of - function mutation in the rpos gene ."], ["currently , most e. coli meningitis studies are done with sp cultures in which the pathogen invasion of \n human bmec is significantly greater than the log phase cultures . in most \n strains of e. coli ,", "rpos plays a \n central role in regulating the sp regulatory genes for protecting cells against \n starvation and stress damage .", "rpos , the major sp regulator , has also been shown \n to regulate the expression of microbial virulence genes in various bacteria \n including e. coli k1 ( o157 : h7 ) , salmonella \n typhimurium , shigella flexneri , yersinia enterocolitica , vibrio \n cholera , and borrelia burgdorfer ,   [ 5 , 53 , 54 ] .", "surprisingly , however , rpos was found to be inactive in meningitic \n strain e44 .", "the current proteomic studies may provide an answer to the \n long - standing question regarding the sp gene regulation in e44 . combining the \n proteomics analysis , virulence determination , and the stress survival studies \n of the iber mutant br2", ", we have \n demonstrated for the first time that iber serves as an rpos - like regulator to \n control gene expression that is critical for stress - resistance and cell \n survival in the sp of e44 .", "iber is not a structural homologue of   rpos as iber \n and rpos do not share any significant sequence homology .", "rpos ( also known as  , \n  , or katf ) is a global regulator in e. coli , which is the second principal  subunit after the major  factor . in e44", ", however , iber appears not to be a master regulator on the \n basis of its genomic prevalence and functional spectrum .", "the prevalence of the \n gimalocus carrying iber is highly dependent on the origin \n of the strain and on the subgroup it belongs to ( a , b1 , b2 , and d ) ( 4 ) . in all \n the studies , where the presence of this locushas been analyzed ,", "gima was found to be restricted to the b2 \n subgroup , a subgroup that includes strains with the highest virulence in mice \n and the highest level of virulence determinants ( 4 ) .", "our proteomic studies showed that a limited number \n of genes were regulated by iber , suggesting that iber is a regulator with a \n narrow functional spectrum .", "in other e. \n coli strains , either pathogenic or probiotic strains , functional \n heterogeneity of rpos in stress tolerance was widely observed [ 5 , 53 , 55 ] . \n", "those studies have shown that some e. \n coli strains can maintain their stress tolerance capability or \n significantly modulate their stress resistance phenotype independent of their rpos genotypes .", "such adaptation processes \n compromising the rpos - dependent stress responses may have significant impact on \n bacterial survival in environments , as well as in the host 's stomach and \n intestine [ 53 , 55 ] .", "iber , a regulator in the gima regulon , may contribute to \n bacterial virulence adaptation process in e44 to complement the functional \n deficiency of rpos .", "another significant finding of our proteomic studies is that \n iber in meningitic strain e44 is able to upregulate tnaa , which is controlled \n by rpos in other e. coli strains \n .", "the virulence determination of the tnaa mutant showed that tnaa and its product indole were required for e44 invasion \n of human bmec .", "the generation of indole , via the tryptophanase activity of \n tnaa , was also observed during the formation of biofilm in e. coli and other bacteria [ 28 , 56 ] .", "in addition to the initiation \n of indole - mediated signaling , tnaa ( tryptophanase ) is able to catabolize \n tryptophan , cysteine , and serine to pyruvate [ 29 , 56 ] .", "the three proteins \n significantly upregulated by iber are tnaa , lpda , and ompc , all of which are \n directly or indirectly involved in pyruvate metabolism .", "ompc , an \n osmotically regulated porin , may facilitate nutrient uptake . on the other \n hand , the three operons ( ptnipkc , cgldtec , and gcxkrci ) in the gima regulon may also directly or indirectly \n contribute to pyruvate metabolism by converting dihydroxyacetone , glycerol , and \n glycerate to pyruvate .", "it has been shown that the capability to catabolize \n carbon source is an important parameter in the ability to persist and compete \n in stationary phase .", "this raises the possibility that signaling by indole , \n which is regulated by iber via tnaa , may play a critical role in the pathways that \n prepare the pathogens for a nutrient - poor environment ( e.g. , cns ) when the \n carbon source becomes limited for energy production ."]]}
{"article_id": "PMC3168080", "article_text": ["today , education has been assigned as an industry for human resource development for different walks of life .", "the students constitute the heart of the education system and they are the most affected ones by the strengths and weaknesses of the education system .", "the students form an identical subgroup compared to others in the society . some of the most common risks faced by a student during his / her college days are psychological , emotional , social , academic , and career risks .", "stress is a very familiar condition faced by the students when they are unable to bear the risks involved in higher education .", "the main sources of stress are academic and time concerns , fear of failure , classroom interactions , and economic issues . apart from this", "the parents have unlimited expectation from their children and therefore they impose their own desires on them . the impact of these influences results in a number of students reporting emotional problems , anxiety , stress , and other neurotic problems .", "they are often found in frightening , abusive , depressing , threatening , competitive , unpredictable , and confusing situations . the percentage of suicides is more among the college students compared to the other ones and these suicides", "majority of the college students strive and try to handle the stress in a positive way . whereas , some of the students do not know , the ways to cope up with their problems and ; therefore , adopt unhealthy ways .", "the coping could be at an emotional , cognitive , or social support seeking level .", "the emotional responsiveness grows out of the simple reflexes of planful emotional responsive behaviour , by the age of two or three years .", "kopp ( 1989 ) described five types of behaviour : visual or physical avoidance , distraction , self - soothing , problem - oriented , and care - eliciting .", "a coping style can be effective at one age but may be totally unsuitable at another age . in the similar way , a particular coping style may be totally appropriate in one situation while inappropriate in another .", "this may be determined to a large part of cultural expectations on the other hand , individual vulnerabilities set limits to ones coping .", "therefore , it is mandatory to understand that how coping ways adopted by college students differ from other students who experience the significant stress ; their perception of handling the psycho - social problems effectively , might help in the planning of intervention services .", "thus , the present study was made to understand the ways in which college students differ in coping with significant stress experienced by the students and to examine the different ways of coping strategies adopted by them .", "the present study examines the coping patterns adopted by the junior college students with respect to stress in the academic area , and to study the gender difference in coping patterns among junior college students .", "the universe considered for the study was , i puc and ii puc students , who were studying either in bachelor of arts , science , or commerce . in the investigation process , a coping checklist with 70 diverse coping pattern , prepared by rao et al . , was used .", "yesno format and they described a broad range of behavioural , emotional , and cognitive response that may be used to handle stress ; and a socio - demographic data sheet was also used . in order to compute the difference between the means of two groups ( male and female ) , frequency , percentage , and t - test", "were used ; and to compare the frequencies of certain variables in the two groups , chi - square was used .", "the study was conducted on i puc and ii puc college students . with regard to the socio - demographic characteristics of the college students ,", "it was observed that majority of the students were aged between 1718 years , and number of females was slightly more than males . with respect to the religion ,", "a majority of the students were hindu , and the least were islamic , compared to the students belonging to other religion ; because most of the people from islam do not tend to pursue higher education .", "with regard to birth order of the child , majority of the students were middle born .", "it was also noted that , a majority of the respondents spoke other languages at home than kannada .", "this proves that respondents families belong to various states , but they have settled here .", "it was seen that most of the students were from nuclear families , which is a very common trend in urban areas . with respect to education background ,", "a majority of the students were from well - educated families , with their fathers being postgraduate and mothers being educated up to higher secondary .", "the studies also revealed that , the fathers of the respondents were businessmen , whereas mothers were housewives . however , approximately one - third of the respondents mothers were employed .", "the coping strategies adopted by college students with regard to stressful events , i.e. , academic area were assessed .", "the results revealed that most of the students ( approximately 90% ) used appraisal - focused coping strategies such as ,  going over the problem again and again  ,  trying to understand it , to make the situation light  , and  refuse to get too serious about it  .", "also , majority of the college students reported that they frequently adopt problem - solving coping strategies such as ,  knowing what has to be done , so doubling the efforts and trying harder to make things work  ,  analyzing the problem bit by bit  ,", " seeking reassurance and emotional support from family members  , and  coming up with different solutions to the problem  . in the current study", ", it has been observed that the students used a combination of appraisal - focused and problem - focused coping strategies . whereas , study by lazarus revealed that , problem - focused and emotion - focused coping strategies were used in virtually every stressful encounter . in the present study , it was found that the students used appraisal - focused coping strategies such as ,  analyzing the situation  ,  cognitively redefining  , and  cognitively avoiding the situation  .", "this proves that the students in adolescence stage were still confused and were not able to choose those coping strategies which were of positive approach to encounter any stress .", "the present study revealed that the students resorted to the coping strategies such as  seeking sexual comfort ( 24% )  ,  taking analgesics or minor tranquilizers without medical advice ( 15% )  ,  to make themselves feel better by having a drink or two ( 10% )  , and  to make themselves feel better by taking mood elevating drugs ( 10% )  .", "these findings were supported by the study by o. languhlin ( 1984 ) , who also reported that resorting to drugs , alcohol , tranquilizers , and sex are more characteristics of inefficient coopers .", "the findings by mccormack ( 1996 ) , stated that 23% to 36% of the students advocate drinking when under stress ; however , even a small percentage of students using this coping strategy may form a high risk group . in the current study ,", "the sample comprised college students belonging to 1 puc and ii puc , who were still in adolescence stage and these students were not fully exposed to the evils of the society . in the study ,", "distribution of the coping strategies adopted by the college students was examined with respect to their gender background .", "the results also revealed that the students irrespective of their gender , used combination of problem - focused and emotion - focused coping strategies .", "it was found that , out of 70 items , 16 significant responses were used by the students .", "the coping strategies adopted by female students were ,  talking to a friend who can do something about the problem  ,  analyzing the problem bit by bit  ,  consoling themselves that things are not that bad and could have been worse  ,  visit places of worship , go on a pilgrimage  ; while male students resorted to coping strategies such as ,  seeking reassurance and emotional support from family members ", ",  engage in vigorous physical exercises  ,  talking to a friend who can do something about the problem  , and  taking a big chance or doing something very risky  .", "it was observed that the female students used emotion - focused coping strategies and sought more social support than males .", "these findings were supported by the study done by frydenberg and lewis , ( 1991 ) and reported that females sought more social support and employed strategies related to ,  hoping for the best  and  wishful thinking  , than males . also , hobfall , et al . , ( 1994 ) reported that when faced with problems of professional and interpersonal situations , support seeking was employed more by women than men .", "the findings by mike and thoits ( 1993 ) suggested that men experience a stoic style of responding to stressors and women have an emotional expressive style .", "the distribution of coping strategies adopted by college students was examined with respect to different faculties , such as arts , science , and commerce .", "it was observed that the students used a combination of appraisal - focused and problem - focused coping strategies . out of 70 responses ,", "the frequently used coping strategies by the students belonging to different faculties were problem - focused , for example ,  pray to god,and  knowing what has to be done , so doubling the efforts and trying harder to make things work  , were displayed by more than 90% of the college students .", "majority ( 90% ) of the students belonging to science and arts faculties and 75% from commerce , used appraisal - focused coping strategies such as ,  console yourself that the things are not at all that bad , it could be worse  ,  make light of the situation , refused to get too serious about it  ,  refusing to believe that it happened  . also , 43% of the students from commerce and science faculties used emotion - focused coping strategies , whereas 35% from arts faculty used emotional - focused coping strategies , such as ,  preparing themselves for the worst to come  , ", "feeling that other people are responsible for what has happened  , and  trying to feel better by eating / nibbling  .", "overall , the total coping repertoires of students belonging to arts , science , and commerce faculties were 37 , 38 , and 35 , respectively . with respect to gender ,", "the total coping repertoire for male and female students , were 36 and 37 , respectively .", "murthy ( 1995 ) found in her study of working women that they have a coping repertoire of approximately 21 .", "karen ( 2000 ) reported that the total coping repertoire of unsuccessful college students was 37 .", "the study was conducted on i puc and ii puc college students . with regard to the socio - demographic characteristics of the college students ,", "it was observed that majority of the students were aged between 1718 years , and number of females was slightly more than males . with respect to the religion ,", "a majority of the students were hindu , and the least were islamic , compared to the students belonging to other religion ; because most of the people from islam do not tend to pursue higher education .", "with regard to birth order of the child , majority of the students were middle born .", "it was also noted that , a majority of the respondents spoke other languages at home than kannada .", "this proves that respondents families belong to various states , but they have settled here .", "it was seen that most of the students were from nuclear families , which is a very common trend in urban areas . with respect to education background ,", "a majority of the students were from well - educated families , with their fathers being postgraduate and mothers being educated up to higher secondary .", "the studies also revealed that , the fathers of the respondents were businessmen , whereas mothers were housewives . however , approximately one - third of the respondents mothers were employed .", "the coping strategies adopted by college students with regard to stressful events , i.e. , academic area were assessed .", "the results revealed that most of the students ( approximately 90% ) used appraisal - focused coping strategies such as ,  going over the problem again and again  ,  trying to understand it , to make the situation light  , and  refuse to get too serious about it  .", "also , majority of the college students reported that they frequently adopt problem - solving coping strategies such as ,  knowing what has to be done , so doubling the efforts and trying harder to make things work  ,  analyzing the problem bit by bit  ,  seeking reassurance and emotional support from family members  , and  coming up with different solutions to the problem  . in the current study", ", it has been observed that the students used a combination of appraisal - focused and problem - focused coping strategies . whereas , study by lazarus revealed that , problem - focused and emotion - focused coping strategies were used in virtually every stressful encounter . in the present study , it was found that the students used appraisal - focused coping strategies such as ,  analyzing the situation  ,  cognitively redefining  , and  cognitively avoiding the situation  .", "this proves that the students in adolescence stage were still confused and were not able to choose those coping strategies which were of positive approach to encounter any stress .", "the present study revealed that the students resorted to the coping strategies such as  seeking sexual comfort ( 24% )  ,  taking analgesics or minor tranquilizers without medical advice ( 15% )  ,  to make themselves feel better by having a drink or two ( 10% )  , and  to make themselves feel better by taking mood elevating drugs ( 10% )  .", "these findings were supported by the study by o. languhlin ( 1984 ) , who also reported that resorting to drugs , alcohol , tranquilizers , and sex are more characteristics of inefficient coopers .", "the findings by mccormack ( 1996 ) , stated that 23% to 36% of the students advocate drinking when under stress ; however , even a small percentage of students using this coping strategy may form a high risk group . in the current study ,", "the sample comprised college students belonging to 1 puc and ii puc , who were still in adolescence stage and these students were not fully exposed to the evils of the society . in the study ,", "distribution of the coping strategies adopted by the college students was examined with respect to their gender background .", "the results also revealed that the students irrespective of their gender , used combination of problem - focused and emotion - focused coping strategies .", "it was found that , out of 70 items , 16 significant responses were used by the students .", "the coping strategies adopted by female students were ,  talking to a friend who can do something about the problem ", ",  analyzing the problem bit by bit  ,  consoling themselves that things are not that bad and could have been worse  ,  visit places of worship , go on a pilgrimage  ; while male students resorted to coping strategies such as ,  seeking reassurance and emotional support from family members ", ",  engage in vigorous physical exercises  ,  talking to a friend who can do something about the problem  , and  taking a big chance or doing something very risky  .", "it was observed that the female students used emotion - focused coping strategies and sought more social support than males .", "these findings were supported by the study done by frydenberg and lewis , ( 1991 ) and reported that females sought more social support and employed strategies related to ,  hoping for the best  and  wishful thinking  , than males . also , hobfall , et al . ,", "( 1994 ) reported that when faced with problems of professional and interpersonal situations , support seeking was employed more by women than men .", "the findings by mike and thoits ( 1993 ) suggested that men experience a stoic style of responding to stressors and women have an emotional expressive style .", "the distribution of coping strategies adopted by college students was examined with respect to different faculties , such as arts , science , and commerce .", "it was observed that the students used a combination of appraisal - focused and problem - focused coping strategies . out of 70 responses ,", "the frequently used coping strategies by the students belonging to different faculties were problem - focused , for example ,  pray to god,and  knowing what has to be done , so doubling the efforts and trying harder to make things work  , were displayed by more than 90% of the college students .", "majority ( 90% ) of the students belonging to science and arts faculties and 75% from commerce , used appraisal - focused coping strategies such as ,  console yourself that the things are not at all that bad , it could be worse  ,  make light of the situation , refused to get too serious about it  ,  refusing to believe that it happened  . also , 43% of the students from commerce and science faculties used emotion - focused coping strategies , whereas 35% from arts faculty used emotional - focused coping strategies , such as ,  preparing themselves for the worst to come  ,  feeling that other people are responsible for what has happened  , and  trying to feel better by eating / nibbling  .", "overall , the total coping repertoires of students belonging to arts , science , and commerce faculties were 37 , 38 , and 35 , respectively . with respect to gender ,", "the total coping repertoire for male and female students , were 36 and 37 , respectively .", "murthy ( 1995 ) found in her study of working women that they have a coping repertoire of approximately 21 .", "karen ( 2000 ) reported that the total coping repertoire of unsuccessful college students was 37 ."], "abstract_text": ["<S> the objective of this study was to examine the coping patterns followed by the junior college students . </S>", "<S> further , an extensive effort was done to study the gender differences in coping patterns used by the students . </S>", "<S> this study was conducted in christ college , bangalore and on the first and second - year students of pre - university studying in either of the branches ( bachelor of arts , science , or commerce ) . </S>", "<S> a total of 120 samples were collected from study population of junior college students using the random sampling method . </S>", "<S> the sample comprised , 40 students from each group of arts , science , and commerce , including both of the sexes . </S>", "<S> the tools such as , socio - demographic data sheet and coping checklist , were used . </S>", "<S> the study findings revealed that majority of the students adopted emotion- and problem - focused coping strategies . </S>", "<S> most of the female students adopted emotion - focused coping strategies , whereas the male students mostly used problem - focused coping strategies . </S>"], "labels": null, "section_names": ["INTRODUCUTION", "MATERIALS AND METHODS", "RESULTS AND DISCUSSION", "Socio-demographic profile of college students", "Coping strategies adopted by college students"], "sections": [["today , education has been assigned as an industry for human resource development for different walks of life .", "the students constitute the heart of the education system and they are the most affected ones by the strengths and weaknesses of the education system .", "the students form an identical subgroup compared to others in the society . some of the most common risks faced by a student during his / her college days are psychological , emotional , social , academic , and career risks .", "stress is a very familiar condition faced by the students when they are unable to bear the risks involved in higher education .", "the main sources of stress are academic and time concerns , fear of failure , classroom interactions , and economic issues . apart from this", "the parents have unlimited expectation from their children and therefore they impose their own desires on them . the impact of these influences results in a number of students reporting emotional problems , anxiety , stress , and other neurotic problems .", "they are often found in frightening , abusive , depressing , threatening , competitive , unpredictable , and confusing situations . the percentage of suicides is more among the college students compared to the other ones and these suicides", "majority of the college students strive and try to handle the stress in a positive way . whereas , some of the students do not know , the ways to cope up with their problems and ; therefore , adopt unhealthy ways .", "the coping could be at an emotional , cognitive , or social support seeking level .", "the emotional responsiveness grows out of the simple reflexes of planful emotional responsive behaviour , by the age of two or three years .", "kopp ( 1989 ) described five types of behaviour : visual or physical avoidance , distraction , self - soothing , problem - oriented , and care - eliciting .", "a coping style can be effective at one age but may be totally unsuitable at another age . in the similar way , a particular coping style may be totally appropriate in one situation while inappropriate in another .", "this may be determined to a large part of cultural expectations on the other hand , individual vulnerabilities set limits to ones coping .", "therefore , it is mandatory to understand that how coping ways adopted by college students differ from other students who experience the significant stress ; their perception of handling the psycho - social problems effectively , might help in the planning of intervention services .", "thus , the present study was made to understand the ways in which college students differ in coping with significant stress experienced by the students and to examine the different ways of coping strategies adopted by them ."], ["the present study examines the coping patterns adopted by the junior college students with respect to stress in the academic area , and to study the gender difference in coping patterns among junior college students .", "the universe considered for the study was , i puc and ii puc students , who were studying either in bachelor of arts , science , or commerce . in the investigation process , a coping checklist with 70 diverse coping pattern , prepared by rao et al . , was used .", "yesno format and they described a broad range of behavioural , emotional , and cognitive response that may be used to handle stress ; and a socio - demographic data sheet was also used . in order to compute the difference between the means of two groups ( male and female ) , frequency , percentage , and t - test", "were used ; and to compare the frequencies of certain variables in the two groups , chi - square was used ."], ["the study was conducted on i puc and ii puc college students . with regard to the socio - demographic characteristics of the college students ,", "it was observed that majority of the students were aged between 1718 years , and number of females was slightly more than males . with respect to the religion ,", "a majority of the students were hindu , and the least were islamic , compared to the students belonging to other religion ; because most of the people from islam do not tend to pursue higher education .", "with regard to birth order of the child , majority of the students were middle born .", "it was also noted that , a majority of the respondents spoke other languages at home than kannada .", "this proves that respondents families belong to various states , but they have settled here .", "it was seen that most of the students were from nuclear families , which is a very common trend in urban areas . with respect to education background ,", "a majority of the students were from well - educated families , with their fathers being postgraduate and mothers being educated up to higher secondary .", "the studies also revealed that , the fathers of the respondents were businessmen , whereas mothers were housewives . however , approximately one - third of the respondents mothers were employed .", "the coping strategies adopted by college students with regard to stressful events , i.e. , academic area were assessed .", "the results revealed that most of the students ( approximately 90% ) used appraisal - focused coping strategies such as ,  going over the problem again and again  ,  trying to understand it , to make the situation light  , and  refuse to get too serious about it  .", "also , majority of the college students reported that they frequently adopt problem - solving coping strategies such as ,  knowing what has to be done , so doubling the efforts and trying harder to make things work  ,  analyzing the problem bit by bit  ,", " seeking reassurance and emotional support from family members  , and  coming up with different solutions to the problem  . in the current study", ", it has been observed that the students used a combination of appraisal - focused and problem - focused coping strategies . whereas , study by lazarus revealed that , problem - focused and emotion - focused coping strategies were used in virtually every stressful encounter . in the present study , it was found that the students used appraisal - focused coping strategies such as ,  analyzing the situation  ,  cognitively redefining  , and  cognitively avoiding the situation  .", "this proves that the students in adolescence stage were still confused and were not able to choose those coping strategies which were of positive approach to encounter any stress .", "the present study revealed that the students resorted to the coping strategies such as  seeking sexual comfort ( 24% )  ,  taking analgesics or minor tranquilizers without medical advice ( 15% )  ,  to make themselves feel better by having a drink or two ( 10% )  , and  to make themselves feel better by taking mood elevating drugs ( 10% )  .", "these findings were supported by the study by o. languhlin ( 1984 ) , who also reported that resorting to drugs , alcohol , tranquilizers , and sex are more characteristics of inefficient coopers .", "the findings by mccormack ( 1996 ) , stated that 23% to 36% of the students advocate drinking when under stress ; however , even a small percentage of students using this coping strategy may form a high risk group . in the current study ,", "the sample comprised college students belonging to 1 puc and ii puc , who were still in adolescence stage and these students were not fully exposed to the evils of the society . in the study ,", "distribution of the coping strategies adopted by the college students was examined with respect to their gender background .", "the results also revealed that the students irrespective of their gender , used combination of problem - focused and emotion - focused coping strategies .", "it was found that , out of 70 items , 16 significant responses were used by the students .", "the coping strategies adopted by female students were ,  talking to a friend who can do something about the problem  ,  analyzing the problem bit by bit  ,  consoling themselves that things are not that bad and could have been worse  ,  visit places of worship , go on a pilgrimage  ; while male students resorted to coping strategies such as ,  seeking reassurance and emotional support from family members ", ",  engage in vigorous physical exercises  ,  talking to a friend who can do something about the problem  , and  taking a big chance or doing something very risky  .", "it was observed that the female students used emotion - focused coping strategies and sought more social support than males .", "these findings were supported by the study done by frydenberg and lewis , ( 1991 ) and reported that females sought more social support and employed strategies related to ,  hoping for the best  and  wishful thinking  , than males . also , hobfall , et al . , ( 1994 ) reported that when faced with problems of professional and interpersonal situations , support seeking was employed more by women than men .", "the findings by mike and thoits ( 1993 ) suggested that men experience a stoic style of responding to stressors and women have an emotional expressive style .", "the distribution of coping strategies adopted by college students was examined with respect to different faculties , such as arts , science , and commerce .", "it was observed that the students used a combination of appraisal - focused and problem - focused coping strategies . out of 70 responses ,", "the frequently used coping strategies by the students belonging to different faculties were problem - focused , for example ,  pray to god,and  knowing what has to be done , so doubling the efforts and trying harder to make things work  , were displayed by more than 90% of the college students .", "majority ( 90% ) of the students belonging to science and arts faculties and 75% from commerce , used appraisal - focused coping strategies such as ,  console yourself that the things are not at all that bad , it could be worse  ,  make light of the situation , refused to get too serious about it  ,  refusing to believe that it happened  . also , 43% of the students from commerce and science faculties used emotion - focused coping strategies , whereas 35% from arts faculty used emotional - focused coping strategies , such as ,  preparing themselves for the worst to come  , ", "feeling that other people are responsible for what has happened  , and  trying to feel better by eating / nibbling  .", "overall , the total coping repertoires of students belonging to arts , science , and commerce faculties were 37 , 38 , and 35 , respectively . with respect to gender ,", "the total coping repertoire for male and female students , were 36 and 37 , respectively .", "murthy ( 1995 ) found in her study of working women that they have a coping repertoire of approximately 21 .", "karen ( 2000 ) reported that the total coping repertoire of unsuccessful college students was 37 ."], ["the study was conducted on i puc and ii puc college students . with regard to the socio - demographic characteristics of the college students ,", "it was observed that majority of the students were aged between 1718 years , and number of females was slightly more than males . with respect to the religion ,", "a majority of the students were hindu , and the least were islamic , compared to the students belonging to other religion ; because most of the people from islam do not tend to pursue higher education .", "with regard to birth order of the child , majority of the students were middle born .", "it was also noted that , a majority of the respondents spoke other languages at home than kannada .", "this proves that respondents families belong to various states , but they have settled here .", "it was seen that most of the students were from nuclear families , which is a very common trend in urban areas . with respect to education background ,", "a majority of the students were from well - educated families , with their fathers being postgraduate and mothers being educated up to higher secondary .", "the studies also revealed that , the fathers of the respondents were businessmen , whereas mothers were housewives . however , approximately one - third of the respondents mothers were employed ."], ["the coping strategies adopted by college students with regard to stressful events , i.e. , academic area were assessed .", "the results revealed that most of the students ( approximately 90% ) used appraisal - focused coping strategies such as ,  going over the problem again and again  ,  trying to understand it , to make the situation light  , and  refuse to get too serious about it  .", "also , majority of the college students reported that they frequently adopt problem - solving coping strategies such as ,  knowing what has to be done , so doubling the efforts and trying harder to make things work  ,  analyzing the problem bit by bit  ,  seeking reassurance and emotional support from family members  , and  coming up with different solutions to the problem  . in the current study", ", it has been observed that the students used a combination of appraisal - focused and problem - focused coping strategies . whereas , study by lazarus revealed that , problem - focused and emotion - focused coping strategies were used in virtually every stressful encounter . in the present study , it was found that the students used appraisal - focused coping strategies such as ,  analyzing the situation  ,  cognitively redefining  , and  cognitively avoiding the situation  .", "this proves that the students in adolescence stage were still confused and were not able to choose those coping strategies which were of positive approach to encounter any stress .", "the present study revealed that the students resorted to the coping strategies such as  seeking sexual comfort ( 24% )  ,  taking analgesics or minor tranquilizers without medical advice ( 15% )  ,  to make themselves feel better by having a drink or two ( 10% )  , and  to make themselves feel better by taking mood elevating drugs ( 10% )  .", "these findings were supported by the study by o. languhlin ( 1984 ) , who also reported that resorting to drugs , alcohol , tranquilizers , and sex are more characteristics of inefficient coopers .", "the findings by mccormack ( 1996 ) , stated that 23% to 36% of the students advocate drinking when under stress ; however , even a small percentage of students using this coping strategy may form a high risk group . in the current study ,", "the sample comprised college students belonging to 1 puc and ii puc , who were still in adolescence stage and these students were not fully exposed to the evils of the society . in the study ,", "distribution of the coping strategies adopted by the college students was examined with respect to their gender background .", "the results also revealed that the students irrespective of their gender , used combination of problem - focused and emotion - focused coping strategies .", "it was found that , out of 70 items , 16 significant responses were used by the students .", "the coping strategies adopted by female students were ,  talking to a friend who can do something about the problem ", ",  analyzing the problem bit by bit  ,  consoling themselves that things are not that bad and could have been worse  ,  visit places of worship , go on a pilgrimage  ; while male students resorted to coping strategies such as ,  seeking reassurance and emotional support from family members ", ",  engage in vigorous physical exercises  ,  talking to a friend who can do something about the problem  , and  taking a big chance or doing something very risky  .", "it was observed that the female students used emotion - focused coping strategies and sought more social support than males .", "these findings were supported by the study done by frydenberg and lewis , ( 1991 ) and reported that females sought more social support and employed strategies related to ,  hoping for the best  and  wishful thinking  , than males . also , hobfall , et al . ,", "( 1994 ) reported that when faced with problems of professional and interpersonal situations , support seeking was employed more by women than men .", "the findings by mike and thoits ( 1993 ) suggested that men experience a stoic style of responding to stressors and women have an emotional expressive style .", "the distribution of coping strategies adopted by college students was examined with respect to different faculties , such as arts , science , and commerce .", "it was observed that the students used a combination of appraisal - focused and problem - focused coping strategies . out of 70 responses ,", "the frequently used coping strategies by the students belonging to different faculties were problem - focused , for example ,  pray to god,and  knowing what has to be done , so doubling the efforts and trying harder to make things work  , were displayed by more than 90% of the college students .", "majority ( 90% ) of the students belonging to science and arts faculties and 75% from commerce , used appraisal - focused coping strategies such as ,  console yourself that the things are not at all that bad , it could be worse  ,  make light of the situation , refused to get too serious about it  ,  refusing to believe that it happened  . also , 43% of the students from commerce and science faculties used emotion - focused coping strategies , whereas 35% from arts faculty used emotional - focused coping strategies , such as ,  preparing themselves for the worst to come  ,  feeling that other people are responsible for what has happened  , and  trying to feel better by eating / nibbling  .", "overall , the total coping repertoires of students belonging to arts , science , and commerce faculties were 37 , 38 , and 35 , respectively . with respect to gender ,", "the total coping repertoire for male and female students , were 36 and 37 , respectively .", "murthy ( 1995 ) found in her study of working women that they have a coping repertoire of approximately 21 .", "karen ( 2000 ) reported that the total coping repertoire of unsuccessful college students was 37 ."]]}
{"article_id": "PMC4495251", "article_text": ["dermatomyositis ( dm ) is an uncommon idiopathic inflammatory myopathy that primarily affects skeletal muscle and skin with well - characterized cutaneous findings .", "it has been well documented that dm carries an increased risk of malignancy and can present as a paraneoplastic syndrome to multiple types of underlying malignancies [ 3 , 4 ] .", "however , 1530% of cases of dm are manifestations of paraneoplastic syndromes of an underlying malignancy . here , we present a case of concurrent dm and breast cancer to highlight a rare presentation , progression and regression of symptoms , as well as key components of diagnosis and treatment .", "the patient is a 47-year - old polish premenopausal female who presented with complaints of a non - tender palpable left breast mass for 4 months at an outside institution .", "she underwent a diagnostic mammogram and ultrasound that reported a 2-cm irregular , hypoechoic mass at 12 o'clock .", "there was a 2.2-cm enhancing mass in the left breast at 12:00 that was consistent with the patient 's biopsy - proven malignancy ( fig . ", ", she underwent a core needle biopsy , which revealed a poorly differentiated triple - negative invasive ductal carcinoma ( fig . ", "there is a 1.9  1.4 cm irregular mass in the left breast at 1112:00 6 cm from the nipple . \n", "there was a 2.2 cm enhancing mass in the left breast at 12:00 that was consistent with the patient 's biopsy - proven malignancy . \n", "high power view of the left breast core biopsy ( b ) . left breast ultrasound .", "there is a 1.9  1.4 cm irregular mass in the left breast at 1112:00 6 cm from the nipple .", "the circled area revealed a 2-cm irregular mass with biopsy clip noted . left breast on mri .", "there was a 2.2 cm enhancing mass in the left breast at 12:00 that was consistent with the patient 's biopsy - proven malignancy .", "two weeks after the biopsy , she complained of new onset pruritic , erythematous rash on her anterior chest , face and back of the hands ( fig . ", "she was complaining of diffuse myalgias , worse during menstruation . owing to her worsening symptoms which included cuticular hypertrophy of the hands with associated erythema over the metacarpophalangeal and proximal interphalangeal joints bilaterally ( fig . ", "5c ) , she was transferred to the bellevue hospital breast clinic and referred to the rheumatology clinic for management of her symptoms .", "initial laboratories revealed moderately elevated laboratory values ( table  1 ) . at this time", ", she was presumptively diagnosed with paraneoplastic dm secondary to invasive ductal carcinoma of the left breast and treated with 20 mg prednisone daily .", "her persistent and slowly progressing symptoms despite aggressive treatments prompted our decision to proceed with surgical intervention in hopes of preventing progression of her musculoskeletal manifestations .", "postoperatively , she experienced rapid improvement in both cutaneous and musculoskeletal manifestations with visible clearing of her rash and return of her strength .", "she was maintained on prednisone with excellent symptomatic control . on her 6-month visit , we noted resolution of most of her rash , periorbital edema as well as myalgias and muscle weakness ( fig . ", "table  1laboratory values prior to the surgery , 3 weeks postoperatively and 6 weeks postoperatively.preop3 weeks postop6 weeks postopwhite blood cell ( 10)9.312.67.8creatine kinase ( u / l)2183302160aldolase ( u / l)17.45.3myoglobin247ast ( u / l)1177744alt ( u / l)539130 \n figure  5:image of patient with facial rash and periorbital edema that initially developed during her workup ( a )", ". images of the worsening rash on her chest and hands that initially developed during her workup ( b and c ) . \n", "image of patient with facial rash and periorbital edema that initially developed during her workup ( a ) .", "images of the worsening rash on her chest and hands that initially developed during her workup ( b and c ) .", "dm typically presents with progressive , symmetrical , proximal muscle weakness and characteristic skin lesions such as helitrope rash , gottron 's papules , gottron 's sign , the v - sign and shawl sign .", "additional cutaneous manifestations that have become more commonly recognized include vasculopathic changes ( i.e. telangiectasias and livedo reticularis ) , cuticular overgrowth (  mechanic 's hands ) and poikiloderma . initial presentation follows a bimodal distribution between ages 515 and 4564 and tends to progress over a 3- to 6-month period before the patient will seek medical attention .", "contrary to this typical presentation , the above patient experienced rapid progression of symptoms over a much shorter period and predominately reported skin manifestations with subsequent rapid development of muscular deterioration .", "the diagnosis and classification of dm commonly follows the criteria set forth by bohan and peter in 1975 ( table  2 ) [ 1 , 2 ] .", "initial evaluation for suspected dm should include such investigations as serum creatine kinase , aldolase , aspartate aminotransferase , alanine aminotransferase and/or lactate dehydrogenase .", "serum creatine kinase is the most sensitive muscle enzyme in the acute phase of the disease as it is released in the serum during muscle damage .", "serum inflammatory markers ( e.g. erythrocyte sedimentation rate and c - reactive protein ) may also be elevated during the active phase of the disease .", "table  2bohan and peter criteria for diagnosis of dermatomyositis.individual criteria \n symmetric proximal muscle weaknessmuscle biopsy evidence of myositisincrease in serum skeletal muscle enzymescharacteristic electromyographic patternstypical rash of dermatomyositisdiagnostic criteria \n definitive5 plus any three of 14probably5 plus any two of 14possible5 plus any one of 14 bohan and peter criteria for diagnosis of dermatomyositis .", "symmetric proximal muscle weakness muscle biopsy evidence of myositis increase in serum skeletal muscle enzymes characteristic electromyographic patterns typical rash of dermatomyositis definitive5 plus any three of 14 probably5 plus any two of 14 possible5 plus any one of 14 previous studies have shown that up to 30% of dm patients had underlying malignancies [ 6 , 7 ] .", "malignancies in dm patients have been found to be highest in those over 45 years of age , commonly diagnosed within the first year of initial presentation , and most strongly associated with lung , ovarian , gastric , pancreatic and colorectal origin .", "malignancy can precede , occur concurrently or develop after the diagnosis of dm . in our patient , the symptoms of dm occurred 2 weeks following her diagnosis of breast cancer .", "the goals of therapy in patients with dm are to improve the patient 's ability to carry out activities of daily living by increasing muscle strength and improving myalgias as well as treating the extramuscular manifestations ( including rash and arthralgias ) .", "there has been report of variable influence of the treatment of the underlying malignancy on the clinical course of the dm [ 9 , 10 ] . in our patient ,", "the complete surgical excision of the underlying breast cancer resulted in dramatic improvement in both her cutaneous manifestations and her musculosckeletal symptoms .", "given the high frequency of malignancy in patients with a diagnosis of dm , any woman over the age of 45 with newly diagnosed dm should prompt a thorough physical examination , including a breast examination , as well as further workup for other malignancies ( e.g. ovarian or colorectal cancer ) .", "early diagnosis of dm as a paraneoplastic syndrome allows for initiation of systemic therapy for symptom control and also treatment of the underlying malignancy itself , which may aid in control of the dm symptoms ."], "abstract_text": ["<S> breast cancer is the most common cancer in women in the usa , with the lifetime incidence of 1 in 8 women . </S>", "<S> dermatomyositis ( dm ) is an uncommon idiopathic inflammatory myopathy that can manifest as a paraneoplastic syndrome of an underlying malignancy . here </S>", "<S> , we report a case of a patient who presented with breast cancer and dm symptoms . </S>", "<S> the patient 's rash and muscle weakness progressed during the workup of her breast cancer , while she was already started on medical treatment of these symptoms with oral prednisone . her cutaneous and musculoskeletal improved dramatically following the treatment of her breast cancer . </S>", "<S> our case report describes the rapid progression and regression of her symptoms emphasizing the benefit of early diagnosis and treatment of dm as well as the underlying breast cancer . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONFLICT OF INTEREST STATEMENT"], "sections": [["dermatomyositis ( dm ) is an uncommon idiopathic inflammatory myopathy that primarily affects skeletal muscle and skin with well - characterized cutaneous findings .", "it has been well documented that dm carries an increased risk of malignancy and can present as a paraneoplastic syndrome to multiple types of underlying malignancies [ 3 , 4 ] .", "however , 1530% of cases of dm are manifestations of paraneoplastic syndromes of an underlying malignancy . here , we present a case of concurrent dm and breast cancer to highlight a rare presentation , progression and regression of symptoms , as well as key components of diagnosis and treatment ."], ["the patient is a 47-year - old polish premenopausal female who presented with complaints of a non - tender palpable left breast mass for 4 months at an outside institution .", "she underwent a diagnostic mammogram and ultrasound that reported a 2-cm irregular , hypoechoic mass at 12 o'clock .", "there was a 2.2-cm enhancing mass in the left breast at 12:00 that was consistent with the patient 's biopsy - proven malignancy ( fig . ", ", she underwent a core needle biopsy , which revealed a poorly differentiated triple - negative invasive ductal carcinoma ( fig . ", "there is a 1.9  1.4 cm irregular mass in the left breast at 1112:00 6 cm from the nipple . \n", "there was a 2.2 cm enhancing mass in the left breast at 12:00 that was consistent with the patient 's biopsy - proven malignancy . \n", "high power view of the left breast core biopsy ( b ) . left breast ultrasound .", "there is a 1.9  1.4 cm irregular mass in the left breast at 1112:00 6 cm from the nipple .", "the circled area revealed a 2-cm irregular mass with biopsy clip noted . left breast on mri .", "there was a 2.2 cm enhancing mass in the left breast at 12:00 that was consistent with the patient 's biopsy - proven malignancy .", "two weeks after the biopsy , she complained of new onset pruritic , erythematous rash on her anterior chest , face and back of the hands ( fig . ", "she was complaining of diffuse myalgias , worse during menstruation . owing to her worsening symptoms which included cuticular hypertrophy of the hands with associated erythema over the metacarpophalangeal and proximal interphalangeal joints bilaterally ( fig . ", "5c ) , she was transferred to the bellevue hospital breast clinic and referred to the rheumatology clinic for management of her symptoms .", "initial laboratories revealed moderately elevated laboratory values ( table  1 ) . at this time", ", she was presumptively diagnosed with paraneoplastic dm secondary to invasive ductal carcinoma of the left breast and treated with 20 mg prednisone daily .", "her persistent and slowly progressing symptoms despite aggressive treatments prompted our decision to proceed with surgical intervention in hopes of preventing progression of her musculoskeletal manifestations .", "postoperatively , she experienced rapid improvement in both cutaneous and musculoskeletal manifestations with visible clearing of her rash and return of her strength .", "she was maintained on prednisone with excellent symptomatic control . on her 6-month visit , we noted resolution of most of her rash , periorbital edema as well as myalgias and muscle weakness ( fig . ", "table  1laboratory values prior to the surgery , 3 weeks postoperatively and 6 weeks postoperatively.preop3 weeks postop6 weeks postopwhite blood cell ( 10)9.312.67.8creatine kinase ( u / l)2183302160aldolase ( u / l)17.45.3myoglobin247ast ( u / l)1177744alt ( u / l)539130 \n figure  5:image of patient with facial rash and periorbital edema that initially developed during her workup ( a )", ". images of the worsening rash on her chest and hands that initially developed during her workup ( b and c ) . \n", "image of patient with facial rash and periorbital edema that initially developed during her workup ( a ) .", "images of the worsening rash on her chest and hands that initially developed during her workup ( b and c ) ."], ["dm typically presents with progressive , symmetrical , proximal muscle weakness and characteristic skin lesions such as helitrope rash , gottron 's papules , gottron 's sign , the v - sign and shawl sign .", "additional cutaneous manifestations that have become more commonly recognized include vasculopathic changes ( i.e. telangiectasias and livedo reticularis ) , cuticular overgrowth (  mechanic 's hands ) and poikiloderma . initial presentation follows a bimodal distribution between ages 515 and 4564 and tends to progress over a 3- to 6-month period before the patient will seek medical attention .", "contrary to this typical presentation , the above patient experienced rapid progression of symptoms over a much shorter period and predominately reported skin manifestations with subsequent rapid development of muscular deterioration .", "the diagnosis and classification of dm commonly follows the criteria set forth by bohan and peter in 1975 ( table  2 ) [ 1 , 2 ] .", "initial evaluation for suspected dm should include such investigations as serum creatine kinase , aldolase , aspartate aminotransferase , alanine aminotransferase and/or lactate dehydrogenase .", "serum creatine kinase is the most sensitive muscle enzyme in the acute phase of the disease as it is released in the serum during muscle damage .", "serum inflammatory markers ( e.g. erythrocyte sedimentation rate and c - reactive protein ) may also be elevated during the active phase of the disease .", "table  2bohan and peter criteria for diagnosis of dermatomyositis.individual criteria \n symmetric proximal muscle weaknessmuscle biopsy evidence of myositisincrease in serum skeletal muscle enzymescharacteristic electromyographic patternstypical rash of dermatomyositisdiagnostic criteria \n definitive5 plus any three of 14probably5 plus any two of 14possible5 plus any one of 14 bohan and peter criteria for diagnosis of dermatomyositis .", "symmetric proximal muscle weakness muscle biopsy evidence of myositis increase in serum skeletal muscle enzymes characteristic electromyographic patterns typical rash of dermatomyositis definitive5 plus any three of 14 probably5 plus any two of 14 possible5 plus any one of 14 previous studies have shown that up to 30% of dm patients had underlying malignancies [ 6 , 7 ] .", "malignancies in dm patients have been found to be highest in those over 45 years of age , commonly diagnosed within the first year of initial presentation , and most strongly associated with lung , ovarian , gastric , pancreatic and colorectal origin .", "malignancy can precede , occur concurrently or develop after the diagnosis of dm . in our patient , the symptoms of dm occurred 2 weeks following her diagnosis of breast cancer .", "the goals of therapy in patients with dm are to improve the patient 's ability to carry out activities of daily living by increasing muscle strength and improving myalgias as well as treating the extramuscular manifestations ( including rash and arthralgias ) .", "there has been report of variable influence of the treatment of the underlying malignancy on the clinical course of the dm [ 9 , 10 ] . in our patient ,", "the complete surgical excision of the underlying breast cancer resulted in dramatic improvement in both her cutaneous manifestations and her musculosckeletal symptoms .", "given the high frequency of malignancy in patients with a diagnosis of dm , any woman over the age of 45 with newly diagnosed dm should prompt a thorough physical examination , including a breast examination , as well as further workup for other malignancies ( e.g. ovarian or colorectal cancer ) .", "early diagnosis of dm as a paraneoplastic syndrome allows for initiation of systemic therapy for symptom control and also treatment of the underlying malignancy itself , which may aid in control of the dm symptoms ."], [""]]}
{"article_id": "PMC4515785", "article_text": ["taken together , malaria ( caused by plasmodium species ) , human african trypanosomiasis ( hat , caused by trypanosoma brucei ) , chagas disease ( caused by t. \n cruzi ) , and leishmaniasis ( caused by leishmania species ) represent diseases with an estimated combined burden of \n over 87 million disability - adjusted life years .", "while progress has certainly been made toward the discovery \n of new hits , much work remains to translate these hits into promising \n chemical lead series that may be advanced for drug development .", "target \n repurposing  , wherein inhibitors of human homologues of essential \n parasite proteins are assessed for efficacy against parasite cells .", "we have recently initiated the repurposing of \n a variety of kinase inhibitors for parasitic diseases and have also described the use of established human phosphodiesterase \n inhibitors as starting points for lead discovery .", "we have described the discovery of neu-617 ( 1 ) , a \n derivative of the approved human cancer drug lapatinib ( 2 ) , which acts as a potent , orally bioavailable growth inhibitor of t. brucei that showed a modest effect in a mouse model of \n bloodstream infection .", "based on the close phylogenetic relationship \n between the kinomes of the trypanosomatid parasites ( t. brucei , t. cruzi , and leishmania major ) , we hypothesized that all the pathogens \n would be susceptible to these inhibitors .", "we have therefore tested 1 , along with the other analogs synthesized during the course \n of our hit - to - lead optimization program , against the two other kinetoplastid \n parasites . based on our previous observation that related chemotypes \n", "show activity against plasmodium falciparum , we also screened this set of compounds against \n cultures of drug - sensitive and drug - resistant strains of malaria . \n in doing so , we noted differential structure  activity relationships \n for these compounds growth inhibitory properties against each \n of the parasites , thus launching a multiparasite optimization campaign \n from the same lead series . in this paper", ", we describe these \n cross - pathogen assay results and \n outline our evaluation of replacements for the quinazoline scaffold \n of 1 for each of the parasites .", "we posit that such a \n cross - screening approach can be a highly fruitful method for identification \n of new protozoan parasite growth inhibitors .", "first , \n in order to ascertain antiparasitic activities of our 66 \n previously reported analogs , we measured \n their growth inhibitory activities against t. cruzi ( intracellular amastigotes ) , l. major ( promastigote \n and intracellular amastigote life stages ) , and p. falciparum ( d6 , drug sensitive strain ) . the structures of the most potent compounds \n for each parasite are shown in figure 1 and \n table 1 , and the complete set of screening \n data for these is tabulated in the supporting \n information ( tables s1s7 ) . program progression from 2 to four", "sem values within 50% . * % inh at \n 10 m . control compounds : t. brucei , suramin ; t. cruzi , benznidazole ; l. major , amphotericin b ; p. falciparum , chloroquine . from these \n experiments", ", we observed that 15 morpholinosulfonamide \n compounds were identified with activity against l. major promastigotes : seven of these were submicromolar inhibitors and \n two were also submicromolar against intracellular amastigotes ( the \n most relevant life stage for human infections of leishmaniasis ) .", "the \n des - fluoro analog neu-551 ( 3 ) showed the highest activity \n against promastigotes ( ec50 = 0.50 m ) .", "we initially \n tested compounds against t. cruzi at a single concentration \n and found that 32 compounds inhibited proliferation > 65% at 10 \n m \n concentrations ; of these 32 compounds , the homopiperazinyl sulfonamide - substituted \n tail was preferred ( neu-628 ( 4 ) , ec50 = 0.51 \n m ) .", "the active compounds against p. falciparum ( drug - sensitive d6 strain ) all contained a basic nitrogen at the \n end of the tail group with neu-627 ( 5 ) being the most \n potent with an ec50 of 27 nm . though we note that the number \n of headgroup replacement analogs tested was far smaller and less diverse \n than the tail variations , the lapatinib headgroup 3-chloro-4-((3-fluorobenzyl)oxy)aniline \n showed optimal potency against all of the parasites , except for both leishmania life stages .", "previous efforts focused on \n the  head  ( 4-benzyloxyanilines ) \n and  tail  ( denoted as r in figure 1 ) regions of lapatinib , while maintaining the central quinazoline \n scaffold ( highlighted in blue in figure 1 ) . \n", "thus , we planned a broader evaluation of other bicyclic aromatic replacements \n for the quinazoline scaffold that would test the important features \n of the chemotype for activity .", "these scaffold replacements were selected \n for exploration of the requisite nitrogen atom positioning in the \n heteroaromatic ring ( see the heterocycle precursors highlighted in \n figure 2 , dihalides 69 ) .", "in addition , the cyanoquinoline and thienopyrimidine scaffolds \n ( dihalides 1012 ) were selected in \n order to include other established tyrosine kinase inhibitor chemotypes . for a matched exploration of these scaffolds", ", we elected to maintain \n the four permutations of head / tail combinations that displayed the \n most potency in each parasite ( shown in figure 1 ) in the new analogs that we prepared .", "we first set out to synthesize \n the requisite dihalogenated scaffolds \n shown in figure 2 .", "the syntheses of 6- and \n 7-bromo-4-chloroquinolines 6a and 6b were \n carried out via the gould ", "was devised to draw out essential interactions \n with either nitrogen of the quinazoline scaffold .", "the requisite cyanoquinoline template 12 was prepared \n using established protocols similar to that of the 4-chloroquinolines . though few examples of cinnoline or phthalazine \n based kinase inhibitors \n exist in the literature , we opted to prepare analogues utilizing both heterocycles \n to test the required positioning of the two nitrogen atoms on the \n scaffold .", "preparation of 6-bromo-4-chlorocinnoline 8a commenced with the bromination of o - aminoacetophenone 13 followed by diazotization of the amine and in situ cyclization to the 6-bromocinnolin-4(1h)-one 15 ( scheme 1 ) .", "we hypothesized that this \n side reaction occurred via chloride displacement under the acidic \n conditions of the reaction ( scheme s1 , see supporting \n information ) .", "a 10:1 product ratio of 8a to 8b could be achieved through the use of thf as solvent and \n 3 equiv of pocl3 .", "the regiomeric 7-bromo-4-chlorocinnoline 8c was prepared in a similar fashion starting instead with \n the acylation of 3-bromoaniline 16 to 2-amino-4-bromoacetophenone 17 , followed by diazotization and chlorination .", "reagents and conditions : ( a ) \n nbs , ch2cl2 , 10 c  23 \n c , o.n . ;", "( b ) nano2 , aq . hcl , h2o , 75 c , \n o.n . ; ( c ) pocl3 , thf , reflux , 2 h ; ( d ) ( i ) bcl3 , ( ch2cl)2 , 0 c ; ( ii ) alcl3 , ch3cn , 80 c , o.n . ; ( iii ) aq . hcl , 80 c , \n 30 min ; ( b ) nano2 , aq . hcl , h2o , 75 c , \n o.n . ; ( c ) pocl3 , thf , reflux , 2 h. the 7-bromo-1-chlorophthalazine template 9a was synthesized \n as shown in scheme 2 .", "aryl bromination of phthalide 19 was carried out in acidic medium with nbs to produce a \n separable mixture of 4- and 6-bromophthalide 20 .", "benzylic \n bromination was achieved using a modification of an established procedure , and the resulting dibromides 21a , b were converted to the corresponding bromophthalazinone 22 upon treatment with hydrazine . as with 15 , 22a was converted to the dichlorinated side product 9b on treatment with neat pocl3 , though dilution \n minimized this side reaction .", "acetonitrile with 3 equiv of pocl3 provided a > 10:1 ratio of the desired chlorination product 9a .", "the 6-bromo-1-chlorophthalazine scaffold 9c could be synthesized in an identical manner starting with the commercially \n available 5-bromophthalide 20b .", "reagents \n and conditions : ( a ) \n nbs , h2so4 , cf3co2h , 23 \n c , 40 h ; ( b ) nbs , aibn , chcl3 , reflux , 1 h ; ( c ) n2h4 , i - proh , reflux , 1.5 h ; ( d ) pocl3 , ch3cn , reflux , 3 h. amination \n of the dihalogenated quinoline , isoquinoline , cyanoquinoline , \n and thienopyrimidine scaffolds was carried out by applying methods \n similar to those we previously reported ( scheme 3 ) .", "however , low yields ( 20% ) \n resulted when these conditions were applied to the cinnoline and phthalazine \n templates .", "switching the solvent to toluene and using 4 equiv of amine \n improved yields to 5478% .", "suzuki couplings using the requisite \n boronates and 57 mol % pd(pph3)4 or \n 1 mol % pd(oac)2 provided the final analogs", ". yields of \n the cinnoline or phthalazine products were drastically improved by \n switching to 2.5 mol % pdcl2(pph3)2 or 5 mol % pd(oac)2 with extended reaction times .", "a higher \n catalyst loading of pd(oac)2 was employed in lieu of pdcl2(pph3)2 in situations where the pph3o byproduct could not be separated from the products .", "reagents and \n conditions : ( a ) 1.1 equiv of amine , i - proh , reflux , o.n . ; ( b ) 4 equiv \n of amine , toluene , reflux , 2.5 h ; ( c ) 4 equiv of amine , toluene , 50 \n c , o.n . ; ( d ) arbpin , et3n , pd(oac)2 , 1:1 \n h2o / etoh , mw , 120 c , 13 h ; ( e ) arbpin , et3n , pdcl2(pph3)2 , 1:1 h2o / etoh , mw , 120 c , 1 h ; ( f ) arbpin , aq .", "na2co3 , pd(pph3)4 , 3:2 glyme / etoh , \n n2 , 85 c , 712 h. with this set of new analogs that are matched to the previously \n described antiparasitic agents , we set \n out measuring these analogs activities against t. \n brucei , t. cruzi , l. major , and p. falciparum , and counter - screened against \n host cells ( nih 3t3 and hepg2 cell lines ) using assays that we previously \n reported .", "a \n small number of compounds showed activity against nih 3t3 cells ; these \n are summarized in the supporting information , table s8 .", "all sem values \n within 25% unless \n noted otherwise . * % inh at 5 m .", "compound 1 remained the most potent t. brucei growth inhibitor among this set of analogs .", "a pair of quinoline - based \n compounds , 50 and 52 , each with a basic \n aliphatic amine on the tail group show double digit nanomolar potency \n ( 79 and 87 nm , respectively ) .", "the potency of these compounds seems \n to be driven by the aliphatic amine in the tail region rather than \n by the placement of nitrogen atoms in the scaffold , because the analogous \n isoquinoline analogue 58 is approximately equipotent \n ( 100 nm ) to 50 .", "the quinoline analogs 50 , 51 , and 52 displayed submicromolar potency \n against intracellular amastigotes \n of t. cruzi .", "most other analogs showed a complete \n loss of activity , perhaps due to issues with penetration into both \n the host cell and the parasite .", "( with intracellular parasites , we \n note that compensating effects from interactions with host cell targets \n can not be ruled out . )", "however , the 3-cyanoquinoline analogue 83 , shows a 5-fold improvement in potency over the matched \n original quinazoline analog 4 . in the headgroup region", ", \n the 3-fluoro substituent affords a 10-fold increase in potency over \n the matched des - fluoro analog ( cf .", "the quinoline 51 displayed a slight drop in potency \n compared with the quinazoline 4 , and the matched isoquinoline 59 is 10-fold less potent .", "taken together with the cyanoquinoline \n analog 83 , this data demonstrates the apparent importance \n of two h - bond acceptor motifs in the scaffold . in the case of l. major", ", 47 showed \n a 2.5-fold increase in potency from the matched quinazoline analogue 3 ( 0.20 versus 0.50 m respectively ) against the promastigote \n life stage of l. major .", "the corresponding isoquinoline \n compound 55 showed a complete loss in activity , reinforcing \n the essentiality of the n1 atom of the quinazoline .", "interestingly , \n there was no activity observed against the amastigote life stage of \n the parasite for 47 .", "in contrast , the 7-substituted quinoline 52 displayed submicromolar potency against both life stages \n ( 0.40 m against promastigotes and 0.89 m versus amastigotes ) . \n", "compared with the quinazoline compound 4 , the analogous \n cinnoline 68 displayed increased potency against amastigotes \n ( 0.24 m ) albeit with a significant decrease in potency against \n the promastigote form .", "this lack of correlation of compounds between \n promastigote and amastigote life stages of the parasite has been reported \n by us and others recently .", "other compounds displaying \n modest potency include 76 and 91 , both containing \n the n - methylhomopiperazinyl sulfonamide tail . in p. falciparum cultures , three isoquinoline \n compounds with a basic amine at", "the terminus of the tail region showed \n modest potency highlighted by 57 being nearly equipotent \n to the parent quinazoline 5 ( 40 vs 27 nm ) .", "though the \n matching quinoline 49 showed a complete loss in activity , \n its regioisomer 50 was slightly more active than 5 ( ec50 = 19 nm ) .", "the four quinoline analogs without \n a basic amine on the tail group were within 2-fold of 5 with 48 being the most potent with an ec50 = 16 nm .", "similarly , all four thienopyrimidine analogs with a basic \n tail group amine ( 8992 ) were potent , \n within 4-fold of 5 . among these compounds there was no \n preference for either thienopyridine isomer ; the n - methylpiperazinyl tail group was 2.5- to 4-fold more potent than \n the n - methylhomopiperazinyl tail on each scaffold . among the phthalazines , 74 and 76 , both \n with a basic nitrogen on the tail and substituted at the 6-position \n ( regiomeric to 5 ) were equipotent to 5 . \n also requiring a basic amine on the tail region , the cinnoline analogs \n with piperazinyl and homopiperazinyl moieties ( 6568 ) showed modest to high potency against p. falciparum d6 strain .", "excitingly , the 7-substituted isomers ( 66 and 68 ) were highly potent antiplasmodial agents ( 14 \n and 3 nm , respectively ) . since we observed that the most potent \n compounds against p. falciparum were 4-aminoquinolines \n ( similar to the prototypical \n antimalarial agent chloroquine ) , we tested them against drug resistant \n parasite strains w2 and c235 .", "we were gratified to observe that all \n of the compounds tested are similarly active against drug resistant \n strains of p. falciparum ( table s9 , supporting information ) , suggesting that despite the shared \n scaffold , these analogs likely display a mechanism of action or mechanism \n of resistance that differs from those of chloroquine .", "our studies \n demonstrate which of these scaffolds are the most promising \n for further optimization on a parasite - by - parasite basis .", ", it is clear that the quinoline scaffold \n is significantly active across all four pathogens .", "we note that cinnoline \n ( c ) and quinoline ( a ) generally provide the most potent antiplasmodial \n agents , and the 7-position substitutions are slightly favored in both \n of these templates . for t. brucei ,", "the 7-substituted \n quinolines and 6-substituted isoquinolines appear optimal ( noting \n that , despite the differential numbering of the scaffolds , these two \n particular scaffolds present the same relative regiochemistry ) .", "analogous \n diagrams for the other pathogens are also provided in figure 3 ( and a diagram for leishmania promastigotes \n is in the supporting information ( figure \n s1 ) .", "a summary of the preferred bicyclic scaffold for each parasite \n is shown in figure 3e , noting that none appear \n to be markedly better than the others against leishmania intracellular amastigotes .", "bar chart showing range of potencies for each \n scaffold ( as labeled \n in tables 2 and 3 ) for \n ( a ) malaria , ( b ) t. brucei , ( c ) l. major ( amastigotes ) , and ( d ) t. cruzi .", "regiochemical \n information is shown by the suffix number ( e.g. , scaffold a-7 is the \n 7-substituted quinoline scaffold ) .", "quinolines are shown in blue , isoquinolines \n in green , cinnolines in yellow , phthalazines in orange , cyanoquinolines \n in red , thieno[3,2-d]pyrimidines in purple , thieno[2,3-d]pyrimidines in light gray , and quinazolines in dark gray . \n", "for all parasites except for t. brucei , we also \n note a preference for tail substituents with a basic amine present . \n", "considering only the most  potent  compounds ( ec50  0.1 m for the extracellular parasites t. brucei and p. falciparum ; ec50  1 m for the intracellular parasites l. major and t. cruzi ) , we note that the n - methylpiperizine and n - methylhomopiperazine are \n preferred among analogs that were tested , over the less - basic morpholine \n or nonbasic morpholinosulfonamide substituents ( table 4 ) . at this point", ", we can not discern whether this is an effect \n that is mediated by the biological target(s ) involved in proliferation \n inhibition , by parasite permeation , or by a combination of both ; this \n will be a matter for further investigation .", " potent  compounds \n are ec50  0.1 m .  potent  compounds \n are ec50  1 m . we have previously observed that molecules bearing \n these head and \n tail combinations possess problematic physicochemical properties , \n likely due to the molecular size and lipophilicity .", "the intent of \n the present work was to explore the central scaffold functionality , \n rather than to address these shortcomings .", "nonetheless , we selected \n five representative compounds from among the most potent to be tested \n for their physicochemical properties ( table 5 ) .", "not unexpectedly , compounds tested were > 99% plasma protein \n bound \n with limited thermodynamic aqueous solubility ( < 1 m ) .", "such \n properties are undoubtedly a result of the high molecular weights \n and clogp values ; these issues remain the focus of ongoing efforts , \n which are now focused on specific scaffolds for each pathogen . in summary , through a cross - parasite \n screening campaign of existing \n quinazoline t. brucei proliferation inhibitors , we \n uncovered new hits against three other protozoan parasites . encouraged \n by that initial success , we focused on exploring the central heterocycle \n scaffold , an exercise that has uncovered additional highly potent \n compounds .", "scaffold variations have revealed heteroatom positioning \n essential for activity within this chemotype , while variations to \n regiochemical attachment points have introduced a new set of regioisomers \n with multiparasite potency .", "thus , we have established a new lead series \n for each of these protozoan parasites , which can be now advanced in \n parallel for drug discovery against four different parasitic diseases . \n with this in mind ,", "further optimization of physicochemical properties \n and cellular selectivity of each is ongoing , with a specific focus \n on the  head  and  tail  regions .", "( st . louis , mo ) , fisher scientific , \n frontier scientific services , inc .", "( newark , de ) , or matrix scientific \n ( columbia , sc ) and used as received .", "boronic acids and esters and \n aniline reagents were purchased , except for those whose syntheses \n are listed in the supporting information .", "reaction solvents were dried by passage through alumina columns \n on a purification system manufactured by innovative technology ( newburyport , \n ma ) .", "nmr spectra were obtained with varian nmr systems , operating \n at 400 or 500 mhz for h acquisitions as noted .", "lcms analysis \n was performed using a waters alliance reverse - phase hplc , with single - wavelength \n uv  visible detector and lct premier time - of - flight mass spectrometer \n ( electrospray ionization ) .", "all newly synthesized compounds that were \n submitted for biological testing were deemed > 95% pure by lcms \n analysis \n ( uv and esi - ms detection ) prior to submission for biological testing . \n", "preparative lcms was performed on a waters fraction lynx system with \n a waters micromass zq mass spectrometer ( electrospray ionization ) \n and a single - wavelength uv ", "yields reported \n for products obtained by preparative hplc represent the amount of \n pure material isolated ; impure fractions were not repurified . in a flame - dried \n 250 ml round - bottom flask", "were added 6-bromocinnolin-4(1h)-one ( 1.00 g , 4.44 mmol ) , anhydrous tetrahydrofuran ( 45 ml ) , and \n phosphorus oxychloride ( 1.25 ml , 13.41 mmol ) .", "the mixture was refluxed \n for 1 h at which point a deep green / blue solution had resulted .", "the \n solution was cooled to 0 c and was quenched by the dropwise \n addition of sat .", "the mixture was allowed \n to warm to room temperature and stir for an additional 1 h. water \n ( 50 ml ) was added , and the mixture was extracted with dichloromethane \n ( 3  100 ml ) .", "nahco3 ( 50 ml ) , washed with brine ( 50 ml ) , dried over \n na2so4 , concentrated on to silica , and purified \n by flash column chromatography using a gradient of 15% methanol \n in dichloromethane to yield an inseparable 10:1 mixture of 8a and 8b as a brown solid in 85% yield .", "h \n nmr ( 500 mhz , cdcl3 )  9.36 ( s , 1 h ) , 8.43 ( d , j = 8.8 hz , 1 h ) , 8.36 ( d , j = 2.0 hz , \n 1 h ) , 7.98 ( dd , j = 9.3 , 2.0 hz , 1 h ) .", "lcms found \n 242.9 [ m + h ] . in a flame - dried \n 25 ml round - bottom flask", "were added 7-bromocinnolin-4(1h)-one ( 166 mg , 0.74 mmol ) , anhydrous tetrahydrofuran ( 7 ml ) , and \n phosphorus oxychloride ( 0.2 ml , 2.15 mmol ) .", "the mixture was refluxed \n for 1 h at which point a deep green / blue solution had resulted .", "the \n solution was cooled to 0 c and was quenched by the dropwise \n addition of sat .", ". the mixture was allowed \n to warm to room temperature and stir for an additional 1 h. water \n ( 12 ml ) was added , and the mixture was extracted with dichloromethane \n ( 3  25 ml ) .", "nahco3 ( 20 ml ) , washed with brine ( 20 ml ) , and dried \n over na2so4 to yield 8c as a dark \n brown solid in 92% yield .", "h nmr ( 500 mhz , cdcl3 )  9.39 ( s , 1 h ) , 8.76 ( d , j = 2.0 hz , 1 \n h ) , 8.09 ( d , j = 9.3 hz , 1 h ) , 7.95 ( dd , j = 9.0 , 1.7 hz , 1 h ) .", "lcms found 242.9 [ m + h ] . in a \n flame - dried 25 ml round - bottom flask were added 7-bromophthalazin-1(2h)-one ( 205 mg , 0.91 mmol ) , anhydrous acetonitrile ( 9 ml ) , \n and phosphorus oxychloride ( 0.3 ml , 3.22 mmol ) .", "the mixture was refluxed \n for 2 h , then cooled to 0 c , diluted with dichloromethane ( 20 \n ml ) , and quenched with a dropwise addition of sat .", "after 1 h , the layers were separated \n and the aqueous was extracted with dichloromethane ( 2  30 ml ) . \n the combined organic layers were washed with sat .", "nahco3 ( 25 ml ) , washed with brine ( 20 ml ) , dried over na2so4 , and concentrated to yield 9a as an orange solid \n in 91% yield .", "h nmr ( 500 mhz , cdcl3 )  \n 9.45 ( s , 1 h ) , 8.498.51 ( m , 1 h ) , 8.10 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.91 ( d , j = 8.8 hz , 1 h ) . \n", "lcms found 242.9 [ m + h ] . in a \n flame - dried 50 ml round - bottom flask were added 6-bromophthalazin-1(2h)-one ( 402 mg , 1.78 mmol ) , anhydrous acetonitrile ( 18 ml ) , \n and phosphorus oxychloride ( 0.5 ml , 5.36 mmol ) .", "the mixture was refluxed \n for 2 h , then cooled to 0 c , diluted with dichloromethane ( 40 \n ml ) , and quenched with a dropwise addition of sat .", "the biphasic mixture was stirred vigorously and allowed \n to warm to room temperature . after 1 h , the layers were separated , \n and the aqueous was extracted with dichloromethane ( 2  50 ml ) . \n the combined organic layers were washed with sat .", "nahco3 ( 40 ml ) , washed with brine ( 30 ml ) , dried over na2so4 , and concentrated to yield 29c as a yellow solid \n in 94% yield .", "h nmr ( 500 mhz , cdcl3 )  \n 9.39 ( s , 1 h ) , 8.178.21 ( m , 2 h ) , 8.10 ( dd , j = 8.8 , 2.0 hz , 1 h ) .", "in a 250 ml round - bottom \n flask were added 1-(2-amino-5-bromophenyl)ethanone \n ( 8.34 g , 39.0 mmol ) , water ( 30 ml ) , and conc .", "the mixture was cooled to 0 c in an ice bath \n and allowed to stir for 15 min until a suspension resulted .", "aqueous \n sodium nitrite ( 2 m , 20 ml , 40.0 mmol ) was then added dropwise with \n an addition funnel .", "the resulting solution was allowed to warm to \n room temperature over 1.5 h and was stirred at room temperature overnight , \n then refluxed for 6 h. the mixture was cooled to room temperature , \n water ( 200 ml ) was added , and the mixture was extracted with ethyl \n acetate ( 3  200 ml ) .", "the combined organic layers were then washed \n with brine ( 50 ml ) , dried over sodium sulfate , filtered , and concentrated \n onto silica .", "the crude product was then purified by flash column chromatography \n using a gradient of 110% methanol in dichloromethane to yield 15 as a dark brown solid in 82% yield .", "h nmr ( 500 \n mhz , dmso - d6 )  14.09 ( br .", "s. , 1 \n h ) , 8.09 ( d , j = 2.2 hz , 1 h ) , 7.92 ( dd , j = 8.8 , 2.2 hz , 1 h ) , 7.79 ( s , 1 h ) , 7.71 ( d , j = 9.1 hz , 1 h ) .", "in a 50 ml round - bottom \n flask were added 1-(2-amino-4-bromophenyl)ethanone \n ( 712 mg , 3.33 mmol ) , water ( 3 ml ) , and conc .", "the mixture was cooled to 0 c in an ice bath and \n allowed to stir for 15 min until a suspension resulted .", "aqueous sodium \n nitrite ( 2 m , 1.84 ml , 3.68 mmol ) was then added dropwise with an \n addition funnel .", "the resulting solution was allowed to warm to room \n temperature over 1.5 h and was stirred at room temperature overnight , \n then refluxed for 6 h. the mixture was cooled to room temperature , \n water ( 35 ml ) was added , and the mixture was extracted with ethyl \n acetate ( 3  40 ml ) .", "the combined organic layers were then washed \n with brine ( 20 ml ) , dried over sodium sulfate , filtered , and concentrated \n onto silica .", "the crude product was then purified by flash column chromatography \n using a gradient of 2050% ethyl acetate in hexanes , then ethyl \n acetate to yield 18 as a light brown solid in 26% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 13.49 ( s , 1 h ) , 7.92 ( d , j = 8.8 hz , 1 h ) , 7.76 ( s , \n 1 h ) , 7.73 ( d , j = 2.0 hz , 1 h ) , 7.53 ( dd , j = 8.8 , 2.0 hz , 1 h ) .", "in a 100 ml round - bottom flask was dissolved isobenzofuran-1(3h)-one ( 4.01 g , 29.9 mmol ) in trifluoroacetic acid ( 14 ml , \n 182 mmol ) and sulfuric acid ( 6.5 ml , 122 mmol ) .", "n - bromosuccinimide ( 7.95 g , 1.49 mmol ) was added portionwise over \n 8 h , and the solution was stirred at room temperature for an additional \n 87 h. the solution was diluted with water ( 40 ml ) and ethyl acetate \n ( 40 ml ) .", "the organic layer was separated , \n and the aqueous layer was extracted with ethyl acetate ( 3  50 \n ml ) .", "the combined organic layers were washed with brine ( 25 ml ) , dried \n over na2so4 , and concentrated onto silica .", "the \n crude product was then purified by flash column chromatography using \n 1020% ethyl acetate in hexanes to yield 20a as \n white solid in 57% yield .", "h nmr ( 500 mhz , cdcl3 )  7.98 ( d , j = 1.5 hz , 1 h ) , 7.77 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.40 ( d , j = 8.3 \n hz , 1 h ) , 5.27 ( s , 2 h ) .", "lcms found 212.9 [ m + h ] . in a 50 ml round - bottom \n flask", "n - bromosuccinimide \n ( 958 mg , 5.38 mmol ) , 2,2-azobis(2-methylpropionitrile ) ( 75 \n mg , 0.46 mmol ) , and chloroform ( 23 ml ) . the mixture was refluxed for \n 2.5 h , then cooled to room temperature and quenched with sat .", "the organic layer was removed , washed with \n water ( 20 ml ) , washed with brine ( 15 ml ) , and concentrated onto silica . \n", "the crude product was purified by flash column chromatography using \n a gradient of 510% ethyl acetate in hexanes to yield 21a as a white solid in 61% yield .", "h nmr ( 500 \n mhz , cdcl3 )  8.06 ( d , j = 1.5 hz , \n 1 h ) , 7.90 ( dd , j = 8.1 , 1.7 hz , 1 h ) , 7.52 ( d , j = 8.3 hz , 1 h ) , 7.37 ( s , 1 h ) .", "were \n added 5-bromoisobenzofuran-1(3h)-one ( 499 mg , 2.34 \n mmol ) , n - bromosuccinimide \n ( 421 mg , 2.37 mmol ) , 2,2-azobis(2-methylpropionitrile ) ( 38 \n mg , 0.23 mmol ) , and chloroform ( 10 ml ) .", "the mixture was refluxed for \n 2.5 h , then cooled to room temperature and quenched with sat .", "the organic layer was removed , washed with \n water ( 10 ml ) , washed with brine ( 5 ml ) , and concentrated onto silica . \n", "the crude product was purified by flash column chromatography using \n 10% ethyl acetate in hexanes to yield 21b as a white \n solid in 49% yield .", "h nmr ( 500 mhz , cdcl3 ) \n  7.747.83 ( m , 3 h ) , 7.36 ( s , 1 h ) .", "gcms found 289.8 \n [ m ] . in a 25 ml round - bottom flask", "was dissolved \n 3,6-dibromoisobenzofuran-1(3h)-one ( 143 mg , 0.49 \n mmol ) in ethanol ( 5 ml ) .", "hydrazine \n monohydrate ( 0.12 ml , 2.48 mmol ) was then added via a syringe , and \n the solution was refluxed for 1.5 h. the solution was cooled to room \n temperature , and ice water ( 15 ml ) was added to the reaction mixture . \n", "the precipitate was vacuum filtered and dried under a vacuum overnight \n to yield 22a as a white solid in 56% yield .", "h nmr ( 500 mhz , dmso - d6 )  12.82 \n ( br .", "s. , 1 h ) , 8.39 ( s , 1 h ) , 8.30 ( d , j = 2.0 hz , \n 1 h ) , 8.11 ( dd , j = 8.5 , 2.2 hz , 1 h ) , 7.90 ( d , j = 8.3 hz , 1 h ) .", "in a 25 ml round - bottom flask was dissolved 3,5-dibromoisobenzofuran-1(3h)-one ( 302 mg , 1.04 mmol ) in ethanol ( 10 ml ) .", "hydrazine \n monohydrate ( 0.25 ml , 5.18 mmol ) was then added via a syringe , and \n the solution was refluxed for 1.5 h. the solution was cooled to room \n temperature , and ice water ( 30 ml ) was added to the reaction mixture . \n", "the precipitate was vacuum filtered and dried under a vacuum overnight \n to yield 22b as a white solid in 73% yield .", "h nmr ( 500 mhz , dmso - d6 )  12.78 \n ( br .", "s. , 1 h ) , 8.33 ( s , 1 h ) , 8.23 ( d , j = 2.0 hz , \n 1 h ) , 8.12 ( d , j = 8.3 hz , 1 h ) , 8.00 ( dd , j = 8.3 , 2.0 hz , 1 h ) .", "to a solution of the appropriate \n aryl chloride ( 1 equiv ) in 2-propanol ( 0.15 m ) was added 3-chloro-4-((3-fluorobenzyl)oxy)aniline \n or 4-(benzyloxy)-3-chloroaniline ( 1.1 equiv ) .", "the formed precipitate was collected by vacuum \n filtration to obtain the desired products . to a solution of the appropriate \n aryl chloride ( 1 equiv ) in 2-propanol ( 0.15 m )", "the combined organic layers were washed with water , washed with brine , \n dried over na2so4 , and concentrated .", "the crude \n products were purified by flash column chromatography to obtain the \n desired products .", "a solution of the appropriate 4-chlorocinnoline \n ( 1 equiv ) and 3-chloro-4-((3-fluorobenzyl)oxy)aniline \n or 4-(benzyloxy)-3-chloroaniline ( 4 equiv ) in toluene ( 0.1 m ) was \n refluxed for 2.5 h and cooled to room temperature .", "triethylamine ( 4 \n equiv ) was added , and the mixture was returned to reflux for an additional \n 30 min .", "the mixture was cooled back to room temperature , and the formed \n yellow precipitate was vacuum filtered , washed with ethyl acetate , \n concentrated onto silica , and purified by flash column chromatography .", "a solution of the appropriate 1-chlorophthalazine ( 1 equiv ) and \n 3-chloro-4-((3-fluorobenzyl)oxy)aniline or 4-(benzyloxy)-3-chloroaniline \n ( 4 equiv ) in anhydrous toluene ( 0.2 m ) was heated at 50 c overnight . \n", "the combined \n organic layers were washed with brine , dried over na2so4 , concentrated onto silica , and purified by flash column chromatography . synthesized by general procedure b. flash \n column chromatography : 2050% ethyl acetate in hexanes to yield 23 as a light brown solid in 90% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  8.99 ( s , 1 \n h ) , 8.63 ( s , 1 h ) , 8.46 ( d , j = 5.4 hz , 1 h ) , 7.80 \n ( d , j = 1.5 hz , 2 h ) , 7.437.51 ( m , 2 h ) , \n 7.277.36 ( m , 4 h ) , 7.19 ( td , j = 8.7 , 2.2 \n hz , 1 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.26 ( s , 2 h ) . \n lcms found 456.8 [ m + h ] .", "synthesized by general procedure b. flash \n column chromatography : 2070% ethyl acetate in hexanes to yield 24 as a brown solid in 70% yield .", "h nmr ( 500 mhz , \n dmso - d6 )  8.98 ( s , 1 h ) , 8.63 ( s , \n 1 h ) , 8.45 ( d , j = 5.4 hz , 1 h ) , 7.80 ( s , 2 h ) , 7.50 \n ( d , j = 7.3 hz , 2 h ) , 7.397.46 ( m , 3 h ) , \n 7.35 ( t , j = 7.3 hz , 1 h ) , 7.30 ( s , 2 h ) , 6.79 ( d , j = 5.4 hz , 1 h ) , 5.23 ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 2050% ethyl acetate in hexanes to yield 25 as a tan colored solid in 82% yield .", "h nmr \n ( 399 mhz , dmso - d6 )  9.05 ( s , 1 \n h ) , 8.43 ( d , j = 5.1 hz , 1 h ) , 8.31 ( d , j = 8.8 hz , 1 h ) , 8.05 ( d , j = 2.2 hz , 1 h ) , 7.67 \n ( dd , j = 8.8 , 2.2 hz , 1 h ) , 7.417.52 ( m , \n 2 h ) , 7.257.37 ( m , 4 h ) , 7.18 ( td , j = 8.6 , \n 2.6 hz , 1 h ) , 6.76 ( d , j = 5.9 hz , 1 h ) , 5.25 ( s , \n 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 2050% ethyl acetate in hexanes to yield 26 as an off - white solid in 83% yield .", "h nmr ( 500 \n mhz , dmso - d6 )  9.04 ( s , 1 h ) , 8.43 \n ( d , j = 4.9 hz , 1 h ) , 8.31 ( d , j = 9.3 hz , 1 h ) , 8.05 ( d , j = 2.0 hz , 1 h ) , 7.67 \n ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.49 ( d , j = 6.8 hz , 2 h ) , 7.397.46 ( m , 3 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.30 ( s , 2 h ) , 6.75 ( d , j = 5.4 \n hz , 1 h ) , 5.23 ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 27 as a pale red - brown solid in 97% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  9.24 ( s , 1 \n h ) , 8.80 ( s , 1 h ) , 8.07 ( d , j = 2.4 hz , 1 h ) , 8.02 \n ( d , j = 5.9 hz , 1 h ) , 7.82 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 7.727.79 ( m , 2 h ) , 7.417.49 \n ( m , 1 h ) , 7.277.35 ( m , 2 h ) , 7.137.23 ( m , 3 h ) , 5.21 \n ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 28 as a light red solid in 93% yield .", "h nmr ( 500 \n mhz , dmso - d6 )  9.22 ( s , 1 h ) , 8.80 \n ( s , 1 h ) , 8.07 ( d , j = 2.4 hz , 1 h ) , 8.02 ( d , j = 5.9 hz , 1 h ) , 7.81 ( dd , j = 8.8 , 1.5 \n hz , 1 h ) , 7.737.78 ( m , 2 h ) , 7.48 ( d , j = \n 7.3 hz , 2 h ) , 7.40 ( t , j = 7.6 hz , 2 h ) , 7.33 ( t , j = 7.3 hz , 1 h ) , 7.21 ( d , j = 8.8 hz , \n 1 h ) , 7.15 ( d , j = 5.9 hz , 1 h ) , 5.18 ( s , 2 h ) . lcms \n found 439.2 [ m + h ] .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 29 as a salmon colored solid in 91% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  9.27 ( s , 1 \n h ) , 8.45 ( d , j = 8.8 hz , 1 h ) , 8.08 ( dd , j = 12.0 , 2.2 hz , 2 h ) , 8.01 ( d , j = 5.4 \n hz , 1 h ) , 7.75 ( ddd , j = 13.8 , 9.2 , 2.4 hz , 2 h ) , \n 7.46 ( m , j = 5.9 hz , 1 h ) , 7.277.35 ( m , 2 \n h ) , 7.107.23 ( m , 3 h ) , 5.22 ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 30 as a burnt orange solid in 81% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  9.24 ( s , 1 \n h ) , 8.45 ( d , j = 8.8 hz , 1 h ) , 8.10 ( d , j = 2.0 hz , 1 h ) , 8.05 ( d , j = 2.4 hz , 1 h ) , 8.01 \n ( d , j = 5.9 hz , 1 h ) , 7.76 ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.72 ( dd , j = 8.8 , 2.4 hz , \n 1 h ) , 7.48 ( d , j = 6.8 hz , 2 h ) , 7.41 ( t , j = 7.3 hz , 2 h ) , 7.34 ( t , j = 7.8 hz , \n 1 h ) , 7.21 ( d , j = 9.3 hz , 1 h ) , 7.13 ( d , j = 5.9 hz , 1 h ) , 5.19 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield an \n inseparable 10:1 mixture of 31 and dichloro side product \n as a vibrant yellow solid in 54% yield .", "h nmr ( 500 mhz , \n dmso - d6 )  9.33 ( s , 1 h ) , 8.78 ( s , \n 1 h ) , 8.70 ( d , j = 1.5 hz , 1 h ) , 8.14 ( d , j = 8.8 hz , 1 h ) , 7.97 ( dd , j = 9.3 , 1.5 \n hz , 1 h ) , 7.54 ( d , j = 2.0 hz , 1 h ) , 7.48 ( td , j = 8.3 , 6.3 hz , 1 h ) , 7.40 ( dd , j = 8.8 , \n 2.4 hz , 1 h ) , 7.307.37 ( m , 3 h ) , 7.20 ( td , j = 9.3 , 2.0 hz , 1 h ) , 5.29 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield an \n inseparable 10:1 mixture of 32 and dichloro side product \n as a yellow solid in 68% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.32 ( s , 1 h ) , 8.78 ( s , 1 h ) , 8.71 \n ( d , j = 2.0 hz , 1 h ) , 8.14 ( d , j = 9.3 hz , 1 h ) , 7.97 ( dd , j = 9.0 , 1.7 hz , 1 h ) , \n 7.477.55 ( m , 3 h ) , 7.44 ( t , j = 7.6 hz , 2 \n h ) , 7.337.41 ( m , 3 h ) , 5.26 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield 33 as a brown solid in 55% yield with 84% purity .", "h nmr ( 500 mhz , dmso - d6 )  9.43 \n ( s , 1 h ) , 8.76 ( s , 1 h ) , 8.41 ( d , j = 2.0 hz , 1 h ) , \n 8.35 ( d , j = 9.3 hz , 1 h ) , 7.90 ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.55 ( d , j = 2.4 hz , 1 h ) , \n 7.49 ( m , j = 6.3 hz , 1 h ) , 7.41 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.317.37 ( m , 3 h ) , 7.20 ( td , j = 8.4 , 2.7 hz , 1 h ) , 5.29 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield 34 as a metallic bronze colored solid in 57% yield with 85% \n purity .", "h nmr ( 500 mhz , dmso - d6 )  9.42 ( s , 1 h ) , 8.75 ( s , 1 h ) , 8.41 ( d , j = 2.0 hz , 1 h ) , 8.35 ( d , j = 8.8 hz , 1 h ) , 7.90 \n ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.54 ( d , j = 2.4 hz , 1 h ) , 7.51 ( d , j = 7.3 hz , 2 h ) , 7.43 \n ( t , j = 7.6 hz , 2 h ) , 7.40 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.337.38 ( m , 2 h ) , 5.26 ( s , 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 520% ethyl acetate in dichloromethane \n to yield 35 as a yellow solid in 69% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.21 \n ( s , 1 h ) , 9.13 ( s , 1 h ) , 8.87 ( s , 1 h ) , 8.16 ( d , j = 2.4 hz , 1 h ) , 8.11 ( dd , j = 8.8 , 1.5 hz , 1 h ) , \n 7.99 ( d , j = 8.8 hz , 1 h ) , 7.81 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 7.9 , 6.1 hz , \n 1 h ) , 7.297.36 ( m , 2 h ) , 7.25 ( d , j = 9.3 \n hz , 1 h ) , 7.17 ( td , j = 8.7 , 2.2 hz , 1 h ) , 5.24 ( s , \n 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 520% ethyl acetate in dichloromethane \n to yield 36 as a light greenish brown solid in 78% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 9.20 ( s , 1 h ) , 9.14 ( s , 1 h ) , 8.88 ( s , 1 h ) , 8.15 ( d , j = 2.4 hz , 1 h ) , 8.13 ( dd , j = 8.5 , 1.7 hz , 1 h ) , \n 8.00 ( d , j = 8.8 hz , 1 h ) , 7.80 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.50 ( d , j = 7.3 hz , 2 h ) , \n 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.27 ( d , j = 8.8 hz , 1 h ) , 5.21 \n ( s , 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 520% ethyl acetate in dichloromethane \n to yield 37 as a light brown solid in 64% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 9.42 ( s , 1 h ) , 9.09 ( s , 1 h ) , 8.62 ( d , j = 9.3 hz , \n 1 h ) , 8.33 ( d , j = 2.0 hz , 1 h ) , 8.19 ( d , j = 2.4 hz , 1 h ) , 8.16 ( dd , j = 9.0 , 2.2 \n hz , 1 h ) , 7.83 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 7.8 , 5.9 hz , 1 h ) , 7.32 ( m , j = 7.3 \n hz , 2 h ) , 7.25 ( d , j = 9.3 hz , 1 h ) , 7.17 ( td , j = 8.5 , 2.0 hz , 1 h ) , 5.24 ( s , 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 530% ethyl acetate in dichloromethane \n to yield 38 as a green - gray colored solid in 75% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 9.25 ( s , 1 h ) , 9.09 ( s , 1 h ) , 8.51 ( d , j = 8.8 hz , \n 1 h ) , 8.33 ( d , j = 2.0 hz , 1 h ) , 8.17 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 8.14 ( d , j = 2.9 \n hz , 1 h ) , 7.78 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.49 ( d , j = 6.8 hz , 2 h ) , 7.42 ( t , j = 7.6 hz , \n 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.26 ( d , j = 8.8 hz , 1 h ) , 5.21 ( s , 2 h ) .", "synthesized by general procedure a , collected \n as a yellow solid in 80% yield .", "h nmr ( 400 mhz , dmso - d6 )  9.03 ( s , 1h ) , 8.85 ( br , s , 1h ) , 8.23 \n ( d , j = 8.8 hz , 1h ) , 7.73 ( d , j = \n 8.8 , 1h ) , 7.59 ( d , j = 2.4 , 1h ) , 7.457.50 \n ( m , 1h ) , 7.307.39 ( m , 4h ) , 7.187.22 ( m , 1h ) , 5.30 \n ( s , 2h ) .", "synthesized by general procedure a , collected \n as a yellow solid in 52% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.02 ( s , 1h ) , 8.82 ( br , s , 1h ) , \n 8.22 ( d , j = 9.0 hz , 1h ) , 7.72 ( d , j = 8.5 hz , 1h ) , 7.57 ( s , 1h ) , 7.5 ( d , j = 7.0 hz , \n 2h ) , 7.43 ( t , j = 7.5 hz , 2h ) , 7.35 ( m , 3h ) , 5.26 \n ( s , 2h ) .", "h nmr ( 400 mhz , dmso - d6 )  ppm \n 10.64 ( s , 1 h ) , 8.71 ( s , 1 h ) , 7.87 ( d , j = 2.9 hz , \n 1 h ) , 7.74 ( s , 1 h ) , 7.57 ( dd , j = 8.8 , 2.2 hz , 1 \n h ) , 7.47 ( m , 1 h ) , 7.31 ( m , 3 h ) , 7.19 ( td , j = 8.1 , \n 2.2 hz , 1 h ) , 5.28 ( s , 2 h ) .", "h nmr ( 500 mhz , dmso - d6 )  ppm \n 9.7 1 ( s , 1 h ) , 8.54 ( s , 1 h ) , 7.89 ( d , j = 2.0 hz , \n 1 h ) , 7.68 ( s , 1 h ) , 7.58 ( dd , j = 8.8 , 2.9 hz , 1 \n h ) , 7.49 ( d , j = 7.8 hz , 2 h ) , 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.26 \n ( d , j = 8.8 hz , 1 h ) , 5.23 ( s , 2 h ) .", "h nmr ( 500 mhz , dmso - d6 )  9.80 ( s , 1h ) , 8.49 ( s , 1h ) , 8.12 \n ( s , 1h ) , 8.02 ( d , j = 2.4 hz , 1h ) , 7.67 ( dd , j = 2.7 , 9.03 hz , 1h ) , 7.437.49 ( m , 1h ) , 7.247.35 \n ( m , 3h ) , 7.18 ( dt , j = 2.4 , 8.6 hz , 1h ) , 5.24 ( s , \n 2h ) .", "h nmr ( 500 mhz , dmso - d6 )  ppm \n 9.94 ( s , 1 h ) , 8.49 ( s , 1 h ) , 8.19 ( s , 1 h ) , 8.02 ( d , j = 2.4 hz , 1 h ) , 7.68 ( dd , j = 8.8 , 2.4 hz , 1 h ) , \n 7.49 ( m , 2 h ) , 7.41 ( m , 2 h ) , 7.34 ( m , 1 h ) , 7.27 ( d , j = 9.3 hz , 1 h ) , 5.21 ( s , 2 h ) .", "lcms found 445.9 [ m + h ] . in a 25 \n ml microwave vial equipped with a stir bar", "were added aryl bromide \n ( 1 equiv ) , boronic ester ( 1.3 equiv ) , 1:1 water / ethanol ( 0.04 m ) , \n triethylamine ( 3 equiv ) , and palladium(ii ) acetate ( 0.01 m in acetone , \n 1 mol % ) .", "the vial was sealed with a septum , and the contents were \n irradiated to and held at 120 c with stirring for 1 h. the reaction \n mixture was cooled to room temperature , diluted with water ( 8 ml ) , \n and extracted with dichloromethane ( 3  8 ml ) .", "naoh ( 1 m , 2  5 ml ) , water ( 5 ml ) , \n and brine ( 5 ml ) .", "the crude product was \n purified by flash column chromatography . in a 25 \n ml microwave vial equipped with a stir bar", "were added aryl bromide \n ( 1 equiv ) , boronic ester ( 1.3 equiv ) , 1:1 water / ethanol ( 0.04 m ) , \n triethylamine ( 3 equiv ) , and bis(triphenylphosphine)palladium(ii ) \n chloride ( 2.5 mol % ) .", "the vial was sealed with a septum , and the contents \n were irradiated to and held at 120 c with stirring for 1 h. \n the reaction mixture was cooled to room temperature , diluted with \n water ( 8 ml ) , and extracted with dichloromethane ( 3  8 ml ) . \n", "naoh ( 1 m , 2  \n 5 ml ) , water ( 5 ml ) , and brine ( 5 ml ) .", "the organic layer was then \n dried over na2so4 and concentrated on to silica . \n", "the crude product was purified by flash column chromatography . in a 25 \n ml microwave vial equipped with a stir bar", "were added aryl bromide \n ( 1 equiv ) , boronic ester ( 1.3 equiv ) , 1:1 water / ethanol ( 0.04 m ) , \n triethylamine ( 3 equiv ) , and palladium(ii ) acetate ( 5 mol % ) .", "the \n vial was sealed with a septum , and the contents were irradiated to \n and held at 120 c with stirring for 3 h. the reaction mixture \n was cooled to room temperature , diluted with water ( 8 ml ) , and extracted \n with dichloromethane ( 3  8 ml ) .", "naoh ( 1 m , 2  5 ml ) , water ( 5 ml ) , and \n brine ( 5 ml ) .", "the crude product was \n purified by flash column chromatography . to a solution \n of the appropriate aryl iodide ( 1 equiv ) in 3:2 dimethoxyethane / ethanol \n ( 0.05 m )", "2 m na2co3 ( 6 equiv ) , and pd(pph3)4 ( 5 mol % ) .", "the mixture was purged with nitrogen and \n heated at 85 c for 7 h. the mixture was cooled to room temperature \n and filtered , and the filtrate was concentrated .", "the residue was dissolved \n in ethyl acetate , washed with water , washed with brine , dried over \n na2so4 , and purified by flash column chromatography . to a solution \n of the appropriate aryl bromide ( 1 equiv ) in 3:2 dimethoxyethane / ethanol \n ( 0.05 m )", "2 m na2co3 ( 6 equiv ) , and pd(pph3)4 ( 7 mol % ) .", "the mixture was heated at 85 c for \n 12 h , then cooled to room temperature , and the solvents were removed \n under reduced pressure .", "the residue was purified by silica column \n chromatography ( hexanes / ethyl acetate ) and then by reverse phase chromatography \n ( water / acetonitrile ) unless otherwise mentioned . synthesized by general procedure a. flash \n column chromatography : 25% methanol in dichloromethane , isolated \n as a yellow solid in 27% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.10 ( br .", "s. , 1 h ) , 8.61 ( d , j = 2.0 hz , 1 h ) , 8.43 ( d , j = 5.4 hz , \n 1 h ) , 8.04 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.92 ( d , j = 8.8 hz , 1 h ) , 7.457.52 ( m , 2 h ) , 7.39 ( t , j = 7.8 hz , 1 h ) , 7.297.37 ( m , 6 h ) , 7.20 ( td , j = 8.8 , 2.0 hz , 1 h ) , 7.00 ( dd , j = 8.1 , \n 1.7 hz , 1 h ) , 6.77 ( d , j = 5.4 hz , 1 h ) , 5.28 ( s , \n 2 h ) , 3.753.81 ( m , 4 h ) , 3.193.26 ( m , 4 h ) . lcms found \n 540.1 [ m + h ] . synthesized by general procedure a. flash \n column chromatography : 25% methanol in dichloromethane , isolated \n as a yellow solid in 24% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 8.99 ( br .", "s. , 1 h ) , 8.47 ( d , j = 5.4 hz , 1 h ) , 8.41 ( d , j = 8.8 hz , \n 1 h ) , 8.12 ( d , j = 2.0 hz , 1 h ) , 7.88 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 7.457.51 ( m , 2 h ) , 7.267.40 \n ( m , 7 h ) , 7.20 ( td , j = 8.8 , 2.4 hz , 1 h ) , 7.02 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 6.76 ( d , j = 5.4 \n hz , 1 h ) , 5.27 ( s , 2 h ) , 3.743.82 ( m , 4 h ) , 3.203.27 \n ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 05% methanol in dichloromethane , isolated \n as a tan colored solid in 65% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.14 ( s , 1 h ) , 8.78 ( d , j = 2.0 hz , 1 h ) , 8.47 ( d , j = 5.4 hz , \n 1 h ) , 8.17 ( d , j = 8.8 hz , 2 h ) , 8.11 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.98 ( d , j = 8.8 \n hz , 1 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.51 ( d , j = 7.3 hz , 2 h ) , 7.48 ( d , j = 1.5 hz , \n 1 h ) , 7.43 ( t , j = 7.6 hz , 2 h ) , 7.327.39 \n ( m , 3 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.25 ( s , 2 h ) , \n 3.613.70 ( m , 4 h ) , 2.882.97 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 25% methanol in dichloromethane , isolated \n as a tan colored solid in 63% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.02 ( s , 1 h ) , 8.50 ( m , j = 5.4 hz , 2 h ) , 8.25 ( d , j = 1.5 hz , \n 1 h ) , 8.18 ( d , j = 8.8 hz , 2 h ) , 7.97 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 7.87 ( d , j = 8.3 \n hz , 2 h ) , 7.51 ( d , j = 7.3 hz , 2 h ) , 7.47 ( d , j = 2.0 hz , 1 h ) , 7.44 ( t , j = 7.6 hz , \n 2 h ) , 7.36 ( m , j = 6.8 hz , 1 h ) , 7.307.34 \n ( m , 2 h ) , 6.79 ( d , j = 5.4 hz , 1 h ) , 5.24 ( s , 2 h ) , \n 3.623.70 ( m , 4 h ) , 2.902.97 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 510% methanol in dichloromethane , isolated \n as a yellow solid in 40% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.19 ( br .", "s. , 1 h ) , 8.78 ( d , j = 2.0 hz , 1 h ) , 8.47 ( d , j = 5.4 hz , \n 1 h ) , 8.16 ( d , j = 8.8 hz , 2 h ) , 8.12 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.98 ( d , j = 8.8 \n hz , 1 h ) , 7.87 ( d , j = 8.8 hz , 2 h ) , 7.457.52 \n ( m , 2 h ) , 7.307.38 ( m , 4 h ) , 7.20 ( td , j = \n 8.7 , 2.7 hz , 1 h ) , 6.81 ( d , j = 5.4 hz , 1 h ) , 5.28 \n ( s , 2 h ) , 2.95 ( br . s. , 4 h ) , 2.352.43 ( m , 4 h ) , 2.15 ( s , \n 3 h ) .", "synthesized by general procedure a. flash \n column chromatography : 510% methanol in dichloromethane , isolated \n as a yellow solid in 33% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.10 ( br .", "s. , 1 h ) , 8.50 ( d , j = 8.3 hz , 2 h ) , 8.25 ( d , j = 2.0 hz , \n 1 h ) , 8.16 ( d , j = 8.3 hz , 2 h ) , 7.97 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.87 ( d , j = 8.3 \n hz , 2 h ) , 7.457.52 ( m , 2 h ) , 7.297.38 ( m , 4 h ) , 7.20 \n ( td , j = 8.5 , 2.0 hz , 1 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.28 ( s , 2 h ) , 2.96 ( br . s. , 4 h ) , 2.40 ( br .", "then \n prep hplc : 595% acetonitrile in water , isolated as a yellow \n solid in 10% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 8.77 ( d , j = 2.0 hz , 1 \n h ) , 8.46 ( d , j = 5.4 hz , 1 h ) , 8.27 ( s , 1 h ) , 8.078.15 \n ( m , 3 h ) , 7.97 ( d , j = 8.8 hz , 1 h ) , 7.92 ( d , j = 8.3 hz , 2 h ) , 7.457.52 ( m , 2 h ) , 7.307.38 \n ( m , 4 h ) , 7.20 ( td , j = 8.5 , 2.4 hz , 1 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.28 ( s , 2 h ) , 3.36 ( m , j = 5.2 , 2.5 , 2.5 hz , 2 h ) , 3.33 ( t , j = 6.3 hz , \n 2 h ) , 2.532.57 ( m , 2 h ) , 2.47 ( m , j = 5.9 \n hz , 2 h ) , 2.22 ( s , 3 h ) , 1.74 ( quin , j = 5.9 hz , \n 2 h ) .", "synthesized by general procedure a. flash \n column chromatography : 520% methanol in dichloromethane , isolated \n as a brown solid in 32% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.06 ( s , 1 h ) , 8.478.52 \n ( m , 2 h ) , 8.23 ( d , j = 2.0 hz , 1 h ) , 8.12 ( d , j = 8.3 hz , 2 h ) , 7.96 ( dd , j = 8.8 , 2.0 \n hz , 1 h ) , 7.91 ( d , j = 8.3 hz , 2 h ) , 7.457.52 \n ( m , 2 h ) , 7.297.37 ( m , 4 h ) , 7.20 ( td , j = \n 8.5 , 2.4 hz , 1 h ) , 6.79 ( d , j = 4.9 hz , 1 h ) , 5.27 \n ( s , 2 h ) , 3.39 ( m , j = 3.4 hz , 2 h ) , 3.313.35 \n ( m , 2 h ) , 2.63 ( br . s. , 2 h ) , 2.58 ( br . s. , 2 h ) , 2.29 ( br . s. , 3 \n h ) , 1.741.83 ( m , 2 h ) .", "synthesized by general procedure a. flash \n column chromatography : 3050% ethyl acetate in hexanes , isolated \n as a biege solid in 59% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.27 ( s , 1 h ) , 8.71 ( s , 1 h ) , \n 8.06 ( d , j = 2.4 hz , 1 h ) , 8.03 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.98 ( d , j = 5.4 hz , 1 h ) , \n 7.88 ( d , j = 8.8 hz , 1 h ) , 7.76 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 8.1 , 6.3 hz , \n 1 h ) , 7.40 ( t , j = 7.8 hz , 1 h ) , 7.297.36 \n ( m , 4 h ) , 7.23 ( d , j = 9.3 hz , 1 h ) , 7.157.21 \n ( m , 2 h ) , 7.02 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 5.23 ( s , \n 2 h ) , 3.753.82 ( m , 4 h ) , 3.203.26 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 3050% ethyl acetate in hexanes , isolated \n as an orange solid in 81% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.21 ( s , 1 h ) , 8.56 ( d , j = 8.8 hz , 1 h ) , 8.13 ( d , j = 2.4 hz , \n 1 h ) , 8.11 ( d , j = 2.0 hz , 1 h ) , 8.00 ( d , j = 5.4 hz , 1 h ) , 7.94 ( dd , j = 8.8 , 1.5 \n hz , 1 h ) , 7.78 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 8.0 , 6.3 hz , 1 h ) , 7.357.41 ( m , 2 h ) , 7.307.35 \n ( m , 2 h ) , 7.28 ( d , j = 7.8 hz , 1 h ) , 7.207.25 \n ( m , 2 h ) , 7.18 ( td , j = 8.8 , 2.4 hz , 1 h ) , 7.03 ( dd , j = 8.1 , 2.2 hz , 1 h ) , 5.23 ( s , 2 h ) , 3.763.81 ( m , \n 4 h ) , 3.213.25 ( m , 4 h ) . lcms found 540.2 [ m + h ] . synthesized by general procedure a. flash \n column chromatography : 2050% ethyl acetate in hexanes , isolated \n as an orange solid in 55% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.35 ( s , 1 h ) , 8.86 ( s , 1 h ) , \n 8.18 ( d , j = 8.8 hz , 2 h ) , 8.12 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 8.04 ( d , j = 2.4 hz , 1 h ) , \n 8.03 ( d , j = 5.9 hz , 1 h ) , 7.96 ( d , j = 8.3 hz , 1 h ) , 7.90 ( d , j = 8.3 hz , 2 h ) , 7.76 \n ( dd , j = 9.3 , 2.9 hz , 1 h ) , 7.50 ( d , j = 6.8 hz , 2 h ) , 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 \n ( t , j = 7.3 hz , 1 h ) , 7.25 ( d , j = 9.3 hz , 1 h ) , 7.22 ( d , j = 5.9 hz , 1 h ) , 5.21 \n ( s , 2 h ) , 3.633.70 ( m , 4 h ) , 2.902.96 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 3050% ethyl acetate in hexanes , isolated \n as an orange solid in 81% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.26 ( s , 1 h ) , 8.65 ( d , j = 9.3 hz , 1 h ) , 8.25 ( d , j = 1.5 hz , \n 1 h ) , 8.16 ( d , j = 8.8 hz , 2 h ) , 8.12 ( d , j = 2.9 hz , 1 h ) , 8.018.07 ( m , 2 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.78 ( dd , j = 9.0 , 2.7 \n hz , 1 h ) , 7.50 ( d , j = 6.8 hz , 2 h ) , 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , \n 1 h ) , 7.27 ( d , j = 5.9 hz , 1 h ) , 7.23 ( d , j = 9.3 hz , 1 h ) , 5.20 ( s , 1 h ) , 3.623.69 ( m , 4 \n h ) , 2.892.97 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : ethyl acetate , isolated as a cream colored \n solid in 53% yield .", "h nmr ( 500 mhz , cdcl3 ) \n  ppm 8.118.14 ( m , 2 h ) , 7.797.89 ( m , 7 h ) , \n 7.54 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.36 ( td , j = 7.8 , 5.9 hz , 1 h ) , 7.28 ( s , 1 h ) , 7.217.27 ( m , \n 2 h ) , 7.18 ( d , j = 5.9 hz , 1 h ) , 7.03 ( td , j = 8.3 , 2.4 hz , 1 h ) , 6.97 ( d , j = 8.8 \n hz , 1 h ) , 5.15 ( s , 2 h ) , 3.08 ( br . s. , 4 h ) , 2.49 ( t , j = 4.6 hz , 4 h ) , 2.28 ( s , 3 h ) .", "synthesized by general procedure a. flash \n column chromatography : ethyl acetate , isolated as a pale orange solid \n in 52% yield .", "h nmr ( 500 mhz , cdcl3 )  \n ppm 8.14 ( d , j = 5.9 hz , 1 h ) , 8.02 ( d , j = 8.8 hz , 1 h ) , 7.95 ( d , j = 2.0 hz , 1 h ) , 7.837.91 \n ( m , 5 h ) , 7.76 ( dd , j = 8.5 , 1.7 hz , 1 h ) , 7.50 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.37 ( td , j = 7.8 , \n 5.9 hz , 1 h ) , 7.227.27 ( m , 2 h ) , 7.21 ( d , j = 5.9 hz , 1 h ) , 7.007.07 ( m , 2 h ) , 6.98 ( d , j = 8.8 hz , 1 h ) , 5.16 ( s , 2 h ) , 3.11 ( br . s. , 4 h ) , 2.52 ( t , j = 4.6 hz , 4 h ) , 2.29 ( s , 3 h ) .", "synthesized by general procedure a. flash \n column chromatography : 5% methanol in dichloromethane , isolated as \n an orange solid in 73% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.36 ( s , 1 h ) , 8.85 ( s , 1 h ) , \n 8.098.14 ( m , 3 h ) , 8.05 ( d , j = 2.4 hz , 1 \n h ) , 8.02 ( d , j = 5.9 hz , 1 h ) , 7.917.97 ( m , \n 3 h ) , 7.77 ( dd , j = 8.8 , 2.9 hz , 1 h ) , 7.47 ( td , j = 8.1 , 6.3 hz , 1 h ) , 7.297.36 ( m , 2 h ) , 7.24 ( d , j = 9.3 hz , 1 h ) , 7.22 ( d , j = 5.4 hz , \n 1 h ) , 7.18 ( td , j = 9.3 , 2.0 hz , 1 h ) , 5.24 ( s , 2 \n h ) , 3.353.38 ( m , 2 h ) , 3.313.35 ( m , 2 h ) , 2.542.58 \n ( m , 2 h ) , 2.472.49 ( m , 2 h ) , 2.23 ( s , 3 h ) , 1.75 ( quin , j = 5.8 hz , 2 h ) .", "synthesized by general procedure a. flash \n column chromatography : 5% methanol in dichloromethane , isolated as \n a pale yellow solid in 68% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.26 ( s , 1 h ) , 8.64 ( d , j = 8.8 hz , 1 h ) , 8.23 ( d , j = 1.5 hz , \n 1 h ) , 8.13 ( d , j = 2.4 hz , 1 h ) , 8.10 ( d , j = 8.3 hz , 2 h ) , 8.008.06 ( m , 2 h ) , 7.92 ( d , j = 8.3 hz , 2 h ) , 7.79 ( dd , j = 9.0 , 2.7 \n hz , 1 h ) , 7.47 ( td , j = 8.3 , 6.3 hz , 1 h ) , 7.297.35 \n ( m , 2 h ) , 7.27 ( d , j = 5.9 hz , 1 h ) , 7.22 ( d , j = 9.3 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.7 \n hz , 1 h ) , 5.23 ( s , 2 h ) , 3.36 ( m , j = 4.9 , 2.4 , 2.4 \n hz , 2 h ) , 3.313.35 ( m , 2 h ) , 2.532.58 ( m , 2 h ) , 2.462.49 \n ( m , 2 h ) , 2.23 ( s , 3 h ) , 1.75 ( quin , j = 5.8 hz , \n 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 210% methanol in dichloromethane , isolated \n as a yellow solid in 67% yield .", "s. , 1 h ) , 8.30 ( s , 1 h ) , 8.26 ( d , j = 4.9 hz , 1 h ) , 7.95 ( dd , j = 9.0 , 1.2 \n hz , 1 h ) , 7.487.59 ( m , 1 h ) , 7.327.41 ( m , 2 h ) , 7.29 \n ( t , j = 7.8 hz , 1 h ) , 7.177.24 ( m , 2 h ) , \n 7.087.17 ( m , 3 h ) , 7.02 ( td , j = 8.4 , 2.2 \n hz , 1 h ) , 6.876.94 ( m , 2 h ) , 5.13 ( s , 2 h ) , 3.763.82 \n ( m , 4 h ) , 3.093.16 ( m , 4 h ) .", "synthesized by general procedure b. flash \n column chromatography : 28% methanol in dichloromethane , then \n 50100% ethyl acetate in hexanes , isolated as a yellow solid \n in 38% yield .", "s. , 1 h ) , 8.31 ( d , j = 8.8 hz , 1 h ) , 7.96 ( d , j = 8.8 hz , \n 1 h ) , 7.387.50 ( m , 3 h ) , 7.237.35 ( m , 5 h ) , 7.15 ( d , j = 8.8 hz , 1 h ) , 7.06 ( m , j = 8.3 , 2.0 \n hz , 2 h ) , 5.24 ( s , 2 h ) , 3.903.96 ( m , 4 h ) , 3.273.32 \n ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 2% methanol and 18% acetone in dichloromethane , \n then 1% methanol in ethyl acetate , isolated as a yellow solid in 8% \n yield .", "s. , 2 h ) , 8.098.40 \n ( m , 4 h ) , 7.91 ( d , j = 7.8 hz , 2 h ) , 7.487.60 \n ( m , 3 h ) , 7.44 ( t , j = 7.6 hz , 3 h ) , 7.37 ( t , j = 7.3 hz , 2 h ) , 5.26 ( s , 2 h ) , 3.613.70 ( m , 4 \n h ) , 2.872.99 ( m , 4 h ) .", "synthesized by general procedure b. flash \n column chromatography : 28% methanol in dichloromethane , then \n 10100% ethyl acetate in dichloromethane , isolated as a yellow \n solid in 71% yield .", "s. , 1 h ) , 8.40 \n ( d , j = 8.8 hz , 1 h ) , 8.05 ( d , j = 8.3 hz , 2 h ) , 8.01 ( d , j = 8.8 hz , 1 h ) , 7.95 \n ( d , j = 8.3 hz , 2 h ) , 7.52 ( d , j = 7.3 hz , 2 h ) , 7.48 ( br .", "s. , 1 h ) , 7.43 ( t , j = \n 7.6 hz , 2 h ) , 7.36 ( t , j = 7.3 hz , 1 h ) , 7.30 ( d , j = 8.8 hz , 1 h ) , 7.17 ( d , j = 8.8 hz , \n 1 h ) , 5.25 ( s , 2 h ) , 3.773.82 ( m , 4 h ) , 3.073.12 ( m , \n 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 510% methanol in dichloromethane , then \n 1030% methanol in ethyl acetate , isolated as a yellow solid \n in 15% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.81 ( s , 1 h ) , 9.01 ( s , 1 h ) , 8.82 ( s , 1 h ) , 8.218.34 \n ( m , 4 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.61 ( d , j = 2.0 hz , 1 h ) , 7.437.53 ( m , 2 h ) , 7.297.40 \n ( m , 3 h ) , 7.20 ( t , j = 8.8 hz , 1 h ) , 5.29 ( s , 2 h ) , \n 2.95 ( br .", "synthesized by general procedure b. flash \n column chromatography : 510% methanol in dichloromethane , isolated \n as a yellow solid in 50% yield .", "s. , \n 1 h ) , 8.39 ( d , j = 8.8 hz , 1 h ) , 7.938.04 \n ( m , 5 h ) , 7.48 ( br . s. , 1 h ) , 7.42 ( td , j = 8.1 , \n 5.9 hz , 1 h ) , 7.31 ( d , j = 7.8 hz , 2 h ) , 7.26 ( d , j = 9.8 hz , 1 h ) , 7.16 ( d , j = 8.8 hz , \n 1 h ) , 7.06 ( td , j = 8.5 , 2.0 hz , 1 h ) , 5.25 ( s , 2 \n h ) , 3.483.52 ( m , 2 h ) , 3.46 ( t , j = 6.3 hz , \n 2 h ) , 2.732.77 ( m , 2 h ) , 2.692.73 ( m , 2 h ) , 2.40 ( s , \n 3 h ) , 1.95 ( dt , j = 11.4 , 5.8 hz , 2 h ) . lcms found \n 618.1 [ m + h ] . synthesized by general procedure b. flash \n column chromatography : 210% methanol in dichloromethane , isolated \n as a yellow solid in 61% yield", "s. , 1 h ) , 8.62 ( s , 1 \n h ) , 8.31 ( br .", "s. , 1 h ) , 8.14 ( d , j = 8.8 hz , 1 h ) , \n 8.02 ( d , j = 8.3 hz , 2 h ) , 7.94 ( d , j = 8.8 hz , 2 h ) , 7.48 ( br .", "s. , 1 h ) , 7.387.45 ( m , 2 h ) , 7.237.33 \n ( m , 3 h ) , 7.16 ( d , j = 8.8 hz , 1 h ) , 7.06 ( td , j = 8.4 , 2.2 hz , 1 h ) , 5.25 ( s , 2 h ) , 3.473.50 ( m , \n 2 h ) , 3.45 ( t , j = 6.6 hz , 2 h ) , 2.702.74 \n ( m , 2 h ) , 2.662.70 ( m , 2 h ) , 2.38 ( s , 3 h ) , 1.891.96 \n ( m , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 515% methanol in dichloromethane , isolated \n as a yellow solid in 52% yield .", "s. , \n 1 h ) , 8.39 ( d , j = 8.8 hz , 1 h ) , 7.938.04 \n ( m , 5 h ) , 7.48 ( br . s. , 1 h ) , 7.42 ( td , j = 8.1 , \n 5.9 hz , 1 h ) , 7.31 ( d , j = 7.8 hz , 2 h ) , 7.26 ( d , j = 9.8 hz , 1 h ) , 7.16 ( d , j = 8.8 hz , \n 1 h ) , 7.06 ( td , j = 8.5 , 2.0 hz , 1 h ) , 5.25 ( s , 2 \n h ) , 3.483.52 ( m , 2 h ) , 3.46 ( t , j = 6.3 hz , \n 2 h ) , 2.732.77 ( m , 2 h ) , 2.692.73 ( m , 2 h ) , 2.40 ( s , \n 3 h ) , 1.95 ( dt , j = 11.4 , 5.8 hz , 2 h ) . lcms found \n 632.1 [ m + h ] .", "synthesized by general procedure c. flash \n column chromatography : 4080% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a yellow \n solid in 9% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.27 ( s , 1 h ) , 9.14 ( s , 1 h ) , 8.78 ( s , 1 h ) , \n 8.27 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 8.16 ( d , j = 2.4 hz , 1 h ) , 8.09 ( d , j = 8.3 hz , \n 1 h ) , 7.82 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.407.50 \n ( m , 2 h ) , 7.39 ( s , 1 h ) , 7.307.36 ( m , 3 h ) , 7.27 ( d , j = 8.8 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 \n hz , 1 h ) , 7.07 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 5.25 ( s , \n 2 h ) , 3.763.82 ( m , 4 h ) , 3.223.27 ( m , 4 h ) .", "synthesized by general procedure c. flash \n column chromatography : 4080% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a pale \n yellow solid in 14% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.24 ( s , 1 h ) , 9.16 ( s , 1 h ) , 8.62 \n ( d , j = 8.3 hz , 1 h ) , 8.308.36 ( m , 2 h ) , \n 8.21 ( d , j = 2.4 hz , 1 h ) , 7.84 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.47 ( td , j = 7.8 , 5.9 hz , \n 1 h ) , 7.387.43 ( m , 2 h ) , 7.297.36 ( m , 3 h ) , 7.26 ( d , j = 9.3 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 \n hz , 1 h ) , 7.06 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 5.25 ( s , \n 2 h ) , 3.753.83 ( m , 4 h ) , 3.213.28 ( m , 4 h ) .", "synthesized by general procedure c. flash \n column chromatography : 50100% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a yellow \n solid in 9% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.36 ( s , 1 h ) , 9.18 ( s , 1 h ) , 8.93 ( s , 1 h ) , \n 8.36 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 8.20 ( d , j = 8.3 hz , 2 h ) , 8.17 ( d , j = 8.3 hz , \n 1 h ) , 8.15 ( d , j = 2.9 hz , 1 h ) , 7.94 ( d , j = 8.3 hz , 2 h ) , 7.82 ( dd , j = 9.0 , 2.7 \n hz , 1 h ) , 7.50 ( d , j = 7.3 hz , 2 h ) , 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , \n 1 h ) , 7.30 ( d , j = 9.3 hz , 1 h ) , 5.22 ( s , 2 h ) , 3.633.69 \n ( m , 4 h ) , 2.902.98 ( m , 4 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5100% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a yellow \n solid in 23% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.28 ( s , 1 h ) , 9.19 ( s , 1 h ) , 8.70 ( d , j = 8.8 hz , 1 h ) , 8.45 ( d , j = 2.0 hz , \n 1 h ) , 8.41 ( dd , j = 8.5 , 1.7 hz , 1 h ) , 8.20 ( d , j = 2.4 hz , 1 h ) , 8.18 ( d , j = 8.3 hz , \n 2 h ) , 7.91 ( d , j = 8.3 hz , 2 h ) , 7.83 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.50 ( d , j = 6.8 \n hz , 2 h ) , 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 ( t , j = 6.8 hz , 1 h ) , 7.27 ( d , j = 8.8 hz , \n 1 h ) , 5.21 ( s , 2 h ) , 3.633.69 ( m , 4 h ) , 2.912.97 ( m , \n 4 h ) .", "synthesized by general procedure \n b. flash column chromatography : 37% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as an \n orange solid in 39% yield .", "s. , 1 h ) , 9.18 ( s , 1 h ) , \n 8.91 ( s , 1 h ) , 8.35 ( d , j = 7.3 hz , 1 h ) , 8.128.21 \n ( m , 6 h ) , 7.93 ( d , j = 8.3 hz , 2 h ) , 7.83 ( d , j = 6.8 hz , 1 h ) , 7.47 ( td , j = 8.3 , 6.3 \n hz , 1 h ) , 7.307.36 ( m , 2 h ) , 7.28 ( d , j = \n 8.8 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 hz , 1 h ) , 5.25 \n ( s , 2 h ) , 2.96 ( br . s. , 4 h ) , 2.352.43 ( m , 4 h ) , 2.15 ( s , \n 3 h ) .", "synthesized by general procedure \n b. flash column chromatography : 310% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as a dull \n yellow solid in 28% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.28 ( s , 1 h ) , 9.20 ( s , 1 h ) , 8.70 \n ( d , j = 8.3 hz , 1 h ) , 8.47 ( d , j = 2.0 hz , 1 h ) , 8.42 ( dd , j = 8.5 , 1.7 hz , 1 h ) , \n 8.21 ( d , j = 2.9 hz , 1 h ) , 8.168.20 ( m , 3 \n h ) , 7.91 ( d , j = 8.3 hz , 2 h ) , 7.84 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.48 ( td , j = 8.3 , \n 6.3 hz , 1 h ) , 7.307.36 ( m , 2 h ) , 7.27 ( d , j = 9.3 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.7 hz , 1 h ) , \n 5.25 ( s , 2 h ) , 2.96 ( br . s. , 4 h ) , 2.39 ( t , j = 4.6 \n hz , 4 h ) , 2.15 ( s , 3 h ) .", "synthesized by general procedure \n b. flash column chromatography : 510% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as an \n orange solid in 29% yield .", "h nmr ( 500 mhz , meoh - d4 )  8.94 ( s , 1 h ) , 8.73 ( s , 1 h ) , 8.25 \n ( s , 2 h ) , 8.20 ( dd , j = 8.5 , 1.2 hz , 1 h ) , 8.008.06 \n ( m , 3 h ) , 7.95 ( d , j = 8.3 hz , 2 h ) , 7.88 ( d , j = 2.4 hz , 1 h ) , 7.59 ( dd , j = 8.8 , 2.4 \n hz , 1 h ) , 7.38 ( td , j = 8.1 , 5.9 hz , 1 h ) , 7.28 ( d , j = 7.8 hz , 1 h ) , 7.23 ( d , j = 9.8 hz , \n 1 h ) , 6.997.06 ( m , 2 h ) , 5.18 ( s , 2 h ) , 3.603.66 ( m , \n 2 h ) , 3.47 ( t , j = 6.6 hz , 2 h ) , 3.223.28 \n ( m , 4 h ) , 2.77 ( s , 3 h ) , 2.142.21 ( m , 2 h ) .", "synthesized by general procedure \n b. flash column chromatography : 515% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as a light \n yellow solid in 29% yield .", "h nmr ( 500 mhz , meoh - d4 )  8.99 ( s , 1 h ) , 8.51 ( d , j = 8.3 hz , 1 h ) , 8.25 ( s , 1 h ) , 8.178.22 ( m , 2 h ) , 7.97 ( s , \n 4 h ) , 7.91 ( d , j = 2.9 hz , 1 h ) , 7.587.63 \n ( m , 1 h ) , 7.39 ( td , j = 7.8 , 5.9 hz , 1 h ) , 7.29 ( d , j = 7.8 hz , 1 h ) , 7.24 ( d , j = 9.3 hz , \n 1 h ) , 7.007.08 ( m , 2 h ) , 5.20 ( s , 2 h ) , 3.543.59 ( m , \n 2 h ) , 3.47 ( t , j = 6.6 hz , 2 h ) , 2.973.04 \n ( m , 4 h ) , 2.60 ( s , 3 h ) , 2.06 ( quin , j = 6.3 hz , \n 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 030% methanol in dichloromethane , isolated \n as a yellow solid in 20% yield .", "h nmr ( 400 mhz , dmso - d6 )  9.91 ( br , s , 1h ) , 8.73 ( s , 1h ) , 8.55 \n ( s , 1h ) , 8.18 ( d , j = 8.8 hz , 1h ) , 7.98 ( d , j = 8.8 hz , 1h ) , 7.55 ( d , j = 2.4 hz , 1h ) , \n 7.457.50 ( m , 1h ) , 7.377.41 ( m , 1h ) , 7.307.35 \n ( m , 6h ) , 7.177.22 ( m , 1h ) , 7.02 ( d , j = 8.0 \n hz , 1h ) , 5.29 ( s , 2h ) , 3.763.79 ( m , 4h ) , 3.203.23 \n ( m , 4h ) . lcms found 565.0 [ m + h ] . synthesized by general procedure d. flash \n column chromatography : 020% ethyl acetate in hexanes , isolated \n as a yellow solid in 50% yield .", "h nmr ( 400 mhz , dmso - d6 )  9.99 ( s , 1h ) , 8.87 ( s , 1h ) , 8.55 \n ( s , 1h ) , 8.24 ( d , j = 8.8 hz , 1h ) , 8.14 ( d , j = 8.0 hz , 2h ) , 8.01 ( d , j = 8.8 , 1h ) , \n 7.85 ( d , j = 8.4 hz , 2h ) , 7.467.51 ( m , 3h ) , \n 7.39 ( t , j = 7.2 hz , 2h ) , 7.297.34 ( m , 3h ) , \n 5.22 ( s , 2h ) , 3.603.63 ( m , 4h ) , 2.872.89 ( m , 4h ) . \n lcms found 611.1 [ m + h ] . synthesized by general procedure d. flash \n column chromatography : 030% methanol in dichloromethane , isolated \n as a yellow solid in 16% yield .", "h nmr ( 400 mhz , dmso - d6 )  10.01 ( br , s , 1h ) , 8.89 ( s , 1h ) , \n 8.59 ( s , 1h ) , 8.27 ( d , j = 8.8 hz , 1h ) , 8.16 ( d , j = 8.0 hz , 2h ) , 8.05 ( d , j = 8.8 hz , 1h ) , \n 7.88 ( d , j = 8.0 hz , 2h ) , 7.55 ( br , s , 1h ) , 7.457.70 \n ( m , 1h ) , 7.317.35 ( m 4h ) , 7.177.21 ( m , 1h ) , 5.30 ( s , \n 2h ) , 2.95 ( br , s , 4h ) , 2.38 ( br , 4h ) , 2.14 ( s , 3h ) .", "synthesized by general procedure d. flash \n column chromatography : 030% methanol in dichloromethane , isolated \n as a yellow solid in 32% yield .", "h nmr ( 400 mhz , cdcl3 )  8.71 ( s , 1h ) , 8.14 ( d , j = 8.8 \n hz , 1h ) , 8.08 ( d , j = 2.4 hz , 1h ) , 7.99 ( dd , j = 8.8 , 2.2 hz , 1h ) , 7.807.82 ( m , 2h ) , 7.66 ( d , j = 8.8 hz , 2h ) , 7.47 ( s , 1h ) , 7.41 ( d , j = 2 hz , 1h ) , 7.367.39 ( m , 1h ) , 7.23 ( s , 1h ) , 7.197.20 \n ( m , 1h ) , 7.17 ( d , j = 2 hz , 1h ) , 7.05 ( dd , j = 8.0,2.4 hz , 1h ) , 7.01 ( d , j = 8.8 hz , \n 1h ) , 5.19 ( s , 2h ) , 3.363.40 ( m , 4h ) , 2.562.62 ( m , \n 4h ) , 2.33 ( s , 3h ) , 1.811.85 ( m , 2h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 12% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n ppm 9.68 ( s , 1 h ) , 8.59 ( s , 1 h ) , 8.01 ( d , j = 2.4 \n hz , 1 h ) , 7.94 ( s , 1 h ) , 7.67 ( dd , j = 8.8 , 2.4 hz , \n 1 h ) , 7.47 ( m , 1 h ) , 7.40 ( m , 1 h ) , 7.37 ( d , j = \n 7.8 hz , 1 h ) , 7.32 ( m , 2 h ) , 7.25 ( m , 2 h ) , 7.18 ( td , j = 9.3 , 2.4 hz , 1 h ) , 7.08 ( dd , j = 7.8 , 2.4 hz , \n 1 h ) , 5.25 ( s , 2 h ) , 3.78 ( m , 4 h ) , 3.23 ( m , 4 h ) , lcms found 547.0 , \n [ m + h ] .", "h nmr ( 500 mhz , dmso - d6 )  9.64 ( s , 1h ) , 8.49 ( s , 1h ) , 8.18 \n ( s , 1h ) , 8.05 ( d , j = 2.93 hz , 1h ) , 7.71 ( dd , j = 2.44 , 8.79 hz , 1h ) , 7.447.50 ( m , 1h ) , 7.137.41 \n ( m , 7h ) , 7.04 ( dd , j = 1.95 , 8.30 hz , 1h ) , 5.25 ( s , \n 2h ) , 3.78 ( t , j = 4.7 hz , 4h ) , 3.21 ( t , j = 4.7 hz , 4h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 8% yield .", "h nmr ( 400 mhz , dmso - d6 )  \n ppm 8.61 ( s , 1 h ) , 9.83 ( s , 1 h ) , 8.15 ( m , 3 h ) , 7.98 ( s , 1 h ) , 7.88 \n ( d , j = 8.8 hz , 2 h ) , 7.65 ( m , 1 h ) , 7.49 ( m , 2 h ) , \n 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 ( m , 1 h ) , 7.27 ( d , j = 8.8 hz , 1 h ) , 5.23 ( s , 2 h ) , 3.65 ( m , 4 h ) , 2.94 ( m , \n 4 h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 40% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n ppm 9.79 ( s , 1 h ) , 8.52 ( s , 1 h ) , 8.41 ( s , 1 h ) , 8.03 ( d , j = 2.0 hz , 1 h ) , 7.97 ( d , j = 8.3 hz , \n 2 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.68 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.49 ( d , j = 7.8 \n hz , 2 h ) , 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.29 ( d , j = 9.3 hz , \n 1 h ) , 5.22 ( s , 2 h ) , 3.65 ( m , 4 h ) , 2.94 ( m , 4 h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 36% yield .", "h nmr ( 400 mhz , dmso - d6 )  \n ppm 9.84 ( s , 1 h ) , 8.62 ( s , 1 h ) , 8.14 ( m , 3 h ) , 7.99 ( d , j = 2.2 hz , 1 h ) , 7.87 ( d , j = 8.8 hz , \n 2 h ) , 7.66 ( dd , j = 9.2 , 2.6 hz , 1 h ) , 7.47 ( m , 1 \n h ) , 7.32 ( m , 2 h ) , 7.26 ( d , j = 8.8 hz , 1 h ) , 7.19 \n ( td , j = 8.8 , 2.2 hz , 1 h ) , 5.26 ( s , 2 h ) , 2.95 ( m , \n 4 h ) , 2.38 ( m , 4 h ) , 2.14 ( s , 3 h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 25% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n ppm 9.79 ( s , 1 h ) , 8.52 ( s , 1 h ) , 8.41 ( s , 1 h ) , 8.05 ( d , j = 2.4 hz , 1 h ) , 7.95 ( d , j = 8.3 hz , \n 2 h ) , 7.87 ( d , j = 8.3 hz , 2 h ) , 7.69 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( m , 1 h ) , 7.32 ( m , 2 h ) , 7.28 \n ( d , j = 8.8 hz , 1 h ) , 7.18 ( td , j = 8.5 , 2.4 hz , 1 h ) , 5.24 ( s , 2 h ) , 2.95 ( m , 4 h ) , 2.37 ( m , 4 h ) , \n 2.14 ( s , 3 h ) .", "synthesized by general procedure e , isolated as a pale yellow solid \n in 4% yield .", "the product was separated by silica column chromatography \n ( dichloromethane / methanol ) and purified by hplc to obtain the formic \n acid salt .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.82 ( s , 1 h ) , 8.61 ( s , 1 h ) , 8.22 ( s , 1 h ) , \n 8.068.13 ( m , 3 h ) , 7.99 ( d , j = 2.4 hz , 1 \n h ) , 7.91 ( d , j = 8.8 hz , 2 h ) , 7.66 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.47 ( td , j = 8.3 , \n 6.3 hz , 1 h ) , 7.297.36 ( m , 2 h ) , 7.26 ( d , j = 8.8 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 hz , 1 h ) , \n 5.26 ( s , 2 h ) , 3.35 ( dt , j = 5.1 , 2.3 hz , 2 h ) , 3.32 \n ( t , j = 6.1 hz , 2 h ) , 2.532.58 ( m , 2 h ) , \n 2.48 ( d , j = 5.9 hz , 2 h ) , 2.23 ( s , 3 h ) , 1.74 ( quin , j = 5.9 hz , 2 h ) .", "synthesized by general procedure e , isolated as a pale yellow solid \n in 10% yield .", "the product was separated by silica column chromatography \n ( dichloromethane / methanol ) and purified by hplc to obtain the formic \n acid salt .", "h nmr ( 500 mhz , cdcl3/cd3od )  ppm 8.46 ( s , 1 h ) , 8.10 ( s , 1 h ) , 7.89 ( m , 5 h ) , 7.60 \n ( m , 1 h ) , 7.39 ( m , 1 h ) , 7.28 ( d , j = 7.8 hz , 1 h ) , \n 7.24 ( d , j = 9.3 hz , 1 h ) , 7.04 ( m , 2 h ) , 5.20 ( s , \n 2 h ) , 3.57 ( m , 2 h ) , 3.45 ( t , j = 6.6 hz , 2 h ) , 3.05 \n ( m , 4 h ) , 2.63 ( s , 3 h ) , 2.08 ( m , 2 h ) ."], "abstract_text": ["<S> tropical protozoal infections are \n a significant cause of morbidity \n and mortality worldwide ; four in particular ( human african trypanosomiasis \n ( hat ) , chagas disease , cutaneous leishmaniasis , and malaria ) have \n an estimated combined burden of over 87 million disability - adjusted \n life years . </S>", "<S> new drugs are needed for each of these diseases . </S>", "<S> building \n on the previous identification of neu-617 ( 1 ) as a potent \n and nontoxic inhibitor of proliferation for the hat pathogen ( trypanosoma brucei ) , we have now tested this class of analogs \n against other protozoal species : t. cruzi ( chagas \n disease ) , leishmania major ( cutaneous leishmaniasis ) , \n and plasmodium falciparum ( malaria ) . </S>", "<S> based on hits \n identified in this screening campaign , we describe the preparation \n of several replacements for the quinazoline scaffold and report these \n inhibitors biological activities against these parasites . \n in doing this , we have identified several potent proliferation inhibitors \n for each pathogen , such as 4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-6-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)quinoline-3-carbonitrile \n ( neu-924 , 83 ) for t. cruzi and n-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)cinnolin-4-amine \n ( neu-1017 , 68 ) for l. major and p. falciparum . </S>"], "labels": null, "section_names": ["Introduction", "Results", "Discussion and Conclusions", "Experimental\nSection"], "sections": [["taken together , malaria ( caused by plasmodium species ) , human african trypanosomiasis ( hat , caused by trypanosoma brucei ) , chagas disease ( caused by t. \n cruzi ) , and leishmaniasis ( caused by leishmania species ) represent diseases with an estimated combined burden of \n over 87 million disability - adjusted life years .", "while progress has certainly been made toward the discovery \n of new hits , much work remains to translate these hits into promising \n chemical lead series that may be advanced for drug development .", "target \n repurposing  , wherein inhibitors of human homologues of essential \n parasite proteins are assessed for efficacy against parasite cells .", "we have recently initiated the repurposing of \n a variety of kinase inhibitors for parasitic diseases and have also described the use of established human phosphodiesterase \n inhibitors as starting points for lead discovery .", "we have described the discovery of neu-617 ( 1 ) , a \n derivative of the approved human cancer drug lapatinib ( 2 ) , which acts as a potent , orally bioavailable growth inhibitor of t. brucei that showed a modest effect in a mouse model of \n bloodstream infection .", "based on the close phylogenetic relationship \n between the kinomes of the trypanosomatid parasites ( t. brucei , t. cruzi , and leishmania major ) , we hypothesized that all the pathogens \n would be susceptible to these inhibitors .", "we have therefore tested 1 , along with the other analogs synthesized during the course \n of our hit - to - lead optimization program , against the two other kinetoplastid \n parasites . based on our previous observation that related chemotypes \n", "show activity against plasmodium falciparum , we also screened this set of compounds against \n cultures of drug - sensitive and drug - resistant strains of malaria . \n in doing so , we noted differential structure  activity relationships \n for these compounds growth inhibitory properties against each \n of the parasites , thus launching a multiparasite optimization campaign \n from the same lead series . in this paper", ", we describe these \n cross - pathogen assay results and \n outline our evaluation of replacements for the quinazoline scaffold \n of 1 for each of the parasites .", "we posit that such a \n cross - screening approach can be a highly fruitful method for identification \n of new protozoan parasite growth inhibitors ."], ["first , \n in order to ascertain antiparasitic activities of our 66 \n previously reported analogs , we measured \n their growth inhibitory activities against t. cruzi ( intracellular amastigotes ) , l. major ( promastigote \n and intracellular amastigote life stages ) , and p. falciparum ( d6 , drug sensitive strain ) . the structures of the most potent compounds \n for each parasite are shown in figure 1 and \n table 1 , and the complete set of screening \n data for these is tabulated in the supporting \n information ( tables s1s7 ) . program progression from 2 to four", "sem values within 50% . * % inh at \n 10 m . control compounds : t. brucei , suramin ; t. cruzi , benznidazole ; l. major , amphotericin b ; p. falciparum , chloroquine . from these \n experiments", ", we observed that 15 morpholinosulfonamide \n compounds were identified with activity against l. major promastigotes : seven of these were submicromolar inhibitors and \n two were also submicromolar against intracellular amastigotes ( the \n most relevant life stage for human infections of leishmaniasis ) .", "the \n des - fluoro analog neu-551 ( 3 ) showed the highest activity \n against promastigotes ( ec50 = 0.50 m ) .", "we initially \n tested compounds against t. cruzi at a single concentration \n and found that 32 compounds inhibited proliferation > 65% at 10 \n m \n concentrations ; of these 32 compounds , the homopiperazinyl sulfonamide - substituted \n tail was preferred ( neu-628 ( 4 ) , ec50 = 0.51 \n m ) .", "the active compounds against p. falciparum ( drug - sensitive d6 strain ) all contained a basic nitrogen at the \n end of the tail group with neu-627 ( 5 ) being the most \n potent with an ec50 of 27 nm . though we note that the number \n of headgroup replacement analogs tested was far smaller and less diverse \n than the tail variations , the lapatinib headgroup 3-chloro-4-((3-fluorobenzyl)oxy)aniline \n showed optimal potency against all of the parasites , except for both leishmania life stages .", "previous efforts focused on \n the  head  ( 4-benzyloxyanilines ) \n and  tail  ( denoted as r in figure 1 ) regions of lapatinib , while maintaining the central quinazoline \n scaffold ( highlighted in blue in figure 1 ) . \n", "thus , we planned a broader evaluation of other bicyclic aromatic replacements \n for the quinazoline scaffold that would test the important features \n of the chemotype for activity .", "these scaffold replacements were selected \n for exploration of the requisite nitrogen atom positioning in the \n heteroaromatic ring ( see the heterocycle precursors highlighted in \n figure 2 , dihalides 69 ) .", "in addition , the cyanoquinoline and thienopyrimidine scaffolds \n ( dihalides 1012 ) were selected in \n order to include other established tyrosine kinase inhibitor chemotypes . for a matched exploration of these scaffolds", ", we elected to maintain \n the four permutations of head / tail combinations that displayed the \n most potency in each parasite ( shown in figure 1 ) in the new analogs that we prepared .", "we first set out to synthesize \n the requisite dihalogenated scaffolds \n shown in figure 2 .", "the syntheses of 6- and \n 7-bromo-4-chloroquinolines 6a and 6b were \n carried out via the gould ", "was devised to draw out essential interactions \n with either nitrogen of the quinazoline scaffold .", "the requisite cyanoquinoline template 12 was prepared \n using established protocols similar to that of the 4-chloroquinolines . though few examples of cinnoline or phthalazine \n based kinase inhibitors \n exist in the literature , we opted to prepare analogues utilizing both heterocycles \n to test the required positioning of the two nitrogen atoms on the \n scaffold .", "preparation of 6-bromo-4-chlorocinnoline 8a commenced with the bromination of o - aminoacetophenone 13 followed by diazotization of the amine and in situ cyclization to the 6-bromocinnolin-4(1h)-one 15 ( scheme 1 ) .", "we hypothesized that this \n side reaction occurred via chloride displacement under the acidic \n conditions of the reaction ( scheme s1 , see supporting \n information ) .", "a 10:1 product ratio of 8a to 8b could be achieved through the use of thf as solvent and \n 3 equiv of pocl3 .", "the regiomeric 7-bromo-4-chlorocinnoline 8c was prepared in a similar fashion starting instead with \n the acylation of 3-bromoaniline 16 to 2-amino-4-bromoacetophenone 17 , followed by diazotization and chlorination .", "reagents and conditions : ( a ) \n nbs , ch2cl2 , 10 c  23 \n c , o.n . ;", "( b ) nano2 , aq . hcl , h2o , 75 c , \n o.n . ; ( c ) pocl3 , thf , reflux , 2 h ; ( d ) ( i ) bcl3 , ( ch2cl)2 , 0 c ; ( ii ) alcl3 , ch3cn , 80 c , o.n . ; ( iii ) aq . hcl , 80 c , \n 30 min ; ( b ) nano2 , aq . hcl , h2o , 75 c , \n o.n . ; ( c ) pocl3 , thf , reflux , 2 h. the 7-bromo-1-chlorophthalazine template 9a was synthesized \n as shown in scheme 2 .", "aryl bromination of phthalide 19 was carried out in acidic medium with nbs to produce a \n separable mixture of 4- and 6-bromophthalide 20 .", "benzylic \n bromination was achieved using a modification of an established procedure , and the resulting dibromides 21a , b were converted to the corresponding bromophthalazinone 22 upon treatment with hydrazine . as with 15 , 22a was converted to the dichlorinated side product 9b on treatment with neat pocl3 , though dilution \n minimized this side reaction .", "acetonitrile with 3 equiv of pocl3 provided a > 10:1 ratio of the desired chlorination product 9a .", "the 6-bromo-1-chlorophthalazine scaffold 9c could be synthesized in an identical manner starting with the commercially \n available 5-bromophthalide 20b .", "reagents \n and conditions : ( a ) \n nbs , h2so4 , cf3co2h , 23 \n c , 40 h ; ( b ) nbs , aibn , chcl3 , reflux , 1 h ; ( c ) n2h4 , i - proh , reflux , 1.5 h ; ( d ) pocl3 , ch3cn , reflux , 3 h. amination \n of the dihalogenated quinoline , isoquinoline , cyanoquinoline , \n and thienopyrimidine scaffolds was carried out by applying methods \n similar to those we previously reported ( scheme 3 ) .", "however , low yields ( 20% ) \n resulted when these conditions were applied to the cinnoline and phthalazine \n templates .", "switching the solvent to toluene and using 4 equiv of amine \n improved yields to 5478% .", "suzuki couplings using the requisite \n boronates and 57 mol % pd(pph3)4 or \n 1 mol % pd(oac)2 provided the final analogs", ". yields of \n the cinnoline or phthalazine products were drastically improved by \n switching to 2.5 mol % pdcl2(pph3)2 or 5 mol % pd(oac)2 with extended reaction times .", "a higher \n catalyst loading of pd(oac)2 was employed in lieu of pdcl2(pph3)2 in situations where the pph3o byproduct could not be separated from the products .", "reagents and \n conditions : ( a ) 1.1 equiv of amine , i - proh , reflux , o.n . ; ( b ) 4 equiv \n of amine , toluene , reflux , 2.5 h ; ( c ) 4 equiv of amine , toluene , 50 \n c , o.n . ; ( d ) arbpin , et3n , pd(oac)2 , 1:1 \n h2o / etoh , mw , 120 c , 13 h ; ( e ) arbpin , et3n , pdcl2(pph3)2 , 1:1 h2o / etoh , mw , 120 c , 1 h ; ( f ) arbpin , aq .", "na2co3 , pd(pph3)4 , 3:2 glyme / etoh , \n n2 , 85 c , 712 h. with this set of new analogs that are matched to the previously \n described antiparasitic agents , we set \n out measuring these analogs activities against t. \n brucei , t. cruzi , l. major , and p. falciparum , and counter - screened against \n host cells ( nih 3t3 and hepg2 cell lines ) using assays that we previously \n reported .", "a \n small number of compounds showed activity against nih 3t3 cells ; these \n are summarized in the supporting information , table s8 .", "all sem values \n within 25% unless \n noted otherwise . * % inh at 5 m ."], ["compound 1 remained the most potent t. brucei growth inhibitor among this set of analogs .", "a pair of quinoline - based \n compounds , 50 and 52 , each with a basic \n aliphatic amine on the tail group show double digit nanomolar potency \n ( 79 and 87 nm , respectively ) .", "the potency of these compounds seems \n to be driven by the aliphatic amine in the tail region rather than \n by the placement of nitrogen atoms in the scaffold , because the analogous \n isoquinoline analogue 58 is approximately equipotent \n ( 100 nm ) to 50 .", "the quinoline analogs 50 , 51 , and 52 displayed submicromolar potency \n against intracellular amastigotes \n of t. cruzi .", "most other analogs showed a complete \n loss of activity , perhaps due to issues with penetration into both \n the host cell and the parasite .", "( with intracellular parasites , we \n note that compensating effects from interactions with host cell targets \n can not be ruled out . )", "however , the 3-cyanoquinoline analogue 83 , shows a 5-fold improvement in potency over the matched \n original quinazoline analog 4 . in the headgroup region", ", \n the 3-fluoro substituent affords a 10-fold increase in potency over \n the matched des - fluoro analog ( cf .", "the quinoline 51 displayed a slight drop in potency \n compared with the quinazoline 4 , and the matched isoquinoline 59 is 10-fold less potent .", "taken together with the cyanoquinoline \n analog 83 , this data demonstrates the apparent importance \n of two h - bond acceptor motifs in the scaffold . in the case of l. major", ", 47 showed \n a 2.5-fold increase in potency from the matched quinazoline analogue 3 ( 0.20 versus 0.50 m respectively ) against the promastigote \n life stage of l. major .", "the corresponding isoquinoline \n compound 55 showed a complete loss in activity , reinforcing \n the essentiality of the n1 atom of the quinazoline .", "interestingly , \n there was no activity observed against the amastigote life stage of \n the parasite for 47 .", "in contrast , the 7-substituted quinoline 52 displayed submicromolar potency against both life stages \n ( 0.40 m against promastigotes and 0.89 m versus amastigotes ) . \n", "compared with the quinazoline compound 4 , the analogous \n cinnoline 68 displayed increased potency against amastigotes \n ( 0.24 m ) albeit with a significant decrease in potency against \n the promastigote form .", "this lack of correlation of compounds between \n promastigote and amastigote life stages of the parasite has been reported \n by us and others recently .", "other compounds displaying \n modest potency include 76 and 91 , both containing \n the n - methylhomopiperazinyl sulfonamide tail . in p. falciparum cultures , three isoquinoline \n compounds with a basic amine at", "the terminus of the tail region showed \n modest potency highlighted by 57 being nearly equipotent \n to the parent quinazoline 5 ( 40 vs 27 nm ) .", "though the \n matching quinoline 49 showed a complete loss in activity , \n its regioisomer 50 was slightly more active than 5 ( ec50 = 19 nm ) .", "the four quinoline analogs without \n a basic amine on the tail group were within 2-fold of 5 with 48 being the most potent with an ec50 = 16 nm .", "similarly , all four thienopyrimidine analogs with a basic \n tail group amine ( 8992 ) were potent , \n within 4-fold of 5 . among these compounds there was no \n preference for either thienopyridine isomer ; the n - methylpiperazinyl tail group was 2.5- to 4-fold more potent than \n the n - methylhomopiperazinyl tail on each scaffold . among the phthalazines , 74 and 76 , both \n with a basic nitrogen on the tail and substituted at the 6-position \n ( regiomeric to 5 ) were equipotent to 5 . \n also requiring a basic amine on the tail region , the cinnoline analogs \n with piperazinyl and homopiperazinyl moieties ( 6568 ) showed modest to high potency against p. falciparum d6 strain .", "excitingly , the 7-substituted isomers ( 66 and 68 ) were highly potent antiplasmodial agents ( 14 \n and 3 nm , respectively ) . since we observed that the most potent \n compounds against p. falciparum were 4-aminoquinolines \n ( similar to the prototypical \n antimalarial agent chloroquine ) , we tested them against drug resistant \n parasite strains w2 and c235 .", "we were gratified to observe that all \n of the compounds tested are similarly active against drug resistant \n strains of p. falciparum ( table s9 , supporting information ) , suggesting that despite the shared \n scaffold , these analogs likely display a mechanism of action or mechanism \n of resistance that differs from those of chloroquine .", "our studies \n demonstrate which of these scaffolds are the most promising \n for further optimization on a parasite - by - parasite basis .", ", it is clear that the quinoline scaffold \n is significantly active across all four pathogens .", "we note that cinnoline \n ( c ) and quinoline ( a ) generally provide the most potent antiplasmodial \n agents , and the 7-position substitutions are slightly favored in both \n of these templates . for t. brucei ,", "the 7-substituted \n quinolines and 6-substituted isoquinolines appear optimal ( noting \n that , despite the differential numbering of the scaffolds , these two \n particular scaffolds present the same relative regiochemistry ) .", "analogous \n diagrams for the other pathogens are also provided in figure 3 ( and a diagram for leishmania promastigotes \n is in the supporting information ( figure \n s1 ) .", "a summary of the preferred bicyclic scaffold for each parasite \n is shown in figure 3e , noting that none appear \n to be markedly better than the others against leishmania intracellular amastigotes .", "bar chart showing range of potencies for each \n scaffold ( as labeled \n in tables 2 and 3 ) for \n ( a ) malaria , ( b ) t. brucei , ( c ) l. major ( amastigotes ) , and ( d ) t. cruzi .", "regiochemical \n information is shown by the suffix number ( e.g. , scaffold a-7 is the \n 7-substituted quinoline scaffold ) .", "quinolines are shown in blue , isoquinolines \n in green , cinnolines in yellow , phthalazines in orange , cyanoquinolines \n in red , thieno[3,2-d]pyrimidines in purple , thieno[2,3-d]pyrimidines in light gray , and quinazolines in dark gray . \n", "for all parasites except for t. brucei , we also \n note a preference for tail substituents with a basic amine present . \n", "considering only the most  potent  compounds ( ec50  0.1 m for the extracellular parasites t. brucei and p. falciparum ; ec50  1 m for the intracellular parasites l. major and t. cruzi ) , we note that the n - methylpiperizine and n - methylhomopiperazine are \n preferred among analogs that were tested , over the less - basic morpholine \n or nonbasic morpholinosulfonamide substituents ( table 4 ) . at this point", ", we can not discern whether this is an effect \n that is mediated by the biological target(s ) involved in proliferation \n inhibition , by parasite permeation , or by a combination of both ; this \n will be a matter for further investigation .", " potent  compounds \n are ec50  0.1 m .  potent  compounds \n are ec50  1 m . we have previously observed that molecules bearing \n these head and \n tail combinations possess problematic physicochemical properties , \n likely due to the molecular size and lipophilicity .", "the intent of \n the present work was to explore the central scaffold functionality , \n rather than to address these shortcomings .", "nonetheless , we selected \n five representative compounds from among the most potent to be tested \n for their physicochemical properties ( table 5 ) .", "not unexpectedly , compounds tested were > 99% plasma protein \n bound \n with limited thermodynamic aqueous solubility ( < 1 m ) .", "such \n properties are undoubtedly a result of the high molecular weights \n and clogp values ; these issues remain the focus of ongoing efforts , \n which are now focused on specific scaffolds for each pathogen . in summary , through a cross - parasite \n screening campaign of existing \n quinazoline t. brucei proliferation inhibitors , we \n uncovered new hits against three other protozoan parasites . encouraged \n by that initial success , we focused on exploring the central heterocycle \n scaffold , an exercise that has uncovered additional highly potent \n compounds .", "scaffold variations have revealed heteroatom positioning \n essential for activity within this chemotype , while variations to \n regiochemical attachment points have introduced a new set of regioisomers \n with multiparasite potency .", "thus , we have established a new lead series \n for each of these protozoan parasites , which can be now advanced in \n parallel for drug discovery against four different parasitic diseases . \n with this in mind ,", "further optimization of physicochemical properties \n and cellular selectivity of each is ongoing , with a specific focus \n on the  head  and  tail  regions ."], ["( st . louis , mo ) , fisher scientific , \n frontier scientific services , inc .", "( newark , de ) , or matrix scientific \n ( columbia , sc ) and used as received .", "boronic acids and esters and \n aniline reagents were purchased , except for those whose syntheses \n are listed in the supporting information .", "reaction solvents were dried by passage through alumina columns \n on a purification system manufactured by innovative technology ( newburyport , \n ma ) .", "nmr spectra were obtained with varian nmr systems , operating \n at 400 or 500 mhz for h acquisitions as noted .", "lcms analysis \n was performed using a waters alliance reverse - phase hplc , with single - wavelength \n uv  visible detector and lct premier time - of - flight mass spectrometer \n ( electrospray ionization ) .", "all newly synthesized compounds that were \n submitted for biological testing were deemed > 95% pure by lcms \n analysis \n ( uv and esi - ms detection ) prior to submission for biological testing . \n", "preparative lcms was performed on a waters fraction lynx system with \n a waters micromass zq mass spectrometer ( electrospray ionization ) \n and a single - wavelength uv ", "yields reported \n for products obtained by preparative hplc represent the amount of \n pure material isolated ; impure fractions were not repurified . in a flame - dried \n 250 ml round - bottom flask", "were added 6-bromocinnolin-4(1h)-one ( 1.00 g , 4.44 mmol ) , anhydrous tetrahydrofuran ( 45 ml ) , and \n phosphorus oxychloride ( 1.25 ml , 13.41 mmol ) .", "the mixture was refluxed \n for 1 h at which point a deep green / blue solution had resulted .", "the \n solution was cooled to 0 c and was quenched by the dropwise \n addition of sat .", "the mixture was allowed \n to warm to room temperature and stir for an additional 1 h. water \n ( 50 ml ) was added , and the mixture was extracted with dichloromethane \n ( 3  100 ml ) .", "nahco3 ( 50 ml ) , washed with brine ( 50 ml ) , dried over \n na2so4 , concentrated on to silica , and purified \n by flash column chromatography using a gradient of 15% methanol \n in dichloromethane to yield an inseparable 10:1 mixture of 8a and 8b as a brown solid in 85% yield .", "h \n nmr ( 500 mhz , cdcl3 )  9.36 ( s , 1 h ) , 8.43 ( d , j = 8.8 hz , 1 h ) , 8.36 ( d , j = 2.0 hz , \n 1 h ) , 7.98 ( dd , j = 9.3 , 2.0 hz , 1 h ) .", "lcms found \n 242.9 [ m + h ] . in a flame - dried \n 25 ml round - bottom flask", "were added 7-bromocinnolin-4(1h)-one ( 166 mg , 0.74 mmol ) , anhydrous tetrahydrofuran ( 7 ml ) , and \n phosphorus oxychloride ( 0.2 ml , 2.15 mmol ) .", "the mixture was refluxed \n for 1 h at which point a deep green / blue solution had resulted .", "the \n solution was cooled to 0 c and was quenched by the dropwise \n addition of sat .", ". the mixture was allowed \n to warm to room temperature and stir for an additional 1 h. water \n ( 12 ml ) was added , and the mixture was extracted with dichloromethane \n ( 3  25 ml ) .", "nahco3 ( 20 ml ) , washed with brine ( 20 ml ) , and dried \n over na2so4 to yield 8c as a dark \n brown solid in 92% yield .", "h nmr ( 500 mhz , cdcl3 )  9.39 ( s , 1 h ) , 8.76 ( d , j = 2.0 hz , 1 \n h ) , 8.09 ( d , j = 9.3 hz , 1 h ) , 7.95 ( dd , j = 9.0 , 1.7 hz , 1 h ) .", "lcms found 242.9 [ m + h ] . in a \n flame - dried 25 ml round - bottom flask were added 7-bromophthalazin-1(2h)-one ( 205 mg , 0.91 mmol ) , anhydrous acetonitrile ( 9 ml ) , \n and phosphorus oxychloride ( 0.3 ml , 3.22 mmol ) .", "the mixture was refluxed \n for 2 h , then cooled to 0 c , diluted with dichloromethane ( 20 \n ml ) , and quenched with a dropwise addition of sat .", "after 1 h , the layers were separated \n and the aqueous was extracted with dichloromethane ( 2  30 ml ) . \n the combined organic layers were washed with sat .", "nahco3 ( 25 ml ) , washed with brine ( 20 ml ) , dried over na2so4 , and concentrated to yield 9a as an orange solid \n in 91% yield .", "h nmr ( 500 mhz , cdcl3 )  \n 9.45 ( s , 1 h ) , 8.498.51 ( m , 1 h ) , 8.10 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.91 ( d , j = 8.8 hz , 1 h ) . \n", "lcms found 242.9 [ m + h ] . in a \n flame - dried 50 ml round - bottom flask were added 6-bromophthalazin-1(2h)-one ( 402 mg , 1.78 mmol ) , anhydrous acetonitrile ( 18 ml ) , \n and phosphorus oxychloride ( 0.5 ml , 5.36 mmol ) .", "the mixture was refluxed \n for 2 h , then cooled to 0 c , diluted with dichloromethane ( 40 \n ml ) , and quenched with a dropwise addition of sat .", "the biphasic mixture was stirred vigorously and allowed \n to warm to room temperature . after 1 h , the layers were separated , \n and the aqueous was extracted with dichloromethane ( 2  50 ml ) . \n the combined organic layers were washed with sat .", "nahco3 ( 40 ml ) , washed with brine ( 30 ml ) , dried over na2so4 , and concentrated to yield 29c as a yellow solid \n in 94% yield .", "h nmr ( 500 mhz , cdcl3 )  \n 9.39 ( s , 1 h ) , 8.178.21 ( m , 2 h ) , 8.10 ( dd , j = 8.8 , 2.0 hz , 1 h ) .", "in a 250 ml round - bottom \n flask were added 1-(2-amino-5-bromophenyl)ethanone \n ( 8.34 g , 39.0 mmol ) , water ( 30 ml ) , and conc .", "the mixture was cooled to 0 c in an ice bath \n and allowed to stir for 15 min until a suspension resulted .", "aqueous \n sodium nitrite ( 2 m , 20 ml , 40.0 mmol ) was then added dropwise with \n an addition funnel .", "the resulting solution was allowed to warm to \n room temperature over 1.5 h and was stirred at room temperature overnight , \n then refluxed for 6 h. the mixture was cooled to room temperature , \n water ( 200 ml ) was added , and the mixture was extracted with ethyl \n acetate ( 3  200 ml ) .", "the combined organic layers were then washed \n with brine ( 50 ml ) , dried over sodium sulfate , filtered , and concentrated \n onto silica .", "the crude product was then purified by flash column chromatography \n using a gradient of 110% methanol in dichloromethane to yield 15 as a dark brown solid in 82% yield .", "h nmr ( 500 \n mhz , dmso - d6 )  14.09 ( br .", "s. , 1 \n h ) , 8.09 ( d , j = 2.2 hz , 1 h ) , 7.92 ( dd , j = 8.8 , 2.2 hz , 1 h ) , 7.79 ( s , 1 h ) , 7.71 ( d , j = 9.1 hz , 1 h ) .", "in a 50 ml round - bottom \n flask were added 1-(2-amino-4-bromophenyl)ethanone \n ( 712 mg , 3.33 mmol ) , water ( 3 ml ) , and conc .", "the mixture was cooled to 0 c in an ice bath and \n allowed to stir for 15 min until a suspension resulted .", "aqueous sodium \n nitrite ( 2 m , 1.84 ml , 3.68 mmol ) was then added dropwise with an \n addition funnel .", "the resulting solution was allowed to warm to room \n temperature over 1.5 h and was stirred at room temperature overnight , \n then refluxed for 6 h. the mixture was cooled to room temperature , \n water ( 35 ml ) was added , and the mixture was extracted with ethyl \n acetate ( 3  40 ml ) .", "the combined organic layers were then washed \n with brine ( 20 ml ) , dried over sodium sulfate , filtered , and concentrated \n onto silica .", "the crude product was then purified by flash column chromatography \n using a gradient of 2050% ethyl acetate in hexanes , then ethyl \n acetate to yield 18 as a light brown solid in 26% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 13.49 ( s , 1 h ) , 7.92 ( d , j = 8.8 hz , 1 h ) , 7.76 ( s , \n 1 h ) , 7.73 ( d , j = 2.0 hz , 1 h ) , 7.53 ( dd , j = 8.8 , 2.0 hz , 1 h ) .", "in a 100 ml round - bottom flask was dissolved isobenzofuran-1(3h)-one ( 4.01 g , 29.9 mmol ) in trifluoroacetic acid ( 14 ml , \n 182 mmol ) and sulfuric acid ( 6.5 ml , 122 mmol ) .", "n - bromosuccinimide ( 7.95 g , 1.49 mmol ) was added portionwise over \n 8 h , and the solution was stirred at room temperature for an additional \n 87 h. the solution was diluted with water ( 40 ml ) and ethyl acetate \n ( 40 ml ) .", "the organic layer was separated , \n and the aqueous layer was extracted with ethyl acetate ( 3  50 \n ml ) .", "the combined organic layers were washed with brine ( 25 ml ) , dried \n over na2so4 , and concentrated onto silica .", "the \n crude product was then purified by flash column chromatography using \n 1020% ethyl acetate in hexanes to yield 20a as \n white solid in 57% yield .", "h nmr ( 500 mhz , cdcl3 )  7.98 ( d , j = 1.5 hz , 1 h ) , 7.77 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.40 ( d , j = 8.3 \n hz , 1 h ) , 5.27 ( s , 2 h ) .", "lcms found 212.9 [ m + h ] . in a 50 ml round - bottom \n flask", "n - bromosuccinimide \n ( 958 mg , 5.38 mmol ) , 2,2-azobis(2-methylpropionitrile ) ( 75 \n mg , 0.46 mmol ) , and chloroform ( 23 ml ) . the mixture was refluxed for \n 2.5 h , then cooled to room temperature and quenched with sat .", "the organic layer was removed , washed with \n water ( 20 ml ) , washed with brine ( 15 ml ) , and concentrated onto silica . \n", "the crude product was purified by flash column chromatography using \n a gradient of 510% ethyl acetate in hexanes to yield 21a as a white solid in 61% yield .", "h nmr ( 500 \n mhz , cdcl3 )  8.06 ( d , j = 1.5 hz , \n 1 h ) , 7.90 ( dd , j = 8.1 , 1.7 hz , 1 h ) , 7.52 ( d , j = 8.3 hz , 1 h ) , 7.37 ( s , 1 h ) .", "were \n added 5-bromoisobenzofuran-1(3h)-one ( 499 mg , 2.34 \n mmol ) , n - bromosuccinimide \n ( 421 mg , 2.37 mmol ) , 2,2-azobis(2-methylpropionitrile ) ( 38 \n mg , 0.23 mmol ) , and chloroform ( 10 ml ) .", "the mixture was refluxed for \n 2.5 h , then cooled to room temperature and quenched with sat .", "the organic layer was removed , washed with \n water ( 10 ml ) , washed with brine ( 5 ml ) , and concentrated onto silica . \n", "the crude product was purified by flash column chromatography using \n 10% ethyl acetate in hexanes to yield 21b as a white \n solid in 49% yield .", "h nmr ( 500 mhz , cdcl3 ) \n  7.747.83 ( m , 3 h ) , 7.36 ( s , 1 h ) .", "gcms found 289.8 \n [ m ] . in a 25 ml round - bottom flask", "was dissolved \n 3,6-dibromoisobenzofuran-1(3h)-one ( 143 mg , 0.49 \n mmol ) in ethanol ( 5 ml ) .", "hydrazine \n monohydrate ( 0.12 ml , 2.48 mmol ) was then added via a syringe , and \n the solution was refluxed for 1.5 h. the solution was cooled to room \n temperature , and ice water ( 15 ml ) was added to the reaction mixture . \n", "the precipitate was vacuum filtered and dried under a vacuum overnight \n to yield 22a as a white solid in 56% yield .", "h nmr ( 500 mhz , dmso - d6 )  12.82 \n ( br .", "s. , 1 h ) , 8.39 ( s , 1 h ) , 8.30 ( d , j = 2.0 hz , \n 1 h ) , 8.11 ( dd , j = 8.5 , 2.2 hz , 1 h ) , 7.90 ( d , j = 8.3 hz , 1 h ) .", "in a 25 ml round - bottom flask was dissolved 3,5-dibromoisobenzofuran-1(3h)-one ( 302 mg , 1.04 mmol ) in ethanol ( 10 ml ) .", "hydrazine \n monohydrate ( 0.25 ml , 5.18 mmol ) was then added via a syringe , and \n the solution was refluxed for 1.5 h. the solution was cooled to room \n temperature , and ice water ( 30 ml ) was added to the reaction mixture . \n", "the precipitate was vacuum filtered and dried under a vacuum overnight \n to yield 22b as a white solid in 73% yield .", "h nmr ( 500 mhz , dmso - d6 )  12.78 \n ( br .", "s. , 1 h ) , 8.33 ( s , 1 h ) , 8.23 ( d , j = 2.0 hz , \n 1 h ) , 8.12 ( d , j = 8.3 hz , 1 h ) , 8.00 ( dd , j = 8.3 , 2.0 hz , 1 h ) .", "to a solution of the appropriate \n aryl chloride ( 1 equiv ) in 2-propanol ( 0.15 m ) was added 3-chloro-4-((3-fluorobenzyl)oxy)aniline \n or 4-(benzyloxy)-3-chloroaniline ( 1.1 equiv ) .", "the formed precipitate was collected by vacuum \n filtration to obtain the desired products . to a solution of the appropriate \n aryl chloride ( 1 equiv ) in 2-propanol ( 0.15 m )", "the combined organic layers were washed with water , washed with brine , \n dried over na2so4 , and concentrated .", "the crude \n products were purified by flash column chromatography to obtain the \n desired products .", "a solution of the appropriate 4-chlorocinnoline \n ( 1 equiv ) and 3-chloro-4-((3-fluorobenzyl)oxy)aniline \n or 4-(benzyloxy)-3-chloroaniline ( 4 equiv ) in toluene ( 0.1 m ) was \n refluxed for 2.5 h and cooled to room temperature .", "triethylamine ( 4 \n equiv ) was added , and the mixture was returned to reflux for an additional \n 30 min .", "the mixture was cooled back to room temperature , and the formed \n yellow precipitate was vacuum filtered , washed with ethyl acetate , \n concentrated onto silica , and purified by flash column chromatography .", "a solution of the appropriate 1-chlorophthalazine ( 1 equiv ) and \n 3-chloro-4-((3-fluorobenzyl)oxy)aniline or 4-(benzyloxy)-3-chloroaniline \n ( 4 equiv ) in anhydrous toluene ( 0.2 m ) was heated at 50 c overnight . \n", "the combined \n organic layers were washed with brine , dried over na2so4 , concentrated onto silica , and purified by flash column chromatography . synthesized by general procedure b. flash \n column chromatography : 2050% ethyl acetate in hexanes to yield 23 as a light brown solid in 90% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  8.99 ( s , 1 \n h ) , 8.63 ( s , 1 h ) , 8.46 ( d , j = 5.4 hz , 1 h ) , 7.80 \n ( d , j = 1.5 hz , 2 h ) , 7.437.51 ( m , 2 h ) , \n 7.277.36 ( m , 4 h ) , 7.19 ( td , j = 8.7 , 2.2 \n hz , 1 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.26 ( s , 2 h ) . \n lcms found 456.8 [ m + h ] .", "synthesized by general procedure b. flash \n column chromatography : 2070% ethyl acetate in hexanes to yield 24 as a brown solid in 70% yield .", "h nmr ( 500 mhz , \n dmso - d6 )  8.98 ( s , 1 h ) , 8.63 ( s , \n 1 h ) , 8.45 ( d , j = 5.4 hz , 1 h ) , 7.80 ( s , 2 h ) , 7.50 \n ( d , j = 7.3 hz , 2 h ) , 7.397.46 ( m , 3 h ) , \n 7.35 ( t , j = 7.3 hz , 1 h ) , 7.30 ( s , 2 h ) , 6.79 ( d , j = 5.4 hz , 1 h ) , 5.23 ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 2050% ethyl acetate in hexanes to yield 25 as a tan colored solid in 82% yield .", "h nmr \n ( 399 mhz , dmso - d6 )  9.05 ( s , 1 \n h ) , 8.43 ( d , j = 5.1 hz , 1 h ) , 8.31 ( d , j = 8.8 hz , 1 h ) , 8.05 ( d , j = 2.2 hz , 1 h ) , 7.67 \n ( dd , j = 8.8 , 2.2 hz , 1 h ) , 7.417.52 ( m , \n 2 h ) , 7.257.37 ( m , 4 h ) , 7.18 ( td , j = 8.6 , \n 2.6 hz , 1 h ) , 6.76 ( d , j = 5.9 hz , 1 h ) , 5.25 ( s , \n 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 2050% ethyl acetate in hexanes to yield 26 as an off - white solid in 83% yield .", "h nmr ( 500 \n mhz , dmso - d6 )  9.04 ( s , 1 h ) , 8.43 \n ( d , j = 4.9 hz , 1 h ) , 8.31 ( d , j = 9.3 hz , 1 h ) , 8.05 ( d , j = 2.0 hz , 1 h ) , 7.67 \n ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.49 ( d , j = 6.8 hz , 2 h ) , 7.397.46 ( m , 3 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.30 ( s , 2 h ) , 6.75 ( d , j = 5.4 \n hz , 1 h ) , 5.23 ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 27 as a pale red - brown solid in 97% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  9.24 ( s , 1 \n h ) , 8.80 ( s , 1 h ) , 8.07 ( d , j = 2.4 hz , 1 h ) , 8.02 \n ( d , j = 5.9 hz , 1 h ) , 7.82 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 7.727.79 ( m , 2 h ) , 7.417.49 \n ( m , 1 h ) , 7.277.35 ( m , 2 h ) , 7.137.23 ( m , 3 h ) , 5.21 \n ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 28 as a light red solid in 93% yield .", "h nmr ( 500 \n mhz , dmso - d6 )  9.22 ( s , 1 h ) , 8.80 \n ( s , 1 h ) , 8.07 ( d , j = 2.4 hz , 1 h ) , 8.02 ( d , j = 5.9 hz , 1 h ) , 7.81 ( dd , j = 8.8 , 1.5 \n hz , 1 h ) , 7.737.78 ( m , 2 h ) , 7.48 ( d , j = \n 7.3 hz , 2 h ) , 7.40 ( t , j = 7.6 hz , 2 h ) , 7.33 ( t , j = 7.3 hz , 1 h ) , 7.21 ( d , j = 8.8 hz , \n 1 h ) , 7.15 ( d , j = 5.9 hz , 1 h ) , 5.18 ( s , 2 h ) . lcms \n found 439.2 [ m + h ] .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 29 as a salmon colored solid in 91% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  9.27 ( s , 1 \n h ) , 8.45 ( d , j = 8.8 hz , 1 h ) , 8.08 ( dd , j = 12.0 , 2.2 hz , 2 h ) , 8.01 ( d , j = 5.4 \n hz , 1 h ) , 7.75 ( ddd , j = 13.8 , 9.2 , 2.4 hz , 2 h ) , \n 7.46 ( m , j = 5.9 hz , 1 h ) , 7.277.35 ( m , 2 \n h ) , 7.107.23 ( m , 3 h ) , 5.22 ( s , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 1030% ethyl acetate in hexanes to yield 30 as a burnt orange solid in 81% yield .", "h nmr \n ( 500 mhz , dmso - d6 )  9.24 ( s , 1 \n h ) , 8.45 ( d , j = 8.8 hz , 1 h ) , 8.10 ( d , j = 2.0 hz , 1 h ) , 8.05 ( d , j = 2.4 hz , 1 h ) , 8.01 \n ( d , j = 5.9 hz , 1 h ) , 7.76 ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.72 ( dd , j = 8.8 , 2.4 hz , \n 1 h ) , 7.48 ( d , j = 6.8 hz , 2 h ) , 7.41 ( t , j = 7.3 hz , 2 h ) , 7.34 ( t , j = 7.8 hz , \n 1 h ) , 7.21 ( d , j = 9.3 hz , 1 h ) , 7.13 ( d , j = 5.9 hz , 1 h ) , 5.19 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield an \n inseparable 10:1 mixture of 31 and dichloro side product \n as a vibrant yellow solid in 54% yield .", "h nmr ( 500 mhz , \n dmso - d6 )  9.33 ( s , 1 h ) , 8.78 ( s , \n 1 h ) , 8.70 ( d , j = 1.5 hz , 1 h ) , 8.14 ( d , j = 8.8 hz , 1 h ) , 7.97 ( dd , j = 9.3 , 1.5 \n hz , 1 h ) , 7.54 ( d , j = 2.0 hz , 1 h ) , 7.48 ( td , j = 8.3 , 6.3 hz , 1 h ) , 7.40 ( dd , j = 8.8 , \n 2.4 hz , 1 h ) , 7.307.37 ( m , 3 h ) , 7.20 ( td , j = 9.3 , 2.0 hz , 1 h ) , 5.29 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield an \n inseparable 10:1 mixture of 32 and dichloro side product \n as a yellow solid in 68% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.32 ( s , 1 h ) , 8.78 ( s , 1 h ) , 8.71 \n ( d , j = 2.0 hz , 1 h ) , 8.14 ( d , j = 9.3 hz , 1 h ) , 7.97 ( dd , j = 9.0 , 1.7 hz , 1 h ) , \n 7.477.55 ( m , 3 h ) , 7.44 ( t , j = 7.6 hz , 2 \n h ) , 7.337.41 ( m , 3 h ) , 5.26 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield 33 as a brown solid in 55% yield with 84% purity .", "h nmr ( 500 mhz , dmso - d6 )  9.43 \n ( s , 1 h ) , 8.76 ( s , 1 h ) , 8.41 ( d , j = 2.0 hz , 1 h ) , \n 8.35 ( d , j = 9.3 hz , 1 h ) , 7.90 ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.55 ( d , j = 2.4 hz , 1 h ) , \n 7.49 ( m , j = 6.3 hz , 1 h ) , 7.41 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.317.37 ( m , 3 h ) , 7.20 ( td , j = 8.4 , 2.7 hz , 1 h ) , 5.29 ( s , 2 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5% methanol in dichloromethane to yield 34 as a metallic bronze colored solid in 57% yield with 85% \n purity .", "h nmr ( 500 mhz , dmso - d6 )  9.42 ( s , 1 h ) , 8.75 ( s , 1 h ) , 8.41 ( d , j = 2.0 hz , 1 h ) , 8.35 ( d , j = 8.8 hz , 1 h ) , 7.90 \n ( dd , j = 9.0 , 2.2 hz , 1 h ) , 7.54 ( d , j = 2.4 hz , 1 h ) , 7.51 ( d , j = 7.3 hz , 2 h ) , 7.43 \n ( t , j = 7.6 hz , 2 h ) , 7.40 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.337.38 ( m , 2 h ) , 5.26 ( s , 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 520% ethyl acetate in dichloromethane \n to yield 35 as a yellow solid in 69% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.21 \n ( s , 1 h ) , 9.13 ( s , 1 h ) , 8.87 ( s , 1 h ) , 8.16 ( d , j = 2.4 hz , 1 h ) , 8.11 ( dd , j = 8.8 , 1.5 hz , 1 h ) , \n 7.99 ( d , j = 8.8 hz , 1 h ) , 7.81 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 7.9 , 6.1 hz , \n 1 h ) , 7.297.36 ( m , 2 h ) , 7.25 ( d , j = 9.3 \n hz , 1 h ) , 7.17 ( td , j = 8.7 , 2.2 hz , 1 h ) , 5.24 ( s , \n 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 520% ethyl acetate in dichloromethane \n to yield 36 as a light greenish brown solid in 78% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 9.20 ( s , 1 h ) , 9.14 ( s , 1 h ) , 8.88 ( s , 1 h ) , 8.15 ( d , j = 2.4 hz , 1 h ) , 8.13 ( dd , j = 8.5 , 1.7 hz , 1 h ) , \n 8.00 ( d , j = 8.8 hz , 1 h ) , 7.80 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.50 ( d , j = 7.3 hz , 2 h ) , \n 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.27 ( d , j = 8.8 hz , 1 h ) , 5.21 \n ( s , 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 520% ethyl acetate in dichloromethane \n to yield 37 as a light brown solid in 64% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 9.42 ( s , 1 h ) , 9.09 ( s , 1 h ) , 8.62 ( d , j = 9.3 hz , \n 1 h ) , 8.33 ( d , j = 2.0 hz , 1 h ) , 8.19 ( d , j = 2.4 hz , 1 h ) , 8.16 ( dd , j = 9.0 , 2.2 \n hz , 1 h ) , 7.83 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 7.8 , 5.9 hz , 1 h ) , 7.32 ( m , j = 7.3 \n hz , 2 h ) , 7.25 ( d , j = 9.3 hz , 1 h ) , 7.17 ( td , j = 8.5 , 2.0 hz , 1 h ) , 5.24 ( s , 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 530% ethyl acetate in dichloromethane \n to yield 38 as a green - gray colored solid in 75% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n 9.25 ( s , 1 h ) , 9.09 ( s , 1 h ) , 8.51 ( d , j = 8.8 hz , \n 1 h ) , 8.33 ( d , j = 2.0 hz , 1 h ) , 8.17 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 8.14 ( d , j = 2.9 \n hz , 1 h ) , 7.78 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.49 ( d , j = 6.8 hz , 2 h ) , 7.42 ( t , j = 7.6 hz , \n 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.26 ( d , j = 8.8 hz , 1 h ) , 5.21 ( s , 2 h ) .", "synthesized by general procedure a , collected \n as a yellow solid in 80% yield .", "h nmr ( 400 mhz , dmso - d6 )  9.03 ( s , 1h ) , 8.85 ( br , s , 1h ) , 8.23 \n ( d , j = 8.8 hz , 1h ) , 7.73 ( d , j = \n 8.8 , 1h ) , 7.59 ( d , j = 2.4 , 1h ) , 7.457.50 \n ( m , 1h ) , 7.307.39 ( m , 4h ) , 7.187.22 ( m , 1h ) , 5.30 \n ( s , 2h ) .", "synthesized by general procedure a , collected \n as a yellow solid in 52% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.02 ( s , 1h ) , 8.82 ( br , s , 1h ) , \n 8.22 ( d , j = 9.0 hz , 1h ) , 7.72 ( d , j = 8.5 hz , 1h ) , 7.57 ( s , 1h ) , 7.5 ( d , j = 7.0 hz , \n 2h ) , 7.43 ( t , j = 7.5 hz , 2h ) , 7.35 ( m , 3h ) , 5.26 \n ( s , 2h ) .", "h nmr ( 400 mhz , dmso - d6 )  ppm \n 10.64 ( s , 1 h ) , 8.71 ( s , 1 h ) , 7.87 ( d , j = 2.9 hz , \n 1 h ) , 7.74 ( s , 1 h ) , 7.57 ( dd , j = 8.8 , 2.2 hz , 1 \n h ) , 7.47 ( m , 1 h ) , 7.31 ( m , 3 h ) , 7.19 ( td , j = 8.1 , \n 2.2 hz , 1 h ) , 5.28 ( s , 2 h ) .", "h nmr ( 500 mhz , dmso - d6 )  ppm \n 9.7 1 ( s , 1 h ) , 8.54 ( s , 1 h ) , 7.89 ( d , j = 2.0 hz , \n 1 h ) , 7.68 ( s , 1 h ) , 7.58 ( dd , j = 8.8 , 2.9 hz , 1 \n h ) , 7.49 ( d , j = 7.8 hz , 2 h ) , 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.26 \n ( d , j = 8.8 hz , 1 h ) , 5.23 ( s , 2 h ) .", "h nmr ( 500 mhz , dmso - d6 )  9.80 ( s , 1h ) , 8.49 ( s , 1h ) , 8.12 \n ( s , 1h ) , 8.02 ( d , j = 2.4 hz , 1h ) , 7.67 ( dd , j = 2.7 , 9.03 hz , 1h ) , 7.437.49 ( m , 1h ) , 7.247.35 \n ( m , 3h ) , 7.18 ( dt , j = 2.4 , 8.6 hz , 1h ) , 5.24 ( s , \n 2h ) .", "h nmr ( 500 mhz , dmso - d6 )  ppm \n 9.94 ( s , 1 h ) , 8.49 ( s , 1 h ) , 8.19 ( s , 1 h ) , 8.02 ( d , j = 2.4 hz , 1 h ) , 7.68 ( dd , j = 8.8 , 2.4 hz , 1 h ) , \n 7.49 ( m , 2 h ) , 7.41 ( m , 2 h ) , 7.34 ( m , 1 h ) , 7.27 ( d , j = 9.3 hz , 1 h ) , 5.21 ( s , 2 h ) .", "lcms found 445.9 [ m + h ] . in a 25 \n ml microwave vial equipped with a stir bar", "were added aryl bromide \n ( 1 equiv ) , boronic ester ( 1.3 equiv ) , 1:1 water / ethanol ( 0.04 m ) , \n triethylamine ( 3 equiv ) , and palladium(ii ) acetate ( 0.01 m in acetone , \n 1 mol % ) .", "the vial was sealed with a septum , and the contents were \n irradiated to and held at 120 c with stirring for 1 h. the reaction \n mixture was cooled to room temperature , diluted with water ( 8 ml ) , \n and extracted with dichloromethane ( 3  8 ml ) .", "naoh ( 1 m , 2  5 ml ) , water ( 5 ml ) , \n and brine ( 5 ml ) .", "the crude product was \n purified by flash column chromatography . in a 25 \n ml microwave vial equipped with a stir bar", "were added aryl bromide \n ( 1 equiv ) , boronic ester ( 1.3 equiv ) , 1:1 water / ethanol ( 0.04 m ) , \n triethylamine ( 3 equiv ) , and bis(triphenylphosphine)palladium(ii ) \n chloride ( 2.5 mol % ) .", "the vial was sealed with a septum , and the contents \n were irradiated to and held at 120 c with stirring for 1 h. \n the reaction mixture was cooled to room temperature , diluted with \n water ( 8 ml ) , and extracted with dichloromethane ( 3  8 ml ) . \n", "naoh ( 1 m , 2  \n 5 ml ) , water ( 5 ml ) , and brine ( 5 ml ) .", "the organic layer was then \n dried over na2so4 and concentrated on to silica . \n", "the crude product was purified by flash column chromatography . in a 25 \n ml microwave vial equipped with a stir bar", "were added aryl bromide \n ( 1 equiv ) , boronic ester ( 1.3 equiv ) , 1:1 water / ethanol ( 0.04 m ) , \n triethylamine ( 3 equiv ) , and palladium(ii ) acetate ( 5 mol % ) .", "the \n vial was sealed with a septum , and the contents were irradiated to \n and held at 120 c with stirring for 3 h. the reaction mixture \n was cooled to room temperature , diluted with water ( 8 ml ) , and extracted \n with dichloromethane ( 3  8 ml ) .", "naoh ( 1 m , 2  5 ml ) , water ( 5 ml ) , and \n brine ( 5 ml ) .", "the crude product was \n purified by flash column chromatography . to a solution \n of the appropriate aryl iodide ( 1 equiv ) in 3:2 dimethoxyethane / ethanol \n ( 0.05 m )", "2 m na2co3 ( 6 equiv ) , and pd(pph3)4 ( 5 mol % ) .", "the mixture was purged with nitrogen and \n heated at 85 c for 7 h. the mixture was cooled to room temperature \n and filtered , and the filtrate was concentrated .", "the residue was dissolved \n in ethyl acetate , washed with water , washed with brine , dried over \n na2so4 , and purified by flash column chromatography . to a solution \n of the appropriate aryl bromide ( 1 equiv ) in 3:2 dimethoxyethane / ethanol \n ( 0.05 m )", "2 m na2co3 ( 6 equiv ) , and pd(pph3)4 ( 7 mol % ) .", "the mixture was heated at 85 c for \n 12 h , then cooled to room temperature , and the solvents were removed \n under reduced pressure .", "the residue was purified by silica column \n chromatography ( hexanes / ethyl acetate ) and then by reverse phase chromatography \n ( water / acetonitrile ) unless otherwise mentioned . synthesized by general procedure a. flash \n column chromatography : 25% methanol in dichloromethane , isolated \n as a yellow solid in 27% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.10 ( br .", "s. , 1 h ) , 8.61 ( d , j = 2.0 hz , 1 h ) , 8.43 ( d , j = 5.4 hz , \n 1 h ) , 8.04 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.92 ( d , j = 8.8 hz , 1 h ) , 7.457.52 ( m , 2 h ) , 7.39 ( t , j = 7.8 hz , 1 h ) , 7.297.37 ( m , 6 h ) , 7.20 ( td , j = 8.8 , 2.0 hz , 1 h ) , 7.00 ( dd , j = 8.1 , \n 1.7 hz , 1 h ) , 6.77 ( d , j = 5.4 hz , 1 h ) , 5.28 ( s , \n 2 h ) , 3.753.81 ( m , 4 h ) , 3.193.26 ( m , 4 h ) . lcms found \n 540.1 [ m + h ] . synthesized by general procedure a. flash \n column chromatography : 25% methanol in dichloromethane , isolated \n as a yellow solid in 24% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 8.99 ( br .", "s. , 1 h ) , 8.47 ( d , j = 5.4 hz , 1 h ) , 8.41 ( d , j = 8.8 hz , \n 1 h ) , 8.12 ( d , j = 2.0 hz , 1 h ) , 7.88 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 7.457.51 ( m , 2 h ) , 7.267.40 \n ( m , 7 h ) , 7.20 ( td , j = 8.8 , 2.4 hz , 1 h ) , 7.02 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 6.76 ( d , j = 5.4 \n hz , 1 h ) , 5.27 ( s , 2 h ) , 3.743.82 ( m , 4 h ) , 3.203.27 \n ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 05% methanol in dichloromethane , isolated \n as a tan colored solid in 65% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.14 ( s , 1 h ) , 8.78 ( d , j = 2.0 hz , 1 h ) , 8.47 ( d , j = 5.4 hz , \n 1 h ) , 8.17 ( d , j = 8.8 hz , 2 h ) , 8.11 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.98 ( d , j = 8.8 \n hz , 1 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.51 ( d , j = 7.3 hz , 2 h ) , 7.48 ( d , j = 1.5 hz , \n 1 h ) , 7.43 ( t , j = 7.6 hz , 2 h ) , 7.327.39 \n ( m , 3 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.25 ( s , 2 h ) , \n 3.613.70 ( m , 4 h ) , 2.882.97 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 25% methanol in dichloromethane , isolated \n as a tan colored solid in 63% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.02 ( s , 1 h ) , 8.50 ( m , j = 5.4 hz , 2 h ) , 8.25 ( d , j = 1.5 hz , \n 1 h ) , 8.18 ( d , j = 8.8 hz , 2 h ) , 7.97 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 7.87 ( d , j = 8.3 \n hz , 2 h ) , 7.51 ( d , j = 7.3 hz , 2 h ) , 7.47 ( d , j = 2.0 hz , 1 h ) , 7.44 ( t , j = 7.6 hz , \n 2 h ) , 7.36 ( m , j = 6.8 hz , 1 h ) , 7.307.34 \n ( m , 2 h ) , 6.79 ( d , j = 5.4 hz , 1 h ) , 5.24 ( s , 2 h ) , \n 3.623.70 ( m , 4 h ) , 2.902.97 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 510% methanol in dichloromethane , isolated \n as a yellow solid in 40% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.19 ( br .", "s. , 1 h ) , 8.78 ( d , j = 2.0 hz , 1 h ) , 8.47 ( d , j = 5.4 hz , \n 1 h ) , 8.16 ( d , j = 8.8 hz , 2 h ) , 8.12 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.98 ( d , j = 8.8 \n hz , 1 h ) , 7.87 ( d , j = 8.8 hz , 2 h ) , 7.457.52 \n ( m , 2 h ) , 7.307.38 ( m , 4 h ) , 7.20 ( td , j = \n 8.7 , 2.7 hz , 1 h ) , 6.81 ( d , j = 5.4 hz , 1 h ) , 5.28 \n ( s , 2 h ) , 2.95 ( br . s. , 4 h ) , 2.352.43 ( m , 4 h ) , 2.15 ( s , \n 3 h ) .", "synthesized by general procedure a. flash \n column chromatography : 510% methanol in dichloromethane , isolated \n as a yellow solid in 33% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.10 ( br .", "s. , 1 h ) , 8.50 ( d , j = 8.3 hz , 2 h ) , 8.25 ( d , j = 2.0 hz , \n 1 h ) , 8.16 ( d , j = 8.3 hz , 2 h ) , 7.97 ( dd , j = 8.8 , 2.0 hz , 1 h ) , 7.87 ( d , j = 8.3 \n hz , 2 h ) , 7.457.52 ( m , 2 h ) , 7.297.38 ( m , 4 h ) , 7.20 \n ( td , j = 8.5 , 2.0 hz , 1 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.28 ( s , 2 h ) , 2.96 ( br . s. , 4 h ) , 2.40 ( br .", "then \n prep hplc : 595% acetonitrile in water , isolated as a yellow \n solid in 10% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 8.77 ( d , j = 2.0 hz , 1 \n h ) , 8.46 ( d , j = 5.4 hz , 1 h ) , 8.27 ( s , 1 h ) , 8.078.15 \n ( m , 3 h ) , 7.97 ( d , j = 8.8 hz , 1 h ) , 7.92 ( d , j = 8.3 hz , 2 h ) , 7.457.52 ( m , 2 h ) , 7.307.38 \n ( m , 4 h ) , 7.20 ( td , j = 8.5 , 2.4 hz , 1 h ) , 6.80 ( d , j = 5.4 hz , 1 h ) , 5.28 ( s , 2 h ) , 3.36 ( m , j = 5.2 , 2.5 , 2.5 hz , 2 h ) , 3.33 ( t , j = 6.3 hz , \n 2 h ) , 2.532.57 ( m , 2 h ) , 2.47 ( m , j = 5.9 \n hz , 2 h ) , 2.22 ( s , 3 h ) , 1.74 ( quin , j = 5.9 hz , \n 2 h ) .", "synthesized by general procedure a. flash \n column chromatography : 520% methanol in dichloromethane , isolated \n as a brown solid in 32% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.06 ( s , 1 h ) , 8.478.52 \n ( m , 2 h ) , 8.23 ( d , j = 2.0 hz , 1 h ) , 8.12 ( d , j = 8.3 hz , 2 h ) , 7.96 ( dd , j = 8.8 , 2.0 \n hz , 1 h ) , 7.91 ( d , j = 8.3 hz , 2 h ) , 7.457.52 \n ( m , 2 h ) , 7.297.37 ( m , 4 h ) , 7.20 ( td , j = \n 8.5 , 2.4 hz , 1 h ) , 6.79 ( d , j = 4.9 hz , 1 h ) , 5.27 \n ( s , 2 h ) , 3.39 ( m , j = 3.4 hz , 2 h ) , 3.313.35 \n ( m , 2 h ) , 2.63 ( br . s. , 2 h ) , 2.58 ( br . s. , 2 h ) , 2.29 ( br . s. , 3 \n h ) , 1.741.83 ( m , 2 h ) .", "synthesized by general procedure a. flash \n column chromatography : 3050% ethyl acetate in hexanes , isolated \n as a biege solid in 59% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.27 ( s , 1 h ) , 8.71 ( s , 1 h ) , \n 8.06 ( d , j = 2.4 hz , 1 h ) , 8.03 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 7.98 ( d , j = 5.4 hz , 1 h ) , \n 7.88 ( d , j = 8.8 hz , 1 h ) , 7.76 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 8.1 , 6.3 hz , \n 1 h ) , 7.40 ( t , j = 7.8 hz , 1 h ) , 7.297.36 \n ( m , 4 h ) , 7.23 ( d , j = 9.3 hz , 1 h ) , 7.157.21 \n ( m , 2 h ) , 7.02 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 5.23 ( s , \n 2 h ) , 3.753.82 ( m , 4 h ) , 3.203.26 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 3050% ethyl acetate in hexanes , isolated \n as an orange solid in 81% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.21 ( s , 1 h ) , 8.56 ( d , j = 8.8 hz , 1 h ) , 8.13 ( d , j = 2.4 hz , \n 1 h ) , 8.11 ( d , j = 2.0 hz , 1 h ) , 8.00 ( d , j = 5.4 hz , 1 h ) , 7.94 ( dd , j = 8.8 , 1.5 \n hz , 1 h ) , 7.78 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( td , j = 8.0 , 6.3 hz , 1 h ) , 7.357.41 ( m , 2 h ) , 7.307.35 \n ( m , 2 h ) , 7.28 ( d , j = 7.8 hz , 1 h ) , 7.207.25 \n ( m , 2 h ) , 7.18 ( td , j = 8.8 , 2.4 hz , 1 h ) , 7.03 ( dd , j = 8.1 , 2.2 hz , 1 h ) , 5.23 ( s , 2 h ) , 3.763.81 ( m , \n 4 h ) , 3.213.25 ( m , 4 h ) . lcms found 540.2 [ m + h ] . synthesized by general procedure a. flash \n column chromatography : 2050% ethyl acetate in hexanes , isolated \n as an orange solid in 55% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.35 ( s , 1 h ) , 8.86 ( s , 1 h ) , \n 8.18 ( d , j = 8.8 hz , 2 h ) , 8.12 ( dd , j = 8.8 , 1.5 hz , 1 h ) , 8.04 ( d , j = 2.4 hz , 1 h ) , \n 8.03 ( d , j = 5.9 hz , 1 h ) , 7.96 ( d , j = 8.3 hz , 1 h ) , 7.90 ( d , j = 8.3 hz , 2 h ) , 7.76 \n ( dd , j = 9.3 , 2.9 hz , 1 h ) , 7.50 ( d , j = 6.8 hz , 2 h ) , 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 \n ( t , j = 7.3 hz , 1 h ) , 7.25 ( d , j = 9.3 hz , 1 h ) , 7.22 ( d , j = 5.9 hz , 1 h ) , 5.21 \n ( s , 2 h ) , 3.633.70 ( m , 4 h ) , 2.902.96 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 3050% ethyl acetate in hexanes , isolated \n as an orange solid in 81% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.26 ( s , 1 h ) , 8.65 ( d , j = 9.3 hz , 1 h ) , 8.25 ( d , j = 1.5 hz , \n 1 h ) , 8.16 ( d , j = 8.8 hz , 2 h ) , 8.12 ( d , j = 2.9 hz , 1 h ) , 8.018.07 ( m , 2 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.78 ( dd , j = 9.0 , 2.7 \n hz , 1 h ) , 7.50 ( d , j = 6.8 hz , 2 h ) , 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , \n 1 h ) , 7.27 ( d , j = 5.9 hz , 1 h ) , 7.23 ( d , j = 9.3 hz , 1 h ) , 5.20 ( s , 1 h ) , 3.623.69 ( m , 4 \n h ) , 2.892.97 ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : ethyl acetate , isolated as a cream colored \n solid in 53% yield .", "h nmr ( 500 mhz , cdcl3 ) \n  ppm 8.118.14 ( m , 2 h ) , 7.797.89 ( m , 7 h ) , \n 7.54 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.36 ( td , j = 7.8 , 5.9 hz , 1 h ) , 7.28 ( s , 1 h ) , 7.217.27 ( m , \n 2 h ) , 7.18 ( d , j = 5.9 hz , 1 h ) , 7.03 ( td , j = 8.3 , 2.4 hz , 1 h ) , 6.97 ( d , j = 8.8 \n hz , 1 h ) , 5.15 ( s , 2 h ) , 3.08 ( br . s. , 4 h ) , 2.49 ( t , j = 4.6 hz , 4 h ) , 2.28 ( s , 3 h ) .", "synthesized by general procedure a. flash \n column chromatography : ethyl acetate , isolated as a pale orange solid \n in 52% yield .", "h nmr ( 500 mhz , cdcl3 )  \n ppm 8.14 ( d , j = 5.9 hz , 1 h ) , 8.02 ( d , j = 8.8 hz , 1 h ) , 7.95 ( d , j = 2.0 hz , 1 h ) , 7.837.91 \n ( m , 5 h ) , 7.76 ( dd , j = 8.5 , 1.7 hz , 1 h ) , 7.50 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.37 ( td , j = 7.8 , \n 5.9 hz , 1 h ) , 7.227.27 ( m , 2 h ) , 7.21 ( d , j = 5.9 hz , 1 h ) , 7.007.07 ( m , 2 h ) , 6.98 ( d , j = 8.8 hz , 1 h ) , 5.16 ( s , 2 h ) , 3.11 ( br . s. , 4 h ) , 2.52 ( t , j = 4.6 hz , 4 h ) , 2.29 ( s , 3 h ) .", "synthesized by general procedure a. flash \n column chromatography : 5% methanol in dichloromethane , isolated as \n an orange solid in 73% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.36 ( s , 1 h ) , 8.85 ( s , 1 h ) , \n 8.098.14 ( m , 3 h ) , 8.05 ( d , j = 2.4 hz , 1 \n h ) , 8.02 ( d , j = 5.9 hz , 1 h ) , 7.917.97 ( m , \n 3 h ) , 7.77 ( dd , j = 8.8 , 2.9 hz , 1 h ) , 7.47 ( td , j = 8.1 , 6.3 hz , 1 h ) , 7.297.36 ( m , 2 h ) , 7.24 ( d , j = 9.3 hz , 1 h ) , 7.22 ( d , j = 5.4 hz , \n 1 h ) , 7.18 ( td , j = 9.3 , 2.0 hz , 1 h ) , 5.24 ( s , 2 \n h ) , 3.353.38 ( m , 2 h ) , 3.313.35 ( m , 2 h ) , 2.542.58 \n ( m , 2 h ) , 2.472.49 ( m , 2 h ) , 2.23 ( s , 3 h ) , 1.75 ( quin , j = 5.8 hz , 2 h ) .", "synthesized by general procedure a. flash \n column chromatography : 5% methanol in dichloromethane , isolated as \n a pale yellow solid in 68% yield .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.26 ( s , 1 h ) , 8.64 ( d , j = 8.8 hz , 1 h ) , 8.23 ( d , j = 1.5 hz , \n 1 h ) , 8.13 ( d , j = 2.4 hz , 1 h ) , 8.10 ( d , j = 8.3 hz , 2 h ) , 8.008.06 ( m , 2 h ) , 7.92 ( d , j = 8.3 hz , 2 h ) , 7.79 ( dd , j = 9.0 , 2.7 \n hz , 1 h ) , 7.47 ( td , j = 8.3 , 6.3 hz , 1 h ) , 7.297.35 \n ( m , 2 h ) , 7.27 ( d , j = 5.9 hz , 1 h ) , 7.22 ( d , j = 9.3 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.7 \n hz , 1 h ) , 5.23 ( s , 2 h ) , 3.36 ( m , j = 4.9 , 2.4 , 2.4 \n hz , 2 h ) , 3.313.35 ( m , 2 h ) , 2.532.58 ( m , 2 h ) , 2.462.49 \n ( m , 2 h ) , 2.23 ( s , 3 h ) , 1.75 ( quin , j = 5.8 hz , \n 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 210% methanol in dichloromethane , isolated \n as a yellow solid in 67% yield .", "s. , 1 h ) , 8.30 ( s , 1 h ) , 8.26 ( d , j = 4.9 hz , 1 h ) , 7.95 ( dd , j = 9.0 , 1.2 \n hz , 1 h ) , 7.487.59 ( m , 1 h ) , 7.327.41 ( m , 2 h ) , 7.29 \n ( t , j = 7.8 hz , 1 h ) , 7.177.24 ( m , 2 h ) , \n 7.087.17 ( m , 3 h ) , 7.02 ( td , j = 8.4 , 2.2 \n hz , 1 h ) , 6.876.94 ( m , 2 h ) , 5.13 ( s , 2 h ) , 3.763.82 \n ( m , 4 h ) , 3.093.16 ( m , 4 h ) .", "synthesized by general procedure b. flash \n column chromatography : 28% methanol in dichloromethane , then \n 50100% ethyl acetate in hexanes , isolated as a yellow solid \n in 38% yield .", "s. , 1 h ) , 8.31 ( d , j = 8.8 hz , 1 h ) , 7.96 ( d , j = 8.8 hz , \n 1 h ) , 7.387.50 ( m , 3 h ) , 7.237.35 ( m , 5 h ) , 7.15 ( d , j = 8.8 hz , 1 h ) , 7.06 ( m , j = 8.3 , 2.0 \n hz , 2 h ) , 5.24 ( s , 2 h ) , 3.903.96 ( m , 4 h ) , 3.273.32 \n ( m , 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 2% methanol and 18% acetone in dichloromethane , \n then 1% methanol in ethyl acetate , isolated as a yellow solid in 8% \n yield .", "s. , 2 h ) , 8.098.40 \n ( m , 4 h ) , 7.91 ( d , j = 7.8 hz , 2 h ) , 7.487.60 \n ( m , 3 h ) , 7.44 ( t , j = 7.6 hz , 3 h ) , 7.37 ( t , j = 7.3 hz , 2 h ) , 5.26 ( s , 2 h ) , 3.613.70 ( m , 4 \n h ) , 2.872.99 ( m , 4 h ) .", "synthesized by general procedure b. flash \n column chromatography : 28% methanol in dichloromethane , then \n 10100% ethyl acetate in dichloromethane , isolated as a yellow \n solid in 71% yield .", "s. , 1 h ) , 8.40 \n ( d , j = 8.8 hz , 1 h ) , 8.05 ( d , j = 8.3 hz , 2 h ) , 8.01 ( d , j = 8.8 hz , 1 h ) , 7.95 \n ( d , j = 8.3 hz , 2 h ) , 7.52 ( d , j = 7.3 hz , 2 h ) , 7.48 ( br .", "s. , 1 h ) , 7.43 ( t , j = \n 7.6 hz , 2 h ) , 7.36 ( t , j = 7.3 hz , 1 h ) , 7.30 ( d , j = 8.8 hz , 1 h ) , 7.17 ( d , j = 8.8 hz , \n 1 h ) , 5.25 ( s , 2 h ) , 3.773.82 ( m , 4 h ) , 3.073.12 ( m , \n 4 h ) .", "synthesized by general procedure a. flash \n column chromatography : 510% methanol in dichloromethane , then \n 1030% methanol in ethyl acetate , isolated as a yellow solid \n in 15% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.81 ( s , 1 h ) , 9.01 ( s , 1 h ) , 8.82 ( s , 1 h ) , 8.218.34 \n ( m , 4 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.61 ( d , j = 2.0 hz , 1 h ) , 7.437.53 ( m , 2 h ) , 7.297.40 \n ( m , 3 h ) , 7.20 ( t , j = 8.8 hz , 1 h ) , 5.29 ( s , 2 h ) , \n 2.95 ( br .", "synthesized by general procedure b. flash \n column chromatography : 510% methanol in dichloromethane , isolated \n as a yellow solid in 50% yield .", "s. , \n 1 h ) , 8.39 ( d , j = 8.8 hz , 1 h ) , 7.938.04 \n ( m , 5 h ) , 7.48 ( br . s. , 1 h ) , 7.42 ( td , j = 8.1 , \n 5.9 hz , 1 h ) , 7.31 ( d , j = 7.8 hz , 2 h ) , 7.26 ( d , j = 9.8 hz , 1 h ) , 7.16 ( d , j = 8.8 hz , \n 1 h ) , 7.06 ( td , j = 8.5 , 2.0 hz , 1 h ) , 5.25 ( s , 2 \n h ) , 3.483.52 ( m , 2 h ) , 3.46 ( t , j = 6.3 hz , \n 2 h ) , 2.732.77 ( m , 2 h ) , 2.692.73 ( m , 2 h ) , 2.40 ( s , \n 3 h ) , 1.95 ( dt , j = 11.4 , 5.8 hz , 2 h ) . lcms found \n 618.1 [ m + h ] . synthesized by general procedure b. flash \n column chromatography : 210% methanol in dichloromethane , isolated \n as a yellow solid in 61% yield", "s. , 1 h ) , 8.62 ( s , 1 \n h ) , 8.31 ( br .", "s. , 1 h ) , 8.14 ( d , j = 8.8 hz , 1 h ) , \n 8.02 ( d , j = 8.3 hz , 2 h ) , 7.94 ( d , j = 8.8 hz , 2 h ) , 7.48 ( br .", "s. , 1 h ) , 7.387.45 ( m , 2 h ) , 7.237.33 \n ( m , 3 h ) , 7.16 ( d , j = 8.8 hz , 1 h ) , 7.06 ( td , j = 8.4 , 2.2 hz , 1 h ) , 5.25 ( s , 2 h ) , 3.473.50 ( m , \n 2 h ) , 3.45 ( t , j = 6.6 hz , 2 h ) , 2.702.74 \n ( m , 2 h ) , 2.662.70 ( m , 2 h ) , 2.38 ( s , 3 h ) , 1.891.96 \n ( m , 2 h ) .", "synthesized by general procedure b. flash \n column chromatography : 515% methanol in dichloromethane , isolated \n as a yellow solid in 52% yield .", "s. , \n 1 h ) , 8.39 ( d , j = 8.8 hz , 1 h ) , 7.938.04 \n ( m , 5 h ) , 7.48 ( br . s. , 1 h ) , 7.42 ( td , j = 8.1 , \n 5.9 hz , 1 h ) , 7.31 ( d , j = 7.8 hz , 2 h ) , 7.26 ( d , j = 9.8 hz , 1 h ) , 7.16 ( d , j = 8.8 hz , \n 1 h ) , 7.06 ( td , j = 8.5 , 2.0 hz , 1 h ) , 5.25 ( s , 2 \n h ) , 3.483.52 ( m , 2 h ) , 3.46 ( t , j = 6.3 hz , \n 2 h ) , 2.732.77 ( m , 2 h ) , 2.692.73 ( m , 2 h ) , 2.40 ( s , \n 3 h ) , 1.95 ( dt , j = 11.4 , 5.8 hz , 2 h ) . lcms found \n 632.1 [ m + h ] .", "synthesized by general procedure c. flash \n column chromatography : 4080% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a yellow \n solid in 9% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.27 ( s , 1 h ) , 9.14 ( s , 1 h ) , 8.78 ( s , 1 h ) , \n 8.27 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 8.16 ( d , j = 2.4 hz , 1 h ) , 8.09 ( d , j = 8.3 hz , \n 1 h ) , 7.82 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.407.50 \n ( m , 2 h ) , 7.39 ( s , 1 h ) , 7.307.36 ( m , 3 h ) , 7.27 ( d , j = 8.8 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 \n hz , 1 h ) , 7.07 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 5.25 ( s , \n 2 h ) , 3.763.82 ( m , 4 h ) , 3.223.27 ( m , 4 h ) .", "synthesized by general procedure c. flash \n column chromatography : 4080% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a pale \n yellow solid in 14% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.24 ( s , 1 h ) , 9.16 ( s , 1 h ) , 8.62 \n ( d , j = 8.3 hz , 1 h ) , 8.308.36 ( m , 2 h ) , \n 8.21 ( d , j = 2.4 hz , 1 h ) , 7.84 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.47 ( td , j = 7.8 , 5.9 hz , \n 1 h ) , 7.387.43 ( m , 2 h ) , 7.297.36 ( m , 3 h ) , 7.26 ( d , j = 9.3 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 \n hz , 1 h ) , 7.06 ( dd , j = 8.3 , 2.0 hz , 1 h ) , 5.25 ( s , \n 2 h ) , 3.753.83 ( m , 4 h ) , 3.213.28 ( m , 4 h ) .", "synthesized by general procedure c. flash \n column chromatography : 50100% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a yellow \n solid in 9% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.36 ( s , 1 h ) , 9.18 ( s , 1 h ) , 8.93 ( s , 1 h ) , \n 8.36 ( dd , j = 8.3 , 1.5 hz , 1 h ) , 8.20 ( d , j = 8.3 hz , 2 h ) , 8.17 ( d , j = 8.3 hz , \n 1 h ) , 8.15 ( d , j = 2.9 hz , 1 h ) , 7.94 ( d , j = 8.3 hz , 2 h ) , 7.82 ( dd , j = 9.0 , 2.7 \n hz , 1 h ) , 7.50 ( d , j = 7.3 hz , 2 h ) , 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , \n 1 h ) , 7.30 ( d , j = 9.3 hz , 1 h ) , 5.22 ( s , 2 h ) , 3.633.69 \n ( m , 4 h ) , 2.902.98 ( m , 4 h ) .", "synthesized by general procedure c. flash \n column chromatography : 5100% ethyl acetate in hexanes , then \n prep hplc 3050% acetonitrile in water , isolated as a yellow \n solid in 23% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.28 ( s , 1 h ) , 9.19 ( s , 1 h ) , 8.70 ( d , j = 8.8 hz , 1 h ) , 8.45 ( d , j = 2.0 hz , \n 1 h ) , 8.41 ( dd , j = 8.5 , 1.7 hz , 1 h ) , 8.20 ( d , j = 2.4 hz , 1 h ) , 8.18 ( d , j = 8.3 hz , \n 2 h ) , 7.91 ( d , j = 8.3 hz , 2 h ) , 7.83 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.50 ( d , j = 6.8 \n hz , 2 h ) , 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 ( t , j = 6.8 hz , 1 h ) , 7.27 ( d , j = 8.8 hz , \n 1 h ) , 5.21 ( s , 2 h ) , 3.633.69 ( m , 4 h ) , 2.912.97 ( m , \n 4 h ) .", "synthesized by general procedure \n b. flash column chromatography : 37% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as an \n orange solid in 39% yield .", "s. , 1 h ) , 9.18 ( s , 1 h ) , \n 8.91 ( s , 1 h ) , 8.35 ( d , j = 7.3 hz , 1 h ) , 8.128.21 \n ( m , 6 h ) , 7.93 ( d , j = 8.3 hz , 2 h ) , 7.83 ( d , j = 6.8 hz , 1 h ) , 7.47 ( td , j = 8.3 , 6.3 \n hz , 1 h ) , 7.307.36 ( m , 2 h ) , 7.28 ( d , j = \n 8.8 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 hz , 1 h ) , 5.25 \n ( s , 2 h ) , 2.96 ( br . s. , 4 h ) , 2.352.43 ( m , 4 h ) , 2.15 ( s , \n 3 h ) .", "synthesized by general procedure \n b. flash column chromatography : 310% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as a dull \n yellow solid in 28% yield .", "h nmr ( 500 mhz , dmso - d6 )  9.28 ( s , 1 h ) , 9.20 ( s , 1 h ) , 8.70 \n ( d , j = 8.3 hz , 1 h ) , 8.47 ( d , j = 2.0 hz , 1 h ) , 8.42 ( dd , j = 8.5 , 1.7 hz , 1 h ) , \n 8.21 ( d , j = 2.9 hz , 1 h ) , 8.168.20 ( m , 3 \n h ) , 7.91 ( d , j = 8.3 hz , 2 h ) , 7.84 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.48 ( td , j = 8.3 , \n 6.3 hz , 1 h ) , 7.307.36 ( m , 2 h ) , 7.27 ( d , j = 9.3 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.7 hz , 1 h ) , \n 5.25 ( s , 2 h ) , 2.96 ( br . s. , 4 h ) , 2.39 ( t , j = 4.6 \n hz , 4 h ) , 2.15 ( s , 3 h ) .", "synthesized by general procedure \n b. flash column chromatography : 510% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as an \n orange solid in 29% yield .", "h nmr ( 500 mhz , meoh - d4 )  8.94 ( s , 1 h ) , 8.73 ( s , 1 h ) , 8.25 \n ( s , 2 h ) , 8.20 ( dd , j = 8.5 , 1.2 hz , 1 h ) , 8.008.06 \n ( m , 3 h ) , 7.95 ( d , j = 8.3 hz , 2 h ) , 7.88 ( d , j = 2.4 hz , 1 h ) , 7.59 ( dd , j = 8.8 , 2.4 \n hz , 1 h ) , 7.38 ( td , j = 8.1 , 5.9 hz , 1 h ) , 7.28 ( d , j = 7.8 hz , 1 h ) , 7.23 ( d , j = 9.8 hz , \n 1 h ) , 6.997.06 ( m , 2 h ) , 5.18 ( s , 2 h ) , 3.603.66 ( m , \n 2 h ) , 3.47 ( t , j = 6.6 hz , 2 h ) , 3.223.28 \n ( m , 4 h ) , 2.77 ( s , 3 h ) , 2.142.21 ( m , 2 h ) .", "synthesized by general procedure \n b. flash column chromatography : 515% methanol in dichloromethane , \n then prep hplc 595% acetonitrile in water , isolated as a light \n yellow solid in 29% yield .", "h nmr ( 500 mhz , meoh - d4 )  8.99 ( s , 1 h ) , 8.51 ( d , j = 8.3 hz , 1 h ) , 8.25 ( s , 1 h ) , 8.178.22 ( m , 2 h ) , 7.97 ( s , \n 4 h ) , 7.91 ( d , j = 2.9 hz , 1 h ) , 7.587.63 \n ( m , 1 h ) , 7.39 ( td , j = 7.8 , 5.9 hz , 1 h ) , 7.29 ( d , j = 7.8 hz , 1 h ) , 7.24 ( d , j = 9.3 hz , \n 1 h ) , 7.007.08 ( m , 2 h ) , 5.20 ( s , 2 h ) , 3.543.59 ( m , \n 2 h ) , 3.47 ( t , j = 6.6 hz , 2 h ) , 2.973.04 \n ( m , 4 h ) , 2.60 ( s , 3 h ) , 2.06 ( quin , j = 6.3 hz , \n 2 h ) .", "synthesized by general procedure d. flash \n column chromatography : 030% methanol in dichloromethane , isolated \n as a yellow solid in 20% yield .", "h nmr ( 400 mhz , dmso - d6 )  9.91 ( br , s , 1h ) , 8.73 ( s , 1h ) , 8.55 \n ( s , 1h ) , 8.18 ( d , j = 8.8 hz , 1h ) , 7.98 ( d , j = 8.8 hz , 1h ) , 7.55 ( d , j = 2.4 hz , 1h ) , \n 7.457.50 ( m , 1h ) , 7.377.41 ( m , 1h ) , 7.307.35 \n ( m , 6h ) , 7.177.22 ( m , 1h ) , 7.02 ( d , j = 8.0 \n hz , 1h ) , 5.29 ( s , 2h ) , 3.763.79 ( m , 4h ) , 3.203.23 \n ( m , 4h ) . lcms found 565.0 [ m + h ] . synthesized by general procedure d. flash \n column chromatography : 020% ethyl acetate in hexanes , isolated \n as a yellow solid in 50% yield .", "h nmr ( 400 mhz , dmso - d6 )  9.99 ( s , 1h ) , 8.87 ( s , 1h ) , 8.55 \n ( s , 1h ) , 8.24 ( d , j = 8.8 hz , 1h ) , 8.14 ( d , j = 8.0 hz , 2h ) , 8.01 ( d , j = 8.8 , 1h ) , \n 7.85 ( d , j = 8.4 hz , 2h ) , 7.467.51 ( m , 3h ) , \n 7.39 ( t , j = 7.2 hz , 2h ) , 7.297.34 ( m , 3h ) , \n 5.22 ( s , 2h ) , 3.603.63 ( m , 4h ) , 2.872.89 ( m , 4h ) . \n lcms found 611.1 [ m + h ] . synthesized by general procedure d. flash \n column chromatography : 030% methanol in dichloromethane , isolated \n as a yellow solid in 16% yield .", "h nmr ( 400 mhz , dmso - d6 )  10.01 ( br , s , 1h ) , 8.89 ( s , 1h ) , \n 8.59 ( s , 1h ) , 8.27 ( d , j = 8.8 hz , 1h ) , 8.16 ( d , j = 8.0 hz , 2h ) , 8.05 ( d , j = 8.8 hz , 1h ) , \n 7.88 ( d , j = 8.0 hz , 2h ) , 7.55 ( br , s , 1h ) , 7.457.70 \n ( m , 1h ) , 7.317.35 ( m 4h ) , 7.177.21 ( m , 1h ) , 5.30 ( s , \n 2h ) , 2.95 ( br , s , 4h ) , 2.38 ( br , 4h ) , 2.14 ( s , 3h ) .", "synthesized by general procedure d. flash \n column chromatography : 030% methanol in dichloromethane , isolated \n as a yellow solid in 32% yield .", "h nmr ( 400 mhz , cdcl3 )  8.71 ( s , 1h ) , 8.14 ( d , j = 8.8 \n hz , 1h ) , 8.08 ( d , j = 2.4 hz , 1h ) , 7.99 ( dd , j = 8.8 , 2.2 hz , 1h ) , 7.807.82 ( m , 2h ) , 7.66 ( d , j = 8.8 hz , 2h ) , 7.47 ( s , 1h ) , 7.41 ( d , j = 2 hz , 1h ) , 7.367.39 ( m , 1h ) , 7.23 ( s , 1h ) , 7.197.20 \n ( m , 1h ) , 7.17 ( d , j = 2 hz , 1h ) , 7.05 ( dd , j = 8.0,2.4 hz , 1h ) , 7.01 ( d , j = 8.8 hz , \n 1h ) , 5.19 ( s , 2h ) , 3.363.40 ( m , 4h ) , 2.562.62 ( m , \n 4h ) , 2.33 ( s , 3h ) , 1.811.85 ( m , 2h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 12% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n ppm 9.68 ( s , 1 h ) , 8.59 ( s , 1 h ) , 8.01 ( d , j = 2.4 \n hz , 1 h ) , 7.94 ( s , 1 h ) , 7.67 ( dd , j = 8.8 , 2.4 hz , \n 1 h ) , 7.47 ( m , 1 h ) , 7.40 ( m , 1 h ) , 7.37 ( d , j = \n 7.8 hz , 1 h ) , 7.32 ( m , 2 h ) , 7.25 ( m , 2 h ) , 7.18 ( td , j = 9.3 , 2.4 hz , 1 h ) , 7.08 ( dd , j = 7.8 , 2.4 hz , \n 1 h ) , 5.25 ( s , 2 h ) , 3.78 ( m , 4 h ) , 3.23 ( m , 4 h ) , lcms found 547.0 , \n [ m + h ] .", "h nmr ( 500 mhz , dmso - d6 )  9.64 ( s , 1h ) , 8.49 ( s , 1h ) , 8.18 \n ( s , 1h ) , 8.05 ( d , j = 2.93 hz , 1h ) , 7.71 ( dd , j = 2.44 , 8.79 hz , 1h ) , 7.447.50 ( m , 1h ) , 7.137.41 \n ( m , 7h ) , 7.04 ( dd , j = 1.95 , 8.30 hz , 1h ) , 5.25 ( s , \n 2h ) , 3.78 ( t , j = 4.7 hz , 4h ) , 3.21 ( t , j = 4.7 hz , 4h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 8% yield .", "h nmr ( 400 mhz , dmso - d6 )  \n ppm 8.61 ( s , 1 h ) , 9.83 ( s , 1 h ) , 8.15 ( m , 3 h ) , 7.98 ( s , 1 h ) , 7.88 \n ( d , j = 8.8 hz , 2 h ) , 7.65 ( m , 1 h ) , 7.49 ( m , 2 h ) , \n 7.42 ( t , j = 7.3 hz , 2 h ) , 7.35 ( m , 1 h ) , 7.27 ( d , j = 8.8 hz , 1 h ) , 5.23 ( s , 2 h ) , 3.65 ( m , 4 h ) , 2.94 ( m , \n 4 h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 40% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n ppm 9.79 ( s , 1 h ) , 8.52 ( s , 1 h ) , 8.41 ( s , 1 h ) , 8.03 ( d , j = 2.0 hz , 1 h ) , 7.97 ( d , j = 8.3 hz , \n 2 h ) , 7.88 ( d , j = 8.3 hz , 2 h ) , 7.68 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.49 ( d , j = 7.8 \n hz , 2 h ) , 7.42 ( t , j = 7.6 hz , 2 h ) , 7.35 ( t , j = 7.3 hz , 1 h ) , 7.29 ( d , j = 9.3 hz , \n 1 h ) , 5.22 ( s , 2 h ) , 3.65 ( m , 4 h ) , 2.94 ( m , 4 h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 36% yield .", "h nmr ( 400 mhz , dmso - d6 )  \n ppm 9.84 ( s , 1 h ) , 8.62 ( s , 1 h ) , 8.14 ( m , 3 h ) , 7.99 ( d , j = 2.2 hz , 1 h ) , 7.87 ( d , j = 8.8 hz , \n 2 h ) , 7.66 ( dd , j = 9.2 , 2.6 hz , 1 h ) , 7.47 ( m , 1 \n h ) , 7.32 ( m , 2 h ) , 7.26 ( d , j = 8.8 hz , 1 h ) , 7.19 \n ( td , j = 8.8 , 2.2 hz , 1 h ) , 5.26 ( s , 2 h ) , 2.95 ( m , \n 4 h ) , 2.38 ( m , 4 h ) , 2.14 ( s , 3 h ) .", "synthesized \n by general procedure e , isolated as a yellow solid in 25% yield .", "h nmr ( 500 mhz , dmso - d6 )  \n ppm 9.79 ( s , 1 h ) , 8.52 ( s , 1 h ) , 8.41 ( s , 1 h ) , 8.05 ( d , j = 2.4 hz , 1 h ) , 7.95 ( d , j = 8.3 hz , \n 2 h ) , 7.87 ( d , j = 8.3 hz , 2 h ) , 7.69 ( dd , j = 8.8 , 2.4 hz , 1 h ) , 7.47 ( m , 1 h ) , 7.32 ( m , 2 h ) , 7.28 \n ( d , j = 8.8 hz , 1 h ) , 7.18 ( td , j = 8.5 , 2.4 hz , 1 h ) , 5.24 ( s , 2 h ) , 2.95 ( m , 4 h ) , 2.37 ( m , 4 h ) , \n 2.14 ( s , 3 h ) .", "synthesized by general procedure e , isolated as a pale yellow solid \n in 4% yield .", "the product was separated by silica column chromatography \n ( dichloromethane / methanol ) and purified by hplc to obtain the formic \n acid salt .", "h nmr ( 500 mhz , dmso - d6 )  ppm 9.82 ( s , 1 h ) , 8.61 ( s , 1 h ) , 8.22 ( s , 1 h ) , \n 8.068.13 ( m , 3 h ) , 7.99 ( d , j = 2.4 hz , 1 \n h ) , 7.91 ( d , j = 8.8 hz , 2 h ) , 7.66 ( dd , j = 9.0 , 2.7 hz , 1 h ) , 7.47 ( td , j = 8.3 , \n 6.3 hz , 1 h ) , 7.297.36 ( m , 2 h ) , 7.26 ( d , j = 8.8 hz , 1 h ) , 7.18 ( td , j = 8.7 , 2.2 hz , 1 h ) , \n 5.26 ( s , 2 h ) , 3.35 ( dt , j = 5.1 , 2.3 hz , 2 h ) , 3.32 \n ( t , j = 6.1 hz , 2 h ) , 2.532.58 ( m , 2 h ) , \n 2.48 ( d , j = 5.9 hz , 2 h ) , 2.23 ( s , 3 h ) , 1.74 ( quin , j = 5.9 hz , 2 h ) .", "synthesized by general procedure e , isolated as a pale yellow solid \n in 10% yield .", "the product was separated by silica column chromatography \n ( dichloromethane / methanol ) and purified by hplc to obtain the formic \n acid salt .", "h nmr ( 500 mhz , cdcl3/cd3od )  ppm 8.46 ( s , 1 h ) , 8.10 ( s , 1 h ) , 7.89 ( m , 5 h ) , 7.60 \n ( m , 1 h ) , 7.39 ( m , 1 h ) , 7.28 ( d , j = 7.8 hz , 1 h ) , \n 7.24 ( d , j = 9.3 hz , 1 h ) , 7.04 ( m , 2 h ) , 5.20 ( s , \n 2 h ) , 3.57 ( m , 2 h ) , 3.45 ( t , j = 6.6 hz , 2 h ) , 3.05 \n ( m , 4 h ) , 2.63 ( s , 3 h ) , 2.08 ( m , 2 h ) ."]]}
{"article_id": "PMC4430798", "article_text": ["\n acute appendicitis is considered as the most frequent cause of acute abdomen in young patients with a slight predominance in males .", "although its clinical diagnosis is simple , 20 - 30% of appendicitis cases are reported to be gangrenous or perforated because of delayed diagnosis , which can lead to increased morbidity and mortality . \n \n", "additionally , up to 10% of patients especially older ones , immunocompromised patients , and pregnant women are diagnosed with atypical presentations such as appendiceal mass which requires long hospital admissions , intravenous antibiotics , and often needs percutaneous drainage . \n \n", "using colonoscopy for the diagnosis and treatment of asymptomatic acute appendicitis and appendiceal abscess are rare . in unusual appendicitis cases , especially in the presence of abdominal comorbidities , colonoscopy is used ; although colonoscopy is not considered as the first line intervention because of complications risk such as bleeding or perforation . furthermore non - invasive tests such as ultrasonography and computed tomography with more sensitivity can be used .", "we report an atypical case of appendiceal abscess , which was diagnosed during colonoscopy . \n", "\n a 30-year - old man referred for evaluation to our center with epigastric pain from three days prior to his evaluation .", "the pain was nonspecific and colicky distributed in periumblical region , did not radiate to any site and was unrelated to bowel movements .", "he had experienced also nausea , anorexia and intermittent vomiting . \n \n there was a history of marijuana and alcohol usage from several years ago . upon physical examination , he was ill with restlessness .", "other laboratory values such as hemoglobin , platelets , serum amylase , erythrocyte sedimentation rate , aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , and electrolytes were normal . \n \n his workup on admission including ; chest x - ray , abdominal x - ray , and abdominal ultrasonography were reported normal .", "an upper gastrointestinal endoscopy was performed after four days of admission that was normal . \n \n within second week after admission ,", "the appendiceal orifice was discovered in the floor of cecum which was draining pus into the lumen ( figure 1 ) . \n ", "purulent discharge from the appendiceal orifice before ( up ) and after ( down ) pressing by forceps ( arrow ) \n result of the biopsies of the mucosa at the base of the appendix showed mild nonspecific inflammation with some necrotic materials and staphylococcus organism .", "the biopsies of the mucosa at terminal ileum , left and right colon demonstrated mild nonspecific inflammation . \n \n", "a second abdominal ultrasonography was performed that showed a fluid collection ( about 70 40 mm ) in favor of appendiceal abscess in right lower quadrant ( figure 2 ) .", "the patient was scheduled for spiral abdomen - pelvic computed tomographic scan which showed a hypodense well defined collection with air fluid level ( about 70 40 30 mm ) in right lower quadrant .", "the collection was extended to pelvic cavity associated with inflammation of the surrounding fat in favor of appendiceal abscess ( figure 3 ) . \n ", "ultrasonography showing a fluid collection in favor of appendiceal abscess   computed tomographic scan showing a hypodense well defined collection with air fluid level associated with inflammation of surrounding fat in favor of appendiceal abscess ( arrow ) . \n", "one week after antibiotic therapy a third abdominal ultrasonography was performed that showed a smaller fluid collection ( about 40 15 mm ) around the appendix .", "three weeks after antibiotic therapy the patient was asymptomatic with normal white blood cell count .", "on the 5th week after antibiotic therapy , exploratory laparotomy did not show any mass or collection in the appendiceal region and elective appendectomy was performed .", "after 3 months of follow - up the patient was well without any complaint or physical finding . \n", "\n acute appendicitis is one of the most frequent causes of acute abdomen . despite the use of scores in the radiological evaluation , and laparoscopic advances ,", "in a cohort study of patients with appendiceal abscess or phlegmon due to acute appendicitis concluded that initial conservative treatment should be considered . \n \n", "colonoscopy is reported as a diagnostic and therapeutic approach for delayed or atypical presentations of acute appendicitis .", "our patient is one of these unusual cases among whom complicated acute appendicitis was incidentally diagnosed by colonoscopy . \n \n", "although the appendiceal orifice is routinely examined during colonoscopy , however there are only few published reports describing acute appendicitis diagnosis in the appendiceal orifice . \n \n in a review article ,", "khawaja fi recommended that colonoscopy should only be done in selected patients with atypical appendicitis or uncertain diagnosis of appendiceal perforation . \n \n", "benatta ma reported an atypical case of pericecal appendiceal abscess diagnosed and treated at colonoscopy , while initial presentation was without any suggestive symptoms of appendicitis . \n \n", "wade ad et al reported an atypical case of appendicitis in a patient with ulcerative colitis diagnosed during colonoscopy . in this report", "christoffersen mmw et al reported an atypical case of periappendicular abscess diagnosed and treated at colonoscopy , while the patient was admitted for colonoscopy polyp control without any suggestive symptoms . \n \n", "kawahara y et al reported a case of appendiceal abscess with draining pus from appendiceal into the lumen diagnosed at colonoscopy and ct scan . in this report because of spontaneous drainage of abscess into the lumen , the appendiceal abscess had resolved by the time of surgery . \n \n", "kapral c et al . reported a case of pericecal abscess that treated by drainage at colonoscopy and antibiotic therapy .", "they intubated the appendix by sphincteroltomy catheter which was placed into the abscess cavity . \n \n", "petro m reported a case of the earliest stage of acute appendicitis in an asymptomatic patient undergoing colonoscopy for evaluation of colorectal cancer . \n \n", "for evaluation of the role of colonoscopy in the diagnosis of atypical appendicitis , a retrospective study in 21 patients performed by chang hs et al .", "some of acute appendicitis cases are reported to be complicated , due to delayed diagnosis .", "this unusual case highlights a patient with an appendiceal abscess diagnosed by colonoscopy . because of spontaneous drainage of abscess into the lumen of colon and antibiotic therapy ,", "although the diagnostic and therapeutic role of colonoscopy in these patients is uncertain , however , it s use can be selected in special situations in the future . \n"], "abstract_text": ["<S> although complicated acute appendicitis is occasionally observed in general practice , the diagnosis of appendiceal abscess by colonoscopy is unusual . </S>", "<S> we report a case of appendiceal abscess with spontaneous drainage into the lumen of colon diagnosed during colonoscopy and resolved by the time of surgery . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONFLICT OF INTEREST"], "sections": [["\n acute appendicitis is considered as the most frequent cause of acute abdomen in young patients with a slight predominance in males .", "although its clinical diagnosis is simple , 20 - 30% of appendicitis cases are reported to be gangrenous or perforated because of delayed diagnosis , which can lead to increased morbidity and mortality . \n \n", "additionally , up to 10% of patients especially older ones , immunocompromised patients , and pregnant women are diagnosed with atypical presentations such as appendiceal mass which requires long hospital admissions , intravenous antibiotics , and often needs percutaneous drainage . \n \n", "using colonoscopy for the diagnosis and treatment of asymptomatic acute appendicitis and appendiceal abscess are rare . in unusual appendicitis cases , especially in the presence of abdominal comorbidities , colonoscopy is used ; although colonoscopy is not considered as the first line intervention because of complications risk such as bleeding or perforation . furthermore non - invasive tests such as ultrasonography and computed tomography with more sensitivity can be used .", "we report an atypical case of appendiceal abscess , which was diagnosed during colonoscopy . \n"], ["\n a 30-year - old man referred for evaluation to our center with epigastric pain from three days prior to his evaluation .", "the pain was nonspecific and colicky distributed in periumblical region , did not radiate to any site and was unrelated to bowel movements .", "he had experienced also nausea , anorexia and intermittent vomiting . \n \n there was a history of marijuana and alcohol usage from several years ago . upon physical examination , he was ill with restlessness .", "other laboratory values such as hemoglobin , platelets , serum amylase , erythrocyte sedimentation rate , aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , and electrolytes were normal . \n \n his workup on admission including ; chest x - ray , abdominal x - ray , and abdominal ultrasonography were reported normal .", "an upper gastrointestinal endoscopy was performed after four days of admission that was normal . \n \n within second week after admission ,", "the appendiceal orifice was discovered in the floor of cecum which was draining pus into the lumen ( figure 1 ) . \n ", "purulent discharge from the appendiceal orifice before ( up ) and after ( down ) pressing by forceps ( arrow ) \n result of the biopsies of the mucosa at the base of the appendix showed mild nonspecific inflammation with some necrotic materials and staphylococcus organism .", "the biopsies of the mucosa at terminal ileum , left and right colon demonstrated mild nonspecific inflammation . \n \n", "a second abdominal ultrasonography was performed that showed a fluid collection ( about 70 40 mm ) in favor of appendiceal abscess in right lower quadrant ( figure 2 ) .", "the patient was scheduled for spiral abdomen - pelvic computed tomographic scan which showed a hypodense well defined collection with air fluid level ( about 70 40 30 mm ) in right lower quadrant .", "the collection was extended to pelvic cavity associated with inflammation of the surrounding fat in favor of appendiceal abscess ( figure 3 ) . \n ", "ultrasonography showing a fluid collection in favor of appendiceal abscess   computed tomographic scan showing a hypodense well defined collection with air fluid level associated with inflammation of surrounding fat in favor of appendiceal abscess ( arrow ) . \n", "one week after antibiotic therapy a third abdominal ultrasonography was performed that showed a smaller fluid collection ( about 40 15 mm ) around the appendix .", "three weeks after antibiotic therapy the patient was asymptomatic with normal white blood cell count .", "on the 5th week after antibiotic therapy , exploratory laparotomy did not show any mass or collection in the appendiceal region and elective appendectomy was performed .", "after 3 months of follow - up the patient was well without any complaint or physical finding . \n"], ["\n acute appendicitis is one of the most frequent causes of acute abdomen . despite the use of scores in the radiological evaluation , and laparoscopic advances ,", "in a cohort study of patients with appendiceal abscess or phlegmon due to acute appendicitis concluded that initial conservative treatment should be considered . \n \n", "colonoscopy is reported as a diagnostic and therapeutic approach for delayed or atypical presentations of acute appendicitis .", "our patient is one of these unusual cases among whom complicated acute appendicitis was incidentally diagnosed by colonoscopy . \n \n", "although the appendiceal orifice is routinely examined during colonoscopy , however there are only few published reports describing acute appendicitis diagnosis in the appendiceal orifice . \n \n in a review article ,", "khawaja fi recommended that colonoscopy should only be done in selected patients with atypical appendicitis or uncertain diagnosis of appendiceal perforation . \n \n", "benatta ma reported an atypical case of pericecal appendiceal abscess diagnosed and treated at colonoscopy , while initial presentation was without any suggestive symptoms of appendicitis . \n \n", "wade ad et al reported an atypical case of appendicitis in a patient with ulcerative colitis diagnosed during colonoscopy . in this report", "christoffersen mmw et al reported an atypical case of periappendicular abscess diagnosed and treated at colonoscopy , while the patient was admitted for colonoscopy polyp control without any suggestive symptoms . \n \n", "kawahara y et al reported a case of appendiceal abscess with draining pus from appendiceal into the lumen diagnosed at colonoscopy and ct scan . in this report because of spontaneous drainage of abscess into the lumen , the appendiceal abscess had resolved by the time of surgery . \n \n", "kapral c et al . reported a case of pericecal abscess that treated by drainage at colonoscopy and antibiotic therapy .", "they intubated the appendix by sphincteroltomy catheter which was placed into the abscess cavity . \n \n", "petro m reported a case of the earliest stage of acute appendicitis in an asymptomatic patient undergoing colonoscopy for evaluation of colorectal cancer . \n \n", "for evaluation of the role of colonoscopy in the diagnosis of atypical appendicitis , a retrospective study in 21 patients performed by chang hs et al .", "some of acute appendicitis cases are reported to be complicated , due to delayed diagnosis .", "this unusual case highlights a patient with an appendiceal abscess diagnosed by colonoscopy . because of spontaneous drainage of abscess into the lumen of colon and antibiotic therapy ,", "although the diagnostic and therapeutic role of colonoscopy in these patients is uncertain , however , it s use can be selected in special situations in the future . \n"], [""]]}
{"article_id": "PMC3133851", "article_text": ["they possess binding sites specific to carbohydrates and have the capacity to interact with molecules of biological fluids and those present in cellular surfaces .", "lectins can substitute natural linkers and activate cellular responses through different roads of intracellular signalling or endocytosis of formed compounds [ 3 , 4 ] .", "camaratu or camaratuba bean ( cratylia mollis ) is forage of the northeast semi - arid region from brazil .", "molecular forms of c. mollis seed lectin ( cramoll 1 , cramoll 2 , cramoll 3 , and cramoll 4 ) have been highly purified and characterized ( de souza et al . ,", "cramoll 1 , cramoll 2 , and cramoll 4 specifically bind glucose / mannose , while cramoll 3 is a galactose - specific glycoprotein", ". preparations containing isoforms ( cramoll 1,4 ) and isolectins ( cramoll 1,2,3 ) , as well as cramoll 1 , have been studied in structural analyses and for the most several biotechnological applications ( de souza et al . , 2003 ) [ 612 ] .   the use of nuclear technique for the evaluation of biomolecules and medicines although old is not widely used even nowadays . since the discovery of the element technetium , in 1947", "scintigraphy imaging provides a dimensional method for exactly locating gamma emitters in a noninvasive procedure under in vivo conditions . for the characterization of drugs , and biomolecules , molecular imaging techniques are extremely helpful to follow biodistribution in experimental animal studies . in this study", ", the isolectins cramol 1,4 were radiolabeled with tc-99 m for the evaluation of the biodistribution and in order to forecast a possible use as radiopharmaceutical in healing process .", "the labeling processes were made using 150  l of cramol ( isoform 1,4 ) solution incubated with stannous chloride ( sncl2 ) solutions ( 80  l / ml ) ( sigma - aldrich ) for 20 minutes at room temperature .", "then , this solution was incubated with 100  ci ( approximately 300  l ) of   technetium-99 m   ( ipen / cnen ) for other 10 minutes in order to label the cramol 1,4 ( here just called cramol ) with tc-99 m . in order to characterize the labeled cramol , thin - layer chromatography ( tlc ) was made using   paper whatman no .", "the tlc was performed using 2  l of a labeled sample in acetone ( proquimios ) as a mobile phase .", "the radioactivity of the strips was verified in a gamma counter ( packard , cobra ii ) as described in table 1 .", "the institutional review board and the animal ethics committee approved the study protocol for this study . the labeled samples ( 3,7  mbq/0,2  ml )", "planar images were obtained 1 hour after injection at a millennium gamma camera ( ge healthcare , cleveland , usa ) .", "counts were acquired for 5  min in a 15% window centered at 140  kev ( figure 1 ) .", "then , animals were sacrificed , their were organs removed and weighted , and the radioactivity uptake was counted in a gamma counter ( packard - cobra ii ) .", "results were expressed as the percentage of injected dose per organ and percent of injected dose per gram of tissue ( table 2 , figures 2 and 3 ) .", ", the results showed that the acetone can be used for this purpose . in a triplicate test ,", "the free pertechnetate was eluted to the top of the chromatograms , and the cramol stayed in the bottom .", "moreover , the results observed in the chromatogram elucidated the question about the efficacy of the labeling process , corroborating that the cramol was efficiently labeled with tc-99 m .", "tc - cramol scintigraphy 1  h after injection shows liver , kidneys , and bladder .", "as we can see , the great part of the lectin was processed by the liver .", "this is absolutely normal especially for injectable drugs , since almost all the drugs are first processed by the hepatic system ( figure 2 ) . otherwise , when dissecting , weighting and counting   the organs , a suspicious data appeared .  ", "as a protein , we were expecting that its clearance would be made by the kidney , and in fact , it occurred .", "however , the rate between the bowel and the kidney is over the double . in this case , the clearance time would be reduced and the reabsorption of the drug could be facilitated by the microvilli of the bowel . in this case  ", "a dosage error could occur or even a toxic reaction due its accumulation in the bowel .", "in both cases ,   further studies should be made to clarify this mechanism   and the relevance of that information in a developed drug based on lectin similar to the cramol 1,4 .", "it is interesting to notice , analyzing both figures 1 and 2 , that the lectin can not pass the hematoencephalic barrier . and in this case , the lectin does not represent a risk for the brain function .", "the radiolabeling of the cramol showed that this lectin has an uncommon behavior specially related to the clearance mechanism that we believe must be more studied .", "it also showed that the nuclear technique can be very helpful for the development of drugs and medicines ."], "abstract_text": ["<S> the cramoll 1,4 is a well - studied lectin . </S>", "<S> however , few studies about its biodistribution have been done before . in this study , we radiolabeled   the cramol 1,4 with tc-99 m and analyzed the biodistribution . </S>", "<S> the results showed that the cramol has an abnormal uptake by the bowel with reflections on its clearance mechanism . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results and Discussion", "4. Conclusion"], "sections": [["they possess binding sites specific to carbohydrates and have the capacity to interact with molecules of biological fluids and those present in cellular surfaces .", "lectins can substitute natural linkers and activate cellular responses through different roads of intracellular signalling or endocytosis of formed compounds [ 3 , 4 ] .", "camaratu or camaratuba bean ( cratylia mollis ) is forage of the northeast semi - arid region from brazil .", "molecular forms of c. mollis seed lectin ( cramoll 1 , cramoll 2 , cramoll 3 , and cramoll 4 ) have been highly purified and characterized ( de souza et al . ,", "cramoll 1 , cramoll 2 , and cramoll 4 specifically bind glucose / mannose , while cramoll 3 is a galactose - specific glycoprotein", ". preparations containing isoforms ( cramoll 1,4 ) and isolectins ( cramoll 1,2,3 ) , as well as cramoll 1 , have been studied in structural analyses and for the most several biotechnological applications ( de souza et al . , 2003 ) [ 612 ] .   the use of nuclear technique for the evaluation of biomolecules and medicines although old is not widely used even nowadays . since the discovery of the element technetium , in 1947", "scintigraphy imaging provides a dimensional method for exactly locating gamma emitters in a noninvasive procedure under in vivo conditions . for the characterization of drugs , and biomolecules , molecular imaging techniques are extremely helpful to follow biodistribution in experimental animal studies . in this study", ", the isolectins cramol 1,4 were radiolabeled with tc-99 m for the evaluation of the biodistribution and in order to forecast a possible use as radiopharmaceutical in healing process ."], ["the labeling processes were made using 150  l of cramol ( isoform 1,4 ) solution incubated with stannous chloride ( sncl2 ) solutions ( 80  l / ml ) ( sigma - aldrich ) for 20 minutes at room temperature .", "then , this solution was incubated with 100  ci ( approximately 300  l ) of   technetium-99 m   ( ipen / cnen ) for other 10 minutes in order to label the cramol 1,4 ( here just called cramol ) with tc-99 m . in order to characterize the labeled cramol , thin - layer chromatography ( tlc ) was made using   paper whatman no .", "the tlc was performed using 2  l of a labeled sample in acetone ( proquimios ) as a mobile phase .", "the radioactivity of the strips was verified in a gamma counter ( packard , cobra ii ) as described in table 1 .", "the institutional review board and the animal ethics committee approved the study protocol for this study . the labeled samples ( 3,7  mbq/0,2  ml )", "planar images were obtained 1 hour after injection at a millennium gamma camera ( ge healthcare , cleveland , usa ) .", "counts were acquired for 5  min in a 15% window centered at 140  kev ( figure 1 ) .", "then , animals were sacrificed , their were organs removed and weighted , and the radioactivity uptake was counted in a gamma counter ( packard - cobra ii ) .", "results were expressed as the percentage of injected dose per organ and percent of injected dose per gram of tissue ( table 2 , figures 2 and 3 ) ."], [", the results showed that the acetone can be used for this purpose . in a triplicate test ,", "the free pertechnetate was eluted to the top of the chromatograms , and the cramol stayed in the bottom .", "moreover , the results observed in the chromatogram elucidated the question about the efficacy of the labeling process , corroborating that the cramol was efficiently labeled with tc-99 m .", "tc - cramol scintigraphy 1  h after injection shows liver , kidneys , and bladder .", "as we can see , the great part of the lectin was processed by the liver .", "this is absolutely normal especially for injectable drugs , since almost all the drugs are first processed by the hepatic system ( figure 2 ) . otherwise , when dissecting , weighting and counting   the organs , a suspicious data appeared .  ", "as a protein , we were expecting that its clearance would be made by the kidney , and in fact , it occurred .", "however , the rate between the bowel and the kidney is over the double . in this case , the clearance time would be reduced and the reabsorption of the drug could be facilitated by the microvilli of the bowel . in this case  ", "a dosage error could occur or even a toxic reaction due its accumulation in the bowel .", "in both cases ,   further studies should be made to clarify this mechanism   and the relevance of that information in a developed drug based on lectin similar to the cramol 1,4 .", "it is interesting to notice , analyzing both figures 1 and 2 , that the lectin can not pass the hematoencephalic barrier . and in this case , the lectin does not represent a risk for the brain function ."], ["the radiolabeling of the cramol showed that this lectin has an uncommon behavior specially related to the clearance mechanism that we believe must be more studied .", "it also showed that the nuclear technique can be very helpful for the development of drugs and medicines ."]]}
{"article_id": "PMC3913824", "article_text": ["the comingling of swine from numerous premises with varied management practices and their interaction with large numbers of exhibitors and visitors make agricultural fairs an ideal setting for the intra- and inter - species transmission of influenza a viruses ( iavs ) between swine and human populations .", "the frequency with which intra- and inter - species iav transmission occurs in these settings is likely due to a myriad of factors , including but not limited to , management practices , iav strain , and animal and/or human population immunity .", "swine is a host species in which reassortment of iav genomic segments may lead to emergent novel strains , since they are susceptible to infection from swine , human and avian influenza a viruses .", "for this reason , limiting the bidirectional zoonotic transmission of these viruses at agricultural fairs is important for public and animal health .", "the association between human and swine influenza was reported after respiratory disease similar to the human disease was noted in pigs during the 1918 human spanish flu pandemic .", "iav subsequently became established in the united states swine population with the relatedness of the swine and human viruses being established in 1931 . for nearly 80 years", ", classical swine influenza h1n1 virus was the dominant endemic iav strain in the north america swine population . in 1998 ,", "triple reassortant h3n2 iavs containing polymerase basic 1 ( pb1 ) , hemagglutinin ( ha ) and neuraminidase ( na ) gene segments from human iav lineages , polymerase basic 2 ( pb2 ) and polymerase acidic ( pa ) genes from avian lineages , and nucleoprotein ( np ) , matrix ( m ) and non - structural ( ns ) gene segments from swine lineages , emerged in north american swine .", "subsequently , this lineage became established in us and canadian swine herds and has resulted in an increased rate of genetic and antigenic change among swine - origin iavs .", "reported cases of humans contracting iav infections directly or indirectly from pigs have been historically sporadic and these variant iavs showed limited capability for sustained human - to - human transmission . however , the emergence of the influenza a(h1n1)pdm09 virus , a strain containing gene segments from north american and european swine lineages , illustrated the pandemic potential of swine lineage iavs crossing the species barrier to humans . while a(h1n1)pdm09 rapidly spread worldwide and became endemic in the human population , sequencing of this virus has to date failed to elucidate any virulence or adaptation markers that would explain its human - to - human transmission efficiency , highlighting our inability to predict iavs with pandemic potential .", "while the origin of the a(h1n1)pdm09 virus remains unknown , the virus was introduced into the north american swine population in 2009 and has since reassorted with other swine - origin iavs .", "epidemiological data show that zoonotic transmission of iav from swine to humans has been documented at unprecedented levels in recent years .", "more than 320 human cases of infection with variant iavs were reported to the centers for disease control 20112012 and likely thousands more h3n2v cases went unreported during those years .", "these zoonotic iavs contained seven genes from contemporary north american swine lineage iav and one gene ( m ) derived from the h1n1pdm09 virus .", "the majority of the cases were epidemiologically linked to swine exposure occurring at agricultural fairs across several states . within ohio , 107 h3n2v cases documented during 2012 resulted in eleven hospitalizations and one fatality .", "we recovered iav from exhibition swine at 10 of 40 ( 25% ) ohio fairs sampled during 2012 .", "genomic analyses of h3n2 iav isolates recovered from pigs at one agricultural fair in the state during 2012 demonstrated > 99% nucleotide similarity to h3n2v isolates recovered from concurrent human cases , providing molecular confirmation of zoonotic iav transmission .", "this record number of variant influenza a cases created the need for a  one health ' effort to minimize intra- and bidirectional inter - species iav transmission at swine exhibitions .", "the reason to prevent iav infections among swine at fairs is clear ; however , the paucity of scientific evidence makes it difficult for veterinary officials to make sound recommendations to protect public and animal health . in the present study", ", we investigate fair specific risk factors contributing to the emergence of influenza a virus in exhibition swine that could be altered to mitigate the risk of iav transmission in these settings .", "as part of an ongoing active iav surveillance project , swine nasal swabs and associated metadata about management practices were collected at 40 ohio fairs in 2012 .", "sample size was selected to provide a 95% probability of detecting iav infection if greater than 15% of the pigs at each fair were infected .", "all pigs sampled in this study were from junior fair market swine shows occurring at agricultural fairs .", "for the outcome of interest , a fair was considered positive if viable iav was recovered from one or more pigs at the fair .", "data collection focused on fair level variables possibly contributing to the presence or absence of iav in the pigs at each exhibition .", "junior fair shows are limited to local exhibitors approximately nine years of age through 19 years of age participating in 4-h , ffa or another youth organization , whereas open - class shows are generally open to all participants regardless of age , affiliation or residence .", "classification of swine included market swine ( pigs bred , raised and intended for food purposes ) and breeding swine ( gilts , sows and/or boars being raised for breeding purposes ) .", "terminal swine shows are those in which all participating livestock are consigned to harvest immediately following the exhibition and partial terminal shows usually require the champion animals to be harvested following the exhibition and other pigs may or may not go to harvest . to account for the variability of arrival and departure procedures among fairs enrolled in this study , the length of the swine exhibition was defined as the number of days between the required arrival deadline for the pigs and the day the pigs were sampled .", "study team members calculated the area per pig ( ft / pig ) from the recorded size of the pens and the number of pigs per pen . while on the fairgrounds , study team members also documented if there was an easily identifiable and operational hand - wash and/or hand - sanitizer station within close proximity to the swine barn(s ) .", "these sanitation stations were used by study team members to determine if they were functional .", "additional variables included the number of pigs at the fair , number of swine exhibitors , fair attendance ( number of people ) and vaccine requirements , all of which were reported to the study team by the fair organizers .", "fair officials also reported if there was a commingling event , such as a pre - fair animal identification or weighing session , during the weeks or months prior to the fair .", "exhibition directors also reported if there were other pigs besides the exhibition swine on the fairgrounds ( i.e. , petting zoo , pig races , educational displays ) .", "the commercial swine inventory was retrieved from united states department of agriculture 's 2007 census of agriculture .", "the county human population was defined as the value reported in the 2010 us census report .", "weather data were collected from the national weather service 's weather station nearest to each fairground .", "data were analyzed using stata version 11.1 ( statacorp , college station , tx , usa ) .", "univariate analysis was performed to calculate unadjusted odd ratios to identify factors contributing to the presence of iav in pigs at fairs .", "exact logistic regression was used for multivariable modeling using a forward stepwise model building approach .", "influenza a virus was recovered from pigs at 10 of the 40 fairs included in the investigation . the presence or absence of iav infection among the pigs at the fairs", "could not be associated with county population , county swine inventory and number of people attending the fair ( table 1 ) . all iav - positive fairs and 27 of 30 ( 90% ) negative fairs were mixed sex ( barrows and gilts ) market swine exhibitions .", "the average space per pig at the studied fairs was 12.8 ft / pig . properly functioning hand - wash and/or hand - sanitizer stations were available at 25 of 40 ( 62.5% ) fairs .", "average daily mean temperature was almost 4  c higher for fairs with iav - positive pigs ( table 1 ) .", "pre - fair tag - in and/or weigh - in events were rather common with 23 out of 40 ( 57.5% ) of the enrolled fairs holding one of these events .", "for every increase of 20 pigs at a fair , the odds ratio of iav infection in the pigs increased by 1.27 times ( table 2 ) .", "the results of the current study provide the first look at fair - level risk factors associated with iav infections in swine at agricultural fairs .", "while it is likely impossible to completely prevent iav transmission at swine exhibitions , these data can be used to develop and evaluate mitigation strategies to reduce the impact of intra- and inter - species iav infections at swine exhibitions .", "just like all other agriculture biosecurity programs , mitigation strategies which are practical , user - friendly , low cost and do not dramatically alter the fair experience for exhibitors and visitors are most likely to be considered , implemented and maintained .", "not surprisingly , larger pig shows appear to be more likely to have iav - infected pigs than smaller swine exhibitions ( table 2 ) .", "larger swine exhibitions tend to also have open - class and breeding swine shows in addition to junior market shows . while open - class shows were common among our studied fairs ( 16 of the 40 ) , only 5 of the 40 ( 12.5% ) fairs in this study had a breeding show ; 4 of those 5 ( 80% ) fairs had iav - infected pigs at the fair .", "the small number of fairs with breeding shows in this study makes analysis of this risk factor problematic ; however , the finding is enough to warrant concern given that breeding swine are intended to leave the exhibition and enter a herd for progeny production .", "this fair - to - farm movement of pigs is a disease introduction risk for the receiving herd and a potential method to disseminate iav strains across a larger geographic area .", "previous research has shown that environmental stressors ( heat , lack of space , noise ) on pigs can affect the course of various diseases in commercial swine operations .", "the average space per pig at the studied fairs was well above 68  ft / finishing pig common throughout the swine industry .", "the results indicate that heat stress could be a contributing factor to iav infections in exhibition swine ; however , caution must be used when interpreting this result because the vast majority of the fairs with iav - positive pigs occurred in a 4-week period during the middle of summer .", "this trend of mid - summer iav activity in ohio 's exhibition swine was also observed in the previous three years and could be more related to animal and/or people movement between these fairs than the weather .", "environmental temperature failed to meet the selection criteria for inclusion in the final multivariable model .", "while the majority of fairs had hand hygiene stations , their presence at fairs was not linked to the occurrence of iav among pigs at the respective fairs .", "the large number of h3n2v infections linked to swine exposure at agricultural fairs during 2012 suggests that hand hygiene stations also had minimal impact on zoonotic transmission of iav in these settings .", "however , hand hygiene stations are critically important in protecting human health by controlling zoonotic diseases transmitted via direct contact at these venues .", "this would limit the time for iav to spread among susceptible pigs and decrease the time humans are exposed to iav - infected pigs .", "the length of the exhibitions in the current study was similar between iav - positive and -negative fairs .", "active recruitment of fairs with more diverse management practices is needed to study the impact of a shortened swine exhibition .", "no matter the length of the exhibition time , the disposition of the pigs following the show must be considered .", "the majority of fairs in this study ( 65% ) had terminal junior market shows .", "the practice of having a terminal show was not associated with decreased odds of iav ; however , sending all the pigs to harvest at the end of each fair is expected to help protect subsequent fairs by decreasing the potential for fair - to - fair spread of iav .", "mandated vaccinations were almost non - existent with only one fair in the current study requiring the pigs be vaccinated for erysipelothrix rhusiopathiae prior to the fair ; no fairs required the pigs to receive iav vaccination before arrival .", "use of iav vaccine in exhibition swine to decrease the risk of iav infections in swine and humans has been one of the most debated topics following the h3n2v outbreak of 2012 .", "there are currently several commercially available swine influenza vaccines in the united states , all of which are universally indicated to reduce clinical signs of disease in pigs but appear to have limited efficacy against 2012 h3n2v strains . although their impact on intra- and inter - species transmission dynamics remains unclear , it is expected that iav vaccines will impart at least partial immunity to circulating strains of iav , which should decrease viral shedding during a fair .", "an unintended consequence of iav vaccine use may be vaccine - associated enhanced respiratory disease , which has been reported in swine vaccinated with swine influenza virus vaccines that are mismatched to circulating strains .", "furthermore , decrease of clinical signs may hamper recognition and response to active iav infections in exhibition pigs .", "some of the major pitfalls of mandated iav vaccination lie with the practical application of vaccines in this setting .", "the logistics of vaccine distribution to swine exhibitors prior to the fair becomes difficult because most exhibition swine are raised by youth exhibitors in small , dispersed herds ( < 10 pigs per herd ) .", "commercial vaccines are usually sold in 50 doses per bottle adding to the cost per vaccinated pig in these small herds . because youth exhibitors and their family members may not be proficient at administering vaccines", ", agriculture education advisors often volunteer to assist the youth with this task , a practice that is time - consuming and increases the risk of infectious agents being transmitted farm to farm . additionally , problematic", "is that most iav vaccines labeled for swine require a booster dose given 24 weeks later to provide optimal protection , which can be difficult for youth exhibitors and their family members to accomplish . tagging or weighing events", "are frequently used as a way for exhibitors to declare ownership of their pig(s ) prior to the fair .", "these pre - fair events could provide an opportunity for mass vaccination of pigs prior to the fair , but the application of such events can facilitate disease spread between animals . even in properly vaccinated pigs , the immunity stimulated by current iav vaccines", "the strains used for commercial swine influenza vaccines are irregularly updated and the constant genetic and antigenic change occurring in contemporary swine - origin iavs makes viral antigens unpredictable and difficult vaccine targets .", "the reason for the increase in the number of reported h3n2v cases during 20112013 remains unclear , but the strain of iav is thought to be a major contributing factor .", "the swine - origin h3n2 iav isolates recovered from these human cases contains the matrix gene from the a(h1n1)pdm09 virus , a recently emerged genomic constellation that increases replication and transmission in cell culture and animal models . epidemiological data modeling", "indicate that children are most susceptible to h3n2v , likely due to lack of strain - specific immunity . additionally , current seasonal trivalent inactivated iav vaccine provides little to no protection against h3n2v strains .", "the limited ability for human - to - human transmission of h3n2v has minimized the impact of these recent zoonotic transmission events , but the outbreak has illustrated the importance of swine exhibitions in zoonotic iav transmission .", "it is nearly impossible to predict the iav strain that will infect the swine at fairs because iav reassortment events and novel strain generation frequently occur in modern swine populations .", "agricultural fairs provide a pathway for human exposure to these ever changing viruses ; thus , blanket iav prevention , without regard for strain , is needed for swine at fairs to decrease zoonotic iav transmission and protect public health . while iav can infect pigs at any fair , the data presented here indicate that special attention should be paid to large pig shows where the likelihood of iav among the pigs is much higher .", "the results presented herein are based on one year of data from a limited geographic area of the united states .", "additional assessments of swine exhibitions in multiple states across several years are needed to provide more comprehensive evaluations of risk factors contributing to the problem .", "these data provide a critical first step toward developing effective iav mitigation strategies in swine populations that benefit fairs , exhibitors , visitors and the swine industry .", "this information will serve as a baseline for measuring the acceptance and effectiveness as mitigation strategies are developed and implemented ."], "abstract_text": ["<S> influenza a virus infections occurring in exhibition swine populations at agricultural fairs during 2012 served as a source of h3n2 variant influenza a viruses transmitted to humans resulting in more than 300 documented cases . prior to the outbreak , </S>", "<S> this investigation was initiated to identify fair - level risk factors contributing to influenza a virus infections in pigs at agricultural fairs . as part of an ongoing active surveillance program , nasal swabs and associated fair - level metadata </S>", "<S> were collected from pigs at 40 junior fair market swine shows held in ohio during the 2012 fair season . </S>", "<S> analyses of the data show that the adjusted odds of having influenza a virus - infected pigs at a fair were 1.27 ( 95% confidential interval ( ci ) : 1.041.66 ) higher for every 20 pig increase in the size of the swine show . </S>", "<S> additionally , four of the five fairs that hosted breeding swine shows in addition to their junior fair market swine shows had pigs test positive for influenza a virus . while the current study was limited to 40 fairs within one state , the findings provided insight for veterinary and public health officials developing mitigation strategies to decrease the intra- and inter - species transmission of influenza a virus at fairs . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["the comingling of swine from numerous premises with varied management practices and their interaction with large numbers of exhibitors and visitors make agricultural fairs an ideal setting for the intra- and inter - species transmission of influenza a viruses ( iavs ) between swine and human populations .", "the frequency with which intra- and inter - species iav transmission occurs in these settings is likely due to a myriad of factors , including but not limited to , management practices , iav strain , and animal and/or human population immunity .", "swine is a host species in which reassortment of iav genomic segments may lead to emergent novel strains , since they are susceptible to infection from swine , human and avian influenza a viruses .", "for this reason , limiting the bidirectional zoonotic transmission of these viruses at agricultural fairs is important for public and animal health .", "the association between human and swine influenza was reported after respiratory disease similar to the human disease was noted in pigs during the 1918 human spanish flu pandemic .", "iav subsequently became established in the united states swine population with the relatedness of the swine and human viruses being established in 1931 . for nearly 80 years", ", classical swine influenza h1n1 virus was the dominant endemic iav strain in the north america swine population . in 1998 ,", "triple reassortant h3n2 iavs containing polymerase basic 1 ( pb1 ) , hemagglutinin ( ha ) and neuraminidase ( na ) gene segments from human iav lineages , polymerase basic 2 ( pb2 ) and polymerase acidic ( pa ) genes from avian lineages , and nucleoprotein ( np ) , matrix ( m ) and non - structural ( ns ) gene segments from swine lineages , emerged in north american swine .", "subsequently , this lineage became established in us and canadian swine herds and has resulted in an increased rate of genetic and antigenic change among swine - origin iavs .", "reported cases of humans contracting iav infections directly or indirectly from pigs have been historically sporadic and these variant iavs showed limited capability for sustained human - to - human transmission . however , the emergence of the influenza a(h1n1)pdm09 virus , a strain containing gene segments from north american and european swine lineages , illustrated the pandemic potential of swine lineage iavs crossing the species barrier to humans . while a(h1n1)pdm09 rapidly spread worldwide and became endemic in the human population , sequencing of this virus has to date failed to elucidate any virulence or adaptation markers that would explain its human - to - human transmission efficiency , highlighting our inability to predict iavs with pandemic potential .", "while the origin of the a(h1n1)pdm09 virus remains unknown , the virus was introduced into the north american swine population in 2009 and has since reassorted with other swine - origin iavs .", "epidemiological data show that zoonotic transmission of iav from swine to humans has been documented at unprecedented levels in recent years .", "more than 320 human cases of infection with variant iavs were reported to the centers for disease control 20112012 and likely thousands more h3n2v cases went unreported during those years .", "these zoonotic iavs contained seven genes from contemporary north american swine lineage iav and one gene ( m ) derived from the h1n1pdm09 virus .", "the majority of the cases were epidemiologically linked to swine exposure occurring at agricultural fairs across several states . within ohio , 107 h3n2v cases documented during 2012 resulted in eleven hospitalizations and one fatality .", "we recovered iav from exhibition swine at 10 of 40 ( 25% ) ohio fairs sampled during 2012 .", "genomic analyses of h3n2 iav isolates recovered from pigs at one agricultural fair in the state during 2012 demonstrated > 99% nucleotide similarity to h3n2v isolates recovered from concurrent human cases , providing molecular confirmation of zoonotic iav transmission .", "this record number of variant influenza a cases created the need for a  one health ' effort to minimize intra- and bidirectional inter - species iav transmission at swine exhibitions .", "the reason to prevent iav infections among swine at fairs is clear ; however , the paucity of scientific evidence makes it difficult for veterinary officials to make sound recommendations to protect public and animal health . in the present study", ", we investigate fair specific risk factors contributing to the emergence of influenza a virus in exhibition swine that could be altered to mitigate the risk of iav transmission in these settings ."], ["as part of an ongoing active iav surveillance project , swine nasal swabs and associated metadata about management practices were collected at 40 ohio fairs in 2012 .", "sample size was selected to provide a 95% probability of detecting iav infection if greater than 15% of the pigs at each fair were infected .", "all pigs sampled in this study were from junior fair market swine shows occurring at agricultural fairs .", "for the outcome of interest , a fair was considered positive if viable iav was recovered from one or more pigs at the fair .", "data collection focused on fair level variables possibly contributing to the presence or absence of iav in the pigs at each exhibition .", "junior fair shows are limited to local exhibitors approximately nine years of age through 19 years of age participating in 4-h , ffa or another youth organization , whereas open - class shows are generally open to all participants regardless of age , affiliation or residence .", "classification of swine included market swine ( pigs bred , raised and intended for food purposes ) and breeding swine ( gilts , sows and/or boars being raised for breeding purposes ) .", "terminal swine shows are those in which all participating livestock are consigned to harvest immediately following the exhibition and partial terminal shows usually require the champion animals to be harvested following the exhibition and other pigs may or may not go to harvest . to account for the variability of arrival and departure procedures among fairs enrolled in this study , the length of the swine exhibition was defined as the number of days between the required arrival deadline for the pigs and the day the pigs were sampled .", "study team members calculated the area per pig ( ft / pig ) from the recorded size of the pens and the number of pigs per pen . while on the fairgrounds , study team members also documented if there was an easily identifiable and operational hand - wash and/or hand - sanitizer station within close proximity to the swine barn(s ) .", "these sanitation stations were used by study team members to determine if they were functional .", "additional variables included the number of pigs at the fair , number of swine exhibitors , fair attendance ( number of people ) and vaccine requirements , all of which were reported to the study team by the fair organizers .", "fair officials also reported if there was a commingling event , such as a pre - fair animal identification or weighing session , during the weeks or months prior to the fair .", "exhibition directors also reported if there were other pigs besides the exhibition swine on the fairgrounds ( i.e. , petting zoo , pig races , educational displays ) .", "the commercial swine inventory was retrieved from united states department of agriculture 's 2007 census of agriculture .", "the county human population was defined as the value reported in the 2010 us census report .", "weather data were collected from the national weather service 's weather station nearest to each fairground .", "data were analyzed using stata version 11.1 ( statacorp , college station , tx , usa ) .", "univariate analysis was performed to calculate unadjusted odd ratios to identify factors contributing to the presence of iav in pigs at fairs .", "exact logistic regression was used for multivariable modeling using a forward stepwise model building approach ."], ["influenza a virus was recovered from pigs at 10 of the 40 fairs included in the investigation . the presence or absence of iav infection among the pigs at the fairs", "could not be associated with county population , county swine inventory and number of people attending the fair ( table 1 ) . all iav - positive fairs and 27 of 30 ( 90% ) negative fairs were mixed sex ( barrows and gilts ) market swine exhibitions .", "the average space per pig at the studied fairs was 12.8 ft / pig . properly functioning hand - wash and/or hand - sanitizer stations were available at 25 of 40 ( 62.5% ) fairs .", "average daily mean temperature was almost 4  c higher for fairs with iav - positive pigs ( table 1 ) .", "pre - fair tag - in and/or weigh - in events were rather common with 23 out of 40 ( 57.5% ) of the enrolled fairs holding one of these events .", "for every increase of 20 pigs at a fair , the odds ratio of iav infection in the pigs increased by 1.27 times ( table 2 ) ."], ["the results of the current study provide the first look at fair - level risk factors associated with iav infections in swine at agricultural fairs .", "while it is likely impossible to completely prevent iav transmission at swine exhibitions , these data can be used to develop and evaluate mitigation strategies to reduce the impact of intra- and inter - species iav infections at swine exhibitions .", "just like all other agriculture biosecurity programs , mitigation strategies which are practical , user - friendly , low cost and do not dramatically alter the fair experience for exhibitors and visitors are most likely to be considered , implemented and maintained .", "not surprisingly , larger pig shows appear to be more likely to have iav - infected pigs than smaller swine exhibitions ( table 2 ) .", "larger swine exhibitions tend to also have open - class and breeding swine shows in addition to junior market shows . while open - class shows were common among our studied fairs ( 16 of the 40 ) , only 5 of the 40 ( 12.5% ) fairs in this study had a breeding show ; 4 of those 5 ( 80% ) fairs had iav - infected pigs at the fair .", "the small number of fairs with breeding shows in this study makes analysis of this risk factor problematic ; however , the finding is enough to warrant concern given that breeding swine are intended to leave the exhibition and enter a herd for progeny production .", "this fair - to - farm movement of pigs is a disease introduction risk for the receiving herd and a potential method to disseminate iav strains across a larger geographic area .", "previous research has shown that environmental stressors ( heat , lack of space , noise ) on pigs can affect the course of various diseases in commercial swine operations .", "the average space per pig at the studied fairs was well above 68  ft / finishing pig common throughout the swine industry .", "the results indicate that heat stress could be a contributing factor to iav infections in exhibition swine ; however , caution must be used when interpreting this result because the vast majority of the fairs with iav - positive pigs occurred in a 4-week period during the middle of summer .", "this trend of mid - summer iav activity in ohio 's exhibition swine was also observed in the previous three years and could be more related to animal and/or people movement between these fairs than the weather .", "environmental temperature failed to meet the selection criteria for inclusion in the final multivariable model .", "while the majority of fairs had hand hygiene stations , their presence at fairs was not linked to the occurrence of iav among pigs at the respective fairs .", "the large number of h3n2v infections linked to swine exposure at agricultural fairs during 2012 suggests that hand hygiene stations also had minimal impact on zoonotic transmission of iav in these settings .", "however , hand hygiene stations are critically important in protecting human health by controlling zoonotic diseases transmitted via direct contact at these venues .", "this would limit the time for iav to spread among susceptible pigs and decrease the time humans are exposed to iav - infected pigs .", "the length of the exhibitions in the current study was similar between iav - positive and -negative fairs .", "active recruitment of fairs with more diverse management practices is needed to study the impact of a shortened swine exhibition .", "no matter the length of the exhibition time , the disposition of the pigs following the show must be considered .", "the majority of fairs in this study ( 65% ) had terminal junior market shows .", "the practice of having a terminal show was not associated with decreased odds of iav ; however , sending all the pigs to harvest at the end of each fair is expected to help protect subsequent fairs by decreasing the potential for fair - to - fair spread of iav .", "mandated vaccinations were almost non - existent with only one fair in the current study requiring the pigs be vaccinated for erysipelothrix rhusiopathiae prior to the fair ; no fairs required the pigs to receive iav vaccination before arrival .", "use of iav vaccine in exhibition swine to decrease the risk of iav infections in swine and humans has been one of the most debated topics following the h3n2v outbreak of 2012 .", "there are currently several commercially available swine influenza vaccines in the united states , all of which are universally indicated to reduce clinical signs of disease in pigs but appear to have limited efficacy against 2012 h3n2v strains . although their impact on intra- and inter - species transmission dynamics remains unclear , it is expected that iav vaccines will impart at least partial immunity to circulating strains of iav , which should decrease viral shedding during a fair .", "an unintended consequence of iav vaccine use may be vaccine - associated enhanced respiratory disease , which has been reported in swine vaccinated with swine influenza virus vaccines that are mismatched to circulating strains .", "furthermore , decrease of clinical signs may hamper recognition and response to active iav infections in exhibition pigs .", "some of the major pitfalls of mandated iav vaccination lie with the practical application of vaccines in this setting .", "the logistics of vaccine distribution to swine exhibitors prior to the fair becomes difficult because most exhibition swine are raised by youth exhibitors in small , dispersed herds ( < 10 pigs per herd ) .", "commercial vaccines are usually sold in 50 doses per bottle adding to the cost per vaccinated pig in these small herds . because youth exhibitors and their family members may not be proficient at administering vaccines", ", agriculture education advisors often volunteer to assist the youth with this task , a practice that is time - consuming and increases the risk of infectious agents being transmitted farm to farm . additionally , problematic", "is that most iav vaccines labeled for swine require a booster dose given 24 weeks later to provide optimal protection , which can be difficult for youth exhibitors and their family members to accomplish . tagging or weighing events", "are frequently used as a way for exhibitors to declare ownership of their pig(s ) prior to the fair .", "these pre - fair events could provide an opportunity for mass vaccination of pigs prior to the fair , but the application of such events can facilitate disease spread between animals . even in properly vaccinated pigs , the immunity stimulated by current iav vaccines", "the strains used for commercial swine influenza vaccines are irregularly updated and the constant genetic and antigenic change occurring in contemporary swine - origin iavs makes viral antigens unpredictable and difficult vaccine targets .", "the reason for the increase in the number of reported h3n2v cases during 20112013 remains unclear , but the strain of iav is thought to be a major contributing factor .", "the swine - origin h3n2 iav isolates recovered from these human cases contains the matrix gene from the a(h1n1)pdm09 virus , a recently emerged genomic constellation that increases replication and transmission in cell culture and animal models . epidemiological data modeling", "indicate that children are most susceptible to h3n2v , likely due to lack of strain - specific immunity . additionally , current seasonal trivalent inactivated iav vaccine provides little to no protection against h3n2v strains .", "the limited ability for human - to - human transmission of h3n2v has minimized the impact of these recent zoonotic transmission events , but the outbreak has illustrated the importance of swine exhibitions in zoonotic iav transmission .", "it is nearly impossible to predict the iav strain that will infect the swine at fairs because iav reassortment events and novel strain generation frequently occur in modern swine populations .", "agricultural fairs provide a pathway for human exposure to these ever changing viruses ; thus , blanket iav prevention , without regard for strain , is needed for swine at fairs to decrease zoonotic iav transmission and protect public health . while iav can infect pigs at any fair , the data presented here indicate that special attention should be paid to large pig shows where the likelihood of iav among the pigs is much higher .", "the results presented herein are based on one year of data from a limited geographic area of the united states .", "additional assessments of swine exhibitions in multiple states across several years are needed to provide more comprehensive evaluations of risk factors contributing to the problem .", "these data provide a critical first step toward developing effective iav mitigation strategies in swine populations that benefit fairs , exhibitors , visitors and the swine industry .", "this information will serve as a baseline for measuring the acceptance and effectiveness as mitigation strategies are developed and implemented ."]]}
{"article_id": "PMC5024144", "article_text": ["turmeric ( curcuma longa l. ) of family zingiberaceae is industrially as well as pharmaceutically important crop extensively cultivated in india .", "it is a vegetatively propagated , polyploid crop commonly known as  golden spice  . because of its multipurpose use as food preservative , spice , therapeutic agent and natural dye , it is acquiring more importance in cosmetics , food and pharmaceutical industries , . in turmeric number of cultivars", "has been developed with different characteristics like high curcumin content , high essential oil content , rhizome yield and disease tolerance etc .", "but their asexual mode of reproduction and poor genetic makeup has creates confusion towards authentic identification .", "single nucleotide polymorphism ( snp ) study could be useful to solve these problems by comparing sequence of each variety with each other . in this current study", ", we conducted reference based transcriptome assembly of suvarna , a well - known turmeric cultivar which could latter on used to get potential snps by comparing with other varieties towards proper identification .", "fresh mature rhizomes of suvarna variety has been collected from centre for biotechnology , siksha o anusandhan university , bhubaneswar , india .", "these rhizomes were washed properly with distilled water and suspended in rna later solution for further analysis .", "rna extraction and library preparation has been done by using illumina truseq rna library tool kit .", "75  bp paired - end sequencing has been done using illumina nextseq500 sequencing platform which developed5 gb of raw data .", "we have precisely given detail information about the reference based transcriptome assembly of suvarna cultivar ( table 1 ) .", "the transcriptome assembly of suvarna developed 42994 numbers of transcripts , with median transcript length 773 and n50 value 1216 .", "we annotated the transcripts using various databases like go , kegg , kog , plantcyc etc .", "as per our understanding transcriptome analysis of cultivar suvarna has been done for the first time and this result could be efficiently used for development of markers such as ssrs and snps for identification of suvarna from its closely similar turmeric cultivars .", "fresh mature rhizomes of suvarna variety has been collected from centre for biotechnology , siksha o anusandhan university , bhubaneswar , india .", "these rhizomes were washed properly with distilled water and suspended in rna later solution for further analysis .", "rna extraction and library preparation has been done by using illumina truseq rna library tool kit .", "75  bp paired - end sequencing has been done using illumina nextseq500 sequencing platform which developed5 gb of raw data .", "we have precisely given detail information about the reference based transcriptome assembly of suvarna cultivar ( table 1 ) .", "the transcriptome assembly of suvarna developed 42994 numbers of transcripts , with median transcript length 773 and n50 value 1216 .", "we annotated the transcripts using various databases like go , kegg , kog , plantcyc etc .", "as per our understanding transcriptome analysis of cultivar suvarna has been done for the first time and this result could be efficiently used for development of markers such as ssrs and snps for identification of suvarna from its closely similar turmeric cultivars ."], "abstract_text": ["<S> turmeric is an economically valued crop , because of its utility in the food , pharmaceutical industries and ayurvedic medicine , attracts the attention in many areas of research work . in the present study , we executed resequencing through transcriptome assembly of the turmeric cultivar suvarna ( cl_suv_10 ) . </S>", "<S> resequencing of suvarna variety has generated 5 gbases raw data with 75  bp paired - end sequence . </S>", "<S> the raw data has been submitted to sra database of ncbi with accession number srr4042181 . </S>", "<S> reads were assembled using cufflinks-2.2.1 tool which ended up with 42994 numbers of transcripts . </S>", "<S> the length of transcripts ranged from 83 to15565 , with a n50 value 1216 and median transcript length 773 . </S>", "<S> the transcripts were annotated through number of databases . </S>", "<S> for the first time transcriptome profiling of cultivar suvarna has been done , which could help towards identification of single nucleotide polymorphisms ( snps ) between suvarna and other turmeric cultivars for its authentic identification . </S>"], "labels": null, "section_names": ["Direct link to deposited data", "Introduction", "Experimental design, materials and methods", "Plant material", "RNA extraction and transcriptome sequencing", "Transcriptome assembly and annotation"], "sections": [[""], ["turmeric ( curcuma longa l. ) of family zingiberaceae is industrially as well as pharmaceutically important crop extensively cultivated in india .", "it is a vegetatively propagated , polyploid crop commonly known as  golden spice  . because of its multipurpose use as food preservative , spice , therapeutic agent and natural dye , it is acquiring more importance in cosmetics , food and pharmaceutical industries , . in turmeric number of cultivars", "has been developed with different characteristics like high curcumin content , high essential oil content , rhizome yield and disease tolerance etc .", "but their asexual mode of reproduction and poor genetic makeup has creates confusion towards authentic identification .", "single nucleotide polymorphism ( snp ) study could be useful to solve these problems by comparing sequence of each variety with each other . in this current study", ", we conducted reference based transcriptome assembly of suvarna , a well - known turmeric cultivar which could latter on used to get potential snps by comparing with other varieties towards proper identification ."], ["fresh mature rhizomes of suvarna variety has been collected from centre for biotechnology , siksha o anusandhan university , bhubaneswar , india .", "these rhizomes were washed properly with distilled water and suspended in rna later solution for further analysis .", "rna extraction and library preparation has been done by using illumina truseq rna library tool kit .", "75  bp paired - end sequencing has been done using illumina nextseq500 sequencing platform which developed5 gb of raw data .", "we have precisely given detail information about the reference based transcriptome assembly of suvarna cultivar ( table 1 ) .", "the transcriptome assembly of suvarna developed 42994 numbers of transcripts , with median transcript length 773 and n50 value 1216 .", "we annotated the transcripts using various databases like go , kegg , kog , plantcyc etc .", "as per our understanding transcriptome analysis of cultivar suvarna has been done for the first time and this result could be efficiently used for development of markers such as ssrs and snps for identification of suvarna from its closely similar turmeric cultivars ."], ["fresh mature rhizomes of suvarna variety has been collected from centre for biotechnology , siksha o anusandhan university , bhubaneswar , india .", "these rhizomes were washed properly with distilled water and suspended in rna later solution for further analysis ."], ["rna extraction and library preparation has been done by using illumina truseq rna library tool kit ."], ["75  bp paired - end sequencing has been done using illumina nextseq500 sequencing platform which developed5 gb of raw data .", "we have precisely given detail information about the reference based transcriptome assembly of suvarna cultivar ( table 1 ) .", "the transcriptome assembly of suvarna developed 42994 numbers of transcripts , with median transcript length 773 and n50 value 1216 .", "we annotated the transcripts using various databases like go , kegg , kog , plantcyc etc .", "as per our understanding transcriptome analysis of cultivar suvarna has been done for the first time and this result could be efficiently used for development of markers such as ssrs and snps for identification of suvarna from its closely similar turmeric cultivars ."]]}
{"article_id": "PMC4083521", "article_text": ["the sense of coherence ( soc ) , which was proposed by antonovsky is based on the salutogenic model of health .", "the soc is composed of three factors ; ( 1 ) the stimuli derived from internal and external environments in the course of living are structured , predictable , and explicable ( comprehensibility ) ; ( 2 ) resources are available to meet the demands posed by these stimuli ( manageability ) ; and ( 3 ) such demands are challenges , worthy of investment and engagement ( meaningfulness ) .", "antonovsky defined the soc as a personality dimension that is hypothesized to influence stress recognition style , facilitate stress management , and contribute to overall well - being .", "people with a strong soc have a high ability to cope with stress and maintain good physical and mental health . to the best of our knowledge", ", six studies have investigated the association between soc , job stress , and mental health .", "findings from these studies suggest that soc modified the effect of job stress on mental health . to the best of our knowledge", ", only four studies have investigated the relationship between the soc and depressive symptoms , one used the brief job stress questionnaire and the other three used general health questionnaire .", "these studies suggested that a weak soc was a strong predictor of mental distress , including depressive symptoms .", "nurses are exposed to high - stress work environments , including irregular work schedules , shift work , and interaction with patients and other hospital staff members .", "therefore , preventing mental distress , including depressive state , is an important issue for nurses .", "the aim of this study was to investigate the relationship between depressive state , job stress , and soc among nurses in japanese general hospital .", "in february 2013 , supervisors distributed a questionnaire to all nurses ( n = 710 ) in a general hospital with 611 beds in an urban area in japan .", "nurse specialties included intensive care , pediatrics , surgery , oncology , and emergency medicine .", "an explanation of the nature of the survey accompanied the questionnaire , which was anonymous and voluntary .", "the study was approved by both the local direction board and the committee for the prevention of physical disease and mental illness among health care workers at the general hospital .", "the questionnaire collected data on age , hours of work ( full - time or part - time ) , shift work , overtime hours per week , and job rank ( manager , middle manager or staff nurse ) .", "we measured job stress using the japanese version of the effort - reward imbalance ( eri ) scale ( 23 items ) translated by tsutsumi et al .", "the eri consists of three subscales ; effort ( 6 items ) , reward ( 11 items ) and over - commitment ( 6 items ) .", "the reward subscale is further divided into three subgroups : esteem , job security , and promotion .", "the eri model indicates that job stress is related to high effort with low reward .", "four eri ratios ( eri , effort - esteem imbalance , effort - promotion imbalance , and effort - job security imbalance ) were calculated based on the total scores according to tsutsumi et al . the soc scale consisted of 29 items assessing comprehensibility , manageability , and meaningfulness .", "scores for each item ranged from 1 to 7 points , and the total score was calculated as the soc score .", "we measured depressive state using the k6 short screening questionnaire that was developed in accordance with the world health organization translation guidelines .", "the k6 consists of six items on depression and anxiety , each of which is measured on a 5-point scale ( 0 - 4 ) .", "higher scores indicate a more depressive state . the k6 was translated into japanese and showed good validity with the diagnostic and statistical manual of mental disorders , fourth edition , mood , and anxiety disorders in a community sample .", "we used pearson 's correlation to investigate the relationship among age , work related - factors , job stress , soc , and depressive state . to examine factors with a significant effect on depressive state ,", "stepwise multiple regression analyses were conducted with the k6 total score as the dependent variable and variables related to the depressive state as independent variables .", "completed questionnaires were returned by 420 out of 710 nurses ( response rate , 59.2% ) .", "male nurses were excluded from the analysis because only 28 of the 42 ( 66.7% ) male nurses responded .", "subjects with missing values for job stress , soc , or depressive state were also excluded ( n = 43 ) . the final sample for analysis consisted of 348 female nurses ( 52.1% ) , including nurses who were managers and middle managers .", "overtime was reported in hours per week , which was voluntary but limited to 45 h / month .", "shift work categories included :  no shift work ,   shift work with rotation to night shift ,  or ", "characteristics of study subjects table 2 shows the pearson 's correlation coefficients between age , overtime hours , eri ratios , soc , and the depressive state .", "depressive state moderately correlated with three eri ratios , over - commitment , and soc .", "pearson 's coreration between work environments , eri ratios , over - commitment , soc and depressive state table 3 shows the influence of work - related factors , eri ratios , and soc on depressive state .", "soc , over - commitment , effort - esteem ratio , and age were significantly correlated with depressive state (  = 0.46 , p < 0.001 ;  = 0.27 , p < 0.001 ;  = 0.16 , p < 0.001 ,  = 0.10 , p < 0.01 , respectively ) .", "the coefficient of multiple determination ( r ) was 0.56 ( f = 106.56 p < 0.001 ) .", "we found that soc , over - commitment , effort - esteem ratio , and age were predictors of depressive state among female nurses in the general hospital .", "the soc was inversely associated with depressive state , which was similar to previous studies .", "the present findings contribute to the literature investigating the relationship between depressive state , job stress , and soc .", "strength of our study is that we investigated the independent contribution of three ratios of eri ( effort - esteem , effort - job security , and effort - promotion ) , over - commitment , and soc to the depressive state . according to our previous study ,", "the present findings suggest that a depressive state of nurses was associated with not only job stress but also both over - commitment and soc .", "age correlated positively with soc [ table 2 ] , which was also similar to a previous study by harri who suggested that soc tends to increase with age .", "previous studies suggested that an increasing soc of workers may reduce negative job stress responses and mental health problems .", "intervention support enhancing the soc could be effective in preventing nurses from experiencing a depressive state .", "first , the sample size was small and included nurses from only one general hospital .", "our findings provide insight into some factors associated with a depressive state among nurses in a general hospital . from a practical perspective", ", the influence of soc on a depressive state should be considered for health care professionals .", "intervention support such as group cognitive psychotherapy to strengthen comprehensibility , manageability , and meaningfulness may help nurses cope better with job stress and reduce their risk of depression ."], "abstract_text": ["<S> background : people with a strong sense of coherence ( soc ) have a high ability to cope with stress and maintain good physical and mental health.aims:the aim of this study was to investigate the relationship between depressive state , job stress , and soc among nurses in a japanese general hospital.materials and methods : a self - reporting survey was conducted among 348 female nurses in a general hospital . </S>", "<S> job stress was measured using the japanese version of the effort - reward imbalance ( eri ) scale . </S>", "<S> depressive state was assessed by the k6 scale . </S>", "<S> soc was assessed with the soc scale , which includes 29 items . </S>", "<S> stepwise multiple regression analysis was conducted to examine factors that significantly affect depressive state.results:soc , over - commitment , effort - esteem ratio , and age were significantly correlated with the depressive state (  = 0.46 , p < </S>", "<S> 0.001 ;  = 0.27 , p < 0.001 ;  = 0.16 , p < 0.001 ;  = 0.10 , p < 0.001 , respectively).conclusions : soc may have a major influence on the depressive state among female nurses in a japanese general hospital . from a practical perspective </S>", "<S> , health care professionals should try to enhance the soc of nurses . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["the sense of coherence ( soc ) , which was proposed by antonovsky is based on the salutogenic model of health .", "the soc is composed of three factors ; ( 1 ) the stimuli derived from internal and external environments in the course of living are structured , predictable , and explicable ( comprehensibility ) ; ( 2 ) resources are available to meet the demands posed by these stimuli ( manageability ) ; and ( 3 ) such demands are challenges , worthy of investment and engagement ( meaningfulness ) .", "antonovsky defined the soc as a personality dimension that is hypothesized to influence stress recognition style , facilitate stress management , and contribute to overall well - being .", "people with a strong soc have a high ability to cope with stress and maintain good physical and mental health . to the best of our knowledge", ", six studies have investigated the association between soc , job stress , and mental health .", "findings from these studies suggest that soc modified the effect of job stress on mental health . to the best of our knowledge", ", only four studies have investigated the relationship between the soc and depressive symptoms , one used the brief job stress questionnaire and the other three used general health questionnaire .", "these studies suggested that a weak soc was a strong predictor of mental distress , including depressive symptoms .", "nurses are exposed to high - stress work environments , including irregular work schedules , shift work , and interaction with patients and other hospital staff members .", "therefore , preventing mental distress , including depressive state , is an important issue for nurses .", "the aim of this study was to investigate the relationship between depressive state , job stress , and soc among nurses in japanese general hospital ."], ["in february 2013 , supervisors distributed a questionnaire to all nurses ( n = 710 ) in a general hospital with 611 beds in an urban area in japan .", "nurse specialties included intensive care , pediatrics , surgery , oncology , and emergency medicine .", "an explanation of the nature of the survey accompanied the questionnaire , which was anonymous and voluntary .", "the study was approved by both the local direction board and the committee for the prevention of physical disease and mental illness among health care workers at the general hospital .", "the questionnaire collected data on age , hours of work ( full - time or part - time ) , shift work , overtime hours per week , and job rank ( manager , middle manager or staff nurse ) .", "we measured job stress using the japanese version of the effort - reward imbalance ( eri ) scale ( 23 items ) translated by tsutsumi et al .", "the eri consists of three subscales ; effort ( 6 items ) , reward ( 11 items ) and over - commitment ( 6 items ) .", "the reward subscale is further divided into three subgroups : esteem , job security , and promotion .", "the eri model indicates that job stress is related to high effort with low reward .", "four eri ratios ( eri , effort - esteem imbalance , effort - promotion imbalance , and effort - job security imbalance ) were calculated based on the total scores according to tsutsumi et al . the soc scale consisted of 29 items assessing comprehensibility , manageability , and meaningfulness .", "scores for each item ranged from 1 to 7 points , and the total score was calculated as the soc score .", "we measured depressive state using the k6 short screening questionnaire that was developed in accordance with the world health organization translation guidelines .", "the k6 consists of six items on depression and anxiety , each of which is measured on a 5-point scale ( 0 - 4 ) .", "higher scores indicate a more depressive state . the k6 was translated into japanese and showed good validity with the diagnostic and statistical manual of mental disorders , fourth edition , mood , and anxiety disorders in a community sample .", "we used pearson 's correlation to investigate the relationship among age , work related - factors , job stress , soc , and depressive state . to examine factors with a significant effect on depressive state ,", "stepwise multiple regression analyses were conducted with the k6 total score as the dependent variable and variables related to the depressive state as independent variables ."], ["completed questionnaires were returned by 420 out of 710 nurses ( response rate , 59.2% ) .", "male nurses were excluded from the analysis because only 28 of the 42 ( 66.7% ) male nurses responded .", "subjects with missing values for job stress , soc , or depressive state were also excluded ( n = 43 ) . the final sample for analysis consisted of 348 female nurses ( 52.1% ) , including nurses who were managers and middle managers .", "overtime was reported in hours per week , which was voluntary but limited to 45 h / month .", "shift work categories included :  no shift work ,   shift work with rotation to night shift ,  or ", "characteristics of study subjects table 2 shows the pearson 's correlation coefficients between age , overtime hours , eri ratios , soc , and the depressive state .", "depressive state moderately correlated with three eri ratios , over - commitment , and soc .", "pearson 's coreration between work environments , eri ratios , over - commitment , soc and depressive state table 3 shows the influence of work - related factors , eri ratios , and soc on depressive state .", "soc , over - commitment , effort - esteem ratio , and age were significantly correlated with depressive state (  = 0.46 , p < 0.001 ;  = 0.27 , p < 0.001 ;  = 0.16 , p < 0.001 ,  = 0.10 , p < 0.01 , respectively ) .", "the coefficient of multiple determination ( r ) was 0.56 ( f = 106.56 p < 0.001 ) ."], ["we found that soc , over - commitment , effort - esteem ratio , and age were predictors of depressive state among female nurses in the general hospital .", "the soc was inversely associated with depressive state , which was similar to previous studies .", "the present findings contribute to the literature investigating the relationship between depressive state , job stress , and soc .", "strength of our study is that we investigated the independent contribution of three ratios of eri ( effort - esteem , effort - job security , and effort - promotion ) , over - commitment , and soc to the depressive state . according to our previous study ,", "the present findings suggest that a depressive state of nurses was associated with not only job stress but also both over - commitment and soc .", "age correlated positively with soc [ table 2 ] , which was also similar to a previous study by harri who suggested that soc tends to increase with age .", "previous studies suggested that an increasing soc of workers may reduce negative job stress responses and mental health problems .", "intervention support enhancing the soc could be effective in preventing nurses from experiencing a depressive state .", "first , the sample size was small and included nurses from only one general hospital ."], ["our findings provide insight into some factors associated with a depressive state among nurses in a general hospital . from a practical perspective", ", the influence of soc on a depressive state should be considered for health care professionals .", "intervention support such as group cognitive psychotherapy to strengthen comprehensibility , manageability , and meaningfulness may help nurses cope better with job stress and reduce their risk of depression ."]]}
{"article_id": "PMC3574462", "article_text": ["huntington 's disease ( hd ) is an autosomal dominant , progressive neurodegenerative disorder characterized by chorea , subcortical dementia and psychiatric symptoms .", "the causal hd gene has been identified as it15 , which has cag repeats in the open reading frame ( orf , in exon 1 ) just following the 5end of the gene .", "it leads to the translation of a protein , huntingtin , whose function is unknown .", "huntingtin forms intraneuronal inclusion bodies which lead to neuronal dysfunction followed by neuronal death due to apoptosis .", "the therapeutic strategies for hd are diverse , ranging from the classical drug therapies to the modern molecular strategies .", "one of the treatment options is the use of drugs with neuroprotective potential , like lithium .", "lithium has demonstrated diverse molecular effects reversing well - described pathophysiological changes such as increased oxidative stress , apoptosis , inflammation , environmental stress , glial dysfunction , neurotrophic factor deficiency , excitotoxicity as well as mitochondrial and endoplasmic reticulum disruption .", "lithium acts by inhibiting a protein- kinase called glycogen synthase kinase 3 ( gsk3 ) that has important actions on the intracellular signal transmission by protein phosphorylation .", "inhibition of this enzyme appears to have a neuroprotector effect in neurodegenerative diseases such as amyotrophic lateral sclerosis , spinocerebellar ataxia type 1 and huntington 's disease .", "we report three patients with huntington 's disease , who showed good response to treatment with low dose lithium .", "mrs . a , a 48-year - old homemaker , presented in 2008 with a three - year history of involuntary movements of the trunk and the hands , which had worsened over the previous 7 - 8 months .", "family history was not available . on examination , ocular saccades were slow ; she had generalized choreiform movements involving hands legs , lips and the trunk ; dystonia of hands and feet and ataxic gait .", "a genetic analysis for huntington 's disease was performed after obtaining written informed consent which showed cag repeat expansion .", "patient was started on lithium 150 mg , once daily , along with carbamazepine 200 mg twice a day .", "patient is on regular follow up for the last four years and is maintaining the same improvement .", "b , a 58-year - old male , presented in 2001 with a history of abnormal movements .", ", the movements were disabling , irregular , would worsen with anxiety and disappear during sleep .", "patient had a significant family history of hd ; involving both parents and 7 out of 9 siblings , totally 9 affected first - degree relatives . on examination , patient was noted to have severe generalized chorea involving the hands , legs , trunk and the neck .", "patient was admitted in 2009 with a worsening of movements over the previous 6 months and weight loss of about 6 kg .", "all investigations including prostate specific antigen and chest x - ray were normal and the weight loss was attributed to nutritional causes .", "family members reported that the patient was able to eat well and had no behavioral symptoms .", "the patient has been on follow up for the last three years and maintains status quo .", "c , a 51-year - old lady presented in 2002 with history of psychiatric symptoms for the previous 8 years .", "onset was sudden with symptoms of being anxious , accusing the family members of neglect , and being violent toward family members .", "patient also had history of non - rhythmic quasi - purposive movements of hands which she attributed to anxiety .", "family history revealed that her mother had movement disorder and an early death . on examination , patient c had choreic movements of the limbs , anxiety and depressive symptoms .", "a diagnosis of hd was made which was confirmed by genetic analysis , which showed expanded cag repeats in the huntingtin 's locus .", "patient was initially started on carbamazepine 400 mg and clonazepam 4 mg / day . over the next one year", ", patient developed psychotic symptoms like delusions of reference and persecution toward her family members and was also started on quetiapine up to 300 mg .", "two years later , patient had sudden deterioration in symptoms with worsening of chorea and onset of cognitive decline  memory disturbances and apraxia , which made her completely dependent for all activities of daily living . during this time , she also developed significant mood swings .", "for the last 3 years , patient has been on a combination of lithium 300 mg , carbamazepine 800 mg , quetiapine 300 mg and clonazepam 2 mg / day .", "patient 's husband reported no further deterioration over 3 years in cognition or movements in the patient .", "however , she continued to be dependent on husband for activities of daily living and eventually died due to cardiac arrest .", "mrs . a , a 48-year - old homemaker , presented in 2008 with a three - year history of involuntary movements of the trunk and the hands , which had worsened over the previous 7 - 8 months .", "family history was not available . on examination , ocular saccades were slow ; she had generalized choreiform movements involving hands legs , lips and the trunk ; dystonia of hands and feet and ataxic gait .", "a genetic analysis for huntington 's disease was performed after obtaining written informed consent which showed cag repeat expansion .", "patient was started on lithium 150 mg , once daily , along with carbamazepine 200 mg twice a day .", "patient is on regular follow up for the last four years and is maintaining the same improvement .", "b , a 58-year - old male , presented in 2001 with a history of abnormal movements .", ", the movements were disabling , irregular , would worsen with anxiety and disappear during sleep .", "patient had a significant family history of hd ; involving both parents and 7 out of 9 siblings , totally 9 affected first - degree relatives . on examination ,", "patient was noted to have severe generalized chorea involving the hands , legs , trunk and the neck .", "patient was admitted in 2009 with a worsening of movements over the previous 6 months and weight loss of about 6 kg .", "all investigations including prostate specific antigen and chest x - ray were normal and the weight loss was attributed to nutritional causes .", "family members reported that the patient was able to eat well and had no behavioral symptoms .", "the patient has been on follow up for the last three years and maintains status quo .", "c , a 51-year - old lady presented in 2002 with history of psychiatric symptoms for the previous 8 years .", "onset was sudden with symptoms of being anxious , accusing the family members of neglect , and being violent toward family members .", "patient also had history of non - rhythmic quasi - purposive movements of hands which she attributed to anxiety .", "family history revealed that her mother had movement disorder and an early death . on examination ,", "a diagnosis of hd was made which was confirmed by genetic analysis , which showed expanded cag repeats in the huntingtin 's locus .", "patient was initially started on carbamazepine 400 mg and clonazepam 4 mg / day . over the next one year", ", patient developed psychotic symptoms like delusions of reference and persecution toward her family members and was also started on quetiapine up to 300 mg .", "two years later , patient had sudden deterioration in symptoms with worsening of chorea and onset of cognitive decline  memory disturbances and apraxia , which made her completely dependent for all activities of daily living . during this time , she also developed significant mood swings .", "for the last 3 years , patient has been on a combination of lithium 300 mg , carbamazepine 800 mg , quetiapine 300 mg and clonazepam 2 mg / day .", "patient 's husband reported no further deterioration over 3 years in cognition or movements in the patient .", "however , she continued to be dependent on husband for activities of daily living and eventually died due to cardiac arrest .", "huntington 's disease ( hd ) is an autosomal dominant progressive neurodegenerative disorder caused by an expanded polyglutamine ( poly q ) tract within huntingtin protein and is characterized by neuronal loss mainly in the striatum and the cortex .", "genetically , hd is caused by a mutation in the gene located on chromosome4p 16.3 .", "the disease - causing mutation in the first exon of huntingtin ( htt ) gene is the expansion of a polymorphic cag repeat that is completely penetrant once it has reached 40 triplets .", "neuropathologically , the mutant huntington protein ( mhtt ) abnormally interacts with a variety of proteins and accumulates in the neuronal nucleus forming an inclusion body which is the hallmark of hd and leads to eventual cell death .", "in addition to the well - documented use of lithium as a mood stabilizing drug , primarily in the treatment of bipolar disorder , recent in vitro and in vivo studies in animal models have increasingly indicated that lithium can be used in the treatment of chronic neurodegenerative diseases like huntington 's disease .", "various pharmacological and gene manipulation studies support the notion that lithium 's main mechanisms of action may be its ability to inhibit gsk3[68 ] as well as induce brain - derived neurotrophic factor - mediated ( bdnf ) signaling , leading to enhanced cell survival by alteration of a wide variety of downstream effectors .", "lithium is shown to promote the action of anti - apoptotic proteins , such as heat - shock protein ( hsp ) , insulin like growth factor ( igf ) , vascular endothelial growth factor ( vegf ) and other neurotrophic factors .", "lithium can directly or indirectly modulate proteins involved in neuronal survival / differentiation which may account for its neuroprotective effects.[1012 ] by inhibiting n - methyl - d - aspartate receptor - mediated calcium influx , lithium also contributes to calcium homeostasis and suppresses calcium - dependent activation of pro - apoptotic signaling pathways .", "lithium is found to enhance autophagy of toxic aggregates in huntington 's disease in various models of hd .", "it acts by increasing mtor - independent autophagy by inhibiting inositol monophosphatase ( impase ) and reducing inositol and ip3 levels .", "induction of autophagy by lithium leads to enhanced clearance of mutant huntingtin fragments associated with huntington 's disease ( hd ) .", "these mechanisms might allow therapeutic doses of lithium to protect neuronal cells from diverse insults that would otherwise lead to massive cell death .", "lithium also stimulates proliferation of stem cells , including neuronal and astroglial progenitor cell proliferation in the sub ventricular zone , hippocampus , striatum and forebrain protecting against apoptosis .", "the stimulation of endogenous neural stem cells and neurogenesis may explain why lithium increases brain cell density and volume in patients with bipolar disorder .", "in this report , we have described the use of lithium to prevent the progression of the huntington 's disease .", "all patients reported here were started on low doses of lithium after explaining the rationale , possible side - effects and off label use of the drug .", "the patients were on regular follow up and reported no worsening of either psychiatric symptoms or choreic movements for a significant period .", "in these patients , lithium was started in order to alleviate the distress caused by the choreic movements and mood swings .", "though rating of the movements was not done , there appears be some benefit in prescribing lithium to patients with huntington 's disease for slowing the progression of chorea and preventing mood swings .", "this report signifies the use of lithium as a neuroprotective agent . it may be beneficial to explore the possibility of use of other psychotropic drugs in the treatment of neurodegenerative disorders"], "abstract_text": ["<S> huntington 's disease is characterized by choreic movements , psychiatric disorders , striatal atrophy with selective small neuronal loss , and autosomal dominant inheritance . the genetic abnormality is cag expansion in huntingtin gene . </S>", "<S> newer therapeutic strategies are evolving to treat this progressive disorder . </S>", "<S> the neuroprotective agents are one such group of drugs being tried . </S>", "<S> lithium has been used to treat huntington 's disease in the past due to its neuroprotective effects . </S>", "<S> though the precise mechanism of action is not clear , lithium can directly or indirectly modulate proteins involved in neuronal survival / differentiation which may account for its neuroprotective effects . </S>", "<S> we report three patients with huntington 's disease in whom lithium prevented the progression of chorea and also helped stabilize mood . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORTS", "Case 1", "Case 2", "Case 3", "DISCUSSION"], "sections": [["huntington 's disease ( hd ) is an autosomal dominant , progressive neurodegenerative disorder characterized by chorea , subcortical dementia and psychiatric symptoms .", "the causal hd gene has been identified as it15 , which has cag repeats in the open reading frame ( orf , in exon 1 ) just following the 5end of the gene .", "it leads to the translation of a protein , huntingtin , whose function is unknown .", "huntingtin forms intraneuronal inclusion bodies which lead to neuronal dysfunction followed by neuronal death due to apoptosis .", "the therapeutic strategies for hd are diverse , ranging from the classical drug therapies to the modern molecular strategies .", "one of the treatment options is the use of drugs with neuroprotective potential , like lithium .", "lithium has demonstrated diverse molecular effects reversing well - described pathophysiological changes such as increased oxidative stress , apoptosis , inflammation , environmental stress , glial dysfunction , neurotrophic factor deficiency , excitotoxicity as well as mitochondrial and endoplasmic reticulum disruption .", "lithium acts by inhibiting a protein- kinase called glycogen synthase kinase 3 ( gsk3 ) that has important actions on the intracellular signal transmission by protein phosphorylation .", "inhibition of this enzyme appears to have a neuroprotector effect in neurodegenerative diseases such as amyotrophic lateral sclerosis , spinocerebellar ataxia type 1 and huntington 's disease .", "we report three patients with huntington 's disease , who showed good response to treatment with low dose lithium ."], ["mrs . a , a 48-year - old homemaker , presented in 2008 with a three - year history of involuntary movements of the trunk and the hands , which had worsened over the previous 7 - 8 months .", "family history was not available . on examination , ocular saccades were slow ; she had generalized choreiform movements involving hands legs , lips and the trunk ; dystonia of hands and feet and ataxic gait .", "a genetic analysis for huntington 's disease was performed after obtaining written informed consent which showed cag repeat expansion .", "patient was started on lithium 150 mg , once daily , along with carbamazepine 200 mg twice a day .", "patient is on regular follow up for the last four years and is maintaining the same improvement .", "b , a 58-year - old male , presented in 2001 with a history of abnormal movements .", ", the movements were disabling , irregular , would worsen with anxiety and disappear during sleep .", "patient had a significant family history of hd ; involving both parents and 7 out of 9 siblings , totally 9 affected first - degree relatives . on examination , patient was noted to have severe generalized chorea involving the hands , legs , trunk and the neck .", "patient was admitted in 2009 with a worsening of movements over the previous 6 months and weight loss of about 6 kg .", "all investigations including prostate specific antigen and chest x - ray were normal and the weight loss was attributed to nutritional causes .", "family members reported that the patient was able to eat well and had no behavioral symptoms .", "the patient has been on follow up for the last three years and maintains status quo .", "c , a 51-year - old lady presented in 2002 with history of psychiatric symptoms for the previous 8 years .", "onset was sudden with symptoms of being anxious , accusing the family members of neglect , and being violent toward family members .", "patient also had history of non - rhythmic quasi - purposive movements of hands which she attributed to anxiety .", "family history revealed that her mother had movement disorder and an early death . on examination , patient c had choreic movements of the limbs , anxiety and depressive symptoms .", "a diagnosis of hd was made which was confirmed by genetic analysis , which showed expanded cag repeats in the huntingtin 's locus .", "patient was initially started on carbamazepine 400 mg and clonazepam 4 mg / day . over the next one year", ", patient developed psychotic symptoms like delusions of reference and persecution toward her family members and was also started on quetiapine up to 300 mg .", "two years later , patient had sudden deterioration in symptoms with worsening of chorea and onset of cognitive decline  memory disturbances and apraxia , which made her completely dependent for all activities of daily living . during this time , she also developed significant mood swings .", "for the last 3 years , patient has been on a combination of lithium 300 mg , carbamazepine 800 mg , quetiapine 300 mg and clonazepam 2 mg / day .", "patient 's husband reported no further deterioration over 3 years in cognition or movements in the patient .", "however , she continued to be dependent on husband for activities of daily living and eventually died due to cardiac arrest ."], ["mrs . a , a 48-year - old homemaker , presented in 2008 with a three - year history of involuntary movements of the trunk and the hands , which had worsened over the previous 7 - 8 months .", "family history was not available . on examination , ocular saccades were slow ; she had generalized choreiform movements involving hands legs , lips and the trunk ; dystonia of hands and feet and ataxic gait .", "a genetic analysis for huntington 's disease was performed after obtaining written informed consent which showed cag repeat expansion .", "patient was started on lithium 150 mg , once daily , along with carbamazepine 200 mg twice a day .", "patient is on regular follow up for the last four years and is maintaining the same improvement ."], ["b , a 58-year - old male , presented in 2001 with a history of abnormal movements .", ", the movements were disabling , irregular , would worsen with anxiety and disappear during sleep .", "patient had a significant family history of hd ; involving both parents and 7 out of 9 siblings , totally 9 affected first - degree relatives . on examination ,", "patient was noted to have severe generalized chorea involving the hands , legs , trunk and the neck .", "patient was admitted in 2009 with a worsening of movements over the previous 6 months and weight loss of about 6 kg .", "all investigations including prostate specific antigen and chest x - ray were normal and the weight loss was attributed to nutritional causes .", "family members reported that the patient was able to eat well and had no behavioral symptoms .", "the patient has been on follow up for the last three years and maintains status quo ."], ["c , a 51-year - old lady presented in 2002 with history of psychiatric symptoms for the previous 8 years .", "onset was sudden with symptoms of being anxious , accusing the family members of neglect , and being violent toward family members .", "patient also had history of non - rhythmic quasi - purposive movements of hands which she attributed to anxiety .", "family history revealed that her mother had movement disorder and an early death . on examination ,", "a diagnosis of hd was made which was confirmed by genetic analysis , which showed expanded cag repeats in the huntingtin 's locus .", "patient was initially started on carbamazepine 400 mg and clonazepam 4 mg / day . over the next one year", ", patient developed psychotic symptoms like delusions of reference and persecution toward her family members and was also started on quetiapine up to 300 mg .", "two years later , patient had sudden deterioration in symptoms with worsening of chorea and onset of cognitive decline  memory disturbances and apraxia , which made her completely dependent for all activities of daily living . during this time , she also developed significant mood swings .", "for the last 3 years , patient has been on a combination of lithium 300 mg , carbamazepine 800 mg , quetiapine 300 mg and clonazepam 2 mg / day .", "patient 's husband reported no further deterioration over 3 years in cognition or movements in the patient .", "however , she continued to be dependent on husband for activities of daily living and eventually died due to cardiac arrest ."], ["huntington 's disease ( hd ) is an autosomal dominant progressive neurodegenerative disorder caused by an expanded polyglutamine ( poly q ) tract within huntingtin protein and is characterized by neuronal loss mainly in the striatum and the cortex .", "genetically , hd is caused by a mutation in the gene located on chromosome4p 16.3 .", "the disease - causing mutation in the first exon of huntingtin ( htt ) gene is the expansion of a polymorphic cag repeat that is completely penetrant once it has reached 40 triplets .", "neuropathologically , the mutant huntington protein ( mhtt ) abnormally interacts with a variety of proteins and accumulates in the neuronal nucleus forming an inclusion body which is the hallmark of hd and leads to eventual cell death .", "in addition to the well - documented use of lithium as a mood stabilizing drug , primarily in the treatment of bipolar disorder , recent in vitro and in vivo studies in animal models have increasingly indicated that lithium can be used in the treatment of chronic neurodegenerative diseases like huntington 's disease .", "various pharmacological and gene manipulation studies support the notion that lithium 's main mechanisms of action may be its ability to inhibit gsk3[68 ] as well as induce brain - derived neurotrophic factor - mediated ( bdnf ) signaling , leading to enhanced cell survival by alteration of a wide variety of downstream effectors .", "lithium is shown to promote the action of anti - apoptotic proteins , such as heat - shock protein ( hsp ) , insulin like growth factor ( igf ) , vascular endothelial growth factor ( vegf ) and other neurotrophic factors .", "lithium can directly or indirectly modulate proteins involved in neuronal survival / differentiation which may account for its neuroprotective effects.[1012 ] by inhibiting n - methyl - d - aspartate receptor - mediated calcium influx , lithium also contributes to calcium homeostasis and suppresses calcium - dependent activation of pro - apoptotic signaling pathways .", "lithium is found to enhance autophagy of toxic aggregates in huntington 's disease in various models of hd .", "it acts by increasing mtor - independent autophagy by inhibiting inositol monophosphatase ( impase ) and reducing inositol and ip3 levels .", "induction of autophagy by lithium leads to enhanced clearance of mutant huntingtin fragments associated with huntington 's disease ( hd ) .", "these mechanisms might allow therapeutic doses of lithium to protect neuronal cells from diverse insults that would otherwise lead to massive cell death .", "lithium also stimulates proliferation of stem cells , including neuronal and astroglial progenitor cell proliferation in the sub ventricular zone , hippocampus , striatum and forebrain protecting against apoptosis .", "the stimulation of endogenous neural stem cells and neurogenesis may explain why lithium increases brain cell density and volume in patients with bipolar disorder .", "in this report , we have described the use of lithium to prevent the progression of the huntington 's disease .", "all patients reported here were started on low doses of lithium after explaining the rationale , possible side - effects and off label use of the drug .", "the patients were on regular follow up and reported no worsening of either psychiatric symptoms or choreic movements for a significant period .", "in these patients , lithium was started in order to alleviate the distress caused by the choreic movements and mood swings .", "though rating of the movements was not done , there appears be some benefit in prescribing lithium to patients with huntington 's disease for slowing the progression of chorea and preventing mood swings .", "this report signifies the use of lithium as a neuroprotective agent . it may be beneficial to explore the possibility of use of other psychotropic drugs in the treatment of neurodegenerative disorders"]]}
{"article_id": "PMC3463188", "article_text": ["  walking ability is impaired in patients with peripheral arterial disease ( pad ) due to decreased blood flow to the skeletal muscle tissue used during walking .", "it has been demonstrated that walking distance is associated with the degree of impairment in the affected leg as measured by the ankle - brachial index ( abi ) and the time to onset of claudication pain [ 16 ] .", "likewise , it has been shown that walking exercise in patients with pad prolongs the onset of claudication pain thus allowing the patient to walk longer but does not change the abi [ 79 ] .  ", "recently , several investigators have used near - infrared spectroscopy ( nirs ) to dynamically study the effect of walking on oxygen desaturation of the gastrocnemius muscle of the impaired leg during exercise [ 2 , 1014 ] .", "investigators noted a marked decline in skeletal muscle tissue oxygenation ( sto2 ) during walking exercise [ 2 , 1014 ] .", "gardner et al . also identified that the rate of decline in calf muscle sto2 was significantly associated with initial claudication time and absolute claudication time in patients with pad .", "three of the above studies examined the changes in tissue oxygenation , using nirs , in patients with pad before and after an exercise training program [ 1214 ] .", "used a pretest - post - test design and reported greater exercise duration and lower mean exercise sto2 in 15 patients after a 3-month treadmill and calf exercise training program .", "studied 55 patients with pad who were given the option to participate in a structured or unstructured home exercise program for 34  2 weeks . at the end of the study , those patients in the structured exercise program had an increased oxyhemoglobin area under the curve when compared to baseline ; similar changes were not identified in the unstructured exercise group .", "lastly , tew et al .   studied calf - tissue oxygenation and treadmill walking response time after randomization to 12 weeks of arm - crank ergometry or a control group in 57 patients with intermittent claudication .", "investigators identified that walking distance and time to nadir sto2 increased in those assigned to arm - cranking and there were no changes in the control group .", "no studies however compared leg muscle responses after a traditional walking program compared to a walking - with - poles program of rehabilitation in patients with claudication .  ", "the purpose of this study was to determine if there were differences in calf muscle sto2 parameters before and after 12 weeks of a traditional walking program versus a walking - with - poles exercise program .", "we reasoned that by using the poles to offload the legs , patients would have less claudication pain and be able to walk longer distance thus achieving a greater training effect .", "we hypothesized that the calf muscle tissue deoxygenation would be less in both groups after 12 weeks of exercise training than at baseline and that the decrease would be greater in those patients assigned to the walking - with - poles group .", "these data represent a subanalysis of data from a larger clinical trial comparing traditional walking to walking with poles .", "patients with pad were screened and recruited to participate in a randomized clinical trial designed to compare the effects of a traditional walking training program versus walking - with - poles exercise training program .", "a total of 2296 patients were screened for eligibility , 146 enrolled in the study .", "patients were recruited using radio and newspaper advertising , posted fliers , and letters of invitation sent to patients at the university and va hospitals . for the purposes of this analysis", ", we included only those participants with an abi of 0.90 ( n = 91 patients ) .", "of those 91 patients , 3 did not have sto2 analyses on their baseline progressive treadmill test due to equipment malfunction and 3 had questionable data resulting in a final sample of 85 patients .", "of those 85 patients , 45 were assigned to the walking - with - poles group and 40 patients were assigned to the traditional walking group .  ", "the major study was a randomized clinical trial to examine the effects of a traditional walking program compared to walking with poles on walking endurance and perceived physical function in patients with pad .", "our primary outcome focused on changes the patients realized walking on the constant work rate treadmill test whereas this analysis used data from the progressive treadmill test . at two minutes on the constant work rate test , the speed and incline increased to 85% of the patient 's workload on the progressive treadmill test resulting in a sudden decline in sto2 . due to the gentle nature of the progressive treadmill protocol used , we were able to detect more subtle changes in sto2 and onset of claudication pain .   ", "patients were randomized using computer - generated permuted blocks and group assignment was managed by the study statistician .", "the training programs for both groups were identical except that one group exercised with poles and the other group did not .", "patients assigned to the walking - with - poles group received training on the use of the poles prior to beginning the exercise program .", "additionally , patients were coached on proper poling mechanics during the training period if needed .", "the training program incorporated interval training whereby exercise consisted mostly of low - to - moderate intensity training at the start of the program and progressed to moderate - to - high intensity .", "exercise intensity was determined by heart rate associated with a percentage of oxygen uptake obtained during the metabolic exercise treadmill tests .", "exercise duration and intensity was adjusted every three weeks . during the first weeks , patients walked for 30 minutes ( 20% light intensity , 60% moderate intensity , and 20% hard intensity ) . by weeks 1012 , patients walked for 55 minutes ( 10% light intensity , 45% moderate intensity , and 45% hard intensity ) .", "light intensity was defined as 2544% peak vo2 , moderate intensity was 4559% peak vo2 , and hard intensity was 6084% vo2 peak .", "the program was designed so that patients walked on the treadmill two days / week and outdoors or in the hospital corridors one day / week . the exercise interval was adjusted based on the patient 's pain tolerance .", "for example , some patients could only walk for one to two minutes at the higher intensities . in order for them to achieve 20% total time at the harder intensities ,", "demographic information , height , weight , comorbid conditions , and medication history were obtained from the patient and reviewed in the patient medical record .", "the abi was measured in the more severely affected leg at baseline and 12 weeks after training .", "the location of the signal was recorded and used consistently throughout the study for that individual subject .", "after the patient rested comfortably in a supine position for 15 minutes , doppler ultrasound was used to measure the systolic pressure in the right and left arms and in the ankle of the most severely affected leg . the arm with the highest systolic pressure", "patients were tested using a gentle treadmill protocol that was developed for patients with pad [ 6 , 16 ] .", "increases in percent grade occurred every 30 seconds , and , after the first 6 minutes , speed increased every 3 minutes .", "initial claudication time was defined as the time during the treadmill test when the subject experienced pain in the affected leg . the absolute walking time was defined as the time walked by the patient on the progressive treadmill test .", "peak oxygen uptake was measured using the medgraphics cpx / d system ( medical graphics corp , st .", "the metabolic cart was calibrated using a 3  l syringe prior to each test and the analyzers were calibrated using references gasses and room air .", "patients breathed through a mouthpiece and their nose was clipped with a standard nose clip .", "oxygen uptake was averaged over 30  s and the highest value at peak exercise was recorded .", "skeletal muscle tissue oxygenation was measured prior to , during , and after exercise using the nirs spectrometer ( inspectra 325 , hutchinson technology , inc . ,", "a 25  mm probe attached to an optical cable was used to noninvasively measure the percentage of hemoglobin oxygen saturation of the tissue beneath the near - infrared light .", "the inspectra machine was calibrated prior to each test according to known high and low reference standards .", "the nirs probe was attached to the medial section of the gastrocnemius muscle of the patient 's most severely affected leg .", "the probe was placed into a specially designed adhesive housing unit to reduce ambient light .", "the probe was then secured to the leg to avoid excessive motion and possible tripping .", "the sto2 values used in this analysis were the sto2 value recorded after 2 minutes of standing prior to exercise ( sto2 rest ) , time walked when the patient reached the nadir sto2 value ( time to nadir sto2 value ) , sto2 value at peak exercise ( sto2 peak ) , and absolute and relative drop in sto2 ( absolute drop in sto2 = rest sto2  minimal sto2 value , relative drop in sto2 = [ rest sto2  nadir sto2 value]/rest sto2 )", ". ratings of perceived leg pain were obtained every minute using the borg ratio scale .", "the initial claudication time is the time that patients either notified the staff of the onset of pain or the time they first reported pain when asked every minute .  ", "the two groups were compared at baseline using the t - test for independent samples and mann whitney u test .", "regression analyses were used to compute the initial decline in slopes for sto2 values ( sto2 value regressed over time ) .", "pearson r correlations were completed to examine relationships between the variables . paired t - tests", "independent t - tests were used to examine differences between groups . intent - to - treat analyses using the baseline value carried forward were completed for the comparisons between groups .", "the sample consisted of primarily older ( age = 69.4  9.2 years ) men .", "over one - third of the patients were current smokers , half were diabetic , and the majority of patients received treatment for hypertension and dyslipidemia ( table 1 ) .", "patients were generally unfit with a peak oxygen uptake of 14.3  2.9  ml  kg  min. the walking - with - poles group was older than the traditional walking group .", "calf muscle oxygenation decreased from 56  17% prior to the treadmill test to 16  18% at peak exercise ( 71% relative decline from rest to peak exercise ) .", "patients walked 4.45  3.67 minutes before experiencing claudication pain and 4.39  3.99 minutes when the sto2 reached its nadir value .", "there was no difference between the time elapsed when the sto2 reached its nadir value and pain onset ( p = .87 ) .", "although all patients reported claudication pain , exercise was not limited by claudication pain for all patients .", "therefore , we examined the onset of claudication pain and time to nadir sto2 for those whose exercise was limited by claudication pain only ( n = 72 ) .", "patients walked 3.35  2.57 minutes before experiencing claudication pain and 3.67  3.49 minutes when the sto2 reached its nadir value .", "there was no difference between the time elapsed when the sto2 reached its nadir value and pain onset ( p = .40 ) .", "after exercise , the time for the sto2 to return to half of its resting value was 1.3  1.3  min and 2.2  2.1 minutes to return to resting values . the initial claudication time and absolute walking time", "were not associated with the sto2 values at rest ( table 2 ) but were moderately related to the time elapsed prior to reaching the nadir sto2 value ( r = .42 , p < .001 ; r = .58 , p = .001 , resp . ) .", "there was also a small to moderate relationship between onset of claudication pain and the exercise slope decline in sto2 ( r = .28 , p = .011 ) .", "relationships were similar between initial claudication time and absolute walking time and the time elapsed prior to reaching the nadir sto2 values when the analysis was restricted to those whose exercise was limited by claudication ( r = 48 , p  .001 and r = .47 , p < .001 ) .", "data comparing groups after exercise training were analyzed using intent - to - treat analysis for all patients . the change in time elapsed", "prior to reaching nadir sto2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks ( r = .65 , p < .001 ) and initial claudication time at 12 weeks ( r = .33 , p = .002 ) .", "analysis of covariance was completed to compare the groups with age as the covariate since there was a difference in age at baseline ( table 3 ) .", "the change in the time elapsed prior to reaching nadir sto2 values ( p = .002 ) and absolute walking time ( p = .002 ) were greater in the traditional walking group when compared to the walking - with - poles group .", "there was , however , no difference in the change in initial claudication time between the two groups ( p = .38 ) .  ", "when the analyses were restricted to those who completed the exercise training only ( n = 71 ) , the change in time elapsed prior to reaching nadir sto2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks ( r = .57 , p < .001 ) and initial claudication time at 12 weeks ( r = .32 , p = .004 ) . data from a sample patient are depicted in figure 1 .", "recovery time was unchanged when baseline and 12-week values were compared ( baseline = 2.45  2.17  min , 12  wk = 2.19  1.94  min , p = .81 ) .", "after 12 weeks of exercise training , within - group changes in initial claudication time , absolute claudication time and time elapsed prior to reaching nadir sto2 values increased ( table 4 . ) the time elapsed prior to reaching nadir sto2 values increased more in the traditional walking group when compared to the walking - with - poles group ( walking group = 4.01  5.13 minutes , walking - with - poles group = .89  3.56 minutes , p = .009 ) .", "similarly , absolute walking time increased more in the traditional walking group than in the walking - with - poles group ( walking group = 3.97  3.14 minutes , walking - with - poles group = 2.24  2.41 minutes , p = .017 ) .", "there was no difference between the two groups in initial claudication time ( walking group = 1.16  3.64 minutes , walking - with - poles group = .85  3.46 minutes , p = .69 ) . since there was a significant difference in age between the two groups , age", "no differences in recovery time were noted between the traditional walking and walking - with - poles groups at the 12-week follow - up time period ( traditional walking recovery time = 1.26  1.43  min , walking with poles = 1.41  1.76  min , p = .70 ) .", "no significant differences were identified in peak oxygen consumption or abi within or between the two groups from baseline to 12 weeks using intent - to - treat analysis or restricting the analysis to those who completed the training only .", "  there are three major findings of this study : ( 1 ) the time elapsed before reaching nadir sto2 values is associated with initial claudication time and absolute walking time , ( 2 ) there was no difference in the time associated with the nadir of sto2 and the onset of claudication pain , and ( 3 ) the time to nadir sto2 of the gastrocnemius muscle was greater after 12 weeks of exercise training in the traditional walking group when compared to the walking with poles group .    as bauer notes , during higher work rates and apparent blood flow limitation , muscle oxygen desaturation becomes more rapid in pad subjects .", "thus , one would expect that oxygen desaturation would be associated with onset of claudication and walking duration .", "our findings reflect these assumptions and were similar to those reported by gardner et al .", ", that is , shorter time to reach nadir sto2 values were associated with shorter initial claudication time and total walking duration .", "second , in gardner 's study , relationships between initial claudication time ( r = .339 ) , absolute walking time ( r = .68 ) and measured time to minimal exercise sto2 were similar to our relationships between initial claudication time ( r = .42 ) , absolute walking time ( r = .58 ) and measured distance to nadir sto2 .", "these findings suggest that our sample patients were similar to patients in other studies [ 2 , 19 ] .  ", "exercise - associated decline in sto2 of the gastrocnemius muscle was less after 12 weeks of exercise training when compared to baseline .", "wang and colleagues recently demonstrated in 17 subjects with pad who completed a 24-week walking program that an increase in the number of capillaries in contact with type iix and iia calf muscle fibers was related to an improved pain - free walking time ( r = .69 and r = .62 , p < .05 ) .", "investigators hypothesized that the ratio of capillaries to muscle fibers affects oxygen supply thus delaying the mismatch between supply and demand .", "both report greater exercise sto2 area under the curve postexercise training when compared to preexercise training . in our study ,", "an increase in the time to nadir sto2 values was associated with delayed initial claudication time and improved absolute walking time .", "our findings provide further evidence that walking exercise improves tissue muscle oxygenation in patients with pad .", "likewise , our findings support the possibility that the onset of claudication and walking duration are linked by the decline in tissue oxygenation .", "the onset of claudication and the time elapsed prior to reaching the nadir sto2 values were nearly identical .", "thus , the patient 's perception of pain is a good indicator of sto2 in the calf muscle .", "exercise - induced decline in gastrocnemius muscle sto2 was less in patients assigned to the traditional walking program than in patients assigned to the walking - with - poles exercise training program .", "we had originally hypothesized that a walking training program with poles for patients would be advantageous because the poles would  offload  the legs during periods of claudication allowing additional walking exercise .", "indeed , oakley et al .   investigated the immediate effects of walking with nordic poles on 20 patients with pad and found that claudication distance increased from 77  m without the poles to 130  m with the poles ( p < .0001 ) and the level of claudication pain also decreased ( p = .0002 ) .", "our group has also previously reported that patients had decreased calf pain when walking with poles .", "oakley et al .   compared muscle histology of 36 patients randomly assigned to 12 weeks of treadmill training , strength training or control .", "after the 12 weeks , only the treadmill group had histological changes in the muscle .", "the increase in muscle ischemia with treadmill training was associated with a marked increase in muscle damage .", "hiatt suggests that skeletal muscle dysfunction and associated muscle metabolic state can be ameliorated with treadmill exercise training .", "tew et al .   provides evidence that even when the exercising muscle is not the calf muscle , tissue oxygenation can improve .", "clearly , the patients assigned to the traditional walking group experienced a prolonged exercise time before experiencing claudication pain , prolonged their absolute walking time and increased the amount of time exercising until reaching nadir values . in the walking - with - poles group", ", there were no changes over time when an intent - to - treat approach was used .", "however , when the analysis was restricted to those who completed the training program only , there was a difference in absolute walking time and a trend toward increasing time to initial claudication pain onset as well as nadir sto2 .", "the explanation for these findings may be that leg metabolic changes in the calf muscle occurred to a greater extent in those assigned to the walking - with - poles group or we may not have had enough statistical power to detect changes that were present .", "another explanation for this finding may be that the intensity of our training program was based on oxygen uptake associated with heart rate response to exercise . with the addition of the upper body to the exercise regime for patients assigned to the walking - with - poles group", "thus , the work accomplished by the leg muscles may have been less even though overall exercise intensity was consistent between the two groups of patients . both groups trained on the treadmill and outdoors .", "patients assigned to the walking with poles group were unable to use the handrails on the treadmill for balance ; added anxiety may have increased the cardiovascular response thus keeping the overall work accomplished lower in some patients .  ", "first , although age was not a contributing factor to the differences between the groups , the walking - with - poles group was older than the traditional walking group .", "we can not exclude that the differences in exercise tolerance and willingness to push limits may differ between someone who is 67 years old ( mean age for walking group ) and someone who is 72 years old ( mean age for walking - with - poles group ) .", "this five - year age difference may have influenced our outcomes in ways that we did not measure .", "next , the sample was primarily men and thus can not be generalized to women .", "additionally , in order to include only those with compressible vessels , we excluded 17 patients who were initially randomized to our study .", "lastly , although all of our patients experienced claudication pain , not all patients stopped exercise due to claudication pain . including patients that did not stop exercise due to claudication pain", "additional studies examining different walking intensities in patients with peripheral arterial disease should be tested .", "further , it may be that use of walking poles early in the rehabilitative effort may augment traditional walking training later by assisting patients to train longer early on .", "in summary , we conclude that there is a significant decline in sto2 in the gastrocnemius muscle during walking exercise in patients with peripheral arterial disease reflecting muscle tissue ischemia .", "contrary to our hypothesis , after 12 weeks of exercise training , absolute walking time and time to reach nadir muscle tissue oxygenation were prolonged in the patients assigned to the traditional walking program more than those assigned to a walking - with - poles exercise training program reflecting greater tissue perfusion and enhancement of arteriovenous oxygen extraction .", "the precise mechanism for these differences remains unknown but these findings may provide some insight to tissue adaptations to exercise stimulus and may therefore be useful to pad rehabilitation programs ."], "abstract_text": ["<S> this randomized trial proposed to determine if there were differences in calf muscle sto2 parameters in patients before and after 12 weeks of a traditional walking or walking - with - poles exercise program . </S>", "<S> data were collected on 85 patients who were randomized to a traditional walking program ( n = 40 ) or walking - with - poles program ( n = 45 ) of exercise training . </S>", "<S> patients walked for 3 times weekly for 12 weeks . </S>", "<S> seventy - one patients completed both the baseline and the 12-week follow - up progressive treadmill tests ( n = 36 traditional walking and n = 35 walking - with - poles ) . using the near - infrared spectroscopy measures , sto2 was measured prior to , during , and after exercise . at baseline , </S>", "<S> calf muscle oxygenation decreased from 56  17% prior to the treadmill test to 16  18% at peak exercise . </S>", "<S> the time elapsed prior to reaching nadir sto2 values increased more in the traditional walking group when compared to the walking - with - poles group . likewise , </S>", "<S> absolute walking time increased more in the traditional walking group than in the walking - with - poles group . </S>", "<S> tissue oxygenation decline during treadmill testing was less for patients assigned to a 12-week traditional walking program when compared to those assigned to a 12-week walking - with - poles program . in conclusion , </S>", "<S> the 12-week traditional walking program was superior to walking - with - poles in improving tissue deoxygenation in patients with pad . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["  walking ability is impaired in patients with peripheral arterial disease ( pad ) due to decreased blood flow to the skeletal muscle tissue used during walking .", "it has been demonstrated that walking distance is associated with the degree of impairment in the affected leg as measured by the ankle - brachial index ( abi ) and the time to onset of claudication pain [ 16 ] .", "likewise , it has been shown that walking exercise in patients with pad prolongs the onset of claudication pain thus allowing the patient to walk longer but does not change the abi [ 79 ] .  ", "recently , several investigators have used near - infrared spectroscopy ( nirs ) to dynamically study the effect of walking on oxygen desaturation of the gastrocnemius muscle of the impaired leg during exercise [ 2 , 1014 ] .", "investigators noted a marked decline in skeletal muscle tissue oxygenation ( sto2 ) during walking exercise [ 2 , 1014 ] .", "gardner et al . also identified that the rate of decline in calf muscle sto2 was significantly associated with initial claudication time and absolute claudication time in patients with pad .", "three of the above studies examined the changes in tissue oxygenation , using nirs , in patients with pad before and after an exercise training program [ 1214 ] .", "used a pretest - post - test design and reported greater exercise duration and lower mean exercise sto2 in 15 patients after a 3-month treadmill and calf exercise training program .", "studied 55 patients with pad who were given the option to participate in a structured or unstructured home exercise program for 34  2 weeks . at the end of the study , those patients in the structured exercise program had an increased oxyhemoglobin area under the curve when compared to baseline ; similar changes were not identified in the unstructured exercise group .", "lastly , tew et al .   studied calf - tissue oxygenation and treadmill walking response time after randomization to 12 weeks of arm - crank ergometry or a control group in 57 patients with intermittent claudication .", "investigators identified that walking distance and time to nadir sto2 increased in those assigned to arm - cranking and there were no changes in the control group .", "no studies however compared leg muscle responses after a traditional walking program compared to a walking - with - poles program of rehabilitation in patients with claudication .  ", "the purpose of this study was to determine if there were differences in calf muscle sto2 parameters before and after 12 weeks of a traditional walking program versus a walking - with - poles exercise program .", "we reasoned that by using the poles to offload the legs , patients would have less claudication pain and be able to walk longer distance thus achieving a greater training effect .", "we hypothesized that the calf muscle tissue deoxygenation would be less in both groups after 12 weeks of exercise training than at baseline and that the decrease would be greater in those patients assigned to the walking - with - poles group .", "these data represent a subanalysis of data from a larger clinical trial comparing traditional walking to walking with poles ."], ["patients with pad were screened and recruited to participate in a randomized clinical trial designed to compare the effects of a traditional walking training program versus walking - with - poles exercise training program .", "a total of 2296 patients were screened for eligibility , 146 enrolled in the study .", "patients were recruited using radio and newspaper advertising , posted fliers , and letters of invitation sent to patients at the university and va hospitals . for the purposes of this analysis", ", we included only those participants with an abi of 0.90 ( n = 91 patients ) .", "of those 91 patients , 3 did not have sto2 analyses on their baseline progressive treadmill test due to equipment malfunction and 3 had questionable data resulting in a final sample of 85 patients .", "of those 85 patients , 45 were assigned to the walking - with - poles group and 40 patients were assigned to the traditional walking group .  ", "the major study was a randomized clinical trial to examine the effects of a traditional walking program compared to walking with poles on walking endurance and perceived physical function in patients with pad .", "our primary outcome focused on changes the patients realized walking on the constant work rate treadmill test whereas this analysis used data from the progressive treadmill test . at two minutes on the constant work rate test , the speed and incline increased to 85% of the patient 's workload on the progressive treadmill test resulting in a sudden decline in sto2 . due to the gentle nature of the progressive treadmill protocol used , we were able to detect more subtle changes in sto2 and onset of claudication pain .   ", "patients were randomized using computer - generated permuted blocks and group assignment was managed by the study statistician .", "the training programs for both groups were identical except that one group exercised with poles and the other group did not .", "patients assigned to the walking - with - poles group received training on the use of the poles prior to beginning the exercise program .", "additionally , patients were coached on proper poling mechanics during the training period if needed .", "the training program incorporated interval training whereby exercise consisted mostly of low - to - moderate intensity training at the start of the program and progressed to moderate - to - high intensity .", "exercise intensity was determined by heart rate associated with a percentage of oxygen uptake obtained during the metabolic exercise treadmill tests .", "exercise duration and intensity was adjusted every three weeks . during the first weeks , patients walked for 30 minutes ( 20% light intensity , 60% moderate intensity , and 20% hard intensity ) . by weeks 1012 , patients walked for 55 minutes ( 10% light intensity , 45% moderate intensity , and 45% hard intensity ) .", "light intensity was defined as 2544% peak vo2 , moderate intensity was 4559% peak vo2 , and hard intensity was 6084% vo2 peak .", "the program was designed so that patients walked on the treadmill two days / week and outdoors or in the hospital corridors one day / week . the exercise interval was adjusted based on the patient 's pain tolerance .", "for example , some patients could only walk for one to two minutes at the higher intensities . in order for them to achieve 20% total time at the harder intensities ,", "demographic information , height , weight , comorbid conditions , and medication history were obtained from the patient and reviewed in the patient medical record .", "the abi was measured in the more severely affected leg at baseline and 12 weeks after training .", "the location of the signal was recorded and used consistently throughout the study for that individual subject .", "after the patient rested comfortably in a supine position for 15 minutes , doppler ultrasound was used to measure the systolic pressure in the right and left arms and in the ankle of the most severely affected leg . the arm with the highest systolic pressure", "patients were tested using a gentle treadmill protocol that was developed for patients with pad [ 6 , 16 ] .", "increases in percent grade occurred every 30 seconds , and , after the first 6 minutes , speed increased every 3 minutes .", "initial claudication time was defined as the time during the treadmill test when the subject experienced pain in the affected leg . the absolute walking time was defined as the time walked by the patient on the progressive treadmill test .", "peak oxygen uptake was measured using the medgraphics cpx / d system ( medical graphics corp , st .", "the metabolic cart was calibrated using a 3  l syringe prior to each test and the analyzers were calibrated using references gasses and room air .", "patients breathed through a mouthpiece and their nose was clipped with a standard nose clip .", "oxygen uptake was averaged over 30  s and the highest value at peak exercise was recorded .", "skeletal muscle tissue oxygenation was measured prior to , during , and after exercise using the nirs spectrometer ( inspectra 325 , hutchinson technology , inc . ,", "a 25  mm probe attached to an optical cable was used to noninvasively measure the percentage of hemoglobin oxygen saturation of the tissue beneath the near - infrared light .", "the inspectra machine was calibrated prior to each test according to known high and low reference standards .", "the nirs probe was attached to the medial section of the gastrocnemius muscle of the patient 's most severely affected leg .", "the probe was placed into a specially designed adhesive housing unit to reduce ambient light .", "the probe was then secured to the leg to avoid excessive motion and possible tripping .", "the sto2 values used in this analysis were the sto2 value recorded after 2 minutes of standing prior to exercise ( sto2 rest ) , time walked when the patient reached the nadir sto2 value ( time to nadir sto2 value ) , sto2 value at peak exercise ( sto2 peak ) , and absolute and relative drop in sto2 ( absolute drop in sto2 = rest sto2  minimal sto2 value , relative drop in sto2 = [ rest sto2  nadir sto2 value]/rest sto2 )", ". ratings of perceived leg pain were obtained every minute using the borg ratio scale .", "the initial claudication time is the time that patients either notified the staff of the onset of pain or the time they first reported pain when asked every minute .  ", "the two groups were compared at baseline using the t - test for independent samples and mann whitney u test .", "regression analyses were used to compute the initial decline in slopes for sto2 values ( sto2 value regressed over time ) .", "pearson r correlations were completed to examine relationships between the variables . paired t - tests", "independent t - tests were used to examine differences between groups . intent - to - treat analyses using the baseline value carried forward were completed for the comparisons between groups ."], ["the sample consisted of primarily older ( age = 69.4  9.2 years ) men .", "over one - third of the patients were current smokers , half were diabetic , and the majority of patients received treatment for hypertension and dyslipidemia ( table 1 ) .", "patients were generally unfit with a peak oxygen uptake of 14.3  2.9  ml  kg  min. the walking - with - poles group was older than the traditional walking group .", "calf muscle oxygenation decreased from 56  17% prior to the treadmill test to 16  18% at peak exercise ( 71% relative decline from rest to peak exercise ) .", "patients walked 4.45  3.67 minutes before experiencing claudication pain and 4.39  3.99 minutes when the sto2 reached its nadir value .", "there was no difference between the time elapsed when the sto2 reached its nadir value and pain onset ( p = .87 ) .", "although all patients reported claudication pain , exercise was not limited by claudication pain for all patients .", "therefore , we examined the onset of claudication pain and time to nadir sto2 for those whose exercise was limited by claudication pain only ( n = 72 ) .", "patients walked 3.35  2.57 minutes before experiencing claudication pain and 3.67  3.49 minutes when the sto2 reached its nadir value .", "there was no difference between the time elapsed when the sto2 reached its nadir value and pain onset ( p = .40 ) .", "after exercise , the time for the sto2 to return to half of its resting value was 1.3  1.3  min and 2.2  2.1 minutes to return to resting values . the initial claudication time and absolute walking time", "were not associated with the sto2 values at rest ( table 2 ) but were moderately related to the time elapsed prior to reaching the nadir sto2 value ( r = .42 , p < .001 ; r = .58 , p = .001 , resp . ) .", "there was also a small to moderate relationship between onset of claudication pain and the exercise slope decline in sto2 ( r = .28 , p = .011 ) .", "relationships were similar between initial claudication time and absolute walking time and the time elapsed prior to reaching the nadir sto2 values when the analysis was restricted to those whose exercise was limited by claudication ( r = 48 , p  .001 and r = .47 , p < .001 ) .", "data comparing groups after exercise training were analyzed using intent - to - treat analysis for all patients . the change in time elapsed", "prior to reaching nadir sto2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks ( r = .65 , p < .001 ) and initial claudication time at 12 weeks ( r = .33 , p = .002 ) .", "analysis of covariance was completed to compare the groups with age as the covariate since there was a difference in age at baseline ( table 3 ) .", "the change in the time elapsed prior to reaching nadir sto2 values ( p = .002 ) and absolute walking time ( p = .002 ) were greater in the traditional walking group when compared to the walking - with - poles group .", "there was , however , no difference in the change in initial claudication time between the two groups ( p = .38 ) .  ", "when the analyses were restricted to those who completed the exercise training only ( n = 71 ) , the change in time elapsed prior to reaching nadir sto2 values between baseline and 12 weeks was related to the change in absolute walking time between baseline and 12 weeks ( r = .57 , p < .001 ) and initial claudication time at 12 weeks ( r = .32 , p = .004 ) . data from a sample patient are depicted in figure 1 .", "recovery time was unchanged when baseline and 12-week values were compared ( baseline = 2.45  2.17  min , 12  wk = 2.19  1.94  min , p = .81 ) .", "after 12 weeks of exercise training , within - group changes in initial claudication time , absolute claudication time and time elapsed prior to reaching nadir sto2 values increased ( table 4 . ) the time elapsed prior to reaching nadir sto2 values increased more in the traditional walking group when compared to the walking - with - poles group ( walking group = 4.01  5.13 minutes , walking - with - poles group = .89  3.56 minutes , p = .009 ) .", "similarly , absolute walking time increased more in the traditional walking group than in the walking - with - poles group ( walking group = 3.97  3.14 minutes , walking - with - poles group = 2.24  2.41 minutes , p = .017 ) .", "there was no difference between the two groups in initial claudication time ( walking group = 1.16  3.64 minutes , walking - with - poles group = .85  3.46 minutes , p = .69 ) . since there was a significant difference in age between the two groups , age", "no differences in recovery time were noted between the traditional walking and walking - with - poles groups at the 12-week follow - up time period ( traditional walking recovery time = 1.26  1.43  min , walking with poles = 1.41  1.76  min , p = .70 ) .", "no significant differences were identified in peak oxygen consumption or abi within or between the two groups from baseline to 12 weeks using intent - to - treat analysis or restricting the analysis to those who completed the training only ."], ["  there are three major findings of this study : ( 1 ) the time elapsed before reaching nadir sto2 values is associated with initial claudication time and absolute walking time , ( 2 ) there was no difference in the time associated with the nadir of sto2 and the onset of claudication pain , and ( 3 ) the time to nadir sto2 of the gastrocnemius muscle was greater after 12 weeks of exercise training in the traditional walking group when compared to the walking with poles group .    as bauer notes , during higher work rates and apparent blood flow limitation , muscle oxygen desaturation becomes more rapid in pad subjects .", "thus , one would expect that oxygen desaturation would be associated with onset of claudication and walking duration .", "our findings reflect these assumptions and were similar to those reported by gardner et al .", ", that is , shorter time to reach nadir sto2 values were associated with shorter initial claudication time and total walking duration .", "second , in gardner 's study , relationships between initial claudication time ( r = .339 ) , absolute walking time ( r = .68 ) and measured time to minimal exercise sto2 were similar to our relationships between initial claudication time ( r = .42 ) , absolute walking time ( r = .58 ) and measured distance to nadir sto2 .", "these findings suggest that our sample patients were similar to patients in other studies [ 2 , 19 ] .  ", "exercise - associated decline in sto2 of the gastrocnemius muscle was less after 12 weeks of exercise training when compared to baseline .", "wang and colleagues recently demonstrated in 17 subjects with pad who completed a 24-week walking program that an increase in the number of capillaries in contact with type iix and iia calf muscle fibers was related to an improved pain - free walking time ( r = .69 and r = .62 , p < .05 ) .", "investigators hypothesized that the ratio of capillaries to muscle fibers affects oxygen supply thus delaying the mismatch between supply and demand .", "both report greater exercise sto2 area under the curve postexercise training when compared to preexercise training . in our study ,", "an increase in the time to nadir sto2 values was associated with delayed initial claudication time and improved absolute walking time .", "our findings provide further evidence that walking exercise improves tissue muscle oxygenation in patients with pad .", "likewise , our findings support the possibility that the onset of claudication and walking duration are linked by the decline in tissue oxygenation .", "the onset of claudication and the time elapsed prior to reaching the nadir sto2 values were nearly identical .", "thus , the patient 's perception of pain is a good indicator of sto2 in the calf muscle .", "exercise - induced decline in gastrocnemius muscle sto2 was less in patients assigned to the traditional walking program than in patients assigned to the walking - with - poles exercise training program .", "we had originally hypothesized that a walking training program with poles for patients would be advantageous because the poles would  offload  the legs during periods of claudication allowing additional walking exercise .", "indeed , oakley et al .   investigated the immediate effects of walking with nordic poles on 20 patients with pad and found that claudication distance increased from 77  m without the poles to 130  m with the poles ( p < .0001 ) and the level of claudication pain also decreased ( p = .0002 ) .", "our group has also previously reported that patients had decreased calf pain when walking with poles .", "oakley et al .   compared muscle histology of 36 patients randomly assigned to 12 weeks of treadmill training , strength training or control .", "after the 12 weeks , only the treadmill group had histological changes in the muscle .", "the increase in muscle ischemia with treadmill training was associated with a marked increase in muscle damage .", "hiatt suggests that skeletal muscle dysfunction and associated muscle metabolic state can be ameliorated with treadmill exercise training .", "tew et al .   provides evidence that even when the exercising muscle is not the calf muscle , tissue oxygenation can improve .", "clearly , the patients assigned to the traditional walking group experienced a prolonged exercise time before experiencing claudication pain , prolonged their absolute walking time and increased the amount of time exercising until reaching nadir values . in the walking - with - poles group", ", there were no changes over time when an intent - to - treat approach was used .", "however , when the analysis was restricted to those who completed the training program only , there was a difference in absolute walking time and a trend toward increasing time to initial claudication pain onset as well as nadir sto2 .", "the explanation for these findings may be that leg metabolic changes in the calf muscle occurred to a greater extent in those assigned to the walking - with - poles group or we may not have had enough statistical power to detect changes that were present .", "another explanation for this finding may be that the intensity of our training program was based on oxygen uptake associated with heart rate response to exercise . with the addition of the upper body to the exercise regime for patients assigned to the walking - with - poles group", "thus , the work accomplished by the leg muscles may have been less even though overall exercise intensity was consistent between the two groups of patients . both groups trained on the treadmill and outdoors .", "patients assigned to the walking with poles group were unable to use the handrails on the treadmill for balance ; added anxiety may have increased the cardiovascular response thus keeping the overall work accomplished lower in some patients .  ", "first , although age was not a contributing factor to the differences between the groups , the walking - with - poles group was older than the traditional walking group .", "we can not exclude that the differences in exercise tolerance and willingness to push limits may differ between someone who is 67 years old ( mean age for walking group ) and someone who is 72 years old ( mean age for walking - with - poles group ) .", "this five - year age difference may have influenced our outcomes in ways that we did not measure .", "next , the sample was primarily men and thus can not be generalized to women .", "additionally , in order to include only those with compressible vessels , we excluded 17 patients who were initially randomized to our study .", "lastly , although all of our patients experienced claudication pain , not all patients stopped exercise due to claudication pain . including patients that did not stop exercise due to claudication pain", "additional studies examining different walking intensities in patients with peripheral arterial disease should be tested .", "further , it may be that use of walking poles early in the rehabilitative effort may augment traditional walking training later by assisting patients to train longer early on ."], ["in summary , we conclude that there is a significant decline in sto2 in the gastrocnemius muscle during walking exercise in patients with peripheral arterial disease reflecting muscle tissue ischemia .", "contrary to our hypothesis , after 12 weeks of exercise training , absolute walking time and time to reach nadir muscle tissue oxygenation were prolonged in the patients assigned to the traditional walking program more than those assigned to a walking - with - poles exercise training program reflecting greater tissue perfusion and enhancement of arteriovenous oxygen extraction .", "the precise mechanism for these differences remains unknown but these findings may provide some insight to tissue adaptations to exercise stimulus and may therefore be useful to pad rehabilitation programs ."]]}
{"article_id": "PMC56898", "article_text": ["access to automated dna sequencing technology has made possible the rapid generation and analysis of gene transcripts expressed in organisms via expressed sequence tags ( ests ) .", "this information has helped to identify those genes expressed in particular stages of development and in specialized tissues or organs .", "novel gene products and target leads for therapeutic intervention can also be gleaned rapidly from ests . a more detailed understanding of the molecular interactions between symbionts , whether pathogenic or mutualistic , is also possible with this approach . for a sequence isolated from interacting symbionts , determining its cellular role ( or roles )", "we refer to this challenge as ' the problem ' : given a sequence x expressed in an interaction between species a and b , did x originate from a or b ?", "various solutions are readily conceived , each with merits and faults . here , we show that a comparative lexical analysis of word counts ( specifically , hexamer frequencies ) , previously used to detect library contamination in sequencing projects , provides a powerful computational basis to infer a transcript 's species of origin . experimentally , one can attempt to solve the problem by hybridizing a clone ( as probe ) to genomic dna ( target ) from both species and determining to which target the probe hybridizes .", "however , if a sequence is highly conserved in the two taxa , hybridization stringency conditions can influence the outcome considerably . for high - throughput est sequence analysis ,", "source verification by hybridization is impractical in terms of time and reagents . as an alternative to in vitro hybridization , several computational solutions are possible .", "were the genome sequence of both species completely determined , one could simply use sequence similarity searching . however , most plant hosts and their microbial symbionts have little or no genomic sequence data available , which makes this approach very unreliable .", "strong similarity to a sequence from one organism does not preclude the possibility that a similar sequence is present in the other species .", "exploiting this fact may seem a viable solution to the problem , as it has proven suitable for predicting the presence of introns among exons in genomic dna .", "however , it really is not practical , because of the need to know the reading frame for translation of a messenger rna into an amino acid .", "est data are of notoriously unreliable quality , sometimes having a large proportion of ambiguous bases , and sometimes having single base - pair insertions or deletions , which disrupt a reading frame .", "word counting is less prone to these sources of error , and uses information intrinsic to biases in codon usage by counting codon pairs as hexamers in a sliding window , whereas codons are read in non - overlapping , tiled windows .", "an intuitive approach to the problem that examines sequence composition is to compare the guanine and cytosine ( gc ) base content of a sequence with other sequences from the species being studied .", "when two species ' genomes have different gc content , this method can be very useful .", "in a recent investigation , for instance , sequences from the stramenopile plant pathogen phytophthora sojae and its soybean ( glycine max ) host showed a 20% difference in mean gc content . the origin of a number of sequences could readily be identified this way , but a large proportion could not , because of considerable overlap in the distributions ' tails .", "counting frequencies of gc is simple word counting , where the word size k is 1/2 : only two semi - words , g / c and a / t are counted .", "an alternative approach to determining the origin of a sequence is suggested by previous work on analysis of word counts , or k - tuple frequencies , which was intended as a means of evaluating a library for contamination when sequencing from a single model organism .", "the word - counting method provides distinct advantages over other computational methods . unlike sequence - similarity searching", ", it does not require that the full protein - coding content of both genomes be known for reasonable inferences to be made .", "further , word counting is sensitive to biases in codon usage and gc content commonly observed when comparing taxa , but does not require knowledge of the reading frame for amino - acid translation .", "that is , the underlying differences between the two organisms that result in base composition or codon usage biases can also be detected by counting words .", "unlike gc analysis , lexical analysis establishes a clear threshold above or below which we can infer the species of origin , and a confidence level for an inference can readily be assigned .", "dunning 's likelihood - ratio test of word dissimilarities   also has the appealing property of being non - parametric , having no assumption of normality for the underlying frequency distribution , which makes it statistically powerful . dunning   demonstrated that unreliable results can be obtained from parametric tests , such as  , particularly in such cases as lexical analysis . in the experiments detailed below , we first validate the word - counting method on sequences whose", "origin and function are known , then compare it with ability to diagnose the origin of sequences with distributions of gc content .", "we examine sequences from pathogenic interactions between species from the genus phytophthora and the plant hosts g. max and medicago truncatula , then apply the word - counting approach to sequences from two microbial mutualists in association with m. truncatula , the arbuscular mycorrhizal zygomycete glomus versiforme , and the nitrogen - fixing bacterium sinorhizobium meliloti .", "validation sequence accession numbers , gene names , and comparison results appear in table 1 .", "the word - counting method was generally quite reliable when tested against sequences of known origin , being wrong in 3 cases out of 50 ; a phosphate transporter from g. versiforme and two in planta - induced genes from phytophthora infestans were misidentified as plant sequences .", "this indicates a failure rate of 6% - all false negatives under the null hypothesis that a transcript originates from the plant host .", "performance of the method was not influenced by whether the isolated source of a sequence was an mrna or dna molecule , as indicated by the column labeled ' mrna ? ' .", "distributions of gc content are approximately normal in two of three cases studied , those of axenic p. sojae cultures ( figure 1 ) . for sequences from infected plant cultures ,", "roughly 25% of a total of 927 infected g. max sequences contain less than 50% gc ; most of these are likely to be plant transcripts .", "this is a considerably greater number than for axenic p. sojae cultures , in which fewer than 5% of mycelia and zoospore isolates contain less than 50% gc .", "several properties of cumulative distribution functions warrant comment , to help explain similar plots from word dissimilarity comparisons ( figures 1b,2a ) .", "the median of a distribution occurs where the function reaches a cumulative probability of 0.5 .", "medians from all three p. sojae libraries are similar , varying by less than 4% gc ( figure 1b ) .", "other moments of the distributions are readily apparent ; the variance is inversely related to the slope at the median value of the function", ". a useful property of cumulative distribution functions is that any point on the y axis gives the integrated area ( cumulative probability ) under the curve .", "we use this property to establish experiment - wide false - positive and false - negative rates ( figure 2a ) . in this case ,", "calibration curves from hexamer dissimilarity tests , shown in figure 2b as solid black lines for plant and dashed black lines for stramenopile training sequences are approximately normal .", "the medians differ considerably , with only about 10% percent overlap in the two distributions ' tails about the neutral t - value of zero . superimposed", "are comparison curves from p. sojae test sets ( figure 2b ) , which parallel the gc content curves in figure 1b but show slightly less variance .", "axenic sequences are clearly more like stramenopiles ( b1 ) than plants ( a1 ) in hexamer composition , with all but a small percentage having positive t values .", "plant - like sequences are as abundant in the mixed library as detected by gc content , about 23% . as expected , the two methods agree , having positively correlated values for gc and t ( r = 0.852 , p < 10 , v = 2,641 ) . looking in more detail at the paired dissimilarity values ( figure 3 ) ,", "the magnitudes of dissimilarity are also apparent , with longer sequences having larger dissimilarity values .", "blastx similarity searches against the protein sequences in nr , a non - redundant library of proteins   revealed that none of the 12 plant - like mycelial transcripts significantly resemble known proteins ( e > 10 ) . among the top ten most plant - like transcripts from the infected g. max library", ", three had no significant matches , four matched putative arabidopsis thaliana proteins , and three matched known g. max proteins : cytochrome p450 ( accession af022460 , e < 10 ) , methylglyoxalase ( accession p46417 , e < 10 ) , and a ripening related protein ( accession af127110 , e < 10 ) .", "thus , the majority of the most plant - like transcripts in the infected soybean library strongly resemble characterized plant sequences .", "analysis results from all p. sojae and mixed - culture transcripts are available as additional data files , grouped by the library from which transcripts were sequenced .", "figure 4 shows that calibration curves from comparing plant and microbial symbiont training sets have good separation and minimal overlap ( about 10% ) in two of three cases , but not for training set b2 , comprised of zygomycetes and chytridiomycetes , which overlaps considerably with plants ( figure 4b ) .", "when comparing between plants and bacteria , the error rates are  = 0.052 and  = 0.084 , much lower than when comparing plants ( a2 , medicago ) with fungi ( b2 , zygomycetes and chytridiomycetes ) .", "error rates for comparing stramenopiles and p. infestans ests with plants are as in figure 2 (  = 0.088 ,  = 0.032 ) . also shown in figure 4", "all resemble calibration curves from plant sequences , having similar medians and slightly less variance than the plant calibration curves .", "comparison curves show that the great majority of test sequences are more plant - like than otherwise , with 20% or less resembling microbial symbionts more closely than plants", ". a greater proportion of microbial sequences is present in the m. truncatula - g .", "versiforme interaction library ( 20% , figure 4b ) than in the p. medicaginis - infected m. truncatula library ( 5% , figure 4a ) .", "however , long 's root - hair enriched library ( mtrhe )   had a greater proportion of putative microbial sequences present ( 7% and 25% ) than any of the libraries isolated from symbiont - associated cultures .", "the axenic and nodulating root libraries had the smallest portion of putative microbial transcripts ( < 2% , figure 4c ) , with the axenic library closely resembling nodulating root libraries .", "the method of preparing a library can affect the proportion of plant and non - plant sequences , as discussed below .", "paired dissimilarity values in figure 5 show in greater detail which sequences are more or less like plant and symbiont .", "considerable variation in the degree of dissimilarity to both training sets is clear , largely due to variation in the length of sequences within test sets .", "d(b ) in figure 4 , most sequences lie above the identity function , and resemble the plant host more closely than the microbial symbiont .", "mycorrhizal test sequences are more difficult to differentiate than sequences from the rhizobacterial or pathogenic associations , as seen by the diminished variation about the identity function in mycorrhizal comparisons ( figure 5b ) , contrasted with comparisons from pathogen - infected and nodulating root libraries ( figures 5a and c , respectively ) .", "analysis results from all m. truncatula and mixed - culture transcripts are available as additional data files , grouped by the library from which transcripts were sequenced , and sorted from the least plant - like transcripts to the most plant - like .", "clearly , the word - counting approach provides a reliable solution to the problem of source identification with known confidence , and has several significant advantages .", "the reliability of the method is best justified in terms of the favorable validation test results , and is further corroborated by agreement with an analysis of gc content . in test cases", "where the correct answer is known a priori , results were correct within error rates expected from overlap in training sets .", "( recall that  = 0.088 for comparisons between plants and stramenopiles , and  = 0.052 for comparisons between plants and bacteria . ) unlike gc content , the problem is clearly resolved by word counting with a threshold value of t = 0 , and with statistical rigor , because false - positive and false - negative rates for a set of comparisons are readily computed from cumulative distributions of dissimilarity between two training sets .", "optimal statistical power ( minimal false - negative rate ) is ensured when using a likelihood - ratio test statistic , as demonstrated by the pearson - neyman theorem .", "rather , it is sufficient that the training set be related to , but not necessarily congeners of , the species from which sequences are being compared .", "sequences from several species of the genus phytophthora were correctly distinguished from plant and bacterial sequences , and three genes from agrobacterium tumefaciens were correctly identified as representing a bacterial sequence .", "however , several caveats warrant prudence . transcribed sequences that do not encode proteins , but rather catalytic single - stranded rnas such as transfer and ribosomal rnas , should be treated independently because they are more highly conserved across taxa than messenger rnas . also , filtering or trimming of low - complexity repeat regions , such as poly(a ) or poly(t ) tracts , is helpful because comparison results can be influenced by the abundance of a single hexamer .", "early in our investigations , using one set of training sequences obtained from directionally cloned p.", "it eventually became clear that , as the p. infestans sequences were all single - pass reads from the 5 ' end of a clone generated with the t3 primer , few sequences complementary to the 3 ' end of the mrna sequence were present in the training set .", "large amounts of the poly(t ) hexamer would be expected when sequencing reverse complements of mrnas obtained from 3 ' sequences generated with the t7 primer .", "as a result , any sequence that contained a poly(t ) tract tended to resemble the plant sequences .", "further , because the error rates for an inference depend on the degree to which calibration curves overlap , the best results are obtained where overlap is minimal . despite these caveats , word counting presents a viable solution to the problem .", "the p. sojae - infected g. max library provides a clear example of contrast in both hexamer composition and gc content , resulting in readily diagnosed origins .", "for clear separation between the two species to appear , the two must differ in composition and a detectable proportion of transcripts from each species must be present in the library . to be detectable , the proportion of transcripts present from a particular species must be greater than the error rate obtained from calibration curves . though these criteria are true for the infected g. max library ( t < 0 for < 25% of 927 transcripts ) , they do not appear to be true for the m. truncatula libraries we analyzed ( t < 0 for 8099% of 8903,017 transcripts ) . in the p. medicaginis interaction library", "the p. sojae - infected library was prepared two days after infection , using a susceptible plant host strain , so as to maximize the number of pathogen transcripts present in the host tissue .", "medicaginis - infected library was prepared ten days after infection and individual plants varied in their degree of susceptibility ( c. vance , unpublished data ) .", "plants were also inoculated in a different manner : ground mycelia were dissolved in sterile water and incubated , and the resulting inoculum was pipetted onto the soil surface , rather than the plant .", "these differences in how tissues were cultured prior to library preparation could have produced the disparate abundance of plant transcripts , though both libraries were prepared from plant tissues infected with phytophthora . for mycorrhizal root libraries", ", we might explain the relative lack of symbiont sequences as resulting simply from a relative lack of transcripts in the host tissue .", "we might therefore expect that most of the transcripts therein originate from the plant host . confounding this result ,", "the error rates in this comparison are the greatest among all the comparisons we performed , most likely because the evolutionary distance between fungi ( zygomycetes and chytridiomycetes ) and plants is the least among comparisons . also , zygomycete protein - coding sequences are rare in genbank , which resulted in a small training set for these fungi , and may have amplified any biases .", "the high false - negative rate probably led to a failure to detect some symbiont transcripts . in nodulating root libraries", ", we do not expect to observe an abundance of bacterial transcripts , because bacteria generally do not form polyadenylated mrnas . as the protocols used to extract and purify mrnas from tissue lysate for the libraries cited in this study all relied on the presence of polyadenylation sites", "the abundance of putative microbial symbiont transcripts among sequences from a pure plant root library is difficult to interpret .", "the predicted portion of microbial transcripts was greater in the axenic root - hair enriched library than in mixed cultures .", "error rates were greatest for comparisons between training sets from plant and pooled zygomycete and chytridiomycete sequences .", "the 13% false - positive rate does not completely explain why about 15% of root - hair enriched transcripts resemble fungal hexamer composition more closely than plants , and warrants further study .", "care had been taken to avoid contaminating plant tissue cultures by culturing seedlings in covered plates .", "because of concern that ethylene accumulation in covered plates could improperly stimulate nodulation - related gene expression , seedlings were treated with ag2so4 , an inhibitor of the plants ' response to ethylene .", "inhibition of the ethylene response could have resulted in synthesis of transcripts that are uncharacteristic of plant roots .", "analysis of another axenic root - hair enriched library , particularly one provided a carbon source to identify potential contaminants , and not treated with an inhibitor of ethylene response , would be an informative test .", "the transcripts identified as most and least like plant or symbiont might also be studied in more detail as candidate participants in symbiosis .", "symbiotic interactions , whether pathogenic or mutualistic , present novel challenges to both plant hosts and the biologists who study them . computational approaches , in concert with experimental verification , can help resolve these challenges .", "to characterize hexamer frequencies in plant hosts and their microbial symbionts , we collected sets of training sequences from public databases and edited them for quality .", "training sets were chosen to be representative of , but obtained independently from , taxa participating in symbiotic associations for which a diagnosis of origin would be made . because the species being compared are represented unevenly in public sequence databases , taxa were chosen so that roughly the same number of genes were analyzed in each training set , rather than simply to maximize the numbers of species or sequences present .", "training sets represent protein - coding sequences from three taxonomic groupings : plants ( a1 , medicago and glycine spp . ) , either fungi ( b2 , zygomycetes and chytridiomycetes ) or stramenopiles ( b1 ) , including ests from p. infestans , and bacteria ( b3 , rhizobium , sinorhizobium and bradyrhizobium ) .", "we performed pairwise comparisons with two different , taxon - specific training sets ( a and b ) to infer the origin of a transcript .", "training sets were obtained by querying the genbank database using the entrez retrieval tool . a preliminary query by taxon name obtained all available nucleotide sequences from that taxon , then the limits option excluded ests , stss ( sequence - tagged sites ) , gsss ( genome survey sequences ) , working draft sequences , and patented sequences from the query set . organellar ( mitochondrial and chloroplast ) dna was also excluded via the limits option .", "a query term to require that a sequence contain a protein - coding region ( cds ) was also added , which excluded ribosomal and transfer rna sequences .", "the results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query .", "( changing slightly the composition of training sets between those dates did not notably affect the experimental outcome . ) following a previously established protocol , we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . in this protocol", ", we resampled each training set 40 times by random partitioning into training ( for hexamer counts ) and test calculation pools . to control for any bias introduced by length variation ,", "a program randomly clipped 300 nucleotide fragments for word counting . as a result , one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate ; independent replicates contained different , randomly chosen training sequences and 300 nucleotide fragments .", "values of the test statistic from 40 resampled replicates were pooled for calibration purposes . as with the original protocol , we pooled the resulting test statistic distributions , normalized them as cumulative distributions , and then evaluated them for overlap .", "we call the resulting comparisons ' calibration curves ' , as they are not used directly to make inferences , but rather indirectly , to evaluate the degree of separation in hexamer counts from different taxa", ". overlap of calibration curves should be minimal to yield the most statistically powerful results possible .", "due to considerable overlap of calibration curves between taxonomically general , inclusive training sets ( that is , all eudicots , all fungi and miscellaneous eukaryotes , and all eubacteria , data not shown ) , we opted to work with specific training sets that included only the most species - specific sequences available , while maintaining approximately equal sample sizes across taxa .", "the most challenging case was that of the arbuscular mycorrhizal fungi , for which very few protein - coding sequences are available . to increase the amount of data in this training set ( b2 ) without biasing sample sizes , we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences , and compared this training set with a set from a single plant genus , medicago ( a2 ) .", "we chose this option , rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes , because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes , based on 18s ribosomal rna sequence data .", "that is , the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes , which resemble more closely the ancestral state in modern lineages . starting with a full set of sequences , we filtered for high - quality sequences by trimming regions having extensive ambiguous bases ( n - rich ) and poly(a ) or poly(t ) regions .", "thus , we trimmed poly(a ) and poly(t ) sites to minimize the cases in which a test sequence resembles one training set more closely than the other , simply by virtue of having an abundance of the hexamer aaaaaa or tttttt .", "similarly , test results obtained from short or n - rich sequences can be difficult to interpret .", "we allowed no more than one n per hexamer and trimmed poly(a ) or poly(t ) tracts longer than 13 nucleotides . to accommodate for possible sequence chimeras ,", "those sequences found to contain an internal poly(a ) or poly(t ) segment longer than 13 nucleotides were partitioned into two fragments , and the longer of the two fragments was used in analysis , provided its length was at least 300 nucleotides . after trimming ,", "we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn .", "all blast searches used default parameters and low - complexity filtering with the programs dust or seg .", "the decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves ( data not shown ) , likely a result of divergent evolutionary rates between protein - coding and non - coding sequences .", "chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes .", "table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening .", "to test the validity of word counting as a solution to the problem , we identified a set of 50 gene sequences from plants ( m. truncatula and g. max ) , oomycetes ( phytophthora ) , zygomycetes ( glomus versiforme ) , and bacteria ( sinorhizobium meliloti and agrobacterium tumefaciens ) , for which the function and origin have been characterized experimentally .", "we chose genes known to play a role in plant - microbe interactions , as well as genes that are found across taxa .", "we withheld these sequences , and partial transcripts of the same genes , from training sets prior to comparative lexical analysis , and calculated hexamer dissimilarities for each of the three training sets as described below . to diagnose the species of origin for sequences expressed in symbiotic cultures , we collected sequences generated by distinct est sequencing projects from the genbank database .", "sequences from pathogenic interactions originated from cultures of a species from the genus phytophthora with its plant host , such as p. sojae and soybean ( g. max ) isolated from inoculated hypocotyls two days after infection   and p. medicaginis and m. truncatula isolated from infected roots 10 days after infection ( c. vance , unpublished data ) .", "sequences expressed during mutualistic interactions were obtained from cultures with m. truncatula and mycorrhizal ( glomus versiforme ; m.j .", "harrison , unpublished data ) or rhizobacterial ( s. meliloti ; k. vandenbosch , unpublished data ) endosymbionts several days after inoculation .", "sequences expressed in pure , axenic cultures from p. sojae mycelia and zoospores   and from sterile , uninoculated m. truncatula roots   provided a basis for comparison in which no foreign transcripts were expected . to maximize the reliability of diagnostic comparisons , we screened test sequences for high quality as for training sequences , and for low similarity to e. coli , chloroplast and mitochondrial genes , and non - coding rna transcripts ( ribosomal and transfer rnas ) .", "independent blastn comparisons identified sequences having very high similarity ( e < 10 ) to vector sequences or moderately high similarity ( e < 10 ) to non - nuclear or non - coding sequences obtained from genbank .", "we wrote a perl program ( countgc.pl ) that calculates the gc base content of a sequence as the portion of guanine and cytosine residues among all unambiguous ( non - n ) nucleotides in a sequence . the hist method in r ,", "version 1.1.1   aggregated continuous percentages into discrete histogram bins , using bin sizes of 2% difference in gc , with inclusive lower bin boundaries and exclusive upper bounds ; the lm method tested for linear correlation of the dissimilarity test statistic t with gc .", "used a likelihood - ratio test to determine whether word frequencies from a particular sequence more closely resemble the frequency distribution of control data sets from the taxon being sequenced or a distantly related outgroup .", "they computed a test statistic t(a , b , x ) for each sequence x as the difference of log - likelihood ratio dissimilarity measures , d(a , x ) = -2log(a , x ) , for two data sets , a control set a and an outgroup b , such that t(a , b , x ) = d(a , x ) - d(b , x ) .", "a negative value for t indicates that the sequence more closely resembles words from a ; conversely , a positive value indicates a likely contaminant related to b. ( dissimilarity is conceptually related to distance . however , dissimilarity does not measure distance because it does not possess the mathematical properties of a distance metric . ) unlike the calculation of calibration curves , in which 300-nucleotide subsequences are randomly resampled , hexamer dissimilarity is measured over the whole length of a test sequence when inferring a transcript 's origin .", "originally , the investigators used the null hypothesis that no difference exists for dissimilarity measures between the two data sets , or that t(a , b , x ) = 0 .", "tested two alternative hypotheses : that t < 0 , being more like a , or t > 0 , like b. lexical analysis using pentamers or heptamers yields similar error rates and very highly correlated values for the test result ( not shown ) . because white et al .  ", "reported the best results were obtained using hexamers , and because a word size of six nucleotides corresponds to the size of a dicodon , we chose to analyze hexamer frequencies . to use longer words", "requires more training data , because the number of possible words increases exponentially with increasing word size .", "use of shorter words may be adequate for some applications and will be investigated in future work . though we used white 's word - counting methods , we did make slight modifications .", "we simplified one program ( called hybridize ) to compute individual dissimilarity values , rather than paired differences ; a patch that details how to modify the c program is available ( see hyb2dis.txt in additional data files ) .", "more importantly , we amended the null hypothesis and interpreted calibration curves to test for statistically significant dissimilarity differences . though the likelihood - ratio test statistic indicates the magnitude of similarity to a or b , we do not know what values for t are significant with known confidence .", "when testing hypotheses , one can make two types of error : type i , or false positives , and type ii , false negatives .", "the false - positive rate is denoted  and false - negative rate . we determine  and  from overlap in the calibration curves . inferring error rates from calibration curves", "is justified because we know the correct answer and determine the error rate via resampling , as with bootstrap methods to infer error rates or confidence intervals .", "we are interested in knowing from which of two organisms a sequence originated , and are reasonably confident that it came from either one or the other .", "thus , we assume it came from one and test whether we have evidence to refute this assumption .", "the null hypothesis here is that sequence x is from a. alternatively , it might be from b. evaluating the calibration curve overlap at t = 0 quantifies the associated error rates .", "the cumulative distribution function ( cdf ) of taxon b specifies  where cdfb intersects 0 ; the cdf from a specifies  as 1-cdfa(0 )", ". we can thus resolve the problem with known confidence p : p ( t > 0 ) = . all other computations were performed as described previously .", "software used for lexical analysis was obtained via anonymous ftp from the tigr software ftp site .", "to characterize hexamer frequencies in plant hosts and their microbial symbionts , we collected sets of training sequences from public databases and edited them for quality .", "training sets were chosen to be representative of , but obtained independently from , taxa participating in symbiotic associations for which a diagnosis of origin would be made . because the species being compared are represented unevenly in public sequence databases , taxa were chosen so that roughly the same number of genes were analyzed in each training set , rather than simply to maximize the numbers of species or sequences present .", "training sets represent protein - coding sequences from three taxonomic groupings : plants ( a1 , medicago and glycine spp . ) , either fungi ( b2 , zygomycetes and chytridiomycetes ) or stramenopiles ( b1 ) , including ests from p. infestans , and bacteria ( b3 , rhizobium , sinorhizobium and bradyrhizobium ) .", "we performed pairwise comparisons with two different , taxon - specific training sets ( a and b ) to infer the origin of a transcript .", "training sets were obtained by querying the genbank database using the entrez retrieval tool . a preliminary query by taxon name obtained all available nucleotide sequences from that taxon , then the limits option excluded ests , stss ( sequence - tagged sites ) , gsss ( genome survey sequences ) , working draft sequences , and patented sequences from the query set . organellar ( mitochondrial and chloroplast ) dna was also excluded via the limits option .", "a query term to require that a sequence contain a protein - coding region ( cds ) was also added , which excluded ribosomal and transfer rna sequences .", "the results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query .", "( changing slightly the composition of training sets between those dates did not notably affect the experimental outcome . ) following a previously established protocol , we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . in this protocol", ", we resampled each training set 40 times by random partitioning into training ( for hexamer counts ) and test calculation pools . to control for any bias introduced by length variation ,", "a program randomly clipped 300 nucleotide fragments for word counting . as a result , one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate ; independent replicates contained different , randomly chosen training sequences and 300 nucleotide fragments .", "values of the test statistic from 40 resampled replicates were pooled for calibration purposes . as with the original protocol , we pooled the resulting test statistic distributions , normalized them as cumulative distributions , and then evaluated them for overlap .", "we call the resulting comparisons ' calibration curves ' , as they are not used directly to make inferences , but rather indirectly , to evaluate the degree of separation in hexamer counts from different taxa", ". overlap of calibration curves should be minimal to yield the most statistically powerful results possible .", "due to considerable overlap of calibration curves between taxonomically general , inclusive training sets ( that is , all eudicots , all fungi and miscellaneous eukaryotes , and all eubacteria , data not shown ) , we opted to work with specific training sets that included only the most species - specific sequences available , while maintaining approximately equal sample sizes across taxa .", "the most challenging case was that of the arbuscular mycorrhizal fungi , for which very few protein - coding sequences are available . to increase the amount of data in this training set ( b2 ) without biasing sample sizes , we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences , and compared this training set with a set from a single plant genus , medicago ( a2 ) .", "we chose this option , rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes , because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes , based on 18s ribosomal rna sequence data .", "that is , the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes , which resemble more closely the ancestral state in modern lineages . starting with a full set of sequences , we filtered for high - quality sequences by trimming regions having extensive ambiguous bases ( n - rich ) and poly(a ) or poly(t ) regions .", "thus , we trimmed poly(a ) and poly(t ) sites to minimize the cases in which a test sequence resembles one training set more closely than the other , simply by virtue of having an abundance of the hexamer aaaaaa or tttttt .", "similarly , test results obtained from short or n - rich sequences can be difficult to interpret .", "we allowed no more than one n per hexamer and trimmed poly(a ) or poly(t ) tracts longer than 13 nucleotides . to accommodate for possible sequence chimeras ,", "those sequences found to contain an internal poly(a ) or poly(t ) segment longer than 13 nucleotides were partitioned into two fragments , and the longer of the two fragments was used in analysis , provided its length was at least 300 nucleotides . after trimming ,", "we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn .", "all blast searches used default parameters and low - complexity filtering with the programs dust or seg .", "the decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves ( data not shown ) , likely a result of divergent evolutionary rates between protein - coding and non - coding sequences .", "chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes .", "table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening .", "to test the validity of word counting as a solution to the problem , we identified a set of 50 gene sequences from plants ( m. truncatula and g. max ) , oomycetes ( phytophthora ) , zygomycetes ( glomus versiforme ) , and bacteria ( sinorhizobium meliloti and agrobacterium tumefaciens ) , for which the function and origin have been characterized experimentally .", "we chose genes known to play a role in plant - microbe interactions , as well as genes that are found across taxa .", "we withheld these sequences , and partial transcripts of the same genes , from training sets prior to comparative lexical analysis , and calculated hexamer dissimilarities for each of the three training sets as described below .", "to characterize hexamer frequencies in plant hosts and their microbial symbionts , we collected sets of training sequences from public databases and edited them for quality .", "training sets were chosen to be representative of , but obtained independently from , taxa participating in symbiotic associations for which a diagnosis of origin would be made . because the species being compared are represented unevenly in public sequence databases , taxa were chosen so that roughly the same number of genes were analyzed in each training set , rather than simply to maximize the numbers of species or sequences present .", "training sets represent protein - coding sequences from three taxonomic groupings : plants ( a1 , medicago and glycine spp . ) , either fungi ( b2 , zygomycetes and chytridiomycetes ) or stramenopiles ( b1 ) , including ests from p. infestans , and bacteria ( b3 , rhizobium , sinorhizobium and bradyrhizobium ) .", "we performed pairwise comparisons with two different , taxon - specific training sets ( a and b ) to infer the origin of a transcript .", "training sets were obtained by querying the genbank database using the entrez retrieval tool . a preliminary query by taxon name obtained all available nucleotide sequences from that taxon , then the limits option excluded ests , stss ( sequence - tagged sites ) , gsss ( genome survey sequences ) , working draft sequences , and patented sequences from the query set . organellar ( mitochondrial and chloroplast ) dna was also excluded via the limits option .", "a query term to require that a sequence contain a protein - coding region ( cds ) was also added , which excluded ribosomal and transfer rna sequences .", "the results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query .", "( changing slightly the composition of training sets between those dates did not notably affect the experimental outcome . ) following a previously established protocol , we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . in this protocol", ", we resampled each training set 40 times by random partitioning into training ( for hexamer counts ) and test calculation pools . to control for any bias introduced by length variation ,", "a program randomly clipped 300 nucleotide fragments for word counting . as a result , one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate ; independent replicates contained different , randomly chosen training sequences and 300 nucleotide fragments .", "values of the test statistic from 40 resampled replicates were pooled for calibration purposes . as with the original protocol , we pooled the resulting test statistic distributions , normalized them as cumulative distributions , and then evaluated them for overlap .", "we call the resulting comparisons ' calibration curves ' , as they are not used directly to make inferences , but rather indirectly , to evaluate the degree of separation in hexamer counts from different taxa", ". overlap of calibration curves should be minimal to yield the most statistically powerful results possible .", "due to considerable overlap of calibration curves between taxonomically general , inclusive training sets ( that is , all eudicots , all fungi and miscellaneous eukaryotes , and all eubacteria , data not shown ) , we opted to work with specific training sets that included only the most species - specific sequences available , while maintaining approximately equal sample sizes across taxa .", "the most challenging case was that of the arbuscular mycorrhizal fungi , for which very few protein - coding sequences are available . to increase the amount of data in this training set ( b2 ) without biasing sample sizes , we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences , and compared this training set with a set from a single plant genus , medicago ( a2 ) .", "we chose this option , rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes , because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes , based on 18s ribosomal rna sequence data .", "that is , the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes , which resemble more closely the ancestral state in modern lineages .", "starting with a full set of sequences , we filtered for high - quality sequences by trimming regions having extensive ambiguous bases ( n - rich ) and poly(a ) or poly(t ) regions .", "thus , we trimmed poly(a ) and poly(t ) sites to minimize the cases in which a test sequence resembles one training set more closely than the other , simply by virtue of having an abundance of the hexamer aaaaaa or tttttt .", "similarly , test results obtained from short or n - rich sequences can be difficult to interpret .", "we allowed no more than one n per hexamer and trimmed poly(a ) or poly(t ) tracts longer than 13 nucleotides . to accommodate for possible sequence chimeras ,", "those sequences found to contain an internal poly(a ) or poly(t ) segment longer than 13 nucleotides were partitioned into two fragments , and the longer of the two fragments was used in analysis , provided its length was at least 300 nucleotides . after trimming , we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn .", "all blast searches used default parameters and low - complexity filtering with the programs dust or seg .", "the decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves ( data not shown ) , likely a result of divergent evolutionary rates between protein - coding and non - coding sequences .", "chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes .", "table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening .", "to test the validity of word counting as a solution to the problem , we identified a set of 50 gene sequences from plants ( m. truncatula and g. max ) , oomycetes ( phytophthora ) , zygomycetes ( glomus versiforme ) , and bacteria ( sinorhizobium meliloti and agrobacterium tumefaciens ) , for which the function and origin have been characterized experimentally .", "we chose genes known to play a role in plant - microbe interactions , as well as genes that are found across taxa .", "we withheld these sequences , and partial transcripts of the same genes , from training sets prior to comparative lexical analysis , and calculated hexamer dissimilarities for each of the three training sets as described below .", "to diagnose the species of origin for sequences expressed in symbiotic cultures , we collected sequences generated by distinct est sequencing projects from the genbank database .", "sequences from pathogenic interactions originated from cultures of a species from the genus phytophthora with its plant host , such as p. sojae and soybean ( g. max ) isolated from inoculated hypocotyls two days after infection   and p. medicaginis and m. truncatula isolated from infected roots 10 days after infection ( c. vance , unpublished data ) .", "sequences expressed during mutualistic interactions were obtained from cultures with m. truncatula and mycorrhizal ( glomus versiforme ; m.j .", "harrison , unpublished data ) or rhizobacterial ( s. meliloti ; k. vandenbosch , unpublished data ) endosymbionts several days after inoculation", ". sequences expressed in pure , axenic cultures from p. sojae mycelia and zoospores   and from sterile , uninoculated m. truncatula roots   provided a basis for comparison in which no foreign transcripts were expected . to maximize the reliability of diagnostic comparisons , we screened test sequences for high quality as for training sequences , and for low similarity to e. coli , chloroplast and mitochondrial genes , and non - coding rna transcripts ( ribosomal and transfer rnas ) .", "independent blastn comparisons identified sequences having very high similarity ( e < 10 ) to vector sequences or moderately high similarity ( e < 10 ) to non - nuclear or non - coding sequences obtained from genbank .", "we wrote a perl program ( countgc.pl ) that calculates the gc base content of a sequence as the portion of guanine and cytosine residues among all unambiguous ( non - n ) nucleotides in a sequence . the hist method in r ,", "version 1.1.1   aggregated continuous percentages into discrete histogram bins , using bin sizes of 2% difference in gc , with inclusive lower bin boundaries and exclusive upper bounds ; the lm method tested for linear correlation of the dissimilarity test statistic t with gc .", "white et al .   used a likelihood - ratio test to determine whether word frequencies from a particular sequence more closely resemble the frequency distribution of control data sets from the taxon being sequenced or a distantly related outgroup .", "they computed a test statistic t(a , b , x ) for each sequence x as the difference of log - likelihood ratio dissimilarity measures , d(a , x ) = -2log(a , x ) , for two data sets , a control set a and an outgroup b , such that t(a , b , x ) = d(a , x ) - d(b , x ) .", "a negative value for t indicates that the sequence more closely resembles words from a ; conversely , a positive value indicates a likely contaminant related to b. ( dissimilarity is conceptually related to distance . however , dissimilarity does not measure distance because it does not possess the mathematical properties of a distance metric . ) unlike the calculation of calibration curves , in which 300-nucleotide subsequences are randomly resampled , hexamer dissimilarity is measured over the whole length of a test sequence when inferring a transcript 's origin .", "originally , the investigators used the null hypothesis that no difference exists for dissimilarity measures between the two data sets , or that t(a , b , x ) = 0 .", "white et al .   tested two alternative hypotheses : that t < 0 , being more like a , or t > 0 , like b. lexical analysis using pentamers or heptamers yields similar error rates and very highly correlated values for the test result ( not shown ) . because white et al .  ", "reported the best results were obtained using hexamers , and because a word size of six nucleotides corresponds to the size of a dicodon , we chose to analyze hexamer frequencies . to use longer words requires more training data , because the number of possible words increases exponentially with increasing word size .", "use of shorter words may be adequate for some applications and will be investigated in future work . though we used white 's word - counting methods , we did make slight modifications .", "we simplified one program ( called hybridize ) to compute individual dissimilarity values , rather than paired differences ; a patch that details how to modify the c program is available ( see hyb2dis.txt in additional data files ) .", "more importantly , we amended the null hypothesis and interpreted calibration curves to test for statistically significant dissimilarity differences . though the likelihood - ratio test statistic indicates the magnitude of similarity to a or b , we do not know what values for t are significant with known confidence .", "when testing hypotheses , one can make two types of error : type i , or false positives , and type ii , false negatives .", "the false - positive rate is denoted  and false - negative rate . we determine  and  from overlap in the calibration curves . inferring error rates from calibration curves", "is justified because we know the correct answer and determine the error rate via resampling , as with bootstrap methods to infer error rates or confidence intervals .", "we are interested in knowing from which of two organisms a sequence originated , and are reasonably confident that it came from either one or the other .", "thus , we assume it came from one and test whether we have evidence to refute this assumption .", "the null hypothesis here is that sequence x is from a. alternatively , it might be from b. evaluating the calibration curve overlap at t = 0 quantifies the associated error rates .", "the cumulative distribution function ( cdf ) of taxon b specifies  where cdfb intersects 0 ; the cdf from a specifies  as 1-cdfa(0 )", ". we can thus resolve the problem with known confidence p : p ( t > 0 ) = . all other computations were performed as described previously .", "software used for lexical analysis was obtained via anonymous ftp from the tigr software ftp site .", "the following files are available for download : countgc.pl : perl script used to compute gc content of sequences analyzed .", "hyb2dis.txt : patch file that converts white 's hybridize program to compute individual dissimilarity values .", "training sets ( glycinemedicago.txt,rhizobia.txt , stramenopiles.txt , zygochytrid.txt ) : fasta - formatted text files that contain the sequences used for calibration and comparison .", "test sets ( psojaeha.txt , psojaemy.txt , psojaezo.txt , mtrhe.txt , dsir.txt , mham.txt , kv0.txt , kv2.txt , kv3.txt ) : fasta - formatted text files containing transcripts analyzed , edited for quality .", "test results ( psojaeha.dat , psojaemy.dat , psojaezo.dat , mtrhe-a1b1.dat , mtrhe-a2b2.dat , dsir.dat , mham.dat , kv0.dat , kv2.dat , kv3.dat ) : text files that contain transcript analysis results , sorted from least to most plant - like .", "fasta - formatted text files containing transcripts analyzed , edited for quality . text files that contain transcript analysis results , sorted from least to most plant - like .", "we thank callum bell , mark gijzen , maria harrison , tom kepler , deb samac , and bruno sobral for valued discussions and feedback . comments from b. m. tyler and an anonymous reviewer on an earlier version of this work greatly enhanced its presentation .", "distribution of gc content in pure and mixed - culture libraries . ( a ) probability densities for histogram bin sizes of 0.02 ( 2% ) in base content .", "( a ) calculation of statistical parameters from cdfs a and b. overlap in the upper tail of cdfa with cdfb and the lower tail of cdfb with cdfa are likely regions for error .", "we find the false - positive rate  where 1 - cdfa intersects 0 [ cdfa(0)= 1 -  ] , and the false - negative rate  where cdfb crosses 0 . also shown", "are the medians (  ) for each distribution , where cdf( ) = 0.5 .", ", solid black line ) and stramenopile plus p. infestans est ( b1 , dashed black line ) training sequences .", "superimposed distributions of test results show dissimilarity differences for infected g. max ( green ) and axenic p. sojae mycelial and zoospore sequences ( blue and cyan , respectively ) .", "each point corresponds to an expressed tag from either ( a ) infected g. max or ( b ) axenic p. sojae mycelial or ( c ) zoospore sequences , compared with plant ( a1 ) and stramenopile plus p. infestans est training sequences ( b1 ) .", "the identity function indicates equal dissimilarity to both training sets , t = d(a ) - d(b)= 0 .", "calibration curves compare plant training sets ( a1 and a2 , solid black lines ) with one of three microbial symbiont training sets ( broken black lines ) : ( a ) stramenopile and p. infestans est sequences ( b1 ) ; ( b ) pooled zygomycete and chytridiomycete coding sequences ( b2 ) ; and ( c ) sequences from the genera rhizobium , sinorhizobium and bradyrhizobium ( b3 ) .", "cumulative distributions of test results from m. truncatula axenic and microbial symbiont mixed cultures appear in each panel ( colored lines ) .", "each point indicates the dissimilarity of a test sequence compared with a plant training set ( a1 or a2 ) and one of three microbial symbiont training sets : ( a ) stramenopile and p. infestans est sequences ( b1 ) ; ( b ) pooled zygomycete and chytridiomycete coding sequences ( b2 ) ; and ( c ) sequences from the genera rhizobium , sinorhizobium and bradyrhizobium ( b3 ) . sequences from m. truncatula axenic ( green ) and microbial symbiont mixed culture libraries are represented in each panel .", "number of sequences ( n ) and nucleotides ( nt ) , as raw , trimmed ( removed n - rich regions , poly(a ) and poly(t ) sites ) , and screened sequences ( removed ribosomal , chloroplast , and mitochondrial dna and remaining sequences shorter than 300 nucleotides ) .", "number of est sequences ( n ) and nucleotides ( nt ) as raw , trimmed ( limited lengths of n - rich regions , poly(a ) and poly(t ) sites ) , and screened ( removed ribosomal , chloroplast , and mitochondrial dna , and remaining sequences shorter than 300 nt ) sequences ."], "abstract_text": ["<S> backgroundmost organisms have developed ways to recognize and interact with other species . </S>", "<S> symbiotic interactions range from pathogenic to mutualistic </S>", "<S> . some molecular mechanisms of interspecific interaction are well understood , but many remain to be discovered . expressed sequence tags ( ests ) from cultures of interacting symbionts can help identify transcripts that regulate symbiosis , but present a unique challenge for functional analysis . given a sequence expressed in an interaction between two symbionts , the challenge is to determine from which organism the transcript originated . for high - throughput sequencing from interaction cultures , </S>", "<S> a reliable computational approach is needed . </S>", "<S> previous investigations into gc nucleotide content and comparative similarity searching provide provisional solutions , but a comparative lexical analysis , which uses a likelihood - ratio test of hexamer counts , is more powerful.resultsvalidation with genes whose origin and function are known yielded 94% accuracy . </S>", "<S> microbial ( non - plant ) transcripts comprised 75% of a phytophthora </S>", "<S> sojae - infected soybean ( glycine max cv harasoy ) library , contrasted with 15% or less in root tissue libraries of medicago truncatula from axenic , phytophthora medicaginis - infected , mycorrhizal , and rhizobacterial treatments . </S>", "<S> mycorrhizal libraries contained about 23% microbial transcripts ; an axenic plant library contained a similar proportion of putative microbial transcripts.conclusionscomparative lexical analysis offers numerous advantages over alternative approaches . </S>", "<S> many of the transcripts isolated from mixed cultures were of unknown function , suggesting specificity to symbiotic metabolism and therefore candidates likely to be interesting for further functional investigation . </S>", "<S> future investigations will determine whether the abundance of non - plant transcripts in a pure plant library indicates procedural artifacts , horizontally transferred genes , or other phenomena . </S>"], "labels": null, "section_names": ["Background", "Results", "Discussion", "Methods and materials", "Training sequences", "Calibration", "Data quality", "Validation", "Test sequences", "Base content", "Comparative lexical analysis", "Additional data files", "Supplementary Material", "Acknowledgements", "Figures and Tables"], "sections": [["access to automated dna sequencing technology has made possible the rapid generation and analysis of gene transcripts expressed in organisms via expressed sequence tags ( ests ) .", "this information has helped to identify those genes expressed in particular stages of development and in specialized tissues or organs .", "novel gene products and target leads for therapeutic intervention can also be gleaned rapidly from ests . a more detailed understanding of the molecular interactions between symbionts , whether pathogenic or mutualistic , is also possible with this approach . for a sequence isolated from interacting symbionts , determining its cellular role ( or roles )", "we refer to this challenge as ' the problem ' : given a sequence x expressed in an interaction between species a and b , did x originate from a or b ?", "various solutions are readily conceived , each with merits and faults . here , we show that a comparative lexical analysis of word counts ( specifically , hexamer frequencies ) , previously used to detect library contamination in sequencing projects , provides a powerful computational basis to infer a transcript 's species of origin . experimentally , one can attempt to solve the problem by hybridizing a clone ( as probe ) to genomic dna ( target ) from both species and determining to which target the probe hybridizes .", "however , if a sequence is highly conserved in the two taxa , hybridization stringency conditions can influence the outcome considerably . for high - throughput est sequence analysis ,", "source verification by hybridization is impractical in terms of time and reagents . as an alternative to in vitro hybridization , several computational solutions are possible .", "were the genome sequence of both species completely determined , one could simply use sequence similarity searching . however , most plant hosts and their microbial symbionts have little or no genomic sequence data available , which makes this approach very unreliable .", "strong similarity to a sequence from one organism does not preclude the possibility that a similar sequence is present in the other species .", "exploiting this fact may seem a viable solution to the problem , as it has proven suitable for predicting the presence of introns among exons in genomic dna .", "however , it really is not practical , because of the need to know the reading frame for translation of a messenger rna into an amino acid .", "est data are of notoriously unreliable quality , sometimes having a large proportion of ambiguous bases , and sometimes having single base - pair insertions or deletions , which disrupt a reading frame .", "word counting is less prone to these sources of error , and uses information intrinsic to biases in codon usage by counting codon pairs as hexamers in a sliding window , whereas codons are read in non - overlapping , tiled windows .", "an intuitive approach to the problem that examines sequence composition is to compare the guanine and cytosine ( gc ) base content of a sequence with other sequences from the species being studied .", "when two species ' genomes have different gc content , this method can be very useful .", "in a recent investigation , for instance , sequences from the stramenopile plant pathogen phytophthora sojae and its soybean ( glycine max ) host showed a 20% difference in mean gc content . the origin of a number of sequences could readily be identified this way , but a large proportion could not , because of considerable overlap in the distributions ' tails .", "counting frequencies of gc is simple word counting , where the word size k is 1/2 : only two semi - words , g / c and a / t are counted .", "an alternative approach to determining the origin of a sequence is suggested by previous work on analysis of word counts , or k - tuple frequencies , which was intended as a means of evaluating a library for contamination when sequencing from a single model organism .", "the word - counting method provides distinct advantages over other computational methods . unlike sequence - similarity searching", ", it does not require that the full protein - coding content of both genomes be known for reasonable inferences to be made .", "further , word counting is sensitive to biases in codon usage and gc content commonly observed when comparing taxa , but does not require knowledge of the reading frame for amino - acid translation .", "that is , the underlying differences between the two organisms that result in base composition or codon usage biases can also be detected by counting words .", "unlike gc analysis , lexical analysis establishes a clear threshold above or below which we can infer the species of origin , and a confidence level for an inference can readily be assigned .", "dunning 's likelihood - ratio test of word dissimilarities   also has the appealing property of being non - parametric , having no assumption of normality for the underlying frequency distribution , which makes it statistically powerful . dunning   demonstrated that unreliable results can be obtained from parametric tests , such as  , particularly in such cases as lexical analysis . in the experiments detailed below , we first validate the word - counting method on sequences whose", "origin and function are known , then compare it with ability to diagnose the origin of sequences with distributions of gc content .", "we examine sequences from pathogenic interactions between species from the genus phytophthora and the plant hosts g. max and medicago truncatula , then apply the word - counting approach to sequences from two microbial mutualists in association with m. truncatula , the arbuscular mycorrhizal zygomycete glomus versiforme , and the nitrogen - fixing bacterium sinorhizobium meliloti ."], ["validation sequence accession numbers , gene names , and comparison results appear in table 1 .", "the word - counting method was generally quite reliable when tested against sequences of known origin , being wrong in 3 cases out of 50 ; a phosphate transporter from g. versiforme and two in planta - induced genes from phytophthora infestans were misidentified as plant sequences .", "this indicates a failure rate of 6% - all false negatives under the null hypothesis that a transcript originates from the plant host .", "performance of the method was not influenced by whether the isolated source of a sequence was an mrna or dna molecule , as indicated by the column labeled ' mrna ? ' .", "distributions of gc content are approximately normal in two of three cases studied , those of axenic p. sojae cultures ( figure 1 ) . for sequences from infected plant cultures ,", "roughly 25% of a total of 927 infected g. max sequences contain less than 50% gc ; most of these are likely to be plant transcripts .", "this is a considerably greater number than for axenic p. sojae cultures , in which fewer than 5% of mycelia and zoospore isolates contain less than 50% gc .", "several properties of cumulative distribution functions warrant comment , to help explain similar plots from word dissimilarity comparisons ( figures 1b,2a ) .", "the median of a distribution occurs where the function reaches a cumulative probability of 0.5 .", "medians from all three p. sojae libraries are similar , varying by less than 4% gc ( figure 1b ) .", "other moments of the distributions are readily apparent ; the variance is inversely related to the slope at the median value of the function", ". a useful property of cumulative distribution functions is that any point on the y axis gives the integrated area ( cumulative probability ) under the curve .", "we use this property to establish experiment - wide false - positive and false - negative rates ( figure 2a ) . in this case ,", "calibration curves from hexamer dissimilarity tests , shown in figure 2b as solid black lines for plant and dashed black lines for stramenopile training sequences are approximately normal .", "the medians differ considerably , with only about 10% percent overlap in the two distributions ' tails about the neutral t - value of zero . superimposed", "are comparison curves from p. sojae test sets ( figure 2b ) , which parallel the gc content curves in figure 1b but show slightly less variance .", "axenic sequences are clearly more like stramenopiles ( b1 ) than plants ( a1 ) in hexamer composition , with all but a small percentage having positive t values .", "plant - like sequences are as abundant in the mixed library as detected by gc content , about 23% . as expected , the two methods agree , having positively correlated values for gc and t ( r = 0.852 , p < 10 , v = 2,641 ) . looking in more detail at the paired dissimilarity values ( figure 3 ) ,", "the magnitudes of dissimilarity are also apparent , with longer sequences having larger dissimilarity values .", "blastx similarity searches against the protein sequences in nr , a non - redundant library of proteins   revealed that none of the 12 plant - like mycelial transcripts significantly resemble known proteins ( e > 10 ) . among the top ten most plant - like transcripts from the infected g. max library", ", three had no significant matches , four matched putative arabidopsis thaliana proteins , and three matched known g. max proteins : cytochrome p450 ( accession af022460 , e < 10 ) , methylglyoxalase ( accession p46417 , e < 10 ) , and a ripening related protein ( accession af127110 , e < 10 ) .", "thus , the majority of the most plant - like transcripts in the infected soybean library strongly resemble characterized plant sequences .", "analysis results from all p. sojae and mixed - culture transcripts are available as additional data files , grouped by the library from which transcripts were sequenced .", "figure 4 shows that calibration curves from comparing plant and microbial symbiont training sets have good separation and minimal overlap ( about 10% ) in two of three cases , but not for training set b2 , comprised of zygomycetes and chytridiomycetes , which overlaps considerably with plants ( figure 4b ) .", "when comparing between plants and bacteria , the error rates are  = 0.052 and  = 0.084 , much lower than when comparing plants ( a2 , medicago ) with fungi ( b2 , zygomycetes and chytridiomycetes ) .", "error rates for comparing stramenopiles and p. infestans ests with plants are as in figure 2 (  = 0.088 ,  = 0.032 ) . also shown in figure 4", "all resemble calibration curves from plant sequences , having similar medians and slightly less variance than the plant calibration curves .", "comparison curves show that the great majority of test sequences are more plant - like than otherwise , with 20% or less resembling microbial symbionts more closely than plants", ". a greater proportion of microbial sequences is present in the m. truncatula - g .", "versiforme interaction library ( 20% , figure 4b ) than in the p. medicaginis - infected m. truncatula library ( 5% , figure 4a ) .", "however , long 's root - hair enriched library ( mtrhe )   had a greater proportion of putative microbial sequences present ( 7% and 25% ) than any of the libraries isolated from symbiont - associated cultures .", "the axenic and nodulating root libraries had the smallest portion of putative microbial transcripts ( < 2% , figure 4c ) , with the axenic library closely resembling nodulating root libraries .", "the method of preparing a library can affect the proportion of plant and non - plant sequences , as discussed below .", "paired dissimilarity values in figure 5 show in greater detail which sequences are more or less like plant and symbiont .", "considerable variation in the degree of dissimilarity to both training sets is clear , largely due to variation in the length of sequences within test sets .", "d(b ) in figure 4 , most sequences lie above the identity function , and resemble the plant host more closely than the microbial symbiont .", "mycorrhizal test sequences are more difficult to differentiate than sequences from the rhizobacterial or pathogenic associations , as seen by the diminished variation about the identity function in mycorrhizal comparisons ( figure 5b ) , contrasted with comparisons from pathogen - infected and nodulating root libraries ( figures 5a and c , respectively ) .", "analysis results from all m. truncatula and mixed - culture transcripts are available as additional data files , grouped by the library from which transcripts were sequenced , and sorted from the least plant - like transcripts to the most plant - like ."], ["clearly , the word - counting approach provides a reliable solution to the problem of source identification with known confidence , and has several significant advantages .", "the reliability of the method is best justified in terms of the favorable validation test results , and is further corroborated by agreement with an analysis of gc content . in test cases", "where the correct answer is known a priori , results were correct within error rates expected from overlap in training sets .", "( recall that  = 0.088 for comparisons between plants and stramenopiles , and  = 0.052 for comparisons between plants and bacteria . ) unlike gc content , the problem is clearly resolved by word counting with a threshold value of t = 0 , and with statistical rigor , because false - positive and false - negative rates for a set of comparisons are readily computed from cumulative distributions of dissimilarity between two training sets .", "optimal statistical power ( minimal false - negative rate ) is ensured when using a likelihood - ratio test statistic , as demonstrated by the pearson - neyman theorem .", "rather , it is sufficient that the training set be related to , but not necessarily congeners of , the species from which sequences are being compared .", "sequences from several species of the genus phytophthora were correctly distinguished from plant and bacterial sequences , and three genes from agrobacterium tumefaciens were correctly identified as representing a bacterial sequence .", "however , several caveats warrant prudence . transcribed sequences that do not encode proteins , but rather catalytic single - stranded rnas such as transfer and ribosomal rnas , should be treated independently because they are more highly conserved across taxa than messenger rnas . also , filtering or trimming of low - complexity repeat regions , such as poly(a ) or poly(t ) tracts , is helpful because comparison results can be influenced by the abundance of a single hexamer .", "early in our investigations , using one set of training sequences obtained from directionally cloned p.", "it eventually became clear that , as the p. infestans sequences were all single - pass reads from the 5 ' end of a clone generated with the t3 primer , few sequences complementary to the 3 ' end of the mrna sequence were present in the training set .", "large amounts of the poly(t ) hexamer would be expected when sequencing reverse complements of mrnas obtained from 3 ' sequences generated with the t7 primer .", "as a result , any sequence that contained a poly(t ) tract tended to resemble the plant sequences .", "further , because the error rates for an inference depend on the degree to which calibration curves overlap , the best results are obtained where overlap is minimal . despite these caveats , word counting presents a viable solution to the problem .", "the p. sojae - infected g. max library provides a clear example of contrast in both hexamer composition and gc content , resulting in readily diagnosed origins .", "for clear separation between the two species to appear , the two must differ in composition and a detectable proportion of transcripts from each species must be present in the library . to be detectable , the proportion of transcripts present from a particular species must be greater than the error rate obtained from calibration curves . though these criteria are true for the infected g. max library ( t < 0 for < 25% of 927 transcripts ) , they do not appear to be true for the m. truncatula libraries we analyzed ( t < 0 for 8099% of 8903,017 transcripts ) . in the p. medicaginis interaction library", "the p. sojae - infected library was prepared two days after infection , using a susceptible plant host strain , so as to maximize the number of pathogen transcripts present in the host tissue .", "medicaginis - infected library was prepared ten days after infection and individual plants varied in their degree of susceptibility ( c. vance , unpublished data ) .", "plants were also inoculated in a different manner : ground mycelia were dissolved in sterile water and incubated , and the resulting inoculum was pipetted onto the soil surface , rather than the plant .", "these differences in how tissues were cultured prior to library preparation could have produced the disparate abundance of plant transcripts , though both libraries were prepared from plant tissues infected with phytophthora . for mycorrhizal root libraries", ", we might explain the relative lack of symbiont sequences as resulting simply from a relative lack of transcripts in the host tissue .", "we might therefore expect that most of the transcripts therein originate from the plant host . confounding this result ,", "the error rates in this comparison are the greatest among all the comparisons we performed , most likely because the evolutionary distance between fungi ( zygomycetes and chytridiomycetes ) and plants is the least among comparisons . also , zygomycete protein - coding sequences are rare in genbank , which resulted in a small training set for these fungi , and may have amplified any biases .", "the high false - negative rate probably led to a failure to detect some symbiont transcripts . in nodulating root libraries", ", we do not expect to observe an abundance of bacterial transcripts , because bacteria generally do not form polyadenylated mrnas . as the protocols used to extract and purify mrnas from tissue lysate for the libraries cited in this study all relied on the presence of polyadenylation sites", "the abundance of putative microbial symbiont transcripts among sequences from a pure plant root library is difficult to interpret .", "the predicted portion of microbial transcripts was greater in the axenic root - hair enriched library than in mixed cultures .", "error rates were greatest for comparisons between training sets from plant and pooled zygomycete and chytridiomycete sequences .", "the 13% false - positive rate does not completely explain why about 15% of root - hair enriched transcripts resemble fungal hexamer composition more closely than plants , and warrants further study .", "care had been taken to avoid contaminating plant tissue cultures by culturing seedlings in covered plates .", "because of concern that ethylene accumulation in covered plates could improperly stimulate nodulation - related gene expression , seedlings were treated with ag2so4 , an inhibitor of the plants ' response to ethylene .", "inhibition of the ethylene response could have resulted in synthesis of transcripts that are uncharacteristic of plant roots .", "analysis of another axenic root - hair enriched library , particularly one provided a carbon source to identify potential contaminants , and not treated with an inhibitor of ethylene response , would be an informative test .", "the transcripts identified as most and least like plant or symbiont might also be studied in more detail as candidate participants in symbiosis .", "symbiotic interactions , whether pathogenic or mutualistic , present novel challenges to both plant hosts and the biologists who study them . computational approaches , in concert with experimental verification , can help resolve these challenges ."], ["to characterize hexamer frequencies in plant hosts and their microbial symbionts , we collected sets of training sequences from public databases and edited them for quality .", "training sets were chosen to be representative of , but obtained independently from , taxa participating in symbiotic associations for which a diagnosis of origin would be made . because the species being compared are represented unevenly in public sequence databases , taxa were chosen so that roughly the same number of genes were analyzed in each training set , rather than simply to maximize the numbers of species or sequences present .", "training sets represent protein - coding sequences from three taxonomic groupings : plants ( a1 , medicago and glycine spp . ) , either fungi ( b2 , zygomycetes and chytridiomycetes ) or stramenopiles ( b1 ) , including ests from p. infestans , and bacteria ( b3 , rhizobium , sinorhizobium and bradyrhizobium ) .", "we performed pairwise comparisons with two different , taxon - specific training sets ( a and b ) to infer the origin of a transcript .", "training sets were obtained by querying the genbank database using the entrez retrieval tool . a preliminary query by taxon name obtained all available nucleotide sequences from that taxon , then the limits option excluded ests , stss ( sequence - tagged sites ) , gsss ( genome survey sequences ) , working draft sequences , and patented sequences from the query set . organellar ( mitochondrial and chloroplast ) dna was also excluded via the limits option .", "a query term to require that a sequence contain a protein - coding region ( cds ) was also added , which excluded ribosomal and transfer rna sequences .", "the results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query .", "( changing slightly the composition of training sets between those dates did not notably affect the experimental outcome . ) following a previously established protocol , we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . in this protocol", ", we resampled each training set 40 times by random partitioning into training ( for hexamer counts ) and test calculation pools . to control for any bias introduced by length variation ,", "a program randomly clipped 300 nucleotide fragments for word counting . as a result , one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate ; independent replicates contained different , randomly chosen training sequences and 300 nucleotide fragments .", "values of the test statistic from 40 resampled replicates were pooled for calibration purposes . as with the original protocol , we pooled the resulting test statistic distributions , normalized them as cumulative distributions , and then evaluated them for overlap .", "we call the resulting comparisons ' calibration curves ' , as they are not used directly to make inferences , but rather indirectly , to evaluate the degree of separation in hexamer counts from different taxa", ". overlap of calibration curves should be minimal to yield the most statistically powerful results possible .", "due to considerable overlap of calibration curves between taxonomically general , inclusive training sets ( that is , all eudicots , all fungi and miscellaneous eukaryotes , and all eubacteria , data not shown ) , we opted to work with specific training sets that included only the most species - specific sequences available , while maintaining approximately equal sample sizes across taxa .", "the most challenging case was that of the arbuscular mycorrhizal fungi , for which very few protein - coding sequences are available . to increase the amount of data in this training set ( b2 ) without biasing sample sizes , we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences , and compared this training set with a set from a single plant genus , medicago ( a2 ) .", "we chose this option , rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes , because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes , based on 18s ribosomal rna sequence data .", "that is , the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes , which resemble more closely the ancestral state in modern lineages . starting with a full set of sequences , we filtered for high - quality sequences by trimming regions having extensive ambiguous bases ( n - rich ) and poly(a ) or poly(t ) regions .", "thus , we trimmed poly(a ) and poly(t ) sites to minimize the cases in which a test sequence resembles one training set more closely than the other , simply by virtue of having an abundance of the hexamer aaaaaa or tttttt .", "similarly , test results obtained from short or n - rich sequences can be difficult to interpret .", "we allowed no more than one n per hexamer and trimmed poly(a ) or poly(t ) tracts longer than 13 nucleotides . to accommodate for possible sequence chimeras ,", "those sequences found to contain an internal poly(a ) or poly(t ) segment longer than 13 nucleotides were partitioned into two fragments , and the longer of the two fragments was used in analysis , provided its length was at least 300 nucleotides . after trimming ,", "we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn .", "all blast searches used default parameters and low - complexity filtering with the programs dust or seg .", "the decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves ( data not shown ) , likely a result of divergent evolutionary rates between protein - coding and non - coding sequences .", "chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes .", "table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening .", "to test the validity of word counting as a solution to the problem , we identified a set of 50 gene sequences from plants ( m. truncatula and g. max ) , oomycetes ( phytophthora ) , zygomycetes ( glomus versiforme ) , and bacteria ( sinorhizobium meliloti and agrobacterium tumefaciens ) , for which the function and origin have been characterized experimentally .", "we chose genes known to play a role in plant - microbe interactions , as well as genes that are found across taxa .", "we withheld these sequences , and partial transcripts of the same genes , from training sets prior to comparative lexical analysis , and calculated hexamer dissimilarities for each of the three training sets as described below . to diagnose the species of origin for sequences expressed in symbiotic cultures , we collected sequences generated by distinct est sequencing projects from the genbank database .", "sequences from pathogenic interactions originated from cultures of a species from the genus phytophthora with its plant host , such as p. sojae and soybean ( g. max ) isolated from inoculated hypocotyls two days after infection   and p. medicaginis and m. truncatula isolated from infected roots 10 days after infection ( c. vance , unpublished data ) .", "sequences expressed during mutualistic interactions were obtained from cultures with m. truncatula and mycorrhizal ( glomus versiforme ; m.j .", "harrison , unpublished data ) or rhizobacterial ( s. meliloti ; k. vandenbosch , unpublished data ) endosymbionts several days after inoculation .", "sequences expressed in pure , axenic cultures from p. sojae mycelia and zoospores   and from sterile , uninoculated m. truncatula roots   provided a basis for comparison in which no foreign transcripts were expected . to maximize the reliability of diagnostic comparisons , we screened test sequences for high quality as for training sequences , and for low similarity to e. coli , chloroplast and mitochondrial genes , and non - coding rna transcripts ( ribosomal and transfer rnas ) .", "independent blastn comparisons identified sequences having very high similarity ( e < 10 ) to vector sequences or moderately high similarity ( e < 10 ) to non - nuclear or non - coding sequences obtained from genbank .", "we wrote a perl program ( countgc.pl ) that calculates the gc base content of a sequence as the portion of guanine and cytosine residues among all unambiguous ( non - n ) nucleotides in a sequence . the hist method in r ,", "version 1.1.1   aggregated continuous percentages into discrete histogram bins , using bin sizes of 2% difference in gc , with inclusive lower bin boundaries and exclusive upper bounds ; the lm method tested for linear correlation of the dissimilarity test statistic t with gc .", "used a likelihood - ratio test to determine whether word frequencies from a particular sequence more closely resemble the frequency distribution of control data sets from the taxon being sequenced or a distantly related outgroup .", "they computed a test statistic t(a , b , x ) for each sequence x as the difference of log - likelihood ratio dissimilarity measures , d(a , x ) = -2log(a , x ) , for two data sets , a control set a and an outgroup b , such that t(a , b , x ) = d(a , x ) - d(b , x ) .", "a negative value for t indicates that the sequence more closely resembles words from a ; conversely , a positive value indicates a likely contaminant related to b. ( dissimilarity is conceptually related to distance . however , dissimilarity does not measure distance because it does not possess the mathematical properties of a distance metric . ) unlike the calculation of calibration curves , in which 300-nucleotide subsequences are randomly resampled , hexamer dissimilarity is measured over the whole length of a test sequence when inferring a transcript 's origin .", "originally , the investigators used the null hypothesis that no difference exists for dissimilarity measures between the two data sets , or that t(a , b , x ) = 0 .", "tested two alternative hypotheses : that t < 0 , being more like a , or t > 0 , like b. lexical analysis using pentamers or heptamers yields similar error rates and very highly correlated values for the test result ( not shown ) . because white et al .  ", "reported the best results were obtained using hexamers , and because a word size of six nucleotides corresponds to the size of a dicodon , we chose to analyze hexamer frequencies . to use longer words", "requires more training data , because the number of possible words increases exponentially with increasing word size .", "use of shorter words may be adequate for some applications and will be investigated in future work . though we used white 's word - counting methods , we did make slight modifications .", "we simplified one program ( called hybridize ) to compute individual dissimilarity values , rather than paired differences ; a patch that details how to modify the c program is available ( see hyb2dis.txt in additional data files ) .", "more importantly , we amended the null hypothesis and interpreted calibration curves to test for statistically significant dissimilarity differences . though the likelihood - ratio test statistic indicates the magnitude of similarity to a or b , we do not know what values for t are significant with known confidence .", "when testing hypotheses , one can make two types of error : type i , or false positives , and type ii , false negatives .", "the false - positive rate is denoted  and false - negative rate . we determine  and  from overlap in the calibration curves . inferring error rates from calibration curves", "is justified because we know the correct answer and determine the error rate via resampling , as with bootstrap methods to infer error rates or confidence intervals .", "we are interested in knowing from which of two organisms a sequence originated , and are reasonably confident that it came from either one or the other .", "thus , we assume it came from one and test whether we have evidence to refute this assumption .", "the null hypothesis here is that sequence x is from a. alternatively , it might be from b. evaluating the calibration curve overlap at t = 0 quantifies the associated error rates .", "the cumulative distribution function ( cdf ) of taxon b specifies  where cdfb intersects 0 ; the cdf from a specifies  as 1-cdfa(0 )", ". we can thus resolve the problem with known confidence p : p ( t > 0 ) = . all other computations were performed as described previously .", "software used for lexical analysis was obtained via anonymous ftp from the tigr software ftp site ."], ["to characterize hexamer frequencies in plant hosts and their microbial symbionts , we collected sets of training sequences from public databases and edited them for quality .", "training sets were chosen to be representative of , but obtained independently from , taxa participating in symbiotic associations for which a diagnosis of origin would be made . because the species being compared are represented unevenly in public sequence databases , taxa were chosen so that roughly the same number of genes were analyzed in each training set , rather than simply to maximize the numbers of species or sequences present .", "training sets represent protein - coding sequences from three taxonomic groupings : plants ( a1 , medicago and glycine spp . ) , either fungi ( b2 , zygomycetes and chytridiomycetes ) or stramenopiles ( b1 ) , including ests from p. infestans , and bacteria ( b3 , rhizobium , sinorhizobium and bradyrhizobium ) .", "we performed pairwise comparisons with two different , taxon - specific training sets ( a and b ) to infer the origin of a transcript .", "training sets were obtained by querying the genbank database using the entrez retrieval tool . a preliminary query by taxon name obtained all available nucleotide sequences from that taxon , then the limits option excluded ests , stss ( sequence - tagged sites ) , gsss ( genome survey sequences ) , working draft sequences , and patented sequences from the query set . organellar ( mitochondrial and chloroplast ) dna was also excluded via the limits option .", "a query term to require that a sequence contain a protein - coding region ( cds ) was also added , which excluded ribosomal and transfer rna sequences .", "the results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query .", "( changing slightly the composition of training sets between those dates did not notably affect the experimental outcome . ) following a previously established protocol , we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . in this protocol", ", we resampled each training set 40 times by random partitioning into training ( for hexamer counts ) and test calculation pools . to control for any bias introduced by length variation ,", "a program randomly clipped 300 nucleotide fragments for word counting . as a result , one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate ; independent replicates contained different , randomly chosen training sequences and 300 nucleotide fragments .", "values of the test statistic from 40 resampled replicates were pooled for calibration purposes . as with the original protocol , we pooled the resulting test statistic distributions , normalized them as cumulative distributions , and then evaluated them for overlap .", "we call the resulting comparisons ' calibration curves ' , as they are not used directly to make inferences , but rather indirectly , to evaluate the degree of separation in hexamer counts from different taxa", ". overlap of calibration curves should be minimal to yield the most statistically powerful results possible .", "due to considerable overlap of calibration curves between taxonomically general , inclusive training sets ( that is , all eudicots , all fungi and miscellaneous eukaryotes , and all eubacteria , data not shown ) , we opted to work with specific training sets that included only the most species - specific sequences available , while maintaining approximately equal sample sizes across taxa .", "the most challenging case was that of the arbuscular mycorrhizal fungi , for which very few protein - coding sequences are available . to increase the amount of data in this training set ( b2 ) without biasing sample sizes , we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences , and compared this training set with a set from a single plant genus , medicago ( a2 ) .", "we chose this option , rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes , because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes , based on 18s ribosomal rna sequence data .", "that is , the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes , which resemble more closely the ancestral state in modern lineages . starting with a full set of sequences , we filtered for high - quality sequences by trimming regions having extensive ambiguous bases ( n - rich ) and poly(a ) or poly(t ) regions .", "thus , we trimmed poly(a ) and poly(t ) sites to minimize the cases in which a test sequence resembles one training set more closely than the other , simply by virtue of having an abundance of the hexamer aaaaaa or tttttt .", "similarly , test results obtained from short or n - rich sequences can be difficult to interpret .", "we allowed no more than one n per hexamer and trimmed poly(a ) or poly(t ) tracts longer than 13 nucleotides . to accommodate for possible sequence chimeras ,", "those sequences found to contain an internal poly(a ) or poly(t ) segment longer than 13 nucleotides were partitioned into two fragments , and the longer of the two fragments was used in analysis , provided its length was at least 300 nucleotides . after trimming ,", "we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn .", "all blast searches used default parameters and low - complexity filtering with the programs dust or seg .", "the decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves ( data not shown ) , likely a result of divergent evolutionary rates between protein - coding and non - coding sequences .", "chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes .", "table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening .", "to test the validity of word counting as a solution to the problem , we identified a set of 50 gene sequences from plants ( m. truncatula and g. max ) , oomycetes ( phytophthora ) , zygomycetes ( glomus versiforme ) , and bacteria ( sinorhizobium meliloti and agrobacterium tumefaciens ) , for which the function and origin have been characterized experimentally .", "we chose genes known to play a role in plant - microbe interactions , as well as genes that are found across taxa .", "we withheld these sequences , and partial transcripts of the same genes , from training sets prior to comparative lexical analysis , and calculated hexamer dissimilarities for each of the three training sets as described below ."], ["to characterize hexamer frequencies in plant hosts and their microbial symbionts , we collected sets of training sequences from public databases and edited them for quality .", "training sets were chosen to be representative of , but obtained independently from , taxa participating in symbiotic associations for which a diagnosis of origin would be made . because the species being compared are represented unevenly in public sequence databases , taxa were chosen so that roughly the same number of genes were analyzed in each training set , rather than simply to maximize the numbers of species or sequences present .", "training sets represent protein - coding sequences from three taxonomic groupings : plants ( a1 , medicago and glycine spp . ) , either fungi ( b2 , zygomycetes and chytridiomycetes ) or stramenopiles ( b1 ) , including ests from p. infestans , and bacteria ( b3 , rhizobium , sinorhizobium and bradyrhizobium ) .", "we performed pairwise comparisons with two different , taxon - specific training sets ( a and b ) to infer the origin of a transcript .", "training sets were obtained by querying the genbank database using the entrez retrieval tool . a preliminary query by taxon name obtained all available nucleotide sequences from that taxon , then the limits option excluded ests , stss ( sequence - tagged sites ) , gsss ( genome survey sequences ) , working draft sequences , and patented sequences from the query set . organellar ( mitochondrial and chloroplast ) dna was also excluded via the limits option .", "a query term to require that a sequence contain a protein - coding region ( cds ) was also added , which excluded ribosomal and transfer rna sequences .", "the results consisted of all sequences that contain a nuclear protein - coding sequence available for that taxon at the time of the query .", "( changing slightly the composition of training sets between those dates did not notably affect the experimental outcome . ) following a previously established protocol , we used a resampling procedure to evaluate the degree of overlap between distributions of hexamer composition obtained from comparing two training sets . in this protocol", ", we resampled each training set 40 times by random partitioning into training ( for hexamer counts ) and test calculation pools . to control for any bias introduced by length variation ,", "a program randomly clipped 300 nucleotide fragments for word counting . as a result , one random 300 nucleotide fragment from each training sequence was present in the training set during a single resampling replicate ; independent replicates contained different , randomly chosen training sequences and 300 nucleotide fragments .", "values of the test statistic from 40 resampled replicates were pooled for calibration purposes . as with the original protocol , we pooled the resulting test statistic distributions , normalized them as cumulative distributions , and then evaluated them for overlap .", "we call the resulting comparisons ' calibration curves ' , as they are not used directly to make inferences , but rather indirectly , to evaluate the degree of separation in hexamer counts from different taxa", ". overlap of calibration curves should be minimal to yield the most statistically powerful results possible .", "due to considerable overlap of calibration curves between taxonomically general , inclusive training sets ( that is , all eudicots , all fungi and miscellaneous eukaryotes , and all eubacteria , data not shown ) , we opted to work with specific training sets that included only the most species - specific sequences available , while maintaining approximately equal sample sizes across taxa .", "the most challenging case was that of the arbuscular mycorrhizal fungi , for which very few protein - coding sequences are available . to increase the amount of data in this training set ( b2 ) without biasing sample sizes , we pooled sequences from all species in the zygomycetes with all available chytridiomycete coding sequences , and compared this training set with a set from a single plant genus , medicago ( a2 ) .", "we chose this option , rather than including an arbitrary subset of sequences from the ascomycetes and basidiomycetes , because zygomycetes and chytridiomycetes have diverged from their common ancestor less recently than the ascomycetes and basidiomycetes , based on 18s ribosomal rna sequence data .", "that is , the ascomycetes and basidiomycetes are more highly derived from the common fungal ancestor than zygomycetes and chytridiomycetes , which resemble more closely the ancestral state in modern lineages ."], ["starting with a full set of sequences , we filtered for high - quality sequences by trimming regions having extensive ambiguous bases ( n - rich ) and poly(a ) or poly(t ) regions .", "thus , we trimmed poly(a ) and poly(t ) sites to minimize the cases in which a test sequence resembles one training set more closely than the other , simply by virtue of having an abundance of the hexamer aaaaaa or tttttt .", "similarly , test results obtained from short or n - rich sequences can be difficult to interpret .", "we allowed no more than one n per hexamer and trimmed poly(a ) or poly(t ) tracts longer than 13 nucleotides . to accommodate for possible sequence chimeras ,", "those sequences found to contain an internal poly(a ) or poly(t ) segment longer than 13 nucleotides were partitioned into two fragments , and the longer of the two fragments was used in analysis , provided its length was at least 300 nucleotides . after trimming , we screened all remaining sequences of 300 nt or longer for similarity to escherichia coli using blastn .", "all blast searches used default parameters and low - complexity filtering with the programs dust or seg .", "the decision to exclude non - coding rna sequences from training sets was informed by the appearance of bimodal distributions of hexamer frequencies and a large degree of overlap between calibration curves ( data not shown ) , likely a result of divergent evolutionary rates between protein - coding and non - coding sequences .", "chloroplast and mitochondrial sequences were eliminated to avoid complications due to variation in codon usage between nuclear and organellar genomes .", "table 2 summarizes counts of sequences and nucleotides in training sets before and after trimming and screening ."], ["to test the validity of word counting as a solution to the problem , we identified a set of 50 gene sequences from plants ( m. truncatula and g. max ) , oomycetes ( phytophthora ) , zygomycetes ( glomus versiforme ) , and bacteria ( sinorhizobium meliloti and agrobacterium tumefaciens ) , for which the function and origin have been characterized experimentally .", "we chose genes known to play a role in plant - microbe interactions , as well as genes that are found across taxa .", "we withheld these sequences , and partial transcripts of the same genes , from training sets prior to comparative lexical analysis , and calculated hexamer dissimilarities for each of the three training sets as described below ."], ["to diagnose the species of origin for sequences expressed in symbiotic cultures , we collected sequences generated by distinct est sequencing projects from the genbank database .", "sequences from pathogenic interactions originated from cultures of a species from the genus phytophthora with its plant host , such as p. sojae and soybean ( g. max ) isolated from inoculated hypocotyls two days after infection   and p. medicaginis and m. truncatula isolated from infected roots 10 days after infection ( c. vance , unpublished data ) .", "sequences expressed during mutualistic interactions were obtained from cultures with m. truncatula and mycorrhizal ( glomus versiforme ; m.j .", "harrison , unpublished data ) or rhizobacterial ( s. meliloti ; k. vandenbosch , unpublished data ) endosymbionts several days after inoculation", ". sequences expressed in pure , axenic cultures from p. sojae mycelia and zoospores   and from sterile , uninoculated m. truncatula roots   provided a basis for comparison in which no foreign transcripts were expected . to maximize the reliability of diagnostic comparisons , we screened test sequences for high quality as for training sequences , and for low similarity to e. coli , chloroplast and mitochondrial genes , and non - coding rna transcripts ( ribosomal and transfer rnas ) .", "independent blastn comparisons identified sequences having very high similarity ( e < 10 ) to vector sequences or moderately high similarity ( e < 10 ) to non - nuclear or non - coding sequences obtained from genbank ."], ["we wrote a perl program ( countgc.pl ) that calculates the gc base content of a sequence as the portion of guanine and cytosine residues among all unambiguous ( non - n ) nucleotides in a sequence . the hist method in r ,", "version 1.1.1   aggregated continuous percentages into discrete histogram bins , using bin sizes of 2% difference in gc , with inclusive lower bin boundaries and exclusive upper bounds ; the lm method tested for linear correlation of the dissimilarity test statistic t with gc ."], ["white et al .   used a likelihood - ratio test to determine whether word frequencies from a particular sequence more closely resemble the frequency distribution of control data sets from the taxon being sequenced or a distantly related outgroup .", "they computed a test statistic t(a , b , x ) for each sequence x as the difference of log - likelihood ratio dissimilarity measures , d(a , x ) = -2log(a , x ) , for two data sets , a control set a and an outgroup b , such that t(a , b , x ) = d(a , x ) - d(b , x ) .", "a negative value for t indicates that the sequence more closely resembles words from a ; conversely , a positive value indicates a likely contaminant related to b. ( dissimilarity is conceptually related to distance . however , dissimilarity does not measure distance because it does not possess the mathematical properties of a distance metric . ) unlike the calculation of calibration curves , in which 300-nucleotide subsequences are randomly resampled , hexamer dissimilarity is measured over the whole length of a test sequence when inferring a transcript 's origin .", "originally , the investigators used the null hypothesis that no difference exists for dissimilarity measures between the two data sets , or that t(a , b , x ) = 0 .", "white et al .   tested two alternative hypotheses : that t < 0 , being more like a , or t > 0 , like b. lexical analysis using pentamers or heptamers yields similar error rates and very highly correlated values for the test result ( not shown ) . because white et al .  ", "reported the best results were obtained using hexamers , and because a word size of six nucleotides corresponds to the size of a dicodon , we chose to analyze hexamer frequencies . to use longer words requires more training data , because the number of possible words increases exponentially with increasing word size .", "use of shorter words may be adequate for some applications and will be investigated in future work . though we used white 's word - counting methods , we did make slight modifications .", "we simplified one program ( called hybridize ) to compute individual dissimilarity values , rather than paired differences ; a patch that details how to modify the c program is available ( see hyb2dis.txt in additional data files ) .", "more importantly , we amended the null hypothesis and interpreted calibration curves to test for statistically significant dissimilarity differences . though the likelihood - ratio test statistic indicates the magnitude of similarity to a or b , we do not know what values for t are significant with known confidence .", "when testing hypotheses , one can make two types of error : type i , or false positives , and type ii , false negatives .", "the false - positive rate is denoted  and false - negative rate . we determine  and  from overlap in the calibration curves . inferring error rates from calibration curves", "is justified because we know the correct answer and determine the error rate via resampling , as with bootstrap methods to infer error rates or confidence intervals .", "we are interested in knowing from which of two organisms a sequence originated , and are reasonably confident that it came from either one or the other .", "thus , we assume it came from one and test whether we have evidence to refute this assumption .", "the null hypothesis here is that sequence x is from a. alternatively , it might be from b. evaluating the calibration curve overlap at t = 0 quantifies the associated error rates .", "the cumulative distribution function ( cdf ) of taxon b specifies  where cdfb intersects 0 ; the cdf from a specifies  as 1-cdfa(0 )", ". we can thus resolve the problem with known confidence p : p ( t > 0 ) = . all other computations were performed as described previously .", "software used for lexical analysis was obtained via anonymous ftp from the tigr software ftp site ."], ["the following files are available for download : countgc.pl : perl script used to compute gc content of sequences analyzed .", "hyb2dis.txt : patch file that converts white 's hybridize program to compute individual dissimilarity values .", "training sets ( glycinemedicago.txt,rhizobia.txt , stramenopiles.txt , zygochytrid.txt ) : fasta - formatted text files that contain the sequences used for calibration and comparison .", "test sets ( psojaeha.txt , psojaemy.txt , psojaezo.txt , mtrhe.txt , dsir.txt , mham.txt , kv0.txt , kv2.txt , kv3.txt ) : fasta - formatted text files containing transcripts analyzed , edited for quality .", "test results ( psojaeha.dat , psojaemy.dat , psojaezo.dat , mtrhe-a1b1.dat , mtrhe-a2b2.dat , dsir.dat , mham.dat , kv0.dat , kv2.dat , kv3.dat ) : text files that contain transcript analysis results , sorted from least to most plant - like ."], ["fasta - formatted text files containing transcripts analyzed , edited for quality . text files that contain transcript analysis results , sorted from least to most plant - like ."], ["we thank callum bell , mark gijzen , maria harrison , tom kepler , deb samac , and bruno sobral for valued discussions and feedback . comments from b. m. tyler and an anonymous reviewer on an earlier version of this work greatly enhanced its presentation ."], ["distribution of gc content in pure and mixed - culture libraries . ( a ) probability densities for histogram bin sizes of 0.02 ( 2% ) in base content .", "( a ) calculation of statistical parameters from cdfs a and b. overlap in the upper tail of cdfa with cdfb and the lower tail of cdfb with cdfa are likely regions for error .", "we find the false - positive rate  where 1 - cdfa intersects 0 [ cdfa(0)= 1 -  ] , and the false - negative rate  where cdfb crosses 0 . also shown", "are the medians (  ) for each distribution , where cdf( ) = 0.5 .", ", solid black line ) and stramenopile plus p. infestans est ( b1 , dashed black line ) training sequences .", "superimposed distributions of test results show dissimilarity differences for infected g. max ( green ) and axenic p. sojae mycelial and zoospore sequences ( blue and cyan , respectively ) .", "each point corresponds to an expressed tag from either ( a ) infected g. max or ( b ) axenic p. sojae mycelial or ( c ) zoospore sequences , compared with plant ( a1 ) and stramenopile plus p. infestans est training sequences ( b1 ) .", "the identity function indicates equal dissimilarity to both training sets , t = d(a ) - d(b)= 0 .", "calibration curves compare plant training sets ( a1 and a2 , solid black lines ) with one of three microbial symbiont training sets ( broken black lines ) : ( a ) stramenopile and p. infestans est sequences ( b1 ) ; ( b ) pooled zygomycete and chytridiomycete coding sequences ( b2 ) ; and ( c ) sequences from the genera rhizobium , sinorhizobium and bradyrhizobium ( b3 ) .", "cumulative distributions of test results from m. truncatula axenic and microbial symbiont mixed cultures appear in each panel ( colored lines ) .", "each point indicates the dissimilarity of a test sequence compared with a plant training set ( a1 or a2 ) and one of three microbial symbiont training sets : ( a ) stramenopile and p. infestans est sequences ( b1 ) ; ( b ) pooled zygomycete and chytridiomycete coding sequences ( b2 ) ; and ( c ) sequences from the genera rhizobium , sinorhizobium and bradyrhizobium ( b3 ) . sequences from m. truncatula axenic ( green ) and microbial symbiont mixed culture libraries are represented in each panel .", "number of sequences ( n ) and nucleotides ( nt ) , as raw , trimmed ( removed n - rich regions , poly(a ) and poly(t ) sites ) , and screened sequences ( removed ribosomal , chloroplast , and mitochondrial dna and remaining sequences shorter than 300 nucleotides ) .", "number of est sequences ( n ) and nucleotides ( nt ) as raw , trimmed ( limited lengths of n - rich regions , poly(a ) and poly(t ) sites ) , and screened ( removed ribosomal , chloroplast , and mitochondrial dna , and remaining sequences shorter than 300 nt ) sequences ."]]}
{"article_id": "PMC3985683", "article_text": ["the transition - metal - catalyzed \n amination of aryl halides has become \n one of the most powerful methods to construct arylamines .", "the development of methods to couple aryl halides \n with amines has been studied extensively in the past decade .", "palladium - catalyzed reactions occur with a broad scope of both coupling \n partners , high functional group tolerance , and good selectivity for \n monoarylation of the amine nucleophile .", "both the palladium metal and the \n ligands used for this reaction are costly , and the ligand is difficult \n to recycle . for these types of reasons ,", "much interest has been \n paid recently \n to develop reactions catalyzed by first - row metal complexes that are \n typically catalyzed by precious metals .", "for example , copper complexes \n have been studied extensively for the coupling of aryl iodides , and \n to a lesser extent aryl bromides , with nitrogen nucleophiles . yet ,", "the scope of such coupling with electron - rich \n bromoarenes , ortho - substituted bromoarenes , unactivated aryl chlorides , \n and heteroaryl halides is limited .  ", "many of the first - row metals \n are more electropositive than those \n of the second row and form electron - rich low - valent species .", "indeed , \n nickel complexes have been shown to catalyze the amination of aryl \n chlorides and phenol derivatives ( scheme 1 ) .", "such nickel - catalyzed amination reactions could be more practical \n than palladium - catalyzed amination reactions on large scale because \n the metal is much less expensive , and much simpler ligands on the \n nickel center are needed for the catalyst to react with aryl chlorides . \n for example", ", hidai showed over 40 years ago that ni(pph3)4 adds phenyl chloride at room temperature to form ( pph3)2ni(ph)(cl ) , whereas pd(pph3)4 does not react with aryl chlorides .", "however , the scope of the nickel - catalyzed amination of aryl halides \n has been limited to the coupling of secondary alkylamines and arylamines .", "primary alkylamines , one of the most significant classes of amines \n for such cross coupling , have been shown to couple only with activated \n aryl chlorides .", "in addition to the limitations on the scope of the coupling \n of \n aryl halides with amines , the mechanism of the coupling of aryl halides \n with amines catalyzed by nickel complexes has not been studied .", "many \n papers cite the potential of ni(i ) intermediates , either as part of a one - electron redox event or as part \n of a mechanism involving ni(i ) and ni(iii ) intermediates . in contrast , \n the mechanism for the palladium - catalyzed amination of aryl electrophiles \n has been studied in detail , including the kinetic behavior of the \n catalytic reaction and stoichiometric reactions of isolated intermediates .", "such studies have not been conducted on nickel - catalyzed couplings \n to form c ", "n bonds , but are particularly important if one is \n to determine how to create catalysts of nickel that are as long - lived \n as those of palladium and that react with similarly broad scope . here , we present the first nickel - catalyzed amination of unactivated \n aryl chlorides with primary aliphatic amines .", "the reaction occurs \n with aryl and heteroaryl chlorides and bromides catalyzed by a single - component \n binap - ligated nickel(0 ) precursor .", "kinetic studies have been conducted \n on the reaction , and studies on the relative rates of the reaction \n with various isolated nickel complexes have been conducted .", "these \n studies rule out a catalytic cycle occurring through a ni(i ) halide \n intermediate and are consistent with a ni(0)/ni(ii ) catalytic cycle \n for the amination of aryl chlorides and aryl bromides , which react \n with different ni(0 ) species .", "we initiated our studies \n of nickel - catalyzed amination of aryl \n chlorides with primary aliphatic amines by studying the reaction between \n 3-chloroanisole and octylamine .", "we investigated nickel complexes generated \n in situ from ni(cod)2 and various phosphine ligands as \n catalysts .", "these reactions were conducted \n with naobu as base in toluene at 50 c . \n", "reactions catalyzed by the combination of ni(cod)2 and \n monophosphines , such as pph3 and pcy3 , afforded \n only trace amounts of the desired amine product ( entries 14 ) . \n", "similar results ( < 5% conversion and < 2% yield ) were obtained \n for reactions catalyzed by the combination of ni(cod)2 ( 1 \n mol % ) and dppe , dppp , or dppb ( 1 or 2 mol % ) ( entries 510 ) . \n", "however , reactions catalyzed by the combination of ni(cod)2 and dppf or binap gave significant amounts of product ( 1667% , \n entries 1114 ) . in particular , the reaction catalyzed by the \n single component catalyst ( binap)ni(-nc - ph ) afforded \n the product in 92% yield ( entry 15 ) .", "an explanation for the high activity \n of this catalyst will be revealed during the presentation of our mechanistic \n studies .", "conditions : 3-chloroanisole \n ( 0.400 \n mmol ) , octylamine ( 0.600 mmol ) , naobu \n ( 0.600 mmol ) , 24 h. conversion and yield were determined by gc analysis \n using dodecane as internal standard .", "[ ni ] = ( binap)ni(-nc - ph ) . with an active catalyst in hand and reliable \n conditions identified for this ni - catalyzed amination , we studied \n the scope of aryl chlorides that undergo these amination reactions , \n and the results are summarized in table 2 . \n in general , a range of electron - rich ( 2a2e ) , electron - neutral ( 2h ) , and electron - deficient \n ( 2i2l ) aryl chlorides coupled with \n octylamine in high yields ( 7696% ) under the developed conditions \n with 1 mol % of nickel complex 1 . for very electron - rich \n aryl chlorides ,", "4 mol % of the catalyst was required to obtain modest \n to good yields ( 2f and 2 g ) .", "for these two \n reactions , aryl chlorides were not fully converted , and only trace \n amounts ( < 5% ) of biaryl compounds were detected by gc analysis . \n", "mono - ortho - substituted aryl chlorides reacted in \n high yields ( 2a , 2c2e , 2h , and 2j ) .", "however , di - ortho - substituted aryl chlorides , such as 2-chloro-1,3-dimethylbenzene , \n did not couple with octylamine under the developed conditions .", "conditions : arcl ( 0.400 mmol ) , octylamine \n ( 0.600 mmol ) , ( binap)ni(-nc - ph ) ( 4.0 mol , \n 1 mol % ) , naobu ( 0.600 mmol ) , toluene \n ( 1 ml ) ; temperature , 50 c ; reaction time , 24 h. ( binap)ni(-nc - ph ) \n ( 16.0 mol , 4 mol % ) .", "the scope of primary amines was studied by conducting the amination \n reactions of 3-chloroanisole catalyzed by 2 mol % complex 1 .", "the results of this study are summarized in table 3 . in general , a variety of primary aliphatic amines underwent \n this amination process in good to excellent isolated yields ( 7596% ) .", "conditions : 3-chloroanisole ( 0.400 \n mmol ) , primary amine ( 0.600 mmol ) , ( binap)ni(-nc - ph ) \n ( 8.0 mol , 2 mol % ) , naobu ( 0.600 \n mmol ) , toluene ( 1 ml ) ; temperature , 50 c ; reaction time , 24 \n h. ( binap)ni(-nc - ph ) \n ( 16.0 mol , 4 mol % ) .", "the steric bulk of the alkyl group of the primary amines significantly \n influenced the yields of these reactions .", "the reactions of an amine \n containing a primary alkyl group ( 2 m ) afforded the product \n in higher yield than the reactions of amines containing a secondary \n alkyl group ( 2n and 2o ) , although the primary \n amines containing a secondary alkyl group still coupled with aryl \n chlorides in good yield .", "however , the amination of 3-chloroanisole \n with primary amines containing a tertiary alkyl group , for example , tert - butylamime and amylamine , gave only trace amounts of \n the desired products .", "amines containing aryl ( 2p ) , heteroaryl \n ( 2q and 2r ) , allyl ( 2s ) , and \n cyano ( 2 t ) groups reacted in high yields ( 9096% ) . \n", "primary amines bearing 1,3-dioxolane ( 2u and 2v ) and morpholine ( 2w ) moieties also reacted in high \n yields ( 8388% ) .", "the reaction of n - isopropylethylenediamine , \n a substrate containing both a primary and a secondary amine , occurred \n selectively with the primary amine , leaving the secondary amine intact \n ( 2x ) .", "nitrogen - containing heterocycles containing \n amino substituents \n are of particular importance for pharmaceutical applications . however , \n the c  n coupling reactions between nitrogen - containing heteroaryl \n electrophiles and primary amines are challenging , presumably because \n of the coordinating properties of both pyridines and primary amines . \n", "nickel catalysts have been reported for the amination of heteroaryl \n chlorides , but the amine nucleophiles are limited to secondary amines .", "cross - coupling reactions between nitrogen - containing \n heteroaryl \n chlorides and primary amines catalyzed by 2 mol % of complex 1 are summarized in table 4 .", "the scope \n of heteroaryl chlorides that underwent this amination reaction encompassed \n a range of 2-pyridyl ( 3a3f ) , 3-pyridyl \n ( 3g3l ) , and 4-pyridyl ( 3m3p ) chlorides containing electron - donating groups \n ( me and ome ) and electron - withdrawing groups ( cn and cf3 ) .", "in general , these reactions afforded the corresponding heteroaryl \n alkylamine in good to excellent isolated yields ( 5796% , average \n yield : 81% ) .", "this amination also occurred in high yields with 2-chloroquinoline \n ( 3q ) and 2-chloropyrazine ( 3r ) .", "the scope of primary amines that underwent reactions with chloropyridines \n was similar to that of the reactions of 3- chloroanisole described \n in table 3 .", "conditions : 3-chloroanisole ( 0.400 \n mmol ) , primary amine ( 0.600 mmol ) , ( binap)ni(-nc - ph ) \n ( 8.0 mol , 2 mol % ) , naobu ( 0.600 \n mmol ) , toluene ( 1 ml ) ; temperature , 50 c ; reaction time , 24 \n h. ( binap)ni(-nc - ph ) \n ( 16.0 mol , 4 mol % ) . compared \n to the well - established palladium - catalyzed amination of aryl bromides , \n", "the nickel - catalyzed amination of aryl bromides has been less studied , \n and conditions for coupling of aryl bromides with amines by nickel \n catalysts have not been identified .", "only two examples of nickel - catalyzed \n amination of aryl bromides have been published , and these two reactions occurred in poor yields ( eqs 1 and 2 ) .", "having identified conditions \n for the nickel - catalyzed amination of aryl chlorides , we tested these \n conditions for the coupling of aryl bromides with primary amines .", "the amination of aryl bromides catalyzed by benzonitrile complex 1 in toluene with naobu base \n occurred in good yields in some cases , but the scope of the reaction \n was limited .", "bromobenzene and 4-bromobenzotrifluoride reacted with \n octylamine in high gc - yields ( 98% and 94% , repectively ) under conditions \n listed in table 2 with 3 mol % of complex 1 ( eq 3 ) .", "however , 3-bromoanisole coupled \n with 2-(1,3-dioxolan-2-yl)ethan-1-amine in only 55% gc - yield , whereas \n the reaction of the same amine with 3-chloroanisole afforded the desired \n product in 87% isolated yield ( eq 4 ) .", "this \n limited scope for the amination of aryl bromides may stem from the \n faster thermal decomposition of arylnickel(ii ) bromide species to \n nickel(i)3 bromide , relative to the rate of decomposition \n of arylnickel(ii ) chloride species .", "the \n catalyst precursor employed in this study is a single - component nickel(0 ) \n complex ( binap)ni(-nc - ph ) ( 1 ) .", "previously , \n we reported the synthesis of this nickel complex by substitution of \n the 1,5-cyclooctadiene ( cod ) in ni(cod)2 by binap , and \n benzonitrile . to avoid using the thermally \n labile , air- and moisture - sensitive ni(cod)2 , we developed \n a new synthesis ( scheme 2 ) of this complex \n starting from the air - stable nickel precursor ( dme)nicl2 .", "reaction of ( dme)nicl2 with 1 equiv of binap in thf \n at 50 c afforded ( binap)nicl2 , which was then reduced \n by zinc metal in the presence of cod .", "p nmr analysis \n of this reaction indicated a clean formation of ( binap)ni(cod ) .", "the \n byproduct zncl2 was removed by washing the reaction mixture \n with degassed aqueous naoh . at this stage ,", "the residue was \n suspended in toluene and then allowed to react with benzonitrile . \n", "arylnickel halide complexes are important intermediates \n in nickel - catalyzed cross - coupling reactions . in general , arylnickel \n halides ligated by monophosphines are synthesized by oxidative addition \n reactions of aryl halides to ni(pr3)x ( r = alkyl or aryl ) , but arylnickel \n halides ligated by bisphosphines are prepared by ligand substitution \n reactions between monophosphine - ligated arylnickel halide complexes \n with bisphosphines .", "the direct oxidative \n addition of aryl halides to bisphosphine - ligated ni(0 ) has rarely \n been investigated , mainly due to the lack of bisphosphine - ligated \n ni(0 ) precursors that can react with aryl halides at ambient temperature . \n", "recently , we reported the synthesis of binap - ligated arylnickel halide \n complexes by reactions of aryl halides with ( binap)ni(cod ) in the \n presence of catalytic amounts of benzonitrile , and we identified ( binap)ni(-nc - ph ) as the active ni(0 ) species . in our communication on the nickel - catalyzed asymmetric -arylation \n of ketones , we reported the single reaction of electron - deficient \n 4-chlorobenzotrifluoride with this newly defined ni(0 ) precursor 1 and no isolation of the products . here", "we show the results \n of the reactions of complex 1 with electronically varied \n aryl chlorides .", "electron - neutral and electron - deficient aryl \n chlorides reacted smoothly with ni(0 ) complex 1 in thf \n at room temperature to afford the arylnickel(ii ) chloride complexes 47 in 7689% isolated yields ( eq 5 ) .", "these complexes are stable in the solid state \n at ambient temperature for at least six months without noticeable \n decomposition . however , upon standing in thf solution at room temperature \n for about 48 h , they fully decomposed to [ ( binap)ni(-cl)]2 ( 8) with concomitant release of biaryls , as \n detected by gc - ms analysis .", "however , the reaction \n of electron - rich 4-chloroanisole with complex 1 in thf \n did not form the arylnickel chloride complex resulting from simple \n oxidative addition , as determined by p nmr spectroscopy . \n instead ,", "p nmr signals corresponding to two mutually trans phosphine resonances ( doublets at 24.2 and 14.4 ppm \n with jpp = 352 hz ) were observed .", "56single - crystal x - ray diffraction of the isolated \n material ( figure 1 ) showed that this product \n is complex 9 ( eq 7 ) .", "complex 9 contains a coordinated monophosphine ligand ph2p(c6h4 - 4-ome ) and a six - membered nickelacyclic \n structure .", "nickelacyclic complex 9 contains a small dihedral \n angle between the two naphthyl groups of 63 , and the nickel \n center consists of a distorted square planar geometry .", "complex 9 is formed by p  c bond cleavage of the binap backbone \n after the initial formation of binap - ligated arylnickel chloride complex \n ( binap)nicl(c6h4 - 4-ome ) .", "c \n bond cleavage of the binap backbone \n in a binap - ligated phenylpalladium bromide complex was reported by \n our group previously .", "all the ellipsoids are \n drawn at the 50% probability and all the \n hydrogen atoms are omitted for clarity .", "complex 9 was tested as a catalyst for the amination \n of 4-chloroanisole with octylamine under the conditions described \n in table 2 .", "only trace ( < 5% by gc analysis ) \n amounts of the amination product were formed .", "thus , the formation \n of catalytically inactive complex 9 by the reaction of \n 4-chloroanisole and nickel precursor 1 explained the \n high catalyst loading required for the amination of electron - rich \n 4-chloroanisole .", "the oxidation state of the nickel intermediates \n in the \n amination of aryl halides with amines has been ambiguous , and pathways \n involving both ni(0)/ni(ii ) and ni(i)/ni(iii ) couples have been proposed \n for these reactions . with isolated ,", "binap - ligated \n ni(0 ) , ni(i ) halide , and arni(ii ) halide complexes in hand , we were \n able to assess precisely the competence of these complexes to be intermediates \n in the amination reactions . to do so", ", we conducted the reaction \n of the isolated ( binap)nicl(c6h4 - 4-cf3 ) ( 7 ) with octylamine and naobu in the presence of 1 equiv of binap to trap a ni(0 ) product \n ( a , scheme 3 ) .", "this reaction afforded n - octyl-4-(trifluoromethyl)aniline in quantitative yield \n in less than 5 min at room temperature , as indicated by f nmr spectroscopic analysis .", "in addition , we conducted the amination \n reaction of 4-chlorobenzotrifluoride with octylamine in the presence \n of naobu catalyzed by 2 mol % of ( binap)nicl(c6h4 - 4-cf3 ) at room temperature ( b , scheme 3 ) .", "full conversion of 4-chlorobenzotrifluoride was \n achieved in 2 h , and this reaction afforded n - octyl-4-(trifluoromethyl)aniline \n in 91% isolated yield . the same reaction catalyzed by 2 mol % of", "[ ( binap)ni--cl]2 did not afford any amination product ( c , scheme 3 ) .", "these results show clearly that the binap - ligated \n arylnickel(ii ) chloride compound is kinetically and chemically competent \n to be an intermediate in the catalytic process and", "the binap - ligated \n nickel(i ) halide is not . to identify the resting \n state of the catalyst in this nickel - catalyzed \n amination reaction", ", we monitored by p{h } \n nmr spectroscopy the reaction of 4-chlorobenzotrifluoride with octylamine in the presence of naobu catalyzed by 2 mol % of ( binap)ni(-nc - ph ) \n ( 1 ) at room temperature . during the first 50% conversion \n of 4-chlorobenzotrifluoride ,", "however , during the second 50% conversion , a new species \n corresponding to a p{h } nmr resonance at \n  31.7 ppm gradually accumulated .", "this new species is ( binap)2ni , as determined by independently preparing this complex \n by reduction of ( dme)nicl2 with zn metal in the presence \n of 2 equiv of binap in thf and comparing the p nmr chemical \n shift . to test the catalytic activity of ( binap)2ni for this amination process", ", we conducted the reaction of 4-chlorobenzotrifluoride \n with octylamine catalyzed by 5 mol % of ( binap)2ni in the \n presence of naobu at room temperature .", "this reaction did \n not afford detectable amounts of the amination product ( a , scheme 4 ) .", "this result is consistent with the early report \n that ( binap)2ni does not undergo oxidative addition with \n aryl chlorides or bromides at ambient temperatures . the same reaction catalyzed by 5 mol % ( binap)2ni in \n the presence of a catalytic amount of phcn ( 30 mol % ) afforded the \n amination product in 84% yield in 20 h ( b , scheme 4 ) . for comparison", ", we conducted the same reaction catalyzed \n by only 2 mol % ( binap)ni(-nc - ph ) .", "this reaction \n afforded the amination product in almost quantitative yield in 2 h \n at room temperature ( c , scheme 4 ) .", "thus , ( binap)ni(-nc - ph ) is a more active catalyst than the combination of \n ( binap)2ni and phcn . to assess the origin of the \n effect of benzonitrile on the rate \n of the catalytic reaction", ", we performed the reaction of ( binap)2ni with benzonitrile and the reaction of ( binap)ni(-nc - ph ) with binap .", "the reaction of ( binap)2ni \n with 10 equiv of benzonitrile did not form detectable amounts of ( binap)ni(-nc - ph ) , as determined by p nmr spectroscopy \n ( d , scheme 4 ) .", "the reaction of ( binap)ni(-nc - ph ) with 1 equiv of binap afforded ( binap)2ni quantitatively at ambient temperature ( e , scheme 3 ) .", "based on these stoichiometric reactions and the catalytic \n reactions in scheme 3 , we conclude that the \n formation of ( binap)2ni is reversible in the presence of \n benzonitrile , but the equilibrium lies largely on the side of ( binap)2ni and free nitrile .", "the formation of bis(amine)-ligated palladium complexes \n was reported \n as one possible pathway for deactivation of the catalyst in the palladium - catalyzed \n amination of aryl halides .", "formation of \n analogous bis(amine ) complexes of nickel was proposed to explain the \n much lower reactivity of primary aliphatic amines than that of cyclic \n secondary aliphatic amines and anilines in the nickel - catalyzed amination \n reaction . however , these bis(amine)-ligated \n nickel complexes have never been prepared or characterized . in the \n nickel - catalyzed amination of aryl chlorides with primary amines catalyzed \n by ( binap)ni(-nc - ph ) , we observed the formation \n of ( binap)2ni .", "the stoichiometry of this conversion indicates \n that there are nickel complexes lacking binap ligand in the reaction \n mixture .", "thus , we studied the ligand substitution reactions between \n primary aliphatic amines and binap - ligated ni(0 ) and binap - ligated \n ni(ii ) complexes .", "the reaction of ( binap)ni(-nc - ph ) with 50 equiv of n - butylamine in toluene \n did not form any new nickel species , and free binap was not detected \n by p nmr spectroscopy ( a , scheme 5 ) .", "this result is consistent with the affinity of the electron - rich \n ni(0 ) center for softer , -accepting ligands .", "however , the reaction of ( binap)nicl(c6h4 - 4-cn ) with 50 equiv of n - butylamine afforded \n free binap and trans-(n - butylamine)2nicl(c6h4 - 4-cn ) ( 10 ) ( b , \n scheme 5 ) .", "complex 10 was isolated \n in 76% yield . with the bis(amine)-ligated complex 10 in hand", ", we \n tested its potential to serve as an intermediate in the amination \n reaction .", "we conducted the reaction of 10 with 2 equiv \n of naobu in the presence or in the absence \n of 1 equiv of binap ( c , scheme 5 ) .", "this result \n indicates that the formation of bis(amine)-ligated nickel complexes \n is one possible pathway for decomposition of the nickel catalyst . after identifying the resting state of the nickel catalyst and \n possible paths for catalyst deactivation", ", we sought to identify the \n rate - limiting step for this nickel - catalyzed amination process .", "to \n do so , we measured the dependence of the initial rates of the amination \n reaction on the concentrations of each reagent under conditions in \n which the other reagents were present in large excess .", "we chose \n the reaction of 4-chlorobenzotrifluoride with octylamine in the presence \n of naobu for the kinetic studies because \n this reaction occurred at ambient temperature , and we were able to \n monitor the reaction conveniently by both p and f nmr spectroscopy .", "furthermore , no background nucleophilic \n aromatic substitution reaction was observed for this aryl chloride \n and octylamine in the presence of naobu . for the initial rates to be a valid method for kinetic \n analysis \n of this reaction", ", all reagents must be soluble in the reaction medium , \n and no induction period can be present .", "the single - component catalyst \n precursor 1 is only marginally soluble in the reaction \n medium .", "thus , we used ( binap)nicl(c6h4 - 4-cf3 ) ( 7 ) as the source of nickel for our kinetic \n experiments because it dissolves in seconds once mixed with other \n reagents .", "benzonitrile was added to the reaction mixture to trap the \n binap - ligated ni(0 ) species to form ( binap)ni(-nc - ph ) , \n which had been identified as the catalyst resting state . in the absence \n of benzonitrile , the irreversible formation of ( binap)2ni occurs quickly .", "benzonitrile ( 10 equiv relative to complex 7 ) was added so that catalyst decomposition was not observed \n by p nmr spectroscopy over the time of the reaction .", "the kinetic behavior was assessed for the coupling of 4-chlorobenzotrifluoride \n with octylamine catalyzed by the combination of complex 7 in the presence of phcn and naobu at \n room temperature ( eq 8) .", "the initial rates were \n measured by f nmr spectroscopy using 4-ocf3-anisole as the internal standard .", "the rate of the amination reaction \n was determined by measuring the formation of n - octyl-4-(trifluoromethyl)aniline \n with respect to time .", "the plots of concentrations of n - octyl-4-(trifluoromethyl)aniline vs time at various concentrations \n of each reagent are shown in figures s1s5 in the supporting information .", "the plots of the initial \n rates of the reaction vs the concentrations of the catalyst , aryl \n chloride , and benzonitrile are shown in figure 2.8 the kinetic analysis showed that this nickel - catalyzed \n amination reaction of aryl chlorides is first order in the nickel \n catalyst , first order in aryl chloride , and inverse first order in \n phcn .", "the remaining reagents , naobu and \n amine , did not affect the initial rate of the reaction .", "these data \n indicate that oxidative addition of the aryl chloride to ( binap)ni(0 ) , \n formed by reversible dissociation of phcn from the catalyst resting \n state ( binap)ni(-nc - ph ) , is the turnover - limiting \n step in the amination reaction .", "plots of initial rates vs concentration \n of catalyst , aryl chloride , \n and benzonitrile for the amination of 4-chlorobenzotrifluoride ( 0.101.0 \n m ) with n - octylamine ( 0.30 m ) catalyzed by ( binap)nicl(c6h4 - 4-cf3 ) ( 0.00600.018 m ) in \n the presence of benzonitrile ( 0.0900.180 m ) and naobu ( 0.30 m ) at room temperature .", "the mechanism of palladium - catalyzed cross - coupling reactions with \n aryl halides as electrophiles has been extensively studied .", "these \n studies reveal that the halide identity has a dramatic effect on the \n mechanism for the oxidative addition of aryl halides to pd(0 ) species . \n", "the additions of the less reactive aryl chlorides occurs through lower - coordinated \n pd(0 ) intermediate than the additions of more reactive aryl iodides .", "however , such mechanistic data has not been \n gained for the nickel - catalyzed amination of aryl halides . for the \n nickel - catalyzed enantioselective -arylation of ketones , we \n found that aryl chlorides react in much higher yields and with much \n higher enantioselectivities than aryl bromides , and we attributed \n this dramatic effect of halide on the reaction yield and enantioselectivity \n to the faster decomposition of the arylnickel(ii ) bromide intermediate \n relative to the arylnickel(ii ) chloride intermediate to their corresponding \n ni(i ) halide complexes .", "because \n the ni(0 ) complex 1 ligated by binap and phcn catalyzes \n the amination of both aryl chlorides and aryl bromides , we can compare \n directly the kinetics and mechanism of the amination reactions of \n aryl chlorides and aryl bromides catalyzed by this nickel system . \n", "to do so , we conducted kinetic experiments on the amination of aryl \n bromides that were similar to those we conducted on the amination \n of aryl chlorides", ". we monitored by p nmr spectroscopy \n the reaction of \n 4-bromobenzotrifluoride with octylamine catalyzed by ( binap)ni(-nc - ph ) in the presence of naobu at room temperature . as was the case in the reactions of chloroarenes , \n ( binap)ni(-nc - ph )", "was identified as the resting \n state of the catalyst in the reaction of 4-bromobenzotrifluoride with \n octylamine .", "kinetic studies were conducted on the reaction of \n 4-bromobenzotrifluoride \n with octylamine catalyzed by ( binap)nibr(c6h4 - 4-cf3 ) ( 11 ) in the presence of added phcn \n ( eq 8) .", "the initial rates were measured for \n various concentrations of each reagent while keeping the other reagents \n in excess .", "the plots of concentrations of n - octyl-4-(trifluoromethyl)aniline \n vs time at various concentrations of each reagent are shown in figures s7s11 .", "the plots of the initial \n rates of the reaction vs the concentrations of the catalyst , aryl \n chloride , and benzonitrile are shown in figure 3 .", "plots of initial rates \n vs concentration of catalyst , aryl bromide , \n and benzonitrile for the amination of 4-bromobenzotrifluoride ( 0.100.80 \n m ) with n - octylamine ( 0.30 m ) catalyzed by ( binap)nicl(c6h4 - 4-cf3 ) ( 0.00600.018 m ) in \n the presence of benzonitrile ( 0.0900.180 m ) and naobu ( 0.30 m ) at room temperature . like the amination of 4-chlorobenzotrifluoride ,", "the amination \n of \n 4-bromobenzotrifluoride with octylamine is first order in the nickel \n catalyst , first order in aryl halide , zero order in naobu , and zero order in amine .", "in contrast to the reaction \n of 4-chlorobenzotrifluoride , which is inverse first order in phcn , \n the amination of 4-bromobenzotrifluoride is zero order in phcn .", "this \n order in phcn , in combination with the first order in aryl bromide , \n indicates that the bromoarene reacts directly with ( binap)ni(-nc - ph ) during the turnover - limiting step of the amination \n of the bromoarene without dissociation of phcn .", "the different reactivities \n of aryl halides with binap - ligated nickel complexes of different coordination \n numbers parallels a trend in oxidative addition to pd(0 ) complexes \n noted in the introduction to this section . based on the stoichiometric reactions of \n the isolated binap - ligated arylnickel halides with primary amines \n in the presence of base and the conducted kinetic studies , we propose \n a catalytic cycle depicted in scheme 6 for \n the nickel - catalyzed amination of aryl chlorides and aryl bromides \n reported in this paper .", "the observed first order rate - dependence on \n the concentrations of both the catalyst and aryl halides indicates \n that the oxidative addition of aryl halides to binap - ligated ni(0 ) \n species to form ( binap)niar(x ) ( x = cl or br ) is the turnover - limiting \n step for both amination processes .", "this binap - ligated arylnickel(ii ) \n halide complex subsequently reacts with primary amine and naobu to form an arylnickel amido complex , followed \n by reductive elimination of the n - alkyl aniline product \n in the presence of phcn to regenerate ( binap)ni(-nc - ph ) , which was confirmed as the catalyst resting state .", "interestingly , different rate - dependences on the concentration \n of phcn were observed for this nickel - catalyzed amination of aryl \n chlorides and aryl bromides .", "the reaction of aryl chlorides is inverse \n first order in phcn , whereas the reaction of aryl bromides is zero \n order in phcn .", "these reaction orders indicate that aryl chlorides \n and aryl bromides react with different nickel(0 ) species .", "the aryl \n chlorides react after reversible dissociation of phcn from ( binap)ni(-nc - ph ) to form the lower - coordinate nickel(0 ) species ( binap)ni(0 ) \n and ultimately the binap - ligated arylnickel(ii ) chloride complex . \n", "however , the aryl bromides react directly with ( binap)ni(-nc - ph ) to form binap - ligated arylnickel(ii ) bromide complex \n and the nitrile dissociates during or immediately after the oxidative \n addition process .", "one could imagine the zero - order dependence on added \n phcn could indicate that dissociation of this ligand is rate limiting , \n but the first - order dependence of the reaction on the aryl bromide \n is inconsistent with this proposal .", "in addition to the steps \n within the catalytic cycle that form the \n arylamine product , there are several pathways that generate catalytically \n inactive nickel species .", "first , the reaction of ( binap)niar(cl ) with \n amine rnh2 forms the catalytically inactive ( rnh2)2niar(cl ) and free binap .", "the released free binap in \n the reaction mixture traps the ( binap)ni(0 ) fragment generated by \n dissociation of phcn from ( binap)ni(-nc - ph ) or \n reductive elimination of n - alkyl aniline from the \n binap - ligated arylnickel(ii ) amido complex to form ( binap)2ni .", "( binap)2ni does not undergo oxidative addition of \n the aryl halide and does not catalyze the amination reaction .", "second , thermal decomposition of ( binap)niar(x ) \n ( ar = electron - neutral and electron - rich aryl groups ) affords the \n catalytically inactive [ ( binap)ni(-x)]2 with concomitant \n formation of biaryl .", "c bond cleavage of the binap backbone is a potential \n pathway for the deactivation of the nickel catalyst .", "indeed , this \n complex was observed to accumulate ( approximately 6.4 mm of the 16 \n mm concentration of initial catalyst ) during the reaction of 4-chloroanisole \n with octylamine . to gain \n information on the \n relative rates of the amination of aryl chlorides and aryl bromides \n and the effect of temperature and concentration of phcn on the selectivity \n of the amination reaction for an aryl chloride versus an aryl bromide", ", \n we conducted a series of reactions with an aryl halide containing \n both chloride and bromide and a mixture of aryl halides in the presence \n of various amounts of phcn at room temperature or 50 c .", "the amination of 1-bromo-4-chlorobenzene with \n octylamine catalyzed \n by 3 mol % of ( binap)ni(-nc - ph ) at 50 c occurred \n selectively at the c  br bond , and the product of the reaction \n at the c  cl bond was not detected by gc - ms analysis .", "( a , scheme 7 ) . however , side - by - side reactions of chlorobenzene \n and bromobenzene under the same conditions occurred in the same gc \n yield ( 98% ) ( b , scheme 7 ) .", "the amination \n of equal amounts of 1-bromo-4-butylbenzene and 4-chlorotoluene \n with octylamine catalyzed by 3 mol % of ( binap)ni(-nc - ph ) was conducted at both room temperature and 50 c ( c , \n scheme 7 ) . at both temperatures ,", "these reactions \n occurred in similar yields , but with different selectivity . at room \n temperature ,", "1-bromo-4-butylbenzene was converted selectively over \n 4-chlorotoluene , and a product ratio of 3:1 was obtained . however , \n at 50 c , 1-bromo-4-butylbenzene and 4-chlorotoluene converted \n equally .", "br \n bond in 1-bromo-4-chlorobenzene ( a , scheme 7 ) , the decreased selectivity for amination of the c  br bond \n over the c  cl bond in different haloarenes might stem from \n irreversible or nearly irreversible binding of the haloarene to ni(0 ) \n to form an arene -complex prior to cleavage of the carbon  halogen \n bond .", "this preferential intramolecular oxidative addition has recently \n been observed for the ni - catalyzed kumada cross - coupling reactions \n that are part of chain - growth polymerizations . to evaluate the effect of phcn on the selectivity \n between the amination \n of an aryl chloride and an aryl bromide", ", we conducted the amination \n of equal amounts of 1-bromo-4-butylbenzene and 4-chlorotoluene with \n octylamine catalyzed by 3 mol % of ( binap)ni(-nc - ph ) \n in the presence of 0.9 equiv of phcn ( relative to octylamine ) ( d , \n scheme 6 ) .", "because our kinetic studies revealed \n different dependences of the reaction rate on phcn for this nickel - catalyzed \n amination ( inverse first order and zero order on phcn for the reaction \n of aryl chloride and aryl bromide , respectively ) , we considered that \n an increased selectivity for the conversion of aryl bromide over aryl \n chloride would be observed for reactions conducted with higher concentrations \n of phcn than for reactions conducted with lower concentrations of \n phcn .", "consistent with this prediction , the selectivity was higher \n ( 30:1 \n for the reaction at room temperature and 9:1 for the reaction at 50 \n c ) for the amination of 1-bromo-4-butylbenzene versus the amination \n of 4-chlorotoluene for reactions run with higher concentrations ( from \n 0.004 to 0.18 m ) of benzonitrile at both room temperature and 50 c \n for 24 h. these data show that the difference in ni(0 ) species that \n adds the different aryl halides can be used to beneficially affect \n selectivity .", "several first - row metal complexes catalyze cross \n coupling reactions , \n and many catalyze the coupling of certain classes of chloroarenes . \n", "however , no first row metal complex has been shown to catalyze the \n coupling of primary alkylamines with unactivated chloroarenes .", "copper \n complexes couple bromoarenes and iodoarenes with primary amines , but \n they do not catalyze the coupling of chloroarenes with primary amines .", "previously , the combination of ni(cod)2 and dppf was \n shown to couple an electron - poor chloroarene with a primary amine , \n but this catalyst did not couple unactivated chloroarenes with primary \n amines .", "a set of preliminary studies \n we conducted have shown that the complexes ( dppf)ni(cod ) or ( dppf)2ni generated in situ by the reaction of ni(cod)2 and dppf do not add aryl chlorides at ambient temperature , and the \n reaction of chloroarenes with ( dppf)ni(cod ) or ( dppf)2ni \n at elevated temperatures forms ( dppf)ni(i)cl and biaryl .", "this \n formation of ( dppf)2ni(0 ) and ( dppf)ni(i)cl at \n the elevated temperatures required for oxidative addition of chloroarene \n to occur , likely prevented productive coupling reactions with unactivated \n chloroarenes .", "these observations further illustrate the importance \n of the nitrile - ligated precursor with the general formula l2ni(-nc - ph ) .", "nickel complexes of n - heterocyclic carbenes have \n been shown to couple aryl chlorides and sulfonates with secondary \n amines and with arylamines , but not primary alkylamines .", "mechanistic \n studies on the reactions of secondary amines catalyzed by the complexes \n of n - heterocyclic carbenes are too limited to determine \n the origin of their lack of reactivity with primary amines , but recent \n studies have shown that the arylnickel halide complexes containing \n hindered n - heterocyclic carbenes are not formed by \n the reaction of ( imes)2ni(0 ) with chlorobenzene , bromobenzene , \n and iodobenzene . instead , ni(i ) species \n are formed , and this formation of ni(i ) complexes could either constitute \n the pathway for catalyst decomposition or a mechanism for reaction \n with this catalyst involving odd - electron nickel intermediates .", "we have shown that nickel complexes containing \n common aryl bisphosphine \n ligands can catalyze the coupling of aryl chlorides and bromides with \n primary amines .", "in particular , we have shown that the mixed ni(0 ) \n species ( binap)ni(-nc - ph ) containing one bisphosphine \n and one side - bound nitrile is a particularly active catalyst for the \n coupling of chloroarenes with primary amines and is more active than \n the related ( binap)2ni catalyst . a range of electronically \n varied aryl halides and nitrogen - containing heteroaryl chlorides , \n including pyridine , quinoline , and isoquinoline derivatives , couple \n with a variety of aliphatic primary amines in high isolated yields . \n several aspects of the current system lead to the ability of this \n first - row metal system to catalyze this class of amination reaction . \n in general , we attribute the high yields for these amination reactions \n to the facile oxidative addition of aryl chlorides and heteroaryl \n chlorides to the ( binap)ni(-nc - ph ) precatalyst \n at ambient temperatures . detailed studies on the mechanism of \n the amination reaction", "the following conclusions about the mechanism of this \n coupling process have been drawn from these studies : ( 1 ) the \n oxidative addition of aryl chlorides to ( binap)ni(-nc - ph ) , together with the stoichiometric reactions of ( binap)niar(cl ) \n with primary aliphatic amines in the presence of base , support a reaction \n pathway involving a ni(0)/ni(ii ) couple in the catalytic cycle .", "( 2 ) the oxidative addition of aryl chlorides and heteroaryl chlorides \n to the single - component nickel precursor ( binap)ni(-nc - ph ) at ambient temperatures prevents the formation of inactive \n ni species . ( 3 ) ni(i ) intermediates , which have been observed \n in oxidative \n addition studies to phosphine - ligated ni(0 ) complexes , appear to result \n from decomposition of the ni(ii ) product of oxidative addition as \n opposed to forming from one - electron pathways for oxidative addition \n to ni(0 ) .", "( 4 ) the reaction of ( binap)ni(-nc - ph ) with electron - rich \n aryl chlorides , such as 4-chloroanisole , leads to the p  c bond \n cleavage of the backbone of binap and forms the nickel complex 9 containing two monophosphine ligands .", "the formation of the \n catalytically inactive complex 9 explains the high catalyst \n loading required for the amination of electron - rich aryl chlorides .", "( 5 ) mechanistic and kinetic studies revealed that the turnover - limiting \n step of these nickel - catalyzed amination reactions of aryl chlorides \n is oxidative addition of the aryl chloride to ( binap)ni(0 ) , which \n is formed by dissociation of phcn from the catalyst resting state \n ( binap)ni(-nc - ph ) .", "( 6 ) mechanistic and kinetic \n studies imply that the turnover - limiting \n step of the reactions of aryl bromides is also oxidative addition , \n but that aryl bromides react with a different nickel species , of composition \n ( binap)ni(-nc - ph ) containing a bound nitrile .", "( 7 ) the formation of catalytically inactive bis(amine)-ligated \n ni(ii ) species ( rnh2)2niar(cl ) , ni(i ) species \n [ ( binap)ni(-cl)]2 , and bis - binap - ligated ni(0 ) species \n ( binap)2ni are the major pathways for catalyst inactivation", ". further studies to improve the lifetime of the ni - bisphosphine \n catalysts , to determine the mechanism by which the phosphine - ligated \n ni(i ) species form as a means to prevent this detrimental path , and \n to determine if ni(i ) complexes are generated during oxidative addition \n of haloarenes will be the subject of future work ."], "abstract_text": ["<S> first - row metal complexes \n often undergo undesirable one - electron \n redox processes during two - electron steps of catalytic cycles . </S>", "<S> we \n report the amination of aryl chlorides and bromides with primary aliphatic \n amines catalyzed by a well - defined , single - component nickel precursor \n ( binap)ni(2-nc - ph ) ( binap = 2,2-bis(biphenylphosphino)-1,1-binaphthalene ) \n that minimizes the formation of ni(i ) species and ( binap)2ni . </S>", "<S> the scope of the reaction encompasses electronically varied aryl \n chlorides and nitrogen - containing heteroaryl chlorides , including \n pyridine , quinoline , and isoquinoline derivatives . </S>", "<S> mechanistic studies \n support the catalytic cycle involving a ni(0)/ni(ii ) couple for this \n nickel - catalyzed amination and are inconsistent with a ni(i ) halide \n intermediate . monitoring the reaction mixture by 31p nmr \n spectroscopy identified ( binap)ni(2-nc - ph ) as the \n resting state of the catalyst in the amination of both aryl chlorides \n and bromides . </S>", "<S> kinetic studies showed that the amination of aryl chlorides \n and bromides is first order in both catalyst and aryl halide and zero \n order in base and amine . </S>", "<S> the reaction of a representative aryl chloride \n is inverse first order in phcn , but the reaction of a representative \n aryl bromide is zero order in phcn . </S>", "<S> this difference in the order of \n the reaction in phcn indicates that the aryl chloride reacts with \n ( binap)ni(0 ) , formed by dissociation phcn from ( binap)ni(2-nc - ph ) , but the aryl bromide directly reacts with ( binap)ni(2-nc - ph ) . </S>", "<S> the overall kinetic behavior is consistent with turnover - limiting \n oxidative addition of the aryl halide to ni(0 ) . </S>", "<S> several pathways for \n catalyst decomposition were identified , such as the formation of the \n catalytically inactive bis(amine)-ligated arylnickel(ii ) chloride , \n ( binap)2ni(0 ) , and the ni(i ) species [ ( binap)ni(-cl)]2 . by using a well - defined nickel complex as catalyst , the \n formation of ( binap)2ni(0 ) is avoided and the formation \n of the ni(i ) species [ ( binap)ni(-cl)]2 is minimized . </S>"], "labels": null, "section_names": ["Introduction", "Results and Discussion", "Conclusions"], "sections": [["the transition - metal - catalyzed \n amination of aryl halides has become \n one of the most powerful methods to construct arylamines .", "the development of methods to couple aryl halides \n with amines has been studied extensively in the past decade .", "palladium - catalyzed reactions occur with a broad scope of both coupling \n partners , high functional group tolerance , and good selectivity for \n monoarylation of the amine nucleophile .", "both the palladium metal and the \n ligands used for this reaction are costly , and the ligand is difficult \n to recycle . for these types of reasons ,", "much interest has been \n paid recently \n to develop reactions catalyzed by first - row metal complexes that are \n typically catalyzed by precious metals .", "for example , copper complexes \n have been studied extensively for the coupling of aryl iodides , and \n to a lesser extent aryl bromides , with nitrogen nucleophiles . yet ,", "the scope of such coupling with electron - rich \n bromoarenes , ortho - substituted bromoarenes , unactivated aryl chlorides , \n and heteroaryl halides is limited .  ", "many of the first - row metals \n are more electropositive than those \n of the second row and form electron - rich low - valent species .", "indeed , \n nickel complexes have been shown to catalyze the amination of aryl \n chlorides and phenol derivatives ( scheme 1 ) .", "such nickel - catalyzed amination reactions could be more practical \n than palladium - catalyzed amination reactions on large scale because \n the metal is much less expensive , and much simpler ligands on the \n nickel center are needed for the catalyst to react with aryl chlorides . \n for example", ", hidai showed over 40 years ago that ni(pph3)4 adds phenyl chloride at room temperature to form ( pph3)2ni(ph)(cl ) , whereas pd(pph3)4 does not react with aryl chlorides .", "however , the scope of the nickel - catalyzed amination of aryl halides \n has been limited to the coupling of secondary alkylamines and arylamines .", "primary alkylamines , one of the most significant classes of amines \n for such cross coupling , have been shown to couple only with activated \n aryl chlorides .", "in addition to the limitations on the scope of the coupling \n of \n aryl halides with amines , the mechanism of the coupling of aryl halides \n with amines catalyzed by nickel complexes has not been studied .", "many \n papers cite the potential of ni(i ) intermediates , either as part of a one - electron redox event or as part \n of a mechanism involving ni(i ) and ni(iii ) intermediates . in contrast , \n the mechanism for the palladium - catalyzed amination of aryl electrophiles \n has been studied in detail , including the kinetic behavior of the \n catalytic reaction and stoichiometric reactions of isolated intermediates .", "such studies have not been conducted on nickel - catalyzed couplings \n to form c ", "n bonds , but are particularly important if one is \n to determine how to create catalysts of nickel that are as long - lived \n as those of palladium and that react with similarly broad scope . here , we present the first nickel - catalyzed amination of unactivated \n aryl chlorides with primary aliphatic amines .", "the reaction occurs \n with aryl and heteroaryl chlorides and bromides catalyzed by a single - component \n binap - ligated nickel(0 ) precursor .", "kinetic studies have been conducted \n on the reaction , and studies on the relative rates of the reaction \n with various isolated nickel complexes have been conducted .", "these \n studies rule out a catalytic cycle occurring through a ni(i ) halide \n intermediate and are consistent with a ni(0)/ni(ii ) catalytic cycle \n for the amination of aryl chlorides and aryl bromides , which react \n with different ni(0 ) species ."], ["we initiated our studies \n of nickel - catalyzed amination of aryl \n chlorides with primary aliphatic amines by studying the reaction between \n 3-chloroanisole and octylamine .", "we investigated nickel complexes generated \n in situ from ni(cod)2 and various phosphine ligands as \n catalysts .", "these reactions were conducted \n with naobu as base in toluene at 50 c . \n", "reactions catalyzed by the combination of ni(cod)2 and \n monophosphines , such as pph3 and pcy3 , afforded \n only trace amounts of the desired amine product ( entries 14 ) . \n", "similar results ( < 5% conversion and < 2% yield ) were obtained \n for reactions catalyzed by the combination of ni(cod)2 ( 1 \n mol % ) and dppe , dppp , or dppb ( 1 or 2 mol % ) ( entries 510 ) . \n", "however , reactions catalyzed by the combination of ni(cod)2 and dppf or binap gave significant amounts of product ( 1667% , \n entries 1114 ) . in particular , the reaction catalyzed by the \n single component catalyst ( binap)ni(-nc - ph ) afforded \n the product in 92% yield ( entry 15 ) .", "an explanation for the high activity \n of this catalyst will be revealed during the presentation of our mechanistic \n studies .", "conditions : 3-chloroanisole \n ( 0.400 \n mmol ) , octylamine ( 0.600 mmol ) , naobu \n ( 0.600 mmol ) , 24 h. conversion and yield were determined by gc analysis \n using dodecane as internal standard .", "[ ni ] = ( binap)ni(-nc - ph ) . with an active catalyst in hand and reliable \n conditions identified for this ni - catalyzed amination , we studied \n the scope of aryl chlorides that undergo these amination reactions , \n and the results are summarized in table 2 . \n in general , a range of electron - rich ( 2a2e ) , electron - neutral ( 2h ) , and electron - deficient \n ( 2i2l ) aryl chlorides coupled with \n octylamine in high yields ( 7696% ) under the developed conditions \n with 1 mol % of nickel complex 1 . for very electron - rich \n aryl chlorides ,", "4 mol % of the catalyst was required to obtain modest \n to good yields ( 2f and 2 g ) .", "for these two \n reactions , aryl chlorides were not fully converted , and only trace \n amounts ( < 5% ) of biaryl compounds were detected by gc analysis . \n", "mono - ortho - substituted aryl chlorides reacted in \n high yields ( 2a , 2c2e , 2h , and 2j ) .", "however , di - ortho - substituted aryl chlorides , such as 2-chloro-1,3-dimethylbenzene , \n did not couple with octylamine under the developed conditions .", "conditions : arcl ( 0.400 mmol ) , octylamine \n ( 0.600 mmol ) , ( binap)ni(-nc - ph ) ( 4.0 mol , \n 1 mol % ) , naobu ( 0.600 mmol ) , toluene \n ( 1 ml ) ; temperature , 50 c ; reaction time , 24 h. ( binap)ni(-nc - ph ) \n ( 16.0 mol , 4 mol % ) .", "the scope of primary amines was studied by conducting the amination \n reactions of 3-chloroanisole catalyzed by 2 mol % complex 1 .", "the results of this study are summarized in table 3 . in general , a variety of primary aliphatic amines underwent \n this amination process in good to excellent isolated yields ( 7596% ) .", "conditions : 3-chloroanisole ( 0.400 \n mmol ) , primary amine ( 0.600 mmol ) , ( binap)ni(-nc - ph ) \n ( 8.0 mol , 2 mol % ) , naobu ( 0.600 \n mmol ) , toluene ( 1 ml ) ; temperature , 50 c ; reaction time , 24 \n h. ( binap)ni(-nc - ph ) \n ( 16.0 mol , 4 mol % ) .", "the steric bulk of the alkyl group of the primary amines significantly \n influenced the yields of these reactions .", "the reactions of an amine \n containing a primary alkyl group ( 2 m ) afforded the product \n in higher yield than the reactions of amines containing a secondary \n alkyl group ( 2n and 2o ) , although the primary \n amines containing a secondary alkyl group still coupled with aryl \n chlorides in good yield .", "however , the amination of 3-chloroanisole \n with primary amines containing a tertiary alkyl group , for example , tert - butylamime and amylamine , gave only trace amounts of \n the desired products .", "amines containing aryl ( 2p ) , heteroaryl \n ( 2q and 2r ) , allyl ( 2s ) , and \n cyano ( 2 t ) groups reacted in high yields ( 9096% ) . \n", "primary amines bearing 1,3-dioxolane ( 2u and 2v ) and morpholine ( 2w ) moieties also reacted in high \n yields ( 8388% ) .", "the reaction of n - isopropylethylenediamine , \n a substrate containing both a primary and a secondary amine , occurred \n selectively with the primary amine , leaving the secondary amine intact \n ( 2x ) .", "nitrogen - containing heterocycles containing \n amino substituents \n are of particular importance for pharmaceutical applications . however , \n the c  n coupling reactions between nitrogen - containing heteroaryl \n electrophiles and primary amines are challenging , presumably because \n of the coordinating properties of both pyridines and primary amines . \n", "nickel catalysts have been reported for the amination of heteroaryl \n chlorides , but the amine nucleophiles are limited to secondary amines .", "cross - coupling reactions between nitrogen - containing \n heteroaryl \n chlorides and primary amines catalyzed by 2 mol % of complex 1 are summarized in table 4 .", "the scope \n of heteroaryl chlorides that underwent this amination reaction encompassed \n a range of 2-pyridyl ( 3a3f ) , 3-pyridyl \n ( 3g3l ) , and 4-pyridyl ( 3m3p ) chlorides containing electron - donating groups \n ( me and ome ) and electron - withdrawing groups ( cn and cf3 ) .", "in general , these reactions afforded the corresponding heteroaryl \n alkylamine in good to excellent isolated yields ( 5796% , average \n yield : 81% ) .", "this amination also occurred in high yields with 2-chloroquinoline \n ( 3q ) and 2-chloropyrazine ( 3r ) .", "the scope of primary amines that underwent reactions with chloropyridines \n was similar to that of the reactions of 3- chloroanisole described \n in table 3 .", "conditions : 3-chloroanisole ( 0.400 \n mmol ) , primary amine ( 0.600 mmol ) , ( binap)ni(-nc - ph ) \n ( 8.0 mol , 2 mol % ) , naobu ( 0.600 \n mmol ) , toluene ( 1 ml ) ; temperature , 50 c ; reaction time , 24 \n h. ( binap)ni(-nc - ph ) \n ( 16.0 mol , 4 mol % ) . compared \n to the well - established palladium - catalyzed amination of aryl bromides , \n", "the nickel - catalyzed amination of aryl bromides has been less studied , \n and conditions for coupling of aryl bromides with amines by nickel \n catalysts have not been identified .", "only two examples of nickel - catalyzed \n amination of aryl bromides have been published , and these two reactions occurred in poor yields ( eqs 1 and 2 ) .", "having identified conditions \n for the nickel - catalyzed amination of aryl chlorides , we tested these \n conditions for the coupling of aryl bromides with primary amines .", "the amination of aryl bromides catalyzed by benzonitrile complex 1 in toluene with naobu base \n occurred in good yields in some cases , but the scope of the reaction \n was limited .", "bromobenzene and 4-bromobenzotrifluoride reacted with \n octylamine in high gc - yields ( 98% and 94% , repectively ) under conditions \n listed in table 2 with 3 mol % of complex 1 ( eq 3 ) .", "however , 3-bromoanisole coupled \n with 2-(1,3-dioxolan-2-yl)ethan-1-amine in only 55% gc - yield , whereas \n the reaction of the same amine with 3-chloroanisole afforded the desired \n product in 87% isolated yield ( eq 4 ) .", "this \n limited scope for the amination of aryl bromides may stem from the \n faster thermal decomposition of arylnickel(ii ) bromide species to \n nickel(i)3 bromide , relative to the rate of decomposition \n of arylnickel(ii ) chloride species .", "the \n catalyst precursor employed in this study is a single - component nickel(0 ) \n complex ( binap)ni(-nc - ph ) ( 1 ) .", "previously , \n we reported the synthesis of this nickel complex by substitution of \n the 1,5-cyclooctadiene ( cod ) in ni(cod)2 by binap , and \n benzonitrile . to avoid using the thermally \n labile , air- and moisture - sensitive ni(cod)2 , we developed \n a new synthesis ( scheme 2 ) of this complex \n starting from the air - stable nickel precursor ( dme)nicl2 .", "reaction of ( dme)nicl2 with 1 equiv of binap in thf \n at 50 c afforded ( binap)nicl2 , which was then reduced \n by zinc metal in the presence of cod .", "p nmr analysis \n of this reaction indicated a clean formation of ( binap)ni(cod ) .", "the \n byproduct zncl2 was removed by washing the reaction mixture \n with degassed aqueous naoh . at this stage ,", "the residue was \n suspended in toluene and then allowed to react with benzonitrile . \n", "arylnickel halide complexes are important intermediates \n in nickel - catalyzed cross - coupling reactions . in general , arylnickel \n halides ligated by monophosphines are synthesized by oxidative addition \n reactions of aryl halides to ni(pr3)x ( r = alkyl or aryl ) , but arylnickel \n halides ligated by bisphosphines are prepared by ligand substitution \n reactions between monophosphine - ligated arylnickel halide complexes \n with bisphosphines .", "the direct oxidative \n addition of aryl halides to bisphosphine - ligated ni(0 ) has rarely \n been investigated , mainly due to the lack of bisphosphine - ligated \n ni(0 ) precursors that can react with aryl halides at ambient temperature . \n", "recently , we reported the synthesis of binap - ligated arylnickel halide \n complexes by reactions of aryl halides with ( binap)ni(cod ) in the \n presence of catalytic amounts of benzonitrile , and we identified ( binap)ni(-nc - ph ) as the active ni(0 ) species . in our communication on the nickel - catalyzed asymmetric -arylation \n of ketones , we reported the single reaction of electron - deficient \n 4-chlorobenzotrifluoride with this newly defined ni(0 ) precursor 1 and no isolation of the products . here", "we show the results \n of the reactions of complex 1 with electronically varied \n aryl chlorides .", "electron - neutral and electron - deficient aryl \n chlorides reacted smoothly with ni(0 ) complex 1 in thf \n at room temperature to afford the arylnickel(ii ) chloride complexes 47 in 7689% isolated yields ( eq 5 ) .", "these complexes are stable in the solid state \n at ambient temperature for at least six months without noticeable \n decomposition . however , upon standing in thf solution at room temperature \n for about 48 h , they fully decomposed to [ ( binap)ni(-cl)]2 ( 8) with concomitant release of biaryls , as \n detected by gc - ms analysis .", "however , the reaction \n of electron - rich 4-chloroanisole with complex 1 in thf \n did not form the arylnickel chloride complex resulting from simple \n oxidative addition , as determined by p nmr spectroscopy . \n instead ,", "p nmr signals corresponding to two mutually trans phosphine resonances ( doublets at 24.2 and 14.4 ppm \n with jpp = 352 hz ) were observed .", "56single - crystal x - ray diffraction of the isolated \n material ( figure 1 ) showed that this product \n is complex 9 ( eq 7 ) .", "complex 9 contains a coordinated monophosphine ligand ph2p(c6h4 - 4-ome ) and a six - membered nickelacyclic \n structure .", "nickelacyclic complex 9 contains a small dihedral \n angle between the two naphthyl groups of 63 , and the nickel \n center consists of a distorted square planar geometry .", "complex 9 is formed by p  c bond cleavage of the binap backbone \n after the initial formation of binap - ligated arylnickel chloride complex \n ( binap)nicl(c6h4 - 4-ome ) .", "c \n bond cleavage of the binap backbone \n in a binap - ligated phenylpalladium bromide complex was reported by \n our group previously .", "all the ellipsoids are \n drawn at the 50% probability and all the \n hydrogen atoms are omitted for clarity .", "complex 9 was tested as a catalyst for the amination \n of 4-chloroanisole with octylamine under the conditions described \n in table 2 .", "only trace ( < 5% by gc analysis ) \n amounts of the amination product were formed .", "thus , the formation \n of catalytically inactive complex 9 by the reaction of \n 4-chloroanisole and nickel precursor 1 explained the \n high catalyst loading required for the amination of electron - rich \n 4-chloroanisole .", "the oxidation state of the nickel intermediates \n in the \n amination of aryl halides with amines has been ambiguous , and pathways \n involving both ni(0)/ni(ii ) and ni(i)/ni(iii ) couples have been proposed \n for these reactions . with isolated ,", "binap - ligated \n ni(0 ) , ni(i ) halide , and arni(ii ) halide complexes in hand , we were \n able to assess precisely the competence of these complexes to be intermediates \n in the amination reactions . to do so", ", we conducted the reaction \n of the isolated ( binap)nicl(c6h4 - 4-cf3 ) ( 7 ) with octylamine and naobu in the presence of 1 equiv of binap to trap a ni(0 ) product \n ( a , scheme 3 ) .", "this reaction afforded n - octyl-4-(trifluoromethyl)aniline in quantitative yield \n in less than 5 min at room temperature , as indicated by f nmr spectroscopic analysis .", "in addition , we conducted the amination \n reaction of 4-chlorobenzotrifluoride with octylamine in the presence \n of naobu catalyzed by 2 mol % of ( binap)nicl(c6h4 - 4-cf3 ) at room temperature ( b , scheme 3 ) .", "full conversion of 4-chlorobenzotrifluoride was \n achieved in 2 h , and this reaction afforded n - octyl-4-(trifluoromethyl)aniline \n in 91% isolated yield . the same reaction catalyzed by 2 mol % of", "[ ( binap)ni--cl]2 did not afford any amination product ( c , scheme 3 ) .", "these results show clearly that the binap - ligated \n arylnickel(ii ) chloride compound is kinetically and chemically competent \n to be an intermediate in the catalytic process and", "the binap - ligated \n nickel(i ) halide is not . to identify the resting \n state of the catalyst in this nickel - catalyzed \n amination reaction", ", we monitored by p{h } \n nmr spectroscopy the reaction of 4-chlorobenzotrifluoride with octylamine in the presence of naobu catalyzed by 2 mol % of ( binap)ni(-nc - ph ) \n ( 1 ) at room temperature . during the first 50% conversion \n of 4-chlorobenzotrifluoride ,", "however , during the second 50% conversion , a new species \n corresponding to a p{h } nmr resonance at \n  31.7 ppm gradually accumulated .", "this new species is ( binap)2ni , as determined by independently preparing this complex \n by reduction of ( dme)nicl2 with zn metal in the presence \n of 2 equiv of binap in thf and comparing the p nmr chemical \n shift . to test the catalytic activity of ( binap)2ni for this amination process", ", we conducted the reaction of 4-chlorobenzotrifluoride \n with octylamine catalyzed by 5 mol % of ( binap)2ni in the \n presence of naobu at room temperature .", "this reaction did \n not afford detectable amounts of the amination product ( a , scheme 4 ) .", "this result is consistent with the early report \n that ( binap)2ni does not undergo oxidative addition with \n aryl chlorides or bromides at ambient temperatures . the same reaction catalyzed by 5 mol % ( binap)2ni in \n the presence of a catalytic amount of phcn ( 30 mol % ) afforded the \n amination product in 84% yield in 20 h ( b , scheme 4 ) . for comparison", ", we conducted the same reaction catalyzed \n by only 2 mol % ( binap)ni(-nc - ph ) .", "this reaction \n afforded the amination product in almost quantitative yield in 2 h \n at room temperature ( c , scheme 4 ) .", "thus , ( binap)ni(-nc - ph ) is a more active catalyst than the combination of \n ( binap)2ni and phcn . to assess the origin of the \n effect of benzonitrile on the rate \n of the catalytic reaction", ", we performed the reaction of ( binap)2ni with benzonitrile and the reaction of ( binap)ni(-nc - ph ) with binap .", "the reaction of ( binap)2ni \n with 10 equiv of benzonitrile did not form detectable amounts of ( binap)ni(-nc - ph ) , as determined by p nmr spectroscopy \n ( d , scheme 4 ) .", "the reaction of ( binap)ni(-nc - ph ) with 1 equiv of binap afforded ( binap)2ni quantitatively at ambient temperature ( e , scheme 3 ) .", "based on these stoichiometric reactions and the catalytic \n reactions in scheme 3 , we conclude that the \n formation of ( binap)2ni is reversible in the presence of \n benzonitrile , but the equilibrium lies largely on the side of ( binap)2ni and free nitrile .", "the formation of bis(amine)-ligated palladium complexes \n was reported \n as one possible pathway for deactivation of the catalyst in the palladium - catalyzed \n amination of aryl halides .", "formation of \n analogous bis(amine ) complexes of nickel was proposed to explain the \n much lower reactivity of primary aliphatic amines than that of cyclic \n secondary aliphatic amines and anilines in the nickel - catalyzed amination \n reaction . however , these bis(amine)-ligated \n nickel complexes have never been prepared or characterized . in the \n nickel - catalyzed amination of aryl chlorides with primary amines catalyzed \n by ( binap)ni(-nc - ph ) , we observed the formation \n of ( binap)2ni .", "the stoichiometry of this conversion indicates \n that there are nickel complexes lacking binap ligand in the reaction \n mixture .", "thus , we studied the ligand substitution reactions between \n primary aliphatic amines and binap - ligated ni(0 ) and binap - ligated \n ni(ii ) complexes .", "the reaction of ( binap)ni(-nc - ph ) with 50 equiv of n - butylamine in toluene \n did not form any new nickel species , and free binap was not detected \n by p nmr spectroscopy ( a , scheme 5 ) .", "this result is consistent with the affinity of the electron - rich \n ni(0 ) center for softer , -accepting ligands .", "however , the reaction of ( binap)nicl(c6h4 - 4-cn ) with 50 equiv of n - butylamine afforded \n free binap and trans-(n - butylamine)2nicl(c6h4 - 4-cn ) ( 10 ) ( b , \n scheme 5 ) .", "complex 10 was isolated \n in 76% yield . with the bis(amine)-ligated complex 10 in hand", ", we \n tested its potential to serve as an intermediate in the amination \n reaction .", "we conducted the reaction of 10 with 2 equiv \n of naobu in the presence or in the absence \n of 1 equiv of binap ( c , scheme 5 ) .", "this result \n indicates that the formation of bis(amine)-ligated nickel complexes \n is one possible pathway for decomposition of the nickel catalyst . after identifying the resting state of the nickel catalyst and \n possible paths for catalyst deactivation", ", we sought to identify the \n rate - limiting step for this nickel - catalyzed amination process .", "to \n do so , we measured the dependence of the initial rates of the amination \n reaction on the concentrations of each reagent under conditions in \n which the other reagents were present in large excess .", "we chose \n the reaction of 4-chlorobenzotrifluoride with octylamine in the presence \n of naobu for the kinetic studies because \n this reaction occurred at ambient temperature , and we were able to \n monitor the reaction conveniently by both p and f nmr spectroscopy .", "furthermore , no background nucleophilic \n aromatic substitution reaction was observed for this aryl chloride \n and octylamine in the presence of naobu . for the initial rates to be a valid method for kinetic \n analysis \n of this reaction", ", all reagents must be soluble in the reaction medium , \n and no induction period can be present .", "the single - component catalyst \n precursor 1 is only marginally soluble in the reaction \n medium .", "thus , we used ( binap)nicl(c6h4 - 4-cf3 ) ( 7 ) as the source of nickel for our kinetic \n experiments because it dissolves in seconds once mixed with other \n reagents .", "benzonitrile was added to the reaction mixture to trap the \n binap - ligated ni(0 ) species to form ( binap)ni(-nc - ph ) , \n which had been identified as the catalyst resting state . in the absence \n of benzonitrile , the irreversible formation of ( binap)2ni occurs quickly .", "benzonitrile ( 10 equiv relative to complex 7 ) was added so that catalyst decomposition was not observed \n by p nmr spectroscopy over the time of the reaction .", "the kinetic behavior was assessed for the coupling of 4-chlorobenzotrifluoride \n with octylamine catalyzed by the combination of complex 7 in the presence of phcn and naobu at \n room temperature ( eq 8) .", "the initial rates were \n measured by f nmr spectroscopy using 4-ocf3-anisole as the internal standard .", "the rate of the amination reaction \n was determined by measuring the formation of n - octyl-4-(trifluoromethyl)aniline \n with respect to time .", "the plots of concentrations of n - octyl-4-(trifluoromethyl)aniline vs time at various concentrations \n of each reagent are shown in figures s1s5 in the supporting information .", "the plots of the initial \n rates of the reaction vs the concentrations of the catalyst , aryl \n chloride , and benzonitrile are shown in figure 2.8 the kinetic analysis showed that this nickel - catalyzed \n amination reaction of aryl chlorides is first order in the nickel \n catalyst , first order in aryl chloride , and inverse first order in \n phcn .", "the remaining reagents , naobu and \n amine , did not affect the initial rate of the reaction .", "these data \n indicate that oxidative addition of the aryl chloride to ( binap)ni(0 ) , \n formed by reversible dissociation of phcn from the catalyst resting \n state ( binap)ni(-nc - ph ) , is the turnover - limiting \n step in the amination reaction .", "plots of initial rates vs concentration \n of catalyst , aryl chloride , \n and benzonitrile for the amination of 4-chlorobenzotrifluoride ( 0.101.0 \n m ) with n - octylamine ( 0.30 m ) catalyzed by ( binap)nicl(c6h4 - 4-cf3 ) ( 0.00600.018 m ) in \n the presence of benzonitrile ( 0.0900.180 m ) and naobu ( 0.30 m ) at room temperature .", "the mechanism of palladium - catalyzed cross - coupling reactions with \n aryl halides as electrophiles has been extensively studied .", "these \n studies reveal that the halide identity has a dramatic effect on the \n mechanism for the oxidative addition of aryl halides to pd(0 ) species . \n", "the additions of the less reactive aryl chlorides occurs through lower - coordinated \n pd(0 ) intermediate than the additions of more reactive aryl iodides .", "however , such mechanistic data has not been \n gained for the nickel - catalyzed amination of aryl halides . for the \n nickel - catalyzed enantioselective -arylation of ketones , we \n found that aryl chlorides react in much higher yields and with much \n higher enantioselectivities than aryl bromides , and we attributed \n this dramatic effect of halide on the reaction yield and enantioselectivity \n to the faster decomposition of the arylnickel(ii ) bromide intermediate \n relative to the arylnickel(ii ) chloride intermediate to their corresponding \n ni(i ) halide complexes .", "because \n the ni(0 ) complex 1 ligated by binap and phcn catalyzes \n the amination of both aryl chlorides and aryl bromides , we can compare \n directly the kinetics and mechanism of the amination reactions of \n aryl chlorides and aryl bromides catalyzed by this nickel system . \n", "to do so , we conducted kinetic experiments on the amination of aryl \n bromides that were similar to those we conducted on the amination \n of aryl chlorides", ". we monitored by p nmr spectroscopy \n the reaction of \n 4-bromobenzotrifluoride with octylamine catalyzed by ( binap)ni(-nc - ph ) in the presence of naobu at room temperature . as was the case in the reactions of chloroarenes , \n ( binap)ni(-nc - ph )", "was identified as the resting \n state of the catalyst in the reaction of 4-bromobenzotrifluoride with \n octylamine .", "kinetic studies were conducted on the reaction of \n 4-bromobenzotrifluoride \n with octylamine catalyzed by ( binap)nibr(c6h4 - 4-cf3 ) ( 11 ) in the presence of added phcn \n ( eq 8) .", "the initial rates were measured for \n various concentrations of each reagent while keeping the other reagents \n in excess .", "the plots of concentrations of n - octyl-4-(trifluoromethyl)aniline \n vs time at various concentrations of each reagent are shown in figures s7s11 .", "the plots of the initial \n rates of the reaction vs the concentrations of the catalyst , aryl \n chloride , and benzonitrile are shown in figure 3 .", "plots of initial rates \n vs concentration of catalyst , aryl bromide , \n and benzonitrile for the amination of 4-bromobenzotrifluoride ( 0.100.80 \n m ) with n - octylamine ( 0.30 m ) catalyzed by ( binap)nicl(c6h4 - 4-cf3 ) ( 0.00600.018 m ) in \n the presence of benzonitrile ( 0.0900.180 m ) and naobu ( 0.30 m ) at room temperature . like the amination of 4-chlorobenzotrifluoride ,", "the amination \n of \n 4-bromobenzotrifluoride with octylamine is first order in the nickel \n catalyst , first order in aryl halide , zero order in naobu , and zero order in amine .", "in contrast to the reaction \n of 4-chlorobenzotrifluoride , which is inverse first order in phcn , \n the amination of 4-bromobenzotrifluoride is zero order in phcn .", "this \n order in phcn , in combination with the first order in aryl bromide , \n indicates that the bromoarene reacts directly with ( binap)ni(-nc - ph ) during the turnover - limiting step of the amination \n of the bromoarene without dissociation of phcn .", "the different reactivities \n of aryl halides with binap - ligated nickel complexes of different coordination \n numbers parallels a trend in oxidative addition to pd(0 ) complexes \n noted in the introduction to this section . based on the stoichiometric reactions of \n the isolated binap - ligated arylnickel halides with primary amines \n in the presence of base and the conducted kinetic studies , we propose \n a catalytic cycle depicted in scheme 6 for \n the nickel - catalyzed amination of aryl chlorides and aryl bromides \n reported in this paper .", "the observed first order rate - dependence on \n the concentrations of both the catalyst and aryl halides indicates \n that the oxidative addition of aryl halides to binap - ligated ni(0 ) \n species to form ( binap)niar(x ) ( x = cl or br ) is the turnover - limiting \n step for both amination processes .", "this binap - ligated arylnickel(ii ) \n halide complex subsequently reacts with primary amine and naobu to form an arylnickel amido complex , followed \n by reductive elimination of the n - alkyl aniline product \n in the presence of phcn to regenerate ( binap)ni(-nc - ph ) , which was confirmed as the catalyst resting state .", "interestingly , different rate - dependences on the concentration \n of phcn were observed for this nickel - catalyzed amination of aryl \n chlorides and aryl bromides .", "the reaction of aryl chlorides is inverse \n first order in phcn , whereas the reaction of aryl bromides is zero \n order in phcn .", "these reaction orders indicate that aryl chlorides \n and aryl bromides react with different nickel(0 ) species .", "the aryl \n chlorides react after reversible dissociation of phcn from ( binap)ni(-nc - ph ) to form the lower - coordinate nickel(0 ) species ( binap)ni(0 ) \n and ultimately the binap - ligated arylnickel(ii ) chloride complex . \n", "however , the aryl bromides react directly with ( binap)ni(-nc - ph ) to form binap - ligated arylnickel(ii ) bromide complex \n and the nitrile dissociates during or immediately after the oxidative \n addition process .", "one could imagine the zero - order dependence on added \n phcn could indicate that dissociation of this ligand is rate limiting , \n but the first - order dependence of the reaction on the aryl bromide \n is inconsistent with this proposal .", "in addition to the steps \n within the catalytic cycle that form the \n arylamine product , there are several pathways that generate catalytically \n inactive nickel species .", "first , the reaction of ( binap)niar(cl ) with \n amine rnh2 forms the catalytically inactive ( rnh2)2niar(cl ) and free binap .", "the released free binap in \n the reaction mixture traps the ( binap)ni(0 ) fragment generated by \n dissociation of phcn from ( binap)ni(-nc - ph ) or \n reductive elimination of n - alkyl aniline from the \n binap - ligated arylnickel(ii ) amido complex to form ( binap)2ni .", "( binap)2ni does not undergo oxidative addition of \n the aryl halide and does not catalyze the amination reaction .", "second , thermal decomposition of ( binap)niar(x ) \n ( ar = electron - neutral and electron - rich aryl groups ) affords the \n catalytically inactive [ ( binap)ni(-x)]2 with concomitant \n formation of biaryl .", "c bond cleavage of the binap backbone is a potential \n pathway for the deactivation of the nickel catalyst .", "indeed , this \n complex was observed to accumulate ( approximately 6.4 mm of the 16 \n mm concentration of initial catalyst ) during the reaction of 4-chloroanisole \n with octylamine . to gain \n information on the \n relative rates of the amination of aryl chlorides and aryl bromides \n and the effect of temperature and concentration of phcn on the selectivity \n of the amination reaction for an aryl chloride versus an aryl bromide", ", \n we conducted a series of reactions with an aryl halide containing \n both chloride and bromide and a mixture of aryl halides in the presence \n of various amounts of phcn at room temperature or 50 c .", "the amination of 1-bromo-4-chlorobenzene with \n octylamine catalyzed \n by 3 mol % of ( binap)ni(-nc - ph ) at 50 c occurred \n selectively at the c  br bond , and the product of the reaction \n at the c  cl bond was not detected by gc - ms analysis .", "( a , scheme 7 ) . however , side - by - side reactions of chlorobenzene \n and bromobenzene under the same conditions occurred in the same gc \n yield ( 98% ) ( b , scheme 7 ) .", "the amination \n of equal amounts of 1-bromo-4-butylbenzene and 4-chlorotoluene \n with octylamine catalyzed by 3 mol % of ( binap)ni(-nc - ph ) was conducted at both room temperature and 50 c ( c , \n scheme 7 ) . at both temperatures ,", "these reactions \n occurred in similar yields , but with different selectivity . at room \n temperature ,", "1-bromo-4-butylbenzene was converted selectively over \n 4-chlorotoluene , and a product ratio of 3:1 was obtained . however , \n at 50 c , 1-bromo-4-butylbenzene and 4-chlorotoluene converted \n equally .", "br \n bond in 1-bromo-4-chlorobenzene ( a , scheme 7 ) , the decreased selectivity for amination of the c  br bond \n over the c  cl bond in different haloarenes might stem from \n irreversible or nearly irreversible binding of the haloarene to ni(0 ) \n to form an arene -complex prior to cleavage of the carbon  halogen \n bond .", "this preferential intramolecular oxidative addition has recently \n been observed for the ni - catalyzed kumada cross - coupling reactions \n that are part of chain - growth polymerizations . to evaluate the effect of phcn on the selectivity \n between the amination \n of an aryl chloride and an aryl bromide", ", we conducted the amination \n of equal amounts of 1-bromo-4-butylbenzene and 4-chlorotoluene with \n octylamine catalyzed by 3 mol % of ( binap)ni(-nc - ph ) \n in the presence of 0.9 equiv of phcn ( relative to octylamine ) ( d , \n scheme 6 ) .", "because our kinetic studies revealed \n different dependences of the reaction rate on phcn for this nickel - catalyzed \n amination ( inverse first order and zero order on phcn for the reaction \n of aryl chloride and aryl bromide , respectively ) , we considered that \n an increased selectivity for the conversion of aryl bromide over aryl \n chloride would be observed for reactions conducted with higher concentrations \n of phcn than for reactions conducted with lower concentrations of \n phcn .", "consistent with this prediction , the selectivity was higher \n ( 30:1 \n for the reaction at room temperature and 9:1 for the reaction at 50 \n c ) for the amination of 1-bromo-4-butylbenzene versus the amination \n of 4-chlorotoluene for reactions run with higher concentrations ( from \n 0.004 to 0.18 m ) of benzonitrile at both room temperature and 50 c \n for 24 h. these data show that the difference in ni(0 ) species that \n adds the different aryl halides can be used to beneficially affect \n selectivity .", "several first - row metal complexes catalyze cross \n coupling reactions , \n and many catalyze the coupling of certain classes of chloroarenes . \n", "however , no first row metal complex has been shown to catalyze the \n coupling of primary alkylamines with unactivated chloroarenes .", "copper \n complexes couple bromoarenes and iodoarenes with primary amines , but \n they do not catalyze the coupling of chloroarenes with primary amines .", "previously , the combination of ni(cod)2 and dppf was \n shown to couple an electron - poor chloroarene with a primary amine , \n but this catalyst did not couple unactivated chloroarenes with primary \n amines .", "a set of preliminary studies \n we conducted have shown that the complexes ( dppf)ni(cod ) or ( dppf)2ni generated in situ by the reaction of ni(cod)2 and dppf do not add aryl chlorides at ambient temperature , and the \n reaction of chloroarenes with ( dppf)ni(cod ) or ( dppf)2ni \n at elevated temperatures forms ( dppf)ni(i)cl and biaryl .", "this \n formation of ( dppf)2ni(0 ) and ( dppf)ni(i)cl at \n the elevated temperatures required for oxidative addition of chloroarene \n to occur , likely prevented productive coupling reactions with unactivated \n chloroarenes .", "these observations further illustrate the importance \n of the nitrile - ligated precursor with the general formula l2ni(-nc - ph ) .", "nickel complexes of n - heterocyclic carbenes have \n been shown to couple aryl chlorides and sulfonates with secondary \n amines and with arylamines , but not primary alkylamines .", "mechanistic \n studies on the reactions of secondary amines catalyzed by the complexes \n of n - heterocyclic carbenes are too limited to determine \n the origin of their lack of reactivity with primary amines , but recent \n studies have shown that the arylnickel halide complexes containing \n hindered n - heterocyclic carbenes are not formed by \n the reaction of ( imes)2ni(0 ) with chlorobenzene , bromobenzene , \n and iodobenzene . instead , ni(i ) species \n are formed , and this formation of ni(i ) complexes could either constitute \n the pathway for catalyst decomposition or a mechanism for reaction \n with this catalyst involving odd - electron nickel intermediates ."], ["we have shown that nickel complexes containing \n common aryl bisphosphine \n ligands can catalyze the coupling of aryl chlorides and bromides with \n primary amines .", "in particular , we have shown that the mixed ni(0 ) \n species ( binap)ni(-nc - ph ) containing one bisphosphine \n and one side - bound nitrile is a particularly active catalyst for the \n coupling of chloroarenes with primary amines and is more active than \n the related ( binap)2ni catalyst . a range of electronically \n varied aryl halides and nitrogen - containing heteroaryl chlorides , \n including pyridine , quinoline , and isoquinoline derivatives , couple \n with a variety of aliphatic primary amines in high isolated yields . \n several aspects of the current system lead to the ability of this \n first - row metal system to catalyze this class of amination reaction . \n in general , we attribute the high yields for these amination reactions \n to the facile oxidative addition of aryl chlorides and heteroaryl \n chlorides to the ( binap)ni(-nc - ph ) precatalyst \n at ambient temperatures . detailed studies on the mechanism of \n the amination reaction", "the following conclusions about the mechanism of this \n coupling process have been drawn from these studies : ( 1 ) the \n oxidative addition of aryl chlorides to ( binap)ni(-nc - ph ) , together with the stoichiometric reactions of ( binap)niar(cl ) \n with primary aliphatic amines in the presence of base , support a reaction \n pathway involving a ni(0)/ni(ii ) couple in the catalytic cycle .", "( 2 ) the oxidative addition of aryl chlorides and heteroaryl chlorides \n to the single - component nickel precursor ( binap)ni(-nc - ph ) at ambient temperatures prevents the formation of inactive \n ni species . ( 3 ) ni(i ) intermediates , which have been observed \n in oxidative \n addition studies to phosphine - ligated ni(0 ) complexes , appear to result \n from decomposition of the ni(ii ) product of oxidative addition as \n opposed to forming from one - electron pathways for oxidative addition \n to ni(0 ) .", "( 4 ) the reaction of ( binap)ni(-nc - ph ) with electron - rich \n aryl chlorides , such as 4-chloroanisole , leads to the p  c bond \n cleavage of the backbone of binap and forms the nickel complex 9 containing two monophosphine ligands .", "the formation of the \n catalytically inactive complex 9 explains the high catalyst \n loading required for the amination of electron - rich aryl chlorides .", "( 5 ) mechanistic and kinetic studies revealed that the turnover - limiting \n step of these nickel - catalyzed amination reactions of aryl chlorides \n is oxidative addition of the aryl chloride to ( binap)ni(0 ) , which \n is formed by dissociation of phcn from the catalyst resting state \n ( binap)ni(-nc - ph ) .", "( 6 ) mechanistic and kinetic \n studies imply that the turnover - limiting \n step of the reactions of aryl bromides is also oxidative addition , \n but that aryl bromides react with a different nickel species , of composition \n ( binap)ni(-nc - ph ) containing a bound nitrile .", "( 7 ) the formation of catalytically inactive bis(amine)-ligated \n ni(ii ) species ( rnh2)2niar(cl ) , ni(i ) species \n [ ( binap)ni(-cl)]2 , and bis - binap - ligated ni(0 ) species \n ( binap)2ni are the major pathways for catalyst inactivation", ". further studies to improve the lifetime of the ni - bisphosphine \n catalysts , to determine the mechanism by which the phosphine - ligated \n ni(i ) species form as a means to prevent this detrimental path , and \n to determine if ni(i ) complexes are generated during oxidative addition \n of haloarenes will be the subject of future work ."]]}
{"article_id": "PMC2704519", "article_text": ["children diagnosed with jia were referred by pediatricians to our immuno - ophthalmology unit for ophthalmic examination .", "children 10 years old or younger presenting to our clinic with intraocular inflammation were referred for pediatric consult to verify a possible jia association . included in this study", "is a cohort of 172 children with jia who were examined with the slit lamp at least on three consecutive visits and followed for a minimum of 3 years and a maximum of 15 years after the original referral visit . at the time of initial examination", "all were diagnosed by pediatricians as having jia either prior to their initial ophthalmic examination ( 152 patients ) or after pediatric consult was sought following the initial detection of intraocular inflammation ( 20 patients ) .", "all 172 children fulfilled the criteria for the diagnosis of jia : children with arthritis of at least 3 months duration , less than 16 years old at the time of first arthritic manifestation and without any other detectable cause for the arthritis .", "history of possible systemic disease and ocular manifestations were carefully reviewed with the patients and parents .", "all patients underwent an ocular examination which included assessment of the visual acuity using the snellen charts or the illiterate e charts and/or familiar pictures for the young verbal children . in a few of the very young and pre - verbal children ,", "the pattern of fixation for near and distance and the elicitation of optokinetic nystagmus were used to assess their visual functions ( benezra and rose 1990 ) . slit lamp biomicroscopy , thorough fundus examination and assessment of refractive errors in both eyes were performed in all cases .", "the intraocular pressure ( iop ) using the goldman applanation tonometer was obtained in cooperative children . in a few of the very young children with early ocular complications the iop assessment , fundus examination and refraction data", "ocular movement abnormalities , the presence or absence of strabismus and the binocular functions were also routinely assessed .", "ocular medical treatment consisted on instillations of corticosteroids and cycloplegic eye drops when the intraocular inflammation was confined to the anterior segment .", "the frequency of eye drops instillations in these cases was arbitrarily modulated for every affected eye according to the intensity of inflammatory reaction in the aqueous humor as assessed at the slit lamp .", "systemic corticosteroids ( up to 1.0 mg / kg / day ) and/or methotrexate ( up to 25 mg once a week ) were prescribed if the intermediate and posterior segments demonstrated active inflammatory process or at the advice of the treating rheumatologist for the control of severe arthritic manifestations .", "complete blood count ( cbc ) , sedimentation rate ( esr ) , c reactive protein ( crp ) , urine culture , kidney and liver function tests were performed in all cases . according to the clinical observations and the results of these routine preliminary examinations , a tailored individual more arduous battery of tests", "the type of intraocular inflammation ( uveitis ) was classified according to the anatomical site of the major inflammatory manifestations following the intraocular inflammation society guidelines ( forrester et al 1998 ; benezra et al 2000 ) .", "the intraocular inflammation was further subdivided to whether the disease was strictly ocular or it was associated with a systemic disease .", "systemic disease associations were determined according to established sets of criteria ( bloch - michel and nussenblatt 1987 ; weiner and benezra 1991 ; dollfus 1998 ; forrester et al 1998 ; benezra et al 2000 ; benezra et al 2005 ) .", "history of possible systemic disease and ocular manifestations were carefully reviewed with the patients and parents .", "all patients underwent an ocular examination which included assessment of the visual acuity using the snellen charts or the illiterate e charts and/or familiar pictures for the young verbal children . in a few of the very young and pre - verbal children ,", "the pattern of fixation for near and distance and the elicitation of optokinetic nystagmus were used to assess their visual functions ( benezra and rose 1990 ) . slit lamp biomicroscopy , thorough fundus examination and assessment of refractive errors in both eyes were performed in all cases .", "the intraocular pressure ( iop ) using the goldman applanation tonometer was obtained in cooperative children . in a few of the very young children with early ocular complications the iop assessment , fundus examination and refraction data", "ocular movement abnormalities , the presence or absence of strabismus and the binocular functions were also routinely assessed .", "ocular medical treatment consisted on instillations of corticosteroids and cycloplegic eye drops when the intraocular inflammation was confined to the anterior segment .", "the frequency of eye drops instillations in these cases was arbitrarily modulated for every affected eye according to the intensity of inflammatory reaction in the aqueous humor as assessed at the slit lamp .", "systemic corticosteroids ( up to 1.0 mg / kg / day ) and/or methotrexate ( up to 25 mg once a week ) were prescribed if the intermediate and posterior segments demonstrated active inflammatory process or at the advice of the treating rheumatologist for the control of severe arthritic manifestations .", "complete blood count ( cbc ) , sedimentation rate ( esr ) , c reactive protein ( crp ) , urine culture , kidney and liver function tests were performed in all cases . according to the clinical observations and the results of these routine preliminary examinations , a tailored individual more arduous battery of tests", "the type of intraocular inflammation ( uveitis ) was classified according to the anatomical site of the major inflammatory manifestations following the intraocular inflammation society guidelines ( forrester et al 1998 ; benezra et al 2000 ) .", "the intraocular inflammation was further subdivided to whether the disease was strictly ocular or it was associated with a systemic disease .", "systemic disease associations were determined according to established sets of criteria ( bloch - michel and nussenblatt 1987 ; weiner and benezra 1991 ; dollfus 1998 ; forrester et al 1998 ; benezra et al 2000 ; benezra et al 2005 ) .", "of the 172 children with jia , 152 were referred by pediatricians for ocular examination after the diagnosis of jia was ascertained . in 20 children ,", "intraocular inflammation was the presenting symptom while jia disease was established later by a pediatrician .", "of the 152 children with the presenting symptom of arthritis , 18 ( 11.8 % ) had the systemic type of jia , 87 ( 57.2% ) had the pauciarticular type and 47 ( 30.9% ) had the polyarticular type .", "of the 152 children presenting with all types of jia arthritic manifestations , in 14 children ( 9.2% ) , the intraocular inflammation ( uveitis ) was diagnosed during the first ophthalmic examination ( 12/14 had clinical evidence for the pauciarticular type of jia ) .", "all these 14 children had a recent history ( 3 to 9 months ) of arthritic disease .", "ten additional children developed uveitis less than three years of the initial ocular examination and , in 7 children the uveitis was detected 3 to 7 years later ( table 1 ) . among the 20 children presenting initially with uveitis , 19 developed clinical manifestations compatible with the pauciarticular type of jia .", "a systemic type of jia was confirmed two months after the diagnosis of uveitis in one out of these 20 children ( table 2 ) .", "thus , out of a total of 172 children with jia , 51 children had an associated intraocular inflammation ( uveitis ) .", "thirty nine of these 51 children ( 76.5% ) had the pauciarticular type of jia . in 32 of these 39 children ( 82% ) ,", "the uveitis was either the presenting symptom or it was detected during the initial ophthalmic examination . on initial examination ,", "the intraocular inflammation processes were confined to the anterior segment in 45 of the 51 ( 88.2% ) affected children . in 5 of the affected children ( 9.8% ) ,", "a few cells were also observed in the anterior vitreous and in one child ( 2% ) , a marked vitritis with peripheral retinal vasculitis was observed in one eye . on", "further follow up however , only in 4 out of the 51 patients ( 7.8% ) , the inflammatory processes remained strictly confined to the anterior segment .", "thus , with time , a tendency towards the involvement of the intermediate and posterior segments was observed .", "on last ophthalmic examination ( 3 to 15 years after initial diagnosis ) , in 22 patients ( 43.1% ) the inflammatory signs were detected in all eye segments ( pan uveitis ) and in 24 children ( 47% ) , the inflammatory reaction involved the anterior and intermediate segments . when the intraocular inflammation was initially diagnosed , it was uniocular in 29 of the 51 children ( 56.9% ) . on", "further follow up , the intraocular inflammation remained uniocular only in 8 of the 51 patients ( 15.7% ) while in 43 children ( 84.3% ) there was involvement of both eyes .", "when both eyes were affected , the intensity of intraocular inflammation was unequal in the majority of patients .", "the eye with more severe inflammation showed invariably a poorer visual acuity and more prominent secondary ocular complications .", "the ocular complications observed in the 94 affected eyes of the 51 children are illustrated in table 3 .", "more severe and early complications were observed in girls manifesting the intraocular inflammatory processes when 4 years of age or younger . in 46 of the 94 eyes with uveitis ( 48% ) , a band - shaped keratopathy developed . in most , the keratopathy was mild and did not interfere with vision . in 5 eyes ( 5.3% ) , the keratopathy was dense , encroached on the visual axis and affected the visual acuity .", "an increased iop above 24mmhg and possible development of glaucoma was detected in 24 of the 94 , ( 25% ) affected eyes .", "medical treatment consisting of beta blockers and carbonic anhydrase inhibitor drops along with a decrease in the instillation frequency of corticosteroid eye drops induced a decrease of iop below 20 mmhg in all cases .", "lens opacity interfering with the visual acuity ( cataract ) was recorded in 56 of the 94 affected eyes ( 59.6% ) . in eyes developing severe lens changes ( dense cataract ) accompanied by band keratopathy , marked impairment of the visual functions interfering with the daily activity and/or inducing amblyopia", "it was assessed that the lens opacity and/or the ensuing amblyopia were the main cause for the decrease of vision to a level lower than 0.05 ( 6/120 or 20/400 ) ( table 4 ) .", "twenty nine of these eyes underwent lensectomy combined with anterior vitrectomy while in one case , the parents declined surgical intervention .", "an intraocular lens ( iol ) was implanted in 19 eyes ( pseudophakia ) while 10 eyes remained aphakic .", "glasses or contact lenses were prescribed for the correction of aphakia in these 10 eyes .", "the reported incidences of this association vary markedly with lower incidences and less severe ocular disease being reported more recently ( sherry et al 1991 ; oren et al 2001 ; sim et al 2006 ; saurenmann et al 2007 )", ". a close look at these published differences reveals that the severity of ocular affection in the various studies is probably associated with population selection and referral trends in specific medical environments .", "it appears that the severity of ocular complications is higher when the study authors are ophthalmologists .", "this tendency is increased further when the papers originate from tertiary clinics specializing in uveitis and surgery of its complications .", "noteworthy is the fact that 20 of the 51 children with jia and uveitis in our cohort presented with severe intraocular inflammation . the diagnosis of jia was made only after these children were referred specifically for pediatric examination to assess the possible presence of jia . in most of these 20 children", "thus , a study regarding the incidence of uveitis in jia carried out by pediatricians will not include these children biasing the incidence as well as the severity of ocular inflammation .", "concomitant with other reports ( sim et al 2006 ; grassi et al 2007 ; henligenhaus et al 2007 ) , we observed in our present study that the highest associated incidence of arthritis and ocular manifestations is found in the group of children with the pauciarticular type .", "we have also noted that , in many cases , the articular and ocular clinical disease severity do not parallel . in many children , we observed severe ocular exacerbations during quiescent periods of arthritic manifestations and vice versa . an interesting aspect of our study is the observation that during the initial phase of the ocular disease , the inflammation is strictly restricted to the anterior segment .", "later during the follow up however , the intermediate and posterior segments become involved in the intraocular inflammatory processes . these observations may result from a  spill over  of the chronically stimulated inflammatory cells from the anterior segment to the posterior structures  dragging  them to react following the release of inflammatory cytokines . the major points of clinical interest which can be derived from our study", "are : \n thirty nine of the 51 children with uveitis in our study group ( 76.5% ) had the pauciarticular type of jia.the majority of children with pauciarticular jia developing intraocular inflammation harbor signs of ocular disease either before or shortly after the development of arthritic manifestations.most children with polyarticular or systemic jia types do not present signs of intraocular inflammation during the first three years following the initial arthritic manifestations.the potential for a child with jia to develop de novo ocular inflammation , at any time during the ophthalmic follow up , is low .", "the majority of children within all jia types who devolop an associated uveitis harbor ocular signs on initial examination .", "therefore , frequent ophthalmic visits for all children with jia seems unjustified.nearly all children presenting with ocular disease and developing later jia characteristics , had a mild arthritis involving only one or two joints .", "thirty nine of the 51 children with uveitis in our study group ( 76.5% ) had the pauciarticular type of jia . the majority of children with pauciarticular jia developing intraocular inflammation harbor signs of ocular disease either before or shortly after the development of arthritic manifestations .", "most children with polyarticular or systemic jia types do not present signs of intraocular inflammation during the first three years following the initial arthritic manifestations .", "the potential for a child with jia to develop de novo ocular inflammation , at any time during the ophthalmic follow up , is low .", "the majority of children within all jia types who devolop an associated uveitis harbor ocular signs on initial examination", "nearly all children presenting with ocular disease and developing later jia characteristics , had a mild arthritis involving only one or two joints . from our present study observations , it can be concluded that rigorous and very frequent ophthalmic exams for jia children are not obligatory if during the first ophthalmic examination ocular inflammatory signs are not detected . furthermore , from our experience , a rigorous time frame for  regular  follow up visits for these children as suggested ( rosenberg and oen 1986 ) seems unnecessary and , in our opinion , may be counterproductive for the following reasons : ( a ) uveitis may be detected during the ophthalmic examination only if the inflammatory signs are already present , ( b ) there are no  predictive signs  for the potential development of intraocular inflammation in these children , and ( c ) an intraocular inflammation may develop 24 or 48 hours after the  regular  periodic visit . in these cases , parents may be reluctant to seek again ophthalmic consultation ( even if they suspect a problem ) until serious complications develop .", "therefore , education of the parents about the ocular disease , its potential evident signs and symptoms should be the main line of conduct for follow up instead of regular frequent ophthalmic visits .", "a drop of vision affecting the child s daily behavior can be noticed by parents .", "also , parents can be taught to perform a visual acuity test at home when playing with the child .", "these measures in our experience are more effective , practical , easily carried out and have the highest possible cost / benefit ratio . in this study", "we confirm , as published earlier ( benezra and cohen 2000 ) and also reported by others ( wolf et al 1987 ; akduman et al 1997 ; woreta et al 2007 ) that , severe complications with loss of vision are still to be feared in children with jia who are developing uveitis .", "most severe ocular complications have been observed in children 4 years of age or younger who presented first with ocular disease or had an ocular involvement detected during the initial ophthalmic examination .", "if not treated early , loss of vision may ensue in these cases even if successful control of the intraocular inflammation is achieved but proper anti amblyopic measures are not applied early .", "last but not least , in the management of jra associated ocular inflammation , surgery should be considered despite the potential for complications . in properly selected cases ,", "early surgery may even be considered in children with severe visual impairment in one eye due to band keratopathy , seclusion of the pupil by inflammatory membranes and/or dense cataract . in these cases , adequate surgical procedure of the cataract combined with intraocular lens implantation is to be considered and can result in good restoration of vision ( benezra and cohen 2000 ; khotaniemi and penttila 2006 ) ."], "abstract_text": ["<S> objective : assess the incidence of intraocular inflammation ( uveitis ) and ocular complications in children with various types of jia in a single cohort of patients.patients:included are 172 children ( 35 boys and 137 girls ) diagnosed with jia . </S>", "<S> all underwent thorough initial ophthalmologic examination and were followed for a minimum of 3 years.results:of 172 children with jia , 152 ( 88.4% ) presented with arthritis . </S>", "<S> uveitis was detected in 14 of the152 children ( 9.2% ) during the first ophthalmic examination . in 17 additional patients of this group ( 11.2% ) , uveitis developed during the follow up period of up to 15 years . </S>", "<S> twenty children out of the total of 172 ( 11.6% ) presented initially with uveitis . in children developing uveitis before or along with arthritic manifestations , </S>", "<S> the ocular disease was chronic with a high rate of secondary complications ( band keratopathy , glaucoma , posterior synechiae and cataract ) . in all affected eyes </S>", "<S> the initial ocular inflammation was typically confined to the anterior segment . </S>", "<S> on longer follow up however , most children developed binocular disease and posterior segment involvement . </S>", "<S> dense cataract and amblyopia were the major cause of severe visual disabilities.conclusion:pauciarticular jia is associated with intraocular inflammation ( uveitis ) early during the arthritic disease course . </S>", "<S> the ocular disease course is unpredictable </S>", "<S> . therefore education of parents regarding its signs and symptoms is of utmost importance . to preserve functional vision , </S>", "<S> secondary ocular complications and amblyopia should be avoided . </S>"], "labels": null, "section_names": ["Patients and methods", "Clinical examination", "Ocular medical treatment", "Laboratory tests", "Classification of uveitis", "Results", "Discussion"], "sections": [["children diagnosed with jia were referred by pediatricians to our immuno - ophthalmology unit for ophthalmic examination .", "children 10 years old or younger presenting to our clinic with intraocular inflammation were referred for pediatric consult to verify a possible jia association . included in this study", "is a cohort of 172 children with jia who were examined with the slit lamp at least on three consecutive visits and followed for a minimum of 3 years and a maximum of 15 years after the original referral visit . at the time of initial examination", "all were diagnosed by pediatricians as having jia either prior to their initial ophthalmic examination ( 152 patients ) or after pediatric consult was sought following the initial detection of intraocular inflammation ( 20 patients ) .", "all 172 children fulfilled the criteria for the diagnosis of jia : children with arthritis of at least 3 months duration , less than 16 years old at the time of first arthritic manifestation and without any other detectable cause for the arthritis .", "history of possible systemic disease and ocular manifestations were carefully reviewed with the patients and parents .", "all patients underwent an ocular examination which included assessment of the visual acuity using the snellen charts or the illiterate e charts and/or familiar pictures for the young verbal children . in a few of the very young and pre - verbal children ,", "the pattern of fixation for near and distance and the elicitation of optokinetic nystagmus were used to assess their visual functions ( benezra and rose 1990 ) . slit lamp biomicroscopy , thorough fundus examination and assessment of refractive errors in both eyes were performed in all cases .", "the intraocular pressure ( iop ) using the goldman applanation tonometer was obtained in cooperative children . in a few of the very young children with early ocular complications the iop assessment , fundus examination and refraction data", "ocular movement abnormalities , the presence or absence of strabismus and the binocular functions were also routinely assessed .", "ocular medical treatment consisted on instillations of corticosteroids and cycloplegic eye drops when the intraocular inflammation was confined to the anterior segment .", "the frequency of eye drops instillations in these cases was arbitrarily modulated for every affected eye according to the intensity of inflammatory reaction in the aqueous humor as assessed at the slit lamp .", "systemic corticosteroids ( up to 1.0 mg / kg / day ) and/or methotrexate ( up to 25 mg once a week ) were prescribed if the intermediate and posterior segments demonstrated active inflammatory process or at the advice of the treating rheumatologist for the control of severe arthritic manifestations .", "complete blood count ( cbc ) , sedimentation rate ( esr ) , c reactive protein ( crp ) , urine culture , kidney and liver function tests were performed in all cases . according to the clinical observations and the results of these routine preliminary examinations , a tailored individual more arduous battery of tests", "the type of intraocular inflammation ( uveitis ) was classified according to the anatomical site of the major inflammatory manifestations following the intraocular inflammation society guidelines ( forrester et al 1998 ; benezra et al 2000 ) .", "the intraocular inflammation was further subdivided to whether the disease was strictly ocular or it was associated with a systemic disease .", "systemic disease associations were determined according to established sets of criteria ( bloch - michel and nussenblatt 1987 ; weiner and benezra 1991 ; dollfus 1998 ; forrester et al 1998 ; benezra et al 2000 ; benezra et al 2005 ) ."], ["history of possible systemic disease and ocular manifestations were carefully reviewed with the patients and parents .", "all patients underwent an ocular examination which included assessment of the visual acuity using the snellen charts or the illiterate e charts and/or familiar pictures for the young verbal children . in a few of the very young and pre - verbal children ,", "the pattern of fixation for near and distance and the elicitation of optokinetic nystagmus were used to assess their visual functions ( benezra and rose 1990 ) . slit lamp biomicroscopy , thorough fundus examination and assessment of refractive errors in both eyes were performed in all cases .", "the intraocular pressure ( iop ) using the goldman applanation tonometer was obtained in cooperative children . in a few of the very young children with early ocular complications the iop assessment , fundus examination and refraction data", "ocular movement abnormalities , the presence or absence of strabismus and the binocular functions were also routinely assessed ."], ["ocular medical treatment consisted on instillations of corticosteroids and cycloplegic eye drops when the intraocular inflammation was confined to the anterior segment .", "the frequency of eye drops instillations in these cases was arbitrarily modulated for every affected eye according to the intensity of inflammatory reaction in the aqueous humor as assessed at the slit lamp .", "systemic corticosteroids ( up to 1.0 mg / kg / day ) and/or methotrexate ( up to 25 mg once a week ) were prescribed if the intermediate and posterior segments demonstrated active inflammatory process or at the advice of the treating rheumatologist for the control of severe arthritic manifestations ."], ["complete blood count ( cbc ) , sedimentation rate ( esr ) , c reactive protein ( crp ) , urine culture , kidney and liver function tests were performed in all cases . according to the clinical observations and the results of these routine preliminary examinations , a tailored individual more arduous battery of tests"], ["the type of intraocular inflammation ( uveitis ) was classified according to the anatomical site of the major inflammatory manifestations following the intraocular inflammation society guidelines ( forrester et al 1998 ; benezra et al 2000 ) .", "the intraocular inflammation was further subdivided to whether the disease was strictly ocular or it was associated with a systemic disease .", "systemic disease associations were determined according to established sets of criteria ( bloch - michel and nussenblatt 1987 ; weiner and benezra 1991 ; dollfus 1998 ; forrester et al 1998 ; benezra et al 2000 ; benezra et al 2005 ) ."], ["of the 172 children with jia , 152 were referred by pediatricians for ocular examination after the diagnosis of jia was ascertained . in 20 children ,", "intraocular inflammation was the presenting symptom while jia disease was established later by a pediatrician .", "of the 152 children with the presenting symptom of arthritis , 18 ( 11.8 % ) had the systemic type of jia , 87 ( 57.2% ) had the pauciarticular type and 47 ( 30.9% ) had the polyarticular type .", "of the 152 children presenting with all types of jia arthritic manifestations , in 14 children ( 9.2% ) , the intraocular inflammation ( uveitis ) was diagnosed during the first ophthalmic examination ( 12/14 had clinical evidence for the pauciarticular type of jia ) .", "all these 14 children had a recent history ( 3 to 9 months ) of arthritic disease .", "ten additional children developed uveitis less than three years of the initial ocular examination and , in 7 children the uveitis was detected 3 to 7 years later ( table 1 ) . among the 20 children presenting initially with uveitis , 19 developed clinical manifestations compatible with the pauciarticular type of jia .", "a systemic type of jia was confirmed two months after the diagnosis of uveitis in one out of these 20 children ( table 2 ) .", "thus , out of a total of 172 children with jia , 51 children had an associated intraocular inflammation ( uveitis ) .", "thirty nine of these 51 children ( 76.5% ) had the pauciarticular type of jia . in 32 of these 39 children ( 82% ) ,", "the uveitis was either the presenting symptom or it was detected during the initial ophthalmic examination . on initial examination ,", "the intraocular inflammation processes were confined to the anterior segment in 45 of the 51 ( 88.2% ) affected children . in 5 of the affected children ( 9.8% ) ,", "a few cells were also observed in the anterior vitreous and in one child ( 2% ) , a marked vitritis with peripheral retinal vasculitis was observed in one eye . on", "further follow up however , only in 4 out of the 51 patients ( 7.8% ) , the inflammatory processes remained strictly confined to the anterior segment .", "thus , with time , a tendency towards the involvement of the intermediate and posterior segments was observed .", "on last ophthalmic examination ( 3 to 15 years after initial diagnosis ) , in 22 patients ( 43.1% ) the inflammatory signs were detected in all eye segments ( pan uveitis ) and in 24 children ( 47% ) , the inflammatory reaction involved the anterior and intermediate segments . when the intraocular inflammation was initially diagnosed , it was uniocular in 29 of the 51 children ( 56.9% ) . on", "further follow up , the intraocular inflammation remained uniocular only in 8 of the 51 patients ( 15.7% ) while in 43 children ( 84.3% ) there was involvement of both eyes .", "when both eyes were affected , the intensity of intraocular inflammation was unequal in the majority of patients .", "the eye with more severe inflammation showed invariably a poorer visual acuity and more prominent secondary ocular complications .", "the ocular complications observed in the 94 affected eyes of the 51 children are illustrated in table 3 .", "more severe and early complications were observed in girls manifesting the intraocular inflammatory processes when 4 years of age or younger . in 46 of the 94 eyes with uveitis ( 48% ) , a band - shaped keratopathy developed . in most , the keratopathy was mild and did not interfere with vision . in 5 eyes ( 5.3% ) , the keratopathy was dense , encroached on the visual axis and affected the visual acuity .", "an increased iop above 24mmhg and possible development of glaucoma was detected in 24 of the 94 , ( 25% ) affected eyes .", "medical treatment consisting of beta blockers and carbonic anhydrase inhibitor drops along with a decrease in the instillation frequency of corticosteroid eye drops induced a decrease of iop below 20 mmhg in all cases .", "lens opacity interfering with the visual acuity ( cataract ) was recorded in 56 of the 94 affected eyes ( 59.6% ) . in eyes developing severe lens changes ( dense cataract ) accompanied by band keratopathy , marked impairment of the visual functions interfering with the daily activity and/or inducing amblyopia", "it was assessed that the lens opacity and/or the ensuing amblyopia were the main cause for the decrease of vision to a level lower than 0.05 ( 6/120 or 20/400 ) ( table 4 ) .", "twenty nine of these eyes underwent lensectomy combined with anterior vitrectomy while in one case , the parents declined surgical intervention .", "an intraocular lens ( iol ) was implanted in 19 eyes ( pseudophakia ) while 10 eyes remained aphakic .", "glasses or contact lenses were prescribed for the correction of aphakia in these 10 eyes ."], ["the reported incidences of this association vary markedly with lower incidences and less severe ocular disease being reported more recently ( sherry et al 1991 ; oren et al 2001 ; sim et al 2006 ; saurenmann et al 2007 )", ". a close look at these published differences reveals that the severity of ocular affection in the various studies is probably associated with population selection and referral trends in specific medical environments .", "it appears that the severity of ocular complications is higher when the study authors are ophthalmologists .", "this tendency is increased further when the papers originate from tertiary clinics specializing in uveitis and surgery of its complications .", "noteworthy is the fact that 20 of the 51 children with jia and uveitis in our cohort presented with severe intraocular inflammation . the diagnosis of jia was made only after these children were referred specifically for pediatric examination to assess the possible presence of jia . in most of these 20 children", "thus , a study regarding the incidence of uveitis in jia carried out by pediatricians will not include these children biasing the incidence as well as the severity of ocular inflammation .", "concomitant with other reports ( sim et al 2006 ; grassi et al 2007 ; henligenhaus et al 2007 ) , we observed in our present study that the highest associated incidence of arthritis and ocular manifestations is found in the group of children with the pauciarticular type .", "we have also noted that , in many cases , the articular and ocular clinical disease severity do not parallel . in many children , we observed severe ocular exacerbations during quiescent periods of arthritic manifestations and vice versa . an interesting aspect of our study is the observation that during the initial phase of the ocular disease , the inflammation is strictly restricted to the anterior segment .", "later during the follow up however , the intermediate and posterior segments become involved in the intraocular inflammatory processes . these observations may result from a  spill over  of the chronically stimulated inflammatory cells from the anterior segment to the posterior structures  dragging  them to react following the release of inflammatory cytokines . the major points of clinical interest which can be derived from our study", "are : \n thirty nine of the 51 children with uveitis in our study group ( 76.5% ) had the pauciarticular type of jia.the majority of children with pauciarticular jia developing intraocular inflammation harbor signs of ocular disease either before or shortly after the development of arthritic manifestations.most children with polyarticular or systemic jia types do not present signs of intraocular inflammation during the first three years following the initial arthritic manifestations.the potential for a child with jia to develop de novo ocular inflammation , at any time during the ophthalmic follow up , is low .", "the majority of children within all jia types who devolop an associated uveitis harbor ocular signs on initial examination .", "therefore , frequent ophthalmic visits for all children with jia seems unjustified.nearly all children presenting with ocular disease and developing later jia characteristics , had a mild arthritis involving only one or two joints .", "thirty nine of the 51 children with uveitis in our study group ( 76.5% ) had the pauciarticular type of jia . the majority of children with pauciarticular jia developing intraocular inflammation harbor signs of ocular disease either before or shortly after the development of arthritic manifestations .", "most children with polyarticular or systemic jia types do not present signs of intraocular inflammation during the first three years following the initial arthritic manifestations .", "the potential for a child with jia to develop de novo ocular inflammation , at any time during the ophthalmic follow up , is low .", "the majority of children within all jia types who devolop an associated uveitis harbor ocular signs on initial examination", "nearly all children presenting with ocular disease and developing later jia characteristics , had a mild arthritis involving only one or two joints . from our present study observations , it can be concluded that rigorous and very frequent ophthalmic exams for jia children are not obligatory if during the first ophthalmic examination ocular inflammatory signs are not detected . furthermore , from our experience , a rigorous time frame for  regular  follow up visits for these children as suggested ( rosenberg and oen 1986 ) seems unnecessary and , in our opinion , may be counterproductive for the following reasons : ( a ) uveitis may be detected during the ophthalmic examination only if the inflammatory signs are already present , ( b ) there are no  predictive signs  for the potential development of intraocular inflammation in these children , and ( c ) an intraocular inflammation may develop 24 or 48 hours after the  regular  periodic visit . in these cases , parents may be reluctant to seek again ophthalmic consultation ( even if they suspect a problem ) until serious complications develop .", "therefore , education of the parents about the ocular disease , its potential evident signs and symptoms should be the main line of conduct for follow up instead of regular frequent ophthalmic visits .", "a drop of vision affecting the child s daily behavior can be noticed by parents .", "also , parents can be taught to perform a visual acuity test at home when playing with the child .", "these measures in our experience are more effective , practical , easily carried out and have the highest possible cost / benefit ratio . in this study", "we confirm , as published earlier ( benezra and cohen 2000 ) and also reported by others ( wolf et al 1987 ; akduman et al 1997 ; woreta et al 2007 ) that , severe complications with loss of vision are still to be feared in children with jia who are developing uveitis .", "most severe ocular complications have been observed in children 4 years of age or younger who presented first with ocular disease or had an ocular involvement detected during the initial ophthalmic examination .", "if not treated early , loss of vision may ensue in these cases even if successful control of the intraocular inflammation is achieved but proper anti amblyopic measures are not applied early .", "last but not least , in the management of jra associated ocular inflammation , surgery should be considered despite the potential for complications . in properly selected cases ,", "early surgery may even be considered in children with severe visual impairment in one eye due to band keratopathy , seclusion of the pupil by inflammatory membranes and/or dense cataract . in these cases , adequate surgical procedure of the cataract combined with intraocular lens implantation is to be considered and can result in good restoration of vision ( benezra and cohen 2000 ; khotaniemi and penttila 2006 ) ."]]}
{"article_id": "PMC3137977", "article_text": ["increasing cytosolic calcium ion ( ca ) concentration is essential for the contraction of smooth muscle cells .", "the increase results from the influx of ca through the plasma membrane and release of ca from intracellular stores , mainly the sarcoplasmic reticulum ( sr ) [ 14 ] .", "phenylephrine ( pe ) or angiotensin ii ( ang ii ) induces receptor - coupled g protein - induced phosphoinositide 3-kinase ( pi3k ) and phospholipase c ( plc ) activation , resulting in ca - dependent vasoconstriction in smooth muscle cells [ 510 ] .", "pi3k activity facilitates the production of 3-phosphorylated phosphoinositides such as phosphatidylinositol 3-phosphate ( pi(3)p ) , phosphatidylinositol ( 3,4)-bisphosphate ( pi(3,4)p2 ) , and phosphatidylinositol ( 3,4,5)-triphosphate ( pi(3,4,5)p3 ) [ 11 , 12 ] . among these", ", pi(3,4,5)p3 stimulates the l - type ca channel ( 12 ) , a voltage - dependent ca channel that plays an important role in the regulation of vascular tone [ 5 , 12 , 13 ] .", "activated plc is an effector in the stimulation of ca release from the endoplasmic reticulum ( er ) or the sr [ 1416 ] .", "plc hydrolyzes phosphatidylinositol 4,5-bisphosphate ( pip2 ) into diacylglycerol ( dag ) and inositol ( 3 , 4 , 5)-triphosphate ( ip3 ) .", "the latter is released as a soluble structure into the cytosol , where it binds to ip3 receptors in the sr [ 15 , 17 ] .", "this binding process increases the cytosolic ca concentration and smooth muscle constriction [ 15 , 16 ] .  ", "the herb cinnamomi ramulus ( cr ) has traditionally been used in asia and europe to treat maladies involving blood circulation and inflammation . in one study ,", "an aqueous extract of cr ameliorated sucrose - induced blood pressure elevation in spontaneously hypertensive rats .", "recently , we reported that cr ethanol extract ( cre ) reduces vascular contraction through the inhibition of voltage - dependent ca channels .", "the present study explored the suggestion that the vasodilatory effect of cre is related to ca - dependent mechanisms in rat aorta .", "all animals were provided with food and water ad libitum and allowed to adapt to the experimental conditions ( temperature , 21  2c ; humidity , 5060% ) for 1 week .", "rabbit polyclonal antibodies against phosphorylated plc ( pplc ) , -actin , and anti - rabbit secondary antibody were purchased from santa cruz biotechnology ( santa cruz , calif , usa ) .", "pi3k / p85 antibody was purchased from cell signaling technology ( beverly , mass , usa ) .", "pe , ang ii , verapamil , nifedipine , fpl64176 , 2 , 4 , 6-trimethyl - n-(meta-3-trifluoromethyl - phenyl)-benzenesulfonamide ( m-3m3fbs ) , u73122 and caffeine were purchased from sigma - aldrich ( st . louis , mo , usa ) .", "cr ( twigs of cinnamomum cassia blume ) collected in china in november 2009 was purchased from humanherb ( gyeongsan , korea ) .", "a voucher specimen ( cre08 ) has been deposited in the college of oriental medicine , dongguk university . dried cr ( 100  g )", "was extracted with 500  ml of 70% ethanol by heating at 75c for 3  h. the extract was filtered through whatman filter paper ( whatman international , maidstone , uk ) to remove the insoluble materials .", "after filtration , the extracts were concentrated by rotary evaporation using a model vv2000 apparatus ( heidolph , walpersdorfer , germany ) at a temperature of 75c and then dried using a model fd8508s freeze dryer ( ilshin , busan , korea ) .", "the ec50 value of 0.1  mg / ml cre was used in all experiments . in a previous research , cinnamaldehyde and coumarin", "also , cinnamaldehyde was known as major active compound of cr for vasodilation , antitumor , and antifungal activity .", "all procedures were performed according to protocols approved by the institutional animal care and use committee of dongguk university .", "briefly , rats were sacrificed and their thoracic aortas were immediately excised and immersed in ice - cold krebs solution ( 115.0  mm nacl , 4.7  mm kcl , 2.5  mm cacl2 , 1.2  mm mgcl2 , 25.0  mm nahco3 , 1.2  mm kh2po4 , and 10.0  mm dextrose ) .", "the aortas were cleaned of all adherent connective tissue and cut into 3  mm long ring segments .", "endothelium was removed from the internal surface of each segment by gentle rubbing with forceps .", "one triangle was anchored to a stationary support and the other was connected to a ft03 isometric force transducer ( grass , quincy , mass , usa ) .", "each vessel ring was incubated in a water - jacketed organ bath ( 10  ml ) that was maintained at 37c and aerated with a mixture of 95% o2 and 5% co2 .", "each ring was stretched passively by imposing the optimal resting tension of approximately 2.0  g , which was maintained throughout the experiment .", "each endothelium - free aortic ring was allowed to equilibrate in the organ bath for at least 50  min before the experiment involving the contractile response to 5  m ang ii , 1  m pe , 10  m fpl64176 , 5  g / ml m-3m3fbs , or 30  mm caffeine .", "endothelium - free rings were used because preliminary experiments ( data not shown ) established that cre relaxes vascular constriction in an endothelium - independent manner .", "the denudation of endothelium was assessed by treating the rings with 1  m acetylcholine .", "isometric tension was recorded using a powerlab/8sp computerized data acquisition system ( adinstruments , castle hill , nsw , australia ) .", "the influence of cre on extracellular ca influx was studied in ca - free krebs solution .", "after equilibration of the ring in ca - free krebs solution containing 60 mm kcl , cumulative doses of cacl2 were added ( 0.3 , 0.6 , 1 , 1.5 , 2.5 , 5 , and 10  mm , in order ) with preincubation of cre in organ bath .", "the cacl2 dose - dependent maximum constriction of the aortic ring with 60  mm kcl in ca - free krebs solution was expressed as 100% . to determine the influence of cre on ca influx through the l - type ca channel", ", aortic rings were pretreated with nifedipine or verapamil before pe contraction , and were preincubated with cre before contraction by fpl64176 . to investigate the inhibitory effect of cre on intracellular ca release by pe in ca - free conditions , and by caffeine in normal krebs solution ,", "the transient contraction of cre preincubated aortic rings was measured . to further investigate the relationship with the plc pathway ,", "aortic rings were constricted with m-3m3fbs , and were preincubated with u73122 prior to contraction by pe .", "a previously described protocol   was used for preparation of protein extract with some modifications .", "briefly , endothelium - free aortic rings were contracted with 1  m pe or 5  g / ml m-3m3fbs , and then treated with cre for 30  min .", "the aortic rings were quick frozen by immersion in acetone containing 10% trichloroacetic acid ( tca ) and 10  mm dithiothreitol ( dtt ) precooled to 80c .", "when used , recovered samples were homogenized in buffer containing 320  mm sucrose , 50  mm tris , 1  mm edta , 1% triton x-100 , 1  mm dtt , and the following protease inhibitors : leupeptin ( 10  g / ml ) , trypsin ( 10  g / ml ) , aprotinin ( 2  g / ml ) , or phenylmethylsulphonyl fluoride ( 100  g / ml ) .", "the protein samples were electrophoresed and the resolved proteins were transferred to a nitrocellulose membrane .", "the membrane was incubated with primary antibodies and then treated with horseradish peroxidase - conjugated anti - rabbit igg as a secondary antibody .", "all bands were detected using an enhanced chemiluminescence system ( amersham biosciences , buckinghamshire , uk ) .", "each set of experiments was done at least three times and results are presented as the mean  sd . the statistical significance of differences between mean values was assessed with student 's t - test or anova .", "ang ii increases the intracellular ca concentration in vascular smooth muscle cells through a sequence of events following activation of ang ii type 1 receptor and l - type calcium channels [ 6 , 7 ] .", "pe- or ang ii - induced contraction was significantly dilated by 53.6  7.8% and 66.0  5.4% , respectively , as compared to maximal tension ( figure 1 ) , indicating that that cre - mediated vasodilation may be related to decreased intracellular ca concentration . to determine the influence of cre on ca influx , the change of contraction was measured by adding cacl2 in an accumulative manner ( 0.3 , 0.6 , 1 , 1.5 , 2.5 , 5 , and 10  mm , in order ) before and after cre pretreatment in ca - free krebs solution containing 60  mm kcl .", "the vasoconstriction rate at the aforementioned cacl2 concentrations were 7.7  8.1% , 23  16% , 44  16.7% , 59  17% , 72  14% , 88.4  9.4% , and 100% , respectively , ( the latter represents the maximum contraction value of the aortic ring , achieved at 10  mm cacl2 ) .", "these contractions were significantly reduced by 1.9  2.2% , 5.8  4.1% , 8.3  6.3% , 11.4  4.4% , 22.3  1.9% , 24.8  6.2% , and 29.7  3.8% ( same respective cacl2 concentrations ) with cre - pretreatment ( figure 2 ) . to discern the effect of cre on the l - type calcium channel , the influence of the l - type calcium channel blocker nifedipine ( 100  m ) or verapamil ( 1  m ) , and the l - type calcium channel activator fpl64176 ( 10  m ) on vasorelaxation of cre against pe - induced contraction of aortic rings was measured .", "pretreatment of aortic rings with nifedipine or verapamil significantly inhibited the relaxant effect of cre ( figure 3(a ) ) .", "previous studies have shown that fpl64176 increases extracellular ca entry , thereby enhancing the cytosolic ca concentration [ 23 , 24 ] .", "presently , fpl64176 induced contraction , which plateaued at 3.75  0.22  g in 30  min , was inhibited by 2.4  0.1  g with preincubation of cre ( figure 3(b ) ) . to assess whether cre is involved in ca release - mediated vasoconstriction from intracellular stores , the transient contraction by pe or caffeine was examined in cre preincubated aortic rings .", "preincubation reduced the magnitude of contraction by pe from 0.32  0.6  g to 0.1  0.6  g ( figure 4(a ) ) . the transient contraction induced by 30  mm caffeine", "was also reduced by cre pretreatment ( figure 4(b ) ) . to evaluate whether the relaxant effect of cre was involved in the plc pathway , the plc pathway inhibitor u73122 and activator m-3m3fbs were used .", "u73122 pretreatment significantly inhibited the relaxant effect of cre on pe - induced contraction from 56.9  2.7% to 8.5  1.3% ( figure 5(a ) ) .", "m-3m3fbs ( 5  g / ml)-induced contraction was relaxed significantly with cre treatment ( figure 5(b ) ) .", "pi3k is directly activated by g - protein for generation of pip3 , which eventually stimulates l - type calcium channels in vascular myocytes [ 5 , 12 ] .", ", ip3 stimulates intracellular ca release from the sr for vasoconstriction [ 15 , 17 ] .", "presently , pe and m-3m3fbs increased the expression levels of pi3k ( 4.5  0.38 and 2.0  0.32 , resp . ) and pplc ( 4.1  0.30 and 2.5  0.14 , resp . ) , which were significantly decreased by cre . pe - induced pi3k expression was decreased by 2.8  0.70 at 50  g / ml and by 0.2  0.6 at 100  g / ml . pplc expression was also decreased by 2.0  0.30 at 50  g / ml and by 1.8  0.1 at 100  g / ml ( figure 6(a ) ) .", "m-3m3fbs - induced pi3k expression was downregulated by 0.6  0.1 at 50  g / ml and by 0.5  0.2 at 100  g / ml , and pplc expression was decreased by 1.4  0.1 at 50  g / ml and by 0.3  0.1 at 100  g / ml ( figure 6(b ) ) .", "its bark and twig are known as cinnamomi cortex ( cc ) and cinnamomi ramulus ( cr ) , respectively .", "cc inhibits helicobacter pylori   and ameliorates sucrose - induced blood pressure elevation in spontaneously hypertensive rats .", "furthermore , cre exerts an endothelium - independent vasodilatory response through inhibition of voltage - dependent ca channels .", "the present study investigated the vasodilatory effect of cre resulting from the inhibition of both ca influx and release in rat aorta .  ", "pe or ang ii stimulate plc isoforms to generate ip3 through the activation of g proteins , causing release of activator ca from sr [ 9 , 10 , 1416 ] . presently , cre markedly and similarly relaxed aortic rings that were precontracted with pe or angii .", "these results suggest that cre - mediated vasodilation may be involved in the regulation of ca mobilization . to assess this ,", "firstly , whether cre actually inhibits extracellular ca influx or not , we measured the tension of aortic rings by accumulative addition of cacl2 in ca - free krebs solution containing 60  mm kcl .", "preincubation with cre significantly reduced rat aortic contraction by addition of cacl2 , , indicating inhibition of ca influx .", "nifedipine ( 100  m ) and verapamil ( 1  m ) pretreatment inhibited the vasodilative effect of cre on pe - induced constricted aortic rings from 54.9  4.1% to 15.3  2.1% and 13.9  5.2% , respectively .", "in addition , preincubation with cre reduced the contraction of aortic ring by 10  m fpl64176 .", "these results support the suggestion that the vasodilative effect of cre is related to the inhibition of l - type calcium channel in the cell membrane .  ", "the sr is the major source of ca release into the cytosol [ 1 , 16 , 17 ] .", "this ca release is induced by the ip3 second messenger , which is generated by plc activation .", "ca release from the sr is considered to be the initial mechanism in agonists such as pe- and ang ii - induced vasoconstriction [ 6 , 17 ] .", "pe - induced transient constriction is dependent on ca release from the sr through the ip3 signal pathway in ca - free krebs solution ; however , caffeine is dependent on ca - induced ca release from the sr [ 27 , 28 ] . to demonstrate the effects of cre on ca release from the sr ,", "transient contractions induced by pe in ca - free krebs solution and induced by caffeine in normal krebs solution were investigated .", "cre significantly reduced the magnitudes of transient contraction by pe and caffeine , suggesting cre inhibits ca release from the sr by blocking the ip3-induced ca release and ca - induced ca release mechanisms .", "pretreatment with the plc inhibitor u73122 significantly reduced the vasorelaxation of cre on pe - induced vasoconstriction , and cre relaxed m-3m3fbs - induced vasoconstriction .", "additionally , we analyzed the expression levels of the intracellular signaling regulator proteins pi3k and plc .", "pi3k generates various 3-phosphorylated phosphoinositides through activation by g - proteins , especially pi(3,4,5)p3 stimulates the l - type ca channel that plays an important role in the regulation of vascular tone [ 8 , 11 , 12 ] . on the other hand , plc formats the two potent second messengers ip3 and dag", "especially , ip3 induces the activation of ip3 receptor on the sr membrane , opening a calcium channel , resulting in the release of ca into the cytosol [ 14 , 17 ] .", "presently , pe- or m-3m3fbs - induced phosphorylation of plc and upregulation of pi3k / p85 protein expression were inhibited by cre ( figure 6 ) .", "the collective data supports the idea that cre dilates vascular contraction through the inhibition of both ca influx via the l - type ca channel and ip3-induced ca release from the sr .   in conclusion", ", the data supports the vasorelaxation of cre through the inhibition of ca influx and ca release .", "therefore , cre may be useful as a drug for the treatment and prevention of high blood pressure associated with ca - dependent contraction of smooth muscle ."], "abstract_text": ["<S> contraction of vascular smooth muscle cells depends on the induction of cytosolic calcium ion ( ca2 + ) due to either ca2 + influx through voltage - gated ca2 + channels or to receptor - mediated ca2 + release from the sarcoplasmic reticulum . </S>", "<S> the present study investigated the vasorelaxation effect of cinnamomi ramulus ethanol extract ( cre ) and the possible mechanisms in rat aorta . </S>", "<S> cre ( 0.1  mg / ml ) relaxed vasoconstriction induced by phenylephrine ( pe ; 1  m ) and angiotensin ii ( 5  m ) . </S>", "<S> preincubation with cre significantly reduced the rat aortic contraction by addition of cacl2 in ca2 + -free krebs solution and fpl64176 ( 10  m ) . </S>", "<S> pretreatment with nifedipine ( 100  m ) or verapamil ( 1  m ) significantly reduced the cre - mediated vasorelaxation of pe - induced vascular contraction . </S>", "<S> in addition , cre also relaxed the vascular contraction caused by m-3m3fbs ( 5  g / ml ) , but u73122 ( 10  m ) significantly inhibited the vasorelaxation of pe precontracted aortic rings . </S>", "<S> furthermore , cre significantly reduced the magnitude of pe- and caffeine ( 30  mm)-induced transient contraction . in vascular strips , </S>", "<S> cre downregulated the expression levels of phosphorylated plc and phosphoinositide 3-kinase elevated by pe or m-3m3fbs . </S>", "<S> these results suggest that cre relaxes vascular smooth muscle through the inhibition of both ca2 + influx via l - type ca2 + channel and inositol triphosphate - induced ca2 + release from the sarcoplasmic reticulum . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion and Conclusions"], "sections": [["increasing cytosolic calcium ion ( ca ) concentration is essential for the contraction of smooth muscle cells .", "the increase results from the influx of ca through the plasma membrane and release of ca from intracellular stores , mainly the sarcoplasmic reticulum ( sr ) [ 14 ] .", "phenylephrine ( pe ) or angiotensin ii ( ang ii ) induces receptor - coupled g protein - induced phosphoinositide 3-kinase ( pi3k ) and phospholipase c ( plc ) activation , resulting in ca - dependent vasoconstriction in smooth muscle cells [ 510 ] .", "pi3k activity facilitates the production of 3-phosphorylated phosphoinositides such as phosphatidylinositol 3-phosphate ( pi(3)p ) , phosphatidylinositol ( 3,4)-bisphosphate ( pi(3,4)p2 ) , and phosphatidylinositol ( 3,4,5)-triphosphate ( pi(3,4,5)p3 ) [ 11 , 12 ] . among these", ", pi(3,4,5)p3 stimulates the l - type ca channel ( 12 ) , a voltage - dependent ca channel that plays an important role in the regulation of vascular tone [ 5 , 12 , 13 ] .", "activated plc is an effector in the stimulation of ca release from the endoplasmic reticulum ( er ) or the sr [ 1416 ] .", "plc hydrolyzes phosphatidylinositol 4,5-bisphosphate ( pip2 ) into diacylglycerol ( dag ) and inositol ( 3 , 4 , 5)-triphosphate ( ip3 ) .", "the latter is released as a soluble structure into the cytosol , where it binds to ip3 receptors in the sr [ 15 , 17 ] .", "this binding process increases the cytosolic ca concentration and smooth muscle constriction [ 15 , 16 ] .  ", "the herb cinnamomi ramulus ( cr ) has traditionally been used in asia and europe to treat maladies involving blood circulation and inflammation . in one study ,", "an aqueous extract of cr ameliorated sucrose - induced blood pressure elevation in spontaneously hypertensive rats .", "recently , we reported that cr ethanol extract ( cre ) reduces vascular contraction through the inhibition of voltage - dependent ca channels .", "the present study explored the suggestion that the vasodilatory effect of cre is related to ca - dependent mechanisms in rat aorta ."], ["all animals were provided with food and water ad libitum and allowed to adapt to the experimental conditions ( temperature , 21  2c ; humidity , 5060% ) for 1 week .", "rabbit polyclonal antibodies against phosphorylated plc ( pplc ) , -actin , and anti - rabbit secondary antibody were purchased from santa cruz biotechnology ( santa cruz , calif , usa ) .", "pi3k / p85 antibody was purchased from cell signaling technology ( beverly , mass , usa ) .", "pe , ang ii , verapamil , nifedipine , fpl64176 , 2 , 4 , 6-trimethyl - n-(meta-3-trifluoromethyl - phenyl)-benzenesulfonamide ( m-3m3fbs ) , u73122 and caffeine were purchased from sigma - aldrich ( st . louis , mo , usa ) .", "cr ( twigs of cinnamomum cassia blume ) collected in china in november 2009 was purchased from humanherb ( gyeongsan , korea ) .", "a voucher specimen ( cre08 ) has been deposited in the college of oriental medicine , dongguk university . dried cr ( 100  g )", "was extracted with 500  ml of 70% ethanol by heating at 75c for 3  h. the extract was filtered through whatman filter paper ( whatman international , maidstone , uk ) to remove the insoluble materials .", "after filtration , the extracts were concentrated by rotary evaporation using a model vv2000 apparatus ( heidolph , walpersdorfer , germany ) at a temperature of 75c and then dried using a model fd8508s freeze dryer ( ilshin , busan , korea ) .", "the ec50 value of 0.1  mg / ml cre was used in all experiments . in a previous research , cinnamaldehyde and coumarin", "also , cinnamaldehyde was known as major active compound of cr for vasodilation , antitumor , and antifungal activity .", "all procedures were performed according to protocols approved by the institutional animal care and use committee of dongguk university .", "briefly , rats were sacrificed and their thoracic aortas were immediately excised and immersed in ice - cold krebs solution ( 115.0  mm nacl , 4.7  mm kcl , 2.5  mm cacl2 , 1.2  mm mgcl2 , 25.0  mm nahco3 , 1.2  mm kh2po4 , and 10.0  mm dextrose ) .", "the aortas were cleaned of all adherent connective tissue and cut into 3  mm long ring segments .", "endothelium was removed from the internal surface of each segment by gentle rubbing with forceps .", "one triangle was anchored to a stationary support and the other was connected to a ft03 isometric force transducer ( grass , quincy , mass , usa ) .", "each vessel ring was incubated in a water - jacketed organ bath ( 10  ml ) that was maintained at 37c and aerated with a mixture of 95% o2 and 5% co2 .", "each ring was stretched passively by imposing the optimal resting tension of approximately 2.0  g , which was maintained throughout the experiment .", "each endothelium - free aortic ring was allowed to equilibrate in the organ bath for at least 50  min before the experiment involving the contractile response to 5  m ang ii , 1  m pe , 10  m fpl64176 , 5  g / ml m-3m3fbs , or 30  mm caffeine .", "endothelium - free rings were used because preliminary experiments ( data not shown ) established that cre relaxes vascular constriction in an endothelium - independent manner .", "the denudation of endothelium was assessed by treating the rings with 1  m acetylcholine .", "isometric tension was recorded using a powerlab/8sp computerized data acquisition system ( adinstruments , castle hill , nsw , australia ) .", "the influence of cre on extracellular ca influx was studied in ca - free krebs solution .", "after equilibration of the ring in ca - free krebs solution containing 60 mm kcl , cumulative doses of cacl2 were added ( 0.3 , 0.6 , 1 , 1.5 , 2.5 , 5 , and 10  mm , in order ) with preincubation of cre in organ bath .", "the cacl2 dose - dependent maximum constriction of the aortic ring with 60  mm kcl in ca - free krebs solution was expressed as 100% . to determine the influence of cre on ca influx through the l - type ca channel", ", aortic rings were pretreated with nifedipine or verapamil before pe contraction , and were preincubated with cre before contraction by fpl64176 . to investigate the inhibitory effect of cre on intracellular ca release by pe in ca - free conditions , and by caffeine in normal krebs solution ,", "the transient contraction of cre preincubated aortic rings was measured . to further investigate the relationship with the plc pathway ,", "aortic rings were constricted with m-3m3fbs , and were preincubated with u73122 prior to contraction by pe .", "a previously described protocol   was used for preparation of protein extract with some modifications .", "briefly , endothelium - free aortic rings were contracted with 1  m pe or 5  g / ml m-3m3fbs , and then treated with cre for 30  min .", "the aortic rings were quick frozen by immersion in acetone containing 10% trichloroacetic acid ( tca ) and 10  mm dithiothreitol ( dtt ) precooled to 80c .", "when used , recovered samples were homogenized in buffer containing 320  mm sucrose , 50  mm tris , 1  mm edta , 1% triton x-100 , 1  mm dtt , and the following protease inhibitors : leupeptin ( 10  g / ml ) , trypsin ( 10  g / ml ) , aprotinin ( 2  g / ml ) , or phenylmethylsulphonyl fluoride ( 100  g / ml ) .", "the protein samples were electrophoresed and the resolved proteins were transferred to a nitrocellulose membrane .", "the membrane was incubated with primary antibodies and then treated with horseradish peroxidase - conjugated anti - rabbit igg as a secondary antibody .", "all bands were detected using an enhanced chemiluminescence system ( amersham biosciences , buckinghamshire , uk ) .", "each set of experiments was done at least three times and results are presented as the mean  sd . the statistical significance of differences between mean values was assessed with student 's t - test or anova ."], ["ang ii increases the intracellular ca concentration in vascular smooth muscle cells through a sequence of events following activation of ang ii type 1 receptor and l - type calcium channels [ 6 , 7 ] .", "pe- or ang ii - induced contraction was significantly dilated by 53.6  7.8% and 66.0  5.4% , respectively , as compared to maximal tension ( figure 1 ) , indicating that that cre - mediated vasodilation may be related to decreased intracellular ca concentration . to determine the influence of cre on ca influx , the change of contraction was measured by adding cacl2 in an accumulative manner ( 0.3 , 0.6 , 1 , 1.5 , 2.5 , 5 , and 10  mm , in order ) before and after cre pretreatment in ca - free krebs solution containing 60  mm kcl .", "the vasoconstriction rate at the aforementioned cacl2 concentrations were 7.7  8.1% , 23  16% , 44  16.7% , 59  17% , 72  14% , 88.4  9.4% , and 100% , respectively , ( the latter represents the maximum contraction value of the aortic ring , achieved at 10  mm cacl2 ) .", "these contractions were significantly reduced by 1.9  2.2% , 5.8  4.1% , 8.3  6.3% , 11.4  4.4% , 22.3  1.9% , 24.8  6.2% , and 29.7  3.8% ( same respective cacl2 concentrations ) with cre - pretreatment ( figure 2 ) . to discern the effect of cre on the l - type calcium channel , the influence of the l - type calcium channel blocker nifedipine ( 100  m ) or verapamil ( 1  m ) , and the l - type calcium channel activator fpl64176 ( 10  m ) on vasorelaxation of cre against pe - induced contraction of aortic rings was measured .", "pretreatment of aortic rings with nifedipine or verapamil significantly inhibited the relaxant effect of cre ( figure 3(a ) ) .", "previous studies have shown that fpl64176 increases extracellular ca entry , thereby enhancing the cytosolic ca concentration [ 23 , 24 ] .", "presently , fpl64176 induced contraction , which plateaued at 3.75  0.22  g in 30  min , was inhibited by 2.4  0.1  g with preincubation of cre ( figure 3(b ) ) . to assess whether cre is involved in ca release - mediated vasoconstriction from intracellular stores , the transient contraction by pe or caffeine was examined in cre preincubated aortic rings .", "preincubation reduced the magnitude of contraction by pe from 0.32  0.6  g to 0.1  0.6  g ( figure 4(a ) ) . the transient contraction induced by 30  mm caffeine", "was also reduced by cre pretreatment ( figure 4(b ) ) . to evaluate whether the relaxant effect of cre was involved in the plc pathway , the plc pathway inhibitor u73122 and activator m-3m3fbs were used .", "u73122 pretreatment significantly inhibited the relaxant effect of cre on pe - induced contraction from 56.9  2.7% to 8.5  1.3% ( figure 5(a ) ) .", "m-3m3fbs ( 5  g / ml)-induced contraction was relaxed significantly with cre treatment ( figure 5(b ) ) .", "pi3k is directly activated by g - protein for generation of pip3 , which eventually stimulates l - type calcium channels in vascular myocytes [ 5 , 12 ] .", ", ip3 stimulates intracellular ca release from the sr for vasoconstriction [ 15 , 17 ] .", "presently , pe and m-3m3fbs increased the expression levels of pi3k ( 4.5  0.38 and 2.0  0.32 , resp . ) and pplc ( 4.1  0.30 and 2.5  0.14 , resp . ) , which were significantly decreased by cre . pe - induced pi3k expression was decreased by 2.8  0.70 at 50  g / ml and by 0.2  0.6 at 100  g / ml . pplc expression was also decreased by 2.0  0.30 at 50  g / ml and by 1.8  0.1 at 100  g / ml ( figure 6(a ) ) .", "m-3m3fbs - induced pi3k expression was downregulated by 0.6  0.1 at 50  g / ml and by 0.5  0.2 at 100  g / ml , and pplc expression was decreased by 1.4  0.1 at 50  g / ml and by 0.3  0.1 at 100  g / ml ( figure 6(b ) ) ."], ["its bark and twig are known as cinnamomi cortex ( cc ) and cinnamomi ramulus ( cr ) , respectively .", "cc inhibits helicobacter pylori   and ameliorates sucrose - induced blood pressure elevation in spontaneously hypertensive rats .", "furthermore , cre exerts an endothelium - independent vasodilatory response through inhibition of voltage - dependent ca channels .", "the present study investigated the vasodilatory effect of cre resulting from the inhibition of both ca influx and release in rat aorta .  ", "pe or ang ii stimulate plc isoforms to generate ip3 through the activation of g proteins , causing release of activator ca from sr [ 9 , 10 , 1416 ] . presently , cre markedly and similarly relaxed aortic rings that were precontracted with pe or angii .", "these results suggest that cre - mediated vasodilation may be involved in the regulation of ca mobilization . to assess this ,", "firstly , whether cre actually inhibits extracellular ca influx or not , we measured the tension of aortic rings by accumulative addition of cacl2 in ca - free krebs solution containing 60  mm kcl .", "preincubation with cre significantly reduced rat aortic contraction by addition of cacl2 , , indicating inhibition of ca influx .", "nifedipine ( 100  m ) and verapamil ( 1  m ) pretreatment inhibited the vasodilative effect of cre on pe - induced constricted aortic rings from 54.9  4.1% to 15.3  2.1% and 13.9  5.2% , respectively .", "in addition , preincubation with cre reduced the contraction of aortic ring by 10  m fpl64176 .", "these results support the suggestion that the vasodilative effect of cre is related to the inhibition of l - type calcium channel in the cell membrane .  ", "the sr is the major source of ca release into the cytosol [ 1 , 16 , 17 ] .", "this ca release is induced by the ip3 second messenger , which is generated by plc activation .", "ca release from the sr is considered to be the initial mechanism in agonists such as pe- and ang ii - induced vasoconstriction [ 6 , 17 ] .", "pe - induced transient constriction is dependent on ca release from the sr through the ip3 signal pathway in ca - free krebs solution ; however , caffeine is dependent on ca - induced ca release from the sr [ 27 , 28 ] . to demonstrate the effects of cre on ca release from the sr ,", "transient contractions induced by pe in ca - free krebs solution and induced by caffeine in normal krebs solution were investigated .", "cre significantly reduced the magnitudes of transient contraction by pe and caffeine , suggesting cre inhibits ca release from the sr by blocking the ip3-induced ca release and ca - induced ca release mechanisms .", "pretreatment with the plc inhibitor u73122 significantly reduced the vasorelaxation of cre on pe - induced vasoconstriction , and cre relaxed m-3m3fbs - induced vasoconstriction .", "additionally , we analyzed the expression levels of the intracellular signaling regulator proteins pi3k and plc .", "pi3k generates various 3-phosphorylated phosphoinositides through activation by g - proteins , especially pi(3,4,5)p3 stimulates the l - type ca channel that plays an important role in the regulation of vascular tone [ 8 , 11 , 12 ] . on the other hand , plc formats the two potent second messengers ip3 and dag", "especially , ip3 induces the activation of ip3 receptor on the sr membrane , opening a calcium channel , resulting in the release of ca into the cytosol [ 14 , 17 ] .", "presently , pe- or m-3m3fbs - induced phosphorylation of plc and upregulation of pi3k / p85 protein expression were inhibited by cre ( figure 6 ) .", "the collective data supports the idea that cre dilates vascular contraction through the inhibition of both ca influx via the l - type ca channel and ip3-induced ca release from the sr .   in conclusion", ", the data supports the vasorelaxation of cre through the inhibition of ca influx and ca release .", "therefore , cre may be useful as a drug for the treatment and prevention of high blood pressure associated with ca - dependent contraction of smooth muscle ."]]}
{"article_id": "PMC4913566", "article_text": ["after an information campaign in horse journals and at meetings , 278 samples of maple tree leaves , sprouts , and seeds collected by horse owners in 2014 were sent to utrecht university ( uu ) and rikiltwageningen ur ( figs 1 , 2 , 3 ) . the samples were taken from the ground , packed in plastic , and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection .", "they were classified by an experienced botanist at the species level ( acer pseudoplatanus , acer platanoides , or acer campestre ) on the basis of the samples and accompanying photographs sent by owners .", "sprouts were only used in this study upon positive classification by means of accompanying leaves , seeds or both . the minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves .", "owners were asked to complete a questionnaire with information about date of collection , address of the pasture , species of maple tree ( in their opinion ) , signalment ( name , breed and age ) of the horse(s ) , and presence of disease ( am ) in horses that were grazed in the pastures where the material was collected .", "if the pasture had contained horses with am , details about the disease ( including date of onset and the final outcome ) , laboratory results and name of the veterinarian were provided .", "inclusion criteria to confirm am were recumbency , dark colored urine , high serum activity of creatine kinase ( ck , either too high to measure in practice , or > 100,000 iu / l in a laboratory ) and postmortem examination , which was performed in 4 cases . exclusion criterion for am was lack of ck analysis unless postmortem examination was performed .", "the geographic location of the pastures where the acer seeds , sprouts and leaves were sampled . \n", "a , b , c leaves of acer pseudoplatanus , acer campestre and acer platanoides .", "hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry ( lc  ms / ms).1 \n  seed samples ( 50 g , as received ) were ground in a retsch mill , and leaf samples ( 50100 g ) were cryogenically milled into a powder , using liquid nitrogen .", "one gram of homogenized sample material was extracted with 10 ml of milliq water ( mechanical shaker , 1 h ) .", "after centrifugation ( 5000 g , 10c , 10 min ) , 10 l of the clear extract was injected into an lc  ms / ms system , acquiring 2 transitions ( m / z 142.174.1 and 142.196.0 ) .", "hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions , against an analytical reference standard ( shypoglycin a2 ) dissolved in milliq water .", "( residuals < 20% ) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg", "/ l , corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts .", "the accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf ( 1 mg / kg ) and seed ( 10 mg / kg ) samples in 5fold dilutions .", "the corresponding repeatabilities ( relative standard deviation , rsd ) were 11% and 7% , respectively .", "the rsd obtained for repetitive analysis ( n = 5 ) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% .", "recoveries obtained for hypoglycin a in spiked seed samples ( 10 mg / kg ) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395%", ". samples with incomplete questionnaire data on relevant facts , such as address and obtainable data about the disease if present , were excluded from further statistical analysis ( 9 samples ) .", "if multiple samples from 1 pasture were submitted , which happened often , the mean result of the analyses per sample type ( sprout , seed or leaf ) were counted as 1 result in further statistical analysis .", "samples that could not be unambiguously assigned to an acer species ( 6 ) , samples that were not fresh ( 17 ) , and samples that contained material other than seeds , sprouts and leaves ( 10 ) also were excluded from the study .", "this approach resulted in 143 usable samples , 30 from the spring and 113 from the autumn of 2014 .", "the samples were grouped by whether they came from pastures grazed by healthy horses or horses with am ( nonam versus am pastures ) . mean concentrations of hypoglycin a were compared for the type of sample ( seed versus sprout versus leaf ) , season ( spring versus autumn ) and disease status for the pasture ( nonam versus am ) .", "statistical analysis was performed using univariate generalized linear models with multiple comparisons , corrected with a posthoc bonferroni test.3 \n  control for normal distribution was performed .", "after an information campaign in horse journals and at meetings , 278 samples of maple tree leaves , sprouts , and seeds collected by horse owners in 2014 were sent to utrecht university ( uu ) and rikiltwageningen ur ( figs 1 , 2 , 3 ) . the samples were taken from the ground , packed in plastic , and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection .", "they were classified by an experienced botanist at the species level ( acer pseudoplatanus , acer platanoides , or acer campestre ) on the basis of the samples and accompanying photographs sent by owners .", "sprouts were only used in this study upon positive classification by means of accompanying leaves , seeds or both . the minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves .", "owners were asked to complete a questionnaire with information about date of collection , address of the pasture , species of maple tree ( in their opinion ) , signalment ( name , breed and age ) of the horse(s ) , and presence of disease ( am ) in horses that were grazed in the pastures where the material was collected .", "if the pasture had contained horses with am , details about the disease ( including date of onset and the final outcome ) , laboratory results and name of the veterinarian were provided .", "inclusion criteria to confirm am were recumbency , dark colored urine , high serum activity of creatine kinase ( ck , either too high to measure in practice , or > 100,000 iu / l in a laboratory ) and postmortem examination , which was performed in 4 cases . exclusion criterion for am was lack of ck analysis unless postmortem examination was performed .", "the geographic location of the pastures where the acer seeds , sprouts and leaves were sampled . \n", "a , b , c leaves of acer pseudoplatanus , acer campestre and acer platanoides .", "hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry ( lc  ms / ms).1 \n  seed samples ( 50 g , as received ) were ground in a retsch mill , and leaf samples ( 50100 g ) were cryogenically milled into a powder , using liquid nitrogen .", "one gram of homogenized sample material was extracted with 10 ml of milliq water ( mechanical shaker , 1 h ) .", "after centrifugation ( 5000 g , 10c , 10 min ) , 10 l of the clear extract was injected into an lc  ms / ms system , acquiring 2 transitions ( m / z 142.174.1 and 142.196.0 ) .", "hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions , against an analytical reference standard ( shypoglycin a2 ) dissolved in milliq water .", "quantification was performed by multilevel calibration . a consistent linear relationship ( residuals < 20% ) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg / l , corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts .", "the accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf ( 1 mg / kg ) and seed ( 10 mg / kg ) samples in 5fold dilutions .", "the corresponding repeatabilities ( relative standard deviation , rsd ) were 11% and 7% , respectively .", "the rsd obtained for repetitive analysis ( n = 5 ) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% .", "recoveries obtained for hypoglycin a in spiked seed samples ( 10 mg / kg ) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395% .", "samples with incomplete questionnaire data on relevant facts , such as address and obtainable data about the disease if present , were excluded from further statistical analysis ( 9 samples ) . if multiple samples from 1 pasture were submitted , which happened often , the mean result of the analyses per sample type ( sprout , seed or leaf ) were counted as 1 result in further statistical analysis .", "samples that could not be unambiguously assigned to an acer species ( 6 ) , samples that were not fresh ( 17 ) , and samples that contained material other than seeds , sprouts and leaves ( 10 ) also were excluded from the study .", "this approach resulted in 143 usable samples , 30 from the spring and 113 from the autumn of 2014 .", "the samples were grouped by whether they came from pastures grazed by healthy horses or horses with am ( nonam versus am pastures ) .", "mean concentrations of hypoglycin a were compared for the type of sample ( seed versus sprout versus leaf ) , season ( spring versus autumn ) and disease status for the pasture ( nonam versus am ) .", "statistical analysis was performed using univariate generalized linear models with multiple comparisons , corrected with a posthoc bonferroni test.3 \n  control for normal distribution was performed . a p value of .05 was set as statistically significant .", "in total , 143 of the collected samples ( 93 seed samples , 14 sprout samples , and 36 leave samples ) from a total of 81 locations were included ( fig 1 ) . none of the acer platanoides ( n = 11 ) and acer campestre ( n = 13 ) samples ( 29 samples of seeds , 11 of leaves and 1 of sprouts ) contained detectable concentrations of hypoglycin a. in contrast , most acer pseudoplatanus leaves , seeds , and sprouts contained the toxin . in 2 samples of seeds and 6 samples of leaves ,", "no hypoglycin a was found ( ie , concentration below limit of quantification [ 1 mg / kg for leaves and 10 mg / kg for seeds ] ) .", "the factor season was not statistically significant ( p = .841 ) and was excluded from further statistical analysis .", "the mean  sd concentrations of hypoglycin a in the samples are presented in table 1 .", "hypoglycin a concentrations differed significantly among the sample types ( seeds and sprouts , p < .001 ; seeds and leaves , p < .001 ; sprouts and leaves , p = .000 ) .", "concentrations were significantly higher in seeds from the am group than in seeds from the nonam group ( seeds , 856  677 versus 456  358 mg / kg ; p = .039 ) . in sprouts , ( 1365  795 mg /", "kg versus 1097  310 , p = .493 ) and leaves ( 31  49 versus 37  34 mg / kg , p = .819 ) this was not the case and when values for all seeds , sprouts , and leaves were combined the difference between nonam and am was not significant either ( p = .127 ) ; ( table 1 ) .", "the concentration ( mg / kg ) of hypoglycin a in samples of acer pseudoplatanus taken from pastures or paddocks grazed by healthy horses and horses with atypical myopathy ( am ) concentration significantly different between pastures used by healthy and am horses ( p = 0.039 ) .", "there were 16 horses ( providing 29 samples ) with am of which 11 died and 5 survived .", "there was no relationship between the concentration of hypoglycin a in the samples and outcome of the disease ( p = .250 ) .", ", acer negundo ( box elder tree ) and acer pseudoplatanus ( sycamore maple ) have been associated with am.15 , 17 \n  the species acer campestre ( hedge maple ) and acer platanoides ( norway maple ) did not contain detectable hypoglycin a concentrations , and thus it is probably safe , in terms of am , to grow these species in or around pastures and paddocks . in 1973 , semiquantitative measurements ( notdetected , trace , weak , moderate , and strong ) also indicated that these tree species were free of hypoglycin a.18 \n  the most common maple tree in the netherlands is acer pseudoplatanus .", "we found that the leaves , sprouts , and seeds contained measurable concentrations of hypoglycin a. although hypoglycin a concentrations were significantly lower in seeds collected from pastures grazed by healthy horses than in samples from pastures grazed by am horses , concentrations were too variable to allow predictions to be made as to whether or not the presence of an individual tree would be a threat to horses grazing in its vicinity .", "our results are consistent with a study that found hypoglycin a concentrations in seeds to be highly variable among trees on the same or different farms.17 hypoglycin a concentrations in our study were similar to those previously reported.17 in the previous study , a mean concentration of 690 mg / kg was found with variation between 40 and 2810 mg / kg and in our study a mean of 856 mg / kg with variation between 25 and 3683 mg / kg was found", ". the question arises as to why not all horses grazing pastures containing or surrounded by acer pseudoplatanus develop am .", "the most likely explanation might relate to differences in the amount of seeds , sprouts , and leaves ingested , other components of the diet , and the sensitivity of the individual horse .", "the availability of seeds , leaves , and sprouts largely is determined by the season and weather , with changes in wind and temperature loosening the attachment of leaves and seeds.6 , 10 , 15 , 19 indeed , inclement weather or high winds have been reported to precede clinical signs of am.6 , 12 , 15 strong winds can disperse leaves and seeds over a long distance , perhaps depositing them in pastures that do not contain acer trees .", "all of these factors could explain why acer trees are not always found in the paddock or pastures grazed by horses with am.8 \n  our results suggest that , per kilogram , sprouts are most dangerous to horses , followed by seeds , with leaves being potentially least harmful .", "data show that there are more cases of am in autumn than in spring.8 because sprouts are more common in spring and seeds are more common in autumn , it would appear that horses eat more seeds than sprouts either because more seeds are available or because they prefer seeds to sprouts .", "however , whether horses eat seeds or sprouts is to a large extent determined by the availability of other feedstuff.20 in general , spring pasture contains more and better grass than autumn pasture , and thus horses may have less reason to eat other feedstuff.20 \n  the finding that the concentration of hypoglycin a is high in sprouts suggests that horse owners should be alert to the presence of acer sprouts in pastures .", "owners should be advised to mow areas of pasture with maple sprouts and to remove the mown material .", "a factor that needs further investigation is tree stress.20 , 21 tree stress or abiotic stress may increase the hypoglycin a concentration in seeds .", "several owners who sent in samples did not return the questionnaire or did not complete it fully .", "moreover , if there was doubt ( after consultation with the owner 's veterinarian ) about whether a horse suffered from am or not , data for samples collected from that horse 's paddock or pasture were not used .", "results of the samples of 1 type ( seed , sprout , or leaf ) originating from 1 pasture were pooled and the mean concentration of these was treated as 1 sample .", "because toxin concentrations are highly variable among trees on the same farm , it would have been better to have taken more samples of all materials .", "the seeds were fresh frozen and not desiccated before freezing and changes in toxin concentrations as a result of artificial freezing could have occurred in the spring samples in the same fashion as occurs naturally in autumn . ideally all cases would have been confirmed by analyzing blood or urine for madd or toxin or toxin metabolites . because doing so was not always possible , the described inclusion criteria were used to make the diagnosis . in conclusion , it is important to know the tree species when giving advice regarding the toxicity of maple trees and the potential risk for horses , because some acer species contain hypoglycin a and others do not .", "unfortunately , the amount of hypoglycin a in seeds is so variable that a reliable prediction of the occurrence of am can not be made for individual farms .", "therefore , it is not worthwhile to measure hypoglycin concentrations in individual seed samples in order to predict am risk .", "however , in all cases in which no hypoglycin a was found , no clinical signs of am were observed .", "thus , the most important recommendation is that owners should prevent horses from eating seeds , sprouts , leaves , or any combination of these from acer pseudoplatanus ( sycamore maple ) , whereas the seeds of acer campestre ( hedge maple ) and acer platanoides ( norway maple ) appear to be safe for horses .", "steps to prevent the ingestion of toxin include moving the horse to a safer pasture , decreasing the size of the pasture ( away from the trees ) , blowing away seeds and leaves , or mowing and removing sprouts ."], "abstract_text": ["<S> backgroundatypical myopathy ( am ) in horses is caused by the plant toxin hypoglycin a , which in europe typically is found in the sycamore maple tree ( acer pseudoplatanus ) . </S>", "<S> owners are concerned about whether their horses are in danger if they graze near maple trees.hypothesis/objectivesto measure hypoglycin a in the most common maple tree species in the netherlands , and to determine whether concentration of toxin is a predictor of am in horses.methodsa total of 278 samples of maple tree leaves , sprouts , and seeds were classified by species . </S>", "<S> mean concentrations of hypoglycin a were compared for the type of sample , the season and the occurrence of am in the pasture ( nonam versus am ) . </S>", "<S> statistical analysis was performed using generalized a linear model ( spps22).resultsalmost all acer pseudoplatanus samples contained hypoglycin a , with concentrations differing significantly among sources ( p < .001 ) . </S>", "<S> concentrations were significantly higher in seeds from the am group than in seeds from the nonam group ( 856  677 and 456  358 mg / kg , respectively ; p = .039 ) . in sprouts and leaves this was not the case . </S>", "<S> acer platanoides and acer campestre samples did not contain detectable concentrations of hypoglycin a.conclusions and clinical importance \n acer platanoides and campestre seem to be safe around paddocks and pastures , whereas almost all acer pseudoplatanus samples contained hypoglycin a. in all am cases , acer pseudoplatanus was found . despite significantly higher concentration of hypoglycin a in seeds of pastures where am has occurred , individual prediction of am can not be made by measuring these concentrations because of the high standard deviation . </S>"], "labels": null, "section_names": ["Materials and Methods", "Collection of Leaves, Seeds, and Sprouts", "Chemical Analysis", "Selection and Grouping of Samples", "Statistical Analysis", "Results", "Discussion", "Supporting information"], "sections": [["after an information campaign in horse journals and at meetings , 278 samples of maple tree leaves , sprouts , and seeds collected by horse owners in 2014 were sent to utrecht university ( uu ) and rikiltwageningen ur ( figs 1 , 2 , 3 ) . the samples were taken from the ground , packed in plastic , and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection .", "they were classified by an experienced botanist at the species level ( acer pseudoplatanus , acer platanoides , or acer campestre ) on the basis of the samples and accompanying photographs sent by owners .", "sprouts were only used in this study upon positive classification by means of accompanying leaves , seeds or both . the minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves .", "owners were asked to complete a questionnaire with information about date of collection , address of the pasture , species of maple tree ( in their opinion ) , signalment ( name , breed and age ) of the horse(s ) , and presence of disease ( am ) in horses that were grazed in the pastures where the material was collected .", "if the pasture had contained horses with am , details about the disease ( including date of onset and the final outcome ) , laboratory results and name of the veterinarian were provided .", "inclusion criteria to confirm am were recumbency , dark colored urine , high serum activity of creatine kinase ( ck , either too high to measure in practice , or > 100,000 iu / l in a laboratory ) and postmortem examination , which was performed in 4 cases . exclusion criterion for am was lack of ck analysis unless postmortem examination was performed .", "the geographic location of the pastures where the acer seeds , sprouts and leaves were sampled . \n", "a , b , c leaves of acer pseudoplatanus , acer campestre and acer platanoides .", "hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry ( lc  ms / ms).1 \n  seed samples ( 50 g , as received ) were ground in a retsch mill , and leaf samples ( 50100 g ) were cryogenically milled into a powder , using liquid nitrogen .", "one gram of homogenized sample material was extracted with 10 ml of milliq water ( mechanical shaker , 1 h ) .", "after centrifugation ( 5000 g , 10c , 10 min ) , 10 l of the clear extract was injected into an lc  ms / ms system , acquiring 2 transitions ( m / z 142.174.1 and 142.196.0 ) .", "hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions , against an analytical reference standard ( shypoglycin a2 ) dissolved in milliq water .", "( residuals < 20% ) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg", "/ l , corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts .", "the accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf ( 1 mg / kg ) and seed ( 10 mg / kg ) samples in 5fold dilutions .", "the corresponding repeatabilities ( relative standard deviation , rsd ) were 11% and 7% , respectively .", "the rsd obtained for repetitive analysis ( n = 5 ) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% .", "recoveries obtained for hypoglycin a in spiked seed samples ( 10 mg / kg ) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395%", ". samples with incomplete questionnaire data on relevant facts , such as address and obtainable data about the disease if present , were excluded from further statistical analysis ( 9 samples ) .", "if multiple samples from 1 pasture were submitted , which happened often , the mean result of the analyses per sample type ( sprout , seed or leaf ) were counted as 1 result in further statistical analysis .", "samples that could not be unambiguously assigned to an acer species ( 6 ) , samples that were not fresh ( 17 ) , and samples that contained material other than seeds , sprouts and leaves ( 10 ) also were excluded from the study .", "this approach resulted in 143 usable samples , 30 from the spring and 113 from the autumn of 2014 .", "the samples were grouped by whether they came from pastures grazed by healthy horses or horses with am ( nonam versus am pastures ) . mean concentrations of hypoglycin a were compared for the type of sample ( seed versus sprout versus leaf ) , season ( spring versus autumn ) and disease status for the pasture ( nonam versus am ) .", "statistical analysis was performed using univariate generalized linear models with multiple comparisons , corrected with a posthoc bonferroni test.3 \n  control for normal distribution was performed ."], ["after an information campaign in horse journals and at meetings , 278 samples of maple tree leaves , sprouts , and seeds collected by horse owners in 2014 were sent to utrecht university ( uu ) and rikiltwageningen ur ( figs 1 , 2 , 3 ) . the samples were taken from the ground , packed in plastic , and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection .", "they were classified by an experienced botanist at the species level ( acer pseudoplatanus , acer platanoides , or acer campestre ) on the basis of the samples and accompanying photographs sent by owners .", "sprouts were only used in this study upon positive classification by means of accompanying leaves , seeds or both . the minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves .", "owners were asked to complete a questionnaire with information about date of collection , address of the pasture , species of maple tree ( in their opinion ) , signalment ( name , breed and age ) of the horse(s ) , and presence of disease ( am ) in horses that were grazed in the pastures where the material was collected .", "if the pasture had contained horses with am , details about the disease ( including date of onset and the final outcome ) , laboratory results and name of the veterinarian were provided .", "inclusion criteria to confirm am were recumbency , dark colored urine , high serum activity of creatine kinase ( ck , either too high to measure in practice , or > 100,000 iu / l in a laboratory ) and postmortem examination , which was performed in 4 cases . exclusion criterion for am was lack of ck analysis unless postmortem examination was performed .", "the geographic location of the pastures where the acer seeds , sprouts and leaves were sampled . \n", "a , b , c leaves of acer pseudoplatanus , acer campestre and acer platanoides ."], ["hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry ( lc  ms / ms).1 \n  seed samples ( 50 g , as received ) were ground in a retsch mill , and leaf samples ( 50100 g ) were cryogenically milled into a powder , using liquid nitrogen .", "one gram of homogenized sample material was extracted with 10 ml of milliq water ( mechanical shaker , 1 h ) .", "after centrifugation ( 5000 g , 10c , 10 min ) , 10 l of the clear extract was injected into an lc  ms / ms system , acquiring 2 transitions ( m / z 142.174.1 and 142.196.0 ) .", "hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions , against an analytical reference standard ( shypoglycin a2 ) dissolved in milliq water .", "quantification was performed by multilevel calibration . a consistent linear relationship ( residuals < 20% ) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg / l , corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts .", "the accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf ( 1 mg / kg ) and seed ( 10 mg / kg ) samples in 5fold dilutions .", "the corresponding repeatabilities ( relative standard deviation , rsd ) were 11% and 7% , respectively .", "the rsd obtained for repetitive analysis ( n = 5 ) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% .", "recoveries obtained for hypoglycin a in spiked seed samples ( 10 mg / kg ) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395% ."], ["samples with incomplete questionnaire data on relevant facts , such as address and obtainable data about the disease if present , were excluded from further statistical analysis ( 9 samples ) . if multiple samples from 1 pasture were submitted , which happened often , the mean result of the analyses per sample type ( sprout , seed or leaf ) were counted as 1 result in further statistical analysis .", "samples that could not be unambiguously assigned to an acer species ( 6 ) , samples that were not fresh ( 17 ) , and samples that contained material other than seeds , sprouts and leaves ( 10 ) also were excluded from the study .", "this approach resulted in 143 usable samples , 30 from the spring and 113 from the autumn of 2014 .", "the samples were grouped by whether they came from pastures grazed by healthy horses or horses with am ( nonam versus am pastures ) .", "mean concentrations of hypoglycin a were compared for the type of sample ( seed versus sprout versus leaf ) , season ( spring versus autumn ) and disease status for the pasture ( nonam versus am ) ."], ["statistical analysis was performed using univariate generalized linear models with multiple comparisons , corrected with a posthoc bonferroni test.3 \n  control for normal distribution was performed . a p value of .05 was set as statistically significant ."], ["in total , 143 of the collected samples ( 93 seed samples , 14 sprout samples , and 36 leave samples ) from a total of 81 locations were included ( fig 1 ) . none of the acer platanoides ( n = 11 ) and acer campestre ( n = 13 ) samples ( 29 samples of seeds , 11 of leaves and 1 of sprouts ) contained detectable concentrations of hypoglycin a. in contrast , most acer pseudoplatanus leaves , seeds , and sprouts contained the toxin . in 2 samples of seeds and 6 samples of leaves ,", "no hypoglycin a was found ( ie , concentration below limit of quantification [ 1 mg / kg for leaves and 10 mg / kg for seeds ] ) .", "the factor season was not statistically significant ( p = .841 ) and was excluded from further statistical analysis .", "the mean  sd concentrations of hypoglycin a in the samples are presented in table 1 .", "hypoglycin a concentrations differed significantly among the sample types ( seeds and sprouts , p < .001 ; seeds and leaves , p < .001 ; sprouts and leaves , p = .000 ) .", "concentrations were significantly higher in seeds from the am group than in seeds from the nonam group ( seeds , 856  677 versus 456  358 mg / kg ; p = .039 ) . in sprouts , ( 1365  795 mg /", "kg versus 1097  310 , p = .493 ) and leaves ( 31  49 versus 37  34 mg / kg , p = .819 ) this was not the case and when values for all seeds , sprouts , and leaves were combined the difference between nonam and am was not significant either ( p = .127 ) ; ( table 1 ) .", "the concentration ( mg / kg ) of hypoglycin a in samples of acer pseudoplatanus taken from pastures or paddocks grazed by healthy horses and horses with atypical myopathy ( am ) concentration significantly different between pastures used by healthy and am horses ( p = 0.039 ) .", "there were 16 horses ( providing 29 samples ) with am of which 11 died and 5 survived .", "there was no relationship between the concentration of hypoglycin a in the samples and outcome of the disease ( p = .250 ) ."], [", acer negundo ( box elder tree ) and acer pseudoplatanus ( sycamore maple ) have been associated with am.15 , 17 \n  the species acer campestre ( hedge maple ) and acer platanoides ( norway maple ) did not contain detectable hypoglycin a concentrations , and thus it is probably safe , in terms of am , to grow these species in or around pastures and paddocks . in 1973 , semiquantitative measurements ( notdetected , trace , weak , moderate , and strong ) also indicated that these tree species were free of hypoglycin a.18 \n  the most common maple tree in the netherlands is acer pseudoplatanus .", "we found that the leaves , sprouts , and seeds contained measurable concentrations of hypoglycin a. although hypoglycin a concentrations were significantly lower in seeds collected from pastures grazed by healthy horses than in samples from pastures grazed by am horses , concentrations were too variable to allow predictions to be made as to whether or not the presence of an individual tree would be a threat to horses grazing in its vicinity .", "our results are consistent with a study that found hypoglycin a concentrations in seeds to be highly variable among trees on the same or different farms.17 hypoglycin a concentrations in our study were similar to those previously reported.17 in the previous study , a mean concentration of 690 mg / kg was found with variation between 40 and 2810 mg / kg and in our study a mean of 856 mg / kg with variation between 25 and 3683 mg / kg was found", ". the question arises as to why not all horses grazing pastures containing or surrounded by acer pseudoplatanus develop am .", "the most likely explanation might relate to differences in the amount of seeds , sprouts , and leaves ingested , other components of the diet , and the sensitivity of the individual horse .", "the availability of seeds , leaves , and sprouts largely is determined by the season and weather , with changes in wind and temperature loosening the attachment of leaves and seeds.6 , 10 , 15 , 19 indeed , inclement weather or high winds have been reported to precede clinical signs of am.6 , 12 , 15 strong winds can disperse leaves and seeds over a long distance , perhaps depositing them in pastures that do not contain acer trees .", "all of these factors could explain why acer trees are not always found in the paddock or pastures grazed by horses with am.8 \n  our results suggest that , per kilogram , sprouts are most dangerous to horses , followed by seeds , with leaves being potentially least harmful .", "data show that there are more cases of am in autumn than in spring.8 because sprouts are more common in spring and seeds are more common in autumn , it would appear that horses eat more seeds than sprouts either because more seeds are available or because they prefer seeds to sprouts .", "however , whether horses eat seeds or sprouts is to a large extent determined by the availability of other feedstuff.20 in general , spring pasture contains more and better grass than autumn pasture , and thus horses may have less reason to eat other feedstuff.20 \n  the finding that the concentration of hypoglycin a is high in sprouts suggests that horse owners should be alert to the presence of acer sprouts in pastures .", "owners should be advised to mow areas of pasture with maple sprouts and to remove the mown material .", "a factor that needs further investigation is tree stress.20 , 21 tree stress or abiotic stress may increase the hypoglycin a concentration in seeds .", "several owners who sent in samples did not return the questionnaire or did not complete it fully .", "moreover , if there was doubt ( after consultation with the owner 's veterinarian ) about whether a horse suffered from am or not , data for samples collected from that horse 's paddock or pasture were not used .", "results of the samples of 1 type ( seed , sprout , or leaf ) originating from 1 pasture were pooled and the mean concentration of these was treated as 1 sample .", "because toxin concentrations are highly variable among trees on the same farm , it would have been better to have taken more samples of all materials .", "the seeds were fresh frozen and not desiccated before freezing and changes in toxin concentrations as a result of artificial freezing could have occurred in the spring samples in the same fashion as occurs naturally in autumn . ideally all cases would have been confirmed by analyzing blood or urine for madd or toxin or toxin metabolites . because doing so was not always possible , the described inclusion criteria were used to make the diagnosis . in conclusion , it is important to know the tree species when giving advice regarding the toxicity of maple trees and the potential risk for horses , because some acer species contain hypoglycin a and others do not .", "unfortunately , the amount of hypoglycin a in seeds is so variable that a reliable prediction of the occurrence of am can not be made for individual farms .", "therefore , it is not worthwhile to measure hypoglycin concentrations in individual seed samples in order to predict am risk .", "however , in all cases in which no hypoglycin a was found , no clinical signs of am were observed .", "thus , the most important recommendation is that owners should prevent horses from eating seeds , sprouts , leaves , or any combination of these from acer pseudoplatanus ( sycamore maple ) , whereas the seeds of acer campestre ( hedge maple ) and acer platanoides ( norway maple ) appear to be safe for horses .", "steps to prevent the ingestion of toxin include moving the horse to a safer pasture , decreasing the size of the pasture ( away from the trees ) , blowing away seeds and leaves , or mowing and removing sprouts ."], [""]]}
{"article_id": "PMC1885947", "article_text": ["cell migration requires the rearrangement of adhesions and cytoskeleton in response to extracellular stimuli to form a leading edge that allows directed locomotion . in most cell types ,", "migration - related adhesive structures are composed of integrin heterodimers and intracellular associated proteins clustered in plaques termed focal complexes and their more mature variant , focal adhesions .", "in addition to these adhesive structures , dcs also assemble distinctive adhesions named podosomes that are characteristic of cells of the myeloid lineage including macrophages and osteoclasts [ 4 , 5 ] .", "the molecular composition of podosomes is similar to that of focal complexes and focal adhesions .", "however , they have a unique organization with actin filaments bundled in discrete foci forming a conical core containing distinctive elements such as gelsolin , cortactin , the actin - nucleating factor arp 2/3 complex , and the scaffolding protein wasp   surrounded by a ring of integrins and integrin - associated proteins [ 4 , 5 ] .", "wasp is an essential component of podosomes , where it activates the arp2/3 complex inducing actin polymerization .", "wip has also been detected in the core of podosomes in endothelial cells expressing egfp - wip , and although there is evidence indicating that wip plays a major role in regulating the activity of wasp family proteins and the dynamics of actin filaments [ 8 , 9 ] , it remains unknown how wip participates in the assembly of podosomes .", "to study the role of wip in podosome formation , the distribution of f - actin and vinculin was examined in wild - type and wip dcs by confocal microscopy .", "as expected , podosomes were grouped in clusters in wild - type dcs ( figures 1a and 1c ) .", "those few podosomes formed in wip dcs displayed an abnormal structure having less discrete actin cores and disorganized vinculin rings around f - actin ( figure  1d ) .", "large focal contacts containing vinculin comprised the most striking adhesion structures in wip dcs ( figures 1b and 1e ) , replacing podosomes as the major points of contact with the substratum", ". the critical involvement of wip in dc podosome formation is also supported by the presence of endogenous wip in the core of podosomes in wild - type cells ( figure  1f ) .", "our data suggest that the key role of wip in podosome formation is in the organization of f - actin and the clustering of integrins and associated proteins .", "the absence of wip had a profound effect on the organization of f - actin within dcs . while the bulk of f - actin in wild - type dcs was associated with the core of podosomes ( table 1 ,", "see figure  s1a in the supplemental data available online ) , in wip dcs it distributed among various anomalous structures including actin cables ( table 1 , figure  s1b ) , large fused actin aggregates , commonly with a circular shape ( table 1 , figure  s1c ) , and extensive membrane ruffles ( table 1 , figure  s1d ) .", "clustering of podosomal integrins and the integrin - associated proteins paxillin and talin was also affected . in wild - type dcs , 2-integrins formed rings around the f - actin cores of podosomes ( figure  s2a ) as previously described .", "in contrast , wip dcs failed to cluster 2-integrins into rings , and instead they formed small integrin - containing focal contacts scattered through the basal surface and around the periphery of the cell ( figure  s2b ) or large plaques associated with anomalous actin - rich aggregates ( figure  s2c ) .", "facs analysis showed that wild - type and wip dcs expressed equivalent levels of 1- and 2-integrins ( figure  s3 ) , indicating that wip does not affect integrin expression .", "paxillin and talin , which normally array around the podosome core in wild - type dcs   ( figures s2d and s2 g ) , in wip dcs localized to focal adhesions ( figures s2e and s2h ) containing 1 integrins ( data not shown ) or formed amorphous clusters colocalizing with the anomalous actin aggregates ( figures s2f and s2i ) .", "the shift of adhesion composition and structure from podosomes ( short half - life in the range of 30 s to 5 min ) to large focal contacts ( long half life in the range of 30 to 60 min ) in wip dcs had a negative impact on adhesion turnover ( figure  1 g ) .", "as previously described , interference reflection microscopy ( irm ) analyses showed podosomes in wild - type dcs rapidly assembled and disassembled , giving a high turnover index ( figure  1 g ) . in wip dcs , although multiple transient and unstable lamellar protrusions were developed at the cell periphery , the major adhesion sites in contact with the substratum were highly stable with a significantly lower turnover index compared to wild - type ( figure  1 g ) .", "interestingly , we found a decrease in podosome turnover in wild - type dcs plated on fibronectin ( fn ) and icam-1 , indicating that integrin ligands promote podosome stability ( figure  1 g ) .", "this stability of adhesion on integrin ligands correlates with an increase in the percentage of wild - type dcs with well - defined podosomes and small focal contacts ( figure  1a ) and accumulation of podosomal f - actin , integrins , and associated proteins ( data not shown ) .", "in contrast , no significant differences were detected in turnover index of adhesions between wip dcs plated on different substrata ( figure  1 g ) .", "the presence of integrin ligands failed to induce major changes in the composition of adhesions of wip  dcs ( figure  1b ) except for an increase in the  size of the focal adhesions . taken together , our data indicate that wip plays a key role in regulating dc adhesion structure and dynamics on biologically relevant substrata , processes crucial for the migratory capacity of cells .", "stabilization of the leading edge in the direction of dc movement is provided by podosomes [ 13 , 14 ] .", "migrating wild - type dcs clearly polarized , forming a patent leading edge , with the rear of the cell retracting at a similar rate , allowing net translocation of the cell body .", "in contrast , wip dcs failed to develop a major leading front and instead formed multiple simultaneous and unstable lateral lamellae and ruffles ( figures 2a2e ; movies s1 and s2 ) .", "cortactin is an arp2/3 activator that plays a major role in the formation of podosomes [ 15 , 16 ] , establishment of cell polarization , and also generation of cellular cortical protrusions .", "it has been proposed that cortactin synergises with the wasp family of proteins to induce actin polymerization , and it is thought that the actin filament meshwork triggered by the n - wasp / cortactin pair may have a different organization from actin meshworks triggered by n - wasp or cortactin alone .", "importantly , wip has been shown to interact with cortactin and induce cortactin - mediated actin polymerization .", "our results show that in the absence of wip , cortactin is displaced from podosome cores where it normally colocalizes with f - actin ( figure  2f ) and is instead associated with random lamellar protrusions and ruffles over the cell periphery ( figure  2 g ) .", "in addition , we found there was a higher proportion of cortactin associated with the cytoskeletal fraction ( triton insoluble ) in adherent wip dcs compared to wild - type dcs ( figure  2h ) . taken together ,", "these results show that in the absence of wip , association of cortactin with the cytoskeleton is deregulated and associated with the formation of random cortical protrusions and loss of dc polarity .", "this suggests that the wasp / wip complex may couple with cortactin to induce a type of meshwork of actin filaments and associated proteins that allow the clustering of 2-integrins during the assembly of podosomes and dc polarization . in the absence of wip", ", cortactin can not be recruited to form a complex with wasp , discrete actin foci in podosomes fail to form , and instead , cortactin codistributes with actin filaments at the cell margin , resulting in the random protrusions and reduced motility ( data not shown ) observed in wip dcs .", "we have previously shown that wasp dcs display equivalent anomalies in 2-integrin clustering   and focal contact formation to that observed in wip dcs .", "also we find here that like wasp dcs , wip dcs fail to polarize and form and stabilize consistent leading edges .", "recently , it has been proposed that wip contributes to the stabilization of wasp expression and the regulation of wasp - mediated actin rearrangement [ 8 , 22 ] .", "we find here that wip dcs express low levels of wasp at the protein level ( figure  3a ) , as recently described for wip t - lymphocytes .", "levels of wip expression were not affected by the lack of wasp in wasp dcs ( figure  3b ) . since mrna levels of wasp in wip dcs were comparable to those of wild - type dcs ( figure  3c )", "the levels of expression of the wip binding partners cortactin , nck , and crkl was not affected in wip dcs ( figure  s4 ) .", "it has been predicted that wip may prevent the exposure of wasp family proteins to proteases , because the 25-residue wasp binding motif from wip wraps around the wip binding domain of n - wasp .", "wasp , but not n - wasp , is a substrate for the protease calpain in platelets , and we have recently found that calpain also cleaves wasp in migrating dcs .", "our present results show that wip prevents wasp cleavage by calpain in dcs since inhibition of this protease in wip dcs resulted in major recovery of wasp expression ( figures 3d and 3e ) .", "this suggests that wip may play a crucial role in regulating the accessibility of wasp for calpain cleavage , which takes place during dissolution of podosomes . a role for wip in the maintenance of wasp levels", "is also suggested by recent findings showing that in was patients , mutations that prevent wasp binding to wip result in diminished expression of wasp [ 24 , 25 ] .", "we suggest that the low levels of wasp in these was patients may be the result of extensive calpain - mediated cleavage because of failure of wasp - wip interaction .", "surprisingly , the recovery of wasp levels in wip dcs by treatment with calpain inhibitors was not sufficient to induce podosome formation , and large focal contacts remained the major adhesion structures ( figure  4a ) .", "in addition , forced expression of egfp - wasp in wip dcs treated with calpain inhibitors alone or in conjunction with proteasome inhibitors   failed to restore podosome formation , and egfp - wasp remained diffusely distributed in the cytoplasm or associated with anomalous f - actin aggregates that could contain vinculin forming abnormal plaques colocalizing with f - actin ( figure  4b , top and middle ) .", "this contrasts with the observed recovery of podosome formation and dynamics in wasp dcs ( where wip levels are normal ) after the expression of egfp - wasp ( figure  4b , bottom ) [ 11 , 13 ] .", "we excluded the possibility that wip binding is essential for the intrinsic actin polymerizing activity of wasp because specific wasp mutants ( wasp a134 t and wasp r138p ) that interrupt wip binding [ 24 , 27 ] showed an actin polymerizing activity equivalent to wild - type wasp ( figure  4d ) .", "these results indicate that wip not only protects wasp from calpain cleavage but also facilitates the localization of wasp to sites of actin polymerization and the organization of integrins , vinculin , and other integrin - associated proteins in circular clusters . the mechanisms by which wip regulates wasp activity to allow podosome formation are yet to be fully elucidated .", "it is possible that wasp / wip interactions may directly facilitate the recruitment of wasp to cell - surface receptors .", "alternatively , wip may facilitate the bridging of wasp to these receptors by mediating the interaction with other adaptor proteins   or may regulate the function of regulatory proteins such as kinases , known to affect wasp activity [ 30 , 31 ] .", "our data show that wip by itself is not sufficient to promote podosome assembly because wasp dcs , which express normal levels of wip , also lack podosomes and assemble large focal contacts and disorganized actin clumps instead .", "it is tempting to speculate that the cellular responses ( of dc ) to external soluble ligands such as chemoattractants would recruit wasp / wip complexes coupled to integrin - associated proteins   to trigger the assembly of podosomes at appropriate sites in the developing leading edge of the cell .", "these complexes would provide a cytoskeletal platform that regulates the accurate clustering and activation of 2-integrins , facilitating their function .", "taken together , our results suggest that wasp and wip work as a functional unit in a cellular context as has been previously proposed [ 8 , 9 ] to regulate podosome formation and dynamics during the establishment of cell polarity as seen in the chemotactic response to extracellular stimuli . in summary , we have shown that in dcs wip is essential for podosome formation and cell polarity by preventing the extensive degradation of wasp by calpain and by facilitating the recruitment of wasp to discrete foci to form the core of podosomes .", "the tight coupling between wasp and wip is therefore a key contributor to normal podosome dynamics and likely of major importance to the cell trafficking behavior of dcs ."], "abstract_text": ["<S> summarythe wiskott - aldrich syndrome protein ( wasp ) is an adaptor protein that is essential for podosome formation in hematopoietic cells [ 1 ] . given that 80% of identified wiskott - aldrich syndrome patients result from mutations in the binding site for wasp - interacting - protein ( wip ) [ 2 ] , we examined the possible role of wip in the regulation of podosome architecture and cell motility in dendritic cells ( dcs ) . </S>", "<S> our results show that wip is essential both for the formation of actin cores containing wasp and cortactin and for the organization of integrin and integrin - associated proteins in circular arrays , specific characteristics of podosome structure . </S>", "<S> we also found that wip is essential for the maintenance of the high turnover of adhesions and polarity in dcs . </S>", "<S> wip exerts these functions by regulating calpain - mediated cleavage of wasp and by facilitating the localization of wasp to sites of actin polymerization at podosomes . </S>", "<S> taken together , our results indicate that wip is critical for the regulation of both the stability and localization of wasp in migrating dcs and suggest that wasp and wip operate as a functional unit to control dc motility in response to changes in the extracellular environment . </S>"], "labels": null, "section_names": ["Results and Discussion"], "sections": [["cell migration requires the rearrangement of adhesions and cytoskeleton in response to extracellular stimuli to form a leading edge that allows directed locomotion . in most cell types ,", "migration - related adhesive structures are composed of integrin heterodimers and intracellular associated proteins clustered in plaques termed focal complexes and their more mature variant , focal adhesions .", "in addition to these adhesive structures , dcs also assemble distinctive adhesions named podosomes that are characteristic of cells of the myeloid lineage including macrophages and osteoclasts [ 4 , 5 ] .", "the molecular composition of podosomes is similar to that of focal complexes and focal adhesions .", "however , they have a unique organization with actin filaments bundled in discrete foci forming a conical core containing distinctive elements such as gelsolin , cortactin , the actin - nucleating factor arp 2/3 complex , and the scaffolding protein wasp   surrounded by a ring of integrins and integrin - associated proteins [ 4 , 5 ] .", "wasp is an essential component of podosomes , where it activates the arp2/3 complex inducing actin polymerization .", "wip has also been detected in the core of podosomes in endothelial cells expressing egfp - wip , and although there is evidence indicating that wip plays a major role in regulating the activity of wasp family proteins and the dynamics of actin filaments [ 8 , 9 ] , it remains unknown how wip participates in the assembly of podosomes .", "to study the role of wip in podosome formation , the distribution of f - actin and vinculin was examined in wild - type and wip dcs by confocal microscopy .", "as expected , podosomes were grouped in clusters in wild - type dcs ( figures 1a and 1c ) .", "those few podosomes formed in wip dcs displayed an abnormal structure having less discrete actin cores and disorganized vinculin rings around f - actin ( figure  1d ) .", "large focal contacts containing vinculin comprised the most striking adhesion structures in wip dcs ( figures 1b and 1e ) , replacing podosomes as the major points of contact with the substratum", ". the critical involvement of wip in dc podosome formation is also supported by the presence of endogenous wip in the core of podosomes in wild - type cells ( figure  1f ) .", "our data suggest that the key role of wip in podosome formation is in the organization of f - actin and the clustering of integrins and associated proteins .", "the absence of wip had a profound effect on the organization of f - actin within dcs . while the bulk of f - actin in wild - type dcs was associated with the core of podosomes ( table 1 ,", "see figure  s1a in the supplemental data available online ) , in wip dcs it distributed among various anomalous structures including actin cables ( table 1 , figure  s1b ) , large fused actin aggregates , commonly with a circular shape ( table 1 , figure  s1c ) , and extensive membrane ruffles ( table 1 , figure  s1d ) .", "clustering of podosomal integrins and the integrin - associated proteins paxillin and talin was also affected . in wild - type dcs , 2-integrins formed rings around the f - actin cores of podosomes ( figure  s2a ) as previously described .", "in contrast , wip dcs failed to cluster 2-integrins into rings , and instead they formed small integrin - containing focal contacts scattered through the basal surface and around the periphery of the cell ( figure  s2b ) or large plaques associated with anomalous actin - rich aggregates ( figure  s2c ) .", "facs analysis showed that wild - type and wip dcs expressed equivalent levels of 1- and 2-integrins ( figure  s3 ) , indicating that wip does not affect integrin expression .", "paxillin and talin , which normally array around the podosome core in wild - type dcs   ( figures s2d and s2 g ) , in wip dcs localized to focal adhesions ( figures s2e and s2h ) containing 1 integrins ( data not shown ) or formed amorphous clusters colocalizing with the anomalous actin aggregates ( figures s2f and s2i ) .", "the shift of adhesion composition and structure from podosomes ( short half - life in the range of 30 s to 5 min ) to large focal contacts ( long half life in the range of 30 to 60 min ) in wip dcs had a negative impact on adhesion turnover ( figure  1 g ) .", "as previously described , interference reflection microscopy ( irm ) analyses showed podosomes in wild - type dcs rapidly assembled and disassembled , giving a high turnover index ( figure  1 g ) . in wip dcs , although multiple transient and unstable lamellar protrusions were developed at the cell periphery , the major adhesion sites in contact with the substratum were highly stable with a significantly lower turnover index compared to wild - type ( figure  1 g ) .", "interestingly , we found a decrease in podosome turnover in wild - type dcs plated on fibronectin ( fn ) and icam-1 , indicating that integrin ligands promote podosome stability ( figure  1 g ) .", "this stability of adhesion on integrin ligands correlates with an increase in the percentage of wild - type dcs with well - defined podosomes and small focal contacts ( figure  1a ) and accumulation of podosomal f - actin , integrins , and associated proteins ( data not shown ) .", "in contrast , no significant differences were detected in turnover index of adhesions between wip dcs plated on different substrata ( figure  1 g ) .", "the presence of integrin ligands failed to induce major changes in the composition of adhesions of wip  dcs ( figure  1b ) except for an increase in the  size of the focal adhesions . taken together , our data indicate that wip plays a key role in regulating dc adhesion structure and dynamics on biologically relevant substrata , processes crucial for the migratory capacity of cells .", "stabilization of the leading edge in the direction of dc movement is provided by podosomes [ 13 , 14 ] .", "migrating wild - type dcs clearly polarized , forming a patent leading edge , with the rear of the cell retracting at a similar rate , allowing net translocation of the cell body .", "in contrast , wip dcs failed to develop a major leading front and instead formed multiple simultaneous and unstable lateral lamellae and ruffles ( figures 2a2e ; movies s1 and s2 ) .", "cortactin is an arp2/3 activator that plays a major role in the formation of podosomes [ 15 , 16 ] , establishment of cell polarization , and also generation of cellular cortical protrusions .", "it has been proposed that cortactin synergises with the wasp family of proteins to induce actin polymerization , and it is thought that the actin filament meshwork triggered by the n - wasp / cortactin pair may have a different organization from actin meshworks triggered by n - wasp or cortactin alone .", "importantly , wip has been shown to interact with cortactin and induce cortactin - mediated actin polymerization .", "our results show that in the absence of wip , cortactin is displaced from podosome cores where it normally colocalizes with f - actin ( figure  2f ) and is instead associated with random lamellar protrusions and ruffles over the cell periphery ( figure  2 g ) .", "in addition , we found there was a higher proportion of cortactin associated with the cytoskeletal fraction ( triton insoluble ) in adherent wip dcs compared to wild - type dcs ( figure  2h ) . taken together ,", "these results show that in the absence of wip , association of cortactin with the cytoskeleton is deregulated and associated with the formation of random cortical protrusions and loss of dc polarity .", "this suggests that the wasp / wip complex may couple with cortactin to induce a type of meshwork of actin filaments and associated proteins that allow the clustering of 2-integrins during the assembly of podosomes and dc polarization . in the absence of wip", ", cortactin can not be recruited to form a complex with wasp , discrete actin foci in podosomes fail to form , and instead , cortactin codistributes with actin filaments at the cell margin , resulting in the random protrusions and reduced motility ( data not shown ) observed in wip dcs .", "we have previously shown that wasp dcs display equivalent anomalies in 2-integrin clustering   and focal contact formation to that observed in wip dcs .", "also we find here that like wasp dcs , wip dcs fail to polarize and form and stabilize consistent leading edges .", "recently , it has been proposed that wip contributes to the stabilization of wasp expression and the regulation of wasp - mediated actin rearrangement [ 8 , 22 ] .", "we find here that wip dcs express low levels of wasp at the protein level ( figure  3a ) , as recently described for wip t - lymphocytes .", "levels of wip expression were not affected by the lack of wasp in wasp dcs ( figure  3b ) . since mrna levels of wasp in wip dcs were comparable to those of wild - type dcs ( figure  3c )", "the levels of expression of the wip binding partners cortactin , nck , and crkl was not affected in wip dcs ( figure  s4 ) .", "it has been predicted that wip may prevent the exposure of wasp family proteins to proteases , because the 25-residue wasp binding motif from wip wraps around the wip binding domain of n - wasp .", "wasp , but not n - wasp , is a substrate for the protease calpain in platelets , and we have recently found that calpain also cleaves wasp in migrating dcs .", "our present results show that wip prevents wasp cleavage by calpain in dcs since inhibition of this protease in wip dcs resulted in major recovery of wasp expression ( figures 3d and 3e ) .", "this suggests that wip may play a crucial role in regulating the accessibility of wasp for calpain cleavage , which takes place during dissolution of podosomes . a role for wip in the maintenance of wasp levels", "is also suggested by recent findings showing that in was patients , mutations that prevent wasp binding to wip result in diminished expression of wasp [ 24 , 25 ] .", "we suggest that the low levels of wasp in these was patients may be the result of extensive calpain - mediated cleavage because of failure of wasp - wip interaction .", "surprisingly , the recovery of wasp levels in wip dcs by treatment with calpain inhibitors was not sufficient to induce podosome formation , and large focal contacts remained the major adhesion structures ( figure  4a ) .", "in addition , forced expression of egfp - wasp in wip dcs treated with calpain inhibitors alone or in conjunction with proteasome inhibitors   failed to restore podosome formation , and egfp - wasp remained diffusely distributed in the cytoplasm or associated with anomalous f - actin aggregates that could contain vinculin forming abnormal plaques colocalizing with f - actin ( figure  4b , top and middle ) .", "this contrasts with the observed recovery of podosome formation and dynamics in wasp dcs ( where wip levels are normal ) after the expression of egfp - wasp ( figure  4b , bottom ) [ 11 , 13 ] .", "we excluded the possibility that wip binding is essential for the intrinsic actin polymerizing activity of wasp because specific wasp mutants ( wasp a134 t and wasp r138p ) that interrupt wip binding [ 24 , 27 ] showed an actin polymerizing activity equivalent to wild - type wasp ( figure  4d ) .", "these results indicate that wip not only protects wasp from calpain cleavage but also facilitates the localization of wasp to sites of actin polymerization and the organization of integrins , vinculin , and other integrin - associated proteins in circular clusters . the mechanisms by which wip regulates wasp activity to allow podosome formation are yet to be fully elucidated .", "it is possible that wasp / wip interactions may directly facilitate the recruitment of wasp to cell - surface receptors .", "alternatively , wip may facilitate the bridging of wasp to these receptors by mediating the interaction with other adaptor proteins   or may regulate the function of regulatory proteins such as kinases , known to affect wasp activity [ 30 , 31 ] .", "our data show that wip by itself is not sufficient to promote podosome assembly because wasp dcs , which express normal levels of wip , also lack podosomes and assemble large focal contacts and disorganized actin clumps instead .", "it is tempting to speculate that the cellular responses ( of dc ) to external soluble ligands such as chemoattractants would recruit wasp / wip complexes coupled to integrin - associated proteins   to trigger the assembly of podosomes at appropriate sites in the developing leading edge of the cell .", "these complexes would provide a cytoskeletal platform that regulates the accurate clustering and activation of 2-integrins , facilitating their function .", "taken together , our results suggest that wasp and wip work as a functional unit in a cellular context as has been previously proposed [ 8 , 9 ] to regulate podosome formation and dynamics during the establishment of cell polarity as seen in the chemotactic response to extracellular stimuli . in summary , we have shown that in dcs wip is essential for podosome formation and cell polarity by preventing the extensive degradation of wasp by calpain and by facilitating the recruitment of wasp to discrete foci to form the core of podosomes .", "the tight coupling between wasp and wip is therefore a key contributor to normal podosome dynamics and likely of major importance to the cell trafficking behavior of dcs ."]]}
{"article_id": "PMC3015912", "article_text": ["ventriculoperitoneal ( vp ) shunting is commonly utilized as a diversion procedure for patients with hydrocephalus .", "multiple techniques for initial placement of the catheters have been described , including conventional minilaparotomy , peel - away introducer sheaths , as well as full laparoscopic approaches .", "vp shunts fail for a variety of reasons , including failure secondary to disconnection of abdominal tubing with migration of the tubing , most commonly further into the peritoneal cavity ; this occurs in 2.5% to 4% of shunts placed according to current published series .", "initial shunt failure rates continue to be high , with a reported failure rate of 31% at 6 months and an infection rate of 7% .", "common presenting features of shunt failure include headache , mental status changes , and vomiting .", "laparoscopy avoids laparotomy and may provide improved confirmation of shunt functionality and placement at the time of revision .", "a 31-year - old obese female was initially treated for pseudotumor cerebri with placement of a lumbar peritoneal shunt in 2003 .", "after 5 failed revisions for nonfunction , a ventriculoperitoneal shunt was placed in september 2007 .", "the patient subsequently presented 11 days postoperatively with headaches , dizziness , and formation of a seroma in her right upper quadrant .", "ct scan revealed the vp catheter tubing coiled in the subcutaneous tissue of the right upper quadrant .", "four 5-mm titanium clips were placed on the catheter in an attempt to prevent repeated migration of the tubing .", "one month later , the patient presented with recurrence of headaches , nausea , and right upper quadrant abdominal pain .", "ct scan was again obtained , revealing that the shunt tubing had migrated back into the subcutaneous tissue ( figure 1 ) .", "a laparoscopic approach was again utilitzed for retrieval and attempted improved fixation of the vp shunt .", "adhesions were taken down at the original entry site of the shunt , revealing a 2-cm fascial defect in the abdominal wall with the shunt tubing coiled within it ( figure 2 ) .", "the tubing was brought down through the defect out of the subcutaneous space and into the peritoneal cavity ( figure 3 ) .", "one port was upsized to 10 mm , and the tubing was externalized through the port and secured to a 3-cm x 10-cm piece of ptfe mesh with 30 prolene sutures ( figure 4 ) .", "csf flow was confirmed , and the tubing attached to the mesh was reintroduced into the peritoneal cavity .", "the mesh was secured to the abdominal wall by using spiral tacks and 0 gore - tex sutures ( figure 5 ) .", "the separate 2-cm fascial defect was repaired with a 10-cm x 10-cm piece of ptfe mesh placed as an onlay with the vp shunt exiting from its inferior border .", "it was secured with spiral tacks and 0 gore - tex sutures for transabdominal fixation ( figure 6 ) .", "the patient was seen in follow - up 7 months postprocedure and reported no recurrence of her symptoms .", "traditionally , laparotomy was necessary for both peritoneal shunt placement as well as subsequent shunt revisions .", "the advent of laparoscopy has afforded a minimally invasive approach that has resulted in decreased patient morbidity related to shunt placement .", "retrieval and repositioning of a displaced vp shunt in the peritoneal cavity using laparoscopic techniques has been previously described . in the present case ,", "our subsequent laparoscopic approach utilized ptfe mesh , well described in the use of laparoscopic ventral hernia repair , to fixate the tubing to the abdominal wall to prevent further tubing migration .", "this approach also allowed us to simultaneously repair the fascial defect associated with the shunt entry site into the peritoneal cavity , allowing us to reap the benefits of decreased recurrence and decreased patient morbidity associated with this technique .", "the technique described was well tolerated without complication and resulted in improvement of clinical symptoms ."], "abstract_text": ["<S> a 31-year - old female with a history of pseudotumor cerebri presented with headache and abdominal discomfort after placement of a ventriculoperitoneal ( vp ) shunt . </S>", "<S> the vp shunt was placed after prior failure and revision of a lumbar peritoneal shunt . </S>", "<S> computed tomography demonstrated shunt migration into the subcutaneous tissue . </S>", "<S> laparoscopy was used to reposition the vp shunt , directing the shunt toward the pelvis . </S>", "<S> the patient presented for further evaluation one month later , at which point the shunt was shown to have migrated into the subcutaneous tissue once again . </S>", "<S> laparoscopy was again used to reposition the shunt and affix it to the abdominal wall by using polytetrafluoroethylene ( ptfe ) mesh . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["ventriculoperitoneal ( vp ) shunting is commonly utilized as a diversion procedure for patients with hydrocephalus .", "multiple techniques for initial placement of the catheters have been described , including conventional minilaparotomy , peel - away introducer sheaths , as well as full laparoscopic approaches .", "vp shunts fail for a variety of reasons , including failure secondary to disconnection of abdominal tubing with migration of the tubing , most commonly further into the peritoneal cavity ; this occurs in 2.5% to 4% of shunts placed according to current published series .", "initial shunt failure rates continue to be high , with a reported failure rate of 31% at 6 months and an infection rate of 7% .", "common presenting features of shunt failure include headache , mental status changes , and vomiting .", "laparoscopy avoids laparotomy and may provide improved confirmation of shunt functionality and placement at the time of revision ."], ["a 31-year - old obese female was initially treated for pseudotumor cerebri with placement of a lumbar peritoneal shunt in 2003 .", "after 5 failed revisions for nonfunction , a ventriculoperitoneal shunt was placed in september 2007 .", "the patient subsequently presented 11 days postoperatively with headaches , dizziness , and formation of a seroma in her right upper quadrant .", "ct scan revealed the vp catheter tubing coiled in the subcutaneous tissue of the right upper quadrant .", "four 5-mm titanium clips were placed on the catheter in an attempt to prevent repeated migration of the tubing .", "one month later , the patient presented with recurrence of headaches , nausea , and right upper quadrant abdominal pain .", "ct scan was again obtained , revealing that the shunt tubing had migrated back into the subcutaneous tissue ( figure 1 ) .", "a laparoscopic approach was again utilitzed for retrieval and attempted improved fixation of the vp shunt .", "adhesions were taken down at the original entry site of the shunt , revealing a 2-cm fascial defect in the abdominal wall with the shunt tubing coiled within it ( figure 2 ) .", "the tubing was brought down through the defect out of the subcutaneous space and into the peritoneal cavity ( figure 3 ) .", "one port was upsized to 10 mm , and the tubing was externalized through the port and secured to a 3-cm x 10-cm piece of ptfe mesh with 30 prolene sutures ( figure 4 ) .", "csf flow was confirmed , and the tubing attached to the mesh was reintroduced into the peritoneal cavity .", "the mesh was secured to the abdominal wall by using spiral tacks and 0 gore - tex sutures ( figure 5 ) .", "the separate 2-cm fascial defect was repaired with a 10-cm x 10-cm piece of ptfe mesh placed as an onlay with the vp shunt exiting from its inferior border .", "it was secured with spiral tacks and 0 gore - tex sutures for transabdominal fixation ( figure 6 ) .", "the patient was seen in follow - up 7 months postprocedure and reported no recurrence of her symptoms ."], ["traditionally , laparotomy was necessary for both peritoneal shunt placement as well as subsequent shunt revisions .", "the advent of laparoscopy has afforded a minimally invasive approach that has resulted in decreased patient morbidity related to shunt placement .", "retrieval and repositioning of a displaced vp shunt in the peritoneal cavity using laparoscopic techniques has been previously described . in the present case ,", "our subsequent laparoscopic approach utilized ptfe mesh , well described in the use of laparoscopic ventral hernia repair , to fixate the tubing to the abdominal wall to prevent further tubing migration .", "this approach also allowed us to simultaneously repair the fascial defect associated with the shunt entry site into the peritoneal cavity , allowing us to reap the benefits of decreased recurrence and decreased patient morbidity associated with this technique .", "the technique described was well tolerated without complication and resulted in improvement of clinical symptoms ."]]}
{"article_id": "PMC3546243", "article_text": ["ventilator - associated pneumonia ( vap ) is defined as pneumonia occurring more than 48 hours after patients have been intubated and received mechanical ventilation .", "vap is the most common nosocomial infection in the intensive care unit ( icu ) with an incidence ranging from 8% to 28% in intubated mechanically ventilated patients .", "ventilator - associated pneumonia complicates the course of patients receiving mechanical ventilation inspite of major advances in techniques for its diagnosis and treatment . in the absence of a gold standard , vap", "is assumed to be diagnosed more accurately by bronchoscopic sampling and microbiological cultures of the lower respiratory tract .", "bronchoscopy , being invasive , is not uncommonly associated with complications , especially in patients on high respiratory supports .", "this has paved the way for less invasive tests such as endotracheal aspirates ( eta ) and quantitative eta cultures with a threshold of 10 to 10 bacteria per milliliter of exudates that is considered as optimal for the microbiological confirmation of vap.[46 ] the american thoracic society ( ats ) guidelines recommend that quantitative cultures can be performed on eta or samples collected either bronchoscopically or nonbronchosopically .", "more importantly , recent small trials have repeatedly shown that there is no advantage of bronchoscopic cultures over quantitative eta cultures when mortality was considered as the end - point further strengthening the case for quantitative eta as a diagnostic tool.[811 ] detection of causative organisms and their antibiotic susceptibility is crucial for diagnosis of vap in order to initiate the appropriate antibiotic treatment thereby reducing the adverse effects of inadequate antibiotic treatment on the patient prognosis .", "hence , the present study is undertaken to isolate , identify and quantitate bacteria and to perform the antibiotic susceptibility testing from the endotracheal aspirates of the clinically suspected patients of vap .", "the prospective study was carried out during the period from january 2010 to december 2010 in department of microbiology and medical intensive care unit ( micu ) on 54 patients with clinical suspicion of ventilator - associated pneumonia .", "the approval of the institutional review board was obtained during the planning phase of the study and each patient ( or his / her caregivers ) gave informed consent prior to participation in the study .", "all the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study .", "the diagnosis of vap was established using clinical pulmonary infection score ( cpis ) , which was evaluated on a daily basis until the patient was on ventilator support .", "clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts .", "endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .", "the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .", "chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions .", "only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .", "the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .", "samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min", ". 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .", "the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .", "all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .", "growth of any organism below the threshold was assumed to be due to colonization or contamination .", "organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .", "escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .", "isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test .", "as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .", "isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method .", "all the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study .", "the diagnosis of vap was established using clinical pulmonary infection score ( cpis ) , which was evaluated on a daily basis until the patient was on ventilator support .", "clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts .", "endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .", "the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .", "chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions . only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .", "the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .", "samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min", ". 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .", "the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .", "all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .", "growth of any organism below the threshold was assumed to be due to colonization or contamination .", "organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .", "escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .", "isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .", "isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method .", "endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .", "the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .", "chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions .", "only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .", "the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .", "samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min", ". 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .", "the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .", "all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .", "growth of any organism below the threshold was assumed to be due to colonization or contamination .", "organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .", "escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .", "isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .", "isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method .", "this prospective study was done in the period from january 2010 to december 2010 in our hospital icu .", "the meansd age of patients was 46.418.45 years ( range 18 to 86 years ) , having a predominance of male population .", "bimodal distribution of age was observed with the first peak between 18 - 28 years of age group and second peak at around 59 years of age(28.89% ) .", "distribution of total number of cases was done depending on the duration of mechanical ventilation into  early - onset vap  developing within 4 days of intubation and  late - onset vap  developing after 4 days of intubation .", "the percentage of patients with early onset vap was 39.62% and late onset vap was 60.38% .", "in our study , acinetobacter baumannii caused predominantly bilateral and right sided pneumonia whereas pseudomonas aeruginosa caused more of bilateral bronchopneumonia [ table 1 ] .", "chest x - ray finding in relation to the organism isolated of 53 patients diagnosed as vap based on a cpis score of more than six , 51 ( 96.23 % ) patients had monomicrobial infection and 2 ( 3.77% ) had polymicrobial infection . among the 27 isolates of acinetobacter baumannii , 1 ( 3.70% ) was resistant to all group of antibiotics tested in the study , including carbapenems .", "other 25 ( 92.59% ) isolates of acinetobacter baumannii were resistant to cotrimoxazole , ciprofloxacin and amikacin , 24 ( 88.89% ) to imipenem , 23 ( 85.18% ) to ceftazidime , 11 ( 40.74% ) to doxycycline , 10 ( 37.04% ) to piperacillin - tazobactam .", "all the 27 ( 100% ) isolates of acinetobacter baumannii were multidrug resistant ( mdr ) i.e. resistant to three or more class of antibiotics .", "pseudomonas aeruginosa was resistant to gentamicin ( 100% ) , aztreonam ( 88.23% ) , ciprofloxacin and amikacin ( 82.35% ) , imipenem ( 47.06% ) , ceftazidime ( 35.29% ) and piperacillin - tazobactam ( 23.53% ) .", "figure 1 shows that late - onset vap was more common than early - onset vap . among the early onset", "vap common organisms isolated were acinetobacter baumannii , pseudomonas aeruginosa , klebsiella pneumoniae , citrobacter freundii and staphylococcus aureus while in case of late onset vap common organisms were acinetobacter baumannii , pseudomonas aeruginosa and methicillin - resistant staphylococcus aureus .", "organisms causing vap ( early and late onset vap ) eight ( 47.06% ) of the 17 pseudomonas aeruginosa isolates were mbl producers . among 27 isolates of acinetobacter baumannii ,", "12 ( 44.44% ) were ampc producers while 14 ( 51.85% ) were mbl producing strains .", "the co - existence of ampc and mbl was reported in 9 ( 33.33% ) isolates of acinetobacter baumannii and 2 ( 11.76% ) isolates of pseudomonas aeruginosa [ figure 2 ] .", "different enzymes produced by the isolated strains in the present study it was found that the mortality rate was 45.28%.rate of mortality was less in early - onset vap ( 23.80% ) as compared to late onset vap group ( 59.37% ) and the difference was found to be statistically significant ( x1=6.473 ; df=1 ; p=0.011 ) .", "out of 53 cases of ventilator - associated pneumonia , 16 ( 30.19% ) were female and 37 ( 69.81% ) were male .", "aetiological agents widely differ according to the population of the patients in the intensive care unit , duration of hospital stay and prior antimicrobial therapy .", "the increase of acinetobacter baumannii ( 49.09% ) infections is due to its great resistance to the environment which enables it to spread , its limited virulence and its extraordinary ability to develop resistance to all the antimicrobials and spread by aerosols .", "one of them was methicillin - resistant staphylococcus aureus ( mrsa ) , but mrsa is generally associated with late onset vap .", "this may be due the fact that this case showed mixed infection with predominant organism being citrobacter freundii ( 1.8  10 cfu / ml ) while mrsa was showing 2.1  10 cfu / ml in quantitative cultures .", "secondly , the patient had history of prior antibiotic therapy which increases risk of mrsa .", "found x - ray pattern in acinetobacter baumannii vap is non - specific . in half of the cases it causes lung infiltration and a diffuse bilateral pattern", "some enzymes of pseudomonas aeruginosa have invasive properties , causing thrombosis of pulmonary vessels and pulmonary infarction ; this produces nodular bilateral lesions , predominantly in inferior lobes , and it is such a characteristic image that it should make the presence of pseudomonas aeruginosa suspected .", "the antibiotic resistance pattern of nonfermenters was almost the same in both early- and late - onset vap .", "many of the early - onset vap cases had the risk factors such as prior antibiotic therapy and current hospitalization for five days or more for infection with mdr pathogens .", "that could be the reason for almost similar susceptibility pattern of the isolates from early- and late - onset vap .", "even the american thoracic society guidelines support the same reasoning by suggesting that patients with early - onset vap who have received prior antibiotics or who had the prior hospitalization within the past 90 days are at greater risk for colonization and infection with mdr pathogens and should be treated similarly to patients with late - onset vap .", "ampc beta - lactamases hydrolyze cephamycins and are not inhibited by commercially available beta - lactamase inhibitors .", "bacteria , mostly klebsiella pneumoniae and e. coli , producing plasmid - mediated ampc beta - lactamases have been responsible for nosocomial outbreaks of infection and colonization .", "twelve ( 44.44% ) out of 27 isolates of acinetobacter baumannii have shown production of ampc beta lactamase enzyme , and no other enterobacteriaceae showed production of ampc beta lactamase .", "presently , there is concern about the acquisition of plasmid - mediated metallo - beta lactamases active against carbapenems , penicillins and cephalosporins . in this study", ", 14 ( 51.85% ) isolates of acinetobacter baumannii and 8 ( 47.06% ) isolates of pseudomonas aeruginosa were plasmid - mediated metallo - beta lactamases enzyme producing strains detected by imipenem - edta combined disc method which corresponds to a study were metallo - beta lactamases were produced by 21.74% of acinetobacter spp . and 50% of pseudomonas aeruginosa .", "early - onset vap in our study was found to be 39.62% while in various study it was found to be around 40% .", "the low incidence of early onset vap in our study may be due to antibiotic use before admission to the icu .", "studies have shown that previous antibiotic use decreases early - onset vap but markedly increases multidrug - resistant ( mdr ) pathogens , which is also reflected in our study . in the present study , it was found that the mortality rate was 45.28% which was in accordance with previous studies such as those by gupta a et al .", "the bacteriological approach for the management of vap avoids the problem of overtreatment by separating colonizers from infecting pathogens .", "this study showed that quantitative culture of eta is a useful test for early diagnosis of vap .", "the antibiotic susceptibility pattern of these isolates will also help the clinicians to choose the appropriate antimicrobial agents for prophylactic as well as treatment purposes ."], "abstract_text": ["<S> background : ventilator - associated pneumonia ( vap ) is a common complication of ventilatory support for patients with acute respiratory failure and is associated with increased morbidity and mortality.aim of the study : the present study was undertaken to do quantitative cultures of aerobic bacteria , perform the antibiotic susceptibility testing from the endotracheal aspirates and clinical outcome of the clinically suspected patients of vap.methods:a prospective study was performed over a period of one year in a tertiary care hospital , enrolling patients on mechanical ventilation ( mv ) for 48 hr . endotracheal aspirates ( eta ) were collected from patients with suspected vap , and direct gram 's stain criteria was used to accept the sample . </S>", "<S> quantitative cultures of eta were performed with the threshold for microbiological diagnosis of vap was taken as 105 colony forming units ( cfu)/ml.results : out of 53 cases , 2 ( 3.77% ) were polymicrobial . </S>", "<S> multidrug resistant bacteria , mainly acinetobacter baumannii 49.09% ( 27/55 ) and pseudomonas aeruginosa 30.91% ( 17/55 ) were the most common pathogens isolated . </S>", "<S> metallo - beta lactamases ( mbls ) was produced by 47.06% ( 8/17 ) of pseudomonas aeruginosa and 62.96% ( 17/27 ) of acinetobacter baumannii.conclusion:the bacteriological approach for the management of vap helps the clinicians in choosing the appropriate antibiotics . </S>", "<S> this study showed that quantitative cultures of endotracheal aspirate at a cutoff point of 105 cfu / ml is one of the alternative to bronchoscopy in the diagnosis of clinically suspected ventilator associated pneumonia . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Inclusion criteria", "Exclusion criteria", "Collection of endotracheal aspirates", "Microbiological processing", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["ventilator - associated pneumonia ( vap ) is defined as pneumonia occurring more than 48 hours after patients have been intubated and received mechanical ventilation .", "vap is the most common nosocomial infection in the intensive care unit ( icu ) with an incidence ranging from 8% to 28% in intubated mechanically ventilated patients .", "ventilator - associated pneumonia complicates the course of patients receiving mechanical ventilation inspite of major advances in techniques for its diagnosis and treatment . in the absence of a gold standard , vap", "is assumed to be diagnosed more accurately by bronchoscopic sampling and microbiological cultures of the lower respiratory tract .", "bronchoscopy , being invasive , is not uncommonly associated with complications , especially in patients on high respiratory supports .", "this has paved the way for less invasive tests such as endotracheal aspirates ( eta ) and quantitative eta cultures with a threshold of 10 to 10 bacteria per milliliter of exudates that is considered as optimal for the microbiological confirmation of vap.[46 ] the american thoracic society ( ats ) guidelines recommend that quantitative cultures can be performed on eta or samples collected either bronchoscopically or nonbronchosopically .", "more importantly , recent small trials have repeatedly shown that there is no advantage of bronchoscopic cultures over quantitative eta cultures when mortality was considered as the end - point further strengthening the case for quantitative eta as a diagnostic tool.[811 ] detection of causative organisms and their antibiotic susceptibility is crucial for diagnosis of vap in order to initiate the appropriate antibiotic treatment thereby reducing the adverse effects of inadequate antibiotic treatment on the patient prognosis .", "hence , the present study is undertaken to isolate , identify and quantitate bacteria and to perform the antibiotic susceptibility testing from the endotracheal aspirates of the clinically suspected patients of vap ."], ["the prospective study was carried out during the period from january 2010 to december 2010 in department of microbiology and medical intensive care unit ( micu ) on 54 patients with clinical suspicion of ventilator - associated pneumonia .", "the approval of the institutional review board was obtained during the planning phase of the study and each patient ( or his / her caregivers ) gave informed consent prior to participation in the study .", "all the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study .", "the diagnosis of vap was established using clinical pulmonary infection score ( cpis ) , which was evaluated on a daily basis until the patient was on ventilator support .", "clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts .", "endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .", "the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .", "chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions .", "only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .", "the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .", "samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min", ". 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .", "the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .", "all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .", "growth of any organism below the threshold was assumed to be due to colonization or contamination .", "organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .", "escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .", "isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test .", "as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .", "isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method ."], ["all the patients 18 years and above who were under mechanical ventilation for more than 48 hours and clinically suspected of having contracted vap were included in this study .", "the diagnosis of vap was established using clinical pulmonary infection score ( cpis ) , which was evaluated on a daily basis until the patient was on ventilator support .", "clinically diagnosed ventilator - associated pneumonia were observed and clinical parameters were recorded from their medical records and bedside charts ."], ["endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .", "the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .", "chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions . only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing .", "the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .", "samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min", ". 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .", "the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .", "all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .", "growth of any organism below the threshold was assumed to be due to colonization or contamination .", "organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .", "escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .", "isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .", "isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method ."], ["endotracheal aspirate ( 1 ml ) was collected under aseptic precaution after 48 hours of intubation whenever patient was suspected to have developed vap in micu .", "the eta was collected using a 22-inch ramson 's 12 f suction catheter with a mucus extractor , which was gently introduced through the endotracheal tube for a distance of approximately 25- 26 cm .", "chest vibration or percussion for 10 min was used to increase the retrieved volume ( 1 ml ) in case the patient produced very little secretions .", "only 1 eta sample was collected from each patient and was immediately taken to the laboratory for prossessing ."], ["the aspirate specimens showing presence of < 10 squamous epithelial cells per low power field or organisms seen under oil immersion in the entire field on gram stain were included in the study .", "samples were homogenized by vortexing for 1 min and centrifuged at 3000 rpm for 10 min", ". 1 ml of sample was diluted in 10 ml of 0.9% sterile saline solution with final log dilutions of 10 , 10 , 10 , 10 , 10 , 10 .", "the samples were then plated on sheep blood agar ( sba ) , chocolate agar ( ca ) , and macconkey agar ( ma ) by using 4 mm nichrome wire loop ( hi - media , mumbai , india ) , which holds 0.01 ml of solution .", "all plates were incubated overnight at 37c and chocolate agar plates at 37c in 5% co2 incubator .", "growth of any organism below the threshold was assumed to be due to colonization or contamination .", "organisms were identified and the antimicrobial susceptibility tests of the following drugs were determined by the kirby - bauer disc diffusion method : erythromycin ( e ) ( 15 g ) , clindamycin ( cd ) ( 10 g ) , cotrimoxazole ( co ) ( 25 g ) , cephalexin ( cp ) ( 5 g ) , linezolid ( lz ) ( 30 g ) , doxycycline ( do ) ( 30 g ) , ciprofloxacin ( cip ) ( 5g ) , ceftazidime ( caz ) ( 30 g ) , amoxyclav ( amc ) ( 30 g ) , vancomycin ( va ) ( 30 g ) , amikacin ( ak ) ( 30 g ) , imipenem ( i ) ( 10 g ) , cefotaxime ( ce ) ( 30 g ) , piperacillin - tazobactam ( pt ) ( 100 g/10 g ) ( hi - media laboratories , mumbai ) .", "escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 27853 were used as quality control strains .", "isolates showing reduced susceptibility to either ceftazidime ( 30 g ) or cefotaxime ( 30 g ) discs were considered asscreen positive for ampc beta lactamases and selected for detection of plasmid - mediated ampc by the ampc disc test . as per clsi 2011 guidelines , when using the new interpretive criteria , routine esbl testing is no longer necessary before reporting results ( ie , it is no longer necessary to edit results for cephalosporins , aztreonam , or penicillins to resistant ) .", "isolates showing reduced susceptibility to imipenem were selected for detection of metallo - beta lactamases ( mbl ) enzymes by imipenem - edta combined disc method ."], ["this prospective study was done in the period from january 2010 to december 2010 in our hospital icu .", "the meansd age of patients was 46.418.45 years ( range 18 to 86 years ) , having a predominance of male population .", "bimodal distribution of age was observed with the first peak between 18 - 28 years of age group and second peak at around 59 years of age(28.89% ) .", "distribution of total number of cases was done depending on the duration of mechanical ventilation into  early - onset vap  developing within 4 days of intubation and  late - onset vap  developing after 4 days of intubation .", "the percentage of patients with early onset vap was 39.62% and late onset vap was 60.38% .", "in our study , acinetobacter baumannii caused predominantly bilateral and right sided pneumonia whereas pseudomonas aeruginosa caused more of bilateral bronchopneumonia [ table 1 ] .", "chest x - ray finding in relation to the organism isolated of 53 patients diagnosed as vap based on a cpis score of more than six , 51 ( 96.23 % ) patients had monomicrobial infection and 2 ( 3.77% ) had polymicrobial infection . among the 27 isolates of acinetobacter baumannii , 1 ( 3.70% ) was resistant to all group of antibiotics tested in the study , including carbapenems .", "other 25 ( 92.59% ) isolates of acinetobacter baumannii were resistant to cotrimoxazole , ciprofloxacin and amikacin , 24 ( 88.89% ) to imipenem , 23 ( 85.18% ) to ceftazidime , 11 ( 40.74% ) to doxycycline , 10 ( 37.04% ) to piperacillin - tazobactam .", "all the 27 ( 100% ) isolates of acinetobacter baumannii were multidrug resistant ( mdr ) i.e. resistant to three or more class of antibiotics .", "pseudomonas aeruginosa was resistant to gentamicin ( 100% ) , aztreonam ( 88.23% ) , ciprofloxacin and amikacin ( 82.35% ) , imipenem ( 47.06% ) , ceftazidime ( 35.29% ) and piperacillin - tazobactam ( 23.53% ) .", "figure 1 shows that late - onset vap was more common than early - onset vap . among the early onset", "vap common organisms isolated were acinetobacter baumannii , pseudomonas aeruginosa , klebsiella pneumoniae , citrobacter freundii and staphylococcus aureus while in case of late onset vap common organisms were acinetobacter baumannii , pseudomonas aeruginosa and methicillin - resistant staphylococcus aureus .", "organisms causing vap ( early and late onset vap ) eight ( 47.06% ) of the 17 pseudomonas aeruginosa isolates were mbl producers . among 27 isolates of acinetobacter baumannii ,", "12 ( 44.44% ) were ampc producers while 14 ( 51.85% ) were mbl producing strains .", "the co - existence of ampc and mbl was reported in 9 ( 33.33% ) isolates of acinetobacter baumannii and 2 ( 11.76% ) isolates of pseudomonas aeruginosa [ figure 2 ] .", "different enzymes produced by the isolated strains in the present study it was found that the mortality rate was 45.28%.rate of mortality was less in early - onset vap ( 23.80% ) as compared to late onset vap group ( 59.37% ) and the difference was found to be statistically significant ( x1=6.473 ; df=1 ; p=0.011 ) ."], ["out of 53 cases of ventilator - associated pneumonia , 16 ( 30.19% ) were female and 37 ( 69.81% ) were male .", "aetiological agents widely differ according to the population of the patients in the intensive care unit , duration of hospital stay and prior antimicrobial therapy .", "the increase of acinetobacter baumannii ( 49.09% ) infections is due to its great resistance to the environment which enables it to spread , its limited virulence and its extraordinary ability to develop resistance to all the antimicrobials and spread by aerosols .", "one of them was methicillin - resistant staphylococcus aureus ( mrsa ) , but mrsa is generally associated with late onset vap .", "this may be due the fact that this case showed mixed infection with predominant organism being citrobacter freundii ( 1.8  10 cfu / ml ) while mrsa was showing 2.1  10 cfu / ml in quantitative cultures .", "secondly , the patient had history of prior antibiotic therapy which increases risk of mrsa .", "found x - ray pattern in acinetobacter baumannii vap is non - specific . in half of the cases it causes lung infiltration and a diffuse bilateral pattern", "some enzymes of pseudomonas aeruginosa have invasive properties , causing thrombosis of pulmonary vessels and pulmonary infarction ; this produces nodular bilateral lesions , predominantly in inferior lobes , and it is such a characteristic image that it should make the presence of pseudomonas aeruginosa suspected .", "the antibiotic resistance pattern of nonfermenters was almost the same in both early- and late - onset vap .", "many of the early - onset vap cases had the risk factors such as prior antibiotic therapy and current hospitalization for five days or more for infection with mdr pathogens .", "that could be the reason for almost similar susceptibility pattern of the isolates from early- and late - onset vap .", "even the american thoracic society guidelines support the same reasoning by suggesting that patients with early - onset vap who have received prior antibiotics or who had the prior hospitalization within the past 90 days are at greater risk for colonization and infection with mdr pathogens and should be treated similarly to patients with late - onset vap .", "ampc beta - lactamases hydrolyze cephamycins and are not inhibited by commercially available beta - lactamase inhibitors .", "bacteria , mostly klebsiella pneumoniae and e. coli , producing plasmid - mediated ampc beta - lactamases have been responsible for nosocomial outbreaks of infection and colonization .", "twelve ( 44.44% ) out of 27 isolates of acinetobacter baumannii have shown production of ampc beta lactamase enzyme , and no other enterobacteriaceae showed production of ampc beta lactamase .", "presently , there is concern about the acquisition of plasmid - mediated metallo - beta lactamases active against carbapenems , penicillins and cephalosporins . in this study", ", 14 ( 51.85% ) isolates of acinetobacter baumannii and 8 ( 47.06% ) isolates of pseudomonas aeruginosa were plasmid - mediated metallo - beta lactamases enzyme producing strains detected by imipenem - edta combined disc method which corresponds to a study were metallo - beta lactamases were produced by 21.74% of acinetobacter spp . and 50% of pseudomonas aeruginosa .", "early - onset vap in our study was found to be 39.62% while in various study it was found to be around 40% .", "the low incidence of early onset vap in our study may be due to antibiotic use before admission to the icu .", "studies have shown that previous antibiotic use decreases early - onset vap but markedly increases multidrug - resistant ( mdr ) pathogens , which is also reflected in our study . in the present study , it was found that the mortality rate was 45.28% which was in accordance with previous studies such as those by gupta a et al ."], ["the bacteriological approach for the management of vap avoids the problem of overtreatment by separating colonizers from infecting pathogens .", "this study showed that quantitative culture of eta is a useful test for early diagnosis of vap .", "the antibiotic susceptibility pattern of these isolates will also help the clinicians to choose the appropriate antimicrobial agents for prophylactic as well as treatment purposes ."]]}
{"article_id": "PMC3977090", "article_text": ["alpha1-adrenergic receptors ( 1-ars ) are a heterogeneous family of g - protein - coupled receptors that present in most human and animal tissues , and there are considerable variations in the expression levels of 1-ar subtypes in various tissues of different species [ 14 ] .", "1-ars play important roles in regulating physiological and pathological responses mediated by catecholamines , particularly in the cardiovascular and urinary systems .", "in addition to mediating catecholamine - induced vasoconstriction , the 1-ars in vascular wall have been shown to promote proliferation and hypertrophy of arterial smooth muscle cells and adventitial fibroblasts [ 5 , 6 ] . in the urinary system ,", "the demonstration of 1-ar expression in human prostate , bladder muscle , and smooth muscles has led to the treatment of bladder outlet obstruction and ureteral stones , via blocking these receptors [ 7 , 8 ] .", "of the three 1-ar subtypes ( 1a , 1b , and 1d ) , the 1d - ar has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [ 911 ] .", "however , recent experiments performed in 1d - ar knockout models suggest that this 1-ar subtype plays an important role in the overall regulation of blood pressure", ". additionally , armenia et al . reported that 1a- and 1d - ars are the major functional subtypes of renal 1-ars in both normal and streptozotocin - induced diabetic sprague - dawley rats . using in situ hybridization , kurooka et al . identified the gene expression of all three 1-ar subtypes in human kidney cortex", "they found that intense 1-ar mrna staining was apparent especially in the smooth muscle of arterial walls , whereas weak staining of each of the 1-ar mrnas was observed in the glomeruli and renal tubules .", "more recently , the presence and distribution of subtypes in human renal pelvis and calyces were evaluated , and 1d - ar was most dense in both followed by 1a and 1b .", "however , the expression and distribution patterns of 1d - ar in normal and diabetic rat kidneys remain unknown . in the present study , we examined mrna levels of three 1-ar subtypes in the kidney cortex of normal zucker lean ( zl ) and zucker diabetic ( zd ) rats by microarray and real - time pcr analyses .", "confocal immunofluorescence microscopy was performed to evaluate the distribution of 1d - ar in the kidney of zucker rats .", "furthermore , we investigated whether activation of peroxisome proliferator - activated receptor- ( ppar ) or ppar , known renoprotective intervention in animal models of type 2 diabetes , would affect renal expression of 1-ar subtypes .", "six - week - old male zucker lean ( zl ) and zucker diabetic fatty ( zd ) rats were purchased from charles river laboratories ( wilmington , ma , usa ) .", "rats were housed in a temperature - controlled room with a 12  :  12-hour light - dark cycle and free access to purina 5008 rat chow and water .", "blood glucose was monitored using the accu - chek glucometer by tail - vein blood sampling . in a previous study in which we characterized the time course of blood glucose in this model", ", we showed that blood glucose of the zd rats began to increase at week 8 , reached a peak at week 12 , and remained at this higher level thereafter .", "the rats were housed in the animal care facility at the morehouse school of medicine that is aaalac accredited .", "all animal protocols were approved by the institutional animal care and use committee and were in accordance with the requirements stated in the national institutes of health guide for the care and use laboratory animals . for fenofibrate treatment , 12-week - old zucker rats were divided into 3 experimental groups : vehicle- ( 0.5% carboxymethylcellulose ig ) treated zl , vehicle - treated zd , or fenofibrate- ( 150 ", "mg / kg / day ig ) treated zd ( f - zd ) rats for 10 weeks . for rosiglitazone treatment ,", "12-week - old zd animals were treated with rosiglitazone ( 10  mg / kg / day in drinking water ) or vehicles for 8 weeks .", "glomeruli were isolated from the kidney cortex of zl and zd rats at the age of 7 , 12 , and 2022 weeks , respectively , by a modified procedure as described previously .", "briefly , the rats were anesthetized and the kidneys were rapidly removed and placed in hanks ' balanced salt solution ( hbss ) at ph  7.4 . the renal cortex was dissected and cut into small pieces with a surgical blade .", "glomeruli were isolated by passing the tissues successively through calibrated sieves ( pore size : 200 , 125 , and 65  m ) and rinsed with hbss .", "isolated glomeruli , collected on the 65  m sieve , were resuspended in hbss .", "total rna was prepared from isolated glomeruli or kidney cortex by using ultrapure trizol reagent according to the manufacturer 's instructions ( gibco - brl , grand island , ny ) .", "the quality of the rna samples was assessed using the agilent 2100 bioanalyzer ( g2938a ) .", "seven hundred and fifty nanograms of total rna per sample were used for crna synthesis and amplification .", "cyanine-3-(cy3- ) labeled crna was purified and hybridized to agilent whole rat genome 44k oligo microarray chips ( p / n g2519f-14870 , agilent technologies ) according to the manufacturer 's instructions .", "the processed microarrays were scanned with the agilent g2565ba dna microarray scanner ( p / n g2505-a ) .", "the scanned images were analyzed with agilent feature software ( version 9.5.1.1 ) using default parameters .", "the resulting text files were loaded into the agilent genespring gx software ( version 7.3 ) for further analysis .", "significantly differentially expressed -adrenergic receptor genes among zl , zd , and f - zd groups were identified by a threshold of 2 fold change and p  0.05 .", "reverse transcription was performed on equal amounts of total rna by using random hexanucleotide primers to produce a cdna library for each sample .", "real - time pcr reactions were run on an icycler iq real - time pcr detection system by using taqman universal pcr master mix ( applied biosystems , p / n 4304437 ) .", "1a- , 1b- , and 1d - ar and -actin gene - specific taqman probe and primer sets were obtained from applied biosystems as assays - on - demand ( aod ) gene expression products .", "the aod identification numbers were rn00567876 for 1a - ar , rn01471343 for 1b - ar , rn00577931 for 1d - ar , and 4331182 for rat -actin", "each sample was run in triplicate , and the comparative threshold cycle ( ct ) method was used to quantify fold increase ( 2 ) compared with controls . for immunofluorescent staining , 5  m thick cryostat sections of oct - embedded kidney samples were used . to study the localization of 1d - ar in the rat kidney", ", the sections were incubated with a mixture of two antibodies overnight : rabbit anti-1d - ar antibody ( 1  :  100 , sigma - aldrich , st .", "louis , mo ) , mouse anti--smooth muscle actin ( -sma , 1  :  100 , santa cruz biotechnology , dallas , tx ) , or goat anti - kidney injury molecule-1 ( kim-1 , 1  :  100 , r&d systems , minneapolis , mn ) . as a negative control ,", "the sections were exposed to nonimmune igg ( in replacement of primary antibodies ) with the same secondary antibodies , and no specific staining occurred .", "comparisons among multiple groups were performed by one - way anova and newman - keuls post hoc test .", "we performed microarray analysis to assess gene expression levels of -adrenergic receptor subtypes in the kidney cortex of 22-week - old normal zl ( blood glucose : 108  8  mg / dl ) and diabetic zd ( blood glucose : 425  35  mg / dl ) rats .", "the expression profile of each experimental group was determined in three animals per group .  ", "table 1 shows gene expression levels of 1- and 2-adrenergic receptor subtypes detected in rat kidney tissue .", "the rank order of expression levels of the three 1-ar mrnas in normal rat kidney cortex was 1b > 1d > 1a . among these genes", "compared to normal zl controls , renal cortical 1d - ar gene was increased by 553% in 22-week - old zd rats ( table 1 ) . to verify the relative transcript levels of 1a- , 1b- , and 1d - ar subtypes derived from the microarray experiment , we performed quantitative real - time pcr ( qpcr ) assay .", "the relative expression levels of 1a- , 1b- , and 1d - ar mrnas normalized against -actin in rat kidney cortex are shown in figure 1 . in consistency with the microarray results , qpcr analysis showed that renal cortical 1d mrna was increased by 16-fold in 22-week - old zd rats compared to zl controls .", "we have previously shown that ppar activation protects against kidney injury in zucker diabetic fatty rats [ 15 , 17 ] . here , we further examined the effect of ppar activation on the expression level of 1-ar mrnas in the diabetic kidneys .", "gene microarray analysis revealed that ppar activation inhibits the upregulation of 1d gene in the diabetic kidneys .", "chronic administration of fenofibrate , a ppar agonist , resulted in a decrease in 1d - ar mrna by 64% compared to vehicle - treated zd animals ( table 1 ) .", "qpcr analysis confirmed a reduction of 1d - ar mrna in the kidney of f - zd rats ( figure 1 ) .", "in contrast , both microarray and qpcr analyses indicated that mrna expression of 1a- and 1b - ar subtypes was not affected by fenofibrate in the diabetic kidneys .", "additionally , the effect of ppar activation on gene expression of 1-ar subtypes was examined in the zd rats after rosiglitazone treatment .", "compared to vehicle - treated zd rats , renal cortical 1d - ar mrna was significantly lower when rosiglitazone was administered for 8 weeks ( figure 2 ) .", "similar to ppar activation , 1a- and 1b - ar mrna expression in the diabetic rats was not affected by rosiglitazone treatment . in a previous study", ", we showed that blood glucose levels were not different between normal zl and diabetic zd rats at the age of 7 weeks .", "blood glucose of the zd rats began to increase at week 8 , reached a peak at week 12 , and remained at this higher level thereafter . to analyze the temporal pattern of renal expression of 1d - ar receptors , renal cortical and glomerular 1d - ar mrna levels", "were compared in the zucker rats at the ages of 7 , 12 , and 2022 weeks . as shown in figure 3 , zd rats at week 7", "had slightly lower 1d - ar mrna level in the glomeruli compared to their zl littermates , whereas renal cortical 1d - ar mrnas were not different between the two groups . at the age of 12 , both renal", "glomerular ( 2.4-fold ) and cortical ( 1.7-fold ) 1d - ar mrna levels were significantly higher in the zd animals , which were further increased by 3.4-fold and 12.9-fold , respectively , at the age of 2022 weeks .", "we performed immunofluorescence staining to correlate 1d - ar gene expression results with its protein level and distribution in the kidney of zucker animals .", "as expected , 1d - ar protein was clearly detected in the renal arteries and arterioles in both normal and diabetic animals ( figure 4 ) .", "the intense staining was primarily in the smooth muscle of renal arterial walls as evidenced by its colocalization with -sma . in normal zl rats ,", "weak 1d - ar staining was also detected in the glomeruli , whereas there was no obvious staining in the renal tubules ( figure 4 ) . in consistency with the mrna expression results ,", "immunofluorescence staining identified increased 1d - ar signal in the glomeruli of diabetic rats , which was partially colocalized with -sma ( figure 4 ) .", "intense tubulointerstitial staining of 1d - ar was apparent in the diabetic kidneys . as shown in figure 5 ,", "1d - ar was expressed in both tubular epithelial cells and activated interstitial fibroblasts , which was positive for -sma staining . to further characterize 1d - ar expression in renal tubules of diabetic animals", ", we performed double staining for 1d - ar and kim-1 , a sensitive tubular injury marker .", "dual labeling revealed a spatial relationship between kim-1 and 1d - ar in the diabetic kidneys .", "as shown in figure 6 , virtually all dilated tubules expressing 1d - ar were also kim-1-positive , suggesting that 1d - ar was expressed in the injured dedifferentiated proximal tubules . in these tubules , 1d - ar expression was predominantly cytoplasmic , whereas kim-1 staining was prominent at the apical membrane .", "in this study , the expression and distribution of 1d - ar mrna and protein were determined by the gene microarray , qpcr , and confocal immunofluorescence analyses .", "although mrna expression of all three 1-ar subtypes ( 1b > 1d > 1a ) was detected in rat kidney cortex , only 1d - ar gene was massively upregulated in the diabetic animals .", "moreover , diabetes - related increase in 1d - ar mrna was inhibited when the zd rats were treated with fenofibrate or rosiglitazone .", "immunostaining for 1d - ar confirmed that intense 1d - ar staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys .", "weak 1d - ar protein staining was detected in the glomeruli of normal zl controls , but there was no obvious staining in the normal tubular epithelium . in consistency with the gene expression results ,", "1d - ar protein was significantly increased in the glomeruli and proximal tubules of diabetic animals .", "the expression of 1-ar subtype mrnas has previously been studied in various animal and human organs , and the predominant subtype mrna expressed differs among species and organs .", "for example , kurooka et al . reported that 1a - ar gene was detected more than 1b - ar or 1d - ar in human kidney cortex .", "in contrast , karabacak et al .   recently evaluated 1-ar subtype protein expression in human renal pelvis and calyx tissues and found that 1d - ar was most dense in both followed by 1a- and 1b - ar subtypes , respectively , where the rate of 1b - ar was significantly lower than the other two . in the rat kidney", ", it was reported that the 1b - ar is predominant when detected by a radioligand binding assay   and rnase protection assay . in consistency with these findings", ", we confirmed the expression of all three 1-ar subtype mrnas ( 1b > 1d > 1a ) in rat kidney cortex by microarray and qpcr analyses .", "of the three 1-ar subtypes , the 1d - ar has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [ 911 ] .", "however , recent experiments performed in 1d - ar knockout models suggest that this 1-ar subtype plays an important role in the overall regulation of blood pressure .", "moreover , armenia et al . reported that 1a- and 1d - ars are the major functional subtypes of renal 1-ars in both normal and streptozotocin - induced sprague - dawley rats .", "interestingly , among the three 1-ar subtypes detected in rat kidney cortex , we found that 1d - ar mrna was markedly increased by 16-fold in the diabetic kidneys .", "additionally , diabetes - associated upregulation of 1d - ar mrna expression was inhibited when the diabetic animals were treated with ppar agonists , known renoprotective interventions .", "therefore , previous evidences and present results strongly suggest that 1d - ar may play an important role in renal physiology and/or pathophysiology .", "diabetic nephropathy is characterized by a series of ultrastructural changes , including glomerular and tubular hypertrophy , mesangial expansion , glomerulosclerosis , and tubulointerstitial fibrosis .", "we have previously demonstrated a progressive loss of renal function in the diabetic animals as evidenced by an increase in urinary protein to creatinine ratio in the zd rats between the ages of 7 and 20 weeks . in the current study", ", we further observed a gradual increase in cortical and glomerular 1d - ar mrna expression during disease progression in the diabetic animals .", "we speculate that increased 1d - ar may play a role in the development of diabetic renal hypertrophy and fibrosis .", "in fact , a role for 1d - ar in vascular hypertrophy and remodeling has been suggested by a recent report that a decrease in the lumen area and an increase in the wall thickness of arteries in hypoxic pulmonary hypertension were strongly inhibited in 1d - ar knockout mice . in this study , we evaluated the expression and distribution of 1d - ar protein in the kidneys of zl and zd rats by immunofluorescence staining .", "as expected , intense 1d - ar staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys .", "compared to low expression level of 1d - ar protein in the glomeruli of normal zl controls , zd rats demonstrate a significant increase in 1d - ar signal . moreover , zd kidneys displayed strong tubulointerstitial 1d - ar signal within fibrotic areas .", "a colocalization of 1d - ar with -sma indicates that 1d - ar expressing interstitial cells are myofibroblasts .", "future work in the field could be necessary to establish the exact role of 1d - ar in activation and proliferation of renal interstitial fibroblasts .", "another interesting novelfinding in our set of data reported here is the 1d - ar expression in tubular epithelial cells of diabetic kidneys .", "although there was no obvious staining in the normal tubules , 1d - ar staining was apparent in the dilated tubules in the fibrotic areas . to further characterize 1d - ar expression in renal tubules of diabetic animals , the spatial relationship between tubular 1d - ar and kim-1 , a tubular injury marker ,", "the selective increased expression by dedifferentiated epithelial cells and activated interstitial fibroblasts supports the potential importance of 1d - ar in renal tubulointerstitial injury .", "moreover , recent studies on the pulmonary circulation suggest that catecholamines may participate in the excessive muscularization and fibrosis of the arteries through erk1/2 signaling pathway in hypoxic pulmonary hypertension [ 5 , 2022 ] .", "therefore , further studies are warranted to evaluate the functional consequences of 1d - ar induction in diabetic kidney injury . in summary ,", "the current study highlights mrna expression of the three 1-ar subtypes in rat kidney cortex .", "an increase in renal expression of 1d - ar mrna and protein was associated with glomerular and tubulointerstitial injury in diabetic nephropathy .", "chronic treatment with ppar agonists prevents the increase in 1d - ar mrna in the diabetic kidneys .", "these findings may provide new insights into the prevention and early management of diabetic kidney disease ."], "abstract_text": ["<S> alpha1d - adrenergic receptor ( 1d - ar ) plays important roles in regulating physiological and pathological responses mediated by catecholamines , particularly in the cardiovascular and urinary systems . </S>", "<S> the present study was designed to investigate the expression profile of 1d - ar in the diabetic kidneys and its modulation by activation of peroxisome proliferator - activated receptors ( ppars ) . </S>", "<S> 12-week - old zucker lean ( zl ) and zucker diabetic fatty ( zd ) rats were treated with fenofibrate or rosiglitazone for 810 weeks . </S>", "<S> gene microarray , real - time pcr , and confocal immunofluorescence microscopy were performed to assess mrna and protein expression of 1d - ar in rat kidney tissue . </S>", "<S> using microarray , we found that 1d - ar gene was dramatically upregulated in 22-week - old zd rats compared to zl controls . </S>", "<S> quantitative pcr analysis verified a 16-fold increase in 1d - ar mrna in renal cortex from zd animals compared to normal controls . </S>", "<S> chronic treatment with fenofibrate or rosiglitazone reduced renal cortical 1d - ar gene . </S>", "<S> immunofluorescence staining confirmed that 1d - ar protein was induced in the glomeruli and tubules of diabetic rats . </S>", "<S> moreover , dual immunostaining for 1d - ar and kidney injury molecule-1 indicated that 1d - ar was expressed in dedifferentiated proximal tubules of diabetic zucker rats . </S>", "<S> taken together , our results show that 1d - ar expression is upregulated in the diabetic kidneys . </S>", "<S> ppar activation suppressed renal expression of 1d - ar in diabetic nephropathy . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["alpha1-adrenergic receptors ( 1-ars ) are a heterogeneous family of g - protein - coupled receptors that present in most human and animal tissues , and there are considerable variations in the expression levels of 1-ar subtypes in various tissues of different species [ 14 ] .", "1-ars play important roles in regulating physiological and pathological responses mediated by catecholamines , particularly in the cardiovascular and urinary systems .", "in addition to mediating catecholamine - induced vasoconstriction , the 1-ars in vascular wall have been shown to promote proliferation and hypertrophy of arterial smooth muscle cells and adventitial fibroblasts [ 5 , 6 ] . in the urinary system ,", "the demonstration of 1-ar expression in human prostate , bladder muscle , and smooth muscles has led to the treatment of bladder outlet obstruction and ureteral stones , via blocking these receptors [ 7 , 8 ] .", "of the three 1-ar subtypes ( 1a , 1b , and 1d ) , the 1d - ar has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [ 911 ] .", "however , recent experiments performed in 1d - ar knockout models suggest that this 1-ar subtype plays an important role in the overall regulation of blood pressure", ". additionally , armenia et al . reported that 1a- and 1d - ars are the major functional subtypes of renal 1-ars in both normal and streptozotocin - induced diabetic sprague - dawley rats . using in situ hybridization , kurooka et al . identified the gene expression of all three 1-ar subtypes in human kidney cortex", "they found that intense 1-ar mrna staining was apparent especially in the smooth muscle of arterial walls , whereas weak staining of each of the 1-ar mrnas was observed in the glomeruli and renal tubules .", "more recently , the presence and distribution of subtypes in human renal pelvis and calyces were evaluated , and 1d - ar was most dense in both followed by 1a and 1b .", "however , the expression and distribution patterns of 1d - ar in normal and diabetic rat kidneys remain unknown . in the present study , we examined mrna levels of three 1-ar subtypes in the kidney cortex of normal zucker lean ( zl ) and zucker diabetic ( zd ) rats by microarray and real - time pcr analyses .", "confocal immunofluorescence microscopy was performed to evaluate the distribution of 1d - ar in the kidney of zucker rats .", "furthermore , we investigated whether activation of peroxisome proliferator - activated receptor- ( ppar ) or ppar , known renoprotective intervention in animal models of type 2 diabetes , would affect renal expression of 1-ar subtypes ."], ["six - week - old male zucker lean ( zl ) and zucker diabetic fatty ( zd ) rats were purchased from charles river laboratories ( wilmington , ma , usa ) .", "rats were housed in a temperature - controlled room with a 12  :  12-hour light - dark cycle and free access to purina 5008 rat chow and water .", "blood glucose was monitored using the accu - chek glucometer by tail - vein blood sampling . in a previous study in which we characterized the time course of blood glucose in this model", ", we showed that blood glucose of the zd rats began to increase at week 8 , reached a peak at week 12 , and remained at this higher level thereafter .", "the rats were housed in the animal care facility at the morehouse school of medicine that is aaalac accredited .", "all animal protocols were approved by the institutional animal care and use committee and were in accordance with the requirements stated in the national institutes of health guide for the care and use laboratory animals . for fenofibrate treatment , 12-week - old zucker rats were divided into 3 experimental groups : vehicle- ( 0.5% carboxymethylcellulose ig ) treated zl , vehicle - treated zd , or fenofibrate- ( 150 ", "mg / kg / day ig ) treated zd ( f - zd ) rats for 10 weeks . for rosiglitazone treatment ,", "12-week - old zd animals were treated with rosiglitazone ( 10  mg / kg / day in drinking water ) or vehicles for 8 weeks .", "glomeruli were isolated from the kidney cortex of zl and zd rats at the age of 7 , 12 , and 2022 weeks , respectively , by a modified procedure as described previously .", "briefly , the rats were anesthetized and the kidneys were rapidly removed and placed in hanks ' balanced salt solution ( hbss ) at ph  7.4 . the renal cortex was dissected and cut into small pieces with a surgical blade .", "glomeruli were isolated by passing the tissues successively through calibrated sieves ( pore size : 200 , 125 , and 65  m ) and rinsed with hbss .", "isolated glomeruli , collected on the 65  m sieve , were resuspended in hbss .", "total rna was prepared from isolated glomeruli or kidney cortex by using ultrapure trizol reagent according to the manufacturer 's instructions ( gibco - brl , grand island , ny ) .", "the quality of the rna samples was assessed using the agilent 2100 bioanalyzer ( g2938a ) .", "seven hundred and fifty nanograms of total rna per sample were used for crna synthesis and amplification .", "cyanine-3-(cy3- ) labeled crna was purified and hybridized to agilent whole rat genome 44k oligo microarray chips ( p / n g2519f-14870 , agilent technologies ) according to the manufacturer 's instructions .", "the processed microarrays were scanned with the agilent g2565ba dna microarray scanner ( p / n g2505-a ) .", "the scanned images were analyzed with agilent feature software ( version 9.5.1.1 ) using default parameters .", "the resulting text files were loaded into the agilent genespring gx software ( version 7.3 ) for further analysis .", "significantly differentially expressed -adrenergic receptor genes among zl , zd , and f - zd groups were identified by a threshold of 2 fold change and p  0.05 .", "reverse transcription was performed on equal amounts of total rna by using random hexanucleotide primers to produce a cdna library for each sample .", "real - time pcr reactions were run on an icycler iq real - time pcr detection system by using taqman universal pcr master mix ( applied biosystems , p / n 4304437 ) .", "1a- , 1b- , and 1d - ar and -actin gene - specific taqman probe and primer sets were obtained from applied biosystems as assays - on - demand ( aod ) gene expression products .", "the aod identification numbers were rn00567876 for 1a - ar , rn01471343 for 1b - ar , rn00577931 for 1d - ar , and 4331182 for rat -actin", "each sample was run in triplicate , and the comparative threshold cycle ( ct ) method was used to quantify fold increase ( 2 ) compared with controls . for immunofluorescent staining , 5  m thick cryostat sections of oct - embedded kidney samples were used . to study the localization of 1d - ar in the rat kidney", ", the sections were incubated with a mixture of two antibodies overnight : rabbit anti-1d - ar antibody ( 1  :  100 , sigma - aldrich , st .", "louis , mo ) , mouse anti--smooth muscle actin ( -sma , 1  :  100 , santa cruz biotechnology , dallas , tx ) , or goat anti - kidney injury molecule-1 ( kim-1 , 1  :  100 , r&d systems , minneapolis , mn ) . as a negative control ,", "the sections were exposed to nonimmune igg ( in replacement of primary antibodies ) with the same secondary antibodies , and no specific staining occurred .", "comparisons among multiple groups were performed by one - way anova and newman - keuls post hoc test ."], ["we performed microarray analysis to assess gene expression levels of -adrenergic receptor subtypes in the kidney cortex of 22-week - old normal zl ( blood glucose : 108  8  mg / dl ) and diabetic zd ( blood glucose : 425  35  mg / dl ) rats .", "the expression profile of each experimental group was determined in three animals per group .  ", "table 1 shows gene expression levels of 1- and 2-adrenergic receptor subtypes detected in rat kidney tissue .", "the rank order of expression levels of the three 1-ar mrnas in normal rat kidney cortex was 1b > 1d > 1a . among these genes", "compared to normal zl controls , renal cortical 1d - ar gene was increased by 553% in 22-week - old zd rats ( table 1 ) . to verify the relative transcript levels of 1a- , 1b- , and 1d - ar subtypes derived from the microarray experiment , we performed quantitative real - time pcr ( qpcr ) assay .", "the relative expression levels of 1a- , 1b- , and 1d - ar mrnas normalized against -actin in rat kidney cortex are shown in figure 1 . in consistency with the microarray results , qpcr analysis showed that renal cortical 1d mrna was increased by 16-fold in 22-week - old zd rats compared to zl controls .", "we have previously shown that ppar activation protects against kidney injury in zucker diabetic fatty rats [ 15 , 17 ] . here , we further examined the effect of ppar activation on the expression level of 1-ar mrnas in the diabetic kidneys .", "gene microarray analysis revealed that ppar activation inhibits the upregulation of 1d gene in the diabetic kidneys .", "chronic administration of fenofibrate , a ppar agonist , resulted in a decrease in 1d - ar mrna by 64% compared to vehicle - treated zd animals ( table 1 ) .", "qpcr analysis confirmed a reduction of 1d - ar mrna in the kidney of f - zd rats ( figure 1 ) .", "in contrast , both microarray and qpcr analyses indicated that mrna expression of 1a- and 1b - ar subtypes was not affected by fenofibrate in the diabetic kidneys .", "additionally , the effect of ppar activation on gene expression of 1-ar subtypes was examined in the zd rats after rosiglitazone treatment .", "compared to vehicle - treated zd rats , renal cortical 1d - ar mrna was significantly lower when rosiglitazone was administered for 8 weeks ( figure 2 ) .", "similar to ppar activation , 1a- and 1b - ar mrna expression in the diabetic rats was not affected by rosiglitazone treatment . in a previous study", ", we showed that blood glucose levels were not different between normal zl and diabetic zd rats at the age of 7 weeks .", "blood glucose of the zd rats began to increase at week 8 , reached a peak at week 12 , and remained at this higher level thereafter . to analyze the temporal pattern of renal expression of 1d - ar receptors , renal cortical and glomerular 1d - ar mrna levels", "were compared in the zucker rats at the ages of 7 , 12 , and 2022 weeks . as shown in figure 3 , zd rats at week 7", "had slightly lower 1d - ar mrna level in the glomeruli compared to their zl littermates , whereas renal cortical 1d - ar mrnas were not different between the two groups . at the age of 12 , both renal", "glomerular ( 2.4-fold ) and cortical ( 1.7-fold ) 1d - ar mrna levels were significantly higher in the zd animals , which were further increased by 3.4-fold and 12.9-fold , respectively , at the age of 2022 weeks .", "we performed immunofluorescence staining to correlate 1d - ar gene expression results with its protein level and distribution in the kidney of zucker animals .", "as expected , 1d - ar protein was clearly detected in the renal arteries and arterioles in both normal and diabetic animals ( figure 4 ) .", "the intense staining was primarily in the smooth muscle of renal arterial walls as evidenced by its colocalization with -sma . in normal zl rats ,", "weak 1d - ar staining was also detected in the glomeruli , whereas there was no obvious staining in the renal tubules ( figure 4 ) . in consistency with the mrna expression results ,", "immunofluorescence staining identified increased 1d - ar signal in the glomeruli of diabetic rats , which was partially colocalized with -sma ( figure 4 ) .", "intense tubulointerstitial staining of 1d - ar was apparent in the diabetic kidneys . as shown in figure 5 ,", "1d - ar was expressed in both tubular epithelial cells and activated interstitial fibroblasts , which was positive for -sma staining . to further characterize 1d - ar expression in renal tubules of diabetic animals", ", we performed double staining for 1d - ar and kim-1 , a sensitive tubular injury marker .", "dual labeling revealed a spatial relationship between kim-1 and 1d - ar in the diabetic kidneys .", "as shown in figure 6 , virtually all dilated tubules expressing 1d - ar were also kim-1-positive , suggesting that 1d - ar was expressed in the injured dedifferentiated proximal tubules . in these tubules , 1d - ar expression was predominantly cytoplasmic , whereas kim-1 staining was prominent at the apical membrane ."], ["in this study , the expression and distribution of 1d - ar mrna and protein were determined by the gene microarray , qpcr , and confocal immunofluorescence analyses .", "although mrna expression of all three 1-ar subtypes ( 1b > 1d > 1a ) was detected in rat kidney cortex , only 1d - ar gene was massively upregulated in the diabetic animals .", "moreover , diabetes - related increase in 1d - ar mrna was inhibited when the zd rats were treated with fenofibrate or rosiglitazone .", "immunostaining for 1d - ar confirmed that intense 1d - ar staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys .", "weak 1d - ar protein staining was detected in the glomeruli of normal zl controls , but there was no obvious staining in the normal tubular epithelium . in consistency with the gene expression results ,", "1d - ar protein was significantly increased in the glomeruli and proximal tubules of diabetic animals .", "the expression of 1-ar subtype mrnas has previously been studied in various animal and human organs , and the predominant subtype mrna expressed differs among species and organs .", "for example , kurooka et al . reported that 1a - ar gene was detected more than 1b - ar or 1d - ar in human kidney cortex .", "in contrast , karabacak et al .   recently evaluated 1-ar subtype protein expression in human renal pelvis and calyx tissues and found that 1d - ar was most dense in both followed by 1a- and 1b - ar subtypes , respectively , where the rate of 1b - ar was significantly lower than the other two . in the rat kidney", ", it was reported that the 1b - ar is predominant when detected by a radioligand binding assay   and rnase protection assay . in consistency with these findings", ", we confirmed the expression of all three 1-ar subtype mrnas ( 1b > 1d > 1a ) in rat kidney cortex by microarray and qpcr analyses .", "of the three 1-ar subtypes , the 1d - ar has been the least studied due to difficulties in obtaining significant expression levels and poor coupling to membrane signals due to its intracellular localization [ 911 ] .", "however , recent experiments performed in 1d - ar knockout models suggest that this 1-ar subtype plays an important role in the overall regulation of blood pressure .", "moreover , armenia et al . reported that 1a- and 1d - ars are the major functional subtypes of renal 1-ars in both normal and streptozotocin - induced sprague - dawley rats .", "interestingly , among the three 1-ar subtypes detected in rat kidney cortex , we found that 1d - ar mrna was markedly increased by 16-fold in the diabetic kidneys .", "additionally , diabetes - associated upregulation of 1d - ar mrna expression was inhibited when the diabetic animals were treated with ppar agonists , known renoprotective interventions .", "therefore , previous evidences and present results strongly suggest that 1d - ar may play an important role in renal physiology and/or pathophysiology .", "diabetic nephropathy is characterized by a series of ultrastructural changes , including glomerular and tubular hypertrophy , mesangial expansion , glomerulosclerosis , and tubulointerstitial fibrosis .", "we have previously demonstrated a progressive loss of renal function in the diabetic animals as evidenced by an increase in urinary protein to creatinine ratio in the zd rats between the ages of 7 and 20 weeks . in the current study", ", we further observed a gradual increase in cortical and glomerular 1d - ar mrna expression during disease progression in the diabetic animals .", "we speculate that increased 1d - ar may play a role in the development of diabetic renal hypertrophy and fibrosis .", "in fact , a role for 1d - ar in vascular hypertrophy and remodeling has been suggested by a recent report that a decrease in the lumen area and an increase in the wall thickness of arteries in hypoxic pulmonary hypertension were strongly inhibited in 1d - ar knockout mice . in this study , we evaluated the expression and distribution of 1d - ar protein in the kidneys of zl and zd rats by immunofluorescence staining .", "as expected , intense 1d - ar staining was apparent especially in the smooth muscle of arterial walls in both normal and diabetic kidneys .", "compared to low expression level of 1d - ar protein in the glomeruli of normal zl controls , zd rats demonstrate a significant increase in 1d - ar signal . moreover , zd kidneys displayed strong tubulointerstitial 1d - ar signal within fibrotic areas .", "a colocalization of 1d - ar with -sma indicates that 1d - ar expressing interstitial cells are myofibroblasts .", "future work in the field could be necessary to establish the exact role of 1d - ar in activation and proliferation of renal interstitial fibroblasts .", "another interesting novelfinding in our set of data reported here is the 1d - ar expression in tubular epithelial cells of diabetic kidneys .", "although there was no obvious staining in the normal tubules , 1d - ar staining was apparent in the dilated tubules in the fibrotic areas . to further characterize 1d - ar expression in renal tubules of diabetic animals , the spatial relationship between tubular 1d - ar and kim-1 , a tubular injury marker ,", "the selective increased expression by dedifferentiated epithelial cells and activated interstitial fibroblasts supports the potential importance of 1d - ar in renal tubulointerstitial injury .", "moreover , recent studies on the pulmonary circulation suggest that catecholamines may participate in the excessive muscularization and fibrosis of the arteries through erk1/2 signaling pathway in hypoxic pulmonary hypertension [ 5 , 2022 ] .", "therefore , further studies are warranted to evaluate the functional consequences of 1d - ar induction in diabetic kidney injury . in summary ,", "the current study highlights mrna expression of the three 1-ar subtypes in rat kidney cortex .", "an increase in renal expression of 1d - ar mrna and protein was associated with glomerular and tubulointerstitial injury in diabetic nephropathy .", "chronic treatment with ppar agonists prevents the increase in 1d - ar mrna in the diabetic kidneys .", "these findings may provide new insights into the prevention and early management of diabetic kidney disease ."]]}
{"article_id": "PMC4621941", "article_text": ["while the occurrence of dental caries has decreased during the last decades , researchers had focused on non - carious lesions , including erosion \n .", "dental erosion is an acid - induced tooth loss not involving microorganisms caused by external and/or internal acids \n .", "recent studies indicate a meaningful increase in dental erosion prevalence , especially in dentin , due to modifications in diet , lifestyle and socioeconomic status \n\n\n\n\n .", "early signs of erosive tooth loss have been found in children and young people \n\n\n .", "considering that the modification in population habits and the decrease of acid exposure are very tough , alternatives to reduce the progression of tooth erosive loss have been investigated .", "naf is one of the most tested fluoride salt , whose mechanism of action against erosion is based on the deposition of caf2-like layer on the surface promoting an additional barrier that inhibits the contact of the acid with the tooth \n\n\n\n\n . however , the anti - erosive effect of naf on dentin is limited since its effect is only seen when the demineralized organic matrix ( dom ) is preserved \n\n\n .", "nevertheless , some loss of the dom by enzymatic activity is expected in the clinical situation , especially in patients with eating disorders \n .", "therefore , the use of other fluoride salts is likely to supply the lack of action of conventional fluoride ( naf ) on dentin , and consequently , it could be more effective against erosion .", "accordingly , titanium tetrafluoride ( tif4 ) has been widely studied against tooth erosive demineralization since 1997 \n , demonstrating the enamel erosion - inhibiting effect \n\n\n\n\n\n\n\n\n . on the other hand ,", "generally , tif4 has a similar effect as naf on the prevention of dentin erosion when it is applied as varnish \n\n\n , or a better effect than naf when applied as high f concentrated solution \n\n\n .", "therefore , the protective effect of tif4 on dentin erosion is still in debate . considering that the application of professional fluoride , such as varnish , is not often done , patients at high risk of erosion would benefit from other alternatives to increase the frequency of fluoride exposure .", "accordingly , the daily application of mouthrinses with low concentration of f , as those containing sncl2/amf / naf marketed in europe ( erosion protection ) , has shown some protective effect against enamel and dentin erosion in vitro and in situ \n\n\n\n .", "therefore , the use of low concentrated tif4 mouthrinse by the patient could be a good alternative ; however , there is a clinical limitation due to the low ph of the solution , which might cause some side effects in oral cavity , since it has cytotoxic effect on fibroblasts \n .", "this study hypothesized that the formulation of an experimental solution containing both naf and tif4 would increase the ph , allowing its use in the clinical situation without losing its protective effect against dentin erosion compared to tif4 alone and the commercial solution erosion protection .", "the null hypothesis is that there is no significant difference in the protective effect against dentin erosive loss among the tested fluoride mouthrinses .", "ninety dentin samples , which were stored in 0.1% thymol/0.9 % nacl solution during the preparation phase , were cut from bovine dental roots .", "the root was separated from the crown using a water - cooled diamond saw and a cutting machine ( isomet low speed saw , buehler , lake bluff , il , usa ) , and embedded in pre-30 self - polymerized acrylic resin in cylindrical shape to facilitate the handling .", "thereafter , they were serially flattened with water - cooled abrasive discs ( 320 , 600 , and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , and finally polished with felt paper wetted with a diamond solution ( 1 m thickness of particles ; buehler , lake bluff , il , usa ) on a rotating polishing machine ( arotec sa ind .", "after polishing , the samples were cleaned in an ultrasonic device with deionized water for 2 min .", "the reference areas on the polished dentin surface were marked with two parallel lines made with a scalpel blade , 1.0 mm apart .", "small drilling was also done on the outer area of the dentin surface to allow the correct position of the sample in the profilometric system .", "prior to the experiment , the baseline profile was measured and two layers of nail varnish ( colorama , com .", ", so paulo , sp , brazil ) were applied on 2/3 of the control surface ( sound surfaces ) , leaving only 1/3 central of the exposed dentin ( 1.0 mm x 5 mm ) .", "dentin samples were randomly allocated to each of the six treatment groups ( n=15 ) : 1 ) commercial sncl2/naf / amf solution ( 800 ppm sn , 500 ppm f , ph 4.5 , erosion protection , gaba int . ag ,", "basel , switzerland , positive control ) ; 2 ) experimental 0.0815% tif4 solution ( 315 ppm ti , 500 ppm f , ph 2.5 ) ; 3 ) experimental 0.105% naf solution ( 500 ppm f , ph 4.5 adjusted with phosphoric acid ) ; 4 ) experimental 0.042% naf+0.049% tif4 solution ( naf- 190 ppm f , tif4  190 ppm ti and 300 ppm f , ph 4.4 ) ; 5 ) experimental 0.063%", "naf+0.036% tif4 solution ( naf  285 ppm f , tif4  140 ppm ti and 220 ppm f ; ph 4.5 ) ; 6 ) no treatment ( untreated , negative control ) .", "all solutions had approximately 500 ppm f based on the calculation obtained from the salts concentrations diluted in deionised water , and their ph was measured using a ph electrode .", "the experimental fluoride solutions were prepared using the analytical grade reagents from sigma - aldrich ( st .", "the fluoride treatments were performed twice a day ( immediately after the first and the last erosive challenges of the day ; v=0.5 ml / sample ) for 1 min , during 7 days of erosive challenges .", "erosive challenges took place by immersion in a freshly opened bottle of soft drink ( sprite zero , coca - cola company spal , porto real , rj , brazil , ph 2.6 , 30 ml / sample ) four times a day for 90 s each , at 25c .", "then , the samples were rinsed with deionized water ( 5 s ) and exposed to artificial saliva ( ph 6.8 , 30 ml / samples , 25c ) for 2h between the erosive challenges and overnight .", "the artificial saliva ( v=500 ml ) consisted of 0.001 g ascorbic acid , 0.015 g glucose , 0.290 g nacl , 0.085 g cacl2 , 0.080 g nh4cl , 0.635 g kcl , 0.080 g nascn , 0.165 g kh2po4 , 0.100 g carbamide and 0.170 g na2po4 , and it was daily renewed \n .", "dentin erosive loss ( m ) was quantitatively determined by a contact profilometer ( mahr perthometer , gttingen , lower saxony , germany ) before ( baseline ) and after 7 days of experiment . for the profilometric measurement", ", the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) .", "five profile measurements were performed at exactly the same sites as the baseline measurement , at intervals of 0.5 mm . to achieve this outcome ,", "the dentin samples presented the identification marks ( small drillings made with drill 1/4 ) and were inserted into a metal device , allowing the stylus to be accurately repositioned at each measurement .", "baseline and final profiles were done and compared using the software mahrsurf cxr20 ( mahr , gttingen , lower saxony , germany ) .", "the scans were superposed and the average depth of the under curve area was calculated ( m ) \n . for a better understanding of the treatments effect , the prevention fraction ( % ) of each treatment", "the software graphpad instat version 2.0 for windows ( graphpad software , la jolla , ca , usa ) was used for the statistical analysis .", "the assumptions of equality of variances and normal distribution of data were checked using the bartlett and kolmogorov - smirnov tests , respectively .", "once the homogeneity was not achieved , the data from dentin loss ( m ) were analyzed using kruskal - wallis followed by dunn s test .", "ninety dentin samples , which were stored in 0.1% thymol/0.9 % nacl solution during the preparation phase , were cut from bovine dental roots .", "the root was separated from the crown using a water - cooled diamond saw and a cutting machine ( isomet low speed saw , buehler , lake bluff , il , usa ) , and embedded in pre-30 self - polymerized acrylic resin in cylindrical shape to facilitate the handling .", "thereafter , they were serially flattened with water - cooled abrasive discs ( 320 , 600 , and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , and finally polished with felt paper wetted with a diamond solution ( 1 m thickness of particles ; buehler , lake bluff , il , usa ) on a rotating polishing machine ( arotec sa ind .", "after polishing , the samples were cleaned in an ultrasonic device with deionized water for 2 min .", "the reference areas on the polished dentin surface were marked with two parallel lines made with a scalpel blade , 1.0 mm apart .", "small drilling was also done on the outer area of the dentin surface to allow the correct position of the sample in the profilometric system .", "prior to the experiment , the baseline profile was measured and two layers of nail varnish ( colorama , com .", ", so paulo , sp , brazil ) were applied on 2/3 of the control surface ( sound surfaces ) , leaving only 1/3 central of the exposed dentin ( 1.0 mm x 5 mm ) .", "dentin samples were randomly allocated to each of the six treatment groups ( n=15 ) : 1 ) commercial sncl2/naf / amf solution ( 800 ppm sn , 500 ppm f , ph 4.5 , erosion protection , gaba int . ag ,", "basel , switzerland , positive control ) ; 2 ) experimental 0.0815% tif4 solution ( 315 ppm ti , 500 ppm f , ph 2.5 ) ; 3 ) experimental 0.105% naf solution ( 500 ppm f , ph 4.5 adjusted with phosphoric acid ) ; 4 ) experimental 0.042% naf+0.049% tif4 solution ( naf- 190 ppm f , tif4  190 ppm ti and 300 ppm f , ph 4.4 ) ; 5 ) experimental 0.063% naf+0.036% tif4 solution ( naf  285 ppm f , tif4  140 ppm ti and 220 ppm f ; ph 4.5 ) ; 6 ) no treatment ( untreated , negative control ) .", "all solutions had approximately 500 ppm f based on the calculation obtained from the salts concentrations diluted in deionised water , and their ph was measured using a ph electrode .", "the experimental fluoride solutions were prepared using the analytical grade reagents from sigma - aldrich ( st .", "the fluoride treatments were performed twice a day ( immediately after the first and the last erosive challenges of the day ; v=0.5 ml / sample ) for 1 min , during 7 days of erosive challenges .", "erosive challenges took place by immersion in a freshly opened bottle of soft drink ( sprite zero , coca - cola company spal , porto real , rj , brazil , ph 2.6 , 30 ml / sample ) four times a day for 90 s each , at 25c .", "then , the samples were rinsed with deionized water ( 5 s ) and exposed to artificial saliva ( ph 6.8 , 30 ml / samples , 25c ) for 2h between the erosive challenges and overnight .", "the artificial saliva ( v=500 ml ) consisted of 0.001 g ascorbic acid , 0.015 g glucose , 0.290 g nacl , 0.085 g cacl2 , 0.080 g nh4cl , 0.635 g kcl , 0.080 g nascn , 0.165 g kh2po4 , 0.100 g carbamide and 0.170 g na2po4 , and it was daily renewed \n .", "dentin erosive loss ( m ) was quantitatively determined by a contact profilometer ( mahr perthometer , gttingen , lower saxony , germany ) before ( baseline ) and after 7 days of experiment . for the profilometric measurement", ", the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) .", "five profile measurements were performed at exactly the same sites as the baseline measurement , at intervals of 0.5 mm . to achieve this outcome ,", "the dentin samples presented the identification marks ( small drillings made with drill 1/4 ) and were inserted into a metal device , allowing the stylus to be accurately repositioned at each measurement .", "baseline and final profiles were done and compared using the software mahrsurf cxr20 ( mahr , gttingen , lower saxony , germany ) .", "the scans were superposed and the average depth of the under curve area was calculated ( m ) \n . for a better understanding of the treatments effect , the prevention fraction ( % ) of each treatment", "the software graphpad instat version 2.0 for windows ( graphpad software , la jolla , ca , usa ) was used for the statistical analysis .", "the assumptions of equality of variances and normal distribution of data were checked using the bartlett and kolmogorov - smirnov tests , respectively .", "once the homogeneity was not achieved , the data from dentin loss ( m ) were analyzed using kruskal - wallis followed by dunn s test .", "all experimental mouthrinses promoted significantly lower dentin erosive loss when compared to the negative control ( p<0.0001 ) , except naf solution ( prevention fraction of 13.3% ; p>0.05 ) .", "the best anti - erosive effect was found for experimental solutions containing 0.0815% tif4 ( prevention fraction of 100% ) and 0.042% naf+0.049% tif4 ( 58.3% ) .", "sncl2/naf / amf ( erosion protection , 52% ) and 0.063% naf+0.036% tif4 ( 40% ) did not significantly differ from 0.042% naf+0.049% tif4 and naf alone , but both were less effective than tif4 alone . the median values ( minimum - maximum ) of dentin erosive loss for each group", "table 1median ( minimum - maximum ) of the dentin erosive loss for different groupssolutionsmedian ( min ; max)erosion protection(positive control)0.86 ( 0.67 ; 1.85 ) \n tif4 ( 0.0815%)-0.19 ( -0.45 ; -0.05 ) \n naf ( 0.105%)1.56 ( 1.01 ; 2.38 ) \n naf+tif4 ( 0.042%+0.049%)0.75 ( 0.21 ; 1.59 ) \n naf+tif4 ( 0.063%+0.036%)1.08 ( 0.59 ; 1.66 ) \n negative control1.80 ( 1.23 ; 4.94 ) \n * negative value means increase of the surface ( deposition)different letters show significant differences among the groups ; min = minimum ; max = maximum ( p<0.0001 ) \n  * negative value means increase of the surface ( deposition ) different letters show significant differences among the groups ; min = minimum ; max = maximum ( p<0.0001 )", "considering the increase of dental erosion s prevalence \n\n\n\n\n\n\n\n\n , the attention has been focused on the development of preventive approaches to reduce the progression of this dental condition .", "the present study investigated the protective effect of the daily application of solution containing tif4/naf . the null hypothesis tested in this study", "was rejected because the tested fluoride mouthrinses had a significantly different effect among them against dentin erosive loss .", "the solution containing pure tif4 showed the best protective effect , differing from all other groups except from a specific combination of tif4 and naf .", "this new approach would benefit patients with high risk of erosion presenting gingival recession due to periodontal disease , brushing habits ( abrasion ) or / and occlusal disorders ( abfraction ) .", "the dentin , in these cases , may be likely exposed to extrinsic acid sources from the diet , and therefore , susceptible to the development of erosion . the present study aimed to simulate the home - care application of low - concentrated fluoride ( 500 ppm f ) solution , after two meals ( morning and evening ) , in periods in which the patient could perform a rinse after the daily hygiene habit .", "the idea behind the combination of two fluorides , tif4 and naf , into an experimental mouthrinse is based on the fact that pure tif4 has low ph , impairing its clinical use .", "the addition of naf to tif4 solution was able to increase its ph to a suitable value to be applied in vivo and to be compared with commercial products . in this study ,", "tif4 alone reduced in 100% dentin loss , which might be due to the deposition of acid - resistant surface layer rich in caf2 , titanium dioxide and hydrated titanium phosphate ( unpublished data ) .", "the protocol of tif4 application tested in this study has not been applied in previous studies , since most of them tested high concentrated tif4 solution applied at once \n\n\n\n\n\n\n\n\n\n\n .", "the addition of naf into tif4 solution decreased the protective effect , considering the percentage of prevention fraction , due to a likely lower precipitation of ti and f salts .", "however , one of the combinations ( 0.042% naf+0.049% tif4 ) was still statistically similar to pure tif4 solution .", "the present results in respect to the daily application of fluoride mouthrinses are more promising than those found for a unique application of a product with high concentration of fluoride , as varnish , against dentin erosion and erosion - abrasion \n\n\n .", "the findings suggest the importance of a frequent low concentrated fluoride exposure rather than a unique application of a high concentrated fluoride product . a recent study was conducted in enamel showing similar results \n", "however , only one of the combinations ( 0.042% naf+0.049% tif4 ) was effective in reducing enamel erosive loss ( 41% preventive fraction ) , while the other one did not differ from the negative control .", "generally , the tested fluoride mouthrinses had better impact on dentin compared to enamel , which might be explained by the differences in the composition between the dental tissues . in case of dentin ,", "the effect of the combinations of tif4 and naf was similar to those provided by a commercial fluoride solution ( positive control ) , which has been widely used in europe for prevention of tooth erosion .", "the preventive fraction found by the application of erosion protection in the present study was similar to a previous in situ study performed by other research group \n . based on this finding", ", we can speculate that the acid - resistance of ti and f precipitates found for our experimental solutions are similar to tin and fluoride precipitates produced by the application of erosion protection on dentin . on the other hand , naf solution presented the worst performance , not differing from the negative control .", "it is widely known that naf is ineffective to protect against tooth erosion \n\n\n\n\n\n\n\n\n  especially in case of dentin , in which its effect depends on the presence of the dom \n\n\n . in this study", ", the dom was not removed , but it would be interesting to test the effect of the experimental fluoride solutions on dentin without dom .", "further studies should also test the effect of the experimental fluoride solutions on both dentin erosion and brushing abrasion to check the stability of the protective effect faced by two different challenges ( chemical and mechanical ) .", "another point to consider is that erosion in dentin is very complex due to the role of dom in the progression of erosive loss \n .", "therefore , erosive loss is difficult to be quantified , since the quality of the remaining organic layer may interfere with the profilometric measurement .", "therefore , to generate reliable data , the profiles must be measured with the samples immersed 100% in water or without dom .", "we have decided to perform the analysis with dom under 100% humidity , since in previous study we have not found differences in the comparison between tif4 and naf varnishes in the profile analysis of dentin with or without dom \n .", "future studies , including in situ and in vivo models , must be performed to confirm the findings , since saliva can be able to buffer the ph of the fluoride solutions , which might lead to different results .", "under the conditions of the present study , we can conclude that the daily application of an experimental mouthrinse containing a specific combination of tif4 and naf has the ability to reduce dentin erosion in vitro , and may be a good alternative for high - risk populations ."], "abstract_text": ["<S> objective this in vitro study assessed the anti - erosive effect of experimental mouthrinses containing tif4 and naf on dentin erosive loss.material and methods bovine dentin specimens were randomly allocated into the groups ( n=15 ) : 1 ) sncl2/naf / amf ( erosion protection/gaba , ph 4.5 , positive control ) ; 2 ) experimental solution with 0.0815% tif4 ( ph 2.5 ) ; 3 ) 0.105% naf ( ph 4.5 ) ; 4 ) 0.042% naf+0.049% tif4 ( ph 4.4 ) ; 5 ) 0.063% naf+0.036% tif4 ( ph 4.5 ) ; 6 ) no treatment ( negative control ) . </S>", "<S> each specimen was cyclically demineralized ( sprite zero , ph 2.6 , 4x90 s / day ) and exposed to artificial saliva between the erosive challenges for 7 days . the treatment with the fluoride solutions </S>", "<S> was done 2x60 s / day , immediately after the first and the last erosive challenges of the day . </S>", "<S> dentin erosive loss was measured by profilometry ( m ) . </S>", "<S> the data were analyzed using kruskal wallis / dunn tests ( p<0.05).results mouthrinses containing tif4 or sn / f were able to show some protective effect against dentin erosive loss compared to negative control . </S>", "<S> the best anti - erosive effect was found for experimental solution containing 0.0815% tif4 ( 100% reduction in dentin loss ) , followed by 0.042% naf+0.049% tif4 ( 58.3% ) , sncl2/naf / amf ( 52% ) and 0.063% naf+0.036% tif4 ( 40% ) . </S>", "<S> naf solution ( 13.3% ) did not significantly differ from control.conclusion the daily application of experimental mouthrinse containing tif4 and naf has the ability to reduce dentin erosion , as well as erosion protection and tif4 alone . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIAL AND METHODS", "Preparation of the dentin samples", "Fluoride treatment", "Erosive challenges", "Profile measurement", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["while the occurrence of dental caries has decreased during the last decades , researchers had focused on non - carious lesions , including erosion \n .", "dental erosion is an acid - induced tooth loss not involving microorganisms caused by external and/or internal acids \n .", "recent studies indicate a meaningful increase in dental erosion prevalence , especially in dentin , due to modifications in diet , lifestyle and socioeconomic status \n\n\n\n\n .", "early signs of erosive tooth loss have been found in children and young people \n\n\n .", "considering that the modification in population habits and the decrease of acid exposure are very tough , alternatives to reduce the progression of tooth erosive loss have been investigated .", "naf is one of the most tested fluoride salt , whose mechanism of action against erosion is based on the deposition of caf2-like layer on the surface promoting an additional barrier that inhibits the contact of the acid with the tooth \n\n\n\n\n . however , the anti - erosive effect of naf on dentin is limited since its effect is only seen when the demineralized organic matrix ( dom ) is preserved \n\n\n .", "nevertheless , some loss of the dom by enzymatic activity is expected in the clinical situation , especially in patients with eating disorders \n .", "therefore , the use of other fluoride salts is likely to supply the lack of action of conventional fluoride ( naf ) on dentin , and consequently , it could be more effective against erosion .", "accordingly , titanium tetrafluoride ( tif4 ) has been widely studied against tooth erosive demineralization since 1997 \n , demonstrating the enamel erosion - inhibiting effect \n\n\n\n\n\n\n\n\n . on the other hand ,", "generally , tif4 has a similar effect as naf on the prevention of dentin erosion when it is applied as varnish \n\n\n , or a better effect than naf when applied as high f concentrated solution \n\n\n .", "therefore , the protective effect of tif4 on dentin erosion is still in debate . considering that the application of professional fluoride , such as varnish , is not often done , patients at high risk of erosion would benefit from other alternatives to increase the frequency of fluoride exposure .", "accordingly , the daily application of mouthrinses with low concentration of f , as those containing sncl2/amf / naf marketed in europe ( erosion protection ) , has shown some protective effect against enamel and dentin erosion in vitro and in situ \n\n\n\n .", "therefore , the use of low concentrated tif4 mouthrinse by the patient could be a good alternative ; however , there is a clinical limitation due to the low ph of the solution , which might cause some side effects in oral cavity , since it has cytotoxic effect on fibroblasts \n .", "this study hypothesized that the formulation of an experimental solution containing both naf and tif4 would increase the ph , allowing its use in the clinical situation without losing its protective effect against dentin erosion compared to tif4 alone and the commercial solution erosion protection .", "the null hypothesis is that there is no significant difference in the protective effect against dentin erosive loss among the tested fluoride mouthrinses ."], ["ninety dentin samples , which were stored in 0.1% thymol/0.9 % nacl solution during the preparation phase , were cut from bovine dental roots .", "the root was separated from the crown using a water - cooled diamond saw and a cutting machine ( isomet low speed saw , buehler , lake bluff , il , usa ) , and embedded in pre-30 self - polymerized acrylic resin in cylindrical shape to facilitate the handling .", "thereafter , they were serially flattened with water - cooled abrasive discs ( 320 , 600 , and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , and finally polished with felt paper wetted with a diamond solution ( 1 m thickness of particles ; buehler , lake bluff , il , usa ) on a rotating polishing machine ( arotec sa ind .", "after polishing , the samples were cleaned in an ultrasonic device with deionized water for 2 min .", "the reference areas on the polished dentin surface were marked with two parallel lines made with a scalpel blade , 1.0 mm apart .", "small drilling was also done on the outer area of the dentin surface to allow the correct position of the sample in the profilometric system .", "prior to the experiment , the baseline profile was measured and two layers of nail varnish ( colorama , com .", ", so paulo , sp , brazil ) were applied on 2/3 of the control surface ( sound surfaces ) , leaving only 1/3 central of the exposed dentin ( 1.0 mm x 5 mm ) .", "dentin samples were randomly allocated to each of the six treatment groups ( n=15 ) : 1 ) commercial sncl2/naf / amf solution ( 800 ppm sn , 500 ppm f , ph 4.5 , erosion protection , gaba int . ag ,", "basel , switzerland , positive control ) ; 2 ) experimental 0.0815% tif4 solution ( 315 ppm ti , 500 ppm f , ph 2.5 ) ; 3 ) experimental 0.105% naf solution ( 500 ppm f , ph 4.5 adjusted with phosphoric acid ) ; 4 ) experimental 0.042% naf+0.049% tif4 solution ( naf- 190 ppm f , tif4  190 ppm ti and 300 ppm f , ph 4.4 ) ; 5 ) experimental 0.063%", "naf+0.036% tif4 solution ( naf  285 ppm f , tif4  140 ppm ti and 220 ppm f ; ph 4.5 ) ; 6 ) no treatment ( untreated , negative control ) .", "all solutions had approximately 500 ppm f based on the calculation obtained from the salts concentrations diluted in deionised water , and their ph was measured using a ph electrode .", "the experimental fluoride solutions were prepared using the analytical grade reagents from sigma - aldrich ( st .", "the fluoride treatments were performed twice a day ( immediately after the first and the last erosive challenges of the day ; v=0.5 ml / sample ) for 1 min , during 7 days of erosive challenges .", "erosive challenges took place by immersion in a freshly opened bottle of soft drink ( sprite zero , coca - cola company spal , porto real , rj , brazil , ph 2.6 , 30 ml / sample ) four times a day for 90 s each , at 25c .", "then , the samples were rinsed with deionized water ( 5 s ) and exposed to artificial saliva ( ph 6.8 , 30 ml / samples , 25c ) for 2h between the erosive challenges and overnight .", "the artificial saliva ( v=500 ml ) consisted of 0.001 g ascorbic acid , 0.015 g glucose , 0.290 g nacl , 0.085 g cacl2 , 0.080 g nh4cl , 0.635 g kcl , 0.080 g nascn , 0.165 g kh2po4 , 0.100 g carbamide and 0.170 g na2po4 , and it was daily renewed \n .", "dentin erosive loss ( m ) was quantitatively determined by a contact profilometer ( mahr perthometer , gttingen , lower saxony , germany ) before ( baseline ) and after 7 days of experiment . for the profilometric measurement", ", the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) .", "five profile measurements were performed at exactly the same sites as the baseline measurement , at intervals of 0.5 mm . to achieve this outcome ,", "the dentin samples presented the identification marks ( small drillings made with drill 1/4 ) and were inserted into a metal device , allowing the stylus to be accurately repositioned at each measurement .", "baseline and final profiles were done and compared using the software mahrsurf cxr20 ( mahr , gttingen , lower saxony , germany ) .", "the scans were superposed and the average depth of the under curve area was calculated ( m ) \n . for a better understanding of the treatments effect , the prevention fraction ( % ) of each treatment", "the software graphpad instat version 2.0 for windows ( graphpad software , la jolla , ca , usa ) was used for the statistical analysis .", "the assumptions of equality of variances and normal distribution of data were checked using the bartlett and kolmogorov - smirnov tests , respectively .", "once the homogeneity was not achieved , the data from dentin loss ( m ) were analyzed using kruskal - wallis followed by dunn s test ."], ["ninety dentin samples , which were stored in 0.1% thymol/0.9 % nacl solution during the preparation phase , were cut from bovine dental roots .", "the root was separated from the crown using a water - cooled diamond saw and a cutting machine ( isomet low speed saw , buehler , lake bluff , il , usa ) , and embedded in pre-30 self - polymerized acrylic resin in cylindrical shape to facilitate the handling .", "thereafter , they were serially flattened with water - cooled abrasive discs ( 320 , 600 , and 1200 grades of al2o3 papers ; buehler , lake bluff , il , usa ) , and finally polished with felt paper wetted with a diamond solution ( 1 m thickness of particles ; buehler , lake bluff , il , usa ) on a rotating polishing machine ( arotec sa ind .", "after polishing , the samples were cleaned in an ultrasonic device with deionized water for 2 min .", "the reference areas on the polished dentin surface were marked with two parallel lines made with a scalpel blade , 1.0 mm apart .", "small drilling was also done on the outer area of the dentin surface to allow the correct position of the sample in the profilometric system .", "prior to the experiment , the baseline profile was measured and two layers of nail varnish ( colorama , com .", ", so paulo , sp , brazil ) were applied on 2/3 of the control surface ( sound surfaces ) , leaving only 1/3 central of the exposed dentin ( 1.0 mm x 5 mm ) ."], ["dentin samples were randomly allocated to each of the six treatment groups ( n=15 ) : 1 ) commercial sncl2/naf / amf solution ( 800 ppm sn , 500 ppm f , ph 4.5 , erosion protection , gaba int . ag ,", "basel , switzerland , positive control ) ; 2 ) experimental 0.0815% tif4 solution ( 315 ppm ti , 500 ppm f , ph 2.5 ) ; 3 ) experimental 0.105% naf solution ( 500 ppm f , ph 4.5 adjusted with phosphoric acid ) ; 4 ) experimental 0.042% naf+0.049% tif4 solution ( naf- 190 ppm f , tif4  190 ppm ti and 300 ppm f , ph 4.4 ) ; 5 ) experimental 0.063% naf+0.036% tif4 solution ( naf  285 ppm f , tif4  140 ppm ti and 220 ppm f ; ph 4.5 ) ; 6 ) no treatment ( untreated , negative control ) .", "all solutions had approximately 500 ppm f based on the calculation obtained from the salts concentrations diluted in deionised water , and their ph was measured using a ph electrode .", "the experimental fluoride solutions were prepared using the analytical grade reagents from sigma - aldrich ( st .", "the fluoride treatments were performed twice a day ( immediately after the first and the last erosive challenges of the day ; v=0.5 ml / sample ) for 1 min , during 7 days of erosive challenges ."], ["erosive challenges took place by immersion in a freshly opened bottle of soft drink ( sprite zero , coca - cola company spal , porto real , rj , brazil , ph 2.6 , 30 ml / sample ) four times a day for 90 s each , at 25c .", "then , the samples were rinsed with deionized water ( 5 s ) and exposed to artificial saliva ( ph 6.8 , 30 ml / samples , 25c ) for 2h between the erosive challenges and overnight .", "the artificial saliva ( v=500 ml ) consisted of 0.001 g ascorbic acid , 0.015 g glucose , 0.290 g nacl , 0.085 g cacl2 , 0.080 g nh4cl , 0.635 g kcl , 0.080 g nascn , 0.165 g kh2po4 , 0.100 g carbamide and 0.170 g na2po4 , and it was daily renewed \n ."], ["dentin erosive loss ( m ) was quantitatively determined by a contact profilometer ( mahr perthometer , gttingen , lower saxony , germany ) before ( baseline ) and after 7 days of experiment . for the profilometric measurement", ", the nail varnish was carefully removed using a scalpel and acetone solution ( 1:1 water ) .", "five profile measurements were performed at exactly the same sites as the baseline measurement , at intervals of 0.5 mm . to achieve this outcome ,", "the dentin samples presented the identification marks ( small drillings made with drill 1/4 ) and were inserted into a metal device , allowing the stylus to be accurately repositioned at each measurement .", "baseline and final profiles were done and compared using the software mahrsurf cxr20 ( mahr , gttingen , lower saxony , germany ) .", "the scans were superposed and the average depth of the under curve area was calculated ( m ) \n . for a better understanding of the treatments effect , the prevention fraction ( % ) of each treatment"], ["the software graphpad instat version 2.0 for windows ( graphpad software , la jolla , ca , usa ) was used for the statistical analysis .", "the assumptions of equality of variances and normal distribution of data were checked using the bartlett and kolmogorov - smirnov tests , respectively .", "once the homogeneity was not achieved , the data from dentin loss ( m ) were analyzed using kruskal - wallis followed by dunn s test ."], ["all experimental mouthrinses promoted significantly lower dentin erosive loss when compared to the negative control ( p<0.0001 ) , except naf solution ( prevention fraction of 13.3% ; p>0.05 ) .", "the best anti - erosive effect was found for experimental solutions containing 0.0815% tif4 ( prevention fraction of 100% ) and 0.042% naf+0.049% tif4 ( 58.3% ) .", "sncl2/naf / amf ( erosion protection , 52% ) and 0.063% naf+0.036% tif4 ( 40% ) did not significantly differ from 0.042% naf+0.049% tif4 and naf alone , but both were less effective than tif4 alone . the median values ( minimum - maximum ) of dentin erosive loss for each group", "table 1median ( minimum - maximum ) of the dentin erosive loss for different groupssolutionsmedian ( min ; max)erosion protection(positive control)0.86 ( 0.67 ; 1.85 ) \n tif4 ( 0.0815%)-0.19 ( -0.45 ; -0.05 ) \n naf ( 0.105%)1.56 ( 1.01 ; 2.38 ) \n naf+tif4 ( 0.042%+0.049%)0.75 ( 0.21 ; 1.59 ) \n naf+tif4 ( 0.063%+0.036%)1.08 ( 0.59 ; 1.66 ) \n negative control1.80 ( 1.23 ; 4.94 ) \n * negative value means increase of the surface ( deposition)different letters show significant differences among the groups ; min = minimum ; max = maximum ( p<0.0001 ) \n  * negative value means increase of the surface ( deposition ) different letters show significant differences among the groups ; min = minimum ; max = maximum ( p<0.0001 )"], ["considering the increase of dental erosion s prevalence \n\n\n\n\n\n\n\n\n , the attention has been focused on the development of preventive approaches to reduce the progression of this dental condition .", "the present study investigated the protective effect of the daily application of solution containing tif4/naf . the null hypothesis tested in this study", "was rejected because the tested fluoride mouthrinses had a significantly different effect among them against dentin erosive loss .", "the solution containing pure tif4 showed the best protective effect , differing from all other groups except from a specific combination of tif4 and naf .", "this new approach would benefit patients with high risk of erosion presenting gingival recession due to periodontal disease , brushing habits ( abrasion ) or / and occlusal disorders ( abfraction ) .", "the dentin , in these cases , may be likely exposed to extrinsic acid sources from the diet , and therefore , susceptible to the development of erosion . the present study aimed to simulate the home - care application of low - concentrated fluoride ( 500 ppm f ) solution , after two meals ( morning and evening ) , in periods in which the patient could perform a rinse after the daily hygiene habit .", "the idea behind the combination of two fluorides , tif4 and naf , into an experimental mouthrinse is based on the fact that pure tif4 has low ph , impairing its clinical use .", "the addition of naf to tif4 solution was able to increase its ph to a suitable value to be applied in vivo and to be compared with commercial products . in this study ,", "tif4 alone reduced in 100% dentin loss , which might be due to the deposition of acid - resistant surface layer rich in caf2 , titanium dioxide and hydrated titanium phosphate ( unpublished data ) .", "the protocol of tif4 application tested in this study has not been applied in previous studies , since most of them tested high concentrated tif4 solution applied at once \n\n\n\n\n\n\n\n\n\n\n .", "the addition of naf into tif4 solution decreased the protective effect , considering the percentage of prevention fraction , due to a likely lower precipitation of ti and f salts .", "however , one of the combinations ( 0.042% naf+0.049% tif4 ) was still statistically similar to pure tif4 solution .", "the present results in respect to the daily application of fluoride mouthrinses are more promising than those found for a unique application of a product with high concentration of fluoride , as varnish , against dentin erosion and erosion - abrasion \n\n\n .", "the findings suggest the importance of a frequent low concentrated fluoride exposure rather than a unique application of a high concentrated fluoride product . a recent study was conducted in enamel showing similar results \n", "however , only one of the combinations ( 0.042% naf+0.049% tif4 ) was effective in reducing enamel erosive loss ( 41% preventive fraction ) , while the other one did not differ from the negative control .", "generally , the tested fluoride mouthrinses had better impact on dentin compared to enamel , which might be explained by the differences in the composition between the dental tissues . in case of dentin ,", "the effect of the combinations of tif4 and naf was similar to those provided by a commercial fluoride solution ( positive control ) , which has been widely used in europe for prevention of tooth erosion .", "the preventive fraction found by the application of erosion protection in the present study was similar to a previous in situ study performed by other research group \n . based on this finding", ", we can speculate that the acid - resistance of ti and f precipitates found for our experimental solutions are similar to tin and fluoride precipitates produced by the application of erosion protection on dentin . on the other hand , naf solution presented the worst performance , not differing from the negative control .", "it is widely known that naf is ineffective to protect against tooth erosion \n\n\n\n\n\n\n\n\n  especially in case of dentin , in which its effect depends on the presence of the dom \n\n\n . in this study", ", the dom was not removed , but it would be interesting to test the effect of the experimental fluoride solutions on dentin without dom .", "further studies should also test the effect of the experimental fluoride solutions on both dentin erosion and brushing abrasion to check the stability of the protective effect faced by two different challenges ( chemical and mechanical ) .", "another point to consider is that erosion in dentin is very complex due to the role of dom in the progression of erosive loss \n .", "therefore , erosive loss is difficult to be quantified , since the quality of the remaining organic layer may interfere with the profilometric measurement .", "therefore , to generate reliable data , the profiles must be measured with the samples immersed 100% in water or without dom .", "we have decided to perform the analysis with dom under 100% humidity , since in previous study we have not found differences in the comparison between tif4 and naf varnishes in the profile analysis of dentin with or without dom \n .", "future studies , including in situ and in vivo models , must be performed to confirm the findings , since saliva can be able to buffer the ph of the fluoride solutions , which might lead to different results ."], ["under the conditions of the present study , we can conclude that the daily application of an experimental mouthrinse containing a specific combination of tif4 and naf has the ability to reduce dentin erosion in vitro , and may be a good alternative for high - risk populations ."]]}
{"article_id": "PMC2778347", "article_text": ["allergic diseases are among the most common chronic diseases throughout the world   and the prevalence of atopic diseases in childhood has significantly increased during the past several decades [ 2 , 3 ] .", "although there is a general consensus on the importance of a genetic predisposition for atopic diseases , only changes in environmental factors can explain this increase [ 46 ] . there is a strong association between sensitisation and symptoms of allergic diseases although this association is not absolute .", "the  allergy march  refers to the natural history of sensitisation to allergens and symptoms of eczema , asthma , and rhinitis , which is characterized by a typical sequence of sensitisation and manifestations of symptoms that appear during a certain age period [ 7 , 8 ] .", "food allergy and atopic eczema during the first years of life have been considered risk factors for subsequent asthma and rhinitis caused by indoor and outdoor inhalant allergens [ 5 , 9 , 10 ] .", "however , allergy - like symptoms in childhood like wheezing and eczema need not have an atopic background [ 11 , 12 ] .", "wheezing episodes in young children are often transient and associated with viral respiratory infections [ 13 , 14 ] .", "thus , better predictors of disease development and diagnostic markers for allergic disease are needed to give proper treatment and valuable advice , especially concerning environmental control to parents of young children .", "the present study was carried out in order to evaluate the diagnostic performance of phadiatop infant , an in vitro test designed to detect allergen - specific ige antibodies known to be relevant in the development of ige - sensitisation in early childhood .", "the study was conducted retrospectively in consecutively included children , below the age of five years and admitted to bkl voksentoppen , rikshospitalet , oslo , during a period of twelve months .", "the children were referred from paediatricians and other paediatric departments at hospitals in norway and thus selected for more severe allergic symptoms .", "frozen - serum samples from 122 children were analysed and clinical data documented in the patients ' records were compiled for the study .", "the study conformed to the principles of helsinki 's declaration and was approved by the local ethics committee .", "skin prick test ( spt ) was performed according to standard procedure at the hospital .", "glycerinated allergen extract ( soluprick   alk - abello , denmark   and a standard lancet with 1 mm tip and a shoulder ( alk - abello , denmark ) was used .  ", "histamine chloride 10 mg / ml was used as positive - control and as negative - control glycerol .", "the reaction was recorded as 3 + when the reaction was equal to the histamine control , 2 + when half the histamine control , and 4 + when double the histamine control . the panel included egg white , cow 's milk , cod fish , peanut , hazel nut , wheat , house dust mite , cat dander , birch , timothy , cladosporium , and latex .", "total and allergen - specific ige antibodies were determined in immunocap   100 ( phadia ab , sweden ) at the hospital laboratory when appropriate according to clinical findings .", "serum samples were sent to phadia ab , uppsala , sweden , for determination of specific ige antibodies using immunocap phadiatop , fx5 ( a food mixture of milk , egg white , fish , peanut , wheat , and soy bean ) , and immunocap phadiatop infant using immunocap100 ( phadia ab , uppsala , sweden ) .", "phadiatop infant is designed to detect allergen - specific ige antibodies to food and inhalant allergens , relevant in the development of ige - mediated disease in young children .", "the following allergens are included in the test : hen 's egg , cow 's milk , peanut , shrimp , cat epithelium and dander , dog dander , house dust mite , common silver birch , timothy , ragweed , and wall pellitory .", "the laboratory analyses were performed in a blinded manner and the results of phadiatop and fx5 were sent back to the investigating physician .", "atopy was defined as a constitutional disposition to produce ige antibodies to common allergens whether the patients had clinical symptoms or not , as judged by the investigator .", "the study was designed to make the diagnosis with best possible available tools ( case history , spt , and allergen - specific ige results ) to evaluate the clinical performance of phadiatop infant . a preliminary diagnosis whether a child was atopic or not was assessed by the investigating physician in retrospect , taking into account clinical history and diagnostic procedures , which includes available spt and allergen - specific ige results , noted in the patient records . in the final diagnosis of the atopic state , the laboratory results from phadiatop and fx5 were also taken into consideration in order to get comparable data of allergen - specific ige sensitisation on all children .", "this final diagnosis was used as the reference for calculation of the diagnostic performance of phadiatop infant .", "quantitative variables are described in appropriate measures of localisation and dispersion , quantitatively variables presented by counts and percentages .", "the diagnostic performance of phadiatop infant was evaluated by calculating sensitivity , specificity , negative and positive predictive values ( npv , ppv ) , respectively , with 95% confidence intervals .", "the subgroups divided by age ,   < 2 years and   2 years were compared with regard to the diagnostic performance of phadiatop infant using descriptive statistics .", "the demographic characteristics of the children classified according to the final diagnosis of atopy / nonatopy or inconclusive diagnosis are shown in table 1 .", "thirty - eight ( 31% ) subjects in the study population were girls and 84 ( 69% ) were boys with a median age of 2.7 years .", "of the 122 children , 86 ( 70% ) were atopic , 26 girls and 60 boys , which is a commonly found gender distribution among atopic children at that age .", "only 18% of the children presented with wheezing as a single symptom and the majority of these children were nonatopic , 55% below 2 years and 40% above this age , respectively .", "eczema as a single symptom was the most prevalent symptom among the children below two years of age ( 49% ) and 63% were classified as atopic . in the older age group of children , eczema and/or wheezing in combination with other allergic symptoms dominated ( 41% ) and", "other allergy - like symptoms such as rhinitis , rhinoconjunctivitis , anaphylaxis , and gastrointestinal symptoms were registered in 31 ( 26% ) children , out of whom 22 ( 71% ) were older than 2 years ( data not shown ) .", "eighty - three of the children ( 68% ) reported at least one first degree relative , with about the same proportion for the atopic as for the nonatopic children , 71% and 61% , respectively ( data not shown ) .", "the diagnostic performance characteristics of phadiatop infant in this study population with a prevalence of 70% are presented in table 2 .", "the sensitivity calculated for the whole group of children was 98% ( 95% ci : 92100% ) and the specificity 89% ( 95% ci : 7497% ) .  ", "the ppv and npv values were 95% ( 95% ci : 8999% ) and 94% ( 95% ci : 8099% ) , respectively .", "the diagnostic performance of the test was found to be similar when the children were separated in the two age groups , below or above two years , but due to small numbers of children in the separated age groups , the calculated values are not presented .", "symptoms of allergic disease in young children are generally unspecific and the diagnosis without objective tests could be an arbitrary process .", "the paediatric section of the european academy of allergy and clinical immunology has recently published a position paper with recommendations on allergy testing in children to improve the identification of allergy and quality of care .", "an earlier published study has shown that 76 out of 147 children could not be classified as having an ige - mediated disease or not , based on case history and physical examination alone .", "similar results were found in a recently published study , where measurements of ige - antibodies , added to case history and physical examinations , highly improved the discrimination between ige- and non - ige - mediated diseases in young children .", "the results from our study confirm these findings and suggest that phadiatop infant could be a useful tool for discrimination between atopy / non - atopy .", "a positive phadiatop infant test should however be followed by allergen - specific antibody testing to a selected panel to identify the offending allergen(s ) [ 19 , 20 ] .", "the test seems to be at least as useful among the youngest children , below two years , as among children at 24 years of age .", "the youngest child in the study was 6 months , which confirms findings from other publications that allergen - specific ige - antibodies can be detected early in life [ 17 , 18 , 21 ] .", "these findings support the value of testing children with allergy - like symptoms at an early age .", "family history has been considered an important risk factor for atopy and has been widely used for prediction of allergy and allergic diseases .", "the results from our study are in good agreement with the notion that allergy / atopy is extremely commonly found . on the other hand ,", "the fact that 30% of the atopic children did not have a family history of allergy agrees with investigators claiming that a great portion of newly diagnosed allergics / atopics do not have a family history of allergy / atopy .", "thus it could be stated that family history is no longer practical in predicting allergic disease .", "this study population represents a selected cohort as voksentoppen is a hospital having referred patients with allergy - like symptoms and allergic diseases from general paediatricians in the whole country , often of a more severe character .", "however , other studies have shown that phadiatop infant could be applied in populations with a lower prevalence of atopy , still demonstrating good performance characteristics [ 17 , 18 , 21 ] .", "the clinical appearance of allergy in our study is in agreement with the concept of the allergy march and supports what has been reported earlier from other studies [ 7 , 8 ] .", "thus , eczema was not common in the nonatopic group of children and only one of the 31 children with eczema was classified as nonatopic . the progression from eczema to other allergic problems", "eighty - six percent of the children with eczema and wheezing in combination with other symptoms were found in the older age group and as many as 82% were classified as atopic .", "the majority of children , all ages , presenting with wheezing were nonatopic ( 73% ) .", "many infants and children who wheeze have transient conditions associated with diminished airway function and do not have an increased risk of asthma later in life .", "however , children with persistent wheezing , starting during the first years of life , and with an atopic heredity , should be considered being at risk for asthma later during childhood [ 11 , 22 , 23 ] .", "therefore , an early diagnosis of ige sensitisation may be important for the choice of treatment to wheezing toddlers .", "children with allergic symptoms usually present at a general paediatrician who needs to discriminate which patients have to be sent to an allergist for further evaluation .", "rule - out tests with a high discriminating potential are suggested to have a gateway function to fulfil this differentiation and an important role to prevent the march of allergic children from the first to secondary level of care .", "the present study shows that phadiatop infant has a diagnostic performance with a high sensitivity and specificity .", "thus , the test could be recommended as a complement to the clinical information in the differential diagnosis on ige - mediated disease in young children with allergy - like symptoms .", "furthermore , the test could be useful in assisting primary care physicians in selecting atopic children at an early stage for further intervention or referral to an allergist .", "an early correct diagnosis will thus allow for better management and a possibility to delay or even prevent the onset of asthma in children with eczema and the avoidance of further deterioration of lung function in children with asthma [ 16 , 25 ] ."], "abstract_text": ["<S> \n background and objective . </S>", "<S> allergy - like symptoms such as wheezing and eczema are common in young children and an early diagnosis is important to initiate correct management . the objective of this study was to evaluate the diagnostic performance of phadiatop infant , an in vitro test for determination of early sensitisation to food and inhalant allergens . </S>", "<S> patients and methods . </S>", "<S> the study was conducted , retrospectively , using frozen sera from 122 children ( median age 2.7 years ) admitted to the hospital with suspected allergic symptoms . </S>", "<S> the doctor 's diagnosis atopic / nonatopic was based on routinely used procedures such as clinical evaluation , spt , total and allergen - specific ige antibodies . </S>", "<S> the performance of phadiatop infant was evaluated in a blinded manner against this diagnosis . </S>", "<S> results . </S>", "<S> eighty - four of the 86 children classified as atopic showed a positive phadiatop infant test . </S>", "<S> thirty - six were classified as nonatopic , 32 of who had a negative test . with a prevalence of atopy of 70% in this population , this gives a sensitivity of 98% , a specificity of 89% , and a positive and negative predictive value of 95% and 94% , respectively . </S>", "<S> conclusion . </S>", "<S> the results from the present study suggest that phadiatop infant could be recommended as a complement to the clinical information in the differential diagnosis on ige - mediated disease in young children with allergy - like symptoms . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Patients and Methods", "3. Results", "4. Discussion"], "sections": [["allergic diseases are among the most common chronic diseases throughout the world   and the prevalence of atopic diseases in childhood has significantly increased during the past several decades [ 2 , 3 ] .", "although there is a general consensus on the importance of a genetic predisposition for atopic diseases , only changes in environmental factors can explain this increase [ 46 ] . there is a strong association between sensitisation and symptoms of allergic diseases although this association is not absolute .", "the  allergy march  refers to the natural history of sensitisation to allergens and symptoms of eczema , asthma , and rhinitis , which is characterized by a typical sequence of sensitisation and manifestations of symptoms that appear during a certain age period [ 7 , 8 ] .", "food allergy and atopic eczema during the first years of life have been considered risk factors for subsequent asthma and rhinitis caused by indoor and outdoor inhalant allergens [ 5 , 9 , 10 ] .", "however , allergy - like symptoms in childhood like wheezing and eczema need not have an atopic background [ 11 , 12 ] .", "wheezing episodes in young children are often transient and associated with viral respiratory infections [ 13 , 14 ] .", "thus , better predictors of disease development and diagnostic markers for allergic disease are needed to give proper treatment and valuable advice , especially concerning environmental control to parents of young children .", "the present study was carried out in order to evaluate the diagnostic performance of phadiatop infant , an in vitro test designed to detect allergen - specific ige antibodies known to be relevant in the development of ige - sensitisation in early childhood ."], ["the study was conducted retrospectively in consecutively included children , below the age of five years and admitted to bkl voksentoppen , rikshospitalet , oslo , during a period of twelve months .", "the children were referred from paediatricians and other paediatric departments at hospitals in norway and thus selected for more severe allergic symptoms .", "frozen - serum samples from 122 children were analysed and clinical data documented in the patients ' records were compiled for the study .", "the study conformed to the principles of helsinki 's declaration and was approved by the local ethics committee .", "skin prick test ( spt ) was performed according to standard procedure at the hospital .", "glycerinated allergen extract ( soluprick   alk - abello , denmark   and a standard lancet with 1 mm tip and a shoulder ( alk - abello , denmark ) was used .  ", "histamine chloride 10 mg / ml was used as positive - control and as negative - control glycerol .", "the reaction was recorded as 3 + when the reaction was equal to the histamine control , 2 + when half the histamine control , and 4 + when double the histamine control . the panel included egg white , cow 's milk , cod fish , peanut , hazel nut , wheat , house dust mite , cat dander , birch , timothy , cladosporium , and latex .", "total and allergen - specific ige antibodies were determined in immunocap   100 ( phadia ab , sweden ) at the hospital laboratory when appropriate according to clinical findings .", "serum samples were sent to phadia ab , uppsala , sweden , for determination of specific ige antibodies using immunocap phadiatop , fx5 ( a food mixture of milk , egg white , fish , peanut , wheat , and soy bean ) , and immunocap phadiatop infant using immunocap100 ( phadia ab , uppsala , sweden ) .", "phadiatop infant is designed to detect allergen - specific ige antibodies to food and inhalant allergens , relevant in the development of ige - mediated disease in young children .", "the following allergens are included in the test : hen 's egg , cow 's milk , peanut , shrimp , cat epithelium and dander , dog dander , house dust mite , common silver birch , timothy , ragweed , and wall pellitory .", "the laboratory analyses were performed in a blinded manner and the results of phadiatop and fx5 were sent back to the investigating physician .", "atopy was defined as a constitutional disposition to produce ige antibodies to common allergens whether the patients had clinical symptoms or not , as judged by the investigator .", "the study was designed to make the diagnosis with best possible available tools ( case history , spt , and allergen - specific ige results ) to evaluate the clinical performance of phadiatop infant . a preliminary diagnosis whether a child was atopic or not was assessed by the investigating physician in retrospect , taking into account clinical history and diagnostic procedures , which includes available spt and allergen - specific ige results , noted in the patient records . in the final diagnosis of the atopic state , the laboratory results from phadiatop and fx5 were also taken into consideration in order to get comparable data of allergen - specific ige sensitisation on all children .", "this final diagnosis was used as the reference for calculation of the diagnostic performance of phadiatop infant .", "quantitative variables are described in appropriate measures of localisation and dispersion , quantitatively variables presented by counts and percentages .", "the diagnostic performance of phadiatop infant was evaluated by calculating sensitivity , specificity , negative and positive predictive values ( npv , ppv ) , respectively , with 95% confidence intervals .", "the subgroups divided by age ,   < 2 years and   2 years were compared with regard to the diagnostic performance of phadiatop infant using descriptive statistics ."], ["the demographic characteristics of the children classified according to the final diagnosis of atopy / nonatopy or inconclusive diagnosis are shown in table 1 .", "thirty - eight ( 31% ) subjects in the study population were girls and 84 ( 69% ) were boys with a median age of 2.7 years .", "of the 122 children , 86 ( 70% ) were atopic , 26 girls and 60 boys , which is a commonly found gender distribution among atopic children at that age .", "only 18% of the children presented with wheezing as a single symptom and the majority of these children were nonatopic , 55% below 2 years and 40% above this age , respectively .", "eczema as a single symptom was the most prevalent symptom among the children below two years of age ( 49% ) and 63% were classified as atopic . in the older age group of children , eczema and/or wheezing in combination with other allergic symptoms dominated ( 41% ) and", "other allergy - like symptoms such as rhinitis , rhinoconjunctivitis , anaphylaxis , and gastrointestinal symptoms were registered in 31 ( 26% ) children , out of whom 22 ( 71% ) were older than 2 years ( data not shown ) .", "eighty - three of the children ( 68% ) reported at least one first degree relative , with about the same proportion for the atopic as for the nonatopic children , 71% and 61% , respectively ( data not shown ) .", "the diagnostic performance characteristics of phadiatop infant in this study population with a prevalence of 70% are presented in table 2 .", "the sensitivity calculated for the whole group of children was 98% ( 95% ci : 92100% ) and the specificity 89% ( 95% ci : 7497% ) .  ", "the ppv and npv values were 95% ( 95% ci : 8999% ) and 94% ( 95% ci : 8099% ) , respectively .", "the diagnostic performance of the test was found to be similar when the children were separated in the two age groups , below or above two years , but due to small numbers of children in the separated age groups , the calculated values are not presented ."], ["symptoms of allergic disease in young children are generally unspecific and the diagnosis without objective tests could be an arbitrary process .", "the paediatric section of the european academy of allergy and clinical immunology has recently published a position paper with recommendations on allergy testing in children to improve the identification of allergy and quality of care .", "an earlier published study has shown that 76 out of 147 children could not be classified as having an ige - mediated disease or not , based on case history and physical examination alone .", "similar results were found in a recently published study , where measurements of ige - antibodies , added to case history and physical examinations , highly improved the discrimination between ige- and non - ige - mediated diseases in young children .", "the results from our study confirm these findings and suggest that phadiatop infant could be a useful tool for discrimination between atopy / non - atopy .", "a positive phadiatop infant test should however be followed by allergen - specific antibody testing to a selected panel to identify the offending allergen(s ) [ 19 , 20 ] .", "the test seems to be at least as useful among the youngest children , below two years , as among children at 24 years of age .", "the youngest child in the study was 6 months , which confirms findings from other publications that allergen - specific ige - antibodies can be detected early in life [ 17 , 18 , 21 ] .", "these findings support the value of testing children with allergy - like symptoms at an early age .", "family history has been considered an important risk factor for atopy and has been widely used for prediction of allergy and allergic diseases .", "the results from our study are in good agreement with the notion that allergy / atopy is extremely commonly found . on the other hand ,", "the fact that 30% of the atopic children did not have a family history of allergy agrees with investigators claiming that a great portion of newly diagnosed allergics / atopics do not have a family history of allergy / atopy .", "thus it could be stated that family history is no longer practical in predicting allergic disease .", "this study population represents a selected cohort as voksentoppen is a hospital having referred patients with allergy - like symptoms and allergic diseases from general paediatricians in the whole country , often of a more severe character .", "however , other studies have shown that phadiatop infant could be applied in populations with a lower prevalence of atopy , still demonstrating good performance characteristics [ 17 , 18 , 21 ] .", "the clinical appearance of allergy in our study is in agreement with the concept of the allergy march and supports what has been reported earlier from other studies [ 7 , 8 ] .", "thus , eczema was not common in the nonatopic group of children and only one of the 31 children with eczema was classified as nonatopic . the progression from eczema to other allergic problems", "eighty - six percent of the children with eczema and wheezing in combination with other symptoms were found in the older age group and as many as 82% were classified as atopic .", "the majority of children , all ages , presenting with wheezing were nonatopic ( 73% ) .", "many infants and children who wheeze have transient conditions associated with diminished airway function and do not have an increased risk of asthma later in life .", "however , children with persistent wheezing , starting during the first years of life , and with an atopic heredity , should be considered being at risk for asthma later during childhood [ 11 , 22 , 23 ] .", "therefore , an early diagnosis of ige sensitisation may be important for the choice of treatment to wheezing toddlers .", "children with allergic symptoms usually present at a general paediatrician who needs to discriminate which patients have to be sent to an allergist for further evaluation .", "rule - out tests with a high discriminating potential are suggested to have a gateway function to fulfil this differentiation and an important role to prevent the march of allergic children from the first to secondary level of care .", "the present study shows that phadiatop infant has a diagnostic performance with a high sensitivity and specificity .", "thus , the test could be recommended as a complement to the clinical information in the differential diagnosis on ige - mediated disease in young children with allergy - like symptoms .", "furthermore , the test could be useful in assisting primary care physicians in selecting atopic children at an early stage for further intervention or referral to an allergist .", "an early correct diagnosis will thus allow for better management and a possibility to delay or even prevent the onset of asthma in children with eczema and the avoidance of further deterioration of lung function in children with asthma [ 16 , 25 ] ."]]}
{"article_id": "PMC4840430", "article_text": ["mainly for safety reasons , many patients and their families are looking for a seizure detection system that is efficient , comfortable , and easy to use .", "this implies that such a system should have a high detection sensitivity ( detect as many seizures as possible ) and low false alarm rate ; it should be unobtrusive , wireless , and in case of daytime use practically unnoticeable ; and it should allow long - term use in a home situation without the presence of professionals . alarming for safety reasons or reassurance", "is already used in fall detectors for the elderly , babyphones ( reacting to sound or movement ) , and glucose monitors for persons with diabetes , .", "in addition to being used for alarming , a detection system could allow offline storage or online streaming of selected data , allowing follow - up on treatment efficacy or summoning emergency support , respectively .", "our team is interested in the long - term observation of patients with epilepsy and detection of their seizures , .", "the focus lies on those convulsive seizures that can be dangerous because of their intensity ( possible injuries ) , duration , or serious consequences such as brain damage , autonomic dysregulation , or suffocation .", "clonic and clonic seizures with patients remaining in one place , their bed , mainly because of the need for supervision at night and because seizures often occur during sleep . as sensors need to be as unobtrusive as possible but still effective , small wireless accelerometers ( measuring the acceleration of movement ) are used and attached to the wrists and ankles of the patient .", "the goal of this study was to test the efficacy of the varia ( video , accelerometry , and radar - induced activity recording ) system and algorithms for tonic  clonic and clonic seizures compared with that of semicontinuous monitoring by professional caregivers ( keeping an eye on multiple patients ) , as well as the independence , robustness , comfort , and user - friendliness of the system .", "we therefore measure long term in a home environment without uncomfortable and stigmatizing electroencephalography ( eeg ) electrodes rather than in a video / eeg monitoring unit , and the system therefore needs to be able to store the data and to allow visual verification of detected events .", "two patients were measured for one month in their rooms in an epilepsy center in flanders , belgium .", "as they returned home for the weekends and as the caregivers needed to get acquainted with the recording system , they were measured for a mean of 12 nights per patient .", "patient 1 is an 8-year - old girl with epilepsy e.c.i . and different types of seizures that typically last no more than 3  min : tonic  clonic seizures with or without faltering respiration , tonic seizures , and clonic seizures .", "she also exhibits myoclonic jerks manifesting as ( head ) nods or facial contractions with eye deviation .", "seizures are noticed based on semicontinuous video monitoring and when a scream or breathing problem is heard .", "patient 2 is a 9-year - old boy with lesional epilepsy due to herpes encephalitis , who is kept in bed with a restraining belt around the waist .", "clonic seizures of various duration , tonic , clonic , and myoclonic seizures and ( series of ) spasms .", "the caregivers use a babyphone and semicontinuous video monitoring to keep an eye on him .", "approval by the medical ethical commission of the antwerp university hospital , belgium and signed informed consent forms from all parents were obtained prior to inclusion in the study , which was performed in accordance with the 1964 declaration of helsinki .", "the system uses video , accelerometry ( acm ) , and radar - induced activity recording and is therefore named varia .", "it consists of an axis m1011 camera ( axis communications ab , sweden ) and a hygrosens rad - mod motion sensor based on the radar principle ( b  +  b thermo - technik gmbh , germany ) .", "the camera and radar are attached to a tripod that is placed close to the patient 's bed .", "the radar is added for detection of movements in the direction of the sensor and for measurement through sheets .", "radar technology is also used for detection of falling , wandering , sitting , or getting up , so we expect it to detect large and slow epileptic and nonepileptic movements , also those caused by other persons in the room .", "four shimmer sensors with integrated three - axis acm ( shimmer , ireland ) , streaming wireless ( bluetooth ) communication standards , are adjusted to allow recording of more than 10  h before batteries need recharging and are put in elastic bracelets worn around wrists and ankles . a laptop with a software application developed in labview ( national instruments corporation , us ) stores all movement data recorded by either camera , radar , or acm ( fig .", "the technical aspects of the recording system will be published in more detail by bonroy et al . and are briefly described in the supplementary material .", "clonic and clonic seizures based on a large database of video / electroencephalography ( eeg ) data were used .", "results of a patient - specific ( algorithm trained only on data of the patient itself ) , a nonpatient - specific ( algorithm trained on data not including those of the patient itself ) , and a semipatient - specific ( algorithm trained on data also including those of the patient itself ) approach were compared with the notes of professional caregivers . for a full description on how to implement these methodologies in practice", "professional caregivers were asked to write down all seizures the patients had , with time and description in a form of a clinical report .", "they recognize epileptic seizures ( one month training and yearly tuition ) , are awake during the whole night , know the patients , and are semicontinuously watching on average four patients with active epilepsy in front of a video screen , only leaving if one of them or one of the other ( maximum ) twelve patients need care . the events detected with the algorithms", "were visually inspected by a pediatric video / eeg specialist and a pediatric neurologist using the screening tool which is a graphical user interface developed in matlab ( mathworks inc . , us ) ( fig .", "2 ) . the screening tool displays information on the number of movement events and on their duration .", "the acm signals , a distribution graph , and the video images are depicted for each event . also , radar and video signals are shown , as well as the fraction ( percentage ) of movement within the event .", "the graph at the lower left corner sets out amplitude of all events based on acm signals and length of all events based on the three detection modalities . in other words", ", it shows the intensity and duration of the movement . after the application of each of the three developed seizure detection algorithms and during postprocessing , the following logical reasoning was used : rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient , we assume the seizure may have been noticed 5  min before or after the actual time in the notes.rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance.rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement.rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm.rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient , we assume the seizure may have been noticed 5  min before or after the actual time in the notes .", "rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance .", "rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement .", "rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm .", "rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "true positives ( tp ) are detected seizures with the extra seizures detected by the algorithm but not the caregiver mentioned in between brackets .", "false negatives ( fn ) are seizures reported by the caregiver but not detected by the algorithm . to calculate sensitivity , the formula tp  /  ( tp  +  fn ) was used .", "false positives ( fp ) are detections that were not seizures , which in some occasions were multiple alarms in one movement event . false detection rate ( fdr ) is calculated as the number of fp per night .", "two patients were measured for one month in their rooms in an epilepsy center in flanders , belgium .", "as they returned home for the weekends and as the caregivers needed to get acquainted with the recording system , they were measured for a mean of 12 nights per patient .", "patient 1 is an 8-year - old girl with epilepsy e.c.i . and different types of seizures that typically last no more than 3  min : tonic  clonic seizures with or without faltering respiration , tonic seizures , and clonic seizures .", "she also exhibits myoclonic jerks manifesting as ( head ) nods or facial contractions with eye deviation .", "seizures are noticed based on semicontinuous video monitoring and when a scream or breathing problem is heard .", "patient 2 is a 9-year - old boy with lesional epilepsy due to herpes encephalitis , who is kept in bed with a restraining belt around the waist .", "clonic seizures of various duration , tonic , clonic , and myoclonic seizures and ( series of ) spasms .", "the caregivers use a babyphone and semicontinuous video monitoring to keep an eye on him .", "approval by the medical ethical commission of the antwerp university hospital , belgium and signed informed consent forms from all parents were obtained prior to inclusion in the study , which was performed in accordance with the 1964 declaration of helsinki .", "the system uses video , accelerometry ( acm ) , and radar - induced activity recording and is therefore named varia .", "it consists of an axis m1011 camera ( axis communications ab , sweden ) and a hygrosens rad - mod motion sensor based on the radar principle ( b  +  b thermo - technik gmbh , germany ) .", "the camera and radar are attached to a tripod that is placed close to the patient 's bed .", "the radar is added for detection of movements in the direction of the sensor and for measurement through sheets .", "radar technology is also used for detection of falling , wandering , sitting , or getting up , so we expect it to detect large and slow epileptic and nonepileptic movements , also those caused by other persons in the room .", "four shimmer sensors with integrated three - axis acm ( shimmer , ireland ) , streaming wireless ( bluetooth ) communication standards , are adjusted to allow recording of more than 10  h before batteries need recharging and are put in elastic bracelets worn around wrists and ankles . a laptop with a software application developed in labview ( national instruments corporation , us ) stores all movement data recorded by either camera , radar , or acm ( fig .", "the technical aspects of the recording system will be published in more detail by bonroy et al . and are briefly described in the supplementary material .", "the algorithms developed by milosevic et al .   for tonic  clonic and clonic seizures based on a large database of video / electroencephalography ( eeg ) data were used .", "results of a patient - specific ( algorithm trained only on data of the patient itself ) , a nonpatient - specific ( algorithm trained on data not including those of the patient itself ) , and a semipatient - specific ( algorithm trained on data also including those of the patient itself ) approach were compared with the notes of professional caregivers . for a full description on how to implement these methodologies in practice", "professional caregivers were asked to write down all seizures the patients had , with time and description in a form of a clinical report .", "they recognize epileptic seizures ( one month training and yearly tuition ) , are awake during the whole night , know the patients , and are semicontinuously watching on average four patients with active epilepsy in front of a video screen , only leaving if one of them or one of the other ( maximum ) twelve patients need care .", "the events detected with the algorithms were visually inspected by a pediatric video / eeg specialist and a pediatric neurologist using the screening tool which is a graphical user interface developed in matlab ( mathworks inc . , us ) ( fig .", "2 ) . the screening tool displays information on the number of movement events and on their duration .", "the acm signals , a distribution graph , and the video images are depicted for each event . also , radar and video signals are shown , as well as the fraction ( percentage ) of movement within the event .", "the graph at the lower left corner sets out amplitude of all events based on acm signals and length of all events based on the three detection modalities . in other words , it shows the intensity and duration of the movement . after the application of each of the three developed seizure detection algorithms and during postprocessing , the following logical reasoning was used : rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient", ", we assume the seizure may have been noticed 5  min before or after the actual time in the notes.rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance.rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement.rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm.rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient , we assume the seizure may have been noticed 5  min before or after the actual time in the notes .", "rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance .", "rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement .", "rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm .", "rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "true positives ( tp ) are detected seizures with the extra seizures detected by the algorithm but not the caregiver mentioned in between brackets .", "false negatives ( fn ) are seizures reported by the caregiver but not detected by the algorithm . to calculate sensitivity , the formula tp  /  ( tp  +  fn ) was used . false positives ( fp ) are detections that were not seizures , which in some occasions were multiple alarms in one movement event", ". false detection rate ( fdr ) is calculated as the number of fp per night .", "monitoring for 15 nights for patient 1 and nine nights for patient 2 resulted in 9586 movement events . in this first validation , 322 events ( 3.4% or 15  min ) were discarded before algorithm application because the data of one or more accelerometers lasted less than 1  s or was completely missing . the results of the nonpatient - specific and semipatient - specific approaches can be found in table 1 .", "the nonpatient - specific approach resulted in a sensitivity of 90% and fdr of one per night for patient 1 and a sensitivity of 33% and fdr of almost two per night for patient 2 .", "the semipatient - specific approach gave somewhat better results with a sensitivity of 93% and fdr of a little over one per night for patient 1 and a sensitivity of 40% and fdr of one per night for patient 2 .", "results of the patient - specific approach are not depicted as it was immediately clear that many seizures were missed , many false alarms occurred , and the approach was not stable in between trials ( the results depended on which data were used for algorithm training ) .", "in this work , we validated algorithms for the detection of tonic  clonic and clonic epileptic seizures with a new ( wireless ) system using video , wireless accelerometry ( acm ) , and radar - induced activity recording , which is placed in a home environment for a prolonged period of time .", "three methodologies were tested : patient - specific , nonpatient - specific , and semipatient - specific algorithms .", "it seems that combining a large amount of data of many ( other ) patients as in the last two methodologies , instead of using a limited amount of data of one specific patient as in the first approach , yields better classification results .", "the semipatient - specific algorithm gave the best results , including 13 extra seizures detected ( 31% ) and eight seizures missed ( 19% ) compared with professional caregivers ' observations .", "of these eight , four were not even recorded in any of the movement data .", "possible explanations include a temporary defect in the system , mistakes in reporting by the caregiver ( one seizure was reported at 5:20  a.m. but found at 6:20  a.m. ) , or short seizures involving only the head ( not attached to an acm ) .", "the limitations of our setup are discomfort ( four accelerometers ) , noncontinuously recorded data ( only movement events ) , and lack of gold standard ( no electroencephalography or eeg ) .", "they are not able to detect the tonic phase of tonic  clonic seizures ( which involves muscle contraction rather than limb movement ) , so if the clonic phase is too short , the seizure will be missed : the alarm is set after 7  s of positive detection , so if the clonic phase lasts 6  s and this whole part is detected , the alarm will not be set off . finally , asymmetric tonic ", "clonic or clonic seizures during which the movements of the limbs are not synchronized and seizures with a low frequency of clonic jerks are often missed , since the spatial and frequency content of these seizures differ from the majority of seizures in the training set ( a large database of wired acm data synchronized with video / eeg ) . fig .", "we compared the results of the semipatient - specific algorithm with those of the screening tool of bonroy et al .  ", "( submitted ) where the most intense and longest movement events were visually inspected for seizures using the same datasets and the graphical user interface of fig .", "2 . the screening tool can be used before a diagnosis is made to quickly and retrospectively look at a night and report abnormal nocturnal behavior , including seizures . once the patient is diagnosed ( with epilepsy )", "this implies that we have to reduce the false detection rate ( fdr ) and increase the sensitivity .", "the comparison results , presented in table 2 , show an increase of sensitivity from 58% to 66% ( five seizures were missed , but 14 more were detected ) and a decrease of fdr per night from 15 to 0.7 . because the method of bonroy et al", ". selected a percentage of  most abnormal events irrespective of the number of movements or seizures that night , this automatically resulted in more false positives , so comparison of fdr is not relevant .", "moreover , 11 of the 16 false positives in our approach were due to a system failure ( broken channel or high - frequency noise ) , hence clustered alarms can be used to indicate this kind of events and reduce the number of false alarms .", "other publications reported on body - attached accelerometry for tonic  clonic and clonic seizure detection .", "lockman et al .   reached a sensitivity of 87% testing 6 patients with ( only ) 8 seizures ; however , patterson et al .   only obtained a sensitivity of 31%", "it is difficult to compare with this study and obtained results , however , mainly because the gold standard video / eeg was not used to annotate the data .", "furthermore , we did not review all video data ( only where the algorithm or caregiver indicated a seizure ) , and some of the seizures reported by the caregivers were not even found in the ( movement ) data .", "although setup without eeg is less optimal for seizure detection performance , it is optimal for testing the independence and robustness of the system and the patient - friendliness of the system in a long - term home environment . also , the second part of the gold standard , namely video , is present in our system .", "furthermore , a major advantage of our system is that it allows video storage for retrospective analysis . of the commercially available seizure detection systems reported by van de vel et al .", ", some keep a log of the detected seizures : epiwatcher   stores the time and duration of up to 20 detections , smartwatch , ,   keeps the time , duration , and movement patterns , epicare ,   saves the time and duration of an unspecified number of detections , and the mobile phone application epdetect   logs the raw accelerometer data .", "some studies report on video - based seizure detection ( so without acm ) , but a disadvantage is the difficulty to measure through bed linen , , . in order to improve the accuracy of the device", ", we are convinced that a multimodal approach is needed , and literature research as well as user questioning has persuaded us that the most important signals to be monitored are movement ( often an early manifestation of seizures ) and heart rate changes ( indicating clinically relevant seizures and part of the pathophysiological mechanism leading to sudden unexpected death in epilepsy or sudep ) .", "it is clear that the number of sensors should be reduced to one or two at the most .", "attachment of a seizure detection device to the upper arm is less comfortable than to the lower arm   and can easily move when for example changing clothes or not tightly attached ; so for the moment , we will continue our research with wrist - attached devices .", "detection of respiration changes is certainly useful , but these changes occur probably almost simultaneously with heart rhythm changes , respiration is a very slow signal , and heart rhythm changes are very easy to detect , which is why more studies focus on cardiac- than respiration - based detection , .", "oxygen saturation measurement and electrodermal activity ( eda ) seem promising , but changes are detected more slowly than heart rhythm changes , , and eda is measured using electrodes ( as are ecg and emg ) , which cause potential skin irritation , and there is a risk of losing the signal due to electrodes falling off or a bad contact with the skin , .", "furthermore , emg and respiration can not be measured at the wrist , and oxygen saturation can not be measured yet without adding a finger cuff , even though reflectance oximetry might be an option in the future .", "all of these arguments lead us to focus our future research on movement detection by acm in combination with heart rate detection by ppg ( photoplethysmography ) .", "the algorithm should be robust and independent of the number and functionality of used sensors , so that there is no rejection of the data , and to be able to use our future device patient - independently , we will be concentrating on adaptive ( learning ) algorithms .", "not only efficacy of the device will be important but also comfort ( including the reduction of sensors ) , user - friendliness ( less setup steps ) , and therapeutic ( e.g. , medication changes ) and social ( e.g. , quality of life including the impact of false alarms ) impact .", "therapeutic impact can be examined by using the logging function of a seizure detection device , but in order to look into social impact , the device obviously needs to allow online use ( with real - time alarming ) .", "the latter brings along additional challenges such as the need for fast computation time , low energy consumption , large storage capacity , and miniaturization of the device , which we need to address .", "finally , it would be interesting to compare the performance with that of commercially available seizure detection bracelets using accelerometry , such as smartwatch or epicare free , also tested in a home environment .", "this study demonstrates the first clinical results of an algorithm for the detection of tonic  clonic and clonic seizures tested on two patients with epilepsy monitored in a long - term home environment with the varia system ( video , accelerometry , and radar - induced activity recording ) developed by our team .", "compared with the professional caregivers ' observations , a mean sensitivity of 61.97% and false detection rate of 1.44 per night were obtained with the nonpatient - specific approach and a mean sensitivity of 66.87% and false detection rate of 1.16 per night with the semipatient - specific approach .", "we believe that , while privacy protection needs to be taken into account , the addition of video to accelerometry allows a seizure detection system not only to alarm the family or ( informal ) caregiver but also allows retrospective ( offline ) or emergency ( online ) verification of detected events .", "much work remains to develop a multimodal , learning , comfortable , and user - friendly device ."], "abstract_text": ["<S> purposethe aim of our study was to test the efficacy of the varia system ( video , accelerometry , and radar - induced activity recording ) and validation of accelerometry - based detection algorithms for nocturnal tonic  clonic and clonic seizures developed by our team.methodswe present the results of two patients with tonic  clonic and clonic seizures , measured for about one month in a home environment with four wireless accelerometers ( acm ) attached to wrists and ankles . </S>", "<S> the algorithms were developed using wired acm data synchronized with the gold standard video-/electroencephalography ( eeg ) and then run offline on the wireless acm signals . </S>", "<S> detection of seizures was compared with semicontinuous monitoring by professional caregivers ( keeping an eye on multiple patients).resultsthe best result for the two patients was obtained with the semipatient - specific algorithm which was developed using all patients with tonic  clonic and clonic seizures in our database with wired acm . </S>", "<S> it gave a mean sensitivity of 66.87% and false detection rate of 1.16 per night . </S>", "<S> this included 13 extra seizures detected ( 31% ) compared with professional caregivers ' observations.conclusionwhile the algorithms were previously validated in a controlled video / eeg monitoring unit with wired sensors , we now show the first results of long - term , wireless testing in a home environment . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Patients", "Measurement", "Analysis", "Results", "Discussion", "Conclusion", "Supplementary data", "Conflict of interest"], "sections": [["mainly for safety reasons , many patients and their families are looking for a seizure detection system that is efficient , comfortable , and easy to use .", "this implies that such a system should have a high detection sensitivity ( detect as many seizures as possible ) and low false alarm rate ; it should be unobtrusive , wireless , and in case of daytime use practically unnoticeable ; and it should allow long - term use in a home situation without the presence of professionals . alarming for safety reasons or reassurance", "is already used in fall detectors for the elderly , babyphones ( reacting to sound or movement ) , and glucose monitors for persons with diabetes , .", "in addition to being used for alarming , a detection system could allow offline storage or online streaming of selected data , allowing follow - up on treatment efficacy or summoning emergency support , respectively .", "our team is interested in the long - term observation of patients with epilepsy and detection of their seizures , .", "the focus lies on those convulsive seizures that can be dangerous because of their intensity ( possible injuries ) , duration , or serious consequences such as brain damage , autonomic dysregulation , or suffocation .", "clonic and clonic seizures with patients remaining in one place , their bed , mainly because of the need for supervision at night and because seizures often occur during sleep . as sensors need to be as unobtrusive as possible but still effective , small wireless accelerometers ( measuring the acceleration of movement ) are used and attached to the wrists and ankles of the patient .", "the goal of this study was to test the efficacy of the varia ( video , accelerometry , and radar - induced activity recording ) system and algorithms for tonic  clonic and clonic seizures compared with that of semicontinuous monitoring by professional caregivers ( keeping an eye on multiple patients ) , as well as the independence , robustness , comfort , and user - friendliness of the system .", "we therefore measure long term in a home environment without uncomfortable and stigmatizing electroencephalography ( eeg ) electrodes rather than in a video / eeg monitoring unit , and the system therefore needs to be able to store the data and to allow visual verification of detected events ."], ["two patients were measured for one month in their rooms in an epilepsy center in flanders , belgium .", "as they returned home for the weekends and as the caregivers needed to get acquainted with the recording system , they were measured for a mean of 12 nights per patient .", "patient 1 is an 8-year - old girl with epilepsy e.c.i . and different types of seizures that typically last no more than 3  min : tonic  clonic seizures with or without faltering respiration , tonic seizures , and clonic seizures .", "she also exhibits myoclonic jerks manifesting as ( head ) nods or facial contractions with eye deviation .", "seizures are noticed based on semicontinuous video monitoring and when a scream or breathing problem is heard .", "patient 2 is a 9-year - old boy with lesional epilepsy due to herpes encephalitis , who is kept in bed with a restraining belt around the waist .", "clonic seizures of various duration , tonic , clonic , and myoclonic seizures and ( series of ) spasms .", "the caregivers use a babyphone and semicontinuous video monitoring to keep an eye on him .", "approval by the medical ethical commission of the antwerp university hospital , belgium and signed informed consent forms from all parents were obtained prior to inclusion in the study , which was performed in accordance with the 1964 declaration of helsinki .", "the system uses video , accelerometry ( acm ) , and radar - induced activity recording and is therefore named varia .", "it consists of an axis m1011 camera ( axis communications ab , sweden ) and a hygrosens rad - mod motion sensor based on the radar principle ( b  +  b thermo - technik gmbh , germany ) .", "the camera and radar are attached to a tripod that is placed close to the patient 's bed .", "the radar is added for detection of movements in the direction of the sensor and for measurement through sheets .", "radar technology is also used for detection of falling , wandering , sitting , or getting up , so we expect it to detect large and slow epileptic and nonepileptic movements , also those caused by other persons in the room .", "four shimmer sensors with integrated three - axis acm ( shimmer , ireland ) , streaming wireless ( bluetooth ) communication standards , are adjusted to allow recording of more than 10  h before batteries need recharging and are put in elastic bracelets worn around wrists and ankles . a laptop with a software application developed in labview ( national instruments corporation , us ) stores all movement data recorded by either camera , radar , or acm ( fig .", "the technical aspects of the recording system will be published in more detail by bonroy et al . and are briefly described in the supplementary material .", "clonic and clonic seizures based on a large database of video / electroencephalography ( eeg ) data were used .", "results of a patient - specific ( algorithm trained only on data of the patient itself ) , a nonpatient - specific ( algorithm trained on data not including those of the patient itself ) , and a semipatient - specific ( algorithm trained on data also including those of the patient itself ) approach were compared with the notes of professional caregivers . for a full description on how to implement these methodologies in practice", "professional caregivers were asked to write down all seizures the patients had , with time and description in a form of a clinical report .", "they recognize epileptic seizures ( one month training and yearly tuition ) , are awake during the whole night , know the patients , and are semicontinuously watching on average four patients with active epilepsy in front of a video screen , only leaving if one of them or one of the other ( maximum ) twelve patients need care . the events detected with the algorithms", "were visually inspected by a pediatric video / eeg specialist and a pediatric neurologist using the screening tool which is a graphical user interface developed in matlab ( mathworks inc . , us ) ( fig .", "2 ) . the screening tool displays information on the number of movement events and on their duration .", "the acm signals , a distribution graph , and the video images are depicted for each event . also , radar and video signals are shown , as well as the fraction ( percentage ) of movement within the event .", "the graph at the lower left corner sets out amplitude of all events based on acm signals and length of all events based on the three detection modalities . in other words", ", it shows the intensity and duration of the movement . after the application of each of the three developed seizure detection algorithms and during postprocessing , the following logical reasoning was used : rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient , we assume the seizure may have been noticed 5  min before or after the actual time in the notes.rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance.rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement.rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm.rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient , we assume the seizure may have been noticed 5  min before or after the actual time in the notes .", "rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance .", "rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement .", "rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm .", "rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "true positives ( tp ) are detected seizures with the extra seizures detected by the algorithm but not the caregiver mentioned in between brackets .", "false negatives ( fn ) are seizures reported by the caregiver but not detected by the algorithm . to calculate sensitivity , the formula tp  /  ( tp  +  fn ) was used .", "false positives ( fp ) are detections that were not seizures , which in some occasions were multiple alarms in one movement event . false detection rate ( fdr ) is calculated as the number of fp per night ."], ["two patients were measured for one month in their rooms in an epilepsy center in flanders , belgium .", "as they returned home for the weekends and as the caregivers needed to get acquainted with the recording system , they were measured for a mean of 12 nights per patient .", "patient 1 is an 8-year - old girl with epilepsy e.c.i . and different types of seizures that typically last no more than 3  min : tonic  clonic seizures with or without faltering respiration , tonic seizures , and clonic seizures .", "she also exhibits myoclonic jerks manifesting as ( head ) nods or facial contractions with eye deviation .", "seizures are noticed based on semicontinuous video monitoring and when a scream or breathing problem is heard .", "patient 2 is a 9-year - old boy with lesional epilepsy due to herpes encephalitis , who is kept in bed with a restraining belt around the waist .", "clonic seizures of various duration , tonic , clonic , and myoclonic seizures and ( series of ) spasms .", "the caregivers use a babyphone and semicontinuous video monitoring to keep an eye on him .", "approval by the medical ethical commission of the antwerp university hospital , belgium and signed informed consent forms from all parents were obtained prior to inclusion in the study , which was performed in accordance with the 1964 declaration of helsinki ."], ["the system uses video , accelerometry ( acm ) , and radar - induced activity recording and is therefore named varia .", "it consists of an axis m1011 camera ( axis communications ab , sweden ) and a hygrosens rad - mod motion sensor based on the radar principle ( b  +  b thermo - technik gmbh , germany ) .", "the camera and radar are attached to a tripod that is placed close to the patient 's bed .", "the radar is added for detection of movements in the direction of the sensor and for measurement through sheets .", "radar technology is also used for detection of falling , wandering , sitting , or getting up , so we expect it to detect large and slow epileptic and nonepileptic movements , also those caused by other persons in the room .", "four shimmer sensors with integrated three - axis acm ( shimmer , ireland ) , streaming wireless ( bluetooth ) communication standards , are adjusted to allow recording of more than 10  h before batteries need recharging and are put in elastic bracelets worn around wrists and ankles . a laptop with a software application developed in labview ( national instruments corporation , us ) stores all movement data recorded by either camera , radar , or acm ( fig .", "the technical aspects of the recording system will be published in more detail by bonroy et al . and are briefly described in the supplementary material ."], ["the algorithms developed by milosevic et al .   for tonic  clonic and clonic seizures based on a large database of video / electroencephalography ( eeg ) data were used .", "results of a patient - specific ( algorithm trained only on data of the patient itself ) , a nonpatient - specific ( algorithm trained on data not including those of the patient itself ) , and a semipatient - specific ( algorithm trained on data also including those of the patient itself ) approach were compared with the notes of professional caregivers . for a full description on how to implement these methodologies in practice", "professional caregivers were asked to write down all seizures the patients had , with time and description in a form of a clinical report .", "they recognize epileptic seizures ( one month training and yearly tuition ) , are awake during the whole night , know the patients , and are semicontinuously watching on average four patients with active epilepsy in front of a video screen , only leaving if one of them or one of the other ( maximum ) twelve patients need care .", "the events detected with the algorithms were visually inspected by a pediatric video / eeg specialist and a pediatric neurologist using the screening tool which is a graphical user interface developed in matlab ( mathworks inc . , us ) ( fig .", "2 ) . the screening tool displays information on the number of movement events and on their duration .", "the acm signals , a distribution graph , and the video images are depicted for each event . also , radar and video signals are shown , as well as the fraction ( percentage ) of movement within the event .", "the graph at the lower left corner sets out amplitude of all events based on acm signals and length of all events based on the three detection modalities . in other words , it shows the intensity and duration of the movement . after the application of each of the three developed seizure detection algorithms and during postprocessing , the following logical reasoning was used : rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient", ", we assume the seizure may have been noticed 5  min before or after the actual time in the notes.rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance.rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement.rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm.rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "rule 1 : a margin of error of 5  min has been taken into account for the seizure reporting . as the caregiver might write down the seizure after having attended to the patient , we assume the seizure may have been noticed 5  min before or after the actual time in the notes .", "rule 2 : only seizures longer than 10  s are considered as possibly dangerous and therefore candidates for the alarm system , hence shorter seizures are not counted when calculating detection performance .", "rule 3 : an alarm is set after 7  s of continuous positive detection .", "clonic seizures is not typically detected by acm ; the tonic phase involves muscle contraction rather than limb movement .", "rule 4 : an alarm within 1  min after a previous one is ignored .", "a future real - time alarm system should allow the system to be turned off by the caregiver when checking on an alarm , and 1  min is considered too short for the caregiver to have checked on and left the patient after the first alarm .", "rule 5 : alarms caused by the caregiver waking or handling the patient ( medication administration ) and installing ( evening ) or removing ( morning ) the system and acm are ignored .", "again , at these times , the future real - time alarm system should be turned off by the caregiver .", "true positives ( tp ) are detected seizures with the extra seizures detected by the algorithm but not the caregiver mentioned in between brackets .", "false negatives ( fn ) are seizures reported by the caregiver but not detected by the algorithm . to calculate sensitivity , the formula tp  /  ( tp  +  fn ) was used . false positives ( fp ) are detections that were not seizures , which in some occasions were multiple alarms in one movement event", ". false detection rate ( fdr ) is calculated as the number of fp per night ."], ["monitoring for 15 nights for patient 1 and nine nights for patient 2 resulted in 9586 movement events . in this first validation , 322 events ( 3.4% or 15  min ) were discarded before algorithm application because the data of one or more accelerometers lasted less than 1  s or was completely missing . the results of the nonpatient - specific and semipatient - specific approaches can be found in table 1 .", "the nonpatient - specific approach resulted in a sensitivity of 90% and fdr of one per night for patient 1 and a sensitivity of 33% and fdr of almost two per night for patient 2 .", "the semipatient - specific approach gave somewhat better results with a sensitivity of 93% and fdr of a little over one per night for patient 1 and a sensitivity of 40% and fdr of one per night for patient 2 .", "results of the patient - specific approach are not depicted as it was immediately clear that many seizures were missed , many false alarms occurred , and the approach was not stable in between trials ( the results depended on which data were used for algorithm training ) ."], ["in this work , we validated algorithms for the detection of tonic  clonic and clonic epileptic seizures with a new ( wireless ) system using video , wireless accelerometry ( acm ) , and radar - induced activity recording , which is placed in a home environment for a prolonged period of time .", "three methodologies were tested : patient - specific , nonpatient - specific , and semipatient - specific algorithms .", "it seems that combining a large amount of data of many ( other ) patients as in the last two methodologies , instead of using a limited amount of data of one specific patient as in the first approach , yields better classification results .", "the semipatient - specific algorithm gave the best results , including 13 extra seizures detected ( 31% ) and eight seizures missed ( 19% ) compared with professional caregivers ' observations .", "of these eight , four were not even recorded in any of the movement data .", "possible explanations include a temporary defect in the system , mistakes in reporting by the caregiver ( one seizure was reported at 5:20  a.m. but found at 6:20  a.m. ) , or short seizures involving only the head ( not attached to an acm ) .", "the limitations of our setup are discomfort ( four accelerometers ) , noncontinuously recorded data ( only movement events ) , and lack of gold standard ( no electroencephalography or eeg ) .", "they are not able to detect the tonic phase of tonic  clonic seizures ( which involves muscle contraction rather than limb movement ) , so if the clonic phase is too short , the seizure will be missed : the alarm is set after 7  s of positive detection , so if the clonic phase lasts 6  s and this whole part is detected , the alarm will not be set off . finally , asymmetric tonic ", "clonic or clonic seizures during which the movements of the limbs are not synchronized and seizures with a low frequency of clonic jerks are often missed , since the spatial and frequency content of these seizures differ from the majority of seizures in the training set ( a large database of wired acm data synchronized with video / eeg ) . fig .", "we compared the results of the semipatient - specific algorithm with those of the screening tool of bonroy et al .  ", "( submitted ) where the most intense and longest movement events were visually inspected for seizures using the same datasets and the graphical user interface of fig .", "2 . the screening tool can be used before a diagnosis is made to quickly and retrospectively look at a night and report abnormal nocturnal behavior , including seizures . once the patient is diagnosed ( with epilepsy )", "this implies that we have to reduce the false detection rate ( fdr ) and increase the sensitivity .", "the comparison results , presented in table 2 , show an increase of sensitivity from 58% to 66% ( five seizures were missed , but 14 more were detected ) and a decrease of fdr per night from 15 to 0.7 . because the method of bonroy et al", ". selected a percentage of  most abnormal events irrespective of the number of movements or seizures that night , this automatically resulted in more false positives , so comparison of fdr is not relevant .", "moreover , 11 of the 16 false positives in our approach were due to a system failure ( broken channel or high - frequency noise ) , hence clustered alarms can be used to indicate this kind of events and reduce the number of false alarms .", "other publications reported on body - attached accelerometry for tonic  clonic and clonic seizure detection .", "lockman et al .   reached a sensitivity of 87% testing 6 patients with ( only ) 8 seizures ; however , patterson et al .   only obtained a sensitivity of 31%", "it is difficult to compare with this study and obtained results , however , mainly because the gold standard video / eeg was not used to annotate the data .", "furthermore , we did not review all video data ( only where the algorithm or caregiver indicated a seizure ) , and some of the seizures reported by the caregivers were not even found in the ( movement ) data .", "although setup without eeg is less optimal for seizure detection performance , it is optimal for testing the independence and robustness of the system and the patient - friendliness of the system in a long - term home environment . also , the second part of the gold standard , namely video , is present in our system .", "furthermore , a major advantage of our system is that it allows video storage for retrospective analysis . of the commercially available seizure detection systems reported by van de vel et al .", ", some keep a log of the detected seizures : epiwatcher   stores the time and duration of up to 20 detections , smartwatch , ,   keeps the time , duration , and movement patterns , epicare ,   saves the time and duration of an unspecified number of detections , and the mobile phone application epdetect   logs the raw accelerometer data .", "some studies report on video - based seizure detection ( so without acm ) , but a disadvantage is the difficulty to measure through bed linen , , . in order to improve the accuracy of the device", ", we are convinced that a multimodal approach is needed , and literature research as well as user questioning has persuaded us that the most important signals to be monitored are movement ( often an early manifestation of seizures ) and heart rate changes ( indicating clinically relevant seizures and part of the pathophysiological mechanism leading to sudden unexpected death in epilepsy or sudep ) .", "it is clear that the number of sensors should be reduced to one or two at the most .", "attachment of a seizure detection device to the upper arm is less comfortable than to the lower arm   and can easily move when for example changing clothes or not tightly attached ; so for the moment , we will continue our research with wrist - attached devices .", "detection of respiration changes is certainly useful , but these changes occur probably almost simultaneously with heart rhythm changes , respiration is a very slow signal , and heart rhythm changes are very easy to detect , which is why more studies focus on cardiac- than respiration - based detection , .", "oxygen saturation measurement and electrodermal activity ( eda ) seem promising , but changes are detected more slowly than heart rhythm changes , , and eda is measured using electrodes ( as are ecg and emg ) , which cause potential skin irritation , and there is a risk of losing the signal due to electrodes falling off or a bad contact with the skin , .", "furthermore , emg and respiration can not be measured at the wrist , and oxygen saturation can not be measured yet without adding a finger cuff , even though reflectance oximetry might be an option in the future .", "all of these arguments lead us to focus our future research on movement detection by acm in combination with heart rate detection by ppg ( photoplethysmography ) .", "the algorithm should be robust and independent of the number and functionality of used sensors , so that there is no rejection of the data , and to be able to use our future device patient - independently , we will be concentrating on adaptive ( learning ) algorithms .", "not only efficacy of the device will be important but also comfort ( including the reduction of sensors ) , user - friendliness ( less setup steps ) , and therapeutic ( e.g. , medication changes ) and social ( e.g. , quality of life including the impact of false alarms ) impact .", "therapeutic impact can be examined by using the logging function of a seizure detection device , but in order to look into social impact , the device obviously needs to allow online use ( with real - time alarming ) .", "the latter brings along additional challenges such as the need for fast computation time , low energy consumption , large storage capacity , and miniaturization of the device , which we need to address .", "finally , it would be interesting to compare the performance with that of commercially available seizure detection bracelets using accelerometry , such as smartwatch or epicare free , also tested in a home environment ."], ["this study demonstrates the first clinical results of an algorithm for the detection of tonic  clonic and clonic seizures tested on two patients with epilepsy monitored in a long - term home environment with the varia system ( video , accelerometry , and radar - induced activity recording ) developed by our team .", "compared with the professional caregivers ' observations , a mean sensitivity of 61.97% and false detection rate of 1.44 per night were obtained with the nonpatient - specific approach and a mean sensitivity of 66.87% and false detection rate of 1.16 per night with the semipatient - specific approach .", "we believe that , while privacy protection needs to be taken into account , the addition of video to accelerometry allows a seizure detection system not only to alarm the family or ( informal ) caregiver but also allows retrospective ( offline ) or emergency ( online ) verification of detected events .", "much work remains to develop a multimodal , learning , comfortable , and user - friendly device ."], [""], [""]]}
{"article_id": "PMC4204268", "article_text": ["angiomyolipoma ( aml ) is a benign mesenchymal tumour consisting of varying amounts of mature adipose tissue , smooth muscle , and thick walled blood vessels .", "it is relatively rare benign tumour appearing in about 0.3% of general population and accounts for 3% of solid renal masses .", "extra renal amls ( eramls ) are rare tumours that present as incidentalomas upon imaging for other conditions .", "case reports of extra renal aml are rare with < 60 reported cases since they were first described by friis and hjortrup in 1982 .", "our case described one of the largest ever reported myolipomas , which despite its size , can be rather asymptomatic .", "magnetic resonance imaging ( mri ) revealed 9.5 cm  8 cm  2 cm right adrenal mass [ figure 1 ] .", "the tumour was predominantly hyperintense with signal intensity similar to fat on t1/t2-weighted images and lost signal on the fat - suppressed sequences .", "a well - encapsulated , firm globular mass separate from the right kidney and no definable adrenal was found [ figures 2 and 3 ] .", "gross post - operative evaluation of the adrenal mass showed approximately 10 cm  8 cm  2 cm greyish white solid , smooth , and firm mass [ figure 4 ] .", "histological examination revealed benign adrenal cortical elements , with a predominance of mature benign adipose tissue interspersed with pockets of haemopoietic ( erythro - myeloid ) intermediates , and megakaryocytes which was confirmatory for the diagnosis of adrenal aml .", "angiomyolipoma is a rare clinical entity mostly involving kidney and is part of the group of tumour known as tumours of perivascular epithelioid cell origin .", "the most widely accepted theory of origin is adrenocortical cell metaplasia in response to stimuli , such as necrosis , inflammation , infection or stress ( meyer et al . ) .", "the presence of perivascular epithelioid cells is characteristic of aml since these cells show immunoreactivity for muscle markers ( epithelial membrane antigen , keratin , vimentin , desmin , and actin ) and hmb-45 .", "positive immunoreactivity for hmb-45 , a monoclonal antibody , is characteristic of amls and can be used to differentiate amls from other similar appearing lesions .", "one is isolated aml , and the other is that is associated with tuberous sclerosis .", "most patients of isolated cases are in the age group of 27 - 72 years , mean age being 43 years . although the majority of eramls are benign , two cases of metastatic and recurrent eramls have been reported .", "the demonstration of fat density ( hypodense ) within an adrenal mass by computed tomography is virtually diagnostic of aml .", "mri is sometimes required to demonstrate origin of the tumour , to define the tissue planes when the tumour is large and heterogeneous , and to distinguish benign from malignant lesions by comparing signal intensity ratios of adrenal to liver .", "a small , < 5 cm , asymptomatic myelolipoma could be followed - up over 1 - 2 year period with imaging controls . on the contrary , asymptomatic lesion or a large > 5 cm myelolipoma", "should be surgically excised , since there are reports of spontaneous rupture and haemorrhage of the mass presented with life - threatening cardiovascular shock .", "unstable patients may benefit from emergent tumour embolisation and a subsequently staged surgical resection . since it is a benign disease , its prognosis is good .", "nevertheless , follow - up is recommended because of atypical morphology . currently , there is no agreed protocol on follow - up but an ultrasound three to 6 months following the surgery with annual clinical examination for large tumours is recommended .", "the adrenal myolipoma is a rare urological entity , which seems to increase its frequency , probably due to causes affecting primarily the function and physiology of the adrenals .", "ever since its introduction by gagner in 1992 , laparoscopic adrenalectomy has become the standard of care for the treatment of functioning and non - functioning adrenal tumours .", "many authors have found a decrease in perioperative morbidity and convalescence after this procedure when compared with open surgery ."], "abstract_text": ["<S> angiomyolipoma ( aml ) is a rare mesenchymal tumour arising from perivascular epithelioid cells . </S>", "<S> it is most commonly seen in kidney , but rarely aml can arise in extra renal sites . </S>", "<S> adrenal aml is a very rare clinical entity , and very few cases have been reported so far . </S>", "<S> we present our experience with a 43-year - old female , who presented with right flank pain . </S>", "<S> magnetic resonance imaging showed a right adrenal mass . </S>", "<S> laparoscopic adrenelectomy was performed , and the histopathology report confirmed the diagnosis of aml . </S>", "<S> patient was discharged uneventfully . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["angiomyolipoma ( aml ) is a benign mesenchymal tumour consisting of varying amounts of mature adipose tissue , smooth muscle , and thick walled blood vessels .", "it is relatively rare benign tumour appearing in about 0.3% of general population and accounts for 3% of solid renal masses .", "extra renal amls ( eramls ) are rare tumours that present as incidentalomas upon imaging for other conditions .", "case reports of extra renal aml are rare with < 60 reported cases since they were first described by friis and hjortrup in 1982 .", "our case described one of the largest ever reported myolipomas , which despite its size , can be rather asymptomatic ."], ["magnetic resonance imaging ( mri ) revealed 9.5 cm  8 cm  2 cm right adrenal mass [ figure 1 ] .", "the tumour was predominantly hyperintense with signal intensity similar to fat on t1/t2-weighted images and lost signal on the fat - suppressed sequences .", "a well - encapsulated , firm globular mass separate from the right kidney and no definable adrenal was found [ figures 2 and 3 ] .", "gross post - operative evaluation of the adrenal mass showed approximately 10 cm  8 cm  2 cm greyish white solid , smooth , and firm mass [ figure 4 ] .", "histological examination revealed benign adrenal cortical elements , with a predominance of mature benign adipose tissue interspersed with pockets of haemopoietic ( erythro - myeloid ) intermediates , and megakaryocytes which was confirmatory for the diagnosis of adrenal aml ."], ["angiomyolipoma is a rare clinical entity mostly involving kidney and is part of the group of tumour known as tumours of perivascular epithelioid cell origin .", "the most widely accepted theory of origin is adrenocortical cell metaplasia in response to stimuli , such as necrosis , inflammation , infection or stress ( meyer et al . ) .", "the presence of perivascular epithelioid cells is characteristic of aml since these cells show immunoreactivity for muscle markers ( epithelial membrane antigen , keratin , vimentin , desmin , and actin ) and hmb-45 .", "positive immunoreactivity for hmb-45 , a monoclonal antibody , is characteristic of amls and can be used to differentiate amls from other similar appearing lesions .", "one is isolated aml , and the other is that is associated with tuberous sclerosis .", "most patients of isolated cases are in the age group of 27 - 72 years , mean age being 43 years . although the majority of eramls are benign , two cases of metastatic and recurrent eramls have been reported .", "the demonstration of fat density ( hypodense ) within an adrenal mass by computed tomography is virtually diagnostic of aml .", "mri is sometimes required to demonstrate origin of the tumour , to define the tissue planes when the tumour is large and heterogeneous , and to distinguish benign from malignant lesions by comparing signal intensity ratios of adrenal to liver .", "a small , < 5 cm , asymptomatic myelolipoma could be followed - up over 1 - 2 year period with imaging controls . on the contrary , asymptomatic lesion or a large > 5 cm myelolipoma", "should be surgically excised , since there are reports of spontaneous rupture and haemorrhage of the mass presented with life - threatening cardiovascular shock .", "unstable patients may benefit from emergent tumour embolisation and a subsequently staged surgical resection . since it is a benign disease , its prognosis is good .", "nevertheless , follow - up is recommended because of atypical morphology . currently , there is no agreed protocol on follow - up but an ultrasound three to 6 months following the surgery with annual clinical examination for large tumours is recommended ."], ["the adrenal myolipoma is a rare urological entity , which seems to increase its frequency , probably due to causes affecting primarily the function and physiology of the adrenals .", "ever since its introduction by gagner in 1992 , laparoscopic adrenalectomy has become the standard of care for the treatment of functioning and non - functioning adrenal tumours .", "many authors have found a decrease in perioperative morbidity and convalescence after this procedure when compared with open surgery ."]]}
{"article_id": "PMC3917400", "article_text": ["a 67-year - old male presented to the emergency eye centre ( eec ) complaining of a painful right eye of 5-day duration .", "clinical examination revealed a visual acuity ( va ) of 6/9 with minimal conjunctival injection , anterior chamber ( ac ) cells 1 + and posterior synechiae ( ps ) .", "a diagnosis of anterior uveitis was made and the patient was commenced on topical prednisolone 1% and topical cyclopentolate 1% .", "the response to this treatment was good and the steroids were slowly tapered over 1 month ; however , before cessation of treatment , the anterior segment inflammation worsened .", "the patient represented with va 6/36 and ac cells 3 + , extensive ps and a posterior vitritis .", "2 , the topical treatment was increased and an urgent referral was made to the uveitis service . at review ,", "4 weeks after presentation , the anterior and posterior segment inflammation remained and a white mass was noted in the peripheral nasal retina [ fig . 1 upper plate ] , almost at the ora serrata .", "a diagnosis of presumed toxoplasma chorioretinitis was made and treatment with pyrimethamine and sulfadiazine followed by oral clindamycin was initiated . on further questioning , it was discovered that there was the possibility of a previous pulmonary tuberculosis ( tb ) infection .", "he had had no previous treatment for active tuberculosis . in view of concern over possible reactivation of tb with an increase in oral steroids ,", "quantiferon and induced sputum sampling were also performed ( all negative ) and the dose of prednisolone was increased to 60 mg daily .", "blood cultures were also negative and there was no serological evidence of toxoplasma infection ( toxoplasma latex < 16 ) .", "( upper plate ) 10 mhz b - scan ultrasonography of subretinal mass ( nasal peripheral retina - white arrow ) ( middle plate ) hematoxylin and eosin stained section of the retinal biopsy showing brown fungal hyphae ( black arrows ) coursing through the abscess.(lower plate ) masson fontana stain showing a positive melanin brown reaction .", "thin arrows showing fungal septae and thick arrows indicate dichotomous branching of hyphae the patient responded to oral steroids and clindamycin , with a reduction in intraocular inflammation and improved va of 6/18 after 2 weeks treatment .", "however , when the oral prednisolone was gradually reduced and the clindamycin stopped ( after 5 weeks treatment ) , there was an increase in intraocular inflammation and va reduced to 3/60 .", "a diagnostic pars plana vitrectomy was therefore performed with vitreous sampling , which revealed a chronic vitritis without infectious agent or neoplasia .", "no herpes family viruses or toxoplasma organisms were identified on polymerase chain reaction testing . in order to clinch the diagnosis , combined cataract surgery , retinal biopsy , and intraocular oil tamponade was performed .", "the nasal sub - retinal mass was biopsied using a 23-gauge ( 23 g ) vitrector hand piece and samples sent to histopathology and microbiology .", "close inspection showed brown pigmented septate branching hyphae that stained strongly with masson fontana melanin stain and with a periodic acid schiff ( pas ) and silver stain [ fig .", "the presence of a melanin containing , septate , branching , and mycelial fungus on histopathology was diagnostic of phaeohyphomycosis infection , on morphology alone .", "prolonged fungal culture of the biopsy was negative and 18s ribosomal subunit polymerase chain reaction ( pcr ) was also negative ( most likely due to the presence of pcr inhibitors in the sample ) .", "the absence of a culture and pcr result did not permit precise speciation of the phaeohyphomycosis fungus .", "the patient remained under joint care and was treated with antifungal therapy for 6 months ; however , no primary source of fungal infection was identified . following retinal biopsy", "the patient developed a retinal fold secondary to proliferative vitreoretinopathy ( pvr ) , which extended from the biopsy site to the optic disc .", "vitrectomy , removal of silicone oil , epiretinal membrane peel , subretinal pvr removal and retinectomy were performed with laser retinopexy and intraocular gas tamponade .", "unfortunately , the patient required two further operations for rhegmatogenous retinal detachment . currently , the retina is flat with heavy silicone oil tamponade and the va is 2/60 .", "further retinal biopsy taken during vitrectomy for retinal detachment revealed no fungal elements on microscopy and intravitreal amphotericin b was also injected perioperatively .", "phaeohyphomycosis is a collective term that describes infection caused by darkly pigmented mycelial fungi , a feature secondary to the presence of melanin within their cell walls .", "the most common phaeohyphomycosis genera pathogenic to humans include exophilia , phialophora , wangiella , bipolaris , exserohilum , cladophialophora .", "morphology by histopathology is sufficient to make a diagnosis of phaehyphomycosis , characterized by branching , septate hyphae with confirmation of melanin by tinctorial staining ( masson fontana or an equivalent ) .", "whilst histopathology permits a clear diagnosis of phaeohyphomycosis , the precise species does require pcr and cultures .", "infection is presumed to be as a result of implantation following superficial trauma and exposure to contaminated soils or organic matter .", "systemic infections result either from direct invasion from a more superficial site , such as the paranasal sinuses or by means of haematogenous dissemination .", "the central nervous system is most frequently affected but other sites including lung , bone , and joint or peritoneum may also be involved .", ", primary cerebral phaeohyphomycosis seems to ignore this rationale with more than half of cases being fully immunocompetent .", "melanin within the cell walls of dematiaceous fungi may provide the reasons for this disparity ; it has been extensively investigated as a virulence factor .", "however , this is the first time a subretinal abscess caused by phaeohyphomycosis has been documented in the medical literature .", "the most plausible theory involves a haematogenous route ( metastatic infection ) transmission from a peripheral nidus to the subretinal space , which in turn initiated a posterior and anterior segment uveitis .", "patients with underlying lung disease appear to be at a increased risk of pulmonary phaeohyphomycosis .", "it is possible that , in this patient , the lung parenchyma may have been a source of infection , in the setting of chronic , low level immune suppression / modulation by the methotrexate therapy with reactivation of a previous self - limiting respiratory phaeomycotic infection , with subsequent dissemination .", "posterior segment pathology is usually diagnosed by characteristic clinical signs , extensive systemic investigations and non - invasive ocular investigations such as fluorescein angiography .", "however , it is not uncommon for vitreoretinal surgeons to aid in the diagnosis of a posterior uveitis of unknown aetiology by sampling vitreous , retina or choroidal tissue , leading to increased diagnostic accuracy and prompt treatment .", "reported 13 cases of retinochoroidal biopsy in unclear uveitis seven of the cases tissue biopsy helped direct specific treatment .", "intraocular lymphoma , recurrence of lymphoblastic leukaemia , chronic scleritis , toxoplasmosis , and metastasis were diagnoses confirmed as a result of the tissue biopsies performed .", "malignancy was also excluded in several cases , which allowed for a trial of medications such as oral steroids and anticytomegalovirus agents . in this case , it is difficult to postulate whether the final va and outcome have been improved by surgical intervention .", "it is clear that the patients intraocular inflammation was increasing rapidly and only high dose steroids subdued the response . without sub - retinal biopsy ,", "the specific treatment of this patient 's phaeohyphomycosis with antifungal medication would not have been possible and years of treatment for idiopathic uveitis may have ensued ."], "abstract_text": ["<S> a 67-year - old former gold miner with rheumatoid arthritis , treated with steroids and methotrexate , presented to eye casualty with a painful right eye . </S>", "<S> examination revealed an anterior uveitis and despite an initial response to topical steroids , the intraocular inflammation worsened with anterior and posterior uveitis development . </S>", "<S> re - examination showed a white mass in the peripheral nasal retina initially suspected of being active toxoplasmosis infection and anti - toxoplasmosis treatment commenced . </S>", "<S> after improvement and tapering of this treatment , the intraocular inflammation reoccurred . </S>", "<S> cytopathological examination of a pars plana vitrectomy obtained vitreous sample that showed a non - diagnostic non - infectious chronic vitritis . </S>", "<S> the vitreoretinal surgeons elected to do a direct biopsy of the white subretinal mass in the peripheral nasal area . </S>", "<S> this revealed , quite unexpectedly , an abscess containing pigmented phaeohyphomycosis fungi . </S>", "<S> this case report documents the multidisciplinary approach that assisted in clinching a final diagnosis and the role of sub - retinal biopsy in this unprecedented scenario . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 67-year - old male presented to the emergency eye centre ( eec ) complaining of a painful right eye of 5-day duration .", "clinical examination revealed a visual acuity ( va ) of 6/9 with minimal conjunctival injection , anterior chamber ( ac ) cells 1 + and posterior synechiae ( ps ) .", "a diagnosis of anterior uveitis was made and the patient was commenced on topical prednisolone 1% and topical cyclopentolate 1% .", "the response to this treatment was good and the steroids were slowly tapered over 1 month ; however , before cessation of treatment , the anterior segment inflammation worsened .", "the patient represented with va 6/36 and ac cells 3 + , extensive ps and a posterior vitritis .", "2 , the topical treatment was increased and an urgent referral was made to the uveitis service . at review ,", "4 weeks after presentation , the anterior and posterior segment inflammation remained and a white mass was noted in the peripheral nasal retina [ fig . 1 upper plate ] , almost at the ora serrata .", "a diagnosis of presumed toxoplasma chorioretinitis was made and treatment with pyrimethamine and sulfadiazine followed by oral clindamycin was initiated . on further questioning , it was discovered that there was the possibility of a previous pulmonary tuberculosis ( tb ) infection .", "he had had no previous treatment for active tuberculosis . in view of concern over possible reactivation of tb with an increase in oral steroids ,", "quantiferon and induced sputum sampling were also performed ( all negative ) and the dose of prednisolone was increased to 60 mg daily .", "blood cultures were also negative and there was no serological evidence of toxoplasma infection ( toxoplasma latex < 16 ) .", "( upper plate ) 10 mhz b - scan ultrasonography of subretinal mass ( nasal peripheral retina - white arrow ) ( middle plate ) hematoxylin and eosin stained section of the retinal biopsy showing brown fungal hyphae ( black arrows ) coursing through the abscess.(lower plate ) masson fontana stain showing a positive melanin brown reaction .", "thin arrows showing fungal septae and thick arrows indicate dichotomous branching of hyphae the patient responded to oral steroids and clindamycin , with a reduction in intraocular inflammation and improved va of 6/18 after 2 weeks treatment .", "however , when the oral prednisolone was gradually reduced and the clindamycin stopped ( after 5 weeks treatment ) , there was an increase in intraocular inflammation and va reduced to 3/60 .", "a diagnostic pars plana vitrectomy was therefore performed with vitreous sampling , which revealed a chronic vitritis without infectious agent or neoplasia .", "no herpes family viruses or toxoplasma organisms were identified on polymerase chain reaction testing . in order to clinch the diagnosis , combined cataract surgery , retinal biopsy , and intraocular oil tamponade was performed .", "the nasal sub - retinal mass was biopsied using a 23-gauge ( 23 g ) vitrector hand piece and samples sent to histopathology and microbiology .", "close inspection showed brown pigmented septate branching hyphae that stained strongly with masson fontana melanin stain and with a periodic acid schiff ( pas ) and silver stain [ fig .", "the presence of a melanin containing , septate , branching , and mycelial fungus on histopathology was diagnostic of phaeohyphomycosis infection , on morphology alone .", "prolonged fungal culture of the biopsy was negative and 18s ribosomal subunit polymerase chain reaction ( pcr ) was also negative ( most likely due to the presence of pcr inhibitors in the sample ) .", "the absence of a culture and pcr result did not permit precise speciation of the phaeohyphomycosis fungus .", "the patient remained under joint care and was treated with antifungal therapy for 6 months ; however , no primary source of fungal infection was identified . following retinal biopsy", "the patient developed a retinal fold secondary to proliferative vitreoretinopathy ( pvr ) , which extended from the biopsy site to the optic disc .", "vitrectomy , removal of silicone oil , epiretinal membrane peel , subretinal pvr removal and retinectomy were performed with laser retinopexy and intraocular gas tamponade .", "unfortunately , the patient required two further operations for rhegmatogenous retinal detachment . currently , the retina is flat with heavy silicone oil tamponade and the va is 2/60 .", "further retinal biopsy taken during vitrectomy for retinal detachment revealed no fungal elements on microscopy and intravitreal amphotericin b was also injected perioperatively ."], ["phaeohyphomycosis is a collective term that describes infection caused by darkly pigmented mycelial fungi , a feature secondary to the presence of melanin within their cell walls .", "the most common phaeohyphomycosis genera pathogenic to humans include exophilia , phialophora , wangiella , bipolaris , exserohilum , cladophialophora .", "morphology by histopathology is sufficient to make a diagnosis of phaehyphomycosis , characterized by branching , septate hyphae with confirmation of melanin by tinctorial staining ( masson fontana or an equivalent ) .", "whilst histopathology permits a clear diagnosis of phaeohyphomycosis , the precise species does require pcr and cultures .", "infection is presumed to be as a result of implantation following superficial trauma and exposure to contaminated soils or organic matter .", "systemic infections result either from direct invasion from a more superficial site , such as the paranasal sinuses or by means of haematogenous dissemination .", "the central nervous system is most frequently affected but other sites including lung , bone , and joint or peritoneum may also be involved .", ", primary cerebral phaeohyphomycosis seems to ignore this rationale with more than half of cases being fully immunocompetent .", "melanin within the cell walls of dematiaceous fungi may provide the reasons for this disparity ; it has been extensively investigated as a virulence factor .", "however , this is the first time a subretinal abscess caused by phaeohyphomycosis has been documented in the medical literature .", "the most plausible theory involves a haematogenous route ( metastatic infection ) transmission from a peripheral nidus to the subretinal space , which in turn initiated a posterior and anterior segment uveitis .", "patients with underlying lung disease appear to be at a increased risk of pulmonary phaeohyphomycosis .", "it is possible that , in this patient , the lung parenchyma may have been a source of infection , in the setting of chronic , low level immune suppression / modulation by the methotrexate therapy with reactivation of a previous self - limiting respiratory phaeomycotic infection , with subsequent dissemination .", "posterior segment pathology is usually diagnosed by characteristic clinical signs , extensive systemic investigations and non - invasive ocular investigations such as fluorescein angiography .", "however , it is not uncommon for vitreoretinal surgeons to aid in the diagnosis of a posterior uveitis of unknown aetiology by sampling vitreous , retina or choroidal tissue , leading to increased diagnostic accuracy and prompt treatment .", "reported 13 cases of retinochoroidal biopsy in unclear uveitis seven of the cases tissue biopsy helped direct specific treatment .", "intraocular lymphoma , recurrence of lymphoblastic leukaemia , chronic scleritis , toxoplasmosis , and metastasis were diagnoses confirmed as a result of the tissue biopsies performed .", "malignancy was also excluded in several cases , which allowed for a trial of medications such as oral steroids and anticytomegalovirus agents . in this case , it is difficult to postulate whether the final va and outcome have been improved by surgical intervention .", "it is clear that the patients intraocular inflammation was increasing rapidly and only high dose steroids subdued the response . without sub - retinal biopsy ,", "the specific treatment of this patient 's phaeohyphomycosis with antifungal medication would not have been possible and years of treatment for idiopathic uveitis may have ensued ."]]}
{"article_id": "PMC3877458", "article_text": ["in 1978 , primary health care was seen as the solution to the inadequate illness management systems that had developed throughout the world .", "it was hoped that phc would address some of the major inequalities in health that were observed by a balanced system of treatment and disease prevention . the world health organization ( who ) envisaged that phc would take place as close as possible to where people live and work and be the first element of a continuing health care process .", "additionally , health service collaboration and multi - professional partnerships were expected to replace professional boundaries and competition . to sum up", ", phc used family physician as the most important ways to increase equitable access and utilization of public health services . in any case , family physician , with the key elements of first contact , comprehensiveness , continuity , and coordination of patient care , is central to the health of any health care system .", "recent government initiatives are supporting general practice to deliver health care through a variety of means and personnel .", "these initiatives have included expanding the roles of nurses in general practice and enabling better access to allied health services through general practitioners ( gps ) .", "evidence clearly indicates the effectiveness of teams in improving the outcomes , especially in chronic disease .", "now , people in most of the countries use professional and educated nurses [ called nurse practitioner ( np ) ] besides family physician or are under their supervision for diagnosis , treatment , and specifically health education of the family . in accordance with the international council of nurses definition ,", " a nurse practitioner is a registered nurse who has acquired the expert knowledge base , complex decision - making skills , and clinical competencies for expanded practice , the characteristics of which are shaped by the context and/or country in which s / he is credentialed to practice .", " the royal college of nursing ( rcn ) defines an np as a registered nurse who has undertaken a specific course of study of at least first - degree ( honors ) level and who : \n makes professionally autonomous decision , for which he or she is accountable;works collaboratively with other health care professionals;provides counseling and health education;screens patient for disease risk factors and early signs of illness ; andhas the authority to admit or discharge patients from their caseload , and refers patients to other health care providers as appropriate . \n ", "makes professionally autonomous decision , for which he or she is accountable ; works collaboratively with other health care professionals ; provides counseling and health education ; screens patient for disease risk factors and early signs of illness ; and has the authority to admit or discharge patients from their caseload , and refers patients to other health care providers as appropriate . in new reform toward family physician in iran ,", "there is not enough attention toward nurses , their services , and specifically their role in education and counseling .", "according to the health - for - all goals , the central concern of nurses in primary care should be the prevention of the disease by educating individuals and families on healthy lifestyles , and teaching community health workers and traditional birth practitioners to carry out many of the functions that nurses themselves have normally performed .", "whereas a big part of the family physician program has not been realized in iran yet ( especially in big cities with a population of over 20,000 ) , most graduate nurses are now working in the school of nursing and midwifery , hospitals , and ambulatory clinics with limited responsibilities .", "in addition , most graduate nurses in our community can play an important role in preventing medicine and family physician team . consequently , in spite of their ability and capacity , they have a brief role in our family physician team & their job description .", "the main goal of this study was to review the role and situation of nps in the new reform for better use of their capabilities in health care services .", "this is a qualitative , comparative ( role of nurses in family physicians team ) , and cross - sectional study that was carried out with triangulation method in three stages in 2011 . in the first stage , we conducted a literature review to design a conceptual framework .", "the second stage was a comparative study on four countries that had great experiences in these two fields ( family physician reform and nps ) and included usa , uk , austria , and canada , based on purposive sampling . in the third stage ,", "all experts were completely familiar with the health system and had academic and research experiences in nursing education system in iran .", "the survey on health systems in different countries indicates that many of the health sectors have begun their reforms with two main goals , including quality improvement and cost limiting .", "family physician and referral system are among the few options that can maintain and even upgrade the quality of services , reducing the costs simultaneously . as each health system in each country", "has its specific referral system , iran is now on the verge of implementing a  best fit  referral system reform .", "so , it is crucial to well define the roles of each component , such as nurses , in this system . in the united states ,", "nps are licensed by the state in which they practice , and have a national board certification ( usually through the american nurses credentialing center or american academy of nurse practitioner ) .", "most nps specialize in a particular field of medical care , and there are as many types of nps as there are medical specialties .", "nps provide high - quality , cost - effective individualized care that is comparable to the health care provided by the physicians , and np services are often covered by insurance providers .", "role of nurses in some organizations in canada , the nurses involved in clinics should participate in specific training course of diagnosis and management of health care after registration .", "then , they are allowed to prescribe drugs , and lab and other diagnostic tests .", "having access to patient medical record , nps can reduce unnecessary visits . according to the standards of practice for nps , the college of nurses of ontario states that they offer  the full scope of primary health care practice , including consultation with physicians or other health care professionals when the client requires care beyond the nps scope of practice . ", "further , they are accountable for establishing a consultative relationship with a physician and consultation occurs with a family physician ; however , nps may consult with a specialist physician , if appropriate to the situation and practice setting .", "no legislative or regulatory deterrent related to the scope of practice exists for the nps to refer to specialist physicians . in austria , nurses in nursing home and maternity do some of the medical procedures under the supervision of physicians .", "these nurses are educated , and can treat , inject drugs , and request for some diagnostic tests for their patients . in the united kingdom ,", "the gps monopoly on primary care has been broken by nurse - led services , including nhs direct , which provides 24-h health advice by telephone , and nhs walk - in centers , which treat minor illness and injury .", "practice nurses and nps increasingly substitute for gps in the care of minor illness and routine management of chronic diseases such as asthma , diabetes , and coronary heart disease . in england and wales ,", "the role of the nps had evolved from that of undertaking traditional , medically prescribed nursing tasks in a treatment room to undertaking screening activities and health assessments of new patients , with over 50% of the nps managing chronic disease clinics .", "six experts in two panels were asked to confirm the role of nurses in family physician reform .", "they suggested that special courses are needed to empower our graduated nurses for their new rules .", "meanwhile , using nps as substitution of gps does not seem to be logically correct due to the gps workload and the social and cultural background in iran .", "the survey on health systems in different countries indicates that many of the health sectors have begun their reforms with two main goals , including quality improvement and cost limiting .", "family physician and referral system are among the few options that can maintain and even upgrade the quality of services , reducing the costs simultaneously . as each health system in each country", "has its specific referral system , iran is now on the verge of implementing a  best fit  referral system reform .", "so , it is crucial to well define the roles of each component , such as nurses , in this system .", "in the united states , nps are licensed by the state in which they practice , and have a national board certification ( usually through the american nurses credentialing center or american academy of nurse practitioner ) .", "most nps specialize in a particular field of medical care , and there are as many types of nps as there are medical specialties .", "nps provide high - quality , cost - effective individualized care that is comparable to the health care provided by the physicians , and np services are often covered by insurance providers .", "role of nurses in some organizations in canada , the nurses involved in clinics should participate in specific training course of diagnosis and management of health care after registration .", "then , they are allowed to prescribe drugs , and lab and other diagnostic tests .", "having access to patient medical record , nps can reduce unnecessary visits . according to the standards of practice for nps , the college of nurses of ontario states that they offer  the full scope of primary health care practice , including consultation with physicians or other health care professionals when the client requires care beyond the nps scope of practice . ", "further , they are accountable for establishing a consultative relationship with a physician and consultation occurs with a family physician ; however , nps may consult with a specialist physician , if appropriate to the situation and practice setting .", "no legislative or regulatory deterrent related to the scope of practice exists for the nps to refer to specialist physicians . in austria , nurses in nursing home and maternity do some of the medical procedures under the supervision of physicians .", "these nurses are educated , and can treat , inject drugs , and request for some diagnostic tests for their patients . in the united kingdom ,", "the gps monopoly on primary care has been broken by nurse - led services , including nhs direct , which provides 24-h health advice by telephone , and nhs walk - in centers , which treat minor illness and injury .", "practice nurses and nps increasingly substitute for gps in the care of minor illness and routine management of chronic diseases such as asthma , diabetes , and coronary heart disease . in england and wales ,", "the role of the nps had evolved from that of undertaking traditional , medically prescribed nursing tasks in a treatment room to undertaking screening activities and health assessments of new patients , with over 50% of the nps managing chronic disease clinics .", "six experts in two panels were asked to confirm the role of nurses in family physician reform .", "they suggested that special courses are needed to empower our graduated nurses for their new rules .", "meanwhile , using nps as substitution of gps does not seem to be logically correct due to the gps workload and the social and cultural background in iran .", "according to the who , in most countries , nurses are the most important group of health workers in terms of numbers , closeness to people and their health problems , and understanding of community needs . there is still debate in nursing and medical circles about what the focus of their role should be .", "for example , lauder , sharkey , and reel suggest that nps and gps in rural and remote areas should be interchangeable and that the focus should be on the competency of the person delivering care rather than the right of one discipline ( medicine ) to perform a particular role .", "in contrast , campbell believes that public acceptance of nurses as gp substitutes would be poor , and therefore suggests that a better role for nurses in primary care would be health education and illness prevention . on the other hand , in our country ( iran ) until now , nurses have not got a competent role in health sector reform .", "given that consumers have limited understanding of phc , it is not surprising they do not articulate confidence in nps acting autonomously but rather as complementary to gps , perhaps undertaking initial assessment for triage purposes and providing ongoing management , education , and support under the gps delegation .", "they would also like them to be family oriented and holistic in their practice , supporting their emotional and social needs in the context of their family lives .", "it means that any effort to change nurses roles in health care services should accompany with some social and cultural changes toward their competencies . in iran , whereas a noticeable reform toward  family physician  is going on , redefining nurses role is essential .", "they can perform more active roles in associating with gps in the clinics of family physicians , both in urban and rural areas , even with higher degrees of autonomy .", "their competencies for case management , education , treatment , and follow - up may repair many of the pitfalls we are confronting in health care services .", "of course , if it has to be done , special courses are needed to empower graduated nurses for their new rules .", "meanwhile , substitution of gps with nps does not seem to be a good suggestion due to gp workload and social and cultural background in iran .", "thus , it can be concluded that in order to achieve the goals of family physicians reform and also utilize the maximum capacity of trained nurses , a fixed and definite place must be considered for them in the gp team to provide their services under the supervision of family physicians .", "their active role not only enriches the family physician team , but also might be crucial in patients training , which is currently one of our weaknesses ."], "abstract_text": ["<S> background : most countries use educated nurses called  nurse practitioners  ( nps ) besides the family physicians for diagnosis , treatment , and specifically health education of the family . </S>", "<S> the main goal of this study was to redefine the role of nps for better use of their capabilities in the so - called  family physician reform  in iran.materials and methods : this is a qualitative and comparative study carried out in three stages ( triangulation method ) in 2011 . in the first stage </S>", "<S> , we conducted a literature review to design a conceptual framework . </S>", "<S> the second stage was a comparative study on four countries . in this study , we focused on the role of nps , which in turn helped to redefine this role in the health sector reform of iran . in the third stage , </S>", "<S> two expert panels were involved and the suggested roles were confirmed.results:in the united states , nps are licensed by the state in which they practice and have a national board certification . in canada , nurses involved in clinics should participate in specific training course of diagnosis and management of health care after registration . in austria , </S>", "<S> nurses in nursing homes and maternity do some of the medical procedures under the supervision of the physicians . in the united kingdom , </S>", "<S> nps increasingly substitute for gps in the care of minor illness and routine management of chronic diseases.conclusions:there is still debate in nursing and medical circles about what the focus of the np roles should be . in iran , whereas a noticeable reform toward  family physician  </S>", "<S> is ongoing , redefining the nurses role is essential . </S>", "<S> they can perform more active roles in associating with gps in the clinics of family physicians , both in urban and rural areas , even with higher degrees of autonomy . </S>"], "labels": null, "section_names": ["I", "M", "R", "First stage: Literature review", "Second stage: Comparative study", "Third stage: Expert panels", "D"], "sections": [["in 1978 , primary health care was seen as the solution to the inadequate illness management systems that had developed throughout the world .", "it was hoped that phc would address some of the major inequalities in health that were observed by a balanced system of treatment and disease prevention . the world health organization ( who ) envisaged that phc would take place as close as possible to where people live and work and be the first element of a continuing health care process .", "additionally , health service collaboration and multi - professional partnerships were expected to replace professional boundaries and competition . to sum up", ", phc used family physician as the most important ways to increase equitable access and utilization of public health services . in any case , family physician , with the key elements of first contact , comprehensiveness , continuity , and coordination of patient care , is central to the health of any health care system .", "recent government initiatives are supporting general practice to deliver health care through a variety of means and personnel .", "these initiatives have included expanding the roles of nurses in general practice and enabling better access to allied health services through general practitioners ( gps ) .", "evidence clearly indicates the effectiveness of teams in improving the outcomes , especially in chronic disease .", "now , people in most of the countries use professional and educated nurses [ called nurse practitioner ( np ) ] besides family physician or are under their supervision for diagnosis , treatment , and specifically health education of the family . in accordance with the international council of nurses definition ,", " a nurse practitioner is a registered nurse who has acquired the expert knowledge base , complex decision - making skills , and clinical competencies for expanded practice , the characteristics of which are shaped by the context and/or country in which s / he is credentialed to practice .", " the royal college of nursing ( rcn ) defines an np as a registered nurse who has undertaken a specific course of study of at least first - degree ( honors ) level and who : \n makes professionally autonomous decision , for which he or she is accountable;works collaboratively with other health care professionals;provides counseling and health education;screens patient for disease risk factors and early signs of illness ; andhas the authority to admit or discharge patients from their caseload , and refers patients to other health care providers as appropriate . \n ", "makes professionally autonomous decision , for which he or she is accountable ; works collaboratively with other health care professionals ; provides counseling and health education ; screens patient for disease risk factors and early signs of illness ; and has the authority to admit or discharge patients from their caseload , and refers patients to other health care providers as appropriate . in new reform toward family physician in iran ,", "there is not enough attention toward nurses , their services , and specifically their role in education and counseling .", "according to the health - for - all goals , the central concern of nurses in primary care should be the prevention of the disease by educating individuals and families on healthy lifestyles , and teaching community health workers and traditional birth practitioners to carry out many of the functions that nurses themselves have normally performed .", "whereas a big part of the family physician program has not been realized in iran yet ( especially in big cities with a population of over 20,000 ) , most graduate nurses are now working in the school of nursing and midwifery , hospitals , and ambulatory clinics with limited responsibilities .", "in addition , most graduate nurses in our community can play an important role in preventing medicine and family physician team . consequently , in spite of their ability and capacity , they have a brief role in our family physician team & their job description ."], ["the main goal of this study was to review the role and situation of nps in the new reform for better use of their capabilities in health care services .", "this is a qualitative , comparative ( role of nurses in family physicians team ) , and cross - sectional study that was carried out with triangulation method in three stages in 2011 . in the first stage , we conducted a literature review to design a conceptual framework .", "the second stage was a comparative study on four countries that had great experiences in these two fields ( family physician reform and nps ) and included usa , uk , austria , and canada , based on purposive sampling . in the third stage ,", "all experts were completely familiar with the health system and had academic and research experiences in nursing education system in iran ."], ["the survey on health systems in different countries indicates that many of the health sectors have begun their reforms with two main goals , including quality improvement and cost limiting .", "family physician and referral system are among the few options that can maintain and even upgrade the quality of services , reducing the costs simultaneously . as each health system in each country", "has its specific referral system , iran is now on the verge of implementing a  best fit  referral system reform .", "so , it is crucial to well define the roles of each component , such as nurses , in this system . in the united states ,", "nps are licensed by the state in which they practice , and have a national board certification ( usually through the american nurses credentialing center or american academy of nurse practitioner ) .", "most nps specialize in a particular field of medical care , and there are as many types of nps as there are medical specialties .", "nps provide high - quality , cost - effective individualized care that is comparable to the health care provided by the physicians , and np services are often covered by insurance providers .", "role of nurses in some organizations in canada , the nurses involved in clinics should participate in specific training course of diagnosis and management of health care after registration .", "then , they are allowed to prescribe drugs , and lab and other diagnostic tests .", "having access to patient medical record , nps can reduce unnecessary visits . according to the standards of practice for nps , the college of nurses of ontario states that they offer  the full scope of primary health care practice , including consultation with physicians or other health care professionals when the client requires care beyond the nps scope of practice . ", "further , they are accountable for establishing a consultative relationship with a physician and consultation occurs with a family physician ; however , nps may consult with a specialist physician , if appropriate to the situation and practice setting .", "no legislative or regulatory deterrent related to the scope of practice exists for the nps to refer to specialist physicians . in austria , nurses in nursing home and maternity do some of the medical procedures under the supervision of physicians .", "these nurses are educated , and can treat , inject drugs , and request for some diagnostic tests for their patients . in the united kingdom ,", "the gps monopoly on primary care has been broken by nurse - led services , including nhs direct , which provides 24-h health advice by telephone , and nhs walk - in centers , which treat minor illness and injury .", "practice nurses and nps increasingly substitute for gps in the care of minor illness and routine management of chronic diseases such as asthma , diabetes , and coronary heart disease . in england and wales ,", "the role of the nps had evolved from that of undertaking traditional , medically prescribed nursing tasks in a treatment room to undertaking screening activities and health assessments of new patients , with over 50% of the nps managing chronic disease clinics .", "six experts in two panels were asked to confirm the role of nurses in family physician reform .", "they suggested that special courses are needed to empower our graduated nurses for their new rules .", "meanwhile , using nps as substitution of gps does not seem to be logically correct due to the gps workload and the social and cultural background in iran ."], ["the survey on health systems in different countries indicates that many of the health sectors have begun their reforms with two main goals , including quality improvement and cost limiting .", "family physician and referral system are among the few options that can maintain and even upgrade the quality of services , reducing the costs simultaneously . as each health system in each country", "has its specific referral system , iran is now on the verge of implementing a  best fit  referral system reform .", "so , it is crucial to well define the roles of each component , such as nurses , in this system ."], ["in the united states , nps are licensed by the state in which they practice , and have a national board certification ( usually through the american nurses credentialing center or american academy of nurse practitioner ) .", "most nps specialize in a particular field of medical care , and there are as many types of nps as there are medical specialties .", "nps provide high - quality , cost - effective individualized care that is comparable to the health care provided by the physicians , and np services are often covered by insurance providers .", "role of nurses in some organizations in canada , the nurses involved in clinics should participate in specific training course of diagnosis and management of health care after registration .", "then , they are allowed to prescribe drugs , and lab and other diagnostic tests .", "having access to patient medical record , nps can reduce unnecessary visits . according to the standards of practice for nps , the college of nurses of ontario states that they offer  the full scope of primary health care practice , including consultation with physicians or other health care professionals when the client requires care beyond the nps scope of practice . ", "further , they are accountable for establishing a consultative relationship with a physician and consultation occurs with a family physician ; however , nps may consult with a specialist physician , if appropriate to the situation and practice setting .", "no legislative or regulatory deterrent related to the scope of practice exists for the nps to refer to specialist physicians . in austria , nurses in nursing home and maternity do some of the medical procedures under the supervision of physicians .", "these nurses are educated , and can treat , inject drugs , and request for some diagnostic tests for their patients . in the united kingdom ,", "the gps monopoly on primary care has been broken by nurse - led services , including nhs direct , which provides 24-h health advice by telephone , and nhs walk - in centers , which treat minor illness and injury .", "practice nurses and nps increasingly substitute for gps in the care of minor illness and routine management of chronic diseases such as asthma , diabetes , and coronary heart disease . in england and wales ,", "the role of the nps had evolved from that of undertaking traditional , medically prescribed nursing tasks in a treatment room to undertaking screening activities and health assessments of new patients , with over 50% of the nps managing chronic disease clinics ."], ["six experts in two panels were asked to confirm the role of nurses in family physician reform .", "they suggested that special courses are needed to empower our graduated nurses for their new rules .", "meanwhile , using nps as substitution of gps does not seem to be logically correct due to the gps workload and the social and cultural background in iran ."], ["according to the who , in most countries , nurses are the most important group of health workers in terms of numbers , closeness to people and their health problems , and understanding of community needs . there is still debate in nursing and medical circles about what the focus of their role should be .", "for example , lauder , sharkey , and reel suggest that nps and gps in rural and remote areas should be interchangeable and that the focus should be on the competency of the person delivering care rather than the right of one discipline ( medicine ) to perform a particular role .", "in contrast , campbell believes that public acceptance of nurses as gp substitutes would be poor , and therefore suggests that a better role for nurses in primary care would be health education and illness prevention . on the other hand , in our country ( iran ) until now , nurses have not got a competent role in health sector reform .", "given that consumers have limited understanding of phc , it is not surprising they do not articulate confidence in nps acting autonomously but rather as complementary to gps , perhaps undertaking initial assessment for triage purposes and providing ongoing management , education , and support under the gps delegation .", "they would also like them to be family oriented and holistic in their practice , supporting their emotional and social needs in the context of their family lives .", "it means that any effort to change nurses roles in health care services should accompany with some social and cultural changes toward their competencies . in iran , whereas a noticeable reform toward  family physician  is going on , redefining nurses role is essential .", "they can perform more active roles in associating with gps in the clinics of family physicians , both in urban and rural areas , even with higher degrees of autonomy .", "their competencies for case management , education , treatment , and follow - up may repair many of the pitfalls we are confronting in health care services .", "of course , if it has to be done , special courses are needed to empower graduated nurses for their new rules .", "meanwhile , substitution of gps with nps does not seem to be a good suggestion due to gp workload and social and cultural background in iran .", "thus , it can be concluded that in order to achieve the goals of family physicians reform and also utilize the maximum capacity of trained nurses , a fixed and definite place must be considered for them in the gp team to provide their services under the supervision of family physicians .", "their active role not only enriches the family physician team , but also might be crucial in patients training , which is currently one of our weaknesses ."]]}
{"article_id": "PMC3118535", "article_text": ["despite advancements in renal replacement therapy , acute kidney injury ( aki ) is a frequent complication with severe implications for the critically ill patient . published mortality rates for intensive care unit patients with aki range between 30 and 70% , and aki alone remains an independent risk factor for mortality even after adjustment for demographics and severity of illness [ 1 , 2 ] .", "kidney ischemia - reperfusion injury ( iri ) occurs in various clinical settings including shock , sepsis , organ transplantation , and vascular surgery .", "it has become apparent that clinically much of the high patient mortality can be attributed to the onset of systemic inflammatory response syndrome ( sirs ) and progression to multiple organ failure ( mof ) .   since the 1950s", "pulmonary dysfunction was thought to result from  increased permeability of congested pulmonary capillaries ,  and this was dubbed the  uremic lung  . even in the acute setting , however , kidney dysfunction directly contributes to the onset of remote organ injury .", "for example , increased kidney ischemia time during complex aortic surgery is associated not only with acute and chronic renal failure , but also with an increased incidence of remote organ injury and death [ 46 ] .  ", "clinical and translational laboratory studies have demonstrated the relevance of interactions between the injured kidney and distant organs , and complex mechanisms of crosstalk between injured kidneys and remote organs such as the lungs , liver , heart , gut , brain , and hematologic system have been identified .", "recent data highlights the importance of both the innate and adaptive immune response , activation of proinflammatory cascades , and an alteration of transcriptional events in remote organs during ischemic aki .", "the purpose of this paper is to review emerging concepts of organ crosstalk and recent experimental data regarding the activation and systemic expression of proinflammatory pathways during ischemic aki . for a complete list of abbreviations used in this paper , please see table 1", "kidney iri remains a major cause of aki in both native and transplanted organs and lacks a specific treatment aside from renal replacement therapy .", "the local effects of kidney iri begin in the most vulnerable regions of the organ , after which a cascade of microvascular inflammation propagates . although 25% of cardiac output contributes to renal blood flow , only a fraction ultimately reaches the vasa recta of the renal medulla , with the majority reaching the renal cortex .", "therefore , even slight alterations in total renal blood flow may lead to anoxic injury in the medulla , resulting in tubular dysfunction , salt wasting , and glomerular vasoconstriction through tubuloglomerular feedback [ 8 , 9 ] .  ", "kidney iri activates an inflammatory response which results in endothelial cell activation , leukocyte adhesion and entrapment , and compromised microvascular blood flow .", "inflammation in the postischemic kidney triggers the upregulation of leukocyte adhesion molecules , toll - like receptors , and downstream transcription factors , which all contribute to disruption of the integrity of the renal vascular endothelium .", "adhesion molecules such as integrins and selectins along with proinflammatory cytokines propagate cellular injury not only locally in the renal tubular epithelial cells but also travel to remote organs where genomic markers of injury are upregulated and phenotypic injury occurs .", "unfortunately , selective inhibition of cytokines and adhesion molecules such as tumor necrosis factor- ( tnf- ) and intercellular adhesion molecule ( icam-1 ) have failed to demonstrate global attenuation of both local and remote organ injury during experimental models of ischemic aki [ 1316 ] .", "however , -melanocyte - stimulating hormone ( -msh ) , a cytokine with broad antiinflammatory , anticytotoxic , and antiapoptotic properties , has demonstrated success in treating the inflammatory phenotype in rodents . in this model ,", "treatment with -msh has attenuated both renal and pulmonary injury during ischemic aki [ 13 , 17 ] .", "the largely unsuccessful effort to ameliorate mof with specific anti - inflammatory therapeutics highlights the complexity of the systemic response to kidney iri . from these early experimental studies ,", "it is possible that the multiple inflammatory pathways activated in each organ system represent a unique response to ischemic aki . despite a paucity of data ,", "several different pathophysiologic responses to ischemic aki in remote organs have been identified ( figure 1 ) . in the heart ,", "increased expression of tnf and interleukin-1 ( il-1 ) are associated with myocyte apoptosis . in the brain , increased expression of chemokines including keratinocyte chemoattractant ( kc , a brain il-8 homologue ) and granulocyte colony - stimulating factor ( g - csf ) are seen with increased vascular permeability .", "additional responses in the lungs , gut , and liver have been discovered , and these individual organ responses will be detailed later in this paper .", "we will now focus on cellular mediators , specifically those involved in the innate and adaptive immune system , which may connect local ischemic aki with distant organ injury .", "the innate immune system plays an important role in mediating the inflammatory response during ischemic aki .", "traditionally , innate immunity elicits an immediate , preprogrammed response to tissue injury that lacks immunologic memory .", "it is composed of plasma proteins ( complement ) , cells ( neutrophils , macrophages , and natural killer cells ) , and physical barriers .", "proposed initiators of the innate immune response during iri include the activation of toll - like receptors ( tlrs ) and the release of reactive oxygen species ( nitric oxide and superoxide anion ) and mitochondrial products .", "the complement system , particularly the alternative pathway , is activated , stimulating release of cytokines and subsequent activation of neutrophils , endothelium , and macrophages .  ", "tlrs are a family of transmembrane proteins which serve as major pattern recognition receptors , binding to a wide range of microbial products and endogenous ligands released as a consequence of injury .", "tlr - dependent signaling serves as a rapid response mechanism to local tissue damage and has been implicated in early activation of the innate immune response during kidney iri [ 11 , 20 ] .", "renal tubular epithelial cells constitutively express tlr2 and tlr4 , and iri results in selective upregulation of these tlrs .", "experimental studies of kidney iri have demonstrated attenuation of renal structural and functional injury in both tlr2/ and tlr4/mice , implicating tlr2 and tlr4 signaling in   renal damage [ 21 , 22 ] .", "tlr2 may also play a role in transplantation tolerance by decreasing the infiltration of t cells , dendritic cells , and macrophages .  ", "t cells may act during early iri despite lacking alloantigenic stimulation , which challenges the traditional function of t cells as exclusive mediators of the adaptive immune response .", "significant evidence exists to support the role of t cells in gut , heart , and lung ischemia - reperfusion injury [ 2428 ] .", "likewise , experimental data also points toward antigen - independent t - cell activation in ischemic aki through cytokines secreted by macrophages and dendritic cells , chemokines , oxygen free radicals and the complement system [ 7 , 19 , 29 ] .  ", "for example , mice deficient in cd4 + and cd8 + cells and athymic mice demonstrate protection from experimental ischemic aki ; however , only adoptive transfer of t cells of the cd4 + type restores this injury [ 30 , 31 ] .", "not all t cells , however , perpetuate inflammatory damage during ischemic aki ; some populations of t cells confer renal protection after iri , particularly the th2 phenotype of cd4 + t cells and t regulatory cells .", "additional recent experimental data has characterized specific populations of t lymphocytes trafficking to remote organs that may facilitate organ crosstalk during ischemic aki . in a rodent model of kidney iri ,", "a distinct influx of cd3 + ( t ) cells with a predominant cd8 + t cell subpopulation was identified 24  hours after experimental kidney iri .", "these pulmonary t cells acquired increased expression of activation markers suggesting that ischemic aki induced trafficking of activated t cells which contributed to pulmonary injury , specifically apoptosis .", "the specific role of subpopulations of t cells in the pathogenesis of ischemic aki remains a stimulating topic of current and future investigation , both in the innate and adaptive immune response to kidney iri .", "the adaptive immune system relies on the specificity of antigen receptors on both b and t cells that respond to millions of various antigenic molecular structures .", "once stimulated by an antigen , b cells ( humoral immunity ) produce specific antibodies , perform opsonization to facilitate phagocytosis , and activate the complement system .", "the t - cell receptor ( tcr ) responds to presented antigen peptides by activating macrophages to kill phagocytosed microbes , directly destroying infected cells and by releasing cytokines to promote further response ( cell - mediated immunity ) .", "antigen - dependent t - cell activation has been demonstrated in experimental models of renal iri [ 34 , 35 ] .", "for example , mice with a restricted tcr cell repertoire suffer less injury and promote a far diminished inflammatory response during renal iri .", "additionally , athymic mice that subsequently underwent adoptive transfer with these tcr - restricted t cells failed to restore injury as severely as seen with transfer of wild - type t - cells .", "t cells also play a role in long term renal modification , as an increased number of both activated and effector - memory t cells have been observed in postischemic kidneys as long as 6 weeks after iri .", "these t cells may in fact recognize antigens released during kidney iri and subsequently target the kidney in an autoimmune response , leading to long - term progression of renal dysfunction .", "this hypothesis was demonstrated in a murine adoptive transfer model in which nave mice received t cells from mice who were 6  weeks after kidney iri and subsequently developed albuminuria .", "little is known about the role of b cells in renal iri ; however , b - cell - deficient mice demonstrated renal protection in the early phase of experimental iri .   in summary , both the innate and adaptive immune responses play critical roles in the pathophysiology of ischemic aki . either following antigen stimulation or in the presence of proinflammatory chemokines and oxygen free radicals ,", "t cells undergo early activation and serve as a bridge between adaptive and innate immunity .", "this specific host immune response to kidney iri facilitates distant organ crosstalk along with soluble proinflammatory mediators and will be further discussed in the following paragraphs .", "the combination of aki with acute lung injury ( ali ) remains a formidable challenge to clinicians caring for critically ill patients . in the setting of mof , aki and ali", "occur more frequently together than any other combination of organ systems , and predicted mortality approaches 80% [ 40 , 41 ] .", "this exceedingly high mortality can not be attributed solely to volume overload ; leukocyte trafficking , uremic toxins , and oxidative stress mechanisms all likely contribute to this devastating clinical syndrome . for the purposes of this paper , ali   represents a pao2/fio2 ratio < 300 combined with chest radiograph findings of acute bilateral infiltrates in the absence of elevated cardiac filling pressures , as defined by the america - european consensus conference on acute respiratory distress syndrome ( ards ) .", "while the mechanisms for ischemic aki - induced ali remain incompletely understood , several studies point toward a self - propagating cycle with activation of proinflammatory and proapoptotic pathways ( figure 2 ) .", "aki leads to lung injury and inflammation , and in turn , ali and its attendant hypoxemia and hypercapnia worsened by mechanical ventilation with high positive end - expiratory pressure leads to diminishing renal hemodynamics and function .", "lung injury during ischemic aki features marked pulmonary vascular permeability , erythrocyte sludging in lung capillaries , interstitial edema , focal alveolar hemorrhage , and inflammatory cell infiltration [ 12 , 43 , 44 ] .", "mechanisms for decreased alveolar fluid clearance include downregulation of pulmonary epithelial salt and water transporters including enac , na , k - atpase , and aquaporins during ischemic aki [ 4345 ] .", "this specific response likely contributes to the increased microvascular permeability and clinical pulmonary edema frequently encountered in the setting of ischemic aki and mof .", "experimental studies have identified a distinct pulmonary functional response and genomic signature induced during ischemic aki which differs from that induced by uremia alone .", "a comprehensive genomic map and ontology analysis revealed many of these top differentially expressed genes participating in proinflammatory and proapoptotic pathways .", "genes with early and sustained activation at 6 and 36  hours after ischemic aki included neutrophilic granule protein ( ngp ) , serum amyloid a3 ( saa3 ) , and interleukin 1-receptor type ii ( il-1r2 ) .   further investigation by deng et al .  ", "has also identified an early pulmonary inflammatory response with rapid activation of transcription factors nf-b and ap-1 during ischemic aki . by 4 hours ,", "leukocytes play a fundamental role in the development of ali , and several studies have documented lung leukocyte activation and trafficking during experimental aki with early and sustained neutrophil sequestration [ 46 , 47 ] .", "additional experiments by klein et al . have demonstrated early expression of the neutrophil chemokine keratinocyte - derived chemokine ( kc / cxcl1 ) and macrophage inflammatory protein ( mip-2/cxcl-2 ) along with increased pulmonary myeloperoxidase activity and pulmonary microvascular permeability during ischemic aki .", "the authors also examined interleukin-6 , a neutrophil - recruiting chemokine previously identified as a candidate gene by preferential expression in whole lung tissue during ischemic aki , and determined it was critical for influencing lymphocyte trafficking and pulmonary permeability .  ", "while neutrophils are key mediators in several extrapulmonary models of ali such as sepsis and mesenteric iri , their role in ischemic aki - induced ali remains to be elucidated .", "uremic neutrophils have even demonstrated a protective effect in the setting of ali . clearly , leukocyte trafficking and the innate immune response play a complex and important role in mediating the pulmonary inflammatory response and dysfunction during renal iri . though not traditionally associated with a proinflammatory response ,", "pulmonary apoptosis plays a critical role in the genomic and phenotypic response to ischemic aki .", "apoptosis of pulmonary epithelial and endothelial cells along with delayed leukocyte apoptosis occurs during ali [ 5052 ] .", "pulmonary apoptosis disrupts tethering forces involved in cell - to - cell and cell - to - extracellular matrix interactions , leading to a loss of endothelial barrier function and increased vascular permeability . in a rodent model ,", "our laboratory has characterized caspase - dependent pulmonary apoptosis which is abrogated by the administration of a broad - spectrum caspase inhibitor .", "not only did caspase inhibition attenuate the pulmonary functional injury , but the proapoptotic response to ischemic aki appears to be mediated via a tnf receptor-1 ( tnf - r-1 ) dependent pathway .  ", "our research has also demonstrated t - cell - dependent pulmonary apoptosis during ischemic aki . along with identification of activated t cells trafficking to the lungs during ischemic aki , we have found that pulmonary apoptosis was subsequently attenuated in t - cell - deficient animals .", "further pursuit of the specific pathway for t cell and tnfr1-dependent pulmonary apoptosis remains an active focus of future investigation in our research laboratory .", "cardiovascular collapse is one of the most common causes of death in the setting of aki , yet the mechanisms involved are not entirely understood .", "kelly has demonstrated left ventricular dilation , increased left ventricular end diastolic and end systolic diameter , increased relaxation time , and decreased fractional shortening in an experimental model of ischemic aki", ". mechanisms of cardiac injury during ischemic aki include cardiac myocyte apoptosis and neutrophil infiltration , which have been attributed to increased cardiac and systemic tnf , il-1 , and icam-1 expression [ 56 , 57 ] .", "when kidney ischemic time is decreased , cardiac il-1 and icam expression , along with myocyte necrosis , decreased correspondingly .", "effects of ischemic aki on the central nervous system ( cns ) are evident clinically when mental status changes develop . of note , renal replacement therapy fails to fully correct this cns manifestation of renal failure .", "uremic toxins certainly contribute to many symptoms of encephalopathy ; however , cellular and soluble inflammatory mediators have also been implicated .", "preclinical data identified an increase of kc , g - csf , and glial fibrillary acidic protein ( gfap ) in the cerebral cortex and hippocampus of the brain , which may serve to recruit neutrophils to sites of neuronal damage .", "this may result from either increased local neuronal production or an alteration of the blood - brain barrier ; further research is required to delineate the source .", "a cell - mediated proinflammatory response has also been identified with activation of microglial cells ( brain macrophages ) during ischemic aki .", "ischemic aki is also implicated in oxidative stress , inflammation , apoptosis , and tissue damage in hepatocytes .", "hepatic stellate cells ( hscs ) regulate leukocyte trafficking and the secretion of chemokines such as il-8 , and crosstalk between hscs likely occurs via a c - jun n - terminal kinase pathway .", "oxidative stress during ischemic aki causes hepatic malondialdehyde , an index of lipid peroxidation , to increase while total glutathione , an antioxidant , decreases .", "proinflammatory cytokine tnf expression and hepatic cellular apoptosis is also evident during ischemic aki .  ", "previous investigators and clinicians have labeled the gut as the  motor  of mof because of its ability to amplify the systemic sirs response in the setting of shock and gut hypoperfusion [ 6264 ] .", "these mechanisms include increased intestinal permeability , interactions between host and bacterial pathogens , and propagation of toxins to distant organs via the lymphatic system [ 63 , 65 ] and could potentially play a role during ischemic aki .", "clinical studies have long demonstrated the increased secretion of potassium by the colon and rectum in response to aki , and recent literature linked channel - inducing factor , a potassium channel regulator found in both the kidney and colon , to ischemic aki [ 67 , 68 ] .", "the role of the gut in response to organ crosstalk during ischemic aki remains a fascinating topic of future investigation .", "aki is a frequent complication amongst hospitalized patients , with grave implications in the setting of mof .", "ischemic aki initiates a cascade of proinflammatory pathways , and through the release of soluble mediators and activation of the host innate and adaptive immune systems , it facilitates organ crosstalk and remote organ injury . as our understanding of the postischemic kidney 's role in mediating organ crosstalk continues to evolve , further laboratory investigation into the remote organ response to this devastating injury"], "abstract_text": ["<S> acute kidney injury ( aki ) is a common complication during inpatient hospitalization , and clinical outcomes remain poor despite advancements in renal replacement therapy . </S>", "<S> aki in the setting of multiple organ failure ( mof ) remains a formidable challenge to clinicians and incurs an unacceptably high mortality rate . </S>", "<S> kidney ischemia - reperfusion injury ( iri ) incites a proinflammatory cascade and releases cellular and soluble mediators with systemic implications for remote organ injury . </S>", "<S> evidence from preclinical models cites mechanisms of organ crosstalk during ischemic aki including the expression of cellular adhesion molecules , lymphocyte trafficking , release of proinflammatory cytokines and chemokines , and modification of the host innate and adaptive immune response systems . in this paper </S>", "<S> , the influence of kidney iri on systemic inflammation and distant organ injury will be examined . </S>", "<S> recent experimental data and evolving concepts of organ crosstalk during ischemic aki will also be discussed in detail . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Ischemic AKI", "3. Evolving Concepts of Organ Crosstalk during Ischemic AKI", "4. Conclusion"], "sections": [["despite advancements in renal replacement therapy , acute kidney injury ( aki ) is a frequent complication with severe implications for the critically ill patient . published mortality rates for intensive care unit patients with aki range between 30 and 70% , and aki alone remains an independent risk factor for mortality even after adjustment for demographics and severity of illness [ 1 , 2 ] .", "kidney ischemia - reperfusion injury ( iri ) occurs in various clinical settings including shock , sepsis , organ transplantation , and vascular surgery .", "it has become apparent that clinically much of the high patient mortality can be attributed to the onset of systemic inflammatory response syndrome ( sirs ) and progression to multiple organ failure ( mof ) .   since the 1950s", "pulmonary dysfunction was thought to result from  increased permeability of congested pulmonary capillaries ,  and this was dubbed the  uremic lung  . even in the acute setting , however , kidney dysfunction directly contributes to the onset of remote organ injury .", "for example , increased kidney ischemia time during complex aortic surgery is associated not only with acute and chronic renal failure , but also with an increased incidence of remote organ injury and death [ 46 ] .  ", "clinical and translational laboratory studies have demonstrated the relevance of interactions between the injured kidney and distant organs , and complex mechanisms of crosstalk between injured kidneys and remote organs such as the lungs , liver , heart , gut , brain , and hematologic system have been identified .", "recent data highlights the importance of both the innate and adaptive immune response , activation of proinflammatory cascades , and an alteration of transcriptional events in remote organs during ischemic aki .", "the purpose of this paper is to review emerging concepts of organ crosstalk and recent experimental data regarding the activation and systemic expression of proinflammatory pathways during ischemic aki . for a complete list of abbreviations used in this paper , please see table 1"], ["kidney iri remains a major cause of aki in both native and transplanted organs and lacks a specific treatment aside from renal replacement therapy .", "the local effects of kidney iri begin in the most vulnerable regions of the organ , after which a cascade of microvascular inflammation propagates . although 25% of cardiac output contributes to renal blood flow , only a fraction ultimately reaches the vasa recta of the renal medulla , with the majority reaching the renal cortex .", "therefore , even slight alterations in total renal blood flow may lead to anoxic injury in the medulla , resulting in tubular dysfunction , salt wasting , and glomerular vasoconstriction through tubuloglomerular feedback [ 8 , 9 ] .  ", "kidney iri activates an inflammatory response which results in endothelial cell activation , leukocyte adhesion and entrapment , and compromised microvascular blood flow .", "inflammation in the postischemic kidney triggers the upregulation of leukocyte adhesion molecules , toll - like receptors , and downstream transcription factors , which all contribute to disruption of the integrity of the renal vascular endothelium .", "adhesion molecules such as integrins and selectins along with proinflammatory cytokines propagate cellular injury not only locally in the renal tubular epithelial cells but also travel to remote organs where genomic markers of injury are upregulated and phenotypic injury occurs .", "unfortunately , selective inhibition of cytokines and adhesion molecules such as tumor necrosis factor- ( tnf- ) and intercellular adhesion molecule ( icam-1 ) have failed to demonstrate global attenuation of both local and remote organ injury during experimental models of ischemic aki [ 1316 ] .", "however , -melanocyte - stimulating hormone ( -msh ) , a cytokine with broad antiinflammatory , anticytotoxic , and antiapoptotic properties , has demonstrated success in treating the inflammatory phenotype in rodents . in this model ,", "treatment with -msh has attenuated both renal and pulmonary injury during ischemic aki [ 13 , 17 ] .", "the largely unsuccessful effort to ameliorate mof with specific anti - inflammatory therapeutics highlights the complexity of the systemic response to kidney iri . from these early experimental studies ,", "it is possible that the multiple inflammatory pathways activated in each organ system represent a unique response to ischemic aki . despite a paucity of data ,", "several different pathophysiologic responses to ischemic aki in remote organs have been identified ( figure 1 ) . in the heart ,", "increased expression of tnf and interleukin-1 ( il-1 ) are associated with myocyte apoptosis . in the brain , increased expression of chemokines including keratinocyte chemoattractant ( kc , a brain il-8 homologue ) and granulocyte colony - stimulating factor ( g - csf ) are seen with increased vascular permeability .", "additional responses in the lungs , gut , and liver have been discovered , and these individual organ responses will be detailed later in this paper .", "we will now focus on cellular mediators , specifically those involved in the innate and adaptive immune system , which may connect local ischemic aki with distant organ injury .", "the innate immune system plays an important role in mediating the inflammatory response during ischemic aki .", "traditionally , innate immunity elicits an immediate , preprogrammed response to tissue injury that lacks immunologic memory .", "it is composed of plasma proteins ( complement ) , cells ( neutrophils , macrophages , and natural killer cells ) , and physical barriers .", "proposed initiators of the innate immune response during iri include the activation of toll - like receptors ( tlrs ) and the release of reactive oxygen species ( nitric oxide and superoxide anion ) and mitochondrial products .", "the complement system , particularly the alternative pathway , is activated , stimulating release of cytokines and subsequent activation of neutrophils , endothelium , and macrophages .  ", "tlrs are a family of transmembrane proteins which serve as major pattern recognition receptors , binding to a wide range of microbial products and endogenous ligands released as a consequence of injury .", "tlr - dependent signaling serves as a rapid response mechanism to local tissue damage and has been implicated in early activation of the innate immune response during kidney iri [ 11 , 20 ] .", "renal tubular epithelial cells constitutively express tlr2 and tlr4 , and iri results in selective upregulation of these tlrs .", "experimental studies of kidney iri have demonstrated attenuation of renal structural and functional injury in both tlr2/ and tlr4/mice , implicating tlr2 and tlr4 signaling in   renal damage [ 21 , 22 ] .", "tlr2 may also play a role in transplantation tolerance by decreasing the infiltration of t cells , dendritic cells , and macrophages .  ", "t cells may act during early iri despite lacking alloantigenic stimulation , which challenges the traditional function of t cells as exclusive mediators of the adaptive immune response .", "significant evidence exists to support the role of t cells in gut , heart , and lung ischemia - reperfusion injury [ 2428 ] .", "likewise , experimental data also points toward antigen - independent t - cell activation in ischemic aki through cytokines secreted by macrophages and dendritic cells , chemokines , oxygen free radicals and the complement system [ 7 , 19 , 29 ] .  ", "for example , mice deficient in cd4 + and cd8 + cells and athymic mice demonstrate protection from experimental ischemic aki ; however , only adoptive transfer of t cells of the cd4 + type restores this injury [ 30 , 31 ] .", "not all t cells , however , perpetuate inflammatory damage during ischemic aki ; some populations of t cells confer renal protection after iri , particularly the th2 phenotype of cd4 + t cells and t regulatory cells .", "additional recent experimental data has characterized specific populations of t lymphocytes trafficking to remote organs that may facilitate organ crosstalk during ischemic aki . in a rodent model of kidney iri ,", "a distinct influx of cd3 + ( t ) cells with a predominant cd8 + t cell subpopulation was identified 24  hours after experimental kidney iri .", "these pulmonary t cells acquired increased expression of activation markers suggesting that ischemic aki induced trafficking of activated t cells which contributed to pulmonary injury , specifically apoptosis .", "the specific role of subpopulations of t cells in the pathogenesis of ischemic aki remains a stimulating topic of current and future investigation , both in the innate and adaptive immune response to kidney iri .", "the adaptive immune system relies on the specificity of antigen receptors on both b and t cells that respond to millions of various antigenic molecular structures .", "once stimulated by an antigen , b cells ( humoral immunity ) produce specific antibodies , perform opsonization to facilitate phagocytosis , and activate the complement system .", "the t - cell receptor ( tcr ) responds to presented antigen peptides by activating macrophages to kill phagocytosed microbes , directly destroying infected cells and by releasing cytokines to promote further response ( cell - mediated immunity ) .", "antigen - dependent t - cell activation has been demonstrated in experimental models of renal iri [ 34 , 35 ] .", "for example , mice with a restricted tcr cell repertoire suffer less injury and promote a far diminished inflammatory response during renal iri .", "additionally , athymic mice that subsequently underwent adoptive transfer with these tcr - restricted t cells failed to restore injury as severely as seen with transfer of wild - type t - cells .", "t cells also play a role in long term renal modification , as an increased number of both activated and effector - memory t cells have been observed in postischemic kidneys as long as 6 weeks after iri .", "these t cells may in fact recognize antigens released during kidney iri and subsequently target the kidney in an autoimmune response , leading to long - term progression of renal dysfunction .", "this hypothesis was demonstrated in a murine adoptive transfer model in which nave mice received t cells from mice who were 6  weeks after kidney iri and subsequently developed albuminuria .", "little is known about the role of b cells in renal iri ; however , b - cell - deficient mice demonstrated renal protection in the early phase of experimental iri .   in summary , both the innate and adaptive immune responses play critical roles in the pathophysiology of ischemic aki . either following antigen stimulation or in the presence of proinflammatory chemokines and oxygen free radicals ,", "t cells undergo early activation and serve as a bridge between adaptive and innate immunity .", "this specific host immune response to kidney iri facilitates distant organ crosstalk along with soluble proinflammatory mediators and will be further discussed in the following paragraphs ."], ["the combination of aki with acute lung injury ( ali ) remains a formidable challenge to clinicians caring for critically ill patients . in the setting of mof , aki and ali", "occur more frequently together than any other combination of organ systems , and predicted mortality approaches 80% [ 40 , 41 ] .", "this exceedingly high mortality can not be attributed solely to volume overload ; leukocyte trafficking , uremic toxins , and oxidative stress mechanisms all likely contribute to this devastating clinical syndrome . for the purposes of this paper , ali   represents a pao2/fio2 ratio < 300 combined with chest radiograph findings of acute bilateral infiltrates in the absence of elevated cardiac filling pressures , as defined by the america - european consensus conference on acute respiratory distress syndrome ( ards ) .", "while the mechanisms for ischemic aki - induced ali remain incompletely understood , several studies point toward a self - propagating cycle with activation of proinflammatory and proapoptotic pathways ( figure 2 ) .", "aki leads to lung injury and inflammation , and in turn , ali and its attendant hypoxemia and hypercapnia worsened by mechanical ventilation with high positive end - expiratory pressure leads to diminishing renal hemodynamics and function .", "lung injury during ischemic aki features marked pulmonary vascular permeability , erythrocyte sludging in lung capillaries , interstitial edema , focal alveolar hemorrhage , and inflammatory cell infiltration [ 12 , 43 , 44 ] .", "mechanisms for decreased alveolar fluid clearance include downregulation of pulmonary epithelial salt and water transporters including enac , na , k - atpase , and aquaporins during ischemic aki [ 4345 ] .", "this specific response likely contributes to the increased microvascular permeability and clinical pulmonary edema frequently encountered in the setting of ischemic aki and mof .", "experimental studies have identified a distinct pulmonary functional response and genomic signature induced during ischemic aki which differs from that induced by uremia alone .", "a comprehensive genomic map and ontology analysis revealed many of these top differentially expressed genes participating in proinflammatory and proapoptotic pathways .", "genes with early and sustained activation at 6 and 36  hours after ischemic aki included neutrophilic granule protein ( ngp ) , serum amyloid a3 ( saa3 ) , and interleukin 1-receptor type ii ( il-1r2 ) .   further investigation by deng et al .  ", "has also identified an early pulmonary inflammatory response with rapid activation of transcription factors nf-b and ap-1 during ischemic aki . by 4 hours ,", "leukocytes play a fundamental role in the development of ali , and several studies have documented lung leukocyte activation and trafficking during experimental aki with early and sustained neutrophil sequestration [ 46 , 47 ] .", "additional experiments by klein et al . have demonstrated early expression of the neutrophil chemokine keratinocyte - derived chemokine ( kc / cxcl1 ) and macrophage inflammatory protein ( mip-2/cxcl-2 ) along with increased pulmonary myeloperoxidase activity and pulmonary microvascular permeability during ischemic aki .", "the authors also examined interleukin-6 , a neutrophil - recruiting chemokine previously identified as a candidate gene by preferential expression in whole lung tissue during ischemic aki , and determined it was critical for influencing lymphocyte trafficking and pulmonary permeability .  ", "while neutrophils are key mediators in several extrapulmonary models of ali such as sepsis and mesenteric iri , their role in ischemic aki - induced ali remains to be elucidated .", "uremic neutrophils have even demonstrated a protective effect in the setting of ali . clearly , leukocyte trafficking and the innate immune response play a complex and important role in mediating the pulmonary inflammatory response and dysfunction during renal iri . though not traditionally associated with a proinflammatory response ,", "pulmonary apoptosis plays a critical role in the genomic and phenotypic response to ischemic aki .", "apoptosis of pulmonary epithelial and endothelial cells along with delayed leukocyte apoptosis occurs during ali [ 5052 ] .", "pulmonary apoptosis disrupts tethering forces involved in cell - to - cell and cell - to - extracellular matrix interactions , leading to a loss of endothelial barrier function and increased vascular permeability . in a rodent model ,", "our laboratory has characterized caspase - dependent pulmonary apoptosis which is abrogated by the administration of a broad - spectrum caspase inhibitor .", "not only did caspase inhibition attenuate the pulmonary functional injury , but the proapoptotic response to ischemic aki appears to be mediated via a tnf receptor-1 ( tnf - r-1 ) dependent pathway .  ", "our research has also demonstrated t - cell - dependent pulmonary apoptosis during ischemic aki . along with identification of activated t cells trafficking to the lungs during ischemic aki , we have found that pulmonary apoptosis was subsequently attenuated in t - cell - deficient animals .", "further pursuit of the specific pathway for t cell and tnfr1-dependent pulmonary apoptosis remains an active focus of future investigation in our research laboratory .", "cardiovascular collapse is one of the most common causes of death in the setting of aki , yet the mechanisms involved are not entirely understood .", "kelly has demonstrated left ventricular dilation , increased left ventricular end diastolic and end systolic diameter , increased relaxation time , and decreased fractional shortening in an experimental model of ischemic aki", ". mechanisms of cardiac injury during ischemic aki include cardiac myocyte apoptosis and neutrophil infiltration , which have been attributed to increased cardiac and systemic tnf , il-1 , and icam-1 expression [ 56 , 57 ] .", "when kidney ischemic time is decreased , cardiac il-1 and icam expression , along with myocyte necrosis , decreased correspondingly .", "effects of ischemic aki on the central nervous system ( cns ) are evident clinically when mental status changes develop . of note , renal replacement therapy fails to fully correct this cns manifestation of renal failure .", "uremic toxins certainly contribute to many symptoms of encephalopathy ; however , cellular and soluble inflammatory mediators have also been implicated .", "preclinical data identified an increase of kc , g - csf , and glial fibrillary acidic protein ( gfap ) in the cerebral cortex and hippocampus of the brain , which may serve to recruit neutrophils to sites of neuronal damage .", "this may result from either increased local neuronal production or an alteration of the blood - brain barrier ; further research is required to delineate the source .", "a cell - mediated proinflammatory response has also been identified with activation of microglial cells ( brain macrophages ) during ischemic aki .", "ischemic aki is also implicated in oxidative stress , inflammation , apoptosis , and tissue damage in hepatocytes .", "hepatic stellate cells ( hscs ) regulate leukocyte trafficking and the secretion of chemokines such as il-8 , and crosstalk between hscs likely occurs via a c - jun n - terminal kinase pathway .", "oxidative stress during ischemic aki causes hepatic malondialdehyde , an index of lipid peroxidation , to increase while total glutathione , an antioxidant , decreases .", "proinflammatory cytokine tnf expression and hepatic cellular apoptosis is also evident during ischemic aki .  ", "previous investigators and clinicians have labeled the gut as the  motor  of mof because of its ability to amplify the systemic sirs response in the setting of shock and gut hypoperfusion [ 6264 ] .", "these mechanisms include increased intestinal permeability , interactions between host and bacterial pathogens , and propagation of toxins to distant organs via the lymphatic system [ 63 , 65 ] and could potentially play a role during ischemic aki .", "clinical studies have long demonstrated the increased secretion of potassium by the colon and rectum in response to aki , and recent literature linked channel - inducing factor , a potassium channel regulator found in both the kidney and colon , to ischemic aki [ 67 , 68 ] .", "the role of the gut in response to organ crosstalk during ischemic aki remains a fascinating topic of future investigation ."], ["aki is a frequent complication amongst hospitalized patients , with grave implications in the setting of mof .", "ischemic aki initiates a cascade of proinflammatory pathways , and through the release of soluble mediators and activation of the host innate and adaptive immune systems , it facilitates organ crosstalk and remote organ injury . as our understanding of the postischemic kidney 's role in mediating organ crosstalk continues to evolve , further laboratory investigation into the remote organ response to this devastating injury"]]}
{"article_id": "PMC3658252", "article_text": ["plasma cell leukemia ( pcl ) is a rare disease that represents approximately 4% of plasma cell malignant disorders.1 diagnosis of pcl is established based on kyle s criteria , which includes an absolute plasma cell number accounting for greater than 2000/l or 20% of white blood cell differentiation.2 pcl consists of two variants : primary pcl presents in patients with no previous history of multiple myeloma ( mm ) , while secondary pcl consists of a leukemic transformation in previously recognized mm.3 primary pcl is an extremely resistant , rapidly progressive , fatal disease , with a median overall survival of 6.8 months.4 there is no standard therapeutic strategy because none of the treatment options have been prospectively evaluated .", "we describe herein a successful case of newly diagnosed primary pcl , treated with a regimen that included bortezomib , followed by auto stem cell transplantation ( asct ) and nonmyeloablative allogeneic stem cell transplantation ( allo - sct ) .", "a 37-year - old man with suspected primary pcl was sent from a nearby hospital to our institution in february 2010 .", "table 1 shows the laboratory data of the initial medical examination , and table 2 shows the data on admission to our hospital . the white blood cell count and its differentiation met kyle s criteria .", "the patient had acute heart failure . his chest x - ray revealed cardiomegaly ( cardiothoracic ratio was 59% ) with slight pulmonary congestion .", "amino - terminal pro - brain natriuretic peptide levels were elevated to 811.2 pg / ml .", "this acute heart failure was probably caused by cardiac amyloidosis , as cardiac ultrasound showed a granular sparkling pattern .", "these cells were negative for cyclin d1 , cd3 , cd5 , cd10 , cd20 , cd56 , cd79a , and  chain , and positive for cd138 and  chain .", "no immunoglobulin h / cyclin d1 ( igh / bcl1 ) translocations were found in fluorescence in situ hybridization analysis .", "treatment with a pad regimen ( bortezomib 1.3 mg / m [ days 1 , 4 , 8 , and 11 ] , adriamycin 1.3 mg / m [ days 1 , 4 , 8 , and 11 ] , and dexamethasone 40 mg / body [ days 14 , 811 , and 1518 ] ) was started .", "as soon as the treatment was started , leukemic plasma cell levels rapidly decreased ( figure 1 ) .", "no organ dysfunction was detected , except that serum creatinine levels temporarily increased to 1.21 mg / dl . on day 27 of the first pad cycle", "after three pad cycles , we were unable to assess his bone marrow , because it remained dry tap .", "serum / , a surrogate marker for the treatment effect of this case , became similar to normal levels .", "in addition , the granular sparkling pattern detected by cardiac ultrasound at the time of admission disappeared . the patient was then administered high - dose therapy ( hdt ) + asct . before hdt + asct , his p eripheral blood stem cells containing 10.9  10/kg cd34-positive cells were harvested with high - dose cyclophosphamide .", "he received hdt + asct with a conditioning regimen of high - dose melphalan ( 100 mg / m [ days 3 and 2 ] ) and bortezomib ( 1 mg / m [ days 7 , 4 , + 1 , and + 4 ] ) . at the end of hdt + asct", "sixty - seven days after hdt + asct , the patient received allogeneic bone marrow stem cell transplantation ( allo - bmt ) from his human leukocyte antigen - matched brother , containing 2.1  10/pt kg nuclear cells and 1.44  10/pt kg cd34-positive cells .", "the conditioning regimen consisted of fludarabine ( 30 mg / m [ day 6 to day 2 ] ) and total body irradiation ( 2 gy [ day 1 ] ) .", "graft - versus - host disease ( gvhd ) prophylaxis included tacrolimus and short - term methotrexate .", "twelve months after allo - bmt , the patient s serum / level was maintained at normal levels .", "he remained in a state of remission , with only the complication of mild skin gvhd .", "there are no established therapeutic regimens for pcl . several regimens that include an alkylating agent are sufficient to improve the overall survival ( os ) of pcl patients .", "jimnez - zepeda and domnguez reported a median os of 6.8 months for pcl patients treated with a common chemotherapy regimen consisting of vincristine , adriamycin , and dexamethasone , versus 2 months for those who received melphalan and prednisolone.5 it has also been reported that a combination of intermediate doses of melphalan and dexamethasone obtained the highest level of response in pcl patients .", "however , the median os was only 60 days for the cohort that achieved a partial or complete response.6 by comparison , bortezomib regimens show promise as induction regimens for pcl .", "katodritou et al reported that a combination of bortezomib and dexamethasone prolonged os ( median , 12 months).7 the efficacy of pad has been reported by chan et al.8 similar to the case presented by chan et al , our case , inducted with pad , showed a very impressive clinical course .", "as soon as pad was started , leukemic plasma cell levels rapidly decreased ( figure 1 ) .", "in addition , anemia and platelet depletion improved , and acute heart and kidney dysfunction rapidly recovered .", "after three pad cycles , serum / levels were normalized , without any adverse effects ( figure 2 ) . in the present case , the absolute values of the immunoglobulin / free light chains in the serum before and after therapy had to be very important", "although pad is effective for pcl , it remains to be determined whether long - term survival is improved , because of a lack of long - term follow - up .", "immunomodulatory drugs ( imids ) , such as thalidomide and lenalidomide , are as promising as bortezomib.9 , the reasons we did not choose imids were that lenalidomide was not approved in japan at the onset of the patient s pcl , and we decided to leave an opportunity to use thalidomide at his relapse .", "our case represented a potential curative strategy for pcl , with the safe introduction of allo - immunization with a n onmyeloablative regimen following pad and hdt + asct . for multiple mm , the most common malignant disease in plasma cells ,", "asct is regarded as the standard therapy for newly diagnosed cases in patients younger than 65 years .", "however , few patients who undergo the procedure are free of disease for more than 10 years . on the other hand ,", "allo - sct is a promising therapy . because of the graft - versus - myeloma effect ,", "a lower relapse rate and longer remissions have been reported in patients receiving allo - sct.11 presumably , pcl may be the same as mm .", "however , the high risk of transplant - related mortality ( 30%60% ) is a limitation of the use of allo - sct for mm . to overcome this limitation , maloney et al reported that combining a cytoreductive autograft with a nonmyeloablative allograft lowered transplant - related mortality by approximately 15%.12 meanwhile , ramasamy et al reported that alemtuzumab - based reduced intensity conditioning allogeneic transplantation.13 although progression - free survival of their patient cohort was comparable to previously published data of reduced intensity conditioning allogeneic transplantation in myeloma , there is no plateau on the survival curves , with a significant transplant - related mortality of 21% .", "although nonmyeloablative conditioning reduces transplant - related mortality , it is difficult for patients older than 65 years to use our strategy . for them", "we have a successful case of newly diagnosed primary pcl , treated with a regimen that included bortezomib and asct , followed by nonmyeloablative allo - sct .", "this strategy is promising for pcl , which , though an extremely resistant disease , may become curable ."], "abstract_text": ["<S> plasma cell leukemia ( pcl ) is a rare disease that represents approximately 4% of plasma cell malignant disorders . </S>", "<S> pcl consists of two variants : primary pcl presents in patients with no previous history of multiple myeloma , while secondary pcl consists of a leukemic transformation in a previously recognized multiple myeloma . </S>", "<S> primary pcl is an extremely resistant , rapidly progressive , fatal disease , with a median overall survival of 6.8 months . </S>", "<S> there is no standard therapeutic strategy , because no treatment option has been prospectively evaluated . </S>", "<S> we describe a successful case of newly diagnosed primary pcl , treated with a regimen that included bortezomib , followed by auto stem cell transplantation and nonmyeloablative allogeneic stem cell transplantation . our patient has maintained remission status for over 12 months since undergoing the allogeneic stem cell transplantation . </S>", "<S> this strategy is promising for pcl , which , though an extremely resistant disease , may become curable . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["plasma cell leukemia ( pcl ) is a rare disease that represents approximately 4% of plasma cell malignant disorders.1 diagnosis of pcl is established based on kyle s criteria , which includes an absolute plasma cell number accounting for greater than 2000/l or 20% of white blood cell differentiation.2 pcl consists of two variants : primary pcl presents in patients with no previous history of multiple myeloma ( mm ) , while secondary pcl consists of a leukemic transformation in previously recognized mm.3 primary pcl is an extremely resistant , rapidly progressive , fatal disease , with a median overall survival of 6.8 months.4 there is no standard therapeutic strategy because none of the treatment options have been prospectively evaluated .", "we describe herein a successful case of newly diagnosed primary pcl , treated with a regimen that included bortezomib , followed by auto stem cell transplantation ( asct ) and nonmyeloablative allogeneic stem cell transplantation ( allo - sct ) ."], ["a 37-year - old man with suspected primary pcl was sent from a nearby hospital to our institution in february 2010 .", "table 1 shows the laboratory data of the initial medical examination , and table 2 shows the data on admission to our hospital . the white blood cell count and its differentiation met kyle s criteria .", "the patient had acute heart failure . his chest x - ray revealed cardiomegaly ( cardiothoracic ratio was 59% ) with slight pulmonary congestion .", "amino - terminal pro - brain natriuretic peptide levels were elevated to 811.2 pg / ml .", "this acute heart failure was probably caused by cardiac amyloidosis , as cardiac ultrasound showed a granular sparkling pattern .", "these cells were negative for cyclin d1 , cd3 , cd5 , cd10 , cd20 , cd56 , cd79a , and  chain , and positive for cd138 and  chain .", "no immunoglobulin h / cyclin d1 ( igh / bcl1 ) translocations were found in fluorescence in situ hybridization analysis .", "treatment with a pad regimen ( bortezomib 1.3 mg / m [ days 1 , 4 , 8 , and 11 ] , adriamycin 1.3 mg / m [ days 1 , 4 , 8 , and 11 ] , and dexamethasone 40 mg / body [ days 14 , 811 , and 1518 ] ) was started .", "as soon as the treatment was started , leukemic plasma cell levels rapidly decreased ( figure 1 ) .", "no organ dysfunction was detected , except that serum creatinine levels temporarily increased to 1.21 mg / dl . on day 27 of the first pad cycle", "after three pad cycles , we were unable to assess his bone marrow , because it remained dry tap .", "serum / , a surrogate marker for the treatment effect of this case , became similar to normal levels .", "in addition , the granular sparkling pattern detected by cardiac ultrasound at the time of admission disappeared . the patient was then administered high - dose therapy ( hdt ) + asct . before hdt + asct , his p eripheral blood stem cells containing 10.9  10/kg cd34-positive cells were harvested with high - dose cyclophosphamide .", "he received hdt + asct with a conditioning regimen of high - dose melphalan ( 100 mg / m [ days 3 and 2 ] ) and bortezomib ( 1 mg / m [ days 7 , 4 , + 1 , and + 4 ] ) . at the end of hdt + asct", "sixty - seven days after hdt + asct , the patient received allogeneic bone marrow stem cell transplantation ( allo - bmt ) from his human leukocyte antigen - matched brother , containing 2.1  10/pt kg nuclear cells and 1.44  10/pt kg cd34-positive cells .", "the conditioning regimen consisted of fludarabine ( 30 mg / m [ day 6 to day 2 ] ) and total body irradiation ( 2 gy [ day 1 ] ) .", "graft - versus - host disease ( gvhd ) prophylaxis included tacrolimus and short - term methotrexate .", "twelve months after allo - bmt , the patient s serum / level was maintained at normal levels .", "he remained in a state of remission , with only the complication of mild skin gvhd ."], ["there are no established therapeutic regimens for pcl . several regimens that include an alkylating agent are sufficient to improve the overall survival ( os ) of pcl patients .", "jimnez - zepeda and domnguez reported a median os of 6.8 months for pcl patients treated with a common chemotherapy regimen consisting of vincristine , adriamycin , and dexamethasone , versus 2 months for those who received melphalan and prednisolone.5 it has also been reported that a combination of intermediate doses of melphalan and dexamethasone obtained the highest level of response in pcl patients .", "however , the median os was only 60 days for the cohort that achieved a partial or complete response.6 by comparison , bortezomib regimens show promise as induction regimens for pcl .", "katodritou et al reported that a combination of bortezomib and dexamethasone prolonged os ( median , 12 months).7 the efficacy of pad has been reported by chan et al.8 similar to the case presented by chan et al , our case , inducted with pad , showed a very impressive clinical course .", "as soon as pad was started , leukemic plasma cell levels rapidly decreased ( figure 1 ) .", "in addition , anemia and platelet depletion improved , and acute heart and kidney dysfunction rapidly recovered .", "after three pad cycles , serum / levels were normalized , without any adverse effects ( figure 2 ) . in the present case , the absolute values of the immunoglobulin / free light chains in the serum before and after therapy had to be very important", "although pad is effective for pcl , it remains to be determined whether long - term survival is improved , because of a lack of long - term follow - up .", "immunomodulatory drugs ( imids ) , such as thalidomide and lenalidomide , are as promising as bortezomib.9 , the reasons we did not choose imids were that lenalidomide was not approved in japan at the onset of the patient s pcl , and we decided to leave an opportunity to use thalidomide at his relapse .", "our case represented a potential curative strategy for pcl , with the safe introduction of allo - immunization with a n onmyeloablative regimen following pad and hdt + asct . for multiple mm , the most common malignant disease in plasma cells ,", "asct is regarded as the standard therapy for newly diagnosed cases in patients younger than 65 years .", "however , few patients who undergo the procedure are free of disease for more than 10 years . on the other hand ,", "allo - sct is a promising therapy . because of the graft - versus - myeloma effect ,", "a lower relapse rate and longer remissions have been reported in patients receiving allo - sct.11 presumably , pcl may be the same as mm .", "however , the high risk of transplant - related mortality ( 30%60% ) is a limitation of the use of allo - sct for mm . to overcome this limitation , maloney et al reported that combining a cytoreductive autograft with a nonmyeloablative allograft lowered transplant - related mortality by approximately 15%.12 meanwhile , ramasamy et al reported that alemtuzumab - based reduced intensity conditioning allogeneic transplantation.13 although progression - free survival of their patient cohort was comparable to previously published data of reduced intensity conditioning allogeneic transplantation in myeloma , there is no plateau on the survival curves , with a significant transplant - related mortality of 21% .", "although nonmyeloablative conditioning reduces transplant - related mortality , it is difficult for patients older than 65 years to use our strategy . for them", "we have a successful case of newly diagnosed primary pcl , treated with a regimen that included bortezomib and asct , followed by nonmyeloablative allo - sct .", "this strategy is promising for pcl , which , though an extremely resistant disease , may become curable ."]]}
{"article_id": "PMC3649509", "article_text": ["fitz  hugh  curtis syndrome is an extra - pelvic manifestation of pelvic inflammatory disease ( pid ) and is characterized by perihepatic adhesions between liver capsule and diaphragm or anterior peritoneal surface(13 ) .", "most fitz  hugh  curtis syndrome patients are women of child bearing age and rarely has the syndrome been reported in males .", "the predominant symptom is pain in the right upper quadrant , which may be confused with biliary disease", "an abdominal computed tomography ( ct ) scan may reveal subcapsular enhancement of the liver in arterial phase ( 4 ) . we herein report a case of fitz  hugh  curtis syndrome in a male patient that was diagnosed via laparoscopy .", "a 29 year - old african american male with russel - silver dwarfism presented with one day history of diarrhea , nausea , vomiting , right side abdominal pain , and abdominal distention .", "the pain was constant , gradually increasing in severity , and not related to food intake .", "his medical history was significant for russel - silver dwarfism , calcium deficiency , cardiomegaly , and bilateral testicular implants for undescended testicles .", "the patient was sexually active only with his girlfriend and denied any history of sexually transmitted disease . on examination patient was afebrile and his vitals were stable .", "laboratory workup revealed white blood cell ( wbc ) count of 14,000/l with normal liver function tests .", "ct scan of the abdomen and pelvis showed a small amount of free fluid in pelvis ; the proximal appendix appeared normal , however the distal appendix was not visualized .", "the liver capsule appeared normal and there was no subcapsular fluid collection ( figure 1 ) .", "ct scan showing normal appearing liver capsule and no perihepatic fluid collection the patient was admitted to the surgical service .", "he was made nil per os ( npo ) , and placed on intravenous fluids and pain medication .", "he was refusing any surgical intervention at this point . on hospital day 5 , a repeat abdominal ct scan demonstrated a normal appearing liver , small bowel , large bowel , and appendix , with a mild increase in pelvic free fluid .", "as the patient 's symptoms did not improve with conservative management , he ultimately agreed to undergo a diagnostic laparoscopy , and was taken to the operating room on hospital day 9 .", "the small bowel was run in a retrograde fashion starting at the caecum , and no stricture , mass , or perforation was noticed .", "appendectomy was not performed as per patient'swishes . following the procedure , the patient reported complete resolution of his symptoms .", "his diet was gradually advanced , which he tolerated well , and was discharged on post operative day 2 .", "fitz -hugh  curtis syndrome was first described in 1920 by carlos stajano . in the 1930 's thomas fitz ", "hugh and arthur curtis also described the syndrome and made a connection between right upper quadrant pain following a pelvic infection and violin  string like perihepatic adhesions(5 ) . the first case of gonococcal perihepatitis in a male", "the incidence ranges from 4% to 14% in women with pid , but is as high as 27% in adolescents with pid , whose less mature genitourinary tract anatomy makes them more susceptible to infection(1 ) .", "the inflammation of the liver capsule has been attributed to the direct bacterial spread from an infected fallopian tube via the right paracolic gutter . in men , hematogenous and lymphatic spread to liver", "the predominant symptoms are right upper quadrant pain , tenderness , and pleuritic right sided chest pain(2 ) .", "these symptoms can pose diagnostic challenges as they may be confused with biliary tract symptoms . in a clinical setting ,", "the diagnosis is adequately established by excluding other possible causes of right upper quadrant pain . on laboratory examination ,", "white blood cell count can be elevated in nearly half of the patients , while liver function tests are normal in most patients . because urethral cultures frequently fail to demonstrate the presence of gonorrhea and chlamydia , the serologic microimmunofluorescence antibody test is helpful in diagnosis ( 2 ) .", "ct scan may show subcapsular fluid collection , thickening of hepatic capsule in the arterial phase , and wedging enhancement of the involved liver parenchyma in more than 50% of patients .", "most cases of fitz  hugh  curtis syndrome are managed with antibiotics against gonorrhea and chlamydia .", "it occurs mostly in premenopausal women , however , cases in males have also been reported ."], "abstract_text": ["<S> fitz  hugh  curtis syndrome is a condition characterized by inflammation of the liver capsule with concomitant pelvic inflammation without involvement of liver parenchyma . </S>", "<S> it is classically seen in young women who present with sharp , pleuritic right upper quadrant pain , usually but not always accompanied by symptoms of pelvic inflammatory disease ( pid ) and is frequently confused with biliary tract disease . rarely the syndrome has been reported in males , hematogenous and lymphatic spread to liver is thought to be the underlying mechanism . </S>", "<S> serological tests and computed tomography ( ct ) scan may aid in diagnosis of fitz  hugh  curtis syndrome . </S>", "<S> definitive diagnosis is made by laparoscopy , which provides both diagnostic and therapeutic benefits . </S>", "<S> we report a case of fitz  hugh  curtis syndrome in a young male patient , which was diagnosed and treated by laparoscopy . </S>", "<S> we also include a review of the literature . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["fitz  hugh  curtis syndrome is an extra - pelvic manifestation of pelvic inflammatory disease ( pid ) and is characterized by perihepatic adhesions between liver capsule and diaphragm or anterior peritoneal surface(13 ) .", "most fitz  hugh  curtis syndrome patients are women of child bearing age and rarely has the syndrome been reported in males .", "the predominant symptom is pain in the right upper quadrant , which may be confused with biliary disease", "an abdominal computed tomography ( ct ) scan may reveal subcapsular enhancement of the liver in arterial phase ( 4 ) . we herein report a case of fitz  hugh  curtis syndrome in a male patient that was diagnosed via laparoscopy ."], ["a 29 year - old african american male with russel - silver dwarfism presented with one day history of diarrhea , nausea , vomiting , right side abdominal pain , and abdominal distention .", "the pain was constant , gradually increasing in severity , and not related to food intake .", "his medical history was significant for russel - silver dwarfism , calcium deficiency , cardiomegaly , and bilateral testicular implants for undescended testicles .", "the patient was sexually active only with his girlfriend and denied any history of sexually transmitted disease . on examination patient was afebrile and his vitals were stable .", "laboratory workup revealed white blood cell ( wbc ) count of 14,000/l with normal liver function tests .", "ct scan of the abdomen and pelvis showed a small amount of free fluid in pelvis ; the proximal appendix appeared normal , however the distal appendix was not visualized .", "the liver capsule appeared normal and there was no subcapsular fluid collection ( figure 1 ) .", "ct scan showing normal appearing liver capsule and no perihepatic fluid collection the patient was admitted to the surgical service .", "he was made nil per os ( npo ) , and placed on intravenous fluids and pain medication .", "he was refusing any surgical intervention at this point . on hospital day 5 , a repeat abdominal ct scan demonstrated a normal appearing liver , small bowel , large bowel , and appendix , with a mild increase in pelvic free fluid .", "as the patient 's symptoms did not improve with conservative management , he ultimately agreed to undergo a diagnostic laparoscopy , and was taken to the operating room on hospital day 9 .", "the small bowel was run in a retrograde fashion starting at the caecum , and no stricture , mass , or perforation was noticed .", "appendectomy was not performed as per patient'swishes . following the procedure , the patient reported complete resolution of his symptoms .", "his diet was gradually advanced , which he tolerated well , and was discharged on post operative day 2 ."], ["fitz -hugh  curtis syndrome was first described in 1920 by carlos stajano . in the 1930 's thomas fitz ", "hugh and arthur curtis also described the syndrome and made a connection between right upper quadrant pain following a pelvic infection and violin  string like perihepatic adhesions(5 ) . the first case of gonococcal perihepatitis in a male", "the incidence ranges from 4% to 14% in women with pid , but is as high as 27% in adolescents with pid , whose less mature genitourinary tract anatomy makes them more susceptible to infection(1 ) .", "the inflammation of the liver capsule has been attributed to the direct bacterial spread from an infected fallopian tube via the right paracolic gutter . in men , hematogenous and lymphatic spread to liver", "the predominant symptoms are right upper quadrant pain , tenderness , and pleuritic right sided chest pain(2 ) .", "these symptoms can pose diagnostic challenges as they may be confused with biliary tract symptoms . in a clinical setting ,", "the diagnosis is adequately established by excluding other possible causes of right upper quadrant pain . on laboratory examination ,", "white blood cell count can be elevated in nearly half of the patients , while liver function tests are normal in most patients . because urethral cultures frequently fail to demonstrate the presence of gonorrhea and chlamydia , the serologic microimmunofluorescence antibody test is helpful in diagnosis ( 2 ) .", "ct scan may show subcapsular fluid collection , thickening of hepatic capsule in the arterial phase , and wedging enhancement of the involved liver parenchyma in more than 50% of patients .", "most cases of fitz  hugh  curtis syndrome are managed with antibiotics against gonorrhea and chlamydia .", "it occurs mostly in premenopausal women , however , cases in males have also been reported ."]]}
{"article_id": "PMC2847166", "article_text": ["chemical shifts are now routinely used as a source of local conformational restraints in the structure determination of proteins by nmr , due mostly to the widespread use of programs such as talos ( cornilescu et al .", "1999 ) and shiftor / preditor ( neal et al . 2006 ; berjanskii et al . 2006 ) .", "these programs share a common approach and output similar data ; both search a database that correlates local patterns of chemical shifts with local conformation , and both provide backbone dihedral angle restraints for individual residues .", "this approach has been very successful , but has some limitations in the stringent criteria needed for selecting proteins or protein fragments to populate the database , i.e. only those with both highly reliable chemical shift and structural data can be included .", "although this may seem adequate  for example , the talos database contains 186 proteins subdivided into over 24,000 tripeptide fragments ( http://spin.niddk.nih.gov/nmrpipe/talos/)the sequence / conformation search space is very large , and the database coverage is unevenly distributed . as a result , rare combinations of amino - acid and conformation may be under - represented in the database , leading to significant under - prediction and even to errors outside of the heavily populated regions of the ramachandran map .", "we have adopted an alternative approach to extracting structural data from chemical shifts based on our simshiftdb algorithm .", "the original simshift was designed to test for structural similarities between proteins in a pair wise manner using chemical shifts to supplement sequence data .", "simshift showed improved ability to detect distant structural relationships when compared to state - of - the - art methods based on the sequence alone .", "a natural further development of pairwise comparison was to adapt the simshift algorithm for database searching , resulting in simshiftdb ( ginzinger et al .", ", simshiftdb provides a list of matching proteins in the database , scored by a measure of statistical significance . in effect", "it searches a synthetic chemical shift database of 13,000 proteins based on the astral library ( chandonia et al .", "the matching sequence can be of any length , and structurally similar regions can be found ranging from small , locally similar fragments up to full domains . in principle , any structural alignment method can also be used to make predictions of local conformation by extracting torsion angles from matching regions of the target proteins , and it is this implementation of the simshiftdb algorithm we present here .", "we benchmark the program against talos as a standard for current methods and hhpred , a sequence search method based on hidden markov models ( sding et al .", "we show that simshiftdb can significantly increase the amount of information that can be derived from chemical shifts .", "2007a ) routine for standardizing chemical shift referencing to produce a pipeline for analysis of chemical shift data .", "full details of the simshiftdb algorithm have been published previously ( see ginzinger et al .", "2007b ) , but it is worthwhile giving a brief overview here ( see fig .  1 ) .", "given a target protein , simshiftdb analyzes each possible pairing of the target protein with one of the template protein structures in the database .", "here we use the proteins from the astral database ( version 1.71 , filtered for 95% sequence identity ) to build the simshiftdb template database . as simshiftdb", "is based on the comparison of chemical shift data , the chemical shifts for all entries in the template database are back - calculated from the three - dimensional structure using shiftx ( neal et al . 2003 ) . for each combination of the target protein with a template from the database ,", "top : an example for a set of local similarities for a target protein ( s ) and a template protein ( t ) .", "bottom : combination of a subset of local similarities to yield a self - consistent final alignmentstep 1 : local similarities are found by looking for high scoring combinations of parts of the target protein sequence ( s ) with parts of the template protein sequence ( t ) .", "for example , block b in the figure shows that the chemical shifts of the target protein sequence from index xmin to xmax are similar to the chemical shifts of the template protein sequence from index ymin to ymax .", "the similarity is calculated by summing the pairwise scores of the residues in the similar region in analogy to a pairwise sequence alignment .", "the pairwise similarity scores are given by the so - called chemical shift substitution matrices , which give a score for each combination of two residues with associated chemical shifts ( for more details see ginzinger 2008).step 2 : the set of local similarities from step 1 is taken as an input for step 2 , where the most significant combination of blocks is identified , according to a statistical model of alignment scores ( karlin and altschul 1993 ) .", "additionally , two blocks have to fulfill two constraints for their combination to be considered : an explanation of the simshift algorithm .", "top : an example for a set of local similarities for a target protein ( s ) and a template protein ( t ) .", "bottom : combination of a subset of local similarities to yield a self - consistent final alignment step 1 : local similarities are found by looking for high scoring combinations of parts of the target protein sequence ( s ) with parts of the template protein sequence ( t ) . fig . ", "for example , block b in the figure shows that the chemical shifts of the target protein sequence from index xmin to xmax are similar to the chemical shifts of the template protein sequence from index ymin to ymax .", "the similarity is calculated by summing the pairwise scores of the residues in the similar region in analogy to a pairwise sequence alignment .", "the pairwise similarity scores are given by the so - called chemical shift substitution matrices , which give a score for each combination of two residues with associated chemical shifts ( for more details see ginzinger 2008 ) .", "step 2 : the set of local similarities from step 1 is taken as an input for step 2 , where the most significant combination of blocks is identified , according to a statistical model of alignment scores ( karlin and altschul 1993 ) .", "additionally , two blocks have to fulfill two constraints for their combination to be considered : blocks may not overlap in the target or in the template protein ; this would otherwise result in an ambiguous alignment.as the three - dimensional structure of the template protein is known , we further require that the euclidean distance between the end of the first block and the beginning of the second block may be bridged ( according to chemical restraints ) by the relevant sequence of amino acids in the target protein .", "blocks may not overlap in the target or in the template protein ; this would otherwise result in an ambiguous alignment . as the three - dimensional structure of the template protein", "is known , we further require that the euclidean distance between the end of the first block and the beginning of the second block may be bridged ( according to chemical restraints ) by the relevant sequence of amino acids in the target protein . finally , we calculate an e - value for the optimal combination of blocks .", "this e - value represents the number of alignments of equal or better quality , which are expected to occur by chance , given the distribution of the amino acids with associated chemical shifts in the target protein and the template database .", "additionally , the e - value takes the size of the template database into account . according to the following evaluation , an e - value of < 10 guarantees a high quality alignment .", "to test the performance of simshiftdb , a benchmark set has to be defined for which both chemical shifts and the three - dimensional structure of the protein are available .", "the bmrb ( seavey et al . 1991 ) is the main public repository for chemical shift data .", "however , there is no consistent mapping to the structural databases , making it difficult to relate structural with chemical shift information reliably .", "therefore a mapping between bmrb and astral is calculated based on amino acid sequence similarity .", "every entry in the benchmark set has to fulfill the following constraints : a 100% sequence match to an astral entry.at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) .", "at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) . to identify protein structures corresponding to the respective bmrb entries , a blast - search ( altschul et al . 1990 ) against the sequences from the astral database is conducted for each bmrb entry . if the full bmrb sequence can be matched without gaps against an astral sequence , the corresponding astral structure is assigned to the bmrb entry .", "as some entries in bmrb match more than one sequence in astral , one representative structure has to be chosen .", "2004 ) provided for each astral entry , thereby selecting the structure with the best resolution . through this procedure", "a benchmark set containing 144 entries was derived . when calculating the similarity score for two residues , simshiftdb is restricted to at most three chemical shifts from the following list : h , h ,", "n , c , c , and c. thus it is important to select a combination of shifts to extract maximum information , and a priority for replacing missing shifts . to identify the most successful strategy , we tested all possible priorities for the six atom types , resulting in 6 ! ", "the most successful priority was : c  >  c  >  h  >  c  >  h  >  n. this is the default priority , and is used in the following analysis . to evaluate the prediction accuracy", ", we applied the program to all entries in the benchmark set , using all proteins from the simshiftdb template database as potential templates .", "subsequently , we used all alignments that achieved an e - value better than 10 to infer torsion angles for the target residue from the associated template residues . if residues of the target were mapped multiple times , we based the prediction on the highest scoring alignment .", "it is important to evaluate the performance of simshiftdb as a function of sequence similarity ; therefore 9 evaluations were performed with the maximum allowed sequence similarity in the evaluated alignments set between 20 and 100% .", "about 70% of all torsion angles predicted using simshiftdb have a high accuracy ( 15 error ) .", "another 10 to 20% of the predicted angles have an error of less than 30. therefore simshiftdb yields accurate results in 85 to 90% of the evaluated predictions . through the use of the chemical shifts ,", "this performance is nearly independent of the percentage of sequence identity in the respective alignments.fig . ", "2evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists also of interest is the performance of simshiftdb on different types of secondary structure .", "figures  3 and 4 show the difference between the secondary structure content in all predictions versus that in high quality and erroneous predictions , respectively .", "it can be seen that predictions for sheet ( for both high quality and erroneous predictions ) match well with the percentage observed in all predictions , and this match is largely independent of sequence similarity .", "in contrast , the percentage of high quality predictions in helix increases with decreasing sequence similarity , whereas the corresponding percentage in coil regions decreases . for erroneous predictions", "this seems logical , as the structures for coil regions are less reliable , and predictions are clearly harder to make than for secondary structure .", "this test shows empirically that simshiftdb has no significant bias when comparing performance in predicting helix versus sheet .", "the independence of sheet predictions from sequence similarity indicates that the chemical shift data is more diagnostic for sheet than for helix.fig . ", "3difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "4difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . ", "3 difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "the lines show the overall secondary structure content in all predictions difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig .  3 to show empirically that simshiftdb uses the information in the chemical shift data to yield more sensitive alignments , especially in the case of low sequence similarity , we compare simshiftdb to hhsearch ( sding 2005 ) .", "hhsearch , a sensitive search tool based on hidden markov models , calculates alignments between proteins using the primary sequence complemented by sequence - based predictions of secondary structure .", "2005 ) , a protein structure prediction method based on hhsearch alignments , ranked second best in the casp7 ( battey et al .", "additionally , it is freely available for download and gives the user the possibility to define arbitrary template databases . therefore it is perfectly suited to serve as a reference for purely sequence - based methods . to compare the performance of simshiftdb and hhsearch we used the benchmark set defined in the previous section . for both methods we ran each target protein against the simshiftdb template database and used alignments achieving an e - value better than 10 to predict torsion angles for the residues in the target protein .", "if residues were mapped multiple times , the prediction was based on the highest scoring alignment .", "the following notation is used for the presentation of the results :- a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5.two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30.missing predictions are treated as predictions with an error > 30. a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5. two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30. missing predictions are treated as predictions with an error > 30. figure  5 shows the results of this evaluation for alignments with maximal sequence identities ranging from 10 to 100% .", "there is a clear trend for simshiftdb to outperform hhsearch as the sequence identity decreases , demonstrating that simshiftdb uses the structural information in chemical shifts to improve alignments.fig . ", "5simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality .", "the brown line corresponds to the number of torsion angles predicted ( right axis ) simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality .", "the brown line corresponds to the number of torsion angles predicted ( right axis ) it is important to compare simshiftdb to the most prominent method for predicting torsion angles from chemical shifts , namely talos ( cornilescu et al .", "again we used the benchmark set defined earlier , and made torsion angle predictions based on simshiftdb alignments that achieved an e - value better than 10 .", "we then compared the quality of each torsion angle prediction to the corresponding talos prediction .", "figure  6 shows that simshiftdb outperforms talos in at least 30% of all cases .", "it should be noted that there is likely to be a significant bias towards talos in these results , as many members of the benchmark set will have been calculated using talos restraints.fig . ", "6simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . ", "5 simshiftdb predictions compared to the respective talos predictions using the same notation as in fig .  5", "to test the performance of simshiftdb , a benchmark set has to be defined for which both chemical shifts and the three - dimensional structure of the protein are available .", "the bmrb ( seavey et al . 1991 ) is the main public repository for chemical shift data .", "however , there is no consistent mapping to the structural databases , making it difficult to relate structural with chemical shift information reliably .", "therefore a mapping between bmrb and astral is calculated based on amino acid sequence similarity .", "every entry in the benchmark set has to fulfill the following constraints : a 100% sequence match to an astral entry.at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) .", "at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) . to identify protein structures corresponding to the respective bmrb entries , a blast - search ( altschul et al . 1990 ) against the sequences from the astral database is conducted for each bmrb entry . if the full bmrb sequence can be matched without gaps against an astral sequence , the corresponding astral structure is assigned to the bmrb entry .", "as some entries in bmrb match more than one sequence in astral , one representative structure has to be chosen .", "2004 ) provided for each astral entry , thereby selecting the structure with the best resolution . through this procedure", "when calculating the similarity score for two residues , simshiftdb is restricted to at most three chemical shifts from the following list : h , h , n , c , c , and c. thus it is important to select a combination of shifts to extract maximum information , and a priority for replacing missing shifts . to identify the most successful strategy , we tested all possible priorities for the six atom types , resulting in 6 !  =  720 evaluations .", "the most successful priority was : c  >  c  >  h  >  c  >  h  >  n. this is the default priority , and is used in the following analysis . to evaluate the prediction accuracy", ", we applied the program to all entries in the benchmark set , using all proteins from the simshiftdb template database as potential templates .", "subsequently , we used all alignments that achieved an e - value better than 10 to infer torsion angles for the target residue from the associated template residues . if residues of the target were mapped multiple times , we based the prediction on the highest scoring alignment .", "it is important to evaluate the performance of simshiftdb as a function of sequence similarity ; therefore 9 evaluations were performed with the maximum allowed sequence similarity in the evaluated alignments set between 20 and 100% .", "about 70% of all torsion angles predicted using simshiftdb have a high accuracy ( 15 error ) .", "another 10 to 20% of the predicted angles have an error of less than 30. therefore simshiftdb yields accurate results in 85 to 90% of the evaluated predictions . through the use of the chemical shifts ,", "this performance is nearly independent of the percentage of sequence identity in the respective alignments.fig . ", "2evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists also of interest is the performance of simshiftdb on different types of secondary structure .", "figures  3 and 4 show the difference between the secondary structure content in all predictions versus that in high quality and erroneous predictions , respectively .", "it can be seen that predictions for sheet ( for both high quality and erroneous predictions ) match well with the percentage observed in all predictions , and this match is largely independent of sequence similarity .", "in contrast , the percentage of high quality predictions in helix increases with decreasing sequence similarity , whereas the corresponding percentage in coil regions decreases . for erroneous predictions", "this seems logical , as the structures for coil regions are less reliable , and predictions are clearly harder to make than for secondary structure .", "this test shows empirically that simshiftdb has no significant bias when comparing performance in predicting helix versus sheet .", "the independence of sheet predictions from sequence similarity indicates that the chemical shift data is more diagnostic for sheet than for helix.fig . ", "3difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "4difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . ", "3 difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "the lines show the overall secondary structure content in all predictions difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig .  3", "to show empirically that simshiftdb uses the information in the chemical shift data to yield more sensitive alignments , especially in the case of low sequence similarity , we compare simshiftdb to hhsearch ( sding 2005 ) .", "hhsearch , a sensitive search tool based on hidden markov models , calculates alignments between proteins using the primary sequence complemented by sequence - based predictions of secondary structure .", "2005 ) , a protein structure prediction method based on hhsearch alignments , ranked second best in the casp7 ( battey et al . 2007 ) experiment .", "additionally , it is freely available for download and gives the user the possibility to define arbitrary template databases .", "therefore it is perfectly suited to serve as a reference for purely sequence - based methods . to compare the performance of simshiftdb and hhsearch we used the benchmark set defined in the previous section .", "for both methods we ran each target protein against the simshiftdb template database and used alignments achieving an e - value better than 10 to predict torsion angles for the residues in the target protein .", "if residues were mapped multiple times , the prediction was based on the highest scoring alignment .", "the following notation is used for the presentation of the results :- a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5.two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30.missing predictions are treated as predictions with an error > 30. a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5. two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30. missing predictions are treated as predictions with an error > 30. figure  5 shows the results of this evaluation for alignments with maximal sequence identities ranging from 10 to 100% .", "there is a clear trend for simshiftdb to outperform hhsearch as the sequence identity decreases , demonstrating that simshiftdb uses the structural information in chemical shifts to improve alignments.fig . ", "5simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality .", "the brown line corresponds to the number of torsion angles predicted ( right axis ) simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality .", "it is important to compare simshiftdb to the most prominent method for predicting torsion angles from chemical shifts , namely talos ( cornilescu et al . 1999 ) .", "again we used the benchmark set defined earlier , and made torsion angle predictions based on simshiftdb alignments that achieved an e - value better than 10 .", "we then compared the quality of each torsion angle prediction to the corresponding talos prediction .", "figure  6 shows that simshiftdb outperforms talos in at least 30% of all cases .", "it should be noted that there is likely to be a significant bias towards talos in these results , as many members of the benchmark set will have been calculated using talos restraints.fig . ", "6simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . ", "5 simshiftdb predictions compared to the respective talos predictions using the same notation as in fig .  5", "we have presented simshiftdb and shown that the program is able to sensitively extract structural information from chemical shift data .", "this information is to a certain extent complementary to that from currently available tools . on one hand", "simshiftdb shows its strength especially in cases of low sequence similarity , which underlines the advantage of including chemical shift information in the alignment algorithm . on the other hand", ", we were able to show that one - third of the predictions by simshiftdb clearly have a higher quality than the corresponding talos predictions , and this is largely independent of sequence similarity .", "the main advantage of simshiftdb is derived from its superior coverage of the search space , due to the large and quickly adaptable template database .", "simshiftdb outperforms talos especially in those cases where talos finds no predictions classified as  good  according to its selection criteria .", "simshiftdb and talos are therefore complementary , and can be used in parallel to increase the number of available predictions .", "ph1500c is a 78-residue homo - hexameric domain currently under investigation in our laboratories , and was chosen because it shows no significant sequence similarity to proteins of known structure .", "we used simshiftdb to search for templates matching ph1500c , using several different chemical shift priorities .", "the search identifies several templates at e - values around 10 , which correspond to the region g17-f40 of ph1500c and consist of three - stranded -meander linked by two tight turns ( fig . ", "the consensus of simshiftdb predictions shows the first turn to be a standard type i -turn , while the second is a less commonly observed 5-residue -turn .", "an example template agrees very well with all structural data available in this region ( fig . ", "7 ) , and suggests local conformational details such as sidechain positions and local hydrogen bonding networks.fig . ", "the left panel shows a representative template structure found in a simshiftdb search for ph1500c ( 1p22 , residues a392-w415 ) .", "residues shown in green are predicted by talos to be in the -region of ramachandran space and the residue in yellow in the + -region , while for residues in white there is no prediction .", "only one talos prediction is made for the six residues outside of canonical secondary structure , i.e. the two turns and the -bulge in the first strand .", "the right panel shows the structure calculated with all available nmr data , showing very good agreement to the template .", "the coloring is as above , with the addition of blue residues for residues in the -region of ramachandran space .", "a conserved sidechain hydrogen - bond acceptor ( t101 in ph1500c ) is shown in grey , highlighting the ability of simshiftdb searches to reveal fine structural detailsfig . ", "8the simshiftdb web - interface at the center for applied molecular engineering an example simshiftdb search result .", "the left panel shows a representative template structure found in a simshiftdb search for ph1500c ( 1p22 , residues a392-w415 ) .", "residues shown in green are predicted by talos to be in the -region of ramachandran space and the residue in yellow in the + -region , while for residues in white there is no prediction .", "only one talos prediction is made for the six residues outside of canonical secondary structure , i.e. the two turns and the -bulge in the first strand .", "the right panel shows the structure calculated with all available nmr data , showing very good agreement to the template .", "the coloring is as above , with the addition of blue residues for residues in the -region of ramachandran space .", "a conserved sidechain hydrogen - bond acceptor ( t101 in ph1500c ) is shown in grey , highlighting the ability of simshiftdb searches to reveal fine structural details the simshiftdb web - interface at the center for applied molecular engineering this example highlights the major difference in the simshiftdb and talos approaches , i.e. the length of the template structures found by simshiftdb when compared to the tripeptides used to make talos predictions .", "the second difference is the use of the e - value as a continuous measure of quality , rather than a discrete selection criterion based on a consensus of the ten best hits . in some cases there", "may be only one or two templates found for any region of the protein , but low e - value scores can nevertheless allow predictions with high confidence .", "we have established an accuracy of above 85% for simshiftdb predictions , based on our benchmark set of proteins .", "this may at first glance compare poorly to talos , where an accuracy of 9798% is reported .", "however , it must be considered that this value is based on single simshiftdb predictions , rather than the consensus of 10 predictions .", "also , it is worth noting that talos is very accurate within secondary structure , and therefore the 23% of errors must be concentrated in the smaller fraction of other predictions . in our experience", ", these errors often result from predictions made out of structural context ; e.g. for a residue in a -turn based on tripeptides from a helix .", "the wider context provided by simshiftdb results should therefore add both to the confidence of its predictions and those from talos .", "the optimum chemical shift priority found for simshiftdb searches is somewhat surprising in that it contains h , which is not generally regarded as containing much structural information .", "perhaps this is due to some complementarities of the information from h and that from other shifts .", "it is worth noting , though , that the difference between the best priorities is small , and it may be worth testing a range of priorities .", "this is easily possible ; although the program searches a database of 13,000 protein structures , an average simshiftdb run takes only 30 seconds on a standard laptop ( intel t2500 , 2.0  ghz , 1  gb ram ) .", "the different results are comparable using the calculated e - values , thereby enabling the user to select the most promising result ."], "abstract_text": ["<S> we present simshiftdb , a new program to extract conformational data from protein chemical shifts using structural alignments . </S>", "<S> the alignments are obtained in searches of a large database containing 13,000 structures and corresponding back - calculated chemical shifts . </S>", "<S> simshiftdb makes use of chemical shift data to provide accurate results even in the case of low sequence similarity , and with even coverage of the conformational search space . </S>", "<S> we compare simshiftdb to hhsearch , a state - of - the - art sequence - based search tool , and to talos , the current standard tool for the task . </S>", "<S> we show that for a significant fraction of the predicted similarities , simshiftdb outperforms the other two methods . </S>", "<S> particularly , the high coverage afforded by the larger database often allows predictions to be made for residues not involved in canonical secondary structure , where talos predictions are both less frequent and more error prone . </S>", "<S> thus simshiftdb can be seen as a complement to currently available methods . </S>"], "labels": null, "section_names": ["Introduction", "Implementation", "Results", "The benchmark set", "Evaluation of prediction accuracy", "Comparison to HHsearch", "Comparison to TALOS", "Discussion"], "sections": [["chemical shifts are now routinely used as a source of local conformational restraints in the structure determination of proteins by nmr , due mostly to the widespread use of programs such as talos ( cornilescu et al .", "1999 ) and shiftor / preditor ( neal et al . 2006 ; berjanskii et al . 2006 ) .", "these programs share a common approach and output similar data ; both search a database that correlates local patterns of chemical shifts with local conformation , and both provide backbone dihedral angle restraints for individual residues .", "this approach has been very successful , but has some limitations in the stringent criteria needed for selecting proteins or protein fragments to populate the database , i.e. only those with both highly reliable chemical shift and structural data can be included .", "although this may seem adequate  for example , the talos database contains 186 proteins subdivided into over 24,000 tripeptide fragments ( http://spin.niddk.nih.gov/nmrpipe/talos/)the sequence / conformation search space is very large , and the database coverage is unevenly distributed . as a result , rare combinations of amino - acid and conformation may be under - represented in the database , leading to significant under - prediction and even to errors outside of the heavily populated regions of the ramachandran map .", "we have adopted an alternative approach to extracting structural data from chemical shifts based on our simshiftdb algorithm .", "the original simshift was designed to test for structural similarities between proteins in a pair wise manner using chemical shifts to supplement sequence data .", "simshift showed improved ability to detect distant structural relationships when compared to state - of - the - art methods based on the sequence alone .", "a natural further development of pairwise comparison was to adapt the simshift algorithm for database searching , resulting in simshiftdb ( ginzinger et al .", ", simshiftdb provides a list of matching proteins in the database , scored by a measure of statistical significance . in effect", "it searches a synthetic chemical shift database of 13,000 proteins based on the astral library ( chandonia et al .", "the matching sequence can be of any length , and structurally similar regions can be found ranging from small , locally similar fragments up to full domains . in principle , any structural alignment method can also be used to make predictions of local conformation by extracting torsion angles from matching regions of the target proteins , and it is this implementation of the simshiftdb algorithm we present here .", "we benchmark the program against talos as a standard for current methods and hhpred , a sequence search method based on hidden markov models ( sding et al .", "we show that simshiftdb can significantly increase the amount of information that can be derived from chemical shifts .", "2007a ) routine for standardizing chemical shift referencing to produce a pipeline for analysis of chemical shift data ."], ["full details of the simshiftdb algorithm have been published previously ( see ginzinger et al .", "2007b ) , but it is worthwhile giving a brief overview here ( see fig .  1 ) .", "given a target protein , simshiftdb analyzes each possible pairing of the target protein with one of the template protein structures in the database .", "here we use the proteins from the astral database ( version 1.71 , filtered for 95% sequence identity ) to build the simshiftdb template database . as simshiftdb", "is based on the comparison of chemical shift data , the chemical shifts for all entries in the template database are back - calculated from the three - dimensional structure using shiftx ( neal et al . 2003 ) . for each combination of the target protein with a template from the database ,", "top : an example for a set of local similarities for a target protein ( s ) and a template protein ( t ) .", "bottom : combination of a subset of local similarities to yield a self - consistent final alignmentstep 1 : local similarities are found by looking for high scoring combinations of parts of the target protein sequence ( s ) with parts of the template protein sequence ( t ) .", "for example , block b in the figure shows that the chemical shifts of the target protein sequence from index xmin to xmax are similar to the chemical shifts of the template protein sequence from index ymin to ymax .", "the similarity is calculated by summing the pairwise scores of the residues in the similar region in analogy to a pairwise sequence alignment .", "the pairwise similarity scores are given by the so - called chemical shift substitution matrices , which give a score for each combination of two residues with associated chemical shifts ( for more details see ginzinger 2008).step 2 : the set of local similarities from step 1 is taken as an input for step 2 , where the most significant combination of blocks is identified , according to a statistical model of alignment scores ( karlin and altschul 1993 ) .", "additionally , two blocks have to fulfill two constraints for their combination to be considered : an explanation of the simshift algorithm .", "top : an example for a set of local similarities for a target protein ( s ) and a template protein ( t ) .", "bottom : combination of a subset of local similarities to yield a self - consistent final alignment step 1 : local similarities are found by looking for high scoring combinations of parts of the target protein sequence ( s ) with parts of the template protein sequence ( t ) . fig . ", "for example , block b in the figure shows that the chemical shifts of the target protein sequence from index xmin to xmax are similar to the chemical shifts of the template protein sequence from index ymin to ymax .", "the similarity is calculated by summing the pairwise scores of the residues in the similar region in analogy to a pairwise sequence alignment .", "the pairwise similarity scores are given by the so - called chemical shift substitution matrices , which give a score for each combination of two residues with associated chemical shifts ( for more details see ginzinger 2008 ) .", "step 2 : the set of local similarities from step 1 is taken as an input for step 2 , where the most significant combination of blocks is identified , according to a statistical model of alignment scores ( karlin and altschul 1993 ) .", "additionally , two blocks have to fulfill two constraints for their combination to be considered : blocks may not overlap in the target or in the template protein ; this would otherwise result in an ambiguous alignment.as the three - dimensional structure of the template protein is known , we further require that the euclidean distance between the end of the first block and the beginning of the second block may be bridged ( according to chemical restraints ) by the relevant sequence of amino acids in the target protein .", "blocks may not overlap in the target or in the template protein ; this would otherwise result in an ambiguous alignment . as the three - dimensional structure of the template protein", "is known , we further require that the euclidean distance between the end of the first block and the beginning of the second block may be bridged ( according to chemical restraints ) by the relevant sequence of amino acids in the target protein . finally , we calculate an e - value for the optimal combination of blocks .", "this e - value represents the number of alignments of equal or better quality , which are expected to occur by chance , given the distribution of the amino acids with associated chemical shifts in the target protein and the template database .", "additionally , the e - value takes the size of the template database into account . according to the following evaluation , an e - value of < 10 guarantees a high quality alignment ."], ["to test the performance of simshiftdb , a benchmark set has to be defined for which both chemical shifts and the three - dimensional structure of the protein are available .", "the bmrb ( seavey et al . 1991 ) is the main public repository for chemical shift data .", "however , there is no consistent mapping to the structural databases , making it difficult to relate structural with chemical shift information reliably .", "therefore a mapping between bmrb and astral is calculated based on amino acid sequence similarity .", "every entry in the benchmark set has to fulfill the following constraints : a 100% sequence match to an astral entry.at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) .", "at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) . to identify protein structures corresponding to the respective bmrb entries , a blast - search ( altschul et al . 1990 ) against the sequences from the astral database is conducted for each bmrb entry . if the full bmrb sequence can be matched without gaps against an astral sequence , the corresponding astral structure is assigned to the bmrb entry .", "as some entries in bmrb match more than one sequence in astral , one representative structure has to be chosen .", "2004 ) provided for each astral entry , thereby selecting the structure with the best resolution . through this procedure", "a benchmark set containing 144 entries was derived . when calculating the similarity score for two residues , simshiftdb is restricted to at most three chemical shifts from the following list : h , h ,", "n , c , c , and c. thus it is important to select a combination of shifts to extract maximum information , and a priority for replacing missing shifts . to identify the most successful strategy , we tested all possible priorities for the six atom types , resulting in 6 ! ", "the most successful priority was : c  >  c  >  h  >  c  >  h  >  n. this is the default priority , and is used in the following analysis . to evaluate the prediction accuracy", ", we applied the program to all entries in the benchmark set , using all proteins from the simshiftdb template database as potential templates .", "subsequently , we used all alignments that achieved an e - value better than 10 to infer torsion angles for the target residue from the associated template residues . if residues of the target were mapped multiple times , we based the prediction on the highest scoring alignment .", "it is important to evaluate the performance of simshiftdb as a function of sequence similarity ; therefore 9 evaluations were performed with the maximum allowed sequence similarity in the evaluated alignments set between 20 and 100% .", "about 70% of all torsion angles predicted using simshiftdb have a high accuracy ( 15 error ) .", "another 10 to 20% of the predicted angles have an error of less than 30. therefore simshiftdb yields accurate results in 85 to 90% of the evaluated predictions . through the use of the chemical shifts ,", "this performance is nearly independent of the percentage of sequence identity in the respective alignments.fig . ", "2evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists also of interest is the performance of simshiftdb on different types of secondary structure .", "figures  3 and 4 show the difference between the secondary structure content in all predictions versus that in high quality and erroneous predictions , respectively .", "it can be seen that predictions for sheet ( for both high quality and erroneous predictions ) match well with the percentage observed in all predictions , and this match is largely independent of sequence similarity .", "in contrast , the percentage of high quality predictions in helix increases with decreasing sequence similarity , whereas the corresponding percentage in coil regions decreases . for erroneous predictions", "this seems logical , as the structures for coil regions are less reliable , and predictions are clearly harder to make than for secondary structure .", "this test shows empirically that simshiftdb has no significant bias when comparing performance in predicting helix versus sheet .", "the independence of sheet predictions from sequence similarity indicates that the chemical shift data is more diagnostic for sheet than for helix.fig . ", "3difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "4difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . ", "3 difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "the lines show the overall secondary structure content in all predictions difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig .  3 to show empirically that simshiftdb uses the information in the chemical shift data to yield more sensitive alignments , especially in the case of low sequence similarity , we compare simshiftdb to hhsearch ( sding 2005 ) .", "hhsearch , a sensitive search tool based on hidden markov models , calculates alignments between proteins using the primary sequence complemented by sequence - based predictions of secondary structure .", "2005 ) , a protein structure prediction method based on hhsearch alignments , ranked second best in the casp7 ( battey et al .", "additionally , it is freely available for download and gives the user the possibility to define arbitrary template databases . therefore it is perfectly suited to serve as a reference for purely sequence - based methods . to compare the performance of simshiftdb and hhsearch we used the benchmark set defined in the previous section . for both methods we ran each target protein against the simshiftdb template database and used alignments achieving an e - value better than 10 to predict torsion angles for the residues in the target protein .", "if residues were mapped multiple times , the prediction was based on the highest scoring alignment .", "the following notation is used for the presentation of the results :- a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5.two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30.missing predictions are treated as predictions with an error > 30. a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5. two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30. missing predictions are treated as predictions with an error > 30. figure  5 shows the results of this evaluation for alignments with maximal sequence identities ranging from 10 to 100% .", "there is a clear trend for simshiftdb to outperform hhsearch as the sequence identity decreases , demonstrating that simshiftdb uses the structural information in chemical shifts to improve alignments.fig . ", "5simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality .", "the brown line corresponds to the number of torsion angles predicted ( right axis ) simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality .", "the brown line corresponds to the number of torsion angles predicted ( right axis ) it is important to compare simshiftdb to the most prominent method for predicting torsion angles from chemical shifts , namely talos ( cornilescu et al .", "again we used the benchmark set defined earlier , and made torsion angle predictions based on simshiftdb alignments that achieved an e - value better than 10 .", "we then compared the quality of each torsion angle prediction to the corresponding talos prediction .", "figure  6 shows that simshiftdb outperforms talos in at least 30% of all cases .", "it should be noted that there is likely to be a significant bias towards talos in these results , as many members of the benchmark set will have been calculated using talos restraints.fig . ", "6simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . ", "5 simshiftdb predictions compared to the respective talos predictions using the same notation as in fig .  5"], ["to test the performance of simshiftdb , a benchmark set has to be defined for which both chemical shifts and the three - dimensional structure of the protein are available .", "the bmrb ( seavey et al . 1991 ) is the main public repository for chemical shift data .", "however , there is no consistent mapping to the structural databases , making it difficult to relate structural with chemical shift information reliably .", "therefore a mapping between bmrb and astral is calculated based on amino acid sequence similarity .", "every entry in the benchmark set has to fulfill the following constraints : a 100% sequence match to an astral entry.at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) .", "at least 100 residues with associated chemical shifts ( to exclude very short protein fragments ; e.g. single helices ) . to identify protein structures corresponding to the respective bmrb entries , a blast - search ( altschul et al . 1990 ) against the sequences from the astral database is conducted for each bmrb entry . if the full bmrb sequence can be matched without gaps against an astral sequence , the corresponding astral structure is assigned to the bmrb entry .", "as some entries in bmrb match more than one sequence in astral , one representative structure has to be chosen .", "2004 ) provided for each astral entry , thereby selecting the structure with the best resolution . through this procedure"], ["when calculating the similarity score for two residues , simshiftdb is restricted to at most three chemical shifts from the following list : h , h , n , c , c , and c. thus it is important to select a combination of shifts to extract maximum information , and a priority for replacing missing shifts . to identify the most successful strategy , we tested all possible priorities for the six atom types , resulting in 6 !  =  720 evaluations .", "the most successful priority was : c  >  c  >  h  >  c  >  h  >  n. this is the default priority , and is used in the following analysis . to evaluate the prediction accuracy", ", we applied the program to all entries in the benchmark set , using all proteins from the simshiftdb template database as potential templates .", "subsequently , we used all alignments that achieved an e - value better than 10 to infer torsion angles for the target residue from the associated template residues . if residues of the target were mapped multiple times , we based the prediction on the highest scoring alignment .", "it is important to evaluate the performance of simshiftdb as a function of sequence similarity ; therefore 9 evaluations were performed with the maximum allowed sequence similarity in the evaluated alignments set between 20 and 100% .", "about 70% of all torsion angles predicted using simshiftdb have a high accuracy ( 15 error ) .", "another 10 to 20% of the predicted angles have an error of less than 30. therefore simshiftdb yields accurate results in 85 to 90% of the evaluated predictions . through the use of the chemical shifts ,", "this performance is nearly independent of the percentage of sequence identity in the respective alignments.fig . ", "2evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists evaluation of torsion angle predictions calculated using simshiftdb , separated according to the maximum allowed sequence identity between query and target .", "the boxes show the percentage of dihedral angle predictions with an error of 30. the red part of each box shows the percentage of predictions with an error of 15. the brown line represents the percentage of residues from the test set for which a prediction exists also of interest is the performance of simshiftdb on different types of secondary structure .", "figures  3 and 4 show the difference between the secondary structure content in all predictions versus that in high quality and erroneous predictions , respectively .", "it can be seen that predictions for sheet ( for both high quality and erroneous predictions ) match well with the percentage observed in all predictions , and this match is largely independent of sequence similarity .", "in contrast , the percentage of high quality predictions in helix increases with decreasing sequence similarity , whereas the corresponding percentage in coil regions decreases . for erroneous predictions", "this seems logical , as the structures for coil regions are less reliable , and predictions are clearly harder to make than for secondary structure .", "this test shows empirically that simshiftdb has no significant bias when comparing performance in predicting helix versus sheet .", "the independence of sheet predictions from sequence similarity indicates that the chemical shift data is more diagnostic for sheet than for helix.fig . ", "3difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "4difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig . ", "3 difference between secondary structure content in high quality predictions versus the content for all predictions .", "the bars show the difference in helix , sheet and coil content ( red , yellow and grey , respectively ) .", "the lines show the overall secondary structure content in all predictions difference between secondary structure content in erroneous predictions versus the content for all predictions using the same scheme as in fig .  3"], ["to show empirically that simshiftdb uses the information in the chemical shift data to yield more sensitive alignments , especially in the case of low sequence similarity , we compare simshiftdb to hhsearch ( sding 2005 ) .", "hhsearch , a sensitive search tool based on hidden markov models , calculates alignments between proteins using the primary sequence complemented by sequence - based predictions of secondary structure .", "2005 ) , a protein structure prediction method based on hhsearch alignments , ranked second best in the casp7 ( battey et al . 2007 ) experiment .", "additionally , it is freely available for download and gives the user the possibility to define arbitrary template databases .", "therefore it is perfectly suited to serve as a reference for purely sequence - based methods . to compare the performance of simshiftdb and hhsearch we used the benchmark set defined in the previous section .", "for both methods we ran each target protein against the simshiftdb template database and used alignments achieving an e - value better than 10 to predict torsion angles for the residues in the target protein .", "if residues were mapped multiple times , the prediction was based on the highest scoring alignment .", "the following notation is used for the presentation of the results :- a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5.two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30.missing predictions are treated as predictions with an error > 30. a simshiftdb prediction is called better if it has an error of 30 and the corresponding hhsearch prediction has an error which is worse by more than 5. two predictions are called equal if both have an error of 30 and the difference between the errors is less than 5 , or both predictions have an error > 30. missing predictions are treated as predictions with an error > 30. figure  5 shows the results of this evaluation for alignments with maximal sequence identities ranging from 10 to 100% .", "there is a clear trend for simshiftdb to outperform hhsearch as the sequence identity decreases , demonstrating that simshiftdb uses the structural information in chemical shifts to improve alignments.fig . ", "5simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality .", "the brown line corresponds to the number of torsion angles predicted ( right axis ) simshiftdb predictions compared to the respective hhsearch predictions , separated according to the maximum allowed sequence identity between query and target .", "the red line shows the percentage of predictions where simshiftdb performs better , the yellow line show the percentage where simshiftdb is either better or gives a result of equal quality ."], ["it is important to compare simshiftdb to the most prominent method for predicting torsion angles from chemical shifts , namely talos ( cornilescu et al . 1999 ) .", "again we used the benchmark set defined earlier , and made torsion angle predictions based on simshiftdb alignments that achieved an e - value better than 10 .", "we then compared the quality of each torsion angle prediction to the corresponding talos prediction .", "figure  6 shows that simshiftdb outperforms talos in at least 30% of all cases .", "it should be noted that there is likely to be a significant bias towards talos in these results , as many members of the benchmark set will have been calculated using talos restraints.fig . ", "6simshiftdb predictions compared to the respective talos predictions using the same notation as in fig . ", "5 simshiftdb predictions compared to the respective talos predictions using the same notation as in fig .  5"], ["we have presented simshiftdb and shown that the program is able to sensitively extract structural information from chemical shift data .", "this information is to a certain extent complementary to that from currently available tools . on one hand", "simshiftdb shows its strength especially in cases of low sequence similarity , which underlines the advantage of including chemical shift information in the alignment algorithm . on the other hand", ", we were able to show that one - third of the predictions by simshiftdb clearly have a higher quality than the corresponding talos predictions , and this is largely independent of sequence similarity .", "the main advantage of simshiftdb is derived from its superior coverage of the search space , due to the large and quickly adaptable template database .", "simshiftdb outperforms talos especially in those cases where talos finds no predictions classified as  good  according to its selection criteria .", "simshiftdb and talos are therefore complementary , and can be used in parallel to increase the number of available predictions .", "ph1500c is a 78-residue homo - hexameric domain currently under investigation in our laboratories , and was chosen because it shows no significant sequence similarity to proteins of known structure .", "we used simshiftdb to search for templates matching ph1500c , using several different chemical shift priorities .", "the search identifies several templates at e - values around 10 , which correspond to the region g17-f40 of ph1500c and consist of three - stranded -meander linked by two tight turns ( fig . ", "the consensus of simshiftdb predictions shows the first turn to be a standard type i -turn , while the second is a less commonly observed 5-residue -turn .", "an example template agrees very well with all structural data available in this region ( fig . ", "7 ) , and suggests local conformational details such as sidechain positions and local hydrogen bonding networks.fig . ", "the left panel shows a representative template structure found in a simshiftdb search for ph1500c ( 1p22 , residues a392-w415 ) .", "residues shown in green are predicted by talos to be in the -region of ramachandran space and the residue in yellow in the + -region , while for residues in white there is no prediction .", "only one talos prediction is made for the six residues outside of canonical secondary structure , i.e. the two turns and the -bulge in the first strand .", "the right panel shows the structure calculated with all available nmr data , showing very good agreement to the template .", "the coloring is as above , with the addition of blue residues for residues in the -region of ramachandran space .", "a conserved sidechain hydrogen - bond acceptor ( t101 in ph1500c ) is shown in grey , highlighting the ability of simshiftdb searches to reveal fine structural detailsfig . ", "8the simshiftdb web - interface at the center for applied molecular engineering an example simshiftdb search result .", "the left panel shows a representative template structure found in a simshiftdb search for ph1500c ( 1p22 , residues a392-w415 ) .", "residues shown in green are predicted by talos to be in the -region of ramachandran space and the residue in yellow in the + -region , while for residues in white there is no prediction .", "only one talos prediction is made for the six residues outside of canonical secondary structure , i.e. the two turns and the -bulge in the first strand .", "the right panel shows the structure calculated with all available nmr data , showing very good agreement to the template .", "the coloring is as above , with the addition of blue residues for residues in the -region of ramachandran space .", "a conserved sidechain hydrogen - bond acceptor ( t101 in ph1500c ) is shown in grey , highlighting the ability of simshiftdb searches to reveal fine structural details the simshiftdb web - interface at the center for applied molecular engineering this example highlights the major difference in the simshiftdb and talos approaches , i.e. the length of the template structures found by simshiftdb when compared to the tripeptides used to make talos predictions .", "the second difference is the use of the e - value as a continuous measure of quality , rather than a discrete selection criterion based on a consensus of the ten best hits . in some cases there", "may be only one or two templates found for any region of the protein , but low e - value scores can nevertheless allow predictions with high confidence .", "we have established an accuracy of above 85% for simshiftdb predictions , based on our benchmark set of proteins .", "this may at first glance compare poorly to talos , where an accuracy of 9798% is reported .", "however , it must be considered that this value is based on single simshiftdb predictions , rather than the consensus of 10 predictions .", "also , it is worth noting that talos is very accurate within secondary structure , and therefore the 23% of errors must be concentrated in the smaller fraction of other predictions . in our experience", ", these errors often result from predictions made out of structural context ; e.g. for a residue in a -turn based on tripeptides from a helix .", "the wider context provided by simshiftdb results should therefore add both to the confidence of its predictions and those from talos .", "the optimum chemical shift priority found for simshiftdb searches is somewhat surprising in that it contains h , which is not generally regarded as containing much structural information .", "perhaps this is due to some complementarities of the information from h and that from other shifts .", "it is worth noting , though , that the difference between the best priorities is small , and it may be worth testing a range of priorities .", "this is easily possible ; although the program searches a database of 13,000 protein structures , an average simshiftdb run takes only 30 seconds on a standard laptop ( intel t2500 , 2.0  ghz , 1  gb ram ) .", "the different results are comparable using the calculated e - values , thereby enabling the user to select the most promising result ."]]}
{"article_id": "PMC3216393", "article_text": ["sutureless clear corneal incisions are now arguably the most common incisions made to perform cataract surgery with phacoemulsification , replacing scleral tunnel and limbal incisions .", "there are significant differences in the effects of clear corneal and scleral or limbal incisions , related to anatomical and physiological differences in the respective structures where the incisions are made .", "the cornea is comprised of a regular lamellar structure of collagen fibrils that stretch from limbus to limbus , arranged in a lattice formation ; this is what provides the primary structural support of the cornea and accounts for its transparency .", "vascular arcades are present , providing a potential source of fibroblasts . the differences in the healing effects of incisions at the limbus and the cornea have been previously discussed in the literature [ 24 ] .", "limbal incisions appear to heal more quickly and are more resistant to deformation pressure than those in the cornea .", "clear corneal incisions also appear to increase the likelihood of endophthalmitis , potentially for the reasons above . in short , there are no disadvantages to a limbal incision in terms of surgical safety . from a structural point of view", "it is known that incisions for cataract surgery will induce a flattening effect when made on ( or near ) the steep axis of the cornea .", "this effect is positively correlated with incision size ( larger incisions generating more astigmatism , all other things being equal ) and location ( scleral or limbal incision inducing less astigmatism than clear corneal ) , though for small incisions the effect of location appears less critical [ 6 , 7 ] . wound construction also appears to have an effect , with square incisions reported to affect astigmatism the least .", "data related to surgically induced astigmatism in the recent literature is primarily related to clear corneal incisions .", "the data reported here summarize the astigmatic changes produced with a small ( 2.2  mm ) square posterior limbal incision .", "a retrospective patient chart review was conducted at one site ( pe ) for a study of toric and spherical iols . those eyes with preoperative and postoperative keratometry results", "eyes were excluded if they had irregular ( nonorthogonal ) corneal astigmatism or if they had previous corneal surgery .  ", "this obviated the need for a specific informed consent or irb approval for the data collection undertaken here .", "in addition , all patients sign an acknowledgement that their deidentified phi data may be used for research purposes when they are seen in the practice .  ", "comparative surgically induced astigmatism data were obtained from one of the authors ( w. hill ) from a website specifically designed to collect preoperative and postoperative keratometry data for the purposes of calculating surgically induced astigmatism .", "this site provides the necessary spreadsheet and instructions to surgeons for the calculation of sia .", "an option allows any surgeon who uses the spreadsheet to upload their data to the website for the purposes of aggregate analysis .", "again , no phi was collected so informed consent was not required for this data set .", "results from ph and the aggregate data from wh were tabulated in microsoft excel and analyzed using statistica statistical software .", "differences between groups were calculated with t - tests and analysis of variance ( anova ) tests , with significance at p < 0.05 . for the patients at one site ( p. ernest )", "the data were corroborated with an automated keratometry reading and the automated keratometry feature of the iolmaster ( carl zeiss meditec , jena , germany ) .", "the automated keratometry readings were repeated on all patients postoperatively , and the comparison to the preoperative automated keratometry was used to calculate sia .", "the surgical technique employed in the time period in which the retrospective data were collected was constant for all patients .", "all incisions were temporal , 2.2  mm square posterior limbal incisions , using a technique previously described in the literature .", "surgical technique was not described in the aggregate sia data collected off the web , but incision type ( e.g. , clear corneal , limbal ) was generally indicated , and the size of the incision was noted .", "these variables were collected so that surgeons could submit different preoperative and postoperative data sets for different incision sizes and locations .", "the review of available clinical records from pe yielded a total of 38 eyes that both met the criteria for inclusion and had available postoperative keratometry data for analysis .", "surgically induced astigmatism was calculated as the vector difference between preoperative and postoperative anterior corneal astigmatism as measured with automated keratometry .", "the sia in this cohort averaged 0.25  d with a standard deviation of 0.14  d.   a total of 1,712 eyes were available in the aggregate sia data from w. hill , from 51 different surgeons .", "the data were filtered to remove duplicates , include only incision sizes of 2.2  mm , and exclude those records where the incision type was not indicated .", "where there were not at least 20 surgeries in any incision category ( size and location ) for a surgeon , those data were also deleted .", "after this data - screening step , 246 surgeries from 5 surgeons remained for comparative analysis .  ", "average preoperative keratometry was not statistically significantly different between surgeons ( p = 0.41 ) ; means ranged from 43.8  d to 44.5  d. the magnitude of preoperative corneal cylinder was also not statistically significantly different between surgeons ( p = 0.13 ) ; means ranged from 0.79  d to 1.04  d. \n table 1 contains a summary of the surgically induced astigmatism by surgeon", ". looking at the aggregate clear corneal incision data versus the ernest posterior limbal data , there was a statistically significant difference in the surgically induced astigmatism by incision type ( p < 0.001 ) . inside the clear corneal group", "as a post hoc test , the ernest data were compared to the surgeon with the lowest average sia from the clear corneal incision group ( surgeon 5 ) ; there was a statistically significantly lower mean sia in the ernest cohort ( p = 0.001 ) .", "note , too , that the standard deviation of the ernest cohort is less than half that of the cohorts of 4 of the 5 other surgeons , and 40% lower than surgeon 5 despite having less than half the sample size . \n", "surgeons 1 to 5 used 2.2  mm clear corneal incisions , while pe used a 2.2  mm square posterior limbal incision .", "there was a statistically significant difference in sia by surgeon ( p < 0.001 ) .", "post hoc testing showed that the ernest data yielded a statistically significantly lower sia than the other surgeons ' data .", "the results suggest that the magnitude and the variability of surgically induced astigmatism with small incision surgery ( 2.2  mm ) is significantly lower if a posterior limbal incision is used instead of a clear corneal incision .", "results for the clear corneal incisions used here as comparative data are consistent with those recently reported in the literature .", "wilczynski et al . reported sia values of 0.50  0.25  d with 1.7  mm and 1.8  mm surgical incisions .", "holland reported sia values of 0.6  0.4  d for 2.4  mm clear corneal incisions .", "masket reported that sia results were somewhat lower with his 2.2  mm clear corneal incisions ( 0.35  0.21  d ) but it is worth noting that his incision was started with a groove at the temporal limbus .", "some of the variability of sia data from surgeons other than ernest is likely related to differences in technique between surgeons .", "in addition , intrasurgeon variability may be slightly higher if surgeons did not measure their postoperative keratometry 1 month or more after surgery , as there is some change in keratometry expected in that first month .", "the minimization of the magnitude ( and variability ) of surgically induced astigmatism is important for modern day cataract surgery , particularly with the use of toric and multifocal intraocular lenses", "residual astigmatism after surgery will reduce the likelihood of spectacle independence for distance vision for these patients .", "minimizing astigmatism for multifocal iols is equally important , as even small amounts of residual astigmatism can compromise the outcome with regard to uncorrected visual acuity at distance .", "surgeons interested in reducing the magnitude and variability of induced astigmatism at the time of cataract surgery may want to consider the use of a 2.2  mm square posterior limbal incision ."], "abstract_text": ["<S> \n purpose . to compare the surgically induced astigmatism from clear corneal and square posterior limbal incisions at the time of cataract surgery </S>", "<S> . methods . </S>", "<S> surgically induced astigmatism was calculated for a set of eyes after cataract surgery using a temporal 2.2  mm square posterior limbal incision . </S>", "<S> results were compared to similar available data from surgeons using clear corneal incisions of similar size . </S>", "<S> results . </S>", "<S> preoperative corneal astigmatism averaged 1.0  d and was not significantly different between the incision types . surgically induced astigmatism with </S>", "<S> the 2.2  mm posterior limbal incision averaged 0.25  0.14  d , significantly lower in magnitude than the aggregate \n surgically induced astigmatism produced by the 2.2  mm clear corneal \n incision ( 0.68  0.49  d ) . conclusion . the 2.2  mm \n square posterior limbal incision induced significantly less , and \n significantly less variable , surgically induced astigmatism \n relative to a similar - sized clear corneal incision . </S>", "<S> this is likely \n to improve refractive outcomes , particularly important with regard \n to premium intraocular lenses . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Patients and Methods", "3. Results", "4. Discussion"], "sections": [["sutureless clear corneal incisions are now arguably the most common incisions made to perform cataract surgery with phacoemulsification , replacing scleral tunnel and limbal incisions .", "there are significant differences in the effects of clear corneal and scleral or limbal incisions , related to anatomical and physiological differences in the respective structures where the incisions are made .", "the cornea is comprised of a regular lamellar structure of collagen fibrils that stretch from limbus to limbus , arranged in a lattice formation ; this is what provides the primary structural support of the cornea and accounts for its transparency .", "vascular arcades are present , providing a potential source of fibroblasts . the differences in the healing effects of incisions at the limbus and the cornea have been previously discussed in the literature [ 24 ] .", "limbal incisions appear to heal more quickly and are more resistant to deformation pressure than those in the cornea .", "clear corneal incisions also appear to increase the likelihood of endophthalmitis , potentially for the reasons above . in short , there are no disadvantages to a limbal incision in terms of surgical safety . from a structural point of view", "it is known that incisions for cataract surgery will induce a flattening effect when made on ( or near ) the steep axis of the cornea .", "this effect is positively correlated with incision size ( larger incisions generating more astigmatism , all other things being equal ) and location ( scleral or limbal incision inducing less astigmatism than clear corneal ) , though for small incisions the effect of location appears less critical [ 6 , 7 ] . wound construction also appears to have an effect , with square incisions reported to affect astigmatism the least .", "data related to surgically induced astigmatism in the recent literature is primarily related to clear corneal incisions .", "the data reported here summarize the astigmatic changes produced with a small ( 2.2  mm ) square posterior limbal incision ."], ["a retrospective patient chart review was conducted at one site ( pe ) for a study of toric and spherical iols . those eyes with preoperative and postoperative keratometry results", "eyes were excluded if they had irregular ( nonorthogonal ) corneal astigmatism or if they had previous corneal surgery .  ", "this obviated the need for a specific informed consent or irb approval for the data collection undertaken here .", "in addition , all patients sign an acknowledgement that their deidentified phi data may be used for research purposes when they are seen in the practice .  ", "comparative surgically induced astigmatism data were obtained from one of the authors ( w. hill ) from a website specifically designed to collect preoperative and postoperative keratometry data for the purposes of calculating surgically induced astigmatism .", "this site provides the necessary spreadsheet and instructions to surgeons for the calculation of sia .", "an option allows any surgeon who uses the spreadsheet to upload their data to the website for the purposes of aggregate analysis .", "again , no phi was collected so informed consent was not required for this data set .", "results from ph and the aggregate data from wh were tabulated in microsoft excel and analyzed using statistica statistical software .", "differences between groups were calculated with t - tests and analysis of variance ( anova ) tests , with significance at p < 0.05 . for the patients at one site ( p. ernest )", "the data were corroborated with an automated keratometry reading and the automated keratometry feature of the iolmaster ( carl zeiss meditec , jena , germany ) .", "the automated keratometry readings were repeated on all patients postoperatively , and the comparison to the preoperative automated keratometry was used to calculate sia .", "the surgical technique employed in the time period in which the retrospective data were collected was constant for all patients .", "all incisions were temporal , 2.2  mm square posterior limbal incisions , using a technique previously described in the literature .", "surgical technique was not described in the aggregate sia data collected off the web , but incision type ( e.g. , clear corneal , limbal ) was generally indicated , and the size of the incision was noted .", "these variables were collected so that surgeons could submit different preoperative and postoperative data sets for different incision sizes and locations ."], ["the review of available clinical records from pe yielded a total of 38 eyes that both met the criteria for inclusion and had available postoperative keratometry data for analysis .", "surgically induced astigmatism was calculated as the vector difference between preoperative and postoperative anterior corneal astigmatism as measured with automated keratometry .", "the sia in this cohort averaged 0.25  d with a standard deviation of 0.14  d.   a total of 1,712 eyes were available in the aggregate sia data from w. hill , from 51 different surgeons .", "the data were filtered to remove duplicates , include only incision sizes of 2.2  mm , and exclude those records where the incision type was not indicated .", "where there were not at least 20 surgeries in any incision category ( size and location ) for a surgeon , those data were also deleted .", "after this data - screening step , 246 surgeries from 5 surgeons remained for comparative analysis .  ", "average preoperative keratometry was not statistically significantly different between surgeons ( p = 0.41 ) ; means ranged from 43.8  d to 44.5  d. the magnitude of preoperative corneal cylinder was also not statistically significantly different between surgeons ( p = 0.13 ) ; means ranged from 0.79  d to 1.04  d. \n table 1 contains a summary of the surgically induced astigmatism by surgeon", ". looking at the aggregate clear corneal incision data versus the ernest posterior limbal data , there was a statistically significant difference in the surgically induced astigmatism by incision type ( p < 0.001 ) . inside the clear corneal group", "as a post hoc test , the ernest data were compared to the surgeon with the lowest average sia from the clear corneal incision group ( surgeon 5 ) ; there was a statistically significantly lower mean sia in the ernest cohort ( p = 0.001 ) .", "note , too , that the standard deviation of the ernest cohort is less than half that of the cohorts of 4 of the 5 other surgeons , and 40% lower than surgeon 5 despite having less than half the sample size . \n", "surgeons 1 to 5 used 2.2  mm clear corneal incisions , while pe used a 2.2  mm square posterior limbal incision .", "there was a statistically significant difference in sia by surgeon ( p < 0.001 ) .", "post hoc testing showed that the ernest data yielded a statistically significantly lower sia than the other surgeons ' data ."], ["the results suggest that the magnitude and the variability of surgically induced astigmatism with small incision surgery ( 2.2  mm ) is significantly lower if a posterior limbal incision is used instead of a clear corneal incision .", "results for the clear corneal incisions used here as comparative data are consistent with those recently reported in the literature .", "wilczynski et al . reported sia values of 0.50  0.25  d with 1.7  mm and 1.8  mm surgical incisions .", "holland reported sia values of 0.6  0.4  d for 2.4  mm clear corneal incisions .", "masket reported that sia results were somewhat lower with his 2.2  mm clear corneal incisions ( 0.35  0.21  d ) but it is worth noting that his incision was started with a groove at the temporal limbus .", "some of the variability of sia data from surgeons other than ernest is likely related to differences in technique between surgeons .", "in addition , intrasurgeon variability may be slightly higher if surgeons did not measure their postoperative keratometry 1 month or more after surgery , as there is some change in keratometry expected in that first month .", "the minimization of the magnitude ( and variability ) of surgically induced astigmatism is important for modern day cataract surgery , particularly with the use of toric and multifocal intraocular lenses", "residual astigmatism after surgery will reduce the likelihood of spectacle independence for distance vision for these patients .", "minimizing astigmatism for multifocal iols is equally important , as even small amounts of residual astigmatism can compromise the outcome with regard to uncorrected visual acuity at distance .", "surgeons interested in reducing the magnitude and variability of induced astigmatism at the time of cataract surgery may want to consider the use of a 2.2  mm square posterior limbal incision ."]]}
{"article_id": "PMC3504390", "article_text": ["the embryonic development of the cerebellum derives from the hindbrain from the fifth week , originating from the thickening of the dorsal alar plates .", "anatomically it is formed by a median portion , the vermis , connected to two large lateral masses , the cerebellar hemispheres .", "the cisterna magna is located in the posterior fossa and occupies the space between the underside of the cerebellum , the posterior side of the bulb and the roof of the fourth ventricle .", "it continues caudally with the spinal subarachnoid space and binds to the fourth ventricle through the opening median . at ultrasonography", "the cerebellar hemispheres are usually low to moderately echogenic , connected above by an echogenic cerebellar tentorium .", "the vermis is a highly echogenic structure , which can be recognized as an oval density in the midline of the axial and sagittal plans .", "the cerebellum and cisterna magna can be observed on ultrasound around 11 weeks , but the formation of the cerebellar vermis ends around 18 weeks .", "for this reason , we must be careful in early diagnosis of the posterior fossa anomalies before this period , due to the possibility of a pathological image simulation [ 3 , 4 ] .", "the measurement of the transverse cerebellar diameter by two - dimensional ultrasonography ( 2dus ) is a very reliable parameter in the evaluation and early detection of intrauterine growth restriction . on the other hand , an adequate evaluation of the posterior fossa abnormalities", "allows the diagnosis of the cisterna magna as the dandy - walker complex , megacisterna magna , and blake 's cyst .", "the three - dimensional ultrasonography ( 3dus ) appeared as an important tool in the evaluation of fetal central nervous system in the late 1990s , because of the development of high resolution endocavitary volumetric transducers .", "currently , with the development of new software like volume contrast imaging allows adequate evaluation of posterior fossa anomalies as well as the biometry of the cerebellar vermis [ 7 , 8 ] . the three - dimensional extended imaging software ( 3d xi - medison , seoul , korea ) is composed of three programs : multislice view , volume ct view , and oblique view .", "the multislice consists of multiple sequential and adjacent planes arranged in the set screen ( sagittal , axial , and coronal ) . the reference plane , number of images arranged in the screen ( 1  1 , 2  1 , 3  2 , 4  3 , or 6  2 ) , orientation and rotation of the image , the magnitude of the magnification , and depth and range between the planes ( 0.5 to 5.0  mm ) can be adjusted according to the region of interest .", "a preliminary study evaluated the application of the multislice view in fetal central nervous system anomalies , showing potential benefits .", "up to date there are no studies evaluating the measures of the fetal cerebellum and cisterna magna by 3dus using the multislice view .", "the objective of this study is to compare the measurements of transverse cerebellar and anterior - posterior cisterna magna diameter obtained by 2d and 3dus using the software 3d xi in normal pregnant women between 18 and 24 weeks .", "a prospective cross - sectional study between march 2010 and february 2011 , involving 69 normal pregnant women between 18 and 24 weeks was performed .", "this study was approved by the research ethics committee of the irmandade da santa casa de misericrdia de so paulo , brazil , and the patients who agreed to participate signed a term of consent .", "this study was carried out at the department of obstetrics and gynecology , faculty of medical sciences of santa casa of so paulo ( fcmscsp ) .", "the patients were randomly selected , and all evaluation made by a single examiner ( fsbb ) , with five years experience in obstetric ultrasound .", "the examinations were performed on a sonoace x8 ( samsung - medison , seoul , korea ) device equipped with multifrequency volumetric convex transducer ( 37  mhz ) .", "the criteria for inclusion were ( 1 ) unique pregnancy with live fetus and ( 2 ) gestational age evaluated by last menstrual period and confirmed by ultrasound performed until the 14th week ( crown - rump length - crl : 484  mm ) .", "exclusion criteria were ( 1 ) pregnant women carrying fetuses with structural anomalies detected at the time of the examination and ( 2 ) pregnant women carrying chronic diseases that would interfere with fetal growth .", "initially , a realtime 2d evaluation was performed in order to evaluate the biometry , morphology , and quantification of amniotic fluid volume . for the 2d measurement of transverse cerebellar and anterior - posterior cisterna magna", "diameter , it was performed a modified transversal slice of the fetal head slightly angled , through the thalamus , cerebellar hemispheres , cisterna magna , cave of septum pellucidum , the occipital bone , and nuchal fold .  ", "an insonation angle of the occipital bone was chosen , taking care that it was focused on an angle of 30. it was performed a single measurement of transverse cerebellar and antero - posterior of the fetal cisterna magna diameter in each mother , and this image is saved in the memory of the device .", "the three - dimensional volume acquisition was performed on the same 2d plane in which was performed the measurements of the transverse cerebellar diameter and anterior - posterior cisterna magna , to encompass the entire fetal skull ( roi - region of interest ) ( figure 1(a ) ) . in order to standardize all 3d measurements , the following preset was used on the device : scanning3d static ; display mode  three - dimensional extended imaging ( multislice view ) ; scanning speed  slow ; angle scanning70 ; overall gain of the device50% .", "after the three - dimensional scanning , the image was displayed in the multiplanar mode ( axial , sagittal , and coronal ) ( figure 1(b ) ) .", "the volumes were saved in the device memory and then stored on compact discs ( cds ) and transferred to a personal computer .", "the analyses were performed offline in the same apparatus in a time period of 30 to 120 days after the volumetric capture . for measurements of the transverse cerebellar and anterior - posterior cisterna magna diameters the program multislice view on 3  2 ( two rows and three columns ) was chosen , with slice thickness of 0.5  mm ( figure 1(c ) ) .", "the buttons 3 and 4 of the device were maneuvered , for navigation between tomographic slices , until in one of them the image of the cerebellum disappeared in higher plan than that of the posterior fossa ( figure 1(d ) ) . from this point , the 1  1 ( one row and one column ) option was selected and the number of the image was recorded . with the presence of only one tomographic slice on the screen , the button 4 of the apparatus was rotated clockwise or counterclockwise , depending on the fetus position , leading the exposition of a lower plan , successively until the outline of the cerebellum began to appear .", "following , each plan of the cerebellum and the cisterna magna was measured , with a difference of 0.5  mm between them to the normal cerebellum outline disappeared and the bone of the skull base could be observed . on average , measurements of the transverse cerebellar diameter and the antero - posterior diameter of the cisterna magna", "were performed in 25 consecutive plans in each volume , being considered as the value of the measurement of the cerebellum and the cisterna magna by 3d xi method the highest value obtained .", "the average time for manipulation of the 3d volume measurement of the cerebellum and the cisterna magna was 180 seconds .", "the data were transferred to an excel 2007 ( microsoft corp , redmond , wa , usa ) spreadsheet and analyzed by the statistical package for social sciences ( spss inc . , chicago , il , usa ) version 19.0 for windows .", "for the 2d and 3d measurements of the transverse cerebellar diameter and antero - posterior cisterna magna , mean , median , maximum , and minimum values were calculated . and for the 3d measurements percentiles 5 , 10 , 90 , and 95 were also calculated in each gestational age evaluated .", "to evaluate the difference between the two techniques the wilcoxon test was used . to evaluate the correlation of 2d and 3d , measurements of the transverse cerebellar diameter and antero - posterior cisterna magna as well as measurements of biparietal diameter ( bpd ) and head circumference ( hc ) according to the gestational age , the spearman 's correlation coefficient ( r ) was used . in cases of high correlation , polynomial regression models , with adjustments by the coefficient of determination ( r )", "the first examiner ( fsbb ) held a second 2d and 3d measures in 8 cases randomly selected 7 days after the first . for interobserver reproducibility ,", "a second examiner ( lsvf ) , with the same experience in obstetric ultrasound performed a third measure of these same 8 cases , and the results were armored one of the other . for this purpose , to calculate the reproducibility intraclass correlation coefficient ( icc ) and", "it is considered poor correlation icc < 0.40 ; satisfactory iccs between 0.40 and 0.75 and excellent icc  0.75 .", "the bland - altman plots evaluate the average of measurements performed by one or two examiners plotted against the difference of their mean values with standard deviation 1.96 ( limits of agreement ) . in all analysis we used a significance level ( p ) < 0.05 .", "the 69 patients initially selected met the criteria for inclusion and exclusion , being allocated in the final statistical analysis .", "the age of the pregnant women ranged from 17 to 41 years , with an average of 26.93 years ( standard deviation 6.10 years ) .", "the number of pregnancies ranged from 1 to 6 , with an average of 2.19 pregnancies ( standard deviation of 1.32 pregnancies ) .", "the average transverse and anterior - posterior cerebellum and cisterna magna diameter by 3dus ranged from 9.23  3.16  mm ( 18.0629.34 ) and 6.62  1.41  mm ( 4.1910.45 ) , respectively .", "the average transverse diameter and antero - posterior cerebellum and cisterna magna by 2dus ranged from 22.33  3.16  mm ( 18.1429.10 ) and 5.60  1.33  mm ( 3.189.32 ) , respectively .", "it was observed that on average the measurements obtained by 3dus were significantly higher , 0.76 and 1.02  mm for the length of the cerebellum and cisterna magna , respectively ( p < 0.001 ) ( tables 1 and 2 ) .", "there was a high correlation between the measurement of transverse cerebellar diameter by 3dus with gestational age ( r = 0.940 , p < 0.001 ) as well as measures of dbp ( r = 0.927 , p < 0.001 ) and hc ( r = 0.938 , p < 0.001 ) .", "the equation that best represented the correlation between the extent of the transverse cerebellar diameter and gestational age was of a second degree : dtc = 7.231 + 1.851  ga  0.027  ga2 ( r = 0879 ) . for the measurement of antero - posterior diameter of the cisterna magna ,", "a low correlation between gestational age , bpd , and hc ( r = 0.462 , p < 0.001 ; r = 0.430 , p < 0,001 ; r = 0.517 , p < 0.001 , resp . ) ( figure 2 ) was observed .", "the low correlation between the antero - posterior diameter of the cisterna magna and gestational age did not allow the construction of polynomial regression models .", "tables 3 and 4 show the percentiles 5 , 10 , 90 , and 95 for measurements of the transverse and anterior - posterior cerebellum and cisterna magna diameter at each gestational age evaluated . an icc > 0.66 for all intra- and interobserver measurements of the cerebellum and cisterna magna length by 3dus was observed , with the exception of the intraobserver measurement of the length of cisterna magna ( icc = 0.792 , 0.668 , 0.691 , and 0.287 , resp . ) .", "the mean differences as well the limits of agreement for intraobserver and interobserver reproducibility of the cerebellum and cisterna magna length by 3dus were 0.16  mm ( limits of agreement , 0.79 ; 1.11 ) , 0.13  mm ( limits of agreement , 1.22 ; 0.95 ) , 0.08  mm ( limits of agreement , 0.67 ; 0.82 ) , and 0.12  mm ( limits of agreement , 0.69 ; 0.93 ) , respectively ( figures 3 and 4 ) .", "the posterior fossa cystic malformations include abnormalities of the meninges ( arachnoid cyst , megacisterna magna ) and cerebellum ( dandy - walker malformation and variants ) .", "the prenatal diagnosis of these malformations is of great importance for an adequate followup of pregnancy as well as the relatives counseling .", "the postnatal results are in general very bad in the dandy - walker malformation and variants , mainly as result of associated anomalies .", "the postnatal result of megacisterna magna is better , especially if isolated . in this study", ", we compared the measurements of the transverse and antero - posterior cerebellum and cisterna magna diameters by 2d and 3dus between 18 and 24 weeks .", "this interval was defined by the fact that the transverse cerebellar diameter showed the highest correlation with gestational age , its measurement in millimeters is similar to the gestational age in weeks .", "furthermore , the diagnosis of agenesis of the vermis , especially the partial can not be performed before 18 weeks . in relation to the antero - posterior diameter of the cisterna magna , in general , in this period the ultrasound of the second trimester for malformations evaluation will be performed , its normal measure being less than 10  mm . in this study", "we observed that the transverse cerebellar and anterior - posterior cisterna magna diameters by 2d and 3dus increased from 18 to 24 weeks , however , the cisterna magna measurements by 3dus showed low correlation with the gestational age . in a cross - sectional study carried out by vinkesteijn et al .   with 360 normal pregnant women between 17 and 34 weeks", ", the average of fetal transverse cerebellar diameter by 2dus was 22.1  mm from 18 to 24 weeks , very similar to our results obtained using 2dus ( 22.33  mm ) . in another cross - sectional study carried out by koktener et al .   with 194 normal pregnant women between 16 and 24 weeks", ", the mean antero - posterior diameter of the cisterna magna by 2dus was 4.83  mm , lower than that obtained in our study ( 5.60  mm ) .", "the test to prove that the antero - posterior diameter of the cisterna magna increased from 18 to 24 weeks is important because the fixed value of 10  mm for the indication of normality can not be real in more advanced gestational ages .", "we believe that this low correlation measurement of the cisterna magna by 3dus and gestational age is due to a greater difficulty in identifying the edges of the structure as it progresses toward the occipital bone .", "in general , the mean diameters of the transverse and anterior - posterior cerebellum and cisterna magna by 2dus were lower than those obtained by 3dus . in studies evaluating the volume of fetal cerebellum by 3dus", ", there was also an increase in this parameter with the gestational age , both by multiplanar and virtual organ computer - aided analysis ( vocal ) methods [ 1820 ] .", "there are no studies evaluating the volume of the fetal cisterna magna by 3dus throughout gestation .", "other studies using other softwares such as three - dimensional volume contrast imaging c - plane have shown a positive correlation between the biometrics of the vermis and gestational age [ 821 ] .", "we observed in this study a statistically significant difference in the measurement of transverse and anterior - posterior cerebellum and cisterna magna by 2d and 3dus using the 3d xi ( multislice view ) software .", "the program multislice view ( samsung - medison , seoul , korea ) allows the evaluation of multiple plans of sequential and adjacent images arranged on the screen , being the thickness of the slices determined by the operator . in this study", ", we evaluated an average of 25 plans with 0.5  mm thickness between them , from the plan of the transverse cerebellar diameter measure to a plan in which the usual outline was lost and the bone of the skull base could be observed .", "we used as the final value the greatest measure found , unlike the 2dus in which we used a single measure .", "we believe that the measures taken by 3dus should be more reliable because it corrects any small displacement of the sound beam transducer , which can compromise the 2d measure . however , the biggest inconvenience of this technique , which makes its application in clinical practice difficult , is the long time required to perform the measurements , an average 180 seconds .", "we noticed in this study an icc value > 0.66 for all length measurements of the fetal cerebellum and cisterna magna through 3dus , except for the intraobserver measure of the cisterna magna ( icc = 0.287 ) .", "such a result could assume a low reproducibility of the new method ; however , by bland - altman plot it was observed that the intraobserver mean difference was similar to the cerebellum and cisterna magna ( 0.16 and 0.13  mm , resp . ) .", "we believe that the low value of icc is due to the small number of cases we evaluated , and as this is a laborious technique with average rating of 25 plans per volume , with a mean time of 180 seconds , it may have contributed to this low value of icc .", "however , the real applicability of the method can only be testified in further studies evaluating pathological cases of fetal central nervous system .", "we believe that the great importance of this study is the evaluation of borderline cases , such as an anterior - posterior diameter of the fetal cisterna magna of 9.0  mm and a transverse cerebellar diameter of 16  mm at 18 weeks of gestational age . in these cases the 3dus with the program multislice view can help in decision making between normal and pathological cases , modifying the prenatal , and counseling of the parents . in summary , this was the first study that compared the measurements of the transverse and antero - posterior fetal cerebellum and cisterna magna diameters by 2d- and 3dus using the 3d xi ( multislice view ) software .", "the measurements the length of the fetal cerebellum and cisterna magna by 3dus were significantly higher than those obtained by 2dus .", "the length measurement of the cisterna magna by 3dus showed low correlation with gestational age .", "measures the length of the cerebellum and cisterna magna by 3dus proved to be reproducible ."], "abstract_text": ["<S> to compare the fetal cerebellum and cisterna magna length measurements by means of two- ( 2dus ) and three - dimensional ( 3dus ) ultrasonography using the three - dimensional extended imaging ( 3d xi ) , a cross - sectional study with 69 healthy pregnant women between 18 and 24 weeks was performed . for the measurements by 2dus , </S>", "<S> the axial planes were used and for the 3dus a sequence of adjacent axial slices ( multislice view ) . to evaluate the difference between the two techniques </S>", "<S> , we used the wilcoxon test . to evaluate the correlation between the cerebellum and cisterna magna length measurements and the gestational age </S>", "<S> , we used the spearman correlation coefficient ( r ) . for the calculation of reproducibility </S>", "<S> , we used the intraclass correlation coefficient ( icc ) . </S>", "<S> the mean of the transverse and anterior - posterior diameter of cerebellum and cisterna magna by 3dus was 9.23 and 6.62  mm , respectively . </S>", "<S> it was observed that the average of the measurements obtained by 3dus was significantly higher , 0.76 and 1.02  mm for the length of the cerebellum and cisterna magna , respectively ( p < 0.001 ) . </S>", "<S> there was a high correlation between the length measurement of the cerebellum 3d ( r = 0.940 , p < 0.001 ) , but low correlation of cisterna magna 3d ( r = 0.462 , p = 0.080 ) with the gestational age . </S>", "<S> there was good intra- and interobserver reproducibility for the cerebellum and cisterna magna 3d with icc = 0.792 \n , 0.668 , 0.691 , and 0.287 , respectively . </S>", "<S> the measurements of the fetal cerebellum and cisterna magna length by 3dus using the software 3d xi were significantly higher than those obtained by 2dus . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["the embryonic development of the cerebellum derives from the hindbrain from the fifth week , originating from the thickening of the dorsal alar plates .", "anatomically it is formed by a median portion , the vermis , connected to two large lateral masses , the cerebellar hemispheres .", "the cisterna magna is located in the posterior fossa and occupies the space between the underside of the cerebellum , the posterior side of the bulb and the roof of the fourth ventricle .", "it continues caudally with the spinal subarachnoid space and binds to the fourth ventricle through the opening median . at ultrasonography", "the cerebellar hemispheres are usually low to moderately echogenic , connected above by an echogenic cerebellar tentorium .", "the vermis is a highly echogenic structure , which can be recognized as an oval density in the midline of the axial and sagittal plans .", "the cerebellum and cisterna magna can be observed on ultrasound around 11 weeks , but the formation of the cerebellar vermis ends around 18 weeks .", "for this reason , we must be careful in early diagnosis of the posterior fossa anomalies before this period , due to the possibility of a pathological image simulation [ 3 , 4 ] .", "the measurement of the transverse cerebellar diameter by two - dimensional ultrasonography ( 2dus ) is a very reliable parameter in the evaluation and early detection of intrauterine growth restriction . on the other hand , an adequate evaluation of the posterior fossa abnormalities", "allows the diagnosis of the cisterna magna as the dandy - walker complex , megacisterna magna , and blake 's cyst .", "the three - dimensional ultrasonography ( 3dus ) appeared as an important tool in the evaluation of fetal central nervous system in the late 1990s , because of the development of high resolution endocavitary volumetric transducers .", "currently , with the development of new software like volume contrast imaging allows adequate evaluation of posterior fossa anomalies as well as the biometry of the cerebellar vermis [ 7 , 8 ] . the three - dimensional extended imaging software ( 3d xi - medison , seoul , korea ) is composed of three programs : multislice view , volume ct view , and oblique view .", "the multislice consists of multiple sequential and adjacent planes arranged in the set screen ( sagittal , axial , and coronal ) . the reference plane , number of images arranged in the screen ( 1  1 , 2  1 , 3  2 , 4  3 , or 6  2 ) , orientation and rotation of the image , the magnitude of the magnification , and depth and range between the planes ( 0.5 to 5.0  mm ) can be adjusted according to the region of interest .", "a preliminary study evaluated the application of the multislice view in fetal central nervous system anomalies , showing potential benefits .", "up to date there are no studies evaluating the measures of the fetal cerebellum and cisterna magna by 3dus using the multislice view .", "the objective of this study is to compare the measurements of transverse cerebellar and anterior - posterior cisterna magna diameter obtained by 2d and 3dus using the software 3d xi in normal pregnant women between 18 and 24 weeks ."], ["a prospective cross - sectional study between march 2010 and february 2011 , involving 69 normal pregnant women between 18 and 24 weeks was performed .", "this study was approved by the research ethics committee of the irmandade da santa casa de misericrdia de so paulo , brazil , and the patients who agreed to participate signed a term of consent .", "this study was carried out at the department of obstetrics and gynecology , faculty of medical sciences of santa casa of so paulo ( fcmscsp ) .", "the patients were randomly selected , and all evaluation made by a single examiner ( fsbb ) , with five years experience in obstetric ultrasound .", "the examinations were performed on a sonoace x8 ( samsung - medison , seoul , korea ) device equipped with multifrequency volumetric convex transducer ( 37  mhz ) .", "the criteria for inclusion were ( 1 ) unique pregnancy with live fetus and ( 2 ) gestational age evaluated by last menstrual period and confirmed by ultrasound performed until the 14th week ( crown - rump length - crl : 484  mm ) .", "exclusion criteria were ( 1 ) pregnant women carrying fetuses with structural anomalies detected at the time of the examination and ( 2 ) pregnant women carrying chronic diseases that would interfere with fetal growth .", "initially , a realtime 2d evaluation was performed in order to evaluate the biometry , morphology , and quantification of amniotic fluid volume . for the 2d measurement of transverse cerebellar and anterior - posterior cisterna magna", "diameter , it was performed a modified transversal slice of the fetal head slightly angled , through the thalamus , cerebellar hemispheres , cisterna magna , cave of septum pellucidum , the occipital bone , and nuchal fold .  ", "an insonation angle of the occipital bone was chosen , taking care that it was focused on an angle of 30. it was performed a single measurement of transverse cerebellar and antero - posterior of the fetal cisterna magna diameter in each mother , and this image is saved in the memory of the device .", "the three - dimensional volume acquisition was performed on the same 2d plane in which was performed the measurements of the transverse cerebellar diameter and anterior - posterior cisterna magna , to encompass the entire fetal skull ( roi - region of interest ) ( figure 1(a ) ) . in order to standardize all 3d measurements , the following preset was used on the device : scanning3d static ; display mode  three - dimensional extended imaging ( multislice view ) ; scanning speed  slow ; angle scanning70 ; overall gain of the device50% .", "after the three - dimensional scanning , the image was displayed in the multiplanar mode ( axial , sagittal , and coronal ) ( figure 1(b ) ) .", "the volumes were saved in the device memory and then stored on compact discs ( cds ) and transferred to a personal computer .", "the analyses were performed offline in the same apparatus in a time period of 30 to 120 days after the volumetric capture . for measurements of the transverse cerebellar and anterior - posterior cisterna magna diameters the program multislice view on 3  2 ( two rows and three columns ) was chosen , with slice thickness of 0.5  mm ( figure 1(c ) ) .", "the buttons 3 and 4 of the device were maneuvered , for navigation between tomographic slices , until in one of them the image of the cerebellum disappeared in higher plan than that of the posterior fossa ( figure 1(d ) ) . from this point , the 1  1 ( one row and one column ) option was selected and the number of the image was recorded . with the presence of only one tomographic slice on the screen , the button 4 of the apparatus was rotated clockwise or counterclockwise , depending on the fetus position , leading the exposition of a lower plan , successively until the outline of the cerebellum began to appear .", "following , each plan of the cerebellum and the cisterna magna was measured , with a difference of 0.5  mm between them to the normal cerebellum outline disappeared and the bone of the skull base could be observed . on average , measurements of the transverse cerebellar diameter and the antero - posterior diameter of the cisterna magna", "were performed in 25 consecutive plans in each volume , being considered as the value of the measurement of the cerebellum and the cisterna magna by 3d xi method the highest value obtained .", "the average time for manipulation of the 3d volume measurement of the cerebellum and the cisterna magna was 180 seconds .", "the data were transferred to an excel 2007 ( microsoft corp , redmond , wa , usa ) spreadsheet and analyzed by the statistical package for social sciences ( spss inc . , chicago , il , usa ) version 19.0 for windows .", "for the 2d and 3d measurements of the transverse cerebellar diameter and antero - posterior cisterna magna , mean , median , maximum , and minimum values were calculated . and for the 3d measurements percentiles 5 , 10 , 90 , and 95 were also calculated in each gestational age evaluated .", "to evaluate the difference between the two techniques the wilcoxon test was used . to evaluate the correlation of 2d and 3d , measurements of the transverse cerebellar diameter and antero - posterior cisterna magna as well as measurements of biparietal diameter ( bpd ) and head circumference ( hc ) according to the gestational age , the spearman 's correlation coefficient ( r ) was used . in cases of high correlation , polynomial regression models , with adjustments by the coefficient of determination ( r )", "the first examiner ( fsbb ) held a second 2d and 3d measures in 8 cases randomly selected 7 days after the first . for interobserver reproducibility ,", "a second examiner ( lsvf ) , with the same experience in obstetric ultrasound performed a third measure of these same 8 cases , and the results were armored one of the other . for this purpose , to calculate the reproducibility intraclass correlation coefficient ( icc ) and", "it is considered poor correlation icc < 0.40 ; satisfactory iccs between 0.40 and 0.75 and excellent icc  0.75 .", "the bland - altman plots evaluate the average of measurements performed by one or two examiners plotted against the difference of their mean values with standard deviation 1.96 ( limits of agreement ) . in all analysis we used a significance level ( p ) < 0.05 ."], ["the 69 patients initially selected met the criteria for inclusion and exclusion , being allocated in the final statistical analysis .", "the age of the pregnant women ranged from 17 to 41 years , with an average of 26.93 years ( standard deviation 6.10 years ) .", "the number of pregnancies ranged from 1 to 6 , with an average of 2.19 pregnancies ( standard deviation of 1.32 pregnancies ) .", "the average transverse and anterior - posterior cerebellum and cisterna magna diameter by 3dus ranged from 9.23  3.16  mm ( 18.0629.34 ) and 6.62  1.41  mm ( 4.1910.45 ) , respectively .", "the average transverse diameter and antero - posterior cerebellum and cisterna magna by 2dus ranged from 22.33  3.16  mm ( 18.1429.10 ) and 5.60  1.33  mm ( 3.189.32 ) , respectively .", "it was observed that on average the measurements obtained by 3dus were significantly higher , 0.76 and 1.02  mm for the length of the cerebellum and cisterna magna , respectively ( p < 0.001 ) ( tables 1 and 2 ) .", "there was a high correlation between the measurement of transverse cerebellar diameter by 3dus with gestational age ( r = 0.940 , p < 0.001 ) as well as measures of dbp ( r = 0.927 , p < 0.001 ) and hc ( r = 0.938 , p < 0.001 ) .", "the equation that best represented the correlation between the extent of the transverse cerebellar diameter and gestational age was of a second degree : dtc = 7.231 + 1.851  ga  0.027  ga2 ( r = 0879 ) . for the measurement of antero - posterior diameter of the cisterna magna ,", "a low correlation between gestational age , bpd , and hc ( r = 0.462 , p < 0.001 ; r = 0.430 , p < 0,001 ; r = 0.517 , p < 0.001 , resp . ) ( figure 2 ) was observed .", "the low correlation between the antero - posterior diameter of the cisterna magna and gestational age did not allow the construction of polynomial regression models .", "tables 3 and 4 show the percentiles 5 , 10 , 90 , and 95 for measurements of the transverse and anterior - posterior cerebellum and cisterna magna diameter at each gestational age evaluated . an icc > 0.66 for all intra- and interobserver measurements of the cerebellum and cisterna magna length by 3dus was observed , with the exception of the intraobserver measurement of the length of cisterna magna ( icc = 0.792 , 0.668 , 0.691 , and 0.287 , resp . ) .", "the mean differences as well the limits of agreement for intraobserver and interobserver reproducibility of the cerebellum and cisterna magna length by 3dus were 0.16  mm ( limits of agreement , 0.79 ; 1.11 ) , 0.13  mm ( limits of agreement , 1.22 ; 0.95 ) , 0.08  mm ( limits of agreement , 0.67 ; 0.82 ) , and 0.12  mm ( limits of agreement , 0.69 ; 0.93 ) , respectively ( figures 3 and 4 ) ."], ["the posterior fossa cystic malformations include abnormalities of the meninges ( arachnoid cyst , megacisterna magna ) and cerebellum ( dandy - walker malformation and variants ) .", "the prenatal diagnosis of these malformations is of great importance for an adequate followup of pregnancy as well as the relatives counseling .", "the postnatal results are in general very bad in the dandy - walker malformation and variants , mainly as result of associated anomalies .", "the postnatal result of megacisterna magna is better , especially if isolated . in this study", ", we compared the measurements of the transverse and antero - posterior cerebellum and cisterna magna diameters by 2d and 3dus between 18 and 24 weeks .", "this interval was defined by the fact that the transverse cerebellar diameter showed the highest correlation with gestational age , its measurement in millimeters is similar to the gestational age in weeks .", "furthermore , the diagnosis of agenesis of the vermis , especially the partial can not be performed before 18 weeks . in relation to the antero - posterior diameter of the cisterna magna , in general , in this period the ultrasound of the second trimester for malformations evaluation will be performed , its normal measure being less than 10  mm . in this study", "we observed that the transverse cerebellar and anterior - posterior cisterna magna diameters by 2d and 3dus increased from 18 to 24 weeks , however , the cisterna magna measurements by 3dus showed low correlation with the gestational age . in a cross - sectional study carried out by vinkesteijn et al .   with 360 normal pregnant women between 17 and 34 weeks", ", the average of fetal transverse cerebellar diameter by 2dus was 22.1  mm from 18 to 24 weeks , very similar to our results obtained using 2dus ( 22.33  mm ) . in another cross - sectional study carried out by koktener et al .   with 194 normal pregnant women between 16 and 24 weeks", ", the mean antero - posterior diameter of the cisterna magna by 2dus was 4.83  mm , lower than that obtained in our study ( 5.60  mm ) .", "the test to prove that the antero - posterior diameter of the cisterna magna increased from 18 to 24 weeks is important because the fixed value of 10  mm for the indication of normality can not be real in more advanced gestational ages .", "we believe that this low correlation measurement of the cisterna magna by 3dus and gestational age is due to a greater difficulty in identifying the edges of the structure as it progresses toward the occipital bone .", "in general , the mean diameters of the transverse and anterior - posterior cerebellum and cisterna magna by 2dus were lower than those obtained by 3dus . in studies evaluating the volume of fetal cerebellum by 3dus", ", there was also an increase in this parameter with the gestational age , both by multiplanar and virtual organ computer - aided analysis ( vocal ) methods [ 1820 ] .", "there are no studies evaluating the volume of the fetal cisterna magna by 3dus throughout gestation .", "other studies using other softwares such as three - dimensional volume contrast imaging c - plane have shown a positive correlation between the biometrics of the vermis and gestational age [ 821 ] .", "we observed in this study a statistically significant difference in the measurement of transverse and anterior - posterior cerebellum and cisterna magna by 2d and 3dus using the 3d xi ( multislice view ) software .", "the program multislice view ( samsung - medison , seoul , korea ) allows the evaluation of multiple plans of sequential and adjacent images arranged on the screen , being the thickness of the slices determined by the operator . in this study", ", we evaluated an average of 25 plans with 0.5  mm thickness between them , from the plan of the transverse cerebellar diameter measure to a plan in which the usual outline was lost and the bone of the skull base could be observed .", "we used as the final value the greatest measure found , unlike the 2dus in which we used a single measure .", "we believe that the measures taken by 3dus should be more reliable because it corrects any small displacement of the sound beam transducer , which can compromise the 2d measure . however , the biggest inconvenience of this technique , which makes its application in clinical practice difficult , is the long time required to perform the measurements , an average 180 seconds .", "we noticed in this study an icc value > 0.66 for all length measurements of the fetal cerebellum and cisterna magna through 3dus , except for the intraobserver measure of the cisterna magna ( icc = 0.287 ) .", "such a result could assume a low reproducibility of the new method ; however , by bland - altman plot it was observed that the intraobserver mean difference was similar to the cerebellum and cisterna magna ( 0.16 and 0.13  mm , resp . ) .", "we believe that the low value of icc is due to the small number of cases we evaluated , and as this is a laborious technique with average rating of 25 plans per volume , with a mean time of 180 seconds , it may have contributed to this low value of icc .", "however , the real applicability of the method can only be testified in further studies evaluating pathological cases of fetal central nervous system .", "we believe that the great importance of this study is the evaluation of borderline cases , such as an anterior - posterior diameter of the fetal cisterna magna of 9.0  mm and a transverse cerebellar diameter of 16  mm at 18 weeks of gestational age . in these cases the 3dus with the program multislice view can help in decision making between normal and pathological cases , modifying the prenatal , and counseling of the parents . in summary , this was the first study that compared the measurements of the transverse and antero - posterior fetal cerebellum and cisterna magna diameters by 2d- and 3dus using the 3d xi ( multislice view ) software .", "the measurements the length of the fetal cerebellum and cisterna magna by 3dus were significantly higher than those obtained by 2dus .", "the length measurement of the cisterna magna by 3dus showed low correlation with gestational age .", "measures the length of the cerebellum and cisterna magna by 3dus proved to be reproducible ."]]}
{"article_id": "PMC3816861", "article_text": ["the protocol for this cross - sectional study was reviewed and approved by the institutional ethical committee for clinical research at the geneva university hospitals .", "all patients who had received a whole pancreas or an islet of langerhans transplant at the geneva university hospitals and who had regular follow - ups at our institution were asked to participate in the study .", "patients were required to have at least partial graft function ( as determined by positive c - peptide levels ) .", "fifteen islet transplant recipients were enrolled in the islet group , which was further subdivided into an insulin - independent group ( ii - isl ) and an insulin - requiring group ( ir - isl ) .", "ten healthy nondiabetic volunteers were recruited to serve as control subjects . finally , five patients with type 1 diabetes awaiting pancreas or islet transplant , all of whom demonstrated no c - peptide production at the time of study inclusion , served as negative control subjects .", "written informed consent was obtained from all patients and control subjects prior to their participation in the study .", "demographics and population characteristics all samples were obtained from patients during either their annual posttransplantation follow - up or one of their regularly scheduled outpatient consultations .", "the serum was labeled using the same anonymous codes assigned to the tubes at the time of the test .", "an arginine stimulation test was performed on all subjects to determine basal and stimulated pancreatic hormone levels .", "blood was drawn at 10 , 0 , 2 , 3 , 4 , 5 , 7 , and 10 min from intravenous injection of a 5 g arginine bolus .", "patients who were back on insulin were required to discontinue slow - acting insulin at least 24 h prior to the test and rapid - acting insulin at least 6 h before the test .", "serum levels of insulin , c - peptide , total proinsulin , and intact proinsulin ( ipi ) were then measured using commercially available elisas as described below .", "acute insulin response ( air ) was calculated as the mean of the three highest values between 2 and 5 min minus the mean of the basal values at 10 and 0 min .", "the area under the curve ( auc ) between 0 and 10 min postinjection for insulin and c - peptide was calculated by the trapezoidal rule with the mean of the baseline values subtracted .", "proinsulin fragments , including split-32,33 proinsulin , des-31,32 proinsulin , split-65,66 proinsulin , and des-64,65 proinsulin , were obtained by subtracting ipi from total proinsulin .", "ratios of insulin to total proinsulin and insulin [ insulin/(total proinsulin+insulin ) ] as well as proinsulin fragments to total proinsulin ( proinsulin fragments / total proinsulin ) were calculated to quantify the rate of proinsulin processing .", "ratios representing peak stimulation were calculated as the mean of ratios for the three highest time points between 2 and 5 min after arginine stimulation .", "insulin/(total proinsulin+insulin ) ratio represents the quantity of fully processed insulin with respect to all circulating forms of the hormone .", "commercially available elisas were used to measure hormone levels for insulin , c - peptide , total proinsulin , and ipi .", "the following kits were used : insulin ( mercodia , uppsala , sweden ) , c - peptide ( mercodia ) , total proinsulin ( millipore , zug , switzerland ) , and ipi ( millipore ) .", "concentrations were determined using a standard curve with high- and low - level control subjects .", "cross - reactivities of the assays were provided by the manufacturer and are as follows : the insulin assay cross - reacts 98% with des-64,65 proinsulin , < 0.01% with c - peptide and proinsulin , and < 0.05% with des-31,32 proinsulin and split-32,33 proinsulin ; the c - peptide elisa cross - reacts < 0.0006% with insulin , < 1.8% with proinsulin , 10% with split-65,66", "proinsulin , 74% with des-64,65 proinsulin , 3% with des-31,32 proinsulin , and 2% with split-32,33 proinsulin ; the total proinsulin elisa cross - reacts 100% with intact and des-31,32 proinsulin , and 81% with des-64,65 proinsulin ; the ipi elisa cross - reacts 36% with des-64,54 proinsulin , and it shows no cross - reactivity with des-31,32 proinsulin .", "the lower limits of detection of the assays were provided by the manufacturer and are as follows : the insulin assay has a detection limit of 6", "pmol / l , the total proinsulin assay has a limit of 0.5 pmol / l , and the ipi assay has a lower limit of 0.1 pmol / l .", "all statistical analyses were performed using prism 6 software ( graphpad software , la jolla , ca ) .", "for all analyses , values obtained from healthy subjects were used as control subjects . for data that were normally distributed ,", "student t test was used to compare two groups , and one - way anova was used to compare between more than two groups . for data that had non - gaussian distributions , we used the mann - whitney test to compare between two groups and the kruskal - wallis test to compare between more than two groups .", "all samples were obtained from patients during either their annual posttransplantation follow - up or one of their regularly scheduled outpatient consultations .", "the serum was labeled using the same anonymous codes assigned to the tubes at the time of the test .", "an arginine stimulation test was performed on all subjects to determine basal and stimulated pancreatic hormone levels .", "blood was drawn at 10 , 0 , 2 , 3 , 4 , 5 , 7 , and 10 min from intravenous injection of a 5 g arginine bolus .", "patients who were back on insulin were required to discontinue slow - acting insulin at least 24 h prior to the test and rapid - acting insulin at least 6 h before the test .", "serum levels of insulin , c - peptide , total proinsulin , and intact proinsulin ( ipi ) were then measured using commercially available elisas as described below . acute insulin response ( air )", "was calculated as the mean of the three highest values between 2 and 5 min minus the mean of the basal values at 10 and 0 min .", "the area under the curve ( auc ) between 0 and 10 min postinjection for insulin and c - peptide was calculated by the trapezoidal rule with the mean of the baseline values subtracted .", "proinsulin fragments , including split-32,33 proinsulin , des-31,32 proinsulin , split-65,66 proinsulin , and des-64,65 proinsulin , were obtained by subtracting ipi from total proinsulin .", "ratios of insulin to total proinsulin and insulin [ insulin/(total proinsulin+insulin ) ] as well as proinsulin fragments to total proinsulin ( proinsulin fragments / total proinsulin ) were calculated to quantify the rate of proinsulin processing .", "ratios representing peak stimulation were calculated as the mean of ratios for the three highest time points between 2 and 5 min after arginine stimulation .", "insulin/(total proinsulin+insulin ) ratio represents the quantity of fully processed insulin with respect to all circulating forms of the hormone .", "commercially available elisas were used to measure hormone levels for insulin , c - peptide , total proinsulin , and ipi .", "the following kits were used : insulin ( mercodia , uppsala , sweden ) , c - peptide ( mercodia ) , total proinsulin ( millipore , zug , switzerland ) , and ipi ( millipore ) .", "concentrations were determined using a standard curve with high- and low - level control subjects .", "cross - reactivities of the assays were provided by the manufacturer and are as follows : the insulin assay cross - reacts 98% with des-64,65 proinsulin , < 0.01% with c - peptide and proinsulin , and < 0.05% with des-31,32 proinsulin and split-32,33 proinsulin ; the c - peptide elisa cross - reacts <", "0.0006% with insulin , < 1.8% with proinsulin , 10% with split-65,66 proinsulin , 74% with des-64,65 proinsulin , 3% with des-31,32 proinsulin , and 2% with split-32,33 proinsulin ; the total proinsulin elisa cross - reacts 100% with intact and des-31,32 proinsulin , and 81% with des-64,65 proinsulin ; the ipi elisa cross - reacts 36% with des-64,54 proinsulin , and it shows no cross - reactivity with des-31,32 proinsulin .", "the lower limits of detection of the assays were provided by the manufacturer and are as follows : the insulin assay has a detection limit of 6", "pmol / l , the c - peptide elisa has a limit of 15 pmol / l , the total proinsulin assay has a limit of 0.5 pmol / l , and the ipi assay has a lower limit of 0.1 pmol / l .", "all statistical analyses were performed using prism 6 software ( graphpad software , la jolla , ca ) . for all analyses , values obtained from healthy subjects were used as control subjects . for data that were normally distributed ,", "student t test was used to compare two groups , and one - way anova was used to compare between more than two groups . for data that had non - gaussian distributions , we used the mann - whitney test to compare between two groups and the kruskal - wallis test to compare between more than two groups .", "subjects in the healthy control group were slightly younger than the rest of the study population ( p < 0.01 ) .", "islet recipients exhibited higher fasting glycemia and hba1c levels than other groups ( p < 0.05 ) .", "nonetheless , hba1c levels remained in the normal range for the ii - isl group .", "creatinine levels were elevated in the pancreas and ir - isl groups with respect to the ii - isl and healthy control groups ( p < 0.005 vs. control , p < 0.05 vs. ii - isl ) .", "figure 1a and b shows the insulin and c - peptide response curves for all five groups .", "the insulin and c - peptide response curves maintain the same overall shape in all groups , with the exception of the diabetic control subjects who demonstrate no response to arginine stimulation .", "insulin levels were higher in pancreas recipients but lower in islet recipients compared with healthy control subjects .", "insulin levels in the pancreas group were significantly higher than those of the healthy control group .", "airs for healthy control subjects , ir - isl , ii - isl , and pancreas groups were 217  163 , 23  13 , 125  90 , and 266", "the c - peptide acute responses in the same groups were 717  362 , 91  53 , 278  124 , and 550  244 pmol / l , respectively ( supplementary table 1 ) . mean acute c - peptide responses were similar in the healthy control group and pancreas group but significantly lower in the islet groups .", "the ir - isl transplantees showed a significantly decreased acute insulin and c - peptide response with respect to all other groups .", "there was no significant difference in air between the ii - isl and control groups .", "as expected , the diabetic control group showed negligible insulin and c - peptide levels .", "a : mean insulin levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group .", "b : mean c - peptide levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group .", "auc values demonstrated similar distributions to acute responses in the various study groups ( supplementary table 1 ) .", "auc values for insulin response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 1,332  959 , 152  91 , 774  528 , and 1,540  707 pmol ", "these values were higher in pancreas recipients and lower in islet recipients compared with healthy control subjects .", "auc values for c - peptide response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 4,955  3,158 , 630  428 , 1,843  875 , and 3,859  2,061 pmol ", "the auc of c - peptide for the ir - isl group was significantly lower than in all other groups .", "mean values for c - peptide and total proinsulin before and after arginine stimulation are shown in fig .", "these figures show that total proinsulin levels are reduced in the ir - isl group with respect to all other groups and follow a distribution similar to that of c - peptide . a : box plots of basal c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isr group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of stimulated c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) .", "the healthy control group demonstrated lower basal proinsulin - processing rates than the pancreas ( p < 0.01 ) and ii - isl ( p < 0.05 ) groups [ 74  14% vs. 83  7% vs. 86  12% , respectively , for insulin/(total proinsulin+insulin ) and 54  25% vs. 67 ", "18% vs. 76  16% , respectively , for proinsulin fragments / total proinsulin ] .", "the ir - isl group displayed a basal insulin/(total proinsulin+insulin ) rate of 75  14% and a basal proinsulin fragments / total proinsulin rate of 54  25% .", "a : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) .", "i / tp+i , insulin / total proinsulin+insulin ; pf / tp , proinsulin fragments / total proinsulin .", "3b . interestingly , healthy control subjects , pancreas recipients , and ii - isl recipients all attain similarly elevated processing ratios after stimulation ( 93  2% vs. 93 ", "3% vs. 93  5% , respectively , for insulin/(total proinsulin+insulin ) and 80  7% vs. 77 ", "9% vs. 76  12% , respectively , for proinsulin fragments / total proinsulin ) , while ir - isl transplant recipients ratios do not demonstrate a comparable increase .", "although ir - isl proinsulin - processing ratios do increase slightly after stimulation to 87  4% and 58  18% for insulin/(total proinsulin + insulin ) and proinsulin fragments / total proinsulin , respectively , these values remain significantly lower than all other groups ( p < 0.01 vs. control and pancreas groups , p < 0.05 vs. ii - isl ) .", "subjects in the healthy control group were slightly younger than the rest of the study population ( p < 0.01 ) .", "islet recipients exhibited higher fasting glycemia and hba1c levels than other groups ( p < 0.05 ) .", "nonetheless , hba1c levels remained in the normal range for the ii - isl group .", "creatinine levels were elevated in the pancreas and ir - isl groups with respect to the ii - isl and healthy control groups ( p < 0.005 vs. control , p < 0.05 vs. ii - isl ) .", "figure 1a and b shows the insulin and c - peptide response curves for all five groups .", "the insulin and c - peptide response curves maintain the same overall shape in all groups , with the exception of the diabetic control subjects who demonstrate no response to arginine stimulation .", "insulin levels were higher in pancreas recipients but lower in islet recipients compared with healthy control subjects .", "insulin levels in the pancreas group were significantly higher than those of the healthy control group .", "airs for healthy control subjects , ir - isl , ii - isl , and pancreas groups were 217  163 , 23  13 , 125  90 , and 266  129 pmol / l , respectively .", "the c - peptide acute responses in the same groups were 717  362 , 91  53 , 278  124 , and 550  244 pmol / l , respectively ( supplementary table 1 ) . mean acute c - peptide responses were similar in the healthy control group and pancreas group but significantly lower in the islet groups .", "the ir - isl transplantees showed a significantly decreased acute insulin and c - peptide response with respect to all other groups .", "there was no significant difference in air between the ii - isl and control groups .", "as expected , the diabetic control group showed negligible insulin and c - peptide levels .", "a : mean insulin levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group .", "b : mean c - peptide levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group", "auc values demonstrated similar distributions to acute responses in the various study groups ( supplementary table 1 ) .", "auc values for insulin response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 1,332  959 , 152  91 , 774  528 , and 1,540  707 pmol ", "these values were higher in pancreas recipients and lower in islet recipients compared with healthy control subjects .", "auc values for c - peptide response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 4,955  3,158 , 630  428 , 1,843  875 , and 3,859  2,061 pmol ", "the auc of c - peptide for the ir - isl group was significantly lower than in all other groups .", "mean values for c - peptide and total proinsulin before and after arginine stimulation are shown in fig .", "these figures show that total proinsulin levels are reduced in the ir - isl group with respect to all other groups and follow a distribution similar to that of c - peptide . a : box plots of basal c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isr group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of stimulated c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) .", "the healthy control group demonstrated lower basal proinsulin - processing rates than the pancreas ( p < 0.01 ) and ii - isl ( p < 0.05 ) groups [ 74  14% vs. 83  7% vs. 86  12% , respectively , for insulin/(total proinsulin+insulin ) and 54  25% vs. 67 ", "18% vs. 76  16% , respectively , for proinsulin fragments / total proinsulin ] .", "the ir - isl group displayed a basal insulin/(total proinsulin+insulin ) rate of 75  14% and a basal proinsulin fragments / total proinsulin rate of 54  25% .", "a : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) .", "i / tp+i , insulin / total proinsulin+insulin ; pf / tp , proinsulin fragments / total proinsulin .", "3b . interestingly , healthy control subjects , pancreas recipients , and ii - isl recipients all attain similarly elevated processing ratios after stimulation ( 93  2% vs. 93 ", "3% vs. 93  5% , respectively , for insulin/(total proinsulin+insulin ) and 80  7% vs. 77 ", "9% vs. 76  12% , respectively , for proinsulin fragments / total proinsulin ) , while ir - isl transplant recipients ratios do not demonstrate a comparable increase .", "although ir - isl proinsulin - processing ratios do increase slightly after stimulation to 87  4% and 58  18% for insulin/(total proinsulin + insulin ) and proinsulin fragments / total proinsulin , respectively , these values remain significantly lower than all other groups ( p < 0.01 vs. control and pancreas groups , p < 0.05 vs. ii - isl ) .", "this study explores for the first time proinsulin processing in islet transplant recipients , not only in comparison with healthy control subjects , but also with respect to pancreas transplant recipients .", "we have shown , first , that the exposure to arginine stimulation is in fact similar in ii - isl transplant recipients and healthy control subjects .", "as expected , recipients of pancreas transplants with systemic venous drainage demonstrate higher insulin response to arginine than healthy control subjects .", "second , there is a noticeable difference in the rate of basal proinsulin processing comparing ii - isl and ir - isl transplant recipients .", "ii - isl patients have proinsulin - processing ratios similar to those of pancreas transplant recipients , which are significantly higher than those of healthy control and ir - isl patients .", "third , after stimulation , ii - isl , pancreas , and healthy control subjects all increase their proinsulin - processing ratios to a similar level .", "in contrast , ir - isl patients are unable to achieve the same increase in proinsulin - processing rate .", "these data suggest that fully functional islet grafts are able to attain optimal proinsulin processing when stimulated but are required to sustain high levels of processing even during a metabolic fasting state in order to maintain glycemic control .", "islet grafts requiring supplementation with insulin therapy , on the other hand , demonstrate low basal proinsulin - processing rates , which are unable to reach optimal levels even when stimulated .", "interestingly , total proinsulin levels in this group ( ir - isl ) are low compared with other groups both before and after stimulation . however , the ratio of proinsulin to insulin in the ir - isl group after stimulation is disproportionately high .", "this suggests that partially functional islet grafts have a defect in proinsulin processing , which manifests itself as relative and not absolute hyperproinsulinemia .", "it should be noted that the calculation of proinsulin fragments may be slightly biased owing to elisa cross - reactivities . in theory ,", "when we calculate proinsulin fragments as proinsulin fragments = total proinsulin  ipi , we are assuming that all of the des-64,65 proinsulin has been measured by the total proinsulin assay and that none of it has been measured by the intact assay .", "in reality , we measure only 81% of des-64,65 with the total proinsulin assay and 36% with the ipi assay .", ". however , since the pc2 pathway responsible for the formation of des-64,65 proinsulin is not the primary pathway for the production of insulin , unmeasured levels of des-64,65 proinsulin are relatively insignificant with respect to des-31,32 proinsulin levels .", "it should also be noted that clearance of proinsulin and proinsulin fragments could be altered by diminished renal function . as seen in table 1 , our ir - isl group demonstrated creatinine levels that were elevated compared with healthy control subjects .", "however , this difference was not significant comparing the ir - isl group to ii - isl and pancreas groups .", "in contrast , the reduced processing ratios observed in the ir - isl group are significantly lower than all other groups .", "both absolute and relative hyperproinsulinemia have already been observed in settings of impaired glucose metabolism ( 1214 ) .", "first , it has been suggested that dysfunctional -cells present a fundamental defect in the pathway responsible for processing of proinsulin to insulin ( 18 ) .", "it is possible in the case of islet transplantation that manipulation of islets , introduction of -cells into a foreign microenvironment , or immunosuppressive drugs may render these cells inherently dysfunctional .", "a second theory suggests that hyperproinsulinemia occurs because of increased demand on -cells , which leads to insufficient time to complete proinsulin processing intracellularly before granule secretion occurs ( 15,19,20 ) .", "it seems reasonable to suppose that a combination of insulin resistance and decreased -cell mass could lead to unattainable insulin needs and eventual -cell deterioration via exhaustion .", "our data show that islet grafts initially present increased basal proinsulin processing with respect to healthy control subjects and that this processing deteriorates as graft function declines , a phenomenon that more readily supports the latter theory .", "we can not , however , exclude the effect of prolonged glucotoxicity on -cell function as an additional possible explanation for the observed processing defects .", "although our two islet groups had similar fasting glucose levels , hba1c was slightly increased in the ir - isl group , indicating that chronic glucotoxicity may have played a role in the functional deterioration of -cells within this subgroup .", "ultimately , a longitudinal study on the same or similar populations might help to verify this hypothesis .", "two studies have previously investigated proinsulin processing in islet transplant recipients ( 16,22 ) . however , neither study used arginine stimulation to explore hormonal processing when under simulated metabolic stress and neither study compared islet and pancreas transplant recipients .", "it is interesting to note that these studies produced conflicting data and presented contrasting conclusions .", "we found that functional islet grafts display increased processing and relatively lower proinsulinemia at rest , with an inability to increase their processing rates further when stimulated .", "these grafts are essentially behaving the same during the metabolic fasting state as fully stimulated healthy control subjects .", "as islet grafts lose function , however , processing  even during the fasting state  becomes less effective and proinsulinemia increases .", "these findings directly correlate with the findings of mcdonald et al . and are supported by the data of klimek at al . , which include mostly ir - isl transplant recipients . two other groups , fiorina et al .", "it would seem that there is a threshold level of demand beyond which -cells can no longer process proinsulin effectively . as stated earlier , we presume this to be due to an increased demand on decreased -cell mass . further investigation is warranted to explain the exact mechanism responsible for the increased basal proinsulin processing seen in ii - isl grafts .", "again , a longitudinal study of these patients might help shed more light on the issue .", "no study previously analyzed the stimulated hormonal response of pancreas grafts with respect to islet grafts .", "our data show that pancreas grafts behave quite similarly to healthy control subjects but with elevated overall hormone levels .", "this is undoubtedly a reflection of the systemic venous pancreatic drainage of these patients , in whom there is no hepatic first - pass metabolism of pancreatic hormonal secretions ( 23 ) .", "pancreas grafts tend to show similar patterns of proinsulin processing to fully functional islet grafts .", "whether this is peculiar to systemically drained pancreata can not be elucidated from this data .", "this study used arginine stimulation rather than oral or intravenous glucose tolerance tests to test secretory reserve in our patient populations .", "arginine stimulation provides the advantage of not inducing hyperglycemia , thus reducing the confounding influence of glucotoxicity to a minimum .", "other investigators have found the intravenous glucose tolerance tests to be more robust and a better indicator of -cell status ( 24 ) , but this method does not account for host insulin resistance . in summary , ii - isl transplant recipients can maintain basal metabolic parameters similar to healthy control subjects at the cost of a higher rate of basal proinsulin processing .", "ir - isl transplant recipients demonstrate both lower insulin response and lower basal rates of proinsulin processing , which remain suboptimal even after arginine stimulation ( i.e. , loss of graft function is associated with less effective processing and relative hyperproinsulinemia )", ". finally , the higher airs of pancreas transplant recipients are a reflection of systemic venous drainage of endocrine secretions ."], "abstract_text": ["<S> objective-cells have demonstrated altered proinsulin processing after islet transplantation . </S>", "<S> we compare -cell metabolic responses and proinsulin processing in pancreas and islet transplant recipients with respect to healthy control subjects.research design and methodswe studied 15 islet and 32 pancreas transplant recipients . </S>", "<S> islet subjects were subdivided into insulin - requiring ( ir - isl , n = 6 ) and insulin - independent ( ii - isl , n = 9 ) groups . </S>", "<S> ten healthy subjects served as control subjects . </S>", "<S> subjects were administered an intravenous arginine stimulation test , and insulin , c - peptide , total proinsulin , intact proinsulin , and proinsulin fragment levels were determined from serum samples . acute insulin response ( air ) and proinsulin </S>", "<S> processing rates were calculated.resultswe found that basal insulin and c - peptide levels were higher in the pancreas group than in all other groups . </S>", "<S> ii - isl patients had basal insulin and c - peptide levels similar to healthy control subjects . </S>", "<S> the ir - isl group had significantly lower airs than all other groups . </S>", "<S> basal processing rates were higher in the pancreas and ii - isl groups than in healthy control subjects and the ir - isl group . </S>", "<S> after arginine stimulation , all groups had elevated processing rates , with the exception of the ir - isl group.conclusionsour data suggest that ii - isl transplant recipients can maintain basal metabolic parameters similar to healthy control subjects at the cost of a higher rate of proinsulin processing . </S>", "<S> ir - isl transplant recipients , on the other hand , demonstrate both lower insulin response and lower basal rates of proinsulin processing even after arginine stimulation . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Sample collection", "Arginine stimulation test", "Assays", "Statistical analysis", "RESULTS", "Demographic characteristics", "Insulin and C-peptide levels", "Proinsulinemia", "Basal proinsulin processing", "Stimulated proinsulin processing", "CONCLUSIONS", "Supplementary Material"], "sections": [["the protocol for this cross - sectional study was reviewed and approved by the institutional ethical committee for clinical research at the geneva university hospitals .", "all patients who had received a whole pancreas or an islet of langerhans transplant at the geneva university hospitals and who had regular follow - ups at our institution were asked to participate in the study .", "patients were required to have at least partial graft function ( as determined by positive c - peptide levels ) .", "fifteen islet transplant recipients were enrolled in the islet group , which was further subdivided into an insulin - independent group ( ii - isl ) and an insulin - requiring group ( ir - isl ) .", "ten healthy nondiabetic volunteers were recruited to serve as control subjects . finally , five patients with type 1 diabetes awaiting pancreas or islet transplant , all of whom demonstrated no c - peptide production at the time of study inclusion , served as negative control subjects .", "written informed consent was obtained from all patients and control subjects prior to their participation in the study .", "demographics and population characteristics all samples were obtained from patients during either their annual posttransplantation follow - up or one of their regularly scheduled outpatient consultations .", "the serum was labeled using the same anonymous codes assigned to the tubes at the time of the test .", "an arginine stimulation test was performed on all subjects to determine basal and stimulated pancreatic hormone levels .", "blood was drawn at 10 , 0 , 2 , 3 , 4 , 5 , 7 , and 10 min from intravenous injection of a 5 g arginine bolus .", "patients who were back on insulin were required to discontinue slow - acting insulin at least 24 h prior to the test and rapid - acting insulin at least 6 h before the test .", "serum levels of insulin , c - peptide , total proinsulin , and intact proinsulin ( ipi ) were then measured using commercially available elisas as described below .", "acute insulin response ( air ) was calculated as the mean of the three highest values between 2 and 5 min minus the mean of the basal values at 10 and 0 min .", "the area under the curve ( auc ) between 0 and 10 min postinjection for insulin and c - peptide was calculated by the trapezoidal rule with the mean of the baseline values subtracted .", "proinsulin fragments , including split-32,33 proinsulin , des-31,32 proinsulin , split-65,66 proinsulin , and des-64,65 proinsulin , were obtained by subtracting ipi from total proinsulin .", "ratios of insulin to total proinsulin and insulin [ insulin/(total proinsulin+insulin ) ] as well as proinsulin fragments to total proinsulin ( proinsulin fragments / total proinsulin ) were calculated to quantify the rate of proinsulin processing .", "ratios representing peak stimulation were calculated as the mean of ratios for the three highest time points between 2 and 5 min after arginine stimulation .", "insulin/(total proinsulin+insulin ) ratio represents the quantity of fully processed insulin with respect to all circulating forms of the hormone .", "commercially available elisas were used to measure hormone levels for insulin , c - peptide , total proinsulin , and ipi .", "the following kits were used : insulin ( mercodia , uppsala , sweden ) , c - peptide ( mercodia ) , total proinsulin ( millipore , zug , switzerland ) , and ipi ( millipore ) .", "concentrations were determined using a standard curve with high- and low - level control subjects .", "cross - reactivities of the assays were provided by the manufacturer and are as follows : the insulin assay cross - reacts 98% with des-64,65 proinsulin , < 0.01% with c - peptide and proinsulin , and < 0.05% with des-31,32 proinsulin and split-32,33 proinsulin ; the c - peptide elisa cross - reacts < 0.0006% with insulin , < 1.8% with proinsulin , 10% with split-65,66", "proinsulin , 74% with des-64,65 proinsulin , 3% with des-31,32 proinsulin , and 2% with split-32,33 proinsulin ; the total proinsulin elisa cross - reacts 100% with intact and des-31,32 proinsulin , and 81% with des-64,65 proinsulin ; the ipi elisa cross - reacts 36% with des-64,54 proinsulin , and it shows no cross - reactivity with des-31,32 proinsulin .", "the lower limits of detection of the assays were provided by the manufacturer and are as follows : the insulin assay has a detection limit of 6", "pmol / l , the total proinsulin assay has a limit of 0.5 pmol / l , and the ipi assay has a lower limit of 0.1 pmol / l .", "all statistical analyses were performed using prism 6 software ( graphpad software , la jolla , ca ) .", "for all analyses , values obtained from healthy subjects were used as control subjects . for data that were normally distributed ,", "student t test was used to compare two groups , and one - way anova was used to compare between more than two groups . for data that had non - gaussian distributions , we used the mann - whitney test to compare between two groups and the kruskal - wallis test to compare between more than two groups ."], ["all samples were obtained from patients during either their annual posttransplantation follow - up or one of their regularly scheduled outpatient consultations .", "the serum was labeled using the same anonymous codes assigned to the tubes at the time of the test ."], ["an arginine stimulation test was performed on all subjects to determine basal and stimulated pancreatic hormone levels .", "blood was drawn at 10 , 0 , 2 , 3 , 4 , 5 , 7 , and 10 min from intravenous injection of a 5 g arginine bolus .", "patients who were back on insulin were required to discontinue slow - acting insulin at least 24 h prior to the test and rapid - acting insulin at least 6 h before the test .", "serum levels of insulin , c - peptide , total proinsulin , and intact proinsulin ( ipi ) were then measured using commercially available elisas as described below . acute insulin response ( air )", "was calculated as the mean of the three highest values between 2 and 5 min minus the mean of the basal values at 10 and 0 min .", "the area under the curve ( auc ) between 0 and 10 min postinjection for insulin and c - peptide was calculated by the trapezoidal rule with the mean of the baseline values subtracted .", "proinsulin fragments , including split-32,33 proinsulin , des-31,32 proinsulin , split-65,66 proinsulin , and des-64,65 proinsulin , were obtained by subtracting ipi from total proinsulin .", "ratios of insulin to total proinsulin and insulin [ insulin/(total proinsulin+insulin ) ] as well as proinsulin fragments to total proinsulin ( proinsulin fragments / total proinsulin ) were calculated to quantify the rate of proinsulin processing .", "ratios representing peak stimulation were calculated as the mean of ratios for the three highest time points between 2 and 5 min after arginine stimulation .", "insulin/(total proinsulin+insulin ) ratio represents the quantity of fully processed insulin with respect to all circulating forms of the hormone ."], ["commercially available elisas were used to measure hormone levels for insulin , c - peptide , total proinsulin , and ipi .", "the following kits were used : insulin ( mercodia , uppsala , sweden ) , c - peptide ( mercodia ) , total proinsulin ( millipore , zug , switzerland ) , and ipi ( millipore ) .", "concentrations were determined using a standard curve with high- and low - level control subjects .", "cross - reactivities of the assays were provided by the manufacturer and are as follows : the insulin assay cross - reacts 98% with des-64,65 proinsulin , < 0.01% with c - peptide and proinsulin , and < 0.05% with des-31,32 proinsulin and split-32,33 proinsulin ; the c - peptide elisa cross - reacts <", "0.0006% with insulin , < 1.8% with proinsulin , 10% with split-65,66 proinsulin , 74% with des-64,65 proinsulin , 3% with des-31,32 proinsulin , and 2% with split-32,33 proinsulin ; the total proinsulin elisa cross - reacts 100% with intact and des-31,32 proinsulin , and 81% with des-64,65 proinsulin ; the ipi elisa cross - reacts 36% with des-64,54 proinsulin , and it shows no cross - reactivity with des-31,32 proinsulin .", "the lower limits of detection of the assays were provided by the manufacturer and are as follows : the insulin assay has a detection limit of 6", "pmol / l , the c - peptide elisa has a limit of 15 pmol / l , the total proinsulin assay has a limit of 0.5 pmol / l , and the ipi assay has a lower limit of 0.1 pmol / l ."], ["all statistical analyses were performed using prism 6 software ( graphpad software , la jolla , ca ) . for all analyses , values obtained from healthy subjects were used as control subjects . for data that were normally distributed ,", "student t test was used to compare two groups , and one - way anova was used to compare between more than two groups . for data that had non - gaussian distributions , we used the mann - whitney test to compare between two groups and the kruskal - wallis test to compare between more than two groups ."], ["subjects in the healthy control group were slightly younger than the rest of the study population ( p < 0.01 ) .", "islet recipients exhibited higher fasting glycemia and hba1c levels than other groups ( p < 0.05 ) .", "nonetheless , hba1c levels remained in the normal range for the ii - isl group .", "creatinine levels were elevated in the pancreas and ir - isl groups with respect to the ii - isl and healthy control groups ( p < 0.005 vs. control , p < 0.05 vs. ii - isl ) .", "figure 1a and b shows the insulin and c - peptide response curves for all five groups .", "the insulin and c - peptide response curves maintain the same overall shape in all groups , with the exception of the diabetic control subjects who demonstrate no response to arginine stimulation .", "insulin levels were higher in pancreas recipients but lower in islet recipients compared with healthy control subjects .", "insulin levels in the pancreas group were significantly higher than those of the healthy control group .", "airs for healthy control subjects , ir - isl , ii - isl , and pancreas groups were 217  163 , 23  13 , 125  90 , and 266", "the c - peptide acute responses in the same groups were 717  362 , 91  53 , 278  124 , and 550  244 pmol / l , respectively ( supplementary table 1 ) . mean acute c - peptide responses were similar in the healthy control group and pancreas group but significantly lower in the islet groups .", "the ir - isl transplantees showed a significantly decreased acute insulin and c - peptide response with respect to all other groups .", "there was no significant difference in air between the ii - isl and control groups .", "as expected , the diabetic control group showed negligible insulin and c - peptide levels .", "a : mean insulin levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group .", "b : mean c - peptide levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group .", "auc values demonstrated similar distributions to acute responses in the various study groups ( supplementary table 1 ) .", "auc values for insulin response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 1,332  959 , 152  91 , 774  528 , and 1,540  707 pmol ", "these values were higher in pancreas recipients and lower in islet recipients compared with healthy control subjects .", "auc values for c - peptide response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 4,955  3,158 , 630  428 , 1,843  875 , and 3,859  2,061 pmol ", "the auc of c - peptide for the ir - isl group was significantly lower than in all other groups .", "mean values for c - peptide and total proinsulin before and after arginine stimulation are shown in fig .", "these figures show that total proinsulin levels are reduced in the ir - isl group with respect to all other groups and follow a distribution similar to that of c - peptide . a : box plots of basal c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isr group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of stimulated c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) .", "the healthy control group demonstrated lower basal proinsulin - processing rates than the pancreas ( p < 0.01 ) and ii - isl ( p < 0.05 ) groups [ 74  14% vs. 83  7% vs. 86  12% , respectively , for insulin/(total proinsulin+insulin ) and 54  25% vs. 67 ", "18% vs. 76  16% , respectively , for proinsulin fragments / total proinsulin ] .", "the ir - isl group displayed a basal insulin/(total proinsulin+insulin ) rate of 75  14% and a basal proinsulin fragments / total proinsulin rate of 54  25% .", "a : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) .", "i / tp+i , insulin / total proinsulin+insulin ; pf / tp , proinsulin fragments / total proinsulin .", "3b . interestingly , healthy control subjects , pancreas recipients , and ii - isl recipients all attain similarly elevated processing ratios after stimulation ( 93  2% vs. 93 ", "3% vs. 93  5% , respectively , for insulin/(total proinsulin+insulin ) and 80  7% vs. 77 ", "9% vs. 76  12% , respectively , for proinsulin fragments / total proinsulin ) , while ir - isl transplant recipients ratios do not demonstrate a comparable increase .", "although ir - isl proinsulin - processing ratios do increase slightly after stimulation to 87  4% and 58  18% for insulin/(total proinsulin + insulin ) and proinsulin fragments / total proinsulin , respectively , these values remain significantly lower than all other groups ( p < 0.01 vs. control and pancreas groups , p < 0.05 vs. ii - isl ) ."], ["subjects in the healthy control group were slightly younger than the rest of the study population ( p < 0.01 ) .", "islet recipients exhibited higher fasting glycemia and hba1c levels than other groups ( p < 0.05 ) .", "nonetheless , hba1c levels remained in the normal range for the ii - isl group .", "creatinine levels were elevated in the pancreas and ir - isl groups with respect to the ii - isl and healthy control groups ( p < 0.005 vs. control , p < 0.05 vs. ii - isl ) ."], ["figure 1a and b shows the insulin and c - peptide response curves for all five groups .", "the insulin and c - peptide response curves maintain the same overall shape in all groups , with the exception of the diabetic control subjects who demonstrate no response to arginine stimulation .", "insulin levels were higher in pancreas recipients but lower in islet recipients compared with healthy control subjects .", "insulin levels in the pancreas group were significantly higher than those of the healthy control group .", "airs for healthy control subjects , ir - isl , ii - isl , and pancreas groups were 217  163 , 23  13 , 125  90 , and 266  129 pmol / l , respectively .", "the c - peptide acute responses in the same groups were 717  362 , 91  53 , 278  124 , and 550  244 pmol / l , respectively ( supplementary table 1 ) . mean acute c - peptide responses were similar in the healthy control group and pancreas group but significantly lower in the islet groups .", "the ir - isl transplantees showed a significantly decreased acute insulin and c - peptide response with respect to all other groups .", "there was no significant difference in air between the ii - isl and control groups .", "as expected , the diabetic control group showed negligible insulin and c - peptide levels .", "a : mean insulin levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group .", "b : mean c - peptide levels during arginine stimulation of healthy control group , ir - isl group , ii - isl group , pancreas group , and diabetic control group", "auc values demonstrated similar distributions to acute responses in the various study groups ( supplementary table 1 ) .", "auc values for insulin response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 1,332  959 , 152  91 , 774  528 , and 1,540  707 pmol ", "these values were higher in pancreas recipients and lower in islet recipients compared with healthy control subjects .", "auc values for c - peptide response in healthy control subjects , ir - isl , ii - isl , and pancreas groups were 4,955  3,158 , 630  428 , 1,843  875 , and 3,859  2,061 pmol ", "the auc of c - peptide for the ir - isl group was significantly lower than in all other groups ."], ["mean values for c - peptide and total proinsulin before and after arginine stimulation are shown in fig .", "these figures show that total proinsulin levels are reduced in the ir - isl group with respect to all other groups and follow a distribution similar to that of c - peptide . a : box plots of basal c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isr group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of stimulated c - peptide and total proinsulin levels in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) ."], ["the healthy control group demonstrated lower basal proinsulin - processing rates than the pancreas ( p < 0.01 ) and ii - isl ( p < 0.05 ) groups [ 74  14% vs. 83  7% vs. 86  12% , respectively , for insulin/(total proinsulin+insulin ) and 54  25% vs. 67 ", "18% vs. 76  16% , respectively , for proinsulin fragments / total proinsulin ] .", "the ir - isl group displayed a basal insulin/(total proinsulin+insulin ) rate of 75  14% and a basal proinsulin fragments / total proinsulin rate of 54  25% .", "a : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group before arginine injection ( basal values ) .", "b : box plots of the proinsulin - processing ratios [ ( insulin / total proinsulin+insulin ) and ( proinsulin fragments / total proinsulin ) ] in healthy control group , ir - isl group , ii - isl group , and pancreas group during peak stimulation after arginine injection ( stimulated values ) .", "data are expressed as median ( solid line ) , interquartile range ( box ) , and range ( whiskers ) .", "i / tp+i , insulin / total proinsulin+insulin ; pf / tp , proinsulin fragments / total proinsulin ."], ["3b . interestingly , healthy control subjects , pancreas recipients , and ii - isl recipients all attain similarly elevated processing ratios after stimulation ( 93  2% vs. 93 ", "3% vs. 93  5% , respectively , for insulin/(total proinsulin+insulin ) and 80  7% vs. 77 ", "9% vs. 76  12% , respectively , for proinsulin fragments / total proinsulin ) , while ir - isl transplant recipients ratios do not demonstrate a comparable increase .", "although ir - isl proinsulin - processing ratios do increase slightly after stimulation to 87  4% and 58  18% for insulin/(total proinsulin + insulin ) and proinsulin fragments / total proinsulin , respectively , these values remain significantly lower than all other groups ( p < 0.01 vs. control and pancreas groups , p < 0.05 vs. ii - isl ) ."], ["this study explores for the first time proinsulin processing in islet transplant recipients , not only in comparison with healthy control subjects , but also with respect to pancreas transplant recipients .", "we have shown , first , that the exposure to arginine stimulation is in fact similar in ii - isl transplant recipients and healthy control subjects .", "as expected , recipients of pancreas transplants with systemic venous drainage demonstrate higher insulin response to arginine than healthy control subjects .", "second , there is a noticeable difference in the rate of basal proinsulin processing comparing ii - isl and ir - isl transplant recipients .", "ii - isl patients have proinsulin - processing ratios similar to those of pancreas transplant recipients , which are significantly higher than those of healthy control and ir - isl patients .", "third , after stimulation , ii - isl , pancreas , and healthy control subjects all increase their proinsulin - processing ratios to a similar level .", "in contrast , ir - isl patients are unable to achieve the same increase in proinsulin - processing rate .", "these data suggest that fully functional islet grafts are able to attain optimal proinsulin processing when stimulated but are required to sustain high levels of processing even during a metabolic fasting state in order to maintain glycemic control .", "islet grafts requiring supplementation with insulin therapy , on the other hand , demonstrate low basal proinsulin - processing rates , which are unable to reach optimal levels even when stimulated .", "interestingly , total proinsulin levels in this group ( ir - isl ) are low compared with other groups both before and after stimulation . however , the ratio of proinsulin to insulin in the ir - isl group after stimulation is disproportionately high .", "this suggests that partially functional islet grafts have a defect in proinsulin processing , which manifests itself as relative and not absolute hyperproinsulinemia .", "it should be noted that the calculation of proinsulin fragments may be slightly biased owing to elisa cross - reactivities . in theory ,", "when we calculate proinsulin fragments as proinsulin fragments = total proinsulin  ipi , we are assuming that all of the des-64,65 proinsulin has been measured by the total proinsulin assay and that none of it has been measured by the intact assay .", "in reality , we measure only 81% of des-64,65 with the total proinsulin assay and 36% with the ipi assay .", ". however , since the pc2 pathway responsible for the formation of des-64,65 proinsulin is not the primary pathway for the production of insulin , unmeasured levels of des-64,65 proinsulin are relatively insignificant with respect to des-31,32 proinsulin levels .", "it should also be noted that clearance of proinsulin and proinsulin fragments could be altered by diminished renal function . as seen in table 1 , our ir - isl group demonstrated creatinine levels that were elevated compared with healthy control subjects .", "however , this difference was not significant comparing the ir - isl group to ii - isl and pancreas groups .", "in contrast , the reduced processing ratios observed in the ir - isl group are significantly lower than all other groups .", "both absolute and relative hyperproinsulinemia have already been observed in settings of impaired glucose metabolism ( 1214 ) .", "first , it has been suggested that dysfunctional -cells present a fundamental defect in the pathway responsible for processing of proinsulin to insulin ( 18 ) .", "it is possible in the case of islet transplantation that manipulation of islets , introduction of -cells into a foreign microenvironment , or immunosuppressive drugs may render these cells inherently dysfunctional .", "a second theory suggests that hyperproinsulinemia occurs because of increased demand on -cells , which leads to insufficient time to complete proinsulin processing intracellularly before granule secretion occurs ( 15,19,20 ) .", "it seems reasonable to suppose that a combination of insulin resistance and decreased -cell mass could lead to unattainable insulin needs and eventual -cell deterioration via exhaustion .", "our data show that islet grafts initially present increased basal proinsulin processing with respect to healthy control subjects and that this processing deteriorates as graft function declines , a phenomenon that more readily supports the latter theory .", "we can not , however , exclude the effect of prolonged glucotoxicity on -cell function as an additional possible explanation for the observed processing defects .", "although our two islet groups had similar fasting glucose levels , hba1c was slightly increased in the ir - isl group , indicating that chronic glucotoxicity may have played a role in the functional deterioration of -cells within this subgroup .", "ultimately , a longitudinal study on the same or similar populations might help to verify this hypothesis .", "two studies have previously investigated proinsulin processing in islet transplant recipients ( 16,22 ) . however , neither study used arginine stimulation to explore hormonal processing when under simulated metabolic stress and neither study compared islet and pancreas transplant recipients .", "it is interesting to note that these studies produced conflicting data and presented contrasting conclusions .", "we found that functional islet grafts display increased processing and relatively lower proinsulinemia at rest , with an inability to increase their processing rates further when stimulated .", "these grafts are essentially behaving the same during the metabolic fasting state as fully stimulated healthy control subjects .", "as islet grafts lose function , however , processing  even during the fasting state  becomes less effective and proinsulinemia increases .", "these findings directly correlate with the findings of mcdonald et al . and are supported by the data of klimek at al . , which include mostly ir - isl transplant recipients . two other groups , fiorina et al .", "it would seem that there is a threshold level of demand beyond which -cells can no longer process proinsulin effectively . as stated earlier , we presume this to be due to an increased demand on decreased -cell mass . further investigation is warranted to explain the exact mechanism responsible for the increased basal proinsulin processing seen in ii - isl grafts .", "again , a longitudinal study of these patients might help shed more light on the issue .", "no study previously analyzed the stimulated hormonal response of pancreas grafts with respect to islet grafts .", "our data show that pancreas grafts behave quite similarly to healthy control subjects but with elevated overall hormone levels .", "this is undoubtedly a reflection of the systemic venous pancreatic drainage of these patients , in whom there is no hepatic first - pass metabolism of pancreatic hormonal secretions ( 23 ) .", "pancreas grafts tend to show similar patterns of proinsulin processing to fully functional islet grafts .", "whether this is peculiar to systemically drained pancreata can not be elucidated from this data .", "this study used arginine stimulation rather than oral or intravenous glucose tolerance tests to test secretory reserve in our patient populations .", "arginine stimulation provides the advantage of not inducing hyperglycemia , thus reducing the confounding influence of glucotoxicity to a minimum .", "other investigators have found the intravenous glucose tolerance tests to be more robust and a better indicator of -cell status ( 24 ) , but this method does not account for host insulin resistance . in summary , ii - isl transplant recipients can maintain basal metabolic parameters similar to healthy control subjects at the cost of a higher rate of basal proinsulin processing .", "ir - isl transplant recipients demonstrate both lower insulin response and lower basal rates of proinsulin processing , which remain suboptimal even after arginine stimulation ( i.e. , loss of graft function is associated with less effective processing and relative hyperproinsulinemia )", ". finally , the higher airs of pancreas transplant recipients are a reflection of systemic venous drainage of endocrine secretions ."], [""]]}
{"article_id": "PMC3181707", "article_text": ["we have demonstrated that , melatonin is a bioprecursor of hypnotic acetyl metabolites produced by enzymatic acetylation of melatonin and 2-oxomelatonin under the control of acetyltransferases , most probably the nat enzymes . in 1994 , in our laboratory , we developed a specific and highly sensitive gas chromatography - mass spectrometry ( gc - ms ) method to assay , simultaneously and distinct ! ) , plasma concentrations of endogenous melatonin ( d0melatonin ) and exogenous melatonin ( d7-melatonin ) , in which 7 atoms of h have been substituted by 7 atoms of deuterium .", "using the same human volunteers ( 12 young subjects in june 1994 and 12 elderly subjects in october 1994 ) , we determined the pharmacokinetics of exogenous d7-melatonin , when given orally and intravenously , and the kinetics of the pineal secretion of endogenous d0-melatonin . ,   the results shown in figure 2 led to the following conclusions : secretion of melatonin by the pineal gland occurs only during the night .", "pharmacokinetic analysis shows that the rate of melatonin secretion by the pineal gland is constant throughout the whole nocturnal pineal melatonin production , for the same subject .", "the beginning and end of melatonin secretion from the pineal gland are the same for each subject , whatever the season and night , length .", "duration of melatonin pineal secretion is between 7.5 and 8 h. therefore , melatonin secretion and sleep are contemporaneous .", "there is a large interindividual variability in the amount , of melatonin released in plasma by the pineal gland during the night in young and old subjects alike .", "results of previous related studies show that melatonin secretion , and therefore the presence of melatonin in the central nervous system ( cns ) , is necessary for the induction and maintenance of nocturnal sleep . however , the presence of melatonin in the cns is insufficient for the induction and maintenance of sleep . indeed , figure 3 and table", "i show results of observations in chicks in an alternate light ( l)-dark ( d ) program ( l / d , 12 h:12 h ) , in which the light phase lasted from 8.00 am until 8.00 pm .", "when melatonin was administered intramuscularly ( pectoralis major muscle ) during the light phase from 2.00 pm to 8.00 pm , the chicks did not .", "the absence of a hypnotic effect during the light phase correlated with the very low level of nat activity in the pineal glands of chicks measured at the same times .", "in contrast , when chicks were observed in a 7-day permanent light , program ( l / l , 12 h:12 h ) , during which nat activity level was constantly higher , the administration of melatonin induced a significant , hypnotic effect .", "the duration of sleep ( between 4 and 5 h ) was much greater than that , observed with diazepam ( between 1 and 2 h ) when it .", "was administered intramuscularly at the same dose ( 1 m per 100 g body weight , at 2.00 pm ) .", "these results lead to the following conclusions : the simultaneous presence of melatonin and nat in the cns ( pineal gland ) is a necessary and sufficient condition for the induction and maintenance of sleep .", "in contrast to the classic so - called  hypnotic drugs  ( eg , benzodiazepines , barbiturates , zopiclone , and zolpidem ) , melatonin does not have direct , hypnotic properties related to its chemical structure .", "during the development of the gc - ms method for the assay of melatonin in plasma , our attention was focussed on the chemical reactivity of melatonin at position 3 , which allows cyciization of the side chain after acylation .", "this proceeds by nucleophilic attack and leads to a fluoroacyl--carboline ( figure 4 ) . considering our previous observations", ", we assumed that melatonin undergoes enzymatic acetylation during the night , under the control of nat , and that this leads to an n - acetyl--carboline , which we call carbo2 .", "we conclude that melatonin is a bioprecursor of hypnotic acetyl metabolites , such as carbo2 .", "chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase ,", "they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min .", "figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) .", "gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) .", "we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low .", "the results are presented in table iii , together with some reference compounds . the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect .", "in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced   sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep .", "chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase ,", "they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min .", "figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) .", "gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) .", "we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program .", "the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low .", "the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect .", "in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced   sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep .", "we have evidenced the role played by melatonin in both inducing and maintaining nocturnal sleep .", "melatonin is the bioprecursor of hypnotic acetyl metabolites , such as carbo2 , which result from the enzymatic acetylation of melatonin ( and 2-oxomelatonin ) by nat .", "since insomnia and sleep disorders may be due to a lack of nat enzymes in the pineal gland , a therapeutic approach to sleep disorders could be suggested .", "patients with insomnia may be treated by administering hypnotic acetyl metabolites of melatonin or their synthetic analogs ."], "abstract_text": ["<S> pharmacokinetic studies of melatonin in young and elderly human volunteers , and the measurement of hypnotic effects in chicks under alternate light - dark or permanent light conditions , show that melatonin is a bioprecursor of hypnotic acetyl metabolites produced by the enzymatic acetylation of both melatonin and 2-oxomelatonin under the control of serotonin n - acetyltransferases ( nats ) , which are present in the pineal gland . </S>", "<S> the acetyl metabolite of melatonin , which we call carbo2 , is an n - acetyl--carboline . </S>", "<S> the electroencephalographs ( eeg ) architecture of the sleep produced by this compound is similar to thai of physiological sleep , and is characterized by the significant proportion of slow - wave deep sleep and rapid eye movement sleep . </S>", "<S> this is in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds . </S>", "<S> since insomnia and sleep disorders are believed to be due to a lack of nat enzymes in the pineal gland , a new therapeutic approach of sleep disorders by administration of such hypnotic acetyl metabolites of melatonin , or synthetic analogs thereof , can be en visaged . </S>"], "labels": null, "section_names": ["Pineal melatonin secretion in humans", "Hypnotic effect of melatonin and NAT in the CNS", "Melatonin: a bioprecursor of hypnotic metabolites", "Acetylation of melatonin in chick pineal glands", "Synthesis of carbo2", "Hypnotic activity of carbo2", "Conclusion"], "sections": [["we have demonstrated that , melatonin is a bioprecursor of hypnotic acetyl metabolites produced by enzymatic acetylation of melatonin and 2-oxomelatonin under the control of acetyltransferases , most probably the nat enzymes . in 1994 , in our laboratory , we developed a specific and highly sensitive gas chromatography - mass spectrometry ( gc - ms ) method to assay , simultaneously and distinct ! ) , plasma concentrations of endogenous melatonin ( d0melatonin ) and exogenous melatonin ( d7-melatonin ) , in which 7 atoms of h have been substituted by 7 atoms of deuterium .", "using the same human volunteers ( 12 young subjects in june 1994 and 12 elderly subjects in october 1994 ) , we determined the pharmacokinetics of exogenous d7-melatonin , when given orally and intravenously , and the kinetics of the pineal secretion of endogenous d0-melatonin . ,   the results shown in figure 2 led to the following conclusions : secretion of melatonin by the pineal gland occurs only during the night .", "pharmacokinetic analysis shows that the rate of melatonin secretion by the pineal gland is constant throughout the whole nocturnal pineal melatonin production , for the same subject .", "the beginning and end of melatonin secretion from the pineal gland are the same for each subject , whatever the season and night , length .", "duration of melatonin pineal secretion is between 7.5 and 8 h. therefore , melatonin secretion and sleep are contemporaneous .", "there is a large interindividual variability in the amount , of melatonin released in plasma by the pineal gland during the night in young and old subjects alike ."], ["results of previous related studies show that melatonin secretion , and therefore the presence of melatonin in the central nervous system ( cns ) , is necessary for the induction and maintenance of nocturnal sleep . however , the presence of melatonin in the cns is insufficient for the induction and maintenance of sleep . indeed , figure 3 and table", "i show results of observations in chicks in an alternate light ( l)-dark ( d ) program ( l / d , 12 h:12 h ) , in which the light phase lasted from 8.00 am until 8.00 pm .", "when melatonin was administered intramuscularly ( pectoralis major muscle ) during the light phase from 2.00 pm to 8.00 pm , the chicks did not .", "the absence of a hypnotic effect during the light phase correlated with the very low level of nat activity in the pineal glands of chicks measured at the same times .", "in contrast , when chicks were observed in a 7-day permanent light , program ( l / l , 12 h:12 h ) , during which nat activity level was constantly higher , the administration of melatonin induced a significant , hypnotic effect .", "the duration of sleep ( between 4 and 5 h ) was much greater than that , observed with diazepam ( between 1 and 2 h ) when it .", "was administered intramuscularly at the same dose ( 1 m per 100 g body weight , at 2.00 pm ) .", "these results lead to the following conclusions : the simultaneous presence of melatonin and nat in the cns ( pineal gland ) is a necessary and sufficient condition for the induction and maintenance of sleep .", "in contrast to the classic so - called  hypnotic drugs  ( eg , benzodiazepines , barbiturates , zopiclone , and zolpidem ) , melatonin does not have direct , hypnotic properties related to its chemical structure ."], ["during the development of the gc - ms method for the assay of melatonin in plasma , our attention was focussed on the chemical reactivity of melatonin at position 3 , which allows cyciization of the side chain after acylation .", "this proceeds by nucleophilic attack and leads to a fluoroacyl--carboline ( figure 4 ) . considering our previous observations", ", we assumed that melatonin undergoes enzymatic acetylation during the night , under the control of nat , and that this leads to an n - acetyl--carboline , which we call carbo2 .", "we conclude that melatonin is a bioprecursor of hypnotic acetyl metabolites , such as carbo2 .", "chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase ,", "they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min .", "figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) .", "gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) .", "we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low .", "the results are presented in table iii , together with some reference compounds . the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect .", "in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced   sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep ."], ["chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase ,", "they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min .", "figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) .", "gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) ."], ["we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program ."], ["the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low .", "the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect .", "in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced   sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep ."], ["we have evidenced the role played by melatonin in both inducing and maintaining nocturnal sleep .", "melatonin is the bioprecursor of hypnotic acetyl metabolites , such as carbo2 , which result from the enzymatic acetylation of melatonin ( and 2-oxomelatonin ) by nat .", "since insomnia and sleep disorders may be due to a lack of nat enzymes in the pineal gland , a therapeutic approach to sleep disorders could be suggested .", "patients with insomnia may be treated by administering hypnotic acetyl metabolites of melatonin or their synthetic analogs ."]]}
{"article_id": "PMC3809381", "article_text": ["\n over the next several decades , the number of americans living to advanced ages will increase substantially .", "although many individuals will age in relatively good health , a growing number will encounter challenges associated with the burdens of chronic conditions and associated disabilities [ 13 ] .", "this is especially so for the large numbers of women who will continue to outlive their male counterparts and likely live those additional years with chronic illnesses requiring day - to - day management [ 4 , 5 ] .", "further , with a dramatic increase of female  baby boomers  with obesity - related chronic conditions , accompanied by reduced fertility rates among this rapidly aging cohort , the additive or multiplicative effects of living with one or more chronic conditions are likely to result in a diminution of ( 1 ) individuals ' capacity to adequately care for themselves , ( 2 ) caregivers to serve as efficient resources , and ( 3 ) healthcare providers to give adequate attention and guidance to complex patients with multiple chronic conditions ( mccs ) . in line with the millions of older women struggling to manage the symptoms associated with chronic disease , there is growing recognition about the importance of self - care behavior , which is supported by strong epidemiological documentation regarding the positive association of self - care and health outcomes [ 710 ] .", "however , this issue transcends women 's exposure to and understanding of pertinent information and their development of self - care skills .", "older women with chronic conditions also need to assess their surrounding resources to develop the confidence and efficacy necessary to initiate and maintain self - care behavior [ 1113 ] .", "self - care behavior are intended to draw upon one 's physical , social , and healthcare environments to compensate for , or delay , physical limitations and chronic conditions from progressing into more severe disabilities .", "further , self - care skills include ( 1 ) identifying strategies that enable older women to adopt and institute appropriate self - care behavior such as getting adequate exercise , eating healthy , or managing medications [ 15 , 16 ] ; ( 2 ) interacting with healthcare providers to obtain resources and referrals necessary to manage the progression of chronic conditions [ 10 , 17 , 18 ] ; and ( 3 ) locating social and community resources to become more educated about their conditions and alleviate the stressors and frustration acting as barriers to self - care behavior [ 6 , 19 ] .   while a growing literature has identified general disparities related to self - care among women with chronic conditions based on their race / ethnicity [ 2022 ] , socioeconomic status [ 2224 ] , and residential rurality [ 22 , 25 ] , the extent to which self - care disparities exist based on these and other sociodemographics requires further investigation .", "similarly , the role of education an indicator of socioeconomic status , healthcare access , and health behavior engagement emphasizes its importance when assessing disparities issues [ 26 , 27 ] .", "additional efforts are needed to examine the influence of sociodemographics on self - care skills and behavior among aging women , especially in the presence of perceptions about healthcare - related factors .", "healthcare provider - patient interactions can foster self - care behavior   although such interactions can also have a less - recognized negative effect on disease self - management .", "women can feel frustrated and helpless when their physicians do not fully explain or clarify the causes of their disease or ways how to best manage their illness .", "some patients report not having enough time to address their concerns , or that their physicians simply would not listen to them .", "conversely , among patients with diabetes , those who have good communication with their physicians report feeling more involved in decision - making efforts to manage their condition more effectively . also , patients who report their physicians provided adequate information about their conditions were more likely to better self - manage their illnesses .  ", "healthcare professionals often encounter challenges to address their female patients ' mcc alongside existing barriers to self - care behavior in their home or community environments .", "regardless of the source or cause of these barriers , competing demands on the side of either the patient or provider have potential to create a recursive relationship resulting in disconnect , miscommunication , frustration , and fewer self - care practices .", "these occurrences may inevitably contribute to decreased health outcomes and rapid chronic disease progression , which highlights the importance of support mechanisms available to the patient .", "effective self - care support mechanisms and resources identified to promote self - care behavior include traditional in - person , familial , and community support systems ( e.g. , support groups and faith - based organizations ) [ 6 , 3135 ] ; however , an emergence in technology - based support mechanisms has been shown to enable individuals with chronic conditions to access and utilize self - care information , despite traditional barriers [ 18 , 3639 ] .", "further , the active seeking of self - care support and resources has been shown to enhance self - care behavior among individuals with chronic conditions [ 12 , 38 , 40 ] .", "the preferred and/or utilized support mechanisms differ by population subgroup and type of chronic condition .", "evidence shows that racial / ethnic minorities with chronic conditions report increased in - person support mechanisms compared to their non - hispanic white counterparts [ 41 , 42 ] .", "conversely , those residing in rural areas have shown improvement in their self - care behavior through the use of internet - based support mechanisms , which may be critical to overcome traditional challenges associated with geographic isolation , less healthcare resources , and longer travel distances to healthcare services [ 25 , 44 ] . the advancing study of self - care behavior has identified factors influencing the adoption and maintenance of self - care behavior for different populations and people of all ages , with more recent attention paid to lifestyle relative to disease self - care behavior [ 17 , 45 , 46 ] . to advance the translation of research to practice", ", this secondary data analysis assesses issues surrounding self - care barriers , healthcare - related frustrations , and perceived supports among middle - aged and older adults with one or more chronic conditions . in an effort to further understand the multilevel influences on perceived barriers to self - care , this study will examine the roles of healthcare frustrations and doctor - patient interactions alongside other simultaneously occurring contextual factors ( see figure 1 ) .", "more specifically , the purposes of this study were to ( 1 ) describe sociodemographic variables , health indicators , healthcare - related frustrations , and perceptions of physician support among middle - aged and older adult women with one or more chronic conditions and ( 2 ) identify these factors ' association with reporting the need to help learning how to take better care of their health among this female population .", "\n the national council on aging ( ncoa ) , with support from atlantic philanthropies and the california healthcare foundation ( chcf ) , commissioned the ncoa chronic care survey , which offers unique insight into the lives of americans with chronic health conditions .", "the ncoa chronic care survey is a nationally representative probability survey of 960 community - dwelling men and women aged 44 years and older with at least one chronic condition .", "lake research partners utilized telephone - based interviewing to collect data using random digit dialing ( rdd ) sampling techniques , which oversampled those aged 65 and older and hispanics / latinos .", "the dataset was weighted by age , race , and region to reflect the overall population of americans 44 + with chronic condition(s ) .", "margin of error is greater when analyzing smaller subgroups within the sample .   to be eligible for inclusion in the ncoa chronic care survey , participants had to report having at least one chronic condition at the time of the study .", "participants were screened for chronic condition(s ) with the following question(s ) :  have you ever been told by a doctor , nurse , or other health professional that you have ( name of chronic condition ) ?  chronic conditions included in the screening process included heart disease , cancer , stroke , diabetes , arthritis , asthma , hypertension or high blood pressure , emphysema , chronic bronchitis , depression , anxiety , and others . only participants who reported  yes  to at least one of these items", "of the 960 adults age 44 years and older in this sample , only women were included in study analyses ( n = 427 ; 44.5% ) . of these women , an additional 140 cases ( 32.8% )", "more specifically , cases were omitted for incomplete data on rurality ( n = 48 ) , frustration with the healthcare system ( n = 25 ) , perceived physician support ( n = 18 ) , self - reported chronic conditions ( n = 17 ) , marital status ( n = 8) , using the internet for general support ( n = 7 ) , race / ethnicity ( n = 6 ) , education ( n = 4 ) , perceived barriers to self - care ( n = 4 ) , and activity limitations ( n = 3 ) .", "the analytic sample for this study contained 287 community - dwelling women , aged 44 years and older who self - reported having at least one chronic condition .", "when comparing characteristics of women omitted from the study ( n = 140 ) with those in the analytic sample ( n = 287 ) , a significantly larger proportion of the analytic sample was younger (  = 4.98 , p = 0.026 ) , non - hispanic white (  = 4.60 , p = 0.032 ) , and married (  = 11.78 , p = 0.001 ) .", "participants were asked to self - report their perceived barriers to self - care using an item intended to measure their need for help to learn how to take better care of their health .", "more specifically , participants were asked to rate their level of agreement to the following statement :  i need help learning how to take better care of my health in a way that works for me and my life . ", "responses were scored on a 5-point likert - type scale ranging from  strongly disagree  to  strongly agree .  based on the frequency distribution , participant responses were then dichotomized into two categories :  disagree  ( scored 0 ) and  agree  ( scored 1 ) .", "participants were asked to report their frustrations with healthcare interactions using items intended to measure their feelings about repeatedly having to describe their conditions at each doctor visit , the self - care instructions they received from the healthcare provider , the time spent interacting with the healthcare provider , and having a friend or family member attend physician 's visits with them . for example , participants were asked to rate their level of agreement to statements like  how often do you feel tired of describing your same conditions and problems every time you go to a hospital or doctor 's office ?  and  how often do you wish you had a friend or family member who could go to the doctor with you ?", " responses were scored using a 3-point likert - type scale with categories of  never  ( scored 0 ) ,  occasionally ", "the healthcare - related frustration scale ( ranging from 0 to 12 ) was created using these six items .", "all items loaded on one factor and the items were summed into a single - composite score (  = 0.766 ) .", "higher scores for the healthcare - related frustration scale indicate a higher level of frustration with healthcare interactions . based on the frequency distribution ,", "the highest tertile ( i.e. , representing the highest frustration levels ) served as the referent group .", "participants were asked to self - report the degree to which their physician engages them in treatment - related problem - solving / decision - making , refers them to other healthcare services and professionals , and asks if they understand their medications and associated regimens .", "for example , participants were asked to rate their level of agreement to statements like  how often does your physician ask for your ideas about how you can take care of your health problems ?  and  how often does your physician talk to other doctors and nurses who are taking care of you ?", " responses were scored using a 5-point likert - type scale with categories of  never  ( scored 1 ) ,  rarely  ( scored 2 ) ,  occasionally ", "( scored 3 ) ,  frequently  ( scored 4 ) , and  always  ( scored 5 ) .", "the perceived physician support scale ( ranging from 6 to 30 ) was created using these six items .", "all items loaded on one factor and the items were summed into a single - composite score (  = 0.776 ) .", "higher scores for the perceived physician support scale indicate a higher level of perceived physician support . based on the frequency distribution , this scale was converted into tertiles for the analytic purposes .", "the lowest tertile ( i.e. , representing the lowest perceived support levels ) served as the referent group .", "participants were asked to self - report aspects of their current health status using items intended to measure activity limitations , the number of prescription medications taken regularly each day , and the number of physician visits in the previous 12 months .", "participants were asked  are you limited in any way in any activities because of physical , mental , or emotional problems ?", " responses were scored as  no  ( scored 0 ) or  yes  ( scored 1 ) .", "participants were also asked  in the past 12 months , how many times have you , yourself made a doctor visit ? ", "participants were asked to self - report their perceptions about their use of the internet for general support related to managing their health problems .", "participants were asked  how much do you rely on the internet for ongoing help and support with your health problems ?  responses were scored on a 4-point likert - type scale with categories of  not at all  ( scored 0 ) ,  a little  ( scored 1 ) ,  some  ( scored 2 ) , and  a lot ", "based on the frequency distribution , participant responses were then dichotomized into two categories :  no  ( scored 0 ; indicating that they do not rely on the internet at all ) and  yes  ( scored 1 ; indicating that they rely on the internet at least a little ) .", "participants were also asked  how often do you feel you get the help and support you need to improve your health and manage your health problems ?  responses were scored using a 5-point likert - type scale with categories of  never  ( scored 0 ) ,  rarely  ( scored 1 ) ,  occasionally  ( scored 2 ) ,  frequently  ( scored 3 ) , and  always ", "sociodemographic variables in this study included age ( i.e. , 4464 years , 65 + years ) ; race / ethnicity ( i.e. , non - hispanic white , non - white ) ; education level ( i.e. , high school or less , some college or more ) ; marital status ( i.e. , unmarried , married ) ; and residential rurality ( i.e. , urban , suburban , and rural ) .  ", "frequencies were calculated for all major study variables , which were initially examined in relationship to participants ' age group ( 4464 years , 65 + years ) and whether they reported needing help learning how to take better care of their health ( yes or no ) .", "pearson 's  tests were performed to assess the independence between the dependent variable and categorized independent variables .", "logistic regression was performed to examine how sociodemographics , health indicators , perceived support , and frustrations were associated with reporting the need to help learning how to take better care of their health ( i.e. , not needing help served as the referent group ) .", "sample characteristics of study participants are presented in table 1 . of the 287 females participating in this study ,", "over 65% of participants were between the ages of 44 and 64 years and 34.4% were aged 65 years and older .", "respondents were disproportionately non - hispanic white ( 88.5% ) , married ( 69.0% ) , and had an education level of some college or more ( 61.7% ) .", "fifty percent of the study population resided in suburban areas , 25.2% resided in urban areas , and 24.8% resided in rural areas .", "approximately 33% of participants reported being limited from activities because of physical , mental , or emotional problems . on average , participants reported taking 3.67 ( 3.83 ) medications daily and visiting a physician 3.07 ( 1.94 ) times in the previous 12 months . over 45% of participants reported relying on the internet for ongoing help and support to manage their health problems , and 67% of participants reported frequently or always getting the help and support they need to improve their health and manage their health problems . compared to women aged 65 years and older , a significantly larger proportion of participants aged 4464 years had some college education or more (  = 7.14 , p = 0.008 ) and relied on the internet for ongoing support to manage their health problems (  = 21.15 , p < 0.001 ) .", "a significantly larger proportion of participants who reported needing help learning how to better care for their health problems were non - white (  = 4.78 , p = 0.029 ) and had a high school education or less (  = 4.23 , p = 0.040 ) .   ", "healthcare - related frustration scale characteristics are presented in table 2 . of those who reported healthcare - related frustrations ,", "16.4% reported frequently wishing their doctor had more time to spend talking to them ; 16.0% reported frequently feeling tired of describing their same conditions and problems every time they go to a hospital or doctor 's office ; 8.7% reported frequently wishing they had a friend or family member who could go to the doctor with them ; and 8.3% reported frequently being tired of feeling on their own when it comes to taking care of their health problems .", "fewer respondents ( 5.6% ) reported frequently feeling their doctor does not realize what it is really like for them at home trying to take care of their health problems or ( 5.2% ) frequently leaving the hospital or doctor 's office feeling confused about what they should do . on average", "conversely , when comparing frustrations by whether the participant reported needing help learning how to better care for their health problems , those needing help reported significantly higher scores on the healthcare - related frustration scale ( t = 4.79 , p < 0.001 ) and higher frustration levels for five of the six individual scale items ( t = 25.94 , p < 0.001 ) .  ", "perceived physician support scale characteristics are presented in table 3 . of those who reported receiving physician support ,", "50.9% reported their physician always helped them get an appointment they needed ; 46.5% reported their physician always asked if they understood their medications when their doctor prescribed them ; 15.7% reported their physician always talked to other doctors and nurses who were taking care of them ; and another 15.7% reported their physician always made plans to contact them after a visit to see how they were doing .", "fewer respondents ( 13.3% ) reported their physician always told them about other people who could help them with their health problems or ( 13.2% ) their physician always asked for their ideas about how they can take care of their health problems . on average , participants scored 18.70 ( 5.84 ) on the perceived physician support scale .", "conversely , when comparing support by whether the participant reported needing help learning how to better care for their health problems , a significantly smaller proportion of those needing help reported asked for their ideas about how they can take care of their health problems (  = 10.48 , p = 0.033 ) . \n table 4 displays the results of the logistic regression analysis explaining factors associated with participants reporting they need help learning how to better care for their health problems ( i.e. , not needing help served as the referent group ) .", "participants who were non - white were significantly more likely to report needing help learning how to better care for their health problems ( compared to non - hispanic whites , or = 2.26 , p = 0.049 ) , whereas those with some college or more were significantly less likely to report needing help learning how to better care for their health problems ( compared to those with high school or less education , or = 0.55 , p = 0.044 ) .", "compared to participants with the highest level ( tertile ) of healthcare - related frustrations , those with middle ( or = 0.17 , p < 0.001 ) and lowest ( or = 0.44 , p = 0.017 ) frustration levels were significantly less likely to report needing help learning how to better care for their health problems , respectively . compared to participants with the lowest level ( tertile ) of perceived physician support , those with the highest level of perceived support were significantly less likely to report needing help learning how to better care for their health problems ( or = 0.49 , p = 0.033 ) .", "despite our concerns that the majority of older women would likely experience self - care problems [ 4850 ] , our analyses revealed that only about one - third of the women in our sample reported needing help learning how to take better care of their chronic conditions . but", "consistent with health disparities in the epidemiology of chronic illnesses [ 22 , 24 ] , persistent health disparities related to self - care behavior were noted among minorities and those with less education .", "the rural healthcare disparity often reported in other studies [ 25 , 5153 ] was not observed in this study , nor were unmarried women disadvantaged relative to their married counterparts .", "these findings suggest middle - aged and older women in this sample were comparable in terms of having a variety of social supports for learning how to take care of their health .   additionally , despite the previous assumption that older women might be disadvantaged relative to younger baby - boomers , no significant differences in help needed based on age group were reported .", "similarly , our proxy measures for disease magnitude and severity ( i.e. , number of medications , physician visits , and limitations in activities ) did not differentiate those needing help .", "however , the existence of an age - based digital divide was observed [ 5456 ] , with older women less likely to use online / technology as a resource for chronic condition self - management .", "further investigation is needed to examine differences in the types of methods / strategies in which these older women engage when caring for their health conditions outside of the healthcare setting .  ", "this study helps elucidate the complex relationships among contextual factors , healthcare frustrations , and patient - provider interactions and support and points to potential opportunities for intervention .", "the lack of a significant relationship between age and healthcare - related frustrations or perceptions about physician support may be attributed to the fact that the sample was selected based on the presence of chronic conditions .", "however , the lack of a significant relationship is consistent with the fact that participation in community - based disease self - management programs is not limited to those of only older ages ; rather , program enrollment is based on the participant 's chronic disease status .  ", "this study reveals two strong modifiable correlates of women needing help learning how to care for their chronic conditions , even after controlling for sociodemographic and health status indicators in multivariate analyses : healthcare - related frustrations and perceived physician support .", "the majority of identified frustrations were significantly related to middle - aged and older women 's perceptions of needing help learning how to care for their health , which is supported in other studies [ 8 , 16 , 58 , 59 ] .", "the recent emphasis on patient - centered care and medical homes   is designed to help reduce such frustrations and hence can be expected to help boost women 's self - efficacy to care for their own health conditions .", "additionally , perceived physician support was another significant factor for knowing how to self - manage chronic conditions , especially in terms of patient activation as exemplified by wanting physicians to  ask for your ideas about how you can take care of your health problems .", " this reinforces previous research about the importance of perceived physician support for motivating patients to engage in healthier lifestyles and recommended medical regimens [ 9 , 31 , 61 , 62 ] . from the health professional point of view , fostering beliefs of patient support can be accomplished through continuing education units ( ceus ) or expanded emphasis during medical school training to develop strategies in which clinicians can engage and implement to be supportive , listen to their patients , and solicit their patients ' thoughts so they have an active role in their healthcare team [ 29 , 63 ] . from the patient point of view , widely available evidence - based self - management programs include elements within their curricula to teach older women how they can more effectively communicate with their healthcare providers . considering perspectives from each side of the healthcare interaction", "is essential to improve self - management both within and outside of the healthcare setting , which has implications for reducing disease mismanagement , unnecessary healthcare utilization ( e.g. , emergency room use ) , and medical costs .", "this secondary dataset did not contain all variables necessary to fully contextualize barriers and challenges associated with chronic disease self - care behavior among this aging population .", "however , this national study contained many variables of interest to address the current research gaps in knowledge about associations of healthcare frustrations , physician support , and self - care needs to chronic disease management .  ", "substantial numbers of participants were lost due to incomplete data on particular scale items , resulting in an analytical sample that was systematically different from the full sample ( e.g. , younger , married , and more white ) and potentially limited the generalizability of study findings .", "this is especially true in that the reduction of cases in the analytic sample reduced the potential proportion of older adults in the study from 42.1% to 33.4% , which may especially influence generalizability of findings to populations aged 65 years and older .", "study participants reported a variety of chronic conditions , but subanalyses based on disease type were not performed because the sample size was inadequate to make such comparisons . recognizing that needed self - care behavior may differ based on women 's particular condition diagnoses , disease stage , and the number of comorbidities in which they are diagnosed , future studies should strive to compare about barriers to self - care , perceived physician support , and frustrations with the healthcare system by their chronic condition profile .", "further , because women 's ability to cope with and adjust to their disease self - care may differ based on their available resources and socioeconomic status , future studies should examine such variables to determine their relationship with self - care disparities .", "another study limitation reducing the ability to widely generalize findings to the greater female community was the self - report and cross - sectional nature of these data .", "however , the sample was derived from random digit dialing and included items that deeply explore the challenges and frustrations with chronic condition self - management , which are not typically seen in other studies that investigate correlations between self - care behavior and other healthcare or physical health indicators .", "thus , this study contributes to a fuller understanding of the complex interrelationships that exist between self - care strategies , provider - patient interactions , and policies / programs in community contexts .", "the current study adds to the existing science base by examining barriers to self - care with a new lens , exploring healthcare - related frustrations and perceptions of physician support , and how these perceptions relate to various life domains , including diverse health status and sociodemographic contexts . identifying these common and unique challenges and correlates of these challenges , with a representative national population ,", "advance our current knowledge about self - care issues among middle - aged and older women .", "learning more about healthcare - related frustrations of and self - management supports utilized by middle - aged and older women has the potential to help program deliverers , healthcare providers , and health agencies provide the services and resources that women with chronic conditions want and think are helpful .", "findings from this investigation has potential to inform and guide modifications in the implementation and dissemination of evidence - based programs for older women to better match individuals with programs that meet their needs ."], "abstract_text": ["<S> previous research emphasizes the importance of reducing healthcare frustrations and enhancing physician supports to help patients engage in recommended healthcare regimens . </S>", "<S> however , less is known about how these factors are associated with aging women 's knowledge about self - care behavior . </S>", "<S> this study examined the sociodemographics , health indicators , healthcare - related frustrations , and perceptions of physician support associated with middle - aged and older adult females ' self - reported need for help to learn how to take better care of their health . </S>", "<S> data were analyzed from 287 females with one or more chronic conditions who completed the national council on aging ( ncoa ) chronic care survey . </S>", "<S> a logistic regression model was developed . </S>", "<S> women who were non - white ( or = 2.26 , p = 0.049 ) were more likely to need help learning how to better manage their health . </S>", "<S> those who had some college education or more ( or = 0.55 , p = 0.044 ) and lower healthcare - related frustrations ( or = 0.44 , p = 0.017 ) and perceived to have more physician support ( or = 0.49 , p = 0.033 ) were less likely to need help learning how to better manage their health . </S>", "<S> findings can inform the planning , implementation , assessment , and dissemination of evidence - based self - management programs for middle - aged and older women within and outside of clinical settings . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Limitations", "6. Conclusion"], "sections": [["\n over the next several decades , the number of americans living to advanced ages will increase substantially .", "although many individuals will age in relatively good health , a growing number will encounter challenges associated with the burdens of chronic conditions and associated disabilities [ 13 ] .", "this is especially so for the large numbers of women who will continue to outlive their male counterparts and likely live those additional years with chronic illnesses requiring day - to - day management [ 4 , 5 ] .", "further , with a dramatic increase of female  baby boomers  with obesity - related chronic conditions , accompanied by reduced fertility rates among this rapidly aging cohort , the additive or multiplicative effects of living with one or more chronic conditions are likely to result in a diminution of ( 1 ) individuals ' capacity to adequately care for themselves , ( 2 ) caregivers to serve as efficient resources , and ( 3 ) healthcare providers to give adequate attention and guidance to complex patients with multiple chronic conditions ( mccs ) . in line with the millions of older women struggling to manage the symptoms associated with chronic disease , there is growing recognition about the importance of self - care behavior , which is supported by strong epidemiological documentation regarding the positive association of self - care and health outcomes [ 710 ] .", "however , this issue transcends women 's exposure to and understanding of pertinent information and their development of self - care skills .", "older women with chronic conditions also need to assess their surrounding resources to develop the confidence and efficacy necessary to initiate and maintain self - care behavior [ 1113 ] .", "self - care behavior are intended to draw upon one 's physical , social , and healthcare environments to compensate for , or delay , physical limitations and chronic conditions from progressing into more severe disabilities .", "further , self - care skills include ( 1 ) identifying strategies that enable older women to adopt and institute appropriate self - care behavior such as getting adequate exercise , eating healthy , or managing medications [ 15 , 16 ] ; ( 2 ) interacting with healthcare providers to obtain resources and referrals necessary to manage the progression of chronic conditions [ 10 , 17 , 18 ] ; and ( 3 ) locating social and community resources to become more educated about their conditions and alleviate the stressors and frustration acting as barriers to self - care behavior [ 6 , 19 ] .   while a growing literature has identified general disparities related to self - care among women with chronic conditions based on their race / ethnicity [ 2022 ] , socioeconomic status [ 2224 ] , and residential rurality [ 22 , 25 ] , the extent to which self - care disparities exist based on these and other sociodemographics requires further investigation .", "similarly , the role of education an indicator of socioeconomic status , healthcare access , and health behavior engagement emphasizes its importance when assessing disparities issues [ 26 , 27 ] .", "additional efforts are needed to examine the influence of sociodemographics on self - care skills and behavior among aging women , especially in the presence of perceptions about healthcare - related factors .", "healthcare provider - patient interactions can foster self - care behavior   although such interactions can also have a less - recognized negative effect on disease self - management .", "women can feel frustrated and helpless when their physicians do not fully explain or clarify the causes of their disease or ways how to best manage their illness .", "some patients report not having enough time to address their concerns , or that their physicians simply would not listen to them .", "conversely , among patients with diabetes , those who have good communication with their physicians report feeling more involved in decision - making efforts to manage their condition more effectively . also , patients who report their physicians provided adequate information about their conditions were more likely to better self - manage their illnesses .  ", "healthcare professionals often encounter challenges to address their female patients ' mcc alongside existing barriers to self - care behavior in their home or community environments .", "regardless of the source or cause of these barriers , competing demands on the side of either the patient or provider have potential to create a recursive relationship resulting in disconnect , miscommunication , frustration , and fewer self - care practices .", "these occurrences may inevitably contribute to decreased health outcomes and rapid chronic disease progression , which highlights the importance of support mechanisms available to the patient .", "effective self - care support mechanisms and resources identified to promote self - care behavior include traditional in - person , familial , and community support systems ( e.g. , support groups and faith - based organizations ) [ 6 , 3135 ] ; however , an emergence in technology - based support mechanisms has been shown to enable individuals with chronic conditions to access and utilize self - care information , despite traditional barriers [ 18 , 3639 ] .", "further , the active seeking of self - care support and resources has been shown to enhance self - care behavior among individuals with chronic conditions [ 12 , 38 , 40 ] .", "the preferred and/or utilized support mechanisms differ by population subgroup and type of chronic condition .", "evidence shows that racial / ethnic minorities with chronic conditions report increased in - person support mechanisms compared to their non - hispanic white counterparts [ 41 , 42 ] .", "conversely , those residing in rural areas have shown improvement in their self - care behavior through the use of internet - based support mechanisms , which may be critical to overcome traditional challenges associated with geographic isolation , less healthcare resources , and longer travel distances to healthcare services [ 25 , 44 ] . the advancing study of self - care behavior has identified factors influencing the adoption and maintenance of self - care behavior for different populations and people of all ages , with more recent attention paid to lifestyle relative to disease self - care behavior [ 17 , 45 , 46 ] . to advance the translation of research to practice", ", this secondary data analysis assesses issues surrounding self - care barriers , healthcare - related frustrations , and perceived supports among middle - aged and older adults with one or more chronic conditions . in an effort to further understand the multilevel influences on perceived barriers to self - care , this study will examine the roles of healthcare frustrations and doctor - patient interactions alongside other simultaneously occurring contextual factors ( see figure 1 ) .", "more specifically , the purposes of this study were to ( 1 ) describe sociodemographic variables , health indicators , healthcare - related frustrations , and perceptions of physician support among middle - aged and older adult women with one or more chronic conditions and ( 2 ) identify these factors ' association with reporting the need to help learning how to take better care of their health among this female population ."], ["\n the national council on aging ( ncoa ) , with support from atlantic philanthropies and the california healthcare foundation ( chcf ) , commissioned the ncoa chronic care survey , which offers unique insight into the lives of americans with chronic health conditions .", "the ncoa chronic care survey is a nationally representative probability survey of 960 community - dwelling men and women aged 44 years and older with at least one chronic condition .", "lake research partners utilized telephone - based interviewing to collect data using random digit dialing ( rdd ) sampling techniques , which oversampled those aged 65 and older and hispanics / latinos .", "the dataset was weighted by age , race , and region to reflect the overall population of americans 44 + with chronic condition(s ) .", "margin of error is greater when analyzing smaller subgroups within the sample .   to be eligible for inclusion in the ncoa chronic care survey , participants had to report having at least one chronic condition at the time of the study .", "participants were screened for chronic condition(s ) with the following question(s ) :  have you ever been told by a doctor , nurse , or other health professional that you have ( name of chronic condition ) ?  chronic conditions included in the screening process included heart disease , cancer , stroke , diabetes , arthritis , asthma , hypertension or high blood pressure , emphysema , chronic bronchitis , depression , anxiety , and others . only participants who reported  yes  to at least one of these items", "of the 960 adults age 44 years and older in this sample , only women were included in study analyses ( n = 427 ; 44.5% ) . of these women , an additional 140 cases ( 32.8% )", "more specifically , cases were omitted for incomplete data on rurality ( n = 48 ) , frustration with the healthcare system ( n = 25 ) , perceived physician support ( n = 18 ) , self - reported chronic conditions ( n = 17 ) , marital status ( n = 8) , using the internet for general support ( n = 7 ) , race / ethnicity ( n = 6 ) , education ( n = 4 ) , perceived barriers to self - care ( n = 4 ) , and activity limitations ( n = 3 ) .", "the analytic sample for this study contained 287 community - dwelling women , aged 44 years and older who self - reported having at least one chronic condition .", "when comparing characteristics of women omitted from the study ( n = 140 ) with those in the analytic sample ( n = 287 ) , a significantly larger proportion of the analytic sample was younger (  = 4.98 , p = 0.026 ) , non - hispanic white (  = 4.60 , p = 0.032 ) , and married (  = 11.78 , p = 0.001 ) .", "participants were asked to self - report their perceived barriers to self - care using an item intended to measure their need for help to learn how to take better care of their health .", "more specifically , participants were asked to rate their level of agreement to the following statement :  i need help learning how to take better care of my health in a way that works for me and my life . ", "responses were scored on a 5-point likert - type scale ranging from  strongly disagree  to  strongly agree .  based on the frequency distribution , participant responses were then dichotomized into two categories :  disagree  ( scored 0 ) and  agree  ( scored 1 ) .", "participants were asked to report their frustrations with healthcare interactions using items intended to measure their feelings about repeatedly having to describe their conditions at each doctor visit , the self - care instructions they received from the healthcare provider , the time spent interacting with the healthcare provider , and having a friend or family member attend physician 's visits with them . for example , participants were asked to rate their level of agreement to statements like  how often do you feel tired of describing your same conditions and problems every time you go to a hospital or doctor 's office ?  and  how often do you wish you had a friend or family member who could go to the doctor with you ?", " responses were scored using a 3-point likert - type scale with categories of  never  ( scored 0 ) ,  occasionally ", "the healthcare - related frustration scale ( ranging from 0 to 12 ) was created using these six items .", "all items loaded on one factor and the items were summed into a single - composite score (  = 0.766 ) .", "higher scores for the healthcare - related frustration scale indicate a higher level of frustration with healthcare interactions . based on the frequency distribution ,", "the highest tertile ( i.e. , representing the highest frustration levels ) served as the referent group .", "participants were asked to self - report the degree to which their physician engages them in treatment - related problem - solving / decision - making , refers them to other healthcare services and professionals , and asks if they understand their medications and associated regimens .", "for example , participants were asked to rate their level of agreement to statements like  how often does your physician ask for your ideas about how you can take care of your health problems ?  and  how often does your physician talk to other doctors and nurses who are taking care of you ?", " responses were scored using a 5-point likert - type scale with categories of  never  ( scored 1 ) ,  rarely  ( scored 2 ) ,  occasionally ", "( scored 3 ) ,  frequently  ( scored 4 ) , and  always  ( scored 5 ) .", "the perceived physician support scale ( ranging from 6 to 30 ) was created using these six items .", "all items loaded on one factor and the items were summed into a single - composite score (  = 0.776 ) .", "higher scores for the perceived physician support scale indicate a higher level of perceived physician support . based on the frequency distribution , this scale was converted into tertiles for the analytic purposes .", "the lowest tertile ( i.e. , representing the lowest perceived support levels ) served as the referent group .", "participants were asked to self - report aspects of their current health status using items intended to measure activity limitations , the number of prescription medications taken regularly each day , and the number of physician visits in the previous 12 months .", "participants were asked  are you limited in any way in any activities because of physical , mental , or emotional problems ?", " responses were scored as  no  ( scored 0 ) or  yes  ( scored 1 ) .", "participants were also asked  in the past 12 months , how many times have you , yourself made a doctor visit ? ", "participants were asked to self - report their perceptions about their use of the internet for general support related to managing their health problems .", "participants were asked  how much do you rely on the internet for ongoing help and support with your health problems ?  responses were scored on a 4-point likert - type scale with categories of  not at all  ( scored 0 ) ,  a little  ( scored 1 ) ,  some  ( scored 2 ) , and  a lot ", "based on the frequency distribution , participant responses were then dichotomized into two categories :  no  ( scored 0 ; indicating that they do not rely on the internet at all ) and  yes  ( scored 1 ; indicating that they rely on the internet at least a little ) .", "participants were also asked  how often do you feel you get the help and support you need to improve your health and manage your health problems ?  responses were scored using a 5-point likert - type scale with categories of  never  ( scored 0 ) ,  rarely  ( scored 1 ) ,  occasionally  ( scored 2 ) ,  frequently  ( scored 3 ) , and  always ", "sociodemographic variables in this study included age ( i.e. , 4464 years , 65 + years ) ; race / ethnicity ( i.e. , non - hispanic white , non - white ) ; education level ( i.e. , high school or less , some college or more ) ; marital status ( i.e. , unmarried , married ) ; and residential rurality ( i.e. , urban , suburban , and rural ) .  ", "frequencies were calculated for all major study variables , which were initially examined in relationship to participants ' age group ( 4464 years , 65 + years ) and whether they reported needing help learning how to take better care of their health ( yes or no ) .", "pearson 's  tests were performed to assess the independence between the dependent variable and categorized independent variables .", "logistic regression was performed to examine how sociodemographics , health indicators , perceived support , and frustrations were associated with reporting the need to help learning how to take better care of their health ( i.e. , not needing help served as the referent group ) ."], ["sample characteristics of study participants are presented in table 1 . of the 287 females participating in this study ,", "over 65% of participants were between the ages of 44 and 64 years and 34.4% were aged 65 years and older .", "respondents were disproportionately non - hispanic white ( 88.5% ) , married ( 69.0% ) , and had an education level of some college or more ( 61.7% ) .", "fifty percent of the study population resided in suburban areas , 25.2% resided in urban areas , and 24.8% resided in rural areas .", "approximately 33% of participants reported being limited from activities because of physical , mental , or emotional problems . on average , participants reported taking 3.67 ( 3.83 ) medications daily and visiting a physician 3.07 ( 1.94 ) times in the previous 12 months . over 45% of participants reported relying on the internet for ongoing help and support to manage their health problems , and 67% of participants reported frequently or always getting the help and support they need to improve their health and manage their health problems . compared to women aged 65 years and older , a significantly larger proportion of participants aged 4464 years had some college education or more (  = 7.14 , p = 0.008 ) and relied on the internet for ongoing support to manage their health problems (  = 21.15 , p < 0.001 ) .", "a significantly larger proportion of participants who reported needing help learning how to better care for their health problems were non - white (  = 4.78 , p = 0.029 ) and had a high school education or less (  = 4.23 , p = 0.040 ) .   ", "healthcare - related frustration scale characteristics are presented in table 2 . of those who reported healthcare - related frustrations ,", "16.4% reported frequently wishing their doctor had more time to spend talking to them ; 16.0% reported frequently feeling tired of describing their same conditions and problems every time they go to a hospital or doctor 's office ; 8.7% reported frequently wishing they had a friend or family member who could go to the doctor with them ; and 8.3% reported frequently being tired of feeling on their own when it comes to taking care of their health problems .", "fewer respondents ( 5.6% ) reported frequently feeling their doctor does not realize what it is really like for them at home trying to take care of their health problems or ( 5.2% ) frequently leaving the hospital or doctor 's office feeling confused about what they should do . on average", "conversely , when comparing frustrations by whether the participant reported needing help learning how to better care for their health problems , those needing help reported significantly higher scores on the healthcare - related frustration scale ( t = 4.79 , p < 0.001 ) and higher frustration levels for five of the six individual scale items ( t = 25.94 , p < 0.001 ) .  ", "perceived physician support scale characteristics are presented in table 3 . of those who reported receiving physician support ,", "50.9% reported their physician always helped them get an appointment they needed ; 46.5% reported their physician always asked if they understood their medications when their doctor prescribed them ; 15.7% reported their physician always talked to other doctors and nurses who were taking care of them ; and another 15.7% reported their physician always made plans to contact them after a visit to see how they were doing .", "fewer respondents ( 13.3% ) reported their physician always told them about other people who could help them with their health problems or ( 13.2% ) their physician always asked for their ideas about how they can take care of their health problems . on average , participants scored 18.70 ( 5.84 ) on the perceived physician support scale .", "conversely , when comparing support by whether the participant reported needing help learning how to better care for their health problems , a significantly smaller proportion of those needing help reported asked for their ideas about how they can take care of their health problems (  = 10.48 , p = 0.033 ) . \n table 4 displays the results of the logistic regression analysis explaining factors associated with participants reporting they need help learning how to better care for their health problems ( i.e. , not needing help served as the referent group ) .", "participants who were non - white were significantly more likely to report needing help learning how to better care for their health problems ( compared to non - hispanic whites , or = 2.26 , p = 0.049 ) , whereas those with some college or more were significantly less likely to report needing help learning how to better care for their health problems ( compared to those with high school or less education , or = 0.55 , p = 0.044 ) .", "compared to participants with the highest level ( tertile ) of healthcare - related frustrations , those with middle ( or = 0.17 , p < 0.001 ) and lowest ( or = 0.44 , p = 0.017 ) frustration levels were significantly less likely to report needing help learning how to better care for their health problems , respectively . compared to participants with the lowest level ( tertile ) of perceived physician support , those with the highest level of perceived support were significantly less likely to report needing help learning how to better care for their health problems ( or = 0.49 , p = 0.033 ) ."], ["despite our concerns that the majority of older women would likely experience self - care problems [ 4850 ] , our analyses revealed that only about one - third of the women in our sample reported needing help learning how to take better care of their chronic conditions . but", "consistent with health disparities in the epidemiology of chronic illnesses [ 22 , 24 ] , persistent health disparities related to self - care behavior were noted among minorities and those with less education .", "the rural healthcare disparity often reported in other studies [ 25 , 5153 ] was not observed in this study , nor were unmarried women disadvantaged relative to their married counterparts .", "these findings suggest middle - aged and older women in this sample were comparable in terms of having a variety of social supports for learning how to take care of their health .   additionally , despite the previous assumption that older women might be disadvantaged relative to younger baby - boomers , no significant differences in help needed based on age group were reported .", "similarly , our proxy measures for disease magnitude and severity ( i.e. , number of medications , physician visits , and limitations in activities ) did not differentiate those needing help .", "however , the existence of an age - based digital divide was observed [ 5456 ] , with older women less likely to use online / technology as a resource for chronic condition self - management .", "further investigation is needed to examine differences in the types of methods / strategies in which these older women engage when caring for their health conditions outside of the healthcare setting .  ", "this study helps elucidate the complex relationships among contextual factors , healthcare frustrations , and patient - provider interactions and support and points to potential opportunities for intervention .", "the lack of a significant relationship between age and healthcare - related frustrations or perceptions about physician support may be attributed to the fact that the sample was selected based on the presence of chronic conditions .", "however , the lack of a significant relationship is consistent with the fact that participation in community - based disease self - management programs is not limited to those of only older ages ; rather , program enrollment is based on the participant 's chronic disease status .  ", "this study reveals two strong modifiable correlates of women needing help learning how to care for their chronic conditions , even after controlling for sociodemographic and health status indicators in multivariate analyses : healthcare - related frustrations and perceived physician support .", "the majority of identified frustrations were significantly related to middle - aged and older women 's perceptions of needing help learning how to care for their health , which is supported in other studies [ 8 , 16 , 58 , 59 ] .", "the recent emphasis on patient - centered care and medical homes   is designed to help reduce such frustrations and hence can be expected to help boost women 's self - efficacy to care for their own health conditions .", "additionally , perceived physician support was another significant factor for knowing how to self - manage chronic conditions , especially in terms of patient activation as exemplified by wanting physicians to  ask for your ideas about how you can take care of your health problems .", " this reinforces previous research about the importance of perceived physician support for motivating patients to engage in healthier lifestyles and recommended medical regimens [ 9 , 31 , 61 , 62 ] . from the health professional point of view , fostering beliefs of patient support can be accomplished through continuing education units ( ceus ) or expanded emphasis during medical school training to develop strategies in which clinicians can engage and implement to be supportive , listen to their patients , and solicit their patients ' thoughts so they have an active role in their healthcare team [ 29 , 63 ] . from the patient point of view , widely available evidence - based self - management programs include elements within their curricula to teach older women how they can more effectively communicate with their healthcare providers . considering perspectives from each side of the healthcare interaction", "is essential to improve self - management both within and outside of the healthcare setting , which has implications for reducing disease mismanagement , unnecessary healthcare utilization ( e.g. , emergency room use ) , and medical costs ."], ["this secondary dataset did not contain all variables necessary to fully contextualize barriers and challenges associated with chronic disease self - care behavior among this aging population .", "however , this national study contained many variables of interest to address the current research gaps in knowledge about associations of healthcare frustrations , physician support , and self - care needs to chronic disease management .  ", "substantial numbers of participants were lost due to incomplete data on particular scale items , resulting in an analytical sample that was systematically different from the full sample ( e.g. , younger , married , and more white ) and potentially limited the generalizability of study findings .", "this is especially true in that the reduction of cases in the analytic sample reduced the potential proportion of older adults in the study from 42.1% to 33.4% , which may especially influence generalizability of findings to populations aged 65 years and older .", "study participants reported a variety of chronic conditions , but subanalyses based on disease type were not performed because the sample size was inadequate to make such comparisons . recognizing that needed self - care behavior may differ based on women 's particular condition diagnoses , disease stage , and the number of comorbidities in which they are diagnosed , future studies should strive to compare about barriers to self - care , perceived physician support , and frustrations with the healthcare system by their chronic condition profile .", "further , because women 's ability to cope with and adjust to their disease self - care may differ based on their available resources and socioeconomic status , future studies should examine such variables to determine their relationship with self - care disparities .", "another study limitation reducing the ability to widely generalize findings to the greater female community was the self - report and cross - sectional nature of these data .", "however , the sample was derived from random digit dialing and included items that deeply explore the challenges and frustrations with chronic condition self - management , which are not typically seen in other studies that investigate correlations between self - care behavior and other healthcare or physical health indicators .", "thus , this study contributes to a fuller understanding of the complex interrelationships that exist between self - care strategies , provider - patient interactions , and policies / programs in community contexts ."], ["the current study adds to the existing science base by examining barriers to self - care with a new lens , exploring healthcare - related frustrations and perceptions of physician support , and how these perceptions relate to various life domains , including diverse health status and sociodemographic contexts . identifying these common and unique challenges and correlates of these challenges , with a representative national population ,", "advance our current knowledge about self - care issues among middle - aged and older women .", "learning more about healthcare - related frustrations of and self - management supports utilized by middle - aged and older women has the potential to help program deliverers , healthcare providers , and health agencies provide the services and resources that women with chronic conditions want and think are helpful .", "findings from this investigation has potential to inform and guide modifications in the implementation and dissemination of evidence - based programs for older women to better match individuals with programs that meet their needs ."]]}
{"article_id": "PMC4297883", "article_text": ["patients with schizophrenia ( scz ) exhibit a wide variety of cognitive deficits , particularly with memory ( barch and ceaser , 2012 ; ranganath et al . , 2008 ) .", "these deficits are predictive of overall functional outcome and clinical remission ( bodnar et al . , 2008 ; green , 2006 ; green et al . , 2004 ; kahn and keefe , 2013 ) , and it has recently been suggested that schizophrenia should be viewed primarily as a cognitive disorder ( kahn and keefe , 2013 ) .", "understanding the nature of the memory deficits is therefore a critical goal when moving towards improved clinical interventions in scz .", "when considering episodic memory , one area in which patients with scz have a substantial deficit is with source monitoring ( identifying the context in which a stimulus was encountered ; johnson et al . , 1993 ) .", "for successful source monitoring it is necessary to bind elements of a memory together into a memory trace , along with their context ( spatial context , temporal context , etc . ) .", "patients demonstrate source monitoring deficits even when stimulus recognition is preserved ( danion et al . , 1999 ;", "this deficit in source monitoring is in many ways not surprising given that patients with scz also demonstrate difficulties with relational or associative memory ( binding items together during memory encoding , and later recalling which stimuli were presented together ) , while object memory is not as severely impaired ( achim et al .", "most typically , source monitoring problems in scz are considered in the context of attributing events from internal ( self ) to external sources .", "patients with predominant hallucinations and thought disorder have a greater tendency or bias to attribute internally generated events to an external source , while still being able to correctly identify externally generated stimuli ( brunelin et al .", "this bias for scz to misattribute internal sources has been observed in the absence of recognition memory deficits or false positive responses ( fisher et al . , 2008 ) .", "interestingly , in a repetitive magnetic stimulation trial of low frequency ( 1  hz , inhibitory ) stimulation to the left temporal parietal junction over 5 consecutive days not only improved auditory hallucinations but also resulted in an improvement in source monitoring compared to sham stimulation ( brunelin et al . , 2006 ) .", "the improvement in auditory hallucination was marginally correlated with the improvement in source monitoring , further suggesting the relationship between source monitoring and hallucinations .", "patients with schizophrenia have also been found to have a deficit in source memory ( remembering the context in which experimental stimuli occurred ; brebion et al . , 2002 ;", "the deficit in source memory has been related to deficits in binding contextual cues together into a holistic memory representation ( diaz - asper et al . , 2008 ; waters et al . , 2004 ) , which is an essential component of source memory .", "source memory errors are present in scz even for short - term recognition ( when source information is tested immediately , minimizing the need to recollect information stored in long - term memory ) , suggesting that source memory deficits in scz may be related to encoding errors rather than problems in recognition ( achim et al . , 2011 ) .", "( 2009 ) performed a list learning task in scz and found that patients who hallucinate had more intrusions ( indicating a word was part of the memory set when it was not ) than non - hallucinating patients .", "this finding was related to source misattribution ( patients attributing an internally generated stimulus to an external source , the original memory set ) .", "overall , scz appears to have a noteworthy deficit in source memory which is likely intricately related to other memory and cognitive processes , such as contextual binding and episodic memory . within healthy controls ,", "source memory involves a range of cortical regions known to be involved in episodic memory , including the medial temporal lobes , prefrontal cortex , and parietal cortex . increased hippocampal activity has been related to trials in which the source was correctly identified ( davachi et al . , 2003 ; ranganath et al . ,", "2004 ) , probably due to the role of the hippocampus in relational binding ( davachi , 2006 ) .", "one of the first fmri studies to examine source memory found greater left prefrontal activity for source memory and for old  new recognition ( nolde et al . , 1998 ) , with subsequent studies finding activity in the left lateral prefrontal cortex associated with source memory for a variety of stimuli types ( mitchell and johnson , 2009 ) .", "prefrontal lesions have been found to disrupt the self - initiation of processes which promote feature binding ( stuss and benson , 1986 ) , and left prefrontal damage is associated with deficits in source monitoring ( duarte et al . , 2005 ) .", "prefrontal activity during source recognition may be more involved in the evaluation of source information ( e.g.  does this stimuli fit with source x  ) rather than retrieving source information per se ( mitchell et al . ,", "activity in medial parietal areas ( intraparietal sulcus and precuneus ) has been suggested to be present regardless of the type of source information being assessed ( uncapher et al . , 2006 ) , while activity in lateral parietal areas may be more dependent on how well the information has been encoded ( wheeler and buckner , 2004 ) and/or to attentional processes ( cabeza , 2008 ) . examining declines in source memory with age", "has proven fruitful for examining structural and functional correlates of source memory , with evidence that age - related decline in source memory is related to decreased activity mainly in the prefrontal and medial temporal lobes ( see mitchell and johnson , 2009 , for review ) .", "( 2006 ) examined source monitoring in scz using a level - of - processing framework .", "patients were presented words with either deep ( semantic ) or shallow ( orthographic ) encoding instructions . during recognition , when participants successfully identified a previously encountered stimuli they were asked under which encoding condition the word was encountered ( the source memory aspect being recalling the context in which the word was encountered , in this case , which encoding condition ) . when contrasting correct vs. incorrect source , both patients and controls activated areas of prefrontal and parietal cortices .", "patients showed activity in the middle and superior temporal gyrus , thalamus , and parahippocampal gyrus , which was correlated with more severe positive and negative symptoms independent of memory performance .", "other neuroimaging studies of source monitoring in scz have focused on deficits related to attributing stimuli as self - generated or externally - generated ( reality monitoring ) .", "deficits in reality monitoring appear to involve the medial prefrontal cortex ( subramaniam et al . , 2012 ; vinogradov et al . , 2008 ; wang et al . , 2011 ) . following computerized training to improve source monitoring ,", "activity was found to be increased in the medial prefrontal cortex ( subramaniam et al . , 2012 ) .", "the purpose of this study was to examine the neural correlates of source memory in schizophrenia . while most previous studies of source memory ( in both controls and schizophrenia ) have used less ecologically valid task ( e.g. identifying the color of the stimuli during encoding ) , we utilized a paradigm involving encounters ( with a person and an object in a specific place ) within a realistic 3d environment ( burgess et al . , 2001 ; king et al . , 2005 ) , which may better evaluate source memory networks used in everyday life .", "we examined participants with early schizophrenia ( within the first 4.5  years of treatment , with 75% of patients within the first 2  years ) thus minimizing potential confounds associated with prolonged illness such as cognitive decline , social isolation and long - term medication effects . during", "fmri scanning a source recognition memory task was employed , which was contrasted with an object memory task . by directly comparing source memory to object memory", ", we can identify regions in the cortex which are specific to source memory compared to object memory and determine if any deficits observed in schizophrenia are source - memory specific .", "we hypothesized that patients would show deficits in source memory relative to object memory and may show compensatory activation in source memory retrieval contrasts .", "furthermore , as deficits in source memory have been associated with auditory hallucinations ( woodward et al . , 2007 )", "we expected to observe relationships between hallucinations and the neural activity of source memory retrieval .", "all participants with scz were treated at the douglas mental health university institute in montreal , canada , at the prevention and early intervention program for psychoses , a specialized service providing treatment to individuals aged 1435  years from a local catchment area .", "individuals with an iq  >  70 who had not taken antipsychotic medication for more than 1  month were consecutively admitted as in- or out - patients .", "patients were assessed with the scale for assessment of positive symptoms ( saps ) ( andreasen , 1984 ) and the scale for assessment of negative symptoms ( sans ) ( andreasen , 1983 ) at numerous time - points following clinic admission ( baseline ; at 1 , 2 , 3 , 6 , 9 , and 12  months ; and every 6  months thereafter ) .", "( 2003 ) or visit http://www.douglas.qc.ca/pages/view?section_id=165 for more details . for the neuroimaging study , only individuals aged 1830  years with no previous history of neurological disease , head trauma causing loss of consciousness , or lifetime diagnosis of substance dependence", "twenty - five people with schizophrenia spectrum disorders were recruited , diagnosed according to the structured clinical interview for dsm - iv ( first et al . , 1997 ) and confirmed between two senior research psychiatrists ( a.m. and r.j . ) .", "twenty - four healthy controls were recruited through advertisements in local newspapers and were included only if they had no current or previous history of any axis i disorders , neurological diseases , or head trauma causing loss of consciousness , and no first - degree family members with schizophrenia or related schizophrenia - spectrum disorders .", "all patients provided written informed consent , and the study was approved by the research ethics boards of the douglas hospital research centre and the montreal neurological institute .", "participants performed an encoding task ( outside the mri ) using a modified version of the virtual city developed by burgess and colleagues ( burgess et al .", ", 2001 ; king et al . , 2005 ) , created using 3ds max ( 3ds max , 2011 ) and unity software ( unity , 2011 ) .", "participants navigated through a 3d virtual city , following a path indicated by green arrows to the site of an encounter ( an encoding trial , a character at a location ) . after approaching within five virtual meters of the character , the participant 's view was frozen and the character stepped aside , and a life - sized object appeared on a small table displayed to the right .", "participants were instructed to pay careful attention to the object , character and location , and try to remember for a later memory test .", "after a 5  s study delay the person and object disappeared , and participants then followed the arrows to the next encounter .", "there were a total of 20 encounters , each with a unique person , location and object .", "a total of 80 recognition trials were administered , in which participants were shown an image consisting of a typical viewpoint encountered within the virtual city ( in one of 20 places where encounters occurred ) , with a person in the center of the screen , and two objects , one on each side of the person .", "participants were then asked one of four possible recognition questions : ( 1 ) person ( which object was paired with this person ) , ( 2 ) place ( which object did you view in this location ) , ( 3 ) object ( which object was viewed in the city ; the other object was new ) , and ( 4 ) bright ( which object is brighter in appearance ) .", "participants responded on an mri - compatible button box to indicate which item ( left or right ) was selected .", "for the person condition , the place was not associated with either object , while in the place condition the person was not associated with either object .", "the person and place conditions were designed to access source memory ( in what context was an object encountered ) while the object condition assesses object memory ( old vs. new ) .", "including two source memory conditions allows us to better understand if the observed activity is modality specific .", "the bright condition was not considered in this analysis as we were specifically interested in source vs. object memory .", "images ( with encoding question ) were presented on the screen for 8000  ms , with a 20008000  ms isi ( in 100  ms increments ) , with an average trial length of 13  s. stimuli were presented and results were recorded through e - prime 1.0 software .", "1 . a practice route was designed to allow participants to become familiar with the arrow key and mouse , and to practice following the arrows and encounter two characters with objects in independent locales .", "participants also practiced answering two of each of the four forced - choice recognition questions regarding the objects collected .", "echo - planar images were collected on a siemens 3  t tim trio mri ( tr  =  2000  ms , te  =  30  ms , flip angle  =  90 , 36 slices of 4  mm thick , 64    64  voxel plane with an fov of 256  mm 4  mm slices ) .", "each bold run was preceded by 4 volumes that were later discarded to allow a magnetic steady state .", "the anatomical scan was an mprage ( tr  =  2300  ms , te  =  2.98  ms , fov 256 , 1    1    1  mm  voxels , flip angle  =  9 ) and lasted 5.21  min .", "data analysis was conducted using spm 8 ( wellcome department of cognitive neurology , london , uk ) .", "data was motion corrected by realigning to the 3rd tr , normalized to the icbm template ( and resampled at 2    2    2  mm  voxel size ) and smoothed with an 8  mm isotropic gaussian kernel . the general linear model was implemented by convolving a standard hemodynamic response function and its first temporal derivative and dispersion .", "events were defined based upon recognition question ( person , place , object , or bright ) , with incorrect answers modeled as distinct event types and excluded from further analysis .", "accuracy data was not available for three controls and four scz patients due to technical problems during initial data collection . for these participants ,", "contrasts were person vs. object and place vs. object ( both performed bidirectionally ) to identify voxels which are differentially activated by source or object memory .", "a second level analysis was performed separately for each group ( controls or scz ) using a one sample t - test .", "corrections for multiple comparisons were performed at the cluster level using an individual voxel threshold of p  <  0.001 ( uncorrected ) . a monte - carlo simulation of 1000 iterations ( slotnick et al", ". , 2003 ) resulted in a cluster extent threshold of 49 resampled voxels . to test for differences between groups ,", "an independent - samples t - test was performed , using the contrast  value derived for each participant from the above contrasts .", "given that between - group differences often have smaller effect sizes , we used a slightly more liberal single - voxel threshold of p  <  0.005 , but corrected to p  <  0.01 at the cluster level ( resulting in an extent threshold of 97  voxels ) . a regression analysis in patients was performed to examine the relationship between source memory and clinical symptoms .", "as patients were stabilized and undergoing treatment at the time of scanning , few patients were actively experiencing positive symptoms as of the assessment closest to the date of scanning . as a result", ", the data did not possess sufficient variability for a regression analysis ( the majority of cases had global scores of 0 or 1 on the saps at closest assessment ) .", "furthermore , positive symptoms are often highly responsive to treatment ( malla et al . , 2006 ; robinson et al .", ", 1999 ) . as such any patient displaying continued positive symptoms may represent treatment resistant patients or those with a more severe illness , rather than relate to the symptoms themselves per se .", "instead , we utilized scores from the assessment at the initial visit to the clinic , prior to treatment onset .", "the presence of specific symptoms at initial clinical assessment was considered as a proxy of how prone to those symptoms each patient may be .", "we propose that the pre - treatment ratings best represent the underlying neurobiology and clinical characteristics of individual patients , as they show their symptom characteristics in an untreated state of illness .", "while it is not possible to make several such assessments prior to treatment onset ( which would best capture the potential symptom profile of each patient ) , such an approach may allow for a data exploration which separates patients who are prone to certain symptoms ( such as hallucinations ) from those who have experienced less or never experienced those symptoms while in an untreated state .", "as such , this can be conceptualized as a  trait based approach to symptom evaluation . saps scores for global hallucinations and global thought disorder were entered into a whole - brain regression model with the person  >  object and place  >  object contrasts . the global delusion score was not included as most patients were highly delusional at baseline . in order to account for differences in time since initial diagnosis ,", "the interval between baseline assessment and mri scan ( in days ) was entered as a covariate in the second - level regression analysis .", "cluster threshold for the regression was 49  voxels at p  <  0.001 uncorrected ( voxel threshold ) .", "there were no significant differences in age or gender distribution between groups , though patients had a marginally significant lower parental ses and significantly fewer years of education .", "accuracy in the person , place and object conditions was assessed using independent samples t - test .", "schizophrenia patients had significantly lower accuracy in the person condition , t(40 )  =  2.247 , p  =  0.03 , but not for place , t(40 )  =  0.44 , p  =  0.66 , or object , t(40 )  =  0.07 , p  =  0.95 .", "numerous cortical regions showed significant increases in neural activity for retrieval of source memory over retrieval of object memory , similar to the pattern observed in previous studies using a similar paradigm ( burgess et al .", ", 2001 ; king et al . , 2005 ) . activated regions in the source memory contrasts ( person  >  object and place  >  object ) included bilateral activity around the parietal occipital sulcus ( including the precuneus and retrosplenial cortex ) extending into the superior parietal cortex , left inferior frontal gyrus ( vlpfc ) , fusiform gyrus bilaterally , and the occipital cortex .", "activity in the person  >  object contrast included the head of the caudate nucleus , the right inferior frontal ( vlpfc ) , and the medial aspect of the superior frontal gyrus .", "object  >  place showed widespread activity in the medial frontal cortex and parietal cortices ( supramarginal gyrus bilaterally ) and smaller clusters in the frontal and occipital cortices .", "for the object  >  person contrast , activity was observed in the left and right angular gyrus .", "2 . when examining source vs. object retrieval contrasts in scz , a smaller number of significantly activated voxels were observed relative to the activity maps of controls . in the person ", ">  object contrast only smaller clusters in the head of the caudate and occipital cortex were significant .", "in place  >  object , mainly posterior activity was observed ( e.g. parietal occipital sulcus and fusiform cortex ) . in both", "the object  >  person and object  >  place , scz showed a right inferior frontal ( vlpfc ) activity which was not observed in controls .", "2 . when comparing between groups , controls demonstrated regions of greater differences between conditions than scz for person  >  object condition ( including bilaterally in the precuneus and superior parietal , and left and right inferior frontal in the vlpfc ) and for place  >  object ( bilaterally in the superior parietal , and the left precuneus ) .", "schizophrenia patients had greater differences between conditions than controls when considering place  >  object across a wide range of areas .", "the group comparison was run using contrast  values , which can be positive ( e.g. if place  >  object ) or negative ( e.g. if object  >  place ) .", "thus , while we observed greater activity in scz than control in the place  >  object contrast , it is not clear from the activation maps if such a difference is due to changes in activity in either object or place . in order to visualize the results for each condition ,", "beta values were extracted for an roi of 11  voxels ( 9 in - plane voxels surrounding the selected voxel and one above and one below ) .", "rois were selected to represent a range of representative patterns of activity across the brain ( e.g. regions in which controls showed differences from scz in both person  >  object and place  >  object , and regions in which scz showed  greater  activity in place  >  object ) .", "which controls had greater differences between conditions than scz , controls are showing increased activity to the source memory conditions ( person and/or place ) relative to object , while  values in scz do not show any such differentiation .", "interestingly , in regions in which we observed  greater  differences between conditions ( in this case , place  >  object ) in scz than controls , we again observe little differences between source memory and object memory in scz .", "thus , it is not that patients with scz are showing greater activity in the place condition , but that they are failing to modulate brain activity in the same way as control participants .", "results of the regression analysis with positive symptoms are presented in table 6 and fig .", "we observed significant negative correlations with the global hallucination score from the saps ( at first assessment ) and place  >  object contrast , in the right midtemporal gyrus , left prefrontal cortex , and right cerebellum ( in a region noted in at least one lesional case study to be associated with source memory deficits ; tamagni et al . ,", "2010 ) . in order to examine the relationship between recognition memory performance and neural activity , spearman 's correlations were run on global hallucination score , memory performance in the place and object conditions , and  values from the three clusters .", "while hallucinations did not significantly correlate with performance in either condition ( place , rho  =  .228 , p  =  0.32 ; object , rho  =  0.28 , p  =  0.22 ) , there was a marginally significant correlation between performance in the object condition and  value in the dlpfc cluster ( rho  =  0.38 , p  =  0.088 ) .", "this study examined differences in the neural correlates of source memory in patients with schizophrenia and controls .", "we examined this issue using a virtual reality paradigm , which has improved ecological validity when assessing the  source  of a memory , as compared to other studies which have used less ecologically valid tasks .", "furthermore , we examined a group of patients , who were within the first 4.5  years of treatment avoiding potential issues associated with illness chronicity such as cognitive decline , prolonged exposure to medications ( although medication exposure remains an issue ) , social isolation , and sedentary lifestyle ( pelletier et al . , 2005 ) . while patients with scz demonstrated some activation in the source memory contrasts , the extent and magnitude of activity were substantially less than those in controls . even in regions in which the difference appeared to be in the directions of scz  >  controls", ", it seems to be the case that controls differentiate between source and object memory while patients do not .", "as discussed below , this finding may be true of other forms of memory , and may therefore be representative of the underlying deficit across a range of memory subtypes .", "more specifically , patients with scz may fail to activate cortical regions which facilitate elaborative memory processes .", "this is consistent with previous findings of relatively intact object memory in scz ( achim et al .", "2009 ) , but deficits in source memory ( johnson et al . , 1993 ) and associative memory ( achim and lepage , 2003 ) .", "it may be that the vr environment minimizes these behavioral differences , in that the place condition may be easier than person as locations were more distinct from each other relative to the 3d rendered characters .", "alternatively , our recent - onset sample may have better preserved function than the more enduring schizophrenia samples often examined . however , this lack of behavioral difference has a positive aspect , in that it removes performance as a potentially major confound in the fmri analysis . had patients with schizophrenia demonstrated profound deficits in performance , it would beg the question if any observed neural activity differences were due to disease pathology or simply related to poor performance ( and as such would be similar to poor performing healthy controls ) .", "we did observe a performance difference in the person condition , and interestingly very little significant activity in the schizophrenia group for person  >  object .", "however , some activations observed in the group analysis , particularly in the posterior regions , were present in both person  >  object and place  >  object , suggesting that these differences were not at all modality specific .", "negative correlations were observed between the difference in neural activity in place  >  object and global hallucinations , measured at intake baseline using the saps .", "this suggests that the propensity of an individual to experience hallucination may modulate differences between source and object memory in these areas .", "we did not observe significant correlations with global thought disorder , which is not surprising as hallucinations but not thought disorder are generally associated with source memory ( woodward et al . , 2007 ) .", "while these regions did not directly overlap activity observed in the healthy control group , the left frontal cluster was proximal to significant activity in place  >  object in controls , while the middle - temporal cluster was proximal to significant voxels in the object  >  place clusters in controls , suggesting that these regions are at least similar to those observed in healthy controls .", "however , the lack of direct overlap and given the nature of the result ( greater difference with more hallucinations ) , it is possible that these regions represent malfunctioning cortical regions which are more active in patients who have experienced hallucinations as part of their disorder .", "this over - activation within these regions when considering source information may play an important role in generating hallucinations , which are essentially the misattribution of internally generated stimuli to an external source .", "at least one study has found relationships between the left prefrontal cortex and the right temporal cortex while patients are actively experiencing hallucinations ( hoffman et al . , 2011 ) , and", "a meta - analysis suggests that these regions among others are frequently active during hallucination ( jardri et al . , 2011 ) . as such , the regions found to be significantly active in our regression are consistent with the existing literature on the neurobiology of hallucinations .", "while many studies examining hallucinations have utilized either general  state based measurements ( approximately how much the participant is hallucinating in general at the time ) or direct analysis of overall activity during hallucinations , we have attempted to utilize a ", "our sample of patients early in treatment makes such an approach possible with minimal confounding for chronicity ( which we attempted to control for by including length of treatment as a covariate in the regression ) .", "our results overall suggest a relationship between mal - adaptive neural activity related to source memory retrieval and how prone participants are to hallucinatory symptoms , further verifying the relationship between source memory and hallucinations ( woodward et al . , 2007 ) . however , some limitations must be considered as well .", "while we have attempted to use baseline scores as a  trait measure of how prone an individual is to hallucinations , it is well known that clinical symptoms can vary across time . as such , the appropriate method for evaluating ", "trait symptoms is to take repeated symptom measurements over time ( mathalon and ford , 2012 ) .", "it would be preferable to have several assessments of pre - treatment symptoms to have a complete picture of the symptom profile of a given patient , but this is not possible as it would require delaying treatment . as such we utilized the best available measure to assess pre - treatment symptom profile , which produced results which are consistent with the existing literature on those symptoms .", "however , it is important to remain considerate of the limitations of our measures when considering these results .", "source memory can be considered a form of associative / relational memory , as participants are binding elements together during encoding and storing these elements together as part of a larger whole .", "a deficit in source memory can be viewed as a failure to associate a memory item with its context . in the case of this study in particular ,", "our source memory paradigm is not far removed from studies examining associative memory ( achim et al . , 2007 ; achim and lepage , 2005 ; murray and ranganath , 2007 ) , which is known to be more impaired in scz than object memory ( achim and lepage , 2003 ) .", "some studies have suggested that patients with schizophrenia do not properly differentiate between associative and item memory ( achim et al .", "2012 ) , in keeping with suggestions that source memory impairments are part of associative memory impairments ( achim et al . , 2011 ) .", "our present study focused on source retrieval , and as such we can not definitively determine if the differences observed in our contrasts are driven by deficits in encoding ( in that the information was not properly moved into memory ) or retrieval ( in that the memories may have been encoded but are not properly accessed ) , or a combination of both ( which seems likely given the plurality of evidence for memory deficits in scz ) .", "( 2011 ) found deficits in source memory even when using short term recall , minimizing the need for retrieval of information from long - term memory .", "this suggests that at least part of the source memory deficit in scz is related to problems with encoding .", "our finding of an overall pattern of lack of differentiation between conditions may be a fairly consistent finding in the cognitive neuroscience of memory in scz , regardless if one considers activity during encoding or retrieval / recognition .", "core regions required for task performance may be relativity intact in scz , while deficits in activity will be observed in ", "that is to say , the deficits in memory ( and possibly cognition in general ) are related to a lack of engagement of extended cortical regions .", "these extended regions are not critically required for minimal task performance , but instead serve to enhance performance ( such as regions involved in cognitive control processes ) .", "healthy controls will tend to utilize such areas automatically , while patients with schizophrenia will fail to do so .", "for example , patients with scz have been shown to have impairments in initiating elaborative encoding processes which may be beneficial during associative encoding ( brebion et al .", "however , when patients are specifically instructed to utilize effective encoding strategies they show an improvement in memory performance , demonstrating that patients with scz can utilize such encoding strategies when specifically instructed but fail to do so spontaneously ( brebion et al . , 1997 ) .", "this pattern of results is similar to that in patients with prefrontal cortical lesions ( alexander et al . , 2003 ) .", "bonner - jackson et al . ( 2008 ) examined memory strategies in schizophrenia by contrasting intentional but unstructured encoding ( simply instructing participant to memorize words ) with an externally imposed deep encoding strategy ( having participants perform an abstract / concrete judgement on words , deep semantic encoding which facilitates memory encoding ; craik and lockhart , 1972 ) .", "they observed group    encoding interactions in several regions , including the left inferior frontal gyrus and precuneus , with the most common finding being a difference between controls and scz in the incidental , unstructured encoding condition .", "for example , in the left inferior frontal cortex , scz patients showed no activity for unstructured encoding , but substantial activity for deep encoding .", "that is to say , when scz patients were provided a structured encoding strategy they activated this region , but failed to do so spontaneously .", "likewise , controls showed significantly better memory performance than scz patients for the deep encoding condition .", "further support for the importance in strategic and/or cognitive control comes from findings of changes and/or normalization of activity in scz following cognitive training ( hooker et al . , 2012 ; penades et al . ,", "within both encoding and recognition studies in schizophrenia , the most prevalent finding is a decrease in the extent or magnitude of activity in scz patients relative to controls , although many studies also report findings in the direction of scz  >  controls ( ragland et al . , 2009 ) .", "while it is likely that scz often shows compensatory networks or inefficient over - activation during cognitive tasks , it can be difficult to judge from many of the published studies on cognition in scz .", "many papers report differences in group activities without also reporting the parameter estimates which accompany those changes .", "if we had done so in this study ( by only presenting the activation maps in fig .", "3 and not the beta values ) we may have concluded that scz showed greater activity in some regions for place vs. object and concluded that this was compensatory of maladaptive over - activation . however , when examining the beta values , we realize that this is not the case but instead these are regions where controls show greater activity for object over place ( often in the form of a decrease in activity for place ) .", "this highlights the importance of carefully examining parameter estimates when performing between - group comparisons .", "it is possible that in at least some cases in the existing literature , the so - called ", "compensatory activity may instead be a lack of activity relative to the control group , such as was the case in this study .", "such ambiguity and misinterpretation can be avoided if studies fully report parameter estimates for contrasts which differ between groups .", "we have reported the results of a study looking at source memory retrieval in scz using a virtual reality environment on a group of patients relatively early into treatment .", "while controls activated a large , extensive network for source relative to object retrieval , scz showed a marked reduction in activity .", "this reduction was borne out in the group comparison , and appears to be related to the fact that patients with scz are failing to activate these regions or differentiate between object and source retrieval . despite the large - scale group differences , patients were still able to perform the source retrieval tasks , suggesting that at least some of the  core system involved in source retrieval is intact . instead , we propose that the observed differences are related to  supporting regions which are not critical to task performance but instead facilitate source retrieval .", "that is , patients may generally fail to engage extended cortical networks which facilitate task performance and facilitate overall cognitive functioning ."], "abstract_text": ["<S> source memory , the ability to identify the context in which a memory occurred , is impaired in schizophrenia and has been related to clinical symptoms such as hallucinations . </S>", "<S> the neurobiological underpinnings of this deficit are not well understood . </S>", "<S> twenty - five patients with recent onset schizophrenia ( within the first 4.5  years of treatment ) and twenty - four healthy controls completed a source memory task . </S>", "<S> participants navigated through a 3d virtual city , and had 20 encounters of an object with a person at a place . </S>", "<S> functional magnetic resonance imaging was performed during a subsequent forced - choice recognition test . </S>", "<S> two objects were presented and participants were asked to either identify which object was seen ( new vs. old object recognition ) , or identify which of the two old objects was associated with either the person or the place being presented ( source memory recognition ) . source memory was examined by contrasting person or place with object . </S>", "<S> both patients and controls demonstrated significant neural activity to source memory relative to object memory , though activity in controls was much more widespread . </S>", "<S> group differences were observed in several regions , including the medial parietal and cingulate cortex , lateral frontal lobes and right superior temporal gyrus . </S>", "<S> patients with schizophrenia did not differentiate between source and object memory in these regions . </S>", "<S> positive correlations with hallucination proneness were observed in the left frontal and right middle temporal cortices and cerebellum . </S>", "<S> patients with schizophrenia have a deficit in the neural circuits which facilitate source memory , which may underlie both the deficits in this domain and be related to auditory hallucinations . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusions"], "sections": [["patients with schizophrenia ( scz ) exhibit a wide variety of cognitive deficits , particularly with memory ( barch and ceaser , 2012 ; ranganath et al . , 2008 ) .", "these deficits are predictive of overall functional outcome and clinical remission ( bodnar et al . , 2008 ; green , 2006 ; green et al . , 2004 ; kahn and keefe , 2013 ) , and it has recently been suggested that schizophrenia should be viewed primarily as a cognitive disorder ( kahn and keefe , 2013 ) .", "understanding the nature of the memory deficits is therefore a critical goal when moving towards improved clinical interventions in scz .", "when considering episodic memory , one area in which patients with scz have a substantial deficit is with source monitoring ( identifying the context in which a stimulus was encountered ; johnson et al . , 1993 ) .", "for successful source monitoring it is necessary to bind elements of a memory together into a memory trace , along with their context ( spatial context , temporal context , etc . ) .", "patients demonstrate source monitoring deficits even when stimulus recognition is preserved ( danion et al . , 1999 ;", "this deficit in source monitoring is in many ways not surprising given that patients with scz also demonstrate difficulties with relational or associative memory ( binding items together during memory encoding , and later recalling which stimuli were presented together ) , while object memory is not as severely impaired ( achim et al .", "most typically , source monitoring problems in scz are considered in the context of attributing events from internal ( self ) to external sources .", "patients with predominant hallucinations and thought disorder have a greater tendency or bias to attribute internally generated events to an external source , while still being able to correctly identify externally generated stimuli ( brunelin et al .", "this bias for scz to misattribute internal sources has been observed in the absence of recognition memory deficits or false positive responses ( fisher et al . , 2008 ) .", "interestingly , in a repetitive magnetic stimulation trial of low frequency ( 1  hz , inhibitory ) stimulation to the left temporal parietal junction over 5 consecutive days not only improved auditory hallucinations but also resulted in an improvement in source monitoring compared to sham stimulation ( brunelin et al . , 2006 ) .", "the improvement in auditory hallucination was marginally correlated with the improvement in source monitoring , further suggesting the relationship between source monitoring and hallucinations .", "patients with schizophrenia have also been found to have a deficit in source memory ( remembering the context in which experimental stimuli occurred ; brebion et al . , 2002 ;", "the deficit in source memory has been related to deficits in binding contextual cues together into a holistic memory representation ( diaz - asper et al . , 2008 ; waters et al . , 2004 ) , which is an essential component of source memory .", "source memory errors are present in scz even for short - term recognition ( when source information is tested immediately , minimizing the need to recollect information stored in long - term memory ) , suggesting that source memory deficits in scz may be related to encoding errors rather than problems in recognition ( achim et al . , 2011 ) .", "( 2009 ) performed a list learning task in scz and found that patients who hallucinate had more intrusions ( indicating a word was part of the memory set when it was not ) than non - hallucinating patients .", "this finding was related to source misattribution ( patients attributing an internally generated stimulus to an external source , the original memory set ) .", "overall , scz appears to have a noteworthy deficit in source memory which is likely intricately related to other memory and cognitive processes , such as contextual binding and episodic memory . within healthy controls ,", "source memory involves a range of cortical regions known to be involved in episodic memory , including the medial temporal lobes , prefrontal cortex , and parietal cortex . increased hippocampal activity has been related to trials in which the source was correctly identified ( davachi et al . , 2003 ; ranganath et al . ,", "2004 ) , probably due to the role of the hippocampus in relational binding ( davachi , 2006 ) .", "one of the first fmri studies to examine source memory found greater left prefrontal activity for source memory and for old  new recognition ( nolde et al . , 1998 ) , with subsequent studies finding activity in the left lateral prefrontal cortex associated with source memory for a variety of stimuli types ( mitchell and johnson , 2009 ) .", "prefrontal lesions have been found to disrupt the self - initiation of processes which promote feature binding ( stuss and benson , 1986 ) , and left prefrontal damage is associated with deficits in source monitoring ( duarte et al . , 2005 ) .", "prefrontal activity during source recognition may be more involved in the evaluation of source information ( e.g.  does this stimuli fit with source x  ) rather than retrieving source information per se ( mitchell et al . ,", "activity in medial parietal areas ( intraparietal sulcus and precuneus ) has been suggested to be present regardless of the type of source information being assessed ( uncapher et al . , 2006 ) , while activity in lateral parietal areas may be more dependent on how well the information has been encoded ( wheeler and buckner , 2004 ) and/or to attentional processes ( cabeza , 2008 ) . examining declines in source memory with age", "has proven fruitful for examining structural and functional correlates of source memory , with evidence that age - related decline in source memory is related to decreased activity mainly in the prefrontal and medial temporal lobes ( see mitchell and johnson , 2009 , for review ) .", "( 2006 ) examined source monitoring in scz using a level - of - processing framework .", "patients were presented words with either deep ( semantic ) or shallow ( orthographic ) encoding instructions . during recognition , when participants successfully identified a previously encountered stimuli they were asked under which encoding condition the word was encountered ( the source memory aspect being recalling the context in which the word was encountered , in this case , which encoding condition ) . when contrasting correct vs. incorrect source , both patients and controls activated areas of prefrontal and parietal cortices .", "patients showed activity in the middle and superior temporal gyrus , thalamus , and parahippocampal gyrus , which was correlated with more severe positive and negative symptoms independent of memory performance .", "other neuroimaging studies of source monitoring in scz have focused on deficits related to attributing stimuli as self - generated or externally - generated ( reality monitoring ) .", "deficits in reality monitoring appear to involve the medial prefrontal cortex ( subramaniam et al . , 2012 ; vinogradov et al . , 2008 ; wang et al . , 2011 ) . following computerized training to improve source monitoring ,", "activity was found to be increased in the medial prefrontal cortex ( subramaniam et al . , 2012 ) .", "the purpose of this study was to examine the neural correlates of source memory in schizophrenia . while most previous studies of source memory ( in both controls and schizophrenia ) have used less ecologically valid task ( e.g. identifying the color of the stimuli during encoding ) , we utilized a paradigm involving encounters ( with a person and an object in a specific place ) within a realistic 3d environment ( burgess et al . , 2001 ; king et al . , 2005 ) , which may better evaluate source memory networks used in everyday life .", "we examined participants with early schizophrenia ( within the first 4.5  years of treatment , with 75% of patients within the first 2  years ) thus minimizing potential confounds associated with prolonged illness such as cognitive decline , social isolation and long - term medication effects . during", "fmri scanning a source recognition memory task was employed , which was contrasted with an object memory task . by directly comparing source memory to object memory", ", we can identify regions in the cortex which are specific to source memory compared to object memory and determine if any deficits observed in schizophrenia are source - memory specific .", "we hypothesized that patients would show deficits in source memory relative to object memory and may show compensatory activation in source memory retrieval contrasts .", "furthermore , as deficits in source memory have been associated with auditory hallucinations ( woodward et al . , 2007 )", "we expected to observe relationships between hallucinations and the neural activity of source memory retrieval ."], ["all participants with scz were treated at the douglas mental health university institute in montreal , canada , at the prevention and early intervention program for psychoses , a specialized service providing treatment to individuals aged 1435  years from a local catchment area .", "individuals with an iq  >  70 who had not taken antipsychotic medication for more than 1  month were consecutively admitted as in- or out - patients .", "patients were assessed with the scale for assessment of positive symptoms ( saps ) ( andreasen , 1984 ) and the scale for assessment of negative symptoms ( sans ) ( andreasen , 1983 ) at numerous time - points following clinic admission ( baseline ; at 1 , 2 , 3 , 6 , 9 , and 12  months ; and every 6  months thereafter ) .", "( 2003 ) or visit http://www.douglas.qc.ca/pages/view?section_id=165 for more details . for the neuroimaging study , only individuals aged 1830  years with no previous history of neurological disease , head trauma causing loss of consciousness , or lifetime diagnosis of substance dependence", "twenty - five people with schizophrenia spectrum disorders were recruited , diagnosed according to the structured clinical interview for dsm - iv ( first et al . , 1997 ) and confirmed between two senior research psychiatrists ( a.m. and r.j . ) .", "twenty - four healthy controls were recruited through advertisements in local newspapers and were included only if they had no current or previous history of any axis i disorders , neurological diseases , or head trauma causing loss of consciousness , and no first - degree family members with schizophrenia or related schizophrenia - spectrum disorders .", "all patients provided written informed consent , and the study was approved by the research ethics boards of the douglas hospital research centre and the montreal neurological institute .", "participants performed an encoding task ( outside the mri ) using a modified version of the virtual city developed by burgess and colleagues ( burgess et al .", ", 2001 ; king et al . , 2005 ) , created using 3ds max ( 3ds max , 2011 ) and unity software ( unity , 2011 ) .", "participants navigated through a 3d virtual city , following a path indicated by green arrows to the site of an encounter ( an encoding trial , a character at a location ) . after approaching within five virtual meters of the character , the participant 's view was frozen and the character stepped aside , and a life - sized object appeared on a small table displayed to the right .", "participants were instructed to pay careful attention to the object , character and location , and try to remember for a later memory test .", "after a 5  s study delay the person and object disappeared , and participants then followed the arrows to the next encounter .", "there were a total of 20 encounters , each with a unique person , location and object .", "a total of 80 recognition trials were administered , in which participants were shown an image consisting of a typical viewpoint encountered within the virtual city ( in one of 20 places where encounters occurred ) , with a person in the center of the screen , and two objects , one on each side of the person .", "participants were then asked one of four possible recognition questions : ( 1 ) person ( which object was paired with this person ) , ( 2 ) place ( which object did you view in this location ) , ( 3 ) object ( which object was viewed in the city ; the other object was new ) , and ( 4 ) bright ( which object is brighter in appearance ) .", "participants responded on an mri - compatible button box to indicate which item ( left or right ) was selected .", "for the person condition , the place was not associated with either object , while in the place condition the person was not associated with either object .", "the person and place conditions were designed to access source memory ( in what context was an object encountered ) while the object condition assesses object memory ( old vs. new ) .", "including two source memory conditions allows us to better understand if the observed activity is modality specific .", "the bright condition was not considered in this analysis as we were specifically interested in source vs. object memory .", "images ( with encoding question ) were presented on the screen for 8000  ms , with a 20008000  ms isi ( in 100  ms increments ) , with an average trial length of 13  s. stimuli were presented and results were recorded through e - prime 1.0 software .", "1 . a practice route was designed to allow participants to become familiar with the arrow key and mouse , and to practice following the arrows and encounter two characters with objects in independent locales .", "participants also practiced answering two of each of the four forced - choice recognition questions regarding the objects collected .", "echo - planar images were collected on a siemens 3  t tim trio mri ( tr  =  2000  ms , te  =  30  ms , flip angle  =  90 , 36 slices of 4  mm thick , 64    64  voxel plane with an fov of 256  mm 4  mm slices ) .", "each bold run was preceded by 4 volumes that were later discarded to allow a magnetic steady state .", "the anatomical scan was an mprage ( tr  =  2300  ms , te  =  2.98  ms , fov 256 , 1    1    1  mm  voxels , flip angle  =  9 ) and lasted 5.21  min .", "data analysis was conducted using spm 8 ( wellcome department of cognitive neurology , london , uk ) .", "data was motion corrected by realigning to the 3rd tr , normalized to the icbm template ( and resampled at 2    2    2  mm  voxel size ) and smoothed with an 8  mm isotropic gaussian kernel . the general linear model was implemented by convolving a standard hemodynamic response function and its first temporal derivative and dispersion .", "events were defined based upon recognition question ( person , place , object , or bright ) , with incorrect answers modeled as distinct event types and excluded from further analysis .", "accuracy data was not available for three controls and four scz patients due to technical problems during initial data collection . for these participants ,", "contrasts were person vs. object and place vs. object ( both performed bidirectionally ) to identify voxels which are differentially activated by source or object memory .", "a second level analysis was performed separately for each group ( controls or scz ) using a one sample t - test .", "corrections for multiple comparisons were performed at the cluster level using an individual voxel threshold of p  <  0.001 ( uncorrected ) . a monte - carlo simulation of 1000 iterations ( slotnick et al", ". , 2003 ) resulted in a cluster extent threshold of 49 resampled voxels . to test for differences between groups ,", "an independent - samples t - test was performed , using the contrast  value derived for each participant from the above contrasts .", "given that between - group differences often have smaller effect sizes , we used a slightly more liberal single - voxel threshold of p  <  0.005 , but corrected to p  <  0.01 at the cluster level ( resulting in an extent threshold of 97  voxels ) . a regression analysis in patients was performed to examine the relationship between source memory and clinical symptoms .", "as patients were stabilized and undergoing treatment at the time of scanning , few patients were actively experiencing positive symptoms as of the assessment closest to the date of scanning . as a result", ", the data did not possess sufficient variability for a regression analysis ( the majority of cases had global scores of 0 or 1 on the saps at closest assessment ) .", "furthermore , positive symptoms are often highly responsive to treatment ( malla et al . , 2006 ; robinson et al .", ", 1999 ) . as such any patient displaying continued positive symptoms may represent treatment resistant patients or those with a more severe illness , rather than relate to the symptoms themselves per se .", "instead , we utilized scores from the assessment at the initial visit to the clinic , prior to treatment onset .", "the presence of specific symptoms at initial clinical assessment was considered as a proxy of how prone to those symptoms each patient may be .", "we propose that the pre - treatment ratings best represent the underlying neurobiology and clinical characteristics of individual patients , as they show their symptom characteristics in an untreated state of illness .", "while it is not possible to make several such assessments prior to treatment onset ( which would best capture the potential symptom profile of each patient ) , such an approach may allow for a data exploration which separates patients who are prone to certain symptoms ( such as hallucinations ) from those who have experienced less or never experienced those symptoms while in an untreated state .", "as such , this can be conceptualized as a  trait based approach to symptom evaluation . saps scores for global hallucinations and global thought disorder were entered into a whole - brain regression model with the person  >  object and place  >  object contrasts . the global delusion score was not included as most patients were highly delusional at baseline . in order to account for differences in time since initial diagnosis ,", "the interval between baseline assessment and mri scan ( in days ) was entered as a covariate in the second - level regression analysis .", "cluster threshold for the regression was 49  voxels at p  <  0.001 uncorrected ( voxel threshold ) ."], ["there were no significant differences in age or gender distribution between groups , though patients had a marginally significant lower parental ses and significantly fewer years of education .", "accuracy in the person , place and object conditions was assessed using independent samples t - test .", "schizophrenia patients had significantly lower accuracy in the person condition , t(40 )  =  2.247 , p  =  0.03 , but not for place , t(40 )  =  0.44 , p  =  0.66 , or object , t(40 )  =  0.07 , p  =  0.95 .", "numerous cortical regions showed significant increases in neural activity for retrieval of source memory over retrieval of object memory , similar to the pattern observed in previous studies using a similar paradigm ( burgess et al .", ", 2001 ; king et al . , 2005 ) . activated regions in the source memory contrasts ( person  >  object and place  >  object ) included bilateral activity around the parietal occipital sulcus ( including the precuneus and retrosplenial cortex ) extending into the superior parietal cortex , left inferior frontal gyrus ( vlpfc ) , fusiform gyrus bilaterally , and the occipital cortex .", "activity in the person  >  object contrast included the head of the caudate nucleus , the right inferior frontal ( vlpfc ) , and the medial aspect of the superior frontal gyrus .", "object  >  place showed widespread activity in the medial frontal cortex and parietal cortices ( supramarginal gyrus bilaterally ) and smaller clusters in the frontal and occipital cortices .", "for the object  >  person contrast , activity was observed in the left and right angular gyrus .", "2 . when examining source vs. object retrieval contrasts in scz , a smaller number of significantly activated voxels were observed relative to the activity maps of controls . in the person ", ">  object contrast only smaller clusters in the head of the caudate and occipital cortex were significant .", "in place  >  object , mainly posterior activity was observed ( e.g. parietal occipital sulcus and fusiform cortex ) . in both", "the object  >  person and object  >  place , scz showed a right inferior frontal ( vlpfc ) activity which was not observed in controls .", "2 . when comparing between groups , controls demonstrated regions of greater differences between conditions than scz for person  >  object condition ( including bilaterally in the precuneus and superior parietal , and left and right inferior frontal in the vlpfc ) and for place  >  object ( bilaterally in the superior parietal , and the left precuneus ) .", "schizophrenia patients had greater differences between conditions than controls when considering place  >  object across a wide range of areas .", "the group comparison was run using contrast  values , which can be positive ( e.g. if place  >  object ) or negative ( e.g. if object  >  place ) .", "thus , while we observed greater activity in scz than control in the place  >  object contrast , it is not clear from the activation maps if such a difference is due to changes in activity in either object or place . in order to visualize the results for each condition ,", "beta values were extracted for an roi of 11  voxels ( 9 in - plane voxels surrounding the selected voxel and one above and one below ) .", "rois were selected to represent a range of representative patterns of activity across the brain ( e.g. regions in which controls showed differences from scz in both person  >  object and place  >  object , and regions in which scz showed  greater  activity in place  >  object ) .", "which controls had greater differences between conditions than scz , controls are showing increased activity to the source memory conditions ( person and/or place ) relative to object , while  values in scz do not show any such differentiation .", "interestingly , in regions in which we observed  greater  differences between conditions ( in this case , place  >  object ) in scz than controls , we again observe little differences between source memory and object memory in scz .", "thus , it is not that patients with scz are showing greater activity in the place condition , but that they are failing to modulate brain activity in the same way as control participants .", "results of the regression analysis with positive symptoms are presented in table 6 and fig .", "we observed significant negative correlations with the global hallucination score from the saps ( at first assessment ) and place  >  object contrast , in the right midtemporal gyrus , left prefrontal cortex , and right cerebellum ( in a region noted in at least one lesional case study to be associated with source memory deficits ; tamagni et al . ,", "2010 ) . in order to examine the relationship between recognition memory performance and neural activity , spearman 's correlations were run on global hallucination score , memory performance in the place and object conditions , and  values from the three clusters .", "while hallucinations did not significantly correlate with performance in either condition ( place , rho  =  .228 , p  =  0.32 ; object , rho  =  0.28 , p  =  0.22 ) , there was a marginally significant correlation between performance in the object condition and  value in the dlpfc cluster ( rho  =  0.38 , p  =  0.088 ) ."], ["this study examined differences in the neural correlates of source memory in patients with schizophrenia and controls .", "we examined this issue using a virtual reality paradigm , which has improved ecological validity when assessing the  source  of a memory , as compared to other studies which have used less ecologically valid tasks .", "furthermore , we examined a group of patients , who were within the first 4.5  years of treatment avoiding potential issues associated with illness chronicity such as cognitive decline , prolonged exposure to medications ( although medication exposure remains an issue ) , social isolation , and sedentary lifestyle ( pelletier et al . , 2005 ) . while patients with scz demonstrated some activation in the source memory contrasts , the extent and magnitude of activity were substantially less than those in controls . even in regions in which the difference appeared to be in the directions of scz  >  controls", ", it seems to be the case that controls differentiate between source and object memory while patients do not .", "as discussed below , this finding may be true of other forms of memory , and may therefore be representative of the underlying deficit across a range of memory subtypes .", "more specifically , patients with scz may fail to activate cortical regions which facilitate elaborative memory processes .", "this is consistent with previous findings of relatively intact object memory in scz ( achim et al .", "2009 ) , but deficits in source memory ( johnson et al . , 1993 ) and associative memory ( achim and lepage , 2003 ) .", "it may be that the vr environment minimizes these behavioral differences , in that the place condition may be easier than person as locations were more distinct from each other relative to the 3d rendered characters .", "alternatively , our recent - onset sample may have better preserved function than the more enduring schizophrenia samples often examined . however , this lack of behavioral difference has a positive aspect , in that it removes performance as a potentially major confound in the fmri analysis . had patients with schizophrenia demonstrated profound deficits in performance , it would beg the question if any observed neural activity differences were due to disease pathology or simply related to poor performance ( and as such would be similar to poor performing healthy controls ) .", "we did observe a performance difference in the person condition , and interestingly very little significant activity in the schizophrenia group for person  >  object .", "however , some activations observed in the group analysis , particularly in the posterior regions , were present in both person  >  object and place  >  object , suggesting that these differences were not at all modality specific .", "negative correlations were observed between the difference in neural activity in place  >  object and global hallucinations , measured at intake baseline using the saps .", "this suggests that the propensity of an individual to experience hallucination may modulate differences between source and object memory in these areas .", "we did not observe significant correlations with global thought disorder , which is not surprising as hallucinations but not thought disorder are generally associated with source memory ( woodward et al . , 2007 ) .", "while these regions did not directly overlap activity observed in the healthy control group , the left frontal cluster was proximal to significant activity in place  >  object in controls , while the middle - temporal cluster was proximal to significant voxels in the object  >  place clusters in controls , suggesting that these regions are at least similar to those observed in healthy controls .", "however , the lack of direct overlap and given the nature of the result ( greater difference with more hallucinations ) , it is possible that these regions represent malfunctioning cortical regions which are more active in patients who have experienced hallucinations as part of their disorder .", "this over - activation within these regions when considering source information may play an important role in generating hallucinations , which are essentially the misattribution of internally generated stimuli to an external source .", "at least one study has found relationships between the left prefrontal cortex and the right temporal cortex while patients are actively experiencing hallucinations ( hoffman et al . , 2011 ) , and", "a meta - analysis suggests that these regions among others are frequently active during hallucination ( jardri et al . , 2011 ) . as such , the regions found to be significantly active in our regression are consistent with the existing literature on the neurobiology of hallucinations .", "while many studies examining hallucinations have utilized either general  state based measurements ( approximately how much the participant is hallucinating in general at the time ) or direct analysis of overall activity during hallucinations , we have attempted to utilize a ", "our sample of patients early in treatment makes such an approach possible with minimal confounding for chronicity ( which we attempted to control for by including length of treatment as a covariate in the regression ) .", "our results overall suggest a relationship between mal - adaptive neural activity related to source memory retrieval and how prone participants are to hallucinatory symptoms , further verifying the relationship between source memory and hallucinations ( woodward et al . , 2007 ) . however , some limitations must be considered as well .", "while we have attempted to use baseline scores as a  trait measure of how prone an individual is to hallucinations , it is well known that clinical symptoms can vary across time . as such , the appropriate method for evaluating ", "trait symptoms is to take repeated symptom measurements over time ( mathalon and ford , 2012 ) .", "it would be preferable to have several assessments of pre - treatment symptoms to have a complete picture of the symptom profile of a given patient , but this is not possible as it would require delaying treatment . as such we utilized the best available measure to assess pre - treatment symptom profile , which produced results which are consistent with the existing literature on those symptoms .", "however , it is important to remain considerate of the limitations of our measures when considering these results .", "source memory can be considered a form of associative / relational memory , as participants are binding elements together during encoding and storing these elements together as part of a larger whole .", "a deficit in source memory can be viewed as a failure to associate a memory item with its context . in the case of this study in particular ,", "our source memory paradigm is not far removed from studies examining associative memory ( achim et al . , 2007 ; achim and lepage , 2005 ; murray and ranganath , 2007 ) , which is known to be more impaired in scz than object memory ( achim and lepage , 2003 ) .", "some studies have suggested that patients with schizophrenia do not properly differentiate between associative and item memory ( achim et al .", "2012 ) , in keeping with suggestions that source memory impairments are part of associative memory impairments ( achim et al . , 2011 ) .", "our present study focused on source retrieval , and as such we can not definitively determine if the differences observed in our contrasts are driven by deficits in encoding ( in that the information was not properly moved into memory ) or retrieval ( in that the memories may have been encoded but are not properly accessed ) , or a combination of both ( which seems likely given the plurality of evidence for memory deficits in scz ) .", "( 2011 ) found deficits in source memory even when using short term recall , minimizing the need for retrieval of information from long - term memory .", "this suggests that at least part of the source memory deficit in scz is related to problems with encoding .", "our finding of an overall pattern of lack of differentiation between conditions may be a fairly consistent finding in the cognitive neuroscience of memory in scz , regardless if one considers activity during encoding or retrieval / recognition .", "core regions required for task performance may be relativity intact in scz , while deficits in activity will be observed in ", "that is to say , the deficits in memory ( and possibly cognition in general ) are related to a lack of engagement of extended cortical regions .", "these extended regions are not critically required for minimal task performance , but instead serve to enhance performance ( such as regions involved in cognitive control processes ) .", "healthy controls will tend to utilize such areas automatically , while patients with schizophrenia will fail to do so .", "for example , patients with scz have been shown to have impairments in initiating elaborative encoding processes which may be beneficial during associative encoding ( brebion et al .", "however , when patients are specifically instructed to utilize effective encoding strategies they show an improvement in memory performance , demonstrating that patients with scz can utilize such encoding strategies when specifically instructed but fail to do so spontaneously ( brebion et al . , 1997 ) .", "this pattern of results is similar to that in patients with prefrontal cortical lesions ( alexander et al . , 2003 ) .", "bonner - jackson et al . ( 2008 ) examined memory strategies in schizophrenia by contrasting intentional but unstructured encoding ( simply instructing participant to memorize words ) with an externally imposed deep encoding strategy ( having participants perform an abstract / concrete judgement on words , deep semantic encoding which facilitates memory encoding ; craik and lockhart , 1972 ) .", "they observed group    encoding interactions in several regions , including the left inferior frontal gyrus and precuneus , with the most common finding being a difference between controls and scz in the incidental , unstructured encoding condition .", "for example , in the left inferior frontal cortex , scz patients showed no activity for unstructured encoding , but substantial activity for deep encoding .", "that is to say , when scz patients were provided a structured encoding strategy they activated this region , but failed to do so spontaneously .", "likewise , controls showed significantly better memory performance than scz patients for the deep encoding condition .", "further support for the importance in strategic and/or cognitive control comes from findings of changes and/or normalization of activity in scz following cognitive training ( hooker et al . , 2012 ; penades et al . ,", "within both encoding and recognition studies in schizophrenia , the most prevalent finding is a decrease in the extent or magnitude of activity in scz patients relative to controls , although many studies also report findings in the direction of scz  >  controls ( ragland et al . , 2009 ) .", "while it is likely that scz often shows compensatory networks or inefficient over - activation during cognitive tasks , it can be difficult to judge from many of the published studies on cognition in scz .", "many papers report differences in group activities without also reporting the parameter estimates which accompany those changes .", "if we had done so in this study ( by only presenting the activation maps in fig .", "3 and not the beta values ) we may have concluded that scz showed greater activity in some regions for place vs. object and concluded that this was compensatory of maladaptive over - activation . however , when examining the beta values , we realize that this is not the case but instead these are regions where controls show greater activity for object over place ( often in the form of a decrease in activity for place ) .", "this highlights the importance of carefully examining parameter estimates when performing between - group comparisons .", "it is possible that in at least some cases in the existing literature , the so - called ", "compensatory activity may instead be a lack of activity relative to the control group , such as was the case in this study .", "such ambiguity and misinterpretation can be avoided if studies fully report parameter estimates for contrasts which differ between groups ."], ["we have reported the results of a study looking at source memory retrieval in scz using a virtual reality environment on a group of patients relatively early into treatment .", "while controls activated a large , extensive network for source relative to object retrieval , scz showed a marked reduction in activity .", "this reduction was borne out in the group comparison , and appears to be related to the fact that patients with scz are failing to activate these regions or differentiate between object and source retrieval . despite the large - scale group differences , patients were still able to perform the source retrieval tasks , suggesting that at least some of the  core system involved in source retrieval is intact . instead , we propose that the observed differences are related to  supporting regions which are not critical to task performance but instead facilitate source retrieval .", "that is , patients may generally fail to engage extended cortical networks which facilitate task performance and facilitate overall cognitive functioning ."]]}
{"article_id": "PMC2199216", "article_text": ["ras is a multifunctional signaling molecule acting as an essential component of signal transduction pathways that regulate cellular physiology ( campbell et al .", "members of the ras family of proteins that are constitutively activated by point mutations play a major role in the onset of a large number of human cancers , including those originating from skeletal muscle tissue ( yoo et al .", "these skeletal muscle tumors are incapable of differentiation and thus do not withdraw from the cell cycle . up to 35% of these tumors", "contain activating ras point mutations , suggesting a major involvement of ras in rhabdomyosarcomas ( stratton et al .", "constitutively active ras mutants stimulate secretion of growth and angiogenic factors ( rak et al .", "1995 ) , potentially allowing neoplastic cells to overcome growth restrictions in their normal tissue environment . in skeletal muscle cells ,", "activated ras mutants have been shown to promote secretion of an unidentified factor that can repress myogenic differentiation and may participate in the development of rhabdomyosarcomas ( weyman and wolfman 1997 ) . of particular interest is the observation that cultured human embryonal rhabdomyosarcoma cells express the fgf-2 gene and produce biologically active fgf-2 ( schweigerer et al .", "release of fgf-2 may stimulate the growth and neovascularization of human rhabdomyosarcomas and contribute to tumor development .", "although ectopic expression of oncogenic ha - ras in myogenic cell lines represses terminal differentiation , it is not reported to elicit a proliferative response ( olson et al .", "1987 ; konieczny et al . 1989 ; weyman and wolfman 1997 ) . from these studies", ", it has been concluded that ras inhibits muscle differentiation without affecting proliferative response pathways .", "fgfs are likely candidates for such factors since they play critical roles in regulation of skeletal muscle differentiation in cultured cells ( linkhart et al .", "1981 ; allen et al . 1985 ; kardami et al . 1985 ; seed and hauschka 1988 ; rando and blau 1994 ; flanagan - steet et al .", "2000 ) , in skeletal muscle development in vivo ( flanagan - steet et al . 2000 ) , and in skeletal muscle regeneration ( anderson et al . 1995 ; floss et al .", "mm14 myoblasts express fgf-1 , -2 , -6 , and -7 but are absolutely dependent on exogenously supplied fgfs to repress myogenesis and promote cell proliferation ( clegg et al .", "fgf-2 is one of four fgfs that do not possess signal peptides and do not use the classical er / golgi - dependent secretory pathways for export from the cell ( florkiewicz et al .", "since ectopically expressed ha - ras can repress differentiation of mm14 cells ( fedorov et al . 1998 ) , we asked if ha - ras was capable of stimulating proliferation in mm14 cells . here", "we report that constitutively active ras stimulates mm14 myoblast proliferation via a novel mechanism that is dependent on export of endogenously produced fgf-2 and subsequent release or activation of the exported fgf-2 .", "moreover , we also found that the signaling pathways used by oncogenic ras to stimulate proliferation and repress differentiation in myogenic cells are distinct and mediated independently .", "mouse mm14 cells ( lim and hauschka 1984 ) were cultured as described previously ( kudla et al .", "baf3/fr1 cells , a baf3 cell clone stably expressing fgf receptor ( fgfr)-1 , was cultured as described by ornitz et al . 1992 .", "wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells ( ornitz et al .", "human recombinant fgf-2 was purified from a yeast strain expressing this growth factor ( rapraeger et al .", "heparin , nacl , and naclo3 were purchased from sigma - aldrich . a monoclonal anti ", "1993 ) and anticysteine - rich fgfr control antibody ( zuber et al . 1997 ) were used as described previously ( hannon et al .", "dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously ( kudla et al .", "equivalent dna concentrations were maintained by the addition of a pbssk+ ( stratagene ) plasmid .", "the pdcr - h - ras ( g12v ) expression vector encoding a constitutively active mutant of human ha - ras , rasg12v ( white et al .", "mm14 cells were grown on 6-well plates to a density of 5  10 and transfected with the indicated expression vectors or control plasmids .", "cells were trypsinized ( 0.05% trypsin , 0.53 mm edta ) and replated at clonal density ( 1,000 cells per 10-cm plate ) 1 h after transfection .", "the cells were maintained in the presence or absence of fgf-2 ( 0.3 nm unless otherwise indicated ) , cultured for 36 h , then processed for -galactosidase histochemistry as described elsewhere ( sanes et al .", "a differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection .", "the reporter contained the firefly luciferase gene driven by a muscle - specific promoter ( msp ; human anti - cardiac actin promoter ) ( kudla et al .", "transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf .", "cells were then washed once with pbs ( ph 7.2 ) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin .", "baf3/fr1 cells ( 10 cells per well in 24-well plates ) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue .", "mouse mm14 cells ( lim and hauschka 1984 ) were cultured as described previously ( kudla et al .", "baf3/fr1 cells , a baf3 cell clone stably expressing fgf receptor ( fgfr)-1 , was cultured as described by ornitz et al . 1992 .", "wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells ( ornitz et al .", "human recombinant fgf-2 was purified from a yeast strain expressing this growth factor ( rapraeger et al .", "heparin , nacl , and naclo3 were purchased from sigma - aldrich . a monoclonal anti ", "1993 ) and anticysteine - rich fgfr control antibody ( zuber et al . 1997 ) were used as described previously ( hannon et al .", "dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously ( kudla et al .", "equivalent dna concentrations were maintained by the addition of a pbssk+ ( stratagene ) plasmid .", "the pdcr - h - ras ( g12v ) expression vector encoding a constitutively active mutant of human ha - ras , rasg12v ( white et al .", "mm14 cells were grown on 6-well plates to a density of 5  10 and transfected with the indicated expression vectors or control plasmids .", "cells were trypsinized ( 0.05% trypsin , 0.53 mm edta ) and replated at clonal density ( 1,000 cells per 10-cm plate ) 1 h after transfection .", "the cells were maintained in the presence or absence of fgf-2 ( 0.3 nm unless otherwise indicated ) , cultured for 36 h , then processed for -galactosidase histochemistry as described elsewhere ( sanes et al .", "a differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection .", "the reporter contained the firefly luciferase gene driven by a muscle - specific promoter ( msp ; human anti - cardiac actin promoter ) ( kudla et al .", "to determine their fgf-2 export capabilities , transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf .", "cells were then washed once with pbs ( ph 7.2 ) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin . the medium was then collected and filtered through a 0.2-m filter .", "baf3/fr1 cells ( 10 cells per well in 24-well plates ) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue .", "mm14 cells are absolutely dependent on exogenously supplied fgfs to repress myogenesis and promote proliferation , yet they express several fgfs ( hannon et al . 1996 ) , suggesting that the endogenously produced fgfs are inaccessible to fgfr-1 .", "in addition , we have established that distinct fgfr-1 signaling pathways mediate the proliferative and differentiation inhibitory responses in mm14 cells ( kudla et al .", "ectopic ha - ras expression stimulated proliferation and repressed differentiation of mm14 cells in the absence of exogenous fgf ( fig .", "1 ) . activated ras appears to replace only fgf - dependent signaling events since mm14 cells transfected with oncogenic ras were unable to proliferate in growth medium with reduced ( 2.5% ) serum ( data not shown ) .", "we hypothesized that ha - ras may induce fgf export or secretion of endogenously produced fgfs and therefore treated mm14 cells expressing rasg12v with agents that block fgf signaling .", "addition of suramin , a negatively charged polysulfonated binaphthyl urea used as a general heparin - binding growth factor antagonist ( lozano et al .", "1998 ) , to cells ectopically expressing oncogenic ras inhibited proliferation in a dose - dependent manner ( fig .", "fgf-2 antibody completely abolished the capacity of ha - ras to stimulate proliferation ( fig .", "2 a ) , whereas addition of a control monoclonal antibody had no effect ( fig .", "unexpectedly , we found that the ability of ha - ras to stimulate proliferation appears dependent on extracellularly supplied fgf-2 .", "fgf signaling is dependent on heparan sulfate , which involves the interaction of heparan sulfate with both fgf and the fgfr tyrosine kinases ( rapraeger et al .", "in addition , heparan sulfate proteoglycans ( hspgs ) participate in fgf storage , sequestration , and release ( rifkin and moscatelli 1989 ) .", "treatment of mm14 cells with sodium chlorate , a reversible inhibitor of intracellular sulfation , prevents fgf binding and induces terminal differentiation ( rapraeger et al .", "incubation of oncogenic ras - transfected mm14 cells with heparitinase ( not shown ) or sodium chlorate significantly decreases cell proliferation ( fig .", "3 a ) . both heparitinase ( not shown ) and chlorate - induced inhibition of mm14 cell proliferation was rescued by addition of heparin ( 50 g / ml ) , indicating that the effect is heparan sulfate specific ( fig .", "3 a ) . surprisingly , addition of 600 pm fgf-2 to chlorate - treated ha - ras  transfected cells promoted proliferation , ameliorating the inhibitory chlorate effect ( fig .", "this was unexpected since addition of fgf-2 had no effect on chlorate - treated parental mm14 cells or mm14 cells transfected with a pcdna3 vector control ( fig .", "3 b ) . the requirement for hspgs and the ability to overcome this requirement with high concentrations of exogenously", "added fgf-2 suggests a more complicated role for hspgs in addition to their known requirement for signaling . taken together , our data demonstrate that rasg12v induces proliferation of skeletal muscle cells and that induction of proliferation requires an autocrine fgf-2 response .", "little is known regarding the mechanisms involved in fgf-2 export since fgf-2 has no signal peptide sequence and is not secreted through the established golgi - dependent secretory pathway ( mignatti et al .", "1992 ) . instead , an unusual atp - dependent pathway that includes the na / k - atpase appears to be involved ( florkiewicz et al .", "whether oncogenic ras is directly involved in regulating fgf-2 export , we asked if mm14 cells expressing ha - ras exhibited increased levels of extracellular fgf-2 .", "although transfection of mm14 cells with fgf-2 results in export of biologically active fgf-2 ( hannon et al .", "1996 ) , this extracellular fgf-2 can not be detected in the tissue culture media , presumably due to its strong association with membrane - bound and extracellular matrix  associated heparan sulfate .", "one assay utilizes heparin treatment of mm14 cells to release bound fgf-2 , which is then assayed on baf3 cells expressing fgfr-1 ( baf3/fr1 ) .", "baf3 cells are pre - b cells that undergo apoptosis after interleukin 3 withdrawal and do not express either fgfrs or hspgs . as such , these cells are unresponsive to fgfs , unless they ectopically express fgfrs and heparin is added as an hspg substitute ( ornitz et al .", "we found that both ras - g12v and control ( pcdna3)-transfected mm14 cells release similar levels of factor(s ) that support baf3/fr1 survival and promote baf3/fr1 proliferation ( fig .", "a second assay involves cotransfection of mm14 cells with a construct encoding an fgf-2luciferase fusion protein and either ras - g12v or a control vector .", "the results from this assay are indistinguishable from the baf3/fr1 cell assay , suggesting that similar levels of fgf-2 are exported by control and ha - ras  transfected cells ( data not shown ) .", "we conclude that oncogenic ras does not affect the level of fgf-2 export from mm14 cells .", "although mm14 cells produce fgf-2 and export fgf-2 that can be recovered in an active form , this fgf-2 is not normally available to the cells ( hannon et al .", "thus , our data suggest that exported fgf-2 is normally retained in an inactive form on the cell surface .", "we propose that ha - ras  activates  this inactive extracellular pool of fgf-2 either by promoting its release from hspgs or by providing a mechanism for fgf-2 to gain access to cell surface fgfr-1 .", "although the mechanisms involved are not understood , the ability of the ha - ras mutant to promote proliferation is dependent on exogenous fgf-2 and subsequent fgf-2mediated signaling events .", "the ability of oncogenic ras to inhibit skeletal muscle differentiation has been well documented , but the ras effector mediating repression of differentiation is not known ( ramocki et al .", "we wanted to determine if the ability of ras to effectively inhibit mm14 differentiation was dependent on extracellular fgf-2 , as is the proliferation response .", "fgf-2 antibody did not affect the ability of ras - g12v to repress myogenesis ( fig .", "these data are consistent with our results published previously , which demonstrate that independent fgf signaling events mediate repression of differentiation and proliferation ( kudla et al .", "thus , similar to fgfr , ha - ras appears to utilize independent signaling mechanisms to repress terminal differentiation and promote proliferation . although the downstream signaling events that mediate the repression of myogenesis by ras are not understood , it is well documented that oncogenic ras stimulates secretion of growth factors and angiogenic factors ( rak et al .", "moreover , ectopic expression of activated ras releases a factor that represses myogenic differentiation ( weyman and wolfman 1997 ) .", "data presented in this report argue that oncogenic ras may be involved in the activation or release of extracellularly localized fgf-2 that is normally sequestered in a biologically inactive state .", "it is noteworthy that all of the extracellular activity observed is neutralized by a specific blocking fgf-2 monoclonal antibody , since proliferating mm14 cells synthesize fgf-1 , -2 , -6 , and -7 ( hannon et al .", "1996 ) . although detectable , fgf-2 mrna is present at extremely low concentrations in many adult tissues despite the presence of high levels of fgf-2 activity , suggesting a mechanism for retaining or storing fgf-2 in a biologically inert form ( baird et al .", "oncogenic ras appears to be involved in activating or releasing inactive , extracellularly localized fgf-2 .", "the fgf-2 produced by skeletal muscle cells can not be detected in the tissue culture medium , forming the basis for previous conclusions that the ras - secreted myogenic inhibitory factor was not an fgf ( weyman and wolfman 1997 ) .", "the ras - dependent proliferation factor we identified is released by a heparin wash and its activity is abrogated by treatment with either chlorate , heparitinase , suramin , or a monoclonal anti  fgf-2 antibody , implying that the proliferation factor is fgf-2 .", "together with the prevalence of oncogenic ras mutants in rhabdomyosarcomas and the involvement of fgfs in regulation of myogenesis , our data suggest that fgf-2 may be a critical factor for supporting ras - dependent growth of rhabdomyosarcomas ."], "abstract_text": ["<S> constitutively activated ras proteins are associated with a large number of human cancers , including those originating from skeletal muscle tissue . in this study , we show that ectopic expression of oncogenic ras stimulates proliferation of the mm14 skeletal muscle satellite cell line in the absence of exogenously added fibroblast growth factors ( fgfs ) . </S>", "<S> mm14 cells express fgf-1 , -2 , -6 , and -7 and produce fgf protein , yet they are dependent on exogenously supplied fgfs to both maintain proliferation and repress terminal differentiation . </S>", "<S> thus , the fgfs produced by these cells are either inaccessible or inactive , since the endogenous fgfs elicit no detectable biological response . </S>", "<S> oncogenic ras - induced proliferation is abolished by addition of an anti  fgf-2 blocking antibody , suramin , or treatment with either sodium chlorate or heparitinase , demonstrating an autocrine requirement for fgf-2 . </S>", "<S> oncogenic ras does not appear to alter cellular export rates of fgf-2 , which does not possess an nh2-terminal or internal signal peptide . </S>", "<S> however , oncogenic ras does appear to be involved in releasing or activating inactive , extracellularly sequestered fgf-2 . </S>", "<S> surprisingly , inhibiting the autocrine fgf-2 required for proliferation has no effect on oncogenic ras - mediated repression of muscle - specific gene expression . </S>", "<S> we conclude that oncogenic ras - induced proliferation of skeletal muscle cells is mediated via a unique and novel mechanism that is distinct from ras - induced repression of terminal differentiation and involves activation of extracellularly localized , inactive fgf-2 . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Cell Culture", "DNA Transfection", "Clonal Growth Assay", "Muscle-specific Promoter Assay", "FGF-2 Export Assay", "Results and Discussion"], "sections": [["ras is a multifunctional signaling molecule acting as an essential component of signal transduction pathways that regulate cellular physiology ( campbell et al .", "members of the ras family of proteins that are constitutively activated by point mutations play a major role in the onset of a large number of human cancers , including those originating from skeletal muscle tissue ( yoo et al .", "these skeletal muscle tumors are incapable of differentiation and thus do not withdraw from the cell cycle . up to 35% of these tumors", "contain activating ras point mutations , suggesting a major involvement of ras in rhabdomyosarcomas ( stratton et al .", "constitutively active ras mutants stimulate secretion of growth and angiogenic factors ( rak et al .", "1995 ) , potentially allowing neoplastic cells to overcome growth restrictions in their normal tissue environment . in skeletal muscle cells ,", "activated ras mutants have been shown to promote secretion of an unidentified factor that can repress myogenic differentiation and may participate in the development of rhabdomyosarcomas ( weyman and wolfman 1997 ) . of particular interest is the observation that cultured human embryonal rhabdomyosarcoma cells express the fgf-2 gene and produce biologically active fgf-2 ( schweigerer et al .", "release of fgf-2 may stimulate the growth and neovascularization of human rhabdomyosarcomas and contribute to tumor development .", "although ectopic expression of oncogenic ha - ras in myogenic cell lines represses terminal differentiation , it is not reported to elicit a proliferative response ( olson et al .", "1987 ; konieczny et al . 1989 ; weyman and wolfman 1997 ) . from these studies", ", it has been concluded that ras inhibits muscle differentiation without affecting proliferative response pathways .", "fgfs are likely candidates for such factors since they play critical roles in regulation of skeletal muscle differentiation in cultured cells ( linkhart et al .", "1981 ; allen et al . 1985 ; kardami et al . 1985 ; seed and hauschka 1988 ; rando and blau 1994 ; flanagan - steet et al .", "2000 ) , in skeletal muscle development in vivo ( flanagan - steet et al . 2000 ) , and in skeletal muscle regeneration ( anderson et al . 1995 ; floss et al .", "mm14 myoblasts express fgf-1 , -2 , -6 , and -7 but are absolutely dependent on exogenously supplied fgfs to repress myogenesis and promote cell proliferation ( clegg et al .", "fgf-2 is one of four fgfs that do not possess signal peptides and do not use the classical er / golgi - dependent secretory pathways for export from the cell ( florkiewicz et al .", "since ectopically expressed ha - ras can repress differentiation of mm14 cells ( fedorov et al . 1998 ) , we asked if ha - ras was capable of stimulating proliferation in mm14 cells . here", "we report that constitutively active ras stimulates mm14 myoblast proliferation via a novel mechanism that is dependent on export of endogenously produced fgf-2 and subsequent release or activation of the exported fgf-2 .", "moreover , we also found that the signaling pathways used by oncogenic ras to stimulate proliferation and repress differentiation in myogenic cells are distinct and mediated independently ."], ["mouse mm14 cells ( lim and hauschka 1984 ) were cultured as described previously ( kudla et al .", "baf3/fr1 cells , a baf3 cell clone stably expressing fgf receptor ( fgfr)-1 , was cultured as described by ornitz et al . 1992 .", "wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells ( ornitz et al .", "human recombinant fgf-2 was purified from a yeast strain expressing this growth factor ( rapraeger et al .", "heparin , nacl , and naclo3 were purchased from sigma - aldrich . a monoclonal anti ", "1993 ) and anticysteine - rich fgfr control antibody ( zuber et al . 1997 ) were used as described previously ( hannon et al .", "dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously ( kudla et al .", "equivalent dna concentrations were maintained by the addition of a pbssk+ ( stratagene ) plasmid .", "the pdcr - h - ras ( g12v ) expression vector encoding a constitutively active mutant of human ha - ras , rasg12v ( white et al .", "mm14 cells were grown on 6-well plates to a density of 5  10 and transfected with the indicated expression vectors or control plasmids .", "cells were trypsinized ( 0.05% trypsin , 0.53 mm edta ) and replated at clonal density ( 1,000 cells per 10-cm plate ) 1 h after transfection .", "the cells were maintained in the presence or absence of fgf-2 ( 0.3 nm unless otherwise indicated ) , cultured for 36 h , then processed for -galactosidase histochemistry as described elsewhere ( sanes et al .", "a differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection .", "the reporter contained the firefly luciferase gene driven by a muscle - specific promoter ( msp ; human anti - cardiac actin promoter ) ( kudla et al .", "transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf .", "cells were then washed once with pbs ( ph 7.2 ) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin .", "baf3/fr1 cells ( 10 cells per well in 24-well plates ) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue ."], ["mouse mm14 cells ( lim and hauschka 1984 ) were cultured as described previously ( kudla et al .", "baf3/fr1 cells , a baf3 cell clone stably expressing fgf receptor ( fgfr)-1 , was cultured as described by ornitz et al . 1992 .", "wehi3 cells were purchased from the american type culture collection and baf3/fr1 cells ( ornitz et al .", "human recombinant fgf-2 was purified from a yeast strain expressing this growth factor ( rapraeger et al .", "heparin , nacl , and naclo3 were purchased from sigma - aldrich . a monoclonal anti ", "1993 ) and anticysteine - rich fgfr control antibody ( zuber et al . 1997 ) were used as described previously ( hannon et al ."], ["dna was transiently transfected into mm14 cells by a calcium phosphate dna precipitation method as described previously ( kudla et al .", "equivalent dna concentrations were maintained by the addition of a pbssk+ ( stratagene ) plasmid .", "the pdcr - h - ras ( g12v ) expression vector encoding a constitutively active mutant of human ha - ras , rasg12v ( white et al ."], ["mm14 cells were grown on 6-well plates to a density of 5  10 and transfected with the indicated expression vectors or control plasmids .", "cells were trypsinized ( 0.05% trypsin , 0.53 mm edta ) and replated at clonal density ( 1,000 cells per 10-cm plate ) 1 h after transfection .", "the cells were maintained in the presence or absence of fgf-2 ( 0.3 nm unless otherwise indicated ) , cultured for 36 h , then processed for -galactosidase histochemistry as described elsewhere ( sanes et al ."], ["a differentiation - sensitive muscle - specific reporter activity assay was used to determine the extent of mm14 differentiation after transient transfection .", "the reporter contained the firefly luciferase gene driven by a muscle - specific promoter ( msp ; human anti - cardiac actin promoter ) ( kudla et al ."], ["to determine their fgf-2 export capabilities , transfected cells were plated at 10 cells per well in 6-well plates and incubated for 36 h in growth media without fgf .", "cells were then washed once with pbs ( ph 7.2 ) and incubated for 1 h at room temperature in 1 ml of baf3/fr1 growth medium supplemented with 50 g / ml of heparin . the medium was then collected and filtered through a 0.2-m filter .", "baf3/fr1 cells ( 10 cells per well in 24-well plates ) were incubated in the collected medium for 72 h. the number of living cells in each well was quantified by counting the number of cells that exclude trypan blue ."], ["mm14 cells are absolutely dependent on exogenously supplied fgfs to repress myogenesis and promote proliferation , yet they express several fgfs ( hannon et al . 1996 ) , suggesting that the endogenously produced fgfs are inaccessible to fgfr-1 .", "in addition , we have established that distinct fgfr-1 signaling pathways mediate the proliferative and differentiation inhibitory responses in mm14 cells ( kudla et al .", "ectopic ha - ras expression stimulated proliferation and repressed differentiation of mm14 cells in the absence of exogenous fgf ( fig .", "1 ) . activated ras appears to replace only fgf - dependent signaling events since mm14 cells transfected with oncogenic ras were unable to proliferate in growth medium with reduced ( 2.5% ) serum ( data not shown ) .", "we hypothesized that ha - ras may induce fgf export or secretion of endogenously produced fgfs and therefore treated mm14 cells expressing rasg12v with agents that block fgf signaling .", "addition of suramin , a negatively charged polysulfonated binaphthyl urea used as a general heparin - binding growth factor antagonist ( lozano et al .", "1998 ) , to cells ectopically expressing oncogenic ras inhibited proliferation in a dose - dependent manner ( fig .", "fgf-2 antibody completely abolished the capacity of ha - ras to stimulate proliferation ( fig .", "2 a ) , whereas addition of a control monoclonal antibody had no effect ( fig .", "unexpectedly , we found that the ability of ha - ras to stimulate proliferation appears dependent on extracellularly supplied fgf-2 .", "fgf signaling is dependent on heparan sulfate , which involves the interaction of heparan sulfate with both fgf and the fgfr tyrosine kinases ( rapraeger et al .", "in addition , heparan sulfate proteoglycans ( hspgs ) participate in fgf storage , sequestration , and release ( rifkin and moscatelli 1989 ) .", "treatment of mm14 cells with sodium chlorate , a reversible inhibitor of intracellular sulfation , prevents fgf binding and induces terminal differentiation ( rapraeger et al .", "incubation of oncogenic ras - transfected mm14 cells with heparitinase ( not shown ) or sodium chlorate significantly decreases cell proliferation ( fig .", "3 a ) . both heparitinase ( not shown ) and chlorate - induced inhibition of mm14 cell proliferation was rescued by addition of heparin ( 50 g / ml ) , indicating that the effect is heparan sulfate specific ( fig .", "3 a ) . surprisingly , addition of 600 pm fgf-2 to chlorate - treated ha - ras  transfected cells promoted proliferation , ameliorating the inhibitory chlorate effect ( fig .", "this was unexpected since addition of fgf-2 had no effect on chlorate - treated parental mm14 cells or mm14 cells transfected with a pcdna3 vector control ( fig .", "3 b ) . the requirement for hspgs and the ability to overcome this requirement with high concentrations of exogenously", "added fgf-2 suggests a more complicated role for hspgs in addition to their known requirement for signaling . taken together , our data demonstrate that rasg12v induces proliferation of skeletal muscle cells and that induction of proliferation requires an autocrine fgf-2 response .", "little is known regarding the mechanisms involved in fgf-2 export since fgf-2 has no signal peptide sequence and is not secreted through the established golgi - dependent secretory pathway ( mignatti et al .", "1992 ) . instead , an unusual atp - dependent pathway that includes the na / k - atpase appears to be involved ( florkiewicz et al .", "whether oncogenic ras is directly involved in regulating fgf-2 export , we asked if mm14 cells expressing ha - ras exhibited increased levels of extracellular fgf-2 .", "although transfection of mm14 cells with fgf-2 results in export of biologically active fgf-2 ( hannon et al .", "1996 ) , this extracellular fgf-2 can not be detected in the tissue culture media , presumably due to its strong association with membrane - bound and extracellular matrix  associated heparan sulfate .", "one assay utilizes heparin treatment of mm14 cells to release bound fgf-2 , which is then assayed on baf3 cells expressing fgfr-1 ( baf3/fr1 ) .", "baf3 cells are pre - b cells that undergo apoptosis after interleukin 3 withdrawal and do not express either fgfrs or hspgs . as such , these cells are unresponsive to fgfs , unless they ectopically express fgfrs and heparin is added as an hspg substitute ( ornitz et al .", "we found that both ras - g12v and control ( pcdna3)-transfected mm14 cells release similar levels of factor(s ) that support baf3/fr1 survival and promote baf3/fr1 proliferation ( fig .", "a second assay involves cotransfection of mm14 cells with a construct encoding an fgf-2luciferase fusion protein and either ras - g12v or a control vector .", "the results from this assay are indistinguishable from the baf3/fr1 cell assay , suggesting that similar levels of fgf-2 are exported by control and ha - ras  transfected cells ( data not shown ) .", "we conclude that oncogenic ras does not affect the level of fgf-2 export from mm14 cells .", "although mm14 cells produce fgf-2 and export fgf-2 that can be recovered in an active form , this fgf-2 is not normally available to the cells ( hannon et al .", "thus , our data suggest that exported fgf-2 is normally retained in an inactive form on the cell surface .", "we propose that ha - ras  activates  this inactive extracellular pool of fgf-2 either by promoting its release from hspgs or by providing a mechanism for fgf-2 to gain access to cell surface fgfr-1 .", "although the mechanisms involved are not understood , the ability of the ha - ras mutant to promote proliferation is dependent on exogenous fgf-2 and subsequent fgf-2mediated signaling events .", "the ability of oncogenic ras to inhibit skeletal muscle differentiation has been well documented , but the ras effector mediating repression of differentiation is not known ( ramocki et al .", "we wanted to determine if the ability of ras to effectively inhibit mm14 differentiation was dependent on extracellular fgf-2 , as is the proliferation response .", "fgf-2 antibody did not affect the ability of ras - g12v to repress myogenesis ( fig .", "these data are consistent with our results published previously , which demonstrate that independent fgf signaling events mediate repression of differentiation and proliferation ( kudla et al .", "thus , similar to fgfr , ha - ras appears to utilize independent signaling mechanisms to repress terminal differentiation and promote proliferation . although the downstream signaling events that mediate the repression of myogenesis by ras are not understood , it is well documented that oncogenic ras stimulates secretion of growth factors and angiogenic factors ( rak et al .", "moreover , ectopic expression of activated ras releases a factor that represses myogenic differentiation ( weyman and wolfman 1997 ) .", "data presented in this report argue that oncogenic ras may be involved in the activation or release of extracellularly localized fgf-2 that is normally sequestered in a biologically inactive state .", "it is noteworthy that all of the extracellular activity observed is neutralized by a specific blocking fgf-2 monoclonal antibody , since proliferating mm14 cells synthesize fgf-1 , -2 , -6 , and -7 ( hannon et al .", "1996 ) . although detectable , fgf-2 mrna is present at extremely low concentrations in many adult tissues despite the presence of high levels of fgf-2 activity , suggesting a mechanism for retaining or storing fgf-2 in a biologically inert form ( baird et al .", "oncogenic ras appears to be involved in activating or releasing inactive , extracellularly localized fgf-2 .", "the fgf-2 produced by skeletal muscle cells can not be detected in the tissue culture medium , forming the basis for previous conclusions that the ras - secreted myogenic inhibitory factor was not an fgf ( weyman and wolfman 1997 ) .", "the ras - dependent proliferation factor we identified is released by a heparin wash and its activity is abrogated by treatment with either chlorate , heparitinase , suramin , or a monoclonal anti  fgf-2 antibody , implying that the proliferation factor is fgf-2 .", "together with the prevalence of oncogenic ras mutants in rhabdomyosarcomas and the involvement of fgfs in regulation of myogenesis , our data suggest that fgf-2 may be a critical factor for supporting ras - dependent growth of rhabdomyosarcomas ."]]}
{"article_id": "PMC2837472", "article_text": ["lipomas represent uncommon neoplasms of the oral cavity ; only 1% to 5% of cases occur at this site . based on their histopathological features of conspicuous multiple components", ", lipomas can be divided into some subclasses [ 2 , 3 ] , and the most common is the fibrolipoma [ 1 , 3 , 4 ] .", "cases reporting on lipoma with osteo / chondroid differentiaion were only 16 cases as a result of retrieving literature from 1960 to 2008 .", "further , other names of this subclassification exist in literature ; osteo / chondrolipoma , ossifying / osseous lipoma , lipoma with chondro / osseous metaplasia , and lipoma with cartilaginous / osseous change . the obscure etiology of osseous / chondroid differentiaion led to confusion of the name . to clarify the etiology of osseous / chondroid differentiaion in lipoma , the authors report additional 2 cases of oral lipoma / fibrolipoma with osseous and/or chondroid differentiation and describe the clinical , histopathological , and immunohistological features of these .", "review of the literature and clinicopathological analysis from the files of the oral pathology department , nihon university school of dentistry at matsudo from 1995 to 2007 of these cases were performed and compared .", "two cases that had been diagnosed as lipoma with osteo / chondroid differentiation were retrieved from the files mentioned earlier . in these cases ,", "sections of 2 cases were deparaffinized in xylene and dehydrated in tris - buffered saline ( ph 7.6 ) .", "primary antibodies against e29 ( epithelial membrane antigen ( ema ) ) , qbend 10 ( cd34 class ii ) , ki - s5 ( ki-67 antigen ) , 5.8a ( myo d1 ) were purchased from a commercial source ( dako , denmark ) , and sc-12488 ( runx-2 ) , sc-20095 ( sox-9 ) , sc-71992 ( s-100  chain ) , sc-71993 ( s-100  chain ) , sc-55520 ( fgf-1 ) , and sc-57494 ( vegf ) were purchased from a commercial source ( santa cruz biotechnology , inc , usa ) . for detection of the antigen ,", "the dextran polymer method ( chem mate envision kit , dako , denmark ) was used . to improve detection , the deparaffinized sections were pretreated by microwave heating with citrate buffer ( ph 6 ) .", "the primary antibodies were generally used at a dilution of 1 : 50 , and the incubation time was 1 hour at room temperature .", "positive controls consisted of specimens of schwannoma for s-100  and  ; squamous cell carcinoma for ema , cd34 and ki-67 ; ossifying fibroma for fgf-1 and runx-2 , normal lung tissue for sox-9 ; and inflammatory granulation tissue for myod-1 . as a negative control , mouse igg1 ( ki-67 , vegf , cd34 , s-100  ) , igg2a ( ema , s-100  ) and igg2b ( fgf-1 ) , goat igg ( runx-2 ) and rabbit igg ( sox-9 ) were used instead of the primary antibodies .", "at least 500 nuclei were counted in 5 high - power ( x400 ) fields and ki-67 labeling index was calculated .", "mean intratumor microvessel density was obtained by calculating the average counts of these 5 fields .", "the protocol was approved by the committee on studies involving human beings of nihon university school of dentistry at matsudo ( ec 05 - 002 ) .", "clinical data were retrieved from patient records , and all cases were reviewed microscopically and subclassified .", "the literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed .", "\n case 1a 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly .", "the patient 's mother first noticed a tiny nodule when the patient was 6 months old .", "examination showed a well - defined hard nodular mass , approximately 16    16    9  mm in size , which was sharply demarcated within the muscles of the tongue and freely mobile .", "a yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation .", "clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia .", "gross examination showed a yellowish soft to hard smooth mass measuring 16    15    12  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component .", "chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue ( figure 1 ) .", "pathological diagnosis was made as lipoma with chondroid differentiation . \n  a 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly", ". the patient 's mother first noticed a tiny nodule when the patient was 6 months old .", "examination showed a well - defined hard nodular mass , approximately 16    16    9  mm in size , which was sharply demarcated within the muscles of the tongue and freely mobile .", "a yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation .", "clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia .", "gross examination showed a yellowish soft to hard smooth mass measuring 16    15    12  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component .", "chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue ( figure 1 ) .", "case 2a 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface , which appeared the sense of incompatibility 2 months prior to consultation .", "examination showed a well- defined hard nodular mass , approximately 5  mm in diameter , and freely mobile , and covered with mucosa of normal aspect and color .", "there was no tenderness , no sign of inflammation . to retrieve more - detailed relativity with the surrounding tissue ,", "ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within .", "gross examination showed a yellowish soft to hard smooth mass measuring 9    5  5  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells .", "a focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion ( figure 2 ) .", "pathological diagnosis was made as fibrolipoma with osseous / chondroid differentiation . \n  a 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface , which appeared the sense of incompatibility 2 months prior to consultation .", "examination showed a well- defined hard nodular mass , approximately 5  mm in diameter , and freely mobile , and covered with mucosa of normal aspect and color .", "there was no tenderness , no sign of inflammation . to retrieve more - detailed relativity with the surrounding tissue", ", ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within .", "gross examination showed a yellowish soft to hard smooth mass measuring 9    5  5  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells .", "a focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion ( figure 2 ) .", "the patients made an uneventful recovery , and after 15 years ( case 1 ) and 1-year ( case 2 ) follow - up , there was no sign of recurrence .", "all chondrocytes were stained for s-100  &  , some for runx-2 ( figure 3(a ) ) and some of the outer layer for sox-9 .", "spindle cells were divided into 2 groups ; peripheral spindle cells around osteo / chondroid tissue and distant spindle cells inside the myxoid area .", "peripheral spindle cells around chondroid tissue stained diffusely for s-100  &  and sox-9 ( figure 3(b ) ) , though peripheral spindle cells around osteoid tissue only stained for runx-2 ( figure 3(c ) ) .", "distant spindle cells inside the myxoid area stained diffusely for s-100  and focally for s-100  , myod , sox-9 , and cd34 .", "as for the number of vessels detected by cd34 , there were 5.0 vessels in this tumor , and there was no differences among the inside areas .", "none of the cases stained for either ema or fgf-1 or with vegf . between 1995 and 2007 ,", "the files for all cases of oral lipoma at this department showed 909 cases of nonepithelial tumors in the oral soft tissue .", "of all the lipoma cases , 27 occurred in males and 19 in females ; their mean age was 53.8 years ( range : 2872 years ) .", "most lesions were located in the buccal mucosa ( n = 16 ) , margin of tongue ( n = 10 ) , lower lip ( n = 7 ) , gingiva ( n = 4 ) ,   and others .", "microscopically , 28 cases ( 61.0% ) were classified as lipoma and 18 ( 39.0% ) as fibrolipoma .", "the literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed .", "fourteen reports ( including 16 cases ) were described , and our cases are number 17 and 18 . of all cases ,", "10 occurred in males and 8 in females ; their mean age was 52.4 years ( range : 2181 years ) .", "most lesions were situated in the tongue ( n = 6 ) , lower lip ( n = 5 ) and other areas .", "microscopically , 6 cases ( 33.3% ) had an osseous component , 11 cases ( 61.1% ) had a chondroid component , and 1 case ( our case , 5.6% ) had an osseous / chondroid component .", "\n\t\t\t\t according to our review of the english - language literature , which included our cases , 6 cases were described as lipoma with osseous component [ 57 , 9 , 14 , 15 ] , 11 cases as chondroid component [ 4 , 813 , 16 , 17 ] and 1 case as fibrolipoma with osseous / chondroid components .", "our case 2 is namely the first report of fibrolipoma with osseous / chondroid differentiation .", "osteolipomas are less common than chondrolipomas   in the whole body , and almost the same tendency was described in oral lesion .", "lipoma with osseous / chondroid differentiaion is extremely rare and occurs mainly in large long - standing lipomas [ 15 , 19 ] .", "case 1 also showed 28-year duration , and this might cause the osseous / chondroid to change into lipoma . as with many tumors ,", "further indistinct etiology of osseous / chondroid change in lipoma has been discussed and most researchers mentioned that their origin is from different types of undifferentiated mesenchymal cells .", "the hypothesis is that the neoplastic transformation occurs in multipotential undifferentiated mesenchymal cells that later differentiate into lipoblasts , chondroblasts , or osteoblasts and fibroblasts .", "another hypothesis is that only the adipose cells have a neoplastic transformation , and that the cartilage and bone is produced by differentiation of undifferentiated mesenchymal cells of stroma in chondroblasts or osteoblasts .", "immunohistochemical results in this study indicated that there was no proliferative characteristic in mesenchymal ( spindle ) cells with small positive rates by ki-67 .", "chondrocytes and peripheral spindle cells around chondroid tissue with positive findings for s-100  ,  ,   and sox-9 had chondromatous characteristics .", "sox-9 belongs to the sox ( sry - related high - mobility group box ) family of transcription factors and is a key regulator of developmental processes including chondrogenesis .", "runx-2 is essential for skeletal mineralization when it stimulates osteoblast differentiation of mesenchymal stem cell and mature chondrocyte differentiation .", "positive findings for runx-2 in spindle cells around osteoid components and some of chondrocytes indicated osteoblast / chondrocyte characteristics of them .", "these characteristics are synthesized and thought to be differentiation on account of small positive rate for ki-67 and negative findings for vegf and fgf-1 in this study .", "osseous / chondroid formation occurred in the central part of these tumors in which a small amount of vessels existed .", "multipotential undifferentiated mesenchymal cells had undergone differentiation to osteoblast / chondroblast by topical modification , such as nutrition disorder or asphyxia .", "the bone formation adjacent to chondroid tissue should not be dystrophic calcification because of its positive findings for runx-2 in spindle cells surrounding chondrocytes .", "these immunohistochemical results supported the conclusion that endochondral ossification and/or perichondral ossification had occurred in these tumor . these results supported lipoma with osseous / chondroid differentiation .", "lipomas are benign mesenchymal neoplasms composed of mature adipocytes and are rare soft tissue tumors with a 2.2% incidence rate of the whole body .", "a 20% incidence rate of lipoma has been reported in the head and neck region .", "a 5% incidence rate in oral lesion of this study showed lower than in the head and neck region , but this rate is almost the same tendency compared to previous reports [ 1 , 20 , 21 ] .", "the average age of the patient of lipoma was 53.8 years old , and male - to - female ratio was 1.4 : 1.0 in this study .", "our study supports the current reports that lipomas are generally more common in males than in females ( soft tissue ) , and occurred in adult patients most often between the ages of 40 and 60 years [ 1 , 3 ] .", "the predominant locations were the buccal mucosa and the margin of the tongue and the lower lip , where there is plenty of adipose tissue .", "the patients ' characteristics and location distribution , cheek and tongue , were similar to previous reports [ 1 , 20 , 22 ] .", "microscopically , 28 cases ( 61.0% ) were classified as lipoma and 18 ( 39.0% ) as fibrolipoma .", "fujimara   have reported , but our case 2 is the first report of fibrolipoma with osseous / chondroid differentiation .", "the two cases this reports contributed to pile up of rare lipoma / fibrolipoma with osseous / chondroid differentiation case , and , in addition , immunohistochemical result is contributed to the clarification of the etiology ."], "abstract_text": ["<S> the purpose of this study was to determine the clinicopathological and immunohistochemical features of lipoma / fibrolipoma with rare occasions as osseous and/or chondroid differentiation in the oral cavity . </S>", "<S> two cases of the tumors , who presented with a painless , relatively hard mass on the oral mucosa , were studied . </S>", "<S> these were consisted of a well - circumscribed \n mass of fatty tissue with chondroid and significant fibrous component intermixed with the lobules of fat cells with chondroid and woven bone \n component , respectively . </S>", "<S> immunohistochemical study revealed that peripheral spindle cells around chondroid tissue stained diffusely for s-100  &  and sox-9 , though peripheral spindle cells around osteoid tissue only stained for runx-2 . according to review of the literature , </S>", "<S> lipoma / fibrolipoma with osseous and/or chondroid differentiation was 18 cases . </S>", "<S> also fibrolipoma with osseous and chondroid differentiation is the first to be reported here . </S>", "<S> these results indicated that the cartilage / bone is produced by differentiation of undifferentiated mesenchymal cells of stroma . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["lipomas represent uncommon neoplasms of the oral cavity ; only 1% to 5% of cases occur at this site . based on their histopathological features of conspicuous multiple components", ", lipomas can be divided into some subclasses [ 2 , 3 ] , and the most common is the fibrolipoma [ 1 , 3 , 4 ] .", "cases reporting on lipoma with osteo / chondroid differentiaion were only 16 cases as a result of retrieving literature from 1960 to 2008 .", "further , other names of this subclassification exist in literature ; osteo / chondrolipoma , ossifying / osseous lipoma , lipoma with chondro / osseous metaplasia , and lipoma with cartilaginous / osseous change . the obscure etiology of osseous / chondroid differentiaion led to confusion of the name . to clarify the etiology of osseous / chondroid differentiaion in lipoma , the authors report additional 2 cases of oral lipoma / fibrolipoma with osseous and/or chondroid differentiation and describe the clinical , histopathological , and immunohistological features of these .", "review of the literature and clinicopathological analysis from the files of the oral pathology department , nihon university school of dentistry at matsudo from 1995 to 2007 of these cases were performed and compared ."], ["two cases that had been diagnosed as lipoma with osteo / chondroid differentiation were retrieved from the files mentioned earlier . in these cases ,", "sections of 2 cases were deparaffinized in xylene and dehydrated in tris - buffered saline ( ph 7.6 ) .", "primary antibodies against e29 ( epithelial membrane antigen ( ema ) ) , qbend 10 ( cd34 class ii ) , ki - s5 ( ki-67 antigen ) , 5.8a ( myo d1 ) were purchased from a commercial source ( dako , denmark ) , and sc-12488 ( runx-2 ) , sc-20095 ( sox-9 ) , sc-71992 ( s-100  chain ) , sc-71993 ( s-100  chain ) , sc-55520 ( fgf-1 ) , and sc-57494 ( vegf ) were purchased from a commercial source ( santa cruz biotechnology , inc , usa ) . for detection of the antigen ,", "the dextran polymer method ( chem mate envision kit , dako , denmark ) was used . to improve detection , the deparaffinized sections were pretreated by microwave heating with citrate buffer ( ph 6 ) .", "the primary antibodies were generally used at a dilution of 1 : 50 , and the incubation time was 1 hour at room temperature .", "positive controls consisted of specimens of schwannoma for s-100  and  ; squamous cell carcinoma for ema , cd34 and ki-67 ; ossifying fibroma for fgf-1 and runx-2 , normal lung tissue for sox-9 ; and inflammatory granulation tissue for myod-1 . as a negative control , mouse igg1 ( ki-67 , vegf , cd34 , s-100  ) , igg2a ( ema , s-100  ) and igg2b ( fgf-1 ) , goat igg ( runx-2 ) and rabbit igg ( sox-9 ) were used instead of the primary antibodies .", "at least 500 nuclei were counted in 5 high - power ( x400 ) fields and ki-67 labeling index was calculated .", "mean intratumor microvessel density was obtained by calculating the average counts of these 5 fields .", "the protocol was approved by the committee on studies involving human beings of nihon university school of dentistry at matsudo ( ec 05 - 002 ) .", "clinical data were retrieved from patient records , and all cases were reviewed microscopically and subclassified .", "the literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed ."], ["\n case 1a 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly .", "the patient 's mother first noticed a tiny nodule when the patient was 6 months old .", "examination showed a well - defined hard nodular mass , approximately 16    16    9  mm in size , which was sharply demarcated within the muscles of the tongue and freely mobile .", "a yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation .", "clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia .", "gross examination showed a yellowish soft to hard smooth mass measuring 16    15    12  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component .", "chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue ( figure 1 ) .", "pathological diagnosis was made as lipoma with chondroid differentiation . \n  a 28-year - old woman presented with a painless relatively hard mass on the dorsal surface in the midline of the tongue that had recently grew slightly", ". the patient 's mother first noticed a tiny nodule when the patient was 6 months old .", "examination showed a well - defined hard nodular mass , approximately 16    16    9  mm in size , which was sharply demarcated within the muscles of the tongue and freely mobile .", "a yellowish tinge was visible through the overlying mucous membrane and the lesion was firm on palpation .", "clinical diagnosis of lipoma with calculus was made and the tumor was completely excised from the tongue under general anesthesia .", "gross examination showed a yellowish soft to hard smooth mass measuring 16    15    12  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of fatty tissue with a cellular chondroid component .", "chondromatous nodules within uniform adipose tissue were seen in large areas of mature fat cells supported by fibrous connective tissue ( figure 1 ) .", "case 2a 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface , which appeared the sense of incompatibility 2 months prior to consultation .", "examination showed a well- defined hard nodular mass , approximately 5  mm in diameter , and freely mobile , and covered with mucosa of normal aspect and color .", "there was no tenderness , no sign of inflammation . to retrieve more - detailed relativity with the surrounding tissue ,", "ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within .", "gross examination showed a yellowish soft to hard smooth mass measuring 9    5  5  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells .", "a focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion ( figure 2 ) .", "pathological diagnosis was made as fibrolipoma with osseous / chondroid differentiation . \n  a 59-year - old man presented with a relatively painless hard mass on the left side of the lower labial vestibule surface , which appeared the sense of incompatibility 2 months prior to consultation .", "examination showed a well- defined hard nodular mass , approximately 5  mm in diameter , and freely mobile , and covered with mucosa of normal aspect and color .", "there was no tenderness , no sign of inflammation . to retrieve more - detailed relativity with the surrounding tissue", ", ct was taken and showed a lesion in the left side of the lower lip that appeared to be a small mass with areas of little calcification within .", "gross examination showed a yellowish soft to hard smooth mass measuring 9    5  5  mm in size .", "microscopically , the tumor consisted of a well - circumscribed mass of mature fat cells supported by fibrous connective tissue septa and myxoid tissue characterized by spindle cells .", "a focal island of consecutive chondroid and woven bone component was surrounded by spindle / fusiform - shaped mesenchymal cells throughout the lesion ( figure 2 ) .", "the patients made an uneventful recovery , and after 15 years ( case 1 ) and 1-year ( case 2 ) follow - up , there was no sign of recurrence .", "all chondrocytes were stained for s-100  &  , some for runx-2 ( figure 3(a ) ) and some of the outer layer for sox-9 .", "spindle cells were divided into 2 groups ; peripheral spindle cells around osteo / chondroid tissue and distant spindle cells inside the myxoid area .", "peripheral spindle cells around chondroid tissue stained diffusely for s-100  &  and sox-9 ( figure 3(b ) ) , though peripheral spindle cells around osteoid tissue only stained for runx-2 ( figure 3(c ) ) .", "distant spindle cells inside the myxoid area stained diffusely for s-100  and focally for s-100  , myod , sox-9 , and cd34 .", "as for the number of vessels detected by cd34 , there were 5.0 vessels in this tumor , and there was no differences among the inside areas .", "none of the cases stained for either ema or fgf-1 or with vegf . between 1995 and 2007 ,", "the files for all cases of oral lipoma at this department showed 909 cases of nonepithelial tumors in the oral soft tissue .", "of all the lipoma cases , 27 occurred in males and 19 in females ; their mean age was 53.8 years ( range : 2872 years ) .", "most lesions were located in the buccal mucosa ( n = 16 ) , margin of tongue ( n = 10 ) , lower lip ( n = 7 ) , gingiva ( n = 4 ) ,   and others .", "microscopically , 28 cases ( 61.0% ) were classified as lipoma and 18 ( 39.0% ) as fibrolipoma .", "the literature from 1960 to 2008 of lipoma with osseous / chondroid differentiation was reviewed .", "fourteen reports ( including 16 cases ) were described , and our cases are number 17 and 18 . of all cases ,", "10 occurred in males and 8 in females ; their mean age was 52.4 years ( range : 2181 years ) .", "most lesions were situated in the tongue ( n = 6 ) , lower lip ( n = 5 ) and other areas .", "microscopically , 6 cases ( 33.3% ) had an osseous component , 11 cases ( 61.1% ) had a chondroid component , and 1 case ( our case , 5.6% ) had an osseous / chondroid component ."], ["\n\t\t\t\t according to our review of the english - language literature , which included our cases , 6 cases were described as lipoma with osseous component [ 57 , 9 , 14 , 15 ] , 11 cases as chondroid component [ 4 , 813 , 16 , 17 ] and 1 case as fibrolipoma with osseous / chondroid components .", "our case 2 is namely the first report of fibrolipoma with osseous / chondroid differentiation .", "osteolipomas are less common than chondrolipomas   in the whole body , and almost the same tendency was described in oral lesion .", "lipoma with osseous / chondroid differentiaion is extremely rare and occurs mainly in large long - standing lipomas [ 15 , 19 ] .", "case 1 also showed 28-year duration , and this might cause the osseous / chondroid to change into lipoma . as with many tumors ,", "further indistinct etiology of osseous / chondroid change in lipoma has been discussed and most researchers mentioned that their origin is from different types of undifferentiated mesenchymal cells .", "the hypothesis is that the neoplastic transformation occurs in multipotential undifferentiated mesenchymal cells that later differentiate into lipoblasts , chondroblasts , or osteoblasts and fibroblasts .", "another hypothesis is that only the adipose cells have a neoplastic transformation , and that the cartilage and bone is produced by differentiation of undifferentiated mesenchymal cells of stroma in chondroblasts or osteoblasts .", "immunohistochemical results in this study indicated that there was no proliferative characteristic in mesenchymal ( spindle ) cells with small positive rates by ki-67 .", "chondrocytes and peripheral spindle cells around chondroid tissue with positive findings for s-100  ,  ,   and sox-9 had chondromatous characteristics .", "sox-9 belongs to the sox ( sry - related high - mobility group box ) family of transcription factors and is a key regulator of developmental processes including chondrogenesis .", "runx-2 is essential for skeletal mineralization when it stimulates osteoblast differentiation of mesenchymal stem cell and mature chondrocyte differentiation .", "positive findings for runx-2 in spindle cells around osteoid components and some of chondrocytes indicated osteoblast / chondrocyte characteristics of them .", "these characteristics are synthesized and thought to be differentiation on account of small positive rate for ki-67 and negative findings for vegf and fgf-1 in this study .", "osseous / chondroid formation occurred in the central part of these tumors in which a small amount of vessels existed .", "multipotential undifferentiated mesenchymal cells had undergone differentiation to osteoblast / chondroblast by topical modification , such as nutrition disorder or asphyxia .", "the bone formation adjacent to chondroid tissue should not be dystrophic calcification because of its positive findings for runx-2 in spindle cells surrounding chondrocytes .", "these immunohistochemical results supported the conclusion that endochondral ossification and/or perichondral ossification had occurred in these tumor . these results supported lipoma with osseous / chondroid differentiation .", "lipomas are benign mesenchymal neoplasms composed of mature adipocytes and are rare soft tissue tumors with a 2.2% incidence rate of the whole body .", "a 20% incidence rate of lipoma has been reported in the head and neck region .", "a 5% incidence rate in oral lesion of this study showed lower than in the head and neck region , but this rate is almost the same tendency compared to previous reports [ 1 , 20 , 21 ] .", "the average age of the patient of lipoma was 53.8 years old , and male - to - female ratio was 1.4 : 1.0 in this study .", "our study supports the current reports that lipomas are generally more common in males than in females ( soft tissue ) , and occurred in adult patients most often between the ages of 40 and 60 years [ 1 , 3 ] .", "the predominant locations were the buccal mucosa and the margin of the tongue and the lower lip , where there is plenty of adipose tissue .", "the patients ' characteristics and location distribution , cheek and tongue , were similar to previous reports [ 1 , 20 , 22 ] .", "microscopically , 28 cases ( 61.0% ) were classified as lipoma and 18 ( 39.0% ) as fibrolipoma .", "fujimara   have reported , but our case 2 is the first report of fibrolipoma with osseous / chondroid differentiation .", "the two cases this reports contributed to pile up of rare lipoma / fibrolipoma with osseous / chondroid differentiation case , and , in addition , immunohistochemical result is contributed to the clarification of the etiology ."]]}
{"article_id": "PMC3114617", "article_text": ["leydig cell tumours are rare stromal tumours of the testis accounting for 13% of testicular neoplasms .", "they exhibit a peak incidence in the preadolescent as well as in the older ( > 50 years ) age groups [ 1 , 2 , 3 , 4 ] .", "the diagnosis of a malignant leydig cell tumour is not always easy since no definite histological criteria for malignancy exist .", "a 35-year - old man presented to our hospital with a 3-month history of a painless left testicular mass .", "blood concentrations of chorionic gonadotrophin , fetoprotein and human placental lactogen were within the reference range .", "a left radical orchidectomy was performed and the specimen submitted for histopathological examination . on microscopic examination , a malignant leydig cell tumour was found .", "the cells displayed acidophilic cytoplasm , intranuclear inclusions and increased mitotic activity ( > 3/10 hpf ) .", "many cells with large atypical pleomorphic nuclei could also be observed ( fig . 1 , fig .", "2 ) . there was no angiolymphatic invasion , foci of necrosis or extension beyond the capsule of the testis .", "the immunohistochemical study showed that the tumour cells were positive for vimentin , melan a and inhibin , and negative for ckae1 , ckae3 , s100p , p63 , cea , afp and actin .", "3 ) . retroperitoneal lymph node dissection was suggested , but the patient declined further surgery .", "leydig cell tumours are rare and only about 3% of them are bilateral . about 1520% of the patients already present with metastatic disease , particularly in the lymph nodes , lung and liver", ". these tumours may be hormonally active , and gynaecomastia is seen in 30% of the cases [ 2 , 3 , 6 ] .", "leydig cell tumours in an undescended testis may exhibit only manifestation of endocrinological disorders such as gynaecomastia , impotence and loss of libido .", "however , among the 480 cases of leydig cell tumours reported in the literature , only 20 cases were associated with cryptorchidism , and there is no evidence that undescended testes are more prone to develop leydig cell tumours [ 2 , 5 , 7 ] .", "features associated with malignancy include a large tumour size ( 5.7 cm ) , nuclear atypia , a mitotic count of > 3/10 hpf , foci of necrosis , angiolymphatic invasion , infiltrative margins , dna aneuploidy and an increased expression of ki67/mib-1 and p53 [ 2 , 3 , 8 , 9 , 10 ] .", "our patient was young and did not fit into either of the known age incidence peaks .", "microscopic features such as the severe nuclear atypia and increased mitotic activity ( > 3/10 hpf ) favoured the diagnosis of malignancy . however , there was no angiolymphatic invasion or extension beyond the capsule of the testis . also , the small size of the tumour was remarkable ."], "abstract_text": ["<S> leydig cell tumour is a benign testicular non - germ cell tumour , and malignant transformation is rare . </S>", "<S> we report a case of a 35-year - old man who came to our hospital with a painless left testicular mass measuring 1.2  1 cm . </S>", "<S> histological evaluation of the tumour showed features of a malignant leydig cell tumour but no infiltration beyond the capsule or metastasis . </S>", "<S> the small size of the tumour was remarkable . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["leydig cell tumours are rare stromal tumours of the testis accounting for 13% of testicular neoplasms .", "they exhibit a peak incidence in the preadolescent as well as in the older ( > 50 years ) age groups [ 1 , 2 , 3 , 4 ] .", "the diagnosis of a malignant leydig cell tumour is not always easy since no definite histological criteria for malignancy exist ."], ["a 35-year - old man presented to our hospital with a 3-month history of a painless left testicular mass .", "blood concentrations of chorionic gonadotrophin , fetoprotein and human placental lactogen were within the reference range .", "a left radical orchidectomy was performed and the specimen submitted for histopathological examination . on microscopic examination , a malignant leydig cell tumour was found .", "the cells displayed acidophilic cytoplasm , intranuclear inclusions and increased mitotic activity ( > 3/10 hpf ) .", "many cells with large atypical pleomorphic nuclei could also be observed ( fig . 1 , fig .", "2 ) . there was no angiolymphatic invasion , foci of necrosis or extension beyond the capsule of the testis .", "the immunohistochemical study showed that the tumour cells were positive for vimentin , melan a and inhibin , and negative for ckae1 , ckae3 , s100p , p63 , cea , afp and actin .", "3 ) . retroperitoneal lymph node dissection was suggested , but the patient declined further surgery ."], ["leydig cell tumours are rare and only about 3% of them are bilateral . about 1520% of the patients already present with metastatic disease , particularly in the lymph nodes , lung and liver", ". these tumours may be hormonally active , and gynaecomastia is seen in 30% of the cases [ 2 , 3 , 6 ] .", "leydig cell tumours in an undescended testis may exhibit only manifestation of endocrinological disorders such as gynaecomastia , impotence and loss of libido .", "however , among the 480 cases of leydig cell tumours reported in the literature , only 20 cases were associated with cryptorchidism , and there is no evidence that undescended testes are more prone to develop leydig cell tumours [ 2 , 5 , 7 ] .", "features associated with malignancy include a large tumour size ( 5.7 cm ) , nuclear atypia , a mitotic count of > 3/10 hpf , foci of necrosis , angiolymphatic invasion , infiltrative margins , dna aneuploidy and an increased expression of ki67/mib-1 and p53 [ 2 , 3 , 8 , 9 , 10 ] .", "our patient was young and did not fit into either of the known age incidence peaks .", "microscopic features such as the severe nuclear atypia and increased mitotic activity ( > 3/10 hpf ) favoured the diagnosis of malignancy . however , there was no angiolymphatic invasion or extension beyond the capsule of the testis . also , the small size of the tumour was remarkable ."]]}
{"article_id": "PMC3560552", "article_text": ["the prevalences of asthma , allergic rhinitis and allergic eye disease in antalya , on the south coast of turkey , have been reported as 8.2% , 10.8% and 7.5% , respectively .", "allergic diseases are most likely due to complex interactions between largely unknown genetic and environmental factors [ 36 ] .", "the micro - array techniques for the detection of specific ige have improved the diagnostic procedures for allergic diseases .", "a detailed knowledge of the sensitization pattern may have relevant implications for the prescription of specific immunotherapy .", "a number of epidemiologic studies have also supported a relationship between allergic rhinoconjunctivitis and diet , hygiene , and life - style , suggesting that environmental factors also impact the development of allergic rhinoconjunctivitis .", "apoptosis is an active physiological process that can cause an inflammatory reaction and tissue damage , and is fundamental to maturation and homeostasis in the immune system .", "it can be induced passively , through lack of essential survival signals , or actively , through ligand - induced trimerization of specific death receptors of the tumor necrosis factor ( tnf ) receptor family , such as fas , the tnf receptor , or the tnf - related apoptosis - inducing ligand ( trail ) receptor .", "trail also is able to prevent apoptosis through the actions of its decoy receptors , dcr-1 and dcr-2 .", "various regulators of trail include fadd , iaps , bcl-2s , p53 , and flips .", "trail is present in cells involved in asthma , including eosinophils , mast cells , fibroblasts , and airway epithelial cells .", "it is expressed in airway remodeling and may be linked with the pathways of transforming growth factor beta1 , which is thought to cause damage to the epithelium .", "the repair process of the epithelium is hindered as a result of increased apoptosis induced by tgf - beta1 , which overlaps with the pathways of trail .", "trail is also seen as the basis for a  miracle  drug for cancer because of its ability to selectively kill cancer cells .", "it has previously been reported that negative selection of t cells in the thymus is controlled by trail .", "for example , mice deficient in trail had a severe defect in thymic deletion of t cells and were hypersensitive to collagen - induced arthritis .", "mast cells activation through fc epsilon ri cross - linking has a pivotal role in the initiation of allergic reactions .", "ige - dependent activation increases trail - induced caspase-8 and caspase-3 cleavage , and regulates human mast cell apoptosis by fine - tuning anti - apoptotic and pro - apoptotic factors .", "our study aimed to identify the role of strail in the pathophysiology of allergic rhinoconjunctivitis , and to explore whether allergen - specific subcutaneous immunotherapy treatment of allergic rhinoconjunctivitis patients altered any observed effect of strail .", "the study was conducted in antalya , turkey between 9 january 2009 and 28 january 2010 .", "the study was approved by the local ethics committee , and written consent was obtained from all patients and healthy volunteers .", "all patients were followed in the immunology and allergy clinic of antalya education and research hospital .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity , ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically .", "the first group of 25 patients included 11 male and 14 female subjects with allergic rhinoconjunctivitis , having a combined mean age of 38.5612.03 years .", "the first group included 2 measurements in the same patients ; group - ia represents data recorded before subcutaneous allergen - specific immunotherapy , and group - ib shows data recorded 12 months after the subcutaneous allergen - specific immunotherapy .", "all patients received immunotherapy every 4 weeks . the symptoms and severity of allergic reactions were recorded before and after treatment .", "a second group of 25 healthy individuals ( 11 male and 14 female ) ( group ii ) had a mean age of 38.2312.21 years ( table 1 ) .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically .", "blood samples were collected into 5 ml plain vacutainer tubes and centrifuged at 3000  g for 10 min .", "serum ige levels , hepatitis markers ( hbs ag , anti hbs , anti hcv ) were evaluated in all patients .", "total and specific ige levels were enumerated by fluoroenzyme immunoassay ( immunocap - feia ) using an immunocap kit ( phar - macia , uppsala , sweden ) .", "values above 100 ku / l and 0.35 ku / l for total and specific ige levels were considered abnormal .", "serum strail levels in all individuals ( patients and healthy controls ) were measured by a sandwich enzyme - linked immunosorbent assay ( diaclone , france ) .", "skin prick tests on the forearm were performed in all patients , using standardized latex extract containing high ammonia natural rubber latex , and a full set of 10 common allergens .", "in addition , venom spt was performed on 1 patient based on the subject s clinical history .", "student s t test was used for comparison of controls and patient groups ( group ia and group ib ) .", "the study was conducted in antalya , turkey between 9 january 2009 and 28 january 2010 .", "the study was approved by the local ethics committee , and written consent was obtained from all patients and healthy volunteers .", "all patients were followed in the immunology and allergy clinic of antalya education and research hospital .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity , ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically .", "the first group of 25 patients included 11 male and 14 female subjects with allergic rhinoconjunctivitis , having a combined mean age of 38.5612.03 years .", "the first group included 2 measurements in the same patients ; group - ia represents data recorded before subcutaneous allergen - specific immunotherapy , and group - ib shows data recorded 12 months after the subcutaneous allergen - specific immunotherapy .", "all patients received immunotherapy every 4 weeks . the symptoms and severity of allergic reactions were recorded before and after treatment .", "a second group of 25 healthy individuals ( 11 male and 14 female ) ( group ii ) had a mean age of 38.2312.21 years ( table 1 ) .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically .", "blood samples were collected into 5 ml plain vacutainer tubes and centrifuged at 3000  g for 10 min .", "serum ige levels , hepatitis markers ( hbs ag , anti hbs , anti hcv ) were evaluated in all patients .", "total and specific ige levels were enumerated by fluoroenzyme immunoassay ( immunocap - feia ) using an immunocap kit ( phar - macia , uppsala , sweden ) .", "values above 100 ku / l and 0.35 ku / l for total and specific ige levels were considered abnormal .", "serum strail levels in all individuals ( patients and healthy controls ) were measured by a sandwich enzyme - linked immunosorbent assay ( diaclone , france ) .", "skin prick tests on the forearm were performed in all patients , using standardized latex extract containing high ammonia natural rubber latex , and a full set of 10 common allergens .", "in addition , venom spt was performed on 1 patient based on the subject s clinical history .", "student s t test was used for comparison of controls and patient groups ( group ia and group ib ) .", "in this clinical follow - up study , 25 patients were already receiving subcutaneous immunotherapy , and these subjects were included for further analysis at our clinic .", "the mean ige levels were : group ia  699.505 iu / ml ; group ib  164.115 iu / ml ; and group ii ", "41.08 iu / ml . there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0/0005 ) .", "prick tests in all patients in group i were detected in mite , olive and grass allergy .", "these results correlated with specific ige , and hepatitis markers were negative in all patients .", "as shown in figure 1 , significant difference was seen in the mean values of strail in allergic rhinitis patients before immunotherapy ( n=25 ; 939.85352.52 pg / ml ) , afterc immunotherapy ( n=25 ; 628.93170.5 pg / ml ) and healthy controls ( n=25 ; 612.64135.6 pg / ml ) .", "there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0.001 ) .", "while there was a statistically significant difference between the pre - treated group and control group ( p<0.0001 ) , there was no difference between the post - treated group and healthy individuals ( p=0.801 ) .", "as shown in table 3 , there was a statistically significant difference between the values of the research group before and after immunotherapy ( grasses mixture , barley mixture , oleaauropeae , d. pteronyssinus , d. farinae )", "in this clinical follow - up study , 25 patients were already receiving subcutaneous immunotherapy , and these subjects were included for further analysis at our clinic .", "the mean ige levels were : group ia  699.505 iu / ml ; group ib  164.115 iu / ml ; and group ii ", "41.08 iu / ml . there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0/0005 ) .", "prick tests in all patients in group i were detected in mite , olive and grass allergy .", "these results correlated with specific ige , and hepatitis markers were negative in all patients .", "as shown in figure 1 , significant difference was seen in the mean values of strail in allergic rhinitis patients before immunotherapy ( n=25 ; 939.85352.52 pg / ml ) , afterc immunotherapy ( n=25 ; 628.93170.5 pg / ml ) and healthy controls ( n=25 ; 612.64135.6 pg / ml ) .", "there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0.001 ) .", "while there was a statistically significant difference between the pre - treated group and control group ( p<0.0001 ) , there was no difference between the post - treated group and healthy individuals ( p=0.801 ) .", "as shown in table 3 , there was a statistically significant difference between the values of the research group before and after immunotherapy ( grasses mixture , barley mixture , oleaauropeae , d. pteronyssinus , d. farinae )", "allergen - specific immunotherapy has been used in the management of allergic diseases for nearly 100 years .", "the results of our previous study suggest that in our region 51.8% of the allergens determined by prick test are mite and 42.3% are pollens", ". pollen positivity rate of the cases that had immune - therapy was 61.8% , and mite positivity rate was 60.4% .", "allergens are known to exhibit regional variation in expression , which suggests that the allergen profiles and skin prick tests should be designed with reference to individual locales .", "allergen - specific immunotherapy treatment significantly reduced the nasal symptom score across all group i patients studied . in this study ,", "spt was a statistically significant difference between the values of the research group before and after allergen - specific subcutaneous immunotherapy ( grasses mixture , barley mixture , oleaauropeae , d. pteronyssinus , d. farinae ) .", "our earlier studies provided a novel perspective on severe persistent allergic asthma and the effect of anti - ige treatment , using as markers serum soluble tnf - related apoptosis - inducing ligand , total antioxidant capacity , hydrogen peroxide , malondialdehyde , ceruloplasmine oxidase activity , high sensitive c reactive protein and total nitric oxide concentrations measurements [ 1316 ] . in our previous study we found that the trail levels in variances of the patients who had the effective anti ige treatment were significantly lower than the healthy controls .", "engagement of the fas / fasl system has not yet been shown to contribute to increase apoptosis .", "recent interest has focused on the molecule trail , which is involved in the pathophysiology of different diseases , including cancer , diabetes mellitus , autoimmune diseases , and inflammation [ 1721 ] .", "trail also present in cells that involved in asthma including eosinophils , mast cells , fibroblasts , and airway epithelial cells .", "it is expressed in airway remodeling and may be linked with the pathways of transforming growth factor - beta ( tgf- ) , which is thought to cause damage to the epithelium .", "the repair process of the epithelium is hindered as a result of increased apoptosis induced by tgf - beta , which overlaps with the pathways of trail .", "these results reflect the different mechanism(s ) in the pathogenesis of allergic diseases by the regulation of apoptosis .", "desloratadine ( dcl ) is a non - sedating antihistamine approved for the treatment of allergic rhinitis .", "patients in groups i - a and b had a dcl usage history during the exacerbation phase from may to november .", "blood samples from the patients were obtained in january , when they would be expected to have fewer allergic symptoms .", "mast cells ( mc ) play a key role in allergy and are involved in several chronic inflammatory diseases . furthermore , they are involved in innate immunity and in tissue repair [ 2427 ] .", "the regulation of mc numbers , as of any other normal cells , depends on both their generation rate and survival time within tissues .", "the critical event in allergic reactions is allergen - induced crosslinking of specific ige molecules bound to fc_ri receptors on the mc surface , which triggers mc degranulation and release of inflammatory mediators .", "non - ige - mediated activation may also contribute to continued degranulation of mc during the late phase of allergic reactions .", "allergic mechanism are involved in the increased susceptibility of human mc to trail - induced apoptosis after ige - dependent activation . in this study ,", "ige levels had a statistically significant difference between the values of the research group before and after allergen - specific subcutaneous immunotherapy .", "taken together , our results and those of others , suggest that characterization of the specific receptor systems activated , and the pro - inflammatory factors regulated , by trail in vivo may lead to the development of novel therapeutic strategies for diseases as diverse as infection , autoimmunity , and allergy ."], "abstract_text": ["<S> summarybackgroundallergic rhinitis is a common health problem affecting the immune system . </S>", "<S> the homeostasis of the immune system is regulated by apoptosis . in this study , </S>", "<S> serum circulating soluble trail levels of allergic rhinoconjunctivitis patients before and after allergen - specific immunotherapy were evaluated.material/methodsthe strail levels of pre- and post - treated allergic rhinoconjunctivitis patients ( n=25 ) were compared to age- and sex - matched healthy individuals ( n=25 ) . </S>", "<S> strail levels were measured by elisa . the skin prick test ( spt ) </S>", "<S> results were recorded before and after treatment.resultsthe strail levels between the pre - treated and control groups were significantly different ( p<0.0001 ) . </S>", "<S> however , there was no significant difference between the post - treated group and healthy individuals ( p=0,801 ) . </S>", "<S> spt was a statistically significant difference between the values of the research group before and after immunotherapy ( grasses mixture , barley mixture , oleaauropeae , d. pteronyssinus , d. farinae).conclusionsthe strail levels were decreased after allergen - specific immunotherapy to healthy levels and may be of use as a marker of efficacy of immunotherapy in allergic rhinoconjunctivitis patients . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Patients", "Laboratory investigations", "Skin-Prick Test (SPT)", "Statistical analysis", "Results", "Laboratory findings", "Skin-Prick Test (SPT)", "Discussion", "Conclusions"], "sections": [["the prevalences of asthma , allergic rhinitis and allergic eye disease in antalya , on the south coast of turkey , have been reported as 8.2% , 10.8% and 7.5% , respectively .", "allergic diseases are most likely due to complex interactions between largely unknown genetic and environmental factors [ 36 ] .", "the micro - array techniques for the detection of specific ige have improved the diagnostic procedures for allergic diseases .", "a detailed knowledge of the sensitization pattern may have relevant implications for the prescription of specific immunotherapy .", "a number of epidemiologic studies have also supported a relationship between allergic rhinoconjunctivitis and diet , hygiene , and life - style , suggesting that environmental factors also impact the development of allergic rhinoconjunctivitis .", "apoptosis is an active physiological process that can cause an inflammatory reaction and tissue damage , and is fundamental to maturation and homeostasis in the immune system .", "it can be induced passively , through lack of essential survival signals , or actively , through ligand - induced trimerization of specific death receptors of the tumor necrosis factor ( tnf ) receptor family , such as fas , the tnf receptor , or the tnf - related apoptosis - inducing ligand ( trail ) receptor .", "trail also is able to prevent apoptosis through the actions of its decoy receptors , dcr-1 and dcr-2 .", "various regulators of trail include fadd , iaps , bcl-2s , p53 , and flips .", "trail is present in cells involved in asthma , including eosinophils , mast cells , fibroblasts , and airway epithelial cells .", "it is expressed in airway remodeling and may be linked with the pathways of transforming growth factor beta1 , which is thought to cause damage to the epithelium .", "the repair process of the epithelium is hindered as a result of increased apoptosis induced by tgf - beta1 , which overlaps with the pathways of trail .", "trail is also seen as the basis for a  miracle  drug for cancer because of its ability to selectively kill cancer cells .", "it has previously been reported that negative selection of t cells in the thymus is controlled by trail .", "for example , mice deficient in trail had a severe defect in thymic deletion of t cells and were hypersensitive to collagen - induced arthritis .", "mast cells activation through fc epsilon ri cross - linking has a pivotal role in the initiation of allergic reactions .", "ige - dependent activation increases trail - induced caspase-8 and caspase-3 cleavage , and regulates human mast cell apoptosis by fine - tuning anti - apoptotic and pro - apoptotic factors .", "our study aimed to identify the role of strail in the pathophysiology of allergic rhinoconjunctivitis , and to explore whether allergen - specific subcutaneous immunotherapy treatment of allergic rhinoconjunctivitis patients altered any observed effect of strail ."], ["the study was conducted in antalya , turkey between 9 january 2009 and 28 january 2010 .", "the study was approved by the local ethics committee , and written consent was obtained from all patients and healthy volunteers .", "all patients were followed in the immunology and allergy clinic of antalya education and research hospital .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity , ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically .", "the first group of 25 patients included 11 male and 14 female subjects with allergic rhinoconjunctivitis , having a combined mean age of 38.5612.03 years .", "the first group included 2 measurements in the same patients ; group - ia represents data recorded before subcutaneous allergen - specific immunotherapy , and group - ib shows data recorded 12 months after the subcutaneous allergen - specific immunotherapy .", "all patients received immunotherapy every 4 weeks . the symptoms and severity of allergic reactions were recorded before and after treatment .", "a second group of 25 healthy individuals ( 11 male and 14 female ) ( group ii ) had a mean age of 38.2312.21 years ( table 1 ) .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically .", "blood samples were collected into 5 ml plain vacutainer tubes and centrifuged at 3000  g for 10 min .", "serum ige levels , hepatitis markers ( hbs ag , anti hbs , anti hcv ) were evaluated in all patients .", "total and specific ige levels were enumerated by fluoroenzyme immunoassay ( immunocap - feia ) using an immunocap kit ( phar - macia , uppsala , sweden ) .", "values above 100 ku / l and 0.35 ku / l for total and specific ige levels were considered abnormal .", "serum strail levels in all individuals ( patients and healthy controls ) were measured by a sandwich enzyme - linked immunosorbent assay ( diaclone , france ) .", "skin prick tests on the forearm were performed in all patients , using standardized latex extract containing high ammonia natural rubber latex , and a full set of 10 common allergens .", "in addition , venom spt was performed on 1 patient based on the subject s clinical history .", "student s t test was used for comparison of controls and patient groups ( group ia and group ib ) ."], ["the study was conducted in antalya , turkey between 9 january 2009 and 28 january 2010 .", "the study was approved by the local ethics committee , and written consent was obtained from all patients and healthy volunteers .", "all patients were followed in the immunology and allergy clinic of antalya education and research hospital .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity , ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically .", "the first group of 25 patients included 11 male and 14 female subjects with allergic rhinoconjunctivitis , having a combined mean age of 38.5612.03 years .", "the first group included 2 measurements in the same patients ; group - ia represents data recorded before subcutaneous allergen - specific immunotherapy , and group - ib shows data recorded 12 months after the subcutaneous allergen - specific immunotherapy .", "all patients received immunotherapy every 4 weeks . the symptoms and severity of allergic reactions were recorded before and after treatment .", "a second group of 25 healthy individuals ( 11 male and 14 female ) ( group ii ) had a mean age of 38.2312.21 years ( table 1 ) .", "subjects with kidney disease , heart disease , liver disease , diabetes mellitus , cancer status , obesity ( body mass index ( bmi ) 30 kgm ) , and autoimmune disease were excluded clinically and serologically ."], ["blood samples were collected into 5 ml plain vacutainer tubes and centrifuged at 3000  g for 10 min .", "serum ige levels , hepatitis markers ( hbs ag , anti hbs , anti hcv ) were evaluated in all patients .", "total and specific ige levels were enumerated by fluoroenzyme immunoassay ( immunocap - feia ) using an immunocap kit ( phar - macia , uppsala , sweden ) .", "values above 100 ku / l and 0.35 ku / l for total and specific ige levels were considered abnormal .", "serum strail levels in all individuals ( patients and healthy controls ) were measured by a sandwich enzyme - linked immunosorbent assay ( diaclone , france ) ."], ["skin prick tests on the forearm were performed in all patients , using standardized latex extract containing high ammonia natural rubber latex , and a full set of 10 common allergens .", "in addition , venom spt was performed on 1 patient based on the subject s clinical history ."], ["student s t test was used for comparison of controls and patient groups ( group ia and group ib ) ."], ["in this clinical follow - up study , 25 patients were already receiving subcutaneous immunotherapy , and these subjects were included for further analysis at our clinic .", "the mean ige levels were : group ia  699.505 iu / ml ; group ib  164.115 iu / ml ; and group ii ", "41.08 iu / ml . there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0/0005 ) .", "prick tests in all patients in group i were detected in mite , olive and grass allergy .", "these results correlated with specific ige , and hepatitis markers were negative in all patients .", "as shown in figure 1 , significant difference was seen in the mean values of strail in allergic rhinitis patients before immunotherapy ( n=25 ; 939.85352.52 pg / ml ) , afterc immunotherapy ( n=25 ; 628.93170.5 pg / ml ) and healthy controls ( n=25 ; 612.64135.6 pg / ml ) .", "there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0.001 ) .", "while there was a statistically significant difference between the pre - treated group and control group ( p<0.0001 ) , there was no difference between the post - treated group and healthy individuals ( p=0.801 ) .", "as shown in table 3 , there was a statistically significant difference between the values of the research group before and after immunotherapy ( grasses mixture , barley mixture , oleaauropeae , d. pteronyssinus , d. farinae )"], ["in this clinical follow - up study , 25 patients were already receiving subcutaneous immunotherapy , and these subjects were included for further analysis at our clinic .", "the mean ige levels were : group ia  699.505 iu / ml ; group ib  164.115 iu / ml ; and group ii ", "41.08 iu / ml . there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0/0005 ) .", "prick tests in all patients in group i were detected in mite , olive and grass allergy .", "these results correlated with specific ige , and hepatitis markers were negative in all patients .", "as shown in figure 1 , significant difference was seen in the mean values of strail in allergic rhinitis patients before immunotherapy ( n=25 ; 939.85352.52 pg / ml ) , afterc immunotherapy ( n=25 ; 628.93170.5 pg / ml ) and healthy controls ( n=25 ; 612.64135.6 pg / ml ) .", "there was a statistically significant difference between the values of the research group before and after immunotherapy ( p=0.001 ) .", "while there was a statistically significant difference between the pre - treated group and control group ( p<0.0001 ) , there was no difference between the post - treated group and healthy individuals ( p=0.801 ) ."], ["as shown in table 3 , there was a statistically significant difference between the values of the research group before and after immunotherapy ( grasses mixture , barley mixture , oleaauropeae , d. pteronyssinus , d. farinae )"], ["allergen - specific immunotherapy has been used in the management of allergic diseases for nearly 100 years .", "the results of our previous study suggest that in our region 51.8% of the allergens determined by prick test are mite and 42.3% are pollens", ". pollen positivity rate of the cases that had immune - therapy was 61.8% , and mite positivity rate was 60.4% .", "allergens are known to exhibit regional variation in expression , which suggests that the allergen profiles and skin prick tests should be designed with reference to individual locales .", "allergen - specific immunotherapy treatment significantly reduced the nasal symptom score across all group i patients studied . in this study ,", "spt was a statistically significant difference between the values of the research group before and after allergen - specific subcutaneous immunotherapy ( grasses mixture , barley mixture , oleaauropeae , d. pteronyssinus , d. farinae ) .", "our earlier studies provided a novel perspective on severe persistent allergic asthma and the effect of anti - ige treatment , using as markers serum soluble tnf - related apoptosis - inducing ligand , total antioxidant capacity , hydrogen peroxide , malondialdehyde , ceruloplasmine oxidase activity , high sensitive c reactive protein and total nitric oxide concentrations measurements [ 1316 ] . in our previous study we found that the trail levels in variances of the patients who had the effective anti ige treatment were significantly lower than the healthy controls .", "engagement of the fas / fasl system has not yet been shown to contribute to increase apoptosis .", "recent interest has focused on the molecule trail , which is involved in the pathophysiology of different diseases , including cancer , diabetes mellitus , autoimmune diseases , and inflammation [ 1721 ] .", "trail also present in cells that involved in asthma including eosinophils , mast cells , fibroblasts , and airway epithelial cells .", "it is expressed in airway remodeling and may be linked with the pathways of transforming growth factor - beta ( tgf- ) , which is thought to cause damage to the epithelium .", "the repair process of the epithelium is hindered as a result of increased apoptosis induced by tgf - beta , which overlaps with the pathways of trail .", "these results reflect the different mechanism(s ) in the pathogenesis of allergic diseases by the regulation of apoptosis .", "desloratadine ( dcl ) is a non - sedating antihistamine approved for the treatment of allergic rhinitis .", "patients in groups i - a and b had a dcl usage history during the exacerbation phase from may to november .", "blood samples from the patients were obtained in january , when they would be expected to have fewer allergic symptoms .", "mast cells ( mc ) play a key role in allergy and are involved in several chronic inflammatory diseases . furthermore , they are involved in innate immunity and in tissue repair [ 2427 ] .", "the regulation of mc numbers , as of any other normal cells , depends on both their generation rate and survival time within tissues .", "the critical event in allergic reactions is allergen - induced crosslinking of specific ige molecules bound to fc_ri receptors on the mc surface , which triggers mc degranulation and release of inflammatory mediators .", "non - ige - mediated activation may also contribute to continued degranulation of mc during the late phase of allergic reactions .", "allergic mechanism are involved in the increased susceptibility of human mc to trail - induced apoptosis after ige - dependent activation . in this study ,", "ige levels had a statistically significant difference between the values of the research group before and after allergen - specific subcutaneous immunotherapy ."], ["taken together , our results and those of others , suggest that characterization of the specific receptor systems activated , and the pro - inflammatory factors regulated , by trail in vivo may lead to the development of novel therapeutic strategies for diseases as diverse as infection , autoimmunity , and allergy ."]]}
{"article_id": "PMC4421156", "article_text": ["calcimimetics provide an alternative approach to the traditional therapy with active vitamin d and phosphate binders for the management of secondary hyperparathyroidism ( shpt ) . type ii calcimimetics are positive allosteric modulators of the parathyroid gland calcium - sensing receptor ( car ) that increase its sensitivity to serum calcium , so that lower concentrations of calcium are sufficient to mediate signalling through the car .", "this suggests that , despite the reduced car expression observed in hyperplastic parathyroid tissues , calcimimetics have the potential to control even advanced disease by enhancing the function of the remaining car [ 47 ] .", "in addition , calcimimetics have the potential to influence both car expression and car functions that have been implicated in the progression of disease , particularly effects on parathyroid hormone ( pth ) , cell proliferation and hyperplasia .", "these characteristics suggest that targeting the car presents a therapeutic option for the stabilization of early disease , as well as remaining a viable therapeutic target in the face of advancing disease .", "in addition to being effective in patients with mild to moderate disease , calcimimetics may be the most viable option for chronic kidney disease ( ckd ) patients with advanced hyperparathyroidism , who are often refractory to standard calcitriol therapy [ 810 ] .", "the data below summarize preclinical findings providing a rationale for the clinical use of calcimimetics .", "administration of cinacalcet or the calcimimetic r-568 to 5/6 nephrectomized rats prevented hyperplasia or increased car expression .", "interestingly , in an in vitro study , the calcimimetic cinacalcet suppressed pth secretion in human parathyroid cells with advanced hyperparathyroidism , even though car expression was significantly diminished .", "expression of vitamin d receptor ( vdr ) mrna is reduced in the parathyroid gland of hypocalcaemic rats compared with normocalcaemic rats . because the vdr is a transcriptional repressor of pth", "recent studies indicate that type ii calcimimetics , in the presence of low concentrations of calcium , can up - regulate vdr mrna .", "rats injected with a calcimimetic ( r-568 , 1 mg / kg intravenously 3 and 6 h before euthanasia ) exhibited an increase in vdr mrna expression compared with controls ( p < 0.001 ) , with a marked increase observed when r-568 and calcitriol were coadministered ( figure 1 ) .", "rats were treated with r-568 ( 1 mg / kg intravenously 3 and 6 h before euthanasia ; n = 22 ) , calcitriol ( 10 pmol / kg intraperitoneally every 30 min for 5.5 h before euthanasia ; n = 22 ) , or a combination of r-568 and calcitriol ( n = 22 ) before euthanasia and measurement of vdr mrna by quantitative real - time polymerase chain reaction . a control group ( n = 20 ) received no treatment . mean serum concentrations of ionized calcium and pth are also included .", "data are mean  standard error . adapted with permission from rodriguez et al . .", "these data indicate that , in addition to regulating pth levels and restoring car density , calcimimetics can up - regulate vdr expression in parathyroid cells .", "this effect of calcimimetics on vdr expression is of clinical importance because it may result in an additive interaction between calcimimetic and vitamin d sterol therapy . although parathyroid cells are relatively quiescent under normal conditions , the mineral and hormonal imbalances characteristic of shpt , particularly low serum calcium and high serum phosphorus , drive cell proliferation leading to polyclonal and oligoclonal cell expansion .", "the importance of maintaining calcium homeostasis is evidenced in weanling rats fed a low - calcium diet for 10 days , in which the number of proliferating cell nuclear antigen ( pcna)-positive parathyroid cells was increased sixfold compared with controls .", "calcimimetics , in the presence of low serum calcium , can hold proliferation in check , as evidenced by decreased parathyroid cell proliferation in uraemic rats following treatment   and their ability to cause regression of advanced hyperplasia in some experimental animal models .", "long - term administration of the calcimimetic cinacalcet significantly reduced the number of proliferating ( pcna - positive ) cells and glandular hyperplasia , as measured by weight , in 5/6 nephrectomized rats .", "this effect was parathyroid tissue - specific and was due to a direct effect on cell growth , rather than to induction of apoptosis .", "taken together , the mechanistic data presented here , and reviewed in greater detail in another article in this supplement ( riccardi and martin ) , suggest that calcimimetics have the potential to retard shpt disease progression . preclinical studies in uraemic rats indicate that calcimimetics can modify the defining parameters in shpt by significantly lowering pth and blood - ionized calcium in a dose - dependent manner without inducing significant changes in serum phosphate .", "administration of cinacalcet or the calcimimetic r-568 to 5/6 nephrectomized rats prevented hyperplasia or increased car expression .", "interestingly , in an in vitro study , the calcimimetic cinacalcet suppressed pth secretion in human parathyroid cells with advanced hyperparathyroidism , even though car expression was significantly diminished .", "expression of vitamin d receptor ( vdr ) mrna is reduced in the parathyroid gland of hypocalcaemic rats compared with normocalcaemic rats . because the vdr is a transcriptional repressor of pth", "recent studies indicate that type ii calcimimetics , in the presence of low concentrations of calcium , can up - regulate vdr mrna .", "rats injected with a calcimimetic ( r-568 , 1 mg / kg intravenously 3 and 6 h before euthanasia ) exhibited an increase in vdr mrna expression compared with controls ( p < 0.001 ) , with a marked increase observed when r-568 and calcitriol were coadministered ( figure 1 ) .", "rats were treated with r-568 ( 1 mg / kg intravenously 3 and 6 h before euthanasia ; n = 22 ) , calcitriol ( 10 pmol / kg intraperitoneally every 30 min for 5.5 h before euthanasia ; n = 22 ) , or a combination of r-568 and calcitriol ( n = 22 ) before euthanasia and measurement of vdr mrna by quantitative real - time polymerase chain reaction . a control group ( n = 20 ) received no treatment . mean serum concentrations of ionized calcium and pth are also included .", "p < 0.01 versus control ; p < 0.05 versus calcimimetic ; p < 0.05 versus control .", "data are mean  standard error . adapted with permission from rodriguez et al . .", "these data indicate that , in addition to regulating pth levels and restoring car density , calcimimetics can up - regulate vdr expression in parathyroid cells .", "this effect of calcimimetics on vdr expression is of clinical importance because it may result in an additive interaction between calcimimetic and vitamin d sterol therapy .", "although parathyroid cells are relatively quiescent under normal conditions , the mineral and hormonal imbalances characteristic of shpt , particularly low serum calcium and high serum phosphorus , drive cell proliferation leading to polyclonal and oligoclonal cell expansion .", "the importance of maintaining calcium homeostasis is evidenced in weanling rats fed a low - calcium diet for 10 days , in which the number of proliferating cell nuclear antigen ( pcna)-positive parathyroid cells was increased sixfold compared with controls .", "calcimimetics , in the presence of low serum calcium , can hold proliferation in check , as evidenced by decreased parathyroid cell proliferation in uraemic rats following treatment   and their ability to cause regression of advanced hyperplasia in some experimental animal models .", "long - term administration of the calcimimetic cinacalcet significantly reduced the number of proliferating ( pcna - positive ) cells and glandular hyperplasia , as measured by weight , in 5/6 nephrectomized rats .", "this effect was parathyroid tissue - specific and was due to a direct effect on cell growth , rather than to induction of apoptosis .", "taken together , the mechanistic data presented here , and reviewed in greater detail in another article in this supplement ( riccardi and martin ) , suggest that calcimimetics have the potential to retard shpt disease progression .", "preclinical studies in uraemic rats indicate that calcimimetics can modify the defining parameters in shpt by significantly lowering pth and blood - ionized calcium in a dose - dependent manner without inducing significant changes in serum phosphate .", "one of the primary treatment goals of shpt is to restore mineral and hormone balance .", "the national kidney foundation kidney disease outcomes quality initiative ( kdoqi ) has established recommendations regarding levels of serum calcium , phosphorus , calcium  phosphorus product ( ca  p ) , and intact pth ( ipth ) for patients based on their degree of kidney function . as kidney function decreases ,", "mineral homeostasis is increasingly perturbed , resulting in increased pth , serum phosphorus , and ca  p. as this dysregulation progresses , the response to standard therapy worsens .", "consequently , pth can become high enough to increase mortality and impede adequate control of phosphate [ 1921 ] .", "these findings underscore the need for new or adjunct treatments with improved efficacy over current modalities that can stabilize disease progression during a long - term course when initiated early , but can also maintain effectiveness in patients with more severe disease . because of the ability of calcimimetics , demonstrated in animal studies , to modulate disease - related processes through multiple mechanisms of action implicated in the development of disease , this therapeutic modality appears to be well suited for use in combination with moderate doses of vitamin d as the primary mode of treatment in shpt management .", "as described earlier , calcimimetics up - regulate car and vdr expression and inhibit parathyroid cell proliferation and hyperplasia .", "these multiple mechanisms of action may allow calcimimetics to ( 1 ) maintain effectiveness during long - term therapy ; ( 2 ) maintain enhanced efficacy in patients with severe disease despite decreased car and vdr expression and ( 3 ) halt development of severe disease if initiated early in patients with mild or moderate shpt .", "phase 3 clinical trials in shpt patients with advanced disease have shown that cinacalcet decreased pth , as well as serum calcium , phosphorus and ca  p .", "analysis of the data from these trials has been performed to investigate the efficacy of cinacalcet in the treatment of both early- and later - stage shpt and to investigate the potential of long - term cinacalcet therapy to stabilize disease progression .", "results of these analyses , presented below , suggest that calcimimetics have the ability to address these unmet therapeutic needs .", "a post - hoc analysis of the combined results of three 26-week , placebo - controlled , phase 3 studies evaluating the efficacy and safety of a once - daily dose of cinacalcet for the treatment of shpt demonstrated the impact of therapy initiated in early or advanced disease .", "these studies included adult patients on maintenance dialysis who had ipth levels 300 pg / ml despite standard treatment with active vitamin d and phosphate binders . patients received standard therapy in combination with cinacalcet ( n = 546 ) or placebo ( n = 408 ) .", "patients included in the analysis were divided into eight subgroups according to their baseline ipth level , which was used as a marker of disease severity ( 300 to > 1000 pg / ml ) .", "findings from this analysis indicated that the use of cinacalcet ( 30 mg titrated up to 180 mg ) significantly decreased both ipth and ca  p levels regardless of disease severity at baseline , compared with standard therapy ( figure 2 ) .", "the progressive nature of shpt , even when patients receive standard therapy , is evidenced by positive changes from baseline in ipth seen in these patients ( figure 2a ) .", "furthermore , the effect of cinacalcet on ca  p ( figure 2b ) and serum phosphorus levels was more pronounced in patients with more severe disease , suggesting a more significant impact of cinacalcet on bone turnover in these patients", ". percentage change in ( a ) ipth and ( b ) ca  p by disease severity .", "patients were treated with cinacalcet ( beginning at 30 mg with possible titration up to 180 mg ; n = 546 ) or placebo ( n = 408 ) for 26 weeks .", "patients were divided into groups according to baseline ipth levels ( pg / ml ) .", "however , although these data demonstrate that cinacalcet is effective in reducing serum ipth and ca  p , even in patients with severe disease , these patients remain less likely to achieve their kdoqi targets than patients with less severe disease .", "after 6 months of treatment with cinacalcet , 76% of patients with a baseline ipth of 300500 pg / ml achieved a serum ipth level of < 300 pg / ml compared with 55% of patients with a baseline ipth of 500800 pg / ml and 16% of patients with a baseline ipth of > 800 pg / ml . similarly , 54% of patients with a baseline ipth of 300500 pg / ml achieved an ipth of < 300 pg / ml and their ca  p target ( < 55 mg / dl ) , compared with 36% and 9% of patients in the 500800 and > 800 mg / ml groups , respectively .", "these data suggest that early treatment of shpt provides the best opportunity to maintain control of serum pth , calcium , phosphorus and ca  p. treatment with cinacalcet for up to 1 year resulted in increased attainment of kdoqi targets compared with standard therapy .", "after 12 months , 66% of patients on dialysis receiving cinacalcet achieved an ipth of 300 pg / ml compared with 15% in the placebo group . similarly", ", 55% of patients receiving cinacalcet achieved their ca  p target ( < 55 mg / dl ) compared with 43% in the placebo group .", "furthermore , the proportion of patients able to achieve both an ipth of 300 pg / ml and their ca  p target was considerably higher in the cinacalcet group than in the placebo group ; 42% of patients in the cinacalcet group achieved both targets simultaneously , whereas only 8% of patients in the placebo group reached both targets ( figure 3a ) .", "proportion of patients achieving serum ipth 300 pg / ml and ca  p < 55 mg / dl during treatment with either cinacalcet ( n = 122 ) or placebo ( n = 126 ) . ( a ) achievement of serum ipth 300 pg / ml and ca  p < 55 mg / dl after 1 year .", "( b ) maintenance of serum ipth 300 pg / ml after 6 and 12 months of treatment .", "( c ) maintenance of combined serum ipth and ca  p target after 6 and 12 months of treatment . adapted with permission from frazo et al . .", "in this study , treatment with cinacalcet not only improved the ability of patients to achieve their kdoqi targets , but also their ability to maintain these targets .", "seventy - one percent of cinacalcet - treated patients were able to achieve an ipth of 300 pg / ml after 6 months of cinacalcet treatment .", "of the group who achieved an ipth of 300 pg / ml at 6 months , 82% also maintained an ipth of 300 pg / ml after 1 year .", "in contrast , in the standard care group , 13% of patients achieved an ipth concentration of 300 pg / ml , with 53% of this group continuing to maintain an ipth 300 pg / ml after 1 year ( figure 3b ) .", "cinacalcet also improved maintenance of the combined ipth and ca  p targets . in the cinacalcet group", ", 52% of patients reached the combined target after 6 months compared with 7% in the standard care group .", "of these patients , 59% in the cinacalcet group maintained the combined target after 1 year compared with just 22% in the standard care group ( figure 3c ) .", "these data demonstrate that cinacalcet effectively reduces and maintains the four critical disease biomarkers associated with shpt in ckd patients .", "cinacalcet , therefore , reduces the shifting in and out of targets often observed in patients using standard therapy .", "open - label extension studies have evaluated the ability of cinacalcet to stabilize disease in shpt patients for up to 4 years [ 2628 ] .", "these analyses were based on a series of double - blind randomized clinical trials in which patients received standard care treatment in combination with either placebo or cinacalcet for 26 or 52 weeks , after which patients could receive open - label cinacalcet treatment .", "in one such open - label extension study , serum ipth was found to progressively increase in patients treated with standard care for 52 weeks .", "after open - label cinacalcet treatment was started , serum ipth was rapidly reduced to a level matching that of cinacalcet - treated patients .", "( n = 59 ) had achieved an ipth of 300 pg / ml at each study visit .", "serum ca  p did not increase during this period . in a separate study , a limited number of patients ( n = 21 ) taking cinacalcet were followed up for as long as 4 years . a substantial upward trend in ipth", "was noted in control patients who received standard therapy during the first year of treatment in this study , before switching to cinacalcet therapy , with levels increasing from approximately 600850 pg / ml . in these patients ,", "cinacalcet reduced and maintained ipth levels throughout the follow - up period ( figure 4 ) .", "after 4 years , the mean serum ipth was 396 pg / ml , 65% of patients achieved a serum ipth of 300 pg / ml , and the mean serum ca  p was 50.5 mg / dl .", "these findings again highlight the progressive nature of shpt in patients receiving standard therapy as first - line treatment , as well as the inability of traditional therapies to effectively control or stabilize disease in both the short and long term . these and other clinical observations discussed in this section are also consistent with preclinical evidence supporting the car as a therapeutic target to optimize the achievement of treatment goals and slow or prevent disease progression .", "patients received double - blind treatment with either cinacalcet ( n = 9 ) or placebo ( n = 12 ) for 1 year followed by a 3-year open - label extension treatment with cinacalcet .", "calcimimetics are highly specific therapeutic agents that effectively lower concentrations of plasma pth and partially correct disturbances in mineral metabolism that are characteristic of shpt .", "the mechanism of action involves allosteric modification of the car , rendering it more sensitive to calcium signalling , thereby helping counteract the decreased car expression and prevent and halt the progression of parathyroid hyperplasia .", "in addition to effects on car signalling , new experimental studies indicate that calcimimetics may act to increase vdr expression .", "these studies raise the possibility that treatment with calcimimetics might improve the sensitivity of the parathyroid glands to calcitriol and vitamin d analogues , allowing reduced doses to be used and thus minimizing the risk of hypercalcaemia and hyperphosphataemia .", "preclinical mechanistic studies with calcimimetics have suggested that this modality may arrest disease progression , even in patients with advanced disease at treatment onset , and stabilize shpt , thereby allowing improved maintenance of kdoqi targets .", ", cinacalcet was found to be effective for the reduction and long - term maintenance of ipth , calcium and phosphorus levels in patients with shpt .", "cinacalcet has been shown to allow more patients to achieve and maintain kdoqi targets compared with standard therapy , and extended use ( up to 4 years ) has been shown to be effective and well tolerated ."], "abstract_text": ["<S> standard therapy for secondary hyperparathyroidism ( shpt ) includes dietary calcium supplementation , active vitamin d , and phosphate binders ; however , these are often insufficient to allow patients to achieve their serum parathyroid hormone ( pth ) , calcium and calcium  phosphorus product ( ca  p ) targets . </S>", "<S> recent preclinical studies have demonstrated that treatment with type ii calcimimetics that increase the sensitivity of the calcium - sensing receptor ( car ) to calcium can reverse the alterations in car and vitamin d receptor expression and parathyroid cell proliferation that are associated with shpt . </S>", "<S> these data suggest that calcimimetic treatment could stabilize disease progression and improve maintenance of treatment goals . in clinical trials involving shpt patients , </S>", "<S> the calcimimetic cinacalcet has been shown to decrease pth , calcium , phosphorus and ca  p. significant improvements were seen regardless of initial disease severity , and benefits were maintained over the course of long - term therapy ( up to 4 years ) , indicating effective disease stabilization . in conclusion , </S>", "<S> preclinical and clinical data provide both theoretical and empirical support for the use of calcimimetics in moderate and advanced shpt to effectively stabilize disease . </S>"], "labels": null, "section_names": ["Stabilization of secondary hyperparathyroidism with calcimimetics: preclinical evidence", "CaR expression", "Vitamin D receptor expression", "Parathyroid cell proliferation and parathyroid hyperplasia", "Unmet medical need in SHPT management", "Effect of cinacalcet on disease stabilization in clinical trials", "Conclusions"], "sections": [["calcimimetics provide an alternative approach to the traditional therapy with active vitamin d and phosphate binders for the management of secondary hyperparathyroidism ( shpt ) . type ii calcimimetics are positive allosteric modulators of the parathyroid gland calcium - sensing receptor ( car ) that increase its sensitivity to serum calcium , so that lower concentrations of calcium are sufficient to mediate signalling through the car .", "this suggests that , despite the reduced car expression observed in hyperplastic parathyroid tissues , calcimimetics have the potential to control even advanced disease by enhancing the function of the remaining car [ 47 ] .", "in addition , calcimimetics have the potential to influence both car expression and car functions that have been implicated in the progression of disease , particularly effects on parathyroid hormone ( pth ) , cell proliferation and hyperplasia .", "these characteristics suggest that targeting the car presents a therapeutic option for the stabilization of early disease , as well as remaining a viable therapeutic target in the face of advancing disease .", "in addition to being effective in patients with mild to moderate disease , calcimimetics may be the most viable option for chronic kidney disease ( ckd ) patients with advanced hyperparathyroidism , who are often refractory to standard calcitriol therapy [ 810 ] .", "the data below summarize preclinical findings providing a rationale for the clinical use of calcimimetics .", "administration of cinacalcet or the calcimimetic r-568 to 5/6 nephrectomized rats prevented hyperplasia or increased car expression .", "interestingly , in an in vitro study , the calcimimetic cinacalcet suppressed pth secretion in human parathyroid cells with advanced hyperparathyroidism , even though car expression was significantly diminished .", "expression of vitamin d receptor ( vdr ) mrna is reduced in the parathyroid gland of hypocalcaemic rats compared with normocalcaemic rats . because the vdr is a transcriptional repressor of pth", "recent studies indicate that type ii calcimimetics , in the presence of low concentrations of calcium , can up - regulate vdr mrna .", "rats injected with a calcimimetic ( r-568 , 1 mg / kg intravenously 3 and 6 h before euthanasia ) exhibited an increase in vdr mrna expression compared with controls ( p < 0.001 ) , with a marked increase observed when r-568 and calcitriol were coadministered ( figure 1 ) .", "rats were treated with r-568 ( 1 mg / kg intravenously 3 and 6 h before euthanasia ; n = 22 ) , calcitriol ( 10 pmol / kg intraperitoneally every 30 min for 5.5 h before euthanasia ; n = 22 ) , or a combination of r-568 and calcitriol ( n = 22 ) before euthanasia and measurement of vdr mrna by quantitative real - time polymerase chain reaction . a control group ( n = 20 ) received no treatment . mean serum concentrations of ionized calcium and pth are also included .", "data are mean  standard error . adapted with permission from rodriguez et al . .", "these data indicate that , in addition to regulating pth levels and restoring car density , calcimimetics can up - regulate vdr expression in parathyroid cells .", "this effect of calcimimetics on vdr expression is of clinical importance because it may result in an additive interaction between calcimimetic and vitamin d sterol therapy . although parathyroid cells are relatively quiescent under normal conditions , the mineral and hormonal imbalances characteristic of shpt , particularly low serum calcium and high serum phosphorus , drive cell proliferation leading to polyclonal and oligoclonal cell expansion .", "the importance of maintaining calcium homeostasis is evidenced in weanling rats fed a low - calcium diet for 10 days , in which the number of proliferating cell nuclear antigen ( pcna)-positive parathyroid cells was increased sixfold compared with controls .", "calcimimetics , in the presence of low serum calcium , can hold proliferation in check , as evidenced by decreased parathyroid cell proliferation in uraemic rats following treatment   and their ability to cause regression of advanced hyperplasia in some experimental animal models .", "long - term administration of the calcimimetic cinacalcet significantly reduced the number of proliferating ( pcna - positive ) cells and glandular hyperplasia , as measured by weight , in 5/6 nephrectomized rats .", "this effect was parathyroid tissue - specific and was due to a direct effect on cell growth , rather than to induction of apoptosis .", "taken together , the mechanistic data presented here , and reviewed in greater detail in another article in this supplement ( riccardi and martin ) , suggest that calcimimetics have the potential to retard shpt disease progression . preclinical studies in uraemic rats indicate that calcimimetics can modify the defining parameters in shpt by significantly lowering pth and blood - ionized calcium in a dose - dependent manner without inducing significant changes in serum phosphate ."], ["administration of cinacalcet or the calcimimetic r-568 to 5/6 nephrectomized rats prevented hyperplasia or increased car expression .", "interestingly , in an in vitro study , the calcimimetic cinacalcet suppressed pth secretion in human parathyroid cells with advanced hyperparathyroidism , even though car expression was significantly diminished ."], ["expression of vitamin d receptor ( vdr ) mrna is reduced in the parathyroid gland of hypocalcaemic rats compared with normocalcaemic rats . because the vdr is a transcriptional repressor of pth", "recent studies indicate that type ii calcimimetics , in the presence of low concentrations of calcium , can up - regulate vdr mrna .", "rats injected with a calcimimetic ( r-568 , 1 mg / kg intravenously 3 and 6 h before euthanasia ) exhibited an increase in vdr mrna expression compared with controls ( p < 0.001 ) , with a marked increase observed when r-568 and calcitriol were coadministered ( figure 1 ) .", "rats were treated with r-568 ( 1 mg / kg intravenously 3 and 6 h before euthanasia ; n = 22 ) , calcitriol ( 10 pmol / kg intraperitoneally every 30 min for 5.5 h before euthanasia ; n = 22 ) , or a combination of r-568 and calcitriol ( n = 22 ) before euthanasia and measurement of vdr mrna by quantitative real - time polymerase chain reaction . a control group ( n = 20 ) received no treatment . mean serum concentrations of ionized calcium and pth are also included .", "p < 0.01 versus control ; p < 0.05 versus calcimimetic ; p < 0.05 versus control .", "data are mean  standard error . adapted with permission from rodriguez et al . .", "these data indicate that , in addition to regulating pth levels and restoring car density , calcimimetics can up - regulate vdr expression in parathyroid cells .", "this effect of calcimimetics on vdr expression is of clinical importance because it may result in an additive interaction between calcimimetic and vitamin d sterol therapy ."], ["although parathyroid cells are relatively quiescent under normal conditions , the mineral and hormonal imbalances characteristic of shpt , particularly low serum calcium and high serum phosphorus , drive cell proliferation leading to polyclonal and oligoclonal cell expansion .", "the importance of maintaining calcium homeostasis is evidenced in weanling rats fed a low - calcium diet for 10 days , in which the number of proliferating cell nuclear antigen ( pcna)-positive parathyroid cells was increased sixfold compared with controls .", "calcimimetics , in the presence of low serum calcium , can hold proliferation in check , as evidenced by decreased parathyroid cell proliferation in uraemic rats following treatment   and their ability to cause regression of advanced hyperplasia in some experimental animal models .", "long - term administration of the calcimimetic cinacalcet significantly reduced the number of proliferating ( pcna - positive ) cells and glandular hyperplasia , as measured by weight , in 5/6 nephrectomized rats .", "this effect was parathyroid tissue - specific and was due to a direct effect on cell growth , rather than to induction of apoptosis .", "taken together , the mechanistic data presented here , and reviewed in greater detail in another article in this supplement ( riccardi and martin ) , suggest that calcimimetics have the potential to retard shpt disease progression .", "preclinical studies in uraemic rats indicate that calcimimetics can modify the defining parameters in shpt by significantly lowering pth and blood - ionized calcium in a dose - dependent manner without inducing significant changes in serum phosphate ."], ["one of the primary treatment goals of shpt is to restore mineral and hormone balance .", "the national kidney foundation kidney disease outcomes quality initiative ( kdoqi ) has established recommendations regarding levels of serum calcium , phosphorus , calcium  phosphorus product ( ca  p ) , and intact pth ( ipth ) for patients based on their degree of kidney function . as kidney function decreases ,", "mineral homeostasis is increasingly perturbed , resulting in increased pth , serum phosphorus , and ca  p. as this dysregulation progresses , the response to standard therapy worsens .", "consequently , pth can become high enough to increase mortality and impede adequate control of phosphate [ 1921 ] .", "these findings underscore the need for new or adjunct treatments with improved efficacy over current modalities that can stabilize disease progression during a long - term course when initiated early , but can also maintain effectiveness in patients with more severe disease . because of the ability of calcimimetics , demonstrated in animal studies , to modulate disease - related processes through multiple mechanisms of action implicated in the development of disease , this therapeutic modality appears to be well suited for use in combination with moderate doses of vitamin d as the primary mode of treatment in shpt management .", "as described earlier , calcimimetics up - regulate car and vdr expression and inhibit parathyroid cell proliferation and hyperplasia .", "these multiple mechanisms of action may allow calcimimetics to ( 1 ) maintain effectiveness during long - term therapy ; ( 2 ) maintain enhanced efficacy in patients with severe disease despite decreased car and vdr expression and ( 3 ) halt development of severe disease if initiated early in patients with mild or moderate shpt .", "phase 3 clinical trials in shpt patients with advanced disease have shown that cinacalcet decreased pth , as well as serum calcium , phosphorus and ca  p .", "analysis of the data from these trials has been performed to investigate the efficacy of cinacalcet in the treatment of both early- and later - stage shpt and to investigate the potential of long - term cinacalcet therapy to stabilize disease progression .", "results of these analyses , presented below , suggest that calcimimetics have the ability to address these unmet therapeutic needs ."], ["a post - hoc analysis of the combined results of three 26-week , placebo - controlled , phase 3 studies evaluating the efficacy and safety of a once - daily dose of cinacalcet for the treatment of shpt demonstrated the impact of therapy initiated in early or advanced disease .", "these studies included adult patients on maintenance dialysis who had ipth levels 300 pg / ml despite standard treatment with active vitamin d and phosphate binders . patients received standard therapy in combination with cinacalcet ( n = 546 ) or placebo ( n = 408 ) .", "patients included in the analysis were divided into eight subgroups according to their baseline ipth level , which was used as a marker of disease severity ( 300 to > 1000 pg / ml ) .", "findings from this analysis indicated that the use of cinacalcet ( 30 mg titrated up to 180 mg ) significantly decreased both ipth and ca  p levels regardless of disease severity at baseline , compared with standard therapy ( figure 2 ) .", "the progressive nature of shpt , even when patients receive standard therapy , is evidenced by positive changes from baseline in ipth seen in these patients ( figure 2a ) .", "furthermore , the effect of cinacalcet on ca  p ( figure 2b ) and serum phosphorus levels was more pronounced in patients with more severe disease , suggesting a more significant impact of cinacalcet on bone turnover in these patients", ". percentage change in ( a ) ipth and ( b ) ca  p by disease severity .", "patients were treated with cinacalcet ( beginning at 30 mg with possible titration up to 180 mg ; n = 546 ) or placebo ( n = 408 ) for 26 weeks .", "patients were divided into groups according to baseline ipth levels ( pg / ml ) .", "however , although these data demonstrate that cinacalcet is effective in reducing serum ipth and ca  p , even in patients with severe disease , these patients remain less likely to achieve their kdoqi targets than patients with less severe disease .", "after 6 months of treatment with cinacalcet , 76% of patients with a baseline ipth of 300500 pg / ml achieved a serum ipth level of < 300 pg / ml compared with 55% of patients with a baseline ipth of 500800 pg / ml and 16% of patients with a baseline ipth of > 800 pg / ml . similarly , 54% of patients with a baseline ipth of 300500 pg / ml achieved an ipth of < 300 pg / ml and their ca  p target ( < 55 mg / dl ) , compared with 36% and 9% of patients in the 500800 and > 800 mg / ml groups , respectively .", "these data suggest that early treatment of shpt provides the best opportunity to maintain control of serum pth , calcium , phosphorus and ca  p. treatment with cinacalcet for up to 1 year resulted in increased attainment of kdoqi targets compared with standard therapy .", "after 12 months , 66% of patients on dialysis receiving cinacalcet achieved an ipth of 300 pg / ml compared with 15% in the placebo group . similarly", ", 55% of patients receiving cinacalcet achieved their ca  p target ( < 55 mg / dl ) compared with 43% in the placebo group .", "furthermore , the proportion of patients able to achieve both an ipth of 300 pg / ml and their ca  p target was considerably higher in the cinacalcet group than in the placebo group ; 42% of patients in the cinacalcet group achieved both targets simultaneously , whereas only 8% of patients in the placebo group reached both targets ( figure 3a ) .", "proportion of patients achieving serum ipth 300 pg / ml and ca  p < 55 mg / dl during treatment with either cinacalcet ( n = 122 ) or placebo ( n = 126 ) . ( a ) achievement of serum ipth 300 pg / ml and ca  p < 55 mg / dl after 1 year .", "( b ) maintenance of serum ipth 300 pg / ml after 6 and 12 months of treatment .", "( c ) maintenance of combined serum ipth and ca  p target after 6 and 12 months of treatment . adapted with permission from frazo et al . .", "in this study , treatment with cinacalcet not only improved the ability of patients to achieve their kdoqi targets , but also their ability to maintain these targets .", "seventy - one percent of cinacalcet - treated patients were able to achieve an ipth of 300 pg / ml after 6 months of cinacalcet treatment .", "of the group who achieved an ipth of 300 pg / ml at 6 months , 82% also maintained an ipth of 300 pg / ml after 1 year .", "in contrast , in the standard care group , 13% of patients achieved an ipth concentration of 300 pg / ml , with 53% of this group continuing to maintain an ipth 300 pg / ml after 1 year ( figure 3b ) .", "cinacalcet also improved maintenance of the combined ipth and ca  p targets . in the cinacalcet group", ", 52% of patients reached the combined target after 6 months compared with 7% in the standard care group .", "of these patients , 59% in the cinacalcet group maintained the combined target after 1 year compared with just 22% in the standard care group ( figure 3c ) .", "these data demonstrate that cinacalcet effectively reduces and maintains the four critical disease biomarkers associated with shpt in ckd patients .", "cinacalcet , therefore , reduces the shifting in and out of targets often observed in patients using standard therapy .", "open - label extension studies have evaluated the ability of cinacalcet to stabilize disease in shpt patients for up to 4 years [ 2628 ] .", "these analyses were based on a series of double - blind randomized clinical trials in which patients received standard care treatment in combination with either placebo or cinacalcet for 26 or 52 weeks , after which patients could receive open - label cinacalcet treatment .", "in one such open - label extension study , serum ipth was found to progressively increase in patients treated with standard care for 52 weeks .", "after open - label cinacalcet treatment was started , serum ipth was rapidly reduced to a level matching that of cinacalcet - treated patients .", "( n = 59 ) had achieved an ipth of 300 pg / ml at each study visit .", "serum ca  p did not increase during this period . in a separate study , a limited number of patients ( n = 21 ) taking cinacalcet were followed up for as long as 4 years . a substantial upward trend in ipth", "was noted in control patients who received standard therapy during the first year of treatment in this study , before switching to cinacalcet therapy , with levels increasing from approximately 600850 pg / ml . in these patients ,", "cinacalcet reduced and maintained ipth levels throughout the follow - up period ( figure 4 ) .", "after 4 years , the mean serum ipth was 396 pg / ml , 65% of patients achieved a serum ipth of 300 pg / ml , and the mean serum ca  p was 50.5 mg / dl .", "these findings again highlight the progressive nature of shpt in patients receiving standard therapy as first - line treatment , as well as the inability of traditional therapies to effectively control or stabilize disease in both the short and long term . these and other clinical observations discussed in this section are also consistent with preclinical evidence supporting the car as a therapeutic target to optimize the achievement of treatment goals and slow or prevent disease progression .", "patients received double - blind treatment with either cinacalcet ( n = 9 ) or placebo ( n = 12 ) for 1 year followed by a 3-year open - label extension treatment with cinacalcet ."], ["calcimimetics are highly specific therapeutic agents that effectively lower concentrations of plasma pth and partially correct disturbances in mineral metabolism that are characteristic of shpt .", "the mechanism of action involves allosteric modification of the car , rendering it more sensitive to calcium signalling , thereby helping counteract the decreased car expression and prevent and halt the progression of parathyroid hyperplasia .", "in addition to effects on car signalling , new experimental studies indicate that calcimimetics may act to increase vdr expression .", "these studies raise the possibility that treatment with calcimimetics might improve the sensitivity of the parathyroid glands to calcitriol and vitamin d analogues , allowing reduced doses to be used and thus minimizing the risk of hypercalcaemia and hyperphosphataemia .", "preclinical mechanistic studies with calcimimetics have suggested that this modality may arrest disease progression , even in patients with advanced disease at treatment onset , and stabilize shpt , thereby allowing improved maintenance of kdoqi targets .", ", cinacalcet was found to be effective for the reduction and long - term maintenance of ipth , calcium and phosphorus levels in patients with shpt .", "cinacalcet has been shown to allow more patients to achieve and maintain kdoqi targets compared with standard therapy , and extended use ( up to 4 years ) has been shown to be effective and well tolerated ."]]}
{"article_id": "PMC1479889", "article_text": ["many neurodegenerative disorders are characterized by \n their presence in neural tissue of aberrant protein aggregates \n ( see table 1 ) . in general , these aggregates arise after the modification of a \n native protein . that modification could result in a conformational \n change of the native protein that promotes the aberrant \n aggregation .", "the most studied model of this mechanism has been the prion \n protein where a change from an alpha helix to beta sheet structure \n facilitates the polymerization of a protein with a different \n conformation that appears to have a cytotoxic effect . in a similar way", ", conformational changes between a native protein \n and its aberrant protein counterpart with capacity for \n self - assembly have been studied in many neurodegenerative \n diseases . among the most common techniques used for these analyses \n", "are x - ray diffraction , nuclear magnetic resonance , circular \n dichroism , or fourier - transformed infrared spectroscopy . \n similarly , to the case of prion protein , in some disorders a \n change from alpha helix to beta sheet conformation has been \n suggested to cause protein aggregation ( table 1 ) , \n probably because in alpha helix , intramolecular hydrogen bonds \n could occur whereas intramolecular hydrogen bonds are facilitated \n in beta sheet conformation , facilitating protein aggregation . \n", "however , there is one case , the formation of aberrant aggregates \n of tau , where the aggregated protein contains also a high \n proportion of alpha helix structure .", "although , in some cases , like that of prion protein , the formation \n of aberrant aggregates of protein could result in a toxic effect \n in the affected neurons , in other cases , like huntingtin \n aggregates , the formation of the aberrant aggregates could be a \n survival response of the affected neurons .", "in other \n neurodegenerative diseases , it will be of interest to know if \n protein aggregation is synonymous of cell toxicity or not .", "protein conformation could also play a role in a possible toxic \n mechanism . in this way", ", a protein with a high proportion of alpha \n helix and hydrophobic regions could be inserted in cell membrane \n promoting toxic effects .", "additionally , the presence of \n aberrant polymers could affect the protein degradation cell machinery ( the proteasome complex ) , decreasing its activity \n and promoting a toxic effect .", "recently , some good reviews have been published on protein \n aggregation and neurodegenerative disorders [ 6 , 7 ] . in this \n review", "we will mainly focus on those aggregates assembled from tau \n protein , aggregates that could be present in the neurological \n disorders known as tauopathies ( for a review see ) ( table 1 ) .", "it has been described that large amounts of native or unmodified \n tau protein were enough to promote tau assembly into fibrillar \n polymers resembling those found in ad [ 912 ] .", "thus , \n obviously , an increase in tau concentration will favour the \n formation of tau polymers .", "recently , it has been reported that not \n all the brain areas have a similar amount of tau protein \n .", "thus , it suggests a different probability in the formation of tau polymers in different brain regions .", "the amount of tau will be the consequence of its synthesis and its \n degradation .", "changes in transcription have been indicated for \n other proteins related to neurodegenerative disorders , where a tata binding protein may play a role .", "tau degradation may \n take place through the proteasome complex [ 15 , 16 ] and it has \n been suggested that such degradation could be regulated by \n posttranslational modifications occurring in tau molecule , like \n its phosphorylation .", "also , tau degradation by other proteases could be regulated by its level of phosphorylation \n .", "it should be also indicated that in some cases like parkinson 's disease or lafora disease , mutations in the e3 \n ubiquitin ligases like parkin   or malin   will \n result in the appearance of aberrant protein aggregates . a conformational change , that could be followed by antibodies that \n react with tau molecule after that conformational change \n [ 2124 ] has been also suggested to be required for the \n transition tau monomer - tau polymer . also , it has been suggested that different posttranslational \n modifications like phosphorylation , glycation , \n or truncation , may play a role in the formation of tau \n polymers . due to the alternative splicing of its heterogenous ( or nuclear ) \n rna , different tau isoforms could be expressed and , therefore , \n different tau aggregates with different tau isoforms in different \n tauopathies could occur , but we will not discuss this point here . \n for further information see .", ", it has been suggested that removal of the amino \n and/or carboxy terminal regions , leaving the tubulin binding \n region will facilitate tau polymerization [ 21 , 22 , 28 ] .", "some work has been done in vitro   and in vivo [ 30 , 31 ] about a possible role of tau phosphorylation on tau \n assembly , suggesting that in some conditions tau phosphorylation \n may increase the capacity of tau for its self - assembly .", "not only \n an increase in serine / threonine phosphorylation of tau could \n regulate its aggregation but also an increase in tau tyrosine \n phosphorylation may increase the formation of tau aggregates \n . this assembly process may involve the formation of oligomers   , filaments , and aggregates of filaments \n ( tangle - like structures ) . in the formation of these aggregates of \n filaments , glycation may play a role . the possible relation between phosphorylation and tau aggregation \n", "has been studied in transgenic mice expressing human tau bearing \n some mutations found in human fronto - temporal dementia linked to \n chromosome 17 ( ftdp-17 ) . in this mouse ,", "tau \n phosphorylation mainly occurs by gsk3 . when a specific \n inhibitor of this kinase , lithium , was given to the transgenic \n mice no tau phosphorylation was found , and in addition no \n aggregation of the protein was detected   suggesting a \n correlation between tau phosphorylation and aggregation in this \n model .", "this result was supported by an additional experiment using \n another mouse also expressing human tau with a ftdp-17 mutation \n .", "alternatively , protein chaperones , acting on tau or in phosphotau , \n could modify the level of tau aggregation , examples could be the \n protein 14 - 3 - 3 , musashi-1   , or pin-1 \n [ 37 , 38 ] .", "the chaperone associated ubiquitin ligase chip could \n be able to target phosphotau for proteasomal degradation \n [ 16 , 18 ] .", "it has been described that tau binding to microtubules is regulated by the level of tau phosphorylation at some specific sites .", "it is known that hyperphosphorylated tau binds with less affinity to microtubules resulting in the decrease in the interaction with microtubules , in a decrease of \n microtubule stability , and probably in a microtubule dysfunction inside the cell that could result in a toxic effect \n .", "also , tau phosphorylation could result , as indicated above , in a decrease of its proteolysis .", "more recently , it has been indicated that expression of a tau mutant ( p301l ) \n could result in an increase of its phosphorylation , since once it is phosphorylated , that mutation can prevent the binding of those phosphatases involved in its dephosphorylation .", "this \n phosphotau could have a decreased capacity for microtubule binding \n and it could be toxic for the cell . additionally , it has been \n indicated that hyperphosphorylated tau can cause \n neurodegeneration , in the absence of large tau aggregates \n . on the other hand , only the overexpression of wild - type human tau in a mouse is sufficient to cause tau phosphorylation , aggregation , and neural toxicity . on the other hand", ", it has been suggested that tau phosphorylation may represent a \n protective function in ad .", "sometime ago , the development of tau pathology , related with \n dementia in ad , was clearly described by braak and braak \n  by following the development of neurofibrillary lesions at different stages of the disease . also , the formation of \n neurofibrillary ( tau aggregates ) pathology within those neurons of \n hippocampus and cerebral cortex affected at different stages was \n found .", "these neurons could degenerate yielding extracellular ghost \n tangles ( enft ) . in the hippocampus", ", an inverse relation has been found between the number of enft and the number \n of surviving neurons [ 4851 ] .", "it suggests that neurons that \n degenerate , have previously developed tau aggregates . on the other \n hand", ", it has been indicated that neurons bearing neurofibrillary \n lesions could survive for a long period of time , and , by comparing with other neurodegenerative disorders , like huntington \n disease , it can be suggested that tau aggregates could protect against neurodegeneration by sequestering toxic ( phospho ? ) \n monomeric tau that could be present in a high amount inside a cell \n in pathological conditions . also , it has been suggested , using a \n transgenic mouse model , that behavioural ( memory ) deficits could be unrelated to the formation of tau polymers , \n", "although , more recently , the discussion of those experiments suggested that hyperphosphorylated , aggregated tau intermediates could be the ones that cause neurodegeneration . in this way , the implication of different types of protein aggregates in neurodegeneration has been extensively \n discussed [ 19 , 54 ] . a possibility about the existence of neurotoxic tau intermediate aggregates in human tauopathies", "is \n based in the fact that patients with ftdp-17 show an extensive \n neurodegeneration with a high level of tau phosphorylation but \n with a low number of tangles . in any case , even if the formation of tau aggregates has a \n protective function for the neurons , that function is not working \n well , as described by braak and braak , and afterwards by delacourte et al , indicating a correlation between \n progression of tau pathology and progression of the disease .", "this \n idea is supported by those experiments indicating that neural loss \n and neurofibrillary tangle number increase in parallel with the \n progression of the disease .", "similar results have been described in other neurological disorders like brain encelphalopathies , where the formation of aberrant polymers are related to the onset of neurodegeneration ; whereas this is far from clear in other disorders like \n huntington disease .", "examples of neurological diseases where aberrant protein \n aggregates are found , and the suggested conformations in the \n aberrant aggregates are indicated ."], "abstract_text": ["<S> protein aggregation takes place in many neurodegenerative \n disorders . however , there is a controversy about the possible \n toxicity of these protein aggregates . in this review , </S>", "<S> this \n controversy is discussed , focussing on the tau aggregation that \n takes place in those disorders known as tauopathies . </S>"], "labels": null, "section_names": ["INTRODUCTION", "TAU AGGREGATION", "TOXICITY OF PHOSPHOTAU", "TOXICITY OF AGGREGATED TAU", "Figures and Tables"], "sections": [["many neurodegenerative disorders are characterized by \n their presence in neural tissue of aberrant protein aggregates \n ( see table 1 ) . in general , these aggregates arise after the modification of a \n native protein . that modification could result in a conformational \n change of the native protein that promotes the aberrant \n aggregation .", "the most studied model of this mechanism has been the prion \n protein where a change from an alpha helix to beta sheet structure \n facilitates the polymerization of a protein with a different \n conformation that appears to have a cytotoxic effect . in a similar way", ", conformational changes between a native protein \n and its aberrant protein counterpart with capacity for \n self - assembly have been studied in many neurodegenerative \n diseases . among the most common techniques used for these analyses \n", "are x - ray diffraction , nuclear magnetic resonance , circular \n dichroism , or fourier - transformed infrared spectroscopy . \n similarly , to the case of prion protein , in some disorders a \n change from alpha helix to beta sheet conformation has been \n suggested to cause protein aggregation ( table 1 ) , \n probably because in alpha helix , intramolecular hydrogen bonds \n could occur whereas intramolecular hydrogen bonds are facilitated \n in beta sheet conformation , facilitating protein aggregation . \n", "however , there is one case , the formation of aberrant aggregates \n of tau , where the aggregated protein contains also a high \n proportion of alpha helix structure .", "although , in some cases , like that of prion protein , the formation \n of aberrant aggregates of protein could result in a toxic effect \n in the affected neurons , in other cases , like huntingtin \n aggregates , the formation of the aberrant aggregates could be a \n survival response of the affected neurons .", "in other \n neurodegenerative diseases , it will be of interest to know if \n protein aggregation is synonymous of cell toxicity or not .", "protein conformation could also play a role in a possible toxic \n mechanism . in this way", ", a protein with a high proportion of alpha \n helix and hydrophobic regions could be inserted in cell membrane \n promoting toxic effects .", "additionally , the presence of \n aberrant polymers could affect the protein degradation cell machinery ( the proteasome complex ) , decreasing its activity \n and promoting a toxic effect .", "recently , some good reviews have been published on protein \n aggregation and neurodegenerative disorders [ 6 , 7 ] . in this \n review", "we will mainly focus on those aggregates assembled from tau \n protein , aggregates that could be present in the neurological \n disorders known as tauopathies ( for a review see ) ( table 1 ) ."], ["it has been described that large amounts of native or unmodified \n tau protein were enough to promote tau assembly into fibrillar \n polymers resembling those found in ad [ 912 ] .", "thus , \n obviously , an increase in tau concentration will favour the \n formation of tau polymers .", "recently , it has been reported that not \n all the brain areas have a similar amount of tau protein \n .", "thus , it suggests a different probability in the formation of tau polymers in different brain regions .", "the amount of tau will be the consequence of its synthesis and its \n degradation .", "changes in transcription have been indicated for \n other proteins related to neurodegenerative disorders , where a tata binding protein may play a role .", "tau degradation may \n take place through the proteasome complex [ 15 , 16 ] and it has \n been suggested that such degradation could be regulated by \n posttranslational modifications occurring in tau molecule , like \n its phosphorylation .", "also , tau degradation by other proteases could be regulated by its level of phosphorylation \n .", "it should be also indicated that in some cases like parkinson 's disease or lafora disease , mutations in the e3 \n ubiquitin ligases like parkin   or malin   will \n result in the appearance of aberrant protein aggregates . a conformational change , that could be followed by antibodies that \n react with tau molecule after that conformational change \n [ 2124 ] has been also suggested to be required for the \n transition tau monomer - tau polymer . also , it has been suggested that different posttranslational \n modifications like phosphorylation , glycation , \n or truncation , may play a role in the formation of tau \n polymers . due to the alternative splicing of its heterogenous ( or nuclear ) \n rna , different tau isoforms could be expressed and , therefore , \n different tau aggregates with different tau isoforms in different \n tauopathies could occur , but we will not discuss this point here . \n for further information see .", ", it has been suggested that removal of the amino \n and/or carboxy terminal regions , leaving the tubulin binding \n region will facilitate tau polymerization [ 21 , 22 , 28 ] .", "some work has been done in vitro   and in vivo [ 30 , 31 ] about a possible role of tau phosphorylation on tau \n assembly , suggesting that in some conditions tau phosphorylation \n may increase the capacity of tau for its self - assembly .", "not only \n an increase in serine / threonine phosphorylation of tau could \n regulate its aggregation but also an increase in tau tyrosine \n phosphorylation may increase the formation of tau aggregates \n . this assembly process may involve the formation of oligomers   , filaments , and aggregates of filaments \n ( tangle - like structures ) . in the formation of these aggregates of \n filaments , glycation may play a role . the possible relation between phosphorylation and tau aggregation \n", "has been studied in transgenic mice expressing human tau bearing \n some mutations found in human fronto - temporal dementia linked to \n chromosome 17 ( ftdp-17 ) . in this mouse ,", "tau \n phosphorylation mainly occurs by gsk3 . when a specific \n inhibitor of this kinase , lithium , was given to the transgenic \n mice no tau phosphorylation was found , and in addition no \n aggregation of the protein was detected   suggesting a \n correlation between tau phosphorylation and aggregation in this \n model .", "this result was supported by an additional experiment using \n another mouse also expressing human tau with a ftdp-17 mutation \n .", "alternatively , protein chaperones , acting on tau or in phosphotau , \n could modify the level of tau aggregation , examples could be the \n protein 14 - 3 - 3 , musashi-1   , or pin-1 \n [ 37 , 38 ] .", "the chaperone associated ubiquitin ligase chip could \n be able to target phosphotau for proteasomal degradation \n [ 16 , 18 ] ."], ["it has been described that tau binding to microtubules is regulated by the level of tau phosphorylation at some specific sites .", "it is known that hyperphosphorylated tau binds with less affinity to microtubules resulting in the decrease in the interaction with microtubules , in a decrease of \n microtubule stability , and probably in a microtubule dysfunction inside the cell that could result in a toxic effect \n .", "also , tau phosphorylation could result , as indicated above , in a decrease of its proteolysis .", "more recently , it has been indicated that expression of a tau mutant ( p301l ) \n could result in an increase of its phosphorylation , since once it is phosphorylated , that mutation can prevent the binding of those phosphatases involved in its dephosphorylation .", "this \n phosphotau could have a decreased capacity for microtubule binding \n and it could be toxic for the cell . additionally , it has been \n indicated that hyperphosphorylated tau can cause \n neurodegeneration , in the absence of large tau aggregates \n . on the other hand , only the overexpression of wild - type human tau in a mouse is sufficient to cause tau phosphorylation , aggregation , and neural toxicity . on the other hand", ", it has been suggested that tau phosphorylation may represent a \n protective function in ad ."], ["sometime ago , the development of tau pathology , related with \n dementia in ad , was clearly described by braak and braak \n  by following the development of neurofibrillary lesions at different stages of the disease . also , the formation of \n neurofibrillary ( tau aggregates ) pathology within those neurons of \n hippocampus and cerebral cortex affected at different stages was \n found .", "these neurons could degenerate yielding extracellular ghost \n tangles ( enft ) . in the hippocampus", ", an inverse relation has been found between the number of enft and the number \n of surviving neurons [ 4851 ] .", "it suggests that neurons that \n degenerate , have previously developed tau aggregates . on the other \n hand", ", it has been indicated that neurons bearing neurofibrillary \n lesions could survive for a long period of time , and , by comparing with other neurodegenerative disorders , like huntington \n disease , it can be suggested that tau aggregates could protect against neurodegeneration by sequestering toxic ( phospho ? ) \n monomeric tau that could be present in a high amount inside a cell \n in pathological conditions . also , it has been suggested , using a \n transgenic mouse model , that behavioural ( memory ) deficits could be unrelated to the formation of tau polymers , \n", "although , more recently , the discussion of those experiments suggested that hyperphosphorylated , aggregated tau intermediates could be the ones that cause neurodegeneration . in this way , the implication of different types of protein aggregates in neurodegeneration has been extensively \n discussed [ 19 , 54 ] . a possibility about the existence of neurotoxic tau intermediate aggregates in human tauopathies", "is \n based in the fact that patients with ftdp-17 show an extensive \n neurodegeneration with a high level of tau phosphorylation but \n with a low number of tangles . in any case , even if the formation of tau aggregates has a \n protective function for the neurons , that function is not working \n well , as described by braak and braak , and afterwards by delacourte et al , indicating a correlation between \n progression of tau pathology and progression of the disease .", "this \n idea is supported by those experiments indicating that neural loss \n and neurofibrillary tangle number increase in parallel with the \n progression of the disease .", "similar results have been described in other neurological disorders like brain encelphalopathies , where the formation of aberrant polymers are related to the onset of neurodegeneration ; whereas this is far from clear in other disorders like \n huntington disease ."], ["examples of neurological diseases where aberrant protein \n aggregates are found , and the suggested conformations in the \n aberrant aggregates are indicated ."]]}
{"article_id": "PMC3205614", "article_text": ["only those contraceptive methods are new in which synthetic steroid is used . regarding the current status of growth ,", "the world population is doubled every 54 years ; however , in poor countries , population is doubled in less than 20 years . to complete and preserve personal health during pregnancy ,", "the optimal use of contraceptive methods is effective , and planning is necessary before pregnancy .", "the rate of pregnancy in women with a high potency for fertility who use no contraceptive method is 90% during one year .", "therefore , conscious and accurate decision making for pregnancy and precise care play an important role in decreasing the maternal mortality .", "induced abortion that can be the important complication followed by unwanted pregnancy play a significant role in the incidence of infection , fever , risk of the next premature delivery , low birth weight , and infertility [ 1 , 4 ] .", "pregnancy is a temporary crisis that creates deep mental , physical , and behavioral changes in a woman .", "conscious and accurate decision making for pregnancy , continuation , and precise care play an important role in declining the maternal mortality . according to the studies by world health organization , close to one - third of the pregnancies in the third world countries", "considering the fact that induced abortion may create some significant complications such as infection , septic shock , fever , risk of the next premature delivery , low birth weight , and infertility , it can also cause some anatomic complications resulting from the surgery such as uterus , bladder , and intestinal rupture [ 1 , 4 ] .", "planning for pregnancy can help the safety of childbirth , and unplanned pregnancies increase the maternal mortality . despite the attempts by the authors in health centers , some unwanted pregnancies occur that can danger the women .", "therefore , we decided to evaluate some factors related to unwanted pregnancy in women referred to health centers .", "\n\t\t\t\t it was a cross - sectional study on 400 randomly pregnant women , who were referred to several clinics and health center in ahwaz city during 2010 .", "data was collected through interview and filling up a designed questionnaire containing demographic characteristics , fertility , and so forth .", "the mean age of women with unwanted pregnancy was 27.5  5.7 years , and in women with intended pregnancy was 24.6  4.5 years .", "the percentage of the older women ( 35 years ) in unwanted pregnancy was 3 times of intended pregnancy which was statistically significant .", "most of pregnant women lived in urban area ( 70.1% ) , and the percentage of rural women who intended pregnancy was higher ( 35.6% ) .", "the educational level in the subjects was 36% in secondary school and 44% in high school .", "37% of subjects with unwanted pregnancy were illiterate and primary school ( table 1 ) .", "the findings have shown that most of pregnant women were housewives ( 82% ) , and their husbands had self - employment ( 40% ) , and 7.1% of subjects with unwanted pregnancy had unemployed husband .", "half of the women with unwanted pregnancy had low economic status , while , in women with intended pregnancy , this rate was only 20% . according to the interviews , good relationship with", "regarding fertility characteristics , the findings showed that more than half of the women with unwanted pregnancy were in the third trimester 54% , ( figure 1 ) .", "80% of unwanted pregnancy had more than two times pregnancies , but the percentage in intended pregnancy was 38% .", "this study showed that more than half of the pregnant women had used one of the contraceptive methods before the recent pregnancy , and 30% had used natural ( interrupted ) methods .", "the most percentage of women with unwanted pregnancy used unreliable methods like interrupted method ( 59.1% ) .", "the incidence of pregnancy followed by the consumption of contraceptive pills in women unwanted pregnancy was 16% .", "this study has shown that 26% of women who wish to become pregnant had more knowledge and performance about pregnancy health 26% , and the low levels of knowledge and performance were more observed in unwanted pregnancy .", "\n\t\t\t\t the incidence of unwanted pregnancy is different in the world , but it has the same undesired outcomes . our study has shown that prevalence of unwanted pregnancy was 26% , and similar conducted studies indicated that the incidence rate of unplanned pregnancy was 25% , 30% , 43% , and 52% [ 69 ] . in this study ,", "relative decrease in the incidence of unwanted pregnancy in our country in comparison with the other countries may be due to the hopeful success of health centers .", "considering the point that intended pregnancy which results in a healthy childbirth from a healthy mother as an aim of midwifery science [ 10 , 11 ] , pregnancy must be occurred based on the accurate and conscious decision and according to the physical , mental , economic , social , and cultural status [ 2 , 3 ] .", "alenova in a study after evaluating the activities of consultation clinics guiding women for that the prevention of pregnancy states that prevention of unwanted pregnancy is more necessary in aged women , and it becomes more vital with the increase of age .", "mohammadloo in his study declares that more than half of the unwanted pregnancies occur in women more than 30 years .", "one of the causes of not intending pregnancy is the age of more than 35 years that has been noted in other studies too [ 6 , 8 , 13 , 14 ] . in the present study ,", "low education has a close relationship with increased incidence of unwanted pregnancy , and , in a study by bennett et al .", "most of the women have the ability to become pregnant for at least three decades in their life , but most of men are potentially fertile all over their life . in our study , the rate of unwanted pregnancy was higher in individuals with more number of children .", "also , in similar studies , the increased prevalence of unwanted pregnancy is observed with an increase in the number of children , from 7.9% in childless women to 92.8% in women with 4 children or more . in our study ,", "unwanted pregnancy was more observed in employee women , and some other studies have achieved this result too . low income , poverty , unemployed husband , and inappropriate job play significant role in the incidence of unplanned pregnancy [ 7 , 1519 ] .", "a study in zimbabwe has shown that women with unemployed husbands were more exposed to the unwanted pregnancy .", "some studies in africa   and new york   indicated that unwanted pregnancy more occurred in poor , low income , and homeless women , which necessitates more concern about the poor women .", "other studies in thailand   and usa   have found that one of the related factors with unwanted pregnancy is the relationship with the spouse ; also they showed that good relationship with the spouse in women with unwanted pregnancy was less than intended cases .", "a study from london has found that planned pregnancy was more observed in couple with more strengthened union in marriage .", "it seems that couples ' relationship is an important and positive factor which affects the increase of their cooperation in regarding the fertility health .", "it was indicated in this study that , in 20% of women with unwanted pregnancy , no contraceptive method was used , and more than half of them used unreliable methods of which failure was more observed in unwanted pregnancies .", "a study in egypt states that 47% of pregnant women with unplanned pregnancy do not use adequate prevention , and 28.8% encounter with the failure of their contraceptive method . in a study in china", ", it has been indicated that the failure of the contraceptive method has been the main cause of unwanted pregnancy .", "it seems that of the most important educational needs and the most effective attempts to prevent unwanted pregnancy are creating motivations in families , providing necessary facilities , and helping them in selection and accurate use of different methods by holding educational classes in health centers .", "therefore , introducing accurate information about the contraceptive methods acquires a profound understanding , and using this information is very important in planning for the pregnancy . considering the lack of safe methods , educational programs , introducing adequate contraceptive options , and consultation before pregnancy", "76% of world population live in developing countries , 85% of births , 95% of neonatal mortality , and 99% of maternal mortality occur in these countries .", "so , regulating reproduction and adequate planning for pregnancy is significant in these countries .   in conclusion", ", more attempts must be taken to decrease complication of pregnancies such as unwanted cases . in the recent years , it has been proved that , in addition , the population control and educational program for contraceptive methods are important and necessary in preventing unwanted pregnancy . because in iran abortion is illegal consider in the muslim religious , unwanted pregnant women can not do abortion except if physician has diagnosis that mother has complication to continue of her pregnancy or there is intrauterine growth retardation in the first trimester ."], "abstract_text": ["<S> we aimed to find the \n                 prevalence and some factors relating with unwanted \n                 pregnancy . </S>", "<S> methods . it was a \n                 cross - sectional study on 400 randomly pregnant \n                 women , who were referring to different health \n                 centers in ahwaz city during 2010 . </S>", "<S> data was \n                 conducted based on questionnaire , and all the \n                 analysis was performed using spss ( version 17 ) \n                 statistical analysis software . \n                 </S>", "<S> results . </S>", "<S> the prevalence of \n                 unwanted pregnancy was 26% . </S>", "<S> the percentage of \n                 unwanted pregnancy in ages more than 35 years was \n                 approximately three times more than the intended \n                 pregnancy . </S>", "<S> there were significant relationship \n                 between unwanted pregnancy and some variables such \n                 as age , number of pregnancy , number of childbirth , \n                 education status , economic status , husband 's \n                 occupation , and the relationship with the spouse \n                 and contraceptive methods ( p < 0.0001 ) . </S>", "<S> conclusion . </S>", "<S> the \n                     prevalence of unwanted pregnancy was high . </S>", "<S> to \n                     prevent unwanted pregnancy using consultation \n                     services before planning to be pregnancy , it is \n                     necessary to identify the factors relating \n                     with unwanted pregnancy . \n </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["only those contraceptive methods are new in which synthetic steroid is used . regarding the current status of growth ,", "the world population is doubled every 54 years ; however , in poor countries , population is doubled in less than 20 years . to complete and preserve personal health during pregnancy ,", "the optimal use of contraceptive methods is effective , and planning is necessary before pregnancy .", "the rate of pregnancy in women with a high potency for fertility who use no contraceptive method is 90% during one year .", "therefore , conscious and accurate decision making for pregnancy and precise care play an important role in decreasing the maternal mortality .", "induced abortion that can be the important complication followed by unwanted pregnancy play a significant role in the incidence of infection , fever , risk of the next premature delivery , low birth weight , and infertility [ 1 , 4 ] .", "pregnancy is a temporary crisis that creates deep mental , physical , and behavioral changes in a woman .", "conscious and accurate decision making for pregnancy , continuation , and precise care play an important role in declining the maternal mortality . according to the studies by world health organization , close to one - third of the pregnancies in the third world countries", "considering the fact that induced abortion may create some significant complications such as infection , septic shock , fever , risk of the next premature delivery , low birth weight , and infertility , it can also cause some anatomic complications resulting from the surgery such as uterus , bladder , and intestinal rupture [ 1 , 4 ] .", "planning for pregnancy can help the safety of childbirth , and unplanned pregnancies increase the maternal mortality . despite the attempts by the authors in health centers , some unwanted pregnancies occur that can danger the women .", "therefore , we decided to evaluate some factors related to unwanted pregnancy in women referred to health centers ."], ["\n\t\t\t\t it was a cross - sectional study on 400 randomly pregnant women , who were referred to several clinics and health center in ahwaz city during 2010 .", "data was collected through interview and filling up a designed questionnaire containing demographic characteristics , fertility , and so forth ."], ["the mean age of women with unwanted pregnancy was 27.5  5.7 years , and in women with intended pregnancy was 24.6  4.5 years .", "the percentage of the older women ( 35 years ) in unwanted pregnancy was 3 times of intended pregnancy which was statistically significant .", "most of pregnant women lived in urban area ( 70.1% ) , and the percentage of rural women who intended pregnancy was higher ( 35.6% ) .", "the educational level in the subjects was 36% in secondary school and 44% in high school .", "37% of subjects with unwanted pregnancy were illiterate and primary school ( table 1 ) .", "the findings have shown that most of pregnant women were housewives ( 82% ) , and their husbands had self - employment ( 40% ) , and 7.1% of subjects with unwanted pregnancy had unemployed husband .", "half of the women with unwanted pregnancy had low economic status , while , in women with intended pregnancy , this rate was only 20% . according to the interviews , good relationship with", "regarding fertility characteristics , the findings showed that more than half of the women with unwanted pregnancy were in the third trimester 54% , ( figure 1 ) .", "80% of unwanted pregnancy had more than two times pregnancies , but the percentage in intended pregnancy was 38% .", "this study showed that more than half of the pregnant women had used one of the contraceptive methods before the recent pregnancy , and 30% had used natural ( interrupted ) methods .", "the most percentage of women with unwanted pregnancy used unreliable methods like interrupted method ( 59.1% ) .", "the incidence of pregnancy followed by the consumption of contraceptive pills in women unwanted pregnancy was 16% .", "this study has shown that 26% of women who wish to become pregnant had more knowledge and performance about pregnancy health 26% , and the low levels of knowledge and performance were more observed in unwanted pregnancy ."], ["\n\t\t\t\t the incidence of unwanted pregnancy is different in the world , but it has the same undesired outcomes . our study has shown that prevalence of unwanted pregnancy was 26% , and similar conducted studies indicated that the incidence rate of unplanned pregnancy was 25% , 30% , 43% , and 52% [ 69 ] . in this study ,", "relative decrease in the incidence of unwanted pregnancy in our country in comparison with the other countries may be due to the hopeful success of health centers .", "considering the point that intended pregnancy which results in a healthy childbirth from a healthy mother as an aim of midwifery science [ 10 , 11 ] , pregnancy must be occurred based on the accurate and conscious decision and according to the physical , mental , economic , social , and cultural status [ 2 , 3 ] .", "alenova in a study after evaluating the activities of consultation clinics guiding women for that the prevention of pregnancy states that prevention of unwanted pregnancy is more necessary in aged women , and it becomes more vital with the increase of age .", "mohammadloo in his study declares that more than half of the unwanted pregnancies occur in women more than 30 years .", "one of the causes of not intending pregnancy is the age of more than 35 years that has been noted in other studies too [ 6 , 8 , 13 , 14 ] . in the present study ,", "low education has a close relationship with increased incidence of unwanted pregnancy , and , in a study by bennett et al .", "most of the women have the ability to become pregnant for at least three decades in their life , but most of men are potentially fertile all over their life . in our study , the rate of unwanted pregnancy was higher in individuals with more number of children .", "also , in similar studies , the increased prevalence of unwanted pregnancy is observed with an increase in the number of children , from 7.9% in childless women to 92.8% in women with 4 children or more . in our study ,", "unwanted pregnancy was more observed in employee women , and some other studies have achieved this result too . low income , poverty , unemployed husband , and inappropriate job play significant role in the incidence of unplanned pregnancy [ 7 , 1519 ] .", "a study in zimbabwe has shown that women with unemployed husbands were more exposed to the unwanted pregnancy .", "some studies in africa   and new york   indicated that unwanted pregnancy more occurred in poor , low income , and homeless women , which necessitates more concern about the poor women .", "other studies in thailand   and usa   have found that one of the related factors with unwanted pregnancy is the relationship with the spouse ; also they showed that good relationship with the spouse in women with unwanted pregnancy was less than intended cases .", "a study from london has found that planned pregnancy was more observed in couple with more strengthened union in marriage .", "it seems that couples ' relationship is an important and positive factor which affects the increase of their cooperation in regarding the fertility health .", "it was indicated in this study that , in 20% of women with unwanted pregnancy , no contraceptive method was used , and more than half of them used unreliable methods of which failure was more observed in unwanted pregnancies .", "a study in egypt states that 47% of pregnant women with unplanned pregnancy do not use adequate prevention , and 28.8% encounter with the failure of their contraceptive method . in a study in china", ", it has been indicated that the failure of the contraceptive method has been the main cause of unwanted pregnancy .", "it seems that of the most important educational needs and the most effective attempts to prevent unwanted pregnancy are creating motivations in families , providing necessary facilities , and helping them in selection and accurate use of different methods by holding educational classes in health centers .", "therefore , introducing accurate information about the contraceptive methods acquires a profound understanding , and using this information is very important in planning for the pregnancy . considering the lack of safe methods , educational programs , introducing adequate contraceptive options , and consultation before pregnancy", "76% of world population live in developing countries , 85% of births , 95% of neonatal mortality , and 99% of maternal mortality occur in these countries .", "so , regulating reproduction and adequate planning for pregnancy is significant in these countries .   in conclusion", ", more attempts must be taken to decrease complication of pregnancies such as unwanted cases . in the recent years , it has been proved that , in addition , the population control and educational program for contraceptive methods are important and necessary in preventing unwanted pregnancy . because in iran abortion is illegal consider in the muslim religious , unwanted pregnant women can not do abortion except if physician has diagnosis that mother has complication to continue of her pregnancy or there is intrauterine growth retardation in the first trimester ."]]}
{"article_id": "PMC3237148", "article_text": ["at the end of past millennium , flexible bronchoscopy was regarded as one of the most frequently performed procedures by the physicians of multiple disciplines to inspect the airway . in our institute , the fibreoptic bronchoscopies are performed by either the pulmonologists or the anaesthetist . at many places , fibreoptic bronchoscopies are still performed after topical anaesthesia only , without any sedation .", "this simplified approach is safe and results in decreased expenditures . in a retrospective study done by colt et al .", ", intravenous sedation was reported in 50% of the procedures ; however , this technique requires the use of adequate anaesthetic resources and is associated with a low but real morbidity and mortality .", "the intravenous sedation limits dynamic analysis of the airways such as vocal cords , presence of local or diffuse malacia or effects of voluntary cough .", "therefore , a risk  benefit approach comparing the same procedure performed under sedation and under local anaesthesia alone is indicated .", "there are several techniques for anaesthetising the vocal cords and tracheobronchial tree , each with its own potential advantages and disadvantages .", "topical lignocaine applied by the  spray as you go  technique with direct instillation of 4% solutions is used by a few bronchoscopists , while the others use transcricoid route for local anaesthesia of the vocal cords and tracheobronchial mucosa .", "we have compared a transcricoid injection of local anaesthesia with the  spray as you go ", "technique without sedation in patients posted for elective fibreoptic bronchoscopies requiring only bronchoalveolar aspirate for diagnosis and not biopsies of any kind .", "it is a very safe technique which can be performed with or without conscious sedation .", "after obtaining institutional ethical committee approval , 60 patients in the age group 2070 years of either sex , undergoing elective fibreoptic bronchoscopies for diagnostic bronchoalveolar aspirate , were included in this study .", "the patients of the specified age group coming to the bronchoscopy suit , requiring only diagnostic bronchoalveolar lavage over a period of 6 months , were selected and alternatively divided into two groups of 30 each .", "group i patients were given a single transcricoid injection of lignocaine , while in group ii patients lignocaine was used as spray as the bronchoscopist entered inside , after the lignocaine sensitivity test was done in all the patients .", "if any contraindication for transcricoid injection was present , like any local pathology , then these patients were included in the other group . after taking", "an informed written consent and lignocaine sensitivity test was done , the patients were alternatively assigned to different groups .", "all the patients were given injection atropine 0.6 mg intramuscularly , 20 min prior to the procedure .", "nebulisation with 3 ml of 4% lignocaine was done in all the patients for 15 min before starting the procedure .", "the blood pressure , pulse and oxygen saturation ( spo2 ) were recorded before the procedure in both the groups .", "the patency of the nostril was checked , and in the more patent nostril , 2 ml of 2% lignocaine gel was applied in all the patients .", "group i patients received transcricoid injection of 3 ml of 4% lignocaine solution given as a bolus through a 21-g hypodermic needle in the sitting position after confirming its position by aspirating air under aseptic conditions . in group", "ii patients , 2 ml of 4% lignocaine was instilled on to the vocal cords under direct vision after insertion of the bronchoscope .", "further boluses of lignocaine were instilled through the bronchoscope if local anaesthesia was thought to be inadequate in both the groups .", "the assistant as well as the bronchoscopist could not be blinded to the local anaesthetic techniques .", "a single endobronchial procedure was selected and the bronchoscopist was also not changed to allow a fair comparison of the two techniques studied .", "the pulse rate and systolic blood pressure were recorded before the procedure and 5 min after the bronchoscopy .", "the time from the nasal insertion of bronchoscope to reach the carina was recorded in both the groups .", "a bout of coughing was considered as a single episode of cough . the total dose of lignocaine used in both the groups was also noted .", "record of any complication like bleeding from the transcricoid site was made , as well as any other complication if detected was observed and noted .", "thirty min after the procedure , an assistant who was unaware of the patients group was asked to assess any discomfort to the patients , using a 10-cm visual analogue scale ( vas ) .", "the vas score of 0 was considered as no discomfort , 1 as mild , 2 as moderate discomfort and 3 or more as severe discomfort .", "the data were analysed using chi - square test and the p values were calculated .", "group i was transcricoid group and group ii patients received  spray as you go  technique .", "the age of patients in group i ( 51.6614.08 years ) was comparable with that in group ii patients ( 48.2613.32 , p = ns ) .", "as shown in table 1 , mean basal values of systolic blood pressure in both the groups were comparable before starting the procedure . in group", "ii , the systolic blood pressure increased significantly from the baseline when measured 5 min after the procedure ( p<0.02 ) . similarly , as shown in table 2 , the pulse rate was comparable in both the groups before starting the procedure ( p = ns ) , but it increased significantly in group ii when measured 5 min after the procedure .", "the total dose of lignocaine used in group ii ( 372.6624.90 mg ) was significantly higher than that used in group i ( 3149.32 mg , p<0.001 ) as shown in figure 1 .", "the number of coughs in group i ( 40.98 ) was significantly lesser than in group ii ( 4.91.24 , p<0.05 ) , as shown in figure 2 . the mean time to reach", "the carina was significantly shorter in group i ( 57.3312.98 sec ) compared to group ii ( 79.3322.35 sec , p<0.02 ) .", "systolic blood pressure before and 5 min after the procedure pulse rate before and 5 min after the procedure dose of lignocaine used plotted against the no . of patients", "cough episodes plotted against the no . of patients as shown in table 3 , the values of vas score were comparable after 30 min of the procedure in both the groups . there were no cases of haematoma or subcutaneous emphysema when the neck was examined after the procedure in the transcricoid group .", "lignocaine is the most commonly used local anaesthetic agent for fibreoptic bronchoscopy and has a wide margin of safety .", "it has been suggested that the total dose should be limited to 300400 mg as absorption of lignocaine from the respiratory mucosa is known to be rapid .", "the risk of more serious side effects increases when blood concentrations exceed 5 mg / l , with seizures and hallucinations occurring at concentrations of 812 mg / l and cardiorespiratory arrest at 2025 mg / l .", "the peak concentration is influenced by dose per unit weight administered and not by the factors considered likely to influence mucosal absorption from the bronchial tree , such as sputum production , airflow obstruction or cigarette smoking .", "a major proportion of the total dose of lignocaine is required to anaesthetise the nose , pharynx and larynx , with only a small proportion needed for the bronchial tree .", "although most clinical studies have reported non - toxic blood lignocaine concentration associated with bronchoscopy , several have reported concentration in the toxic range ( > 5 mg / l ) .", "the control of coughing is of paramount importance for the quality of a bronchoscopy as this facilitates ease of viewing the bronchial tree and obtaining good biopsy samples .", "activation of the cough centre in the brain stem causes the respiratory muscles to induce cough , the bronchial smooth muscle to cause bronchoconstriction and subsequently the airway submucosal glands to secrete mucus .", "various local anaesthetic techniques can be used to anaesthetise the respiratory mucosa for fibreoptic bronchoscopy .", "it would be safe and not unpleasant for the patient and would at the same time provide acceptable conditions for the bronchoscopist . in our institute", "these patients are malnourished and have deranged liver function tests as well as the pulmonary function tests , hence doing fibreoptic bronchoscopy under local anaesthesia only without sedation is a routine .", "the present study was designed to compare the techniques of applying topical anaesthesia to the respiratory mucosa for elective diagnostic fibreoptic bronchoscopy .", "the bronchoscopist was not changed , to allow a fair comparison of the two techniques studied .", "although the current british thoracic society guidelines provide a consensus statement on the current evidence base without specific guidance on drugs or techniques and without defining methods of sedation , the guidelines recommend offering sedation to all , except where there are contraindications .", "if a centre has experience of performing unsedated diagnostic flexible bronchoscopy , it is reported that patient co - operation is not improved with sedation .", "performed a double - blinded , placebo - controlled trial in 100 patients in a centre that normally performs unsedated bronchoscopy , and could not demonstrate improved patient tolerance , comfort and co - operation with lorazepam .", "tried to identify the common fears of patients undergoing fibreoptic bronchoscopy and also determine whether any factors might contribute to reducing these fears .", "it was found that doctors were more likely to explain the indication for bronchoscopy than how it would be performed .", "they concluded that provision of detailed information about sensations that are likely to be experienced in bronchoscopy could be used to allay some of the common fears .", "improved preparation of patients with lower education , inferior health status and asthma may lead to decreased pain during fibreoptic bronchoscopy .", "although sedation is associated with major complications , sedative drugs are often given immediately before fibreoptic bronchoscopy in the belief that patient 's comfort is improved .", "compared two such regimens with placebo and concluded that routine sedation has little part to play in patients undergoing a single diagnostic procedure .", "the present study compared two techniques of anaesthetising the respiratory mucosa for diagnostic bronchoscopy without sedation .", "the transcricoid method was more effective than the  spray as you go technique  .", "no complication was associated with transcricoid injection and minor bleeding associated with the technique did not interfere with the bronchoscopy .", "they recommended transcricoid technique as a safe method of inducing effective local anaesthesia that is well tolerated by the patient .", "in our study , we nebulised all the patients with 3 ml of 4% lignocaine for 15 min before the procedure .", "the systolic blood pressure and pulse rate were compared between the two groups before the procedure and 5 min after the procedure . the systolic blood pressure and", "the pulse rate were comparable in both the groups before the procedure , but the systolic blood pressure increased significantly in group ii after the procedure ( p<0.02 ) .", "similarly , the pulse rate also increased significantly after the procedure in group ii ( p<0.001 ) .", "the vas score was similar in both the groups , 30 min after the procedure ( p = ns ) . the incidence of side effects was negligible in both the groups .", "the dose of lignocaine used in group ii ( 372.6624.90 mg ) was significantly higher than that used in group i ( 3149.32 mg , p<0.001 ) . the number of coughs in group i", "( 40.98 ) was also significantly lower than in group ii ( 4.91.24 , p<0.05 ) .", "the time to reach the carina was also significantly lesser in group i ( 57.3312.98 sec ) compared to group ii ( 79.3322.35 sec , p<0.02 ) as the transcricoid injection given probably brought about excellent relaxation of the vocal cords , making the introduction of the bronchoscope smooth .", "diagnostic fibreoptic bronchoscopy without sedation with transcricoid injection of lignocaine can be recommended as a safe method of anaesthetising the respiratory mucosa , which is well tolerated by the patients with negligible side effects and provides acceptable conditions for the bronchoscopist as compared to the  spray as you go  technique ."], "abstract_text": ["<S> aim : the aim of the study was to compare transcricoid injection with spray as you go technique for diagnostic fibreoptic bronchoscopy , to perform the procedure without sedation and to record any complication or side effects.methods:sixty patients belonging to the age group 2070 years , undergoing diagnostic bronchoscopy over a period of 6 months , were randomly selected and divided into two groups alternatively to receive 3 ml of 4% lignocaine by a single transcricoid puncture ( group i ) or 2 ml of 4% lignocaine instilled through the bronchoscope on to the vocal cords and further 1 ml of 2% lignocaine into each main bronchus ( group ii ) </S>", "<S> . additional dose of lignocaine as required was given in both the groups . </S>", "<S> all patients were given intramuscular atropine 0.6 mg , 20 min before the procedure . </S>", "<S> nebulisation with 3 ml of 4% lignocaine was given to all patients . </S>", "<S> the time from nasal insertion of the bronchoscope to reach the carina was recorded , and the total dose of lignocaine required in both the groups was calculated and compared . </S>", "<S> the cough episodes during the procedure , systolic blood pressure , and pulse rate were compared before the procedure and 5 min after the procedure in both the groups . </S>", "<S> a010 visual analogue scale ( vas ) was used to assess discomfort 30 min after the procedure.results:the time to reach carina was more in group ii ( p<0.02 ) , and cough episodes were also more in group ii ( p<0.05 ) than in group i. the vitals before the procedure were comparable in both the groups , but 5 min after the procedure the vitals were more stable in group i than in group ii , and the total dose of lignocaine required in group ii was more than in group i ( p<0.001 ) . however , the vas score was comparable in both the groups.conclusion:transcricoid puncture for diagnostic bronchoscopies without sedation was associated with no complication and discomfort and required lesser dose of local anaesthetic with more stable vitals and good conditions for bronchoscopists . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["at the end of past millennium , flexible bronchoscopy was regarded as one of the most frequently performed procedures by the physicians of multiple disciplines to inspect the airway . in our institute , the fibreoptic bronchoscopies are performed by either the pulmonologists or the anaesthetist . at many places , fibreoptic bronchoscopies are still performed after topical anaesthesia only , without any sedation .", "this simplified approach is safe and results in decreased expenditures . in a retrospective study done by colt et al .", ", intravenous sedation was reported in 50% of the procedures ; however , this technique requires the use of adequate anaesthetic resources and is associated with a low but real morbidity and mortality .", "the intravenous sedation limits dynamic analysis of the airways such as vocal cords , presence of local or diffuse malacia or effects of voluntary cough .", "therefore , a risk  benefit approach comparing the same procedure performed under sedation and under local anaesthesia alone is indicated .", "there are several techniques for anaesthetising the vocal cords and tracheobronchial tree , each with its own potential advantages and disadvantages .", "topical lignocaine applied by the  spray as you go  technique with direct instillation of 4% solutions is used by a few bronchoscopists , while the others use transcricoid route for local anaesthesia of the vocal cords and tracheobronchial mucosa .", "we have compared a transcricoid injection of local anaesthesia with the  spray as you go ", "technique without sedation in patients posted for elective fibreoptic bronchoscopies requiring only bronchoalveolar aspirate for diagnosis and not biopsies of any kind .", "it is a very safe technique which can be performed with or without conscious sedation ."], ["after obtaining institutional ethical committee approval , 60 patients in the age group 2070 years of either sex , undergoing elective fibreoptic bronchoscopies for diagnostic bronchoalveolar aspirate , were included in this study .", "the patients of the specified age group coming to the bronchoscopy suit , requiring only diagnostic bronchoalveolar lavage over a period of 6 months , were selected and alternatively divided into two groups of 30 each .", "group i patients were given a single transcricoid injection of lignocaine , while in group ii patients lignocaine was used as spray as the bronchoscopist entered inside , after the lignocaine sensitivity test was done in all the patients .", "if any contraindication for transcricoid injection was present , like any local pathology , then these patients were included in the other group . after taking", "an informed written consent and lignocaine sensitivity test was done , the patients were alternatively assigned to different groups .", "all the patients were given injection atropine 0.6 mg intramuscularly , 20 min prior to the procedure .", "nebulisation with 3 ml of 4% lignocaine was done in all the patients for 15 min before starting the procedure .", "the blood pressure , pulse and oxygen saturation ( spo2 ) were recorded before the procedure in both the groups .", "the patency of the nostril was checked , and in the more patent nostril , 2 ml of 2% lignocaine gel was applied in all the patients .", "group i patients received transcricoid injection of 3 ml of 4% lignocaine solution given as a bolus through a 21-g hypodermic needle in the sitting position after confirming its position by aspirating air under aseptic conditions . in group", "ii patients , 2 ml of 4% lignocaine was instilled on to the vocal cords under direct vision after insertion of the bronchoscope .", "further boluses of lignocaine were instilled through the bronchoscope if local anaesthesia was thought to be inadequate in both the groups .", "the assistant as well as the bronchoscopist could not be blinded to the local anaesthetic techniques .", "a single endobronchial procedure was selected and the bronchoscopist was also not changed to allow a fair comparison of the two techniques studied .", "the pulse rate and systolic blood pressure were recorded before the procedure and 5 min after the bronchoscopy .", "the time from the nasal insertion of bronchoscope to reach the carina was recorded in both the groups .", "a bout of coughing was considered as a single episode of cough . the total dose of lignocaine used in both the groups was also noted .", "record of any complication like bleeding from the transcricoid site was made , as well as any other complication if detected was observed and noted .", "thirty min after the procedure , an assistant who was unaware of the patients group was asked to assess any discomfort to the patients , using a 10-cm visual analogue scale ( vas ) .", "the vas score of 0 was considered as no discomfort , 1 as mild , 2 as moderate discomfort and 3 or more as severe discomfort .", "the data were analysed using chi - square test and the p values were calculated ."], ["group i was transcricoid group and group ii patients received  spray as you go  technique .", "the age of patients in group i ( 51.6614.08 years ) was comparable with that in group ii patients ( 48.2613.32 , p = ns ) .", "as shown in table 1 , mean basal values of systolic blood pressure in both the groups were comparable before starting the procedure . in group", "ii , the systolic blood pressure increased significantly from the baseline when measured 5 min after the procedure ( p<0.02 ) . similarly , as shown in table 2 , the pulse rate was comparable in both the groups before starting the procedure ( p = ns ) , but it increased significantly in group ii when measured 5 min after the procedure .", "the total dose of lignocaine used in group ii ( 372.6624.90 mg ) was significantly higher than that used in group i ( 3149.32 mg , p<0.001 ) as shown in figure 1 .", "the number of coughs in group i ( 40.98 ) was significantly lesser than in group ii ( 4.91.24 , p<0.05 ) , as shown in figure 2 . the mean time to reach", "the carina was significantly shorter in group i ( 57.3312.98 sec ) compared to group ii ( 79.3322.35 sec , p<0.02 ) .", "systolic blood pressure before and 5 min after the procedure pulse rate before and 5 min after the procedure dose of lignocaine used plotted against the no . of patients", "cough episodes plotted against the no . of patients as shown in table 3 , the values of vas score were comparable after 30 min of the procedure in both the groups . there were no cases of haematoma or subcutaneous emphysema when the neck was examined after the procedure in the transcricoid group ."], ["lignocaine is the most commonly used local anaesthetic agent for fibreoptic bronchoscopy and has a wide margin of safety .", "it has been suggested that the total dose should be limited to 300400 mg as absorption of lignocaine from the respiratory mucosa is known to be rapid .", "the risk of more serious side effects increases when blood concentrations exceed 5 mg / l , with seizures and hallucinations occurring at concentrations of 812 mg / l and cardiorespiratory arrest at 2025 mg / l .", "the peak concentration is influenced by dose per unit weight administered and not by the factors considered likely to influence mucosal absorption from the bronchial tree , such as sputum production , airflow obstruction or cigarette smoking .", "a major proportion of the total dose of lignocaine is required to anaesthetise the nose , pharynx and larynx , with only a small proportion needed for the bronchial tree .", "although most clinical studies have reported non - toxic blood lignocaine concentration associated with bronchoscopy , several have reported concentration in the toxic range ( > 5 mg / l ) .", "the control of coughing is of paramount importance for the quality of a bronchoscopy as this facilitates ease of viewing the bronchial tree and obtaining good biopsy samples .", "activation of the cough centre in the brain stem causes the respiratory muscles to induce cough , the bronchial smooth muscle to cause bronchoconstriction and subsequently the airway submucosal glands to secrete mucus .", "various local anaesthetic techniques can be used to anaesthetise the respiratory mucosa for fibreoptic bronchoscopy .", "it would be safe and not unpleasant for the patient and would at the same time provide acceptable conditions for the bronchoscopist . in our institute", "these patients are malnourished and have deranged liver function tests as well as the pulmonary function tests , hence doing fibreoptic bronchoscopy under local anaesthesia only without sedation is a routine .", "the present study was designed to compare the techniques of applying topical anaesthesia to the respiratory mucosa for elective diagnostic fibreoptic bronchoscopy .", "the bronchoscopist was not changed , to allow a fair comparison of the two techniques studied .", "although the current british thoracic society guidelines provide a consensus statement on the current evidence base without specific guidance on drugs or techniques and without defining methods of sedation , the guidelines recommend offering sedation to all , except where there are contraindications .", "if a centre has experience of performing unsedated diagnostic flexible bronchoscopy , it is reported that patient co - operation is not improved with sedation .", "performed a double - blinded , placebo - controlled trial in 100 patients in a centre that normally performs unsedated bronchoscopy , and could not demonstrate improved patient tolerance , comfort and co - operation with lorazepam .", "tried to identify the common fears of patients undergoing fibreoptic bronchoscopy and also determine whether any factors might contribute to reducing these fears .", "it was found that doctors were more likely to explain the indication for bronchoscopy than how it would be performed .", "they concluded that provision of detailed information about sensations that are likely to be experienced in bronchoscopy could be used to allay some of the common fears .", "improved preparation of patients with lower education , inferior health status and asthma may lead to decreased pain during fibreoptic bronchoscopy .", "although sedation is associated with major complications , sedative drugs are often given immediately before fibreoptic bronchoscopy in the belief that patient 's comfort is improved .", "compared two such regimens with placebo and concluded that routine sedation has little part to play in patients undergoing a single diagnostic procedure .", "the present study compared two techniques of anaesthetising the respiratory mucosa for diagnostic bronchoscopy without sedation .", "the transcricoid method was more effective than the  spray as you go technique  .", "no complication was associated with transcricoid injection and minor bleeding associated with the technique did not interfere with the bronchoscopy .", "they recommended transcricoid technique as a safe method of inducing effective local anaesthesia that is well tolerated by the patient .", "in our study , we nebulised all the patients with 3 ml of 4% lignocaine for 15 min before the procedure .", "the systolic blood pressure and pulse rate were compared between the two groups before the procedure and 5 min after the procedure . the systolic blood pressure and", "the pulse rate were comparable in both the groups before the procedure , but the systolic blood pressure increased significantly in group ii after the procedure ( p<0.02 ) .", "similarly , the pulse rate also increased significantly after the procedure in group ii ( p<0.001 ) .", "the vas score was similar in both the groups , 30 min after the procedure ( p = ns ) . the incidence of side effects was negligible in both the groups .", "the dose of lignocaine used in group ii ( 372.6624.90 mg ) was significantly higher than that used in group i ( 3149.32 mg , p<0.001 ) . the number of coughs in group i", "( 40.98 ) was also significantly lower than in group ii ( 4.91.24 , p<0.05 ) .", "the time to reach the carina was also significantly lesser in group i ( 57.3312.98 sec ) compared to group ii ( 79.3322.35 sec , p<0.02 ) as the transcricoid injection given probably brought about excellent relaxation of the vocal cords , making the introduction of the bronchoscope smooth ."], ["diagnostic fibreoptic bronchoscopy without sedation with transcricoid injection of lignocaine can be recommended as a safe method of anaesthetising the respiratory mucosa , which is well tolerated by the patients with negligible side effects and provides acceptable conditions for the bronchoscopist as compared to the  spray as you go  technique ."]]}
{"article_id": "PMC3391619", "article_text": ["charcot - marie - tooth disease ( cmt ) is the most common hereditary motor and sensory neuropathy , and cmt type 1a ( cmt1a ) neuropathy , which is the most common type of cmt , is subject to a gene dosage effect.1 the primary genetic cause of cmt1a is a duplication of pmp22 resulting from the unequal crossover between two homologous repetitive elements that flank the 1.4-mb region of chromosome 17p12.2 a new mechanism responsible for the outbreak of cmt1a - which is related to a nonrecurrent rearrangement has recently been reported.3 cmt1a is also caused by a point mutation in pmp22.4 abnormal axon - schwann cell interactions cause abnormalities in axonal structure and function , but the exact pathogenic mechanism causing cmt1a is unknown.5 one obvious possibility is that the weakness and sensory loss are the result of axonal degeneration ; secondary axonal degeneration is common in cmt1a patients , and its degree determines the patient 's functional disability.6 this finding is consistent with the electrophysiological and clinical findings in patients with cmt1a and in several animal models of demyelinating neuropathy.7,8 axonal dysfunction induced by demyelinating schwann cells can also occur without axonal degeneration.9 the relationship between motor nerve conduction velocities ( mncvs ) and disease disabilities has been the subject of much debate.10 - 14 most cmt1a research groups have found a relatively weak correlation between the severity of muscle weakness and nerve conduction velocities,10 - 12 although some have noted that patients with slower velocities developed more weakness.13,14 moreover , longitudinal studies have shown that the velocities remained unchanged over decades , whereas the compound muscle action potential ( cmap ) amplitudes decreased.15,16 however , the relationship between mncvs , cmaps , and the disease severity has not been investigated in koreans .", "therefore , we determined whether there is a correlation between clinical disabilities and electrophysiological values in korean cmt1a patients with pmp22 duplication .", "we enrolled 167 cmt1a patients ( 93 males , 74 females ) with pmp22 duplication who took part in nerve conduction studies at 20 university hospitals in korea .", "this study was approved by the institutional review board of the ethics committee of the ewha womans university hospital .", "the patients ' clinical information was obtained and analyzed , including assessment of the gender ratio , age at onset , disease duration , motor and sensory impairment , deep tendon reflex , and muscle atrophy .", "the age at onset was determined by asking the patients when their symptoms - such as distal muscle weakness , foot deformity and/or sensory change - first appeared .", "the impairment and progression of cmt was evaluated in these patients using two scales : the functional disability scale ( fds)13 and the cmt neuropathy score ( cmtns).17 disease severity was quantified for each patient using a 9-point fds based on the following criteria : 0 , normal ; 1 , normal but with cramps and fatigability ; 2 , unable to run ; 3 , difficulty walking but still able to walk unaided ; 4 , walking with a cane ; 5 , walking with crutches ; 6 , walking with a walking frame ; 7 , wheelchair bound ; and 8 , bedridden . in addition , we determined the cmtns based on the motor and sensory symptoms , and according to the pain and vibration , muscle strength , and neurophysiologic test results .", "the scores of this test were used to divide the patients into two groups : mild ( 0<cmtns10 ) and moderate or severe ( cmtns11 ) .", "the mncvs and sensory nerve conduction velocities ( sncvs ) for the median , ulnar , peroneal , tibial , and sural nerves were determined in all 167 patients .", "the mncvs of the median and ulnar nerves were determined by stimulating at the elbow and wrist , respectfully , while cmaps were recorded over the abductor pollicis brevis and the adductor digiti quinti , respectively .", "the mncvs of the peroneal and tibial nerves were determined by stimulating at the knee and ankle while recording the cmaps over the extensor digitorum brevis and adductor hallucis , respectively .", "the sncvs and sensory nerve action potentials ( snaps ) were obtained over a finger - wrist segment from the median and ulnar nerves , and they were also recorded for the sural nerves .", "the terminal latency indices ( tlis ) that adjust the distal motor latency for the terminal distance and the proximal mncv were calculated .", "the calculated tli was compared to the tli of the normal population as reported previously.18 tlis were calculated for the median nerves as follows : tli = terminal distance ( mm)/[mncv ( ms)distal motor latency ( ms ) ] .", "11.0 ( spss , chicago , il , usa ) . percentages and means were compared by the chi - square test and student 's t - test , respectively .", "correlations were determined using pearson 's correlation coefficient ( r ) , and probability values of p<0.05 were considered to be indicative of statistical significance .", "we enrolled 167 cmt1a patients ( 93 males , 74 females ) with pmp22 duplication who took part in nerve conduction studies at 20 university hospitals in korea .", "this study was approved by the institutional review board of the ethics committee of the ewha womans university hospital .", "the patients ' clinical information was obtained and analyzed , including assessment of the gender ratio , age at onset , disease duration , motor and sensory impairment , deep tendon reflex , and muscle atrophy .", "the age at onset was determined by asking the patients when their symptoms - such as distal muscle weakness , foot deformity and/or sensory change - first appeared .", "the impairment and progression of cmt was evaluated in these patients using two scales : the functional disability scale ( fds)13 and the cmt neuropathy score ( cmtns).17 disease severity was quantified for each patient using a 9-point fds based on the following criteria : 0 , normal ; 1 , normal but with cramps and fatigability ; 2 , unable to run ; 3 , difficulty walking but still able to walk unaided ; 4 , walking with a cane ; 5 , walking with crutches ; 6 , walking with a walking frame ; 7 , wheelchair bound ; and 8 , bedridden . in addition , we determined the cmtns based on the motor and sensory symptoms , and according to the pain and vibration , muscle strength , and neurophysiologic test results .", "the scores of this test were used to divide the patients into two groups : mild ( 0<cmtns10 ) and moderate or severe ( cmtns11 ) .", "the mncvs and sensory nerve conduction velocities ( sncvs ) for the median , ulnar , peroneal , tibial , and sural nerves were determined in all 167 patients .", "the mncvs of the median and ulnar nerves were determined by stimulating at the elbow and wrist , respectfully , while cmaps were recorded over the abductor pollicis brevis and the adductor digiti quinti , respectively .", "the mncvs of the peroneal and tibial nerves were determined by stimulating at the knee and ankle while recording the cmaps over the extensor digitorum brevis and adductor hallucis , respectively .", "the sncvs and sensory nerve action potentials ( snaps ) were obtained over a finger - wrist segment from the median and ulnar nerves , and they were also recorded for the sural nerves .", "the terminal latency indices ( tlis ) that adjust the distal motor latency for the terminal distance and the proximal mncv were calculated .", "the calculated tli was compared to the tli of the normal population as reported previously.18 tlis were calculated for the median nerves as follows : tli = terminal distance ( mm)/[mncv ( ms)distal motor latency ( ms ) ] .", "11.0 ( spss , chicago , il , usa ) . percentages and means were compared by the chi - square test and student 's t - test , respectively .", "correlations were determined using pearson 's correlation coefficient ( r ) , and probability values of p<0.05 were considered to be indicative of statistical significance .", "the mean age at examination was 36.9 years ( range , 5 - 75 ye - ars ) and the mean age at onset was 15.5 years ( range , 2 - 45 years ) .", "the mean disease duration at the time of examination was 22.3 years ( range , 0 - 57 years ) .", "the disease had manifested before the second decade of life in 69% of the patients .", "twelve individuals ( 7% ) with pmp22 duplication did not complain of any clinical symptoms . muscle weakness and", "atrophy started predominantly in the distal legs , with these symptoms being noted to a lesser extent in the distal upper limbs .", "the paresis in the distal regions of the lower limbs varied from asymptomatic to severe weakness .", "pinprick sensory loss was found in 87 ( 52% ) patients , and proprioception loss was found in 112 ( 67% ) .", "heel gait defects occurred more commonly than toe gait defects ( p<0.001 ) , and high - arched foot deformities were common ( 89% ) .", "both the mncv and sncv of the median nerve were uniformly reduced in the cmt1a patients .", "the cmap of the median nerve was significantly correlated with the disease duration ( fig .", "1a ; p<0.001 ) and the cmap of the ulnar nerve ( fig . 1b ; p<0.01 ) .", "however , no correlations were found between the age at onset and the cmaps in the median and ulnar nerves ( fig .", "the tli of the patients ' median nerve ( 0.310.10 , meansd ; range , 0.13 - 0.72 ) did not differ significantly from the normal values seen in healthy control subjects ( 0.310.04 ; range , 0.23 - 0.43 ) .", "these results suggest that demyelination in cmt1a patients is uniformly distributed between the proximal and distal portions of the nerves .", "the characteristics of the mild ( 0<cmtns10 ) and the moderate or severe ( cmtns11 ) cmtns groups are listed in table 1 .", "the gender ratio was similar in the two groups , and the mean age at onset did not differ significantly between them .", "however , the disease duration at the time of examination was significantly longer in the moderate or severe cmtns group ( p<0.001 ) .", "the patients in the moderate or severe cmtns group were more severely affected than those of the mild cmtns group by muscle weakness ( p<0.001 ) , muscle atrophy ( p<0.001 ) , sensory loss ( p<0.001 ) , the frequency of areflexia ( upper , p<0.001 ; lower , p<0.01 ) , heel and toe gait defects ( p<0.001 ) , and foot deformity ( p<0.05 ) .", "the amplitudes of the evoked median nerve motor responses were more severely affected in the moderate or severe cmtns group than in the mild cmtns group . in contrast , neither the mncvs nor the sncvs differed significantly between the two groups .", "the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were significantly correlated with the cmtns , and the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were correlated with the fds score ( table 2 ) .", "in contrast , the mncv of the median nerve was only correlated with the cmtns ( p<0.05 ) . the snaps and sncvs of the median , ulnar , and sural nerves", "the mean age at examination was 36.9 years ( range , 5 - 75 ye - ars ) and the mean age at onset was 15.5 years ( range , 2 - 45 years ) .", "the mean disease duration at the time of examination was 22.3 years ( range , 0 - 57 years ) .", "the disease had manifested before the second decade of life in 69% of the patients .", "twelve individuals ( 7% ) with pmp22 duplication did not complain of any clinical symptoms . muscle weakness and", "atrophy started predominantly in the distal legs , with these symptoms being noted to a lesser extent in the distal upper limbs .", "the paresis in the distal regions of the lower limbs varied from asymptomatic to severe weakness .", "pinprick sensory loss was found in 87 ( 52% ) patients , and proprioception loss was found in 112 ( 67% ) .", "heel gait defects occurred more commonly than toe gait defects ( p<0.001 ) , and high - arched foot deformities were common ( 89% ) .", "both the mncv and sncv of the median nerve were uniformly reduced in the cmt1a patients .", "the cmap of the median nerve was significantly correlated with the disease duration ( fig .", "1a ; p<0.001 ) and the cmap of the ulnar nerve ( fig . 1b ; p<0.01 ) .", "however , no correlations were found between the age at onset and the cmaps in the median and ulnar nerves ( fig .", "the tli of the patients ' median nerve ( 0.310.10 , meansd ; range , 0.13 - 0.72 ) did not differ significantly from the normal values seen in healthy control subjects ( 0.310.04 ; range , 0.23 - 0.43 ) .", "these results suggest that demyelination in cmt1a patients is uniformly distributed between the proximal and distal portions of the nerves .", "the characteristics of the mild ( 0<cmtns10 ) and the moderate or severe ( cmtns11 ) cmtns groups are listed in table 1 .", "the gender ratio was similar in the two groups , and the mean age at onset did not differ significantly between them .", "however , the disease duration at the time of examination was significantly longer in the moderate or severe cmtns group ( p<0.001 ) .", "the patients in the moderate or severe cmtns group were more severely affected than those of the mild cmtns group by muscle weakness ( p<0.001 ) , muscle atrophy ( p<0.001 ) , sensory loss ( p<0.001 ) , the frequency of areflexia ( upper , p<0.001 ; lower , p<0.01 ) , heel and toe gait defects ( p<0.001 ) , and foot deformity ( p<0.05 ) .", "the amplitudes of the evoked median nerve motor responses were more severely affected in the moderate or severe cmtns group than in the mild cmtns group . in contrast , neither the mncvs nor the sncvs differed significantly between the two groups .", "the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were significantly correlated with the cmtns , and the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were correlated with the fds score ( table 2 ) .", "in contrast , the mncv of the median nerve was only correlated with the cmtns ( p<0.05 ) . the snaps and sncvs of the median , ulnar , and sural nerves", "the present study investigated the correlations between disease disabilities and the electrophysiological findings in korean cmt1a patients with pmp22 duplication , and found that the disabilities were related to the cmaps rather than to the ncvs .", "it has been reported that cmt symptoms progress due to axonal degeneration and loss,5,19 with axonal loss being the major cause of impairment and disability in patients with cmt1a.20 the accentuation of muscle wasting and sensory impairment in the distal extremities was correlated with the corresponding reduction in the cmaps and snaps but not with the decrease in mncv or sncv.10 - 12 our results support the hypothesis that the disease - related disabilities are correlated with the decreased nerve action potentials rather than the conduction velocities .", "it is important to determine how the demyelination of sch - wann cells causes axonal injury , but this remains elusive.21 many hypotheses have been postulated.22 - 25 for example , local biochemical changes in axons , such as decreased neurofilament phosphorylation , increased neurofilament density , and decreased axonal transport due to demyelinating schwann cells , could lead to axonal dysfunction and eventual axonal loss.9 some longitudinal cmt studies have revealed that nerve conduction velocities remain unchanged for more than 10 years , but that the cmaps progressively decrease over the same period.16,26 in the present study we also found an absence of velocity changes over a 10-year period in nine patients despite gradually increasing clinical severities .", "these results suggest that the degree of change in cmap amplitude is useful for predicting the clinical severity of the disease . as a marker of axonal loss ,", "we also found that the clinical manifestations of muscle atrophy were also strongly correlated with the cmaps and axonal loss , which became more pronounced as the disease progressed .", "the disease severity varies considerably among patients with cmt1a.27 attempts to identify the mitigating factors are underway , and these efforts could lead to effective therapeutic approaches for reducing the expression of pmp22.28 pmp22 duplication mainly induces a demyelinating phenotype , while the features of axonal pathology appear concomitantly with advancing age.6 the patients with pmp22 duplication show a great variability of disease severity , ranging from asymptomatic individuals to wheelchair - bound patients .", "these results suggest that other molecular , endogenous , and environmental factors ( e.g. , genetic factors related to the pmp22 expression ) influence the progression of this disease . in the present study , the age at onset was the first decade in 44% of the cmt1a patients and before 20 years of age in 69% .", "the age at onset as an indicator of the cmt disease severity has been the subject of debate.11,12,26 although birouk et al.13 reported an association between the age at onset and the severity of the disorder , others have suggested that the age at onset is not a reliable indicator of disease severity ; the latter is consistent with our findings.11,26 instead , one of the most important disease - modifying factors in our study appeared to be the disease duration .", "a longitudinal study found that axonal involvement progressed in a time - dependent manner , and that axonal degeneration proceeded concomitantly with advancing disease.10,16 the age - dependent reduction in the cmaps , and the increase in axonal degeneration and clinical disability observed in our study were also in good agreement with previous reports.15,29 furthermore , the median nerve tli did not differ between korean cmt1a patients and healthy controls . despite the distal location of the cmt symptoms", ", the nerve conduction velocities slowed uniformly along the proximal and distal segments , which implies a uniform pathological process of demyelination . in conclusion", ", a significant correlation was found between the clinical - electrophysiological manifestations and the physical disabilities among the korean cmt1a patients in this study .", "although our study was cross - sectional in design and involved only a single race , we propose that the clinical disease severity in patients with cmt1a is determined by the extent of axonal dysfunction ."], "abstract_text": ["<S> background and purposecharcot - marie - tooth disease ( cmt ) type 1a ( cmt1a ) is the demyelinating form of cmt that is significantly associated with pmp22 duplication . </S>", "<S> some studies have found that the disease - related disabilities of these patients are correlated with their compound muscle action potentials ( cmaps ) , while others have suggested that they are related to the nerve conduction velocities . in the present study , we investigated the correlations between the disease - related disabilities and the electrophysiological values in a large cohort of korean cmt1a patients.methodswe analyzed 167 cmt1a patients of korean origin with pmp22 duplication using clinical and electrophysiological assessments , including the cmt neuropathy score and the functional disability scale.resultsclinical motor disabilities were significantly correlated with the cmaps but not the motor nerve conduction velocities ( mncvs ) . </S>", "<S> moreover , the observed sensory impairments matched the corresponding reductions in the sensory nerve action potentials ( snaps ) but not with slowing of the sensory nerve conduction velocities ( sncvs ) . </S>", "<S> in addition , cmaps were strongly correlated with the disease duration but not with the age at onset . </S>", "<S> the terminal latency index did not differ between cmt1a patients and healthy controls.conclusionsin cmt1a patients , disease - related disabilities such as muscle wasting and sensory impairment were strongly correlated with cmaps and snaps but not with the mncvs or sncvs . </S>", "<S> therefore , we suggest that the clinical disabilities of cmt patients are determined by the extent of axonal dysfunction . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Patients", "Clinical assessments", "Electrophysiological studies", "Statistical analyses", "Results", "Clinical manifestations", "Electrophysiological findings", "Comparison between the mild CMTNS group and the moderate or severe CMTNS group", "Correlation between disabilities and electrophysiological values", "Discussion"], "sections": [["charcot - marie - tooth disease ( cmt ) is the most common hereditary motor and sensory neuropathy , and cmt type 1a ( cmt1a ) neuropathy , which is the most common type of cmt , is subject to a gene dosage effect.1 the primary genetic cause of cmt1a is a duplication of pmp22 resulting from the unequal crossover between two homologous repetitive elements that flank the 1.4-mb region of chromosome 17p12.2 a new mechanism responsible for the outbreak of cmt1a - which is related to a nonrecurrent rearrangement has recently been reported.3 cmt1a is also caused by a point mutation in pmp22.4 abnormal axon - schwann cell interactions cause abnormalities in axonal structure and function , but the exact pathogenic mechanism causing cmt1a is unknown.5 one obvious possibility is that the weakness and sensory loss are the result of axonal degeneration ; secondary axonal degeneration is common in cmt1a patients , and its degree determines the patient 's functional disability.6 this finding is consistent with the electrophysiological and clinical findings in patients with cmt1a and in several animal models of demyelinating neuropathy.7,8 axonal dysfunction induced by demyelinating schwann cells can also occur without axonal degeneration.9 the relationship between motor nerve conduction velocities ( mncvs ) and disease disabilities has been the subject of much debate.10 - 14 most cmt1a research groups have found a relatively weak correlation between the severity of muscle weakness and nerve conduction velocities,10 - 12 although some have noted that patients with slower velocities developed more weakness.13,14 moreover , longitudinal studies have shown that the velocities remained unchanged over decades , whereas the compound muscle action potential ( cmap ) amplitudes decreased.15,16 however , the relationship between mncvs , cmaps , and the disease severity has not been investigated in koreans .", "therefore , we determined whether there is a correlation between clinical disabilities and electrophysiological values in korean cmt1a patients with pmp22 duplication ."], ["we enrolled 167 cmt1a patients ( 93 males , 74 females ) with pmp22 duplication who took part in nerve conduction studies at 20 university hospitals in korea .", "this study was approved by the institutional review board of the ethics committee of the ewha womans university hospital .", "the patients ' clinical information was obtained and analyzed , including assessment of the gender ratio , age at onset , disease duration , motor and sensory impairment , deep tendon reflex , and muscle atrophy .", "the age at onset was determined by asking the patients when their symptoms - such as distal muscle weakness , foot deformity and/or sensory change - first appeared .", "the impairment and progression of cmt was evaluated in these patients using two scales : the functional disability scale ( fds)13 and the cmt neuropathy score ( cmtns).17 disease severity was quantified for each patient using a 9-point fds based on the following criteria : 0 , normal ; 1 , normal but with cramps and fatigability ; 2 , unable to run ; 3 , difficulty walking but still able to walk unaided ; 4 , walking with a cane ; 5 , walking with crutches ; 6 , walking with a walking frame ; 7 , wheelchair bound ; and 8 , bedridden . in addition , we determined the cmtns based on the motor and sensory symptoms , and according to the pain and vibration , muscle strength , and neurophysiologic test results .", "the scores of this test were used to divide the patients into two groups : mild ( 0<cmtns10 ) and moderate or severe ( cmtns11 ) .", "the mncvs and sensory nerve conduction velocities ( sncvs ) for the median , ulnar , peroneal , tibial , and sural nerves were determined in all 167 patients .", "the mncvs of the median and ulnar nerves were determined by stimulating at the elbow and wrist , respectfully , while cmaps were recorded over the abductor pollicis brevis and the adductor digiti quinti , respectively .", "the mncvs of the peroneal and tibial nerves were determined by stimulating at the knee and ankle while recording the cmaps over the extensor digitorum brevis and adductor hallucis , respectively .", "the sncvs and sensory nerve action potentials ( snaps ) were obtained over a finger - wrist segment from the median and ulnar nerves , and they were also recorded for the sural nerves .", "the terminal latency indices ( tlis ) that adjust the distal motor latency for the terminal distance and the proximal mncv were calculated .", "the calculated tli was compared to the tli of the normal population as reported previously.18 tlis were calculated for the median nerves as follows : tli = terminal distance ( mm)/[mncv ( ms)distal motor latency ( ms ) ] .", "11.0 ( spss , chicago , il , usa ) . percentages and means were compared by the chi - square test and student 's t - test , respectively .", "correlations were determined using pearson 's correlation coefficient ( r ) , and probability values of p<0.05 were considered to be indicative of statistical significance ."], ["we enrolled 167 cmt1a patients ( 93 males , 74 females ) with pmp22 duplication who took part in nerve conduction studies at 20 university hospitals in korea .", "this study was approved by the institutional review board of the ethics committee of the ewha womans university hospital ."], ["the patients ' clinical information was obtained and analyzed , including assessment of the gender ratio , age at onset , disease duration , motor and sensory impairment , deep tendon reflex , and muscle atrophy .", "the age at onset was determined by asking the patients when their symptoms - such as distal muscle weakness , foot deformity and/or sensory change - first appeared .", "the impairment and progression of cmt was evaluated in these patients using two scales : the functional disability scale ( fds)13 and the cmt neuropathy score ( cmtns).17 disease severity was quantified for each patient using a 9-point fds based on the following criteria : 0 , normal ; 1 , normal but with cramps and fatigability ; 2 , unable to run ; 3 , difficulty walking but still able to walk unaided ; 4 , walking with a cane ; 5 , walking with crutches ; 6 , walking with a walking frame ; 7 , wheelchair bound ; and 8 , bedridden . in addition , we determined the cmtns based on the motor and sensory symptoms , and according to the pain and vibration , muscle strength , and neurophysiologic test results .", "the scores of this test were used to divide the patients into two groups : mild ( 0<cmtns10 ) and moderate or severe ( cmtns11 ) ."], ["the mncvs and sensory nerve conduction velocities ( sncvs ) for the median , ulnar , peroneal , tibial , and sural nerves were determined in all 167 patients .", "the mncvs of the median and ulnar nerves were determined by stimulating at the elbow and wrist , respectfully , while cmaps were recorded over the abductor pollicis brevis and the adductor digiti quinti , respectively .", "the mncvs of the peroneal and tibial nerves were determined by stimulating at the knee and ankle while recording the cmaps over the extensor digitorum brevis and adductor hallucis , respectively .", "the sncvs and sensory nerve action potentials ( snaps ) were obtained over a finger - wrist segment from the median and ulnar nerves , and they were also recorded for the sural nerves .", "the terminal latency indices ( tlis ) that adjust the distal motor latency for the terminal distance and the proximal mncv were calculated .", "the calculated tli was compared to the tli of the normal population as reported previously.18 tlis were calculated for the median nerves as follows : tli = terminal distance ( mm)/[mncv ( ms)distal motor latency ( ms ) ] ."], ["11.0 ( spss , chicago , il , usa ) . percentages and means were compared by the chi - square test and student 's t - test , respectively .", "correlations were determined using pearson 's correlation coefficient ( r ) , and probability values of p<0.05 were considered to be indicative of statistical significance ."], ["the mean age at examination was 36.9 years ( range , 5 - 75 ye - ars ) and the mean age at onset was 15.5 years ( range , 2 - 45 years ) .", "the mean disease duration at the time of examination was 22.3 years ( range , 0 - 57 years ) .", "the disease had manifested before the second decade of life in 69% of the patients .", "twelve individuals ( 7% ) with pmp22 duplication did not complain of any clinical symptoms . muscle weakness and", "atrophy started predominantly in the distal legs , with these symptoms being noted to a lesser extent in the distal upper limbs .", "the paresis in the distal regions of the lower limbs varied from asymptomatic to severe weakness .", "pinprick sensory loss was found in 87 ( 52% ) patients , and proprioception loss was found in 112 ( 67% ) .", "heel gait defects occurred more commonly than toe gait defects ( p<0.001 ) , and high - arched foot deformities were common ( 89% ) .", "both the mncv and sncv of the median nerve were uniformly reduced in the cmt1a patients .", "the cmap of the median nerve was significantly correlated with the disease duration ( fig .", "1a ; p<0.001 ) and the cmap of the ulnar nerve ( fig . 1b ; p<0.01 ) .", "however , no correlations were found between the age at onset and the cmaps in the median and ulnar nerves ( fig .", "the tli of the patients ' median nerve ( 0.310.10 , meansd ; range , 0.13 - 0.72 ) did not differ significantly from the normal values seen in healthy control subjects ( 0.310.04 ; range , 0.23 - 0.43 ) .", "these results suggest that demyelination in cmt1a patients is uniformly distributed between the proximal and distal portions of the nerves .", "the characteristics of the mild ( 0<cmtns10 ) and the moderate or severe ( cmtns11 ) cmtns groups are listed in table 1 .", "the gender ratio was similar in the two groups , and the mean age at onset did not differ significantly between them .", "however , the disease duration at the time of examination was significantly longer in the moderate or severe cmtns group ( p<0.001 ) .", "the patients in the moderate or severe cmtns group were more severely affected than those of the mild cmtns group by muscle weakness ( p<0.001 ) , muscle atrophy ( p<0.001 ) , sensory loss ( p<0.001 ) , the frequency of areflexia ( upper , p<0.001 ; lower , p<0.01 ) , heel and toe gait defects ( p<0.001 ) , and foot deformity ( p<0.05 ) .", "the amplitudes of the evoked median nerve motor responses were more severely affected in the moderate or severe cmtns group than in the mild cmtns group . in contrast , neither the mncvs nor the sncvs differed significantly between the two groups .", "the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were significantly correlated with the cmtns , and the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were correlated with the fds score ( table 2 ) .", "in contrast , the mncv of the median nerve was only correlated with the cmtns ( p<0.05 ) . the snaps and sncvs of the median , ulnar , and sural nerves"], ["the mean age at examination was 36.9 years ( range , 5 - 75 ye - ars ) and the mean age at onset was 15.5 years ( range , 2 - 45 years ) .", "the mean disease duration at the time of examination was 22.3 years ( range , 0 - 57 years ) .", "the disease had manifested before the second decade of life in 69% of the patients .", "twelve individuals ( 7% ) with pmp22 duplication did not complain of any clinical symptoms . muscle weakness and", "atrophy started predominantly in the distal legs , with these symptoms being noted to a lesser extent in the distal upper limbs .", "the paresis in the distal regions of the lower limbs varied from asymptomatic to severe weakness .", "pinprick sensory loss was found in 87 ( 52% ) patients , and proprioception loss was found in 112 ( 67% ) .", "heel gait defects occurred more commonly than toe gait defects ( p<0.001 ) , and high - arched foot deformities were common ( 89% ) ."], ["both the mncv and sncv of the median nerve were uniformly reduced in the cmt1a patients .", "the cmap of the median nerve was significantly correlated with the disease duration ( fig .", "1a ; p<0.001 ) and the cmap of the ulnar nerve ( fig . 1b ; p<0.01 ) .", "however , no correlations were found between the age at onset and the cmaps in the median and ulnar nerves ( fig .", "the tli of the patients ' median nerve ( 0.310.10 , meansd ; range , 0.13 - 0.72 ) did not differ significantly from the normal values seen in healthy control subjects ( 0.310.04 ; range , 0.23 - 0.43 ) .", "these results suggest that demyelination in cmt1a patients is uniformly distributed between the proximal and distal portions of the nerves ."], ["the characteristics of the mild ( 0<cmtns10 ) and the moderate or severe ( cmtns11 ) cmtns groups are listed in table 1 .", "the gender ratio was similar in the two groups , and the mean age at onset did not differ significantly between them .", "however , the disease duration at the time of examination was significantly longer in the moderate or severe cmtns group ( p<0.001 ) .", "the patients in the moderate or severe cmtns group were more severely affected than those of the mild cmtns group by muscle weakness ( p<0.001 ) , muscle atrophy ( p<0.001 ) , sensory loss ( p<0.001 ) , the frequency of areflexia ( upper , p<0.001 ; lower , p<0.01 ) , heel and toe gait defects ( p<0.001 ) , and foot deformity ( p<0.05 ) .", "the amplitudes of the evoked median nerve motor responses were more severely affected in the moderate or severe cmtns group than in the mild cmtns group . in contrast , neither the mncvs nor the sncvs differed significantly between the two groups ."], ["the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were significantly correlated with the cmtns , and the cmaps of the median ( p<0.001 ) , ulnar ( p<0.001 ) , peroneal ( p<0.05 ) , and tibial ( p<0.001 ) nerves were correlated with the fds score ( table 2 ) .", "in contrast , the mncv of the median nerve was only correlated with the cmtns ( p<0.05 ) . the snaps and sncvs of the median , ulnar , and sural nerves"], ["the present study investigated the correlations between disease disabilities and the electrophysiological findings in korean cmt1a patients with pmp22 duplication , and found that the disabilities were related to the cmaps rather than to the ncvs .", "it has been reported that cmt symptoms progress due to axonal degeneration and loss,5,19 with axonal loss being the major cause of impairment and disability in patients with cmt1a.20 the accentuation of muscle wasting and sensory impairment in the distal extremities was correlated with the corresponding reduction in the cmaps and snaps but not with the decrease in mncv or sncv.10 - 12 our results support the hypothesis that the disease - related disabilities are correlated with the decreased nerve action potentials rather than the conduction velocities .", "it is important to determine how the demyelination of sch - wann cells causes axonal injury , but this remains elusive.21 many hypotheses have been postulated.22 - 25 for example , local biochemical changes in axons , such as decreased neurofilament phosphorylation , increased neurofilament density , and decreased axonal transport due to demyelinating schwann cells , could lead to axonal dysfunction and eventual axonal loss.9 some longitudinal cmt studies have revealed that nerve conduction velocities remain unchanged for more than 10 years , but that the cmaps progressively decrease over the same period.16,26 in the present study we also found an absence of velocity changes over a 10-year period in nine patients despite gradually increasing clinical severities .", "these results suggest that the degree of change in cmap amplitude is useful for predicting the clinical severity of the disease . as a marker of axonal loss ,", "we also found that the clinical manifestations of muscle atrophy were also strongly correlated with the cmaps and axonal loss , which became more pronounced as the disease progressed .", "the disease severity varies considerably among patients with cmt1a.27 attempts to identify the mitigating factors are underway , and these efforts could lead to effective therapeutic approaches for reducing the expression of pmp22.28 pmp22 duplication mainly induces a demyelinating phenotype , while the features of axonal pathology appear concomitantly with advancing age.6 the patients with pmp22 duplication show a great variability of disease severity , ranging from asymptomatic individuals to wheelchair - bound patients .", "these results suggest that other molecular , endogenous , and environmental factors ( e.g. , genetic factors related to the pmp22 expression ) influence the progression of this disease . in the present study , the age at onset was the first decade in 44% of the cmt1a patients and before 20 years of age in 69% .", "the age at onset as an indicator of the cmt disease severity has been the subject of debate.11,12,26 although birouk et al.13 reported an association between the age at onset and the severity of the disorder , others have suggested that the age at onset is not a reliable indicator of disease severity ; the latter is consistent with our findings.11,26 instead , one of the most important disease - modifying factors in our study appeared to be the disease duration .", "a longitudinal study found that axonal involvement progressed in a time - dependent manner , and that axonal degeneration proceeded concomitantly with advancing disease.10,16 the age - dependent reduction in the cmaps , and the increase in axonal degeneration and clinical disability observed in our study were also in good agreement with previous reports.15,29 furthermore , the median nerve tli did not differ between korean cmt1a patients and healthy controls . despite the distal location of the cmt symptoms", ", the nerve conduction velocities slowed uniformly along the proximal and distal segments , which implies a uniform pathological process of demyelination . in conclusion", ", a significant correlation was found between the clinical - electrophysiological manifestations and the physical disabilities among the korean cmt1a patients in this study .", "although our study was cross - sectional in design and involved only a single race , we propose that the clinical disease severity in patients with cmt1a is determined by the extent of axonal dysfunction ."]]}
{"article_id": "PMC5058989", "article_text": ["a negative balance between the formation and resorption of bone mass can lead to the development of osteoporosis .", "moreover , osteoporosisis characterized by skeletal fragility resulting from reduced bone mass and disrupted bone microarchitecture .", "osteoporosis increases the risk of fracture and thus has been considered a major public health concern . traditionally , the development of osteoporosis is related to several risk factors , such as aging , immobility , hypertension , antihypertensive agents , hyperparathyroidism , menopause , diabetes mellitus , corticosteroid usage , low calcium intake , vitamin d deficiency , and genetic vulnerability .", "vertebral fracture is the most common osteoporotic fracture , but only from one - third to one quarter of the patients with vertebral fracture can be clinically identified .", "furthermore , it is reported that asymptomatic vertebral fractures are associated with future hip fracture by threefold and other nonvertebral fracture by twofold .", "hip fracture is the second common osteoporotic fracture and it may incur substantial healthcare costs resulted from disability . furthermore , the effect of hip fracture on mortality increase can adversely extend up to 10 years or more .", "the mortality following a hip fracture generally increases with the increment of age and is greater in men , but the sex difference declines after age 80 .", "although the rates of hip fracture have declined in the west , the rate is increasing in the developing world .", "it is estimated that > 50% of hip fractures worldwide will occur in asia by 2050 . the functional outcome and increased mortality of osteoporotic fracture", "are heterogeneous and depend on age , activity of daily living prior to fracture , pre - fracture comorbidities , and the cognition .", "hepatitis c virus infection ( hcv ) is a global health problem estimated to affect 170 million people worldwide .", "hepatitis c virus infection is a hepatotropic virus that mainly causes inflammation and fibrosis of the liver .", "it is reported that 20% of hcv - infected patients will progress to liver cirrhosis .", "however , hcv can also cause several extrahepatic manifestations , such as diabetes mellitus , rheumatic disorders , lymphoproliferative disease , cardiovascular events , and cognitive impairment .", "although the role of osteoporosis as a sequence of cirrhosis or advanced liver disease has been thoroughly documented , the effect of hcv exposure on bone mineral density in the absence of advanced liver disease remains debated .", "some scholars have proposed that chronic hcv infection without liver cirrhosis contributes to reduced bone mineral density , whereas other scholars have asserted the opposite . to assess the association between hcv exposure and subsequent development of osteoporosis", ", we conducted a nationwide population - based cohort study by analyzing data from a nationwide medical database , the national health insurance research database ( nhird ) .", "the national health insurance ( nhi ) program , initiated on march 1 , 1995 , provides comprehensive coverage for the medical care of taiwan residents . at the end of 2014 ,", "23.75 million people ( 99.9% of the population ) were enrolled in the program . in cooperation with the bureau of national health insurance ( bnhi ) , the national health research institutes established several data sets for public use , including the longitudinal health insurance database 2000 ( lhid2000 ) , a cohort data set comprising 1,000,000 randomly selected cases from the registry of nhi beneficiaries in 2000 . to maintain confidentiality , personal information , such as patient identification numbers and sensitive personal data ,", "are encrypted in the database , and international classification of diseases , ninth edition , clinical modification ( icd-9-cm ) codes are used for disease classification .", "this study was approved to fulfill the condition for exemption by the institutional review board ( irb ) of china medical university ( cmuh-104-rec2 - 115 ) .", "the irb also specifically waived the consent requirement . from 2000 to 2011 , patients aged 20 years and older with diagnosed hcv infection ( icd-9-cm codes 070.41 , 070.44 , 070.51 , and 070.54 ) identified from the lhid2000 comprised the hcv infection cohort .", "patients with a history of osteoporosis ( icd-9-cm codes 733.0 and 733.1 ) and hepatitis b virus ( hbv ) infection ( icd-9-cm codes 070.20 , 070.22 , 070.30 , and 070.32 ) diagnosed before the index date , those with missing information , and those younger than 20 years were excluded .", "using 1:m case - control studies is to increase the power and to control possible confounding .", "based on the statistical efficiency does not gain much when m  >  4 , we constructed a 1:4 matched cohort study . for each hcv case ,", "4 insurers with no history of hcv exposure , hbv infection , and osteoporosis were assigned to a comparison cohort and frequency matched with the hcv exposure cohorts according to age ( every 5-year span ) , sex , and index year .", "individuals were excluded from the comparison cohort using the same criteria used for the hcv exposure cohort . the primary endpoint in this study", "each participant was followed from the index date until the endpoint , withdrawal from the nhi program , or december 31 , 2011 .", "the baseline characteristics of participant comorbidities were also analyzed ; the comorbidities included diabetes ( icd-9-cm code 250 ) , hypertension ( icd-9-cm codes 401405 ) , hyperlipidemia ( icd-9-cm code 272 ) , heart failure ( icd-9-cm code 428 ) , stroke ( icd-9-cm codes 430438 ) , obesity ( icd-9-cm code 278 ) , and cirrhosis ( icd-9-cm codes 571.2 , 571.5 , and 571.6 ) . the chi - square test and t test for categorical and continuous variables , respectively , were first used to compare the distributions of age , sex , and baseline comorbidities between the hcv exposure and the comparison cohorts .", "the incidence densities of osteoporosis were estimated in person - years for the various risk factors . to estimate the hazard ratios ( hrs ) and 95% confidence intervals ( cis ) of osteoporosis ,", "multivariable models were simultaneously adjusted for age , sex , and the comorbidities of diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis .", "meier estimates were plotted to illustrate the cumulative incidence of osteoporosis , and the log - rank test was performed to examine the difference between the hcv exposure and the comparison cohorts .", "all statistical analyses were performed using the sas package ( version 9.4 for windows ; sas institute , inc , cary , nc ) .", "the national health insurance ( nhi ) program , initiated on march 1 , 1995 , provides comprehensive coverage for the medical care of taiwan residents . at the end of 2014 ,", "23.75 million people ( 99.9% of the population ) were enrolled in the program . in cooperation with the bureau of national health insurance ( bnhi ) , the national health research institutes established several data sets for public use , including the longitudinal health insurance database 2000 ( lhid2000 ) , a cohort data set comprising 1,000,000 randomly selected cases from the registry of nhi beneficiaries in 2000 . to maintain confidentiality , personal information , such as patient identification numbers and sensitive personal data ,", "are encrypted in the database , and international classification of diseases , ninth edition , clinical modification ( icd-9-cm ) codes are used for disease classification .", "this study was approved to fulfill the condition for exemption by the institutional review board ( irb ) of china medical university ( cmuh-104-rec2 - 115 ) .", "from 2000 to 2011 , patients aged 20 years and older with diagnosed hcv infection ( icd-9-cm codes 070.41 , 070.44 , 070.51 , and 070.54 ) identified from the lhid2000 comprised the hcv infection cohort . the date for hcv exposure coding", "patients with a history of osteoporosis ( icd-9-cm codes 733.0 and 733.1 ) and hepatitis b virus ( hbv ) infection ( icd-9-cm codes 070.20 , 070.22 , 070.30 , and 070.32 ) diagnosed before the index date , those with missing information , and those younger than 20 years were excluded . using 1:m case - control studies is to increase the power and to control possible confounding . based on the statistical efficiency", "does not gain much when m  >  4 , we constructed a 1:4 matched cohort study .", "for each hcv case , 4 insurers with no history of hcv exposure , hbv infection , and osteoporosis were assigned to a comparison cohort and frequency matched with the hcv exposure cohorts according to age ( every 5-year span ) , sex , and index year .", "individuals were excluded from the comparison cohort using the same criteria used for the hcv exposure cohort .", "each participant was followed from the index date until the endpoint , withdrawal from the nhi program , or december 31 , 2011 .", "the baseline characteristics of participant comorbidities were also analyzed ; the comorbidities included diabetes ( icd-9-cm code 250 ) , hypertension ( icd-9-cm codes 401405 ) , hyperlipidemia ( icd-9-cm code 272 ) , heart failure ( icd-9-cm code 428 ) , stroke ( icd-9-cm codes 430438 ) , obesity ( icd-9-cm code 278 ) , and cirrhosis ( icd-9-cm codes 571.2 , 571.5 , and 571.6 ) .", "the chi - square test and t test for categorical and continuous variables , respectively , were first used to compare the distributions of age , sex , and baseline comorbidities between the hcv exposure and the comparison cohorts .", "the incidence densities of osteoporosis were estimated in person - years for the various risk factors . to estimate the hazard ratios ( hrs ) and 95% confidence intervals ( cis ) of osteoporosis ,", "multivariable models were simultaneously adjusted for age , sex , and the comorbidities of diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis .", "meier estimates were plotted to illustrate the cumulative incidence of osteoporosis , and the log - rank test was performed to examine the difference between the hcv exposure and the comparison cohorts .", "all statistical analyses were performed using the sas package ( version 9.4 for windows ; sas institute , inc , cary , nc ) .", "of the 51,535 sampled patients , 41,228 and 10,307 were categorized as the comparison and hcv exposure cohorts , respectively ( table 1 ) .", "most patients were aged 50 years ( 61.7% ) , and 54.6% of the patients were women .", "the mean age was 54.1    15.3 years in the hcv exposure cohort and 53.7    15.5 years in the comparison cohort .", "regarding baseline characteristics , the hcv exposure cohort exhibited a higher prevalence of diabetes , hypertension , hyperlipidemia , heart failure , stroke , obesity , and cirrhosis than did the comparison cohort . during the mean follow - up periods of 5.44 and 6.01 years for the hcv exposure and comparison cohorts , respectively , the kaplan ", "meier curve revealed that the cumulative incidence of osteoporosis was higher in the hcv exposure cohort than in the comparison cohort ( figure 1 , log - rank test p  <  0.001 ) .", "demographic characteristics and comorbidities in cohorts with and without hcv exposure cumulative incidence comparison of osteoporosis for patients with ( dashed line ) or without ( solid line ) hcv exposure.hcv  =  hepatitis c virus .", "the overall incidence of osteoporosis in the hcv exposure cohort was higher than that in the comparison cohort ( 8.27 vs 6.19 per 1,000 person - years ; crude hr  =  1.33 , 95% ci  =  1.201.47 ) ( table 2 ) .", "after we adjusted for factors such as age , sex , and comorbidities , namely diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis , the risk of developing osteoporosis was significantly higher in the hcv exposure cohort than in the comparison cohort ( adjusted hr [ ahr ]  =  1.35 ; 95% ci  =  1.211.51 ) . compared with", "patients aged 49 years , the risk of developing osteoporosis was 4.05-fold higher in those aged 50 to 64 years ( 95% ci  =  3.8454.760 ) and 8.82-fold higher in those aged 65 years or older ( 95% ci  =  7.4810.40 ) . in the multivariate model ,", "the risk for osteoporosis was 3.10-fold higher for women than for men ( 95% ci  =  2.813.43 ) and was higher for patients with the comorbidities of hypertension ( ahr  =  1.19 , 95% ci  =  1.071.31 ) , hyperlipidemia ( ahr  =  1.17 , 95% ci  =  1.051.29 ) , and heart failure ( ahr  =  1.23 , 95% ci  =  1.021.49 ) .", "the incidence and hazard ratio for osteoporosis and osteoporosis - associated risk factor the incidence of osteoporosis increased with age , was higher in women than in men , and increased with comorbidity in both cohorts ( table 3 ) .", "the overall risk of osteoporosis related to several variables including age , sex , and presence of comorbidities was compared in the hcv exposure cohort and the comparison cohort .", "the risk of osteoporosis in patients exposed to hcv in all stratifications was higher than that in the comparison cohorts .", "however , the risk of osteoporosis contributed by hcv decreased with age ( aged  49 : ahr  =  1.79 , 95% ci  =  1.322.43 ; aged 5064 : ahr  =  1.36 , 95% ci  =  1.141.62 ; aged  65 : ahr  =  1.23 , 95% ci  =  1.051.44 ) and the presence of comorbidity ( no comorbidity : ahr  =  1.54 , 95% ci  =  1.261.89 ; comorbidity : ahr  =  1.27 , 95% ci  =  1.121.43 ) .", "incidence of osteoporosis by age , sex , and comorbidity and cox model measured hazards ratio for patients with hcv infection compared those without hcv exposure the patients exposed to hcv exhibited a 1.38-fold ( 95% ci  =  1.241.55 ) higher risk of developing osteoporosis compared with the patients who were not exposed to hcv ( table 4 ) .", "the risk of osteoporotic fracture did not differ significantly between patients exposed to hcv and the comparison cohorts ( ahr  =  0.80 , 95% ci  =  0.441.45 ) .", "comparisons of hazard ratios between patients with and without hcv exposure for different outcomes osteoporosis ( or osteoporotic fracture )", "consistent with the literature proposing that hcv seroprevalence peaks after age 55 and that women are predisposed to hcv infection , our results revealed that most patients ( 61.7% ) were aged 50 years and that 54.6% of the patients were women .", "the mean age in the hcv exposure cohort was 54.1    15.3 years . compared with patients who were not exposed to hcv", ", patients who were exposed to hcv tended to have more comorbidities , including diabetes , hypertension , hyperlipidemia , heart failure , stroke , obesity , and cirrhosis .", "our results revealed that the hcv exposure cohort had more comorbidities ; however , the risk of osteoporosis remained higher in the hcv exposure cohort after adjusting for age , sex , and the comorbidities of diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis .", "the mechanism affecting the pathophysiology that causes comorbidities in patients exposed to hcv may include hcv - associated insulin resistance and atherosclerosis .", "hcv may cause diabetes mellitus by directly interfering with insulin signaling and inducing insulin resistance in hepatocytes ; hcv - infected hepatocytes can secrete mediators that induce extrahepatic insulin resistance , notably in the skeletal muscle .", "hepatitis c virus may increase the risk of cardiovascular and cerebrovascular events through atherosclerosis induced by systemic inflammation , chronic endothelial injury , or direct infection of the arterial wall .", "our study revealed that the osteoporosis incidence increased with age and was higher in women than in men .", "the association between osteoporosis and aging has been confirmed in the literature , and the gene expression of the rennin ", "the decreasing rate of bone mineral density is swifter in the early postmenopausal period , which typically begins after age 50 , and women aged 40 to 59 years have the highest risk of developing osteoporosis .", "consistent with our result that women are predisposed to osteoporosis , the us preventive services task force indicated that as many as 1 in 2 postmenopausal women and 1 in 5 men are at risk for osteoporosis - related fracture .", "moreover , in the present study , osteoporosis was associated with hypertension , hyperlipidemia , and heart failure .", "both osteoporosis and hypertension are common among the aging population and may share similar etiologies , such as low calcium intake and levels , vitamin d and vitamin k deficiency , high salt consumption , imbalanced nitric oxide levels , and antihypertensive agents that exert detrimental effects on the skeletal metabolism , strength , and density .", "hyperlipidemia can reduce bone formation and promote bone loss by causing the products of lipid oxidation to accumulate in the subendothelial spaces of the vasculature and bone .", "moreover , hyperlipidemia can induce secondary hyperparathyroidism , thereby impairing bone regeneration and mechanical strength .", "heart failure and osteoporosis share several risk factors , such as aging , smoking , and postmenopausal and antihypertensive agents ; heart failure can also accelerate bone loss by inducing hyperaldosteronism and secondary hyperparathyroidism . however , the effect of obesity on bone metabolism is controversial .", "obesity is traditionally regarded as a protective factor for osteoporosis , conferring a positive mechanical loading on bone formation .", "for example , both osteoblasts and adipocytes are derived from a common mesenchymal stem cell and agents inhibiting adipogenesis - stimulated osteoblast differentiation and vice versa .", "furthermore , the reduced bone formation caused by aging is usually accompanied by adipogenesis in bone marrow cavities .", "moreover , elevated oxidative stress is common in people with obesity and osteoporosis . to our knowledge", ", this is the first population - based study to assess the relation between hcv exposure and the incidences of osteoporosis and osteoporotic fracture .", "our statistical analyses benefited from the use of a nationwide database and the 12-year observation of participants selected from a representative cohort comprising 1,000,000 residents covered by the nhi program .", "hansen et al conducted a large - scale population - based study to explore the association between hcv exposure and all - site fracture , but omitted discussing osteoporosis development .", "they concluded that the risk of fracture equally increased in patients exposed to hcv ( chronic or cleared infections ) , and the major determinants of fracture in such patients are lifestyle - related factors , such as alcohol and drug abuse , which substantially increases fracture risk .", "by contrast , our epidemiological study demonstrated that hcv exposure increases the risk of osteoporosis and the detrimental effect of hcv on osteoporotic fracture was not obvious .", "several pathogenic mechanisms are involved in bone mineral density loss in patients exposed to hcv .", "first , fibronectin can infiltrate the bone matrix to enhance matrix mineralization , reducing its production by the liver .", "second , insulin - like growth factor 1 , involved in osteoblast differentiation and proliferation , is produced by the liver and is reduced in patients with chronic liver disease .", "third , the receptor  activator ratio of nuclear factor kappa ligand and osteoprotegerin is higher in patients with chronic liver disease , enhancing bone resorption .", "fourth , interleukin-6 is increased by chronic hcv infection and can activate osteoclasts to increase bone resorption .", "by contrast , the association between hcv and osteoporosis may be due to shared risk factors since the prevalence of important osteoporosis risk factors was higher in the hcv exposed patients as compared to the patients without hcv exposure .", "however , it is reasonable to conclude that the increased risk of osteoporosis observed in hcv exposed patients was more likely to be due to the effect of their hcv status since the possible confounding effect of osteoporosis risk factors has been significantly minimized in our study .", "moreover , the osteoporosis risk contributed by hcv decreased with the increment of age may be due to the absence or low prevalence of osteoporosis - associated comorbidities in the younger patients .", "the results of our subgroup analyses in which we excluded patients with important comorbidities at baseline also confirmed the validity of our results .", "this finding , coupled with the results of the subgroup analyses , affirms the possible causal association between hcv and osteoporosis , and suggested that hcv may be a possible risk factor for osteoporosis .", "nevertheless , hcv may be less influential than other risk factors , such as hypertension , hyperlipidemia , and heart failure , in contributing to the development of osteoporosis .", "first , the large - scale national database provided statistical benefits to our longitudinal study to evaluate the association between hcv and osteoporosis .", "second , the recruited subjects were a stable population and 99% of the residents in taiwan have been covered by the nhi program .", "first , we could not ascertain the patients viremic status , such as the viral loads or the genotypes of the hcv .", "nevertheless , all patients in our case cohort exhibited positive anti - hcv antibodies , which mean recent or past hcv exposure .", "moreover , the effect of antiviral therapy on the stage of liver fibrosis or the grade of liver inflammation could not be ascertained in our study .", "therefore , we could not prove the ameliorating effect of antiviral therapy on bone mineral density through the arrest of liver cirrhosis even though the association between osteoporotic fracture and cirrhosis has been thoroughly documented .", ". however , to enhance the accuracy of diagnosis , we only recruited patients who received medical care for osteoporosis > 3 separate visits . moreover , the bnhi organizes regular audits performed by medical experts to ensure the accuracy of insurance claim codes in taiwan .", "finally , we can not clarify the temporal association between hcv exposure and osteoporosis since the date of hcv exposure could not be ascertained . in conclusion", ", this nationwide population - based cohort study concludes that hcv exposure increases the risk of developing subsequent osteoporosis , but no detrimental effect on osteoporotic fracture was observed .", "furthermore , hcv may be less influential than other risk factors , such as hypertension , hyperlipidemia , and heart failure , in contributing to the development of osteoporosis ."], "abstract_text": ["<S> abstractthe effect of hepatitis c virus ( hcv ) exposure on bone mineral density without advanced liver disease remains debated . </S>", "<S> thus , we assessed the relation between hcv exposure and the risk of osteoporosis.from 2000 to 2011 , patients aged > 20 years with hcv exposure were identified from the longitudinal health insurance database 2000 . of the 51,535 sampled patients , </S>", "<S> 41,228 and 10,307 patients were categorized as the comparison and the hcv exposure cohorts , respectively.the overall incidence of osteoporosis in the hcv exposure cohort was higher than in the comparison cohort ( 8.27 vs 6.19 per 1000 person - years ; crude hazard ratio  =  1.33 , 95% confidence interval  =  1.201.47 ) . </S>", "<S> the incidence of osteoporosis , higher in women than in men , increased with age and comorbidity of hypertension , hyperlipidemia , and heart failure . </S>", "<S> the risk of developing osteoporosis was significantly higher in the hcv exposure cohort than in the comparison cohort after adjusting for age , sex , diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis . </S>", "<S> however , the risk of osteoporosis contributed by hcv decreased with age and the presence of comorbidity . </S>", "<S> furthermore , the risk of osteoporotic fracture did not differ significantly between patients exposed to hcv and the comparison cohorts.hcv increases the risk of osteoporosis , but no detrimental effect on osteoporotic fracture was observed in this study . furthermore , hcv may be less influential than other risk factors , such as hypertension , hyperlipidemia , and heart failure , in contributing to the development of osteoporosis . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Data Source", "Participants", "Outcome and Comorbidities", "Statistical Analyses", "RESULTS", "DISCUSSION"], "sections": [["a negative balance between the formation and resorption of bone mass can lead to the development of osteoporosis .", "moreover , osteoporosisis characterized by skeletal fragility resulting from reduced bone mass and disrupted bone microarchitecture .", "osteoporosis increases the risk of fracture and thus has been considered a major public health concern . traditionally , the development of osteoporosis is related to several risk factors , such as aging , immobility , hypertension , antihypertensive agents , hyperparathyroidism , menopause , diabetes mellitus , corticosteroid usage , low calcium intake , vitamin d deficiency , and genetic vulnerability .", "vertebral fracture is the most common osteoporotic fracture , but only from one - third to one quarter of the patients with vertebral fracture can be clinically identified .", "furthermore , it is reported that asymptomatic vertebral fractures are associated with future hip fracture by threefold and other nonvertebral fracture by twofold .", "hip fracture is the second common osteoporotic fracture and it may incur substantial healthcare costs resulted from disability . furthermore , the effect of hip fracture on mortality increase can adversely extend up to 10 years or more .", "the mortality following a hip fracture generally increases with the increment of age and is greater in men , but the sex difference declines after age 80 .", "although the rates of hip fracture have declined in the west , the rate is increasing in the developing world .", "it is estimated that > 50% of hip fractures worldwide will occur in asia by 2050 . the functional outcome and increased mortality of osteoporotic fracture", "are heterogeneous and depend on age , activity of daily living prior to fracture , pre - fracture comorbidities , and the cognition .", "hepatitis c virus infection ( hcv ) is a global health problem estimated to affect 170 million people worldwide .", "hepatitis c virus infection is a hepatotropic virus that mainly causes inflammation and fibrosis of the liver .", "it is reported that 20% of hcv - infected patients will progress to liver cirrhosis .", "however , hcv can also cause several extrahepatic manifestations , such as diabetes mellitus , rheumatic disorders , lymphoproliferative disease , cardiovascular events , and cognitive impairment .", "although the role of osteoporosis as a sequence of cirrhosis or advanced liver disease has been thoroughly documented , the effect of hcv exposure on bone mineral density in the absence of advanced liver disease remains debated .", "some scholars have proposed that chronic hcv infection without liver cirrhosis contributes to reduced bone mineral density , whereas other scholars have asserted the opposite . to assess the association between hcv exposure and subsequent development of osteoporosis", ", we conducted a nationwide population - based cohort study by analyzing data from a nationwide medical database , the national health insurance research database ( nhird ) ."], ["the national health insurance ( nhi ) program , initiated on march 1 , 1995 , provides comprehensive coverage for the medical care of taiwan residents . at the end of 2014 ,", "23.75 million people ( 99.9% of the population ) were enrolled in the program . in cooperation with the bureau of national health insurance ( bnhi ) , the national health research institutes established several data sets for public use , including the longitudinal health insurance database 2000 ( lhid2000 ) , a cohort data set comprising 1,000,000 randomly selected cases from the registry of nhi beneficiaries in 2000 . to maintain confidentiality , personal information , such as patient identification numbers and sensitive personal data ,", "are encrypted in the database , and international classification of diseases , ninth edition , clinical modification ( icd-9-cm ) codes are used for disease classification .", "this study was approved to fulfill the condition for exemption by the institutional review board ( irb ) of china medical university ( cmuh-104-rec2 - 115 ) .", "the irb also specifically waived the consent requirement . from 2000 to 2011 , patients aged 20 years and older with diagnosed hcv infection ( icd-9-cm codes 070.41 , 070.44 , 070.51 , and 070.54 ) identified from the lhid2000 comprised the hcv infection cohort .", "patients with a history of osteoporosis ( icd-9-cm codes 733.0 and 733.1 ) and hepatitis b virus ( hbv ) infection ( icd-9-cm codes 070.20 , 070.22 , 070.30 , and 070.32 ) diagnosed before the index date , those with missing information , and those younger than 20 years were excluded .", "using 1:m case - control studies is to increase the power and to control possible confounding .", "based on the statistical efficiency does not gain much when m  >  4 , we constructed a 1:4 matched cohort study . for each hcv case ,", "4 insurers with no history of hcv exposure , hbv infection , and osteoporosis were assigned to a comparison cohort and frequency matched with the hcv exposure cohorts according to age ( every 5-year span ) , sex , and index year .", "individuals were excluded from the comparison cohort using the same criteria used for the hcv exposure cohort . the primary endpoint in this study", "each participant was followed from the index date until the endpoint , withdrawal from the nhi program , or december 31 , 2011 .", "the baseline characteristics of participant comorbidities were also analyzed ; the comorbidities included diabetes ( icd-9-cm code 250 ) , hypertension ( icd-9-cm codes 401405 ) , hyperlipidemia ( icd-9-cm code 272 ) , heart failure ( icd-9-cm code 428 ) , stroke ( icd-9-cm codes 430438 ) , obesity ( icd-9-cm code 278 ) , and cirrhosis ( icd-9-cm codes 571.2 , 571.5 , and 571.6 ) . the chi - square test and t test for categorical and continuous variables , respectively , were first used to compare the distributions of age , sex , and baseline comorbidities between the hcv exposure and the comparison cohorts .", "the incidence densities of osteoporosis were estimated in person - years for the various risk factors . to estimate the hazard ratios ( hrs ) and 95% confidence intervals ( cis ) of osteoporosis ,", "multivariable models were simultaneously adjusted for age , sex , and the comorbidities of diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis .", "meier estimates were plotted to illustrate the cumulative incidence of osteoporosis , and the log - rank test was performed to examine the difference between the hcv exposure and the comparison cohorts .", "all statistical analyses were performed using the sas package ( version 9.4 for windows ; sas institute , inc , cary , nc ) ."], ["the national health insurance ( nhi ) program , initiated on march 1 , 1995 , provides comprehensive coverage for the medical care of taiwan residents . at the end of 2014 ,", "23.75 million people ( 99.9% of the population ) were enrolled in the program . in cooperation with the bureau of national health insurance ( bnhi ) , the national health research institutes established several data sets for public use , including the longitudinal health insurance database 2000 ( lhid2000 ) , a cohort data set comprising 1,000,000 randomly selected cases from the registry of nhi beneficiaries in 2000 . to maintain confidentiality , personal information , such as patient identification numbers and sensitive personal data ,", "are encrypted in the database , and international classification of diseases , ninth edition , clinical modification ( icd-9-cm ) codes are used for disease classification .", "this study was approved to fulfill the condition for exemption by the institutional review board ( irb ) of china medical university ( cmuh-104-rec2 - 115 ) ."], ["from 2000 to 2011 , patients aged 20 years and older with diagnosed hcv infection ( icd-9-cm codes 070.41 , 070.44 , 070.51 , and 070.54 ) identified from the lhid2000 comprised the hcv infection cohort . the date for hcv exposure coding", "patients with a history of osteoporosis ( icd-9-cm codes 733.0 and 733.1 ) and hepatitis b virus ( hbv ) infection ( icd-9-cm codes 070.20 , 070.22 , 070.30 , and 070.32 ) diagnosed before the index date , those with missing information , and those younger than 20 years were excluded . using 1:m case - control studies is to increase the power and to control possible confounding . based on the statistical efficiency", "does not gain much when m  >  4 , we constructed a 1:4 matched cohort study .", "for each hcv case , 4 insurers with no history of hcv exposure , hbv infection , and osteoporosis were assigned to a comparison cohort and frequency matched with the hcv exposure cohorts according to age ( every 5-year span ) , sex , and index year .", "individuals were excluded from the comparison cohort using the same criteria used for the hcv exposure cohort ."], ["each participant was followed from the index date until the endpoint , withdrawal from the nhi program , or december 31 , 2011 .", "the baseline characteristics of participant comorbidities were also analyzed ; the comorbidities included diabetes ( icd-9-cm code 250 ) , hypertension ( icd-9-cm codes 401405 ) , hyperlipidemia ( icd-9-cm code 272 ) , heart failure ( icd-9-cm code 428 ) , stroke ( icd-9-cm codes 430438 ) , obesity ( icd-9-cm code 278 ) , and cirrhosis ( icd-9-cm codes 571.2 , 571.5 , and 571.6 ) ."], ["the chi - square test and t test for categorical and continuous variables , respectively , were first used to compare the distributions of age , sex , and baseline comorbidities between the hcv exposure and the comparison cohorts .", "the incidence densities of osteoporosis were estimated in person - years for the various risk factors . to estimate the hazard ratios ( hrs ) and 95% confidence intervals ( cis ) of osteoporosis ,", "multivariable models were simultaneously adjusted for age , sex , and the comorbidities of diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis .", "meier estimates were plotted to illustrate the cumulative incidence of osteoporosis , and the log - rank test was performed to examine the difference between the hcv exposure and the comparison cohorts .", "all statistical analyses were performed using the sas package ( version 9.4 for windows ; sas institute , inc , cary , nc ) ."], ["of the 51,535 sampled patients , 41,228 and 10,307 were categorized as the comparison and hcv exposure cohorts , respectively ( table 1 ) .", "most patients were aged 50 years ( 61.7% ) , and 54.6% of the patients were women .", "the mean age was 54.1    15.3 years in the hcv exposure cohort and 53.7    15.5 years in the comparison cohort .", "regarding baseline characteristics , the hcv exposure cohort exhibited a higher prevalence of diabetes , hypertension , hyperlipidemia , heart failure , stroke , obesity , and cirrhosis than did the comparison cohort . during the mean follow - up periods of 5.44 and 6.01 years for the hcv exposure and comparison cohorts , respectively , the kaplan ", "meier curve revealed that the cumulative incidence of osteoporosis was higher in the hcv exposure cohort than in the comparison cohort ( figure 1 , log - rank test p  <  0.001 ) .", "demographic characteristics and comorbidities in cohorts with and without hcv exposure cumulative incidence comparison of osteoporosis for patients with ( dashed line ) or without ( solid line ) hcv exposure.hcv  =  hepatitis c virus .", "the overall incidence of osteoporosis in the hcv exposure cohort was higher than that in the comparison cohort ( 8.27 vs 6.19 per 1,000 person - years ; crude hr  =  1.33 , 95% ci  =  1.201.47 ) ( table 2 ) .", "after we adjusted for factors such as age , sex , and comorbidities , namely diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis , the risk of developing osteoporosis was significantly higher in the hcv exposure cohort than in the comparison cohort ( adjusted hr [ ahr ]  =  1.35 ; 95% ci  =  1.211.51 ) . compared with", "patients aged 49 years , the risk of developing osteoporosis was 4.05-fold higher in those aged 50 to 64 years ( 95% ci  =  3.8454.760 ) and 8.82-fold higher in those aged 65 years or older ( 95% ci  =  7.4810.40 ) . in the multivariate model ,", "the risk for osteoporosis was 3.10-fold higher for women than for men ( 95% ci  =  2.813.43 ) and was higher for patients with the comorbidities of hypertension ( ahr  =  1.19 , 95% ci  =  1.071.31 ) , hyperlipidemia ( ahr  =  1.17 , 95% ci  =  1.051.29 ) , and heart failure ( ahr  =  1.23 , 95% ci  =  1.021.49 ) .", "the incidence and hazard ratio for osteoporosis and osteoporosis - associated risk factor the incidence of osteoporosis increased with age , was higher in women than in men , and increased with comorbidity in both cohorts ( table 3 ) .", "the overall risk of osteoporosis related to several variables including age , sex , and presence of comorbidities was compared in the hcv exposure cohort and the comparison cohort .", "the risk of osteoporosis in patients exposed to hcv in all stratifications was higher than that in the comparison cohorts .", "however , the risk of osteoporosis contributed by hcv decreased with age ( aged  49 : ahr  =  1.79 , 95% ci  =  1.322.43 ; aged 5064 : ahr  =  1.36 , 95% ci  =  1.141.62 ; aged  65 : ahr  =  1.23 , 95% ci  =  1.051.44 ) and the presence of comorbidity ( no comorbidity : ahr  =  1.54 , 95% ci  =  1.261.89 ; comorbidity : ahr  =  1.27 , 95% ci  =  1.121.43 ) .", "incidence of osteoporosis by age , sex , and comorbidity and cox model measured hazards ratio for patients with hcv infection compared those without hcv exposure the patients exposed to hcv exhibited a 1.38-fold ( 95% ci  =  1.241.55 ) higher risk of developing osteoporosis compared with the patients who were not exposed to hcv ( table 4 ) .", "the risk of osteoporotic fracture did not differ significantly between patients exposed to hcv and the comparison cohorts ( ahr  =  0.80 , 95% ci  =  0.441.45 ) .", "comparisons of hazard ratios between patients with and without hcv exposure for different outcomes osteoporosis ( or osteoporotic fracture )"], ["consistent with the literature proposing that hcv seroprevalence peaks after age 55 and that women are predisposed to hcv infection , our results revealed that most patients ( 61.7% ) were aged 50 years and that 54.6% of the patients were women .", "the mean age in the hcv exposure cohort was 54.1    15.3 years . compared with patients who were not exposed to hcv", ", patients who were exposed to hcv tended to have more comorbidities , including diabetes , hypertension , hyperlipidemia , heart failure , stroke , obesity , and cirrhosis .", "our results revealed that the hcv exposure cohort had more comorbidities ; however , the risk of osteoporosis remained higher in the hcv exposure cohort after adjusting for age , sex , and the comorbidities of diabetes , hypertension , hyperlipidemia , heart failure , stroke , and cirrhosis .", "the mechanism affecting the pathophysiology that causes comorbidities in patients exposed to hcv may include hcv - associated insulin resistance and atherosclerosis .", "hcv may cause diabetes mellitus by directly interfering with insulin signaling and inducing insulin resistance in hepatocytes ; hcv - infected hepatocytes can secrete mediators that induce extrahepatic insulin resistance , notably in the skeletal muscle .", "hepatitis c virus may increase the risk of cardiovascular and cerebrovascular events through atherosclerosis induced by systemic inflammation , chronic endothelial injury , or direct infection of the arterial wall .", "our study revealed that the osteoporosis incidence increased with age and was higher in women than in men .", "the association between osteoporosis and aging has been confirmed in the literature , and the gene expression of the rennin ", "the decreasing rate of bone mineral density is swifter in the early postmenopausal period , which typically begins after age 50 , and women aged 40 to 59 years have the highest risk of developing osteoporosis .", "consistent with our result that women are predisposed to osteoporosis , the us preventive services task force indicated that as many as 1 in 2 postmenopausal women and 1 in 5 men are at risk for osteoporosis - related fracture .", "moreover , in the present study , osteoporosis was associated with hypertension , hyperlipidemia , and heart failure .", "both osteoporosis and hypertension are common among the aging population and may share similar etiologies , such as low calcium intake and levels , vitamin d and vitamin k deficiency , high salt consumption , imbalanced nitric oxide levels , and antihypertensive agents that exert detrimental effects on the skeletal metabolism , strength , and density .", "hyperlipidemia can reduce bone formation and promote bone loss by causing the products of lipid oxidation to accumulate in the subendothelial spaces of the vasculature and bone .", "moreover , hyperlipidemia can induce secondary hyperparathyroidism , thereby impairing bone regeneration and mechanical strength .", "heart failure and osteoporosis share several risk factors , such as aging , smoking , and postmenopausal and antihypertensive agents ; heart failure can also accelerate bone loss by inducing hyperaldosteronism and secondary hyperparathyroidism . however , the effect of obesity on bone metabolism is controversial .", "obesity is traditionally regarded as a protective factor for osteoporosis , conferring a positive mechanical loading on bone formation .", "for example , both osteoblasts and adipocytes are derived from a common mesenchymal stem cell and agents inhibiting adipogenesis - stimulated osteoblast differentiation and vice versa .", "furthermore , the reduced bone formation caused by aging is usually accompanied by adipogenesis in bone marrow cavities .", "moreover , elevated oxidative stress is common in people with obesity and osteoporosis . to our knowledge", ", this is the first population - based study to assess the relation between hcv exposure and the incidences of osteoporosis and osteoporotic fracture .", "our statistical analyses benefited from the use of a nationwide database and the 12-year observation of participants selected from a representative cohort comprising 1,000,000 residents covered by the nhi program .", "hansen et al conducted a large - scale population - based study to explore the association between hcv exposure and all - site fracture , but omitted discussing osteoporosis development .", "they concluded that the risk of fracture equally increased in patients exposed to hcv ( chronic or cleared infections ) , and the major determinants of fracture in such patients are lifestyle - related factors , such as alcohol and drug abuse , which substantially increases fracture risk .", "by contrast , our epidemiological study demonstrated that hcv exposure increases the risk of osteoporosis and the detrimental effect of hcv on osteoporotic fracture was not obvious .", "several pathogenic mechanisms are involved in bone mineral density loss in patients exposed to hcv .", "first , fibronectin can infiltrate the bone matrix to enhance matrix mineralization , reducing its production by the liver .", "second , insulin - like growth factor 1 , involved in osteoblast differentiation and proliferation , is produced by the liver and is reduced in patients with chronic liver disease .", "third , the receptor  activator ratio of nuclear factor kappa ligand and osteoprotegerin is higher in patients with chronic liver disease , enhancing bone resorption .", "fourth , interleukin-6 is increased by chronic hcv infection and can activate osteoclasts to increase bone resorption .", "by contrast , the association between hcv and osteoporosis may be due to shared risk factors since the prevalence of important osteoporosis risk factors was higher in the hcv exposed patients as compared to the patients without hcv exposure .", "however , it is reasonable to conclude that the increased risk of osteoporosis observed in hcv exposed patients was more likely to be due to the effect of their hcv status since the possible confounding effect of osteoporosis risk factors has been significantly minimized in our study .", "moreover , the osteoporosis risk contributed by hcv decreased with the increment of age may be due to the absence or low prevalence of osteoporosis - associated comorbidities in the younger patients .", "the results of our subgroup analyses in which we excluded patients with important comorbidities at baseline also confirmed the validity of our results .", "this finding , coupled with the results of the subgroup analyses , affirms the possible causal association between hcv and osteoporosis , and suggested that hcv may be a possible risk factor for osteoporosis .", "nevertheless , hcv may be less influential than other risk factors , such as hypertension , hyperlipidemia , and heart failure , in contributing to the development of osteoporosis .", "first , the large - scale national database provided statistical benefits to our longitudinal study to evaluate the association between hcv and osteoporosis .", "second , the recruited subjects were a stable population and 99% of the residents in taiwan have been covered by the nhi program .", "first , we could not ascertain the patients viremic status , such as the viral loads or the genotypes of the hcv .", "nevertheless , all patients in our case cohort exhibited positive anti - hcv antibodies , which mean recent or past hcv exposure .", "moreover , the effect of antiviral therapy on the stage of liver fibrosis or the grade of liver inflammation could not be ascertained in our study .", "therefore , we could not prove the ameliorating effect of antiviral therapy on bone mineral density through the arrest of liver cirrhosis even though the association between osteoporotic fracture and cirrhosis has been thoroughly documented .", ". however , to enhance the accuracy of diagnosis , we only recruited patients who received medical care for osteoporosis > 3 separate visits . moreover , the bnhi organizes regular audits performed by medical experts to ensure the accuracy of insurance claim codes in taiwan .", "finally , we can not clarify the temporal association between hcv exposure and osteoporosis since the date of hcv exposure could not be ascertained . in conclusion", ", this nationwide population - based cohort study concludes that hcv exposure increases the risk of developing subsequent osteoporosis , but no detrimental effect on osteoporotic fracture was observed .", "furthermore , hcv may be less influential than other risk factors , such as hypertension , hyperlipidemia , and heart failure , in contributing to the development of osteoporosis ."]]}
{"article_id": "PMC5314909", "article_text": ["each disaster leaves an imprint on the affected population , a singular  signature .  a critical unmet need in the field of disaster mental / behavioral health is the capability to tailor mental health and psychosocial support ( mhpss ) to the unique constellation of psychological risk factors operating within each disaster event .", "we have developed and introduced trauma signature ( tsig ) analysis to the fields of disaster mental / behavioral health and disaster public health in response to this identified need .", "we define tsig analysis in the following manner : trauma signature ( tsig ) analysis is an evidence - based method that examines the interrelationship between population exposure to a disaster , extreme event , or complex emergency and the interrelated physical and psychological consequences for the purpose of providing timely , actionable guidance for effective mental health and psychosocial support ( mhpss)or disaster behavioral health ( dbh ) * \n support  that is organically tailored and targeted to the defining features of the event . according to shultz et al .", "2013 ( in press ) ,  tsig examines the extent to which disaster survivors were exposed to empirically - documented risk factors for psychological distress and mental health disorders . grounded on the disaster ecology model , tsig is premised on the assumption that each disaster exposes the affected population to a novel pattern of traumatizing hazards , loss , and change .", "this singular  signature  of exposure risks is a predictor ( or series of predictors ) of the psychosocial and mental health consequences .", "disaster - specific analysis is important because , as kessler and team have documented across a spectrum of international disasters ,  secondary stressors unique to a particular disaster situation have more impact than the disasters themselves  in determining the prevalence of post - disaster mental disorders .  too frequently , mhpss response to disasters is unguided , uncoordinated , and unscientific .", "for example , in our tsig analysis of the 2010 haiti earthquake it was evident that mhpss was never prioritized .", "the disaster behavioral health response that was cobbled together in haiti evidenced many serious deficits ( table 1 ) including : self - deployment and arrival on - scene of many uninvited personnel , lack of coordination among response teams , provision of non - evidence - informed services loosely labeled as  psychosocial ,  provision of services such as critical incident stress debriefing ( cisd ) that are known to be ineffective and potentially harmful for survivors , provision of services that were not culturally - adapted or language - appropriate for haiti , premature cessation of many services , and minimal or no program evaluation . from the perspective of responders , there was a lack of pre - deployment guidance about the specific stressors that would be encountered , and lack of on - scene psychological support , leading to high rates of traumatization among response personnel .", "disaster - specific risk factors can be identified from disaster situation reports issued in the early aftermath allowing mhpss response to be tailored and timed to the defining features of the event .", "it is time to infuse science into mhpss response ; we need evidence - based support and intervention .", "optimal mhpss response that incorporates tsig analysis can be summarized as a sequence of six steps ( see also figure  1 ) : 1 ) initial tsig analysis to guide response based on the defining features of the disaster or potentially - traumatizing event ( pte ) , 2 ) tsig - guided preparation of responders for what to expect and deployment of appropriate response assets , 3 ) delivery and evaluation of evidence - informed early interventions , 4 ) implementation of onsite validated disaster behavioral health needs assessment , 5 ) identification and intervention with individuals at high risk for disaster - related psychological distress , impairment , or psychopathology , and 6 ) continuous monitoring of responder and survivor mental health throughout recovery with comprehensive (  end - to - end  ) evaluation of the mhpss response .", "optimal mental health and psychosocial support ( mhpss ) : six sequential steps while the components that are uniquely contributed by tsig analysis are primarily incorporated into steps 1 and 2 , in order to achieve optimal mhpss response , significant quality improvement is needed and desirable for steps 36 . an electronic appendix , entitled  operationalizing tsig  ( www.landesbioscience.com/journals/disasterhealth/commart-sup.pdf ) provides details for each of the six steps in the categories of description , rationale , operationalization , unmet needs , research questions , and opportunities .", "the initial tsig analysis is the first step of six that comprise an optimal evidence - based mhpss response ( summarized in figure  1 ) . as illustrated in figure  2 ,", "initial tsig analysis consists of these elements : 1 ) characterization of the affected communities ( geographic scope , numbers affected , demographics ) ; 2 ) real - time collection and synthesis of information from disaster situation reports (  sitreps  ) issued regularly in the early days and weeks post - impact ; 3 ) identification and data collection from disaster monitoring and scientific resources specific to the disaster event ( e.g. , united states geological survey ( usgs ) earthquake data on the haiti earthquake , national hurricane center ( nhc ) data on superstorm sandy ) ; 4 ) consultation with disaster sciences subject matter experts ( e.g. , usgs geophysicists for haiti earthquake , nhc meteorologists for superstorm sandy ) ; 5 ) construction of a hazard profile based on open - source data developed in the preceding steps ; 6 ) review and integration of the scientific literature on evidence - based risk factors for psychological distress and psychopathology for persons exposed to the event - specific hazards ; 7 ) incorporation of information on disaster stressors identified anecdotally in media and social media accounts of the event ; 8) creation of an event - specific stressor / risk factor matrix that is cross - referenced with the evidence - based literature ( stressors are enumerated by disaster phase within the categories of exposure to hazard , loss , and change ) , ccounts , cross - referenced with the evidence - based literature ; and 9 ) generation of a tsig summary for the disaster based on the estimated psychological severity of exposures to hazards , loss , and change . figure  2 .", "initial post - impact trauma signature analysis several key points in this process require more explanation . initial tsig analysis is triggered when a disaster alert or warning is issued , or when disaster strikes without warning .", "local disaster response is activated immediately and higher - level response ( regional , national , international ) is brought into play as needed depending upon the scale of the event . for major events ,", "publicly - accessible disaster situation reports ( sitreps ) are generated and hosted on websites such as the global disaster alert and coordination system ( gdacs ) , united nations office for the coordination of humanitarian assistance , and reliefweb within the early hours or days , hazard profile . to create the hazard profile that provides a scientifically - sound description of the event", ", tsig begins with review of disaster data including sitreps from governmental and ngo agencies and available data from disaster monitoring and scientific resources .", "subject matter experts ( smes ) may be contacted to assure that the event is correctly described from a disaster sciences perspective .", "this step describes the physical forces of harm that impact the human population in harm s way in order to characterize  exposure to hazard .", " disaster epidemiologic data are also collected on numbers of deaths , injuries , displaced , affected .", "the next step involves matching the scientific event description to the ever - expanding literature on psychological risk factors ( for distress , disorder , and psychiatric diagnosis ) .", "this is summarized in a  stressor matrix  : risk factors are arrayed by disaster phase for categories of exposure to hazard , loss , and change . news media and social media feeds", "may also be incorporated at this stage to enrich the list of stressors . trauma signature summary .", "the final step in the initial tsig analysis involves the construction of a summary table of salient psychological risk factors , grouped into categories of exposure to hazard , loss , and change with an estimate of the  exposure severity  for each risk factor .", "to demonstrate the feasibility of conducting tsig analysis , we developed a series of disaster case studies , beginning with post hoc analyses of historical disasters and progressing to real - time analyses of unfolding events .", "our first tsig case study was performed on data from the 2010 haiti earthquake which we described as  a potent example of the rare catastrophic event where all major risk factors for psychological distress and impairment are prominent and compounding . ", "our tsig case studies have been wide - ranging , including examples of natural , human - generated , and hybrid disasters , and complex emergencies .", "in addition to tsig analyses for the 2010 haiti earthquake , we performed analyses on other natural disaster events including the 2011 us , super tornado outbreak , 2009 and 2011 river floods in the upper midwest , and 2012 superstorm sandy . in the realm of hybrid ( interacting natural and human - generated components ) , we carefully analyzed the 2011 great east japan disaster .", "the 2010 deepwater horizon oil spill provided an application of tsig for an anthropogenic ( human - generated ) event . in the realm of complex emergencies / humanitarian crises , we have examined patterns of internal displacement in colombia and the russia - georgia conflict in south ossetia . with each case study , the research literature database of evidence - based research studies is expanded .", "the case studies have provided an open invitation for colleagues with interest in a particular disaster event to join with us both in authorship and in advancing the tsig methodology .", "tsig developers and co - authors have presented a series of invited papers , workshops , and institutes to introduce the methodology and actively seek feedback .", "currently , an international cadre of co - authors and co - developers spans five continents .", "however , at present we are soliciting additional disaster case studies , consulting with subject matter experts , creating the structure for the literature database , and launching an internet - based delphi process in 2013 to develop expert consensus regarding the methodology .", "we will then seek opportunities to partner with lead response agencies in real - world , real - time applications of the tsig process .", "once refined , our intention is to develop a practical system that can help infuse evidence - based science into the decision - making process for matching mhpss response to the defining features of the disaster event .", "tsig is designed to contribute to the creation of mhpss response that increasingly approaches the attributes of optimal response outlined in table 2 ."], "abstract_text": ["<S> trauma signature ( tsig ) analysis is an evidence - based method that examines the interrelationship between population exposure to a disaster , extreme event , or complex emergency , and the inter - related physical and psychological consequences for the purpose of providing timely , actionable guidance for effective mental health and psychosocial support that is organically tailored and targeted to the defining features of the event . \n\n </S>", "<S> a series of tsig case studies has been published since 2011 and tsig analyses of recent disasters are in process . </S>", "<S> disaster health intends to expedite and feature novel tsig research focusing on late - breaking disaster events . \n at the current stage of development , </S>", "<S> expert consensus is sought for refining the tsig methodology using a delphi process . </S>", "<S> the overarching goal is to create a fully operational system to provide timely guidance for adapting disaster behavioral health support to the salient psychological risk factors in each disaster . </S>"], "labels": null, "section_names": ["Introduction and Rationale for Trauma Signature (TSIG) Analysis", "TSIG Analysis in the Context of Evidence-Based MHPSS Disaster Response", "Framework for the Initial TSIG Analysis", "TSIG Case Studies", "Ongoing Development and Refinement of the TSIG Model", "Supplementary Material", "Disclosure of Potential Conflicts of Interest"], "sections": [["each disaster leaves an imprint on the affected population , a singular  signature .  a critical unmet need in the field of disaster mental / behavioral health is the capability to tailor mental health and psychosocial support ( mhpss ) to the unique constellation of psychological risk factors operating within each disaster event .", "we have developed and introduced trauma signature ( tsig ) analysis to the fields of disaster mental / behavioral health and disaster public health in response to this identified need .", "we define tsig analysis in the following manner : trauma signature ( tsig ) analysis is an evidence - based method that examines the interrelationship between population exposure to a disaster , extreme event , or complex emergency and the interrelated physical and psychological consequences for the purpose of providing timely , actionable guidance for effective mental health and psychosocial support ( mhpss)or disaster behavioral health ( dbh ) * \n support  that is organically tailored and targeted to the defining features of the event . according to shultz et al .", "2013 ( in press ) ,  tsig examines the extent to which disaster survivors were exposed to empirically - documented risk factors for psychological distress and mental health disorders . grounded on the disaster ecology model , tsig is premised on the assumption that each disaster exposes the affected population to a novel pattern of traumatizing hazards , loss , and change .", "this singular  signature  of exposure risks is a predictor ( or series of predictors ) of the psychosocial and mental health consequences .", "disaster - specific analysis is important because , as kessler and team have documented across a spectrum of international disasters ,  secondary stressors unique to a particular disaster situation have more impact than the disasters themselves  in determining the prevalence of post - disaster mental disorders .  too frequently , mhpss response to disasters is unguided , uncoordinated , and unscientific .", "for example , in our tsig analysis of the 2010 haiti earthquake it was evident that mhpss was never prioritized .", "the disaster behavioral health response that was cobbled together in haiti evidenced many serious deficits ( table 1 ) including : self - deployment and arrival on - scene of many uninvited personnel , lack of coordination among response teams , provision of non - evidence - informed services loosely labeled as  psychosocial ,  provision of services such as critical incident stress debriefing ( cisd ) that are known to be ineffective and potentially harmful for survivors , provision of services that were not culturally - adapted or language - appropriate for haiti , premature cessation of many services , and minimal or no program evaluation . from the perspective of responders , there was a lack of pre - deployment guidance about the specific stressors that would be encountered , and lack of on - scene psychological support , leading to high rates of traumatization among response personnel .", "disaster - specific risk factors can be identified from disaster situation reports issued in the early aftermath allowing mhpss response to be tailored and timed to the defining features of the event .", "it is time to infuse science into mhpss response ; we need evidence - based support and intervention ."], ["optimal mhpss response that incorporates tsig analysis can be summarized as a sequence of six steps ( see also figure  1 ) : 1 ) initial tsig analysis to guide response based on the defining features of the disaster or potentially - traumatizing event ( pte ) , 2 ) tsig - guided preparation of responders for what to expect and deployment of appropriate response assets , 3 ) delivery and evaluation of evidence - informed early interventions , 4 ) implementation of onsite validated disaster behavioral health needs assessment , 5 ) identification and intervention with individuals at high risk for disaster - related psychological distress , impairment , or psychopathology , and 6 ) continuous monitoring of responder and survivor mental health throughout recovery with comprehensive (  end - to - end  ) evaluation of the mhpss response .", "optimal mental health and psychosocial support ( mhpss ) : six sequential steps while the components that are uniquely contributed by tsig analysis are primarily incorporated into steps 1 and 2 , in order to achieve optimal mhpss response , significant quality improvement is needed and desirable for steps 36 . an electronic appendix , entitled  operationalizing tsig  ( www.landesbioscience.com/journals/disasterhealth/commart-sup.pdf ) provides details for each of the six steps in the categories of description , rationale , operationalization , unmet needs , research questions , and opportunities ."], ["the initial tsig analysis is the first step of six that comprise an optimal evidence - based mhpss response ( summarized in figure  1 ) . as illustrated in figure  2 ,", "initial tsig analysis consists of these elements : 1 ) characterization of the affected communities ( geographic scope , numbers affected , demographics ) ; 2 ) real - time collection and synthesis of information from disaster situation reports (  sitreps  ) issued regularly in the early days and weeks post - impact ; 3 ) identification and data collection from disaster monitoring and scientific resources specific to the disaster event ( e.g. , united states geological survey ( usgs ) earthquake data on the haiti earthquake , national hurricane center ( nhc ) data on superstorm sandy ) ; 4 ) consultation with disaster sciences subject matter experts ( e.g. , usgs geophysicists for haiti earthquake , nhc meteorologists for superstorm sandy ) ; 5 ) construction of a hazard profile based on open - source data developed in the preceding steps ; 6 ) review and integration of the scientific literature on evidence - based risk factors for psychological distress and psychopathology for persons exposed to the event - specific hazards ; 7 ) incorporation of information on disaster stressors identified anecdotally in media and social media accounts of the event ; 8) creation of an event - specific stressor / risk factor matrix that is cross - referenced with the evidence - based literature ( stressors are enumerated by disaster phase within the categories of exposure to hazard , loss , and change ) , ccounts , cross - referenced with the evidence - based literature ; and 9 ) generation of a tsig summary for the disaster based on the estimated psychological severity of exposures to hazards , loss , and change . figure  2 .", "initial post - impact trauma signature analysis several key points in this process require more explanation . initial tsig analysis is triggered when a disaster alert or warning is issued , or when disaster strikes without warning .", "local disaster response is activated immediately and higher - level response ( regional , national , international ) is brought into play as needed depending upon the scale of the event . for major events ,", "publicly - accessible disaster situation reports ( sitreps ) are generated and hosted on websites such as the global disaster alert and coordination system ( gdacs ) , united nations office for the coordination of humanitarian assistance , and reliefweb within the early hours or days , hazard profile . to create the hazard profile that provides a scientifically - sound description of the event", ", tsig begins with review of disaster data including sitreps from governmental and ngo agencies and available data from disaster monitoring and scientific resources .", "subject matter experts ( smes ) may be contacted to assure that the event is correctly described from a disaster sciences perspective .", "this step describes the physical forces of harm that impact the human population in harm s way in order to characterize  exposure to hazard .", " disaster epidemiologic data are also collected on numbers of deaths , injuries , displaced , affected .", "the next step involves matching the scientific event description to the ever - expanding literature on psychological risk factors ( for distress , disorder , and psychiatric diagnosis ) .", "this is summarized in a  stressor matrix  : risk factors are arrayed by disaster phase for categories of exposure to hazard , loss , and change . news media and social media feeds", "may also be incorporated at this stage to enrich the list of stressors . trauma signature summary .", "the final step in the initial tsig analysis involves the construction of a summary table of salient psychological risk factors , grouped into categories of exposure to hazard , loss , and change with an estimate of the  exposure severity  for each risk factor ."], ["to demonstrate the feasibility of conducting tsig analysis , we developed a series of disaster case studies , beginning with post hoc analyses of historical disasters and progressing to real - time analyses of unfolding events .", "our first tsig case study was performed on data from the 2010 haiti earthquake which we described as  a potent example of the rare catastrophic event where all major risk factors for psychological distress and impairment are prominent and compounding . ", "our tsig case studies have been wide - ranging , including examples of natural , human - generated , and hybrid disasters , and complex emergencies .", "in addition to tsig analyses for the 2010 haiti earthquake , we performed analyses on other natural disaster events including the 2011 us , super tornado outbreak , 2009 and 2011 river floods in the upper midwest , and 2012 superstorm sandy . in the realm of hybrid ( interacting natural and human - generated components ) , we carefully analyzed the 2011 great east japan disaster .", "the 2010 deepwater horizon oil spill provided an application of tsig for an anthropogenic ( human - generated ) event . in the realm of complex emergencies / humanitarian crises , we have examined patterns of internal displacement in colombia and the russia - georgia conflict in south ossetia . with each case study , the research literature database of evidence - based research studies is expanded .", "the case studies have provided an open invitation for colleagues with interest in a particular disaster event to join with us both in authorship and in advancing the tsig methodology .", "tsig developers and co - authors have presented a series of invited papers , workshops , and institutes to introduce the methodology and actively seek feedback .", "currently , an international cadre of co - authors and co - developers spans five continents ."], ["however , at present we are soliciting additional disaster case studies , consulting with subject matter experts , creating the structure for the literature database , and launching an internet - based delphi process in 2013 to develop expert consensus regarding the methodology .", "we will then seek opportunities to partner with lead response agencies in real - world , real - time applications of the tsig process .", "once refined , our intention is to develop a practical system that can help infuse evidence - based science into the decision - making process for matching mhpss response to the defining features of the disaster event .", "tsig is designed to contribute to the creation of mhpss response that increasingly approaches the attributes of optimal response outlined in table 2 ."], [""], [""]]}
{"article_id": "PMC4681915", "article_text": ["cerebrovascular diseases ( cvds ) are associated with balance disturbances , dependency in \n performing daily living activities , and limited ambulation . as life expectancy increases", "spasticity is a speed - dependent resistance to the passive motion of muscles and tendons , \n developing secondary to enhanced stretch reflexes in patients with upper motor neuron \n lesions .", "spasticity is a major problem in rehabilitation programs , and it hampers patient \n progress and may trigger severe complications2 , 3 . quantitative assessment of spasticity is \n critical when gauging responses to medical treatment and physical therapy , and when making a \n prognosis . at present , rather limited clinical scoring systems of spasticity , and \n biomechanical and electrophysiological data , are used .", "electroneuromyographic evaluation of \n the spinal reflex organization allows detailed follow - up of physiological changes .", "this measure , \n and also the f reflex , assessed using various techniques , are employed to quantitate and \n explore spasticity4 .", "transcutaneous electrical nerve stimulation ( tens ) is a rehabilitative form of physical \n therapy used to treat pain .", "various types of nerve fiber are stimulated at different \n frequencies , wavelengths , and amplitudes .", "tens delays the h reflex \n through the 1a fibers , which mediate presynaptic inhibition8 . in the present study", ", we evaluated the utility of tens for patients with spasticity due to \n cvd , who were undergoing rehabilitation , was evaluated using clinical scoring systems and \n electrophysiological assessment .", "this study also aimed to explore whether tens affects \n spasticity electrophysiologically , and the extent of the effects on clinical features .", "patients hospitalized in ankara physical therapy and rehabilitation training and research \n hospital , who were participating in a rehabilitation program for hemiplegia with spasticity \n in the lower extremity , were recruited .", "patients who had received any treatment for spasticity , who lacked spasticity , who had a \n history of any systemic disease that might cause peripheral neuropathy , or symptoms of \n radiculopathy in the lower extremities , who had suffered strokes less than 30 days prior , \n who had a prior history of cerebrovascular disease , who had upper motor neuron damage on the \n non - hemiplegic side , who could not co - operate , or who were obese , were excluded .", "aphasic and \n geriatric patients were also excluded ; because their co - operation was lacking .", "patients with spasticity of at least ashworth grade 1 in the lower extremities were \n included . the active motor power and spasticity of all patients", "reflex , and superficial and deep \n sensorial examinations were performed . the time taken to walk 10  m by patients who could \n walk was measured before and after tens . after a clinical evaluation", ", emg examinations were initially performed on healthy controls \n and , later , on the affected sides of patients .", "all electrophysiological work was performed \n in a calm , intermediately lit room with patients in the supine position .", "their feet extended \n from the examination bed , commencing at the ankle joints , each of which was in the 90 \n neutral position ; the knee joint was at 180 of extension .", "all patients were asked to relax \n their muscles as much as possible during the electrophysiological study . the h reflexes and \n m responses of the gastrocnemius - soleus muscle were electrophysiologically recorded on both \n the healthy and hemiplegic sides .", "subsequently , a single 30  min session of tens was applied \n to the hemiplegic lower extremity .", "ten minutes later , the h reflexes and m responses were \n re - assessed and compared with the pre - tens data .", "a nihon kohden neuropack  four - channel emg platform was used for measurements in the \n single - channel mode .", "the active \n electrode was placed on the gastrocnemius - soleus muscle , in a slightly medial position , \n 14  cm proximal to the junction of the achilles tendon and the heel .", "the reference electrode \n was placed at the junction of the tendon and the heel , and a velcro ground electrode was \n placed between the active electrode and the stimulator .", "the latter electrode was placed on \n the fossae popliteal , to stimulate the tibial nerve , and the cathode was placed across the \n active electrode .", "the stimulus intensity \n was increased in steps of 2 ma , commencing at zero , and the combined action potentials of \n the soleus muscle were recorded .", "the \n peak - to - peak amplitudes and latencies of the h and m responses were measured .", "the latencies \n were assessed at the point of separation of each response from the baseline .", "the \n sensitivity , low frequency filter , and high frequency filter were 2  mv / dv , 200  hz , and 3 \n khz , respectively .", "stimuli were created randomly , \n and delivered to the popliteal fossa with the posterior tibial nerve lying proximal to the \n cathode . a rectangular flow 1  ms in duration , delivered every 2  s , served as the stimulus . \n", "the sweep speed and sensitivity were 10  ms and 2  mv / dv , respectively .", "subsequently , the largest h wave was determined by \n varying the strength of the stimulus with the location of the stimulator unchanged .", "after all electrophysiological work was completed , and all wave amplitudes calculated , the \n h / m maximum amplitude ratio was calculated for each patient by dividing the maximal \n amplitude of the h reflex by that of the m responses from both the healthy and affected \n sides .", "in addition , to evaluate alpha motor neuron excitability and the extent of \n spasticity , the slopes of the rising sides of the h and m amplitude - stimulus severity slopes \n were calculated using excel and the h slope , m slope , and h slope / m slope values were \n obtained .", "tens was performed in the supine position with the feet hanging in a neutral manner . \n", "electrodes were placed bilaterally on the tibialis nerve , between the tendon of the muscle \n and the medial malleolus ; thus the gastrocnemius muscle was included .", "the recording \n electrodes used in the electrophysiological study were not removed . a systems 200e tens platform was used .", "this equipment has two output channels the flow \n intensity in each channel can be adjusted independently .", "the frequency was 50  hz and the \n pulse width was 100  ms in normal stimulation .", "the current intensity was not allowed to \n exceed a mean of 50 ma and no contractions were noted .", "electrophysiological data were collected before and after tens , the same as \n for the patient group .", "controls were positioned the same as patients , and tens was applied \n for 30  min .", "electrophysiological data were gathered from only the right lower extremities of \n the controls and the pre- and post - tens values were compared .", "the  , wilcoxon signed - rank , and kruskal - wallis tests , were used , as \n appropriate , to evaluate the experimental parameters .", "the patient group with hemiplegia secondary to cvd numbered 27 [ 12 females ( 44.4% ) and 15 \n males ( 55.6% ) ] . a total of 24 healthy subjects [ 10 females ( 41.7% ) and 14 males ( 58.3% ) ] \n served as controls .", "the mean ages of the patients and controls were 60.9312.8 and \n 49.886.85  years , respectively .", "the mean heights of the patients and controls were \n 160.858.99 and 165.38 + 8.56  cm , respectively . the mean age , but", "fourteen ( 51.9% ) and 13 ( 48.l% ) patients respectively had left- and right - side hemiplegia . \n", "thromboembolic and \n hemorrhagic stroke were the principal causes of hemiplegia in 21 ( 77.8% ) and 6 ( 22.2% ) \n patients , respectively .", "superficial sensory nerve \n examinations revealed hypoesthesia in 10 patients ( 37% ) , but 17 were normal ( 63% ) . the deep \n tendon reflexes ( dtrs ) on the hemiplegic sides were normal in the upper extremity of one \n patient ( 3.7% ) but hyperactive in 26 ( 96.3% ) ; the lower extremity on the affected side was \n hyperactive in all patients . on the healthy sides ,", "the dtrs were normative in both the upper \n and lower extremities of 26 ( 96.3% ) patients , and hyperactive in one ( 3.7% ) .", "no significant \n difference in the mean pre- and post - tens lower - extremity dtrs on the hemiplegic side was \n found ( p>0.05 ) .", "the values of the spasticity parameters before and after tens differed significantly in the \n patient group ( table 1table 1.change in the spasticity between pre- and post - tensashworth 1ashworth 2ashworth 3pre - tens10 ( 37%)13 ( 48.2%)4 ( 14.8%)post - tens14 ( 51.9%)11 ( 40.7%)2 ( 7.4% ) all p values < 0.05 ) .", "fourteen patients were able to walk , either with support or independently , but 13 were not . \n", "after tens , 11 patients \n exhibited increased walking speeds and 3 reduced speeds . when patient hemiplegic and healthy lower extremities were electrophysiologically compared , \n the mean maxima of the h reflex , the h / m ratio , the h slope , and h slope / m slope ratio , were \n significantly greater on the affected side .", "in addition , the mean m response amplitude was \n significantly different ( greater on the healthy side ) .", "the differences in the h / m maximum \n and h slope / m slope ratios were particularly marked ( table 2table 2.electrophysiological variables of the healthy and affected sides of the \n patientsvariablehealthy sideaffected sideh max amp ( mv)1,669.851559.792,899.701,698.69h max lat ( msec)31.212.1831.082.28 m max amp ( mv)8,735.892,820.527,439.892,825.16 m max lat ( msec)4.900.444.930.40h / m max amp ( mv)0.190.160.420.22h slope0.010.0090.030.22 m slope0.030.0060.030.006h / m slope0.40.320.910.43 ) .", "the h maximum amplitude values were higher on the spastic hemiplegic side than on the \n healthy side .", "the h / m ratio , h slope , and h slope / m slope ratio were higher on the affected \n side .", "however , the mean m amplitude was higher on the normal than on the hemiplegic side . \n the mean h reflex amplitude , the h / m amplitude ratio , the h maximum latency , the h slope , \n and the h / m slope ratio , differed significantly pre- and post - tens on the hemiplegic \n side .", "the post - tens electrophysiological changes in the control group were similar to those in \n the patient group .", "the h maximum amplitude , the h / m ratio , the m maximum amplitude , and the \n m slope ratio , decreased , whereas the h and m maximum latencies increased , with statistical \n significance .", "the duration of disease was positively associated with the mean h maximum amplitude , the \n h / m ratio , and the h / m slope ratio ; however , no association was statistically significant . \n", "the h maximum latency was lowest in patients with disease durations greater than 100 \n days .", "higher mean h maximum amplitudes , h / m ratios , and h slopes evident as spasticity worsened . \n", "however , only the mean h slope differed significantly among the spasticity subgroups , with \n significant differences being found between subgroups i and iii , and ii and iii , in parallel \n with worsening of spasticity .", "tens significantly reduced spasticity scores and increased the walking speed . on the \n spastic side , tens significantly reduced the m amplitude and increased the h reflex \n amplitude , the h / m maximum amplitude ratio , the h slope , and the h slope / m slope ratio . \n also , the mean patient h reflex amplitude , the h / m ratio , the h slope , and the h slope / m \n slope ratio , decreased significantly , and the h reflex maximum latency increased , after \n tens . in controls , tens significantly decreased the h maximum amplitude , the h / m ratio , the \n m maximum amplitude , and the m slope ratio , and lengthened the h and m maximum latencies . \n the h maximum latency was the only variable affected by lesional duration .", "the mean h \n maximum amplitude , the h / m ratio , and the mean h slope increased as spasticity worsened on \n the ashworth scale .", "explored changes in the spasticity of patients with medulla spinalis injuries \n after tens , and found there were short - term reductions in spasticity9 .", "robinson et al . applied 20  min electrical stimulation to \n the quadriceps muscles of patients with medulla spinalis injuries and reported a significant \n decrease in leg spasticity10 .", "cho et al . \n found that a single session of high - frequency tens significantly improved the spasticity \n ( for less than 1 day ) of chronic stroke patients .", "similarly , tens reduced the spasticity of stroke \n patients , and repeat tens reduced the hyperactive tension reflexes of the plantar flexors , \n and the passive resistance to plantar flexor movement11 . in a study that included acute stroke patients , tens applied to \n acupuncture points for 3 weeks decreased plantar flexor spasticity and increased \n dorsiflexion of the ankle joint . in chronic stroke patients", ", however , ankle joint \n dorsoflexion was strengthened by stimulation , and muscle co - contraction was decreased . in \n addition", "repeated skin stimulation was \n advocated , to increase the number of cortical fields represented12 . in a study that included chronic stroke patients , \n", "addition of tens to a 20-session rehabilitation program decreased spasticity by 30% and \n markedly improved muscle strength13 .", "park \n et al . showed that balance , walking , and the functional activity of chronic stroke patients \n improved after 30 tens sessions ( each 30  min in duration ) , and a combination of exercise and \n tens increased the activation of pathways associated with proprioception and balance14 . in the present study , a significant \n decrease in spasticity and an increase in walking speed were evident after acute hemiplegic \n the patients in a rehabilitation program received a single session of tens . fisher reported that the m amplitudes of patients with first - motor neuron diseases \n decreased , after tens , compared to the amplitudes of polyneuropathic patients15 .", "angel and hoffman , who first showed that \n the h / m ratio was increased in spastic patients , reported mean ratios of 9.17 and 0.48 in \n the extremities of control and spastic groups , respectively .", "the excitability of alpha motor \n neurons increased when various physiopathological mechanisms were in play ; therefore , the \n responses to stimuli carried by 1a afferent fibers increased16 .", "garcia - mullin and mayer reported mean h / m ratios of 0.33 and 0.46 \n on the healthy and paretic sides , respectively , of spastic hemiplegic stroke patients in \n whom the disease duration was longer than 90 days17 .", "bakhtiary showed that spasticity was reduced by electrical stimulation , but no significant \n change in the h reflex amplitude was evident . also , the h / m maximum ratio did not change , \n and this was attributed to muscle weakness caused by stimulation18 .", "gaft et al . noted significant decreases in both \n spasticity and the h reflex amplitude after stimulation . in the present study", ", significant \n differences were found in the m response and h reflex amplitude between the healthy and \n affected sides of spastic the hemiplegic stroke patients .", "the mean maximum amplitudes of the \n h reflexes of the triceps surae were 1,669 and 2,899  mv on the healthy and paretic sides , \n respectively , and the difference was significant .", "the difference in the h / m ratio between \n the healthy and plegic sides was also significant19 .", "huang et al . reported that the h / m maximum and h2/h1 ratios were not adequate for defining \n the extent of spasticity as assessed by the ashworth scale .", "the mean h / m maximum and h2/h1 \n values increased in stroke patients , compared to healthy controls , but the m maximum ratio \n did not significantly differ between the groups .", "the h2/h1 and m maximum ratios did not \n significantly correlate with other measures of spasticity .", "in contrast to other previous \n studies , huang et al . concluded that the mas value correlated with the h / m ratio20 . in the present study , we noted a decrease \n in the m amplitude on the spastic side was observed , whereas the h reflex amplitude and the \n h / m maximum amplitude ratio , increased .", "higashi et al . found that the change in the h slope / m slope ratio was more significant on \n the spastic than the healthy side , and no significant difference in the h / m maxima of the \n two sides was evident .", "as the h slope / m slope graph is bell - shaped , such data are more \n compatible with the brunnstrom stage of hemiplegic patients than are other indicators . in \n conclusion", ", the h slope/ m slope ratio is primarily useful for objectively evaluating the \n extent of spasticity , and the excitability of the motor neuron pool of the spastic sides of \n hemiplegic patients21 .", "measured the excitability of the motor neuron pool and compared the h / m ratios , the h \n maximum amplitude , and the h slope / m slope ratio , in spastic and normal individuals , and \n found there were marked differences between the two groups .", "they concluded , the h slope / m \n slope ratio was particularly useful for evaluating motor neuron pool excitability22 .", "in the present study , steeper h slope and \n greater h slope / m slope ratio on the spastic ( compared to healthy ) side of hemiplegic \n patients prior to tens were observed .", "one study evaluating the electrophysiological components of the h reflex and f wave \n compared the pre- and post - tens h reflexes , and f wave latencies and amplitudes , and \n reported the reduction in spasticity was associated with electrophysiologically proven \n decreases in the h reflex amplitude , the f wave amplitude , and the h / m and f / m ratios23 . in another study , tens was applied \n segmentally and heterosegmentally to the median nerve of the wrist , and to the common \n peroneal nerve ( placebo ) .", "the h reflex latencies increased in 75% of the patients in the \n experimental groups , but the h amplitude did not change significantly .", "aydin et al . compared tens with baclofen , the commonest drug prescribed to treat spasticity \n in patients with medulla spinalis injuries .", "significant improvements in all parameters , \n except pain , were evident in the tens group , and they were associated with decreases in the \n h amplitude and h / m ratio , and the m response .", "the electrophysiological and clinical changes \n did not differ significantly between the tens and baclofen group25 .", "tekeoglu et al . also reported that tens reduced \n spasticity in stroke patients , and improved their capacity to engage in activities of daily \n living , concluding that tens effectively improved motor function26 . in the present study , a single session of tens resulted in significant clinical inhibition \n of spasticity ; however , lower extremity strength did not improve .", "on the spastic side , tens \n significantly decreased the mean h reflex amplitude , the h / m ratio , the h slope , and the h \n slope / m slope ratio ; and increased the h reflex maximum latency .", "this study had some \n limitation walking speed was not measured after several tens applications . in conclusion ,", "tens for hemiplegic patients with spastic lower extremities due to cvd \n markedly improved clinical parameters and significantly changed electrophysiological \n variables .", "the results of this study suggest that tens is effective when used to manage \n spasticity ."], "abstract_text": ["<S> to investigate whether transcutaneous electrical nerve stimulation ( tens ) mitigates the \n spasticity of hemiplegic stroke patients , as assessed by electrophysiological variables , \n and the effects , if any , on the clinical appearance of spasticity . </S>", "<S> [ subjects and methods ] \n twenty - seven subjects who had acute hemiplegia and 24 healthy people as the control group , \n were enrolled in this study . </S>", "<S> some of the acute cerebrovascular disease patients could \n walk . </S>", "<S> subjects who did not have spasticity , who were taking antispasticity medicine , or \n had a previous episode of cerebrovascular disease were excluded . </S>", "<S> the walking speed of the \n patients was recorded before and after tens . </S>", "<S> emg examinations were performed on the \n healthy controls and in the affected side of the patients . </S>", "<S> a 30-minute single session of \n tens was applied to lower extremity . at 10 minutes after tens </S>", "<S> , the emg examinations were \n repeated . </S>", "<S> [ results ] a statistically significant decrease in the spasticity variables , and \n increased walking speed were found post - tens . </S>", "<S> the lower m amplitude and higher h reflex \n amplitude , h / m maximum amplitude ratio , h slope , and h slope / m slope ratio on the spastic \n side were found to be statistically significant . </S>", "<S> [ conclusion ] tens application for \n hemiplegic patients with spastic lower extremities due to cerebrovascular disease resulted \n in marked improvement in clinical scales of spasticity and significant changes in the \n electrophysiological variables . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["cerebrovascular diseases ( cvds ) are associated with balance disturbances , dependency in \n performing daily living activities , and limited ambulation . as life expectancy increases", "spasticity is a speed - dependent resistance to the passive motion of muscles and tendons , \n developing secondary to enhanced stretch reflexes in patients with upper motor neuron \n lesions .", "spasticity is a major problem in rehabilitation programs , and it hampers patient \n progress and may trigger severe complications2 , 3 . quantitative assessment of spasticity is \n critical when gauging responses to medical treatment and physical therapy , and when making a \n prognosis . at present , rather limited clinical scoring systems of spasticity , and \n biomechanical and electrophysiological data , are used .", "electroneuromyographic evaluation of \n the spinal reflex organization allows detailed follow - up of physiological changes .", "this measure , \n and also the f reflex , assessed using various techniques , are employed to quantitate and \n explore spasticity4 .", "transcutaneous electrical nerve stimulation ( tens ) is a rehabilitative form of physical \n therapy used to treat pain .", "various types of nerve fiber are stimulated at different \n frequencies , wavelengths , and amplitudes .", "tens delays the h reflex \n through the 1a fibers , which mediate presynaptic inhibition8 . in the present study", ", we evaluated the utility of tens for patients with spasticity due to \n cvd , who were undergoing rehabilitation , was evaluated using clinical scoring systems and \n electrophysiological assessment .", "this study also aimed to explore whether tens affects \n spasticity electrophysiologically , and the extent of the effects on clinical features ."], ["patients hospitalized in ankara physical therapy and rehabilitation training and research \n hospital , who were participating in a rehabilitation program for hemiplegia with spasticity \n in the lower extremity , were recruited .", "patients who had received any treatment for spasticity , who lacked spasticity , who had a \n history of any systemic disease that might cause peripheral neuropathy , or symptoms of \n radiculopathy in the lower extremities , who had suffered strokes less than 30 days prior , \n who had a prior history of cerebrovascular disease , who had upper motor neuron damage on the \n non - hemiplegic side , who could not co - operate , or who were obese , were excluded .", "aphasic and \n geriatric patients were also excluded ; because their co - operation was lacking .", "patients with spasticity of at least ashworth grade 1 in the lower extremities were \n included . the active motor power and spasticity of all patients", "reflex , and superficial and deep \n sensorial examinations were performed . the time taken to walk 10  m by patients who could \n walk was measured before and after tens . after a clinical evaluation", ", emg examinations were initially performed on healthy controls \n and , later , on the affected sides of patients .", "all electrophysiological work was performed \n in a calm , intermediately lit room with patients in the supine position .", "their feet extended \n from the examination bed , commencing at the ankle joints , each of which was in the 90 \n neutral position ; the knee joint was at 180 of extension .", "all patients were asked to relax \n their muscles as much as possible during the electrophysiological study . the h reflexes and \n m responses of the gastrocnemius - soleus muscle were electrophysiologically recorded on both \n the healthy and hemiplegic sides .", "subsequently , a single 30  min session of tens was applied \n to the hemiplegic lower extremity .", "ten minutes later , the h reflexes and m responses were \n re - assessed and compared with the pre - tens data .", "a nihon kohden neuropack  four - channel emg platform was used for measurements in the \n single - channel mode .", "the active \n electrode was placed on the gastrocnemius - soleus muscle , in a slightly medial position , \n 14  cm proximal to the junction of the achilles tendon and the heel .", "the reference electrode \n was placed at the junction of the tendon and the heel , and a velcro ground electrode was \n placed between the active electrode and the stimulator .", "the latter electrode was placed on \n the fossae popliteal , to stimulate the tibial nerve , and the cathode was placed across the \n active electrode .", "the stimulus intensity \n was increased in steps of 2 ma , commencing at zero , and the combined action potentials of \n the soleus muscle were recorded .", "the \n peak - to - peak amplitudes and latencies of the h and m responses were measured .", "the latencies \n were assessed at the point of separation of each response from the baseline .", "the \n sensitivity , low frequency filter , and high frequency filter were 2  mv / dv , 200  hz , and 3 \n khz , respectively .", "stimuli were created randomly , \n and delivered to the popliteal fossa with the posterior tibial nerve lying proximal to the \n cathode . a rectangular flow 1  ms in duration , delivered every 2  s , served as the stimulus . \n", "the sweep speed and sensitivity were 10  ms and 2  mv / dv , respectively .", "subsequently , the largest h wave was determined by \n varying the strength of the stimulus with the location of the stimulator unchanged .", "after all electrophysiological work was completed , and all wave amplitudes calculated , the \n h / m maximum amplitude ratio was calculated for each patient by dividing the maximal \n amplitude of the h reflex by that of the m responses from both the healthy and affected \n sides .", "in addition , to evaluate alpha motor neuron excitability and the extent of \n spasticity , the slopes of the rising sides of the h and m amplitude - stimulus severity slopes \n were calculated using excel and the h slope , m slope , and h slope / m slope values were \n obtained .", "tens was performed in the supine position with the feet hanging in a neutral manner . \n", "electrodes were placed bilaterally on the tibialis nerve , between the tendon of the muscle \n and the medial malleolus ; thus the gastrocnemius muscle was included .", "the recording \n electrodes used in the electrophysiological study were not removed . a systems 200e tens platform was used .", "this equipment has two output channels the flow \n intensity in each channel can be adjusted independently .", "the frequency was 50  hz and the \n pulse width was 100  ms in normal stimulation .", "the current intensity was not allowed to \n exceed a mean of 50 ma and no contractions were noted .", "electrophysiological data were collected before and after tens , the same as \n for the patient group .", "controls were positioned the same as patients , and tens was applied \n for 30  min .", "electrophysiological data were gathered from only the right lower extremities of \n the controls and the pre- and post - tens values were compared .", "the  , wilcoxon signed - rank , and kruskal - wallis tests , were used , as \n appropriate , to evaluate the experimental parameters ."], ["the patient group with hemiplegia secondary to cvd numbered 27 [ 12 females ( 44.4% ) and 15 \n males ( 55.6% ) ] . a total of 24 healthy subjects [ 10 females ( 41.7% ) and 14 males ( 58.3% ) ] \n served as controls .", "the mean ages of the patients and controls were 60.9312.8 and \n 49.886.85  years , respectively .", "the mean heights of the patients and controls were \n 160.858.99 and 165.38 + 8.56  cm , respectively . the mean age , but", "fourteen ( 51.9% ) and 13 ( 48.l% ) patients respectively had left- and right - side hemiplegia . \n", "thromboembolic and \n hemorrhagic stroke were the principal causes of hemiplegia in 21 ( 77.8% ) and 6 ( 22.2% ) \n patients , respectively .", "superficial sensory nerve \n examinations revealed hypoesthesia in 10 patients ( 37% ) , but 17 were normal ( 63% ) . the deep \n tendon reflexes ( dtrs ) on the hemiplegic sides were normal in the upper extremity of one \n patient ( 3.7% ) but hyperactive in 26 ( 96.3% ) ; the lower extremity on the affected side was \n hyperactive in all patients . on the healthy sides ,", "the dtrs were normative in both the upper \n and lower extremities of 26 ( 96.3% ) patients , and hyperactive in one ( 3.7% ) .", "no significant \n difference in the mean pre- and post - tens lower - extremity dtrs on the hemiplegic side was \n found ( p>0.05 ) .", "the values of the spasticity parameters before and after tens differed significantly in the \n patient group ( table 1table 1.change in the spasticity between pre- and post - tensashworth 1ashworth 2ashworth 3pre - tens10 ( 37%)13 ( 48.2%)4 ( 14.8%)post - tens14 ( 51.9%)11 ( 40.7%)2 ( 7.4% ) all p values < 0.05 ) .", "fourteen patients were able to walk , either with support or independently , but 13 were not . \n", "after tens , 11 patients \n exhibited increased walking speeds and 3 reduced speeds . when patient hemiplegic and healthy lower extremities were electrophysiologically compared , \n the mean maxima of the h reflex , the h / m ratio , the h slope , and h slope / m slope ratio , were \n significantly greater on the affected side .", "in addition , the mean m response amplitude was \n significantly different ( greater on the healthy side ) .", "the differences in the h / m maximum \n and h slope / m slope ratios were particularly marked ( table 2table 2.electrophysiological variables of the healthy and affected sides of the \n patientsvariablehealthy sideaffected sideh max amp ( mv)1,669.851559.792,899.701,698.69h max lat ( msec)31.212.1831.082.28 m max amp ( mv)8,735.892,820.527,439.892,825.16 m max lat ( msec)4.900.444.930.40h / m max amp ( mv)0.190.160.420.22h slope0.010.0090.030.22 m slope0.030.0060.030.006h / m slope0.40.320.910.43 ) .", "the h maximum amplitude values were higher on the spastic hemiplegic side than on the \n healthy side .", "the h / m ratio , h slope , and h slope / m slope ratio were higher on the affected \n side .", "however , the mean m amplitude was higher on the normal than on the hemiplegic side . \n the mean h reflex amplitude , the h / m amplitude ratio , the h maximum latency , the h slope , \n and the h / m slope ratio , differed significantly pre- and post - tens on the hemiplegic \n side .", "the post - tens electrophysiological changes in the control group were similar to those in \n the patient group .", "the h maximum amplitude , the h / m ratio , the m maximum amplitude , and the \n m slope ratio , decreased , whereas the h and m maximum latencies increased , with statistical \n significance .", "the duration of disease was positively associated with the mean h maximum amplitude , the \n h / m ratio , and the h / m slope ratio ; however , no association was statistically significant . \n", "the h maximum latency was lowest in patients with disease durations greater than 100 \n days .", "higher mean h maximum amplitudes , h / m ratios , and h slopes evident as spasticity worsened . \n", "however , only the mean h slope differed significantly among the spasticity subgroups , with \n significant differences being found between subgroups i and iii , and ii and iii , in parallel \n with worsening of spasticity ."], ["tens significantly reduced spasticity scores and increased the walking speed . on the \n spastic side , tens significantly reduced the m amplitude and increased the h reflex \n amplitude , the h / m maximum amplitude ratio , the h slope , and the h slope / m slope ratio . \n also , the mean patient h reflex amplitude , the h / m ratio , the h slope , and the h slope / m \n slope ratio , decreased significantly , and the h reflex maximum latency increased , after \n tens . in controls , tens significantly decreased the h maximum amplitude , the h / m ratio , the \n m maximum amplitude , and the m slope ratio , and lengthened the h and m maximum latencies . \n the h maximum latency was the only variable affected by lesional duration .", "the mean h \n maximum amplitude , the h / m ratio , and the mean h slope increased as spasticity worsened on \n the ashworth scale .", "explored changes in the spasticity of patients with medulla spinalis injuries \n after tens , and found there were short - term reductions in spasticity9 .", "robinson et al . applied 20  min electrical stimulation to \n the quadriceps muscles of patients with medulla spinalis injuries and reported a significant \n decrease in leg spasticity10 .", "cho et al . \n found that a single session of high - frequency tens significantly improved the spasticity \n ( for less than 1 day ) of chronic stroke patients .", "similarly , tens reduced the spasticity of stroke \n patients , and repeat tens reduced the hyperactive tension reflexes of the plantar flexors , \n and the passive resistance to plantar flexor movement11 . in a study that included acute stroke patients , tens applied to \n acupuncture points for 3 weeks decreased plantar flexor spasticity and increased \n dorsiflexion of the ankle joint . in chronic stroke patients", ", however , ankle joint \n dorsoflexion was strengthened by stimulation , and muscle co - contraction was decreased . in \n addition", "repeated skin stimulation was \n advocated , to increase the number of cortical fields represented12 . in a study that included chronic stroke patients , \n", "addition of tens to a 20-session rehabilitation program decreased spasticity by 30% and \n markedly improved muscle strength13 .", "park \n et al . showed that balance , walking , and the functional activity of chronic stroke patients \n improved after 30 tens sessions ( each 30  min in duration ) , and a combination of exercise and \n tens increased the activation of pathways associated with proprioception and balance14 . in the present study , a significant \n decrease in spasticity and an increase in walking speed were evident after acute hemiplegic \n the patients in a rehabilitation program received a single session of tens . fisher reported that the m amplitudes of patients with first - motor neuron diseases \n decreased , after tens , compared to the amplitudes of polyneuropathic patients15 .", "angel and hoffman , who first showed that \n the h / m ratio was increased in spastic patients , reported mean ratios of 9.17 and 0.48 in \n the extremities of control and spastic groups , respectively .", "the excitability of alpha motor \n neurons increased when various physiopathological mechanisms were in play ; therefore , the \n responses to stimuli carried by 1a afferent fibers increased16 .", "garcia - mullin and mayer reported mean h / m ratios of 0.33 and 0.46 \n on the healthy and paretic sides , respectively , of spastic hemiplegic stroke patients in \n whom the disease duration was longer than 90 days17 .", "bakhtiary showed that spasticity was reduced by electrical stimulation , but no significant \n change in the h reflex amplitude was evident . also , the h / m maximum ratio did not change , \n and this was attributed to muscle weakness caused by stimulation18 .", "gaft et al . noted significant decreases in both \n spasticity and the h reflex amplitude after stimulation . in the present study", ", significant \n differences were found in the m response and h reflex amplitude between the healthy and \n affected sides of spastic the hemiplegic stroke patients .", "the mean maximum amplitudes of the \n h reflexes of the triceps surae were 1,669 and 2,899  mv on the healthy and paretic sides , \n respectively , and the difference was significant .", "the difference in the h / m ratio between \n the healthy and plegic sides was also significant19 .", "huang et al . reported that the h / m maximum and h2/h1 ratios were not adequate for defining \n the extent of spasticity as assessed by the ashworth scale .", "the mean h / m maximum and h2/h1 \n values increased in stroke patients , compared to healthy controls , but the m maximum ratio \n did not significantly differ between the groups .", "the h2/h1 and m maximum ratios did not \n significantly correlate with other measures of spasticity .", "in contrast to other previous \n studies , huang et al . concluded that the mas value correlated with the h / m ratio20 . in the present study , we noted a decrease \n in the m amplitude on the spastic side was observed , whereas the h reflex amplitude and the \n h / m maximum amplitude ratio , increased .", "higashi et al . found that the change in the h slope / m slope ratio was more significant on \n the spastic than the healthy side , and no significant difference in the h / m maxima of the \n two sides was evident .", "as the h slope / m slope graph is bell - shaped , such data are more \n compatible with the brunnstrom stage of hemiplegic patients than are other indicators . in \n conclusion", ", the h slope/ m slope ratio is primarily useful for objectively evaluating the \n extent of spasticity , and the excitability of the motor neuron pool of the spastic sides of \n hemiplegic patients21 .", "measured the excitability of the motor neuron pool and compared the h / m ratios , the h \n maximum amplitude , and the h slope / m slope ratio , in spastic and normal individuals , and \n found there were marked differences between the two groups .", "they concluded , the h slope / m \n slope ratio was particularly useful for evaluating motor neuron pool excitability22 .", "in the present study , steeper h slope and \n greater h slope / m slope ratio on the spastic ( compared to healthy ) side of hemiplegic \n patients prior to tens were observed .", "one study evaluating the electrophysiological components of the h reflex and f wave \n compared the pre- and post - tens h reflexes , and f wave latencies and amplitudes , and \n reported the reduction in spasticity was associated with electrophysiologically proven \n decreases in the h reflex amplitude , the f wave amplitude , and the h / m and f / m ratios23 . in another study , tens was applied \n segmentally and heterosegmentally to the median nerve of the wrist , and to the common \n peroneal nerve ( placebo ) .", "the h reflex latencies increased in 75% of the patients in the \n experimental groups , but the h amplitude did not change significantly .", "aydin et al . compared tens with baclofen , the commonest drug prescribed to treat spasticity \n in patients with medulla spinalis injuries .", "significant improvements in all parameters , \n except pain , were evident in the tens group , and they were associated with decreases in the \n h amplitude and h / m ratio , and the m response .", "the electrophysiological and clinical changes \n did not differ significantly between the tens and baclofen group25 .", "tekeoglu et al . also reported that tens reduced \n spasticity in stroke patients , and improved their capacity to engage in activities of daily \n living , concluding that tens effectively improved motor function26 . in the present study , a single session of tens resulted in significant clinical inhibition \n of spasticity ; however , lower extremity strength did not improve .", "on the spastic side , tens \n significantly decreased the mean h reflex amplitude , the h / m ratio , the h slope , and the h \n slope / m slope ratio ; and increased the h reflex maximum latency .", "this study had some \n limitation walking speed was not measured after several tens applications . in conclusion ,", "tens for hemiplegic patients with spastic lower extremities due to cvd \n markedly improved clinical parameters and significantly changed electrophysiological \n variables .", "the results of this study suggest that tens is effective when used to manage \n spasticity ."]]}
{"article_id": "PMC3876890", "article_text": ["proteinase - activated receptors ( pars ) belong to a family of g - protein - coupled receptors with seven transmembrane domains activated via proteolytic cleavage by serine proteinases .", "a total of four pars have been identified and cloned . among them , par-1 [ 2 , 3 ] , par-3 , and par-4   are targets for thrombin , trypsin , and cathepsin g , whereas par-2 is resistant to thrombin but can be activated by trypsin , mast cell tryptase [ 6 , 7 ] , neutrophil elastase , and insect - derived proteinase . pars are expressed by various cells involved in inflammatory and immunological responses , such as vascular endothelial cells , epithelial cells , mast cells , t cells , monocyte , eosinophils , and neutrophils [ 10 , 11 ] . in these cells ,", "activation of pars affects their main functions such as proliferation , degranulation , and release of inflammatory mediators [ 10 , 11 ] . in our previous study , we have showed the expression of par-1 , par-2 , and par-3 on t cells , and thrombin- , trypsin- , and tryptase - induced interleukin ( il-6 ) release from t cells .", "it has also been reported that cytoplasmic free calcium and phospholipase c and protein kinase c activation are increased in t - leukemic cell lines following stimulation with thrombin or the thrombin receptor agonist peptide .", "thrombin and thrombin receptor agonist also enhanced cd69 expression and il-2 productions by cross - linking t cell receptors in both jurkat t cells and peripheral blood lymphocytes .", "we , therefore , anticipated that thrombin , trypsin , and tryptase might induce tnf release from t cells through pars .", "tnf is a major proinflammatory cytokine that is thought to be important in the pathogenesis of asthma , food allergy , ocular allergy , and atopic dermatitis .", "it has been reported that the increased number of tnf expressing cells and levels of tnf is observed in the bronchoalveolar lavage ( bal ) and in the airways of asthmatics .", "inhaled tnf increases airway responsiveness to methacholine in asthmatic subjects associated with a sputum neutrophilia .", "since pars , tnf , and t cells all play roles in inflammation , we believe , there must be some linkages between them .", "the aim of the present study is to investigate roles of thrombin , tryptase , trypsin , elastase , and agonist peptides of pars in the secretion of tnf from purified human t cells and subtypes of t cells .", "\n human thrombin , trypsin ( specific activity : 10,000  baee  u / mg protein ) , soybean trypsin inhibitor ( sbti ) , and bovine serum albumin ( bsa , fraction v ) were purchased from sigma ( st louis , mo , usa ) .", "recombinant hirudin and human neutrophil elastase ( specific activity : 20 meo - suc - ala - ala - pro - val - pna u / mg protein ) were obtained from calbiochem ( san diego , ca , usa ) .", "recombinant human lung  tryptase ( specific activity : 1,000  n cbz - l - lysine thiobenzyl ester u / mg protein ) was from promega ( madison , wi , usa ) .", "agonist peptides of pars , and their reverse forms , and par-2 antagonist peptide fsllry - nh2 were synthesized in cl bio - scientific inc .", "the sequences of the active and reverse peptides were par-1 , sfllr - nh2 and rllfs - nh2 , tfllrn - nh2 and nrllft - nh2 ; par-2 , sligkv - nh2 and vkgils - nh2 as well as trans cinnamoyl ( tc)-ligrlo - nh2 and tc - olrgil - nh2 ; par-3 , tfrgap - nh2 and pagrft - nh2 .", "rpmi 1640 and newborn calf serum ( ncs ) were obtained from gibco ( carlsbad , ca , usa ) .", "pe - conjugated mouse anti - human cd3 monoclonal antibody , pe - conjugated goat - anti rabbit igg , and tnf opteia elisa kits were purchased from bd pharmingen ( san jose , ca , usa ) .", "trizol reagent and sybr green i stain were purchased from invitrogen ( carlsbad , ca , usa ) .", "cellular activation of signaling kits for extracellular signal - regulated kinase ( erk ) , 2-(2-diamino)-3-methoxyphenyl-4h-1-benzopyran-4-one ( pd98059 ) , akt , pi3k , and p38 2-(4-morpholinyl)-8-phenyl-4h-1-benzopyran-4-one ( ly294002 ) was purchased from cell signaling technology ( beverly , ma , usa ) .", "exscript rt reagent kit and sybr premix ex taq ( perfect real time ) were obtained from takara ( dalian , china ) .", "rabbit anti - human par-1 and rabbit anti - huamn par-2 polyclonal antibodies were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) .", "fitc - conjugated mouse anti - human cd4 monoclonal , pe - conjugated mouse anti - human cd8 monoclonal , percp - cy5.5-conjugated mouse anti - human tnf monoclonal , fitc - conjugated mouse anti - human ifn monoclonal , pe - conjugated mouse anti - human il-4 monoclonal , apc - conjugated mouse anti - human cd25 monoclonal , and apc - conjugated mouse anti - human il-17 monoclonal antibodies were purchased from ebioscience .", "all other reagents were of analytic grade and obtained from sigma ( st louis , mo , usa ) .", "human t cells were isolated from peripheral blood mononuclear cells ( pbmcs ) by a macs system with t cell isolation kit i according to the manufacturer 's protocol . in brief", ", pbmcs were isolated from fresh blood donated by healthy volunteers , 100  ml from each individual per visit .", "the informed consent from each volunteer and agreement with the ethical committee of the first affiliated hospital of nanjing medical university were obtained . after being separated from red blood cells by ficoll - paque density gradient ,", "pbmcs were collected and incubated with microbead - linked anti - cd3 monoclonal antibody for 15  min at 8c .", "cd3 + t cells were separated from other cells by passing through a magnetic cell separation system . for purity analysis", ", the cells were resuspended in pbs and incubated with pe - conjugated monoclonal antibody against human cd3 for 1  h. the purity of t cells was consistently more than 95% and cell viability was more than 98% .", "the purified cd3 + t cells were then used for the further cell challenge tests .", "t cells were cultured in 24-well culture plates at a density of 5  10cells / well in rpmi 1640 medium containing 10% ncs at 37c for 2  h with 5% co2 , respectively .", "the culture supernatants were then removed and cells were washed twice with fresh serum - free rpmi 1640 medium at 300  g for 10  min . for challenge experiments ,", "cells were exposed to various doses of thrombin ( 0.013.0  g / ml , 1  u = 0.5  g , 1  u / ml = 5.6  nm , u = nih unit ) , trypsin ( 0.010.3  g / ml , 1  g / ml = 42  nm ) , tryptase ( 0.252.0  g / ml , 1  g / ml = 7.4  nm ) , and elastase ( 0.010.3  g / ml , 1  g / ml = 34  nm , 1  u / ml = 1700  nm ) with or without their inhibitors ; and to agonist peptides of par-1 , par-2 and par-3 ( all at 0.1100  m ) and their reverse peptides , respectively , for 16  h before the culture , supernatants were harvested and stored at 40c till use .", "quantitative expression of tnf mrnas in t cells was determined by real - time pcr following the manufacture 's protocol . briefly , after synthesizing cdna from the total rna by using exscripttm rt reagent kit , real - time pcr was performed by using sybr premix ex taq on the abi prism 7000 sequence detection system ( perkin elmer applied systems , foster city , ca , usa ) .", "each reaction contains 12.5  l of 2  sybr green master mix , 1  l of 10  m of primers , 1  l of the cdna , to a total volume of 25  l .", "the thermal cycling conditions included an initial denaturation step at 50c for 2  min , 95c for 10  min ; 40 cycles at 95c for 15  s , annealing temperatures at 60c for 30  s , and extension at 72c for 30  s. the sequences of pcr primers for tnf and -actin were 5-ccccagggacctctctctaatc-3 ( forward ) and 5-ggtttgctacaacatgggctaca-3 ( reverse ) ; 5-aggggccggactcgtcatact-3 ( forward ) , and 5-ggcggcaacaccatgtaccct-3 ( reverse ) , respectively .", "consequently , at the end of the pcr cycles , specificities of the amplification products were controlled by dissociation curve analysis .", "expression of mrna in each sample was finally determined after correction with -actin expression . the gene specific threshold cycle ( ct ) for each sample ( ct ) was corrected by subtracting the ct for the housekeeping gene -actin .", "untreated controls were chosen as the reference samples , and the ct for all experimental samples was subtracted by the ct for the control samples ( ct ) .", "t cells were preincubated with 50  m of pd98059 , 20  m of ly294002 , or medium alone for 30  min before adding thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 30  min , 2  h , or 6  h. the cells were lysed in a buffer containing 20  mm of tris - hcl ( ph 7.4 ) , 137  mm of nacl , 10% glycerol , 1% triton x-100 , 2  mm of edta , 25  mm of -glycerophosphate , 2  mm of sodium pyrophosphate , and 0.5  mm of dithiothreitol at 4c for 30  min .", "cell debris was removed by centrifugation of the lysate at 12,000  g for 10  min .", "the supernatants were mixed with equal volumes of 2x sodium dodecyl sulphate ( sds ) sample buffer and heated to 100c for 10  min .", "an equal volume of sample was fractionated by sds - page on a 10% acrylamide gel and transferred onto polyvinylidene difluoride ( pvdf ) membranes with a bio - rad transfer system , according to the manufacturer 's instructions . after blocking nonspecific binding sites with 5% bsa in tbst ( 50  mm of tris , 0.15  m of nacl , 0.1% tween 20 , ph 7.6 ) for 1  h", ", membranes were probed with phospho - erk1/2 , phospho - akt , phospho - p38 , or phospho - pi3k antibodies at 4c overnight , followed by incubation with hrp - conjugated secondary antibodies .", "immunoreactive bands were visualized by using enhanced chemiluminescence reagents according to the manufacturer 's protocol .", "densitometry analysis of immunoblots was carried out using quantity one software ( bio - rad , usa ) .", "the levels of tnf in culture supernatants were measured with opteia elisa kits according to the manufacturer 's instructions .", "the plates were read on a plate reader ( molecular devices , menlo park , ca ) with the softmax data analysis program .", "the minimum detectable concentration of tnf was 2.2  pg / ml . to test the par1 and 2 expressions after treatment of trypsin and thrombin", ", isolated t cells were pelleted by centrifugation at 450  g for 10  min after cells were stimulated with thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 16  h. for par1 and par2 staining , cells were incubated with rabbit anti - human par1 or par2 antibodies at 37c for 1  h. after washing , cells were incubated with pe - conjugated goat anti - rabbit igg antibody 37c for 45  min .", "after washing , cells were analyzed on a fluorescence - activated cell sorting ( facs ) arial flow cytometer with celldevia software ( bd biosciences , usa ) . to test the secretion of tnf from subtypes of t cells", ", isolated t cells were pelleted by centrifugation at 450  g for 10  min and then fixed and permeabilized by using a cell fixation / permeabilization kit ( bd pharmingen ) . briefly", ", thoroughly resuspended cells were added in 100  l of bd cytofix / cytoperm solution and incubated for 20  min at 4c .", "cells were then incubated with fluorescence labeled anti - human cd4 , cd8 , cd25 , tnf , ifn , il-4 , and il-17 monoclonal antibodies or isotope control , respectively ( at a final concentration of 4  g / ml ) at 4c for 30  min .", "after washing , cells were analyzed on a fluorescence - activated cell sorting ( facs ) arial flow cytometer with celldevia software ( bd biosciences , usa ) .", "the purity of t cells was consistently more than 95% ( date was shown in supplementary material , figure s1 ) .", "it has been shown that thrombin , trypsin , and tryptase can induce proinflammatory cytokine il-6 release from t cells , but little is known of serine proteinase - induced tnf release from t cells . here , we showed that thrombin at concentrations of 1.0 and 3.0  g / ml provoked tnf release from t cells following 16  h incubation period in a dose - dependent manner .", "approximately up to 2.5-fold increase in tnf release was observed when t cells were incubated with thrombin for 16  h. at 6  h following incubation , data ( not shown ) on both basal and induced tnf release from t cells were inconsistent .", "this is most likely due to the limitation of the assay sensitivity and relatively low secretion of tnf .", "par-1 agonist peptides , sfllr - nh2 at the concentration of 100  m and tfllrn - nh2 at the concentration of 5  m , induced a significant release of tnf at 16  h following incubation .", "however , rllfs - nh2 , a reverse peptide of sfllr - nh2 , and nrllft - nh2 , a reverse peptide of tfllrn - nh2 , had little effect on release of tnf from t cells ( figure 1(a ) ) .", "hirudin , a specific thrombin inhibitor , was able to inhibit thrombin - induced secretion of tnf .", "approximately up to 82.4% inhibition of thrombin - induced secretion of tnf was observed when 3.0  g / ml of thrombin and 10  u / ml of hirudin were added to t cells for 16  h. hirudin alone at the concentrations tested had little effect on tnf secretion from t cells .", "sch 79797 , a par-1 antagonist at the concentration of 1  m , inhibited 89% thrombin - induced tnf release from t cells ( figure 1(a ) ) .", "similarly , trypsin at the concentration of 0.3  g / ml induced 2.3-fold increase in tnf release from t cells at 16  h ( figure 1(b ) ) .", "however , tryptase at the concentrations up to 2  g / ml and elastase at the concentrations up to 6  u / ml had little effect on tnf release from t cells ( data not shown ) .", "inhibitors of trypsin , sbti at the concentrations of 10 and 30  g / ml , eliminated 0.3  g / ml trypsin - induced tnf release by a value up to 94.8 and 94.2% , respectively .", "sbti alone at the concentrations tested had little effect on tnf secretion from t cells .", "sch 79797 , a par-1 antagonist at the concentration of 1  m , inhibited 96.8% trypsin - induced tnf release from t cells ( figure 1(b ) ) .", "sligkv , an agonist peptide of par-2 and tfrgap - nh2 , an agonist peptide of par-3 at the concentrations up to 100  m , did not appear to have any effect on tnf release from t cells ( data not shown ) . in order to confirm the findings above", ", we investigated the influence of the serine proteinases on the expression of tnf mrna in t cells .", "it was found that the expression of tnf mrna was upregulated when t cells were incubated with thrombin at 1 and 3  g / ml for 2 and 6  h. the maximum enhanced expression of tnf mrna was 4.2-fold over baseline control ( figure 2(a ) ) after 6  h incubation .", "hirudin , a specific thrombin inhibitor at the concentration of 3  u / ml , completely abolished thrombin - induced upregulated expression of tnf mrna after 6  h incubation ( figure 2(b ) ) .", "trypsin at the concentration of 0.3  g / ml also induced increased expression of tnf mrna by a value up to approximately 4.0-fold in t cells ( figure 2(a ) ) , which was completely blocked by sbti ( figure 2(b ) ) .", "similarly , sch 79797 at the concentration of 1  m inhibited both thrombin- and trypsin - induced upregulated expression of tnf mrna in t cells by a value up to 72 and 72.5% , respectively ( figure 2(b ) ) .", "sfllr - nh2 at the concentration of 100  m and tfllrn - nh2 at the concentration of 5  m significantly increase the expression of tnf mrna at 2 and 6  h following incubation ( figure 2(a ) ) .", "but rllfs - nh2 , a reverse peptide of sfllr - nh2 , and nrllft - nh2 , a reverse peptide of tfllrn - nh2 , had little effect on expression of tnf mrna in t cells ( data not shown ) .", "at the same time , neither thrombin nor trypsin showed obvious effect on the expression of par-1 and par-2 ( data not shown ) .", "it is wellknown that there are numerous subtypes of t cells and each of them has distinctive functions .", "we , therefore , investigated subtypes of t cells by flow cytometer analysis in order to determine the subtypes that upregulate tnf in response to trypsin or thrombin .", "the results showed that trypsin and thrombin induced upregulated expression of tnf in cd4 + t cells , but not cd8 + t cells , following 16  h incubation period . among cd4 + t cells , trypsin and thrombin enhanced tnf expression in il-4 + or cd25 + t cells , but not in ifn+ or il-17 + t cells .", "sch 79797 was able to inhibit enhanced tnf expression induced by trypsin and thrombin ( figures 3(a ) and 3(b ) ) . in order to examine signal transduction pathways of thrombin and trypsin", ", t cells were preincubated with pd98059 , ly294002 , or medium alone for 30  min before adding thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 16  h. following 16  h incubation period , pd98059 an inhibitor of mapk pathway , and ly294002 , an inhibitor of pi3k , completely blocked thrombin- and trypsin - induced release of tnf ( figure 4(a ) ) . furthermore , pd98059 inhibited thrombin- and trypsin - induced upregulation of expression of tnf mrna by a value up to 91.2 and 98.6% , and ly294002 eliminated thrombin- and trypsin - induced expression of tnf mrna by 95.5 and 83.2% in t cells following 6  h incubation ( figure 4(b ) ) .", "3  g / ml ) induced enhanced phosphorylation of erk1/2 in t cells following 0.5 , 2 , and 6  h incubation periods .", "however , thrombin and trypsin did not significantly affect phosphorylation of p38 in t cells following 0.5 , 2 , and 6  h incubation periods ( date was shown in supplementary material , figure s2 ) .", "pd98059 was able to completely block thrombin- and trypsin - induced phosphorylation of erk1/2 when it was preincubated with t cells for 30  min .", "thrombin at a concentration of 3  g / ml and trypsin at a concentration of 0.3  g / ml induced significantly increased phosphorylation of akt in t cells following 0.5 , 2 , and 6  h incubation periods .", "however , thrombin and trypsin did not significantly affect phosphorylation of pi3k in t cells following 0.5 , 2 , and 6  h incubation periods ( date was shown in supplementary material , figure s3 ) .", "ly294002 was able to block thrombin- and trypsin - induced phosphorylation of akt when it was incubated with t cells for 30  min .", "we discovered in the present study that serine proteinases thrombin and trypsin , but not tryptase induced tnf release from human t cells .", "since tnf is a potent proinflammatory cytokine , our observation is likely to add some novel information for , understanding of actions of serine proteinases in causing inflammation . as little as 1.0  g / ml of thrombin was able to induce significant tnf release from t cells , suggesting this proteinase is a potent secretagogue of tnf .", "this concentration of thrombin should be easily achieved in blood , particularly when the processes of platelet aggregation and coagulation are initiated .", "inhibition of thrombin - induced tnf release by a specific inhibitor of thrombin and hirudin indicates that action of thrombin on t cells was dependent on the enzymatic activity of this serine proteinase .", "there are 3 receptors for thrombin on cells , including par-1 , par-3 , and par-4 [ 2 , 3 ] .", "since par-1 agonist peptides sfllr - nh2 , and tfllrn - nh2 , but not par-3 agonist peptide tfrgap - nh2 were capable of stimulating tnf release , a par-1 antagonist sch 79797   almost completely abolished thrombin - induced tnf release from t cells , and purified human t cells do not express par-4 ; the action of thrombin on t cells is most likely through activation of par-1 .", "our previous report which found thrombin - induced il-6 secretion from human peripheral blood t cells may support our current findings . while little information is available on induction of tnf release from t cells by trypsin , the ability of trypsin to stimulate il-6 secretion from t cells   may support the anticipation that trypsin is capable of inducing cytokine release from t cells .", "as little as 0.3  g / ml of trypsin was able to provoke tnf secretion from t cells proved that it is a potent stimulus of tnf release .", "as for thrombin , inhibitor of trypsin sbti was able to inhibit trypsin - induced tnf release from t cells , indicating that an intact catalytic site is required for the serine proteinase to stimulate tnf release .", "since par-1 is one of three receptors of trypsin , par-1 agonist peptides sfllr - nh2 and tfllrn - nh2 are capable of stimulating tnf release from t cells , and sch 79797 almost completely abolished trypsin - induced tnf release from t cells , the action of trypsin on t cells is most likely through activation of par-1 .", "since par-2 agonist peptide sligkv - nh2 and tryptase are not capable of stimulating tnf release from t cells , the action of trypsin on t cells is not likely through activation of par-2 .", "trypsin- and thrombin - induced upregulated expression of tnf was observed in cd4 + , il-4 + or cd25 + t cells , indicating that il-4 + , and cd25 + t cells are major sources of tnf .", "while little information on the relationship between cd25 + t cells and tnf is available , a study which found that the percentage of cd4(+)cd25(+ ) t cells were significantly high , but the percentage of foxp3(+ ) cells were low in allergic rhinitis patients , and that il-4 , il-5 , and tnf levels in nasal lavage fluids were high indicates that the increased tnf release may be from cd4(+)cd25(+ ) , nonregulatory t cells .", "we believe that the current study is the first work that demonstrates coexpression of cd25 and tnf in the subtype of cd4(+ ) t cells .", "similarly , we clearly found that il-4 + t cells express enhanced tnf , though little information on co - expression of il-4 and tnf in t cells is available .", "this finding implicates that trypsin and thrombin may be involved in the inflammation through induction of tnf release from il-4 + or cd25 + t cells .", "it was demonstrated that nickel - specific cd4 + t cell lines   and th17 cells   corelease il-17 and tnf , but trypsin- and thrombin - induced tnf release appears not from il-17 + t cells as tnf expression in il-17 + t cells was not upregulated by these two proteinases .", "mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release from highly purified t cells , as pd98059 , an inhibitor of mapk / erk pathway , almost completely blocked thrombin- and trypsin - provoked phophorylation of erk and tnf release . while little information on signaling pathways associated with par-1 signaling in purified t cells is available , the previous reports that par-1 agonists activated mapk / erk and p38 mapk signaling pathways in dermal   and cardiac fibroblasts   may support our current observation that mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release .", "in addition , pi3k / akt signaling pathway seems also to be involved in thrombin and trypsin induced tnf secretion , as ly294002 an inhibitor of pi3k / akt signaling pathway partially diminished thrombin and trypsin induced tnf secretion and completely abolished thrombin and trypsin provoked phosphorylation of akt .", "this finding is in the same line with the report , which showed that thrombin stimulated enhance pi3k activity in hamster embryonic fibroblasts , but different from our previous report , which showed that thrombin did not enhanced pi3k activity in human dermal fibroblasts .", "the discrepancy between these studies may be due to the difference in cell origin and species .", "tnf is a member of a growing family of peptide mediators comprising at least 19 cytokines , including lymphotoxin- , fas ligand , and cd40 ligand .", "the family is now considered as central mediators of a broad range of biological activities in protective immune responses against a variety of infectious pathogens . on the other hand", ", tnf also exerts host - damaging effects in sepsis and autoimmune disease [ 30 , 31 ] .", "these findings indicate that tnf is one of key mediators of inflammation ; therefore , our current study is of importance in understanding tnf - related inflammation and the mechanism of proteinase - induced cytokine production in t cells .", "in conclusion , it is discovered in the present study that serine proteinases thrombin and trypsin are potent stimuli of tnf secretion from highly purified t cells .", "their actions on t cells depend on their enzymatic activities and are likely through activation of par-1 .", "stimulation of tnf secretion from t cells by serine proteinases further proved that these proteinases are actively involved in the pathogenesis of inflammation and regulation of immune response in man ."], "abstract_text": ["<S> serine proteinases have been recognized as playing an important role in inflammation via proteinase activated receptors ( pars ) . </S>", "<S> however , little is known about the influence of serine proteinases and pars on tnf secretion from highly purified t cells . </S>", "<S> we challenged t cells from human peripheral blood with serine proteinases and agonist peptides of pars and measured the levels of tnf in culture supernatants by elisa . </S>", "<S> the results showed that thrombin and trypsin , but not tryptase , stimulated approximately up to 2.5-fold increase in tnf release from t cells following 16 h incubation . </S>", "<S> proteinase inhibitors and par-1 antagonist sch 79797 almost completely abolished thrombin- and trypsin - induced tnf release from t cells . </S>", "<S> agonist peptides of par-1 , but not par-2 induced tnf release from t cells . </S>", "<S> moreover , trypsin- and thrombin - induced upregulated expression of tnf was observed in cd4 + , il-4 + , or cd25 + t cells , but not in ifn+ or il-17 + t cells . </S>", "<S> the signaling pathways mapk / erk and pi3k / akt are involved in the thrombin- and trypsin - induced tnf release from t cells . in conclusion , </S>", "<S> thrombin and trypsin can induce tnf release from il-4 + and cd25 + t cells through activation of par-1 and therefore contribute to regulation of immune response and inflammation of the body . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["proteinase - activated receptors ( pars ) belong to a family of g - protein - coupled receptors with seven transmembrane domains activated via proteolytic cleavage by serine proteinases .", "a total of four pars have been identified and cloned . among them , par-1 [ 2 , 3 ] , par-3 , and par-4   are targets for thrombin , trypsin , and cathepsin g , whereas par-2 is resistant to thrombin but can be activated by trypsin , mast cell tryptase [ 6 , 7 ] , neutrophil elastase , and insect - derived proteinase . pars are expressed by various cells involved in inflammatory and immunological responses , such as vascular endothelial cells , epithelial cells , mast cells , t cells , monocyte , eosinophils , and neutrophils [ 10 , 11 ] . in these cells ,", "activation of pars affects their main functions such as proliferation , degranulation , and release of inflammatory mediators [ 10 , 11 ] . in our previous study , we have showed the expression of par-1 , par-2 , and par-3 on t cells , and thrombin- , trypsin- , and tryptase - induced interleukin ( il-6 ) release from t cells .", "it has also been reported that cytoplasmic free calcium and phospholipase c and protein kinase c activation are increased in t - leukemic cell lines following stimulation with thrombin or the thrombin receptor agonist peptide .", "thrombin and thrombin receptor agonist also enhanced cd69 expression and il-2 productions by cross - linking t cell receptors in both jurkat t cells and peripheral blood lymphocytes .", "we , therefore , anticipated that thrombin , trypsin , and tryptase might induce tnf release from t cells through pars .", "tnf is a major proinflammatory cytokine that is thought to be important in the pathogenesis of asthma , food allergy , ocular allergy , and atopic dermatitis .", "it has been reported that the increased number of tnf expressing cells and levels of tnf is observed in the bronchoalveolar lavage ( bal ) and in the airways of asthmatics .", "inhaled tnf increases airway responsiveness to methacholine in asthmatic subjects associated with a sputum neutrophilia .", "since pars , tnf , and t cells all play roles in inflammation , we believe , there must be some linkages between them .", "the aim of the present study is to investigate roles of thrombin , tryptase , trypsin , elastase , and agonist peptides of pars in the secretion of tnf from purified human t cells and subtypes of t cells ."], ["\n human thrombin , trypsin ( specific activity : 10,000  baee  u / mg protein ) , soybean trypsin inhibitor ( sbti ) , and bovine serum albumin ( bsa , fraction v ) were purchased from sigma ( st louis , mo , usa ) .", "recombinant hirudin and human neutrophil elastase ( specific activity : 20 meo - suc - ala - ala - pro - val - pna u / mg protein ) were obtained from calbiochem ( san diego , ca , usa ) .", "recombinant human lung  tryptase ( specific activity : 1,000  n cbz - l - lysine thiobenzyl ester u / mg protein ) was from promega ( madison , wi , usa ) .", "agonist peptides of pars , and their reverse forms , and par-2 antagonist peptide fsllry - nh2 were synthesized in cl bio - scientific inc .", "the sequences of the active and reverse peptides were par-1 , sfllr - nh2 and rllfs - nh2 , tfllrn - nh2 and nrllft - nh2 ; par-2 , sligkv - nh2 and vkgils - nh2 as well as trans cinnamoyl ( tc)-ligrlo - nh2 and tc - olrgil - nh2 ; par-3 , tfrgap - nh2 and pagrft - nh2 .", "rpmi 1640 and newborn calf serum ( ncs ) were obtained from gibco ( carlsbad , ca , usa ) .", "pe - conjugated mouse anti - human cd3 monoclonal antibody , pe - conjugated goat - anti rabbit igg , and tnf opteia elisa kits were purchased from bd pharmingen ( san jose , ca , usa ) .", "trizol reagent and sybr green i stain were purchased from invitrogen ( carlsbad , ca , usa ) .", "cellular activation of signaling kits for extracellular signal - regulated kinase ( erk ) , 2-(2-diamino)-3-methoxyphenyl-4h-1-benzopyran-4-one ( pd98059 ) , akt , pi3k , and p38 2-(4-morpholinyl)-8-phenyl-4h-1-benzopyran-4-one ( ly294002 ) was purchased from cell signaling technology ( beverly , ma , usa ) .", "exscript rt reagent kit and sybr premix ex taq ( perfect real time ) were obtained from takara ( dalian , china ) .", "rabbit anti - human par-1 and rabbit anti - huamn par-2 polyclonal antibodies were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) .", "fitc - conjugated mouse anti - human cd4 monoclonal , pe - conjugated mouse anti - human cd8 monoclonal , percp - cy5.5-conjugated mouse anti - human tnf monoclonal , fitc - conjugated mouse anti - human ifn monoclonal , pe - conjugated mouse anti - human il-4 monoclonal , apc - conjugated mouse anti - human cd25 monoclonal , and apc - conjugated mouse anti - human il-17 monoclonal antibodies were purchased from ebioscience .", "all other reagents were of analytic grade and obtained from sigma ( st louis , mo , usa ) .", "human t cells were isolated from peripheral blood mononuclear cells ( pbmcs ) by a macs system with t cell isolation kit i according to the manufacturer 's protocol . in brief", ", pbmcs were isolated from fresh blood donated by healthy volunteers , 100  ml from each individual per visit .", "the informed consent from each volunteer and agreement with the ethical committee of the first affiliated hospital of nanjing medical university were obtained . after being separated from red blood cells by ficoll - paque density gradient ,", "pbmcs were collected and incubated with microbead - linked anti - cd3 monoclonal antibody for 15  min at 8c .", "cd3 + t cells were separated from other cells by passing through a magnetic cell separation system . for purity analysis", ", the cells were resuspended in pbs and incubated with pe - conjugated monoclonal antibody against human cd3 for 1  h. the purity of t cells was consistently more than 95% and cell viability was more than 98% .", "the purified cd3 + t cells were then used for the further cell challenge tests .", "t cells were cultured in 24-well culture plates at a density of 5  10cells / well in rpmi 1640 medium containing 10% ncs at 37c for 2  h with 5% co2 , respectively .", "the culture supernatants were then removed and cells were washed twice with fresh serum - free rpmi 1640 medium at 300  g for 10  min . for challenge experiments ,", "cells were exposed to various doses of thrombin ( 0.013.0  g / ml , 1  u = 0.5  g , 1  u / ml = 5.6  nm , u = nih unit ) , trypsin ( 0.010.3  g / ml , 1  g / ml = 42  nm ) , tryptase ( 0.252.0  g / ml , 1  g / ml = 7.4  nm ) , and elastase ( 0.010.3  g / ml , 1  g / ml = 34  nm , 1  u / ml = 1700  nm ) with or without their inhibitors ; and to agonist peptides of par-1 , par-2 and par-3 ( all at 0.1100  m ) and their reverse peptides , respectively , for 16  h before the culture , supernatants were harvested and stored at 40c till use .", "quantitative expression of tnf mrnas in t cells was determined by real - time pcr following the manufacture 's protocol . briefly , after synthesizing cdna from the total rna by using exscripttm rt reagent kit , real - time pcr was performed by using sybr premix ex taq on the abi prism 7000 sequence detection system ( perkin elmer applied systems , foster city , ca , usa ) .", "each reaction contains 12.5  l of 2  sybr green master mix , 1  l of 10  m of primers , 1  l of the cdna , to a total volume of 25  l .", "the thermal cycling conditions included an initial denaturation step at 50c for 2  min , 95c for 10  min ; 40 cycles at 95c for 15  s , annealing temperatures at 60c for 30  s , and extension at 72c for 30  s. the sequences of pcr primers for tnf and -actin were 5-ccccagggacctctctctaatc-3 ( forward ) and 5-ggtttgctacaacatgggctaca-3 ( reverse ) ; 5-aggggccggactcgtcatact-3 ( forward ) , and 5-ggcggcaacaccatgtaccct-3 ( reverse ) , respectively .", "consequently , at the end of the pcr cycles , specificities of the amplification products were controlled by dissociation curve analysis .", "expression of mrna in each sample was finally determined after correction with -actin expression . the gene specific threshold cycle ( ct ) for each sample ( ct ) was corrected by subtracting the ct for the housekeeping gene -actin .", "untreated controls were chosen as the reference samples , and the ct for all experimental samples was subtracted by the ct for the control samples ( ct ) .", "t cells were preincubated with 50  m of pd98059 , 20  m of ly294002 , or medium alone for 30  min before adding thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 30  min , 2  h , or 6  h. the cells were lysed in a buffer containing 20  mm of tris - hcl ( ph 7.4 ) , 137  mm of nacl , 10% glycerol , 1% triton x-100 , 2  mm of edta , 25  mm of -glycerophosphate , 2  mm of sodium pyrophosphate , and 0.5  mm of dithiothreitol at 4c for 30  min .", "cell debris was removed by centrifugation of the lysate at 12,000  g for 10  min .", "the supernatants were mixed with equal volumes of 2x sodium dodecyl sulphate ( sds ) sample buffer and heated to 100c for 10  min .", "an equal volume of sample was fractionated by sds - page on a 10% acrylamide gel and transferred onto polyvinylidene difluoride ( pvdf ) membranes with a bio - rad transfer system , according to the manufacturer 's instructions . after blocking nonspecific binding sites with 5% bsa in tbst ( 50  mm of tris , 0.15  m of nacl , 0.1% tween 20 , ph 7.6 ) for 1  h", ", membranes were probed with phospho - erk1/2 , phospho - akt , phospho - p38 , or phospho - pi3k antibodies at 4c overnight , followed by incubation with hrp - conjugated secondary antibodies .", "immunoreactive bands were visualized by using enhanced chemiluminescence reagents according to the manufacturer 's protocol .", "densitometry analysis of immunoblots was carried out using quantity one software ( bio - rad , usa ) .", "the levels of tnf in culture supernatants were measured with opteia elisa kits according to the manufacturer 's instructions .", "the plates were read on a plate reader ( molecular devices , menlo park , ca ) with the softmax data analysis program .", "the minimum detectable concentration of tnf was 2.2  pg / ml . to test the par1 and 2 expressions after treatment of trypsin and thrombin", ", isolated t cells were pelleted by centrifugation at 450  g for 10  min after cells were stimulated with thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 16  h. for par1 and par2 staining , cells were incubated with rabbit anti - human par1 or par2 antibodies at 37c for 1  h. after washing , cells were incubated with pe - conjugated goat anti - rabbit igg antibody 37c for 45  min .", "after washing , cells were analyzed on a fluorescence - activated cell sorting ( facs ) arial flow cytometer with celldevia software ( bd biosciences , usa ) . to test the secretion of tnf from subtypes of t cells", ", isolated t cells were pelleted by centrifugation at 450  g for 10  min and then fixed and permeabilized by using a cell fixation / permeabilization kit ( bd pharmingen ) . briefly", ", thoroughly resuspended cells were added in 100  l of bd cytofix / cytoperm solution and incubated for 20  min at 4c .", "cells were then incubated with fluorescence labeled anti - human cd4 , cd8 , cd25 , tnf , ifn , il-4 , and il-17 monoclonal antibodies or isotope control , respectively ( at a final concentration of 4  g / ml ) at 4c for 30  min .", "after washing , cells were analyzed on a fluorescence - activated cell sorting ( facs ) arial flow cytometer with celldevia software ( bd biosciences , usa ) ."], ["the purity of t cells was consistently more than 95% ( date was shown in supplementary material , figure s1 ) .", "it has been shown that thrombin , trypsin , and tryptase can induce proinflammatory cytokine il-6 release from t cells , but little is known of serine proteinase - induced tnf release from t cells . here , we showed that thrombin at concentrations of 1.0 and 3.0  g / ml provoked tnf release from t cells following 16  h incubation period in a dose - dependent manner .", "approximately up to 2.5-fold increase in tnf release was observed when t cells were incubated with thrombin for 16  h. at 6  h following incubation , data ( not shown ) on both basal and induced tnf release from t cells were inconsistent .", "this is most likely due to the limitation of the assay sensitivity and relatively low secretion of tnf .", "par-1 agonist peptides , sfllr - nh2 at the concentration of 100  m and tfllrn - nh2 at the concentration of 5  m , induced a significant release of tnf at 16  h following incubation .", "however , rllfs - nh2 , a reverse peptide of sfllr - nh2 , and nrllft - nh2 , a reverse peptide of tfllrn - nh2 , had little effect on release of tnf from t cells ( figure 1(a ) ) .", "hirudin , a specific thrombin inhibitor , was able to inhibit thrombin - induced secretion of tnf .", "approximately up to 82.4% inhibition of thrombin - induced secretion of tnf was observed when 3.0  g / ml of thrombin and 10  u / ml of hirudin were added to t cells for 16  h. hirudin alone at the concentrations tested had little effect on tnf secretion from t cells .", "sch 79797 , a par-1 antagonist at the concentration of 1  m , inhibited 89% thrombin - induced tnf release from t cells ( figure 1(a ) ) .", "similarly , trypsin at the concentration of 0.3  g / ml induced 2.3-fold increase in tnf release from t cells at 16  h ( figure 1(b ) ) .", "however , tryptase at the concentrations up to 2  g / ml and elastase at the concentrations up to 6  u / ml had little effect on tnf release from t cells ( data not shown ) .", "inhibitors of trypsin , sbti at the concentrations of 10 and 30  g / ml , eliminated 0.3  g / ml trypsin - induced tnf release by a value up to 94.8 and 94.2% , respectively .", "sbti alone at the concentrations tested had little effect on tnf secretion from t cells .", "sch 79797 , a par-1 antagonist at the concentration of 1  m , inhibited 96.8% trypsin - induced tnf release from t cells ( figure 1(b ) ) .", "sligkv , an agonist peptide of par-2 and tfrgap - nh2 , an agonist peptide of par-3 at the concentrations up to 100  m , did not appear to have any effect on tnf release from t cells ( data not shown ) . in order to confirm the findings above", ", we investigated the influence of the serine proteinases on the expression of tnf mrna in t cells .", "it was found that the expression of tnf mrna was upregulated when t cells were incubated with thrombin at 1 and 3  g / ml for 2 and 6  h. the maximum enhanced expression of tnf mrna was 4.2-fold over baseline control ( figure 2(a ) ) after 6  h incubation .", "hirudin , a specific thrombin inhibitor at the concentration of 3  u / ml , completely abolished thrombin - induced upregulated expression of tnf mrna after 6  h incubation ( figure 2(b ) ) .", "trypsin at the concentration of 0.3  g / ml also induced increased expression of tnf mrna by a value up to approximately 4.0-fold in t cells ( figure 2(a ) ) , which was completely blocked by sbti ( figure 2(b ) ) .", "similarly , sch 79797 at the concentration of 1  m inhibited both thrombin- and trypsin - induced upregulated expression of tnf mrna in t cells by a value up to 72 and 72.5% , respectively ( figure 2(b ) ) .", "sfllr - nh2 at the concentration of 100  m and tfllrn - nh2 at the concentration of 5  m significantly increase the expression of tnf mrna at 2 and 6  h following incubation ( figure 2(a ) ) .", "but rllfs - nh2 , a reverse peptide of sfllr - nh2 , and nrllft - nh2 , a reverse peptide of tfllrn - nh2 , had little effect on expression of tnf mrna in t cells ( data not shown ) .", "at the same time , neither thrombin nor trypsin showed obvious effect on the expression of par-1 and par-2 ( data not shown ) .", "it is wellknown that there are numerous subtypes of t cells and each of them has distinctive functions .", "we , therefore , investigated subtypes of t cells by flow cytometer analysis in order to determine the subtypes that upregulate tnf in response to trypsin or thrombin .", "the results showed that trypsin and thrombin induced upregulated expression of tnf in cd4 + t cells , but not cd8 + t cells , following 16  h incubation period . among cd4 + t cells , trypsin and thrombin enhanced tnf expression in il-4 + or cd25 + t cells , but not in ifn+ or il-17 + t cells .", "sch 79797 was able to inhibit enhanced tnf expression induced by trypsin and thrombin ( figures 3(a ) and 3(b ) ) . in order to examine signal transduction pathways of thrombin and trypsin", ", t cells were preincubated with pd98059 , ly294002 , or medium alone for 30  min before adding thrombin 3.0  g / ml , trypsin 0.3  g / ml , or medium alone for 16  h. following 16  h incubation period , pd98059 an inhibitor of mapk pathway , and ly294002 , an inhibitor of pi3k , completely blocked thrombin- and trypsin - induced release of tnf ( figure 4(a ) ) . furthermore , pd98059 inhibited thrombin- and trypsin - induced upregulation of expression of tnf mrna by a value up to 91.2 and 98.6% , and ly294002 eliminated thrombin- and trypsin - induced expression of tnf mrna by 95.5 and 83.2% in t cells following 6  h incubation ( figure 4(b ) ) .", "3  g / ml ) induced enhanced phosphorylation of erk1/2 in t cells following 0.5 , 2 , and 6  h incubation periods .", "however , thrombin and trypsin did not significantly affect phosphorylation of p38 in t cells following 0.5 , 2 , and 6  h incubation periods ( date was shown in supplementary material , figure s2 ) .", "pd98059 was able to completely block thrombin- and trypsin - induced phosphorylation of erk1/2 when it was preincubated with t cells for 30  min .", "thrombin at a concentration of 3  g / ml and trypsin at a concentration of 0.3  g / ml induced significantly increased phosphorylation of akt in t cells following 0.5 , 2 , and 6  h incubation periods .", "however , thrombin and trypsin did not significantly affect phosphorylation of pi3k in t cells following 0.5 , 2 , and 6  h incubation periods ( date was shown in supplementary material , figure s3 ) .", "ly294002 was able to block thrombin- and trypsin - induced phosphorylation of akt when it was incubated with t cells for 30  min ."], ["we discovered in the present study that serine proteinases thrombin and trypsin , but not tryptase induced tnf release from human t cells .", "since tnf is a potent proinflammatory cytokine , our observation is likely to add some novel information for , understanding of actions of serine proteinases in causing inflammation . as little as 1.0  g / ml of thrombin was able to induce significant tnf release from t cells , suggesting this proteinase is a potent secretagogue of tnf .", "this concentration of thrombin should be easily achieved in blood , particularly when the processes of platelet aggregation and coagulation are initiated .", "inhibition of thrombin - induced tnf release by a specific inhibitor of thrombin and hirudin indicates that action of thrombin on t cells was dependent on the enzymatic activity of this serine proteinase .", "there are 3 receptors for thrombin on cells , including par-1 , par-3 , and par-4 [ 2 , 3 ] .", "since par-1 agonist peptides sfllr - nh2 , and tfllrn - nh2 , but not par-3 agonist peptide tfrgap - nh2 were capable of stimulating tnf release , a par-1 antagonist sch 79797   almost completely abolished thrombin - induced tnf release from t cells , and purified human t cells do not express par-4 ; the action of thrombin on t cells is most likely through activation of par-1 .", "our previous report which found thrombin - induced il-6 secretion from human peripheral blood t cells may support our current findings . while little information is available on induction of tnf release from t cells by trypsin , the ability of trypsin to stimulate il-6 secretion from t cells   may support the anticipation that trypsin is capable of inducing cytokine release from t cells .", "as little as 0.3  g / ml of trypsin was able to provoke tnf secretion from t cells proved that it is a potent stimulus of tnf release .", "as for thrombin , inhibitor of trypsin sbti was able to inhibit trypsin - induced tnf release from t cells , indicating that an intact catalytic site is required for the serine proteinase to stimulate tnf release .", "since par-1 is one of three receptors of trypsin , par-1 agonist peptides sfllr - nh2 and tfllrn - nh2 are capable of stimulating tnf release from t cells , and sch 79797 almost completely abolished trypsin - induced tnf release from t cells , the action of trypsin on t cells is most likely through activation of par-1 .", "since par-2 agonist peptide sligkv - nh2 and tryptase are not capable of stimulating tnf release from t cells , the action of trypsin on t cells is not likely through activation of par-2 .", "trypsin- and thrombin - induced upregulated expression of tnf was observed in cd4 + , il-4 + or cd25 + t cells , indicating that il-4 + , and cd25 + t cells are major sources of tnf .", "while little information on the relationship between cd25 + t cells and tnf is available , a study which found that the percentage of cd4(+)cd25(+ ) t cells were significantly high , but the percentage of foxp3(+ ) cells were low in allergic rhinitis patients , and that il-4 , il-5 , and tnf levels in nasal lavage fluids were high indicates that the increased tnf release may be from cd4(+)cd25(+ ) , nonregulatory t cells .", "we believe that the current study is the first work that demonstrates coexpression of cd25 and tnf in the subtype of cd4(+ ) t cells .", "similarly , we clearly found that il-4 + t cells express enhanced tnf , though little information on co - expression of il-4 and tnf in t cells is available .", "this finding implicates that trypsin and thrombin may be involved in the inflammation through induction of tnf release from il-4 + or cd25 + t cells .", "it was demonstrated that nickel - specific cd4 + t cell lines   and th17 cells   corelease il-17 and tnf , but trypsin- and thrombin - induced tnf release appears not from il-17 + t cells as tnf expression in il-17 + t cells was not upregulated by these two proteinases .", "mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release from highly purified t cells , as pd98059 , an inhibitor of mapk / erk pathway , almost completely blocked thrombin- and trypsin - provoked phophorylation of erk and tnf release . while little information on signaling pathways associated with par-1 signaling in purified t cells is available , the previous reports that par-1 agonists activated mapk / erk and p38 mapk signaling pathways in dermal   and cardiac fibroblasts   may support our current observation that mapk / erk pathway is the signaling pathway that is most likely involved in the thrombin- and trypsin - induced tnf release .", "in addition , pi3k / akt signaling pathway seems also to be involved in thrombin and trypsin induced tnf secretion , as ly294002 an inhibitor of pi3k / akt signaling pathway partially diminished thrombin and trypsin induced tnf secretion and completely abolished thrombin and trypsin provoked phosphorylation of akt .", "this finding is in the same line with the report , which showed that thrombin stimulated enhance pi3k activity in hamster embryonic fibroblasts , but different from our previous report , which showed that thrombin did not enhanced pi3k activity in human dermal fibroblasts .", "the discrepancy between these studies may be due to the difference in cell origin and species .", "tnf is a member of a growing family of peptide mediators comprising at least 19 cytokines , including lymphotoxin- , fas ligand , and cd40 ligand .", "the family is now considered as central mediators of a broad range of biological activities in protective immune responses against a variety of infectious pathogens . on the other hand", ", tnf also exerts host - damaging effects in sepsis and autoimmune disease [ 30 , 31 ] .", "these findings indicate that tnf is one of key mediators of inflammation ; therefore , our current study is of importance in understanding tnf - related inflammation and the mechanism of proteinase - induced cytokine production in t cells ."], ["in conclusion , it is discovered in the present study that serine proteinases thrombin and trypsin are potent stimuli of tnf secretion from highly purified t cells .", "their actions on t cells depend on their enzymatic activities and are likely through activation of par-1 .", "stimulation of tnf secretion from t cells by serine proteinases further proved that these proteinases are actively involved in the pathogenesis of inflammation and regulation of immune response in man ."]]}
{"article_id": "PMC5021505", "article_text": ["relapsing chronic inflammation found in the gut of individuals affected by inflammatory bowel diseases ( ibd ) is a result of several overlapping factors , including dysregulation of the immune response to the enteric microbiota , genetic susceptibility , and environmental factors [ 1 , 2 ] .", "reactive oxygen ( ros ) and nitrogen ( rns ) species generated by inflammatory cells during an immune response create oxidative stress and are considered as important factors contributing to the pathogenesis of ibd .", "lymphocytes , neutrophils , and macrophages activated during the gut inflammation produce high amounts of ros / rns destroying surrounding tissue .", "although oxidative stress is a major factor in the inflamed tissue leading towards necrosis , dna damage , and carcinogenesis , certain amounts of ros and other free radicals have an indispensable role in regulating different cell signalling pathways .", "in addition , some immune cells , namely , the macrophages and neutrophils , use ros to combat the microorganisms responsible for the infection .", "current therapies of ibd , including immunosuppressive drugs , antibiotics , and biological drugs , are efficient in controlling the course of the disease .", "however , for many affected individuals , conventional therapy becomes an inadequate choice for long - term treatment because of significant side effects and risk factors , such as increased cancer risk , development of tuberculosis , and heart failure [ 57 ] . in recent years", ", hyperbaric oxygen ( hbo2 ) therapy has been introduced as a possible additional treatment for ibd patients , especially in the case of refractory disease when the standard therapy is ineffective .", "hbo2 involves exposure to 100% oxygen under pressure greater than 1 atmosphere of absolute pressure ( atm ) .", "it is a well - established procedure frequently applied in the medical practice , especially effective in treating wounds of various aetiologies [ 811 ] .", "hbo2 increases blood and tissue oxygen saturation resulting also in enhanced production of ros and rns .", "previous studies have verified that the clinical efficacy of hbo2 derives from modulation of intracellular transduction cascades , leading to synthesis of growth factors which promote wound healing , neoangiogenesis , and ameliorates postischemic and postinflammatory injuries .", "additional investigations on the mechanism underlying hbo2-induced wound healing have revealed a central role of hypoxia inducible factor-1 alpha ( hif-1 ) as transcriptional regulator of genes involved in angiogenesis , energy metabolism , and cell proliferation [ 9 , 14 , 15 ] . a further important function attributed to the hif-1 is modulation of the immune responses , including the helper t - cell differentiation towards regulatory ( treg ) versus th17 phenotype [ 16 , 17 ] and its strong anti - inflammatory activity in the gastrointestinal mucosa and hypoxic epithelium as a result of the transactivation of specific genes encoding for barrier - protective elements such as mucins [ 1820 ] .", "hypoxia and ros / rns can induce stabilization of hif-1 leading to the activation of the hypoxia signal transduction pathway . increased oxidative stress in the gut mucosa", "has been verified in humans suffering from ulcerative colitis [ 22 , 23 ] , as well as in experimentally induced colitis in animals [ 24 , 25 ] .", "a recent study revealed increased activity of antioxidative enzymes and reduced oxidative stress in the inflamed gut mucosa following hbo2 exposure ; however , specific mechanisms inducing activation of antioxidative enzymes in the inflamed colonic tissue upon hbo2 remain unknown . since there is evidence that the intracellular redox status is in a close correlation with the inflammatory microenvironment , and it can also be changed by hbo2 , the aim of this study was to investigate the effects of hbo2 on the mrna expression of hif-1 , proinflammatory cytokines , and antioxidative enzymes in the gut and peripheral lymphoid organs of balb / c mice with dss - induced colitis . an additional aim was to assess the activity of antioxidative enzymes and whether hif-1 gene expression regulation during the gut inflammation and hbo2 treatment correlates with the changes in antioxidative and proinflammatory gene expression .", "our findings reveal that hbo2 treatment may effectively modulate the intestinal milieu in inflammatory conditions involving hif-1-mediated regulation of antioxidative gene expression .", "balb / c mice , obtained from charles river ( calco , italy ) , were bred at the animal facility of the medical faculty osijek ( croatia ) .", "mice were provided with standard rodent chow ( mucedola , settimo milanese , italy ) and water ad libitum .", "the experimental facility was maintained at 22  2c , 55  5% humidity , and 12-hour light / dark cycle .", "all procedures involving live animals were conducted in accordance with the european guidelines for the care and use of laboratory animals ( directive 86/609/eec ) and were approved by the local ethical committee ( faculty of medicine , university of osijek ) and croatian ministry of agriculture . for each experiment male mice at the age of 1012 weeks were randomized into 4 groups ( n = 4 - 5 mice / group / experiment ) : control mice ( ctrl ) , control mice undergoing hbo2 ( ctrl + hbo2 ) , mice receiving dextran sodium sulphate ( dss ) , and dss treated mice undergoing hbo2 ( dss + hbo2 ) .", "the average body weight of the mice at the time of inclusion into the study was 22.8  0.4  g. colitis was induced by 5%  ( w / v ) of dss ( mr 36.00050.000 , mp biomedicals , illkrich , france ) in drinking water ad libitum for 7 consecutive days [ 26 , 27 ] .", "the hbo2 treatment was initiated at day 1 and was administered twice a day , 12 hours apart , until the end of experiment ( the last session was applied in the morning of day 8 ; 15 sessions in total ) . during one hbo2 session ,", "mice were exposed to 100% o2 for 60 minutes at 2.4 bars with addition of 15 minutes for gradual compression and decompression .", "mice were sacrificed by cervical dislocation on day 8 , after the morning hbo2 session .", "disease activity index ( dai ) was assessed by daily measurement and scoring of animal body weight loss , stool consistency , and the presence of occult or gross blood per rectum .", "measurements were performed at the same time each day until the end of experiment ( day 8) .", "dai was determined as a sum of body weight loss score ( 0 , none ; 1 , 15% loss ; 2 , 510% ; 3 , 1015% ; 4 , > 15% ) , stool consistency score ( 0 , normal ; 2 , loose stool ; 4 , diarrhea ) , and score of occult / gross bleeding ( 0 , normal ; 2 , occult bleeding ; 4 , gross hematochezia ) .", "mice were sacrificed by cervical dislocation on day 8 following the last morning hbo2 treatment .", "colons , mesenteric lymph nodes ( mln ) , and spleens were collected for further analysis .", "colonic tissue was removed immediately after the animals were sacrificed , washed in pbs , and fixed in 4% paraformaldehyde .", "after 72 hours fixed tissue was embedded in paraffin and cut into a series of 6  m thick sections .", "histological evaluation was preformed according to modified geboes score as follows : \n grade 0 ( structural ( architectural changes ) ) : \n 0 : no abnormality ,  1 : mild abnormality ,  2 : mild or moderate diffuse or multifocal abnormalities ,  3 : severe diffuse or multifocal abnormalities . \n ", "2 : mild or moderate diffuse or multifocal abnormalities , 3 : severe diffuse or multifocal abnormalities . \n", "grade 1 ( chronic inflammatory infiltrate ) : \n 0 : no increase ,  1 : mild but unequivocal increase ,  2 : moderate increase ,  3 : marked increase . \n  1 : mild but unequivocal increase , 2 : moderate increase , \n grade 2 ( lamina propria leukocytes ) : \n 0 : no increase ,  1 : mild but unequivocal increase ,  2 : moderate increase ,  3 : marked increase . \n  1 : mild but unequivocal increase , 2 : moderate increase , \n grade 3 ( intraepithelial neutrophils ) : \n 0 : none ,  1 : < 5% crypts involved ,  2 : < 50% crypts involved ,  3 : > 50% crypts involved . \n  1 : < 5% crypts involved , 2 : < 50% crypts involved , 3 : > 50% crypts involved . \n grade 4 ( crypt destruction ) : \n 0 : none ,  1 : probable , local excess of neutrophils in part of crypt ,  2 : probable , marked attenuation . \n  1 : probable , local excess of neutrophils in part of crypt , 2 : probable , marked attenuation", ". \n grade 0 ( structural ( architectural changes ) ) : \n 3 : unequivocal crypt destruction . \n  3 : unequivocal crypt destruction . \n", "grade 5 ( erosion or ulceration ) : \n 0 : no erosion , ulceration , or granulation tissue ,  1 : recovering epithelium + adjacent inflammation ,  2 : probable erosion focally stripped ,  3 : unequivocal erosion ,  4 : ulcer or granulation tissue.slides were analysed using light microscopy at magnifications 40x , 100x , 200x , and 400x and photographed at 200x ( olympus bx50 microscope , olympus c-5050 digital camera , and quickphoto pro imaging software ( promicra s.r.o . ,", "prague , czech republic ) ) . 0 : no erosion , ulceration , or granulation tissue , 1 : recovering epithelium + adjacent inflammation , 2 : probable erosion focally stripped , 3 : unequivocal erosion , 4 : ulcer or granulation tissue .", "after isolation , the colon was cleaned of intestinal content and freed of fat tissue , washed in dmem ( sigma aldrich , steinheim , germany ) , cut into 5  cm long pieces , and , while shaken at 100  rpm , incubated at 37c for 20 minutes in dmem with 25  mm edta .", "the tissue was then thoroughly washed in pbs buffer for at least 5 times , cut into 2  mm long strips , and digested in dmem containing 5  u / ml dnase ( roche , mannheim , germany ) and collagenase ii ( gibco , paisley , uk ) at 37c for 20  min . following this ,", "supernatant was removed , and the previous step was repeated until complete digestion of the tissue was achieved .", "the supernatant was then filtered through a 100  m sized filter ; dmem + 2% fbs ( sigma aldrich , steinheim , germany ) was added and centrifuged at 800  g for 10  min at room temperature .", "cells were resuspended in 5  ml of 40% percoll ( ge healthcare , uppsala , uk ) , overlaid on 4  ml of 80% percoll , and centrifuged at 900  g/20  min/4c .", "lymphocytes were isolated from the mesenteric lymph nodes ( mln ) and spleen by teasing apart the organs between the frosted ends of two microscopic slides .", "the cells were incubated with a mixture of pe anti - cd4 ( clone gk1.5 , exbio antibodies ) , fitc anti - b220 ( clone ra3 - 6b2 , obtained from the american type culture collection and conjugated with fitc using standard procedures ) , and apc anti - cd3 antibodies ( clone 145 - 2c11 , exbio antibodies ) and the other panel with percp anti - cd45 ( clone 30-f11 , bd biosciences ) , fitc anti - gr-1 ( clone rb6 - 8c5 , bd biosciences ) , and pe anti - f4/80 ( clone bm8 , stemcell technologies inc . ) or pe anti - cd4 ( clone mem-241 , exbio antibodies ) and percp anti - cd8 ( clone mem-31 , exbio antibodies ) antibodies .", "dead cells were excluded based on 7-aminoactinomycin d ( 7-aad ) ( applichem , darmstadt , germany ) staining .", "at least 20,000 live cells were collected by a bd facs canto ii cytometer ( facs canto ii , becton dickinson , san jose , ca , usa ) and analysed using the flowlogic software ( inivai technologies , mentone , australia ) . to assess hydrogen peroxide ( h2o2 ) and peroxynitrite ( onoo ) level , 10 lymphocytes isolated from mln and spleens were incubated for 30  min on + 4c with 10  m dichlorofluorescein diacetate ( dcf - da ) ( biomol , hamburg , germany ) , washed for 5  min at 400  g on + 4c , and analysed with the facs canto ii .", "following this , cells were stimulated with 100  nm phorbol 12-myristate 13-acetate ( pma , calbiochem , darmstadt , germany ) , incubated for 30  min , and analysed for the second time . at each measurement minimum of 10,000 target cells", "colon , mln , and spleen samples were isolated , snap frozen in liquid nitrogen , and stored at 80c till analysis .", "rna purity and concentration was assessed by nanophotometer p - class p330 - 30 ( implen , munich , germany ) . in order to purify rna from all polysaccharides , including dss ,", "an additional purification step using 8  m licl was performed , followed by the standard genomic dna purification step using deoxyribonuclease i kit ( sigma aldrich , st louis , mo , usa ) .", "one microgram of rna was used for cdna synthesis by high capacity cdna kit with rnase inhibitor ( applied biosystems , foster city , ca , usa ) .", "real - time pcr was performed on cfx96 system ( bio rad , singapore ) to assess relative expression of catalase ( cat ) , glutathione peroxidase 1 ( gpx1 ) , superoxide dismutase 1 ( sod1 ) , hif-1 , il-1 , il-2 , and il-6 .", "except for the primers for il-6 gene published by jeong et al . , all other primers were custom made using primer 3 software .", "messenger rna expression was determined using ssofast evagreen supermix ( bio rad , singapore ) .", "tissue powder was additionally homogenized in 100  mm phosphate buffer solution ( ph 7.0 ) containing 1  mm edta ( 1  :  10 , w / v ) using ultra turrax t10 homogenizer ( ika , staufen , germany ) while kept on ice .", "tissue homogenates were sonicated for 30 seconds on ice in three 10 seconds intervals and then centrifuged at 20,000  g for 15 minutes at 4c .", "cat , gpx , and sod enzyme activities were determined using a lambda 25 uv - vis spectrophotometer equipped with uv winlab 6.0 software package ( perkin elmer for the better , massachusetts , usa ) .", "catalase ( ec 1.11.1.6 ) activity was estimated spectrophotometrically using h2o2 as a substrate . the reaction mixture consisted of 10  mm h2o2 in 50  mm phosphate buffer ph 7.0 .", "changes in absorbance of the reaction mixture were measured at 240  nm during 2 minutes after the sample addition .", "one unit of activity corresponds to the loss of 1  mol of h2o2 per minute .", "cat activity was calculated using molar extinction coefficient (  = 0.04  mm  cm ) and expressed as u  mg protein . to assess glutathione peroxidase ( ec 1.11.1.9 ) activity a modified method described by wendel   using h2o2 as a substrate was employed .", "gpx activity was determined indirectly by measuring the rate of nadph oxidation to nadp+ , accompanied by a decrease in absorbance at 340  nm .", "the assay mixture consisted of 50  mm phosphate buffer with 0.4  mm edta and 1  mm sodium azide ( ph 7.0 ) , 0.12  mm nadph , 3.2 units of gr , 1  mm glutathione , and 0.0007% ( w / w ) h2o2 in a total volume of 1.55  ml .", "one unit catalyses the oxidation by h2o2 of 1.0  mole of reduced glutathione to oxidized glutathione per minute at ph 7.0 and 25c .", "gpx activity was calculated using molar extinction coefficient for nadph (  = 6.220  mm  cm ) and expressed as u  mg protein .", "superoxide dismutase ( ec 1.15.1.1 . ) activity was determined using cytochrome c ( 0.05  mm ) as an inhibitory molecule in pbs buffer saline with 0.1  mm edta in system xanthine ( 1  mm)/xanthine oxidase ( 50  u ) by flohe method . total soluble protein concentration in protein extracts was determined by bradford reagent ( sigma aldrich , steinheim , germany ) following manufacturer 's protocol and using bovine serum albumin as a standard .", "were tested by one - way anova or kruskal - wallis test followed by the holm - sidak / tukey or dunn 's post hoc multiple comparison procedure , respectively ( sigma plot 11.0 , sigmastat inc .", ", the student t - test and mann - whitney u statistic were used to compare the differences between the two groups in the case of normally distributed variables and variables that violated assumption of normality , respectively .", "spearman 's correlations were calculated where appropriate ( sigma plot 11.0 , sigmastat inc . ) .", "dai results were analysed by two - way anova and bonferroni post hoc test ( graphpad prism 5.0 , graphpad software , inc . , la jolla , ca , usa ) . a p value of < 0.05 was considered statistically significant for all procedures .", "all data are presented as mean values  standard error of mean ( s.e.m . ) .", "in order to determine the effects of hbo2 on the course of acute colitis , balb / c mice were exposed to 5% dss in the drinking water ad libitum and daily monitored for body weight , stool consistency , and occult / gross rectal bleeding to calculate dai . in this", "study the dss treatment induced substantial weight loss , rectal bleeding , loose stool , and colon shortening resulting in significantly higher dai compared to the control ( ctrl ) group , starting from day 3 until the end of the experiment ( p < 0.01 ; figure 1(a ) ) .", "mice that received dss and underwent hbo2 treatment ( dss + hbo2 group ) also presented with significantly higher dai compared to the ctrl group ; however , in this group of mice hbo2 treatment significantly reduced dai compared to the dss mice , starting from day 5 throughout day 8 ( days 5 and 8 p < 0.01 ; days 6 and 7 p < 0.05 ; figure 1(a ) ) .", "in addition , average colon length in the dss group of mice was significantly shorter ( 8.14  0.23  cm ) compared to the ctrl group ( 13.88  0.45  cm ; p < 0.0001 ) , while this effect was significantly ameliorated by hbo2 treatment in the dss + hbo2 group ( 11.34  0.38  cm ) compared to the dss group ( p = 0.0001 , figure 1(b ) ) .", "dss induced colitis resulted in significant body mass loss compared to ctrl group , irrespective of hbo2 treatment ( 11.89  0.03% and 8.24  0.01% in the dss and dss + hbo2 group , resp . ;", "ctrl and ctrl + hbo2 gained body mass during the experiment , 8.25  0.2% and 0.33  0.01% , respectively .", "histological assessment of the colon revealed severe inflammation and ulceration extending into the deep portions of the mucosa with loss of crypts and with increased number of lamina propria leukocytes in dss group of mice .", "we also found structural changes of mucosa in the colonic tissue of dss + hbo2 mice but with reduced infiltration of inflammatory cells and decreased crypt distortion .", "when compared to the dss + hbo2 group , the dss group had significantly higher total histological score as well as individual scores ( see modified geboes score in section 2.3 , figure 1(d ) , p < 0.001 ) , except for the intraepithelial neutrophil infiltration score ( p = 0.104 ) .", "distribution of inflammatory cells among the peripheral lymphoid organs , including gr-1 leukocytes ( monocytes and neutrophils ) , f4/80 leukocytes ( monocytes ) , cd3 t lymphocytes , and b220 b lymphocytes ( figure 2 ) , was assessed at the end of the experiment . in the mln", ", frequencies of gr-1 cells did not differ among the experimental groups , while dss induced a significant increase in f4/80 and decrease in cd3 cell frequencies ( p < 0.05 and p = 0.032 , resp . ; figure 2(b ) ) .", "these findings in the dss group were accompanied by a b - cell increase which was not statistically significant .", "hbo2 alone had no effect on the cell frequencies in mln of control mice , whereas it substantially ameliorated these changes in mice with dss - induced colitis ( dss + hbo2 group ) but without reaching statistical significance . in the spleen", ", gr-1 cell frequencies were significantly decreased in the dss group compared to ctrl ( p = 0.006 ) and ctrl + hbo2 groups ( p < 0.001 ; figure 2(a ) ) .", "hbo2 treatment reversed gr-1 cell frequencies to control values in the dss + hbo2 group ( p = 0.056 ; figure 2(a ) ) .", "in addition , the dss mice showed reduced frequencies of b220 lymphocytes compared to the ctrl group ( p = 0.016 ) , and hbo2 treatment abolished these effects in the dss + hbo2 group ( p = 0.034 ; figure 2(c ) ) .", "in addition , our study revealed that dss - induced immune responses in the mln and the spleen were significantly dampened by hbo2 treatment .", "frequency of cd4 cells among the colon lamina propria lymphocytes of dss and dss + hbo2 groups was significantly increased compared to the ctrl group ( p = 0.015 and p = 0.047 , resp . ) , while the frequency of cd8 cells was significantly increased only in the dss group when compared to the ctrl group ( p = 0.011 ; figure 3 ) .", "inflammatory conditions have been known to include the enhanced production of ros and other oxidative mediators that may affect transcriptional regulation via hif-1. to investigate the role of hif-1 in the regulation of the antioxidative response / capacity during dss - induced colitis and hbo2 treatment , hif-1 , cat , gpx1 , and sod1 mrna expressions were determined using quantitative pcr method .", "mrna expression was significantly changed by the hbo2 treatment and the inflammatory microenvironment in the gut mucosa .", "dss - induced colitis resulted in significant upregulation of hif-1 gene in colonic mucosa ( p = 0.008 for dss group compared to ctrl ) , and the hbo2 treatment further increased hif-1 mrna expression in the dss + hbo2 group ( p = 0.028 compared to ctrl ; figure 3(a ) ) .", "in addition , the activity of hif-1 protein was indirectly confirmed by measuring mrna expression of well - established hif-1 target genes , vegf and pgk1 .", "both genes showed strong positive correlation to the hif-1 mrna ( supplementary figure 1 ) ( see supplementary material available online at http://dx.doi.org/10.1155/2016/7141430 ) .", "there was also a tendency for upregulation of hif-1 gene in mln and spleens of the dss group and its downregulation via hbo2 in the dss + hbo2 group ( figure 4(a ) ) ; however , these changes did not reach statistical significance .", "inflammation during dss - induced colitis and the hbo2 treatment also induced significant changes in mrna expression of target antioxidative genes .", "dss - treated mice presented with significant downregulation of the cat gene in the colon compared to the ctrl group ( p = 0.031 ) , while there was a significant upregulation of cat gene in the spleen of the dss + hbo2 mice compared to the ctrl group ( p = 0.026 ; figure 4(b ) ) . in the colon , gpx1 mrna expression", "was increased in the dss ( p = 0.034 ) and the dss + hbo2 ( p = 0.003 ) group compared to ctrl group .", "the upregulation was even greater in mice with dss - induced colitis that underwent the hbo2 treatment ( dss + hbo2 group ; figure 4(b ) ) .", "sod1 mrna expression was significantly reduced in the colon of the dss + hbo2 group compared to ctrl ( p = 0.008 ) and ctrl + hbo2 ( p = 0.007 ) groups .", "similar changes in sod1 gene expression were also found in the mln of the dss + hbo2 group ( p = 0.025 compared to ctrl ; figure 4(b ) ) .", "to summarize , colitis resulted in gpx1 gene upregulation and cat gene downregulation , while hbo2 downregulated sod1 and further upregulated gpx1 in a tissue - specific manner . to examine the possible role of hif-1 in transcriptional control of antioxidative genes in the colon ,", "the results revealed a strong negative correlation between hif-1 and sod1 ( r = 0.651 , p = 0.001 ) and a positive correlation of hif-1 to the gpx1 gene ( r = 0.750 , p < 0.001 ) , while there was no significant correlation between the hif-1 and the cat gene in the colonic tissue ( figure 4(c ) ) .", "the early phase of inflammation is mediated by several proinflammatory mediators , which prompted us to assess how hbo2 treatment affects their production .", "we found that gut mucosa inflammation was accompanied with a significant increase in il-1 and il-6 gene expression . in the case of il-6 gene , this was significant for dss and dss + hbo2 groups compared to the ctrl + hbo2 group ( p = 0.024 and p = 0.021 , resp . ;", "furthermore , il-6 gene was significantly upregulated in the mln of the dss group ( p = 0.001 compared to ctrl ) , and hbo2 reduced its expression almost to control values in the dss + hbo2 group ( p = 0.016 compared to dss ; figure 5(a ) ) .", "il-1 mrna expression in the colonic mucosa was significantly increased during inflammation in dss group compared to ctrl ( p = 0.014 ) and ctrl + hbo2 groups ( p = 0.041 ) .", "il-1 mrna levels in the colon of the dss + hbo2 group did not significantly differ from the control groups , suggesting that hbo2 treatment blocked the increase of il-1 gene expression in the inflamed mucosa . \n", "il-2 gene was significantly upregulated in the mln of the dss group compared to the ctrl group ( p = 0.003 ) , and hbo2 treatment resulted in its significant downregulation in the dss + hbo2 group ( p = 0.032 ) .", "similarly , il-2 gene was significantly downregulated in the spleen of mice from the dss + hbo2 group compared to the ctrl group ( p = 0.025 ) .", "in addition , there was a strong positive correlation between hif-1 and il-6 gene ( r = 0.749 , p < 0.001 ; figure 5(b ) ) , while there was no correlation between hif-1 and il-1 or il-2 genes in the colonic tissue ( figure 5(b ) ) .", "in addition to their mrna expression , we also tested the enzymatic activity of antioxidative enzymes .", "we found that both the inflammation and the hbo2 treatment per se were able to change the activity of antioxidative enzymes in the colonic mucosa and the peripheral lymphoid organs ( mln and spleen ) . in spleen hbo2 treatment per", "se induced significant increase of sod activity ( p = 0.040 compared to ctrl ) .", "mice with dss - induced colitis presented with significantly increased activity of sod ( p = 0.012 compared to ctrl ) in the colon and reduced cat activity in mln and spleen ( p = 0.023 and p = 0.032 , resp . ) compared to ctrl group .", "treatment did not change sod activity in the inflamed colonic mucosa , which was comparable to the levels found in dss and ctrl + hbo2 groups ( significantly increased compared to ctrl , p = 0.010 ) . on the other hand , hbo2 treatment significantly increased cat ( p = 0.020 ) and gpx ( p = 0.001 ) activities in the spleens of the dss + hbo2 .", "immune cells at the site of inflammation and in the peripheral lymphoid organs are an important source of ros .", "therefore we assessed the basal levels of intracellular h2o2 and onoo and their production upon pma - induced activation in the lymphocytes isolated from mln and spleens of the mice from all experimental groups ( figure 6 ) .", "basal h2o2 and onoo production in the mln was not significantly different among the groups , except for the lymphocytes from the dss + hbo2 group which presented with a significant increase of h2o2 and onoo levels compared to the ctrl group ( p = 0.033 ) .", "pma stimulation resulted in increased intracellular h2o2 and onoo production , although statistically significant only for ctrl ( p = 0.031 ) and dss + hbo2 ( p = 0.012 ) groups . in the spleen ,", "hbo2 increased lymphocyte h2o2 and onoo production in ctrl + hbo2 and dss + hbo2 groups ( p = 0.004 and p = 0.007 compared to the ctrl ; and p = 0.005 and p = 0.009 compared to the dss group ) .", "their production after pma - induced activation was decreased in all experimental groups except the ctrl group ; however , this effect reached statistical significance only in the ctrl + hbo2 group ( p = 0.018 compared to unstimulated lymphocytes ) .", "in the present study , the experimental model of dss - induced colitis in balb / c mice was employed to explore the effects of hbo2 on the antioxidative enzymes , transcription factor hif-1 , and proinflammatory cytokine genes during colonic inflammation and their role in modulating the course of the disease via hbo2 treatment .", "the most important findings are that ( a ) hbo2 significantly reduces symptoms and severity of dss - induced colitis , as evidenced by clinical appearance , contraction of the immune cell expansion and mobilization , and reversal of il-1 , il-2 , and il-6 gene expression ; ( b ) hbo2 modulates the expression of antioxidative enzyme genes and enzyme activities during colitis ; and ( c ) hbo2 enhances hif-1 mrna expression in the inflamed colonic tissue which is in a strong correlation with gpx1 , sod1 , and il-6 mrna expression .", "several previous studies in animals and humans demonstrated the positive effects of hbo2 treatment in influencing the severity of colitis and reducing gut mucosa inflammation [ 27 , 34 ] ; however , data on the precise underlying mechanisms are scarce .", "considerably more data on the beneficial anti - inflammatory effects of hbo2 are available for other conditions such as septic shock , ischemia / reperfusion injuries , and atherogenesis , where the previous studies reported reduced proinflammatory cytokine expression , suppressed development of th cells , shrinking of spleen and lymph nodes , decreased responses to antigens , and reduced frequencies of circulating leukocytes [ 3542 ] .", "although this is the first animal study investigating the effects of hbo2 performed on dss - induced colitis in balb / c mice and correlating it with the immune cell frequencies , our results are in line with previous findings on the changes associated with dss - induced acute immune response , as well as on the effects of hbo2 on the antioxidative enzyme activities determined in other animal models , such as tnbs and acetic acid induced colitis in rats [ 27 , 43 , 44 ] . during colitis mice presented with decreased t and b cell frequencies in the spleen and reduced t cell frequencies in the mln , suggesting that lymphocytes are recruited from the peripheral lymphoid organs and probably migrate to the inflamed colonic mucosa .", "one element of the beneficial effect of hbo2 may be linked to normalized t and b cells frequency in the mln and spleen of mice with dss - induced colitis after hyperbaric treatment ( figures 1 and 2 ) . by measuring cd4 and cd8 lymphocyte in colon", "we confirm our hypothesis of t - cell recruitment from the peripheral lymphoid organs and their migration to the inflamed colonic mucosa , as well as immunomodulatory effect of hbo2 . in our model hbo2", "did not affect cell frequencies in the peripheral lymphoid organs of control mice , in contrast to previous findings where hbo2 treatment per se was able to change lymphocyte subset populations in the spleen .", "for a long time macrophages and neutrophils have been considered as immune cells exclusively producing proinflammatory cytokines , chemokines , and large amounts of ros / rns contributing to aggravated inflammation .", "we have found decreased spleen gr-1 cell frequencies during colitis and their normalization upon hbo2 treatment ( figure 2 ) .", "in addition , we showed increased mln frequencies of f4/80 cells in dss group , while hbo2 treatment reversed their frequencies almost to control values .", "these findings indicate that hbo2 can modulate distribution of phagocytes by retaining neutrophils in the spleen and instigating macrophage migration towards the site of inflammation , in agreement with previous findings describing inhibited neutrophil infiltration into the gut of mice with dss - induced colitis . furthermore , hbo2 treatment alone did not change the expression of proinflammatory cytokines in the colon , mln , or spleen of the control mice ; however , dss - induced colitis resulted in a significant il-1 and il-6 gene upregulation in the colonic tissue and il-2 gene upregulation in the mln ( figure 5 ) .", "consistent with previous studies , hbo2 treatment abolished these effects , further confirming its anti - inflammatory potential [ 4749 ] .", "several animal studies on the effects of hbo2 on the experimental colitis reported an increased antioxidative capacity and changes in antioxidative enzyme activity [ 24 , 25 ] .", "it has been proposed that an optimal hbo2 treatment could generate ros which would function primarily as intermediates in the antioxidative signalling pathways leading to increased expression of antioxidative enzymes , reduced inflammation , and ameliorated colitis symptoms but would not further damage the colonic tissue [ 50 , 51 ] .", ". showed that 24 hours after a two - hour hbo2 treatment at 2 bars in rats oxidative stress is not elevated , as evidenced by assessing ferric reducing antioxidant power ability of plasma ( frap ) and thiobarbituric acid reactive substances ( tbars ) level .", "in addition , a recent study also suggests that ros produced by nadph oxidase complex are important mediators inducing anti - inflammatory response in autoimmune diseases .", "data on the cat mrna level during dss - induced colitis and upon hbo2 treatment were not available prior to this study .", "we found that cat mrna expression is tissue and treatment specific ( figure 4 ) .", "colitis resulted in a significant downregulation of cat mrna expression in the colonic mucosa , and the hbo2 treatment induced its upregulation in the spleen of dss + hbo2 group of mice .", "these results were largely in accordance with our finding on enzymatic catalase activity that was decreased in all measured tissues in the dss group and reversed to control values in the spleen of dss + hbo2 group ( table 2 ) , as well as with a previous study demonstrating decreased catalase activity in colonic tissue upon dss treatment .", "this is also in line with a study on skin transplanted balb / c mice where hbo2 treatment increased catalase , gpx , and sod activity in the spleen .", "furthermore , upregulation of protein and mrna catalase levels 14 days after hbo2 treatment , but not after 7 days , was also observed in the ulcer tissue of patients with diabetic foot , indicating a time - course for the effect of hbo2 to prevail .", "we found that gpx1 mrna level was upregulated in the colon of dss treated mice , irrespective of the hbo2 treatment , and there were no significant differences in the gpx1 mrna expression in mln and spleen .", "in contrast to our findings on mrna expression , colon gpx enzyme activity was slightly reduced in dss + hbo2 group compared to other groups , which is consistent with previous results obtained in acetic acid induced colitis in rats receiving combined hbo2 and ozone treatment . however , other reports indicate decreased gpx and sod activity in the inflamed distal colon mucosa and the plasma of rats with acetic acid induced colitis , and hbo2 normalized gpx but not sod activity in the colon .", "the observed discrepancies in the results may be related to the differences in experimental models used among the studies . in addition , in our study hbo2 treatment induced enhanced gpx and sod activity in the spleen of dss mice which is in contrast to reduced sod1 mrna expression and might be explained by additional sod2 and sod3 function in regulation of antioxidative capacity .", "although intracellular h2o2 and onoo levels were slightly increased during inflammation and hbo2 treatment in the mln and hbo2 per se increased its level in spleen , impaired lymphocyte function was not observed . intensive research on the beneficial wound healing effects of hbo2 revealed its capacity to induce neovascularization , reduce oedema , decrease leukocyte adhesion , stimulate fibroblast expansion , and inhibit bacterial growth [ 14 , 57 ]", ". some of these processes are transcriptionally regulated by hif-1 , namely , the vascular endothelial growth factor ( vegf ) expression , regulatory t lymphocyte differentiation , and preservation of epithelial thigh junction integrity .", "in addition , previous studies employing conditional deletion of epithelial hif-1 or pharmacologic activation of hif-1 in a murine model of colitis demonstrated a protective role for hif-1 in colitis .", "it has also been shown that hif-1 increases expression of barrier - protective genes ( multidrug resistance gene-1 , intestinal trefoil factor , cd73 ) , decreases tnf mrna expression , and enhances antimicrobial activity by transcribing beta - defensin 1 . in the present study we found increased expression of hif-1 gene in inflamed colonic tissue , and", "hif-1 gene expression was changed ( upregulated ) by the inflammation while hbo2 treatment showed a tendency to reverse this increase .", "these data suggest involvement of different mechanisms controlling hif-1 gene expression at the site of inflammation ( colon ) and the peripheral lymphoid organs ( mln and spleen ) , responsible for the initiation of the immune response and the t / b - cell expansion and differentiation , respectively .", "we also demonstrated a strong positive correlation between hif-1 and gpx1 mrna levels in the colon ( figure 4(b ) ) .", "this is in line with in vitro studies where overexpressed hif-1 in colorectal cancer cells resulted in enhanced gpx1 expression through tgf-ri / smad2/erk1/2/hif-1 signalling cascade , suggesting transcriptional regulation of gpx1 by hif-1 . in the present study we found strong negative correlation between hif-1 and sod1 mrna expression", "this is in accordance with a previous study showing that docosahexaenoic acid downregulates sod1 gene transcription through an hre - mediated mechanism ( hre , hypoxia - response element ) , involving hif signalling in human cancer cells ; thus our results indicate similar mechanism involved in sod1 control in the murine colon mucosa in vivo during colitis and hbo2 .", "previous studies revealed that hif-1 mediated transcriptional regulation of different proinflammatory cytokines and growth factor genes are tissue and cell specific and include regulation trough alternative splicing , mrna stability , and interactions with other transcription factors like nf-b [ 6669 ] . in our study we found a strong correlation between hif-1 and il-6 mrna levels suggesting involvement of hif-1 in transcriptional regulation of il-6 gene during colonic inflammation and hbo2 . in conclusion , our results confirmed that hbo2 exerts an anti - inflammatory effect on dss - induced colitis in mice , and this effect at least involves hif-1 and antioxidative genes expression regulation ( as outlined in figure 7 ) .", "however , further studies are necessary to identify the cells that may contribute to or are influenced by the effects upon hbo2 treatment ."], "abstract_text": ["<S> reactive oxygen species ( ros ) and nitrogen species have an indispensable role in regulating cell signalling pathways , including transcriptional control via hypoxia inducible factor-1 ( hif-1 ) . </S>", "<S> hyperbaric oxygenation treatment ( hbo2 ) increases tissue oxygen content and leads to enhanced ros production . in the present study </S>", "<S> dss - induced colitis has been employed in balb / c mice as an experimental model of gut mucosa inflammation to investigate the effects of hbo2 on hif-1 , antioxidative enzyme , and proinflammatory cytokine genes during the colonic inflammation . here </S>", "<S> we report that hbo2 significantly reduces severity of dss - induced colitis , as evidenced by the clinical features , histological assessment , impaired immune cell expansion and mobilization , and reversal of il-1 , il-2 , and il-6 gene expression . </S>", "<S> gene expression and antioxidative enzyme activity were changed by the hbo2 and the inflammatory microenvironment in the gut mucosa . </S>", "<S> strong correlation of hif-1 mrna level to gpx1 , sod1 , and il-6 mrna expression suggests involvement of hif-1 in transcriptional regulation of these genes during colonic inflammation and hbo2 . </S>", "<S> this is further confirmed by a strong correlation of hif-1 with known target genes vegf and pgk1 . </S>", "<S> results demonstrate that hbo2 has an anti - inflammatory effect in dss - induced colitis in mice , and this effect is at least partly dependent on expression of hif-1 and antioxidative genes . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["relapsing chronic inflammation found in the gut of individuals affected by inflammatory bowel diseases ( ibd ) is a result of several overlapping factors , including dysregulation of the immune response to the enteric microbiota , genetic susceptibility , and environmental factors [ 1 , 2 ] .", "reactive oxygen ( ros ) and nitrogen ( rns ) species generated by inflammatory cells during an immune response create oxidative stress and are considered as important factors contributing to the pathogenesis of ibd .", "lymphocytes , neutrophils , and macrophages activated during the gut inflammation produce high amounts of ros / rns destroying surrounding tissue .", "although oxidative stress is a major factor in the inflamed tissue leading towards necrosis , dna damage , and carcinogenesis , certain amounts of ros and other free radicals have an indispensable role in regulating different cell signalling pathways .", "in addition , some immune cells , namely , the macrophages and neutrophils , use ros to combat the microorganisms responsible for the infection .", "current therapies of ibd , including immunosuppressive drugs , antibiotics , and biological drugs , are efficient in controlling the course of the disease .", "however , for many affected individuals , conventional therapy becomes an inadequate choice for long - term treatment because of significant side effects and risk factors , such as increased cancer risk , development of tuberculosis , and heart failure [ 57 ] . in recent years", ", hyperbaric oxygen ( hbo2 ) therapy has been introduced as a possible additional treatment for ibd patients , especially in the case of refractory disease when the standard therapy is ineffective .", "hbo2 involves exposure to 100% oxygen under pressure greater than 1 atmosphere of absolute pressure ( atm ) .", "it is a well - established procedure frequently applied in the medical practice , especially effective in treating wounds of various aetiologies [ 811 ] .", "hbo2 increases blood and tissue oxygen saturation resulting also in enhanced production of ros and rns .", "previous studies have verified that the clinical efficacy of hbo2 derives from modulation of intracellular transduction cascades , leading to synthesis of growth factors which promote wound healing , neoangiogenesis , and ameliorates postischemic and postinflammatory injuries .", "additional investigations on the mechanism underlying hbo2-induced wound healing have revealed a central role of hypoxia inducible factor-1 alpha ( hif-1 ) as transcriptional regulator of genes involved in angiogenesis , energy metabolism , and cell proliferation [ 9 , 14 , 15 ] . a further important function attributed to the hif-1 is modulation of the immune responses , including the helper t - cell differentiation towards regulatory ( treg ) versus th17 phenotype [ 16 , 17 ] and its strong anti - inflammatory activity in the gastrointestinal mucosa and hypoxic epithelium as a result of the transactivation of specific genes encoding for barrier - protective elements such as mucins [ 1820 ] .", "hypoxia and ros / rns can induce stabilization of hif-1 leading to the activation of the hypoxia signal transduction pathway . increased oxidative stress in the gut mucosa", "has been verified in humans suffering from ulcerative colitis [ 22 , 23 ] , as well as in experimentally induced colitis in animals [ 24 , 25 ] .", "a recent study revealed increased activity of antioxidative enzymes and reduced oxidative stress in the inflamed gut mucosa following hbo2 exposure ; however , specific mechanisms inducing activation of antioxidative enzymes in the inflamed colonic tissue upon hbo2 remain unknown . since there is evidence that the intracellular redox status is in a close correlation with the inflammatory microenvironment , and it can also be changed by hbo2 , the aim of this study was to investigate the effects of hbo2 on the mrna expression of hif-1 , proinflammatory cytokines , and antioxidative enzymes in the gut and peripheral lymphoid organs of balb / c mice with dss - induced colitis . an additional aim was to assess the activity of antioxidative enzymes and whether hif-1 gene expression regulation during the gut inflammation and hbo2 treatment correlates with the changes in antioxidative and proinflammatory gene expression .", "our findings reveal that hbo2 treatment may effectively modulate the intestinal milieu in inflammatory conditions involving hif-1-mediated regulation of antioxidative gene expression ."], ["balb / c mice , obtained from charles river ( calco , italy ) , were bred at the animal facility of the medical faculty osijek ( croatia ) .", "mice were provided with standard rodent chow ( mucedola , settimo milanese , italy ) and water ad libitum .", "the experimental facility was maintained at 22  2c , 55  5% humidity , and 12-hour light / dark cycle .", "all procedures involving live animals were conducted in accordance with the european guidelines for the care and use of laboratory animals ( directive 86/609/eec ) and were approved by the local ethical committee ( faculty of medicine , university of osijek ) and croatian ministry of agriculture . for each experiment male mice at the age of 1012 weeks were randomized into 4 groups ( n = 4 - 5 mice / group / experiment ) : control mice ( ctrl ) , control mice undergoing hbo2 ( ctrl + hbo2 ) , mice receiving dextran sodium sulphate ( dss ) , and dss treated mice undergoing hbo2 ( dss + hbo2 ) .", "the average body weight of the mice at the time of inclusion into the study was 22.8  0.4  g. colitis was induced by 5%  ( w / v ) of dss ( mr 36.00050.000 , mp biomedicals , illkrich , france ) in drinking water ad libitum for 7 consecutive days [ 26 , 27 ] .", "the hbo2 treatment was initiated at day 1 and was administered twice a day , 12 hours apart , until the end of experiment ( the last session was applied in the morning of day 8 ; 15 sessions in total ) . during one hbo2 session ,", "mice were exposed to 100% o2 for 60 minutes at 2.4 bars with addition of 15 minutes for gradual compression and decompression .", "mice were sacrificed by cervical dislocation on day 8 , after the morning hbo2 session .", "disease activity index ( dai ) was assessed by daily measurement and scoring of animal body weight loss , stool consistency , and the presence of occult or gross blood per rectum .", "measurements were performed at the same time each day until the end of experiment ( day 8) .", "dai was determined as a sum of body weight loss score ( 0 , none ; 1 , 15% loss ; 2 , 510% ; 3 , 1015% ; 4 , > 15% ) , stool consistency score ( 0 , normal ; 2 , loose stool ; 4 , diarrhea ) , and score of occult / gross bleeding ( 0 , normal ; 2 , occult bleeding ; 4 , gross hematochezia ) .", "mice were sacrificed by cervical dislocation on day 8 following the last morning hbo2 treatment .", "colons , mesenteric lymph nodes ( mln ) , and spleens were collected for further analysis .", "colonic tissue was removed immediately after the animals were sacrificed , washed in pbs , and fixed in 4% paraformaldehyde .", "after 72 hours fixed tissue was embedded in paraffin and cut into a series of 6  m thick sections .", "histological evaluation was preformed according to modified geboes score as follows : \n grade 0 ( structural ( architectural changes ) ) : \n 0 : no abnormality ,  1 : mild abnormality ,  2 : mild or moderate diffuse or multifocal abnormalities ,  3 : severe diffuse or multifocal abnormalities . \n ", "2 : mild or moderate diffuse or multifocal abnormalities , 3 : severe diffuse or multifocal abnormalities . \n", "grade 1 ( chronic inflammatory infiltrate ) : \n 0 : no increase ,  1 : mild but unequivocal increase ,  2 : moderate increase ,  3 : marked increase . \n  1 : mild but unequivocal increase , 2 : moderate increase , \n grade 2 ( lamina propria leukocytes ) : \n 0 : no increase ,  1 : mild but unequivocal increase ,  2 : moderate increase ,  3 : marked increase . \n  1 : mild but unequivocal increase , 2 : moderate increase , \n grade 3 ( intraepithelial neutrophils ) : \n 0 : none ,  1 : < 5% crypts involved ,  2 : < 50% crypts involved ,  3 : > 50% crypts involved . \n  1 : < 5% crypts involved , 2 : < 50% crypts involved , 3 : > 50% crypts involved . \n grade 4 ( crypt destruction ) : \n 0 : none ,  1 : probable , local excess of neutrophils in part of crypt ,  2 : probable , marked attenuation . \n  1 : probable , local excess of neutrophils in part of crypt , 2 : probable , marked attenuation", ". \n grade 0 ( structural ( architectural changes ) ) : \n 3 : unequivocal crypt destruction . \n  3 : unequivocal crypt destruction . \n", "grade 5 ( erosion or ulceration ) : \n 0 : no erosion , ulceration , or granulation tissue ,  1 : recovering epithelium + adjacent inflammation ,  2 : probable erosion focally stripped ,  3 : unequivocal erosion ,  4 : ulcer or granulation tissue.slides were analysed using light microscopy at magnifications 40x , 100x , 200x , and 400x and photographed at 200x ( olympus bx50 microscope , olympus c-5050 digital camera , and quickphoto pro imaging software ( promicra s.r.o . ,", "prague , czech republic ) ) . 0 : no erosion , ulceration , or granulation tissue , 1 : recovering epithelium + adjacent inflammation , 2 : probable erosion focally stripped , 3 : unequivocal erosion , 4 : ulcer or granulation tissue .", "after isolation , the colon was cleaned of intestinal content and freed of fat tissue , washed in dmem ( sigma aldrich , steinheim , germany ) , cut into 5  cm long pieces , and , while shaken at 100  rpm , incubated at 37c for 20 minutes in dmem with 25  mm edta .", "the tissue was then thoroughly washed in pbs buffer for at least 5 times , cut into 2  mm long strips , and digested in dmem containing 5  u / ml dnase ( roche , mannheim , germany ) and collagenase ii ( gibco , paisley , uk ) at 37c for 20  min . following this ,", "supernatant was removed , and the previous step was repeated until complete digestion of the tissue was achieved .", "the supernatant was then filtered through a 100  m sized filter ; dmem + 2% fbs ( sigma aldrich , steinheim , germany ) was added and centrifuged at 800  g for 10  min at room temperature .", "cells were resuspended in 5  ml of 40% percoll ( ge healthcare , uppsala , uk ) , overlaid on 4  ml of 80% percoll , and centrifuged at 900  g/20  min/4c .", "lymphocytes were isolated from the mesenteric lymph nodes ( mln ) and spleen by teasing apart the organs between the frosted ends of two microscopic slides .", "the cells were incubated with a mixture of pe anti - cd4 ( clone gk1.5 , exbio antibodies ) , fitc anti - b220 ( clone ra3 - 6b2 , obtained from the american type culture collection and conjugated with fitc using standard procedures ) , and apc anti - cd3 antibodies ( clone 145 - 2c11 , exbio antibodies ) and the other panel with percp anti - cd45 ( clone 30-f11 , bd biosciences ) , fitc anti - gr-1 ( clone rb6 - 8c5 , bd biosciences ) , and pe anti - f4/80 ( clone bm8 , stemcell technologies inc . ) or pe anti - cd4 ( clone mem-241 , exbio antibodies ) and percp anti - cd8 ( clone mem-31 , exbio antibodies ) antibodies .", "dead cells were excluded based on 7-aminoactinomycin d ( 7-aad ) ( applichem , darmstadt , germany ) staining .", "at least 20,000 live cells were collected by a bd facs canto ii cytometer ( facs canto ii , becton dickinson , san jose , ca , usa ) and analysed using the flowlogic software ( inivai technologies , mentone , australia ) . to assess hydrogen peroxide ( h2o2 ) and peroxynitrite ( onoo ) level , 10 lymphocytes isolated from mln and spleens were incubated for 30  min on + 4c with 10  m dichlorofluorescein diacetate ( dcf - da ) ( biomol , hamburg , germany ) , washed for 5  min at 400  g on + 4c , and analysed with the facs canto ii .", "following this , cells were stimulated with 100  nm phorbol 12-myristate 13-acetate ( pma , calbiochem , darmstadt , germany ) , incubated for 30  min , and analysed for the second time . at each measurement minimum of 10,000 target cells", "colon , mln , and spleen samples were isolated , snap frozen in liquid nitrogen , and stored at 80c till analysis .", "rna purity and concentration was assessed by nanophotometer p - class p330 - 30 ( implen , munich , germany ) . in order to purify rna from all polysaccharides , including dss ,", "an additional purification step using 8  m licl was performed , followed by the standard genomic dna purification step using deoxyribonuclease i kit ( sigma aldrich , st louis , mo , usa ) .", "one microgram of rna was used for cdna synthesis by high capacity cdna kit with rnase inhibitor ( applied biosystems , foster city , ca , usa ) .", "real - time pcr was performed on cfx96 system ( bio rad , singapore ) to assess relative expression of catalase ( cat ) , glutathione peroxidase 1 ( gpx1 ) , superoxide dismutase 1 ( sod1 ) , hif-1 , il-1 , il-2 , and il-6 .", "except for the primers for il-6 gene published by jeong et al . , all other primers were custom made using primer 3 software .", "messenger rna expression was determined using ssofast evagreen supermix ( bio rad , singapore ) .", "tissue powder was additionally homogenized in 100  mm phosphate buffer solution ( ph 7.0 ) containing 1  mm edta ( 1  :  10 , w / v ) using ultra turrax t10 homogenizer ( ika , staufen , germany ) while kept on ice .", "tissue homogenates were sonicated for 30 seconds on ice in three 10 seconds intervals and then centrifuged at 20,000  g for 15 minutes at 4c .", "cat , gpx , and sod enzyme activities were determined using a lambda 25 uv - vis spectrophotometer equipped with uv winlab 6.0 software package ( perkin elmer for the better , massachusetts , usa ) .", "catalase ( ec 1.11.1.6 ) activity was estimated spectrophotometrically using h2o2 as a substrate . the reaction mixture consisted of 10  mm h2o2 in 50  mm phosphate buffer ph 7.0 .", "changes in absorbance of the reaction mixture were measured at 240  nm during 2 minutes after the sample addition .", "one unit of activity corresponds to the loss of 1  mol of h2o2 per minute .", "cat activity was calculated using molar extinction coefficient (  = 0.04  mm  cm ) and expressed as u  mg protein . to assess glutathione peroxidase ( ec 1.11.1.9 ) activity a modified method described by wendel   using h2o2 as a substrate was employed .", "gpx activity was determined indirectly by measuring the rate of nadph oxidation to nadp+ , accompanied by a decrease in absorbance at 340  nm .", "the assay mixture consisted of 50  mm phosphate buffer with 0.4  mm edta and 1  mm sodium azide ( ph 7.0 ) , 0.12  mm nadph , 3.2 units of gr , 1  mm glutathione , and 0.0007% ( w / w ) h2o2 in a total volume of 1.55  ml .", "one unit catalyses the oxidation by h2o2 of 1.0  mole of reduced glutathione to oxidized glutathione per minute at ph 7.0 and 25c .", "gpx activity was calculated using molar extinction coefficient for nadph (  = 6.220  mm  cm ) and expressed as u  mg protein .", "superoxide dismutase ( ec 1.15.1.1 . ) activity was determined using cytochrome c ( 0.05  mm ) as an inhibitory molecule in pbs buffer saline with 0.1  mm edta in system xanthine ( 1  mm)/xanthine oxidase ( 50  u ) by flohe method . total soluble protein concentration in protein extracts was determined by bradford reagent ( sigma aldrich , steinheim , germany ) following manufacturer 's protocol and using bovine serum albumin as a standard .", "were tested by one - way anova or kruskal - wallis test followed by the holm - sidak / tukey or dunn 's post hoc multiple comparison procedure , respectively ( sigma plot 11.0 , sigmastat inc .", ", the student t - test and mann - whitney u statistic were used to compare the differences between the two groups in the case of normally distributed variables and variables that violated assumption of normality , respectively .", "spearman 's correlations were calculated where appropriate ( sigma plot 11.0 , sigmastat inc . ) .", "dai results were analysed by two - way anova and bonferroni post hoc test ( graphpad prism 5.0 , graphpad software , inc . , la jolla , ca , usa ) . a p value of < 0.05 was considered statistically significant for all procedures .", "all data are presented as mean values  standard error of mean ( s.e.m . ) ."], ["in order to determine the effects of hbo2 on the course of acute colitis , balb / c mice were exposed to 5% dss in the drinking water ad libitum and daily monitored for body weight , stool consistency , and occult / gross rectal bleeding to calculate dai . in this", "study the dss treatment induced substantial weight loss , rectal bleeding , loose stool , and colon shortening resulting in significantly higher dai compared to the control ( ctrl ) group , starting from day 3 until the end of the experiment ( p < 0.01 ; figure 1(a ) ) .", "mice that received dss and underwent hbo2 treatment ( dss + hbo2 group ) also presented with significantly higher dai compared to the ctrl group ; however , in this group of mice hbo2 treatment significantly reduced dai compared to the dss mice , starting from day 5 throughout day 8 ( days 5 and 8 p < 0.01 ; days 6 and 7 p < 0.05 ; figure 1(a ) ) .", "in addition , average colon length in the dss group of mice was significantly shorter ( 8.14  0.23  cm ) compared to the ctrl group ( 13.88  0.45  cm ; p < 0.0001 ) , while this effect was significantly ameliorated by hbo2 treatment in the dss + hbo2 group ( 11.34  0.38  cm ) compared to the dss group ( p = 0.0001 , figure 1(b ) ) .", "dss induced colitis resulted in significant body mass loss compared to ctrl group , irrespective of hbo2 treatment ( 11.89  0.03% and 8.24  0.01% in the dss and dss + hbo2 group , resp . ;", "ctrl and ctrl + hbo2 gained body mass during the experiment , 8.25  0.2% and 0.33  0.01% , respectively .", "histological assessment of the colon revealed severe inflammation and ulceration extending into the deep portions of the mucosa with loss of crypts and with increased number of lamina propria leukocytes in dss group of mice .", "we also found structural changes of mucosa in the colonic tissue of dss + hbo2 mice but with reduced infiltration of inflammatory cells and decreased crypt distortion .", "when compared to the dss + hbo2 group , the dss group had significantly higher total histological score as well as individual scores ( see modified geboes score in section 2.3 , figure 1(d ) , p < 0.001 ) , except for the intraepithelial neutrophil infiltration score ( p = 0.104 ) .", "distribution of inflammatory cells among the peripheral lymphoid organs , including gr-1 leukocytes ( monocytes and neutrophils ) , f4/80 leukocytes ( monocytes ) , cd3 t lymphocytes , and b220 b lymphocytes ( figure 2 ) , was assessed at the end of the experiment . in the mln", ", frequencies of gr-1 cells did not differ among the experimental groups , while dss induced a significant increase in f4/80 and decrease in cd3 cell frequencies ( p < 0.05 and p = 0.032 , resp . ; figure 2(b ) ) .", "these findings in the dss group were accompanied by a b - cell increase which was not statistically significant .", "hbo2 alone had no effect on the cell frequencies in mln of control mice , whereas it substantially ameliorated these changes in mice with dss - induced colitis ( dss + hbo2 group ) but without reaching statistical significance . in the spleen", ", gr-1 cell frequencies were significantly decreased in the dss group compared to ctrl ( p = 0.006 ) and ctrl + hbo2 groups ( p < 0.001 ; figure 2(a ) ) .", "hbo2 treatment reversed gr-1 cell frequencies to control values in the dss + hbo2 group ( p = 0.056 ; figure 2(a ) ) .", "in addition , the dss mice showed reduced frequencies of b220 lymphocytes compared to the ctrl group ( p = 0.016 ) , and hbo2 treatment abolished these effects in the dss + hbo2 group ( p = 0.034 ; figure 2(c ) ) .", "in addition , our study revealed that dss - induced immune responses in the mln and the spleen were significantly dampened by hbo2 treatment .", "frequency of cd4 cells among the colon lamina propria lymphocytes of dss and dss + hbo2 groups was significantly increased compared to the ctrl group ( p = 0.015 and p = 0.047 , resp . ) , while the frequency of cd8 cells was significantly increased only in the dss group when compared to the ctrl group ( p = 0.011 ; figure 3 ) .", "inflammatory conditions have been known to include the enhanced production of ros and other oxidative mediators that may affect transcriptional regulation via hif-1. to investigate the role of hif-1 in the regulation of the antioxidative response / capacity during dss - induced colitis and hbo2 treatment , hif-1 , cat , gpx1 , and sod1 mrna expressions were determined using quantitative pcr method .", "mrna expression was significantly changed by the hbo2 treatment and the inflammatory microenvironment in the gut mucosa .", "dss - induced colitis resulted in significant upregulation of hif-1 gene in colonic mucosa ( p = 0.008 for dss group compared to ctrl ) , and the hbo2 treatment further increased hif-1 mrna expression in the dss + hbo2 group ( p = 0.028 compared to ctrl ; figure 3(a ) ) .", "in addition , the activity of hif-1 protein was indirectly confirmed by measuring mrna expression of well - established hif-1 target genes , vegf and pgk1 .", "both genes showed strong positive correlation to the hif-1 mrna ( supplementary figure 1 ) ( see supplementary material available online at http://dx.doi.org/10.1155/2016/7141430 ) .", "there was also a tendency for upregulation of hif-1 gene in mln and spleens of the dss group and its downregulation via hbo2 in the dss + hbo2 group ( figure 4(a ) ) ; however , these changes did not reach statistical significance .", "inflammation during dss - induced colitis and the hbo2 treatment also induced significant changes in mrna expression of target antioxidative genes .", "dss - treated mice presented with significant downregulation of the cat gene in the colon compared to the ctrl group ( p = 0.031 ) , while there was a significant upregulation of cat gene in the spleen of the dss + hbo2 mice compared to the ctrl group ( p = 0.026 ; figure 4(b ) ) . in the colon , gpx1 mrna expression", "was increased in the dss ( p = 0.034 ) and the dss + hbo2 ( p = 0.003 ) group compared to ctrl group .", "the upregulation was even greater in mice with dss - induced colitis that underwent the hbo2 treatment ( dss + hbo2 group ; figure 4(b ) ) .", "sod1 mrna expression was significantly reduced in the colon of the dss + hbo2 group compared to ctrl ( p = 0.008 ) and ctrl + hbo2 ( p = 0.007 ) groups .", "similar changes in sod1 gene expression were also found in the mln of the dss + hbo2 group ( p = 0.025 compared to ctrl ; figure 4(b ) ) .", "to summarize , colitis resulted in gpx1 gene upregulation and cat gene downregulation , while hbo2 downregulated sod1 and further upregulated gpx1 in a tissue - specific manner . to examine the possible role of hif-1 in transcriptional control of antioxidative genes in the colon ,", "the results revealed a strong negative correlation between hif-1 and sod1 ( r = 0.651 , p = 0.001 ) and a positive correlation of hif-1 to the gpx1 gene ( r = 0.750 , p < 0.001 ) , while there was no significant correlation between the hif-1 and the cat gene in the colonic tissue ( figure 4(c ) ) .", "the early phase of inflammation is mediated by several proinflammatory mediators , which prompted us to assess how hbo2 treatment affects their production .", "we found that gut mucosa inflammation was accompanied with a significant increase in il-1 and il-6 gene expression . in the case of il-6 gene , this was significant for dss and dss + hbo2 groups compared to the ctrl + hbo2 group ( p = 0.024 and p = 0.021 , resp . ;", "furthermore , il-6 gene was significantly upregulated in the mln of the dss group ( p = 0.001 compared to ctrl ) , and hbo2 reduced its expression almost to control values in the dss + hbo2 group ( p = 0.016 compared to dss ; figure 5(a ) ) .", "il-1 mrna expression in the colonic mucosa was significantly increased during inflammation in dss group compared to ctrl ( p = 0.014 ) and ctrl + hbo2 groups ( p = 0.041 ) .", "il-1 mrna levels in the colon of the dss + hbo2 group did not significantly differ from the control groups , suggesting that hbo2 treatment blocked the increase of il-1 gene expression in the inflamed mucosa . \n", "il-2 gene was significantly upregulated in the mln of the dss group compared to the ctrl group ( p = 0.003 ) , and hbo2 treatment resulted in its significant downregulation in the dss + hbo2 group ( p = 0.032 ) .", "similarly , il-2 gene was significantly downregulated in the spleen of mice from the dss + hbo2 group compared to the ctrl group ( p = 0.025 ) .", "in addition , there was a strong positive correlation between hif-1 and il-6 gene ( r = 0.749 , p < 0.001 ; figure 5(b ) ) , while there was no correlation between hif-1 and il-1 or il-2 genes in the colonic tissue ( figure 5(b ) ) .", "in addition to their mrna expression , we also tested the enzymatic activity of antioxidative enzymes .", "we found that both the inflammation and the hbo2 treatment per se were able to change the activity of antioxidative enzymes in the colonic mucosa and the peripheral lymphoid organs ( mln and spleen ) . in spleen hbo2 treatment per", "se induced significant increase of sod activity ( p = 0.040 compared to ctrl ) .", "mice with dss - induced colitis presented with significantly increased activity of sod ( p = 0.012 compared to ctrl ) in the colon and reduced cat activity in mln and spleen ( p = 0.023 and p = 0.032 , resp . ) compared to ctrl group .", "treatment did not change sod activity in the inflamed colonic mucosa , which was comparable to the levels found in dss and ctrl + hbo2 groups ( significantly increased compared to ctrl , p = 0.010 ) . on the other hand , hbo2 treatment significantly increased cat ( p = 0.020 ) and gpx ( p = 0.001 ) activities in the spleens of the dss + hbo2 .", "immune cells at the site of inflammation and in the peripheral lymphoid organs are an important source of ros .", "therefore we assessed the basal levels of intracellular h2o2 and onoo and their production upon pma - induced activation in the lymphocytes isolated from mln and spleens of the mice from all experimental groups ( figure 6 ) .", "basal h2o2 and onoo production in the mln was not significantly different among the groups , except for the lymphocytes from the dss + hbo2 group which presented with a significant increase of h2o2 and onoo levels compared to the ctrl group ( p = 0.033 ) .", "pma stimulation resulted in increased intracellular h2o2 and onoo production , although statistically significant only for ctrl ( p = 0.031 ) and dss + hbo2 ( p = 0.012 ) groups . in the spleen ,", "hbo2 increased lymphocyte h2o2 and onoo production in ctrl + hbo2 and dss + hbo2 groups ( p = 0.004 and p = 0.007 compared to the ctrl ; and p = 0.005 and p = 0.009 compared to the dss group ) .", "their production after pma - induced activation was decreased in all experimental groups except the ctrl group ; however , this effect reached statistical significance only in the ctrl + hbo2 group ( p = 0.018 compared to unstimulated lymphocytes ) ."], ["in the present study , the experimental model of dss - induced colitis in balb / c mice was employed to explore the effects of hbo2 on the antioxidative enzymes , transcription factor hif-1 , and proinflammatory cytokine genes during colonic inflammation and their role in modulating the course of the disease via hbo2 treatment .", "the most important findings are that ( a ) hbo2 significantly reduces symptoms and severity of dss - induced colitis , as evidenced by clinical appearance , contraction of the immune cell expansion and mobilization , and reversal of il-1 , il-2 , and il-6 gene expression ; ( b ) hbo2 modulates the expression of antioxidative enzyme genes and enzyme activities during colitis ; and ( c ) hbo2 enhances hif-1 mrna expression in the inflamed colonic tissue which is in a strong correlation with gpx1 , sod1 , and il-6 mrna expression .", "several previous studies in animals and humans demonstrated the positive effects of hbo2 treatment in influencing the severity of colitis and reducing gut mucosa inflammation [ 27 , 34 ] ; however , data on the precise underlying mechanisms are scarce .", "considerably more data on the beneficial anti - inflammatory effects of hbo2 are available for other conditions such as septic shock , ischemia / reperfusion injuries , and atherogenesis , where the previous studies reported reduced proinflammatory cytokine expression , suppressed development of th cells , shrinking of spleen and lymph nodes , decreased responses to antigens , and reduced frequencies of circulating leukocytes [ 3542 ] .", "although this is the first animal study investigating the effects of hbo2 performed on dss - induced colitis in balb / c mice and correlating it with the immune cell frequencies , our results are in line with previous findings on the changes associated with dss - induced acute immune response , as well as on the effects of hbo2 on the antioxidative enzyme activities determined in other animal models , such as tnbs and acetic acid induced colitis in rats [ 27 , 43 , 44 ] . during colitis mice presented with decreased t and b cell frequencies in the spleen and reduced t cell frequencies in the mln , suggesting that lymphocytes are recruited from the peripheral lymphoid organs and probably migrate to the inflamed colonic mucosa .", "one element of the beneficial effect of hbo2 may be linked to normalized t and b cells frequency in the mln and spleen of mice with dss - induced colitis after hyperbaric treatment ( figures 1 and 2 ) . by measuring cd4 and cd8 lymphocyte in colon", "we confirm our hypothesis of t - cell recruitment from the peripheral lymphoid organs and their migration to the inflamed colonic mucosa , as well as immunomodulatory effect of hbo2 . in our model hbo2", "did not affect cell frequencies in the peripheral lymphoid organs of control mice , in contrast to previous findings where hbo2 treatment per se was able to change lymphocyte subset populations in the spleen .", "for a long time macrophages and neutrophils have been considered as immune cells exclusively producing proinflammatory cytokines , chemokines , and large amounts of ros / rns contributing to aggravated inflammation .", "we have found decreased spleen gr-1 cell frequencies during colitis and their normalization upon hbo2 treatment ( figure 2 ) .", "in addition , we showed increased mln frequencies of f4/80 cells in dss group , while hbo2 treatment reversed their frequencies almost to control values .", "these findings indicate that hbo2 can modulate distribution of phagocytes by retaining neutrophils in the spleen and instigating macrophage migration towards the site of inflammation , in agreement with previous findings describing inhibited neutrophil infiltration into the gut of mice with dss - induced colitis . furthermore , hbo2 treatment alone did not change the expression of proinflammatory cytokines in the colon , mln , or spleen of the control mice ; however , dss - induced colitis resulted in a significant il-1 and il-6 gene upregulation in the colonic tissue and il-2 gene upregulation in the mln ( figure 5 ) .", "consistent with previous studies , hbo2 treatment abolished these effects , further confirming its anti - inflammatory potential [ 4749 ] .", "several animal studies on the effects of hbo2 on the experimental colitis reported an increased antioxidative capacity and changes in antioxidative enzyme activity [ 24 , 25 ] .", "it has been proposed that an optimal hbo2 treatment could generate ros which would function primarily as intermediates in the antioxidative signalling pathways leading to increased expression of antioxidative enzymes , reduced inflammation , and ameliorated colitis symptoms but would not further damage the colonic tissue [ 50 , 51 ] .", ". showed that 24 hours after a two - hour hbo2 treatment at 2 bars in rats oxidative stress is not elevated , as evidenced by assessing ferric reducing antioxidant power ability of plasma ( frap ) and thiobarbituric acid reactive substances ( tbars ) level .", "in addition , a recent study also suggests that ros produced by nadph oxidase complex are important mediators inducing anti - inflammatory response in autoimmune diseases .", "data on the cat mrna level during dss - induced colitis and upon hbo2 treatment were not available prior to this study .", "we found that cat mrna expression is tissue and treatment specific ( figure 4 ) .", "colitis resulted in a significant downregulation of cat mrna expression in the colonic mucosa , and the hbo2 treatment induced its upregulation in the spleen of dss + hbo2 group of mice .", "these results were largely in accordance with our finding on enzymatic catalase activity that was decreased in all measured tissues in the dss group and reversed to control values in the spleen of dss + hbo2 group ( table 2 ) , as well as with a previous study demonstrating decreased catalase activity in colonic tissue upon dss treatment .", "this is also in line with a study on skin transplanted balb / c mice where hbo2 treatment increased catalase , gpx , and sod activity in the spleen .", "furthermore , upregulation of protein and mrna catalase levels 14 days after hbo2 treatment , but not after 7 days , was also observed in the ulcer tissue of patients with diabetic foot , indicating a time - course for the effect of hbo2 to prevail .", "we found that gpx1 mrna level was upregulated in the colon of dss treated mice , irrespective of the hbo2 treatment , and there were no significant differences in the gpx1 mrna expression in mln and spleen .", "in contrast to our findings on mrna expression , colon gpx enzyme activity was slightly reduced in dss + hbo2 group compared to other groups , which is consistent with previous results obtained in acetic acid induced colitis in rats receiving combined hbo2 and ozone treatment . however , other reports indicate decreased gpx and sod activity in the inflamed distal colon mucosa and the plasma of rats with acetic acid induced colitis , and hbo2 normalized gpx but not sod activity in the colon .", "the observed discrepancies in the results may be related to the differences in experimental models used among the studies . in addition , in our study hbo2 treatment induced enhanced gpx and sod activity in the spleen of dss mice which is in contrast to reduced sod1 mrna expression and might be explained by additional sod2 and sod3 function in regulation of antioxidative capacity .", "although intracellular h2o2 and onoo levels were slightly increased during inflammation and hbo2 treatment in the mln and hbo2 per se increased its level in spleen , impaired lymphocyte function was not observed . intensive research on the beneficial wound healing effects of hbo2 revealed its capacity to induce neovascularization , reduce oedema , decrease leukocyte adhesion , stimulate fibroblast expansion , and inhibit bacterial growth [ 14 , 57 ]", ". some of these processes are transcriptionally regulated by hif-1 , namely , the vascular endothelial growth factor ( vegf ) expression , regulatory t lymphocyte differentiation , and preservation of epithelial thigh junction integrity .", "in addition , previous studies employing conditional deletion of epithelial hif-1 or pharmacologic activation of hif-1 in a murine model of colitis demonstrated a protective role for hif-1 in colitis .", "it has also been shown that hif-1 increases expression of barrier - protective genes ( multidrug resistance gene-1 , intestinal trefoil factor , cd73 ) , decreases tnf mrna expression , and enhances antimicrobial activity by transcribing beta - defensin 1 . in the present study we found increased expression of hif-1 gene in inflamed colonic tissue , and", "hif-1 gene expression was changed ( upregulated ) by the inflammation while hbo2 treatment showed a tendency to reverse this increase .", "these data suggest involvement of different mechanisms controlling hif-1 gene expression at the site of inflammation ( colon ) and the peripheral lymphoid organs ( mln and spleen ) , responsible for the initiation of the immune response and the t / b - cell expansion and differentiation , respectively .", "we also demonstrated a strong positive correlation between hif-1 and gpx1 mrna levels in the colon ( figure 4(b ) ) .", "this is in line with in vitro studies where overexpressed hif-1 in colorectal cancer cells resulted in enhanced gpx1 expression through tgf-ri / smad2/erk1/2/hif-1 signalling cascade , suggesting transcriptional regulation of gpx1 by hif-1 . in the present study we found strong negative correlation between hif-1 and sod1 mrna expression", "this is in accordance with a previous study showing that docosahexaenoic acid downregulates sod1 gene transcription through an hre - mediated mechanism ( hre , hypoxia - response element ) , involving hif signalling in human cancer cells ; thus our results indicate similar mechanism involved in sod1 control in the murine colon mucosa in vivo during colitis and hbo2 .", "previous studies revealed that hif-1 mediated transcriptional regulation of different proinflammatory cytokines and growth factor genes are tissue and cell specific and include regulation trough alternative splicing , mrna stability , and interactions with other transcription factors like nf-b [ 6669 ] . in our study we found a strong correlation between hif-1 and il-6 mrna levels suggesting involvement of hif-1 in transcriptional regulation of il-6 gene during colonic inflammation and hbo2 . in conclusion , our results confirmed that hbo2 exerts an anti - inflammatory effect on dss - induced colitis in mice , and this effect at least involves hif-1 and antioxidative genes expression regulation ( as outlined in figure 7 ) .", "however , further studies are necessary to identify the cells that may contribute to or are influenced by the effects upon hbo2 treatment ."]]}
{"article_id": "PMC4870547", "article_text": ["sexuality in women is a complex issue with physiological , psychological , and cultural components .", "determination of sexual partner involvement , intimate relationships , and past abusive relationships is crucial in female sexual dysfunction ( fsd ) ( 1 , 2 ) .", "sexual dysfunction is more prevalent among women compared to men ( 43% vs. 31% ) ( 3 - 5 ) . in 1960 ,", "masters and johnson studied and reported both on healthy sexual function and sexual dysfunction for the first time .", "they described four phases of the human sexual response cycle , as follows : excitement , plateau , orgasm , and resolution .", "nowadays , the accepted classification of fsd consists of disturbances in desire , including hypoactive sexual desire disorders ( hsdd ) , sexual aversion disorders , sexual arousal disorders , orgasmic disorders , and sexual pain disorders , including dyspareunia and vaginismus ( 3 , 6 - 9 ) .", "fsd can further be classified as primary or secondary and persistent or situational ( 10 ) .", "intimate sexual function is one of the best predictors of quality of life , and sexual dysfunction causes many problems for couples .", "some researchers have found that the frequency of sexual relationship and sexual satisfaction are positively associated with marital satisfaction ( 11 , 12 ) . among other factors , illicit drug dependency changes", ". moreover , opioids act on a variety of neurotransmitters in the brain , including endorphins .", "furthermore , morphine has been found to inhibit sexual behavior in a dose - dependent manner through a complex pharmacological domain ( 13 , 14 ) .", ", most studies on drug abuse and sexual performance have focused on addicted populations , and have usually considered men .", "although many studies have looked at the frequency of sexual dysfunction in women , limited research has focused on the impact of addiction on spouse sexual function . to the best of our knowledge", ", few studies have been conducted on the effect of males opium dependency on their wives sexuality .", "thus , the present cross - sectional study aimed to assess the prevalence of fsd among women with addicted partners . we hypothesized that substance dependency in males would affect their wives sexual function .", "this cross - sectional controlled study was conducted on 340 women of reproductive age ( 25 - 50 years old ) selected through convenience sampling .", "the case group included 160 women whose husbands were opioid dependent . some of them were the wives of opioid - dependent males hospitalized in the addiction treatment units of teaching hospitals affiliated with the shiraz university of medical sciences , shiraz , iran .", "other women were encountered in centers where opioid - dependent males spouses were referred for group therapies .", "in contrast , the control group ( n = 160 ) included women whose spouses were not opioid dependent ; they were selected from the general population referring to the clinics of the shiraz university of medical sciences . after giving informed consent ,", "the participants were interviewed privately according to the dsm - iv - tr criteria by a senior female medical student who was one of the researchers and had been properly trained regarding the nature of the study .", "the interview focused on hsdd , sexual arousal disorder , orgasmic disorder , and sexual pain disorder .", "in addition , the subjects demographic information , such as age , level of education , marital status , and duration of marriage , was obtained using a self - constructed questionnaire designed by the authors . to include the women in the study", "the exclusion criteria of the study were a history of substance use disorders , gynecological disorders ( gyn ) , and chronic medical disease that has been shown to affect sexual functioning ( e.g. hypertension , diabetes mellitus , chronic kidney disease , cancer , spinal cord injury , lupus , fibromyalgia , chronic pain and chronic depression ) .", "it should be noted that the questionnaires were anonymous and participation in the study was voluntary .", "the chi - square test was used for categorical data , while the t - test was utilized for continuous data .", "the protocol for this study was approved by the ethics committee of the shiraz university of medical sciences , shiraz , iran .", "out of the 340 women who were selected , 5 in the case group and 15 in the control group were excluded due to missing data .", "the mean ages of the case and the control group were 36.35 and 33.2 years , respectively . in this study , statistical significance was defined as p < 0.05 .", "it should be noted that in terms of demographic characteristics , there was no significant difference between the case and the control group ( p > 0.05 ) , so , we could use further statistical methods on the data .", "table 2 shows the frequency of sexual dysfunction in the two study groups . according to the results ,", "approximately more than 50% of the participants had problems in at least one domain of sexual function . moreover ,", "the main fsd observed in the case women was hsdd ( 75.46.9% ) followed by sexual aversion disorder ( 73.46.2% ) , sexual arousal disorder ( 65 .40.9% ) , orgasmic disorder ( 59.37.1% ) , and sexual pain disorder ( 45.28.5% ) .", "furthermore , 75 out of the 155 women in the case group ( 46.9% ) and 16 out of the 145 women in the control group ( 10% ) had hsdd , and the difference between the two groups was statistically significant ( p < 0.05 ) .", "in addition , 73 women in the case group ( 46.2% ) and 20 in the control group ( 12.6% ) had sexual aversion disorder , with a significant difference between the two groups ( p < 0.05 ) . in this study ,", "this disorder was a significant problem affecting a considerable number of women in both the case and control groups ( table 2 ) .", "this high prevalence may have resulted from various causes . although studies have shown that many factors , such as age and level of education , affect sexual function ( 15 - 17 ) , the findings of the current study revealed no relationship between the demographic characteristics , that is , age , duration of marriage , and level of education , and sexual dysfunction . as can be seen in table 2 ,", "the frequency of hsdd and aversive sexual desire disorder was more common in the case group than the control group , and the difference was statistically significant ( p < 0.05 ) .", "the high frequency of hsdd among the addicts spouses exhibited in this study was in line with the study performed by noori et al .", "however , noori et al . included no control groups in their study , and consequently , it was not possible to compare the characteristics of the subjects with those of women whose husbands were not addicted .", "the findings of the present study showed that the addicts spouses suffered from hsdd significantly more than the control group .", "another finding was the higher frequency of orgasmic disorder in the case group ; however , this difference was not statistically significant . the higher rate of orgasmic disorder among the addicts spouses obtained in this research was in line with the study by noori et al .", "in contrast to our research , in one study conducted on 2626 women in iran in 2006 , 31.5% of the subjects ( 759 ) suffered from sexual disorder , and the most prevalent disorder was orgasmic disorder , followed by desire disorder ( 19 ) . in the dsm - iv - tr , sexual desire disorders are divided into two classes , as follows : 1 ) hsdd characterized by a deficiency or lack of sexual fantasies and desire for sexual activity and 2 ) sexual aversion disorder characterized by an aversion to and avoidance of genital contact with a sexual partner .", "no single cause of hsdd has been defined ; however , physiological , psychological , and sociocultural factors that contribute to female sexual desire may all be important in its development ( 20 , 21 ) .", "master and johnson s linear model of sexual response does not always work for females .", "some factors , such as emotional intimacy and relationship satisfaction , may change this model .", "studies have shown that the motivating factors for female sexual desire are very complicated . sexual desire and", "the presence or absence of orgasm could result from multiple cultural and environmental factors , as well as from interpersonal and intrapersonal distresses , and are greatly affected by emotional intimacy ( 2 ) . since addiction in the family could be the origin of many stressors and disputes , such stresses and interpersonal turmoil could have a decisive role in decreasing females sexual desire toward their addicted husbands .", "researchers have found that sexual response phases in women are a combination of mental and physical responses which overlap with one another ( 2 , 3 ) .", "in general , women have diverse reasons to initiate or agree to have sex with their partners .", "sexual motivation in females is far more complicated than just the presence or absence of sexual desire and is characterized by thinking or fantasizing about sex and longing to have sex .", "moreover , the decision to be sexual may originate from a conscious wish for emotional closeness or result from seduction or a suggestion from a partner .", "addicted couples often have conflicts over money and drugs ; so that love gradually flies out of the window , and most often these couples relationships end at a sad , bitter point ( 11 , 22 , 23 ) . hence , this kind of relationship is expected to have a negative impact on sexuality .", "of course , further studies are needed in order for better characterization and understanding of fsd epidemiology .", "one of the limitations of this study was the difficulty of gaining access to the case group sample and persuading them to cooperate with the researchers .", "in addition , when they agreed to take part in the study , the interviewer had to meet them out of their group", ". moreover , the subjects might have answered the questions conservatively due to the particular nature of the study subject , that is , sexual behavior , in iranian culture .", "another limitation of the study was a lack of control of other contributing factors , such as the economic status of the family and sexual disorders and duration of opioid dependency in addicted husbands , which are assumed to have an effect on their wives sexual dysfunctions ."], "abstract_text": ["<S> backgroundopiate abuse in males has significant effects on their sexual functions . </S>", "<S> in contrast , sexuality in females is a multidimensional issue that can strongly be affected by several factors in their partners . </S>", "<S> however , only a limited number of studies have assessed the role of males opioid dependency in their female partners sexual function.objectivesthe present study aimed to evaluate the effect of males opioid dependency on their wives sexual function compared to the sexual function of the females whose husbands were not opioid dependent.patients and methodsthis study included 340 women who were selected through convenience sampling and divided into a control ( females whose husbands were not opioid dependent ) and a case group ( women whose husbands were opioid dependent ) . </S>", "<S> the data were collected through an interview according to the dsm - iv - r criteria for female sexual dysfunctions by a senior female medical student who was one of the researchers . </S>", "<S> finally , the data were entered into the spss statistical software ( v. 15 ) and analyzed using the t - test and chi - square test.resultsaccording to the results , the frequency of hypoactive sexual desire disorder and sexual aversion disorder in the control group was significantly higher than that of the case group ( p < 0.05).conclusionsthe results showed that having an addicted husband could strongly affect some sexual domains in women . </S>", "<S> it could change the pattern of desire and motivation for sexual contact in females and alter their attitude toward the sexual relationship , thereby causing disturbances in the females normal sexual function . </S>"], "labels": null, "section_names": ["1. Background", "2. Objectives", "3. Patients and Methods", "4. Results", "5. Discussion"], "sections": [["sexuality in women is a complex issue with physiological , psychological , and cultural components .", "determination of sexual partner involvement , intimate relationships , and past abusive relationships is crucial in female sexual dysfunction ( fsd ) ( 1 , 2 ) .", "sexual dysfunction is more prevalent among women compared to men ( 43% vs. 31% ) ( 3 - 5 ) . in 1960 ,", "masters and johnson studied and reported both on healthy sexual function and sexual dysfunction for the first time .", "they described four phases of the human sexual response cycle , as follows : excitement , plateau , orgasm , and resolution .", "nowadays , the accepted classification of fsd consists of disturbances in desire , including hypoactive sexual desire disorders ( hsdd ) , sexual aversion disorders , sexual arousal disorders , orgasmic disorders , and sexual pain disorders , including dyspareunia and vaginismus ( 3 , 6 - 9 ) .", "fsd can further be classified as primary or secondary and persistent or situational ( 10 ) .", "intimate sexual function is one of the best predictors of quality of life , and sexual dysfunction causes many problems for couples .", "some researchers have found that the frequency of sexual relationship and sexual satisfaction are positively associated with marital satisfaction ( 11 , 12 ) . among other factors , illicit drug dependency changes", ". moreover , opioids act on a variety of neurotransmitters in the brain , including endorphins .", "furthermore , morphine has been found to inhibit sexual behavior in a dose - dependent manner through a complex pharmacological domain ( 13 , 14 ) ."], [", most studies on drug abuse and sexual performance have focused on addicted populations , and have usually considered men .", "although many studies have looked at the frequency of sexual dysfunction in women , limited research has focused on the impact of addiction on spouse sexual function . to the best of our knowledge", ", few studies have been conducted on the effect of males opium dependency on their wives sexuality .", "thus , the present cross - sectional study aimed to assess the prevalence of fsd among women with addicted partners . we hypothesized that substance dependency in males would affect their wives sexual function ."], ["this cross - sectional controlled study was conducted on 340 women of reproductive age ( 25 - 50 years old ) selected through convenience sampling .", "the case group included 160 women whose husbands were opioid dependent . some of them were the wives of opioid - dependent males hospitalized in the addiction treatment units of teaching hospitals affiliated with the shiraz university of medical sciences , shiraz , iran .", "other women were encountered in centers where opioid - dependent males spouses were referred for group therapies .", "in contrast , the control group ( n = 160 ) included women whose spouses were not opioid dependent ; they were selected from the general population referring to the clinics of the shiraz university of medical sciences . after giving informed consent ,", "the participants were interviewed privately according to the dsm - iv - tr criteria by a senior female medical student who was one of the researchers and had been properly trained regarding the nature of the study .", "the interview focused on hsdd , sexual arousal disorder , orgasmic disorder , and sexual pain disorder .", "in addition , the subjects demographic information , such as age , level of education , marital status , and duration of marriage , was obtained using a self - constructed questionnaire designed by the authors . to include the women in the study", "the exclusion criteria of the study were a history of substance use disorders , gynecological disorders ( gyn ) , and chronic medical disease that has been shown to affect sexual functioning ( e.g. hypertension , diabetes mellitus , chronic kidney disease , cancer , spinal cord injury , lupus , fibromyalgia , chronic pain and chronic depression ) .", "it should be noted that the questionnaires were anonymous and participation in the study was voluntary .", "the chi - square test was used for categorical data , while the t - test was utilized for continuous data .", "the protocol for this study was approved by the ethics committee of the shiraz university of medical sciences , shiraz , iran ."], ["out of the 340 women who were selected , 5 in the case group and 15 in the control group were excluded due to missing data .", "the mean ages of the case and the control group were 36.35 and 33.2 years , respectively . in this study , statistical significance was defined as p < 0.05 .", "it should be noted that in terms of demographic characteristics , there was no significant difference between the case and the control group ( p > 0.05 ) , so , we could use further statistical methods on the data .", "table 2 shows the frequency of sexual dysfunction in the two study groups . according to the results ,", "approximately more than 50% of the participants had problems in at least one domain of sexual function . moreover ,", "the main fsd observed in the case women was hsdd ( 75.46.9% ) followed by sexual aversion disorder ( 73.46.2% ) , sexual arousal disorder ( 65 .40.9% ) , orgasmic disorder ( 59.37.1% ) , and sexual pain disorder ( 45.28.5% ) .", "furthermore , 75 out of the 155 women in the case group ( 46.9% ) and 16 out of the 145 women in the control group ( 10% ) had hsdd , and the difference between the two groups was statistically significant ( p < 0.05 ) .", "in addition , 73 women in the case group ( 46.2% ) and 20 in the control group ( 12.6% ) had sexual aversion disorder , with a significant difference between the two groups ( p < 0.05 ) . in this study ,"], ["this disorder was a significant problem affecting a considerable number of women in both the case and control groups ( table 2 ) .", "this high prevalence may have resulted from various causes . although studies have shown that many factors , such as age and level of education , affect sexual function ( 15 - 17 ) , the findings of the current study revealed no relationship between the demographic characteristics , that is , age , duration of marriage , and level of education , and sexual dysfunction . as can be seen in table 2 ,", "the frequency of hsdd and aversive sexual desire disorder was more common in the case group than the control group , and the difference was statistically significant ( p < 0.05 ) .", "the high frequency of hsdd among the addicts spouses exhibited in this study was in line with the study performed by noori et al .", "however , noori et al . included no control groups in their study , and consequently , it was not possible to compare the characteristics of the subjects with those of women whose husbands were not addicted .", "the findings of the present study showed that the addicts spouses suffered from hsdd significantly more than the control group .", "another finding was the higher frequency of orgasmic disorder in the case group ; however , this difference was not statistically significant . the higher rate of orgasmic disorder among the addicts spouses obtained in this research was in line with the study by noori et al .", "in contrast to our research , in one study conducted on 2626 women in iran in 2006 , 31.5% of the subjects ( 759 ) suffered from sexual disorder , and the most prevalent disorder was orgasmic disorder , followed by desire disorder ( 19 ) . in the dsm - iv - tr , sexual desire disorders are divided into two classes , as follows : 1 ) hsdd characterized by a deficiency or lack of sexual fantasies and desire for sexual activity and 2 ) sexual aversion disorder characterized by an aversion to and avoidance of genital contact with a sexual partner .", "no single cause of hsdd has been defined ; however , physiological , psychological , and sociocultural factors that contribute to female sexual desire may all be important in its development ( 20 , 21 ) .", "master and johnson s linear model of sexual response does not always work for females .", "some factors , such as emotional intimacy and relationship satisfaction , may change this model .", "studies have shown that the motivating factors for female sexual desire are very complicated . sexual desire and", "the presence or absence of orgasm could result from multiple cultural and environmental factors , as well as from interpersonal and intrapersonal distresses , and are greatly affected by emotional intimacy ( 2 ) . since addiction in the family could be the origin of many stressors and disputes , such stresses and interpersonal turmoil could have a decisive role in decreasing females sexual desire toward their addicted husbands .", "researchers have found that sexual response phases in women are a combination of mental and physical responses which overlap with one another ( 2 , 3 ) .", "in general , women have diverse reasons to initiate or agree to have sex with their partners .", "sexual motivation in females is far more complicated than just the presence or absence of sexual desire and is characterized by thinking or fantasizing about sex and longing to have sex .", "moreover , the decision to be sexual may originate from a conscious wish for emotional closeness or result from seduction or a suggestion from a partner .", "addicted couples often have conflicts over money and drugs ; so that love gradually flies out of the window , and most often these couples relationships end at a sad , bitter point ( 11 , 22 , 23 ) . hence , this kind of relationship is expected to have a negative impact on sexuality .", "of course , further studies are needed in order for better characterization and understanding of fsd epidemiology .", "one of the limitations of this study was the difficulty of gaining access to the case group sample and persuading them to cooperate with the researchers .", "in addition , when they agreed to take part in the study , the interviewer had to meet them out of their group", ". moreover , the subjects might have answered the questions conservatively due to the particular nature of the study subject , that is , sexual behavior , in iranian culture .", "another limitation of the study was a lack of control of other contributing factors , such as the economic status of the family and sexual disorders and duration of opioid dependency in addicted husbands , which are assumed to have an effect on their wives sexual dysfunctions ."]]}
{"article_id": "PMC3015535", "article_text": ["laparoscopic surgery has gained widespread acceptance in a variety of procedures , ranging from gastric fundoplication to cholecystectomy .", "although laparoscopic appendectomy ( la ) is more expensive than is open appendectomy ( oa ) due to the costs of the disposable equipment , and can be more technically challenging in children , the overall cost of the operation has been shown to be similar to that of open appendectomy .", "the real cost savings of la are due to the shorter postoperative length of hospital stay ( los ) and the infrequent postoperative complications . the ultimate reduction in postoperative hospital costs to make la", "most cost - effective would be to perform the procedure as same - day surgery or so - called fast - track surgery . because it has been our observation that many children can be discharged within 24 hours after a laparoscopic appendectomy , we reviewed our records to determine its feasibility .", "a retrospective chart review was performed on 79 children who underwent a laparoscopic operation for the suspected diagnosis of acute appendicitis over a 3-year period between july 1997 and july 2000 .", "prior to the operation , if the diagnosis of acute appendicitis was unclear , the patients were evaluated with further laboratory and diagnostic testing , which included an abdominal computed tomography scan or ultrasound evaluation , or both of these , in select patients .", "laboratory information included white blood count and urinalysis , and depending on the circumstances , might also include a beta human chorionic gonadotropin and rapid streptococcal screen , as well as other tests as indicated .", "all patients underwent the operation while under general anesthesia , and all received a single dose of a broad - spectrum antibiotic preoperatively for wound infection prophylaxis .", "the standard laparoscopic operative technique was used , with either a 5-mm or 12-mm port introduced at the umbilicus after inducing pneumoperitoneum with carbon dioxide . a 0- or 30-degree laparoscopic telescope", "was then introduced through this port , and the abdominal and pelvic cavities were directly visualized with the laparoscope to inspect for other possible pathologies .", "next , two 5-mm ports were introduced under direct vision , one in the suprapubic region or the left lower quadrant and another in the right iliac fossa or in the right upper quadrant .", "the appendix was identified and grasped by its distal end to fully expose the entire organ and its attached structures .", "the appendiceal artery was isolated , ligated , and divided or cauterized . at that point ,", "a staple line was fired across the base of the appendix by using an endoscopic stapler , or endoscopic loops were used to isolate the base of the appendix , and it was divided .", "if the appendix was particularly enlarged or friable , it was removed from the abdomen through the umbilical incision with the use of an endoscopic bag .", "all ports were removed under direct visualization , and the fascia was reapproximated to prevent future incisional hernia occurrences .", "if any evidence of localized or generalized peritonitis was visualized during the operation , patients were sent home on oral antibiotics , usually a 5-day course of amoxicillin plus clavulanate .", "if the appendix was perforated prior to removal , patients were placed on triple antibiotic coverage intravenously , which included ampicillin , gentamicin , and clindamycin for 5 days while in the hospital .", "if any evidence of abscess formation or peritoneal soilage was noted , the abdominal cavity was vigorously lavaged with normal saline .", "postoperative analgesia was obtained usually with ketorolac tromethamine and acetaminophen . to be discharged home , patients had to be tolerating oral liquids , be afebrile , and free of nausea and vomiting .", "seventy - nine children ( 44 boys and 35 girls ) between 2 to 17 years of age ( mean , 11 years ) underwent la . in 4 ( 5% ) children , all with perforated appendicitis , the la was converted to an open appendectomy , secondary to technical difficulties in completing the operation laparoscopically . at operation , 51 ( 64.5% )", "had acute appendicitis , 22 ( 27.8% ) had perforated appendicitis , 4 ( 5% ) had ruptured ovarian cysts , and 2 ( 2.5% ) had no obvious pathology .", "total los for all 79 patients was a median of 58 hours , and median postoperative los was 35 hours .", "complications included wound infection , 2 ( 2.5% ) ; abdominal abscess , 4 ( 5% ) ; drug rash , 2 ; and epididymoorchitis , 1 .", "all but one complication ( drug rash , 1 ) occurred in the perforated group . in the 57 ( 72% ) children without perforated appendicitis , the total los was a median of 42 hours , while median postoperative los was only 28 hours .", "thirty - two ( 56% ) of the children without perforated appendicitis went home in 24 hours following la .", "no significant morbidity occurred in the nonperforated group ( drug rash , 1 ; fever > 24 hrs , 3 ) ; and no readmissions or reoperations were necessary on follow - up .", "although most appendectomies , especially in children , are not done laparoscopically , many studies have shown that laparoscopic appendectomy ( la ) is at least as good as open appendectomy ( oa ) , with several benefits , including less postoperative pain and shorter lengths of stay ( los ) .", "the disadvantages of la , which include increased operative time and increased cost of equipment , are easily offset by the decreased postoperative recovery time and the apparent decreased incidence of postoperative complications . additionally , it has been suggested that with increasing operative experience the operative time required for la will decrease significantly .", "lastly , it has been suggested that even if patients are not discharged from the hospital soon enough after la to make a significant difference between the cost of la versus oa , la has a much shorter recovery time and returns patients to a productive lifestyle sooner , thus justifying la .", "although most children are not working , the care - givers or parents can return to work sooner , when their child goes back to school .", "adult series have documented a decreased incidence of postoperative complications and a decreased incidence of wound infection after la .", ", in our series , 2 patients had wound infection and 4 patients developed abscesses , which is a similar complication rate to that in other series . in all instances , these complications arose in the perforated group .", "although perforation was at first considered a contraindication for la , it has now been utilized successfully in the management of acute appendicitis as well as perforated appendicitis .", "it has been suggested that thorough lavage of the abdominal cavity after appendectomy can help to decrease the incidence of abscess formation , and this is a practice that we utilize and is facilitated by the use of laparoscopy .", "certainly , the laparoscopic approach facilitates the complete irrigation of the abdominal cavity and identification of all loculated collections .", "an additional benefit of laparoscopic surgery is that it leads to greater accuracy of diagnosis , especially in teenage female patients with suspected appendicitis . in cases such as obesity and mental retardation , the diagnosis of appendicitis", "laparoscopy can be used to delineate the source of abdominal pain when the diagnosis of appendicitis is suspected but not certain . in 4 of our patients with the presumed diagnosis of appendicitis ,", "intraoperative visualization revealed normal appendices , and ruptured ovarian cysts were identified as the source of their pain . obviously , all four patients were female .", "others have also found an increased preponderance of unclear diagnoses in the female population . in our patients ,", "the appendix was always removed at the time of operation , despite the fact that occasionally ( in 4 patients ) the gross appearance of the appendix was normal . in the past", ", it has been unclear whether it would be of any benefit to the patient to remove a healthy organ , but it has been argued that with advances in laparoscopy and its proven benefit , there is no justification for leaving a visually normal appendix in place . additionally , microscopic evidence of early appendicitis is occasionally seen . in our study , it was noted that 2 visually normal appendices were found to have microscopic evidence of appendicitis .", "outpatient surgery has been widely accepted in a variety of procedures , and many are done laparoscopically . the idea that an appendectomy can be done in the pediatric patient as outpatient surgery is not a new one . in 1993 ,", "ramesh and gallard suggested early discharge after open appendectomy , even within 24 hours . in another study ,", "velhote et al also found that most children could be sent home within 24 hours after appendectomy . in that study", ", the appendectomy was performed through a standard gridiron incision of 2 cm or less .", "brosseuk and bathe suggested laparoscopic appendectomy as outpatient surgery in 1999 , and they advocated early discharge in both perforated and simple appendicitis .", "the fact that all major complications in our study occurred in perforated cases would argue against these cases being performed as same - day surgery , although others have disputed this approach . in our treatment protocol", ", perforated appendicitis would not be placed on a fast - track surgery list because these patients usually receive inpatient intravenous antibiotic therapy for 72 hours .", "we suggest that laparoscopic appendectomy is a safe and effective treatment in the pediatric population , and that in cases of nonperforated appendicitis this may be performed as a fast - track or short - stay procedure .", "we believe that this not only is more convenient for patients and their families , but it also adds to the overall cost - effectiveness of laparoscopic appendectomy .", "our findings are based on a retrospective review of our charts over the past 3 years , and this is an obvious criticism of the study . in no instance was it prospectively decided that the child would be discharged within 24 hours .", "in fact , a small number of children met exclusion criteria due to delays in discharge not related to their medical condition .", "we made our best attempt to identify when the order was given for the child to be released rather than the actual time that the child left the hospital .", "however , the documentation was occasionally unclear , and these children were excluded if it could not be ascertained exactly when the order was given or when they left the hospital .", "we believe that in cases of simple appendicitis , there is no reason not to dismiss a child within 24 hours if the above - mentioned discharge criteria are met .", "we anticipate that in the future , a greater percentage of our pediatric patients will be discharged within 24 hours , and we feel that in cases of simple appendicitis , laparoscopic appendectomy can be done as fast - track or short - stay surgery .", "it may be safely performed as fast - track or same - day surgery , in select children without perforated appendicitis , with a postoperative stay of 24 hours in the majority of such patients ."], "abstract_text": ["<S> background : laparoscopic surgery has reduced the length of hospital stay for common operations like cholecystectomy , gastric fundoplication , and appendectomy . </S>", "<S> we have noticed a reduction in length of hospital stay for children undergoing laparoscopic appendectomy . </S>", "<S> we , therefore , looked at our data to assess whether laparoscopic appendectomy in children could be performed as fast - track or same - day surgery ( 24-hour postoperative stay).methods : we performed a retrospective review of the records of all children who underwent laparoscopic appendectomy for suspected appendicitis during a 3-year period ( 7/97 to 7/00).results : laparoscopic appendectomy was performed in 79 children ( 44 boys and 35 girls ) , between 2 to 17 years of age ( mean , 11 years ) . in 4 ( 5% ) children with perforated appendicitis </S>", "<S> , the laparoscopic appendectomy was converted to an open appendectomy . at operation , 51 ( 64.5% ) had acute appendicitis , 22 ( 27.8% ) had perforated appendicitis , 4 ( 5% ) had ruptured ovarian cysts , and 2 ( 2.5% ) had no pathology . </S>", "<S> the median operative time was 54 minutes . </S>", "<S> total length of stay for all 79 patients was a median of 58 hours , and median postoperative los was 35 hours . </S>", "<S> complications included wound infection ( 2 ) , abdominal abscess ( 4 ) , drug rash ( 2 ) , and epididymo - orchitis ( 1 ) . in 57 ( 72% ) children without perforated appendicitis , the total length of hospital stay was a median of 42 hours , while median postoperative length of stay was only 28 hours . </S>", "<S> thirty - two ( 56% ) children went home in < 24 hours following laparoscopic appendectomy . </S>", "<S> no significant morbidity was noted in the nonperforated group ( drug rash,1 ; fever > 24 hrs , 3 ) ; and no readmissions or reoperations were necessary on follow-up.conclusion:laparoscopic appendectomy is safe and effective for treating children with appendicitis . </S>", "<S> laparoscopic appendectomy may be safely performed as fast - track or same - day surgery , in select children without perforated appendicitis , with a postoperative stay of 24 hours . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["laparoscopic surgery has gained widespread acceptance in a variety of procedures , ranging from gastric fundoplication to cholecystectomy .", "although laparoscopic appendectomy ( la ) is more expensive than is open appendectomy ( oa ) due to the costs of the disposable equipment , and can be more technically challenging in children , the overall cost of the operation has been shown to be similar to that of open appendectomy .", "the real cost savings of la are due to the shorter postoperative length of hospital stay ( los ) and the infrequent postoperative complications . the ultimate reduction in postoperative hospital costs to make la", "most cost - effective would be to perform the procedure as same - day surgery or so - called fast - track surgery . because it has been our observation that many children can be discharged within 24 hours after a laparoscopic appendectomy , we reviewed our records to determine its feasibility ."], ["a retrospective chart review was performed on 79 children who underwent a laparoscopic operation for the suspected diagnosis of acute appendicitis over a 3-year period between july 1997 and july 2000 .", "prior to the operation , if the diagnosis of acute appendicitis was unclear , the patients were evaluated with further laboratory and diagnostic testing , which included an abdominal computed tomography scan or ultrasound evaluation , or both of these , in select patients .", "laboratory information included white blood count and urinalysis , and depending on the circumstances , might also include a beta human chorionic gonadotropin and rapid streptococcal screen , as well as other tests as indicated .", "all patients underwent the operation while under general anesthesia , and all received a single dose of a broad - spectrum antibiotic preoperatively for wound infection prophylaxis .", "the standard laparoscopic operative technique was used , with either a 5-mm or 12-mm port introduced at the umbilicus after inducing pneumoperitoneum with carbon dioxide . a 0- or 30-degree laparoscopic telescope", "was then introduced through this port , and the abdominal and pelvic cavities were directly visualized with the laparoscope to inspect for other possible pathologies .", "next , two 5-mm ports were introduced under direct vision , one in the suprapubic region or the left lower quadrant and another in the right iliac fossa or in the right upper quadrant .", "the appendix was identified and grasped by its distal end to fully expose the entire organ and its attached structures .", "the appendiceal artery was isolated , ligated , and divided or cauterized . at that point ,", "a staple line was fired across the base of the appendix by using an endoscopic stapler , or endoscopic loops were used to isolate the base of the appendix , and it was divided .", "if the appendix was particularly enlarged or friable , it was removed from the abdomen through the umbilical incision with the use of an endoscopic bag .", "all ports were removed under direct visualization , and the fascia was reapproximated to prevent future incisional hernia occurrences .", "if any evidence of localized or generalized peritonitis was visualized during the operation , patients were sent home on oral antibiotics , usually a 5-day course of amoxicillin plus clavulanate .", "if the appendix was perforated prior to removal , patients were placed on triple antibiotic coverage intravenously , which included ampicillin , gentamicin , and clindamycin for 5 days while in the hospital .", "if any evidence of abscess formation or peritoneal soilage was noted , the abdominal cavity was vigorously lavaged with normal saline .", "postoperative analgesia was obtained usually with ketorolac tromethamine and acetaminophen . to be discharged home , patients had to be tolerating oral liquids , be afebrile , and free of nausea and vomiting ."], ["seventy - nine children ( 44 boys and 35 girls ) between 2 to 17 years of age ( mean , 11 years ) underwent la . in 4 ( 5% ) children , all with perforated appendicitis , the la was converted to an open appendectomy , secondary to technical difficulties in completing the operation laparoscopically . at operation , 51 ( 64.5% )", "had acute appendicitis , 22 ( 27.8% ) had perforated appendicitis , 4 ( 5% ) had ruptured ovarian cysts , and 2 ( 2.5% ) had no obvious pathology .", "total los for all 79 patients was a median of 58 hours , and median postoperative los was 35 hours .", "complications included wound infection , 2 ( 2.5% ) ; abdominal abscess , 4 ( 5% ) ; drug rash , 2 ; and epididymoorchitis , 1 .", "all but one complication ( drug rash , 1 ) occurred in the perforated group . in the 57 ( 72% ) children without perforated appendicitis , the total los was a median of 42 hours , while median postoperative los was only 28 hours .", "thirty - two ( 56% ) of the children without perforated appendicitis went home in 24 hours following la .", "no significant morbidity occurred in the nonperforated group ( drug rash , 1 ; fever > 24 hrs , 3 ) ; and no readmissions or reoperations were necessary on follow - up ."], ["although most appendectomies , especially in children , are not done laparoscopically , many studies have shown that laparoscopic appendectomy ( la ) is at least as good as open appendectomy ( oa ) , with several benefits , including less postoperative pain and shorter lengths of stay ( los ) .", "the disadvantages of la , which include increased operative time and increased cost of equipment , are easily offset by the decreased postoperative recovery time and the apparent decreased incidence of postoperative complications . additionally , it has been suggested that with increasing operative experience the operative time required for la will decrease significantly .", "lastly , it has been suggested that even if patients are not discharged from the hospital soon enough after la to make a significant difference between the cost of la versus oa , la has a much shorter recovery time and returns patients to a productive lifestyle sooner , thus justifying la .", "although most children are not working , the care - givers or parents can return to work sooner , when their child goes back to school .", "adult series have documented a decreased incidence of postoperative complications and a decreased incidence of wound infection after la .", ", in our series , 2 patients had wound infection and 4 patients developed abscesses , which is a similar complication rate to that in other series . in all instances , these complications arose in the perforated group .", "although perforation was at first considered a contraindication for la , it has now been utilized successfully in the management of acute appendicitis as well as perforated appendicitis .", "it has been suggested that thorough lavage of the abdominal cavity after appendectomy can help to decrease the incidence of abscess formation , and this is a practice that we utilize and is facilitated by the use of laparoscopy .", "certainly , the laparoscopic approach facilitates the complete irrigation of the abdominal cavity and identification of all loculated collections .", "an additional benefit of laparoscopic surgery is that it leads to greater accuracy of diagnosis , especially in teenage female patients with suspected appendicitis . in cases such as obesity and mental retardation , the diagnosis of appendicitis", "laparoscopy can be used to delineate the source of abdominal pain when the diagnosis of appendicitis is suspected but not certain . in 4 of our patients with the presumed diagnosis of appendicitis ,", "intraoperative visualization revealed normal appendices , and ruptured ovarian cysts were identified as the source of their pain . obviously , all four patients were female .", "others have also found an increased preponderance of unclear diagnoses in the female population . in our patients ,", "the appendix was always removed at the time of operation , despite the fact that occasionally ( in 4 patients ) the gross appearance of the appendix was normal . in the past", ", it has been unclear whether it would be of any benefit to the patient to remove a healthy organ , but it has been argued that with advances in laparoscopy and its proven benefit , there is no justification for leaving a visually normal appendix in place . additionally , microscopic evidence of early appendicitis is occasionally seen . in our study , it was noted that 2 visually normal appendices were found to have microscopic evidence of appendicitis .", "outpatient surgery has been widely accepted in a variety of procedures , and many are done laparoscopically . the idea that an appendectomy can be done in the pediatric patient as outpatient surgery is not a new one . in 1993 ,", "ramesh and gallard suggested early discharge after open appendectomy , even within 24 hours . in another study ,", "velhote et al also found that most children could be sent home within 24 hours after appendectomy . in that study", ", the appendectomy was performed through a standard gridiron incision of 2 cm or less .", "brosseuk and bathe suggested laparoscopic appendectomy as outpatient surgery in 1999 , and they advocated early discharge in both perforated and simple appendicitis .", "the fact that all major complications in our study occurred in perforated cases would argue against these cases being performed as same - day surgery , although others have disputed this approach . in our treatment protocol", ", perforated appendicitis would not be placed on a fast - track surgery list because these patients usually receive inpatient intravenous antibiotic therapy for 72 hours .", "we suggest that laparoscopic appendectomy is a safe and effective treatment in the pediatric population , and that in cases of nonperforated appendicitis this may be performed as a fast - track or short - stay procedure .", "we believe that this not only is more convenient for patients and their families , but it also adds to the overall cost - effectiveness of laparoscopic appendectomy .", "our findings are based on a retrospective review of our charts over the past 3 years , and this is an obvious criticism of the study . in no instance was it prospectively decided that the child would be discharged within 24 hours .", "in fact , a small number of children met exclusion criteria due to delays in discharge not related to their medical condition .", "we made our best attempt to identify when the order was given for the child to be released rather than the actual time that the child left the hospital .", "however , the documentation was occasionally unclear , and these children were excluded if it could not be ascertained exactly when the order was given or when they left the hospital .", "we believe that in cases of simple appendicitis , there is no reason not to dismiss a child within 24 hours if the above - mentioned discharge criteria are met .", "we anticipate that in the future , a greater percentage of our pediatric patients will be discharged within 24 hours , and we feel that in cases of simple appendicitis , laparoscopic appendectomy can be done as fast - track or short - stay surgery ."], ["it may be safely performed as fast - track or same - day surgery , in select children without perforated appendicitis , with a postoperative stay of 24 hours in the majority of such patients ."]]}
{"article_id": "PMC138946", "article_text": ["a major effort in the nucleocytoplasmic transport field has been directed towards the analysis of all members of the importin  family , in particular with the aim of identifying specific transport cargoes .", "this has led to the characterization of a large number of related proteins ( both importins and exportins ) in all eukaryotic species analyzed .", "new members have primarily been identified by sequence homology or biochemically via their interaction with the small gtpase ran .", "strikingly , the roles of many of these receptors is conserved from yeast to humans .", "there are 14 putative members of the importin  family in the completed saccharomyces cerevisiae genome , nine of which have been shown to function as importins and four as exportins .", "the genes encoding yeast transport receptors are dispersed throughout the genome and none of them contains introns .", "little is known about the gross structure of the genes encoding these nuclear transport receptors , and our knowledge of the chromosomal localization or the organization of the individual genes encoding members of this family is very poor .", "the relative molecular masses of members of importin--like proteins vary between 90 kda and 130 kda , but all are characterized by an acidic isoelectric point .", "the overall sequence similarity between various transport receptors is low ( less than 20% amino acid identity ) and , in many cases , is restricted to the amino - terminal domain .", "work mainly on importin  has demonstrated that these receptors bind rangtp via the amino - terminal domain and cargo via the carboxy - terminal domain . to permit shuttling through the nuclear pore complex ( npc )", ", transport receptors also contain one or multiple binding domains for components of the npcs , called nucleoporins .", "truncation studies using importin  indicate that the binding site for nucleoporins containing fxfg repeats ( in the single - letter amino acid code , where x is , in many cases , a small polar residue or glycine ) is located in an amino - terminal / central region of importin  ( residues 152 - 352 ) .", "this was recently confirmed in the crystal structure of an amino - terminal fragment of importin  in a complex with five fxfg nucleoporin repeats .", "additional information comes from the crystal structures of importin  and of transportin 1 ( also known as karyopherin 2 ) , which were solved either in a complex with rangtp , with a cargo , or in the free form .", "overall , the structures of importin--like receptors are characterized by a very similar series of helical heat repeats ( 19 in importin  and 18 in transportin 1 ; figure 1 ) .", "heat repeats are approximately 40 residues in length and are found in many eukaryotic proteins such as the pr65/a subunit of protein phosphatase 2a .", "the fundamental repeat unit is a right - handed superhelical structure consisting of a hairpin made up of two  helices , named a and b , separated by a sharp turn .", "each hairpin is connected to the next by a linker region . in transportin 1 ,", "almost all linkers contain a third helix , but there are very few linker helices in importin . in both receptors , one turn is extended into a long acidic loop , which has been suggested to be important for rangtp - mediated cargo release .", "full - length importin  complexed with the importin--binding domain of importin  ( ibb ) forms a snail - like superhelical structure wrapping tightly around the ibb domain .", "the structure of the uncomplexed amino terminus of importin  reveals a different superhelical architecture with a much steeper helical pitch than the cargo - bound or rangtp - bound forms .", "this suggests that importin  undergoes twisted conformational changes in its heat - repeat helix stacking , which could be essential for the regulation of cargo binding and release and/or for protein interactions during the translocation through the npc .", "although the sequence homology is limited , it is expected that exportins will fold in a similar way to the reported importin structures .", "it still remains unclear , however , why rangtp is required for binding of cargo to exportins but causes cargo dissociation from importins .", "structure of importin . ( a ) importin  is composed of 19 helical - repeat motifs ( heat repeats ) .", "each consists of an a and a b helix connected by a short turn , which in heat-8 is replaced by an acidic loop critical for the regulation of substrate binding and release .", "the heat repeats 1 - 8 are required for high - affinity binding to rangtp .", "the importin--binding ( ibb ) domain of importin  interacts mainly with residues located in repeats 7 - 19 of importin  .", "the binding site for nucleoporins of the npc is located between residues 152 and 352 , corresponding to repeats 4 - 8 . on the basis of the crystal structure ,", "the a helices of heat repeats 5 and 6 and a region between heat repeats 6 and 7 are thought to be critical for recognition of the fxfg motif .", "( b ) structure of importin  bound to the ibb domain of importin  ( adapted from ) .", "importin  ( yellow ) forms a superhelical structure that wraps like a snail around the ibb domain ( blue ) .", "the 19 heat repeats share a common core of 21 residues , comprising the a helix with about three turns and the b helix with about four turns .", "the helices critical for the interaction with fxfg - repeat nucleoporins   are in green .", "note the acidic loop , which contacts both rangtp and the ibb domain ( white arrow ) .", "a major function of transport factors of the importin  family is to mediate the transport between the nucleus and cytoplasm of macromolecules that contain nuclear import or export signals . to this end ,", ", they can be found in the nucleus , at the npc or in the cytoplasm . at present , very little is known about the tissue distribution of this family of proteins .", "all members have the ability to recognize and bind specific cargoes , either directly or via adaptor molecules , to bind rangtp and to interact with nucleoporins at the npc .", "interactions between the proteins of the importin  family and nucleoporin repeats have been shown both in vitro   and in vivo .", "these interactions contribute to the import or export of importin  family members and their cargoes through the central transporter of the npc .", "import and export are multistep processes that are initiated by the recognition of nuclear localization signals ( nlss ) and nuclear export signals ( ness ) .", "the most thoroughly studied import signals are the ' classical ' and the bipartite nlss , first identified in sv40 large t antigen and nucleoplasmin , respectively .", "their transport is mediated by importin  , the first - characterized member of this protein family .", "additional importin--dependent adaptors in vertebrates include snurportin 1 ( involved in import of m3g - capped small nuclear ribonucleoproteins , snrnps ) and xrip , ( involved in the import of replication protein a , rpa ) .", "importin  can also form a complex with another importin--like factor , importin 7 , in order to transport the linker histone h1 into the nucleus .", "in addition , importin  is able to interact directly with a large variety of different cargoes , including the t - cell protein tyrosine phosphatase , the human immunodeficency virus ( hiv ) tat and rev proteins , human t - cell leukemia virus rex protein , ribosomal proteins l23a , s7 , and l5 , cyclin b1 , smad   and the parathyroid - hormone - related protein .", "table 1 details known importins , exportins and their cargoes and adaptors . proteins in the importin--like family and their cargoes abbreviations : h.s .", ", homo sapiens ; s.c . , saccharomyces cerevisiae ; x.l . , xenopus laevis .", "genbank accession numbers are given in square brackets for each protein , and alternative names are in parenthesis .", "the import of ribosomal proteins seems to rely on at least partially redundant mechanisms . in yeast ,", "/ yrb4p has been shown to be an important mediator of ribosomal - protein import , but the related protein kap121p", "/ pse1p can functionally substitute for kap123p in vivo . in mammalian cells , at least four importin--like transport factors are able to mediate import of ribosomal proteins .", "interestingly , both the -like importin receptor binding ( bib ) domain and some ribosomal proteins can be imported by any of the four receptors importin  , transportin 1 , importin 5 or importin 7 .", "the yeast importin kap104p mediates nuclear import of the mrna - binding proteins nab2p and nab4p .", "its vertebrate homolog , transportin 1 ( kap2 ) , also mediates import of the rna - binding proteins hnrnp a1 and hnrnp f , but also of ribosomal proteins .", "mammalian transportin - sr mediates nuclear import of a group of abundant arginine / serine - rich proteins , which are essential pre - mrna splicing factors . in yeast ,", "the tata - binding protein ( tbp ) is imported into the nucleus by kap114p .", "exportin 1 ( crm1p , xpo1p ) has been identified in both yeast and higher eukaryotes as an export receptor for leucine - rich nes - containing proteins .", "human exportin 1 has been found to export protein kinase inhibitor  ( pki ) and hiv rev , ib , snurportin 1 , htlv rex , the small nuclear rna , cyclin b1   and the transcription factor nf - at4 .", "targets for exportin in s. cerevisiae include the transcription factors yap1p , and ace2p , the mitogen - activated protein kinase hog1p , and the heat - shock protein ssb1p .", "msn5p was first identified as a yeast exportin that exports the phosphorylated form of the transcription factor pho4p .", "interestingly , msn5p was recently shown to function as an import receptor for the trimeric rpa , suggesting that individual members of the importin--like protein family can function both as import and export receptors .", "the first cellular rna export receptor to be discovered , named exportin - t in higher eukaryotes or los1p in yeast , mediates nuclear export of trnas . many of the nuclear transport factors identified in s. cerevisiae are not essential for viability , even though they transport essential cargoes .", "this phenomenon can be explained by the fact that cargoes can use alternative transport pathways .", "this is probably best exemplified in the import pathway of ribosomal proteins , which is mediated by at least four different import receptors ( see above ) .", "nuclear transport mediated by importins and exportins is strongly directional in vivo : importins bind their cargo in the cytoplasm and transfer it to the nucleus , whereas exportins interact with their substrates in the nucleus and mediate their export to the cytoplasm ( figure 2 ) .", "protein translocation through the npc is thought to occur by an essentially similar mechanism for all importin--related receptors , except for the fact that , in some situations , additional adaptors are required to bridge the cargo - receptor interaction .", "this is mediated by importin  together with its adaptor importin  , which binds both the nls - containing cargo and importin  in the cytoplasm . after a trimeric importin -importin -nls complex is formed , importin  mediates docking at the npc .", "in the presence of rangdp and free gtp , this trimeric complex translocates through the npc .", "translocation is terminated by binding of rangtp to importin  , which releases the complex from the npc and dissociates importin  from importin  ( reviewed in ) .", "free importin  has a lower affinity for the nls cargo , and release from importin  is therefore believed to trigger release of the nls cargo as well .", "thereafter , the importin -rangtp complex , and importin  bound to its exportin ( cas ) and rangtp , are re - exported to the cytoplasm for another round of import . a schematic representation of nuclear import and export cycles through the npc .", "the cargo - loaded importin translocates through the npc into the nucleus , where the cargo is dissociated from the importin by binding of importin to rangtp .", "the importin - rangtp complex recycles back to the cytoplasm , where rangtp hydrolysis is stimulated by rangap and ranbp1 ; this frees the importin for the next round of import .", "exportins bind their export substrates in the nucleus , forming a trimeric cargo - exportin - rangtp complex .", "this complex is exported from the nucleus and dissociated in the cytoplasm by hydrolysis of rangtp to rangdp and inorganic phosphate ( pi ) .", "this releases the export substrate , and the exportin is recycled back into the nucleus . for details , see text .", "transport in the reverse direction , mediated by exportins , is regulated in a converse manner ( figure 2 ) .", "a paradigm for transport out of the nucleus is the export of leucine - rich nes - containing proteins .", "exportin 1 binds to substrates containing a leucine - rich nes in the nucleus , forming a trimeric complex with rangtp .", "this complex is then transferred to the cytoplasm by a mechanism involving binding of exportin 1 to the npc . once in the cytoplasm ,", "gtp hydrolysis results in dissociation of ran from the complex , allowing exportin 1 to release its cargo .", "free exportin 1 re - enters the nucleus to bind and export additional cargo molecules .", "as illustrated in these examples , the rangtp cycle plays a key role in conferring directionality to nucleocytoplasmic transport events   and rangtp acts as a marker of the nuclear compartment for both nuclear import and export ( figure 2 ; reviewed in ) .", "remarkably , this model predicts that only a single molecule of gtp is hydrolyzed per import / export cycle ; it strictly requires that rangtp is highly enriched in the nucleus .", "it is thought that a steep rangtp - rangdp gradient is generated by the cellular compartmentalization of regulators of the ran cycle .", "specifically , the guanine - nucleotide exchange factor of ran ( rangef or rcc1 ) , which regenerates rangtp is nuclear and bound to chromatin . in contrast , the main gtpase - activating protein ( rangap ) , and the ran - binding proteins , ranbp1 and ranbp2 , which stimulate gtp hydrolysis by ran , are found in the cytoplasm .", "this asymmetric distribution predicts that ran is present mainly in the gtp - bound form in the nucleus , whereas ran is immediately converted to a gdp - bound state in the cytoplasm .", "recently , it was suggested that in addition to the ran cycle , the npc itself could provide an additional mechanism to ensure transport directionality .", "given that several nucleoporins implicated in binding to importins and exportins have distinctive locations in the structure of the npc , the asymmetric design of the npc may also be important to efficiently drive nuclear import and export .", "nuclear transport mediated by importins and exportins is strongly directional in vivo : importins bind their cargo in the cytoplasm and transfer it to the nucleus , whereas exportins interact with their substrates in the nucleus and mediate their export to the cytoplasm ( figure 2 ) .", "protein translocation through the npc is thought to occur by an essentially similar mechanism for all importin--related receptors , except for the fact that , in some situations , additional adaptors are required to bridge the cargo - receptor interaction .", "this is mediated by importin  together with its adaptor importin  , which binds both the nls - containing cargo and importin  in the cytoplasm . after a trimeric importin -importin -nls complex is formed , importin  mediates docking at the npc . in the presence of rangdp and free gtp ,", "translocation is terminated by binding of rangtp to importin  , which releases the complex from the npc and dissociates importin  from importin  ( reviewed in ) .", "free importin  has a lower affinity for the nls cargo , and release from importin  is therefore believed to trigger release of the nls cargo as well .", "thereafter , the importin -rangtp complex , and importin  bound to its exportin ( cas ) and rangtp , are re - exported to the cytoplasm for another round of import . a schematic representation of nuclear import and export cycles through the npc .", "the cargo - loaded importin translocates through the npc into the nucleus , where the cargo is dissociated from the importin by binding of importin to rangtp .", "the importin - rangtp complex recycles back to the cytoplasm , where rangtp hydrolysis is stimulated by rangap and ranbp1 ; this frees the importin for the next round of import .", "exportins bind their export substrates in the nucleus , forming a trimeric cargo - exportin - rangtp complex .", "this complex is exported from the nucleus and dissociated in the cytoplasm by hydrolysis of rangtp to rangdp and inorganic phosphate ( pi ) .", "this releases the export substrate , and the exportin is recycled back into the nucleus . for details , see text .", "transport in the reverse direction , mediated by exportins , is regulated in a converse manner ( figure 2 ) .", "a paradigm for transport out of the nucleus is the export of leucine - rich nes - containing proteins .", "exportin 1 binds to substrates containing a leucine - rich nes in the nucleus , forming a trimeric complex with rangtp .", "this complex is then transferred to the cytoplasm by a mechanism involving binding of exportin 1 to the npc .", "once in the cytoplasm , gtp hydrolysis results in dissociation of ran from the complex , allowing exportin 1 to release its cargo .", "free exportin 1 re - enters the nucleus to bind and export additional cargo molecules .", "as illustrated in these examples , the rangtp cycle plays a key role in conferring directionality to nucleocytoplasmic transport events   and rangtp acts as a marker of the nuclear compartment for both nuclear import and export ( figure 2 ; reviewed in ) .", "remarkably , this model predicts that only a single molecule of gtp is hydrolyzed per import / export cycle ; it strictly requires that rangtp is highly enriched in the nucleus .", "it is thought that a steep rangtp - rangdp gradient is generated by the cellular compartmentalization of regulators of the ran cycle .", "specifically , the guanine - nucleotide exchange factor of ran ( rangef or rcc1 ) , which regenerates rangtp is nuclear and bound to chromatin . in contrast , the main gtpase - activating protein ( rangap ) , and the ran - binding proteins , ranbp1 and ranbp2 , which stimulate gtp hydrolysis by ran , are found in the cytoplasm .", "this asymmetric distribution predicts that ran is present mainly in the gtp - bound form in the nucleus , whereas ran is immediately converted to a gdp - bound state in the cytoplasm .", "recently , it was suggested that in addition to the ran cycle , the npc itself could provide an additional mechanism to ensure transport directionality .", "given that several nucleoporins implicated in binding to importins and exportins have distinctive locations in the structure of the npc , the asymmetric design of the npc may also be important to efficiently drive nuclear import and export .", "despite the large amount of progress that has been made in the nucleocytoplasmic transport field in recent years , many important questions remain unsolved .", "many import and export receptors have now been characterized and their first cargoes have been identified .", "the further characterization of new transport receptors and adaptors , and the identification of new import and export substrates , will lead to a more complete picture of nucleocytoplasmic transport .", "a big challenge for the future will be to understand how translocation through the npc occurs , and how nucleocytoplasmic transport is regulated .", "to gain insight into these questions , a quantitative analysis of interactions between transport receptors and nucleoporins will be required .", "it will be also interesting to see whether an increasing affinity gradient of receptors for nucleoporins along the npc exists and , if so , whether it makes an important contribution towards the direction of transport .", "other issues that remain to be solved include the structural differences between members of the importin and exportin family .", "how does rangtp dissociate import complexes in the nucleus but promote binding of export cargoes to exportins ?", "a key mechanistic topic that is poorly understood is the export of rnas and rnps .", "although a trna - export factor has been identified , the mechanism of rrna or mrna export is still poorly understood .", "several proteins in s. cerevisiae , such as mex67p and yra1p , and their metazoan counterparts tap and aly , have been indicated to play an important role in mrna export .", "mex67p / tap does not belong to the family of importin--like proteins , suggesting that there are alternative translocation pathways through the npc ."], "abstract_text": ["<S> the importin--like family of nuclear transport receptors mediates the transport of a large number of substrates between the cytoplasm and the nucleus . </S>", "<S> the family is made up of importins and exportins which shuttle between the nucleus and the cytoplasm and are regulated by the small gtpase ran . </S>"], "labels": null, "section_names": ["Gene organization and evolutionary history", "Characteristic structural features", "Localization and function", "Mechanism and regulation", "Frontiers"], "sections": [["a major effort in the nucleocytoplasmic transport field has been directed towards the analysis of all members of the importin  family , in particular with the aim of identifying specific transport cargoes .", "this has led to the characterization of a large number of related proteins ( both importins and exportins ) in all eukaryotic species analyzed .", "new members have primarily been identified by sequence homology or biochemically via their interaction with the small gtpase ran .", "strikingly , the roles of many of these receptors is conserved from yeast to humans .", "there are 14 putative members of the importin  family in the completed saccharomyces cerevisiae genome , nine of which have been shown to function as importins and four as exportins .", "the genes encoding yeast transport receptors are dispersed throughout the genome and none of them contains introns .", "little is known about the gross structure of the genes encoding these nuclear transport receptors , and our knowledge of the chromosomal localization or the organization of the individual genes encoding members of this family is very poor ."], ["the relative molecular masses of members of importin--like proteins vary between 90 kda and 130 kda , but all are characterized by an acidic isoelectric point .", "the overall sequence similarity between various transport receptors is low ( less than 20% amino acid identity ) and , in many cases , is restricted to the amino - terminal domain .", "work mainly on importin  has demonstrated that these receptors bind rangtp via the amino - terminal domain and cargo via the carboxy - terminal domain . to permit shuttling through the nuclear pore complex ( npc )", ", transport receptors also contain one or multiple binding domains for components of the npcs , called nucleoporins .", "truncation studies using importin  indicate that the binding site for nucleoporins containing fxfg repeats ( in the single - letter amino acid code , where x is , in many cases , a small polar residue or glycine ) is located in an amino - terminal / central region of importin  ( residues 152 - 352 ) .", "this was recently confirmed in the crystal structure of an amino - terminal fragment of importin  in a complex with five fxfg nucleoporin repeats .", "additional information comes from the crystal structures of importin  and of transportin 1 ( also known as karyopherin 2 ) , which were solved either in a complex with rangtp , with a cargo , or in the free form .", "overall , the structures of importin--like receptors are characterized by a very similar series of helical heat repeats ( 19 in importin  and 18 in transportin 1 ; figure 1 ) .", "heat repeats are approximately 40 residues in length and are found in many eukaryotic proteins such as the pr65/a subunit of protein phosphatase 2a .", "the fundamental repeat unit is a right - handed superhelical structure consisting of a hairpin made up of two  helices , named a and b , separated by a sharp turn .", "each hairpin is connected to the next by a linker region . in transportin 1 ,", "almost all linkers contain a third helix , but there are very few linker helices in importin . in both receptors , one turn is extended into a long acidic loop , which has been suggested to be important for rangtp - mediated cargo release .", "full - length importin  complexed with the importin--binding domain of importin  ( ibb ) forms a snail - like superhelical structure wrapping tightly around the ibb domain .", "the structure of the uncomplexed amino terminus of importin  reveals a different superhelical architecture with a much steeper helical pitch than the cargo - bound or rangtp - bound forms .", "this suggests that importin  undergoes twisted conformational changes in its heat - repeat helix stacking , which could be essential for the regulation of cargo binding and release and/or for protein interactions during the translocation through the npc .", "although the sequence homology is limited , it is expected that exportins will fold in a similar way to the reported importin structures .", "it still remains unclear , however , why rangtp is required for binding of cargo to exportins but causes cargo dissociation from importins .", "structure of importin . ( a ) importin  is composed of 19 helical - repeat motifs ( heat repeats ) .", "each consists of an a and a b helix connected by a short turn , which in heat-8 is replaced by an acidic loop critical for the regulation of substrate binding and release .", "the heat repeats 1 - 8 are required for high - affinity binding to rangtp .", "the importin--binding ( ibb ) domain of importin  interacts mainly with residues located in repeats 7 - 19 of importin  .", "the binding site for nucleoporins of the npc is located between residues 152 and 352 , corresponding to repeats 4 - 8 . on the basis of the crystal structure ,", "the a helices of heat repeats 5 and 6 and a region between heat repeats 6 and 7 are thought to be critical for recognition of the fxfg motif .", "( b ) structure of importin  bound to the ibb domain of importin  ( adapted from ) .", "importin  ( yellow ) forms a superhelical structure that wraps like a snail around the ibb domain ( blue ) .", "the 19 heat repeats share a common core of 21 residues , comprising the a helix with about three turns and the b helix with about four turns .", "the helices critical for the interaction with fxfg - repeat nucleoporins   are in green .", "note the acidic loop , which contacts both rangtp and the ibb domain ( white arrow ) ."], ["a major function of transport factors of the importin  family is to mediate the transport between the nucleus and cytoplasm of macromolecules that contain nuclear import or export signals . to this end ,", ", they can be found in the nucleus , at the npc or in the cytoplasm . at present , very little is known about the tissue distribution of this family of proteins .", "all members have the ability to recognize and bind specific cargoes , either directly or via adaptor molecules , to bind rangtp and to interact with nucleoporins at the npc .", "interactions between the proteins of the importin  family and nucleoporin repeats have been shown both in vitro   and in vivo .", "these interactions contribute to the import or export of importin  family members and their cargoes through the central transporter of the npc .", "import and export are multistep processes that are initiated by the recognition of nuclear localization signals ( nlss ) and nuclear export signals ( ness ) .", "the most thoroughly studied import signals are the ' classical ' and the bipartite nlss , first identified in sv40 large t antigen and nucleoplasmin , respectively .", "their transport is mediated by importin  , the first - characterized member of this protein family .", "additional importin--dependent adaptors in vertebrates include snurportin 1 ( involved in import of m3g - capped small nuclear ribonucleoproteins , snrnps ) and xrip , ( involved in the import of replication protein a , rpa ) .", "importin  can also form a complex with another importin--like factor , importin 7 , in order to transport the linker histone h1 into the nucleus .", "in addition , importin  is able to interact directly with a large variety of different cargoes , including the t - cell protein tyrosine phosphatase , the human immunodeficency virus ( hiv ) tat and rev proteins , human t - cell leukemia virus rex protein , ribosomal proteins l23a , s7 , and l5 , cyclin b1 , smad   and the parathyroid - hormone - related protein .", "table 1 details known importins , exportins and their cargoes and adaptors . proteins in the importin--like family and their cargoes abbreviations : h.s .", ", homo sapiens ; s.c . , saccharomyces cerevisiae ; x.l . , xenopus laevis .", "genbank accession numbers are given in square brackets for each protein , and alternative names are in parenthesis .", "the import of ribosomal proteins seems to rely on at least partially redundant mechanisms . in yeast ,", "/ yrb4p has been shown to be an important mediator of ribosomal - protein import , but the related protein kap121p", "/ pse1p can functionally substitute for kap123p in vivo . in mammalian cells , at least four importin--like transport factors are able to mediate import of ribosomal proteins .", "interestingly , both the -like importin receptor binding ( bib ) domain and some ribosomal proteins can be imported by any of the four receptors importin  , transportin 1 , importin 5 or importin 7 .", "the yeast importin kap104p mediates nuclear import of the mrna - binding proteins nab2p and nab4p .", "its vertebrate homolog , transportin 1 ( kap2 ) , also mediates import of the rna - binding proteins hnrnp a1 and hnrnp f , but also of ribosomal proteins .", "mammalian transportin - sr mediates nuclear import of a group of abundant arginine / serine - rich proteins , which are essential pre - mrna splicing factors . in yeast ,", "the tata - binding protein ( tbp ) is imported into the nucleus by kap114p .", "exportin 1 ( crm1p , xpo1p ) has been identified in both yeast and higher eukaryotes as an export receptor for leucine - rich nes - containing proteins .", "human exportin 1 has been found to export protein kinase inhibitor  ( pki ) and hiv rev , ib , snurportin 1 , htlv rex , the small nuclear rna , cyclin b1   and the transcription factor nf - at4 .", "targets for exportin in s. cerevisiae include the transcription factors yap1p , and ace2p , the mitogen - activated protein kinase hog1p , and the heat - shock protein ssb1p .", "msn5p was first identified as a yeast exportin that exports the phosphorylated form of the transcription factor pho4p .", "interestingly , msn5p was recently shown to function as an import receptor for the trimeric rpa , suggesting that individual members of the importin--like protein family can function both as import and export receptors .", "the first cellular rna export receptor to be discovered , named exportin - t in higher eukaryotes or los1p in yeast , mediates nuclear export of trnas . many of the nuclear transport factors identified in s. cerevisiae are not essential for viability , even though they transport essential cargoes .", "this phenomenon can be explained by the fact that cargoes can use alternative transport pathways .", "this is probably best exemplified in the import pathway of ribosomal proteins , which is mediated by at least four different import receptors ( see above ) .", "nuclear transport mediated by importins and exportins is strongly directional in vivo : importins bind their cargo in the cytoplasm and transfer it to the nucleus , whereas exportins interact with their substrates in the nucleus and mediate their export to the cytoplasm ( figure 2 ) .", "protein translocation through the npc is thought to occur by an essentially similar mechanism for all importin--related receptors , except for the fact that , in some situations , additional adaptors are required to bridge the cargo - receptor interaction .", "this is mediated by importin  together with its adaptor importin  , which binds both the nls - containing cargo and importin  in the cytoplasm . after a trimeric importin -importin -nls complex is formed , importin  mediates docking at the npc .", "in the presence of rangdp and free gtp , this trimeric complex translocates through the npc .", "translocation is terminated by binding of rangtp to importin  , which releases the complex from the npc and dissociates importin  from importin  ( reviewed in ) .", "free importin  has a lower affinity for the nls cargo , and release from importin  is therefore believed to trigger release of the nls cargo as well .", "thereafter , the importin -rangtp complex , and importin  bound to its exportin ( cas ) and rangtp , are re - exported to the cytoplasm for another round of import . a schematic representation of nuclear import and export cycles through the npc .", "the cargo - loaded importin translocates through the npc into the nucleus , where the cargo is dissociated from the importin by binding of importin to rangtp .", "the importin - rangtp complex recycles back to the cytoplasm , where rangtp hydrolysis is stimulated by rangap and ranbp1 ; this frees the importin for the next round of import .", "exportins bind their export substrates in the nucleus , forming a trimeric cargo - exportin - rangtp complex .", "this complex is exported from the nucleus and dissociated in the cytoplasm by hydrolysis of rangtp to rangdp and inorganic phosphate ( pi ) .", "this releases the export substrate , and the exportin is recycled back into the nucleus . for details , see text .", "transport in the reverse direction , mediated by exportins , is regulated in a converse manner ( figure 2 ) .", "a paradigm for transport out of the nucleus is the export of leucine - rich nes - containing proteins .", "exportin 1 binds to substrates containing a leucine - rich nes in the nucleus , forming a trimeric complex with rangtp .", "this complex is then transferred to the cytoplasm by a mechanism involving binding of exportin 1 to the npc . once in the cytoplasm ,", "gtp hydrolysis results in dissociation of ran from the complex , allowing exportin 1 to release its cargo .", "free exportin 1 re - enters the nucleus to bind and export additional cargo molecules .", "as illustrated in these examples , the rangtp cycle plays a key role in conferring directionality to nucleocytoplasmic transport events   and rangtp acts as a marker of the nuclear compartment for both nuclear import and export ( figure 2 ; reviewed in ) .", "remarkably , this model predicts that only a single molecule of gtp is hydrolyzed per import / export cycle ; it strictly requires that rangtp is highly enriched in the nucleus .", "it is thought that a steep rangtp - rangdp gradient is generated by the cellular compartmentalization of regulators of the ran cycle .", "specifically , the guanine - nucleotide exchange factor of ran ( rangef or rcc1 ) , which regenerates rangtp is nuclear and bound to chromatin . in contrast , the main gtpase - activating protein ( rangap ) , and the ran - binding proteins , ranbp1 and ranbp2 , which stimulate gtp hydrolysis by ran , are found in the cytoplasm .", "this asymmetric distribution predicts that ran is present mainly in the gtp - bound form in the nucleus , whereas ran is immediately converted to a gdp - bound state in the cytoplasm .", "recently , it was suggested that in addition to the ran cycle , the npc itself could provide an additional mechanism to ensure transport directionality .", "given that several nucleoporins implicated in binding to importins and exportins have distinctive locations in the structure of the npc , the asymmetric design of the npc may also be important to efficiently drive nuclear import and export ."], ["nuclear transport mediated by importins and exportins is strongly directional in vivo : importins bind their cargo in the cytoplasm and transfer it to the nucleus , whereas exportins interact with their substrates in the nucleus and mediate their export to the cytoplasm ( figure 2 ) .", "protein translocation through the npc is thought to occur by an essentially similar mechanism for all importin--related receptors , except for the fact that , in some situations , additional adaptors are required to bridge the cargo - receptor interaction .", "this is mediated by importin  together with its adaptor importin  , which binds both the nls - containing cargo and importin  in the cytoplasm . after a trimeric importin -importin -nls complex is formed , importin  mediates docking at the npc . in the presence of rangdp and free gtp ,", "translocation is terminated by binding of rangtp to importin  , which releases the complex from the npc and dissociates importin  from importin  ( reviewed in ) .", "free importin  has a lower affinity for the nls cargo , and release from importin  is therefore believed to trigger release of the nls cargo as well .", "thereafter , the importin -rangtp complex , and importin  bound to its exportin ( cas ) and rangtp , are re - exported to the cytoplasm for another round of import . a schematic representation of nuclear import and export cycles through the npc .", "the cargo - loaded importin translocates through the npc into the nucleus , where the cargo is dissociated from the importin by binding of importin to rangtp .", "the importin - rangtp complex recycles back to the cytoplasm , where rangtp hydrolysis is stimulated by rangap and ranbp1 ; this frees the importin for the next round of import .", "exportins bind their export substrates in the nucleus , forming a trimeric cargo - exportin - rangtp complex .", "this complex is exported from the nucleus and dissociated in the cytoplasm by hydrolysis of rangtp to rangdp and inorganic phosphate ( pi ) .", "this releases the export substrate , and the exportin is recycled back into the nucleus . for details , see text .", "transport in the reverse direction , mediated by exportins , is regulated in a converse manner ( figure 2 ) .", "a paradigm for transport out of the nucleus is the export of leucine - rich nes - containing proteins .", "exportin 1 binds to substrates containing a leucine - rich nes in the nucleus , forming a trimeric complex with rangtp .", "this complex is then transferred to the cytoplasm by a mechanism involving binding of exportin 1 to the npc .", "once in the cytoplasm , gtp hydrolysis results in dissociation of ran from the complex , allowing exportin 1 to release its cargo .", "free exportin 1 re - enters the nucleus to bind and export additional cargo molecules .", "as illustrated in these examples , the rangtp cycle plays a key role in conferring directionality to nucleocytoplasmic transport events   and rangtp acts as a marker of the nuclear compartment for both nuclear import and export ( figure 2 ; reviewed in ) .", "remarkably , this model predicts that only a single molecule of gtp is hydrolyzed per import / export cycle ; it strictly requires that rangtp is highly enriched in the nucleus .", "it is thought that a steep rangtp - rangdp gradient is generated by the cellular compartmentalization of regulators of the ran cycle .", "specifically , the guanine - nucleotide exchange factor of ran ( rangef or rcc1 ) , which regenerates rangtp is nuclear and bound to chromatin . in contrast , the main gtpase - activating protein ( rangap ) , and the ran - binding proteins , ranbp1 and ranbp2 , which stimulate gtp hydrolysis by ran , are found in the cytoplasm .", "this asymmetric distribution predicts that ran is present mainly in the gtp - bound form in the nucleus , whereas ran is immediately converted to a gdp - bound state in the cytoplasm .", "recently , it was suggested that in addition to the ran cycle , the npc itself could provide an additional mechanism to ensure transport directionality .", "given that several nucleoporins implicated in binding to importins and exportins have distinctive locations in the structure of the npc , the asymmetric design of the npc may also be important to efficiently drive nuclear import and export ."], ["despite the large amount of progress that has been made in the nucleocytoplasmic transport field in recent years , many important questions remain unsolved .", "many import and export receptors have now been characterized and their first cargoes have been identified .", "the further characterization of new transport receptors and adaptors , and the identification of new import and export substrates , will lead to a more complete picture of nucleocytoplasmic transport .", "a big challenge for the future will be to understand how translocation through the npc occurs , and how nucleocytoplasmic transport is regulated .", "to gain insight into these questions , a quantitative analysis of interactions between transport receptors and nucleoporins will be required .", "it will be also interesting to see whether an increasing affinity gradient of receptors for nucleoporins along the npc exists and , if so , whether it makes an important contribution towards the direction of transport .", "other issues that remain to be solved include the structural differences between members of the importin and exportin family .", "how does rangtp dissociate import complexes in the nucleus but promote binding of export cargoes to exportins ?", "a key mechanistic topic that is poorly understood is the export of rnas and rnps .", "although a trna - export factor has been identified , the mechanism of rrna or mrna export is still poorly understood .", "several proteins in s. cerevisiae , such as mex67p and yra1p , and their metazoan counterparts tap and aly , have been indicated to play an important role in mrna export .", "mex67p / tap does not belong to the family of importin--like proteins , suggesting that there are alternative translocation pathways through the npc ."]]}
{"article_id": "PMC4848105", "article_text": ["musculoskeletal conditions , or disorders of the muscles and bones , are the most common causes of chronic disability in the world . in the us alone , disease of the musculoskeletal system was the primary diagnosis during 83,228,000 office visits in 2009 ( 8.0% of total visits ) .", "a stroke can also cause many of the same physical deficiencies seen in musculoskeletal conditions .", "approximately 2.6% of all americans over the age of 20 have had a stroke , and each year 795,000 more people suffer a stroke ( 1 every 40 seconds ) .", "the most common non - medication treatment for musculoskeletal conditions and stroke is rehabilitation . for patients", ", rehabilitation is a tedious chore that involves many months of treatment , resulting in low compliance .", "rehabilitation regimens combine home - based exercises with therapist - monitored sessions ; during the latter , the therapist assesses the patient 's capabilities and adjusts the exercise tasks accordingly . previous research on upper body rehabilitation has shown that positive functional outcomes are achieved from programs that emphasize task - oriented , repetitive training exercises combined with biofeedback , .", "thus , virtual reality ( vr ) rehabilitation systems have been developed that can repetitively simulate these task - oriented training exercises , .", "vr rehabilitation has been shown to be successful for improving upper body function in stroke patients , most likely because the interesting and engaging virtual tasks encourage increased repetition .", "some vr rehabilitation programs have also been designed to be used without the supervision of a physiotherapist . however", "a previous study demonstrated that subjects trained with an adaptive vr rehabilitation system had improved upper body functionality when compared to subjects trained with conventional rehabilitation .", "robotic rehabilitation systems , designed to address the limitations of traditional physiotherapy , have been developed primarily for upper extremity rehabilitation of stroke patients .", "these robots generally consist of an arm attachment with multiple actuated degrees of freedom that is affixed to the patient and guides them through different exercise tasks through the use of an immersive visual environment .", "studies have shown that patients achieved significant motor recovery and improved upper extremity functionality after participating in robotic rehabilitation .", "some shortcomings of the current rehabilitation robots are their limited availability and portability , and their high cost ( $ 50,000 usd ) ; as a result , few patients can benefit from robotic rehabilitation .", "pneumatic actuators have previously been used in upper body robotic rehabilitation systems because they are lightweight , strong , and mechanically compliant to human anatomy . in terms of control strategies , impedance control and admittance control", "have been used to ensure the safety of the subjects while they interact with the robot . in these control laws ,", "the controller uses both the position of the robot 's joints and the force produced by the actuators in order to limit the amount of force applied to the subject . in terms of efficacy", ", previous research has suggested that pneumatically actuated rehabilitation robots can be used by patients with movement disabilities or stroke to improve their motor function .", "the primary drawbacks of pneumatic actuators are that they are difficult to control due to non - linear behavior during actuation , and that they require an external supply of compressed air .", "the overall objective of this research was to create an adaptive home - use robotic rehabilitation system for the upper body .", "the specific goals for the system were to collect data from the subject in real - time using simple and portable sensors , to create a customized exercise task for the subject by adapting a standard exercise task , and to build a simple and non - rigid wearable robot that will provide subject - specific resistance training .", "this paper presents results from comparison testing between the customized exercise tasks and the standard exercise tasks , as well as preliminary testing of a pneumatically actuated prototype for the wearable robot .", "a microsoft \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor and an electromyograph ( emg ) system were combined with custom software written in c++ in order to collect data from the subject in real time .", "data was obtained from the microsoft \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } using official microsoft software ( \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sdk version 1.6 , microsoft , redmond , wa , usa ) .", "the 3d joint position data ( mediolateral , anteroposterior , and vertical axes ) for 20 joints per subject was acquired at the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } native sampling frequency of 30 hz .", "the data was subsequently smoothed using the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } smoothing algorithm and adjusted based on the height and angle of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor .", "simultaneously , a wireless surface emg system ( cometa , milan , italy ) was used to collect electrical signals at 1000 hz from two electrodes , one on the subject 's biceps muscle and one on the lateral head of the subject 's triceps muscle .", "the emg signals ' linear envelopes were processed in real - time using a previously described method \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm bandpass~filter}=10~{\\rm hz}\\hbox{--}500~{\\rm hz};~{\\rm lowpass~filter}=30~{\\rm hz})$ \n \\end{document } \n .", "the emg data were normalized to the subject 's maximum voluntary contraction ( % mvc ) . a 3d visual environment ( ve ) previously created using custom software in c++ and opengl   was modified to display the data from the sensors as well as the exercise task ( see section iii ) .", "1 , part a ) where the joint center data collected from the subject by the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } was displayed as a skeleton figure .", "the subject navigated the ve by using leg gestures to indicate directional arrows that rotated and translated the environment .", "separate from the ve , an additional window displayed the color video data from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } as well as the real - time emg data ( fig .", "1 , part b ) . this window was used to ensure that the subject remained positioned in front of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor and that the emg sensors were functioning properly . \n", "1.visual environment : a ) virtual room , b ) video and emg data . visual environment : a ) virtual room , b ) video and emg data .", "1 ) and the subject used his right hand to follow the sphere as closely as possible as it moved around the ve in a repeating cyclical 2d pattern .", "the 2d pattern was projected in the 3d visual environment as perpendicular to the subject . from the subject 's view", ", the pattern was constrained to an orthogonal plane directly in front of his viewpoint .", "the distance to this plane was held constant . as the subject turned and moved within the environment , the pattern turned at the same rate and moved with the subject . in this way", ", the 2d pattern always appeared in front and perpendicular to the subject 's viewpoint .", "biofeedback was used to aid the subject 's accuracy ; the sphere changed color and the volume of music playing in the background increased as the subject 's hand approached the center of the sphere . for data , the 3d position of the sphere , the 3d position of the subject 's hand , and the subject 's biceps and triceps emg signals were recorded simultaneously during the exercise tasks . the protocol for creating the customized exercise tasks , described previously by barzilay and wolf , consisted of generating an inverse model of the subject , which was modeled by a neural network .", "the subject first completed 10 full cycles of a visual follow training exercise task , or approximately 3 minutes of training .", "the subject 's performance during this training exercise ( in terms of the 3d position of the hand , the 3d velocity of the hand , and the biceps and triceps emg signals ) for all 10 cycles was averaged together and smoothed to create one full cycle of the subject 's performance for each of the 8 measured variables .", "the averaged 3d position and 3d velocity ( 6 variables ) of the sphere during the training exercise task were used as targets to train the neural network .", "after the neural network was trained , it was used to simulate the subject 's individual performance during a standard exercise task . averaged emg data from 10 healthy subjects performing the standard exercise task were used as the additional inputs to the neural network .", "the wearable robot prototype consisted of the test platform , the pneumatics assembly ( and associated electronic components ) , and the control architecture .", "a test platform was built to model the human arm and elbow joint ; in this way , the basic prototype and the control algorithms for the wearable robot could be developed and validated in a safe environment without a risk of injury . the platform was designed to replicate the mass properties \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm total~mass}=1.6~{\\rm kg})$ \n \\end{document } , inertial properties \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm i}=0.1~{\\rm kg}{^{\\ast}}{\\rm m}^{2})$ \n \\end{document } , and dimensions of the human arm . the test platform consisted of two \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 7\\times 7\\times 30~{\\rm cm}$ \n \\end{document } aluminum cages ( simulating the upper arm and forearm segments ) connected together by two ball bearing assemblies to simulate an elbow joint ( fig .", "the upper arm cage was bolted to a wooden base while the forearm cage was allowed to swing freely up to 45\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } in either direction about the elbow joint . in future iterations ,", "the primary requirements for the assembly were that the components be both non - rigid and highly compliant as the robot will eventually be worn on the subject 's arm .", "as such , mckibben - type pneumatic artificial muscles ( pams ) were chosen as the actuation components ; as an additional benefit , these actuators closely replicate the behavior of natural muscles .", "two festo dmsp-10 - 120 pams ( festo , denkendorf , germany ) were attached to the upper arm cage of the test platform to replicate the biceps and triceps muscles ( fig .", "the muscles were rigidly anchored to the base of the upper arm cage and then connected to the forearm cage at the elbow joint with non - elastic metal wire ( fig .", ", actuation of the upper arm cage was accomplished by transforming the force from the antagonistic pams into torque about the joint , as in the human arm .", "each pam was independently controlled by using two 2/2 normally closed festo mhj-10 pneumatic valves ( festo , denkendorf , germany ) , one for inlet and one for outlet .", "motorola mpx5700 pressure sensors ( motorola solutions , schaumburg , il , usa ) measured pressure in the pams and output an analog signal to the microcontroller . finally , an austriamicrosystems as5145 magnetic encoder ( ams ag , unterpremstatten , austria ) was used to determine the position of the elbow joint , and an arduino uno board ( sparkfun , boulder , ca , usa ) was used as the microcontroller for the entire prototype . in order to identify the robot 's passive dynamic properties ,", "an impulse torque disturbance was applied to the joint and the angular position was recorded with respect to time .", "the system was modeled as a standard 2nd order linear system , with the following equation for the angular position:\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(s\\right)=\\thinspace{{m_{in}(s)}\\over{is^{2}+2crs+2kr}}\\quad=\\quad{{^{\\textstyle m_{in}(s)}\\!\\bigl/\\!_{\\textstyle i}\\over{s^{2}+\\left(^{\\textstyle 2cr}\\!\\bigl/\\!_{\\textstyle i}\\right)s+{^{\\textstyle 2kr}\\!\\bigl/\\!_{\\textstyle i}}}}}\\eqno{\\hbox{(1)}}$$ \n \\end{document } where \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c$ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ k$ \n \\end{document } are the damping and spring coefficients of the moveable parts of the robot prototype and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ r$ \n \\end{document } is the distance from the center of the joint to the attachments of the non - elastic metal wire ( fig .", "2 ) . the response in time to an impulse in the form of\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ m_{in}\\left(t\\right)=t\\delta ( t)\\eqno{\\hbox{(2)}}$$ \n \\end{document } therefore leads to the following equation ( in the time domain via the inverse laplace transform):\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(t\\right)=l^{-1}\\left\\{{{^{\\textstyle t}\\!\\bigl/\\!_{\\textstyle i}}\\over{s^{2}+\\left(^{\\textstyle 2cr}\\!\\bigl/\\!_{\\textstyle", "i}\\right)s+{^{\\textstyle 2kr}\\!\\bigl/\\!_{\\textstyle i}}}}\\right\\}\\!.\\eqno{\\hbox{(3)}}$$ \n \\end{document } \n \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{d}$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } were defined as follows:\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\eqalignno{\\omega_{n}^{2}=&\\,{^{\\textstyle \\rm 2kr}\\!\\bigl/\\!_{\\textstyle \\rm i}},\\quad{2\\zeta\\omega}_{n}^{2}={^{\\textstyle \\rm 2cr}\\!\\bigl/\\!{\\textstyle \\rm i}},\\;\\;{\\rm and}\\cr\\omega_{d}=&\\,\\omega_{n}\\sqrt{1-\\zeta^{2}}.&{\\hbox{(4)}}}$$ \n \\end{document } as such , the resultant response was \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(t\\right)=\\thinspace{{t}\\over{i\\omega_{d}}}{\\rm sin}(\\omega_{d}t)e^{-\\omega_{n}\\zeta t}\\!.\\eqno{\\hbox{(5)}}$$ \n \\end{document } the damping coefficient \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } was assumed to be relatively small \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( \\zeta\\leq 0.1)$ \n \\end{document } and therefore the difference between \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{d}$ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } was neglected .", "the final approximated function was \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(t\\right)=\\thinspace a\\cdot{\\rm sin}(\\omega_{n}t)e^{-\\omega_{n}\\zeta t}.\\eqno{\\hbox{(6)}}$$ \n \\end{document } the control architecture for the robot was based on pressure control of the pams .", "the pams were controlled through pulse - width modulation ( pwm ) of the pressure valves .", "a simplified dynamic equation for the robot was used for analysis due to the complexity of modeling the highly non - linear behavior of the pams .", "the dynamic equation used for the control architecture was \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i{\\mathddot{\\theta}}=\\tau_{dist}+\\thinspace\\tau_{cont},\\eqno{\\hbox{(7)}}$$ \n \\end{document } where \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\tau_{dist}$ \n \\end{document } is the disturbance torque due to dampening , friction , gravity , spring constant , and so on , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\tau_{cont}$ \n \\end{document } is the control torque applied by the pams and controlled using the sensors , microprocessor , and valves .", "the control torque applied by the pams was modeled as \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\tau_{control}=\\left(f_{left}-f_{right}\\right)\\cdot r\\eqno{\\hbox{(8)}}$$ \n \\end{document } and a simple proportional loop was implemented ( fig .", "the pwm parameters that determined the duty cycle for the valves were chosen so as not to exceed the limits of the valves , which had a minimum switching time of 1 msec ( 1000 hz ) . a 16 msec ( 60 hz ) pwm period ( divided into 10% duty cycles ) and a 600 hz switching frequency", "were selected , which resulted in a minimum switching time of 1.6 msec for each 10% duty cycle . in terms of the control rule for the pams , when the pressure error was positive \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm reference}>{\\rm measured})$ \n \\end{document } , the inlet valve duty cycle was determined as:\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ duty\\thinspace cycle = k_{pressure}\\cdot\\delta pressure\\eqno{\\hbox{(9)}}$$ \n \\end{document } and the outlet duty cycle remained closed ( 0% ) .", "two different experiments were performed for the assessment of the subject data collection and visual environment .", "the first experiment focused on the accuracy of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor , while the second experiment focused on a comparison of the customized exercise task .", "10 young , healthy subjects \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm age}\\!:27.1~{\\rm years}\\pm{2.9},~{\\rm height}\\!:128.6~{\\rm cm}\\pm{7.9},~{\\rm male}={6})$ \n \\end{document } with no history of upper body impairment volunteered to participate in this study .", "the subjects wore minimal clothing on their upper bodies to allow for placement of 25 reflective markers on their torsos and arms .", "each subject completed three trials of two different upper body exercises : 1 ) a range - of - motion ( rom ) exercise where the subject slowly raised both of his arms , elbow joints extended , from the sides of his torso to 45\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } above the transverse plane and back down , three repetitions per trial , and 2 ) a visual follow task , where the subject used his right hand to follow a moving sphere flying in a 3d figure 8 pattern in the ve for a total of 45 seconds .", "data was collected during these exercise tasks using both the robotic rehabilitation system and a 10 camera opto - electronic vicon mx motion capture system ( vicon , oxford , uk ) as a gold - standard for validation . for the robotic rehabilitation system ,", "3d joint center data was recorded for the wrist , elbow , and shoulder joints of the right arm using the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sdk version 1.5 and custom c++ software .", "additionally , a synchronization signal was sent from the robotic rehabilitation system to indicate to the vicon system when data were being collected . for the vicon system ,", "the reflective markers were placed on the subjects according to the vicon upper body model marker set .", "vicon nexus software ( version 1.8.2 ) was used to record the position of the markers during the exercise tasks as well as the synchronization signal .", "the data was post - processing in vicon nexus to obtain the joint center locations of the right wrist , elbow , and gleno - humeral ( shoulder ) joint .", "analysis of the data was completed in matlab r2012a ( mathworks , natick , ma , usa ) .", "a homogenous transformation was used to align the coordinate systems of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } and vicon measurement systems . to find the best parameters for the homogenous transformation , a minimum cost maximal matching optimization was used ( cost function : average distance between a small , randomized set of paired points from each system ) .", "the data was then resampled to a common frame rate of 120 hz , and the synchronized subset of the data extracted . the pearson 's correlation ( r )", "coefficient and the root - mean - square error ( rmse ) were calculated between the resampled and synchronized vicon and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } data for each trial .", "twelve young , healthy subjects \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm age}\\!:29.8~{\\rm years}\\pm{4.2},~{\\rm height}\\!:169.0~{\\rm cm}\\pm 9.3,~{\\rm male}={5})$ \n \\end{document } with no history of upper body impairment volunteered to participate in this study .", "the subjects wore close - fitting short sleeve shirts to allow for the placement of the two emg electrodes on their biceps and triceps .", "each subject was first asked to complete 10 full cycles of one of two different training exercise tasks , either a vertical double figure 8 pattern ( fig .", "4 , part a )   or a vertical half figure 8 pattern ( fig .", "4 , part b ) . after the neural network was trained , a related exercise task , consisting of either a full horizontal figure 8 pattern ( fig .", "4 , part a )   or half of a horizontal double figure 8 pattern ( fig .", "4 , part b ) , was given to the neural network as input data ( combined with the healthy emg data for that particular task ) , and a customized exercise task was output from the neural network .", "the subject then completed 10 full cycles of this customized output exercise task ( custom ) , as well as 10 full cycles of the unmodified original task used as input to the neural network ( standard ) ( fig .", "4 ) . a full exercise set consisted of the three linked exercise tasks ( training , custom , standard ) , and each subject completed 4 full sets ( two of each type of set ) , for a total of 12 exercise tasks per subject . \n fig .", "analysis of the data was completed in matlab r2012a ( mathworks , natick , ma , usa ) . for the custom and standard exercise tasks , the root - mean - square error ( rmse )", "was calculated between the subject 's hand and the sphere for both position and velocity .", "for the emg signals , the peak value and the area under the linear envelope were calculated .", "finally , the data from the subjects were merged , and paired \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t$ \n \\end{document}-tests ( two - tailed , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\alpha=0.05 $ \n \\end{document } ) were conducted between the standard exercise data and the corresponding custom exercise data in the set for the aforementioned variables . for the system identification ,", "the final approximated function ( 6 ) was tested experimentally by applying an external impulse torque disturbance to the robot and then recording the measurements from the encoder in the elbow joint ; this test measured the passive properties of the robot because the valves were sealed after the initial impulse .", "the experiment was conducted five separate times , and the approximations for \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ a$ \n \\end{document } were determined using the  nlinfit  function in matlab . to test the pressure control algorithm ,", "continuous sine wave pressure reference input signals were sent to the robot prototype at 60 hz .", "sine wave frequencies of 0.25 hz , 0.5 hz , 0.75 hz , and 1 hz were tested ( \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm gain}=0.10 $ \n \\end{document } for all tests ) . during the tests ,", "after the trials , the rmse and pearson 's correlation coefficient ( r ) were calculated between the reference pressure and the measured pressure . as a comparison to the passive system identification testing ,", "an external impulse torque disturbance was applied to the robot when the control algorithm was active , and the measurements from the encoder in the elbow joint were subsequently recorded .", "this test measured the closed - loop properties of the robot because control algorithm was active and the valves were open after the initial impulse .", "the experiment was conducted 15 separate times , and the approximations for \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ a$ \n \\end{document } were again determined using the  nlinfit  function in matlab .", ", a subject moved his right arm from full extension to full flexion at different speeds .", "the angle of the subject 's right elbow was continuously updated in real - time based on the 3d position of the subject 's shoulder , elbow , and wrist joints .", "this angle was then converted into a voltage signal ( between 0 and 5 volts ) that corresponded to the relative angle of the elbow ( between a minimum of 35\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } when the hand touched the shoulder , to a maximum of 175\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } when the elbow was fully extended ) .", "any angle measured beyond these limits was considered either 0 or 5 volts , depending on which limit was exceeded .", "a national instruments usb-6218 ( bnc ) data acquisition device ( national instruments corporation , austin , tx , usa ) was connected to the rehabilitation system , and the elbow angle voltage was sent as an output analog signal from this device .", "this signal was then connected to the analog input of the arduino uno to provide a real - time position reference signal for the robot prototype based on the subject 's elbow angle .", "this position signal was then converted into two corresponding pressure reference input signals for the pams , where 0 volts corresponded to the minimum \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { -}45^{\\circ})$ \n \\end{document } and 5 volts corresponded to the maximum \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { + } { 45}^{\\circ})$ \n \\end{document } allowable angle of the robot from the neutral position ( 0\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } ) . in this way , the subject could actuate the robot by changing the angle of his elbow .", "five 30-second duration tests were conducted . during the tests , the real - time angle of the subject 's elbow ( measured by the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } ) and the real - time angle of the robotic elbow joint ( measured by the encoder ) were simultaneously recorded .", "after the tests , the angle data were resampled and independently normalized on a scale of 0 to 1 .", "finally , the rmse and pearson 's correlation coefficient ( r ) were calculated between the normalized angle of the subject 's elbow and the normalized angle of the robotic elbow joint .", "the results from the validation testing between the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } data and the vicon data are presented for all trials of the 10 subjects for the right arm joints ( wrist , elbow , and shoulder ) by exercise task in table i. for the rom task , the largest rmse was observed for the shoulder joint . for the visual follow task , the largest rmse was observed for the elbow joint , then the wrist joint , and last the shoulder joint .", "furthermore , the rmse for the elbow and shoulder joints was greater during the visual follow task when compared to the rom task , while the rmse for the shoulder joint was greater during the rom task . \n", "table ivicon - kinect comparisons by joint and task vicon - kinect comparisons by joint and task a comparison of the standard and custom exercise tasks by exercise set is presented in table ii .", "the subjects had greater rmse for the position and the velocity variables during the custom tasks ( as compared to the standard tasks ) for both sets . \n", "table iikinematic and emg variables by set kinematic and emg variables by set the results from the external impulse response tests of the robot prototype are presented in table iii for \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ a$ \n \\end{document } , derived from the final approximated function ( 6 ) .", "the joint angle measured by the encoder was then compared to the predicted joint angle , which was calculated by using function ( 6 ) and the experimentally - determined function parameters . \n", "table iiiimpulse response the average values for the five passive properties tests were \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta=0.064 $ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}=15.01~{\\rm rad}/{\\rm sec}$ \n \\end{document}. compared to the predicted joint angle , the average rmse was 0.23\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } with a high correlation \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm r}=0.99)$ \n \\end{document}. the average values for the fifteen active properties tests were \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta=0.13 $ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}=14.84~{\\rm rad}/{\\rm sec}$ \n \\end{document}. compared to the predicted joint angle , the average rmse was 0.90\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } with a high correlation \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm r}=0.97)$ \n \\end{document}. the results from the pressure control algorithm tests are presented in table iv , figs . 5 and 6 . \n", "5.reference vs. measured pressure in the pams by frequency for sine wave input signals . \n fig .", "table ivref - meas comparisons by frequency and pam reference vs. measured pressure in the pams by frequency for sine wave input signals .", "ref - meas comparisons by frequency and pam the continuous joint angles during the pressure control algorithm tests are shown in fig .", "for the 0.25 hz and 0.5 hz continuous sine wave pressure reference input signals , the measured pressure accurately followed the reference pressure for both pams \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm r}=0.99,~{\\rm", "average~rmse}=0.093~{\\rm bar})$ \n \\end{document}. however , for the 0.75 hz and 1 hz input signals , the measured pressure did not follow the reference pressure during pressurization when the reference pressure was greater than 2.5 bars , indicating saturation of the pams .", "the range for the joint angles was 38.8\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 0.25 hz , 39.0\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 0.5 hz , 51.9\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 0.75 hz , and 53.6\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 1 hz .", "the wearable robot prototype was able to accurately mimic the angle of the subject 's elbow in real - time .", "the rmse between the normalized position of the subject 's elbow and the normalized position of the robot elbow ranged from 0.073 to 0.103 ( average 0.088 ) , and the correlation ranged from 0.94 to 0.97 ( average 0.96 , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm p}<0.001 $ \n \\end{document } ) .", "the overall objective of this research was to create an adaptive home - use robotic rehabilitation system for the upper body .", "this paper presented results from comparison testing between different exercise tasks and from preliminary testing of a pneumatically actuated prototype for the wearable robot , all of which demonstrate that this robotic rehabilitation system achieved the stated objective .", "the first specific goal for this research was to collect data from a subject in real - time using simple and portable sensors . during the subject testing ,", "the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor measured the 3d position and 3d velocity of the subject 's hand at 30 hz .", "experimental testing was conducted to determine the accuracy of the joint center measurements from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } as compared to the vicon gold - standard measurements during two upper - body exercise tasks ( table i ) .", "the results presented in table i show that the largest rmse for any joint during either task was 35.8 mm , while the smallest was 25.5 mm .", "these results are similar to a previous study comparing the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm\\thinspace}$ \n \\end{document } measurements to vicon measurements for upper body exercise tasks .", "clark et al . showed that between the two systems , there was approximately 42.9 mm difference during a lateral reaching task and 13.1 mm difference during a forward reaching task for hand measurements .", "these results suggest that the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } measurements are sufficiently accurate for clinical significance , and therefore the robotic rehabilitation system is sufficiently accurate for measurements of rehabilitation exercises . since traditional physiotherapy regimens for the upper body are managed by physiotherapists , easily identified visual landmarks are used to quantify the success of the exercises .", "one study , focused on rehabilitation after a thrower 's elbow injury , used visual quantification landmarks such as  extend elbow to full extension ", ",  raise arm to shoulder level  and  stand with shoulder abducted 90\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document }  to describe the exercises .", "the current standard is to evaluate the exercises based on visual observation ( which can result in substantial errors when compared to a quantitative system like vicon ) . as such , rmse of 35 mm to 40 mm in joint center measurements", "can be considered insignificant for clinical purposes because visual observation error in a clinical setting has been shown to be approximately 10% and this rmse is less than 10% of the movement range .", "therefore , the joint center measurements from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor are accurate enough to quantify rehabilitation exercises of the upper body , and are an improvement to the current standard of visual observation by a physiotherapist .", "the second specific goal of the system was to create a customized exercise task for each subject .", "a neural network was created using the subject 's training exercise data and was then used to modify a standard exercise task into a custom exercise task . for both sets of exercises ,", "the subjects had greater rmse for the position and the velocity variables during the custom tasks ; in other words , during the custom task , their kinematic performance was worse in terms of accuracy to the instructed task .", "these results show that the subjects were less able to replicate the custom task , suggesting that the custom task was more difficult to complete .", "this may be a result of the unconstrained nature of the neural network used to create the custom task .", "no output constraints were placed on the neural network , and the researchers did not manually alter the output . as a result ,", "consequently , the subjects were continually reacting to the unpredictable trajectory of the moving sphere during the custom task but were able to anticipate the trajectory of the standard task , thereby making the standard task easier to complete . adding constraints to the neural network", "previous successful adaptive rehabilitation systems ,   used trained physiotherapists to adapt the rehabilitation tasks to the subjects based on the quantitative data .", "therefore , the therapist acted as a constraint on the output of the systems , and could modify the output exercise task to achieve the desired goal .", "future iterations of the system will include constraints on the neural network and/or a trained physiotherapist who can modify the output task in order to ensure that the custom task is an anticipatory task , not a reactionary task .", "both the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor and the emg system are easily transportable and simple to use after minimal training .", "thus , it is feasible to use the entire rehabilitation system in a home setting , as the only additional requirements are a computer and a display device like a television .", "the third specific goal for the system was to build a simple and non - rigid wearable robot that will provide subject - specific resistance training .", "the results for the wearable robot prototype demonstrate the feasibility of using pams for actuation .", "table iii shows that the exoskeleton can be modeled as a second order linear system described by ( 6 ) .", "the measured damping coefficient \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta=0.064 $ \n \\end{document } was low , suggesting that it might be difficult to control the robot .", "however , it is likely that a human elbow will have a higher damping coefficient than the test platform and therefore will be more controllable . furthermore , the measured damping coefficient was higher during the closed - loop comparison testing of the robot \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( \\zeta=0.13)$ \n \\end{document } , showing that the simple pressure control algorithm increased the damping of the robot .", "overall , these results indicate that a robust controller can be built that will control the robot .", "the first controller developed for the robot was based on pressure control of the pams .", "the results from the pressure control algorithm tests indicate that there was saturation of the pams at 0.75 hz and 1 hz during pressurization ; the valves did not pressurize the pams fast enough to follow the reference signal .", "this saturation most likely occurred because the reference pressure input signals were both high amplitude and high frequency , and as such required a high change rate ( 7 bar / second ) of the pressure inside the pams ( corresponding to an angle change of 100\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}/{\\rm second}$ \n \\end{document } ) . as the pressure rose inside the valves , the pressure differential between the valves and the pressure source decreased , thereby decreasing the flow rate to the valves . eventually , the decreased flow rate was insufficient to achieve the high change rate required by the reference pressure input signals , and saturation occurred .", "this trend was not observed during depressurization because it would only occur near 0 bar , and the pressure reference input signals did not reach this level .", "while the motion bandwidths of normal human arm movements are centered at approximately 2 hz , most of these movements are relatively low amplitude and would not require high pressure change rates of the pams .", "nevertheless , future versions of the wearable robot will use a higher pressure source to increase the pressure differential ( and therefore the flow rate ) of the pams so that the robot can follow reference pressure input signals of at least 5 hz .", "additionally , force sensors will be added to the robot so that advanced control algorithms can be implemented , such as impedance or admittance control , in order to more accurately control the pneumatically actuated robot .", "the control performance of the wearable robot did not account for additional loading from the subject . in a wearable robot ,", "the subject would also be exerting force on the robot ( both assistive and resistive force ) .", "this force would need to be measured and compensated by the controller in order to accurately control the position of the robot .", "future prototypes of the robot arm should include sensors to measure the force exerted on the robot by the subject .", "the results from the tests of the combined system indicate that the pressure loop can successfully follow low amplitude pressure reference input signals at frequencies greater than 1 hz . during these tests ,", "saturation was not observed when the subject actuated the robot with a low amplitude reference input signal greater than 1 hz .", "varying the rate of change of the elbow angle also did not affect the accuracy of the robot , as it was able to follow the subject 's slow and fast movements .", "due to a slightly noisy input signal ( caused by small inaccuracies in the measured elbow angle from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } ) and insufficient damping in the control algorithm , the robot sometimes oscillated about the desired angle .", "furthermore , this test demonstrates the feasibility of the entire robotic rehabilitation system because the subject was able to actuate the wearable robot using only the movement of his elbow .", "future iterations of the entire system will implement real - time smoothing of the elbow angle reference signal in order to compensate for the small inaccuracies in the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } measurements , and the damping terms in the control algorithm will be increased .", "this study described a promising home - use robotic rehabilitation system comprised of low - cost components .", "further research is necessary to create a commercial version of this system , specifically on the prototype of the wearable robot .", "however , the data collection component of the system could be used in the near future for large scale research studies .", "the immediate next steps are to create a gui for the system to increase usability and to optimize the model generation code for speed and accuracy .", "afterwards , this part of the rehabilitation system could be used by a variety of end - users for large scale research studies on rehabilitation exercises .", "given the low cost and easy portability of the microsoft \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor , these studies should include home - based exercise components .", "concurrently , the next prototype of the wearable robot should be developed with the goal of having a fully functioning wearable prototype within two years .", "a limitation of this study is that the current system does not provide force feedback to the subjects .", "the wearable robot was designed to provide force feedback during the exercise tasks , but the current prototype was not tested on human subjects .", "future iterations of the robot prototype should be designed for wearability and human safety so that it can be worn by the subjects in order to provide force feedback .", "the addition of force feedback to the system should significantly enhance the results as the subject will have additional biofeedback information to help them complete the rehabilitation exercises ."], "abstract_text": ["<S> robotic rehabilitation systems have been developed to treat musculoskeletal conditions , but limited availability prevents most patients from using them . </S>", "<S> the objective of this paper was to create a home - use robotic rehabilitation system . </S>", "<S> data were obtained in real time from a microsoft \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } and a wireless surface electromyograph system . </S>", "<S> results from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor were compared to a standard motion capture system . </S>", "<S> a subject completed visual follow exercise tasks in a 3-d visual environment . </S>", "<S> data from two training exercises were used to generate a neural network , which was then used to simulate the subject 's individual performance . </S>", "<S> the subjects completed both the exercise task output from the neural network ( custom ) , and the unmodified task ( standard ) . </S>", "<S> in addition , a wearable arm robot prototype was built . </S>", "<S> basic system identification was completed , and a control algorithm for the robot based on pressure control was designed and tested . </S>", "<S> the subjects had greater root - mean - square error for position and velocity variables during the custom exercise tasks . </S>", "<S> these results suggest that the custom task was difficult to complete , possibly because the neural network was unconstrained . </S>", "<S> finally , the robot prototype was able to mimic changes in a subject 's elbow angle in real time , demonstrating the feasibility of the robotic rehabilitation system . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusion"], "sections": [["musculoskeletal conditions , or disorders of the muscles and bones , are the most common causes of chronic disability in the world . in the us alone , disease of the musculoskeletal system was the primary diagnosis during 83,228,000 office visits in 2009 ( 8.0% of total visits ) .", "a stroke can also cause many of the same physical deficiencies seen in musculoskeletal conditions .", "approximately 2.6% of all americans over the age of 20 have had a stroke , and each year 795,000 more people suffer a stroke ( 1 every 40 seconds ) .", "the most common non - medication treatment for musculoskeletal conditions and stroke is rehabilitation . for patients", ", rehabilitation is a tedious chore that involves many months of treatment , resulting in low compliance .", "rehabilitation regimens combine home - based exercises with therapist - monitored sessions ; during the latter , the therapist assesses the patient 's capabilities and adjusts the exercise tasks accordingly . previous research on upper body rehabilitation has shown that positive functional outcomes are achieved from programs that emphasize task - oriented , repetitive training exercises combined with biofeedback , .", "thus , virtual reality ( vr ) rehabilitation systems have been developed that can repetitively simulate these task - oriented training exercises , .", "vr rehabilitation has been shown to be successful for improving upper body function in stroke patients , most likely because the interesting and engaging virtual tasks encourage increased repetition .", "some vr rehabilitation programs have also been designed to be used without the supervision of a physiotherapist . however", "a previous study demonstrated that subjects trained with an adaptive vr rehabilitation system had improved upper body functionality when compared to subjects trained with conventional rehabilitation .", "robotic rehabilitation systems , designed to address the limitations of traditional physiotherapy , have been developed primarily for upper extremity rehabilitation of stroke patients .", "these robots generally consist of an arm attachment with multiple actuated degrees of freedom that is affixed to the patient and guides them through different exercise tasks through the use of an immersive visual environment .", "studies have shown that patients achieved significant motor recovery and improved upper extremity functionality after participating in robotic rehabilitation .", "some shortcomings of the current rehabilitation robots are their limited availability and portability , and their high cost ( $ 50,000 usd ) ; as a result , few patients can benefit from robotic rehabilitation .", "pneumatic actuators have previously been used in upper body robotic rehabilitation systems because they are lightweight , strong , and mechanically compliant to human anatomy . in terms of control strategies , impedance control and admittance control", "have been used to ensure the safety of the subjects while they interact with the robot . in these control laws ,", "the controller uses both the position of the robot 's joints and the force produced by the actuators in order to limit the amount of force applied to the subject . in terms of efficacy", ", previous research has suggested that pneumatically actuated rehabilitation robots can be used by patients with movement disabilities or stroke to improve their motor function .", "the primary drawbacks of pneumatic actuators are that they are difficult to control due to non - linear behavior during actuation , and that they require an external supply of compressed air .", "the overall objective of this research was to create an adaptive home - use robotic rehabilitation system for the upper body .", "the specific goals for the system were to collect data from the subject in real - time using simple and portable sensors , to create a customized exercise task for the subject by adapting a standard exercise task , and to build a simple and non - rigid wearable robot that will provide subject - specific resistance training .", "this paper presents results from comparison testing between the customized exercise tasks and the standard exercise tasks , as well as preliminary testing of a pneumatically actuated prototype for the wearable robot ."], ["a microsoft \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor and an electromyograph ( emg ) system were combined with custom software written in c++ in order to collect data from the subject in real time .", "data was obtained from the microsoft \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } using official microsoft software ( \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sdk version 1.6 , microsoft , redmond , wa , usa ) .", "the 3d joint position data ( mediolateral , anteroposterior , and vertical axes ) for 20 joints per subject was acquired at the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } native sampling frequency of 30 hz .", "the data was subsequently smoothed using the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } smoothing algorithm and adjusted based on the height and angle of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor .", "simultaneously , a wireless surface emg system ( cometa , milan , italy ) was used to collect electrical signals at 1000 hz from two electrodes , one on the subject 's biceps muscle and one on the lateral head of the subject 's triceps muscle .", "the emg signals ' linear envelopes were processed in real - time using a previously described method \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm bandpass~filter}=10~{\\rm hz}\\hbox{--}500~{\\rm hz};~{\\rm lowpass~filter}=30~{\\rm hz})$ \n \\end{document } \n .", "the emg data were normalized to the subject 's maximum voluntary contraction ( % mvc ) . a 3d visual environment ( ve ) previously created using custom software in c++ and opengl   was modified to display the data from the sensors as well as the exercise task ( see section iii ) .", "1 , part a ) where the joint center data collected from the subject by the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } was displayed as a skeleton figure .", "the subject navigated the ve by using leg gestures to indicate directional arrows that rotated and translated the environment .", "separate from the ve , an additional window displayed the color video data from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } as well as the real - time emg data ( fig .", "1 , part b ) . this window was used to ensure that the subject remained positioned in front of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor and that the emg sensors were functioning properly . \n", "1.visual environment : a ) virtual room , b ) video and emg data . visual environment : a ) virtual room , b ) video and emg data .", "1 ) and the subject used his right hand to follow the sphere as closely as possible as it moved around the ve in a repeating cyclical 2d pattern .", "the 2d pattern was projected in the 3d visual environment as perpendicular to the subject . from the subject 's view", ", the pattern was constrained to an orthogonal plane directly in front of his viewpoint .", "the distance to this plane was held constant . as the subject turned and moved within the environment , the pattern turned at the same rate and moved with the subject . in this way", ", the 2d pattern always appeared in front and perpendicular to the subject 's viewpoint .", "biofeedback was used to aid the subject 's accuracy ; the sphere changed color and the volume of music playing in the background increased as the subject 's hand approached the center of the sphere . for data , the 3d position of the sphere , the 3d position of the subject 's hand , and the subject 's biceps and triceps emg signals were recorded simultaneously during the exercise tasks . the protocol for creating the customized exercise tasks , described previously by barzilay and wolf , consisted of generating an inverse model of the subject , which was modeled by a neural network .", "the subject first completed 10 full cycles of a visual follow training exercise task , or approximately 3 minutes of training .", "the subject 's performance during this training exercise ( in terms of the 3d position of the hand , the 3d velocity of the hand , and the biceps and triceps emg signals ) for all 10 cycles was averaged together and smoothed to create one full cycle of the subject 's performance for each of the 8 measured variables .", "the averaged 3d position and 3d velocity ( 6 variables ) of the sphere during the training exercise task were used as targets to train the neural network .", "after the neural network was trained , it was used to simulate the subject 's individual performance during a standard exercise task . averaged emg data from 10 healthy subjects performing the standard exercise task were used as the additional inputs to the neural network .", "the wearable robot prototype consisted of the test platform , the pneumatics assembly ( and associated electronic components ) , and the control architecture .", "a test platform was built to model the human arm and elbow joint ; in this way , the basic prototype and the control algorithms for the wearable robot could be developed and validated in a safe environment without a risk of injury . the platform was designed to replicate the mass properties \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm total~mass}=1.6~{\\rm kg})$ \n \\end{document } , inertial properties \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm i}=0.1~{\\rm kg}{^{\\ast}}{\\rm m}^{2})$ \n \\end{document } , and dimensions of the human arm . the test platform consisted of two \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ 7\\times 7\\times 30~{\\rm cm}$ \n \\end{document } aluminum cages ( simulating the upper arm and forearm segments ) connected together by two ball bearing assemblies to simulate an elbow joint ( fig .", "the upper arm cage was bolted to a wooden base while the forearm cage was allowed to swing freely up to 45\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } in either direction about the elbow joint . in future iterations ,", "the primary requirements for the assembly were that the components be both non - rigid and highly compliant as the robot will eventually be worn on the subject 's arm .", "as such , mckibben - type pneumatic artificial muscles ( pams ) were chosen as the actuation components ; as an additional benefit , these actuators closely replicate the behavior of natural muscles .", "two festo dmsp-10 - 120 pams ( festo , denkendorf , germany ) were attached to the upper arm cage of the test platform to replicate the biceps and triceps muscles ( fig .", "the muscles were rigidly anchored to the base of the upper arm cage and then connected to the forearm cage at the elbow joint with non - elastic metal wire ( fig .", ", actuation of the upper arm cage was accomplished by transforming the force from the antagonistic pams into torque about the joint , as in the human arm .", "each pam was independently controlled by using two 2/2 normally closed festo mhj-10 pneumatic valves ( festo , denkendorf , germany ) , one for inlet and one for outlet .", "motorola mpx5700 pressure sensors ( motorola solutions , schaumburg , il , usa ) measured pressure in the pams and output an analog signal to the microcontroller . finally , an austriamicrosystems as5145 magnetic encoder ( ams ag , unterpremstatten , austria ) was used to determine the position of the elbow joint , and an arduino uno board ( sparkfun , boulder , ca , usa ) was used as the microcontroller for the entire prototype . in order to identify the robot 's passive dynamic properties ,", "an impulse torque disturbance was applied to the joint and the angular position was recorded with respect to time .", "the system was modeled as a standard 2nd order linear system , with the following equation for the angular position:\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(s\\right)=\\thinspace{{m_{in}(s)}\\over{is^{2}+2crs+2kr}}\\quad=\\quad{{^{\\textstyle m_{in}(s)}\\!\\bigl/\\!_{\\textstyle i}\\over{s^{2}+\\left(^{\\textstyle 2cr}\\!\\bigl/\\!_{\\textstyle i}\\right)s+{^{\\textstyle 2kr}\\!\\bigl/\\!_{\\textstyle i}}}}}\\eqno{\\hbox{(1)}}$$ \n \\end{document } where \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ c$ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ k$ \n \\end{document } are the damping and spring coefficients of the moveable parts of the robot prototype and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ r$ \n \\end{document } is the distance from the center of the joint to the attachments of the non - elastic metal wire ( fig .", "2 ) . the response in time to an impulse in the form of\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ m_{in}\\left(t\\right)=t\\delta ( t)\\eqno{\\hbox{(2)}}$$ \n \\end{document } therefore leads to the following equation ( in the time domain via the inverse laplace transform):\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(t\\right)=l^{-1}\\left\\{{{^{\\textstyle t}\\!\\bigl/\\!_{\\textstyle i}}\\over{s^{2}+\\left(^{\\textstyle 2cr}\\!\\bigl/\\!_{\\textstyle", "i}\\right)s+{^{\\textstyle 2kr}\\!\\bigl/\\!_{\\textstyle i}}}}\\right\\}\\!.\\eqno{\\hbox{(3)}}$$ \n \\end{document } \n \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{d}$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } were defined as follows:\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\eqalignno{\\omega_{n}^{2}=&\\,{^{\\textstyle \\rm 2kr}\\!\\bigl/\\!_{\\textstyle \\rm i}},\\quad{2\\zeta\\omega}_{n}^{2}={^{\\textstyle \\rm 2cr}\\!\\bigl/\\!{\\textstyle \\rm i}},\\;\\;{\\rm and}\\cr\\omega_{d}=&\\,\\omega_{n}\\sqrt{1-\\zeta^{2}}.&{\\hbox{(4)}}}$$ \n \\end{document } as such , the resultant response was \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(t\\right)=\\thinspace{{t}\\over{i\\omega_{d}}}{\\rm sin}(\\omega_{d}t)e^{-\\omega_{n}\\zeta t}\\!.\\eqno{\\hbox{(5)}}$$ \n \\end{document } the damping coefficient \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } was assumed to be relatively small \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( \\zeta\\leq 0.1)$ \n \\end{document } and therefore the difference between \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{d}$ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } was neglected .", "the final approximated function was \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\theta\\left(t\\right)=\\thinspace a\\cdot{\\rm sin}(\\omega_{n}t)e^{-\\omega_{n}\\zeta t}.\\eqno{\\hbox{(6)}}$$ \n \\end{document } the control architecture for the robot was based on pressure control of the pams .", "the pams were controlled through pulse - width modulation ( pwm ) of the pressure valves .", "a simplified dynamic equation for the robot was used for analysis due to the complexity of modeling the highly non - linear behavior of the pams .", "the dynamic equation used for the control architecture was \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ i{\\mathddot{\\theta}}=\\tau_{dist}+\\thinspace\\tau_{cont},\\eqno{\\hbox{(7)}}$$ \n \\end{document } where \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\tau_{dist}$ \n \\end{document } is the disturbance torque due to dampening , friction , gravity , spring constant , and so on , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\tau_{cont}$ \n \\end{document } is the control torque applied by the pams and controlled using the sensors , microprocessor , and valves .", "the control torque applied by the pams was modeled as \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ \\tau_{control}=\\left(f_{left}-f_{right}\\right)\\cdot r\\eqno{\\hbox{(8)}}$$ \n \\end{document } and a simple proportional loop was implemented ( fig .", "the pwm parameters that determined the duty cycle for the valves were chosen so as not to exceed the limits of the valves , which had a minimum switching time of 1 msec ( 1000 hz ) . a 16 msec ( 60 hz ) pwm period ( divided into 10% duty cycles ) and a 600 hz switching frequency", "were selected , which resulted in a minimum switching time of 1.6 msec for each 10% duty cycle . in terms of the control rule for the pams , when the pressure error was positive \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm reference}>{\\rm measured})$ \n \\end{document } , the inlet valve duty cycle was determined as:\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ $ duty\\thinspace cycle = k_{pressure}\\cdot\\delta pressure\\eqno{\\hbox{(9)}}$$ \n \\end{document } and the outlet duty cycle remained closed ( 0% ) .", "two different experiments were performed for the assessment of the subject data collection and visual environment .", "the first experiment focused on the accuracy of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor , while the second experiment focused on a comparison of the customized exercise task .", "10 young , healthy subjects \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm age}\\!:27.1~{\\rm years}\\pm{2.9},~{\\rm height}\\!:128.6~{\\rm cm}\\pm{7.9},~{\\rm male}={6})$ \n \\end{document } with no history of upper body impairment volunteered to participate in this study .", "the subjects wore minimal clothing on their upper bodies to allow for placement of 25 reflective markers on their torsos and arms .", "each subject completed three trials of two different upper body exercises : 1 ) a range - of - motion ( rom ) exercise where the subject slowly raised both of his arms , elbow joints extended , from the sides of his torso to 45\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } above the transverse plane and back down , three repetitions per trial , and 2 ) a visual follow task , where the subject used his right hand to follow a moving sphere flying in a 3d figure 8 pattern in the ve for a total of 45 seconds .", "data was collected during these exercise tasks using both the robotic rehabilitation system and a 10 camera opto - electronic vicon mx motion capture system ( vicon , oxford , uk ) as a gold - standard for validation . for the robotic rehabilitation system ,", "3d joint center data was recorded for the wrist , elbow , and shoulder joints of the right arm using the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sdk version 1.5 and custom c++ software .", "additionally , a synchronization signal was sent from the robotic rehabilitation system to indicate to the vicon system when data were being collected . for the vicon system ,", "the reflective markers were placed on the subjects according to the vicon upper body model marker set .", "vicon nexus software ( version 1.8.2 ) was used to record the position of the markers during the exercise tasks as well as the synchronization signal .", "the data was post - processing in vicon nexus to obtain the joint center locations of the right wrist , elbow , and gleno - humeral ( shoulder ) joint .", "analysis of the data was completed in matlab r2012a ( mathworks , natick , ma , usa ) .", "a homogenous transformation was used to align the coordinate systems of the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } and vicon measurement systems . to find the best parameters for the homogenous transformation , a minimum cost maximal matching optimization was used ( cost function : average distance between a small , randomized set of paired points from each system ) .", "the data was then resampled to a common frame rate of 120 hz , and the synchronized subset of the data extracted . the pearson 's correlation ( r )", "coefficient and the root - mean - square error ( rmse ) were calculated between the resampled and synchronized vicon and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } data for each trial .", "twelve young , healthy subjects \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm age}\\!:29.8~{\\rm years}\\pm{4.2},~{\\rm height}\\!:169.0~{\\rm cm}\\pm 9.3,~{\\rm male}={5})$ \n \\end{document } with no history of upper body impairment volunteered to participate in this study .", "the subjects wore close - fitting short sleeve shirts to allow for the placement of the two emg electrodes on their biceps and triceps .", "each subject was first asked to complete 10 full cycles of one of two different training exercise tasks , either a vertical double figure 8 pattern ( fig .", "4 , part a )   or a vertical half figure 8 pattern ( fig .", "4 , part b ) . after the neural network was trained , a related exercise task , consisting of either a full horizontal figure 8 pattern ( fig .", "4 , part a )   or half of a horizontal double figure 8 pattern ( fig .", "4 , part b ) , was given to the neural network as input data ( combined with the healthy emg data for that particular task ) , and a customized exercise task was output from the neural network .", "the subject then completed 10 full cycles of this customized output exercise task ( custom ) , as well as 10 full cycles of the unmodified original task used as input to the neural network ( standard ) ( fig .", "4 ) . a full exercise set consisted of the three linked exercise tasks ( training , custom , standard ) , and each subject completed 4 full sets ( two of each type of set ) , for a total of 12 exercise tasks per subject . \n fig .", "analysis of the data was completed in matlab r2012a ( mathworks , natick , ma , usa ) . for the custom and standard exercise tasks , the root - mean - square error ( rmse )", "was calculated between the subject 's hand and the sphere for both position and velocity .", "for the emg signals , the peak value and the area under the linear envelope were calculated .", "finally , the data from the subjects were merged , and paired \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ t$ \n \\end{document}-tests ( two - tailed , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\alpha=0.05 $ \n \\end{document } ) were conducted between the standard exercise data and the corresponding custom exercise data in the set for the aforementioned variables . for the system identification ,", "the final approximated function ( 6 ) was tested experimentally by applying an external impulse torque disturbance to the robot and then recording the measurements from the encoder in the elbow joint ; this test measured the passive properties of the robot because the valves were sealed after the initial impulse .", "the experiment was conducted five separate times , and the approximations for \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ a$ \n \\end{document } were determined using the  nlinfit  function in matlab . to test the pressure control algorithm ,", "continuous sine wave pressure reference input signals were sent to the robot prototype at 60 hz .", "sine wave frequencies of 0.25 hz , 0.5 hz , 0.75 hz , and 1 hz were tested ( \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm gain}=0.10 $ \n \\end{document } for all tests ) . during the tests ,", "after the trials , the rmse and pearson 's correlation coefficient ( r ) were calculated between the reference pressure and the measured pressure . as a comparison to the passive system identification testing ,", "an external impulse torque disturbance was applied to the robot when the control algorithm was active , and the measurements from the encoder in the elbow joint were subsequently recorded .", "this test measured the closed - loop properties of the robot because control algorithm was active and the valves were open after the initial impulse .", "the experiment was conducted 15 separate times , and the approximations for \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ a$ \n \\end{document } were again determined using the  nlinfit  function in matlab .", ", a subject moved his right arm from full extension to full flexion at different speeds .", "the angle of the subject 's right elbow was continuously updated in real - time based on the 3d position of the subject 's shoulder , elbow , and wrist joints .", "this angle was then converted into a voltage signal ( between 0 and 5 volts ) that corresponded to the relative angle of the elbow ( between a minimum of 35\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } when the hand touched the shoulder , to a maximum of 175\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } when the elbow was fully extended ) .", "any angle measured beyond these limits was considered either 0 or 5 volts , depending on which limit was exceeded .", "a national instruments usb-6218 ( bnc ) data acquisition device ( national instruments corporation , austin , tx , usa ) was connected to the rehabilitation system , and the elbow angle voltage was sent as an output analog signal from this device .", "this signal was then connected to the analog input of the arduino uno to provide a real - time position reference signal for the robot prototype based on the subject 's elbow angle .", "this position signal was then converted into two corresponding pressure reference input signals for the pams , where 0 volts corresponded to the minimum \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { -}45^{\\circ})$ \n \\end{document } and 5 volts corresponded to the maximum \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { + } { 45}^{\\circ})$ \n \\end{document } allowable angle of the robot from the neutral position ( 0\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } ) . in this way , the subject could actuate the robot by changing the angle of his elbow .", "five 30-second duration tests were conducted . during the tests , the real - time angle of the subject 's elbow ( measured by the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } ) and the real - time angle of the robotic elbow joint ( measured by the encoder ) were simultaneously recorded .", "after the tests , the angle data were resampled and independently normalized on a scale of 0 to 1 .", "finally , the rmse and pearson 's correlation coefficient ( r ) were calculated between the normalized angle of the subject 's elbow and the normalized angle of the robotic elbow joint ."], ["the results from the validation testing between the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } data and the vicon data are presented for all trials of the 10 subjects for the right arm joints ( wrist , elbow , and shoulder ) by exercise task in table i. for the rom task , the largest rmse was observed for the shoulder joint . for the visual follow task , the largest rmse was observed for the elbow joint , then the wrist joint , and last the shoulder joint .", "furthermore , the rmse for the elbow and shoulder joints was greater during the visual follow task when compared to the rom task , while the rmse for the shoulder joint was greater during the rom task . \n", "table ivicon - kinect comparisons by joint and task vicon - kinect comparisons by joint and task a comparison of the standard and custom exercise tasks by exercise set is presented in table ii .", "the subjects had greater rmse for the position and the velocity variables during the custom tasks ( as compared to the standard tasks ) for both sets . \n", "table iikinematic and emg variables by set kinematic and emg variables by set the results from the external impulse response tests of the robot prototype are presented in table iii for \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta$ \n \\end{document } , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}$ \n \\end{document } , and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ a$ \n \\end{document } , derived from the final approximated function ( 6 ) .", "the joint angle measured by the encoder was then compared to the predicted joint angle , which was calculated by using function ( 6 ) and the experimentally - determined function parameters . \n", "table iiiimpulse response the average values for the five passive properties tests were \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta=0.064 $ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}=15.01~{\\rm rad}/{\\rm sec}$ \n \\end{document}. compared to the predicted joint angle , the average rmse was 0.23\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } with a high correlation \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm r}=0.99)$ \n \\end{document}. the average values for the fifteen active properties tests were \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta=0.13 $ \n \\end{document } and \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\omega_{n}=14.84~{\\rm rad}/{\\rm sec}$ \n \\end{document}. compared to the predicted joint angle , the average rmse was 0.90\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } with a high correlation \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm r}=0.97)$ \n \\end{document}. the results from the pressure control algorithm tests are presented in table iv , figs . 5 and 6 . \n", "5.reference vs. measured pressure in the pams by frequency for sine wave input signals . \n fig .", "table ivref - meas comparisons by frequency and pam reference vs. measured pressure in the pams by frequency for sine wave input signals .", "ref - meas comparisons by frequency and pam the continuous joint angles during the pressure control algorithm tests are shown in fig .", "for the 0.25 hz and 0.5 hz continuous sine wave pressure reference input signals , the measured pressure accurately followed the reference pressure for both pams \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( { \\rm r}=0.99,~{\\rm", "average~rmse}=0.093~{\\rm bar})$ \n \\end{document}. however , for the 0.75 hz and 1 hz input signals , the measured pressure did not follow the reference pressure during pressurization when the reference pressure was greater than 2.5 bars , indicating saturation of the pams .", "the range for the joint angles was 38.8\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 0.25 hz , 39.0\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 0.5 hz , 51.9\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 0.75 hz , and 53.6\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document } at 1 hz .", "the wearable robot prototype was able to accurately mimic the angle of the subject 's elbow in real - time .", "the rmse between the normalized position of the subject 's elbow and the normalized position of the robot elbow ranged from 0.073 to 0.103 ( average 0.088 ) , and the correlation ranged from 0.94 to 0.97 ( average 0.96 , \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm p}<0.001 $ \n \\end{document } ) ."], ["the overall objective of this research was to create an adaptive home - use robotic rehabilitation system for the upper body .", "this paper presented results from comparison testing between different exercise tasks and from preliminary testing of a pneumatically actuated prototype for the wearable robot , all of which demonstrate that this robotic rehabilitation system achieved the stated objective .", "the first specific goal for this research was to collect data from a subject in real - time using simple and portable sensors . during the subject testing ,", "the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor measured the 3d position and 3d velocity of the subject 's hand at 30 hz .", "experimental testing was conducted to determine the accuracy of the joint center measurements from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } as compared to the vicon gold - standard measurements during two upper - body exercise tasks ( table i ) .", "the results presented in table i show that the largest rmse for any joint during either task was 35.8 mm , while the smallest was 25.5 mm .", "these results are similar to a previous study comparing the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm\\thinspace}$ \n \\end{document } measurements to vicon measurements for upper body exercise tasks .", "clark et al . showed that between the two systems , there was approximately 42.9 mm difference during a lateral reaching task and 13.1 mm difference during a forward reaching task for hand measurements .", "these results suggest that the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } measurements are sufficiently accurate for clinical significance , and therefore the robotic rehabilitation system is sufficiently accurate for measurements of rehabilitation exercises . since traditional physiotherapy regimens for the upper body are managed by physiotherapists , easily identified visual landmarks are used to quantify the success of the exercises .", "one study , focused on rehabilitation after a thrower 's elbow injury , used visual quantification landmarks such as  extend elbow to full extension ", ",  raise arm to shoulder level  and  stand with shoulder abducted 90\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}$ \n \\end{document }  to describe the exercises .", "the current standard is to evaluate the exercises based on visual observation ( which can result in substantial errors when compared to a quantitative system like vicon ) . as such , rmse of 35 mm to 40 mm in joint center measurements", "can be considered insignificant for clinical purposes because visual observation error in a clinical setting has been shown to be approximately 10% and this rmse is less than 10% of the movement range .", "therefore , the joint center measurements from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor are accurate enough to quantify rehabilitation exercises of the upper body , and are an improvement to the current standard of visual observation by a physiotherapist .", "the second specific goal of the system was to create a customized exercise task for each subject .", "a neural network was created using the subject 's training exercise data and was then used to modify a standard exercise task into a custom exercise task . for both sets of exercises ,", "the subjects had greater rmse for the position and the velocity variables during the custom tasks ; in other words , during the custom task , their kinematic performance was worse in terms of accuracy to the instructed task .", "these results show that the subjects were less able to replicate the custom task , suggesting that the custom task was more difficult to complete .", "this may be a result of the unconstrained nature of the neural network used to create the custom task .", "no output constraints were placed on the neural network , and the researchers did not manually alter the output . as a result ,", "consequently , the subjects were continually reacting to the unpredictable trajectory of the moving sphere during the custom task but were able to anticipate the trajectory of the standard task , thereby making the standard task easier to complete . adding constraints to the neural network", "previous successful adaptive rehabilitation systems ,   used trained physiotherapists to adapt the rehabilitation tasks to the subjects based on the quantitative data .", "therefore , the therapist acted as a constraint on the output of the systems , and could modify the output exercise task to achieve the desired goal .", "future iterations of the system will include constraints on the neural network and/or a trained physiotherapist who can modify the output task in order to ensure that the custom task is an anticipatory task , not a reactionary task .", "both the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor and the emg system are easily transportable and simple to use after minimal training .", "thus , it is feasible to use the entire rehabilitation system in a home setting , as the only additional requirements are a computer and a display device like a television .", "the third specific goal for the system was to build a simple and non - rigid wearable robot that will provide subject - specific resistance training .", "the results for the wearable robot prototype demonstrate the feasibility of using pams for actuation .", "table iii shows that the exoskeleton can be modeled as a second order linear system described by ( 6 ) .", "the measured damping coefficient \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ \\zeta=0.064 $ \n \\end{document } was low , suggesting that it might be difficult to control the robot .", "however , it is likely that a human elbow will have a higher damping coefficient than the test platform and therefore will be more controllable . furthermore , the measured damping coefficient was higher during the closed - loop comparison testing of the robot \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ( \\zeta=0.13)$ \n \\end{document } , showing that the simple pressure control algorithm increased the damping of the robot .", "overall , these results indicate that a robust controller can be built that will control the robot .", "the first controller developed for the robot was based on pressure control of the pams .", "the results from the pressure control algorithm tests indicate that there was saturation of the pams at 0.75 hz and 1 hz during pressurization ; the valves did not pressurize the pams fast enough to follow the reference signal .", "this saturation most likely occurred because the reference pressure input signals were both high amplitude and high frequency , and as such required a high change rate ( 7 bar / second ) of the pressure inside the pams ( corresponding to an angle change of 100\\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ ^{\\circ}/{\\rm second}$ \n \\end{document } ) . as the pressure rose inside the valves , the pressure differential between the valves and the pressure source decreased , thereby decreasing the flow rate to the valves . eventually , the decreased flow rate was insufficient to achieve the high change rate required by the reference pressure input signals , and saturation occurred .", "this trend was not observed during depressurization because it would only occur near 0 bar , and the pressure reference input signals did not reach this level .", "while the motion bandwidths of normal human arm movements are centered at approximately 2 hz , most of these movements are relatively low amplitude and would not require high pressure change rates of the pams .", "nevertheless , future versions of the wearable robot will use a higher pressure source to increase the pressure differential ( and therefore the flow rate ) of the pams so that the robot can follow reference pressure input signals of at least 5 hz .", "additionally , force sensors will be added to the robot so that advanced control algorithms can be implemented , such as impedance or admittance control , in order to more accurately control the pneumatically actuated robot .", "the control performance of the wearable robot did not account for additional loading from the subject . in a wearable robot ,", "the subject would also be exerting force on the robot ( both assistive and resistive force ) .", "this force would need to be measured and compensated by the controller in order to accurately control the position of the robot .", "future prototypes of the robot arm should include sensors to measure the force exerted on the robot by the subject .", "the results from the tests of the combined system indicate that the pressure loop can successfully follow low amplitude pressure reference input signals at frequencies greater than 1 hz . during these tests ,", "saturation was not observed when the subject actuated the robot with a low amplitude reference input signal greater than 1 hz .", "varying the rate of change of the elbow angle also did not affect the accuracy of the robot , as it was able to follow the subject 's slow and fast movements .", "due to a slightly noisy input signal ( caused by small inaccuracies in the measured elbow angle from the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } ) and insufficient damping in the control algorithm , the robot sometimes oscillated about the desired angle .", "furthermore , this test demonstrates the feasibility of the entire robotic rehabilitation system because the subject was able to actuate the wearable robot using only the movement of his elbow .", "future iterations of the entire system will implement real - time smoothing of the elbow angle reference signal in order to compensate for the small inaccuracies in the \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } measurements , and the damping terms in the control algorithm will be increased ."], ["this study described a promising home - use robotic rehabilitation system comprised of low - cost components .", "further research is necessary to create a commercial version of this system , specifically on the prototype of the wearable robot .", "however , the data collection component of the system could be used in the near future for large scale research studies .", "the immediate next steps are to create a gui for the system to increase usability and to optimize the model generation code for speed and accuracy .", "afterwards , this part of the rehabilitation system could be used by a variety of end - users for large scale research studies on rehabilitation exercises .", "given the low cost and easy portability of the microsoft \\documentclass[12pt]{minimal } \n \\usepackage{amsmath } \n \\usepackage{wasysym } \n \\usepackage{amsfonts } \n \\usepackage{amssymb } \n \\usepackage{amsbsy } \n \\usepackage{upgreek } \n \\usepackage{mathrsfs } \n \\setlength{\\oddsidemargin}{-69pt } \n \\begin{document } \n } { } $ { \\rm kinect}^{\\rm tm}$ \n \\end{document } sensor , these studies should include home - based exercise components .", "concurrently , the next prototype of the wearable robot should be developed with the goal of having a fully functioning wearable prototype within two years .", "a limitation of this study is that the current system does not provide force feedback to the subjects .", "the wearable robot was designed to provide force feedback during the exercise tasks , but the current prototype was not tested on human subjects .", "future iterations of the robot prototype should be designed for wearability and human safety so that it can be worn by the subjects in order to provide force feedback .", "the addition of force feedback to the system should significantly enhance the results as the subject will have additional biofeedback information to help them complete the rehabilitation exercises ."]]}
{"article_id": "PMC4535111", "article_text": ["international diabetic federation estimated that in india there are 66.8 million patients in 2013 , with nearly 50% undiagnosed .", "diabetes was related to one million deaths and mortality was higher under age of 60 years . by 2030 there will more than 79 million diabetics , making it one of the major public health challenge to the country .", "observed retinopathy in 28.9% , nephropathy in 32.5% , neuropathy in 30.1% , cardio - atherosclerotic diseases in 19.2% and peripheral vascular diseases in 18.1% diabetic patients .", "the investigators of a1 cheive study observed high prevalence of macrovascular and microvascular complications among the indian diabetic patients .", "different studies conducted in countries like united states have observed the positive association of patient 's knowledge regarding the disease and self care to the treatment compliance .", "hence , this study was planned to assess knowledge about diabetes and its correlation with pharmacological and non - pharmacological compliance , among the diabetics attending a rural health center from sangli district , maharashtra ( india ) .", "this was a cross - sectional study conducted in a rural health center attached to a medical college from sangli district of maharashtra , india .", "the study population was adult type-2 diabetes patients on oral hypoglycemic drugs for at least 6 months attending the rural health centre .", "the patients attending for routine check up or drug re - prescription and consenting to participate were included in the study .", "minors , patients on insulin , hospitalized patients , patients who had undertook any sensitization program other than routine counseling by physician and those who can not read in the local language were excluded from the study .", "prevalence of knowledge regarding diabetes in the pilot study was 52.71% ; considering level of significance as 5% and error 15% ;", "study instrument was a pretested , prevalidated , self - administered questionnaire with good test - retest reliability ( spearman correlation coefficient , r = 0.81 ) and internal consistency ( cronbach 's  , r = 0.78 ) .", "it was developed in marathi language with the help of subject experts and published literature and finalized after the pilot study .", "it consists of initial section with general information of patient like age , gender etc .", ", the occupations reported by patients were classified as sedentary work , moderate work and heavy work .", "the second section consisted of questions regarding knowledge of patients about diabetes and its complications and the answers were scored .", "those scoring under 50% , 5075% and more than 75% were considered to be having poor , moderate and good knowledge , respectively .", "pharmacological compliance was self - reported , with participants reporting to have missed more than two doses in last 15 days were considered as non - compliant .", "due to lack of exact definition of compliance to the non - pharmacological management , a scale was developed with equal importance to diet modifications and physical exercises .", "the maximum possible score was 12 and the participants scoring above 9 ( 75% ) were considered as compliant to non - pharmacological management .", "mean , standard deviation , percentage , chi - square and binary logistic regression were applied .", "data from the pilot study and incomplete questionnaires were not included in the final analysis .", "out of 329 questionnaires collected , 307 were complete and hence used in the final analysis .", "two hundred twenty - three ( 72.6% ) participants were male while 84 ( 27.4% ) were female [ table 1 ] . the mean age of study participants was 55.6 years ( range 3585 years and standard deviation  12.22 years ) .", "two hundred ninety - two ( 95.1% ) participants were married . while considering the educational status , 106 ( 34.5% )", "had studied up to secondary school and 201 ( 65.5% ) had attended college . among the participants : 208 ( 67.8% ) , 56 ( 18.2% ) and 43 ( 14% ) were sedentary , moderate and heavy workers , respectively .", "the mean morbidity with diabetes was 10.7 years ( range : 1 to 44 years and ; standard deviation : 10.02 years ) .", "age group and gender distribution of the participants the pharmacological compliance was reported by 234 ( 76.2% ) participants .", "the mean score for the non - pharmacological compliance was 8.35 ( standard deviation : 2.8 ) . based on preset criteria of score , 156 ( 50.8% ) participants were considered to compliant to the non - pharmacological management .", "the mean score for knowledge regarding diabetes was 14.82 ( standard deviation : 3.5 ) . only 29 ( 9.4% ) participants had good knowledge , whereas 219 ( 71.3% ) had moderate and 59 ( 19.2% ) participants had poor knowledge .", "age was not associated with the knowledge ( chi - square 5.47 , p = 0.49 ) , however , higher percentage of older age group ( 75 yrs ) participants had poor knowledge .", "gender was associated with knowledge ( chi - square 10.78 , p = 0.005 ) ; higher percentage of male participants ( 81.6% ) had moderate to good knowledge as compared to females ( 78.6% ) . marital status and occupation", "education was not associated with the knowledge ( chi - square 2.23 , p = 0.33 ) , however higher percentage of participants who had attended college had moderate to good knowledge .", "pharmacological compliance was associated with knowledge . among the participants with good compliance , 88.5% had moderate to good knowledge as compared to only 56.2% participants with poor compliance .", "the moderate to good knowledge was present in 87.8% participants with good compliance as compared to only 73.5% with poor compliance [ table 2 ] .", "association between knowledge regarding diabetes with pharmacological compliance and non - pharmacological management compliance binary logistic regression was applied with knowledge regarding diabetes as dependent variable , while age , gender , education , occupation , pharmacological compliance and non - pharmacological management compliance as independent variables .", "the chance accuracy rate for the model is 83.6% , which was greater than calculated chance accuracy rate ( 67.3% ) .", "pharmacological and non - pharmacological management compliance were the highly significant predictors , while education and age were also significant predictors for knowledge regarding diabetes [ table 3 ] .", "binary logistic regression model - association of age , gender , education occupation , pharmacological compliance and non - pharmacological management compliance with knowledge regarding to diabetes", "in the current study , we observed that the patients compliant with pharmacological and non - pharmacological management had good knowledge regarding diabetes .", "al - qazaz hkh et al . , ( 2010 ) , observed significant correlations between the knowledge about diabetes and adherence to the treatment in subjects from penang .", "when carried out a study at al - makhfia governmental diabetes primary healthcare clinic in nablus , palestine in 2012 , observed that diabetic patients with high knowledge were less likely to be non - adherent .", "chaudhary et al . , from multan ( pakistan ) in 2010 , observed that male gender , higher education and older age ( > 40 years ) were associated with the knowledge regarding diabetes mellitus .", "however , age groups used by them were only two : < 40 years and > 40 years , while in the current study four age groups were considered with lowest being 3550 years .", "abdella and mohammad studied awareness of diabetic patients about their illness and associated complications in ethiopia ( 2012 ) ; majority of patients ( 67% ) had good knowledge on actions to be taken on the occurrence of acute complications and reasons for developing acute complications . while we observed that less than 10% participants had good knowledge .", "however , there is difference in the questionnaire used in both the studies and most of the participants from abdella study had undergone sensitization unlike the current study subjects .", "( bangladesh ) 2013 , observed that 19% respondents had poor knowledge , while 68% had average and 13% had good knowledge .", "they also deduced that male gender and higher educated people are likely to have better knowledge regarding diabetes .", "conducted a study on adults in tamaka village , kolar ( india ) in 2009 .", "observed that male gender , higher education and younger age of respondents was significantly associated with level of knowledge , while type of occupation is not associated with it .", "bansal et al . observed compliance to medication in 82.5% patients from rural area of ludhiana district , india .", "rao et al . observed that 83.6% patients from southern karnataka were on regular medication .", "santhanakrishnan et al . observed compliance to oral hypoglycemic agents in 76% subjects , dietary modifications in 81.4% and physical activity in 37% subjects from puducherry , india . while in the current study , pharmacological compliance", "was reported by 76.2% participants and compliance to the non - pharmacological management was reported by 50.8% participants .", "being a self - administered questionnaire reporting bias can not be totally eliminated ; there may be over - estimation of compliance .", "confounders like economical state , availability of medications , counseling by health professionals , exposure to information through media , additional use of ayurvedic / homeopathic / other indigenous systems of medicines etc .", ", may be present and are not considered in the current study . only self - reported compliance was considered without confirming .", ". however , many of the patients have poor knowledge regarding the disease , the situation is worse among the females .", "lower level of literacy and old age hinder the presence of knowledge regarding the diabetes .", "seminars , counseling sessions and workshop should be arranged periodically for diabetic patients to increase their awareness regarding diabetes disease in total per say ."], "abstract_text": ["<S> introduction : diabetes is an important public health problem of india . </S>", "<S> studies have shown that increase in patients knowledge regarding the disease results in better compliance to treatment and decrease in complications . </S>", "<S> this study was planned to assess the knowledge about diabetes and its correlation with pharmacological and non - pharmacological compliance , among the diabetic patients attending rural health center from sangli district , maharashtra ( india).materials and methods : the study was conducted during september to november 2014 . </S>", "<S> the study subjects were all willing adult patients with type ii diabetes mellitus attending a selected rural hospital . </S>", "<S> the study tool was pretested and self - administered questionnaire . </S>", "<S> analysis was done using microsoft excel and spss-22.results:total study participants were 307 in number , with the mean age of 55.6 years . </S>", "<S> the mean morbidity with diabetes was 10.7 years . </S>", "<S> only 23.8% had good knowledge regarding diabetes , while 19.2% participants had poor knowledge . </S>", "<S> knowledge was significantly associated with the compliance to the pharmacological and non - pharmacological management.conclusion:although most of the patients were suffering with diabetes for many years there is lack of knowledge regarding the disease and self care . </S>", "<S> the compliance to the management of diabetes was better in patients with good knowledge . </S>", "<S> seminars , counseling sessions and workshop should be arranged periodically for diabetic patients to increase their awareness . </S>"], "labels": null, "section_names": ["Introduction", "Material and Methods", "Results", "Discussion", "Conclusion"], "sections": [["international diabetic federation estimated that in india there are 66.8 million patients in 2013 , with nearly 50% undiagnosed .", "diabetes was related to one million deaths and mortality was higher under age of 60 years . by 2030 there will more than 79 million diabetics , making it one of the major public health challenge to the country .", "observed retinopathy in 28.9% , nephropathy in 32.5% , neuropathy in 30.1% , cardio - atherosclerotic diseases in 19.2% and peripheral vascular diseases in 18.1% diabetic patients .", "the investigators of a1 cheive study observed high prevalence of macrovascular and microvascular complications among the indian diabetic patients .", "different studies conducted in countries like united states have observed the positive association of patient 's knowledge regarding the disease and self care to the treatment compliance .", "hence , this study was planned to assess knowledge about diabetes and its correlation with pharmacological and non - pharmacological compliance , among the diabetics attending a rural health center from sangli district , maharashtra ( india ) ."], ["this was a cross - sectional study conducted in a rural health center attached to a medical college from sangli district of maharashtra , india .", "the study population was adult type-2 diabetes patients on oral hypoglycemic drugs for at least 6 months attending the rural health centre .", "the patients attending for routine check up or drug re - prescription and consenting to participate were included in the study .", "minors , patients on insulin , hospitalized patients , patients who had undertook any sensitization program other than routine counseling by physician and those who can not read in the local language were excluded from the study .", "prevalence of knowledge regarding diabetes in the pilot study was 52.71% ; considering level of significance as 5% and error 15% ;", "study instrument was a pretested , prevalidated , self - administered questionnaire with good test - retest reliability ( spearman correlation coefficient , r = 0.81 ) and internal consistency ( cronbach 's  , r = 0.78 ) .", "it was developed in marathi language with the help of subject experts and published literature and finalized after the pilot study .", "it consists of initial section with general information of patient like age , gender etc .", ", the occupations reported by patients were classified as sedentary work , moderate work and heavy work .", "the second section consisted of questions regarding knowledge of patients about diabetes and its complications and the answers were scored .", "those scoring under 50% , 5075% and more than 75% were considered to be having poor , moderate and good knowledge , respectively .", "pharmacological compliance was self - reported , with participants reporting to have missed more than two doses in last 15 days were considered as non - compliant .", "due to lack of exact definition of compliance to the non - pharmacological management , a scale was developed with equal importance to diet modifications and physical exercises .", "the maximum possible score was 12 and the participants scoring above 9 ( 75% ) were considered as compliant to non - pharmacological management .", "mean , standard deviation , percentage , chi - square and binary logistic regression were applied .", "data from the pilot study and incomplete questionnaires were not included in the final analysis ."], ["out of 329 questionnaires collected , 307 were complete and hence used in the final analysis .", "two hundred twenty - three ( 72.6% ) participants were male while 84 ( 27.4% ) were female [ table 1 ] . the mean age of study participants was 55.6 years ( range 3585 years and standard deviation  12.22 years ) .", "two hundred ninety - two ( 95.1% ) participants were married . while considering the educational status , 106 ( 34.5% )", "had studied up to secondary school and 201 ( 65.5% ) had attended college . among the participants : 208 ( 67.8% ) , 56 ( 18.2% ) and 43 ( 14% ) were sedentary , moderate and heavy workers , respectively .", "the mean morbidity with diabetes was 10.7 years ( range : 1 to 44 years and ; standard deviation : 10.02 years ) .", "age group and gender distribution of the participants the pharmacological compliance was reported by 234 ( 76.2% ) participants .", "the mean score for the non - pharmacological compliance was 8.35 ( standard deviation : 2.8 ) . based on preset criteria of score , 156 ( 50.8% ) participants were considered to compliant to the non - pharmacological management .", "the mean score for knowledge regarding diabetes was 14.82 ( standard deviation : 3.5 ) . only 29 ( 9.4% ) participants had good knowledge , whereas 219 ( 71.3% ) had moderate and 59 ( 19.2% ) participants had poor knowledge .", "age was not associated with the knowledge ( chi - square 5.47 , p = 0.49 ) , however , higher percentage of older age group ( 75 yrs ) participants had poor knowledge .", "gender was associated with knowledge ( chi - square 10.78 , p = 0.005 ) ; higher percentage of male participants ( 81.6% ) had moderate to good knowledge as compared to females ( 78.6% ) . marital status and occupation", "education was not associated with the knowledge ( chi - square 2.23 , p = 0.33 ) , however higher percentage of participants who had attended college had moderate to good knowledge .", "pharmacological compliance was associated with knowledge . among the participants with good compliance , 88.5% had moderate to good knowledge as compared to only 56.2% participants with poor compliance .", "the moderate to good knowledge was present in 87.8% participants with good compliance as compared to only 73.5% with poor compliance [ table 2 ] .", "association between knowledge regarding diabetes with pharmacological compliance and non - pharmacological management compliance binary logistic regression was applied with knowledge regarding diabetes as dependent variable , while age , gender , education , occupation , pharmacological compliance and non - pharmacological management compliance as independent variables .", "the chance accuracy rate for the model is 83.6% , which was greater than calculated chance accuracy rate ( 67.3% ) .", "pharmacological and non - pharmacological management compliance were the highly significant predictors , while education and age were also significant predictors for knowledge regarding diabetes [ table 3 ] .", "binary logistic regression model - association of age , gender , education occupation , pharmacological compliance and non - pharmacological management compliance with knowledge regarding to diabetes"], ["in the current study , we observed that the patients compliant with pharmacological and non - pharmacological management had good knowledge regarding diabetes .", "al - qazaz hkh et al . , ( 2010 ) , observed significant correlations between the knowledge about diabetes and adherence to the treatment in subjects from penang .", "when carried out a study at al - makhfia governmental diabetes primary healthcare clinic in nablus , palestine in 2012 , observed that diabetic patients with high knowledge were less likely to be non - adherent .", "chaudhary et al . , from multan ( pakistan ) in 2010 , observed that male gender , higher education and older age ( > 40 years ) were associated with the knowledge regarding diabetes mellitus .", "however , age groups used by them were only two : < 40 years and > 40 years , while in the current study four age groups were considered with lowest being 3550 years .", "abdella and mohammad studied awareness of diabetic patients about their illness and associated complications in ethiopia ( 2012 ) ; majority of patients ( 67% ) had good knowledge on actions to be taken on the occurrence of acute complications and reasons for developing acute complications . while we observed that less than 10% participants had good knowledge .", "however , there is difference in the questionnaire used in both the studies and most of the participants from abdella study had undergone sensitization unlike the current study subjects .", "( bangladesh ) 2013 , observed that 19% respondents had poor knowledge , while 68% had average and 13% had good knowledge .", "they also deduced that male gender and higher educated people are likely to have better knowledge regarding diabetes .", "conducted a study on adults in tamaka village , kolar ( india ) in 2009 .", "observed that male gender , higher education and younger age of respondents was significantly associated with level of knowledge , while type of occupation is not associated with it .", "bansal et al . observed compliance to medication in 82.5% patients from rural area of ludhiana district , india .", "rao et al . observed that 83.6% patients from southern karnataka were on regular medication .", "santhanakrishnan et al . observed compliance to oral hypoglycemic agents in 76% subjects , dietary modifications in 81.4% and physical activity in 37% subjects from puducherry , india . while in the current study , pharmacological compliance", "was reported by 76.2% participants and compliance to the non - pharmacological management was reported by 50.8% participants .", "being a self - administered questionnaire reporting bias can not be totally eliminated ; there may be over - estimation of compliance .", "confounders like economical state , availability of medications , counseling by health professionals , exposure to information through media , additional use of ayurvedic / homeopathic / other indigenous systems of medicines etc .", ", may be present and are not considered in the current study . only self - reported compliance was considered without confirming ."], [". however , many of the patients have poor knowledge regarding the disease , the situation is worse among the females .", "lower level of literacy and old age hinder the presence of knowledge regarding the diabetes .", "seminars , counseling sessions and workshop should be arranged periodically for diabetic patients to increase their awareness regarding diabetes disease in total per say ."]]}
{"article_id": "PMC2040486", "article_text": ["lactic acid was first found and described in sour milk by the swedish chemist karl wilhelm scheele ( 17421786 ) in 1780  .", "the swedish chemist jns jakob berzelius ( 17791848 ) found lactic acid in fluid extracted from meat in 1808  [ 2 , 3 ] , and the german chemist justus von liebig ( 18031873 ) , who established the world 's first school of chemistry at giessen , proved that lactic acid was always present in muscular tissue of dead organisms  . in 1859 , emil heinrich du bois - reymond ( 18181896 ) published several articles on the influence of lactic acid on muscle contraction  [ 59 ] .", "araki and zillessen found that if they interrupted oxygen supply to muscles in mammals and birds , lactic acid was formed and increased  [ 1014 ] .", "this was the first demonstration of the relationship between tissue hypoxia and the formation of lactate .", "the occurrence of increased lactic acid in blood ( hyperlactataemia ) nowadays reflects severe illness , in which the increased blood lactate levels may result from both anaerobic and aerobic production or from a  decreased clearance .", "it was the german physician  chemist johann joseph scherer who first demonstrated the occurrence of lactic acid in human blood under pathological conditions after death in 1843 and 1851  [ 15 , 17 ] , and carl folwarczny in 1858 who first demonstrated lactic acid in blood of a  living patient . in this article", "we wish to honour scherer 's forgotten observations and describe the influence of his finding on further research on lactic acid at the end of the 19th century .", "born on 18 march 1814 in aschaffenburg , germany , scherer studied medicine , chemistry , geology and mineralogy at the university of wrzburg between 1833 and 1836 .", "he obtained his phd in medicine and surgery in 1838 with a  thesis entitled  versuche ber die wirkung einiger gifte auf verscheidene thierclassen ", "he practised medicine in wipfeld , but inspired by the chemist ernst von bibra ( 18061878 ) he completed his studies in chemistry at the university of munich between 18381840  . in 1840", "he was employed at the laboratory of justus liebig at giessen , and became professor at the medical faculty in 1842 , professor of organic chemistry in 1847 , and later professor of general , anorganic and pharmaceutical chemistry .", "his work especially concerned quantitative research on blood and urine in pathological conditions . in 1843", "he published his book  chemische und mikroskopische untersuchungen zur pathologie angestellt an den kliniken des julius - hospitales zu wrzburg ( chemical and microscopic investigations of pathology carried out at the julius clinic at wrzburg )   ( fig .  1 )", ", in which he described 72 case reports , giving details on clinical course , diagnosis , and results obtained during autopsy and analysis of body fluids .", "in one chapter in his 1843 book entitled ' untersuchungen von krankhaften stoffen bei der i m winter 18421843 in wrzburg und der umgegend herrschenden puerperal - fieber - epidemie ' ( investigations of pathological substances obtained during the epidemic of puerperal fever which occurred in the winter of 18421843 in and around wrzburg ) scherer described the cases of seven young women who all died peripartum .", "one of the women , the 23-year - old primipara eva rumpel , gave birth to a  healthy child on 9 january 1843 .", "the same night she developed a  painfully swollen abdomen and became ill , feverish , and sweaty , with rapid pulse and severe thirst .", "the next evening she deteriorated , became delirious , with anxious breathing , a  tense abdomen , cold extremities and rapid pulse , finally losing consciousness .", "a.m. , 36  h after the onset of the first symptoms , she died . during autopsy", ", severe purulent endometritis , vaginal pus , pulmonary oedema , and shock liver and shock spleen were found .", "the blood that was obtained directly from the heart was chemically analysed , in which lactic acid was found .", "most likely this unfortunate woman had died from a  fulminant septic shock caused by group a  haemolytic streptococci ( streptococcus pyogenes ) .", "another patient , the 28-year - old , 7 months pregnant ( second pregnancy ) margaretha glck , was , after being icteric , nauseous , vomiting and complaining about epigastric pain for 8 days , admitted to the lying - in birth clinic on 6 february 1843 .", "four days later she was transferred to the hospital with severe nosebleeds and generalised exanthema or purpura . in the evening she suffered from severe gastric bleeding and epistaxis , showing rapid pulse , cold extremities and dizziness . the next morning", ", she was transferred back to the birth clinic , where she gave birth to a  premature child ( 30 weeks ) and suffered from a  severe post - partum fluxus .", "she was again transferred to the hospital with the following symptoms : cold clammy skin , tachycardia , severe lochia and persistent exanthema or purpura , but without signs of an acute abdomen . during the night of february 11", "autopsy revealed a  small intracerebral haematoma , normal lungs without pulmonary oedema , ascites and an anaemic , foul smelling uterus filled with purulent and decayed tissue and pus .", "blood was also obtained directly from the heart during autopsy and lactic acid was found . in this case", "we could think of a  haemorrhagic shock and cerebral haemorrhage due to clotting disorders possibly resulting from either acute fatty liver of pregnancy / hellp syndrome , idiopathic thrombocytopenic purpura , thrombotic microangiopathy ( ttp / hus ) or dic .", "in the conclusions of his 1843 book , scherer attached high importance to the fact that he found lactic acid in cases of puerperal fever , which he had not found before in healthy persons .", "he held the opinion that lactic acid was formed in blood during bodily deterioration in severe diseases like puerperal fever .", "lactic acid was thus described for the first time in human blood and was demonstrated for the first time as a  symptom of septic and haemorrhagic shock . in the same period a  junior obstetrician in vienna , ignaz philipp semmelweis ( 18181865 ) , discovered in 1847 that physicians carried infectious particles on their hands from the mortuary to the obstetrical clinic , causing puerperal fever and puerperal sepsis , and he introduced a  successful method for its prevention .", "louis pasteur ( 18221894 ) found in 1879 that infection with streptococci was the most important cause of puerperal fever  .", "scherer worked closely with the famous pathologist rudolf virchow ( 18211902 ) on several projects ( fig .  2 ) . in 1851", "virchow performed an autopsy on a  patient who had died from leukaemia and offered scherer blood from this patient for analysis .", "the results of this analysis were published the same year in the  verhandlungen der physikalisch - medicinischen gesellschaft in wrzburg  .", "virchow and scherer had previously studied the spleens of patients who died from leukaemia , and were curious if they could find the same results in the blood .", "scherer reached the conclusion that : the blood of this patient contains :  ameisensure , essigsure und milchsure , die gleichfalls von mir schon frher als in der milzflssigkeit vorkommend bezeichnet wurden ", "( formic acid , acetic acid , and lactic acid , as also found by me previously in fluids from the spleen ) . \n", "2johann joseph scherer ( left ) and rudolf virchow ( right ) in 1849 johann joseph scherer ( left ) and rudolf virchow ( right ) in 1849", "scherer 's observations inspired others to conduct further research , primarily in patients with leukaemia  [ 1922 ] , but also in patients with other conditions and diseases and in animal experiments with dogs and rabbits  .", "while scherer found lactic acid in blood obtained after death during autopsy , mosler and krner   mention an observation made by carl folwarczny , published in the allgemeinen wiener medicinischen zeitung in 1858 , where blood was withdrawn from a  leukaemia patient during life , analysed according to scherer 's method , and found positive for lactic acid . in addition , carl folwarczny described in 1863 in his ' handbuch der physiologischen chemie '   that lactic acid can be found in the blood of patients with leukaemia , septicaemia ( pyaemia ) and in conditions leading to septicaemia like puerperal fever , the latter probably after scherer 's observations . in an extensive article , the berliner physician georg salomon  , who had serious doubts that the occurrence of lactic acid in blood was mostly related to leukaemia , proved in 1878 that lactic acid was also present in the blood of patients who were suffering and died from other diseases .", "he studied blood obtained during autopsy from cadavers , but also blood from patients obtained by bloodletting or cupping , and in some cases he compared the blood before and after death .", "he was able to demonstrate lactic acid in the blood of patients suffering from leukaemia , ( pernicious ) anaemia , congestive heart failure , chronic obstructive pulmonary disease , pleuritis , pericarditis , pneumonia and several solid malignant tumours .", "gaglio   is often erroneously mentioned as the first author to find lactic acid in blood  [ 2729 ] .", "he was able to demonstrate lactic acid in fresh arterial blood withdrawn from dogs and rabbits after bloodletting .", "both gaglio and berlinerblau , however , neglected previous research , as indignantly described by salomon in 1888 [  ich erlaube mir , den inhalt meiner arbeiten , die von gaglio nur ganz flchtig , von berlinerblau gar nicht berhrt sind , in krze zu reproduciren ", "( i take the liberty of summarizing the contents of my work , which was mentioned only briefly by gaglio and not at all by berlinerblau ) ]  .", "the japanese chemist trasaburo araki showed that the amount of lactic acid in exhausted muscle results from muscle activation  .", "irisawa  , inspired by the results obtained by salomon and gaglio , obtained fresh blood of 11 dying patients with serious conditions . in six cases he found hyperlactataemia , in four cases normal values .", "he speculated on the aetiology of hyperlactataemia , the most plausible cause being the severe hypoxia during the dying process . in an experiment in which he made a  dog anaemic for several days", ", he found a  rise in lactic acid levels during the time leading up to death . in cambridge ( uk ) ,", "walter morley fletcher ( 18731933 ) and frederick gowland hopkins ( 18611947 ) worked together on the metabolic changes occurring in muscular contractions and rigor mortis under anaerobic conditions , and found that lactate was the product of carbohydrate metabolism  .", "their classic 1907 paper demonstrated rigorously that muscle contraction is accompanied by the anaerobic formation of lactic acid , which is removed aerobically , at a  rate depending on the level of exposure to oxygen  .", "poul astrup and john severingshaus mentioned scherer 's 1851 article as first demonstration of lactic acid in blood , but overlooked the 1843 cases and folwarczny 's work  . in conclusion , scherer 's 1843 case reports   should be cited as the first description of lactic acid in human blood and also as the first demonstration of lactic acid as a  pathological finding in septic and haemorrhagic shock .", "folwarczny , in 1858 , was the first to demonstrate lactic acid in blood in a  living patient ."], "abstract_text": ["<S> lactic acid was first found and described in sour milk by karl wilhelm scheele ( 17421786 ) in 1780 . the german physician  </S>", "<S> chemist johann joseph scherer ( 18411869 ) demonstrated the occurrence of lactic acid in human blood under pathological conditions in 1843 and 1851 . in this article </S>", "<S> we honour the forgotten observations by scherer and describe the influence of scherer 's finding on further research on lactic acid at the end of the 19th century . </S>", "<S> we conclude that scherer 's 1843 case reports should be cited as the first description of lactic acid in human blood after death and also as the first demonstration of lactic acid as a  pathological finding in septic and haemorrhagic shock . </S>", "<S> carl folwarczny was , in 1858 , the first to demonstrate lactic acid in blood in a  living patient . </S>"], "labels": null, "section_names": ["Introduction", "Biography of Johann Joseph Scherer", "The 1843 cases", "The 1851 article", "Further research"], "sections": [["lactic acid was first found and described in sour milk by the swedish chemist karl wilhelm scheele ( 17421786 ) in 1780  .", "the swedish chemist jns jakob berzelius ( 17791848 ) found lactic acid in fluid extracted from meat in 1808  [ 2 , 3 ] , and the german chemist justus von liebig ( 18031873 ) , who established the world 's first school of chemistry at giessen , proved that lactic acid was always present in muscular tissue of dead organisms  . in 1859 , emil heinrich du bois - reymond ( 18181896 ) published several articles on the influence of lactic acid on muscle contraction  [ 59 ] .", "araki and zillessen found that if they interrupted oxygen supply to muscles in mammals and birds , lactic acid was formed and increased  [ 1014 ] .", "this was the first demonstration of the relationship between tissue hypoxia and the formation of lactate .", "the occurrence of increased lactic acid in blood ( hyperlactataemia ) nowadays reflects severe illness , in which the increased blood lactate levels may result from both anaerobic and aerobic production or from a  decreased clearance .", "it was the german physician  chemist johann joseph scherer who first demonstrated the occurrence of lactic acid in human blood under pathological conditions after death in 1843 and 1851  [ 15 , 17 ] , and carl folwarczny in 1858 who first demonstrated lactic acid in blood of a  living patient . in this article", "we wish to honour scherer 's forgotten observations and describe the influence of his finding on further research on lactic acid at the end of the 19th century ."], ["born on 18 march 1814 in aschaffenburg , germany , scherer studied medicine , chemistry , geology and mineralogy at the university of wrzburg between 1833 and 1836 .", "he obtained his phd in medicine and surgery in 1838 with a  thesis entitled  versuche ber die wirkung einiger gifte auf verscheidene thierclassen ", "he practised medicine in wipfeld , but inspired by the chemist ernst von bibra ( 18061878 ) he completed his studies in chemistry at the university of munich between 18381840  . in 1840", "he was employed at the laboratory of justus liebig at giessen , and became professor at the medical faculty in 1842 , professor of organic chemistry in 1847 , and later professor of general , anorganic and pharmaceutical chemistry .", "his work especially concerned quantitative research on blood and urine in pathological conditions . in 1843", "he published his book  chemische und mikroskopische untersuchungen zur pathologie angestellt an den kliniken des julius - hospitales zu wrzburg ( chemical and microscopic investigations of pathology carried out at the julius clinic at wrzburg )   ( fig .  1 )", ", in which he described 72 case reports , giving details on clinical course , diagnosis , and results obtained during autopsy and analysis of body fluids ."], ["in one chapter in his 1843 book entitled ' untersuchungen von krankhaften stoffen bei der i m winter 18421843 in wrzburg und der umgegend herrschenden puerperal - fieber - epidemie ' ( investigations of pathological substances obtained during the epidemic of puerperal fever which occurred in the winter of 18421843 in and around wrzburg ) scherer described the cases of seven young women who all died peripartum .", "one of the women , the 23-year - old primipara eva rumpel , gave birth to a  healthy child on 9 january 1843 .", "the same night she developed a  painfully swollen abdomen and became ill , feverish , and sweaty , with rapid pulse and severe thirst .", "the next evening she deteriorated , became delirious , with anxious breathing , a  tense abdomen , cold extremities and rapid pulse , finally losing consciousness .", "a.m. , 36  h after the onset of the first symptoms , she died . during autopsy", ", severe purulent endometritis , vaginal pus , pulmonary oedema , and shock liver and shock spleen were found .", "the blood that was obtained directly from the heart was chemically analysed , in which lactic acid was found .", "most likely this unfortunate woman had died from a  fulminant septic shock caused by group a  haemolytic streptococci ( streptococcus pyogenes ) .", "another patient , the 28-year - old , 7 months pregnant ( second pregnancy ) margaretha glck , was , after being icteric , nauseous , vomiting and complaining about epigastric pain for 8 days , admitted to the lying - in birth clinic on 6 february 1843 .", "four days later she was transferred to the hospital with severe nosebleeds and generalised exanthema or purpura . in the evening she suffered from severe gastric bleeding and epistaxis , showing rapid pulse , cold extremities and dizziness . the next morning", ", she was transferred back to the birth clinic , where she gave birth to a  premature child ( 30 weeks ) and suffered from a  severe post - partum fluxus .", "she was again transferred to the hospital with the following symptoms : cold clammy skin , tachycardia , severe lochia and persistent exanthema or purpura , but without signs of an acute abdomen . during the night of february 11", "autopsy revealed a  small intracerebral haematoma , normal lungs without pulmonary oedema , ascites and an anaemic , foul smelling uterus filled with purulent and decayed tissue and pus .", "blood was also obtained directly from the heart during autopsy and lactic acid was found . in this case", "we could think of a  haemorrhagic shock and cerebral haemorrhage due to clotting disorders possibly resulting from either acute fatty liver of pregnancy / hellp syndrome , idiopathic thrombocytopenic purpura , thrombotic microangiopathy ( ttp / hus ) or dic .", "in the conclusions of his 1843 book , scherer attached high importance to the fact that he found lactic acid in cases of puerperal fever , which he had not found before in healthy persons .", "he held the opinion that lactic acid was formed in blood during bodily deterioration in severe diseases like puerperal fever .", "lactic acid was thus described for the first time in human blood and was demonstrated for the first time as a  symptom of septic and haemorrhagic shock . in the same period a  junior obstetrician in vienna , ignaz philipp semmelweis ( 18181865 ) , discovered in 1847 that physicians carried infectious particles on their hands from the mortuary to the obstetrical clinic , causing puerperal fever and puerperal sepsis , and he introduced a  successful method for its prevention .", "louis pasteur ( 18221894 ) found in 1879 that infection with streptococci was the most important cause of puerperal fever  ."], ["scherer worked closely with the famous pathologist rudolf virchow ( 18211902 ) on several projects ( fig .  2 ) . in 1851", "virchow performed an autopsy on a  patient who had died from leukaemia and offered scherer blood from this patient for analysis .", "the results of this analysis were published the same year in the  verhandlungen der physikalisch - medicinischen gesellschaft in wrzburg  .", "virchow and scherer had previously studied the spleens of patients who died from leukaemia , and were curious if they could find the same results in the blood .", "scherer reached the conclusion that : the blood of this patient contains :  ameisensure , essigsure und milchsure , die gleichfalls von mir schon frher als in der milzflssigkeit vorkommend bezeichnet wurden ", "( formic acid , acetic acid , and lactic acid , as also found by me previously in fluids from the spleen ) . \n", "2johann joseph scherer ( left ) and rudolf virchow ( right ) in 1849 johann joseph scherer ( left ) and rudolf virchow ( right ) in 1849"], ["scherer 's observations inspired others to conduct further research , primarily in patients with leukaemia  [ 1922 ] , but also in patients with other conditions and diseases and in animal experiments with dogs and rabbits  .", "while scherer found lactic acid in blood obtained after death during autopsy , mosler and krner   mention an observation made by carl folwarczny , published in the allgemeinen wiener medicinischen zeitung in 1858 , where blood was withdrawn from a  leukaemia patient during life , analysed according to scherer 's method , and found positive for lactic acid . in addition , carl folwarczny described in 1863 in his ' handbuch der physiologischen chemie '   that lactic acid can be found in the blood of patients with leukaemia , septicaemia ( pyaemia ) and in conditions leading to septicaemia like puerperal fever , the latter probably after scherer 's observations . in an extensive article , the berliner physician georg salomon  , who had serious doubts that the occurrence of lactic acid in blood was mostly related to leukaemia , proved in 1878 that lactic acid was also present in the blood of patients who were suffering and died from other diseases .", "he studied blood obtained during autopsy from cadavers , but also blood from patients obtained by bloodletting or cupping , and in some cases he compared the blood before and after death .", "he was able to demonstrate lactic acid in the blood of patients suffering from leukaemia , ( pernicious ) anaemia , congestive heart failure , chronic obstructive pulmonary disease , pleuritis , pericarditis , pneumonia and several solid malignant tumours .", "gaglio   is often erroneously mentioned as the first author to find lactic acid in blood  [ 2729 ] .", "he was able to demonstrate lactic acid in fresh arterial blood withdrawn from dogs and rabbits after bloodletting .", "both gaglio and berlinerblau , however , neglected previous research , as indignantly described by salomon in 1888 [  ich erlaube mir , den inhalt meiner arbeiten , die von gaglio nur ganz flchtig , von berlinerblau gar nicht berhrt sind , in krze zu reproduciren ", "( i take the liberty of summarizing the contents of my work , which was mentioned only briefly by gaglio and not at all by berlinerblau ) ]  .", "the japanese chemist trasaburo araki showed that the amount of lactic acid in exhausted muscle results from muscle activation  .", "irisawa  , inspired by the results obtained by salomon and gaglio , obtained fresh blood of 11 dying patients with serious conditions . in six cases he found hyperlactataemia , in four cases normal values .", "he speculated on the aetiology of hyperlactataemia , the most plausible cause being the severe hypoxia during the dying process . in an experiment in which he made a  dog anaemic for several days", ", he found a  rise in lactic acid levels during the time leading up to death . in cambridge ( uk ) ,", "walter morley fletcher ( 18731933 ) and frederick gowland hopkins ( 18611947 ) worked together on the metabolic changes occurring in muscular contractions and rigor mortis under anaerobic conditions , and found that lactate was the product of carbohydrate metabolism  .", "their classic 1907 paper demonstrated rigorously that muscle contraction is accompanied by the anaerobic formation of lactic acid , which is removed aerobically , at a  rate depending on the level of exposure to oxygen  .", "poul astrup and john severingshaus mentioned scherer 's 1851 article as first demonstration of lactic acid in blood , but overlooked the 1843 cases and folwarczny 's work  . in conclusion , scherer 's 1843 case reports   should be cited as the first description of lactic acid in human blood and also as the first demonstration of lactic acid as a  pathological finding in septic and haemorrhagic shock .", "folwarczny , in 1858 , was the first to demonstrate lactic acid in blood in a  living patient ."]]}
{"article_id": "PMC4268563", "article_text": ["panax ginseng ( ginseng ) has been used traditionally in eastern asia over thousands of years .", "it has been used orally to treat various diseases including hypertension , diabetes mellitus , liver and kidney dysfunction , mental disorders , and postmenopausal disorders .", "in addition , topical applications have also been used to heal wounds and reduce skin inflammation . in the past few decades", ", it has been proved that ginseng extracts actually show a wide range of effects against human diseases .", "their potential therapeutic effects have been mainly attributed to its immunomodulatory , neuroprotective , antioxidative , antitumor , and hepatoprotective activities .", "ginseng contains a number of active ingredients including ginsenosides , polysaccharides , phytosterols , peptides , polyacetylenes , fatty acids , and polyacetylenic alcohols , which have different effects on carbohydrate and lipid metabolism , cognition , angiogenesis , and the neuroendocrine , immune , cardiovascular , and central nervous systems . among the active constituents of ginseng ,", "ginsenosides are known to be the major biologically active components of ginseng and the most widely studied .", "several studies have shown that ginsenosides play important roles in the pharmacological effects of ginseng .", ". however , ginseng contains other constituents , including ginsenoyne , phenolic compounds , polyacetylenes , sesquiterpenes , methoxypyrazine , alkylpyrazine derivatives , sesquiterpene alcohols , panasinsanols , and -carboline .", "white ginseng is peeled , dried , ginseng root and red ginseng is produced by steaming fresh ginseng root at 98100c for 23  h , and then drying until the moisture content is < 15% .", "red and white ginseng have both been shown to have immunomodulatory , anti - inflammatory , antioxidant , and antiatopic activities . moreover ,", "red ginseng has been reported to have more potent pharmacological activities than white ginseng in some respects [ 2022 ] .", "the differences in biological activities between red and white ginseng are caused by the chemical changes of ginsenosides after the steaming process .", "steaming partially converts the original ginsenosides to deglycoslated derivatives . as a result , the species and amounts of ginsenosides are quite different based on the processing method used .", "chu et  al   showed that a total of 53 and 43 compounds were tentatively identified in white ginseng and red ginseng samples , respectively .", "the featured compounds are mainly malonyl ginsenosides in white ginseng , and decarboxyl products of mal - ginsenosides and the dehydrated compounds from polar ginsenosides were characteristic in red ginseng .", "it is interesting that ginsenosides show a wide variety of biological activities , although the absorption rates from orally administered intact ginsenosides are very low . in the human intestinal tract ,", "thus , the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . in this context", "several studies showed that the transformation of ginsenosides into deglycosylated ginsenosides is needed to increase ginseng 's effectiveness in  vivo \n .", "abnormal changes in skin color induce significant cosmetic problems with a negative effect on quality of life .", "there are two groups of pigmentary disorders : disorders of the quantitative and qualitative distribution of normal pigment and the abnormal presence of exogenous or endogenous pigments in the skin .", "hyperpigmentation - related diseases include melasma , lentigines , nevus , ephelis , freckles , postinflammatory hyperpigmentation , and age spots .", "postinflammatory hyperpigmentation appears in many skin conditions , including acne , eczema , and contact dermatitis .", "skin color is determined by various factors including melanin content , oxygenation state of hemoglobin in capillary vessels , carotenoid content , water content , and organization of collagen fibers in the dermis . among these factors , melanin is the major determinant of skin color . in this context ,", "melanogenesis is a biochemical pathway responsible for melanin synthesis that is controlled by complex regulatory mechanisms .", "melanogenesis occurs in melanocytes confined in separate cytoplasmic organelles called melanosomes , which contain key enzymes of melanogenesis .", "differences in skin color are related to the size , number , shape , and distribution of melanosomes , whereas melanocyte density typically remains relatively constant  .", "although tyrosinase is the key regulatory enzyme of melanogenesis , tyrosinase - related protein ( trp)-1 , dopachrome tautomerase ( dct / trp2 ) , and melanosomal matrix proteins ( pmel17 , mart-1 ) carry out important roles in regulating melanogenesis .", "the genes of tyrosinase , trp-1 , and dct contain common transcription starting sites , the microphthalmia - associated transcription factor ( mitf ) binding sites .", "the intracellular signal transduction pathways of protein kinase c , cyclic amp ( camp ) , and nitrogen oxide are involved in the regulation of melanogenesis .", "various endogenous and exogenous factors , such as estrogen and ultraviolet ( uv ) radiation , affect melanogenesis via signal transduction pathways .", "these endogenous / exogenous factors exert their actions directly on melanocytes or indirectly via surrounding skin cells .", "melanocytes , keratinocytes , dermal fibroblasts , and other skin cells communicate with each other by factors that are secreted and cell  cell contacts .", "it has been shown that the interactions between keratinocytes and melanocytes are critical in the regulation of melanogenesis .", "in addition , dermal factors have been found to be involved in the regulation of melanogenesis . at the same time , stimulated melanocytes secret a number of signal molecules targeting not only keratinocytes but also skin immune cells .", "soluble factors released by melanocytes include proinflammatory cytokines and chemokines such as interleukin ( il)-1/1 , il-6 , il-8 il-10 , tumor necrosis factor ( tnf)- , transforming growth factor ( tgf)- , catecholamines , eicosanoids , serotonin , -melanocyte stimulating factor ( -msh ) , and nitric oxide ( no ) .", "a variety of hypopigmenting agents including hydroquinone , arbutin , tretinoin , kojic acid , azelaic acid , vitamin c , n - acetylglucosamine , niacinamide , linoleic acid , ellagic acid , methimazole , dioic acid , soy extract , licorice extract , rucinol , and glycolic acid have been used alone or in combination to treat abnormal hyperpigmentation .", "these agents can interfere with the pigmentation process at several different steps of skin pigmentation .", "however , the treatment of hyperpigmented conditions still remains challenging and the results are often discouraging .", "thus there is a need for novel skin - whitening agents that are highly effective and tolerable . in this article", ", we review recent reports investigating the skin - whitening effect of ginseng and its components and the underlying mechanisms of action , and then discuss their potential as candidates for novel skin - whitening agents .", "p.  ginseng is one of the most widely used medicinal plants in traditional oriental medicine . over thousands of years", ", it has been used to improve the overall condition of skin , as well as to treat a wide variety of diseases . however", ", genuine scientific approaches to clarify the efficacy of ginseng in skin have only been made in recent years .", "several reports have shown that ginseng extract , powder , or some other constituents could inhibit melanogenesis in  vitro and in  vivo . table  1 summarizes the direct effects of ginseng and its components on skin color and key enzymes involved in melanogenesis .", "song et  al reported that red ginseng powder improved melasma in a human clinical trial .", "they orally administered korean red ginseng powder for 24 weeks to female patients with melasma .", "after 24 weeks , the melasma area and severity index score decreased and melasma quality of life scale showed improvement in 91% of patients .", "in addition , 74% of the patients showed some improvement on the patient- and investigator - rated global improvement scales .", "most of reports investigating the antimelanogenic effect of ginseng were conducted in  vitro used purified tyrosinase or melanocyte cell lines . in melan - a cells treated with ethanol extract of ginseng seeds , melanin content and", "in addition to the crude extract or powder , several studies tested the effects of specific constituents of ginseng . the phenol compounds inhibited tyrosinase activity while ginsenoside prevents uvb - induced intracellular increase of reactive oxygen species [ 4648 ] . in some reports , ginsenosides alone exerted antimelanogenic effects .", "aglycone of ginsenoside rh4 inhibited melanin synthesis in b16 melanoma cells , possibly by involvement of protein kinase a pathway .", "it significantly reduced melanin content and tyrosinase activity in -msh and forskolin - stimulated b16 melanoma cells .", "it reduced the camp level and camp response - element binding protein level in b16 melanoma cells , and this might be responsible for the downregulation of mitf and tyrosinase .", "in addition , ginsenoside rb1 inhibited melanogenesis through the inhibition of tyrosinase activity in -msh - stimulated b16 cells in a dose - dependent manner .", "the crude methanol extract of the fresh leaves of p.  ginseng showed inhibitory activity on mushroom tyrosinase , and p - coumaric acid was characterized as the principal tyrosinase inhibitor in the extract .", "p - coumaric acid inhibited melanogenesis in b16f10 melanoma cells stimulated by -msh , and was suggested to interfere with melanogenesis by its structural similarity with tyrosine .", "interestingly , p - coumaric acid showed weaker inhibition against mushroom tyrosinase but more strongly inhibited human or murine tyrosinase in comparison with kojic acid and arbutin .", "enzyme kinetics analysis indicate that p - coumaric acid is a mixed type ( for tyrosine ) or competitive inhibitor ( for dopa ) of human tyrosinase .", "in addition , p - coumaric acid potently inhibits melanogenesis in human epidermal melanocytes exposed to uvb .", "cinnamic acid , one of the major components of cinnamomum cassia blume , is found in the root and seed of p.  ginseng \n .", "cinnamic acid significantly reduced melanin production , tyrosinase activity , and tyrosinase expression in the melan - a cells .", "in addition , cinnamic acid showed depigmenting activity on the uvb - tanned skin of brown guinea pigs .", "it is already known that the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . although the contents of total ginsenosides in red ginseng and fermented red ginseng using lactobacillus brevis were not significantly different , the ginsenoside metabolite content was higher in fermented red ginseng compared to red ginseng .", "the tyrosinase inhibitory activity of fermented red ginseng extract was more potent compared with red ginseng extract in a test using mushroom tyrosinase .", "as reviewed above , crude extract or some components of ginseng and its components showed antimelanogenic activities by direct inhibition on key enzymes of melanogenesis , such as tyrosinase .", "moreover , ginseng and its components could exert antimelanogenic activity via action on the several factors related in melanocyte physiology . among a large number of soluble factors produced from melanocytes , keratinocytes , fibroblasts , and immune cells in skin , adrenocorticotropic hormone ( acth ) , -msh , endothelin-1 , prostaglandin e2 , prostaglandin f2 , no , and histamine are well - known stimulators of melanogenesis [ 37,5963 ] .", "il-1/1 and granulocyte - macrophage colony - stimulating factor ( gm - csf ) stimulate melanogenesis , while il-6 , tgf-1 , and tnf- downregulate melanin production .", "gm - csf has been reported to be involved in regulating the proliferation and differentiation of epidermal melanocytes .", "treatment of melan - a cells with conditioned media from uv - irradiated sp-1 keratinocytes increased melanocyte proliferation , and the proliferative effect of the conditioned media was blocked by anti - gm - csf antibody treatment . when uv - irradiated sp-1 keratinocytes were treated with red ginseng extract or saponin of red ginseng , the increased melanocyte proliferation by the conditioned media was blocked . in that report ,", "red ginseng extract or saponin of red ginseng treatment decreased the expression of gm - csf induced by uv - b irradiation in sp-1 keratinocytes .", "as mentioned above , inflammatory cytokines such as il-1 and tnf- take part in the regulation of melanogenesis .", "ginseng extracts and ginsenosides have been reported to have anti - inflammatory activities in several different studies .", "ginsenosides inhibit different inducer - activated signaling protein kinases and transcription factor nuclear factor ( nf)-b , and then decrease the production of proinflammatory cytokines and mediators of inflammation .", "korean red ginseng extracts decreased tnf- and il-8 production in lipopolysaccharide ( lps)-stimulated hacat keratinocytes and show radical scavenging and antioxidant activity in human dermal fibroblasts .", "these findings suggest that ginseng extracts and ginsenosides might affect melanogenesis through their anti - inflammatory activities . the effect of ginseng on no production is still questionable .", "sun ginseng , a new processed ginseng prepared by steaming at high temperature , reduced uv - b - induced cell damage and decreased no production by inhibition of inducible no synthase mrna synthesis in hacat keratinocytes and human dermal fibroblasts .", "red ginseng marc oil inhibited inducible no synthase and cyclooxygenase-2 via nf-b and p38 pathways in lps - stimulated raw264.7 cells .", "in addition , ginsenjilinol , a protopanaxatriol - type saponin obtained from the roots of p.  ginseng , shows inhibitory activity on no production in lps - stimulated raw264.7 cells .", "by contrast , there are some controversial reports that ginseng extract enhanced no production or no signaling .", "hong et  al reported that ginseng extract administration stimulated nongenomic endothelial no synthase activation and enhanced no production in spontaneously hypertensive rats . in another report , water extract of korean red ginseng exerted vasoprotective effects through augmentation of no production by inhibiting arginase .", "therefore , the effect of ginseng on melanogenesis via no signaling remains to be clarified by further study .", "considerable numbers of immune cells including langerhans cells , macrophages , mast cells , and t cells are working actively in skin tissue . because the immunostimulatory activities of many ginsenosides are known , it is not surprising that ginsenosides could enhance the reactivity of skin immune cells . in a recent paper ,", "a cream containing 0.1% ginsenoside f1 ( a metabolite of ginsenoside rg1 ) showed a significant whitening effect on artificially tanned human skin .", "however , ginsenoside f1 did not directly inhibit mrna expression of tyrosinase or dct in normal human epidermal melanocytes .", "instead , ginsenoside f1 enhanced production of il-13 from human epidermal  t cells , and il-13 significantly reduced the mrna expression and protein amount of both tyrosinase and dct resulting in visible brightening of normal human epidermal melanocyte pellets .", "these results suggest that ginsenosides might be able to regulate melanogenesis via their effect on skin immune cells .", "recently , several reports have shown that extract , powder , or some constituents of ginseng could inhibit melanogenesis in  vivo or in  vitro", ". the underlying mechanisms of the antimelanogenic effect of ginseng or its components included the direct inhibition of key enzymes of melanogenesis ( tyrosinase and dct ) , inhibition of transcription factors ( mitf , nf-b ) or signaling pathways ( protein kinase a pathway and protein kinase c pathway ) involved in melanogenesis , decreasing the production of inducers of melanogenesis ( camp , gm - csf ) , and enhancing production of antimelanogenic factor ( il-13 ) . fig . ", "although issues surrounding the antimelanogenic activity of ginseng still remain controversial , especially in its effect on the production of proinflammatory cytokines and no , these recent findings suggest that ginseng and its constituents might be potential candidates for novel skin - whitening agents ."], "abstract_text": ["<S> abnormal changes in skin color induce significant cosmetic problems and affect quality of life . </S>", "<S> there are two groups of abnormal change in skin color ; hyperpigmentation and hypopigmentation . </S>", "<S> hyperpigmentation , darkening skin color by excessive pigmentation , is a major concern for asian people with yellow  brown skin . </S>", "<S> a variety of hypopigmenting agents have been used , but treating the hyperpigmented condition is still challenging and the results are often discouraging . </S>", "<S> panax ginseng has been used traditionally in eastern asia to treat various diseases , due to its immunomodulatory , neuroprotective , antioxidative , and antitumor activities . recently </S>", "<S> , several reports have shown that extract , powder , or some constituents of ginseng could inhibit melanogenesis in  vivo or in  vitro . </S>", "<S> the underlying mechanisms of antimelanogenic properties in ginseng or its components include the direct inhibition of key enzymes of melanogenesis , inhibition of transcription factors or signaling pathways involved in melanogenesis , decreasing production of inducers of melanogenesis , and enhancing production of antimelanogenic factor . </S>", "<S> although there still remain some controversial issues surrounding the antimelanogenic activity of ginseng , especially in its effect on production of proinflammatory cytokines and nitric oxide , these recent findings suggest that ginseng and its constituents might be potential candidates for novel skin whitening agents . </S>"], "labels": null, "section_names": ["Introduction", "Effects of ginseng and its components on melanogenesis", "Effects of ginseng and its components on factors related to melanogenesis", "Conclusion", "Conflicts of interest"], "sections": [["panax ginseng ( ginseng ) has been used traditionally in eastern asia over thousands of years .", "it has been used orally to treat various diseases including hypertension , diabetes mellitus , liver and kidney dysfunction , mental disorders , and postmenopausal disorders .", "in addition , topical applications have also been used to heal wounds and reduce skin inflammation . in the past few decades", ", it has been proved that ginseng extracts actually show a wide range of effects against human diseases .", "their potential therapeutic effects have been mainly attributed to its immunomodulatory , neuroprotective , antioxidative , antitumor , and hepatoprotective activities .", "ginseng contains a number of active ingredients including ginsenosides , polysaccharides , phytosterols , peptides , polyacetylenes , fatty acids , and polyacetylenic alcohols , which have different effects on carbohydrate and lipid metabolism , cognition , angiogenesis , and the neuroendocrine , immune , cardiovascular , and central nervous systems . among the active constituents of ginseng ,", "ginsenosides are known to be the major biologically active components of ginseng and the most widely studied .", "several studies have shown that ginsenosides play important roles in the pharmacological effects of ginseng .", ". however , ginseng contains other constituents , including ginsenoyne , phenolic compounds , polyacetylenes , sesquiterpenes , methoxypyrazine , alkylpyrazine derivatives , sesquiterpene alcohols , panasinsanols , and -carboline .", "white ginseng is peeled , dried , ginseng root and red ginseng is produced by steaming fresh ginseng root at 98100c for 23  h , and then drying until the moisture content is < 15% .", "red and white ginseng have both been shown to have immunomodulatory , anti - inflammatory , antioxidant , and antiatopic activities . moreover ,", "red ginseng has been reported to have more potent pharmacological activities than white ginseng in some respects [ 2022 ] .", "the differences in biological activities between red and white ginseng are caused by the chemical changes of ginsenosides after the steaming process .", "steaming partially converts the original ginsenosides to deglycoslated derivatives . as a result , the species and amounts of ginsenosides are quite different based on the processing method used .", "chu et  al   showed that a total of 53 and 43 compounds were tentatively identified in white ginseng and red ginseng samples , respectively .", "the featured compounds are mainly malonyl ginsenosides in white ginseng , and decarboxyl products of mal - ginsenosides and the dehydrated compounds from polar ginsenosides were characteristic in red ginseng .", "it is interesting that ginsenosides show a wide variety of biological activities , although the absorption rates from orally administered intact ginsenosides are very low . in the human intestinal tract ,", "thus , the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . in this context", "several studies showed that the transformation of ginsenosides into deglycosylated ginsenosides is needed to increase ginseng 's effectiveness in  vivo \n .", "abnormal changes in skin color induce significant cosmetic problems with a negative effect on quality of life .", "there are two groups of pigmentary disorders : disorders of the quantitative and qualitative distribution of normal pigment and the abnormal presence of exogenous or endogenous pigments in the skin .", "hyperpigmentation - related diseases include melasma , lentigines , nevus , ephelis , freckles , postinflammatory hyperpigmentation , and age spots .", "postinflammatory hyperpigmentation appears in many skin conditions , including acne , eczema , and contact dermatitis .", "skin color is determined by various factors including melanin content , oxygenation state of hemoglobin in capillary vessels , carotenoid content , water content , and organization of collagen fibers in the dermis . among these factors , melanin is the major determinant of skin color . in this context ,", "melanogenesis is a biochemical pathway responsible for melanin synthesis that is controlled by complex regulatory mechanisms .", "melanogenesis occurs in melanocytes confined in separate cytoplasmic organelles called melanosomes , which contain key enzymes of melanogenesis .", "differences in skin color are related to the size , number , shape , and distribution of melanosomes , whereas melanocyte density typically remains relatively constant  .", "although tyrosinase is the key regulatory enzyme of melanogenesis , tyrosinase - related protein ( trp)-1 , dopachrome tautomerase ( dct / trp2 ) , and melanosomal matrix proteins ( pmel17 , mart-1 ) carry out important roles in regulating melanogenesis .", "the genes of tyrosinase , trp-1 , and dct contain common transcription starting sites , the microphthalmia - associated transcription factor ( mitf ) binding sites .", "the intracellular signal transduction pathways of protein kinase c , cyclic amp ( camp ) , and nitrogen oxide are involved in the regulation of melanogenesis .", "various endogenous and exogenous factors , such as estrogen and ultraviolet ( uv ) radiation , affect melanogenesis via signal transduction pathways .", "these endogenous / exogenous factors exert their actions directly on melanocytes or indirectly via surrounding skin cells .", "melanocytes , keratinocytes , dermal fibroblasts , and other skin cells communicate with each other by factors that are secreted and cell  cell contacts .", "it has been shown that the interactions between keratinocytes and melanocytes are critical in the regulation of melanogenesis .", "in addition , dermal factors have been found to be involved in the regulation of melanogenesis . at the same time , stimulated melanocytes secret a number of signal molecules targeting not only keratinocytes but also skin immune cells .", "soluble factors released by melanocytes include proinflammatory cytokines and chemokines such as interleukin ( il)-1/1 , il-6 , il-8 il-10 , tumor necrosis factor ( tnf)- , transforming growth factor ( tgf)- , catecholamines , eicosanoids , serotonin , -melanocyte stimulating factor ( -msh ) , and nitric oxide ( no ) .", "a variety of hypopigmenting agents including hydroquinone , arbutin , tretinoin , kojic acid , azelaic acid , vitamin c , n - acetylglucosamine , niacinamide , linoleic acid , ellagic acid , methimazole , dioic acid , soy extract , licorice extract , rucinol , and glycolic acid have been used alone or in combination to treat abnormal hyperpigmentation .", "these agents can interfere with the pigmentation process at several different steps of skin pigmentation .", "however , the treatment of hyperpigmented conditions still remains challenging and the results are often discouraging .", "thus there is a need for novel skin - whitening agents that are highly effective and tolerable . in this article", ", we review recent reports investigating the skin - whitening effect of ginseng and its components and the underlying mechanisms of action , and then discuss their potential as candidates for novel skin - whitening agents ."], ["p.  ginseng is one of the most widely used medicinal plants in traditional oriental medicine . over thousands of years", ", it has been used to improve the overall condition of skin , as well as to treat a wide variety of diseases . however", ", genuine scientific approaches to clarify the efficacy of ginseng in skin have only been made in recent years .", "several reports have shown that ginseng extract , powder , or some other constituents could inhibit melanogenesis in  vitro and in  vivo . table  1 summarizes the direct effects of ginseng and its components on skin color and key enzymes involved in melanogenesis .", "song et  al reported that red ginseng powder improved melasma in a human clinical trial .", "they orally administered korean red ginseng powder for 24 weeks to female patients with melasma .", "after 24 weeks , the melasma area and severity index score decreased and melasma quality of life scale showed improvement in 91% of patients .", "in addition , 74% of the patients showed some improvement on the patient- and investigator - rated global improvement scales .", "most of reports investigating the antimelanogenic effect of ginseng were conducted in  vitro used purified tyrosinase or melanocyte cell lines . in melan - a cells treated with ethanol extract of ginseng seeds , melanin content and", "in addition to the crude extract or powder , several studies tested the effects of specific constituents of ginseng . the phenol compounds inhibited tyrosinase activity while ginsenoside prevents uvb - induced intracellular increase of reactive oxygen species [ 4648 ] . in some reports , ginsenosides alone exerted antimelanogenic effects .", "aglycone of ginsenoside rh4 inhibited melanin synthesis in b16 melanoma cells , possibly by involvement of protein kinase a pathway .", "it significantly reduced melanin content and tyrosinase activity in -msh and forskolin - stimulated b16 melanoma cells .", "it reduced the camp level and camp response - element binding protein level in b16 melanoma cells , and this might be responsible for the downregulation of mitf and tyrosinase .", "in addition , ginsenoside rb1 inhibited melanogenesis through the inhibition of tyrosinase activity in -msh - stimulated b16 cells in a dose - dependent manner .", "the crude methanol extract of the fresh leaves of p.  ginseng showed inhibitory activity on mushroom tyrosinase , and p - coumaric acid was characterized as the principal tyrosinase inhibitor in the extract .", "p - coumaric acid inhibited melanogenesis in b16f10 melanoma cells stimulated by -msh , and was suggested to interfere with melanogenesis by its structural similarity with tyrosine .", "interestingly , p - coumaric acid showed weaker inhibition against mushroom tyrosinase but more strongly inhibited human or murine tyrosinase in comparison with kojic acid and arbutin .", "enzyme kinetics analysis indicate that p - coumaric acid is a mixed type ( for tyrosine ) or competitive inhibitor ( for dopa ) of human tyrosinase .", "in addition , p - coumaric acid potently inhibits melanogenesis in human epidermal melanocytes exposed to uvb .", "cinnamic acid , one of the major components of cinnamomum cassia blume , is found in the root and seed of p.  ginseng \n .", "cinnamic acid significantly reduced melanin production , tyrosinase activity , and tyrosinase expression in the melan - a cells .", "in addition , cinnamic acid showed depigmenting activity on the uvb - tanned skin of brown guinea pigs .", "it is already known that the pharmacological actions of these ginsenosides have been closely related to their biotransformation by human intestinal bacteria . although the contents of total ginsenosides in red ginseng and fermented red ginseng using lactobacillus brevis were not significantly different , the ginsenoside metabolite content was higher in fermented red ginseng compared to red ginseng .", "the tyrosinase inhibitory activity of fermented red ginseng extract was more potent compared with red ginseng extract in a test using mushroom tyrosinase ."], ["as reviewed above , crude extract or some components of ginseng and its components showed antimelanogenic activities by direct inhibition on key enzymes of melanogenesis , such as tyrosinase .", "moreover , ginseng and its components could exert antimelanogenic activity via action on the several factors related in melanocyte physiology . among a large number of soluble factors produced from melanocytes , keratinocytes , fibroblasts , and immune cells in skin , adrenocorticotropic hormone ( acth ) , -msh , endothelin-1 , prostaglandin e2 , prostaglandin f2 , no , and histamine are well - known stimulators of melanogenesis [ 37,5963 ] .", "il-1/1 and granulocyte - macrophage colony - stimulating factor ( gm - csf ) stimulate melanogenesis , while il-6 , tgf-1 , and tnf- downregulate melanin production .", "gm - csf has been reported to be involved in regulating the proliferation and differentiation of epidermal melanocytes .", "treatment of melan - a cells with conditioned media from uv - irradiated sp-1 keratinocytes increased melanocyte proliferation , and the proliferative effect of the conditioned media was blocked by anti - gm - csf antibody treatment . when uv - irradiated sp-1 keratinocytes were treated with red ginseng extract or saponin of red ginseng , the increased melanocyte proliferation by the conditioned media was blocked . in that report ,", "red ginseng extract or saponin of red ginseng treatment decreased the expression of gm - csf induced by uv - b irradiation in sp-1 keratinocytes .", "as mentioned above , inflammatory cytokines such as il-1 and tnf- take part in the regulation of melanogenesis .", "ginseng extracts and ginsenosides have been reported to have anti - inflammatory activities in several different studies .", "ginsenosides inhibit different inducer - activated signaling protein kinases and transcription factor nuclear factor ( nf)-b , and then decrease the production of proinflammatory cytokines and mediators of inflammation .", "korean red ginseng extracts decreased tnf- and il-8 production in lipopolysaccharide ( lps)-stimulated hacat keratinocytes and show radical scavenging and antioxidant activity in human dermal fibroblasts .", "these findings suggest that ginseng extracts and ginsenosides might affect melanogenesis through their anti - inflammatory activities . the effect of ginseng on no production is still questionable .", "sun ginseng , a new processed ginseng prepared by steaming at high temperature , reduced uv - b - induced cell damage and decreased no production by inhibition of inducible no synthase mrna synthesis in hacat keratinocytes and human dermal fibroblasts .", "red ginseng marc oil inhibited inducible no synthase and cyclooxygenase-2 via nf-b and p38 pathways in lps - stimulated raw264.7 cells .", "in addition , ginsenjilinol , a protopanaxatriol - type saponin obtained from the roots of p.  ginseng , shows inhibitory activity on no production in lps - stimulated raw264.7 cells .", "by contrast , there are some controversial reports that ginseng extract enhanced no production or no signaling .", "hong et  al reported that ginseng extract administration stimulated nongenomic endothelial no synthase activation and enhanced no production in spontaneously hypertensive rats . in another report , water extract of korean red ginseng exerted vasoprotective effects through augmentation of no production by inhibiting arginase .", "therefore , the effect of ginseng on melanogenesis via no signaling remains to be clarified by further study .", "considerable numbers of immune cells including langerhans cells , macrophages , mast cells , and t cells are working actively in skin tissue . because the immunostimulatory activities of many ginsenosides are known , it is not surprising that ginsenosides could enhance the reactivity of skin immune cells . in a recent paper ,", "a cream containing 0.1% ginsenoside f1 ( a metabolite of ginsenoside rg1 ) showed a significant whitening effect on artificially tanned human skin .", "however , ginsenoside f1 did not directly inhibit mrna expression of tyrosinase or dct in normal human epidermal melanocytes .", "instead , ginsenoside f1 enhanced production of il-13 from human epidermal  t cells , and il-13 significantly reduced the mrna expression and protein amount of both tyrosinase and dct resulting in visible brightening of normal human epidermal melanocyte pellets .", "these results suggest that ginsenosides might be able to regulate melanogenesis via their effect on skin immune cells ."], ["recently , several reports have shown that extract , powder , or some constituents of ginseng could inhibit melanogenesis in  vivo or in  vitro", ". the underlying mechanisms of the antimelanogenic effect of ginseng or its components included the direct inhibition of key enzymes of melanogenesis ( tyrosinase and dct ) , inhibition of transcription factors ( mitf , nf-b ) or signaling pathways ( protein kinase a pathway and protein kinase c pathway ) involved in melanogenesis , decreasing the production of inducers of melanogenesis ( camp , gm - csf ) , and enhancing production of antimelanogenic factor ( il-13 ) . fig . ", "although issues surrounding the antimelanogenic activity of ginseng still remain controversial , especially in its effect on the production of proinflammatory cytokines and no , these recent findings suggest that ginseng and its constituents might be potential candidates for novel skin - whitening agents ."], [""]]}
{"article_id": "PMC3824354", "article_text": ["chronic kidney disease ( ckd ) is associated with a number of comorbidities and prognosis is poor , because many patients experience progression to end - stage renal disease .", "also , in the majority of patients disease progression may be altered due to more or less suitable immunosuppressive protocol treatments and therapeutic approaches .", "the mechanisms of injury underlying progression are blurred , but traditional opinion pointed to an association between decline in renal function with the degree of proteinuria and histological findings of glomerulosclerosis and interstitial fibrosis [ 2 , 3 ] .", "kidney injury molecule-1 ( kim-1 ) is a new specific biomarker of proximal tubule injury that can be measured both in urine and kidney tissue samples .", "it is an apoptotic - cell phagocytosis and scavenger receptor that is most highly upregulated in proximal tubular epithelium in acute and chronic kidney injury .", "also , much attention has been paid to its possible pathophysiological role in modulation of tubular damage and repair [ 57 ] . besides a close relationship between tissue kim-1 expression and urinary kim-1 concentration ,", "our previous six - month prospective pilot study showed that kim-1 expression in tissue correlated better with tin features and renal function in different chronic kidney diseases than with urinary kim-1 concentration .", "therefore , that investigation was the basis of this long - term retrospective study , where only kim-1 tissue expression as a predictor of long - term kidney function was examined .  ", "the aim of this retrospective investigation was to evaluate ( 1 ) possible associations between tissue kim-1 expression and tubulointerstitial ( tin ) inflammation , atrophy , and fibrosis in different chronic kidney diseases and ( 2 ) possible associations between tissue kim-1 expression and some demographic and laboratory parameters as well as kidney function and proteinuria at the time of biopsy and 6 , 12 , 24 , and 36 months later .", "the retrospective study included 60 patients ( 27 men ) of mean age 34.42  12.15 years ( range 1859 years ) , who were hospitalized in the clinical center of serbia for kidney biopsy from 2006 to 2009 .", "indications for kidney biopsy were nephrotic syndrome , pathologic proteinuria without nephrotic syndrome , and abnormal urinary sediment ( erythrocyturia and leukocyturia ) in several samples .", "pathohistological analysis revealed minimal change glomerulonephritis ( mcgn ) in two patients , non - iga gn in nine , iga gn in six , membranous and membranoproliferative gn in seven patients each , focal glomerulosclerosis in eleven , lupus nephritis in ten , and crescentic gn in eight patients .", "after diagnosis , the participants were treated according to established protocols for each type of gn with immunological and nonimmunological therapy ( nonsalt diets , statins , angiotensin - converting - enzyme - ace inhibitors , and angiotensin receptor type 1 blockers atb ) .  ", "the institutional review committee approved our study protocol therebys following local biomedical research regulations ( irb no .", "a complete blood count ; serum urea and creatinine , albumin ; lipid status , and urine sediment were determined in blood and urine samples collected 1 day before kidney biopsy as well proteinuria / day . only samples with a sterile urine culture were processed .", "urine sediments with more than 3 rbc / hpf or 5 wbc / hpf were defined as clinically significant erythrocyturia or leukocyturia .", "estimated glomerular filtration rate ( egfr ) was calculated with a shortened version of the modification of diet in renal disease ( mdrd ; patients 18 years ) .", "alteration in tin were assessed after routine hematoxylin - eosin staining by a qualified nephropathologist who was not familiar with the preset histopathologic or clinical diagnosis , nor the identity of patients .", "inflammation activity was then scored numerically ( 0 : no inflammation , 1 : slightly marked inflammation , 2 : moderate inflammation , and 3 : strong inflammation ) as well as atrophy and fibrosis advancement ( 0 : no atrophy and no fibrosis , 1 : slightly marked atrophy and fibrosis , 2 : moderate atrophy and fibrosis , 3 : very pronounced atrophy and fibrosis ) .", "after deparaffinization tissue samples were rehydrated and stained immunohistochemically for kim-1 using the tissue kim-1/tim-1 kit ( r & d systems inc . , minneapolis , mn , usa ) with contrasting hematoxylin and eosin staining .", "a tubule was considered to be kim-1 positive if it contained at least one kim-1 positive cell regardless of whether the kim-1 expression was cytoplasmic or apical .", "kim-1 staining was scored semiquantitatively by estimating the percentage of cortical tubules expressing kim-1 per field ( the complete biopsy area was scored , with a minimum of five fields ; in controls 30 fields were scored ) .", "unaffected parts of kidneys from patients with renal cell carcinoma were used as the negative control .", "the 6- , 12- , 24- , and 36-month followup for all patients included measurement of serum creatinine concentration , egfr , and proteinuria .  ", "data are presented as mean 1 standard deviation ( sd ) and the range of values .", "the one - sample kolmogorov - smirnov test was used to check for normal distribution of the variables .", "relationships between variables were estimated using pearson 's rank and spearmen 's correlation tests as appropriate .", "independent predictors of renal function 6 , 12 , 24 , and 36 months after the kidney biopsy were identified by stepwise multivariant regression analysis .", "\n table 2 presents the demographic , clinical , and laboratory data for the patients at the time of kidney biopsy .", "tissue kim-1 expression was significantly induced in all kidney biopsies except in one patient with mcgn .", "tissue kim-1 had a typically apical localization , but it could also be found in the cytoplasm with no apparent affinity for the apical membrane of tubulocytes . kim-1", "positive tubules were located mainly in the tin regions affected by inflammation or fibrosis but were not observed in regions with end - stage fibrosis . kim-1", "univariate analysis showed significant positive associations between tissue kim-1 expression and age ( r = 0.313 ; p = 0.015 ) , tin inflammation ( r = 0.4563 ; p = 0.004 ) , tin fibrosis ( r = 0.317 ; p = 0.021 ) , and hemoglobin ( r = 0.440 ; p = 0.001 ) as well as negative associations with albumin concentration ( r = 0.376 ; p = 0.011 ) and egfr ( r = 0.572 ; p < 0.001 ) at the time of biopsy and 6 ( r = 0.442 ; p = 0.002 ) , 24 ( r = 0.398 ; p = 0.012 ) , and 36 ( r = 0.412 ; p = 0.015 ) months later .", "tissue kim-1 expression correlated significantly only with proteinuria / day 6 months after biopsy ( r = 0.394 ; p = 0.026 ) but not with proteinuria at the time of biopsy and 12 , 24 , and 36 months later .  \n", "table 3 gives the correlation coefficients between egfr at the time of biopsy and 6 , 12 , 24 , and 36 months later and the examined variables .", "multivariant stepwise regression analysis showed that the best predictor of kidney function at the time of biopsy ( egfr 0 ) was tin inflammation ( p < 0.001 ) , as well as of egfr12 ( p = 0.015 ) , egfr 24 ( p = 0.039 ) , and egfr 36 ( p = 0.012 ) .", "however tissue kim-1 expression was the best predictor of egfr 6 ( p = 0.016 ) along with tin inflammation ( p = 0.016 ) ( table 4 ) .  ", "figure 2 presents the linear correlation between tin inflammation ( p < 0.001 ) , tissue kim-1 expression ( p < 0.001 ) , and egfr 6 .", "in our previous 6-month prospective study , we found higher urine kim-1 concentrations in twenty chronic renal patients than in control subjects and a significant positive correlation between urine kim-1 level and kim-1 expression in tissue .", "although , urine kim-1 content reflected tissue kim-1 expression , tissue kim-1 correlated better with tin inflammation and fibrosis as well as with kidney function at the time of biopsy and 3 and 6 months later .", "therefore , we retrospectively examined sixty patients with different kidney diseases in order to evaluate the importance of tissue kim-1 expression in predicting kidney function in the ensuing 36 months after kidney biopsy . in the present study tissue", "in addition , kim-1 positive tubules were located in parts of the cortex that were imbued with inflammatory infiltrate and/or fibrotic changes but not in completely atrophic areas .", "it is well known that the expression of kim-1 in tubules is associated with inflammation and tin damage .", "double - labeling studies have shown simultaneous expression of kim-1 and markers of prefibrotic changes , repair , and chemotaxis [ 10 , 11 ] .", "the study of van timmeren et al .   also demonstrated a connection between kim-1 expression in tissue , interstitial fibrosis , and macrophage accumulation .", ".   first showed that tubular epithelial cells expressing kim-1 on their surface could act as phagocytes for apoptotic and necrotic renal epithelial cells .", "kim-1 is expressed predominantly in renal tubules , which , judging by the simultaneous expression of markers of dedifferentiation , are currently under the influence of agents that cause injury , but is absent from completely atrophic tubules .", "controversy remains about the function of kim-1 : is it actively regulating the inflammatory process ? or is its expression just a response to damage , attempted recovery , and/or repair ? .", "the present study revealed a significant negative correlation between tissue kim-1 expression and all estimated gfr at the time of kidney biopsy and 6 , 24 , and 36 months later .", "however multivariate analysis pointed to tin inflammation , but neither tin fibrosis nor proteinuria , as the best predictor of kidney function at biopsy and 6 , 12 , 24 , and 36 months afterwards .", "however , along with tin inflammation , tissue kim-1 expression was the best predictor of kidney function 6 months after biopsy . interstitial infiltration of inflammatory cells can be seen in a variety of immune and nonimmune kidney diseases and is thought to play a significant role in tin damage and fibrosis . upon exposure to high protein concentrations ,", "renal tubule cells produce a host of chemokines , vasoactive mediators , and adhesion molecules , which may contribute to interstitial fibrosis .", "protocol immunosuppressive therapy and treatment with ace inhibitors and at blockers for chronic renal patients with different chronic immune and nonimmune - mediated kidney disorders proven by biopsy have the strongest effects during the first 6 months of therapy .", "the severity of pretreatment tin damage predicts a dulled response to renoprotective intervention , with a worse long - term renal outcome .", "therefore , we can speculate that patients with less interstitial inflammation at the time of kidney biopsy will have a more favorable response to treatment and better kidney function afterwards . also , the finding that kim-1 was the strongest predictor of kidney function 6 months after biopsy along with tin inflammation may support the hypothesis about its potential role in the development of interstitial fibrosis and an unsatisfactory treatment response .", "association between higher urinary kim-1 levels and poorer kidney function was found in some human studies in native [ 10 , 14 ] and transplanted kidneys   but data on human tissue kim-1 expression are scarce .", "zhang et al .   found that kim-1 expression in transplant biopsies is a sensitive measure of cell injury .", "in addition , they showed that more intense kim-1 staining predicts a better graft outcome over the ensuing 18 months and speculated that its level of expression may be an indicator of graft function recovery .", "it is well known that proteinuria is an ominous biomarker of progressive kidney disease   and our study revealed that only proteinuria 6 months after kidney biopsy correlated negatively with kidney function 24 and 36 months after the biopsies and positively with kim-1 expression in tissue .", "earlier investigations [ 10 , 14 , 17 ] have yielded conflicting data regarding the relationship between urinary kim-1 level or tissue kim-1 expression and proteinuria .", "one explanation may be that proteinuria is not always accompanied by tin damage and a progressive decline in renal function [ 18 , 19 ] .", "data regarding the selectivity of proteinuria were not available for the purposes of this study .", "the present study exposed a strong association between kim-1 tissue staining and tin inflammation and fibrosis but not with tin atrophy .", "although multivariate analysis pointed to tin inflammation , as the best predictor of kidney function at biopsy and 6 , 12 , 24 , and 36 months later , tissue kim-1 expression is one of the best predictor of kidney function 6 months after biopsy , the time when treatment effects are the strongest .", "therefore , we can speculate that kim-1 has a potential role in the development of interstitial fibrosis and poor treatment responses .", "the major limitation of this investigation is the relatively small number of patients examined but it could be the basis for a larger prospective follow - up study for evaluation of urinary kim-1 concentration and tissue kim-1 expression in patients with various chronic and acute renal diseases ."], "abstract_text": ["<S> \n objectives . retrospective study was designed to examine the importance of tissue kidney injury molecule-1 ( kim-1 ) expression in predicting kidney function in sixty patients ( 27 males ) aged 34.15  12.23 years with different kidney diseases over three years after kidney biopsy . </S>", "<S> materials and methods . </S>", "<S> tissue kim-1 expression was determined immunohistochemically and kim-1 staining was scored semiquantitatively , as well as tubulointerstitialis ( tin ) , inflammation , atrophy , and fibrosis . kidney function ( mdrd formula ) and </S>", "<S> proteinuria / day were evaluated at the time of biopsy ( gfr0 ) and 6 , 12 , 24 , and 36 months later . </S>", "<S> results . significantly positive correlations between tissue kim-1 expression and age ( r = 0.313 ) , tin inflammation ( r = 0.456 ) , fibrosis ( r = 0.317 ) , and </S>", "<S> proteinuria at 6 months ( r = 0.394 ) as well as negative correlations with gfr0 ( r = 0.572 ) , gfr6 ( r = 0.442 ) , gfr24 ( r = 0.398 ) , and gfr36 ( r = 0.412 ) were found . meanwhile , tin inflammation was the best predictor of all measured kidney functions during three years , while tissue kim-1 expression ( p = 0.016 ) was a predictor only at 6 months after biopsy . </S>", "<S> conclusion . </S>", "<S> tissue kim-1 expression significantly predicts kidney function solely at 6 months after biopsy , when the effects of immune and nonimmune treatments are the strongest . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["chronic kidney disease ( ckd ) is associated with a number of comorbidities and prognosis is poor , because many patients experience progression to end - stage renal disease .", "also , in the majority of patients disease progression may be altered due to more or less suitable immunosuppressive protocol treatments and therapeutic approaches .", "the mechanisms of injury underlying progression are blurred , but traditional opinion pointed to an association between decline in renal function with the degree of proteinuria and histological findings of glomerulosclerosis and interstitial fibrosis [ 2 , 3 ] .", "kidney injury molecule-1 ( kim-1 ) is a new specific biomarker of proximal tubule injury that can be measured both in urine and kidney tissue samples .", "it is an apoptotic - cell phagocytosis and scavenger receptor that is most highly upregulated in proximal tubular epithelium in acute and chronic kidney injury .", "also , much attention has been paid to its possible pathophysiological role in modulation of tubular damage and repair [ 57 ] . besides a close relationship between tissue kim-1 expression and urinary kim-1 concentration ,", "our previous six - month prospective pilot study showed that kim-1 expression in tissue correlated better with tin features and renal function in different chronic kidney diseases than with urinary kim-1 concentration .", "therefore , that investigation was the basis of this long - term retrospective study , where only kim-1 tissue expression as a predictor of long - term kidney function was examined .  ", "the aim of this retrospective investigation was to evaluate ( 1 ) possible associations between tissue kim-1 expression and tubulointerstitial ( tin ) inflammation , atrophy , and fibrosis in different chronic kidney diseases and ( 2 ) possible associations between tissue kim-1 expression and some demographic and laboratory parameters as well as kidney function and proteinuria at the time of biopsy and 6 , 12 , 24 , and 36 months later ."], ["the retrospective study included 60 patients ( 27 men ) of mean age 34.42  12.15 years ( range 1859 years ) , who were hospitalized in the clinical center of serbia for kidney biopsy from 2006 to 2009 .", "indications for kidney biopsy were nephrotic syndrome , pathologic proteinuria without nephrotic syndrome , and abnormal urinary sediment ( erythrocyturia and leukocyturia ) in several samples .", "pathohistological analysis revealed minimal change glomerulonephritis ( mcgn ) in two patients , non - iga gn in nine , iga gn in six , membranous and membranoproliferative gn in seven patients each , focal glomerulosclerosis in eleven , lupus nephritis in ten , and crescentic gn in eight patients .", "after diagnosis , the participants were treated according to established protocols for each type of gn with immunological and nonimmunological therapy ( nonsalt diets , statins , angiotensin - converting - enzyme - ace inhibitors , and angiotensin receptor type 1 blockers atb ) .  ", "the institutional review committee approved our study protocol therebys following local biomedical research regulations ( irb no .", "a complete blood count ; serum urea and creatinine , albumin ; lipid status , and urine sediment were determined in blood and urine samples collected 1 day before kidney biopsy as well proteinuria / day . only samples with a sterile urine culture were processed .", "urine sediments with more than 3 rbc / hpf or 5 wbc / hpf were defined as clinically significant erythrocyturia or leukocyturia .", "estimated glomerular filtration rate ( egfr ) was calculated with a shortened version of the modification of diet in renal disease ( mdrd ; patients 18 years ) .", "alteration in tin were assessed after routine hematoxylin - eosin staining by a qualified nephropathologist who was not familiar with the preset histopathologic or clinical diagnosis , nor the identity of patients .", "inflammation activity was then scored numerically ( 0 : no inflammation , 1 : slightly marked inflammation , 2 : moderate inflammation , and 3 : strong inflammation ) as well as atrophy and fibrosis advancement ( 0 : no atrophy and no fibrosis , 1 : slightly marked atrophy and fibrosis , 2 : moderate atrophy and fibrosis , 3 : very pronounced atrophy and fibrosis ) .", "after deparaffinization tissue samples were rehydrated and stained immunohistochemically for kim-1 using the tissue kim-1/tim-1 kit ( r & d systems inc . , minneapolis , mn , usa ) with contrasting hematoxylin and eosin staining .", "a tubule was considered to be kim-1 positive if it contained at least one kim-1 positive cell regardless of whether the kim-1 expression was cytoplasmic or apical .", "kim-1 staining was scored semiquantitatively by estimating the percentage of cortical tubules expressing kim-1 per field ( the complete biopsy area was scored , with a minimum of five fields ; in controls 30 fields were scored ) .", "unaffected parts of kidneys from patients with renal cell carcinoma were used as the negative control .", "the 6- , 12- , 24- , and 36-month followup for all patients included measurement of serum creatinine concentration , egfr , and proteinuria .  ", "data are presented as mean 1 standard deviation ( sd ) and the range of values .", "the one - sample kolmogorov - smirnov test was used to check for normal distribution of the variables .", "relationships between variables were estimated using pearson 's rank and spearmen 's correlation tests as appropriate .", "independent predictors of renal function 6 , 12 , 24 , and 36 months after the kidney biopsy were identified by stepwise multivariant regression analysis ."], ["\n table 2 presents the demographic , clinical , and laboratory data for the patients at the time of kidney biopsy .", "tissue kim-1 expression was significantly induced in all kidney biopsies except in one patient with mcgn .", "tissue kim-1 had a typically apical localization , but it could also be found in the cytoplasm with no apparent affinity for the apical membrane of tubulocytes . kim-1", "positive tubules were located mainly in the tin regions affected by inflammation or fibrosis but were not observed in regions with end - stage fibrosis . kim-1", "univariate analysis showed significant positive associations between tissue kim-1 expression and age ( r = 0.313 ; p = 0.015 ) , tin inflammation ( r = 0.4563 ; p = 0.004 ) , tin fibrosis ( r = 0.317 ; p = 0.021 ) , and hemoglobin ( r = 0.440 ; p = 0.001 ) as well as negative associations with albumin concentration ( r = 0.376 ; p = 0.011 ) and egfr ( r = 0.572 ; p < 0.001 ) at the time of biopsy and 6 ( r = 0.442 ; p = 0.002 ) , 24 ( r = 0.398 ; p = 0.012 ) , and 36 ( r = 0.412 ; p = 0.015 ) months later .", "tissue kim-1 expression correlated significantly only with proteinuria / day 6 months after biopsy ( r = 0.394 ; p = 0.026 ) but not with proteinuria at the time of biopsy and 12 , 24 , and 36 months later .  \n", "table 3 gives the correlation coefficients between egfr at the time of biopsy and 6 , 12 , 24 , and 36 months later and the examined variables .", "multivariant stepwise regression analysis showed that the best predictor of kidney function at the time of biopsy ( egfr 0 ) was tin inflammation ( p < 0.001 ) , as well as of egfr12 ( p = 0.015 ) , egfr 24 ( p = 0.039 ) , and egfr 36 ( p = 0.012 ) .", "however tissue kim-1 expression was the best predictor of egfr 6 ( p = 0.016 ) along with tin inflammation ( p = 0.016 ) ( table 4 ) .  ", "figure 2 presents the linear correlation between tin inflammation ( p < 0.001 ) , tissue kim-1 expression ( p < 0.001 ) , and egfr 6 ."], ["in our previous 6-month prospective study , we found higher urine kim-1 concentrations in twenty chronic renal patients than in control subjects and a significant positive correlation between urine kim-1 level and kim-1 expression in tissue .", "although , urine kim-1 content reflected tissue kim-1 expression , tissue kim-1 correlated better with tin inflammation and fibrosis as well as with kidney function at the time of biopsy and 3 and 6 months later .", "therefore , we retrospectively examined sixty patients with different kidney diseases in order to evaluate the importance of tissue kim-1 expression in predicting kidney function in the ensuing 36 months after kidney biopsy . in the present study tissue", "in addition , kim-1 positive tubules were located in parts of the cortex that were imbued with inflammatory infiltrate and/or fibrotic changes but not in completely atrophic areas .", "it is well known that the expression of kim-1 in tubules is associated with inflammation and tin damage .", "double - labeling studies have shown simultaneous expression of kim-1 and markers of prefibrotic changes , repair , and chemotaxis [ 10 , 11 ] .", "the study of van timmeren et al .   also demonstrated a connection between kim-1 expression in tissue , interstitial fibrosis , and macrophage accumulation .", ".   first showed that tubular epithelial cells expressing kim-1 on their surface could act as phagocytes for apoptotic and necrotic renal epithelial cells .", "kim-1 is expressed predominantly in renal tubules , which , judging by the simultaneous expression of markers of dedifferentiation , are currently under the influence of agents that cause injury , but is absent from completely atrophic tubules .", "controversy remains about the function of kim-1 : is it actively regulating the inflammatory process ? or is its expression just a response to damage , attempted recovery , and/or repair ? .", "the present study revealed a significant negative correlation between tissue kim-1 expression and all estimated gfr at the time of kidney biopsy and 6 , 24 , and 36 months later .", "however multivariate analysis pointed to tin inflammation , but neither tin fibrosis nor proteinuria , as the best predictor of kidney function at biopsy and 6 , 12 , 24 , and 36 months afterwards .", "however , along with tin inflammation , tissue kim-1 expression was the best predictor of kidney function 6 months after biopsy . interstitial infiltration of inflammatory cells can be seen in a variety of immune and nonimmune kidney diseases and is thought to play a significant role in tin damage and fibrosis . upon exposure to high protein concentrations ,", "renal tubule cells produce a host of chemokines , vasoactive mediators , and adhesion molecules , which may contribute to interstitial fibrosis .", "protocol immunosuppressive therapy and treatment with ace inhibitors and at blockers for chronic renal patients with different chronic immune and nonimmune - mediated kidney disorders proven by biopsy have the strongest effects during the first 6 months of therapy .", "the severity of pretreatment tin damage predicts a dulled response to renoprotective intervention , with a worse long - term renal outcome .", "therefore , we can speculate that patients with less interstitial inflammation at the time of kidney biopsy will have a more favorable response to treatment and better kidney function afterwards . also , the finding that kim-1 was the strongest predictor of kidney function 6 months after biopsy along with tin inflammation may support the hypothesis about its potential role in the development of interstitial fibrosis and an unsatisfactory treatment response .", "association between higher urinary kim-1 levels and poorer kidney function was found in some human studies in native [ 10 , 14 ] and transplanted kidneys   but data on human tissue kim-1 expression are scarce .", "zhang et al .   found that kim-1 expression in transplant biopsies is a sensitive measure of cell injury .", "in addition , they showed that more intense kim-1 staining predicts a better graft outcome over the ensuing 18 months and speculated that its level of expression may be an indicator of graft function recovery .", "it is well known that proteinuria is an ominous biomarker of progressive kidney disease   and our study revealed that only proteinuria 6 months after kidney biopsy correlated negatively with kidney function 24 and 36 months after the biopsies and positively with kim-1 expression in tissue .", "earlier investigations [ 10 , 14 , 17 ] have yielded conflicting data regarding the relationship between urinary kim-1 level or tissue kim-1 expression and proteinuria .", "one explanation may be that proteinuria is not always accompanied by tin damage and a progressive decline in renal function [ 18 , 19 ] .", "data regarding the selectivity of proteinuria were not available for the purposes of this study ."], ["the present study exposed a strong association between kim-1 tissue staining and tin inflammation and fibrosis but not with tin atrophy .", "although multivariate analysis pointed to tin inflammation , as the best predictor of kidney function at biopsy and 6 , 12 , 24 , and 36 months later , tissue kim-1 expression is one of the best predictor of kidney function 6 months after biopsy , the time when treatment effects are the strongest .", "therefore , we can speculate that kim-1 has a potential role in the development of interstitial fibrosis and poor treatment responses .", "the major limitation of this investigation is the relatively small number of patients examined but it could be the basis for a larger prospective follow - up study for evaluation of urinary kim-1 concentration and tissue kim-1 expression in patients with various chronic and acute renal diseases ."]]}
{"article_id": "PMC4196304", "article_text": ["these range from various slow - growing benign tumors and low - grade malignancies to fast growing high - grade malignancies .", "angiofibromas are one such group of tumors arising in this region , which present as swellings . of these", ", nasal angiofibromas ( nas ) are the most common and nas occurring in the region other than the nose are called as extranasopharyngeal angiofibromas ( ena ) .", "a 54-year - old male reported to our department with history of swelling over the right side of face since 5 years .", "the patient reported insidious onset of swelling with a gradual progress to the present size .", "he also reported mild , intermittent , and dull pain in the region of swelling .", "clinical examination confirmed a solitary , oval , well - defined swelling measuring approximately 7  5 cm in the preauricular region [ figure 1 ] .", "it extended superioinferiorly from the level of outer canthus of the eye to inferior border of mandible and anterioposteriorly extended from 2 cm in front of anterior border of ramus to the mastoid notch [ figure 1 ] .", "on palpation , the swelling was firm , non - tender , free from skin , and mobile over the underlying structures .", "ultrasonography revealed a large lobulated solid mass of 6  4 cm in the right parotid region with multiple vessels with low velocity flow and without any areas of calcification or cystic changes .", "findings were suggestive of a low - grade vascular tumor . computed tomography scan with contrast medium revealed a large well - circumscribed , solitary isodense lesion measuring 6.36  4.39 cm [ figure 2 ] .", "it was observed to be extending anteriorly over the superficial lobe of the parotid gland , with anterior displacement of parotid gland .", "fnac revealed numerous vascular channels with occasional singly scattered and single cluster of plump spindle cells .", "ct- scan transverse section classical superficial parotidectomy was performed under general anesthesia using a modified appiani incision .", "tumor mass was dissected anteriorly and excised along with the superficial lobe of parotid gland [ figure 3 ] .", "facial nerve was located using the anterograde method and all the branches of facial nerve were preserved .", "follow up , facial nerve function was intact and no sign of reoccurrence was observed .", "histopathological sections revealed connective tissue with numerous vascular spaces of variable sizes ranging from small capillary - like vessels to partly lined vessels . around the endothelial cells ,", "cells were plump or stellate - shaped with mild inflammatory cell response and composed predominantly of plasma cells and a few lymphoid follicles [ figure 4 ] .", "photomicrograph shows numerous endothelial lined blood capillaries with collagen fibres interspersed with fibroblasts and fibrocytes ( h&e stain , 40 ) .", "( b ) photomicrograph shows endothelial lined blood vessels with connective tissue wall in the background of collagen fibres interspersed with fibroblasts and fibrocytes ( h&e stain , 400 ) photomicrograph of the sections showing the blood vessels positive for cd 34 ( ihc stain , 100 )", "various soft tissue tumors , both benign and malignant , present as swelling in the parotid region . clinical examination confirmed a smooth - surfaced , solitary , oval , firm , well - defined swelling measuring approximately 7  5 cm in the preauricular region .", "based on the clinical findings of location , consistency and borders a differential diagnosis of pleomorphic adenoma , solitary fibrous tumor ( sft ) , low - grade fibrosarcoma , and lipoma was made . as the most common tumors found in the region are parotid tumors , a provisional diagnosis of mixed parotid tumor was made . however , fnac and contrast - enhanced ct scan were in favor of a tumor with vascular component , shifting the differential diagnosis towards tumors with a vascular component presenting as slow - growing , circumscribed firm swellings .", "histopathological examination of the excised lesion revealed features suggestive of angiofibroma and had to be differentiated from lesions with similar features .", "the differential diagnosis included low - grade vascular lesions , spindle cell neoplasms , comprising of a large range of benign and low - grade malignant soft tissue lesions , including cellular angiofibroma , sft , low - grade fibromyxoid sarcoma ( lgfms ) , low - grade myxofibrosarcoma , myxoid liposarcoma , giant cell angiofibroma ( gca ) , angiomyolipoma , and angiomyofibroblastoma .", "cellular angiofibroma , usually occurs in the pelviperineal region , which would be an unusual location for the tumors reported herein ; however , cases have been reported to have occurred in the buccal mucosa , raising a diagnostic possibility .", "cellular angiofibroma shows more rounded , non - branching vessels , often of medium size with thicker walls .", "the tumors are generally more uniformly cellular , composed of lesional cells with short , stubby nuclei resembling those of spindle cell lipoma .", "sfts occur in a variety of locations in the head and neck region ; myxoid examples may be particularly difficult to identify and should be considered in the differential diagnosis .", "microscopically , besides the so - called  patternless  architecture , there is pronounced regional variation in cellularity , prominent thick collagen bundles and characteristic branching ectatic staghorn vessels , which are not accompanied by the abundant smaller - sized vessels present in angiofibroma of soft tissue . in addition , the tumor cells in sft express strong and diffuse cd34 in most cases .", "this entity lacks the innumerable , evenly distributed , arborizing thin - walled vessels characteristic of angiofibroma .", "lgfms is a distinctive fibroblastic malignant neoplasm characterized by a peculiar tendency to give rise to very late metastases .", "histologically , it shows alternating collagenous and myxoid areas with a usually swirling or whorled growth pattern , a frequently lobular appearance , and deceptively bland spindle cell morphology .", "the lesions tend to be more hypocellular , with a fibrous component that has uniform collagen deposition rather than the fibrillary or coarsely banded collagen fibers in angiofibroma of soft tissue . although lgfms may contain arcades of thin - walled vessels , vascularity is usually not prominent .", "usually occurring in subcutaneous tissues of the extremities , it shows distinctive histologic features including a lobulated growth pattern with infiltrative margins and fusiform , round , or stellate tumor cells with frequently slightly eosinophilic cytoplasm and hyperchromatic atypical or pleomorphic nuclei .", "obvious features of malignancy are usually present , characteristic elongated curvilinear vessels with perivascular hypercellularity , these features bear little or no resemblance to the rich vascular network of angiofibroma .", "myxoid liposarcoma contains a prominent plexiform network of thin - walled capillaries ( which has been referred to as a  chicken wire  or ", "however , it also shows scattered univacuolar and multivacuolar lipoblasts throughout , as well as stromal mucin pools not seen in angiofibroma .", "gca has been reported as a benign mesenchymal tumor with 2 cases originating in the buccal mucosa .", "although benign , the lesion has potential for local reoccurrence , especially after incomplete resection .", "histopathologically , it has similar presentation to that of sft , with presence of multinucleated giant cells not seen in angiofibroma .", "angiomyolipoma differs from angiofibromas because of the presence of prominent muscular arteries while angiomyofibroblastoma differs because of the presence of fibrovascular component with a loose myxoid fibroelement .", "enas have interstitial stromal tissue predominance with less vascular elements such as that of long - standing na .", "other distinctive histologic features are angiofibroma of soft tissue , consisting of a vaguely lobular , variably cellular proliferation of bland , uniform spindle cells set in an abundant , variably myxoid or collagenous extracellular matrix with numerous small , and thin - walled , branching blood vessels .", "the complexity of the vascular pattern , often also including larger vessels of varying size and shape , is the most noticeable feature .", "immunohistochemical analyses may show that stromal cells have strong cytoplasmic reactivity for vimentin and are generally immunonegative for smooth muscle actin and desmin .", "histopathology coupled with immunohistochemistry usually confirms the diagnosis in favor of angiofibroma . in our case ,", "the tissue specimen showed features consistent with the diagnosis of ena . in 1980 , de vincentiis and pinelli reviewed a series of 704 cases of angiofibroma and found that 13 cases manifested outside the nasopharynx , thus suggesting that extra nasopharyngeal localization of this tumor is a possible although rare occurrence . a review article by windfuhr and remmert in 2004 summarized 65 patients with enas .", "the most common site reported for enas is the maxillary sinus followed by the ethmoid , nasal cavity , and nasal septum .", "enas are also reported to originate from the ethmoid sinus , nasal cavity , nasal septum , larynx , sphenoid sinus , cheek , conjunctiva , oropharynx , retromolar area , middle turbinate , inferior turbinate , and tonsil .", "other rare sites for enas occurrence have also been described - external nose , hard palate , external ear , lacrimal sac , carotid bifurcation , oesophagus , trachea , facial nerve , middle cranial fossa , and infratemporal fossa .", "the most common age of incidence was the second decade ( 46% ) in comparison to 80% of patients with nas .", "enas have a higher mean age , i.e , 22 years compared to nas , which have a mean age of 18 years .", "this lesion has been described as different from that of the classical nasopharyngeal variety in being more common in females , older individuals with an early presentation , and being capsulated with poor vascularity and infrequent recurrence . unlike nas , enas present non - specific and numerous symptoms .", "imaging modalities such as ct scan , mri , ct angiography , and ultrasonography are required for optimal preoperative evaluation of nas and enas .", "imaging of nas with a contrast agent leads to diffuse and usually homogenous involvement in ct and mri scans with strong contrast enhancement .", "in contrast , enas enhances moderate amount of contrast or none due to its weak vascular involvement . surgical excision is the prime modality of treatment for enas ; radiotherapy maybe applied for non - resectable lesions .", "occurrence of enas , although rare in the maxillofacial region , should be considered in differential diagnosis of swellings in this region ."], "abstract_text": ["<S> angiofibromas are rare , benign , locally invasive vascular tumors , which represent 0.05 - 0.5% of all head and neck tumors . </S>", "<S> most frequent site of occurrence is the posterior nasopharynx , called as nasopharyngeal angiofibromas ( na ) , when these arise outside the nasopharyngeal region they are termed as extranasopharyngeal angiofibromas ( ena ) . </S>", "<S> only 65 cases of ena have been reported , and the most common site has been reported to be maxilla followed by ethmoids . </S>", "<S> other unusual sites of occurrence reported so far in literature are nasal cavity , nasal septum , larynx , sphenoid sinus , pterygomaxillary fissure , infratemporal fossa , cheek , oropharynx , retromolar area , middle turbinate , inferior turbinate , and tonsil . </S>", "<S> ena arising from the superficial lobe of parotid gland has not been reported in the literature so far and this case is the first to be reported . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["these range from various slow - growing benign tumors and low - grade malignancies to fast growing high - grade malignancies .", "angiofibromas are one such group of tumors arising in this region , which present as swellings . of these", ", nasal angiofibromas ( nas ) are the most common and nas occurring in the region other than the nose are called as extranasopharyngeal angiofibromas ( ena ) ."], ["a 54-year - old male reported to our department with history of swelling over the right side of face since 5 years .", "the patient reported insidious onset of swelling with a gradual progress to the present size .", "he also reported mild , intermittent , and dull pain in the region of swelling .", "clinical examination confirmed a solitary , oval , well - defined swelling measuring approximately 7  5 cm in the preauricular region [ figure 1 ] .", "it extended superioinferiorly from the level of outer canthus of the eye to inferior border of mandible and anterioposteriorly extended from 2 cm in front of anterior border of ramus to the mastoid notch [ figure 1 ] .", "on palpation , the swelling was firm , non - tender , free from skin , and mobile over the underlying structures .", "ultrasonography revealed a large lobulated solid mass of 6  4 cm in the right parotid region with multiple vessels with low velocity flow and without any areas of calcification or cystic changes .", "findings were suggestive of a low - grade vascular tumor . computed tomography scan with contrast medium revealed a large well - circumscribed , solitary isodense lesion measuring 6.36  4.39 cm [ figure 2 ] .", "it was observed to be extending anteriorly over the superficial lobe of the parotid gland , with anterior displacement of parotid gland .", "fnac revealed numerous vascular channels with occasional singly scattered and single cluster of plump spindle cells .", "ct- scan transverse section classical superficial parotidectomy was performed under general anesthesia using a modified appiani incision .", "tumor mass was dissected anteriorly and excised along with the superficial lobe of parotid gland [ figure 3 ] .", "facial nerve was located using the anterograde method and all the branches of facial nerve were preserved .", "follow up , facial nerve function was intact and no sign of reoccurrence was observed .", "histopathological sections revealed connective tissue with numerous vascular spaces of variable sizes ranging from small capillary - like vessels to partly lined vessels . around the endothelial cells ,", "cells were plump or stellate - shaped with mild inflammatory cell response and composed predominantly of plasma cells and a few lymphoid follicles [ figure 4 ] .", "photomicrograph shows numerous endothelial lined blood capillaries with collagen fibres interspersed with fibroblasts and fibrocytes ( h&e stain , 40 ) .", "( b ) photomicrograph shows endothelial lined blood vessels with connective tissue wall in the background of collagen fibres interspersed with fibroblasts and fibrocytes ( h&e stain , 400 ) photomicrograph of the sections showing the blood vessels positive for cd 34 ( ihc stain , 100 )"], ["various soft tissue tumors , both benign and malignant , present as swelling in the parotid region . clinical examination confirmed a smooth - surfaced , solitary , oval , firm , well - defined swelling measuring approximately 7  5 cm in the preauricular region .", "based on the clinical findings of location , consistency and borders a differential diagnosis of pleomorphic adenoma , solitary fibrous tumor ( sft ) , low - grade fibrosarcoma , and lipoma was made . as the most common tumors found in the region are parotid tumors , a provisional diagnosis of mixed parotid tumor was made . however , fnac and contrast - enhanced ct scan were in favor of a tumor with vascular component , shifting the differential diagnosis towards tumors with a vascular component presenting as slow - growing , circumscribed firm swellings .", "histopathological examination of the excised lesion revealed features suggestive of angiofibroma and had to be differentiated from lesions with similar features .", "the differential diagnosis included low - grade vascular lesions , spindle cell neoplasms , comprising of a large range of benign and low - grade malignant soft tissue lesions , including cellular angiofibroma , sft , low - grade fibromyxoid sarcoma ( lgfms ) , low - grade myxofibrosarcoma , myxoid liposarcoma , giant cell angiofibroma ( gca ) , angiomyolipoma , and angiomyofibroblastoma .", "cellular angiofibroma , usually occurs in the pelviperineal region , which would be an unusual location for the tumors reported herein ; however , cases have been reported to have occurred in the buccal mucosa , raising a diagnostic possibility .", "cellular angiofibroma shows more rounded , non - branching vessels , often of medium size with thicker walls .", "the tumors are generally more uniformly cellular , composed of lesional cells with short , stubby nuclei resembling those of spindle cell lipoma .", "sfts occur in a variety of locations in the head and neck region ; myxoid examples may be particularly difficult to identify and should be considered in the differential diagnosis .", "microscopically , besides the so - called  patternless  architecture , there is pronounced regional variation in cellularity , prominent thick collagen bundles and characteristic branching ectatic staghorn vessels , which are not accompanied by the abundant smaller - sized vessels present in angiofibroma of soft tissue . in addition , the tumor cells in sft express strong and diffuse cd34 in most cases .", "this entity lacks the innumerable , evenly distributed , arborizing thin - walled vessels characteristic of angiofibroma .", "lgfms is a distinctive fibroblastic malignant neoplasm characterized by a peculiar tendency to give rise to very late metastases .", "histologically , it shows alternating collagenous and myxoid areas with a usually swirling or whorled growth pattern , a frequently lobular appearance , and deceptively bland spindle cell morphology .", "the lesions tend to be more hypocellular , with a fibrous component that has uniform collagen deposition rather than the fibrillary or coarsely banded collagen fibers in angiofibroma of soft tissue . although lgfms may contain arcades of thin - walled vessels , vascularity is usually not prominent .", "usually occurring in subcutaneous tissues of the extremities , it shows distinctive histologic features including a lobulated growth pattern with infiltrative margins and fusiform , round , or stellate tumor cells with frequently slightly eosinophilic cytoplasm and hyperchromatic atypical or pleomorphic nuclei .", "obvious features of malignancy are usually present , characteristic elongated curvilinear vessels with perivascular hypercellularity , these features bear little or no resemblance to the rich vascular network of angiofibroma .", "myxoid liposarcoma contains a prominent plexiform network of thin - walled capillaries ( which has been referred to as a  chicken wire  or ", "however , it also shows scattered univacuolar and multivacuolar lipoblasts throughout , as well as stromal mucin pools not seen in angiofibroma .", "gca has been reported as a benign mesenchymal tumor with 2 cases originating in the buccal mucosa .", "although benign , the lesion has potential for local reoccurrence , especially after incomplete resection .", "histopathologically , it has similar presentation to that of sft , with presence of multinucleated giant cells not seen in angiofibroma .", "angiomyolipoma differs from angiofibromas because of the presence of prominent muscular arteries while angiomyofibroblastoma differs because of the presence of fibrovascular component with a loose myxoid fibroelement .", "enas have interstitial stromal tissue predominance with less vascular elements such as that of long - standing na .", "other distinctive histologic features are angiofibroma of soft tissue , consisting of a vaguely lobular , variably cellular proliferation of bland , uniform spindle cells set in an abundant , variably myxoid or collagenous extracellular matrix with numerous small , and thin - walled , branching blood vessels .", "the complexity of the vascular pattern , often also including larger vessels of varying size and shape , is the most noticeable feature .", "immunohistochemical analyses may show that stromal cells have strong cytoplasmic reactivity for vimentin and are generally immunonegative for smooth muscle actin and desmin .", "histopathology coupled with immunohistochemistry usually confirms the diagnosis in favor of angiofibroma . in our case ,", "the tissue specimen showed features consistent with the diagnosis of ena . in 1980 , de vincentiis and pinelli reviewed a series of 704 cases of angiofibroma and found that 13 cases manifested outside the nasopharynx , thus suggesting that extra nasopharyngeal localization of this tumor is a possible although rare occurrence . a review article by windfuhr and remmert in 2004 summarized 65 patients with enas .", "the most common site reported for enas is the maxillary sinus followed by the ethmoid , nasal cavity , and nasal septum .", "enas are also reported to originate from the ethmoid sinus , nasal cavity , nasal septum , larynx , sphenoid sinus , cheek , conjunctiva , oropharynx , retromolar area , middle turbinate , inferior turbinate , and tonsil .", "other rare sites for enas occurrence have also been described - external nose , hard palate , external ear , lacrimal sac , carotid bifurcation , oesophagus , trachea , facial nerve , middle cranial fossa , and infratemporal fossa .", "the most common age of incidence was the second decade ( 46% ) in comparison to 80% of patients with nas .", "enas have a higher mean age , i.e , 22 years compared to nas , which have a mean age of 18 years .", "this lesion has been described as different from that of the classical nasopharyngeal variety in being more common in females , older individuals with an early presentation , and being capsulated with poor vascularity and infrequent recurrence . unlike nas , enas present non - specific and numerous symptoms .", "imaging modalities such as ct scan , mri , ct angiography , and ultrasonography are required for optimal preoperative evaluation of nas and enas .", "imaging of nas with a contrast agent leads to diffuse and usually homogenous involvement in ct and mri scans with strong contrast enhancement .", "in contrast , enas enhances moderate amount of contrast or none due to its weak vascular involvement . surgical excision is the prime modality of treatment for enas ; radiotherapy maybe applied for non - resectable lesions .", "occurrence of enas , although rare in the maxillofacial region , should be considered in differential diagnosis of swellings in this region ."]]}
{"article_id": "PMC3043761", "article_text": ["atherosclerotic renal artery hypertension is reported in almost 7% of adults older than 65 years1 ) and is associated with cardiovascular events , and may double risk of mortality2 ) . in the 1990s , radiological angioplasty began to replace surgical revascularization .", "galaria et al . showed that percutaneous and open renal revascularization had equivalent long - term functional outcomes3 ) . over time , the less - invasive procedure became more accessible .", "most cases of atherosclerotic renovascular disease ( arvd ) are located in the ostium , and are extensions of calcified aortic plaques4 ) .", "stent placement might also provide additional force to increase the rates of technical success5 ) and to reduce the rate of restenosis at 6 months after the initial procedure6 ) .", "intervention with stents has become a standard procedure ; in patients with stenosis of the renal artery , placement of a stent is likely to be the initial form of treatment .", "however , there is limited evidence to support revascularization over medical therapy for patients with atherosclerotic renal artery stenosis7 , 8) .", "retrospective studies previously reported that renal artery lesions might progress to severe stenosis and ultimately to renal artery occlusion9 ) .", "michael et al . performed a prospective study that reported that the progression of renal artery disease was a frequent occurrence with an annual rate of progression of renal artery lesions reported to be 7%10 ) .", "however , at the time of these studies , statins were being used in fewer patients .", "a retrospective study of the effects of statins on the progression of arvd has shown that the use of statins reduces the risk of progression and the development of arvd11 ) .", "another prospective population - based study reported that the progression to significant arvd was observed in only 4.0% during 8 years of follow - up ( annualized rate , 0.5% per year ) , and no case of arvd progressed to occlusion12 ) .", "essential hypertension and clinically silent renal artery stenosis often coexist , and essential hypertension also coexists with renovascular hypertension13 ) .", "renal injury distal to an atherosclerotic renovascular obstruction is due to multiple intrinsic factors producing parenchymal tissue injury ( fig .", "non - traditional mediators of arvd such as inflammatory pathways , reactive oxygen species production , ischemia / reperfusion damage and modulation of matrix turnover have been proposed as causes of the renal failure related to arvd15 ) .", "this complexity of the pathophysiology might explain why the severity of the stenosis is not correlated with renal dysfunction .", "clinical data support dissociation of improved renal artery patency from clinical outcome in patients with atherosclerotic renal artery stenosis ; this may illustrate the effects of irreversible injury on the post stenotic kidney . a prospective clinical study reported by wright et al . showed absence of a correlation between renal artery anatomy and baseline renal function or functional outcome , and a good correlation between renal functional outcome and proteinuria", "; these findings suggest that renal parenchymal damage is a major determinant of renal dysfunction and outcome rather than the severity of the renal artery stenosis in arvd16 ) .", "reported that the mean arterial pressure did not decrease by 10 mmhg or more after revascularization and renal function declined in most patients with arvd that had high resistance - index values before revascularization17 ) .", "chrysochou et al . showed a correlation between baseline proteinuria and decline in estimated glomerular filtration rate ( gfr ) with time after revascularization18 ) .", "therefore , post stenotic renal injury can lead to renal parenchymal injury reflected by high intrarenal resistance and/or the presence of proteinuria .", "proteinuria and other factors involved in intrarenal resistance are predictors of a poor outcome after renal revascularization in arvd", ". when patients with chronic kidney disease undergo renal angioplasty with / without stent placement , the response with regard to renal function can be negative as well as positive .", "positive responses include improvement of renal function , and stabilization or attenuation of declining renal function , as expected .", "possible causes of these adverse outcomes include contrast induced nephropathy , atheroembolism , and restenosis or stent thrombosis19 )", ". the administration of contrast might increase the risk of acute renal dysfunction , especially in patients that have preexisting renal impairment .", "an ex vivo study reported that each manipulation of atheroma specimens , from simply advancing the guidewire through the atherosclerotic lesion to positioning and deploying the wallstent , releases thousands of fragments , and that these atherosclerotic fragments are of sufficient size to create vascular occlusion and initiate significant renal parenchymal damage20 ) .", "the nature of this disorder suggests that a systemic approach is necessary to provide cardiovascular protection .", "previously , statin therapy was discussed as a method of altering the natural history of arvd11 ) .", "angiotensin - converting enzyme inhibitors and angiotensin receptor blockers effectively reduce blood pressure in patients with renovascular disease . in addition , a population - based cohort study of over 3,500 patients with arvd in canada found that angiotensin inhibitors could cause acute renal toxicity in a small subset of vulnerable patients ; however , they still improved the cardiovascular and renal outcomes in patients with arvd , but at the expense of acute renal toxicity21 ) . recently", "three randomized controlled trials have been performed : the stent placement and blood pressure and lipid - lowering for the prevention of progression of renal dysfunction caused by atherosclerotic ostial stenosis of the renal artery ( star ) trial , the angioplasty and stenting for renal artery lesions ( astral ) trial , and the cardiovascular outcomes in renal artherosclerotic lesions ( coral ) trial ( table 1 ) .", "two large randomized trials of intervention vs. medical therapy showed negative results for the intervention .", "the star trial was a european multicenter trial that enrolled 140 patients with ostial renal artery stenosis greater than 50% , blood pressure controlled to less than 140/90 mmhg , and creatinine clearance 15 to 80 ml / min7 ) .", "all patients received angiotensin blocking agents and a statin , regardless of their lipid levels .", "after a 2-year interventional period , no difference was observed in the decline of renal function , the degree of blood pressure control , and the rates of cardiovascular morbidity and death .", "investigators in the astral trial enrolled 806 patients with at least one stenotic renal artery considered suitable for balloon angioplasty , stenting , or both in their international , multicenter trial8 ) .", "the mean estimated gfr was 40 ml / min , and most of the patients were on statin and angiotensin blocking therapy . at a mean follow - up of 33.6 months", ", no difference was noted between the treatment groups in the decline of renal function or blood pressure control , and the renal function worsened slightly in both groups .", "most of the enrolled patients in the two clinical trials had relatively asymptomatic atherosclerotic renal artery stenosis .", "therefore , the practice of indiscriminately performing revascularization , without strong evidence , is no longer acceptable .", "the coral trial is an ongoing multicenter randomized controlled trial in the united states22 ) .", "it is still enrolling patients that have drug - resistant hypertension or a gfr < 60 ml / min .", "it is using a standardized medical protocol to control blood pressure , and embolic protection devices during procedures are encouraged .", "thus far , intervention has not been recommended if renal function has remained stable over the past 6 to 12 months and if hypertension can be controlled medically . according to a clinical classification of atherosclerotic renal artery stenosis with guidelines for vascular intervention of surveillance published in 2008 by the atherosclerotic peripheral vascular symposium23 ) , additional recommendations favoring medical therapy were as follows : very advanced age and/or limited life expectancy , extensive co - morbidities that make revascularization too risky , high risk for or previous experience with atheroembolic disease , and other concomitant renal parenchymal diseases that cause progressive renal dysfunction ( e.g. , interstitial nephritis , diabetic nephropathy)23 ) .", "they also recommended factors favoring medical therapy and intervention as follows : progressive decline in gfr during treatment of systemic hypertension , failure to achieve adequate blood pressure control with optimal medical therapy ( medical failure ) , rapid or recurrent decline in the gfr in association with a reduction in systemic pressure , decline in the gfr during therapy with angiotensin - converting enzyme inhibitors or angiotensin receptor blockers , and recurrent congestive heart failure in a patient in whom the adequacy of left ventricular function does not provide an explanation23 ) .", "the best evidence supporting intervention is for bilateral stenosis with \" flash \" pulmonary edema , but the evidence is from retrospective studies24 - 26 ) .", "the aim of treatment for arvd should include the prevention of cardiovascular and/or renal events .", "treatment of patients with arvd should include consideration of the following factors : age , co - morbidity , blood pressure , renal function , and kidney size .", "the results of two recent studies suggest that patients with arvd and stable renal function can be controlled medically .", "intervention should be considered only in patients with arvd and rapidly progressive cardiac or renal dysfunction .", "the results of the coral and the post hoc analysis of astral might provide additional evidence for revascularization ."], "abstract_text": ["<S> atherosclerotic renovascular hypertension is a form of secondary hypertension due to renal artery stenosis . after the introduction of medical therapy such as with statins and angiotensin blocking agents </S>", "<S> , it has been considered a very slowly progressive disease . in the 1990s , </S>", "<S> surgical methods were compared to radiological intervention and showed no additional benefits . </S>", "<S> recent clinical data also demonstrate that in cases of relatively stable atherosclerotic renovascular disease , medical therapy is as effective as other interventions with regard to patient outcomes . in this paper </S>", "<S> the recent clinical outcomes are reviewed . </S>"], "labels": null, "section_names": ["Introduction", "Natural history", "Dissociation between arterial patency and function", "Factors favoring medical therapy", "Conclusion"], "sections": [["atherosclerotic renal artery hypertension is reported in almost 7% of adults older than 65 years1 ) and is associated with cardiovascular events , and may double risk of mortality2 ) . in the 1990s , radiological angioplasty began to replace surgical revascularization .", "galaria et al . showed that percutaneous and open renal revascularization had equivalent long - term functional outcomes3 ) . over time , the less - invasive procedure became more accessible .", "most cases of atherosclerotic renovascular disease ( arvd ) are located in the ostium , and are extensions of calcified aortic plaques4 ) .", "stent placement might also provide additional force to increase the rates of technical success5 ) and to reduce the rate of restenosis at 6 months after the initial procedure6 ) .", "intervention with stents has become a standard procedure ; in patients with stenosis of the renal artery , placement of a stent is likely to be the initial form of treatment .", "however , there is limited evidence to support revascularization over medical therapy for patients with atherosclerotic renal artery stenosis7 , 8) ."], ["retrospective studies previously reported that renal artery lesions might progress to severe stenosis and ultimately to renal artery occlusion9 ) .", "michael et al . performed a prospective study that reported that the progression of renal artery disease was a frequent occurrence with an annual rate of progression of renal artery lesions reported to be 7%10 ) .", "however , at the time of these studies , statins were being used in fewer patients .", "a retrospective study of the effects of statins on the progression of arvd has shown that the use of statins reduces the risk of progression and the development of arvd11 ) .", "another prospective population - based study reported that the progression to significant arvd was observed in only 4.0% during 8 years of follow - up ( annualized rate , 0.5% per year ) , and no case of arvd progressed to occlusion12 ) ."], ["essential hypertension and clinically silent renal artery stenosis often coexist , and essential hypertension also coexists with renovascular hypertension13 ) .", "renal injury distal to an atherosclerotic renovascular obstruction is due to multiple intrinsic factors producing parenchymal tissue injury ( fig .", "non - traditional mediators of arvd such as inflammatory pathways , reactive oxygen species production , ischemia / reperfusion damage and modulation of matrix turnover have been proposed as causes of the renal failure related to arvd15 ) .", "this complexity of the pathophysiology might explain why the severity of the stenosis is not correlated with renal dysfunction .", "clinical data support dissociation of improved renal artery patency from clinical outcome in patients with atherosclerotic renal artery stenosis ; this may illustrate the effects of irreversible injury on the post stenotic kidney . a prospective clinical study reported by wright et al . showed absence of a correlation between renal artery anatomy and baseline renal function or functional outcome , and a good correlation between renal functional outcome and proteinuria", "; these findings suggest that renal parenchymal damage is a major determinant of renal dysfunction and outcome rather than the severity of the renal artery stenosis in arvd16 ) .", "reported that the mean arterial pressure did not decrease by 10 mmhg or more after revascularization and renal function declined in most patients with arvd that had high resistance - index values before revascularization17 ) .", "chrysochou et al . showed a correlation between baseline proteinuria and decline in estimated glomerular filtration rate ( gfr ) with time after revascularization18 ) .", "therefore , post stenotic renal injury can lead to renal parenchymal injury reflected by high intrarenal resistance and/or the presence of proteinuria .", "proteinuria and other factors involved in intrarenal resistance are predictors of a poor outcome after renal revascularization in arvd", ". when patients with chronic kidney disease undergo renal angioplasty with / without stent placement , the response with regard to renal function can be negative as well as positive .", "positive responses include improvement of renal function , and stabilization or attenuation of declining renal function , as expected .", "possible causes of these adverse outcomes include contrast induced nephropathy , atheroembolism , and restenosis or stent thrombosis19 )", ". the administration of contrast might increase the risk of acute renal dysfunction , especially in patients that have preexisting renal impairment .", "an ex vivo study reported that each manipulation of atheroma specimens , from simply advancing the guidewire through the atherosclerotic lesion to positioning and deploying the wallstent , releases thousands of fragments , and that these atherosclerotic fragments are of sufficient size to create vascular occlusion and initiate significant renal parenchymal damage20 ) ."], ["the nature of this disorder suggests that a systemic approach is necessary to provide cardiovascular protection .", "previously , statin therapy was discussed as a method of altering the natural history of arvd11 ) .", "angiotensin - converting enzyme inhibitors and angiotensin receptor blockers effectively reduce blood pressure in patients with renovascular disease . in addition , a population - based cohort study of over 3,500 patients with arvd in canada found that angiotensin inhibitors could cause acute renal toxicity in a small subset of vulnerable patients ; however , they still improved the cardiovascular and renal outcomes in patients with arvd , but at the expense of acute renal toxicity21 ) . recently", "three randomized controlled trials have been performed : the stent placement and blood pressure and lipid - lowering for the prevention of progression of renal dysfunction caused by atherosclerotic ostial stenosis of the renal artery ( star ) trial , the angioplasty and stenting for renal artery lesions ( astral ) trial , and the cardiovascular outcomes in renal artherosclerotic lesions ( coral ) trial ( table 1 ) .", "two large randomized trials of intervention vs. medical therapy showed negative results for the intervention .", "the star trial was a european multicenter trial that enrolled 140 patients with ostial renal artery stenosis greater than 50% , blood pressure controlled to less than 140/90 mmhg , and creatinine clearance 15 to 80 ml / min7 ) .", "all patients received angiotensin blocking agents and a statin , regardless of their lipid levels .", "after a 2-year interventional period , no difference was observed in the decline of renal function , the degree of blood pressure control , and the rates of cardiovascular morbidity and death .", "investigators in the astral trial enrolled 806 patients with at least one stenotic renal artery considered suitable for balloon angioplasty , stenting , or both in their international , multicenter trial8 ) .", "the mean estimated gfr was 40 ml / min , and most of the patients were on statin and angiotensin blocking therapy . at a mean follow - up of 33.6 months", ", no difference was noted between the treatment groups in the decline of renal function or blood pressure control , and the renal function worsened slightly in both groups .", "most of the enrolled patients in the two clinical trials had relatively asymptomatic atherosclerotic renal artery stenosis .", "therefore , the practice of indiscriminately performing revascularization , without strong evidence , is no longer acceptable .", "the coral trial is an ongoing multicenter randomized controlled trial in the united states22 ) .", "it is still enrolling patients that have drug - resistant hypertension or a gfr < 60 ml / min .", "it is using a standardized medical protocol to control blood pressure , and embolic protection devices during procedures are encouraged .", "thus far , intervention has not been recommended if renal function has remained stable over the past 6 to 12 months and if hypertension can be controlled medically . according to a clinical classification of atherosclerotic renal artery stenosis with guidelines for vascular intervention of surveillance published in 2008 by the atherosclerotic peripheral vascular symposium23 ) , additional recommendations favoring medical therapy were as follows : very advanced age and/or limited life expectancy , extensive co - morbidities that make revascularization too risky , high risk for or previous experience with atheroembolic disease , and other concomitant renal parenchymal diseases that cause progressive renal dysfunction ( e.g. , interstitial nephritis , diabetic nephropathy)23 ) .", "they also recommended factors favoring medical therapy and intervention as follows : progressive decline in gfr during treatment of systemic hypertension , failure to achieve adequate blood pressure control with optimal medical therapy ( medical failure ) , rapid or recurrent decline in the gfr in association with a reduction in systemic pressure , decline in the gfr during therapy with angiotensin - converting enzyme inhibitors or angiotensin receptor blockers , and recurrent congestive heart failure in a patient in whom the adequacy of left ventricular function does not provide an explanation23 ) .", "the best evidence supporting intervention is for bilateral stenosis with \" flash \" pulmonary edema , but the evidence is from retrospective studies24 - 26 ) ."], ["the aim of treatment for arvd should include the prevention of cardiovascular and/or renal events .", "treatment of patients with arvd should include consideration of the following factors : age , co - morbidity , blood pressure , renal function , and kidney size .", "the results of two recent studies suggest that patients with arvd and stable renal function can be controlled medically .", "intervention should be considered only in patients with arvd and rapidly progressive cardiac or renal dysfunction .", "the results of the coral and the post hoc analysis of astral might provide additional evidence for revascularization ."]]}
{"article_id": "PMC4134796", "article_text": ["tuberculosis ( tb ) caused by mycobacterium tuberculosis ( m. tb ) continues to be a significant global health problem , affecting millions of people worldwide [ 1 , 2 ] .", "it is a prevalent infectious disease in china , with 250,000 deaths from tb annually and 6 million active tb patients at present .", "the global incidence of tb is raising due to coinfection with the human immunodeficiency virus ( hiv ) and the emergence of multidrug - resistant ( mdr ) m. tb strains [ 5 , 6 ] . according to the report of world health organization ( who )", ", m. tb will cause 1 billion new cases and about 35 million deaths worldwide by 2020 .", "therefore , effective treatment and control strategies are urgently needed to counteract the global threat of tb .", "the current vaccine against tb , m. bovis bacilli - calmette - gurin ( bcg ) , is a live attenuated vaccine which has been widely used throughout the world for many decades .", "bcg protects children efficiently against miliary and meningeal tb , but protective efficiency against pulmonary tb in adults has been found to vary highly from 0% to 80% .", "much effort has been devoted to improving bcg efficacy by genetic engineering technology because of its strong immunostimulatory properties and proven safety for human use [ 9 , 10 ] .", "recombinant bcg ( rbcg ) expressing different immunodominant antigens of m. tb , such as secreted antigens ( ag85b , ag85c , esat-6 , etc . ) or latency associated antigens ( -crystallin , rv2659c , rv3407 and rv1733c , etc . ) , have been tested as candidate vaccines against tb and are demonstrated to have an enhanced ability to induce th1 immune response and protection against m. tb challenge in animal models [ 11 , 12 ] .", "also , it is definitely no doubt that doses of antigens could subtly influence the magnitude of host immune response as well as protection efficacy , no matter antigen is administered in the form of rbcg , protein , dna , or rna .", "we have previously reported the construction of a m. tb fura gene operator / promoter ( pfura)-based differential expression system , from which it is feasible to express target antigens of interest in a modular fashion .", "m. tb chimeric antigen ag856a2 , which is coded by a recombinant ag85a gene with 2 copies of esat-6 gene inserted at the acc i site of ag85a ( see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2014/196124 ) , shows improved immunogenicity in mice when it is inoculated intramuscularly as a dna vaccine .", "for the current study , we selected two rbcg strains overexpressing the same chimeric antigen ag856a2 at the maximum difference : rbcg186 and rbcg486 overexpressing the fusion protein under control of the wild - type or the optimized double - mutated fura promoters , respectively .", "we tested their efficacy as vaccines in c57bl/6 mice , comparing immune response and protection against m. tb challenge .", "the results showed that mice vaccinated with rbcg186 or rbcg486 generally induced higher antigen - specific effector and memory immune responses , as well as protective efficacies compared to mice vaccinated with the parent bcg strain .", "however , the two rbcg strains between themselves , which expressed the chimeric antigen ag856a2 at different levels , induced different antigen - specific ifn- production and comparable number of m. tb - specific cd4 t cells expressing il-2 . and the protective efficacies imposed by the two rbcg strains displayed no significant differences although higher protection was observed in rbcg486 vaccinated mice than that in rbcg186 vaccinated mice .", "female specific pathogen - free ( spf ) c57bl/6 mice aged 68  wks were purchased from shanghai slac laboratory animal co. , ltd .", "( shanghai , china ) and kept under spf conditions with food and water ad libitum until challenge .", "infected mice were maintained in a biosafety level 3 ( bsl-3 ) biocontainment animal facility .", "all animal experiment protocols were approved by chinese science academy committee on care and use of laboratory animals and were performed according to the guidelines of the laboratory animal ethical board of shanghai public health clinical center . \n", "bcg and its derivative recombinant strains were grown in liquid middlebrook 7h9 broth ( bd difco , usa ) supplemented with 10% oleic acid - albumin - dextrose - catalase enrichment ( oadc , bd difco , usa ) , 0.2% glycerol , and 0.05% tween 80 .", "cultures in the exponential phase were frozen and stored at 80c . when required , kanamycin was added at a final concentration of 50 or 20  g / ml for e. coli or mycobacteria , respectively .", "two rbcg strains overexpressing m. tb chimeric immunodominant antigen ag856a2 at different levels were constructed as previously described .", "briefly , the ag856a2 coding gene , which is a recombinant ag85a gene with 2 copies of esat-6 gene inserted in its acc i site , was amplified from the plasmid template of dna vaccine hg856a and then cloned into mycobacterial differential expression vectors pmfa11 and pmfa41 under control of the prototypical and double - mutated ( mutations : initial codon change from gtg to aug and 6  bp substitution at upstream at - rich region ) fura promoter , respectively .", "the resulting constructs were electroporated into bcg - danish competent cells and selected on middlebrook 7h11 agar with kanamycin .", "the rbcg transformants were grown to midexponential phase in complete middlebrook 7h9 broth and then verified the recombinant protein expression by routine western - blotting assay .", "mice were vaccinated subcutaneously ( s.c . ) with 2  10 colony - forming units ( cfu ) of bcg or rbcgs in 100  l saline .", "eight weeks after vaccination , groups of 6 mice were either sacrificed for assessment of antigen - specific t cell responses in splenocytes or exposed to an aerosol of virulent m. tb h37rv strain to deposit an inhaled dose of 100200 cfu per lung by an inhalation exposure system ( glas - col , usa ) .", "ifn- elispot assay kit ( bd biosciences , usa ) was used as described by the manufacturer .", "plates were coated with anti - ifn- mab overnight at 4c and then blocked with rpmi 1640 medium containing 10% fetal bovine serum ( fbs ) for 1  h at room temperature .", "splenocytes ( 2.5  10 cells / well ) from immunized mice were isolated , plated , and cultured with 10  g / ml ppd ( statens serum institute , denmark ) or 2  g / ml recombinant ag85a , 6  g / ml recombinant esat-6 to provide stimulation at 37c , 5% co2 for 20  h. after washing the plates with pbs - t20 ( 1  pbs , ph 7.4 , 0.05% tween 20 ) , biotinylated anti - ifn- was added for 2  h at room temperature .", "streptavidin - hrp was added for 45  min , and the color was developed with 3-amino-9-ethylcarbazole ( aec ) substrate ( bd biosciences ) .", "an immunospot analyzer ( cellular technology , usa ) was used to count the spots .", "splenocytes ( 2  10 cells / well ) isolated at 8  weeks after immunization were plated in 96-well plates and stimulated with 10  g / ml ppd for 14  h in the presence of 1  g / ml anti - cd28 ( bd biosciences ) and subsequently incubated for an additional 5  h at 37c following the addition of 0.5  l / ml monensin / golgistop ( bd biosciences ) .", "following overnight incubation at 4c , the cells were washed in facs buffer ( pbs containing 0.1% sodium azide and 1% fbs ) and subsequently stained for 30  min at 4c for surface markers with mabs as indicated using anti - cd3-pacific blue , anti - cd8-fitc , and anti - cd44-v500 ( all from bd biosciences ) .", "cells were then washed in facs buffer , fixed , permeabilized using the cytofix / cytoperm kit ( bd biosciences ) according to the manufacturer 's instructions and stained intracellularly for 30  min at 4c using anti - ifn--apc - cy7 , anti - tnf--percp - cy5.5 , and anti - il-2-apc mabs ( all from bd biosciences ) .", "cells were subsequently washed , resuspended in facs buffer , and then analyzed by multiparameter flow cytometry using a bd facsaria flow cytometer ( bd biosciences ) . for each sample ,", "at least 300,000 events were collected and responses were analyzed using flowjo software ( tree star , usa ) .", "five weeks after infection , mice were sacrificed and the mycobacterial burden was determined by plating homogenates of lung , excluding right postcaval lobe , and entire spleen onto middlebrook 7h11 agar plates supplemented with 10% oadc enrichment and a 4-antibiotic mixture ( 40  u / ml polymycin b , 4  g / ml amphotericin , 50  g / ml carbenicillin , 2  g / ml trimethoprim ) that prevents growth of contaminating microorganisms .", "plates were incubated at 37c for 3  weeks in semisealed plastic bags and then cfu were counted and expressed as log10 cfu per organ .", "then , the embedded lung lobes were sectioned in thickness of 5  m , stained with haematoxylin and eosin ( h & e ) and photographed using a olympus ckx41 microscope ( olympus , japan ) fitted with an olympus dp20 camera connected to a computer .", "the image pro plus program ( media cybernetics , usa ) was utilized to objectively assess the level of inflammation present in each image .", "the mean percent of area inflamed was quantified averaging from three to five lung sections of each of the different groups of mice .", "the antibodies were rabbit polyclonal anti - mouse tnf- ( abcam , uk ) , rabbit polyclonal ifn- antibodies ( invitrogen , usa ) , and rabbit polyclonal anti - mouse inos antibody ( cayman chemical , usa ) .", "all sections were examined by light microscopy , and the expression of tnf- , ifn- , or inos was semiquantified by intensity of positive signal using image pro plus software .", "immune responses , protective efficacies , and histopathological staining were tested by one - way anova followed by tukey 's multiple comparison tests of the means .", "we have previously developed a novel mycobacterial differential expression system ( pmfa series ) based on the m. tb fura gene operator / promoter ( pfura ) or its derivatives .", "ag856a2 was cloned into two of these plasmids , pmfa11 and pmfa41 , which drives low and high gene expression under the control of the wild - type and modified fura promoters , respectively . by transformation of bcg", ", we obtained two strains , rbcg186 and rbcg486 , which drove correspondingly low and high expression of chimeric immunodominant antigen ag856a2 ( figure 1 , upper panel ) .", "quantification of the band intensities of western - blot indicated that rbcg486 roughly expressed > 3-fold of ag856a2 than rbcg186 did ( figure 1 , lower panel ) .", "eight weeks after vaccination , elispot assay of splenocytes showed that more cells in the rbcg486-vaccinated mice expressed ag85a - specific ifn- compared to those of rbcg186- and bcg - vaccinated mice ( figure 2 , left panel ) .", "also , significantly elevated numbers of splenocytes expressed esat-6-specific ifn- in both rbcg186- and rbcg486-vaccinated mice compared to that of bcg - vaccinated mice ( figure 2 , middle panel ) .", "additionally , esat-6-specific ifn- was induced at much higher level in rbcg486-vaccinated mice compared to rbcg186 group ( figure 2 , middle panel ) .", "a similar pattern of ppd - specific ifn- responses as ag85a - specific response was also observed but the difference was not statistically significant , regarding to the comparisons of rbcg486-vaccinated mice and other immunized groups ( figure 2 , right panel ) .", "we used flow cytometry to measure the capacity of m. tb - specific cd4 t cells from spleens of vaccinated mice producing cytokines ifn- , tnf- , and il-2 at single cell level after stimulation in vitro with ppd .", "the cytokine - producing cd3cd4 cells were classified into seven subpopulations based on their production of ifn- , tnf- , and il-2 in any combination ( figure 3(a ) ) .", "significantly increased frequencies of ppd - specific il-2 cd4 t cells were identified in rbcg - vaccinated mice , whereas increased frequencies of ifn- cells were identified in bcg - vaccinated mice even though statistically insignificant ( figure 3(a ) ) .", "the pie chart of this data clarified the dominance of il-2 cd4 t cells in rbcg - vaccinated mice , while ifn- cd4 t cells dominated the responses of bcg - vaccinated mice ( figure 3(b ) ) .", "rbcg and bcg - vaccination did not differ in their ability to induce m. tb - specific cd4 t cells producing other combinations of cytokines ( p > 0.05 ) . in accordance , we also observed higher integrated mean fluorescence intensities ( imfi = % frequency  mfi ) of il-2 in il-2-producing cd4 t cells , even though it is statistically insignificant ( figure 3(c ) ) . in general , rbcg induced higher antigen - specific cytokine responses as compared to bcg ( figures 2 and 3 ) , and rbcg486 induced higher antigen - specific ifn- response ( figure 2 ) and comparable frequency of m. tb - specific cd4 t cells expressing il-2 ( figure 3 ) .", "then , we further compared the protective efficacies of rbcg486 , rbcg186 , and bcg against m. tb - challenge .", "as shown in figure 4(a ) , 5  weeks after challenge all vaccinated mice had a significantly reduced bacillary load in lungs , when compared to the saline - treated mice .", "vaccination with bcg and rbcg186 resulted in a comparable reduction in bacillary load ( figure 4 ) . however , even though rbcg486 vaccination induced a significantly greater protection when compared to the bcg - vaccinated mice , it showed no difference of protection when compared to the rbcg186-vaccinated mice ( figure 4(a ) ) .", "the bacillary loads in spleens shared the similar pattern as those in lungs , with rbcg486-vaccinated mice having far fewer bacilli when compared to the saline - treated or bcg - vaccinated mice and having comparable bacilli compared to the rbcg186-vaccinated mice ( figure 4(b ) ) .", "five weeks after challenge , m. tb infection caused severe pathology changes in saline - treated mice , with about 24.3% of the tissue showing extensive multifocal granulomatous infiltration , characterized by numerous foamy macrophages surrounded by inflammatory cells ( figure 5 ) .", "however , all the vaccinated groups of mice had significantly reduced pulmonary granulomatous consolidation compared to the unvaccinated mice ( i.e. , 13.42% consolidation in bcg - vaccinated group , 7.24% in rbcg186-vaccinated group , and 4.87% in rbcg486-vaccinated group ) .", "the rbcg - vaccinated mice showed the mildest pathology , and all of the mice in these two groups had mainly well - preserved alveolar spaces with only a few scattered areas of diffused infiltration ( figure 5 ) .", "immunohistochemical staining of the lung tissues showed the presence of tnf- , ifn- , and inos in all groups of infected mice and staining was strongest in the granulomatous lesions compared to that in the nongranulomatous areas .", "five weeks after infection , a very high level of tnf- was observed in the lungs of saline - treated mice ( figure 6(a ) ) ; tnf- staining was extensive in necrotic areas within the advanced coalescent granulomas .", "vaccination with bcg resulted in the reduced amounts of tnf- expression , even though statistically insignificant .", "in contrast , rbcg - vaccinated mice , especially rbcg486-vaccinated mice , showed only a little weak staining for tnf- and this was restricted primarily to the granuloma core ( figure 6(a ) ) .", "similar patterns of ifn- and inos staining were also observed except that there was relatively much weaker staining in the lungs of ( r)bcg - vaccinated mice compared to the saline - treated mice ( figures 6(b ) and 6(c ) ) .", "similar pattern of tnf- , ifn- , and inos staining was also observed in the infected spleens of vaccinated mice , with the highest staining in saline - treated mice , moderate staining in bcg - vaccinated mice , and the lowest staining in rbcg - vaccinated mice ( figure  s2 ) .", "during the past decades , great efforts have been focused on modifications of the current bcg vaccine to develop new anti - tb vaccine candidates . some modified rbcg strains , such as rbcg30 and rbcgurec::hly ,", "have been demonstrated to yield improved protection against m. tb infection in experimental animal model compared to the existing bcg vaccine and have entered into clinical trial .", "nevertheless , it is promising to keep on optimization of bcg protective immune if two points are being issued .", "one is the fact that the best immunodominant antigen for tb should be precisely defined .", "another is that the expression levels of such antigens should be optimal enough to elicit effective immune responses . here , we constructed two rbcg strains overexpressing immunodominant chimeric antigen ag856a2 at varying levels depending upon the strengths of the different fura promoters   and then compared the cellular immune response and protection in mice induced by these two rbcg strains . one way to improve bcg efficacy", "is to overexpress mycobacterial immunodominant antigens to induce optimal host immune responses in the life cycle of bcg within host [ 12 , 19 ] .", "this kind of strategy reflects that the doses of antigens are one of pivotal factors influencing the protective efficacies of vaccines .", "demonstrated that protective efficiency of tb subunit vaccines is highly dependent on the antigen dose .", "they vaccinated mice with different doses of fusion protein ag85b - tb10.4 which were emulsified in adjuvant ic31 , and the higher immune response and protective efficacy were only observed when the antigen was administered in proper doses , and decreasing or increasing of the antigen dose would dramatically dwarf the protection efficacies of the antigens . in our study ,", "the cognate antigen ag856a2 in rbcg186 and rbcg486 was expressed under the control of promoters pfura and pfurama ( figure s1 ) .", "these two promoters , by their nature , were verified to have varied promoter activities , with pfura the lower one and pfurama the higher one , and were consequently used to develop the rbcg strains overexpressing chimeric antigen ag856a2 at different levels , with lower expression in rbcg186 and higher expression in rbcg486 ( figure 1 ) . and different ag856a2 antigen loading in rbcgs resulted in differential host immune responses , with the higher antigen - specific effector immune response in the rbcg486-vaccinated mice as validated through in vitro ifn- elispot assay ( figure 2 ) .", "however , we did not observe the significant differences in the qualities of m. tb - specific cd4 t cells coexpressing ifn- , tnf- , and il-2 ( figure 3 ) , nor the protection efficacies and lung inflammations , between the two groups of rbcgs - vaccinated mice ( figures 4 and 5 ) .", "interestingly , subtly higher percent of polyfunctional cd4 t cells ( ifn-il-2tnf- ) was observed in bcg - vaccinated mice compared to other groups of mice ( figure 3(a ) ) ; however , the protective efficacy elicited by bcg vaccination is not that effective as rbcgs ( figure 4 ) . this contradictory result could be explained with the fact that the lower imfi values of ifn- , il-2 , and tnf- in bcg - vaccinated mice were observed ( see the case of il-2 in figure 3(c ) as representative ) .", "mfi provides one measure of the quality of the immune response since the cells that are more actively producing cytokine stain more brightly , thus the lower imfi values of cytokines reflected poor quality although mildly higher frequency of polyfunctional cd4 t cells was seen in bcg - vaccinated mice , and this further emphasizes that not only the magnitude but also the quality of vaccine - induced t cells responses are critical to guide development of effective immunization strategies . in addition , higher il-2 secretion , both in the levels of percentage and imfi , were seen in the rbcg486-vaccinated mice than that of bcg group ; this data support our recent findings that il-2 production in the spleens of vaccinated mice after vaccination can predict vaccine efficacy ( kang h , et al .", "immunology , 2014 ; in press ) . the same explanation might also be used to account for the fact that although the saline - treated mice showed high numbers of cells producing tnf- , the imfi is relative low ( data not shown ) .", "the quality of t cell response has significant effect on the establishment of protective memory . as with the phenotypic heterogeneous nature of t cells", "thus , in addition to monitoring exclusively the ifn- response after vaccination , researchers have been focusing on the coexpression of more cytokines at single cell level through flow cytometry technique [ 24 , 25 ] .", "the rbcg186 or rbcg486 , at least at the time we tested , induced much higher frequencies of il-2 cd4 t cells responding to ppd stimulation in splenocytes compared to the saline - treated or bcg - vaccinated mice after vaccination , which was further confirmed by higher il-2 production when cytokine concentration was measured as imfi value ( figure 3 ) .", "although il-2 has little direct effector function , it has the ability to expand effector functions of other t cells . in the linear model of differentiation for cd4 th1 cells , il-2 cd4 t cells belong to memory cells and have the potential to differentiate into ifn--producing cells after recalling by the relevant antigens .", "thus , rbcg186 and rbcg486 , because of the incorporation of chimeric antigen ag856a2 , enhance the memory capacity of host to m. tb pathogen .", "however , we did not detect any differences of cd4 t cells between rbcg186-vaccinated and rbcg486-vaccinated mice .", "this may attribute to the short vaccination time window we chose , or the real differences lies in other functions of t cells which is beyond the scope of the t cell functions currently tested and may need to be further exploited in the future .", "those relevant th1 cytokines ( e.g. , tnf- and ifn- ) , in a larger extent , function through activation of macrophages . tnf- and ifn- synergistically inhibit the growth of m. tb in macrophages through stimulating the production of reactive nitrogen intermediates ( rnis ) [ 27 , 28 ] . as for rnis , inos is the vital enzyme involved for the production of rnis [ 29 , 30 ] .", "tnf- , ifn- , and inos give proper containment of m. tb in the early stage . at later stage of infection when inhibition or killing of m. tb is well established ,", "their levels of expression will go down to a reasonable value ; otherwise immune - pathological response would happen .", "rbcgs , especially rbcg486 , induced enhanced protection against m. tb infection in this study ( figure 4 ) .", "consistent with the protective efficacy , the inflammation responses in the infected lungs alleviated greatly in rbcgs - vaccinated mice after infection ( figure 5 ) .", "when measuring the expression levels of inflammatory molecules , the rbcgs - vaccinated mice also displayed reduced levels of expression which were in accordance with the remissive granulomatous inflammation ( figures 5 and 6 ) ."], "abstract_text": ["<S> one approach for improving bcg efficacy is to utilize bcg as vehicle to develop recombinant bcg ( rbcg ) strains overexpressing mycobacterium tuberculosis ( m. tb ) antigens . </S>", "<S> also expression level of a candidate antigen should impact the final t cell responses conferred by rbcg . in this study , based on our previously constructed differential expression system , we developed two rbcg strains overexpressing m. tb chimeric antigen ag856a2 ( coding a recombinant ag85a with 2 copies of esat-6 inserted at acc i site of ag85a ) at differential levels under the control of the subtly modified fura promoters . </S>", "<S> these two rbcg strains were used to vaccinate c57bl/6 mice and exploit dose of incorporated antigen in rbcg to optimize immune response and protective efficiency against m. tb challenge in mouse model . </S>", "<S> the results showed that rbcg strains overexpressing ag856a2 at differential levels induced different antigen - specific ifn- production and comparable number of m. tb - specific cd4 t cells expressing il-2 . </S>", "<S> m. tb challenge experiment showed that rbcg strains afforded enhanced but comparable immune protection characterized by reduced bacillary load , lung pathology , and inflammation . </S>", "<S> these results suggested that the dose of antigens incorporated in rbcg can impact t cell immune responses but imposed no significantly differential protective efficacies . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["tuberculosis ( tb ) caused by mycobacterium tuberculosis ( m. tb ) continues to be a significant global health problem , affecting millions of people worldwide [ 1 , 2 ] .", "it is a prevalent infectious disease in china , with 250,000 deaths from tb annually and 6 million active tb patients at present .", "the global incidence of tb is raising due to coinfection with the human immunodeficiency virus ( hiv ) and the emergence of multidrug - resistant ( mdr ) m. tb strains [ 5 , 6 ] . according to the report of world health organization ( who )", ", m. tb will cause 1 billion new cases and about 35 million deaths worldwide by 2020 .", "therefore , effective treatment and control strategies are urgently needed to counteract the global threat of tb .", "the current vaccine against tb , m. bovis bacilli - calmette - gurin ( bcg ) , is a live attenuated vaccine which has been widely used throughout the world for many decades .", "bcg protects children efficiently against miliary and meningeal tb , but protective efficiency against pulmonary tb in adults has been found to vary highly from 0% to 80% .", "much effort has been devoted to improving bcg efficacy by genetic engineering technology because of its strong immunostimulatory properties and proven safety for human use [ 9 , 10 ] .", "recombinant bcg ( rbcg ) expressing different immunodominant antigens of m. tb , such as secreted antigens ( ag85b , ag85c , esat-6 , etc . ) or latency associated antigens ( -crystallin , rv2659c , rv3407 and rv1733c , etc . ) , have been tested as candidate vaccines against tb and are demonstrated to have an enhanced ability to induce th1 immune response and protection against m. tb challenge in animal models [ 11 , 12 ] .", "also , it is definitely no doubt that doses of antigens could subtly influence the magnitude of host immune response as well as protection efficacy , no matter antigen is administered in the form of rbcg , protein , dna , or rna .", "we have previously reported the construction of a m. tb fura gene operator / promoter ( pfura)-based differential expression system , from which it is feasible to express target antigens of interest in a modular fashion .", "m. tb chimeric antigen ag856a2 , which is coded by a recombinant ag85a gene with 2 copies of esat-6 gene inserted at the acc i site of ag85a ( see figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2014/196124 ) , shows improved immunogenicity in mice when it is inoculated intramuscularly as a dna vaccine .", "for the current study , we selected two rbcg strains overexpressing the same chimeric antigen ag856a2 at the maximum difference : rbcg186 and rbcg486 overexpressing the fusion protein under control of the wild - type or the optimized double - mutated fura promoters , respectively .", "we tested their efficacy as vaccines in c57bl/6 mice , comparing immune response and protection against m. tb challenge .", "the results showed that mice vaccinated with rbcg186 or rbcg486 generally induced higher antigen - specific effector and memory immune responses , as well as protective efficacies compared to mice vaccinated with the parent bcg strain .", "however , the two rbcg strains between themselves , which expressed the chimeric antigen ag856a2 at different levels , induced different antigen - specific ifn- production and comparable number of m. tb - specific cd4 t cells expressing il-2 . and the protective efficacies imposed by the two rbcg strains displayed no significant differences although higher protection was observed in rbcg486 vaccinated mice than that in rbcg186 vaccinated mice ."], ["female specific pathogen - free ( spf ) c57bl/6 mice aged 68  wks were purchased from shanghai slac laboratory animal co. , ltd .", "( shanghai , china ) and kept under spf conditions with food and water ad libitum until challenge .", "infected mice were maintained in a biosafety level 3 ( bsl-3 ) biocontainment animal facility .", "all animal experiment protocols were approved by chinese science academy committee on care and use of laboratory animals and were performed according to the guidelines of the laboratory animal ethical board of shanghai public health clinical center . \n", "bcg and its derivative recombinant strains were grown in liquid middlebrook 7h9 broth ( bd difco , usa ) supplemented with 10% oleic acid - albumin - dextrose - catalase enrichment ( oadc , bd difco , usa ) , 0.2% glycerol , and 0.05% tween 80 .", "cultures in the exponential phase were frozen and stored at 80c . when required , kanamycin was added at a final concentration of 50 or 20  g / ml for e. coli or mycobacteria , respectively .", "two rbcg strains overexpressing m. tb chimeric immunodominant antigen ag856a2 at different levels were constructed as previously described .", "briefly , the ag856a2 coding gene , which is a recombinant ag85a gene with 2 copies of esat-6 gene inserted in its acc i site , was amplified from the plasmid template of dna vaccine hg856a and then cloned into mycobacterial differential expression vectors pmfa11 and pmfa41 under control of the prototypical and double - mutated ( mutations : initial codon change from gtg to aug and 6  bp substitution at upstream at - rich region ) fura promoter , respectively .", "the resulting constructs were electroporated into bcg - danish competent cells and selected on middlebrook 7h11 agar with kanamycin .", "the rbcg transformants were grown to midexponential phase in complete middlebrook 7h9 broth and then verified the recombinant protein expression by routine western - blotting assay .", "mice were vaccinated subcutaneously ( s.c . ) with 2  10 colony - forming units ( cfu ) of bcg or rbcgs in 100  l saline .", "eight weeks after vaccination , groups of 6 mice were either sacrificed for assessment of antigen - specific t cell responses in splenocytes or exposed to an aerosol of virulent m. tb h37rv strain to deposit an inhaled dose of 100200 cfu per lung by an inhalation exposure system ( glas - col , usa ) .", "ifn- elispot assay kit ( bd biosciences , usa ) was used as described by the manufacturer .", "plates were coated with anti - ifn- mab overnight at 4c and then blocked with rpmi 1640 medium containing 10% fetal bovine serum ( fbs ) for 1  h at room temperature .", "splenocytes ( 2.5  10 cells / well ) from immunized mice were isolated , plated , and cultured with 10  g / ml ppd ( statens serum institute , denmark ) or 2  g / ml recombinant ag85a , 6  g / ml recombinant esat-6 to provide stimulation at 37c , 5% co2 for 20  h. after washing the plates with pbs - t20 ( 1  pbs , ph 7.4 , 0.05% tween 20 ) , biotinylated anti - ifn- was added for 2  h at room temperature .", "streptavidin - hrp was added for 45  min , and the color was developed with 3-amino-9-ethylcarbazole ( aec ) substrate ( bd biosciences ) .", "an immunospot analyzer ( cellular technology , usa ) was used to count the spots .", "splenocytes ( 2  10 cells / well ) isolated at 8  weeks after immunization were plated in 96-well plates and stimulated with 10  g / ml ppd for 14  h in the presence of 1  g / ml anti - cd28 ( bd biosciences ) and subsequently incubated for an additional 5  h at 37c following the addition of 0.5  l / ml monensin / golgistop ( bd biosciences ) .", "following overnight incubation at 4c , the cells were washed in facs buffer ( pbs containing 0.1% sodium azide and 1% fbs ) and subsequently stained for 30  min at 4c for surface markers with mabs as indicated using anti - cd3-pacific blue , anti - cd8-fitc , and anti - cd44-v500 ( all from bd biosciences ) .", "cells were then washed in facs buffer , fixed , permeabilized using the cytofix / cytoperm kit ( bd biosciences ) according to the manufacturer 's instructions and stained intracellularly for 30  min at 4c using anti - ifn--apc - cy7 , anti - tnf--percp - cy5.5 , and anti - il-2-apc mabs ( all from bd biosciences ) .", "cells were subsequently washed , resuspended in facs buffer , and then analyzed by multiparameter flow cytometry using a bd facsaria flow cytometer ( bd biosciences ) . for each sample ,", "at least 300,000 events were collected and responses were analyzed using flowjo software ( tree star , usa ) .", "five weeks after infection , mice were sacrificed and the mycobacterial burden was determined by plating homogenates of lung , excluding right postcaval lobe , and entire spleen onto middlebrook 7h11 agar plates supplemented with 10% oadc enrichment and a 4-antibiotic mixture ( 40  u / ml polymycin b , 4  g / ml amphotericin , 50  g / ml carbenicillin , 2  g / ml trimethoprim ) that prevents growth of contaminating microorganisms .", "plates were incubated at 37c for 3  weeks in semisealed plastic bags and then cfu were counted and expressed as log10 cfu per organ .", "then , the embedded lung lobes were sectioned in thickness of 5  m , stained with haematoxylin and eosin ( h & e ) and photographed using a olympus ckx41 microscope ( olympus , japan ) fitted with an olympus dp20 camera connected to a computer .", "the image pro plus program ( media cybernetics , usa ) was utilized to objectively assess the level of inflammation present in each image .", "the mean percent of area inflamed was quantified averaging from three to five lung sections of each of the different groups of mice .", "the antibodies were rabbit polyclonal anti - mouse tnf- ( abcam , uk ) , rabbit polyclonal ifn- antibodies ( invitrogen , usa ) , and rabbit polyclonal anti - mouse inos antibody ( cayman chemical , usa ) .", "all sections were examined by light microscopy , and the expression of tnf- , ifn- , or inos was semiquantified by intensity of positive signal using image pro plus software .", "immune responses , protective efficacies , and histopathological staining were tested by one - way anova followed by tukey 's multiple comparison tests of the means ."], ["we have previously developed a novel mycobacterial differential expression system ( pmfa series ) based on the m. tb fura gene operator / promoter ( pfura ) or its derivatives .", "ag856a2 was cloned into two of these plasmids , pmfa11 and pmfa41 , which drives low and high gene expression under the control of the wild - type and modified fura promoters , respectively . by transformation of bcg", ", we obtained two strains , rbcg186 and rbcg486 , which drove correspondingly low and high expression of chimeric immunodominant antigen ag856a2 ( figure 1 , upper panel ) .", "quantification of the band intensities of western - blot indicated that rbcg486 roughly expressed > 3-fold of ag856a2 than rbcg186 did ( figure 1 , lower panel ) .", "eight weeks after vaccination , elispot assay of splenocytes showed that more cells in the rbcg486-vaccinated mice expressed ag85a - specific ifn- compared to those of rbcg186- and bcg - vaccinated mice ( figure 2 , left panel ) .", "also , significantly elevated numbers of splenocytes expressed esat-6-specific ifn- in both rbcg186- and rbcg486-vaccinated mice compared to that of bcg - vaccinated mice ( figure 2 , middle panel ) .", "additionally , esat-6-specific ifn- was induced at much higher level in rbcg486-vaccinated mice compared to rbcg186 group ( figure 2 , middle panel ) .", "a similar pattern of ppd - specific ifn- responses as ag85a - specific response was also observed but the difference was not statistically significant , regarding to the comparisons of rbcg486-vaccinated mice and other immunized groups ( figure 2 , right panel ) .", "we used flow cytometry to measure the capacity of m. tb - specific cd4 t cells from spleens of vaccinated mice producing cytokines ifn- , tnf- , and il-2 at single cell level after stimulation in vitro with ppd .", "the cytokine - producing cd3cd4 cells were classified into seven subpopulations based on their production of ifn- , tnf- , and il-2 in any combination ( figure 3(a ) ) .", "significantly increased frequencies of ppd - specific il-2 cd4 t cells were identified in rbcg - vaccinated mice , whereas increased frequencies of ifn- cells were identified in bcg - vaccinated mice even though statistically insignificant ( figure 3(a ) ) .", "the pie chart of this data clarified the dominance of il-2 cd4 t cells in rbcg - vaccinated mice , while ifn- cd4 t cells dominated the responses of bcg - vaccinated mice ( figure 3(b ) ) .", "rbcg and bcg - vaccination did not differ in their ability to induce m. tb - specific cd4 t cells producing other combinations of cytokines ( p > 0.05 ) . in accordance , we also observed higher integrated mean fluorescence intensities ( imfi = % frequency  mfi ) of il-2 in il-2-producing cd4 t cells , even though it is statistically insignificant ( figure 3(c ) ) . in general , rbcg induced higher antigen - specific cytokine responses as compared to bcg ( figures 2 and 3 ) , and rbcg486 induced higher antigen - specific ifn- response ( figure 2 ) and comparable frequency of m. tb - specific cd4 t cells expressing il-2 ( figure 3 ) .", "then , we further compared the protective efficacies of rbcg486 , rbcg186 , and bcg against m. tb - challenge .", "as shown in figure 4(a ) , 5  weeks after challenge all vaccinated mice had a significantly reduced bacillary load in lungs , when compared to the saline - treated mice .", "vaccination with bcg and rbcg186 resulted in a comparable reduction in bacillary load ( figure 4 ) . however , even though rbcg486 vaccination induced a significantly greater protection when compared to the bcg - vaccinated mice , it showed no difference of protection when compared to the rbcg186-vaccinated mice ( figure 4(a ) ) .", "the bacillary loads in spleens shared the similar pattern as those in lungs , with rbcg486-vaccinated mice having far fewer bacilli when compared to the saline - treated or bcg - vaccinated mice and having comparable bacilli compared to the rbcg186-vaccinated mice ( figure 4(b ) ) .", "five weeks after challenge , m. tb infection caused severe pathology changes in saline - treated mice , with about 24.3% of the tissue showing extensive multifocal granulomatous infiltration , characterized by numerous foamy macrophages surrounded by inflammatory cells ( figure 5 ) .", "however , all the vaccinated groups of mice had significantly reduced pulmonary granulomatous consolidation compared to the unvaccinated mice ( i.e. , 13.42% consolidation in bcg - vaccinated group , 7.24% in rbcg186-vaccinated group , and 4.87% in rbcg486-vaccinated group ) .", "the rbcg - vaccinated mice showed the mildest pathology , and all of the mice in these two groups had mainly well - preserved alveolar spaces with only a few scattered areas of diffused infiltration ( figure 5 ) .", "immunohistochemical staining of the lung tissues showed the presence of tnf- , ifn- , and inos in all groups of infected mice and staining was strongest in the granulomatous lesions compared to that in the nongranulomatous areas .", "five weeks after infection , a very high level of tnf- was observed in the lungs of saline - treated mice ( figure 6(a ) ) ; tnf- staining was extensive in necrotic areas within the advanced coalescent granulomas .", "vaccination with bcg resulted in the reduced amounts of tnf- expression , even though statistically insignificant .", "in contrast , rbcg - vaccinated mice , especially rbcg486-vaccinated mice , showed only a little weak staining for tnf- and this was restricted primarily to the granuloma core ( figure 6(a ) ) .", "similar patterns of ifn- and inos staining were also observed except that there was relatively much weaker staining in the lungs of ( r)bcg - vaccinated mice compared to the saline - treated mice ( figures 6(b ) and 6(c ) ) .", "similar pattern of tnf- , ifn- , and inos staining was also observed in the infected spleens of vaccinated mice , with the highest staining in saline - treated mice , moderate staining in bcg - vaccinated mice , and the lowest staining in rbcg - vaccinated mice ( figure  s2 ) ."], ["during the past decades , great efforts have been focused on modifications of the current bcg vaccine to develop new anti - tb vaccine candidates . some modified rbcg strains , such as rbcg30 and rbcgurec::hly ,", "have been demonstrated to yield improved protection against m. tb infection in experimental animal model compared to the existing bcg vaccine and have entered into clinical trial .", "nevertheless , it is promising to keep on optimization of bcg protective immune if two points are being issued .", "one is the fact that the best immunodominant antigen for tb should be precisely defined .", "another is that the expression levels of such antigens should be optimal enough to elicit effective immune responses . here , we constructed two rbcg strains overexpressing immunodominant chimeric antigen ag856a2 at varying levels depending upon the strengths of the different fura promoters   and then compared the cellular immune response and protection in mice induced by these two rbcg strains . one way to improve bcg efficacy", "is to overexpress mycobacterial immunodominant antigens to induce optimal host immune responses in the life cycle of bcg within host [ 12 , 19 ] .", "this kind of strategy reflects that the doses of antigens are one of pivotal factors influencing the protective efficacies of vaccines .", "demonstrated that protective efficiency of tb subunit vaccines is highly dependent on the antigen dose .", "they vaccinated mice with different doses of fusion protein ag85b - tb10.4 which were emulsified in adjuvant ic31 , and the higher immune response and protective efficacy were only observed when the antigen was administered in proper doses , and decreasing or increasing of the antigen dose would dramatically dwarf the protection efficacies of the antigens . in our study ,", "the cognate antigen ag856a2 in rbcg186 and rbcg486 was expressed under the control of promoters pfura and pfurama ( figure s1 ) .", "these two promoters , by their nature , were verified to have varied promoter activities , with pfura the lower one and pfurama the higher one , and were consequently used to develop the rbcg strains overexpressing chimeric antigen ag856a2 at different levels , with lower expression in rbcg186 and higher expression in rbcg486 ( figure 1 ) . and different ag856a2 antigen loading in rbcgs resulted in differential host immune responses , with the higher antigen - specific effector immune response in the rbcg486-vaccinated mice as validated through in vitro ifn- elispot assay ( figure 2 ) .", "however , we did not observe the significant differences in the qualities of m. tb - specific cd4 t cells coexpressing ifn- , tnf- , and il-2 ( figure 3 ) , nor the protection efficacies and lung inflammations , between the two groups of rbcgs - vaccinated mice ( figures 4 and 5 ) .", "interestingly , subtly higher percent of polyfunctional cd4 t cells ( ifn-il-2tnf- ) was observed in bcg - vaccinated mice compared to other groups of mice ( figure 3(a ) ) ; however , the protective efficacy elicited by bcg vaccination is not that effective as rbcgs ( figure 4 ) . this contradictory result could be explained with the fact that the lower imfi values of ifn- , il-2 , and tnf- in bcg - vaccinated mice were observed ( see the case of il-2 in figure 3(c ) as representative ) .", "mfi provides one measure of the quality of the immune response since the cells that are more actively producing cytokine stain more brightly , thus the lower imfi values of cytokines reflected poor quality although mildly higher frequency of polyfunctional cd4 t cells was seen in bcg - vaccinated mice , and this further emphasizes that not only the magnitude but also the quality of vaccine - induced t cells responses are critical to guide development of effective immunization strategies . in addition , higher il-2 secretion , both in the levels of percentage and imfi , were seen in the rbcg486-vaccinated mice than that of bcg group ; this data support our recent findings that il-2 production in the spleens of vaccinated mice after vaccination can predict vaccine efficacy ( kang h , et al .", "immunology , 2014 ; in press ) . the same explanation might also be used to account for the fact that although the saline - treated mice showed high numbers of cells producing tnf- , the imfi is relative low ( data not shown ) .", "the quality of t cell response has significant effect on the establishment of protective memory . as with the phenotypic heterogeneous nature of t cells", "thus , in addition to monitoring exclusively the ifn- response after vaccination , researchers have been focusing on the coexpression of more cytokines at single cell level through flow cytometry technique [ 24 , 25 ] .", "the rbcg186 or rbcg486 , at least at the time we tested , induced much higher frequencies of il-2 cd4 t cells responding to ppd stimulation in splenocytes compared to the saline - treated or bcg - vaccinated mice after vaccination , which was further confirmed by higher il-2 production when cytokine concentration was measured as imfi value ( figure 3 ) .", "although il-2 has little direct effector function , it has the ability to expand effector functions of other t cells . in the linear model of differentiation for cd4 th1 cells , il-2 cd4 t cells belong to memory cells and have the potential to differentiate into ifn--producing cells after recalling by the relevant antigens .", "thus , rbcg186 and rbcg486 , because of the incorporation of chimeric antigen ag856a2 , enhance the memory capacity of host to m. tb pathogen .", "however , we did not detect any differences of cd4 t cells between rbcg186-vaccinated and rbcg486-vaccinated mice .", "this may attribute to the short vaccination time window we chose , or the real differences lies in other functions of t cells which is beyond the scope of the t cell functions currently tested and may need to be further exploited in the future .", "those relevant th1 cytokines ( e.g. , tnf- and ifn- ) , in a larger extent , function through activation of macrophages . tnf- and ifn- synergistically inhibit the growth of m. tb in macrophages through stimulating the production of reactive nitrogen intermediates ( rnis ) [ 27 , 28 ] . as for rnis , inos is the vital enzyme involved for the production of rnis [ 29 , 30 ] .", "tnf- , ifn- , and inos give proper containment of m. tb in the early stage . at later stage of infection when inhibition or killing of m. tb is well established ,", "their levels of expression will go down to a reasonable value ; otherwise immune - pathological response would happen .", "rbcgs , especially rbcg486 , induced enhanced protection against m. tb infection in this study ( figure 4 ) .", "consistent with the protective efficacy , the inflammation responses in the infected lungs alleviated greatly in rbcgs - vaccinated mice after infection ( figure 5 ) .", "when measuring the expression levels of inflammatory molecules , the rbcgs - vaccinated mice also displayed reduced levels of expression which were in accordance with the remissive granulomatous inflammation ( figures 5 and 6 ) ."]]}
{"article_id": "PMC3638331", "article_text": ["a 6-year - old boy presented with a history of penetrating injury to the upper left eyelid sustained while diving head - first into a pond . on presentation , he could only perceive hand movements very close to his face . since a computed tomography ( ct )", "scan done at this time did not reveal the presence of any foreign body , the patient was treated for orbital cellulitis .", "one month later , he returned reporting discharge from the eyelid wound , which had formed a fistula .", "a repeat ct scan showed an abscess with an empty sinus tract but no distinguishable foreign body [ fig . 1 ] .", "the patient subsequently developed septic arthritis of the left hip joint , which was treated separately .", "subsequent re - evaluation of the patient 's left eye revealed a queried perception of light , relative afferent pupillary defect , optic disc pallor , and inferotemporal displacement of the globe .", "a superonasal quadrant granulation mass was observed from the non - healing wound , but elevation and depression of the globe in adduction were minimally restricted .", "re - evaluation of the ct scan showed a single hypodense superomedial lesion corresponding to the previously noted sinus tract .", "a superomedial orbitotomy resulted in the removal of 4 wooden foreign bodies , each approximately 1.5 inches in length . by post - operative day 5", ", the patient 's edema and proptosis had resolved . at 1-month follow - up ,", "the eyelid wound was completely healed , and left eye visual acuity had improved to 20/120 .", "axial ( a ) and coronal ( b ) ct of a 6-year - old boy with a history of penetrating injury shows an empty abscess tract without obvious foreign body .", "subsequent orbitotomy recovers four wooden fragments ( d ) a 12-year - old boy presented to an outside hospital with fever , vomiting , and a prominent right eye with a painful , swollen lower lid .", "he had a history of a penetrating injury sustained while diving head - first into a river 12 days previously .", "after 1 week of treatment with systemic antibiotics , the patient presented to our institution with continued swelling at the injury site .", "he had an indurated swelling of the lower lid with a discharging sinus , mechanical ptosis , diplopia , eccentric upward displacement of the globe , and 3-mm of proptosis .", "repeat ct scan showed a hypodense body surrounded by secondary granulomatous inflammation [ fig . 2 ] .", "post - operatively , the patient showed symptomatic improvement with a reduction in proptosis and a visual acuity of 20/30 in the affected eye [ fig . 3 ] .", "coronal ( a ) and axial ( b ) ct scans taken one week after initial ct showed no evidence of retained foreign body .", "ct now shows a hypodense body surrounded by secondary granulomatous inflammation pre - operative ( a ) and post - operative ( b ) photographs of 12-year - old boy with a history of penetrating injury by a 2 inch wooden foreign body ( c ) sustained while pond - diving", "a 6-year - old boy presented with a history of penetrating injury to the upper left eyelid sustained while diving head - first into a pond . on presentation , he could only perceive hand movements very close to his face . since a computed tomography ( ct )", "scan done at this time did not reveal the presence of any foreign body , the patient was treated for orbital cellulitis .", "one month later , he returned reporting discharge from the eyelid wound , which had formed a fistula .", "a repeat ct scan showed an abscess with an empty sinus tract but no distinguishable foreign body [ fig . 1 ] .", "the patient subsequently developed septic arthritis of the left hip joint , which was treated separately .", "subsequent re - evaluation of the patient 's left eye revealed a queried perception of light , relative afferent pupillary defect , optic disc pallor , and inferotemporal displacement of the globe .", "a superonasal quadrant granulation mass was observed from the non - healing wound , but elevation and depression of the globe in adduction were minimally restricted .", "re - evaluation of the ct scan showed a single hypodense superomedial lesion corresponding to the previously noted sinus tract .", "a superomedial orbitotomy resulted in the removal of 4 wooden foreign bodies , each approximately 1.5 inches in length . by post - operative day 5", ", the patient 's edema and proptosis had resolved . at 1-month follow - up ,", "the eyelid wound was completely healed , and left eye visual acuity had improved to 20/120 .", "axial ( a ) and coronal ( b ) ct of a 6-year - old boy with a history of penetrating injury shows an empty abscess tract without obvious foreign body .", "a 12-year - old boy presented to an outside hospital with fever , vomiting , and a prominent right eye with a painful , swollen lower lid .", "he had a history of a penetrating injury sustained while diving head - first into a river 12 days previously .", "after 1 week of treatment with systemic antibiotics , the patient presented to our institution with continued swelling at the injury site .", "he had an indurated swelling of the lower lid with a discharging sinus , mechanical ptosis , diplopia , eccentric upward displacement of the globe , and 3-mm of proptosis .", "repeat ct scan showed a hypodense body surrounded by secondary granulomatous inflammation [ fig . 2 ] .", "post - operatively , the patient showed symptomatic improvement with a reduction in proptosis and a visual acuity of 20/30 in the affected eye [ fig . 3 ] .", "coronal ( a ) and axial ( b ) ct scans taken one week after initial ct showed no evidence of retained foreign body .", "ct now shows a hypodense body surrounded by secondary granulomatous inflammation pre - operative ( a ) and post - operative ( b ) photographs of 12-year - old boy with a history of penetrating injury by a 2 inch wooden foreign body ( c ) sustained while pond - diving", "the injuries sustained in the cases we present are troubling since swimming and diving in rivers , ponds , and other natural bodies of water is a common pastime of children in rural areas worldwide .", "penetration injuries by wooden foreign bodies embedded in shallow riverbeds have not been previously recognized as a risk of outdoor swimming and diving . however , in our experience these types of injuries represent a true threat to vision .", "this danger is compounded by the fact that retained organic foreign bodies are not often immediately apparent on imaging studies , making prompt removal difficult .", "previous studies report that wood , depending on its level of hydration , can be isodense to either air or orbital fat on ct scan . in our two cases ,", "ct scans taken within a few days of injury did not show a distinct foreign body , but only edema and signs of inflammation .", "only after the foreign body became infected did a discharging sinus cavity form and become evident on ct scans . while nasr et al .", "found magnetic resonance imaging ( mri ) to be slightly better at identifying organic foreign bodies , other studies recommend using ct scans for the initial identification of wooden foreign bodies , so the best imaging method remains controversial . as in our case , serial imaging in patients with a high - risk mechanism of injury may be a useful non - invasive method of identifying a retained wooden orbital foreign body .", "unfortunately , orbital exploration does not always identify the presence of a wooden orbital foreign body . as in our two cases ,", "these fragments are often not lodged in a tissue , but embedded within orbital fat .", "they are thus mobile within the orbital cavity until becoming enclosed within a discharging sinus .", "thus , even after a negative orbital exploration , it is important to keep a high index of suspicion for a retained wooden orbital foreign body . in summary", ", ophthalmologists attending to cases of penetrating orbital injuries sustained during river and pond diving should maintain a high index of suspicion for a retained wooden orbital foreign body and realize that neither orbital exploration nor imaging may be sufficient for diagnosis .", "consideration should be given to serial imaging in the event of a negative initial scan .", "a thorough history as to the mechanism of injury , a meticulous exam , and frequent follow - up visits are also of utmost importance ."], "abstract_text": ["<S> diving into lakes and ponds is a common activity of rural children . </S>", "<S> we present two cases of penetrating orbital injuries from plant matter sustained in this manner . </S>", "<S> such injuries pose a particular challenge because wooden foreign bodies are often missed during orbital exploration , and current imaging modalities can not reliably identify retained organic material . </S>", "<S> when a patient presents with orbital penetration after a high - risk mechanism of injury , such as freshwater diving , the clinician must maintain a very high index of suspicion for retained wooden foreign body . </S>"], "labels": null, "section_names": ["Case Reports", "Case 1", "Case 2", "Discussion"], "sections": [["a 6-year - old boy presented with a history of penetrating injury to the upper left eyelid sustained while diving head - first into a pond . on presentation , he could only perceive hand movements very close to his face . since a computed tomography ( ct )", "scan done at this time did not reveal the presence of any foreign body , the patient was treated for orbital cellulitis .", "one month later , he returned reporting discharge from the eyelid wound , which had formed a fistula .", "a repeat ct scan showed an abscess with an empty sinus tract but no distinguishable foreign body [ fig . 1 ] .", "the patient subsequently developed septic arthritis of the left hip joint , which was treated separately .", "subsequent re - evaluation of the patient 's left eye revealed a queried perception of light , relative afferent pupillary defect , optic disc pallor , and inferotemporal displacement of the globe .", "a superonasal quadrant granulation mass was observed from the non - healing wound , but elevation and depression of the globe in adduction were minimally restricted .", "re - evaluation of the ct scan showed a single hypodense superomedial lesion corresponding to the previously noted sinus tract .", "a superomedial orbitotomy resulted in the removal of 4 wooden foreign bodies , each approximately 1.5 inches in length . by post - operative day 5", ", the patient 's edema and proptosis had resolved . at 1-month follow - up ,", "the eyelid wound was completely healed , and left eye visual acuity had improved to 20/120 .", "axial ( a ) and coronal ( b ) ct of a 6-year - old boy with a history of penetrating injury shows an empty abscess tract without obvious foreign body .", "subsequent orbitotomy recovers four wooden fragments ( d ) a 12-year - old boy presented to an outside hospital with fever , vomiting , and a prominent right eye with a painful , swollen lower lid .", "he had a history of a penetrating injury sustained while diving head - first into a river 12 days previously .", "after 1 week of treatment with systemic antibiotics , the patient presented to our institution with continued swelling at the injury site .", "he had an indurated swelling of the lower lid with a discharging sinus , mechanical ptosis , diplopia , eccentric upward displacement of the globe , and 3-mm of proptosis .", "repeat ct scan showed a hypodense body surrounded by secondary granulomatous inflammation [ fig . 2 ] .", "post - operatively , the patient showed symptomatic improvement with a reduction in proptosis and a visual acuity of 20/30 in the affected eye [ fig . 3 ] .", "coronal ( a ) and axial ( b ) ct scans taken one week after initial ct showed no evidence of retained foreign body .", "ct now shows a hypodense body surrounded by secondary granulomatous inflammation pre - operative ( a ) and post - operative ( b ) photographs of 12-year - old boy with a history of penetrating injury by a 2 inch wooden foreign body ( c ) sustained while pond - diving"], ["a 6-year - old boy presented with a history of penetrating injury to the upper left eyelid sustained while diving head - first into a pond . on presentation , he could only perceive hand movements very close to his face . since a computed tomography ( ct )", "scan done at this time did not reveal the presence of any foreign body , the patient was treated for orbital cellulitis .", "one month later , he returned reporting discharge from the eyelid wound , which had formed a fistula .", "a repeat ct scan showed an abscess with an empty sinus tract but no distinguishable foreign body [ fig . 1 ] .", "the patient subsequently developed septic arthritis of the left hip joint , which was treated separately .", "subsequent re - evaluation of the patient 's left eye revealed a queried perception of light , relative afferent pupillary defect , optic disc pallor , and inferotemporal displacement of the globe .", "a superonasal quadrant granulation mass was observed from the non - healing wound , but elevation and depression of the globe in adduction were minimally restricted .", "re - evaluation of the ct scan showed a single hypodense superomedial lesion corresponding to the previously noted sinus tract .", "a superomedial orbitotomy resulted in the removal of 4 wooden foreign bodies , each approximately 1.5 inches in length . by post - operative day 5", ", the patient 's edema and proptosis had resolved . at 1-month follow - up ,", "the eyelid wound was completely healed , and left eye visual acuity had improved to 20/120 .", "axial ( a ) and coronal ( b ) ct of a 6-year - old boy with a history of penetrating injury shows an empty abscess tract without obvious foreign body ."], ["a 12-year - old boy presented to an outside hospital with fever , vomiting , and a prominent right eye with a painful , swollen lower lid .", "he had a history of a penetrating injury sustained while diving head - first into a river 12 days previously .", "after 1 week of treatment with systemic antibiotics , the patient presented to our institution with continued swelling at the injury site .", "he had an indurated swelling of the lower lid with a discharging sinus , mechanical ptosis , diplopia , eccentric upward displacement of the globe , and 3-mm of proptosis .", "repeat ct scan showed a hypodense body surrounded by secondary granulomatous inflammation [ fig . 2 ] .", "post - operatively , the patient showed symptomatic improvement with a reduction in proptosis and a visual acuity of 20/30 in the affected eye [ fig . 3 ] .", "coronal ( a ) and axial ( b ) ct scans taken one week after initial ct showed no evidence of retained foreign body .", "ct now shows a hypodense body surrounded by secondary granulomatous inflammation pre - operative ( a ) and post - operative ( b ) photographs of 12-year - old boy with a history of penetrating injury by a 2 inch wooden foreign body ( c ) sustained while pond - diving"], ["the injuries sustained in the cases we present are troubling since swimming and diving in rivers , ponds , and other natural bodies of water is a common pastime of children in rural areas worldwide .", "penetration injuries by wooden foreign bodies embedded in shallow riverbeds have not been previously recognized as a risk of outdoor swimming and diving . however , in our experience these types of injuries represent a true threat to vision .", "this danger is compounded by the fact that retained organic foreign bodies are not often immediately apparent on imaging studies , making prompt removal difficult .", "previous studies report that wood , depending on its level of hydration , can be isodense to either air or orbital fat on ct scan . in our two cases ,", "ct scans taken within a few days of injury did not show a distinct foreign body , but only edema and signs of inflammation .", "only after the foreign body became infected did a discharging sinus cavity form and become evident on ct scans . while nasr et al .", "found magnetic resonance imaging ( mri ) to be slightly better at identifying organic foreign bodies , other studies recommend using ct scans for the initial identification of wooden foreign bodies , so the best imaging method remains controversial . as in our case , serial imaging in patients with a high - risk mechanism of injury may be a useful non - invasive method of identifying a retained wooden orbital foreign body .", "unfortunately , orbital exploration does not always identify the presence of a wooden orbital foreign body . as in our two cases ,", "these fragments are often not lodged in a tissue , but embedded within orbital fat .", "they are thus mobile within the orbital cavity until becoming enclosed within a discharging sinus .", "thus , even after a negative orbital exploration , it is important to keep a high index of suspicion for a retained wooden orbital foreign body . in summary", ", ophthalmologists attending to cases of penetrating orbital injuries sustained during river and pond diving should maintain a high index of suspicion for a retained wooden orbital foreign body and realize that neither orbital exploration nor imaging may be sufficient for diagnosis .", "consideration should be given to serial imaging in the event of a negative initial scan .", "a thorough history as to the mechanism of injury , a meticulous exam , and frequent follow - up visits are also of utmost importance ."]]}
{"article_id": "PMC5433125", "article_text": ["postcataract surgery endophthalmitis is a relatively uncommon but devastating complication of modern cataract surgery , and there is not complete consensus regarding the ideal practice patterns for prophylaxis .", "the european society of cataract and refractive surgeons ( escrs ) prospective multi - center interventional trial ( and many other case  control , cross - sectional , and longitudinal studies ) supported the hypothesis that the use of icc at the conclusion of cataract surgery reduces the incidence of postoperative endophthalmitis .", "it is estimated that two to four cases of endophthalmitis per 1000 surgeries can be avoided if surgeons adopt the use of icc , and the absence of an icc prophylactic regimen at a dose of 1 mg/0.1 ml was associated with an almost 5-fold increase in the risk of postoperative endophthalmitis . the injection is expedient , painless , and achieves high antibiotic concentrations in the immediate postoperative period . in addition , intracameral injection may be particularly effective in patients who sustain posterior capsule rupture and would otherwise incur an increased the risk of endophthalmitis . when considering the implementation of any intervention , cost - effectiveness is another important variable .", "estimated the cost - effectiveness ratio for icc to be $ 1403 per case of postoperative endophthalmitis prevented .", "many commonly used topical antibiotics are not cost - effective compared with icc , even under optimistic assumptions about their efficacy .", "another economic analysis comparing different prophylaxis regimens concluded that icc provided the best cost - effectiveness ratio .", "therefore , the decision to adopt this therapy is supported by studies demonstrating efficacy and cost - effectiveness . the final variable that must be included when", "one weakness inherent in any meticulously performed clinical trial is that the results may not be generalizable to a more diverse patient population and varying scenarios of care .", "most clinical trials make great effort to reduce the risk of enrollment in the study , and therefore the safety profile reported may be a  best - case scenario . ", "the purpose of this review is to provide an evidence - based review and synthesis of the literature regarding the risks of icc when administered for the prevention of postcataract surgery endophthalmitis , thereby informing decisions regarding best practices for cataract surgery .", "the categories of risk to be reviewed are : \n risk of anaphylaxis , especially in penicillin or cephalosporin allergic patientsrisk of toxicity at routine clinical dosesrisk of toxicity at increased doses due to compounding errorsidiosyncratic reactions , including toxic anterior segment syndrome ( tass ) . \n ", "risk of anaphylaxis , especially in penicillin or cephalosporin allergic patients risk of toxicity at routine clinical doses risk of toxicity at increased doses due to compounding errors idiosyncratic reactions , including toxic anterior segment syndrome ( tass ) .", "we performed a systematic review of the existing scientific literature using pubmed and google scholar .", "the key words used were  cefuroxime ,   endophthalmitis  and  cataract surgeries .", "all articles were read in full by both authors with the exception of two that were available only as abstracts .", "the highest level of evidence for each aspect of the intervention was assigned utilizing the oxford center for evidence - based medicine guidelines .", "cross - reactivity between penicillins and most second- and all third- and fourth - generation cephalosporins is negligible .", "it is generally considered safe to administer a cephalosporin with a side chain that is structurally dissimilar to that of penicillin . in patients with a documented ige - mediated reaction to penicillin , use of cephalosporins with a similar side chain", "however , cephalosporins with different side chains ( such as cefuroxime ) may be given .", "it should be noted that the escrs study excluded patients with penicillin or cephalosporin allergy , as did several other large studies .", "icc injection during cataract surgery was well tolerated in a prospective study of forty penicillin - allergic patients with a negative preoperative cefuroxime skin test .", "the use of icc in patients with penicillin allergy was explored between 2004 and 2012 in a case  control registry study ; the control group was the cohort of patients undergoing cataract surgery under a hospital policy of excluding patients with self - reported penicillin allergy .", "after a critical review of the literature and pilot study in 817 patients with reported penicillin allergy , this policy was altered , and all patients without a specific history of cephalosporin anaphylactic reaction were administered icc . out of 13,592 subsequent cataract surgeries , there were no reported cases of anaphylaxis or allergic reaction .", "this study is limited by the registry design , which may underreport or miscategorize adverse events .", "a similar longitudinal observational study by shorstein et al . reported a decreasing endophthalmitis rate after instituting a standard icc protocol ; there were no reports of anaphylaxis or allergic reaction among 12,609 surgeries , but some patients received other intracameral antibiotics , and it is unclear if penicillin - allergic patients routinely received cefuroxime or other antibiotics such as moxifloxacin or vancomycin .", "control study comparing the endophthalmitis rates before and after instituting a standard icc regimen ; patients with cefuroxime allergy were excluded , but penicillin - allergic patients received icc .", "there were no cases of anaphylaxis among 2289 patients receiving icc , but the prevalence of penicillin allergy was not reported .", "there are two case reports of an anaphylactic reaction in penicillin or cephalosporin allergic patients who received icc .", "the number of patients from these centers that had received icc were not reported , preventing an estimation of the incidence of this complication .", "adverse effects that have rarely been reported with routine clinical doses include serous macular detachment , cystoid macular edema ( cme ) , increased central foveal thickness , decreased best - corrected distance visual acuity , anterior chamber inflammation , and vitritis .", "however , a prospective study found that icc at the standard dose of 1 mg/0.1 ml did not have a statistically significant effect on postoperative macular thickness compared with nonadministration of intracameral antibiotic ; although , this study was under - powered to detect rare events .", "the escrs study and other longitudinal cohort studies were not designed to assess safety or adverse events as primary endpoints , but a large number of patients enrolled without a reported increase in these adverse events suggests that they are not associated , are masked by confounding factors , or are exceedingly rare .", "a lingering concern for some ophthalmologists is the risk of ocular toxicity attributable to inadvertent exposure to elevated concentrations ( typically due to compounding errors ) .", "high doses of cefuroxime are associated with anterior and posterior segment inflammation with fibrin formation , corneal edema , elevated intraocular pressure , serous macular detachment , cme , hemorrhagic retinal infarction , and reduced rod photoreceptor cell function by electroretinography .", "table 1 summarizes the reported complications related to inadvertent administration of increased concentrations or volumes of cefuroxime .", "a limitation in assigning risk based on studies such as this is the lack of incidence data ; it is unknown how many patients have received increased doses of icc , and therefore , the rate of these adverse events is unknown .", "the incidence rate of compounding errors for icc is also unknown and is presumably highly dependent on local factors such as the mechanisms of medication preparation and quality control measures .", "adverse events attributed to cefuroxime compounding errors tass after cataract surgery has been reported in association with the intracameral use of cefuroxime .", "reported an ongoing cluster of tass cases at a single center , with a resolution of the outbreak after discontinuing icc in favor of intracameral moxifloxacin .", "as expected , there was no  rechallenge  in affected patients to determine conclusively if cefuroxime or other factor contributed to tass .", "there was also no reported analysis of cefuroxime concentration to ascertain the potential role of dilution errors or other factors related to preparation .", "no other studies were found that studied the relationship of cefuroxime to tass , or the effects of specific interventions to reduce the incidence of tass related to cefuroxime .", "cross - reactivity between penicillins and most second- and all third- and fourth - generation cephalosporins is negligible .", "it is generally considered safe to administer a cephalosporin with a side chain that is structurally dissimilar to that of penicillin . in patients with a documented ige - mediated reaction to penicillin , use of cephalosporins with a similar side chain", "however , cephalosporins with different side chains ( such as cefuroxime ) may be given .", "it should be noted that the escrs study excluded patients with penicillin or cephalosporin allergy , as did several other large studies .", "icc injection during cataract surgery was well tolerated in a prospective study of forty penicillin - allergic patients with a negative preoperative cefuroxime skin test .", "the use of icc in patients with penicillin allergy was explored between 2004 and 2012 in a case  control registry study ; the control group was the cohort of patients undergoing cataract surgery under a hospital policy of excluding patients with self - reported penicillin allergy .", "after a critical review of the literature and pilot study in 817 patients with reported penicillin allergy , this policy was altered , and all patients without a specific history of cephalosporin anaphylactic reaction were administered icc . out of 13,592 subsequent cataract surgeries , there were no reported cases of anaphylaxis or allergic reaction .", "this study is limited by the registry design , which may underreport or miscategorize adverse events .", "a similar longitudinal observational study by shorstein et al . reported a decreasing endophthalmitis rate after instituting a standard icc protocol ; there were no reports of anaphylaxis or allergic reaction among 12,609 surgeries , but some patients received other intracameral antibiotics , and it is unclear if penicillin - allergic patients routinely received cefuroxime or other antibiotics such as moxifloxacin or vancomycin .", "control study comparing the endophthalmitis rates before and after instituting a standard icc regimen ; patients with cefuroxime allergy were excluded , but penicillin - allergic patients received icc .", "there were no cases of anaphylaxis among 2289 patients receiving icc , but the prevalence of penicillin allergy was not reported .", "there are two case reports of an anaphylactic reaction in penicillin or cephalosporin allergic patients who received icc .", "the number of patients from these centers that had received icc were not reported , preventing an estimation of the incidence of this complication .", "adverse effects that have rarely been reported with routine clinical doses include serous macular detachment , cystoid macular edema ( cme ) , increased central foveal thickness , decreased best - corrected distance visual acuity , anterior chamber inflammation , and vitritis .", "however , a prospective study found that icc at the standard dose of 1 mg/0.1 ml did not have a statistically significant effect on postoperative macular thickness compared with nonadministration of intracameral antibiotic ; although , this study was under - powered to detect rare events .", "the escrs study and other longitudinal cohort studies were not designed to assess safety or adverse events as primary endpoints , but a large number of patients enrolled without a reported increase in these adverse events suggests that they are not associated , are masked by confounding factors , or are exceedingly rare .", "a lingering concern for some ophthalmologists is the risk of ocular toxicity attributable to inadvertent exposure to elevated concentrations ( typically due to compounding errors ) .", "high doses of cefuroxime are associated with anterior and posterior segment inflammation with fibrin formation , corneal edema , elevated intraocular pressure , serous macular detachment , cme , hemorrhagic retinal infarction , and reduced rod photoreceptor cell function by electroretinography .", "table 1 summarizes the reported complications related to inadvertent administration of increased concentrations or volumes of cefuroxime . a limitation in assigning risk based on studies such as this is the lack of incidence data ; it is unknown how many patients have received increased doses of icc , and therefore , the rate of these adverse events is unknown .", "the incidence rate of compounding errors for icc is also unknown and is presumably highly dependent on local factors such as the mechanisms of medication preparation and quality control measures .", "tass after cataract surgery has been reported in association with the intracameral use of cefuroxime .", "reported an ongoing cluster of tass cases at a single center , with a resolution of the outbreak after discontinuing icc in favor of intracameral moxifloxacin .", "as expected , there was no  rechallenge  in affected patients to determine conclusively if cefuroxime or other factor contributed to tass .", "there was also no reported analysis of cefuroxime concentration to ascertain the potential role of dilution errors or other factors related to preparation .", "no other studies were found that studied the relationship of cefuroxime to tass , or the effects of specific interventions to reduce the incidence of tass related to cefuroxime .", "the efficacy of icc as a prophylaxis for postcataract surgery endophthalmitis has been well established , but concerns regarding the risks of this intervention remain .", "the consensus of the literature from systemic administration of antibiotics is that the risk of cross - reactivity between second - generation cephalosporins ( such as cefuroxime ) and penicillin is very low .", "our review of the literature supports a low rate of anaphylactic reactions , even among penicillin - allergic patients . a postal survey conducted among consultant ophthalmic surgeons working in the national health service ophthalmic departments in england revealed that of 262 consultants , 103 ( 37% ) used cefuroxime in patients allergic to penicillin .", "we propose that an acceptable practice pattern would be to consider icc in all cataract surgery patients , including those with a history of penicillin allergy ; administration to those patients with cephalosporin allergy may be considered , but skin testing may be indicated to identify those patients that are at increased risk of anaphylaxis and should not receive icc .", "alternative intracameral antibiotics , such as vancomycin and moxifloxacin , may reduce the risk of anaphylaxis in cefuroxime - allergic patients , but the efficacy of those interventions has been less well established .", "the latest survey of members of the american society of cataract and refractive surgeons revealed that 30% of the united states ophthalmologists were utilizing intracameral antibiotics , compared to 70% of european respondents .", "however , many us cataract surgeons believe that intra - cameral antibiotics are unnecessary , based on concerns about the methodology of the escrs and other studies , or that the absolute benefits do not outweigh the risks of dilution errors and toxicity . not surprisingly , a clinical study demonstrated that the mathematical accuracy of a dilution protocol does not ensure dosage accuracy in a real - world clinical scenario .", "the authors suggest that a commercial preparation would likely reduce the risk of dilution errors , but commercial preparations of intracameral antibiotic agents may not be financially viable in all health care environments .", "implementation of icc should include measures to audit and provide quality assurance of the cefuroxime dilution protocol .", "half of the risk - benefit equation of icc has been solved to the satisfaction of most clinicians ; the efficacy is clear .", "the remaining barriers to more widespread adoption include concerns about risks such as anaphylaxis , dilution errors , and toxicity ( especially with noncommercial preparation ) and finally , the additional costs of this therapy .", "since endophthalmitis is a rare event , even a slight increase in the risk of prophylactic therapy may negate the potential benefits .", "our review and synthesis of the literature regarding the risks of cefuroxime therapy support the following recommendations : \n cefuroxime may be used safely in patients with penicillin allergy ( level 2a evidence)efforts to reduce the risk of dilution errors may include the use of a commercially prepared product , or strict quality assurance measures ( level 4 evidence)routine measures to reduce tass should also apply to the use of cefuroxime ( level 5 evidence ) ; even though , there is no proven association . \n ", "cefuroxime may be used safely in patients with penicillin allergy ( level 2a evidence ) efforts to reduce the risk of dilution errors may include the use of a commercially prepared product , or strict quality assurance measures ( level 4 evidence ) routine measures to reduce tass should also apply to the use of cefuroxime ( level 5 evidence ) ; even though , there is no proven association .", "future studies may further define the cost - effectiveness of icc , and continued efforts to reduce the risk attributable to dilution errors are indicated .", "finally , comparative studies of the efficacy and safety of cefuroxime compared to other intracameral antibiotics are needed to help define the optimal endophthalmitis prophylaxis regimen for our patients undergoing cataract surgery ."], "abstract_text": ["<S> background : endophthalmitis after cataract surgery is a rare but vision - threatening complication . </S>", "<S> intracameral cefuroxime ( icc ) has been reported to be effective at reducing the risk , but concerns regarding the risks associated with this intervention remain.methods:systematic review and synthesis of the literature on icc , with a focus on the risks of therapy.results:level 2a evidence was found to support the use of cefuroxime in penicillin - allergic patients . compounding or dilutional errors are associated with ocular toxicity , but the incidence and risk of this occurrence are unknown . </S>", "<S> level 4 evidence supports interventions that reduce the risk of dilutional errors . </S>", "<S> the association of cefuroxime injection with toxic anterior segment syndrome ( tass ) is not established ; level 5 evidence supports standard measures to reduce the incidence of tass related to cefuroxime administration.conclusion:cefuroxime can be administered safely to penicillin - allergic patients , and steps should be taken to reduce the risk of compounding or dilutional errors to avoid negating the benefits of this intervention . </S>", "<S> recommended practice patterns for endophthalmitis prophylaxis should consider the risks and benefits of icc . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Risk of anaphylaxis", "Risks of cefuroxime toxicity at standard doses", "Risks of cefuroxime toxicity at increased doses", "Toxic anterior segment syndrome and other idiosyncratic adverse events", "Discussion", "Conclusion", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["postcataract surgery endophthalmitis is a relatively uncommon but devastating complication of modern cataract surgery , and there is not complete consensus regarding the ideal practice patterns for prophylaxis .", "the european society of cataract and refractive surgeons ( escrs ) prospective multi - center interventional trial ( and many other case  control , cross - sectional , and longitudinal studies ) supported the hypothesis that the use of icc at the conclusion of cataract surgery reduces the incidence of postoperative endophthalmitis .", "it is estimated that two to four cases of endophthalmitis per 1000 surgeries can be avoided if surgeons adopt the use of icc , and the absence of an icc prophylactic regimen at a dose of 1 mg/0.1 ml was associated with an almost 5-fold increase in the risk of postoperative endophthalmitis . the injection is expedient , painless , and achieves high antibiotic concentrations in the immediate postoperative period . in addition , intracameral injection may be particularly effective in patients who sustain posterior capsule rupture and would otherwise incur an increased the risk of endophthalmitis . when considering the implementation of any intervention , cost - effectiveness is another important variable .", "estimated the cost - effectiveness ratio for icc to be $ 1403 per case of postoperative endophthalmitis prevented .", "many commonly used topical antibiotics are not cost - effective compared with icc , even under optimistic assumptions about their efficacy .", "another economic analysis comparing different prophylaxis regimens concluded that icc provided the best cost - effectiveness ratio .", "therefore , the decision to adopt this therapy is supported by studies demonstrating efficacy and cost - effectiveness . the final variable that must be included when", "one weakness inherent in any meticulously performed clinical trial is that the results may not be generalizable to a more diverse patient population and varying scenarios of care .", "most clinical trials make great effort to reduce the risk of enrollment in the study , and therefore the safety profile reported may be a  best - case scenario . ", "the purpose of this review is to provide an evidence - based review and synthesis of the literature regarding the risks of icc when administered for the prevention of postcataract surgery endophthalmitis , thereby informing decisions regarding best practices for cataract surgery .", "the categories of risk to be reviewed are : \n risk of anaphylaxis , especially in penicillin or cephalosporin allergic patientsrisk of toxicity at routine clinical dosesrisk of toxicity at increased doses due to compounding errorsidiosyncratic reactions , including toxic anterior segment syndrome ( tass ) . \n ", "risk of anaphylaxis , especially in penicillin or cephalosporin allergic patients risk of toxicity at routine clinical doses risk of toxicity at increased doses due to compounding errors idiosyncratic reactions , including toxic anterior segment syndrome ( tass ) ."], ["we performed a systematic review of the existing scientific literature using pubmed and google scholar .", "the key words used were  cefuroxime ,   endophthalmitis  and  cataract surgeries .", "all articles were read in full by both authors with the exception of two that were available only as abstracts .", "the highest level of evidence for each aspect of the intervention was assigned utilizing the oxford center for evidence - based medicine guidelines ."], ["cross - reactivity between penicillins and most second- and all third- and fourth - generation cephalosporins is negligible .", "it is generally considered safe to administer a cephalosporin with a side chain that is structurally dissimilar to that of penicillin . in patients with a documented ige - mediated reaction to penicillin , use of cephalosporins with a similar side chain", "however , cephalosporins with different side chains ( such as cefuroxime ) may be given .", "it should be noted that the escrs study excluded patients with penicillin or cephalosporin allergy , as did several other large studies .", "icc injection during cataract surgery was well tolerated in a prospective study of forty penicillin - allergic patients with a negative preoperative cefuroxime skin test .", "the use of icc in patients with penicillin allergy was explored between 2004 and 2012 in a case  control registry study ; the control group was the cohort of patients undergoing cataract surgery under a hospital policy of excluding patients with self - reported penicillin allergy .", "after a critical review of the literature and pilot study in 817 patients with reported penicillin allergy , this policy was altered , and all patients without a specific history of cephalosporin anaphylactic reaction were administered icc . out of 13,592 subsequent cataract surgeries , there were no reported cases of anaphylaxis or allergic reaction .", "this study is limited by the registry design , which may underreport or miscategorize adverse events .", "a similar longitudinal observational study by shorstein et al . reported a decreasing endophthalmitis rate after instituting a standard icc protocol ; there were no reports of anaphylaxis or allergic reaction among 12,609 surgeries , but some patients received other intracameral antibiotics , and it is unclear if penicillin - allergic patients routinely received cefuroxime or other antibiotics such as moxifloxacin or vancomycin .", "control study comparing the endophthalmitis rates before and after instituting a standard icc regimen ; patients with cefuroxime allergy were excluded , but penicillin - allergic patients received icc .", "there were no cases of anaphylaxis among 2289 patients receiving icc , but the prevalence of penicillin allergy was not reported .", "there are two case reports of an anaphylactic reaction in penicillin or cephalosporin allergic patients who received icc .", "the number of patients from these centers that had received icc were not reported , preventing an estimation of the incidence of this complication .", "adverse effects that have rarely been reported with routine clinical doses include serous macular detachment , cystoid macular edema ( cme ) , increased central foveal thickness , decreased best - corrected distance visual acuity , anterior chamber inflammation , and vitritis .", "however , a prospective study found that icc at the standard dose of 1 mg/0.1 ml did not have a statistically significant effect on postoperative macular thickness compared with nonadministration of intracameral antibiotic ; although , this study was under - powered to detect rare events .", "the escrs study and other longitudinal cohort studies were not designed to assess safety or adverse events as primary endpoints , but a large number of patients enrolled without a reported increase in these adverse events suggests that they are not associated , are masked by confounding factors , or are exceedingly rare .", "a lingering concern for some ophthalmologists is the risk of ocular toxicity attributable to inadvertent exposure to elevated concentrations ( typically due to compounding errors ) .", "high doses of cefuroxime are associated with anterior and posterior segment inflammation with fibrin formation , corneal edema , elevated intraocular pressure , serous macular detachment , cme , hemorrhagic retinal infarction , and reduced rod photoreceptor cell function by electroretinography .", "table 1 summarizes the reported complications related to inadvertent administration of increased concentrations or volumes of cefuroxime .", "a limitation in assigning risk based on studies such as this is the lack of incidence data ; it is unknown how many patients have received increased doses of icc , and therefore , the rate of these adverse events is unknown .", "the incidence rate of compounding errors for icc is also unknown and is presumably highly dependent on local factors such as the mechanisms of medication preparation and quality control measures .", "adverse events attributed to cefuroxime compounding errors tass after cataract surgery has been reported in association with the intracameral use of cefuroxime .", "reported an ongoing cluster of tass cases at a single center , with a resolution of the outbreak after discontinuing icc in favor of intracameral moxifloxacin .", "as expected , there was no  rechallenge  in affected patients to determine conclusively if cefuroxime or other factor contributed to tass .", "there was also no reported analysis of cefuroxime concentration to ascertain the potential role of dilution errors or other factors related to preparation .", "no other studies were found that studied the relationship of cefuroxime to tass , or the effects of specific interventions to reduce the incidence of tass related to cefuroxime ."], ["cross - reactivity between penicillins and most second- and all third- and fourth - generation cephalosporins is negligible .", "it is generally considered safe to administer a cephalosporin with a side chain that is structurally dissimilar to that of penicillin . in patients with a documented ige - mediated reaction to penicillin , use of cephalosporins with a similar side chain", "however , cephalosporins with different side chains ( such as cefuroxime ) may be given .", "it should be noted that the escrs study excluded patients with penicillin or cephalosporin allergy , as did several other large studies .", "icc injection during cataract surgery was well tolerated in a prospective study of forty penicillin - allergic patients with a negative preoperative cefuroxime skin test .", "the use of icc in patients with penicillin allergy was explored between 2004 and 2012 in a case  control registry study ; the control group was the cohort of patients undergoing cataract surgery under a hospital policy of excluding patients with self - reported penicillin allergy .", "after a critical review of the literature and pilot study in 817 patients with reported penicillin allergy , this policy was altered , and all patients without a specific history of cephalosporin anaphylactic reaction were administered icc . out of 13,592 subsequent cataract surgeries , there were no reported cases of anaphylaxis or allergic reaction .", "this study is limited by the registry design , which may underreport or miscategorize adverse events .", "a similar longitudinal observational study by shorstein et al . reported a decreasing endophthalmitis rate after instituting a standard icc protocol ; there were no reports of anaphylaxis or allergic reaction among 12,609 surgeries , but some patients received other intracameral antibiotics , and it is unclear if penicillin - allergic patients routinely received cefuroxime or other antibiotics such as moxifloxacin or vancomycin .", "control study comparing the endophthalmitis rates before and after instituting a standard icc regimen ; patients with cefuroxime allergy were excluded , but penicillin - allergic patients received icc .", "there were no cases of anaphylaxis among 2289 patients receiving icc , but the prevalence of penicillin allergy was not reported .", "there are two case reports of an anaphylactic reaction in penicillin or cephalosporin allergic patients who received icc .", "the number of patients from these centers that had received icc were not reported , preventing an estimation of the incidence of this complication ."], ["adverse effects that have rarely been reported with routine clinical doses include serous macular detachment , cystoid macular edema ( cme ) , increased central foveal thickness , decreased best - corrected distance visual acuity , anterior chamber inflammation , and vitritis .", "however , a prospective study found that icc at the standard dose of 1 mg/0.1 ml did not have a statistically significant effect on postoperative macular thickness compared with nonadministration of intracameral antibiotic ; although , this study was under - powered to detect rare events .", "the escrs study and other longitudinal cohort studies were not designed to assess safety or adverse events as primary endpoints , but a large number of patients enrolled without a reported increase in these adverse events suggests that they are not associated , are masked by confounding factors , or are exceedingly rare ."], ["a lingering concern for some ophthalmologists is the risk of ocular toxicity attributable to inadvertent exposure to elevated concentrations ( typically due to compounding errors ) .", "high doses of cefuroxime are associated with anterior and posterior segment inflammation with fibrin formation , corneal edema , elevated intraocular pressure , serous macular detachment , cme , hemorrhagic retinal infarction , and reduced rod photoreceptor cell function by electroretinography .", "table 1 summarizes the reported complications related to inadvertent administration of increased concentrations or volumes of cefuroxime . a limitation in assigning risk based on studies such as this is the lack of incidence data ; it is unknown how many patients have received increased doses of icc , and therefore , the rate of these adverse events is unknown .", "the incidence rate of compounding errors for icc is also unknown and is presumably highly dependent on local factors such as the mechanisms of medication preparation and quality control measures ."], ["tass after cataract surgery has been reported in association with the intracameral use of cefuroxime .", "reported an ongoing cluster of tass cases at a single center , with a resolution of the outbreak after discontinuing icc in favor of intracameral moxifloxacin .", "as expected , there was no  rechallenge  in affected patients to determine conclusively if cefuroxime or other factor contributed to tass .", "there was also no reported analysis of cefuroxime concentration to ascertain the potential role of dilution errors or other factors related to preparation .", "no other studies were found that studied the relationship of cefuroxime to tass , or the effects of specific interventions to reduce the incidence of tass related to cefuroxime ."], ["the efficacy of icc as a prophylaxis for postcataract surgery endophthalmitis has been well established , but concerns regarding the risks of this intervention remain .", "the consensus of the literature from systemic administration of antibiotics is that the risk of cross - reactivity between second - generation cephalosporins ( such as cefuroxime ) and penicillin is very low .", "our review of the literature supports a low rate of anaphylactic reactions , even among penicillin - allergic patients . a postal survey conducted among consultant ophthalmic surgeons working in the national health service ophthalmic departments in england revealed that of 262 consultants , 103 ( 37% ) used cefuroxime in patients allergic to penicillin .", "we propose that an acceptable practice pattern would be to consider icc in all cataract surgery patients , including those with a history of penicillin allergy ; administration to those patients with cephalosporin allergy may be considered , but skin testing may be indicated to identify those patients that are at increased risk of anaphylaxis and should not receive icc .", "alternative intracameral antibiotics , such as vancomycin and moxifloxacin , may reduce the risk of anaphylaxis in cefuroxime - allergic patients , but the efficacy of those interventions has been less well established .", "the latest survey of members of the american society of cataract and refractive surgeons revealed that 30% of the united states ophthalmologists were utilizing intracameral antibiotics , compared to 70% of european respondents .", "however , many us cataract surgeons believe that intra - cameral antibiotics are unnecessary , based on concerns about the methodology of the escrs and other studies , or that the absolute benefits do not outweigh the risks of dilution errors and toxicity . not surprisingly , a clinical study demonstrated that the mathematical accuracy of a dilution protocol does not ensure dosage accuracy in a real - world clinical scenario .", "the authors suggest that a commercial preparation would likely reduce the risk of dilution errors , but commercial preparations of intracameral antibiotic agents may not be financially viable in all health care environments .", "implementation of icc should include measures to audit and provide quality assurance of the cefuroxime dilution protocol ."], ["half of the risk - benefit equation of icc has been solved to the satisfaction of most clinicians ; the efficacy is clear .", "the remaining barriers to more widespread adoption include concerns about risks such as anaphylaxis , dilution errors , and toxicity ( especially with noncommercial preparation ) and finally , the additional costs of this therapy .", "since endophthalmitis is a rare event , even a slight increase in the risk of prophylactic therapy may negate the potential benefits .", "our review and synthesis of the literature regarding the risks of cefuroxime therapy support the following recommendations : \n cefuroxime may be used safely in patients with penicillin allergy ( level 2a evidence)efforts to reduce the risk of dilution errors may include the use of a commercially prepared product , or strict quality assurance measures ( level 4 evidence)routine measures to reduce tass should also apply to the use of cefuroxime ( level 5 evidence ) ; even though , there is no proven association . \n ", "cefuroxime may be used safely in patients with penicillin allergy ( level 2a evidence ) efforts to reduce the risk of dilution errors may include the use of a commercially prepared product , or strict quality assurance measures ( level 4 evidence ) routine measures to reduce tass should also apply to the use of cefuroxime ( level 5 evidence ) ; even though , there is no proven association .", "future studies may further define the cost - effectiveness of icc , and continued efforts to reduce the risk attributable to dilution errors are indicated .", "finally , comparative studies of the efficacy and safety of cefuroxime compared to other intracameral antibiotics are needed to help define the optimal endophthalmitis prophylaxis regimen for our patients undergoing cataract surgery ."], [""], [""]]}
{"article_id": "PMC4450692", "article_text": ["thyroid cancer is the most common endocrine neoplasia and accounts for 1% of all human cancers ( 1 ) .", "medullary thyroid carcinoma ( mtc ) is a rare neoplasm of the calcitonin - secreting thyroid cells and accounts for 510% of all thyroid cancers .", "mtc can occurs as sporadically ( 75% ) or as a part of the autosomal dominantly inherited forms ( 25% ) , ( 2 , 3 ) . the inherited type of mtc can be divided in three clinically distinct forms : multiple endocrine neoplasia type2a ( men2a ) , multiple endocrine neoplasia type2b ( men2b ) , and familial mtc ( fmtc ) ( 4 ) .", "men2a is defined by mtc , bilateral pheochromocytoma , and multiple tumors of the parathyroid glands ( parathyroid adenomas , primary hyperparathyroidism or hpt ) , within a single patient or family .", "men2b is characterized by the early development of an aggressive form of mtc in all affected individuals ( typically during the first year of life ) , pheochromocytoma , the absence of hyperparathyroidism , and visible physical stigmata such as raised bumps on the lips and tongue , ganglioneuromas of the intestine , marfanoid body habitus with skeletal deformations .", "fmtc is characterized by a strong predisposition to mtc in families with a very low incidence of other endocrinopathies related to men2 ( 1 , 5 ) . the molecular pathology of inherited mtcs is constitutive of ret ( rearranged during transfection ) proto - oncogene .", "the ret gene is located on chromosome 10q11.2 , consisting of 21 exons and encodes a tyrosine kinase receptor .", "this complex plays a critical role in cell proliferation and differentiation of tissues derived from neural crest cells , such as c - cells of thyroid gland ( 5 , 6 ) .", "ret is a tyrosine kinase protein and includes three domains ; a large extracellular domain containing a cysteine - rich region and a series of cadherin homology domains , a trans - membrane domain , and an intracellular tyrosine kinase domain , required for ret phosphorylation and downstream signaling ( 5 , 7 ) .", "this protein is activated by binding a soluble ligand of the glial cell - line - derived neurotrophic factor ( gdnf ) family and requires a co - receptor of the gdnf family receptors  ( gfr ) ( 8) .", "the majority of men2a families are associated with one of the six point mutations in conserved cysteine residues in exon10 ( codons 609 , 611 , 618 , and 620 ) or exon11 ( codons 630 , 634 ) in the extracellular domain of the ret ( 2 , 5 , 810 , 12 ) . in fmtc cases ,", "ret mutations are mainly detected in the same six codons as for men2a and also in exon13 ( codon 768 ) , or in exon15 ( codon 891 ) , in the intracellular region of ret ( 5 , 8 , 12 ) .", "mutation of codon 918 in ret exon16 in the tyrosine kinase domain , accounts for the vast majority of patients with men2b ( 5 , 8) . for the first time , ret polymorphisms g691s and s904s were described in 1994 ( 13 , 14 ) .", "recent studies have found g691s in exon11 , s904s single neucleotide polymorphisms in exon15 of the ret pathway to be associated with the risk of developing sporadic mtc ( smtc ) ( 7 ) .", "it seems , two of these ret snps ( g691s and s904s ) may modify the age at onset of mtc tumor in family members ( 5 , 15 ) but fabienne lesueur et al . have rejected this hypothesis ( 7 ) . in the present study , we report the frequency of g691s / s904s haplotype in iranian mtc patients and their relatives .", "over the last ten years , one hundred ninety mtc patients , were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening .", "this study has been approved by the ethics committee of cellular and molecular research center , research institute for endocrine sciences , shahid beheshti university of medical sciences .", "a questionnaire was used to collect information about age , sex , and history of neoplasia . if any mutation was found in patients , their first - degree relatives were invited for screening .", "121 first - degree relatives from 31 index cases comprised 57 males and 64 females , with meansd age 29.0916.34 years .", "genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c .", "for identification of ret variant 691 ( rs1799939 , codon 691 of exon 11 , ggt > agt , gly > ser ) and 904 ( rs1800863 , codon 904 of exon 15 , tcc > tcg , ser > ser , ) , dna samples were amplified using the polymerase chain reaction ( pcr ) and the specific oligonucleotides primers ( table1 ) .", "the exon11 running profile was constituted initial denaturation at 96 c for 1min and , followed by 30 cycles with 96 c for 45s , annealing at 60 c for 1min dependent on the primer sequences , and extension at 72c for 45s , with a final extension at 72 c for 10 min .", "sequences of oligonucleotide primers used for pcr amplification of exons 11 , 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and , followed by 35 cycles of 94c for 30s , annealing starting at 56c for 1min , 72c for 1min , and a final extension at 72c for 10min .", "the amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method ( abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit , applied biosystems , california , usa ) . to detect the ret mutations and snps , sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast .", "over the last ten years , one hundred ninety mtc patients , were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening .", "this study has been approved by the ethics committee of cellular and molecular research center , research institute for endocrine sciences , shahid beheshti university of medical sciences .", "a questionnaire was used to collect information about age , sex , and history of neoplasia . if any mutation was found in patients , their first - degree relatives were invited for screening .", "121 first - degree relatives from 31 index cases comprised 57 males and 64 females , with meansd age 29.0916.34 years .", "genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c .", "for identification of ret variant 691 ( rs1799939 , codon 691 of exon 11 , ggt > agt , gly > ser ) and 904 ( rs1800863 , codon 904 of exon 15 , tcc > tcg , ser > ser , ) , dna samples were amplified using the polymerase chain reaction ( pcr ) and the specific oligonucleotides primers ( table1 ) .", "the exon11 running profile was constituted initial denaturation at 96 c for 1min and , followed by 30 cycles with 96 c for 45s , annealing at 60 c for 1min dependent on the primer sequences , and extension at 72c for 45s , with a final extension at 72 c for 10 min .", "sequences of oligonucleotide primers used for pcr amplification of exons 11 , 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and , followed by 35 cycles of 94c for 30s , annealing starting at 56c for 1min , 72c for 1min , and a final extension at 72c for 10min .", "the amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method ( abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit , applied biosystems , california , usa ) .", "to detect the ret mutations and snps , sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast .", "among 311 participants , 18 dna samples did not have enough quality , so were excluded from the study .", "we analyzed germline dna mutations of ret gene in 181 iranian mtc patients and 112 relatives , with a total of 293 members .", "the patients included , 33 fmtc , 6 men2a , 2 men2b , 1 pheochromocytoma and 145 apparently smtc cases ( table 2 ) .", "the frequency of females in our population was a little higher than males , but this ratio was not significant ( 160 vs. 133 , p > 0.05 ) .", "specifically , we found 2 germline polymorphisms of ret gene at codon691 ( ggt > agt , exon 11 , rs1799939 ) that causes glycine to serine amino acid substitution , and codon 904 ( tcc > tcg , exon 15 , rs1800863 ) that does not lead to an ami - no acid alteration .", "furthermore , the allele frequencies of each of these snps were similar in all patients and relatives ( 21.5% and 10.75% , respectively ) .", "the frequency of patients with medullary thyroid carcinoma as the g691s and s904s variants were in complete linkage disequilibrium , so the results were grouped together and referred to as g691s / s904s haplotype ( fig.1 ) .", "the analysis of g691s / s904s ret gene haplotype showed that 104 of 181 ( 57.45% ) mtc patients and 55 of 112 ( 49.1% ) relatives had this haplotype .", "in particular , 82 mtcs and 47 relatives were heterozygous and 22 mtcs and 8 relatives were homozygous for the g691s / s904s haplotype ( table 3 ) .", "there was not significant correlation between age of diagnosis and the presence of g691s / s904s haplotype .", "the frequency of g691s and s904s variants in the population the frequency distribution of g691s and s904s variants among mtc patients and their relatives", "in this study , we found two variants of ret gene , g691s ( ggt > agt , exon 11 , rs1799939 ) and s904s ( tcc > tcg , exon 15 , rs1800863 ) that are cosegregated together as a haplotype , suggesting that these polymorphisms are in linkage disequilibrium with each other .", "the assessment of the occurrence of two polymorphic changes g691s , and s904s in our population is similar to the study performed by lucieli ceolin et al .", "unfortunately , in our study we did not have control group to compared with healthy population , but some studies have demonstrated that the ret variant g691s is more frequent in mtc patients than in the general population ( 16 , 17 ) .", "it has also been postulated that such variants are relatively common in the population which may confer a much higher attributable risk in the general population than mutations in high penetrance cancer susceptibility genes ( 5 , 11 ) .", "some studies support our data that both g691s and s904s snps are associated with sporadic mtc and men2a ( 5 , 7 , 18 ) .", "two studies have shown that this haplotype may modify the age at onset of mtc tumor in family members ( 5 , 15 ) , however , this hypothesis is controversial ( 7 ) . the ret - g691s polymorphism has been suggested as a genetic modifier in men2a ( 15 ) .", "overall , these data strengthen the potential role of the ret - g691s polymorphism in mtc .", "one of the probable mechanisms is that the g691s polymorphism seems to enhance ret on - cogenicity of other distinct mutations affecting ret ( 1820 ) , on the other hands , bases exchange in the dna molecule could create a new alternative splicing site , leading to the synthesis of a truncated protein , erroneous ligand binding , micro - rna binding , change of structure and mrna stability as well as a number of copies , and also the change in the secondary structure of proteins ( 16 ) .", "the ret gene mutations are a critical factor in patient management , e.g. to decide on total or partial thyroidectomy or the age at which the child should be subject to surgery ( 6 ) .", "ret gene mutations are very important in mtc patient management , especially in decision making for total or partial thyroidectomy or the age at which the child should be subject to surgery . in the case of inherited mutations ,", "those who test negative for germline mutations will be reassured ; those who test positive for germline mutations will be screened for calcitonin levels and will be offered a prophylactic thyroidectomy depending on the complete clinical and biochemical picture .", "this data showed the frequency of g691s / s904s haplotype among iranian mtc patients and their family , for the first time .", "even though g691s / s904s haplotype are not considered as oncogenic mutations at this time , its functional role should be investigated .", "further researches on this issue can contribute toward clarifying the prevalence of this haplotype and its probable modifying effect on the phenotypic characteristic of mtc .", "ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors ."], "abstract_text": ["<S> abstractbackgroundmedullary thyroid carcinoma ( mtc ) occurs in both sporadic ( 75% ) and hereditary ( 25% ) forms . </S>", "<S> the missense mutations of the rearranged during transfection ( ret ) proto - oncogene in mtc development have been well demonstrated . </S>", "<S> the aim of this study was to investigate frequency of g691s / s904s haplotype in mtc patients and their relatives.methodsin this research 293 participants were studied , including 181 patients ( 102 female , 79 male ) and 112 their relatives ( 58 female , 54 male ) . </S>", "<S> genomic dna was extracted from peripheral blood leucocytes using the standard salting out / proteinase k method . </S>", "<S> nucleotide change detection was performed using pcr and direct dna sequencing methods.resultsaccording to dna sequencing results , 159 individuals ( 104 patients , 55 relatives ) had both g691s ( rs1799939 ) missense mutation in exon11 and s904s ( rs1800863 ) synonymous mutation in exon 15 of ret proto - oncogene . </S>", "<S> the allele frequency of g691s / s904s haplotype was 21.15% in patients and 10.75% in their relatives.conclusionthe obtained data showed the frequency of g691s / s904s ret gene haplotype among iranian mtc patients and their relatives . </S>", "<S> the g691s and s904s nucleotide changes were in complete linkage disequilibrium , so the results were grouped together and referred to as g691s / s904s haplotype . </S>", "<S> further analysis is need to demonstrate the association between this haplotype and mtc development . </S>"], "labels": null, "section_names": ["Introduction", "Material and Methods", "Samples", "Genomic DNA extraction", "PCR assay", "Bioinformatics and Statistical analysis", "Results", "Discussion", "Conclusion", "Ethical considerations"], "sections": [["thyroid cancer is the most common endocrine neoplasia and accounts for 1% of all human cancers ( 1 ) .", "medullary thyroid carcinoma ( mtc ) is a rare neoplasm of the calcitonin - secreting thyroid cells and accounts for 510% of all thyroid cancers .", "mtc can occurs as sporadically ( 75% ) or as a part of the autosomal dominantly inherited forms ( 25% ) , ( 2 , 3 ) . the inherited type of mtc can be divided in three clinically distinct forms : multiple endocrine neoplasia type2a ( men2a ) , multiple endocrine neoplasia type2b ( men2b ) , and familial mtc ( fmtc ) ( 4 ) .", "men2a is defined by mtc , bilateral pheochromocytoma , and multiple tumors of the parathyroid glands ( parathyroid adenomas , primary hyperparathyroidism or hpt ) , within a single patient or family .", "men2b is characterized by the early development of an aggressive form of mtc in all affected individuals ( typically during the first year of life ) , pheochromocytoma , the absence of hyperparathyroidism , and visible physical stigmata such as raised bumps on the lips and tongue , ganglioneuromas of the intestine , marfanoid body habitus with skeletal deformations .", "fmtc is characterized by a strong predisposition to mtc in families with a very low incidence of other endocrinopathies related to men2 ( 1 , 5 ) . the molecular pathology of inherited mtcs is constitutive of ret ( rearranged during transfection ) proto - oncogene .", "the ret gene is located on chromosome 10q11.2 , consisting of 21 exons and encodes a tyrosine kinase receptor .", "this complex plays a critical role in cell proliferation and differentiation of tissues derived from neural crest cells , such as c - cells of thyroid gland ( 5 , 6 ) .", "ret is a tyrosine kinase protein and includes three domains ; a large extracellular domain containing a cysteine - rich region and a series of cadherin homology domains , a trans - membrane domain , and an intracellular tyrosine kinase domain , required for ret phosphorylation and downstream signaling ( 5 , 7 ) .", "this protein is activated by binding a soluble ligand of the glial cell - line - derived neurotrophic factor ( gdnf ) family and requires a co - receptor of the gdnf family receptors  ( gfr ) ( 8) .", "the majority of men2a families are associated with one of the six point mutations in conserved cysteine residues in exon10 ( codons 609 , 611 , 618 , and 620 ) or exon11 ( codons 630 , 634 ) in the extracellular domain of the ret ( 2 , 5 , 810 , 12 ) . in fmtc cases ,", "ret mutations are mainly detected in the same six codons as for men2a and also in exon13 ( codon 768 ) , or in exon15 ( codon 891 ) , in the intracellular region of ret ( 5 , 8 , 12 ) .", "mutation of codon 918 in ret exon16 in the tyrosine kinase domain , accounts for the vast majority of patients with men2b ( 5 , 8) . for the first time , ret polymorphisms g691s and s904s were described in 1994 ( 13 , 14 ) .", "recent studies have found g691s in exon11 , s904s single neucleotide polymorphisms in exon15 of the ret pathway to be associated with the risk of developing sporadic mtc ( smtc ) ( 7 ) .", "it seems , two of these ret snps ( g691s and s904s ) may modify the age at onset of mtc tumor in family members ( 5 , 15 ) but fabienne lesueur et al . have rejected this hypothesis ( 7 ) . in the present study , we report the frequency of g691s / s904s haplotype in iranian mtc patients and their relatives ."], ["over the last ten years , one hundred ninety mtc patients , were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening .", "this study has been approved by the ethics committee of cellular and molecular research center , research institute for endocrine sciences , shahid beheshti university of medical sciences .", "a questionnaire was used to collect information about age , sex , and history of neoplasia . if any mutation was found in patients , their first - degree relatives were invited for screening .", "121 first - degree relatives from 31 index cases comprised 57 males and 64 females , with meansd age 29.0916.34 years .", "genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c .", "for identification of ret variant 691 ( rs1799939 , codon 691 of exon 11 , ggt > agt , gly > ser ) and 904 ( rs1800863 , codon 904 of exon 15 , tcc > tcg , ser > ser , ) , dna samples were amplified using the polymerase chain reaction ( pcr ) and the specific oligonucleotides primers ( table1 ) .", "the exon11 running profile was constituted initial denaturation at 96 c for 1min and , followed by 30 cycles with 96 c for 45s , annealing at 60 c for 1min dependent on the primer sequences , and extension at 72c for 45s , with a final extension at 72 c for 10 min .", "sequences of oligonucleotide primers used for pcr amplification of exons 11 , 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and , followed by 35 cycles of 94c for 30s , annealing starting at 56c for 1min , 72c for 1min , and a final extension at 72c for 10min .", "the amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method ( abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit , applied biosystems , california , usa ) . to detect the ret mutations and snps , sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast ."], ["over the last ten years , one hundred ninety mtc patients , were referred to cellular and molecular research center of shahid beheshti university of medical sciences in order to ret genetic screening .", "this study has been approved by the ethics committee of cellular and molecular research center , research institute for endocrine sciences , shahid beheshti university of medical sciences .", "a questionnaire was used to collect information about age , sex , and history of neoplasia . if any mutation was found in patients , their first - degree relatives were invited for screening .", "121 first - degree relatives from 31 index cases comprised 57 males and 64 females , with meansd age 29.0916.34 years ."], ["genomic dna was extracted from peripheral blood leucocytes using standard salting out / proteinase k method and stored at -20c ."], ["for identification of ret variant 691 ( rs1799939 , codon 691 of exon 11 , ggt > agt , gly > ser ) and 904 ( rs1800863 , codon 904 of exon 15 , tcc > tcg , ser > ser , ) , dna samples were amplified using the polymerase chain reaction ( pcr ) and the specific oligonucleotides primers ( table1 ) .", "the exon11 running profile was constituted initial denaturation at 96 c for 1min and , followed by 30 cycles with 96 c for 45s , annealing at 60 c for 1min dependent on the primer sequences , and extension at 72c for 45s , with a final extension at 72 c for 10 min .", "sequences of oligonucleotide primers used for pcr amplification of exons 11 , 15 of the ret pcr reaction for exon 15 was constituted initial denaturation at 94c for 3min and , followed by 35 cycles of 94c for 30s , annealing starting at 56c for 1min , 72c for 1min , and a final extension at 72c for 10min .", "the amplimers were confirmed for the presence or absence of mutations by direct dna sequencing method ( abi 3100 genetic analyzer and big dye terminator v3.1 cycle sequencing kit , applied biosystems , california , usa ) ."], ["to detect the ret mutations and snps , sequences were analyzed by chromas 2.33 software and were also compared with the reference ret gene sequence using web based tool ncbi blast ."], ["among 311 participants , 18 dna samples did not have enough quality , so were excluded from the study .", "we analyzed germline dna mutations of ret gene in 181 iranian mtc patients and 112 relatives , with a total of 293 members .", "the patients included , 33 fmtc , 6 men2a , 2 men2b , 1 pheochromocytoma and 145 apparently smtc cases ( table 2 ) .", "the frequency of females in our population was a little higher than males , but this ratio was not significant ( 160 vs. 133 , p > 0.05 ) .", "specifically , we found 2 germline polymorphisms of ret gene at codon691 ( ggt > agt , exon 11 , rs1799939 ) that causes glycine to serine amino acid substitution , and codon 904 ( tcc > tcg , exon 15 , rs1800863 ) that does not lead to an ami - no acid alteration .", "furthermore , the allele frequencies of each of these snps were similar in all patients and relatives ( 21.5% and 10.75% , respectively ) .", "the frequency of patients with medullary thyroid carcinoma as the g691s and s904s variants were in complete linkage disequilibrium , so the results were grouped together and referred to as g691s / s904s haplotype ( fig.1 ) .", "the analysis of g691s / s904s ret gene haplotype showed that 104 of 181 ( 57.45% ) mtc patients and 55 of 112 ( 49.1% ) relatives had this haplotype .", "in particular , 82 mtcs and 47 relatives were heterozygous and 22 mtcs and 8 relatives were homozygous for the g691s / s904s haplotype ( table 3 ) .", "there was not significant correlation between age of diagnosis and the presence of g691s / s904s haplotype .", "the frequency of g691s and s904s variants in the population the frequency distribution of g691s and s904s variants among mtc patients and their relatives"], ["in this study , we found two variants of ret gene , g691s ( ggt > agt , exon 11 , rs1799939 ) and s904s ( tcc > tcg , exon 15 , rs1800863 ) that are cosegregated together as a haplotype , suggesting that these polymorphisms are in linkage disequilibrium with each other .", "the assessment of the occurrence of two polymorphic changes g691s , and s904s in our population is similar to the study performed by lucieli ceolin et al .", "unfortunately , in our study we did not have control group to compared with healthy population , but some studies have demonstrated that the ret variant g691s is more frequent in mtc patients than in the general population ( 16 , 17 ) .", "it has also been postulated that such variants are relatively common in the population which may confer a much higher attributable risk in the general population than mutations in high penetrance cancer susceptibility genes ( 5 , 11 ) .", "some studies support our data that both g691s and s904s snps are associated with sporadic mtc and men2a ( 5 , 7 , 18 ) .", "two studies have shown that this haplotype may modify the age at onset of mtc tumor in family members ( 5 , 15 ) , however , this hypothesis is controversial ( 7 ) . the ret - g691s polymorphism has been suggested as a genetic modifier in men2a ( 15 ) .", "overall , these data strengthen the potential role of the ret - g691s polymorphism in mtc .", "one of the probable mechanisms is that the g691s polymorphism seems to enhance ret on - cogenicity of other distinct mutations affecting ret ( 1820 ) , on the other hands , bases exchange in the dna molecule could create a new alternative splicing site , leading to the synthesis of a truncated protein , erroneous ligand binding , micro - rna binding , change of structure and mrna stability as well as a number of copies , and also the change in the secondary structure of proteins ( 16 ) .", "the ret gene mutations are a critical factor in patient management , e.g. to decide on total or partial thyroidectomy or the age at which the child should be subject to surgery ( 6 ) .", "ret gene mutations are very important in mtc patient management , especially in decision making for total or partial thyroidectomy or the age at which the child should be subject to surgery . in the case of inherited mutations ,", "those who test negative for germline mutations will be reassured ; those who test positive for germline mutations will be screened for calcitonin levels and will be offered a prophylactic thyroidectomy depending on the complete clinical and biochemical picture ."], ["this data showed the frequency of g691s / s904s haplotype among iranian mtc patients and their family , for the first time .", "even though g691s / s904s haplotype are not considered as oncogenic mutations at this time , its functional role should be investigated .", "further researches on this issue can contribute toward clarifying the prevalence of this haplotype and its probable modifying effect on the phenotypic characteristic of mtc ."], ["ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors ."]]}
{"article_id": "PMC3277061", "article_text": ["after initial establishment and derivation of human embryonic stem cells ( hesc ; thomson et al . , 1998 ; reubinoff et al . , 2000 )", "first the infection free status of the donors has to be addressed , in europe couples are tested before any fertility treatment is offered , but the cells themselves have to be tested , too ( hovatta , 2011 ) .", "second , optimized good manufacturing practice ( gmp ) compliant systems must be implemented for derivation , scaling - up , banking of cells , and their corresponding quality assurance controls ( unger et al . , 2008 ; ausubel et al . , 2011 ) .", "the culture systems currently encounter the problem of suboptimal quality of xeno - free culture constituents . thus strategies are needed to overcome this difficulty ( sidhu et al . , 2008 ) .", "steps have been taken ; initially , hesc were grown on irradiated mouse feeders , later human fore - skin fibroblast were used ( hovatta et al . , 2003 ) , now we can use gmp compliant coating substrates specially designed for hesc growth ( rodin et al . ,", "steps were also taken for the generation defined xeno - free gmp compliant medium for derivation and for expansion ( ludwig et al . , 2006 ;", "the potential of somatic cell reprogramming via expression of specific transcription factors and thus the generation of hesc induced pluripotent stem cells ( hipsc ; takahashi and yamanaka , 2006 ; takahashi et al . , 2007 ) has the advantage that they could be generated from the recipient patients own cells .", "there is no deep understanding of the effects that the reprogramming events have ; for instance on extracellular signaling ( okita et al . , 2011 ) , and the way that this could lead to immune reaction .", "hence fast reactivity is already present in healthy individuals for controlling any rapidly amplifying cells ( dhodapkar et al . , 2010 ) .", "un - silenced expression of the reprogramming factor oct-4 might then cause undesired immunoreactivity on the transplanted cells .", "immunoreactivity toward graft - derived hipsc of the same genetic background was also shown in animal models ( zhao et al . , 2011 ) .", "for successful reprogramming of somatic cells , many epigenetic changes must occur in an adequate manner .", "dna methylation changes during reprogramming must occur in important developmental and oncogenic regions , which increases the oncogenic risk of the reprogrammed cells ( doi et al .", "there is an additional risk for abnormalities and high tumorigenic potential , especially if c - myc is used as one of the transcription factors ( okita et al . , 2007 ) .", "also , genetic and epigenetic stability and large - scale genomic rearrangements after reprogramming and subsequent culture ( kim et al .", ", 2010 ; gore et al . , 2011 ; hussein et al . , 2011 ; lister et al . , 2011 )", "it is also important to address the safety of long - term culture , as shown recently ; the occurrence of chromosomal rearrangements in long - term culture of 125 hesc and 11 hipsc ( amps et al . , 2011 ) .", "there is a consensus that undifferentiated pluripotent stem cells ( psc ) will not be used directly in any clinical transplantations procedure , but instead their psc derived differentiated cells recently , results using hesc derived dopaminergic neurons have shown correct phenotype differentiation and grafting potential given by no tumor formation , maintenance of the grafted cells , and functional recovery in parkinsonian animal models in mice , rats , and monkeys ( kriks et al . , 2011 ) .", "the protocols designed for this cell replacement assay were optimal regarding the phenotype , quantity of the cells , functionality , and immunological properties .", "integration of transplanted cells was achieved when single cells were transplanted , the use of proper biodegradable scaffolds must also be considered .", "in addition to this initial report regarding the neural lineage , differentiation protocols for other cell types are needed . even if transplantation in animal models is successful , it is important to generate safety strategies before clinical trials to appropriately remove undifferentiated psc from their psc derived therapeutic cells .", "strategies such as inserting suicide gene ( drobyski et al . , 2003 ; uchibori et al . , 2009", ") might have controversial outcomes under clinical trials given their safety ( yi et al .", "alternatively , strategies such as removal of undifferentiated cells using antibodies might be safer ( tang et al . , 2011 ) .", "as discussed earlier , an optimal engraftment and cell replacement strategy should account for a minimal immune reaction in the recipient .", "this immune reaction occurs because the immune system of the recipient recognizes the grafted cells as foreign material or mismatched cellular components and thus generates a cascade of events that ultimately results in destruction and rejection of the grafted cells .", "this destruction can also compromise the recipient s immune status ( petersen et al . , 1975 ) .", "immunoreactivity toward the graft is mainly caused by t cell response toward unmatched major histocompatibility complex ( mhc ) ; in humans called human leukocyte antigen ( hla ) .", "if the profile is unmatched , it will result in rejection ( lechler et al . , 2005 ) .", "this rejection can occur via direct allorecognition of the donor antigen presenting cells ( apc ) or via indirect recognition of apoptotic cells ingested by the recipients apc , in both cases apcs presenting unmatched mhcs ( walsh et al . , 2004 ) .", "several groups have studied mhc profiles of hesc and their differentiated cells ( swijnenburg et al . , 2008 ; pearl et al . , 2011 ) .", "findings are that undifferentiated cells express mhc i antigens , though at low levels compared with somatic cells ; but they do not express mhc ii molecules ( drukker et al . , 2002 , 2006 ; li et al . , 2004 ) .", "during in vitro differentiation toward germ lineages , embryoid body ( eb ) formation , or teratoma formation mhc i expression increases dramatically ( drukker and benvenisty , 2004 )", ". also culture methods of hesc can change antigen expression levels ( rajala et al . , 2010 ) .", "careful selection of culture conditions , both for the undifferentiated hesc and for their differentiated derivatives is needed .", "human embryonic stem cells adopts the expression of non - human cell surface markers if exposed to such substances during culture ( martin et al . , 2005 ; hisamatsu - sakamoto et al . , 2008 )", "hence , optimal culture conditions must be xeno - free from the initial derivation and onward .", "these culture conditions must be carefully analyzed and scientific consensus must be achieved in order to raise current methodologies .", "challenges with the immunoreactivity of the transplantable cells could be addressed by rigorous immunosuppressive treatments . unfortunately , this is not desired , since there is a clear correlation between the length and intensity of exposure to immunosuppressive therapy and post - transplant risk of malignancy and tumor aggressiveness ( gutierrez - dalmau and campistol , 2007 ) .", "an interesting solution is costimulatory blockage of t cell response ( grinnemo et al . , 2006 , 2008 ; swijnenburg et al . , 2008 ; pearl et al . , 2011", ". this immunosuppression strategy will generate tolerance to the grafted cells and thus increase graft survival ; initial pharmaceutical agents have been developed and pending clinical applications to the fda are to give in the near future more information .", "in this mini - review we highlighted the most important areas to be considered under a cell replacement therapy .", "the possibility of using hipsc derived therapeutic cells in cell replacement therapies requires still long - term studies in non - human animal models addressing the questions of immunogenicity , epigenetic and genetic stability of these cells , and the optimized differentiation of the cells .", "the importance of profiling immunogenic markers as part of the stem - ness characterization and profiling of cells allocated in stem cell banks must be consider .", "such information has to be well protected so that it will not be lost in any given situation .", "adequate culture conditions , supporting correct immunogenicity of the cells under a transplantation assay is also required .", "next , the management of immunosuppression schemes must aim to a minimal time influencing the immunological status of the recipient . from all the information obtained ,", "these profiles can then be used in combination with methodologies focused at monitoring the status of the transplanted cells . in a given scenario that undesired cells persist in the transplant , adequate counteracting actions have immediately to be taken .", "such possibilities have to be tested and the removal of undesired effects confirmed before starting cell transplantations .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."], "abstract_text": ["<S> human embryonic stem cells ( hesc ) and induced pluripotent stem cells ( hipsc ) are an attractive cell source for regenerative medicine . </S>", "<S> these cells can be expanded to vast numbers and can be differentiated to many desired pluripotent stem cells ( psc ) derived therapeutic cells . </S>", "<S> cell replacement bears promises , but also challenges </S>", "<S> . the introduction of exogenous cells in a recipient must address several different topics ; its safety , the exclusion of tumor formation , the immunological response and possible rejection , the cells cleanliness and their biological quality , and quantity representing the functionality of the psc derived therapeutic cells . </S>", "<S> tumor formation requires the removal of any psc remaining after differentiation . </S>", "<S> immunological rejection can be addressed with immunomodulation of the cells and the recipient . </S>", "<S> cleanliness can be optimized using good manufacturing practice quality systems . at last </S>", "<S> , the functionality of the cells must be tested in in vitro and in animal models . after addressing these challenges , </S>", "<S> precise strategies are developed to monitor the status of the cells at different times and in case of undesired results , corresponding counteracting strategies must exist before any clinical attempt . </S>"], "labels": null, "section_names": ["Culture Conditions of Pluripotent Cells", "Reprogramming and hiPSC", "Pluripotent Stem Cell Differentiation", "The Immunogenic Profiles", "Immunosuppression", "Discussion", "Conflict of Interest Statement"], "sections": [["after initial establishment and derivation of human embryonic stem cells ( hesc ; thomson et al . , 1998 ; reubinoff et al . , 2000 )", "first the infection free status of the donors has to be addressed , in europe couples are tested before any fertility treatment is offered , but the cells themselves have to be tested , too ( hovatta , 2011 ) .", "second , optimized good manufacturing practice ( gmp ) compliant systems must be implemented for derivation , scaling - up , banking of cells , and their corresponding quality assurance controls ( unger et al . , 2008 ; ausubel et al . , 2011 ) .", "the culture systems currently encounter the problem of suboptimal quality of xeno - free culture constituents . thus strategies are needed to overcome this difficulty ( sidhu et al . , 2008 ) .", "steps have been taken ; initially , hesc were grown on irradiated mouse feeders , later human fore - skin fibroblast were used ( hovatta et al . , 2003 ) , now we can use gmp compliant coating substrates specially designed for hesc growth ( rodin et al . ,", "steps were also taken for the generation defined xeno - free gmp compliant medium for derivation and for expansion ( ludwig et al . , 2006 ;"], ["the potential of somatic cell reprogramming via expression of specific transcription factors and thus the generation of hesc induced pluripotent stem cells ( hipsc ; takahashi and yamanaka , 2006 ; takahashi et al . , 2007 ) has the advantage that they could be generated from the recipient patients own cells .", "there is no deep understanding of the effects that the reprogramming events have ; for instance on extracellular signaling ( okita et al . , 2011 ) , and the way that this could lead to immune reaction .", "hence fast reactivity is already present in healthy individuals for controlling any rapidly amplifying cells ( dhodapkar et al . , 2010 ) .", "un - silenced expression of the reprogramming factor oct-4 might then cause undesired immunoreactivity on the transplanted cells .", "immunoreactivity toward graft - derived hipsc of the same genetic background was also shown in animal models ( zhao et al . , 2011 ) .", "for successful reprogramming of somatic cells , many epigenetic changes must occur in an adequate manner .", "dna methylation changes during reprogramming must occur in important developmental and oncogenic regions , which increases the oncogenic risk of the reprogrammed cells ( doi et al .", "there is an additional risk for abnormalities and high tumorigenic potential , especially if c - myc is used as one of the transcription factors ( okita et al . , 2007 ) .", "also , genetic and epigenetic stability and large - scale genomic rearrangements after reprogramming and subsequent culture ( kim et al .", ", 2010 ; gore et al . , 2011 ; hussein et al . , 2011 ; lister et al . , 2011 )", "it is also important to address the safety of long - term culture , as shown recently ; the occurrence of chromosomal rearrangements in long - term culture of 125 hesc and 11 hipsc ( amps et al . , 2011 ) ."], ["there is a consensus that undifferentiated pluripotent stem cells ( psc ) will not be used directly in any clinical transplantations procedure , but instead their psc derived differentiated cells recently , results using hesc derived dopaminergic neurons have shown correct phenotype differentiation and grafting potential given by no tumor formation , maintenance of the grafted cells , and functional recovery in parkinsonian animal models in mice , rats , and monkeys ( kriks et al . , 2011 ) .", "the protocols designed for this cell replacement assay were optimal regarding the phenotype , quantity of the cells , functionality , and immunological properties .", "integration of transplanted cells was achieved when single cells were transplanted , the use of proper biodegradable scaffolds must also be considered .", "in addition to this initial report regarding the neural lineage , differentiation protocols for other cell types are needed . even if transplantation in animal models is successful , it is important to generate safety strategies before clinical trials to appropriately remove undifferentiated psc from their psc derived therapeutic cells .", "strategies such as inserting suicide gene ( drobyski et al . , 2003 ; uchibori et al . , 2009", ") might have controversial outcomes under clinical trials given their safety ( yi et al .", "alternatively , strategies such as removal of undifferentiated cells using antibodies might be safer ( tang et al . , 2011 ) ."], ["as discussed earlier , an optimal engraftment and cell replacement strategy should account for a minimal immune reaction in the recipient .", "this immune reaction occurs because the immune system of the recipient recognizes the grafted cells as foreign material or mismatched cellular components and thus generates a cascade of events that ultimately results in destruction and rejection of the grafted cells .", "this destruction can also compromise the recipient s immune status ( petersen et al . , 1975 ) .", "immunoreactivity toward the graft is mainly caused by t cell response toward unmatched major histocompatibility complex ( mhc ) ; in humans called human leukocyte antigen ( hla ) .", "if the profile is unmatched , it will result in rejection ( lechler et al . , 2005 ) .", "this rejection can occur via direct allorecognition of the donor antigen presenting cells ( apc ) or via indirect recognition of apoptotic cells ingested by the recipients apc , in both cases apcs presenting unmatched mhcs ( walsh et al . , 2004 ) .", "several groups have studied mhc profiles of hesc and their differentiated cells ( swijnenburg et al . , 2008 ; pearl et al . , 2011 ) .", "findings are that undifferentiated cells express mhc i antigens , though at low levels compared with somatic cells ; but they do not express mhc ii molecules ( drukker et al . , 2002 , 2006 ; li et al . , 2004 ) .", "during in vitro differentiation toward germ lineages , embryoid body ( eb ) formation , or teratoma formation mhc i expression increases dramatically ( drukker and benvenisty , 2004 )", ". also culture methods of hesc can change antigen expression levels ( rajala et al . , 2010 ) .", "careful selection of culture conditions , both for the undifferentiated hesc and for their differentiated derivatives is needed .", "human embryonic stem cells adopts the expression of non - human cell surface markers if exposed to such substances during culture ( martin et al . , 2005 ; hisamatsu - sakamoto et al . , 2008 )", "hence , optimal culture conditions must be xeno - free from the initial derivation and onward .", "these culture conditions must be carefully analyzed and scientific consensus must be achieved in order to raise current methodologies ."], ["challenges with the immunoreactivity of the transplantable cells could be addressed by rigorous immunosuppressive treatments . unfortunately , this is not desired , since there is a clear correlation between the length and intensity of exposure to immunosuppressive therapy and post - transplant risk of malignancy and tumor aggressiveness ( gutierrez - dalmau and campistol , 2007 ) .", "an interesting solution is costimulatory blockage of t cell response ( grinnemo et al . , 2006 , 2008 ; swijnenburg et al . , 2008 ; pearl et al . , 2011", ". this immunosuppression strategy will generate tolerance to the grafted cells and thus increase graft survival ; initial pharmaceutical agents have been developed and pending clinical applications to the fda are to give in the near future more information ."], ["in this mini - review we highlighted the most important areas to be considered under a cell replacement therapy .", "the possibility of using hipsc derived therapeutic cells in cell replacement therapies requires still long - term studies in non - human animal models addressing the questions of immunogenicity , epigenetic and genetic stability of these cells , and the optimized differentiation of the cells .", "the importance of profiling immunogenic markers as part of the stem - ness characterization and profiling of cells allocated in stem cell banks must be consider .", "such information has to be well protected so that it will not be lost in any given situation .", "adequate culture conditions , supporting correct immunogenicity of the cells under a transplantation assay is also required .", "next , the management of immunosuppression schemes must aim to a minimal time influencing the immunological status of the recipient . from all the information obtained ,", "these profiles can then be used in combination with methodologies focused at monitoring the status of the transplanted cells . in a given scenario that undesired cells persist in the transplant , adequate counteracting actions have immediately to be taken .", "such possibilities have to be tested and the removal of undesired effects confirmed before starting cell transplantations ."], ["the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ."]]}
{"article_id": "PMC2629952", "article_text": ["to report two cases of femtosecond laser - assisted small incision deep lamellar endothelial keratoplasty ( dlek ) for patients with corneal endothelial decompensation by fuchs dystrophy and glaucoma", "irvine , ca ) with 15 khz of repetition rate , was used for a 9.5 mm diameter by 400 m thickness donor corneal lamellar dissection .", "in case 1 , the graft was clear and compact without interface haze , orbscan showed smooth and regular corneal surface , specular microscopy was unremarkable without sign of corneal endothelial damage , and optical coherence tomography showed uniform graft well attached to recipient stroma with minimal interface reflection at 2 months postoperation . in case 2 , the graft was clear and compact with minimal interface haze at 1 month postoperation .", "femtosecond laser - assisted small incision dlek was safe and technically feasible in our cases ; however , further evaluation is required to determine long - term effects .", "the donor corneoscleral button was mounted in a dedicated artificial anterior chamber ( bausch & lomb , st .", "this was to assure a donor corneal thickness between 500 and 565 m , in order to approximate a graft thickness of 100 to 165 m . the femtosecond laser ( intralase ; intralase corp . , irvine , ca ) was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness .", "femtosecond laser parameter settings were as follows : raster energy 3.6 j , spot separation 10 m , line separation 10 m , side cut energy 3.8 j , spot size 2.4 m , and repetition rate 15 khz .", "after laser application , stromal and side cut adhesions were fully released and the anterior corneal flap was fully lifted .", "the donor corneoscleral button was then placed in medium and transported to the operating room , where the anterior flap was excised with scissors and an 8.0 mm trephine ( katena products , inc . , denville , nj ) was used to punch out the posterior donor corneoscleral button . the recipient corneal lamellar dissection and graft transplantation", "was performed as previously described.5,6 briefly , following an 8.0 mm epithelial marking and 5.0 mm scleral incision , a deep lamellar pocket was created to approximately 75 - 85% of corneal thickness .", "the posterior recipient disc was then excised with cindy scissors ( bausch & lomb ) using the 8.0 mm epithelial mark as a template .", "the excised posterior recipient disc was removed from the lamellar pocket and spread over recipient corneal epithelium to verify size .", "an 8.0 mm pre - prepared posterior donor disc was then folded , endothelial side inside , with a layer of viscoelastic ( healon ; pharmacia , peapack , nj ) coating the endothelium and inserted into the anterior chamber through the small incision using forceps .", "the folded donor disc was opened and attached to the recipient stromal bed by injecting air underneath the graft .", "the scleral wound was closed with 2 or 3 interrupted 10 - 0 nylon sutures and air was replaced with balanced salt solution to normalize intraocular pressure ( iop ) .", "femtosecond laser - assisted small incision dlek was performed in the right eye of a 73-year - old male with fuchs dystrophy ( fig .", "1 ) . preoperative uncorrected visual acuity ( ucva ) and best spectacle corrected visual acuity ( bscva ) was 20/100 and 20/80 respectively and manifest refraction was + 0.75 -1.7530 .", "donor corneoscleral button lamellar dissection was performed using intralase femtosecond laser ( intralase corp . ) , set at a depth of 400 m and flap diameter of 9.5 mm .", "ucva and bscva was 20/150 and 20/100 respectively and manifest refraction was + 2.75 -1.0050 .", "corneal topography showed smooth and regular corneal surface and specular microscopy was unremarkable without sign of corneal endothelial damage ( fig .", "optical coherence tomography ( oct ) findings showed compact and uniform graft , well attached to the recipient stromal bed with minimal interface reflection ( fig .", "3 ) . femtosecond laser - assisted small incision dlek was performed in the left eye of a 94-year - old female with corneal edema , glaucoma , and dry type age - related macular degeneration ( armd ) .", "her preoperative ucva and bscva was 20/400 and 20/200 respectively and her manifest refraction was -1.00 -1.7595 .", "iop was well controlled with topical medication ( cosopt ; merck & co. , whitehouse station , nj ) .", "postoperation , the graft was clear and compact , iop was 19 mmhg , and ucva was 20/400 .", "at one month , the graft was clear and compact with minimal interface haze ; iop measured 31 mmhg .", "anti - glaucoma medication ( alphagan ; allergan , irvine , ca ) was added and topical steroid tapered .", "we were not able to perform further examinations due to follow - up loss at two months postoperation .", "all methods adhered to the declaration of helsinki of the world medical association for research involving human subjects .", "the donor corneoscleral button was mounted in a dedicated artificial anterior chamber ( bausch & lomb , st .", "this was to assure a donor corneal thickness between 500 and 565 m , in order to approximate a graft thickness of 100 to 165 m . the femtosecond laser ( intralase ; intralase corp . , irvine , ca ) was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness .", "femtosecond laser parameter settings were as follows : raster energy 3.6 j , spot separation 10 m , line separation 10 m , side cut energy 3.8 j , spot size 2.4 m , and repetition rate 15 khz .", "after laser application , stromal and side cut adhesions were fully released and the anterior corneal flap was fully lifted .", "the donor corneoscleral button was then placed in medium and transported to the operating room , where the anterior flap was excised with scissors and an 8.0 mm trephine ( katena products , inc . , denville , nj ) was used to punch out the posterior donor corneoscleral button . the recipient corneal lamellar dissection and graft transplantation", "was performed as previously described.5,6 briefly , following an 8.0 mm epithelial marking and 5.0 mm scleral incision , a deep lamellar pocket was created to approximately 75 - 85% of corneal thickness .", "the posterior recipient disc was then excised with cindy scissors ( bausch & lomb ) using the 8.0 mm epithelial mark as a template .", "the excised posterior recipient disc was removed from the lamellar pocket and spread over recipient corneal epithelium to verify size .", "an 8.0 mm pre - prepared posterior donor disc was then folded , endothelial side inside , with a layer of viscoelastic ( healon ; pharmacia , peapack , nj ) coating the endothelium and inserted into the anterior chamber through the small incision using forceps .", "the folded donor disc was opened and attached to the recipient stromal bed by injecting air underneath the graft .", "the scleral wound was closed with 2 or 3 interrupted 10 - 0 nylon sutures and air was replaced with balanced salt solution to normalize intraocular pressure ( iop ) .", "femtosecond laser - assisted small incision dlek was performed in the right eye of a 73-year - old male with fuchs dystrophy ( fig .", "preoperative uncorrected visual acuity ( ucva ) and best spectacle corrected visual acuity ( bscva ) was 20/100 and 20/80 respectively and manifest refraction was + 0.75 -1.7530 .", "donor corneoscleral button lamellar dissection was performed using intralase femtosecond laser ( intralase corp . ) , set at a depth of 400 m and flap diameter of 9.5 mm .", "ucva and bscva was 20/150 and 20/100 respectively and manifest refraction was + 2.75 -1.0050 .", "corneal topography showed smooth and regular corneal surface and specular microscopy was unremarkable without sign of corneal endothelial damage ( fig .", "2b and c ) . optical coherence tomography ( oct ) findings showed compact and uniform graft , well attached to the recipient stromal bed with minimal interface reflection ( fig .", "femtosecond laser - assisted small incision dlek was performed in the left eye of a 94-year - old female with corneal edema , glaucoma , and dry type age - related macular degeneration ( armd ) .", "her preoperative ucva and bscva was 20/400 and 20/200 respectively and her manifest refraction was -1.00 -1.7595 .", "iop was well controlled with topical medication ( cosopt ; merck & co. , whitehouse station , nj ) .", "postoperation , the graft was clear and compact , iop was 19 mmhg , and ucva was 20/400 .", "at one month , the graft was clear and compact with minimal interface haze ; iop measured 31 mmhg .", "anti - glaucoma medication ( alphagan ; allergan , irvine , ca ) was added and topical steroid tapered .", "we were not able to perform further examinations due to follow - up loss at two months postoperation .", "all methods adhered to the declaration of helsinki of the world medical association for research involving human subjects .", "posterior keratoplasty refers to removal of diseased posterior corneal layers ( endothelium , descemet 's membrane , and posterior stroma ) and replacement by partial - thickness donor tissue .", "this procedure can be an attractive alternative to penetrating keratoplasty ( pkp ) in treating corneal endothelial diseases such as fuchs dystrophy and aphakic and pseudophakic bullous keratopathy.13 - 15 the endothelial replacement technique via scleral incision was first described by melles and later modified by terry and ousley to ' deep lamellar endothelial keratoplasty ' ( dlek).3,4 recent modification includes introducing the folded donor disc through the a small incision.6 one problem in performing dlek is the technical difficulty associated with manual lamellar dissection .", "it is not only time consuming , but also has the potential risk of corneal perforation.8 moreover , even with uncomplicated dissection , the plane may not be smooth and uniform , which results in interface scarring and reduction in potential best vision.6,7 in a recent paper , azar and coauthors described microkeratome - assisted posterior keratoplasty.16 given that laser - assisted in situ keratomileusis ( lasik ) shows almost no interface haze on the cornea,17,18 this technique 's potential advantage is less interface scarring than manual dissection .", "however , microkeratome - assisted lamellar dissection does have the risk of corneal perforation in deeper dissection .", "recently , the femtosecond laser was introduced in refractive surgery fields for lasik flap creation . according to recent papers", ", it demonstrated improved flap uniformity and better flap thickness predictability than mechanical microkeratomes.9 - 12,19 similar to the microkeratome , femtosecond laser technology has evolved .", "after initial procedures with a 2 khz laser in 1996 , 15 khz ( 2003 ) , 30 khz ( 2005 ) , and soon after , 60 khz ( 2006 ) engines were introduced .", "pulses are scanned and placed in a vertical pattern for trephination ( side ) cuts or in a spiral or raster ( zigzag ) pattern to achieve lamellar cuts .", "the smoothness of optical surfaces is determined by programmable parameters : energy per pulse , separation of adjacent laser spots ( spot separation ) , and raster pattern row spacing ( line separation ) .", "the closer the laser spots , the less energy required.20 the intralase system scans tissue at a repetition rate which depends on laser engine capacity .", "higher repetition rates allow use of lower pulse energy and closer spot / line separation settings , which results in a smoother lamellar interface , an easier anterior corneal flap lift , and a faster procedure .", "the newer 60 khz engine permits a line / spot separation down to 66 m and energy less than 1 j per pulse , which may show superior efficiency for harvesting the posterior corneal disk.21 one concern during preoperative femtosecond laser - assisted donor eye posterior lamellar discs ( plds ) preparation is the possibility of endothelial cell loss ( ecl ) .", "ecl has been reported at 4% after 150 to 200 m thick endothelial side plds preparations , and did not appear to be caused by laser pulse energy.22 another in vitro study reported 4.3% versus 7.7% ecl after 30 khz laser lamellar cutting compared to 15 khz laser for horizontal lamellar cuts at corneal depth of 400 m and 9.5 mm diameter.23 favorable outcomes are consistent with histological results showing adjacent thermal damage to be on the order of 1   m.24 clinical results from case 1 showed relatively intact postoperative endothelium status , which was comparable to previous reports .", "moreover , the femtosecond laser is expected to be applied in full - thickness pkp .", "cuts are customizable to achieve different graft geometric configurations , potentially allowing for sutureless , self - adhesive , or shaped keratoplasty .", "two or more side / lamellar cut segments can be combined to create patterns for shaped keratoplasty , including top - hat ( larger diameter cut posteriorly ) , mushroom ( larger diameter cut anteriorly ) , zigzag , and christmas tree patterns.25 peripheral wound edge shaping can provide stronger healing by increasing surface area , reduce corneal astigmatism by decreasing suture number , reduce donor - host topographic distortion disparity , and quicken visual recovery .", "following we describe small incision dlek by femtosecond laser for donor corneal lamellar dissection . before performing femtosecond laser - assisted donor lamellar dissection , we measured donor corneal thickness by ultrasound pachymetry .", "if corneal thickness was thicker than 565 m , glycerin was applied to dehydrate the cornea , and avoid a thinner graft than intended , as well as to ascertain graft thickness of 100 to 165 m for transplantation .", "the laser was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness .", "afterwards , two possible methods to obtain the posterior donor disc were used . in the first method ,", "this bi - hinged donor corneoscleral button is then placed in medium , transported to the operating room , and punched out with an 8.0 mm trephine .", "separation of the anterior and posterior donor corneal disc follows . in the second method , stromal and side cut adhesions", "this donor corneoscleral button is placed in medium and transported ; following , the anterior flap is excised using scissors and the posterior part is punched out with an 8.0 mm trephine . in our cases", "although the disadvantage is using scissors for anterior cap removal , there are other advantages . by removing the anterior cap ,", "it is easier to visualize the posterior donor button edge , which may reduce a decentered donor cut .", "furthermore , anterior and posterior donor disc separation is much easier , since the anterior flap is fully lifted and removed before trephination .", "case 1 clinical results showed a clear and compact graft without interface haze at two months postoperation .", "these findings were further confirmed in oct , which showed a clear and uniform graft with minimal interface reflection and good graft attachment . despite good clinical findings , postop visual acuity was not as expected .", "however , the patient was satisfied and his vision fluctuation ( previously worse in the morning ) was eliminated .", "considering findings and short - term follow - up , we may expect further improvement with time .", "however , postop visual acuity did not improve because of low visual potential from dry armd and glaucoma .", "unfortunately , this patient had an iop increase at one month postoperation and was unable to follow - up .", "visual acuity after either pkp or dlek is mainly a function of retinal macular potential and cornea optical quality . in studies of pkp in young patients with keratoconus , postoperative visual results", "are uniformly better compared to results in older fuchs ' dystrophy patients , despite similar graft problems of high or irregular astigmatism.26 this suggests that older maculas , may play a role in visual loss after any form of corneal transplantation , including dlek .", "in contrast to pkp where the cornea contributes to refractive visual loss , often resulting from high or irregular astigmatism , in dlek , topography is usually normal and corneal visual loss is most likely attributed to the donor - recipient stromal interface .", "the interface in dlek may account , on average , for 1 line of visual loss compared to the individual macular potential vision.27 other potential corneal factors include increased corneal thickness and posterior corneal curvature .", "how this irregularity influences visual acuity requires further investigation . due to lack of long term follow - up and small case number", ", we can not fully determine potential advantages of ' femtosecond laser - assisted small incision dlek ' .", "however , these cases demonstrate graft uniformity , technical feasibility , and potential application of femtosecond laser in lamellar keratoplasty .", "further evaluation with longer follow - up and more cases would reveal potential advantages . unfortunately , the use of femtosecond laser in recipient corneas is currently limited by the institutional review board .", "also the femtosecond laser is not approved for cuts deeper than 400 m ; therefore recipient cornea posterior lip creation requires scissors .", "hopefully in the near future , we can use the femtosecond laser and perform both donor and recipient corneal lamellar dissection ."], "abstract_text": ["<S> purposeto report two cases of femtosecond laser - assisted small incision deep lamellar endothelial keratoplasty ( dlek ) for patients with corneal endothelial decompensation by fuchs dystrophy and glaucomamethodsfemtosecond laser ( intralase ; intralase corp . , </S>", "<S> irvine , ca ) with 15 khz of repetition rate , was used for a 9.5 mm diameter by 400 m thickness donor corneal lamellar dissection.resultsin case 1 , the graft was clear and compact without interface haze , orbscan showed smooth and regular corneal surface , specular microscopy was unremarkable without sign of corneal endothelial damage , and optical coherence tomography showed uniform graft well attached to recipient stroma with minimal interface reflection at 2 months postoperation . in case 2 , the graft was clear and compact with minimal interface haze at 1 month postoperation . </S>", "<S> femtosecond laser - assisted small incision dlek was safe and technically feasible in our cases ; however , further evaluation is required to determine long - term effects . </S>"], "labels": null, "section_names": ["Purpose", "Methods", "Results", "Case Report", "Surgical Procedure", "Case 1", "Case 2", "Discussion"], "sections": [["to report two cases of femtosecond laser - assisted small incision deep lamellar endothelial keratoplasty ( dlek ) for patients with corneal endothelial decompensation by fuchs dystrophy and glaucoma"], ["irvine , ca ) with 15 khz of repetition rate , was used for a 9.5 mm diameter by 400 m thickness donor corneal lamellar dissection ."], ["in case 1 , the graft was clear and compact without interface haze , orbscan showed smooth and regular corneal surface , specular microscopy was unremarkable without sign of corneal endothelial damage , and optical coherence tomography showed uniform graft well attached to recipient stroma with minimal interface reflection at 2 months postoperation . in case 2 , the graft was clear and compact with minimal interface haze at 1 month postoperation .", "femtosecond laser - assisted small incision dlek was safe and technically feasible in our cases ; however , further evaluation is required to determine long - term effects ."], ["the donor corneoscleral button was mounted in a dedicated artificial anterior chamber ( bausch & lomb , st .", "this was to assure a donor corneal thickness between 500 and 565 m , in order to approximate a graft thickness of 100 to 165 m . the femtosecond laser ( intralase ; intralase corp . , irvine , ca ) was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness .", "femtosecond laser parameter settings were as follows : raster energy 3.6 j , spot separation 10 m , line separation 10 m , side cut energy 3.8 j , spot size 2.4 m , and repetition rate 15 khz .", "after laser application , stromal and side cut adhesions were fully released and the anterior corneal flap was fully lifted .", "the donor corneoscleral button was then placed in medium and transported to the operating room , where the anterior flap was excised with scissors and an 8.0 mm trephine ( katena products , inc . , denville , nj ) was used to punch out the posterior donor corneoscleral button . the recipient corneal lamellar dissection and graft transplantation", "was performed as previously described.5,6 briefly , following an 8.0 mm epithelial marking and 5.0 mm scleral incision , a deep lamellar pocket was created to approximately 75 - 85% of corneal thickness .", "the posterior recipient disc was then excised with cindy scissors ( bausch & lomb ) using the 8.0 mm epithelial mark as a template .", "the excised posterior recipient disc was removed from the lamellar pocket and spread over recipient corneal epithelium to verify size .", "an 8.0 mm pre - prepared posterior donor disc was then folded , endothelial side inside , with a layer of viscoelastic ( healon ; pharmacia , peapack , nj ) coating the endothelium and inserted into the anterior chamber through the small incision using forceps .", "the folded donor disc was opened and attached to the recipient stromal bed by injecting air underneath the graft .", "the scleral wound was closed with 2 or 3 interrupted 10 - 0 nylon sutures and air was replaced with balanced salt solution to normalize intraocular pressure ( iop ) .", "femtosecond laser - assisted small incision dlek was performed in the right eye of a 73-year - old male with fuchs dystrophy ( fig .", "1 ) . preoperative uncorrected visual acuity ( ucva ) and best spectacle corrected visual acuity ( bscva ) was 20/100 and 20/80 respectively and manifest refraction was + 0.75 -1.7530 .", "donor corneoscleral button lamellar dissection was performed using intralase femtosecond laser ( intralase corp . ) , set at a depth of 400 m and flap diameter of 9.5 mm .", "ucva and bscva was 20/150 and 20/100 respectively and manifest refraction was + 2.75 -1.0050 .", "corneal topography showed smooth and regular corneal surface and specular microscopy was unremarkable without sign of corneal endothelial damage ( fig .", "optical coherence tomography ( oct ) findings showed compact and uniform graft , well attached to the recipient stromal bed with minimal interface reflection ( fig .", "3 ) . femtosecond laser - assisted small incision dlek was performed in the left eye of a 94-year - old female with corneal edema , glaucoma , and dry type age - related macular degeneration ( armd ) .", "her preoperative ucva and bscva was 20/400 and 20/200 respectively and her manifest refraction was -1.00 -1.7595 .", "iop was well controlled with topical medication ( cosopt ; merck & co. , whitehouse station , nj ) .", "postoperation , the graft was clear and compact , iop was 19 mmhg , and ucva was 20/400 .", "at one month , the graft was clear and compact with minimal interface haze ; iop measured 31 mmhg .", "anti - glaucoma medication ( alphagan ; allergan , irvine , ca ) was added and topical steroid tapered .", "we were not able to perform further examinations due to follow - up loss at two months postoperation .", "all methods adhered to the declaration of helsinki of the world medical association for research involving human subjects ."], ["the donor corneoscleral button was mounted in a dedicated artificial anterior chamber ( bausch & lomb , st .", "this was to assure a donor corneal thickness between 500 and 565 m , in order to approximate a graft thickness of 100 to 165 m . the femtosecond laser ( intralase ; intralase corp . , irvine , ca ) was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness .", "femtosecond laser parameter settings were as follows : raster energy 3.6 j , spot separation 10 m , line separation 10 m , side cut energy 3.8 j , spot size 2.4 m , and repetition rate 15 khz .", "after laser application , stromal and side cut adhesions were fully released and the anterior corneal flap was fully lifted .", "the donor corneoscleral button was then placed in medium and transported to the operating room , where the anterior flap was excised with scissors and an 8.0 mm trephine ( katena products , inc . , denville , nj ) was used to punch out the posterior donor corneoscleral button . the recipient corneal lamellar dissection and graft transplantation", "was performed as previously described.5,6 briefly , following an 8.0 mm epithelial marking and 5.0 mm scleral incision , a deep lamellar pocket was created to approximately 75 - 85% of corneal thickness .", "the posterior recipient disc was then excised with cindy scissors ( bausch & lomb ) using the 8.0 mm epithelial mark as a template .", "the excised posterior recipient disc was removed from the lamellar pocket and spread over recipient corneal epithelium to verify size .", "an 8.0 mm pre - prepared posterior donor disc was then folded , endothelial side inside , with a layer of viscoelastic ( healon ; pharmacia , peapack , nj ) coating the endothelium and inserted into the anterior chamber through the small incision using forceps .", "the folded donor disc was opened and attached to the recipient stromal bed by injecting air underneath the graft .", "the scleral wound was closed with 2 or 3 interrupted 10 - 0 nylon sutures and air was replaced with balanced salt solution to normalize intraocular pressure ( iop ) ."], ["femtosecond laser - assisted small incision dlek was performed in the right eye of a 73-year - old male with fuchs dystrophy ( fig .", "preoperative uncorrected visual acuity ( ucva ) and best spectacle corrected visual acuity ( bscva ) was 20/100 and 20/80 respectively and manifest refraction was + 0.75 -1.7530 .", "donor corneoscleral button lamellar dissection was performed using intralase femtosecond laser ( intralase corp . ) , set at a depth of 400 m and flap diameter of 9.5 mm .", "ucva and bscva was 20/150 and 20/100 respectively and manifest refraction was + 2.75 -1.0050 .", "corneal topography showed smooth and regular corneal surface and specular microscopy was unremarkable without sign of corneal endothelial damage ( fig .", "2b and c ) . optical coherence tomography ( oct ) findings showed compact and uniform graft , well attached to the recipient stromal bed with minimal interface reflection ( fig ."], ["femtosecond laser - assisted small incision dlek was performed in the left eye of a 94-year - old female with corneal edema , glaucoma , and dry type age - related macular degeneration ( armd ) .", "her preoperative ucva and bscva was 20/400 and 20/200 respectively and her manifest refraction was -1.00 -1.7595 .", "iop was well controlled with topical medication ( cosopt ; merck & co. , whitehouse station , nj ) .", "postoperation , the graft was clear and compact , iop was 19 mmhg , and ucva was 20/400 .", "at one month , the graft was clear and compact with minimal interface haze ; iop measured 31 mmhg .", "anti - glaucoma medication ( alphagan ; allergan , irvine , ca ) was added and topical steroid tapered .", "we were not able to perform further examinations due to follow - up loss at two months postoperation .", "all methods adhered to the declaration of helsinki of the world medical association for research involving human subjects ."], ["posterior keratoplasty refers to removal of diseased posterior corneal layers ( endothelium , descemet 's membrane , and posterior stroma ) and replacement by partial - thickness donor tissue .", "this procedure can be an attractive alternative to penetrating keratoplasty ( pkp ) in treating corneal endothelial diseases such as fuchs dystrophy and aphakic and pseudophakic bullous keratopathy.13 - 15 the endothelial replacement technique via scleral incision was first described by melles and later modified by terry and ousley to ' deep lamellar endothelial keratoplasty ' ( dlek).3,4 recent modification includes introducing the folded donor disc through the a small incision.6 one problem in performing dlek is the technical difficulty associated with manual lamellar dissection .", "it is not only time consuming , but also has the potential risk of corneal perforation.8 moreover , even with uncomplicated dissection , the plane may not be smooth and uniform , which results in interface scarring and reduction in potential best vision.6,7 in a recent paper , azar and coauthors described microkeratome - assisted posterior keratoplasty.16 given that laser - assisted in situ keratomileusis ( lasik ) shows almost no interface haze on the cornea,17,18 this technique 's potential advantage is less interface scarring than manual dissection .", "however , microkeratome - assisted lamellar dissection does have the risk of corneal perforation in deeper dissection .", "recently , the femtosecond laser was introduced in refractive surgery fields for lasik flap creation . according to recent papers", ", it demonstrated improved flap uniformity and better flap thickness predictability than mechanical microkeratomes.9 - 12,19 similar to the microkeratome , femtosecond laser technology has evolved .", "after initial procedures with a 2 khz laser in 1996 , 15 khz ( 2003 ) , 30 khz ( 2005 ) , and soon after , 60 khz ( 2006 ) engines were introduced .", "pulses are scanned and placed in a vertical pattern for trephination ( side ) cuts or in a spiral or raster ( zigzag ) pattern to achieve lamellar cuts .", "the smoothness of optical surfaces is determined by programmable parameters : energy per pulse , separation of adjacent laser spots ( spot separation ) , and raster pattern row spacing ( line separation ) .", "the closer the laser spots , the less energy required.20 the intralase system scans tissue at a repetition rate which depends on laser engine capacity .", "higher repetition rates allow use of lower pulse energy and closer spot / line separation settings , which results in a smoother lamellar interface , an easier anterior corneal flap lift , and a faster procedure .", "the newer 60 khz engine permits a line / spot separation down to 66 m and energy less than 1 j per pulse , which may show superior efficiency for harvesting the posterior corneal disk.21 one concern during preoperative femtosecond laser - assisted donor eye posterior lamellar discs ( plds ) preparation is the possibility of endothelial cell loss ( ecl ) .", "ecl has been reported at 4% after 150 to 200 m thick endothelial side plds preparations , and did not appear to be caused by laser pulse energy.22 another in vitro study reported 4.3% versus 7.7% ecl after 30 khz laser lamellar cutting compared to 15 khz laser for horizontal lamellar cuts at corneal depth of 400 m and 9.5 mm diameter.23 favorable outcomes are consistent with histological results showing adjacent thermal damage to be on the order of 1   m.24 clinical results from case 1 showed relatively intact postoperative endothelium status , which was comparable to previous reports .", "moreover , the femtosecond laser is expected to be applied in full - thickness pkp .", "cuts are customizable to achieve different graft geometric configurations , potentially allowing for sutureless , self - adhesive , or shaped keratoplasty .", "two or more side / lamellar cut segments can be combined to create patterns for shaped keratoplasty , including top - hat ( larger diameter cut posteriorly ) , mushroom ( larger diameter cut anteriorly ) , zigzag , and christmas tree patterns.25 peripheral wound edge shaping can provide stronger healing by increasing surface area , reduce corneal astigmatism by decreasing suture number , reduce donor - host topographic distortion disparity , and quicken visual recovery .", "following we describe small incision dlek by femtosecond laser for donor corneal lamellar dissection . before performing femtosecond laser - assisted donor lamellar dissection , we measured donor corneal thickness by ultrasound pachymetry .", "if corneal thickness was thicker than 565 m , glycerin was applied to dehydrate the cornea , and avoid a thinner graft than intended , as well as to ascertain graft thickness of 100 to 165 m for transplantation .", "the laser was then used to create a corneal flap of 9.5 mm diameter and 400 m thickness .", "afterwards , two possible methods to obtain the posterior donor disc were used . in the first method ,", "this bi - hinged donor corneoscleral button is then placed in medium , transported to the operating room , and punched out with an 8.0 mm trephine .", "separation of the anterior and posterior donor corneal disc follows . in the second method , stromal and side cut adhesions", "this donor corneoscleral button is placed in medium and transported ; following , the anterior flap is excised using scissors and the posterior part is punched out with an 8.0 mm trephine . in our cases", "although the disadvantage is using scissors for anterior cap removal , there are other advantages . by removing the anterior cap ,", "it is easier to visualize the posterior donor button edge , which may reduce a decentered donor cut .", "furthermore , anterior and posterior donor disc separation is much easier , since the anterior flap is fully lifted and removed before trephination .", "case 1 clinical results showed a clear and compact graft without interface haze at two months postoperation .", "these findings were further confirmed in oct , which showed a clear and uniform graft with minimal interface reflection and good graft attachment . despite good clinical findings , postop visual acuity was not as expected .", "however , the patient was satisfied and his vision fluctuation ( previously worse in the morning ) was eliminated .", "considering findings and short - term follow - up , we may expect further improvement with time .", "however , postop visual acuity did not improve because of low visual potential from dry armd and glaucoma .", "unfortunately , this patient had an iop increase at one month postoperation and was unable to follow - up .", "visual acuity after either pkp or dlek is mainly a function of retinal macular potential and cornea optical quality . in studies of pkp in young patients with keratoconus , postoperative visual results", "are uniformly better compared to results in older fuchs ' dystrophy patients , despite similar graft problems of high or irregular astigmatism.26 this suggests that older maculas , may play a role in visual loss after any form of corneal transplantation , including dlek .", "in contrast to pkp where the cornea contributes to refractive visual loss , often resulting from high or irregular astigmatism , in dlek , topography is usually normal and corneal visual loss is most likely attributed to the donor - recipient stromal interface .", "the interface in dlek may account , on average , for 1 line of visual loss compared to the individual macular potential vision.27 other potential corneal factors include increased corneal thickness and posterior corneal curvature .", "how this irregularity influences visual acuity requires further investigation . due to lack of long term follow - up and small case number", ", we can not fully determine potential advantages of ' femtosecond laser - assisted small incision dlek ' .", "however , these cases demonstrate graft uniformity , technical feasibility , and potential application of femtosecond laser in lamellar keratoplasty .", "further evaluation with longer follow - up and more cases would reveal potential advantages . unfortunately , the use of femtosecond laser in recipient corneas is currently limited by the institutional review board .", "also the femtosecond laser is not approved for cuts deeper than 400 m ; therefore recipient cornea posterior lip creation requires scissors .", "hopefully in the near future , we can use the femtosecond laser and perform both donor and recipient corneal lamellar dissection ."]]}
{"article_id": "PMC4363873", "article_text": ["the prevalence of diabetes has explosively increased worldwide , leading to an increase in the number of patients who suffer from diabetic vascular complications such as diabetic nephropathy ( dn ) .", "dn is not only the leading cause of end - stage kidney disease but also a significant risk factor for cardiovascular disease .", "although the treatment for dn is important to improve patients ' prognosis , the current treatment remains suboptimal and therefore novel approaches for dn are urgently needed .", "dipeptidyl peptidase-4 inhibitors ( dpp4i ) have been recently introduced in clinic as a new oral hypoglycemic agent .", "dpp4 is a serine exopeptidase and processes the substrates that have either n - terminal proline or alanine including glucagon - like peptide-1 ( glp-1 ) and glucose - dependent insulinotropic polypeptide ( gip ) known as incretin hormones .", "these incretin hormones are secreted from the small intestine after foods intake and induce the release of insulin from the pancreatic beta cells in the islets of langerhans .", "in addition to the reduction in the blood glucose level , it has been reported that dpp4i possesses properties and can protect the cardiovascular system , kidney , liver , and bone   from injuries .", "since it has been reported that receptors for incretins were not expressed within glomerulus , protective effects of dpp4i for kidney might depend on another substrate for dpp4 .", "for instance , stromal cell - derived factor-1 ( sdf-1 ) , one of the substrates for dpp4 , can be stabilized by dpp4i . because sdf-1 is the most important protein for the recruitment and homing of bone marrow - derived regenerative stem cells , increased levels of sdf-1 through dpp4 inhibition", "has reportedly increased the intragraft number of progenitor cells that had contributed to the recovery from ischemia - reperfusion lung injury throughout the mammalian system .", "it has also been reported that dpp4i decreased the levels of urinary albumin excretion in diabetic patients [ 9 , 10 ] ; however , the mechanism behind how dpp4i ameliorates kidney injuries is not yet clear .", "pituitary adenylate cyclase - activating polypeptide ( pacap ) is one of the substrates of dpp4 .", "pacap belongs to the glucagon superfamily of peptides and was originally purified from sheep hypothalamus in 1989 .", "pacap is able to potentiate cyclic adenosine monophosphate ( camp ) production in pituitary cells and has a diverse array of biological functions , particularly neuroprotective and general cytoprotective roles , such as anti - apoptosis and anti - inflammation .", "although the highest concentrations are observed in the nervous system , a wide variety of tissues , such as heart , pancreas , liver , and kidney , produce pacap ; secreted pacap also has protective effects on the different types of tissues and cells through the three different receptors : pac1 , vpac1 , and vpac2 .", "pacap was found to exist in two forms : 38-amino - acid , a major form , and 27-amino - acid , a short one , truncated at c - terminal and to a much lesser extent in blood stream , respectively .", "recently , it has been reported that dpp4 degraded pacap ( 127 ) and ( 138 ) to form pacap ( 327 ) and ( 338 ) .", "it has been reported that pacap has protective effects in the kidney against various insults , including ischemia / reperfusion injury , drug - induced nephrotoxicity , and myeloma light chain - induced nephropathy .", "pacap treatment in streptozotocin - induced diabetic animals decreased cytokine expression and prevented kidney injuries .", "these results indicated that pacap has protective roles in the kidney ; however , it is unknown which cell types pacap affects and how it decreases cytokine expression .", "therefore , in this study , we investigated the effects of pacap on kidney cells and the mechanisms of how pacap decreases the expression of inflammatory cytokines .", "lipopolysaccharide ( lps ) and u-73122 ( phospholipase c ( plc ) inhibitor ) were obtained from sigma aldrich and h89 ( protein kinase a ( pka ) inhibitor ) was obtained from cell signaling .", "cultured mouse podocytes , transformed by ectopic expression of cyclin - dependent kinase 4 , were kindly provided by dr .", "podocytes were maintained in roswell park memorial institute ( rpmi ) 1640 medium containing 10% fetal bovine serum ( fbs , sigma aldrich , usa ) , penicillin ( 100  u / ml ) , and streptomycin ( 100  u / ml , sigma aldrich , usa ) .", "all procedures were performed in accordance with the guidelines of the research center for animal life science of chiba university of medical science .", "six- to nine - week - old male c57bl/6j mice were purchased from clea japan ( japan ) and housed in cages and maintained on a 12  h light/12  h dark cycle .", "the glomeruli were isolated by dynabeads ( invitrogen , norway ) perfusion technique as previously described .", "the kidney tissues were dissected from the mouse , fixed in oct compound , and stored at 80c until use .", "several 6  m thick frozen sections were prepared and fixed with ice - cold methanol , air - dried for 30  min at room temperature , and then blocked with blocking buffer containing 2% bovine serum albumin ( bsa ) and 0.05% tween-20 in pbs . after washing with tween in pbs ( pbst ; 0.1% tween-20 in pbs ) , the slides were coincubated with vpac1 antibody ( 1  :  50 dilution , santa cruz : sc-30019 ) , podocalyxin ( 1  :  200 dilution , r&d systems : mab1556 ) , and toll - like receptor 4 ( 1  :  50 dilution , santa cruz : sc-10741 )", ". the slides were imaged by the axio observer d1 ( zeiss ) or with a confocal laser scanning microscope ( leica lsm5 pascal ) .", "the podocytes were cultured in lab - tek ii chamber slides ( nalge nunc international ) , deprived of serum for 24  h , and stimulated with 100  ng / ml lps for 1  h. then the podocytes were fixed in ice - cold methanol for 10  min , rinsed with pbs , and incubated in a blocking buffer containing 0.5% bsa and 0.25% tween-20 in pbs for 1  h at room temperature .", "the slides were then incubated with an anti - nuclear factor - kappa b ( nf-b ) antibody ( 1  :  50 , santa cruz : sc-372 ) overnight at 4c , washed several times with pbs , and then incubated with 1  :  1000 dilution of fluorescent - conjugated secondary antibody ( alexa fluor 488 goat anti - rabbit igg , invitrogen ) for 1  h at room temperature .", "the slides were then washed with pbst , nuclear - stained with hoechst 33342 , and mounted with a fluorescence mounting medium . for the evaluation of immunostaining for nf-b ,", "the cells that had accumulated nf-b in their nuclei were counted at 10 randomly selected areas .", "the total rna was extracted using the purelink rna mini kit ( ambion : 12183 - 018a ) according to the manufacturer 's protocols .", "two micrograms of total rna were reverse - transcribed with the superscript iii reverse transcriptase kit ( invitrogen : 18080 ) .", "the complementary dna product was then subjected to pcr using the system with different pairs of oligonucleotide primers that were shown in supplemental table 1 , available online at http://dx.doi.org/10.1155/2015/727152 .", "the cycling conditions were as follows : a denaturation step at 94c for 30  s followed by 30 cycles , annealing at 55c for both pac1 and -actin and 58c for vpac1 for 30 s , and elongation at 72c for 30  s with the final extension at 72c for 7  min .", "the pcr products were separated by gel electrophoresis on a 2% agarose gel with ethidium bromide ; the signals were quantified using a chemidoc mp imagelab pcsystem ( bio - rad ) .", "quantitative pcr was performed in the 7500 fast real - time pcr system ( applied biosystems ) using the fast sybr green master mix ( applied biosystems : 4385612 ) .", "pcr conditions were set for incubation at 95c for 20  s followed by 40 cycles of 3  s at 95c and 30  s at 60c .", "podocytes were lysed in a sds sample buffer containing 0.5  m tris - hcl , 10% sds , glycerol , bromophenol blue , and 3% 2-mercaptoethanol .", "they were boiled at 95c for 10  min , and then the protein was fractionated on 10%15% polyacrylamide gels ( e - pagel , atto corporation , japan ) .", "the protein was transferred to pvdf membranes ( immobilon - p transfer membrane ) ; the membranes were blocked for 1  h at room temperature to block the nonspecific binding of the protein and incubated for 18  h at 4c with primary antibodies .", "the primary antibodies that were used were as follows : anti - vpac1 antibody ( 1  :  200 dilution , santa cruz : sc-30019 ) , anti - phospho - p44/42 mapk ( extracellular signal - regulated kinase ( erk1/2 ) ) antibody ( 1  :  1000 dilution , cell - signaling : number 9106 ) , and anti - p44/42 mapk ( erk1/2 ) antibody ( 1  :  1000 dilution , cell - signaling : number 9102 ) .", "the blots were then washed and incubated with second antibodies , peroxidase - conjugated anti - rabbit immunoglobulins ( 1  :  2500 dilution , ge healthcare ) , or goat anti - mouse igg - hrp ( 1  :  2500 dilution , santa cruz : sc-2055 ) for 1  h at room temperature .", "after washing for several times , the antibody binding sites were visualized using an ecl western blotting detection system ( ge healthcare : rpn2106 ) .", "the blots were quantified using a chemidoc mp imagelab pcsystem ( bio - rad ) .", "podocytes were plated in 96-well dishes at 50% confluency and transfected with 10  ng per well camp response element- ( cre- ) lux construct that contained consecutive camp response element by lipofectamine ltx and plus reagents ( invitrogen ) . at", "six hours after transfection , cells were deprived of serum for 48  h. and then , the cells were stimulated with pacap for 1  h. the luciferase activities in the cell lysate were measured in a 1420 arvo sx multilabel counter ( wallac , inc . , gaithersburg , md ) using the dual luciferase reporter assay system ( promega , madison , wi ) .", "to correct for potential variation in transfection efficiency , prl - tk vector ( promega ) was cotransfected in all experiments .", "statistical analyses were done using sas 9.3 and/or microsoft office excel by student 's unpaired t - test .", "pacap works as a ligand and binds with specific receptors in order to transduce intracellular signals .", "therefore , we first examined in which cell types pacap transduces intracellular signal within the glomeruli .", "reverse transcriptase pcr revealed that mrna for vpac1 but not pac1 was detected in glomeruli as shown in figure 1(a ) .", "immunohistochemistry revealed that vpac1 was primarily expressed in podocalyxin - positive cells ( figures 1(b ) and 1(c ) ) .", "complete absence of signal was observed when primary antibodies were omitted ( data not shown ) .", "rt - pcr ( figure 1(a ) ) and western blotting ( figure 1(d ) ) also confirmed that the vpac1 mrna and protein were expressed not only in isolated glomeruli but also in cultured podocytes .", "because vpac1 , a pacap receptor , was expressed on podocytes , we next examined whether pacap acted on podocytes .", "it has been reported that pacap binds with vpac1 , which is coupled with gs protein and induces rapid camp production , which ultimately activates the pka pathway .", "ten nm pacap significantly increased the cellular contents of camp in a time dependent manner ( supplemental figure 1 ) . increased levels of camp in the presence of pacap", "was associated with increased levels of camp responsive element promoter activities ( figure 2(a ) ) and phosphorylated camp response element binding protein ( creb ) ( figure 2(b ) ) .", "these results indicated that pacap primarily sent signals to glomerular podocytes , especially activating the camp / pka pathway in the podocytes .", "it has been reported that pacap also played an anti - inflammatory role in peripheral and central tissues .", "toll - like receptors ( tlrs ) are the principal mediators of innate immunity and are reportedly activated by bacterial endotoxins .", "the tlr4 proteins were localized to podocytes and endothelial cells by immunohistochemistry as shown in figure 3 .", "as shown in figure 4 , lps , a ligand of tlr4 , significantly increased the expression of il-6 and mcp-1 . in the presence of pacap , the increased expressions of il-6 and mcp-1 were significantly attenuated .", "the expression of tlr2 , tlr4 , and myeloid differentiation primary response gene 88 ( myd88 ) , a tlr adaptor protein , but not that of il-1 receptor associated kinase-1 ( irak1 ) , a down steam signaling molecule of myd88 , was decreased in the presence of pacap .", "it has been reported that pacap activates not only adenylate cyclase ( ac ) , which eventually activates the pka signaling pathway , but also plc , which leads to an increase in intracellular calcium signaling .", "then , we examined the effect of h89 , an inhibitor of the pka signaling pathway , and of u-73122 , a known plc inhibitor , on the increased levels of mcp-1 .", "mcp-1 expression was reversed by h-89 but not by u-73122 as shown in figure 5 .", "these results indicated that pacap suppressed the expression of mcp-1 through the camp / pka - dependent signaling pathway .", "it has been reported that lps can activate tlr4 and subsequently the myd88 transfers signals by activating nf-b and mitogen - activated protein kinase , which eventually results in the expression of proinflammatory cytokines .", "therefore , we examined the effects of pacap on the nf-b transnuclear localization and phosphorylation of erk .", "figures 6 and 7 showed that , in the presence of lps , nf-b transnuclear localization and phosphorylation of erk were significantly increased and pacap significantly ameliorated both .", "in the present study , we reported that vpac1 , a pacap receptor , was exclusively expressed in glomerular podocytes .", "pacap , a substrate for dpp4 , activated the camp / pka signaling pathway in cultured podocytes and inhibited the expression of inflammatory cytokines , which were induced by lps / tlr4 signaling .", "pacap was first identified 25 years ago and has become one of the most studied neuropeptides [ 11 , 12 ] .", "pacap is expressed not only throughout the nervous system but also in peripheral tissues , such as the gastrointestinal tract , the endocrine tissues , and the urinary tract .", "in addition to widespread expression of pacap , it reportedly has a variety of biological functions that are primarily neuroprotective and general cytoprotective roles through the specific receptors , such as pac1 , vpac1 , and vpac2 .", "for instance , pacap has protected the kidney from ischemia - induced kidney injuries , myeloma injuries , cisplatin - induced renal failure , cyclosporine a - induced nephrotoxicity , and dn .", "these renoprotective actions appeared to depend on the anti - inflammatory actions on circulating cells , glomerular cells , and tubular cells .", "the kidney is injured by a wide variety of insults leading to chronic kidney diseases ( ckd ) . among ckd", "reported that pacap - treated streptozotocin - induced diabetic mice had fewer histological changes , such as pas - positive areas within the glomeruli , tubular damage , and arteriolar hyalinosis compared with the controls .", "pacap treatment also decreased the expression of a number of cytokines that were upregulated in diabetic conditions .", "however , the specific cell types that are affected by pacap directly have not been reported .", "jean cr reported that vpac1 , but not pac1 and vpac2 , was expressed in human glomeruli . in agreement with this previous report", ", we found that vpac1 was expressed within the glomerulus and localized its expression to podocytes .", "recent studies of podocyte - expressed genes have considerably enhanced our knowledge of the molecular mechanisms of glomerular filtration . a number of podocyte - expressed genes , such as nephrotic syndrome type-1 ( nphs-1 ) , nephrotic syndrome type-2 ( nphs-2 ) ; actin - related proteins such as -actinin 4 , inverted formin 2 , and cd2-associated protein ( cd2ap ) ; and cytoplasmic signaling molecules , such as phospholipase c , epsilon-1 ( plce1 ) and transient receptor potential cation channel , subfamily c , member 6 ( trpc6 ) , were all involved in maintaining the cytoskeletal dynamics .", "therefore , among the glomerular cells , the podocytes are becoming the most highlighted cell type in the field of glomerular research of most glomerular diseases .", "thus , it is intriguing that pacap may give signals to podocytes . because it has been reported that pacap has anti - inflammatory effects in a wide variety of disease models , we focused on the anti - inflammatory effects of pacap on cultured podocytes . among the inflammatory signals ,", "tlr , a sensor in the innate immune system , has been highlighted in the development of dn .", "it has been reported that tlr2 and tlr4 have been expressed in podocytes and activated in diabetic conditions .", "activated tlrs have been reported to induce proinflammatory cytokines in podocytes and a disorganized podocyte cytoskeletal structure   which eventually led to proteinuria .", "therefore , our finding about the inhibition of podocytes ' tlr - related signaling in the presence pacap is significant in terms of treating dn .", "indeed , it has been recently reported that the treatment of pacap effectively counteracted diabetes induced podocyte injury in vivo .", "tlr is a conserved family of pattern recognition receptors , which is triggered by microbial pathogens , fatty acids , uric acids , oxidative stress , and high glucose .", "when tlr is activated , it recruits different adaptor molecules , such as myd88 and irak-1 . activated myd88-dependent or myd88-independent pathway engages the activation of erk and nf-b signaling , which results in inflammatory cytokines . because pacap attenuated both activation of erk and nf-b in our case", "it has reported that administration of pacap attenuated the expression of tlrs and its adaptor protein in kidney . in agreement with these previous reports , lps - induced inflammation", "related genes , such as mcp-1 and il-6 , and tlr signals related genes , such as tlr2 , tlr4 , and myd88 but not irak-1 were suppressed by pacap in this study .", "it has been reported that lps - induced expression of tlr2 has been suppressed by ciprofloxacin through the production of prostaglandin ( pg ) e2 but not through pka in monocyte / macrophages . because pge2 was reportedly induced in the presence of pacap , ns398 , an inhibitor of pge2 , was tested and was not able to reverse the effects of pacap , which attenuated the tlr4 expression ( data not shown ) .", "thus , the precise mechanisms of how pacap inhibits the expression of tlrs and myd88 need to be further analyzed .", "dpp4i has been introduced in clinic and has become one of the most promising options to treat diabetic patients owing to their effectiveness in glucose lowering and low risk of hypoglycemia and weight gain .", "dpp4i stabilizes its substrates and incretins ( glp-1 and gip ) , which are the main substrates for lowering blood glucose . beyond the hypoglycemic action", ", it has been reported that dpp4i has numerous potential benefits in diabetic vascular complications , including dn .", "the possible renoprotective effects of dpp4i include the reduction of oxidative stress and anti - inflammation and the improvement of endothelial dysfunctions through incretin - dependent and -independent pathways .", "since it has been reported that glp-1 receptor was not detected in glomerulus   and we were also able to detect neither glp-1 receptor nor gip receptor both in glomerulus and cultured podocyte ( supplemental figure 2 : expression of glp-1 receptor and gip receptor were evaluated by rt - pcr ) , incretin independent pathway might have roles in protecting against dn .", "pacap is n - terminally truncated by dpp4 , and it has been reported that dpp4-degraded pacap loses its insulinotropic effects . because the n - terminal of pacap is a high - affinity site for vpac1 binding , dpp4-degraded pacap may reduce the signals through vpac1 .", "we also confirmed that the treatment of linagliptin , dpp4i , inhibited the degradation of pacap which secreted from cultured cells ( supplemental figure 3 : linagliptin protect pacap from degradation ) . in this study", ", we demonstrated that pacap had anti - inflammatory effects on podocytes , leading to an assumption that dpp4i protects kidney from injuries through the stabilization of pacap .", "nevertheless , we have not confirmed that pacap has protective roles in vivo . because the half - life of pacap injected into mice and humans is between 2 and 10  min due to enzymatic degradation", ", truncated pacap is required to be produced in order to get pacap more stable in vivo without losing receptor activating effects .", "in conclusion , we have shown that pacap has anti - inflammatory effects on glomerular podocytes . because treatment options for dn are still limited , pacap may be a good candidate for prevention / attenuation of dn . however , more study is definitely needed to prove this possibility ."], "abstract_text": ["<S> diabetic nephropathy ( dn ) is a leading cause of end - stage kidney disease ; however , there are few treatment options . </S>", "<S> inflammation plays a crucial role in the initiation and/or progression of dn . </S>", "<S> pituitary adenylate cyclase - activating polypeptide ( pacap ) is a neuropeptide , which was originally isolated from the ovine hypothalamus and reportedly has diverse biological functions . </S>", "<S> it has been reported that pacap has renoprotective effects in different models of kidney pathology . however , the specific cell types within the kidney that are protected by pacap have not yet been reported . in this study , we localized vpac1 , one of the pacap receptors , to glomerular podocytes , which also reportedly has crucial roles not only in glomerular physiology but also in pathology . </S>", "<S> pacap was effective in the downregulation of proinflammatory cytokines , such as monocyte chemoattractant protein-1 ( mcp-1 ) and interleukin-6 , which had been induced by the activation of toll - like receptor ( tlr ) with lipopolysaccharide . </S>", "<S> pacap also had downregulated the expression of mcp-1 through the protein kinase a signaling pathway ; this led to the attenuation of the activation of extracellular signal - regulated kinase and nuclear factor - kappa b signaling . </S>", "<S> our results suggested that pacap could be a possible treatment option for dn through the use of anti - inflammation effects on glomerular podocytes . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["the prevalence of diabetes has explosively increased worldwide , leading to an increase in the number of patients who suffer from diabetic vascular complications such as diabetic nephropathy ( dn ) .", "dn is not only the leading cause of end - stage kidney disease but also a significant risk factor for cardiovascular disease .", "although the treatment for dn is important to improve patients ' prognosis , the current treatment remains suboptimal and therefore novel approaches for dn are urgently needed .", "dipeptidyl peptidase-4 inhibitors ( dpp4i ) have been recently introduced in clinic as a new oral hypoglycemic agent .", "dpp4 is a serine exopeptidase and processes the substrates that have either n - terminal proline or alanine including glucagon - like peptide-1 ( glp-1 ) and glucose - dependent insulinotropic polypeptide ( gip ) known as incretin hormones .", "these incretin hormones are secreted from the small intestine after foods intake and induce the release of insulin from the pancreatic beta cells in the islets of langerhans .", "in addition to the reduction in the blood glucose level , it has been reported that dpp4i possesses properties and can protect the cardiovascular system , kidney , liver , and bone   from injuries .", "since it has been reported that receptors for incretins were not expressed within glomerulus , protective effects of dpp4i for kidney might depend on another substrate for dpp4 .", "for instance , stromal cell - derived factor-1 ( sdf-1 ) , one of the substrates for dpp4 , can be stabilized by dpp4i . because sdf-1 is the most important protein for the recruitment and homing of bone marrow - derived regenerative stem cells , increased levels of sdf-1 through dpp4 inhibition", "has reportedly increased the intragraft number of progenitor cells that had contributed to the recovery from ischemia - reperfusion lung injury throughout the mammalian system .", "it has also been reported that dpp4i decreased the levels of urinary albumin excretion in diabetic patients [ 9 , 10 ] ; however , the mechanism behind how dpp4i ameliorates kidney injuries is not yet clear .", "pituitary adenylate cyclase - activating polypeptide ( pacap ) is one of the substrates of dpp4 .", "pacap belongs to the glucagon superfamily of peptides and was originally purified from sheep hypothalamus in 1989 .", "pacap is able to potentiate cyclic adenosine monophosphate ( camp ) production in pituitary cells and has a diverse array of biological functions , particularly neuroprotective and general cytoprotective roles , such as anti - apoptosis and anti - inflammation .", "although the highest concentrations are observed in the nervous system , a wide variety of tissues , such as heart , pancreas , liver , and kidney , produce pacap ; secreted pacap also has protective effects on the different types of tissues and cells through the three different receptors : pac1 , vpac1 , and vpac2 .", "pacap was found to exist in two forms : 38-amino - acid , a major form , and 27-amino - acid , a short one , truncated at c - terminal and to a much lesser extent in blood stream , respectively .", "recently , it has been reported that dpp4 degraded pacap ( 127 ) and ( 138 ) to form pacap ( 327 ) and ( 338 ) .", "it has been reported that pacap has protective effects in the kidney against various insults , including ischemia / reperfusion injury , drug - induced nephrotoxicity , and myeloma light chain - induced nephropathy .", "pacap treatment in streptozotocin - induced diabetic animals decreased cytokine expression and prevented kidney injuries .", "these results indicated that pacap has protective roles in the kidney ; however , it is unknown which cell types pacap affects and how it decreases cytokine expression .", "therefore , in this study , we investigated the effects of pacap on kidney cells and the mechanisms of how pacap decreases the expression of inflammatory cytokines ."], ["lipopolysaccharide ( lps ) and u-73122 ( phospholipase c ( plc ) inhibitor ) were obtained from sigma aldrich and h89 ( protein kinase a ( pka ) inhibitor ) was obtained from cell signaling .", "cultured mouse podocytes , transformed by ectopic expression of cyclin - dependent kinase 4 , were kindly provided by dr .", "podocytes were maintained in roswell park memorial institute ( rpmi ) 1640 medium containing 10% fetal bovine serum ( fbs , sigma aldrich , usa ) , penicillin ( 100  u / ml ) , and streptomycin ( 100  u / ml , sigma aldrich , usa ) .", "all procedures were performed in accordance with the guidelines of the research center for animal life science of chiba university of medical science .", "six- to nine - week - old male c57bl/6j mice were purchased from clea japan ( japan ) and housed in cages and maintained on a 12  h light/12  h dark cycle .", "the glomeruli were isolated by dynabeads ( invitrogen , norway ) perfusion technique as previously described .", "the kidney tissues were dissected from the mouse , fixed in oct compound , and stored at 80c until use .", "several 6  m thick frozen sections were prepared and fixed with ice - cold methanol , air - dried for 30  min at room temperature , and then blocked with blocking buffer containing 2% bovine serum albumin ( bsa ) and 0.05% tween-20 in pbs . after washing with tween in pbs ( pbst ; 0.1% tween-20 in pbs ) , the slides were coincubated with vpac1 antibody ( 1  :  50 dilution , santa cruz : sc-30019 ) , podocalyxin ( 1  :  200 dilution , r&d systems : mab1556 ) , and toll - like receptor 4 ( 1  :  50 dilution , santa cruz : sc-10741 )", ". the slides were imaged by the axio observer d1 ( zeiss ) or with a confocal laser scanning microscope ( leica lsm5 pascal ) .", "the podocytes were cultured in lab - tek ii chamber slides ( nalge nunc international ) , deprived of serum for 24  h , and stimulated with 100  ng / ml lps for 1  h. then the podocytes were fixed in ice - cold methanol for 10  min , rinsed with pbs , and incubated in a blocking buffer containing 0.5% bsa and 0.25% tween-20 in pbs for 1  h at room temperature .", "the slides were then incubated with an anti - nuclear factor - kappa b ( nf-b ) antibody ( 1  :  50 , santa cruz : sc-372 ) overnight at 4c , washed several times with pbs , and then incubated with 1  :  1000 dilution of fluorescent - conjugated secondary antibody ( alexa fluor 488 goat anti - rabbit igg , invitrogen ) for 1  h at room temperature .", "the slides were then washed with pbst , nuclear - stained with hoechst 33342 , and mounted with a fluorescence mounting medium . for the evaluation of immunostaining for nf-b ,", "the cells that had accumulated nf-b in their nuclei were counted at 10 randomly selected areas .", "the total rna was extracted using the purelink rna mini kit ( ambion : 12183 - 018a ) according to the manufacturer 's protocols .", "two micrograms of total rna were reverse - transcribed with the superscript iii reverse transcriptase kit ( invitrogen : 18080 ) .", "the complementary dna product was then subjected to pcr using the system with different pairs of oligonucleotide primers that were shown in supplemental table 1 , available online at http://dx.doi.org/10.1155/2015/727152 .", "the cycling conditions were as follows : a denaturation step at 94c for 30  s followed by 30 cycles , annealing at 55c for both pac1 and -actin and 58c for vpac1 for 30 s , and elongation at 72c for 30  s with the final extension at 72c for 7  min .", "the pcr products were separated by gel electrophoresis on a 2% agarose gel with ethidium bromide ; the signals were quantified using a chemidoc mp imagelab pcsystem ( bio - rad ) .", "quantitative pcr was performed in the 7500 fast real - time pcr system ( applied biosystems ) using the fast sybr green master mix ( applied biosystems : 4385612 ) .", "pcr conditions were set for incubation at 95c for 20  s followed by 40 cycles of 3  s at 95c and 30  s at 60c .", "podocytes were lysed in a sds sample buffer containing 0.5  m tris - hcl , 10% sds , glycerol , bromophenol blue , and 3% 2-mercaptoethanol .", "they were boiled at 95c for 10  min , and then the protein was fractionated on 10%15% polyacrylamide gels ( e - pagel , atto corporation , japan ) .", "the protein was transferred to pvdf membranes ( immobilon - p transfer membrane ) ; the membranes were blocked for 1  h at room temperature to block the nonspecific binding of the protein and incubated for 18  h at 4c with primary antibodies .", "the primary antibodies that were used were as follows : anti - vpac1 antibody ( 1  :  200 dilution , santa cruz : sc-30019 ) , anti - phospho - p44/42 mapk ( extracellular signal - regulated kinase ( erk1/2 ) ) antibody ( 1  :  1000 dilution , cell - signaling : number 9106 ) , and anti - p44/42 mapk ( erk1/2 ) antibody ( 1  :  1000 dilution , cell - signaling : number 9102 ) .", "the blots were then washed and incubated with second antibodies , peroxidase - conjugated anti - rabbit immunoglobulins ( 1  :  2500 dilution , ge healthcare ) , or goat anti - mouse igg - hrp ( 1  :  2500 dilution , santa cruz : sc-2055 ) for 1  h at room temperature .", "after washing for several times , the antibody binding sites were visualized using an ecl western blotting detection system ( ge healthcare : rpn2106 ) .", "the blots were quantified using a chemidoc mp imagelab pcsystem ( bio - rad ) .", "podocytes were plated in 96-well dishes at 50% confluency and transfected with 10  ng per well camp response element- ( cre- ) lux construct that contained consecutive camp response element by lipofectamine ltx and plus reagents ( invitrogen ) . at", "six hours after transfection , cells were deprived of serum for 48  h. and then , the cells were stimulated with pacap for 1  h. the luciferase activities in the cell lysate were measured in a 1420 arvo sx multilabel counter ( wallac , inc . , gaithersburg , md ) using the dual luciferase reporter assay system ( promega , madison , wi ) .", "to correct for potential variation in transfection efficiency , prl - tk vector ( promega ) was cotransfected in all experiments .", "statistical analyses were done using sas 9.3 and/or microsoft office excel by student 's unpaired t - test ."], ["pacap works as a ligand and binds with specific receptors in order to transduce intracellular signals .", "therefore , we first examined in which cell types pacap transduces intracellular signal within the glomeruli .", "reverse transcriptase pcr revealed that mrna for vpac1 but not pac1 was detected in glomeruli as shown in figure 1(a ) .", "immunohistochemistry revealed that vpac1 was primarily expressed in podocalyxin - positive cells ( figures 1(b ) and 1(c ) ) .", "complete absence of signal was observed when primary antibodies were omitted ( data not shown ) .", "rt - pcr ( figure 1(a ) ) and western blotting ( figure 1(d ) ) also confirmed that the vpac1 mrna and protein were expressed not only in isolated glomeruli but also in cultured podocytes .", "because vpac1 , a pacap receptor , was expressed on podocytes , we next examined whether pacap acted on podocytes .", "it has been reported that pacap binds with vpac1 , which is coupled with gs protein and induces rapid camp production , which ultimately activates the pka pathway .", "ten nm pacap significantly increased the cellular contents of camp in a time dependent manner ( supplemental figure 1 ) . increased levels of camp in the presence of pacap", "was associated with increased levels of camp responsive element promoter activities ( figure 2(a ) ) and phosphorylated camp response element binding protein ( creb ) ( figure 2(b ) ) .", "these results indicated that pacap primarily sent signals to glomerular podocytes , especially activating the camp / pka pathway in the podocytes .", "it has been reported that pacap also played an anti - inflammatory role in peripheral and central tissues .", "toll - like receptors ( tlrs ) are the principal mediators of innate immunity and are reportedly activated by bacterial endotoxins .", "the tlr4 proteins were localized to podocytes and endothelial cells by immunohistochemistry as shown in figure 3 .", "as shown in figure 4 , lps , a ligand of tlr4 , significantly increased the expression of il-6 and mcp-1 . in the presence of pacap , the increased expressions of il-6 and mcp-1 were significantly attenuated .", "the expression of tlr2 , tlr4 , and myeloid differentiation primary response gene 88 ( myd88 ) , a tlr adaptor protein , but not that of il-1 receptor associated kinase-1 ( irak1 ) , a down steam signaling molecule of myd88 , was decreased in the presence of pacap .", "it has been reported that pacap activates not only adenylate cyclase ( ac ) , which eventually activates the pka signaling pathway , but also plc , which leads to an increase in intracellular calcium signaling .", "then , we examined the effect of h89 , an inhibitor of the pka signaling pathway , and of u-73122 , a known plc inhibitor , on the increased levels of mcp-1 .", "mcp-1 expression was reversed by h-89 but not by u-73122 as shown in figure 5 .", "these results indicated that pacap suppressed the expression of mcp-1 through the camp / pka - dependent signaling pathway .", "it has been reported that lps can activate tlr4 and subsequently the myd88 transfers signals by activating nf-b and mitogen - activated protein kinase , which eventually results in the expression of proinflammatory cytokines .", "therefore , we examined the effects of pacap on the nf-b transnuclear localization and phosphorylation of erk .", "figures 6 and 7 showed that , in the presence of lps , nf-b transnuclear localization and phosphorylation of erk were significantly increased and pacap significantly ameliorated both ."], ["in the present study , we reported that vpac1 , a pacap receptor , was exclusively expressed in glomerular podocytes .", "pacap , a substrate for dpp4 , activated the camp / pka signaling pathway in cultured podocytes and inhibited the expression of inflammatory cytokines , which were induced by lps / tlr4 signaling .", "pacap was first identified 25 years ago and has become one of the most studied neuropeptides [ 11 , 12 ] .", "pacap is expressed not only throughout the nervous system but also in peripheral tissues , such as the gastrointestinal tract , the endocrine tissues , and the urinary tract .", "in addition to widespread expression of pacap , it reportedly has a variety of biological functions that are primarily neuroprotective and general cytoprotective roles through the specific receptors , such as pac1 , vpac1 , and vpac2 .", "for instance , pacap has protected the kidney from ischemia - induced kidney injuries , myeloma injuries , cisplatin - induced renal failure , cyclosporine a - induced nephrotoxicity , and dn .", "these renoprotective actions appeared to depend on the anti - inflammatory actions on circulating cells , glomerular cells , and tubular cells .", "the kidney is injured by a wide variety of insults leading to chronic kidney diseases ( ckd ) . among ckd", "reported that pacap - treated streptozotocin - induced diabetic mice had fewer histological changes , such as pas - positive areas within the glomeruli , tubular damage , and arteriolar hyalinosis compared with the controls .", "pacap treatment also decreased the expression of a number of cytokines that were upregulated in diabetic conditions .", "however , the specific cell types that are affected by pacap directly have not been reported .", "jean cr reported that vpac1 , but not pac1 and vpac2 , was expressed in human glomeruli . in agreement with this previous report", ", we found that vpac1 was expressed within the glomerulus and localized its expression to podocytes .", "recent studies of podocyte - expressed genes have considerably enhanced our knowledge of the molecular mechanisms of glomerular filtration . a number of podocyte - expressed genes , such as nephrotic syndrome type-1 ( nphs-1 ) , nephrotic syndrome type-2 ( nphs-2 ) ; actin - related proteins such as -actinin 4 , inverted formin 2 , and cd2-associated protein ( cd2ap ) ; and cytoplasmic signaling molecules , such as phospholipase c , epsilon-1 ( plce1 ) and transient receptor potential cation channel , subfamily c , member 6 ( trpc6 ) , were all involved in maintaining the cytoskeletal dynamics .", "therefore , among the glomerular cells , the podocytes are becoming the most highlighted cell type in the field of glomerular research of most glomerular diseases .", "thus , it is intriguing that pacap may give signals to podocytes . because it has been reported that pacap has anti - inflammatory effects in a wide variety of disease models , we focused on the anti - inflammatory effects of pacap on cultured podocytes . among the inflammatory signals ,", "tlr , a sensor in the innate immune system , has been highlighted in the development of dn .", "it has been reported that tlr2 and tlr4 have been expressed in podocytes and activated in diabetic conditions .", "activated tlrs have been reported to induce proinflammatory cytokines in podocytes and a disorganized podocyte cytoskeletal structure   which eventually led to proteinuria .", "therefore , our finding about the inhibition of podocytes ' tlr - related signaling in the presence pacap is significant in terms of treating dn .", "indeed , it has been recently reported that the treatment of pacap effectively counteracted diabetes induced podocyte injury in vivo .", "tlr is a conserved family of pattern recognition receptors , which is triggered by microbial pathogens , fatty acids , uric acids , oxidative stress , and high glucose .", "when tlr is activated , it recruits different adaptor molecules , such as myd88 and irak-1 . activated myd88-dependent or myd88-independent pathway engages the activation of erk and nf-b signaling , which results in inflammatory cytokines . because pacap attenuated both activation of erk and nf-b in our case", "it has reported that administration of pacap attenuated the expression of tlrs and its adaptor protein in kidney . in agreement with these previous reports , lps - induced inflammation", "related genes , such as mcp-1 and il-6 , and tlr signals related genes , such as tlr2 , tlr4 , and myd88 but not irak-1 were suppressed by pacap in this study .", "it has been reported that lps - induced expression of tlr2 has been suppressed by ciprofloxacin through the production of prostaglandin ( pg ) e2 but not through pka in monocyte / macrophages . because pge2 was reportedly induced in the presence of pacap , ns398 , an inhibitor of pge2 , was tested and was not able to reverse the effects of pacap , which attenuated the tlr4 expression ( data not shown ) .", "thus , the precise mechanisms of how pacap inhibits the expression of tlrs and myd88 need to be further analyzed .", "dpp4i has been introduced in clinic and has become one of the most promising options to treat diabetic patients owing to their effectiveness in glucose lowering and low risk of hypoglycemia and weight gain .", "dpp4i stabilizes its substrates and incretins ( glp-1 and gip ) , which are the main substrates for lowering blood glucose . beyond the hypoglycemic action", ", it has been reported that dpp4i has numerous potential benefits in diabetic vascular complications , including dn .", "the possible renoprotective effects of dpp4i include the reduction of oxidative stress and anti - inflammation and the improvement of endothelial dysfunctions through incretin - dependent and -independent pathways .", "since it has been reported that glp-1 receptor was not detected in glomerulus   and we were also able to detect neither glp-1 receptor nor gip receptor both in glomerulus and cultured podocyte ( supplemental figure 2 : expression of glp-1 receptor and gip receptor were evaluated by rt - pcr ) , incretin independent pathway might have roles in protecting against dn .", "pacap is n - terminally truncated by dpp4 , and it has been reported that dpp4-degraded pacap loses its insulinotropic effects . because the n - terminal of pacap is a high - affinity site for vpac1 binding , dpp4-degraded pacap may reduce the signals through vpac1 .", "we also confirmed that the treatment of linagliptin , dpp4i , inhibited the degradation of pacap which secreted from cultured cells ( supplemental figure 3 : linagliptin protect pacap from degradation ) . in this study", ", we demonstrated that pacap had anti - inflammatory effects on podocytes , leading to an assumption that dpp4i protects kidney from injuries through the stabilization of pacap .", "nevertheless , we have not confirmed that pacap has protective roles in vivo . because the half - life of pacap injected into mice and humans is between 2 and 10  min due to enzymatic degradation", ", truncated pacap is required to be produced in order to get pacap more stable in vivo without losing receptor activating effects ."], ["in conclusion , we have shown that pacap has anti - inflammatory effects on glomerular podocytes . because treatment options for dn are still limited , pacap may be a good candidate for prevention / attenuation of dn . however , more study is definitely needed to prove this possibility ."]]}
{"article_id": "PMC5314861", "article_text": ["opsoclonus - myoclonus - ataxia syndrome ( omas ) , also called  kinsbourne syndrome  or ", "dancing eye syndrome ,  is a serious , rare , and often chronic neurological disorder .", "omas consists of three main symptoms : opsoclonus ( conjugate , multidirectional , chaotic eye movements ) , myoclonus ( nonepileptic limb jerking that can also involve the head and face ) and truncal ataxia , which cause gait imbalance .", "omas is generally a paraneoplastic or parainfectious entity , but in children , it is most commonly associated with occult neuroblastoma ( nb ) in about 50% of cases and between 2% and 3% of children with nb have omas .", "although most patients with nb and omas have good survival rates , 70%80% of these children will have long - term neurologic sequelae . in pediatric age , omas may be associated with neuroblastic tumors ( nb , ganglioneuroblastoma , or ganglioneuroma ) .", "the diagnosis of omas may be difficult in some patients and should be considered even when only some of the features are present .", "international consensus has described three of the following four diagnostic criteria should be present to describe the typical syndrome : ( 1 ) opsoclonus , ( 2 ) myoclonus / ataxia , ( 3 ) behavioral change and/or sleep disturbance , and ( 4 ) nb . we present a retrospective study of five children presented to the pediatric oncology clinic ( poc ) of all india institute of medical sciences ( aiims ) with a diagnosis of opsoclonus - myoclonus syndrome ( oms ) .", "the objective of this study was to describe the clinical profile and outcome of this disorder .", "the medical records of all children presented to poc , department of pediatrics , aiims , with a diagnosis of opsoclonus - myoclonus were retrieved and reviewed .", "the diagnosis of opsoclonus - myoclonus was based on a constellation of any three of the four clinical features : opsoclonus , myoclonus , ataxia , and encephalopathy / irritability / behavioral change / sleep disturbance .", "outcome was assessed on follow - up by direct assessment and by telephonic communication ( in one patient ) .", "a total of six patients with a diagnosis of opsoclonus - myoclonus were admitted over 4-year period [ table 1 ] .", "opsoclonus , myoclonus , ataxia , and encephalopathy / behavioral abnormalities ( irritability / sleep disturbance ) were present in all children at presentation .", "the duration of symptoms at the time of presentation was in the range of 610 months .", "clinical profile and outcome in six children with opsoclonus - myoclonus syndrome the reasons for the delay in diagnosis included misdiagnosis by peripheral physicians and delayed referral to our center .", "all children had paraneoplastic opsoclonus - myoclonus ( two left paravertebral ganglioneuroblastoma , two left paravertebral nb , one left paravertebral plus left psoas muscle ganglioneuroblastoma , and one right paravertebral ganglioneuroblastoma ) [ figures 13 ] .", "the diagnosis of tumor was made on contrast - enhanced computed tomography scan ( ct ) and magnetic resonance imaging ( mri ) of the chest , abdomen , and pelvis ( 3 ct scan and 3 mri ) .", "tests for urinary excretion of vanillylmandelic acid ( vma ) were negative in all cases .", "iodine-131 metaiodobenzylguanidine scintigraphy scan ( i-131 mibg ) was performed in all cases , but it detected mibg concentrating tumor only in two cases .", "( case 2 ) ( original ) axial section showing well - defined enhancing soft - tissue mass lesion ( 3.5 cm  2 cm  1.4 cm ) seen in the left upper psoas muscle extending into left neural foramen in between l2 and l3 vertebra up to dura mater ( a and b ) ( case 3 ) axial section and coronal section showing right paravertebral mass of size 1.5 cm  0.6 cm  2.4 cm at the level of d6 to d8 vertebral body ( a and b ) ( case 5 ) axial and coronal section showing well - defined left paravertebral mass lesion 2 cm  1.8 cm  2.9 cm at the level of d6 to d8 vertebral body an initial diagnosis of omas was made in all children .", "three children received injection adrenocorticotropic hormone ( acth ) ; two children received injection acth plus injection methylprednisolone followed by oral steroids over 4 weeks .", "one child ( case 1 ) also received intravenous immunoglobulin ( ivig ) and oral clonazepam and risperidone in view of abnormal behavior ( excessive irritability , biting , and head banging ) .", "one child ( case 5 ) had spontaneous improvement and complete recovery without specific immunomodulator therapy .", "of the six cases with paraneoplastic oms , four cases were treated with surgical resection of tumor and chemotherapy . in one child (", "case 5 ) , treatment was deferred by parents but on telephonic communication that the child was symptoms free and healthy at 9 months after presentation to us . in another case (", "case 6 ) , she received only injection acth , surgery , and chemotherapy were denied by parents .", "after follow - up of 24 months , the child was asymptomatic , but ct of the abdomen revealed mass of same size that was documented 2 years back .", "one child ( case 3 ) died of febrile encephalopathy ( not related to disease ) .", "other names for oms include omas , paraneoplastic opsoclonus - myoclonus ataxia , kinsbourne syndrome , myoclonic encephalopathy of infants , dancing eyes - dancing feet syndrome , and dancing eyes syndrome . in our study , all six children were between 2 and 3 years of age , similar to the trend seen in other reports , in which oma was uncommonly diagnosed before 1 year of age . one possible explanation for the low incidence in infancy may be due to ability to develop specific antibodies is less in younger infants .", "we observed , alike to previously published studies , a similar median age at clinical presentation , acute / subacute onset of presentation , association of nb , and response to immunomodulator therapy .", "we have found poor sensitivity of urine vma in our case series with similar findings reported by brunklaus et al .", "we also observed poorer sensitivities for mibg scintigraphy scan in contrast with the reported high sensitivity ( up to 95% ) for detection of nb as compared to an abdominal ct scan / mri . in adults , oms", "is seen in relation to malignancies of the breast and lung ( small cell carcinoma ) , in association with antibodies which are directed against an rna binding antigen from the anti - hu antibody .", "this antibody is not found in children in oms of nb . in children , nb which presents with oms is more mature", ", shows a favorable histology , and absence of n - myc oncogene amplification than tumors which occur without symptoms of oms .", "oms , the most frequent paraneoplastic syndrome in pediatric age group , remains a challenge for treatment .", "various immunomodulatory therapies have been used including steroids , ivig , cyclophosphamide , and more recently , rituximab .", "patients with nb and oma have been reported to have excellent survival . according to children 's cancer group data ,", "the 3-year survival rate for children with nonmetastatic nb and oma was 100% ( reported from 675 patients who were diagnosed between 1980 and 1994 ) in compared to 77% in non - oma .", "described a case series of 11 patients ( largest case series from india ) with a diagnosis of opsoclonus - myoclonus ( of the 11 , 4 had paraneoplastic etiology ) and concluded that children with paraneoplastic opsoclonus had more relapses and had a poor outcome as compared to an idiopathic group .", ", in our series , all six children had paraneoplastic oms , and all had good outcome . this tumor is known to have spontaneous regression .", "our one child ( case 5 ) had spontaneous symptomatic resolution , and another child ( case 6 ) is doing well without surgery and chemotherapy .", "we have seen a good therapeutic response with immunomodulators , including acth , ivig , and corticosteroids .", "because of the small number of patients , it is difficult to compare these therapies .", "no one had relapse in our series contradicting to those reported by tate et al .", "( up to 52% ) could be due to the small number of patients in our study . in most children with nb ,", "the characteristic feature is the response of this syndrome to corticosteroids and acth and the resolution of the neurological signs when the nb is treated .", "children often develop lifelong neurologic sequelae that impair motor , cognitive , language , and behavioral developments .", "observed that in older children , late effects are less likely seen because basic motor and cognition have already been formed .", "rudnick et al . reported that children with more advanced stage disease had better outcomes with regard to neurologic sequelae . one possible explanation for this association may be that patients with advanced stage disease require more intensive therapy that usually includes chemotherapy .", "russo et al . suggested that chemotherapy may improve neurologic outcome in children with oma and nb , possibly due to its immunosuppressive effects . in our study , five children with oms responded with acth and steroids ; one child recovered spontaneously .", "the outcome was good in our all children , whereas in the study by tate et al . , the outcome was independent of etiology .", "other studies have also observed better outcome of idiopathic opsoclonus - myoclonus . we did not observe progressive developmental and behavioral problems in our patients , which could be due to small number of patients and short follow - up .", "oms is a rare disorder , but it affects children more frequently than adults and exhibits an excellent rate of survival . screening for an occult nb is necessary in all children with this syndrome .", "oms ( paraneoplastic ) had a good outcome without significant neurological deficits in our experience ."], "abstract_text": ["<S> the opsoclonus - myoclonus ataxia syndrome ( omas ) also called  kinsbourne syndrome  or  </S>", "<S> dancing eye syndrome  is a rare but serious disorder characterized by opsoclonus , myoclonus , and ataxia , along with extreme irritability and behavioural changes . </S>", "<S> data on its epidemiology , clinical features , and outcome are limited worldwide . </S>", "<S> the aim of the study was to evaluate the clinical profile and outcome of children with omas . </S>", "<S> a retrospective data of all children presented to pediatric oncology clinic with a diagnosis of opsoclonus - myoclonus from 2013 to 2016 were collected . </S>", "<S> 6 patients with a diagnosis of omas were presented over a 4-year period . </S>", "<S> all 6 cases had paraneoplastic etiology . </S>", "<S> all children had good outcome without any relapse . </S>", "<S> paraneoplastic opsoclonus myoclonus had a good outcome in our experience . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusion", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["opsoclonus - myoclonus - ataxia syndrome ( omas ) , also called  kinsbourne syndrome  or ", "dancing eye syndrome ,  is a serious , rare , and often chronic neurological disorder .", "omas consists of three main symptoms : opsoclonus ( conjugate , multidirectional , chaotic eye movements ) , myoclonus ( nonepileptic limb jerking that can also involve the head and face ) and truncal ataxia , which cause gait imbalance .", "omas is generally a paraneoplastic or parainfectious entity , but in children , it is most commonly associated with occult neuroblastoma ( nb ) in about 50% of cases and between 2% and 3% of children with nb have omas .", "although most patients with nb and omas have good survival rates , 70%80% of these children will have long - term neurologic sequelae . in pediatric age , omas may be associated with neuroblastic tumors ( nb , ganglioneuroblastoma , or ganglioneuroma ) .", "the diagnosis of omas may be difficult in some patients and should be considered even when only some of the features are present .", "international consensus has described three of the following four diagnostic criteria should be present to describe the typical syndrome : ( 1 ) opsoclonus , ( 2 ) myoclonus / ataxia , ( 3 ) behavioral change and/or sleep disturbance , and ( 4 ) nb . we present a retrospective study of five children presented to the pediatric oncology clinic ( poc ) of all india institute of medical sciences ( aiims ) with a diagnosis of opsoclonus - myoclonus syndrome ( oms ) .", "the objective of this study was to describe the clinical profile and outcome of this disorder ."], ["the medical records of all children presented to poc , department of pediatrics , aiims , with a diagnosis of opsoclonus - myoclonus were retrieved and reviewed .", "the diagnosis of opsoclonus - myoclonus was based on a constellation of any three of the four clinical features : opsoclonus , myoclonus , ataxia , and encephalopathy / irritability / behavioral change / sleep disturbance .", "outcome was assessed on follow - up by direct assessment and by telephonic communication ( in one patient ) ."], ["a total of six patients with a diagnosis of opsoclonus - myoclonus were admitted over 4-year period [ table 1 ] .", "opsoclonus , myoclonus , ataxia , and encephalopathy / behavioral abnormalities ( irritability / sleep disturbance ) were present in all children at presentation .", "the duration of symptoms at the time of presentation was in the range of 610 months .", "clinical profile and outcome in six children with opsoclonus - myoclonus syndrome the reasons for the delay in diagnosis included misdiagnosis by peripheral physicians and delayed referral to our center .", "all children had paraneoplastic opsoclonus - myoclonus ( two left paravertebral ganglioneuroblastoma , two left paravertebral nb , one left paravertebral plus left psoas muscle ganglioneuroblastoma , and one right paravertebral ganglioneuroblastoma ) [ figures 13 ] .", "the diagnosis of tumor was made on contrast - enhanced computed tomography scan ( ct ) and magnetic resonance imaging ( mri ) of the chest , abdomen , and pelvis ( 3 ct scan and 3 mri ) .", "tests for urinary excretion of vanillylmandelic acid ( vma ) were negative in all cases .", "iodine-131 metaiodobenzylguanidine scintigraphy scan ( i-131 mibg ) was performed in all cases , but it detected mibg concentrating tumor only in two cases .", "( case 2 ) ( original ) axial section showing well - defined enhancing soft - tissue mass lesion ( 3.5 cm  2 cm  1.4 cm ) seen in the left upper psoas muscle extending into left neural foramen in between l2 and l3 vertebra up to dura mater ( a and b ) ( case 3 ) axial section and coronal section showing right paravertebral mass of size 1.5 cm  0.6 cm  2.4 cm at the level of d6 to d8 vertebral body ( a and b ) ( case 5 ) axial and coronal section showing well - defined left paravertebral mass lesion 2 cm  1.8 cm  2.9 cm at the level of d6 to d8 vertebral body an initial diagnosis of omas was made in all children .", "three children received injection adrenocorticotropic hormone ( acth ) ; two children received injection acth plus injection methylprednisolone followed by oral steroids over 4 weeks .", "one child ( case 1 ) also received intravenous immunoglobulin ( ivig ) and oral clonazepam and risperidone in view of abnormal behavior ( excessive irritability , biting , and head banging ) .", "one child ( case 5 ) had spontaneous improvement and complete recovery without specific immunomodulator therapy .", "of the six cases with paraneoplastic oms , four cases were treated with surgical resection of tumor and chemotherapy . in one child (", "case 5 ) , treatment was deferred by parents but on telephonic communication that the child was symptoms free and healthy at 9 months after presentation to us . in another case (", "case 6 ) , she received only injection acth , surgery , and chemotherapy were denied by parents .", "after follow - up of 24 months , the child was asymptomatic , but ct of the abdomen revealed mass of same size that was documented 2 years back .", "one child ( case 3 ) died of febrile encephalopathy ( not related to disease ) ."], ["other names for oms include omas , paraneoplastic opsoclonus - myoclonus ataxia , kinsbourne syndrome , myoclonic encephalopathy of infants , dancing eyes - dancing feet syndrome , and dancing eyes syndrome . in our study , all six children were between 2 and 3 years of age , similar to the trend seen in other reports , in which oma was uncommonly diagnosed before 1 year of age . one possible explanation for the low incidence in infancy may be due to ability to develop specific antibodies is less in younger infants .", "we observed , alike to previously published studies , a similar median age at clinical presentation , acute / subacute onset of presentation , association of nb , and response to immunomodulator therapy .", "we have found poor sensitivity of urine vma in our case series with similar findings reported by brunklaus et al .", "we also observed poorer sensitivities for mibg scintigraphy scan in contrast with the reported high sensitivity ( up to 95% ) for detection of nb as compared to an abdominal ct scan / mri . in adults , oms", "is seen in relation to malignancies of the breast and lung ( small cell carcinoma ) , in association with antibodies which are directed against an rna binding antigen from the anti - hu antibody .", "this antibody is not found in children in oms of nb . in children , nb which presents with oms is more mature", ", shows a favorable histology , and absence of n - myc oncogene amplification than tumors which occur without symptoms of oms .", "oms , the most frequent paraneoplastic syndrome in pediatric age group , remains a challenge for treatment .", "various immunomodulatory therapies have been used including steroids , ivig , cyclophosphamide , and more recently , rituximab .", "patients with nb and oma have been reported to have excellent survival . according to children 's cancer group data ,", "the 3-year survival rate for children with nonmetastatic nb and oma was 100% ( reported from 675 patients who were diagnosed between 1980 and 1994 ) in compared to 77% in non - oma .", "described a case series of 11 patients ( largest case series from india ) with a diagnosis of opsoclonus - myoclonus ( of the 11 , 4 had paraneoplastic etiology ) and concluded that children with paraneoplastic opsoclonus had more relapses and had a poor outcome as compared to an idiopathic group .", ", in our series , all six children had paraneoplastic oms , and all had good outcome . this tumor is known to have spontaneous regression .", "our one child ( case 5 ) had spontaneous symptomatic resolution , and another child ( case 6 ) is doing well without surgery and chemotherapy .", "we have seen a good therapeutic response with immunomodulators , including acth , ivig , and corticosteroids .", "because of the small number of patients , it is difficult to compare these therapies .", "no one had relapse in our series contradicting to those reported by tate et al .", "( up to 52% ) could be due to the small number of patients in our study . in most children with nb ,", "the characteristic feature is the response of this syndrome to corticosteroids and acth and the resolution of the neurological signs when the nb is treated .", "children often develop lifelong neurologic sequelae that impair motor , cognitive , language , and behavioral developments .", "observed that in older children , late effects are less likely seen because basic motor and cognition have already been formed .", "rudnick et al . reported that children with more advanced stage disease had better outcomes with regard to neurologic sequelae . one possible explanation for this association may be that patients with advanced stage disease require more intensive therapy that usually includes chemotherapy .", "russo et al . suggested that chemotherapy may improve neurologic outcome in children with oma and nb , possibly due to its immunosuppressive effects . in our study , five children with oms responded with acth and steroids ; one child recovered spontaneously .", "the outcome was good in our all children , whereas in the study by tate et al . , the outcome was independent of etiology .", "other studies have also observed better outcome of idiopathic opsoclonus - myoclonus . we did not observe progressive developmental and behavioral problems in our patients , which could be due to small number of patients and short follow - up ."], ["oms is a rare disorder , but it affects children more frequently than adults and exhibits an excellent rate of survival . screening for an occult nb is necessary in all children with this syndrome .", "oms ( paraneoplastic ) had a good outcome without significant neurological deficits in our experience ."], [""], [""]]}
{"article_id": "PMC3085288", "article_text": ["to the surprise and deep disappointment of all involved in the treatment of lung cancer , several large trials did not demonstrate any benefit of tyrosine kinase inhibitors ( tkis ) as an addition to chemotherapy [ 13 ] .", "basic and clinical research then focused on mutations of the gene for epidermal growth factor receptor ( egfr ) as a predictive factor for response to monotherapy with tkis and to development of new compounds with broader and/or irreversible inhibition .   the biological basis for the negative experience with combined treatment", "gefitinib and erlotinib met all three standard criteria for inclusion in a combination with chemotherapy : activity as monotherapy , different mechanism of action , and different toxicity .", "why , then , did the combination not work ? as explained in a recent editorial", ", we believe that the cells of tumors sensitive to tkis are pushed into the g-0 phase of the cell cycle and therefore become resistant to cytotoxic drugs", ". if antagonism between the two classes of drugs is really the biological basis for the aforementioned negative experience , then an optimal combination of tkis and chemotherapy should be in an intermittent , rather than a continuous schedule .", "this brief report presents a single - institution experience on intermittent chemotherapy and tki in a small series of patients with advanced adenocarcinoma of the lung .", "our hypothesis was that intermittent treatment would lead to superior time to progression , when compared to experience with chemotherapy alone .", "if confirmed , such a result would be a solid basis for a randomised clinical trial .", "patients eligible for the trial were chemonave with microscopically confirmed adenocarcinoma of the lung , had stage iii b ( wet ) or iv according to uicc - tnm classification ( 6th edition ) , had smoking history of less than 10 packs in years , had an ecog performance status 0 or 1 , and had adequate parameters of hematological , liver , and renal function to receive cisplatin - based chemotherapy . in the absence of neurological symptoms , patients with brain metastases were eligible and were treated with brain irradiation only in case of intracranial progression .", "all patients had their diagnosis confirmed by biopsy or cytology . at the time when the trial was initiated , testing for egfr mutations was not available .  ", "within three weeks prior to treatment , the precise extent of the disease was determined by chest x - ray and ct scanning of the chest , upper abdomen , and brain . since 2008 ,", "pet - ct scanning has been available and included in the initial diagnostics and in followup .", "the treatment started with four cycles of intermittent chemotherapy and erlotinib according to the following schedule : day 1 : gemcitabine 1250  mg / m in 30-minute infusion , day 2 : cisplatin 75  mg / m , with appropriate hydration and antiemetics , day 4 : gemcitabine 1250  mg / m in 30-minute infusion , days 515 : erlotinib 150  mg daily p.o .", "the number of cycles depended on tolerance to cisplatin - based chemotherapy and was determined individually . immediately after the last cycle", ", patients continued with erlotinib 150  mg / m daily continuously until progression or unacceptable toxicity .", "definition of complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , and progression followed the recist criteria .  ", "the first evaluation of response was done during the third cycle of intermittent therapy , with confirmation of response during the fifth cycle .", "control radiological examinations were repeated every 2 months for chest x - ray , every 4 months for ct , and at 6 and 12 months for pet - ct ( only patients who had this examination during their initial diagnostics ) . in october 2010 , all biopsy samples were reviewed , and specimens with more than 10% of tumour tissue were analyzed .", "genomic dna was extracted from formalin - fixed , paraffin - embedded tissue sections using qiaamp dna ffpe tissue kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .", "quantification of extracted dna was done on qubit fluorometer ( invitrogen , carlsbad , usa ) . to detect egfr gene - activating mutations , we used therascreen egfr29 mutation kit ( dxs diagnostics , qiagen , manchester , uk ) .", "all realtime pcr reactions were performed in a 25  l final volume on abi 7500 instrument ( applied biosystems , carlsbad , usa ) .", "after standard chemotherapy for metastatic nonsmall cell carcinoma , the expected ttp is 5 months .", "the size of this single - arm nonrandomised trial of intermittent therapy was based on the assumption of 9 months as the median time to progression ( ttp ) .", "to obtain such a result with a confidence interval of 612 months , we planned to recruit 40 patients .", "the investigators strictly followed recommendations of the helsinki declaration ( 1964 , with later amendments ) and of the european council convention on protection of human rights in bio - medicine , as accepted in oviedo in 1997 .", "the protocol was approved by the institutional review board ( institute of oncology , ljubljana ) and by the national committee for medical ethics , ministry of health , republic of slovenia .", "one patient was later found to have metastatic carcinoma of the pancreas rather than primary lung cancer and was excluded from all further analyses .   with 12 patients each ,", "twelve patients were never - smokers , and most were in good general condition ( ps 0 - 1 for 21 patients ) . with the exception of a single patient with  wet ", "bone metastases were the most common site of metastatic disease , followed by pleura / pericardium , contralateral lung metastases , and liver .", "two or more sites of metastatic disease were documented in 4 and 12 patients , respectively ( table 1 ) .", "three patients had only cytological diagnosis , and an additional 3 had biopsy samples too small to allow for analysis of egfr mutations in tumor cells .", "of the 18 adequate samples , 8 were positive for egfr gene - activating mutations .", "the actual number of cycles of intermittent therapy was from 1 to 6 cycles ( median : 4 cycles ) . due to early progression ,", "7 patients were still on maintenance treatment with erlotinib , and an additional patient stopped treatment with erlotinib after 12 months in pet - ct confirmed complete remission ( figures 1 and 2 ) . for the remaining patients , median total duration of treatment was 10 months . during the initial phase ,", "side effects of maintenance with erlotinib were skin toxicity ( grade 3 : 1  pt ; grade 2 : 11  pts ) and diarrhea ( grade 2 in 1  pt ) .", "all patients are evaluable for response , and no patient has been lost to followup . for the whole group of 24 patients , complete remission ( cr ) was seen in 5 pts ; partial remission ( pr ) in 9  pts ( response rate 58% ) , minimal response or stable disease ( sd ) in 8  pts , and progression in 2  pts . a clear and statistically significant ( p <", ".05 ) correlation was seen between the presence of activating egfr mutations and response . among the 8 patients who were positive for egfr gene - activating mutations , 4 complete and", "no cr and only 2 pr were seen among the 10 patients negative for mutations ( table 2 ) .  ", "for the whole group , median time to progression ( ttp ) was 13.4 months , and median overall survival ( os ) was 23 months .", "median ttp and os for this group was 21.5 months and 24.2 months , respectively .", "for patients without egfr mutations , ttp was 5 months , and os was 7 months ( table 2 and figures 3 and 4 ) .", "this clinical trial was launched at a time when routine testing for egfr gene - activating mutations was not yet available .", "selection of patients for a combination of chemotherapy and erlotinib was made on the basis of classical histopathology ( adenocarcinoma ) and smoking status .  ", "since testing for egfr gene mutations is now available , it is clear that patients with activating mutations are those who really benefit from tkis . in addition ,", "standard first - line treatment for patients with activating egfr mutations is now monotherapy with a tki [ 6 , 7 ] .", "since continuing a trial with the same selection criteria and without considering the status of egfr gene activating mutations was not justified , the research group made a decision to close the trial and analyse the experience . in order to get a longer interval for intermittent erlotinib , gemcitabine was given on days 1 and 4 of the cycle . when compared to the standard day 1 and day 8 schedule", ", this minor modification in timing of cytotoxic drugs did not have any adverse effect on the tolerance to treatment .", "clearly , other platin - based schedules which apply chemotherapy on a 3-weekly basis ( such as pemetrexed - cisplatin or paclitaxel - carboplatin ) can offer an even longer interval for tkis and might be considered for future trials of intermittent treatment .  ", "two other groups recently reported promising experience with intermittent chemotherapy and tkis . in a trial from the usa ,", "two schedules of intermittent treatment were tested . in combination with pemetrexed ( 500  mg / m on day 1 ) , erlotinib", "was given either as a pulse application in a high dose ( range : 800 to 1400  mg ) given on days 2 , 9 and 16 , or in lower doses ( 150250  mg daily ) on days 2 to 16 .", "while tolerance to this treatment was good , the small number and heterogeneity of patients recruited into this trial do not allow for any clear conclusion regarding the effectiveness of intermittent treatment . of more importance", "this study from asia compared gemcitabine and either cisplatin or carboplatin to a schedule with addition of intermittent application of erlotinib ( 150  mg on days 14 to 28 of the cycle ) and reported significantly superior ttp with the intermittent schedule .", "their experience is most valuable but may not be of direct relevance for the rest of the world , due to the well - known differences in sensitivity of lung cancer to tkis between asian and caucasian patients .   despite its small size ,", "our trial can offer valuable experience for further research on optimisation of treatment with combinations of chemotherapy and tkis .", "looking at the whole series of patients , we can conclude that intermittent chemotherapy and erlotinib is a treatment of very low toxicity .", "it is also clear that the efficacy of treatment is closely related to the presence or absence of egfr gene - activating mutations .  ", "the most important finding is the excellent response rate with a substantial proportion of complete responses and prolonged ttp and os for patients positive for egfr gene - activating mutations . for many years , the maximal expectation of a patient with metastatic nonsmall cell lung cancer was a partial remission of relatively short duration in the range of 5 to 9 months . with intermittent treatment ,", "while the number of patients in our trial is small and any definitive conclusion would be premature , we nevertheless believe that further research of intermittent therapy for patients positive for egfr gene - activating mutations is warranted . a randomised trial comparing first - line tki as monotherapy to the intermittent schedule should clarify the real value of this new approach ."], "abstract_text": ["<S> \n background . intermittent application of chemotherapy and tyrosine kinase inhibitors may avoid antagonism between the two classes of drugs . </S>", "<S> this hypothesis was tested in a phase ii clinical trial . </S>", "<S> patients and methods . </S>", "<S> eligible patients were nonsmokers or light smokers , chemo - nave , with metastatic adenocarcinoma of the lung . </S>", "<S> treatment : 4 to 6 cycles of gemcitabine 1250  mg / m2 on days 1 and 4 , cisplatin 75  mg / m2 on day 2 , and erlotnib 150 mg daily on days 515 , followed by erlotinib as maintenance . results . </S>", "<S> 24 patients entered the trial . </S>", "<S> four pts had grade 3 toxicity . </S>", "<S> complete remission ( cr ) and partial remission ( pr ) were seen in 5  pts and 9  pts , respectively ( response rate 58% ) . </S>", "<S> median time to progression ( ttp ) was 13.4 months and median overall survival ( os ) was 23 months . when compared to patients with negative or unknown status of egfr mutations , </S>", "<S> 8 patients with egfr gene activating mutations had significantly superior experience : 4 cr and 4 pr , with median ttp 21.5 months and os 24.2 months ( p < .05 ) . conclusions . </S>", "<S> intermittent schedule with gemcitabine , cisplatin and erlotinib has mild toxicity . for patients who are positive for egfr gene activating mutations </S>", "<S> , this treatment offers excellent response rate , time to progression and survival . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Patients and Methods", "3. Results", "4. Discussion and Conclusions", " Conflict of Interests"], "sections": [["to the surprise and deep disappointment of all involved in the treatment of lung cancer , several large trials did not demonstrate any benefit of tyrosine kinase inhibitors ( tkis ) as an addition to chemotherapy [ 13 ] .", "basic and clinical research then focused on mutations of the gene for epidermal growth factor receptor ( egfr ) as a predictive factor for response to monotherapy with tkis and to development of new compounds with broader and/or irreversible inhibition .   the biological basis for the negative experience with combined treatment", "gefitinib and erlotinib met all three standard criteria for inclusion in a combination with chemotherapy : activity as monotherapy , different mechanism of action , and different toxicity .", "why , then , did the combination not work ? as explained in a recent editorial", ", we believe that the cells of tumors sensitive to tkis are pushed into the g-0 phase of the cell cycle and therefore become resistant to cytotoxic drugs", ". if antagonism between the two classes of drugs is really the biological basis for the aforementioned negative experience , then an optimal combination of tkis and chemotherapy should be in an intermittent , rather than a continuous schedule .", "this brief report presents a single - institution experience on intermittent chemotherapy and tki in a small series of patients with advanced adenocarcinoma of the lung .", "our hypothesis was that intermittent treatment would lead to superior time to progression , when compared to experience with chemotherapy alone .", "if confirmed , such a result would be a solid basis for a randomised clinical trial ."], ["patients eligible for the trial were chemonave with microscopically confirmed adenocarcinoma of the lung , had stage iii b ( wet ) or iv according to uicc - tnm classification ( 6th edition ) , had smoking history of less than 10 packs in years , had an ecog performance status 0 or 1 , and had adequate parameters of hematological , liver , and renal function to receive cisplatin - based chemotherapy . in the absence of neurological symptoms , patients with brain metastases were eligible and were treated with brain irradiation only in case of intracranial progression .", "all patients had their diagnosis confirmed by biopsy or cytology . at the time when the trial was initiated , testing for egfr mutations was not available .  ", "within three weeks prior to treatment , the precise extent of the disease was determined by chest x - ray and ct scanning of the chest , upper abdomen , and brain . since 2008 ,", "pet - ct scanning has been available and included in the initial diagnostics and in followup .", "the treatment started with four cycles of intermittent chemotherapy and erlotinib according to the following schedule : day 1 : gemcitabine 1250  mg / m in 30-minute infusion , day 2 : cisplatin 75  mg / m , with appropriate hydration and antiemetics , day 4 : gemcitabine 1250  mg / m in 30-minute infusion , days 515 : erlotinib 150  mg daily p.o .", "the number of cycles depended on tolerance to cisplatin - based chemotherapy and was determined individually . immediately after the last cycle", ", patients continued with erlotinib 150  mg / m daily continuously until progression or unacceptable toxicity .", "definition of complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , and progression followed the recist criteria .  ", "the first evaluation of response was done during the third cycle of intermittent therapy , with confirmation of response during the fifth cycle .", "control radiological examinations were repeated every 2 months for chest x - ray , every 4 months for ct , and at 6 and 12 months for pet - ct ( only patients who had this examination during their initial diagnostics ) . in october 2010 , all biopsy samples were reviewed , and specimens with more than 10% of tumour tissue were analyzed .", "genomic dna was extracted from formalin - fixed , paraffin - embedded tissue sections using qiaamp dna ffpe tissue kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .", "quantification of extracted dna was done on qubit fluorometer ( invitrogen , carlsbad , usa ) . to detect egfr gene - activating mutations , we used therascreen egfr29 mutation kit ( dxs diagnostics , qiagen , manchester , uk ) .", "all realtime pcr reactions were performed in a 25  l final volume on abi 7500 instrument ( applied biosystems , carlsbad , usa ) .", "after standard chemotherapy for metastatic nonsmall cell carcinoma , the expected ttp is 5 months .", "the size of this single - arm nonrandomised trial of intermittent therapy was based on the assumption of 9 months as the median time to progression ( ttp ) .", "to obtain such a result with a confidence interval of 612 months , we planned to recruit 40 patients .", "the investigators strictly followed recommendations of the helsinki declaration ( 1964 , with later amendments ) and of the european council convention on protection of human rights in bio - medicine , as accepted in oviedo in 1997 .", "the protocol was approved by the institutional review board ( institute of oncology , ljubljana ) and by the national committee for medical ethics , ministry of health , republic of slovenia ."], ["one patient was later found to have metastatic carcinoma of the pancreas rather than primary lung cancer and was excluded from all further analyses .   with 12 patients each ,", "twelve patients were never - smokers , and most were in good general condition ( ps 0 - 1 for 21 patients ) . with the exception of a single patient with  wet ", "bone metastases were the most common site of metastatic disease , followed by pleura / pericardium , contralateral lung metastases , and liver .", "two or more sites of metastatic disease were documented in 4 and 12 patients , respectively ( table 1 ) .", "three patients had only cytological diagnosis , and an additional 3 had biopsy samples too small to allow for analysis of egfr mutations in tumor cells .", "of the 18 adequate samples , 8 were positive for egfr gene - activating mutations .", "the actual number of cycles of intermittent therapy was from 1 to 6 cycles ( median : 4 cycles ) . due to early progression ,", "7 patients were still on maintenance treatment with erlotinib , and an additional patient stopped treatment with erlotinib after 12 months in pet - ct confirmed complete remission ( figures 1 and 2 ) . for the remaining patients , median total duration of treatment was 10 months . during the initial phase ,", "side effects of maintenance with erlotinib were skin toxicity ( grade 3 : 1  pt ; grade 2 : 11  pts ) and diarrhea ( grade 2 in 1  pt ) .", "all patients are evaluable for response , and no patient has been lost to followup . for the whole group of 24 patients , complete remission ( cr ) was seen in 5 pts ; partial remission ( pr ) in 9  pts ( response rate 58% ) , minimal response or stable disease ( sd ) in 8  pts , and progression in 2  pts . a clear and statistically significant ( p <", ".05 ) correlation was seen between the presence of activating egfr mutations and response . among the 8 patients who were positive for egfr gene - activating mutations , 4 complete and", "no cr and only 2 pr were seen among the 10 patients negative for mutations ( table 2 ) .  ", "for the whole group , median time to progression ( ttp ) was 13.4 months , and median overall survival ( os ) was 23 months .", "median ttp and os for this group was 21.5 months and 24.2 months , respectively .", "for patients without egfr mutations , ttp was 5 months , and os was 7 months ( table 2 and figures 3 and 4 ) ."], ["this clinical trial was launched at a time when routine testing for egfr gene - activating mutations was not yet available .", "selection of patients for a combination of chemotherapy and erlotinib was made on the basis of classical histopathology ( adenocarcinoma ) and smoking status .  ", "since testing for egfr gene mutations is now available , it is clear that patients with activating mutations are those who really benefit from tkis . in addition ,", "standard first - line treatment for patients with activating egfr mutations is now monotherapy with a tki [ 6 , 7 ] .", "since continuing a trial with the same selection criteria and without considering the status of egfr gene activating mutations was not justified , the research group made a decision to close the trial and analyse the experience . in order to get a longer interval for intermittent erlotinib , gemcitabine was given on days 1 and 4 of the cycle . when compared to the standard day 1 and day 8 schedule", ", this minor modification in timing of cytotoxic drugs did not have any adverse effect on the tolerance to treatment .", "clearly , other platin - based schedules which apply chemotherapy on a 3-weekly basis ( such as pemetrexed - cisplatin or paclitaxel - carboplatin ) can offer an even longer interval for tkis and might be considered for future trials of intermittent treatment .  ", "two other groups recently reported promising experience with intermittent chemotherapy and tkis . in a trial from the usa ,", "two schedules of intermittent treatment were tested . in combination with pemetrexed ( 500  mg / m on day 1 ) , erlotinib", "was given either as a pulse application in a high dose ( range : 800 to 1400  mg ) given on days 2 , 9 and 16 , or in lower doses ( 150250  mg daily ) on days 2 to 16 .", "while tolerance to this treatment was good , the small number and heterogeneity of patients recruited into this trial do not allow for any clear conclusion regarding the effectiveness of intermittent treatment . of more importance", "this study from asia compared gemcitabine and either cisplatin or carboplatin to a schedule with addition of intermittent application of erlotinib ( 150  mg on days 14 to 28 of the cycle ) and reported significantly superior ttp with the intermittent schedule .", "their experience is most valuable but may not be of direct relevance for the rest of the world , due to the well - known differences in sensitivity of lung cancer to tkis between asian and caucasian patients .   despite its small size ,", "our trial can offer valuable experience for further research on optimisation of treatment with combinations of chemotherapy and tkis .", "looking at the whole series of patients , we can conclude that intermittent chemotherapy and erlotinib is a treatment of very low toxicity .", "it is also clear that the efficacy of treatment is closely related to the presence or absence of egfr gene - activating mutations .  ", "the most important finding is the excellent response rate with a substantial proportion of complete responses and prolonged ttp and os for patients positive for egfr gene - activating mutations . for many years , the maximal expectation of a patient with metastatic nonsmall cell lung cancer was a partial remission of relatively short duration in the range of 5 to 9 months . with intermittent treatment ,", "while the number of patients in our trial is small and any definitive conclusion would be premature , we nevertheless believe that further research of intermittent therapy for patients positive for egfr gene - activating mutations is warranted . a randomised trial comparing first - line tki as monotherapy to the intermittent schedule should clarify the real value of this new approach ."], [""]]}
{"article_id": "PMC4214302", "article_text": ["using metagenomic deep sequencing , we analyzed fecal samples from 180 infants and children ages 7 days96 months ( mean 18.7 months ) in tunisia who had unexplained diarrhea that tested negative for rotavirus , norovirus , astrovirus , sapovirus , adenovirus types 40 and 41 , and aichi virus by reverse transcription pcr ( 7 ) .", "the fecal supernatants were filtered through a 0.45-m filter ( millipore , darmstadt , germany ) to remove bacterium - sized particles , and the filtrates were digested with a mixture of dnases ( turbo dnase from ambion , carlsbad , ca , usa ; baseline - zero from epicenter , madison , wi , usa ; and benzonase from novagen , san diego , ca , usa ) and rnase ( fermentas , pittsburgh , pa , usa ) to digest unprotected nucleic acids . enriched viral nucleic acids ( rna and dna ) were then extracted and amplified by using scriptseq v2 rna - seq library preparation kit ( epicenter ) and analyzed in pools of 10 specimens in 2 illumina miseq run of 250-bp end reads , yielding 20,693,619 unique sequences .", "we compared the illumina sequences with the genbank nonredundant protein databases using blastx ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . using a blastx e - score cutoff of 10", ", we identified , in decreasing frequency , sequences related to the mammalian viruses : sapovirus ( 120,177 reads ) , anelloviridae ( 14,841 reads ) , parechovirus ( 10,557 reads ) , norovirus ( 4,551 reads ) , enterovirus ( 3,857 reads ) , circoviridae ( 2,127 reads ) , group a rotavirus ( 839 reads ) , adeno - associated virus ( 812 reads ) , picobirnavirus ( 274 reads ) , bufavirus ( 168 reads ) , wu polyomavirus ( 136 reads ) , bocavirus ( 62 reads ) , adenovirus ( 58 reads ) , papillomavirus ( 22 reads ) , cosavirus ( 20 reads ) , group c rotavirus ( 17 reads ) , human astrovirus 1 ( 14 reads ) , salivirus ( 4 reads ) , and aichi virus ( 2 reads ) .", "one pool showed a single read encoding a parvovirus - like protein segment with high levels of genetic similarity ( blastx e - score of 5  10 ) to the nonstructural protein ( ns ) 1 of rat parvovirus ( genbank accession no .", "was then identified by using pcr and underwent further deep sequencing as above , generating 11 more parovirus sequences .", "no other eukaryotic viral sequences were identified from 260,000 unique sequence reads from this patient .", "the near complete parvovirus genome was then acquired by filling genome gaps by pcr and amplifying 5 and 3 extremities using race ( rapid amplification of cdna ends , life technologies ) .", "we named this virus tusavirus 1 for tunisian stool - associated parvovirus . \n  a nearly complete 4,424-bp genome ( tusavirus 1 , genbank accession no .", "kj495710 ) was successfully acquired with partial 5 untranslated region ( 243 bp ) , complete ns1 open reading frame ( 625 aa ) , complete viral protein ( vp ) 1 open reading frame ( 715 aa ) , and a partial 3 untranslated region ( 68 bp ) .", "tusavirus has a potential upstream start codon mss in a weaker kozak consensus sequence than maq ( figure , panel a ) , which we selected as the start codon . the walker loop gpattgks [ gxxxxgk(t / s ) ] , which is an atp- or gtp - binding motif , was found in the ns1 .", "potential splicing signals to express vp1 were identified on the basis of alignments to other protoparvoviruses and classic rna splicing motifs ( figure , panel a ) .", "the phospholipase a2 ( pla2 ) motif was identified in vp1 n - termini with expected calcium - binding site and catalytic residues .", "the methionine codon of vp2 was located upstream of glycine - rich sequence ( gggaraggvg ) .", "an unusual serine - rich sequence ( sssdsgpsss ) was also seen near vp1 n - termini .", "the alignment of the pla2 regions of representatives of 5 protoparvovirus species show the calcium - binding region and catalytic residues in tusavirus .", "pairwise sliding window of percentage nucleotide similarity of tusavirus aligns with the genetically closest kilham rat parvovirus .", "b ) phylogenetic trees generated with nonstructural protein ( ns ) 1 and vp1 of tusavirus and of the 5 international committee on taxonomy of viruses  designated species in the protoparvovirus genus .", "fpv , feline parvovirus ; mev , mink enteritis virus ; cpv , canine parvovirus ; rapv , raccoon parvovirus ; rpv1 , rat parvovirus 1 ; mvmp , minute virus of mice , prototype ; hapv , hamster parvovirus ; ppv - kr , porcine parvovirus kresse ; simian bupv , simian bufavirus ; bupv1 , bufavirus 1 ; bupv2 , bufavirus 2 ; amdv , aleutian mink disease virus ; gfav , gray fox amdovirus ; b19v - lali , human parvovirus b19-lali .", "bootstrap values ( based on 100 replicates ) for each node are given if > 70% .", "protein sequence alignments were made by using clustalx version 2.0.3 ( http://www.clustal.org ) with the default settings ; a phylogenetic tree with 100 bootstrap resamples of the alignment datasets was generated by using the neighbor - joining method based on the jones - taylor - thornton matrix - based model in mega5 ( http://www.megasoftware.net ) .", "phylogenetic analysis showed that tusavirus 1 was distinct from known members of the protoparvovirus genus ( figure , panel b ) .", "vp1 and vp2 shared identities of 39% and 37% , respectively , to those of kilham rat parvovirus .", "according to the international committee on taxonomy of viruses , the members of the same parvovirus genus should share > 30% and members of the same species > 85% aa identity in ns1 .", "the protoparvovirus genus currently comprises species infecting carnivores , rodents , pigs , and humans ( 1 ) .", "tusavirus 1 is proposed as prototype for primate protoparvovirus 2 species that would join bufaviruses as human viruses in this genus .", "we used a nested pcr targeting ns1 to determine the prevalence of this virus in the 180 tunisia diarrhea samples .", "primers tusa - f1 ( 5-gaagaagctggaaactgtggtca-3 ) and tusa - r1 ( 5-ctcgtctttctcccaggcatct-3 ) were used for the first round of pcr , and primers tusa - f2 ( 5-attgctccaacaccagtcatca-3 ) and tusa - r2 ( 5-tctggtctggtccaatcttcttc-3 ) for the second round of pcr .", "the pcr conditions were : 95c for 5 min , 35 cycles 95c for 30 s , 52c or 51c ( for the first or second round , respectively ) for 30 s , and 72c for 1 min , a final extension at 72c for 10 min .", "no samples except the one initially detected by deep sequencing were pcr positive , yielding a low prevalence of 0.56% ( 1/180 ) in this tunisian population .", "we detected fecal shedding of a previously uncharacterized parvovirus in a child with unexplained diarrhea .", "the 18-month - old girl showed twice daily liquid and greenish feces over 3 days but no fever ( 37.4c ) , vomiting , or dehydration . to identify other viral infections in this patient ,", "no other mammalian virus was detected , suggesting a possible role for tusavirus 1 in this patient s gastrointestinal illness , although the lack of testing for pathogenic bacteria and parasites does not enable us to exclude these alternative explanations .", "control pcr studies of unexplained diarrhea and serologic tests , are needed to define the prevalence and disease association of this new parvovirus species in different age groups and populations ."], "abstract_text": ["<S> a divergent parvovirus genome was the only eukaryotic viral sequence detected in feces of a tunisian child with unexplained diarrhea . </S>", "<S> tusavirus 1 shared 44% and 39% identity with the nonstructural protein 1 and viral protein 1 , respectively , of the closest genome , kilham rat parvovirus , indicating presence of a new human viral species in the protoparvovirus genus . </S>"], "labels": null, "section_names": ["The Study", "Conclusions"], "sections": [["using metagenomic deep sequencing , we analyzed fecal samples from 180 infants and children ages 7 days96 months ( mean 18.7 months ) in tunisia who had unexplained diarrhea that tested negative for rotavirus , norovirus , astrovirus , sapovirus , adenovirus types 40 and 41 , and aichi virus by reverse transcription pcr ( 7 ) .", "the fecal supernatants were filtered through a 0.45-m filter ( millipore , darmstadt , germany ) to remove bacterium - sized particles , and the filtrates were digested with a mixture of dnases ( turbo dnase from ambion , carlsbad , ca , usa ; baseline - zero from epicenter , madison , wi , usa ; and benzonase from novagen , san diego , ca , usa ) and rnase ( fermentas , pittsburgh , pa , usa ) to digest unprotected nucleic acids . enriched viral nucleic acids ( rna and dna ) were then extracted and amplified by using scriptseq v2 rna - seq library preparation kit ( epicenter ) and analyzed in pools of 10 specimens in 2 illumina miseq run of 250-bp end reads , yielding 20,693,619 unique sequences .", "we compared the illumina sequences with the genbank nonredundant protein databases using blastx ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . using a blastx e - score cutoff of 10", ", we identified , in decreasing frequency , sequences related to the mammalian viruses : sapovirus ( 120,177 reads ) , anelloviridae ( 14,841 reads ) , parechovirus ( 10,557 reads ) , norovirus ( 4,551 reads ) , enterovirus ( 3,857 reads ) , circoviridae ( 2,127 reads ) , group a rotavirus ( 839 reads ) , adeno - associated virus ( 812 reads ) , picobirnavirus ( 274 reads ) , bufavirus ( 168 reads ) , wu polyomavirus ( 136 reads ) , bocavirus ( 62 reads ) , adenovirus ( 58 reads ) , papillomavirus ( 22 reads ) , cosavirus ( 20 reads ) , group c rotavirus ( 17 reads ) , human astrovirus 1 ( 14 reads ) , salivirus ( 4 reads ) , and aichi virus ( 2 reads ) .", "one pool showed a single read encoding a parvovirus - like protein segment with high levels of genetic similarity ( blastx e - score of 5  10 ) to the nonstructural protein ( ns ) 1 of rat parvovirus ( genbank accession no .", "was then identified by using pcr and underwent further deep sequencing as above , generating 11 more parovirus sequences .", "no other eukaryotic viral sequences were identified from 260,000 unique sequence reads from this patient .", "the near complete parvovirus genome was then acquired by filling genome gaps by pcr and amplifying 5 and 3 extremities using race ( rapid amplification of cdna ends , life technologies ) .", "we named this virus tusavirus 1 for tunisian stool - associated parvovirus . \n  a nearly complete 4,424-bp genome ( tusavirus 1 , genbank accession no .", "kj495710 ) was successfully acquired with partial 5 untranslated region ( 243 bp ) , complete ns1 open reading frame ( 625 aa ) , complete viral protein ( vp ) 1 open reading frame ( 715 aa ) , and a partial 3 untranslated region ( 68 bp ) .", "tusavirus has a potential upstream start codon mss in a weaker kozak consensus sequence than maq ( figure , panel a ) , which we selected as the start codon . the walker loop gpattgks [ gxxxxgk(t / s ) ] , which is an atp- or gtp - binding motif , was found in the ns1 .", "potential splicing signals to express vp1 were identified on the basis of alignments to other protoparvoviruses and classic rna splicing motifs ( figure , panel a ) .", "the phospholipase a2 ( pla2 ) motif was identified in vp1 n - termini with expected calcium - binding site and catalytic residues .", "the methionine codon of vp2 was located upstream of glycine - rich sequence ( gggaraggvg ) .", "an unusual serine - rich sequence ( sssdsgpsss ) was also seen near vp1 n - termini .", "the alignment of the pla2 regions of representatives of 5 protoparvovirus species show the calcium - binding region and catalytic residues in tusavirus .", "pairwise sliding window of percentage nucleotide similarity of tusavirus aligns with the genetically closest kilham rat parvovirus .", "b ) phylogenetic trees generated with nonstructural protein ( ns ) 1 and vp1 of tusavirus and of the 5 international committee on taxonomy of viruses  designated species in the protoparvovirus genus .", "fpv , feline parvovirus ; mev , mink enteritis virus ; cpv , canine parvovirus ; rapv , raccoon parvovirus ; rpv1 , rat parvovirus 1 ; mvmp , minute virus of mice , prototype ; hapv , hamster parvovirus ; ppv - kr , porcine parvovirus kresse ; simian bupv , simian bufavirus ; bupv1 , bufavirus 1 ; bupv2 , bufavirus 2 ; amdv , aleutian mink disease virus ; gfav , gray fox amdovirus ; b19v - lali , human parvovirus b19-lali .", "bootstrap values ( based on 100 replicates ) for each node are given if > 70% .", "protein sequence alignments were made by using clustalx version 2.0.3 ( http://www.clustal.org ) with the default settings ; a phylogenetic tree with 100 bootstrap resamples of the alignment datasets was generated by using the neighbor - joining method based on the jones - taylor - thornton matrix - based model in mega5 ( http://www.megasoftware.net ) .", "phylogenetic analysis showed that tusavirus 1 was distinct from known members of the protoparvovirus genus ( figure , panel b ) .", "vp1 and vp2 shared identities of 39% and 37% , respectively , to those of kilham rat parvovirus .", "according to the international committee on taxonomy of viruses , the members of the same parvovirus genus should share > 30% and members of the same species > 85% aa identity in ns1 .", "the protoparvovirus genus currently comprises species infecting carnivores , rodents , pigs , and humans ( 1 ) .", "tusavirus 1 is proposed as prototype for primate protoparvovirus 2 species that would join bufaviruses as human viruses in this genus .", "we used a nested pcr targeting ns1 to determine the prevalence of this virus in the 180 tunisia diarrhea samples .", "primers tusa - f1 ( 5-gaagaagctggaaactgtggtca-3 ) and tusa - r1 ( 5-ctcgtctttctcccaggcatct-3 ) were used for the first round of pcr , and primers tusa - f2 ( 5-attgctccaacaccagtcatca-3 ) and tusa - r2 ( 5-tctggtctggtccaatcttcttc-3 ) for the second round of pcr .", "the pcr conditions were : 95c for 5 min , 35 cycles 95c for 30 s , 52c or 51c ( for the first or second round , respectively ) for 30 s , and 72c for 1 min , a final extension at 72c for 10 min .", "no samples except the one initially detected by deep sequencing were pcr positive , yielding a low prevalence of 0.56% ( 1/180 ) in this tunisian population ."], ["we detected fecal shedding of a previously uncharacterized parvovirus in a child with unexplained diarrhea .", "the 18-month - old girl showed twice daily liquid and greenish feces over 3 days but no fever ( 37.4c ) , vomiting , or dehydration . to identify other viral infections in this patient ,", "no other mammalian virus was detected , suggesting a possible role for tusavirus 1 in this patient s gastrointestinal illness , although the lack of testing for pathogenic bacteria and parasites does not enable us to exclude these alternative explanations .", "control pcr studies of unexplained diarrhea and serologic tests , are needed to define the prevalence and disease association of this new parvovirus species in different age groups and populations ."]]}
{"article_id": "PMC5013738", "article_text": ["it may be regarded as the localized equivalent of the systemic inflammatory response seen after any trigger of inflammation , which recently has been described as systemic inflammatory response syndrome .", "the complex nature of surgical infections , the multifaceted aspects of treatment , and the complexity of intensive care unit ( icu ) support makes evaluation of new diagnostic and therapeutic advances in this field very difficult .", "mannheim peritonitis index ( mpi ) is a scoring system used in peritonitis which is simple and cost - effective .", "mpi was originally derived from data collected from 1253 patients with peritonitis treated between 1963 and 1979 and was developed by discriminant analysis of 17 possible risk factors , by wacha , 8 of these were of prognostic relevance and was currently employed widely for predicting mortality from peritonitis .", "the information is collected at the time of admission and first laparotomy [ tables 1 and 2 ] .", "the study was done in fifty patients with peritonitis who presented at government medical college , amritsar , and were operated after taking informed consent .", "patients were followed up for the final outcome , and results were analyzed with the score of mpi .", "in this study , majority of patients belonged to age group of 21 - 50 years .", "the youngest patient was 8-year - old female , whereas to oldest was a 65-year - old male .", "there were 82% male patients and 18% female patients with the male to female ratio being 4.6:1 [ figure 1 ] .", "it was further found that mortality for female patients was 55.56% , and for male patients , it was 9.76% . in patients with localized peritonitis , there was no mortality as compared to diffuse peritonitis which had mortality of 20.45% .", "majority of the patients presented to hospital after 24 h ( 86% ) , and most commonly during 48 - 72 h ( 44% ) .", "the mortality of patients was highest ( 66% ) who were presented to hospital after 72 h , and it was 33% who were presented between 48 and 72 h. there was no mortality for patients who presented within 48 h. the mortality of patients who had clear , cloudy , and fecal character of exudates was 12.5% , 15% , and 100% , respectively . for patients with no malignancy ,", "patients having no organ failure preoperatively , mortality was 7.14% as compared to patients having organ failure , mortality was 75% [ table 3 ] .", "summary of risk factors of mannheim peritonitis index and mortality the study showed that mortality steadily increases with increase in mpi score showing statistically significant results [ p < 0.001 , figure 2 ] .", "various postoperative complications seen in the study group were wound sepsis , wound dehiscence , acute renal failure , pneumonitis , acute respiratory distress syndrome , and shock .", "the study also showed that morbidity steadily increases with mpi score [ p < 0.001 , figure 3 ] .", "mortality and mannheim peritonitis index score morbidity and mannheim peritonitis index score for a score of 27 , the sensitivity was 66.67% , specificity was 100% , and positive predictive value for mortality is 100% at an accuracy of 94% [ p < 0.001 , table 4 ] . sensitivity and specificity of mannheim peritonitis index score 27", "peritonitis , inflammation of serosal membrane lining the abdominal cavity and abdominal viscera , is associated with high mortality rate .", "peritonitis remains a hot spot for the surgeons despite advancements in surgical technique and intensive care treatment .", "various factors such as age , sex , duration , site of perforation , extent of peritonitis , and delay in surgical intervention are associated with morbidity and mortality .", "bacteria and digestive enzymes act on the peritoneal serosal surface lead to enzymatic digestion and necrosis and an outpouring of serum protein and electrolytes from the blood to the cavity .", "there is formation of exudate rich in granulocytes , which may be diffuse or confined to an abscess .", "systemically , there is paralysis of the bowel , hemoconcentration occurs , and alterations of the cardiac output due to the shift of fluids and later acidosis .", "intrapulmonary shunting , hypo- or hypercapnia , hypoxemia , progressive azotemia , acute tubular necrosis , weight loss by protein consumption , fall of body temperature , loss of heat production , and exhaustion are other complications that may lead to the death of the patient , if the process is not interrupted .", "a successful outcome depends on early surgical intervention , source control , and exclusive intraoperative peritoneal lavage .", "it may also depend on exact recognition of the seriousness of the disease , an accurate assessment , and classification of the patient 's risks . despite the surgical treatment and sophisticated icus , last generation antibiotics and a better understanding of pathophysiology of peritonitis mortality rate are still very high , i.e. , 1020% even in good institutions .", "early prognostic evaluation is necessary to select high - risk patients for more aggressive therapeutic procedures such as radical debridement , lavage systems , open management , and planned reoperations .", "classification of the severity of disease means the definition of groups by risk , and it is of confirmed value in both controlled and uncontrolled clinical trials . an accurate risk index classification is only method to settle a standard of comparison between groups of patient and different treatment methods which would allow further prospective adequate comparative study .", "the mpi has a definite score for an individual , which has a good accuracy .", "mpi can be used to define risk groups as well as predict outcome in patients with peritonitis , as indicated by various studies .", "showed patients who obtained < 21 points and more than 29 points , mortality rate was 6% and 50% , respectively .", "showed mortality at score of above 21 and 29 of 60% and 100% , whereas fgger et al . showed below 21 , no mortality ; between 21 and 29 , mortality was 29% ; and more than 29 , it was 100% .", "studies showed mortality among patients who obtained < 21 points varied between 0% and 2.3% , in the 2129-point group between 3.85% and 60% , and in patients with score of > 29  between 15% and 100% .", "billing 's meta - analysis showed the following mean mortality rates in the groups with < 21 points , between 21 and 29 points , and above 29 points : 2.3% ( 011% ) , 22.5% ( 10.650% ) , and 59.1% ( 4187% ) , respectively .", "other studies have also shown statistically significant relation of morbidity and mortality with increasing mpi score .", "yoshiko and masayuki showed patients with mpi score of 26 or less have mortality of 3.8% , whereas score of 26 or more had mortality of 41% .", "such difference in cutoff values might be due to different demographics and therapeutic options offered to patients at different institutions . in our study ,", "score of 27 showed the sensitivity , specificity , and positive predictive value at 66.67% , 100% , and 100% , respectively , at an accuracy of 94% . in our study showed mortality rate with score < 21 , 21 - 27 , and > 27 was 0% , 27.28% , and 100% , respectively .", "similarly , morbidity rate for patients with a score < 21 , 21 - 27 , and > 27 was 13.33% , 65.71% , and 100% , respectively .", "the predictions resulting from mpi were reliable , indicating stratification of risk groups can be done by probability intervals .", "study showed that age > 50 years out of a total of 79.3% death was seen in 85.2% and survival in 67.6% and p = 0.04 , thus showing that age > 50 years is a significant risk factor . in our study , patients < 50 years , mortality rate is 13.16% , and for patients more than 50 years , the mortality rate is 33.33% although it did not reach statistically significant values ( p = 0.113 ) .", "wittmann showed in his study , a high mortality rate ( 50% ) when the diagnosis of peritonitis was made after 48 h. the observed high frequency of patients with preoperative peritonitis duration longer than 24 h ( 65.5% ) was correlated with high death rate .", "our study also showed similar results with mortality of patients who presented between 48 and 72 h was 33% and > 72 h was 66% as compared to no mortality who presented within 24 and 48 h ( p < 0.001 ) .", "a study by basnet and sharma , out of the fifty patients included in the study , 21 ( 42% ) were female and 29 ( 58% ) were male .", "about 24.1% of the males and 19% of females requiring relaparotomy expired in the study , making female sex not a bad prognosticator in their study ( p = 0.100 ) .", "mortality for female patients was 55.56% as compared to male patients having mortality of 9.76% ( p = 0.001 ) .", "the study by notash et al . had mortality 17.5% mostly due to organ failure and septicemia .", "our study also showed organ failure , a significant risk factor as patients having organ failure had mortality of 75% as compared to no organ failure having mortality of 7.14% ( p < 0.001 ) .", "another study concluded that with age more than 50 years , late presentation , diffuse peritonitis , purulent exudate , noncolonic pathology , and organic failure associated with high mpi score and more mortality .", "this study proves that mpi scoring system is a simple and effective tool for assessing the morbidity and mortality in patients with peritonitis with statistically significant results .", "duration of pain > 24 h , organ failure on admission , female sex , and feculent exudate were found to be independently significant factors in predicting the mortality .", "such patients should be monitored closely with close attention to be given to support the vital systems . to conclude , mpi is a simple and reliable tool in predicting the outcome in cases of peritonitis ."], "abstract_text": ["<S> introduction : peritonitis is one of the most common problems in general surgery practice with high mortality rate </S>", "<S> . one of the reasons for high mortality is that peritonitis affects the general condition and leads to complications causing multiple organ failure , renal failure , sepsis , and postoperative ventilatory support . </S>", "<S> early prognostic evaluation of patients with peritonitis is desirable to select high - risk patients for intensive management and also to provide a reliable objective classification of severity and operative risk.patients and methods : fifty cases with diagnosis of peritonitis coming to government medical college , amritsar , were studied . </S>", "<S> stratification of these patients was done according to mannheim peritonitis index ( mpi ) , and their outcome was examined.results:mortality steadily increases with increase in mpi score . for patients with a score < 21 , </S>", "<S> the mortality rate was 0% ; for score 2127 , it was 27.28% ; and for score > 27 , it was 100% ( p </S>", "<S> < 0.001 ) . </S>", "<S> for patients with a score < 21 , the morbidity rate was 13.33% ; for score 21 - 27 , it was 65.71% ; and for score > 27 , it was 100% ( p < 0.001 ) . </S>", "<S> duration of pain > 24 h , organ failure on admission , female sex , and feculent exudate were found to be independently significant factors in predicting the mortality among the study population . for a score of 27 , </S>", "<S> the sensitivity was 66.67% , specificity was 100% , and positive predictive value for mortality is 100% at an accuracy of 94%.conclusion : this study proves that mpi scoring system is a simple and effective tool for assessing the morbidity and mortality in patients with peritonitis . </S>"], "labels": null, "section_names": ["I", "P", "R", "D", "C", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["it may be regarded as the localized equivalent of the systemic inflammatory response seen after any trigger of inflammation , which recently has been described as systemic inflammatory response syndrome .", "the complex nature of surgical infections , the multifaceted aspects of treatment , and the complexity of intensive care unit ( icu ) support makes evaluation of new diagnostic and therapeutic advances in this field very difficult .", "mannheim peritonitis index ( mpi ) is a scoring system used in peritonitis which is simple and cost - effective .", "mpi was originally derived from data collected from 1253 patients with peritonitis treated between 1963 and 1979 and was developed by discriminant analysis of 17 possible risk factors , by wacha , 8 of these were of prognostic relevance and was currently employed widely for predicting mortality from peritonitis .", "the information is collected at the time of admission and first laparotomy [ tables 1 and 2 ] ."], ["the study was done in fifty patients with peritonitis who presented at government medical college , amritsar , and were operated after taking informed consent .", "patients were followed up for the final outcome , and results were analyzed with the score of mpi ."], ["in this study , majority of patients belonged to age group of 21 - 50 years .", "the youngest patient was 8-year - old female , whereas to oldest was a 65-year - old male .", "there were 82% male patients and 18% female patients with the male to female ratio being 4.6:1 [ figure 1 ] .", "it was further found that mortality for female patients was 55.56% , and for male patients , it was 9.76% . in patients with localized peritonitis , there was no mortality as compared to diffuse peritonitis which had mortality of 20.45% .", "majority of the patients presented to hospital after 24 h ( 86% ) , and most commonly during 48 - 72 h ( 44% ) .", "the mortality of patients was highest ( 66% ) who were presented to hospital after 72 h , and it was 33% who were presented between 48 and 72 h. there was no mortality for patients who presented within 48 h. the mortality of patients who had clear , cloudy , and fecal character of exudates was 12.5% , 15% , and 100% , respectively . for patients with no malignancy ,", "patients having no organ failure preoperatively , mortality was 7.14% as compared to patients having organ failure , mortality was 75% [ table 3 ] .", "summary of risk factors of mannheim peritonitis index and mortality the study showed that mortality steadily increases with increase in mpi score showing statistically significant results [ p < 0.001 , figure 2 ] .", "various postoperative complications seen in the study group were wound sepsis , wound dehiscence , acute renal failure , pneumonitis , acute respiratory distress syndrome , and shock .", "the study also showed that morbidity steadily increases with mpi score [ p < 0.001 , figure 3 ] .", "mortality and mannheim peritonitis index score morbidity and mannheim peritonitis index score for a score of 27 , the sensitivity was 66.67% , specificity was 100% , and positive predictive value for mortality is 100% at an accuracy of 94% [ p < 0.001 , table 4 ] . sensitivity and specificity of mannheim peritonitis index score 27"], ["peritonitis , inflammation of serosal membrane lining the abdominal cavity and abdominal viscera , is associated with high mortality rate .", "peritonitis remains a hot spot for the surgeons despite advancements in surgical technique and intensive care treatment .", "various factors such as age , sex , duration , site of perforation , extent of peritonitis , and delay in surgical intervention are associated with morbidity and mortality .", "bacteria and digestive enzymes act on the peritoneal serosal surface lead to enzymatic digestion and necrosis and an outpouring of serum protein and electrolytes from the blood to the cavity .", "there is formation of exudate rich in granulocytes , which may be diffuse or confined to an abscess .", "systemically , there is paralysis of the bowel , hemoconcentration occurs , and alterations of the cardiac output due to the shift of fluids and later acidosis .", "intrapulmonary shunting , hypo- or hypercapnia , hypoxemia , progressive azotemia , acute tubular necrosis , weight loss by protein consumption , fall of body temperature , loss of heat production , and exhaustion are other complications that may lead to the death of the patient , if the process is not interrupted .", "a successful outcome depends on early surgical intervention , source control , and exclusive intraoperative peritoneal lavage .", "it may also depend on exact recognition of the seriousness of the disease , an accurate assessment , and classification of the patient 's risks . despite the surgical treatment and sophisticated icus , last generation antibiotics and a better understanding of pathophysiology of peritonitis mortality rate are still very high , i.e. , 1020% even in good institutions .", "early prognostic evaluation is necessary to select high - risk patients for more aggressive therapeutic procedures such as radical debridement , lavage systems , open management , and planned reoperations .", "classification of the severity of disease means the definition of groups by risk , and it is of confirmed value in both controlled and uncontrolled clinical trials . an accurate risk index classification is only method to settle a standard of comparison between groups of patient and different treatment methods which would allow further prospective adequate comparative study .", "the mpi has a definite score for an individual , which has a good accuracy .", "mpi can be used to define risk groups as well as predict outcome in patients with peritonitis , as indicated by various studies .", "showed patients who obtained < 21 points and more than 29 points , mortality rate was 6% and 50% , respectively .", "showed mortality at score of above 21 and 29 of 60% and 100% , whereas fgger et al . showed below 21 , no mortality ; between 21 and 29 , mortality was 29% ; and more than 29 , it was 100% .", "studies showed mortality among patients who obtained < 21 points varied between 0% and 2.3% , in the 2129-point group between 3.85% and 60% , and in patients with score of > 29  between 15% and 100% .", "billing 's meta - analysis showed the following mean mortality rates in the groups with < 21 points , between 21 and 29 points , and above 29 points : 2.3% ( 011% ) , 22.5% ( 10.650% ) , and 59.1% ( 4187% ) , respectively .", "other studies have also shown statistically significant relation of morbidity and mortality with increasing mpi score .", "yoshiko and masayuki showed patients with mpi score of 26 or less have mortality of 3.8% , whereas score of 26 or more had mortality of 41% .", "such difference in cutoff values might be due to different demographics and therapeutic options offered to patients at different institutions . in our study ,", "score of 27 showed the sensitivity , specificity , and positive predictive value at 66.67% , 100% , and 100% , respectively , at an accuracy of 94% . in our study showed mortality rate with score < 21 , 21 - 27 , and > 27 was 0% , 27.28% , and 100% , respectively .", "similarly , morbidity rate for patients with a score < 21 , 21 - 27 , and > 27 was 13.33% , 65.71% , and 100% , respectively .", "the predictions resulting from mpi were reliable , indicating stratification of risk groups can be done by probability intervals .", "study showed that age > 50 years out of a total of 79.3% death was seen in 85.2% and survival in 67.6% and p = 0.04 , thus showing that age > 50 years is a significant risk factor . in our study , patients < 50 years , mortality rate is 13.16% , and for patients more than 50 years , the mortality rate is 33.33% although it did not reach statistically significant values ( p = 0.113 ) .", "wittmann showed in his study , a high mortality rate ( 50% ) when the diagnosis of peritonitis was made after 48 h. the observed high frequency of patients with preoperative peritonitis duration longer than 24 h ( 65.5% ) was correlated with high death rate .", "our study also showed similar results with mortality of patients who presented between 48 and 72 h was 33% and > 72 h was 66% as compared to no mortality who presented within 24 and 48 h ( p < 0.001 ) .", "a study by basnet and sharma , out of the fifty patients included in the study , 21 ( 42% ) were female and 29 ( 58% ) were male .", "about 24.1% of the males and 19% of females requiring relaparotomy expired in the study , making female sex not a bad prognosticator in their study ( p = 0.100 ) .", "mortality for female patients was 55.56% as compared to male patients having mortality of 9.76% ( p = 0.001 ) .", "the study by notash et al . had mortality 17.5% mostly due to organ failure and septicemia .", "our study also showed organ failure , a significant risk factor as patients having organ failure had mortality of 75% as compared to no organ failure having mortality of 7.14% ( p < 0.001 ) .", "another study concluded that with age more than 50 years , late presentation , diffuse peritonitis , purulent exudate , noncolonic pathology , and organic failure associated with high mpi score and more mortality .", "this study proves that mpi scoring system is a simple and effective tool for assessing the morbidity and mortality in patients with peritonitis with statistically significant results .", "duration of pain > 24 h , organ failure on admission , female sex , and feculent exudate were found to be independently significant factors in predicting the mortality ."], ["such patients should be monitored closely with close attention to be given to support the vital systems . to conclude , mpi is a simple and reliable tool in predicting the outcome in cases of peritonitis ."], [""], [""]]}
{"article_id": "PMC3647415", "article_text": ["during 20092010 , serum samples from patients with suspected dengue were received by the fdoh for dengue diagnostic testing ; the samples came from 16 of florida s 67counties .", "specific , real - time rt - pcr ( 8) and igm anti - denv elisa ( 9 ) . samples with highly positive rt - pcr results were spread onto cultured ae .", "albopictus c6/36 cells , and the presence of virus and genome were confirmed by immunofluorescence ( 10 ) and rt - pcr , respectively ( 11 ) .", "isolates were further propagated and viral rna was extracted from culture supernatants by using the universal biorobot system ( qiagen , valencia , ca , usa ) .", "the envelope glycoprotein ( e ) gene was amplified ( technical appendix ) , and the e gene open - reading frame ( 1,485 bp ) was sequenced .", "multiple sequence alignments were performed by using the muscle module available in mega 5 ( www.megasoftware.net ) .", "evolutionary history was inferred by using maximum likelihood and phylogenetic trees constructed by using neighbor - joining methods .", "evolutionary distances were computed , and several e gene sequences from genbank were included in the phylogenetic tree to support tree topology by genotype ( technical appendix ) . in 2009 , five denv-1positive cases were identified by rt - pcr in key west .", "subsequently , in 2010 , the fdoh tested 195 serum samples by real - time rt - pcr .", "fifty - six ( 29% ) samples were positive for denv rna : denv-1 ( 37 [ 66% ] samples ) , denv-2 ( 13 [ 23% ] samples ) , denv-3 ( 3 [ 5% ] samples ) , and denv-4 ( 3 [ 5% ] samples ) .", "monroe county submitted 73 serum samples , of which 31 ( 42% ) had results positive for recent dengue infection : denv-1 was detected in 22 by rt - pcr , and 9 had positive igm anti - denv elisa results .", "none of the denv-1 patients from monroe county had a history of recent travel to a dengue - endemic region before the onset of symptoms .", "fifteen other florida counties submitted serum samples : 13 counties submitted < 10 specimens , 1 submitted 2030 specimens , and 1 submitted > 60specimens .", "denv-1 was found in 15 serum samples from 6 of these counties ; however , all the patients had a history of recent international travel .", "we sequenced the e gene of 12 denv-1 strains isolated in florida during 20092010 to determine their genetic relatedness ; of the 12 strains , 8 were from key west and 1 each was from dade , broward , orange , and pinellas  counties .", "in addition , 23 denv-1 e sequences published in genbank , including the 2010 key west isolates obtained from a blood donor and a mosquito pool ( 6,7 ) , were used to construct a maximum - likelihood phylogenetic tree .", "this phylogenetic analysis showed that all the florida denv-1 isolates belong to the american  african genotype ( genotype v ) ( 12,13 ) together with other viruses isolated throughout the americas ( figure 1 ) .  ", "maximum - likelihood phylogenetic tree of dengue virus type 1 , including isolates from key west , florida , usa , and representative isolates from 5 genotypes with global geographic distribution .", "solid circles , 8 key west viruses ( monroe county ) isolated during 20092010 ; solid diamonds , isolates from other florida counties ( dade , pinellas , orange , and broward counties ) .", "each taxon represents a single virus isolate and is labeled with the geographic origin and collection year .", "thirty - six envelope glycoprotein gene sequences obtained from genbank were included to support tree topology and identify genotypes . all genotypes except the american  african genotype ( v ) have been collapsed .", "key west denv-1 viruses grouped among central american viruses , which configure a distinct lineage separate from the caribbean viruses .", "this divergence between the central american and caribbean lineages is well supported by high bootstrap values .", "moreover , the key west and monroe county viruses grouped together and indicated a distinct sublineage supported by a high bootstrap value ( 99% ) , separating them from viruses isolated in dade , orange , pinellas , and broward counties that were more closely related to other central american viruses ( figure 1 ) .", "one 2009 isolate ( iq425062 ) from a key west patient is related to this group , suggesting a separate introduction of denv-1 in key west .", "the sequence similarity between the 2009 and 2010 key west strains was < 0.9% ; however , the evolutionary distance and taxa positions between the 2009 and 2010 strains presented in the phylogenetic tree suggests that the 2010 strains diverged from the 2009 strains .", "the observed differences between e gene sequences for the key west strains ( 20092010 ) and the rest of the strains in this phylogeny were < 2.1% with the other florida strains , < 1.2% with central american strains , and < 4.8% with the rest of the american  african genotype .", "evolutionary distances and the topology of the central american lineage suggest this lineage is the genetic origin of the florida denv-1 strain .", "most viruses isolated in monroe county diverged from the central american lineage into a distinct sublineage  the key west denv-1 strain associated with the 20092010 outbreak .", "the high level of genetic similarity among the viruses isolated in monroe county , their close evolutionary distances , and the lack of recent international travel for the case - patients suggest endemic transmission and microevolution of this denv .", "conversely , the scattered and separate phylogenetic positioning of virus strains from patients with travel - associated cases from other florida counties indicates a different origin from the majority of key west isolates .", "although the 2009 broward isolate ( jq425067 ) is positioned near the central american lineage , the low bootstrap value ( 53% ) does not support lineage ancestry .", "the epidemiologic and phylogenetic evidence suggests that the 2010 cases appeared to be a continuation of the 2009 outbreak . unlike cases along the texas  mexico border ( 14 ) ,", "in addition , denv-1 was detected in a blood donation from the monroe county in 2010 , further supporting local transmission of denv ( 7 ) .", "collectively , these findings indicate that endemic denv-1 was transmitted in key west over a period of > 2 years .", "table showing the dengue virus type 1 ( denv-1 ) envelope gene reverse transcription pcr and another showing denv-1 strains used in a study of the genetic relatedness of dengue viruses in key west , florida , usa , 20092010 ."], "abstract_text": ["<S> sequencing of dengue virus type 1 ( denv-1 ) strains isolated in key west / monroe county , florida , indicate endemic transmission for > 2 years of a distinct and predominant sublineage of the american  african genotype . </S>", "<S> denv-1 strains isolated elsewhere in florida grouped within a separate central american lineage . </S>", "<S> findings indicate endemic transmission of denv into the continental united states . </S>"], "labels": null, "section_names": ["The Study", "Conclusions", "None"], "sections": [["during 20092010 , serum samples from patients with suspected dengue were received by the fdoh for dengue diagnostic testing ; the samples came from 16 of florida s 67counties .", "specific , real - time rt - pcr ( 8) and igm anti - denv elisa ( 9 ) . samples with highly positive rt - pcr results were spread onto cultured ae .", "albopictus c6/36 cells , and the presence of virus and genome were confirmed by immunofluorescence ( 10 ) and rt - pcr , respectively ( 11 ) .", "isolates were further propagated and viral rna was extracted from culture supernatants by using the universal biorobot system ( qiagen , valencia , ca , usa ) .", "the envelope glycoprotein ( e ) gene was amplified ( technical appendix ) , and the e gene open - reading frame ( 1,485 bp ) was sequenced .", "multiple sequence alignments were performed by using the muscle module available in mega 5 ( www.megasoftware.net ) .", "evolutionary history was inferred by using maximum likelihood and phylogenetic trees constructed by using neighbor - joining methods .", "evolutionary distances were computed , and several e gene sequences from genbank were included in the phylogenetic tree to support tree topology by genotype ( technical appendix ) . in 2009 , five denv-1positive cases were identified by rt - pcr in key west .", "subsequently , in 2010 , the fdoh tested 195 serum samples by real - time rt - pcr .", "fifty - six ( 29% ) samples were positive for denv rna : denv-1 ( 37 [ 66% ] samples ) , denv-2 ( 13 [ 23% ] samples ) , denv-3 ( 3 [ 5% ] samples ) , and denv-4 ( 3 [ 5% ] samples ) .", "monroe county submitted 73 serum samples , of which 31 ( 42% ) had results positive for recent dengue infection : denv-1 was detected in 22 by rt - pcr , and 9 had positive igm anti - denv elisa results .", "none of the denv-1 patients from monroe county had a history of recent travel to a dengue - endemic region before the onset of symptoms .", "fifteen other florida counties submitted serum samples : 13 counties submitted < 10 specimens , 1 submitted 2030 specimens , and 1 submitted > 60specimens .", "denv-1 was found in 15 serum samples from 6 of these counties ; however , all the patients had a history of recent international travel .", "we sequenced the e gene of 12 denv-1 strains isolated in florida during 20092010 to determine their genetic relatedness ; of the 12 strains , 8 were from key west and 1 each was from dade , broward , orange , and pinellas  counties .", "in addition , 23 denv-1 e sequences published in genbank , including the 2010 key west isolates obtained from a blood donor and a mosquito pool ( 6,7 ) , were used to construct a maximum - likelihood phylogenetic tree .", "this phylogenetic analysis showed that all the florida denv-1 isolates belong to the american  african genotype ( genotype v ) ( 12,13 ) together with other viruses isolated throughout the americas ( figure 1 ) .  ", "maximum - likelihood phylogenetic tree of dengue virus type 1 , including isolates from key west , florida , usa , and representative isolates from 5 genotypes with global geographic distribution .", "solid circles , 8 key west viruses ( monroe county ) isolated during 20092010 ; solid diamonds , isolates from other florida counties ( dade , pinellas , orange , and broward counties ) .", "each taxon represents a single virus isolate and is labeled with the geographic origin and collection year .", "thirty - six envelope glycoprotein gene sequences obtained from genbank were included to support tree topology and identify genotypes . all genotypes except the american  african genotype ( v ) have been collapsed .", "key west denv-1 viruses grouped among central american viruses , which configure a distinct lineage separate from the caribbean viruses .", "this divergence between the central american and caribbean lineages is well supported by high bootstrap values .", "moreover , the key west and monroe county viruses grouped together and indicated a distinct sublineage supported by a high bootstrap value ( 99% ) , separating them from viruses isolated in dade , orange , pinellas , and broward counties that were more closely related to other central american viruses ( figure 1 ) .", "one 2009 isolate ( iq425062 ) from a key west patient is related to this group , suggesting a separate introduction of denv-1 in key west .", "the sequence similarity between the 2009 and 2010 key west strains was < 0.9% ; however , the evolutionary distance and taxa positions between the 2009 and 2010 strains presented in the phylogenetic tree suggests that the 2010 strains diverged from the 2009 strains .", "the observed differences between e gene sequences for the key west strains ( 20092010 ) and the rest of the strains in this phylogeny were < 2.1% with the other florida strains , < 1.2% with central american strains , and < 4.8% with the rest of the american  african genotype ."], ["evolutionary distances and the topology of the central american lineage suggest this lineage is the genetic origin of the florida denv-1 strain .", "most viruses isolated in monroe county diverged from the central american lineage into a distinct sublineage  the key west denv-1 strain associated with the 20092010 outbreak .", "the high level of genetic similarity among the viruses isolated in monroe county , their close evolutionary distances , and the lack of recent international travel for the case - patients suggest endemic transmission and microevolution of this denv .", "conversely , the scattered and separate phylogenetic positioning of virus strains from patients with travel - associated cases from other florida counties indicates a different origin from the majority of key west isolates .", "although the 2009 broward isolate ( jq425067 ) is positioned near the central american lineage , the low bootstrap value ( 53% ) does not support lineage ancestry .", "the epidemiologic and phylogenetic evidence suggests that the 2010 cases appeared to be a continuation of the 2009 outbreak . unlike cases along the texas  mexico border ( 14 ) ,", "in addition , denv-1 was detected in a blood donation from the monroe county in 2010 , further supporting local transmission of denv ( 7 ) .", "collectively , these findings indicate that endemic denv-1 was transmitted in key west over a period of > 2 years ."], ["table showing the dengue virus type 1 ( denv-1 ) envelope gene reverse transcription pcr and another showing denv-1 strains used in a study of the genetic relatedness of dengue viruses in key west , florida , usa , 20092010 ."]]}
{"article_id": "PMC4427820", "article_text": ["corneal epithelium is able to alter its thickness to mask subepithelial stromal irregularities and maintain a smooth anterior surface of the eye .", "laser refractive surgery such as lasik alters the anterior corneal contour and leads to remodeling of corneal epithelium .", "we estimated the extent of the epithelial remodeling after lasik with a mathematical model in a previous study .", "however , in that study , we did not have the capability to measure the corneal epithelial thickness to validate the model directly .", "instead , we constructed the model constants based on regression in manifest refraction after lasik .", "direct measurement of epithelial thickness change may help to better understand corneal epithelial remodeling after lasik and improve lasik ablation patterns with less regression and surgery - induced aberrations .", "previous studies used confocal microscopy to measure epithelial thickness , but the number of points measured was limited   and the measurement was time consuming . very high - frequency digital ultrasound was also used to map corneal epithelium and stromal thickness [ 4 , 5 ] . however , because ultrasound can not pass through air , this technique required immersing the cornea in a fluid bath .", "though both confocal microscopy and very high - frequency digital ultrasound are feasible in measuring corneal epithelial thickness , they are not often used in routine lasik because they required touching the cornea .", "optical coherence tomography ( oct ) is a noncontact imaging technique based on principles of low - coherence interferometry .", "the current generation of oct is based on fourier - domain technique [ 6 , 7 ] . in a recent article", ", we demonstrated that a commercial fourier - domain oct could automatically map the corneal epithelial thickness with good repeatability . in this study , we use this algorithm to map corneal epithelial thickness for eyes before and after myopic lasik surgeries .", "the changes in epithelial thickness were used to validate the smoothing model we mentioned above .", "this prospective observational study was conducted at doheny eye institute , los angeles , ca .", "the lasik subjects enrolled in this study had no history of eye surgery and were comprehensively examined to exclude any eye diseases including dry eye .", "soft contact lenses wearers were asked to stop wearing contact lenses at least two weeks prior to lasik .", "the treatment of study subjects was in accordance with the tenets of the declaration of helsinki .", "the laser settings were based on manifest and cycloplegic refractions calculated at the corneal plane .", "the lasik flap was created with a 60  khz femtosecond laser ( intralase , abbott medical optics , santa ana , ca ) .", "the femtosecond flap thickness was programmed to 110  m with a diameter of 9.0  mm and a 70-degree angled side cut .", "the stromal ablations were performed with visx star s4 ir customvue excimer laser ( abbott medical optics , santa ana , ca ) .", "the optical zone was set to 6.5  mm in diameter centered on the pupil center with blend / transition zones up to 8.0  mm . to measure epithelial thickness before and after lasik , a commercial fourier - domain oct system ( rtvue , optovue inc . ,", "fremont , ca ) with a speed of 26,000 axial scans per second was used .", "a  pachymetry + cpwr  scan pattern ( 6 mm scan diameter , 8 radials , 1024 axial - scans each , repeated 5 times ) centered at the pupil center was used to map the cornea .", "the subjects were asked to look straight ahead and fixate on the internal fixation target of the oct system .", "the oct scan pattern was repeated 2 times on each eye during the same visit .", "the algorithm was described in a previous article   and was available in the commercial rtvue software .", "the average epithelial thickness over the central 1  mm diameter , 1~2  mm , 2~3  mm , 3~4  mm , 4~5  mm , and 5~6  mm annular zones , was used in the analysis .", "correlation of lasik - induced change in epithelial thickness with the amount of spherical equivalent of lasik correction was investigated . a smoothing constant  ", "based on our epithelial smoothing model , the change in epithelial thickness could be calculated by applying a first - order , 2-dimensional butterworth low - pass filter to the ablation profile .", "the cutoff frequency of the butterworth filter was the reciprocal of the smoothing constant . if we simulated the ablation profile for 1  d special myopic lasik using munnerlyn algorithm   and assumed the optical zone diameter to be 6.5  mm , the smoothing contact could be estimated .", "the smoothing constant has a unit of length and can be thought of as the radius over which epithelial smoothing occurs .", "ablation simulations were performed using matlab software version 5.3 ( the mathworks , inc . ,", "paired t - test was used to compare the difference of preoperative and postoperative epithelial thickness .", "generalized estimating equation was used to account for the intereye correlation in the variance of t - test .", "statistical analysis was performed using the microsoft excel and sas 9.1 ( sas institute inc . , cary , nc , usa ) .", "nineteen eyes ( 10 right eyes , 9 left eyes ) from 11 myopic lasik patients ( 6 women , 5 men ) were analyzed in the study .", "the spherical equivalent of lasik correction ranged from 1.69  d to 6.75  d ( mean : 4.39  1.63  d ) .", "corneal epithelium was thicker on the inferior side compared to that on the superior side both before ( figure 1(a ) ) and after lasik ( figure 1(b ) ) .", "the average central epithelial thickness was measured to be 52.6  4.1  m ( 40.9~60.6  m ) before lasik and 56.2  4.3  m ( 50.0~65.5  m ) 3 months after lasik ( p = 0.013 , figure 2 ) .", "the average epithelial thickness at 5~6  mm annular zone was 51.6  6.6  m ( 39.6~67.4  m ) before lasik and 54.8  4.3  m ( 49.8~68.0  m ) 3 months after lasik ( p = 0.024 , figure 2 ) .", "the epithelial thickening reached maximum at about 4  mm diameter and tapered off toward the peripheral ( figure 3 ) .", "the change in average central epithelial thickness was significantly correlated with lasik spherical equivalent setting ( figure 4 ) .", "the slope of 1.15 indicated that , for every diopter of myopic lasik correction , the central epithelial thickness increased by 1.15  m , which corresponded to a smoothing constant of about 0.46  mm . based on this smoothing constant", "pattern ; that is , the maximum epithelial thickening occurred at an annular area around the center ( figure 5 ) .", "previous studies have demonstrated that it is feasible to use oct to measure the thickness of different layers of the cornea , such as the epithelium , stroma , and lasik flap using time - domain oct . however , the axial resolution of the earlier oct systems was low and the manual computer - caliper measurement was time consuming . in this study , we used newer fourier - domain oct which has faster scan rate and higher axial resolution with automated measurement of epithelial thickness .", "the finding agreed with previous oct studies [ 8 , 12 ] as well as results with very high - frequency digital ultrasound .", "the asymmetry might be caused by the movement of upper eyelids during blinking [ 4 , 13 ] .", "similar inferior / superior asymmetry in the thickness of corneal epithelium was also found after lasik ( figure 1(b ) ) .", "the average central epithelial thickening was about 3.6  m at the 3-month followup for a mean spherical equivalent correction of 4.39  d. this finding is in agreement with previous studies that demonstrated the correlation between central epithelial thickening and the amount of myopia correction [ 5 , 1419 ] . using confocal microscopy , spadea et al .   demonstrated that epithelial thickness increased within the first week after lasik , with a maximum increase of approximately 6.5  m by the third month for a mean spherical equivalent correction of 10.48  d. using very high - frequency digital ultrasound , reinstein et al . found that there was a central approximately 5  mm zone of epithelial thickening of up to 7.5  m 1 year after lasik for a mean spherical equivalent correction of 3.34  d. in a recent study where a similar oct system was used , kanellopoulos and asimellis   found that increases in central ( 0~2  mm diameter ) , midperipheral ( 5  mm diameter ) , and overall mean epithelial thickness appeared to be in almost a linear correlation with the amount of targeted myopic correction ( 1.39  m / d for midperipheral region ) .", "the authors hypothesized that epithelial hyperplasia might be caused by a thinned cornea which was biomechanically unstable .", "this hypothesis was also supported by a study of epithelial thickness changes after collagen cross - linking ( cxl ) .", "however , the epithelial thickness changes could actually be explained by the simultaneous topography - guided ablation to reduce the cone . in other words ,", "the epithelial thickness changes could be a response to focal curvature changes   in addition to corneal biomechanical properties .", "the average central epithelial thickening was significantly correlated with lasik spherical equivalent setting . for every diopter of", "the 1.15  m per diopter epithelial thickening was matched to a smoothing constant of 0.46  mm .", "this value was larger than our previous estimate ( 0.32  mm )   probably because of the mix of spherical astigmatism subjects in this dataset .", "our results showed more epithelial thickening centrally ( 1  mm diameter zone ) than that paracentrally ( 5 - 6  mm annular zone ) .", "this does not agree with a previous study with very high - frequency digital ultrasound   where epithelial thinning was observed between the 5.6  mm and 8  mm diameters except superiorly .", "we speculated that the discrepancy may be caused by larger optical zone ( 6.5  mm ) and use of transition zone ( up to 8.0  mm diameter ) in our study compared to the 6  mm optical zone used in the previous study . in figure 2 , if we could measure epithelial thickness beyond 6  mm diameter and the plot could be extrapolated , epithelial thinning would have been observed at the periphery .", "however , because we did not have access to the proprietary ablation profiles of the laser companies nor had our current oct system the ability to measure a larger area , we could not provide a more concrete explanation for the disagreement . on the other hand , both clinical data ( figure 3 ) and simulation ( figure 5 ) showed that the maximum epithelial thickening occurred at an annular area about 3~4  mm in diameter , not at the center .", "the difference was more obvious on the average epithelial change map from the clinical data than the simulation .", "we speculated the reason being that the actual lasik ablation pattern might precompensate for the laser - induced spherical aberration , which meant that the actual ablation at the paracentral area would be deeper than munnerlyn 's algorithm used in the simulation which did not account for spherical aberrations .", "in addition , after the compensatory remodeling of corneal epithelium to surface curvature changes , the area with increased epithelial thickening was most likely to be annular because of the ring shape of spherical aberration .", "one limitation of this study was that it only included epithelial thickness measurements 3 months after lasik .", "however , central epithelial thickening has been reported 1 year   up to 7 years   after excimer laser ablation , all of which found no statistically significant change in central epithelial thickness after 3 months .", "therefore , it may be reasonable to assume that the epithelial thickness is stable 3 months after lasik .", "it also should be pointed out that , besides focal corneal curvature , changes in corneal biomechanical properties , such as dry eye   and cross - linking [ 23 , 24 ] , may result in corneal remodeling as well .", "therefore , it is important to take multiple factors into consideration if they are mixed . in summary ,", "fourier - domain oct was demonstrated to be a valuable tool for noncontact measurements of corneal epithelial thicknesses change caused by lasik .", "the maximum epithelial thickening occurred at an annular area about 3~4  mm in diameter .", "however , wider scans are needed to measure epithelial thickness change toward the edge of the ablation zone ."], "abstract_text": ["<S> \n purpose . to quantify corneal epithelial thickness changes after myopic lasik by oct </S>", "<S> . methods . </S>", "<S> epithelial thickness before and after myopic lasik were measured by a fourier - domain oct system . </S>", "<S> average central ( within 1  mm diameter ) and paracentral epithelial thickness ( 5~6  mm diameter ) before and after lasik were compared . </S>", "<S> correlation between central epithelial thickness change and laser spherical equivalent setting was evaluated . </S>", "<S> an epithelial smoothing constant was estimated based on a mathematical model published previously . </S>", "<S> results . </S>", "<S> nineteen eyes from 11 subjects were included in the study . </S>", "<S> eyes had myopic lasik ranging from 1.69  d to 6.75  d spherical equivalent . </S>", "<S> the average central epithelial thickness was 52.6  4.1  m before lasik and 56.2  4.3  m 3 months after lasik ( p = 0.002 ) . </S>", "<S> the average paracentral epithelial thickness was 51.6  6.6  m before lasik and 54.8  4.3  m 3 months after lasik ( p = 0.007 ) . </S>", "<S> the change in average central epithelial thickness was correlated with laser spherical equivalent ( r2 = 0.40 , p = 0.028 ) . </S>", "<S> the epithelial smoothing constant was estimated to be 0.46  mm . </S>", "<S> conclusions . corneal epithelial thickens </S>", "<S> centrally and paracentrally after myopic lasik . the extent of epithelial remodeling correlated with the amount of lasik correction and could be predicted by a mathematical model . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["corneal epithelium is able to alter its thickness to mask subepithelial stromal irregularities and maintain a smooth anterior surface of the eye .", "laser refractive surgery such as lasik alters the anterior corneal contour and leads to remodeling of corneal epithelium .", "we estimated the extent of the epithelial remodeling after lasik with a mathematical model in a previous study .", "however , in that study , we did not have the capability to measure the corneal epithelial thickness to validate the model directly .", "instead , we constructed the model constants based on regression in manifest refraction after lasik .", "direct measurement of epithelial thickness change may help to better understand corneal epithelial remodeling after lasik and improve lasik ablation patterns with less regression and surgery - induced aberrations .", "previous studies used confocal microscopy to measure epithelial thickness , but the number of points measured was limited   and the measurement was time consuming . very high - frequency digital ultrasound was also used to map corneal epithelium and stromal thickness [ 4 , 5 ] . however , because ultrasound can not pass through air , this technique required immersing the cornea in a fluid bath .", "though both confocal microscopy and very high - frequency digital ultrasound are feasible in measuring corneal epithelial thickness , they are not often used in routine lasik because they required touching the cornea .", "optical coherence tomography ( oct ) is a noncontact imaging technique based on principles of low - coherence interferometry .", "the current generation of oct is based on fourier - domain technique [ 6 , 7 ] . in a recent article", ", we demonstrated that a commercial fourier - domain oct could automatically map the corneal epithelial thickness with good repeatability . in this study , we use this algorithm to map corneal epithelial thickness for eyes before and after myopic lasik surgeries .", "the changes in epithelial thickness were used to validate the smoothing model we mentioned above ."], ["this prospective observational study was conducted at doheny eye institute , los angeles , ca .", "the lasik subjects enrolled in this study had no history of eye surgery and were comprehensively examined to exclude any eye diseases including dry eye .", "soft contact lenses wearers were asked to stop wearing contact lenses at least two weeks prior to lasik .", "the treatment of study subjects was in accordance with the tenets of the declaration of helsinki .", "the laser settings were based on manifest and cycloplegic refractions calculated at the corneal plane .", "the lasik flap was created with a 60  khz femtosecond laser ( intralase , abbott medical optics , santa ana , ca ) .", "the femtosecond flap thickness was programmed to 110  m with a diameter of 9.0  mm and a 70-degree angled side cut .", "the stromal ablations were performed with visx star s4 ir customvue excimer laser ( abbott medical optics , santa ana , ca ) .", "the optical zone was set to 6.5  mm in diameter centered on the pupil center with blend / transition zones up to 8.0  mm . to measure epithelial thickness before and after lasik , a commercial fourier - domain oct system ( rtvue , optovue inc . ,", "fremont , ca ) with a speed of 26,000 axial scans per second was used .", "a  pachymetry + cpwr  scan pattern ( 6 mm scan diameter , 8 radials , 1024 axial - scans each , repeated 5 times ) centered at the pupil center was used to map the cornea .", "the subjects were asked to look straight ahead and fixate on the internal fixation target of the oct system .", "the oct scan pattern was repeated 2 times on each eye during the same visit .", "the algorithm was described in a previous article   and was available in the commercial rtvue software .", "the average epithelial thickness over the central 1  mm diameter , 1~2  mm , 2~3  mm , 3~4  mm , 4~5  mm , and 5~6  mm annular zones , was used in the analysis .", "correlation of lasik - induced change in epithelial thickness with the amount of spherical equivalent of lasik correction was investigated . a smoothing constant  ", "based on our epithelial smoothing model , the change in epithelial thickness could be calculated by applying a first - order , 2-dimensional butterworth low - pass filter to the ablation profile .", "the cutoff frequency of the butterworth filter was the reciprocal of the smoothing constant . if we simulated the ablation profile for 1  d special myopic lasik using munnerlyn algorithm   and assumed the optical zone diameter to be 6.5  mm , the smoothing contact could be estimated .", "the smoothing constant has a unit of length and can be thought of as the radius over which epithelial smoothing occurs .", "ablation simulations were performed using matlab software version 5.3 ( the mathworks , inc . ,", "paired t - test was used to compare the difference of preoperative and postoperative epithelial thickness .", "generalized estimating equation was used to account for the intereye correlation in the variance of t - test .", "statistical analysis was performed using the microsoft excel and sas 9.1 ( sas institute inc . , cary , nc , usa ) ."], ["nineteen eyes ( 10 right eyes , 9 left eyes ) from 11 myopic lasik patients ( 6 women , 5 men ) were analyzed in the study .", "the spherical equivalent of lasik correction ranged from 1.69  d to 6.75  d ( mean : 4.39  1.63  d ) .", "corneal epithelium was thicker on the inferior side compared to that on the superior side both before ( figure 1(a ) ) and after lasik ( figure 1(b ) ) .", "the average central epithelial thickness was measured to be 52.6  4.1  m ( 40.9~60.6  m ) before lasik and 56.2  4.3  m ( 50.0~65.5  m ) 3 months after lasik ( p = 0.013 , figure 2 ) .", "the average epithelial thickness at 5~6  mm annular zone was 51.6  6.6  m ( 39.6~67.4  m ) before lasik and 54.8  4.3  m ( 49.8~68.0  m ) 3 months after lasik ( p = 0.024 , figure 2 ) .", "the epithelial thickening reached maximum at about 4  mm diameter and tapered off toward the peripheral ( figure 3 ) .", "the change in average central epithelial thickness was significantly correlated with lasik spherical equivalent setting ( figure 4 ) .", "the slope of 1.15 indicated that , for every diopter of myopic lasik correction , the central epithelial thickness increased by 1.15  m , which corresponded to a smoothing constant of about 0.46  mm . based on this smoothing constant", "pattern ; that is , the maximum epithelial thickening occurred at an annular area around the center ( figure 5 ) ."], ["previous studies have demonstrated that it is feasible to use oct to measure the thickness of different layers of the cornea , such as the epithelium , stroma , and lasik flap using time - domain oct . however , the axial resolution of the earlier oct systems was low and the manual computer - caliper measurement was time consuming . in this study , we used newer fourier - domain oct which has faster scan rate and higher axial resolution with automated measurement of epithelial thickness .", "the finding agreed with previous oct studies [ 8 , 12 ] as well as results with very high - frequency digital ultrasound .", "the asymmetry might be caused by the movement of upper eyelids during blinking [ 4 , 13 ] .", "similar inferior / superior asymmetry in the thickness of corneal epithelium was also found after lasik ( figure 1(b ) ) .", "the average central epithelial thickening was about 3.6  m at the 3-month followup for a mean spherical equivalent correction of 4.39  d. this finding is in agreement with previous studies that demonstrated the correlation between central epithelial thickening and the amount of myopia correction [ 5 , 1419 ] . using confocal microscopy , spadea et al .   demonstrated that epithelial thickness increased within the first week after lasik , with a maximum increase of approximately 6.5  m by the third month for a mean spherical equivalent correction of 10.48  d. using very high - frequency digital ultrasound , reinstein et al . found that there was a central approximately 5  mm zone of epithelial thickening of up to 7.5  m 1 year after lasik for a mean spherical equivalent correction of 3.34  d. in a recent study where a similar oct system was used , kanellopoulos and asimellis   found that increases in central ( 0~2  mm diameter ) , midperipheral ( 5  mm diameter ) , and overall mean epithelial thickness appeared to be in almost a linear correlation with the amount of targeted myopic correction ( 1.39  m / d for midperipheral region ) .", "the authors hypothesized that epithelial hyperplasia might be caused by a thinned cornea which was biomechanically unstable .", "this hypothesis was also supported by a study of epithelial thickness changes after collagen cross - linking ( cxl ) .", "however , the epithelial thickness changes could actually be explained by the simultaneous topography - guided ablation to reduce the cone . in other words ,", "the epithelial thickness changes could be a response to focal curvature changes   in addition to corneal biomechanical properties .", "the average central epithelial thickening was significantly correlated with lasik spherical equivalent setting . for every diopter of", "the 1.15  m per diopter epithelial thickening was matched to a smoothing constant of 0.46  mm .", "this value was larger than our previous estimate ( 0.32  mm )   probably because of the mix of spherical astigmatism subjects in this dataset .", "our results showed more epithelial thickening centrally ( 1  mm diameter zone ) than that paracentrally ( 5 - 6  mm annular zone ) .", "this does not agree with a previous study with very high - frequency digital ultrasound   where epithelial thinning was observed between the 5.6  mm and 8  mm diameters except superiorly .", "we speculated that the discrepancy may be caused by larger optical zone ( 6.5  mm ) and use of transition zone ( up to 8.0  mm diameter ) in our study compared to the 6  mm optical zone used in the previous study . in figure 2 , if we could measure epithelial thickness beyond 6  mm diameter and the plot could be extrapolated , epithelial thinning would have been observed at the periphery .", "however , because we did not have access to the proprietary ablation profiles of the laser companies nor had our current oct system the ability to measure a larger area , we could not provide a more concrete explanation for the disagreement . on the other hand , both clinical data ( figure 3 ) and simulation ( figure 5 ) showed that the maximum epithelial thickening occurred at an annular area about 3~4  mm in diameter , not at the center .", "the difference was more obvious on the average epithelial change map from the clinical data than the simulation .", "we speculated the reason being that the actual lasik ablation pattern might precompensate for the laser - induced spherical aberration , which meant that the actual ablation at the paracentral area would be deeper than munnerlyn 's algorithm used in the simulation which did not account for spherical aberrations .", "in addition , after the compensatory remodeling of corneal epithelium to surface curvature changes , the area with increased epithelial thickening was most likely to be annular because of the ring shape of spherical aberration .", "one limitation of this study was that it only included epithelial thickness measurements 3 months after lasik .", "however , central epithelial thickening has been reported 1 year   up to 7 years   after excimer laser ablation , all of which found no statistically significant change in central epithelial thickness after 3 months .", "therefore , it may be reasonable to assume that the epithelial thickness is stable 3 months after lasik .", "it also should be pointed out that , besides focal corneal curvature , changes in corneal biomechanical properties , such as dry eye   and cross - linking [ 23 , 24 ] , may result in corneal remodeling as well .", "therefore , it is important to take multiple factors into consideration if they are mixed . in summary ,", "fourier - domain oct was demonstrated to be a valuable tool for noncontact measurements of corneal epithelial thicknesses change caused by lasik .", "the maximum epithelial thickening occurred at an annular area about 3~4  mm in diameter .", "however , wider scans are needed to measure epithelial thickness change toward the edge of the ablation zone ."]]}
{"article_id": "PMC3703355", "article_text": ["this is particularly the case of the health of people living in contaminated site(s ) ( cs ) which is affected by the legacy of past industrialization and current industrial activities , often in absence of environmental remediation .", "european community legislation addresses the concept of cs only in the context of the thematic strategy for soil protection   and the soil framework directive proposed by the european commission ( ec ) in 2006 ; for details on the legal framework and definitions at eu level refer to who 2013 . according to 2007 estimates ,", "soil contamination requiring clean up is present in approximately 250,000 sites in the european environment agency ( eea ) member countries .", "although main polluting sources may vary across europe , industrial production and commercial activities , oil industry and waste disposal and treatment are reported to be the major ones .", "national reports indicate that heavy metals and mineral oils are the main soil contaminants , while mineral oils and chlorinated hydrocarbons are the most frequent pollutants found in groundwater .", "a general definition , following the public health perspective , is  areas hosting or having hosted human activities which have produced or might produce environmental contamination of soil , surface or groundwater , air , and food chain , resulting or being able to result in human health impacts  . given this definition , an area affected by a single chemical contamination of a single environmental matrix ( e.g. , the soil contamination caused by a given pesticide ) and a large area with soil , water , air , and food chain contamination by multiple chemicals ( e.g. , the contamination caused by long - term emissions of a petrochemical complex ) can be both considered contaminated sites .  ", "there are several approaches and methods for assessing the health impact of national priority contaminated sites ( npcs ) . on the one hand", ", one can apply risk assessment techniques , where available data on the degree of contamination ( typically measures of concentration of specific hazardous chemical agents ) are used to quantitatively estimate the risk of occurrence of health endpoints causally associated with the agents ; on the other hand , epidemiological approaches applicable to npcss involve the inclusive collection of available health information of resident population , and various agents and exposures . in these assessments ,", "a first descriptive level is based on epidemiological tools that do not require an ad hoc collection of data and aims at describing the health profile of populations documenting ascertained or suspected associations with local environmental risks and the potentiality to provide efficient answers .", "more detailed analyses can be carried out , at a higher level of definition , by collecting specific data on health outcomes and/or on exposure .  ", "sentieri project ( epidemiological study of residents in italian contaminated sites ) [ 6 , 7 ] is an example of first level descriptive approach adopting an ecological study design , looking at the aggregate population level rather than at individual level .", "sentieri project is a national project developed to evaluate the health profile of populations residing in the italian sites of national interest for environmental remediation - national priority contaminated sites ( npcs ) .", "these sites were labelled as sites of national interest because of their substantial contamination , documented in qualitative and/or quantitative terms , and the consequent potential impact on the health of residents .", "the methods proposed under the approach exemplified by sentieri can be generalized and applied to other npcss .  ", "this paper 's objective is to present the rationale and methods underlying sentieri project and to describe data and resources required to apply a similar approach in other countries .", "when studying how the environment can adversely affect human health , it is usually very difficult to identify clear cause - effect relationships because they are characterized by multicausality with different strengths of association .", "in addition , these relationships are influenced by individual factors ( e.g. , genetic , diet , life - style , occupation , and socioeconomic status ) that can also have a role on both exposure level and disease development .", "it describes the health profile of residents in contaminated sites through small area analysis by applying the multistep procedure described in the following sections .", "a selection of epidemiologic terms are defined in glossary to facilitate the reading of the paper .", "as a first step , npcss to be studied should be chosen , and the criteria adopted to define npcs / npcss are clearly indicated . the npcs selection will depend on the aims of the study to be undertaken , on the availability of the npccs - related information , and on any other consideration researchers would make and consider appropriate .", "in many instances , npcss to be studied are chosen by third parties , such as an environmental authority , by public concern , and media pressure .", "it is advisable that the criteria used throughout this phase are clearly stated . in most instances npcss", "are characterized by the presence of numerous and different environmental sources of contamination possibly leading to human exposures .", "all the available npcs data should be collected and described in a standardized , homogeneous way .", "geographical characteristics , extension of the contaminated area , and demographic information about residents potentially affected should be listed .", "detailed description of contamination characteristics should be included as well as the presence of industries and all other human activities that have contributed to the environmental deterioration of the npcs .", "researchers will specify the sources used for this task : scientific reports , acts , and so on .", "populations at risk can be identified as people living in the neighborhood of npcss sources or living in areas defined as contaminated .", "typically , the distance from the areas affected by contamination is used , but also dispersion modelling results are used . in the latter case , the definition of the areas most affected by contamination , and the consequent identification of at - risk populations , depends on the accuracy of the model .", "there are several models used to evaluate the areas affected by contaminants ; their implementation and improvement depend on the available information on several parameters .", "for example , in the case of emissions into the air from oil refineries , the parameters to be considered should be characteristics of emission sources : for example , height , flow rate , composition of emissions , exit temperature , local orography , and meteorological conditions .", "areas characterised by contamination processes different from direct industrial emissions require other parameters . in the case of landfills", "many early disposal sites did not have liners to trap rainwater that percolate through the landfill , and some newer landfills have liners that leak .", "the percolating water leaches toxic chemicals ( e.g. , from batteries , electronic equipment , and discarded household chemicals ) that can contaminate soil and groundwater in ways that make it difficult to adequately identify the population at risk .", "furthermore , in defining contaminated areas in case of complex industrial contamination , it should be considered that populations can experience several routes of exposure , mainly through inhalation of pollutants emitted into the atmosphere , and through ingestion when contaminants are accumulated in soil , water , and food chain .   for the reference population", "the same data of the area units under study are needed : cases and populations stratified by gender and age categories .", "the reference population should be selected considering two different needs : ( 1 ) it should be comparable to the studied populations for factors that can affect the health profile with the exception of the contamination at study", " the differences in the health profile between the compared populations should be ideally due only to the differences in environmental exposures , namely , to the contamination ; ( 2 ) it should be sufficiently numerous to obtain stable reference rates also for rare diseases . these two needs have opposite requirements , as the first one is usually negatively correlated with the dimension of the population , while the second one is positively correlated with the dimension of the population .", "usually one or two populations among the following are selected as reference population : national , and regional , local ( i.e. , a population composed of populations residing in the neighbourhood of the contaminated area ) .", "the aims of the study will imply a sound outcomes selection to include the ones for which environmental exposure / s ( see environmental data ) is suspected or ascertained to play an etiologic role . the possible health impact from environmental exposures", "is measured in terms of mortality , morbidity , incidence of neoplastic diseases , and so forth .", "general considerations about the quality of available information and data as well as intrinsic limitations of the selected outcome measures should be described and discussed .", "health indicator characteristics should be carefully examined and multiple aspects considered taking into account the inherent uncertainty .", "sources of national or local routinely collected data , spatial and temporal coverage , and quality aspects are all of extreme importance for the validity of study results and their usefulness in terms of general knowledge and public health relevance . an appropriate length of the period under study", "will make research results and conclusions more informative for diseases with long latency times ; precision of the epidemiological parameters will also improve with a longer study period .  ", "the specific value of small - area analysis is that it permits the examination of data for population which tend to be more homogeneous in character and in their environmental circumstances than the larger and more widely spread populations .  ", "the smallest territorial unit that can be used in small area studies depends on data availability that may vary in different countries .", "for example , in italy small area studies can be carried out at the census tract ( average of 200 residents ) and municipality levels . in 2001", "about 70% of the italian municipalities included less than 5,000 residents ; this compares with great britain , where small area studies can be carried out at the following levels : enumeration districts400 residents , electoral wards5,100 inhabitants , and postcode sectors6,600 people . in geographical studies of environment and health , confounding from social and economic factors may occur .", "to control such confounding effect , standardisation techniques have been extensively used since the mid-1990s . to account for possible confounding from socioeconomic factors in sentieri project ,", "deprivation can be defined as  a state of observable and demonstrable disadvantage relative to the local community or the wider society or nation to which an individual , family , or groups belong  .", "deprivation indices are area - based measures of material and social disadvantageous circumstances , that is , indicators of relative deprivation at population level .", "a detailed discussion on the use and critical aspects of the application of di in small area studies of environment and health can be found in an open access systematic review .   when performing epidemiologic studies , there is a risk for researchers to become data - driven .", "this can be the case when commenting results for causes showing an increase , possibly on the basis of statistical significance . as an example , sentieri dealt with the complexity of the relation between area contamination and health effects . for each npcs", "studied the project focused on those causes identified a priori from the epidemiological evidence of their association with selected environmental exposures .", "it is suggested that researchers use environmental contaminants information considering the available level of details . in sentieri possible relevant exposures", "were abstracted from legislative decrees , that is , administrative sources defining npcss boundaries and coded on a productive sectors basis ( i.e. , petrochemicals and/or refineries , harbours areas , etc . ) .", "while for some npcss information on specific chemical contaminants was available , for others only productive plants were listed .", "this is to point out that researchers should be able to adapt this approach to their specific situation .", "when studying factors that determine changes in the occurrence of a given health condition or its predictive factors , it should always be kept in mind that most diseases have a multiple etiology", ". therefore also exposures different from those specifically present in npcss should be considered , as well as every known cause or risk factor , such as smoking habits , alcohol consumption or other sources independent from those at study , that is , socioeconomic factors and occupational exposures , and air pollution due to combustion of fuels for transport activities and domestic heating .", "the latter aspects are particularly relevant when studying complex industrial settings close or overlapping to urban highly populated areas .", "once the environmental exposures of interest identified , researchers should examine the updated scientific literature to evaluate the associated health effects .", "this apparently easy task is in fact quite demanding , because by browsing the literature different kinds of publications are collected : handbooks , original articles , letters to scientific journals , multicentric studies , editorials , reviews , meta - analyses , and so on .  ", "therefore , the first decision to be taken is about the  relevance  to give to the collected material and how to use it to define the strength of the association between the specific health outcome and the environmental exposure / s that characterize / s the npcss .  ", "sources expressing the epidemiological community consensus , evaluating scientific evidence by means of standardized criteria , and weighting the study design and the occurrence of biased results were considered most important ( i.e. , iarc monographs , who publications , european environment agency reports , handbooks of environmental , and occupational medicine ) . they were followed in the hierarchy by quantitative meta - analyses .", "consistency among sources was a criterion used to classify the strength of the causal association .  ", "this process was performed considering not only the environmental exposures , but also those risk factors previously mentioned ( smoking habits , alcohol consumption , air pollution , socioeconomic aspects , and occupational exposures ) .", "literature sources were presented in the final report in a tabular form to let the reader follow the entire process of evaluation for each cause combined with different exposures . on the basis of explicit criteria", "the strength of the causal association for each cause - exposure combination was classified ( matrix of a priori evidence evaluation ) .", "expertise in environmental and occupational epidemiology , statistics , and public health clinical medicine , toxicology , and analytical chemistry are needed to tackle this complex environmental issue .", "all data about contamination sources and characterization of contamination should be collected and examined to identify the contamination diffusion and finally define the areas and populations possibly affected by contaminants .", "sentieri project studied populations residing in the sites of national interest for environmental remediation ( national priority contaminated sites  npcss ) .   in sentieri", "the production activities and sources of contamination listed in the decrees defining the sites ' boundaries were used as a proxy for environmental residential exposures ; they are coded as chemical industries , petrochemicals and refineries , steel plants , power plants , mines and/or quarries , harbour areas , and asbestos or other mineral fibres , landfills , and incinerators . for each npcs contamination data", "the study population comprised residents in 44 npcss ; each one included one or more municipalities , a total of 5.5 million inhabitants in the 44 npcss , about 10% of the italian population at the time of 2001 census .", "in sentieri project the italian 2001 standard population was used as a reference to calculate crude and standardized rates .", "each outcome and cause analysed should be characterized to define its specific contribution in evaluating the possible health impact of contamination . for this aim , mortality , morbidity ( e.g. , hospital discharge records ) , cancer incidence , and", "congenital malformations prevalence could be of major interest . as described in the previous section , for each outcome a matrix of a priori evaluation", "each outcome can be considered usable for the sentieri approach if its validity is previously verified , and appropriate epidemiological parameters can be estimated .", "briefly , sentieri studied mortality in 44 npcss using data at municipality level ( 19952002 ) , calculating indicators such as crude and standardized rates ( italian 2001 standard population as reference ) and standardized mortality ratio ( smr ) , using regional comparison rates , both crude ( smr ) and adjusted for an ad hoc deprivation index ( smr i d ) . to control for confounding from social and economic factors an ad hoc deprivation index was built and applied to the smr estimates in sentieri project ( sentieri di ) .", "the deprivation index was constructed using the 2001 national census variables representing the following socioeconomic domains : education , unemployment , dwelling ownership , and overcrowding .", "the epidemiological evidence was examined on the basis of explicit criteria to build a matrix of the a priori epidemiological evaluation of the strength of the causal association for each combination of selected outcome and environmental exposure . for details concerning the a priori evaluation", "firstly a multidisciplinary group of researchers ( see the previous section ) draws up the list of causes to be submitted to the evidence evaluation . ", "secondly , the epidemiologists classify the strength of the causal association of each outcome / exposure combination .", "firstly a multidisciplinary group of researchers ( see the previous section ) draws up the list of causes to be submitted to the evidence evaluation .", "secondly , the epidemiologists classify the strength of the causal association of each outcome / exposure combination . to complete this phase in sentieri project ,", "the epidemiologists developed a procedure to examine the epidemiological literature published from 1998 to 2009 on the health risk of populations living in npcss .", "the epidemiologists examined each cause of death / exposure combination ( mortality was the first outcome to be analyzed ) in terms of strength of causal inference .  environmental exposures ", "the former were fixed on the basis of the possible sources of contamination in npcss listed in the decrees that defined each site boundaries ( e.g. , chemical industry , steel plants ) ; the  other exposures  were the environmental , lifestyle and occupational most important known etiological factors : air pollution , active and passive smoking , alcohol intake , occupational exposures , and socioeconomic status .", "the procedure finally led to classify the evidence of the causal association into three categories : sufficient to infer the presence of a causal association , limited to infer the presence of a causal association , and inadequate to infer the presence or the absence of a causal association .", "the criteria adopted for the classification are reported in table 1 . at the end of the second phase , a matrix of epidemiological a priori evidence about the strength of each outcome - cause / environmental exposure or outcome - cause/other exposure  causal association was prepared ( example in table 2 ) .", "specific causes with a certain level of strength of causal association with the environmental exposures present in each npcs were reported and discussed in detail in sentieri . in order to have a general description of the residents ' health profile , main broad groups of causes of death", "the assessment and appropriate consideration of previous studies performed on the same npcs , if any , ameliorate the level of knowledge , reducing scientific uncertainties about the heath impact of contamination and facilitating the process of identification and implementation of remediation interventions .", "when more than one npcs are studied , a homogeneous way of presenting and discussing results can make the study results clearer and more readable .  ", "it included a summary description of population and contamination data : specific sections dedicated to ( a ) results by gender ( general health profile and specific , a priori selected causes ) ; ( b ) previous studies carried out in the investigated area ; and ( c ) a conclusive paragraph with suggestions for further scientific and/or remediation priorities as well as recommendations for public health interventions", ".   in single sites examples of recommendations aimed at a better description and clarification of the observed health effects include the investigation of the respiratory diseases prevalence in children , the conduction of occupational and residential cohort studies , and health surveillance activities as well as exposure assessment and biological monitoring investigations .  ", "an example is the one of sassuolo - scandiano npcs presented in a following paragraph .", "the presence of asbestos or asbestiform fibres was the motivation for including six npcss in the  national environmental remediation programme .", " in five of these sites increases in malignant pleural neoplasm mortality were observed ; in four of them the excess was in both genders . in four out of six other sites where in addition to asbestos other sources of environmental pollution", "most causes of death analyzed in sentieri have multifactorial etiology ; furthermore in most npcss multiple sources of different pollutants are present , sometimes concurrently with air pollution from urban areas : in these cases , drawing conclusions on the association between environmental exposures and specific health outcomes might be a hard task . notwithstanding these difficulties , in a number of cases", "the attribution could be possible on the basis of the increases observed in both genders and in different age classes , and the exclusion of a major role of occupational exposures was thus allowed ( in italy most of the workforce in industrial setting is still represented by males ) .", "for example , a role of emissions from refineries and petrochemical plants was hypothesized for the observed increases in mortality from lung cancer and respiratory diseases in two npcss ; a role of emissions from metal industries was suggested to explain increased mortality from respiratory diseases in two other sites . in six npcss an etiological role of air pollution in the raise of congenital anomalies and perinatal disorders was suggested , and a causal role of heavy metals , pah 's , and halogenated compounds was suspected for mortality from renal failure in six sites . according to the 2001 census ,", "the npcs sassuolo - scandiano includes 6 municipalities with a 102,811 overall population . the perimetration decree of this npcs lists the presence of pottery manufacturing plants , an environmental exposure that sentieri qualifies as  c  ( plants producing chemicals and/or chemical products ) .", "sentieri results among the main causes of death show in this npcs some excesses for all causes and circulatory and respiratory systems diseases in men .", "some excesses are also observed for digestive system diseases in women ( table 3 ) . for the causes of death with an a priori sufficient or limited evidence of causal association with the environmental exposure in this npcs , an excess for respiratory diseases and asthma", "was observed in men ( table 4 ) , and for congenital anomalies ( malformations ) in all age classes , both genders ( table 5 ) .", "previous studies in this npcs showed that lead , a metal used in the pottery production , polluted subsoil , surface , and ground waters . notwithstanding", "a decrease in occupational exposure between the beginning of the seventies and half of the nineties , in 1995 , the lead levels remained high . at the beginning of the eighties , lead exposure levels in children were high ; during the second half of the nineties a sharp decrease was observed , and blood concentrations were lower than the 10  g/100 ml limit . according to sentieri , the evidence of the causal association between occupation and respiratory system diseases and asthma was classified as sufficient .  ", "since silicosis was not separately analyzed in sentieri and its risk is known to increase in pottery production , as observed in the cohort study of workers compensated for silicosis in italy , it was probably conducive to the observed respiratory diseases excess in this npcs .  ", "the occupational exposure to lead in pottery production might have contributed to the mortality excess due to parkinson 's disease and hypertension .", "a hypertension excess was pointed out for both genders ( men : smr = 192 ( 90% c.i .", "data acquisition for the appraisal of the present lead environmental pollution and occupational exposure is suggested .", "the mortality for causes of death with a priori sufficient or limited evidence of causal association with the environmental exposure showed , for the period 19952002 , 3,508 excess deaths for all causes , corresponding to 439 deaths / year ; the number of excess deaths was 1,321 for respiratory diseases , 898 for lung cancer , and 588 for pleural neoplasms .", "when considering excess mortality with no restriction to causes of death with a priori sufficient or limited evidence of causal association with the environmental exposure , the number of excess deaths for all causes was 9,969 ( smr 102.5 , about 1,200 excess deaths / year ) ; the excess was 4,309 for all neoplasms", "( about 538 excess deaths / year ) , 1 887 for circulatory system diseases , and 600 for respiratory system diseases .", "the ecological approach used in sentieri does not allow to draw firm conclusions on the causal relationships between residential exposure and health status in residents in a contaminated site ; furthermore , the causal inference might be complicated for causes with multifactorial etiology in areas with multiple sources of different pollutants and concurrent presence of air pollution from urban areas .", "notwithstanding these difficulties , in a number of instances , the a priori evaluation of the epidemiological evidence as carried out in sentieri reinforced the findings and strengthened the case of an etiological role to some environmental exposures .", "this has varying degrees of persuasiveness for example , an increased lung cancer and respiratory disease risk was observed in sites hosting refineries and petrochemical plants , suggesting the need for further studies ; the ascertained exposure - disease association between pleural neoplasm mortality and asbestos was confirmed in sites with documented presence of asbestos and asbestos - like fibres .", "another aspect which could increase the persuasiveness of environmental - related health effects is the identification of raised health risks in children living in contaminated sites . in the 44 combined npcss , among children", "0 - 1 year old , mortality from all causes and from perinatal conditions was , respectively , 4% ( 3,328 cases ) and 5% higher ( 1,903 cases ) than the italian reference population , and the overall mortality was significantly increased in one or more age groups ( 0 - 1 , 014 , and 019 years ) among children living in 11 ( 25% ) npcss .", "the value of an ecological study like sentieri should be measured , as recently suggested , against the baseline level of knowledge ; in this context sentieri contribution can be considered high given the absence of systematic and standardized epidemiological investigations of the health impact of residents in npcss .  ", "exposure ascertainment is a key phase in ecological environmental investigations ; the exposures affecting the study population should ideally be described in detail , while in practice a number of limitations affect this crucial aspect in most studies . in some investigations", "the exposure / s is a time - bound event in a limited geographical area , leading to a point source emission of a limited number of contaminants whose nature has been identified and whose toxicological properties can be partially known .", "events such as the explosion of seveso in 1976   and bhopal in 1984 belong to this category .", "more frequently the environment has been progressively and perhaps surreptitiously contaminated by a heterogeneous mixture of pollutants originating from industry ( often a variety of industrial activities ) or waste treatment / disposal activities . in this case", "several environmental matrices are contaminated over a period of years , leading to multiple sources of exposure to a variety of exogenous agents , possibly changing qualitatively or quantitatively overtime .", "for example , in sentieri project the sources of environmental exposures were abstracted from the legislative decrees defining sites ' boundaries , and chemical industry , petrochemicals and refineries plants , steel plants , power plants , mines and/or quarries , harbour areas , asbestos or other mineral fibres , landfills , and incinerators subsequently coded .", "such data are insufficient to give a full picture of space and time distribution and variability of the exposures . in addition , for most npcss no information is available on sources of exposure that can have a health impact , such as concurrent air pollution from road traffic and exposures in the occupational setting .  ", "another limitation in exposure ascertainment lies in the implicit assumption that all residents in the area under investigation experience the same exposures , while exposure variability is likely to be substantial , due to many factors ( e.g. , concentration of contaminants and their diffusion to soil and water , distance of residence from polluting sources ) .", "the possible consequences of such nondifferential exposure misclassification are complex , and direction of the resulting bias is not predictable .", "an additional limitation in exposure ascertainment derives from the territorial size and the population dimension of the areas at study for which vital statistics are available .", "whatever the administrative boundaries are , they hardly correspond to the distribution of environmental pollutants , so that the misclassification of exposure ( and loss of statistical power ) is common . as far as outcome measures", ". however , the analysis of hospital discharge records , ad hoc registry data of specific pathologies ( e.g. , cancer , congenital malformations ) can give a better picture of the health profile of residents in npcss .  ", "each vital statistics is able to provide information only about the events that is designed to record , and databases need to be validated for use in epidemiological studies . in the majority of countries", "death rates from all causes are unlikely to be biased because reporting the event of death is exhaustive .", "therefore the overall mortality , which is an important indicator of conditions of life , can be analyzed with confidence . in italy mortality data", "are available for the whole country , and validity of cause of death certification has been documented for specific diseases [ 1922 ] . in most european countries hospital discharge records ( hdrs ) are indicators of hospital activity , and their main use is for administrative purposes : validity when employed in ecologic studies", "some italian reports [ 24 , 25 ] comment on some critical aspects of this novel utilization of hdrs .", "the analysis of cancer incidence and congenital malformations data in environmental epidemiology investigations can be considered , subject to validity evaluation . in italy in the context of contaminated sites congenital anomalies have been used in a descriptive study   and the investigation of cancer incidence has been planned .", "mortality statistics and hospital discharge statistics have been using the succeeding versions of the international classification of diseases ( icd ) that guarantees homogeneity and comparability of results obtained in different places .", "however , particularly for cancer , the resolution power of the icd is far lower than that of classifications based on pathological diagnosis such as the succeeding versions of the international classification of diseases for oncology ( icd  o ) .", "some associations between environmental agents and cancer are limited to specific pathological variants of the disease ( e.g. , wood dust and adenocarcinoma of the nose and nasal sinuses ) ; for populations served by cancer registries where the histological type of cancer is systematically recorded this problem is solved . in environmental health studies factors as socioeconomic status , occupational exposures , and individual lifestyles can have an etiologic role on the health effects under study thus possibly confounding the exposure - disease relationships .", "socioeconomic status is a determinant of health and disease . since the mid-1990s ecologic studies of environment and health in uk adjusted for deprivation using census data ; for a review refer to pasetto 2010 . to account for deprivation in sentieri project mortality data", "were analyzed both crude and adjusted   based on an ad hoc deprivation index ( sentieri di ) .  ", "occupational exposures are potential confounders in ecological studies of environment and health ; individual based studies may be needed to disentangle environmental and occupational risk .  ", "the present paper referred as a common thread to sentieri project , in its main components of mortality study and a priori evaluation of the epidemiological evidence .", "its major strengths are the standardization of the mortality analysis and npcss classification in terms of environmental exposure which allow the study of all npcss in one country ; the a priori evidence evaluation to comment and interpret study results is a key characterizing element of the project .", "additional assets are that the mortality analysis can be updated and other vital statistics data can be analysed ; also the a priori evidence evaluation can be brought up to date following the established criteria and procedures .", "the sentieri approach allows the description of the health status of populations living in the italian npcss .", "furthermore , it is suitable for an overall analysis of data from different npcss and comparative analysis of data from npcss with the same contamination sources .", "the sentieri approach is , in its essence , a tool to describe the health profile of residents in npcss to document ascertained or suspected associations with local environmental risks ; it does not require an ad hoc data collection .", "the approach can also be of value for health surveillance activities in npcss ( possibly analysing different outcomes ) ; in addition it can contribute to etiological evaluation of cause - effect associations if additional data from biomonitoring investigations , risk assessment studies , and individual - based epidemiological studies are available .", "the links between environmental exposures and health effects depend on the environmental pollutants and diseases being considered but are also influenced by factors such as genetic constitution , age , nutrition and lifestyles , occupation , and socioeconomic factors such as poverty and level of education .", "identifying these relationships is therefore challenging ; however , the effort is often worthwhile as it may help to redefine priorities and unlock resources .", "other strengths of the sentieri approach are that the a priori evidence evaluation and mortality analysis can be updated ; other health outcomes in addition to mortality can be analyzed , for example , cancer incidence , morbidity , and adverse reproductive effects ; also the a priori evaluation can be carried out for environmental exposures different from the ones in sentieri project . notwithstanding the remarkably laborious activities required to set up a national project such as sentieri", ", major benefits in terms of quality and quantity of findings , and a favourable cost / gain balance can be expected inasmuch as this becomes a permanent system of epidemiological observation on health of residents in npcss ."], "abstract_text": ["<S> sentieri project ( epidemiological study of residents in italian contaminated sites ) studied mortality in the sites of national interest for environmental remediation ( national priority contaminated sites  </S>", "<S> npcss ) . </S>", "<S> sentieri described mortality of residents in npcsss , and it specifically focused on causes of death for which environmental exposure is suspected or ascertained to play an etiologic role . </S>", "<S> the epidemiological evidence of the causal association was classified a priori into one of these three categories : sufficient ( s ) , limited ( l ) , and inadequate ( i ) . </S>", "<S> mortality in the period 19952002 was studied for 63 single or grouped causes at the municipal level by computing : crude rate , standardized rate , standardized mortality ratios ( smr ) , and smr adjusted for an ad hoc deprivation index . </S>", "<S> regional populations were used as references for smr calculations and 90% ci accompanied smr values . </S>", "<S> the deprivation index was constructed using 2001 national census variables for the following socioeconomic domains : education , unemployment , dwelling ownership , and overcrowding . </S>", "<S> sentieri results will allow the priorities setting in remediation intervention so as to prevent adverse health effects from environmental exposure . </S>", "<S> this paper 's objective is to present the rationale , methods , advantages , and limitations underlying sentieri project and to describe data and resources required to apply a similar approach in other countries . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Rationale", "3. The Study of the Health Profile of Residents in NPCSs: What to Do and Why", "4. The Study of the Health Profile of Residents in NPCSs: SENTIERI Project", "5. The Study of the Health Profile of Residents in NPCSs: SENTIERI ProjectMain Findings and Results for a Single Site and for All NPCSs Combined", "6. Discussion", "7. Concluding Remarks"], "sections": [["this is particularly the case of the health of people living in contaminated site(s ) ( cs ) which is affected by the legacy of past industrialization and current industrial activities , often in absence of environmental remediation .", "european community legislation addresses the concept of cs only in the context of the thematic strategy for soil protection   and the soil framework directive proposed by the european commission ( ec ) in 2006 ; for details on the legal framework and definitions at eu level refer to who 2013 . according to 2007 estimates ,", "soil contamination requiring clean up is present in approximately 250,000 sites in the european environment agency ( eea ) member countries .", "although main polluting sources may vary across europe , industrial production and commercial activities , oil industry and waste disposal and treatment are reported to be the major ones .", "national reports indicate that heavy metals and mineral oils are the main soil contaminants , while mineral oils and chlorinated hydrocarbons are the most frequent pollutants found in groundwater .", "a general definition , following the public health perspective , is  areas hosting or having hosted human activities which have produced or might produce environmental contamination of soil , surface or groundwater , air , and food chain , resulting or being able to result in human health impacts  . given this definition , an area affected by a single chemical contamination of a single environmental matrix ( e.g. , the soil contamination caused by a given pesticide ) and a large area with soil , water , air , and food chain contamination by multiple chemicals ( e.g. , the contamination caused by long - term emissions of a petrochemical complex ) can be both considered contaminated sites .  ", "there are several approaches and methods for assessing the health impact of national priority contaminated sites ( npcs ) . on the one hand", ", one can apply risk assessment techniques , where available data on the degree of contamination ( typically measures of concentration of specific hazardous chemical agents ) are used to quantitatively estimate the risk of occurrence of health endpoints causally associated with the agents ; on the other hand , epidemiological approaches applicable to npcss involve the inclusive collection of available health information of resident population , and various agents and exposures . in these assessments ,", "a first descriptive level is based on epidemiological tools that do not require an ad hoc collection of data and aims at describing the health profile of populations documenting ascertained or suspected associations with local environmental risks and the potentiality to provide efficient answers .", "more detailed analyses can be carried out , at a higher level of definition , by collecting specific data on health outcomes and/or on exposure .  ", "sentieri project ( epidemiological study of residents in italian contaminated sites ) [ 6 , 7 ] is an example of first level descriptive approach adopting an ecological study design , looking at the aggregate population level rather than at individual level .", "sentieri project is a national project developed to evaluate the health profile of populations residing in the italian sites of national interest for environmental remediation - national priority contaminated sites ( npcs ) .", "these sites were labelled as sites of national interest because of their substantial contamination , documented in qualitative and/or quantitative terms , and the consequent potential impact on the health of residents .", "the methods proposed under the approach exemplified by sentieri can be generalized and applied to other npcss .  ", "this paper 's objective is to present the rationale and methods underlying sentieri project and to describe data and resources required to apply a similar approach in other countries ."], ["when studying how the environment can adversely affect human health , it is usually very difficult to identify clear cause - effect relationships because they are characterized by multicausality with different strengths of association .", "in addition , these relationships are influenced by individual factors ( e.g. , genetic , diet , life - style , occupation , and socioeconomic status ) that can also have a role on both exposure level and disease development .", "it describes the health profile of residents in contaminated sites through small area analysis by applying the multistep procedure described in the following sections .", "a selection of epidemiologic terms are defined in glossary to facilitate the reading of the paper ."], ["as a first step , npcss to be studied should be chosen , and the criteria adopted to define npcs / npcss are clearly indicated . the npcs selection will depend on the aims of the study to be undertaken , on the availability of the npccs - related information , and on any other consideration researchers would make and consider appropriate .", "in many instances , npcss to be studied are chosen by third parties , such as an environmental authority , by public concern , and media pressure .", "it is advisable that the criteria used throughout this phase are clearly stated . in most instances npcss", "are characterized by the presence of numerous and different environmental sources of contamination possibly leading to human exposures .", "all the available npcs data should be collected and described in a standardized , homogeneous way .", "geographical characteristics , extension of the contaminated area , and demographic information about residents potentially affected should be listed .", "detailed description of contamination characteristics should be included as well as the presence of industries and all other human activities that have contributed to the environmental deterioration of the npcs .", "researchers will specify the sources used for this task : scientific reports , acts , and so on .", "populations at risk can be identified as people living in the neighborhood of npcss sources or living in areas defined as contaminated .", "typically , the distance from the areas affected by contamination is used , but also dispersion modelling results are used . in the latter case , the definition of the areas most affected by contamination , and the consequent identification of at - risk populations , depends on the accuracy of the model .", "there are several models used to evaluate the areas affected by contaminants ; their implementation and improvement depend on the available information on several parameters .", "for example , in the case of emissions into the air from oil refineries , the parameters to be considered should be characteristics of emission sources : for example , height , flow rate , composition of emissions , exit temperature , local orography , and meteorological conditions .", "areas characterised by contamination processes different from direct industrial emissions require other parameters . in the case of landfills", "many early disposal sites did not have liners to trap rainwater that percolate through the landfill , and some newer landfills have liners that leak .", "the percolating water leaches toxic chemicals ( e.g. , from batteries , electronic equipment , and discarded household chemicals ) that can contaminate soil and groundwater in ways that make it difficult to adequately identify the population at risk .", "furthermore , in defining contaminated areas in case of complex industrial contamination , it should be considered that populations can experience several routes of exposure , mainly through inhalation of pollutants emitted into the atmosphere , and through ingestion when contaminants are accumulated in soil , water , and food chain .   for the reference population", "the same data of the area units under study are needed : cases and populations stratified by gender and age categories .", "the reference population should be selected considering two different needs : ( 1 ) it should be comparable to the studied populations for factors that can affect the health profile with the exception of the contamination at study", " the differences in the health profile between the compared populations should be ideally due only to the differences in environmental exposures , namely , to the contamination ; ( 2 ) it should be sufficiently numerous to obtain stable reference rates also for rare diseases . these two needs have opposite requirements , as the first one is usually negatively correlated with the dimension of the population , while the second one is positively correlated with the dimension of the population .", "usually one or two populations among the following are selected as reference population : national , and regional , local ( i.e. , a population composed of populations residing in the neighbourhood of the contaminated area ) .", "the aims of the study will imply a sound outcomes selection to include the ones for which environmental exposure / s ( see environmental data ) is suspected or ascertained to play an etiologic role . the possible health impact from environmental exposures", "is measured in terms of mortality , morbidity , incidence of neoplastic diseases , and so forth .", "general considerations about the quality of available information and data as well as intrinsic limitations of the selected outcome measures should be described and discussed .", "health indicator characteristics should be carefully examined and multiple aspects considered taking into account the inherent uncertainty .", "sources of national or local routinely collected data , spatial and temporal coverage , and quality aspects are all of extreme importance for the validity of study results and their usefulness in terms of general knowledge and public health relevance . an appropriate length of the period under study", "will make research results and conclusions more informative for diseases with long latency times ; precision of the epidemiological parameters will also improve with a longer study period .  ", "the specific value of small - area analysis is that it permits the examination of data for population which tend to be more homogeneous in character and in their environmental circumstances than the larger and more widely spread populations .  ", "the smallest territorial unit that can be used in small area studies depends on data availability that may vary in different countries .", "for example , in italy small area studies can be carried out at the census tract ( average of 200 residents ) and municipality levels . in 2001", "about 70% of the italian municipalities included less than 5,000 residents ; this compares with great britain , where small area studies can be carried out at the following levels : enumeration districts400 residents , electoral wards5,100 inhabitants , and postcode sectors6,600 people . in geographical studies of environment and health , confounding from social and economic factors may occur .", "to control such confounding effect , standardisation techniques have been extensively used since the mid-1990s . to account for possible confounding from socioeconomic factors in sentieri project ,", "deprivation can be defined as  a state of observable and demonstrable disadvantage relative to the local community or the wider society or nation to which an individual , family , or groups belong  .", "deprivation indices are area - based measures of material and social disadvantageous circumstances , that is , indicators of relative deprivation at population level .", "a detailed discussion on the use and critical aspects of the application of di in small area studies of environment and health can be found in an open access systematic review .   when performing epidemiologic studies , there is a risk for researchers to become data - driven .", "this can be the case when commenting results for causes showing an increase , possibly on the basis of statistical significance . as an example , sentieri dealt with the complexity of the relation between area contamination and health effects . for each npcs", "studied the project focused on those causes identified a priori from the epidemiological evidence of their association with selected environmental exposures .", "it is suggested that researchers use environmental contaminants information considering the available level of details . in sentieri possible relevant exposures", "were abstracted from legislative decrees , that is , administrative sources defining npcss boundaries and coded on a productive sectors basis ( i.e. , petrochemicals and/or refineries , harbours areas , etc . ) .", "while for some npcss information on specific chemical contaminants was available , for others only productive plants were listed .", "this is to point out that researchers should be able to adapt this approach to their specific situation .", "when studying factors that determine changes in the occurrence of a given health condition or its predictive factors , it should always be kept in mind that most diseases have a multiple etiology", ". therefore also exposures different from those specifically present in npcss should be considered , as well as every known cause or risk factor , such as smoking habits , alcohol consumption or other sources independent from those at study , that is , socioeconomic factors and occupational exposures , and air pollution due to combustion of fuels for transport activities and domestic heating .", "the latter aspects are particularly relevant when studying complex industrial settings close or overlapping to urban highly populated areas .", "once the environmental exposures of interest identified , researchers should examine the updated scientific literature to evaluate the associated health effects .", "this apparently easy task is in fact quite demanding , because by browsing the literature different kinds of publications are collected : handbooks , original articles , letters to scientific journals , multicentric studies , editorials , reviews , meta - analyses , and so on .  ", "therefore , the first decision to be taken is about the  relevance  to give to the collected material and how to use it to define the strength of the association between the specific health outcome and the environmental exposure / s that characterize / s the npcss .  ", "sources expressing the epidemiological community consensus , evaluating scientific evidence by means of standardized criteria , and weighting the study design and the occurrence of biased results were considered most important ( i.e. , iarc monographs , who publications , european environment agency reports , handbooks of environmental , and occupational medicine ) . they were followed in the hierarchy by quantitative meta - analyses .", "consistency among sources was a criterion used to classify the strength of the causal association .  ", "this process was performed considering not only the environmental exposures , but also those risk factors previously mentioned ( smoking habits , alcohol consumption , air pollution , socioeconomic aspects , and occupational exposures ) .", "literature sources were presented in the final report in a tabular form to let the reader follow the entire process of evaluation for each cause combined with different exposures . on the basis of explicit criteria", "the strength of the causal association for each cause - exposure combination was classified ( matrix of a priori evidence evaluation ) .", "expertise in environmental and occupational epidemiology , statistics , and public health clinical medicine , toxicology , and analytical chemistry are needed to tackle this complex environmental issue ."], ["all data about contamination sources and characterization of contamination should be collected and examined to identify the contamination diffusion and finally define the areas and populations possibly affected by contaminants .", "sentieri project studied populations residing in the sites of national interest for environmental remediation ( national priority contaminated sites  npcss ) .   in sentieri", "the production activities and sources of contamination listed in the decrees defining the sites ' boundaries were used as a proxy for environmental residential exposures ; they are coded as chemical industries , petrochemicals and refineries , steel plants , power plants , mines and/or quarries , harbour areas , and asbestos or other mineral fibres , landfills , and incinerators . for each npcs contamination data", "the study population comprised residents in 44 npcss ; each one included one or more municipalities , a total of 5.5 million inhabitants in the 44 npcss , about 10% of the italian population at the time of 2001 census .", "in sentieri project the italian 2001 standard population was used as a reference to calculate crude and standardized rates .", "each outcome and cause analysed should be characterized to define its specific contribution in evaluating the possible health impact of contamination . for this aim , mortality , morbidity ( e.g. , hospital discharge records ) , cancer incidence , and", "congenital malformations prevalence could be of major interest . as described in the previous section , for each outcome a matrix of a priori evaluation", "each outcome can be considered usable for the sentieri approach if its validity is previously verified , and appropriate epidemiological parameters can be estimated .", "briefly , sentieri studied mortality in 44 npcss using data at municipality level ( 19952002 ) , calculating indicators such as crude and standardized rates ( italian 2001 standard population as reference ) and standardized mortality ratio ( smr ) , using regional comparison rates , both crude ( smr ) and adjusted for an ad hoc deprivation index ( smr i d ) . to control for confounding from social and economic factors an ad hoc deprivation index was built and applied to the smr estimates in sentieri project ( sentieri di ) .", "the deprivation index was constructed using the 2001 national census variables representing the following socioeconomic domains : education , unemployment , dwelling ownership , and overcrowding .", "the epidemiological evidence was examined on the basis of explicit criteria to build a matrix of the a priori epidemiological evaluation of the strength of the causal association for each combination of selected outcome and environmental exposure . for details concerning the a priori evaluation", "firstly a multidisciplinary group of researchers ( see the previous section ) draws up the list of causes to be submitted to the evidence evaluation . ", "secondly , the epidemiologists classify the strength of the causal association of each outcome / exposure combination .", "firstly a multidisciplinary group of researchers ( see the previous section ) draws up the list of causes to be submitted to the evidence evaluation .", "secondly , the epidemiologists classify the strength of the causal association of each outcome / exposure combination . to complete this phase in sentieri project ,", "the epidemiologists developed a procedure to examine the epidemiological literature published from 1998 to 2009 on the health risk of populations living in npcss .", "the epidemiologists examined each cause of death / exposure combination ( mortality was the first outcome to be analyzed ) in terms of strength of causal inference .  environmental exposures ", "the former were fixed on the basis of the possible sources of contamination in npcss listed in the decrees that defined each site boundaries ( e.g. , chemical industry , steel plants ) ; the  other exposures  were the environmental , lifestyle and occupational most important known etiological factors : air pollution , active and passive smoking , alcohol intake , occupational exposures , and socioeconomic status .", "the procedure finally led to classify the evidence of the causal association into three categories : sufficient to infer the presence of a causal association , limited to infer the presence of a causal association , and inadequate to infer the presence or the absence of a causal association .", "the criteria adopted for the classification are reported in table 1 . at the end of the second phase , a matrix of epidemiological a priori evidence about the strength of each outcome - cause / environmental exposure or outcome - cause/other exposure  causal association was prepared ( example in table 2 ) ."], ["specific causes with a certain level of strength of causal association with the environmental exposures present in each npcs were reported and discussed in detail in sentieri . in order to have a general description of the residents ' health profile , main broad groups of causes of death", "the assessment and appropriate consideration of previous studies performed on the same npcs , if any , ameliorate the level of knowledge , reducing scientific uncertainties about the heath impact of contamination and facilitating the process of identification and implementation of remediation interventions .", "when more than one npcs are studied , a homogeneous way of presenting and discussing results can make the study results clearer and more readable .  ", "it included a summary description of population and contamination data : specific sections dedicated to ( a ) results by gender ( general health profile and specific , a priori selected causes ) ; ( b ) previous studies carried out in the investigated area ; and ( c ) a conclusive paragraph with suggestions for further scientific and/or remediation priorities as well as recommendations for public health interventions", ".   in single sites examples of recommendations aimed at a better description and clarification of the observed health effects include the investigation of the respiratory diseases prevalence in children , the conduction of occupational and residential cohort studies , and health surveillance activities as well as exposure assessment and biological monitoring investigations .  ", "an example is the one of sassuolo - scandiano npcs presented in a following paragraph .", "the presence of asbestos or asbestiform fibres was the motivation for including six npcss in the  national environmental remediation programme .", " in five of these sites increases in malignant pleural neoplasm mortality were observed ; in four of them the excess was in both genders . in four out of six other sites where in addition to asbestos other sources of environmental pollution", "most causes of death analyzed in sentieri have multifactorial etiology ; furthermore in most npcss multiple sources of different pollutants are present , sometimes concurrently with air pollution from urban areas : in these cases , drawing conclusions on the association between environmental exposures and specific health outcomes might be a hard task . notwithstanding these difficulties , in a number of cases", "the attribution could be possible on the basis of the increases observed in both genders and in different age classes , and the exclusion of a major role of occupational exposures was thus allowed ( in italy most of the workforce in industrial setting is still represented by males ) .", "for example , a role of emissions from refineries and petrochemical plants was hypothesized for the observed increases in mortality from lung cancer and respiratory diseases in two npcss ; a role of emissions from metal industries was suggested to explain increased mortality from respiratory diseases in two other sites . in six npcss an etiological role of air pollution in the raise of congenital anomalies and perinatal disorders was suggested , and a causal role of heavy metals , pah 's , and halogenated compounds was suspected for mortality from renal failure in six sites . according to the 2001 census ,", "the npcs sassuolo - scandiano includes 6 municipalities with a 102,811 overall population . the perimetration decree of this npcs lists the presence of pottery manufacturing plants , an environmental exposure that sentieri qualifies as  c  ( plants producing chemicals and/or chemical products ) .", "sentieri results among the main causes of death show in this npcs some excesses for all causes and circulatory and respiratory systems diseases in men .", "some excesses are also observed for digestive system diseases in women ( table 3 ) . for the causes of death with an a priori sufficient or limited evidence of causal association with the environmental exposure in this npcs , an excess for respiratory diseases and asthma", "was observed in men ( table 4 ) , and for congenital anomalies ( malformations ) in all age classes , both genders ( table 5 ) .", "previous studies in this npcs showed that lead , a metal used in the pottery production , polluted subsoil , surface , and ground waters . notwithstanding", "a decrease in occupational exposure between the beginning of the seventies and half of the nineties , in 1995 , the lead levels remained high . at the beginning of the eighties , lead exposure levels in children were high ; during the second half of the nineties a sharp decrease was observed , and blood concentrations were lower than the 10  g/100 ml limit . according to sentieri , the evidence of the causal association between occupation and respiratory system diseases and asthma was classified as sufficient .  ", "since silicosis was not separately analyzed in sentieri and its risk is known to increase in pottery production , as observed in the cohort study of workers compensated for silicosis in italy , it was probably conducive to the observed respiratory diseases excess in this npcs .  ", "the occupational exposure to lead in pottery production might have contributed to the mortality excess due to parkinson 's disease and hypertension .", "a hypertension excess was pointed out for both genders ( men : smr = 192 ( 90% c.i .", "data acquisition for the appraisal of the present lead environmental pollution and occupational exposure is suggested .", "the mortality for causes of death with a priori sufficient or limited evidence of causal association with the environmental exposure showed , for the period 19952002 , 3,508 excess deaths for all causes , corresponding to 439 deaths / year ; the number of excess deaths was 1,321 for respiratory diseases , 898 for lung cancer , and 588 for pleural neoplasms .", "when considering excess mortality with no restriction to causes of death with a priori sufficient or limited evidence of causal association with the environmental exposure , the number of excess deaths for all causes was 9,969 ( smr 102.5 , about 1,200 excess deaths / year ) ; the excess was 4,309 for all neoplasms", "( about 538 excess deaths / year ) , 1 887 for circulatory system diseases , and 600 for respiratory system diseases ."], ["the ecological approach used in sentieri does not allow to draw firm conclusions on the causal relationships between residential exposure and health status in residents in a contaminated site ; furthermore , the causal inference might be complicated for causes with multifactorial etiology in areas with multiple sources of different pollutants and concurrent presence of air pollution from urban areas .", "notwithstanding these difficulties , in a number of instances , the a priori evaluation of the epidemiological evidence as carried out in sentieri reinforced the findings and strengthened the case of an etiological role to some environmental exposures .", "this has varying degrees of persuasiveness for example , an increased lung cancer and respiratory disease risk was observed in sites hosting refineries and petrochemical plants , suggesting the need for further studies ; the ascertained exposure - disease association between pleural neoplasm mortality and asbestos was confirmed in sites with documented presence of asbestos and asbestos - like fibres .", "another aspect which could increase the persuasiveness of environmental - related health effects is the identification of raised health risks in children living in contaminated sites . in the 44 combined npcss , among children", "0 - 1 year old , mortality from all causes and from perinatal conditions was , respectively , 4% ( 3,328 cases ) and 5% higher ( 1,903 cases ) than the italian reference population , and the overall mortality was significantly increased in one or more age groups ( 0 - 1 , 014 , and 019 years ) among children living in 11 ( 25% ) npcss .", "the value of an ecological study like sentieri should be measured , as recently suggested , against the baseline level of knowledge ; in this context sentieri contribution can be considered high given the absence of systematic and standardized epidemiological investigations of the health impact of residents in npcss .  ", "exposure ascertainment is a key phase in ecological environmental investigations ; the exposures affecting the study population should ideally be described in detail , while in practice a number of limitations affect this crucial aspect in most studies . in some investigations", "the exposure / s is a time - bound event in a limited geographical area , leading to a point source emission of a limited number of contaminants whose nature has been identified and whose toxicological properties can be partially known .", "events such as the explosion of seveso in 1976   and bhopal in 1984 belong to this category .", "more frequently the environment has been progressively and perhaps surreptitiously contaminated by a heterogeneous mixture of pollutants originating from industry ( often a variety of industrial activities ) or waste treatment / disposal activities . in this case", "several environmental matrices are contaminated over a period of years , leading to multiple sources of exposure to a variety of exogenous agents , possibly changing qualitatively or quantitatively overtime .", "for example , in sentieri project the sources of environmental exposures were abstracted from the legislative decrees defining sites ' boundaries , and chemical industry , petrochemicals and refineries plants , steel plants , power plants , mines and/or quarries , harbour areas , asbestos or other mineral fibres , landfills , and incinerators subsequently coded .", "such data are insufficient to give a full picture of space and time distribution and variability of the exposures . in addition , for most npcss no information is available on sources of exposure that can have a health impact , such as concurrent air pollution from road traffic and exposures in the occupational setting .  ", "another limitation in exposure ascertainment lies in the implicit assumption that all residents in the area under investigation experience the same exposures , while exposure variability is likely to be substantial , due to many factors ( e.g. , concentration of contaminants and their diffusion to soil and water , distance of residence from polluting sources ) .", "the possible consequences of such nondifferential exposure misclassification are complex , and direction of the resulting bias is not predictable .", "an additional limitation in exposure ascertainment derives from the territorial size and the population dimension of the areas at study for which vital statistics are available .", "whatever the administrative boundaries are , they hardly correspond to the distribution of environmental pollutants , so that the misclassification of exposure ( and loss of statistical power ) is common . as far as outcome measures", ". however , the analysis of hospital discharge records , ad hoc registry data of specific pathologies ( e.g. , cancer , congenital malformations ) can give a better picture of the health profile of residents in npcss .  ", "each vital statistics is able to provide information only about the events that is designed to record , and databases need to be validated for use in epidemiological studies . in the majority of countries", "death rates from all causes are unlikely to be biased because reporting the event of death is exhaustive .", "therefore the overall mortality , which is an important indicator of conditions of life , can be analyzed with confidence . in italy mortality data", "are available for the whole country , and validity of cause of death certification has been documented for specific diseases [ 1922 ] . in most european countries hospital discharge records ( hdrs ) are indicators of hospital activity , and their main use is for administrative purposes : validity when employed in ecologic studies", "some italian reports [ 24 , 25 ] comment on some critical aspects of this novel utilization of hdrs .", "the analysis of cancer incidence and congenital malformations data in environmental epidemiology investigations can be considered , subject to validity evaluation . in italy in the context of contaminated sites congenital anomalies have been used in a descriptive study   and the investigation of cancer incidence has been planned .", "mortality statistics and hospital discharge statistics have been using the succeeding versions of the international classification of diseases ( icd ) that guarantees homogeneity and comparability of results obtained in different places .", "however , particularly for cancer , the resolution power of the icd is far lower than that of classifications based on pathological diagnosis such as the succeeding versions of the international classification of diseases for oncology ( icd  o ) .", "some associations between environmental agents and cancer are limited to specific pathological variants of the disease ( e.g. , wood dust and adenocarcinoma of the nose and nasal sinuses ) ; for populations served by cancer registries where the histological type of cancer is systematically recorded this problem is solved . in environmental health studies factors as socioeconomic status , occupational exposures , and individual lifestyles can have an etiologic role on the health effects under study thus possibly confounding the exposure - disease relationships .", "socioeconomic status is a determinant of health and disease . since the mid-1990s ecologic studies of environment and health in uk adjusted for deprivation using census data ; for a review refer to pasetto 2010 . to account for deprivation in sentieri project mortality data", "were analyzed both crude and adjusted   based on an ad hoc deprivation index ( sentieri di ) .  ", "occupational exposures are potential confounders in ecological studies of environment and health ; individual based studies may be needed to disentangle environmental and occupational risk .  ", "the present paper referred as a common thread to sentieri project , in its main components of mortality study and a priori evaluation of the epidemiological evidence .", "its major strengths are the standardization of the mortality analysis and npcss classification in terms of environmental exposure which allow the study of all npcss in one country ; the a priori evidence evaluation to comment and interpret study results is a key characterizing element of the project .", "additional assets are that the mortality analysis can be updated and other vital statistics data can be analysed ; also the a priori evidence evaluation can be brought up to date following the established criteria and procedures ."], ["the sentieri approach allows the description of the health status of populations living in the italian npcss .", "furthermore , it is suitable for an overall analysis of data from different npcss and comparative analysis of data from npcss with the same contamination sources .", "the sentieri approach is , in its essence , a tool to describe the health profile of residents in npcss to document ascertained or suspected associations with local environmental risks ; it does not require an ad hoc data collection .", "the approach can also be of value for health surveillance activities in npcss ( possibly analysing different outcomes ) ; in addition it can contribute to etiological evaluation of cause - effect associations if additional data from biomonitoring investigations , risk assessment studies , and individual - based epidemiological studies are available .", "the links between environmental exposures and health effects depend on the environmental pollutants and diseases being considered but are also influenced by factors such as genetic constitution , age , nutrition and lifestyles , occupation , and socioeconomic factors such as poverty and level of education .", "identifying these relationships is therefore challenging ; however , the effort is often worthwhile as it may help to redefine priorities and unlock resources .", "other strengths of the sentieri approach are that the a priori evidence evaluation and mortality analysis can be updated ; other health outcomes in addition to mortality can be analyzed , for example , cancer incidence , morbidity , and adverse reproductive effects ; also the a priori evaluation can be carried out for environmental exposures different from the ones in sentieri project . notwithstanding the remarkably laborious activities required to set up a national project such as sentieri", ", major benefits in terms of quality and quantity of findings , and a favourable cost / gain balance can be expected inasmuch as this becomes a permanent system of epidemiological observation on health of residents in npcss ."]]}
{"article_id": "PMC3214355", "article_text": ["the study population included 1177 patients who underwent different types of cardiac surgeries with cardiopulmonary bypass method at sina private hospital ( isfahan , iran ) , between april 2008 and september 2009 .", "all patients underwent a cardiovascular diagnostic procedure requiring the use of a contrast agent for left ventriculography , coronary or carotid angiography , and thoracic and/or abdominal aorta angiography before their surgeries .", "thus , the patients requiring emergency dialysis therapy or those that underwent the off - pump method surgery were excluded .", "data for the studied patients including demographics , clinical characteristics , laboratory data , medical treatments , angiographic data , ( including amount and type of the contrast agent used ) , comorbid conditions and cardiac procedural data ( including its timing in relation to the preceding angiography ) , were collected by reviewing hospital record files in a computerized database .", "the study was approved by the research ethics committee of isfahan university of medical sciences , isfahan sina heart center .", "the contrast agent used were meglumine compound 76% ( urografin , bayer , usa ) as hyperosmolar , iopromide 370 ( ultravisit , berlex montville , usa , mfd . in : germany ) and iohexol 300 ( iopaque , iran or omnipaque 300 , usa ) as low - osmolar contrast agents .", "use of nonsteroidal anti - inflammatory agents and other nephrotoxic drugs were avoided or withheld for 48 hours before angiography .", "postoperative renal risk was assessed using 2 different risk scores of a simplified predictive index for renal replacement therapy after cardiac surgery and a risk score for prediction of contrast - induced nephropathy.9 the main outcome of interest was the incidence of postoperative arf , defined as postoperative serum creatinine > 2 times baseline and/or the need for renal replacement therapy .", "the mean value of baseline creatinine was 0.99 mg / dl.6 the obtained results are presented as mean  sd , frequency , and percentage .", "comparisons were made using chi - square test or fisher 's exact test for categorical variables and nonparametric test for continuous variables .", "the amount of contrast media was entered into the model as both a linear ( continuous variable ) and a dichotomous term ( median or less and greater than median value ) .", "multivariable logistic regression analysis was used to identify independent predictors of arf using variables found to have marginal association ( p > 0.10 ) on univariate analysis .", "odds ratio ( or ) and 95% confidence interval ( ci ) were constructed to provide an estimate of adjusted risk posed by post - procedural arf .", "comparisons were made using chi - square test or fisher 's exact test for categorical variables and nonparametric test for continuous variables .", "the amount of contrast media was entered into the model as both a linear ( continuous variable ) and a dichotomous term ( median or less and greater than median value ) .", "multivariable logistic regression analysis was used to identify independent predictors of arf using variables found to have marginal association ( p > 0.10 ) on univariate analysis .", "odds ratio ( or ) and 95% confidence interval ( ci ) were constructed to provide an estimate of adjusted risk posed by post - procedural arf .", "comparisons were made using chi - square test or fisher 's exact test for categorical variables and nonparametric test for continuous variables .", "the amount of contrast media was entered into the model as both a linear ( continuous variable ) and a dichotomous term ( median or less and greater than median value ) .", "multivariable logistic regression analysis was used to identify independent predictors of arf using variables found to have marginal association ( p > 0.10 ) on univariate analysis .", "odds ratio ( or ) and 95% confidence interval ( ci ) were constructed to provide an estimate of adjusted risk posed by post - procedural arf .", "of 1177 patients who underwent different types of cardiac surgeries after angiography , arf occurred in 465 subjects , 2 of these patients needed dialysis .", "mean dose of hyperosmolar and low - osmolar contrast agents used overall were 1.23 and 1.21 ml / kg , respectively .", "baseline clinical features between the two cohorts with and without postoperative arf are listed in table 1 .", "postoperative arf was more prevalent in older and male patients ( p = 0.003 and 0.002 , respectively ) , and those with a higher history of hypertension(p = 0.012 ) , but prevalence of other risk factors as well as left ventricular ejection fraction were similar in both group of patients .", "there was a significant difference between last pre and post- operative creatinine in both groups ( p < 0.001 ) .", "patients with arf were also more likely to have undergone combined coronary artery bypass grafting and valve surgery the same day as angiography and also underwent more postoperative intra - aortic balloon pump insertion ( iabp ) ( table 2 ) .", "the patients with arf were more likely to have received higher doses of both hyper - osmolar and low - osmolar contrast agents compared to mean doses ( p = 0.17 and 0.035 , respectively ) .", "however , the time interval between cardiac surgery and last catheterization was not different between the patients with and without arf ( p > 0.05 ) ( table 2 ) . clinical and demographic characteristics of the studied population contrast agent and cardiac surgery data overall postoperative peak creatinine was highest on day 0 ( figure 2 ) , then decreased and remained significantly unchanged after this period ( p < 0.05 ) .", "however , overall prevalence of acute renal failure during follow - up period ( figure 2 ) had a changeable trend and had the highest rates in days 1 ( 53.57% ) and 6 ( 52.17% ) ( p < 0.05 ) .", "relationship between the time interval flanked by angiography and surgery and postoperative serum creatinine relationship between the time interval flanked by angiography and surgery and postoperative acute renal failure independent correlates of postoperative arf in a multivariate model are listed in table 3 .", "combined coronary artery bypass grafting ( cabg ) and valve surgery was the strongest predictor of postoperative arf ( or : 4.976 , p = 0.002 ) , followed by intra - aortic balloon pump insertion ( or : 6.890 , p = 0.009 ) and higher dose of contrast agent ( or : 1.446 , p = 0.031 ) .", "the major findings of the present study were that the higher dose of the used contrast agents besides some other determinants such as combined coronary bypass and valvular surgery as well as iabp insertion could effectively predict post - procedure acute arf .", "on the other hand , the use of high dose of contrast agent was strongly related to the incidence of arf after combined cardiac surgery .", "in fact , our data confirmed findings of other investigations that arf after cardiac surgery was associated with the amount of prescribed contrast agents . in a similar study by ranucci and his colleagues,6 cardiac surgery performed on the day of cardiac catheterization and higher dose of contrast agent", "although these investigators demonstrated a correlation between the time interval of cardiac catheterization and surgical procedure with the incidence of acute arf , we could not demonstrate this relationship in our multivariable regression model . in another study by del duca", "et al4 cardiac catheterization performed within 5 days before operation , baseline glomerular filtration rate of less than 60 ml / min and prolonged cardiopulmonary bypass duration , were significant risk factors for acute renal failure after cardiac surgery . moreover , other studies established similar clinical and surgical characteristics associated with increased risk of arf after cardiac surgery , including age,168 baseline serum creatinine,10 diabetes mellitus,1113 congestive heart failure,1012 emergency surgery,1114 concomitant valve surgery with coronary artery bypass grafting,11 preoperative use of iabp,14 and low cardiac output syndrome in the postoperative course.1415 however , these studies consistently failed to account for the influence of the timing between previous angiography and cardiac surgery and the amount of the contrast agent used in this event .", "multiple mechanisms have been suggested to contribute to the renal damage following cardiac diagnostic procedures , including non - pulsatile flow , embolization , and trauma to the blood constituents , hypothermia , and activation of known inflammatory pathways.16 iodinated contrast as a common contrast agent , after causing a brief period of vasodilation , can cause sustained intrarenal vasoconstriction and ischemic injury .", "the ischemic injury sets off a cascade of events largely driven by oxidative injury , causing death of renal tubular cells .", "if a sufficient mass of nephron units are affected , then a recognizable rise in serum creatinine will occur.1718 therefore , minimizing the amount of contrast agent during preceding angiography is recommended .", "similar studies need to be conducted regarding alternate imaging modalities that do not use iodine - based contrast agents to obtain information about ventricular function , valvular dysfunction and aortic pathologic characteristics.6 finally , strategies to prevent contrast - induced nephropathy during angiography that is continued in the postoperative period may also have the potential to decrease arf after early cardiac surgery .", "mms designed the study and drafted this manuscript ; mg carried out the study and drafted the tables ; pn carried out the study and drafted the tables ; hsh helped in designing the study ; hms helped in final drafting ; ak helped in designing and carried out the study ; pms data analysis ; and nf data collection ."], "abstract_text": ["<S> background : there is limited data about the influence of timing of cardiac surgery in relation to diagnostic angiography and/or the impact of the amount of contrast media used during angiography on the occurance of acute renal failure ( arf ) . </S>", "<S> therefore , in the present study the effect of the time interval between diagnostic angiography and cardiac surgery and also the amount of contrast media used during the diagnostic procedure on the incidence of arf after cardiac surgery was investigated.methods:data of 1177 patients who underwent different types of cardiac surgeries after cardiac catheterization were prospectively examined . </S>", "<S> the influence of time interval between cardiac catheterization and surgery as well as the amount of contrast agent on postoperative arf were assessed using multivariable logistic regression.results:the patients who progressed to arf were more likely to have received a higher dose of contrast agent compared to the mean dose . however , the time interval between cardiac surgery and last catheterization was not significantly different between the patients with and without arf ( p = 0.05 ) . overall , postoperative peak creatinine was highest on day 0 , then decreased and remained significantly unchanged after this period . </S>", "<S> overall prevalence of acute renal failure during follow - up period had a changeable trend and had the highest rates in days 1 ( 53.57% ) and 6 ( 52.17% ) after surgery . </S>", "<S> combined coronary bypass and valve surgery were the strongest predictor of postoperative arf ( or : 4.976 , ci = 1.613 - 15.355 and p = 0.002 ) , followed by intra - aortic balloon pump insertion ( or : 6.890 , ci = 1.482 - 32.032 and p = 0.009 ) and usage of higher doses of contrast media agent ( or : 1.446 , ci = 1.033 - 2.025 and p = 0.031).conclusions : minimizing the amount of contrast agent has a potential role in reducing the incidence of postoperative arf in patients undergoing cardiac surgery , but delaying cardiac surgery after exposure to these agents might not have this protective effect . </S>"], "labels": null, "section_names": ["Methods", "None", "Statistical analyses", "Results", "Discussion", "Authors Contributions"], "sections": [["the study population included 1177 patients who underwent different types of cardiac surgeries with cardiopulmonary bypass method at sina private hospital ( isfahan , iran ) , between april 2008 and september 2009 .", "all patients underwent a cardiovascular diagnostic procedure requiring the use of a contrast agent for left ventriculography , coronary or carotid angiography , and thoracic and/or abdominal aorta angiography before their surgeries .", "thus , the patients requiring emergency dialysis therapy or those that underwent the off - pump method surgery were excluded .", "data for the studied patients including demographics , clinical characteristics , laboratory data , medical treatments , angiographic data , ( including amount and type of the contrast agent used ) , comorbid conditions and cardiac procedural data ( including its timing in relation to the preceding angiography ) , were collected by reviewing hospital record files in a computerized database .", "the study was approved by the research ethics committee of isfahan university of medical sciences , isfahan sina heart center .", "the contrast agent used were meglumine compound 76% ( urografin , bayer , usa ) as hyperosmolar , iopromide 370 ( ultravisit , berlex montville , usa , mfd . in : germany ) and iohexol 300 ( iopaque , iran or omnipaque 300 , usa ) as low - osmolar contrast agents .", "use of nonsteroidal anti - inflammatory agents and other nephrotoxic drugs were avoided or withheld for 48 hours before angiography .", "postoperative renal risk was assessed using 2 different risk scores of a simplified predictive index for renal replacement therapy after cardiac surgery and a risk score for prediction of contrast - induced nephropathy.9 the main outcome of interest was the incidence of postoperative arf , defined as postoperative serum creatinine > 2 times baseline and/or the need for renal replacement therapy .", "the mean value of baseline creatinine was 0.99 mg / dl.6 the obtained results are presented as mean  sd , frequency , and percentage .", "comparisons were made using chi - square test or fisher 's exact test for categorical variables and nonparametric test for continuous variables .", "the amount of contrast media was entered into the model as both a linear ( continuous variable ) and a dichotomous term ( median or less and greater than median value ) .", "multivariable logistic regression analysis was used to identify independent predictors of arf using variables found to have marginal association ( p > 0.10 ) on univariate analysis .", "odds ratio ( or ) and 95% confidence interval ( ci ) were constructed to provide an estimate of adjusted risk posed by post - procedural arf ."], ["comparisons were made using chi - square test or fisher 's exact test for categorical variables and nonparametric test for continuous variables .", "the amount of contrast media was entered into the model as both a linear ( continuous variable ) and a dichotomous term ( median or less and greater than median value ) .", "multivariable logistic regression analysis was used to identify independent predictors of arf using variables found to have marginal association ( p > 0.10 ) on univariate analysis .", "odds ratio ( or ) and 95% confidence interval ( ci ) were constructed to provide an estimate of adjusted risk posed by post - procedural arf ."], ["comparisons were made using chi - square test or fisher 's exact test for categorical variables and nonparametric test for continuous variables .", "the amount of contrast media was entered into the model as both a linear ( continuous variable ) and a dichotomous term ( median or less and greater than median value ) .", "multivariable logistic regression analysis was used to identify independent predictors of arf using variables found to have marginal association ( p > 0.10 ) on univariate analysis .", "odds ratio ( or ) and 95% confidence interval ( ci ) were constructed to provide an estimate of adjusted risk posed by post - procedural arf ."], ["of 1177 patients who underwent different types of cardiac surgeries after angiography , arf occurred in 465 subjects , 2 of these patients needed dialysis .", "mean dose of hyperosmolar and low - osmolar contrast agents used overall were 1.23 and 1.21 ml / kg , respectively .", "baseline clinical features between the two cohorts with and without postoperative arf are listed in table 1 .", "postoperative arf was more prevalent in older and male patients ( p = 0.003 and 0.002 , respectively ) , and those with a higher history of hypertension(p = 0.012 ) , but prevalence of other risk factors as well as left ventricular ejection fraction were similar in both group of patients .", "there was a significant difference between last pre and post- operative creatinine in both groups ( p < 0.001 ) .", "patients with arf were also more likely to have undergone combined coronary artery bypass grafting and valve surgery the same day as angiography and also underwent more postoperative intra - aortic balloon pump insertion ( iabp ) ( table 2 ) .", "the patients with arf were more likely to have received higher doses of both hyper - osmolar and low - osmolar contrast agents compared to mean doses ( p = 0.17 and 0.035 , respectively ) .", "however , the time interval between cardiac surgery and last catheterization was not different between the patients with and without arf ( p > 0.05 ) ( table 2 ) . clinical and demographic characteristics of the studied population contrast agent and cardiac surgery data overall postoperative peak creatinine was highest on day 0 ( figure 2 ) , then decreased and remained significantly unchanged after this period ( p < 0.05 ) .", "however , overall prevalence of acute renal failure during follow - up period ( figure 2 ) had a changeable trend and had the highest rates in days 1 ( 53.57% ) and 6 ( 52.17% ) ( p < 0.05 ) .", "relationship between the time interval flanked by angiography and surgery and postoperative serum creatinine relationship between the time interval flanked by angiography and surgery and postoperative acute renal failure independent correlates of postoperative arf in a multivariate model are listed in table 3 .", "combined coronary artery bypass grafting ( cabg ) and valve surgery was the strongest predictor of postoperative arf ( or : 4.976 , p = 0.002 ) , followed by intra - aortic balloon pump insertion ( or : 6.890 , p = 0.009 ) and higher dose of contrast agent ( or : 1.446 , p = 0.031 ) ."], ["the major findings of the present study were that the higher dose of the used contrast agents besides some other determinants such as combined coronary bypass and valvular surgery as well as iabp insertion could effectively predict post - procedure acute arf .", "on the other hand , the use of high dose of contrast agent was strongly related to the incidence of arf after combined cardiac surgery .", "in fact , our data confirmed findings of other investigations that arf after cardiac surgery was associated with the amount of prescribed contrast agents . in a similar study by ranucci and his colleagues,6 cardiac surgery performed on the day of cardiac catheterization and higher dose of contrast agent", "although these investigators demonstrated a correlation between the time interval of cardiac catheterization and surgical procedure with the incidence of acute arf , we could not demonstrate this relationship in our multivariable regression model . in another study by del duca", "et al4 cardiac catheterization performed within 5 days before operation , baseline glomerular filtration rate of less than 60 ml / min and prolonged cardiopulmonary bypass duration , were significant risk factors for acute renal failure after cardiac surgery . moreover , other studies established similar clinical and surgical characteristics associated with increased risk of arf after cardiac surgery , including age,168 baseline serum creatinine,10 diabetes mellitus,1113 congestive heart failure,1012 emergency surgery,1114 concomitant valve surgery with coronary artery bypass grafting,11 preoperative use of iabp,14 and low cardiac output syndrome in the postoperative course.1415 however , these studies consistently failed to account for the influence of the timing between previous angiography and cardiac surgery and the amount of the contrast agent used in this event .", "multiple mechanisms have been suggested to contribute to the renal damage following cardiac diagnostic procedures , including non - pulsatile flow , embolization , and trauma to the blood constituents , hypothermia , and activation of known inflammatory pathways.16 iodinated contrast as a common contrast agent , after causing a brief period of vasodilation , can cause sustained intrarenal vasoconstriction and ischemic injury .", "the ischemic injury sets off a cascade of events largely driven by oxidative injury , causing death of renal tubular cells .", "if a sufficient mass of nephron units are affected , then a recognizable rise in serum creatinine will occur.1718 therefore , minimizing the amount of contrast agent during preceding angiography is recommended .", "similar studies need to be conducted regarding alternate imaging modalities that do not use iodine - based contrast agents to obtain information about ventricular function , valvular dysfunction and aortic pathologic characteristics.6 finally , strategies to prevent contrast - induced nephropathy during angiography that is continued in the postoperative period may also have the potential to decrease arf after early cardiac surgery ."], ["mms designed the study and drafted this manuscript ; mg carried out the study and drafted the tables ; pn carried out the study and drafted the tables ; hsh helped in designing the study ; hms helped in final drafting ; ak helped in designing and carried out the study ; pms data analysis ; and nf data collection ."]]}
{"article_id": "PMC2771154", "article_text": ["colorectal cancer ( crc ) is one of the most common human malignancies in western countries .", "colorectal adenomas have high malignancy potential when they are large in diameter and/or present with severe dysplasia and/or a villous component .", "colonoscopic polypectomy has been documented to significantly reduce the incidence of colorectal cancer [ 3 , 4 ] .", "therefore , the identification of factors associated with the development of colorectal adenoma represents a major goal in colorectal cancer prevention .", "they could indeed allow the selection of individuals at risk of crc who may benefit from a screening by colonoscopy .", "the adenoma - carcinoma sequence suggests that colorectal adenomas and adenocarcinomas share common environmental and genetic risk factors .", "an increased risk of colorectal tumors has been found in relatives of patients with large adenomas [ 5 , 6 ] .", "a case - control study had suggested that family history of colorectal cancer influenced only the growth of adenomas or their malignant transformation . however , relatively few epidemiologic studies explored genetic risk factors in colorectal adenomas .", "we investigated , through a case - control study , the relation between polymorphisms within a series of candidate genes involved in colorectal tumorigenesis and putatively in the formation and the development of colorectal adenomas such carcinogen metabolism enzymes , methylation enzymes , dna repair genes , oncogenes and tumor suppressor genes .", "the genetics of adenomas ( geade ) study is a case - control and family study of patients with high - risk adenomas ( 10 mm ) .", "the data were obtained from 18 participating gastroenterology units of general hospitals in france . from september 1995 to march 2000 , 306 consecutive patients with newly diagnosed colorectal large adenoma ( la ) were enrolled in the study .", "patients with personal cancer history , familial adenomatous polyposis , established hereditary nonpolyposis colorectal cancer or inflammatory bowel disease were excluded . to distinguish genetic factors involved in the occurrence of adenomas or in their growth , 307 cases with small adenomas ( with a diameter smaller than 0.5 cm ) ( sa ) and 572 polyp - free controls ( with normal colonoscopy ) ( pf )", "reason for referral , family history of crc , completeness of colonoscopy were registered for all patients and controls .", "two pf per la cases were selected as controls within over 2000 pf for matching on age , gender , and geographic area .", "blood specimens were obtained at time of colonoscopy and those patients who presented with a polyp were included only when histological examination revealed the adenomatous nature of the lesion . as polyps were totally removed during colonoscopy , their natural evolution could not be scored .", "after longitudinally section , half of the tumor material was fixed for histologic analysis and half was frozen for molecular characterization .", "twenty individuals had to be excluded because of insufficient tumor material : 11 patients with la , 5 with sa , and 4 pf .", "the final groups contained 295 patients with la , 302 with sa , and 568 pf as controls .", "all patients and controls signed an informed consent after approval of the study by an ethic committee for biomedical research ( le kremlin - bictre ) and the database was declared to the national committee commission nationale de linformatique et des liberts ( cnil ) .", "genes have been selected for their role in colorectal tumorigenesis and for the presence of frequent neutral polymorphisms .", "the polymorphisms of mmp1 , mmp3 , and mmp7 gene promoters , that are responsible for the degradation of extracellular matrix components , had been previously studied and were not considered in the present analysis .", "three genes , ts , ugt1a1 , and mdr1 , are implicated in folates , bilirubin metabolism , and transport of xenobiotic respectively .", "all these pathways are suspected to play a role in cancer occurrence or in the transformation of benign lesions , folates by interfering with dna synthesis and repair , bilirubin by its known antioxydant properties , and transport of xenobiotic by its detoxication function .", "case - control reports   exemplified the relevance of these three genes in addition to hras1 .", "three additional pathways have been secondary studied including the wnt pathway ( apc , ctnnb1 , axin2 ) , the p53 pathway ( tp53 , bax ) , the tgfb pathway ( tgfbr2 ) , the main mmr and ber genes ( msh2 , mlh1 , myh ) , and card15 as part of the family with lrr domains .", "all these genes have been strongly assessed for their major role in oncogenesis , cycle cell control , apoptosis regulation , cell adhesion and migration , proliferation , dna repair as their main functions .", "the vntr of hras1 was studied by conventional electrophoresis after pcr amplification on 1.2% agarose gels at 2 volts / cm for 1518 hours .", "hardy - weinberg proportions were tested for each polymorphism . linkage disequilibrium ( ld ) between pairwise loci", "genotype - specific odds ratio ( or ) and 95% confidence intervals ( cis ) were computed using unconditional logistic regression adjusted on matching factors ; wald test was used to assess the global effect of each polymorphism .", "the homozygous genotype for the more frequent allele among controls was set as the reference class .", "the association was further examined using the combination test , that allows the analysis of all possible combinations of snps within a given gene or tightly linked genes to test their association with the disease .", "for each snps combination , the method computes a statistic test contrasting the genotypic ( or haplotypic ) distribution between cases and controls .", "because all these tests are correlated ( many of them are nested in each other and the snps are likely to be in ld ) , a permutation procedure has been implemented which displays a significance level adequately adjusted for multiple testing .", "first , we used the famhap12 software to apply this method by performing haplotypic tests .", "then the combintest ( jannot , personal communication ) was used to perform genotypic tests .", "the method was extended to the test of combined polymorphisms on different genes that may act interactively .", "to avoid multiple tests biases , we chose a three - step strategy that minimizes the number of comparisons .", "first , we considered two - by - two combinations of polymorphisms chosen on their plausibility to interact with each other , for instance , apc and myh both responsible , when mutated , for adenomatous polyposis .", "finally , as a combination of polymorphisms may have an effect even if the genes are not suspected to interact with each other and are not involved in the same pathway , all possible combinations were considered if the first two steps did not reveal any significant association .", "when several combinations of polymorphisms were significant within a given test , it was possible to test nested combinations using chi - square calculation to determine whether a given polymorphism adds a significant contribution to the association found .", "the average age was very similar in patients ( 62 years for la and 61 years for sa ) and in pf controls ( 61 years ) .", "the sex ratio ( male / female ) was quite similar in la ( 1.7 ) and in pf controls ( 1.5 ) , slightly lower in sa ( 1.2 ) because of the absence of matching . \n", "table 1 describes the characteristics of the polymorphisms studied and the allele frequencies in controls .", "the distribution of genotypes in controls was consistent with hardy - weinberg proportions for all polymorphisms except for tp53.3 ( p = .0013 without correction for multiple testing ) . a similar departure from h - w proportions (", "p = .010 ) was found in the hapmap european population ( http://www.ncbi.nlm.nih.gov/projects/snp/ ) for this polymorphism .", "all polymorphisms within a same gene were moderately or not found in ld except myh ; myh.1 and myh.2 are in quasicomplete disequilibrium ( d = 1 and r = 0.99 ) .", "analysis of single polymorphisms for the different comparisons , la versus pf , la versus sa , and sa versus pf did not reveal any association for most of the genes studied .", "the p - values found to be less than .05 were obtained for apc.1 , mdr1.2 , and axin2.9 .", "the results of the haplotypic and genotypic combination tests performed for each gene or gene combination are shown in table 4 .", "when considering each gene separately , only apc displayed a significant difference between small and large adenomas ( p = .014 in haplotypic analysis and p = .07 in genotypic analysis ) .", "as apc.1 alone and the combination apc.1-apc.2 appeared both significant , we tested whether apc.2 provided a significant contribution to the association , which was not the case (  = 2.12 , 1df ) showing that the association was solely due to the apc.1 ( p.glu1317gln ) polymorphism .", "the combination apc - myh appeared also significant with a global p - value of .045 .", "however , the only significant combination was apc.1-apc.2 already found , showing that myh does not provide any additional contribution to the association between apc and adenomas .", "no difference was found for the other two comparisons , that is , sa versus pf and la versus pf . finally , the analysis of all possible combinations did not provide any significant result .", "we investigated the role of candidate gene polymorphisms in a case - control study of patients with large adenomatous polyps ( n = 295 ) , compared to patients with small adenomatous polyps ( n = 302 ) and polyp - free controls ( n = 568 ) .", "the 38 polymorphisms studied belonged to 14 genes possibly involved in increasing of colorectal cancer risk .", "the reason for comparing these different groups of patients was that different genes might be involved in the different steps of carcinogenesis . using this case - control study", ", we had shown that mmp polymorphism was most probably involved in the occurrence , but not in the growth of polyps . using the combination test", ", we had shown that the effect was due to a specific combination of mmp1-mmp3 polymorphisms which was not found using logistic regression .", "indeed , a major property of this test is that it allows the detection of polymorphisms with low marginal effects .", "multiple testing may be a limitation of studies in which several polymorphisms in several genes are tested .", "the combination test allows a correction for multiple tests on tightly linked markers , which other methods of correction such as bonferroni , benjamini - hochberg , or fdr ( false discovery rate ) do not . in the present study", ", we could show that the positive results obtained for axin2 and mdr1 when analyzing single polymorphisms were false positives as these results were not confirmed when using the combination test . for independent markers , there was no need for correcting for multiple testing as our results were essentially negative .", "our study did not give evidence for an effect of any of the gene polymorphisms studied except the p.glu1317gly variant of the apc gene .", "some studies reported an effect of this variant in patients with multiple adenomas or colorectal cancer [ 1517 ] , and some others found an effect in adenomas but not in cancer [ 18 , 19 ] .", "more recently , hahnloser et al .   reported an effect of this variant in a group of patients with a small number of adenomas ( 1 to 3 lifetime adenomas ) and in a group of crc cases .", "none of these studies considered the size of adenomas and could differentiate adenomas at high - risk from those at low - risk of cancer .", "our results favour the hypothesis that the p.glu1317gly variant would influence the growth and not the occurrence of adenomas , and would have thus indirectly an influence on colorectal cancer risk .", "nevertheless , although the three groups are paired on gender and geographic origin , it is not stated if other known risk factors would influence adenoma growth .", "it is thus important to plan a specific multivariate analysis when building a replication sample . regarding the ts polymorphisms", "chen et al .   found a significant effect of the 3r allele of ts.2 , particularly of the 3r/3r genotype ( or = 3.3 ) . in our study ,", "the or associated with 3r/3r genotype was 1.43 ( ci 0.942.16 ) for la versus pf and 1.10 ( ci 0.681.78 ) for la versus sa .", "on the other hand , ulrich et al .   found no marginal effect with any of these two polymorphisms but found a significant interaction of ts.2 with folate intake .", "similarly , the most recent studies [ 23 , 24 ] found no marginal effect of either polymorphism but a slight interaction with folate or vitamin b6 intake . the groups ' sizes in our study were of the same order of magnitude as in the study of chen et al . ,", "but the three other studies which did not show any marginal effect included larger groups ( more than 500 individuals in each group ) . given the size of our population , we could a priori detect an or of 1.7 with a power of at least 80% .", "it is highly probable that the ts gene , which produces a key enzyme in folate metabolism , has an effect only in interaction with dietary exposures , which would explain why this effect was not found in our study .", "moreover a new snp has been recently identified in the second repeat of the 3r allele leading to split the allele 3r in 3rc and 3rg alleles as a tri - allelic polymorphism .", "this variant could explain these discrepant results since patients with 3rc/3rc genotype have a transcriptional activity of ts comparable to those with 2r/2r genotype . in our study ,", "the problem of multiple testing was adequately controlled for each test performed by the permutation procedure implemented in the combination test . in the last analysis step ,", "the tremendous number of comparisons resulted in a drastic correction of each p - value , which considerably lowered the testing power .", "thus , our negative results certainly do not definitively demonstrate the absence of specific combination influencing colorectal tumorigenesis , but more probably that , if it exists , this ( of these ) effect(s ) is ( are ) low and could be found through the initial ( and further ) genome - wide association studies of colorectal cancer testing much larger numbers of patients and controls [ 26 , 27 ] ."], "abstract_text": ["<S> predisposition to sporadic colorectal tumours is influenced by genes with minor phenotypic effects . </S>", "<S> a case - control study was set up on 295 patients treated for a large adenoma matched with polyp - free individuals on gender , age , and geographic origin in a 1 : 2 proportion . </S>", "<S> a second group of 302 patients treated for a small adenoma was also characterized to distinguish effects on adenoma occurrence and growth . </S>", "<S> we focussed the study on 38 single nucleotide polymorphisms ( snps ) encompassing 14 genes involved in colorectal carcinogenesis . </S>", "<S> effect of snps was tested using unconditional logistic regression . </S>", "<S> comparisons were made for haplotypes within a given gene and for biologically relevant genes combinations using the combination test . </S>", "<S> the apc p.glu1317gly variant appeared to influence the adenoma growth ( p = .04 , exact test ) but not its occurrence . </S>", "<S> this result needs to be replicated and genome - wide association studies may be necessary to fully identify low - penetrance alleles involved in early stages of colorectal tumorigenesis . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["colorectal cancer ( crc ) is one of the most common human malignancies in western countries .", "colorectal adenomas have high malignancy potential when they are large in diameter and/or present with severe dysplasia and/or a villous component .", "colonoscopic polypectomy has been documented to significantly reduce the incidence of colorectal cancer [ 3 , 4 ] .", "therefore , the identification of factors associated with the development of colorectal adenoma represents a major goal in colorectal cancer prevention .", "they could indeed allow the selection of individuals at risk of crc who may benefit from a screening by colonoscopy .", "the adenoma - carcinoma sequence suggests that colorectal adenomas and adenocarcinomas share common environmental and genetic risk factors .", "an increased risk of colorectal tumors has been found in relatives of patients with large adenomas [ 5 , 6 ] .", "a case - control study had suggested that family history of colorectal cancer influenced only the growth of adenomas or their malignant transformation . however , relatively few epidemiologic studies explored genetic risk factors in colorectal adenomas .", "we investigated , through a case - control study , the relation between polymorphisms within a series of candidate genes involved in colorectal tumorigenesis and putatively in the formation and the development of colorectal adenomas such carcinogen metabolism enzymes , methylation enzymes , dna repair genes , oncogenes and tumor suppressor genes ."], ["the genetics of adenomas ( geade ) study is a case - control and family study of patients with high - risk adenomas ( 10 mm ) .", "the data were obtained from 18 participating gastroenterology units of general hospitals in france . from september 1995 to march 2000 , 306 consecutive patients with newly diagnosed colorectal large adenoma ( la ) were enrolled in the study .", "patients with personal cancer history , familial adenomatous polyposis , established hereditary nonpolyposis colorectal cancer or inflammatory bowel disease were excluded . to distinguish genetic factors involved in the occurrence of adenomas or in their growth , 307 cases with small adenomas ( with a diameter smaller than 0.5 cm ) ( sa ) and 572 polyp - free controls ( with normal colonoscopy ) ( pf )", "reason for referral , family history of crc , completeness of colonoscopy were registered for all patients and controls .", "two pf per la cases were selected as controls within over 2000 pf for matching on age , gender , and geographic area .", "blood specimens were obtained at time of colonoscopy and those patients who presented with a polyp were included only when histological examination revealed the adenomatous nature of the lesion . as polyps were totally removed during colonoscopy , their natural evolution could not be scored .", "after longitudinally section , half of the tumor material was fixed for histologic analysis and half was frozen for molecular characterization .", "twenty individuals had to be excluded because of insufficient tumor material : 11 patients with la , 5 with sa , and 4 pf .", "the final groups contained 295 patients with la , 302 with sa , and 568 pf as controls .", "all patients and controls signed an informed consent after approval of the study by an ethic committee for biomedical research ( le kremlin - bictre ) and the database was declared to the national committee commission nationale de linformatique et des liberts ( cnil ) .", "genes have been selected for their role in colorectal tumorigenesis and for the presence of frequent neutral polymorphisms .", "the polymorphisms of mmp1 , mmp3 , and mmp7 gene promoters , that are responsible for the degradation of extracellular matrix components , had been previously studied and were not considered in the present analysis .", "three genes , ts , ugt1a1 , and mdr1 , are implicated in folates , bilirubin metabolism , and transport of xenobiotic respectively .", "all these pathways are suspected to play a role in cancer occurrence or in the transformation of benign lesions , folates by interfering with dna synthesis and repair , bilirubin by its known antioxydant properties , and transport of xenobiotic by its detoxication function .", "case - control reports   exemplified the relevance of these three genes in addition to hras1 .", "three additional pathways have been secondary studied including the wnt pathway ( apc , ctnnb1 , axin2 ) , the p53 pathway ( tp53 , bax ) , the tgfb pathway ( tgfbr2 ) , the main mmr and ber genes ( msh2 , mlh1 , myh ) , and card15 as part of the family with lrr domains .", "all these genes have been strongly assessed for their major role in oncogenesis , cycle cell control , apoptosis regulation , cell adhesion and migration , proliferation , dna repair as their main functions .", "the vntr of hras1 was studied by conventional electrophoresis after pcr amplification on 1.2% agarose gels at 2 volts / cm for 1518 hours .", "hardy - weinberg proportions were tested for each polymorphism . linkage disequilibrium ( ld ) between pairwise loci", "genotype - specific odds ratio ( or ) and 95% confidence intervals ( cis ) were computed using unconditional logistic regression adjusted on matching factors ; wald test was used to assess the global effect of each polymorphism .", "the homozygous genotype for the more frequent allele among controls was set as the reference class .", "the association was further examined using the combination test , that allows the analysis of all possible combinations of snps within a given gene or tightly linked genes to test their association with the disease .", "for each snps combination , the method computes a statistic test contrasting the genotypic ( or haplotypic ) distribution between cases and controls .", "because all these tests are correlated ( many of them are nested in each other and the snps are likely to be in ld ) , a permutation procedure has been implemented which displays a significance level adequately adjusted for multiple testing .", "first , we used the famhap12 software to apply this method by performing haplotypic tests .", "then the combintest ( jannot , personal communication ) was used to perform genotypic tests .", "the method was extended to the test of combined polymorphisms on different genes that may act interactively .", "to avoid multiple tests biases , we chose a three - step strategy that minimizes the number of comparisons .", "first , we considered two - by - two combinations of polymorphisms chosen on their plausibility to interact with each other , for instance , apc and myh both responsible , when mutated , for adenomatous polyposis .", "finally , as a combination of polymorphisms may have an effect even if the genes are not suspected to interact with each other and are not involved in the same pathway , all possible combinations were considered if the first two steps did not reveal any significant association .", "when several combinations of polymorphisms were significant within a given test , it was possible to test nested combinations using chi - square calculation to determine whether a given polymorphism adds a significant contribution to the association found ."], ["the average age was very similar in patients ( 62 years for la and 61 years for sa ) and in pf controls ( 61 years ) .", "the sex ratio ( male / female ) was quite similar in la ( 1.7 ) and in pf controls ( 1.5 ) , slightly lower in sa ( 1.2 ) because of the absence of matching . \n", "table 1 describes the characteristics of the polymorphisms studied and the allele frequencies in controls .", "the distribution of genotypes in controls was consistent with hardy - weinberg proportions for all polymorphisms except for tp53.3 ( p = .0013 without correction for multiple testing ) . a similar departure from h - w proportions (", "p = .010 ) was found in the hapmap european population ( http://www.ncbi.nlm.nih.gov/projects/snp/ ) for this polymorphism .", "all polymorphisms within a same gene were moderately or not found in ld except myh ; myh.1 and myh.2 are in quasicomplete disequilibrium ( d = 1 and r = 0.99 ) .", "analysis of single polymorphisms for the different comparisons , la versus pf , la versus sa , and sa versus pf did not reveal any association for most of the genes studied .", "the p - values found to be less than .05 were obtained for apc.1 , mdr1.2 , and axin2.9 .", "the results of the haplotypic and genotypic combination tests performed for each gene or gene combination are shown in table 4 .", "when considering each gene separately , only apc displayed a significant difference between small and large adenomas ( p = .014 in haplotypic analysis and p = .07 in genotypic analysis ) .", "as apc.1 alone and the combination apc.1-apc.2 appeared both significant , we tested whether apc.2 provided a significant contribution to the association , which was not the case (  = 2.12 , 1df ) showing that the association was solely due to the apc.1 ( p.glu1317gln ) polymorphism .", "the combination apc - myh appeared also significant with a global p - value of .045 .", "however , the only significant combination was apc.1-apc.2 already found , showing that myh does not provide any additional contribution to the association between apc and adenomas .", "no difference was found for the other two comparisons , that is , sa versus pf and la versus pf . finally , the analysis of all possible combinations did not provide any significant result ."], ["we investigated the role of candidate gene polymorphisms in a case - control study of patients with large adenomatous polyps ( n = 295 ) , compared to patients with small adenomatous polyps ( n = 302 ) and polyp - free controls ( n = 568 ) .", "the 38 polymorphisms studied belonged to 14 genes possibly involved in increasing of colorectal cancer risk .", "the reason for comparing these different groups of patients was that different genes might be involved in the different steps of carcinogenesis . using this case - control study", ", we had shown that mmp polymorphism was most probably involved in the occurrence , but not in the growth of polyps . using the combination test", ", we had shown that the effect was due to a specific combination of mmp1-mmp3 polymorphisms which was not found using logistic regression .", "indeed , a major property of this test is that it allows the detection of polymorphisms with low marginal effects .", "multiple testing may be a limitation of studies in which several polymorphisms in several genes are tested .", "the combination test allows a correction for multiple tests on tightly linked markers , which other methods of correction such as bonferroni , benjamini - hochberg , or fdr ( false discovery rate ) do not . in the present study", ", we could show that the positive results obtained for axin2 and mdr1 when analyzing single polymorphisms were false positives as these results were not confirmed when using the combination test . for independent markers , there was no need for correcting for multiple testing as our results were essentially negative .", "our study did not give evidence for an effect of any of the gene polymorphisms studied except the p.glu1317gly variant of the apc gene .", "some studies reported an effect of this variant in patients with multiple adenomas or colorectal cancer [ 1517 ] , and some others found an effect in adenomas but not in cancer [ 18 , 19 ] .", "more recently , hahnloser et al .   reported an effect of this variant in a group of patients with a small number of adenomas ( 1 to 3 lifetime adenomas ) and in a group of crc cases .", "none of these studies considered the size of adenomas and could differentiate adenomas at high - risk from those at low - risk of cancer .", "our results favour the hypothesis that the p.glu1317gly variant would influence the growth and not the occurrence of adenomas , and would have thus indirectly an influence on colorectal cancer risk .", "nevertheless , although the three groups are paired on gender and geographic origin , it is not stated if other known risk factors would influence adenoma growth .", "it is thus important to plan a specific multivariate analysis when building a replication sample . regarding the ts polymorphisms", "chen et al .   found a significant effect of the 3r allele of ts.2 , particularly of the 3r/3r genotype ( or = 3.3 ) . in our study ,", "the or associated with 3r/3r genotype was 1.43 ( ci 0.942.16 ) for la versus pf and 1.10 ( ci 0.681.78 ) for la versus sa .", "on the other hand , ulrich et al .   found no marginal effect with any of these two polymorphisms but found a significant interaction of ts.2 with folate intake .", "similarly , the most recent studies [ 23 , 24 ] found no marginal effect of either polymorphism but a slight interaction with folate or vitamin b6 intake . the groups ' sizes in our study were of the same order of magnitude as in the study of chen et al . ,", "but the three other studies which did not show any marginal effect included larger groups ( more than 500 individuals in each group ) . given the size of our population , we could a priori detect an or of 1.7 with a power of at least 80% .", "it is highly probable that the ts gene , which produces a key enzyme in folate metabolism , has an effect only in interaction with dietary exposures , which would explain why this effect was not found in our study .", "moreover a new snp has been recently identified in the second repeat of the 3r allele leading to split the allele 3r in 3rc and 3rg alleles as a tri - allelic polymorphism .", "this variant could explain these discrepant results since patients with 3rc/3rc genotype have a transcriptional activity of ts comparable to those with 2r/2r genotype . in our study ,", "the problem of multiple testing was adequately controlled for each test performed by the permutation procedure implemented in the combination test . in the last analysis step ,", "the tremendous number of comparisons resulted in a drastic correction of each p - value , which considerably lowered the testing power .", "thus , our negative results certainly do not definitively demonstrate the absence of specific combination influencing colorectal tumorigenesis , but more probably that , if it exists , this ( of these ) effect(s ) is ( are ) low and could be found through the initial ( and further ) genome - wide association studies of colorectal cancer testing much larger numbers of patients and controls [ 26 , 27 ] ."]]}
{"article_id": "PMC2988937", "article_text": ["a 56-year - old man suffering from diabetes and hypertension was admitted to the neurology department in a hospital elsewhere with signs of epileptic seizure . after a brief period ,", "a diagnostic laparotomy with gastrotomy was performed , at which over 3 liters of blood were found , but no active bleeding .", "the site of origin of hemorrhage was suspected at the gastroesophageal junction , although no peptic ulcer could be discovered .", "gastroscopy was performed 1 day later , demonstrating a large , ulcerating tumor at the cardia , of which biopsies were taken . because the patient was now diagnosed with a bleeding tumor at the gastroesophageal junction , he was transferred to the icu of the canisius - wilhelmina hospital in nijmegen ( in formation with the radboud university hospital comprising the esophagus centre eastern netherlands , scon ) .", "a computed tomography ( ct ) scan was performed , showing a large tumor at the gastroesophageal junction , extending towards the pancreatic tail ( fig .", "a second gastroscopy with new biopsies , 8 days later , proved the same results . at the time of the third gastroscopy ,", "a second ct scan revealed a remarkable decline of the entire process , emanating from the pancreatic tail as a pseudocyst ( fig .", "our patient experienced hypovolemic shock following massive gastrointestinal hemorrhage caused by a burst - through of a pancreatic pseudocyst to the stomach .", "the incidence of upper gastrointestinal hemorrhage in the netherlands is 45 - 70 per 100,000 per year , most of which are caused by peptic ulcers of the stomach and duodenum .", "a pseudocyst of the pancreas consists of one or several collections of pancreatic fluid , surrounded by inflamed pancreatic tissues .", "the wall is formed by the enclosing structures , i.e. stomach , transverse colon or omentum . the pseudocyst is usually connected to the pancreatic duct .", "the distinctive feature between a genuine cyst and a pseudocyst is the lack of an epithelial layer .", "pseudocysts of the pancreas arise as a result of episodes of acute pancreatitis or blunt force abdominal trauma .", "the cause is often disruption or obstruction of the pancreatic duct , with subsequent accumulation of pancreatic fluids , containing high concentrations of pancreatic enzymes . in acute pancreatitis , ( pseudo)cyst and necrosis", "can be difficult to distinguish by contrast - enhanced ct scan , whereas magnetic resonance imaging ( mri ) is distinctive .", "pseudocysts can damage adjacent arteries by means of mechanical pressure and digestion by pancreatic enzymes , hereby causing the formation of pseudoaneurysm with possible subsequent hemorrhage into the pancreatic duct .", "usually , this involves the splenic artery , but also the gastroepiploic arteries and gastroduodenal branches can be affected .", "the old motto is to drain pseudocysts larger than 6 cm or existing longer than 6 weeks , although literature on the subject scarcely supports this .", "the possibilities for drainage are to marsupialize the pseudocyst through laparotomy or laparoscopy to the stomach or jejunum , or to perform resection of the pancreatic tail", "pseudoaneurysms or hemorrhages are eligible for endovascular embolization , and transgastric drainage of cysts is contraindicated during hemorrhages , unless embolization is performed first .", "case series and case reports in the available literature on hemorrhaging pseudocysts all advocate endovascular embolization , followed by resection of the pancreatic tail [ 5 , 8 , 9 , 10 , 11 , 12 , 13 ] . our patient was diagnosed with hemorrhaging pseudocyst 16 days after admission and initial laparotomy .", "because of the improving clinical condition and decline of the pseudocyst on ct scan , it was decided to pursue a ", "the initial plan was to perform pancreatic tail resection after clinical recovery ; however , ultrasonography and ct scan revealed a rapid decline and full disappearance of the pseudocyst .", "our patient made a full recovery and has adjusted his life style . in all case reports ,", "operative intervention is advocated , precipitated by endovascular embolization in case of serious hemodynamic instability .", "one case series of 16 patients reports a 62% mortality rate , in which endovascular embolization is combined with pancreatic tail resection .", "there is evidence to support the role of angiography in case of acute upper gastrointestinal hemorrhage , simultaneously offering the possibility for endovascular treatment [ 2 , 5 , 8 , 9 , 10 , 11 , 12 , 13 ] .", "embolizing the splenic artery does not inevitably necessitate splenectomy , for the remaining blood supply to the spleen is routed through the vasa brevia , which should be sufficient .", "furthermore , the course of our patient proves that surgical intervention such as pancreatic tail resection is not always an absolute necessity .", "however , surgery is nowadays not the only option for treatment , as possibilities in radiological endovascular diagnostics and techniques are increasing and will eventually be the first step in treating acute upper gastrointestinal hemorrhage ."], "abstract_text": ["<S> acute upper gastrointestinal tract hemorrhage represents a frequent morbidity which can be localized and treated endoscopically . when endoscopic treatment alone is failing , radiological or surgical treatment may be warranted . </S>", "<S> a case history will be presented regarding a rare cause of intestinal hemorrhage with an extraordinary course of illness . </S>"], "labels": null, "section_names": ["Case Report", "Discussion", "Conclusion"], "sections": [["a 56-year - old man suffering from diabetes and hypertension was admitted to the neurology department in a hospital elsewhere with signs of epileptic seizure . after a brief period ,", "a diagnostic laparotomy with gastrotomy was performed , at which over 3 liters of blood were found , but no active bleeding .", "the site of origin of hemorrhage was suspected at the gastroesophageal junction , although no peptic ulcer could be discovered .", "gastroscopy was performed 1 day later , demonstrating a large , ulcerating tumor at the cardia , of which biopsies were taken . because the patient was now diagnosed with a bleeding tumor at the gastroesophageal junction , he was transferred to the icu of the canisius - wilhelmina hospital in nijmegen ( in formation with the radboud university hospital comprising the esophagus centre eastern netherlands , scon ) .", "a computed tomography ( ct ) scan was performed , showing a large tumor at the gastroesophageal junction , extending towards the pancreatic tail ( fig .", "a second gastroscopy with new biopsies , 8 days later , proved the same results . at the time of the third gastroscopy ,", "a second ct scan revealed a remarkable decline of the entire process , emanating from the pancreatic tail as a pseudocyst ( fig .", "our patient experienced hypovolemic shock following massive gastrointestinal hemorrhage caused by a burst - through of a pancreatic pseudocyst to the stomach ."], ["the incidence of upper gastrointestinal hemorrhage in the netherlands is 45 - 70 per 100,000 per year , most of which are caused by peptic ulcers of the stomach and duodenum .", "a pseudocyst of the pancreas consists of one or several collections of pancreatic fluid , surrounded by inflamed pancreatic tissues .", "the wall is formed by the enclosing structures , i.e. stomach , transverse colon or omentum . the pseudocyst is usually connected to the pancreatic duct .", "the distinctive feature between a genuine cyst and a pseudocyst is the lack of an epithelial layer .", "pseudocysts of the pancreas arise as a result of episodes of acute pancreatitis or blunt force abdominal trauma .", "the cause is often disruption or obstruction of the pancreatic duct , with subsequent accumulation of pancreatic fluids , containing high concentrations of pancreatic enzymes . in acute pancreatitis , ( pseudo)cyst and necrosis", "can be difficult to distinguish by contrast - enhanced ct scan , whereas magnetic resonance imaging ( mri ) is distinctive .", "pseudocysts can damage adjacent arteries by means of mechanical pressure and digestion by pancreatic enzymes , hereby causing the formation of pseudoaneurysm with possible subsequent hemorrhage into the pancreatic duct .", "usually , this involves the splenic artery , but also the gastroepiploic arteries and gastroduodenal branches can be affected .", "the old motto is to drain pseudocysts larger than 6 cm or existing longer than 6 weeks , although literature on the subject scarcely supports this .", "the possibilities for drainage are to marsupialize the pseudocyst through laparotomy or laparoscopy to the stomach or jejunum , or to perform resection of the pancreatic tail", "pseudoaneurysms or hemorrhages are eligible for endovascular embolization , and transgastric drainage of cysts is contraindicated during hemorrhages , unless embolization is performed first .", "case series and case reports in the available literature on hemorrhaging pseudocysts all advocate endovascular embolization , followed by resection of the pancreatic tail [ 5 , 8 , 9 , 10 , 11 , 12 , 13 ] . our patient was diagnosed with hemorrhaging pseudocyst 16 days after admission and initial laparotomy .", "because of the improving clinical condition and decline of the pseudocyst on ct scan , it was decided to pursue a ", "the initial plan was to perform pancreatic tail resection after clinical recovery ; however , ultrasonography and ct scan revealed a rapid decline and full disappearance of the pseudocyst .", "our patient made a full recovery and has adjusted his life style . in all case reports ,", "operative intervention is advocated , precipitated by endovascular embolization in case of serious hemodynamic instability .", "one case series of 16 patients reports a 62% mortality rate , in which endovascular embolization is combined with pancreatic tail resection .", "there is evidence to support the role of angiography in case of acute upper gastrointestinal hemorrhage , simultaneously offering the possibility for endovascular treatment [ 2 , 5 , 8 , 9 , 10 , 11 , 12 , 13 ] .", "embolizing the splenic artery does not inevitably necessitate splenectomy , for the remaining blood supply to the spleen is routed through the vasa brevia , which should be sufficient .", "furthermore , the course of our patient proves that surgical intervention such as pancreatic tail resection is not always an absolute necessity ."], ["however , surgery is nowadays not the only option for treatment , as possibilities in radiological endovascular diagnostics and techniques are increasing and will eventually be the first step in treating acute upper gastrointestinal hemorrhage ."]]}
{"article_id": "PMC3177388", "article_text": ["today , implant therapy is regarded as an extremely reliable approach to replace missing teeth .", "the introduction of osseointegrated implants in dentistry represented a turning point in dental clinical practice.1 as a general principle in implant surgery implant surfaces should be surrounded by alveolar bone.2 following loss of teeth , natural process of bone resorption occurs.3 sometimes due to prosthetic or anatomical limitations of alveolar ridge , it is not possible to insert implants in bone appropriately.2 and also , while preparing implant sites in narrow ridges , dehiscence or fenestration defects may occur frequently that threaten the survival of implants.4 successful implant treatment depends on sufficient bone volume at the implant surface.5 insufficient amount of supporting bone , will limit the effectiveness of osseointegrated dental implants3 and will have an adverse effect on dental implant prognosis.6 several clinical studies have shown that to ensure long term success of implants , at least one millimetre thickness of bone in buccal surface and lingual surface of implant is essential .", "if the implant surface is not completely covered with bone , it will result in gingival recession , unpleasant appearance , problems in keeping good oral hygiene and ultimately increases the risk of infection around the implant.7 implants can be placed in alveolar ridges with defects including dehiscence , intraalveolar defects , and fenestration.8 several methods are presented to reconstruct the destroyed alveolar bone which include osteoconduction , osteoinduction and guided bone regeneration ( gbr).6 many surgical techniques are introduced to enhance alveolar bone volume for placing implants that include various grafting techniques , bone - building using emineral method , and expanding the bone and guided tissue regeneration ( gtr).5 among these techniques , gbr is widely used .", "gbr procedures performed in dental implants enable clinicians to increase the width and height of defected alveolar ridges or to treat fenestrations and dehiscence around implants .", "many researchers have reported predictable results in the simultaneous use of gbr technique when placing implants.7 in most cases of gbr , membranes are supported by protective materials consisting allografts , synthetic materials and xenografts.6 using autograft in augmentation has always been considered as a gold standard , but limited access to autogenous sources , particularly in the areas inside the mouth , prolongation of surgical procedures and bacterial contamination complication have always been considered as the limiting factors in autogenous transplantation . in addition , general surgical risks such as infection , bleeding , pain and swelling , damage to inferior alveolar nerve and the adjacent teeth should also be noted .", "therefore , using biomaterials on its own or in combination with autogenous bone is very common.9 dfdba can be named as one of the allograft materials which has osteoinductive potential , because this substance contains some major bone morphogenetic proteins ( bmps ) of donor tissue matrix . against this view", ", many recent reports have shown that augmentation with dfdba is not os - teoinductive , because it does not contain the necessary bmps to induce bone formation .", "in the united states , emineralised bone matrix ( dbm ) is considered a transplantable tissue and therefore , is regulated primarily by the american association of tissue banks.10 treatment of dehiscence and fenestration lesions with gbr technique and placing of implants simultaneously have predictable results.11 recent clinical studies have proven that the use of bone substitute materials together with placing implants led to successful coverage of pre - exposed implant surfaces.12", "this prospective clinical trial animal study was conducted in professor torabinejad dental research centre isfahan university of medical sciences , isfahan , iran .", "samples dissection was carried out in the department of materials science and engineering , sharif university of technology , tehran , iran . studied population included three healthy adult iranian dogs that were chosen by convenient method .", "inclusion criteria included healthy 2 to 4 years old dogs that held all the teeth . at the beginning of the study ,", "after examination to confirm their health and having the required criteria ; seven step vaccination was carried out and the dogs were quarantined for 15 days .", "all the mandibular premolars teeth were extracted and then , the area was left to heal for three months . after healing period , the area assessed by periapical radiography in order to ensure full recovery , after preparing two sits on one side of each mandible and three sites on the other side which were more appropriate in terms of size and bone quality to place the implants , dehiscence bone defects , were prepared with dimensions of 5 mm apicocronaly , 4 mm mesiodistaly and 3 mm buccolingual in buccal plate using high - speed handpiece and diamond fissure bur and abundant irrigation .", "10 mm periodontal probes were used to measure dimensions of the artificial dehiscence defects ; all measurements were performed by a specialist investigator .", "euroteknika implants with 10 mm in length , 4.1 mm in diameter and sla surface ( natea system ) inserted into the prepared sites .", "implant stability quotient ( isq ) of each implant was recorded using ostell device ( ostell integration diagnostics , svedalen , sweden ) which was higher than 56 % in all cases , indicating good initial stability of all implants placed .", "after that in two lesions , that was chosen in random , dfdba cenobone , hamanand saz baft , iran and in two other lesions dfdba dembone , pacific coast tissue bank , u.s were placed and the fifth lesion was left empty ; finally , they all were covered by collagen membranes ( bicon , usa ) .", "both dfdba brands were powder in sahape in 0.5 and 1 grams vials the size of the particles were almost similar and of the existing type to treat small lesions .", "dfdba particles were placed in the lesions and were gently pressed against the implant ; the lesion were with particles filled up in a way that all the screws were covered and the primary bone contour was maintained .", "then , the grafted material and surrounded bone were covered completely by collagen membrane . moreover ,", "the gingival flaps were replaced without stretching to submerge the implant and were stitched by polyglycolic acid sutures ( vicryl ) using simple and interrupted loop sutures .", "then , final status of implants were determined by periapical radiography . during the first week after surgery", ", animals were examined several times a day to control the health and complications after the operation and also to ensure the sutures are not opened .", "the animals mouth was washed twice a day using 0.2 chlorhexidine mouthwashes during the first week and thereafter , once a week . after four months of osseointegration period , dogs were anesthetized and periapical radiographs were taken to observe implants situation within the bone . then , crestal incision was done and the implants were exposed and the value of isq was recorded .", "samples were taken and immediately transferred to formaldehyde 10% and kept for 24 hours to be prepared for optical microscope study .", "samples were sectioned by with the ground section method using microtome ( accutom-50 cutting machine , copenhagen , denmark ) .", "sectioned samples were obtained buccolingualy and parallel to the axis of implants in 100 micron thickness .", "sectioned samples prepared with trichro massons staining studied by optical microscope ( olympus , japon ) . using histomorphometric method , the percentage of bone implant contact ( bic ) was cal - culated and type of bone ( lamellar , woven ) was determined at 40 times magnification and recorded by an experienced pathologist using optical microscope . in this method , the area of 2 mm around the implants is evaluated .", "spss 11.5 software was used to analyse data , using one - way anova and pearson correlation and regression tests .", "average bic in cenobone group ( iranian dfdba ) , dembone group ( american dfdba ) and control group were 77.36%  9.96 ( max 89.1% and min 63.4% ) , 78.91%  11.99 ( max 91% and min 56.3% ) and 71.56%  5.61 ( max 75.8% and min 65.2% ) , respectively .", "therewas no significant difference in bic amounts among the three groups ( p = 0.607 ) .", "there was not significant difference in the bic amounts among the three groups defects ( p=0.388 ) .", "comparison of average bic of lamellar bone , woven bone , cenobone and dembone groups defects and control group is presented in figure 1 .", "comparison of average bic of lamellar bone , woven bone , cenobone and dembone groups defects and control group ( lamellar bone p = 0.298 and woven bone p = 0.380 ) . furthermore , there was not meaningful difference in rate of lamellar bone ( p=0.298 ) and woven bone ( p = 0.380 ) formation in defects among the three groups .", "the total sample average isq was 70.83%  6.30 ( max 82.5% and min 61.5% ) .", "average isq in cenobone group , dembone group and control group were 70.29%  7.74 ( max 82.25% and min 61.5% ) , 72.25%  6.81 ( max 82.5% and min 65.25% ) and 69.08%  2.67 ( max 72% and min 66.75% ) , respectively .", "there was no significant difference in isq amounts among the three groups ( p = 0.781 ) .", "there was a positive relationship between total sample bic and isq ( p = 0.004 , r = 0.692 ) .", "histological view under light microscope showes contact of lamellar new bone with implant in cenobone group magnification x40  massons 's trichrom staning .", "histologic view under light microscope showes contact of lamellar new bone with implant in dembone group ( magnification x40  massons 's trichromstaning ) .", "results of the current study indicated that adding dfdba ( dembone and cenobone ) to membrane , on its own did not significantly increase the obtained bic and isq amounts .", "caplanis and colleagues placed , implants in the alveolar defects which treated with membrane on its own and membrane plus dfdba in dogs , the average bic was about 70%13 which is consistent to our study . and also , the bic obtained in this study is consistent with the findings of von arx and colleagues study14 that placed implants in areas grafted with dfdba and other hybrid materials in dogs and gained a high percentage of bic ( 59% to 75% ) . in this study , adding dfdba to the collagen membrane did not lead to strengthen induction of bone formation , and these findings were consistent with the results of other studies that showed adding dfdba to the membrane on its own did not significantly increase the clinical results obtained with the gbr procedure.151920 there were significant differences between the products of bone banks in terms of induction of bone formation .", "some studies found the use of dfdba to have a positive impact to increase bone growth while others considered it not to be beneficial .", "schwartz and colleagues have shown that there is a wide variety of dfdba products on the market which have different inductive capabilities.21 these differences may be related to the origin and methods of preparation of dfdba and if the preparation methods were the same in different bone banks , this would be due to individual donors ages and sexs , disease and injury , medical treatment or genetic differences .", "also , the variations of time between death and the bone extraction , may result in significant loss of the bone inductive ability .", "there are many differences in size and the surface shape of dfdba particles that may affect their inductive ability .", "bone cells distinguish different surface shapes and roughness and this will lead to differences in phenotypic diversity.22 in de vicente and colleagues study , the implants which the bone defects around them were filled with dfdba showed similar bic to the implants which their defects had just covered with collagen membranes.23 therefore , adding dfdba did not have any advantages over membrane on its own , which was in line with other studies.15161920242526 in stentz and colleague 's study,26 using dfdba together with membrane in bone defects around implants improved the healing of the bone density . since they had used radiographic method , the obtained information could not determine the value of bic , which is a better evaluating index for outcome of implant treatment and for these reason , they could not show the dfdba efficiency to increase bic .", "becker and colleagues27 did not find the use of dfdba beneficial for periodontal regeneration and bone regeneration around implants , while abolfazli and colleagues found the use of dfdba ( cenobone ) beneficial to repair periodontal lesions in two or three walls alveolar bone defects and reported its effect on bone - formation being the same as aotogenous bone graft . in some studies", "dfdba was enriched with rhbmp-2 and growth factors and produced better results.2230 the current study results indicated that the relationship between indices of bic and isq was positive and significant . these findings are compatible with huang and colleagues study31 as well as nkenke and colleagues study32 which also found the significant and positive relationship between bic and isq amounts in implants placed in human cadaver bone .", "results of this study are coincident with the findings of other researchers in efficiency of using the resonance frequency analysis method to determine the implants stability3133 and showed the resonance frequency analysis is a suitable and reliable method to determine implant stability .", "adding dfdba ( dembone and cenobone ) , either the american or iranian type , to membrane on its own did not significantly increase the obtained bic and isq with the gbr procedure .", "the resonance frequency analysis is still a suitable and reliable method to determine implant stability ."], "abstract_text": ["<S> background : decalcified freeze - dried bone allograft ( dfdba ) may have the potential to enhance bone formation around dental implants . </S>", "<S> our aim in this study was the evaluation and comparison of two types of dfdba in treatment of dehiscence defects around euroteknika implants in dogs.methods:in this prospective clinical trial animal study , all mandibular premolars of three iranian dogs were extracted . </S>", "<S> after 3 months of healing , fifteen sla type euroteknika dental implants ( natea ) with 4.1 mm diameter and 10 mm length were placed in osteotomy sites with dehiscence defects of 5 mm length , 4 mm width , and 3 mm depth . guided bone regeneration ( gbr ) procedures were performed using cenobone and collagen membrane for six implants , the other six implants received dembone and collagen membrane and the final three implants received only collagen membrane . </S>", "<S> all implants were submerged . </S>", "<S> after 4 months of healing , implants were uncovered and stability ( implant stability quotient ) of all implants was measured . </S>", "<S> then , block biopsies of each implant site were taken and processed for ground sectioning and histomorphometric analysis . </S>", "<S> the data was analyzed by anova and pearson tests . </S>", "<S> p value less than 0.05 was considered to be significant.results:all implants osseointegrated after 4 months . </S>", "<S> the mean values of bone to implant contact for histomorphometric measurements of cenobone , denobone , and control groups were 77.36  9.96% , 78.91  11.9% and 71.56  5.61% respectively , with no significant differences among the various treatment groups . </S>", "<S> the correlation of implant stability quotient and histomorphometric techniques was 0.692.conclusion:in treating of dehiscence defects with gbr technique in this study , adding dfdba did not significantly enhance the percentages of bone - to - implant contact measurements ; and implant stability quotient resonance frequency analysis appeared to be a precise technique . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion"], "sections": [["today , implant therapy is regarded as an extremely reliable approach to replace missing teeth .", "the introduction of osseointegrated implants in dentistry represented a turning point in dental clinical practice.1 as a general principle in implant surgery implant surfaces should be surrounded by alveolar bone.2 following loss of teeth , natural process of bone resorption occurs.3 sometimes due to prosthetic or anatomical limitations of alveolar ridge , it is not possible to insert implants in bone appropriately.2 and also , while preparing implant sites in narrow ridges , dehiscence or fenestration defects may occur frequently that threaten the survival of implants.4 successful implant treatment depends on sufficient bone volume at the implant surface.5 insufficient amount of supporting bone , will limit the effectiveness of osseointegrated dental implants3 and will have an adverse effect on dental implant prognosis.6 several clinical studies have shown that to ensure long term success of implants , at least one millimetre thickness of bone in buccal surface and lingual surface of implant is essential .", "if the implant surface is not completely covered with bone , it will result in gingival recession , unpleasant appearance , problems in keeping good oral hygiene and ultimately increases the risk of infection around the implant.7 implants can be placed in alveolar ridges with defects including dehiscence , intraalveolar defects , and fenestration.8 several methods are presented to reconstruct the destroyed alveolar bone which include osteoconduction , osteoinduction and guided bone regeneration ( gbr).6 many surgical techniques are introduced to enhance alveolar bone volume for placing implants that include various grafting techniques , bone - building using emineral method , and expanding the bone and guided tissue regeneration ( gtr).5 among these techniques , gbr is widely used .", "gbr procedures performed in dental implants enable clinicians to increase the width and height of defected alveolar ridges or to treat fenestrations and dehiscence around implants .", "many researchers have reported predictable results in the simultaneous use of gbr technique when placing implants.7 in most cases of gbr , membranes are supported by protective materials consisting allografts , synthetic materials and xenografts.6 using autograft in augmentation has always been considered as a gold standard , but limited access to autogenous sources , particularly in the areas inside the mouth , prolongation of surgical procedures and bacterial contamination complication have always been considered as the limiting factors in autogenous transplantation . in addition , general surgical risks such as infection , bleeding , pain and swelling , damage to inferior alveolar nerve and the adjacent teeth should also be noted .", "therefore , using biomaterials on its own or in combination with autogenous bone is very common.9 dfdba can be named as one of the allograft materials which has osteoinductive potential , because this substance contains some major bone morphogenetic proteins ( bmps ) of donor tissue matrix . against this view", ", many recent reports have shown that augmentation with dfdba is not os - teoinductive , because it does not contain the necessary bmps to induce bone formation .", "in the united states , emineralised bone matrix ( dbm ) is considered a transplantable tissue and therefore , is regulated primarily by the american association of tissue banks.10 treatment of dehiscence and fenestration lesions with gbr technique and placing of implants simultaneously have predictable results.11 recent clinical studies have proven that the use of bone substitute materials together with placing implants led to successful coverage of pre - exposed implant surfaces.12"], ["this prospective clinical trial animal study was conducted in professor torabinejad dental research centre isfahan university of medical sciences , isfahan , iran .", "samples dissection was carried out in the department of materials science and engineering , sharif university of technology , tehran , iran . studied population included three healthy adult iranian dogs that were chosen by convenient method .", "inclusion criteria included healthy 2 to 4 years old dogs that held all the teeth . at the beginning of the study ,", "after examination to confirm their health and having the required criteria ; seven step vaccination was carried out and the dogs were quarantined for 15 days .", "all the mandibular premolars teeth were extracted and then , the area was left to heal for three months . after healing period , the area assessed by periapical radiography in order to ensure full recovery , after preparing two sits on one side of each mandible and three sites on the other side which were more appropriate in terms of size and bone quality to place the implants , dehiscence bone defects , were prepared with dimensions of 5 mm apicocronaly , 4 mm mesiodistaly and 3 mm buccolingual in buccal plate using high - speed handpiece and diamond fissure bur and abundant irrigation .", "10 mm periodontal probes were used to measure dimensions of the artificial dehiscence defects ; all measurements were performed by a specialist investigator .", "euroteknika implants with 10 mm in length , 4.1 mm in diameter and sla surface ( natea system ) inserted into the prepared sites .", "implant stability quotient ( isq ) of each implant was recorded using ostell device ( ostell integration diagnostics , svedalen , sweden ) which was higher than 56 % in all cases , indicating good initial stability of all implants placed .", "after that in two lesions , that was chosen in random , dfdba cenobone , hamanand saz baft , iran and in two other lesions dfdba dembone , pacific coast tissue bank , u.s were placed and the fifth lesion was left empty ; finally , they all were covered by collagen membranes ( bicon , usa ) .", "both dfdba brands were powder in sahape in 0.5 and 1 grams vials the size of the particles were almost similar and of the existing type to treat small lesions .", "dfdba particles were placed in the lesions and were gently pressed against the implant ; the lesion were with particles filled up in a way that all the screws were covered and the primary bone contour was maintained .", "then , the grafted material and surrounded bone were covered completely by collagen membrane . moreover ,", "the gingival flaps were replaced without stretching to submerge the implant and were stitched by polyglycolic acid sutures ( vicryl ) using simple and interrupted loop sutures .", "then , final status of implants were determined by periapical radiography . during the first week after surgery", ", animals were examined several times a day to control the health and complications after the operation and also to ensure the sutures are not opened .", "the animals mouth was washed twice a day using 0.2 chlorhexidine mouthwashes during the first week and thereafter , once a week . after four months of osseointegration period , dogs were anesthetized and periapical radiographs were taken to observe implants situation within the bone . then , crestal incision was done and the implants were exposed and the value of isq was recorded .", "samples were taken and immediately transferred to formaldehyde 10% and kept for 24 hours to be prepared for optical microscope study .", "samples were sectioned by with the ground section method using microtome ( accutom-50 cutting machine , copenhagen , denmark ) .", "sectioned samples were obtained buccolingualy and parallel to the axis of implants in 100 micron thickness .", "sectioned samples prepared with trichro massons staining studied by optical microscope ( olympus , japon ) . using histomorphometric method , the percentage of bone implant contact ( bic ) was cal - culated and type of bone ( lamellar , woven ) was determined at 40 times magnification and recorded by an experienced pathologist using optical microscope . in this method , the area of 2 mm around the implants is evaluated .", "spss 11.5 software was used to analyse data , using one - way anova and pearson correlation and regression tests ."], ["average bic in cenobone group ( iranian dfdba ) , dembone group ( american dfdba ) and control group were 77.36%  9.96 ( max 89.1% and min 63.4% ) , 78.91%  11.99 ( max 91% and min 56.3% ) and 71.56%  5.61 ( max 75.8% and min 65.2% ) , respectively .", "therewas no significant difference in bic amounts among the three groups ( p = 0.607 ) .", "there was not significant difference in the bic amounts among the three groups defects ( p=0.388 ) .", "comparison of average bic of lamellar bone , woven bone , cenobone and dembone groups defects and control group is presented in figure 1 .", "comparison of average bic of lamellar bone , woven bone , cenobone and dembone groups defects and control group ( lamellar bone p = 0.298 and woven bone p = 0.380 ) . furthermore , there was not meaningful difference in rate of lamellar bone ( p=0.298 ) and woven bone ( p = 0.380 ) formation in defects among the three groups .", "the total sample average isq was 70.83%  6.30 ( max 82.5% and min 61.5% ) .", "average isq in cenobone group , dembone group and control group were 70.29%  7.74 ( max 82.25% and min 61.5% ) , 72.25%  6.81 ( max 82.5% and min 65.25% ) and 69.08%  2.67 ( max 72% and min 66.75% ) , respectively .", "there was no significant difference in isq amounts among the three groups ( p = 0.781 ) .", "there was a positive relationship between total sample bic and isq ( p = 0.004 , r = 0.692 ) .", "histological view under light microscope showes contact of lamellar new bone with implant in cenobone group magnification x40  massons 's trichrom staning .", "histologic view under light microscope showes contact of lamellar new bone with implant in dembone group ( magnification x40  massons 's trichromstaning ) ."], ["results of the current study indicated that adding dfdba ( dembone and cenobone ) to membrane , on its own did not significantly increase the obtained bic and isq amounts .", "caplanis and colleagues placed , implants in the alveolar defects which treated with membrane on its own and membrane plus dfdba in dogs , the average bic was about 70%13 which is consistent to our study . and also , the bic obtained in this study is consistent with the findings of von arx and colleagues study14 that placed implants in areas grafted with dfdba and other hybrid materials in dogs and gained a high percentage of bic ( 59% to 75% ) . in this study , adding dfdba to the collagen membrane did not lead to strengthen induction of bone formation , and these findings were consistent with the results of other studies that showed adding dfdba to the membrane on its own did not significantly increase the clinical results obtained with the gbr procedure.151920 there were significant differences between the products of bone banks in terms of induction of bone formation .", "some studies found the use of dfdba to have a positive impact to increase bone growth while others considered it not to be beneficial .", "schwartz and colleagues have shown that there is a wide variety of dfdba products on the market which have different inductive capabilities.21 these differences may be related to the origin and methods of preparation of dfdba and if the preparation methods were the same in different bone banks , this would be due to individual donors ages and sexs , disease and injury , medical treatment or genetic differences .", "also , the variations of time between death and the bone extraction , may result in significant loss of the bone inductive ability .", "there are many differences in size and the surface shape of dfdba particles that may affect their inductive ability .", "bone cells distinguish different surface shapes and roughness and this will lead to differences in phenotypic diversity.22 in de vicente and colleagues study , the implants which the bone defects around them were filled with dfdba showed similar bic to the implants which their defects had just covered with collagen membranes.23 therefore , adding dfdba did not have any advantages over membrane on its own , which was in line with other studies.15161920242526 in stentz and colleague 's study,26 using dfdba together with membrane in bone defects around implants improved the healing of the bone density . since they had used radiographic method , the obtained information could not determine the value of bic , which is a better evaluating index for outcome of implant treatment and for these reason , they could not show the dfdba efficiency to increase bic .", "becker and colleagues27 did not find the use of dfdba beneficial for periodontal regeneration and bone regeneration around implants , while abolfazli and colleagues found the use of dfdba ( cenobone ) beneficial to repair periodontal lesions in two or three walls alveolar bone defects and reported its effect on bone - formation being the same as aotogenous bone graft . in some studies", "dfdba was enriched with rhbmp-2 and growth factors and produced better results.2230 the current study results indicated that the relationship between indices of bic and isq was positive and significant . these findings are compatible with huang and colleagues study31 as well as nkenke and colleagues study32 which also found the significant and positive relationship between bic and isq amounts in implants placed in human cadaver bone .", "results of this study are coincident with the findings of other researchers in efficiency of using the resonance frequency analysis method to determine the implants stability3133 and showed the resonance frequency analysis is a suitable and reliable method to determine implant stability ."], ["adding dfdba ( dembone and cenobone ) , either the american or iranian type , to membrane on its own did not significantly increase the obtained bic and isq with the gbr procedure .", "the resonance frequency analysis is still a suitable and reliable method to determine implant stability ."]]}
{"article_id": "PMC4262749", "article_text": ["the goal to achieve ideal pediatric drug therapy is a worldwide challenge for regulatory bodies and clinicians .", "children are treated with medicines not tested for safety and efficacy and are frequently supported by the low quality of evidence . in the absence of standard prescribing information , clinicians prescribe drugs in an off - label manner .", "off - label use means use of medicine which is outside the terms of product license with respect to dose , route of administration , indication , or age .", "off - label medicine use among children represents an important health issue as the effects and health risks may be unexpected .", "various national and international studies have been published about the amount of and problems associated with off - label medicines in children .", "the magnitude of off - label prescribing is accounted to be between 18% and 60% in infants but it may be up to 90% in neonates [ 39 ] .", "most of these studies have been conducted outside india and may not be applicable as hospitalization and prescribing patterns differ based on culture , healthcare infrastructure , and health policies .", "only one previous study has been conducted in india which reported 50.62% of off - label prescribing based on british national formulary .", "currently there is no study conducted in india based on national formulary of india ( nfi ) .", "hence , the objective of the research study was to quantify off - label use based on nfi , various predictors and discusses some strategies to monitor it .", "the prospective observational study was carried out at a tertiary care hospital in ahmedabad ( india ) for the period of six months .", "all the pediatric patients in ages between 0 and 12 years receiving at least one medication and admitted in pediatric ward were included in the study .", "the pediatric ward was of 60 bed size and attended by 10 academic pediatricians and 28 postgraduate students .", "patients were not considered if they were in age of more than 12 years , not taking any prescription medication and undergoing surgery .", "demographics , clinical characteristics , and medication usage were obtained from the medical records using predefined paper case record forms .", "the data collection was carried out by one of the researchers ( mms ) who discussed each of the medicines with attending pediatrician and clinical pharmacologist ( dr ) .", "drugs were entered into the database using the world health organization anatomical therapeutic chemical ( who - atc ) classification system .", "we utilized national formulary of india ( nfi , 4th edition , 2011 ) which is an official publication of indian pharmacopoeia commission , ministry of health and family welfare , government of india .", "categories for off - label use were allocated for each medicine according to the reason(s ) why their use was deemed off - label when compared to the terms of the product labeling for that medicine in nfi .", "we followed a published algorithm and used these dimensions to determine whether there is strong scientific evidence for frequently prescribed off - label medicines   ( table 4 ) .", "variables such as age , number of medications , number of diagnoses , and length of hospital stay were regarded as continuous and expressed as mean with standard deviation ( sd ) .", "the odds ratio ( or ) with 95% confidence interval ( ci ) was used to determine the predictors for off - label prescribing .", "the data were analyzed using statistical package for the social sciences ( spss inc . ,", "this study was reported according to the strengthening the reporting of observational studies in epidemiology ( strobe ) guidelines .", "the study included a total of 320 patients admitted in pediatric general ward of the public teaching hospital over a period of six months .", "the mean age was 2.73 years ( range = 0.1 to 12 years and standard deviation : 3.09 ) .", "the most common age group was 0 to 1 years representing 43% of total sample size . on average", "each patient had 1.2 number of diagnosis and 48 patients ( mean = 2.5 ) had more than one diagnosis during hospitalization . the majority of patients suffered from respiratory diseases ( 33% ) , central nervous system diseases ( 16.5% ) , and gastrointestinal disease ( 11% ) .", "a total of 1645 medications were prescribed to the study cohort during hospitalization , with a total hospital stay of 1743 days .", "this constituted the mean number of five ( sd = 2 ) medications prescribed per patient and 5.48 ( sd = 3.62 ) days of hospital stay .", "the number of medications and hospital stay ranged from one to 13 and one to 33 days , respectively .", "the majority of patients received antibiotics , cough , and cold preparations , antipyretics , inhaled corticosteroids , bronchodilators , and antiepileptic drugs .", "medications were mostly administered by oral route ( 40% ) and intravenous route ( 35% ) followed by inhalation route ( 25% ) .", "of the 320 patients included in the study , 310 ( 97% ) patients received at least one off - label medication .", "a total of 1645 medications were administered during the hospitalization ; 1152 ( 70% ) medicines were prescribed in off - label manner when its usage was validated with national formulary of india , 2011 , for five different off - label categories .", "patients received on average ( sd ) 3.66 ( 2.13 ) off - label medicines within a range of one to 10 .", "the most common cause of off - label prescribing 893 ( 63% ) was due to higher dose use ( category 1 ) than stated for particular pediatric patients .", "off - label medicines use for age ( category 2 ) and indication ( category 3 ) was found to be 282 ( 19.8% ) and 145 ( 10.2% ) , respectively .", "those medicines which had no pediatric information ( category 5 ) were 76 ( 5.3% ) .", "use of ipratropium , salbutamol , and dextromethorphan was common for different dose and age limits .", "lorazepam ( seizures ) and ondansetron ( nausea and vomiting ) were frequently used for off - label indication .", "the study also attempted to distribute the receipt of off - label medicines in different age groups as shown in table 2 .", "the extent off - label prescribing was highest ( 76% ) in age group of more than 1 to 2 years , followed by age group of more than 1 to 12 months which accounted for 69% of off - label use .", "off - label use in dose ( category 1 ) was consistent among the patients in age of 0 to 6 years . the use of medicines in patients for restricted age limits ( category 2 ) was highest in 112-month age group .", "indication ( category 3 ) and absence of pediatric information ( category 5 ) were most prominent in 612-year patients", ". highest proportion of off - label medicines were prescribed in anti - infectives for systemic use 389 ( 73% ) and respiratory system 378 ( 82% ) as shown in table 3 .", "the amount of off - label prescribing in nervous system and alimentary tract and metabolism system was 220 ( 53% ) and 85 ( 86% ) , respectively .", "majority of off - label medicines prescribed were ceftriaxone , amikacin , amoxicillin , and vancomycin in antibiotics class and inhaled corticosteroids , ipratropium , salbutamol , chlorpheniramine , phenylephrine corresponding to respiratory system .", "blood and blood forming agents 16 ( 89% ) , hormonal preparation 32 ( 80% ) , and cardiovascular medicines 24 ( 71% ) had substantial high amount of off - label use .", "adrenaline , nifedipine , prednisolone , and dexamethasone were also used in off - label manner .", "antiparasitic products 8 ( 16% ) had minimum off - label use . antibiotics ( 75% ) and inhaled corticosteroids ( 79% ) were mostly prescribed in higher doses ( category 1 ) than recommended . in respiratory system , many medicines ( 39.5% ) were prescribed below age limits ( category 2 ) as indicated in the formulary .", "medicines used for unapproved indication ( category 3 ) largely confined to alimentary tract and metabolism system and blood and blood forming agents .", "medicines which had no pediatric information were predominantly in antihypertensive drug class . on multivariate regression analysis as shown in table 5 , we found that pediatric patients in age of 0 to 2 years ( or 1.68 , 95% ci , 1.262.24 , ) were more likely to receive off - label medicines than any other age group .", "female patients ( or 1.41 , 95% ci , 1.131.77 ) received substantially high amount of off - label medicines compared to male .", "hospital stay of six to 10 days ( or 1.91 , 95% ci , 1.332.75 ) also carried higher risk of off - label prescription .", "using the data of the pediatric ward of a tertiary care hospital , we found that 70% of medicines were prescribed in off - label manner .", "the magnitude off - label prescribing is substantial higher than reported in the recent studies [ 1416 ] .", "cuzzolin et al . performed a literature review of published studies on off - label and unlicensed drug use in children .", "seven studies involved neonatal intensive care units ( nicus ) , fifteen studies included pediatric wards , and twelve studies were conducted in community setting .", "cuzzolin et al . found off - label prescribing in pediatric ward ranging between 18 and 60% .", "various reasons for different rates of off - label use are off - label classification methods , sample sizes , pediatrician 's prescribing habits , in - house treatment protocols , and diseases characteristics most importantly pediatric drug regulation .", "patients in ages between 0 and 2 years are most likely to receive off - label medicines and had highest representation in study sample .", "previous studies also established that age group of 0 to 2 years is the highest recipient of off - label prescriptions [ 16 , 17 ] .", "this is mainly due to absence of specific dosing guidelines and route administration in 0-to-2-year age group in nfi . of the medicines prescribed during the period", ", it was observed that antibiotics , respiratory medicines , and nervous system medicines were frequently prescribed in off - label manner .", "several studies had shown high rate of off - label prescribing in respiratory , antibiotics , analgesics , and antiepileptics .", "about 82% of medicines in respiratory system were off - label which is more than what is reported in studies in us ( n = 312 million , 70% )   and portugal ( n = 500 , 77% ) .", "if asthma therapies are considered , inhaled corticosteroids are frequently prescribed ( 30.7% of all prescriptions ) .", "the mainstay therapy for asthma is inhaled corticosteroids ( ics ) , but guidelines often do not give specific recommendations for upper doses limits specially in children .", "various combinations of antihistaminics , decongestants , and/or analgesics were prescribed to patients in off - label doses for common cold .", "still , the off - label use of paracetamol is substantial , mainly due to off - label classification for dose or age .", "although paracetamol is normally considered safe in pediatrics care , a previous cochrane review pointed out that there is limited evidence regarding the efficacy and safety for paracetamol in the treatment of fever in children .", "lorazepam and ondansetron were widely prescribed for off - label indication but are supported by well conducted clinical studies [ 27 , 28 ] .", "better medicines for children  to improve monitoring medicine safety in the pediatrics and highlighted its concern on off - label medicines .", "the strict drug approval procedure is the way to ensure quality data on quality , safety , and efficacy for different pediatric age groups . despite many regulatory amendments and policies , we still have apathetic outlook to pediatric clinical trials .", "the pharmaceutical companies should be convinced to have appropriate pediatric information in the label and they are not being permitted to market drugs likely to be used in children without suitable pediatric labeling .", "indian drug regulatory authorities should also develop pediatric specific drug development regulation so that the tendency to market medicines without pediatric specific data is discouraged .", "this might ensure pediatric labeling for new medicines yet to be introduced in the market ; but it is unlikely that drug companies will carry out trials to confirm pediatric use for drugs already marketed and used in children , although in an off - label manner .", "alternatively , the situation can be improved when prescribers report their pediatric experiences with different off - label medicines in form research articles or discussion at scientific platforms . when medicines are used as off - label ,", "each patient is unique and risk - benefit pertaining to him should be assessed by high quality evidence .", "the doctor needs to be updated with latest evidence which could be accomplished by using several useful drug compendia like drugdex , clinical pharmacology , and so forth .", "only such focused and coordinated actions would make sure that children 's right to safe , cost - effective , and quality medicines would be realized .", "based on national formulary of india , our data suggest that magnitude of off - label prescribing in pediatric inpatients is considerable higher than reported in some of the countries .", "dose discrepancy and use in restricted age limits were identified as main contributor to off - label prescribing .", "there is need for strict drug regulation for pediatric population to ensure safety and effectiveness of pharmacotherapy .", "further studies are needed to examine why there are inadequate dosing guidelines and generation of more clinical data especially in respiratory medicines .", "understanding various risk factors and spectrum of off - label medicine use can assist developing prevention strategies ."], "abstract_text": ["<S> \n background . in the absence of standard pediatric prescribing information </S>", "<S> , clinicians often use medicines in an off - label way . </S>", "<S> many studies have been published across the globe reporting different rates of off - label use . </S>", "<S> there is currently no study based on indian drug formulary </S>", "<S> . methods . </S>", "<S> the prospective observational study included pediatric patients in ages between 0 and 12 years admitted in a tertiary care hospital . </S>", "<S> off - label use was assessed using the national formulary of india ( nfi ) . </S>", "<S> predictors of off - label use were determined by logistic regression . results . of the 1645 medications prescribed , 1152 ( 70% ) were off - label based on 14 possible off - label categories . </S>", "<S> off - label medicines were mainly due to dose difference and use in restricted age limits as indicated in nfi . </S>", "<S> respiratory medicines ( 82% ) , anti - infectives ( 73% ) , and nervous system medicines ( 53% ) had higher off - label use . </S>", "<S> important predictors of off - label prescribing were pediatric patients in age of 0 to 2 years ( or 1.68 , 95% ci ; p < 0.001 ) and hospital stay of six to 10 days ( or 1.91 , 95% ci ; p < 0.001 ) . conclusion . </S>", "<S> off - label prescribing is common among pediatric patients . </S>", "<S> there is need to generate more quality data on the safety and efficacy of off - label medicines to rationalize pediatric pharmacotherapy . </S>"], "labels": null, "section_names": ["1. Introduction ", "2. Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["the goal to achieve ideal pediatric drug therapy is a worldwide challenge for regulatory bodies and clinicians .", "children are treated with medicines not tested for safety and efficacy and are frequently supported by the low quality of evidence . in the absence of standard prescribing information , clinicians prescribe drugs in an off - label manner .", "off - label use means use of medicine which is outside the terms of product license with respect to dose , route of administration , indication , or age .", "off - label medicine use among children represents an important health issue as the effects and health risks may be unexpected .", "various national and international studies have been published about the amount of and problems associated with off - label medicines in children .", "the magnitude of off - label prescribing is accounted to be between 18% and 60% in infants but it may be up to 90% in neonates [ 39 ] .", "most of these studies have been conducted outside india and may not be applicable as hospitalization and prescribing patterns differ based on culture , healthcare infrastructure , and health policies .", "only one previous study has been conducted in india which reported 50.62% of off - label prescribing based on british national formulary .", "currently there is no study conducted in india based on national formulary of india ( nfi ) .", "hence , the objective of the research study was to quantify off - label use based on nfi , various predictors and discusses some strategies to monitor it ."], ["the prospective observational study was carried out at a tertiary care hospital in ahmedabad ( india ) for the period of six months .", "all the pediatric patients in ages between 0 and 12 years receiving at least one medication and admitted in pediatric ward were included in the study .", "the pediatric ward was of 60 bed size and attended by 10 academic pediatricians and 28 postgraduate students .", "patients were not considered if they were in age of more than 12 years , not taking any prescription medication and undergoing surgery .", "demographics , clinical characteristics , and medication usage were obtained from the medical records using predefined paper case record forms .", "the data collection was carried out by one of the researchers ( mms ) who discussed each of the medicines with attending pediatrician and clinical pharmacologist ( dr ) .", "drugs were entered into the database using the world health organization anatomical therapeutic chemical ( who - atc ) classification system .", "we utilized national formulary of india ( nfi , 4th edition , 2011 ) which is an official publication of indian pharmacopoeia commission , ministry of health and family welfare , government of india .", "categories for off - label use were allocated for each medicine according to the reason(s ) why their use was deemed off - label when compared to the terms of the product labeling for that medicine in nfi .", "we followed a published algorithm and used these dimensions to determine whether there is strong scientific evidence for frequently prescribed off - label medicines   ( table 4 ) .", "variables such as age , number of medications , number of diagnoses , and length of hospital stay were regarded as continuous and expressed as mean with standard deviation ( sd ) .", "the odds ratio ( or ) with 95% confidence interval ( ci ) was used to determine the predictors for off - label prescribing .", "the data were analyzed using statistical package for the social sciences ( spss inc . ,", "this study was reported according to the strengthening the reporting of observational studies in epidemiology ( strobe ) guidelines ."], ["the study included a total of 320 patients admitted in pediatric general ward of the public teaching hospital over a period of six months .", "the mean age was 2.73 years ( range = 0.1 to 12 years and standard deviation : 3.09 ) .", "the most common age group was 0 to 1 years representing 43% of total sample size . on average", "each patient had 1.2 number of diagnosis and 48 patients ( mean = 2.5 ) had more than one diagnosis during hospitalization . the majority of patients suffered from respiratory diseases ( 33% ) , central nervous system diseases ( 16.5% ) , and gastrointestinal disease ( 11% ) .", "a total of 1645 medications were prescribed to the study cohort during hospitalization , with a total hospital stay of 1743 days .", "this constituted the mean number of five ( sd = 2 ) medications prescribed per patient and 5.48 ( sd = 3.62 ) days of hospital stay .", "the number of medications and hospital stay ranged from one to 13 and one to 33 days , respectively .", "the majority of patients received antibiotics , cough , and cold preparations , antipyretics , inhaled corticosteroids , bronchodilators , and antiepileptic drugs .", "medications were mostly administered by oral route ( 40% ) and intravenous route ( 35% ) followed by inhalation route ( 25% ) .", "of the 320 patients included in the study , 310 ( 97% ) patients received at least one off - label medication .", "a total of 1645 medications were administered during the hospitalization ; 1152 ( 70% ) medicines were prescribed in off - label manner when its usage was validated with national formulary of india , 2011 , for five different off - label categories .", "patients received on average ( sd ) 3.66 ( 2.13 ) off - label medicines within a range of one to 10 .", "the most common cause of off - label prescribing 893 ( 63% ) was due to higher dose use ( category 1 ) than stated for particular pediatric patients .", "off - label medicines use for age ( category 2 ) and indication ( category 3 ) was found to be 282 ( 19.8% ) and 145 ( 10.2% ) , respectively .", "those medicines which had no pediatric information ( category 5 ) were 76 ( 5.3% ) .", "use of ipratropium , salbutamol , and dextromethorphan was common for different dose and age limits .", "lorazepam ( seizures ) and ondansetron ( nausea and vomiting ) were frequently used for off - label indication .", "the study also attempted to distribute the receipt of off - label medicines in different age groups as shown in table 2 .", "the extent off - label prescribing was highest ( 76% ) in age group of more than 1 to 2 years , followed by age group of more than 1 to 12 months which accounted for 69% of off - label use .", "off - label use in dose ( category 1 ) was consistent among the patients in age of 0 to 6 years . the use of medicines in patients for restricted age limits ( category 2 ) was highest in 112-month age group .", "indication ( category 3 ) and absence of pediatric information ( category 5 ) were most prominent in 612-year patients", ". highest proportion of off - label medicines were prescribed in anti - infectives for systemic use 389 ( 73% ) and respiratory system 378 ( 82% ) as shown in table 3 .", "the amount of off - label prescribing in nervous system and alimentary tract and metabolism system was 220 ( 53% ) and 85 ( 86% ) , respectively .", "majority of off - label medicines prescribed were ceftriaxone , amikacin , amoxicillin , and vancomycin in antibiotics class and inhaled corticosteroids , ipratropium , salbutamol , chlorpheniramine , phenylephrine corresponding to respiratory system .", "blood and blood forming agents 16 ( 89% ) , hormonal preparation 32 ( 80% ) , and cardiovascular medicines 24 ( 71% ) had substantial high amount of off - label use .", "adrenaline , nifedipine , prednisolone , and dexamethasone were also used in off - label manner .", "antiparasitic products 8 ( 16% ) had minimum off - label use . antibiotics ( 75% ) and inhaled corticosteroids ( 79% ) were mostly prescribed in higher doses ( category 1 ) than recommended . in respiratory system , many medicines ( 39.5% ) were prescribed below age limits ( category 2 ) as indicated in the formulary .", "medicines used for unapproved indication ( category 3 ) largely confined to alimentary tract and metabolism system and blood and blood forming agents .", "medicines which had no pediatric information were predominantly in antihypertensive drug class . on multivariate regression analysis as shown in table 5 , we found that pediatric patients in age of 0 to 2 years ( or 1.68 , 95% ci , 1.262.24 , ) were more likely to receive off - label medicines than any other age group .", "female patients ( or 1.41 , 95% ci , 1.131.77 ) received substantially high amount of off - label medicines compared to male .", "hospital stay of six to 10 days ( or 1.91 , 95% ci , 1.332.75 ) also carried higher risk of off - label prescription ."], ["using the data of the pediatric ward of a tertiary care hospital , we found that 70% of medicines were prescribed in off - label manner .", "the magnitude off - label prescribing is substantial higher than reported in the recent studies [ 1416 ] .", "cuzzolin et al . performed a literature review of published studies on off - label and unlicensed drug use in children .", "seven studies involved neonatal intensive care units ( nicus ) , fifteen studies included pediatric wards , and twelve studies were conducted in community setting .", "cuzzolin et al . found off - label prescribing in pediatric ward ranging between 18 and 60% .", "various reasons for different rates of off - label use are off - label classification methods , sample sizes , pediatrician 's prescribing habits , in - house treatment protocols , and diseases characteristics most importantly pediatric drug regulation .", "patients in ages between 0 and 2 years are most likely to receive off - label medicines and had highest representation in study sample .", "previous studies also established that age group of 0 to 2 years is the highest recipient of off - label prescriptions [ 16 , 17 ] .", "this is mainly due to absence of specific dosing guidelines and route administration in 0-to-2-year age group in nfi . of the medicines prescribed during the period", ", it was observed that antibiotics , respiratory medicines , and nervous system medicines were frequently prescribed in off - label manner .", "several studies had shown high rate of off - label prescribing in respiratory , antibiotics , analgesics , and antiepileptics .", "about 82% of medicines in respiratory system were off - label which is more than what is reported in studies in us ( n = 312 million , 70% )   and portugal ( n = 500 , 77% ) .", "if asthma therapies are considered , inhaled corticosteroids are frequently prescribed ( 30.7% of all prescriptions ) .", "the mainstay therapy for asthma is inhaled corticosteroids ( ics ) , but guidelines often do not give specific recommendations for upper doses limits specially in children .", "various combinations of antihistaminics , decongestants , and/or analgesics were prescribed to patients in off - label doses for common cold .", "still , the off - label use of paracetamol is substantial , mainly due to off - label classification for dose or age .", "although paracetamol is normally considered safe in pediatrics care , a previous cochrane review pointed out that there is limited evidence regarding the efficacy and safety for paracetamol in the treatment of fever in children .", "lorazepam and ondansetron were widely prescribed for off - label indication but are supported by well conducted clinical studies [ 27 , 28 ] .", "better medicines for children  to improve monitoring medicine safety in the pediatrics and highlighted its concern on off - label medicines .", "the strict drug approval procedure is the way to ensure quality data on quality , safety , and efficacy for different pediatric age groups . despite many regulatory amendments and policies , we still have apathetic outlook to pediatric clinical trials .", "the pharmaceutical companies should be convinced to have appropriate pediatric information in the label and they are not being permitted to market drugs likely to be used in children without suitable pediatric labeling .", "indian drug regulatory authorities should also develop pediatric specific drug development regulation so that the tendency to market medicines without pediatric specific data is discouraged .", "this might ensure pediatric labeling for new medicines yet to be introduced in the market ; but it is unlikely that drug companies will carry out trials to confirm pediatric use for drugs already marketed and used in children , although in an off - label manner .", "alternatively , the situation can be improved when prescribers report their pediatric experiences with different off - label medicines in form research articles or discussion at scientific platforms . when medicines are used as off - label ,", "each patient is unique and risk - benefit pertaining to him should be assessed by high quality evidence .", "the doctor needs to be updated with latest evidence which could be accomplished by using several useful drug compendia like drugdex , clinical pharmacology , and so forth .", "only such focused and coordinated actions would make sure that children 's right to safe , cost - effective , and quality medicines would be realized ."], ["based on national formulary of india , our data suggest that magnitude of off - label prescribing in pediatric inpatients is considerable higher than reported in some of the countries .", "dose discrepancy and use in restricted age limits were identified as main contributor to off - label prescribing .", "there is need for strict drug regulation for pediatric population to ensure safety and effectiveness of pharmacotherapy .", "further studies are needed to examine why there are inadequate dosing guidelines and generation of more clinical data especially in respiratory medicines .", "understanding various risk factors and spectrum of off - label medicine use can assist developing prevention strategies ."]]}
{"article_id": "PMC4171902", "article_text": ["the role of the thyroid gland in pregnancy and the impact of thyroid disorders on the course of pregnancy and development of the offspring have drawn a considerable interest in the recent years , both in the medical and in the general society . about 2 and 4%", "a growing body of evidence suggests that subclinical hypothyroidism may be associated with in vitro fertilization failure , subfertility , infertility , spontaneous abortion , placental abruption , gestational hypertension , preeclampsia , preterm delivery , postpartum thyroid dysfunction , depression ( including postpartum depression ) , conversion to overt hypothyroidism , and impaired cognitive and psychomotor child development .", "american thyroid association in its recently published guidelines have stated against universal screening of pregnant women for hypothyroidism .", "it seems that prevalence of hypothyroidism is more in asian countries compared to the west . in a recent study by dhanwal et al .", "looking at these data the purpose of the study was to know whether routine screening would prove to be beneficial in our country .", "the study was an out - patient department ( opd ) based prospective cohort , observational study in which 305 women were randomly selected and screened on opd basis by thyroid - stimulating hormone ( tsh ) levels ( cutoff level 0.10 - 2.50 miu / ml ) as per recent guidelines .", "these women were followed till term and subsequent delivery to study the effect on maternal and perinatal outcome .", "known cases of other medical disorders and women who did not give consent for tsh estimation were excluded from the study .", "0.10mu / l , the reference values for tsh were 0.1 - 2.5miu / l , 0.2 - 3.0 miu / l and 0.3 - 3.0 miu / l in the first , second , and third trimesters of pregnancy respectively , considering the recent literature . the statistical difference between variables", "in the study following are the observations made : the mean age of women was 24.46 years ( sd = 2.00 ) . the mean gestational age at time of screening 9.09 weeks .", "( sd = 4.09 ) 38.68% of women were of gestational age less than 9 weeks and 61.31% were with gestational age 913 weeks .", "cut off of 9 weeks was taken to identify those who presented early in first trimester , 55.08% women were nullipara , 31.14% were primipara , 11.47% were para 2 , 2.2% women were para 3 and more . it was seen that 4.9% women were already having symptoms of thyroid diseases ( main symptoms seen were weakness , lethargy , loss of appetite , weight gain ) , 4.2% had history of previous thyroid diseases ( which is either hypo or hyperthyroidism ) , 0.65% women had history of other autoimmune diseases ( apla syndrome ) 4.9% women had history of previous caesarean section , 1.6% women had family history of thyroid diseases , 0.3% had history of irradiation , 1.6% had history of medication ( thyroxine for hypothyroidism ) . on this screening", ". however , the incidence of hypothyroidism in high risk population was 20.58% and in normal population was 6.7% which shows a significant association of thyroid disorders with high risk factors ( p < 0.001 ) . in the euthyroid women 7.2% had an adverse perinatal outcome , 92.7% had normal outcomes .", "this shows statistically significant association abnormal tsh values with adverse pregnancy outcomes ( p < 0.001 ) . considering the route of delivery , 88.85% women had normal delivery , out of them 0.36% were hyperthyroid , 5.5% were hypothyroid rest were euthyroid .", "in abnormal perinatal outcomes 6.2% women had lower segment caesarean section ( lscs ) out of them 73.68% were euthyroid and 26.31% were hypothyroid .", "the main indications for lscs in these cases were fetal distress , maternal cephalopelvic disproportion ( cpd ) , contracted pelvis , and failed induction .", "1.9% had preterm labour , out of them 50% were euthyroid , 50% were hypothyroid .", "in all , there was only one preterm fresh still birth in a euthyroid woman .", "out of 2.2% spontaneous abortions 28.5% were in euthyroid group while 71.4% were in hypothyroid group .", "this study shows significant association between abnormal tsh values and adverse perinatal outcomes ( p < 0.001 ) .", "this prospective screening of thyroid function in a cohort of unselected pregnant women shows that high - risk women ( with a personal or family history of thyroid disorders or a personal history of other autoimmune diseases ) have more significant ( p < 0.001 ) increased risk of hypothyroidism ( subclinical or overt ) during early pregnancy .", "however , testing only the high - risk pregnant women , as the consensus guidelines recommend , would miss about one- of women with hypothyroidism .", "therefore with the growing evidence for an association between maternal subclinical hypothyroidism and adverse pregnancy outcomes but lack of intervention trials showing beneficial effect of thyroxine ( t4 ) in preventing these adverse outcomes , the controversy between targeted high - risk case finding and universal screening continues .", "the consensus guidelines recommend the use of t4 in pregnant women with subclinical hypothyroidism , justified on the basis of potential benefit to risk ratio .", "our study shows that , without universal screening , a significant number of such pregnant women with thyroid dysfunction will not be picked up .", "free thyroxine ( ft4 ) increases with suppression of tsh in response to placental human chorionic gonadotrophin during the first trimester , whereas ft4 tends to decrease in late gestation .", "this is likely to be the cause for the high prevalence of suppressed tsh in this cohort .", "furthermore increased serum thyroid - binding globulin and decreased albumin during pregnancy result in assay - dependent variations in ft4 levels .", "these observations have led to the call for using trimester and assay - specific reference ranges for thyroid function tests in pregnancy .", "if the trimester specific reference range is used , 9.8% pregnant women in this cohort will be considered to have hypothyroidism .", "whereas there will be less of a controversy to use the trimester - specific reference range in titrating the dose of t4 in pregnant women on t4 replacement , further studies are needed to determine the threshold level of tsh at which initiation of t4 replacement should be considered .", "clinical studies have confirmed that the increased requirement for t4 ( or exogenous lt4 ) occurs as early as 4 - 6 weeks of pregnancy .", "such requirements gradually increase through 16 - 20 weeks of pregnancy , and thereafter plateau until time of delivery .", "these data provide the basis for recommending adjustments to thyroid hormone in affected women once pregnant and for the timing of follow - up intervals for tsh in treated patients .", "the levothyroxine ( lt4 ) adjustment , when necessary , should be made as soon as possible after pregnancy is confirmed to reduce the probability of hypothyroidism .", "a prospective , randomized study has recently provided evidence in support of one dose adjustment strategy for women receiving lt4 who are newly pregnant . for women who are euthyroid while receiving once - daily dosing of lt4 ( regardless of amount ) , a recommendation to increase by two additional tablets weekly ( nine tablets per week instead of seven tablets per week ; 29% increase ) can effectively prevent maternal hypothyroidism during the first trimester and mimic gestational physiology .", "this augmented dose should occur immediately after a missed menstrual cycle or suspected pregnancy occurs .", "a separate option is to increase the dosage of daily lt4 by approximately 25 - 30% .", "there is also an uncertainty regarding the most appropriate initial screening test for thyroid dysfunction in pregnancy .", "the consensus guidelines recommend using tsh as the initial test , whereas others have stressed the importance of testing ft4 by highlighting the fact that ft4 ( and ft3 ) is responsible for thyroid hormone action and that maternal hypothyroxinemia ( normal tsh but low ft4 ) is associated with neuropsychological deficit in the offspring . in our study ,", "the cause of maternal hypothyroxemia is not fully understood , but iodine deficiency is thought to be a major factor although urinary iodine was not analyzed in the present cohort , a previous study in this same population has shown that 7 and 40% pregnant women have urinary iodine excretion of less than 50 g / l ( suggestive of dietary iodine deficiency ) and 50 - 100", "nearly one - quarter of hypothyroid women on t4 replacement in this study had raised tsh at their first antenatal visit .", "given the fact that the fetus relies entirely on maternal thyroid hormones for its development until about 13 week of gestation , it is critical to ensure adequate t4 replacement in pregnant women during the first trimester .", "hypothyroid pregnant women on t4 require an increased dose from as early as the fifth week of gestation to maintain optimum t4 replacement .", "some recommend a 30% increase in the t4 dose as soon as the pregnancy is confirmed , with further dose adjustments based on tsh measurements .", "in addition , through education of all hypothyroid women in the reproductive age , every attempt should be made to ensure an adequate t4 replacement before a planned pregnancy .", "so screening of thyroid disorders should be done in early pregnancy . it will help to diagnose the cases at the earliest and to carry out timely intervention to prevent adverse perinatal outcomes .", "but still there is a controversy regarding universal thyroid function screening or high - risk screening in early pregnancy .", "but if we screen only high risk population we would miss 4.6% cases which could have been diagnosed and treated earlier .", "so this study emphasizes high risk screening in early pregnancy but also supports that universal screening should be a part of our screening protocols so that all thyroid disorders are screened and treated at the earliest .", "so in our country we must follow indian thyroid society guidelines which clearly recommend that  all pregnant women should be screened at 1 antenatal visit by measuring tsh levels  , and highlight that  ideally screening should be carried out during prepregnancy evaluation or as soon as pregnancy is confirmed . "], "abstract_text": ["<S> objective : to determine the importance of screening for thyriod disorders in the first trimester of pregnancy.materials and methods : the study was conducted on 305 patients which were were randomly selected and screened on opd basis by tsh levels ( cut off level 0.10 - 2.50 miu / ml).results : in the 305 women screened mean age was 24.46 years , mean gestational age was 9.09 weeks , 89.83% were euthyroid , 9.8%were hypothyroid , 0.32% were hyperthyroid . </S>", "<S> incidence of hypothyroidism in high risk population was 20.58% and in normal population was 6.7% . </S>", "<S> there was significant association of thyroid disorders with high risk factors ( p < 0.001 ) . </S>", "<S> in hypothyroid women 46% had adverse perinatal outcomes and 53.33% had normal outcomes . </S>", "<S> this shows statistically significant association abnormal tsh values with adverse pregnancy outcomes ( p < 0.001 ) . in abnormal perinatal outcomes 6.2% women had caesarean section out of them 73.68% were euthyroid , 26.31% were hypothyroid 1.9% had preterm labour , out of them 50% were euthyroid , 50% were hypothyroid . out of 2.2% spontaneous abortions </S>", "<S> 28.5% were in euthyroid group while 71.4% were in hypothyroid group . </S>", "<S> there was 1 term stillbirth in hypothyroid group . </S>", "<S> this study showed significant association between abnormal thyroid stimulating hormone ( tsh ) values and adverse perinatal outcomes ( p < 0.001).conclusion : there is significant correlation between risk factors and hypothyroidism . </S>", "<S> so high risk screening is mandatory in early pregnancy . </S>", "<S> but if we screen only high risk population we would miss 4.6% cases which could have been diagnosed and treated earlier . </S>", "<S> therefore it is important to screen all pregnant women in the first trimester , it should be made mandatory . </S>"], "labels": null, "section_names": ["I", "M", "R", "D", "C"], "sections": [["the role of the thyroid gland in pregnancy and the impact of thyroid disorders on the course of pregnancy and development of the offspring have drawn a considerable interest in the recent years , both in the medical and in the general society . about 2 and 4%", "a growing body of evidence suggests that subclinical hypothyroidism may be associated with in vitro fertilization failure , subfertility , infertility , spontaneous abortion , placental abruption , gestational hypertension , preeclampsia , preterm delivery , postpartum thyroid dysfunction , depression ( including postpartum depression ) , conversion to overt hypothyroidism , and impaired cognitive and psychomotor child development .", "american thyroid association in its recently published guidelines have stated against universal screening of pregnant women for hypothyroidism .", "it seems that prevalence of hypothyroidism is more in asian countries compared to the west . in a recent study by dhanwal et al .", "looking at these data the purpose of the study was to know whether routine screening would prove to be beneficial in our country ."], ["the study was an out - patient department ( opd ) based prospective cohort , observational study in which 305 women were randomly selected and screened on opd basis by thyroid - stimulating hormone ( tsh ) levels ( cutoff level 0.10 - 2.50 miu / ml ) as per recent guidelines .", "these women were followed till term and subsequent delivery to study the effect on maternal and perinatal outcome .", "known cases of other medical disorders and women who did not give consent for tsh estimation were excluded from the study .", "0.10mu / l , the reference values for tsh were 0.1 - 2.5miu / l , 0.2 - 3.0 miu / l and 0.3 - 3.0 miu / l in the first , second , and third trimesters of pregnancy respectively , considering the recent literature . the statistical difference between variables"], ["in the study following are the observations made : the mean age of women was 24.46 years ( sd = 2.00 ) . the mean gestational age at time of screening 9.09 weeks .", "( sd = 4.09 ) 38.68% of women were of gestational age less than 9 weeks and 61.31% were with gestational age 913 weeks .", "cut off of 9 weeks was taken to identify those who presented early in first trimester , 55.08% women were nullipara , 31.14% were primipara , 11.47% were para 2 , 2.2% women were para 3 and more . it was seen that 4.9% women were already having symptoms of thyroid diseases ( main symptoms seen were weakness , lethargy , loss of appetite , weight gain ) , 4.2% had history of previous thyroid diseases ( which is either hypo or hyperthyroidism ) , 0.65% women had history of other autoimmune diseases ( apla syndrome ) 4.9% women had history of previous caesarean section , 1.6% women had family history of thyroid diseases , 0.3% had history of irradiation , 1.6% had history of medication ( thyroxine for hypothyroidism ) . on this screening", ". however , the incidence of hypothyroidism in high risk population was 20.58% and in normal population was 6.7% which shows a significant association of thyroid disorders with high risk factors ( p < 0.001 ) . in the euthyroid women 7.2% had an adverse perinatal outcome , 92.7% had normal outcomes .", "this shows statistically significant association abnormal tsh values with adverse pregnancy outcomes ( p < 0.001 ) . considering the route of delivery , 88.85% women had normal delivery , out of them 0.36% were hyperthyroid , 5.5% were hypothyroid rest were euthyroid .", "in abnormal perinatal outcomes 6.2% women had lower segment caesarean section ( lscs ) out of them 73.68% were euthyroid and 26.31% were hypothyroid .", "the main indications for lscs in these cases were fetal distress , maternal cephalopelvic disproportion ( cpd ) , contracted pelvis , and failed induction .", "1.9% had preterm labour , out of them 50% were euthyroid , 50% were hypothyroid .", "in all , there was only one preterm fresh still birth in a euthyroid woman .", "out of 2.2% spontaneous abortions 28.5% were in euthyroid group while 71.4% were in hypothyroid group .", "this study shows significant association between abnormal tsh values and adverse perinatal outcomes ( p < 0.001 ) ."], ["this prospective screening of thyroid function in a cohort of unselected pregnant women shows that high - risk women ( with a personal or family history of thyroid disorders or a personal history of other autoimmune diseases ) have more significant ( p < 0.001 ) increased risk of hypothyroidism ( subclinical or overt ) during early pregnancy .", "however , testing only the high - risk pregnant women , as the consensus guidelines recommend , would miss about one- of women with hypothyroidism .", "therefore with the growing evidence for an association between maternal subclinical hypothyroidism and adverse pregnancy outcomes but lack of intervention trials showing beneficial effect of thyroxine ( t4 ) in preventing these adverse outcomes , the controversy between targeted high - risk case finding and universal screening continues .", "the consensus guidelines recommend the use of t4 in pregnant women with subclinical hypothyroidism , justified on the basis of potential benefit to risk ratio .", "our study shows that , without universal screening , a significant number of such pregnant women with thyroid dysfunction will not be picked up .", "free thyroxine ( ft4 ) increases with suppression of tsh in response to placental human chorionic gonadotrophin during the first trimester , whereas ft4 tends to decrease in late gestation .", "this is likely to be the cause for the high prevalence of suppressed tsh in this cohort .", "furthermore increased serum thyroid - binding globulin and decreased albumin during pregnancy result in assay - dependent variations in ft4 levels .", "these observations have led to the call for using trimester and assay - specific reference ranges for thyroid function tests in pregnancy .", "if the trimester specific reference range is used , 9.8% pregnant women in this cohort will be considered to have hypothyroidism .", "whereas there will be less of a controversy to use the trimester - specific reference range in titrating the dose of t4 in pregnant women on t4 replacement , further studies are needed to determine the threshold level of tsh at which initiation of t4 replacement should be considered .", "clinical studies have confirmed that the increased requirement for t4 ( or exogenous lt4 ) occurs as early as 4 - 6 weeks of pregnancy .", "such requirements gradually increase through 16 - 20 weeks of pregnancy , and thereafter plateau until time of delivery .", "these data provide the basis for recommending adjustments to thyroid hormone in affected women once pregnant and for the timing of follow - up intervals for tsh in treated patients .", "the levothyroxine ( lt4 ) adjustment , when necessary , should be made as soon as possible after pregnancy is confirmed to reduce the probability of hypothyroidism .", "a prospective , randomized study has recently provided evidence in support of one dose adjustment strategy for women receiving lt4 who are newly pregnant . for women who are euthyroid while receiving once - daily dosing of lt4 ( regardless of amount ) , a recommendation to increase by two additional tablets weekly ( nine tablets per week instead of seven tablets per week ; 29% increase ) can effectively prevent maternal hypothyroidism during the first trimester and mimic gestational physiology .", "this augmented dose should occur immediately after a missed menstrual cycle or suspected pregnancy occurs .", "a separate option is to increase the dosage of daily lt4 by approximately 25 - 30% .", "there is also an uncertainty regarding the most appropriate initial screening test for thyroid dysfunction in pregnancy .", "the consensus guidelines recommend using tsh as the initial test , whereas others have stressed the importance of testing ft4 by highlighting the fact that ft4 ( and ft3 ) is responsible for thyroid hormone action and that maternal hypothyroxinemia ( normal tsh but low ft4 ) is associated with neuropsychological deficit in the offspring . in our study ,", "the cause of maternal hypothyroxemia is not fully understood , but iodine deficiency is thought to be a major factor although urinary iodine was not analyzed in the present cohort , a previous study in this same population has shown that 7 and 40% pregnant women have urinary iodine excretion of less than 50 g / l ( suggestive of dietary iodine deficiency ) and 50 - 100", "nearly one - quarter of hypothyroid women on t4 replacement in this study had raised tsh at their first antenatal visit .", "given the fact that the fetus relies entirely on maternal thyroid hormones for its development until about 13 week of gestation , it is critical to ensure adequate t4 replacement in pregnant women during the first trimester .", "hypothyroid pregnant women on t4 require an increased dose from as early as the fifth week of gestation to maintain optimum t4 replacement .", "some recommend a 30% increase in the t4 dose as soon as the pregnancy is confirmed , with further dose adjustments based on tsh measurements .", "in addition , through education of all hypothyroid women in the reproductive age , every attempt should be made to ensure an adequate t4 replacement before a planned pregnancy ."], ["so screening of thyroid disorders should be done in early pregnancy . it will help to diagnose the cases at the earliest and to carry out timely intervention to prevent adverse perinatal outcomes .", "but still there is a controversy regarding universal thyroid function screening or high - risk screening in early pregnancy .", "but if we screen only high risk population we would miss 4.6% cases which could have been diagnosed and treated earlier .", "so this study emphasizes high risk screening in early pregnancy but also supports that universal screening should be a part of our screening protocols so that all thyroid disorders are screened and treated at the earliest .", "so in our country we must follow indian thyroid society guidelines which clearly recommend that  all pregnant women should be screened at 1 antenatal visit by measuring tsh levels  , and highlight that  ideally screening should be carried out during prepregnancy evaluation or as soon as pregnancy is confirmed . "]]}
{"article_id": "PMC3635227", "article_text": ["esophageal cancer is a devastating disease with rapidly increasing incidence in western . despite important advances in therapy ,", "> 50% of patients have incurable disease at diagnosis and only 510% of these patients have life expectancy > 5 years .", "dysphagia is the most common symptom that afflicts these patients , causing 70% of complaints , and leading to severe malnutrition and reduced quality of life [ 1 , 2 ] .", "this factor prevents curative treatment in the majority of them , thus making palliative care a more realistic option [ 1 , 3 ] .", "although surgical resection remains the treatment of choice for early cancers , palliative esophagectomy is not recommended due to high mortality rates .", "however , surgical procedures in order to bypass the dietary route with relief of dysphagia have been reported in the literature but have fallen into disuse because of the morbidity and mortality of the procedure itself and advances in endoscopic treatment .", "this paper demonstrates the use of surgical treatment using the laparoscopic approach in the palliative care of a patient with advanced esophageal cancer .", "a 69-year - old male presented with progressive dysphagia , retrosternal pain and weight loss of 10 kg in 2 months .", "the finding was a circumferential lesion from 19 to 25 cm of the incisors , with biopsy diagnosing squamous cell carcinoma .", "radiological examinations showed lymphadenopathy in the celiac trunk and in the mediastinal area . a bronchoscopy diagnosed tracheal infiltration by neoplasia .", "the patient was treated with external radiotherapy ( 13  250 cgy ) with symptom relief and regained performance status ( from 3 to 1 ) . however , after re - staging exams , the persistence of the disease was diagnosed , with dysphagia worsening .", "the technique , as described by lacerda et al . in a previous report , involves the creation of a gastric tube close to 3 cm in width and with an average length of 30 cm ( fig . ", "1 ) and preservation of the right gastroepiploic vessels by laparoscopy . \n figure  1:gastric tube .", "the tube is then passed along a retrosternal path and end - to - side anastomosis using a circular stapler is performed with the cervical esophagus prepared earlier by left cervicotomy .", "a laminar drain is applied in the cervical incision ( fig .  2 ) . \n", "the patient was discharged from the hospital on the fifth post - operative day with oral intake . no leakage or other complication", "the patient survived for 4 months , gained 10 kg in this period and did not refer any dysphagia after the procedure .", "currently , there are several methods for this purpose , including the esophageal stent , endoscopic dilation , radiotherapy ( both external and endoluminal  brachytherapy ) , chemotherapy and laser recanalization ( yag ) .", "the choice of procedure and the response to treatment should be properly related to life expectancy , which has also been related to performance status , weight loss , tumor extension , clinical stage , sex and age .", "dysphagia from inoperable esophageal cancer is a common and complex management problem , and there is no consensus on the ideal treatment approach .", "all methods have their own advantages and disadvantages in rapid or late onset effect in symptom relief as in recurrence .", "no single intervention palliates dysphagia at all time for every patient ; therefore , healthcare providers must assess the risks and benefits of each palliative intervention for individual patients .", "surgical resection is still cited in the literature as a way to palliate esophageal cancer but is restricted to patients with severe risk of complications ( e.g. perforation or incontrollable tumor bleeding ) due to high morbidity and mortality in relation to non - surgical palliative options .", "that there are no randomized controlled studies to compare the addition of surgery to palliative chemotherapy with chemotherapy alone .", "they also state that such studies may become possible and worthwhile if minimal access surgery can be achieved that has reduced complications and better recovery of health - related quality of life than standard open surgery . in 1979 , postlethwait   proposed a technique for esophageal bypass surgery with an isoperistaltic gastric tube , and this method is still used despite its morbidity .", "this report demonstrates that an update in the technique proposed by postlethwait for palliation of esophageal cancer with the use of minimal access surgery is feasible .", "according to the current literature , the bypass procedure in order to palliate dysphagia could be reserved for patients in which the stent has failed or contraindicated .", "high costs can sometimes make the lesser invasive procedure more prohibitive to the patient than surgery itself ."], "abstract_text": ["<S> esophageal cancer is a devastating disease with rapidly increasing incidence in western countries . </S>", "<S> dysphagia is the most common complication , causing severe malnutrition and reduced quality of life . </S>", "<S> a 69-year - old male with persistent esophageal cancer after radiation therapy was subjected to palliative by - pass surgery using a laparoscopic approach . </S>", "<S> due to the advanced stage at diagnosis , palliative treatment was a more realistic option . </S>", "<S> dysphagia is a most distressing symptom of this disease , causing malnutrition and reducing quality of life . </S>", "<S> the goal of palliation is to improve swallowing . </S>", "<S> the most common methods applied are endoscopic stenting , radiation therapy ( external or brachytherapy ) , chemotherapy , yttrium - aluminum - garnet laser rechanneling or endoscopic dilatation . </S>", "<S> palliative surgery is rarely proposed due to morbidity and complications . </S>", "<S> this paper demonstrates an update in the technique proposed by postlethwait in 1979 for palliation of esophageal cancer . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["esophageal cancer is a devastating disease with rapidly increasing incidence in western . despite important advances in therapy ,", "> 50% of patients have incurable disease at diagnosis and only 510% of these patients have life expectancy > 5 years .", "dysphagia is the most common symptom that afflicts these patients , causing 70% of complaints , and leading to severe malnutrition and reduced quality of life [ 1 , 2 ] .", "this factor prevents curative treatment in the majority of them , thus making palliative care a more realistic option [ 1 , 3 ] .", "although surgical resection remains the treatment of choice for early cancers , palliative esophagectomy is not recommended due to high mortality rates .", "however , surgical procedures in order to bypass the dietary route with relief of dysphagia have been reported in the literature but have fallen into disuse because of the morbidity and mortality of the procedure itself and advances in endoscopic treatment .", "this paper demonstrates the use of surgical treatment using the laparoscopic approach in the palliative care of a patient with advanced esophageal cancer ."], ["a 69-year - old male presented with progressive dysphagia , retrosternal pain and weight loss of 10 kg in 2 months .", "the finding was a circumferential lesion from 19 to 25 cm of the incisors , with biopsy diagnosing squamous cell carcinoma .", "radiological examinations showed lymphadenopathy in the celiac trunk and in the mediastinal area . a bronchoscopy diagnosed tracheal infiltration by neoplasia .", "the patient was treated with external radiotherapy ( 13  250 cgy ) with symptom relief and regained performance status ( from 3 to 1 ) . however , after re - staging exams , the persistence of the disease was diagnosed , with dysphagia worsening .", "the technique , as described by lacerda et al . in a previous report , involves the creation of a gastric tube close to 3 cm in width and with an average length of 30 cm ( fig . ", "1 ) and preservation of the right gastroepiploic vessels by laparoscopy . \n figure  1:gastric tube .", "the tube is then passed along a retrosternal path and end - to - side anastomosis using a circular stapler is performed with the cervical esophagus prepared earlier by left cervicotomy .", "a laminar drain is applied in the cervical incision ( fig .  2 ) . \n", "the patient was discharged from the hospital on the fifth post - operative day with oral intake . no leakage or other complication", "the patient survived for 4 months , gained 10 kg in this period and did not refer any dysphagia after the procedure ."], ["currently , there are several methods for this purpose , including the esophageal stent , endoscopic dilation , radiotherapy ( both external and endoluminal  brachytherapy ) , chemotherapy and laser recanalization ( yag ) .", "the choice of procedure and the response to treatment should be properly related to life expectancy , which has also been related to performance status , weight loss , tumor extension , clinical stage , sex and age .", "dysphagia from inoperable esophageal cancer is a common and complex management problem , and there is no consensus on the ideal treatment approach .", "all methods have their own advantages and disadvantages in rapid or late onset effect in symptom relief as in recurrence .", "no single intervention palliates dysphagia at all time for every patient ; therefore , healthcare providers must assess the risks and benefits of each palliative intervention for individual patients .", "surgical resection is still cited in the literature as a way to palliate esophageal cancer but is restricted to patients with severe risk of complications ( e.g. perforation or incontrollable tumor bleeding ) due to high morbidity and mortality in relation to non - surgical palliative options .", "that there are no randomized controlled studies to compare the addition of surgery to palliative chemotherapy with chemotherapy alone .", "they also state that such studies may become possible and worthwhile if minimal access surgery can be achieved that has reduced complications and better recovery of health - related quality of life than standard open surgery . in 1979 , postlethwait   proposed a technique for esophageal bypass surgery with an isoperistaltic gastric tube , and this method is still used despite its morbidity .", "this report demonstrates that an update in the technique proposed by postlethwait for palliation of esophageal cancer with the use of minimal access surgery is feasible .", "according to the current literature , the bypass procedure in order to palliate dysphagia could be reserved for patients in which the stent has failed or contraindicated .", "high costs can sometimes make the lesser invasive procedure more prohibitive to the patient than surgery itself ."]]}
{"article_id": "PMC2612976", "article_text": ["a 35-year - old female presented with a painless , slowly growing \n mass in the right inferior orbit for the past four years .", "she did \n not complain of diminution of visual acuity , diplopia or field \n defect . on examination , visual acuity in both eyes", "the right side demonstrated a solid , non - tender , \n freely mobile mass in the inferior orbit , which became more \n prominent on applying pressure on the upper part of the globe . \n", "the mass measured approximately 20 mm  15 mm and the \n size did not vary with valsalva maneuver , ocular movements \n or posture .", "slit - lamp \n and fundus examination of the eye showed normal anterior \n and posterior segments .", "b - scan ultrasonography revealed an extraconal , \n homogenous lesion in the inferior part of the right orbit . \n", "computed tomography revealed a non - enhancing well- \n defined homogenous mass measuring 20 mm  12 mm in the \n right inferior orbit which was displacing the inferior rectus \n and inferior oblique muscle upwards .", "the mass was separate \n from the above - mentioned muscles and produced no globe \n indentation or displacement [ figure 1a ] .", "a provisional diagnosis of orbital dermoid was made and \n the patient was taken up for an excision biopsy .", "peroperative findings were of a smooth , well - encapsulated \n mass with no attachments to the surrounding muscles .", "on histopathological examination , the gross specimen \n consisted of a smooth well - encapsulated solid mass measuring \n 25 mm in the greatest diameter [ figure 1b ] .", "microscopically , a \n biphasic pattern of tumor cells was seen with areas of closely \n packed spindle cells having fusiform nuclei and eosinophilic \n cytoplasm ( antoni a ) admixed with looser myxoid tissue \n having ovoid cells ( antoni b ) . at places ,", "these tumors cells \n were arranged in palisades and this arrangement of cells \n is referred to as verocay bodies .", "orbital \n schwannoma is a tumor of adulthood usually presenting \n between 20 to 70 years of age .", "schwannomas are usually \n asymptomatic when small and may produce progressive , \n painless proptosis on enlargement", ". a variable combination \n of signs and symptoms may be present due to the variable \n origin and location of the tumor in the orbit.2 displacement \n of the globe is related to the site of the tumor mass .", "most \n schwannomas arise from branches of either the supraorbital \n or supratrochlear nerves and hence produce downward \n displacement of the globe .", "less commonly , the tumor may arise \n from the infraorbital nerve and produce upward displacement \n of globe .", "larger tumors may produce diplopia , particularly \n when they arise from the orbital portion of the third , fourth \n or sixth nerve .", "the growth of the tumor may \n cause compression of the optic nerve with papilledema or \n optic atrophy .", "surgical excision is the treatment of choice and \n the tumor should be removed intact at the earliest to prevent \n compression of the optic nerve.3 incomplete excision can lead \n to recurrence or even intracranial extension .", "highly cellular \n tumors have greater chance of recurrence and malignant \n transformation . therefore an early treatment is indicated \n to avoid the complications related to progressive growth of \n the tumor . in our patient , we were successful in achieving \n the above objectives with timely and complete excision of \n the tumor .", "orbital schwannoma arising from the infraorbital nerve \n is rare and clinical diagnosis is often difficult .", "extensive \n literature search revealed only a few case reports of an orbital \n schwannoma arising from the infraorbital nerve.4 - 10 our \n patient presented with the tumor in this uncommon location . \n", "a solitary schwannoma , though rare , should be considered \n as a preoperative differential diagnosis of a unilateral slow- \n growing orbital mass in an adult and prompt management \n is warranted to prevent development of vision - threatening \n complications ."], "abstract_text": ["<S> a rare case of unilateral orbital schwannoma arising from the \n infraorbital nerve is presented . </S>", "<S> an excision biopsy with complete \n removal of the mass in the inferior orbit was performed . </S>", "<S> a \n definitive diagnosis was made on histopathological examination . \n </S>", "<S> the clinical and histological features of schwannoma are \n discussed . a need for early removal of such tumors </S>", "<S> is \n recommended to prevent complications . </S>"], "labels": null, "section_names": ["Case Report", "Discussion"], "sections": [["a 35-year - old female presented with a painless , slowly growing \n mass in the right inferior orbit for the past four years .", "she did \n not complain of diminution of visual acuity , diplopia or field \n defect . on examination , visual acuity in both eyes", "the right side demonstrated a solid , non - tender , \n freely mobile mass in the inferior orbit , which became more \n prominent on applying pressure on the upper part of the globe . \n", "the mass measured approximately 20 mm  15 mm and the \n size did not vary with valsalva maneuver , ocular movements \n or posture .", "slit - lamp \n and fundus examination of the eye showed normal anterior \n and posterior segments .", "b - scan ultrasonography revealed an extraconal , \n homogenous lesion in the inferior part of the right orbit . \n", "computed tomography revealed a non - enhancing well- \n defined homogenous mass measuring 20 mm  12 mm in the \n right inferior orbit which was displacing the inferior rectus \n and inferior oblique muscle upwards .", "the mass was separate \n from the above - mentioned muscles and produced no globe \n indentation or displacement [ figure 1a ] .", "a provisional diagnosis of orbital dermoid was made and \n the patient was taken up for an excision biopsy .", "peroperative findings were of a smooth , well - encapsulated \n mass with no attachments to the surrounding muscles .", "on histopathological examination , the gross specimen \n consisted of a smooth well - encapsulated solid mass measuring \n 25 mm in the greatest diameter [ figure 1b ] .", "microscopically , a \n biphasic pattern of tumor cells was seen with areas of closely \n packed spindle cells having fusiform nuclei and eosinophilic \n cytoplasm ( antoni a ) admixed with looser myxoid tissue \n having ovoid cells ( antoni b ) . at places ,", "these tumors cells \n were arranged in palisades and this arrangement of cells \n is referred to as verocay bodies ."], ["orbital \n schwannoma is a tumor of adulthood usually presenting \n between 20 to 70 years of age .", "schwannomas are usually \n asymptomatic when small and may produce progressive , \n painless proptosis on enlargement", ". a variable combination \n of signs and symptoms may be present due to the variable \n origin and location of the tumor in the orbit.2 displacement \n of the globe is related to the site of the tumor mass .", "most \n schwannomas arise from branches of either the supraorbital \n or supratrochlear nerves and hence produce downward \n displacement of the globe .", "less commonly , the tumor may arise \n from the infraorbital nerve and produce upward displacement \n of globe .", "larger tumors may produce diplopia , particularly \n when they arise from the orbital portion of the third , fourth \n or sixth nerve .", "the growth of the tumor may \n cause compression of the optic nerve with papilledema or \n optic atrophy .", "surgical excision is the treatment of choice and \n the tumor should be removed intact at the earliest to prevent \n compression of the optic nerve.3 incomplete excision can lead \n to recurrence or even intracranial extension .", "highly cellular \n tumors have greater chance of recurrence and malignant \n transformation . therefore an early treatment is indicated \n to avoid the complications related to progressive growth of \n the tumor . in our patient , we were successful in achieving \n the above objectives with timely and complete excision of \n the tumor .", "orbital schwannoma arising from the infraorbital nerve \n is rare and clinical diagnosis is often difficult .", "extensive \n literature search revealed only a few case reports of an orbital \n schwannoma arising from the infraorbital nerve.4 - 10 our \n patient presented with the tumor in this uncommon location . \n", "a solitary schwannoma , though rare , should be considered \n as a preoperative differential diagnosis of a unilateral slow- \n growing orbital mass in an adult and prompt management \n is warranted to prevent development of vision - threatening \n complications ."]]}
{"article_id": "PMC4857868", "article_text": ["risperidone has been reported to be effective in management of disruptive behaviors , including hyperactivity , irritability , aggression , and temper tantrums . increased appetite , weight gain , headache , and sedation", "literature search revealed only four published reports of risperidone - related bleeding , including hemorrhagic cystitis,1 ) nasal bleeding,2,3 ) and gastrointestinal bleeding.4 ) we hereby describe the first pediatric case of gingival bleeding during risperidone treatment .", "an 11-year - old - girl was referred to our out - patient clinic for her hyperactivity , temper tantrums , sleep problems , and self - injurious behaviors . according to her psychiatric assessment and psychometric evaluation ,", "she was started on risperidone 0.25 mg / day treatment for her disruptive behaviors and two weeks later the dose was increased to 0.5 mg / day .", "one - week after the dose increase , she experienced gingival bleeding when she brushed her teeth .", "risperidone was considered to be the reason for bleeding , therefore we decided to reduce the dose to 0.25 mg / day .", "gingival bleeding ceased within a week . because worsening of her behavioral problems , her mother increased the dose to 0.33 mg / day .", "she experienced gingival bleeding rarely ( twice a month ) and mildly during this dose regime , therefore we stopped the medication .", "we decided to change risperidone to methylphenidate ; however , her parents refused to continue to the treatment because of their worries about the potential side effects .", "she had no history of any bleeding disorder or any other medical condition including current allergies .", "she was not taking any other medication other than risperidone at the time of the development of bleeding .", "there are several case reports about risperidone - related bleeding ; however , to our knowledge , there is no report of gingival bleeding associated with risperidone in the literature .", "we presented a case who experienced gingival bleeding when risperidone dose was increased to 0.5 mg / day , and subsided after decreasing the dose to 0.25 mg / day , suggesting a dose - dependent side - effect .", "however , the chronological relationship between the time of risperidone 0.5 mg / day administration and emergence of bleeding in the absence of an identifiable medical condition suggests risperidone to be the causative agent .", "there was no other agent likely to be the cause of bleeding , except risperidone , and bleeding ceased when the dose was decreased .", "although an etiological relationship between risperidone use and gingival bleeding can not be drawn from a single case , the naranjo probability scale score ( 8) reveals a probable relationship.5 ) the bleeding side effect of risperidone might be caused by several mechanisms , including thrombocytopenia and 5-hydroxytryptamine 2a ( 5-ht2a ) receptor antagonism.3 ) thrombocytopenia is a known adverse effect of atypical antipsychotics and has also been reported during risperidone.6 ) in our reported case platelet count was normal , therefore , gingival bleeding could not be a consequence of thrombocytopenia .", "antagonism of 5-ht2a receptor was suggested to cause bleeding by inhibiting the release of vasoconstrictors from platelets and reducing the platelet aggregation.4 ) therefore , we may speculate that risperidone s high affinity to 5-ht2a receptors might have probably resulted gingival bleeding .", "this case suggests that bleeding associated with risperidone may be a dose - dependent side effect .", "however , these potential explanations may not describe the entire mechanism of this adverse reaction .", "it is also possible that she might have some undetermined predisposing factors for bleeding that is triggered by risperidone .", "there are several reports on gingival bleeding during anti - depressants;7 ) however , this is the first case on gingival bleeding associated with risperidone . although bleeding is a rare side effect , clinicians should be aware that risperidone may cause bleeding .", "risperidone may also increase the risk of bleeding in susceptible patients with a history of coagulation disorders , and with concomitant use of other drugs , including non - steroidal anti - inflammatory drugs , aspirin , or other medications that affect coagulation ."], "abstract_text": ["<S> there are several case reports on risperidone - related bleeding ; however , to our knowledge , there is no report about gingival bleeding associated with risperidone in the literature . </S>", "<S> we presented a case who experienced gingival bleeding when risperidone dose was increased to 0.5 mg / day , and subsided after decreasing the dose to 0.25 mg / day , suggesting a dose - dependent side - effect . </S>", "<S> the bleeding side effect of risperidone might be caused by several mechanisms , including 5-hydroxytryptamine 2a receptor antagonism . </S>", "<S> although bleeding associated with risperidone is rarely reported , clinicians should be aware of this side effect . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE", "DISCUSSION"], "sections": [["risperidone has been reported to be effective in management of disruptive behaviors , including hyperactivity , irritability , aggression , and temper tantrums . increased appetite , weight gain , headache , and sedation", "literature search revealed only four published reports of risperidone - related bleeding , including hemorrhagic cystitis,1 ) nasal bleeding,2,3 ) and gastrointestinal bleeding.4 ) we hereby describe the first pediatric case of gingival bleeding during risperidone treatment ."], ["an 11-year - old - girl was referred to our out - patient clinic for her hyperactivity , temper tantrums , sleep problems , and self - injurious behaviors . according to her psychiatric assessment and psychometric evaluation ,", "she was started on risperidone 0.25 mg / day treatment for her disruptive behaviors and two weeks later the dose was increased to 0.5 mg / day .", "one - week after the dose increase , she experienced gingival bleeding when she brushed her teeth .", "risperidone was considered to be the reason for bleeding , therefore we decided to reduce the dose to 0.25 mg / day .", "gingival bleeding ceased within a week . because worsening of her behavioral problems , her mother increased the dose to 0.33 mg / day .", "she experienced gingival bleeding rarely ( twice a month ) and mildly during this dose regime , therefore we stopped the medication .", "we decided to change risperidone to methylphenidate ; however , her parents refused to continue to the treatment because of their worries about the potential side effects .", "she had no history of any bleeding disorder or any other medical condition including current allergies .", "she was not taking any other medication other than risperidone at the time of the development of bleeding ."], ["there are several case reports about risperidone - related bleeding ; however , to our knowledge , there is no report of gingival bleeding associated with risperidone in the literature .", "we presented a case who experienced gingival bleeding when risperidone dose was increased to 0.5 mg / day , and subsided after decreasing the dose to 0.25 mg / day , suggesting a dose - dependent side - effect .", "however , the chronological relationship between the time of risperidone 0.5 mg / day administration and emergence of bleeding in the absence of an identifiable medical condition suggests risperidone to be the causative agent .", "there was no other agent likely to be the cause of bleeding , except risperidone , and bleeding ceased when the dose was decreased .", "although an etiological relationship between risperidone use and gingival bleeding can not be drawn from a single case , the naranjo probability scale score ( 8) reveals a probable relationship.5 ) the bleeding side effect of risperidone might be caused by several mechanisms , including thrombocytopenia and 5-hydroxytryptamine 2a ( 5-ht2a ) receptor antagonism.3 ) thrombocytopenia is a known adverse effect of atypical antipsychotics and has also been reported during risperidone.6 ) in our reported case platelet count was normal , therefore , gingival bleeding could not be a consequence of thrombocytopenia .", "antagonism of 5-ht2a receptor was suggested to cause bleeding by inhibiting the release of vasoconstrictors from platelets and reducing the platelet aggregation.4 ) therefore , we may speculate that risperidone s high affinity to 5-ht2a receptors might have probably resulted gingival bleeding .", "this case suggests that bleeding associated with risperidone may be a dose - dependent side effect .", "however , these potential explanations may not describe the entire mechanism of this adverse reaction .", "it is also possible that she might have some undetermined predisposing factors for bleeding that is triggered by risperidone .", "there are several reports on gingival bleeding during anti - depressants;7 ) however , this is the first case on gingival bleeding associated with risperidone . although bleeding is a rare side effect , clinicians should be aware that risperidone may cause bleeding .", "risperidone may also increase the risk of bleeding in susceptible patients with a history of coagulation disorders , and with concomitant use of other drugs , including non - steroidal anti - inflammatory drugs , aspirin , or other medications that affect coagulation ."]]}
{"article_id": "PMC4544142", "article_text": ["intestinal obstruction can be a result of mechanical or functional obstruction of the intestines , thereby preventing the normal transit of the products of digestion .", "intestinal pseudo - obstruction ( adynamic ) , characterized by abdominal pain , nausea , vomiting , constipation , and severe abdominal distension , is a clinical syndrome caused by severe impairment in the ability of the intestines to push the food through , in the absence of any lesion in the intestinal lumen .", "secondary pseudo - obstruction is more common than the primary and is commonly associated with neuroleptics , opiates , diabetes mellitus , and severe metabolic illnesses .", "intrathecal baclofen has been reported to cause intestinal pseudo - obstruction and life - threatening constipation .", "though baclofen might sometimes cause constipation , there are only very few reports on intestinal pseudo - obstruction complicating oral baclofen therapy .", "a 50-year - old male , post cervical discectomy ( c3 - 4 ) for prolapsed intervertebral disc with quadriparesis , neurogenic bladder , and spasticity , was on baclofen at a dose of 50 mg / day for 6 months .", "he presented to the emergency department with acute onset of bilious projectile vomiting , abdominal distension , and constipation since 3 days .", "one and half months back , he had a traumatic urethral injury for which open suprapubic cystostomy was performed .", "he had no history of other co - morbidities or drug intake other than baclofen . on examination , his vitals were stable but he was dehydrated .", "plain radiograph of the abdomen revealed dilated large and small bowel loops without any air fluid levels .", "the possibility of adhesive intestinal obstruction was considered and the patient was kept nil per oral with nasogastric aspiration .", "a diagnosis of pseudo - obstruction was considered secondary to long - term baclofen intake , as he had gradually improved over 1 week without any active intervention except for the discontinuation of baclofen since admission .", "the patient was able to tolerate oral diet after 7 days of admission with no recurrence of symptoms .", "currently , at 1 month of follow - up , he is asymptomatic with no recurrences .", "colonic pseudo - obstruction is characterized by distension of the colon with features of colonic obstruction , in the absence of mechanical obstruction .", "primary pseudo - obstruction is rare and is a motility disorder involving the autonomic innervation of the intestinal wall .", "secondary pseudo - obstruction is more common and it has been associated with conditions like severe metabolic illness , diabetes mellitus , myxedema , scleroderma , parkinson 's disease , hyperparathyroidism , and drugs like neuroleptics and opiates .", "baclofen , a derivative of gamma - aminobutyric acid ( gaba ) , is an agonist for the gaba b receptors , and is used to treat spasticity following spinal cord injury , multiple sclerosis , and cerebral palsy .", "tolerance to baclofen mainly develops after many years to intrathecal route , but this beneficial property can be potentially harmful necessitating baclofen withdrawal in patients developing adynamic intestinal obstruction . intestinal pseudo - obstruction has been reported after intrathecal baclofen , but till date , only a single case has been reported in literature describing adynamic intestinal obstruction caused by therapeutic dose of oral baclofen at 20 mg / day for 2 years in a 75-year - old male .", "animal studies have shown that gaba - ergic mechanisms are involved in synaptosomal nitric oxide synthesis resulting in relaxation of rat ileum . in human preparations obtained from patients with malignant tumors , baclofen suppressed both amplitude and frequency of spontaneous and pharmacologically induced contractions in the longitudinal muscle of jejunum , eventually causing a complete block at higher concentrations while not affecting spontaneous motility of circular or longitudinal colon muscles .", "gabaergic neurons , which are predominantly inhibitory interneurons , are distributed throughout the central nervous system and the enteric nervous system in humans .", "hence , baclofen being a competitive gaba b agonist , affects the gastrointestinal function by acting both peripherally in the intestinal tract by inhibiting the myenteric plexus and centrally on brainstem nuclei which coordinate afferent input from and efferent output to the intestine by crossing the blood  brain barrier . in our case", ", we considered the possibilities of baclofen - induced pseudo - obstruction of the intestine and adhesive intestinal obstruction .", "hence , baclofen was discontinued and patient was put on nasogastric decompression . considering the remote possibility of adhesive obstruction , water - soluble contrast enema , colonoscopy , or neostigmine was not tried .", "colonoscopic decompression was planned in case of deterioration , but the patient recovered completely with prompt discontinuation of baclofen , nasogastric decompression , and maintenance of electrolytes . considering the temporal relationship of the occurrence of symptoms after 6 months of baclofen therapy , improvement after withdrawal of the drug , and presence of pharmacological explanation for the occurrence of obstruction and evidence of similar reports earlier , we considered the possibility of baclofen - induced pseudo - obstruction . on causality assessment ,", "baclofen - induced pseudo - obstruction in our case belongs to the  probable / likely  category as per the world health organization - uppsala monitoring centre ( who - umc ) system and to the ", "objective evidence in the form of blood levels of baclofen was not available , and re - challenging was not attempted because intestinal pseudo - obstruction could be fatal .", "the remote possibility of adhesive obstruction is mostly unlikely as the patient recovered with withdrawal of baclofen .", "oral baclofen used for spasticity can cause constipation , but intestinal pseudo - obstruction is a rare possibility and in such cases , prompt discontinuation of the drug can be therapeutic . this rare cause of intestinal pseudo - obstruction needs to be borne in mind to avoid potentially morbid investigations like gastrograffin enema or colonoscopy , which are associated with complications like perforation ."], "abstract_text": ["<S> baclofen is a gamma- aminobutyric acid b ( gaba b ) agonist used for the management of spasticity associated with spinal cord injury . </S>", "<S> oral baclofen might cause constipation , but intestinal pseudo - obstruction is very rare . </S>", "<S> we report a 50-year - old male with spasticity following cervical discectomy ( c3 - 4 ) on oral baclofen for 6 months with intestinal pseudo - obstruction . </S>", "<S> he had undergone open suprapubic cystostomy for traumatic urethral injury , 45 days prior to the presentation and adhesive intestinal obstruction was also considered a possibility . however , there were no air fluid levels on abdominal radiographs and ultrasound abdomen was non - contributory . </S>", "<S> withdrawal of baclofen was therapeutic in this patient . </S>", "<S> this case is being reported to highlight the rare possibility of oral baclofen induced intestinal pseudo - obstruction . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["intestinal obstruction can be a result of mechanical or functional obstruction of the intestines , thereby preventing the normal transit of the products of digestion .", "intestinal pseudo - obstruction ( adynamic ) , characterized by abdominal pain , nausea , vomiting , constipation , and severe abdominal distension , is a clinical syndrome caused by severe impairment in the ability of the intestines to push the food through , in the absence of any lesion in the intestinal lumen .", "secondary pseudo - obstruction is more common than the primary and is commonly associated with neuroleptics , opiates , diabetes mellitus , and severe metabolic illnesses .", "intrathecal baclofen has been reported to cause intestinal pseudo - obstruction and life - threatening constipation .", "though baclofen might sometimes cause constipation , there are only very few reports on intestinal pseudo - obstruction complicating oral baclofen therapy ."], ["a 50-year - old male , post cervical discectomy ( c3 - 4 ) for prolapsed intervertebral disc with quadriparesis , neurogenic bladder , and spasticity , was on baclofen at a dose of 50 mg / day for 6 months .", "he presented to the emergency department with acute onset of bilious projectile vomiting , abdominal distension , and constipation since 3 days .", "one and half months back , he had a traumatic urethral injury for which open suprapubic cystostomy was performed .", "he had no history of other co - morbidities or drug intake other than baclofen . on examination , his vitals were stable but he was dehydrated .", "plain radiograph of the abdomen revealed dilated large and small bowel loops without any air fluid levels .", "the possibility of adhesive intestinal obstruction was considered and the patient was kept nil per oral with nasogastric aspiration .", "a diagnosis of pseudo - obstruction was considered secondary to long - term baclofen intake , as he had gradually improved over 1 week without any active intervention except for the discontinuation of baclofen since admission .", "the patient was able to tolerate oral diet after 7 days of admission with no recurrence of symptoms .", "currently , at 1 month of follow - up , he is asymptomatic with no recurrences ."], ["colonic pseudo - obstruction is characterized by distension of the colon with features of colonic obstruction , in the absence of mechanical obstruction .", "primary pseudo - obstruction is rare and is a motility disorder involving the autonomic innervation of the intestinal wall .", "secondary pseudo - obstruction is more common and it has been associated with conditions like severe metabolic illness , diabetes mellitus , myxedema , scleroderma , parkinson 's disease , hyperparathyroidism , and drugs like neuroleptics and opiates .", "baclofen , a derivative of gamma - aminobutyric acid ( gaba ) , is an agonist for the gaba b receptors , and is used to treat spasticity following spinal cord injury , multiple sclerosis , and cerebral palsy .", "tolerance to baclofen mainly develops after many years to intrathecal route , but this beneficial property can be potentially harmful necessitating baclofen withdrawal in patients developing adynamic intestinal obstruction . intestinal pseudo - obstruction has been reported after intrathecal baclofen , but till date , only a single case has been reported in literature describing adynamic intestinal obstruction caused by therapeutic dose of oral baclofen at 20 mg / day for 2 years in a 75-year - old male .", "animal studies have shown that gaba - ergic mechanisms are involved in synaptosomal nitric oxide synthesis resulting in relaxation of rat ileum . in human preparations obtained from patients with malignant tumors , baclofen suppressed both amplitude and frequency of spontaneous and pharmacologically induced contractions in the longitudinal muscle of jejunum , eventually causing a complete block at higher concentrations while not affecting spontaneous motility of circular or longitudinal colon muscles .", "gabaergic neurons , which are predominantly inhibitory interneurons , are distributed throughout the central nervous system and the enteric nervous system in humans .", "hence , baclofen being a competitive gaba b agonist , affects the gastrointestinal function by acting both peripherally in the intestinal tract by inhibiting the myenteric plexus and centrally on brainstem nuclei which coordinate afferent input from and efferent output to the intestine by crossing the blood  brain barrier . in our case", ", we considered the possibilities of baclofen - induced pseudo - obstruction of the intestine and adhesive intestinal obstruction .", "hence , baclofen was discontinued and patient was put on nasogastric decompression . considering the remote possibility of adhesive obstruction , water - soluble contrast enema , colonoscopy , or neostigmine was not tried .", "colonoscopic decompression was planned in case of deterioration , but the patient recovered completely with prompt discontinuation of baclofen , nasogastric decompression , and maintenance of electrolytes . considering the temporal relationship of the occurrence of symptoms after 6 months of baclofen therapy , improvement after withdrawal of the drug , and presence of pharmacological explanation for the occurrence of obstruction and evidence of similar reports earlier , we considered the possibility of baclofen - induced pseudo - obstruction . on causality assessment ,", "baclofen - induced pseudo - obstruction in our case belongs to the  probable / likely  category as per the world health organization - uppsala monitoring centre ( who - umc ) system and to the ", "objective evidence in the form of blood levels of baclofen was not available , and re - challenging was not attempted because intestinal pseudo - obstruction could be fatal .", "the remote possibility of adhesive obstruction is mostly unlikely as the patient recovered with withdrawal of baclofen ."], ["oral baclofen used for spasticity can cause constipation , but intestinal pseudo - obstruction is a rare possibility and in such cases , prompt discontinuation of the drug can be therapeutic . this rare cause of intestinal pseudo - obstruction needs to be borne in mind to avoid potentially morbid investigations like gastrograffin enema or colonoscopy , which are associated with complications like perforation ."]]}
{"article_id": "PMC3844227", "article_text": ["the incidences were getting higher and higher . increased intramyocellular lipid accumulation plays a very important role in obesity and diabetes , as well as their complications [ 2 , 3 ] .", "this attracted great interest in the effect of accumulated lipid intermediates on insulin resistance [ 4 , 5 ] .", "lipotoxicity was regarded as the link of high levels of lipids with impaired insulin signaling [ 6 , 7 ] .", "this sparked the interest in how excessive lipid affects -oxidation and mitochondrial biogenesis , which may have a great impact on glucose utilization and insulin resistance [ 710 ] .", "carnitine palmitoyl transferase 1 ( cpt1 ) is an important rate - limiting enzyme of mitochondrial -oxidation , by controlling the mitochondrial uptake of long - chain acyl - coas .", "its muscle isoform , cpt1b , is the predominant isoform rich in the heart and skeletal muscles .", "it was suggested that inhibiting cpt1 activity by specific cpt1 inhibitors alleviates insulin resistance in diet - induced obese mice .", "however , there is no study investigating if specific cpt1b deficiency in skeletal muscles had significant impact on lipids and insulin sensitivity .", "therefore , in this study , by establishing a mouse model , we directly explored this issue . with specific knockout of carnitine palmitoyl transferase-1b ( cpt1b ) in skeletal muscles of mice ,", "cpt1b m/ mouse model was established as per previously described [ 12 , 16 , 17 ] by using male c57/bl6 mice ( 12 weeks old ; 2025  g ) with cre - lox technology [ 17 , 18 ] .", "mice were genotyped by standard pcr of tail dna with pcr master mix ( applied biosystems inc . ) .", "twenty mice from each group ( cpt1band cpt1b m/ ) were kept on a 12  h/12  h light / dark cycle , in temperature - steady rooms , and had ad libitum access to water and chow diet .", "all experimental procedures were conducted according to the care and use guide of laboratory animals and were approved by the iaec of the huzhou central hospital .", "general and metabolic profiles were measured at 12 , 18 , 24 , and 30 weeks of age , including the following : body weight , lipids in serum , food intake , body mass , and fat mass , as per related protocols [ 19 , 20 ] .", "skeletal muscle mrna and protein were extracted and purified with standard protocols as per our previous protocols [ 7 , 21 ] .", "rt - pcr was performed for mrna quantization per standard protocols with the reagents from abi system inc .", "quantitative real - time rt - pcr analyses were carried out by using step one real - time pcr system ( applied biosystems inc . ) .", "cycles were 50c for 2  min , a 2  min 95c denaturing step , followed by 50 cycles of 95c denaturation , incubated at 60c for 2  min , and denatured at 95c for 1 sec for the final step .", "the sequences of the primers are as follows : for cpt1b : forward : gac cat agaggc act tct cagcat gg , reverse : gcagca gcttca ggg tttgt ; -actin : forward : gtc ctc tcc caa gtc cac ac , reverse : ggg aga cca aaa gcc ttc at ; pakt forward : taa tac gac tca cta tag ggc caa ggagatcatgc , reverse : gatttaggtgacactatagctccaagctatcgtcc ; glut4 forward : accgtggtccttigctgtgtt , reverse : acc cca atg ttg tac cca aac t. as described previously [ 16 , 22 , 23 ] , a modified mitochondrial cpt1 assay was performed to measure cpt1 activity .", "briefly , quantified mitochondria ( 100  g protein ) from skeletal muscles were incubated with reaction buffer ( 117 mm tris - hcl , 0.28  mm reduced glutathione , 4.4  mm atp , 4.4  mm mgcl2 , 16.7  mm kcl , 2.2  mm kcn , 40  mg / l rotenone , 0.1% bsa , and 50  mm palmitoylcoa ) at 37c for 5  min . initiate the reaction by adding 2  mm [ c]-carnitine ( 0.1  ci ) and queued it 10  min later with 50  l 1.2  mm ice cold hcl .", "[ c]-palmitoyl carnitine was extracted with water saturated butanol and measured via liquid scintillation counting .", "as previously described [ 24 , 25 ] , fao was examined with radiolabeled palmitate fao assay .", "skeletal muscle tissues were homogenized , and mitochondria were isolated and quantified ( 100  g proteins ) .", "palmitate oxidation assay was performed with reaction buffer ( 75.5  mm sucrose , 12.5  mm k2hpo4 , 100  mm kcl , 1.75  mm mgcl2 - 6h2o , 1.75  mm l - carnitine , 0.125  mm l(- ) malic acid , 1.75  mm dtt , 0.07  mm nad+ , 2  mm atp , 10 mm tris - hcl , and 0.07  mm coenzyme a ) .", "the reaction started when 200m [ c]-palmitate-15% bsa ( 1  :  6 ) complex ( 0.04  ci / reaction mixture ) was added and stopped by 3.5  m perchloric acid after incubation at 37c for 30  min .", "co2-trapping medium ( naoh , 0.1  m ) for c radioactivity was measured by liquid scintillation to calculate palmitate oxidation rate .", "after incubation , co2 and c - asps were measured ( ls 6500 ; beckman coulter ) .", "calculations were then performed accordingly .   as previously reported [ 2628 ] , tags , dags , and ceramides were tested with kc - esi - ms and gas chromatography ( applied biosystems inc . ) .", "after the solution was evaporated dry and reconstituted , the samples were analyzed with mass spectrometry ( ms ) .", "the analysis was performed in positive ion mode electrospray ion ( esi - ms ) source and precursor ion scans m / z 264 and 282 ( ceramides ) .", "ceramides were quantified by taking the ratios of the integrated intensity for each subspecies to the intensity of c17:0 .   as described [ 2931 ] ,", "an intraperitoneal glucose tolerance test ( igtt ) and insulin tolerance test ( itt ) were performed on nonanaesthetized mice after 8 hours ' fasting .", "blood glucose was measured with lancet glucometer ( johnson and johnson ) . for igtt , 20% glucose ( 2.0 ", "blood samples were collected from tail vein at 30 , 60 , 90 , and 120  min for glucose levels . for itt", ", glucose blood levels were sampled at 5 , 10 , 15 , 20 , 25 , and 30  min following intraperitoneal injection of human insulin ( 0.75  u 4.5  nmol / kg ; novolin r , novo nordisk ) .   as described previously [ 32 , 33 ]", ", skeletal muscles were homogenized in ice - cold buffer ( 250  mm sucrose , 10  mm tris - hcl , 2  mm edta , and 1  mm atp ( ph 7.4 ) ) . for glucose oxidation ,", "fresh skeletal muscle tissues were homogenized in ice - cold buffer ( 5  mm kcl , 2  mm tris - hcl , 0.5  mm tris base , 0.25  mm mgcl2 - 6h2o , 0.05  mm edta , and 0.05  mm atp ( ph 7.4 ) ) , 400  l homogenate was used and the reaction started when 200  m [ c]-glucose ( 0.1  ci / reaction mixture ) was added .", "co2-trapping medium ( naoh , 0.1  m ) for c radioactivity was measured by liquid scintillation to calculate glucose oxidation and quantified by weight of the skeletal muscle tissue homogenate .", "student 's t - test was used to evaluate the statistical significance of differences between knockout and controls .", "as shown in figure 1(a ) , the mrna expression of cpt1b decreased specifically in skeletal muscles ( rt - pcr ) but not in the heart muscle in cpt1b m/ mice .", "cpt1 activity is barely detectable ( dtnb assay at 412  nm ) in mitochondria ( figure 1(b ) ) for the knockout mice , even up to 10 minutes .", "compared to the mice in cpt1b group , those mice in cpt1b m/ group had similar daily food intake . however , as shown in table 1 , their body weights were lower , starting about 12 weeks of age , along with lower fat mass ( p < 0.05 , resp . ) .", "their lipids levels were higher ( p < 0.05 ) , but their glucose and insulin levels were similar .", "as shown in figure 2(a ) , fao was decreased in isolated mitochondria ( radiolabeled palmitate ) in skeletal muscles of cpt1b m/ mice .", "this was accompanied by elevation of ceramides , tags , and dags ( figures 2(b ) and 2(c ) ) .", "interestingly , for the ceramides , c16 , c18 , and c18:1 had significant increases for cpt1b m/ , but not for c24 or c24:1 .", "both dags and tags are dramatically elevated in cpt1b m/ mice .   as shown in figure 3(a ) , cpt1b m/ mice maintain insulin sensitivity ( insulin tolerance test ) . compared with wild type , glucose tolerance test of cpt1b m/ mice had improved significantly ( as shown in figure 3(b ) ) .", "pyruvate oxidation went up ( figure 3(c ) ) and so did insulin - stimulated pakt and glut4 ( figure 4 ) .", "the relationship of fatty acid oxidation , lipids accumulation , and insulin sensitivity in skeletal muscles has been quite interesting .", "imbalanced fatty acid uptake and fatty acid oxidation ( fao ) have been linked to insulin resistance in muscles , which in turn worsens and complicates obesity and diabetes , as well as their complications [ 7 , 34 , 35 ] .", "our study demonstrated that in mice with conditional knockout cpt1b in skeletal muscles , mitochondrial fatty acid oxidation was depressed dramatically .", "this was accompanied by increased accumulation of lipids in skeletal muscles , such as dags , tags , and ceramides .", "regardless of these changes , insulin tolerance test , glucose tolerance test , and pyruvate oxidation all proved better insulin sensitivity for the knockout mice .", "this is partially consistent with the recent results in high - fat diet - induced obese mice , when given cpt1 inhibitor oxfenicine , as far as the major findings are concerned .", "another study done with different cpt1 inhibitor , etomoxir , suggested that lipids intermediates increased .", "70% of total insulin - induced glucose disposal occurs in skeletal muscles , this may indicate that increased fatty acid intermediates in skeletal muscles could damage insulin sensitivity .", "our study showed that regardless of the characteristics of lipids accumulation , the mice are still insulin sensitive .", "first , due to the knockout of cpt1b in skeletal muscles , the major fuel sources were in deficit .", "this may suggest that signaling pathways related to energy were impaired or injured such as ampk or mtor .", "second , abnormal mitochondrial biogenesis and mitochondrial dysfunction may contribute to insulin resistance and diabetes [ 39 , 40 ] .", "we explored from these aspects the enzymes involved in mitochondrial activity such as -had and citrate synthase , genes related to transcriptions such as nrf ( nuclear respiratory factor ) , and the major nuclear receptor activator pgc1 .", "thirdly , considering the correlation of adaptation of peroxisomal and amino acid to mitochondria , we further explored these changes .", "we will report these results in our next paper and further discuss the mechanism involved . in summary ,", "it suggested that with specific knockout of cpt1b in skeletal muscles , although there were lipids accumulations in skeletal muscles , the insulin sensitivity still remains ."], "abstract_text": ["<S> \n objective . by specific knockout of carnitine palmitoyl transferase 1b ( cpt1b ) in skeletal muscles </S>", "<S> , we explored the effect of cpt1b deficiency on lipids and insulin sensitivity . </S>", "<S> methods . </S>", "<S> mice with specific knockout of cpt1b in skeletal muscles ( cpt1b m/ ) were used for the experiment group , with littermate c57bl/6 as controls ( cpt1b ) . </S>", "<S> general and metabolic profiles were measured and compared between groups . </S>", "<S> mrna expression and cpt1 activity were measured in skeletal muscle tissues and compared between groups . </S>", "<S> mitochondrial fatty acid oxidation ( fao ) , triglycerides ( tags ) , diglycerides ( dags ) , and ceramides were examined in skeletal muscles in two groups . </S>", "<S> phosphorylated akt ( pakt ) and glucose transporter 4 ( glut4 ) were determined with real - time polymerase chain reaction ( rt - pcr ) . </S>", "<S> insulin tolerance test , glucose tolerance test , and pyruvate oxidation were performed in both groups . </S>", "<S> results . </S>", "<S> cpt1b m/ model was successfully established , with impaired muscle cpt1 activity . </S>", "<S> compared with cpt1b mice , cpt1b m/ mice had similar food intake but lower body weight or fat mass and higher lipids but similar glucose or insulin levels . </S>", "<S> their mitochondrial fao of skeletal muscles was impaired . </S>", "<S> there were lipids accumulations ( tags , dags , and ceramides ) in skeletal muscle . </S>", "<S> however , pakt and glut4 , insulin sensitivity , glucose tolerance , and pyruvate oxidation were preserved . </S>", "<S> conclusion . </S>", "<S> skeletal muscle - specific cpt1 deficiency elevates lipotoxic intermediates but preserves insulin sensitivity . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["the incidences were getting higher and higher . increased intramyocellular lipid accumulation plays a very important role in obesity and diabetes , as well as their complications [ 2 , 3 ] .", "this attracted great interest in the effect of accumulated lipid intermediates on insulin resistance [ 4 , 5 ] .", "lipotoxicity was regarded as the link of high levels of lipids with impaired insulin signaling [ 6 , 7 ] .", "this sparked the interest in how excessive lipid affects -oxidation and mitochondrial biogenesis , which may have a great impact on glucose utilization and insulin resistance [ 710 ] .", "carnitine palmitoyl transferase 1 ( cpt1 ) is an important rate - limiting enzyme of mitochondrial -oxidation , by controlling the mitochondrial uptake of long - chain acyl - coas .", "its muscle isoform , cpt1b , is the predominant isoform rich in the heart and skeletal muscles .", "it was suggested that inhibiting cpt1 activity by specific cpt1 inhibitors alleviates insulin resistance in diet - induced obese mice .", "however , there is no study investigating if specific cpt1b deficiency in skeletal muscles had significant impact on lipids and insulin sensitivity .", "therefore , in this study , by establishing a mouse model , we directly explored this issue . with specific knockout of carnitine palmitoyl transferase-1b ( cpt1b ) in skeletal muscles of mice ,"], ["cpt1b m/ mouse model was established as per previously described [ 12 , 16 , 17 ] by using male c57/bl6 mice ( 12 weeks old ; 2025  g ) with cre - lox technology [ 17 , 18 ] .", "mice were genotyped by standard pcr of tail dna with pcr master mix ( applied biosystems inc . ) .", "twenty mice from each group ( cpt1band cpt1b m/ ) were kept on a 12  h/12  h light / dark cycle , in temperature - steady rooms , and had ad libitum access to water and chow diet .", "all experimental procedures were conducted according to the care and use guide of laboratory animals and were approved by the iaec of the huzhou central hospital .", "general and metabolic profiles were measured at 12 , 18 , 24 , and 30 weeks of age , including the following : body weight , lipids in serum , food intake , body mass , and fat mass , as per related protocols [ 19 , 20 ] .", "skeletal muscle mrna and protein were extracted and purified with standard protocols as per our previous protocols [ 7 , 21 ] .", "rt - pcr was performed for mrna quantization per standard protocols with the reagents from abi system inc .", "quantitative real - time rt - pcr analyses were carried out by using step one real - time pcr system ( applied biosystems inc . ) .", "cycles were 50c for 2  min , a 2  min 95c denaturing step , followed by 50 cycles of 95c denaturation , incubated at 60c for 2  min , and denatured at 95c for 1 sec for the final step .", "the sequences of the primers are as follows : for cpt1b : forward : gac cat agaggc act tct cagcat gg , reverse : gcagca gcttca ggg tttgt ; -actin : forward : gtc ctc tcc caa gtc cac ac , reverse : ggg aga cca aaa gcc ttc at ; pakt forward : taa tac gac tca cta tag ggc caa ggagatcatgc , reverse : gatttaggtgacactatagctccaagctatcgtcc ; glut4 forward : accgtggtccttigctgtgtt , reverse : acc cca atg ttg tac cca aac t. as described previously [ 16 , 22 , 23 ] , a modified mitochondrial cpt1 assay was performed to measure cpt1 activity .", "briefly , quantified mitochondria ( 100  g protein ) from skeletal muscles were incubated with reaction buffer ( 117 mm tris - hcl , 0.28  mm reduced glutathione , 4.4  mm atp , 4.4  mm mgcl2 , 16.7  mm kcl , 2.2  mm kcn , 40  mg / l rotenone , 0.1% bsa , and 50  mm palmitoylcoa ) at 37c for 5  min . initiate the reaction by adding 2  mm [ c]-carnitine ( 0.1  ci ) and queued it 10  min later with 50  l 1.2  mm ice cold hcl .", "[ c]-palmitoyl carnitine was extracted with water saturated butanol and measured via liquid scintillation counting .", "as previously described [ 24 , 25 ] , fao was examined with radiolabeled palmitate fao assay .", "skeletal muscle tissues were homogenized , and mitochondria were isolated and quantified ( 100  g proteins ) .", "palmitate oxidation assay was performed with reaction buffer ( 75.5  mm sucrose , 12.5  mm k2hpo4 , 100  mm kcl , 1.75  mm mgcl2 - 6h2o , 1.75  mm l - carnitine , 0.125  mm l(- ) malic acid , 1.75  mm dtt , 0.07  mm nad+ , 2  mm atp , 10 mm tris - hcl , and 0.07  mm coenzyme a ) .", "the reaction started when 200m [ c]-palmitate-15% bsa ( 1  :  6 ) complex ( 0.04  ci / reaction mixture ) was added and stopped by 3.5  m perchloric acid after incubation at 37c for 30  min .", "co2-trapping medium ( naoh , 0.1  m ) for c radioactivity was measured by liquid scintillation to calculate palmitate oxidation rate .", "after incubation , co2 and c - asps were measured ( ls 6500 ; beckman coulter ) .", "calculations were then performed accordingly .   as previously reported [ 2628 ] , tags , dags , and ceramides were tested with kc - esi - ms and gas chromatography ( applied biosystems inc . ) .", "after the solution was evaporated dry and reconstituted , the samples were analyzed with mass spectrometry ( ms ) .", "the analysis was performed in positive ion mode electrospray ion ( esi - ms ) source and precursor ion scans m / z 264 and 282 ( ceramides ) .", "ceramides were quantified by taking the ratios of the integrated intensity for each subspecies to the intensity of c17:0 .   as described [ 2931 ] ,", "an intraperitoneal glucose tolerance test ( igtt ) and insulin tolerance test ( itt ) were performed on nonanaesthetized mice after 8 hours ' fasting .", "blood glucose was measured with lancet glucometer ( johnson and johnson ) . for igtt , 20% glucose ( 2.0 ", "blood samples were collected from tail vein at 30 , 60 , 90 , and 120  min for glucose levels . for itt", ", glucose blood levels were sampled at 5 , 10 , 15 , 20 , 25 , and 30  min following intraperitoneal injection of human insulin ( 0.75  u 4.5  nmol / kg ; novolin r , novo nordisk ) .   as described previously [ 32 , 33 ]", ", skeletal muscles were homogenized in ice - cold buffer ( 250  mm sucrose , 10  mm tris - hcl , 2  mm edta , and 1  mm atp ( ph 7.4 ) ) . for glucose oxidation ,", "fresh skeletal muscle tissues were homogenized in ice - cold buffer ( 5  mm kcl , 2  mm tris - hcl , 0.5  mm tris base , 0.25  mm mgcl2 - 6h2o , 0.05  mm edta , and 0.05  mm atp ( ph 7.4 ) ) , 400  l homogenate was used and the reaction started when 200  m [ c]-glucose ( 0.1  ci / reaction mixture ) was added .", "co2-trapping medium ( naoh , 0.1  m ) for c radioactivity was measured by liquid scintillation to calculate glucose oxidation and quantified by weight of the skeletal muscle tissue homogenate .", "student 's t - test was used to evaluate the statistical significance of differences between knockout and controls ."], ["as shown in figure 1(a ) , the mrna expression of cpt1b decreased specifically in skeletal muscles ( rt - pcr ) but not in the heart muscle in cpt1b m/ mice .", "cpt1 activity is barely detectable ( dtnb assay at 412  nm ) in mitochondria ( figure 1(b ) ) for the knockout mice , even up to 10 minutes .", "compared to the mice in cpt1b group , those mice in cpt1b m/ group had similar daily food intake . however , as shown in table 1 , their body weights were lower , starting about 12 weeks of age , along with lower fat mass ( p < 0.05 , resp . ) .", "their lipids levels were higher ( p < 0.05 ) , but their glucose and insulin levels were similar .", "as shown in figure 2(a ) , fao was decreased in isolated mitochondria ( radiolabeled palmitate ) in skeletal muscles of cpt1b m/ mice .", "this was accompanied by elevation of ceramides , tags , and dags ( figures 2(b ) and 2(c ) ) .", "interestingly , for the ceramides , c16 , c18 , and c18:1 had significant increases for cpt1b m/ , but not for c24 or c24:1 .", "both dags and tags are dramatically elevated in cpt1b m/ mice .   as shown in figure 3(a ) , cpt1b m/ mice maintain insulin sensitivity ( insulin tolerance test ) . compared with wild type , glucose tolerance test of cpt1b m/ mice had improved significantly ( as shown in figure 3(b ) ) .", "pyruvate oxidation went up ( figure 3(c ) ) and so did insulin - stimulated pakt and glut4 ( figure 4 ) ."], ["the relationship of fatty acid oxidation , lipids accumulation , and insulin sensitivity in skeletal muscles has been quite interesting .", "imbalanced fatty acid uptake and fatty acid oxidation ( fao ) have been linked to insulin resistance in muscles , which in turn worsens and complicates obesity and diabetes , as well as their complications [ 7 , 34 , 35 ] .", "our study demonstrated that in mice with conditional knockout cpt1b in skeletal muscles , mitochondrial fatty acid oxidation was depressed dramatically .", "this was accompanied by increased accumulation of lipids in skeletal muscles , such as dags , tags , and ceramides .", "regardless of these changes , insulin tolerance test , glucose tolerance test , and pyruvate oxidation all proved better insulin sensitivity for the knockout mice .", "this is partially consistent with the recent results in high - fat diet - induced obese mice , when given cpt1 inhibitor oxfenicine , as far as the major findings are concerned .", "another study done with different cpt1 inhibitor , etomoxir , suggested that lipids intermediates increased .", "70% of total insulin - induced glucose disposal occurs in skeletal muscles , this may indicate that increased fatty acid intermediates in skeletal muscles could damage insulin sensitivity .", "our study showed that regardless of the characteristics of lipids accumulation , the mice are still insulin sensitive .", "first , due to the knockout of cpt1b in skeletal muscles , the major fuel sources were in deficit .", "this may suggest that signaling pathways related to energy were impaired or injured such as ampk or mtor .", "second , abnormal mitochondrial biogenesis and mitochondrial dysfunction may contribute to insulin resistance and diabetes [ 39 , 40 ] .", "we explored from these aspects the enzymes involved in mitochondrial activity such as -had and citrate synthase , genes related to transcriptions such as nrf ( nuclear respiratory factor ) , and the major nuclear receptor activator pgc1 .", "thirdly , considering the correlation of adaptation of peroxisomal and amino acid to mitochondria , we further explored these changes .", "we will report these results in our next paper and further discuss the mechanism involved . in summary ,", "it suggested that with specific knockout of cpt1b in skeletal muscles , although there were lipids accumulations in skeletal muscles , the insulin sensitivity still remains ."]]}
{"article_id": "PMC4976566", "article_text": ["pelvic organ prolapse ( pop ) is a major healthcare problem in middle - aged and elderly women and is usually accompanied by pelvic floor dysfunction ( pfd ) .", "the introduction of mesh repair in vaginal prolapse surgery showed possible advantageous results in early reports .", "however , pelvic floor structures progressively weaken in the elderly women , contributing to the high rate of recurrence .", "it has been reported that more than 30% of patients undergoing prolapse repair need reoperation .", "recent announcements from the us food and drug administration ( fda ) describe increasing concern for complications after transvaginal mesh ( tvm ) surgery .", "the risk factors for mesh exposure have been explored for decades , but it remains unclear what leads to the mesh exposure and operation failure .", "we analyzed clinical data to identify possible risk factors for mesh exposure to reduce the tvm complication rate and to improve patients quality of life .", "a retrospective study of patients undergoing tvm surgery between january 2004 and december 2012 was conducted in the department of obstetrics and gynecology at the first affiliated hospital of chinese pla general hospital .", "the operative methods included prosima anterior leaf fixation , prolift anterior leaf fixation , small mesh fixation , prosima posterior leaf fixation , prolift total pelvic fixation , tension - free vaginal tape ( tvt ) , and tvt - obturator .", "clinical data were collected including age , menopause status , body mass index ( bmi ) , gravidity and parity , stage of the pop , tvm types , concomitant procedures , operation time , blood loss , postoperative morbidity , and postoperative mesh exposure . only patients with at least 3 months of follow - up time", "the study was approved by the institutional review board of first affiliated hospital of chinese pla general hospital .", "quantitative data were expressed as mean  standard deviation ( minimum , maximum ) while categorical data were expressed as absolute number and rate .", "statistical analysis was performed to analyze the risk factors for mesh exposure using the spss 10.0 software ( spss inc .", "independent sample 's t - test was used for comparisons between the two groups while binary logistic regression was adopted to explore the risk factors for mesh exposure .", "a retrospective study of patients undergoing tvm surgery between january 2004 and december 2012 was conducted in the department of obstetrics and gynecology at the first affiliated hospital of chinese pla general hospital .", "the operative methods included prosima anterior leaf fixation , prolift anterior leaf fixation , small mesh fixation , prosima posterior leaf fixation , prolift total pelvic fixation , tension - free vaginal tape ( tvt ) , and tvt - obturator .", "clinical data were collected including age , menopause status , body mass index ( bmi ) , gravidity and parity , stage of the pop , tvm types , concomitant procedures , operation time , blood loss , postoperative morbidity , and postoperative mesh exposure . only patients with at least 3 months of follow - up time", "the study was approved by the institutional review board of first affiliated hospital of chinese pla general hospital .", "quantitative data were expressed as mean  standard deviation ( minimum , maximum ) while categorical data were expressed as absolute number and rate . statistical analysis was performed to analyze the risk factors for mesh exposure using the spss 10.0 software ( spss inc .", "independent sample 's t - test was used for comparisons between the two groups while binary logistic regression was adopted to explore the risk factors for mesh exposure . a value of p < 0.05 was considered statistically significant .", "data from 218 patients who had received reconstruction pelvic surgery ( rps ) due to pfd from january 2004 to december 2012 data were collected .", "a total of 195 ( 89.4% ) subjects were enrolled and 150 ( 68.8% ) subjects were followed up for more than 2 years .", "the median follow - up time was 35.1  23.6 months ( range : 2.0104.0 months ) .", "the average age was 63.9  11.2 years ( 43.089.0 years , mean age 65.0 years ) .", "the median duration of menopause was 14.8  10.7 years ( range : 035.0 years ) .", "the average gravidity was 3.6  1.6 times ( range : 111 times ) and the average parity was 2.6  1.5 times ( range : 19 times ) .", "seven patients had a history of pelvic surgery , including 3 with hysterectomy plus anterior wall colporrhaphy , 1 with posterior wall colporrhaphy , and 1 with hysterectomy .", "a total of 195 patients underwent transvaginal rps with polypropylene ( pp ) mesh ; 180 ( 92.3% ) received anterior prolapse surgery , 1 ( 0.5% ) received posterior prolapse surgery , and 14 ( 7.2% ) received combined anterior and posterior prolapse surgery .", "a total of 209 transvaginal pp meshes were placed ; 194 in the anterior wall and 15 in the posterior wall .", "concomitantly , transvaginal hysterectomy was performed in 189 ( 96.9% ) cases , high uterosacral ligament suspension in 176 ( 90.2% ) , sacrospinous ligament fixation in 5 ( 2.5% ) , posterior wall colporrhaphy in 130 ( 66.7% ) , perineal repair in 160 ( 82.1% ) , and tvt in 61 ( 31.3% ) .", "the mean operative time was 2.4  1.1 h ( range : 1.04.0 h ) .", "the mean body temperature in the first 3 days after the operation was 37.1  0.3c ( range : 36.838.2c ) .", "the average follow - up time was 35.1  23.6 months ( range : 2104 months ) .", "a successful operation was defined as stage 2 or less by pop quantification ( pop - q ) examination .", "all other patients were found by pop - q to be stage 2 or less up to half a year after the surgery .", "the objective success rate was 98.9% , and no patient was reoperated because of recurrence .", "mesh exposure was defined by criteria established by the international urogynecological association and the international continence society [ figure 1a and 1b ] .", "mesh exposure was found in 32 patients , whose age ranged from 43 to 81 years .", "the mean exposure diameter was 0.6  0.3 cm ( range : 0.31.5 cm ) [ table 1 ] .", "the management of vaginal mesh exposure included regular observation , local application of estrogen ointment and metronidazole suppositories , and removal of exposed mesh .", "most cases gradually healed in half a year . no mesh exposure progression was detected .", "there were eight patients who were admitted for removal of exposed mesh and after mesh resection the vaginal mucosa healed completely .", "( a and b ) mesh exposure was found in the follow - up checkup , as the black arrows show .", "demography of patients with mesh exposure ( n = 32 ) bmi : body mass index ; ci : confidence interval ; sd : standard deviation .", "thirty - two patients with mesh exposure were recruited into the exposed group and the other 163 patients were recruited into the nonexposed group . as shown in table 2 , the median age , the operative time , and the blood loss were comparable in the nonexposed and exposed groups .", "statistical analysis revealed no significant difference for menopausal status , gravidity and parity , bmi , blood loss , age , or postoperative morbidity .", "however , the number of concomitant procedures in the nonexposed group was significantly lower than those in the exposed group .", "sslf : sacrospinous ligament fixation ; hus : high uterosacral ligament suspension ; tvt : tension - free vaginal tape ; tvt - o : tension - free vaginal tape obturator technique ; pop - q : pelvic organ prolapse quantification .", "logistic regression analysis of the risk factors for mesh exposure was performed in 195 patients [ table 3 ] .", "risk factors for mesh exposure in our analysis included operative duration and the concomitant procedures ( p = 0.043 , p = 0.001 ) .", "the relative risk was 1.899 and 0.376 , respectively , for operative time and number of operations .", "other factors such as age , menopausal status , gravidity , parity , bmi , and blood loss were not related to mesh exposure .", "risk factors for mesh exposure bmi : body mass index ; ci : confidence interval ; or : odds ratio .", "data from 218 patients who had received reconstruction pelvic surgery ( rps ) due to pfd from january 2004 to december 2012 data were collected .", "a total of 195 ( 89.4% ) subjects were enrolled and 150 ( 68.8% ) subjects were followed up for more than 2 years .", "the median follow - up time was 35.1  23.6 months ( range : 2.0104.0 months ) .", "the average age was 63.9  11.2 years ( 43.089.0 years , mean age 65.0 years ) .", "the median duration of menopause was 14.8  10.7 years ( range : 035.0 years ) .", "the average gravidity was 3.6  1.6 times ( range : 111 times ) and the average parity was 2.6  1.5 times ( range : 19 times ) .", "seven patients had a history of pelvic surgery , including 3 with hysterectomy plus anterior wall colporrhaphy , 1 with posterior wall colporrhaphy , and 1 with hysterectomy .", "a total of 195 patients underwent transvaginal rps with polypropylene ( pp ) mesh ; 180 ( 92.3% ) received anterior prolapse surgery , 1 ( 0.5% ) received posterior prolapse surgery , and 14 ( 7.2% ) received combined anterior and posterior prolapse surgery .", "a total of 209 transvaginal pp meshes were placed ; 194 in the anterior wall and 15 in the posterior wall .", "concomitantly , transvaginal hysterectomy was performed in 189 ( 96.9% ) cases , high uterosacral ligament suspension in 176 ( 90.2% ) , sacrospinous ligament fixation in 5 ( 2.5% ) , posterior wall colporrhaphy in 130 ( 66.7% ) , perineal repair in 160 ( 82.1% ) , and tvt in 61 ( 31.3% ) .", "the mean operative time was 2.4  1.1 h ( range : 1.04.0 h ) .", "the mean body temperature in the first 3 days after the operation was 37.1  0.3c ( range : 36.838.2c ) .", "the average follow - up time was 35.1  23.6 months ( range : 2104 months ) .", "a successful operation was defined as stage 2 or less by pop quantification ( pop - q ) examination .", "all other patients were found by pop - q to be stage 2 or less up to half a year after the surgery .", "the objective success rate was 98.9% , and no patient was reoperated because of recurrence .", "mesh exposure was defined by criteria established by the international urogynecological association and the international continence society [ figure 1a and 1b ] .", "mesh exposure was found in 32 patients , whose age ranged from 43 to 81 years .", "the mean exposure diameter was 0.6  0.3 cm ( range : 0.31.5 cm ) [ table 1 ] .", "the management of vaginal mesh exposure included regular observation , local application of estrogen ointment and metronidazole suppositories , and removal of exposed mesh .", "most cases gradually healed in half a year . no mesh exposure progression was detected .", "there were eight patients who were admitted for removal of exposed mesh and after mesh resection the vaginal mucosa healed completely .", "( a and b ) mesh exposure was found in the follow - up checkup , as the black arrows show .", "demography of patients with mesh exposure ( n = 32 ) bmi : body mass index ; ci : confidence interval ; sd : standard deviation .", "thirty - two patients with mesh exposure were recruited into the exposed group and the other 163 patients were recruited into the nonexposed group . as shown in table 2 , the median age , the operative time , and the blood loss were comparable in the nonexposed and exposed groups .", "statistical analysis revealed no significant difference for menopausal status , gravidity and parity , bmi , blood loss , age , or postoperative morbidity .", "however , the number of concomitant procedures in the nonexposed group was significantly lower than those in the exposed group .", "sslf : sacrospinous ligament fixation ; hus : high uterosacral ligament suspension ; tvt : tension - free vaginal tape ; tvt - o : tension - free vaginal tape obturator technique ; pop - q : pelvic organ prolapse quantification .", "logistic regression analysis of the risk factors for mesh exposure was performed in 195 patients [ table 3 ] .", "risk factors for mesh exposure in our analysis included operative duration and the concomitant procedures ( p = 0.043 , p = 0.001 ) .", "the relative risk was 1.899 and 0.376 , respectively , for operative time and number of operations .", "other factors such as age , menopausal status , gravidity , parity , bmi , and blood loss were not related to mesh exposure .", "risk factors for mesh exposure bmi : body mass index ; ci : confidence interval ; or : odds ratio .", "epidemiological studies have shown that 29.2% of the pop patients need to be reoperated after traditional operation , especially in advanced pop .", "the synthetic pp mesh has been used more widely in pelvic reconstructive surgery for the past 1020 years .", "however , the related complications of pp mesh have been reported occasionally and some of them are serious and life - threatening .", ", falagas reported that the incidence of mesh exposure rate ranged from 0% to 33% .", "recently , a meta - analysis of 110 studies comprising 11,785 patients reported 10.3% overall rate of mesh exposure . in this study", ", we have found a higher mesh exposure rate in tvm of 15.8% . in this study , we identified risk factors for pp mesh exposure .", "we analyzed the onset of the exposure and found that 59% occurred within 1 year after the operation and 41% beyond 1 year .", "it was also found that 72.7% ( 24/33 ) of mesh exposure occurred within half a year . among them , 39.4% ( 13/33 ) happened within 2 months .", "one year after the operation the onset of exposure was rather low ( 1/31 ) . according to the literature , mesh exposure is more likely to occur in patients older than 70 years due to thinner vaginal mucosa caused by the low estrogen level . in 2005 ,", "achtari et al . reported that patient age was a risk factor for mesh exposure .", "kim et al . reported that the rate of mesh erosion was not related to patient age when comparing a group > 70 years of age to a group < 70 .", "in two recent retrospective cohort studies , which included patients over 80 with pop , no mesh exposure was found after the operation . in our study , mesh exposure was found to occur mainly in patients between 50 and 75 years old .", "the subsequent regression analysis has also not revealed any effect of age for mesh exposure .", "first , we tried to reserve the vaginal mucosa as much as possible during our operation .", "the number of concomitant operation procedures is another contributing factor for mesh exposure . in 2004 , thompson et al .", ". believed that risk factors for mesh exposure were concomitant hysterectomy and inverted t colpotomy while ganj et al . thought the most important factor was the length of the incisions in vagina mucosa and the tension on the incision line", "controversially , stepanian et al . found that concomitant hysterectomy would not increase the risk of mesh exposure . in our experience ,", "exposure was significantly higher in patients who received posterior vaginal wall colporrhaphy , perineorrhaphy , and vaginal tape .", "binary logistic regression analysis showed that the number of concomitant procedures and operative time were risk factors for mesh exposure .", "as the number of concomitant procedures increased , the rate of mesh exposure increased , possibly due to a longer operative time . in this study , we used 3 different types of meshes .", "prosima was smaller and can be adjusted by clipping but is not as evenly placed as prolift .", "ventricular septal defect is maintained for 28 days after prosima placement , which may arouse a local inflammation reaction .", "our results showed that prosima , prolift , and mesh had an exposure rate of 19.1% , 15.9% , and 12.3% , respectively .", "although prosima showed a higher exposure occurrence , statistical analysis did not reveal any difference among them , indicating they share similar exposure hazards .", "it was reported that surgeon 's experience is also related with mesh exposure . in our hospital ,", "the rate of mesh exposure was 14.6% in the first 5 years and 17% in the following 3 years .", "this indicated that the surgeon 's experience might not be a strong protective factor for mesh exposure .", "in addition , it was found that the exposure rate of vaginal tape was only 1.6% , which indicated vaginal tape was safer and less likely to have mesh exposure .", "the warning from us fda regarding tvm indicates improper use of mesh may cause serious safety problems .", "candidates should be carefully chosen , usually patients with advanced pop . furthermore , alternative nonsurgical treatment or autologous tissue reconstructive surgery should be explored .", "access system should be implemented to select eligible surgeons , and a standardized management system should be established for pelvic surgery . in general", "although exposure may occur after surgery , exposure rate was low and easy to manage .", "epidemiological studies have shown that 29.2% of the pop patients need to be reoperated after traditional operation , especially in advanced pop .", "the synthetic pp mesh has been used more widely in pelvic reconstructive surgery for the past 1020 years .", "however , the related complications of pp mesh have been reported occasionally and some of them are serious and life - threatening .", ", falagas reported that the incidence of mesh exposure rate ranged from 0% to 33% .", "recently , a meta - analysis of 110 studies comprising 11,785 patients reported 10.3% overall rate of mesh exposure . in this study", ", we have found a higher mesh exposure rate in tvm of 15.8% . in this study , we identified risk factors for pp mesh exposure .", "we analyzed the onset of the exposure and found that 59% occurred within 1 year after the operation and 41% beyond 1 year .", "it was also found that 72.7% ( 24/33 ) of mesh exposure occurred within half a year . among them , 39.4% ( 13/33 ) happened within 2 months .", "one year after the operation the onset of exposure was rather low ( 1/31 ) .", "according to the literature , mesh exposure is more likely to occur in patients older than 70 years due to thinner vaginal mucosa caused by the low estrogen level . in 2005 ,", "achtari et al . reported that patient age was a risk factor for mesh exposure .", "kim et al . reported that the rate of mesh erosion was not related to patient age when comparing a group > 70 years of age to a group < 70 .", "in two recent retrospective cohort studies , which included patients over 80 with pop , no mesh exposure was found after the operation . in our study , mesh exposure was found to occur mainly in patients between 50 and 75 years old .", "the subsequent regression analysis has also not revealed any effect of age for mesh exposure .", "first , we tried to reserve the vaginal mucosa as much as possible during our operation .", "the number of concomitant operation procedures is another contributing factor for mesh exposure . in 2004 , thompson et al .", "believed that risk factors for mesh exposure were concomitant hysterectomy and inverted t colpotomy while ganj et al . thought the most important factor was the length of the incisions in vagina mucosa and the tension on the incision line", "controversially , stepanian et al . found that concomitant hysterectomy would not increase the risk of mesh exposure . in our experience ,", "exposure was significantly higher in patients who received posterior vaginal wall colporrhaphy , perineorrhaphy , and vaginal tape .", "binary logistic regression analysis showed that the number of concomitant procedures and operative time were risk factors for mesh exposure .", "as the number of concomitant procedures increased , the rate of mesh exposure increased , possibly due to a longer operative time . in this study , we used 3 different types of meshes .", "prosima was smaller and can be adjusted by clipping but is not as evenly placed as prolift .", "ventricular septal defect is maintained for 28 days after prosima placement , which may arouse a local inflammation reaction .", "our results showed that prosima , prolift , and mesh had an exposure rate of 19.1% , 15.9% , and 12.3% , respectively .", "although prosima showed a higher exposure occurrence , statistical analysis did not reveal any difference among them , indicating they share similar exposure hazards .", "it was reported that surgeon 's experience is also related with mesh exposure . in our hospital ,", "the rate of mesh exposure was 14.6% in the first 5 years and 17% in the following 3 years .", "this indicated that the surgeon 's experience might not be a strong protective factor for mesh exposure .", "in addition , it was found that the exposure rate of vaginal tape was only 1.6% , which indicated vaginal tape was safer and less likely to have mesh exposure .", "the warning from us fda regarding tvm indicates improper use of mesh may cause serious safety problems .", "candidates should be carefully chosen , usually patients with advanced pop . furthermore , alternative nonsurgical treatment or autologous tissue reconstructive surgery should be explored .", "access system should be implemented to select eligible surgeons , and a standardized management system should be established for pelvic surgery . in general , our results showed that tvm surgery is beneficial for patients with advanced pop . although exposure may occur after surgery , exposure rate was low and easy to manage ."], "abstract_text": ["<S> background : mesh exposure after surgery continues to be a clinical challenge for urogynecological surgeons . </S>", "<S> the purpose of this study was to explore the risk factors for polypropylene ( pp ) mesh exposure after transvaginal mesh ( tvm ) surgery.methods:this study included 195 patients with advanced pelvic organ prolapse ( pop ) , who underwent tvm from january 2004 to december 2012 at the first affiliated hospital of chinese pla general hospital . </S>", "<S> clinical data were evaluated including patient 's demography , tvm type , concomitant procedures , operation time , blood loss , postoperative morbidity , and mesh exposure . </S>", "<S> mesh exposure was identified through postoperative vaginal examination . </S>", "<S> statistical analysis was performed to identify risk factors for mesh exposure.results:two-hundred and nine transvaginal pp meshes were placed , including 194 in the anterior wall and 15 in the posterior wall . </S>", "<S> concomitant tension - free vaginal tape was performed in 61 cases . </S>", "<S> the mean follow - up time was 35.1  23.6 months . </S>", "<S> pp mesh exposure was identified in 32 cases ( 16.4% ) , with 31 in the anterior wall and 1 in the posterior wall . </S>", "<S> significant difference was found in operating time and concomitant procedures between exposed and nonexposed groups ( f = 7.443 , p = 0.007 ; f = 4.307 , p = 0.039 , respectively ) . </S>", "<S> binary logistic regression revealed that the number of concomitant procedures and operation time were risk factors for mesh exposure ( p = 0.001 , p = 0.043).conclusion : concomitant procedures and increased operating time increase the risk for postoperative mesh exposure in patients undergoing tvm surgery for pop . </S>"], "labels": null, "section_names": ["I", "M", "Research design and trial population", "Statistical analysis", "R", "Patient characteristics", "Surgery", "Postoperative follow-up", "Comparison of risks factors between the two groups", "Logistic regression analysis of the risk factors for mesh exposure", "D", "Mesh exposure rate after RPS with transvaginal mesh", "Age and mesh exposure", "Operation type, concomitant procedure, and mesh exposure", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["pelvic organ prolapse ( pop ) is a major healthcare problem in middle - aged and elderly women and is usually accompanied by pelvic floor dysfunction ( pfd ) .", "the introduction of mesh repair in vaginal prolapse surgery showed possible advantageous results in early reports .", "however , pelvic floor structures progressively weaken in the elderly women , contributing to the high rate of recurrence .", "it has been reported that more than 30% of patients undergoing prolapse repair need reoperation .", "recent announcements from the us food and drug administration ( fda ) describe increasing concern for complications after transvaginal mesh ( tvm ) surgery .", "the risk factors for mesh exposure have been explored for decades , but it remains unclear what leads to the mesh exposure and operation failure .", "we analyzed clinical data to identify possible risk factors for mesh exposure to reduce the tvm complication rate and to improve patients quality of life ."], ["a retrospective study of patients undergoing tvm surgery between january 2004 and december 2012 was conducted in the department of obstetrics and gynecology at the first affiliated hospital of chinese pla general hospital .", "the operative methods included prosima anterior leaf fixation , prolift anterior leaf fixation , small mesh fixation , prosima posterior leaf fixation , prolift total pelvic fixation , tension - free vaginal tape ( tvt ) , and tvt - obturator .", "clinical data were collected including age , menopause status , body mass index ( bmi ) , gravidity and parity , stage of the pop , tvm types , concomitant procedures , operation time , blood loss , postoperative morbidity , and postoperative mesh exposure . only patients with at least 3 months of follow - up time", "the study was approved by the institutional review board of first affiliated hospital of chinese pla general hospital .", "quantitative data were expressed as mean  standard deviation ( minimum , maximum ) while categorical data were expressed as absolute number and rate .", "statistical analysis was performed to analyze the risk factors for mesh exposure using the spss 10.0 software ( spss inc .", "independent sample 's t - test was used for comparisons between the two groups while binary logistic regression was adopted to explore the risk factors for mesh exposure ."], ["a retrospective study of patients undergoing tvm surgery between january 2004 and december 2012 was conducted in the department of obstetrics and gynecology at the first affiliated hospital of chinese pla general hospital .", "the operative methods included prosima anterior leaf fixation , prolift anterior leaf fixation , small mesh fixation , prosima posterior leaf fixation , prolift total pelvic fixation , tension - free vaginal tape ( tvt ) , and tvt - obturator .", "clinical data were collected including age , menopause status , body mass index ( bmi ) , gravidity and parity , stage of the pop , tvm types , concomitant procedures , operation time , blood loss , postoperative morbidity , and postoperative mesh exposure . only patients with at least 3 months of follow - up time", "the study was approved by the institutional review board of first affiliated hospital of chinese pla general hospital ."], ["quantitative data were expressed as mean  standard deviation ( minimum , maximum ) while categorical data were expressed as absolute number and rate . statistical analysis was performed to analyze the risk factors for mesh exposure using the spss 10.0 software ( spss inc .", "independent sample 's t - test was used for comparisons between the two groups while binary logistic regression was adopted to explore the risk factors for mesh exposure . a value of p < 0.05 was considered statistically significant ."], ["data from 218 patients who had received reconstruction pelvic surgery ( rps ) due to pfd from january 2004 to december 2012 data were collected .", "a total of 195 ( 89.4% ) subjects were enrolled and 150 ( 68.8% ) subjects were followed up for more than 2 years .", "the median follow - up time was 35.1  23.6 months ( range : 2.0104.0 months ) .", "the average age was 63.9  11.2 years ( 43.089.0 years , mean age 65.0 years ) .", "the median duration of menopause was 14.8  10.7 years ( range : 035.0 years ) .", "the average gravidity was 3.6  1.6 times ( range : 111 times ) and the average parity was 2.6  1.5 times ( range : 19 times ) .", "seven patients had a history of pelvic surgery , including 3 with hysterectomy plus anterior wall colporrhaphy , 1 with posterior wall colporrhaphy , and 1 with hysterectomy .", "a total of 195 patients underwent transvaginal rps with polypropylene ( pp ) mesh ; 180 ( 92.3% ) received anterior prolapse surgery , 1 ( 0.5% ) received posterior prolapse surgery , and 14 ( 7.2% ) received combined anterior and posterior prolapse surgery .", "a total of 209 transvaginal pp meshes were placed ; 194 in the anterior wall and 15 in the posterior wall .", "concomitantly , transvaginal hysterectomy was performed in 189 ( 96.9% ) cases , high uterosacral ligament suspension in 176 ( 90.2% ) , sacrospinous ligament fixation in 5 ( 2.5% ) , posterior wall colporrhaphy in 130 ( 66.7% ) , perineal repair in 160 ( 82.1% ) , and tvt in 61 ( 31.3% ) .", "the mean operative time was 2.4  1.1 h ( range : 1.04.0 h ) .", "the mean body temperature in the first 3 days after the operation was 37.1  0.3c ( range : 36.838.2c ) .", "the average follow - up time was 35.1  23.6 months ( range : 2104 months ) .", "a successful operation was defined as stage 2 or less by pop quantification ( pop - q ) examination .", "all other patients were found by pop - q to be stage 2 or less up to half a year after the surgery .", "the objective success rate was 98.9% , and no patient was reoperated because of recurrence .", "mesh exposure was defined by criteria established by the international urogynecological association and the international continence society [ figure 1a and 1b ] .", "mesh exposure was found in 32 patients , whose age ranged from 43 to 81 years .", "the mean exposure diameter was 0.6  0.3 cm ( range : 0.31.5 cm ) [ table 1 ] .", "the management of vaginal mesh exposure included regular observation , local application of estrogen ointment and metronidazole suppositories , and removal of exposed mesh .", "most cases gradually healed in half a year . no mesh exposure progression was detected .", "there were eight patients who were admitted for removal of exposed mesh and after mesh resection the vaginal mucosa healed completely .", "( a and b ) mesh exposure was found in the follow - up checkup , as the black arrows show .", "demography of patients with mesh exposure ( n = 32 ) bmi : body mass index ; ci : confidence interval ; sd : standard deviation .", "thirty - two patients with mesh exposure were recruited into the exposed group and the other 163 patients were recruited into the nonexposed group . as shown in table 2 , the median age , the operative time , and the blood loss were comparable in the nonexposed and exposed groups .", "statistical analysis revealed no significant difference for menopausal status , gravidity and parity , bmi , blood loss , age , or postoperative morbidity .", "however , the number of concomitant procedures in the nonexposed group was significantly lower than those in the exposed group .", "sslf : sacrospinous ligament fixation ; hus : high uterosacral ligament suspension ; tvt : tension - free vaginal tape ; tvt - o : tension - free vaginal tape obturator technique ; pop - q : pelvic organ prolapse quantification .", "logistic regression analysis of the risk factors for mesh exposure was performed in 195 patients [ table 3 ] .", "risk factors for mesh exposure in our analysis included operative duration and the concomitant procedures ( p = 0.043 , p = 0.001 ) .", "the relative risk was 1.899 and 0.376 , respectively , for operative time and number of operations .", "other factors such as age , menopausal status , gravidity , parity , bmi , and blood loss were not related to mesh exposure .", "risk factors for mesh exposure bmi : body mass index ; ci : confidence interval ; or : odds ratio ."], ["data from 218 patients who had received reconstruction pelvic surgery ( rps ) due to pfd from january 2004 to december 2012 data were collected .", "a total of 195 ( 89.4% ) subjects were enrolled and 150 ( 68.8% ) subjects were followed up for more than 2 years .", "the median follow - up time was 35.1  23.6 months ( range : 2.0104.0 months ) .", "the average age was 63.9  11.2 years ( 43.089.0 years , mean age 65.0 years ) .", "the median duration of menopause was 14.8  10.7 years ( range : 035.0 years ) .", "the average gravidity was 3.6  1.6 times ( range : 111 times ) and the average parity was 2.6  1.5 times ( range : 19 times ) .", "seven patients had a history of pelvic surgery , including 3 with hysterectomy plus anterior wall colporrhaphy , 1 with posterior wall colporrhaphy , and 1 with hysterectomy ."], ["a total of 195 patients underwent transvaginal rps with polypropylene ( pp ) mesh ; 180 ( 92.3% ) received anterior prolapse surgery , 1 ( 0.5% ) received posterior prolapse surgery , and 14 ( 7.2% ) received combined anterior and posterior prolapse surgery .", "a total of 209 transvaginal pp meshes were placed ; 194 in the anterior wall and 15 in the posterior wall .", "concomitantly , transvaginal hysterectomy was performed in 189 ( 96.9% ) cases , high uterosacral ligament suspension in 176 ( 90.2% ) , sacrospinous ligament fixation in 5 ( 2.5% ) , posterior wall colporrhaphy in 130 ( 66.7% ) , perineal repair in 160 ( 82.1% ) , and tvt in 61 ( 31.3% ) .", "the mean operative time was 2.4  1.1 h ( range : 1.04.0 h ) .", "the mean body temperature in the first 3 days after the operation was 37.1  0.3c ( range : 36.838.2c ) ."], ["the average follow - up time was 35.1  23.6 months ( range : 2104 months ) .", "a successful operation was defined as stage 2 or less by pop quantification ( pop - q ) examination .", "all other patients were found by pop - q to be stage 2 or less up to half a year after the surgery .", "the objective success rate was 98.9% , and no patient was reoperated because of recurrence .", "mesh exposure was defined by criteria established by the international urogynecological association and the international continence society [ figure 1a and 1b ] .", "mesh exposure was found in 32 patients , whose age ranged from 43 to 81 years .", "the mean exposure diameter was 0.6  0.3 cm ( range : 0.31.5 cm ) [ table 1 ] .", "the management of vaginal mesh exposure included regular observation , local application of estrogen ointment and metronidazole suppositories , and removal of exposed mesh .", "most cases gradually healed in half a year . no mesh exposure progression was detected .", "there were eight patients who were admitted for removal of exposed mesh and after mesh resection the vaginal mucosa healed completely .", "( a and b ) mesh exposure was found in the follow - up checkup , as the black arrows show .", "demography of patients with mesh exposure ( n = 32 ) bmi : body mass index ; ci : confidence interval ; sd : standard deviation ."], ["thirty - two patients with mesh exposure were recruited into the exposed group and the other 163 patients were recruited into the nonexposed group . as shown in table 2 , the median age , the operative time , and the blood loss were comparable in the nonexposed and exposed groups .", "statistical analysis revealed no significant difference for menopausal status , gravidity and parity , bmi , blood loss , age , or postoperative morbidity .", "however , the number of concomitant procedures in the nonexposed group was significantly lower than those in the exposed group .", "sslf : sacrospinous ligament fixation ; hus : high uterosacral ligament suspension ; tvt : tension - free vaginal tape ; tvt - o : tension - free vaginal tape obturator technique ; pop - q : pelvic organ prolapse quantification ."], ["logistic regression analysis of the risk factors for mesh exposure was performed in 195 patients [ table 3 ] .", "risk factors for mesh exposure in our analysis included operative duration and the concomitant procedures ( p = 0.043 , p = 0.001 ) .", "the relative risk was 1.899 and 0.376 , respectively , for operative time and number of operations .", "other factors such as age , menopausal status , gravidity , parity , bmi , and blood loss were not related to mesh exposure .", "risk factors for mesh exposure bmi : body mass index ; ci : confidence interval ; or : odds ratio ."], ["epidemiological studies have shown that 29.2% of the pop patients need to be reoperated after traditional operation , especially in advanced pop .", "the synthetic pp mesh has been used more widely in pelvic reconstructive surgery for the past 1020 years .", "however , the related complications of pp mesh have been reported occasionally and some of them are serious and life - threatening .", ", falagas reported that the incidence of mesh exposure rate ranged from 0% to 33% .", "recently , a meta - analysis of 110 studies comprising 11,785 patients reported 10.3% overall rate of mesh exposure . in this study", ", we have found a higher mesh exposure rate in tvm of 15.8% . in this study , we identified risk factors for pp mesh exposure .", "we analyzed the onset of the exposure and found that 59% occurred within 1 year after the operation and 41% beyond 1 year .", "it was also found that 72.7% ( 24/33 ) of mesh exposure occurred within half a year . among them , 39.4% ( 13/33 ) happened within 2 months .", "one year after the operation the onset of exposure was rather low ( 1/31 ) . according to the literature , mesh exposure is more likely to occur in patients older than 70 years due to thinner vaginal mucosa caused by the low estrogen level . in 2005 ,", "achtari et al . reported that patient age was a risk factor for mesh exposure .", "kim et al . reported that the rate of mesh erosion was not related to patient age when comparing a group > 70 years of age to a group < 70 .", "in two recent retrospective cohort studies , which included patients over 80 with pop , no mesh exposure was found after the operation . in our study , mesh exposure was found to occur mainly in patients between 50 and 75 years old .", "the subsequent regression analysis has also not revealed any effect of age for mesh exposure .", "first , we tried to reserve the vaginal mucosa as much as possible during our operation .", "the number of concomitant operation procedures is another contributing factor for mesh exposure . in 2004 , thompson et al .", ". believed that risk factors for mesh exposure were concomitant hysterectomy and inverted t colpotomy while ganj et al . thought the most important factor was the length of the incisions in vagina mucosa and the tension on the incision line", "controversially , stepanian et al . found that concomitant hysterectomy would not increase the risk of mesh exposure . in our experience ,", "exposure was significantly higher in patients who received posterior vaginal wall colporrhaphy , perineorrhaphy , and vaginal tape .", "binary logistic regression analysis showed that the number of concomitant procedures and operative time were risk factors for mesh exposure .", "as the number of concomitant procedures increased , the rate of mesh exposure increased , possibly due to a longer operative time . in this study , we used 3 different types of meshes .", "prosima was smaller and can be adjusted by clipping but is not as evenly placed as prolift .", "ventricular septal defect is maintained for 28 days after prosima placement , which may arouse a local inflammation reaction .", "our results showed that prosima , prolift , and mesh had an exposure rate of 19.1% , 15.9% , and 12.3% , respectively .", "although prosima showed a higher exposure occurrence , statistical analysis did not reveal any difference among them , indicating they share similar exposure hazards .", "it was reported that surgeon 's experience is also related with mesh exposure . in our hospital ,", "the rate of mesh exposure was 14.6% in the first 5 years and 17% in the following 3 years .", "this indicated that the surgeon 's experience might not be a strong protective factor for mesh exposure .", "in addition , it was found that the exposure rate of vaginal tape was only 1.6% , which indicated vaginal tape was safer and less likely to have mesh exposure .", "the warning from us fda regarding tvm indicates improper use of mesh may cause serious safety problems .", "candidates should be carefully chosen , usually patients with advanced pop . furthermore , alternative nonsurgical treatment or autologous tissue reconstructive surgery should be explored .", "access system should be implemented to select eligible surgeons , and a standardized management system should be established for pelvic surgery . in general", "although exposure may occur after surgery , exposure rate was low and easy to manage ."], ["epidemiological studies have shown that 29.2% of the pop patients need to be reoperated after traditional operation , especially in advanced pop .", "the synthetic pp mesh has been used more widely in pelvic reconstructive surgery for the past 1020 years .", "however , the related complications of pp mesh have been reported occasionally and some of them are serious and life - threatening .", ", falagas reported that the incidence of mesh exposure rate ranged from 0% to 33% .", "recently , a meta - analysis of 110 studies comprising 11,785 patients reported 10.3% overall rate of mesh exposure . in this study", ", we have found a higher mesh exposure rate in tvm of 15.8% . in this study , we identified risk factors for pp mesh exposure .", "we analyzed the onset of the exposure and found that 59% occurred within 1 year after the operation and 41% beyond 1 year .", "it was also found that 72.7% ( 24/33 ) of mesh exposure occurred within half a year . among them , 39.4% ( 13/33 ) happened within 2 months .", "one year after the operation the onset of exposure was rather low ( 1/31 ) ."], ["according to the literature , mesh exposure is more likely to occur in patients older than 70 years due to thinner vaginal mucosa caused by the low estrogen level . in 2005 ,", "achtari et al . reported that patient age was a risk factor for mesh exposure .", "kim et al . reported that the rate of mesh erosion was not related to patient age when comparing a group > 70 years of age to a group < 70 .", "in two recent retrospective cohort studies , which included patients over 80 with pop , no mesh exposure was found after the operation . in our study , mesh exposure was found to occur mainly in patients between 50 and 75 years old .", "the subsequent regression analysis has also not revealed any effect of age for mesh exposure .", "first , we tried to reserve the vaginal mucosa as much as possible during our operation ."], ["the number of concomitant operation procedures is another contributing factor for mesh exposure . in 2004 , thompson et al .", "believed that risk factors for mesh exposure were concomitant hysterectomy and inverted t colpotomy while ganj et al . thought the most important factor was the length of the incisions in vagina mucosa and the tension on the incision line", "controversially , stepanian et al . found that concomitant hysterectomy would not increase the risk of mesh exposure . in our experience ,", "exposure was significantly higher in patients who received posterior vaginal wall colporrhaphy , perineorrhaphy , and vaginal tape .", "binary logistic regression analysis showed that the number of concomitant procedures and operative time were risk factors for mesh exposure .", "as the number of concomitant procedures increased , the rate of mesh exposure increased , possibly due to a longer operative time . in this study , we used 3 different types of meshes .", "prosima was smaller and can be adjusted by clipping but is not as evenly placed as prolift .", "ventricular septal defect is maintained for 28 days after prosima placement , which may arouse a local inflammation reaction .", "our results showed that prosima , prolift , and mesh had an exposure rate of 19.1% , 15.9% , and 12.3% , respectively .", "although prosima showed a higher exposure occurrence , statistical analysis did not reveal any difference among them , indicating they share similar exposure hazards .", "it was reported that surgeon 's experience is also related with mesh exposure . in our hospital ,", "the rate of mesh exposure was 14.6% in the first 5 years and 17% in the following 3 years .", "this indicated that the surgeon 's experience might not be a strong protective factor for mesh exposure .", "in addition , it was found that the exposure rate of vaginal tape was only 1.6% , which indicated vaginal tape was safer and less likely to have mesh exposure .", "the warning from us fda regarding tvm indicates improper use of mesh may cause serious safety problems .", "candidates should be carefully chosen , usually patients with advanced pop . furthermore , alternative nonsurgical treatment or autologous tissue reconstructive surgery should be explored .", "access system should be implemented to select eligible surgeons , and a standardized management system should be established for pelvic surgery . in general , our results showed that tvm surgery is beneficial for patients with advanced pop . although exposure may occur after surgery , exposure rate was low and easy to manage ."], [""], [""]]}
{"article_id": "PMC4062987", "article_text": ["5-fluorouracil ( 5-fu ) is a widely used chemotherapeutic drug and cardiac complications are not rare .", "however myocardial infarction , tako - tsubo - like syndrome and cardiogenic shock are also described .", "a 50-year - old man ( weight 70 kg , height 180 cm ) was admitted to a primary care hospital following an episode of syncope . he reported acute orthopnea and an attack of sweating at night accompanied by nausea and vomiting before loss of consciousness for 1 min .", "in the emergency room , patient developed asystole and was immediately and successfully resuscitated for 2 min . afterwards , he was fully oriented without any neurological dysfunction .", "consequently , he was transferred to our heart center for further diagnostic work - up and therapy .", "importantly , patient had no previous history of cardiovascular disease or risk factors , but was diagnosed with colorectal cancer ( ct3 n1 m0 ) and underwent recent neoadjuvant chemotherapy with 5-fu ( 9500 mg by continuous infusion over a period of 120 h ) until 1 day before his acute presentation .", "his physical examination revealed hypotensive blood pressures ( 89/58 mmhg ) and heart rate at rest of 95 bpm .", "the electrocardiogram documented sinus rhythm at 95 bpm with non - significant st - elevation in leads ii , iii , avf .", "high - sensitive troponin t ( 60 ng / l ; upper reference value 14 ng / l ) and creatine kinase - mb fraction ( 0.74 mol / l*s ; upper reference value 0.41 mol / l*s ) were slightly elevated .", "creatinine kinase showed a normal value of 0.93 mol / l*s ( upper reference value 3.17 mol / l*s ) .", "initial transthoracic echocardiography ( tte ) showed a normal sized left ventricle ( left - ventricular end - diastolic diameter : 48 mm , left - ventricular end - diastolic volume : 103 ml ) with global hypokinesia and severely decreased left - ventricular systolic function [ left - ventricular ejection fraction : 16% ; [ figure 1a and b ] .", "transthoracic echocardiography on day 1 ( a ) apical four - chamber view during diastole , ( b ) apical four - chamber view during systole ( there is global hypokinesia with severely impaired thickening of the left ventricular wall in systole ) one day after admission , patient had persistent hypotension with increasing lactate levels as a sign of peripheral malperfusion . on repeat echocardiographic examination , left - ventricular systolic function was found to have further decreased with a left - ventricular ejection fraction of < 10% . due to the progressive cardiogenic shock", ", we decided to treat this condition by the use of an extracorporeal membrane oxygenation ( ecmo ) support by percutaneous implantation .", "on ecmo support , hemodynamic stabilization was evident and medical heart failure treatment including angiotensin - converting enzyme - inhibitor , diuretics and spironolactone was commenced .", "after 4 days with ecmo therapy , left - ventricular ejection fraction improved to 45% and lactate levels were in normal ranges ( < 2", "therefore , ecmo - flow was reduced slowly up to 1.5 l / min . due to the stable lactate levels ( < 2", "the following course was uncomplicated with normalization of blood pressures and no signs of heart failure .", "eight days after admission , he was transferred to the normal ward . left - ventricular ejection fraction was now recovered to a normal value of 60% [ figure 2a and b ] . for confirmation and further differential diagnosis cardiac , magnetic resonance imaging ( mri )", "the mri showed a normal sized left ventricle ( left ventricular end - diastolic volume : 149 ml ; normal value 102 - 235 ml ) with normal ejection fraction of 57% and without any specific wall motion abnormality .", "furthermore , t1-weighted imaging showed pronounced early contrast enhancement , indicating some degree of myocardial hyperemia .", "patient was discharged from hospital in a stable cardiac condition without symptoms of heart failure 11 days after admission .", "transthoracic echocardiography on day 10 ( a ) apical four - chamber view during diastole , ( b ) apical four - chamber view during systole .", "this report describes a patient with no history of cardiovascular disease who experienced acute left - ventricular failure after administration of 5-fu .", "cardiac complications ranging from angina pectoris to cardiogenic shock after chemotherapy with 5-fu are not rare .", "coronary vasospasm , endothelial damage with consequent thrombus formation , increased myocardial oxygen demand , block of myocardial cell metabolism and tako - tsubo - like syndrome with supraphysiologic levels of plasma catecholamines are discussed as potential pathogenetic mechanisms .", "our patient did not report any angina and obstructive coronary lesions were excluded by angiography .", "echocardiography did not reveal an apical ballooning as common in tako - tsubo - like syndrome .", "the cardiac mri of our patient strengthens the concept of an additional temporary myocardial inflammation .", "increased global myocardial edema and evidence of myocardial hyperemia are compatible with a regressive inflammation .", "unfortunately , the mri could not be performed in the acute setting with severely decreased left - ventricular function due to ecmo - therapy and therefore , the extent of myocardial edema and hyperemia might be underestimated .", "treatment of cardiogenic shock is still a therapeutic challenge , especially if coronary artery disease was excluded .", "usually , sympathomimetics are used as first line therapeutics , but several disadvantages in their use have to be taken into account .", "sympathomimetics induced vasoconstriction can lead to perfusion mismatch or even deficit within the microcirculation and metabolic acidosis due to an increased oxygen demand can be observed regularly .", "in addition , some studies give evidence that use of sympathomimetics is directly linked to enhanced systemic inflammatory response due to an increased interleukin-6 expression . in our case with unclear pathogenesis of cardiogenic shock", ", we decided to use a mechanical hemodynamic support ( intraaortic balloon counterpulsation or ecmo ) to avoid above mentioned disadvantages with the use of sympathomimetics , especially with the knowledge about inflammation as a possible cause of 5-fu induced left - ventricular dysfunction .", "recently , intraaortic balloon counterpulsation was shown not to reduce 30-day mortality in patients with cardiogenic shock complicating myocardial infarction . in our case with progressive cardiogenic shock ,", "therefore , we decided to use ecmo - the most potent form of acute cardiorespiratory support , which enables complete relief of cardiac workload and prevents high - dose usage of sympathomimetics .", "cardiogenic shock should be taken into consideration as a possible complication secondary to chemotherapy with 5-fu , even in patients without a history of cardiovascular disease . to the best of our knowledge ,", "this is the first report of an ecmo support in this kind of chemotherapeutic induced cardiogenic shock .", "ecmo support enabled complete relief of cardiac workload and together with medical therapy for heart failure , complete recovery of cardiac function was possible ."], "abstract_text": ["<S> a 50-year - old man with no previous history of cardiovascular disease or risk factors was admitted for syncope and orthopnea . </S>", "<S> importantly , he underwent recent chemotherapy with 5-fluorouracil ( 5-fu ) until 1 day before his acute presentation . in the emergency room , </S>", "<S> patient developed asystole and was successfully resuscitated for 2 min . at coronary angiography , no signs of coronary artery disease were detectable , but transthoracic echocardiography showed a severely decreased left - ventricular systolic function . due to the progressive cardiogenic shock , an extracorporeal membrane oxygenation ( ecmo ) support was used as bridge - to - recovery and to avoid the use of sympathomimetics with their known disadvantages . on ecmo support , hemodynamic stabilization was evident and medical heart failure treatment was commenced . </S>", "<S> left - ventricular function recovered to normal values within a short period of time . </S>", "<S> cardiac complications after chemotherapy with 5-fu are not rare and should be taken into consideration even in acute heart failure with cardiogenic shock . </S>", "<S> ecmo as the most potent form of acute cardiorespiratory support enables complete relief of cardiac workload and therefore recovery of cardiac function . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [["5-fluorouracil ( 5-fu ) is a widely used chemotherapeutic drug and cardiac complications are not rare .", "however myocardial infarction , tako - tsubo - like syndrome and cardiogenic shock are also described ."], ["a 50-year - old man ( weight 70 kg , height 180 cm ) was admitted to a primary care hospital following an episode of syncope . he reported acute orthopnea and an attack of sweating at night accompanied by nausea and vomiting before loss of consciousness for 1 min .", "in the emergency room , patient developed asystole and was immediately and successfully resuscitated for 2 min . afterwards , he was fully oriented without any neurological dysfunction .", "consequently , he was transferred to our heart center for further diagnostic work - up and therapy .", "importantly , patient had no previous history of cardiovascular disease or risk factors , but was diagnosed with colorectal cancer ( ct3 n1 m0 ) and underwent recent neoadjuvant chemotherapy with 5-fu ( 9500 mg by continuous infusion over a period of 120 h ) until 1 day before his acute presentation .", "his physical examination revealed hypotensive blood pressures ( 89/58 mmhg ) and heart rate at rest of 95 bpm .", "the electrocardiogram documented sinus rhythm at 95 bpm with non - significant st - elevation in leads ii , iii , avf .", "high - sensitive troponin t ( 60 ng / l ; upper reference value 14 ng / l ) and creatine kinase - mb fraction ( 0.74 mol / l*s ; upper reference value 0.41 mol / l*s ) were slightly elevated .", "creatinine kinase showed a normal value of 0.93 mol / l*s ( upper reference value 3.17 mol / l*s ) .", "initial transthoracic echocardiography ( tte ) showed a normal sized left ventricle ( left - ventricular end - diastolic diameter : 48 mm , left - ventricular end - diastolic volume : 103 ml ) with global hypokinesia and severely decreased left - ventricular systolic function [ left - ventricular ejection fraction : 16% ; [ figure 1a and b ] .", "transthoracic echocardiography on day 1 ( a ) apical four - chamber view during diastole , ( b ) apical four - chamber view during systole ( there is global hypokinesia with severely impaired thickening of the left ventricular wall in systole ) one day after admission , patient had persistent hypotension with increasing lactate levels as a sign of peripheral malperfusion . on repeat echocardiographic examination , left - ventricular systolic function was found to have further decreased with a left - ventricular ejection fraction of < 10% . due to the progressive cardiogenic shock", ", we decided to treat this condition by the use of an extracorporeal membrane oxygenation ( ecmo ) support by percutaneous implantation .", "on ecmo support , hemodynamic stabilization was evident and medical heart failure treatment including angiotensin - converting enzyme - inhibitor , diuretics and spironolactone was commenced .", "after 4 days with ecmo therapy , left - ventricular ejection fraction improved to 45% and lactate levels were in normal ranges ( < 2", "therefore , ecmo - flow was reduced slowly up to 1.5 l / min . due to the stable lactate levels ( < 2", "the following course was uncomplicated with normalization of blood pressures and no signs of heart failure .", "eight days after admission , he was transferred to the normal ward . left - ventricular ejection fraction was now recovered to a normal value of 60% [ figure 2a and b ] . for confirmation and further differential diagnosis cardiac , magnetic resonance imaging ( mri )", "the mri showed a normal sized left ventricle ( left ventricular end - diastolic volume : 149 ml ; normal value 102 - 235 ml ) with normal ejection fraction of 57% and without any specific wall motion abnormality .", "furthermore , t1-weighted imaging showed pronounced early contrast enhancement , indicating some degree of myocardial hyperemia .", "patient was discharged from hospital in a stable cardiac condition without symptoms of heart failure 11 days after admission .", "transthoracic echocardiography on day 10 ( a ) apical four - chamber view during diastole , ( b ) apical four - chamber view during systole ."], ["this report describes a patient with no history of cardiovascular disease who experienced acute left - ventricular failure after administration of 5-fu .", "cardiac complications ranging from angina pectoris to cardiogenic shock after chemotherapy with 5-fu are not rare .", "coronary vasospasm , endothelial damage with consequent thrombus formation , increased myocardial oxygen demand , block of myocardial cell metabolism and tako - tsubo - like syndrome with supraphysiologic levels of plasma catecholamines are discussed as potential pathogenetic mechanisms .", "our patient did not report any angina and obstructive coronary lesions were excluded by angiography .", "echocardiography did not reveal an apical ballooning as common in tako - tsubo - like syndrome .", "the cardiac mri of our patient strengthens the concept of an additional temporary myocardial inflammation .", "increased global myocardial edema and evidence of myocardial hyperemia are compatible with a regressive inflammation .", "unfortunately , the mri could not be performed in the acute setting with severely decreased left - ventricular function due to ecmo - therapy and therefore , the extent of myocardial edema and hyperemia might be underestimated .", "treatment of cardiogenic shock is still a therapeutic challenge , especially if coronary artery disease was excluded .", "usually , sympathomimetics are used as first line therapeutics , but several disadvantages in their use have to be taken into account .", "sympathomimetics induced vasoconstriction can lead to perfusion mismatch or even deficit within the microcirculation and metabolic acidosis due to an increased oxygen demand can be observed regularly .", "in addition , some studies give evidence that use of sympathomimetics is directly linked to enhanced systemic inflammatory response due to an increased interleukin-6 expression . in our case with unclear pathogenesis of cardiogenic shock", ", we decided to use a mechanical hemodynamic support ( intraaortic balloon counterpulsation or ecmo ) to avoid above mentioned disadvantages with the use of sympathomimetics , especially with the knowledge about inflammation as a possible cause of 5-fu induced left - ventricular dysfunction .", "recently , intraaortic balloon counterpulsation was shown not to reduce 30-day mortality in patients with cardiogenic shock complicating myocardial infarction . in our case with progressive cardiogenic shock ,", "therefore , we decided to use ecmo - the most potent form of acute cardiorespiratory support , which enables complete relief of cardiac workload and prevents high - dose usage of sympathomimetics ."], ["cardiogenic shock should be taken into consideration as a possible complication secondary to chemotherapy with 5-fu , even in patients without a history of cardiovascular disease . to the best of our knowledge ,", "this is the first report of an ecmo support in this kind of chemotherapeutic induced cardiogenic shock .", "ecmo support enabled complete relief of cardiac workload and together with medical therapy for heart failure , complete recovery of cardiac function was possible ."]]}
{"article_id": "PMC3648601", "article_text": ["a summary of methods is provided below and a detailed description of methods is included in the supplementary information .", "the toxin nanosponges were prepared by fusing rbc membrane vesicles on preformed poly(lactic - co - glycolic acid ) ( plga ) nanoparticles through an established extrusion process .", "the size of the nanosponges was obtained from three dynamic light scattering ( dls ) measurements using a malvern zen 3600 zetasizer .", "the morphology of the nanosponges after absorbing toxins was measured by transmission electron microscopy ( tem ) . for preparation of human rbc nanosponges ,", "the rbcs were collected from whole human blood ( bioreclamation ) and the characterization results were shown in fig .", "reconstitution of the lyophilized samples was performed by solubilizing the samples in water and the characterization results were included in fig .", "the in vitro toxin neutralization ability of the nanosponges was examined by mixing 3 g of -toxin with 200 l of 1 mg / ml nanosponges for 30 min , followed by adding into 1.8 ml of 5% purified mouse rbcs .", "the in vitro toxin absorption capacity of the nanosponges was determined through titrating -toxin to a fixed amount of nanosponges .", "the interaction of the nanosponges with cells was examined by a scanning fluorescence microscopy by incubating fluorescent nanosponges and rbc membrane vesicles with human umbilical vein endothelial cells ( huvec ) .", "the in vitro cellular cytotoxicity of nanosponge - sequestered toxins was examined by incubating nanosponges of different concentrations with varied amounts of -toxin , streptolysin - o , and melittin for 30 min , followed by adding to huvecs for 24 hr .", "the in vivo toxin neutralization ability of the nanosponges was tested through subcutaneous injection of the nanosponge / toxin mixture to the flank region of nude mice , followed by histological analyses .", "on - site neutralization of -toxin by the nanosponges was conducted by subcutaneously injecting 50 l of 36 g / ml of -toxin solution , immediately followed by a 100 l injection of 2 mg / ml nanosponges .", "the mice were imaged 3 days later for visualization of skin lesion formation ( fig s11 ) .", "the in vivo detoxification efficacy was tested through intravenous injection of nanosponges before or after administration of a lethal dose of -toxin to icr mice , followed by monitoring the survival rate of the mice .", "for the in vivo hepatotoxicity study , one group of mice was sacrificed on day 3 following the injection of the toxin - bound nanosponges and another group was sacrificed on day 7 ."], "abstract_text": ["<S> detoxification treatments such as toxin - targeted anti - virulence therapy1 , 2 offer ways to cleanse the body of virulence factors that are caused by bacterial infections , venomous injuries , and biological weaponry . because existing detoxification platforms such as antisera3 , monoclonal antibodies4 , small - molecule inhibitors5 , 6 , and molecularly imprinted polymers7 act by targeting the molecular structures of the toxins , customized treatments are required for different diseases . here </S>", "<S> we show a biomimetic toxin nanosponge that functions as a toxin decoy in vivo . </S>", "<S> the nanosponge , which consists of a polymeric nanoparticle core surrounded by red blood cell membranes , absorbs membrane - damaging toxins and diverts them away from their cellular targets . in a mouse model , </S>", "<S> the nanosponges markedly reduce the toxicity of staphylococcal alpha - hemolysin ( -toxin ) and thus improve the survival rate of toxin - challenged mice . </S>", "<S> this biologically inspired toxin nanosponge presents a detoxification treatment that can potentially treat a variety of injuries and diseases caused by pore - forming toxins . </S>"], "labels": null, "section_names": ["Methods", "Supplementary Material"], "sections": [["a summary of methods is provided below and a detailed description of methods is included in the supplementary information .", "the toxin nanosponges were prepared by fusing rbc membrane vesicles on preformed poly(lactic - co - glycolic acid ) ( plga ) nanoparticles through an established extrusion process .", "the size of the nanosponges was obtained from three dynamic light scattering ( dls ) measurements using a malvern zen 3600 zetasizer .", "the morphology of the nanosponges after absorbing toxins was measured by transmission electron microscopy ( tem ) . for preparation of human rbc nanosponges ,", "the rbcs were collected from whole human blood ( bioreclamation ) and the characterization results were shown in fig .", "reconstitution of the lyophilized samples was performed by solubilizing the samples in water and the characterization results were included in fig .", "the in vitro toxin neutralization ability of the nanosponges was examined by mixing 3 g of -toxin with 200 l of 1 mg / ml nanosponges for 30 min , followed by adding into 1.8 ml of 5% purified mouse rbcs .", "the in vitro toxin absorption capacity of the nanosponges was determined through titrating -toxin to a fixed amount of nanosponges .", "the interaction of the nanosponges with cells was examined by a scanning fluorescence microscopy by incubating fluorescent nanosponges and rbc membrane vesicles with human umbilical vein endothelial cells ( huvec ) .", "the in vitro cellular cytotoxicity of nanosponge - sequestered toxins was examined by incubating nanosponges of different concentrations with varied amounts of -toxin , streptolysin - o , and melittin for 30 min , followed by adding to huvecs for 24 hr .", "the in vivo toxin neutralization ability of the nanosponges was tested through subcutaneous injection of the nanosponge / toxin mixture to the flank region of nude mice , followed by histological analyses .", "on - site neutralization of -toxin by the nanosponges was conducted by subcutaneously injecting 50 l of 36 g / ml of -toxin solution , immediately followed by a 100 l injection of 2 mg / ml nanosponges .", "the mice were imaged 3 days later for visualization of skin lesion formation ( fig s11 ) .", "the in vivo detoxification efficacy was tested through intravenous injection of nanosponges before or after administration of a lethal dose of -toxin to icr mice , followed by monitoring the survival rate of the mice .", "for the in vivo hepatotoxicity study , one group of mice was sacrificed on day 3 following the injection of the toxin - bound nanosponges and another group was sacrificed on day 7 ."], [""]]}
{"article_id": "PMC4388973", "article_text": ["hand , foot , and mouth disease ( hfmd ) is a common , acute , and mostly self - limiting enteroviral infection that presents with fever and vesicular lesions on the hands , feet , mouth , and frequently buttocks as characteristic features1,2,3,4 ) .", "although patients with hfmd recover without any sequelae within about 1 week , severe complications occur in a few cases , some of which are life threatening .", "severe complications accompanying hfmd include encephalitis , pulmonary edema / hemorrhage , cardiopulmonary collapse , and myocarditis1,2 ) .", "enterovirus 71 can often lead to a more severe clinical course accompanied by neurologic and cardiopulmonary complications and may rapidly lead to death1,2,3 ) .", "enterovirus 71-induced hfmd developed in korea in 2000 but it was not an epidemic and fatal until 20081,4,5 ) . in the spring of 2009 , a large nationwide outbreak of hfmd caused by enterovirus 71 occurred in korea , and a few cases with encephalitis were fatal1,4,6 ) . a multicenter study in korea performed by the enterovirus complication working group revealed that encephalitis with cardiopulmonary complications of hfmd occurred in 2.4% of patients , including 2 patients ( 1.2% ) who expired1 ) .", "pulmonary hemorrhage in enterovirus 71-infected hfmd patients has been reported in taiwan , china , and malaysia2,7,8,9 ) .", "however , to our knowledge , no cases of hfmd with sudden - onset massive pulmonary hemorrhage have been reported in korea . here", ", we report a fatal case of enterovirus 71-induced hfmd that progressed rapidly with massive pulmonary hemorrhage and convulsion .", "a 12-month - old , previously healthy boy with hfmd was referred to inje university ilsan paik hospital because of 3 episodes febrile convulsions .", "he was born at full term weighing 3.87 kg through normal vaginal delivery by a vietnamese mother .", "he was admitted to a primary hospital with high fever ( up to 39 ) , throat vesicles , and vesicular rashes on his hands and feet for 2 days . on the second hospital day , he had 3 generalized tonic - clonic and atonic convulsions with vertical eyeball deviation within 5 minutes of each other .", "his chest radiography was normal . within 5 hours after admission , he vomited 3 times after meals . besides a body temperature of 38.3 , his other vital signs were stable and he remained conscious .", "desaturation to 76% percutaneous blood oxygen saturation ( spo2 ) and tachycardia to 176 beats / min accompanied by perioral cyanosis occurred suddenly 6 hours after admission .", "his respiration rate was 34 breaths / min , and auscultation disclosed rales on the whole lung field .", "an endotracheal tube was inserted immediately , and a large amount of blood from a pulmonary hemorrhage came out through it .", "comparison of laboratory test results between his initial and pulmonary hemorrhagic status revealed that his hemoglobin decreased from 9.6 to 8.3 mg / dl and his white blood cell count increased from 13,090 to 17,450 cells/l .", "erythrocyte sedimentation rate was slightly increased from 15 to 32 mm / hr , but c - reactive protein level decreased from 1.06 to 0.2 mg / dl .", "prothrombin time ( pt ) and pt international normalized ratio were slightly prolonged ( 16.5 and 1.39 seconds , respectively ) .", "meanwhile , d - dimer ( 2,375.91 ng / ml ) and fibrin degradation product ( 5 - 20 g / ml ) were elevated .", "eight hours after admission , his blood pressure decreased to 82/41 mmhg and heart rate increased to 220 beats / min .", "his body temperature ranged from 38.8 to 40.2. the patient was diagnosed as fatal hfmd with massive pulmonary hemorrhage .", "intravenous immunoglobulin ( ivig , 1 g / kg / dose ) , dexamethasone ( 0.3 mg / kg / day in divided doses every 6 hours ) , and cefotaxime ( 150 mg / kg / day in divided doses every 6 hours ) were administered immediately .", "in addition , leukocyte - depleted red blood cells and fresh frozen plasma were transfused , and intravenous vitamin k and endotracheal epinephrine were administered .", "lumbar puncture was scheduled but was ultimately not performed because of the patient 's unstable vital signs .", "the patient expired 15 hours after admission , and it was within 3 days of fever onset and within 9 hours after intubation .", "we reported it to the korea centers for disease control and prevention ( kcdc ) , sending his urine , serum , nasopharyngeal swab , and endotracheal aspirate .", "enterovirus genome detection was attempted by realtime reverse transcription - polymerase chain reaction ( rt - pcr ) using a taqman system ( applied biosystems , foster city , ca , usa ) . for genotyping ,", "seminested rt - pcr was used to amplify part of the viral protein 1 ( vp1 ) gene of enteroviruses according to the kcdc protocol , and sequencing analysis for the vp1 amplicon was performed by an automatic sequencer and the dnastar software package ( dnastar inc . , madison , wi , usa ) .", "the kcdc isolated enterovirus 71 with subgenotype c4a from the patient 's serum and nasopharyngeal swab .", "bocavirus was also isolated from his nasopharyngeal swab by real - time rt - pcr at our hospital .", "to our knowledge , this is the first reported case of hfmd with sudden - onset massive pulmonary hemorrhage in korea .", "pulmonary edema / hemorrhage is the mysterious hallmark symptom of enteroviral hfmd and can kill a child within 1 day2 ) .", "an epidemic study conducted in taiwan from 1998 to 2005 reported that pulmonary edema / hemorrhage occurred in 43% of patients with severe culture - proven enteroviral hfmd / herpangina2 ) .", "this discrepancy maybe due to different enterovirus virulence or subgenotypes , or differences in susceptibility with respect to ethnicity .", "pulmonary edema / hemorrhage is characterized by respiratory distress , tachypnea , tachycardia , hemoptysis , and rapidly progressing diffuse infiltration or congestion on a chest film2 ) .", "a study on the outbreak in taiwan in 1998 reported that enterovirus 71-infected hfmd patients with pulmonary edema had suddenonset tachypnea , tachycardia , and cyanosis within 1 - 3 days after disease onset , and pulmonary edema led to a rapid death within 12 hours after intubation10 ) .", "the present patient exhibited a change in consciousness , respiratory distress , tachypnea , tachycardia , hemoptysis , rapidly progressing infiltration on a chest film , hypoxemia , and hypotension .", "this patient ultimately expired within 3 days of fever onset and within 9 hours after intubation . in this report , we had applied pulse oxymetry from the beginning , so we could find his desaturation immediately after pulmonary hemorrhage .", "we suggest saturation monitoring for hfmd patients with central nervous system ( cns ) involvements for early detection .", "it may be caused by increased pulmonary vascular permeability as a result of either brainstem encephalitis or a systemic inflammatory response caused by excessive cytokine release2,6 ) .", "cns involvement of enterovirus 71-induced hfmd may also trigger a sympathetic storm , resulting in vasoconstriction with high systemic vascular resistance , passive pulmonary volume loading , and pulmonary edema / hemorrhage8 ) .", "the present patient had several episodes of convulsions , lethargy , and loss of consciousness .", "therefore , we suspected encephalitis , although we were unable to perform a cerebrospinal fluid study because of his unstable vital signs . his suspected encephalitis may be a possible cause of the pulmonary hemorrhage . however , pulmonary edema / hemorrhage without cns involvement occurred in 11% of patients with severe culture - proven enteroviral hfmd / herpangina in an epidemic study in taiwan from 1998 to 20052 ) .", "another possible explanation for the pathogenesis is coinfection with a second virus2 ) , as bocavirus coinfection was detected in the patient .", "however , the presence of both viruses would not result in increased mortality due to hfmd , according to a previous study2 ) . a multicenter study performed by the enterovirus complication working group investigating enterovirus 71-induced hfmd in korea in 2009 reported that an hfmd rash pattern , fever longer than 4 days , peak body temperature > 39 , vomiting , headache , neurologic signs , leukocytosis , serum glucose > 100 mg / dl , and isolated enterovirus 71 may be indicative of poor prognosis during hfmd epidemic periods1,10 ) .", "in addition , young age at disease onset and a history of lethargy are associated with an increased risk of severe hfmd11 ) .", "the present patient was young and presented with hfmd rashes and vesicles , fever up to 39 , vomiting , lethargy , and convulsions as neurologic signs as well as leukocytosis , high serum glucose level , and isolated enterovirus 71 as poor prognostic factors .", "hfmd patients are very frequently approximately 1 year of age , and deaths were reported to occur at around 1 year of age in other studies12,13,14,15 ) . in the 2009 outbreak ,", "2 patients around 1 year of age expired , and the present patient was a 12-month - old baby .", "the gold standard for the diagnosis of enteroviral infection is viral culture and isolation in 2 or more specimens of cerebrospinal fluid , blood , nasopharyngeal swab or secretion , vesicular fluid , or stool to increased diagnostic sensitivity1,4,6,11 ) . in the present report ,", "enterovirus 71 was isolated from both the patient 's serum and nasopharyngeal swab . in the 2009 korean outbreak ,", "the subgenotype of enterovirus 71 was c4a , which was prevalent in china in 2008 . in the present case , the subgenotype of enterovirus 71 was also c4a .", "the administration of high - dose ivig is recommended for severe hfmd as optional treatment , because ivig therapy was effective in many reports4,5,6,7,11 ) .", "ivig neutralizes the virus and has nonspecific antiinflammatory properties11 ) ; this would significantly reduce mortality by attenuating sympathetic activity and cytokine production5 ) .", "furthermore , fluid restriction , inotropic agents , and early intubation with positive - pressure mechanical ventilation are recommended for severe hfmd with pulmonary edema or hemorrhage8 ) .", "pulmonary hemorrhage in enterovirus 71-infected hfmd is very rare ; the present case is likely the first to have occurred in korea .", "the present case of fatal hfmd was rapidly aggravated and the patient expired within 3 days from fever onset .", "therefore , all physicians should pay special attention to infants and young children with hfmd , particularly in enterovirus 71 epidemic areas such as korea , to enable early detection and management of severe hfmd .", "above all , keeping the infected child at home until full recovery and controlling the preschool with infected child are very important for reducing infection6 ) .", "in addition , the development of antiviral agents and vaccines is necessary especially for the control of severe hfmd with complication ."], "abstract_text": ["<S> hand , foot , and mouth disease ( hfmd ) is an acute , mostly self - limiting infection . </S>", "<S> patients usually recover without any sequelae </S>", "<S> . however , a few cases are life threatening , especially those caused by enterovirus 71 ( ev71 ) . </S>", "<S> a 12-month - old boy was admitted to a primary hospital with high fever and vesicular lesions of the mouth , hands , and feet . </S>", "<S> after 3 days , he experienced 3 seizure episodes and was referred to our hospital . on admission , he was conscious and his chest radiograph was normal . however , 6 hours later , he suddenly lost consciousness and had developed a massive pulmonary hemorrhage that continued until his death . </S>", "<S> he experienced several more intermittent seizures , and diffuse infiltration of both lung fields was observed on chest radiography . </S>", "<S> intravenous immunoglobulin , dexamethasone , cefotaxime , leukocyte - depleted red blood cells , fresh frozen plasma , inotropics , vitamin k , and endotracheal epinephrine were administered . </S>", "<S> the patient died 9 hours after intubation , within 3 days from fever onset . </S>", "<S> ev71 subgenotype c4a was isolated retrospectively from serum and nasopharyngeal swab by real - time reverse transcription - polymerase chain reaction . here , </S>", "<S> we report a fatal case of ev71-associated hfmd with sudden - onset massive pulmonary hemorrhage and suspected encephalitis . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion"], "sections": [["hand , foot , and mouth disease ( hfmd ) is a common , acute , and mostly self - limiting enteroviral infection that presents with fever and vesicular lesions on the hands , feet , mouth , and frequently buttocks as characteristic features1,2,3,4 ) .", "although patients with hfmd recover without any sequelae within about 1 week , severe complications occur in a few cases , some of which are life threatening .", "severe complications accompanying hfmd include encephalitis , pulmonary edema / hemorrhage , cardiopulmonary collapse , and myocarditis1,2 ) .", "enterovirus 71 can often lead to a more severe clinical course accompanied by neurologic and cardiopulmonary complications and may rapidly lead to death1,2,3 ) .", "enterovirus 71-induced hfmd developed in korea in 2000 but it was not an epidemic and fatal until 20081,4,5 ) . in the spring of 2009 , a large nationwide outbreak of hfmd caused by enterovirus 71 occurred in korea , and a few cases with encephalitis were fatal1,4,6 ) . a multicenter study in korea performed by the enterovirus complication working group revealed that encephalitis with cardiopulmonary complications of hfmd occurred in 2.4% of patients , including 2 patients ( 1.2% ) who expired1 ) .", "pulmonary hemorrhage in enterovirus 71-infected hfmd patients has been reported in taiwan , china , and malaysia2,7,8,9 ) .", "however , to our knowledge , no cases of hfmd with sudden - onset massive pulmonary hemorrhage have been reported in korea . here", ", we report a fatal case of enterovirus 71-induced hfmd that progressed rapidly with massive pulmonary hemorrhage and convulsion ."], ["a 12-month - old , previously healthy boy with hfmd was referred to inje university ilsan paik hospital because of 3 episodes febrile convulsions .", "he was born at full term weighing 3.87 kg through normal vaginal delivery by a vietnamese mother .", "he was admitted to a primary hospital with high fever ( up to 39 ) , throat vesicles , and vesicular rashes on his hands and feet for 2 days . on the second hospital day , he had 3 generalized tonic - clonic and atonic convulsions with vertical eyeball deviation within 5 minutes of each other .", "his chest radiography was normal . within 5 hours after admission , he vomited 3 times after meals . besides a body temperature of 38.3 , his other vital signs were stable and he remained conscious .", "desaturation to 76% percutaneous blood oxygen saturation ( spo2 ) and tachycardia to 176 beats / min accompanied by perioral cyanosis occurred suddenly 6 hours after admission .", "his respiration rate was 34 breaths / min , and auscultation disclosed rales on the whole lung field .", "an endotracheal tube was inserted immediately , and a large amount of blood from a pulmonary hemorrhage came out through it .", "comparison of laboratory test results between his initial and pulmonary hemorrhagic status revealed that his hemoglobin decreased from 9.6 to 8.3 mg / dl and his white blood cell count increased from 13,090 to 17,450 cells/l .", "erythrocyte sedimentation rate was slightly increased from 15 to 32 mm / hr , but c - reactive protein level decreased from 1.06 to 0.2 mg / dl .", "prothrombin time ( pt ) and pt international normalized ratio were slightly prolonged ( 16.5 and 1.39 seconds , respectively ) .", "meanwhile , d - dimer ( 2,375.91 ng / ml ) and fibrin degradation product ( 5 - 20 g / ml ) were elevated .", "eight hours after admission , his blood pressure decreased to 82/41 mmhg and heart rate increased to 220 beats / min .", "his body temperature ranged from 38.8 to 40.2. the patient was diagnosed as fatal hfmd with massive pulmonary hemorrhage .", "intravenous immunoglobulin ( ivig , 1 g / kg / dose ) , dexamethasone ( 0.3 mg / kg / day in divided doses every 6 hours ) , and cefotaxime ( 150 mg / kg / day in divided doses every 6 hours ) were administered immediately .", "in addition , leukocyte - depleted red blood cells and fresh frozen plasma were transfused , and intravenous vitamin k and endotracheal epinephrine were administered .", "lumbar puncture was scheduled but was ultimately not performed because of the patient 's unstable vital signs .", "the patient expired 15 hours after admission , and it was within 3 days of fever onset and within 9 hours after intubation .", "we reported it to the korea centers for disease control and prevention ( kcdc ) , sending his urine , serum , nasopharyngeal swab , and endotracheal aspirate .", "enterovirus genome detection was attempted by realtime reverse transcription - polymerase chain reaction ( rt - pcr ) using a taqman system ( applied biosystems , foster city , ca , usa ) . for genotyping ,", "seminested rt - pcr was used to amplify part of the viral protein 1 ( vp1 ) gene of enteroviruses according to the kcdc protocol , and sequencing analysis for the vp1 amplicon was performed by an automatic sequencer and the dnastar software package ( dnastar inc . , madison , wi , usa ) .", "the kcdc isolated enterovirus 71 with subgenotype c4a from the patient 's serum and nasopharyngeal swab .", "bocavirus was also isolated from his nasopharyngeal swab by real - time rt - pcr at our hospital ."], ["to our knowledge , this is the first reported case of hfmd with sudden - onset massive pulmonary hemorrhage in korea .", "pulmonary edema / hemorrhage is the mysterious hallmark symptom of enteroviral hfmd and can kill a child within 1 day2 ) .", "an epidemic study conducted in taiwan from 1998 to 2005 reported that pulmonary edema / hemorrhage occurred in 43% of patients with severe culture - proven enteroviral hfmd / herpangina2 ) .", "this discrepancy maybe due to different enterovirus virulence or subgenotypes , or differences in susceptibility with respect to ethnicity .", "pulmonary edema / hemorrhage is characterized by respiratory distress , tachypnea , tachycardia , hemoptysis , and rapidly progressing diffuse infiltration or congestion on a chest film2 ) .", "a study on the outbreak in taiwan in 1998 reported that enterovirus 71-infected hfmd patients with pulmonary edema had suddenonset tachypnea , tachycardia , and cyanosis within 1 - 3 days after disease onset , and pulmonary edema led to a rapid death within 12 hours after intubation10 ) .", "the present patient exhibited a change in consciousness , respiratory distress , tachypnea , tachycardia , hemoptysis , rapidly progressing infiltration on a chest film , hypoxemia , and hypotension .", "this patient ultimately expired within 3 days of fever onset and within 9 hours after intubation . in this report , we had applied pulse oxymetry from the beginning , so we could find his desaturation immediately after pulmonary hemorrhage .", "we suggest saturation monitoring for hfmd patients with central nervous system ( cns ) involvements for early detection .", "it may be caused by increased pulmonary vascular permeability as a result of either brainstem encephalitis or a systemic inflammatory response caused by excessive cytokine release2,6 ) .", "cns involvement of enterovirus 71-induced hfmd may also trigger a sympathetic storm , resulting in vasoconstriction with high systemic vascular resistance , passive pulmonary volume loading , and pulmonary edema / hemorrhage8 ) .", "the present patient had several episodes of convulsions , lethargy , and loss of consciousness .", "therefore , we suspected encephalitis , although we were unable to perform a cerebrospinal fluid study because of his unstable vital signs . his suspected encephalitis may be a possible cause of the pulmonary hemorrhage . however , pulmonary edema / hemorrhage without cns involvement occurred in 11% of patients with severe culture - proven enteroviral hfmd / herpangina in an epidemic study in taiwan from 1998 to 20052 ) .", "another possible explanation for the pathogenesis is coinfection with a second virus2 ) , as bocavirus coinfection was detected in the patient .", "however , the presence of both viruses would not result in increased mortality due to hfmd , according to a previous study2 ) . a multicenter study performed by the enterovirus complication working group investigating enterovirus 71-induced hfmd in korea in 2009 reported that an hfmd rash pattern , fever longer than 4 days , peak body temperature > 39 , vomiting , headache , neurologic signs , leukocytosis , serum glucose > 100 mg / dl , and isolated enterovirus 71 may be indicative of poor prognosis during hfmd epidemic periods1,10 ) .", "in addition , young age at disease onset and a history of lethargy are associated with an increased risk of severe hfmd11 ) .", "the present patient was young and presented with hfmd rashes and vesicles , fever up to 39 , vomiting , lethargy , and convulsions as neurologic signs as well as leukocytosis , high serum glucose level , and isolated enterovirus 71 as poor prognostic factors .", "hfmd patients are very frequently approximately 1 year of age , and deaths were reported to occur at around 1 year of age in other studies12,13,14,15 ) . in the 2009 outbreak ,", "2 patients around 1 year of age expired , and the present patient was a 12-month - old baby .", "the gold standard for the diagnosis of enteroviral infection is viral culture and isolation in 2 or more specimens of cerebrospinal fluid , blood , nasopharyngeal swab or secretion , vesicular fluid , or stool to increased diagnostic sensitivity1,4,6,11 ) . in the present report ,", "enterovirus 71 was isolated from both the patient 's serum and nasopharyngeal swab . in the 2009 korean outbreak ,", "the subgenotype of enterovirus 71 was c4a , which was prevalent in china in 2008 . in the present case , the subgenotype of enterovirus 71 was also c4a .", "the administration of high - dose ivig is recommended for severe hfmd as optional treatment , because ivig therapy was effective in many reports4,5,6,7,11 ) .", "ivig neutralizes the virus and has nonspecific antiinflammatory properties11 ) ; this would significantly reduce mortality by attenuating sympathetic activity and cytokine production5 ) .", "furthermore , fluid restriction , inotropic agents , and early intubation with positive - pressure mechanical ventilation are recommended for severe hfmd with pulmonary edema or hemorrhage8 ) .", "pulmonary hemorrhage in enterovirus 71-infected hfmd is very rare ; the present case is likely the first to have occurred in korea .", "the present case of fatal hfmd was rapidly aggravated and the patient expired within 3 days from fever onset .", "therefore , all physicians should pay special attention to infants and young children with hfmd , particularly in enterovirus 71 epidemic areas such as korea , to enable early detection and management of severe hfmd .", "above all , keeping the infected child at home until full recovery and controlling the preschool with infected child are very important for reducing infection6 ) .", "in addition , the development of antiviral agents and vaccines is necessary especially for the control of severe hfmd with complication ."]]}
{"article_id": "PMC3136113", "article_text": ["since the increase in adiposity is related to general poor health factors such as an increase in type ii diabetes and increased incidence of coronary heart disease , exercise to reduce adiposity is recommended for the ageing population .", "a further consequence of the ageing process is that elderly individuals have an impaired ability to oxidise fatty acids , particularly after a meal . since most individuals eat a meal prior to exercise in order to provide some form of sustenance , what should the meal contain if the exercise priority is to  burn fat  ?", "generally , a high - fat , low - cho meal increases fat oxidation during subsequent exercise [ 46 ] , whereas the ingestion of cho before exercise depresses the rate of fat oxidation due to hyperinsulinemia in the postprandial period . altering the type of cho consumed has been shown to have an effect on the magnitude of hyperinsulinemia and depression of fat oxidation [ 8 , 9 ] postprandial increases in glucose and insulin concentration promote cho oxidation , resulting in decreased fatty acid oxidation .", "wu et al .   found that the amount of fat oxidised was significantly higher during exercise commencing 3  h after consuming a low glycemic index ( lgi ) meal compared with a high glycemic index ( hgi ) meal .", "they also demonstrated that the hgi meal resulted in a greater glycemic and insulinemic response during the postprandial period compared with lgi meal .", "this is supported by stevenson et al .   who investigated the metabolic responses to hgi and lgi mixed meals after 60-minute exercise at 70% vo2max   and found that significant differences in hyperglycemia and hyperinsulinemia can be achieved repeatedly by changing the glycemic index ( gi ) of the cho in a mixed meal .", "they observed that the amount of fat oxidised during the postprandial period following lunch was significantly higher in the lgi than the hgi trial .", "thus , at least for several hours postprandial , both at rest and during exercise , fat utilisation is depressed after hgi compared with lgi meals .", "the effects of different meals ( high fat , hgi , and lgi ) on fat and cho metabolism at rest and during exercise in young subjects have been extensively studied , although this is not the case for elderly individuals . as mentioned previously", ", it is an important health benefit for the elderly to engage in some form of aerobic exercise for improvements in the cardiovascular system and to reduce body fat .", "therefore , the present study was designed to investigate the effects of four different types of meals ( normal , high - fat , hgi , and lgi ) on fat and cho metabolism during exercise in elderly male subjects .", "eight healthy males ( mean  sd , age 63.3  5.2  years , height 168  0.05  cm , body mass 78.1  14.0  kg , body fat   21  5.3% , and vo2  max   36.9  10.4  mlkgmin ) gave informed written consent to participate in the study after gaining approval from the human ethics committee of liverpool john moores university . blood pressure ( dinamap pro series , ge medical systems , florida ) was determined prior to performing any exercise as a screening for hypertension .", "participants reported to the laboratory on five separate occasions . in the first session they were familiarised with the laboratory environment and physiological testing equipment .", "height , body mass , and percent of body fat using dxa were also determined during this session .", "after familiarisation , vo2  max   was determined on a cycle ergometer as described previously .", "after initial physiological measurements , participants reported to the physiology laboratory on four separate occasions , each of which was allocated for the performance of a 30-minutes exercise on a cycle ergometer at 60%   vo2  max   after having a high fat ( hf ) , high carbohydrate hgi ( hgi ) , high carbohydrate low lgi ( lgi ) , and normal ( n ) meal .", "the rationale for 30-minutes exercise reflects a typical aerobic bout of exercise undertaken by such persons in a gym session and is recommended for health purposes .", "the four meals were given to subjects in a counterbalanced design and sessions were separated by at least 3 days .", "to avoid circadian variation , experiments were always performed at the same time of day ( 08:00 am ) after an overnight fast .", "participants completed a 2-day food diary on the day before their first test and were asked to repeat this diet before all subsequent trials .", "in addition , subjects were requested to refrain from drinking alcohol , nor to engage in any kind of strenuous exercise 24 hours before trials .", "participants reported to the physiology laboratory after an overnight fast and remained seated for 20 minutes .", "after this rest period , blood pressure was checked and a blood sample ( 10  ml ) was taken .", "they then consumed one of the meals which were provided in a random , counterbalanced order within 2030 minutes . at 3  h 20  min after the meal , subjects started the exercise protocol that included a 30-minutes cycling at 60% vo2  max  .", "two more venous blood samples were taken , immediately before exercise ( 3 hours after the meal ) , and immediately after exercise in each session . oxygen consumption ( vo2 ) ,", "carbon dioxide output ( vco2 ) , and respiratory exchange ratio ( rer ) were measured breath by breath throughout the exercise .", "participants were provided with one of the four following isoenergetic test meals : ( 1 ) and ( 2 ) hgi and lgi : 65% carbohydrate , 20% fat , and 15% protein , ( 3 ) hf : 65% fat , 20% carbohydrate and 15% protein , or ( 4 ) n : 50% carbohydrate , 35% fat , and 15% protein .", "the glycaemic index values for hgi and lgi were 74.32 and 29.26 , respectively . before the meal , immediately before exercise and immediately after exercise venous blood samples were drawn in a seated position in each session .", "two microhaematocrit tubes ( l. i. p. shipley , england ) and two -haemoglobin microcuvettes ( hemocue ab , ngleholm , sweden ) were filled with whole blood for determination of haematocrit and haemoglobin , respectively .", "plasma was obtained by collecting the blood sample into tubes that had been pretreated with an anti - coagulant ( lithium heparin ) .", "these samples were mixed and immediately centrifuged at 4c for 15 minutes at 1900  g. after centrifugation , plasma was separated and stored at 70c for the subsequent analysis of glucose , glycerol , nonesterified fatty acids ( nefas ) , and b - hydroxybutyrate ( 3-ohb ) .", "the blood was stored at room temperature for 30 minutes before centrifuged at 20c for 15 minutes at 2000  g. serum was stored at 70c for subsequent analysis of insulin .", "nefa , glucose , glycerol , and 3-ohb were analysed using appropriate kits on ilab 300 analyser ( il instrumentation laboratories , warrington , uk ) .", "insulin was assayed by elisa ( drg instruments gmbh , germany ) using a fully automated system ( triturus , grifols , cambridge , uk ) . insulin resistance ( homa2-ir ) and -cell function", "( homa2 % b ) in fasting state were determined using a homeostasis model assessment ( homa - ir ) and were calculated from fasting insulin and fasting glucose .", "all statistical analyses were performed using the software statistical package spss version 12 ( chicago , usa ) .", "one - way anova was employed to evaluate differences in the resting mean values of all the variables measured over the four testing occasions .", "in addition , fat and cho oxidations values during exercise for four trials were compared using one - way anova .", "a two - way anova ( 4  3 ) with repeated measures across meals ( 4 levels ) and conditions ( 3 levels ) was employed to examine the differences in mean values for blood parameters .", "when anova indicated the presence of a significant difference , post hoc comparisons using the bonferroni method were applied to determine pairwise differences .", "no significant main effect of the meals was observed for rates of fat oxidation ( f3,21 = 1.8 ; p = .177 ) , although fat oxidation was demonstrably , but nonsignificantly , higher after hf ( 0.26  0.04  g / min ) than n ( 0.21  0.04  g / min ) , hgi ( 0.22  0.03  g / min ) , and lgi ( 0.19  0.03  g / min ) . the rates of carbohydrate oxidation during exercise were 1.79  0.28 , 1.58  0.22 , and 1.68  0.22 , 1.77  0.21  g / min for n , hf , hgi , and lgi , respectively .", "statistical analysis revealed no significant effect of meal on the rate of carbohydrate oxidation during exercise .", "statistical analysis showed a significant main effect of the meal on nefa concentration ( f3,21 = 39.2 ; p = .001 ) .", "pairwise comparisons revealed a significant difference between nefa responses to n and hf ( p = .02 ) as well as between hgi and lgi meals ( p = .003 ) .", "pre - exercise nefa concentration increased significantly following hf ( from 0.39  0.08 to 0.61  0.08 ", "mmol / l ) and decreased significantly after eating hgi ( from 0.44  0.09 to 0.13  0.02  mmol / l ) and lgi ( from 0.55  0.08 to 0.27  0.07 ", "however , nefa concentration increased significantly in response to exercise only after hgi ( from 0.12  0.02 to 0.36  0.09 ", "mmol / l ) and lgi ( from 0.27  0.06 to 0.64  0.18 ", "figure 1 highlights data from nefa . a significant effect of meal was found for glycerol concentration ( f3,21 = 9.7 ; p = .001 ) .", "pairwise comparisons revealed a significant difference between hf and hgi ( p = .01 ) .", "resting glycerol values were increased significantly after all meals except for hgi ( figure 2 ) .", "moreover , glycerol concentration increased significantly during exercise after all types of meals ( figure 2 ) .", "the statistical analysis revealed a significant effect of the meal on 3-ohb ( f3,21 = 3.6 ; p = .03 ) .", "however , pairwise comparisons did not show any significant difference among the four meals . resting 3-ohb concentration", "a significant effect of meals on glucose concentration was found ( f3,21 = 39.2 ; p = .001 ) .", "postprandial glucose concentration increased following all meals , though the changes were not statistically significant ( figure 4 ) .", "however , glucose concentration decreased significantly ( p < .05 ) from 5.36  0.25 to 4.65  0.08 ", "mmol / l during 30  minutes of exercise after hf and lgi , respectively .", "although anova did not show a main significant effect of either meal or time on insulin concentration , the pre - exercise insulin concentration ( 18.9  1.7  u / ml ) in hgi trial was significantly ( p < .05 ) higher than resting values ( 15.2  1.1  u / ml ) .", "in addition , insulin concentration decreased significantly during exercise after n , hf and hgi ( figure 5 ) . the mean ( se ) resting value of homa2-ir ( insulin resistance ) for all subjects was 0.76  0.06 and that for homa2- ( -cell function ) was 78.5  69 .", "these resting values are indicators of normal insulin resistance and -cell function in our elderly subjects .", "the present study is the first study designed to investigate the effects of pre - exercise mixed meals on fat and carbohydrate metabolism during exercise in elderly individuals , and its principle finding was that in spite of some changes in fat metabolites , the composition of the meal did not result in differences in cho and fat oxidation .  ", "these results are in contrast to the data reported by previous studies in young subjects that observed a rise in fat oxidation after hf due to increases in fatty acid ( fa ) availability and mobilization [ 4 , 6 , 15 , 16 ] .", "moreover , a depression in the rate of fat oxidation following cho ingestion , attributed to hyperinsulinaemia , has also been observed in the postprandial period [ 79 ] .  ", "availability and utilisation of plasma fa decreases after a cho meal partly because the cho - induced rise in insulin inhibits the mobilisation and availability of circulating fa , which reduces fat oxidation possibly by inhibiting the rate of long - chain fatty acids entry into the mitochondria for -oxidation [ 17 , 18 ] . despite cho - induced hyperinsulinemia and suppression of fa after cho ingestion ,", "our results show that the cho oxidation was resistant to alteration during exercise in elderly males .", "several mechanisms , including impaired insulin - stimulated glucose uptake , may have contributed to these results .", "having said that , it should be noted that the homa scores indicate normal insulin resistance and -cell function for these elderly participants .", "the increased release of fas in older individuals , in excess of the energy needs and/or oxidative capacity of respiring tissues , increases the amount of non - oxidised fas .", "excess non - oxidised fas with age may have several adverse metabolic effects including increased glucose production   and impaired insulin - stimulated glucose uptake .", "fas exert their effects through inhibition of pdh with subsequent increased intracellular concentration of glucose-6-phosphate and inhibition of hexokinase , which decreases glucose uptake .", "another factor that may contribute to impaired glucose uptake with ageing is inhibition of glucose transport either due to less availability of the glut-4 transporters or , to the signalling processes for glut-4 vesicle translocation to the plasma membrane .", "these considerations need further exploration . although after the hf meal , nefa , and glycerol concentrations were higher than for the other meals , the fat oxidation rates during exercise were not different .", "evaluation of skeletal muscle samples has revealed that maximal mitochondrial oxidative enzyme activity is lower in older than in young subjects because of both decreased mitochondrial volume density   and mitochondrial function .", "lower activity of enzymes such as ampk , camp , and protein kinase c in elderly individuals results in activation of acc ( acetyl - coa carboxylase ) .", "activation of acc leads to an increase in concentration of malonyl - coa which has an inhibitory effect on cpt - i , thereby inhibiting the entry of long chain fatty acids into mitochondria and resulting in lower fa oxidation .", "lack of changes in fat oxidation after hf was found in spite of increased lipolysis .", "it has been demonstrated that relative to the energy needs of the body , fa release is not impaired in the elderly .", "in fact , fas are released in excess of energy needs in older individuals when compared to younger controls .", "thus , when considered relative to the energy demands of the body or the metabolically active tissue mass , ageing is not associated with impaired fa release which supports our findings for lipolysis .   a higher rate of fat oxidation during submaximal exercise after ingesting lgi foods has been reported in young healthy males when compared to hgi [ 9 , 11 ] . in terms of the effect of gi ,", "the results of the present study were somewhat unexpected since the calculated amount of fat oxidation during 30  minutes of cycling commencing 3 hour after consuming hgi and lgi meals was not significantly different .", "one possible explanation for this discrepancy might be the impaired glucose uptake associated with ageing as previously discussed . in the present study", ", postprandial nefa concentration was increased after hf , which is in agreement with previous studies on young participants [ 6 , 9 ] .", "the increase in plasma nefa concentration which occurred might be a result of tag hydrolysis by endothelial lipoprotein lipase ( lpl ) .", "both hgi and lgi resulted in suppression of nefa concentrations 3 hours after the meal consumption .", "however , nefa concentrations at the end of 30-minutes cycling in both hgi and lgi trials were raised to pre - meal levels .", "higher post - exercise nefa is typical response to exercise - induced decrease in insulin and increase in catecholamines .   ", "the lack of a significant increase in glycerol after hgi might be due to the enhanced secretion of insulin following the hgi meal which would activate the enzyme lpl in adipose tissue .", "the insulin activation of lpl serves to increase tag uptake and storage after a single meal which eventually results in lower glycerol concentration .", "studies in young subjects during low and moderate intensity exercise have demonstrated that increased blood glucose availability suppresses fat utilisation by inhibition of both fat mobilisation and fat oxidation within muscle [ 7 , 18 ] .", "postprandial glycerol concentration was significantly higher after hf than hgi and lgi in elderly subjects which reflects the lack of insulin response to hf in comparison to the other meals .", "these findings are in agreement with those of whitley et al .   and murphy et al .   who reported increases in glycerol after hf in young participants .", "the present study demonstrated that in elderly individuals feeding isoenergetic meals containing different proportions of carbohydrate and fat alters the metabolic variables at rest and during subsequent exercise .", "energy regulation during 30  min of cycling in elderly individuals following isoenergetic meals is associated with a relative increase in fat oxidation and a decrease in carbohydrate oxidation following hf and a corresponding increase in cho oxidation and a decrease following high carbohydrate ( low fat ) meals .", "therefore , based on these finding , it could be concluded that fat and carbohydrate metabolism in elderly individuals in response to different meals at rest and during subsequent exercise are to some extent different from those of young individuals and further studies are warranted to investigate the mechanism / s responsible especially in relation to insulin action and sensitivity .", "what we can state is that , on balance , eating any type of meal 3 hours prior to a 30-minutes bout of exercise is unlikely to significantly impact on so - called  fat burning  in healthy elderly males ."], "abstract_text": ["<S> the present study was designed to investigate the effects of four different meals on fat and cho metabolism during subsequent exercise in elderly males . </S>", "<S> eight healthy males ( age : 63.3  5.2 years ) reported to the physiology laboratory on four separate occasions , each of which was allocated for the performance of a 30-minute exercise on a cycle ergometer at 60% vo2max   after having normal ( n ) , high fat ( hf ) , high carbohydrate high glycaemic index ( hgi ) and high carbohydrate low glycaemic index ( lgi ) meals . </S>", "<S> fat oxidation during exercise after the meals ( hf = 0.26  0.04  g / min ; n = 0.21  0.04  g / min ; hgi = 0.22  0.03  g / min ; lgi = 0.19  0.03  </S>", "<S> g / min ) was not significant ( p > .05 ) , and neither were the rates of carbohydrate oxidation ( n = 1.79  0.28 , hf = 1.58  0.22 , hgi = 1.68  0.22 , and lgi = 1.77  0.21  g / m ) . </S>", "<S> nefa concentration increased after hf ( p < .05 ) but decreased after hgi and lgi ( p < .05 ) . </S>", "<S> glucose concentration decreased as a result of exercise after hf , and lgi ( p < .05 ) whereas insulin concentration decreased significantly during exercise after n , hf , and hgi ( p < .05 ) . </S>", "<S> it can be concluded that , in elderly males , feeding isoenergetic meals containing different proportions of carbohydrate and fat do not significantly alter oxidation of fat and cho during exercise in spite of changes in some circulating metabolites . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["since the increase in adiposity is related to general poor health factors such as an increase in type ii diabetes and increased incidence of coronary heart disease , exercise to reduce adiposity is recommended for the ageing population .", "a further consequence of the ageing process is that elderly individuals have an impaired ability to oxidise fatty acids , particularly after a meal . since most individuals eat a meal prior to exercise in order to provide some form of sustenance , what should the meal contain if the exercise priority is to  burn fat  ?", "generally , a high - fat , low - cho meal increases fat oxidation during subsequent exercise [ 46 ] , whereas the ingestion of cho before exercise depresses the rate of fat oxidation due to hyperinsulinemia in the postprandial period . altering the type of cho consumed has been shown to have an effect on the magnitude of hyperinsulinemia and depression of fat oxidation [ 8 , 9 ] postprandial increases in glucose and insulin concentration promote cho oxidation , resulting in decreased fatty acid oxidation .", "wu et al .   found that the amount of fat oxidised was significantly higher during exercise commencing 3  h after consuming a low glycemic index ( lgi ) meal compared with a high glycemic index ( hgi ) meal .", "they also demonstrated that the hgi meal resulted in a greater glycemic and insulinemic response during the postprandial period compared with lgi meal .", "this is supported by stevenson et al .   who investigated the metabolic responses to hgi and lgi mixed meals after 60-minute exercise at 70% vo2max   and found that significant differences in hyperglycemia and hyperinsulinemia can be achieved repeatedly by changing the glycemic index ( gi ) of the cho in a mixed meal .", "they observed that the amount of fat oxidised during the postprandial period following lunch was significantly higher in the lgi than the hgi trial .", "thus , at least for several hours postprandial , both at rest and during exercise , fat utilisation is depressed after hgi compared with lgi meals .", "the effects of different meals ( high fat , hgi , and lgi ) on fat and cho metabolism at rest and during exercise in young subjects have been extensively studied , although this is not the case for elderly individuals . as mentioned previously", ", it is an important health benefit for the elderly to engage in some form of aerobic exercise for improvements in the cardiovascular system and to reduce body fat .", "therefore , the present study was designed to investigate the effects of four different types of meals ( normal , high - fat , hgi , and lgi ) on fat and cho metabolism during exercise in elderly male subjects ."], ["eight healthy males ( mean  sd , age 63.3  5.2  years , height 168  0.05  cm , body mass 78.1  14.0  kg , body fat   21  5.3% , and vo2  max   36.9  10.4  mlkgmin ) gave informed written consent to participate in the study after gaining approval from the human ethics committee of liverpool john moores university . blood pressure ( dinamap pro series , ge medical systems , florida ) was determined prior to performing any exercise as a screening for hypertension .", "participants reported to the laboratory on five separate occasions . in the first session they were familiarised with the laboratory environment and physiological testing equipment .", "height , body mass , and percent of body fat using dxa were also determined during this session .", "after familiarisation , vo2  max   was determined on a cycle ergometer as described previously .", "after initial physiological measurements , participants reported to the physiology laboratory on four separate occasions , each of which was allocated for the performance of a 30-minutes exercise on a cycle ergometer at 60%   vo2  max   after having a high fat ( hf ) , high carbohydrate hgi ( hgi ) , high carbohydrate low lgi ( lgi ) , and normal ( n ) meal .", "the rationale for 30-minutes exercise reflects a typical aerobic bout of exercise undertaken by such persons in a gym session and is recommended for health purposes .", "the four meals were given to subjects in a counterbalanced design and sessions were separated by at least 3 days .", "to avoid circadian variation , experiments were always performed at the same time of day ( 08:00 am ) after an overnight fast .", "participants completed a 2-day food diary on the day before their first test and were asked to repeat this diet before all subsequent trials .", "in addition , subjects were requested to refrain from drinking alcohol , nor to engage in any kind of strenuous exercise 24 hours before trials .", "participants reported to the physiology laboratory after an overnight fast and remained seated for 20 minutes .", "after this rest period , blood pressure was checked and a blood sample ( 10  ml ) was taken .", "they then consumed one of the meals which were provided in a random , counterbalanced order within 2030 minutes . at 3  h 20  min after the meal , subjects started the exercise protocol that included a 30-minutes cycling at 60% vo2  max  .", "two more venous blood samples were taken , immediately before exercise ( 3 hours after the meal ) , and immediately after exercise in each session . oxygen consumption ( vo2 ) ,", "carbon dioxide output ( vco2 ) , and respiratory exchange ratio ( rer ) were measured breath by breath throughout the exercise .", "participants were provided with one of the four following isoenergetic test meals : ( 1 ) and ( 2 ) hgi and lgi : 65% carbohydrate , 20% fat , and 15% protein , ( 3 ) hf : 65% fat , 20% carbohydrate and 15% protein , or ( 4 ) n : 50% carbohydrate , 35% fat , and 15% protein .", "the glycaemic index values for hgi and lgi were 74.32 and 29.26 , respectively . before the meal , immediately before exercise and immediately after exercise venous blood samples were drawn in a seated position in each session .", "two microhaematocrit tubes ( l. i. p. shipley , england ) and two -haemoglobin microcuvettes ( hemocue ab , ngleholm , sweden ) were filled with whole blood for determination of haematocrit and haemoglobin , respectively .", "plasma was obtained by collecting the blood sample into tubes that had been pretreated with an anti - coagulant ( lithium heparin ) .", "these samples were mixed and immediately centrifuged at 4c for 15 minutes at 1900  g. after centrifugation , plasma was separated and stored at 70c for the subsequent analysis of glucose , glycerol , nonesterified fatty acids ( nefas ) , and b - hydroxybutyrate ( 3-ohb ) .", "the blood was stored at room temperature for 30 minutes before centrifuged at 20c for 15 minutes at 2000  g. serum was stored at 70c for subsequent analysis of insulin .", "nefa , glucose , glycerol , and 3-ohb were analysed using appropriate kits on ilab 300 analyser ( il instrumentation laboratories , warrington , uk ) .", "insulin was assayed by elisa ( drg instruments gmbh , germany ) using a fully automated system ( triturus , grifols , cambridge , uk ) . insulin resistance ( homa2-ir ) and -cell function", "( homa2 % b ) in fasting state were determined using a homeostasis model assessment ( homa - ir ) and were calculated from fasting insulin and fasting glucose .", "all statistical analyses were performed using the software statistical package spss version 12 ( chicago , usa ) .", "one - way anova was employed to evaluate differences in the resting mean values of all the variables measured over the four testing occasions .", "in addition , fat and cho oxidations values during exercise for four trials were compared using one - way anova .", "a two - way anova ( 4  3 ) with repeated measures across meals ( 4 levels ) and conditions ( 3 levels ) was employed to examine the differences in mean values for blood parameters .", "when anova indicated the presence of a significant difference , post hoc comparisons using the bonferroni method were applied to determine pairwise differences ."], ["no significant main effect of the meals was observed for rates of fat oxidation ( f3,21 = 1.8 ; p = .177 ) , although fat oxidation was demonstrably , but nonsignificantly , higher after hf ( 0.26  0.04  g / min ) than n ( 0.21  0.04  g / min ) , hgi ( 0.22  0.03  g / min ) , and lgi ( 0.19  0.03  g / min ) . the rates of carbohydrate oxidation during exercise were 1.79  0.28 , 1.58  0.22 , and 1.68  0.22 , 1.77  0.21  g / min for n , hf , hgi , and lgi , respectively .", "statistical analysis revealed no significant effect of meal on the rate of carbohydrate oxidation during exercise .", "statistical analysis showed a significant main effect of the meal on nefa concentration ( f3,21 = 39.2 ; p = .001 ) .", "pairwise comparisons revealed a significant difference between nefa responses to n and hf ( p = .02 ) as well as between hgi and lgi meals ( p = .003 ) .", "pre - exercise nefa concentration increased significantly following hf ( from 0.39  0.08 to 0.61  0.08 ", "mmol / l ) and decreased significantly after eating hgi ( from 0.44  0.09 to 0.13  0.02  mmol / l ) and lgi ( from 0.55  0.08 to 0.27  0.07 ", "however , nefa concentration increased significantly in response to exercise only after hgi ( from 0.12  0.02 to 0.36  0.09 ", "mmol / l ) and lgi ( from 0.27  0.06 to 0.64  0.18 ", "figure 1 highlights data from nefa . a significant effect of meal was found for glycerol concentration ( f3,21 = 9.7 ; p = .001 ) .", "pairwise comparisons revealed a significant difference between hf and hgi ( p = .01 ) .", "resting glycerol values were increased significantly after all meals except for hgi ( figure 2 ) .", "moreover , glycerol concentration increased significantly during exercise after all types of meals ( figure 2 ) .", "the statistical analysis revealed a significant effect of the meal on 3-ohb ( f3,21 = 3.6 ; p = .03 ) .", "however , pairwise comparisons did not show any significant difference among the four meals . resting 3-ohb concentration", "a significant effect of meals on glucose concentration was found ( f3,21 = 39.2 ; p = .001 ) .", "postprandial glucose concentration increased following all meals , though the changes were not statistically significant ( figure 4 ) .", "however , glucose concentration decreased significantly ( p < .05 ) from 5.36  0.25 to 4.65  0.08 ", "mmol / l during 30  minutes of exercise after hf and lgi , respectively .", "although anova did not show a main significant effect of either meal or time on insulin concentration , the pre - exercise insulin concentration ( 18.9  1.7  u / ml ) in hgi trial was significantly ( p < .05 ) higher than resting values ( 15.2  1.1  u / ml ) .", "in addition , insulin concentration decreased significantly during exercise after n , hf and hgi ( figure 5 ) . the mean ( se ) resting value of homa2-ir ( insulin resistance ) for all subjects was 0.76  0.06 and that for homa2- ( -cell function ) was 78.5  69 .", "these resting values are indicators of normal insulin resistance and -cell function in our elderly subjects ."], ["the present study is the first study designed to investigate the effects of pre - exercise mixed meals on fat and carbohydrate metabolism during exercise in elderly individuals , and its principle finding was that in spite of some changes in fat metabolites , the composition of the meal did not result in differences in cho and fat oxidation .  ", "these results are in contrast to the data reported by previous studies in young subjects that observed a rise in fat oxidation after hf due to increases in fatty acid ( fa ) availability and mobilization [ 4 , 6 , 15 , 16 ] .", "moreover , a depression in the rate of fat oxidation following cho ingestion , attributed to hyperinsulinaemia , has also been observed in the postprandial period [ 79 ] .  ", "availability and utilisation of plasma fa decreases after a cho meal partly because the cho - induced rise in insulin inhibits the mobilisation and availability of circulating fa , which reduces fat oxidation possibly by inhibiting the rate of long - chain fatty acids entry into the mitochondria for -oxidation [ 17 , 18 ] . despite cho - induced hyperinsulinemia and suppression of fa after cho ingestion ,", "our results show that the cho oxidation was resistant to alteration during exercise in elderly males .", "several mechanisms , including impaired insulin - stimulated glucose uptake , may have contributed to these results .", "having said that , it should be noted that the homa scores indicate normal insulin resistance and -cell function for these elderly participants .", "the increased release of fas in older individuals , in excess of the energy needs and/or oxidative capacity of respiring tissues , increases the amount of non - oxidised fas .", "excess non - oxidised fas with age may have several adverse metabolic effects including increased glucose production   and impaired insulin - stimulated glucose uptake .", "fas exert their effects through inhibition of pdh with subsequent increased intracellular concentration of glucose-6-phosphate and inhibition of hexokinase , which decreases glucose uptake .", "another factor that may contribute to impaired glucose uptake with ageing is inhibition of glucose transport either due to less availability of the glut-4 transporters or , to the signalling processes for glut-4 vesicle translocation to the plasma membrane .", "these considerations need further exploration . although after the hf meal , nefa , and glycerol concentrations were higher than for the other meals , the fat oxidation rates during exercise were not different .", "evaluation of skeletal muscle samples has revealed that maximal mitochondrial oxidative enzyme activity is lower in older than in young subjects because of both decreased mitochondrial volume density   and mitochondrial function .", "lower activity of enzymes such as ampk , camp , and protein kinase c in elderly individuals results in activation of acc ( acetyl - coa carboxylase ) .", "activation of acc leads to an increase in concentration of malonyl - coa which has an inhibitory effect on cpt - i , thereby inhibiting the entry of long chain fatty acids into mitochondria and resulting in lower fa oxidation .", "lack of changes in fat oxidation after hf was found in spite of increased lipolysis .", "it has been demonstrated that relative to the energy needs of the body , fa release is not impaired in the elderly .", "in fact , fas are released in excess of energy needs in older individuals when compared to younger controls .", "thus , when considered relative to the energy demands of the body or the metabolically active tissue mass , ageing is not associated with impaired fa release which supports our findings for lipolysis .   a higher rate of fat oxidation during submaximal exercise after ingesting lgi foods has been reported in young healthy males when compared to hgi [ 9 , 11 ] . in terms of the effect of gi ,", "the results of the present study were somewhat unexpected since the calculated amount of fat oxidation during 30  minutes of cycling commencing 3 hour after consuming hgi and lgi meals was not significantly different .", "one possible explanation for this discrepancy might be the impaired glucose uptake associated with ageing as previously discussed . in the present study", ", postprandial nefa concentration was increased after hf , which is in agreement with previous studies on young participants [ 6 , 9 ] .", "the increase in plasma nefa concentration which occurred might be a result of tag hydrolysis by endothelial lipoprotein lipase ( lpl ) .", "both hgi and lgi resulted in suppression of nefa concentrations 3 hours after the meal consumption .", "however , nefa concentrations at the end of 30-minutes cycling in both hgi and lgi trials were raised to pre - meal levels .", "higher post - exercise nefa is typical response to exercise - induced decrease in insulin and increase in catecholamines .   ", "the lack of a significant increase in glycerol after hgi might be due to the enhanced secretion of insulin following the hgi meal which would activate the enzyme lpl in adipose tissue .", "the insulin activation of lpl serves to increase tag uptake and storage after a single meal which eventually results in lower glycerol concentration .", "studies in young subjects during low and moderate intensity exercise have demonstrated that increased blood glucose availability suppresses fat utilisation by inhibition of both fat mobilisation and fat oxidation within muscle [ 7 , 18 ] .", "postprandial glycerol concentration was significantly higher after hf than hgi and lgi in elderly subjects which reflects the lack of insulin response to hf in comparison to the other meals .", "these findings are in agreement with those of whitley et al .   and murphy et al .   who reported increases in glycerol after hf in young participants .", "the present study demonstrated that in elderly individuals feeding isoenergetic meals containing different proportions of carbohydrate and fat alters the metabolic variables at rest and during subsequent exercise .", "energy regulation during 30  min of cycling in elderly individuals following isoenergetic meals is associated with a relative increase in fat oxidation and a decrease in carbohydrate oxidation following hf and a corresponding increase in cho oxidation and a decrease following high carbohydrate ( low fat ) meals .", "therefore , based on these finding , it could be concluded that fat and carbohydrate metabolism in elderly individuals in response to different meals at rest and during subsequent exercise are to some extent different from those of young individuals and further studies are warranted to investigate the mechanism / s responsible especially in relation to insulin action and sensitivity .", "what we can state is that , on balance , eating any type of meal 3 hours prior to a 30-minutes bout of exercise is unlikely to significantly impact on so - called  fat burning  in healthy elderly males ."]]}
{"article_id": "PMC4966200", "article_text": ["physical fitness is the ability that helps people to adapt with their environmental conditions and plays an important role in daily activities .", "it is highly associated to one 's health status and has a striking effect on the quality of life , learning and working efficiency , and physical activity contributions .", "physical fitness is composed of performance - related physical fitness and health - related physical fitness .", "indeed , genetic somehow determines physical fitness , but environmental factors including physical activity levels , socioeconomic status , television viewing , and anthropometric factors highly modify it . since fitness level drops with aging , physical involvement should be highly suggested .", "however , inactive life affects body muscle and functioning ability negatively , and people are vulnerable to chronic diseases as consequences .", "a higher quality of life can be obtained through moderate exercises to develop or maintain physical fitness .", "lamb declared that individuals can successfully cope with challenges in their life via optimal physical fitness .", "physical training program can be effective for athletes and nonathletes in different ages to improve aerobic power , muscle strength , anaerobic power , jumping ability , and local muscle endurance .", "additional strength training and plyometric training in 912-year - old soccer players for 2 years showed a significant improvement of motor performance factors .", "mini - trampoline exercises may need higher levels of involvement in the muscles of lower extremities .", "since mini - trampoline exercises comprise a multicomponent approach , numerous physical factors including strength , body stability , muscle coordinative responses , joint movement amplitudes , and spatial integration can be affected positively .", ". indicated not only balance and strength improvement but also muscular imbalances equalization of the two limbs due to mini - trampoline training .", ". showed improvement in motor performance of participants with intellectual disabilities after applying trampoline training intervention .", "however , rebounding exercise for 11 weeks revealed minimal cardiorespiratory fitness improvement and no significant body composition changes in 17 sedentary women .", "very few studies were conducted to detect specific contributions derived from trampoline exercise as a different training model approach .", "therefore , the purpose of this study was to examine the effect of trampoline training intervention on anthropometric variables and physical fitness components of 1114-year - old male students , iran .", "thirty - eight healthy school - aged male students ( mean age : 12.6 years , standard deviation : 2.1 ) were selected randomly from the junior high schools of ardabil , iran .", "they were divided into two groups of 19 students based on gender , residential school , and no trampoline training experience .", "the first five students were omitted as they had prior experience , and the second five students were excluded because they had failed to attend the exercise sessions consistently [ figure 1 ] .", "the study protocol received approval from the ethics committee of the university of urmia , and it was in accordance with the declaration of helsinki ( last modified in 2000 ) .", "flow chart of the progress through the phases of the study according to the consort statements evaluation process of participants was conducted within a 2-day period by the same researcher .", "the 1 day was assigned to anthropometric measurement and the 2 day for motor ability tests .", "anthropometry and motor ability of the participants were measured before and after the 20 weeks trampoline training intervention .", ", crymych , dyfed , uk ) and weight ( model ds-410 , seiko , tokyo , japan ) were assessed to the nearest 10 mm and 0.1 kg respectively , and body mass index was calculated using following formula : weight ( kg)/(height [ m ] ) .", "moreover , lower extremity girths including thigh and calf were measured on the right side of each participant .", "skinfolds thickness was measured at three body sites ( triceps , subscapula , and suprailiac ) with a harpenden skinfold caliper .", "participants in experimental group trained 20-week , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week .", "although none of the students had balance problems , some important safety guidelines were considered to prevent the possibility of injury .", "all participants were instructed to perform variety of movements with eyes opened including one foot and two - foot jump , hop with rotation in different directions , hand and toe contacts in the air , and alternate sit and stand movement . the instructor started to teach forward and backward somersault when participants were familiar with spatial awareness , spatial orientation , and body control after performing all of the mentioned movements . finally , students were asked to combine individual movements and execute them as a routine throughout the training sessions .", "however , participants in the control group only participated in school physical education classes 2 times / week for 45 min .", "motor ability of all participants was assessed after a short period of warm up using standing long jump and vertical jump based on eurofit test battery .", "each participant standing reach height , tip of middle finger in the hand closest to the wall while participant stood side on to the wall , was marked .", "then , participant used both arms and legs contributions to project the body as high as possible while standing away from the wall . the standing reach height and the jump height difference were the score .", "each participant stood behind the marked line on the ground with swinging arms and bending knees .", "the take - off was done when the arms reached forward to lead participant as far as possible .", "a two - way repeated measures analysis of variance ( anova ) ( condition    trial ) with repeated measures on both factors was used . to further analyze the responses of the groups", "thirty - eight healthy school - aged male students ( mean age : 12.6 years , standard deviation : 2.1 ) were selected randomly from the junior high schools of ardabil , iran .", "they were divided into two groups of 19 students based on gender , residential school , and no trampoline training experience .", "the first five students were omitted as they had prior experience , and the second five students were excluded because they had failed to attend the exercise sessions consistently [ figure 1 ] .", "the study protocol received approval from the ethics committee of the university of urmia , and it was in accordance with the declaration of helsinki ( last modified in 2000 ) .", "flow chart of the progress through the phases of the study according to the consort statements", "the 1 day was assigned to anthropometric measurement and the 2 day for motor ability tests .", "anthropometry and motor ability of the participants were measured before and after the 20 weeks trampoline training intervention .", ", crymych , dyfed , uk ) and weight ( model ds-410 , seiko , tokyo , japan ) were assessed to the nearest 10 mm and 0.1 kg respectively , and body mass index was calculated using following formula : weight ( kg)/(height [ m ] ) . moreover , lower extremity girths including thigh and calf were measured on the right side of each participant .", "skinfolds thickness was measured at three body sites ( triceps , subscapula , and suprailiac ) with a harpenden skinfold caliper .", "participants in experimental group trained 20-week , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week .", "although none of the students had balance problems , some important safety guidelines were considered to prevent the possibility of injury .", "all participants were instructed to perform variety of movements with eyes opened including one foot and two - foot jump , hop with rotation in different directions , hand and toe contacts in the air , and alternate sit and stand movement . the instructor started to teach forward and backward somersault when participants were familiar with spatial awareness , spatial orientation , and body control after performing all of the mentioned movements .", "finally , students were asked to combine individual movements and execute them as a routine throughout the training sessions .", "however , participants in the control group only participated in school physical education classes 2 times / week for 45 min .", "motor ability of all participants was assessed after a short period of warm up using standing long jump and vertical jump based on eurofit test battery .", "each participant standing reach height , tip of middle finger in the hand closest to the wall while participant stood side on to the wall , was marked .", "then , participant used both arms and legs contributions to project the body as high as possible while standing away from the wall . the standing reach height and the jump height difference were the score .", "each participant stood behind the marked line on the ground with swinging arms and bending knees .", "the take - off was done when the arms reached forward to lead participant as far as possible .", ". a two - way repeated measures analysis of variance ( anova ) ( condition    trial ) with repeated measures on both factors was used . to further analyze the responses of the groups", "anthropometric and physical fitness variables of the two groups are reported in tables 1 and 2 .", "the anova repeated measurement test showed a statistically significant main effect of time in calf girth p = 0.001 , fat% p = 0.01 , vertical jump p = 0.001 , and long jump p = 0.001 .", "the anova repeated measurement test revealed a statistically significant main effect of group in fat% p = 0.001 .", "characteristics of the study participants differences between pre- and post - training of anthropometric and physical fitness indicators in the trampoline and control groups post hoc paired t - tests indicated statistical significant differences in trampoline group between the two measurements about calf girth ( t = 4.35 , p = 0.001 ) , fat% ( t = 5.87 , p = 0.001 ) , vertical jump ( t = 5.53 , p = 0.001 ) , and long jump ( t = 10.00 , p = 0.001 ) .", "post hoc paired t - tests showed statistical significant differences in control group between the two measurements concerning calf girth ( t = 5.67 , p = 0.001 ) , vertical jump ( t = 3.88 , p = 0.002 ) , and long jump ( t = 4.22 , p = 0.001 ) .", "the analysis of independent t - test ( pre- to post - subtraction between two groups ) indicated significant differences in fat% p = 0.001 , high jump p = 0.02 , and long jump p = 0.02 .", "this study investigated whether anthropometric measures and motor performance components are changed via trampoline training considering that this is the first study in a relatively longer period . since athletes increase metabolism through practice , our finding indicated significant changes of body fat% as a result of trampoline training that is not consistent with the findings of edin et al .", "indeed , no significance changes of body composition variables in the study of edin et al ( 1990 ) may be attributed to the 11 weeks exercise that is limited in terms of training period compared to our study .", "although trampoline exercises are not basically aerobic , long - term training program made significant changes in body fat% between the two groups .", "lower limb characteristics such as thigh girth and calf girth were improved considering pre - test and post - test differences .", "however , we found no significant difference between experimental and control group in this regard .", "the improved lower limb muscle sizes are related to participant 's growth process , and trampoline training has no significant effect on the hypertrophy of lower limb muscles .", "( 2011 ) reported balance improvement due to mini - trampoline exercises in elderly participants .", "( 2013 ) indicated improvement in motor performance of participants with intellectual disabilities after applying trampoline training intervention .", "our findings are in agreement with those studies in this regard and showed significant improvement of jumping ability in healthy trampoline participants compared to control group .", "this improvement can be attributed to the higher amount of biomechanical stimuli with jumping on a trampoline compared to other forms of activity like running .", "furthermore , body segments coordination due to training programs can help participants to use the ideal motor patterns for performing jumping tests .", "trampoline training stimulates proprioceptive sense that can facilitate motor performance and prevent musculoskeletal injuries as a result of sudden fall in life .", "since the nature of this training intervention is recreational , people in different ages can be involved to promote the quality of their life .", "based on these finding , we can conclude that a 20-week trampoline training with four physical activity sessions / week in 1114-year - old students seem to have a significant effect on body fat% reduction and effective results in terms of anaerobic physical fitness .", "therefore , it is suggested that different training model approach such as trampoline exercises can help students to promote the level of health and motor performance ."], "abstract_text": ["<S> background : physical exercises can influence some anthropometric and fitness components differently . </S>", "<S> the aim of present study was to evaluate how a relatively long - term training program in 11 - 14-year - old male iranian students affects their anthropometric and motor performance measures.methods:measurements were conducted on the anthropometric and fitness components of participants ( n = 28 ) prior to and following the program . </S>", "<S> they trained 20 weeks , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week . </S>", "<S> motor performance of all participants was assessed using standing long jump and vertical jump based on eurofit test battery.results:the analysis of variance ( anova ) repeated measurement test showed a statistically significant main effect of time in calf girth p = 0.001 , fat% p = 0.01 , vertical jump p = 0.001 , and long jump p = 0.001 . </S>", "<S> the anova repeated measurement test revealed a statistically significant main effect of group in fat% p = 0.001 . </S>", "<S> post hoc paired t - tests indicated statistical significant differences in trampoline group between the two measurements about calf girth ( t = 4.35 , p = 0.001 ) , fat% ( t = 5.87 , p = 0.001 ) , vertical jump ( t = 5.53 , p = 0.001 ) , and long jump ( t = 10.00 , p = 0.001).conclusions : we can conclude that 20-week trampoline training with four physical activity sessions / week in 1114-year - old students seems to have a significant effect on body fat% reduction and effective results in terms of anaerobic physical fitness . </S>", "<S> therefore , it is suggested that different training model approach such as trampoline exercises can help students to promote the level of health and motor performance . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "Participants", "Anthropometry and body composition", "Trampoline training", "Physical fitness measures", "Vertical jump test", "Standing long jump test", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSIONS", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["physical fitness is the ability that helps people to adapt with their environmental conditions and plays an important role in daily activities .", "it is highly associated to one 's health status and has a striking effect on the quality of life , learning and working efficiency , and physical activity contributions .", "physical fitness is composed of performance - related physical fitness and health - related physical fitness .", "indeed , genetic somehow determines physical fitness , but environmental factors including physical activity levels , socioeconomic status , television viewing , and anthropometric factors highly modify it . since fitness level drops with aging , physical involvement should be highly suggested .", "however , inactive life affects body muscle and functioning ability negatively , and people are vulnerable to chronic diseases as consequences .", "a higher quality of life can be obtained through moderate exercises to develop or maintain physical fitness .", "lamb declared that individuals can successfully cope with challenges in their life via optimal physical fitness .", "physical training program can be effective for athletes and nonathletes in different ages to improve aerobic power , muscle strength , anaerobic power , jumping ability , and local muscle endurance .", "additional strength training and plyometric training in 912-year - old soccer players for 2 years showed a significant improvement of motor performance factors .", "mini - trampoline exercises may need higher levels of involvement in the muscles of lower extremities .", "since mini - trampoline exercises comprise a multicomponent approach , numerous physical factors including strength , body stability , muscle coordinative responses , joint movement amplitudes , and spatial integration can be affected positively .", ". indicated not only balance and strength improvement but also muscular imbalances equalization of the two limbs due to mini - trampoline training .", ". showed improvement in motor performance of participants with intellectual disabilities after applying trampoline training intervention .", "however , rebounding exercise for 11 weeks revealed minimal cardiorespiratory fitness improvement and no significant body composition changes in 17 sedentary women .", "very few studies were conducted to detect specific contributions derived from trampoline exercise as a different training model approach .", "therefore , the purpose of this study was to examine the effect of trampoline training intervention on anthropometric variables and physical fitness components of 1114-year - old male students , iran ."], ["thirty - eight healthy school - aged male students ( mean age : 12.6 years , standard deviation : 2.1 ) were selected randomly from the junior high schools of ardabil , iran .", "they were divided into two groups of 19 students based on gender , residential school , and no trampoline training experience .", "the first five students were omitted as they had prior experience , and the second five students were excluded because they had failed to attend the exercise sessions consistently [ figure 1 ] .", "the study protocol received approval from the ethics committee of the university of urmia , and it was in accordance with the declaration of helsinki ( last modified in 2000 ) .", "flow chart of the progress through the phases of the study according to the consort statements evaluation process of participants was conducted within a 2-day period by the same researcher .", "the 1 day was assigned to anthropometric measurement and the 2 day for motor ability tests .", "anthropometry and motor ability of the participants were measured before and after the 20 weeks trampoline training intervention .", ", crymych , dyfed , uk ) and weight ( model ds-410 , seiko , tokyo , japan ) were assessed to the nearest 10 mm and 0.1 kg respectively , and body mass index was calculated using following formula : weight ( kg)/(height [ m ] ) .", "moreover , lower extremity girths including thigh and calf were measured on the right side of each participant .", "skinfolds thickness was measured at three body sites ( triceps , subscapula , and suprailiac ) with a harpenden skinfold caliper .", "participants in experimental group trained 20-week , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week .", "although none of the students had balance problems , some important safety guidelines were considered to prevent the possibility of injury .", "all participants were instructed to perform variety of movements with eyes opened including one foot and two - foot jump , hop with rotation in different directions , hand and toe contacts in the air , and alternate sit and stand movement . the instructor started to teach forward and backward somersault when participants were familiar with spatial awareness , spatial orientation , and body control after performing all of the mentioned movements . finally , students were asked to combine individual movements and execute them as a routine throughout the training sessions .", "however , participants in the control group only participated in school physical education classes 2 times / week for 45 min .", "motor ability of all participants was assessed after a short period of warm up using standing long jump and vertical jump based on eurofit test battery .", "each participant standing reach height , tip of middle finger in the hand closest to the wall while participant stood side on to the wall , was marked .", "then , participant used both arms and legs contributions to project the body as high as possible while standing away from the wall . the standing reach height and the jump height difference were the score .", "each participant stood behind the marked line on the ground with swinging arms and bending knees .", "the take - off was done when the arms reached forward to lead participant as far as possible .", "a two - way repeated measures analysis of variance ( anova ) ( condition    trial ) with repeated measures on both factors was used . to further analyze the responses of the groups"], ["thirty - eight healthy school - aged male students ( mean age : 12.6 years , standard deviation : 2.1 ) were selected randomly from the junior high schools of ardabil , iran .", "they were divided into two groups of 19 students based on gender , residential school , and no trampoline training experience .", "the first five students were omitted as they had prior experience , and the second five students were excluded because they had failed to attend the exercise sessions consistently [ figure 1 ] .", "the study protocol received approval from the ethics committee of the university of urmia , and it was in accordance with the declaration of helsinki ( last modified in 2000 ) .", "flow chart of the progress through the phases of the study according to the consort statements"], ["the 1 day was assigned to anthropometric measurement and the 2 day for motor ability tests .", "anthropometry and motor ability of the participants were measured before and after the 20 weeks trampoline training intervention .", ", crymych , dyfed , uk ) and weight ( model ds-410 , seiko , tokyo , japan ) were assessed to the nearest 10 mm and 0.1 kg respectively , and body mass index was calculated using following formula : weight ( kg)/(height [ m ] ) . moreover , lower extremity girths including thigh and calf were measured on the right side of each participant .", "skinfolds thickness was measured at three body sites ( triceps , subscapula , and suprailiac ) with a harpenden skinfold caliper ."], ["participants in experimental group trained 20-week , 1.5 h / session with 10 min rest , in 4 times trampoline training programs per week .", "although none of the students had balance problems , some important safety guidelines were considered to prevent the possibility of injury .", "all participants were instructed to perform variety of movements with eyes opened including one foot and two - foot jump , hop with rotation in different directions , hand and toe contacts in the air , and alternate sit and stand movement . the instructor started to teach forward and backward somersault when participants were familiar with spatial awareness , spatial orientation , and body control after performing all of the mentioned movements .", "finally , students were asked to combine individual movements and execute them as a routine throughout the training sessions .", "however , participants in the control group only participated in school physical education classes 2 times / week for 45 min ."], ["motor ability of all participants was assessed after a short period of warm up using standing long jump and vertical jump based on eurofit test battery ."], ["each participant standing reach height , tip of middle finger in the hand closest to the wall while participant stood side on to the wall , was marked .", "then , participant used both arms and legs contributions to project the body as high as possible while standing away from the wall . the standing reach height and the jump height difference were the score ."], ["each participant stood behind the marked line on the ground with swinging arms and bending knees .", "the take - off was done when the arms reached forward to lead participant as far as possible ."], [". a two - way repeated measures analysis of variance ( anova ) ( condition    trial ) with repeated measures on both factors was used . to further analyze the responses of the groups"], ["anthropometric and physical fitness variables of the two groups are reported in tables 1 and 2 .", "the anova repeated measurement test showed a statistically significant main effect of time in calf girth p = 0.001 , fat% p = 0.01 , vertical jump p = 0.001 , and long jump p = 0.001 .", "the anova repeated measurement test revealed a statistically significant main effect of group in fat% p = 0.001 .", "characteristics of the study participants differences between pre- and post - training of anthropometric and physical fitness indicators in the trampoline and control groups post hoc paired t - tests indicated statistical significant differences in trampoline group between the two measurements about calf girth ( t = 4.35 , p = 0.001 ) , fat% ( t = 5.87 , p = 0.001 ) , vertical jump ( t = 5.53 , p = 0.001 ) , and long jump ( t = 10.00 , p = 0.001 ) .", "post hoc paired t - tests showed statistical significant differences in control group between the two measurements concerning calf girth ( t = 5.67 , p = 0.001 ) , vertical jump ( t = 3.88 , p = 0.002 ) , and long jump ( t = 4.22 , p = 0.001 ) .", "the analysis of independent t - test ( pre- to post - subtraction between two groups ) indicated significant differences in fat% p = 0.001 , high jump p = 0.02 , and long jump p = 0.02 ."], ["this study investigated whether anthropometric measures and motor performance components are changed via trampoline training considering that this is the first study in a relatively longer period . since athletes increase metabolism through practice , our finding indicated significant changes of body fat% as a result of trampoline training that is not consistent with the findings of edin et al .", "indeed , no significance changes of body composition variables in the study of edin et al ( 1990 ) may be attributed to the 11 weeks exercise that is limited in terms of training period compared to our study .", "although trampoline exercises are not basically aerobic , long - term training program made significant changes in body fat% between the two groups .", "lower limb characteristics such as thigh girth and calf girth were improved considering pre - test and post - test differences .", "however , we found no significant difference between experimental and control group in this regard .", "the improved lower limb muscle sizes are related to participant 's growth process , and trampoline training has no significant effect on the hypertrophy of lower limb muscles .", "( 2011 ) reported balance improvement due to mini - trampoline exercises in elderly participants .", "( 2013 ) indicated improvement in motor performance of participants with intellectual disabilities after applying trampoline training intervention .", "our findings are in agreement with those studies in this regard and showed significant improvement of jumping ability in healthy trampoline participants compared to control group .", "this improvement can be attributed to the higher amount of biomechanical stimuli with jumping on a trampoline compared to other forms of activity like running .", "furthermore , body segments coordination due to training programs can help participants to use the ideal motor patterns for performing jumping tests .", "trampoline training stimulates proprioceptive sense that can facilitate motor performance and prevent musculoskeletal injuries as a result of sudden fall in life .", "since the nature of this training intervention is recreational , people in different ages can be involved to promote the quality of their life ."], ["based on these finding , we can conclude that a 20-week trampoline training with four physical activity sessions / week in 1114-year - old students seem to have a significant effect on body fat% reduction and effective results in terms of anaerobic physical fitness .", "therefore , it is suggested that different training model approach such as trampoline exercises can help students to promote the level of health and motor performance ."], [""], [""]]}
{"article_id": "PMC3107716", "article_text": ["optical buffers are recognized as essential components in wavelength routers , in which the packets of data can be stored for resolving packet contention problems and also delay the outgoing packets.1,2 in practice , the optical router patents have been proposed and recorded,35 which can be useful for various applications .", "recently , the promising techniques of microscopic volume trapping and transportation within the add / drop multiplexer have been reported in both theory6 and experiment,7 respectively , in which the transporter is known as an optical tweezer . here , the optical tweezer generation technique has become a powerful tool for the manipulation of micrometer - sized particles . to date", "moreover , the use of dynamic tweezers is now also understood in practical works.810 schulz et al11 have shown that the transfer of trapped atoms between two optical potentials can be performed . in principle , optical tweezers use forces exerted by intensity gradients in the strongly focused beams of light to trap and move the microscopic volumes of matters , in which the other combination of force is induced by the interaction between photons , which is caused by photon scattering effects . in application , the field intensity can be adjusted and tuned , in which the desired gradient field and scattering force can form the suitable trapping force . hence , the appropriate force can be configured for the transmitter / receiver , which can perform the long distance microscopic transportation . in this paper , dynamic optical tweezers / vortices", "are generated using a dark soliton , bright soliton , and gaussian pulse propagating within an add / drop optical multiplexer incorporating two nanoring resonators ( panda ring resonator ) .", "the dynamic behaviors of solitons and gaussian pulses are well described by tasakorn et al.12 by using the proposed system , the transceiver can be integrated and performed by using a single device . here , the use of the transceiver to form the transportation of microscopic volumes of matter , especially13 for molecule transportation in liquid core waveguide,14,15 drug delivery , and dna transportation , in which the buffer is needed before reaching the required destination .", "in operation , the trapping forces are exerted by the intensity gradients in the strongly focused beams of light to trap and move the microscopic volumes of matters , in which the optical forces are customarily defined by the relationship:16 \n ( 1)f = qnmpc here q is a dimensionless efficiency , nm is the refractive index of the suspending medium , c is the speed of light , and p is the incident laser power , measured at the specimen .", "q represents the fraction of power utilized to exert force . for plane wave incidences on a perfectly absorbing particle", "because biological specimens are usually contained in aqueous media , the dependence of f on nm can rarely be exploited to achieve higher trapping forces .", "increasing the laser power is possible , but only over a limited range due to the possibility of optical damage .", "it depends upon the numerical aperture ( na ) , laser wavelength , light polarization state , laser mode structure , relative index of refraction , and the geometry of the particle .", "furthermore , in the rayleigh regime , trapping forces decompose naturally into two components . since , in this limit , the electromagnetic field is uniform across the dielectric , particles can be treated as induced point dipoles .", "the scattering force is given by:16 \n ( 2)fscatt = nmsc , \n ( 3)=83(kr)4r2(m21m2 + 2)2 here is the scattering cross section of a rayleigh sphere with radius r. s is the time averaged poynting vector , n is the index of refraction of the particle , m = n / nm is the relative index , and k = 2nm/ is the wave number of the light .", "the scattering force is proportional to the energy flux and points along the direction of the propagation of the incident light .", "the gradient field ( fgrad ) is the lorentz force acting on the dipole induced by the light field .", "it is given by:16 \n ( 4)fgrad=2e2,where \n ( 5)=nm2r3(m21m2 + 2)is the polarizability of the particle .", "the gradient force is proportional and parallel to the gradient in energy density ( for m > 1 ) .", "the stable trapping requires that the gradient force is in the  direction , which is against the direction of incident light ( dark soliton valley ) .", "it is greater than the scattering force . by increasing the na , when the focal spot size is decreased , the gradient strength is increased,16,17 which happens within a tiny system , for instance , a nanoscale device such as the nanoring resonator . in our proposal ,", "the trapping force is formed by using a dark soliton , in which the valley of the dark soliton is generated and controlled within the panda ring resonator by the control port signals . in this paper", ", we used the same theory of optical trapping and ring resonator , in which the simulation results and applications are differed from the previous work.18 from figure 1 , the output field ( et1 ) at the through port is given by:19 \n ( 6)et1=aei1bei2el2jknl2  [cet1(el2jknl2)2+dei2(el2jknl2)31f(e2ljknl2)2]where \n a \n ( 11)(12),b \n ( 11)(12)1(12)e0l , c \n 1(11)(12)2e0e0l , d \n ( 11)(12)1(11)2(12)e0e0l2 , andf \n ( 11)(12)(11)(12)e0e0l . \n", "( 11)(12)1(12)e0l , \n 1(11)(12)2e0e0l , \n ( 11)(12)1(11)2(12)e0e0l2 , and \n ( 11)(12)(11)(12)e0e0l . here ,", "et and ed represent the optical fields of the through port and drop ports , respectively .", " = kneff is the propagation constant , neff is the effective refractive index of the waveguide , and the circumference of the ring is l = 2r , where r is the radius of the ring . 1 and 2 are the coupling coefficients of the add / drop filters , kn = 2/ is the wave propagation number for in a vacuum , and the waveguide ( ring resonator ) loss is  = 0.5 dbmm .", "the fractional coupler intensity loss is  = 0.1 . in the case of the add / drop device", ", the nonlinear refractive index does not affect the system , therefore , it is neglected .", "the electric fields e0 and e0l are the field circulated within the nanoring at the right and left side of add / drop optical filter .", "the power output ( pt1 ) at through port is written as : \n ( 7)pt1=|et1|2 . the output field (", "et2)at drop port is expressed as:19 \n ( 8)et2=(12)(12)ei2  [(11)(12)12e0ei1el2jknl2+xe0e0lle2i(el2jknl2)21ye0e0l(e2ljknl2)2],where \n x \n ( 12)(11)(11)2(12),y = \n ( 11)(12)(11)(12 ) \n ( 12)(11)(11)2(12 ) , \n ( 11)(12)(11)(12 ) the power output ( pt2 ) at drop port is : \n ( 9)pt2=|et2|2 . in operation , the optical tweezers can be trapped , transported , and stored within the panda ring resonator and wavelength router , which can be used to form the microscopic volume ( molecule ) transportation and drug delivery via the waveguide,18 in which the manipulation of trapped microscopic volumes within the optical tweezers has been reported .", "molecular buffers are devices that can be used to store or delay atoms / molecules for a period of time ( see figure 2 ) , where light intensity and velocity can also be controlled , which was described by rosenberry et al20 and lignie and woerdman,21 available for medical application .", "molecular buffers are a new device , which are operated in the same way as gas buffers.22 the polarizability of the particle is calculated by using equation ( 5 ) , in this case , we assume that the sphere particle is polystyrene ( n = 1.5894 ) and the liquid medium is water ( n = 1.33 ) , and the optical power which is required to trap particles of a certain size / polarizibility is 9.1w , which is the slope as shown in figure 3a . in simulation , the bright soliton with center wavelength at 1.50 m , peak power 2w , pulse 35fs is input into the system via the input port , and the coupling coefficients are given as 0 = 0.5 , 1 = 0.35 , 2 = 0.1 , and 3 = 0.35 , respectively .", "the ring radii are radd = 10 and 30 m , rr = 50 and 100 nm , and rl = 50 and 100 nm , respectively . to date , the evidence of a practical device with a radius of 30 nm has been reported by piyatamrong et al.19 aeff are 0.50 , 0.25 , and 0.25 m . in this case", ", the dynamic tweezers ( gradient fields ) can be in the form of bright solitons , gaussian pulses , and dark solitons , which can be used to trap the required microscopic volume .", "there are four different center wavelengths of tweezers generated ; the dynamical movements are seen in figure 4 , where ( a ) |e1| , ( b ) |e2| , ( c ) |e3| , ( d ) |e4| , ( e ) through port , and ( f ) drop port signals , where in this case all microscopic volumes are received by the drop port . in practice ,", "the fabrication parameters which can be easily controlled are the ring resonator radii instead of the coupling constants .", "the important aspect of the result is that the tunable tweezers can be obtained by tuning ( controlling ) the add ( control ) port input signal , in which the required amount of microscopic volume ( atom / photon / molecule ) can be obtained and seen at the drop / through ports , otherwise they propagate within a panda ring before collapsing / decaying into the waveguide .", "more results of the optical tweezers generated within the panda ring are shown in figure 5 , where in this case , the bright soliton is used as the control port signal to obtain the tunable results .", "the output optical tweezers of the through and drop ports with different coupling constants are as shown in figure 5a , while the different wavelength results are as shown in figure 5b , which can be performed by the selected targets . in application", ", the trapped microscopic volumes ( molecules ) can move into the wavelength router via the through port , while the retrieved microscopic volumes are received via the drop port ( connecting target ) .", "the advantage of the proposed system is that the transmitter and receiver can be fabricated on - chip and alternatively operated by a single device .", "the magnitude of the optical trapping force is the pico newton , which depends upon the relative refractive index of particle , which was given by kumar et al.23 the particle radius was given by hu et al,24 fischer and srensen,25 and nieminen et al,26 which is located in the cavity .", "we have proposed a new system that can be used to trap ( delay ) and transport molecules into an optical waveguide by using optical tweezers , which can be used for drug storage and as a delivery system . by utilizing the reasonable dark soliton input power ,", "the dynamic tweezers can be controlled and stored ( delayed ) within the system before reaching the final destination .", "tweezer amplification is also available by using the nanoring resonators , in which the signals can be modulated via the control port as shown in figures 4b and 5a . in conclusion", ", we have shown that the use of a transceiver for long distance microscopic volume by using the proposed system , in which the drug delivery or molecular communication can be performed via the wavelength router to the required ( connecting ) targets . however , the problems of large microscopic volume and neutral matter may cause a problem , in which the pursuit of new guide pipes and media,27 for instance , nano tubes and specific gases , will be the issue of investigation ."], "abstract_text": ["<S> a novel design of molecular buffer for molecule storage and delivery using a panda ring resonator is proposed . </S>", "<S> the optical vortices can be generated and controlled to form the trapping tools in the same way as the optical tweezers . in theory , the trapping force is formed by the combination between the gradient field and scattering photons , which is reviewed . by using the intense optical vortices generated within the panda ring resonator , the required molecules can be trapped and moved ( transported ) dynamically within the wavelength router or network , ie , a molecular buffer . </S>", "<S> this can be performed within the wavelength router before reaching the required destination . </S>", "<S> the advantage of the proposed system is that a transmitter and receiver can be formed within the same system , which is available for molecule storage and transportation . </S>"], "labels": null, "section_names": ["Introduction", "Theory", "Molecular buffer", "Conclusion"], "sections": [["optical buffers are recognized as essential components in wavelength routers , in which the packets of data can be stored for resolving packet contention problems and also delay the outgoing packets.1,2 in practice , the optical router patents have been proposed and recorded,35 which can be useful for various applications .", "recently , the promising techniques of microscopic volume trapping and transportation within the add / drop multiplexer have been reported in both theory6 and experiment,7 respectively , in which the transporter is known as an optical tweezer . here , the optical tweezer generation technique has become a powerful tool for the manipulation of micrometer - sized particles . to date", "moreover , the use of dynamic tweezers is now also understood in practical works.810 schulz et al11 have shown that the transfer of trapped atoms between two optical potentials can be performed . in principle , optical tweezers use forces exerted by intensity gradients in the strongly focused beams of light to trap and move the microscopic volumes of matters , in which the other combination of force is induced by the interaction between photons , which is caused by photon scattering effects . in application , the field intensity can be adjusted and tuned , in which the desired gradient field and scattering force can form the suitable trapping force . hence , the appropriate force can be configured for the transmitter / receiver , which can perform the long distance microscopic transportation . in this paper , dynamic optical tweezers / vortices", "are generated using a dark soliton , bright soliton , and gaussian pulse propagating within an add / drop optical multiplexer incorporating two nanoring resonators ( panda ring resonator ) .", "the dynamic behaviors of solitons and gaussian pulses are well described by tasakorn et al.12 by using the proposed system , the transceiver can be integrated and performed by using a single device . here , the use of the transceiver to form the transportation of microscopic volumes of matter , especially13 for molecule transportation in liquid core waveguide,14,15 drug delivery , and dna transportation , in which the buffer is needed before reaching the required destination ."], ["in operation , the trapping forces are exerted by the intensity gradients in the strongly focused beams of light to trap and move the microscopic volumes of matters , in which the optical forces are customarily defined by the relationship:16 \n ( 1)f = qnmpc here q is a dimensionless efficiency , nm is the refractive index of the suspending medium , c is the speed of light , and p is the incident laser power , measured at the specimen .", "q represents the fraction of power utilized to exert force . for plane wave incidences on a perfectly absorbing particle", "because biological specimens are usually contained in aqueous media , the dependence of f on nm can rarely be exploited to achieve higher trapping forces .", "increasing the laser power is possible , but only over a limited range due to the possibility of optical damage .", "it depends upon the numerical aperture ( na ) , laser wavelength , light polarization state , laser mode structure , relative index of refraction , and the geometry of the particle .", "furthermore , in the rayleigh regime , trapping forces decompose naturally into two components . since , in this limit , the electromagnetic field is uniform across the dielectric , particles can be treated as induced point dipoles .", "the scattering force is given by:16 \n ( 2)fscatt = nmsc , \n ( 3)=83(kr)4r2(m21m2 + 2)2 here is the scattering cross section of a rayleigh sphere with radius r. s is the time averaged poynting vector , n is the index of refraction of the particle , m = n / nm is the relative index , and k = 2nm/ is the wave number of the light .", "the scattering force is proportional to the energy flux and points along the direction of the propagation of the incident light .", "the gradient field ( fgrad ) is the lorentz force acting on the dipole induced by the light field .", "it is given by:16 \n ( 4)fgrad=2e2,where \n ( 5)=nm2r3(m21m2 + 2)is the polarizability of the particle .", "the gradient force is proportional and parallel to the gradient in energy density ( for m > 1 ) .", "the stable trapping requires that the gradient force is in the  direction , which is against the direction of incident light ( dark soliton valley ) .", "it is greater than the scattering force . by increasing the na , when the focal spot size is decreased , the gradient strength is increased,16,17 which happens within a tiny system , for instance , a nanoscale device such as the nanoring resonator . in our proposal ,", "the trapping force is formed by using a dark soliton , in which the valley of the dark soliton is generated and controlled within the panda ring resonator by the control port signals . in this paper", ", we used the same theory of optical trapping and ring resonator , in which the simulation results and applications are differed from the previous work.18 from figure 1 , the output field ( et1 ) at the through port is given by:19 \n ( 6)et1=aei1bei2el2jknl2  [cet1(el2jknl2)2+dei2(el2jknl2)31f(e2ljknl2)2]where \n a \n ( 11)(12),b \n ( 11)(12)1(12)e0l , c \n 1(11)(12)2e0e0l , d \n ( 11)(12)1(11)2(12)e0e0l2 , andf \n ( 11)(12)(11)(12)e0e0l . \n", "( 11)(12)1(12)e0l , \n 1(11)(12)2e0e0l , \n ( 11)(12)1(11)2(12)e0e0l2 , and \n ( 11)(12)(11)(12)e0e0l . here ,", "et and ed represent the optical fields of the through port and drop ports , respectively .", " = kneff is the propagation constant , neff is the effective refractive index of the waveguide , and the circumference of the ring is l = 2r , where r is the radius of the ring . 1 and 2 are the coupling coefficients of the add / drop filters , kn = 2/ is the wave propagation number for in a vacuum , and the waveguide ( ring resonator ) loss is  = 0.5 dbmm .", "the fractional coupler intensity loss is  = 0.1 . in the case of the add / drop device", ", the nonlinear refractive index does not affect the system , therefore , it is neglected .", "the electric fields e0 and e0l are the field circulated within the nanoring at the right and left side of add / drop optical filter .", "the power output ( pt1 ) at through port is written as : \n ( 7)pt1=|et1|2 . the output field (", "et2)at drop port is expressed as:19 \n ( 8)et2=(12)(12)ei2  [(11)(12)12e0ei1el2jknl2+xe0e0lle2i(el2jknl2)21ye0e0l(e2ljknl2)2],where \n x \n ( 12)(11)(11)2(12),y = \n ( 11)(12)(11)(12 ) \n ( 12)(11)(11)2(12 ) , \n ( 11)(12)(11)(12 ) the power output ( pt2 ) at drop port is : \n ( 9)pt2=|et2|2 . in operation , the optical tweezers can be trapped , transported , and stored within the panda ring resonator and wavelength router , which can be used to form the microscopic volume ( molecule ) transportation and drug delivery via the waveguide,18 in which the manipulation of trapped microscopic volumes within the optical tweezers has been reported ."], ["molecular buffers are devices that can be used to store or delay atoms / molecules for a period of time ( see figure 2 ) , where light intensity and velocity can also be controlled , which was described by rosenberry et al20 and lignie and woerdman,21 available for medical application .", "molecular buffers are a new device , which are operated in the same way as gas buffers.22 the polarizability of the particle is calculated by using equation ( 5 ) , in this case , we assume that the sphere particle is polystyrene ( n = 1.5894 ) and the liquid medium is water ( n = 1.33 ) , and the optical power which is required to trap particles of a certain size / polarizibility is 9.1w , which is the slope as shown in figure 3a . in simulation , the bright soliton with center wavelength at 1.50 m , peak power 2w , pulse 35fs is input into the system via the input port , and the coupling coefficients are given as 0 = 0.5 , 1 = 0.35 , 2 = 0.1 , and 3 = 0.35 , respectively .", "the ring radii are radd = 10 and 30 m , rr = 50 and 100 nm , and rl = 50 and 100 nm , respectively . to date , the evidence of a practical device with a radius of 30 nm has been reported by piyatamrong et al.19 aeff are 0.50 , 0.25 , and 0.25 m . in this case", ", the dynamic tweezers ( gradient fields ) can be in the form of bright solitons , gaussian pulses , and dark solitons , which can be used to trap the required microscopic volume .", "there are four different center wavelengths of tweezers generated ; the dynamical movements are seen in figure 4 , where ( a ) |e1| , ( b ) |e2| , ( c ) |e3| , ( d ) |e4| , ( e ) through port , and ( f ) drop port signals , where in this case all microscopic volumes are received by the drop port . in practice ,", "the fabrication parameters which can be easily controlled are the ring resonator radii instead of the coupling constants .", "the important aspect of the result is that the tunable tweezers can be obtained by tuning ( controlling ) the add ( control ) port input signal , in which the required amount of microscopic volume ( atom / photon / molecule ) can be obtained and seen at the drop / through ports , otherwise they propagate within a panda ring before collapsing / decaying into the waveguide .", "more results of the optical tweezers generated within the panda ring are shown in figure 5 , where in this case , the bright soliton is used as the control port signal to obtain the tunable results .", "the output optical tweezers of the through and drop ports with different coupling constants are as shown in figure 5a , while the different wavelength results are as shown in figure 5b , which can be performed by the selected targets . in application", ", the trapped microscopic volumes ( molecules ) can move into the wavelength router via the through port , while the retrieved microscopic volumes are received via the drop port ( connecting target ) .", "the advantage of the proposed system is that the transmitter and receiver can be fabricated on - chip and alternatively operated by a single device .", "the magnitude of the optical trapping force is the pico newton , which depends upon the relative refractive index of particle , which was given by kumar et al.23 the particle radius was given by hu et al,24 fischer and srensen,25 and nieminen et al,26 which is located in the cavity ."], ["we have proposed a new system that can be used to trap ( delay ) and transport molecules into an optical waveguide by using optical tweezers , which can be used for drug storage and as a delivery system . by utilizing the reasonable dark soliton input power ,", "the dynamic tweezers can be controlled and stored ( delayed ) within the system before reaching the final destination .", "tweezer amplification is also available by using the nanoring resonators , in which the signals can be modulated via the control port as shown in figures 4b and 5a . in conclusion", ", we have shown that the use of a transceiver for long distance microscopic volume by using the proposed system , in which the drug delivery or molecular communication can be performed via the wavelength router to the required ( connecting ) targets . however , the problems of large microscopic volume and neutral matter may cause a problem , in which the pursuit of new guide pipes and media,27 for instance , nano tubes and specific gases , will be the issue of investigation ."]]}
{"article_id": "PMC3533157", "article_text": ["assessment of effects of different assisted reproductive techniques ( art ) like in vitro fertilization ( ivf ) and intra cytoplasmic sperm injection ( icsi ) on speech and language development needs comprehensive studies .", "some studies showed that they are more susceptible to cerebral palsy which causes speech and language defects .", "development of speech and language consists of four periods : prelinguistic , linguistic , school and adolescence period .", "defects in prelinguistic behavior , i.e. physical defect like cleft palate , neurological defect like cerebral palsy or developmental defect like down syndrome produce speech defects . in two studies in belgium and finland in infants of art", "there was no difference between development of these children with infants of normal conception[5 , 6 ] . in another study", "no differences in mental , motor , social and expressive language development were found , and while receptive language development was in the normal range , ivf infants scored lower than control infants .", "one study did not report any excess of neurodevelopmental disorders in ivf / icsi children .", "the majority of studies followed the children during infancy , thereby precluding pertinent conclusion on the risk of neurodevelopmental disorders which express at older ages such as fine manipulation disability or dyslexia .", "one study shows that a large proportions of preterm deliveries in ivf children have increased risk of cerebral palsy .", "regarding the importance of this subject and the lack of a comprehensive study on the prelinguistic behavior of infants of art in iran , this study was designed .", "the aim of this study is to evaluate prelinguistic behavior of 9 month old art infants of royan institute .", "in this descriptive , cross sectional study , 151 term infants of art from royan institute have been evaluated in children 's health and development research center of tehran from august 2007 until august 2009 .", "the research ethics committee of the academic center of education , culture and research ( acecr ) and royan institute approved the study .", "the sampling method was non random sequential with the inclusion criteria of infants conceived through one of the art methods ( ivf / icsi ) , born in term and being resident in tehran .", "preterm born infants were excluded . after signing of research consents by parents , prelinguistic behavior of 151 infants who were born full term ( > 37 weeks ) are assessed .", "this scale is used for assessment of development of speech and language in children from birth to 3 years old .", "it has sensitivity of 90 - 95% for primary detection of speech , language and cognition defects .", "the method of art , sex of infants , age of mother at pregnancy , time and speech and language defect of parents at present and past time also were mentioned in questionnaire .", "the prelinguistic behavior of infants include : crying at birth , different cries , smiling , cooing , babbling , reduplicated babbling and echolalia , which are assessed in this study .", "one - hundred one term infants of art were evaluated . in this study , 76 ( 50.5% ) were male and 75 ( 49.5% ) female .", "there was 129 ( 85.4% ) of infants conceived by icsi and 22 ( 14.5% ) by ivf .", "prelinguistic behavior delay which had been assessed according to elm-2 is shown in table 1 .", "number of infants with delay in reduplicated babbling in icsi method was more than un ivf . \n ", "prevalence of prelinguistic behavior delay in assisted reproductive techniques methods ivf : invitro fertilization ; icsi : intra cytoplasmic sperm injection the prelinguistic behavior delays in different sex and mothers age is shown in table 2 . there was a significant difference only in echolalia delay in the two sexes .", "prevalence of prelinguistic behavior delay in different sexes and mothers age mothers of 118 infants were younger than 35 years and 33 ones were older .", "two studies showed that art infants had normal cognition development [ 11 , 12 ] .", "zhu jl in showed that infertility treatment , especially icsi , may be associated with a slightly delayed cognitive language development .", "another study showed that there was no difference between cognitive development of infants of art and normal infants . in this study infants showed delay in reduplicated babbling and echolalia . in a study by sutcliffe children were assessed with the griffiths mental  development scales .", "icsi children were around the midpoint for the griffiths scales and did not differ significantly for griffiths quotients and suhquotients .", "one study reported significantly lower mental scores in 1 year old infants born after icsi than in age matched infants born after ivf and naturally conceived infants .", "stratification for gender revealed that lower mental development index scores were only found in boys , not in girls . in our study delay of reduplicated babbling and echolalia were more in boys .", "there is no study about effect of mother 's age on speech and language development in infants .", "bonduelle and his collegues in their study , by selection of infants of mothers with the same age exclude affect of this variable . in our study delay of reduplicated", "there was no relation between speech and language defect of parents and prelinguistic behavior of infants .", "limited sample size , difficult access to this group of infants and unwillingness of parents to complete the questionnaires render less reliable results .", "this study was a new study in our country which shows differences between art infants in some prelinguistic behavior such as reduplicated babbling and echolalia .", "this study showed that prelinguistic behavior of art infants are affected by art method , infants sex and mother 's age at the time of pregnancy .", "detection and correction of any defect or delay in this period prevent major speech and language problems .", "more studies with larger sample size to compare art infants with infants of normal conception would be desirable ."], "abstract_text": ["<S> objectivethe aim of this study is assessment of effects of different assisted reproductive techniques ( art ) like in vitro fertilization ( ivf ) and intra cytoplasmic sperm injection ( icsi ) on prelinguistic behavior of infants conceived by these techniques.methodsin this descriptive , cross sectional study , prelinguistic behavior of 151 full term art infants of royan institute have been assessed in children 's health and development research center of tehran from august 2007 until august 2009 . </S>", "<S> questionnaires were completed by parents at 9 months old . </S>", "<S> the questionnaire was standard according to early language milestone scale-2 ( elm-2 ) . </S>", "<S> data were analyzed by spss version 16 and using chi - square test.findingstwenty-two ( 14.5% ) of infants were conceived by ivf and 129 ( 85.4% ) by icsi . </S>", "<S> number of infants with delay in reduplicated babbling in icsi method was more than in ivf . </S>", "<S> there was only a significant difference in echolalia delay in the two sexes . </S>", "<S> echolalia was delayed more in boys . </S>", "<S> delay of reduplicated babbling was more in infants of younger mothers . </S>", "<S> there was no relation between speech and language defect of parents and infants.conclusionthis study showed that prelingustic behavior of art infants are affected by kind of art method , infant sex , and mother 's age at the time of pregnancy . </S>"], "labels": null, "section_names": ["Introduction", "Subjects and Methods", "Findings", "Discussion", "Conclusion", "Conflict of Interest"], "sections": [["assessment of effects of different assisted reproductive techniques ( art ) like in vitro fertilization ( ivf ) and intra cytoplasmic sperm injection ( icsi ) on speech and language development needs comprehensive studies .", "some studies showed that they are more susceptible to cerebral palsy which causes speech and language defects .", "development of speech and language consists of four periods : prelinguistic , linguistic , school and adolescence period .", "defects in prelinguistic behavior , i.e. physical defect like cleft palate , neurological defect like cerebral palsy or developmental defect like down syndrome produce speech defects . in two studies in belgium and finland in infants of art", "there was no difference between development of these children with infants of normal conception[5 , 6 ] . in another study", "no differences in mental , motor , social and expressive language development were found , and while receptive language development was in the normal range , ivf infants scored lower than control infants .", "one study did not report any excess of neurodevelopmental disorders in ivf / icsi children .", "the majority of studies followed the children during infancy , thereby precluding pertinent conclusion on the risk of neurodevelopmental disorders which express at older ages such as fine manipulation disability or dyslexia .", "one study shows that a large proportions of preterm deliveries in ivf children have increased risk of cerebral palsy .", "regarding the importance of this subject and the lack of a comprehensive study on the prelinguistic behavior of infants of art in iran , this study was designed .", "the aim of this study is to evaluate prelinguistic behavior of 9 month old art infants of royan institute ."], ["in this descriptive , cross sectional study , 151 term infants of art from royan institute have been evaluated in children 's health and development research center of tehran from august 2007 until august 2009 .", "the research ethics committee of the academic center of education , culture and research ( acecr ) and royan institute approved the study .", "the sampling method was non random sequential with the inclusion criteria of infants conceived through one of the art methods ( ivf / icsi ) , born in term and being resident in tehran .", "preterm born infants were excluded . after signing of research consents by parents , prelinguistic behavior of 151 infants who were born full term ( > 37 weeks ) are assessed .", "this scale is used for assessment of development of speech and language in children from birth to 3 years old .", "it has sensitivity of 90 - 95% for primary detection of speech , language and cognition defects .", "the method of art , sex of infants , age of mother at pregnancy , time and speech and language defect of parents at present and past time also were mentioned in questionnaire .", "the prelinguistic behavior of infants include : crying at birth , different cries , smiling , cooing , babbling , reduplicated babbling and echolalia , which are assessed in this study ."], ["one - hundred one term infants of art were evaluated . in this study , 76 ( 50.5% ) were male and 75 ( 49.5% ) female .", "there was 129 ( 85.4% ) of infants conceived by icsi and 22 ( 14.5% ) by ivf .", "prelinguistic behavior delay which had been assessed according to elm-2 is shown in table 1 .", "number of infants with delay in reduplicated babbling in icsi method was more than un ivf . \n ", "prevalence of prelinguistic behavior delay in assisted reproductive techniques methods ivf : invitro fertilization ; icsi : intra cytoplasmic sperm injection the prelinguistic behavior delays in different sex and mothers age is shown in table 2 . there was a significant difference only in echolalia delay in the two sexes .", "prevalence of prelinguistic behavior delay in different sexes and mothers age mothers of 118 infants were younger than 35 years and 33 ones were older ."], ["two studies showed that art infants had normal cognition development [ 11 , 12 ] .", "zhu jl in showed that infertility treatment , especially icsi , may be associated with a slightly delayed cognitive language development .", "another study showed that there was no difference between cognitive development of infants of art and normal infants . in this study infants showed delay in reduplicated babbling and echolalia . in a study by sutcliffe children were assessed with the griffiths mental  development scales .", "icsi children were around the midpoint for the griffiths scales and did not differ significantly for griffiths quotients and suhquotients .", "one study reported significantly lower mental scores in 1 year old infants born after icsi than in age matched infants born after ivf and naturally conceived infants .", "stratification for gender revealed that lower mental development index scores were only found in boys , not in girls . in our study delay of reduplicated babbling and echolalia were more in boys .", "there is no study about effect of mother 's age on speech and language development in infants .", "bonduelle and his collegues in their study , by selection of infants of mothers with the same age exclude affect of this variable . in our study delay of reduplicated", "there was no relation between speech and language defect of parents and prelinguistic behavior of infants .", "limited sample size , difficult access to this group of infants and unwillingness of parents to complete the questionnaires render less reliable results ."], ["this study was a new study in our country which shows differences between art infants in some prelinguistic behavior such as reduplicated babbling and echolalia .", "this study showed that prelinguistic behavior of art infants are affected by art method , infants sex and mother 's age at the time of pregnancy .", "detection and correction of any defect or delay in this period prevent major speech and language problems .", "more studies with larger sample size to compare art infants with infants of normal conception would be desirable ."], [""]]}
{"article_id": "PMC3317066", "article_text": ["a number of drugs are currently available to treat malaria ; however , treatment is becoming complicated by drug resistance , toxicity , and high cost .", "recently , drug resistance against the new effective drug , artemisinin , is also emerging [ 4 , 5 ] , and we need new effective drugs to treat malaria .", "therefore , the development of other classes of effective antimalarials , especially compounds that act against novel biochemical targets , is required .", "to develop such compounds , it is very important to characterize the structural and biochemical features of new drug targets . among potential new targets for antimalarial chemotherapy", "proteases are druggable targets , and at present protease inhibitors are now licensed as well as in clinical development to treat different diseases for example osteoporosis , diabetes , cancer , hypertension , and infectious diseases [ 69 ] .", "recent advances , including the sequencing of plasmodium genomes ( http://www.plasmodb.org/ ) and development of new tools for manipulating plasmodium genes , have improved our understanding of the cysteine proteases of parasites .", "therefore , given the importance of the cysteine proteases , this paper will focus on structural - functional relationship of falcipains , major cysteine proteases of p. falciparum .   in cases of malaria ,", "the parasite relies on human hemoglobin hydrolysis to supply amino acids for protein synthesis and to maintain osmotic stability [ 11 , 12 ] .", "cysteine proteases are involved in hemoglobin hydrolysis and have been validated as promising drug targets [ 13 , 14 ] . in a recent report , ch'ng et al .", ", using cysteine protease inhibitors , demonstrate that clan ca cysteine proteases of p. falciparum are also involved in chloroquine - mediated programmed cell death .", "the best characterized plasmodium cysteine proteases are the falcipains , papain family ( clan ca ) enzymes ( figure 1 ) . among the four p. falciparum cysteine proteases , falcipain-2 and falcipain-3 appear to be the principal food vacuolar hemoglobinases [ 1618 ] .", "when the falcipain-2 gene is disrupted , undegraded hemoglobin accumulates in the food vacuole , confirming that this enzyme participates in haemoglobin hydrolysis .", "however , disruption of falcipain-3 could not be achieved , but the gene was readily replaced with a tagged functional copy , indicating that falcipain-3 is essential for erythrocytic parasites .", "falcipain-2 and falcipain-3 share 67% sequence identity and contribute more or less equally to the digestion of hemoglobin in the food vacuole . comparing with other papain family proteases ,", "specifically , this paper will survey available information on structure and function of different domains of falcipain-2 and falcipain-3 and their interaction with inhibitors .", "falcipains are major cysteine protease , named due to the function of a catalytic cysteine , which mediates protein hydrolysis via nucleophilic attack on the carbonyl carbon of a susceptible peptide bond .", "falcipains have two main domains , the prodomain and the mature domain . the prodomain is further divided into different small domains . at the n - terminus of the prodomain ,", "the first 35 amino acids are cytosolic , followed by a 20 amino acid transmembrane domain and a 188 amino acid lumenal domain .", "the inhibitory domain is present in the c - terminal part of the prodomain ( figure 2 ) .", "cysteine proteases of the parasite hydrolyze hemoglobin in the acidic food vacuole [ 20 , 21 ] .", "recently , it has been demonstrated that prodomain has unique motifs which are responsible for targeting the falcipains . using transfection technology and constructing different chimeras with portions of the n - terminal part of prodomain", "fused to green fluorescent protein , it has been shown that the prodomains of falcipain-2 and falcipain-3 were sufficient to target green fluorescent protein to the food vacuole .", "once enzymes are in the er , using the signal from transmembrane domain , the proteins appear to traffic to the plasma membrane . at the plasma membrane falcipain-2 and falcipain-3", "appear to be endocytosed in a process requiring the presence of both cytoplasmic and luminal prodomain trafficking motifs , followed by vesicular transport to the food vacuole .  ", "in summary , serial truncation and deletion studies showed that both a 20-amino acid stretch of the lumenal portion and a 10-amino acid stretch of the cytoplasmic portion of the enzymes were essential for food vacuolar trafficking ( figure 3 ) .", "like many other proteases , falcipains are synthesized as a zymogen , and the prodomain inhibits the activity of the mature enzyme . to investigate how the prodomain regulates the activity of falcipain-2 , pandey et al", ". expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2 .", "it has been found that a c - terminal segment ( leu  asp ) of the prodomain , including two motifs ( erfnin and gnfd ) that are also conserved in cathepsin l subfamily proteases , mediates prodomain inhibitory activity .", "earlier work with other cathepsin l subfamily proteases suggests key roles for conserved hydrophobic amino acids ( phenylalanine and tryptophan ) as well as the erfnin and gnfd motifs in maintaining prodomain structure .", "later , pandey et al . explored the roles of conserved motifs in maintaining prodomain structure by circular dichroism analysis .", "secondary structure was seen in a fragment with potent inhibitory activity ( leu  asp ) , but not in two larger constructs that lacked any sequence downstream of the erfnin and gnfd motifs or in a peptide spanning the erfnin and gnfd motifs .", "these results indicate that , together with erfnin and gnfd motifs , an upstream region including two conserved phe * residues is required for proper folding or maintenance of secondary structure of the prodomain ( figure 2 ) .", "the 3d structures of the mature domain of falcipain-2 and falcipain-3 have been solved [ 25 , 26 ] , and the profalcipain-2 model suggests that conserved residues provide stability to the inhibitory domain ( figure 4(a ) ) .", "modeling , based on the solved structure of procathepsin b and procathepsin l [ 27 , 28 ] , shows that the charged pair arg and glu of the prodomain appear to form a salt bridge ( figure 4(b ) ) .", "further , glu from the gnfd motif may form a separate salt bridge with lys in the mature domain .", "phe may participate in nonpolar interactions and possibly pi stacking interactions , with two tryptophan residues in the mature domain , trp and trp .", "the model also suggests that the prodomain of falcipain-2 binds the mature domain in a manner similar to that of procathepsin k and l , inhibiting catalytic activity by blocking substrate access to the active site ( figure 4(a ) ) .", "they encode short n - terminal extensions of the mature domain , which is unique among described papain family proteases [ 13 , 14 ] .", "looking at the family of c1a cysteine protease sequences on the merops database ( http://merops.sanger.ac.uk/ ) have identified more than 40 members with an n - terminus extension of 12 amino acids at this location .", "notably , 18 of these sequences were those of falcipains and homologues from other plasmodial species", "to better characterize the determinants of folding for falcipain-2 , pandey and sijwali et al . expressed multiple prodomain and mature constructs of the enzyme in e. coli and assessed their abilities to refold by checking their activity [ 23 , 29 ]", "this folding study showed that refolding of the mature domain could be achieved when the mature domain was covalently joined with the n - terminus extension ( figure 5 ) .", "only 12 amino acids of the n - terminus extension ( refolding domain ) are necessary for refolding .", "deletion of 12 amino acids from n - terminus segment of the mature domain yielded a construct incapable of correct folding , but inclusion of this segment in trans allowed folding to active falcipain-2 .", "correct folding also occurred when the catalytic domain was refolded with a separate prodomain - folding domain construct but not with an isolated folding domain peptide [ 23 , 29 ] ( figure 5 ) .", "these results indicated that the prodomain mediates the interaction between the mature and folding domain when they were not covalently bound .", "however , it was not clear from these studies , whether the amino - terminal extension is required only for folding or whether it also plays an essential role in mediating enzyme activity . to determine whether the folding domain was required for activity , pandey et al", "after refolding and purification of the mature domain by anion exchange chromatography , the purified refolded mature domain still showed activity .", "together , these results suggest that the refolding domain is only required for refolding , and once refolding is accomplished , the refolding domain is not required for activity . the n - terminus extensions of different falcipain subfamily enzymes have limited ( 2045% ) sequence identity but are functionally conserved .", "chimeras of the falcipain-2 catalytic domain with the refolding domain of six other plasmodial proteases folded normally and had similar kinetic parameters to those of recombinant falcipain-2 .", "these results showed that refolding domain can be swapped between the plasmodial proteases that harbor the same function .", "the above - mentioned experiment also indicates that all plasmodial cysteine proteases including falcipain-3 also use n - terminus extension as a refolding domain .", "structural analysis further reveals that the folding domain , in fact , has a short but significant element of secondary structure .", "glutamate of the mature domain forms a buried hydrogen bond with tyrosine and a salt bridge with arginine of the refolding domain .", "this suggests that the small folding domain , though shorter than the standard papain family prodomain , still plays a very important role in folding by stabilizing the mature domain by a hydrogen bond and a salt bridge .", "this is analogous to a similar interaction in falcipain-2 , where tryosine of the refolding domain of falcipain-3 provides hydrogen bonding anchor to glutamate of the mature domain of falcipain-3 .", "these results indicate that the folding domains of falcipain-2 and falcipain-3 stabilize the mature domain in a similar fashion .", "falcipain-2 and 3 efficiently hydrolyze human hemoglobin in the acidic food vacuole of parasite [ 17 , 18 ] .", "it has recently been suggested that hemoglobin hydrolysis is not a highly ordered process but rather proceeds with rapid cleavage by falcipains at multiple sites .", "falcipain subfamily proteases contain an unusual motif near the c - terminus , which is present between the highly conserved active site histidine and asparagine residues ( figure 2 ) .", "a motif of identical size ( 14 aa ) is found in all studied proteases of this subfamily of falcipain , although sequence identity is modest . in case of falcipain-2 and falcipain-3 , motifs", "smaller motifs are present in the sequences encoding p. falciparum serine repeat antigens ( 10 aa ) and dipeptidyl peptidase", "i ( 8 aa ) , and larger ( 20 aa ) motifs are present in two other putative p. falciparum dipeptidyl peptidase genes [ 14 , 33 ] .", "to evaluate the function of the unusual c - terminal motif in falcipains , a mutant enzyme was made by deleting that motif lacking 10 amino acids from falcipain-2 , and the biochemical properties of wild and mutant enzymes were compared .", "native page , biacore , and gel filtration studies indicated that the motif mediates specific interactions with hemoglobin .", "in fact , falcipain-2 has relatively higher affinity for methemoglobin ( kd is 0.8  m ) than hemoglobin ( kda is 3.3  m ) .", "it had been demonstrated that several factors contribute to the formation of methemoglobin during malarial infection , including acidic ph of plasmodial food vacuole , oxidative damage in rbc [ 35 , 36 ] , which causes an increase in methemoglobin content to up to 2042% in the plasmodial food vacuole .", "thus , the higher affinity of falcipain-2 for methemoglobin looks like an adaptation to the specific conditions in the infected rbc .", "the enzyme without this motif showed negligible activity against hemoglobin or globin . further , a peptide encoding the motif blocked hemoglobin hydrolysis but not the hydrolysis of casein , indicating that the motif specifically interacts with hemoglobin .", "thus , this study suggests that the motif mediates the most biologically relevant activity of falcipain , the hydrolysis of hemoglobin during trophozoite stage .", "falcipains may also have other functions in erythrocytic malaria parasites , and it would be interesting to explore the role of the motif in hydrolysis of other proteins of potential biological relevance . in the case of other members of the falcipain", "subfamily , it is likely that the encoded motifs share the same functions as the falcipain motif , because a number of members of this subfamily have been also shown to be hemoglobinases .", "the sequence conservation among the motifs of falcipain subfamily proteases is not high , but , as in the case of the n - terminal extension that mediates protein folding , the presence of these unusual motifs in all related proteases probably indicates a conserved function . for genes encoding more distantly related enzymes , including falcipain-1 , dipeptidyl peptidases , and serine repeat antigens , c - terminal motifs vary greatly in sequence and size , and specific functions of predicted motifs are undefined .", "data indicates that falcipain-2 captures hemoglobin via its c - terminal motif before subsequently cleaving the substrate at multiple sites .", "further , the structure of falcipain indicates a protruding configuration for the motif , surrounded by a predominant negative charge , and it may be that charged residues are crucial for interaction with hemoglobin .", "it is proposed that hemoglobin , which has many charged surface residues , first binds at the motif through charge - charge interaction and brings hemoglobin closer to the active site before hydrolysis .", "the function of the motif in mediating catalysis despite its separation from the active site somewhat resembles that of the hemopexin domain of matrix metalloproteases .", "the hemopexin domain is required for both the efficient cleavage of collagen   and the formation of complexes with both inhibitors and proteases to mediate biological activities .", "thus , as is the case for the falcipain-2 motif , the hemopexin domain serves to facilitate biologically relevant protein - substrate and protein - inhibitor interactions .", "however , the hemopexin domain is approximately 20 times larger than the falcipain motif and , unlike the motif , is separated from the catalytic domain by a hinge region .", "therefore , it appears that falcipains and matrix metalloproteases use similar means of biological control , but the specific mechanisms by which the proteases mediate interactions with substrates and inhibitors are different .", "another papain family protease , cathepsin k , forms a pentameric complex with chondroitin sulfate to facilitate hydrolysis of collagen . having analogy to falcipain-2-hemoglobin interaction , structural requirements for complex formation between cathepsin k and chondroitin sulfate", "it is interesting to raise a question , why do falcipains contain a unique motif that mediates the hydrolysis of hemoglobin ?", "this mechanism might not have evolved simply to facilitate hemoglobin hydrolysis , because other papain family enzymes without c - terminus motifs still can hydrolyze hemoglobin .", "it looks like that the utilization of a specific motif at this region to mediate enzyme - substrate interaction is an unusual but conserved feature of plasmodial cysteine proteases .", "it may be possible that an evolutionary bottleneck occurred in ancestral plasmodial cysteine proteases , and those enzymes might not be that efficient in hemoglobin hydrolysis .", "in addition , plasmodial cysteine proteases might have evolved with introduction of specific motif for efficient hemoglobin hydrolysis .", "the interaction of a major hemoglobinase , falcipain-2 , with most biologically relevant substrate , hemoglobin , could be an interesting starting point for the development of an effective drug .", "the structure of falcipain-2 hemoglobin binding domain will guide the design of inhibitors that should interfere with hemoglobin binding .", "there are two major classes of cysteine protease inhibitor , small inhibitors like leupeptin , vinyl sulfones , e64 , and another class known as macromolecular inhibitor .", "these endogenous cysteine protease inhibitors have been described in a number of eukaryotic systems . here", ", we will discuss three major cysteine protease inhibitors , cystatin , chagasin , and falstatin .", "excluding the two unique motifs discussed earlier , the rest of the falcipains are structurally similar to homologous proteases in the papain family .", "cystatin was used as a falcipain inhibitor with the expectation that protein - protein interaction over large surfaces would enhance crystal quality and thus facilitate structural determination .", "cystatins inhibit a wide range of papain family cysteine proteases with high affinity making them ideal candidates for cocrystallization with falcipain .", "falcipain and chicken egg white cystatin formed 1  :  1 complex ( figure 6(a ) ) .", "the inhibitory constants ( ki ) of cystatin for falcipain-2 and falcipain-3 are 6.5  nm and 100  nm , respectively .", "it is interesting that cystatin is a more potent inhibitor of falcipain-2 than falcipain-3 , which suggests that cystatin regulates both the falcipains with different rates .", "the falcipain - cystatin complex shares many features with two known cysteine protease - cystatin structures : cathepsin h - stefin a and papain - stefin b ( the stefins are a subclass within the cystatin superfamily [ 41 , 42 ] ) . these cystatins bind to target proteases in similar orientations , resulting in extensive interactions with the target proteases ( figure 6(b ) ) .", "cystatin largely interacts with falcipain-2 at prime sites where substrate - binding pockets are relatively shallow and less defined , but falcipains also have binding specificity at nonprimed sites .", "although , the binding preference is not as pronounced as that found at the s2 pocket ( figure 6(b ) ) .", "cystatin is only able to access the solvent exposed periphery of the nonprime site of falcipain-2 and majority of its binding occurs at the prime end of the active site . unlike most other cathepsin l - like cysteine proteases ,", "crystal structures indicate that this preference may be due to ile in falcipains , because it creates a small protrusion in the otherwise flat base of the s2 pocket , which restricts aromatic side - chain binding   ( figure 6(b ) ) .", "the falcipain  cystatin complex can be exploited to design potent inhibitors of the malaria parasite .", "a recent study indicates that a peptide based on cystatin binding residues blocked the activity of falcipain-2 and led to accumulation of undegraded hemoglobin in the food vacuole .", "chagasin is a cysteine protease inhibitor that was first identified in trypanosoma cruzi as the physiological regulator of cruzain , the major cysteine protease [ 45 , 46 ] .", "cruzain is also a papain - like ( clan ca ) cysteine protease that is expressed in all stages of t. cruzi life cycle .", "it is a key virulence factor of t. cruzi , the infectious agent responsible for the leading cause of heart disease in latin america , called the chagas disease .", "chagasin is associated with cruzain during its trafficking to specific compartments of the parasite cell , and accumulated evidence suggests that the primary role of chagasin is in posttranslational regulation of protease activity .", "1 binding with falcipain-2 ( figure 7 ) . the protease - binding loops ( bc , de , and fg ) in chagasin form a well - aligned wedge that fills the active site groove of falcipain-2 to obstruct substrate binding ( figure 7 ) .", "the bc loop is one of the three signature motifs that contribute to the inhibition of the cysteine protease .", "this is confirmed by a synthetic peptide corresponding to the bc loop of the chagasin like protein from e. histolytica , which specifically blocked the activity of cysteine proteases .", "it is noteworthy that thr 31 in the bc loop of chagasin binds to the catalytic cys at the falcipain-2 active site by water - mediated hydrogen bonds ( figure 7 ) .", "it is likely that the highly conserved thr serves as a key functional residue among chagasin - like inhibitors .", "another interesting feature of the falcipain-2-chagasin interaction is the highly mobile de loop like in e64 , a strong irreversible inhibitor of cysteine protease , which occupies the nonprime site . in summary ,", "the bc loop , mobile de loop , and the rpw / f motif in the fg loop are the key elements for binding with falcipain-2 .", "disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer , rheumatoid arthritis , osteoporosis , and emphysema . in case of malaria ,", "falciparum parasites express falstatin , a potent inhibitor of falcipains and many other cysteine proteases .", "the stage - specificity of falstatin expression and the effects of anti - falstatin antibodies on parasite development suggest that this inhibitor facilitates a process that also requires proteolytic activity , the invasion of erythrocytes by p. falciparum merozoites .", "falstatin is a competitive and reversible inhibitor of falcipains , as demonstrated by increasing calculated km values but similar vmax values with increasing concentrations of falstatin . to evaluate the mechanism of inhibition of cysteine proteases by falstatin , pandey et al .", "tested the ability of active site - inhibited falcipain-2 to compete with active falcipain-2 for binding with falstatin .", "in contrast to results with the prodomain , the inhibitory effect of falstatin was not affected by the presence of active site inhibited falcipain-2 .", "thus , the binding of falstatin to falcipain-2 appears to be via interaction with the enzyme active site .", "leupeptin , e-64 , and vinyl sulfones are major cysteine protease inhibitors that bind to the active site [ 26 , 48 ] .", "the active sites of both enzymes are located in a cleft between the structurally distinct domain of the papain - like fold .", "the structures of falcipain-2 and falcipain-3 have been determined in complex with these small inhibitors .", "e-64 , and leupeptin interact with residues in the s1 , s2 , and s3 subsites of falcipain-2 and falcipain-3 , corresponding to the p1 , and p2 , and p3 position of the inhibitors ( figure 8) .", "the conserved catalytic residues of falcipain-2 and falcipain-3 ( gln , cys , his , asn , resp . )", "inhibitors display binding modes with their partner enzymes similar to those found in other papain family enzymes .", "the inhibitors bind to the main chain of falcipain-2 and falcipain-3 by glycine ( gly in falcipain-2 and gly in falcipain-3 ) residue that is highly conserved in the s3 subsite of clan ca cysteine proteases ( figure 9 ) . in cocrystallization , these residues form hydrogen bonds with the o and n atoms of the inhibitor backbone . in the case of the falcipain-2 complex with e-64 ,", "enzyme active sites gln , ser , cys , asn , and his are involved in the formation of additional hydrogen bonds with e-64 . in the falcipain-3-leupeptin complex ,", "gln , cys , and asn also act as hydrogen bonding partners to the inhibitor .", "the enzyme - inhibitor complex stabilize by a series of possible hydrophobic interaction using nonpolar region of gly , tyr , gly , leu , ser , leu , asn , and ala in falcipain-2 , and tyro , gly , tyr , ile , and ser in case of falcipain-3 .", "the active site cysteine of falcipain-2 forms a covalent , irreversible hemithioketal with the e-64 epoxy carbon ( figure 9 ) .", "the active site cysteine of falcipain-3 forms a covalent , reversible hemithioacetal with the asymmetric carbonyl carbon of leupeptin ( figure 10 ) . the interaction pattern of e-64 and leupeptin with all papain family enzymes is conserved .", "the carboxyl group of e-64 and carbonyl group of leupeptin occupy the oxyanion hole formed by the conserved catalytic residues . falcipain-2 and falcipain-3 have a clear preference for substrates / inhibitors that contains a leucine at p2 position , and both leupeptin and e64 contain a leu at p2 .", "surprisingly , falcipain-3 has been shown to be much less efficient at hydrolyzing peptide substrates and more difficult to inhibit with small peptidyl - based inhibitors compared with falcipain-2 [ 16 , 51 ] .", "the structure that forms the active site is highly conserved between falcipain-2 and falcipain-3 , and there is no notable movement in the peptide backbone or loop region .", "however , superimposition of falcipain-2 and falcipain-3 structures highlights two important substitutions in the s2 subsite .", "this position of the s2 subsite is known to be a key determiner of the specificity ( figures 9 and 10 ) . in comparison to asp in falcipain-2 ,", "the additional side chain carbon in glu of falcipain-3 , this increased the size as well as flexibility at this position . in case of falcipain-3leupeptin complex , the larger and more flexible residue ( glu ) at the bottom of s2 subsite and bulkier residue ( try ) create a narrow wall of the s2 subsite , as compared to the falcipain  e-64 complex ( figure 10 ) .", "the overall structures of falcipain-3leupeptin and falcipain-2e-64 complexes show a similar mode of binding and inhibition compared with macromolecule inhibitors like chagasin and cystatin .", "like e64 and leupeptin , vinyl sulphones ( mu - leu - hph - vsph ) have been shown to be effective inhibitors of a number of papain - family - like cysteine proteases .", "mu - leu - hph - vsph is a potent irreversible inhibitor of falcipain-2 and falcipain-3 .", "the cocrystallization of falcipain-3 with mu - leu - hph - vsph indicates that inhibitor binds the respective s1 and s3 subsites to form an irreversible , covalent bond with the sulfur of the active site cysteine thiol in enzyme   ( figure 11 ) .", "given the hydrophobic nature of the p1 and p2 substituents in mu - leu - hph - vsph , the active site of falcipain-3 is lined with a number of residues that are able to make nonpolar contacts with their respective inhibitor   ( figure 11 ) . as seen in the crystal structures of falcipain-3 with the above inhibitor , the residue at the bottom of the s2 subsite ( glu ) points out of the pocket to avoid a potentially unfavorable interaction with the bulky phe residue at the p2 position of inhibitor .", "cysteine proteases of the malaria parasite may be targeted for inhibition by vinyl sulphones . indeed , vinyl sulphones - based cysteine protease inhibitors are already being used in preclinical trials for the chagas disease .", "the understanding of the cysteine proteases of malaria parasites has increased markedly in recent years . since cysteine proteases that play an important role in the parasite life cycle by degrading erythrocyte proteins , most notably hemoglobin , are attractive targets for antimalarial chemotherapy . falcipain-2 and falcipain-3 are the best characterized cysteine proteases of malaria parasite , the structure and function of different domains and their interaction with small and macromolecular inhibitors are studied", ". the structure - function study of falcipains and interaction with inhibitors will provide detail insights to develop rational design of inhibitor against falcipains .", "structure - guided approaches should have great role in the design of potent and highly selective inhibitor .", "efforts to optimize current inhibitors based on the structure - function of falcipains are currently underway ."], "abstract_text": ["<S> evidence indicates that cysteine proteases play essential role in malaria parasites ; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria . </S>", "<S> studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis . </S>", "<S> the best characterized plasmodium cysteine proteases are falcipains , which are papain family enzymes . </S>", "<S> falcipain-2 and falcipain-3 are major hemoglobinases of p. falciparum . </S>", "<S> structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain . </S>", "<S> overall , the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria . </S>", "<S> drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Domains of Falcipains", "3. Structural Basis of Inhibition of Falcipains", "4. Summary and Conclusion"], "sections": [["a number of drugs are currently available to treat malaria ; however , treatment is becoming complicated by drug resistance , toxicity , and high cost .", "recently , drug resistance against the new effective drug , artemisinin , is also emerging [ 4 , 5 ] , and we need new effective drugs to treat malaria .", "therefore , the development of other classes of effective antimalarials , especially compounds that act against novel biochemical targets , is required .", "to develop such compounds , it is very important to characterize the structural and biochemical features of new drug targets . among potential new targets for antimalarial chemotherapy", "proteases are druggable targets , and at present protease inhibitors are now licensed as well as in clinical development to treat different diseases for example osteoporosis , diabetes , cancer , hypertension , and infectious diseases [ 69 ] .", "recent advances , including the sequencing of plasmodium genomes ( http://www.plasmodb.org/ ) and development of new tools for manipulating plasmodium genes , have improved our understanding of the cysteine proteases of parasites .", "therefore , given the importance of the cysteine proteases , this paper will focus on structural - functional relationship of falcipains , major cysteine proteases of p. falciparum .   in cases of malaria ,", "the parasite relies on human hemoglobin hydrolysis to supply amino acids for protein synthesis and to maintain osmotic stability [ 11 , 12 ] .", "cysteine proteases are involved in hemoglobin hydrolysis and have been validated as promising drug targets [ 13 , 14 ] . in a recent report , ch'ng et al .", ", using cysteine protease inhibitors , demonstrate that clan ca cysteine proteases of p. falciparum are also involved in chloroquine - mediated programmed cell death .", "the best characterized plasmodium cysteine proteases are the falcipains , papain family ( clan ca ) enzymes ( figure 1 ) . among the four p. falciparum cysteine proteases , falcipain-2 and falcipain-3 appear to be the principal food vacuolar hemoglobinases [ 1618 ] .", "when the falcipain-2 gene is disrupted , undegraded hemoglobin accumulates in the food vacuole , confirming that this enzyme participates in haemoglobin hydrolysis .", "however , disruption of falcipain-3 could not be achieved , but the gene was readily replaced with a tagged functional copy , indicating that falcipain-3 is essential for erythrocytic parasites .", "falcipain-2 and falcipain-3 share 67% sequence identity and contribute more or less equally to the digestion of hemoglobin in the food vacuole . comparing with other papain family proteases ,", "specifically , this paper will survey available information on structure and function of different domains of falcipain-2 and falcipain-3 and their interaction with inhibitors ."], ["falcipains are major cysteine protease , named due to the function of a catalytic cysteine , which mediates protein hydrolysis via nucleophilic attack on the carbonyl carbon of a susceptible peptide bond .", "falcipains have two main domains , the prodomain and the mature domain . the prodomain is further divided into different small domains . at the n - terminus of the prodomain ,", "the first 35 amino acids are cytosolic , followed by a 20 amino acid transmembrane domain and a 188 amino acid lumenal domain .", "the inhibitory domain is present in the c - terminal part of the prodomain ( figure 2 ) .", "cysteine proteases of the parasite hydrolyze hemoglobin in the acidic food vacuole [ 20 , 21 ] .", "recently , it has been demonstrated that prodomain has unique motifs which are responsible for targeting the falcipains . using transfection technology and constructing different chimeras with portions of the n - terminal part of prodomain", "fused to green fluorescent protein , it has been shown that the prodomains of falcipain-2 and falcipain-3 were sufficient to target green fluorescent protein to the food vacuole .", "once enzymes are in the er , using the signal from transmembrane domain , the proteins appear to traffic to the plasma membrane . at the plasma membrane falcipain-2 and falcipain-3", "appear to be endocytosed in a process requiring the presence of both cytoplasmic and luminal prodomain trafficking motifs , followed by vesicular transport to the food vacuole .  ", "in summary , serial truncation and deletion studies showed that both a 20-amino acid stretch of the lumenal portion and a 10-amino acid stretch of the cytoplasmic portion of the enzymes were essential for food vacuolar trafficking ( figure 3 ) .", "like many other proteases , falcipains are synthesized as a zymogen , and the prodomain inhibits the activity of the mature enzyme . to investigate how the prodomain regulates the activity of falcipain-2 , pandey et al", ". expressed constructs encoding different portions of the prodomain and tested their ability to inhibit recombinant mature falcipain-2 .", "it has been found that a c - terminal segment ( leu  asp ) of the prodomain , including two motifs ( erfnin and gnfd ) that are also conserved in cathepsin l subfamily proteases , mediates prodomain inhibitory activity .", "earlier work with other cathepsin l subfamily proteases suggests key roles for conserved hydrophobic amino acids ( phenylalanine and tryptophan ) as well as the erfnin and gnfd motifs in maintaining prodomain structure .", "later , pandey et al . explored the roles of conserved motifs in maintaining prodomain structure by circular dichroism analysis .", "secondary structure was seen in a fragment with potent inhibitory activity ( leu  asp ) , but not in two larger constructs that lacked any sequence downstream of the erfnin and gnfd motifs or in a peptide spanning the erfnin and gnfd motifs .", "these results indicate that , together with erfnin and gnfd motifs , an upstream region including two conserved phe * residues is required for proper folding or maintenance of secondary structure of the prodomain ( figure 2 ) .", "the 3d structures of the mature domain of falcipain-2 and falcipain-3 have been solved [ 25 , 26 ] , and the profalcipain-2 model suggests that conserved residues provide stability to the inhibitory domain ( figure 4(a ) ) .", "modeling , based on the solved structure of procathepsin b and procathepsin l [ 27 , 28 ] , shows that the charged pair arg and glu of the prodomain appear to form a salt bridge ( figure 4(b ) ) .", "further , glu from the gnfd motif may form a separate salt bridge with lys in the mature domain .", "phe may participate in nonpolar interactions and possibly pi stacking interactions , with two tryptophan residues in the mature domain , trp and trp .", "the model also suggests that the prodomain of falcipain-2 binds the mature domain in a manner similar to that of procathepsin k and l , inhibiting catalytic activity by blocking substrate access to the active site ( figure 4(a ) ) .", "they encode short n - terminal extensions of the mature domain , which is unique among described papain family proteases [ 13 , 14 ] .", "looking at the family of c1a cysteine protease sequences on the merops database ( http://merops.sanger.ac.uk/ ) have identified more than 40 members with an n - terminus extension of 12 amino acids at this location .", "notably , 18 of these sequences were those of falcipains and homologues from other plasmodial species", "to better characterize the determinants of folding for falcipain-2 , pandey and sijwali et al . expressed multiple prodomain and mature constructs of the enzyme in e. coli and assessed their abilities to refold by checking their activity [ 23 , 29 ]", "this folding study showed that refolding of the mature domain could be achieved when the mature domain was covalently joined with the n - terminus extension ( figure 5 ) .", "only 12 amino acids of the n - terminus extension ( refolding domain ) are necessary for refolding .", "deletion of 12 amino acids from n - terminus segment of the mature domain yielded a construct incapable of correct folding , but inclusion of this segment in trans allowed folding to active falcipain-2 .", "correct folding also occurred when the catalytic domain was refolded with a separate prodomain - folding domain construct but not with an isolated folding domain peptide [ 23 , 29 ] ( figure 5 ) .", "these results indicated that the prodomain mediates the interaction between the mature and folding domain when they were not covalently bound .", "however , it was not clear from these studies , whether the amino - terminal extension is required only for folding or whether it also plays an essential role in mediating enzyme activity . to determine whether the folding domain was required for activity , pandey et al", "after refolding and purification of the mature domain by anion exchange chromatography , the purified refolded mature domain still showed activity .", "together , these results suggest that the refolding domain is only required for refolding , and once refolding is accomplished , the refolding domain is not required for activity . the n - terminus extensions of different falcipain subfamily enzymes have limited ( 2045% ) sequence identity but are functionally conserved .", "chimeras of the falcipain-2 catalytic domain with the refolding domain of six other plasmodial proteases folded normally and had similar kinetic parameters to those of recombinant falcipain-2 .", "these results showed that refolding domain can be swapped between the plasmodial proteases that harbor the same function .", "the above - mentioned experiment also indicates that all plasmodial cysteine proteases including falcipain-3 also use n - terminus extension as a refolding domain .", "structural analysis further reveals that the folding domain , in fact , has a short but significant element of secondary structure .", "glutamate of the mature domain forms a buried hydrogen bond with tyrosine and a salt bridge with arginine of the refolding domain .", "this suggests that the small folding domain , though shorter than the standard papain family prodomain , still plays a very important role in folding by stabilizing the mature domain by a hydrogen bond and a salt bridge .", "this is analogous to a similar interaction in falcipain-2 , where tryosine of the refolding domain of falcipain-3 provides hydrogen bonding anchor to glutamate of the mature domain of falcipain-3 .", "these results indicate that the folding domains of falcipain-2 and falcipain-3 stabilize the mature domain in a similar fashion .", "falcipain-2 and 3 efficiently hydrolyze human hemoglobin in the acidic food vacuole of parasite [ 17 , 18 ] .", "it has recently been suggested that hemoglobin hydrolysis is not a highly ordered process but rather proceeds with rapid cleavage by falcipains at multiple sites .", "falcipain subfamily proteases contain an unusual motif near the c - terminus , which is present between the highly conserved active site histidine and asparagine residues ( figure 2 ) .", "a motif of identical size ( 14 aa ) is found in all studied proteases of this subfamily of falcipain , although sequence identity is modest . in case of falcipain-2 and falcipain-3 , motifs", "smaller motifs are present in the sequences encoding p. falciparum serine repeat antigens ( 10 aa ) and dipeptidyl peptidase", "i ( 8 aa ) , and larger ( 20 aa ) motifs are present in two other putative p. falciparum dipeptidyl peptidase genes [ 14 , 33 ] .", "to evaluate the function of the unusual c - terminal motif in falcipains , a mutant enzyme was made by deleting that motif lacking 10 amino acids from falcipain-2 , and the biochemical properties of wild and mutant enzymes were compared .", "native page , biacore , and gel filtration studies indicated that the motif mediates specific interactions with hemoglobin .", "in fact , falcipain-2 has relatively higher affinity for methemoglobin ( kd is 0.8  m ) than hemoglobin ( kda is 3.3  m ) .", "it had been demonstrated that several factors contribute to the formation of methemoglobin during malarial infection , including acidic ph of plasmodial food vacuole , oxidative damage in rbc [ 35 , 36 ] , which causes an increase in methemoglobin content to up to 2042% in the plasmodial food vacuole .", "thus , the higher affinity of falcipain-2 for methemoglobin looks like an adaptation to the specific conditions in the infected rbc .", "the enzyme without this motif showed negligible activity against hemoglobin or globin . further , a peptide encoding the motif blocked hemoglobin hydrolysis but not the hydrolysis of casein , indicating that the motif specifically interacts with hemoglobin .", "thus , this study suggests that the motif mediates the most biologically relevant activity of falcipain , the hydrolysis of hemoglobin during trophozoite stage .", "falcipains may also have other functions in erythrocytic malaria parasites , and it would be interesting to explore the role of the motif in hydrolysis of other proteins of potential biological relevance . in the case of other members of the falcipain", "subfamily , it is likely that the encoded motifs share the same functions as the falcipain motif , because a number of members of this subfamily have been also shown to be hemoglobinases .", "the sequence conservation among the motifs of falcipain subfamily proteases is not high , but , as in the case of the n - terminal extension that mediates protein folding , the presence of these unusual motifs in all related proteases probably indicates a conserved function . for genes encoding more distantly related enzymes , including falcipain-1 , dipeptidyl peptidases , and serine repeat antigens , c - terminal motifs vary greatly in sequence and size , and specific functions of predicted motifs are undefined .", "data indicates that falcipain-2 captures hemoglobin via its c - terminal motif before subsequently cleaving the substrate at multiple sites .", "further , the structure of falcipain indicates a protruding configuration for the motif , surrounded by a predominant negative charge , and it may be that charged residues are crucial for interaction with hemoglobin .", "it is proposed that hemoglobin , which has many charged surface residues , first binds at the motif through charge - charge interaction and brings hemoglobin closer to the active site before hydrolysis .", "the function of the motif in mediating catalysis despite its separation from the active site somewhat resembles that of the hemopexin domain of matrix metalloproteases .", "the hemopexin domain is required for both the efficient cleavage of collagen   and the formation of complexes with both inhibitors and proteases to mediate biological activities .", "thus , as is the case for the falcipain-2 motif , the hemopexin domain serves to facilitate biologically relevant protein - substrate and protein - inhibitor interactions .", "however , the hemopexin domain is approximately 20 times larger than the falcipain motif and , unlike the motif , is separated from the catalytic domain by a hinge region .", "therefore , it appears that falcipains and matrix metalloproteases use similar means of biological control , but the specific mechanisms by which the proteases mediate interactions with substrates and inhibitors are different .", "another papain family protease , cathepsin k , forms a pentameric complex with chondroitin sulfate to facilitate hydrolysis of collagen . having analogy to falcipain-2-hemoglobin interaction , structural requirements for complex formation between cathepsin k and chondroitin sulfate", "it is interesting to raise a question , why do falcipains contain a unique motif that mediates the hydrolysis of hemoglobin ?", "this mechanism might not have evolved simply to facilitate hemoglobin hydrolysis , because other papain family enzymes without c - terminus motifs still can hydrolyze hemoglobin .", "it looks like that the utilization of a specific motif at this region to mediate enzyme - substrate interaction is an unusual but conserved feature of plasmodial cysteine proteases .", "it may be possible that an evolutionary bottleneck occurred in ancestral plasmodial cysteine proteases , and those enzymes might not be that efficient in hemoglobin hydrolysis .", "in addition , plasmodial cysteine proteases might have evolved with introduction of specific motif for efficient hemoglobin hydrolysis .", "the interaction of a major hemoglobinase , falcipain-2 , with most biologically relevant substrate , hemoglobin , could be an interesting starting point for the development of an effective drug .", "the structure of falcipain-2 hemoglobin binding domain will guide the design of inhibitors that should interfere with hemoglobin binding ."], ["there are two major classes of cysteine protease inhibitor , small inhibitors like leupeptin , vinyl sulfones , e64 , and another class known as macromolecular inhibitor .", "these endogenous cysteine protease inhibitors have been described in a number of eukaryotic systems . here", ", we will discuss three major cysteine protease inhibitors , cystatin , chagasin , and falstatin .", "excluding the two unique motifs discussed earlier , the rest of the falcipains are structurally similar to homologous proteases in the papain family .", "cystatin was used as a falcipain inhibitor with the expectation that protein - protein interaction over large surfaces would enhance crystal quality and thus facilitate structural determination .", "cystatins inhibit a wide range of papain family cysteine proteases with high affinity making them ideal candidates for cocrystallization with falcipain .", "falcipain and chicken egg white cystatin formed 1  :  1 complex ( figure 6(a ) ) .", "the inhibitory constants ( ki ) of cystatin for falcipain-2 and falcipain-3 are 6.5  nm and 100  nm , respectively .", "it is interesting that cystatin is a more potent inhibitor of falcipain-2 than falcipain-3 , which suggests that cystatin regulates both the falcipains with different rates .", "the falcipain - cystatin complex shares many features with two known cysteine protease - cystatin structures : cathepsin h - stefin a and papain - stefin b ( the stefins are a subclass within the cystatin superfamily [ 41 , 42 ] ) . these cystatins bind to target proteases in similar orientations , resulting in extensive interactions with the target proteases ( figure 6(b ) ) .", "cystatin largely interacts with falcipain-2 at prime sites where substrate - binding pockets are relatively shallow and less defined , but falcipains also have binding specificity at nonprimed sites .", "although , the binding preference is not as pronounced as that found at the s2 pocket ( figure 6(b ) ) .", "cystatin is only able to access the solvent exposed periphery of the nonprime site of falcipain-2 and majority of its binding occurs at the prime end of the active site . unlike most other cathepsin l - like cysteine proteases ,", "crystal structures indicate that this preference may be due to ile in falcipains , because it creates a small protrusion in the otherwise flat base of the s2 pocket , which restricts aromatic side - chain binding   ( figure 6(b ) ) .", "the falcipain  cystatin complex can be exploited to design potent inhibitors of the malaria parasite .", "a recent study indicates that a peptide based on cystatin binding residues blocked the activity of falcipain-2 and led to accumulation of undegraded hemoglobin in the food vacuole .", "chagasin is a cysteine protease inhibitor that was first identified in trypanosoma cruzi as the physiological regulator of cruzain , the major cysteine protease [ 45 , 46 ] .", "cruzain is also a papain - like ( clan ca ) cysteine protease that is expressed in all stages of t. cruzi life cycle .", "it is a key virulence factor of t. cruzi , the infectious agent responsible for the leading cause of heart disease in latin america , called the chagas disease .", "chagasin is associated with cruzain during its trafficking to specific compartments of the parasite cell , and accumulated evidence suggests that the primary role of chagasin is in posttranslational regulation of protease activity .", "1 binding with falcipain-2 ( figure 7 ) . the protease - binding loops ( bc , de , and fg ) in chagasin form a well - aligned wedge that fills the active site groove of falcipain-2 to obstruct substrate binding ( figure 7 ) .", "the bc loop is one of the three signature motifs that contribute to the inhibition of the cysteine protease .", "this is confirmed by a synthetic peptide corresponding to the bc loop of the chagasin like protein from e. histolytica , which specifically blocked the activity of cysteine proteases .", "it is noteworthy that thr 31 in the bc loop of chagasin binds to the catalytic cys at the falcipain-2 active site by water - mediated hydrogen bonds ( figure 7 ) .", "it is likely that the highly conserved thr serves as a key functional residue among chagasin - like inhibitors .", "another interesting feature of the falcipain-2-chagasin interaction is the highly mobile de loop like in e64 , a strong irreversible inhibitor of cysteine protease , which occupies the nonprime site . in summary ,", "the bc loop , mobile de loop , and the rpw / f motif in the fg loop are the key elements for binding with falcipain-2 .", "disturbance of the equilibrium between cysteine proteases and natural inhibitors is a key event in the pathogenesis of cancer , rheumatoid arthritis , osteoporosis , and emphysema . in case of malaria ,", "falciparum parasites express falstatin , a potent inhibitor of falcipains and many other cysteine proteases .", "the stage - specificity of falstatin expression and the effects of anti - falstatin antibodies on parasite development suggest that this inhibitor facilitates a process that also requires proteolytic activity , the invasion of erythrocytes by p. falciparum merozoites .", "falstatin is a competitive and reversible inhibitor of falcipains , as demonstrated by increasing calculated km values but similar vmax values with increasing concentrations of falstatin . to evaluate the mechanism of inhibition of cysteine proteases by falstatin , pandey et al .", "tested the ability of active site - inhibited falcipain-2 to compete with active falcipain-2 for binding with falstatin .", "in contrast to results with the prodomain , the inhibitory effect of falstatin was not affected by the presence of active site inhibited falcipain-2 .", "thus , the binding of falstatin to falcipain-2 appears to be via interaction with the enzyme active site .", "leupeptin , e-64 , and vinyl sulfones are major cysteine protease inhibitors that bind to the active site [ 26 , 48 ] .", "the active sites of both enzymes are located in a cleft between the structurally distinct domain of the papain - like fold .", "the structures of falcipain-2 and falcipain-3 have been determined in complex with these small inhibitors .", "e-64 , and leupeptin interact with residues in the s1 , s2 , and s3 subsites of falcipain-2 and falcipain-3 , corresponding to the p1 , and p2 , and p3 position of the inhibitors ( figure 8) .", "the conserved catalytic residues of falcipain-2 and falcipain-3 ( gln , cys , his , asn , resp . )", "inhibitors display binding modes with their partner enzymes similar to those found in other papain family enzymes .", "the inhibitors bind to the main chain of falcipain-2 and falcipain-3 by glycine ( gly in falcipain-2 and gly in falcipain-3 ) residue that is highly conserved in the s3 subsite of clan ca cysteine proteases ( figure 9 ) . in cocrystallization , these residues form hydrogen bonds with the o and n atoms of the inhibitor backbone . in the case of the falcipain-2 complex with e-64 ,", "enzyme active sites gln , ser , cys , asn , and his are involved in the formation of additional hydrogen bonds with e-64 . in the falcipain-3-leupeptin complex ,", "gln , cys , and asn also act as hydrogen bonding partners to the inhibitor .", "the enzyme - inhibitor complex stabilize by a series of possible hydrophobic interaction using nonpolar region of gly , tyr , gly , leu , ser , leu , asn , and ala in falcipain-2 , and tyro , gly , tyr , ile , and ser in case of falcipain-3 .", "the active site cysteine of falcipain-2 forms a covalent , irreversible hemithioketal with the e-64 epoxy carbon ( figure 9 ) .", "the active site cysteine of falcipain-3 forms a covalent , reversible hemithioacetal with the asymmetric carbonyl carbon of leupeptin ( figure 10 ) . the interaction pattern of e-64 and leupeptin with all papain family enzymes is conserved .", "the carboxyl group of e-64 and carbonyl group of leupeptin occupy the oxyanion hole formed by the conserved catalytic residues . falcipain-2 and falcipain-3 have a clear preference for substrates / inhibitors that contains a leucine at p2 position , and both leupeptin and e64 contain a leu at p2 .", "surprisingly , falcipain-3 has been shown to be much less efficient at hydrolyzing peptide substrates and more difficult to inhibit with small peptidyl - based inhibitors compared with falcipain-2 [ 16 , 51 ] .", "the structure that forms the active site is highly conserved between falcipain-2 and falcipain-3 , and there is no notable movement in the peptide backbone or loop region .", "however , superimposition of falcipain-2 and falcipain-3 structures highlights two important substitutions in the s2 subsite .", "this position of the s2 subsite is known to be a key determiner of the specificity ( figures 9 and 10 ) . in comparison to asp in falcipain-2 ,", "the additional side chain carbon in glu of falcipain-3 , this increased the size as well as flexibility at this position . in case of falcipain-3leupeptin complex , the larger and more flexible residue ( glu ) at the bottom of s2 subsite and bulkier residue ( try ) create a narrow wall of the s2 subsite , as compared to the falcipain  e-64 complex ( figure 10 ) .", "the overall structures of falcipain-3leupeptin and falcipain-2e-64 complexes show a similar mode of binding and inhibition compared with macromolecule inhibitors like chagasin and cystatin .", "like e64 and leupeptin , vinyl sulphones ( mu - leu - hph - vsph ) have been shown to be effective inhibitors of a number of papain - family - like cysteine proteases .", "mu - leu - hph - vsph is a potent irreversible inhibitor of falcipain-2 and falcipain-3 .", "the cocrystallization of falcipain-3 with mu - leu - hph - vsph indicates that inhibitor binds the respective s1 and s3 subsites to form an irreversible , covalent bond with the sulfur of the active site cysteine thiol in enzyme   ( figure 11 ) .", "given the hydrophobic nature of the p1 and p2 substituents in mu - leu - hph - vsph , the active site of falcipain-3 is lined with a number of residues that are able to make nonpolar contacts with their respective inhibitor   ( figure 11 ) . as seen in the crystal structures of falcipain-3 with the above inhibitor , the residue at the bottom of the s2 subsite ( glu ) points out of the pocket to avoid a potentially unfavorable interaction with the bulky phe residue at the p2 position of inhibitor .", "cysteine proteases of the malaria parasite may be targeted for inhibition by vinyl sulphones . indeed , vinyl sulphones - based cysteine protease inhibitors are already being used in preclinical trials for the chagas disease ."], ["the understanding of the cysteine proteases of malaria parasites has increased markedly in recent years . since cysteine proteases that play an important role in the parasite life cycle by degrading erythrocyte proteins , most notably hemoglobin , are attractive targets for antimalarial chemotherapy . falcipain-2 and falcipain-3 are the best characterized cysteine proteases of malaria parasite , the structure and function of different domains and their interaction with small and macromolecular inhibitors are studied", ". the structure - function study of falcipains and interaction with inhibitors will provide detail insights to develop rational design of inhibitor against falcipains .", "structure - guided approaches should have great role in the design of potent and highly selective inhibitor .", "efforts to optimize current inhibitors based on the structure - function of falcipains are currently underway ."]]}
{"article_id": "PMC4393001", "article_text": ["alzheimer 's disease ( ad ) is the most common cause of dementia in the aging population .", "this disease develops over time and leads to significant cognitive deficits affecting memory , insight , judgment , abstraction , and language functions .", "ad affects more than 5 million people in the united states and this number is projected to rise to 35 million by 2050 [ 2 , 3 ] .", "this estimate underscores both the scope of this health care issue for the society as a whole and the need for the development of therapeutic options for these patients .  ", "the diagnosis of this neurodegenerative disease relies on the presence of senile plaques and neurofibrillary tangles in affected brain areas at autopsy .", "these ad hallmark lesions are the results of the pathological deposition of proteins normally present throughout the brain .", "senile plaques are composed of extracellular deposits of beta - amyloid ( a ) derived by proteolytic cleavage from the amyloid precursor protein ( app ) [ 410 ] .", "neurofibrillary tangles , on the other hand , are intracellular bundles of self - assembled tau proteins [ 1138 ] . the formation of both senile plaques and neurofibrillary tangles is associated with progressive and irreversible degeneration of neuronal processes and the loss of synaptic connections [ 3950 ] .    initially , multiple studies focused on defining the characteristics of ad and on the analysis of the composition of senile plaques and neurofibrillary tangles .", "more recently , data have been obtained on the molecular mechanisms that link the formation of these lesions and underlie neurodegeneration and cell death in ad and related disorders .", "calpains are ca - dependent proteases in which activity is dysregulated in ad and other neurodegenerative diseases [ 5154 ] .", "a growing body of evidence suggests that the abnormal activation of calpains might modulate not only the formation of senile plaques and neurofibrillary tangles but also the development of synaptic pathology in ad .", "these data reviewed below position calpains at the crossroads of the mechanisms involved in the formation of the main pathological alterations associated with ad .", "furthermore , these findings underscore the importance of calpains as potential targets to block the activation of the signaling cascades leading to degeneration in ad . in turn", ", this information could be applied to the design of therapeutic options and prevention strategies for this devastating disease .    in this review , we first summarized data on the properties of calpains and the mechanisms underlying their activation . then , we reviewed findings on the dysregulation of calpain in the context of ad and its deleterious consequences for the morphology and function of affected brain areas .", "  calpains constitute a family of ca - dependent cysteine proteases involved in multiple and very diverse cell functions including cell development , proliferation , and differentiation , cell motility , growth cone motility and guidance , apoptosis , learning , and memory , among others [ 51 , 52 ] .", "originally , two members of this family were identified : calpain 1 ( -calpain ) and calpain 2 ( m - calpain ) , also known as conventional or classical calpains .", "more recently , the other 14 members of the calpain family have been identified in mammals [ 51 , 55 ] .", "in contrast to classical calpains that are ubiquitously distributed , the expression of some of these unconventional or nonclassical calpains is tissue specific .", "for example , calpain 3a and calpain 8 are mainly present in skeletal and smooth muscle cells , respectively .", "calpain 11 , on the other hand , is highly expressed in testes , while calpain 13 is concentrated in the lung and skin .", "others , like calpains 5 , 7 , 9 , and 10 have more widespread distributions .", "none of these new members of the calpain family are highly expressed in the central nervous system ( cns ) .", "both calpain 1 and calpain 2 are heterodimers composed of a large catalytic subunit ( ~80  kda ) and a small regulatory subunit ( ~30  kda ) .", "based on this sequence , the catalytic subunit has been divided into four distinct domains ( reviewed in ) .", "domain 1 corresponds to the n - terminal region of this subunit and undergoes autolysis upon ca binding .", "domain 2 contains a catalytic unit formed by cys , his , and asn residues .", "domain 4 has partial homology with calmodulin and contains several ef - hand calcium - binding motifs .", "the regulatory subunit can be divided into two domains , a calmodulin - like domain and a glycine - rich domain .   ", "while both proteases are present in neurons and glial cells , their relative abundance differs .", "calpain 1 is more abundant in neurons , and calpain 2 is prominent in glial cells .", "analysis of the subcellular localization of these enzymes demonstrated that calpain 1 is concentrated in the cell bodies and is also detected in the processes extended by central neurons , although at lower levels .", "calpain 1 immunoreactivity has been also detected in postsynaptic densities as well as in dendritic spines .", "astrocytic processes are also enriched in calpain 2 [ 62 , 63 ] . calpains cleave proteins generating large fragments .", "initially , it was thought that these proteases ' cleavage sites have a leu or a val residue in the p2 position .", "more recently , it has been shown that the cleavage site specificity is determined by conformation rather than amino acid sequence [ 6568 ] .", "the specificities of the substrates for both calpains are very similar but not identical . in vitro studies", "calpain substrates include cytoskeletal proteins ( cadherin , catenin , desmin , dystrophin , gelsolin , filamin , fodrin , microtubule - associated proteins map 1 and map 2 , neurofilament proteins , spectrin , tau , talin , troponin , tubulin , vimentin , and vinculin ) , signal transduction proteins ( calcium / calmodulin - dependent protein kinase , epidermal growth factor ( egf ) kinase , pp60 , protein kinase c , calcineurin , and caspases 3 , 7 , 8 , 9 , 12 , and 14 ) , synaptic proteins ( dynamin 1 , postsynaptic density ( psd ) 95 , n - methyl - d - aspartic acid ( nmda ) glutamate receptors , and metabotropic glutamate receptor glur1 ) , and transcription factors ( p53 ) , among others [ 51 , 53 ] .   calpain activation is tightly regulated to prevent massive proteolytic activity in the cell .", "the binding of ca to either calpain results in conformational changes that initiate their proteolytic activity .", "while calpain 1 is activated in the presence of micromolar concentrations of ca , calpain 2 requires millimolar concentrations .", "the ca requirement for either calpain is significantly higher than the concentration of this ion in living cells .", "this fact has prompted a series of studies on potential mechanisms that could lead to a decrease in the ca requirement for the activation of these proteases in living cells . in vitro studies", "have shown that the interaction of calpains with a series of activator molecules lowers the concentration of ca required to initiate the activation of these proteases . among them , phospholipids ( i.e. , phosphatidylinositol ) are the most studied .", "however , the phospholipid to calpain molar ratio required for lowering the ca requirement for either calpain activation is not likely to be achieved in the cellular context [ 6971 ] .", "nevertheless , it can not be ruled out that higher concentrations of ca are achieved in limited cellular areas where ca is not easily detectable by available methods . in turn", ", these local micromolar or millimolar ca concentrations could trigger calpain activation in distinct subcellular domains .  ", "in addition to ca , calpastatin has a key role in the regulation of calpain .", "calpastatin , a heat - stable protein ranging from ~70 to ~140  kda of apparent molecular weight depending on the cell type , is considered a specific endogenous inhibitor of calpains .", "immunocytochemical analysis showed calpastatin immunoreactivity throughout the dendritic tree in pyramidal neurons and purkinje cells .", "therefore , the ratio calpain / calpastatin plays a key role in the regulation of calpain activity [ 7880 ] .", "the inhibitory effect of calpastatin requires ca - dependent high - affinity binding to three sites of calpain .  ", "the kinases responsible for the phosphorylation of some of these sites have already been identified .", "thus , two of those sites seem to be phosphorylated by protein kinase c ( pkc ) , two by protein kinase a ( pka ) , two by calmodulin kinase ii , one site by casein kinase i , and another by protein kinase g .", "although the role of the phosphorylation of each site in calpain activation has not been completely elucidated , data suggest that phosphorylation mediated by mitogen - activated kinases ( erk / map kinases ) activates these proteases . on the other hand , phosphorylation by pka may decrease their activity [ 82 , 83 ] .", "as briefly reviewed above , calpain activation is a tightly regulated process to prevent deleterious consequences of massive proteolytic activity .", "these regulatory mechanisms seem to decline with aging resulting in an increased calpain activity [ 8487 ] .", "thus , several studies have reported significantly increased activation of calpains in ad brains [ 8894 ] .", "the hyperactivation of calpain in the context of ad is the result of several factors including enhanced intracellular ca concentration and decreased calpastatin levels .", "experiments performed using an ad culture model system showed that oligomeric a induced a significant ( 5-fold ) and instantaneous rise in ca in hippocampal neurons [ 9597 ] .", "these studies also addressed the source of the ca influx leading to calpain activation in the context of ad taking advantage of specific blockers of ca release from the endoplasmic reticulum , the major source of intracellular ca , and bapta , a chelator of extracellular ca .", "the data obtained showed that a induces calpain activation by enhancing extracellular ca influx [ 9597 ] .", "the mechanisms underlying the regulation of the enhanced ca influx and calpain activation in ad have also been studied .", "both nmda receptors and voltage - gated calcium channels ( vgcc ) have been implicated in such regulation in central neurons [ 99101 ] .", "specific nmda receptor inhibitors , mk801 , and the fda approved nmda receptor antagonist memantine   significantly attenuated the initial a-induced increase of ca and blocked a-induced calpain activation in cultured hippocampal neurons [ 9597 ] . in contrast ,", "nimodipine , an l - type vsccs blocker , did not decrease the a-induced activation of calpain [ 9597 ] .", "these data suggest that nmda receptors play an important role in mediating the sustained ca influx and enhanced calpain activity induced by aggregated a. furthermore , these studies suggested that factors that affect nmda receptor - mediated ca influx could modulate calpain activation and its deleterious effects in central neurons .", "recently , it has been shown that cholesterol regulates calpain activity in the context of ad .", "a population - based study demonstrated that not only high cholesterol levels but also moderately elevated cholesterol levels in midlife represent a significant risk factor for ad [ 106 , 107 ] .", "interestingly , cholesterol has been implicated in the susceptibility of cells to ca influx [ 108111 ] .", "thus , elevated membrane cholesterol actually increased the susceptibility of cells to a-induced elevation in ca influx leading to cell death via a calpain - dependent mechanism [ 111 , 112 ] . by regulating the nmda receptor content and changing their localization in membrane microdomains at the synaptic sites", ", cholesterol modulates the ability of a to induce ca influx , leading to calpain activation in hippocampal neurons [ 111 , 112 ] .", "calpastatin also seems to play an important role in the regulation of calpain activation in ad .", "thus , it has been shown that calpastatin is markedly depleted in the cortex of ad brains at late stages of the disease as compared to age - matched controls .", "focal areas of calpastatin depletion have been also detected along dystrophic neurites at early stages of ad .", "on the other hand , no changes in calpastatin levels were detected in neurons less susceptible to neurodegeneration in ad like purkinje cells .", "this decrease in calpastatin levels is the result of the proteolytic activity of caspases and calpain . in turn , the decrease in the ratio calpastatin to calpain causes calpain hyperactivation perpetuating this cellular deleterious effect [ 79 , 113115 ] .", "these findings have been complemented with immunocytochemical studies on the distribution of calpain in ad .", "these studies have shown intense calpain immunoreactivity in dystrophic neurites associated with neurofibrillary tangles , neuritic plaques , and neuropil threads in the hippocampal region and entorhinal cortex of subjects suffering from ad and other tauopathies [ 116118 ] . based on this information ,", "it was hypothesized that the enhanced calpain activity observed in ad brains plays an important role in the formation of senile plaques , neurofibrillary tangles , and synaptic dysfunction in the context of ad ( figure 1 ) .", "these peptides are the result of the sequential proteolytic cleavage of app by two proteases .", "first , -secretase ( -site app - cleaving enzyme ( bace 1 ) ) cuts app at the n - terminus of the a domain leaving a 99-amino - acid - long c - terminal fragment .", "then , the -secretase complex ( presenilin 1 , presenilin 2 , nicastrin , aph1 , and pen2 ) cuts the c - terminus to generate a peptides of 38 to 43 amino acids in length [ 5 , 119121 ] . of these peptides ,", "therefore , increased levels of bace 1 could lead to enhanced production of a , its aggregation , and consequently the activation of signaling pathways associated with the neurodegenerative process .", "thus , a 2-fold increase in bace1 levels has been detected in ad brains [ 127129 ] .", "recently , it has been shown that the calpain / calpastatin system can modulate the pathological deposition of a. calpain activation increases the levels of bace1 in a transgenic mouse model of ad .", "in addition , the bidirectional regulation of the activation of the calpain / calpastatin system and a metabolism has been established . using transgenic mice overexpressing app", ", these authors showed that calpastatin deficiency enhanced calpain activation , a production , and increased mortality . in turn , the increased levels of a lead to enhanced ca influx and increased calpain activation .", "senile plaques are often surrounded by dystrophic neurons that contain tau aggregates known as neurofibrillary tangles .", "many studies have focused on the composition and the mechanisms underlying the formation of tau aggregates .", "those studies have identified two types of tau filaments in the neurofibrillary tangles : straight and paired helical filaments .", "tau hyperphosphorylation has been attributed to the increased activity of several kinases , including cyclin - dependent kinase 5 ( cdk5 ) , glycogen synthase kinase  ( gsk ) , and the mitogen - activated protein kinase ( mapk ) in hippocampal neurons [ 138 , 143145 ] .", "thus , it has been shown that calpain cleaves the inhibitory domain of gsk3 generating two fragments of 40 and 30  kda .", "the latter has a longer half life than p35 and therefore it is a more potent activator of cdk5 .", "experiments using calpeptin , a cell permeable calpain inhibitor , blocked both erk 1 and erk 2 activation .", "conditions that blocked the activation of these kinases induced a decreased in tau phosphorylation and resulted in enhanced neuronal survival in the presence of a . besides tau phosphorylation", ", calpain activation might play a role in tau - mediated neurodegeneration by inducing tau cleavage . in vitro studies", "have shown that both fetal and adult tau isoforms are rapidly proteolyzed by calpains [ 151153 ] .", "on the other hand , tau present in paired - helical filaments is considerably more resistant to proteolysis by these proteases [ 153156 ] .", "these findings suggest that phosphorylation might regulate the susceptibility of tau to calpain - mediated cleavage .", "tau obtained from cultured neurons incubated in the presence of this phosphatase inhibitor was more resistant to calpain cleavage than tau extracted from nontreated control neurons .", "nevertheless , the effect of phosphorylation on calpain - mediated tau cleavage seems to be complex .", "this effect might depend on the site phosphorylated and/or the extent of phosphorylation under pathological conditions since highly phosphorylated fetal isoforms are readily cleaved by calpain .  ", "calpain - mediated tau cleavage seems to play an important role under neurodegenerative conditions [ 157164 ] .", "it has been shown that calpain activation results in the generation of several n - terminal tau fragments .", "one of such tau fragments , of ~20  kda of apparent molecular weight , has been detected in mitochondria present in synaptosomal fractions obtained from ad brains .", "the levels of this tau fragment are partially reduced when cultured neurons are treated with a calpain inhibitor .", "it has been shown that tau fragments containing the 2644 tau amino acids affect mitochondria oxidative phosphorylation acting at the level of the adenine nucleotides translocator contributing to synapse dysfunction .  ", "a smaller tau fragment generated by calpain cleavage has been detected in mature hippocampal neurons treated with aggregated a oligomers [ 160 , 161 ] .", "a-induced tau cleavage mediated by calpain 1 leads to the generation of a neurotoxic 17  kda tau fragment ( tau 45230 ) in cultured hippocampal neurons [ 160 , 161 ] .", "this fragment has also been detected in the neocortex of ad brains as well as in brain areas affected by other tauopathies .", "furthermore , when expressed in neuronal and nonneuronal cell types or in an in vivo drosophila model system , the 17  kda tau fragment produced cell death in the absence of a oligomers [ 160 , 161 , 163 ] .", "these data provided strong evidence for an important role of calpain 1 and the generation of the 17  kda tau fragment in the progression of a-mediated neurodegeneration .", "it is worth noting that calpain 2 activation cleaves tau generating a smaller tau fragment that lacks neurotoxic effects in central neurons .  ", "in addition to the presence of senile plaques and neurofibrillary tangles , ad brains are characterized by a decrease in synaptic contacts .", "this synaptic loss seems to be the best morphological correlate of the functional deficits observed in the mid to late stages of ad [ 39 , 40 ] .", "although no significant decline in synapse number has been detected in the earliest stages of the disease , a stage of synaptic dysfunction seems to precede frank synapse loss [ 41 , 46 , 165 ] . by cleaving proteins in the presynaptic terminals and/or the postsynaptic elements", "changes in proteins involved in synaptic vesicle biogenesis and/or recycling at synaptic terminals are responsible , at least in part , for the synaptic dysfunction detected in ad .", "recently , data obtained using culture and animal models of ad showed that a induced a significant reduction in dynamin 1 levels that preceded synapse loss .", "dynamin 1 pinches off synaptic vesicles , freeing them from the membrane and allowing them to reenter the synaptic vesicle pool to be refilled for future release [ 167 , 168 ] .", "decreased levels of this protein lead to the depletion of synaptic vesicles and the accumulations of invaginated pits at presynaptic membranes adjacent to the synaptic clefts [ 9597 , 169 , 170 ] .", "the a-induced decrease in dynamin 1 is a result , at least partially , of calpain - mediated proteolysis [ 9597 , 171 ] .  ", "thus , it has been shown that calpain activation results in the breakdown of several proteins leading to changes in the ultrastructure of the postsynaptic density ( psd ) .", "these changes in the psd are accompanied by the rapid cleavage of psd-95 and the nmda receptor subunits nr1 and nr2a and 2b [ 172175 ] .", "calpain has been shown to truncate also mglur1 exacerbating nmda - mediated neurotoxicity [ 176178 ] .  ", "an additional mechanism by which calpain activation can result in synaptic dysfunction involved the cleavage of pka leading to a decrease in both the regulatory and the catalytic subunits of this kinase .", "the decrease in pka activity attenuates creb activation impairing memory [ 179 , 180 ] .", "taken together , the data reviewed above strongly suggest that calpains play an important role in the neurodegeneration mechanisms underlying ad in the aging population . based on these data", ", it has been tempting to speculate that calpain inhibition could be a useful tool to prevent neurodegeneration in this disease . to obtain further insights into the beneficial effects of blocking calpain activation , several studies", "have been conducted using experimental approaches to either suppress the expression of these proteases or prevent their abnormal activation .", "early studies using gene deletion of the calpain regulatory subunit by homologous recombinant techniques showed that the depletion of these proteases is embryonic lethal .", "the data obtained showed that indeed the suppression of calpain 1 expression by means of these specific probes prevented oxidative stress - induced cell injury in human hepatic cancer cell lines .", "other studies have assessed the effects of calpain inhibitors on cell death in culture model systems of ad .", "leupeptin and e64 calpain inhibitors had protective effects in those cultures [ 160 , 183186 ] .", "the idea that calpain could be a potential therapeutic target in neurodegenerative diseases has been reinforced by studies performed in vitro and in culture using the calpain inhibitor mdl 28170 .", "those studies suggested a protective effect of calpain inhibitors against excitotoxicity [ 178 , 187 , 188 ] .", "unfortunately , the potential use of known calpain inhibitors as therapeutic tools in ad is limited due to their low cellular penetration , poor selectivity , and kinetics . recently ,", "this benzoylalanine - derived ketoamide is capable of inhibiting calpain in nanomolar concentrations and has improved oral bioavailability , water solubility , and metabolic stability .", "this calpain inhibitor is highly effective in preventing calpain - mediated cleavage of dynamin 1 and tau in cultured hippocampal neurons .", "this inhibitor is effective not only when added prior to the a treatment but also when added simultaneously with a and even when added after a has triggered the neurodegeneration process .", "the calpain inhibitor a-705253 also prevents a oligomer - induced neurodegeneration of the nucleus basalis magnocellularis .", "these experiments raised the possibility that more potent calpain inhibitors could have beneficial effects even at late stages of ad .", "moreover , initial studies using calpain inhibitors in mouse and rat models of ad showed an encouraging recovery of cognitive function in these animals when they were treated with calpain inhibitors at an early age [ 186 , 190 ] . together , these data underscore the potential importance of calpain inhibitors as promising therapeutic tools in ad and related neurodegenerative diseases .  ", "in summary , the data briefly reviewed above provide strong support for the role of calpains in ad .", "they also highlight the tantalizing possibility that these proteases could serve as targets for the development of therapeutic interventions for this disease , one of the main challenges for decades to come in ad research ."], "abstract_text": ["<S> alzheimer 's disease ( ad ) is characterized by the presence of senile plaques and neurofibrillary tangles in the neocortex and hippocampus of ad patients . in addition , a marked decrease in synaptic contacts has been detected in these affected brain areas . due to its prevalence in the aging population , this disease has been the focus of numerous studies . </S>", "<S> the data obtained from those studies suggest that the mechanisms leading to the formation of the hallmark lesions of ad might be linked . </S>", "<S> one of such mechanisms seems to be the dysregulation of calcium homeostasis that results in the abnormal activation of calpains . </S>", "<S> calpains are a family of ca2 + -dependent cysteine proteases that play a key role in multiple cell functions including cell development , differentiation and proliferation , axonal guidance , growth cone motility , and cell death , among others . in this paper , we briefly reviewed data on the structure of these proteases and their regulation under normal conditions . </S>", "<S> we also summarized data underscoring the participation of calpains in the neurodegenerative mechanisms associated with ad . </S>"], "labels": null, "section_names": ["1. Introduction", "2. The Calpain Family", "3. Dysregulation of Calpain Activity in AD and Related Disorders", "4. The Role of Calpain in the Generation of Pathological Manifestations of AD", "5. Future Directions and Concluding Remarks"], "sections": [["alzheimer 's disease ( ad ) is the most common cause of dementia in the aging population .", "this disease develops over time and leads to significant cognitive deficits affecting memory , insight , judgment , abstraction , and language functions .", "ad affects more than 5 million people in the united states and this number is projected to rise to 35 million by 2050 [ 2 , 3 ] .", "this estimate underscores both the scope of this health care issue for the society as a whole and the need for the development of therapeutic options for these patients .  ", "the diagnosis of this neurodegenerative disease relies on the presence of senile plaques and neurofibrillary tangles in affected brain areas at autopsy .", "these ad hallmark lesions are the results of the pathological deposition of proteins normally present throughout the brain .", "senile plaques are composed of extracellular deposits of beta - amyloid ( a ) derived by proteolytic cleavage from the amyloid precursor protein ( app ) [ 410 ] .", "neurofibrillary tangles , on the other hand , are intracellular bundles of self - assembled tau proteins [ 1138 ] . the formation of both senile plaques and neurofibrillary tangles is associated with progressive and irreversible degeneration of neuronal processes and the loss of synaptic connections [ 3950 ] .    initially , multiple studies focused on defining the characteristics of ad and on the analysis of the composition of senile plaques and neurofibrillary tangles .", "more recently , data have been obtained on the molecular mechanisms that link the formation of these lesions and underlie neurodegeneration and cell death in ad and related disorders .", "calpains are ca - dependent proteases in which activity is dysregulated in ad and other neurodegenerative diseases [ 5154 ] .", "a growing body of evidence suggests that the abnormal activation of calpains might modulate not only the formation of senile plaques and neurofibrillary tangles but also the development of synaptic pathology in ad .", "these data reviewed below position calpains at the crossroads of the mechanisms involved in the formation of the main pathological alterations associated with ad .", "furthermore , these findings underscore the importance of calpains as potential targets to block the activation of the signaling cascades leading to degeneration in ad . in turn", ", this information could be applied to the design of therapeutic options and prevention strategies for this devastating disease .    in this review , we first summarized data on the properties of calpains and the mechanisms underlying their activation . then , we reviewed findings on the dysregulation of calpain in the context of ad and its deleterious consequences for the morphology and function of affected brain areas ."], ["  calpains constitute a family of ca - dependent cysteine proteases involved in multiple and very diverse cell functions including cell development , proliferation , and differentiation , cell motility , growth cone motility and guidance , apoptosis , learning , and memory , among others [ 51 , 52 ] .", "originally , two members of this family were identified : calpain 1 ( -calpain ) and calpain 2 ( m - calpain ) , also known as conventional or classical calpains .", "more recently , the other 14 members of the calpain family have been identified in mammals [ 51 , 55 ] .", "in contrast to classical calpains that are ubiquitously distributed , the expression of some of these unconventional or nonclassical calpains is tissue specific .", "for example , calpain 3a and calpain 8 are mainly present in skeletal and smooth muscle cells , respectively .", "calpain 11 , on the other hand , is highly expressed in testes , while calpain 13 is concentrated in the lung and skin .", "others , like calpains 5 , 7 , 9 , and 10 have more widespread distributions .", "none of these new members of the calpain family are highly expressed in the central nervous system ( cns ) .", "both calpain 1 and calpain 2 are heterodimers composed of a large catalytic subunit ( ~80  kda ) and a small regulatory subunit ( ~30  kda ) .", "based on this sequence , the catalytic subunit has been divided into four distinct domains ( reviewed in ) .", "domain 1 corresponds to the n - terminal region of this subunit and undergoes autolysis upon ca binding .", "domain 2 contains a catalytic unit formed by cys , his , and asn residues .", "domain 4 has partial homology with calmodulin and contains several ef - hand calcium - binding motifs .", "the regulatory subunit can be divided into two domains , a calmodulin - like domain and a glycine - rich domain .   ", "while both proteases are present in neurons and glial cells , their relative abundance differs .", "calpain 1 is more abundant in neurons , and calpain 2 is prominent in glial cells .", "analysis of the subcellular localization of these enzymes demonstrated that calpain 1 is concentrated in the cell bodies and is also detected in the processes extended by central neurons , although at lower levels .", "calpain 1 immunoreactivity has been also detected in postsynaptic densities as well as in dendritic spines .", "astrocytic processes are also enriched in calpain 2 [ 62 , 63 ] . calpains cleave proteins generating large fragments .", "initially , it was thought that these proteases ' cleavage sites have a leu or a val residue in the p2 position .", "more recently , it has been shown that the cleavage site specificity is determined by conformation rather than amino acid sequence [ 6568 ] .", "the specificities of the substrates for both calpains are very similar but not identical . in vitro studies", "calpain substrates include cytoskeletal proteins ( cadherin , catenin , desmin , dystrophin , gelsolin , filamin , fodrin , microtubule - associated proteins map 1 and map 2 , neurofilament proteins , spectrin , tau , talin , troponin , tubulin , vimentin , and vinculin ) , signal transduction proteins ( calcium / calmodulin - dependent protein kinase , epidermal growth factor ( egf ) kinase , pp60 , protein kinase c , calcineurin , and caspases 3 , 7 , 8 , 9 , 12 , and 14 ) , synaptic proteins ( dynamin 1 , postsynaptic density ( psd ) 95 , n - methyl - d - aspartic acid ( nmda ) glutamate receptors , and metabotropic glutamate receptor glur1 ) , and transcription factors ( p53 ) , among others [ 51 , 53 ] .   calpain activation is tightly regulated to prevent massive proteolytic activity in the cell .", "the binding of ca to either calpain results in conformational changes that initiate their proteolytic activity .", "while calpain 1 is activated in the presence of micromolar concentrations of ca , calpain 2 requires millimolar concentrations .", "the ca requirement for either calpain is significantly higher than the concentration of this ion in living cells .", "this fact has prompted a series of studies on potential mechanisms that could lead to a decrease in the ca requirement for the activation of these proteases in living cells . in vitro studies", "have shown that the interaction of calpains with a series of activator molecules lowers the concentration of ca required to initiate the activation of these proteases . among them , phospholipids ( i.e. , phosphatidylinositol ) are the most studied .", "however , the phospholipid to calpain molar ratio required for lowering the ca requirement for either calpain activation is not likely to be achieved in the cellular context [ 6971 ] .", "nevertheless , it can not be ruled out that higher concentrations of ca are achieved in limited cellular areas where ca is not easily detectable by available methods . in turn", ", these local micromolar or millimolar ca concentrations could trigger calpain activation in distinct subcellular domains .  ", "in addition to ca , calpastatin has a key role in the regulation of calpain .", "calpastatin , a heat - stable protein ranging from ~70 to ~140  kda of apparent molecular weight depending on the cell type , is considered a specific endogenous inhibitor of calpains .", "immunocytochemical analysis showed calpastatin immunoreactivity throughout the dendritic tree in pyramidal neurons and purkinje cells .", "therefore , the ratio calpain / calpastatin plays a key role in the regulation of calpain activity [ 7880 ] .", "the inhibitory effect of calpastatin requires ca - dependent high - affinity binding to three sites of calpain .  ", "the kinases responsible for the phosphorylation of some of these sites have already been identified .", "thus , two of those sites seem to be phosphorylated by protein kinase c ( pkc ) , two by protein kinase a ( pka ) , two by calmodulin kinase ii , one site by casein kinase i , and another by protein kinase g .", "although the role of the phosphorylation of each site in calpain activation has not been completely elucidated , data suggest that phosphorylation mediated by mitogen - activated kinases ( erk / map kinases ) activates these proteases . on the other hand , phosphorylation by pka may decrease their activity [ 82 , 83 ] ."], ["as briefly reviewed above , calpain activation is a tightly regulated process to prevent deleterious consequences of massive proteolytic activity .", "these regulatory mechanisms seem to decline with aging resulting in an increased calpain activity [ 8487 ] .", "thus , several studies have reported significantly increased activation of calpains in ad brains [ 8894 ] .", "the hyperactivation of calpain in the context of ad is the result of several factors including enhanced intracellular ca concentration and decreased calpastatin levels .", "experiments performed using an ad culture model system showed that oligomeric a induced a significant ( 5-fold ) and instantaneous rise in ca in hippocampal neurons [ 9597 ] .", "these studies also addressed the source of the ca influx leading to calpain activation in the context of ad taking advantage of specific blockers of ca release from the endoplasmic reticulum , the major source of intracellular ca , and bapta , a chelator of extracellular ca .", "the data obtained showed that a induces calpain activation by enhancing extracellular ca influx [ 9597 ] .", "the mechanisms underlying the regulation of the enhanced ca influx and calpain activation in ad have also been studied .", "both nmda receptors and voltage - gated calcium channels ( vgcc ) have been implicated in such regulation in central neurons [ 99101 ] .", "specific nmda receptor inhibitors , mk801 , and the fda approved nmda receptor antagonist memantine   significantly attenuated the initial a-induced increase of ca and blocked a-induced calpain activation in cultured hippocampal neurons [ 9597 ] . in contrast ,", "nimodipine , an l - type vsccs blocker , did not decrease the a-induced activation of calpain [ 9597 ] .", "these data suggest that nmda receptors play an important role in mediating the sustained ca influx and enhanced calpain activity induced by aggregated a. furthermore , these studies suggested that factors that affect nmda receptor - mediated ca influx could modulate calpain activation and its deleterious effects in central neurons .", "recently , it has been shown that cholesterol regulates calpain activity in the context of ad .", "a population - based study demonstrated that not only high cholesterol levels but also moderately elevated cholesterol levels in midlife represent a significant risk factor for ad [ 106 , 107 ] .", "interestingly , cholesterol has been implicated in the susceptibility of cells to ca influx [ 108111 ] .", "thus , elevated membrane cholesterol actually increased the susceptibility of cells to a-induced elevation in ca influx leading to cell death via a calpain - dependent mechanism [ 111 , 112 ] . by regulating the nmda receptor content and changing their localization in membrane microdomains at the synaptic sites", ", cholesterol modulates the ability of a to induce ca influx , leading to calpain activation in hippocampal neurons [ 111 , 112 ] .", "calpastatin also seems to play an important role in the regulation of calpain activation in ad .", "thus , it has been shown that calpastatin is markedly depleted in the cortex of ad brains at late stages of the disease as compared to age - matched controls .", "focal areas of calpastatin depletion have been also detected along dystrophic neurites at early stages of ad .", "on the other hand , no changes in calpastatin levels were detected in neurons less susceptible to neurodegeneration in ad like purkinje cells .", "this decrease in calpastatin levels is the result of the proteolytic activity of caspases and calpain . in turn , the decrease in the ratio calpastatin to calpain causes calpain hyperactivation perpetuating this cellular deleterious effect [ 79 , 113115 ] ."], ["these findings have been complemented with immunocytochemical studies on the distribution of calpain in ad .", "these studies have shown intense calpain immunoreactivity in dystrophic neurites associated with neurofibrillary tangles , neuritic plaques , and neuropil threads in the hippocampal region and entorhinal cortex of subjects suffering from ad and other tauopathies [ 116118 ] . based on this information ,", "it was hypothesized that the enhanced calpain activity observed in ad brains plays an important role in the formation of senile plaques , neurofibrillary tangles , and synaptic dysfunction in the context of ad ( figure 1 ) .", "these peptides are the result of the sequential proteolytic cleavage of app by two proteases .", "first , -secretase ( -site app - cleaving enzyme ( bace 1 ) ) cuts app at the n - terminus of the a domain leaving a 99-amino - acid - long c - terminal fragment .", "then , the -secretase complex ( presenilin 1 , presenilin 2 , nicastrin , aph1 , and pen2 ) cuts the c - terminus to generate a peptides of 38 to 43 amino acids in length [ 5 , 119121 ] . of these peptides ,", "therefore , increased levels of bace 1 could lead to enhanced production of a , its aggregation , and consequently the activation of signaling pathways associated with the neurodegenerative process .", "thus , a 2-fold increase in bace1 levels has been detected in ad brains [ 127129 ] .", "recently , it has been shown that the calpain / calpastatin system can modulate the pathological deposition of a. calpain activation increases the levels of bace1 in a transgenic mouse model of ad .", "in addition , the bidirectional regulation of the activation of the calpain / calpastatin system and a metabolism has been established . using transgenic mice overexpressing app", ", these authors showed that calpastatin deficiency enhanced calpain activation , a production , and increased mortality . in turn , the increased levels of a lead to enhanced ca influx and increased calpain activation .", "senile plaques are often surrounded by dystrophic neurons that contain tau aggregates known as neurofibrillary tangles .", "many studies have focused on the composition and the mechanisms underlying the formation of tau aggregates .", "those studies have identified two types of tau filaments in the neurofibrillary tangles : straight and paired helical filaments .", "tau hyperphosphorylation has been attributed to the increased activity of several kinases , including cyclin - dependent kinase 5 ( cdk5 ) , glycogen synthase kinase  ( gsk ) , and the mitogen - activated protein kinase ( mapk ) in hippocampal neurons [ 138 , 143145 ] .", "thus , it has been shown that calpain cleaves the inhibitory domain of gsk3 generating two fragments of 40 and 30  kda .", "the latter has a longer half life than p35 and therefore it is a more potent activator of cdk5 .", "experiments using calpeptin , a cell permeable calpain inhibitor , blocked both erk 1 and erk 2 activation .", "conditions that blocked the activation of these kinases induced a decreased in tau phosphorylation and resulted in enhanced neuronal survival in the presence of a . besides tau phosphorylation", ", calpain activation might play a role in tau - mediated neurodegeneration by inducing tau cleavage . in vitro studies", "have shown that both fetal and adult tau isoforms are rapidly proteolyzed by calpains [ 151153 ] .", "on the other hand , tau present in paired - helical filaments is considerably more resistant to proteolysis by these proteases [ 153156 ] .", "these findings suggest that phosphorylation might regulate the susceptibility of tau to calpain - mediated cleavage .", "tau obtained from cultured neurons incubated in the presence of this phosphatase inhibitor was more resistant to calpain cleavage than tau extracted from nontreated control neurons .", "nevertheless , the effect of phosphorylation on calpain - mediated tau cleavage seems to be complex .", "this effect might depend on the site phosphorylated and/or the extent of phosphorylation under pathological conditions since highly phosphorylated fetal isoforms are readily cleaved by calpain .  ", "calpain - mediated tau cleavage seems to play an important role under neurodegenerative conditions [ 157164 ] .", "it has been shown that calpain activation results in the generation of several n - terminal tau fragments .", "one of such tau fragments , of ~20  kda of apparent molecular weight , has been detected in mitochondria present in synaptosomal fractions obtained from ad brains .", "the levels of this tau fragment are partially reduced when cultured neurons are treated with a calpain inhibitor .", "it has been shown that tau fragments containing the 2644 tau amino acids affect mitochondria oxidative phosphorylation acting at the level of the adenine nucleotides translocator contributing to synapse dysfunction .  ", "a smaller tau fragment generated by calpain cleavage has been detected in mature hippocampal neurons treated with aggregated a oligomers [ 160 , 161 ] .", "a-induced tau cleavage mediated by calpain 1 leads to the generation of a neurotoxic 17  kda tau fragment ( tau 45230 ) in cultured hippocampal neurons [ 160 , 161 ] .", "this fragment has also been detected in the neocortex of ad brains as well as in brain areas affected by other tauopathies .", "furthermore , when expressed in neuronal and nonneuronal cell types or in an in vivo drosophila model system , the 17  kda tau fragment produced cell death in the absence of a oligomers [ 160 , 161 , 163 ] .", "these data provided strong evidence for an important role of calpain 1 and the generation of the 17  kda tau fragment in the progression of a-mediated neurodegeneration .", "it is worth noting that calpain 2 activation cleaves tau generating a smaller tau fragment that lacks neurotoxic effects in central neurons .  ", "in addition to the presence of senile plaques and neurofibrillary tangles , ad brains are characterized by a decrease in synaptic contacts .", "this synaptic loss seems to be the best morphological correlate of the functional deficits observed in the mid to late stages of ad [ 39 , 40 ] .", "although no significant decline in synapse number has been detected in the earliest stages of the disease , a stage of synaptic dysfunction seems to precede frank synapse loss [ 41 , 46 , 165 ] . by cleaving proteins in the presynaptic terminals and/or the postsynaptic elements", "changes in proteins involved in synaptic vesicle biogenesis and/or recycling at synaptic terminals are responsible , at least in part , for the synaptic dysfunction detected in ad .", "recently , data obtained using culture and animal models of ad showed that a induced a significant reduction in dynamin 1 levels that preceded synapse loss .", "dynamin 1 pinches off synaptic vesicles , freeing them from the membrane and allowing them to reenter the synaptic vesicle pool to be refilled for future release [ 167 , 168 ] .", "decreased levels of this protein lead to the depletion of synaptic vesicles and the accumulations of invaginated pits at presynaptic membranes adjacent to the synaptic clefts [ 9597 , 169 , 170 ] .", "the a-induced decrease in dynamin 1 is a result , at least partially , of calpain - mediated proteolysis [ 9597 , 171 ] .  ", "thus , it has been shown that calpain activation results in the breakdown of several proteins leading to changes in the ultrastructure of the postsynaptic density ( psd ) .", "these changes in the psd are accompanied by the rapid cleavage of psd-95 and the nmda receptor subunits nr1 and nr2a and 2b [ 172175 ] .", "calpain has been shown to truncate also mglur1 exacerbating nmda - mediated neurotoxicity [ 176178 ] .  ", "an additional mechanism by which calpain activation can result in synaptic dysfunction involved the cleavage of pka leading to a decrease in both the regulatory and the catalytic subunits of this kinase .", "the decrease in pka activity attenuates creb activation impairing memory [ 179 , 180 ] ."], ["taken together , the data reviewed above strongly suggest that calpains play an important role in the neurodegeneration mechanisms underlying ad in the aging population . based on these data", ", it has been tempting to speculate that calpain inhibition could be a useful tool to prevent neurodegeneration in this disease . to obtain further insights into the beneficial effects of blocking calpain activation , several studies", "have been conducted using experimental approaches to either suppress the expression of these proteases or prevent their abnormal activation .", "early studies using gene deletion of the calpain regulatory subunit by homologous recombinant techniques showed that the depletion of these proteases is embryonic lethal .", "the data obtained showed that indeed the suppression of calpain 1 expression by means of these specific probes prevented oxidative stress - induced cell injury in human hepatic cancer cell lines .", "other studies have assessed the effects of calpain inhibitors on cell death in culture model systems of ad .", "leupeptin and e64 calpain inhibitors had protective effects in those cultures [ 160 , 183186 ] .", "the idea that calpain could be a potential therapeutic target in neurodegenerative diseases has been reinforced by studies performed in vitro and in culture using the calpain inhibitor mdl 28170 .", "those studies suggested a protective effect of calpain inhibitors against excitotoxicity [ 178 , 187 , 188 ] .", "unfortunately , the potential use of known calpain inhibitors as therapeutic tools in ad is limited due to their low cellular penetration , poor selectivity , and kinetics . recently ,", "this benzoylalanine - derived ketoamide is capable of inhibiting calpain in nanomolar concentrations and has improved oral bioavailability , water solubility , and metabolic stability .", "this calpain inhibitor is highly effective in preventing calpain - mediated cleavage of dynamin 1 and tau in cultured hippocampal neurons .", "this inhibitor is effective not only when added prior to the a treatment but also when added simultaneously with a and even when added after a has triggered the neurodegeneration process .", "the calpain inhibitor a-705253 also prevents a oligomer - induced neurodegeneration of the nucleus basalis magnocellularis .", "these experiments raised the possibility that more potent calpain inhibitors could have beneficial effects even at late stages of ad .", "moreover , initial studies using calpain inhibitors in mouse and rat models of ad showed an encouraging recovery of cognitive function in these animals when they were treated with calpain inhibitors at an early age [ 186 , 190 ] . together , these data underscore the potential importance of calpain inhibitors as promising therapeutic tools in ad and related neurodegenerative diseases .  ", "in summary , the data briefly reviewed above provide strong support for the role of calpains in ad .", "they also highlight the tantalizing possibility that these proteases could serve as targets for the development of therapeutic interventions for this disease , one of the main challenges for decades to come in ad research ."]]}
{"article_id": "PMC5005989", "article_text": ["for many years , the global community has observed the calamitous effects of the human immunodeficiency virus ( hiv ) on the human population .", "hiv was historically viewed as an incurable disease that gradually progressed into acquired immune deficiency syndrome ( aids ) and led to one s untimely death . during his tenure ( 20012008 )", ", united states president george w. bush recognized the devastating impact of hiv as a global health crisis that required immediate action . through bipartisan efforts between president bush and the 108 us congress ,", "the passage of hr 1298 ( public law 10825 ) led to the creation of the president s emergency plan for aids relief , commonly referred to as pepfar .", "the objective of pepfar was to provide $ 15 billion in aid , targeting 12 focus countries in sub - saharan africa : botswana , cote divoire , ethiopia , kenya , mozambique , namibia , nigeria , rwanda , south africa , tanzania , uganda , and zambia . in an empirical research that was conducted on the impact of international humanitarian assistance on combating hiv / aids , azuine et al .", "discovered that the increase of donor assistance in developing countries led to an overall reduction of hiv infection rates .", "pepfar has illustrated the pertinence of an interconnected world and the urgency to embrace those connections during stages of global stress . according to the united nations ,", "the united states has contributed more funding to the fight against hiv / aids than any other country .", "the research presented here provides a better understanding of how well pepfar advanced that fight and whether the allocation of a large amount of dollars for pepfar was crucial in saving lives and decreasing hiv infection rates in sub - saharan africa .", "the general policy research question for this research was to determine whether the pepfar funding allocated to the 12 resource - scarce countries in sub - saharan africa had any relevant effect in reducing the hiv infection rates of males and females between the ages of 15 and 49 .", "an analysis to determine whether the pepfar policy was effective and efficient may influence future global humanitarian assistance provided to other countries by the united states .", "researchers agree that the continent of africa is the focal point of the hiv pandemic and that hiv / aids is a detrimental problem for the global community .", "even though other countries such as guyana , haiti , and vietnam were also recipients of pepfar funding , the majority of the crisis , population affected , and funding allocated for pepfar was concentrated in the above - mentioned 12 african countries .", "the specific focus addressed herein is whether the pepfar funding allocation was effective in meeting its intended purpose of reducing hiv infection rates in the 12 african countries that received funding . to this day", ", africa prevails as the most hiv / aids affected continent in the world , in particular the sub - saharan areas .", "africans account for approximately two thirds of hiv infections and three quarters of deaths associated with hiv from around the world .", "the joint united nations programme on hiv / aids ( unaids ) , united nations children s fund ( unicef ) , and world health organization ( who ) determined that approximately 75% of people in sub - saharan africa between the ages of 15 and 49 are unaware of their hiv status .", "together , hiv and aids have devastating effects on society and the economy . together ,", "hiv and aids create reproductive concerns , reduced revenues , increased healthcare expenses , decreased school enrollment , increased rates of absence at work , disruptions in the family environment , decreased human capital , increases in poverty rates , a lower gross domestic product ( gdp ) , and more orphaned children.[9 - 14 ] due to high levels of poverty , many developing countries in sub - saharan africa do not have the necessary resources or funding to combat the hiv / aids pandemic occurring in their nations .", "the methods that were used to treat and prevent further growth in cases of hiv / aids were education , counseling , hiv testing , antiretroviral medication , and building health care infrastructure . the treatment category of funding from pepfar focused on the amount of monetary support given to an african country for hiv treatment , the purchase of antiretroviral ( arv ) drugs , and the building of health infrastructure .", "one of the most essential components of the pepfar program was the increased access to generic arv medication and therapy .", "the purpose of the arv medication is to reduce the plasma level of hiv viral load.[19 , 20 ] the reduced viral load resulting from arv therapy not only improves the health of the infected individual , but also lowers the risk of passing the disease on to someone else .", "the availability of arv was critical in prolonging the lives of hiv infected individuals and reducing transmission rates .", "the prevention category of pepfar focused on the foundational strategy of preventing the proliferation of hiv .", "this foundation was known as the abc approach : ( a ) abstain , ( b ) be faithful , and ( c ) use condoms .", "there was strong emphasis on the  abstinence from sex before marriage  and  be faithful  aspects , which sometimes generated controversy because of the religious component in the program .", "safe - sex education was also a pertinent program in the fight against hiv / aids .", "the care category of pepfar focused on counseling , testing people for hiv / aids , taking care of orphan children , and palliative care for individuals with hiv / aids .", "since the majority of people in sub - saharan africa are unaware of their hiv status , this negligence could well lead to the rapid spread of the disease through sexual intercourse .", "the final category of pepfar efforts focused on training in health management , increasing health care staffing levels , and strengthening the healthcare management system .", "health management and effective management systems are important because one of the common theories is that the spread of hiv / aids in africa is worsened by high corruption rates and lack of accountability among government officials .", "the rampant level of government corruption may prevent government officials from being effective leaders in addressing the hiv / aids pandemic .", "to this day , africa prevails as the most hiv / aids affected continent in the world , in particular the sub - saharan areas .", "africans account for approximately two thirds of hiv infections and three quarters of deaths associated with hiv from around the world .", "the joint united nations programme on hiv / aids ( unaids ) , united nations children s fund ( unicef ) , and world health organization ( who ) determined that approximately 75% of people in sub - saharan africa between the ages of 15 and 49 are unaware of their hiv status . together ,", "hiv and aids create reproductive concerns , reduced revenues , increased healthcare expenses , decreased school enrollment , increased rates of absence at work , disruptions in the family environment , decreased human capital , increases in poverty rates , a lower gross domestic product ( gdp ) , and more orphaned children.[9 - 14 ] due to high levels of poverty , many developing countries in sub - saharan africa do not have the necessary resources or funding to combat the hiv / aids pandemic occurring in their nations .", "the methods that were used to treat and prevent further growth in cases of hiv / aids were education , counseling , hiv testing , antiretroviral medication , and building health care infrastructure . the treatment category of funding from pepfar focused on the amount of monetary support given to an african country for hiv treatment , the purchase of antiretroviral ( arv ) drugs , and the building of health infrastructure .", "one of the most essential components of the pepfar program was the increased access to generic arv medication and therapy .", "the purpose of the arv medication is to reduce the plasma level of hiv viral load.[19 , 20 ] the reduced viral load resulting from arv therapy not only improves the health of the infected individual , but also lowers the risk of passing the disease on to someone else .", "the availability of arv was critical in prolonging the lives of hiv infected individuals and reducing transmission rates .", "the prevention category of pepfar focused on the foundational strategy of preventing the proliferation of hiv .", "this foundation was known as the abc approach : ( a ) abstain , ( b ) be faithful , and ( c ) use condoms .", "there was strong emphasis on the  abstinence from sex before marriage  and  be faithful  aspects , which sometimes generated controversy because of the religious component in the program .", "safe - sex education was also a pertinent program in the fight against hiv / aids .", "the care category of pepfar focused on counseling , testing people for hiv / aids , taking care of orphan children , and palliative care for individuals with hiv / aids .", "since the majority of people in sub - saharan africa are unaware of their hiv status , this negligence could well lead to the rapid spread of the disease through sexual intercourse .", "the final category of pepfar efforts focused on training in health management , increasing health care staffing levels , and strengthening the healthcare management system .", "health management and effective management systems are important because one of the common theories is that the spread of hiv / aids in africa is worsened by high corruption rates and lack of accountability among government officials .", "the rampant level of government corruption may prevent government officials from being effective leaders in addressing the hiv / aids pandemic .", "based on theoretical evidence , this study expected that the 12 focus countries in africa that received pepfar funding would see a decrease in the hiv infection rates .", "even though arv medications are able to prolong the lives of hiv infected individuals , the hiv infection rates that are calculated by the world bank take into consideration individuals who are taking the arv medication .", "the software used for the collection of hiv infection rates reduced the infectivity rates among people receiving arv treatments .", "table 1 lists the 12 countries in africa that received pepfar funding and table 2 provides the amount of pepfar funding that was received from 2004 through 2008 in millions of us dollars .", "table 3 shows the hiv infection rates of males and females between the ages of 15 and 49 in the 12 countries in africa that received pepfar funding from 2004 through 2008 .", "pepfar focus countries in sub - saharan africa source : united states president s emergency plan for aids relief countries that received pepfar funding ( millions of us dollars ) , 20042008 source : united states president s emergency plan for aids relief hiv infection rates ( % ) of males and females ages 1549 , 20042008 source : united states president s emergency plan for aids relief the following theoretical models were applied to this research : hivrateit= 1 + 2%pepfar per gdpit+ it hivrateit= 1 + 2%pepfar per gdpit + 3%health per gdpit + 4corruptionit + 5gdppcit+ it the dependent variable was hiv infection rates in africa .", "the control independent variables were healthcare expenditure per gdp , corruption rate of a country , and gdp per capita .", "where : \n 1 is the intercept of the models and it is the stochastic error term of the models.hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank).%health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank).gdppcit was the gdp per capita of each country in us dollars ( world bank).%pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar).corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . \n ", "1 is the intercept of the models and it is the stochastic error term of the models .", "hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank ) .", "% health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank ) .", "gdppcit was the gdp per capita of each country in us dollars ( world bank ) .", "% pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar ) .", "corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) .", "this study used panel data from 2002 through 2010 covering the 12 countries that received pepfar funding in the continent of africa . even though pepfar was active from 2004 through 2008 , this research includes two years before ( 2002 and 2003 ) and two years after ( 2009 and 2010 ) in order to increase the ability to estimate the actual effect of receiving pepfar funding . in order to analyze the correlation between pepfar funding and hiv infection rates in africa ,", "this research used stata software version 13 and conducted a fixed - effects panel regression analysis .", "the fixed - effects technique was chosen based on results from the hausman and sargan - hansen tests , which were performed through stata .", "there was the possibilities of both heteroskedasticity and autocorrelation in this study because this analysis included a combination of cross - sectional and time - series data .", "the use of panel data for this research required statistical tests to determine if the regression model did indeed encounter a problem with heteroskedasticity and autocorrelation . as expected , the results of the tests indicated the presence of these problems in the regression model . in order to correct for the heteroskedasticity and autocorrelation problems ,", "this research used the robust standard errors for panel regression with cross - sectional dependence model .", "hivrateit= 1 + 2%pepfar per gdpit + 3%health per gdpit + 4corruptionit + 5gdppcit+ it the dependent variable was hiv infection rates in africa .", "the control independent variables were healthcare expenditure per gdp , corruption rate of a country , and gdp per capita .", "where : \n 1 is the intercept of the models and it is the stochastic error term of the models.hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank).%health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank).gdppcit was the gdp per capita of each country in us dollars ( world bank).%pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar).corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . \n ", "1 is the intercept of the models and it is the stochastic error term of the models .", "hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank ) .", "% health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank ) .", "gdppcit was the gdp per capita of each country in us dollars ( world bank ) .", "% pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar ) .", "corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) .", "this study used panel data from 2002 through 2010 covering the 12 countries that received pepfar funding in the continent of africa . even though pepfar was active from 2004 through 2008 , this research includes two years before ( 2002 and 2003 ) and two years after ( 2009 and 2010 ) in order to increase the ability to estimate the actual effect of receiving pepfar funding . in order to analyze the correlation between pepfar funding and hiv infection rates in africa ,", "this research used stata software version 13 and conducted a fixed - effects panel regression analysis .", "the fixed - effects technique was chosen based on results from the hausman and sargan - hansen tests , which were performed through stata .", "there was the possibilities of both heteroskedasticity and autocorrelation in this study because this analysis included a combination of cross - sectional and time - series data .", "the use of panel data for this research required statistical tests to determine if the regression model did indeed encounter a problem with heteroskedasticity and autocorrelation .", "as expected , the results of the tests indicated the presence of these problems in the regression model . in order to correct for the heteroskedasticity and autocorrelation problems ,", "this research used the robust standard errors for panel regression with cross - sectional dependence model .", "although several components have likely contributed to the declining hiv rates in the pepfar focus countries , the main objective of this research was to determine whether pepfar was an effective factor in lowering the hiv / aids infection rates , and , if so , by how much .", "table 4 provides the results from the fixed - effects panel regression analysis for models 1 and 2 . in model 1 ,", "pepfar funding per gdp was statistically significant at the 0.01 level . in model 2 ,", "pepfar funding per gdp and gdp per capita were statistically significant at the 0.01 level .", "even though this research found correlation between hiv infection rates and pepfar funding , as the adage goes , correlation does not imply causation .", "thus , the results must be considered in light of this caveat , as is the case with all empirical analyses . in model 1 ,", "the results illustrate that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.41 percentage points .", "the within r value for this model was 0.1684 , indicating that 16.84% of the variability in the dependent variable was explained by the independent variables . in model 2 ,", "the results illustrated that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.355 percentage points .", "for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points .", "for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points . the within r value for this model was 0.3733 , indicating that 37.33% of the variability in the dependent variable was explained by the independent variables .", "figure 1 provides graphs depicting a comparison between the amounts of pepfar funding received by the 12 countries in sub - saharan africa and the hiv rates from 2002 through 2010 . even though pepfar funding had steadily increased for each of the countries , most of the hiv infection rates decreased slightly , but remained relatively steady overall .", "comparison of hiv infection rates and pepfar funding the united states provided large monetary support toward the pepfar program .", "even though the results indicated that pepfar was statistically significant , the evaluation of whether pepfar was politically and economically significant remains subjective .", "some may argue that any decrease in hiv infection rates highlights the relevance of the continued efforts of pepfar , while others could argue that the large amount of money invested in pepfar should have generated larger decreases in the hiv infection rates in africa than the rates found in this study .", "based on this research , the overall hiv rate decreased , but the large amount of money invested to combat hiv infections did not drastically lower the hiv infection rates .", "the success of combating hiv / aids involves the collaboration of many public and humanitarian organizations .", "future research should include analysis on the impact of public sector agencies , universities , faith - based organizations , private contractors , and non - governmental organizations in this philanthropic mission .", "the center for global development released data that provided specific dollar amounts on how much pepfar funding was allocated to the different organizations from 2004 through 2006 in the categories of treatment , prevention , care , and other . although the data is sparse , analyzing the data supplied by the center for global development would provide a better indication of the role and impact each sector had in the pepfar program .", "a clear understanding of the global impact with the struggle against hiv / aids and the role that pepfar has played in that struggle is appropriate for future public policies , legislation , and programs . to the degree that it succeeded ,", "the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa .", "the global community is becoming more interconnected and societies are becoming mutually dependent on one another to combat health crises .", "the increase of social , economic , and political globalization broadens the realization that hiv / aids does not recognize race , gender , ethnicity , sexual orientation , or geographic borders .", "pepfar had some impact on the hiv pandemic in the 12 focus countries in sub - saharan africa through increasing humanitarian funding , providing access to cost - effective arv medication , developing improved healthcare infrastructures , and implementing a number of prevention programs .", "even though hiv / aids can not be cured as of yet , there is intrinsic value in preventing the rapid spread of this malady .", "although the programs and allocated funding of pepfar accomplished most of the stated objectives , there remains even more that needs to be accomplished , for hiv / aids continues to persist as a global pandemic presenting significant challenges .", "despite a few limitations , this study was able to analyze and illustrate the relative effectiveness of pepfar funding in lowering the hiv infection rates on the continent of africa , and has hopefully provided valuable insight for future policy considerations . \n", "the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa.on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.36 percentage points.for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points.for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points . \n ", "the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa . on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received ,", "for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points .", "for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points ."], "abstract_text": ["<S> background : hiv and aids continue to have a calamitous effect on individuals living on the continent of africa . u.s . </S>", "<S> president george w. bush implemented the president s emergency plan for aids relief ( pepfar ) with the objective of committing approximately $ 15 billion from 2004 through 2008 to assist with the reduction of the hiv pandemic worldwide . the majority of the pepfar policy and funding focused on 12 countries in sub - saharan africa : botswana , cote divoire , ethiopia , kenya , mozambique , namibia , nigeria , rwanda , south africa , tanzania , uganda , and zambia . </S>", "<S> the policy question this research paper seeks to analyze is whether the pepfar funding ( as a % of gross domestic product ( gdp ) ) allocated to the 12 countries in africa had any effect on the decrease of hiv infection rates of males and females between the ages of 15 and 49.methods:a fixed - effects panel regression analysis was conducted to determine if this association exists . </S>", "<S> this study examined the 12 african countries that received pepfar funding over the years 2002 to 2010 ; even though pepfar was only active from 2004 through 2008 , this research included two years prior and two years after this timeframe in order to better estimate the effect of pepfar funding on hiv reduction.results:the results illustrate that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.355 percentage points.conclusions and global health implications : while the empirical findings in this study suggested that the correlation between pepfar funding and hiv reduction is statistically significant , the practical significance is perhaps less obvious . </S>", "<S> arguably , the reduction rate should be higher given the extent of funding targeted to this project . </S>", "<S> the conclusion of this research provides suggestions on future research and the policy implications of pepfar . </S>"], "labels": null, "section_names": ["Background", "Embracing Connections During Global Stress: Fighting the Pandemic", "Methodology", "Model 1", "Model 2", "Results", "Conclusion and Global Health Implications"], "sections": [["for many years , the global community has observed the calamitous effects of the human immunodeficiency virus ( hiv ) on the human population .", "hiv was historically viewed as an incurable disease that gradually progressed into acquired immune deficiency syndrome ( aids ) and led to one s untimely death . during his tenure ( 20012008 )", ", united states president george w. bush recognized the devastating impact of hiv as a global health crisis that required immediate action . through bipartisan efforts between president bush and the 108 us congress ,", "the passage of hr 1298 ( public law 10825 ) led to the creation of the president s emergency plan for aids relief , commonly referred to as pepfar .", "the objective of pepfar was to provide $ 15 billion in aid , targeting 12 focus countries in sub - saharan africa : botswana , cote divoire , ethiopia , kenya , mozambique , namibia , nigeria , rwanda , south africa , tanzania , uganda , and zambia . in an empirical research that was conducted on the impact of international humanitarian assistance on combating hiv / aids , azuine et al .", "discovered that the increase of donor assistance in developing countries led to an overall reduction of hiv infection rates .", "pepfar has illustrated the pertinence of an interconnected world and the urgency to embrace those connections during stages of global stress . according to the united nations ,", "the united states has contributed more funding to the fight against hiv / aids than any other country .", "the research presented here provides a better understanding of how well pepfar advanced that fight and whether the allocation of a large amount of dollars for pepfar was crucial in saving lives and decreasing hiv infection rates in sub - saharan africa .", "the general policy research question for this research was to determine whether the pepfar funding allocated to the 12 resource - scarce countries in sub - saharan africa had any relevant effect in reducing the hiv infection rates of males and females between the ages of 15 and 49 .", "an analysis to determine whether the pepfar policy was effective and efficient may influence future global humanitarian assistance provided to other countries by the united states .", "researchers agree that the continent of africa is the focal point of the hiv pandemic and that hiv / aids is a detrimental problem for the global community .", "even though other countries such as guyana , haiti , and vietnam were also recipients of pepfar funding , the majority of the crisis , population affected , and funding allocated for pepfar was concentrated in the above - mentioned 12 african countries .", "the specific focus addressed herein is whether the pepfar funding allocation was effective in meeting its intended purpose of reducing hiv infection rates in the 12 african countries that received funding . to this day", ", africa prevails as the most hiv / aids affected continent in the world , in particular the sub - saharan areas .", "africans account for approximately two thirds of hiv infections and three quarters of deaths associated with hiv from around the world .", "the joint united nations programme on hiv / aids ( unaids ) , united nations children s fund ( unicef ) , and world health organization ( who ) determined that approximately 75% of people in sub - saharan africa between the ages of 15 and 49 are unaware of their hiv status .", "together , hiv and aids have devastating effects on society and the economy . together ,", "hiv and aids create reproductive concerns , reduced revenues , increased healthcare expenses , decreased school enrollment , increased rates of absence at work , disruptions in the family environment , decreased human capital , increases in poverty rates , a lower gross domestic product ( gdp ) , and more orphaned children.[9 - 14 ] due to high levels of poverty , many developing countries in sub - saharan africa do not have the necessary resources or funding to combat the hiv / aids pandemic occurring in their nations .", "the methods that were used to treat and prevent further growth in cases of hiv / aids were education , counseling , hiv testing , antiretroviral medication , and building health care infrastructure . the treatment category of funding from pepfar focused on the amount of monetary support given to an african country for hiv treatment , the purchase of antiretroviral ( arv ) drugs , and the building of health infrastructure .", "one of the most essential components of the pepfar program was the increased access to generic arv medication and therapy .", "the purpose of the arv medication is to reduce the plasma level of hiv viral load.[19 , 20 ] the reduced viral load resulting from arv therapy not only improves the health of the infected individual , but also lowers the risk of passing the disease on to someone else .", "the availability of arv was critical in prolonging the lives of hiv infected individuals and reducing transmission rates .", "the prevention category of pepfar focused on the foundational strategy of preventing the proliferation of hiv .", "this foundation was known as the abc approach : ( a ) abstain , ( b ) be faithful , and ( c ) use condoms .", "there was strong emphasis on the  abstinence from sex before marriage  and  be faithful  aspects , which sometimes generated controversy because of the religious component in the program .", "safe - sex education was also a pertinent program in the fight against hiv / aids .", "the care category of pepfar focused on counseling , testing people for hiv / aids , taking care of orphan children , and palliative care for individuals with hiv / aids .", "since the majority of people in sub - saharan africa are unaware of their hiv status , this negligence could well lead to the rapid spread of the disease through sexual intercourse .", "the final category of pepfar efforts focused on training in health management , increasing health care staffing levels , and strengthening the healthcare management system .", "health management and effective management systems are important because one of the common theories is that the spread of hiv / aids in africa is worsened by high corruption rates and lack of accountability among government officials .", "the rampant level of government corruption may prevent government officials from being effective leaders in addressing the hiv / aids pandemic ."], ["to this day , africa prevails as the most hiv / aids affected continent in the world , in particular the sub - saharan areas .", "africans account for approximately two thirds of hiv infections and three quarters of deaths associated with hiv from around the world .", "the joint united nations programme on hiv / aids ( unaids ) , united nations children s fund ( unicef ) , and world health organization ( who ) determined that approximately 75% of people in sub - saharan africa between the ages of 15 and 49 are unaware of their hiv status . together ,", "hiv and aids create reproductive concerns , reduced revenues , increased healthcare expenses , decreased school enrollment , increased rates of absence at work , disruptions in the family environment , decreased human capital , increases in poverty rates , a lower gross domestic product ( gdp ) , and more orphaned children.[9 - 14 ] due to high levels of poverty , many developing countries in sub - saharan africa do not have the necessary resources or funding to combat the hiv / aids pandemic occurring in their nations .", "the methods that were used to treat and prevent further growth in cases of hiv / aids were education , counseling , hiv testing , antiretroviral medication , and building health care infrastructure . the treatment category of funding from pepfar focused on the amount of monetary support given to an african country for hiv treatment , the purchase of antiretroviral ( arv ) drugs , and the building of health infrastructure .", "one of the most essential components of the pepfar program was the increased access to generic arv medication and therapy .", "the purpose of the arv medication is to reduce the plasma level of hiv viral load.[19 , 20 ] the reduced viral load resulting from arv therapy not only improves the health of the infected individual , but also lowers the risk of passing the disease on to someone else .", "the availability of arv was critical in prolonging the lives of hiv infected individuals and reducing transmission rates .", "the prevention category of pepfar focused on the foundational strategy of preventing the proliferation of hiv .", "this foundation was known as the abc approach : ( a ) abstain , ( b ) be faithful , and ( c ) use condoms .", "there was strong emphasis on the  abstinence from sex before marriage  and  be faithful  aspects , which sometimes generated controversy because of the religious component in the program .", "safe - sex education was also a pertinent program in the fight against hiv / aids .", "the care category of pepfar focused on counseling , testing people for hiv / aids , taking care of orphan children , and palliative care for individuals with hiv / aids .", "since the majority of people in sub - saharan africa are unaware of their hiv status , this negligence could well lead to the rapid spread of the disease through sexual intercourse .", "the final category of pepfar efforts focused on training in health management , increasing health care staffing levels , and strengthening the healthcare management system .", "health management and effective management systems are important because one of the common theories is that the spread of hiv / aids in africa is worsened by high corruption rates and lack of accountability among government officials .", "the rampant level of government corruption may prevent government officials from being effective leaders in addressing the hiv / aids pandemic ."], ["based on theoretical evidence , this study expected that the 12 focus countries in africa that received pepfar funding would see a decrease in the hiv infection rates .", "even though arv medications are able to prolong the lives of hiv infected individuals , the hiv infection rates that are calculated by the world bank take into consideration individuals who are taking the arv medication .", "the software used for the collection of hiv infection rates reduced the infectivity rates among people receiving arv treatments .", "table 1 lists the 12 countries in africa that received pepfar funding and table 2 provides the amount of pepfar funding that was received from 2004 through 2008 in millions of us dollars .", "table 3 shows the hiv infection rates of males and females between the ages of 15 and 49 in the 12 countries in africa that received pepfar funding from 2004 through 2008 .", "pepfar focus countries in sub - saharan africa source : united states president s emergency plan for aids relief countries that received pepfar funding ( millions of us dollars ) , 20042008 source : united states president s emergency plan for aids relief hiv infection rates ( % ) of males and females ages 1549 , 20042008 source : united states president s emergency plan for aids relief the following theoretical models were applied to this research : hivrateit= 1 + 2%pepfar per gdpit+ it hivrateit= 1 + 2%pepfar per gdpit + 3%health per gdpit + 4corruptionit + 5gdppcit+ it the dependent variable was hiv infection rates in africa .", "the control independent variables were healthcare expenditure per gdp , corruption rate of a country , and gdp per capita .", "where : \n 1 is the intercept of the models and it is the stochastic error term of the models.hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank).%health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank).gdppcit was the gdp per capita of each country in us dollars ( world bank).%pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar).corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . \n ", "1 is the intercept of the models and it is the stochastic error term of the models .", "hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank ) .", "% health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank ) .", "gdppcit was the gdp per capita of each country in us dollars ( world bank ) .", "% pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar ) .", "corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) .", "this study used panel data from 2002 through 2010 covering the 12 countries that received pepfar funding in the continent of africa . even though pepfar was active from 2004 through 2008 , this research includes two years before ( 2002 and 2003 ) and two years after ( 2009 and 2010 ) in order to increase the ability to estimate the actual effect of receiving pepfar funding . in order to analyze the correlation between pepfar funding and hiv infection rates in africa ,", "this research used stata software version 13 and conducted a fixed - effects panel regression analysis .", "the fixed - effects technique was chosen based on results from the hausman and sargan - hansen tests , which were performed through stata .", "there was the possibilities of both heteroskedasticity and autocorrelation in this study because this analysis included a combination of cross - sectional and time - series data .", "the use of panel data for this research required statistical tests to determine if the regression model did indeed encounter a problem with heteroskedasticity and autocorrelation . as expected , the results of the tests indicated the presence of these problems in the regression model . in order to correct for the heteroskedasticity and autocorrelation problems ,", "this research used the robust standard errors for panel regression with cross - sectional dependence model ."], [""], ["hivrateit= 1 + 2%pepfar per gdpit + 3%health per gdpit + 4corruptionit + 5gdppcit+ it the dependent variable was hiv infection rates in africa .", "the control independent variables were healthcare expenditure per gdp , corruption rate of a country , and gdp per capita .", "where : \n 1 is the intercept of the models and it is the stochastic error term of the models.hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank).%health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank).gdppcit was the gdp per capita of each country in us dollars ( world bank).%pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar).corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . \n ", "1 is the intercept of the models and it is the stochastic error term of the models .", "hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank ) .", "% health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank ) .", "gdppcit was the gdp per capita of each country in us dollars ( world bank ) .", "% pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar ) .", "corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) .", "this study used panel data from 2002 through 2010 covering the 12 countries that received pepfar funding in the continent of africa . even though pepfar was active from 2004 through 2008 , this research includes two years before ( 2002 and 2003 ) and two years after ( 2009 and 2010 ) in order to increase the ability to estimate the actual effect of receiving pepfar funding . in order to analyze the correlation between pepfar funding and hiv infection rates in africa ,", "this research used stata software version 13 and conducted a fixed - effects panel regression analysis .", "the fixed - effects technique was chosen based on results from the hausman and sargan - hansen tests , which were performed through stata .", "there was the possibilities of both heteroskedasticity and autocorrelation in this study because this analysis included a combination of cross - sectional and time - series data .", "the use of panel data for this research required statistical tests to determine if the regression model did indeed encounter a problem with heteroskedasticity and autocorrelation .", "as expected , the results of the tests indicated the presence of these problems in the regression model . in order to correct for the heteroskedasticity and autocorrelation problems ,", "this research used the robust standard errors for panel regression with cross - sectional dependence model ."], ["although several components have likely contributed to the declining hiv rates in the pepfar focus countries , the main objective of this research was to determine whether pepfar was an effective factor in lowering the hiv / aids infection rates , and , if so , by how much .", "table 4 provides the results from the fixed - effects panel regression analysis for models 1 and 2 . in model 1 ,", "pepfar funding per gdp was statistically significant at the 0.01 level . in model 2 ,", "pepfar funding per gdp and gdp per capita were statistically significant at the 0.01 level .", "even though this research found correlation between hiv infection rates and pepfar funding , as the adage goes , correlation does not imply causation .", "thus , the results must be considered in light of this caveat , as is the case with all empirical analyses . in model 1 ,", "the results illustrate that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.41 percentage points .", "the within r value for this model was 0.1684 , indicating that 16.84% of the variability in the dependent variable was explained by the independent variables . in model 2 ,", "the results illustrated that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.355 percentage points .", "for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points .", "for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points . the within r value for this model was 0.3733 , indicating that 37.33% of the variability in the dependent variable was explained by the independent variables .", "figure 1 provides graphs depicting a comparison between the amounts of pepfar funding received by the 12 countries in sub - saharan africa and the hiv rates from 2002 through 2010 . even though pepfar funding had steadily increased for each of the countries , most of the hiv infection rates decreased slightly , but remained relatively steady overall .", "comparison of hiv infection rates and pepfar funding the united states provided large monetary support toward the pepfar program .", "even though the results indicated that pepfar was statistically significant , the evaluation of whether pepfar was politically and economically significant remains subjective .", "some may argue that any decrease in hiv infection rates highlights the relevance of the continued efforts of pepfar , while others could argue that the large amount of money invested in pepfar should have generated larger decreases in the hiv infection rates in africa than the rates found in this study .", "based on this research , the overall hiv rate decreased , but the large amount of money invested to combat hiv infections did not drastically lower the hiv infection rates .", "the success of combating hiv / aids involves the collaboration of many public and humanitarian organizations .", "future research should include analysis on the impact of public sector agencies , universities , faith - based organizations , private contractors , and non - governmental organizations in this philanthropic mission .", "the center for global development released data that provided specific dollar amounts on how much pepfar funding was allocated to the different organizations from 2004 through 2006 in the categories of treatment , prevention , care , and other . although the data is sparse , analyzing the data supplied by the center for global development would provide a better indication of the role and impact each sector had in the pepfar program ."], ["a clear understanding of the global impact with the struggle against hiv / aids and the role that pepfar has played in that struggle is appropriate for future public policies , legislation , and programs . to the degree that it succeeded ,", "the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa .", "the global community is becoming more interconnected and societies are becoming mutually dependent on one another to combat health crises .", "the increase of social , economic , and political globalization broadens the realization that hiv / aids does not recognize race , gender , ethnicity , sexual orientation , or geographic borders .", "pepfar had some impact on the hiv pandemic in the 12 focus countries in sub - saharan africa through increasing humanitarian funding , providing access to cost - effective arv medication , developing improved healthcare infrastructures , and implementing a number of prevention programs .", "even though hiv / aids can not be cured as of yet , there is intrinsic value in preventing the rapid spread of this malady .", "although the programs and allocated funding of pepfar accomplished most of the stated objectives , there remains even more that needs to be accomplished , for hiv / aids continues to persist as a global pandemic presenting significant challenges .", "despite a few limitations , this study was able to analyze and illustrate the relative effectiveness of pepfar funding in lowering the hiv infection rates on the continent of africa , and has hopefully provided valuable insight for future policy considerations . \n", "the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa.on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.36 percentage points.for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points.for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points . \n ", "the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa . on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received ,", "for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points .", "for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points ."]]}
{"article_id": "PMC3245727", "article_text": ["descriptive investigation of a cluster of 5 sfts cases following exposure to an index patient who died from the disease was performed . a 77-year - old male farmer who died from sfts", "five other cases in the cluster included a 32-year - old male intensive care unit ( icu ) physician ( case 1 ) , a 48-year - old male icu consultation physician ( case 2 ) , a 42-year - old younger son of the index patient ( case 3 ) , and a 45-year - old older son of the index patient ( case 4 ) , as well as a 43-year - old male mortuary beautician ( case 5 ) .", "the investigation included a review of circumstances and medical records , specimen collection , virus isolation , real - time polymerase chain reaction for viral rna detection , and sequencing and serological tests ( capture enzyme - linked immunosorbent assay [ elisa ] for immunoglobin [ ig ] m antibody , antigen sandwich elisa for igg antibody , and microneutralization tests [ mnt ] ) .", "as for risk assessment of transmission factors , all contactors of the index patient during the period from the beginning ( 25 september 2010 ) to the end ( 8 october 2010 ) were classified through 3 types of contacts , including blood , droplet , and possible airborne contacts . the investigation was reviewed and approved by the ethics committee of china center for disease control and prevention ( cdc ) , which uses international guidelines to ensure confidentiality , anonymity , and informed consent .", "the index patient was from an sfts - endemic region and first had onset of illness on 25 september 2010 ; was admitted to a local hospital on 28 september with a high fever of 39.5c and vomiting ; and the infection was identified through initial laboratory testing of thrombocytopenia , leukocytopenia , and elevated aspartate aminotransferase level .", "the patient was diagnosed as a suspected sfts case , and transferred to the intensive care unit the next day , where he received ribavirin , dexamethasone , omeprazole , and cryoprecipitation for therapy . on 2 october 2010 ,", "his condition deteriorated with hypersomnia , shortness of breath , and mouth mucosal bleeding . on 3 october", ", he appeared confused and showed dyspnea , then was intubated and mechanical ventilated . on 4 october ,", "the patient was in shock and comatose , developed disseminated intravascular coagulation and multiple organ dysfunction syndrome , and died the next day .", "an important observation was that the index patient had unique hemorrhagic symptoms , including bleeding from mouth mucosa , gastrointestinal lumen , and lungs , which was rare in previously identified sfts patients .", "the patient s fever remained high ( 39c40c ) until death , although lowered somewhat by giving dexamethasone .", "clinical laboratory values ( table 1 ) showed unusually high level of tissue enzymes , blood urea nitrogen , and creatinine , which might indicate impaired liver , heart , and kidney functions .", "consistent with the largely reduced platelet count , the index patient had a significantly elongated activated partial thromboplastin time , which represented a largely impaired coagulation function .", "immediately after death , the corpse was transferred to the patient s home in a local village for a funeral ceremony , then transferred to a crematorium 3 days later .", "laboratory analysis of a cluster of severe fever with thrombocytopenia syndrome patients in china alt , alanine aminotransferase ; ast , aspartate aminotransferase ; ldh , lactate dehydrogenase ; aptt , activated partial thromboplastin time .", " , negative ; + , weak positive ; + + + , strong positive .", "viral rna copies were calculated according to standard reaction curves , which were established using serially diluted in vitro rna transcripts as standard samples .", "virus load was determined based on an average conversion coefficient between virus copies and virus titer presented as 50% tissue culture infective dose ( tcid50/ml ) .", "serum samples collected in the acute phase were subjected to immunoglobin ( ig ) m detection with igm antibody capture enzyme - linked immunosorbent assay ( elisa ) , with horseradish peroxidase  conjugated recombinant severe fever with thrombocytopenia syndrome bunyavirus ( sftsv ) nucleoprotein as the detection agent .", "serum samples collected in the convalescent phase were subjected to igg detection with sandwich elisa by using recombinant sftsv nucleoprotein as the detection agent .", "values for igm antibodies , igg antibodies , and mnt are the reciprocals of the serum dilution .", "risk factors were assessed using logistic regression analysis ; p  <  .05 was considered to be statistically different .", "epidemiologic investigations began with interviews with 5 secondary sfts patients and 58 other individuals who had exposure to the index patient from onset of the illness until cremation .", "we found that all 5 secondary cases had possible blood contact through skin or mucosa .", "case 1 , the local icu doctor , performed the intubation for the index patient , resuscitating the patient before his death .", "case 2 , the consultant doctor , traveled from a nonendemic area to the local hospital for a medical consult with the patient , and the patient s blood dropped on his hand when he was helping an icu nurse draw blood without wearing gloves .", "case 3 , the younger son of the index patient , was a long - distance truck driver not living in the same village , and directly touched the blood flowing from the deceased patient s mouth and nose when he cried on the corpse .", "case 4 , the older son of the index patient , was also a long - distance truck driver , and took care of the dead body and was wiping off the blood from the face of the corpse .", "case 5 , a local mortuary beautician , did the make - up for the corpse with gloves and mask , but took them off twice during the procedure . the above 5 cases all developed the disease 715 days after exposure , with only 3 of them having contact with blood after death . in comparison with the index patient", ", all 5 secondary cases had minor clinical features ( table 1 ) , without obvious hemorrhagic manifestations , so they did not receive dexamethasone therapy . except for the major risk factor of blood contact , droplet contact ( n  =  4 ) , which included 3 medical staff who were involved in the intubation and the index patient s daughter who cared for him before death ,", "in addition , the risk factor of possible airborne transmission ( being in the icu room or funeral room without mask protection ) was also assessed among all 63 exposed individuals . of them , the medical doctors and nurses ( n  =  16 ) were with protection ; and the others were without ( n  =  47 ) , including icu patients who were staying in the same room ( n  =  8) , family members (", "n  =  3 ) , and visitors to the funeral room where the corpse was kept ( n  =  35 ) .", "the multivariate analysis ( logistic regression analysis ) showed that blood contact was the most likely mode of transmission ( p  <  .001 ; table 1 ) .", "it is noticeable that the serum collected from the index patient on the day he died contained a relatively high amount of sftsv , which reached about 3.55    10 viral rna copies / ml ( calculated as 9.67    10 50% tissue culture infective dose [ tcid50]/ml ) .", "this number was about 100  000-fold higher than viral copies detected in the serum samples of the 5 secondary patients , which ranged from 10 to 10 copies / ml ( table 1 ) .", "all 5 patients had virus - specific igm antibodies in the acute phase , and elevated virus - specific immunoglobin g ( igg ) antibodies in the convalescent phase . the index patient , however , had minimal or undetectable levels of virus specific igm antibodies on day 11 after onset of fever ( table 1 ) . compared with the entire genomic sequence of the virus strain isolated from the index patient ,", "cases 2 and 3 were nearly 100% identical , and provided genetic evidence for the epidemic findings on the possible transmission of sftsv from the index patient to secondary patients .", "here , we reported the person - to - person transmission of sftsv with a cluster of 6 sfts patients that occurred in shandong province of eastern china .", "investigation studies revealed that all 5 secondary infected patients had possible contact with the index patient s blood .", "of them , only case 1 ( icu doctor ) had evidence of droplet contact contamination ( during intubation ) but not blood contact , but we included him also as a blood contactor because he performed intubation without protective face shield and goggles , therefore he had an extremely high risk of blood contact through unprotected skin and mucosa .", "the risk assessment revealed that blood contact was the major risk factor for the human - to - human transmission of sftsv .", "additionally , 3 secondary patients who only had contact with the index patient after death were still infected , which indicated that sftsv - infected blood may remain infectious for a long time , even after patient death .", "it is notable that the level of virus copies in the index patient s serum was extremely high , but with no detectable igm or igg antibodies , which suggested that his immune responses to limit the replication of sftsv were severely impaired .", "the patient had no immune system disease , thus the minimal immune responses might be due to the early and sustained application of glucocorticoid , which has a side effect of repressing immune functions ; clinical physicians should take note of this finding .", "clinically , sfts is easily confused with hemorrhagic fever with renal syndrome caused by hantavirus   and human anaplasmosis .", "the hantavirus - specific igm antibodies were not detected in the sfts patients serum samples ( data not show ) . due to the limitation of our study , we were not able to detect rickettsia infection , which was previously reported in outbreaks of nosocomial infections", ". however , the methods we used for diagnosis of sfts patients were well validated , and the human - to - human transmission of sftsv we described in this study could not simply be classified as being nosocomial transmission ; specially , 3 of the 5 secondary cases that occurred in the household .", "additionally , the lessons of person - to - person transmission of sftsv we learned from this study highlighted the necessity of establishing standard precautions for avoiding direct contact and blood - based transmission .", "this work was supported by the china mega - project for infectious diseases ( 2008zx10004 - 001 , 2009zx10004 ) from the ministry of science and technology and ministry of health of the people s republic of china .", "conflicts that the editors consider relevant to the content of the manuscript have been disclosed ."], "abstract_text": ["<S> severe fever with thrombocytopenia syndrome bunyavirus is a newly discovered bunyavirus with high pathogenicity to human . </S>", "<S> the transmission model has been largely uncharacterized . </S>", "<S> investigation on a cluster of severe fever with thrombocytopenia syndrome cases provided evidence of person - to - person transmission through blood contact to the index patient with high serum virus load . </S>"], "labels": null, "section_names": ["METHODS", "RESULTS", "DISCUSSION", "Financial support.", "Potential conflicts of interest."], "sections": [["descriptive investigation of a cluster of 5 sfts cases following exposure to an index patient who died from the disease was performed . a 77-year - old male farmer who died from sfts", "five other cases in the cluster included a 32-year - old male intensive care unit ( icu ) physician ( case 1 ) , a 48-year - old male icu consultation physician ( case 2 ) , a 42-year - old younger son of the index patient ( case 3 ) , and a 45-year - old older son of the index patient ( case 4 ) , as well as a 43-year - old male mortuary beautician ( case 5 ) .", "the investigation included a review of circumstances and medical records , specimen collection , virus isolation , real - time polymerase chain reaction for viral rna detection , and sequencing and serological tests ( capture enzyme - linked immunosorbent assay [ elisa ] for immunoglobin [ ig ] m antibody , antigen sandwich elisa for igg antibody , and microneutralization tests [ mnt ] ) .", "as for risk assessment of transmission factors , all contactors of the index patient during the period from the beginning ( 25 september 2010 ) to the end ( 8 october 2010 ) were classified through 3 types of contacts , including blood , droplet , and possible airborne contacts . the investigation was reviewed and approved by the ethics committee of china center for disease control and prevention ( cdc ) , which uses international guidelines to ensure confidentiality , anonymity , and informed consent ."], ["the index patient was from an sfts - endemic region and first had onset of illness on 25 september 2010 ; was admitted to a local hospital on 28 september with a high fever of 39.5c and vomiting ; and the infection was identified through initial laboratory testing of thrombocytopenia , leukocytopenia , and elevated aspartate aminotransferase level .", "the patient was diagnosed as a suspected sfts case , and transferred to the intensive care unit the next day , where he received ribavirin , dexamethasone , omeprazole , and cryoprecipitation for therapy . on 2 october 2010 ,", "his condition deteriorated with hypersomnia , shortness of breath , and mouth mucosal bleeding . on 3 october", ", he appeared confused and showed dyspnea , then was intubated and mechanical ventilated . on 4 october ,", "the patient was in shock and comatose , developed disseminated intravascular coagulation and multiple organ dysfunction syndrome , and died the next day .", "an important observation was that the index patient had unique hemorrhagic symptoms , including bleeding from mouth mucosa , gastrointestinal lumen , and lungs , which was rare in previously identified sfts patients .", "the patient s fever remained high ( 39c40c ) until death , although lowered somewhat by giving dexamethasone .", "clinical laboratory values ( table 1 ) showed unusually high level of tissue enzymes , blood urea nitrogen , and creatinine , which might indicate impaired liver , heart , and kidney functions .", "consistent with the largely reduced platelet count , the index patient had a significantly elongated activated partial thromboplastin time , which represented a largely impaired coagulation function .", "immediately after death , the corpse was transferred to the patient s home in a local village for a funeral ceremony , then transferred to a crematorium 3 days later .", "laboratory analysis of a cluster of severe fever with thrombocytopenia syndrome patients in china alt , alanine aminotransferase ; ast , aspartate aminotransferase ; ldh , lactate dehydrogenase ; aptt , activated partial thromboplastin time .", " , negative ; + , weak positive ; + + + , strong positive .", "viral rna copies were calculated according to standard reaction curves , which were established using serially diluted in vitro rna transcripts as standard samples .", "virus load was determined based on an average conversion coefficient between virus copies and virus titer presented as 50% tissue culture infective dose ( tcid50/ml ) .", "serum samples collected in the acute phase were subjected to immunoglobin ( ig ) m detection with igm antibody capture enzyme - linked immunosorbent assay ( elisa ) , with horseradish peroxidase  conjugated recombinant severe fever with thrombocytopenia syndrome bunyavirus ( sftsv ) nucleoprotein as the detection agent .", "serum samples collected in the convalescent phase were subjected to igg detection with sandwich elisa by using recombinant sftsv nucleoprotein as the detection agent .", "values for igm antibodies , igg antibodies , and mnt are the reciprocals of the serum dilution .", "risk factors were assessed using logistic regression analysis ; p  <  .05 was considered to be statistically different .", "epidemiologic investigations began with interviews with 5 secondary sfts patients and 58 other individuals who had exposure to the index patient from onset of the illness until cremation .", "we found that all 5 secondary cases had possible blood contact through skin or mucosa .", "case 1 , the local icu doctor , performed the intubation for the index patient , resuscitating the patient before his death .", "case 2 , the consultant doctor , traveled from a nonendemic area to the local hospital for a medical consult with the patient , and the patient s blood dropped on his hand when he was helping an icu nurse draw blood without wearing gloves .", "case 3 , the younger son of the index patient , was a long - distance truck driver not living in the same village , and directly touched the blood flowing from the deceased patient s mouth and nose when he cried on the corpse .", "case 4 , the older son of the index patient , was also a long - distance truck driver , and took care of the dead body and was wiping off the blood from the face of the corpse .", "case 5 , a local mortuary beautician , did the make - up for the corpse with gloves and mask , but took them off twice during the procedure . the above 5 cases all developed the disease 715 days after exposure , with only 3 of them having contact with blood after death . in comparison with the index patient", ", all 5 secondary cases had minor clinical features ( table 1 ) , without obvious hemorrhagic manifestations , so they did not receive dexamethasone therapy . except for the major risk factor of blood contact , droplet contact ( n  =  4 ) , which included 3 medical staff who were involved in the intubation and the index patient s daughter who cared for him before death ,", "in addition , the risk factor of possible airborne transmission ( being in the icu room or funeral room without mask protection ) was also assessed among all 63 exposed individuals . of them , the medical doctors and nurses ( n  =  16 ) were with protection ; and the others were without ( n  =  47 ) , including icu patients who were staying in the same room ( n  =  8) , family members (", "n  =  3 ) , and visitors to the funeral room where the corpse was kept ( n  =  35 ) .", "the multivariate analysis ( logistic regression analysis ) showed that blood contact was the most likely mode of transmission ( p  <  .001 ; table 1 ) .", "it is noticeable that the serum collected from the index patient on the day he died contained a relatively high amount of sftsv , which reached about 3.55    10 viral rna copies / ml ( calculated as 9.67    10 50% tissue culture infective dose [ tcid50]/ml ) .", "this number was about 100  000-fold higher than viral copies detected in the serum samples of the 5 secondary patients , which ranged from 10 to 10 copies / ml ( table 1 ) .", "all 5 patients had virus - specific igm antibodies in the acute phase , and elevated virus - specific immunoglobin g ( igg ) antibodies in the convalescent phase . the index patient , however , had minimal or undetectable levels of virus specific igm antibodies on day 11 after onset of fever ( table 1 ) . compared with the entire genomic sequence of the virus strain isolated from the index patient ,", "cases 2 and 3 were nearly 100% identical , and provided genetic evidence for the epidemic findings on the possible transmission of sftsv from the index patient to secondary patients ."], ["here , we reported the person - to - person transmission of sftsv with a cluster of 6 sfts patients that occurred in shandong province of eastern china .", "investigation studies revealed that all 5 secondary infected patients had possible contact with the index patient s blood .", "of them , only case 1 ( icu doctor ) had evidence of droplet contact contamination ( during intubation ) but not blood contact , but we included him also as a blood contactor because he performed intubation without protective face shield and goggles , therefore he had an extremely high risk of blood contact through unprotected skin and mucosa .", "the risk assessment revealed that blood contact was the major risk factor for the human - to - human transmission of sftsv .", "additionally , 3 secondary patients who only had contact with the index patient after death were still infected , which indicated that sftsv - infected blood may remain infectious for a long time , even after patient death .", "it is notable that the level of virus copies in the index patient s serum was extremely high , but with no detectable igm or igg antibodies , which suggested that his immune responses to limit the replication of sftsv were severely impaired .", "the patient had no immune system disease , thus the minimal immune responses might be due to the early and sustained application of glucocorticoid , which has a side effect of repressing immune functions ; clinical physicians should take note of this finding .", "clinically , sfts is easily confused with hemorrhagic fever with renal syndrome caused by hantavirus   and human anaplasmosis .", "the hantavirus - specific igm antibodies were not detected in the sfts patients serum samples ( data not show ) . due to the limitation of our study , we were not able to detect rickettsia infection , which was previously reported in outbreaks of nosocomial infections", ". however , the methods we used for diagnosis of sfts patients were well validated , and the human - to - human transmission of sftsv we described in this study could not simply be classified as being nosocomial transmission ; specially , 3 of the 5 secondary cases that occurred in the household .", "additionally , the lessons of person - to - person transmission of sftsv we learned from this study highlighted the necessity of establishing standard precautions for avoiding direct contact and blood - based transmission ."], ["this work was supported by the china mega - project for infectious diseases ( 2008zx10004 - 001 , 2009zx10004 ) from the ministry of science and technology and ministry of health of the people s republic of china ."], ["conflicts that the editors consider relevant to the content of the manuscript have been disclosed ."]]}
{"article_id": "PMC3195075", "article_text": ["therefore , the mother 's knowledge about child care influences the nature and quality of care that is given to the child .", "several studies have revealed that the mothers level of education has a positive impact on her knowledge and how she deals with child health care issues.[213 ] our experience in pediatric practice has revealed significant gaps pertaining to child health issues in the mothers knowledge .", "there seems to have been very little improvement in the knowledge of mothers on common child health matters over the years inspite of the many years of girls education in the country .", "this descriptive cross - sectional survey aimed to answer the following questions : ( 1 ) what is the level of mothers knowledge of certain aspects of child health matters ? ( 2 ) what are the main sources of health information and which ones are the most useful ? ( 3 ) is there a correlation between mothers level of knowledge and the number of years spent in formal education ? ( 4 ) in the mother 's view , does the current formal education of girls in schools / colleges have a significant impact on mothers knowledge of child health matters .", "it was reviewed and refined by two other colleagues who have a lot of experience in pediatrics .", "the first part comprised information about mothers nationality , age , work , level of education and the number of children she has in addition to sources of health information and the role of formal school education in child health matters .", "the second part contained 40 statements about different aspects of child health issues including nutrition , immunization , development , accident prevention , certain neonatal and infants problems or behaviors and some common childhood illnesses .", "mothers who attended with their children at the pediatric outpatient clinic of king khalid university hospital in riyadh during the months of july and august , in 2007 , were invited to participate in the study .", "those who agreed were interviewed by a trained non - medical research assistant using the items and statements of the questionnaire as a basis for the structured interview .", "it was indicated clearly to the mothers that participation was voluntary and their non- contribution would have no adverse effect on the quality of care given to their child in the hospital .", "they scored one point for each knowledge question answered correctly and zero for wrong and  do not know  answers .", "the first 20 questionnaires completed were used as a pilot test to evaluate the clarity of language and level of information in the questionnaire .", "data was entered in ms excel and analyzed using spss pc 11.5 version statistical software .", "descriptive statistics , mean and standard deviation and proportion were used to quantify the study and outcome variables .", "demographic data regarding nationality , age , work , education , and number of children are represented in table 1 . as the main source of health information , 295 ( 80% ) cited family members ( grandmothers , mothers , sisters , relatives etc . ) , 30 ( 8.2% ) cited schools , 68 ( 17.2% ) cited tv and radio , 60 ( 16% ) cited journals and magazines , 52 ( 14.2% ) cited friends , colleagues and other members of society , 101 ( 27.1% ) cited books and only 25 ( 7.1% ) cited health care professionals .", "demographic data of the mothers two hundred twenty - five ( 60.6% ) of the mothers believe that formal girls education in schools and colleges does not provide sufficient child health education . on the question of where to obtain useful information of child health , 287 ( 77.6% ) of the mothers supported the increase in instruction on child health in the curriculum in schools , 284 ( 77% ) supported specific courses on child health targeting girls and women , 321 ( 86.8% ) supported the use of the mass media , and 311 ( 84.1% ) supported programs run by health care professionals .", "the mean knowledge score of the total sample was 25 ( out of 40 ) , the minimum score obtained was 14 and the maximum was 36 .", "we found no statistically significant correlation between the total score on mothers knowledge or any of the items on the knowledge questionnaire and mothers level of education .", "table 2 indicates total knowledge score in relation to mothers education , age and number of children .", "the results of mothers responses to individual items on the knowledge questionnaire are indicated in table 3 .", "association between the scores of mother 's knowledge and mother 's age , educational status and number of children mother 's response to the knowledge questionnaire incorrect knowledge on certain specific items of the questionnaire included 131 ( 35.1% ) who believed that on most occasions , fever in children was a symptom of serious disease , 313 ( 83.9% ) believed that the best method to treat a child with a fever was to bathe him / her with cold water or wipe with cold or iced cloth .", "questions on diseases covered by the national vaccination program revealed that 285 ( 76.4% ) believed that mumps was not targeted , 245 ( 65.7% ) believed that tetanus was not targeted , 203 ( 54.4% ) believed that whooping cough ( pertussis ) was not targeted and 241(64.6% ) believed that diphtheria was not targeted by the national vaccination program .", "three - hundred and five ( 81.8% ) of the mothers believed that the best course of action for parents of a child who was due for a scheduled vaccination but has a fever , cough and runny nose ( symptoms of upper respiratory infection ) was to defer the vaccination until he was completely symptom free . on the question of conditions which were regarded as serious and warranted urgent attention by a doctor , 176 ( 47.2% )", "did not see the need for the newborn baby who was sleeping all the time and was not interested in feeding to be seen by a doctor .", "another 82 ( 22% ) disagreed with seeking medical help for a child who cries continuously , does not sleep and who has pain with every movement .", "three - hundred and one ( 80.7% ) of mothers believed that the appearance of jaundice on the first day of the life of a newborn was physiological ( natural ) and so nothing other than more frequent feeding and exposure to sunlight was required .", "it is noteworthy that more than 30% of the mothers believed that a newborn baby who passes stools after each feed , or a newborn who is otherwise healthy but has repetitive movements of the chin , or one who strains and becomes red in the face during defecation , or a breast fed baby who passes stools only once every 5 days , may have a serious illness and needed urgent medical attention . on feeding practices , 200 ( 53.6% ) of", "the mothers believed that there was no harm in giving a child a bottle of milk at bedtime in bed or whenever he cried during sleep ; 90 ( 24.1% ) did not agree that the most acceptable time to start solid food for children was at 6 months of age , 225 ( 60.3% ) agreed with force - feeding when children refused to eat .", "results on the items on safety practices revealed that only 84 ( 22.5% ) of the mothers believed that it was harmless to let babies sleep by their mothers in the same bed .", "one - hundred and ninety ( 50.9% ) thought that it was not harmful to let babies sleep on their tummy in bed ( prone sleep position ) .", "two hundred two ( 59.5% ) believed that putting a baby who crawls in a walker protected him from household accidents .", "two - hundred and five ( 55% ) believed that the best place for a baby / an infant to sit or be carried in a car was the mother 's lap .", "our results revealed that the mean score of the sample which was just above the cut - off score , was an indication of a satisfactory knowledge level .", "if it had been fixed at 20 , the knowledge of more than 90% would have been satisfactory , on the other hand , if it had been raised to 30 the knowledge of only 12% would have been satisfactory . a closer look at some items revealed significant and sometimes serious gaps in the mothers knowledge .", "similar gaps have been reported from different societies.[71521 ] our results indicated that a majority of mothers believed that the best method to treat a child with a fever was to bathe him with cold water or rub him with ice cold cloth .", "this was worse than that reported by al eissa et al where 50% of the parents would use cold water and 7% ice water for the treatment of fever .", "impicciatore et al reported that 9% of the mothers used iced packing to reduce fever .", "knowledge of the diseases covered by the national vaccination program revealed that a high proportion of mothers were not fully informed about certain diseases targeted by the program .", "impicciatore et al reported that only 26% of italian mothers were able to state the vaccinations which were compulsory .", "our results revealed no statistically significant correlation between mother s knowledge score and their level of education , age or number of children .", "this is similar to the results of jan et al ( jeddah , ksa , 2000 ) who found no correlation between parents level of education and safety practices , but contrary to the finding of moawed et al ( riyadh , ksa , 2000 ) who found a statistically significant correlation between mothers knowledge and their practices during infants diarrheal episodes and mothers age , education and birth order .", "it is also not in accord with shawky et al ( jeddah , ksa , 2001 ) who reported on the effect of maternal education on the rate of childhood handicap and found that the risk of having a handicapped child declined sharply with the increase in the level of maternal education .", "one possible explanation of this difference in the results could be the use of different research methodology ( sample selection , data gathering and others ) .", "it is worrying to find that the majority of mothers believed that jaundice on the first day of life of a newborn was physiological ( natural ) and required no more than frequent feeding and exposure to sun light", ". also disturbing is the fact that nearly half did not realize that a newborn baby who slept all the time and was not interested in feeding could be seriously sick and needed to be seen urgently by a doctor ; another quarter did not realize that a child who cried continuously , who did not sleep and was in pain with every movement was sick enough to require urgent medical attention .", "dongre et al from rural india reported that 23% of mothers were not aware of any danger signs in a newborn such as , poor sucking , low birth weight , lethargy/ unconsciousness , rapid / difficult breathing .", "these were indicated as danger signs by 34.4% , 25.8% , 25.5% , 10.3% mothers respectively , while hypothermia and convulsions were referred to as danger signs by 10.3% and 8.6% mothers respectively .", "the majority of mothers cited family members as their main source of health information , which is higher than what was reported by al eissa et al , where some 35% of the parents mentioned friends and relatives as their main source of information .", "only few mothers cited health care professionals as their main source of health information . in the report by impecciatore et al from italy", ", only 42% of the mothers said that the pediatricians had spontaneously spoken to them about vaccination during consultation with a child .", "al eissa et al reported that only a group of 37% of parents cited medical personnel as their source of information .", "these findings reflect a lack of active educational intervention by professionals . it is encouraging to find that more than three - fourths of the mothers believed that it was harmful to let babies sleep with them in the same bed .", "this is contrary to the finding of jan et al on which 75% of the mothers reported sleeping next to their infants in the same bed , though it is difficult to discern whether mothers were compelled to do so by social circumstances or they believed it was safer for their infant .", "around two - thirds believed that schools did not provide sufficient information on child health and more than two - thirds supported the increase of education of child health in the curriculum of schools . according to kolbe ,", "behaviors and attitudes about health that began during childhood were responsible for most of the deaths , illnesses and disability .", "comprehensive health education programs in schools represent one effective way of providing students with the knowledge and skills to prevent health - impairing behavior .", "several previous studies stressed that health education in school curricula was not adequate , and that the knowledge of high school girls and university students on health matters was deficient.[2730 ] furthermore , studies have shown that school teachers were not trained to give health education in schools .", "their college curriculum did not equip them for such a role and so the teachers refrained from teaching health subjects . according to summerfield and others", "hundreds of studies have evaluated health education and concluded that it was effective , but its effectiveness depended upon factors such as teacher training , comprehensiveness of the health program , time available for instruction , family involvement and community support.[253234 ] the responsibility for health education was not to be left solely to schools .", "in addition to all other sectors of the community , health care personnel and other health care institutions should play a major role in health education .", "milaat et al , agble et al and kari et al , reported successful involvement of health care personnel and medical students in targeted health education classes for school children and university students .", "such efforts may prove more effective than formal general health education classes taught by uninterested , unenthusiastic , untrained school teachers .", "every visit to a health care professional or every health care facility or institution by patients / parents should be an opportunity for the delivery of a short course in health education", ". possible limitations of the study are that the sample of mothers selected may not have been representative of the community ; mothers who volunteered to participate in the survey may prove to be different from the non - participants . whether the respondents were serious in completing the questionnaire and whether research assistant had influenced the responses of the mothers to the questionnaires is not known .", "it also revealed that health education in schools was deficient and it also exposed the limited involvement of health care personnel and institutions in health care education .", "there is a need for health education programs that target high school girls , university students , mothers and other caregivers ( e.g. fathers ) .", "these should be delivered by trained personnel in classes , courses , and special sessions . in addition , health care facilities should be reformed to make health education an essential and compulsory part of health care delivery .", "involvement in these educational activities should be a mandatory requirement for the issue of a license to practice ."], "abstract_text": ["<S> background : child care is mostly the responsibility of mothers . </S>", "<S> several studies have revealed that the mothers education has a positive impact on their knowledge and practice in child health matters.objectives:the study was undertaken to assess the level of mothers knowledge on certain aspects of child health care and whether there is any correlation between their level of knowledge and the number of years of formal education they have had.materials and methods : a two - part questionnaire was distributed . </S>", "<S> the first part comprised information about mother 's nationality , age , work , level of education and number of children , in addition to sources of health information and the role of school education in child health matters . </S>", "<S> the second part contained 40 statements about different aspects of child health matters . </S>", "<S> a structured interview with the mothers who attended with their children at the pediatric outpatient clinic of king khalid university hospital in riyadh during july and august 2007 , was conducted by a trained non - medical research assistant using the items and statements of the questionnaire as a base . </S>", "<S> a knowledge score was calculated from the number of correct answers . </S>", "<S> the maximum score was 40 . </S>", "<S> an arbitrary cut - off score of 25 was considered satisfactory.results:three-hundred-seventy-three questionnaires were completed . </S>", "<S> the mean score of the total sample was 25 ( out of 40 ) and the minimum score obtained was 14 , and the maximum 36 . </S>", "<S> fifty - eight percent scored 25 or more . </S>", "<S> scrutiny of individual items on the questionnaire revealed significant and serious gaps in mother 's knowledge . </S>", "<S> no statistically significant correlation was evident between mothers knowledge of child health related matters and level of education , age , or number of children.conclusion : mothers knowledge of child health related matters is deficient . at present , </S>", "<S> knowledge on child health matters taught in schools in the kingdom is inadequate . </S>", "<S> health care institutions play a limited role in health education . </S>", "<S> there should be proper effective practical means of disseminating information on child health matters among mothers in our community . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["therefore , the mother 's knowledge about child care influences the nature and quality of care that is given to the child .", "several studies have revealed that the mothers level of education has a positive impact on her knowledge and how she deals with child health care issues.[213 ] our experience in pediatric practice has revealed significant gaps pertaining to child health issues in the mothers knowledge .", "there seems to have been very little improvement in the knowledge of mothers on common child health matters over the years inspite of the many years of girls education in the country .", "this descriptive cross - sectional survey aimed to answer the following questions : ( 1 ) what is the level of mothers knowledge of certain aspects of child health matters ? ( 2 ) what are the main sources of health information and which ones are the most useful ? ( 3 ) is there a correlation between mothers level of knowledge and the number of years spent in formal education ? ( 4 ) in the mother 's view , does the current formal education of girls in schools / colleges have a significant impact on mothers knowledge of child health matters ."], ["it was reviewed and refined by two other colleagues who have a lot of experience in pediatrics .", "the first part comprised information about mothers nationality , age , work , level of education and the number of children she has in addition to sources of health information and the role of formal school education in child health matters .", "the second part contained 40 statements about different aspects of child health issues including nutrition , immunization , development , accident prevention , certain neonatal and infants problems or behaviors and some common childhood illnesses .", "mothers who attended with their children at the pediatric outpatient clinic of king khalid university hospital in riyadh during the months of july and august , in 2007 , were invited to participate in the study .", "those who agreed were interviewed by a trained non - medical research assistant using the items and statements of the questionnaire as a basis for the structured interview .", "it was indicated clearly to the mothers that participation was voluntary and their non- contribution would have no adverse effect on the quality of care given to their child in the hospital .", "they scored one point for each knowledge question answered correctly and zero for wrong and  do not know  answers .", "the first 20 questionnaires completed were used as a pilot test to evaluate the clarity of language and level of information in the questionnaire .", "data was entered in ms excel and analyzed using spss pc 11.5 version statistical software .", "descriptive statistics , mean and standard deviation and proportion were used to quantify the study and outcome variables ."], ["demographic data regarding nationality , age , work , education , and number of children are represented in table 1 . as the main source of health information , 295 ( 80% ) cited family members ( grandmothers , mothers , sisters , relatives etc . ) , 30 ( 8.2% ) cited schools , 68 ( 17.2% ) cited tv and radio , 60 ( 16% ) cited journals and magazines , 52 ( 14.2% ) cited friends , colleagues and other members of society , 101 ( 27.1% ) cited books and only 25 ( 7.1% ) cited health care professionals .", "demographic data of the mothers two hundred twenty - five ( 60.6% ) of the mothers believe that formal girls education in schools and colleges does not provide sufficient child health education . on the question of where to obtain useful information of child health , 287 ( 77.6% ) of the mothers supported the increase in instruction on child health in the curriculum in schools , 284 ( 77% ) supported specific courses on child health targeting girls and women , 321 ( 86.8% ) supported the use of the mass media , and 311 ( 84.1% ) supported programs run by health care professionals .", "the mean knowledge score of the total sample was 25 ( out of 40 ) , the minimum score obtained was 14 and the maximum was 36 .", "we found no statistically significant correlation between the total score on mothers knowledge or any of the items on the knowledge questionnaire and mothers level of education .", "table 2 indicates total knowledge score in relation to mothers education , age and number of children .", "the results of mothers responses to individual items on the knowledge questionnaire are indicated in table 3 .", "association between the scores of mother 's knowledge and mother 's age , educational status and number of children mother 's response to the knowledge questionnaire incorrect knowledge on certain specific items of the questionnaire included 131 ( 35.1% ) who believed that on most occasions , fever in children was a symptom of serious disease , 313 ( 83.9% ) believed that the best method to treat a child with a fever was to bathe him / her with cold water or wipe with cold or iced cloth .", "questions on diseases covered by the national vaccination program revealed that 285 ( 76.4% ) believed that mumps was not targeted , 245 ( 65.7% ) believed that tetanus was not targeted , 203 ( 54.4% ) believed that whooping cough ( pertussis ) was not targeted and 241(64.6% ) believed that diphtheria was not targeted by the national vaccination program .", "three - hundred and five ( 81.8% ) of the mothers believed that the best course of action for parents of a child who was due for a scheduled vaccination but has a fever , cough and runny nose ( symptoms of upper respiratory infection ) was to defer the vaccination until he was completely symptom free . on the question of conditions which were regarded as serious and warranted urgent attention by a doctor , 176 ( 47.2% )", "did not see the need for the newborn baby who was sleeping all the time and was not interested in feeding to be seen by a doctor .", "another 82 ( 22% ) disagreed with seeking medical help for a child who cries continuously , does not sleep and who has pain with every movement .", "three - hundred and one ( 80.7% ) of mothers believed that the appearance of jaundice on the first day of the life of a newborn was physiological ( natural ) and so nothing other than more frequent feeding and exposure to sunlight was required .", "it is noteworthy that more than 30% of the mothers believed that a newborn baby who passes stools after each feed , or a newborn who is otherwise healthy but has repetitive movements of the chin , or one who strains and becomes red in the face during defecation , or a breast fed baby who passes stools only once every 5 days , may have a serious illness and needed urgent medical attention . on feeding practices , 200 ( 53.6% ) of", "the mothers believed that there was no harm in giving a child a bottle of milk at bedtime in bed or whenever he cried during sleep ; 90 ( 24.1% ) did not agree that the most acceptable time to start solid food for children was at 6 months of age , 225 ( 60.3% ) agreed with force - feeding when children refused to eat .", "results on the items on safety practices revealed that only 84 ( 22.5% ) of the mothers believed that it was harmless to let babies sleep by their mothers in the same bed .", "one - hundred and ninety ( 50.9% ) thought that it was not harmful to let babies sleep on their tummy in bed ( prone sleep position ) .", "two hundred two ( 59.5% ) believed that putting a baby who crawls in a walker protected him from household accidents .", "two - hundred and five ( 55% ) believed that the best place for a baby / an infant to sit or be carried in a car was the mother 's lap ."], ["our results revealed that the mean score of the sample which was just above the cut - off score , was an indication of a satisfactory knowledge level .", "if it had been fixed at 20 , the knowledge of more than 90% would have been satisfactory , on the other hand , if it had been raised to 30 the knowledge of only 12% would have been satisfactory . a closer look at some items revealed significant and sometimes serious gaps in the mothers knowledge .", "similar gaps have been reported from different societies.[71521 ] our results indicated that a majority of mothers believed that the best method to treat a child with a fever was to bathe him with cold water or rub him with ice cold cloth .", "this was worse than that reported by al eissa et al where 50% of the parents would use cold water and 7% ice water for the treatment of fever .", "impicciatore et al reported that 9% of the mothers used iced packing to reduce fever .", "knowledge of the diseases covered by the national vaccination program revealed that a high proportion of mothers were not fully informed about certain diseases targeted by the program .", "impicciatore et al reported that only 26% of italian mothers were able to state the vaccinations which were compulsory .", "our results revealed no statistically significant correlation between mother s knowledge score and their level of education , age or number of children .", "this is similar to the results of jan et al ( jeddah , ksa , 2000 ) who found no correlation between parents level of education and safety practices , but contrary to the finding of moawed et al ( riyadh , ksa , 2000 ) who found a statistically significant correlation between mothers knowledge and their practices during infants diarrheal episodes and mothers age , education and birth order .", "it is also not in accord with shawky et al ( jeddah , ksa , 2001 ) who reported on the effect of maternal education on the rate of childhood handicap and found that the risk of having a handicapped child declined sharply with the increase in the level of maternal education .", "one possible explanation of this difference in the results could be the use of different research methodology ( sample selection , data gathering and others ) .", "it is worrying to find that the majority of mothers believed that jaundice on the first day of life of a newborn was physiological ( natural ) and required no more than frequent feeding and exposure to sun light", ". also disturbing is the fact that nearly half did not realize that a newborn baby who slept all the time and was not interested in feeding could be seriously sick and needed to be seen urgently by a doctor ; another quarter did not realize that a child who cried continuously , who did not sleep and was in pain with every movement was sick enough to require urgent medical attention .", "dongre et al from rural india reported that 23% of mothers were not aware of any danger signs in a newborn such as , poor sucking , low birth weight , lethargy/ unconsciousness , rapid / difficult breathing .", "these were indicated as danger signs by 34.4% , 25.8% , 25.5% , 10.3% mothers respectively , while hypothermia and convulsions were referred to as danger signs by 10.3% and 8.6% mothers respectively .", "the majority of mothers cited family members as their main source of health information , which is higher than what was reported by al eissa et al , where some 35% of the parents mentioned friends and relatives as their main source of information .", "only few mothers cited health care professionals as their main source of health information . in the report by impecciatore et al from italy", ", only 42% of the mothers said that the pediatricians had spontaneously spoken to them about vaccination during consultation with a child .", "al eissa et al reported that only a group of 37% of parents cited medical personnel as their source of information .", "these findings reflect a lack of active educational intervention by professionals . it is encouraging to find that more than three - fourths of the mothers believed that it was harmful to let babies sleep with them in the same bed .", "this is contrary to the finding of jan et al on which 75% of the mothers reported sleeping next to their infants in the same bed , though it is difficult to discern whether mothers were compelled to do so by social circumstances or they believed it was safer for their infant .", "around two - thirds believed that schools did not provide sufficient information on child health and more than two - thirds supported the increase of education of child health in the curriculum of schools . according to kolbe ,", "behaviors and attitudes about health that began during childhood were responsible for most of the deaths , illnesses and disability .", "comprehensive health education programs in schools represent one effective way of providing students with the knowledge and skills to prevent health - impairing behavior .", "several previous studies stressed that health education in school curricula was not adequate , and that the knowledge of high school girls and university students on health matters was deficient.[2730 ] furthermore , studies have shown that school teachers were not trained to give health education in schools .", "their college curriculum did not equip them for such a role and so the teachers refrained from teaching health subjects . according to summerfield and others", "hundreds of studies have evaluated health education and concluded that it was effective , but its effectiveness depended upon factors such as teacher training , comprehensiveness of the health program , time available for instruction , family involvement and community support.[253234 ] the responsibility for health education was not to be left solely to schools .", "in addition to all other sectors of the community , health care personnel and other health care institutions should play a major role in health education .", "milaat et al , agble et al and kari et al , reported successful involvement of health care personnel and medical students in targeted health education classes for school children and university students .", "such efforts may prove more effective than formal general health education classes taught by uninterested , unenthusiastic , untrained school teachers .", "every visit to a health care professional or every health care facility or institution by patients / parents should be an opportunity for the delivery of a short course in health education", ". possible limitations of the study are that the sample of mothers selected may not have been representative of the community ; mothers who volunteered to participate in the survey may prove to be different from the non - participants . whether the respondents were serious in completing the questionnaire and whether research assistant had influenced the responses of the mothers to the questionnaires is not known ."], ["it also revealed that health education in schools was deficient and it also exposed the limited involvement of health care personnel and institutions in health care education .", "there is a need for health education programs that target high school girls , university students , mothers and other caregivers ( e.g. fathers ) .", "these should be delivered by trained personnel in classes , courses , and special sessions . in addition , health care facilities should be reformed to make health education an essential and compulsory part of health care delivery .", "involvement in these educational activities should be a mandatory requirement for the issue of a license to practice ."]]}
{"article_id": "PMC3644838", "article_text": ["indications for covered self - expandable metallic airway stent removal include recurrent mucous plugging of the stent , excessive or recurrent granuloma formation , migration of the stent , stent infection , recurrence of stenosis , stent failure , stent fractures and accomplishment of treatment . usually , it is tedious to remove a stent because of their embedding in the bronchial mucosa and epithelization of the inner surface .", "granuloma formation at the extremities is a relative common occurrence in covered metallic stents possibly resulting from the radial force applied against the airway wall and the friction exerted .", "we report a case of covered self expandable metallic stent removal with a rigid bronchoscope under general anesthesia .", "the case is unique because it had almost all the common complications associated with the stent placement but it was successfully removed without much ado . to the best of our knowledge", "this is the first case of successful removal of a complete stent reported from india .", "a 21 year old female presented with complaints of increasing dyspnoea and cough with pain chest for last two weeks .", "patient had a past history of benign tracheal stenosis which was opened up by endobronchial electro surgery followed by placement of a self expandable metallic airway covered stent .", "x - ray chest done one and a half year back showed stent well in place [ figure 1 ] .", "x - ray chest done one and a half year back showing stent well in place bronchoscopy was done under local anesthesia and a stenosis was observed well above the level of the stent [ figure 2 ] with granulation tissue inside the stent [ figure 3 ] , lower end of the stent was fractured , carina was not visualized and stent was seen entering upper part of right main bronchus [ figure 4 ] .", "the beveled edge of the rigid scope was advanced between the stent wall and the airway mucosa thus creating a space between them .", "the circular extraction loop at the upper end of the metallic stent was grasped with a rigid optical alligator forceps and pulled in the lumen of rigid bronchoscope with continuous winding movements gradually separating the stent from the tracheal wall . immediately after that", "this is one of the rare cases of successful removal of self expandable metallic airway covered stent in benign tracheal stenosis .", "stenosis above the level of the stent granulation tissue inside the stent and at the margins fracture of stent at its lower end ( stent is seen at upper part of right main bronchus , carina is not visualized ) the stent after removal", "the management of patients with tracheal / bronchial strictures of benign etiology can be quite challenging .", "though surgical tracheal sleeve resection and reconstruction remains the gold standard for most benign airway strictures , non - surgical modalities such as laser photocoagulation and resection , balloon dilatation , electrocautery and stenting of the airway have been developed to deal with airway stenosis .", "various inoperable benign airway disorders are considered for airway stenting , like post intubation tracheal stenosis , tracheal burn or trauma , tracheo - broncho - malacia and extrinsic compression of trachea . though silicone prostheses are considered to be first choice in benign diseases", "their use is less suited , especially in airway wall malacia or distal and angular stenosis where sems are preferred .", "the disadvantages of the silicone stents are high migration rate , small lumen to wall thickness ratio and difficulty in clearing the secretions . on the other hand", ", the major drawback for placement of sems in benign situations is their difficult repositioning and removal in situations like excessive granulation tissue , stent fractures and stent migration .", "the complications of stent removal include significant oozing , tracheal mucosal dehiscence , tracheal puncture requiring thoracotomy , retained stent pieces , need for restenting , respiratory failure , tracheotomy , and even death . in our case ,", "various authors have come up with different methods of removal of the stent , like the technique of nashef et al . , which was similar to that of rolling spaghetti on a fork , but much more difficult and at least equally messy .", "they further observed that there may be several fractures of the stent , which have to be removed piece by piece . in our case", "filler et al . , used a metal suction catheter to dissect the stent from the airway wall before extraction with the forceps .", "zakaluzny et al . , employed a rigid suspension laryngotracheoscope and used optical forceps to dissect the stent from the airway wall .", "alligator forceps were then employed to grasp and extract the stent through the rigid scope while the scope was advanced further into the airway .", "we first separated the stent from the tracheal wall and then removed all the works ( scope , forceps and the stent ) in one piece .", "to conclude , this case is unique for many reasons , firstly , it had almost all of the complications that can be associated with stent placement ( stenosis of trachea at the upper end of the stent , extensive granuloma formation , migration of the stent downwards and fracture of the stent at its lower end ) , secondly , we were able to remove the stent in toto [ figure 5 ] and lastly , there were no complications after removal of the stent .", "further studies are needed to re evaluate their removal successfully and safely and to produce better designed and technically superior stents like bio - absorbable stents which may not have to be removed at all ."], "abstract_text": ["<S> covered self expandable metallic airway stents ( sems ) have been used for benign tracheal stenosis , post intubation tracheal stenosis , tracheal burn or trauma , tracheo - broncho - malacia , and extrinsic compression of trachea . </S>", "<S> their placement is considered to be permanent , with open surgery the only way to remove the stent , though there are few cases reports of their removal with the bronchoscope , but the complications after their removal are very high . in our patient , one and a half years after placement of sems , she developed cough with dyspnoea , video bronchoscopy showed stenosis above the level of stent with granulation tissue inside the stent , stent fracture in lower part and stent migration to right main bronchus , thus she had all conceivable complications of stent placement . </S>", "<S> the stent was removed with the help of rigid bronchoscope under general anaesthesia . </S>", "<S> she was discharged the following day . </S>", "<S> the case is being reported because it was unique in having all the possible complications of stent placement , and rare as we could take out the stent in toto . </S>", "<S> thirdly , the stent could be removed without any complication . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["indications for covered self - expandable metallic airway stent removal include recurrent mucous plugging of the stent , excessive or recurrent granuloma formation , migration of the stent , stent infection , recurrence of stenosis , stent failure , stent fractures and accomplishment of treatment . usually , it is tedious to remove a stent because of their embedding in the bronchial mucosa and epithelization of the inner surface .", "granuloma formation at the extremities is a relative common occurrence in covered metallic stents possibly resulting from the radial force applied against the airway wall and the friction exerted .", "we report a case of covered self expandable metallic stent removal with a rigid bronchoscope under general anesthesia .", "the case is unique because it had almost all the common complications associated with the stent placement but it was successfully removed without much ado . to the best of our knowledge", "this is the first case of successful removal of a complete stent reported from india ."], ["a 21 year old female presented with complaints of increasing dyspnoea and cough with pain chest for last two weeks .", "patient had a past history of benign tracheal stenosis which was opened up by endobronchial electro surgery followed by placement of a self expandable metallic airway covered stent .", "x - ray chest done one and a half year back showed stent well in place [ figure 1 ] .", "x - ray chest done one and a half year back showing stent well in place bronchoscopy was done under local anesthesia and a stenosis was observed well above the level of the stent [ figure 2 ] with granulation tissue inside the stent [ figure 3 ] , lower end of the stent was fractured , carina was not visualized and stent was seen entering upper part of right main bronchus [ figure 4 ] .", "the beveled edge of the rigid scope was advanced between the stent wall and the airway mucosa thus creating a space between them .", "the circular extraction loop at the upper end of the metallic stent was grasped with a rigid optical alligator forceps and pulled in the lumen of rigid bronchoscope with continuous winding movements gradually separating the stent from the tracheal wall . immediately after that", "this is one of the rare cases of successful removal of self expandable metallic airway covered stent in benign tracheal stenosis .", "stenosis above the level of the stent granulation tissue inside the stent and at the margins fracture of stent at its lower end ( stent is seen at upper part of right main bronchus , carina is not visualized ) the stent after removal"], ["the management of patients with tracheal / bronchial strictures of benign etiology can be quite challenging .", "though surgical tracheal sleeve resection and reconstruction remains the gold standard for most benign airway strictures , non - surgical modalities such as laser photocoagulation and resection , balloon dilatation , electrocautery and stenting of the airway have been developed to deal with airway stenosis .", "various inoperable benign airway disorders are considered for airway stenting , like post intubation tracheal stenosis , tracheal burn or trauma , tracheo - broncho - malacia and extrinsic compression of trachea . though silicone prostheses are considered to be first choice in benign diseases", "their use is less suited , especially in airway wall malacia or distal and angular stenosis where sems are preferred .", "the disadvantages of the silicone stents are high migration rate , small lumen to wall thickness ratio and difficulty in clearing the secretions . on the other hand", ", the major drawback for placement of sems in benign situations is their difficult repositioning and removal in situations like excessive granulation tissue , stent fractures and stent migration .", "the complications of stent removal include significant oozing , tracheal mucosal dehiscence , tracheal puncture requiring thoracotomy , retained stent pieces , need for restenting , respiratory failure , tracheotomy , and even death . in our case ,", "various authors have come up with different methods of removal of the stent , like the technique of nashef et al . , which was similar to that of rolling spaghetti on a fork , but much more difficult and at least equally messy .", "they further observed that there may be several fractures of the stent , which have to be removed piece by piece . in our case", "filler et al . , used a metal suction catheter to dissect the stent from the airway wall before extraction with the forceps .", "zakaluzny et al . , employed a rigid suspension laryngotracheoscope and used optical forceps to dissect the stent from the airway wall .", "alligator forceps were then employed to grasp and extract the stent through the rigid scope while the scope was advanced further into the airway .", "we first separated the stent from the tracheal wall and then removed all the works ( scope , forceps and the stent ) in one piece .", "to conclude , this case is unique for many reasons , firstly , it had almost all of the complications that can be associated with stent placement ( stenosis of trachea at the upper end of the stent , extensive granuloma formation , migration of the stent downwards and fracture of the stent at its lower end ) , secondly , we were able to remove the stent in toto [ figure 5 ] and lastly , there were no complications after removal of the stent .", "further studies are needed to re evaluate their removal successfully and safely and to produce better designed and technically superior stents like bio - absorbable stents which may not have to be removed at all ."]]}
{"article_id": "PMC3299167", "article_text": ["takayasu 's arteritis ( ta ) is a chronic idiopathic occlusive inflammation of aorta and its major branches predominantly affecting females in over 85% of cases .", "the major clinical finding is loss of palpable pulses in the upper limbs and neck .", "the unsuspected ischemia of vital regional vascular beds may render these patients at risk for inappropriate management .", "this case report describes the management of a trauma patient in whom the diagnosis of ta was established by specific investigations done for the same .", "the role of meticulous monitoring and importance of anticipation of such disease for prompt diagnosis and management is emphasized .", "a 26-year - old female was brought to the emergency room ( er ) with history of motor vehicle accident . on initial evaluation", "her physical examination revealed unrecordable upper limb peripheral pulses and blood pressure , although lower limb pulses were easily palpated .", "the radiological investigations revealed multiple left - sided rib fractures with bilateral pleural collections and small lung contusions .", "after infusion of two liters of lactated ringer 's solution , her blood pressure ( bp measured in right upper limb ) increased to 80/50 mmhg and heart rate ( hr ) was 140/min .", "she also had shaft and inter - trochanteric femur fractures on the right and left side , respectively . focused assessment sonography in trauma ( fast ) was negative and x - ray of the pelvis was normal .", "bilateral chest tubes were inserted and the patient was transferred to the intensive care unit ( icu ) with ongoing resuscitation with fluids , blood products and dopamine infusion ( 7 g / kg / min ) .", "fluid resuscitation was continued to maintain a central venous pressure ( cvp ) of around 10 mmhg .", "invasive arterial blood pressure monitoring was established via the right femoral artery and the bp recorded was 140/90 mmhg .", "dopamine infusion was then discontinued and thereafter the patient was subjected to arterial doppler and computed tomography ( ct ) angiography studies to rule out traumatic aortic rupture as a differential diagnosis .", "arterial doppler showed low - velocity monophasic flow in bilateral axillary , brachial and ulnar arteries .", "the erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) were normal .", "electrocardiography ( ecg ) , 2d echocardiography ( 2d echo ) and funduscopy were normal .", "thoracic epidural catheter was inserted for pain relief with continuous infusion of 0.125% bupivacaine and fentanyl ( 2 mcg / ml ) .", "epidural catheter was removed on 5 day of hospitalization and patient was moved to the ward in a stable condition with advice for regular follow - up and advised to check bp measurements in the lower limb if ever required .", "the occlusive thromboaortopathy ( ta ) is a group of diseases in which there is no or feeble pulses in upper half of body .", "the natural course of disease often gives rise to four main complications : takayasu 's hypertensive ischemic retinopathy , secondary hypertension , aortic regurgitation and aortic or arterial aneurysm .", "most patients with ta present during their child - bearing age , and a high index of suspicion is required to make an early diagnosis .", "patient may be entirely asymptomatic and the incidental finding of unequal pulse and blood pressure in upper and lower limb , bruit and hypertension may prompt further evaluation .", "stroke , congestive heart failure , and rarely ruptured aneurysm may be the heralding events . hemorrhagic shock is the commonest type of shock in trauma patients .", "any hypotensive trauma patient is presumed to be hypovolemic and resuscitative attempts are made with fluid infusion as was with our patient .", "there was no contributory past history of symptoms or signs suggestive of ta in our patient .", "a cvp of around 10 mmhg suggested a normovolemia status unlike the impression got by the low blood pressure ( as recorded in upper limbs ) .", "the presence of tachycardia in our patient could be attributed to pain and anxiety associated with trauma .", "there was no fall in hematocrit from the baseline ( perhaps due to hemo - concentration ) .", "dopamine infusion was started in our patient in an attempt to maintain perfusion and restore blood pressure but was discontinued when invasive monitoring revealed a normal bp .", "normal value of esr and c - reactive protein suggested that the disease was not active at that moment .", "our patient was perhaps asymptomatic for the disease and was incidentally diagnosed during workup after trauma .", "but ta as a differential diagnosis of exclusion may be considered for a pulseless trauma patient .", "normocytic normochromic anemia , hyper - gammaglobulin , and elevated esr may occur , but are not pathognomonic .", "medical treatment includes corticosteroids , anticoagulants and symptomatic therapy for which anti - hypertensive agents , digitalis and antibiotics are frequently used .", "follow - up in cases of ta includes monitoring for disease activity and its complications like hypertension , organ failure , seizures etc .", "long - term complications of corticosteroid use are also to be considered in the follow - up .", "anesthesiologists may encounter these patients in operation theater for anesthesia or in icus or emergency units after trauma or after one of its complications like a ruptured aneurysm .", "tas may be considered as a differential diagnosis in trauma patients , if examination reveals hypotension or unequal peripheral pulses in upper and lower limb in a young adult , provided hypovolemic shock is excluded .", "a thorough detailed physical examination with palpation of all the peripheral pulses as stressed in atls guidelines is reemphasized . following the basic atls guidelines", "would assist clinicians in picking up this discrepancy as part of their routine evaluation for all trauma patients .", "then , the clinician may suspect an atypical cause for hypotension if a blood pressure cuff on an upper extremity is reading a hypotensive measurement but the patient has strong , 2 + pulses in the lower extremities .", "these patients may be issued a special identification card containing the details of the disease and treatment course for future reference ."], "abstract_text": ["<S> hemorrhagic shock is the most common reason to explain the inability to feel pulse in a trauma patient . </S>", "<S> however , clinicians should always suspect atypical causes for differential pulses in this population and takayasu 's arteritis ( ta ) is one such example . </S>", "<S> we report a case of aorto - arteritis in a patient who presented with trauma and was later diagnosed with ta . </S>", "<S> she had blood pressure discrepancy between upper and lower limbs noted upon her initial trauma evaluation . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE HISTORY", "DISCUSSION", "CONCLUSION"], "sections": [["takayasu 's arteritis ( ta ) is a chronic idiopathic occlusive inflammation of aorta and its major branches predominantly affecting females in over 85% of cases .", "the major clinical finding is loss of palpable pulses in the upper limbs and neck .", "the unsuspected ischemia of vital regional vascular beds may render these patients at risk for inappropriate management .", "this case report describes the management of a trauma patient in whom the diagnosis of ta was established by specific investigations done for the same .", "the role of meticulous monitoring and importance of anticipation of such disease for prompt diagnosis and management is emphasized ."], ["a 26-year - old female was brought to the emergency room ( er ) with history of motor vehicle accident . on initial evaluation", "her physical examination revealed unrecordable upper limb peripheral pulses and blood pressure , although lower limb pulses were easily palpated .", "the radiological investigations revealed multiple left - sided rib fractures with bilateral pleural collections and small lung contusions .", "after infusion of two liters of lactated ringer 's solution , her blood pressure ( bp measured in right upper limb ) increased to 80/50 mmhg and heart rate ( hr ) was 140/min .", "she also had shaft and inter - trochanteric femur fractures on the right and left side , respectively . focused assessment sonography in trauma ( fast ) was negative and x - ray of the pelvis was normal .", "bilateral chest tubes were inserted and the patient was transferred to the intensive care unit ( icu ) with ongoing resuscitation with fluids , blood products and dopamine infusion ( 7 g / kg / min ) .", "fluid resuscitation was continued to maintain a central venous pressure ( cvp ) of around 10 mmhg .", "invasive arterial blood pressure monitoring was established via the right femoral artery and the bp recorded was 140/90 mmhg .", "dopamine infusion was then discontinued and thereafter the patient was subjected to arterial doppler and computed tomography ( ct ) angiography studies to rule out traumatic aortic rupture as a differential diagnosis .", "arterial doppler showed low - velocity monophasic flow in bilateral axillary , brachial and ulnar arteries .", "the erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) were normal .", "electrocardiography ( ecg ) , 2d echocardiography ( 2d echo ) and funduscopy were normal .", "thoracic epidural catheter was inserted for pain relief with continuous infusion of 0.125% bupivacaine and fentanyl ( 2 mcg / ml ) .", "epidural catheter was removed on 5 day of hospitalization and patient was moved to the ward in a stable condition with advice for regular follow - up and advised to check bp measurements in the lower limb if ever required ."], ["the occlusive thromboaortopathy ( ta ) is a group of diseases in which there is no or feeble pulses in upper half of body .", "the natural course of disease often gives rise to four main complications : takayasu 's hypertensive ischemic retinopathy , secondary hypertension , aortic regurgitation and aortic or arterial aneurysm .", "most patients with ta present during their child - bearing age , and a high index of suspicion is required to make an early diagnosis .", "patient may be entirely asymptomatic and the incidental finding of unequal pulse and blood pressure in upper and lower limb , bruit and hypertension may prompt further evaluation .", "stroke , congestive heart failure , and rarely ruptured aneurysm may be the heralding events . hemorrhagic shock is the commonest type of shock in trauma patients .", "any hypotensive trauma patient is presumed to be hypovolemic and resuscitative attempts are made with fluid infusion as was with our patient .", "there was no contributory past history of symptoms or signs suggestive of ta in our patient .", "a cvp of around 10 mmhg suggested a normovolemia status unlike the impression got by the low blood pressure ( as recorded in upper limbs ) .", "the presence of tachycardia in our patient could be attributed to pain and anxiety associated with trauma .", "there was no fall in hematocrit from the baseline ( perhaps due to hemo - concentration ) .", "dopamine infusion was started in our patient in an attempt to maintain perfusion and restore blood pressure but was discontinued when invasive monitoring revealed a normal bp .", "normal value of esr and c - reactive protein suggested that the disease was not active at that moment .", "our patient was perhaps asymptomatic for the disease and was incidentally diagnosed during workup after trauma .", "but ta as a differential diagnosis of exclusion may be considered for a pulseless trauma patient .", "normocytic normochromic anemia , hyper - gammaglobulin , and elevated esr may occur , but are not pathognomonic .", "medical treatment includes corticosteroids , anticoagulants and symptomatic therapy for which anti - hypertensive agents , digitalis and antibiotics are frequently used .", "follow - up in cases of ta includes monitoring for disease activity and its complications like hypertension , organ failure , seizures etc .", "long - term complications of corticosteroid use are also to be considered in the follow - up .", "anesthesiologists may encounter these patients in operation theater for anesthesia or in icus or emergency units after trauma or after one of its complications like a ruptured aneurysm ."], ["tas may be considered as a differential diagnosis in trauma patients , if examination reveals hypotension or unequal peripheral pulses in upper and lower limb in a young adult , provided hypovolemic shock is excluded .", "a thorough detailed physical examination with palpation of all the peripheral pulses as stressed in atls guidelines is reemphasized . following the basic atls guidelines", "would assist clinicians in picking up this discrepancy as part of their routine evaluation for all trauma patients .", "then , the clinician may suspect an atypical cause for hypotension if a blood pressure cuff on an upper extremity is reading a hypotensive measurement but the patient has strong , 2 + pulses in the lower extremities .", "these patients may be issued a special identification card containing the details of the disease and treatment course for future reference ."]]}
{"article_id": "PMC4166658", "article_text": ["neurotransmitters ( nts ) are signaling molecules , which play pivotal roles in neuronal communications in the central nervous system [ 1 , 2 ] . it is reported that changes in nts quantitation in several brain regions involve the development of many psychiatric diseases and neurodegenerative diseases [ 3 , 4 ] .", "generally , neurotransmitters are classified into two categories based on their chemical styles : ( i ) the small molecules ( dopamine ( da ) , serotonin ( 5-ht ) , norepinephrine ( ne ) , epinephrine ( ep ) , glutamate ( glu ) , -aminobutyric acid ( gaba ) , histamine , and endocannabinoids and ( ii ) the neuropeptides ( enkephalin , endorphin , and substance p ) . the quantitation of various nts as a small molecule in the brain , especially the aromatic monoamines , should be measured by using high - pressure liquid chromatography ( hplc ) separation coupled with amperometric electrochemical detection ( ecd ) .", "this method has been applied in nts analysis over the last three decades [ 68 ] .", "however , it is still rather difficult to determine different types of nts simultaneously in one sample owing to the limited capability of accommodating changes in the mobile phase composition .", "another difficulty of incorporating this method is that the analytes can only be identified by a stable retention time matching .", "nevertheless , tandem mass spectrometry ( ms / ms ) can provide high specificity due to additional structure information and high sensitivity .", "therefore , it has been commonly used for the quantification of nts in the brain by coupling with both gas chromatography ( gc ) and liquid chromatography ( lc ) [ 5 , 11 , 12 ] .", "owing to various efficiencies and time consumption of derivatization , a simplified sample preparation using liquid chromatography coupled with electrospray tandem mass spectrometry ( esi - ms / ms ) is widely employed to quantify the nts and their metabolites in the brains without derivatization [ 5 , 9 , 13 ] .", "the use of isotope labeled internal standards is vital to the enhanced method performance because the isotope ratio measurements provide a measure of quality control for each analyte by compensating for changes in analyte , retention time , recovery , degradation , and changes in detector responses caused by coeluting contaminants . in this study", ", we developed a sensitive , simple , and simultaneous method to quantify the six major nts such as da , 5-ht , ne , ep , glu , and gaba in mouse brains [ 14 , 15 ] .", "in addition , a tetrahydrobiopterin ( bh4 ) , a vital cofactor for the biosynthesis of the da , 5-ht , and ne , was also measured in the same sample . to establish a novel method for the direct measurement of biologically active levels of bh4 , da , 5-ht , ne ,", "ep , glu , and gaba in the brain samples , the present study was performed using a high efficiency hilic column for bh4 and da , a luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m ) column for 5-ht , ne , ep , glu , and gaba with reversed - phase hplc separation and an esi - ms / ms , which could minimize the sample interferences . at the same time , the multiple reactions monitoring ( mrm ) scan mode was sensitive enough to identify and quantify the bh4 and nts in this new method .", "the ( 6r)-5,6,7,8-tetrahydrobiopterin dihydrochloride , dopamine hydrochloride , serotonin hydrochloride , ( )-norepinephrine , ( )-epinephrine , d - glutamic acid , and -aminobutyric acid were purchased from sigma - aldrich corporation ( st . louis , mo , usa ) .", "internal standards ( is ) with isotope labeling were 2-(3,4-dihydorxyphenyl ) ethyl-1,1,2,2-d4-amine hcl ( dopamine - d4 , 98% at % d ) ; serotonin-  , ,,-d4 creatinine sulfate complex ( serotonin - d4 , 98% at % d ) ; ( )-norepinephrine-2,5,6,,,-d6 hcl ( norepinepherine - d6 , 98% at % d ) ; ( )-epinephrine - d3 ( n - methyl - d3 ) ( epinephrine - d3 , 98% at % d ) ; l - glutamic-2,3,3,4,4-d5 acid ( glutamate - d5 , 98% at % d ) ; 4-aminobutyric-2,2,3,3,4,4-d6 acid ( -aminobutyric acid - d6 , 98% at % d ) .", "epsilon - acetamidocaproic acid ( aaca ) was donated by kuhnil pharmaceuticals ( seoul , korea ) .", "water was purified with a milli - q water purification system ( millipore , bedford , ma , usa ) .", "full - scan positive mass spectra of bh4 and the is ( aaca ) reveal the protonated molecules , [ m + h ] , of m / z 242.1 and 174 , respectively", ". the mass - to - charge ratios of fragments of bh4 after fragmentation were 166 , 107 , and 149 and fragments of is were 114 , 156 , and 79 .", "the most abundant ion in the product ion spectra was 114 for is ( figure 1(a ) ) and 166 for bh4 ( figure 1(b ) ) .", "in parallel , full - scan positive mass spectra of bh2 and the biopterin showed the protonated molecules , [ m + h ] , of m / z 240.0 and 238.0 , respectively .", "the mass - to - charge ratios of fragments were 196.0 , 164.9 , and 168.0 in bh2 and of 177.9 , 193.9 , and 192.0 in biopterin .", "the most abundant ion in the product ion spectra was 196.0 for bh2 ( figure 1(c ) ) and 177.9 for biopterin ( figure 1(d ) ) .", "full - scan positive mass spectra of da and the is ( dopamine - d4 ) showed that m / z of protonated molecules [ m + h ] are 154.1 and 158.1 , respectively .", "the mass - to - charge ratios of fragments of da after fragmentation were 137.0 , 90.9 , and 64.9 and of da - d4 141.0 , 95.0 , and 67.9 .", "the most abundant ion in the product ion spectra was 137.0 for da ( figure 2(a ) ) and 141.0 for is ( figure 2(b ) ) .", "but da mrm had 154.1 to 90.9 due to matrix effects , which increased the mass - to - charge ratio level .", "full - scan positive mass spectra of 5-ht and the iss ( serotonin - d4 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 177.0 and 181.0 , respectively .", "after fragmentation , fragments of 5-ht seen were m / z 160.0 , 114.9 , and 132.0 and fragments of 5-ht - d4  m / z were 164.0 , 118.0 , and 136.0 .", "the most abundant ion in the product ion spectra was at 160.0 for 5-ht ( figure 2(c ) ) and at 164.0 for is ( figure 2(d ) ) .", "full - scan positive mass spectra of ne and the is ( norepinephrine - d6 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 170.1 and 176.1 , respectively .", "after fragmentation , fragments of ne were m / z 152.0 , 107.0 , and 76.9 and fragments of ne - d6  m / z were 158.0 , 111.0 , and 112.0 .", "the most abundant ion in the product ion spectra was at 152.0 for ne ( figure 2(e ) ) and at 158.0 for is ( figure 2(f ) ) .", "but m / z 170.1 to 107.0 and 176.1 to 111.0 was selected for ne and is ( ne - d6 ) mrm due to matrix effects , which increased the mass - to - charge ratio level .", "full - scan positive mass spectra of ep and the is ( epinephrine - d3 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 184.1 and 187.1 , respectively .", "the m / z of fragments of ep after fragmentation were 166.0 , 76.9 , and 107.0 and of ep - d3 169.0 , 76.9 , and 107.0 , respectively .", "the most abundant ion in the product ion spectra was at 166.0 for ep ( figure 3(a ) ) and at 169.0 for is ( figure 3(b ) ) .", "full - scan positive mass spectra of glu and the is ( glutamate - d5 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 148.0 and 153.0 , respectively .", "the mass - to - charge ratios of fragments of glu after fragmentation were 129.0 , 83.9 , and 55.9 and of glu - d5 were 135.0 , 84.8 , and 88.0 , respectively .", "the most abundant ion in the product ion spectra was at 129.0 for glutamate ( figure 3(c ) ) and at 135.0 for is ( figure 3(d ) ) .", "but m / z 148.0 to 84.0 and m / z 153.0 to 88.0 for glutamate and is ( glutamate - d5 ) mrm were selected due to matrix effects , which increased the mass - to - charge ratio level .", "full - scan positive mass spectra of gaba and the is ( gaba - d6 ) showed the protonated molecules , [ m + h ] , of m / z 104.0 and 110.1 , respectively .", "the mass - to - charge ratios of fragments of gaba after fragmentation were 87.0 , 44.9 , and 85.0 and of gaba - d6 were 93.0 , 49.0 , and 91.9 , respectively .", "the most abundant ion in the product ion spectra was at 87.0 for gaba ( figure 3(e ) ) and at 93.0 for is ( figure 3(f ) ) .", "individual stock solution of each nt and isotope - labeled standard was prepared by accurate weighing of each compound ( 1  mg / ml methanol as the stock solution ) .", "the solution of bh4 , da , 5-ht , ne , ep , glu , and gaba was prepared as a stock ( 1  mg / ml of each ) with pure acetonitrile and then diluted with acetonitrile ( 50% ) for each experiment .", "standard solutions of bh4 , da , 5-ht , ne , ep , glu , and gaba for calibration curves were prepared by spiking the blank solution prepared to the appropriate amounts , but added volumes were less than 10% of total dw volume .", "the final yielding concentrations for the standard curve were 10 , 20 , 50 , 100 , 200 , 500 , 1000 , 2000 , 5000 , and 10000  ng / g for bh4 and dopamine . in parallel ,", "the final concentrations for the standard curve were 20 , 50 , 100 , 200 , 500 , 1000 , 2000 , 5000 , and 10000  ng / g for 5-ht , ne , and ep .", "likewise , the final concentrations for the standard curve were 0.2 , 0.5 , 1 , 2 , 5 , 10 , 20 , 50 , 100 , and 200  g / g for glu and gaba .", "the tolerance for reliable detection was 10  ng / g for bh4 and dopamine , 20  ng / g for 5-ht , ne and ep , and 0.2  g / g for glu and gaba .", "the linear ranges and correlation coefficient of the calibration curve were summarized in table 1 .", "icr mice ( male , body weight 2030  g , n = 24 ) , ( daehanbiolink inc . , chungju , south korea ) , were used .", "the mice were kept under a controlled condition ( ambient temperature of 20 to 25c , 12-h light / dark cycle ) .", "nih 's guidelines for animal research were followed for all animal procedures and were approved by institutional animal care and use committee ( iacuc ; dku-12 - 018 ) which adheres to the guidelines issued by the institution of laboratory of animal resources ( ilar ) .", "the specific brain regions of mouse were quickly dissected on an ice bath   and , subsequently , isolated brain tissues were homogenized with acetonitrile ( 1  mg/10  l ) according to the internal standard ( aaca : 100  ng / ml ; dopamine - d4 , serotonin - d4 , norepinephrine - d6 , epinephrine - d3 , glutamate - d5 , and gaba - d6 :  1  g / ml ) . after a thorough homogenization , the bh4 and nts ( da , 5-ht , ne , ep , glu , and gaba ) from brain tissues were extracted by sonication for 60s .", "the homogenates of brain tissue were centrifuged at 12,000  rpm for 10  min at 4c .", "supernatants were carefully transferred to 96-well plates and then injected onto the lc - ms / ms system by an autosampler for subsequent analysis .", "for determination of bh4 and nts in mouse brain tissues , d - water was used as blank matrix .", "the liquid chromatographic system used was the accela system ( thermo fisher scientific inc . ,", "waltham , ma , usa ) , equipped with a nanospace si-2 3133 solvent delivery module as an autosampler ( shiseido inc . ,", "japan ) and connected to discovery max ( thermo fisher scientific , inc . ) quadrupole tandem mass spectrometer coupled with electrospray ionization ( esi - ms / ms ) .", "system control and data analysis were performed using the xcalibur software ( thermo fisher scientific , inc . ) .", "chromatographic separation was achieved using hydrophilic interaction chromatography ( hilic ) sepax polar - imidazole ( 2.1  mm  100  mm , i.d . , 3  m particle size )", "hplc column ( sepax technologies , delaware , usa ) to assay bh4 and dopamine , and luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m particle size ) to assay serotonin , norepinepherine , epinephrine , glautamte , and gaba with a phenomenex c18 guard column ( 4  mm  2  mm , phenomenex ) .", "a nanospace si-2 3004 column oven ( shiseido , japan ) was used online . to assay bh4 and dopamine , the mobile phase consisted of 10  mm ammonium formate ( ph 3 ) in an acetonitrile / water ( 75  :  25 , v / v ) mixture .", "the flow rate was 300  l / min and the injection volume was 5  l . to assay 5-ht , ne , ep , glu , and gaba , the mobile phase consisted of an acetonitrile / water ( 20  :  80 , v / v ) mixture .", "the flow rate was run at 350  l / min and the injection volume was 5  l .", "the electrospray ionization ( esi ) mass spectrometer was operated in the positive ion mode .", "the optimal condition was as follows : the esi needle spray voltage was 4000  v , the sheath gas pressure 35 unit , the auxiliary gas pressure 20 unit , the capillary temperature 206c , the collision gas ( ar ) pressure 1.5  mtorr , the skimmer offset 5  v , and the chrome filter peak width 10  s. scanning was performed in profile mode with the sim width 0.700  fwhm , scan time 0.200  s , and scan width 0.5  da .", "it was successful to qualify bh4 using hydrophilic interaction chromatography ( hilic ) sepax polar - imidazole ( 2.1  mm  100  mm , i.d .", ", 3  m particle size ) hplc column ( sepax technologies , delaware , usa ) .", "moreover , the peaks had a symmetric shape , and we confirmed the lc - ms / ms chromatogram of bh4 , aaca , da , and da - d4 ( figure 4 ) . following the same strategy", ", we analyzed successfully for 5-ht , ne , ep , glu , and gaba by using luna 3u c18 ( 3.0  mm  150  mm , i.d . , 3  m particle size ) .", "the whole validation experiments followed the guideline of  fda ( us ) [ guidance for industry ; handling and retention of ba and be testing samples ] , may 2004 .", "linear regression of the ratio of peak area of bh4 or nts to that of is was done with weighting of 1/x ( least - squares linear regression analysis , where x is the concentration of the analyte ) .", "precision and accuracy were evaluated by three different concentrations of qc solutions : interday precision was evaluated for 5 replicates per a single concentration .", "the value of accuracy was expressed as the mean of 25 replicates of determined concentration from 5 different analytical tests to the qc concentration ( table 2 ) .", "all the values , tables , and figures given in the text are expressed as mean  sd .", "statistical differences between means were evaluated with two - tailed student 's t - test .", "for simple sample preparation , protein precipitation was attempted using acetonitrile . to prevent sample degradation and oxidation , an ascorbic acid with 0.01% ( w / v )", "the peaks of bh4 , dopamine , and is were best when acetonitrile was used for protein precipitation and as an organic solvent of the mobile phase when using the hilic column ( polar - imidazole , 2.0  mm  150  mm ; i.d .", ", 3  m ) ( figure 4 ) . because bh4 and dopamine are easily dissolved in water , they are difficult to match the reverse column ( c18 ) in chromatography analysis", "the other nts ( 5-ht , ne , ep , glu , and gaba ) were matched with a c18 column ( luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m particle size ) ) ( figure 5 ) , but the hilic column could not separate peak of nts clearly .", "the optimized electrospray ionization condition should be sensitive enough to detect bh4 , da , is , bh2 , and biopterin in positive ion detection mode .", "the most abundant protonated ion peaks ( [ m + h ] ) in the q1 mass spectra of bh4 , da , is , bh2 , and biopterin were at 242.1 , 154.1 , 174.0 , 240.0 , and 238.0 , respectively ( figures 1 and 2 ) .", "the product ions in q3 mass spectra and proposed fragmentation patterns were bh4 , which becomes at 2-amino-7,8-dihydropteridin-4(1h)-one of m / z 166.0 by losing propane-1,2-diol .", "da becomes butane-1,2-diol of m / z 90.9 by losing ( e)-3-methylpent-3-en-1-amine ; is , ( e)-n - ethylidenepentan-1-amine of m / z 114.0 by losing both carboxyl and hydroxyl groups ; bh2 , 2-amino-7,8-dihydro-6-(hydroxymethyl)pteridin-4(1h)-one ) of m / z 196.0 by losing propan-2-ol ; biopterin , 2-amino-6-(hydroxymethyl)pteridin-4(1h)-one ) of m / z 196.0 by losing propan-2-ol . also , to confirm separation between bh4 and other biopterins in biological samples , experiments were previously conducted to quantify biopterin , bh2 , and bh4 in a mixed matrix .", "the optimized electrospray ionization condition should be sensitive enough to detect bh4 , da , 5-ht , ne , ep , glu , gaba , and iss with positive ion detection mode .", "the most abundant protonated ion peaks ( [ m + h ] ) in the q1 mass spectra of bh4 , da , 5-ht , ne , ep , glu , gaba , and iss are listed in table 3 .", "the product ions and collision energy in q3 mass spectra of bh4 , da , 5-ht , ne , ep , glu , gaba , and iss were listed in table 3 .", "previously , our lab reported bh4 and dopamine levels in rat brain region . to extend this method to mouse brain regions , we applied it the bh4 and nts in the mouse brain .", "the peak areas of the spiked standard were constructed by subtracting the corresponding areas derived from the matrix . meanwhile , calibration using internal standardization with deuterated analogues was performed . in biological specimen analysis , the isotope - labeled analogues of the targeted analyte are often recommended . due to their similar physicochemical properties ,", "compared to deuterated analogues , the variability during sample preparation and ionization efficiency in the transfer of analytes from liquid to gas could be compensated for , and they could be differentiated ideally by their distinct mass - to - charge ( m / z ) ratios .", "all analytes were subjected to hplc - ms / ms analysis , and their distinct mass - to - charge ( m / z ) ratios were determined .", "there are representative lc - ms / ms chromatograms of bh4 , da , and iss ( aaca and dopamine - d6 ) in the dw matrix ( figure 4 ) . also , there are 5-ht , ne , ep , glu , gaba , and iss lc - ms / ms chromatograms in the dw matrix ( figure 5 ) .", "we tested the newly developed method using olfactory bulb ( ob ) , frontal cortex ( fc ) , hippocampus ( hp ) , striatum ( st ) , hypothalamus ( ht ) , pituitary gland ( pt ) , midbrain ( mb ) , cerebellum ( cb ) , and brainstem ( bs ) from the mice and subsequently confirmed that the quantity of bh4 and nts ( table 4 ) . eight different concentrations from 10 to 2000  ng / g of bh4 , from 10 to 5000  ng / g of da , from 20 to 10000  ng / g of 5-ht , ne , and ep , and from 0.2 to 200  g / g of glu , gaba is plotted against is for the standard curves .", "the correlation coefficients ( r ) , lod , and loq of the standard curve are shown in table 1 .", "the lc - ms / ms methodology was used to measure the levels of bh4 and nts in nine brain regions including ob , fc , hp , st , ht , pt , mb , cb , and bs from mice .", "the newly - developed lc - ms / ms method was used to analyze the quantity of bh4 and nts in mice brain regions ( table 4 ) .", "the endogenous levels of bh4 , da , 5-ht , ne , ep , glu , and gaba were successfully detected and measured in mice brain regions .", "the present study was undertaken in order to describe a sensitive and specific lc - ms / ms method for simultaneous detection of bh4 , da , 5-ht , ne , ep , glu , and gaba from mouse brain tissue .", "the principal advantages of using lc - ms / ms method include a simple purification procedure and a simple chromatographic condition using the mrm scan mode .", "the use of a hilic column overcame the limitations of separating hydrophilic materials . therefore , hilic column could separate bh4 and da from matrix effect with an appropriate retention time .", "the other nts ( 5-ht , ne , ep , glu , and gaba ) were matched well with a luna 3   c18 column .", "so , the current developed method should be very useful for brain tissue works of research , regarding the analysis of the alternation of the levels of bh4 , da , 5-ht , ne , ep , glu , and gaba .", "this new method can enable measurement of bh4 and nts rapidly and accurately in brain tissues .", "previously , bh4 levels have been indirectly calculated by measuring the concentrations of biopterin in biological samples .", "however the limitation of this indirect method is that it is unable to measure the exact bh4 levels owing to rapid oxidation and degradation . to avoid the problem", ", we tested several experimental conditions and found that a low temperature is a critical factor to prevent decomposition of bh4 in the brain tissues extract .", "but the addition of antioxidant ( dte ) and/or acid ( hcl ) to the samples does not affect dramatically the stability of bh4 .", "keeping the treated extracts at 4c is necessary and enough to maintain bh4 stable for 4 hours , which is long enough to finish the analysis of the targets in samples . by using hilic column , bh4 and da", "were separated into single peaks . under other methods , many unknown materials in the biological matrix interfered with the analysis of bh4 and da in the biological samples .", "but the use of a hilic column could overcome the limitation to separating hydrophilic materials .", "so , hilic column could separate successfully the bh4 and da from matrix effect with an appropriate retention time ( figure 4 ) .", "in addition , it could increase the sensitivity , selectivity , and accuracy of bh4 and da in brain samples using mrm scan mode . using luna 3   c18 column", ", 3  m particle size ) , 5-ht , ne , ep , glu , and gaba were separated into single peaks . also , the luna 3   c18 column could separate nts from matrix effect with an appropriate retention time , and the usage of mrm scan mode could increase the sensitivity , selectivity , and accuracy of nts detection in brain samples ( figure 5 ) .", "the levels of bh4 and nts were measured in several brain sections by using the newly - developed experimental method ( table 4 ) .", "the bh4 is an essential cofactor for the aromatic acid hydroxylases , which are essential in the formation of nts ( da , 5-ht , and ne ) , as well as for nitric oxide synthase ( nos ) , a vital enzyme for normal vascular and cardiac nitric oxide .", "therefore , it is necessary to reliably measure the biological concentration of bh4 for the evaluation of various diseases and for screening potential therapeutic candidates in neurological diseases .", "these results showed that the bh4 level was at its highest in olfactory bulb , followed by cerebellum , frontal cortex , striatum , midbrain , pituitary gland , hypothalamus , brainstem , and hippocampus in a decreasing order .", "however , the da level was at its highest in striatum , followed by hypothalamus , midbrain , pituitary gland , and olfactory bulb in a decreasing order .", "interestingly , there were no detectable da in hippocampus , frontal cortex , cerebellum , and brainstem .", "however , the lower limit of quantification in our method is 10  ng / g in samples .", "therefore , even though there are some da transmissions in these regions , the amount of da in hippocampus , brain cortex , and brainstem could be below 10  ng / g .", "these data indicate that the level of bh4 could be distinctly correlated with the level of da in the mouse brain tissue .", "the 5-ht levels were at their highest in midbrain and brainstem , followed by hypothalamus , striatum , hippocampus , frontal cortex , occipital lobe , and cerebellum in a decreasing order .", "the ne level was at its highest in hypothalamus and pituitary gland , followed by brainstem , midbrain , olfactory blub , cerebellum , striatum , and frontal cortex in a decreasing order .", "the ep level was at its highest in hippocampus , followed by hypothalamus , brainstem , midbrain , olfactory blub , frontal cortex , and pituitary gland in a decreasing order .", "however , there was no detectable ep in cerebellum ; the levels of ne and ep have similar order in mouse brain sections .", "the glu level was at its highest in hippocampus , followed by frontal cortex , striatum , hypothalamus , cerebellum , midbrain , brainstem , olfactory blub , and pituitary gland in a decreasing order .", "the gaba level was at its highest in hypothalamus , olfactory bulb , and hippocampus , followed by midbrain , frontal cortex , striatum , cerebellum , striatum , and brainstem in a decreasing order .", "interestingly , the levels of glu and gaba were detected as microgram based units , but others were detected as nanogram based units .", "these results suggested that the neurotransmitters in mouse brain were differentially released to do their function in brain sections .", "however , a simple and rapid liquid chromatography tandem mass spectrometry ( lc - ms / ms ) method has been developed for the determination of bh4 , da , 5-ht , ne , ep , glu , and gaba in mouse brain ; the quantitative determination of endogenous neurotransmitters in brain regions by chromatographic coupled mass spectrometry presented here still has a limitation because of the typical lack of analyte - free matrix .", "there is no analyte - free sample of the authentic matrix ; therefore , we have to use a surrogate matrix containing the authentic analyte .", "a simple and rapid liquid chromatography tandem mass spectrometry ( lc - ms / ms ) method has been developed for the determination of bh4 , da , 5-ht , ne , ep , glu , and gaba in mouse brain using epsilon - acetamidocaproic acid ( aaca ) and isotopically labeled neurotransmitters as an internal standard . although it is clear that further studies are necessary to understand the physiological meaning of the different levels of bh4 and nts , this new method could be applied for tracking the changes of the endogenous bh4 and nts which are affected significantly by various stimuli or in neurodegenerative diseases [ 10 , 23 ] ."], "abstract_text": ["<S> a simple and rapid liquid chromatography tandem mass spectrometry method has been developed for the determination of bh4 , da , 5-ht , ne , ep , glu , and gaba in mouse brain using epsilon - acetamidocaproic acid and isotopically labeled neurotransmitters as internal standards . </S>", "<S> proteins in the samples were precipitated by adding acetonitrile , and then the supernatants were separated by a sepax polar - imidazole ( 2.1  mm  100  mm , i.d . </S>", "<S> , 3  m ) column by adding a mixture of 10  mm ammonium formate in acetonitrile / water ( 75  :  25 , v / v , 300  l / min ) for bh4 and da . to assay 5-ht , ne , ep , glu , and gaba ; a luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m ) column was used by adding a mixture of 1% formic acid in acetonitrile / water ( 20  :  80 , v / v , 350  l / min ) . </S>", "<S> the total chromatographic run time was 5.5  min . </S>", "<S> the method was validated for the analysis of samples . </S>", "<S> the calibration curve was linear between 10 and 2000  ng / g for bh4 ( r2 = 0.995 ) \n , 10 and 5000  ng / g for da ( r2 = 0.997 ) \n , 20 and 10000  ng / g for 5-ht ( r2 = 0.994 ) \n , ne ( r2 = 0.993 ) \n , and ep ( r2 = 0.993 ) \n , and 0.2 and 200  g / g for glu ( r2 = 0.996 ) \n and gaba ( r2 = 0.999 ) \n in the mouse brain tissues . as stated above , lc - ms / ms results were obtained and established to be a useful tool for the quantitative analysis of bh4 , da , 5-ht , ne , ep , glu , and gaba in the experimental rodent brain . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["neurotransmitters ( nts ) are signaling molecules , which play pivotal roles in neuronal communications in the central nervous system [ 1 , 2 ] . it is reported that changes in nts quantitation in several brain regions involve the development of many psychiatric diseases and neurodegenerative diseases [ 3 , 4 ] .", "generally , neurotransmitters are classified into two categories based on their chemical styles : ( i ) the small molecules ( dopamine ( da ) , serotonin ( 5-ht ) , norepinephrine ( ne ) , epinephrine ( ep ) , glutamate ( glu ) , -aminobutyric acid ( gaba ) , histamine , and endocannabinoids and ( ii ) the neuropeptides ( enkephalin , endorphin , and substance p ) . the quantitation of various nts as a small molecule in the brain , especially the aromatic monoamines , should be measured by using high - pressure liquid chromatography ( hplc ) separation coupled with amperometric electrochemical detection ( ecd ) .", "this method has been applied in nts analysis over the last three decades [ 68 ] .", "however , it is still rather difficult to determine different types of nts simultaneously in one sample owing to the limited capability of accommodating changes in the mobile phase composition .", "another difficulty of incorporating this method is that the analytes can only be identified by a stable retention time matching .", "nevertheless , tandem mass spectrometry ( ms / ms ) can provide high specificity due to additional structure information and high sensitivity .", "therefore , it has been commonly used for the quantification of nts in the brain by coupling with both gas chromatography ( gc ) and liquid chromatography ( lc ) [ 5 , 11 , 12 ] .", "owing to various efficiencies and time consumption of derivatization , a simplified sample preparation using liquid chromatography coupled with electrospray tandem mass spectrometry ( esi - ms / ms ) is widely employed to quantify the nts and their metabolites in the brains without derivatization [ 5 , 9 , 13 ] .", "the use of isotope labeled internal standards is vital to the enhanced method performance because the isotope ratio measurements provide a measure of quality control for each analyte by compensating for changes in analyte , retention time , recovery , degradation , and changes in detector responses caused by coeluting contaminants . in this study", ", we developed a sensitive , simple , and simultaneous method to quantify the six major nts such as da , 5-ht , ne , ep , glu , and gaba in mouse brains [ 14 , 15 ] .", "in addition , a tetrahydrobiopterin ( bh4 ) , a vital cofactor for the biosynthesis of the da , 5-ht , and ne , was also measured in the same sample . to establish a novel method for the direct measurement of biologically active levels of bh4 , da , 5-ht , ne ,", "ep , glu , and gaba in the brain samples , the present study was performed using a high efficiency hilic column for bh4 and da , a luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m ) column for 5-ht , ne , ep , glu , and gaba with reversed - phase hplc separation and an esi - ms / ms , which could minimize the sample interferences . at the same time , the multiple reactions monitoring ( mrm ) scan mode was sensitive enough to identify and quantify the bh4 and nts in this new method ."], ["the ( 6r)-5,6,7,8-tetrahydrobiopterin dihydrochloride , dopamine hydrochloride , serotonin hydrochloride , ( )-norepinephrine , ( )-epinephrine , d - glutamic acid , and -aminobutyric acid were purchased from sigma - aldrich corporation ( st . louis , mo , usa ) .", "internal standards ( is ) with isotope labeling were 2-(3,4-dihydorxyphenyl ) ethyl-1,1,2,2-d4-amine hcl ( dopamine - d4 , 98% at % d ) ; serotonin-  , ,,-d4 creatinine sulfate complex ( serotonin - d4 , 98% at % d ) ; ( )-norepinephrine-2,5,6,,,-d6 hcl ( norepinepherine - d6 , 98% at % d ) ; ( )-epinephrine - d3 ( n - methyl - d3 ) ( epinephrine - d3 , 98% at % d ) ; l - glutamic-2,3,3,4,4-d5 acid ( glutamate - d5 , 98% at % d ) ; 4-aminobutyric-2,2,3,3,4,4-d6 acid ( -aminobutyric acid - d6 , 98% at % d ) .", "epsilon - acetamidocaproic acid ( aaca ) was donated by kuhnil pharmaceuticals ( seoul , korea ) .", "water was purified with a milli - q water purification system ( millipore , bedford , ma , usa ) .", "full - scan positive mass spectra of bh4 and the is ( aaca ) reveal the protonated molecules , [ m + h ] , of m / z 242.1 and 174 , respectively", ". the mass - to - charge ratios of fragments of bh4 after fragmentation were 166 , 107 , and 149 and fragments of is were 114 , 156 , and 79 .", "the most abundant ion in the product ion spectra was 114 for is ( figure 1(a ) ) and 166 for bh4 ( figure 1(b ) ) .", "in parallel , full - scan positive mass spectra of bh2 and the biopterin showed the protonated molecules , [ m + h ] , of m / z 240.0 and 238.0 , respectively .", "the mass - to - charge ratios of fragments were 196.0 , 164.9 , and 168.0 in bh2 and of 177.9 , 193.9 , and 192.0 in biopterin .", "the most abundant ion in the product ion spectra was 196.0 for bh2 ( figure 1(c ) ) and 177.9 for biopterin ( figure 1(d ) ) .", "full - scan positive mass spectra of da and the is ( dopamine - d4 ) showed that m / z of protonated molecules [ m + h ] are 154.1 and 158.1 , respectively .", "the mass - to - charge ratios of fragments of da after fragmentation were 137.0 , 90.9 , and 64.9 and of da - d4 141.0 , 95.0 , and 67.9 .", "the most abundant ion in the product ion spectra was 137.0 for da ( figure 2(a ) ) and 141.0 for is ( figure 2(b ) ) .", "but da mrm had 154.1 to 90.9 due to matrix effects , which increased the mass - to - charge ratio level .", "full - scan positive mass spectra of 5-ht and the iss ( serotonin - d4 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 177.0 and 181.0 , respectively .", "after fragmentation , fragments of 5-ht seen were m / z 160.0 , 114.9 , and 132.0 and fragments of 5-ht - d4  m / z were 164.0 , 118.0 , and 136.0 .", "the most abundant ion in the product ion spectra was at 160.0 for 5-ht ( figure 2(c ) ) and at 164.0 for is ( figure 2(d ) ) .", "full - scan positive mass spectra of ne and the is ( norepinephrine - d6 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 170.1 and 176.1 , respectively .", "after fragmentation , fragments of ne were m / z 152.0 , 107.0 , and 76.9 and fragments of ne - d6  m / z were 158.0 , 111.0 , and 112.0 .", "the most abundant ion in the product ion spectra was at 152.0 for ne ( figure 2(e ) ) and at 158.0 for is ( figure 2(f ) ) .", "but m / z 170.1 to 107.0 and 176.1 to 111.0 was selected for ne and is ( ne - d6 ) mrm due to matrix effects , which increased the mass - to - charge ratio level .", "full - scan positive mass spectra of ep and the is ( epinephrine - d3 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 184.1 and 187.1 , respectively .", "the m / z of fragments of ep after fragmentation were 166.0 , 76.9 , and 107.0 and of ep - d3 169.0 , 76.9 , and 107.0 , respectively .", "the most abundant ion in the product ion spectra was at 166.0 for ep ( figure 3(a ) ) and at 169.0 for is ( figure 3(b ) ) .", "full - scan positive mass spectra of glu and the is ( glutamate - d5 ) showed that the mass - to - charge ratios of protonated molecules [ m + h ] were 148.0 and 153.0 , respectively .", "the mass - to - charge ratios of fragments of glu after fragmentation were 129.0 , 83.9 , and 55.9 and of glu - d5 were 135.0 , 84.8 , and 88.0 , respectively .", "the most abundant ion in the product ion spectra was at 129.0 for glutamate ( figure 3(c ) ) and at 135.0 for is ( figure 3(d ) ) .", "but m / z 148.0 to 84.0 and m / z 153.0 to 88.0 for glutamate and is ( glutamate - d5 ) mrm were selected due to matrix effects , which increased the mass - to - charge ratio level .", "full - scan positive mass spectra of gaba and the is ( gaba - d6 ) showed the protonated molecules , [ m + h ] , of m / z 104.0 and 110.1 , respectively .", "the mass - to - charge ratios of fragments of gaba after fragmentation were 87.0 , 44.9 , and 85.0 and of gaba - d6 were 93.0 , 49.0 , and 91.9 , respectively .", "the most abundant ion in the product ion spectra was at 87.0 for gaba ( figure 3(e ) ) and at 93.0 for is ( figure 3(f ) ) .", "individual stock solution of each nt and isotope - labeled standard was prepared by accurate weighing of each compound ( 1  mg / ml methanol as the stock solution ) .", "the solution of bh4 , da , 5-ht , ne , ep , glu , and gaba was prepared as a stock ( 1  mg / ml of each ) with pure acetonitrile and then diluted with acetonitrile ( 50% ) for each experiment .", "standard solutions of bh4 , da , 5-ht , ne , ep , glu , and gaba for calibration curves were prepared by spiking the blank solution prepared to the appropriate amounts , but added volumes were less than 10% of total dw volume .", "the final yielding concentrations for the standard curve were 10 , 20 , 50 , 100 , 200 , 500 , 1000 , 2000 , 5000 , and 10000  ng / g for bh4 and dopamine . in parallel ,", "the final concentrations for the standard curve were 20 , 50 , 100 , 200 , 500 , 1000 , 2000 , 5000 , and 10000  ng / g for 5-ht , ne , and ep .", "likewise , the final concentrations for the standard curve were 0.2 , 0.5 , 1 , 2 , 5 , 10 , 20 , 50 , 100 , and 200  g / g for glu and gaba .", "the tolerance for reliable detection was 10  ng / g for bh4 and dopamine , 20  ng / g for 5-ht , ne and ep , and 0.2  g / g for glu and gaba .", "the linear ranges and correlation coefficient of the calibration curve were summarized in table 1 .", "icr mice ( male , body weight 2030  g , n = 24 ) , ( daehanbiolink inc . , chungju , south korea ) , were used .", "the mice were kept under a controlled condition ( ambient temperature of 20 to 25c , 12-h light / dark cycle ) .", "nih 's guidelines for animal research were followed for all animal procedures and were approved by institutional animal care and use committee ( iacuc ; dku-12 - 018 ) which adheres to the guidelines issued by the institution of laboratory of animal resources ( ilar ) .", "the specific brain regions of mouse were quickly dissected on an ice bath   and , subsequently , isolated brain tissues were homogenized with acetonitrile ( 1  mg/10  l ) according to the internal standard ( aaca : 100  ng / ml ; dopamine - d4 , serotonin - d4 , norepinephrine - d6 , epinephrine - d3 , glutamate - d5 , and gaba - d6 :  1  g / ml ) . after a thorough homogenization , the bh4 and nts ( da , 5-ht , ne , ep , glu , and gaba ) from brain tissues were extracted by sonication for 60s .", "the homogenates of brain tissue were centrifuged at 12,000  rpm for 10  min at 4c .", "supernatants were carefully transferred to 96-well plates and then injected onto the lc - ms / ms system by an autosampler for subsequent analysis .", "for determination of bh4 and nts in mouse brain tissues , d - water was used as blank matrix .", "the liquid chromatographic system used was the accela system ( thermo fisher scientific inc . ,", "waltham , ma , usa ) , equipped with a nanospace si-2 3133 solvent delivery module as an autosampler ( shiseido inc . ,", "japan ) and connected to discovery max ( thermo fisher scientific , inc . ) quadrupole tandem mass spectrometer coupled with electrospray ionization ( esi - ms / ms ) .", "system control and data analysis were performed using the xcalibur software ( thermo fisher scientific , inc . ) .", "chromatographic separation was achieved using hydrophilic interaction chromatography ( hilic ) sepax polar - imidazole ( 2.1  mm  100  mm , i.d . , 3  m particle size )", "hplc column ( sepax technologies , delaware , usa ) to assay bh4 and dopamine , and luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m particle size ) to assay serotonin , norepinepherine , epinephrine , glautamte , and gaba with a phenomenex c18 guard column ( 4  mm  2  mm , phenomenex ) .", "a nanospace si-2 3004 column oven ( shiseido , japan ) was used online . to assay bh4 and dopamine , the mobile phase consisted of 10  mm ammonium formate ( ph 3 ) in an acetonitrile / water ( 75  :  25 , v / v ) mixture .", "the flow rate was 300  l / min and the injection volume was 5  l . to assay 5-ht , ne , ep , glu , and gaba , the mobile phase consisted of an acetonitrile / water ( 20  :  80 , v / v ) mixture .", "the flow rate was run at 350  l / min and the injection volume was 5  l .", "the electrospray ionization ( esi ) mass spectrometer was operated in the positive ion mode .", "the optimal condition was as follows : the esi needle spray voltage was 4000  v , the sheath gas pressure 35 unit , the auxiliary gas pressure 20 unit , the capillary temperature 206c , the collision gas ( ar ) pressure 1.5  mtorr , the skimmer offset 5  v , and the chrome filter peak width 10  s. scanning was performed in profile mode with the sim width 0.700  fwhm , scan time 0.200  s , and scan width 0.5  da .", "it was successful to qualify bh4 using hydrophilic interaction chromatography ( hilic ) sepax polar - imidazole ( 2.1  mm  100  mm , i.d .", ", 3  m particle size ) hplc column ( sepax technologies , delaware , usa ) .", "moreover , the peaks had a symmetric shape , and we confirmed the lc - ms / ms chromatogram of bh4 , aaca , da , and da - d4 ( figure 4 ) . following the same strategy", ", we analyzed successfully for 5-ht , ne , ep , glu , and gaba by using luna 3u c18 ( 3.0  mm  150  mm , i.d . , 3  m particle size ) .", "the whole validation experiments followed the guideline of  fda ( us ) [ guidance for industry ; handling and retention of ba and be testing samples ] , may 2004 .", "linear regression of the ratio of peak area of bh4 or nts to that of is was done with weighting of 1/x ( least - squares linear regression analysis , where x is the concentration of the analyte ) .", "precision and accuracy were evaluated by three different concentrations of qc solutions : interday precision was evaluated for 5 replicates per a single concentration .", "the value of accuracy was expressed as the mean of 25 replicates of determined concentration from 5 different analytical tests to the qc concentration ( table 2 ) .", "all the values , tables , and figures given in the text are expressed as mean  sd .", "statistical differences between means were evaluated with two - tailed student 's t - test ."], ["for simple sample preparation , protein precipitation was attempted using acetonitrile . to prevent sample degradation and oxidation , an ascorbic acid with 0.01% ( w / v )", "the peaks of bh4 , dopamine , and is were best when acetonitrile was used for protein precipitation and as an organic solvent of the mobile phase when using the hilic column ( polar - imidazole , 2.0  mm  150  mm ; i.d .", ", 3  m ) ( figure 4 ) . because bh4 and dopamine are easily dissolved in water , they are difficult to match the reverse column ( c18 ) in chromatography analysis", "the other nts ( 5-ht , ne , ep , glu , and gaba ) were matched with a c18 column ( luna 3   c18 ( 3.0  mm  150  mm , i.d . , 3  m particle size ) ) ( figure 5 ) , but the hilic column could not separate peak of nts clearly .", "the optimized electrospray ionization condition should be sensitive enough to detect bh4 , da , is , bh2 , and biopterin in positive ion detection mode .", "the most abundant protonated ion peaks ( [ m + h ] ) in the q1 mass spectra of bh4 , da , is , bh2 , and biopterin were at 242.1 , 154.1 , 174.0 , 240.0 , and 238.0 , respectively ( figures 1 and 2 ) .", "the product ions in q3 mass spectra and proposed fragmentation patterns were bh4 , which becomes at 2-amino-7,8-dihydropteridin-4(1h)-one of m / z 166.0 by losing propane-1,2-diol .", "da becomes butane-1,2-diol of m / z 90.9 by losing ( e)-3-methylpent-3-en-1-amine ; is , ( e)-n - ethylidenepentan-1-amine of m / z 114.0 by losing both carboxyl and hydroxyl groups ; bh2 , 2-amino-7,8-dihydro-6-(hydroxymethyl)pteridin-4(1h)-one ) of m / z 196.0 by losing propan-2-ol ; biopterin , 2-amino-6-(hydroxymethyl)pteridin-4(1h)-one ) of m / z 196.0 by losing propan-2-ol . also , to confirm separation between bh4 and other biopterins in biological samples , experiments were previously conducted to quantify biopterin , bh2 , and bh4 in a mixed matrix .", "the optimized electrospray ionization condition should be sensitive enough to detect bh4 , da , 5-ht , ne , ep , glu , gaba , and iss with positive ion detection mode .", "the most abundant protonated ion peaks ( [ m + h ] ) in the q1 mass spectra of bh4 , da , 5-ht , ne , ep , glu , gaba , and iss are listed in table 3 .", "the product ions and collision energy in q3 mass spectra of bh4 , da , 5-ht , ne , ep , glu , gaba , and iss were listed in table 3 .", "previously , our lab reported bh4 and dopamine levels in rat brain region . to extend this method to mouse brain regions , we applied it the bh4 and nts in the mouse brain .", "the peak areas of the spiked standard were constructed by subtracting the corresponding areas derived from the matrix . meanwhile , calibration using internal standardization with deuterated analogues was performed . in biological specimen analysis , the isotope - labeled analogues of the targeted analyte are often recommended . due to their similar physicochemical properties ,", "compared to deuterated analogues , the variability during sample preparation and ionization efficiency in the transfer of analytes from liquid to gas could be compensated for , and they could be differentiated ideally by their distinct mass - to - charge ( m / z ) ratios .", "all analytes were subjected to hplc - ms / ms analysis , and their distinct mass - to - charge ( m / z ) ratios were determined .", "there are representative lc - ms / ms chromatograms of bh4 , da , and iss ( aaca and dopamine - d6 ) in the dw matrix ( figure 4 ) . also , there are 5-ht , ne , ep , glu , gaba , and iss lc - ms / ms chromatograms in the dw matrix ( figure 5 ) .", "we tested the newly developed method using olfactory bulb ( ob ) , frontal cortex ( fc ) , hippocampus ( hp ) , striatum ( st ) , hypothalamus ( ht ) , pituitary gland ( pt ) , midbrain ( mb ) , cerebellum ( cb ) , and brainstem ( bs ) from the mice and subsequently confirmed that the quantity of bh4 and nts ( table 4 ) . eight different concentrations from 10 to 2000  ng / g of bh4 , from 10 to 5000  ng / g of da , from 20 to 10000  ng / g of 5-ht , ne , and ep , and from 0.2 to 200  g / g of glu , gaba is plotted against is for the standard curves .", "the correlation coefficients ( r ) , lod , and loq of the standard curve are shown in table 1 .", "the lc - ms / ms methodology was used to measure the levels of bh4 and nts in nine brain regions including ob , fc , hp , st , ht , pt , mb , cb , and bs from mice .", "the newly - developed lc - ms / ms method was used to analyze the quantity of bh4 and nts in mice brain regions ( table 4 ) .", "the endogenous levels of bh4 , da , 5-ht , ne , ep , glu , and gaba were successfully detected and measured in mice brain regions ."], ["the present study was undertaken in order to describe a sensitive and specific lc - ms / ms method for simultaneous detection of bh4 , da , 5-ht , ne , ep , glu , and gaba from mouse brain tissue .", "the principal advantages of using lc - ms / ms method include a simple purification procedure and a simple chromatographic condition using the mrm scan mode .", "the use of a hilic column overcame the limitations of separating hydrophilic materials . therefore , hilic column could separate bh4 and da from matrix effect with an appropriate retention time .", "the other nts ( 5-ht , ne , ep , glu , and gaba ) were matched well with a luna 3   c18 column .", "so , the current developed method should be very useful for brain tissue works of research , regarding the analysis of the alternation of the levels of bh4 , da , 5-ht , ne , ep , glu , and gaba .", "this new method can enable measurement of bh4 and nts rapidly and accurately in brain tissues .", "previously , bh4 levels have been indirectly calculated by measuring the concentrations of biopterin in biological samples .", "however the limitation of this indirect method is that it is unable to measure the exact bh4 levels owing to rapid oxidation and degradation . to avoid the problem", ", we tested several experimental conditions and found that a low temperature is a critical factor to prevent decomposition of bh4 in the brain tissues extract .", "but the addition of antioxidant ( dte ) and/or acid ( hcl ) to the samples does not affect dramatically the stability of bh4 .", "keeping the treated extracts at 4c is necessary and enough to maintain bh4 stable for 4 hours , which is long enough to finish the analysis of the targets in samples . by using hilic column , bh4 and da", "were separated into single peaks . under other methods , many unknown materials in the biological matrix interfered with the analysis of bh4 and da in the biological samples .", "but the use of a hilic column could overcome the limitation to separating hydrophilic materials .", "so , hilic column could separate successfully the bh4 and da from matrix effect with an appropriate retention time ( figure 4 ) .", "in addition , it could increase the sensitivity , selectivity , and accuracy of bh4 and da in brain samples using mrm scan mode . using luna 3   c18 column", ", 3  m particle size ) , 5-ht , ne , ep , glu , and gaba were separated into single peaks . also , the luna 3   c18 column could separate nts from matrix effect with an appropriate retention time , and the usage of mrm scan mode could increase the sensitivity , selectivity , and accuracy of nts detection in brain samples ( figure 5 ) .", "the levels of bh4 and nts were measured in several brain sections by using the newly - developed experimental method ( table 4 ) .", "the bh4 is an essential cofactor for the aromatic acid hydroxylases , which are essential in the formation of nts ( da , 5-ht , and ne ) , as well as for nitric oxide synthase ( nos ) , a vital enzyme for normal vascular and cardiac nitric oxide .", "therefore , it is necessary to reliably measure the biological concentration of bh4 for the evaluation of various diseases and for screening potential therapeutic candidates in neurological diseases .", "these results showed that the bh4 level was at its highest in olfactory bulb , followed by cerebellum , frontal cortex , striatum , midbrain , pituitary gland , hypothalamus , brainstem , and hippocampus in a decreasing order .", "however , the da level was at its highest in striatum , followed by hypothalamus , midbrain , pituitary gland , and olfactory bulb in a decreasing order .", "interestingly , there were no detectable da in hippocampus , frontal cortex , cerebellum , and brainstem .", "however , the lower limit of quantification in our method is 10  ng / g in samples .", "therefore , even though there are some da transmissions in these regions , the amount of da in hippocampus , brain cortex , and brainstem could be below 10  ng / g .", "these data indicate that the level of bh4 could be distinctly correlated with the level of da in the mouse brain tissue .", "the 5-ht levels were at their highest in midbrain and brainstem , followed by hypothalamus , striatum , hippocampus , frontal cortex , occipital lobe , and cerebellum in a decreasing order .", "the ne level was at its highest in hypothalamus and pituitary gland , followed by brainstem , midbrain , olfactory blub , cerebellum , striatum , and frontal cortex in a decreasing order .", "the ep level was at its highest in hippocampus , followed by hypothalamus , brainstem , midbrain , olfactory blub , frontal cortex , and pituitary gland in a decreasing order .", "however , there was no detectable ep in cerebellum ; the levels of ne and ep have similar order in mouse brain sections .", "the glu level was at its highest in hippocampus , followed by frontal cortex , striatum , hypothalamus , cerebellum , midbrain , brainstem , olfactory blub , and pituitary gland in a decreasing order .", "the gaba level was at its highest in hypothalamus , olfactory bulb , and hippocampus , followed by midbrain , frontal cortex , striatum , cerebellum , striatum , and brainstem in a decreasing order .", "interestingly , the levels of glu and gaba were detected as microgram based units , but others were detected as nanogram based units .", "these results suggested that the neurotransmitters in mouse brain were differentially released to do their function in brain sections .", "however , a simple and rapid liquid chromatography tandem mass spectrometry ( lc - ms / ms ) method has been developed for the determination of bh4 , da , 5-ht , ne , ep , glu , and gaba in mouse brain ; the quantitative determination of endogenous neurotransmitters in brain regions by chromatographic coupled mass spectrometry presented here still has a limitation because of the typical lack of analyte - free matrix .", "there is no analyte - free sample of the authentic matrix ; therefore , we have to use a surrogate matrix containing the authentic analyte ."], ["a simple and rapid liquid chromatography tandem mass spectrometry ( lc - ms / ms ) method has been developed for the determination of bh4 , da , 5-ht , ne , ep , glu , and gaba in mouse brain using epsilon - acetamidocaproic acid ( aaca ) and isotopically labeled neurotransmitters as an internal standard . although it is clear that further studies are necessary to understand the physiological meaning of the different levels of bh4 and nts , this new method could be applied for tracking the changes of the endogenous bh4 and nts which are affected significantly by various stimuli or in neurodegenerative diseases [ 10 , 23 ] ."]]}
{"article_id": "PMC4903118", "article_text": ["it plays several important physiological and pathological roles in not only reproductive system but also other systems .", "estrogen disorder results in various diseases , such as endometrial diseases , skeletal diseases , and reproductive system tumors .", "increasing attention has been paid to revealing of the functions of estrogen in physiological and pathological conditions .", "estrogen receptors ( er )  and  , the two well established nuclear estrogen receptors , have different physiological functions depending upon their various distributions .", "lots of evidences show that estrogen induces the proliferation of cancer cells in breast , uterus , and ovarian cancer through er. on the contrary , activation of er can reverse this effect .", "notably , a novel transmembrane estrogen receptor , known as g - couple estrogen receptor ( gper ) , was found .", "er are involved in the initiation , migration , and progression of estrogen - related multiorgan cancers , such as breast cancer , ovarian cancer , prostate cancer , testicular cancer , liver cancer , and lung cancer as well .", "although increasing studied are focused on the roles of gper-1 in different types of cancers , the functions of gper-1 in cancers remain unclear yet .", "characteristics and functions of reproductive system cancer will be summarized and discussed in the present review .", "g - protein - coupled estrogen receptor-1 ( gper-1 ) , a seven - transmembrane - domain receptor localized in cell surface , was first identified in 1996 .", "gper-1 is detected broadly in numerous human tissues , such as breast , prostate , ovary , placenta , subcutaneous adipose , visceral adipose , arteries , vessels , heart , liver , lung and intestine tissues .", "gper-1 is a member of gpcr superfamily , which is structurally unrelated to the classical er and er. there are four transcriptional variants encoding 375 amino acids composing seven transmembrane proteins .", "but gper-1 binding domain exists inside the plasma membranes and the endoplasmic reticulum [ 58 ] .", "estradiol , the major type of estrogen , binds to gper-1 with a high affinity to gper-1 while the other two isoforms , estrone and estriol , have very low binding affinities [ 5 , 9 ] .", "furthermore , numbers of environmental estrogen bind to gper-1 and activate the downstream signaling pathways , such as bisphenol a , genistein , and nonylphenol .", "gper-1-specific compound 1 ( g-1 ) is a specific agonist of gper-1 , which has no function of er and er and was identified using virtual and biomolecular screening in 2006 .", "g-1 has been widely used as a target tool to evaluate the function of gper-1 in different cells and disease models .", "gper-1 mediates both genomic and nongenomic response with its ligands . to date , gper-1 signaling pathways have not been fully elucidated yet .", "the binding ligands of gper-1 , such as estrogen , g-1 , tamoxifen , and ici182,780 , cross the plasma membrane and bind to the gper-1 on endoplasmic reticulum where they activate its  and  subunits and subsequently activate both src and adenylyl cyclase ( ac ) leading to the intracellular camp production .", "the phosphorylation of src induces matrix metalloproteinase ( mmp ) production , which cleaves pro - heparan - bound epidermal growth factor ( pro - hb - egf ) releasing free hb - egf .", "hb - egf binds to the egfr leading to activation of multiple molecules such as ras , pi3k , akt , and erk1/2 .", "the downstream signal of pi3k and akt results in several nuclear receptors activation which is closely related the proliferation and migration of cancer cell .", "gper-1 also binds to the g - couple protein  subunit and activates the phospholipase c ( plc ) , ac , and camp .", "activated plc results in inositol triphosphate ( ip3 ) production , which further binds to its receptor and leads to intracellular calcium mobilization .", "breast cancer is generally classified into estrogen receptor positive ( er+ ) and er negative ( er ) . in clinical practices ,", "the endocrine treatments such as tamoxifen and aromatase are recommended in the er positive breast cancers , while there is no benefit in the er negative cancers .", "gper-1 is widely expressed in both of these breast cancer types and the primary breast cancers .", "recent clinical study results showed that the expression of gper-1 might correlate with clinical and pathological poor outcome biomarkers .", "other results also showed that the expression of gper-1 was inversely correlated with the er expression .", "coexpression of gper-1 and er was found in almost 24% patients with inflammatory breast cancer , while 19% only express er and 46% only express gper-1 .", "the gper-1 mrna levels were significant higher in er positive breast cancer cells compared to er negative cancer cells , and the expression of gper-1 depends on er mrna level .", "interestingly , gper-1 preformed a different proliferation manner in er positive mcf-7 breast cancer cell line .", "g-1 enhanced migration of mcf-7 breast cancer cells by activating erk1/2 and egfr signaling pathway , which is tremendously attenuated by g15 .", "the other evidences also approved that gper-1 is an initiator of tamoxifen resistance in breast cancers [ 1921 ] .", "the promotion roles of gper-1 in cancer cells proliferation and migration may be correlated with the autolysis of calpain 1 , cleavage of cyclin e , or the expression of target gene .", "there are also some studies which found that gper-1 inhibits the growth of er positive mcf-7 cells , which is probably through g - couple  and  subunits activating without camp signal activation [ 2124 ] . however , combination treatment with g-1 and her2 antibody trastuzumab exerted an additive growth inhibitory effect on breast cancer cells .", "thus , gper-1 inhibits er positive breast cancers proliferation which is a potential target for er positive breast cancers and drug - resistant breast cancer .", "deficiency of er in breast cancer is correlated with poor response to endocrine therapy . in er negative breast cancers , gper-1 stimulates the erk1/2 through the egfr / mapk signal cascade , inducing target gene like c - fos expression , which is involved in the progressing of breast malignancies [ 2729 ] .", "estrogen and antiestrogens can also promote the production of the early growth response-1 ( egr-1 ) , connective tissue growth factor ( ctgf ) , and insulin - like growth factor 1 ( igf-1 ) through the gper-1 [ 28 , 30 , 31 ] .", "grp30 activation stimulated breast cancer cells migration through ctgh , cxc receptor ( cxcr1 ) , and notch pathways . furthermore , gper-1 agonist g-1 promoted inflammation in breast cancers .", "gper-1 was reported to affect the deformation of breast glandular structure inducing the malignant transformation of breast tissue .", "gper-1 can also induce expression of cancer - associated fibroblasts ( cafs ) in tumor microenvironment [ 34 , 35 ] . on the contrary", ", a recent study showed that activation of gper-1 by g-1 resulted in g2/m - phase arrest and induction of mitochondrial - related apoptosis .", "the other studies also proved that g-1 treatment suppressed the growth of skbr3 cancer cells and increased the survival rate by inducing the erk1/2 signal activation [ 36 , 37 ] .", "1520% of breast cancers are included in triple negative breast cancers ( tnbc ) , characterized by lack of er , progesterone receptor ( pr ) , and egfr2 ( her-2 )", ". a higher rate of recurrence and aggressive biological features were found in younger females [ 38 , 39 ] .", "gper-1 expression was found in majority of tnbcs patients . in the gper knockdown mice model , the proliferation of tnbcs ,", "these findings suggest that gper plays a key role in putative mechanism for tnbcs and gper might be a therapeutic target for tnbcs .", "snps of gper-1 , histone acetylation , and transcription factor recruitment were significantly associated with tumor size and histological grading [ 42 , 43 ] .", "gper rna as well as gper-1 protein presents in both primary and malignant ovarian tumor tissues . the expression of gper-1 was significantly increased in ovarian carcinomas compared to pericarcinomatous tissues impendent with the expression of egfr , er , and er .", "estrogen and g-1 induce ovarian cancer cell growth responses via egfr - mapk signaling pathways .", "furthermore , gper-1 promoted the migration and invasion of ovarian cancer cells ovcar5 which is characterized by negative er and positive gper by increasing the expression mmp-9 [ 48 , 49 ] .", "atrazine , one of the most common pesticide contaminants , promoted ovarian cancer cells proliferation via induction of erk and expression of estrogen target gene through gper-1 pathway .", "but other studies results showed that g-1 suppressed proliferation and induced apoptosis of human ovarian cancer cells probably through inhibition of cell cycle progression in g2/m - phase in ovarian carcinomas [ 51 , 52 ] . \n", "gper-1 has been shown to be involved in a variety of hormone - dependent cancers .", "it is well understood that estrogens play a critical role in pathological germ cell proliferation in testicular germ cell tumors .", "gper-1 seems to be involved in modulating the growth of estrogen dependent testicular cancer cells .", "estrogen induces the high expression of gper-1 correlated with low levels of er in human testicular carcinoma in situ and seminomas [ 53 , 54 ] .", "bisphenol a , a common environmental estrogen , can also promote the proliferation of testicular seminomas cells through gper-1 .", "the above findings suggested that gper-1 may be a potential therapeutic target [ 56 , 57 ] .", "estrogen has an efficacy for advanced prostate cancer ( pc ) via the mediation of the classical estrogen receptors .", "the effects of er on pc growth and metastases have different mechanisms in different cellular microenvironments .", "the expression of gper-1 is higher in the preneoplastic lesions and normal areas of benign prostate than the basal epithelial cells .", "g-1 , the selectively activating gper-1 , inhibited the growth of multiple pc cells in vitro and in vivo through erk1/2 and c - jun / c - fos signaling pathways , which indicates that the g-1 may be a new option for pc through targeting gper-1 .", "g-1 inhibited castration - resistant phase but had no effect on androgen - sensitive tumors .", "the antitumor effect of g-1 on cr tumors was related to necrosis ( approximately 65% ) accompanied with neutrophils infiltration .", "g-1 can also upregulate neutrophil - related chemokines and inflammation - mediated cytokines in the cr tumors . in one word", "there are other studies which proved that gper-1 induced proliferation , differentiation , and drug resistance of lung cancers [ 63 , 64 ] , thyroid cancers , bladder cancers , and oral squamous carcinomas .", "more studies to reveal the functions and mechanisms of gper-1 in the other system cancers are warranted .", "majority of the study results addressed that activation of gper-1 by estrogen and g-1 results in the downstream signals and target genes activation , which promotes the proliferation , migration , and invasion of cancer cells . and", "this effect is in nonclassical er expression dependent manner in most cancers except for ovarian cancers .", "it is interesting that several other studies showed that g-1 , the special agonist of gper-1 , promoted the expression of gper-1 and inhibited the proliferation of er negative breast cancer cells , ovarian cancer cells , and prostate cancer cells .", "the opposite effects of gper-1 in cancer cells may be associated with the epigenetic of gper-1 , such as the snps and histone acetylation .", "the different cell types , tumor microenvironment , and hormonal level may also affect the functions of gper-1", ". controversies still exist on the gper-1 localization and related signaling pathways , in particular the potential action as proapoptotic mediator . since the function and mechanisms of gper-1 are still unclear , more researches and clinical studies are strongly warranted to clarify the different function and mechanisms in different cancer types and conditions ."], "abstract_text": ["<S> the g - protein - coupled estrogen receptor-1 ( gper-1 ) , also known as gpr30 , is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types . </S>", "<S> the progress of estrogen - related cancer is promoted by gper-1 activation through mitogen - activated protein kinases ( mapk ) , phosphoinositide 3-kinase ( pi3k ) , and phospholipase c ( plc ) signaling pathways . however , </S>", "<S> this promoting effect of gper-1 is nonclassic estrogen receptor ( er ) dependent manner . </S>", "<S> in addition , clinical evidences revealed that gper-1 is associated with estrogen resistance in estrogen - related cancer patients . </S>", "<S> these give a hint that gper-1 may be a novel therapeutic target for the estrogen - related cancers . </S>", "<S> however , preclinical studies also found that gper-1 activation of its special agonist g-1 inhibits cancer cell proliferation . </S>", "<S> this review aims to summarize the characteristics and complex functions of gper-1 in cancers . </S>"], "labels": null, "section_names": ["1. Introduction", "2. The Characteristics of GPER-1", "3. Functions of GPER-1 in Reproductive System Tumors", "4. GPER-1 in Other Tumors", "5. Conclusion"], "sections": [["it plays several important physiological and pathological roles in not only reproductive system but also other systems .", "estrogen disorder results in various diseases , such as endometrial diseases , skeletal diseases , and reproductive system tumors .", "increasing attention has been paid to revealing of the functions of estrogen in physiological and pathological conditions .", "estrogen receptors ( er )  and  , the two well established nuclear estrogen receptors , have different physiological functions depending upon their various distributions .", "lots of evidences show that estrogen induces the proliferation of cancer cells in breast , uterus , and ovarian cancer through er. on the contrary , activation of er can reverse this effect .", "notably , a novel transmembrane estrogen receptor , known as g - couple estrogen receptor ( gper ) , was found .", "er are involved in the initiation , migration , and progression of estrogen - related multiorgan cancers , such as breast cancer , ovarian cancer , prostate cancer , testicular cancer , liver cancer , and lung cancer as well .", "although increasing studied are focused on the roles of gper-1 in different types of cancers , the functions of gper-1 in cancers remain unclear yet .", "characteristics and functions of reproductive system cancer will be summarized and discussed in the present review ."], ["g - protein - coupled estrogen receptor-1 ( gper-1 ) , a seven - transmembrane - domain receptor localized in cell surface , was first identified in 1996 .", "gper-1 is detected broadly in numerous human tissues , such as breast , prostate , ovary , placenta , subcutaneous adipose , visceral adipose , arteries , vessels , heart , liver , lung and intestine tissues .", "gper-1 is a member of gpcr superfamily , which is structurally unrelated to the classical er and er. there are four transcriptional variants encoding 375 amino acids composing seven transmembrane proteins .", "but gper-1 binding domain exists inside the plasma membranes and the endoplasmic reticulum [ 58 ] .", "estradiol , the major type of estrogen , binds to gper-1 with a high affinity to gper-1 while the other two isoforms , estrone and estriol , have very low binding affinities [ 5 , 9 ] .", "furthermore , numbers of environmental estrogen bind to gper-1 and activate the downstream signaling pathways , such as bisphenol a , genistein , and nonylphenol .", "gper-1-specific compound 1 ( g-1 ) is a specific agonist of gper-1 , which has no function of er and er and was identified using virtual and biomolecular screening in 2006 .", "g-1 has been widely used as a target tool to evaluate the function of gper-1 in different cells and disease models .", "gper-1 mediates both genomic and nongenomic response with its ligands . to date , gper-1 signaling pathways have not been fully elucidated yet .", "the binding ligands of gper-1 , such as estrogen , g-1 , tamoxifen , and ici182,780 , cross the plasma membrane and bind to the gper-1 on endoplasmic reticulum where they activate its  and  subunits and subsequently activate both src and adenylyl cyclase ( ac ) leading to the intracellular camp production .", "the phosphorylation of src induces matrix metalloproteinase ( mmp ) production , which cleaves pro - heparan - bound epidermal growth factor ( pro - hb - egf ) releasing free hb - egf .", "hb - egf binds to the egfr leading to activation of multiple molecules such as ras , pi3k , akt , and erk1/2 .", "the downstream signal of pi3k and akt results in several nuclear receptors activation which is closely related the proliferation and migration of cancer cell .", "gper-1 also binds to the g - couple protein  subunit and activates the phospholipase c ( plc ) , ac , and camp .", "activated plc results in inositol triphosphate ( ip3 ) production , which further binds to its receptor and leads to intracellular calcium mobilization ."], ["breast cancer is generally classified into estrogen receptor positive ( er+ ) and er negative ( er ) . in clinical practices ,", "the endocrine treatments such as tamoxifen and aromatase are recommended in the er positive breast cancers , while there is no benefit in the er negative cancers .", "gper-1 is widely expressed in both of these breast cancer types and the primary breast cancers .", "recent clinical study results showed that the expression of gper-1 might correlate with clinical and pathological poor outcome biomarkers .", "other results also showed that the expression of gper-1 was inversely correlated with the er expression .", "coexpression of gper-1 and er was found in almost 24% patients with inflammatory breast cancer , while 19% only express er and 46% only express gper-1 .", "the gper-1 mrna levels were significant higher in er positive breast cancer cells compared to er negative cancer cells , and the expression of gper-1 depends on er mrna level .", "interestingly , gper-1 preformed a different proliferation manner in er positive mcf-7 breast cancer cell line .", "g-1 enhanced migration of mcf-7 breast cancer cells by activating erk1/2 and egfr signaling pathway , which is tremendously attenuated by g15 .", "the other evidences also approved that gper-1 is an initiator of tamoxifen resistance in breast cancers [ 1921 ] .", "the promotion roles of gper-1 in cancer cells proliferation and migration may be correlated with the autolysis of calpain 1 , cleavage of cyclin e , or the expression of target gene .", "there are also some studies which found that gper-1 inhibits the growth of er positive mcf-7 cells , which is probably through g - couple  and  subunits activating without camp signal activation [ 2124 ] . however , combination treatment with g-1 and her2 antibody trastuzumab exerted an additive growth inhibitory effect on breast cancer cells .", "thus , gper-1 inhibits er positive breast cancers proliferation which is a potential target for er positive breast cancers and drug - resistant breast cancer .", "deficiency of er in breast cancer is correlated with poor response to endocrine therapy . in er negative breast cancers , gper-1 stimulates the erk1/2 through the egfr / mapk signal cascade , inducing target gene like c - fos expression , which is involved in the progressing of breast malignancies [ 2729 ] .", "estrogen and antiestrogens can also promote the production of the early growth response-1 ( egr-1 ) , connective tissue growth factor ( ctgf ) , and insulin - like growth factor 1 ( igf-1 ) through the gper-1 [ 28 , 30 , 31 ] .", "grp30 activation stimulated breast cancer cells migration through ctgh , cxc receptor ( cxcr1 ) , and notch pathways . furthermore , gper-1 agonist g-1 promoted inflammation in breast cancers .", "gper-1 was reported to affect the deformation of breast glandular structure inducing the malignant transformation of breast tissue .", "gper-1 can also induce expression of cancer - associated fibroblasts ( cafs ) in tumor microenvironment [ 34 , 35 ] . on the contrary", ", a recent study showed that activation of gper-1 by g-1 resulted in g2/m - phase arrest and induction of mitochondrial - related apoptosis .", "the other studies also proved that g-1 treatment suppressed the growth of skbr3 cancer cells and increased the survival rate by inducing the erk1/2 signal activation [ 36 , 37 ] .", "1520% of breast cancers are included in triple negative breast cancers ( tnbc ) , characterized by lack of er , progesterone receptor ( pr ) , and egfr2 ( her-2 )", ". a higher rate of recurrence and aggressive biological features were found in younger females [ 38 , 39 ] .", "gper-1 expression was found in majority of tnbcs patients . in the gper knockdown mice model , the proliferation of tnbcs ,", "these findings suggest that gper plays a key role in putative mechanism for tnbcs and gper might be a therapeutic target for tnbcs .", "snps of gper-1 , histone acetylation , and transcription factor recruitment were significantly associated with tumor size and histological grading [ 42 , 43 ] .", "gper rna as well as gper-1 protein presents in both primary and malignant ovarian tumor tissues . the expression of gper-1 was significantly increased in ovarian carcinomas compared to pericarcinomatous tissues impendent with the expression of egfr , er , and er .", "estrogen and g-1 induce ovarian cancer cell growth responses via egfr - mapk signaling pathways .", "furthermore , gper-1 promoted the migration and invasion of ovarian cancer cells ovcar5 which is characterized by negative er and positive gper by increasing the expression mmp-9 [ 48 , 49 ] .", "atrazine , one of the most common pesticide contaminants , promoted ovarian cancer cells proliferation via induction of erk and expression of estrogen target gene through gper-1 pathway .", "but other studies results showed that g-1 suppressed proliferation and induced apoptosis of human ovarian cancer cells probably through inhibition of cell cycle progression in g2/m - phase in ovarian carcinomas [ 51 , 52 ] . \n", "gper-1 has been shown to be involved in a variety of hormone - dependent cancers .", "it is well understood that estrogens play a critical role in pathological germ cell proliferation in testicular germ cell tumors .", "gper-1 seems to be involved in modulating the growth of estrogen dependent testicular cancer cells .", "estrogen induces the high expression of gper-1 correlated with low levels of er in human testicular carcinoma in situ and seminomas [ 53 , 54 ] .", "bisphenol a , a common environmental estrogen , can also promote the proliferation of testicular seminomas cells through gper-1 .", "the above findings suggested that gper-1 may be a potential therapeutic target [ 56 , 57 ] .", "estrogen has an efficacy for advanced prostate cancer ( pc ) via the mediation of the classical estrogen receptors .", "the effects of er on pc growth and metastases have different mechanisms in different cellular microenvironments .", "the expression of gper-1 is higher in the preneoplastic lesions and normal areas of benign prostate than the basal epithelial cells .", "g-1 , the selectively activating gper-1 , inhibited the growth of multiple pc cells in vitro and in vivo through erk1/2 and c - jun / c - fos signaling pathways , which indicates that the g-1 may be a new option for pc through targeting gper-1 .", "g-1 inhibited castration - resistant phase but had no effect on androgen - sensitive tumors .", "the antitumor effect of g-1 on cr tumors was related to necrosis ( approximately 65% ) accompanied with neutrophils infiltration .", "g-1 can also upregulate neutrophil - related chemokines and inflammation - mediated cytokines in the cr tumors . in one word"], ["there are other studies which proved that gper-1 induced proliferation , differentiation , and drug resistance of lung cancers [ 63 , 64 ] , thyroid cancers , bladder cancers , and oral squamous carcinomas .", "more studies to reveal the functions and mechanisms of gper-1 in the other system cancers are warranted ."], ["majority of the study results addressed that activation of gper-1 by estrogen and g-1 results in the downstream signals and target genes activation , which promotes the proliferation , migration , and invasion of cancer cells . and", "this effect is in nonclassical er expression dependent manner in most cancers except for ovarian cancers .", "it is interesting that several other studies showed that g-1 , the special agonist of gper-1 , promoted the expression of gper-1 and inhibited the proliferation of er negative breast cancer cells , ovarian cancer cells , and prostate cancer cells .", "the opposite effects of gper-1 in cancer cells may be associated with the epigenetic of gper-1 , such as the snps and histone acetylation .", "the different cell types , tumor microenvironment , and hormonal level may also affect the functions of gper-1", ". controversies still exist on the gper-1 localization and related signaling pathways , in particular the potential action as proapoptotic mediator . since the function and mechanisms of gper-1 are still unclear , more researches and clinical studies are strongly warranted to clarify the different function and mechanisms in different cancer types and conditions ."]]}
{"article_id": "PMC3869051", "article_text": ["ensemble  hi - c protocol to create a method to determine the contacts in an individual nucleus ( fig . 1a , supplementary information ) .", "we used male , mouse , spleenic cd4 t cells , differentiated in vitro to t helper ( th1 ) cells to produce a population of cells ( > 95% cd4 ) , of which 69% have 2n genome content , reflecting mature cell withdrawal from the cell cycle .", "chromatin cross - linking , restriction enzyme ( bgl ii or dpn ii ) digestion , biotin fill - in and ligation were performed in nuclei ( fig .", "1a ) as opposed to ensemble hi - c where ligation is performed after nuclear lysis and dilution of chromatin complexes .", "we then selected individual nuclei under the microscope , placed them in individual tubes , reversed cross - links , and purified biotinylated hi - c ligation junctions on streptavidin - coated beads .", "the captured ligation junctions were then digested with a second restiction enzyme ( alu i ) to fragment the dna , and ligated to customized illumina adapters with unique 3 bp identification tags .", "single cell hi - c libraries were then pcr amplified , size selected and characterized by multiplexed , paired - end sequencing .", "de - multiplexed single cell hi - c libraries were next filtered thoroughly to systematically remove several sources of noise ( extended data fig .", "hi - c in male diploid cells can theoretically give rise to at most two ligation products per autosomal restriction fragment end , and one product per fragment end from the single x chromosome . using bgl ii", ", the total number of distinct mappable fragment - end pairs per single cell can not therefore exceed 1,201,870 ( extended data fig .", "deep sequencing of the single cell hi - c libraries demonstrated that following stringent filtering our current scheme allows recovery of up to 2.5% of this theoretical potential , and has identified at least 1000 distinct hi - c pairings in half ( 37/74 ) of the cells .", "deep sequencing confirmed saturation of the libraries complexity , and allowed elimination of spurious flow cell read pairings and additional biases ( extended data tables 1 - 3 ) .", "based on additional quality metrics we selected ten single cell datasets , containing 11,159 - 30,671 distinct fragment - end pairs for subsequent in - depth analysis ( extended data fig .", "visualization of the single cell maps suggested that despite their inherent sparseness , they clearly reflect hallmarks of chromosomal organization , including frequent cis - contacts along the matrix diagonal and notably , highly clustered trans - chromosomal contacts between specific chromosomes ( fig .", "we used the same population of cd4 th1 cells to generate an ensemble hi - c library .", "sequencing and analysis of 190 million read pairs produced a contact map representing the mean contact enrichments within approximately 10 million nuclei .", "the probability of observing a contact between two chromosomal elements decays with linear distance following a power law regime for distances larger than 100 kb .", "we found similar regimes for the ensemble , individual cells and a pool of 60 single cells ( fig .", "1c ) . moreover , after normalizing the matrices given this canonical trend , comparison of intra - chromosomal interaction intensities for the pool and ensemble , by global correlation analysis of contact enrichment values at 1 mb resolution generates a highly significant correspondence ( fig .", "this is emphasized by the high similarity observed in comparisons of individual chromosomes from ensemble and pooled hi - c maps ( fig .", "despite different experimental procedures and sparse nature of the single cell matrices , the pooled matrix retains the most prominent properties of the ensemble map , confirming the validity of the approach and prompting us to further explore the similarities and differences among the individual cell chromosomal conformations .", "a key architectural feature of ensemble hi - c datasets is their topological domain structure .", "as expected 1403 domains were identified in the th1 cell ensemble hi - c map ( supplementary information table 1 , and supplementary information ) .", "we used the ensemble domains to ask whether the same domain structure can be observed at the single cell level .", "visual inspection of the domain structure overlaid on individual intra - chromosomal contact maps ( fig .", "2a ) , and global statistical analysis of the ratios between intra- and inter - domain contact intensities in individual cells ( approximately 2-fold enrichment on scales of 100 kb to 1 mb , fig .", "2a ) , both supported the idea that domains are observed consistently in the single cell maps . to test whether domain structures are variable between individual cells , we estimated the distributions of intra - domain contact enrichments across cells and compared it to the distributions derived from reshuffled maps .", "we reasoned that cell - to - cell variation in intra - domain contact intensities would result in an increase of the variance of this distribution compared to the expected variance resulting from sampling contacts in uniformly ( shuffled ) intra - domain contacts .", "2c ) , showed that the distributions for the intra - domain enrichments in real cells are not more varied than expected ( kolmogorov - smirnov p < 0.52 ) .", "a similar observation was derived by comparison of the correlations between intra - domain contact enrichments for pairs of real and pairs of reshuffled maps ( extended data fig .", "while this analysis can not quantify variability in the high - resolution internal structure of domains , the data suggests that domain intactness is generally conserved at the single cell level . visual comparison of whole chromosome contact maps ( fig .", "2d ) suggested that unlike intra - domain interactions , inter - domain contacts within single cell chromosomes are structured non - uniformly .", "the maps showed large - scale structures as indicated , for example , by specific insulation points separating chromosomes into two or more mega - domains in a cell - specific fashion . to rule out the possibility that this can be explained by sparse sampling of contacts in each single cell map", "real or randomized ) we quantified the frequency of loci that strongly polarize the matrix into two weakly connected submatrices ( using an insulation score ; supplementary methods ) .", "we confirmed that single cell maps indeed show many more such loci than reshuffled maps ( fig .", "the reshuffled controls made by mixing contacts from different single cell maps , are in fact similar to sparse versions of the ensemble map , which do not show specific structure at the intra - domain level . along similar lines ,", "the correlation in contact intensities between domains on the same chromosome in pairs of single cell maps is lower compared to reshuffled controls ( fig 2f ) . taken together , these data show that domains form a robust and recurrent conformational basis that is evident in each of the single cells", ". however , inter - domain contacts are highly variable between individual cells , suggesting large - scale differences in higher - order chromosome folding that are obscured in ensemble maps , averaged over millions of such structures .", "to determine whether the single cell hi - c data is consistent with unique chromosome conformations we developed a modeling approach to reconstruct the conformations of the single - copy , male x chromosome .", "we used intra - chromosomal contacts as distance restraints and calculated structural models using a simulated annealing protocol to condense a particle - on - a - string representation of individual chromosomes from random initial conformations ( supplementary information ) , to produce both fine - scale and low - resolution models , with backbone particles representing either 50 or 500 kb of the chromosome , respectively . for fine - scale calculations ,", "each intra - chromosomal contact restrained its precise position on the chromosome , while low - resolution calculations combined contacts into larger bins . tests of our simulation protocol demonstrated that restraint density was the most important parameter for modeling ( extended data fig .", "hence , from the ten high - quality single cell datasets , we selected six with the largest numbers of intra - chromosomal x contacts , plus one with a lower number of contacts ( cell-9 ) for contrast .", "repeat calculations starting from random positions generated 200 x chromosome models for each cell at both scales .", "the fine - scale models displayed very low numbers of restraint violations ( extended data fig .", "we introduced an estimated average unit dna distance length to approximate packaging of chromatin fibers ( ~0.15 m/50 kb ) ( supplementary information ) .", "this resulted in models with a mean x chromosome territory diameter of 4.3 m ( range 3.3 - 5.9 m ) , in good agreement with x chromosome paint fish in th1 cells ( fig .", "we confirmed that the restrained points in a single cell are indeed close in the structures calculated from them ( extended data fig .", "interestingly , the single cell distance matrix demonstrates how the network of contacts in a model imparts further structural information beyond the directly observed contacts ( extended data fig .", "comparison of the low - resolution models demonstrated convergence toward a single conformation for each single cell dataset ( fig .", "hierarchical clustering revealed 4 - 5 that were most representative of the data ( fig .", "4a , b ) . highlighting four regions of the x chromosome showed large - scale conformational differences between cells ( fig .", "models created by shuffling hi - c contacts , or combining contacts from two cells resulted in structures smaller and more compact than observed chromosome territories ( extended data fig .", "4c , d ) with many restraints stretched toward or exceeding their upper bounds ( extended data fig .", "these results reaffirm that the variation in single cell contacts is not the result of partial sampling of a single underlying structure .", "we next asked whether despite their cell - to - cell variability , x chromosome structures share common folding properties that could be tested in real cells .", "one such important property , which is often consistent within a cell population , and with multiple potential functional implications , is localisation within the chromosomal territory relative to its surface . to predict loci with consistent positions within their chromosome territory we calculated the structural density along the x chromosome ( supplementary information ) and identified regions with consistently high or low structural density ( fig .", "we chose five such regions ( p1-p5 ) with predicted positions near the surface ( p1 , p2 , p5 ; low structural density ) or inside ( p3 , p4 ; high structural density ) the model x chromosome territories using the 1200 models from the six cells ( extended data fig .", "we then performed double label dna fish with x chromosome paints and p1-p5 bac probes ( fig .", "the distances between dna fish signals and edge of the chromosome territory in over one hundred th1 cells showed that probes p1 , p2 and p5 were indeed found predominantly outside or toward the edge of the chromosome territory , whereas signals for probes p3 and p4 were found at internal positions ( fig .", "these data show that despite highly variable inter - domain structure of the x chromosomal territory , some of its key organisational properties are robustly observed across the cell population .", "overlaying data from trans - chromosomal contacts on the x chromosome models demonstrates that trans - chromosomal contacting regions are strongly enriched toward the inferred surface of the models ( fig .", "these observations prompted us to further explore the structural characteristics of interfaces between chromosomal territories , and the relationships between such interfaces and the domain structure of the territory itself .", "we found that trans - chromosomal contact enrichments of domains vary across cells ( fig .", "4a ) , showing a significant difference between the mean contact enrichment per domain in the real and reshuffled maps ( p < 1.2e-9 , kolmogorov - smirnov test ) .", "the higher variance of the distribution for the real data suggests some domains are more likely to contact elements on other chromosomes .", "previous work has suggested that active genomic regions on the sub - domain scale often loop out of their chromosome territories , which might imply less defined local domain structures and disassociation from their chromosome territory .", "however , our analysis shows that trans - contacting domains retain domain organisation , as demonstrated by the intra - domain contact probabilities within them ( fig .", "trans - contacting domains show slightly reduced contact intensity to other domains on the same chromosome ( fig .", "5c , d ) , consistent with localisation on the interfaces of their territories rather than dissociation from them .", "analyses of ensemble hi - c data have previously shown that active marks correlate with enrichment of trans - chromosomal contacts . using the single cell maps combined with annotation of domains based on their enrichment for histone h3 lysine 4 tri - methylation ( h3k4me3 ) hotspots ( fig .", "4d ) , we tested whether this correlation is the result of low frequency re - localisation of active domains to other chromosome territories ( looping out ) , or from frequent localisation of active domains on territory interfaces . as shown in fig .", "4e , domains with high trans- to cis - chromosomal contact ratios ( excluding intra - domain ) are highly correlated with h3k4me3 enrichment in all cells . however , the data show that domains ( including active ones ) retain their association with the territory in almost all cases .", "very few domains with strong trans - contacts were found to lack association with their own territory ( i.e. , upper left points in graphs in fig .", "4e ) . some of this lack of perfect territory re - localisation can be explained by having two copies of each autosomal domain , but the overall reduction with territory association for trans - contacting domains is much smaller than the 50% expected by this explanation ( reduction estimated at 15 - 20% and 10% for contacts across 1 - 5 mb and 10 mb , respectively , fig .", "4c ) . comparison of active domain localization shows that different active domains are highly trans - contacting in each cell ( extended data fig .", "5e ) . together , these data show that preferential localisation of active domains to territory interfaces is a hallmark of chromosome organisation in all cells .", "active domains maintain their intra - domain organisation , and only partially lose intra - chromosomal contacts with other domains .", "our data are consistent with the concept that chromosomal territories are maintained robustly despite the trans - chromosomal contacts between active domains .", "interestingly , domains associated with lamin - b1 , which are thought to be primarily inactive regions , are also found toward the surface of the models ( fig .", "3h ) . however , these domains are highly anti - correlated with h3k4me3 domains ( spearman s correlation = 0.73 ) and typically depleted of trans - chromosomal contacts ( extended data fig .", "superposition of h3k4me3 , lamin - b1 enriched domains and trans - chromosomal contacts on the x chromosome models illustrates spatial partitioning of the active , trans - contacting regions from those that are lamin - associated , although both types of domains tend toward the surface of the chromosome territory , supporting the above descriptions of differential positioning of domains ( extended data fig .", "ensemble hi - c maps generate a highly complex view of chromosomal contacts , including low intensity contacts between all possible chromosomal pairings . in contrast", ", years of single cell analyses by microscopy have suggested that individual cells have much simpler and discrete chromosome structures involving a limited number of interfaces between spatially constrained chromosomal territories .", "our single cell maps bridge the gap between the genomic and imaging techniques , showing cell - specific clusters of trans - chromosomal contacts associating some pairs of chromosomes , and a lack of contacts between other chromosome pairs ( fig .", "5a , red ) confirming it is not a consequence of sparse contact sampling ( extended data fig .", "trans - chromosomal contact clusters bring pairs of domains together , as shown by comparing the enrichment in trans - contacts between pairs of elements connecting the same two domains and pairs connecting one domain with two different domains ( fig .", "such synergistic contacting preferentially brings together pairs of active domains , with interaction between active and inactive domains being underrepresented ( fig .", "although inactive domains are depleted as a group from trans - chromosomal interactions ( fig .", "4e ) , inactive domains that engage in trans - contacts are more likely to interact with other inactive domains .", "interestingly , analysis of interacting pairs of domains suggests that the number of chromosomes contacted by each chromosome is relatively constant ( less than 30% difference ) despite the > 3-fold change in chromosome size , the total number of trans - chromosomal contacts in the map , or a number of other factors ( fig .", "we note that even though the total number of chromosome - chromosome interfaces per single cell is bounded , the detailed interface between chromosome pairs can involve multiple domain - domain contacts reflecting higher order organization ( extended data fig .", "overall , these results indicate that each chromosome contacts a discrete and fairly constant number of other chromosomes in a single cell , with little dependency on the chromosome size . at the single cell level both", "the microscopic and genomic observations therefore indicate highly defined territory structures , which can be hypothesised to harbor much of the chromosome within the territory , and expose a limited , relatively constant surface area engaged in chromosome - to - chromosome interfaces .", "since these interfaces are highly variable among different cells , their averaging by ensemble hi - c contributes toward the relatively uniform trans - chromosomal contact matrices previously reported .", "we have presented a new experimental strategy to create hi - c contact maps from single cells .", "the approach allows for characterization of thousands of simultaneous contacts occurring in individual cells , and provides unique insights into hi - c technology and 3d chromosomal architecture ( supplementary videos 3 and 4 ) .", "single cell contact maps reflect conservation of domain structure that was recently characterized , but show that inter - domain and trans - chromosomal contact structure is highly variable between individual cells .", "genome - wide statistical analysis and reconstruction of the single copy x chromosome models gave us the opportunity to quantify key features of chromosomal architecture .", "for example , active domains tend to locate on the boundaries of their chromosomal territories in the majority of nuclei , while maintaining associations with other domains on the same chromosome", ". our results do not exclude chromosome territory intermingling , but argue against domains becoming completely immersed in other territories . coupled with previous observations of small and large - scale chromatin mobility a highly dynamic view of chromosomal organization ermerges , where territories are continuously being remodeled , while maintaining some key local ( domain ) and global ( depth from surface ) organisational features .", "male th1 cells were fixed and subjected to modified hi - c , in which nuclei were maintained through restriction enzyme digestion , biotin fill - in labelling and ligation .", "single nuclei were isolated and processed to prepare single cell hi - c libraries for paired - end sequencing .", "sequences were mapped to the mouse genome , and abnormal read pairs were discarded . read pairs that occurred only once ( without duplication ) in the library sequencing were removed .", "we chose 10 single cell datasets for further in - depth analyses based on several quality criteria ( see supplementary information ) . to validate the single cell hi - c procedure , we pooled the single cell hi - c datasets and compared them to ensemble hi - c dataset prepared from approximately 10 million cells essentially as described .", "we created reshuffled datasets by randomly redistributing contacts of the analyzed single cells to create the same number of cells with the same number of contacts in each cell as a control to statistically analyse the variation among single cell datasets .", "we reconstructed three - dimensional x chromosome structure models using restrained molecular dynamics calculations employing a simulated annealing protocol .", "a combination of unambiguous distance restraints from the x intra - chromosomal contacts in the single cell hi - c dataset and anti - distance restraints between regions that were found not to contact each other in the ensemble hi - c dataset was used . to assess the precision and accuracy of the structure generation process we used the protocol to generate synthetic hilbert curve structures , and explored the impact of varying the number of restraints . for pair - wise comparison of the structures ,", "we calculated the root - mean - square deviation ( rmsd ) . to compare the x chromosome models to x chromosome structure in vivo , we selected five loci with consistently high or low structural density in the models , and compared distances between the loci and the x chromosome territory surface in cells ( dna fish ) .", ". a , shown are the numbers of read - pairs in the original and re - sequenced runs , the number of fend - pairs in the original one , the number of fend - pairs when combining the sequences of the two runs , and the addition to fend - pairs the re - sequencing contributed .", "% valid non - amplified are the percentages of singly covered fend - pairs in the original sample that were supported by more read - pairs in the re - sequenced one .", "these fend - pairs were discarded as potential spurious pairs in the original run , but proved by the re - sequencing to be valid pairs .", "this gives a sense of the fraction of valid pairs we discard when removing the read - pairs suspected to be sequencing pairing errors .", "b , shown are fend - pair coverage contingency tables of the original and the re - sequenced runs for the five single - cells . mouse and human nuclei or single cell hi - c samples were mixed in different stages of the experiment ( group a , before fixation ; group b , before library construction [ so all the mouse and human samples in each library have the same identification tag ] ; group c , before library amplification [ so mouse and human samples in each library have different identification tags ] ) .", "we created single cell ( for group a ) or human / mouse two cell ( for groups b and c ) hi - c libraries and analysed them .", "the table shows the percentages of the three possible read - pairs : mouse - mouse ( mm9-mm9 ) , human - human ( hg18-hg18 ) and human - mouse ( hg18-mm9 ) .", "for group a , we selected mouse cells based on morphology . in group a , all six libraries contain almost exclusively mouse - mouse read - pairs with insignificant human - human or mouse - human pairs .", "each group b library has both human - human and mouse - mouse read - pairs as expected , and the number of spurious human - mouse read - pairs is extremely low . in each group", "c library , which was created by amplifying the distinctly tagged human ( c1-c6 ) and mouse ( c7-c12 ) single cell samples in the same tube ( e.g. , c1 and c7 , c2 and c8 , etc . ) , the fractions of foreign pairs ( human reads with a mouse tag and vice versa ) and of spurious pairs ( human - mouse ) were consistently extremely low . to estimate the fraction of foreign and spurious pairs that could have originated simply from mapping a truly pure mouse library to a concatenated human - mouse genome , libraries from pure mouse cells ( group d ) were mapped to such a genome .", "the mean percentages of both foreign and spurious fend - pairs in this lane are the same as those found in the different human - mouse mixed lanes , suggesting there is no inter - cellular contamination .", "phix174 dna library was added to four lanes of single cell hi - c multiplexed libraries . in theory , no mixed mouse - phix174 read - pair is expected , but in fact a small number were detected .", "shown are the fraction of phix174 dna loaded to each lane capacity , the percentage of phix174 read - ends in the lane , and the observed number of read - pairs by type .", "the pairing probability was crudely estimated from these figures , and from it the number of expected spurious mouse - mouse read - pairs was calculated .", "most of these spurious pairs are discarded when the unique identification tags at the beginning of each read - end are matched .", "shown is the estimated number of spurious mouse pairs that coincidently have matching identification tag and are therefore not detected and removed . a , efficiency of biotin labelling at hi - c ligation junctions for two hi - c ligation products , showing 90 - 95% efficiency ( supplementary information ) .", "c , discarding the missed re2 read - pairs removes a uniform  blanket  of non - specific contacts from the map .", "shown is the dependency between the number of fend - pairs in a sample and the estimated number of autosomal fends covered by more than two fend - pairs under different models .", "the binomial model ( grey line ) distributes fend - pairs to fends randomly without any constraint , as if sampling fend - pairs from an infinite number of chromosomes .", "number of fends in each 250 kb genomic bin for bgl ii or dpn ii as re1 .", "tail of bins with few fends is for bins of low mappability and near the chromosomes edges .", "f , median fend length ( distance from re1 to the first upstream re2 ) in each 250 kb genomic bin for bgl ii or dpn ii as re1 .", "g , information on the two restriction enzymes we used for re1 , bgl ii ( 6 cutter , which we used predominantly ) and dpn ii ( 4 cutter , only used for cell-8 ) .", "fends in which their first re2 site starts a non - unique 36 bp sequence are marked as non - unique fends .", "we discarded both blind and non - unique fends and used only the unique fends .", "the number of actual fends in a male mouse genome , which have two copies of each autosome and a single x chromosome are shown as well as the median fragment length ( chromosomes y and mitochondrial genome were ignored throughout the analysis ) .", "h , information on the ten single - cell datasets that successfully passed the quality control filters .", "p - value of the number of autosomal fends with more than two covering fend - pairs was calculated from the binomial model ( panel d and supplementary information ) .", "k , distribution of fend - pair coverage ( number of read - pairs that support each fend - pair ) in the ten single - cell datasets .", "l , distribution of mean contacts per fend calculated for each mappable 1 mbp , normalized by the mean value in each cell , and averaged across autosomal or x chromosomes from the ten single - cells . a , ratios between intra - domain and inter - domain contact enrichments over genomic distance .", "chromosomes are grouped into four groups : group 1 ( chromosomes 1 , 8 , 15 , 16 and x ) , group 2 ( chromosomes 2 , 6 , 10 , 13 and 18 ) , group 3 ( chromosomes 3 , 5 , 11 , 14 and 17 ) and group 4 ( chromosomes 4 , 7 , 9 , 12 , 19 ) .", "the intra- over inter- domain enrichment is persistent in all chromosome groups and does not seem to stem from peculiar chromosomes .", "b , distribution of correlations between intra - domain contact numbers of all domains from pairs of real and reshuffled controls .", "c , distribution of the insulation score at each fend in nine single cell hi - c datasets ( where re1 is bgl ii ; real cells ) is shown in red .", "fifty sets of reshuffled cells were produced ( see supplementary methods ) and their insulation score distribution is shown in black .", "real cells have a heavier tail of highly insulating loci , which is indicative of non - uniform and cell - specific inter - domain contact structure .", "a , results of structure calculations using restraints from a space - filling hilbert curve test structure with 4096 particles and four typical results of structure modelling using different numbers of restraints are shown ( upper panels ) .", "structure calculations of the hilbert curve from random positions using different sets of 1024 restraints ( lower panel ) .", "b , comparison of rmsd values from hilbert curve and single cell x chromosome models .", "structure calculations for hilbert curves were repeated 100 times with variable numbers of restraints as shown .", "the root mean square deviation ( rmsd ) values between 100 models ( precision ) using the indicated number of restrains ( mean + / sd ) are plotted in blue .", "the rmsd values between the original hilbert curve and each of the 100 models ( accuracy ) for the same numbers of restraints are plotted in green ( mean + / sd ) .", "rmsd values from 100 repeated calculations of fine - scale ( 50 kb backbone ) x chromosome structure from the seven single cell datasets are also plotted ( red ; mean + / sd ) .", "the distances between directly restrained positions in fine - scale ( 50 kb backbone ) x chromosome models are shown .", "models for the six single cell datasets ( cell-1 to cell-6 ; red ) show no values exceeding the upper bound ( dashed line ) .", "calculations with six shuffled interaction maps ( created from cell-1 dataset ; blue ) show significant violations .", "structure calculations performed on merged pairs of datasets ( yellow ; all possible combination of cell-1 to cell-4 ) have a few violations and are significantly closer to the upper limit .", "d , comparison of structure - derived distance matrix from 200 fine - scale x chromosome models from cell-1 ( orange ) and its single cell hi - c contacts ( black crosses ) . the orange colour indicates the minimum distance between backbone particles .", "e , comparison or x chromosome structural models for six cells computed using low - resolution ( 500 kb binned ) single - cell hi - c interaction data .", "the bundles shown represent minimised structural alignments of five models from repeat calculations for each cell .", "a , pair - wise comparison of fine - scale x chromosome structural models by rmsd analysis .", "each pixel represents an rmsd value for a pair - wise comparison of two models .", "the order of 200 models in each panel was determined by hierarchical clustering of the rmsd values .", "numbers shown are the mean rmsd values and the standard deviations for all the comparisons for each cell calculated by comparing the hi - c contact particles .", "b , cell - to - cell comparison of 200 fine - scale x chromosome structural models by rmsd analysis .", "each pixel represents an rmsd value for a pair - wise comparison of two models .", "c , fine - scale x chromosome structures calculated from cell-1 and cell-3 datasets , and a structure from the combined dataset .", "d , typical structure calculated using a randomised dataset , where the interacting points for cell-1 have been shuffled with a pairing probability proportional to one over the square root of the sequence separation .", "colours and scale as shown in c. e , distribution of measurements of depth from the surface for five loci p1 - p5 ( fig .", "3e ) in 1200 x chromosome models ( 200 fine - scale models for each of the six cells ) . whiskers on box plots define 10th and 90th percentiles and the outliers are shown as individual dots . a , intra - domain contact enrichment for each quartile of trans - chromosomal contacting domains .", "b , same as a but subtracting the mean quartile enrichment in each genomic distance emphasizing the differences shown in a. c , using the same sets as in a but plotting the enrichment of inter - domain contacts within the same chromosome .", "d , same as c but subtracting the mean quartile enrichment in each genomic distance .", "e , percentage of cells in which high and low h3k4me3 enriched domains are trans - interacting .", "for each cell the domains with top 10th percentile trans intensity were defined as trans - interacting in that cell .", "we then counted for each domain the fraction of cells in which that domain was trans - interacting .", "shown are the distributions of these fractions for h3k4me3 enriched and non - enriched domains ( the top and bottom 25th percentiles , respectively ) .", "f , distribution of the average laminb1-damid enrichment in chromosomal domains , color coded according to the enrichment value .", "g , domains plotted according to their number of trans- and cis - chromosomal ( but excluding intra - domain ) contacts , color - coded as in f. the domain laminb1-damid enrichment and h3k4me3 peak density are highly anti - correlated ( spearman s correlation = 0.73 ) .", "h , intra - domain contact enrichment for high vs. low quartile of domains stratified by their mean laminb1-damid enrichment . error bars indicate 95% confidence intervals . i , using the same sets as in h but plotting the enrichment of inter - domain contacts within the same chromosome", "error bars as in h. j , lamin - b1 domains show a minor decrease in intra - domain contact intensities that might suggest less compacted domains , and significantly increased cis inter - domain contact , maybe due to lack of trans - chromosomal contacts .", "topology of lamin b1 , h3k4me3 and trans - contacts on five - model bundles of low - resolution x chromosome models .", "scale bar , 1 m . a , comparison of observed coverage of the trans - chromosomal 1 mb square bins of each cell ( red lines ) , versus predicted coverage assuming a binomial model ( random uniform distribution of contacts to bins ; black dashed line ) .", "observed coverage is consistently higher than the uniform model , indicating the highly non - random distribution of trans - chromosomal contacts to genomic bins .", "b , trans - chromosomal contact enrichment around observed trans contacts as a function of the contacts total distance on both chromosomes ( manhattan distance in the contact map ) . observed and expected ( by random uniform contact distribution )", "number of contacts are counted around each trans contact , and their ratio is shown for the 9 real cells ( blue ; where re1 is bgl ii ) and reshuffled cells ( red ) , at two different scales .", "c , left panel , trans contacts were classified according to h3k4me3 density of the domains they associate : high and low for top and bottom 25th percentiles , respectively , mid for 25th-75th percentiles . shown is the log ratio of the contingency table counts with the expected counts generated by multiplying the corresponding marginal probabilities for each group ( chi - square test ; p = 5.8e-18 ) . to make sure this phenomena is not caused by the trans enrichment of active domains and depletion of non - active ones , only the top 15th percentile trans enriched domains from each cell were used .", "middle panel , similar to left panel but contacts are classified by their associated domain gene density ( chi - square test ; p = 2.3e-12 ) .", "right panel , similar to left panel but using domains in the top 40th percentile of gene density , classifying by their h3k4me3 density , to test h3k4me3 enrichment beyond gene density ( chi - square test ; p = 3.6e-06 ) . in all cases active / gene - rich domains preferentially interact with each other , although active domains ( high h3k4me3 density ) interact beyond that expected by their gene density .", "a , the number of interacting chromosomes per chromosome is depicted in circles sized according to the number of single cells the value was observed in , while ordering chromosomes by the number of transcription start sites ( tsss ) in each chromosome which is shown by blue bars .", "spearman correlation between the number of tsss and the mean value of the number of interacting chromosomes per chromosome is 0.18 .", "two chromosomes were defined as interacting when they had at least one domain - domain interaction ( see main text ) supported by two or more contacts .", "however , the change is small , and the number of interacting autosomal chromosomes per chromosome ( the plotted value divided by two ) remains between 4 and 6 .", "b , same as a except that chromosomes ordered by the number of active h3k4me3 domains ( the top 25th percentile h3k4me3 peak density domains ) .", "c , same as a except that chromosomes are ordered by the number of non - lad basepairs in the chromosome .", "the fraction of a chromosome covered by lads ranges from 31% to 53% and is correlated with chromosome size ( 0.52 spearman ) .", "thus , chromosome lengths span a range of 3.2 fold change , while their non - lad fraction spans a smaller range of 2.8 fold change .", "the mean number of contacts of each chromosome with others it interacts with is shown for the ten single cells , ordering chromosomes by their size .", "chromosome size is correlated with the number of contacts it has , but the dynamic range of this number is small .", "e , the number of interacting chromosomes per chromosome is depicted in circles sized as the number of single cells the value was observed in , while ordering the ten single cell datasets by the number of trans contacts in each dataset , shown by blue bars . only autosomes are displayed .", "spearman correlations between the number of trans contacts in each dataset and the mean value of the number of interacting chromosomes per chromosome is 0.73 .", "however , the change is small , and the number of interacting autosomal chromosomes per chromosome ( the plotted value divided by two ) remains between 4 and 6 .", "top and bottom 30th percentiles of h3k4me3 peak density domains are marked in light pink and light grey , respectively .", "note the grid - like trans contacts arrangement , and the correspondence between the two large trans contact clusters and the organisation of cis contacts in both chromosomes to large  mega domains  ."], "abstract_text": ["<S> large - scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and dna replication and repair . </S>", "<S> genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously , but do so by averaging chromosome conformations from millions of nuclei . </S>", "<S> here we introduce single cell hi - c , combined with genome - wide statistical analysis and structural modeling of single copy x chromosomes , to show that individual chromosomes maintain domain organisation at the megabase scale , but show variable cell - to - cell chromosome territory structures at larger scales . despite this structural stochasticity , localisation of active gene domains to boundaries of territories </S>", "<S> is a hallmark of chromosomal conformation . </S>", "<S> single cell hi - c data bridge current gaps between genomics and microscopy studies of chromosomes , demonstrating how modular organisation underlies dynamic chromosome structure , and how this structure is probabilistically linked with genome activity patterns . </S>"], "labels": null, "section_names": ["Single cell Hi-C", "Single cell and ensemble Hi-C similarity", "Intra- and inter-domain contacts", "3D structural modeling of X chromosomes", "Domains at the interface", "Online Methods", "Supplementary Material"], "sections": [["ensemble  hi - c protocol to create a method to determine the contacts in an individual nucleus ( fig . 1a , supplementary information ) .", "we used male , mouse , spleenic cd4 t cells , differentiated in vitro to t helper ( th1 ) cells to produce a population of cells ( > 95% cd4 ) , of which 69% have 2n genome content , reflecting mature cell withdrawal from the cell cycle .", "chromatin cross - linking , restriction enzyme ( bgl ii or dpn ii ) digestion , biotin fill - in and ligation were performed in nuclei ( fig .", "1a ) as opposed to ensemble hi - c where ligation is performed after nuclear lysis and dilution of chromatin complexes .", "we then selected individual nuclei under the microscope , placed them in individual tubes , reversed cross - links , and purified biotinylated hi - c ligation junctions on streptavidin - coated beads .", "the captured ligation junctions were then digested with a second restiction enzyme ( alu i ) to fragment the dna , and ligated to customized illumina adapters with unique 3 bp identification tags .", "single cell hi - c libraries were then pcr amplified , size selected and characterized by multiplexed , paired - end sequencing .", "de - multiplexed single cell hi - c libraries were next filtered thoroughly to systematically remove several sources of noise ( extended data fig .", "hi - c in male diploid cells can theoretically give rise to at most two ligation products per autosomal restriction fragment end , and one product per fragment end from the single x chromosome . using bgl ii", ", the total number of distinct mappable fragment - end pairs per single cell can not therefore exceed 1,201,870 ( extended data fig .", "deep sequencing of the single cell hi - c libraries demonstrated that following stringent filtering our current scheme allows recovery of up to 2.5% of this theoretical potential , and has identified at least 1000 distinct hi - c pairings in half ( 37/74 ) of the cells .", "deep sequencing confirmed saturation of the libraries complexity , and allowed elimination of spurious flow cell read pairings and additional biases ( extended data tables 1 - 3 ) .", "based on additional quality metrics we selected ten single cell datasets , containing 11,159 - 30,671 distinct fragment - end pairs for subsequent in - depth analysis ( extended data fig .", "visualization of the single cell maps suggested that despite their inherent sparseness , they clearly reflect hallmarks of chromosomal organization , including frequent cis - contacts along the matrix diagonal and notably , highly clustered trans - chromosomal contacts between specific chromosomes ( fig ."], ["we used the same population of cd4 th1 cells to generate an ensemble hi - c library .", "sequencing and analysis of 190 million read pairs produced a contact map representing the mean contact enrichments within approximately 10 million nuclei .", "the probability of observing a contact between two chromosomal elements decays with linear distance following a power law regime for distances larger than 100 kb .", "we found similar regimes for the ensemble , individual cells and a pool of 60 single cells ( fig .", "1c ) . moreover , after normalizing the matrices given this canonical trend , comparison of intra - chromosomal interaction intensities for the pool and ensemble , by global correlation analysis of contact enrichment values at 1 mb resolution generates a highly significant correspondence ( fig .", "this is emphasized by the high similarity observed in comparisons of individual chromosomes from ensemble and pooled hi - c maps ( fig .", "despite different experimental procedures and sparse nature of the single cell matrices , the pooled matrix retains the most prominent properties of the ensemble map , confirming the validity of the approach and prompting us to further explore the similarities and differences among the individual cell chromosomal conformations ."], ["a key architectural feature of ensemble hi - c datasets is their topological domain structure .", "as expected 1403 domains were identified in the th1 cell ensemble hi - c map ( supplementary information table 1 , and supplementary information ) .", "we used the ensemble domains to ask whether the same domain structure can be observed at the single cell level .", "visual inspection of the domain structure overlaid on individual intra - chromosomal contact maps ( fig .", "2a ) , and global statistical analysis of the ratios between intra- and inter - domain contact intensities in individual cells ( approximately 2-fold enrichment on scales of 100 kb to 1 mb , fig .", "2a ) , both supported the idea that domains are observed consistently in the single cell maps . to test whether domain structures are variable between individual cells , we estimated the distributions of intra - domain contact enrichments across cells and compared it to the distributions derived from reshuffled maps .", "we reasoned that cell - to - cell variation in intra - domain contact intensities would result in an increase of the variance of this distribution compared to the expected variance resulting from sampling contacts in uniformly ( shuffled ) intra - domain contacts .", "2c ) , showed that the distributions for the intra - domain enrichments in real cells are not more varied than expected ( kolmogorov - smirnov p < 0.52 ) .", "a similar observation was derived by comparison of the correlations between intra - domain contact enrichments for pairs of real and pairs of reshuffled maps ( extended data fig .", "while this analysis can not quantify variability in the high - resolution internal structure of domains , the data suggests that domain intactness is generally conserved at the single cell level . visual comparison of whole chromosome contact maps ( fig .", "2d ) suggested that unlike intra - domain interactions , inter - domain contacts within single cell chromosomes are structured non - uniformly .", "the maps showed large - scale structures as indicated , for example , by specific insulation points separating chromosomes into two or more mega - domains in a cell - specific fashion . to rule out the possibility that this can be explained by sparse sampling of contacts in each single cell map", "real or randomized ) we quantified the frequency of loci that strongly polarize the matrix into two weakly connected submatrices ( using an insulation score ; supplementary methods ) .", "we confirmed that single cell maps indeed show many more such loci than reshuffled maps ( fig .", "the reshuffled controls made by mixing contacts from different single cell maps , are in fact similar to sparse versions of the ensemble map , which do not show specific structure at the intra - domain level . along similar lines ,", "the correlation in contact intensities between domains on the same chromosome in pairs of single cell maps is lower compared to reshuffled controls ( fig 2f ) . taken together , these data show that domains form a robust and recurrent conformational basis that is evident in each of the single cells", ". however , inter - domain contacts are highly variable between individual cells , suggesting large - scale differences in higher - order chromosome folding that are obscured in ensemble maps , averaged over millions of such structures ."], ["to determine whether the single cell hi - c data is consistent with unique chromosome conformations we developed a modeling approach to reconstruct the conformations of the single - copy , male x chromosome .", "we used intra - chromosomal contacts as distance restraints and calculated structural models using a simulated annealing protocol to condense a particle - on - a - string representation of individual chromosomes from random initial conformations ( supplementary information ) , to produce both fine - scale and low - resolution models , with backbone particles representing either 50 or 500 kb of the chromosome , respectively . for fine - scale calculations ,", "each intra - chromosomal contact restrained its precise position on the chromosome , while low - resolution calculations combined contacts into larger bins . tests of our simulation protocol demonstrated that restraint density was the most important parameter for modeling ( extended data fig .", "hence , from the ten high - quality single cell datasets , we selected six with the largest numbers of intra - chromosomal x contacts , plus one with a lower number of contacts ( cell-9 ) for contrast .", "repeat calculations starting from random positions generated 200 x chromosome models for each cell at both scales .", "the fine - scale models displayed very low numbers of restraint violations ( extended data fig .", "we introduced an estimated average unit dna distance length to approximate packaging of chromatin fibers ( ~0.15 m/50 kb ) ( supplementary information ) .", "this resulted in models with a mean x chromosome territory diameter of 4.3 m ( range 3.3 - 5.9 m ) , in good agreement with x chromosome paint fish in th1 cells ( fig .", "we confirmed that the restrained points in a single cell are indeed close in the structures calculated from them ( extended data fig .", "interestingly , the single cell distance matrix demonstrates how the network of contacts in a model imparts further structural information beyond the directly observed contacts ( extended data fig .", "comparison of the low - resolution models demonstrated convergence toward a single conformation for each single cell dataset ( fig .", "hierarchical clustering revealed 4 - 5 that were most representative of the data ( fig .", "4a , b ) . highlighting four regions of the x chromosome showed large - scale conformational differences between cells ( fig .", "models created by shuffling hi - c contacts , or combining contacts from two cells resulted in structures smaller and more compact than observed chromosome territories ( extended data fig .", "4c , d ) with many restraints stretched toward or exceeding their upper bounds ( extended data fig .", "these results reaffirm that the variation in single cell contacts is not the result of partial sampling of a single underlying structure .", "we next asked whether despite their cell - to - cell variability , x chromosome structures share common folding properties that could be tested in real cells .", "one such important property , which is often consistent within a cell population , and with multiple potential functional implications , is localisation within the chromosomal territory relative to its surface . to predict loci with consistent positions within their chromosome territory we calculated the structural density along the x chromosome ( supplementary information ) and identified regions with consistently high or low structural density ( fig .", "we chose five such regions ( p1-p5 ) with predicted positions near the surface ( p1 , p2 , p5 ; low structural density ) or inside ( p3 , p4 ; high structural density ) the model x chromosome territories using the 1200 models from the six cells ( extended data fig .", "we then performed double label dna fish with x chromosome paints and p1-p5 bac probes ( fig .", "the distances between dna fish signals and edge of the chromosome territory in over one hundred th1 cells showed that probes p1 , p2 and p5 were indeed found predominantly outside or toward the edge of the chromosome territory , whereas signals for probes p3 and p4 were found at internal positions ( fig .", "these data show that despite highly variable inter - domain structure of the x chromosomal territory , some of its key organisational properties are robustly observed across the cell population ."], ["overlaying data from trans - chromosomal contacts on the x chromosome models demonstrates that trans - chromosomal contacting regions are strongly enriched toward the inferred surface of the models ( fig .", "these observations prompted us to further explore the structural characteristics of interfaces between chromosomal territories , and the relationships between such interfaces and the domain structure of the territory itself .", "we found that trans - chromosomal contact enrichments of domains vary across cells ( fig .", "4a ) , showing a significant difference between the mean contact enrichment per domain in the real and reshuffled maps ( p < 1.2e-9 , kolmogorov - smirnov test ) .", "the higher variance of the distribution for the real data suggests some domains are more likely to contact elements on other chromosomes .", "previous work has suggested that active genomic regions on the sub - domain scale often loop out of their chromosome territories , which might imply less defined local domain structures and disassociation from their chromosome territory .", "however , our analysis shows that trans - contacting domains retain domain organisation , as demonstrated by the intra - domain contact probabilities within them ( fig .", "trans - contacting domains show slightly reduced contact intensity to other domains on the same chromosome ( fig .", "5c , d ) , consistent with localisation on the interfaces of their territories rather than dissociation from them .", "analyses of ensemble hi - c data have previously shown that active marks correlate with enrichment of trans - chromosomal contacts . using the single cell maps combined with annotation of domains based on their enrichment for histone h3 lysine 4 tri - methylation ( h3k4me3 ) hotspots ( fig .", "4d ) , we tested whether this correlation is the result of low frequency re - localisation of active domains to other chromosome territories ( looping out ) , or from frequent localisation of active domains on territory interfaces . as shown in fig .", "4e , domains with high trans- to cis - chromosomal contact ratios ( excluding intra - domain ) are highly correlated with h3k4me3 enrichment in all cells . however , the data show that domains ( including active ones ) retain their association with the territory in almost all cases .", "very few domains with strong trans - contacts were found to lack association with their own territory ( i.e. , upper left points in graphs in fig .", "4e ) . some of this lack of perfect territory re - localisation can be explained by having two copies of each autosomal domain , but the overall reduction with territory association for trans - contacting domains is much smaller than the 50% expected by this explanation ( reduction estimated at 15 - 20% and 10% for contacts across 1 - 5 mb and 10 mb , respectively , fig .", "4c ) . comparison of active domain localization shows that different active domains are highly trans - contacting in each cell ( extended data fig .", "5e ) . together , these data show that preferential localisation of active domains to territory interfaces is a hallmark of chromosome organisation in all cells .", "active domains maintain their intra - domain organisation , and only partially lose intra - chromosomal contacts with other domains .", "our data are consistent with the concept that chromosomal territories are maintained robustly despite the trans - chromosomal contacts between active domains .", "interestingly , domains associated with lamin - b1 , which are thought to be primarily inactive regions , are also found toward the surface of the models ( fig .", "3h ) . however , these domains are highly anti - correlated with h3k4me3 domains ( spearman s correlation = 0.73 ) and typically depleted of trans - chromosomal contacts ( extended data fig .", "superposition of h3k4me3 , lamin - b1 enriched domains and trans - chromosomal contacts on the x chromosome models illustrates spatial partitioning of the active , trans - contacting regions from those that are lamin - associated , although both types of domains tend toward the surface of the chromosome territory , supporting the above descriptions of differential positioning of domains ( extended data fig .", "ensemble hi - c maps generate a highly complex view of chromosomal contacts , including low intensity contacts between all possible chromosomal pairings . in contrast", ", years of single cell analyses by microscopy have suggested that individual cells have much simpler and discrete chromosome structures involving a limited number of interfaces between spatially constrained chromosomal territories .", "our single cell maps bridge the gap between the genomic and imaging techniques , showing cell - specific clusters of trans - chromosomal contacts associating some pairs of chromosomes , and a lack of contacts between other chromosome pairs ( fig .", "5a , red ) confirming it is not a consequence of sparse contact sampling ( extended data fig .", "trans - chromosomal contact clusters bring pairs of domains together , as shown by comparing the enrichment in trans - contacts between pairs of elements connecting the same two domains and pairs connecting one domain with two different domains ( fig .", "such synergistic contacting preferentially brings together pairs of active domains , with interaction between active and inactive domains being underrepresented ( fig .", "although inactive domains are depleted as a group from trans - chromosomal interactions ( fig .", "4e ) , inactive domains that engage in trans - contacts are more likely to interact with other inactive domains .", "interestingly , analysis of interacting pairs of domains suggests that the number of chromosomes contacted by each chromosome is relatively constant ( less than 30% difference ) despite the > 3-fold change in chromosome size , the total number of trans - chromosomal contacts in the map , or a number of other factors ( fig .", "we note that even though the total number of chromosome - chromosome interfaces per single cell is bounded , the detailed interface between chromosome pairs can involve multiple domain - domain contacts reflecting higher order organization ( extended data fig .", "overall , these results indicate that each chromosome contacts a discrete and fairly constant number of other chromosomes in a single cell , with little dependency on the chromosome size . at the single cell level both", "the microscopic and genomic observations therefore indicate highly defined territory structures , which can be hypothesised to harbor much of the chromosome within the territory , and expose a limited , relatively constant surface area engaged in chromosome - to - chromosome interfaces .", "since these interfaces are highly variable among different cells , their averaging by ensemble hi - c contributes toward the relatively uniform trans - chromosomal contact matrices previously reported .", "we have presented a new experimental strategy to create hi - c contact maps from single cells .", "the approach allows for characterization of thousands of simultaneous contacts occurring in individual cells , and provides unique insights into hi - c technology and 3d chromosomal architecture ( supplementary videos 3 and 4 ) .", "single cell contact maps reflect conservation of domain structure that was recently characterized , but show that inter - domain and trans - chromosomal contact structure is highly variable between individual cells .", "genome - wide statistical analysis and reconstruction of the single copy x chromosome models gave us the opportunity to quantify key features of chromosomal architecture .", "for example , active domains tend to locate on the boundaries of their chromosomal territories in the majority of nuclei , while maintaining associations with other domains on the same chromosome", ". our results do not exclude chromosome territory intermingling , but argue against domains becoming completely immersed in other territories . coupled with previous observations of small and large - scale chromatin mobility a highly dynamic view of chromosomal organization ermerges , where territories are continuously being remodeled , while maintaining some key local ( domain ) and global ( depth from surface ) organisational features ."], ["male th1 cells were fixed and subjected to modified hi - c , in which nuclei were maintained through restriction enzyme digestion , biotin fill - in labelling and ligation .", "single nuclei were isolated and processed to prepare single cell hi - c libraries for paired - end sequencing .", "sequences were mapped to the mouse genome , and abnormal read pairs were discarded . read pairs that occurred only once ( without duplication ) in the library sequencing were removed .", "we chose 10 single cell datasets for further in - depth analyses based on several quality criteria ( see supplementary information ) . to validate the single cell hi - c procedure , we pooled the single cell hi - c datasets and compared them to ensemble hi - c dataset prepared from approximately 10 million cells essentially as described .", "we created reshuffled datasets by randomly redistributing contacts of the analyzed single cells to create the same number of cells with the same number of contacts in each cell as a control to statistically analyse the variation among single cell datasets .", "we reconstructed three - dimensional x chromosome structure models using restrained molecular dynamics calculations employing a simulated annealing protocol .", "a combination of unambiguous distance restraints from the x intra - chromosomal contacts in the single cell hi - c dataset and anti - distance restraints between regions that were found not to contact each other in the ensemble hi - c dataset was used . to assess the precision and accuracy of the structure generation process we used the protocol to generate synthetic hilbert curve structures , and explored the impact of varying the number of restraints . for pair - wise comparison of the structures ,", "we calculated the root - mean - square deviation ( rmsd ) . to compare the x chromosome models to x chromosome structure in vivo , we selected five loci with consistently high or low structural density in the models , and compared distances between the loci and the x chromosome territory surface in cells ( dna fish ) ."], [". a , shown are the numbers of read - pairs in the original and re - sequenced runs , the number of fend - pairs in the original one , the number of fend - pairs when combining the sequences of the two runs , and the addition to fend - pairs the re - sequencing contributed .", "% valid non - amplified are the percentages of singly covered fend - pairs in the original sample that were supported by more read - pairs in the re - sequenced one .", "these fend - pairs were discarded as potential spurious pairs in the original run , but proved by the re - sequencing to be valid pairs .", "this gives a sense of the fraction of valid pairs we discard when removing the read - pairs suspected to be sequencing pairing errors .", "b , shown are fend - pair coverage contingency tables of the original and the re - sequenced runs for the five single - cells . mouse and human nuclei or single cell hi - c samples were mixed in different stages of the experiment ( group a , before fixation ; group b , before library construction [ so all the mouse and human samples in each library have the same identification tag ] ; group c , before library amplification [ so mouse and human samples in each library have different identification tags ] ) .", "we created single cell ( for group a ) or human / mouse two cell ( for groups b and c ) hi - c libraries and analysed them .", "the table shows the percentages of the three possible read - pairs : mouse - mouse ( mm9-mm9 ) , human - human ( hg18-hg18 ) and human - mouse ( hg18-mm9 ) .", "for group a , we selected mouse cells based on morphology . in group a , all six libraries contain almost exclusively mouse - mouse read - pairs with insignificant human - human or mouse - human pairs .", "each group b library has both human - human and mouse - mouse read - pairs as expected , and the number of spurious human - mouse read - pairs is extremely low . in each group", "c library , which was created by amplifying the distinctly tagged human ( c1-c6 ) and mouse ( c7-c12 ) single cell samples in the same tube ( e.g. , c1 and c7 , c2 and c8 , etc . ) , the fractions of foreign pairs ( human reads with a mouse tag and vice versa ) and of spurious pairs ( human - mouse ) were consistently extremely low . to estimate the fraction of foreign and spurious pairs that could have originated simply from mapping a truly pure mouse library to a concatenated human - mouse genome , libraries from pure mouse cells ( group d ) were mapped to such a genome .", "the mean percentages of both foreign and spurious fend - pairs in this lane are the same as those found in the different human - mouse mixed lanes , suggesting there is no inter - cellular contamination .", "phix174 dna library was added to four lanes of single cell hi - c multiplexed libraries . in theory , no mixed mouse - phix174 read - pair is expected , but in fact a small number were detected .", "shown are the fraction of phix174 dna loaded to each lane capacity , the percentage of phix174 read - ends in the lane , and the observed number of read - pairs by type .", "the pairing probability was crudely estimated from these figures , and from it the number of expected spurious mouse - mouse read - pairs was calculated .", "most of these spurious pairs are discarded when the unique identification tags at the beginning of each read - end are matched .", "shown is the estimated number of spurious mouse pairs that coincidently have matching identification tag and are therefore not detected and removed . a , efficiency of biotin labelling at hi - c ligation junctions for two hi - c ligation products , showing 90 - 95% efficiency ( supplementary information ) .", "c , discarding the missed re2 read - pairs removes a uniform  blanket  of non - specific contacts from the map .", "shown is the dependency between the number of fend - pairs in a sample and the estimated number of autosomal fends covered by more than two fend - pairs under different models .", "the binomial model ( grey line ) distributes fend - pairs to fends randomly without any constraint , as if sampling fend - pairs from an infinite number of chromosomes .", "number of fends in each 250 kb genomic bin for bgl ii or dpn ii as re1 .", "tail of bins with few fends is for bins of low mappability and near the chromosomes edges .", "f , median fend length ( distance from re1 to the first upstream re2 ) in each 250 kb genomic bin for bgl ii or dpn ii as re1 .", "g , information on the two restriction enzymes we used for re1 , bgl ii ( 6 cutter , which we used predominantly ) and dpn ii ( 4 cutter , only used for cell-8 ) .", "fends in which their first re2 site starts a non - unique 36 bp sequence are marked as non - unique fends .", "we discarded both blind and non - unique fends and used only the unique fends .", "the number of actual fends in a male mouse genome , which have two copies of each autosome and a single x chromosome are shown as well as the median fragment length ( chromosomes y and mitochondrial genome were ignored throughout the analysis ) .", "h , information on the ten single - cell datasets that successfully passed the quality control filters .", "p - value of the number of autosomal fends with more than two covering fend - pairs was calculated from the binomial model ( panel d and supplementary information ) .", "k , distribution of fend - pair coverage ( number of read - pairs that support each fend - pair ) in the ten single - cell datasets .", "l , distribution of mean contacts per fend calculated for each mappable 1 mbp , normalized by the mean value in each cell , and averaged across autosomal or x chromosomes from the ten single - cells . a , ratios between intra - domain and inter - domain contact enrichments over genomic distance .", "chromosomes are grouped into four groups : group 1 ( chromosomes 1 , 8 , 15 , 16 and x ) , group 2 ( chromosomes 2 , 6 , 10 , 13 and 18 ) , group 3 ( chromosomes 3 , 5 , 11 , 14 and 17 ) and group 4 ( chromosomes 4 , 7 , 9 , 12 , 19 ) .", "the intra- over inter- domain enrichment is persistent in all chromosome groups and does not seem to stem from peculiar chromosomes .", "b , distribution of correlations between intra - domain contact numbers of all domains from pairs of real and reshuffled controls .", "c , distribution of the insulation score at each fend in nine single cell hi - c datasets ( where re1 is bgl ii ; real cells ) is shown in red .", "fifty sets of reshuffled cells were produced ( see supplementary methods ) and their insulation score distribution is shown in black .", "real cells have a heavier tail of highly insulating loci , which is indicative of non - uniform and cell - specific inter - domain contact structure .", "a , results of structure calculations using restraints from a space - filling hilbert curve test structure with 4096 particles and four typical results of structure modelling using different numbers of restraints are shown ( upper panels ) .", "structure calculations of the hilbert curve from random positions using different sets of 1024 restraints ( lower panel ) .", "b , comparison of rmsd values from hilbert curve and single cell x chromosome models .", "structure calculations for hilbert curves were repeated 100 times with variable numbers of restraints as shown .", "the root mean square deviation ( rmsd ) values between 100 models ( precision ) using the indicated number of restrains ( mean + / sd ) are plotted in blue .", "the rmsd values between the original hilbert curve and each of the 100 models ( accuracy ) for the same numbers of restraints are plotted in green ( mean + / sd ) .", "rmsd values from 100 repeated calculations of fine - scale ( 50 kb backbone ) x chromosome structure from the seven single cell datasets are also plotted ( red ; mean + / sd ) .", "the distances between directly restrained positions in fine - scale ( 50 kb backbone ) x chromosome models are shown .", "models for the six single cell datasets ( cell-1 to cell-6 ; red ) show no values exceeding the upper bound ( dashed line ) .", "calculations with six shuffled interaction maps ( created from cell-1 dataset ; blue ) show significant violations .", "structure calculations performed on merged pairs of datasets ( yellow ; all possible combination of cell-1 to cell-4 ) have a few violations and are significantly closer to the upper limit .", "d , comparison of structure - derived distance matrix from 200 fine - scale x chromosome models from cell-1 ( orange ) and its single cell hi - c contacts ( black crosses ) . the orange colour indicates the minimum distance between backbone particles .", "e , comparison or x chromosome structural models for six cells computed using low - resolution ( 500 kb binned ) single - cell hi - c interaction data .", "the bundles shown represent minimised structural alignments of five models from repeat calculations for each cell .", "a , pair - wise comparison of fine - scale x chromosome structural models by rmsd analysis .", "each pixel represents an rmsd value for a pair - wise comparison of two models .", "the order of 200 models in each panel was determined by hierarchical clustering of the rmsd values .", "numbers shown are the mean rmsd values and the standard deviations for all the comparisons for each cell calculated by comparing the hi - c contact particles .", "b , cell - to - cell comparison of 200 fine - scale x chromosome structural models by rmsd analysis .", "each pixel represents an rmsd value for a pair - wise comparison of two models .", "c , fine - scale x chromosome structures calculated from cell-1 and cell-3 datasets , and a structure from the combined dataset .", "d , typical structure calculated using a randomised dataset , where the interacting points for cell-1 have been shuffled with a pairing probability proportional to one over the square root of the sequence separation .", "colours and scale as shown in c. e , distribution of measurements of depth from the surface for five loci p1 - p5 ( fig .", "3e ) in 1200 x chromosome models ( 200 fine - scale models for each of the six cells ) . whiskers on box plots define 10th and 90th percentiles and the outliers are shown as individual dots . a , intra - domain contact enrichment for each quartile of trans - chromosomal contacting domains .", "b , same as a but subtracting the mean quartile enrichment in each genomic distance emphasizing the differences shown in a. c , using the same sets as in a but plotting the enrichment of inter - domain contacts within the same chromosome .", "d , same as c but subtracting the mean quartile enrichment in each genomic distance .", "e , percentage of cells in which high and low h3k4me3 enriched domains are trans - interacting .", "for each cell the domains with top 10th percentile trans intensity were defined as trans - interacting in that cell .", "we then counted for each domain the fraction of cells in which that domain was trans - interacting .", "shown are the distributions of these fractions for h3k4me3 enriched and non - enriched domains ( the top and bottom 25th percentiles , respectively ) .", "f , distribution of the average laminb1-damid enrichment in chromosomal domains , color coded according to the enrichment value .", "g , domains plotted according to their number of trans- and cis - chromosomal ( but excluding intra - domain ) contacts , color - coded as in f. the domain laminb1-damid enrichment and h3k4me3 peak density are highly anti - correlated ( spearman s correlation = 0.73 ) .", "h , intra - domain contact enrichment for high vs. low quartile of domains stratified by their mean laminb1-damid enrichment . error bars indicate 95% confidence intervals . i , using the same sets as in h but plotting the enrichment of inter - domain contacts within the same chromosome", "error bars as in h. j , lamin - b1 domains show a minor decrease in intra - domain contact intensities that might suggest less compacted domains , and significantly increased cis inter - domain contact , maybe due to lack of trans - chromosomal contacts .", "topology of lamin b1 , h3k4me3 and trans - contacts on five - model bundles of low - resolution x chromosome models .", "scale bar , 1 m . a , comparison of observed coverage of the trans - chromosomal 1 mb square bins of each cell ( red lines ) , versus predicted coverage assuming a binomial model ( random uniform distribution of contacts to bins ; black dashed line ) .", "observed coverage is consistently higher than the uniform model , indicating the highly non - random distribution of trans - chromosomal contacts to genomic bins .", "b , trans - chromosomal contact enrichment around observed trans contacts as a function of the contacts total distance on both chromosomes ( manhattan distance in the contact map ) . observed and expected ( by random uniform contact distribution )", "number of contacts are counted around each trans contact , and their ratio is shown for the 9 real cells ( blue ; where re1 is bgl ii ) and reshuffled cells ( red ) , at two different scales .", "c , left panel , trans contacts were classified according to h3k4me3 density of the domains they associate : high and low for top and bottom 25th percentiles , respectively , mid for 25th-75th percentiles . shown is the log ratio of the contingency table counts with the expected counts generated by multiplying the corresponding marginal probabilities for each group ( chi - square test ; p = 5.8e-18 ) . to make sure this phenomena is not caused by the trans enrichment of active domains and depletion of non - active ones , only the top 15th percentile trans enriched domains from each cell were used .", "middle panel , similar to left panel but contacts are classified by their associated domain gene density ( chi - square test ; p = 2.3e-12 ) .", "right panel , similar to left panel but using domains in the top 40th percentile of gene density , classifying by their h3k4me3 density , to test h3k4me3 enrichment beyond gene density ( chi - square test ; p = 3.6e-06 ) . in all cases active / gene - rich domains preferentially interact with each other , although active domains ( high h3k4me3 density ) interact beyond that expected by their gene density .", "a , the number of interacting chromosomes per chromosome is depicted in circles sized according to the number of single cells the value was observed in , while ordering chromosomes by the number of transcription start sites ( tsss ) in each chromosome which is shown by blue bars .", "spearman correlation between the number of tsss and the mean value of the number of interacting chromosomes per chromosome is 0.18 .", "two chromosomes were defined as interacting when they had at least one domain - domain interaction ( see main text ) supported by two or more contacts .", "however , the change is small , and the number of interacting autosomal chromosomes per chromosome ( the plotted value divided by two ) remains between 4 and 6 .", "b , same as a except that chromosomes ordered by the number of active h3k4me3 domains ( the top 25th percentile h3k4me3 peak density domains ) .", "c , same as a except that chromosomes are ordered by the number of non - lad basepairs in the chromosome .", "the fraction of a chromosome covered by lads ranges from 31% to 53% and is correlated with chromosome size ( 0.52 spearman ) .", "thus , chromosome lengths span a range of 3.2 fold change , while their non - lad fraction spans a smaller range of 2.8 fold change .", "the mean number of contacts of each chromosome with others it interacts with is shown for the ten single cells , ordering chromosomes by their size .", "chromosome size is correlated with the number of contacts it has , but the dynamic range of this number is small .", "e , the number of interacting chromosomes per chromosome is depicted in circles sized as the number of single cells the value was observed in , while ordering the ten single cell datasets by the number of trans contacts in each dataset , shown by blue bars . only autosomes are displayed .", "spearman correlations between the number of trans contacts in each dataset and the mean value of the number of interacting chromosomes per chromosome is 0.73 .", "however , the change is small , and the number of interacting autosomal chromosomes per chromosome ( the plotted value divided by two ) remains between 4 and 6 .", "top and bottom 30th percentiles of h3k4me3 peak density domains are marked in light pink and light grey , respectively .", "note the grid - like trans contacts arrangement , and the correspondence between the two large trans contact clusters and the organisation of cis contacts in both chromosomes to large  mega domains  ."]]}
{"article_id": "PMC2695811", "article_text": ["oligonucleotides were synthesized on an expedite automated dna / rna synthesizer ( applied biosystems , foster city , ca ) using nucleoside phosphoramidites purchased from glen research ( sterling , va ) .", "all oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis ( page ) and desalted using a c18 sep - pak cartridge ( waters , milford , ma ) .", "histidine - tagged t7 rna polymerase was purified from e. coli strain bl21 containing plasmid pbh161 ( kindly provided by william mcallister , state university of new york , brooklyn ) .", "thermus aquaticus dna polymerase was cloned from total genomic dna and purified as described previously30 .", "m1 rna , the catalytic subunit of rnase p , was obtained from e. coli genomic dna ( sigma - aldrich , st .", "louis , mo ) by pcr amplification and subsequent in vitro transcription , as described previously1 .", "calf intestine phosphatase and t4 polynucleotide kinase were purchased from new england biolabs ( ipswich , ma ) , yeast inorganic pyrophosphatase was from sigma - aldrich , and bovine pancreatic dnase i was from roche applied science ( indianapolis , in ) .", "nucleoside and deoxynucleoside 5-triphosphates , theophylline , and fmn were purchased from sigma - aldrich , [ -p]atp ( 7 ci / pmol ) was from perkin elmer ( waltham , ma ) , and caffeine was from mp biomedicals ( solon , oh ) .", "photinus pyralis ( firefly ) luciferase , saccharomyces cerevisiae adenosine-5-triphosphate sulfurylase , adenosine 5-phosphosulfate , and d - luciferin were from sigma - aldrich .", "bovine calf serum was from omega scientific ( tarzana , ca ) and superasin ( rnase inhibitor ) was from ambion ( austin , tx ) .", "all rna enzymes and substrates were prepared by in vitro transcription in a reaction mixture containing 0.4 m dna template , 0.8 m synthetic oligodeoxynucleotide having the sequence 5-ggactaatacgactcactata-3  ( t7 rna polymerase promoter sequence underlined ) , 2 mm each of the four ntps , 15 u/l t7 rna polymerase , 0.001 u/l inorganic pyrophosphatase , 15 mm mgcl2 , 2 mm spermidine , 5 mm dithiothreitol , and 50 mm tris - hcl ( ph 7.5 ) .", "the mixture was incubated at 37 c for 2 h , quenched by adding an equal volume of 15 mm na2edta , treated with 1 u/l dnase i , and extracted with a 1:1 mixture of phenol : chloroform .", "transcription of m1 rna was performed similarly , except employing a double - stranded dna template that was generated by pcr .", "the a and a  substrates could not be obtained reliably by in vitro transcription due to heterogeneity at the 3  end of the transcripts . instead", ", these substrates were prepared from the corresponding e or e  molecules by cleaving off the b or b  portion using e. coli m1 rna , as described previously1 .", "the external guide sequence rna for cleavage of etheo and efmn had the sequence 5-cguaaguugcggucucacca-3  , and for etheo and efmn had the sequence 5-auauucaugcggucucacca-3  ( nucleotides complementary to the target rna underlined ) .", "for the second pair of etheo and etheo molecules used in the multiplex experiments , the external guide sequence rnas had the sequence 5-cguaguaugcggucuacca-3  and 5-gaauaucauugcggucucacca-3  , respectively .", "the a and a  substrates were [ 5-p]-labeled by first dephosphorylating using calf intestine alkaline phosphatase , then phosphorylating using t4 polynucleotide kinase and [ -p]atp . the labeled substrates were purified by page and desalted using a nensorb 20 cartridge ( nen life sciences , waltham , ma ) .", "rna - catalyzed rna ligation was performed in a reaction mixture containing 5 m e or e  , 0.1 m [ 5-p]-labeled a  or a , 6 m b  or b , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "aliquots were taken at various times and quenched by adding an equal volume of gel - loading buffer containing 50 mm na2edta and 18 m urea .", "the products were separated by page and quantitated using a pharosfx molecular imager ( bio - rad , hercules , ca ) .", "the data were fit to the equation : ft = fmax(a1ek1t)(a2ek2t ) , where ft is the fraction reacted at time t , fmax is the overall maximum extent of the reaction , a1 and k1 are the amplitude and rate of the initial fast phase , and a2 and k2 are the amplitude and rate of the subsequent slow phase , respectively . in the presence of 5 mm theophylline ,", "the reaction catalyzed by etheo exhibited a fast phase with an amplitude of 0.57 and rate constant of 1.4 min , followed by a slow phase with an amplitude of 0.24 and rate constant of 0.044 min ; the reaction catalyzed by etheo had an amplitude of 0.52 and rate constant of 0.59 min in the fast phase , and an amplitude of 0.26 and rate constant of 0.045 min in the slow phase .", "cross - catalytic exponential amplification was performed in a reaction mixture containing 0.02 m each of e and e  , 5 m each of [ 5-p]-labeled a and a  , 5 m each of b and b  , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "the reaction was initiated by mixing equal volumes of two solutions , one containing the enzymes and substrates , and the other containing the mgcl2 and epps buffer .", "aliquots were taken at various times , quenched , and the amounts of newly - synthesized e and e  were quantitated as described above .", "the data were fit to the logistic growth equation , as described in the main text .", "known concentrations of inorganic pyrophosphate or samples taken from the cross - replication reaction were diluted 10-fold into a reaction mixture containing 0.15 g/l luciferase , 0.00045 u/l atp sulfurylase , 10 m adenosine 5-phosphosulfate , 0.5 mm d - luciferin , 25 mm magnesium acetate , 0.1% bovine serum albumin , 1 mm dithiothreitol , 0.4 g/l polyvinylpyrrolidone ( mw 360,000 ) , and 100 mm tris - acetate ( ph 7.75 ) .", "the pyrophosphate standards were prepared in a solution identical to that employed in cross - replication , but lacking the rna enzymes and substrates .", "luminescence was detected using a perkin elmer ls55 luminescence spectrometer operating in bioluminescence mode , with a pmt voltage of 900 v , cycle time of 200 ms , gate time of 180 ms , and delay time of 0 .", "the flash count was set to 1 , the emission filter was fully open , and the emission slit width was 12 nm .", "following addition of the sample to the luciferase mixture , luminescence was monitored for 5 min with a 0.1 s integration time .", "the amount of light generated was linear over a pyrophosphate concentration range of 0.110 m .", "oligonucleotides were synthesized on an expedite automated dna / rna synthesizer ( applied biosystems , foster city , ca ) using nucleoside phosphoramidites purchased from glen research ( sterling , va ) .", "all oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis ( page ) and desalted using a c18 sep - pak cartridge ( waters , milford , ma ) .", "histidine - tagged t7 rna polymerase was purified from e. coli strain bl21 containing plasmid pbh161 ( kindly provided by william mcallister , state university of new york , brooklyn ) .", "thermus aquaticus dna polymerase was cloned from total genomic dna and purified as described previously30 .", "m1 rna , the catalytic subunit of rnase p , was obtained from e. coli genomic dna ( sigma - aldrich , st .", "louis , mo ) by pcr amplification and subsequent in vitro transcription , as described previously1 .", "calf intestine phosphatase and t4 polynucleotide kinase were purchased from new england biolabs ( ipswich , ma ) , yeast inorganic pyrophosphatase was from sigma - aldrich , and bovine pancreatic dnase i was from roche applied science ( indianapolis , in ) .", "nucleoside and deoxynucleoside 5-triphosphates , theophylline , and fmn were purchased from sigma - aldrich , [ -p]atp ( 7 ci / pmol ) was from perkin elmer ( waltham , ma ) , and caffeine was from mp biomedicals ( solon , oh ) .", "photinus pyralis ( firefly ) luciferase , saccharomyces cerevisiae adenosine-5-triphosphate sulfurylase , adenosine 5-phosphosulfate , and d - luciferin were from sigma - aldrich .", "bovine calf serum was from omega scientific ( tarzana , ca ) and superasin ( rnase inhibitor ) was from ambion ( austin , tx ) .", "all rna enzymes and substrates were prepared by in vitro transcription in a reaction mixture containing 0.4 m dna template , 0.8 m synthetic oligodeoxynucleotide having the sequence 5-ggactaatacgactcactata-3  ( t7 rna polymerase promoter sequence underlined ) , 2 mm each of the four ntps , 15 u/l t7 rna polymerase , 0.001 u/l inorganic pyrophosphatase , 15 mm mgcl2 , 2 mm spermidine , 5 mm dithiothreitol , and 50 mm tris - hcl ( ph 7.5 ) .", "the mixture was incubated at 37 c for 2 h , quenched by adding an equal volume of 15 mm na2edta , treated with 1 u/l dnase i , and extracted with a 1:1 mixture of phenol : chloroform .", "transcription of m1 rna was performed similarly , except employing a double - stranded dna template that was generated by pcr .", "the a and a  substrates could not be obtained reliably by in vitro transcription due to heterogeneity at the 3  end of the transcripts . instead", ", these substrates were prepared from the corresponding e or e  molecules by cleaving off the b or b  portion using e. coli m1 rna , as described previously1 .", "the external guide sequence rna for cleavage of etheo and efmn had the sequence 5-cguaaguugcggucucacca-3  , and for etheo and efmn had the sequence 5-auauucaugcggucucacca-3  ( nucleotides complementary to the target rna underlined ) .", "for the second pair of etheo and etheo molecules used in the multiplex experiments , the external guide sequence rnas had the sequence 5-cguaguaugcggucuacca-3  and 5-gaauaucauugcggucucacca-3  , respectively .", "the a and a  substrates were [ 5-p]-labeled by first dephosphorylating using calf intestine alkaline phosphatase , then phosphorylating using t4 polynucleotide kinase and [ -p]atp . the labeled substrates were purified by page and desalted using a nensorb 20 cartridge ( nen life sciences , waltham , ma ) .", "rna - catalyzed rna ligation was performed in a reaction mixture containing 5 m e or e  , 0.1 m [ 5-p]-labeled a  or a , 6 m b  or b , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "aliquots were taken at various times and quenched by adding an equal volume of gel - loading buffer containing 50 mm na2edta and 18 m urea .", "the products were separated by page and quantitated using a pharosfx molecular imager ( bio - rad , hercules , ca ) .", "the data were fit to the equation : ft = fmax(a1ek1t)(a2ek2t ) , where ft is the fraction reacted at time t , fmax is the overall maximum extent of the reaction , a1 and k1 are the amplitude and rate of the initial fast phase , and a2 and k2 are the amplitude and rate of the subsequent slow phase , respectively . in the presence of 5 mm theophylline ,", "the reaction catalyzed by etheo exhibited a fast phase with an amplitude of 0.57 and rate constant of 1.4 min , followed by a slow phase with an amplitude of 0.24 and rate constant of 0.044 min ; the reaction catalyzed by etheo had an amplitude of 0.52 and rate constant of 0.59 min in the fast phase , and an amplitude of 0.26 and rate constant of 0.045 min in the slow phase .", "cross - catalytic exponential amplification was performed in a reaction mixture containing 0.02 m each of e and e  , 5 m each of [ 5-p]-labeled a and a  , 5 m each of b and b  , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "the reaction was initiated by mixing equal volumes of two solutions , one containing the enzymes and substrates , and the other containing the mgcl2 and epps buffer .", "aliquots were taken at various times , quenched , and the amounts of newly - synthesized e and e  were quantitated as described above .", "the data were fit to the logistic growth equation , as described in the main text .", "known concentrations of inorganic pyrophosphate or samples taken from the cross - replication reaction were diluted 10-fold into a reaction mixture containing 0.15 g/l luciferase , 0.00045 u/l atp sulfurylase , 10 m adenosine 5-phosphosulfate , 0.5 mm d - luciferin , 25 mm magnesium acetate , 0.1% bovine serum albumin , 1 mm dithiothreitol , 0.4 g/l polyvinylpyrrolidone ( mw 360,000 ) , and 100 mm tris - acetate ( ph 7.75 ) .", "the pyrophosphate standards were prepared in a solution identical to that employed in cross - replication , but lacking the rna enzymes and substrates .", "luminescence was detected using a perkin elmer ls55 luminescence spectrometer operating in bioluminescence mode , with a pmt voltage of 900 v , cycle time of 200 ms , gate time of 180 ms , and delay time of 0 .", "the flash count was set to 1 , the emission filter was fully open , and the emission slit width was 12 nm .", "following addition of the sample to the luciferase mixture , luminescence was monitored for 5 min with a 0.1 s integration time .", "the amount of light generated was linear over a pyrophosphate concentration range of 0.110 m ."], "abstract_text": ["<S> rna enzymes have been developed that undergo self - sustained replication at a constant temperature in the absence of proteins1 . </S>", "<S> these rna molecules amplify exponentially through a cross - replicative process , whereby two enzymes catalyze each other s synthesis by joining component oligonucleotides . </S>", "<S> other rna enzymes have been made to operate in a ligand - dependent manner by combining a catalytic domain with a ligand - binding domain ( aptamer ) to provide an  aptazyme2,3 . </S>", "<S> the principle of ligand - dependent rna catalysis now has been extended to the cross - replicating rna enzymes so that exponential amplification occurs in the presence , but not the absence , of the cognate ligand . </S>", "<S> the exponential growth rate of the rna depends on the concentration of the ligand , enabling one to determine the concentration of ligand in a sample . </S>", "<S> this process is analogous to quantitative pcr ( qpcr ) , but can be generalized to a wide variety of targets , including proteins and small molecules that are relevant to medical diagnostics and environmental monitoring . </S>"], "labels": null, "section_names": ["Methods", "Materials", "Preparation of aptazymes and substrates", "Individual RNA-catalyzed reactions", "Cross-replication reactions", "Luciferase assays", "Supplementary Material"], "sections": [["oligonucleotides were synthesized on an expedite automated dna / rna synthesizer ( applied biosystems , foster city , ca ) using nucleoside phosphoramidites purchased from glen research ( sterling , va ) .", "all oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis ( page ) and desalted using a c18 sep - pak cartridge ( waters , milford , ma ) .", "histidine - tagged t7 rna polymerase was purified from e. coli strain bl21 containing plasmid pbh161 ( kindly provided by william mcallister , state university of new york , brooklyn ) .", "thermus aquaticus dna polymerase was cloned from total genomic dna and purified as described previously30 .", "m1 rna , the catalytic subunit of rnase p , was obtained from e. coli genomic dna ( sigma - aldrich , st .", "louis , mo ) by pcr amplification and subsequent in vitro transcription , as described previously1 .", "calf intestine phosphatase and t4 polynucleotide kinase were purchased from new england biolabs ( ipswich , ma ) , yeast inorganic pyrophosphatase was from sigma - aldrich , and bovine pancreatic dnase i was from roche applied science ( indianapolis , in ) .", "nucleoside and deoxynucleoside 5-triphosphates , theophylline , and fmn were purchased from sigma - aldrich , [ -p]atp ( 7 ci / pmol ) was from perkin elmer ( waltham , ma ) , and caffeine was from mp biomedicals ( solon , oh ) .", "photinus pyralis ( firefly ) luciferase , saccharomyces cerevisiae adenosine-5-triphosphate sulfurylase , adenosine 5-phosphosulfate , and d - luciferin were from sigma - aldrich .", "bovine calf serum was from omega scientific ( tarzana , ca ) and superasin ( rnase inhibitor ) was from ambion ( austin , tx ) .", "all rna enzymes and substrates were prepared by in vitro transcription in a reaction mixture containing 0.4 m dna template , 0.8 m synthetic oligodeoxynucleotide having the sequence 5-ggactaatacgactcactata-3  ( t7 rna polymerase promoter sequence underlined ) , 2 mm each of the four ntps , 15 u/l t7 rna polymerase , 0.001 u/l inorganic pyrophosphatase , 15 mm mgcl2 , 2 mm spermidine , 5 mm dithiothreitol , and 50 mm tris - hcl ( ph 7.5 ) .", "the mixture was incubated at 37 c for 2 h , quenched by adding an equal volume of 15 mm na2edta , treated with 1 u/l dnase i , and extracted with a 1:1 mixture of phenol : chloroform .", "transcription of m1 rna was performed similarly , except employing a double - stranded dna template that was generated by pcr .", "the a and a  substrates could not be obtained reliably by in vitro transcription due to heterogeneity at the 3  end of the transcripts . instead", ", these substrates were prepared from the corresponding e or e  molecules by cleaving off the b or b  portion using e. coli m1 rna , as described previously1 .", "the external guide sequence rna for cleavage of etheo and efmn had the sequence 5-cguaaguugcggucucacca-3  , and for etheo and efmn had the sequence 5-auauucaugcggucucacca-3  ( nucleotides complementary to the target rna underlined ) .", "for the second pair of etheo and etheo molecules used in the multiplex experiments , the external guide sequence rnas had the sequence 5-cguaguaugcggucuacca-3  and 5-gaauaucauugcggucucacca-3  , respectively .", "the a and a  substrates were [ 5-p]-labeled by first dephosphorylating using calf intestine alkaline phosphatase , then phosphorylating using t4 polynucleotide kinase and [ -p]atp . the labeled substrates were purified by page and desalted using a nensorb 20 cartridge ( nen life sciences , waltham , ma ) .", "rna - catalyzed rna ligation was performed in a reaction mixture containing 5 m e or e  , 0.1 m [ 5-p]-labeled a  or a , 6 m b  or b , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "aliquots were taken at various times and quenched by adding an equal volume of gel - loading buffer containing 50 mm na2edta and 18 m urea .", "the products were separated by page and quantitated using a pharosfx molecular imager ( bio - rad , hercules , ca ) .", "the data were fit to the equation : ft = fmax(a1ek1t)(a2ek2t ) , where ft is the fraction reacted at time t , fmax is the overall maximum extent of the reaction , a1 and k1 are the amplitude and rate of the initial fast phase , and a2 and k2 are the amplitude and rate of the subsequent slow phase , respectively . in the presence of 5 mm theophylline ,", "the reaction catalyzed by etheo exhibited a fast phase with an amplitude of 0.57 and rate constant of 1.4 min , followed by a slow phase with an amplitude of 0.24 and rate constant of 0.044 min ; the reaction catalyzed by etheo had an amplitude of 0.52 and rate constant of 0.59 min in the fast phase , and an amplitude of 0.26 and rate constant of 0.045 min in the slow phase .", "cross - catalytic exponential amplification was performed in a reaction mixture containing 0.02 m each of e and e  , 5 m each of [ 5-p]-labeled a and a  , 5 m each of b and b  , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "the reaction was initiated by mixing equal volumes of two solutions , one containing the enzymes and substrates , and the other containing the mgcl2 and epps buffer .", "aliquots were taken at various times , quenched , and the amounts of newly - synthesized e and e  were quantitated as described above .", "the data were fit to the logistic growth equation , as described in the main text .", "known concentrations of inorganic pyrophosphate or samples taken from the cross - replication reaction were diluted 10-fold into a reaction mixture containing 0.15 g/l luciferase , 0.00045 u/l atp sulfurylase , 10 m adenosine 5-phosphosulfate , 0.5 mm d - luciferin , 25 mm magnesium acetate , 0.1% bovine serum albumin , 1 mm dithiothreitol , 0.4 g/l polyvinylpyrrolidone ( mw 360,000 ) , and 100 mm tris - acetate ( ph 7.75 ) .", "the pyrophosphate standards were prepared in a solution identical to that employed in cross - replication , but lacking the rna enzymes and substrates .", "luminescence was detected using a perkin elmer ls55 luminescence spectrometer operating in bioluminescence mode , with a pmt voltage of 900 v , cycle time of 200 ms , gate time of 180 ms , and delay time of 0 .", "the flash count was set to 1 , the emission filter was fully open , and the emission slit width was 12 nm .", "following addition of the sample to the luciferase mixture , luminescence was monitored for 5 min with a 0.1 s integration time .", "the amount of light generated was linear over a pyrophosphate concentration range of 0.110 m ."], ["oligonucleotides were synthesized on an expedite automated dna / rna synthesizer ( applied biosystems , foster city , ca ) using nucleoside phosphoramidites purchased from glen research ( sterling , va ) .", "all oligonucleotides were purified by denaturing polyacrylamide gel electrophoresis ( page ) and desalted using a c18 sep - pak cartridge ( waters , milford , ma ) .", "histidine - tagged t7 rna polymerase was purified from e. coli strain bl21 containing plasmid pbh161 ( kindly provided by william mcallister , state university of new york , brooklyn ) .", "thermus aquaticus dna polymerase was cloned from total genomic dna and purified as described previously30 .", "m1 rna , the catalytic subunit of rnase p , was obtained from e. coli genomic dna ( sigma - aldrich , st .", "louis , mo ) by pcr amplification and subsequent in vitro transcription , as described previously1 .", "calf intestine phosphatase and t4 polynucleotide kinase were purchased from new england biolabs ( ipswich , ma ) , yeast inorganic pyrophosphatase was from sigma - aldrich , and bovine pancreatic dnase i was from roche applied science ( indianapolis , in ) .", "nucleoside and deoxynucleoside 5-triphosphates , theophylline , and fmn were purchased from sigma - aldrich , [ -p]atp ( 7 ci / pmol ) was from perkin elmer ( waltham , ma ) , and caffeine was from mp biomedicals ( solon , oh ) .", "photinus pyralis ( firefly ) luciferase , saccharomyces cerevisiae adenosine-5-triphosphate sulfurylase , adenosine 5-phosphosulfate , and d - luciferin were from sigma - aldrich .", "bovine calf serum was from omega scientific ( tarzana , ca ) and superasin ( rnase inhibitor ) was from ambion ( austin , tx ) ."], ["all rna enzymes and substrates were prepared by in vitro transcription in a reaction mixture containing 0.4 m dna template , 0.8 m synthetic oligodeoxynucleotide having the sequence 5-ggactaatacgactcactata-3  ( t7 rna polymerase promoter sequence underlined ) , 2 mm each of the four ntps , 15 u/l t7 rna polymerase , 0.001 u/l inorganic pyrophosphatase , 15 mm mgcl2 , 2 mm spermidine , 5 mm dithiothreitol , and 50 mm tris - hcl ( ph 7.5 ) .", "the mixture was incubated at 37 c for 2 h , quenched by adding an equal volume of 15 mm na2edta , treated with 1 u/l dnase i , and extracted with a 1:1 mixture of phenol : chloroform .", "transcription of m1 rna was performed similarly , except employing a double - stranded dna template that was generated by pcr .", "the a and a  substrates could not be obtained reliably by in vitro transcription due to heterogeneity at the 3  end of the transcripts . instead", ", these substrates were prepared from the corresponding e or e  molecules by cleaving off the b or b  portion using e. coli m1 rna , as described previously1 .", "the external guide sequence rna for cleavage of etheo and efmn had the sequence 5-cguaaguugcggucucacca-3  , and for etheo and efmn had the sequence 5-auauucaugcggucucacca-3  ( nucleotides complementary to the target rna underlined ) .", "for the second pair of etheo and etheo molecules used in the multiplex experiments , the external guide sequence rnas had the sequence 5-cguaguaugcggucuacca-3  and 5-gaauaucauugcggucucacca-3  , respectively .", "the a and a  substrates were [ 5-p]-labeled by first dephosphorylating using calf intestine alkaline phosphatase , then phosphorylating using t4 polynucleotide kinase and [ -p]atp . the labeled substrates were purified by page and desalted using a nensorb 20 cartridge ( nen life sciences , waltham , ma ) ."], ["rna - catalyzed rna ligation was performed in a reaction mixture containing 5 m e or e  , 0.1 m [ 5-p]-labeled a  or a , 6 m b  or b , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "aliquots were taken at various times and quenched by adding an equal volume of gel - loading buffer containing 50 mm na2edta and 18 m urea .", "the products were separated by page and quantitated using a pharosfx molecular imager ( bio - rad , hercules , ca ) .", "the data were fit to the equation : ft = fmax(a1ek1t)(a2ek2t ) , where ft is the fraction reacted at time t , fmax is the overall maximum extent of the reaction , a1 and k1 are the amplitude and rate of the initial fast phase , and a2 and k2 are the amplitude and rate of the subsequent slow phase , respectively . in the presence of 5 mm theophylline ,", "the reaction catalyzed by etheo exhibited a fast phase with an amplitude of 0.57 and rate constant of 1.4 min , followed by a slow phase with an amplitude of 0.24 and rate constant of 0.044 min ; the reaction catalyzed by etheo had an amplitude of 0.52 and rate constant of 0.59 min in the fast phase , and an amplitude of 0.26 and rate constant of 0.045 min in the slow phase ."], ["cross - catalytic exponential amplification was performed in a reaction mixture containing 0.02 m each of e and e  , 5 m each of [ 5-p]-labeled a and a  , 5 m each of b and b  , 25 mm mgcl2 , and 50 mm epps ( ph 8.5 ) , which was incubated at 42 c .", "the reaction was initiated by mixing equal volumes of two solutions , one containing the enzymes and substrates , and the other containing the mgcl2 and epps buffer .", "aliquots were taken at various times , quenched , and the amounts of newly - synthesized e and e  were quantitated as described above .", "the data were fit to the logistic growth equation , as described in the main text ."], ["known concentrations of inorganic pyrophosphate or samples taken from the cross - replication reaction were diluted 10-fold into a reaction mixture containing 0.15 g/l luciferase , 0.00045 u/l atp sulfurylase , 10 m adenosine 5-phosphosulfate , 0.5 mm d - luciferin , 25 mm magnesium acetate , 0.1% bovine serum albumin , 1 mm dithiothreitol , 0.4 g/l polyvinylpyrrolidone ( mw 360,000 ) , and 100 mm tris - acetate ( ph 7.75 ) .", "the pyrophosphate standards were prepared in a solution identical to that employed in cross - replication , but lacking the rna enzymes and substrates .", "luminescence was detected using a perkin elmer ls55 luminescence spectrometer operating in bioluminescence mode , with a pmt voltage of 900 v , cycle time of 200 ms , gate time of 180 ms , and delay time of 0 .", "the flash count was set to 1 , the emission filter was fully open , and the emission slit width was 12 nm .", "following addition of the sample to the luciferase mixture , luminescence was monitored for 5 min with a 0.1 s integration time .", "the amount of light generated was linear over a pyrophosphate concentration range of 0.110 m ."], [""]]}
{"article_id": "PMC3725715", "article_text": ["neoadjuvant chemotherapy actually takes an important place in treatment of operable breast cancer in the hope of improving conservative surgery rate of female patients .", "neoadjuvant chemotherapy includes today on target therapies involving the research on expression of specific molecules by tumoral cells . in routine practice , estrogen receptors ( er )", ", progesterone receptors ( pr ) , and human epidermal growth factor receptor 2 ( her2 ) are the common used biomarkers .", "new pharmaceutical molecules , other than er / pr or her2 , are now already evaluated in clinical research as new biomarkers and new target for neoadjuvant therapy .", "fine - needle aspiration cytology ( fnac ) of the breast is wellknown as a safe , effective , economical , and accurate technique for diagnosing palpable breast lesion [ 24 ] .", "this last decade , fnac technique is improved by the development of new cytological methods allowing standardization of fixation and assuring constant results with ancillary tests such as immunocytochemistry and in situ molecular biology .", "also , one of the advantages of fnac is the management of small tissue fragments permitting a repetitive evaluation of the chronological evolution in expression of tumoral biomarkers .", "previously , the role of fnac has been challenged by results obtained with cnb that seems more robust than fnac . in general ,", "nevertheless , cnb carries disadvantage in terms of a long tissue processing time and patient discomfort such as pain ( 1.7% to 3.7% ) , hematoma ( 0.72% ) , and very rarely pneumothorax [ 6 , 7 ] .", "fnac includes more advantages than cnb such as minimal invasiveness and minimal discomfort ( more painless ) that could be interesting for aged or frailty patients with comorbidities . in palpable lesions ,", "fnac could perform repetitively and is a serious candidate for the chronological followup of neoadjuvant chemotherapy response .", "an important quality of fnac is its ability to give rapid diagnostic information equivalent to that of frozen sections . in our experience", ", the result of rapid fnac prior to cnb improves the quality of cnb and gives an immediate diagnosis decreasing anxiety of patient .  ", "other indications of fnac are staging of multiple tumors or suspicious zones and apparition of a new suspicious lesion during neoadjuvant chemotherapy .", "finally , fnac could be an excellent alternative when radiographic screening of breast is not available .  ", "table 1 compares the main advantages and disadvantages of fnac versus cnb . \n other clinical fnac indications . \n", "palpable breast lesion.rapid diagnosis to decrease anxiety of patient.patient with morbidity ( senile , cardiac , diabetic etc.).clinical staging : multiple lesions , suspicious lymph node , and so on .", "apparition of new lesion in patient treated for breast cancer.evaluation of biomarkers.evaluation of biomarker changes following time or metastasis . when cnb technique is not available . \n ", "evaluation of biomarker changes following time or metastasis .   when cnb technique is not available .", "it is well known that the combination of clinical evaluation , mammography , and fnac , called triple diagnosis , gives a precise diagnosis [ 10 , 11 ] .", "yu et al .   recently demonstrated in meta - analysis of 46 studies that fnac had a sensitivity of 92.7% and a specificity of 94.8% except the unsatisfactory samples .", "the roc curve showed an excellent area under the curve of 0.986 , presenting a high level of accuracy . on the other hand ,", "if the fnac result was negative , the probability of breast cancer is approximately 8% .", "these authors concluded that fnac was an accurate material for evaluation on breast malignancy if rigorous criteria are used .", "also , they said that fnac may provide a favourable screening method and permit an improvement of treatment planning .", "therefore , when fnac is unsatisfactory , cnb is required to minimize the probability of a missed malignant diagnosis . in a study of fnac and immediate diagnosis performed in 408 palpable breast lesions , liew et al . in 2010  ", "reached the same conclusions : 98.1% sensitivity , 89.5% specificity , and 95.8% accuracy . in 508 cnb followed by jackman et al .", ", the rate of false negative for all lesions was 4.4% , for microcalcifications alone 1.2% , and for tumoral mass 0.8% .", "these results of cnb are quasi - identical to those obtained with the satisfactory fnac .", "most of false negative fnac results of sampling error or discordance between clinical and histological observations [ 6 , 14 ] . in a comparison between cnb and repeat fnac after an indeterminate diagnosis with fnac , kooistra et al .  ", "suggested that cnb should be performed after an indeterminate fnac to obtain a reliable preoperative diagnosis .", "other authors concluded that , although the fnac is easier to perform , this technique was not efficient for small and nonpalpable lesions or diagnosis of microcalcifications as those for in situ carcinoma .", "these comments are likely true because some preneoplastic lesions are associated with slight cell atypia .", "however , bilous   emphasized that cnb shows also problems with similar lesions such as atypical proliferative lesions ( atypical ductal hyperplasia , in situ lobular neoplasia , etc . ) , cellular fibroepithelial lesions , papillary tumors , mucinous carcinoma , radial scar , spindle cell lesions .", "moreover , fnac interpretation requires serious experience of the cytopathologist and they think that this is one of the main reasons that overall cnb is to be preferred .   a recent japanese study   of 5693 fnac and 7 different laboratories illustrated a great variability between the institutions suggesting likely difference between education of cytologists and the lack of clinical or radiological information ( triple diagnosis ) .", "these last years a new cytological technique , called liquid - based cytology ( lbc ) , has been developed and approved by the food and drug administration .", "briefly , lbc standardises the cell fixation , concentrates epithelial cells , and discards blood cells and/or cell debris that obscure the smear .", "the efficiency of lbc in the breast cytology has been demonstrated by numerous publications .  ", "the main advantage of lbc is surely to adjunct ancillary tests such as immunocytochemistry , flow cytometry , or molecular biology [ 1820 ] .", "compared the er and pr statuses from fnac ( immunocytochemistry ) and cnb ( immunohistochemistry ) , both performed on surgical breast tumors .", "they found that both methods give similar results with a concordance between the 2 tests of 98% for er ( with kappa correlation score = 0.93 ) and 96% for pr ( kappa = 0.91 ) .", ".   demonstrated similar data in a comparative study between primary breast tumours and their metastasis .", "interestingly , the concordance between these both localisations was 81% for er , 65% for pr , and 71% for her2 , suggesting a possibility of biological difference between primary tumors and their metastasis .  ", "other publications showed a long - time storage at 20c and 80c at least 6 months without significant loss of immunoreactivity of pr and ep from breast fnac .", "the cell block cytology is an attractive cytological method for ancillary techniques or long - time cell conservations and consists in putting cells of fnac directly in formol fixative fluid identically at a classical histology ( figures 1 and 2 : er , her2 and fish on cell block cytology ) . briefly , after centrifugation to concentrate cells , the pellet was embedded in a synthetic polymer gel that is then processed in paraffin block that could be cut at 4  m , as classical biopsy slides . with this technique , ferguson et al .  ", "found a concordance rate of 95% for er , 90% for pr , and 88% for her2 .", "similar results were observed by shabaik et al .   with high specificity ( 100% for both ) and lower sensibility ( 85% and 80% resp .", "for er and pr ) . finally , in fnac , false negative er or pr immunostaining exists but false positive tests are very unlikely .", "false negative immunohistochemical results are also observed in cnb : in a retrospective study , seferina et al .  ", "calculated a rate of false negative of 26.5% and a rate of false positive of 63.8% for both er and pr . for her2 , they showed 5.4% for false negative rate and 50% for false positive rate .", "nevertheless , in our experience , this discordance is often associated with the manipulation of cnb before the fixation ( crush artefacts ) or with a defect of fixation as desiccation .", "fortunately the concordance with molecular biology by hybridisation in situ using fish , cish , and sish is very good and can help when her2 is uncertain [ 6 , 25 , 27 ] .", "the fish is accurate for lbc cytology [ 22 , 25 , 28 , 29 ] .", "the extraction of mrna or dna is also feasible from lbc and fnac , allowing all gene expression analyses [ 2932 ] . in our experience ,", "the clinical staging and preoperative lymph node status are important for the evaluation of eligible patient to neoadjuvant therapy . in the axillary lymph node fnac , chang et al .  ", "fnac in lymph node is a cost effective and safe method , false positive is virtually non - existent , and false negative can occur when lymph node is partially involved such as by micrometastase , or isolated tumor cells .   in our experience", ", we improve axillary lymph node fnac / lbc by immunocytochemistry using cytokeratin antibody .  ", "thus , axillary fnac plays a role in staging of advanced cases for systemic and neoadjuvant therapy and in evaluating candidates for sentinel lymph node surgical procedure or axillary lymph node dissection .", "despite the fact that cnb has been progressively replaced by fnac in the investigation of nonpalpable lesions or microcalcifications without a clinical or radiological mass lesion , fnac has yet a role in palpable lesions in the triple diagnosis association and performed by experienced cytologists . in these conditions , fnac is a safe , effective , economical , and accurate technique for breast cancer evaluation .  ", "recently , cytological methods using lbc technology , associated or not with the cellblock cytological technique , improve immunocytological and molecular tests with the same efficiency as classical histology .  ", "if the limits of its indications are well known , fnac still plays a role in the modern oncological practice ."], "abstract_text": ["<S> despite the fact that cnb has been progressively replaced by fnac in the investigation of nonpalpable lesions or microcalcifications without a clinical or radiological mass lesion , fnac has yet a role in palpable lesions provided it is associated with the triple diagnosis and experienced cytologist . in these conditions </S>", "<S> , fnac is a safe , effective , economical , and accurate technique for breast cancer evaluation . </S>", "<S> numerous literature reviews and meta - analyses illustrated the advantages and disadvantages of both methods cnb and fnac . </S>", "<S> the difference does not seem significant when noninformative and unsatisfactory fnac was excluded . </S>", "<S> recently , cytological methods using liquid - based cytology ( lbc ) technology improve immunocytological and molecular tests with the same efficiency as classical immunohistochemistry . \n </S>", "<S> the indications of fnac were , for palpable lesions , relative contraindication of cnb ( elderly or frailty ) , staging of multiple nodules in conjunction or not with cnb , staging of lymph node status , newly appearing lesion in patient under neoadjuvant treatment , decreasing of anxiety with a rapid diagnosis , evaluation of biomarkers and new biomarkers , and chronological evaluation of biomarker following the neoadjuvant therapy response . </S>"], "labels": null, "section_names": ["1. Introduction", "2. What Are the Advantages of FNAC in Comparison with Core-Needle Biopsy (CNB)?", "3. Is the Diagnosis of FNAC Accurate?", "4. What about Biomarkers?", "5. Sentinel Lymph Node Evaluation by FNAC", "6. Conclusions "], "sections": [["neoadjuvant chemotherapy actually takes an important place in treatment of operable breast cancer in the hope of improving conservative surgery rate of female patients .", "neoadjuvant chemotherapy includes today on target therapies involving the research on expression of specific molecules by tumoral cells . in routine practice , estrogen receptors ( er )", ", progesterone receptors ( pr ) , and human epidermal growth factor receptor 2 ( her2 ) are the common used biomarkers .", "new pharmaceutical molecules , other than er / pr or her2 , are now already evaluated in clinical research as new biomarkers and new target for neoadjuvant therapy .", "fine - needle aspiration cytology ( fnac ) of the breast is wellknown as a safe , effective , economical , and accurate technique for diagnosing palpable breast lesion [ 24 ] .", "this last decade , fnac technique is improved by the development of new cytological methods allowing standardization of fixation and assuring constant results with ancillary tests such as immunocytochemistry and in situ molecular biology .", "also , one of the advantages of fnac is the management of small tissue fragments permitting a repetitive evaluation of the chronological evolution in expression of tumoral biomarkers ."], ["previously , the role of fnac has been challenged by results obtained with cnb that seems more robust than fnac . in general ,", "nevertheless , cnb carries disadvantage in terms of a long tissue processing time and patient discomfort such as pain ( 1.7% to 3.7% ) , hematoma ( 0.72% ) , and very rarely pneumothorax [ 6 , 7 ] .", "fnac includes more advantages than cnb such as minimal invasiveness and minimal discomfort ( more painless ) that could be interesting for aged or frailty patients with comorbidities . in palpable lesions ,", "fnac could perform repetitively and is a serious candidate for the chronological followup of neoadjuvant chemotherapy response .", "an important quality of fnac is its ability to give rapid diagnostic information equivalent to that of frozen sections . in our experience", ", the result of rapid fnac prior to cnb improves the quality of cnb and gives an immediate diagnosis decreasing anxiety of patient .  ", "other indications of fnac are staging of multiple tumors or suspicious zones and apparition of a new suspicious lesion during neoadjuvant chemotherapy .", "finally , fnac could be an excellent alternative when radiographic screening of breast is not available .  ", "table 1 compares the main advantages and disadvantages of fnac versus cnb . \n other clinical fnac indications . \n", "palpable breast lesion.rapid diagnosis to decrease anxiety of patient.patient with morbidity ( senile , cardiac , diabetic etc.).clinical staging : multiple lesions , suspicious lymph node , and so on .", "apparition of new lesion in patient treated for breast cancer.evaluation of biomarkers.evaluation of biomarker changes following time or metastasis . when cnb technique is not available . \n ", "evaluation of biomarker changes following time or metastasis .   when cnb technique is not available ."], ["it is well known that the combination of clinical evaluation , mammography , and fnac , called triple diagnosis , gives a precise diagnosis [ 10 , 11 ] .", "yu et al .   recently demonstrated in meta - analysis of 46 studies that fnac had a sensitivity of 92.7% and a specificity of 94.8% except the unsatisfactory samples .", "the roc curve showed an excellent area under the curve of 0.986 , presenting a high level of accuracy . on the other hand ,", "if the fnac result was negative , the probability of breast cancer is approximately 8% .", "these authors concluded that fnac was an accurate material for evaluation on breast malignancy if rigorous criteria are used .", "also , they said that fnac may provide a favourable screening method and permit an improvement of treatment planning .", "therefore , when fnac is unsatisfactory , cnb is required to minimize the probability of a missed malignant diagnosis . in a study of fnac and immediate diagnosis performed in 408 palpable breast lesions , liew et al . in 2010  ", "reached the same conclusions : 98.1% sensitivity , 89.5% specificity , and 95.8% accuracy . in 508 cnb followed by jackman et al .", ", the rate of false negative for all lesions was 4.4% , for microcalcifications alone 1.2% , and for tumoral mass 0.8% .", "these results of cnb are quasi - identical to those obtained with the satisfactory fnac .", "most of false negative fnac results of sampling error or discordance between clinical and histological observations [ 6 , 14 ] . in a comparison between cnb and repeat fnac after an indeterminate diagnosis with fnac , kooistra et al .  ", "suggested that cnb should be performed after an indeterminate fnac to obtain a reliable preoperative diagnosis .", "other authors concluded that , although the fnac is easier to perform , this technique was not efficient for small and nonpalpable lesions or diagnosis of microcalcifications as those for in situ carcinoma .", "these comments are likely true because some preneoplastic lesions are associated with slight cell atypia .", "however , bilous   emphasized that cnb shows also problems with similar lesions such as atypical proliferative lesions ( atypical ductal hyperplasia , in situ lobular neoplasia , etc . ) , cellular fibroepithelial lesions , papillary tumors , mucinous carcinoma , radial scar , spindle cell lesions .", "moreover , fnac interpretation requires serious experience of the cytopathologist and they think that this is one of the main reasons that overall cnb is to be preferred .   a recent japanese study   of 5693 fnac and 7 different laboratories illustrated a great variability between the institutions suggesting likely difference between education of cytologists and the lack of clinical or radiological information ( triple diagnosis ) ."], ["these last years a new cytological technique , called liquid - based cytology ( lbc ) , has been developed and approved by the food and drug administration .", "briefly , lbc standardises the cell fixation , concentrates epithelial cells , and discards blood cells and/or cell debris that obscure the smear .", "the efficiency of lbc in the breast cytology has been demonstrated by numerous publications .  ", "the main advantage of lbc is surely to adjunct ancillary tests such as immunocytochemistry , flow cytometry , or molecular biology [ 1820 ] .", "compared the er and pr statuses from fnac ( immunocytochemistry ) and cnb ( immunohistochemistry ) , both performed on surgical breast tumors .", "they found that both methods give similar results with a concordance between the 2 tests of 98% for er ( with kappa correlation score = 0.93 ) and 96% for pr ( kappa = 0.91 ) .", ".   demonstrated similar data in a comparative study between primary breast tumours and their metastasis .", "interestingly , the concordance between these both localisations was 81% for er , 65% for pr , and 71% for her2 , suggesting a possibility of biological difference between primary tumors and their metastasis .  ", "other publications showed a long - time storage at 20c and 80c at least 6 months without significant loss of immunoreactivity of pr and ep from breast fnac .", "the cell block cytology is an attractive cytological method for ancillary techniques or long - time cell conservations and consists in putting cells of fnac directly in formol fixative fluid identically at a classical histology ( figures 1 and 2 : er , her2 and fish on cell block cytology ) . briefly , after centrifugation to concentrate cells , the pellet was embedded in a synthetic polymer gel that is then processed in paraffin block that could be cut at 4  m , as classical biopsy slides . with this technique , ferguson et al .  ", "found a concordance rate of 95% for er , 90% for pr , and 88% for her2 .", "similar results were observed by shabaik et al .   with high specificity ( 100% for both ) and lower sensibility ( 85% and 80% resp .", "for er and pr ) . finally , in fnac , false negative er or pr immunostaining exists but false positive tests are very unlikely .", "false negative immunohistochemical results are also observed in cnb : in a retrospective study , seferina et al .  ", "calculated a rate of false negative of 26.5% and a rate of false positive of 63.8% for both er and pr . for her2 , they showed 5.4% for false negative rate and 50% for false positive rate .", "nevertheless , in our experience , this discordance is often associated with the manipulation of cnb before the fixation ( crush artefacts ) or with a defect of fixation as desiccation .", "fortunately the concordance with molecular biology by hybridisation in situ using fish , cish , and sish is very good and can help when her2 is uncertain [ 6 , 25 , 27 ] .", "the fish is accurate for lbc cytology [ 22 , 25 , 28 , 29 ] .", "the extraction of mrna or dna is also feasible from lbc and fnac , allowing all gene expression analyses [ 2932 ] . in our experience ,"], ["the clinical staging and preoperative lymph node status are important for the evaluation of eligible patient to neoadjuvant therapy . in the axillary lymph node fnac , chang et al .  ", "fnac in lymph node is a cost effective and safe method , false positive is virtually non - existent , and false negative can occur when lymph node is partially involved such as by micrometastase , or isolated tumor cells .   in our experience", ", we improve axillary lymph node fnac / lbc by immunocytochemistry using cytokeratin antibody .  ", "thus , axillary fnac plays a role in staging of advanced cases for systemic and neoadjuvant therapy and in evaluating candidates for sentinel lymph node surgical procedure or axillary lymph node dissection ."], ["despite the fact that cnb has been progressively replaced by fnac in the investigation of nonpalpable lesions or microcalcifications without a clinical or radiological mass lesion , fnac has yet a role in palpable lesions in the triple diagnosis association and performed by experienced cytologists . in these conditions , fnac is a safe , effective , economical , and accurate technique for breast cancer evaluation .  ", "recently , cytological methods using lbc technology , associated or not with the cellblock cytological technique , improve immunocytological and molecular tests with the same efficiency as classical histology .  ", "if the limits of its indications are well known , fnac still plays a role in the modern oncological practice ."]]}
{"article_id": "PMC3976786", "article_text": ["understanding how and why vector - borne diseases like malaria remain a persistent problem despite being tools for diagnosis and treatment is essential for developing effective control measures for sub - saharan africa .", "temperature is one of the key climatic variables that determine the range of malaria transmission and hence global warming is likely to result in an increase in malaria prone areas especially where temperatures have generally been lower than the optimal range of 2527c for mosquito development .", "furthermore degradation of the environment and social and economic pressures due to population growth may boost the expansion of malaria prone areas .", "population migrations , drug and pesticide resistance , and deterioration of health service delivery systems will also influence the level of malaria transmission .", "the epidemiology of malaria is very complex , involving factors pertaining to the malaria parasites , the insect vectors , the human hosts , and the environment .", "an understanding of the link between malaria transmission , climatic variables , and other human related factors is therefore necessary for developing appropriate measures that will significantly reduce transmission and perhaps eliminate malaria in endemic areas . in most cases these human related risk factors", "the level of risk to human populations living in malaria endemic areas varies markedly across continents and also within countries and different areas within the same countries .", "several studies have shown that malaria vector distribution , transmission rates , and incidence can vary widely over short distances , between neighbouring villages and even within a single settlement , as a result of small area variations in risk factors [ 5 , 6 ] .", "identification and understanding of this variation are important in the detection of high risk groups and for selective targeting of intervention .", "many studies have attempted to identify household and individual level factors associated with malaria . some of the factors studied include access to health facilities , type of housing that people live in , proximity of human settlements to vector breeding sites [ 1012 ] , vector abundance , socioeconomic status , gender , occupation , residential mobility , travel , presence of domestic animals near homesteads , and use of preventive methods such as bed nets .", "information on how these factors interact to expose communities and individuals to malaria infections needs to be investigated systematically in each geographical setting , in conjunction with climate related factors .", "documented information about individual and household risk factors associated with malaria transmission is lacking in botswana . in order to address this paucity of information we assessed household and individual risk factors that may be contributing to malaria transmission in tubu village community in the okavango subdistrict in northern botswana .", "the study was conducted in the context of a larger project , botswana ecohealth project ( bep ) , which is investigating the impacts of hydroclimate change on population health . apart from contributing towards the objectives of bep ,", "the results of this study are relevant to an initiative by the ministry of health in botswana to eliminate malaria by year 2016 .", "the study was conducted in tubu village on the banks of the thaoge river , one of the distributaries of the okavango delta .", "tubu village is located in the okavango subdistrict at an altitude of about 950  m above sea level and between latitude 1935s and longitude 2227e . according to the national central statistics office report of 2011", "the area is subjected to annual flooding but the extent of flooding varies from year to year . the community practices flood recession farming locally known as  molapo  farming .", "they plough their fields along the banks of the river as the flood recedes and in some instances they plant crops taking advantage of a raised water table caused by flooding .", "community development and everyday governance issues pertaining to law and order in the area are conducted at a central place ( locally known as the  kgotla  ) within the village .", "tubu has a village development committee ( vdc ) established as a local government structure to guide development planning and implementation in the village .", "the village also has a primary school , a resident social worker , and a health post staffed with a nurse , nurse aid , and a health education assistant .", "gumare hospital , located 10  km east of the village , offers an array of general health services to the local community .", "the hospital also serves as the district health centre for critically ill patients referred from all district health posts including tubu .", "malaria cases in the okavango subdistrict have been fluctuating since 2005 with unconfirmed cases ranging between 4,686 in 2005 and 10,993 in 2006 and confirmed cases ranging between five ( in 2012 ) and 791 ( in 2006 ) ( ministry of health annual report , 2012 ) .", "the maximum number of deaths between 2005 and 2012 was 16 , recorded in 2006 .", "data from clinic records for the period of 2005 to 2010 indicated that 2009 had the highest with 131 unconfirmed cases and 2005 the lowest with 50 cases .", "however during the same period information on rapid diagnostic tests ( rdts ) was inconsistent and available only for years 2006 , 2008 , and 2010 with 9 , 4 , and 5 cases , respectively .", "pretesting of the questionnaire involved trained field research assistants with higher secondary education qualifications and field supervisors with degree qualifications who eventually administered the final questionnaire .", "the questionnaire had four thematic sections ; first part focused on sociodemographic characteristics such as gender , age , marital status , education level , farming practice , household income , ethnicity , respondent 's relationship to household head , employment status , and occupation and the second part included aspects on how the community or individuals got exposed to mosquito bites due to late night activities , location of homesteads in relation to animal shelters and mosquito breeding sites , visits made to other areas outside tubu village in the last 8 months , and history of malaria episodes in the last 8 months .", "a short period of 8 months was chosen so as to minimise on - recall bias as the time frame included the previous malaria transmission season which fell between the months of october 2011 and june 2012 .", "the third part of the questionnaire focused on malaria prevention methods being practiced and health delivery services for the study area .", "the last part involved general observations made at each homestead during the interview with regard to house structure and use , type of eaves , and vegetation cover surrounding the homesteads . to ensure accuracy and good quality data , pretesting of questionnaires", "was carried out two weeks prior to the actual data collection exercise in etsha 1 , a village with similar environmental and sociodemographic patterns to tubu village .", "following the pretest survey the questionnaire was revised , on the basis of responses given , for improvement of clarity and addition of essential questions that had been omitted in the original questionnaire .", "vague questions in the original questionnaire were either omitted from the final questionnaire or improved .", "the pretest exercise was also used to standardize the manner in which the interviewers would conduct the interviews and to ensure that they had a common understanding of each question . from the household listing compiled for the bep project the target sample size , as determined by raosoft inc .", "however , due to the fact that tubu is a relatively small village a census survey was done consisting of individuals representing all the 71 households .", "the questionnaire was administered at the household level to any person who is 18 years and above with preference being given to the heads of the household if they were present .", "only one person per household was interviewed . during the interviews the respondents answered some of the questions pertaining to children and other members of the same household such as visits outside study area and possession and use of nets .", "numbers 1 to 4 were assigned to individuals from the community who had come to attend the pras for the major bep project .", "all individuals assigned a similar number were grouped together to participate in the malaria risk survey .", "pra technique , using the closed scoring / ranking approach , was used to cross - check on some of the responses from the questionnaire interviews .", "the maximum number of individuals at any one given time during the pra discussions was 16 , thus providing a total ranking capacity ( n ) of 160 .", "the respondents were asked whether they had suffered from malaria within the past 8 months ( the period between october 2011 and june 2012 ) , which included the traditional peak malaria transmission season in botswana .", "mosquito sampling was conducted in houses , in pit traps , and in larval breeding sites located within the study area .", "adult mosquitoes were sampled from pyrethrum space spray knockdowns in 6 selected huts used as bedrooms and from 4 pit traps .", "the physiological conditions of the adult mosquitoes collected from indoors and pit traps were scored as non - blood - fed , blood - fed , half gravid , and full gravid . all adult mosquito specimens , from both adult and larval sampling , were identified to species level by the polymerase chain reaction ( pcr ) technique . a geographic positioning system instrument ( gps )", "was used to determine straight line distances of study homesteads from the nearest mosquito breeding habitats .", "permission to carry out the study was obtained from the ministry of health , republic of botswana ( permit number : ppme 13/18/1 vol .", "meetings with the community leadership and other interested community members were held to introduce the study , clearly explaining its objectives . on the day of the interview verbal consent", "respect for privacy was maintained during adult mosquito sampling in selected rooms used as bedrooms .", "participants were informed that they could refuse to participate in the study and not be prejudiced .", "respondents were assured that all the information captured on the data sheets would be treated with confidentiality and that no answers would be linked to individuals in the final analysis and presentation of findings .", "descriptive statistics were carried out to determine relative frequencies of all the survey variables . some of the variables had provisions for multiple responses .", "appropriate graphs and tables were generated to show differences in the relative frequencies of various variables .", "levels of association between various variables were determined by the pearson  test and fisher 's exact test in situations where the expected frequencies were less than five . where appropriate , p values and confidence intervals ( ci ) for odds ratios ( or ) are shown .", "the level of association between various factors and history of malaria was calculated based on the respondents ' views .", "the maximum number of people residing in a single homestead was 20 and the minimum was one .", "a total of 483 individuals resided in all the 71 study homesteads , thus giving an average household size of seven people .", "about two - thirds of the respondents had some years of schooling ( table 1 ) .", "the majority of households earned an income of less than us$63 per month ( figure 1 ) .", "only 7% of the respondents earned more than us$250 per month . eighty - one point seven percent ( 58 ) of the respondents resided within 1  km from the nearest health facility .", "eighteen - point three percent ( 13 ) resided 1  km to 2  km from the clinic .", "all the 71 respondents indicated that it took them less than an hour to get service at the clinic from the time they joined the queue . at the time of the study ( june 2012 ) 53.5% ( 38 ) of the respondents said that they had suffered from malaria within the past 8 months whilst 46.5% ( 33 ) did not remember ever suffering from malaria in their entire life .", "forty - seven point nine percent ( 34 ) mentioned that at least one member of their family had suffered from malaria within the past 8 months . among family members who had suffered from malaria ,", "73.5% ( 25 ) were above five years old and 26.5% were below the age of five years .", "there was an association between household income and history of a malaria episode ( table 2 ) .", "almost all of those who had experienced a malaria attack were in the lowest bracket for household monthly income . in the whole village", "only two house roof types were observed . these were either of grass or iron sheet .", "a general observation noted that houses in the village were either grass thatched , built of reeds , pole , and mud or home - made bricks ( referred to as traditional hut / house ) or built of bricks and roofed with iron sheets ( referred to as modern hut / house ) . among those structures used as bedrooms by the respondents , 52.1% ( 37 ) were traditional and 47.9% ( 34 ) were modern houses / huts . in traditional houses / huts large eave openings", "were observed in 89.2% ( 33 ) and the rest ( 10.8% ( 4 ) ) had small eave openings . in modern houses 2.9% ( 1 ) had large eave openings and the rest ( 97.1% ( 33 ) ) had virtually no eave openings .", "there was an association between history of malaria episode and use of traditional huts / houses as bedrooms ( table 3 ) .", "majority of individuals who experienced a malaria attack used traditional houses / huts as bedrooms .", "low vegetation cover surrounding homesteads was observed at 81.7% ( 58 ) of the homesteads .", "moderate vegetation cover was observed at 14.1% ( 10 ) of the homesteads whilst only 4.2% ( 3 ) of the homesteads were surrounded by dense vegetation cover .", "individual contact with mosquito bites was categorized into  always ,   sometimes ,   often ,  and  rarely .  out of 98.6% ( 70 ) who experienced mosquito bites , 54.9% ( 39 ) mentioned that they ", "seven percent ( 5 ) and 8.5% ( 6 )  often  and  rarely  got bitten by mosquitoes , respectively .", "no association was found between house eave size and getting bitten by mosquitoes and between house eave size and frequency of bites ( table 2 ) . out of the 70 individuals who experienced mosquito bites 84.5% ( 60 ) indicated that they received the bites in the evening .", "the rest got mosquito bites during daytime ( 1.4% ( 1 ) ) , after dawn ( 1.4% ( 1 ) ) , or throughout the day ( 1.4% ( 1 ) ) .", "no association was found between house eave size and time of mosquito bites ( table 2 ) .", "about 93% ( 66 ) of respondents reported that they opened doors in the morning whilst 7% ( 5 ) during the day .", "ninety - seven point two percent ( 69 ) reported that they closed doors in the evening whilst only 1.4% ( 1 ) indicated that they closed doors after dawn and 1.4% ( 1 ) before dusk .", "eighty - eight point seven percent ( 63 ) mentioned that they were involved in late outdoor activities that included social gatherings ( 28.2% ( 20 ) ) , relaxing outdoors ( 74.6% ( 53 ) ) , preparing meals ( 50.7% ( 36 ) ) , and doing other things ( 8.5% ( 6 ) ) .", "a strong relationship was established between late outdoor activities and history of malaria episode ( table 3 ) .", "most of the outdoor activities were said to be done between 2000  hrs and 2200  hrs by 63.4% ( 45 ) of the respondents .", "a borderline association between the period spent outdoors and history of malaria episodes was established ( table 2 ) .", "ninety - three percent ( 93% ( 66 ) ( of the respondents mentioned that they prepared meals outdoors using firewood .", "only 2.8% ( 2 ) said they used gas for cooking and the remainder ( 4.2% ( 3 ) ) used both gas and firewood .", "eighty - five point nine percent ( 61 ) of the respondents mentioned that they were fulltime farmers . among them", "60.7% ( 37 ) had field structures erected on the farm and these were in the form of mud houses ( 28.2% ( 20 ) ) , brick houses ( 2.8% ( 2 ) ) , or other form of materials ( 21.1% ( 15 ) ) such as the grass - made temporary shelters locally known as the  mathibelo .", " there was no evidence of association between history of malaria episode and being a full time farmer ( table 2 ) .", "seventy - six point one percent ( 54 ) of the respondents indicated that they had other residential homesteads elsewhere outside tubu village .", "seventy - three point two percent ( 52 ) had other homesteads in gumare village , 1.4% ( 1 ) in the cattle post areas , and another 1.4% ( 1 ) in shakawe village , about 150  km from tubu village .", "it is defined as an area designated solely for keeping domestic animals , mainly cattle , goats , and a few donkeys and horses .", "cattle posts are usually located away from defined residential areas . majority ( 69.1% ( 49 ) ) of respondents", "fifty - nine point two percent ( 42 ) of respondents mentioned that they had travelled outside tubu village in the last 8 months .", "ninety - five point two percent ( 40 ) of them had travelled to malaria endemic areas in the north of the village whilst 4.8% ( 2 ) had travelled to non - malaria - endemic areas .", "there was a significant association between travel outside tubu village and history of malaria disease ( table 3 ) .", "twenty - six point eight percent ( 19 ) of respondents mentioned that 8 months prior to the date of the interview they had received visitors who stayed overnight .", "they reported that 94.7% ( 18 ) of the visitors were coming from malaria endemic areas .", "ninety - four point four percent ( 67 ) of the respondents indicated that they owned mosquito nets ( insecticide treated or untreated mosquito nets ) .", "more than half of the respondents who possessed mosquito nets had not experienced any malaria attack indicating an association between previous malaria episode and possession of mosquito nets ( table 3 ) .", "number of nets per household were reported to range from zero ( 2.8% ( 2 ) ) to more than six ( 5.6% ( 4 ) ) , with 39.4% ( 28 ) possessing one or two nets , 33.8% ( 24 ) possessing three or four nets , and 18.3% ( 13 ) possessing five or six nets . however , there was no association between history of malaria attack and the number of mosquito nets owned by a household ( table 2 ) .", "insecticide treated mosquito nets ( itns ) were possessed by 78.9% ( 56 ) of the responses whilst 50.7% ( 36 ) also possessed untreated mosquito nets .", "it was not unusual to find some households with a mixture of insecticide treated and untreated mosquito nets at the same time .", "two point eight percent ( 2 ) of the respondents were not sure of the status ( treated or not treated ) of their mosquito nets indicating poor knowledge on mosquito nets .", "ninety - one point five percent ( 65 ) of the respondents indicated that they always used mosquito nets .", "only 1.4% ( 1 ) mentioned that they used mosquito nets more often whilst 5.6% ( 4 ) sometimes used them and 1.4% ( 1 ) never used mosquito nets .", "usage of mosquito nets , among those who always used them , was reported to be relatively high ( 46.2% ( 30 ) ) during summer and very low ( 3.1% ( 2 ) ) in winter .", "most of the respondents were aware of government 's efforts to control malaria through indoor residual spraying ( irs ) ( 97.2% ( 69 ) ) and distribution of itns [ 97.2% ( 69 ) ] .", "only 2.8% ( 2 ) of the respondents were not aware of what the government did to control malaria in tubu village .", "twenty nine respondents got their mosquito nets from the town council ( figure 2 ) .", "other respondents acquired their mosquito nets from the shops ( 14 ) , both shops and town council ( 14 ) , both clinic and council ( 5 ) , clinic ( 4 ) , and both clinic and shops ( 3 ) .", "other personal malaria prevention methods reported as commonly practiced by the community included taking antimalarial drugs ( 1.4% ( 1 ) ) , clearing of surrounding vegetation ( 12.7% ( 9 ) ) , avoiding stagnant water ( 12.7% ( 9 ) ) , wearing long sleeved clothes ( 2.8% ( 2 ) ) , and use of aerosols ( 2.8% ( 2 ) ) and repellents [ 8.5% ( 6 ) ] .", "a total of 13 individuals ( 4 males and 9 females ) participated in the pra discussions . during the discussions", "this shortage resulted in some household members using emptied maize meal bags to construct  mosquito nets .", "however these self - made nets had disadvantages in that they did not kill mosquitoes since they were not treated with an insecticide .", "furthermore they were deemed uncomfortable as they tended to retain heat and could not fit well around modern beds . in winter", "( june - july ) , most households did not use the nets so often since mosquitoes were not a major problem during that time .", "the group was satisfied with the spraying exercise but expressed concern regarding coverage of areas inaccessible to the spraying teams due to flooding and also the fact that some households refused to have their homes sprayed claiming that the insecticide caused allergic reactions .", "mosquitoes were obtained from larval breeding ( 22 ) and adult sampling ( 64 ) . using the pcr species identification technique all the 86 specimens", "in addition other nonvector mosquitoes , namely , two an , demeilloni and one an .", "apart from anopheles species , more than 200 culicine mosquitoes were also caught resting either indoors or outdoors and breeding in the sites where the vector mosquitoes were found .", "forty - eight point four percent ( 31/64 ) of the anopheles arabiensis mosquitoes caught resting indoors in selected huts were blood - fed and at different stages of their gonotrophic cycle .", "mosquitoes were also observed resting outdoors in animal burrow pits located within the village and close to breeding sites . \n ", "these included vegetated areas at periphery of water bodies , animal hoof prints , open sunlit puddles created by receding floods , and man - made temporary puddles within farming fields .", "six homesteads were located within 100  m from mosquito breeding habitats , 17 were between 100  m and 500  m , 38 were between 500  m and 1000  m , and 10 were between 1000  m and 3000  m ( figure 3 ) .", "previous malaria episode was significantly associated with distance from water bodies ( table 2 ) .", "majority of our study population earned income far less than the botswana national poverty datum line pegged at us$81.94 ( bwp611.30 ) by the ministry of finance and development planning as of 22 april 2013 .", "this is consistent with the country statistics that show that about 44.8% of rural people ( 30.6% in towns ) in botswana are classified as poor .", "such communities have been found to be at greater risk of malaria disease because they are poorer and face higher transmission rates than their urban counterparts .", "malaria parasiteamia was associated with a reduced household socioeconomic status in a rural area in tanzania .", "social and economic factors aggravate the contribution of climate related factors in influencing the malaria burden . for many social and economic reasons the community in tubu village", "according to the world health organisation such sedentary communities living in malaria prone areas and who can not afford to move out of flood - affected areas due to low economic status have an increased chance of acquiring infection .", "this is particularly true for children , pregnant women , and older people , who form the high risk groups .", "other important factors that contributed to individual exposure to mosquito bites were the limited use of personal protection methods such as taking antimalaria tablets and use of aerosols and repellents .", "cost of these prevention tools may have been the limiting factor especially in a community regarded as leaving below the national poverty datum line .", "similar observations were made in nouma , burkina faso , where the use of aerosols was not a popular method perhaps because of the costs associated with such measures .", "minimum use of these simple measures not only affect individual exposure to mosquito bites but is also an indicator of very low socioeconomic status , which in itself has been proposed as an important factor associated with malaria .", "residential house status or structure has some implications in malaria transmission as poorly constructed houses expose individuals to mosquitoes that may find the dwelling more comfortable as a resting place .", "traditional huts / houses with large eave gaps present in tubu village played a role in allowing free movement in and out of the huts / houses and providing suitable shelter for mosquitoes that eventually attacked the bedroom occupants .", "previous studies have shown that traditional grass thatched houses with open eaves and lacking ceilings provided more favourable resting places for mosquitoes and put the occupants at risk of contracting malaria than houses with closed eaves , iron corrugated / asbestos covered roofs , and having ceilings . nevertheless , in poorly constructed houses", "where cooking and sleeping take place in the same room , smoke could repel mosquitoes through the eaves at the time that cooking is taking place [ 9 , 25 ] .", "however the community in tubu village did not benefit from the protective effects of smoke since most of the respondents prepared their meals outdoors .", "our findings indicated that the community was equally exposed to mosquito bites anytime of the night regardless of the size of eaves . therefore closing doors", "early in the evening did not provide any protection since mosquitoes could still freely enter the dwellings through the eaves as was evident from the anopheles and culicinemosquitoes caught resting indoors .", "arabiensis mosquitoes in the village verified that the community was at risk of being bitten by an infected vector mosquito .", "the fact that some study participants spent time in other homesteads away from tubu village did not make any difference since the structure of the dwellings there was the same and most of the alternative homesteads were in the same geographical area with similar climatic and environmental characteristics .", "most of the respondents who reported history of malaria attack also reported that they spent much of their night time outdoors during the peak biting period and therefore could have picked the infection during that period when they are not yet sleeping under a mosquito net .", "arabiensis could also expose individuals in the community who may not necessarily be involved in late outdoor activities .", "arabiensis has been shown to have early biting activities , which peak between 1900  hrs and 2000  hrs , with over 70% of biting activity occurring before 2200  hrs .", "the documented peak biting period coincided very well with the time that most of the individuals in tubu village would be involved in late outdoor activities since the vector mosquito is also known to bite humans both outdoors and indoors . \n ", "arabiensis is an opportunistic species that feeds preferentially on humans in many parts of africa but can be diverted to domestic animals as their density increases . in tubu village", "the presence and number of livestock near or within 3000  m from the homesteads could have some influence on the degree of malaria transmission at community and household levels .", "it is most likely that the risk of getting mosquito bites could have been reduced due to zooprophylaxis   especially in situations where the species predominantly displays zoophagic foraging tendencies .", "however , the benefit of keeping livestock close to human dwellings has been refuted by many authors from studies conducted in the gambia   and the ethiopian highlands .", "it is clear that the association between the presence of livestock as a risk factor and malaria transmission is a complex issue that needs further investigations in botswana , where the ratio of cattle to humans is very high .", "more than half of the respondents had travelled outside tubu village in the last 8 months , to other more intense malaria endemic areas .", "although active screening for malaria parasites was not done among the respondents , the responses to questions crafted to determine malaria risk implications of travel outside the study area indicated that travel to more intense malaria transmission areas exposed individuals to malaria . reported visitors from malaria endemic areas", "could also have played an important role in importing malaria parasites to the village . in a study conducted in urban kisumu , kenya ,", "children who reported spending at least one night per month in a rural area endemic to malaria were found to be at risk of contracting infections , indicating that exposure during rural travel was an important element of risk .", "the risks of acquiring malaria through travel were also observed in an urban area situated in the largest port of the pacific coast in columbia .", "insecticide treated nets ( itns ) have become quite significant as the most practical method of mosquito control by protecting at - risk individuals from mosquito bites and hence malaria infections . in this study participation of different organisations such as the council ,", "clinic ( ministry of health ) , and shops in supplying mosquito nets and the promotion done by the government could have resulted in overwhelming possession and use of mosquito nets by the community .", "similar findings were reported in nouma , burkina faso , and also in an urban area of burkina faso   where it was attributed to regional promotion . reported mosquito net use in tubu village was very high regardless of whether it is treated or not .", "the use of untreated mosquito nets has also been found to have some protective measures against mosquito bites [ 19 , 33 ] . in burkina faso", "mosquito net use was relatively more frequent in summer than winter , thereby conferring protection from mosquito bites during the peak malaria transmission period [ 22 , 32 ] . for an average family size of 6.8 , with some homesteads possessing as many as 6 nets ,", "it is most likely that each individual in the community had the opportunity to use a mosquito net .", "however , actual mosquito net use was not investigated and overreporting by the respondents was possible since ownership does not necessarily translate to actual use .", "innovativeness by the community in  mosquito net  making using waste maize - meal sacks has also been reported by the doane college which implemented the doane nets project in kenya whereby recycled plastic bags were converted into mosquito nets .", "since the quality of material used to make maize - bag  mosquito nets  was of major concern there is need for the ministry of health to promote the initiative and provide the community with the proper netting material for making more comfortable mosquito nets .", ", it shows that the community was aware of the protection given by nets and willing to contribute towards this initiative , thereby rendering the nets intervention more sustainable and not just waiting for handouts .", "irs , in addition to itns , is a mainstay of all national malaria control programmes in southern africa .", "irs is based on the assumption that mosquitoes feed and rest indoors , thereby coming in contact with the sprayed surfaces .", "mosquito survey conducted in tubu village showed that almost half of the vector mosquitoes caught resting indoors were blood - fed , an indication that the community was at risk of potentially infective mosquito bites .", "therefore , more should be done in terms of indoor residual spraying , to deter mosquitoes from seeking indoor shelter .", "in addition the ministry of health should devise strategies to cover those high risk areas for malaria transmission that are considered inaccessible due to flooding .", "although blood - meal analysis was not done , there is a high likelihood that some of the malaria vector mosquitoes might have fed from human beings , thus making the community vulnerable to potentially infective mosquitoes .", "this facilitated prompt treatment of malaria and other diseases . from similar observations made in ghana", "it was suggested that in such situations a higher health education standard could have an additional effect in reducing malaria disease .", "most of the homesteads were surrounded by low vegetation cover not ideal as resting places for mosquitoes . in general , the area does not support thick vegetation and probably this resulted in daytime resting mosquitoes taking shelter in animal burrow pits .", "incidentally , during the pra discussions , the community lamented increased activities of pests such as squirrels and porcupine which create burrow pits that have become convenient resting places for mosquitoes .", "the location of homesteads relative to mosquito breeding habitats was one of the major risk factors for malaria transmission .", "arabiensis infested water bodies , a distance considered to be within the general flight range of most mosquito species .", "such closeness of homesteads from breeding sites , in addition to the presence of animal burrow pits within the village , made the human population vulnerable to mosquito bites .", "year - round sampling through the major bep project established that larval breeding in tubu village took place throughout the year in a variety of habitats created interchangeably by either rainfall or floods ( bep fifth report , 2012 ) , thus exposing the community to year - round mosquito bites .", "the presence of culicine mosquitoes breeding in the same habitats exacerbated the problem of mosquito biting , though the species were of no medical importance in botswana .", "abundance of larvae and larval habitats and proximity of humans to larval habitats have been found to correlate very well with vector mosquito biting rates .", "close proximity of homesteads to vector mosquito breeding sites increased the risk of malaria in many countries including uganda   and ethiopia .", "the ministry of health changed its policy on malaria case management and recording in botswana .", "as from october 2010 to date all suspected malaria cases are not handled at any clinic , tubu included , but promptly referred to gumare district hospital .", "all recordings are done at central / district hospital . retrieving information from the district attendance register of patients originating from tubu village", "was not possible because cases were pooled together onto one district register , with other patients presenting with other diseases .", "furthermore the outcome on malaria patients referred to gumare district hospital is never conveyed back to the clinic of origin . as a result of these anomalies ,", "there is a possibility of both over- or underreporting of malaria episodes by the respondents .", "however a previous study among the same community / individuals showed that the community was well aware of the disease ( 95.6% ) in terms of signs and symptoms ( 88.7% ) and prevention measures ( 98.6% ) and 100% of the respondents who had suspected a malaria attack sought medical treatment at the clinic .", "because of the small sample and population sizes of the study area , the results can only be extrapolated to other villages with similar demographic , ecological , and environmental characteristics and can not be generalized to all malaria prone villages in botswana .", "similar case studies in different malaria prone settings in botswana should be undertaken for comparative purposes .", "in conclusion , socioeconomic status , exposure to mosquito bites through individual nocturnal outdoor activities , and mobility of the community were identified as high risk factors for malaria transmission in tubu village .", "limited use of malaria protective measures such as insecticide treated nets , house structure ( traditional or modern ) , and close location of homesteads in relation to breeding sites exposed individuals to mosquito bites .", "however the number of mosquito nets possessed in each household and the farming status were not considered as risk factors for malaria .", "hut / house eaves used as bedrooms by the respondents did not have any influence on the level , time , and frequency of mosquito bites .", "the overwhelming use of mosquito nets treated , untreated , or home - made proved to be a viable tool for malaria control in the village .", "satisfactory service provided at and the close proximity of the health facility were added advantages to the community .", "furthermore the community benefited from the low vegetation cover surrounding their homesteads , which did not provide enough shelter for even nuisance mosquitoes .", "it is recommended that the ministry of health should develop a health education package encompassing some of the risk factors identified in this study and other indicated similar studies to be conducted in different settings . from our findings the most important issues to be addressed through the health education package include behavioural changes during nocturnal outdoor activities , ways of protecting oneself during visits to other malaria endemic areas , advice on the ideal location of homesteads in relation to mosquito sources , and promoting the use of insecticide treated eave curtains .", "ways of promoting household based control methods , based on these specific risk factors , should be devised .", "the ministry of health should support community initiatives in mosquito net making , in addition to strengthening irs activities in inaccessible flood prone areas of the delta .", "future research should comprehensively look at the role of other potential risk factors , such as zooprophylaxis and firewood smoke , in malaria transmission and on the possibility of incorporating the benefits derived , if any , into community based malaria control initiatives ."], "abstract_text": ["<S> \n this study investigated potential risk factors associated with malaria transmission in tubu village , okavango subdistrict , a malaria endemic area in northern botswana . </S>", "<S> data was derived from a census questionnaire survey , participatory rural appraisal workshop , field observations , and mosquito surveys . </S>", "<S> history of malaria episodes was associated with several factors : household income ( p < 0.05 ) , late outdoor activities ( or = 7.016 ; ci = 1.78627.559 ) , time spent outdoors ( p = 0.051 ) , travel outside study area ( or = 2.70 ; ci = 1.0047.260 ) , nonpossession of insecticide treated nets ( or = 0.892 ; ci = 0.7970.998 ) , hut / house structure ( or = 11.781 ; ci = 3.86835.885 ) , and homestead location from water bodies ( p < 0.05 ) . </S>", "<S> no associations were established between history of malaria episodes and the following factors : being a farmer ( p > 0.05 ) and number of nets possessed ( p > 0.05 ) . </S>", "<S> eave size was not associated with mosquito bites ( p > 0.05 ) , frequency of mosquito bites ( p > 0.05 ) , and time of mosquito bites ( p > 0.05 ) . </S>", "<S> possession of nets was very high ( 94.7% ) . </S>", "<S> close proximity of a health facility and low vegetation cover were added advantages . </S>", "<S> some of the identified risk factors are important for developing effective control and elimination strategies involving the community , with limited resources . \n </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Limitations of the Study", "6. Conclusions"], "sections": [["understanding how and why vector - borne diseases like malaria remain a persistent problem despite being tools for diagnosis and treatment is essential for developing effective control measures for sub - saharan africa .", "temperature is one of the key climatic variables that determine the range of malaria transmission and hence global warming is likely to result in an increase in malaria prone areas especially where temperatures have generally been lower than the optimal range of 2527c for mosquito development .", "furthermore degradation of the environment and social and economic pressures due to population growth may boost the expansion of malaria prone areas .", "population migrations , drug and pesticide resistance , and deterioration of health service delivery systems will also influence the level of malaria transmission .", "the epidemiology of malaria is very complex , involving factors pertaining to the malaria parasites , the insect vectors , the human hosts , and the environment .", "an understanding of the link between malaria transmission , climatic variables , and other human related factors is therefore necessary for developing appropriate measures that will significantly reduce transmission and perhaps eliminate malaria in endemic areas . in most cases these human related risk factors", "the level of risk to human populations living in malaria endemic areas varies markedly across continents and also within countries and different areas within the same countries .", "several studies have shown that malaria vector distribution , transmission rates , and incidence can vary widely over short distances , between neighbouring villages and even within a single settlement , as a result of small area variations in risk factors [ 5 , 6 ] .", "identification and understanding of this variation are important in the detection of high risk groups and for selective targeting of intervention .", "many studies have attempted to identify household and individual level factors associated with malaria . some of the factors studied include access to health facilities , type of housing that people live in , proximity of human settlements to vector breeding sites [ 1012 ] , vector abundance , socioeconomic status , gender , occupation , residential mobility , travel , presence of domestic animals near homesteads , and use of preventive methods such as bed nets .", "information on how these factors interact to expose communities and individuals to malaria infections needs to be investigated systematically in each geographical setting , in conjunction with climate related factors .", "documented information about individual and household risk factors associated with malaria transmission is lacking in botswana . in order to address this paucity of information we assessed household and individual risk factors that may be contributing to malaria transmission in tubu village community in the okavango subdistrict in northern botswana .", "the study was conducted in the context of a larger project , botswana ecohealth project ( bep ) , which is investigating the impacts of hydroclimate change on population health . apart from contributing towards the objectives of bep ,", "the results of this study are relevant to an initiative by the ministry of health in botswana to eliminate malaria by year 2016 ."], ["the study was conducted in tubu village on the banks of the thaoge river , one of the distributaries of the okavango delta .", "tubu village is located in the okavango subdistrict at an altitude of about 950  m above sea level and between latitude 1935s and longitude 2227e . according to the national central statistics office report of 2011", "the area is subjected to annual flooding but the extent of flooding varies from year to year . the community practices flood recession farming locally known as  molapo  farming .", "they plough their fields along the banks of the river as the flood recedes and in some instances they plant crops taking advantage of a raised water table caused by flooding .", "community development and everyday governance issues pertaining to law and order in the area are conducted at a central place ( locally known as the  kgotla  ) within the village .", "tubu has a village development committee ( vdc ) established as a local government structure to guide development planning and implementation in the village .", "the village also has a primary school , a resident social worker , and a health post staffed with a nurse , nurse aid , and a health education assistant .", "gumare hospital , located 10  km east of the village , offers an array of general health services to the local community .", "the hospital also serves as the district health centre for critically ill patients referred from all district health posts including tubu .", "malaria cases in the okavango subdistrict have been fluctuating since 2005 with unconfirmed cases ranging between 4,686 in 2005 and 10,993 in 2006 and confirmed cases ranging between five ( in 2012 ) and 791 ( in 2006 ) ( ministry of health annual report , 2012 ) .", "the maximum number of deaths between 2005 and 2012 was 16 , recorded in 2006 .", "data from clinic records for the period of 2005 to 2010 indicated that 2009 had the highest with 131 unconfirmed cases and 2005 the lowest with 50 cases .", "however during the same period information on rapid diagnostic tests ( rdts ) was inconsistent and available only for years 2006 , 2008 , and 2010 with 9 , 4 , and 5 cases , respectively .", "pretesting of the questionnaire involved trained field research assistants with higher secondary education qualifications and field supervisors with degree qualifications who eventually administered the final questionnaire .", "the questionnaire had four thematic sections ; first part focused on sociodemographic characteristics such as gender , age , marital status , education level , farming practice , household income , ethnicity , respondent 's relationship to household head , employment status , and occupation and the second part included aspects on how the community or individuals got exposed to mosquito bites due to late night activities , location of homesteads in relation to animal shelters and mosquito breeding sites , visits made to other areas outside tubu village in the last 8 months , and history of malaria episodes in the last 8 months .", "a short period of 8 months was chosen so as to minimise on - recall bias as the time frame included the previous malaria transmission season which fell between the months of october 2011 and june 2012 .", "the third part of the questionnaire focused on malaria prevention methods being practiced and health delivery services for the study area .", "the last part involved general observations made at each homestead during the interview with regard to house structure and use , type of eaves , and vegetation cover surrounding the homesteads . to ensure accuracy and good quality data , pretesting of questionnaires", "was carried out two weeks prior to the actual data collection exercise in etsha 1 , a village with similar environmental and sociodemographic patterns to tubu village .", "following the pretest survey the questionnaire was revised , on the basis of responses given , for improvement of clarity and addition of essential questions that had been omitted in the original questionnaire .", "vague questions in the original questionnaire were either omitted from the final questionnaire or improved .", "the pretest exercise was also used to standardize the manner in which the interviewers would conduct the interviews and to ensure that they had a common understanding of each question . from the household listing compiled for the bep project the target sample size , as determined by raosoft inc .", "however , due to the fact that tubu is a relatively small village a census survey was done consisting of individuals representing all the 71 households .", "the questionnaire was administered at the household level to any person who is 18 years and above with preference being given to the heads of the household if they were present .", "only one person per household was interviewed . during the interviews the respondents answered some of the questions pertaining to children and other members of the same household such as visits outside study area and possession and use of nets .", "numbers 1 to 4 were assigned to individuals from the community who had come to attend the pras for the major bep project .", "all individuals assigned a similar number were grouped together to participate in the malaria risk survey .", "pra technique , using the closed scoring / ranking approach , was used to cross - check on some of the responses from the questionnaire interviews .", "the maximum number of individuals at any one given time during the pra discussions was 16 , thus providing a total ranking capacity ( n ) of 160 .", "the respondents were asked whether they had suffered from malaria within the past 8 months ( the period between october 2011 and june 2012 ) , which included the traditional peak malaria transmission season in botswana .", "mosquito sampling was conducted in houses , in pit traps , and in larval breeding sites located within the study area .", "adult mosquitoes were sampled from pyrethrum space spray knockdowns in 6 selected huts used as bedrooms and from 4 pit traps .", "the physiological conditions of the adult mosquitoes collected from indoors and pit traps were scored as non - blood - fed , blood - fed , half gravid , and full gravid . all adult mosquito specimens , from both adult and larval sampling , were identified to species level by the polymerase chain reaction ( pcr ) technique . a geographic positioning system instrument ( gps )", "was used to determine straight line distances of study homesteads from the nearest mosquito breeding habitats .", "permission to carry out the study was obtained from the ministry of health , republic of botswana ( permit number : ppme 13/18/1 vol .", "meetings with the community leadership and other interested community members were held to introduce the study , clearly explaining its objectives . on the day of the interview verbal consent", "respect for privacy was maintained during adult mosquito sampling in selected rooms used as bedrooms .", "participants were informed that they could refuse to participate in the study and not be prejudiced .", "respondents were assured that all the information captured on the data sheets would be treated with confidentiality and that no answers would be linked to individuals in the final analysis and presentation of findings .", "descriptive statistics were carried out to determine relative frequencies of all the survey variables . some of the variables had provisions for multiple responses .", "appropriate graphs and tables were generated to show differences in the relative frequencies of various variables .", "levels of association between various variables were determined by the pearson  test and fisher 's exact test in situations where the expected frequencies were less than five . where appropriate , p values and confidence intervals ( ci ) for odds ratios ( or ) are shown .", "the level of association between various factors and history of malaria was calculated based on the respondents ' views ."], ["the maximum number of people residing in a single homestead was 20 and the minimum was one .", "a total of 483 individuals resided in all the 71 study homesteads , thus giving an average household size of seven people .", "about two - thirds of the respondents had some years of schooling ( table 1 ) .", "the majority of households earned an income of less than us$63 per month ( figure 1 ) .", "only 7% of the respondents earned more than us$250 per month . eighty - one point seven percent ( 58 ) of the respondents resided within 1  km from the nearest health facility .", "eighteen - point three percent ( 13 ) resided 1  km to 2  km from the clinic .", "all the 71 respondents indicated that it took them less than an hour to get service at the clinic from the time they joined the queue . at the time of the study ( june 2012 ) 53.5% ( 38 ) of the respondents said that they had suffered from malaria within the past 8 months whilst 46.5% ( 33 ) did not remember ever suffering from malaria in their entire life .", "forty - seven point nine percent ( 34 ) mentioned that at least one member of their family had suffered from malaria within the past 8 months . among family members who had suffered from malaria ,", "73.5% ( 25 ) were above five years old and 26.5% were below the age of five years .", "there was an association between household income and history of a malaria episode ( table 2 ) .", "almost all of those who had experienced a malaria attack were in the lowest bracket for household monthly income . in the whole village", "only two house roof types were observed . these were either of grass or iron sheet .", "a general observation noted that houses in the village were either grass thatched , built of reeds , pole , and mud or home - made bricks ( referred to as traditional hut / house ) or built of bricks and roofed with iron sheets ( referred to as modern hut / house ) . among those structures used as bedrooms by the respondents , 52.1% ( 37 ) were traditional and 47.9% ( 34 ) were modern houses / huts . in traditional houses / huts large eave openings", "were observed in 89.2% ( 33 ) and the rest ( 10.8% ( 4 ) ) had small eave openings . in modern houses 2.9% ( 1 ) had large eave openings and the rest ( 97.1% ( 33 ) ) had virtually no eave openings .", "there was an association between history of malaria episode and use of traditional huts / houses as bedrooms ( table 3 ) .", "majority of individuals who experienced a malaria attack used traditional houses / huts as bedrooms .", "low vegetation cover surrounding homesteads was observed at 81.7% ( 58 ) of the homesteads .", "moderate vegetation cover was observed at 14.1% ( 10 ) of the homesteads whilst only 4.2% ( 3 ) of the homesteads were surrounded by dense vegetation cover .", "individual contact with mosquito bites was categorized into  always ,   sometimes ,   often ,  and  rarely .  out of 98.6% ( 70 ) who experienced mosquito bites , 54.9% ( 39 ) mentioned that they ", "seven percent ( 5 ) and 8.5% ( 6 )  often  and  rarely  got bitten by mosquitoes , respectively .", "no association was found between house eave size and getting bitten by mosquitoes and between house eave size and frequency of bites ( table 2 ) . out of the 70 individuals who experienced mosquito bites 84.5% ( 60 ) indicated that they received the bites in the evening .", "the rest got mosquito bites during daytime ( 1.4% ( 1 ) ) , after dawn ( 1.4% ( 1 ) ) , or throughout the day ( 1.4% ( 1 ) ) .", "no association was found between house eave size and time of mosquito bites ( table 2 ) .", "about 93% ( 66 ) of respondents reported that they opened doors in the morning whilst 7% ( 5 ) during the day .", "ninety - seven point two percent ( 69 ) reported that they closed doors in the evening whilst only 1.4% ( 1 ) indicated that they closed doors after dawn and 1.4% ( 1 ) before dusk .", "eighty - eight point seven percent ( 63 ) mentioned that they were involved in late outdoor activities that included social gatherings ( 28.2% ( 20 ) ) , relaxing outdoors ( 74.6% ( 53 ) ) , preparing meals ( 50.7% ( 36 ) ) , and doing other things ( 8.5% ( 6 ) ) .", "a strong relationship was established between late outdoor activities and history of malaria episode ( table 3 ) .", "most of the outdoor activities were said to be done between 2000  hrs and 2200  hrs by 63.4% ( 45 ) of the respondents .", "a borderline association between the period spent outdoors and history of malaria episodes was established ( table 2 ) .", "ninety - three percent ( 93% ( 66 ) ( of the respondents mentioned that they prepared meals outdoors using firewood .", "only 2.8% ( 2 ) said they used gas for cooking and the remainder ( 4.2% ( 3 ) ) used both gas and firewood .", "eighty - five point nine percent ( 61 ) of the respondents mentioned that they were fulltime farmers . among them", "60.7% ( 37 ) had field structures erected on the farm and these were in the form of mud houses ( 28.2% ( 20 ) ) , brick houses ( 2.8% ( 2 ) ) , or other form of materials ( 21.1% ( 15 ) ) such as the grass - made temporary shelters locally known as the  mathibelo .", " there was no evidence of association between history of malaria episode and being a full time farmer ( table 2 ) .", "seventy - six point one percent ( 54 ) of the respondents indicated that they had other residential homesteads elsewhere outside tubu village .", "seventy - three point two percent ( 52 ) had other homesteads in gumare village , 1.4% ( 1 ) in the cattle post areas , and another 1.4% ( 1 ) in shakawe village , about 150  km from tubu village .", "it is defined as an area designated solely for keeping domestic animals , mainly cattle , goats , and a few donkeys and horses .", "cattle posts are usually located away from defined residential areas . majority ( 69.1% ( 49 ) ) of respondents", "fifty - nine point two percent ( 42 ) of respondents mentioned that they had travelled outside tubu village in the last 8 months .", "ninety - five point two percent ( 40 ) of them had travelled to malaria endemic areas in the north of the village whilst 4.8% ( 2 ) had travelled to non - malaria - endemic areas .", "there was a significant association between travel outside tubu village and history of malaria disease ( table 3 ) .", "twenty - six point eight percent ( 19 ) of respondents mentioned that 8 months prior to the date of the interview they had received visitors who stayed overnight .", "they reported that 94.7% ( 18 ) of the visitors were coming from malaria endemic areas .", "ninety - four point four percent ( 67 ) of the respondents indicated that they owned mosquito nets ( insecticide treated or untreated mosquito nets ) .", "more than half of the respondents who possessed mosquito nets had not experienced any malaria attack indicating an association between previous malaria episode and possession of mosquito nets ( table 3 ) .", "number of nets per household were reported to range from zero ( 2.8% ( 2 ) ) to more than six ( 5.6% ( 4 ) ) , with 39.4% ( 28 ) possessing one or two nets , 33.8% ( 24 ) possessing three or four nets , and 18.3% ( 13 ) possessing five or six nets . however , there was no association between history of malaria attack and the number of mosquito nets owned by a household ( table 2 ) .", "insecticide treated mosquito nets ( itns ) were possessed by 78.9% ( 56 ) of the responses whilst 50.7% ( 36 ) also possessed untreated mosquito nets .", "it was not unusual to find some households with a mixture of insecticide treated and untreated mosquito nets at the same time .", "two point eight percent ( 2 ) of the respondents were not sure of the status ( treated or not treated ) of their mosquito nets indicating poor knowledge on mosquito nets .", "ninety - one point five percent ( 65 ) of the respondents indicated that they always used mosquito nets .", "only 1.4% ( 1 ) mentioned that they used mosquito nets more often whilst 5.6% ( 4 ) sometimes used them and 1.4% ( 1 ) never used mosquito nets .", "usage of mosquito nets , among those who always used them , was reported to be relatively high ( 46.2% ( 30 ) ) during summer and very low ( 3.1% ( 2 ) ) in winter .", "most of the respondents were aware of government 's efforts to control malaria through indoor residual spraying ( irs ) ( 97.2% ( 69 ) ) and distribution of itns [ 97.2% ( 69 ) ] .", "only 2.8% ( 2 ) of the respondents were not aware of what the government did to control malaria in tubu village .", "twenty nine respondents got their mosquito nets from the town council ( figure 2 ) .", "other respondents acquired their mosquito nets from the shops ( 14 ) , both shops and town council ( 14 ) , both clinic and council ( 5 ) , clinic ( 4 ) , and both clinic and shops ( 3 ) .", "other personal malaria prevention methods reported as commonly practiced by the community included taking antimalarial drugs ( 1.4% ( 1 ) ) , clearing of surrounding vegetation ( 12.7% ( 9 ) ) , avoiding stagnant water ( 12.7% ( 9 ) ) , wearing long sleeved clothes ( 2.8% ( 2 ) ) , and use of aerosols ( 2.8% ( 2 ) ) and repellents [ 8.5% ( 6 ) ] .", "a total of 13 individuals ( 4 males and 9 females ) participated in the pra discussions . during the discussions", "this shortage resulted in some household members using emptied maize meal bags to construct  mosquito nets .", "however these self - made nets had disadvantages in that they did not kill mosquitoes since they were not treated with an insecticide .", "furthermore they were deemed uncomfortable as they tended to retain heat and could not fit well around modern beds . in winter", "( june - july ) , most households did not use the nets so often since mosquitoes were not a major problem during that time .", "the group was satisfied with the spraying exercise but expressed concern regarding coverage of areas inaccessible to the spraying teams due to flooding and also the fact that some households refused to have their homes sprayed claiming that the insecticide caused allergic reactions .", "mosquitoes were obtained from larval breeding ( 22 ) and adult sampling ( 64 ) . using the pcr species identification technique all the 86 specimens", "in addition other nonvector mosquitoes , namely , two an , demeilloni and one an .", "apart from anopheles species , more than 200 culicine mosquitoes were also caught resting either indoors or outdoors and breeding in the sites where the vector mosquitoes were found .", "forty - eight point four percent ( 31/64 ) of the anopheles arabiensis mosquitoes caught resting indoors in selected huts were blood - fed and at different stages of their gonotrophic cycle .", "mosquitoes were also observed resting outdoors in animal burrow pits located within the village and close to breeding sites . \n ", "these included vegetated areas at periphery of water bodies , animal hoof prints , open sunlit puddles created by receding floods , and man - made temporary puddles within farming fields .", "six homesteads were located within 100  m from mosquito breeding habitats , 17 were between 100  m and 500  m , 38 were between 500  m and 1000  m , and 10 were between 1000  m and 3000  m ( figure 3 ) .", "previous malaria episode was significantly associated with distance from water bodies ( table 2 ) ."], ["majority of our study population earned income far less than the botswana national poverty datum line pegged at us$81.94 ( bwp611.30 ) by the ministry of finance and development planning as of 22 april 2013 .", "this is consistent with the country statistics that show that about 44.8% of rural people ( 30.6% in towns ) in botswana are classified as poor .", "such communities have been found to be at greater risk of malaria disease because they are poorer and face higher transmission rates than their urban counterparts .", "malaria parasiteamia was associated with a reduced household socioeconomic status in a rural area in tanzania .", "social and economic factors aggravate the contribution of climate related factors in influencing the malaria burden . for many social and economic reasons the community in tubu village", "according to the world health organisation such sedentary communities living in malaria prone areas and who can not afford to move out of flood - affected areas due to low economic status have an increased chance of acquiring infection .", "this is particularly true for children , pregnant women , and older people , who form the high risk groups .", "other important factors that contributed to individual exposure to mosquito bites were the limited use of personal protection methods such as taking antimalaria tablets and use of aerosols and repellents .", "cost of these prevention tools may have been the limiting factor especially in a community regarded as leaving below the national poverty datum line .", "similar observations were made in nouma , burkina faso , where the use of aerosols was not a popular method perhaps because of the costs associated with such measures .", "minimum use of these simple measures not only affect individual exposure to mosquito bites but is also an indicator of very low socioeconomic status , which in itself has been proposed as an important factor associated with malaria .", "residential house status or structure has some implications in malaria transmission as poorly constructed houses expose individuals to mosquitoes that may find the dwelling more comfortable as a resting place .", "traditional huts / houses with large eave gaps present in tubu village played a role in allowing free movement in and out of the huts / houses and providing suitable shelter for mosquitoes that eventually attacked the bedroom occupants .", "previous studies have shown that traditional grass thatched houses with open eaves and lacking ceilings provided more favourable resting places for mosquitoes and put the occupants at risk of contracting malaria than houses with closed eaves , iron corrugated / asbestos covered roofs , and having ceilings . nevertheless , in poorly constructed houses", "where cooking and sleeping take place in the same room , smoke could repel mosquitoes through the eaves at the time that cooking is taking place [ 9 , 25 ] .", "however the community in tubu village did not benefit from the protective effects of smoke since most of the respondents prepared their meals outdoors .", "our findings indicated that the community was equally exposed to mosquito bites anytime of the night regardless of the size of eaves . therefore closing doors", "early in the evening did not provide any protection since mosquitoes could still freely enter the dwellings through the eaves as was evident from the anopheles and culicinemosquitoes caught resting indoors .", "arabiensis mosquitoes in the village verified that the community was at risk of being bitten by an infected vector mosquito .", "the fact that some study participants spent time in other homesteads away from tubu village did not make any difference since the structure of the dwellings there was the same and most of the alternative homesteads were in the same geographical area with similar climatic and environmental characteristics .", "most of the respondents who reported history of malaria attack also reported that they spent much of their night time outdoors during the peak biting period and therefore could have picked the infection during that period when they are not yet sleeping under a mosquito net .", "arabiensis could also expose individuals in the community who may not necessarily be involved in late outdoor activities .", "arabiensis has been shown to have early biting activities , which peak between 1900  hrs and 2000  hrs , with over 70% of biting activity occurring before 2200  hrs .", "the documented peak biting period coincided very well with the time that most of the individuals in tubu village would be involved in late outdoor activities since the vector mosquito is also known to bite humans both outdoors and indoors . \n ", "arabiensis is an opportunistic species that feeds preferentially on humans in many parts of africa but can be diverted to domestic animals as their density increases . in tubu village", "the presence and number of livestock near or within 3000  m from the homesteads could have some influence on the degree of malaria transmission at community and household levels .", "it is most likely that the risk of getting mosquito bites could have been reduced due to zooprophylaxis   especially in situations where the species predominantly displays zoophagic foraging tendencies .", "however , the benefit of keeping livestock close to human dwellings has been refuted by many authors from studies conducted in the gambia   and the ethiopian highlands .", "it is clear that the association between the presence of livestock as a risk factor and malaria transmission is a complex issue that needs further investigations in botswana , where the ratio of cattle to humans is very high .", "more than half of the respondents had travelled outside tubu village in the last 8 months , to other more intense malaria endemic areas .", "although active screening for malaria parasites was not done among the respondents , the responses to questions crafted to determine malaria risk implications of travel outside the study area indicated that travel to more intense malaria transmission areas exposed individuals to malaria . reported visitors from malaria endemic areas", "could also have played an important role in importing malaria parasites to the village . in a study conducted in urban kisumu , kenya ,", "children who reported spending at least one night per month in a rural area endemic to malaria were found to be at risk of contracting infections , indicating that exposure during rural travel was an important element of risk .", "the risks of acquiring malaria through travel were also observed in an urban area situated in the largest port of the pacific coast in columbia .", "insecticide treated nets ( itns ) have become quite significant as the most practical method of mosquito control by protecting at - risk individuals from mosquito bites and hence malaria infections . in this study participation of different organisations such as the council ,", "clinic ( ministry of health ) , and shops in supplying mosquito nets and the promotion done by the government could have resulted in overwhelming possession and use of mosquito nets by the community .", "similar findings were reported in nouma , burkina faso , and also in an urban area of burkina faso   where it was attributed to regional promotion . reported mosquito net use in tubu village was very high regardless of whether it is treated or not .", "the use of untreated mosquito nets has also been found to have some protective measures against mosquito bites [ 19 , 33 ] . in burkina faso", "mosquito net use was relatively more frequent in summer than winter , thereby conferring protection from mosquito bites during the peak malaria transmission period [ 22 , 32 ] . for an average family size of 6.8 , with some homesteads possessing as many as 6 nets ,", "it is most likely that each individual in the community had the opportunity to use a mosquito net .", "however , actual mosquito net use was not investigated and overreporting by the respondents was possible since ownership does not necessarily translate to actual use .", "innovativeness by the community in  mosquito net  making using waste maize - meal sacks has also been reported by the doane college which implemented the doane nets project in kenya whereby recycled plastic bags were converted into mosquito nets .", "since the quality of material used to make maize - bag  mosquito nets  was of major concern there is need for the ministry of health to promote the initiative and provide the community with the proper netting material for making more comfortable mosquito nets .", ", it shows that the community was aware of the protection given by nets and willing to contribute towards this initiative , thereby rendering the nets intervention more sustainable and not just waiting for handouts .", "irs , in addition to itns , is a mainstay of all national malaria control programmes in southern africa .", "irs is based on the assumption that mosquitoes feed and rest indoors , thereby coming in contact with the sprayed surfaces .", "mosquito survey conducted in tubu village showed that almost half of the vector mosquitoes caught resting indoors were blood - fed , an indication that the community was at risk of potentially infective mosquito bites .", "therefore , more should be done in terms of indoor residual spraying , to deter mosquitoes from seeking indoor shelter .", "in addition the ministry of health should devise strategies to cover those high risk areas for malaria transmission that are considered inaccessible due to flooding .", "although blood - meal analysis was not done , there is a high likelihood that some of the malaria vector mosquitoes might have fed from human beings , thus making the community vulnerable to potentially infective mosquitoes .", "this facilitated prompt treatment of malaria and other diseases . from similar observations made in ghana", "it was suggested that in such situations a higher health education standard could have an additional effect in reducing malaria disease .", "most of the homesteads were surrounded by low vegetation cover not ideal as resting places for mosquitoes . in general , the area does not support thick vegetation and probably this resulted in daytime resting mosquitoes taking shelter in animal burrow pits .", "incidentally , during the pra discussions , the community lamented increased activities of pests such as squirrels and porcupine which create burrow pits that have become convenient resting places for mosquitoes .", "the location of homesteads relative to mosquito breeding habitats was one of the major risk factors for malaria transmission .", "arabiensis infested water bodies , a distance considered to be within the general flight range of most mosquito species .", "such closeness of homesteads from breeding sites , in addition to the presence of animal burrow pits within the village , made the human population vulnerable to mosquito bites .", "year - round sampling through the major bep project established that larval breeding in tubu village took place throughout the year in a variety of habitats created interchangeably by either rainfall or floods ( bep fifth report , 2012 ) , thus exposing the community to year - round mosquito bites .", "the presence of culicine mosquitoes breeding in the same habitats exacerbated the problem of mosquito biting , though the species were of no medical importance in botswana .", "abundance of larvae and larval habitats and proximity of humans to larval habitats have been found to correlate very well with vector mosquito biting rates .", "close proximity of homesteads to vector mosquito breeding sites increased the risk of malaria in many countries including uganda   and ethiopia ."], ["the ministry of health changed its policy on malaria case management and recording in botswana .", "as from october 2010 to date all suspected malaria cases are not handled at any clinic , tubu included , but promptly referred to gumare district hospital .", "all recordings are done at central / district hospital . retrieving information from the district attendance register of patients originating from tubu village", "was not possible because cases were pooled together onto one district register , with other patients presenting with other diseases .", "furthermore the outcome on malaria patients referred to gumare district hospital is never conveyed back to the clinic of origin . as a result of these anomalies ,", "there is a possibility of both over- or underreporting of malaria episodes by the respondents .", "however a previous study among the same community / individuals showed that the community was well aware of the disease ( 95.6% ) in terms of signs and symptoms ( 88.7% ) and prevention measures ( 98.6% ) and 100% of the respondents who had suspected a malaria attack sought medical treatment at the clinic .", "because of the small sample and population sizes of the study area , the results can only be extrapolated to other villages with similar demographic , ecological , and environmental characteristics and can not be generalized to all malaria prone villages in botswana .", "similar case studies in different malaria prone settings in botswana should be undertaken for comparative purposes ."], ["in conclusion , socioeconomic status , exposure to mosquito bites through individual nocturnal outdoor activities , and mobility of the community were identified as high risk factors for malaria transmission in tubu village .", "limited use of malaria protective measures such as insecticide treated nets , house structure ( traditional or modern ) , and close location of homesteads in relation to breeding sites exposed individuals to mosquito bites .", "however the number of mosquito nets possessed in each household and the farming status were not considered as risk factors for malaria .", "hut / house eaves used as bedrooms by the respondents did not have any influence on the level , time , and frequency of mosquito bites .", "the overwhelming use of mosquito nets treated , untreated , or home - made proved to be a viable tool for malaria control in the village .", "satisfactory service provided at and the close proximity of the health facility were added advantages to the community .", "furthermore the community benefited from the low vegetation cover surrounding their homesteads , which did not provide enough shelter for even nuisance mosquitoes .", "it is recommended that the ministry of health should develop a health education package encompassing some of the risk factors identified in this study and other indicated similar studies to be conducted in different settings . from our findings the most important issues to be addressed through the health education package include behavioural changes during nocturnal outdoor activities , ways of protecting oneself during visits to other malaria endemic areas , advice on the ideal location of homesteads in relation to mosquito sources , and promoting the use of insecticide treated eave curtains .", "ways of promoting household based control methods , based on these specific risk factors , should be devised .", "the ministry of health should support community initiatives in mosquito net making , in addition to strengthening irs activities in inaccessible flood prone areas of the delta .", "future research should comprehensively look at the role of other potential risk factors , such as zooprophylaxis and firewood smoke , in malaria transmission and on the possibility of incorporating the benefits derived , if any , into community based malaria control initiatives ."]]}
{"article_id": "PMC3401615", "article_text": ["the prevalence of pancreatic cystic neoplasms ( pcns ) is around 2.5%.1,2 pancreatic cysts are increasingly discovered probably because of the ubiquitous presence of multi - detector computed tomography ( ct ) scans and their increased use to evaluate patients with abdominal complaints .", "endoscopic ultrasound ( eus ) plays a pivotal role in the evaluation of patients with pancreatic cysts but eus and cross - sectional imaging alone have proven to be inaccurate in identifying the exact nature of the cysts .", "there is inadequate information on the natural history of pancreatic cysts but our knowledge base can not be expanded without being able to determine the exact nature of the cyst noninvasively . toward this end ,", "eus with fine needle aspiration ( fna ) along with cyst fluid analysis has been advocated to improve the utility of eus in the diagnosis of pancreatic cysts .", "size > 3 cm and/or the presence of mural nodules appear to be the best indicators of malignant change in patients with the side branch form of intraductal papillary mucinous neoplasm ( sb - ipmn ) and mucinous cystic neoplasms ( mcn).3 eus with cyst fluid aspiration and/or fna can play a role in differentiating pcns .", "this paper will review the role of eus - fna in the evaluation of pcns .", "if the cysts are incidental ( the patient has no symptoms referable to the pancreas ) , the great majority are determined to be neoplastic.4 in one study , the majority of incidentally detected pancreatic cysts ( 58% ) were mucinous and therefore had some malignant potential.4 these data suggest that all incidentally found pancreatic cysts should undergo further evaluation .", "serous cyst adenomas consist of multiple \" micro cysts \" with thin septae coursing through the lesion .", "however , 20% have a dominant macrocystic or even solid component which can result in confusion with mucinous cysts.5,6 mcns are almost exclusively located in the tail of the pancreas and are either unilocular or have only a small number of discrete compartments .", "rarely they will have peripheral ( eggshell ) calcification which is highly predictive of cancer.7 mcn almost never communicate with the pancreatic ductal system .", "the cystic component of the side branch form of ipmn is essentially a dilated side branch(s ) and by definition , is part of the pancreatic duct .", "magnetic resonance imaging ( mri ) is superior in imaging sb - ipmn by virtue of its ability to visualize the pancreatic ductal system .", "secretin stimulated mri / magnetic resonance cholangiopancreatography can be used to highlight the ductal anatomy8 and is commonly used in pancreatic centers but its superiority to non - secretin studies in the evaluation of ipmn has not been proven .", "identification of sb - ipmn with eus can be made by visualizing a dilated side branch(s ) with connection to the main pancreatic duct .", "depending on the plane of imaging , they may appear as a chain of lakes - separate cysts that become confluent when scanning back and forth across the cyst .", "the primary problem with imaging alone in the evaluation of pancreatic cysts is that there are no clearly differentiating features that allow a high degree of diagnostic accuracy .", "studies have shown that imaging alone is inaccurate in differentiating each of the types of cystic neoplasms.9 in a recent study from the group at the academic medical center in amsterdam , the accuracy in identifying the nature of a pancreatic cyst using eus imaging alone was only 23% to 46% amongst a group of expert endosonographers.10 this study used 4 criteria to differentiate cysts : 1 ) septations , 2 ) mural nodules , 3 ) a solid component , and 4 ) communication with the pancreatic duct . as a result of these discouraging reports , eus - guided cyst aspiration and analysis along with cytology have been used to improve diagnostic accuracy ; especially the differentiation between macrocystic serous cyst adenoma , sb - ipmn and mcn .", "difficulties in establishing an accurate diagnosis with imaging alone have prompted investigators to pursue adjunctive measures .", "the idea of using analysis of cyst aspirates to diagnose pcn dates back to 1991.11 the massachusetts general hospital surgical group is credited with advancing the clinical application of this concept12 and brugge13 then applied eus - fna techniques to obtain cyst fluid noninvasively .", "1 to 2 ml of fluid is needed to perform carcinoembryonic antigen ( cea ) analysis . as a result", ", the cyst should be 1 to 2 cm in size to obtain sufficient fluid for cea analysis .", "recently , it has been possible to perform cea on as little as 500 l of fluid ( redpath integrated pathology , pittsburgh , pa , usa ) . because the fluid may be quite viscous , a 19 or 22 gauge needle is preferred .", "intravenous antibiotics are recommended during the procedure with oral antibiotics given for 3 to 5 days afterwards .", "it is recommended that the fluid be sent for cea , amylase and/or lipase , and cytology .", "the rationale for analyzing for amylase or lipase is to aid in determining if there is ductal communication .", "the cooperative pancreatic cyst study group then reported that cyst fluid analysis for cea was the best test to differentiate a mucinous from a nonmucinous cyst.14 however , the sensitivity and specificity for cea is 73% and 84% , respectively , and 25% of mucinous cysts will have a cea level less than 192 ng / ml.14 it may be possible to improve the analysis of cyst fluid by combining cea and molecular analysis ( dna quantity , k - ras mutations and allelic imbalance mutations ) .", "sawhney et al.15 reported a 100% sensitivity of discrimination between mucinous and nonmucinous cysts using a combination of cea and molecular analysis .", "it has also been reported that combining an extracellular mucin stain with cea analysis can provide an accurate discrimination between mucinous and nonmucinous cyst.16 problems with eus - guided cyst aspiration were delineated in a paper by de jong.17 one hundred forty - three consecutive patients with indeterminate pancreatic cysts underwent eus - guided cyst aspiration and the fluid was sent for cea , ca19 - 9 , amylase and cytology .", "only 90% could be punctured ( cyst in inaccessible location or too small ) . of the remaining 128 patients ,", "only 31% had adequate cellularity for analysis and there was sufficient fluid for cea in only 68 ( 49% ) .", "fluid specimens from pancreatic cysts seldom yield cells because few viable cells are shed from the lining .", "the lining epithelium can be patchy which also contributes to the difficulty in obtaining adequate samples .", "the lining of serous cyst adenomas is a glycogen - rich cuboidal epithelium whereas mucinous cysts have a mucin containing columnar epithelium ( mcn is differentiated from ipmn by having ovariantype stroma ) .", "a cytology brush within a 19 gauge needle has been developed in an attempt to improve cytologic yield during eus - guided cyst aspiration ( echobrush ; cook endoscopy , winston - salem , nc , usa ) .", "the needle is passed into the cyst and then the brush is advanced against the inner wall of the cyst . the 1st report of using the cytology brush for pancreatic cyst cytology came from al - haddad et al.18 they studied 10 patients with pancreatic cysts and compared the cellular yield for standard fna compared to brush cytology of the cyst wall . in 7 of 10 patients , the cellular yield and detection of diagnostic cells was superior for the echobrush .", "sendino et al.19 reported results in 30 patients with pancreatic cysts evaluated with the echobrush ( cook endoscopy ) . in 8/30 patients ( 27% )", "however , the brush cytology provided a specific diagnosis in 20/22 ( 91% ) patients in which the technique succeeded .", "the brush technique was superior to fluid aspiration ( 73% vs. 36% ; p=0.08 ) .", "three patients experienced complications ( 10% ) : 1 self limited pancreatitis , 1 self limited bleed , and 1 retroperitoneal bleed resulting in patient death at 30 days .", "a recent report presents better results in obtaining cytology by performing cyst wall puncture ( cwp).20 the technique reported was to advance a 22 gauge needle into the cyst and aspirate all the fluid contents .", "then , without withdrawing the needle , the needle tip is moved back and forth across the residual hypoechoic cyst wall .", "if the cyst wall was not visualized , the needle was moved 2 to 3 mm from the needle tip location where the aspiration was completed . using this technique , the authors reported obtaining material adequate for cytologic assessment in 81% of cysts ( 60/66 ) .", "thirty percent of cysts with cea < 192 ng / ml were proven to be mucinous by cytology . in 67% of cases where the cyst fluid volume was insufficient for cea analysis , cytology demonstrated mucinous epithelium", "the complication rate was 1.45% ( 1 episode of pancreatitis which was graded as mild and self - limited ) .", "an alternative or adjunct to cyst wall cytology might be to directly examine the cyst wall .", "there have been recent reports of using a small confocal microscopy probe ( mauna kea technologies , paris , france ) .", "the cellvizio system is a probe based confocal microscopy system and a very small probe has been developed which can be passed down a 19 gauge needle . like any other optical biopsy system ,", "konda et al.21 reported use of this technology on 18 patients ; 12 of whom had pancreatic cysts .", "imaging succeeded in 17/18 patients and in 10/17 , the images were considered to be high quality .", "future studies will need to address safety issues and determine if a specific diagnosis can be made easily and safely .", "there have also been reports using the optical catheter used in the spyglass system ( boston scientific , natick , ma , usa ) to directly visualize the cyst lining .", "direct visualization of both cysts revealed smooth lining and biopsies revealed a mucinous cystadenoma in both cases . there were no immediate complications in either case but one developed severe pancreatitis 1 month after the procedure .", "identification of masses or nodules emanating from the cyst wall can indicate the presence of cancer or high grade dysplasia.3 however , it is important to make a differentiation between a mural nodule and an aggregate of mucin .", "this distinction was studied by zhong et al.23 who reviewed pathology , eus and ct examinations from 57 patients who had undergone surgical resection of mucinous cysts .", "cancer or high grade dysplasia was found in 23% of cysts with mural nodules verses 3% without nodules ( p=0.02 ) .", "mucin balls accounted for 65% of the intracystic lesions detected by eus and were characterized by being round , having smooth edges and being anechoic in the center with an echogenic rim .", "eus had sensitivity of 75% for the detection of mural nodules compared to 24% for ct .", "aspiration of cyst contents is indicated if identification of the cyst can not be made with imaging alone . however ,", "if there is an associated mass or mural nodule , then the mass itself should be targeted for cytologic examination . while mucinous cysts are the most common indication , cystic degeneration of neuroendocrine tumors and adenocarcinoma as well as solid pseudopapillary neoplasm and acinar - cell cystadenocarcinoma represent cystic neoplasms of the pancreas that present with a solid component . in these cases", "any of the 3 gauges of needles ( 25 , 22 , or 19 ) could be used . in the instance of a mass", "multiple punctures into a cyst is the main risk factor for infection . in the case of a mural nodule , it is best if the cyst fluid can be removed 1st leaving a solid mass to target .", "our practice is to combine fluid aspiration ( performed 1st ) and then the needle is re - directed to the mass .", "we prefer to have the 1st pass stained and interpreted before considering a 2nd pass to minimize the number of passes needed to establish the diagnosis and decrease the rate of complications .", "there has been concern in performing eus - fna of a cyst in the body or tail of the pancreas if malignant transformation is suspected .", "our japanese colleagues have been most vocal in expressing concerns about the potential of needle tract seeding of cancer cells.24,25 a resectable cystic mass in the body of the pancreas should be considered for surgical resection without fna in most circumstances .", "pancreatic cysts are being increasingly recognized and when detected incidentally , they are likely neoplastic and should undergo further evaluation which should include eus .", "the cyst fluid should be sent for cea , amylase and cytology . in the future", ", safe techniques should be developed to improve the cytologic yield because it appears that the exact nature of the lining in mucinous cysts is predictive of their malignant potential.26,27"], "abstract_text": ["<S> incidental pancreatic cysts are being increasingly recognized recently with incremented concern about health and frequent health check - up . </S>", "<S> endoscopic ultrasound ( eus ) has emerged as the principal modality for imaging pancreas for various pancreatic diseases including pancreatic cyst . </S>", "<S> but imaging alone can not accurately identify the exact nature of the pancreatic cyst . </S>", "<S> eus - guided fine needle aspiration is a useful adjunctive procedure to differentiate pancreatic cystic lesions . </S>", "<S> cystic fluid analysis with cytologic evaluation is important to diagnose etiology of pancreatic cystic lesions , helping the clinician to more accurately assess the presence or potential for malignancy . </S>"], "labels": null, "section_names": ["INTRODUCTION", "IMAGING OF PCN", "EUS-FNA FOR PANCREATIC CYSTIC FLUID ASPIRATION", "EUS-FNA FOR CYST WALL MASS", "CONCLUSIONS"], "sections": [["the prevalence of pancreatic cystic neoplasms ( pcns ) is around 2.5%.1,2 pancreatic cysts are increasingly discovered probably because of the ubiquitous presence of multi - detector computed tomography ( ct ) scans and their increased use to evaluate patients with abdominal complaints .", "endoscopic ultrasound ( eus ) plays a pivotal role in the evaluation of patients with pancreatic cysts but eus and cross - sectional imaging alone have proven to be inaccurate in identifying the exact nature of the cysts .", "there is inadequate information on the natural history of pancreatic cysts but our knowledge base can not be expanded without being able to determine the exact nature of the cyst noninvasively . toward this end ,", "eus with fine needle aspiration ( fna ) along with cyst fluid analysis has been advocated to improve the utility of eus in the diagnosis of pancreatic cysts .", "size > 3 cm and/or the presence of mural nodules appear to be the best indicators of malignant change in patients with the side branch form of intraductal papillary mucinous neoplasm ( sb - ipmn ) and mucinous cystic neoplasms ( mcn).3 eus with cyst fluid aspiration and/or fna can play a role in differentiating pcns .", "this paper will review the role of eus - fna in the evaluation of pcns ."], ["if the cysts are incidental ( the patient has no symptoms referable to the pancreas ) , the great majority are determined to be neoplastic.4 in one study , the majority of incidentally detected pancreatic cysts ( 58% ) were mucinous and therefore had some malignant potential.4 these data suggest that all incidentally found pancreatic cysts should undergo further evaluation .", "serous cyst adenomas consist of multiple \" micro cysts \" with thin septae coursing through the lesion .", "however , 20% have a dominant macrocystic or even solid component which can result in confusion with mucinous cysts.5,6 mcns are almost exclusively located in the tail of the pancreas and are either unilocular or have only a small number of discrete compartments .", "rarely they will have peripheral ( eggshell ) calcification which is highly predictive of cancer.7 mcn almost never communicate with the pancreatic ductal system .", "the cystic component of the side branch form of ipmn is essentially a dilated side branch(s ) and by definition , is part of the pancreatic duct .", "magnetic resonance imaging ( mri ) is superior in imaging sb - ipmn by virtue of its ability to visualize the pancreatic ductal system .", "secretin stimulated mri / magnetic resonance cholangiopancreatography can be used to highlight the ductal anatomy8 and is commonly used in pancreatic centers but its superiority to non - secretin studies in the evaluation of ipmn has not been proven .", "identification of sb - ipmn with eus can be made by visualizing a dilated side branch(s ) with connection to the main pancreatic duct .", "depending on the plane of imaging , they may appear as a chain of lakes - separate cysts that become confluent when scanning back and forth across the cyst .", "the primary problem with imaging alone in the evaluation of pancreatic cysts is that there are no clearly differentiating features that allow a high degree of diagnostic accuracy .", "studies have shown that imaging alone is inaccurate in differentiating each of the types of cystic neoplasms.9 in a recent study from the group at the academic medical center in amsterdam , the accuracy in identifying the nature of a pancreatic cyst using eus imaging alone was only 23% to 46% amongst a group of expert endosonographers.10 this study used 4 criteria to differentiate cysts : 1 ) septations , 2 ) mural nodules , 3 ) a solid component , and 4 ) communication with the pancreatic duct . as a result of these discouraging reports , eus - guided cyst aspiration and analysis along with cytology have been used to improve diagnostic accuracy ; especially the differentiation between macrocystic serous cyst adenoma , sb - ipmn and mcn ."], ["difficulties in establishing an accurate diagnosis with imaging alone have prompted investigators to pursue adjunctive measures .", "the idea of using analysis of cyst aspirates to diagnose pcn dates back to 1991.11 the massachusetts general hospital surgical group is credited with advancing the clinical application of this concept12 and brugge13 then applied eus - fna techniques to obtain cyst fluid noninvasively .", "1 to 2 ml of fluid is needed to perform carcinoembryonic antigen ( cea ) analysis . as a result", ", the cyst should be 1 to 2 cm in size to obtain sufficient fluid for cea analysis .", "recently , it has been possible to perform cea on as little as 500 l of fluid ( redpath integrated pathology , pittsburgh , pa , usa ) . because the fluid may be quite viscous , a 19 or 22 gauge needle is preferred .", "intravenous antibiotics are recommended during the procedure with oral antibiotics given for 3 to 5 days afterwards .", "it is recommended that the fluid be sent for cea , amylase and/or lipase , and cytology .", "the rationale for analyzing for amylase or lipase is to aid in determining if there is ductal communication .", "the cooperative pancreatic cyst study group then reported that cyst fluid analysis for cea was the best test to differentiate a mucinous from a nonmucinous cyst.14 however , the sensitivity and specificity for cea is 73% and 84% , respectively , and 25% of mucinous cysts will have a cea level less than 192 ng / ml.14 it may be possible to improve the analysis of cyst fluid by combining cea and molecular analysis ( dna quantity , k - ras mutations and allelic imbalance mutations ) .", "sawhney et al.15 reported a 100% sensitivity of discrimination between mucinous and nonmucinous cysts using a combination of cea and molecular analysis .", "it has also been reported that combining an extracellular mucin stain with cea analysis can provide an accurate discrimination between mucinous and nonmucinous cyst.16 problems with eus - guided cyst aspiration were delineated in a paper by de jong.17 one hundred forty - three consecutive patients with indeterminate pancreatic cysts underwent eus - guided cyst aspiration and the fluid was sent for cea , ca19 - 9 , amylase and cytology .", "only 90% could be punctured ( cyst in inaccessible location or too small ) . of the remaining 128 patients ,", "only 31% had adequate cellularity for analysis and there was sufficient fluid for cea in only 68 ( 49% ) .", "fluid specimens from pancreatic cysts seldom yield cells because few viable cells are shed from the lining .", "the lining epithelium can be patchy which also contributes to the difficulty in obtaining adequate samples .", "the lining of serous cyst adenomas is a glycogen - rich cuboidal epithelium whereas mucinous cysts have a mucin containing columnar epithelium ( mcn is differentiated from ipmn by having ovariantype stroma ) .", "a cytology brush within a 19 gauge needle has been developed in an attempt to improve cytologic yield during eus - guided cyst aspiration ( echobrush ; cook endoscopy , winston - salem , nc , usa ) .", "the needle is passed into the cyst and then the brush is advanced against the inner wall of the cyst . the 1st report of using the cytology brush for pancreatic cyst cytology came from al - haddad et al.18 they studied 10 patients with pancreatic cysts and compared the cellular yield for standard fna compared to brush cytology of the cyst wall . in 7 of 10 patients , the cellular yield and detection of diagnostic cells was superior for the echobrush .", "sendino et al.19 reported results in 30 patients with pancreatic cysts evaluated with the echobrush ( cook endoscopy ) . in 8/30 patients ( 27% )", "however , the brush cytology provided a specific diagnosis in 20/22 ( 91% ) patients in which the technique succeeded .", "the brush technique was superior to fluid aspiration ( 73% vs. 36% ; p=0.08 ) .", "three patients experienced complications ( 10% ) : 1 self limited pancreatitis , 1 self limited bleed , and 1 retroperitoneal bleed resulting in patient death at 30 days .", "a recent report presents better results in obtaining cytology by performing cyst wall puncture ( cwp).20 the technique reported was to advance a 22 gauge needle into the cyst and aspirate all the fluid contents .", "then , without withdrawing the needle , the needle tip is moved back and forth across the residual hypoechoic cyst wall .", "if the cyst wall was not visualized , the needle was moved 2 to 3 mm from the needle tip location where the aspiration was completed . using this technique , the authors reported obtaining material adequate for cytologic assessment in 81% of cysts ( 60/66 ) .", "thirty percent of cysts with cea < 192 ng / ml were proven to be mucinous by cytology . in 67% of cases where the cyst fluid volume was insufficient for cea analysis , cytology demonstrated mucinous epithelium", "the complication rate was 1.45% ( 1 episode of pancreatitis which was graded as mild and self - limited ) .", "an alternative or adjunct to cyst wall cytology might be to directly examine the cyst wall .", "there have been recent reports of using a small confocal microscopy probe ( mauna kea technologies , paris , france ) .", "the cellvizio system is a probe based confocal microscopy system and a very small probe has been developed which can be passed down a 19 gauge needle . like any other optical biopsy system ,", "konda et al.21 reported use of this technology on 18 patients ; 12 of whom had pancreatic cysts .", "imaging succeeded in 17/18 patients and in 10/17 , the images were considered to be high quality .", "future studies will need to address safety issues and determine if a specific diagnosis can be made easily and safely .", "there have also been reports using the optical catheter used in the spyglass system ( boston scientific , natick , ma , usa ) to directly visualize the cyst lining .", "direct visualization of both cysts revealed smooth lining and biopsies revealed a mucinous cystadenoma in both cases . there were no immediate complications in either case but one developed severe pancreatitis 1 month after the procedure ."], ["identification of masses or nodules emanating from the cyst wall can indicate the presence of cancer or high grade dysplasia.3 however , it is important to make a differentiation between a mural nodule and an aggregate of mucin .", "this distinction was studied by zhong et al.23 who reviewed pathology , eus and ct examinations from 57 patients who had undergone surgical resection of mucinous cysts .", "cancer or high grade dysplasia was found in 23% of cysts with mural nodules verses 3% without nodules ( p=0.02 ) .", "mucin balls accounted for 65% of the intracystic lesions detected by eus and were characterized by being round , having smooth edges and being anechoic in the center with an echogenic rim .", "eus had sensitivity of 75% for the detection of mural nodules compared to 24% for ct .", "aspiration of cyst contents is indicated if identification of the cyst can not be made with imaging alone . however ,", "if there is an associated mass or mural nodule , then the mass itself should be targeted for cytologic examination . while mucinous cysts are the most common indication , cystic degeneration of neuroendocrine tumors and adenocarcinoma as well as solid pseudopapillary neoplasm and acinar - cell cystadenocarcinoma represent cystic neoplasms of the pancreas that present with a solid component . in these cases", "any of the 3 gauges of needles ( 25 , 22 , or 19 ) could be used . in the instance of a mass", "multiple punctures into a cyst is the main risk factor for infection . in the case of a mural nodule , it is best if the cyst fluid can be removed 1st leaving a solid mass to target .", "our practice is to combine fluid aspiration ( performed 1st ) and then the needle is re - directed to the mass .", "we prefer to have the 1st pass stained and interpreted before considering a 2nd pass to minimize the number of passes needed to establish the diagnosis and decrease the rate of complications .", "there has been concern in performing eus - fna of a cyst in the body or tail of the pancreas if malignant transformation is suspected .", "our japanese colleagues have been most vocal in expressing concerns about the potential of needle tract seeding of cancer cells.24,25 a resectable cystic mass in the body of the pancreas should be considered for surgical resection without fna in most circumstances ."], ["pancreatic cysts are being increasingly recognized and when detected incidentally , they are likely neoplastic and should undergo further evaluation which should include eus .", "the cyst fluid should be sent for cea , amylase and cytology . in the future", ", safe techniques should be developed to improve the cytologic yield because it appears that the exact nature of the lining in mucinous cysts is predictive of their malignant potential.26,27"]]}
{"article_id": "PMC3640789", "article_text": ["brucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products and can lead to a systemic disease with any organ involvement . in this report , we describe a case of brucellosis - induced avascular necrosis of the hip .", "brucellosis was diagnosed through serological tests , and avascular necrosis of the femoral head was confirmed by pelvic mri .", "the patient was treated with a combination of antimicrobial treatments and referred to the orthopedic service for total hip arthroplasty .", "brucellosis may present with unusual manifestations and should be always taken into consideration , particularly in endemic areas .", "brucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products of infected animals ( 1 ) .", "it is a serious public health problem particularly in endemic areas , and is accompanied by .", "the disease is endemic in iran and has a high prevalence in lorestan province , central iran ( 3 ) .", "brucellosis is a systemic disease that may involve any organ in the body ( 4 ) .", "osteoarticular involvements including arthritis , spondylitis , osteomyelitis , tendonitis , and bursitis occur frequently and are reported in 3085% of patients with brucellosis ( 5 ) .", "the involvement of the large peripheral joints is usually manifested as monoarthritis ( 1 ) . although the hip joints involvement may happen in brucellosis , brucellosis - induced avascular necrosis of the hip", ", we describe a case of brucellosis that complicated by avascular necrosis of the femoral head .", "the patient was a 50-year - old rural farmer and rancher who presented with groin pain for six months before admission .", "he reported no history of trauma , underlying diseases , and steroids and alcohol use . over the previous six months", ", he had experienced symptoms including .fever , night sweats , weakness , fatigue , anorexia , and weight loss of 10 kg .", "the patient was visited by a local therapist ; in addition there was a delay in timely referral due to his addiction to opium .", "the groin pain was his most severe complaint on admission so that he could not bear his weight on the right leg .", "the physical examinations on admission showed stable vital signs , there was no organomegaly , and the right hip was in flexion and external rotation position so that any active and passive motion increased the pain .", "the results of laboratory study were as follows : complete blood count ( cbc ) : nl , liver function test ( lft ) : nl , rheamatic factor ( rf ) : neg , antinuclear antibody ( ana ) : neg , blood culture : neg , elevated erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) : positive , and standard agglutination test for brucellosis : positive ( table 1 ) .", "pelvic radiography and magnetic resonance imaging ( mri ) were administered showing avascular necrosis of right femoral head ( fig . 1 , 2 ) .", "the patient was subsequently treated with standard antibrucellosis regimen : streptomycine 1gr / d i m for three weeks , doxycycline 100mg / bid po , and rifampin 600mg / d po .", ".despite the improvement in the patient s general condition and laboratory evidence showing infection control , the patient was suffered from right groin pain and limitation of motion in the hip joint .", "although osteoarticular involvements , especially arthritis in the large peripheral joints , are among the most common manifestations of brucellosis , no joint destruction has been reported in many studies ( 5 ) . despite few reports on the permanent joint complications of brucellosis - induced peripheral joint arthritis , hip joints pyogenic infection has a poor prognosis , particularly if treatment is delayed ( 6 ) .", "delays in diagnosis and treatment of brucellosis - induced hip arthritis could lead to complications including dislocation and avascular necrosis of the femoral head ( 4 ) .", "although aspiration and examination of the joint fluid were not performed due to lack of synovial effusion on admission , considering the positive serological tests for brucellosis and concurrent avascular necrosis of the femoral head on mri , it seems that the delay in diagnosis and treatment of brucellosis in this patient had led to avascular necrosis of the femoral head .", "finally , the following questions arise : was avascular necrosis caused by increased intra - articular pressure related to the infection or due to direct involvement of the femoral head by the organism ?", "since brucellosis is a systemic infection with a broad clinical spectrum ranging from asymptomatic forms to deaths in severe cases , this disease should be taken into considerations when dealing with any patient with various and nonspecific symptoms in endemic areas with extension of the clinical signs of brucellosis .", "moreover , early diagnosis and long - term treatment and follow - up are of great importance in brucellosis ."], "abstract_text": ["<S> backgroundbrucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products and can lead to a systemic disease with any organ involvement . in this report , we describe a case of brucellosis - induced avascular necrosis of the hip . </S>", "<S> brucellosis was diagnosed through serological tests , and avascular necrosis of the femoral head was confirmed by pelvic mri . </S>", "<S> the patient was treated with a combination of antimicrobial treatments and referred to the orthopedic service for total hip arthroplasty . </S>", "<S> brucellosis may present with unusual manifestations and should be always taken into consideration , particularly in endemic areas . </S>"], "labels": null, "section_names": ["Background", "Introduction", "Case presentation", "Discussion", "Conclusions and Recommendations", "Ethical consideration"], "sections": [["brucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products and can lead to a systemic disease with any organ involvement . in this report , we describe a case of brucellosis - induced avascular necrosis of the hip .", "brucellosis was diagnosed through serological tests , and avascular necrosis of the femoral head was confirmed by pelvic mri .", "the patient was treated with a combination of antimicrobial treatments and referred to the orthopedic service for total hip arthroplasty .", "brucellosis may present with unusual manifestations and should be always taken into consideration , particularly in endemic areas ."], ["brucellosis is a zoonotic disease mostly transmitted to humans through consumption of unpasteurized dairy products of infected animals ( 1 ) .", "it is a serious public health problem particularly in endemic areas , and is accompanied by .", "the disease is endemic in iran and has a high prevalence in lorestan province , central iran ( 3 ) .", "brucellosis is a systemic disease that may involve any organ in the body ( 4 ) .", "osteoarticular involvements including arthritis , spondylitis , osteomyelitis , tendonitis , and bursitis occur frequently and are reported in 3085% of patients with brucellosis ( 5 ) .", "the involvement of the large peripheral joints is usually manifested as monoarthritis ( 1 ) . although the hip joints involvement may happen in brucellosis , brucellosis - induced avascular necrosis of the hip", ", we describe a case of brucellosis that complicated by avascular necrosis of the femoral head ."], ["the patient was a 50-year - old rural farmer and rancher who presented with groin pain for six months before admission .", "he reported no history of trauma , underlying diseases , and steroids and alcohol use . over the previous six months", ", he had experienced symptoms including .fever , night sweats , weakness , fatigue , anorexia , and weight loss of 10 kg .", "the patient was visited by a local therapist ; in addition there was a delay in timely referral due to his addiction to opium .", "the groin pain was his most severe complaint on admission so that he could not bear his weight on the right leg .", "the physical examinations on admission showed stable vital signs , there was no organomegaly , and the right hip was in flexion and external rotation position so that any active and passive motion increased the pain .", "the results of laboratory study were as follows : complete blood count ( cbc ) : nl , liver function test ( lft ) : nl , rheamatic factor ( rf ) : neg , antinuclear antibody ( ana ) : neg , blood culture : neg , elevated erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) : positive , and standard agglutination test for brucellosis : positive ( table 1 ) .", "pelvic radiography and magnetic resonance imaging ( mri ) were administered showing avascular necrosis of right femoral head ( fig . 1 , 2 ) .", "the patient was subsequently treated with standard antibrucellosis regimen : streptomycine 1gr / d i m for three weeks , doxycycline 100mg / bid po , and rifampin 600mg / d po .", ".despite the improvement in the patient s general condition and laboratory evidence showing infection control , the patient was suffered from right groin pain and limitation of motion in the hip joint ."], ["although osteoarticular involvements , especially arthritis in the large peripheral joints , are among the most common manifestations of brucellosis , no joint destruction has been reported in many studies ( 5 ) . despite few reports on the permanent joint complications of brucellosis - induced peripheral joint arthritis , hip joints pyogenic infection has a poor prognosis , particularly if treatment is delayed ( 6 ) .", "delays in diagnosis and treatment of brucellosis - induced hip arthritis could lead to complications including dislocation and avascular necrosis of the femoral head ( 4 ) .", "although aspiration and examination of the joint fluid were not performed due to lack of synovial effusion on admission , considering the positive serological tests for brucellosis and concurrent avascular necrosis of the femoral head on mri , it seems that the delay in diagnosis and treatment of brucellosis in this patient had led to avascular necrosis of the femoral head .", "finally , the following questions arise : was avascular necrosis caused by increased intra - articular pressure related to the infection or due to direct involvement of the femoral head by the organism ?"], ["since brucellosis is a systemic infection with a broad clinical spectrum ranging from asymptomatic forms to deaths in severe cases , this disease should be taken into considerations when dealing with any patient with various and nonspecific symptoms in endemic areas with extension of the clinical signs of brucellosis .", "moreover , early diagnosis and long - term treatment and follow - up are of great importance in brucellosis ."], [""]]}
{"article_id": "PMC5088311", "article_text": ["since the framingham heart study first reported  risk factors  for atherosclerotic cardiovascular disease , numerous efforts have aimed at improving risk assessment in the asymptomatic population . over time , these efforts have resulted in the introduction of framingham risk score ( frs ) and other biomarkers including the use of noninvasive imaging modalities such as coronary calcium scoring with ct scan and carotid imt and plaque measurement .", "while these methods have shown prognostic values independent of risk factors , mainstream medicine is still relying on the frs , which tends to be inaccurate for individualized risk assessment and fails to assess the current status of vascular health .", "moreover , frs and risk factor - based scoring systems are neither designed nor used to monitor response to therapeutic interventions .", "a comprehensive cardiovascular risk assessment requires measurement of risk factors as well as structural and functional markers of the arterial system . for the widespread acceptance and clinical adoption of a new test", ", it must be ( 1 ) incrementally predictive over risk factors , ( 2 ) responsive to therapy , ( 3 ) operator - independent and reproducible , ( 4 ) low - cost and widely accessible in primary care settings , and ( 5 ) posing no significant side effects . in recent years , endothelial function measurement has emerged as a reasonable candidate that could fit the above criteria .", "barometer  of cardiovascular risk , and endothelial dysfunction is the  gateway  to atherosclerotic cardiovascular diseases [ 3 , 4 ] . over the past 20 years", ", a number of noninvasive methods of assessing peripheral endothelial function have been introduced , including ultrasound imaging of brachial flow - mediated dilatation ( fmd ) , fingertip arterial tonometry , fingertip photoplethysmography , and laser doppler flowmetry [ 512 ] .", "a new technique named digital thermal monitoring ( dtm ) has been developed to evaluate endothelial function by measuring vascular reactivity during a 5-minute arm - cuff reactive hyperemia test .", "dtm is the newest addition to the field and monitors fingertip temperature changes to measure vascular reactivity .", "dtm is a noninvasive , automated , and operator - independent test that can be performed both at physicians ' offices and in patients ' homes .", "we and other researchers have previously reported the relationships between dtm and cvd risk factors , coronary calcium score , myocardial perfusion defects , and coronary angiographic findings [ 1320 ] .", "therefore , the present study was conducted on a large registry of 6,084 patients from 18 different clinics to better characterize dtm index of vascular reactivity ( vri ) in relation to patients ' phenotypes .", "the methodology for measuring endothelial function and vascular reactivity using dtm has been previously described [ 2125 ] .", "all dtm tests were performed using a vendys 6000 portable system ( endothelix , houston , tx ) , a pc - based system that fully automates the cuff reactive hyperemia protocol . the general test setup and a sample vendys test report", "blood pressure cuffs were placed on both of the subject 's upper arms , and vendys skin temperature sensors were affixed to both of the subject 's index fingers .", "the software - driven dtm test began with an automated measurement of blood pressure and heart rate obtained from the left arm cuff . following a 5-minute period of patient and temperature stabilization , a 5-minute cuff occlusion ( cuff inflated to 30  mmhg above systolic bp ) of the right arm", "fingertip temperature in the right hand decreased because of the absence of warm circulating blood . when the cuff was released after the 5-minute occlusion , hyperemic blood flow to the forearm and hand", "was restored , and this resulted in a  temperature rebound  in the fingertip that is directly related to the subject 's hyperemic blood flow response , endothelial function , and vascular reactivity [ 21 , 22 ] . using the recorded fingertip temperatures , the ambient temperature of the testing room , the observed slope of temperature decline , and a multivariate bioheat formula , the vendys software calculated and plotted a zero reactivity curve ( zrc ) .", "the zrc served as an internal control and showed the expected temperature rebound curve , if zero vascular reactivity was present and the other variables remained the same .", "in other words , the zrc is the expected temperature curve , if no vasodilatation and subsequent reactive hyperemia had occurred .", "vascular reactivity index ( vri ) was determined by taking the maximum difference between the observed temperature rebound curve and the zrc during the reactive hyperemia period .", "vri ranged from 0.0 to 3.5 and was classified as being indicative of poor ( 0.0 to < 1.0 ) , intermediate ( 1.0 to < 2.0 ) , or good ( 2.0 ) vascular reactivity .", "the vendys dtm test registry includes age , sex , blood pressure , heart rate , vri , and fingertip temperature measurements recorded during dtm tests .", "this study includes a total of 6,084 patients from 18 clinics that volunteered to submit their data to the registry .", "the number of each type of medical practice is as follows : cardiology = 9 , general / family practice = 4 , antiaging = 3 , and internal medicine = 2 .", "vri scores in men and women were compared using unpaired student 's t - test .", "comparisons of categorical data ( e.g. , proportion of subjects with good vri in men versus women ) were performed using fisher 's exact test .", "pairwise correlations were examined using pearson 's correlation coefficient , and correlations between vri and multiple patient characteristics ( i.e. , age , sex , blood pressure , and heart rate ) were evaluated using multiple linear regression analysis .", "p value < 0.05 was considered significant . when performing statistical comparisons , tests with missing data", "cold finger flag  was defined as the condition in which the right finger temperature at start of cuff occlusion ( time 300  s ) is 27c .", "temperatures < 27c often resulted in technically poor results .  sympathetic response flag  was defined as the condition in which left finger temperature continuously declines ( > 0.5c temperature drop over a 5-minute time period ) after right arm - cuff occlusion . when evaluating vri , tests that exhibited  cold finger flag ", "in addition to monitoring temperature at the index finger of the right arm , we studied temperature changes at the index finger of the left ( nonoccluded ) arm and observed interesting signals that are currently under further investigations and not included in the results below .", "overall , the study population had the typical age and sex distribution seen in internal medicine and cardiology clinics .", ", 54% men , 46% women , systolic blood pressure ( sbp ) 138  20  mmhg , diastolic blood pressure ( dbp ) 77  12  mmhg , and heart rate ( hr ) 70  13  bpm . the vri distribution with cumulative percentages", "overall , the vri values exhibited the appearance of a normal distribution , with the exception of a small clustering of vri values at or above zero .", "thirteen percent of vri tests were categorized as poor vascular reactivity ( vri < 1.0 ) , 70% as intermediate ( 1.0  vri < 2.0 ) , and 17% as good ( vri  2.0 ) .", "vri was slightly higher in women than in men ( 1.56  0.58 versus 1.50  0.49 ; p = 0.0001 ) .", "the distribution of poor , intermediate , and good vri in men and women is shown in figure 2(b ) .", "the percentage of good vri was higher in women than in men ( 21% versus 13% ; p < 0.0001 ) . in contrast , men were slightly less likely to have poor vri than women ( 12% versus 14% ; p = 0.03 ) .", "vri was mildly and inversely correlated with age ( r = 0.21 , p < 0.01 ) as illustrated in figure 3 . as shown in figure 4(a ) , poor vri ( < 1.0 ) was most frequent in the oldest age group ( > 70  yrs . , 18% )", "compared with middle age ( 5070  yrs . , 10% ) and younger ( < 50  yrs . , 6%", "however , the distribution of poor , intermediate , and good vri values in this elderly age group ( figure 4(b ) ) was similar to that of the overall study population ( 13% poor , 70% intermediate , and 17% good ) .", "vri was not significantly correlated with sbp , dbp , pulse pressure ( pp ) , or heart rate .", "a trend was seen of higher vri scores in subjects with higher diastolic blood pressure ( r = 0.10 ; p = ns ) . however , none of the blood pressure variables were significantly correlated with vri .", "multiple regression models were built using vri as the dependent variable and age , sex , sbp , dbp , and hr as independent variables .", "as shown in table 2 , age , sex , and diastolic blood pressure were significant but weak predictors of vri .", "this is the largest report to date on any fingertip - based measurement of vascular reactivity and endothelial function [ 7 , 26 ] .", "our analyses showed that vri values derived from dtm followed a near - normal distribution and the reasonable distribution conformed to previously established cutoff values for categorizing vri scores as indicative of poor ( 0.0 to < 1.0 ) , intermediate ( 1.0 to < 2.0 ) , or good ( 2.0 ) vascular reactivity .", "vri was weakly and inversely correlated with age . however , as shown in figure 4(a ) , the frequency of poor vri was three times higher in > 70  y versus < 50  y. this finding was in line with the findings of framingham heart study reported by hamburg et al . .", "nonetheless , as reported by schnabel et al . in a community based study of 5,000 individuals , classical risk factors only accounted for 15.4% of fmd and 13.9% of pat variability .", "this clearly indicates that endothelial function provides a new angle into the status of vascular risk .", "the distribution of weak association between vri and age is in sharp contrast to other vascular tests , including coronary artery calcium , carotid intimal - media thickness , and arterial stiffness , that are strongly and positively associated with age and , therefore , require age specific cutoffs .", "although the highest prevalence of poor vri was found in patients older than 70  y , the distribution of vri values in this elderly population ( figure 4(b ) ) clearly shows a sizable number of good and intermediate scores .", "these findings support the clinical utility of dtm as a test that can differentiate good vascular function from poor vascular function , regardless of patient 's age .", "this is consistent with the sex differences of endothelial function measured by flow - mediated dilation in healthy adults .", "however , the magnitude of the sex difference for vri is not felt to be large enough to warrant establishing sex - specific cutoff values for good , intermediate , and poor vascular reactivity .", "we also observed a trend of higher vri values with higher diastolic bp but no association with systolic bp or pulse pressure .", "it is possible that the subjects with high bp would have been on multiple antihypertensive medications that could have increased their vri scores .", "the results of multivariable analyses showed that sbp and dbp were found to have minimal correlations with age .", "because blood pressure is known to correlate strongly with age in untreated population , bp - lowering medications may have played a significant role in modifying ( leveling ) the relationships in our study population . because our data set was limited by the unknown status of bp medication", "many investigators of peripheral vascular endothelial function refer to fmd as the method of choice and consider it to be a  reference  standard , but it has several physiological and technical issues , including operator - dependency , which may result in excessively high inter- and intraobserver variability , effect of the baseline diameter , low flow - mediated constriction , and reduced arterial wall compliance [ 3234 ] .", "the correlation between fmd and finger - based measurements has been less than strong [ 7 , 26 , 3538 ] .", "the weak or inconsistent correlations between fmd and the finger - based measurements have been explained by the notion that fmd mainly reflects macrovascular reactivity , whereas the finger - based measurements mostly reflect microvascular reactivity . currently , there is no evidence regarding superiority of macrovascular over microvascular reactivity .", "more studies are needed to evaluate the predictive value of each for risk assessment and monitoring response to therapies .", "previous studies showed a significant relationship between poor vri and high framingham risk score as well as high coronary calcium score [ 14 , 18 , 19 ] .", "moreover , one study found that individuals with both poor vri and high framingham risk score had the highest coronary calcium scores .", "although more work is needed to develop clear clinical guidelines to incorporate such physiologic measurements into patient care , there is no doubt that these physiologic data offer a new window to an individualized assessment .", "the fact is that risk factors are population - based factors and do not speak for individual 's susceptibility to the risk factors , nor can they evaluate the current status or activity level of the disease . on the other hand , structural markers such as coronary calcium and carotid imt - plaque", "are good indicators of susceptibility to risk factors and show the effects of past exposure , but they do not show the current status or the activity level of the disease .", "in fact , calcification will not go away with treatments , making it not suitable for monitoring progression and regression .", "measurement of endothelial function and vascular reactivity provides an instant status of the vascular physiology .", "therefore , for a comprehensive assessment of vascular health , one must pay attention to risk factors , structural markers , and functional markers of the disease .", "although almost all cvd patients receive medications and other therapeutic interventions , not all respond to the treatments or respond similarly .", "identifying who responds well and who responds poorly is a major challenge and currently classified as  residual risk .", " budoff et al . reported that vri was significantly higher in patients who received statins and aged garlic extract , compared with those who received statin alone , and that patients who showed a significant improvement in vri had less progression of coronary calcium .", "showed an independent and significant predictive value for endothelial function measurement in patients at high risk for cardiovascular events .", "similarly , rubinshtein et al . showed that poor fingertip - based vascular reactivity measurement with pat significantly predicted poor outcomes .", "together , these data clearly point to the clinical utility of endothelial function in primary and secondary prevention", ". a detailed comparison of fmd , pat , dtm , and other noninvasive cvd risk assessment methods is shown in table 3 .", "the primary limitation was that the vendys registry data do not include information about patients ' clinical conditions or medication use .", "we also did not know when the dtm tests were performed in relation to each individual 's medical history and use of medications that might have affected vascular reactivity .", "strengths of our study include a large sample size and a mixed population of males and females , geographically dispersed and from various outpatient clinics .", "the present study using the largest database of finger - based assessment of endothelial function shows that digital thermal monitoring of vascular reactivity provides meaningful and reproducible physiological variables .", "it also suggests independent roles for vri as new indices of vascular reactivity and endothelial function .", "it is essentially a combination of a blood pressure and a thermometer empowered by intelligent software that can be used both at clinics and at home .", "however , further studies are needed to incorporate these functional measurements into clinical practice guidelines for primary and secondary prevention ."], "abstract_text": ["<S> \n background . </S>", "<S> endothelial function is viewed as a barometer of cardiovascular health and plays a central role in vascular reactivity . </S>", "<S> several studies showed digital thermal monitoring ( dtm ) as a simple noninvasive method to measure vascular reactivity that is correlated with atherosclerosis risk factors and coronary artery disease . </S>", "<S> objectives . to further evaluate the relations between patient characteristics and dtm indices in a large patient registry . methods . </S>", "<S> dtm measures were correlated with age , sex , heart rate , and systolic and diastolic blood pressure in 6084 patients from 18 clinics . results . </S>", "<S> dtm vascular reactivity index ( vri ) was normally distributed and inversely correlated with age ( r = 0.21 , p < 0.0001 ) . </S>", "<S> thirteen percent of vri tests were categorized as poor vascular reactivity ( vri < 1.0 ) , 70 percent as intermediate ( 1.0  vri < 2.0 ) , and 17 percent as good ( vri  2.0 ) . </S>", "<S> poor vri ( < 1.0 ) was noted in 6% of < 50  y , 10% of 5070  y , and 18% of 70  y. in multiple linear regression analyses , age , sex , and diastolic blood pressure were significant but weak predictors of vri . </S>", "<S> conclusions . as the largest database of finger - based vascular reactivity measurement , this report adds to prior findings that vri is a meaningful physiological marker and reflects a high level of residual risk found in patients currently under care . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Limitations", "6. Conclusion"], "sections": [["since the framingham heart study first reported  risk factors  for atherosclerotic cardiovascular disease , numerous efforts have aimed at improving risk assessment in the asymptomatic population . over time , these efforts have resulted in the introduction of framingham risk score ( frs ) and other biomarkers including the use of noninvasive imaging modalities such as coronary calcium scoring with ct scan and carotid imt and plaque measurement .", "while these methods have shown prognostic values independent of risk factors , mainstream medicine is still relying on the frs , which tends to be inaccurate for individualized risk assessment and fails to assess the current status of vascular health .", "moreover , frs and risk factor - based scoring systems are neither designed nor used to monitor response to therapeutic interventions .", "a comprehensive cardiovascular risk assessment requires measurement of risk factors as well as structural and functional markers of the arterial system . for the widespread acceptance and clinical adoption of a new test", ", it must be ( 1 ) incrementally predictive over risk factors , ( 2 ) responsive to therapy , ( 3 ) operator - independent and reproducible , ( 4 ) low - cost and widely accessible in primary care settings , and ( 5 ) posing no significant side effects . in recent years , endothelial function measurement has emerged as a reasonable candidate that could fit the above criteria .", "barometer  of cardiovascular risk , and endothelial dysfunction is the  gateway  to atherosclerotic cardiovascular diseases [ 3 , 4 ] . over the past 20 years", ", a number of noninvasive methods of assessing peripheral endothelial function have been introduced , including ultrasound imaging of brachial flow - mediated dilatation ( fmd ) , fingertip arterial tonometry , fingertip photoplethysmography , and laser doppler flowmetry [ 512 ] .", "a new technique named digital thermal monitoring ( dtm ) has been developed to evaluate endothelial function by measuring vascular reactivity during a 5-minute arm - cuff reactive hyperemia test .", "dtm is the newest addition to the field and monitors fingertip temperature changes to measure vascular reactivity .", "dtm is a noninvasive , automated , and operator - independent test that can be performed both at physicians ' offices and in patients ' homes .", "we and other researchers have previously reported the relationships between dtm and cvd risk factors , coronary calcium score , myocardial perfusion defects , and coronary angiographic findings [ 1320 ] .", "therefore , the present study was conducted on a large registry of 6,084 patients from 18 different clinics to better characterize dtm index of vascular reactivity ( vri ) in relation to patients ' phenotypes ."], ["the methodology for measuring endothelial function and vascular reactivity using dtm has been previously described [ 2125 ] .", "all dtm tests were performed using a vendys 6000 portable system ( endothelix , houston , tx ) , a pc - based system that fully automates the cuff reactive hyperemia protocol . the general test setup and a sample vendys test report", "blood pressure cuffs were placed on both of the subject 's upper arms , and vendys skin temperature sensors were affixed to both of the subject 's index fingers .", "the software - driven dtm test began with an automated measurement of blood pressure and heart rate obtained from the left arm cuff . following a 5-minute period of patient and temperature stabilization , a 5-minute cuff occlusion ( cuff inflated to 30  mmhg above systolic bp ) of the right arm", "fingertip temperature in the right hand decreased because of the absence of warm circulating blood . when the cuff was released after the 5-minute occlusion , hyperemic blood flow to the forearm and hand", "was restored , and this resulted in a  temperature rebound  in the fingertip that is directly related to the subject 's hyperemic blood flow response , endothelial function , and vascular reactivity [ 21 , 22 ] . using the recorded fingertip temperatures , the ambient temperature of the testing room , the observed slope of temperature decline , and a multivariate bioheat formula , the vendys software calculated and plotted a zero reactivity curve ( zrc ) .", "the zrc served as an internal control and showed the expected temperature rebound curve , if zero vascular reactivity was present and the other variables remained the same .", "in other words , the zrc is the expected temperature curve , if no vasodilatation and subsequent reactive hyperemia had occurred .", "vascular reactivity index ( vri ) was determined by taking the maximum difference between the observed temperature rebound curve and the zrc during the reactive hyperemia period .", "vri ranged from 0.0 to 3.5 and was classified as being indicative of poor ( 0.0 to < 1.0 ) , intermediate ( 1.0 to < 2.0 ) , or good ( 2.0 ) vascular reactivity .", "the vendys dtm test registry includes age , sex , blood pressure , heart rate , vri , and fingertip temperature measurements recorded during dtm tests .", "this study includes a total of 6,084 patients from 18 clinics that volunteered to submit their data to the registry .", "the number of each type of medical practice is as follows : cardiology = 9 , general / family practice = 4 , antiaging = 3 , and internal medicine = 2 .", "vri scores in men and women were compared using unpaired student 's t - test .", "comparisons of categorical data ( e.g. , proportion of subjects with good vri in men versus women ) were performed using fisher 's exact test .", "pairwise correlations were examined using pearson 's correlation coefficient , and correlations between vri and multiple patient characteristics ( i.e. , age , sex , blood pressure , and heart rate ) were evaluated using multiple linear regression analysis .", "p value < 0.05 was considered significant . when performing statistical comparisons , tests with missing data", "cold finger flag  was defined as the condition in which the right finger temperature at start of cuff occlusion ( time 300  s ) is 27c .", "temperatures < 27c often resulted in technically poor results .  sympathetic response flag  was defined as the condition in which left finger temperature continuously declines ( > 0.5c temperature drop over a 5-minute time period ) after right arm - cuff occlusion . when evaluating vri , tests that exhibited  cold finger flag ", "in addition to monitoring temperature at the index finger of the right arm , we studied temperature changes at the index finger of the left ( nonoccluded ) arm and observed interesting signals that are currently under further investigations and not included in the results below ."], ["overall , the study population had the typical age and sex distribution seen in internal medicine and cardiology clinics .", ", 54% men , 46% women , systolic blood pressure ( sbp ) 138  20  mmhg , diastolic blood pressure ( dbp ) 77  12  mmhg , and heart rate ( hr ) 70  13  bpm . the vri distribution with cumulative percentages", "overall , the vri values exhibited the appearance of a normal distribution , with the exception of a small clustering of vri values at or above zero .", "thirteen percent of vri tests were categorized as poor vascular reactivity ( vri < 1.0 ) , 70% as intermediate ( 1.0  vri < 2.0 ) , and 17% as good ( vri  2.0 ) .", "vri was slightly higher in women than in men ( 1.56  0.58 versus 1.50  0.49 ; p = 0.0001 ) .", "the distribution of poor , intermediate , and good vri in men and women is shown in figure 2(b ) .", "the percentage of good vri was higher in women than in men ( 21% versus 13% ; p < 0.0001 ) . in contrast , men were slightly less likely to have poor vri than women ( 12% versus 14% ; p = 0.03 ) .", "vri was mildly and inversely correlated with age ( r = 0.21 , p < 0.01 ) as illustrated in figure 3 . as shown in figure 4(a ) , poor vri ( < 1.0 ) was most frequent in the oldest age group ( > 70  yrs . , 18% )", "compared with middle age ( 5070  yrs . , 10% ) and younger ( < 50  yrs . , 6%", "however , the distribution of poor , intermediate , and good vri values in this elderly age group ( figure 4(b ) ) was similar to that of the overall study population ( 13% poor , 70% intermediate , and 17% good ) .", "vri was not significantly correlated with sbp , dbp , pulse pressure ( pp ) , or heart rate .", "a trend was seen of higher vri scores in subjects with higher diastolic blood pressure ( r = 0.10 ; p = ns ) . however , none of the blood pressure variables were significantly correlated with vri .", "multiple regression models were built using vri as the dependent variable and age , sex , sbp , dbp , and hr as independent variables .", "as shown in table 2 , age , sex , and diastolic blood pressure were significant but weak predictors of vri ."], ["this is the largest report to date on any fingertip - based measurement of vascular reactivity and endothelial function [ 7 , 26 ] .", "our analyses showed that vri values derived from dtm followed a near - normal distribution and the reasonable distribution conformed to previously established cutoff values for categorizing vri scores as indicative of poor ( 0.0 to < 1.0 ) , intermediate ( 1.0 to < 2.0 ) , or good ( 2.0 ) vascular reactivity .", "vri was weakly and inversely correlated with age . however , as shown in figure 4(a ) , the frequency of poor vri was three times higher in > 70  y versus < 50  y. this finding was in line with the findings of framingham heart study reported by hamburg et al . .", "nonetheless , as reported by schnabel et al . in a community based study of 5,000 individuals , classical risk factors only accounted for 15.4% of fmd and 13.9% of pat variability .", "this clearly indicates that endothelial function provides a new angle into the status of vascular risk .", "the distribution of weak association between vri and age is in sharp contrast to other vascular tests , including coronary artery calcium , carotid intimal - media thickness , and arterial stiffness , that are strongly and positively associated with age and , therefore , require age specific cutoffs .", "although the highest prevalence of poor vri was found in patients older than 70  y , the distribution of vri values in this elderly population ( figure 4(b ) ) clearly shows a sizable number of good and intermediate scores .", "these findings support the clinical utility of dtm as a test that can differentiate good vascular function from poor vascular function , regardless of patient 's age .", "this is consistent with the sex differences of endothelial function measured by flow - mediated dilation in healthy adults .", "however , the magnitude of the sex difference for vri is not felt to be large enough to warrant establishing sex - specific cutoff values for good , intermediate , and poor vascular reactivity .", "we also observed a trend of higher vri values with higher diastolic bp but no association with systolic bp or pulse pressure .", "it is possible that the subjects with high bp would have been on multiple antihypertensive medications that could have increased their vri scores .", "the results of multivariable analyses showed that sbp and dbp were found to have minimal correlations with age .", "because blood pressure is known to correlate strongly with age in untreated population , bp - lowering medications may have played a significant role in modifying ( leveling ) the relationships in our study population . because our data set was limited by the unknown status of bp medication", "many investigators of peripheral vascular endothelial function refer to fmd as the method of choice and consider it to be a  reference  standard , but it has several physiological and technical issues , including operator - dependency , which may result in excessively high inter- and intraobserver variability , effect of the baseline diameter , low flow - mediated constriction , and reduced arterial wall compliance [ 3234 ] .", "the correlation between fmd and finger - based measurements has been less than strong [ 7 , 26 , 3538 ] .", "the weak or inconsistent correlations between fmd and the finger - based measurements have been explained by the notion that fmd mainly reflects macrovascular reactivity , whereas the finger - based measurements mostly reflect microvascular reactivity . currently , there is no evidence regarding superiority of macrovascular over microvascular reactivity .", "more studies are needed to evaluate the predictive value of each for risk assessment and monitoring response to therapies .", "previous studies showed a significant relationship between poor vri and high framingham risk score as well as high coronary calcium score [ 14 , 18 , 19 ] .", "moreover , one study found that individuals with both poor vri and high framingham risk score had the highest coronary calcium scores .", "although more work is needed to develop clear clinical guidelines to incorporate such physiologic measurements into patient care , there is no doubt that these physiologic data offer a new window to an individualized assessment .", "the fact is that risk factors are population - based factors and do not speak for individual 's susceptibility to the risk factors , nor can they evaluate the current status or activity level of the disease . on the other hand , structural markers such as coronary calcium and carotid imt - plaque", "are good indicators of susceptibility to risk factors and show the effects of past exposure , but they do not show the current status or the activity level of the disease .", "in fact , calcification will not go away with treatments , making it not suitable for monitoring progression and regression .", "measurement of endothelial function and vascular reactivity provides an instant status of the vascular physiology .", "therefore , for a comprehensive assessment of vascular health , one must pay attention to risk factors , structural markers , and functional markers of the disease .", "although almost all cvd patients receive medications and other therapeutic interventions , not all respond to the treatments or respond similarly .", "identifying who responds well and who responds poorly is a major challenge and currently classified as  residual risk .", " budoff et al . reported that vri was significantly higher in patients who received statins and aged garlic extract , compared with those who received statin alone , and that patients who showed a significant improvement in vri had less progression of coronary calcium .", "showed an independent and significant predictive value for endothelial function measurement in patients at high risk for cardiovascular events .", "similarly , rubinshtein et al . showed that poor fingertip - based vascular reactivity measurement with pat significantly predicted poor outcomes .", "together , these data clearly point to the clinical utility of endothelial function in primary and secondary prevention", ". a detailed comparison of fmd , pat , dtm , and other noninvasive cvd risk assessment methods is shown in table 3 ."], ["the primary limitation was that the vendys registry data do not include information about patients ' clinical conditions or medication use .", "we also did not know when the dtm tests were performed in relation to each individual 's medical history and use of medications that might have affected vascular reactivity .", "strengths of our study include a large sample size and a mixed population of males and females , geographically dispersed and from various outpatient clinics ."], ["the present study using the largest database of finger - based assessment of endothelial function shows that digital thermal monitoring of vascular reactivity provides meaningful and reproducible physiological variables .", "it also suggests independent roles for vri as new indices of vascular reactivity and endothelial function .", "it is essentially a combination of a blood pressure and a thermometer empowered by intelligent software that can be used both at clinics and at home .", "however , further studies are needed to incorporate these functional measurements into clinical practice guidelines for primary and secondary prevention ."]]}
{"article_id": "PMC3612216", "article_text": ["a sandwich or laminate technique is one of the methods proposed for composite resin restorations , which was introduced for the first time by mclean , et al . in 1985 . the basic idea behind this technique is to use two different restorative materials for a single restorative procedure so that the maximum physico - mechanical and esthetic properties of these two materials can be exploited simultaneously .", "generally , the first component is a layer of conventional or resin - modified glass - ionomer cement which has drawn attention due to its capacity to form an inherent bond with tooth structures and the resultant better seal and decrease in microleakage ( particularly in dentinal walls ) ; it can also release fluoride and decrease the odds of carious lesions .", "the second component is a layer of composite materials ( including composite resin , compomer , and ormocer ) , which is used to compensate for the limitations and disadvantages of glass - ionomer cements , including weak mechanical properties and inappropriate esthetic appearance .", "it has been reported that the combination of these two materials have different property results in the clinical success of restorations .", "the success of the laminate technique depends on the strength of the bond between the glass - ionomer and the resin composite materials in addition to the strength of the bond between the glass - ionomer cement and dentin . however , there is a relatively weak bond strength between conventional glass - ionomers and composite resin materials , mainly because of the lack of a chemical bond between these two materials and the low cohesive strength of glass - ionomer.[35 ] several studies have evaluated different surface preparation techniques for glass - ionomers , such as the use of different bonding systems and surface etching procedures in order to increase the bond strength between these two materials in the laminate technique .", "etching the surface with phosphoric acid has yielded different results . in a study carried out by sheth , et al . etching the surface of glass - ionomer had no effect on bond strength increase ; however , an increase in bond strength after acid etching has been reported in another study .", "it has also been reported that premature etching of glass - ionomer cement ( before its initial setting reaction ) and failure to use an adhesive resin between the glass - ionomer cement and composite resin increases the odds of restoration failure . in another study , it was demonstrated that the use of a self - etch adhesive system on half - set glass - ionomer cement ( before its initial setting ) increases the bond strength between the glass - ionomer cement and composite resin more than that observed with the use of total - etch systems ( its application after the initial setting of the cement ) .", "it has also been reported that the use of a glass - ionomer - based adhesive system applied after the cement 's initial setting improves the bond strength between the glass - ionomer cement and composite resin compared to that with the use of total - etch adhesive systems under similar conditions .", "a new group of composite resin materials , giomers , have been introduced in less than a decade , which consist of reacted glass - ionomer fillers in a resin matrix ; they are used in cavities in a manner similar to composite resins with the application of an adhesive system . in addition to appropriate esthetic results , easy polishing , fluoride recharging potential and strength , these materials release fluoride which may enhance their antibacterial effects .", "however , no studies to date have evaluated the bond strength between glass - ionomers and giomers ; therefore , the aim of the present study was to evaluate the effect of three surface preparation methods on the shear bond strength of giomer to different surface treated conventional glass - ionomer .", "sixty cylindrical specimens were used in the present in vitro study . in order to prepare the samples ,", "a plastic mold ( with an inner diameter of 6 mm and a height of 4 mm ) was placed on a glass slab .", "then conventional glass - ionomer ( gc fuji ii , gc corporation , tokyo , japan ) was packed into the plastic mold after mixing according to the manufacturer 's instructions .", "another glass slide was used on the other side of the mold to make the free surface of the conventional glass - ionomer smooth .", "then the samples were randomly divided into three groups of 20 . according to the manufacturer", ", the initial setting of gc fuji ii conventional glass - ionomer lasts in 5 minutes and a half , and the net setting time is 2 minutes and a half .", "three groups were compared ; a total - etch adhesive resin or a self - etch giomer - based adhesive resin on set glass - ionomer ; or the self - etch giomer - based adhesive resin on the unset glass - ionomer ( before completion of the initial setting ) . in group 1 the surfaces of the specimens were etched with 37% phosphoric acid ( 3 m espe dental products , st .", "paul , mn , usa ) for 15 seconds after 5 minutes and 30 seconds from the mixing procedure ; the initial setting reaction was confirmed with the use of a sharp dental explorer .", "it should be pointed out that a complete setting of conventional glass - ionomer takes 24 - 72 hours .", "after surface etching , a single bond total - etch adhesive system ( 3 m espe dental products , st .", "paul , mn , usa ) was applied according to the manufacturer 's instructions and light cured [ table 1 ] .", "a second transparent mold , with a diameter of 3 mm and a height of 2 mm , was placed on the conventional glass - ionomer specimen and a giomer restorative , beautifil ii a3-shade ( shofu dental corporation , osaka , japan ) , was packed into the transparent mold and cured using a halogen light - curing unit ( astralis 7 ; ivoclar vivadent , fl-9494 schaan , liechtenstein ) .", "the light - directing probe had a diameter of 8 mm and directed a light ray at an intensity of 700 mw / cm perpendicular to the surface , barely touching the surface , for 40 seconds . a light intensity of 700 mw / cm was confirmed using a radiometer before the start of each experimental session .", "after transparent mold removal , the specimens were cured for another 20 seconds from each direction .", "then the samples were kept in humidity chamber at 37c for 1 hour before being immersed in distilled water at 37c for the next 23 hours . in the next stage ,", "a 500-cycle thermocycling procedure was carried out at 5c/55c  5c with a dwell time of 30 seconds and a transfer time of 10 seconds .", "chemical composition and application mode of adhesives used in group 2 , the procedures were similar to those in group 1 except that fl - bond ii ( shofu dental corporation , osaka , japan ) self - etch adhesive resin was used on set glass - ionomer surface according to the manufacturer 's instructions and light cured [ table 1 ] , without acid etching . in group 3 ,", "all the procedures were similar to those in group 2 except that fl - bond ii adhesive was applied according to the manufacturer 's instructions right after the initial setting and before hardening of glass - ionomer ( 2 minutes and a half after the initiation of mixing , which is equal to the cement 's net setting time ) .", "it should be pointed out that during that time the surface hardness of the cement was sufficient to place the second mold without damaging the cement . in order to measure the shear bond strength ,", "the samples were mounted in cold - cured acrylic resin from the glass - ionomer side and the shear bond strength values of the samples were measured in newton using a universal testing machine ( h5k - s model , hounsfield test equipment , surrey , uk ) at a crosshead speed of 1 mm / min using a 0.5 mm - wide chisel .", "then the shear bond strength values were calculated in mpa by dividing the force ( in newton ) by the surface area ( mm ) of the samples .", "data were analyzed by one - way analysis of variance ( anova ) and pairwise comparisons were made by a tukey test using spss/ win.15 .", "the failure modes were determined under a stereomicroscope ( smz1500 , nikon , tokyo , japan ) at 20 .", "the failure modes were classified as follows : adhesive failure : failure at giomer  glass  ionomer cement interface .", "in addition , to evaluate the interface between the conventional glass - ionomer and giomer in the groups under study , two additional specimens were prepared in each group and subsequent to longitudinal sectioning , they were evaluated under an scanning electron microscope [ sem ] ( tescan , vega ii xmu , brno , czech republic ) [ figure 1 ] .", "scanning electron micrographs of conventional glass - ionomer and giomer interface in the study groups ( mag 500 ) .", "arrows indicate margins of adhesive resin between glass - ionomer and giomer . in the three scanning electron micrographs ,", "the materials which are placed on the right and left sides of the adhesive resin indicate glass - ionomer and giomer , respectively", "shear bond strengths in mpa ( means and standard deviations ) for the study groups are represented in table 2 .", "there were statistically significant differences in shear bond strength values between the study groups ( p < 0.0005 ) .", "pairwise comparisons by a tukey test revealed that there were statistically significant differences in shear bond strengths between group 2 and the two other groups ( p < 0.0005 ) , whereas the bond strength difference between groups 1 and 3 was not statistically significant ( p = 0.609 ) . means and standard deviations ( sd ) of the shear bond strengths ( mpa ) measured in study groups", "moreover , all the cohesive failures were within glass - ionomer cement and there were no cohesive failures inside the giomer .", "failure modes of the study groups sem photomicrographs of the glass - ionomer and giomer interface in the study groups are represented in figure 1 . in the sem photomicrograph in group 1", "[ figure 1a ] the hybrid zone ( the area of adhesive resin penetration into glass - ionomer ) is almost homogeneous but has irregular borders on the glass - ionomer cement side . in the sem photomicrograph in group 2", "[ figure 1b ] the hybrid zone is regular and homogenous and in the sem photomicrograph in group 3 [ figure 1c ] the hybrid zone is irregular and non - homogenous .", "moreover , the use of self - etch adhesive resin ( fl - bond ii ) in groups 2 and 3 resulted in the thicker hybrid zone than total - etch adhesive resin ( single bond ) in group 1 .", "an appropriate bond between glass - ionomer and the superficial resin materials is very important for the success of the sandwich technique . in the present study shear bond strength of conventional glass - ionomer to giomer", "was evaluated using three different surface preparations for glass - ionomer ( use of self - etch adhesive on glass - ionomer with or without complete initial setting reaction and use of total - etch adhesive on glass - ionomer after completion of the initial setting reaction ) . in designing the present study ,", "the use of total - etch adhesive on glass - ionomer without the completion of the initial setting reaction was not considered , because in total - etch adhesives , rinsing after etching and contamination with moisture before completion of the initial setting reaction of glass - ionomer influences the surface integrity of the cement .", "the results of the present study showed that shear bond strength of glass - ionomer to giomer depends on surface preparation . in this context , in the group in which glass - ionomer was set and self - etch adhesive was used , the highest shear bond strength was recorded compared to two other groups , which is consistent with the results of a study carried out by gopikrishna , et al . on glass - ionomer - based adhesives ; in the case of complete setting reaction the bond strength was higher than that in incomplete setting reaction .", "contrary to the results of the present study , knight , et al . reported no statistically significant differences in the bond strength before and after initial setting of glass - ionomer in the co - cure technique , where conventional glass - ionomer , resin - modified glass - ionomer ( rmgi ) , and composite resin were placed in consecutive layers before light - curing procedure .", "the differences in the results of that study and the present study might be attributed to different etching times in the two studies .", "the etching time was 15 seconds in the present study , while in the study done by knight , et al .", "it appears that longer etching time paves the way for greater destruction of glass - ionomer surface by the acid and decreases the bond strength .", "in addition , a different technique was used in the above - mentioned study , i.e. , a layer of rmgi was used over conventional glass - ionomer before placement of composite resin and the curing process was carried out for rmgi and composite resin simultaneously .", "it is well - known that the ph of glass ionomer cements is strongly acidic upon mixing , and it will increase with time , with the most rapid increase during the first 5 - 10 minutes of setting , regardless of the composition of the glass ionomer material . in comparison of groups 2 and 3 , it appears that the acidic ph of unset glass - ionomer prevented complete polymerization of the giomer - based self - etch adhesive and therefore decrease the bond strength .", "in addition , this acidic ph might have an adverse effect on the polymerization of giomer itself . in a study carried out by mohamed - tahir , et al .", "another study has shown that surface hardness of composite resin placed on polyalkenoate glass ( set for 4 minutes ) significantly decreased . moreover ,", "sem observation of the interface in group 3 revealed an inhomogeneous structure and occasionally breakdown of the glass - ionomer surface , which may have affected the bond strength .", "polyacrylic acid prevents polymerization of composite resin and results in composite resin softening ; apparently this effect was intensified in incompletely set glass - ionomer . in comparison of groups 1 and 2", ", it appears that etching the glass - ionomer surface immediately after the initial setting in group 1 in the total - etch system resulted in the destruction of cement surface , crack formation and decrease in bond strength .", "this speculation was confirmed by the sem micrographs of the interface . the irregular interface in group 1 may be an indication of damage and weakening of the glass ionomer surface rather than improved micromechanical interlocking , when compared to group 2 which was treated by a comparatively mild acid , i.e. , the self - etching primer .", "several studies have demonstrated a decrease in bond strength of glass - ionomer to composite resin as a result of etching the glass - ionomer surface .", "it has been reported that etching during the initial setting reaction of glass - ionomer leads to dissolution of weak calcium - polyacrylate rings , with the resultant deterioration of its physical properties . in the total - etch procedure", "however , another reason for a higher bond strength in the self - etch group compared to the total - etch group is the fact that the acidic monomers in the self - etch primer can chemically bond to the calcium in glass - ionomer and increase bond strength .", "in addition , a more appropriate compatibility of giomer with the giomer - based self - etch adhesive might be another reason for a higher bond strength in group 2 ; it has been suggested that the use of adhesives compatible with resin - based restorative materials can decrease deleterious chemical interferences .", "moreover in the self - etch adhesive group , resin penetration occurs simultaneous with the etching process and it is probable that the discrepancy between these two processes is eliminated or minimized .", "another factor that needs to be taken into account is the difference in composition and mechanical properties of the two adhesives ; fl - bond ii is a two - step self - etching adhesive with filler particles while single bond is a mixture of hydrophilic monomers and solvents that contains no fillers .", "it has been suggested that the two - step self - etching adhesives that incorporate a hydrophobic resin as a separate bonding agent may have enhance mechanical properties compared to simplified adhesives . moreover , apart from the composition of resin matrix , addition of filler particles enhances the mechanical strength of the bonding layer and contributes to bond strength . in the present study", ", no differences were observed in bond strength between groups 1 and 3 , and bond strength in both groups was significantly less than that in group 2 .", "regarding failure mode in the present study , the majority of failures were of the cohesive type in glass - ionomer , which is consistent with previous studies .", "this phenomenon might be attributed to lower mechanical properties of glass ionomer cements when compared to resin - based materials and the presence of numerous air inclusion bodies inside glass - ionomer , which act as stress concentration points and probably increase the odds of cohesive failure .", "a disadvantage of sandwich technique is the absence of a chemical bond between conventional glass - ionomer and superficial resin materials ; therefore , researchers have focused on the establishment of a chemical bond between them .", "considering the results of the present study , use of giomer - based self - etch systems not only decreases the time needed for the clinical application but also can result in the establishment of a chemical bond between giomer and conventional glass - ionomer .", "taking into account the limitation of this in vitro study , etching the surface of set glass - ionomer with a total - etch system or placement of self - etch adhesive on the surface of glass - ionomer with incomplete initial setting compromised bonding of giomer to glass - ionomer ."], "abstract_text": ["<S> background : an appropriate bond between glass - ionomer and the superficial resin materials is very important for the success of sandwich technique . the aim of the present in vitro study was to evaluate the effect of three surface treatments of conventional glass - ionomer on its shear bond strength to giomer.materials and methods : sixty cylindrical specimens of a conventional glass - ionomer ( gc fuji ii ) were prepared and randomly divided into three groups ( n = 20 ) . </S>", "<S> the specimens in groups 1 and 2 were treated with total - etch adhesive resin ( single bond ) along with acid etching , and self - etch adhesive resin ( fl - bond ii ) on the set glass - ionomer , respectively . </S>", "<S> specimens in group 3 were treated with self - etch adhesive resin ( fl - bond ii ) before initial setting of the glass - ionomer was complete . </S>", "<S> then a giomer restorative ( beautifil ii ) was added to the specimens . </S>", "<S> subsequent to thermocycling , the specimens were subjected to shear bond strength test . </S>", "<S> failure modes were evaluated under a stereomicroscope . </S>", "<S> data were analyzed by one - way analysis of variance and a post hoc tukey test at a significance level of p < 0.05.results:there were statistically significant differences in bond strengths between the groups ( p < 0.0005 ) . </S>", "<S> differences in bond strengths between group 2 and other groups were significant ( p < 0.0005 ) while the differences between groups 1 and 3 were not significant </S>", "<S> . failures were predominantly of the cohesive type in all the groups.conclusion:based on the results of this study , the use of self - etch adhesive resin ( fl - bond ii ) on the set glass - ionomer yielded the highest bond strength in the glass - ionomer / giomer sandwich technique . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["a sandwich or laminate technique is one of the methods proposed for composite resin restorations , which was introduced for the first time by mclean , et al . in 1985 . the basic idea behind this technique is to use two different restorative materials for a single restorative procedure so that the maximum physico - mechanical and esthetic properties of these two materials can be exploited simultaneously .", "generally , the first component is a layer of conventional or resin - modified glass - ionomer cement which has drawn attention due to its capacity to form an inherent bond with tooth structures and the resultant better seal and decrease in microleakage ( particularly in dentinal walls ) ; it can also release fluoride and decrease the odds of carious lesions .", "the second component is a layer of composite materials ( including composite resin , compomer , and ormocer ) , which is used to compensate for the limitations and disadvantages of glass - ionomer cements , including weak mechanical properties and inappropriate esthetic appearance .", "it has been reported that the combination of these two materials have different property results in the clinical success of restorations .", "the success of the laminate technique depends on the strength of the bond between the glass - ionomer and the resin composite materials in addition to the strength of the bond between the glass - ionomer cement and dentin . however , there is a relatively weak bond strength between conventional glass - ionomers and composite resin materials , mainly because of the lack of a chemical bond between these two materials and the low cohesive strength of glass - ionomer.[35 ] several studies have evaluated different surface preparation techniques for glass - ionomers , such as the use of different bonding systems and surface etching procedures in order to increase the bond strength between these two materials in the laminate technique .", "etching the surface with phosphoric acid has yielded different results . in a study carried out by sheth , et al . etching the surface of glass - ionomer had no effect on bond strength increase ; however , an increase in bond strength after acid etching has been reported in another study .", "it has also been reported that premature etching of glass - ionomer cement ( before its initial setting reaction ) and failure to use an adhesive resin between the glass - ionomer cement and composite resin increases the odds of restoration failure . in another study , it was demonstrated that the use of a self - etch adhesive system on half - set glass - ionomer cement ( before its initial setting ) increases the bond strength between the glass - ionomer cement and composite resin more than that observed with the use of total - etch systems ( its application after the initial setting of the cement ) .", "it has also been reported that the use of a glass - ionomer - based adhesive system applied after the cement 's initial setting improves the bond strength between the glass - ionomer cement and composite resin compared to that with the use of total - etch adhesive systems under similar conditions .", "a new group of composite resin materials , giomers , have been introduced in less than a decade , which consist of reacted glass - ionomer fillers in a resin matrix ; they are used in cavities in a manner similar to composite resins with the application of an adhesive system . in addition to appropriate esthetic results , easy polishing , fluoride recharging potential and strength , these materials release fluoride which may enhance their antibacterial effects .", "however , no studies to date have evaluated the bond strength between glass - ionomers and giomers ; therefore , the aim of the present study was to evaluate the effect of three surface preparation methods on the shear bond strength of giomer to different surface treated conventional glass - ionomer ."], ["sixty cylindrical specimens were used in the present in vitro study . in order to prepare the samples ,", "a plastic mold ( with an inner diameter of 6 mm and a height of 4 mm ) was placed on a glass slab .", "then conventional glass - ionomer ( gc fuji ii , gc corporation , tokyo , japan ) was packed into the plastic mold after mixing according to the manufacturer 's instructions .", "another glass slide was used on the other side of the mold to make the free surface of the conventional glass - ionomer smooth .", "then the samples were randomly divided into three groups of 20 . according to the manufacturer", ", the initial setting of gc fuji ii conventional glass - ionomer lasts in 5 minutes and a half , and the net setting time is 2 minutes and a half .", "three groups were compared ; a total - etch adhesive resin or a self - etch giomer - based adhesive resin on set glass - ionomer ; or the self - etch giomer - based adhesive resin on the unset glass - ionomer ( before completion of the initial setting ) . in group 1 the surfaces of the specimens were etched with 37% phosphoric acid ( 3 m espe dental products , st .", "paul , mn , usa ) for 15 seconds after 5 minutes and 30 seconds from the mixing procedure ; the initial setting reaction was confirmed with the use of a sharp dental explorer .", "it should be pointed out that a complete setting of conventional glass - ionomer takes 24 - 72 hours .", "after surface etching , a single bond total - etch adhesive system ( 3 m espe dental products , st .", "paul , mn , usa ) was applied according to the manufacturer 's instructions and light cured [ table 1 ] .", "a second transparent mold , with a diameter of 3 mm and a height of 2 mm , was placed on the conventional glass - ionomer specimen and a giomer restorative , beautifil ii a3-shade ( shofu dental corporation , osaka , japan ) , was packed into the transparent mold and cured using a halogen light - curing unit ( astralis 7 ; ivoclar vivadent , fl-9494 schaan , liechtenstein ) .", "the light - directing probe had a diameter of 8 mm and directed a light ray at an intensity of 700 mw / cm perpendicular to the surface , barely touching the surface , for 40 seconds . a light intensity of 700 mw / cm was confirmed using a radiometer before the start of each experimental session .", "after transparent mold removal , the specimens were cured for another 20 seconds from each direction .", "then the samples were kept in humidity chamber at 37c for 1 hour before being immersed in distilled water at 37c for the next 23 hours . in the next stage ,", "a 500-cycle thermocycling procedure was carried out at 5c/55c  5c with a dwell time of 30 seconds and a transfer time of 10 seconds .", "chemical composition and application mode of adhesives used in group 2 , the procedures were similar to those in group 1 except that fl - bond ii ( shofu dental corporation , osaka , japan ) self - etch adhesive resin was used on set glass - ionomer surface according to the manufacturer 's instructions and light cured [ table 1 ] , without acid etching . in group 3 ,", "all the procedures were similar to those in group 2 except that fl - bond ii adhesive was applied according to the manufacturer 's instructions right after the initial setting and before hardening of glass - ionomer ( 2 minutes and a half after the initiation of mixing , which is equal to the cement 's net setting time ) .", "it should be pointed out that during that time the surface hardness of the cement was sufficient to place the second mold without damaging the cement . in order to measure the shear bond strength ,", "the samples were mounted in cold - cured acrylic resin from the glass - ionomer side and the shear bond strength values of the samples were measured in newton using a universal testing machine ( h5k - s model , hounsfield test equipment , surrey , uk ) at a crosshead speed of 1 mm / min using a 0.5 mm - wide chisel .", "then the shear bond strength values were calculated in mpa by dividing the force ( in newton ) by the surface area ( mm ) of the samples .", "data were analyzed by one - way analysis of variance ( anova ) and pairwise comparisons were made by a tukey test using spss/ win.15 .", "the failure modes were determined under a stereomicroscope ( smz1500 , nikon , tokyo , japan ) at 20 .", "the failure modes were classified as follows : adhesive failure : failure at giomer  glass  ionomer cement interface .", "in addition , to evaluate the interface between the conventional glass - ionomer and giomer in the groups under study , two additional specimens were prepared in each group and subsequent to longitudinal sectioning , they were evaluated under an scanning electron microscope [ sem ] ( tescan , vega ii xmu , brno , czech republic ) [ figure 1 ] .", "scanning electron micrographs of conventional glass - ionomer and giomer interface in the study groups ( mag 500 ) .", "arrows indicate margins of adhesive resin between glass - ionomer and giomer . in the three scanning electron micrographs ,", "the materials which are placed on the right and left sides of the adhesive resin indicate glass - ionomer and giomer , respectively"], ["shear bond strengths in mpa ( means and standard deviations ) for the study groups are represented in table 2 .", "there were statistically significant differences in shear bond strength values between the study groups ( p < 0.0005 ) .", "pairwise comparisons by a tukey test revealed that there were statistically significant differences in shear bond strengths between group 2 and the two other groups ( p < 0.0005 ) , whereas the bond strength difference between groups 1 and 3 was not statistically significant ( p = 0.609 ) . means and standard deviations ( sd ) of the shear bond strengths ( mpa ) measured in study groups", "moreover , all the cohesive failures were within glass - ionomer cement and there were no cohesive failures inside the giomer .", "failure modes of the study groups sem photomicrographs of the glass - ionomer and giomer interface in the study groups are represented in figure 1 . in the sem photomicrograph in group 1", "[ figure 1a ] the hybrid zone ( the area of adhesive resin penetration into glass - ionomer ) is almost homogeneous but has irregular borders on the glass - ionomer cement side . in the sem photomicrograph in group 2", "[ figure 1b ] the hybrid zone is regular and homogenous and in the sem photomicrograph in group 3 [ figure 1c ] the hybrid zone is irregular and non - homogenous .", "moreover , the use of self - etch adhesive resin ( fl - bond ii ) in groups 2 and 3 resulted in the thicker hybrid zone than total - etch adhesive resin ( single bond ) in group 1 ."], ["an appropriate bond between glass - ionomer and the superficial resin materials is very important for the success of the sandwich technique . in the present study shear bond strength of conventional glass - ionomer to giomer", "was evaluated using three different surface preparations for glass - ionomer ( use of self - etch adhesive on glass - ionomer with or without complete initial setting reaction and use of total - etch adhesive on glass - ionomer after completion of the initial setting reaction ) . in designing the present study ,", "the use of total - etch adhesive on glass - ionomer without the completion of the initial setting reaction was not considered , because in total - etch adhesives , rinsing after etching and contamination with moisture before completion of the initial setting reaction of glass - ionomer influences the surface integrity of the cement .", "the results of the present study showed that shear bond strength of glass - ionomer to giomer depends on surface preparation . in this context , in the group in which glass - ionomer was set and self - etch adhesive was used , the highest shear bond strength was recorded compared to two other groups , which is consistent with the results of a study carried out by gopikrishna , et al . on glass - ionomer - based adhesives ; in the case of complete setting reaction the bond strength was higher than that in incomplete setting reaction .", "contrary to the results of the present study , knight , et al . reported no statistically significant differences in the bond strength before and after initial setting of glass - ionomer in the co - cure technique , where conventional glass - ionomer , resin - modified glass - ionomer ( rmgi ) , and composite resin were placed in consecutive layers before light - curing procedure .", "the differences in the results of that study and the present study might be attributed to different etching times in the two studies .", "the etching time was 15 seconds in the present study , while in the study done by knight , et al .", "it appears that longer etching time paves the way for greater destruction of glass - ionomer surface by the acid and decreases the bond strength .", "in addition , a different technique was used in the above - mentioned study , i.e. , a layer of rmgi was used over conventional glass - ionomer before placement of composite resin and the curing process was carried out for rmgi and composite resin simultaneously .", "it is well - known that the ph of glass ionomer cements is strongly acidic upon mixing , and it will increase with time , with the most rapid increase during the first 5 - 10 minutes of setting , regardless of the composition of the glass ionomer material . in comparison of groups 2 and 3 , it appears that the acidic ph of unset glass - ionomer prevented complete polymerization of the giomer - based self - etch adhesive and therefore decrease the bond strength .", "in addition , this acidic ph might have an adverse effect on the polymerization of giomer itself . in a study carried out by mohamed - tahir , et al .", "another study has shown that surface hardness of composite resin placed on polyalkenoate glass ( set for 4 minutes ) significantly decreased . moreover ,", "sem observation of the interface in group 3 revealed an inhomogeneous structure and occasionally breakdown of the glass - ionomer surface , which may have affected the bond strength .", "polyacrylic acid prevents polymerization of composite resin and results in composite resin softening ; apparently this effect was intensified in incompletely set glass - ionomer . in comparison of groups 1 and 2", ", it appears that etching the glass - ionomer surface immediately after the initial setting in group 1 in the total - etch system resulted in the destruction of cement surface , crack formation and decrease in bond strength .", "this speculation was confirmed by the sem micrographs of the interface . the irregular interface in group 1 may be an indication of damage and weakening of the glass ionomer surface rather than improved micromechanical interlocking , when compared to group 2 which was treated by a comparatively mild acid , i.e. , the self - etching primer .", "several studies have demonstrated a decrease in bond strength of glass - ionomer to composite resin as a result of etching the glass - ionomer surface .", "it has been reported that etching during the initial setting reaction of glass - ionomer leads to dissolution of weak calcium - polyacrylate rings , with the resultant deterioration of its physical properties . in the total - etch procedure", "however , another reason for a higher bond strength in the self - etch group compared to the total - etch group is the fact that the acidic monomers in the self - etch primer can chemically bond to the calcium in glass - ionomer and increase bond strength .", "in addition , a more appropriate compatibility of giomer with the giomer - based self - etch adhesive might be another reason for a higher bond strength in group 2 ; it has been suggested that the use of adhesives compatible with resin - based restorative materials can decrease deleterious chemical interferences .", "moreover in the self - etch adhesive group , resin penetration occurs simultaneous with the etching process and it is probable that the discrepancy between these two processes is eliminated or minimized .", "another factor that needs to be taken into account is the difference in composition and mechanical properties of the two adhesives ; fl - bond ii is a two - step self - etching adhesive with filler particles while single bond is a mixture of hydrophilic monomers and solvents that contains no fillers .", "it has been suggested that the two - step self - etching adhesives that incorporate a hydrophobic resin as a separate bonding agent may have enhance mechanical properties compared to simplified adhesives . moreover , apart from the composition of resin matrix , addition of filler particles enhances the mechanical strength of the bonding layer and contributes to bond strength . in the present study", ", no differences were observed in bond strength between groups 1 and 3 , and bond strength in both groups was significantly less than that in group 2 .", "regarding failure mode in the present study , the majority of failures were of the cohesive type in glass - ionomer , which is consistent with previous studies .", "this phenomenon might be attributed to lower mechanical properties of glass ionomer cements when compared to resin - based materials and the presence of numerous air inclusion bodies inside glass - ionomer , which act as stress concentration points and probably increase the odds of cohesive failure .", "a disadvantage of sandwich technique is the absence of a chemical bond between conventional glass - ionomer and superficial resin materials ; therefore , researchers have focused on the establishment of a chemical bond between them .", "considering the results of the present study , use of giomer - based self - etch systems not only decreases the time needed for the clinical application but also can result in the establishment of a chemical bond between giomer and conventional glass - ionomer ."], ["taking into account the limitation of this in vitro study , etching the surface of set glass - ionomer with a total - etch system or placement of self - etch adhesive on the surface of glass - ionomer with incomplete initial setting compromised bonding of giomer to glass - ionomer ."]]}
{"article_id": "PMC4411904", "article_text": ["planning , evaluating , and budget allocation for prevention programs are contingent upon estimating the size of their target groups .", "population size estimation of the target groups in some context , such as hiv / aids , always entail numerous challenges in countries where such diseases are concentrated in specific sub - populations with stigmatized behaviors & characteristics ( e.g. : injecting drug users ) ( 15 ) .", "traditional sampling methods that are used to estimate the size of these high risk populations , including multiplier , capture - recapture , etc .", ", are very complicated , if not impossible , since locating people with high risk behaviors is difficult , and approaching them directly can greatly reduce response reliability ( 510 ) . estimating social network size of a representative sample , through a general population survey , and asking participants questions about specific high - risk behaviors among their acquaintances ( 1113 ) is one of the best means of gathering information on the sizes of such populations , also called hidden or hard - to - count populations due to the fact that it is not possible to calculate their sizes through regular , direct methods ( 14 )", "one such method that has held the interest of researchers for the past decade is network scale - up ( nsu ) ( 6 ) . in this method ,", "the average social network size of the respondents ( shown by c ) is used as a prerequisite for estimating the size of a high risk population in the society ( shown by e ) based on the mean number of persons with high risk behavior known by respondents ( shown by m ) ( 15 ) . in the past two decades", ", several studies in different countries including the united states , ukraine , brazil , moldova , rwanda , japan , china , and thailand have used this method to determine social network sizes as a pre - requisite for estimating high risk population sizes .", "this difference points to the need for local studies to determine this value and its determinants ( 1421 ) . in iran , the estimated social network size on a national scale is between 308 and 380 .", "however , tehran , as a megacity and the capital of iran , has unique demographic , cultural , and social features , special inter - individual relationship patterns , and different subpopulation proportions .", "in addition , due to the migration of different ethnic groups to this city , various ethnic networks have been formed ( 22 ) . therefore to observe these different characteristics and the technical considerations which are recommended in the nsu method ,", "the present study was designed to examine the social network size of the tehran province residents and the demographic factors affecting it in order to prepare the grounds for estimating the size of hidden populations .", "the social network size was estimated using an indirect approach , that is , respondents were asked how many persons they knew in a certain sub - population within the society , and estimations were made based on the respondents answers . the term  knowing a person  has a specific definition in this study , and involves a certain time span and space ( 23 , 24 ) ; therefore by saying a knows b , it is understood that a knows b by name and face ; a can visit , call or email b whenever he / she wants , and vice versa . moreover ,", "a has contacted b by phone , email or in person at least once within the past 2 years , and b is a resident of tehran province ( 2527 ) . for the total sample size 1029 , we selected 829 persons from tehran and 200 from robat karim , the capital of robat karim county in tehran provincein 2012 .", "the city of tehran was divided into 5 geographical zones : north , south , east , west , and center and the sample size were divided proportional to the population size of these five zones .", "sampling was done in crowded areas , such as streets and parks ; individuals were selected through convenience sampling , and questions were asked in the form of street interviews .", "all participants were 18 or older , and had been residents of tehran province for the past five years . upon entering the survey ,", "participants were briefed on the study objectives , and their informed consent was obtained . in order to determine the social network size , participants were asked questions regarding the number of people in their personal networks using the indirect method , that is , they were asked how many people they each knew in specific subpopulations . after coordination with the ministry of health and the statistical center of iran , twenty - three known populations with clear and available provincial level statistics were selected to be used in this study . in order to improve estimation accuracy , and in view of the findings of similar studies ( 20 , 28 ) , several considerations were taken into account in preparing the final list of the known populations to be used in the present study ; for instance , the size of all selected populations was between 0.1% and 4% of the total population of tehran province .", "moreover , we excluded populations that could potentially induce a transmission error in the estimations for which there was no practical correction method .", "popularity of names in the past three decades was observed in assigning names , and more popular names were used at an almost even distribution . out of the twenty - three initial subpopulations ,", "thirteen were eventually used in the study : first graders , high school graduates , university applicants , university students , married people , divorced people , those who had had normal vaginal deliveries ( nvd ) , those who had had cesarean sections ( c / s ) , primary school staff , people with the first names hamed , abulfazl , sara and marjan . in this study , social network size was estimated using the maximum likelihood estimation ( mle ) method and the equation below : \n c^i  =  j  mijj  ej.t where c^i is the social network size of the respondent i , mij is the number of people that i knows in the known population j , ej is the real size of the known population j , the statistical information for which is available through previous censuses or surveys , and t is the total size of the general population in the survey area ( 11 , 13 ) . to compute the 95% confidence intervals of the social network size , standard error c was calculated through the formula below : ( 29 ) \n se  ci = tcijej this simple model will only work if the following strong assumptions exist ( 11 , 24 ) : \n all respondents have equal opportunity to know each member of the subpopulations under study.all respondents are fully informed about their own social network.all respondents can recall the number of their acquaintances in the subpopulations under study quickly and clearly .", "all respondents can recall the number of their acquaintances in the subpopulations under study quickly and clearly .", "violation of each of these assumptions brings about errors in the results achieved through the network scale - up method .", "one thing that can violate assumption a is the barrier effect , which pertains to barriers causing some respondents to know the subpopulations in the study with stronger or weaker possibility .", "transmission error , on the other hand , can violate assumption b , and that is when all people in a given person s network are similarly likely not to be aware of him / her belonging in a subpopulation .", "estimation effect is another factor that can violate assumption c , and it is due to a lack of precise knowledge of certain details about the people in a high risk social network . in order to control various effects and errors that can bias estimations ,", "the following methods were employed to investigate the sources for these potential errors : 1 ) back estimation of each of the known populations to determine the best groups for estimating the ultimate social network size , and 2 ) variable stratification of the study samples , carrying out all analyses independently in each subgroup , and comparing results with that for the total sample .", "moreover , acquaintances who were among the respondents social network but were not residents of tehran province were eliminated from all estimations . in order to perform the first correction , a preliminary calculation of the social network size", "was done , the size of each known population was assumed unknown ( e ) , and then the size of each subpopulation was estimated through the formula below : at this point , the estimate / real ratio ( e / r ratio ) for each population was calculated by dividing the estimated population sizes by the real sizes throughout the province .", "the next step was to eliminate the first known population in which this ratio was not between 0.5 and 2 .", "after removing the population in which this ratio was the farthest away from the above - mentioned range of 0.5 and 2 , the whole process was repeated one more time .", "the procedure was repeated for each of the populations , starting with the farthest outlier , until none of the calculated ratios was outside of this range .", "the final social network size was computed from the average social network size calculated for people in this stage ( ci ) ( 14 , 24 , 30 ) . in the end ,", "the effect of various demographic and background factors on the social network size of the residents of tehran province was analyzed using a linear regression model .", "a total of 1029 people were interviewed in this study ; 46.7% participants were male .", "the majority ( 42.3% ) was in the 18  25 year age group ; the last educational degree of most participants was high school graduate or bachelor s degree , and they were mostly university students ( table 1 ) .", "demographics of study participants using the thirteen known populations , the social network size was estimated at 200.4 ( ci95% : 188.3 , 212.5 ) , which was used for the back estimation of the known populations in this study .", "the pearson correlation coefficient between the real and estimated sizes of these known populations was 0.44 ( p value = 0.12 ) .", "the e / r ratio was approximately 1 in all but three of the subpopulations ; exceptions were first graders , and nvd , c / s where e / r ratios were 0.40 , 0.43 , and 0.3 respectively , and therefore the social network size of tehran province was estimated by eliminating the subpopulations c / s , first graders and nvds in that order .", "table 2 represents the estimated social network size after excluding each of the above subpopulations , as well as the e / r ratio for populations whose sizes have been calculated based on the estimated network sizes that continue to be out of the desired range . having eliminated c / s , primary school and nvd groups , the estimated cs were 220.8 , 243.4 and 259.1 respectively .", "in addition , the correlation between the real and estimated sizes of remaining groupwas improved significantly in every steps ( r= 0.32 based on groups , 0.51 , 0.72 and 0.82 by dropping c / s , primary school and nvd groups respectively ) . estimate / real ( e / r ) ratio & estimate - real correlation changes after stepwise excluding the known populations with out of range e / r ratio table 3 demonstrates results of applying the back estimation method to subpopulations with estimated sizes within the acceptable range that were used in the final network size estimations .", "back estimations and e / r ratios of all populations are presented in table 4 .", "known populations , source of data , back estimation of their population sizes , and the e / r ratio e / r ratio in separate estimations of social network size for men and women ( before and after excluding the three out of range e / r ratio known groups ) figure 1 represents the scatter plot of the real against estimated sizes of the thirteen known populations , and the same measures after excluding the three abovementioned subpopulations .", "the final network size estimated by using the remaining ten subpopulations was 259.1 ( ci95% : 242.2 , 276 ) .", "real  estimate scatter plot of known population sizes ( before and after excluding the three out of range e / r ratio known groups ) in the univariate analysis , social network size was significantly associated with gender ( p=0.01 ) , age ( p<0.001 ) , and occupation ( p<0.001 ) .", "men had larger networks comparing to women ( 291.8 versus 230.4 ) , and c in younger people was larger than that in older ones ( 328.5 versus 201.1 ) .", "multivariate analysis showed that variables of gender and age impacted social network size ( table 5 ) .", "age was also proved to have a significant impact on the respondents knowing the studied subpopulations ( p < 0.001 ) . comparison of social network size in different sub groups based on different background variables the possibility of knowing a person who had graduated from high school , participated in the university entrance exam , or been admitted into university in the past year was higher in the younger age group ( 56.6% , 63.4% and 39.3% respectively ) ( table 6 ) .", "based on the findings of this study and by using the mle method , the social network size of the residents of tehran province was estimated at 259.1 which were calculated using 10 known populations with an e / r ratio between 0.5 and 2 . age and gender were determinants of peoples network sizes .", "estimated network sizes were different in male and female respondents , and appropriate known populations used for c estimation in these two groups were not equal .", "our experience showed that all subpopulations with known size were not appropriate to be used in the estimation of c. we dropped three sub - populations to improve the internal validity of our size estimations , which also used in other comparable studies ( 11 , 14 , 20 , 22 ) . by eliminating these 3 subpopulations , the social network size", "the national study was primarily biased in a similar manner as well ( 22 ) .", "therefore , estimation of c based on the size of some known subpopulations is a stepwise technique and has to be applied with some considerations ; otherwise the estimated c might have big bias .", "the estimated sizes for known subpopulations c / s , nvd , and first graders were less than half the real sizes , and for the subpopulation of people with the first name marjan the estimated size was more than twice the actual size .", "after all three subpopulations were eventually eliminated , the correlation coefficient was more than double ( 0.82 as opposed to 0.32 ) , and became statistically significant .", "moreover , the estimations for the subpopulation marjan  a persian female first name- fit within the acceptable range , and was closer to 1 .", "this indicated that the social network size estimated by using the ten remaining known populations would yield a more reliable estimate of hidden subpopulations too .", "similar studies confirm likewise that elimination of populations with estimations outside of the range 0.5  1.5increases the correlation coefficient between back estimations and real sizes .", "conducted a research in ukraine to estimate the number of idu s fsw s and msm .", "of the 22 known populations used in this study , 13 fell within the acceptable e / r ratio range , and were used in the final network scale - up calculations .", "the estimated sizes of known populations had a high correlation coefficient with the real sizes using the back estimation technique ( r = 0.912 ) , and this number reached 0.94 after nine of the known populations were eliminated ( 14 ) . in another study conducted on 1554 individuals in the u.s .", "after elimination of two populations with a discrepancy between their real and estimated sizes , the correlation rose to 0.94 ( 11 ) . in the present study ,", "the average e / r ratio using thirteen known populations was calculated to be 1.17 .", "removing each of the outliers brought this ratio closer to 1 , and after eliminating the 3 sub - populations mentioned earlier , the e / r ratio reached 1.09 .", "this figure indicated an average overestimation of about 9% in the calculations . in the ukrainian study ,", "the e / r ratio was 1.65 , which denotes an overestimation of 65% in population size estimations ( 14 ) .", "overestimation seems to be one of the common limitations of the network scale - up method with small subpopulations ( 11 , 30 ) . in the present study ,", "this problem occurred with the subpopulation of people with the first name marjan , which was the smallest subpopulation with only 0.12% of the total population of tehran province .", "since nicknames are common in iran , this overestimation may be because the respondents network includes people called marjan who are registered in the census with a different name ; this can be true with several names .", "after eliminating the c / s subpopulation , the e / r ratio for the subpopulation marjan fell within the acceptable range ( e / r = 2 ) , and after the two subpopulations nvd and first graders were removed , this ratio decreased even further ( e / r = 1.7 ) .", "another common calculation error in the network scale - up method is underestimation of the size of large subpopulations ( 11 , 30 ) .", "the largest subpopulations used in this study were c / s , first graders , married people , and university applicants .", "the first two were underestimated as expected , while the other two had more accurate estimations , probably on account of a more satisfactory transmission and the respondents being more informed on these properties due to the significance that iranians attribute to marriage and university education .", "study on se - roprevalence in the united states , it is difficult for respondents to estimate the number of acquaintances they have in large subpopulations ( 11 ) .", "in addition to the estimation error in the c / s subpopulation , another explanation for this group having the lowest e / r ratio ( 0.34 ) and an e / r ratio lower than 0.5 for the nvd group is transmission error .", "the similar natures of these two subpopulations as well as their connection to gender suggested that respondents gender impacts their awareness of nvd or c / s occurring within their social networks . in order to assess the validity of this theory ,", "network size estimations and back estimations for populations were performed based on the respondents gender . based on our findings ,", "the e / r ratios for known populations were different in male and female res pondents ( table 4 ) .", "the e / r ratios were out of range for the subpopulations c / s , nvd , and first graders when estimated through the networks of male respondents , but this limitation did not exist in case of female respondents .", "the difference seems to be understandable considering the nature of these subpopulations and the iranian culture : an iranian male respondent would be less likely to be aware of childbirth conditions or first graders in his social network compared to a female respondent in a similar situation . in the ukrainian study , similar circumstances were encountered when estimating the number of militiamen , since there was a higher likelihood for male respondents to know someone in this subpopulation on account of their cultural status and social relations compared to women ( 77% vs. 68% ) . therefore , using the militiamen subpopulation to estimate the social network size leads to different results in male and female respondents ( 14 ) .", "barrier effect is another significant factor that can compromise the estimation of population sizes in the network scale - up method .", "one common barrier , which could be a concern in the present study , is the respondents age .", "those in the 18  25 year group were more likely than others to know someone in the subpopulations high school graduates , university applicants , and university students , considering how these categories are age - related .", "based on the results shown in table6 however , barrier effect did not affect the results of this study .", "this is probably because the 18 - 25 year old group is relatively larger in the general population , and our 18 - 25 year old group was proportionately larger too . in the ukrainian study", ", the two subpopulations polish and moldavian were removed due to the risk of barrier effect and consequent estimation errors . in the final stage of the present study ,", "the remaining ten subpopulations were used to estimate the social network size at 259.1 , and this was used as the base for estimating the known population sizes with the most suitable e / r ratio and the highest correlation with their true sizes .", "this number was smaller compared to similar study in the country , carried out by shokoohi et al . in kerman province in 2010 ( c = 303 ) .", "their study was performed on 500 men between 18 and 45 years of age , and the data were collected through interviews .", "the difference between the social network sizes of the two provinces of tehran and kerman seems to be due to the lifestyle specific to metropolitan areas and capital cities that somehow limits social relationships ; one other reason may be that the kerman study was restricted to male samples , who are expected to have more social contacts and consequently larger networks than females in a city like kerman ( 27 ) .", "two estimates were computed by entering 23 known groups in the study using regression - based ( c=308 ) and ratio - based ( c=380 ) methods . according to their results , the ratio - based method , which is also used in the present study , is the recommended approach because of higher internal validity and prediction validity ( 22 ) .", "the ratio of the population size to the total tehran population was beyond the range of 0.1% to 4.0% for 10 of the 23 known groups in the national study ; differences in population compositions and their effect on results can partly explain the different estimates in these two studies .", "moreover , unique social , cultural , political , and economic processes in tehran have created a different pattern of social interactions that restricts inter - individual relationships and limits the social network size .", "social science studies also confirm that in recent years , the nature of urban development in megaci - ties such as tehran has entailed loosening in social ties .", "this is due to concentration of population in metropolitan areas and absence of alternative social practices ( 31 ) . in the ukrainian study ,", "the social network size was estimated at 202 using a network scale - up approach and the mle method , which was also employed by the present study ( 27 ) . in a 2012 study conducted in japan by ezoe et al . with the purpose of estimating the msm population ,", "the network size was estimated to be 363.5 regardless of gender , with 174 being male .", "out of the initial ten known populations used in this study , only three were eventually used to estimate the social network size in the pilot stage : male fire fighter , policemen , and military personnel .", "of the seven eliminated populations , five were removed on account of estimation error , that is , their estimated sizes were not consistent with their real sizes , and two were removed due to transmission error ( 20 ) .", "the social network size was estimated at approximately 291 in the united states , as shown in the 2001 study by mccarty et al . , using four separate national sets of samples .", "the study used the network scale - up method on 29 known populations , three of which were similar to the subpopulations in the present study and those were : first names , people who gave birth within the last year , and victims of car accidents ( 24 ) . the social network size estimated by killworth et al .", "was 286 , which is larger than the present study , and this may be due to the numerous cultural and social differences between iran and the united states ( 11 ) .", "although in the kerman study , there was no significant relationship between the social network size and any of the demographic factors ( age , education , marital status , and occupation ) ( 27 ) ; the results of the present study showed that men comparing to women and younger people comparing older ones had a significantly larger social network , which seems understandable on account of cultural and social considerations .", "in the national study and the chinese study , the social network sizes of men and younger people was larger compared to those of women and other age groups , and this is due to men and young people having wider social circles ( 21 , 22 ) .", "married people had smaller social networks compared to single people in the two studies mentioned above ; this difference was found to exist in the present study as well , although the difference between these two groups was statistically not significant in tehran province ( 248.8 versus 270.1 ) . in this study , there were limitations associated with the network scale - up method .", "firstly , it is unrealistic to expect accurate and flawless estimations , because although there is a precise and specific definition for the term  knowing a person ", ", data collected on social networks is self - reported . in minimize this limitation ,", "the present study attempted to use known populations that were easily recognizable by all respondents , and had clear and uniform definitions for all members of the society .", "furthermore , the time span for the questions was the past year so that respondents could remember the details with fewer errors .", "another issue , which is quite unavoidable , is lack of definite boundaries separating many subpopulations in the respondents points of view .", "researchers are limited to subpopulations for which official statistics exist , and these subpopulations have been assigned specific definitions that are not necessarily consistent with those of various members of the society .", "we intended to select a representative sample of general population in order to minimize different types of selection and information biases .", "although it was not a fully random sample of whole community and there are some methodological considerations , based on the existing experiences in iran and the result of a methodological study that was performed for comparing three popular sampling method in this type of studies ( street - based , telephone - based and home - based interviews ) in this regard , it seems street - based sampling from deferent geographical zones would be a feasible sampling scheme which was used in the national study as well .", "based on the above explanation , it seems that the social network size of tehran s residents is different not only with similar studies in other countries but also with the results of national survey in iran , which might be mainly because of the special social and cultural pattern of communications among different communities .", "we also showed that the c varies considerably in males and females , in young and old people .", "therefore , local estimations for c in different sub - populations are needed to improve the accuracy of nsu estimations .", "ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or fal sification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors ."], "abstract_text": ["<S> abstractbackgroundnetwork scale - up is an indirect method for estimating the size of hidden , hard - to - count or high risk populations . </S>", "<S> social network size estimation is the first step in this method . </S>", "<S> the present study was conducted with the purpose of estimating the social network size of the tehran province residents and its determinants.methodsmaximum likelihood estimation was applied to estimate people s network sizes by using populations of known sizes and the scale - up method . </S>", "<S> respondents were selected from tehran province through convenience sampling in 2012 . out of thirteen selected subpopulations with known size , ten had minimum accuracy which used in our analysis.resultsof the 1029 respondents in this study , 46.7% were male . </S>", "<S> the social network size of tehran province residents was estimated to be 259.1 ( ci95% : 242.2 , 276 ) based on the ten known populations remained in this study . </S>", "<S> this size was 291.8 in men and 230.4 in women . </S>", "<S> younger people ( 1825 years old ) had larger network sizes compared to the other age groups ( p<0.001).conclusionour estimation for social network size of tehran inhabitants was smaller than that previously estimated size for the whole country ( c=380 ) . in addition , we found that the social network of subpopulations was different . </S>", "<S> this difference means that we need local estimations for sub - populations to improve the accuracy of population size estimation using network scale up method . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion", "Ethical considerations"], "sections": [["planning , evaluating , and budget allocation for prevention programs are contingent upon estimating the size of their target groups .", "population size estimation of the target groups in some context , such as hiv / aids , always entail numerous challenges in countries where such diseases are concentrated in specific sub - populations with stigmatized behaviors & characteristics ( e.g. : injecting drug users ) ( 15 ) .", "traditional sampling methods that are used to estimate the size of these high risk populations , including multiplier , capture - recapture , etc .", ", are very complicated , if not impossible , since locating people with high risk behaviors is difficult , and approaching them directly can greatly reduce response reliability ( 510 ) . estimating social network size of a representative sample , through a general population survey , and asking participants questions about specific high - risk behaviors among their acquaintances ( 1113 ) is one of the best means of gathering information on the sizes of such populations , also called hidden or hard - to - count populations due to the fact that it is not possible to calculate their sizes through regular , direct methods ( 14 )", "one such method that has held the interest of researchers for the past decade is network scale - up ( nsu ) ( 6 ) . in this method ,", "the average social network size of the respondents ( shown by c ) is used as a prerequisite for estimating the size of a high risk population in the society ( shown by e ) based on the mean number of persons with high risk behavior known by respondents ( shown by m ) ( 15 ) . in the past two decades", ", several studies in different countries including the united states , ukraine , brazil , moldova , rwanda , japan , china , and thailand have used this method to determine social network sizes as a pre - requisite for estimating high risk population sizes .", "this difference points to the need for local studies to determine this value and its determinants ( 1421 ) . in iran , the estimated social network size on a national scale is between 308 and 380 .", "however , tehran , as a megacity and the capital of iran , has unique demographic , cultural , and social features , special inter - individual relationship patterns , and different subpopulation proportions .", "in addition , due to the migration of different ethnic groups to this city , various ethnic networks have been formed ( 22 ) . therefore to observe these different characteristics and the technical considerations which are recommended in the nsu method ,", "the present study was designed to examine the social network size of the tehran province residents and the demographic factors affecting it in order to prepare the grounds for estimating the size of hidden populations ."], ["the social network size was estimated using an indirect approach , that is , respondents were asked how many persons they knew in a certain sub - population within the society , and estimations were made based on the respondents answers . the term  knowing a person  has a specific definition in this study , and involves a certain time span and space ( 23 , 24 ) ; therefore by saying a knows b , it is understood that a knows b by name and face ; a can visit , call or email b whenever he / she wants , and vice versa . moreover ,", "a has contacted b by phone , email or in person at least once within the past 2 years , and b is a resident of tehran province ( 2527 ) . for the total sample size 1029 , we selected 829 persons from tehran and 200 from robat karim , the capital of robat karim county in tehran provincein 2012 .", "the city of tehran was divided into 5 geographical zones : north , south , east , west , and center and the sample size were divided proportional to the population size of these five zones .", "sampling was done in crowded areas , such as streets and parks ; individuals were selected through convenience sampling , and questions were asked in the form of street interviews .", "all participants were 18 or older , and had been residents of tehran province for the past five years . upon entering the survey ,", "participants were briefed on the study objectives , and their informed consent was obtained . in order to determine the social network size , participants were asked questions regarding the number of people in their personal networks using the indirect method , that is , they were asked how many people they each knew in specific subpopulations . after coordination with the ministry of health and the statistical center of iran , twenty - three known populations with clear and available provincial level statistics were selected to be used in this study . in order to improve estimation accuracy , and in view of the findings of similar studies ( 20 , 28 ) , several considerations were taken into account in preparing the final list of the known populations to be used in the present study ; for instance , the size of all selected populations was between 0.1% and 4% of the total population of tehran province .", "moreover , we excluded populations that could potentially induce a transmission error in the estimations for which there was no practical correction method .", "popularity of names in the past three decades was observed in assigning names , and more popular names were used at an almost even distribution . out of the twenty - three initial subpopulations ,", "thirteen were eventually used in the study : first graders , high school graduates , university applicants , university students , married people , divorced people , those who had had normal vaginal deliveries ( nvd ) , those who had had cesarean sections ( c / s ) , primary school staff , people with the first names hamed , abulfazl , sara and marjan . in this study , social network size was estimated using the maximum likelihood estimation ( mle ) method and the equation below : \n c^i  =  j  mijj  ej.t where c^i is the social network size of the respondent i , mij is the number of people that i knows in the known population j , ej is the real size of the known population j , the statistical information for which is available through previous censuses or surveys , and t is the total size of the general population in the survey area ( 11 , 13 ) . to compute the 95% confidence intervals of the social network size , standard error c was calculated through the formula below : ( 29 ) \n se  ci = tcijej this simple model will only work if the following strong assumptions exist ( 11 , 24 ) : \n all respondents have equal opportunity to know each member of the subpopulations under study.all respondents are fully informed about their own social network.all respondents can recall the number of their acquaintances in the subpopulations under study quickly and clearly .", "all respondents can recall the number of their acquaintances in the subpopulations under study quickly and clearly .", "violation of each of these assumptions brings about errors in the results achieved through the network scale - up method .", "one thing that can violate assumption a is the barrier effect , which pertains to barriers causing some respondents to know the subpopulations in the study with stronger or weaker possibility .", "transmission error , on the other hand , can violate assumption b , and that is when all people in a given person s network are similarly likely not to be aware of him / her belonging in a subpopulation .", "estimation effect is another factor that can violate assumption c , and it is due to a lack of precise knowledge of certain details about the people in a high risk social network . in order to control various effects and errors that can bias estimations ,", "the following methods were employed to investigate the sources for these potential errors : 1 ) back estimation of each of the known populations to determine the best groups for estimating the ultimate social network size , and 2 ) variable stratification of the study samples , carrying out all analyses independently in each subgroup , and comparing results with that for the total sample .", "moreover , acquaintances who were among the respondents social network but were not residents of tehran province were eliminated from all estimations . in order to perform the first correction , a preliminary calculation of the social network size", "was done , the size of each known population was assumed unknown ( e ) , and then the size of each subpopulation was estimated through the formula below : at this point , the estimate / real ratio ( e / r ratio ) for each population was calculated by dividing the estimated population sizes by the real sizes throughout the province .", "the next step was to eliminate the first known population in which this ratio was not between 0.5 and 2 .", "after removing the population in which this ratio was the farthest away from the above - mentioned range of 0.5 and 2 , the whole process was repeated one more time .", "the procedure was repeated for each of the populations , starting with the farthest outlier , until none of the calculated ratios was outside of this range .", "the final social network size was computed from the average social network size calculated for people in this stage ( ci ) ( 14 , 24 , 30 ) . in the end ,", "the effect of various demographic and background factors on the social network size of the residents of tehran province was analyzed using a linear regression model ."], ["a total of 1029 people were interviewed in this study ; 46.7% participants were male .", "the majority ( 42.3% ) was in the 18  25 year age group ; the last educational degree of most participants was high school graduate or bachelor s degree , and they were mostly university students ( table 1 ) .", "demographics of study participants using the thirteen known populations , the social network size was estimated at 200.4 ( ci95% : 188.3 , 212.5 ) , which was used for the back estimation of the known populations in this study .", "the pearson correlation coefficient between the real and estimated sizes of these known populations was 0.44 ( p value = 0.12 ) .", "the e / r ratio was approximately 1 in all but three of the subpopulations ; exceptions were first graders , and nvd , c / s where e / r ratios were 0.40 , 0.43 , and 0.3 respectively , and therefore the social network size of tehran province was estimated by eliminating the subpopulations c / s , first graders and nvds in that order .", "table 2 represents the estimated social network size after excluding each of the above subpopulations , as well as the e / r ratio for populations whose sizes have been calculated based on the estimated network sizes that continue to be out of the desired range . having eliminated c / s , primary school and nvd groups , the estimated cs were 220.8 , 243.4 and 259.1 respectively .", "in addition , the correlation between the real and estimated sizes of remaining groupwas improved significantly in every steps ( r= 0.32 based on groups , 0.51 , 0.72 and 0.82 by dropping c / s , primary school and nvd groups respectively ) . estimate / real ( e / r ) ratio & estimate - real correlation changes after stepwise excluding the known populations with out of range e / r ratio table 3 demonstrates results of applying the back estimation method to subpopulations with estimated sizes within the acceptable range that were used in the final network size estimations .", "back estimations and e / r ratios of all populations are presented in table 4 .", "known populations , source of data , back estimation of their population sizes , and the e / r ratio e / r ratio in separate estimations of social network size for men and women ( before and after excluding the three out of range e / r ratio known groups ) figure 1 represents the scatter plot of the real against estimated sizes of the thirteen known populations , and the same measures after excluding the three abovementioned subpopulations .", "the final network size estimated by using the remaining ten subpopulations was 259.1 ( ci95% : 242.2 , 276 ) .", "real  estimate scatter plot of known population sizes ( before and after excluding the three out of range e / r ratio known groups ) in the univariate analysis , social network size was significantly associated with gender ( p=0.01 ) , age ( p<0.001 ) , and occupation ( p<0.001 ) .", "men had larger networks comparing to women ( 291.8 versus 230.4 ) , and c in younger people was larger than that in older ones ( 328.5 versus 201.1 ) .", "multivariate analysis showed that variables of gender and age impacted social network size ( table 5 ) .", "age was also proved to have a significant impact on the respondents knowing the studied subpopulations ( p < 0.001 ) . comparison of social network size in different sub groups based on different background variables the possibility of knowing a person who had graduated from high school , participated in the university entrance exam , or been admitted into university in the past year was higher in the younger age group ( 56.6% , 63.4% and 39.3% respectively ) ( table 6 ) ."], ["based on the findings of this study and by using the mle method , the social network size of the residents of tehran province was estimated at 259.1 which were calculated using 10 known populations with an e / r ratio between 0.5 and 2 . age and gender were determinants of peoples network sizes .", "estimated network sizes were different in male and female respondents , and appropriate known populations used for c estimation in these two groups were not equal .", "our experience showed that all subpopulations with known size were not appropriate to be used in the estimation of c. we dropped three sub - populations to improve the internal validity of our size estimations , which also used in other comparable studies ( 11 , 14 , 20 , 22 ) . by eliminating these 3 subpopulations , the social network size", "the national study was primarily biased in a similar manner as well ( 22 ) .", "therefore , estimation of c based on the size of some known subpopulations is a stepwise technique and has to be applied with some considerations ; otherwise the estimated c might have big bias .", "the estimated sizes for known subpopulations c / s , nvd , and first graders were less than half the real sizes , and for the subpopulation of people with the first name marjan the estimated size was more than twice the actual size .", "after all three subpopulations were eventually eliminated , the correlation coefficient was more than double ( 0.82 as opposed to 0.32 ) , and became statistically significant .", "moreover , the estimations for the subpopulation marjan  a persian female first name- fit within the acceptable range , and was closer to 1 .", "this indicated that the social network size estimated by using the ten remaining known populations would yield a more reliable estimate of hidden subpopulations too .", "similar studies confirm likewise that elimination of populations with estimations outside of the range 0.5  1.5increases the correlation coefficient between back estimations and real sizes .", "conducted a research in ukraine to estimate the number of idu s fsw s and msm .", "of the 22 known populations used in this study , 13 fell within the acceptable e / r ratio range , and were used in the final network scale - up calculations .", "the estimated sizes of known populations had a high correlation coefficient with the real sizes using the back estimation technique ( r = 0.912 ) , and this number reached 0.94 after nine of the known populations were eliminated ( 14 ) . in another study conducted on 1554 individuals in the u.s .", "after elimination of two populations with a discrepancy between their real and estimated sizes , the correlation rose to 0.94 ( 11 ) . in the present study ,", "the average e / r ratio using thirteen known populations was calculated to be 1.17 .", "removing each of the outliers brought this ratio closer to 1 , and after eliminating the 3 sub - populations mentioned earlier , the e / r ratio reached 1.09 .", "this figure indicated an average overestimation of about 9% in the calculations . in the ukrainian study ,", "the e / r ratio was 1.65 , which denotes an overestimation of 65% in population size estimations ( 14 ) .", "overestimation seems to be one of the common limitations of the network scale - up method with small subpopulations ( 11 , 30 ) . in the present study ,", "this problem occurred with the subpopulation of people with the first name marjan , which was the smallest subpopulation with only 0.12% of the total population of tehran province .", "since nicknames are common in iran , this overestimation may be because the respondents network includes people called marjan who are registered in the census with a different name ; this can be true with several names .", "after eliminating the c / s subpopulation , the e / r ratio for the subpopulation marjan fell within the acceptable range ( e / r = 2 ) , and after the two subpopulations nvd and first graders were removed , this ratio decreased even further ( e / r = 1.7 ) .", "another common calculation error in the network scale - up method is underestimation of the size of large subpopulations ( 11 , 30 ) .", "the largest subpopulations used in this study were c / s , first graders , married people , and university applicants .", "the first two were underestimated as expected , while the other two had more accurate estimations , probably on account of a more satisfactory transmission and the respondents being more informed on these properties due to the significance that iranians attribute to marriage and university education .", "study on se - roprevalence in the united states , it is difficult for respondents to estimate the number of acquaintances they have in large subpopulations ( 11 ) .", "in addition to the estimation error in the c / s subpopulation , another explanation for this group having the lowest e / r ratio ( 0.34 ) and an e / r ratio lower than 0.5 for the nvd group is transmission error .", "the similar natures of these two subpopulations as well as their connection to gender suggested that respondents gender impacts their awareness of nvd or c / s occurring within their social networks . in order to assess the validity of this theory ,", "network size estimations and back estimations for populations were performed based on the respondents gender . based on our findings ,", "the e / r ratios for known populations were different in male and female res pondents ( table 4 ) .", "the e / r ratios were out of range for the subpopulations c / s , nvd , and first graders when estimated through the networks of male respondents , but this limitation did not exist in case of female respondents .", "the difference seems to be understandable considering the nature of these subpopulations and the iranian culture : an iranian male respondent would be less likely to be aware of childbirth conditions or first graders in his social network compared to a female respondent in a similar situation . in the ukrainian study , similar circumstances were encountered when estimating the number of militiamen , since there was a higher likelihood for male respondents to know someone in this subpopulation on account of their cultural status and social relations compared to women ( 77% vs. 68% ) . therefore , using the militiamen subpopulation to estimate the social network size leads to different results in male and female respondents ( 14 ) .", "barrier effect is another significant factor that can compromise the estimation of population sizes in the network scale - up method .", "one common barrier , which could be a concern in the present study , is the respondents age .", "those in the 18  25 year group were more likely than others to know someone in the subpopulations high school graduates , university applicants , and university students , considering how these categories are age - related .", "based on the results shown in table6 however , barrier effect did not affect the results of this study .", "this is probably because the 18 - 25 year old group is relatively larger in the general population , and our 18 - 25 year old group was proportionately larger too . in the ukrainian study", ", the two subpopulations polish and moldavian were removed due to the risk of barrier effect and consequent estimation errors . in the final stage of the present study ,", "the remaining ten subpopulations were used to estimate the social network size at 259.1 , and this was used as the base for estimating the known population sizes with the most suitable e / r ratio and the highest correlation with their true sizes .", "this number was smaller compared to similar study in the country , carried out by shokoohi et al . in kerman province in 2010 ( c = 303 ) .", "their study was performed on 500 men between 18 and 45 years of age , and the data were collected through interviews .", "the difference between the social network sizes of the two provinces of tehran and kerman seems to be due to the lifestyle specific to metropolitan areas and capital cities that somehow limits social relationships ; one other reason may be that the kerman study was restricted to male samples , who are expected to have more social contacts and consequently larger networks than females in a city like kerman ( 27 ) .", "two estimates were computed by entering 23 known groups in the study using regression - based ( c=308 ) and ratio - based ( c=380 ) methods . according to their results , the ratio - based method , which is also used in the present study , is the recommended approach because of higher internal validity and prediction validity ( 22 ) .", "the ratio of the population size to the total tehran population was beyond the range of 0.1% to 4.0% for 10 of the 23 known groups in the national study ; differences in population compositions and their effect on results can partly explain the different estimates in these two studies .", "moreover , unique social , cultural , political , and economic processes in tehran have created a different pattern of social interactions that restricts inter - individual relationships and limits the social network size .", "social science studies also confirm that in recent years , the nature of urban development in megaci - ties such as tehran has entailed loosening in social ties .", "this is due to concentration of population in metropolitan areas and absence of alternative social practices ( 31 ) . in the ukrainian study ,", "the social network size was estimated at 202 using a network scale - up approach and the mle method , which was also employed by the present study ( 27 ) . in a 2012 study conducted in japan by ezoe et al . with the purpose of estimating the msm population ,", "the network size was estimated to be 363.5 regardless of gender , with 174 being male .", "out of the initial ten known populations used in this study , only three were eventually used to estimate the social network size in the pilot stage : male fire fighter , policemen , and military personnel .", "of the seven eliminated populations , five were removed on account of estimation error , that is , their estimated sizes were not consistent with their real sizes , and two were removed due to transmission error ( 20 ) .", "the social network size was estimated at approximately 291 in the united states , as shown in the 2001 study by mccarty et al . , using four separate national sets of samples .", "the study used the network scale - up method on 29 known populations , three of which were similar to the subpopulations in the present study and those were : first names , people who gave birth within the last year , and victims of car accidents ( 24 ) . the social network size estimated by killworth et al .", "was 286 , which is larger than the present study , and this may be due to the numerous cultural and social differences between iran and the united states ( 11 ) .", "although in the kerman study , there was no significant relationship between the social network size and any of the demographic factors ( age , education , marital status , and occupation ) ( 27 ) ; the results of the present study showed that men comparing to women and younger people comparing older ones had a significantly larger social network , which seems understandable on account of cultural and social considerations .", "in the national study and the chinese study , the social network sizes of men and younger people was larger compared to those of women and other age groups , and this is due to men and young people having wider social circles ( 21 , 22 ) .", "married people had smaller social networks compared to single people in the two studies mentioned above ; this difference was found to exist in the present study as well , although the difference between these two groups was statistically not significant in tehran province ( 248.8 versus 270.1 ) . in this study , there were limitations associated with the network scale - up method .", "firstly , it is unrealistic to expect accurate and flawless estimations , because although there is a precise and specific definition for the term  knowing a person ", ", data collected on social networks is self - reported . in minimize this limitation ,", "the present study attempted to use known populations that were easily recognizable by all respondents , and had clear and uniform definitions for all members of the society .", "furthermore , the time span for the questions was the past year so that respondents could remember the details with fewer errors .", "another issue , which is quite unavoidable , is lack of definite boundaries separating many subpopulations in the respondents points of view .", "researchers are limited to subpopulations for which official statistics exist , and these subpopulations have been assigned specific definitions that are not necessarily consistent with those of various members of the society .", "we intended to select a representative sample of general population in order to minimize different types of selection and information biases .", "although it was not a fully random sample of whole community and there are some methodological considerations , based on the existing experiences in iran and the result of a methodological study that was performed for comparing three popular sampling method in this type of studies ( street - based , telephone - based and home - based interviews ) in this regard , it seems street - based sampling from deferent geographical zones would be a feasible sampling scheme which was used in the national study as well ."], ["based on the above explanation , it seems that the social network size of tehran s residents is different not only with similar studies in other countries but also with the results of national survey in iran , which might be mainly because of the special social and cultural pattern of communications among different communities .", "we also showed that the c varies considerably in males and females , in young and old people .", "therefore , local estimations for c in different sub - populations are needed to improve the accuracy of nsu estimations ."], ["ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or fal sification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors ."]]}
{"article_id": "PMC4310945", "article_text": ["benign prostatic hyperplasia ( bph ) is a highly prevalent disease affecting middle aged and elderly men ( 1 ) . in the united states ,", "bph is the most common benign tumor in men and is the most typically the disorder that reduces the quality of life ( qol ) in men , causing lower urinary tract symptoms ( luts ) ( 2 ) .", "bph could be accepted as one of the processes of aging , rather than a life threatening disease , however , and as korea is rapidly becoming an aging society , the qol becomes a major issue among koreans .", "it seems certain that , before long , bph will emerge as one of the main priorities of concern for health care service authorities , as well as the government .", "according to the report in the united states , approximately 4.5 million visits were made to physicians ' offices for a primary diagnosis of bph , and almost 8 million visits were made with a primary or secondary diagnosis of bph in the year 2000 ( 3 ) .", "the direct cost of bph treatment was estimated to be 1.1 billion usd , excluding outpatient pharmaceuticals and nutritional supplements or herbal medications .", "there have been some population surveys on the prevalence of bph in korea ( 4 , 5 , 6 ) , but the scale of those studies was small , and the issue of cost for bph treatment was not investigated .", "therefore , in the present study , the authors aimed to estimate the amount of health care utilization due to bph by surveying the records of patients who visited the designated medical institutions by the health insurance review and assessment service ( hira ) of korea . the hira monitors and analyzes reimbursement records from the korean national health insurance ( nhi ) and medical aid . because almost the entire nation is required to join the nhi ( approximately 96.6% of the population ) and medical aid ( approximately 3.4% ) , hira records cover all citizens ( approximately 49 million people ) in korea .", "we also aimed to find any seasonal or regional variation in the cost of health care utilization due to bph , as well as to examine economic burdens on the health insurance system and the pattern of practice in the management of bph .", "lastly , this nationwide , large - scale survey was conducted to offer the most reliable and objective epidemiological data in order to provide fundamental information for authorities formulating health care policy to improve the national health - related qol .", "we evaluated data from the hira from a span of 5 yr ( 2004 to 2008 ) .", "data consisted of the patient 's age , residence at diagnosis , treatment cost for bph ( medical and surgical ) , time to surgery from medication , emergency visits , etc .", "the hira is a non - profit governmental organization established to review medical fees and to evaluate the appropriateness of health care benefits that beneficiaries receive .", "the hira database contains reimbursement records from all medical facilities ( ~5 - 6 million inpatient - visits per year in about 1,100 hospitals and 25,000 private clinics ) in korea ( 7 ) . the criteria to divide the residence at diagnosis into rural and urban depended on the locations of the clinics or hospitals which were visited by patients .", "in hira database , small and medium sized cities and farm villages are equivalent to the rural area , while large cities more than megalopolis correspond to the urban area ( 8) .", "we used all claims records of outpatient visits or hospital admissions of patients aged 20 yr or older , diagnosed with bph ( international classification of diseases [ icd]-10 diagnostic code : n40 ) as the primary diagnosis .", "we then assessed the seasonal and annualpatterns of hospital visits and duration of treatment due to bph , as well as the changes in the amount of insurance payment for the diagnosis and treatment of bph , during the research period .", "hospital visit ( hv ) meant the number of days that the patients visited the hospital due to bph and duration of treatment ( dt ) indicated the period of prescription if the patients received medications due to bph .", "total amount of insurance payment ( taip ) gave the information of the total cost of national insurance payment for bph within one year , and per capitainsurance payment ( pcip ) signified calculated values using this total amount of insurance payment and national population data ( 9 , 10 ) .", "age - specific number of hv and dt were presented , and differences over the years were analyzed with a poisson analysis and the relative risk ( rr ) was estimated .", "rr was the calculated specific value which indicated the age - specific relative difference of hv , dt , and pcip based on the value of 1 .", "a time series analysis was performed to explore the seasonal and annual variation of the amount of hv , dt , taip , and pcip .", "the durbin - watson test was used to evaluate self - correlations of error . to remove the self - correlations of error", "a poisson regression analysis was done to estimate the rr of annual variation , age difference , number of hv , and dt for the pcip .", "age - specific pcip was analyzed after age - standardization using data from the 2005 census ( 9 , 10 ) , obtained from the korean national statistical office .", "all hypotheses were evaluated in a 2-sided manner , and p value of < 0.05 was considered significant .", "this study was exempted from review and evaluation by the institutional review board of the boramae medical center , seoul .", "this study was exempted from review and evaluation by the institutional review board of the boramae medical center , seoul .", "the total number of hv from 2004 to 2008 was 12,088,995 . the number of visits increased with time and reached the almost 1.7 fold in 2008 compared to 2004 ( table 1 ) .", "after a logarithmic transformation to remove an increasing tendency of the range of fluctuation , the linear pattern persisted .", "the total dt almost doubled from 3,463,520 days in 2004 to 6,598,787 days in 2008 ( table 1 ) .", "1c ) . in the time series analysis using the stepwise autoregressive process , the amount of hvincreased every winter which demonstrated seasonality , repetition of a variation of patterns in a 12-month cycle ( fig . 2a , p=0.005 ) .", "patients in the 20s showed a decrease in hv from year to year , those in the 30s showed an increase until 2006 and then a decrease , those in the 40s showed an increase throughout the study period .", "after a poisson regression analysis , we found that hv were different depending on the age groups , years , and type of visit ( inpatient or outpatient ) ( p<0.001 ) .", "the interaction between age groups and years was significant ( p<0.001 ) , which meant that the variation of hv by year was significantly different depending on the age groups .", "estimated rr is presented in table 2 . for patients in the 20s and 30s ,", "hv significantly decreased 0.78 and 0.96 times , respectively , compared with that of the previous year . on the other hand ,", "in patients in the 40s and above , hv significantly increased 1.10 , 1.13 , 1.17 , and 1.16 times compared to the previous year .", "there was 11.45 times more outpatient hv than inpatient hv in the same age group and year.dt markedly increased in patients in the 60s and 80s ( fig .", "the variation of dt by years was significantly different according to the age groups . per capita insurance payment ( pcip ) increased with increasing age and with times ( table 3 and fig .", "the rate of increase was steeper in the age groups older than the 50s compared to the other age groups . in a significance test for the regression coefficient", ", we found that the pcip were different depending on the age groups and years ( p<0.001 ) .", "the interaction between age groups and years was significant ( p<0.001 ) , which implied that the variation of pcip by years was significantly different across the age groups .", "estimated rr from the poisson regression analysis is presented in the table 3 . in patients in the 20s ,", ", in patients in the 30s or older , pcip significantly increased 1.05 , 1.20 , 1.21 , 1.21 , and 1.20 times compared with the previous year .", "there was 2.93 times more outpatient pcip than inpatient pcip in the same age group and year .", "there was no regional difference between urban and rural areas in terms of hv count , dt , and taip due to bph .", "there is not a single developed country in which the importance of care due to bph is not growing .", "the main contributors of the rising prevalence of bph in korea are suspected to be the elevated standard of living , westernized diet with increased consumption of animal fat , and aging population ( 11 ) . although the fact that the prevalence of bph increases with age is widely accepted and presumed to apply to the korean society , the rate of increase in this study is noteworthy and greater than expected . in the us ,", "the total rate of bph procedures increased significantly after 2002 and was driven by a marked increase of new minimally invasive surgical technologies ( mist ) ( 12 , 13 ) .", "our data also shows a remarkable elevation in medical costs over a relatively short period of time , which is suspected to be due to not only a sharp upsurge of disease prevalence but also an increase in physicians ' interests , along with the expanded release of new bph drugs and the increase in patients ' treatment - seeking behaviors ( 13 ) . in this study", ", we demonstrated that the demand for bph treatment nearly doubled throughout the 5 yr of study period .", "the dt was 3462273 days in 2004 and increased by 1.9-fold , to 6598787 days , in 2008 .", "the hv was 1827996 days in 2004 and increased by 1.7-fold , to 3057111 days , in 2008 .", "this rapid growth happened irrespective of regional differences such as whether the area is urban or rural , and regardless of practice patterns by urologists or non - urologists , whether inpatient or outpatient .", "that was the reason why potentially bph patients in the elderly were more diagnosed and treated after recent large - scale public advertisements and medical education about bph . in other words , in the past , elderly patients suffering from voiding problem due to bph did not know the cause of the problem and the solving methods . but these days , awareness of bph was wide spread and people could easily access the medical knowledge , elderly bph patients had more chance to receive the proper medical care ( 14 ) . by analyzing the hospital visits , we found that the frequency of hospital visits started to increase in the autumn and peaked in december in a similar pattern every year from 2004 to 2008 . from february to april , when the temperature was warmer , the number of hospital visits was fewer .", "the difference between the number of hospital visits in the colder season and the warmer season reached 1.2 fold .", "in korea , the annual mean temperature ranges from 10 to 16 degrees celsius except in the high mountain areas and islands . the warmest month is august , whereas january is the coldest one .", "the monthly mean temperature ranges from 23 to 27 in august and from -6 to + 7 degrees celsius in january ( 15 ) .", "a majority of urologists have experienced the seasonal variation and rising tendency of bph incidence in the winter , and such a phenomenon has been confirmed with solid evidence based on a large population data ( 16 , 17 ) .", "it is presumed that our body is too slow to adapt to the sudden change in temperature in the winter ( 18 , 19 ) .", "in addition , alcohol consumption rapidly increases at the end of the year in korea , deteriorating luts in bph patients and occasionally leading to the most serious complication of bph : acute urinary retention ( aur ) ( 20 ) .", "higher rates of cold medicine intake could be another explanation for worsening bph symptoms in the winter .", "alpha - adrenergic agonists , which are a common component of over - the - counter cold medicines , may precipitate urinary retention by enhancing bladder outlet resistance ( 21 , 22 ) .", "the reports are inconsistent and controversy persists on the seasonal variation of testosterone , but in the largest cross - sectional study , that analyzed archival data from 4,462 us veterans aged 32 - 44 yr , there was a report of a seasonal peak of total testosterone in december ( 24 ) .", "the amount of hvincreased every winter which demonstrated statically significant seasonality , but the dt and taip were not showed significant pattern.the reasons were suggested that hv was directly influenced and changed by seasonality , but dt and taip were not .", "for example , even though a patient visited hospital due to aggravating voiding symptoms in winter , the diagnostic procedures or medications were not changed because of season of winter , rather influenced by physician or medical costs . until recently , studies on the health care costs of bph were reported mostly in western societies ( 25 , 26 ) . in the pharmacy and medical claims data obtained from 61 us healthcare plans ,", "77,040 patients aged over 45 yr were examined , and the overall average annual cost of diagnosis and management for bph was 31.4 million usd . based on us census estimates ( july 2003 ) , the direct cost estimate was approximately 3 billion usd for the year after bph diagnosis . through this study", ", we figured out that korean taip in 2008 was 66 million usd ( 1 usd=1,103.36 korean won [ krw ] in 2008 ) which were relatively small expenditure of bph costs compared to the united states .", "however , it is not possible to describe briefly the gap between the two countries , because the medical system of korea and the united states was totally different .", "to explain the discrepancy of medical costs between korea and other countries would be an interesting subject in future research .", "the present study has several limitations that require consideration because of the methodological modality using the insurance claim data , as was the case for several studies reported in similar manner ( 4 , 5 , 26 , 27 ) .", "the insurance claims records might have underestimated the entire bph population if many patients with bph were not diagnosed or treated in health care institutions .", "additionally , only a single claim with a bph diagnosis was used to identify patients with the condition , so the incidence of this condition might be overestimated in the database because a single patient may have visited clinics several times a year .", "this study reflects data only for the 5 yr from 2004 to 2005 , which is a relatively short period of time .", "in addition , we did not have detailed clinical information for each patient , including symptom severity , duration , prostate size , and other medical or surgical history . using the hira data ,", "we did not demonstrated cost of each treatment modalities . however , as was shown in the us ( 12 ) , mist has gained enormous attention from the urologists and the number of mist performed is also increasing in korea .", "additional researches are warranted . despite these limitations , however , this study has important implications .", "this is the first nation - wide , population - based study demonstrating not only seasonal and age - specific variation in the incidence of bph , but also the amount of the economic costs for bph patients in korea .", "therefore , our large - scale data could provide the most reliable information about the incidence of bph and its cost for researchers and authorities formulating health care policy .", "health care utilization due to bph is rapidly increasing in korea and is more remarkable in the elderly population .", "our study is the largest scale population study , as well as the most objective clinical data in korea , so it could provide fundamental epidemiological information on bph ."], "abstract_text": ["<S> using the korean public health insurance database , we analyzed patients diagnosed as benign prostatic hyperplasia ( bph ) from 2004 to 2008 . </S>", "<S> age and year - specific amount and seasonal variation of hospital visits ( hv ) , duration of treatment ( dt ) , the total and per capita amount of insurance payment ( taip , pcip ) were evaluated . </S>", "<S> a total of 12,088,995 hv were studied . </S>", "<S> total hv increased 1.7 times and dt almost doubled in 2008 compared to those in 2004 . </S>", "<S> hv , dt , and taip showed linearly increasing patterns year by year . in a time series analysis </S>", "<S> , hv increased in winter and demonstrated seasonality in a 12-month cycle . in a poisson regression analysis , </S>", "<S> the annual variations of hv , dt , taip , and pcip were different by age groups . in patients </S>", "<S> older than 40 yr , hv significantly increased 1.10 - 1.16 times compared to that of the previous year . </S>", "<S> dt markedly increased in their 60s and 80s patients . </S>", "<S> the rate of increase in pcip was steeper in patients 50 yr and older than in the others.health care utilization due to bph was rapidly increasing in korea and it was remarkable in the elderly population . </S>", "<S> seasonal variation of hv demonstrated that health care utilization increased in winter.graphical abstract </S>"], "labels": null, "section_names": ["Graphical Abstract", "INTRODUCTION", "MATERIALS AND METHODS", "Ethics statement", "RESULTS", "DISCUSSION"], "sections": [[""], ["benign prostatic hyperplasia ( bph ) is a highly prevalent disease affecting middle aged and elderly men ( 1 ) . in the united states ,", "bph is the most common benign tumor in men and is the most typically the disorder that reduces the quality of life ( qol ) in men , causing lower urinary tract symptoms ( luts ) ( 2 ) .", "bph could be accepted as one of the processes of aging , rather than a life threatening disease , however , and as korea is rapidly becoming an aging society , the qol becomes a major issue among koreans .", "it seems certain that , before long , bph will emerge as one of the main priorities of concern for health care service authorities , as well as the government .", "according to the report in the united states , approximately 4.5 million visits were made to physicians ' offices for a primary diagnosis of bph , and almost 8 million visits were made with a primary or secondary diagnosis of bph in the year 2000 ( 3 ) .", "the direct cost of bph treatment was estimated to be 1.1 billion usd , excluding outpatient pharmaceuticals and nutritional supplements or herbal medications .", "there have been some population surveys on the prevalence of bph in korea ( 4 , 5 , 6 ) , but the scale of those studies was small , and the issue of cost for bph treatment was not investigated .", "therefore , in the present study , the authors aimed to estimate the amount of health care utilization due to bph by surveying the records of patients who visited the designated medical institutions by the health insurance review and assessment service ( hira ) of korea . the hira monitors and analyzes reimbursement records from the korean national health insurance ( nhi ) and medical aid . because almost the entire nation is required to join the nhi ( approximately 96.6% of the population ) and medical aid ( approximately 3.4% ) , hira records cover all citizens ( approximately 49 million people ) in korea .", "we also aimed to find any seasonal or regional variation in the cost of health care utilization due to bph , as well as to examine economic burdens on the health insurance system and the pattern of practice in the management of bph .", "lastly , this nationwide , large - scale survey was conducted to offer the most reliable and objective epidemiological data in order to provide fundamental information for authorities formulating health care policy to improve the national health - related qol ."], ["we evaluated data from the hira from a span of 5 yr ( 2004 to 2008 ) .", "data consisted of the patient 's age , residence at diagnosis , treatment cost for bph ( medical and surgical ) , time to surgery from medication , emergency visits , etc .", "the hira is a non - profit governmental organization established to review medical fees and to evaluate the appropriateness of health care benefits that beneficiaries receive .", "the hira database contains reimbursement records from all medical facilities ( ~5 - 6 million inpatient - visits per year in about 1,100 hospitals and 25,000 private clinics ) in korea ( 7 ) . the criteria to divide the residence at diagnosis into rural and urban depended on the locations of the clinics or hospitals which were visited by patients .", "in hira database , small and medium sized cities and farm villages are equivalent to the rural area , while large cities more than megalopolis correspond to the urban area ( 8) .", "we used all claims records of outpatient visits or hospital admissions of patients aged 20 yr or older , diagnosed with bph ( international classification of diseases [ icd]-10 diagnostic code : n40 ) as the primary diagnosis .", "we then assessed the seasonal and annualpatterns of hospital visits and duration of treatment due to bph , as well as the changes in the amount of insurance payment for the diagnosis and treatment of bph , during the research period .", "hospital visit ( hv ) meant the number of days that the patients visited the hospital due to bph and duration of treatment ( dt ) indicated the period of prescription if the patients received medications due to bph .", "total amount of insurance payment ( taip ) gave the information of the total cost of national insurance payment for bph within one year , and per capitainsurance payment ( pcip ) signified calculated values using this total amount of insurance payment and national population data ( 9 , 10 ) .", "age - specific number of hv and dt were presented , and differences over the years were analyzed with a poisson analysis and the relative risk ( rr ) was estimated .", "rr was the calculated specific value which indicated the age - specific relative difference of hv , dt , and pcip based on the value of 1 .", "a time series analysis was performed to explore the seasonal and annual variation of the amount of hv , dt , taip , and pcip .", "the durbin - watson test was used to evaluate self - correlations of error . to remove the self - correlations of error", "a poisson regression analysis was done to estimate the rr of annual variation , age difference , number of hv , and dt for the pcip .", "age - specific pcip was analyzed after age - standardization using data from the 2005 census ( 9 , 10 ) , obtained from the korean national statistical office .", "all hypotheses were evaluated in a 2-sided manner , and p value of < 0.05 was considered significant .", "this study was exempted from review and evaluation by the institutional review board of the boramae medical center , seoul ."], ["this study was exempted from review and evaluation by the institutional review board of the boramae medical center , seoul ."], ["the total number of hv from 2004 to 2008 was 12,088,995 . the number of visits increased with time and reached the almost 1.7 fold in 2008 compared to 2004 ( table 1 ) .", "after a logarithmic transformation to remove an increasing tendency of the range of fluctuation , the linear pattern persisted .", "the total dt almost doubled from 3,463,520 days in 2004 to 6,598,787 days in 2008 ( table 1 ) .", "1c ) . in the time series analysis using the stepwise autoregressive process , the amount of hvincreased every winter which demonstrated seasonality , repetition of a variation of patterns in a 12-month cycle ( fig . 2a , p=0.005 ) .", "patients in the 20s showed a decrease in hv from year to year , those in the 30s showed an increase until 2006 and then a decrease , those in the 40s showed an increase throughout the study period .", "after a poisson regression analysis , we found that hv were different depending on the age groups , years , and type of visit ( inpatient or outpatient ) ( p<0.001 ) .", "the interaction between age groups and years was significant ( p<0.001 ) , which meant that the variation of hv by year was significantly different depending on the age groups .", "estimated rr is presented in table 2 . for patients in the 20s and 30s ,", "hv significantly decreased 0.78 and 0.96 times , respectively , compared with that of the previous year . on the other hand ,", "in patients in the 40s and above , hv significantly increased 1.10 , 1.13 , 1.17 , and 1.16 times compared to the previous year .", "there was 11.45 times more outpatient hv than inpatient hv in the same age group and year.dt markedly increased in patients in the 60s and 80s ( fig .", "the variation of dt by years was significantly different according to the age groups . per capita insurance payment ( pcip ) increased with increasing age and with times ( table 3 and fig .", "the rate of increase was steeper in the age groups older than the 50s compared to the other age groups . in a significance test for the regression coefficient", ", we found that the pcip were different depending on the age groups and years ( p<0.001 ) .", "the interaction between age groups and years was significant ( p<0.001 ) , which implied that the variation of pcip by years was significantly different across the age groups .", "estimated rr from the poisson regression analysis is presented in the table 3 . in patients in the 20s ,", ", in patients in the 30s or older , pcip significantly increased 1.05 , 1.20 , 1.21 , 1.21 , and 1.20 times compared with the previous year .", "there was 2.93 times more outpatient pcip than inpatient pcip in the same age group and year .", "there was no regional difference between urban and rural areas in terms of hv count , dt , and taip due to bph ."], ["there is not a single developed country in which the importance of care due to bph is not growing .", "the main contributors of the rising prevalence of bph in korea are suspected to be the elevated standard of living , westernized diet with increased consumption of animal fat , and aging population ( 11 ) . although the fact that the prevalence of bph increases with age is widely accepted and presumed to apply to the korean society , the rate of increase in this study is noteworthy and greater than expected . in the us ,", "the total rate of bph procedures increased significantly after 2002 and was driven by a marked increase of new minimally invasive surgical technologies ( mist ) ( 12 , 13 ) .", "our data also shows a remarkable elevation in medical costs over a relatively short period of time , which is suspected to be due to not only a sharp upsurge of disease prevalence but also an increase in physicians ' interests , along with the expanded release of new bph drugs and the increase in patients ' treatment - seeking behaviors ( 13 ) . in this study", ", we demonstrated that the demand for bph treatment nearly doubled throughout the 5 yr of study period .", "the dt was 3462273 days in 2004 and increased by 1.9-fold , to 6598787 days , in 2008 .", "the hv was 1827996 days in 2004 and increased by 1.7-fold , to 3057111 days , in 2008 .", "this rapid growth happened irrespective of regional differences such as whether the area is urban or rural , and regardless of practice patterns by urologists or non - urologists , whether inpatient or outpatient .", "that was the reason why potentially bph patients in the elderly were more diagnosed and treated after recent large - scale public advertisements and medical education about bph . in other words , in the past , elderly patients suffering from voiding problem due to bph did not know the cause of the problem and the solving methods . but these days , awareness of bph was wide spread and people could easily access the medical knowledge , elderly bph patients had more chance to receive the proper medical care ( 14 ) . by analyzing the hospital visits , we found that the frequency of hospital visits started to increase in the autumn and peaked in december in a similar pattern every year from 2004 to 2008 . from february to april , when the temperature was warmer , the number of hospital visits was fewer .", "the difference between the number of hospital visits in the colder season and the warmer season reached 1.2 fold .", "in korea , the annual mean temperature ranges from 10 to 16 degrees celsius except in the high mountain areas and islands . the warmest month is august , whereas january is the coldest one .", "the monthly mean temperature ranges from 23 to 27 in august and from -6 to + 7 degrees celsius in january ( 15 ) .", "a majority of urologists have experienced the seasonal variation and rising tendency of bph incidence in the winter , and such a phenomenon has been confirmed with solid evidence based on a large population data ( 16 , 17 ) .", "it is presumed that our body is too slow to adapt to the sudden change in temperature in the winter ( 18 , 19 ) .", "in addition , alcohol consumption rapidly increases at the end of the year in korea , deteriorating luts in bph patients and occasionally leading to the most serious complication of bph : acute urinary retention ( aur ) ( 20 ) .", "higher rates of cold medicine intake could be another explanation for worsening bph symptoms in the winter .", "alpha - adrenergic agonists , which are a common component of over - the - counter cold medicines , may precipitate urinary retention by enhancing bladder outlet resistance ( 21 , 22 ) .", "the reports are inconsistent and controversy persists on the seasonal variation of testosterone , but in the largest cross - sectional study , that analyzed archival data from 4,462 us veterans aged 32 - 44 yr , there was a report of a seasonal peak of total testosterone in december ( 24 ) .", "the amount of hvincreased every winter which demonstrated statically significant seasonality , but the dt and taip were not showed significant pattern.the reasons were suggested that hv was directly influenced and changed by seasonality , but dt and taip were not .", "for example , even though a patient visited hospital due to aggravating voiding symptoms in winter , the diagnostic procedures or medications were not changed because of season of winter , rather influenced by physician or medical costs . until recently , studies on the health care costs of bph were reported mostly in western societies ( 25 , 26 ) . in the pharmacy and medical claims data obtained from 61 us healthcare plans ,", "77,040 patients aged over 45 yr were examined , and the overall average annual cost of diagnosis and management for bph was 31.4 million usd . based on us census estimates ( july 2003 ) , the direct cost estimate was approximately 3 billion usd for the year after bph diagnosis . through this study", ", we figured out that korean taip in 2008 was 66 million usd ( 1 usd=1,103.36 korean won [ krw ] in 2008 ) which were relatively small expenditure of bph costs compared to the united states .", "however , it is not possible to describe briefly the gap between the two countries , because the medical system of korea and the united states was totally different .", "to explain the discrepancy of medical costs between korea and other countries would be an interesting subject in future research .", "the present study has several limitations that require consideration because of the methodological modality using the insurance claim data , as was the case for several studies reported in similar manner ( 4 , 5 , 26 , 27 ) .", "the insurance claims records might have underestimated the entire bph population if many patients with bph were not diagnosed or treated in health care institutions .", "additionally , only a single claim with a bph diagnosis was used to identify patients with the condition , so the incidence of this condition might be overestimated in the database because a single patient may have visited clinics several times a year .", "this study reflects data only for the 5 yr from 2004 to 2005 , which is a relatively short period of time .", "in addition , we did not have detailed clinical information for each patient , including symptom severity , duration , prostate size , and other medical or surgical history . using the hira data ,", "we did not demonstrated cost of each treatment modalities . however , as was shown in the us ( 12 ) , mist has gained enormous attention from the urologists and the number of mist performed is also increasing in korea .", "additional researches are warranted . despite these limitations , however , this study has important implications .", "this is the first nation - wide , population - based study demonstrating not only seasonal and age - specific variation in the incidence of bph , but also the amount of the economic costs for bph patients in korea .", "therefore , our large - scale data could provide the most reliable information about the incidence of bph and its cost for researchers and authorities formulating health care policy .", "health care utilization due to bph is rapidly increasing in korea and is more remarkable in the elderly population .", "our study is the largest scale population study , as well as the most objective clinical data in korea , so it could provide fundamental epidemiological information on bph ."]]}
{"article_id": "PMC5233774", "article_text": ["primary retroperitoneal tumors ( prts ) of vascular origin are a diverse group of rare abdominal neoplasms , both benign and malignant", ". the most frequent malignant tumors are liposarcoma and leiomyosarcoma , while the most often found benign tumors are lipoma , leiomyoma and cavernous hemangioma [ 25 ] .", "hemangiomas are a group of neoplasms originating from vascular tissue where benign tumors prevail . among these capillary hemangioma ,", "cavernous hemangiomas most frequently occur in the liver . other described localizations are skin , muscles , bones , central nervous system and retroperitoneal organs ( intestines , kidneys , adrenal glands , urinary bladder , uterus ) [ 616 ] .", "an interventional staged therapy is becoming more popular with the aim of reducing the diameter of the main hemangioma , especially that surrounded with diffuse hemangiomatosis .", "successful combination of transcatheter arterial embolization ( tae ) prior to the surgery of cavernous hemangioma of the liver was reported recently . nevertheless , the treatment of choice for primary retroperitoneal tumors is still radical surgical resection that leads to recovery .", "surgical techniques used for treatment of prts are : open procedures , laparoscopy , surgical endoscopy and percutaneous radiofrequency ablation [ 8 , 19 ] .", "the present case is a patient with giant retroperitoneal cavernous hemangioma originating from the ilium with atypical clinical course who was referred for surgical treatment .", "a 71-year - old female patient was referred to the department of surgical oncology with a giant retroperitoneal tumor located in the left iliac fossa .", "the patient discovered the abdominal tumor 15 years earlier , but as it did not cause any problems she refused to seek medical help .", "several months prior to admission to the department the first symptoms appeared : left lower limb edema , exertional dyspnea , abdominal distension and anemia with pallor and lowered hemoglobin level .", "physical examination revealed a large , skin modeling , nonpulsatile mass in the left iliac fossa ( fig .", "b  computed tomography scan revealing a large , left - sided retroperitoneal mass when abdominal contrast - enhanced computed tomography ( ct ) was performed , a tumor 20  17  18 cm in diameter lying on the iliac ala was found ( fig .", "an ultrasonography - guided fine needle biopsy was performed , revealing blood cells with necrotic masses and connective tissue which prevented precise diagnosis .", "blood laboratory findings were : anemia ( hb 8.8 g / dl ) , elevated white blood cell count ( 21.81", "g / l ) and high c - reactive protein level ( 100.8 mg / l ) . because of uncertainties regarding the histological type of the tumor , escalation of abdominal pain and left lower limb edema in recent weeks and the patient s strong willingness to remove the tumor , the decision to perform laparotomy was made ( figs .", "2 a , b ) . \n a  giant tumor with sigmoid colon tight on it .", "c  the tumor after resection ( arrow  peduncle connecting the tumor to the ala ) during the surgery a large mass on the ala was found . there were no signs of infiltration of neighboring organs , and the sigmoid colon was tight on the tumor . after separating the sigmoid and identification of the left ureter and iliac vessels", "massive damage to the internal surface of the ala was found in the place where the tumor grew .", "pathologic examination showed a tumor 20  19  16 cm in diameter , weighing 3000 g ( fig .", "histochemically the cells lining the lacunae were stained positively by markers for cd34 ( figs .", "3 a , b ) . \n a  microscopic image with h&e ( enlargement 10 ) . diag .", "staining with markers for cd34 confirm an endothelial origin of the cells lining the lacunae the postoperative follow - up is 3 years . within this time limb edema and", "this is caused by its initially symptomless course and therefore delayed presentation to a physician .", "the most frequent symptoms of retroperitoneal tumors are non - specific abdominal pain ( 51.8% ) and abdominal distension ( 18.7% ) .", "less frequent symptoms include constipation ( 8% ) , fever ( 8% ) , dysuria ( 4.3% ) , lower limb edema ( 6.5% ) , weight loss and cachexia ( 5% ) .", "the time of these symptoms occurrence varies from several months for sarcomas to years for benign tumors .", "patients usually visit a physician when they discover a pathological mass in the abdomen ( 60.4% ) . in the present case her first symptoms ( abdominal distension and limb edema ) occurred 15 years after discovering the tumor . on ct images retroperitoneal tumors present as heterogeneous pathological masses with hypoechogenic areas corresponding to necrotic zones in the tumor .", "we considered an interventional staged therapy with tae for its possible advantage of reduction of the diameter of the main tumor mass , and surrounding diffuse hemangiomatosis , which was reported in the case of cavernous hemangioma of the liver .", "the age of the patient with possible diffuse arteriosclerosis and the necessity to resect the symptomatic tumor were the arguments to abandon the percutaneous option .", "however , one can find publications on treatment of hepatic cavernous hemangioma with percutaneous radiofrequency ablation .", "in the present case the size and localization of the tumor and urgent necessity made the operation a high - risk procedure with possibility of resecting vital structures , such as the sigmoid colon or ureter . in the present case the long - term history of abdominal tumor indicated a benign tumor , while radiological and clinical findings suggested malignant disease .", "the literature review shows that in differential diagnosis of giant abdominal tumors , rare benign neoplasms should be considered in every case .", "radical surgical resection resulted in full recovery in our patient , which corresponds to commonly found conclusions despite the huge diameters and the origin of retroperitoneal tumors [ 20 , 21 ] . in the literature reports we found only 15 cases of retroperitoneal cavernous hemangiomas in kidneys , adrenal glands , urinary bladder , uterus and the retroperitoneal part of the rectum [ 615 ] .", "however , no description of cavernous hemangioma of the ilium was found ; therefore our report seems to be a unique contribution to contemporary knowledge ( tab .", "because of their initially symptomless course , primary retroperitoneal tumors are usually diagnosed in an advanced stage of the disease , which is the reason for many uncertainties regarding safe surgical resection .", "radical surgical resection remains the treatment of choice for primary retroperitoneal cavernous hemangiomas , although the decision of performing such operations should be taken after thorough analysis of indications ( escalation of symptoms ) and contraindications ( risk of damaging vital retroperitoneal organs with massive bleeding ) ."], "abstract_text": ["<S> retroperitoneal hemangiomas are very rare . </S>", "<S> this paper presents the case of a 71-year - old female patient with giant cavernous hemangioma of the retroperitoneum who underwent surgical treatment for abdominal pain and left lower limb edema . </S>", "<S> interventional staged treatment with percutaneous transcatheter arterial embolization prior to surgery was considered . </S>", "<S> radical resection of the tumor was performed , which caused the symptoms to abate . </S>", "<S> additionally a literature review of cases involving cavernous hemangioma in the retroperitoneal space is presented . </S>", "<S> no description of retroperitoneal cavernous hemangioma originating from the bowel was found in the analyzed reports . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Discussion", "Conclusions"], "sections": [["primary retroperitoneal tumors ( prts ) of vascular origin are a diverse group of rare abdominal neoplasms , both benign and malignant", ". the most frequent malignant tumors are liposarcoma and leiomyosarcoma , while the most often found benign tumors are lipoma , leiomyoma and cavernous hemangioma [ 25 ] .", "hemangiomas are a group of neoplasms originating from vascular tissue where benign tumors prevail . among these capillary hemangioma ,", "cavernous hemangiomas most frequently occur in the liver . other described localizations are skin , muscles , bones , central nervous system and retroperitoneal organs ( intestines , kidneys , adrenal glands , urinary bladder , uterus ) [ 616 ] .", "an interventional staged therapy is becoming more popular with the aim of reducing the diameter of the main hemangioma , especially that surrounded with diffuse hemangiomatosis .", "successful combination of transcatheter arterial embolization ( tae ) prior to the surgery of cavernous hemangioma of the liver was reported recently . nevertheless , the treatment of choice for primary retroperitoneal tumors is still radical surgical resection that leads to recovery .", "surgical techniques used for treatment of prts are : open procedures , laparoscopy , surgical endoscopy and percutaneous radiofrequency ablation [ 8 , 19 ] .", "the present case is a patient with giant retroperitoneal cavernous hemangioma originating from the ilium with atypical clinical course who was referred for surgical treatment ."], ["a 71-year - old female patient was referred to the department of surgical oncology with a giant retroperitoneal tumor located in the left iliac fossa .", "the patient discovered the abdominal tumor 15 years earlier , but as it did not cause any problems she refused to seek medical help .", "several months prior to admission to the department the first symptoms appeared : left lower limb edema , exertional dyspnea , abdominal distension and anemia with pallor and lowered hemoglobin level .", "physical examination revealed a large , skin modeling , nonpulsatile mass in the left iliac fossa ( fig .", "b  computed tomography scan revealing a large , left - sided retroperitoneal mass when abdominal contrast - enhanced computed tomography ( ct ) was performed , a tumor 20  17  18 cm in diameter lying on the iliac ala was found ( fig .", "an ultrasonography - guided fine needle biopsy was performed , revealing blood cells with necrotic masses and connective tissue which prevented precise diagnosis .", "blood laboratory findings were : anemia ( hb 8.8 g / dl ) , elevated white blood cell count ( 21.81", "g / l ) and high c - reactive protein level ( 100.8 mg / l ) . because of uncertainties regarding the histological type of the tumor , escalation of abdominal pain and left lower limb edema in recent weeks and the patient s strong willingness to remove the tumor , the decision to perform laparotomy was made ( figs .", "2 a , b ) . \n a  giant tumor with sigmoid colon tight on it .", "c  the tumor after resection ( arrow  peduncle connecting the tumor to the ala ) during the surgery a large mass on the ala was found . there were no signs of infiltration of neighboring organs , and the sigmoid colon was tight on the tumor . after separating the sigmoid and identification of the left ureter and iliac vessels", "massive damage to the internal surface of the ala was found in the place where the tumor grew .", "pathologic examination showed a tumor 20  19  16 cm in diameter , weighing 3000 g ( fig .", "histochemically the cells lining the lacunae were stained positively by markers for cd34 ( figs .", "3 a , b ) . \n a  microscopic image with h&e ( enlargement 10 ) . diag .", "staining with markers for cd34 confirm an endothelial origin of the cells lining the lacunae the postoperative follow - up is 3 years . within this time limb edema and"], ["this is caused by its initially symptomless course and therefore delayed presentation to a physician .", "the most frequent symptoms of retroperitoneal tumors are non - specific abdominal pain ( 51.8% ) and abdominal distension ( 18.7% ) .", "less frequent symptoms include constipation ( 8% ) , fever ( 8% ) , dysuria ( 4.3% ) , lower limb edema ( 6.5% ) , weight loss and cachexia ( 5% ) .", "the time of these symptoms occurrence varies from several months for sarcomas to years for benign tumors .", "patients usually visit a physician when they discover a pathological mass in the abdomen ( 60.4% ) . in the present case her first symptoms ( abdominal distension and limb edema ) occurred 15 years after discovering the tumor . on ct images retroperitoneal tumors present as heterogeneous pathological masses with hypoechogenic areas corresponding to necrotic zones in the tumor .", "we considered an interventional staged therapy with tae for its possible advantage of reduction of the diameter of the main tumor mass , and surrounding diffuse hemangiomatosis , which was reported in the case of cavernous hemangioma of the liver .", "the age of the patient with possible diffuse arteriosclerosis and the necessity to resect the symptomatic tumor were the arguments to abandon the percutaneous option .", "however , one can find publications on treatment of hepatic cavernous hemangioma with percutaneous radiofrequency ablation .", "in the present case the size and localization of the tumor and urgent necessity made the operation a high - risk procedure with possibility of resecting vital structures , such as the sigmoid colon or ureter . in the present case the long - term history of abdominal tumor indicated a benign tumor , while radiological and clinical findings suggested malignant disease .", "the literature review shows that in differential diagnosis of giant abdominal tumors , rare benign neoplasms should be considered in every case .", "radical surgical resection resulted in full recovery in our patient , which corresponds to commonly found conclusions despite the huge diameters and the origin of retroperitoneal tumors [ 20 , 21 ] . in the literature reports we found only 15 cases of retroperitoneal cavernous hemangiomas in kidneys , adrenal glands , urinary bladder , uterus and the retroperitoneal part of the rectum [ 615 ] .", "however , no description of cavernous hemangioma of the ilium was found ; therefore our report seems to be a unique contribution to contemporary knowledge ( tab ."], ["because of their initially symptomless course , primary retroperitoneal tumors are usually diagnosed in an advanced stage of the disease , which is the reason for many uncertainties regarding safe surgical resection .", "radical surgical resection remains the treatment of choice for primary retroperitoneal cavernous hemangiomas , although the decision of performing such operations should be taken after thorough analysis of indications ( escalation of symptoms ) and contraindications ( risk of damaging vital retroperitoneal organs with massive bleeding ) ."]]}
{"article_id": "PMC4375300", "article_text": ["the pathogenesis of gmd in pd patients is related to factors such as weight gain and insulin resistance evoked by peritoneal glucose load [ 13 ] .", "the periodical evaluation of pd patients for gmd is of clinical relevance and should be an element of the surveillance protocol .", "the  gold standard  method used for the diagnosis of gmd defined by the world health organization ( who ) in 1998 uses the level of 2-h post - challenge plasma glucose ( 2-hpg ) in the oral glucose tolerance test ( 75-g ogtt ) .", "criteria for the diagnosis are the following : fasting plasma glucose ( fpg ) 100  mg / dl ( 5.6  mmol / l ) to 125  mg / dl ( 6.9  mmol / l ) ( impaired fasting glucose , ifg ) ; 2-h pg in the 75-g ogtt 140  mg / dl ( 7.8  mmol / l ) to 199  mg / dl ( 11.0  mmol / l ) ( impaired glucose tolerance , igt ) ; 2-h pg in the 75-g ogtt or random 200  mg / dl ( 11.1  mmol / l ) ( dm ) . the utility of the recently recommended use of hba1c in terms of diabetes diagnosis ( diagnostic threshold of 6.5  % ) still remains to be validated .", "the population at a particular risk of glucose metabolism disorders includes renal transplant recipients , pregnant women , and patients treated with peritoneal dialysis . for each of these groups", ", there is a lack of clear guidelines for the detection and diagnosis of gmd .", "therefore , alternative methods for detecting all glucose metabolism disorders ( ifg , igt , and dm ) in the population of high risk of hyperglycemia have been investigated .", "the issue of glucose metabolism disorders ( gmd ) in pd patients has regained interest in recent years .", "this increase in clinical relevance was caused by new evidence that even mild hyperglycemia in the dialysis period can be harmful in the long term , being associated with worse survival and elevated risk of post - transplant diabetes mellitus [ 68 ] .", "treatment with peritoneal dialysis is inherently associated with an additional load of glucose adsorbed from dialysis fluid .", "the effect of glucose deriving from the peritoneal cavity on the incidence of glucose intolerance still evokes some ambiguity , and published data are equivocal [ 13 , 911 ] .", "the recent data suggest that high glucose exposure from dialysis solution may be a risk factor for vascular calcification in non - diabetic pd patients .", "there seem to be some analogies between the occurrence of de novo gmd in patients treated with pd and gestational diabetes ( gdm ) , in which a combination of increased maternal adiposity and naturally occurring human placental hormones produces insulin resistance [ 13 , 14 ] . in both cases ,", "gestational diabetes mellitus is defined as  any degree of glucose intolerance with onset or first recognition during pregnancy  .", "the mechanisms that lead to chronic insulin resistance in gdm are varied as they are in the general population .", "up to 2012 gdm was diagnosed by a two - step approach using a sequential model of universal screening with a 1-h 50-g glucose challenge test ( 50-g gct ) followed by a diagnostic 75-g oral glucose tolerance test ( 75-g ogtt ) for women with a positive screening test [ threshold value 140  mg / dl ( 7.8  mmol / l ) ] .", "the gdm was recognized in 75-g ogtt when the plasma glucose value was 140  mg / dl .", "ada recommendations for 2011 adopted new , stricter criteria for the diagnosis of gestational diabetes and confirmed them for 2012 . according to the current recommendations for all pregnant women between 24 and 28  weeks of pregnancy ,", "gestational diabetes is diagnosed if the fasting plasma glucose is at least 92  mg / dl ( 5.1  mmol / l ) , after 1  h 180  mg / dl ( 10.0  mmol / l ) , and after 2  h 153  mg / dl ( 8.5  mmol / l ) .", "these criteria were based on the results of the hapo study   and recommendations of the research group of the international association of the diabetes and pregnancy study groups ( iadpsg ) .", "there is also a critical discussion concerning the methods and criteria for the new ada 2012 diagnosis of gestational diabetes [ 2023 ] . currently , there are no clear recommendations for diagnosis and classification of glucose disturbances occurring de novo in a population of patients on pd . in our opinion ,", "the who  gold standard  is not appropriate for pd patients because gmd differ from those in the general population , and other screening strategies should be explored .", "considering the mentioned similarities , we aimed to examine the utility of the oral glucose tolerance screening test ( 50-g gct ) for pd patients , which is used in a two - step approach to evaluate gestational diabetes . the first step ", "the 50-g gct  is performed at any time of the day ( the fasting state is not required ) , using 50  g of glucose , and the plasma glucose was measured at 1  h after administration .", "in the current study , we aimed to evaluate the applicability of the 50-g gct for the detection of gmd in non - diabetic continuous ambulatory pd patients with normal fasting glucose levels .", "the study was performed in 20 prevalent patients without history of diabetes before pd treatment onset who had been on a pd program at our institution as their first dialysis modality .", "the median age of patients was 44.19  years [ interquartile range ( iqr ) 34.1556.25 ] , 9 being males and 11 being females , all caucasian with cause of end - stage renal disease ( esrd ) being glomerular disease in 12 patients ( 60  % ) , hypertensive nephropathy in four patients ( 20  % ) , interstitial nephropathy in three patients ( 15  % ) , and polycystic kidney disease in one patient ( 5  % ) .", "the median duration of pd treatment before inclusion in the study was 15.34  months ( iqr 10.821.34 ) . \n", "patients were assessed at the study onset ( on the day of the 50-g gct ) and at the end of pd treatment ( 13 patients ) or at end of the observation period ( december 2012 ) for continuing the pd program ( seven patients ) . at the study onset", ", all patients underwent 50-g gct with a glucose dose of 50  g. the test is performed at any time of the day ( the fasting state is not required ) , using 50  g of glucose , and the glucose is measured at 1  h after the administration .", "interpretation of our 50-g gct results was as follows : glucose < 140  mg / dl  normal value ; 140199  mg / dl  impaired glucose tolerance ( igt ) , 200  mg / dl  diabetes . \n in case of a fasting glucose value 100125 ", "mg / dl , and normal value in 50-g gct  impaired fasting glucose ( ifg ) was diagnosed .", "the 50-g gct was performed in patients with an empty abdomen after the night dwell draining . during the night , all patients used 1.36  % glucose containing dialysis solution .", "the glucose was measured at 1  h after administration ; the fasting state was not required .", "in addition , the following parameters of glucose metabolism were measured : c - peptide , fasting insulin serum concentration , and the glycated hemoglobin level ( hba1c ) .", "the concentrations of c - peptide and insulin were evaluated in serum samples , and hba1c in whole blood using a chemiluminescent microparticle immunoassay ( cmia ) on the architect system ( abbott , usa ) .", "based on fasting insulin and glucose values , insulin resistance ( ir ) was determined according to the homeostasis model assessment insulin resistance ( homa - ir ) .", "they encompassed blood pressure ( bp ) , serum albumin , lipid profiles , hb , crp , liver tests , mean daily peritoneal glucose load , ultrafiltration volume , residual diuresis , weekly dialytic creatinine clearance ( ccr ) , weekly dialytic kt / v , and other laboratory parameters ( uric acid , pth , tsh , fe , tsat , and electrolytes ) .", "systolic and diastolic bp , residual gfr , weekly dialytic ccr , and kt / v were measured by standard methods .", "the estimated peritoneal glucose load was calculated by the product of the volume and the glucose concentration of each exchange .", "the glucose load did not differ between the patients and was similar in all study patients during the follow - up .", "the patients were prospectively followed for a median time of 25.8  months ( iqr 18.9933.57 ) , and 50-g gct was repeated at the end of the observation period . the statistical analysis was performed with r for windows , version 2.15.1 , and medcalc for windows , version 12.3.1.0 .", "clinical characteristics at the study onset are shown in table  1.table  1clinical characteristics at the study onsetvariable \n n \n medianiqr ( interquartile range)fasting glucose level ( mg / dl)2091.583.596glucose level1  h after gct ( mg / dl)20165137.5195hba1c ( % ) 205.55.45.7c - peptide ( ng / ml)207.245.1210.95fasting insulin serum concentration ( u / ml)2010.347.7916.05homa - ir202.131.573.53systolic bp ( mmhg)20135120155.5diastolic bp ( mmhg)2087.58090serum albumin ( g / dl)203.83.653.95total cholesterol ( mg / dl)20196179231hdl cholesterol ( mg / dl)20433852ldl cholesterol ( mg / dl)20128.5105.5143triglycerides ( mg / dl)20164.5130226.5hb ( g / dl)2010.610.111.85crp ( mg / l)202.091.116.69aspat ( iu / l)2017.51421alat ( iu / l)20171225uric acid ( mg / dl)206.35.557.3pth20817490.51,221.5tsh202.191.054.11fe207451.588tsat ( % ) 2025.41933.9ca ( mmol / l)2098.259.2p ( mg / dl)206.44.857.25mean daily peritoneal glucose load ( g/24  h)20120120120ultrafiltration volume ( ml/24  h)201,3007251,500residual diuresis ( ml/24  h)201,1505001,500weekly dialytic ccr200.690.620.75weekly dialytic kt / v202.192.112.7bmi  total ( kg / m)2025.1423.8327.63 clinical characteristics at the study onset 50-g gct revealed gmd in 15 investigated patients ( 75  % ) .", "the most frequent abnormality was igt ( plasma glucose between 140 and 199  mg / dl ) , which occurred in 11 patients ( 55  % ) .", "however , despite normal fasting glucose , in four patients ( 20  % ) , the glucose concentrations at 1  h after oral load were above 200  mg / dl , suggesting a preliminary diabetes diagnosis .", "the other glucose metabolism indicators exhibited little clinical utility for the detection of abnormal glucose tolerance in pd patients .", "the median percentage of hba1c was in the normal range ( 5.5  % ) , exceeding the upper limit only in two patients ( 10  % ) .", "median value of homa - ir was 2.13 , being above 2.6 ( the cutoff for ir ) only in seven patients ( 35  % ) .", "c - peptide concentrations were significantly higher than in non - diabetic healthy individuals ( p  =  0.03 ) .", "a significant correlation was found between homa - ir and c - peptide measurements ( p  =  0.001 ,  0.677 ) .", "homa - ir and c - peptide determinations did not correlate with other investigated parameters , i.e. , serum albumin , crp , lipid profile , glucose load in the dialysis fluid , dialysis dose , ultrafiltration , and residual diuresis .", "patients with gmd were recommended a low - carbohydrate diet , lifestyle modification to promote weight loss , and increased physical activity .", "the adherence was checked at a monthly interval during patients visit to the pd clinic .", "four patients , in whom diabetes was recognized after the first 50-g gct , additionally received short - acting sulphonylurea compounds ( glipizide ) for a few months ( 35 ) , and then , a diet satisfactorily corrected the glucose value .", "the patients were prospectively observed , and after a median time of 25.8  months , the glucose metabolism evaluation was repeated . during the second 50-g gct ,", "igt persisted in eight patients ( 40  % ) , and of particular note , ifg ( 100125  mg / dl ) occurred in four patients ( 20  % ) despite 50-g gct being in the normal range .", "all patients who exhibited the highest abnormalities suggesting diabetes in the first gct when retested at the end of the observation achieved a normal range . \n", "glucose metabolism disorders at study onset and at the end of observation are shown in table  2.table  2glucose metabolism disorders at study onset and at the end of observationgct 50  ggmdstudy onsetend of observationifg0 pts4 ptsfasting glucose level ( mg / dl )  median / iqr124/112153glucose level1  h after gct ( mg / dl )  median / iqr176/152189igt11 pts8 ptsfasting glucose level ( mg / dl )  median / iqr93/839587/78.2590.25glucose level1  h after gct ( mg / dl )  median / iqr167/149184150/146167dm4 pts0 ptsfasting glucose level ( mg / dl )  median / iqr93/83.399.8glucose level1  h after gct ( mg / dl )  median / iq207/204236 glucose metabolism disorders at study onset and at the end of observation", "the occurrence of gmd in 75  % of the study subjects representing a small single - center cohort of prevalent pd patients gives a good illustration of the epidemiological dimension of the problem .", "after the first oral glucose load of 50  g , igt was revealed in 11 subjects ( 55  % ) , and a glucose rise suggesting diabetes appeared in four patients ( 20  % ) .", "the 50-g gct was performed in prevalent continuous ambulatory peritoneal dialysis ( capd ) patients , who had been on dialysis for a median time of 15.34  months .", "surprisingly , the literature on gmd in the pd population is very scanty . in two works published in the 1980s , lindholm et al .", "[ 25 , 26 ] based on their own modification of ogtt ( using 1  g glucose per kg body weight ) carried out in 15 patients concluded that pd treatment up to 1  year does not deteriorate glucose metabolism .", ". published results of ogtt diminished from standard 7550  g glucose load accomplished in six patients being on pd for at least 6  months .", "the test was applied exclusively to detect insulin resistance , and the data on increased glycemic and insulinemic responses were presented . more informative and extensive", "investigation was done by cheng et al .   in 35 nondiabetic patients treated by pd for more than 1  year .", "they challenged patients by standard 75 ogtt and found igt in 31  % . in a larger - scale epidemiological study ,", "insulin resistance was significantly more frequent in pd patients than in hemodialysis and pre - dialysis subjects ( 47 vs. 21 and 26  % , respectively ) . in a study by tatar et al .", ", insulin resistance was an independent risk factor for arterial stiffness in nondiabetic pd patients older than 50  years . in our present single - center study , we found homa - ir values indicating insulin resistance ( > 2.6 ) in seven patients ( 35  % ) .", "the c - peptide levels were significantly higher in the pd group than in healthy individuals and correlated closely with homa - ir .", "it should be mentioned that the elevated c - peptide concentrations in pd patients are mainly a consequence of the fact that 70  % of this peptide is eliminated by the kidney route .", "however , the strong correlation with homa - ir can be treated as a sign of preserved excretory  pancreatic cells capacity .", "commenting on the results of our study , it is worth emphasizing that the study group encompasses low - risk , middle - aged , non - obese subjects , with normal fasting glucose , normal hba1c , and slightly elevated cholesterol level .", "the only more pronounced abnormality which is associated with glucose intolerance in epidemiological investigation was hypertriglyceridemia .", "discovery of gmd in 75  % of patients with such clinical characteristics highlights the clinical significance of this issue .", "we are conscious that 50-g gct was not validated in the pd population , but it seems to be well suited for a person on pd treatment due to its simplicity and convenience .", "a plasma glucose value  140  mg / dl , we gave up on performing the second step 75-g ogtt , because the aim of our study was not to recognize diabetes by who criteria , but reveal a group with special risk of hyperglycemia .", "we treated our test as a screening , not diagnostic method , also taking into account the cost  benefit relationship .", "the 50-g gct allows quick verification of glucose disturbances in patients with an additional daily dose of glucose in the dialysis fluids . in contradistinction to standard 75-g ogtt , it does not require patients to keep coming to the pd clinic at different appointment hours without eating .", "it obviously does not provide a definitive diagnosis . in the next project following these preliminary data", "however , we believe that abnormalities revealed by 50-g gct are already of clinical value alerting the patient and physician . it provides a stimulus for preventive measures , which as shown by our experience can be effective , i.e. , the normalization of the test in four patients with the worst results at the onset . the other positive aspect of the current study is supplying evidence that gmd are not inexorably progressive under pd treatment .", "the maintenance of normal glucose metabolism in pd patients is in the context of obtained results a feasible goal .", "its achievement could bring benefits in terms of longer patient survival and lower risk of post - transplant diabetes development .", "the oral glucose challenge screening gct test appears to be a sensitive instrument for the detection of glucose metabolism disorders in pd patients with normal fasting glucose ."], "abstract_text": ["<S> background \n the aim was to evaluate the clinical utility of the oral glucose tolerance screening test ( 50-g gct  glucose challenge test ) for the detection of glucose metabolism disorders ( gmd ) in peritoneal dialysis ( pd ) patients with normal fasting glucose levels . \n </S>", "<S> methods \n the 50-g gct was performed in 20 prevalent patients without history of diabetes before pd treatment onset , who had been on dialysis for a median time of 15.34  months . </S>", "<S> in addition , other indicators of glucose metabolism were measured : c - peptide , fasting insulin serum concentration , and the glycated hemoglobin level ( hba1c ) . </S>", "<S> the patients were prospectively followed for a median time of 25.8  months . \n </S>", "<S> results50-g gct revealed gmd in 15 studied patients ( 75  % ) impaired glucose tolerance in 11 patients ( 55  % ) and diabetes mellitus in four patients ( 20  % ) . hba1c and insulin resistance , estimated by homeostasis model assessment , </S>", "<S> were elevated in two ( 10  % ) and seven ( 35  % ) patients , respectively . in patients with gmd , dietetic and pharmacologic interventions were performed . </S>", "<S> when the 50-g gct was repeated at the end of the observation period , 12 ( 60  % ) patients reported gmd , with no case of diabetes.conclusion50-g gct appears to be a simple and practical tool for the detection of gmd in pd patients with normal fasting glucose . </S>", "<S> timely therapeutic intervention can effectively inhibit the progression of glucose intolerance during pd treatment . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Results", "Discussion", "Conclusion", "Conflict of interest"], "sections": [["the pathogenesis of gmd in pd patients is related to factors such as weight gain and insulin resistance evoked by peritoneal glucose load [ 13 ] .", "the periodical evaluation of pd patients for gmd is of clinical relevance and should be an element of the surveillance protocol .", "the  gold standard  method used for the diagnosis of gmd defined by the world health organization ( who ) in 1998 uses the level of 2-h post - challenge plasma glucose ( 2-hpg ) in the oral glucose tolerance test ( 75-g ogtt ) .", "criteria for the diagnosis are the following : fasting plasma glucose ( fpg ) 100  mg / dl ( 5.6  mmol / l ) to 125  mg / dl ( 6.9  mmol / l ) ( impaired fasting glucose , ifg ) ; 2-h pg in the 75-g ogtt 140  mg / dl ( 7.8  mmol / l ) to 199  mg / dl ( 11.0  mmol / l ) ( impaired glucose tolerance , igt ) ; 2-h pg in the 75-g ogtt or random 200  mg / dl ( 11.1  mmol / l ) ( dm ) . the utility of the recently recommended use of hba1c in terms of diabetes diagnosis ( diagnostic threshold of 6.5  % ) still remains to be validated .", "the population at a particular risk of glucose metabolism disorders includes renal transplant recipients , pregnant women , and patients treated with peritoneal dialysis . for each of these groups", ", there is a lack of clear guidelines for the detection and diagnosis of gmd .", "therefore , alternative methods for detecting all glucose metabolism disorders ( ifg , igt , and dm ) in the population of high risk of hyperglycemia have been investigated .", "the issue of glucose metabolism disorders ( gmd ) in pd patients has regained interest in recent years .", "this increase in clinical relevance was caused by new evidence that even mild hyperglycemia in the dialysis period can be harmful in the long term , being associated with worse survival and elevated risk of post - transplant diabetes mellitus [ 68 ] .", "treatment with peritoneal dialysis is inherently associated with an additional load of glucose adsorbed from dialysis fluid .", "the effect of glucose deriving from the peritoneal cavity on the incidence of glucose intolerance still evokes some ambiguity , and published data are equivocal [ 13 , 911 ] .", "the recent data suggest that high glucose exposure from dialysis solution may be a risk factor for vascular calcification in non - diabetic pd patients .", "there seem to be some analogies between the occurrence of de novo gmd in patients treated with pd and gestational diabetes ( gdm ) , in which a combination of increased maternal adiposity and naturally occurring human placental hormones produces insulin resistance [ 13 , 14 ] . in both cases ,", "gestational diabetes mellitus is defined as  any degree of glucose intolerance with onset or first recognition during pregnancy  .", "the mechanisms that lead to chronic insulin resistance in gdm are varied as they are in the general population .", "up to 2012 gdm was diagnosed by a two - step approach using a sequential model of universal screening with a 1-h 50-g glucose challenge test ( 50-g gct ) followed by a diagnostic 75-g oral glucose tolerance test ( 75-g ogtt ) for women with a positive screening test [ threshold value 140  mg / dl ( 7.8  mmol / l ) ] .", "the gdm was recognized in 75-g ogtt when the plasma glucose value was 140  mg / dl .", "ada recommendations for 2011 adopted new , stricter criteria for the diagnosis of gestational diabetes and confirmed them for 2012 . according to the current recommendations for all pregnant women between 24 and 28  weeks of pregnancy ,", "gestational diabetes is diagnosed if the fasting plasma glucose is at least 92  mg / dl ( 5.1  mmol / l ) , after 1  h 180  mg / dl ( 10.0  mmol / l ) , and after 2  h 153  mg / dl ( 8.5  mmol / l ) .", "these criteria were based on the results of the hapo study   and recommendations of the research group of the international association of the diabetes and pregnancy study groups ( iadpsg ) .", "there is also a critical discussion concerning the methods and criteria for the new ada 2012 diagnosis of gestational diabetes [ 2023 ] . currently , there are no clear recommendations for diagnosis and classification of glucose disturbances occurring de novo in a population of patients on pd . in our opinion ,", "the who  gold standard  is not appropriate for pd patients because gmd differ from those in the general population , and other screening strategies should be explored .", "considering the mentioned similarities , we aimed to examine the utility of the oral glucose tolerance screening test ( 50-g gct ) for pd patients , which is used in a two - step approach to evaluate gestational diabetes . the first step ", "the 50-g gct  is performed at any time of the day ( the fasting state is not required ) , using 50  g of glucose , and the plasma glucose was measured at 1  h after administration .", "in the current study , we aimed to evaluate the applicability of the 50-g gct for the detection of gmd in non - diabetic continuous ambulatory pd patients with normal fasting glucose levels ."], ["the study was performed in 20 prevalent patients without history of diabetes before pd treatment onset who had been on a pd program at our institution as their first dialysis modality .", "the median age of patients was 44.19  years [ interquartile range ( iqr ) 34.1556.25 ] , 9 being males and 11 being females , all caucasian with cause of end - stage renal disease ( esrd ) being glomerular disease in 12 patients ( 60  % ) , hypertensive nephropathy in four patients ( 20  % ) , interstitial nephropathy in three patients ( 15  % ) , and polycystic kidney disease in one patient ( 5  % ) .", "the median duration of pd treatment before inclusion in the study was 15.34  months ( iqr 10.821.34 ) . \n", "patients were assessed at the study onset ( on the day of the 50-g gct ) and at the end of pd treatment ( 13 patients ) or at end of the observation period ( december 2012 ) for continuing the pd program ( seven patients ) . at the study onset", ", all patients underwent 50-g gct with a glucose dose of 50  g. the test is performed at any time of the day ( the fasting state is not required ) , using 50  g of glucose , and the glucose is measured at 1  h after the administration .", "interpretation of our 50-g gct results was as follows : glucose < 140  mg / dl  normal value ; 140199  mg / dl  impaired glucose tolerance ( igt ) , 200  mg / dl  diabetes . \n in case of a fasting glucose value 100125 ", "mg / dl , and normal value in 50-g gct  impaired fasting glucose ( ifg ) was diagnosed .", "the 50-g gct was performed in patients with an empty abdomen after the night dwell draining . during the night , all patients used 1.36  % glucose containing dialysis solution .", "the glucose was measured at 1  h after administration ; the fasting state was not required .", "in addition , the following parameters of glucose metabolism were measured : c - peptide , fasting insulin serum concentration , and the glycated hemoglobin level ( hba1c ) .", "the concentrations of c - peptide and insulin were evaluated in serum samples , and hba1c in whole blood using a chemiluminescent microparticle immunoassay ( cmia ) on the architect system ( abbott , usa ) .", "based on fasting insulin and glucose values , insulin resistance ( ir ) was determined according to the homeostasis model assessment insulin resistance ( homa - ir ) .", "they encompassed blood pressure ( bp ) , serum albumin , lipid profiles , hb , crp , liver tests , mean daily peritoneal glucose load , ultrafiltration volume , residual diuresis , weekly dialytic creatinine clearance ( ccr ) , weekly dialytic kt / v , and other laboratory parameters ( uric acid , pth , tsh , fe , tsat , and electrolytes ) .", "systolic and diastolic bp , residual gfr , weekly dialytic ccr , and kt / v were measured by standard methods .", "the estimated peritoneal glucose load was calculated by the product of the volume and the glucose concentration of each exchange .", "the glucose load did not differ between the patients and was similar in all study patients during the follow - up .", "the patients were prospectively followed for a median time of 25.8  months ( iqr 18.9933.57 ) , and 50-g gct was repeated at the end of the observation period . the statistical analysis was performed with r for windows , version 2.15.1 , and medcalc for windows , version 12.3.1.0 ."], ["clinical characteristics at the study onset are shown in table  1.table  1clinical characteristics at the study onsetvariable \n n \n medianiqr ( interquartile range)fasting glucose level ( mg / dl)2091.583.596glucose level1  h after gct ( mg / dl)20165137.5195hba1c ( % ) 205.55.45.7c - peptide ( ng / ml)207.245.1210.95fasting insulin serum concentration ( u / ml)2010.347.7916.05homa - ir202.131.573.53systolic bp ( mmhg)20135120155.5diastolic bp ( mmhg)2087.58090serum albumin ( g / dl)203.83.653.95total cholesterol ( mg / dl)20196179231hdl cholesterol ( mg / dl)20433852ldl cholesterol ( mg / dl)20128.5105.5143triglycerides ( mg / dl)20164.5130226.5hb ( g / dl)2010.610.111.85crp ( mg / l)202.091.116.69aspat ( iu / l)2017.51421alat ( iu / l)20171225uric acid ( mg / dl)206.35.557.3pth20817490.51,221.5tsh202.191.054.11fe207451.588tsat ( % ) 2025.41933.9ca ( mmol / l)2098.259.2p ( mg / dl)206.44.857.25mean daily peritoneal glucose load ( g/24  h)20120120120ultrafiltration volume ( ml/24  h)201,3007251,500residual diuresis ( ml/24  h)201,1505001,500weekly dialytic ccr200.690.620.75weekly dialytic kt / v202.192.112.7bmi  total ( kg / m)2025.1423.8327.63 clinical characteristics at the study onset 50-g gct revealed gmd in 15 investigated patients ( 75  % ) .", "the most frequent abnormality was igt ( plasma glucose between 140 and 199  mg / dl ) , which occurred in 11 patients ( 55  % ) .", "however , despite normal fasting glucose , in four patients ( 20  % ) , the glucose concentrations at 1  h after oral load were above 200  mg / dl , suggesting a preliminary diabetes diagnosis .", "the other glucose metabolism indicators exhibited little clinical utility for the detection of abnormal glucose tolerance in pd patients .", "the median percentage of hba1c was in the normal range ( 5.5  % ) , exceeding the upper limit only in two patients ( 10  % ) .", "median value of homa - ir was 2.13 , being above 2.6 ( the cutoff for ir ) only in seven patients ( 35  % ) .", "c - peptide concentrations were significantly higher than in non - diabetic healthy individuals ( p  =  0.03 ) .", "a significant correlation was found between homa - ir and c - peptide measurements ( p  =  0.001 ,  0.677 ) .", "homa - ir and c - peptide determinations did not correlate with other investigated parameters , i.e. , serum albumin , crp , lipid profile , glucose load in the dialysis fluid , dialysis dose , ultrafiltration , and residual diuresis .", "patients with gmd were recommended a low - carbohydrate diet , lifestyle modification to promote weight loss , and increased physical activity .", "the adherence was checked at a monthly interval during patients visit to the pd clinic .", "four patients , in whom diabetes was recognized after the first 50-g gct , additionally received short - acting sulphonylurea compounds ( glipizide ) for a few months ( 35 ) , and then , a diet satisfactorily corrected the glucose value .", "the patients were prospectively observed , and after a median time of 25.8  months , the glucose metabolism evaluation was repeated . during the second 50-g gct ,", "igt persisted in eight patients ( 40  % ) , and of particular note , ifg ( 100125  mg / dl ) occurred in four patients ( 20  % ) despite 50-g gct being in the normal range .", "all patients who exhibited the highest abnormalities suggesting diabetes in the first gct when retested at the end of the observation achieved a normal range . \n", "glucose metabolism disorders at study onset and at the end of observation are shown in table  2.table  2glucose metabolism disorders at study onset and at the end of observationgct 50  ggmdstudy onsetend of observationifg0 pts4 ptsfasting glucose level ( mg / dl )  median / iqr124/112153glucose level1  h after gct ( mg / dl )  median / iqr176/152189igt11 pts8 ptsfasting glucose level ( mg / dl )  median / iqr93/839587/78.2590.25glucose level1  h after gct ( mg / dl )  median / iqr167/149184150/146167dm4 pts0 ptsfasting glucose level ( mg / dl )  median / iqr93/83.399.8glucose level1  h after gct ( mg / dl )  median / iq207/204236 glucose metabolism disorders at study onset and at the end of observation"], ["the occurrence of gmd in 75  % of the study subjects representing a small single - center cohort of prevalent pd patients gives a good illustration of the epidemiological dimension of the problem .", "after the first oral glucose load of 50  g , igt was revealed in 11 subjects ( 55  % ) , and a glucose rise suggesting diabetes appeared in four patients ( 20  % ) .", "the 50-g gct was performed in prevalent continuous ambulatory peritoneal dialysis ( capd ) patients , who had been on dialysis for a median time of 15.34  months .", "surprisingly , the literature on gmd in the pd population is very scanty . in two works published in the 1980s , lindholm et al .", "[ 25 , 26 ] based on their own modification of ogtt ( using 1  g glucose per kg body weight ) carried out in 15 patients concluded that pd treatment up to 1  year does not deteriorate glucose metabolism .", ". published results of ogtt diminished from standard 7550  g glucose load accomplished in six patients being on pd for at least 6  months .", "the test was applied exclusively to detect insulin resistance , and the data on increased glycemic and insulinemic responses were presented . more informative and extensive", "investigation was done by cheng et al .   in 35 nondiabetic patients treated by pd for more than 1  year .", "they challenged patients by standard 75 ogtt and found igt in 31  % . in a larger - scale epidemiological study ,", "insulin resistance was significantly more frequent in pd patients than in hemodialysis and pre - dialysis subjects ( 47 vs. 21 and 26  % , respectively ) . in a study by tatar et al .", ", insulin resistance was an independent risk factor for arterial stiffness in nondiabetic pd patients older than 50  years . in our present single - center study , we found homa - ir values indicating insulin resistance ( > 2.6 ) in seven patients ( 35  % ) .", "the c - peptide levels were significantly higher in the pd group than in healthy individuals and correlated closely with homa - ir .", "it should be mentioned that the elevated c - peptide concentrations in pd patients are mainly a consequence of the fact that 70  % of this peptide is eliminated by the kidney route .", "however , the strong correlation with homa - ir can be treated as a sign of preserved excretory  pancreatic cells capacity .", "commenting on the results of our study , it is worth emphasizing that the study group encompasses low - risk , middle - aged , non - obese subjects , with normal fasting glucose , normal hba1c , and slightly elevated cholesterol level .", "the only more pronounced abnormality which is associated with glucose intolerance in epidemiological investigation was hypertriglyceridemia .", "discovery of gmd in 75  % of patients with such clinical characteristics highlights the clinical significance of this issue .", "we are conscious that 50-g gct was not validated in the pd population , but it seems to be well suited for a person on pd treatment due to its simplicity and convenience .", "a plasma glucose value  140  mg / dl , we gave up on performing the second step 75-g ogtt , because the aim of our study was not to recognize diabetes by who criteria , but reveal a group with special risk of hyperglycemia .", "we treated our test as a screening , not diagnostic method , also taking into account the cost  benefit relationship .", "the 50-g gct allows quick verification of glucose disturbances in patients with an additional daily dose of glucose in the dialysis fluids . in contradistinction to standard 75-g ogtt , it does not require patients to keep coming to the pd clinic at different appointment hours without eating .", "it obviously does not provide a definitive diagnosis . in the next project following these preliminary data", "however , we believe that abnormalities revealed by 50-g gct are already of clinical value alerting the patient and physician . it provides a stimulus for preventive measures , which as shown by our experience can be effective , i.e. , the normalization of the test in four patients with the worst results at the onset . the other positive aspect of the current study is supplying evidence that gmd are not inexorably progressive under pd treatment .", "the maintenance of normal glucose metabolism in pd patients is in the context of obtained results a feasible goal .", "its achievement could bring benefits in terms of longer patient survival and lower risk of post - transplant diabetes development ."], ["the oral glucose challenge screening gct test appears to be a sensitive instrument for the detection of glucose metabolism disorders in pd patients with normal fasting glucose ."], [""]]}
{"article_id": "PMC3320745", "article_text": ["proteomics has emerged as a valuable tool \n for the identification \n of biomarkers associated with human disease including those resulting from very subtle changes in normal cell \n functions and signaling pathways .", "proteomic technology has advanced \n to a state where thousands of proteins can be identified within complex \n samples , yet disease - based studies using fresh or frozen tissues are limited \n by a lack of available specimens for longitudinal clinical investigations . \n", "in contrast , there are millions of archival formalin - fixed , paraffin - embedded \n ( ffpe ) tissues for which the clinical course of disease and response \n to therapy has been established .", "ffpe tissue studies are affected \n greatly by sample collection , tissue processing , and archival time .", "though these factors are difficult to control \n in archival samples , improvements in techniques such as mtraq have \n made relative quantitation of disease biomarkers in ffpe tissue possible .", "improving protein extraction and detection of less abundant protein \n biomarkers is also of critical importance in proteomic analysis .", "protein \n modifications by formaldehyde treatment and histological processing significantly limit the use ffpe tissues for proteomic analyses . \n", "this has prevented proteomic studies of the clinical course of diseases , \n such as prostate and breast cancer , that evolve slowly or where the \n time between treatment and recurrence is long .", "coupling proteomic \n investigations with the retrospective pathology information available \n from archival ffpe tissues would produce a wealth of practical information \n on human diseases .", "some are practical for slide - mounted ffpe tissue , such as \n quantitative fluorescence imaging analysis ( qfia ) , which is reproducible \n and sensitive for specific standardized proteins , or maldi - imaging \n mass spectrometry ( ms ) .", "other encouraging mass spectrometry \n ( ms)-based proteomic studies of ffpe tissues have appeared in the \n recent literature ; however , these investigations have typically been \n restricted to minute tissue specimens , such as those obtained by laser \n capture microdissection .", "further , some studies report high rates of \n false - positive protein identification and are limited to the analysis \n of tryptic digests of ffpe tissues by liquid chromatography  ms \n ( lc  ms ) .", "our laboratory has been studying the reactions \n of formaldehyde \n with proteins and ways to reverse these reactions .", "using proteins in aqueous solution , we demonstrated that \n the majority of protein formaldehyde adducts and cross - links were \n consistently reversed with mild heating following the removal of excess \n formaldehyde by dialysis .", "we then developed \n a tissue surrogate , which consists of one or more proteins that form \n a gel - like plug when treated with formaldehyde at protein concentrations \n exceeding 75 mg / ml . these tissue surrogates have sufficient physical \n integrity to be processed using normal histological methods .", "a variety of extraction buffers and heating \n protocols were examined for their ability to recover proteins from \n tissue surrogates .", "protein recovery was generally modest , and studies \n with multiprotein tissue surrogates revealed extraction bias , meaning \n that the composition of the solubilized proteins did not match that \n of the corresponding tissue surrogate .", "subsequent studies showed that \n the ethanol dehydration step of histology caused most formaldehyde - treated \n proteins to adopt conformations enriched in -sheets , leading \n to the formation of protein aggregates where the -sheets form \n a dense network of intermolecular formaldehyde cross - links .", "we proposed that the difficulty of rehydrating \n these stabilized protein aggregates was the primary impediment to \n recovering proteins from ffpe tissue .", "pressure promotes water \n penetration into the inner core of proteins , \n causing denaturation , whereas heat alone causes protein unfolding \n followed by aggregation .", "consequently , \n we hypothesized that the combined effects of heat and elevated pressure \n would facilitate the rehydration of the highly cross - linked protein \n aggregates in ffpe tissues .", "the increased exposure to water should \n greatly improve protein solubilization while simultaneously promoting \n the reversal of protein  formaldehyde adducts and cross - links . \n", "initial physical studies on tissue surrogates supported this hypothesis and suggested that the effect of pressure was \n to reduce the size of protein aggregates through increased water penetration , \n rather than to increase the rate of reversal of protein  formaldehyde \n adducts and cross - links directly .", "encouraged by these results , we extracted ffpe mouse liver tissues \n with heat augmented by elevated hydrostatic pressure , with the goal \n of reducing extraction bias , improving the recovery of intact proteins , \n and obtaining tryptic digests that more closely resemble those from \n matched fresh - frozen tissue .", "tissue tek oct compound was purchased from \n sakura , usa , amicon ultra 3k centrifugal filters were purchased from \n millipore , and 37% formaldehyde and pierce detergent removal columns \n were obtained from thermo fisher . precast nupage bis - tris 412% \n gels , 2-(n - morpholino)ethanesulfonic acid ", "sds \n running buffer , and the silverquest staining kit were purchased from \n life technologies .", "tissue extracts were \n heated under a pressure of 40,000 psi ( 276 mpa ) using both home - built \n and commercial instruments .", "the home - built instrument consisted of \n a 2-ml capacity ms-1 stainless steel reaction vessel coupled to a \n manually operated high pressure piston screw pump available from high \n pressure equipment company ( erie , pa ) .", "the temperature of the pressure \n vessel was regulated by a eurotherm 2132 temperature controller ( leesburg , \n va ) connected to an aluminum heating collar surrounding the reaction \n vessel .", "the tissue extract ( 2 ml ) was added directly to the reaction \n vessel using a syringe .", "the construction and operation of this pressure \n system has been described in detail previously .", "the commercial instrument was a model nep 2320 barocycler ( pressure \n biosciences ) modified by the manufacturer to hold isobaric pressure \n and to provide temperature control up to 95 c .", "the tissue extract \n ( 2 ml ) was added to a ft500 sample tube , which was capped and placed \n in the pressure vessel of the barocycler .", "the liver from a female balb / c \n mouse was obtained under the secondary use provision from the department \n of laboratory animal medicine of the armed forces institute of pathology \n ( usa ) .", "half of the liver was immediately frozen in tissue - tek oct \n compound , divided into several equal - sized pieces , and stored at 80 \n c .", "the other half was fixed for 48 h at 4 c in 10% formalin . \n", "the fixed liver tissue was washed for 30 min with distilled water \n and dehydrated through a graded series of alcohols ( 70 , 85 , and 100% \n by volume ) and two changes of xylene , 30 min each .", "recovery experiments , 10 m sections of ffpe mouse liver were \n cleared of paraffin by incubating the sections through two changes \n of xylene for 10 min each .", "the sections were rehydrated through a \n series of graded alcohols for 10 min each2 changes each of \n 100% ethanol , 85% ethanol , and 70% ethanol  and then incubated \n in distilled water for a minimum of 30 min , as described previously .", "matched fresh - frozen and ffpe liver tissue sections \n were analyzed after 30 days , and again after 1 year , of storage .", "the ability \n of elevated hydrostatic pressure combined with heat to improve the \n recovery of proteins from ffpe mouse liver tissue was evaluated using \n two heat - based ffpe proteomic protocols recently reported in the literature .", "for each protocol , the experimental procedure was followed exactly \n as published , but with the heating step divided into two arms , or \n variations . in the first arm", ", the ffpe tissue extract was heated at \n the temperature and for the length of time reported in the original \n method . in the second experimental arm ,", "the temperature and length \n of time was identical , but the experiment was performed under a pressure \n of 40,000 psi .", "the two experimental arms were then analyzed using \n identical gel electrophoresis and lc / ms conditions .", "the protein content \n of the tissue extracts was determined using a bca protein assay kit \n ( pierce ) .", "following deparaffinization and \n rehydration , the tissue was cut into small pieces and suspended in \n 36 ml of 50 mm tris - hcl , ph 7 , 2% sds ( extraction buffer 1 , \n eb1 ) as described by shi et al .", "the tissue \n suspension was homogenized with three 5-s cycles of sonication on \n ice using a probe - tip sonicator , and the resulting homogenate was \n split into two equal fractions .", "one fraction ( protocol-1a ) was incubated \n at 100 c for 30 min followed by 80 c for 2 h at atmospheric \n pressure ( 14.7 psi ) in a sand bath .", "the second fraction ( protocol-1p ) \n was incubated at 100 c for 30 min followed by 80 c for \n 2 h at 40,000 psi using our hand - built pressure instrument .", "a 750 \n m section ( approximate thickness ) of matched fresh - frozen liver \n was cut with a razor blade and homogenized in 22.5 ml of eb1 by sonication \n as described above .", "proteins were extracted using two methods : incubation \n of the homogenate in an ice bath for 2.5 h ( protocol-1fi ) or incubation \n at 100 c for 30 min followed by 80 c for 2 h at atmospheric \n pressure using a sand bath ( protocol-1fh ) .", "following deparaffinization and rehydration , the \n tissue was cut into small pieces and suspended in 36 ml of \n 100 mm tris - hcl , ph 8 , 100 mm dtt , 4% sds ( extraction buffer 2 , eb2 ) \n as described by ostasiewicz et al . and \n homogenized with three 5-s cycles of sonication on ice using a probe - tip \n sonicator .", "one fraction ( protocol-2a ) was incubated at 95 c \n for 1 h at atmospheric pressure in a sand bath .", "the second fraction \n ( protocol-2p ) was incubated at 95 c for 1 h at 40,000 psi using \n the barocycler instrument .", "a 750 m section of matched fresh - frozen \n liver was cut with a razor blade and homogenized in 22.5 ml of eb2 \n by sonication as described above .", "proteins were extracted by incubating \n the homogenate at 95 c for 3 min at atmospheric pressure using \n a sand bath ( protocol-2fh ) .", "all ffpe and matched \n fresh - frozen mouse liver tissue extracts ( 40 g each ) were separated \n by sds - page on precast nupage bis - tris 412% gels using 2-(n - morpholino)ethanesulfonic acid ", "the gels were stained using the silverquest silver staining kit and \n documented using an epson v500 photoscanner and annotated in adobe \n photoshop , version 7.1 . each gel lane", "was then divided into 10 bands \n and placed into microcentrifuge tubes . in - gel tryptic digestion was \n carried out as previously described .", "separation of the \n digested peptides was performed using nanocolumns prepared in - house \n ( 75 m i.d . packed with jupiter c18 particles , 5 m , 300 \n  ) connected to an agilent 1100 nanoflow lc system , which \n was used to deliver binary gradient solvents a ( 0.1% formic acid ( fa ) \n in water ) and b ( 0.1% fa in acetonitrile ) . reversed - phase chromatography \n was performed by solubilizing the lyophilized tryptic peptides in \n 10 l of 0.1% trifluoroacetic acid and injecting 7 l \n of sample per analysis .", "after sample injection , a 20-min wash with \n 98% mobile phase a was used to remove any remaining salts from the \n sample .", "peptide elution was accomplished using a linear gradient of \n 2% solvent b to 42% solvent b over 45 min at a constant flow rate \n of 250 nl / min .", "the nanoflow reversed - phase lc column was coupled \n online to a linear ion trap mass spectrometer ( ltq , thermoelectron ) \n using the manufacturer s nanoelectrospray source with an applied \n electrospray potential of 1.75 kv and a capillary transfer tube temperature \n of 185 c .", "the ltq - ms was operated in a data - dependent mode where \n each full ms scan was followed by seven tandem ms scans in which the \n seven most abundant peptide molecular ions detected were dynamically \n selected for ms / ms analysis using a normalized cid energy of 35% . \n a dynamic exclusion of 60-s was applied to reduce redundant selection \n of peptides .", "the ms / ms spectra were analyzed using sequest ( thermoelectron ) \n against a combined uniprot nonredundant mouse proteome database containing \n 36,799 protein sequences . only peptides with conventional tryptic \n termini ( allowing for up to two internal missed cleavages ) possessing \n delta - correlation scores ( cn ) > 0.08 and charge state - dependent cross - correlation ( xcorr ) criteria as follows were considered as \n legitimate identifications : > 1.9 for + 1 charged peptides , > 2.2 \n for \n + 2 charged peptides , and > 3.1 for + 3 charged peptides . a reverse - database \n search , performed using the respective databases , resulted in a calculated \n false - positive rate of < 2% for all samples analyzed .", "ffpe \n and matched fresh - frozen mouse liver tissue stored for 30 days were \n extracted using protocol-1 .", "the fresh liver tissue was completely \n solubilized in the extraction buffer ( eb1 ) using extraction either \n on ice ( protocol-1fi ) or at elevated temperature ( protocol-1fh ) .", "the \n quantity of protein solubilized in the buffer was designated as 100% \n protein recovery in table 1 .", "ffpe mouse liver \n tissue extracted at atmospheric pressure ( protocol-1a ) resulted in \n a protein recovery of only 17% , and a large plug of remaining tissue \n was observed in the extraction vial . in contrast , when the extraction \n was performed at 40,000 psi ( protocol-1p ) , the solubilized protein \n increased to 77% and only a small amount of tissue residue remained \n in the vial .", "ffpe and matching fresh - frozen liver tissue stored for \n 1 year were extracted using protocol-2 .", "the fresh liver tissue was \n completely solubilized in the extraction buffer ( eb2 ) when extracted \n at elevated temperature ( protocol-2fh ) .", "the quantity of protein solubilized \n in the buffer was designated as 100% protein recovery in table 1 .", "the advantage of using elevated pressure was again \n evident , as 79% of the ffpe liver protein was solubilized at elevated \n hydrostatic pressure ( protocol-2p ) while only 18% was recovered at \n ambient pressure ( protocol-2a ) .", "ffpe mouse liver was homogenized \n in extraction buffer and heated with or without elevated pressure . \n", "fresh - frozen tissue was extracted either at atmospheric pressure using \n the indicated extraction condition or on ice for 2.5 h. protocol used for protein extraction \n ( see materials and methods ) .", "tissue was heated at 100 c \n for 30 min ; then the temperature was lowered to 80 c for 2 h. the amount of protein extracted \n from fresh frozen tissue was set to 100% .", "previous studies using ffpe tissue surrogates containing \n several \n proteins indicated that failure to completely \n solubilize the tissue surrogate led to extraction bias , such that \n the composition of the solubilized protein solution differed significantly \n from that of the original surrogate .", "the extraction bias became less \n significant as the percentage of total protein solubilized from the \n tissue surrogate increased .", "thus , the \n 4-fold improvement in protein solubilization realized when the extraction \n was performed at elevated pressure is likely to lead to a protein \n extract that more accurately represents the composition of the original \n ffpe tissue .", "further , the increased amount of recovered protein allows \n a greater number of analytical techniques to be performed .", "while most \n published proteomic studies of ffpe tissue analyze only a few thousand \n cells from microdissected tissue , the use of elevated pressure has the advantage of improving protein \n extraction from whole tissue sections .", "this ability is particularly \n useful in instances where tissue microdissection is not practical \n or when a more global proteomic analysis is desired .", "unbiased protein \n extraction and standardized protocols are particularly important with \n techniques such as reverse phase protein arrays ( rppas ) , where the \n protein components of cell signaling pathways are quantified to direct \n clinical treatment of cancer .", "the \n 30-day - old ffpe mouse liver extracted at 40,000 psi ( protocol-1p ) \n exhibited a number of well resolved high and low molecular weight \n protein bands by sds - page , corresponding to 87% of those seen \n in the matched fresh - frozen tissue extracted on ice ( figure 1 , lanes 2 and 1 , respectively ) .", "the ffpe samples \n extracted at ambient pressure ( protocol-1a ) contained relatively few \n well - resolved protein bands equivalent to 25% of those seen \n in frozen mouse liver ( figure 1 , lane 3 ) .", "similar \n results were seen with 1-year - old ffpe mouse liver extracted at 40,000 \n psi ( protocol-2p ) with well resolved high and low molecular weight \n protein bands corresponding to 74% of those seen in the matched fresh - frozen \n tissue ( not shown ) . when the extraction was performed ambient pressure \n ( protocol-2a ) this value", "1d sds - page of fresh - frozen \n and ffpe mouse liver extracts : lane \n 1 , fresh - frozen tissue ( protocol-1fi ) ; lane m , molecular weight marker ; \n lane 2 , ffpe tissue extracted with heat at 40,000 psi ( protocol-1p ) ; \n lane 3 , ffpe tissue extracted with heat alone ( protocol-1a ) . as top - down ms sequencing technology improves , \n the ability to extract \n and analyze intact proteins from ffpe tissue will become more important . \n", "top - down sequencing facilitates the measurement of combinations of \n modifications , such as phosphorylation and glycosylation , and the \n direct quantitation of specific protein isoforms and splice variants . \n", "few of these measurements are directly obtainable using bottom - up \n proteomic approaches in which proteins are digested into peptides . \n the ability to extract intact proteins from the seemingly inexhaustible \n source of ffpe tissues will increase the diagnostic and prognostic \n efficacy of proteomic - based biomarker discovery by allowing biomarker \n validation using orthogonal methods such as western blotting , immunohistochemistry , \n immunoassays , and structural and interaction proteomics . in this context , \n the > 3-fold increase in the recovery of intact proteins from ffpe \n tissue achieved by using elevated pressure represents a significant \n breakthrough .", "ffpe \n and matched fresh - frozen liver tissue stored for 30 days were extracted \n using protocol-1 .", "ffpe tissue was extracted at ambient pressure ( protocol-1a ) \n or 40,000 psi ( protocol-1p ) , while the matched fresh - frozen liver \n tissue was extracted using protocol-1fi and protocol-1fh .", "the solubilized \n proteins were separated by 1d - page , and each gel lane was excised , \n digested with trypsin , desalted , and analyzed by lc - ms / ms .", "the total \n unique peptide and protein identifications for each tissue type and \n extraction condition are shown in table 1 . \n", "ffpe tissue extracted with heat alone resulted in the identification \n of 5565 unique peptides and 3449 unique proteins .", "the addition of \n elevated hydrostatic pressure significantly improved both the number \n of unique peptides ( 9621 ) and proteins ( 5192 ) identified .", "the number \n of proteins identified from the high pressure - extracted sample was \n comparable to the number of unique proteins identified from fresh - frozen \n tissue , which ranged from 4932 for tissue extracted on ice to 4451 \n for frozen tissue extracted with heat ( table 1 ) .", "the ms results for the 30-day - old ffpe mouse liver extracted \n under elevated pressure ( protocol-1p ) and the matched fresh - frozen \n tissue extracted on ice ( protocol-1fi ) were searched using gominer , \n a gene ontology program .", "the percentages of nuclear , membrane , intracellular , \n and extracellular proteins identified in fresh - frozen and ffpe liver \n were virtually identical ( figure 2a ) , as were \n the results for classification by biological function ( figure 2b ) .", "gene ontology analysis of proteins identified by lc - ms / ms . \n proteins \n identified using fresh - frozen mouse liver ( protocol-1fi ) or ffpe liver \n extracted with heat and elevated pressure ( protocol-1p ) were categorized \n by subcellular localization ( a ) or biological process ( b ) , using gominer \n gene ontology software . to address the effect of long - term storage of the \n ffpe specimens", ", \n the mouse liver samples were investigated after an additional 11 months \n of storage ( 1-year - old sample ) .", "ffpe tissue was extracted at ambient \n pressure ( protocol-2a ) or 40,000 psi ( protocol-2p ) , while the matched \n fresh - frozen liver tissue was extracted at high temperature ( protocol-2fh ) . \n", "the solubilized proteins were separated by 1d - page , and each gel lane \n was excised , digested with trypsin , desalted , and analyzed by lc - ms / ms . \n from the 1-d gel", ", we were able to identify 3492 nonredundant proteins \n in the 1-year - old ffpe liver extracted under elevated pressure , which \n was comparable to the 3415 nonredundant proteins identified in the \n matched fresh - frozen mouse liver extracted at high temperature .", "in \n contrast , only 107 unique proteins were identified in the ffpe tissue \n extracted at ambient pressure .", "figure 3 shows venn diagrams of the unique \n and common ( overlapping ) proteins identified in mouse liver tissue \n extracts prepared by different methods .", "figure 3a compares 30-day - old fresh - frozen liver tissue extracted on ice \n ( protocol-1fi ) versus 100 c for 30 min followed by 80 c \n for 2 h ( protocol-1fh ) . figure 3b compares \n 30-day - old ffpe liver tissue extracted under pressure ( protocol-1p ) \n", "figure 3c compares 1-year - old \n ffpe liver tissue extracted under pressure ( protocol-2p ) versus matched \n fresh - frozen liver extracted at elevated temperature ( protocol-2fh ) . \n", "notably , the common proteins , expressed as a percentage of either \n the ffpe or matched fresh - frozen mouse liver tissue , were 50% \n for all three tissue pairs .", "venn diagrams showing the number of unique and \n common proteins \n identified using lc ms / ms analysis : panel a , fresh - frozen tissue extracted \n on ice ( protocol-1fi ) or with heat ( protocol-1fh ) ; panel b , fresh - frozen \n tissue extracted with heat ( protocol-1fh ) and ffpe mouse liver extracted \n with elevated pressure ( protocol-1p ) ; panel c , fresh - frozen tissue \n extracted with heat ( protocol-2fh ) and ffpe mouse liver extracted \n with elevated pressure ( protocol-2p ) .", "figure 4a is a pie chart \n showing the number \n of unique proteins identified by two or more fully tryptic peptides \n for 30-day - old ffpe mouse liver extracted under pressure ( protocol-1p ) . \n", "the results reveal that 49% of the proteins were identified by two \n peptides , 51% were identified by three or more peptides , and 21% of \n the proteins were identified by five or more peptides .", "figure 4b shows similar results for 1-year - old ffpe mouse \n liver extracted under pressure ( protocol-2p ) , with 57% of the proteins \n identified by two peptides , 41% by three or more peptides , and 15% \n identified by five or more peptides .", "these results are similar to \n those for the matched fresh - frozen mouse liver tissue ( not shown ) . \n", "the complete list of peptides identified in the fresh - frozen and high - pressure - recovered \n ffpe tissue , with their corresponding xcorr values , can be found online \n ( supporting information table 1 ) .", "total number \n of unique proteins identified using lc - ms / ms by two \n or more unique , fully tryptic peptides in ffpe mouse tissue extracted \n with heat and elevated pressure ( 40,000 psi ) : a , 30-day - old ffpe mouse \n liver ( protocol-1p ) ; b , 1-year - old ffpe mouse liver ( protocol-2p ) .", "virtually all protocols reported in the \n literature for the extraction \n of proteins from ffpe tissue use a variation of the heat - induced antigen \n retrieval technique developed by shi and taylor for the recovery of antigenicity in immunohistochemical \n studies .", "this method involves exposing ffpe tissue sections to a buffer \n solution containing a detergent and/or protein denaturant and elevated \n temperatures of 90120 c for a period of 1030 \n min .", "optimal antigen recovery varies \n with the host ffpe tissue , with each type requiring different buffers , \n ph values , buffer additives , and incubation temperatures .", "although the ffpe proteomic literature is still quite limited , it \n is not unreasonable to propose that the same situation applies to \n the proteomic analysis of ffpe tissues .", "elevated hydrostatic pressure \n is not a technique unto itself but rather an adjuvant method that \n can be applied to existing or future ffpe protein extraction protocols . \n while it remains to be shown that this method is useful with every \n tissue fixation and protein extraction protocol , elevated pressure \n acts through purely physical means and \n should be compatible with ffpe protein extraction buffers of any ph \n and containing any detergent , protein denaturant , or other additive .", "this should allow its integration into a wide \n range of protein extraction protocols for ms - based proteomics with \n little to no alteration to downstream sample preparation and analysis . \n", "this was demonstrated in this report by the successful application \n of elevated pressure to two very different published ffpe protein \n extraction protocols .", "an increase in pressure to 40,000 psi , to augment \n heat treatment , improved protein extraction efficiency from ffpe mouse \n liver tissue by approximately 4-fold and increased the number of unique \n proteins identified by up to 30-fold over the published methods used \n at ambient pressure .", "further , the tryptic digests of these pressure - extracted \n tissues resulted in protein profiles that more closely resembled those \n from matched fresh - frozen tissue when analyzed by lc / ms than did those \n extracted with heat alone , while maintaining a false - identification \n rate of < 2% .", "the ability of elevated pressure to significantly \n improve the recovery of intact proteins from ffpe tissues over the \n use of heat alone has great potential for broad application to top - down \n proteomic studies for the identification of disease biomarkers ."], "abstract_text": ["<S> formaldehyde - fixed , paraffin - embedded ( ffpe ) tissue repositories \n represent a valuable resource for the retrospective study of disease \n progression and response to therapy . </S>", "<S> however , the proteomic analysis \n of ffpe tissues has been hampered by formaldehyde - induced protein \n modifications , which reduce protein extraction efficiency and may \n lead to protein misidentification . here , we demonstrate the use of \n heat augmented with high hydrostatic pressure ( 40,000 psi ) as a novel \n method for the recovery of intact proteins from ffpe mouse liver . \n </S>", "<S> when ffpe mouse liver was extracted using heat and elevated pressure , \n there was a 4-fold increase in protein extraction efficiency , a 3-fold \n increase in the extraction of intact proteins , and up to a 30-fold \n increase in the number of nonredundant proteins identified by mass \n spectrometry , compared to matched tissue extracted with heat alone . \n </S>", "<S> more importantly , the number of nonredundant proteins identified in \n the ffpe tissue was nearly identical to that of matched fresh - frozen \n tissue . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results and Discussion", "Conclusion"], "sections": [["proteomics has emerged as a valuable tool \n for the identification \n of biomarkers associated with human disease including those resulting from very subtle changes in normal cell \n functions and signaling pathways .", "proteomic technology has advanced \n to a state where thousands of proteins can be identified within complex \n samples , yet disease - based studies using fresh or frozen tissues are limited \n by a lack of available specimens for longitudinal clinical investigations . \n", "in contrast , there are millions of archival formalin - fixed , paraffin - embedded \n ( ffpe ) tissues for which the clinical course of disease and response \n to therapy has been established .", "ffpe tissue studies are affected \n greatly by sample collection , tissue processing , and archival time .", "though these factors are difficult to control \n in archival samples , improvements in techniques such as mtraq have \n made relative quantitation of disease biomarkers in ffpe tissue possible .", "improving protein extraction and detection of less abundant protein \n biomarkers is also of critical importance in proteomic analysis .", "protein \n modifications by formaldehyde treatment and histological processing significantly limit the use ffpe tissues for proteomic analyses . \n", "this has prevented proteomic studies of the clinical course of diseases , \n such as prostate and breast cancer , that evolve slowly or where the \n time between treatment and recurrence is long .", "coupling proteomic \n investigations with the retrospective pathology information available \n from archival ffpe tissues would produce a wealth of practical information \n on human diseases .", "some are practical for slide - mounted ffpe tissue , such as \n quantitative fluorescence imaging analysis ( qfia ) , which is reproducible \n and sensitive for specific standardized proteins , or maldi - imaging \n mass spectrometry ( ms ) .", "other encouraging mass spectrometry \n ( ms)-based proteomic studies of ffpe tissues have appeared in the \n recent literature ; however , these investigations have typically been \n restricted to minute tissue specimens , such as those obtained by laser \n capture microdissection .", "further , some studies report high rates of \n false - positive protein identification and are limited to the analysis \n of tryptic digests of ffpe tissues by liquid chromatography  ms \n ( lc  ms ) .", "our laboratory has been studying the reactions \n of formaldehyde \n with proteins and ways to reverse these reactions .", "using proteins in aqueous solution , we demonstrated that \n the majority of protein formaldehyde adducts and cross - links were \n consistently reversed with mild heating following the removal of excess \n formaldehyde by dialysis .", "we then developed \n a tissue surrogate , which consists of one or more proteins that form \n a gel - like plug when treated with formaldehyde at protein concentrations \n exceeding 75 mg / ml . these tissue surrogates have sufficient physical \n integrity to be processed using normal histological methods .", "a variety of extraction buffers and heating \n protocols were examined for their ability to recover proteins from \n tissue surrogates .", "protein recovery was generally modest , and studies \n with multiprotein tissue surrogates revealed extraction bias , meaning \n that the composition of the solubilized proteins did not match that \n of the corresponding tissue surrogate .", "subsequent studies showed that \n the ethanol dehydration step of histology caused most formaldehyde - treated \n proteins to adopt conformations enriched in -sheets , leading \n to the formation of protein aggregates where the -sheets form \n a dense network of intermolecular formaldehyde cross - links .", "we proposed that the difficulty of rehydrating \n these stabilized protein aggregates was the primary impediment to \n recovering proteins from ffpe tissue .", "pressure promotes water \n penetration into the inner core of proteins , \n causing denaturation , whereas heat alone causes protein unfolding \n followed by aggregation .", "consequently , \n we hypothesized that the combined effects of heat and elevated pressure \n would facilitate the rehydration of the highly cross - linked protein \n aggregates in ffpe tissues .", "the increased exposure to water should \n greatly improve protein solubilization while simultaneously promoting \n the reversal of protein  formaldehyde adducts and cross - links . \n", "initial physical studies on tissue surrogates supported this hypothesis and suggested that the effect of pressure was \n to reduce the size of protein aggregates through increased water penetration , \n rather than to increase the rate of reversal of protein  formaldehyde \n adducts and cross - links directly .", "encouraged by these results , we extracted ffpe mouse liver tissues \n with heat augmented by elevated hydrostatic pressure , with the goal \n of reducing extraction bias , improving the recovery of intact proteins , \n and obtaining tryptic digests that more closely resemble those from \n matched fresh - frozen tissue ."], ["tissue tek oct compound was purchased from \n sakura , usa , amicon ultra 3k centrifugal filters were purchased from \n millipore , and 37% formaldehyde and pierce detergent removal columns \n were obtained from thermo fisher . precast nupage bis - tris 412% \n gels , 2-(n - morpholino)ethanesulfonic acid ", "sds \n running buffer , and the silverquest staining kit were purchased from \n life technologies .", "tissue extracts were \n heated under a pressure of 40,000 psi ( 276 mpa ) using both home - built \n and commercial instruments .", "the home - built instrument consisted of \n a 2-ml capacity ms-1 stainless steel reaction vessel coupled to a \n manually operated high pressure piston screw pump available from high \n pressure equipment company ( erie , pa ) .", "the temperature of the pressure \n vessel was regulated by a eurotherm 2132 temperature controller ( leesburg , \n va ) connected to an aluminum heating collar surrounding the reaction \n vessel .", "the tissue extract ( 2 ml ) was added directly to the reaction \n vessel using a syringe .", "the construction and operation of this pressure \n system has been described in detail previously .", "the commercial instrument was a model nep 2320 barocycler ( pressure \n biosciences ) modified by the manufacturer to hold isobaric pressure \n and to provide temperature control up to 95 c .", "the tissue extract \n ( 2 ml ) was added to a ft500 sample tube , which was capped and placed \n in the pressure vessel of the barocycler .", "the liver from a female balb / c \n mouse was obtained under the secondary use provision from the department \n of laboratory animal medicine of the armed forces institute of pathology \n ( usa ) .", "half of the liver was immediately frozen in tissue - tek oct \n compound , divided into several equal - sized pieces , and stored at 80 \n c .", "the other half was fixed for 48 h at 4 c in 10% formalin . \n", "the fixed liver tissue was washed for 30 min with distilled water \n and dehydrated through a graded series of alcohols ( 70 , 85 , and 100% \n by volume ) and two changes of xylene , 30 min each .", "recovery experiments , 10 m sections of ffpe mouse liver were \n cleared of paraffin by incubating the sections through two changes \n of xylene for 10 min each .", "the sections were rehydrated through a \n series of graded alcohols for 10 min each2 changes each of \n 100% ethanol , 85% ethanol , and 70% ethanol  and then incubated \n in distilled water for a minimum of 30 min , as described previously .", "matched fresh - frozen and ffpe liver tissue sections \n were analyzed after 30 days , and again after 1 year , of storage .", "the ability \n of elevated hydrostatic pressure combined with heat to improve the \n recovery of proteins from ffpe mouse liver tissue was evaluated using \n two heat - based ffpe proteomic protocols recently reported in the literature .", "for each protocol , the experimental procedure was followed exactly \n as published , but with the heating step divided into two arms , or \n variations . in the first arm", ", the ffpe tissue extract was heated at \n the temperature and for the length of time reported in the original \n method . in the second experimental arm ,", "the temperature and length \n of time was identical , but the experiment was performed under a pressure \n of 40,000 psi .", "the two experimental arms were then analyzed using \n identical gel electrophoresis and lc / ms conditions .", "the protein content \n of the tissue extracts was determined using a bca protein assay kit \n ( pierce ) .", "following deparaffinization and \n rehydration , the tissue was cut into small pieces and suspended in \n 36 ml of 50 mm tris - hcl , ph 7 , 2% sds ( extraction buffer 1 , \n eb1 ) as described by shi et al .", "the tissue \n suspension was homogenized with three 5-s cycles of sonication on \n ice using a probe - tip sonicator , and the resulting homogenate was \n split into two equal fractions .", "one fraction ( protocol-1a ) was incubated \n at 100 c for 30 min followed by 80 c for 2 h at atmospheric \n pressure ( 14.7 psi ) in a sand bath .", "the second fraction ( protocol-1p ) \n was incubated at 100 c for 30 min followed by 80 c for \n 2 h at 40,000 psi using our hand - built pressure instrument .", "a 750 \n m section ( approximate thickness ) of matched fresh - frozen liver \n was cut with a razor blade and homogenized in 22.5 ml of eb1 by sonication \n as described above .", "proteins were extracted using two methods : incubation \n of the homogenate in an ice bath for 2.5 h ( protocol-1fi ) or incubation \n at 100 c for 30 min followed by 80 c for 2 h at atmospheric \n pressure using a sand bath ( protocol-1fh ) .", "following deparaffinization and rehydration , the \n tissue was cut into small pieces and suspended in 36 ml of \n 100 mm tris - hcl , ph 8 , 100 mm dtt , 4% sds ( extraction buffer 2 , eb2 ) \n as described by ostasiewicz et al . and \n homogenized with three 5-s cycles of sonication on ice using a probe - tip \n sonicator .", "one fraction ( protocol-2a ) was incubated at 95 c \n for 1 h at atmospheric pressure in a sand bath .", "the second fraction \n ( protocol-2p ) was incubated at 95 c for 1 h at 40,000 psi using \n the barocycler instrument .", "a 750 m section of matched fresh - frozen \n liver was cut with a razor blade and homogenized in 22.5 ml of eb2 \n by sonication as described above .", "proteins were extracted by incubating \n the homogenate at 95 c for 3 min at atmospheric pressure using \n a sand bath ( protocol-2fh ) .", "all ffpe and matched \n fresh - frozen mouse liver tissue extracts ( 40 g each ) were separated \n by sds - page on precast nupage bis - tris 412% gels using 2-(n - morpholino)ethanesulfonic acid ", "the gels were stained using the silverquest silver staining kit and \n documented using an epson v500 photoscanner and annotated in adobe \n photoshop , version 7.1 . each gel lane", "was then divided into 10 bands \n and placed into microcentrifuge tubes . in - gel tryptic digestion was \n carried out as previously described .", "separation of the \n digested peptides was performed using nanocolumns prepared in - house \n ( 75 m i.d . packed with jupiter c18 particles , 5 m , 300 \n  ) connected to an agilent 1100 nanoflow lc system , which \n was used to deliver binary gradient solvents a ( 0.1% formic acid ( fa ) \n in water ) and b ( 0.1% fa in acetonitrile ) . reversed - phase chromatography \n was performed by solubilizing the lyophilized tryptic peptides in \n 10 l of 0.1% trifluoroacetic acid and injecting 7 l \n of sample per analysis .", "after sample injection , a 20-min wash with \n 98% mobile phase a was used to remove any remaining salts from the \n sample .", "peptide elution was accomplished using a linear gradient of \n 2% solvent b to 42% solvent b over 45 min at a constant flow rate \n of 250 nl / min .", "the nanoflow reversed - phase lc column was coupled \n online to a linear ion trap mass spectrometer ( ltq , thermoelectron ) \n using the manufacturer s nanoelectrospray source with an applied \n electrospray potential of 1.75 kv and a capillary transfer tube temperature \n of 185 c .", "the ltq - ms was operated in a data - dependent mode where \n each full ms scan was followed by seven tandem ms scans in which the \n seven most abundant peptide molecular ions detected were dynamically \n selected for ms / ms analysis using a normalized cid energy of 35% . \n a dynamic exclusion of 60-s was applied to reduce redundant selection \n of peptides .", "the ms / ms spectra were analyzed using sequest ( thermoelectron ) \n against a combined uniprot nonredundant mouse proteome database containing \n 36,799 protein sequences . only peptides with conventional tryptic \n termini ( allowing for up to two internal missed cleavages ) possessing \n delta - correlation scores ( cn ) > 0.08 and charge state - dependent cross - correlation ( xcorr ) criteria as follows were considered as \n legitimate identifications : > 1.9 for + 1 charged peptides , > 2.2 \n for \n + 2 charged peptides , and > 3.1 for + 3 charged peptides . a reverse - database \n search , performed using the respective databases , resulted in a calculated \n false - positive rate of < 2% for all samples analyzed ."], ["ffpe \n and matched fresh - frozen mouse liver tissue stored for 30 days were \n extracted using protocol-1 .", "the fresh liver tissue was completely \n solubilized in the extraction buffer ( eb1 ) using extraction either \n on ice ( protocol-1fi ) or at elevated temperature ( protocol-1fh ) .", "the \n quantity of protein solubilized in the buffer was designated as 100% \n protein recovery in table 1 .", "ffpe mouse liver \n tissue extracted at atmospheric pressure ( protocol-1a ) resulted in \n a protein recovery of only 17% , and a large plug of remaining tissue \n was observed in the extraction vial . in contrast , when the extraction \n was performed at 40,000 psi ( protocol-1p ) , the solubilized protein \n increased to 77% and only a small amount of tissue residue remained \n in the vial .", "ffpe and matching fresh - frozen liver tissue stored for \n 1 year were extracted using protocol-2 .", "the fresh liver tissue was \n completely solubilized in the extraction buffer ( eb2 ) when extracted \n at elevated temperature ( protocol-2fh ) .", "the quantity of protein solubilized \n in the buffer was designated as 100% protein recovery in table 1 .", "the advantage of using elevated pressure was again \n evident , as 79% of the ffpe liver protein was solubilized at elevated \n hydrostatic pressure ( protocol-2p ) while only 18% was recovered at \n ambient pressure ( protocol-2a ) .", "ffpe mouse liver was homogenized \n in extraction buffer and heated with or without elevated pressure . \n", "fresh - frozen tissue was extracted either at atmospheric pressure using \n the indicated extraction condition or on ice for 2.5 h. protocol used for protein extraction \n ( see materials and methods ) .", "tissue was heated at 100 c \n for 30 min ; then the temperature was lowered to 80 c for 2 h. the amount of protein extracted \n from fresh frozen tissue was set to 100% .", "previous studies using ffpe tissue surrogates containing \n several \n proteins indicated that failure to completely \n solubilize the tissue surrogate led to extraction bias , such that \n the composition of the solubilized protein solution differed significantly \n from that of the original surrogate .", "the extraction bias became less \n significant as the percentage of total protein solubilized from the \n tissue surrogate increased .", "thus , the \n 4-fold improvement in protein solubilization realized when the extraction \n was performed at elevated pressure is likely to lead to a protein \n extract that more accurately represents the composition of the original \n ffpe tissue .", "further , the increased amount of recovered protein allows \n a greater number of analytical techniques to be performed .", "while most \n published proteomic studies of ffpe tissue analyze only a few thousand \n cells from microdissected tissue , the use of elevated pressure has the advantage of improving protein \n extraction from whole tissue sections .", "this ability is particularly \n useful in instances where tissue microdissection is not practical \n or when a more global proteomic analysis is desired .", "unbiased protein \n extraction and standardized protocols are particularly important with \n techniques such as reverse phase protein arrays ( rppas ) , where the \n protein components of cell signaling pathways are quantified to direct \n clinical treatment of cancer .", "the \n 30-day - old ffpe mouse liver extracted at 40,000 psi ( protocol-1p ) \n exhibited a number of well resolved high and low molecular weight \n protein bands by sds - page , corresponding to 87% of those seen \n in the matched fresh - frozen tissue extracted on ice ( figure 1 , lanes 2 and 1 , respectively ) .", "the ffpe samples \n extracted at ambient pressure ( protocol-1a ) contained relatively few \n well - resolved protein bands equivalent to 25% of those seen \n in frozen mouse liver ( figure 1 , lane 3 ) .", "similar \n results were seen with 1-year - old ffpe mouse liver extracted at 40,000 \n psi ( protocol-2p ) with well resolved high and low molecular weight \n protein bands corresponding to 74% of those seen in the matched fresh - frozen \n tissue ( not shown ) . when the extraction was performed ambient pressure \n ( protocol-2a ) this value", "1d sds - page of fresh - frozen \n and ffpe mouse liver extracts : lane \n 1 , fresh - frozen tissue ( protocol-1fi ) ; lane m , molecular weight marker ; \n lane 2 , ffpe tissue extracted with heat at 40,000 psi ( protocol-1p ) ; \n lane 3 , ffpe tissue extracted with heat alone ( protocol-1a ) . as top - down ms sequencing technology improves , \n the ability to extract \n and analyze intact proteins from ffpe tissue will become more important . \n", "top - down sequencing facilitates the measurement of combinations of \n modifications , such as phosphorylation and glycosylation , and the \n direct quantitation of specific protein isoforms and splice variants . \n", "few of these measurements are directly obtainable using bottom - up \n proteomic approaches in which proteins are digested into peptides . \n the ability to extract intact proteins from the seemingly inexhaustible \n source of ffpe tissues will increase the diagnostic and prognostic \n efficacy of proteomic - based biomarker discovery by allowing biomarker \n validation using orthogonal methods such as western blotting , immunohistochemistry , \n immunoassays , and structural and interaction proteomics . in this context , \n the > 3-fold increase in the recovery of intact proteins from ffpe \n tissue achieved by using elevated pressure represents a significant \n breakthrough .", "ffpe \n and matched fresh - frozen liver tissue stored for 30 days were extracted \n using protocol-1 .", "ffpe tissue was extracted at ambient pressure ( protocol-1a ) \n or 40,000 psi ( protocol-1p ) , while the matched fresh - frozen liver \n tissue was extracted using protocol-1fi and protocol-1fh .", "the solubilized \n proteins were separated by 1d - page , and each gel lane was excised , \n digested with trypsin , desalted , and analyzed by lc - ms / ms .", "the total \n unique peptide and protein identifications for each tissue type and \n extraction condition are shown in table 1 . \n", "ffpe tissue extracted with heat alone resulted in the identification \n of 5565 unique peptides and 3449 unique proteins .", "the addition of \n elevated hydrostatic pressure significantly improved both the number \n of unique peptides ( 9621 ) and proteins ( 5192 ) identified .", "the number \n of proteins identified from the high pressure - extracted sample was \n comparable to the number of unique proteins identified from fresh - frozen \n tissue , which ranged from 4932 for tissue extracted on ice to 4451 \n for frozen tissue extracted with heat ( table 1 ) .", "the ms results for the 30-day - old ffpe mouse liver extracted \n under elevated pressure ( protocol-1p ) and the matched fresh - frozen \n tissue extracted on ice ( protocol-1fi ) were searched using gominer , \n a gene ontology program .", "the percentages of nuclear , membrane , intracellular , \n and extracellular proteins identified in fresh - frozen and ffpe liver \n were virtually identical ( figure 2a ) , as were \n the results for classification by biological function ( figure 2b ) .", "gene ontology analysis of proteins identified by lc - ms / ms . \n proteins \n identified using fresh - frozen mouse liver ( protocol-1fi ) or ffpe liver \n extracted with heat and elevated pressure ( protocol-1p ) were categorized \n by subcellular localization ( a ) or biological process ( b ) , using gominer \n gene ontology software . to address the effect of long - term storage of the \n ffpe specimens", ", \n the mouse liver samples were investigated after an additional 11 months \n of storage ( 1-year - old sample ) .", "ffpe tissue was extracted at ambient \n pressure ( protocol-2a ) or 40,000 psi ( protocol-2p ) , while the matched \n fresh - frozen liver tissue was extracted at high temperature ( protocol-2fh ) . \n", "the solubilized proteins were separated by 1d - page , and each gel lane \n was excised , digested with trypsin , desalted , and analyzed by lc - ms / ms . \n from the 1-d gel", ", we were able to identify 3492 nonredundant proteins \n in the 1-year - old ffpe liver extracted under elevated pressure , which \n was comparable to the 3415 nonredundant proteins identified in the \n matched fresh - frozen mouse liver extracted at high temperature .", "in \n contrast , only 107 unique proteins were identified in the ffpe tissue \n extracted at ambient pressure .", "figure 3 shows venn diagrams of the unique \n and common ( overlapping ) proteins identified in mouse liver tissue \n extracts prepared by different methods .", "figure 3a compares 30-day - old fresh - frozen liver tissue extracted on ice \n ( protocol-1fi ) versus 100 c for 30 min followed by 80 c \n for 2 h ( protocol-1fh ) . figure 3b compares \n 30-day - old ffpe liver tissue extracted under pressure ( protocol-1p ) \n", "figure 3c compares 1-year - old \n ffpe liver tissue extracted under pressure ( protocol-2p ) versus matched \n fresh - frozen liver extracted at elevated temperature ( protocol-2fh ) . \n", "notably , the common proteins , expressed as a percentage of either \n the ffpe or matched fresh - frozen mouse liver tissue , were 50% \n for all three tissue pairs .", "venn diagrams showing the number of unique and \n common proteins \n identified using lc ms / ms analysis : panel a , fresh - frozen tissue extracted \n on ice ( protocol-1fi ) or with heat ( protocol-1fh ) ; panel b , fresh - frozen \n tissue extracted with heat ( protocol-1fh ) and ffpe mouse liver extracted \n with elevated pressure ( protocol-1p ) ; panel c , fresh - frozen tissue \n extracted with heat ( protocol-2fh ) and ffpe mouse liver extracted \n with elevated pressure ( protocol-2p ) .", "figure 4a is a pie chart \n showing the number \n of unique proteins identified by two or more fully tryptic peptides \n for 30-day - old ffpe mouse liver extracted under pressure ( protocol-1p ) . \n", "the results reveal that 49% of the proteins were identified by two \n peptides , 51% were identified by three or more peptides , and 21% of \n the proteins were identified by five or more peptides .", "figure 4b shows similar results for 1-year - old ffpe mouse \n liver extracted under pressure ( protocol-2p ) , with 57% of the proteins \n identified by two peptides , 41% by three or more peptides , and 15% \n identified by five or more peptides .", "these results are similar to \n those for the matched fresh - frozen mouse liver tissue ( not shown ) . \n", "the complete list of peptides identified in the fresh - frozen and high - pressure - recovered \n ffpe tissue , with their corresponding xcorr values , can be found online \n ( supporting information table 1 ) .", "total number \n of unique proteins identified using lc - ms / ms by two \n or more unique , fully tryptic peptides in ffpe mouse tissue extracted \n with heat and elevated pressure ( 40,000 psi ) : a , 30-day - old ffpe mouse \n liver ( protocol-1p ) ; b , 1-year - old ffpe mouse liver ( protocol-2p ) ."], ["virtually all protocols reported in the \n literature for the extraction \n of proteins from ffpe tissue use a variation of the heat - induced antigen \n retrieval technique developed by shi and taylor for the recovery of antigenicity in immunohistochemical \n studies .", "this method involves exposing ffpe tissue sections to a buffer \n solution containing a detergent and/or protein denaturant and elevated \n temperatures of 90120 c for a period of 1030 \n min .", "optimal antigen recovery varies \n with the host ffpe tissue , with each type requiring different buffers , \n ph values , buffer additives , and incubation temperatures .", "although the ffpe proteomic literature is still quite limited , it \n is not unreasonable to propose that the same situation applies to \n the proteomic analysis of ffpe tissues .", "elevated hydrostatic pressure \n is not a technique unto itself but rather an adjuvant method that \n can be applied to existing or future ffpe protein extraction protocols . \n while it remains to be shown that this method is useful with every \n tissue fixation and protein extraction protocol , elevated pressure \n acts through purely physical means and \n should be compatible with ffpe protein extraction buffers of any ph \n and containing any detergent , protein denaturant , or other additive .", "this should allow its integration into a wide \n range of protein extraction protocols for ms - based proteomics with \n little to no alteration to downstream sample preparation and analysis . \n", "this was demonstrated in this report by the successful application \n of elevated pressure to two very different published ffpe protein \n extraction protocols .", "an increase in pressure to 40,000 psi , to augment \n heat treatment , improved protein extraction efficiency from ffpe mouse \n liver tissue by approximately 4-fold and increased the number of unique \n proteins identified by up to 30-fold over the published methods used \n at ambient pressure .", "further , the tryptic digests of these pressure - extracted \n tissues resulted in protein profiles that more closely resembled those \n from matched fresh - frozen tissue when analyzed by lc / ms than did those \n extracted with heat alone , while maintaining a false - identification \n rate of < 2% .", "the ability of elevated pressure to significantly \n improve the recovery of intact proteins from ffpe tissues over the \n use of heat alone has great potential for broad application to top - down \n proteomic studies for the identification of disease biomarkers ."]]}
{"article_id": "PMC4736196", "article_text": ["type 2 diabetes mellitus ( t2 dm ) is considered a major epidemic of this century .", "it is estimated that its prevalence will increase worldwide from 371 million people in 2013 to 552 million people in 2030 .", "t2 dm is associated with accelerated progression of atherosclerosis , the major cause of vascular complications leading to increased morbidity and mortality .", "chronic , low - grade inflammation has been demonstrated to be involved in the pathogenesis of atherosclerosis in subjects at high risk to develop cardiovascular disease [ 37 ] . among immune cells infiltrating atherosclerotic lesions , polymorphonuclear neutrophil leukocytes with their products", "were reported to have an important role in the development and progression of atherosclerosis [ 811 ] .", "marino and coworkers have recently reported that both circulating and intraplaque polymorphonuclear neutrophil leukocytes from subjects with carotid atherosclerosis are active producers of different inflammatory mediators including the vascular endothelial growth factor ( vegf ) .", "several environmental and genetic factors ( i.e. , hypoxia , hyperglycemia , oxidative stress , ischemia , and gene polymorphisms of vegf ) influence plasma vegf levels [ 1216 ] . among several polymorphisms of the vegf gene ,", "the rs2010963 ( 634c / g polymorphism of the vegf gene ) and few others were reported to affect serum vegf levels [ 1315 ] .", "moreover , rs2010963 was demonstrated to be associated with several disorders , such as diabetic retinopathy , diabetic nephropathy , myocardial infarction , and impaired prognosis in patients with chronic heart failure [ 1315 , 17 ] . despite these findings ,", "however , data about vegf polymorphisms and their possible association with carotid atherosclerosis in patients with diabetes mellitus are limited [ 1820 ] .", "additionally , cimt is highly heritable and associated with stroke and myocardial infarction , making it a promising quantitative intermediate phenotype for genetic studies of vascular disease .", "the present study was thus designed to investigate the association between polymorphisms of the vegf gene ( rs2010963 ) and the kdr gene ( rs2071559 ) and markers of carotid atherosclerosis ( such as carotid intima - media thickness ( cimt ) , the number of affected segments of carotid arteries , and the sum of plaques thickness ) in patients with t2 dm .", "the study protocol was approved by the slovene medical ethics committee in september 2010 ( protocol number 128/09/2010 ) . after an informed consent for the participation in the study", "this cross - sectional study included 595 subjects with t2 dm and 200 subjects without t2 dm ( control group ) .", "they were selected among patients admitted to the diabetes outpatient clinics of the general hospitals murska sobota and slovenj gradec , slovenia .", "subjects in the control group were not allowed to have t2 dm , and they were the staff of the general hospital murska sobota .", "subjects with t2 dm and control subjects were excluded if they had homozygous familial hypercholesterolaemia or a previous cardiovascular event such as myocardial infarction or a cerebral stroke .", "all ultrasound examinations were performed by two experienced doctors blinded to the participants ' diabetes status .", "the cimt , defined as the distance from the leading edge of the lumen - intima interface to the leading edge of the media - adventitia interface , was measured , as described previously .", "plaques were defined as a focal intima - media thickening and divided into 5 types according to their echogenic / echolucent characteristics , as previously described . the interobserver reliability for carotid plaque characterization", "the genomic dna was extracted from 100  l of whole blood using a flexigene dna isolation kit , in accordance with the recommended protocol ( qiagene gmbh , hilden , germany ) . for vegf rs2010963 polymorphism competitive allele specific pcr ( kasp )", "was conducted on an abi step - one system ( applied biosystems , foster city , ca ) .", "the reaction mixture ( 5  l ) contained 2.5  l 2x kaspar reaction mix ( v3 ) , 0.07  l assay mix , 1.43  l of distilled water dnase / rnase - free ( gibco , invitrogen life technologies ) , and 10  ng of extracted genomic dna ( 1  l ) .", "thermal cycling employed the following conditions : hot - start enzyme activation ( 15  min at 94c ) , denaturation ( 20  sec at 94c ) followed by 10 cycles of touchdown over 6557c for 60  sec ( dropping 0.8c per cycle ) , and final 26 cycles ( 20  sec at 94c and 60  sec at annealing temperature 57c ) . for rs2071559 ( kdr ) everything was the same with the exception of thermal conditions . hot - start enzyme activation ( 15  min at 94c ) and denaturation ( 20  sec at 94c ) were followed by 15 cycles of touchdown over 5565c for 60  sec ( dropping 0.8c per cycle ) and final 26 cycles ( 20  sec at 93c and 60  sec at annealing temperature 58c ) .", "in addition , the fasting serum vegf levels were analyzed in 70 subjects with t2 dm and in 33 subjects with t2 dm .", "for the determination of the fasting serum vegf concentration ( isoform vegf 165 ) , a solid phase sandwich elisa using two kinds of high specific antibodies ( hvegf assay kit , ibl co. , ltd .", "the respective cv ( % ) were between 3 and 5.5 for interassay measurements and between 2.6 and 5.3 for intra - assay measurements .", "continuous clinical data were compared using unpaired student 's t - test or analysis of variance ( anova ) .", "a statistical analysis was performed using the spss program for windows version 21 ( spss inc . ,", "patients with t2 dm were older , had a greater waist circumference , and had higher fasting glucose and hba1c levels compared to controls , whereas there were no differences in bmi and systolic and diastolic blood pressure between patients with t2 dm and control subjects ( table 1 ) .", "patients with t2 dm had lower total , hdl , and ldl cholesterol levels and a higher triglyceride level compared to controls ( table 1 ) .", "plasma levels of inflammatory markers ( i.e. , hs - crp and fibrinogen ) were higher in patients with t2 dm compared to controls ( table 1 ) . additionally , there was higher percentage of men , statin therapy , and antihypertensive therapy and lower percentage of smokers in t2 dm group compared to control group ( table 1 ) .", "the genotype distributions in both patients with t2 dm and controls were in hardy - weinberg equilibrium for both vegf gene polymorphisms [ rs2010963 : t2 dm ( genotype frequencies : cc genotype 8.7% , cg genotype 47.1% , and gg genotype 44.2% ;  = 3.48 ; p = 0.06 ) and controls ( genotype frequencies : cc genotype 9% , cg genotype 48% , and gg genotype 43% ;  = 1.46 ; p = 0.22 ) ] .", "the genotype distributions in both patients with t2 dm and controls were in hardy - weinberg equilibrium for the kdr gene polymorphism [ rs2071559 : t2 dm ( genotype frequencies : cc genotype 22.0% , ct genotype 51.9% , and tt genotype 26.1% ;  = 0.97 ; p = 0.33 ) and controls ( genotype frequencies : cc genotype 30.0% , ct genotype 48.0% , and tt genotype 22.0% ;  = 0.63 ; p = 0.23 ) ] .", "no statistically significant differences in the vegf rs2010963 and kdr rs2071559 genotype distribution frequencies were observed between t2 dm patients and controls . the observed minor allele frequency ( maf ) distributions were mostly in agreement with the 1000 genomes project data in the european population .", "the c allele frequency of the vegf rs2010963 showed no significant difference ( p = 0.79 ) between patients with t2 dm and controls ( 32.3% versus 33% ) .", "however , the c allele frequency of the kdr rs2071559 polymorphism was significantly lower ( p = 0.04 ) in t2 dm subjects as compared to the controls ( 49% versus 54% ) .", "higher vegf serum levels were demonstrated in subjects with t2 dm with the cc genotype ( rs2010963 ) compared to those with other ( cg + gg ) genotypes ( table 2 ) . moreover , higher vegf serum levels were found in subjects with the cc genotype ( rs2071559 ) compared to those with other ( ct + tt ) genotypes ( table 2 ) .", "the comparison of atherosclerosis parameters was performed with regard to different genotypes of the vegf polymorphism ( rs2010963 ) upon enrolment . in our study", ", we did not demonstrate any association between the rs2010963 and either cimt , the sum of plaque thickness , the number of involved segments , hscrp or the presence of carotid plaques , or the presence of unstable carotid plaques ( tables 3 and 4 ) .", "we did , however , demonstrate an association between the rs2071559 and either cimt or the sum of plaque thickness in subjects with t2 dm ( table 3 ) .", "in our study , we demonstrated an association between the rs2071559 of kdr and cimt in subjects with t2 dm , whereas we did not demonstrate an association between tested polymorphism of vegf ( rs2010963 ) and cimt .", "variations in the vegf gene were reported to be weakly associated with cimt . none of the single genotyped polymorphisms ( 2578a > c rs699947 ,", "634c > g rs2010963 , and + 936c > t rs3025039 ) were significantly associated with overall imt in the study reported by kangas - kontio and coworkers .", "the haplotype ccc , however , was associated with higher overall cimt in women and the haplotype cct with higher cimt in the internal carotid artery in men .", "additionally , we also demonstrated an association between the rs2071559 of kdr and the sum of plaque thickness in subjects with t2 dm , whereas no association between tested polymorphism of vegf ( rs2010963 ) and markers of carotid atherosclerosis was demonstrated .", "the rs2010963 polymorphism of the vegf gene was not demonstrated to exert a significant influence on the risk of subclinical atherosclerosis manifested by the presence of endothelial dysfunction by brachial artery reactivity and increased cimt in a series of patients with rheumatoid arthritis .", "contrary , the importance of vegf and its receptor ( vegfr1 ) was reported by russell and coworkers .", "in unstable plaques ( cap rupture / thinning ) increased vegf and receptor ( vegfr1 ) staining as well as increased microvessel density was demonstrated in comparison with stable carotid plaques .", "additionally , marino and coworkers have recently reported that both circulating and intraplaque polymorphonuclear neutrophils ( pmn ) from subjects with carotid atherosclerosis are active producers of vegf , il-8 , and elastase . moreover", ", an evidence is provided that these pmn have an increased ability to produce vegf ( at mrna levels ) in comparison to cells from healthy subjects . additionally , increased vegf mrna occurs in both intraplaque and circulating pmn , at rest as well as after stimulation , suggesting that such functional changes are systemic and not limited to cells infiltrating the vascular wall .", "in contrast to these findings , we did not demonstrate an effect of vegf / kdr polymorphisms on the presence of either plaques or unstable plaques , since no difference in genotype distribution was present . in our study , the effect of either rs2071559 of kdr or rs2010963 on vegf serum levels was demonstrated .", "these findings are in accordance with our previous studies in which subjects with recent mi history ( up to 9 months after mi ) were enrolled [ 13 , 16 , 25 ] . moreover , increased plasma vegf levels demonstrated in the stable phase after mi correlated with inflammation cytokines ( il-8 and il-6 ) , but not with atherosclerotic burden .", "in contrast to the minor effect of the rs2071559 of kdr and the absence of the rs2010963 of the vegf , an association of either rs2071559 or rs2010963 with mi has recently been reported in caucasians with t2 dm [ 13 , 16 , 24 ] . our present findings and previous reports are additional evidence that markers of carotid atherosclerosis and atherothrombotic events ( i.e. , mi ) are most probably not regulated via similar genetical / biological mechanisms . to conclude , in our study we demonstrated a minor effect of the rs2071559 of kdr on markers of carotid atherosclerosis ( cimt , sum of plaque thickness ) in subjects with t2 dm , whereas we failed to demonstrate an effect of tested polymorphism of the vegf gene ( rs2010963 ) on markers of carotid atherosclerosis"], "abstract_text": ["<S> \n background . the current study was designed to reveal possible associations between the polymorphisms of the vascular endothelial growth factor ( vegf ) gene ( rs2010963 ) and its receptor , kinase insert domain - containing receptor ( kdr ) gene polymorphism ( rs2071559 ) , and markers of carotid atherosclerosis in patients with type 2 diabetes mellitus ( t2 dm ) . </S>", "<S> patients and methods . </S>", "<S> 595 t2 dm subjects and 200 control subjects were enrolled . the carotid intima - media thickness </S>", "<S> ( cimt ) and plaque characteristics ( presence and structure ) were assessed ultrasonographically . </S>", "<S> biochemical analyses were performed using standard biochemical methods . </S>", "<S> genotyping of vegf / kdr polymorphisms ( rs2010963 , rs2071559 ) was performed using kaspar assays . </S>", "<S> results . </S>", "<S> genotype distributions and allele frequencies of the vegf / kdr polymorphisms ( rs2010963 , rs2071559 ) were not statistically significantly different between diabetic patients and controls . in our study </S>", "<S> , we demonstrated an association between the rs2071559 of kdr and either cimt or the sum of plaque thickness in subjects with t2 dm . </S>", "<S> we did not , however , demonstrate any association between the tested polymorphism of vegf ( rs2010963 ) and either cimt , the sum of plaque thickness , the number of involved segments , hscrp , the presence of carotid plaques , or the presence of unstable carotid plaques . conclusions . in the present study </S>", "<S> , we demonstrated minor effect of the rs2071559 of kdr on markers of carotid atherosclerosis in subjects with t2 dm . </S>"], "labels": null, "section_names": ["1. Introduction ", "2. Material and Methods", "3. Results", "4. Discussion"], "sections": [["type 2 diabetes mellitus ( t2 dm ) is considered a major epidemic of this century .", "it is estimated that its prevalence will increase worldwide from 371 million people in 2013 to 552 million people in 2030 .", "t2 dm is associated with accelerated progression of atherosclerosis , the major cause of vascular complications leading to increased morbidity and mortality .", "chronic , low - grade inflammation has been demonstrated to be involved in the pathogenesis of atherosclerosis in subjects at high risk to develop cardiovascular disease [ 37 ] . among immune cells infiltrating atherosclerotic lesions , polymorphonuclear neutrophil leukocytes with their products", "were reported to have an important role in the development and progression of atherosclerosis [ 811 ] .", "marino and coworkers have recently reported that both circulating and intraplaque polymorphonuclear neutrophil leukocytes from subjects with carotid atherosclerosis are active producers of different inflammatory mediators including the vascular endothelial growth factor ( vegf ) .", "several environmental and genetic factors ( i.e. , hypoxia , hyperglycemia , oxidative stress , ischemia , and gene polymorphisms of vegf ) influence plasma vegf levels [ 1216 ] . among several polymorphisms of the vegf gene ,", "the rs2010963 ( 634c / g polymorphism of the vegf gene ) and few others were reported to affect serum vegf levels [ 1315 ] .", "moreover , rs2010963 was demonstrated to be associated with several disorders , such as diabetic retinopathy , diabetic nephropathy , myocardial infarction , and impaired prognosis in patients with chronic heart failure [ 1315 , 17 ] . despite these findings ,", "however , data about vegf polymorphisms and their possible association with carotid atherosclerosis in patients with diabetes mellitus are limited [ 1820 ] .", "additionally , cimt is highly heritable and associated with stroke and myocardial infarction , making it a promising quantitative intermediate phenotype for genetic studies of vascular disease .", "the present study was thus designed to investigate the association between polymorphisms of the vegf gene ( rs2010963 ) and the kdr gene ( rs2071559 ) and markers of carotid atherosclerosis ( such as carotid intima - media thickness ( cimt ) , the number of affected segments of carotid arteries , and the sum of plaques thickness ) in patients with t2 dm ."], ["the study protocol was approved by the slovene medical ethics committee in september 2010 ( protocol number 128/09/2010 ) . after an informed consent for the participation in the study", "this cross - sectional study included 595 subjects with t2 dm and 200 subjects without t2 dm ( control group ) .", "they were selected among patients admitted to the diabetes outpatient clinics of the general hospitals murska sobota and slovenj gradec , slovenia .", "subjects in the control group were not allowed to have t2 dm , and they were the staff of the general hospital murska sobota .", "subjects with t2 dm and control subjects were excluded if they had homozygous familial hypercholesterolaemia or a previous cardiovascular event such as myocardial infarction or a cerebral stroke .", "all ultrasound examinations were performed by two experienced doctors blinded to the participants ' diabetes status .", "the cimt , defined as the distance from the leading edge of the lumen - intima interface to the leading edge of the media - adventitia interface , was measured , as described previously .", "plaques were defined as a focal intima - media thickening and divided into 5 types according to their echogenic / echolucent characteristics , as previously described . the interobserver reliability for carotid plaque characterization", "the genomic dna was extracted from 100  l of whole blood using a flexigene dna isolation kit , in accordance with the recommended protocol ( qiagene gmbh , hilden , germany ) . for vegf rs2010963 polymorphism competitive allele specific pcr ( kasp )", "was conducted on an abi step - one system ( applied biosystems , foster city , ca ) .", "the reaction mixture ( 5  l ) contained 2.5  l 2x kaspar reaction mix ( v3 ) , 0.07  l assay mix , 1.43  l of distilled water dnase / rnase - free ( gibco , invitrogen life technologies ) , and 10  ng of extracted genomic dna ( 1  l ) .", "thermal cycling employed the following conditions : hot - start enzyme activation ( 15  min at 94c ) , denaturation ( 20  sec at 94c ) followed by 10 cycles of touchdown over 6557c for 60  sec ( dropping 0.8c per cycle ) , and final 26 cycles ( 20  sec at 94c and 60  sec at annealing temperature 57c ) . for rs2071559 ( kdr ) everything was the same with the exception of thermal conditions . hot - start enzyme activation ( 15  min at 94c ) and denaturation ( 20  sec at 94c ) were followed by 15 cycles of touchdown over 5565c for 60  sec ( dropping 0.8c per cycle ) and final 26 cycles ( 20  sec at 93c and 60  sec at annealing temperature 58c ) .", "in addition , the fasting serum vegf levels were analyzed in 70 subjects with t2 dm and in 33 subjects with t2 dm .", "for the determination of the fasting serum vegf concentration ( isoform vegf 165 ) , a solid phase sandwich elisa using two kinds of high specific antibodies ( hvegf assay kit , ibl co. , ltd .", "the respective cv ( % ) were between 3 and 5.5 for interassay measurements and between 2.6 and 5.3 for intra - assay measurements .", "continuous clinical data were compared using unpaired student 's t - test or analysis of variance ( anova ) .", "a statistical analysis was performed using the spss program for windows version 21 ( spss inc . ,"], ["patients with t2 dm were older , had a greater waist circumference , and had higher fasting glucose and hba1c levels compared to controls , whereas there were no differences in bmi and systolic and diastolic blood pressure between patients with t2 dm and control subjects ( table 1 ) .", "patients with t2 dm had lower total , hdl , and ldl cholesterol levels and a higher triglyceride level compared to controls ( table 1 ) .", "plasma levels of inflammatory markers ( i.e. , hs - crp and fibrinogen ) were higher in patients with t2 dm compared to controls ( table 1 ) . additionally , there was higher percentage of men , statin therapy , and antihypertensive therapy and lower percentage of smokers in t2 dm group compared to control group ( table 1 ) .", "the genotype distributions in both patients with t2 dm and controls were in hardy - weinberg equilibrium for both vegf gene polymorphisms [ rs2010963 : t2 dm ( genotype frequencies : cc genotype 8.7% , cg genotype 47.1% , and gg genotype 44.2% ;  = 3.48 ; p = 0.06 ) and controls ( genotype frequencies : cc genotype 9% , cg genotype 48% , and gg genotype 43% ;  = 1.46 ; p = 0.22 ) ] .", "the genotype distributions in both patients with t2 dm and controls were in hardy - weinberg equilibrium for the kdr gene polymorphism [ rs2071559 : t2 dm ( genotype frequencies : cc genotype 22.0% , ct genotype 51.9% , and tt genotype 26.1% ;  = 0.97 ; p = 0.33 ) and controls ( genotype frequencies : cc genotype 30.0% , ct genotype 48.0% , and tt genotype 22.0% ;  = 0.63 ; p = 0.23 ) ] .", "no statistically significant differences in the vegf rs2010963 and kdr rs2071559 genotype distribution frequencies were observed between t2 dm patients and controls . the observed minor allele frequency ( maf ) distributions were mostly in agreement with the 1000 genomes project data in the european population .", "the c allele frequency of the vegf rs2010963 showed no significant difference ( p = 0.79 ) between patients with t2 dm and controls ( 32.3% versus 33% ) .", "however , the c allele frequency of the kdr rs2071559 polymorphism was significantly lower ( p = 0.04 ) in t2 dm subjects as compared to the controls ( 49% versus 54% ) .", "higher vegf serum levels were demonstrated in subjects with t2 dm with the cc genotype ( rs2010963 ) compared to those with other ( cg + gg ) genotypes ( table 2 ) . moreover , higher vegf serum levels were found in subjects with the cc genotype ( rs2071559 ) compared to those with other ( ct + tt ) genotypes ( table 2 ) .", "the comparison of atherosclerosis parameters was performed with regard to different genotypes of the vegf polymorphism ( rs2010963 ) upon enrolment . in our study", ", we did not demonstrate any association between the rs2010963 and either cimt , the sum of plaque thickness , the number of involved segments , hscrp or the presence of carotid plaques , or the presence of unstable carotid plaques ( tables 3 and 4 ) .", "we did , however , demonstrate an association between the rs2071559 and either cimt or the sum of plaque thickness in subjects with t2 dm ( table 3 ) ."], ["in our study , we demonstrated an association between the rs2071559 of kdr and cimt in subjects with t2 dm , whereas we did not demonstrate an association between tested polymorphism of vegf ( rs2010963 ) and cimt .", "variations in the vegf gene were reported to be weakly associated with cimt . none of the single genotyped polymorphisms ( 2578a > c rs699947 ,", "634c > g rs2010963 , and + 936c > t rs3025039 ) were significantly associated with overall imt in the study reported by kangas - kontio and coworkers .", "the haplotype ccc , however , was associated with higher overall cimt in women and the haplotype cct with higher cimt in the internal carotid artery in men .", "additionally , we also demonstrated an association between the rs2071559 of kdr and the sum of plaque thickness in subjects with t2 dm , whereas no association between tested polymorphism of vegf ( rs2010963 ) and markers of carotid atherosclerosis was demonstrated .", "the rs2010963 polymorphism of the vegf gene was not demonstrated to exert a significant influence on the risk of subclinical atherosclerosis manifested by the presence of endothelial dysfunction by brachial artery reactivity and increased cimt in a series of patients with rheumatoid arthritis .", "contrary , the importance of vegf and its receptor ( vegfr1 ) was reported by russell and coworkers .", "in unstable plaques ( cap rupture / thinning ) increased vegf and receptor ( vegfr1 ) staining as well as increased microvessel density was demonstrated in comparison with stable carotid plaques .", "additionally , marino and coworkers have recently reported that both circulating and intraplaque polymorphonuclear neutrophils ( pmn ) from subjects with carotid atherosclerosis are active producers of vegf , il-8 , and elastase . moreover", ", an evidence is provided that these pmn have an increased ability to produce vegf ( at mrna levels ) in comparison to cells from healthy subjects . additionally , increased vegf mrna occurs in both intraplaque and circulating pmn , at rest as well as after stimulation , suggesting that such functional changes are systemic and not limited to cells infiltrating the vascular wall .", "in contrast to these findings , we did not demonstrate an effect of vegf / kdr polymorphisms on the presence of either plaques or unstable plaques , since no difference in genotype distribution was present . in our study , the effect of either rs2071559 of kdr or rs2010963 on vegf serum levels was demonstrated .", "these findings are in accordance with our previous studies in which subjects with recent mi history ( up to 9 months after mi ) were enrolled [ 13 , 16 , 25 ] . moreover , increased plasma vegf levels demonstrated in the stable phase after mi correlated with inflammation cytokines ( il-8 and il-6 ) , but not with atherosclerotic burden .", "in contrast to the minor effect of the rs2071559 of kdr and the absence of the rs2010963 of the vegf , an association of either rs2071559 or rs2010963 with mi has recently been reported in caucasians with t2 dm [ 13 , 16 , 24 ] . our present findings and previous reports are additional evidence that markers of carotid atherosclerosis and atherothrombotic events ( i.e. , mi ) are most probably not regulated via similar genetical / biological mechanisms . to conclude , in our study we demonstrated a minor effect of the rs2071559 of kdr on markers of carotid atherosclerosis ( cimt , sum of plaque thickness ) in subjects with t2 dm , whereas we failed to demonstrate an effect of tested polymorphism of the vegf gene ( rs2010963 ) on markers of carotid atherosclerosis"]]}
{"article_id": "PMC4885187", "article_text": ["hidradenomas are benign cutaneous tumors of sweat gland origin , with the clear cell type constituting the most frequent histologic variety . usually , they are diagnosed in the elderly population , the peak incidence being the fifth to sixth decade .", "we describe a young girl , in whom nodular hidradenoma developed at the age of 6 years .", "hence , our case demonstrates that nodular hidradenoma is a rare differential diagnosis of skin tumors , even in the pediatric age group .", "a 10-year - old girl presented to us with a swelling on the left thigh , which had been progressively enlarging over the preceding 4 years .", "cutaneous examination revealed a solitary erythematous dome - shaped , well - circumscribed tumor , measuring approximately 6 cm  4.5 cm .", "there was spontaneous dripping of straw - colored serous fluid at the rate of 2030 drops / min , the discharge accentuating on pressure .", "biochemical analysis of the serous fluid revealed glucose 25 mg / dl , protein 4 g / dl , sodium 131 mmol / l , and potassium 4.3 mmol / l , almost similar to that of serum , the low concentration of glucose compelling us to think of something else . the mass was excised .", "the solid portion was largely composed of a cell type having finely granular , faintly eosinophilic cytoplasm with a dark - colored round to oval nucleus .", "the cyst was lined by a single layer of cuboidal epithelium without evidence of decapitation secretion [ figure 5 ] .", "solitary dome - shaped , well - circumscribed tumor over the left thigh photomicrograph showing a well - encapsulated mass composed of solid and cystic portions ( h and e , 40 ) photomicrograph showing a well - encapsulated mass with large cystic spaces within it .", "ductal luminal structures can be appreciated ( h and e , 100 ) photomicrograph showing clear cells in the center along with cystic spaces ( h and e , 400 ) note the large cystic cavity within the mass .", "clear cell hidradenoma or eccrine acrospiroma of the skin was first described by liu , in 1949 , as clear cell papillary carcinoma of the skin .", "it was reported under various designations such as nodular hidradenoma , eccrine acrospiroma , solid - cystic hidradenoma , clear cell acrospiroma , clear cell myoepithelioma , and eccrine sweat gland adenoma .", "clinically , nodular hidradenoma presents as a slow - growing , red- , blue- , or brown - colored solitary , 530 mm in size , freely mobile and firm nodule , with an occasional cystic appearance .", "these are most commonly found on the scalp , face , thorax , abdomen , and gluteal region .", "this tumor is found mainly in adults and is excised more commonly in women than in men .", "our patient was a young girl who presented with a gradually increasing swelling on the lower limb , a site that is unusual for the development of nodular hidradenoma .", "literature search showed very few cases of nodular hidradenoma on the lower limb , that too in the pediatric age group .", "however , a case of nodular hidradenoma masquerading as umbilical polyp has been reported in a 1-year - old male child", "our patient complained of serous discharge from the lesion , for the preceding 3 months .", "clinical differential diagnoses for our case were cutaneous lymphoma , dermatofibrosarcoma protuberans , epidermoid cyst , and sweat gland tumor . however , histology reliably excluded all the possibilities .", "it was predominantly composed of a cell type having finely granular , faintly eosinophilic cytoplasm with a dark - colored round to oval nucleus .", "the chronically discharging nature of the tumor in our patient was attributable to the fluid - filled cavity within the lesion .", "biochemical analysis of the fluid was almost consistent with that of serum , however , the low concentration of glucose hinted that it might be normal sweat .", "recently , nuclear grooving has been described as a useful morphological feature to aid in its diagnosis .", "immunohistochemical staining of the tumor cells demonstrates positive staining with antibodies against ck - cam 5.2 , ber - ep4 , p63 , epithelial membrane antigen , s-100 protein , smooth muscle actin , and vimentin .", "however , due to unavailability of resources and financial constraints , the immunohistochemical analysis could not be performed in our case .", "the high rate of local recurrence ( 10% ) and potential for malignant transformation make surgical removal with wide margins as the widely accepted modality of treatment .", "the high recurrence is attributable to incomplete resection and to tumor tissue located between the dermis and subcutaneous tissue .", "these are characterized by increased mitotic activity , angiolymphatic invasion , local extension into deeper tissues , and a dispersed pattern of growth .", "nodular hidradenoma in the pediatric age group is extremely rareit is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limbthe presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting . \n ", "nodular hidradenoma in the pediatric age group is extremely rare it is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limb the presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting .", "nodular hidradenoma in the pediatric age group is extremely rareit is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limbthe presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting . \n ", "nodular hidradenoma in the pediatric age group is extremely rare it is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limb the presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting .", "nodular hidradenoma in the pediatric age group is extremely rareit is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limbthe presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting . \n ", "nodular hidradenoma in the pediatric age group is extremely rare it is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limb the presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting ."], "abstract_text": ["<S> a 10-year - old girl presented with a swelling on her left thigh that was associated with dripping of serous fluid from the lesion . based on histological features of the excised tumor , a diagnosis of nodular hidradenoma was made . </S>", "<S> atypical features including large size of the tumor , location on the lower limb , a weeping presentation , and histological finding of a nodular hidradenoma with an unusually large cystic cavity discharging fluid prompted us to report the case . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "None", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["hidradenomas are benign cutaneous tumors of sweat gland origin , with the clear cell type constituting the most frequent histologic variety . usually , they are diagnosed in the elderly population , the peak incidence being the fifth to sixth decade .", "we describe a young girl , in whom nodular hidradenoma developed at the age of 6 years .", "hence , our case demonstrates that nodular hidradenoma is a rare differential diagnosis of skin tumors , even in the pediatric age group ."], ["a 10-year - old girl presented to us with a swelling on the left thigh , which had been progressively enlarging over the preceding 4 years .", "cutaneous examination revealed a solitary erythematous dome - shaped , well - circumscribed tumor , measuring approximately 6 cm  4.5 cm .", "there was spontaneous dripping of straw - colored serous fluid at the rate of 2030 drops / min , the discharge accentuating on pressure .", "biochemical analysis of the serous fluid revealed glucose 25 mg / dl , protein 4 g / dl , sodium 131 mmol / l , and potassium 4.3 mmol / l , almost similar to that of serum , the low concentration of glucose compelling us to think of something else . the mass was excised .", "the solid portion was largely composed of a cell type having finely granular , faintly eosinophilic cytoplasm with a dark - colored round to oval nucleus .", "the cyst was lined by a single layer of cuboidal epithelium without evidence of decapitation secretion [ figure 5 ] .", "solitary dome - shaped , well - circumscribed tumor over the left thigh photomicrograph showing a well - encapsulated mass composed of solid and cystic portions ( h and e , 40 ) photomicrograph showing a well - encapsulated mass with large cystic spaces within it .", "ductal luminal structures can be appreciated ( h and e , 100 ) photomicrograph showing clear cells in the center along with cystic spaces ( h and e , 400 ) note the large cystic cavity within the mass ."], ["clear cell hidradenoma or eccrine acrospiroma of the skin was first described by liu , in 1949 , as clear cell papillary carcinoma of the skin .", "it was reported under various designations such as nodular hidradenoma , eccrine acrospiroma , solid - cystic hidradenoma , clear cell acrospiroma , clear cell myoepithelioma , and eccrine sweat gland adenoma .", "clinically , nodular hidradenoma presents as a slow - growing , red- , blue- , or brown - colored solitary , 530 mm in size , freely mobile and firm nodule , with an occasional cystic appearance .", "these are most commonly found on the scalp , face , thorax , abdomen , and gluteal region .", "this tumor is found mainly in adults and is excised more commonly in women than in men .", "our patient was a young girl who presented with a gradually increasing swelling on the lower limb , a site that is unusual for the development of nodular hidradenoma .", "literature search showed very few cases of nodular hidradenoma on the lower limb , that too in the pediatric age group .", "however , a case of nodular hidradenoma masquerading as umbilical polyp has been reported in a 1-year - old male child", "our patient complained of serous discharge from the lesion , for the preceding 3 months .", "clinical differential diagnoses for our case were cutaneous lymphoma , dermatofibrosarcoma protuberans , epidermoid cyst , and sweat gland tumor . however , histology reliably excluded all the possibilities .", "it was predominantly composed of a cell type having finely granular , faintly eosinophilic cytoplasm with a dark - colored round to oval nucleus .", "the chronically discharging nature of the tumor in our patient was attributable to the fluid - filled cavity within the lesion .", "biochemical analysis of the fluid was almost consistent with that of serum , however , the low concentration of glucose hinted that it might be normal sweat .", "recently , nuclear grooving has been described as a useful morphological feature to aid in its diagnosis .", "immunohistochemical staining of the tumor cells demonstrates positive staining with antibodies against ck - cam 5.2 , ber - ep4 , p63 , epithelial membrane antigen , s-100 protein , smooth muscle actin , and vimentin .", "however , due to unavailability of resources and financial constraints , the immunohistochemical analysis could not be performed in our case .", "the high rate of local recurrence ( 10% ) and potential for malignant transformation make surgical removal with wide margins as the widely accepted modality of treatment .", "the high recurrence is attributable to incomplete resection and to tumor tissue located between the dermis and subcutaneous tissue .", "these are characterized by increased mitotic activity , angiolymphatic invasion , local extension into deeper tissues , and a dispersed pattern of growth .", "nodular hidradenoma in the pediatric age group is extremely rareit is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limbthe presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting . \n ", "nodular hidradenoma in the pediatric age group is extremely rare it is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limb the presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting ."], ["nodular hidradenoma in the pediatric age group is extremely rareit is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limbthe presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting . \n ", "nodular hidradenoma in the pediatric age group is extremely rare it is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limb the presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting ."], [""], ["nodular hidradenoma in the pediatric age group is extremely rareit is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limbthe presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting . \n ", "nodular hidradenoma in the pediatric age group is extremely rare it is usually located over the head , neck , and trunk . in our case ,", "the lesion was present in the lower limb the presence of an unusually large cystic cavity within the tumoral mass , leading to serous discharge , makes our case even more interesting ."]]}
{"article_id": "PMC2661578", "article_text": ["the gothenburg osteoporosis and obesity determinants ( good ) study was initiated with the aim to determine both environmental and genetic factors involved in the regulation of fat mass as previously described ( 23 ) . to be included in the good study , subjects had to be male , 18 to 20 years of age , and willing to participate in the study", "a total of 1,068 subjects , representative of the general young male population of gothenburg ( 23 ) ( data not shown ) , were enrolled in the study . of the 1,068 subjects ,", "complete growth and weight charts for determination of bmi between 1 and 10 years of age were available for 612 subjects ( growth chart subsample ) .", "dual - energy x - ray absorptiometry analyses were performed on 610 subjects of the growth chart subsample , whereas abdominal ct scans were performed on 201 ( the ct subsample ) of the 612 study subjects in the growth chart subsample .", "the growth chart subsample is representative of the entire good cohort in terms of age ( good 18.9  0.6 years , growth chart subsample 18.9  0.5 years , nonsignificant ) , weight ( good 73.9  11.9 kg , growth chart subsample 73.3  11.5 kg , nonsignificant ) , height ( good 181.4  6.8 cm , growth chart subsample 182.0  6.8 cm , nonsignificant ) , and bmi ( good 22.4  3.2 kg / m , growth chart subsample 22.1  3.1 kg / m , nonsignificant ) .", "the ct subsample is representative of both the growth chart subsample and the entire good cohort in terms of weight , height , and bmi ( ct subsample weight 72.7  11.2 kg , height 181.9  7.0 cm , bmi 21.9  3.0 kg / m , nonsignificant versus both the entire good cohort and the growth chart subsample ) .", "however , the subjects of the ct subsample were slightly younger ( ct subsample 18.7  0.5 years ) than both the subjects of the entire good cohort and the subjects of the growth chart subsample ( p < 0.05 ) .", "the good study was approved by the local ethics committee at gothenburg university . written and oral informed consent was obtained from all the study participants .", "height was measured using a wall - mounted stadiometer and weight was measured to the nearest 0.1 kg as previously described ( 16,23 ) .", "total body fat mass , percentage body fat , total body lean mass , and fat mass of the trunk were assessed using the lunar prodigy dxa ( ge lunar , madison , wi ) .", "a previously described ct technique was used to measure the cross - sectional adipose area of the abdomen ( 16,24,25 ) .", "a single slice at the level of the fourth lumbar vertebra was acquired with a general electric high speed advantage ct system ( version rp2 ; ge medical systems , milwaukee , wi ) .", "subcutaneous and visceral adipose tissue areas were measured , and then the visceral adipose tissue area was divided into an intraperitoneal and a retroperitoneal adipose tissue area .", "growth and weight charts were collected for the subjects of the good study and longitudinal growth was curve fitted according to the infancy - childhood  puberty ( icp ) model ( 26 ) as previously described ( 16,27 ) .", "the icp model represents a widely used model for fitting of human growth data to mathematical functions ( 26 ) .", "the infancy part is represented by an exponential function , the childhood part by a quadratic function , and the pubertal part by a logistic function .", "the model also includes the transformations between the different phases . by using this model , we computed values of height at exact ages .", "body composition , including bmi , is greatly influenced by pubertal stage and thereby pubertal timing ( 28 ) .", "complete growth charts with enough data to determine pubertal timing , requiring several height and weight measurements around the pubertal growth spurt , were not available for the complete growth chart subsample used in the present study .", "we have therefore chosen to use measurements of bmi before and after , but not during , puberty .", "body weights between 1 and 10 years of age were estimated through fitting of the weight curve for each child using smooth splines ( smooth.spline in the r package statistics , the r foundation for statistic computing , vienna , austria ; http://www.r-project.org ) .", "bmi values between 1 and 10 years of age were then calculated from the estimated values of weight and height .", "the z score , the standardization of a variable relative to the investigated population expressed in sds , was calculated using the software spss 15.0 ( spss , chicago , il ) . for each subject ( n = 612 ) in this cohort and for each age , the height was calculated using the icp model .", "these values were then used to calculate the z score for each subject at each age based on the variation ( sd ) in calculated height within the presently investigated population at that specific age .", "the variance of young adult body composition parameters ( r ) explained by bmi or bmi z score changes and the corresponding  coefficients during childhood and adolescence was calculated using linear regression analyses with age - adjusted adult body composition variables as the dependent variable .", "age adjustments were performed because the subjects differed somewhat in age at body composition analysis ( 1820 years of age , mean  sd 18.9  0.5 years ) and were performed between age at fat analysis and the body composition variables .", "all subsequent statistical calculations were performed using the age - adjusted body composition variables . for the calculations in fig .", "for all the statistical analyses , the software spss ( version 15.0 ) was used .", "height was measured using a wall - mounted stadiometer and weight was measured to the nearest 0.1 kg as previously described ( 16,23 ) .", "total body fat mass , percentage body fat , total body lean mass , and fat mass of the trunk were assessed using the lunar prodigy dxa ( ge lunar , madison , wi ) .", "a previously described ct technique was used to measure the cross - sectional adipose area of the abdomen ( 16,24,25 ) .", "a single slice at the level of the fourth lumbar vertebra was acquired with a general electric high speed advantage ct system ( version rp2 ; ge medical systems , milwaukee , wi ) .", "subcutaneous and visceral adipose tissue areas were measured , and then the visceral adipose tissue area was divided into an intraperitoneal and a retroperitoneal adipose tissue area .", "growth and weight charts were collected for the subjects of the good study and longitudinal growth was curve fitted according to the infancy - childhood  puberty ( icp ) model ( 26 ) as previously described ( 16,27 ) .", "the icp model represents a widely used model for fitting of human growth data to mathematical functions ( 26 ) .", "the infancy part is represented by an exponential function , the childhood part by a quadratic function , and the pubertal part by a logistic function .", "the model also includes the transformations between the different phases . by using this model , we computed values of height at exact ages .", "body composition , including bmi , is greatly influenced by pubertal stage and thereby pubertal timing ( 28 ) .", "complete growth charts with enough data to determine pubertal timing , requiring several height and weight measurements around the pubertal growth spurt , were not available for the complete growth chart subsample used in the present study .", "we have therefore chosen to use measurements of bmi before and after , but not during , puberty .", "body weights between 1 and 10 years of age were estimated through fitting of the weight curve for each child using smooth splines ( smooth.spline in the r package statistics , the r foundation for statistic computing , vienna , austria ; http://www.r-project.org ) .", "bmi values between 1 and 10 years of age were then calculated from the estimated values of weight and height .", "the z score , the standardization of a variable relative to the investigated population expressed in sds , was calculated using the software spss 15.0 ( spss , chicago , il ) . for each subject ( n = 612 ) in this cohort and for each age", "these values were then used to calculate the z score for each subject at each age based on the variation ( sd ) in calculated height within the presently investigated population at that specific age .", "the variance of young adult body composition parameters ( r ) explained by bmi or bmi z score changes and the corresponding  coefficients during childhood and adolescence was calculated using linear regression analyses with age - adjusted adult body composition variables as the dependent variable .", "age adjustments were performed because the subjects differed somewhat in age at body composition analysis ( 1820 years of age , mean  sd 18.9  0.5 years ) and were performed between age at fat analysis and the body composition variables .", "all subsequent statistical calculations were performed using the age - adjusted body composition variables . for the calculations in fig .", "for all the statistical analyses , the software spss ( version 15.0 ) was used .", "the present study represents a subsample ( n = 612 [ growth chart subsample ] ) of the original good cohort ( n = 1,068 ) in which subjects with available detailed growth charts from 1 to 10 years of age were included .", "anthropometrics and measurements of fat ( dxa)/adipose tissue areas ( abdominal ct ) of the subjects participating in the present study are presented in table 1 .", "average bmi in this cohort declined during early childhood years , and adiposity rebound ( the lowest bmi ) was reached slightly before 6 years of age , after which it began to increase ( fig .", "2 ) during both early ( age 14 years ) and late ( age 410 years ) childhood were , as expected , clear positive predictors of adult bmi . during adolescence ,", "pubertal stage influences body composition ( 29 ) . because interindividual variations in pubertal onset will confound the calculations during adolescence , the predictive role of bmi for each individual year during adolescence for adult bmi and body composition is not shown . instead", ", the  coefficient for the correlation of the entire adolescence ( age 1019 years ) is shown in fig .", "2 , demonstrating that , as expected , bmi changes during adolescence also predicted adult bmi .", "childhood bmi and variance in body composition and fat variables ( bmi [ a ] and bmi change [ b ] ) during early and late childhood .", "association between childhood bmi between 1 and 10 years of age and young - adult body composition parameters is expressed as accumulated r ( % , c and d ) or  coefficients ( sd in adult body composition parameter per sd in bmi at that age , e and f ) .", "variables of adult body composition and fat distribution have been age adjusted . at , adipose tissue ; ip , intraperitoneal ; sc , subcutaneous .", "correlation between change in standardized bmi during different developmental time periods and adult body composition and fat distribution parameters ( early childhood defined as 14 years , late childhood defined as 410 years , and adolescence defined as 10 years to young adult ) .", "bars indicate  coefficient ( 95% cis ) expressed as sd in investigated adult body composition parameter per sd change in bmi during that growth period .", "variables of adult body composition and fat distribution have been age adjusted . * * * p < 0.01 , * p < 0.05 significant association between the developmental time period and the investigated body composition parameter .", "95% cis are given for the  coefficients , making it possible to evaluate the differences in relative contribution of the three time periods for each dependent parameter .", "at , adipose tissue ; ip , intraperitoneal ; ns , not significant ; sc , subcutaneous .", "bmi alterations during development reflect changes in fat mass , lean mass , or both . to study the role of childhood bmi and bmi changes for the prediction of different adult body compartments , the associations between bmi during development and age - adjusted adult body composition variables were analyzed . the variance ( r ) in adult total body lean mass explained by bmi as well as the corresponding  coefficients for the correlations increased during early childhood , leveled off during late childhood , and increased again during adolescence ( figs .", "the variance in adult bmi and the variance in adult lean mass explained by childhood bmi and the corresponding  coefficients for the correlations were similar until 4 years of age , when they separated distinctly ( fig .", "1c and e ) . the  coefficient for the correlation between change in bmi and adult lean mass was higher during adolescence than during late childhood ( fig .", "2 ) . thus , bmi changes during early childhood and adolescence but not during late childhood were major determinants of young adult lean mass .", "the prediction of young - adult total body fat mass from bmi and bmi changes during different developmental periods followed a different pattern from that of lean mass .", "the variances in total body fat mass and percentage total body fat explained by childhood bmi and the corresponding  coefficients for the correlation were low before 4 years of age , but after 4 years of age , the plotted cumulated variance ( r ) and the corresponding  coefficients for the correlation for young- adult total body fat mass and percentage body fat tightly followed the curve displaying the variance of adult bmi explained ( fig .", "the predictive value of childhood bmi for young - adult percentage total body fat increased substantially during late childhood years , as demonstrated by the fact that the variance in percentage total body fat explained by bmi at 4 years of age was only 1.7% , whereas it was 20.8% at 10 years of age ( fig .", "to analyze fat distribution in detail , we performed abdominal ct scans ( on a subset of the present good sample [ ct subsample ] , n = 201 ) for measurements of abdominal subcutaneous and visceral adipose tissue areas .", "the abdominal ct measurements revealed that the contribution of bmi during development for the explanation of the variance in young - adult subcutaneous adipose tissue area and the corresponding  coefficients for the correlations followed a pattern similar to that of total body fat mass and percentage total body fat , with a distinct increase after 4 years of age ( fig .", "1d and f ) . for both young - adult percentage fat mass and subcutaneous adipose tissue area ,", "the  coefficients for the correlations were of similar magnitude during late childhood and adolescence but clearly higher than seen during early childhood ( fig .", "bmi changes during late childhood and adolescence , but not during early childhood , clearly predicted young - adult total fat mass and the amount of subcutaneous adipose tissue .", "the contribution of bmi during the different developmental periods for the explanation of the variance in amount of young - adult visceral adipose tissue ( intraperitoneal + retroperitoneal adipose tissue areas ) and the corresponding  coefficients for the correlations followed a pattern different and clearly distinct from that of total body fat mass and subcutaneous adipose tissue area .", "bmi and bmi changes during early and late childhood contributed only marginally to the explanation of the variance in intraperitoneal adipose tissue area , and the corresponding  coefficient for the correlation between change in bmi and amount of young - adult intraperitoneal adipose tissue was low ( figs . 1d and f and 2 ) . instead , adolescence was the developmental period when bmi changes started to contribute substantially to the explanation of the variance in amount of adult intraperitoneal adipose tissue , and the corresponding  coefficients for the correlations between change in bmi and amount of young - adult intraperitoneal adipose tissue were then higher than seen during early and late childhood ( fig .", "the results for retroperitoneal adipose tissue were similar as seen for intraperitoneal adipose tissue ( data not shown ) .", "analyses using weight for age during childhood and adolescence showed , in a manner similar to that seen for bmi for age , clear age - dependent associations with adult fat parameters ( data not shown ) .", "in contrast , analyses of height for age did not show any obvious age - dependent association with fat parameters ( data not shown ) .", "changes in bmi during adolescence , but not during early or late childhood , predicted the amount of adult visceral adipose tissue . to further describe the association between developmental changes in bmi and adult fat mass / adipose tissue areas , subjects were divided into groups based on change in bmi z score ( > 1 sd , less than 1 sd , and average bmi z score change ) during the three different developmental time periods ( fig .", "subjects with more than 1 sd increase in bmi z score during late childhood and during adolescence had a clearly higher percentage total body fat mass ( late childhood , + 44% , p < 0.001 ; adolescence , + 60% , p < 0.001 , fig . 3a ) and larger subcutaneous adipose tissue area ( late childhood + 83% , p <", "3b ) than subjects with average change in bmi . in contrast , subjects with more than 1 sd increase in bmi z score during late childhood had unaffected intraperitoneal adipose tissue area compared with subjects with average change in bmi ( fig .", "3c ) . however , for adolescence , subjects with more than 1 sd increase in bmi z score had larger intraperitoneal adipose tissue area ( + 91% , p < 0.001 ) than subjects with unchanged bmi z score ( fig .", "a specific association between high young - adult subcutaneous , but not visceral ( intraperitoneal + retroperitoneal ) , adipose tissue area and increased bmi z score during late childhood is supported by the finding that young - adult visceral adipose tissue area adjusted for total adipose tissue area was clearly reduced in subjects with more than 1 sd increase in bmi z score during late childhood ( fig .", "3d ) . in contrast , an increase in bmi z score of more than 1 sd during adolescence was associated with an increased visceral adipose tissue area adjusted for total adipose tissue area ( fig .", "3d ) . subjects who decreased their bmi with more than 1 sd did not differ from those with no change in bmi z score for any of the investigated fat parameters ( fig .", "thus , the amount of visceral adipose tissue was predicted by large increases in standardized bmi specifically during adolescence , whereas a high amount of subcutaneous adipose tissue area was predicted by increases in bmi during both late childhood and adolescence .", "the impact of developmental bmi changes for percentage total body fat ( a ) , subcutaneous adipose tissue area ( b ) , intraperitoneal adipose tissue area ( c ) , and visceral ( intraperitoneal + retroperitoneal ) adipose tissue adjusted for total abdominal ( visceral + subcutaneous ) adipose tissue area ( d ) .", "subjects with more than 1 sd increase were compared with subjects with average change or less than 1 sd decrease in standardized bmi during early childhood ( 14 years of age ) , late childhood ( 410 years of age ) , and adolescence ( 1019 years of age ) .", "variables of adult body composition and fat distribution have been age adjusted and are expressed as means  sem and were analyzed using anova followed by tukey 's post hoc analysis . * * * p < 0.001 versus average , * * p < 0.01 versus average , # # # p < 0.001 versus less than 1 sd decrease , and # p < 0.05 versus less than 1 sd .", "a : early childhood less than 1 sd , n = 72 , average n = 471 ; more than 1 sd , n = 67 ; late childhood less than 1 sd , n = 75 , average n = 462 ; more than 1 sd , n = 73 ; adolescence less than 1 sd , n = 55 , average n = 500 ; more than 1 sd , n = 55 .", "d : early childhood less than 1 sd , n = 21 , average n = 160 ; more than 1 sd , n = 20 ; late childhood less than 1 sd , n = 22 , average n = 154 ; more than 1 sd , n = 25 ; adolescence less than 1 sd , n = 22 , average n = 164 ; more than 1 sd , n = 15 .", "we next investigated if bmi increases in subjects with low prepubertal bmi had a similar impact on the amount of adult visceral adipose tissue as seen in subjects with a high prepubertal bmi .", "subjects were divided into tertiles according to prepubertal ( 10 years of age ) bmi ( low 15.2  0.8", "kg / m , average 16.7  0.4 kg / m , and high 19.3  1.5 kg / m ) and tertiles of bmi z score change ( low 1.0  0.6 sd , average 0.1  0.2 sd , and high 0.7  0.6 sd ) during adolescence ( fig .", "subjects with greater increase in adolescence bmi had significantly larger visceral adipose tissue areas ( low prepubertal bmi + 56% , average prepubertal bmi + 53% , and high prepubertal bmi + 57% ; p < 0.05 for all ) than subjects with average adolescence bmi increase ( fig .", "bmi increases during adolescence predicted the amount of adult visceral adipose tissue independently of prepubertal bmi .", "bmi increases during adolescence are associated with high adult visceral fat mass in subjects with low , average , and high prepubertal bmi .", "intraperitoneal fat area ( y - axis ) in subjects divided into tertiles according to ( i ) prepubertal bmi ( x - axis ; low , average , and high ) and ( ii ) bmi - z score increase during adolescence ( z - axis ; low , average , and high ) .", "data are expressed as means and were analyzed using anova followed by tukey 's post hoc analysis . * p < 0.05 , * * p < 0.01 versus average bmi z score change during adolescence (  ) ; # # p < 0.01 versus low (  ) .", "n for lowest bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 9 , 19 , and 39 ; for average bmi during adolescence (  ) low prepubertal bmi to high ( left to right ) 26 , 25 , and 16 ; and for high bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 32 , 23 , and 12 .", "average bmi in this cohort declined during early childhood years , and adiposity rebound ( the lowest bmi ) was reached slightly before 6 years of age , after which it began to increase ( fig .", "2 ) during both early ( age 14 years ) and late ( age 410 years ) childhood were , as expected , clear positive predictors of adult bmi . during adolescence ,", "because interindividual variations in pubertal onset will confound the calculations during adolescence , the predictive role of bmi for each individual year during adolescence for adult bmi and body composition is not shown . instead", ", the  coefficient for the correlation of the entire adolescence ( age 1019 years ) is shown in fig .", "2 , demonstrating that , as expected , bmi changes during adolescence also predicted adult bmi .", "childhood bmi and variance in body composition and fat variables ( bmi [ a ] and bmi change [ b ] ) during early and late childhood .", "association between childhood bmi between 1 and 10 years of age and young - adult body composition parameters is expressed as accumulated r ( % , c and d ) or  coefficients ( sd in adult body composition parameter per sd in bmi at that age , e and f ) .", "variables of adult body composition and fat distribution have been age adjusted . at , adipose tissue ; ip , intraperitoneal ; sc , subcutaneous .", "correlation between change in standardized bmi during different developmental time periods and adult body composition and fat distribution parameters ( early childhood defined as 14 years , late childhood defined as 410 years , and adolescence defined as 10 years to young adult ) .", "bars indicate  coefficient ( 95% cis ) expressed as sd in investigated adult body composition parameter per sd change in bmi during that growth period .", "variables of adult body composition and fat distribution have been age adjusted . * * * p < 0.01 , * p < 0.05 significant association between the developmental time period and the investigated body composition parameter .", "95% cis are given for the  coefficients , making it possible to evaluate the differences in relative contribution of the three time periods for each dependent parameter .", "at , adipose tissue ; ip , intraperitoneal ; ns , not significant ; sc , subcutaneous .", "bmi alterations during development reflect changes in fat mass , lean mass , or both . to study the role of childhood bmi and bmi changes for the prediction of different adult body compartments , the associations between bmi during development and age - adjusted adult body composition variables were analyzed . the variance ( r ) in adult total body lean mass explained by bmi as well as the corresponding  coefficients for the correlations increased during early childhood , leveled off during late childhood , and increased again during adolescence ( figs .", "the variance in adult bmi and the variance in adult lean mass explained by childhood bmi and the corresponding  coefficients for the correlations were similar until 4 years of age , when they separated distinctly ( fig .", "the  coefficient for the correlation between change in bmi and adult lean mass was higher during adolescence than during late childhood ( fig .", ", bmi changes during early childhood and adolescence but not during late childhood were major determinants of young adult lean mass .", "the prediction of young - adult total body fat mass from bmi and bmi changes during different developmental periods followed a different pattern from that of lean mass .", "the variances in total body fat mass and percentage total body fat explained by childhood bmi and the corresponding  coefficients for the correlation were low before 4 years of age , but after 4 years of age , the plotted cumulated variance ( r ) and the corresponding  coefficients for the correlation for young- adult total body fat mass and percentage body fat tightly followed the curve displaying the variance of adult bmi explained ( fig .", "the predictive value of childhood bmi for young - adult percentage total body fat increased substantially during late childhood years , as demonstrated by the fact that the variance in percentage total body fat explained by bmi at 4 years of age was only 1.7% , whereas it was 20.8% at 10 years of age ( fig .", "to analyze fat distribution in detail , we performed abdominal ct scans ( on a subset of the present good sample [ ct subsample ] , n = 201 ) for measurements of abdominal subcutaneous and visceral adipose tissue areas .", "the abdominal ct measurements revealed that the contribution of bmi during development for the explanation of the variance in young - adult subcutaneous adipose tissue area and the corresponding  coefficients for the correlations followed a pattern similar to that of total body fat mass and percentage total body fat , with a distinct increase after 4 years of age ( fig .", "1d and f ) . for both young - adult percentage fat mass and subcutaneous adipose tissue area ,", "the  coefficients for the correlations were of similar magnitude during late childhood and adolescence but clearly higher than seen during early childhood ( fig .", "bmi changes during late childhood and adolescence , but not during early childhood , clearly predicted young - adult total fat mass and the amount of subcutaneous adipose tissue .", "the contribution of bmi during the different developmental periods for the explanation of the variance in amount of young - adult visceral adipose tissue ( intraperitoneal + retroperitoneal adipose tissue areas ) and the corresponding  coefficients for the correlations followed a pattern different and clearly distinct from that of total body fat mass and subcutaneous adipose tissue area .", "bmi and bmi changes during early and late childhood contributed only marginally to the explanation of the variance in intraperitoneal adipose tissue area , and the corresponding  coefficient for the correlation between change in bmi and amount of young - adult intraperitoneal adipose tissue was low ( figs .", ", adolescence was the developmental period when bmi changes started to contribute substantially to the explanation of the variance in amount of adult intraperitoneal adipose tissue , and the corresponding  coefficients for the correlations between change in bmi and amount of young - adult intraperitoneal adipose tissue were then higher than seen during early and late childhood ( fig .", "the results for retroperitoneal adipose tissue were similar as seen for intraperitoneal adipose tissue ( data not shown ) .", "analyses using weight for age during childhood and adolescence showed , in a manner similar to that seen for bmi for age , clear age - dependent associations with adult fat parameters ( data not shown ) .", "in contrast , analyses of height for age did not show any obvious age - dependent association with fat parameters ( data not shown ) .", "changes in bmi during adolescence , but not during early or late childhood , predicted the amount of adult visceral adipose tissue .", "to further describe the association between developmental changes in bmi and adult fat mass / adipose tissue areas , subjects were divided into groups based on change in bmi z score ( > 1 sd , less than 1 sd , and average bmi z score change ) during the three different developmental time periods ( fig .", "subjects with more than 1 sd increase in bmi z score during late childhood and during adolescence had a clearly higher percentage total body fat mass ( late childhood , + 44% , p < 0.001 ; adolescence , + 60% , p < 0.001 , fig . 3a ) and larger subcutaneous adipose tissue area ( late childhood + 83% , p < 0.001 ; adolescence , + 138% , p < 0.001 , fig .", "3b ) than subjects with average change in bmi . in contrast , subjects with more than 1 sd increase in bmi z score during late childhood had unaffected intraperitoneal adipose tissue area compared with subjects with average change in bmi ( fig .", "3c ) . however , for adolescence , subjects with more than 1 sd increase in bmi z score had larger intraperitoneal adipose tissue area ( + 91% , p < 0.001 ) than subjects with unchanged bmi z score ( fig .", "a specific association between high young - adult subcutaneous , but not visceral ( intraperitoneal + retroperitoneal ) , adipose tissue area and increased bmi z score during late childhood is supported by the finding that young - adult visceral adipose tissue area adjusted for total adipose tissue area was clearly reduced in subjects with more than 1 sd increase in bmi z score during late childhood ( fig .", "3d ) . in contrast , an increase in bmi z score of more than 1 sd during adolescence was associated with an increased visceral adipose tissue area adjusted for total adipose tissue area ( fig .", "3d ) . subjects who decreased their bmi with more than 1 sd did not differ from those with no change in bmi z score for any of the investigated fat parameters ( fig .", "thus , the amount of visceral adipose tissue was predicted by large increases in standardized bmi specifically during adolescence , whereas a high amount of subcutaneous adipose tissue area was predicted by increases in bmi during both late childhood and adolescence . the impact of developmental bmi changes for percentage total body fat ( a ) , subcutaneous adipose tissue area ( b ) , intraperitoneal adipose tissue area ( c ) , and visceral ( intraperitoneal + retroperitoneal ) adipose tissue adjusted for total abdominal ( visceral + subcutaneous ) adipose tissue area ( d ) .", "subjects with more than 1 sd increase were compared with subjects with average change or less than 1 sd decrease in standardized bmi during early childhood ( 14 years of age ) , late childhood ( 410 years of age ) , and adolescence ( 1019 years of age ) .", "variables of adult body composition and fat distribution have been age adjusted and are expressed as means  sem and were analyzed using anova followed by tukey 's post hoc analysis . * * * p < 0.001 versus average , * * p < 0.01 versus average , # # # p < 0.001 versus less than 1 sd decrease , and # p < 0.05 versus less than 1 sd .", "a : early childhood less than 1 sd , n = 72 , average n = 471 ; more than 1 sd , n = 67 ; late childhood less than 1 sd , n = 75 , average n = 462 ; more than 1 sd , n = 73 ; adolescence less than 1 sd , n = 55 , average n = 500 ; more than 1 sd , n = 55 .", "d : early childhood less than 1 sd , n = 21 , average n = 160 ; more than 1 sd , n = 20 ; late childhood less than 1 sd , n = 22 , average n = 154 ; more than 1 sd , n = 25 ; adolescence less than 1 sd , n = 22 , average n = 164 ; more than 1 sd , n = 15 .", "we next investigated if bmi increases in subjects with low prepubertal bmi had a similar impact on the amount of adult visceral adipose tissue as seen in subjects with a high prepubertal bmi .", "subjects were divided into tertiles according to prepubertal ( 10 years of age ) bmi ( low 15.2  0.8", "kg / m , average 16.7  0.4 kg / m , and high 19.3  1.5 kg / m ) and tertiles of bmi z score change ( low 1.0  0.6 sd , average 0.1  0.2 sd , and high 0.7  0.6 sd ) during adolescence ( fig .", "4 ) . for all three prepubertal bmi groups , subjects with greater increase in adolescence bmi had significantly larger visceral adipose tissue areas ( low prepubertal bmi + 56% , average prepubertal bmi + 53% , and high prepubertal bmi + 57% ;", "p < 0.05 for all ) than subjects with average adolescence bmi increase ( fig .", "bmi increases during adolescence predicted the amount of adult visceral adipose tissue independently of prepubertal bmi .", "bmi increases during adolescence are associated with high adult visceral fat mass in subjects with low , average , and high prepubertal bmi .", "intraperitoneal fat area ( y - axis ) in subjects divided into tertiles according to ( i ) prepubertal bmi ( x - axis ; low , average , and high ) and ( ii ) bmi - z score increase during adolescence ( z - axis ; low , average , and high ) .", "data are expressed as means and were analyzed using anova followed by tukey 's post hoc analysis . * p < 0.05 , * * p < 0.01 versus average bmi z score change during adolescence (  ) ; # # p < 0.01 versus low (  ) .", "n for lowest bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 9 , 19 , and 39 ; for average bmi during adolescence (  ) low prepubertal bmi to high ( left to right ) 26 , 25 , and 16 ; and for high bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 32 , 23 , and 12 .", "both bmi and obesity track strongly from childhood to adulthood ( 79,30,31 ) , but the role for bmi changes during development as predictors of adult body composition and fat distribution is unclear .", "the main objective of the present study was to investigate the association between bmi changes during different developmental periods and young - adult fat distribution in a well - characterized cohort of young - adult men .", "we demonstrate that the amount of adult subcutaneous adipose tissue and visceral adipose tissue was associated with bmi changes during distinct developmental periods .", "we also identified the childhood age after which bmi increases had a clear impact on adult total body fat mass .", "our main finding was that the amount of adult visceral adipose tissue was associated with bmi changes specifically during adolescence , whereas the amount of subcutaneous adipose tissue was associated with bmi changes during both late childhood and adolescence .", "subjects with more than 1 sd increase in bmi z score during adolescence had > 90% greater visceral adipose tissue areas than subjects with average change in bmi z score . these data , hence , demonstrate that bmi increases during adolescence were associated with a larger amount of visceral adipose tissue at young adult age .", "a large amount of visceral fat is a well - known risk factor for cardiovascular disease .", "a recent study demonstrated that although the amount of subcutaneous adipose tissue is associated with the metabolic syndrome , the amount of visceral adipose tissue remains more strongly related and is therefore regarded as an independent risk factor ( 5 ) . increased understanding of how and when subcutaneous and visceral fat compartments", "are predicted might , therefore , add important knowledge for the prevention of cardiovascular disease . in the present study , bmi increases during adolescence in subjects with a low prepubertal bmi had a similar impact on the amount of adult visceral adipose tissue as in subjects with a high prepubertal bmi .", "bmi increases during adolescence predicted the amount of adult visceral adipose tissue independently of prepubertal bmi .", "an increase by more than 1 sd in standardized bmi both during late childhood and during adolescence was , in the present study , associated with 50% more adult percentage total body fat .", "the new delhi birth cohort study also demonstrated that bmi changes during late childhood were associated with anthropometric measures of adult fat mass ( 13 ) .", "the new delhi study represents a large longitudinally followed cohort , but indicators of fat mass and lean mass were only derived from anthropometric measurements and , consequently , the visceral fat could not be determined in that study .", "the differences both in height and weight for age and in nutritional status between the indian children in the new delhi birth cohort and children in the western world ( 13,3234 ) might also limit the interpretations of the data from the new delhi birth cohort into the western world .", "a major strength of the present study is that both the subcutaneous and the visceral adipose tissue areas were measured using abdominal ct scans , giving us the opportunity to determine the predictive value of developmental bmi changes for these two fat depots separately . in a danish study , including men born between 1930 and 1956 , it was shown that subjects with both increases and decreases in bmi between 7 and 13 years of age had a higher risk of obesity in adulthood compared with those who maintained their bmi level ( 35 ) .", "in contrast , in the present cohort of boys born between 1983 and 1985 , increases but not decreases in bmi during growth were associated with increased adult fat mass and bmi . because the subjects in the danish study were born between 1930 and 1956 ,", "it is possible that the decline in bmi during growth is related to environmental factors such as temporary undernourishment among certain subjects during this period ( including world war ii ) .", "although we in the present study have characterized the associations between childhood bmi and adult obesity , it is clear that a substantial part of adult obesity can not be explained by childhood obesity ( 6 ) .", "bmi changes during early childhood and adolescence were , in the present study , predicting adult lean mass , whereas bmi changes during late childhood and adolescence were predicting adult total body fat mass .", "these findings are in accordance with the findings in the new delhi birth cohort study , demonstrating that bmi during early childhood predicts anthropometric measures of adult lean mass more strongly than anthropometric measures of adult fat mass in indian children ( 13 ) .", "both the present study and the new delhi birth cohort study thus support the notion that bmi changes during early childhood are indicators of adult lean mass , whereas bmi changes during late childhood and adolescence are indicators of adult total body fat mass .", "our data demonstrate that 4 years of age was the threshold age for swedish boys after which bmi increases were clearly associated with increased adult total body fat mass .", "it should be emphasized that the present findings are based on association studies and , therefore , should be interpreted with caution .", "the present study demonstrates that the amount of young - adult subcutaneous adipose tissue was associated with bmi changes both during late childhood and adolescence , although young - adult visceral adipose tissue areas were associated with bmi changes specifically during adolescence .", "these findings suggest that avoiding substantial bmi increases during adolescence might , independent of prepubertal bmi , result in lower adult visceral fat mass ."], "abstract_text": ["<S> objectivethe amount of visceral adipose tissue is a risk factor for the metabolic syndrome . </S>", "<S> it is unclear how bmi changes during childhood and adolescence predict adult fat distribution . </S>", "<S> we hypothesized that there are critical periods during development for the prediction of adult subcutaneous and visceral fat mass by bmi changes during childhood and adolescence.research design and methodsdetailed growth charts were retrieved for the men participating in the population - based gothenburg osteoporosis and obesity determinants ( good ) study ( n = 612 ) . </S>", "<S> body composition was analyzed using dual - energy x - ray absorptiometry and adipose tissue areas using abdominal computed tomography at 18 to 20 years of age.resultsthe main finding in the present study was that subjects with increases in bmi z score of more than 1 sd during adolescence had , independent of prepubertal bmi , both larger subcutaneous ( + 138% ; p < 0.001 ) and visceral adipose tissue areas ( + 91% ; p < 0.001 ) than subjects with unchanged bmi z - score . </S>", "<S> in contrast , subjects with increases in bmi z score of more than 1 sd during late childhood had a larger amount of adult subcutaneous adipose tissue ( + 83% ; p < 0.001 ) than subjects with unchanged bmi z score but an unaffected amount of visceral adipose tissue . </S>", "<S> bmi changes during adolescence predict both visceral and subcutaneous adipose tissue of the abdomen , whereas bmi changes during late childhood predict only the subcutaneous adipose tissue.conclusionsthe amount of visceral adipose tissue in young adult men was associated with bmi changes specifically during adolescence , whereas the amount of subcutaneous adipose tissue was associated with bmi changes during both late childhood and adolescence . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Anthropometric measurements.", "Dual-energy X-ray absorptiometry.", "Abdominal CT analyses of cross-sectional adipose tissue areas.", "Estimation of childhood BMI.", "RESULTS", "BMI changes during both early and late childhood as well as during adolescence predicted young adult BMI.", "Adult total body lean mass was predicted by changes in BMI during early childhood and adolescence.", "Adult total body fat mass and subcutaneous adipose tissue area were predicted by changes in BMI during late childhood and adolescence.", "Adult visceral adipose tissue areas were predicted by changes in BMI, specifically during adolescence.", "Changes in BMI ", "BMI increases during adolescence predict the amount of adult visceral adipose tissue independently of prepubertal BMI.", "DISCUSSION"], "sections": [["the gothenburg osteoporosis and obesity determinants ( good ) study was initiated with the aim to determine both environmental and genetic factors involved in the regulation of fat mass as previously described ( 23 ) . to be included in the good study , subjects had to be male , 18 to 20 years of age , and willing to participate in the study", "a total of 1,068 subjects , representative of the general young male population of gothenburg ( 23 ) ( data not shown ) , were enrolled in the study . of the 1,068 subjects ,", "complete growth and weight charts for determination of bmi between 1 and 10 years of age were available for 612 subjects ( growth chart subsample ) .", "dual - energy x - ray absorptiometry analyses were performed on 610 subjects of the growth chart subsample , whereas abdominal ct scans were performed on 201 ( the ct subsample ) of the 612 study subjects in the growth chart subsample .", "the growth chart subsample is representative of the entire good cohort in terms of age ( good 18.9  0.6 years , growth chart subsample 18.9  0.5 years , nonsignificant ) , weight ( good 73.9  11.9 kg , growth chart subsample 73.3  11.5 kg , nonsignificant ) , height ( good 181.4  6.8 cm , growth chart subsample 182.0  6.8 cm , nonsignificant ) , and bmi ( good 22.4  3.2 kg / m , growth chart subsample 22.1  3.1 kg / m , nonsignificant ) .", "the ct subsample is representative of both the growth chart subsample and the entire good cohort in terms of weight , height , and bmi ( ct subsample weight 72.7  11.2 kg , height 181.9  7.0 cm , bmi 21.9  3.0 kg / m , nonsignificant versus both the entire good cohort and the growth chart subsample ) .", "however , the subjects of the ct subsample were slightly younger ( ct subsample 18.7  0.5 years ) than both the subjects of the entire good cohort and the subjects of the growth chart subsample ( p < 0.05 ) .", "the good study was approved by the local ethics committee at gothenburg university . written and oral informed consent was obtained from all the study participants .", "height was measured using a wall - mounted stadiometer and weight was measured to the nearest 0.1 kg as previously described ( 16,23 ) .", "total body fat mass , percentage body fat , total body lean mass , and fat mass of the trunk were assessed using the lunar prodigy dxa ( ge lunar , madison , wi ) .", "a previously described ct technique was used to measure the cross - sectional adipose area of the abdomen ( 16,24,25 ) .", "a single slice at the level of the fourth lumbar vertebra was acquired with a general electric high speed advantage ct system ( version rp2 ; ge medical systems , milwaukee , wi ) .", "subcutaneous and visceral adipose tissue areas were measured , and then the visceral adipose tissue area was divided into an intraperitoneal and a retroperitoneal adipose tissue area .", "growth and weight charts were collected for the subjects of the good study and longitudinal growth was curve fitted according to the infancy - childhood  puberty ( icp ) model ( 26 ) as previously described ( 16,27 ) .", "the icp model represents a widely used model for fitting of human growth data to mathematical functions ( 26 ) .", "the infancy part is represented by an exponential function , the childhood part by a quadratic function , and the pubertal part by a logistic function .", "the model also includes the transformations between the different phases . by using this model , we computed values of height at exact ages .", "body composition , including bmi , is greatly influenced by pubertal stage and thereby pubertal timing ( 28 ) .", "complete growth charts with enough data to determine pubertal timing , requiring several height and weight measurements around the pubertal growth spurt , were not available for the complete growth chart subsample used in the present study .", "we have therefore chosen to use measurements of bmi before and after , but not during , puberty .", "body weights between 1 and 10 years of age were estimated through fitting of the weight curve for each child using smooth splines ( smooth.spline in the r package statistics , the r foundation for statistic computing , vienna , austria ; http://www.r-project.org ) .", "bmi values between 1 and 10 years of age were then calculated from the estimated values of weight and height .", "the z score , the standardization of a variable relative to the investigated population expressed in sds , was calculated using the software spss 15.0 ( spss , chicago , il ) . for each subject ( n = 612 ) in this cohort and for each age , the height was calculated using the icp model .", "these values were then used to calculate the z score for each subject at each age based on the variation ( sd ) in calculated height within the presently investigated population at that specific age .", "the variance of young adult body composition parameters ( r ) explained by bmi or bmi z score changes and the corresponding  coefficients during childhood and adolescence was calculated using linear regression analyses with age - adjusted adult body composition variables as the dependent variable .", "age adjustments were performed because the subjects differed somewhat in age at body composition analysis ( 1820 years of age , mean  sd 18.9  0.5 years ) and were performed between age at fat analysis and the body composition variables .", "all subsequent statistical calculations were performed using the age - adjusted body composition variables . for the calculations in fig .", "for all the statistical analyses , the software spss ( version 15.0 ) was used ."], ["height was measured using a wall - mounted stadiometer and weight was measured to the nearest 0.1 kg as previously described ( 16,23 ) ."], ["total body fat mass , percentage body fat , total body lean mass , and fat mass of the trunk were assessed using the lunar prodigy dxa ( ge lunar , madison , wi ) ."], ["a previously described ct technique was used to measure the cross - sectional adipose area of the abdomen ( 16,24,25 ) .", "a single slice at the level of the fourth lumbar vertebra was acquired with a general electric high speed advantage ct system ( version rp2 ; ge medical systems , milwaukee , wi ) .", "subcutaneous and visceral adipose tissue areas were measured , and then the visceral adipose tissue area was divided into an intraperitoneal and a retroperitoneal adipose tissue area ."], ["growth and weight charts were collected for the subjects of the good study and longitudinal growth was curve fitted according to the infancy - childhood  puberty ( icp ) model ( 26 ) as previously described ( 16,27 ) .", "the icp model represents a widely used model for fitting of human growth data to mathematical functions ( 26 ) .", "the infancy part is represented by an exponential function , the childhood part by a quadratic function , and the pubertal part by a logistic function .", "the model also includes the transformations between the different phases . by using this model , we computed values of height at exact ages .", "body composition , including bmi , is greatly influenced by pubertal stage and thereby pubertal timing ( 28 ) .", "complete growth charts with enough data to determine pubertal timing , requiring several height and weight measurements around the pubertal growth spurt , were not available for the complete growth chart subsample used in the present study .", "we have therefore chosen to use measurements of bmi before and after , but not during , puberty .", "body weights between 1 and 10 years of age were estimated through fitting of the weight curve for each child using smooth splines ( smooth.spline in the r package statistics , the r foundation for statistic computing , vienna , austria ; http://www.r-project.org ) .", "bmi values between 1 and 10 years of age were then calculated from the estimated values of weight and height .", "the z score , the standardization of a variable relative to the investigated population expressed in sds , was calculated using the software spss 15.0 ( spss , chicago , il ) . for each subject ( n = 612 ) in this cohort and for each age", "these values were then used to calculate the z score for each subject at each age based on the variation ( sd ) in calculated height within the presently investigated population at that specific age .", "the variance of young adult body composition parameters ( r ) explained by bmi or bmi z score changes and the corresponding  coefficients during childhood and adolescence was calculated using linear regression analyses with age - adjusted adult body composition variables as the dependent variable .", "age adjustments were performed because the subjects differed somewhat in age at body composition analysis ( 1820 years of age , mean  sd 18.9  0.5 years ) and were performed between age at fat analysis and the body composition variables .", "all subsequent statistical calculations were performed using the age - adjusted body composition variables . for the calculations in fig .", "for all the statistical analyses , the software spss ( version 15.0 ) was used ."], ["the present study represents a subsample ( n = 612 [ growth chart subsample ] ) of the original good cohort ( n = 1,068 ) in which subjects with available detailed growth charts from 1 to 10 years of age were included .", "anthropometrics and measurements of fat ( dxa)/adipose tissue areas ( abdominal ct ) of the subjects participating in the present study are presented in table 1 .", "average bmi in this cohort declined during early childhood years , and adiposity rebound ( the lowest bmi ) was reached slightly before 6 years of age , after which it began to increase ( fig .", "2 ) during both early ( age 14 years ) and late ( age 410 years ) childhood were , as expected , clear positive predictors of adult bmi . during adolescence ,", "pubertal stage influences body composition ( 29 ) . because interindividual variations in pubertal onset will confound the calculations during adolescence , the predictive role of bmi for each individual year during adolescence for adult bmi and body composition is not shown . instead", ", the  coefficient for the correlation of the entire adolescence ( age 1019 years ) is shown in fig .", "2 , demonstrating that , as expected , bmi changes during adolescence also predicted adult bmi .", "childhood bmi and variance in body composition and fat variables ( bmi [ a ] and bmi change [ b ] ) during early and late childhood .", "association between childhood bmi between 1 and 10 years of age and young - adult body composition parameters is expressed as accumulated r ( % , c and d ) or  coefficients ( sd in adult body composition parameter per sd in bmi at that age , e and f ) .", "variables of adult body composition and fat distribution have been age adjusted . at , adipose tissue ; ip , intraperitoneal ; sc , subcutaneous .", "correlation between change in standardized bmi during different developmental time periods and adult body composition and fat distribution parameters ( early childhood defined as 14 years , late childhood defined as 410 years , and adolescence defined as 10 years to young adult ) .", "bars indicate  coefficient ( 95% cis ) expressed as sd in investigated adult body composition parameter per sd change in bmi during that growth period .", "variables of adult body composition and fat distribution have been age adjusted . * * * p < 0.01 , * p < 0.05 significant association between the developmental time period and the investigated body composition parameter .", "95% cis are given for the  coefficients , making it possible to evaluate the differences in relative contribution of the three time periods for each dependent parameter .", "at , adipose tissue ; ip , intraperitoneal ; ns , not significant ; sc , subcutaneous .", "bmi alterations during development reflect changes in fat mass , lean mass , or both . to study the role of childhood bmi and bmi changes for the prediction of different adult body compartments , the associations between bmi during development and age - adjusted adult body composition variables were analyzed . the variance ( r ) in adult total body lean mass explained by bmi as well as the corresponding  coefficients for the correlations increased during early childhood , leveled off during late childhood , and increased again during adolescence ( figs .", "the variance in adult bmi and the variance in adult lean mass explained by childhood bmi and the corresponding  coefficients for the correlations were similar until 4 years of age , when they separated distinctly ( fig .", "1c and e ) . the  coefficient for the correlation between change in bmi and adult lean mass was higher during adolescence than during late childhood ( fig .", "2 ) . thus , bmi changes during early childhood and adolescence but not during late childhood were major determinants of young adult lean mass .", "the prediction of young - adult total body fat mass from bmi and bmi changes during different developmental periods followed a different pattern from that of lean mass .", "the variances in total body fat mass and percentage total body fat explained by childhood bmi and the corresponding  coefficients for the correlation were low before 4 years of age , but after 4 years of age , the plotted cumulated variance ( r ) and the corresponding  coefficients for the correlation for young- adult total body fat mass and percentage body fat tightly followed the curve displaying the variance of adult bmi explained ( fig .", "the predictive value of childhood bmi for young - adult percentage total body fat increased substantially during late childhood years , as demonstrated by the fact that the variance in percentage total body fat explained by bmi at 4 years of age was only 1.7% , whereas it was 20.8% at 10 years of age ( fig .", "to analyze fat distribution in detail , we performed abdominal ct scans ( on a subset of the present good sample [ ct subsample ] , n = 201 ) for measurements of abdominal subcutaneous and visceral adipose tissue areas .", "the abdominal ct measurements revealed that the contribution of bmi during development for the explanation of the variance in young - adult subcutaneous adipose tissue area and the corresponding  coefficients for the correlations followed a pattern similar to that of total body fat mass and percentage total body fat , with a distinct increase after 4 years of age ( fig .", "1d and f ) . for both young - adult percentage fat mass and subcutaneous adipose tissue area ,", "the  coefficients for the correlations were of similar magnitude during late childhood and adolescence but clearly higher than seen during early childhood ( fig .", "bmi changes during late childhood and adolescence , but not during early childhood , clearly predicted young - adult total fat mass and the amount of subcutaneous adipose tissue .", "the contribution of bmi during the different developmental periods for the explanation of the variance in amount of young - adult visceral adipose tissue ( intraperitoneal + retroperitoneal adipose tissue areas ) and the corresponding  coefficients for the correlations followed a pattern different and clearly distinct from that of total body fat mass and subcutaneous adipose tissue area .", "bmi and bmi changes during early and late childhood contributed only marginally to the explanation of the variance in intraperitoneal adipose tissue area , and the corresponding  coefficient for the correlation between change in bmi and amount of young - adult intraperitoneal adipose tissue was low ( figs . 1d and f and 2 ) . instead , adolescence was the developmental period when bmi changes started to contribute substantially to the explanation of the variance in amount of adult intraperitoneal adipose tissue , and the corresponding  coefficients for the correlations between change in bmi and amount of young - adult intraperitoneal adipose tissue were then higher than seen during early and late childhood ( fig .", "the results for retroperitoneal adipose tissue were similar as seen for intraperitoneal adipose tissue ( data not shown ) .", "analyses using weight for age during childhood and adolescence showed , in a manner similar to that seen for bmi for age , clear age - dependent associations with adult fat parameters ( data not shown ) .", "in contrast , analyses of height for age did not show any obvious age - dependent association with fat parameters ( data not shown ) .", "changes in bmi during adolescence , but not during early or late childhood , predicted the amount of adult visceral adipose tissue . to further describe the association between developmental changes in bmi and adult fat mass / adipose tissue areas , subjects were divided into groups based on change in bmi z score ( > 1 sd , less than 1 sd , and average bmi z score change ) during the three different developmental time periods ( fig .", "subjects with more than 1 sd increase in bmi z score during late childhood and during adolescence had a clearly higher percentage total body fat mass ( late childhood , + 44% , p < 0.001 ; adolescence , + 60% , p < 0.001 , fig . 3a ) and larger subcutaneous adipose tissue area ( late childhood + 83% , p <", "3b ) than subjects with average change in bmi . in contrast , subjects with more than 1 sd increase in bmi z score during late childhood had unaffected intraperitoneal adipose tissue area compared with subjects with average change in bmi ( fig .", "3c ) . however , for adolescence , subjects with more than 1 sd increase in bmi z score had larger intraperitoneal adipose tissue area ( + 91% , p < 0.001 ) than subjects with unchanged bmi z score ( fig .", "a specific association between high young - adult subcutaneous , but not visceral ( intraperitoneal + retroperitoneal ) , adipose tissue area and increased bmi z score during late childhood is supported by the finding that young - adult visceral adipose tissue area adjusted for total adipose tissue area was clearly reduced in subjects with more than 1 sd increase in bmi z score during late childhood ( fig .", "3d ) . in contrast , an increase in bmi z score of more than 1 sd during adolescence was associated with an increased visceral adipose tissue area adjusted for total adipose tissue area ( fig .", "3d ) . subjects who decreased their bmi with more than 1 sd did not differ from those with no change in bmi z score for any of the investigated fat parameters ( fig .", "thus , the amount of visceral adipose tissue was predicted by large increases in standardized bmi specifically during adolescence , whereas a high amount of subcutaneous adipose tissue area was predicted by increases in bmi during both late childhood and adolescence .", "the impact of developmental bmi changes for percentage total body fat ( a ) , subcutaneous adipose tissue area ( b ) , intraperitoneal adipose tissue area ( c ) , and visceral ( intraperitoneal + retroperitoneal ) adipose tissue adjusted for total abdominal ( visceral + subcutaneous ) adipose tissue area ( d ) .", "subjects with more than 1 sd increase were compared with subjects with average change or less than 1 sd decrease in standardized bmi during early childhood ( 14 years of age ) , late childhood ( 410 years of age ) , and adolescence ( 1019 years of age ) .", "variables of adult body composition and fat distribution have been age adjusted and are expressed as means  sem and were analyzed using anova followed by tukey 's post hoc analysis . * * * p < 0.001 versus average , * * p < 0.01 versus average , # # # p < 0.001 versus less than 1 sd decrease , and # p < 0.05 versus less than 1 sd .", "a : early childhood less than 1 sd , n = 72 , average n = 471 ; more than 1 sd , n = 67 ; late childhood less than 1 sd , n = 75 , average n = 462 ; more than 1 sd , n = 73 ; adolescence less than 1 sd , n = 55 , average n = 500 ; more than 1 sd , n = 55 .", "d : early childhood less than 1 sd , n = 21 , average n = 160 ; more than 1 sd , n = 20 ; late childhood less than 1 sd , n = 22 , average n = 154 ; more than 1 sd , n = 25 ; adolescence less than 1 sd , n = 22 , average n = 164 ; more than 1 sd , n = 15 .", "we next investigated if bmi increases in subjects with low prepubertal bmi had a similar impact on the amount of adult visceral adipose tissue as seen in subjects with a high prepubertal bmi .", "subjects were divided into tertiles according to prepubertal ( 10 years of age ) bmi ( low 15.2  0.8", "kg / m , average 16.7  0.4 kg / m , and high 19.3  1.5 kg / m ) and tertiles of bmi z score change ( low 1.0  0.6 sd , average 0.1  0.2 sd , and high 0.7  0.6 sd ) during adolescence ( fig .", "subjects with greater increase in adolescence bmi had significantly larger visceral adipose tissue areas ( low prepubertal bmi + 56% , average prepubertal bmi + 53% , and high prepubertal bmi + 57% ; p < 0.05 for all ) than subjects with average adolescence bmi increase ( fig .", "bmi increases during adolescence predicted the amount of adult visceral adipose tissue independently of prepubertal bmi .", "bmi increases during adolescence are associated with high adult visceral fat mass in subjects with low , average , and high prepubertal bmi .", "intraperitoneal fat area ( y - axis ) in subjects divided into tertiles according to ( i ) prepubertal bmi ( x - axis ; low , average , and high ) and ( ii ) bmi - z score increase during adolescence ( z - axis ; low , average , and high ) .", "data are expressed as means and were analyzed using anova followed by tukey 's post hoc analysis . * p < 0.05 , * * p < 0.01 versus average bmi z score change during adolescence (  ) ; # # p < 0.01 versus low (  ) .", "n for lowest bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 9 , 19 , and 39 ; for average bmi during adolescence (  ) low prepubertal bmi to high ( left to right ) 26 , 25 , and 16 ; and for high bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 32 , 23 , and 12 ."], ["average bmi in this cohort declined during early childhood years , and adiposity rebound ( the lowest bmi ) was reached slightly before 6 years of age , after which it began to increase ( fig .", "2 ) during both early ( age 14 years ) and late ( age 410 years ) childhood were , as expected , clear positive predictors of adult bmi . during adolescence ,", "because interindividual variations in pubertal onset will confound the calculations during adolescence , the predictive role of bmi for each individual year during adolescence for adult bmi and body composition is not shown . instead", ", the  coefficient for the correlation of the entire adolescence ( age 1019 years ) is shown in fig .", "2 , demonstrating that , as expected , bmi changes during adolescence also predicted adult bmi .", "childhood bmi and variance in body composition and fat variables ( bmi [ a ] and bmi change [ b ] ) during early and late childhood .", "association between childhood bmi between 1 and 10 years of age and young - adult body composition parameters is expressed as accumulated r ( % , c and d ) or  coefficients ( sd in adult body composition parameter per sd in bmi at that age , e and f ) .", "variables of adult body composition and fat distribution have been age adjusted . at , adipose tissue ; ip , intraperitoneal ; sc , subcutaneous .", "correlation between change in standardized bmi during different developmental time periods and adult body composition and fat distribution parameters ( early childhood defined as 14 years , late childhood defined as 410 years , and adolescence defined as 10 years to young adult ) .", "bars indicate  coefficient ( 95% cis ) expressed as sd in investigated adult body composition parameter per sd change in bmi during that growth period .", "variables of adult body composition and fat distribution have been age adjusted . * * * p < 0.01 , * p < 0.05 significant association between the developmental time period and the investigated body composition parameter .", "95% cis are given for the  coefficients , making it possible to evaluate the differences in relative contribution of the three time periods for each dependent parameter .", "at , adipose tissue ; ip , intraperitoneal ; ns , not significant ; sc , subcutaneous ."], ["bmi alterations during development reflect changes in fat mass , lean mass , or both . to study the role of childhood bmi and bmi changes for the prediction of different adult body compartments , the associations between bmi during development and age - adjusted adult body composition variables were analyzed . the variance ( r ) in adult total body lean mass explained by bmi as well as the corresponding  coefficients for the correlations increased during early childhood , leveled off during late childhood , and increased again during adolescence ( figs .", "the variance in adult bmi and the variance in adult lean mass explained by childhood bmi and the corresponding  coefficients for the correlations were similar until 4 years of age , when they separated distinctly ( fig .", "the  coefficient for the correlation between change in bmi and adult lean mass was higher during adolescence than during late childhood ( fig .", ", bmi changes during early childhood and adolescence but not during late childhood were major determinants of young adult lean mass ."], ["the prediction of young - adult total body fat mass from bmi and bmi changes during different developmental periods followed a different pattern from that of lean mass .", "the variances in total body fat mass and percentage total body fat explained by childhood bmi and the corresponding  coefficients for the correlation were low before 4 years of age , but after 4 years of age , the plotted cumulated variance ( r ) and the corresponding  coefficients for the correlation for young- adult total body fat mass and percentage body fat tightly followed the curve displaying the variance of adult bmi explained ( fig .", "the predictive value of childhood bmi for young - adult percentage total body fat increased substantially during late childhood years , as demonstrated by the fact that the variance in percentage total body fat explained by bmi at 4 years of age was only 1.7% , whereas it was 20.8% at 10 years of age ( fig .", "to analyze fat distribution in detail , we performed abdominal ct scans ( on a subset of the present good sample [ ct subsample ] , n = 201 ) for measurements of abdominal subcutaneous and visceral adipose tissue areas .", "the abdominal ct measurements revealed that the contribution of bmi during development for the explanation of the variance in young - adult subcutaneous adipose tissue area and the corresponding  coefficients for the correlations followed a pattern similar to that of total body fat mass and percentage total body fat , with a distinct increase after 4 years of age ( fig .", "1d and f ) . for both young - adult percentage fat mass and subcutaneous adipose tissue area ,", "the  coefficients for the correlations were of similar magnitude during late childhood and adolescence but clearly higher than seen during early childhood ( fig .", "bmi changes during late childhood and adolescence , but not during early childhood , clearly predicted young - adult total fat mass and the amount of subcutaneous adipose tissue ."], ["the contribution of bmi during the different developmental periods for the explanation of the variance in amount of young - adult visceral adipose tissue ( intraperitoneal + retroperitoneal adipose tissue areas ) and the corresponding  coefficients for the correlations followed a pattern different and clearly distinct from that of total body fat mass and subcutaneous adipose tissue area .", "bmi and bmi changes during early and late childhood contributed only marginally to the explanation of the variance in intraperitoneal adipose tissue area , and the corresponding  coefficient for the correlation between change in bmi and amount of young - adult intraperitoneal adipose tissue was low ( figs .", ", adolescence was the developmental period when bmi changes started to contribute substantially to the explanation of the variance in amount of adult intraperitoneal adipose tissue , and the corresponding  coefficients for the correlations between change in bmi and amount of young - adult intraperitoneal adipose tissue were then higher than seen during early and late childhood ( fig .", "the results for retroperitoneal adipose tissue were similar as seen for intraperitoneal adipose tissue ( data not shown ) .", "analyses using weight for age during childhood and adolescence showed , in a manner similar to that seen for bmi for age , clear age - dependent associations with adult fat parameters ( data not shown ) .", "in contrast , analyses of height for age did not show any obvious age - dependent association with fat parameters ( data not shown ) .", "changes in bmi during adolescence , but not during early or late childhood , predicted the amount of adult visceral adipose tissue ."], ["to further describe the association between developmental changes in bmi and adult fat mass / adipose tissue areas , subjects were divided into groups based on change in bmi z score ( > 1 sd , less than 1 sd , and average bmi z score change ) during the three different developmental time periods ( fig .", "subjects with more than 1 sd increase in bmi z score during late childhood and during adolescence had a clearly higher percentage total body fat mass ( late childhood , + 44% , p < 0.001 ; adolescence , + 60% , p < 0.001 , fig . 3a ) and larger subcutaneous adipose tissue area ( late childhood + 83% , p < 0.001 ; adolescence , + 138% , p < 0.001 , fig .", "3b ) than subjects with average change in bmi . in contrast , subjects with more than 1 sd increase in bmi z score during late childhood had unaffected intraperitoneal adipose tissue area compared with subjects with average change in bmi ( fig .", "3c ) . however , for adolescence , subjects with more than 1 sd increase in bmi z score had larger intraperitoneal adipose tissue area ( + 91% , p < 0.001 ) than subjects with unchanged bmi z score ( fig .", "a specific association between high young - adult subcutaneous , but not visceral ( intraperitoneal + retroperitoneal ) , adipose tissue area and increased bmi z score during late childhood is supported by the finding that young - adult visceral adipose tissue area adjusted for total adipose tissue area was clearly reduced in subjects with more than 1 sd increase in bmi z score during late childhood ( fig .", "3d ) . in contrast , an increase in bmi z score of more than 1 sd during adolescence was associated with an increased visceral adipose tissue area adjusted for total adipose tissue area ( fig .", "3d ) . subjects who decreased their bmi with more than 1 sd did not differ from those with no change in bmi z score for any of the investigated fat parameters ( fig .", "thus , the amount of visceral adipose tissue was predicted by large increases in standardized bmi specifically during adolescence , whereas a high amount of subcutaneous adipose tissue area was predicted by increases in bmi during both late childhood and adolescence . the impact of developmental bmi changes for percentage total body fat ( a ) , subcutaneous adipose tissue area ( b ) , intraperitoneal adipose tissue area ( c ) , and visceral ( intraperitoneal + retroperitoneal ) adipose tissue adjusted for total abdominal ( visceral + subcutaneous ) adipose tissue area ( d ) .", "subjects with more than 1 sd increase were compared with subjects with average change or less than 1 sd decrease in standardized bmi during early childhood ( 14 years of age ) , late childhood ( 410 years of age ) , and adolescence ( 1019 years of age ) .", "variables of adult body composition and fat distribution have been age adjusted and are expressed as means  sem and were analyzed using anova followed by tukey 's post hoc analysis . * * * p < 0.001 versus average , * * p < 0.01 versus average , # # # p < 0.001 versus less than 1 sd decrease , and # p < 0.05 versus less than 1 sd .", "a : early childhood less than 1 sd , n = 72 , average n = 471 ; more than 1 sd , n = 67 ; late childhood less than 1 sd , n = 75 , average n = 462 ; more than 1 sd , n = 73 ; adolescence less than 1 sd , n = 55 , average n = 500 ; more than 1 sd , n = 55 .", "d : early childhood less than 1 sd , n = 21 , average n = 160 ; more than 1 sd , n = 20 ; late childhood less than 1 sd , n = 22 , average n = 154 ; more than 1 sd , n = 25 ; adolescence less than 1 sd , n = 22 , average n = 164 ; more than 1 sd , n = 15 ."], ["we next investigated if bmi increases in subjects with low prepubertal bmi had a similar impact on the amount of adult visceral adipose tissue as seen in subjects with a high prepubertal bmi .", "subjects were divided into tertiles according to prepubertal ( 10 years of age ) bmi ( low 15.2  0.8", "kg / m , average 16.7  0.4 kg / m , and high 19.3  1.5 kg / m ) and tertiles of bmi z score change ( low 1.0  0.6 sd , average 0.1  0.2 sd , and high 0.7  0.6 sd ) during adolescence ( fig .", "4 ) . for all three prepubertal bmi groups , subjects with greater increase in adolescence bmi had significantly larger visceral adipose tissue areas ( low prepubertal bmi + 56% , average prepubertal bmi + 53% , and high prepubertal bmi + 57% ;", "p < 0.05 for all ) than subjects with average adolescence bmi increase ( fig .", "bmi increases during adolescence predicted the amount of adult visceral adipose tissue independently of prepubertal bmi .", "bmi increases during adolescence are associated with high adult visceral fat mass in subjects with low , average , and high prepubertal bmi .", "intraperitoneal fat area ( y - axis ) in subjects divided into tertiles according to ( i ) prepubertal bmi ( x - axis ; low , average , and high ) and ( ii ) bmi - z score increase during adolescence ( z - axis ; low , average , and high ) .", "data are expressed as means and were analyzed using anova followed by tukey 's post hoc analysis . * p < 0.05 , * * p < 0.01 versus average bmi z score change during adolescence (  ) ; # # p < 0.01 versus low (  ) .", "n for lowest bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 9 , 19 , and 39 ; for average bmi during adolescence (  ) low prepubertal bmi to high ( left to right ) 26 , 25 , and 16 ; and for high bmi during adolescence (  ) from low prepubertal bmi to high ( left to right ) 32 , 23 , and 12 ."], ["both bmi and obesity track strongly from childhood to adulthood ( 79,30,31 ) , but the role for bmi changes during development as predictors of adult body composition and fat distribution is unclear .", "the main objective of the present study was to investigate the association between bmi changes during different developmental periods and young - adult fat distribution in a well - characterized cohort of young - adult men .", "we demonstrate that the amount of adult subcutaneous adipose tissue and visceral adipose tissue was associated with bmi changes during distinct developmental periods .", "we also identified the childhood age after which bmi increases had a clear impact on adult total body fat mass .", "our main finding was that the amount of adult visceral adipose tissue was associated with bmi changes specifically during adolescence , whereas the amount of subcutaneous adipose tissue was associated with bmi changes during both late childhood and adolescence .", "subjects with more than 1 sd increase in bmi z score during adolescence had > 90% greater visceral adipose tissue areas than subjects with average change in bmi z score . these data , hence , demonstrate that bmi increases during adolescence were associated with a larger amount of visceral adipose tissue at young adult age .", "a large amount of visceral fat is a well - known risk factor for cardiovascular disease .", "a recent study demonstrated that although the amount of subcutaneous adipose tissue is associated with the metabolic syndrome , the amount of visceral adipose tissue remains more strongly related and is therefore regarded as an independent risk factor ( 5 ) . increased understanding of how and when subcutaneous and visceral fat compartments", "are predicted might , therefore , add important knowledge for the prevention of cardiovascular disease . in the present study , bmi increases during adolescence in subjects with a low prepubertal bmi had a similar impact on the amount of adult visceral adipose tissue as in subjects with a high prepubertal bmi .", "bmi increases during adolescence predicted the amount of adult visceral adipose tissue independently of prepubertal bmi .", "an increase by more than 1 sd in standardized bmi both during late childhood and during adolescence was , in the present study , associated with 50% more adult percentage total body fat .", "the new delhi birth cohort study also demonstrated that bmi changes during late childhood were associated with anthropometric measures of adult fat mass ( 13 ) .", "the new delhi study represents a large longitudinally followed cohort , but indicators of fat mass and lean mass were only derived from anthropometric measurements and , consequently , the visceral fat could not be determined in that study .", "the differences both in height and weight for age and in nutritional status between the indian children in the new delhi birth cohort and children in the western world ( 13,3234 ) might also limit the interpretations of the data from the new delhi birth cohort into the western world .", "a major strength of the present study is that both the subcutaneous and the visceral adipose tissue areas were measured using abdominal ct scans , giving us the opportunity to determine the predictive value of developmental bmi changes for these two fat depots separately . in a danish study , including men born between 1930 and 1956 , it was shown that subjects with both increases and decreases in bmi between 7 and 13 years of age had a higher risk of obesity in adulthood compared with those who maintained their bmi level ( 35 ) .", "in contrast , in the present cohort of boys born between 1983 and 1985 , increases but not decreases in bmi during growth were associated with increased adult fat mass and bmi . because the subjects in the danish study were born between 1930 and 1956 ,", "it is possible that the decline in bmi during growth is related to environmental factors such as temporary undernourishment among certain subjects during this period ( including world war ii ) .", "although we in the present study have characterized the associations between childhood bmi and adult obesity , it is clear that a substantial part of adult obesity can not be explained by childhood obesity ( 6 ) .", "bmi changes during early childhood and adolescence were , in the present study , predicting adult lean mass , whereas bmi changes during late childhood and adolescence were predicting adult total body fat mass .", "these findings are in accordance with the findings in the new delhi birth cohort study , demonstrating that bmi during early childhood predicts anthropometric measures of adult lean mass more strongly than anthropometric measures of adult fat mass in indian children ( 13 ) .", "both the present study and the new delhi birth cohort study thus support the notion that bmi changes during early childhood are indicators of adult lean mass , whereas bmi changes during late childhood and adolescence are indicators of adult total body fat mass .", "our data demonstrate that 4 years of age was the threshold age for swedish boys after which bmi increases were clearly associated with increased adult total body fat mass .", "it should be emphasized that the present findings are based on association studies and , therefore , should be interpreted with caution .", "the present study demonstrates that the amount of young - adult subcutaneous adipose tissue was associated with bmi changes both during late childhood and adolescence , although young - adult visceral adipose tissue areas were associated with bmi changes specifically during adolescence .", "these findings suggest that avoiding substantial bmi increases during adolescence might , independent of prepubertal bmi , result in lower adult visceral fat mass ."]]}
{"article_id": "PMC4296460", "article_text": ["it is estimated that around 50% of the population has one or more sites with 1 mm or more of such root exposure .", "this prevalence rate increases to > 88% in individuals who are 65 years or older .", "key factors in the treatment of gingival recession are to prevent the progressive marginal tissue recession and to facilitate plaque control in the affected area .", "various treatment modalities have been put forth for the correction of gingival recession . these include free gingival autograft , sub - epithelial connective tissue graft ( sctg ) , coronally advanced flap ( caf ) .", "it is seen that the incidence of adverse effects , such as discomfort with or without pain , was directly related to the donor sites of sctg .", "hence despite sctg being the gold standard for root coverage research is always aimed at finding a suitable alternative to this procedure to reduce patient morbidity .", "this procedure has been modified by many others with an intention to improve the stability and predictability of this technique . in a recent 14 year follow - up study pini prato et al .", "hence this procedure is now generally employed with adjuncts such as guided tissue regeneration membrane , emdogain , acellular dermal matrix and platelet rich plasma to enhance its long term stability .", "however , evidence regarding the beneficial impact of these procedures on the stability of root coverage is inconclusive .", "hyaluronic acid ( ha ) is a linear polysaccharide found in extracellular matrices of skin .", "hyaluronan has many structural and physiological functions within tissues , including extracellular and cellular interactions , growth factor interaction and the regulation of osmotic pressure and tissue lubrication , which helps in the maintenance of the structural and homeostatic integrity of tissues .", "one gram of ha can bind up to 6 l of water . as a physical background material", ", it has functions in space filling , lubrication , shock absorption , and protein exclusion .", "according to various clinical studies hyaluronan undergoes degradation in chronically inflamed gingival tissues , whereas the proteoglycan associated sulfated glycosaminoglycans , such as chondroitin 4-sulphate and dermatan sulfate , remain relatively intact .", "ha is a potent anti - inflammatory agent and modulates wound healing due to its ability to scavenge the inflammatory cells - derived reactive oxygen species .", "this study is aimed at testing the efficacy of hyaluronan gel as an adjunct to caf in root coverage .", "this was a randomized clinical trial with split mouth study design , carried out in the postgraduate clinic of department of periodontics , coorg institute of dental sciences , virajpet .", "the study sample included 10 subjects of which seven are male , and three female ( at least 20 sites ) having miller 's class i gingival recession on the canine and premolar region were recruited from department of periodontics , coorg institute of dental sciences .", "the sample size was calculated adjusting the minimum acceptable power of study at 80% and maximum allowable alpha error at 5% .", "the recession defects were designated as experimental and control sites randomly prior to surgery by spin of a coin .", "experimental group includes hyaluronan gel ( gengigel 0.2% gel which is 0.2% hyaluronan gel marketed by ricerfarma pharmaceuticals , milan , italy ) with caf and control group with caf alone .", "this self - funded study was approved by institutional ethical committee , and the procedure and the procedure was explained to all the subjects who provided signed informed consent before participation .", "medical and dental examination was obtained from all the individuals who were followed by complete periodontal examination .", "patients with malaligned teeth , teeth in trauma from occlusion , cervical abrasion and restorations were also excluded .", "subjects were included who are systemically healthy with two or more sites of miller 's class i recession and those who can understand and follow oral hygiene instructions properly . at baseline", "group allocation and sampling site selection were performed by one examiner moogala srinivas ( ms ) with a unc 15 probe and rounded off to the nearest 0.5 mm .", "the rd was assessed at baseline , 1 , 3 , 6 , 12 and 24 weeks , and the pd and cal were recorded at 12 and 24 weeks with the patients consent .", "the patients who were selected and gave their approval for the surgical intervention were educated and motivated with more emphasis on proper oral hygiene maintenance . all the patients underwent the initial phase of treatment of thorough scaling and root planning .", "the sites were randomly assigned to the control group or the test group using a flip coin technique immediately before surgery .", "the surgical area was prepared with adequate anesthesia using 2% lignocaine hcl containing 1:80,000 epinephrine .", "after local anesthesia and before the elevation of the flap , both the exposed root surfaces were gently planned with a sharp gracey no .", "this incision was horizontally extended to the adjacent papillae avoiding the gingival margin of the adjacent teeth .", "two oblique releasing incisions were carried out from the mesial , and distal extremities of the horizontal incision beyond the mucogingival junction [ figure 1a ] .", "a trapezoidal full - thickness flap was raised with a periosteal elevator , until the mucogingival junction .", "then a partial - thickness dissection was carried out apically leaving the underlying periosteum in place .", "in addition a mesiodistal and apical dissection parallel to the vestibular lining mucosa was performed with a blade to release residual muscle tension and to facilitate the passive coronal displacement of the flap .", "( a ) horizontal and vertical incisions given in relation to 23 ; ( b ) hyaluronan gel applied on the root surface of experimental site ; ( c ) suturing done .", "( d ) postoperative 1-week in the control site ; ( e ) postoperative 24 weeks in the experimental site ; ( f ) postoperative 24 weeks in the control site in the experimental group , hyaluronan gel ( gengigel 0.2% ) applied on the root surface using a sterile instrument prior to flap advancement and suturing [ figure 1b ] whereas in the control group the flap was advanced coronally without application of ha gel .", "suturing of oblique releasing incisions was performed , with 5 - 0 ethicon nonresorbable sutures , while the coronal mesial and distal extremities of the flap were secured by two single sutures placed in the interdental areas [ figure 1c ] .", "additional interrupted sutures were applied , when necessary , to close the oblique releasing incisions in the alveolar mucosa . immediately following surgery , use of ice packs", "was recommended for 3 h. all patients were instructed to discontinue tooth brushing , avoid trauma around the surgical site . in the event of pain , the use of ibuprophen + paracetamol ( thrice daily ) was recommended .", "a 0.2% chlorhexidine digluconate solution rinse was prescribed 2 times ( 60 s ) daily for the first 10 days .", "the patients were instructed to clean the surgical sites with a cotton pellet soaked in a 0.2% chlorhexidine digluconate solution twice daily for 10 days .", "three weeks after surgery , the patients were instructed to resume mechanical tooth cleaning of the treated areas using a soft toothbrush with a careful roll technique .", "adverse effects such as inflammation , bleeding on probing , pain and abscess formation were assessed postsurgically by first examiner ( ms ) .", "the patients were recalled for collection of data at 1 , 3 , 6 , 12 and 24 weeks using prefabricated acrylic stent .", "both experimental group and control group sites were evaluated postoperatively at 1 , 3 , 6 , 12 , and 24 weeks after surgery [ figure 1e and f ] .", "stent made of acrylic was used to standardize the direction and position of the probe .", "on each visit , the area was checked for meticulous plaque control , but no subgingival instrumentation was performed until the 6 week .", "a total of 20 buccal gingival recession sites in 10 patients were considered for the study .", "the values obtained were tabulated and statistically analyzed using student 's t - test and repeated measure anova tests .", "all the statistical methods were carried out through the statistical package for the social sciences ( spss ) for windows ( version 16.0 ) .", "the descriptive procedure displays univariate summary statistics for several variables in a single table and calculates standardized values ( z scores ) . using the general linear model procedure , null hypotheses about the effects of both within and between the subject factors", "in addition , the effects of constant covariates and covariate interactions between - subjects factors can be included .", "the patients who were selected and gave their approval for the surgical intervention were educated and motivated with more emphasis on proper oral hygiene maintenance . all the patients underwent the initial phase of treatment of thorough scaling and root planning .", "all the surgical procedures were carried out by one operator . the sites were randomly assigned to the control group or the test group using a flip coin technique immediately before surgery .", "the surgical area was prepared with adequate anesthesia using 2% lignocaine hcl containing 1:80,000 epinephrine .", "after local anesthesia and before the elevation of the flap , both the exposed root surfaces were gently planned with a sharp gracey no .", "this incision was horizontally extended to the adjacent papillae avoiding the gingival margin of the adjacent teeth .", "two oblique releasing incisions were carried out from the mesial , and distal extremities of the horizontal incision beyond the mucogingival junction [ figure 1a ] .", "a trapezoidal full - thickness flap was raised with a periosteal elevator , until the mucogingival junction .", "then a partial - thickness dissection was carried out apically leaving the underlying periosteum in place .", "in addition a mesiodistal and apical dissection parallel to the vestibular lining mucosa was performed with a blade to release residual muscle tension and to facilitate the passive coronal displacement of the flap .", "( a ) horizontal and vertical incisions given in relation to 23 ; ( b ) hyaluronan gel applied on the root surface of experimental site ; ( c ) suturing done .", "( d ) postoperative 1-week in the control site ; ( e ) postoperative 24 weeks in the experimental site ; ( f ) postoperative 24 weeks in the control site in the experimental group , hyaluronan gel ( gengigel 0.2% ) applied on the root surface using a sterile instrument prior to flap advancement and suturing [ figure 1b ] whereas in the control group the flap was advanced coronally without application of ha gel .", "suturing of oblique releasing incisions was performed , with 5 - 0 ethicon nonresorbable sutures , while the coronal mesial and distal extremities of the flap were secured by two single sutures placed in the interdental areas [ figure 1c ] .", "additional interrupted sutures were applied , when necessary , to close the oblique releasing incisions in the alveolar mucosa .", "immediately following surgery , use of ice packs was recommended for 3 h. all patients were instructed to discontinue tooth brushing , avoid trauma around the surgical site . in the event of pain ,", "a 0.2% chlorhexidine digluconate solution rinse was prescribed 2 times ( 60 s ) daily for the first 10 days .", "the patients were instructed to clean the surgical sites with a cotton pellet soaked in a 0.2% chlorhexidine digluconate solution twice daily for 10 days .", "three weeks after surgery , the patients were instructed to resume mechanical tooth cleaning of the treated areas using a soft toothbrush with a careful roll technique .", "adverse effects such as inflammation , bleeding on probing , pain and abscess formation were assessed postsurgically by first examiner ( ms ) .", "the patients were recalled for collection of data at 1 , 3 , 6 , 12 and 24 weeks using prefabricated acrylic stent .", "both experimental group and control group sites were evaluated postoperatively at 1 , 3 , 6 , 12 , and 24 weeks after surgery [ figure 1e and f ] .", "stent made of acrylic was used to standardize the direction and position of the probe . on each visit ,", "the area was checked for meticulous plaque control , but no subgingival instrumentation was performed until the 6 week .", "a total of 20 buccal gingival recession sites in 10 patients were considered for the study .", "the values obtained were tabulated and statistically analyzed using student 's t - test and repeated measure anova tests .", "all the statistical methods were carried out through the statistical package for the social sciences ( spss ) for windows ( version 16.0 ) .", "the descriptive procedure displays univariate summary statistics for several variables in a single table and calculates standardized values ( z scores ) . using the general linear model procedure , null hypotheses about the effects of both within and between the subject factors", "in addition , the effects of constant covariates and covariate interactions between - subjects factors can be included .", "mean probing pocket depth ( ppd ) in experimental sites at baseline was 1.8  0.42 mm , and in control sites it was 2.0  0.47 mm . at the end of 24 weeks", ", mean pd was 1.7 mm  0.82 in experimental sites and 2.0 mm  0.81 mm in control sites [ table 1 and figure 2 ] .", "the overall comparisons between the two groups were not statistically significant ( p = 0.917 ) [ table 2 ] .", "descriptive analysis of two groups mean probing pocket depth values plotted against time for experimental and control groups results of repeated measure anova for ppd for between experimental and control groups clinical attachment level had a significant gain and stability , at 24 weeks follow - up both groups .", "cal gain was significant for both groups , but no intergroup difference was reported at 12 months [ table 3 and figure 3 ] .", "comparison of mean clinical attachment level values between experimental and control groups student 's t - test showing the cal difference between control and experimental groups on an average , experimental site showed initial rd of approximately 3.2 mm  0.78 mm and control sites 2.9 mm  0.73 mm .", "after 24 weeks , mean rd was 1.1 mm  0.99 mm in experimental sites and 1.0 mm  0.66 mm in control sites [ table 4 and figure 4 ] .", "the percentage of root coverage for experimental and control group was 68.33% 28% and 61.67% 30.22% , respectively [ table 5 and figure 5 ] .", "descriptive analysis of two groups mean recession depth values plotted against time for experimental and control groups percentage of root coverage comparison of mean percentage of root coverage between control ( c ) and experimental ( e ) group at 24 weeks , the change in rd was statistically significant ( p = 0.00 ) in both groups [ tables 6 and 7 ] .", "results of repeated measure anova for rd for experimental group results of repeated measure anova for rd for control group", "there are many factors that determine success of root coverage procedures namely , gingival biotype , amount of keratinized tissue , location of the tooth in the arch , prominence of the root , rd and width .", "other than these local factors there are other factors like patient compliance , brushing technique , use of tobacco , etc . , that determine success and long term stability of root coverage procedures .", "it is a linear polysaccharide and one of the principle components of the extracellular matrix , which is biodegradable , biocompatible and nontoxic .", "ha is a macromolecule of several million daltons , consisting of repeating units of d - glucoronic acid ( 1--3 ) n - acetyl d- glucosamine ( 1-4 ) n. exogenous application of hyaluronan has shown to have anti edema , anti - inflammatory and good wound healing properties .", "it has been used extensively in the field of dentistry for its various beneficial applications . in periodontics", "it has been used to treat gingivitis and periodontitis and more recently in the treatment of periodontal intra bony defects .", "the present study was conceived in an effort to merge two simple and cost effective techniques to achieve better root coverage .", "a split mouth study design was considered appropriate to minimize the bias that would arise due to variations in healing pattern among individuals .", "complete root coverage was achieved immediately following the surgical procedures in all cases except one in the experimental group . during the study ,", "after 24 weeks , 100% root coverage was seen in six sites out of which only two were control sites .", "that is , at the end of 24 weeks , 40% of experimental sites demonstrated 100% root coverage [ figure 1e ] , whereas only 20% of the control sites showed complete root coverage [ figure 1f ] .", "this suggests that clinically the root coverage achieved with the use of hyaluronan gel is more predictable at the end of 24 weeks .", "percentage of root coverage in the experimental sites was 68.3% and control sites were 61.6% .", "this is in accordance with the systematic review by chambrone et al . which said that that the mean root coverage for caf alone was ranging from 55.9% to 86.7% .", "the biological properties of hyaluronan as reported by studies by ballini et al . suggests that it promotes regeneration .", "the clinical stability of the root coverage achieved after 24 weeks in this study supports this aspect of previous studies .", "it has been suggested from previous animal studies that there is an increase in hard tissue component of the periodontium following treatment with hyaluronan .", "it promotes wound healing by cell migration and proliferation , facilitating white blood cell infiltration and improving tissue hydration .", "this could contribute to better stability of the root coverage procedures that is seen with the use of hyaluronan .", "hyaluronan as previously mentioned has been extensively studied in the field of dentistry , but to the best of our knowledge , there is no published data on the usage of ha in the treatment of root coverage procedures .", "hence , it is premature at this point to make a comparison of the results of this study with previous studies .", "a longer follow - up is required to know the long - term stability of the treatment procedures .", "it is entirely based on clinical parameters , where in the exact nature of periodontal regeneration achieved could be made only by histological evaluation , studies with large sample size and higher statistical power are needed in the future to explore and establish use of hyaluronan as an adjunct in periodontal surgeries .", "this study shows that hyaluronan affects treatment outcome when used as an adjunct to caf procedure .", "although results are not statistically significant , clinically experimental group shows better results as compared to the control group .", "though caf gives 100% coverage immediately following the surgery only 61 - 64% can be expected 24 weeks after surgery .", "hyaluronan can be easily combined with caf procedure in compromised cases and situations where more stable results are desired ."], "abstract_text": ["<S> background : the purpose of this study was to assess the efficacy of hyaluronic acid ( ha ) in root coverage procedures as an adjunct to coronally advanced flap ( caf ) procedure.materials and methods : this was a randomized clinical trial with split mouth design , where 10 patients with 20 sites of millers class i recession were treated and followed - up for a period of 6 months . </S>", "<S> caf procedure was performed , ha was applied onto the experimental sites before suturing the flap . </S>", "<S> recession depth ( rd ) was measured regularly at baseline 1 , 3 , 6 , 12 , and 24 weeks postoperatively . </S>", "<S> probing pocket depth ( ppd ) and clinical attachment level ( cal ) were also measured along with rd at baseline and 12 and 24 weeks.results:there was a significant change in rd , ppd , cal , and percentage of root coverage in both groups when compared to the baseline values . </S>", "<S> there was no statistically significant difference between experimental and control group in terms of rd ( p = 0.917 ) , ppd ( p = 0.917 ) and cal ( p = 0.761 ) . </S>", "<S> rd was 3.2 mm  0.78 mm in experimental site and control sites 2.9 mm  0.73 mm reduced to 1.1 mm  0.99 mm in experimental sites and 1.0 mm  0.66 mm in control sites . </S>", "<S> though , there is no statistically significant difference root coverage in the experimental group appeared to be clinically more stable compared with the control group after 24 weeks.conclusions:this study suggests that use of ha may improve the clinical outcome of root coverage with caf procedure . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Treatment protocol", "Surgical technique", "Postsurgical care", "Data collection", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["it is estimated that around 50% of the population has one or more sites with 1 mm or more of such root exposure .", "this prevalence rate increases to > 88% in individuals who are 65 years or older .", "key factors in the treatment of gingival recession are to prevent the progressive marginal tissue recession and to facilitate plaque control in the affected area .", "various treatment modalities have been put forth for the correction of gingival recession . these include free gingival autograft , sub - epithelial connective tissue graft ( sctg ) , coronally advanced flap ( caf ) .", "it is seen that the incidence of adverse effects , such as discomfort with or without pain , was directly related to the donor sites of sctg .", "hence despite sctg being the gold standard for root coverage research is always aimed at finding a suitable alternative to this procedure to reduce patient morbidity .", "this procedure has been modified by many others with an intention to improve the stability and predictability of this technique . in a recent 14 year follow - up study pini prato et al .", "hence this procedure is now generally employed with adjuncts such as guided tissue regeneration membrane , emdogain , acellular dermal matrix and platelet rich plasma to enhance its long term stability .", "however , evidence regarding the beneficial impact of these procedures on the stability of root coverage is inconclusive .", "hyaluronic acid ( ha ) is a linear polysaccharide found in extracellular matrices of skin .", "hyaluronan has many structural and physiological functions within tissues , including extracellular and cellular interactions , growth factor interaction and the regulation of osmotic pressure and tissue lubrication , which helps in the maintenance of the structural and homeostatic integrity of tissues .", "one gram of ha can bind up to 6 l of water . as a physical background material", ", it has functions in space filling , lubrication , shock absorption , and protein exclusion .", "according to various clinical studies hyaluronan undergoes degradation in chronically inflamed gingival tissues , whereas the proteoglycan associated sulfated glycosaminoglycans , such as chondroitin 4-sulphate and dermatan sulfate , remain relatively intact .", "ha is a potent anti - inflammatory agent and modulates wound healing due to its ability to scavenge the inflammatory cells - derived reactive oxygen species .", "this study is aimed at testing the efficacy of hyaluronan gel as an adjunct to caf in root coverage ."], ["this was a randomized clinical trial with split mouth study design , carried out in the postgraduate clinic of department of periodontics , coorg institute of dental sciences , virajpet .", "the study sample included 10 subjects of which seven are male , and three female ( at least 20 sites ) having miller 's class i gingival recession on the canine and premolar region were recruited from department of periodontics , coorg institute of dental sciences .", "the sample size was calculated adjusting the minimum acceptable power of study at 80% and maximum allowable alpha error at 5% .", "the recession defects were designated as experimental and control sites randomly prior to surgery by spin of a coin .", "experimental group includes hyaluronan gel ( gengigel 0.2% gel which is 0.2% hyaluronan gel marketed by ricerfarma pharmaceuticals , milan , italy ) with caf and control group with caf alone .", "this self - funded study was approved by institutional ethical committee , and the procedure and the procedure was explained to all the subjects who provided signed informed consent before participation .", "medical and dental examination was obtained from all the individuals who were followed by complete periodontal examination .", "patients with malaligned teeth , teeth in trauma from occlusion , cervical abrasion and restorations were also excluded .", "subjects were included who are systemically healthy with two or more sites of miller 's class i recession and those who can understand and follow oral hygiene instructions properly . at baseline", "group allocation and sampling site selection were performed by one examiner moogala srinivas ( ms ) with a unc 15 probe and rounded off to the nearest 0.5 mm .", "the rd was assessed at baseline , 1 , 3 , 6 , 12 and 24 weeks , and the pd and cal were recorded at 12 and 24 weeks with the patients consent .", "the patients who were selected and gave their approval for the surgical intervention were educated and motivated with more emphasis on proper oral hygiene maintenance . all the patients underwent the initial phase of treatment of thorough scaling and root planning .", "the sites were randomly assigned to the control group or the test group using a flip coin technique immediately before surgery .", "the surgical area was prepared with adequate anesthesia using 2% lignocaine hcl containing 1:80,000 epinephrine .", "after local anesthesia and before the elevation of the flap , both the exposed root surfaces were gently planned with a sharp gracey no .", "this incision was horizontally extended to the adjacent papillae avoiding the gingival margin of the adjacent teeth .", "two oblique releasing incisions were carried out from the mesial , and distal extremities of the horizontal incision beyond the mucogingival junction [ figure 1a ] .", "a trapezoidal full - thickness flap was raised with a periosteal elevator , until the mucogingival junction .", "then a partial - thickness dissection was carried out apically leaving the underlying periosteum in place .", "in addition a mesiodistal and apical dissection parallel to the vestibular lining mucosa was performed with a blade to release residual muscle tension and to facilitate the passive coronal displacement of the flap .", "( a ) horizontal and vertical incisions given in relation to 23 ; ( b ) hyaluronan gel applied on the root surface of experimental site ; ( c ) suturing done .", "( d ) postoperative 1-week in the control site ; ( e ) postoperative 24 weeks in the experimental site ; ( f ) postoperative 24 weeks in the control site in the experimental group , hyaluronan gel ( gengigel 0.2% ) applied on the root surface using a sterile instrument prior to flap advancement and suturing [ figure 1b ] whereas in the control group the flap was advanced coronally without application of ha gel .", "suturing of oblique releasing incisions was performed , with 5 - 0 ethicon nonresorbable sutures , while the coronal mesial and distal extremities of the flap were secured by two single sutures placed in the interdental areas [ figure 1c ] .", "additional interrupted sutures were applied , when necessary , to close the oblique releasing incisions in the alveolar mucosa . immediately following surgery , use of ice packs", "was recommended for 3 h. all patients were instructed to discontinue tooth brushing , avoid trauma around the surgical site . in the event of pain , the use of ibuprophen + paracetamol ( thrice daily ) was recommended .", "a 0.2% chlorhexidine digluconate solution rinse was prescribed 2 times ( 60 s ) daily for the first 10 days .", "the patients were instructed to clean the surgical sites with a cotton pellet soaked in a 0.2% chlorhexidine digluconate solution twice daily for 10 days .", "three weeks after surgery , the patients were instructed to resume mechanical tooth cleaning of the treated areas using a soft toothbrush with a careful roll technique .", "adverse effects such as inflammation , bleeding on probing , pain and abscess formation were assessed postsurgically by first examiner ( ms ) .", "the patients were recalled for collection of data at 1 , 3 , 6 , 12 and 24 weeks using prefabricated acrylic stent .", "both experimental group and control group sites were evaluated postoperatively at 1 , 3 , 6 , 12 , and 24 weeks after surgery [ figure 1e and f ] .", "stent made of acrylic was used to standardize the direction and position of the probe .", "on each visit , the area was checked for meticulous plaque control , but no subgingival instrumentation was performed until the 6 week .", "a total of 20 buccal gingival recession sites in 10 patients were considered for the study .", "the values obtained were tabulated and statistically analyzed using student 's t - test and repeated measure anova tests .", "all the statistical methods were carried out through the statistical package for the social sciences ( spss ) for windows ( version 16.0 ) .", "the descriptive procedure displays univariate summary statistics for several variables in a single table and calculates standardized values ( z scores ) . using the general linear model procedure , null hypotheses about the effects of both within and between the subject factors", "in addition , the effects of constant covariates and covariate interactions between - subjects factors can be included ."], ["the patients who were selected and gave their approval for the surgical intervention were educated and motivated with more emphasis on proper oral hygiene maintenance . all the patients underwent the initial phase of treatment of thorough scaling and root planning ."], ["all the surgical procedures were carried out by one operator . the sites were randomly assigned to the control group or the test group using a flip coin technique immediately before surgery .", "the surgical area was prepared with adequate anesthesia using 2% lignocaine hcl containing 1:80,000 epinephrine .", "after local anesthesia and before the elevation of the flap , both the exposed root surfaces were gently planned with a sharp gracey no .", "this incision was horizontally extended to the adjacent papillae avoiding the gingival margin of the adjacent teeth .", "two oblique releasing incisions were carried out from the mesial , and distal extremities of the horizontal incision beyond the mucogingival junction [ figure 1a ] .", "a trapezoidal full - thickness flap was raised with a periosteal elevator , until the mucogingival junction .", "then a partial - thickness dissection was carried out apically leaving the underlying periosteum in place .", "in addition a mesiodistal and apical dissection parallel to the vestibular lining mucosa was performed with a blade to release residual muscle tension and to facilitate the passive coronal displacement of the flap .", "( a ) horizontal and vertical incisions given in relation to 23 ; ( b ) hyaluronan gel applied on the root surface of experimental site ; ( c ) suturing done .", "( d ) postoperative 1-week in the control site ; ( e ) postoperative 24 weeks in the experimental site ; ( f ) postoperative 24 weeks in the control site in the experimental group , hyaluronan gel ( gengigel 0.2% ) applied on the root surface using a sterile instrument prior to flap advancement and suturing [ figure 1b ] whereas in the control group the flap was advanced coronally without application of ha gel .", "suturing of oblique releasing incisions was performed , with 5 - 0 ethicon nonresorbable sutures , while the coronal mesial and distal extremities of the flap were secured by two single sutures placed in the interdental areas [ figure 1c ] .", "additional interrupted sutures were applied , when necessary , to close the oblique releasing incisions in the alveolar mucosa ."], ["immediately following surgery , use of ice packs was recommended for 3 h. all patients were instructed to discontinue tooth brushing , avoid trauma around the surgical site . in the event of pain ,", "a 0.2% chlorhexidine digluconate solution rinse was prescribed 2 times ( 60 s ) daily for the first 10 days .", "the patients were instructed to clean the surgical sites with a cotton pellet soaked in a 0.2% chlorhexidine digluconate solution twice daily for 10 days .", "three weeks after surgery , the patients were instructed to resume mechanical tooth cleaning of the treated areas using a soft toothbrush with a careful roll technique .", "adverse effects such as inflammation , bleeding on probing , pain and abscess formation were assessed postsurgically by first examiner ( ms ) ."], ["the patients were recalled for collection of data at 1 , 3 , 6 , 12 and 24 weeks using prefabricated acrylic stent .", "both experimental group and control group sites were evaluated postoperatively at 1 , 3 , 6 , 12 , and 24 weeks after surgery [ figure 1e and f ] .", "stent made of acrylic was used to standardize the direction and position of the probe . on each visit ,", "the area was checked for meticulous plaque control , but no subgingival instrumentation was performed until the 6 week ."], ["a total of 20 buccal gingival recession sites in 10 patients were considered for the study .", "the values obtained were tabulated and statistically analyzed using student 's t - test and repeated measure anova tests .", "all the statistical methods were carried out through the statistical package for the social sciences ( spss ) for windows ( version 16.0 ) .", "the descriptive procedure displays univariate summary statistics for several variables in a single table and calculates standardized values ( z scores ) . using the general linear model procedure , null hypotheses about the effects of both within and between the subject factors", "in addition , the effects of constant covariates and covariate interactions between - subjects factors can be included ."], ["mean probing pocket depth ( ppd ) in experimental sites at baseline was 1.8  0.42 mm , and in control sites it was 2.0  0.47 mm . at the end of 24 weeks", ", mean pd was 1.7 mm  0.82 in experimental sites and 2.0 mm  0.81 mm in control sites [ table 1 and figure 2 ] .", "the overall comparisons between the two groups were not statistically significant ( p = 0.917 ) [ table 2 ] .", "descriptive analysis of two groups mean probing pocket depth values plotted against time for experimental and control groups results of repeated measure anova for ppd for between experimental and control groups clinical attachment level had a significant gain and stability , at 24 weeks follow - up both groups .", "cal gain was significant for both groups , but no intergroup difference was reported at 12 months [ table 3 and figure 3 ] .", "comparison of mean clinical attachment level values between experimental and control groups student 's t - test showing the cal difference between control and experimental groups on an average , experimental site showed initial rd of approximately 3.2 mm  0.78 mm and control sites 2.9 mm  0.73 mm .", "after 24 weeks , mean rd was 1.1 mm  0.99 mm in experimental sites and 1.0 mm  0.66 mm in control sites [ table 4 and figure 4 ] .", "the percentage of root coverage for experimental and control group was 68.33% 28% and 61.67% 30.22% , respectively [ table 5 and figure 5 ] .", "descriptive analysis of two groups mean recession depth values plotted against time for experimental and control groups percentage of root coverage comparison of mean percentage of root coverage between control ( c ) and experimental ( e ) group at 24 weeks , the change in rd was statistically significant ( p = 0.00 ) in both groups [ tables 6 and 7 ] .", "results of repeated measure anova for rd for experimental group results of repeated measure anova for rd for control group"], ["there are many factors that determine success of root coverage procedures namely , gingival biotype , amount of keratinized tissue , location of the tooth in the arch , prominence of the root , rd and width .", "other than these local factors there are other factors like patient compliance , brushing technique , use of tobacco , etc . , that determine success and long term stability of root coverage procedures .", "it is a linear polysaccharide and one of the principle components of the extracellular matrix , which is biodegradable , biocompatible and nontoxic .", "ha is a macromolecule of several million daltons , consisting of repeating units of d - glucoronic acid ( 1--3 ) n - acetyl d- glucosamine ( 1-4 ) n. exogenous application of hyaluronan has shown to have anti edema , anti - inflammatory and good wound healing properties .", "it has been used extensively in the field of dentistry for its various beneficial applications . in periodontics", "it has been used to treat gingivitis and periodontitis and more recently in the treatment of periodontal intra bony defects .", "the present study was conceived in an effort to merge two simple and cost effective techniques to achieve better root coverage .", "a split mouth study design was considered appropriate to minimize the bias that would arise due to variations in healing pattern among individuals .", "complete root coverage was achieved immediately following the surgical procedures in all cases except one in the experimental group . during the study ,", "after 24 weeks , 100% root coverage was seen in six sites out of which only two were control sites .", "that is , at the end of 24 weeks , 40% of experimental sites demonstrated 100% root coverage [ figure 1e ] , whereas only 20% of the control sites showed complete root coverage [ figure 1f ] .", "this suggests that clinically the root coverage achieved with the use of hyaluronan gel is more predictable at the end of 24 weeks .", "percentage of root coverage in the experimental sites was 68.3% and control sites were 61.6% .", "this is in accordance with the systematic review by chambrone et al . which said that that the mean root coverage for caf alone was ranging from 55.9% to 86.7% .", "the biological properties of hyaluronan as reported by studies by ballini et al . suggests that it promotes regeneration .", "the clinical stability of the root coverage achieved after 24 weeks in this study supports this aspect of previous studies .", "it has been suggested from previous animal studies that there is an increase in hard tissue component of the periodontium following treatment with hyaluronan .", "it promotes wound healing by cell migration and proliferation , facilitating white blood cell infiltration and improving tissue hydration .", "this could contribute to better stability of the root coverage procedures that is seen with the use of hyaluronan .", "hyaluronan as previously mentioned has been extensively studied in the field of dentistry , but to the best of our knowledge , there is no published data on the usage of ha in the treatment of root coverage procedures .", "hence , it is premature at this point to make a comparison of the results of this study with previous studies .", "a longer follow - up is required to know the long - term stability of the treatment procedures .", "it is entirely based on clinical parameters , where in the exact nature of periodontal regeneration achieved could be made only by histological evaluation , studies with large sample size and higher statistical power are needed in the future to explore and establish use of hyaluronan as an adjunct in periodontal surgeries ."], ["this study shows that hyaluronan affects treatment outcome when used as an adjunct to caf procedure .", "although results are not statistically significant , clinically experimental group shows better results as compared to the control group .", "though caf gives 100% coverage immediately following the surgery only 61 - 64% can be expected 24 weeks after surgery .", "hyaluronan can be easily combined with caf procedure in compromised cases and situations where more stable results are desired ."]]}
{"article_id": "PMC3813259", "article_text": ["ulcerative colitis ( uc ) is characterized by chronic and relapsing inflammation of the intestines , resulting in diarrhea , abdominal pain , and a variety of other symptoms ( 1 ) , and impacts negatively on the quality of life .", "huangqing - tang decoction , described by zhang zhongjing ( 150 to 219 a.d . , in chinese eastern han dynasty ) , has been used for the treatment of uc disease for thousands of years .", "radix scutellariae and radix paeoniae alba are the key ingredient herbs present in this decoction , the combination of radix scutellariae and radix paeoniae alba ( 3:2 g / g ) renders its features , such as the heat - clearing , antiinflammatory , antidiarrheic and antinociceptive effects .", "apparently , it is essential to study the synergistic interaction of radix scutellariae and radix paeoniae alba for elucidating the substantial foundation of huangqing - tang . \n", "radix scutellariae has been widely used in traditional chinese medicine ( tcm ) for the treatment of inflammation , fever , hepatitis , allergic diseases and hypertension , and is comprised of the flavonoids , baicalin ( bg ) , wogonoside ( wg ) , baicalein ( b ) and wogonin ( w ) as the active ingredients ( figure 1 ) ( 24 ) . to date , the pharmacokinetic ( pk ) profiles of bg and wg in plasma , brain and eyes of rats and rabbits have been reported ( 511 ) .", "it is generally assumed that baicalin is poorly absorbed from the gastrointestinal tract in its native form and must be hydrolyzed by microflora enzymes ( bacterial - glucuronidase ) in gut to its aglycone - baicalein , then reconverted back to baicalin .", "similar to baicalin , wogonoside is also firstly metabolized by microflora enzymes and reconverted , and is subsequently excreted mainly in conjugated form following administration of radix scutellariae extract in rat and human .", "baicalin and wogonoside demonstrated bimodal phenomenon in the plasma profile ( 12 , 13 ) .", "after oral administration of baicalein , levels of baicalein were negligible , whereas the glucuronides / sulfates conjugates of baicalein were predominant in the plasma ( 14 , 15 ) . various case reports on herb  drug interactions or herb  herb interactions in - vivo", "have been published in recent years , in addition to some reports on the influence of disease condition on the pharmacokinetic characteristics of drug ( 1619 ) .", ". a better understanding of the pharmacokinetics of herbal interactions and herbs in diseased rats would be helpful in formulating rational dosage regimens ( 20 ) .", "however , to the best of our knowledge , there are no published studies reporting the pharmacokinetics of bg , wg , b , and w after oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts to normal rats , let alone to uc rats .", "the syndrome of the trinitro - benzene - sulfonic acid)tnbs(-induced colonitis model is similar to the spontaneous uc .", "this animal model has been used extensively in studies of colonitis and its complications , including the altered drug pharmacokinetics under the colonitis conditions ( 2126 ) .", "the aim of this study is to explore the pharmacokinetic differences of the four flavonoids after oral administration of pure baicalin , radix scutellariae extract and scutellariae - paeoniae couple extract to rats and to compare the pharmacokinetic parameters of uc rats with those of normal rats .", "\n materials and chemicals \n \n radix scutellariae , radix paeoniae alba were purchased from bozhou medicine company ( anhui , china ) and authenticated by prof .", "feng li from the college of pharmacy , university of traditional chinese medicine ( liaoning , china ) .", "the voucher specimens ( no.20081205 , 20081211 ) have been deposited in liaoning university of tcm .", "zhenqiu zhang ( dept . of chemistry in our institute , liaoning university of tcm ) .", "baicalin and wogonin were purchased from the national institute for the control of pharmaceutical and biological products ( beijing , china ) .", "the solvents used for chromatographic analysis were hplc grade and were purchased from fisher company inc .", "deionized water was prepared in a mill - q academic water purification system ( millipore , bedford , ma , usa ) .", "all the other reagents were of analytical grade and provided by kermel chemical co. ( tianjin , china ) . \n", "apparatus and chromatographic conditions \n  the concentrations of the four flavonoids in plasma were assayed using reverse - phase high performance liquid chromatography ( agilent 1100 series ) equipped with a variable wave length uv detector and pump ( agilent model g1314a vwd ) .", "separation was accomplished on a eclipsel xdb - c18 column ( 250 mm  4.6 mm , 5 m particle size ) .", "the mobile phase was composed of acetonitrile ( a ) : 0.1% phosphoric acid water ( b ) ( 0  10 min , 16 : 84 ; 10  15 min , 22 : 78 ; 15  25 min , 25 : 75 ; 25  30 min , 34 : 64 ; 30  50 min , 42 : 58 ,", "v / v ) at a flow rate of 1.0 ml / min with gradient elution .", "preparation of radix scutellariae and scutellariae - paeoniae couple extracts \n \n radix scutellariae and radix paeoniae alba were mixed in the ratio of 1.5 : 1 g / g and the total weight was 200 g. the mixture was decocted twice by refluxing with 70% ethanol ( 1 : 10 and then 1 : 8 w / v ) for 1 h , and the solution obtained was concentrated to give an extract of 60.5 g. radix scutellariae ( 200 g ) was treated as above to provide an extract of 43.3 g. the dried powder was stored at 4 c before use . for estimation of the administered dose , the concentrations of baicalin , wogonoside , baicalein and wogonin in the extracts and pure baicalin", "the extract powder ( 0.10 g ) was ultrasound with 100 ml 60% methanol for 30 min .", "the contents of bg , b , wg and w were determined to be 17.3 , 7.6 , 2.4 and 1.7 % in scutellariae - paeoniae couple extract and 43.6 , 19.1 , 6.2 and 4.5% in radix scutellariae extract , respectively .", "male sprague  dawley rats , weighing 250 - 280 g , were obtained from the experimental animal department of dalian university ( dalian , china ) .", "animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals ( us national research council , 1996 ) and the related ethics regulations of liaoning university of tcm .", "rats were housed in an air - conditioned animal quarter at a temperature of 22  2 c and a relative humidity of 50  2% .", "the animals were acclimatized to the facilities for five days , and then fasted with free access to water for 24 h prior to each experiment .", "colitis was induced in rats according to the model and method described by morris et al .", "briefly , rats were lightly anesthetized with 10% chloral hydrate solution , and then a medical - grade polyurethane cannula for enteral feeding ( external diameter 2 mm ) was inserted into the anus and the tip was advanced to 8 cm proximal to the anal verge .", "shanghai , china . ) dissolved in 100% ethanol was instilled into the colon through the cannula ( at a dose 80 mg / kg ) .", "following the instillation of the hapten , the rats were maintained in a head - down position for an additional 30 sec to prevent leakage of the intra colonic instillation .", "successful replication of the model of uc was characterized by reduction in activities , apathy and occult blood in stool .", "biosample collection \n  the uc rats were divided into three subgroups randomly , the pure baicalin , radix scutellariae extract and scutellariae - paeoniae couple extract subgroup .", "each group was respectively administrated an oral dose of 200 mg / kg baicalin ( in the form of pure compound or co - administrated mixture , 0.21 g / kg pure baicalin , 0.46 g / kg radix scutellariae extract and 1.16 g / kg scutellariae - paeoniae couple extract ) , all of the compounds were suspended in water and homogenized using ultrasonic technology just prior to dosing .", "blood samples ( 0.5 ml ) were collected into a heparinized tube via the oculi chorioideae vein before drug administration and at 0.083 , 0.25 , 0.5 , 0.75 , 1.0 , 2.0 , 4.0 , 8.0 , 12.0 and 24.0 h after drug administration , and were centrifuged at 4000 rpm for 10 min for the separation of plasma from the red blood cells .", "the supernatant was decanted carefully and was stored at  40 c until analysis . \n", "biosample preparation \n  an aliquot of 15 l arctigenin ( 32.0 m in methanol ) and 10 l hcl solution ( 0.1 mm ) were added into 100 l plasma , and then spiked with methanol 75 l and acetonitrile 100 l by vortex mixing for 5 min .", "an aliquot of 50 l of the supernatant was injected into the hplc system .   known amounts of baicalin , baicalein wogonoside , wogonin and is were added into 100 l of blank plasma to prepare the following series of standards .", "the calibration curves showed good linearity in the range of 0.12331.6 m for baicalin , 0.28873.6 m for wogonoside , 0.1338.50 m for baicalein , 0.0627.9 m for wogonin .", "the extraction recoveries and matrix effects at three quality control ( qc ) concentrations were assayed in sets of six replicates .", "the cvs of the recoveries were less than 20% at low concentration , and less than 15% at medium and high concentrations .", "accuracy and precision of the method were investigated by intra - day and inter - day data via comparing the mean of five replicates of each qc sample .", "the stabilities of baicalin , wogonoside , baicalein and wogonin were determined by analyzing qc samples at three concentrations exposed to encounter during sample storage .", "the corresponding relative errors were less than  6% for samples at three concentrations for each reference standard , respectively .", "results of the stability test showed that under the experimental conditions the samples were stable throughout the testing process .  ", "the plasma concentration vs. time data of baicalin and wogonoside were analyzed by non - compartmental model using the nonlinear least squares regression program winnonlin ( scientific consulting inc . ,", "shanghai , china ) were used to calculate the pharmacokinetic parameters , such as the area under curve ( auc ) , the maximum plasma concentration ( cmax ) and the corresponding time ( tmax ) , the half - life of absorption ( t1/2 ) , etc .", "statistical analysis of pharmacokinetic parameter estimates was performed using a two - tailed nonparametric t - test .", "chemical structures of arctigenin ( a ) , baicalin ( b ) , baicalein ( c ) , wogonoside ( d ) and wogonin ( e ) .", "the selectivity of the method was evaluated by analyzing blank plasma samples prior to administration .", "the chromatograms were free of interfering peaks at the retention times of is ( 34.5 min ) and baicalin ( 20.6 min ) , wogonoside ( 25.9 min ) , baicalein ( 31.9 min ) and wogonin ( 40.8 min ) .", "figure 2 showed the representative chromatograms of blank plasma sample ( a ) , blank plasma sample spiked with baicalin , wogonoside , baicalein , wogonin and is ( b ) , blank plasma sample spiked with is ( c ) , plasma sample ( 1.5 h ) after oral administration of scutellariae - paeoniae couple extract ( d ) . under the established chromatographic condition ,", "no interfering peaks were observed at the elution times of baicalin , wogonoside , baicalein , wogonin and the internal standard .  ", "chromatograms of blank plasma sample ( a ) , plasma sample spiked with baicalin , wogonoside , baicalein , wogonin and is ( b ) , plasma sample spiked with is ( c ) , plasma samples 1.5 h after oral administration of scutellariae - paeoniae couple extracts ( d ) . \n", "pharmacokinetics of baicalin and wogonoside after oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts to rats . \n ", "the mean plasma concentration - time profiles and standard deviation in normal rats and uc rats following the administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts are depicted in figure 3 ( a ) and 3 ( b ) .", "pharmacokinetic parameters resulting from non - compartmental analysis are listed in tables 1 and 2 .  ", "the plasma concentration  time profile of baicalin ( bg ) and wogonoside ( wg ) after oral administration of pure baicalin ( 0.21 g / kg ) , radix scutellariae extract ( 0.46 g / kg ) and scutellariae - paeoniae couple extract ( 1.16 g / kg ) to normal ( a ) and ulcerative colitis ( uc ) ( b ) rats pharmacokinetic differences of baicalin after oral administration of pure baicalin ( 0.21 g / kg ) , radix scutellariae extract ( 0.46 g / kg ) and scutellariae - paeoniae couple extract ( 1.16 g / kg of baicalin ) to normal and uc rats mean  sd , n=10;*p < 0.05 vs. normal - rats pharmacokinetic differences of wogonoside after oral administration of pure baicalin ( 0.21 g / kg ) , radix scutellariae extract ( 0.46 g / kg ) and scutellariae - paeoniae couple ( 1.16 g / kg ) to normal and uc rats mean  s.d . , n=10 ; * p<0.05 vs. normal - rats following oral administration of pure baicalin ( purity > 95.5% ) to rats , wogonoside was detected in all rats plasma in addition to baicalin , the plasma concentration was almost equal to that of baicalin .", "the same result was observed after oral administration of radix scutellariae extract ( 200 mg / kg of baicalin , 28 mg / kg wogonoside ) or scutellariae - paeoniae couple extract ( 200 mg / kg of baicalin , 27 mg / kg of wogonoside ) to rats . the present result suggested that baicalin might convert to wogonoside in the rat body .", "zhenyu zhu et al . ( 4 ) reported that they found a peak ( named m ) with a retention time of about 5 min at m / z 461 was similar to wogonoside ( m / z 461 ) after oral administration of xiaochaihu tang and radix scutellariae extract to rats , which was possibly a methylated product of baicalin .", "methylation generally existed during the drug metabolism in - vivo , and due to the multiple hydroxyl groups on the baicalin benzene ring , the combination of a methyl group with a hydroxyl group was easy .", "so , we tentatively concluded that absorbed baicalin could be methylated to wogonoside in - vivo .", "following oral administration of radix scutellariae or scutellariae - paeoniae couple extracts to rats , baicalein and wogonin with very high concentrations were presented in rat tissues ( 28 ) , however , in the present study , it was below the detection limit in all plasma samples collected for all rats .", "these results appeared to be consistent with previous reports ( 14 , 15 , 29 , 30 ) .", "as described in previous studies , baicalin firstly must be hydrolyzed to baicalein by -glucuronidase in - vivo , which was produced by both intestinal bacteria and intestinal epithelial cells ( 3134 ) .", "thus , baicalin can first enter the intestinal wall as baicalein and is then reconverted to baicalin within the intestinal mucosa and thereby is absorbed into the blood .", "therefore , baicalin in plasma may be derived from both baicalin and baicalein in radix scutellariae or scutellariae - paeoniae .", "the doses of baicalin and baicalein in the extracts were summed to produce the sum dose of baicalin and baicalin which was used to calculate dose - normalized cmax and auc0-t . based on the similar chemical structure between wogonoside and baicalin ,", "it is proposed that the transformation between wogonoside and wogonin are similar to that between baicalin and baicalein in the intestinal wall , the sum dose of wogonoside and wogonin was calculated in the same way .", "consequently , we could only detect baicalin and wogonoside , not baicalein and wogonin in plasma .   as shown in figure 3 and table 1 - 2 ,", "the absorption speed of baicalin and wogonoside in the normal pure baicalin group was quicker than those in the normal radix scutellariae and scutellariae - paeoniae couple groups , tmax for baicalin were : ( 0.087  0.02 ) h , ( 0.27  0.03 ) h and ( 0.28  0.03 ) h ; tmax for wogonoside were ( 0.11  0.02 ) h , ( 0.22  0.09 ) h , ( 0.21  0.08 ) h , respectively .", "but tmax of baicalin and wogonoside were little different among the three groups of uc rats .", "the pharmacokinetic profiles differences of baicalin and wogonoside were reflected in auc(0t ) and cmax .  ", "following oral adminstration scutellariae - paeoniae couple extract to rats , cmax and auc(0t ) of baicalin and wogonoside were higher than the other two forms ( p  0.05 ) .", "compared to the radix scutellariae groups , cmax and auc(0t ) of baicalin and wogonoside in the pure baicalin groups were significantly lower ( p  0.05 ) .", "these chemical components might produce certain pharmacological effects and have impact on the pharmacokinetic profiles of baicalin and wogonoside . under the guidance of the theory of tcm ,", "radix paeoniae alba , used as messenger herb and hematinic herb , could guide the bioactive ingredients of radix scutellariae to the proper tissues and exert a harmonizing effect , including the increase of absorption and bioavailability .", "radix paeoniae alba also could increase the absorption of baicalin and wogonoside through increasing the function of spleen and improving the patients digestive system vitality and immune system .", "thus , compared to pure baicalin and radix scutellariae extract , the cmax and auc(0t ) of baicalin and wogonoside increased remarkably ( p < 0.05 ) when radix scutellariae was mix - decocted with radix paeoniae alba , in contrast , the absorption of pure baicalin was the least .", "the present study demonstrated that herb extract could enhance the bioavailability of baicalin and wogonoside in rat plasma , a significant drug - drug interaction may occur and improve the absorption of baicalin and wogonoside when radix paeoniae alba is administered in combination with radix scutellariae ( 35 ) .   either in uc or in normal rats , baicalin and wogonoside demonstrated bimodal phenomenon in rat plasma after oral administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts ( figure 3 ) .", "the first absorption peaks occurred at about 0.15 - 0.27 h and the second was at about 8  12 h. the concentrations of the second peak ( cmax ) and the values of auc(0t ) dose were lower than which of the first peak .", "the ability of rapid absorption of baicalin and wogonoside may result in the rapid appearance of the first peak .", "baicalin and wogonoside had double - site absorption kinetics , and baicalin and wogonoside undergo enteric circulation and enterohepatic circulation in rats after oral dosing , which may be responsible for the second peak .", "but they were always detected and kept their concentration at a certain range in plasma .", "free drug might bind with plasma protein when it reaches a certain concentration ( 19 ) .", "wang et al . ( 36 ) reported that the binding rate of free baicalin with plasma protein could reach 80% .", "it was not able to maintain a high concentration for a long time because the baicalin would enter into the plasma and then bind with plasma protein .", "when the concentration of free baicalin decreased to a certain concentration , the bound baicalin would dissociate . \n", "comparative pharmacokinetics of baicalin and wogonoside after oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts between uc and normal rats \n  from figure 4 and table 1 - 2 , it was noteworthy that uc rats showed better absorption than normal rats , regardless of oral administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts .", "the absorption speeds of baicalin and wogonoside were very rapid , tmax were approximately 0.13 h. tmax showed no statistically significant difference among the three groups of uc rats .", "cmax and auc(0t ) of baicalin and wogonoside in uc rats were greatly higher than those in normal rats ( p < 0.05 ) .", "after oral administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts to rats , auc(0t ) of baicalin were : ( 41.46  0.62 ) , ( 59.12  6.42 ) and ( 104.87  0.86 ) ( g / ml)h in uc groups vs ( 17.77  0.66 ) , ( 28.04  4.06 ) and ( 49.01  4.61 ) ( g / ml)h in normal groups ; auc(0t ) of wogonoside were : ( 40.86  5.31 ) , ( 57.95  1.91 ) and ( 101.31  3.05 ) ( g / ml)h in the uc groups vs ( 14.67  1.93 ) , ( 22.89  3.17 ) and ( 40.35  4.96 ) g / ml / h in the normal groups , respectively .  ", "plasma concentration - time profiles of baicalin ( bg ) ( a ) and wogonoside ( wg ) ( b ) after oral administration of scutellariae - paeoniae couple extract ( 1.16 g / kg ) to ulcerative colitis ( uc ) and normal rats the significantly higher cmax and auc(0t ) of baicalin and wogonoside in uc rats indicated that ulcerative colitis had an influence on the pharmacokinetic characteristics of baicalin and wogonoside in pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts .", "several factors were likely to be involved in the alteration of the pharmacokinetic behavior of baicalin and wogonoside under the colitis condition , including -glucuronidase , udp - glucuronosyltransferase , mrp2 , intestinal or hepatic metabolic enzymes and bile flow rate , etc .", "trinitrobenzene sulfonic acid ( tnbs)-induced uc condition might alter the formation of glucuronide , glutathione , and sulfate conjugates which played an important role in the metabolism of compounds in rats ( 40 - 43 ) .", "these factors may result in a higher exposure of baicalin and wogonoside after oral doses . as known that intestinal flora plays an important role in the absorption and reabsorption of baicalin and wogonoside , we hypothesized that intestinal bacteria in uc rats would be very rich which lead to increased -glucuronidase activity .", "thus , profound intestinal -glucuronidase activity due to the ulcerative colitis condition may have enabled the rapid conversion of baicalin and wogonoside to baicalein and wogonin which have the propensity to be absorbed more completely leading to higher baicalin and wogonoside exposure after oral administration relative to normal rats .  ", "the findings of this study demonstrated that the pharmacokinetic behaviors of baicalin and wogonoside were remarkably altered in uc rats .", "scutellariae - paeoniae couple extract may exert more effectiveness than pure baicalin or radix scutellariae extract .", "it proves the rationality of using scutellariae - paeoniae couple for the treatment of ulcerative colonitis disease .", "in summary , the results of the present study highlighted that drug  drug interactions , herb  drug interactions and herb  herb interactions always existed and affected the pharmacokinetic behavior of herbal ingredients . statistically significant differences ( p < 0.05 ) in pharmacokinetic parameters of baicalin and wogonoside including cmax and auc(0t ) were obtained among the rats orally administered pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts .", "the huge number of active ingredients in tcm made them suitable for multi - target actions .", "baicalin and wogonoside had a better absorption effect , which would improve the therapeutic efficacy .", "we need to take caution when extrapolating pk and exposure data from healthy animals to diseased animals in designing pharmacological studies ."], "abstract_text": ["<S> the aim of this study was to investigate the pharmacokinetic profiles of baicalin , wogonoside , baicalein and wogonin after oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts were administered and the pharmacokinetics profiles were compared between normal and ulcerative colitis rats . </S>", "<S> the plasma concentrations of the four flavonoids were determined by using a simple and rapid high - performance liquid chromatography method . </S>", "<S> all the rats were divided randomly into two groups ( ulcerative colitis and normal groups ) . </S>", "<S> each group contained three subgroups : pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts subgroup . </S>", "<S> each group received oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts at the same dose of 200 mg / kg baicalin . </S>", "<S> the results showed that wogonoside , possibily as a methylated product of baicalin , was found in plasma after oral administration of pure baicalin or formulas to rats . </S>", "<S> baicalin and wogonoside demonstrated bimodal phenomenon . </S>", "<S> baicalin and wogonoside in scutellariae - paeoniae couple extract had shown better absorption than which in pure baicalin and radix scutellariae extract . whether oral administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts , ulcerative colitis rats showed better absorption than normal rats . for example </S>", "<S> auc(0t ) of baicalin were : ( 41.46  0.62 ) , ( 59.12  6.42 ) and ( 104.87  0.86 ) ( g / ml)h in uc groups vs ( 17.77  0.66 ) , ( 28.04  4.06 ) and ( 49.01  4.61 ) ( g / ml)h in normal groups , respectively . </S>", "<S> the pharmacokinetics properties of the four flavonoids differed between ulcerative colitis and normal rats , including auc(0t ) and cmax ( p < 0.05 ) . </S>"], "labels": null, "section_names": ["Introduction", "Experimental", "Results and Discussion", "Conclusions"], "sections": [["ulcerative colitis ( uc ) is characterized by chronic and relapsing inflammation of the intestines , resulting in diarrhea , abdominal pain , and a variety of other symptoms ( 1 ) , and impacts negatively on the quality of life .", "huangqing - tang decoction , described by zhang zhongjing ( 150 to 219 a.d . , in chinese eastern han dynasty ) , has been used for the treatment of uc disease for thousands of years .", "radix scutellariae and radix paeoniae alba are the key ingredient herbs present in this decoction , the combination of radix scutellariae and radix paeoniae alba ( 3:2 g / g ) renders its features , such as the heat - clearing , antiinflammatory , antidiarrheic and antinociceptive effects .", "apparently , it is essential to study the synergistic interaction of radix scutellariae and radix paeoniae alba for elucidating the substantial foundation of huangqing - tang . \n", "radix scutellariae has been widely used in traditional chinese medicine ( tcm ) for the treatment of inflammation , fever , hepatitis , allergic diseases and hypertension , and is comprised of the flavonoids , baicalin ( bg ) , wogonoside ( wg ) , baicalein ( b ) and wogonin ( w ) as the active ingredients ( figure 1 ) ( 24 ) . to date , the pharmacokinetic ( pk ) profiles of bg and wg in plasma , brain and eyes of rats and rabbits have been reported ( 511 ) .", "it is generally assumed that baicalin is poorly absorbed from the gastrointestinal tract in its native form and must be hydrolyzed by microflora enzymes ( bacterial - glucuronidase ) in gut to its aglycone - baicalein , then reconverted back to baicalin .", "similar to baicalin , wogonoside is also firstly metabolized by microflora enzymes and reconverted , and is subsequently excreted mainly in conjugated form following administration of radix scutellariae extract in rat and human .", "baicalin and wogonoside demonstrated bimodal phenomenon in the plasma profile ( 12 , 13 ) .", "after oral administration of baicalein , levels of baicalein were negligible , whereas the glucuronides / sulfates conjugates of baicalein were predominant in the plasma ( 14 , 15 ) . various case reports on herb  drug interactions or herb  herb interactions in - vivo", "have been published in recent years , in addition to some reports on the influence of disease condition on the pharmacokinetic characteristics of drug ( 1619 ) .", ". a better understanding of the pharmacokinetics of herbal interactions and herbs in diseased rats would be helpful in formulating rational dosage regimens ( 20 ) .", "however , to the best of our knowledge , there are no published studies reporting the pharmacokinetics of bg , wg , b , and w after oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts to normal rats , let alone to uc rats .", "the syndrome of the trinitro - benzene - sulfonic acid)tnbs(-induced colonitis model is similar to the spontaneous uc .", "this animal model has been used extensively in studies of colonitis and its complications , including the altered drug pharmacokinetics under the colonitis conditions ( 2126 ) .", "the aim of this study is to explore the pharmacokinetic differences of the four flavonoids after oral administration of pure baicalin , radix scutellariae extract and scutellariae - paeoniae couple extract to rats and to compare the pharmacokinetic parameters of uc rats with those of normal rats ."], ["\n materials and chemicals \n \n radix scutellariae , radix paeoniae alba were purchased from bozhou medicine company ( anhui , china ) and authenticated by prof .", "feng li from the college of pharmacy , university of traditional chinese medicine ( liaoning , china ) .", "the voucher specimens ( no.20081205 , 20081211 ) have been deposited in liaoning university of tcm .", "zhenqiu zhang ( dept . of chemistry in our institute , liaoning university of tcm ) .", "baicalin and wogonin were purchased from the national institute for the control of pharmaceutical and biological products ( beijing , china ) .", "the solvents used for chromatographic analysis were hplc grade and were purchased from fisher company inc .", "deionized water was prepared in a mill - q academic water purification system ( millipore , bedford , ma , usa ) .", "all the other reagents were of analytical grade and provided by kermel chemical co. ( tianjin , china ) . \n", "apparatus and chromatographic conditions \n  the concentrations of the four flavonoids in plasma were assayed using reverse - phase high performance liquid chromatography ( agilent 1100 series ) equipped with a variable wave length uv detector and pump ( agilent model g1314a vwd ) .", "separation was accomplished on a eclipsel xdb - c18 column ( 250 mm  4.6 mm , 5 m particle size ) .", "the mobile phase was composed of acetonitrile ( a ) : 0.1% phosphoric acid water ( b ) ( 0  10 min , 16 : 84 ; 10  15 min , 22 : 78 ; 15  25 min , 25 : 75 ; 25  30 min , 34 : 64 ; 30  50 min , 42 : 58 ,", "v / v ) at a flow rate of 1.0 ml / min with gradient elution .", "preparation of radix scutellariae and scutellariae - paeoniae couple extracts \n \n radix scutellariae and radix paeoniae alba were mixed in the ratio of 1.5 : 1 g / g and the total weight was 200 g. the mixture was decocted twice by refluxing with 70% ethanol ( 1 : 10 and then 1 : 8 w / v ) for 1 h , and the solution obtained was concentrated to give an extract of 60.5 g. radix scutellariae ( 200 g ) was treated as above to provide an extract of 43.3 g. the dried powder was stored at 4 c before use . for estimation of the administered dose , the concentrations of baicalin , wogonoside , baicalein and wogonin in the extracts and pure baicalin", "the extract powder ( 0.10 g ) was ultrasound with 100 ml 60% methanol for 30 min .", "the contents of bg , b , wg and w were determined to be 17.3 , 7.6 , 2.4 and 1.7 % in scutellariae - paeoniae couple extract and 43.6 , 19.1 , 6.2 and 4.5% in radix scutellariae extract , respectively .", "male sprague  dawley rats , weighing 250 - 280 g , were obtained from the experimental animal department of dalian university ( dalian , china ) .", "animal welfare and experimental procedures were strictly in accordance with the guide for the care and use of laboratory animals ( us national research council , 1996 ) and the related ethics regulations of liaoning university of tcm .", "rats were housed in an air - conditioned animal quarter at a temperature of 22  2 c and a relative humidity of 50  2% .", "the animals were acclimatized to the facilities for five days , and then fasted with free access to water for 24 h prior to each experiment .", "colitis was induced in rats according to the model and method described by morris et al .", "briefly , rats were lightly anesthetized with 10% chloral hydrate solution , and then a medical - grade polyurethane cannula for enteral feeding ( external diameter 2 mm ) was inserted into the anus and the tip was advanced to 8 cm proximal to the anal verge .", "shanghai , china . ) dissolved in 100% ethanol was instilled into the colon through the cannula ( at a dose 80 mg / kg ) .", "following the instillation of the hapten , the rats were maintained in a head - down position for an additional 30 sec to prevent leakage of the intra colonic instillation .", "successful replication of the model of uc was characterized by reduction in activities , apathy and occult blood in stool .", "biosample collection \n  the uc rats were divided into three subgroups randomly , the pure baicalin , radix scutellariae extract and scutellariae - paeoniae couple extract subgroup .", "each group was respectively administrated an oral dose of 200 mg / kg baicalin ( in the form of pure compound or co - administrated mixture , 0.21 g / kg pure baicalin , 0.46 g / kg radix scutellariae extract and 1.16 g / kg scutellariae - paeoniae couple extract ) , all of the compounds were suspended in water and homogenized using ultrasonic technology just prior to dosing .", "blood samples ( 0.5 ml ) were collected into a heparinized tube via the oculi chorioideae vein before drug administration and at 0.083 , 0.25 , 0.5 , 0.75 , 1.0 , 2.0 , 4.0 , 8.0 , 12.0 and 24.0 h after drug administration , and were centrifuged at 4000 rpm for 10 min for the separation of plasma from the red blood cells .", "the supernatant was decanted carefully and was stored at  40 c until analysis . \n", "biosample preparation \n  an aliquot of 15 l arctigenin ( 32.0 m in methanol ) and 10 l hcl solution ( 0.1 mm ) were added into 100 l plasma , and then spiked with methanol 75 l and acetonitrile 100 l by vortex mixing for 5 min .", "an aliquot of 50 l of the supernatant was injected into the hplc system .   known amounts of baicalin , baicalein wogonoside , wogonin and is were added into 100 l of blank plasma to prepare the following series of standards .", "the calibration curves showed good linearity in the range of 0.12331.6 m for baicalin , 0.28873.6 m for wogonoside , 0.1338.50 m for baicalein , 0.0627.9 m for wogonin .", "the extraction recoveries and matrix effects at three quality control ( qc ) concentrations were assayed in sets of six replicates .", "the cvs of the recoveries were less than 20% at low concentration , and less than 15% at medium and high concentrations .", "accuracy and precision of the method were investigated by intra - day and inter - day data via comparing the mean of five replicates of each qc sample .", "the stabilities of baicalin , wogonoside , baicalein and wogonin were determined by analyzing qc samples at three concentrations exposed to encounter during sample storage .", "the corresponding relative errors were less than  6% for samples at three concentrations for each reference standard , respectively .", "results of the stability test showed that under the experimental conditions the samples were stable throughout the testing process .  ", "the plasma concentration vs. time data of baicalin and wogonoside were analyzed by non - compartmental model using the nonlinear least squares regression program winnonlin ( scientific consulting inc . ,", "shanghai , china ) were used to calculate the pharmacokinetic parameters , such as the area under curve ( auc ) , the maximum plasma concentration ( cmax ) and the corresponding time ( tmax ) , the half - life of absorption ( t1/2 ) , etc .", "statistical analysis of pharmacokinetic parameter estimates was performed using a two - tailed nonparametric t - test .", "chemical structures of arctigenin ( a ) , baicalin ( b ) , baicalein ( c ) , wogonoside ( d ) and wogonin ( e ) ."], ["the selectivity of the method was evaluated by analyzing blank plasma samples prior to administration .", "the chromatograms were free of interfering peaks at the retention times of is ( 34.5 min ) and baicalin ( 20.6 min ) , wogonoside ( 25.9 min ) , baicalein ( 31.9 min ) and wogonin ( 40.8 min ) .", "figure 2 showed the representative chromatograms of blank plasma sample ( a ) , blank plasma sample spiked with baicalin , wogonoside , baicalein , wogonin and is ( b ) , blank plasma sample spiked with is ( c ) , plasma sample ( 1.5 h ) after oral administration of scutellariae - paeoniae couple extract ( d ) . under the established chromatographic condition ,", "no interfering peaks were observed at the elution times of baicalin , wogonoside , baicalein , wogonin and the internal standard .  ", "chromatograms of blank plasma sample ( a ) , plasma sample spiked with baicalin , wogonoside , baicalein , wogonin and is ( b ) , plasma sample spiked with is ( c ) , plasma samples 1.5 h after oral administration of scutellariae - paeoniae couple extracts ( d ) . \n", "pharmacokinetics of baicalin and wogonoside after oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts to rats . \n ", "the mean plasma concentration - time profiles and standard deviation in normal rats and uc rats following the administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts are depicted in figure 3 ( a ) and 3 ( b ) .", "pharmacokinetic parameters resulting from non - compartmental analysis are listed in tables 1 and 2 .  ", "the plasma concentration  time profile of baicalin ( bg ) and wogonoside ( wg ) after oral administration of pure baicalin ( 0.21 g / kg ) , radix scutellariae extract ( 0.46 g / kg ) and scutellariae - paeoniae couple extract ( 1.16 g / kg ) to normal ( a ) and ulcerative colitis ( uc ) ( b ) rats pharmacokinetic differences of baicalin after oral administration of pure baicalin ( 0.21 g / kg ) , radix scutellariae extract ( 0.46 g / kg ) and scutellariae - paeoniae couple extract ( 1.16 g / kg of baicalin ) to normal and uc rats mean  sd , n=10;*p < 0.05 vs. normal - rats pharmacokinetic differences of wogonoside after oral administration of pure baicalin ( 0.21 g / kg ) , radix scutellariae extract ( 0.46 g / kg ) and scutellariae - paeoniae couple ( 1.16 g / kg ) to normal and uc rats mean  s.d . , n=10 ; * p<0.05 vs. normal - rats following oral administration of pure baicalin ( purity > 95.5% ) to rats , wogonoside was detected in all rats plasma in addition to baicalin , the plasma concentration was almost equal to that of baicalin .", "the same result was observed after oral administration of radix scutellariae extract ( 200 mg / kg of baicalin , 28 mg / kg wogonoside ) or scutellariae - paeoniae couple extract ( 200 mg / kg of baicalin , 27 mg / kg of wogonoside ) to rats . the present result suggested that baicalin might convert to wogonoside in the rat body .", "zhenyu zhu et al . ( 4 ) reported that they found a peak ( named m ) with a retention time of about 5 min at m / z 461 was similar to wogonoside ( m / z 461 ) after oral administration of xiaochaihu tang and radix scutellariae extract to rats , which was possibly a methylated product of baicalin .", "methylation generally existed during the drug metabolism in - vivo , and due to the multiple hydroxyl groups on the baicalin benzene ring , the combination of a methyl group with a hydroxyl group was easy .", "so , we tentatively concluded that absorbed baicalin could be methylated to wogonoside in - vivo .", "following oral administration of radix scutellariae or scutellariae - paeoniae couple extracts to rats , baicalein and wogonin with very high concentrations were presented in rat tissues ( 28 ) , however , in the present study , it was below the detection limit in all plasma samples collected for all rats .", "these results appeared to be consistent with previous reports ( 14 , 15 , 29 , 30 ) .", "as described in previous studies , baicalin firstly must be hydrolyzed to baicalein by -glucuronidase in - vivo , which was produced by both intestinal bacteria and intestinal epithelial cells ( 3134 ) .", "thus , baicalin can first enter the intestinal wall as baicalein and is then reconverted to baicalin within the intestinal mucosa and thereby is absorbed into the blood .", "therefore , baicalin in plasma may be derived from both baicalin and baicalein in radix scutellariae or scutellariae - paeoniae .", "the doses of baicalin and baicalein in the extracts were summed to produce the sum dose of baicalin and baicalin which was used to calculate dose - normalized cmax and auc0-t . based on the similar chemical structure between wogonoside and baicalin ,", "it is proposed that the transformation between wogonoside and wogonin are similar to that between baicalin and baicalein in the intestinal wall , the sum dose of wogonoside and wogonin was calculated in the same way .", "consequently , we could only detect baicalin and wogonoside , not baicalein and wogonin in plasma .   as shown in figure 3 and table 1 - 2 ,", "the absorption speed of baicalin and wogonoside in the normal pure baicalin group was quicker than those in the normal radix scutellariae and scutellariae - paeoniae couple groups , tmax for baicalin were : ( 0.087  0.02 ) h , ( 0.27  0.03 ) h and ( 0.28  0.03 ) h ; tmax for wogonoside were ( 0.11  0.02 ) h , ( 0.22  0.09 ) h , ( 0.21  0.08 ) h , respectively .", "but tmax of baicalin and wogonoside were little different among the three groups of uc rats .", "the pharmacokinetic profiles differences of baicalin and wogonoside were reflected in auc(0t ) and cmax .  ", "following oral adminstration scutellariae - paeoniae couple extract to rats , cmax and auc(0t ) of baicalin and wogonoside were higher than the other two forms ( p  0.05 ) .", "compared to the radix scutellariae groups , cmax and auc(0t ) of baicalin and wogonoside in the pure baicalin groups were significantly lower ( p  0.05 ) .", "these chemical components might produce certain pharmacological effects and have impact on the pharmacokinetic profiles of baicalin and wogonoside . under the guidance of the theory of tcm ,", "radix paeoniae alba , used as messenger herb and hematinic herb , could guide the bioactive ingredients of radix scutellariae to the proper tissues and exert a harmonizing effect , including the increase of absorption and bioavailability .", "radix paeoniae alba also could increase the absorption of baicalin and wogonoside through increasing the function of spleen and improving the patients digestive system vitality and immune system .", "thus , compared to pure baicalin and radix scutellariae extract , the cmax and auc(0t ) of baicalin and wogonoside increased remarkably ( p < 0.05 ) when radix scutellariae was mix - decocted with radix paeoniae alba , in contrast , the absorption of pure baicalin was the least .", "the present study demonstrated that herb extract could enhance the bioavailability of baicalin and wogonoside in rat plasma , a significant drug - drug interaction may occur and improve the absorption of baicalin and wogonoside when radix paeoniae alba is administered in combination with radix scutellariae ( 35 ) .   either in uc or in normal rats , baicalin and wogonoside demonstrated bimodal phenomenon in rat plasma after oral administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts ( figure 3 ) .", "the first absorption peaks occurred at about 0.15 - 0.27 h and the second was at about 8  12 h. the concentrations of the second peak ( cmax ) and the values of auc(0t ) dose were lower than which of the first peak .", "the ability of rapid absorption of baicalin and wogonoside may result in the rapid appearance of the first peak .", "baicalin and wogonoside had double - site absorption kinetics , and baicalin and wogonoside undergo enteric circulation and enterohepatic circulation in rats after oral dosing , which may be responsible for the second peak .", "but they were always detected and kept their concentration at a certain range in plasma .", "free drug might bind with plasma protein when it reaches a certain concentration ( 19 ) .", "wang et al . ( 36 ) reported that the binding rate of free baicalin with plasma protein could reach 80% .", "it was not able to maintain a high concentration for a long time because the baicalin would enter into the plasma and then bind with plasma protein .", "when the concentration of free baicalin decreased to a certain concentration , the bound baicalin would dissociate . \n", "comparative pharmacokinetics of baicalin and wogonoside after oral administration of pure baicalin , radix scutellariae and scutellariae - paeoniae couple extracts between uc and normal rats \n  from figure 4 and table 1 - 2 , it was noteworthy that uc rats showed better absorption than normal rats , regardless of oral administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts .", "the absorption speeds of baicalin and wogonoside were very rapid , tmax were approximately 0.13 h. tmax showed no statistically significant difference among the three groups of uc rats .", "cmax and auc(0t ) of baicalin and wogonoside in uc rats were greatly higher than those in normal rats ( p < 0.05 ) .", "after oral administration of pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts to rats , auc(0t ) of baicalin were : ( 41.46  0.62 ) , ( 59.12  6.42 ) and ( 104.87  0.86 ) ( g / ml)h in uc groups vs ( 17.77  0.66 ) , ( 28.04  4.06 ) and ( 49.01  4.61 ) ( g / ml)h in normal groups ; auc(0t ) of wogonoside were : ( 40.86  5.31 ) , ( 57.95  1.91 ) and ( 101.31  3.05 ) ( g / ml)h in the uc groups vs ( 14.67  1.93 ) , ( 22.89  3.17 ) and ( 40.35  4.96 ) g / ml / h in the normal groups , respectively .  ", "plasma concentration - time profiles of baicalin ( bg ) ( a ) and wogonoside ( wg ) ( b ) after oral administration of scutellariae - paeoniae couple extract ( 1.16 g / kg ) to ulcerative colitis ( uc ) and normal rats the significantly higher cmax and auc(0t ) of baicalin and wogonoside in uc rats indicated that ulcerative colitis had an influence on the pharmacokinetic characteristics of baicalin and wogonoside in pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts .", "several factors were likely to be involved in the alteration of the pharmacokinetic behavior of baicalin and wogonoside under the colitis condition , including -glucuronidase , udp - glucuronosyltransferase , mrp2 , intestinal or hepatic metabolic enzymes and bile flow rate , etc .", "trinitrobenzene sulfonic acid ( tnbs)-induced uc condition might alter the formation of glucuronide , glutathione , and sulfate conjugates which played an important role in the metabolism of compounds in rats ( 40 - 43 ) .", "these factors may result in a higher exposure of baicalin and wogonoside after oral doses . as known that intestinal flora plays an important role in the absorption and reabsorption of baicalin and wogonoside , we hypothesized that intestinal bacteria in uc rats would be very rich which lead to increased -glucuronidase activity .", "thus , profound intestinal -glucuronidase activity due to the ulcerative colitis condition may have enabled the rapid conversion of baicalin and wogonoside to baicalein and wogonin which have the propensity to be absorbed more completely leading to higher baicalin and wogonoside exposure after oral administration relative to normal rats .  ", "the findings of this study demonstrated that the pharmacokinetic behaviors of baicalin and wogonoside were remarkably altered in uc rats .", "scutellariae - paeoniae couple extract may exert more effectiveness than pure baicalin or radix scutellariae extract .", "it proves the rationality of using scutellariae - paeoniae couple for the treatment of ulcerative colonitis disease ."], ["in summary , the results of the present study highlighted that drug  drug interactions , herb  drug interactions and herb  herb interactions always existed and affected the pharmacokinetic behavior of herbal ingredients . statistically significant differences ( p < 0.05 ) in pharmacokinetic parameters of baicalin and wogonoside including cmax and auc(0t ) were obtained among the rats orally administered pure baicalin , radix scutellariae or scutellariae - paeoniae couple extracts .", "the huge number of active ingredients in tcm made them suitable for multi - target actions .", "baicalin and wogonoside had a better absorption effect , which would improve the therapeutic efficacy .", "we need to take caution when extrapolating pk and exposure data from healthy animals to diseased animals in designing pharmacological studies ."]]}
{"article_id": "PMC4309341", "article_text": ["weekly creatinine clearance ( wccr ) and kt / v are well - known parameters reflecting dialysis \n dose and adequacy , and are applied to adjustment of the dialysis menu in each patient who \n undergoes peritoneal dialysis ( pd ) . however , the limitations of these parameters in \n predicting survival and morbidity have been reported .", "moreover , the collection of urine and peritoneal effluent \n is cumbersome work not only for patients , but also for medical staff .", "estimated ccr ( eccr ) was proposed as an alternative marker for measured total ccr ( tccr ) , \n because it is less expensive and time - consuming .", "but it was also reported that eccr calculated by the \n cockcroft - gault formula substantially overestimated the effect of age on creatinine \n excretion in pd patients .", "estimated glomerular filtration rate ( egfr ) is an alternative marker for measured gfr \n ( mgfr ) , and it might be a possible indicator for prognosis in the general population without \n known cardiovascular disease , diabetes or kidney disease and in ckd patients included in the modification of diet in \n renal disease ( mdrd ) study", ". \n moreover , these prognostic features of egfr might be partially independent of mgfr .", "we reported that egfr was well correlated with tccr in pd patients by cross - sectional \n analysis .", "similar \n correlation of egfr with ccr was reported by khosla , et al . , although the analyzed patients were \n restricted to the population with a difference between two measurements of ccr of less than \n 25% . to clarify the actual utility of egfr in peritoneal dialysis practice", ", we retrospectively \n collected the data from 21 pd patients at ja toride medical center for the period of one \n year .", "patients who underwent pd at ja toride medical center for one year or longer were enrolled \n in the study after their informed consent was obtained .", "pd patients with a treatment period \n of less than a year , and patients who underwent combined pd and hemodialysis ( hd ) therapy \n were excluded . to measure renal ccr ,", "urine was collected for 24 hours ( from the second voiding of the day \n before visiting the hospital to the first voiding on the day of visiting the hospital ) .", "pd \n effluent was also collected for 24 hours on the day before visiting the hospital to measure \n peritoneal ccr .", "the sum of the peritoneal and renal ccr was defined as the measured tccr . \n", "the serum , urine , and effluent cr were measured by the established enzyme method . the formula proposed by the japanese society of nephrology", "was applied for calculation of egfr as follows : egfr ( ml / min/1.73 m ) = 194  serum cr  age \n (  0.739 in females ) protein catabolic rate ( pcr ) was calculated by the formula proposed by teehan as follows : pcr ( g / day ) = 6.25  ( urinary urea excretion + peritoneal urea excretion + 1.39 + 0.15 + \n 0.031  body weight in kg ) .", "pcr was normalized ( divided ) by the measured body weight ( bw ) and described as npcr \n ( g / kg / day ) . according to the guidelines of the japanese ministry of health , labour , and welfare , \n informed consent of the patients", "the study protocol was approved by the ethics \n committee of ja toride medical center .", "the data are shown as means  sd , unless otherwise specified . to observe the correlation of \n two sets of data ,", "the pearson s correlation coefficients were calculated using excel 2007 \n ( microsoft , redmond , wa , u.s.a . ) .", "as table 1table 1characteristics of the studied patientsmen / women16 / 5age ( years)66.6  12.6 ( 4695)capd / apd13 / 8cause of ckddm7cgn7ns4others3treatment period of pd in months45.1  32.4 ( 14118)samples in the data analysis1145.4  1.5 ( 27 ) per patient shows , 21 patients met the inclusion criteria and were included in this study . \n", "the patients comprised 15 men and 6 women with an average age of 66.6  12.6 years ( 4695 \n years old ) , and their average pd period was 45.1  32.4 months ( 14118 months ) .", "their causes \n of ckd were diabetes mellitus ( dm ) in seven patients , chronic glomerulonephritis ( cgn ) in \n seven patients , nephrosclerosis ( ns ) in four patients , and others in three patients . \n", "continuous ambulatory pd ( capd ) was undertaken in 13 patients , and the other eight patients \n received automated pd ( apd ) . during the period of one year from november 2010 to october \n 2011 , 114 data sets of egfr and tccr from the 21 patients were suitable for data analysis . \n according to the analysis , egfr and tccr were correlated ( r. = 0.435 in figure 1figure 1egfr and tccr .", "the open circles in the plotted data were obtained from one patient \n who denied the habitual intake of creatine supplements , such as creatine \n monophosphate , but poorly complied with the diet therapy . ) . however , their correlation was less than that of a previous cross - sectional study \n ( r. = 0.836 ) .", "unexpectedly \n lower egfr levels were observed in one patient ( shown as open circles in figure 1 ) . repeated counseling of the patient revealed \n that his diet fluctuated . as figure 2figure 2tccr", "- egfr difference and npcr ( a ) and total cr excretion and npcr ( b ) in one \n patient who showed fluctuating diet intake .", "more detailed data from the patient , \n exhibited as open circles in figure 1 are \n shown .", "variations in the difference between egfr and tccr were correlated with both \n total cr excretion and npcr .", "shows , tccr - egfr difference and total cr excretion were actually correlated with \n npcr in this patient .", "similar correlations were observed for the difference between egfr and \n tccr with total cr excretion ( r. = 0.821 in figure \n 3figure 3tccr - egfr difference and total cr excretion .", "differences between egfr and tccr were \n well correlated with total cr excretion in total samples . ) and npcr ( r. = 0.636 in figure 4figure 4tccr - egfr and npcr .", "differences between egfr and tccr were correlated with npcr in \n total samples . ) in all the data .", "total cr excretion and npcr were also correlated with each other \n ( r. = 0.516 in figure 5figure 5total cr excretion and npcr .", "total cr excretion and npcr were also correlated with \n each other in total samples . ) .", "the open circles in the plotted data were obtained from one patient \n who denied the habitual intake of creatine supplements , such as creatine \n monophosphate , but poorly complied with the diet therapy . tccr - egfr difference and npcr ( a ) and total cr excretion and npcr ( b ) in one \n patient who showed fluctuating diet intake . more detailed data from the patient , \n exhibited as open circles in figure 1 are \n shown .", "variations in the difference between egfr and tccr were correlated with both \n total cr excretion and npcr . tccr - egfr difference and total cr excretion .", "differences between egfr and tccr were \n well correlated with total cr excretion in total samples .", "total cr excretion and npcr were also correlated with \n each other in total samples .", "this retrospective study with the data collected for a year from 21 pd patients showed that \n the correlation of egfr with tccr was less than that of a previous cross - sectional \n study .", "the difference \n between tccr and egfr was mostly dependent on total cr excretion , as reported in \n pre - dialysis patients , but \n this has never been reported in pd patients . moreover , total cr excretion fluctuated \n according to the npcr .", "consequently , protein intake estimated by npcr may affect the \n difference between tccr and egfr . because creatinine is homogeneously distributed in the body water , serum creatinine level \n is determined by the balance of several factors , such as oral intake , generation in the \n muscle , intrinsic degradation ( metabolic clearance ) and clearance by peritoneal dialysis and \n the kidney ( figure 6figure 6considerable factors determining serum cr levels .", "meats ( per 100 g ) generally contain \n 400460 mg of creatine and creatinine ( see discussion for details ) . ) .", "the amount of creatinine obtained by oral intake is mainly dependent on the amount \n of meat , which generally contains 400450 mg of creatinine and creatine per 100 g , or more \n precisely , 1 mg of creatinine and 23 mg of creatine per 1 g of meat protein . actually , total cr excretion was \n well correlated with npcr in this study ( figure \n 5 ) .", "meanwhile , the cr generation rate in the muscle is relatively stable , because it \n is thought to be related to the muscle mass and activity .", "mainly \n affects the variance in urinary creatinine excretion , especially in the case of preserved \n residual renal function , which has a wider ranging capacity for compensation .", "consequently , \n the serum cr concentration and its calculation product , egfr , are supposed to be more stable \n parameters than ccr by diminishing the preserved capacity in small solutes clearance of the \n kidney .", "the increased difference between tccr and egfr in the higher pcr population could be \n explained by a similar reason , because pcr was positively correlated with total cr \n excretion .", "hence , serum cr and its calculation product , egfr , may be superior to ccr because \n they are less dependent on diet , unlike ccr .", "( per 100 g ) generally contain \n 400460 mg of creatine and creatinine ( see discussion for details ) .", "recently , it has been reported that egfr had a prognostic value for ckd - related \n complications , kidney failure , cardiovascular risk \n factors and \n mortality .", "it is possible \n that egfr has a similar independency and distinct feature as a predictive indicator even in \n pd patients .", "hence , the obtained value can fluctuate \n as a result of several factors , such as patient condition , diet and drugs , which means that \n mgfr should not always be considered a representative value or ", "gold standard  of renal \n function , or rather the \n preserved capacity of the kidney to maintain the serum level of creatinine within a certain \n range under conditions of fluctuating intake may be a more reasonable index for actual \n kidney function .", "although the peritoneal clearance of small solutes represented by \n creatinine should be taken into consideration in pd patients , egfr derived from serum creatinine , age and \n gender may diminish the compensatory fluctuation of small solute clearance in the kidney , \n and provide a more stable level in each pd patient .", "this feature may be valuable in routine \n clinical practice , and it is worth investigating the significance of egfr as a possible \n prognostic factor in pd patients , as reported in pre - dialysis patients , \n .", "this study has some limitations . because the study design was a retrospective analysis at a \n single center , the patient characteristics were divergent with respect to pd modalities , \n exposure periods , residual renal function and adherence to diet therapy .", "in addition , the \n study population was small , and equal amounts of data were not collected for each patient . \n", "therefore , differences between individual patients and the entire study population could not \n be distinguished from the obtained data .", "however , fluctuating cr excretion was certainly \n observed not only in data from individual patient but in all collected data , as shown in \n figures 2 , 3 , 4 , 5 .", "this study revealed the practical profile of egfr , distinct from tccr , as a more stable \n marker for small solutes clearance by dialysis and the kidney .", "a more extended and \n longitudinal study will be needed to verify the actual value of egfr in pd practice ."], "abstract_text": ["<S> objective : the usefulness of estimated glomerular filtration rate may not be \n restricted to pre - dialysis patients , since we reported that estimated glomerular \n filtration rate was well correlated with measured total creatinine clearance in peritoneal \n dialysis patients . to clarify the clinical usefulness of estimated glomerular filtration \n rate as a parameter for peritoneal dialysis adequacy , </S>", "<S> we retrospectively surveyed \n estimated glomerular filtration rate and total creatinine clearance in peritoneal dialysis \n patients treated at ja toride medical center.patients and methods : a total of 114 data sets of estimated glomerular \n filtration rate and total creatinine clearance from 21 pd patients treated at ja toride \n medical center were collected from november 2010 to october 2011 . </S>", "<S> the patients consisted \n of 15 men and six women with an average age of 66.6  12.6 years ( 4695 years old ) . </S>", "<S> the \n average number of samples was 5.4  1.5 ( 2 to 7 ) per patient.results : the collected data showed less correlation of estimated glomerular \n filtration rate and total creatinine clearance ( r. = 0.435 ) than that of a previous \n cross - sectional study ( r. = 0.836 ) . </S>", "<S> as reported in pre - dialysis patients , the differences \n between estimated glomerular filtration rate and total creatinine clearance were \n correlated with total creatinine excretion in urine and pd effluent ( r. = 0.821 ) . </S>", "<S> the \n differences were also correlated with normalized protein catabolic rate , which was one of \n the main determinant factors for total creatinine excretion ( r. = 0.636 ) . </S>", "<S> a similar \n tendency was apparently observed in one patient with poor compliance to diet therapy and \n fluctuating dietary intake . from the analysis of these data , serum creatinine seemed to \n fluctuate less possibly due to compensatory capacity of the residual renal function in \n small solute clearance.conclusions : consequently , estimated glomerular filtration rate was turned \n out to be a more stable parameter than total creatinine clearance , which might be a \n desirable feature in long - term follow - up of peritoneal dialysis patients . </S>"], "labels": null, "section_names": ["Introduction", "Patients and Methods", "Results", "Discussion", "Conclusion"], "sections": [["weekly creatinine clearance ( wccr ) and kt / v are well - known parameters reflecting dialysis \n dose and adequacy , and are applied to adjustment of the dialysis menu in each patient who \n undergoes peritoneal dialysis ( pd ) . however , the limitations of these parameters in \n predicting survival and morbidity have been reported .", "moreover , the collection of urine and peritoneal effluent \n is cumbersome work not only for patients , but also for medical staff .", "estimated ccr ( eccr ) was proposed as an alternative marker for measured total ccr ( tccr ) , \n because it is less expensive and time - consuming .", "but it was also reported that eccr calculated by the \n cockcroft - gault formula substantially overestimated the effect of age on creatinine \n excretion in pd patients .", "estimated glomerular filtration rate ( egfr ) is an alternative marker for measured gfr \n ( mgfr ) , and it might be a possible indicator for prognosis in the general population without \n known cardiovascular disease , diabetes or kidney disease and in ckd patients included in the modification of diet in \n renal disease ( mdrd ) study", ". \n moreover , these prognostic features of egfr might be partially independent of mgfr .", "we reported that egfr was well correlated with tccr in pd patients by cross - sectional \n analysis .", "similar \n correlation of egfr with ccr was reported by khosla , et al . , although the analyzed patients were \n restricted to the population with a difference between two measurements of ccr of less than \n 25% . to clarify the actual utility of egfr in peritoneal dialysis practice", ", we retrospectively \n collected the data from 21 pd patients at ja toride medical center for the period of one \n year ."], ["patients who underwent pd at ja toride medical center for one year or longer were enrolled \n in the study after their informed consent was obtained .", "pd patients with a treatment period \n of less than a year , and patients who underwent combined pd and hemodialysis ( hd ) therapy \n were excluded . to measure renal ccr ,", "urine was collected for 24 hours ( from the second voiding of the day \n before visiting the hospital to the first voiding on the day of visiting the hospital ) .", "pd \n effluent was also collected for 24 hours on the day before visiting the hospital to measure \n peritoneal ccr .", "the sum of the peritoneal and renal ccr was defined as the measured tccr . \n", "the serum , urine , and effluent cr were measured by the established enzyme method . the formula proposed by the japanese society of nephrology", "was applied for calculation of egfr as follows : egfr ( ml / min/1.73 m ) = 194  serum cr  age \n (  0.739 in females ) protein catabolic rate ( pcr ) was calculated by the formula proposed by teehan as follows : pcr ( g / day ) = 6.25  ( urinary urea excretion + peritoneal urea excretion + 1.39 + 0.15 + \n 0.031  body weight in kg ) .", "pcr was normalized ( divided ) by the measured body weight ( bw ) and described as npcr \n ( g / kg / day ) . according to the guidelines of the japanese ministry of health , labour , and welfare , \n informed consent of the patients", "the study protocol was approved by the ethics \n committee of ja toride medical center .", "the data are shown as means  sd , unless otherwise specified . to observe the correlation of \n two sets of data ,", "the pearson s correlation coefficients were calculated using excel 2007 \n ( microsoft , redmond , wa , u.s.a . ) ."], ["as table 1table 1characteristics of the studied patientsmen / women16 / 5age ( years)66.6  12.6 ( 4695)capd / apd13 / 8cause of ckddm7cgn7ns4others3treatment period of pd in months45.1  32.4 ( 14118)samples in the data analysis1145.4  1.5 ( 27 ) per patient shows , 21 patients met the inclusion criteria and were included in this study . \n", "the patients comprised 15 men and 6 women with an average age of 66.6  12.6 years ( 4695 \n years old ) , and their average pd period was 45.1  32.4 months ( 14118 months ) .", "their causes \n of ckd were diabetes mellitus ( dm ) in seven patients , chronic glomerulonephritis ( cgn ) in \n seven patients , nephrosclerosis ( ns ) in four patients , and others in three patients . \n", "continuous ambulatory pd ( capd ) was undertaken in 13 patients , and the other eight patients \n received automated pd ( apd ) . during the period of one year from november 2010 to october \n 2011 , 114 data sets of egfr and tccr from the 21 patients were suitable for data analysis . \n according to the analysis , egfr and tccr were correlated ( r. = 0.435 in figure 1figure 1egfr and tccr .", "the open circles in the plotted data were obtained from one patient \n who denied the habitual intake of creatine supplements , such as creatine \n monophosphate , but poorly complied with the diet therapy . ) . however , their correlation was less than that of a previous cross - sectional study \n ( r. = 0.836 ) .", "unexpectedly \n lower egfr levels were observed in one patient ( shown as open circles in figure 1 ) . repeated counseling of the patient revealed \n that his diet fluctuated . as figure 2figure 2tccr", "- egfr difference and npcr ( a ) and total cr excretion and npcr ( b ) in one \n patient who showed fluctuating diet intake .", "more detailed data from the patient , \n exhibited as open circles in figure 1 are \n shown .", "variations in the difference between egfr and tccr were correlated with both \n total cr excretion and npcr .", "shows , tccr - egfr difference and total cr excretion were actually correlated with \n npcr in this patient .", "similar correlations were observed for the difference between egfr and \n tccr with total cr excretion ( r. = 0.821 in figure \n 3figure 3tccr - egfr difference and total cr excretion .", "differences between egfr and tccr were \n well correlated with total cr excretion in total samples . ) and npcr ( r. = 0.636 in figure 4figure 4tccr - egfr and npcr .", "differences between egfr and tccr were correlated with npcr in \n total samples . ) in all the data .", "total cr excretion and npcr were also correlated with each other \n ( r. = 0.516 in figure 5figure 5total cr excretion and npcr .", "total cr excretion and npcr were also correlated with \n each other in total samples . ) .", "the open circles in the plotted data were obtained from one patient \n who denied the habitual intake of creatine supplements , such as creatine \n monophosphate , but poorly complied with the diet therapy . tccr - egfr difference and npcr ( a ) and total cr excretion and npcr ( b ) in one \n patient who showed fluctuating diet intake . more detailed data from the patient , \n exhibited as open circles in figure 1 are \n shown .", "variations in the difference between egfr and tccr were correlated with both \n total cr excretion and npcr . tccr - egfr difference and total cr excretion .", "differences between egfr and tccr were \n well correlated with total cr excretion in total samples .", "total cr excretion and npcr were also correlated with \n each other in total samples ."], ["this retrospective study with the data collected for a year from 21 pd patients showed that \n the correlation of egfr with tccr was less than that of a previous cross - sectional \n study .", "the difference \n between tccr and egfr was mostly dependent on total cr excretion , as reported in \n pre - dialysis patients , but \n this has never been reported in pd patients . moreover , total cr excretion fluctuated \n according to the npcr .", "consequently , protein intake estimated by npcr may affect the \n difference between tccr and egfr . because creatinine is homogeneously distributed in the body water , serum creatinine level \n is determined by the balance of several factors , such as oral intake , generation in the \n muscle , intrinsic degradation ( metabolic clearance ) and clearance by peritoneal dialysis and \n the kidney ( figure 6figure 6considerable factors determining serum cr levels .", "meats ( per 100 g ) generally contain \n 400460 mg of creatine and creatinine ( see discussion for details ) . ) .", "the amount of creatinine obtained by oral intake is mainly dependent on the amount \n of meat , which generally contains 400450 mg of creatinine and creatine per 100 g , or more \n precisely , 1 mg of creatinine and 23 mg of creatine per 1 g of meat protein . actually , total cr excretion was \n well correlated with npcr in this study ( figure \n 5 ) .", "meanwhile , the cr generation rate in the muscle is relatively stable , because it \n is thought to be related to the muscle mass and activity .", "mainly \n affects the variance in urinary creatinine excretion , especially in the case of preserved \n residual renal function , which has a wider ranging capacity for compensation .", "consequently , \n the serum cr concentration and its calculation product , egfr , are supposed to be more stable \n parameters than ccr by diminishing the preserved capacity in small solutes clearance of the \n kidney .", "the increased difference between tccr and egfr in the higher pcr population could be \n explained by a similar reason , because pcr was positively correlated with total cr \n excretion .", "hence , serum cr and its calculation product , egfr , may be superior to ccr because \n they are less dependent on diet , unlike ccr .", "( per 100 g ) generally contain \n 400460 mg of creatine and creatinine ( see discussion for details ) .", "recently , it has been reported that egfr had a prognostic value for ckd - related \n complications , kidney failure , cardiovascular risk \n factors and \n mortality .", "it is possible \n that egfr has a similar independency and distinct feature as a predictive indicator even in \n pd patients .", "hence , the obtained value can fluctuate \n as a result of several factors , such as patient condition , diet and drugs , which means that \n mgfr should not always be considered a representative value or ", "gold standard  of renal \n function , or rather the \n preserved capacity of the kidney to maintain the serum level of creatinine within a certain \n range under conditions of fluctuating intake may be a more reasonable index for actual \n kidney function .", "although the peritoneal clearance of small solutes represented by \n creatinine should be taken into consideration in pd patients , egfr derived from serum creatinine , age and \n gender may diminish the compensatory fluctuation of small solute clearance in the kidney , \n and provide a more stable level in each pd patient .", "this feature may be valuable in routine \n clinical practice , and it is worth investigating the significance of egfr as a possible \n prognostic factor in pd patients , as reported in pre - dialysis patients , \n .", "this study has some limitations . because the study design was a retrospective analysis at a \n single center , the patient characteristics were divergent with respect to pd modalities , \n exposure periods , residual renal function and adherence to diet therapy .", "in addition , the \n study population was small , and equal amounts of data were not collected for each patient . \n", "therefore , differences between individual patients and the entire study population could not \n be distinguished from the obtained data .", "however , fluctuating cr excretion was certainly \n observed not only in data from individual patient but in all collected data , as shown in \n figures 2 , 3 , 4 , 5 ."], ["this study revealed the practical profile of egfr , distinct from tccr , as a more stable \n marker for small solutes clearance by dialysis and the kidney .", "a more extended and \n longitudinal study will be needed to verify the actual value of egfr in pd practice ."]]}
{"article_id": "PMC4417567", "article_text": ["mesenchymal stem cells ( mscs ) , also referred to as multipotent mesenchymal stromal cells , have been a focus of recent research , partially because they are an extraordinary model for investigating the biological mechanisms that allow a cellular population to generate diverse cell types and because they are a potential tool in cellular therapies for several clinical applications .", "mscs can differentiate into mesenchymal lineages and secrete cytokines and growth factors with paracrine effects that favor the regeneration of damaged tissues [ 1 , 2 ] .", "several studies have demonstrated that mscs possess an immunoregulatory function in vitro and in vivo and that this property suggests clinical applications in the regulation of immunocompetent cell responses [ 2 , 3 ] .", "this review addresses current knowledge of the biological aspects involved in msc immunoregulatory capacity and the clinical focus of these characteristics that allows these cells to be used in the treatment of several diseases with an immune component involved .", "this review culminates with a clinical description of the diseases treated with mscs as a component of cell therapy procedures .", "mscs are adult stem cells that are initially isolated from bone marrow ( bm )   and can generate stromal bm components , such as adipocytes , reticular cells , and osteoblasts , whereas in conjunction with additional cellular components , mscs maintain hematopoiesis .", "mscs proliferate in vitro as adherent , colony - forming cells with a high capacity for self - renewal and proliferation [ 4 , 5 ] . because there is no definite marker of mscs ,", "the international society for cellular therapy has established minimum criteria that these in vitro cell populations must fulfill and certain characteristics to be considered mscs .", "the cells must be positive for cd105 , cd73 , and cd90 , express low levels of mhc - i , and be negative for mhc - ii , cd11b , cd14 , cd34 , cd45 , and cd31 .", "additionally , these cells must be capable of differentiation into osteoblasts , adipocytes , and chondroblasts in vitro [ 5 , 6 ] .", "mscs have been isolated from multiple tissues : skeletal muscle , adipose tissue ( at ) , synovial membranes , dental pulp , periodontal ligaments , cervical tissue , menstrual blood , wharton 's jelly ( wj ) , umbilical cord ( uc ) , umbilical cord blood ( ucb ) , amniotic fluid , placenta ( pl ) , and fetal tissues such as blood , liver , and bm [ 710 ] . in most cases ,", "isolated mscs are heterogeneous in proliferation and differentiation , although all express the characteristic msc marker profile .", "mscs cultivated in vitro possess three biological properties that qualify them for use in cellular therapy : ( a ) broad potential of differentiation , ( b ) secretion of trophic factors that favor tissue remodeling , and ( c ) immunoregulatory properties .", "furthermore , mscs differentiate into different mesodermal lineages ( adipocytes , chondrocytes , osteocytes , fibroblasts , and myocytes ) .", "because of this potential for differentiation , mscs were initially used in the treatment of imperfect osteogenesis   and myocardial damage .", "the benefits observed in these initial cell therapy protocols were thought to be the result of osteogenic and myogenic differentiation .", "the current understanding is that , in addition to diverse mesodermal differentiation capacity , msc benefits arise primarily from the secretion of trophic factors and immunoregulatory capacity [ 13 ] .", "mscs profoundly affect immune response through their interactions with the cellular components of the innate ( natural killer cells ( nk ) ) and adaptive ( dendritic cells ( dcs ) , b lymphocytes , and t lymphocytes ) immune system .", "msc immunoregulation can occur through cellular contact and/or the secretion of diverse factors [ 1317 ] .", "because of these properties , mscs can prevent the inappropriate activation of t lymphocytes and generate a tolerogenic environment during wound repair or stop an immune response during healing , thus contributing to the maintenance of immune homeostasis [ 2 , 3 ] .", "below , we describe the immunoregulatory effects of mscs on specific immune cells with special emphasis on the effect of mscs on t lymphocytes because of their role as effector cells in many diseases with an immune component .", "the phases in which t cells are vulnerable to msc immunoregulation , recognizing from a biological perspective that there are no obvious limits between phases , are described below .  \n", "t lymphocytes express and secrete molecules characteristic of this phase , such as cd25 , cd69 , cd38 , cytotoxic t lymphocyte antigen-4 ( ctla-4 ) , and human leukocyte antigen - dr ( hla - dr ) and in addition the cytokines interferon- ( ifn ) , tumor necrosis factor ( tnf ) , and il-2 , among others . currently , there are contradictory results regarding the effect of mscs on t lymphocyte activation .", "some studies have observed that bm - mscs prevent the expression of the early activation markers cd25 and cd69 in t cells stimulated with phytohemagglutinin ( pha ) [ 19 , 20 ] , whereas other studies describe no effect by bm - mscs on the expression of these molecules [ 16 , 21 ] .", "such contradictory results may be because of differences in the population of t lymphocytes studied . with this understanding , the activation of peripheral blood mononuclear cells ( pbmc ) with pha in the presence of bm - mscs results in lower numbers of cd4 and cd8 that express cd25 , cd38 , and cd69 . employing the identical model ,", "contradictory results report that the activation of pbmc with antibodies in the presence of bm - mscs does not modify the expression of cd25 or cd69 in cd4 and cd8 populations .", "similarly , no change was reported in the expression of cd25 , cd69 , or ctla-4 in studies of populations enriched with cd4 and cd8 lymphocytes activated by alloantigens . however , a recent study using populations enriched with cd3 t lymphocytes activated with anti - cd2/cd3/cd28 in the presence of bm - mscs showed an increase in the expression of cd69 in cd4 and cd8 t lymphocyte populations .", "the effects of mscs on the secretion of cytokines by activated t lymphocytes are also described by contradictory results . some studies", "have demonstrated that the presence of mscs diminishes [ 14 , 21 ] or increases [ 23 , 24 ] significantly the secretion of ifn by activated t lymphocytes .", "nonetheless , it has been described that the effects of mscs on ifn secretion depend on the source of the lymphocyte population studied . in this study", ", the authors demonstrated that the activation of cd3 t lymphocytes with anti - cd3/cd28 in the presence of mscs from adipose tissue resulted in an increase in ifn , which was an effect that was not observed when pbmc were activated with the identical stimulus .", "our laboratory recently demonstrated that the activation of cd3 t cells with antibodies in the presence of bm - mscs , ucb - mscs , and pl - mscs also resulted in an increase in ifn in cocultures .", "this observed effect might be related to the generation of ifn-producing t regulatory cells ( tregs ) .  \n", "the effect of mscs on the proliferation of t lymphocytes is independent of the activation method .", "the first studies to analyze the effects of bm - mscs on the proliferation of t lymphocytes used irradiated mscs that were cocultivated with alloantigen - stimulated pbmc .", "these studies reported that mscs inhibited t - cell proliferation in a dose - dependent fashion [ 13 , 16 , 17 , 19 ] .", "however , in addition to inhibiting alloantigen - induced proliferation , mscs can inhibit proliferation induced by polyclonal activators such as pha [ 14 , 26 ] or anti - cd3/cd28 [ 21 , 23 , 24 ] .", "the immunosuppressive effects of mscs have been analyzed using total populations of pbmc and populations enriched in cd3 , cd4 , or cd8 lymphocytes .", "every case has demonstrated the capacity of mscs to diminish proliferation [ 13 , 16 , 17 , 19 , 21 , 23 , 24 ] .", "immunoregulation has been shown to be independent of the induction of apoptosis [ 16 , 27 ] and is performed through mechanisms dependent and independent of cellular contact . among", "the secreted factors identified are transforming growth factor beta 1 ( tgf1 ) , hepatocyte growth factor ( hgf ) , indoleamine-2,3-dioxygenase ( ido ) , prostaglandin e2 ( pge2 ) , il-10 , and hla - g5 [ 13 , 17 , 28 ] , whereas programmed death - ligand 1 ( pd - l1 ) and hla - g1 are involved in contact - dependent mechanisms ( figure 1 ) [ 2830 ] . whether direct contact between mscs and t lymphocytes is necessary for the inhibition of t - cell proliferation remains controversial .", "some authors have suggested that mscs act via an immunosuppressive mechanism independent of cell - to - cell contact [ 31 , 32 ] , whereas others have indicated that contact is required for efficient immunoregulation [ 24 , 2830 , 33 , 34 ] .", "however , the mechanism of msc immunoregulation appears to depend on cellular populations , mode of activation , and the presence or absence of cell - to - cell contact [ 23 , 32 ] .  \n", "( 3 ) differentiation and effector function . upon activation by the presence of pathogens or signs of damage ,", "helper t cells cd4 ( th0 ) differentiate into one of the following subtypes , depending on the t - cell microenvironment : th1 , th2 , th17 , or tregs .", "each population is characterized by the secretion of a set of cytokines whose function is essential to eliminate pathogens within the organism , resolve inflammation , and maintain immune homeostasis .", "several studies have suggested that mscs modulate the differentiation , function , and balance of these subpopulations and foster the development of an anti - inflammatory immune response ( figure 1 ) [ 14 , 28 , 35 , 36 ] .", "the activation of nave t cells ( cd45ra ) in favorable conditions for the induction of th1 or th2 and in the presence of mscs results in the inhibition of ifn secretion by th1 cells and the increase of il-4 secretion by th2 cells .", "furthermore , mscs inhibit the production of proinflammatory cytokines il-17 , il-22 , ifn , and tnf and the differentiation of nave cd4 lymphocytes to th17 .", "additionally , mscs promote the secretion of il-10 and the expression of the foxp3 transcription factor , thus suggesting differentiation toward tregs ( figure 1 ) .", "similarly , in a murine model , the presence of mscs in the th1 and th17 differentiation processes favors differentiation to cd4cd25foxp3 tregs .", "this effect was not observed when mscs were added to cultures of mature th1 or th17 populations .", "these results indicate that mscs affect the differentiation and function of inflammatory t lymphocyte populations in their capacity to produce proinflammatory cytokines and also in the induction of a tregs phenotype .", "several studies have described the role of mscs in the induction of distinct tregs populations [ 14 , 28 , 3740 ] . in an initial study analyzing the participation of bm - mscs in the differentiation of tregs populations , maccario et al . observed that the presence of allogeneic and autologous mscs , with respect to the responder t lymphocyte population in mixed lymphocyte culture ( cml ) , induced a significant increase in the cd4cd25 t lymphocyte population .", "however , only allogeneic mscs favored an increase in cd4cd25ctla-4 populations . in the same year , a different study observed that pbmc activation by il-2 in the presence of mscs increased the proportion of cd4cd25 t lymphocytes .", "subsequently , prevosto et al . showed that pbmc in coculture with bm - mscs generated a population of cells that could inhibit t lymphocyte proliferation induced by alloantigens or polyclonal activators ( anti - cd3 or pha ) , and this effect required cell - to - cell contact .", "the authors observed that the increase in foxp3 mrna expression occurred only in the tregs populations derived from cd4 lymphocytes .", "mscs can induce and maintain the function and phenotype of tregs derived from cd3 , cd3cd45ro , or cd3cd45ra t lymphocyte populations . beginning with a cd3 population ,", "the authors also demonstrated that the presence of bm - mscs maintained foxp3 expression in tregs .", "furthermore , mscs can induce regulatory t type 1- ( tr1- ) like cells characterized by ifn and il-10 secretion . through an in vivo transplant - induced arteriosclerosis model ( obstructed arteries ) , jui et al .", "demonstrated that the local administration of bm - mscs could prevent this pathology through a local increase in ifn and il-10 . in a subsequent in vitro study , the identical laboratory demonstrated that bm - mscs favored the generation of tr1 lymphocytes with an il-10ifncd4 phenotype mediated by pge2 and ido ( figure 1 ) .", "msc participation in t lymphocyte subpopulation equilibrium has also been observed in human in vivo studies .", "patients who have received mscs for the treatment of gvhd show subsequent increases of cd4cd25foxp3 and tr1 populations and decreases of th17 .", "similarly , the administration of mscs to patients with systemic lupus erythematous induced an increase in cd4cd25foxp3 tregs in the peripheral blood .", "increases in this tregs population have also been observed in kidney transplant patients , which were transplanted with autologous mscs .", "these cells are derived from bm - cd34 cells in vivo and from monocytes stimulated with il-4 and granulocyte macrophage colony - stimulating factor ( gm - csf ) in vitro .", "the primary function of dcs is to process and present antigens to virgin and memory t cells , although they also interact with other immune components such as b lymphocytes and nk cells .", "the individual dcs must mature to initiate an appropriate immune response , and during the maturation process , dcs increase the membrane expression of mhc - ii and t - cell costimulatory molecules cd80 and cd86 .", "mscs can significantly reduce monocyte differentiation into dcs , affecting the upregulation of cd1a , cd40 , cd80 , cd86 , and hla - dr ( figure 2 ) [ 15 , 37 , 4648 ] .", "this reduction is performed through the secretion of factors [ 15 , 46 ] and is a reversible process because these monocytes then differentiate normally at the removal of mscs . when immature dcs ( idcs ) derived from monocytes - msc cocultures were activated with lipopolysaccharide ( lps ) to induce their final differentiation , they expressed lower levels of the maturation marker cd83 and costimulatory molecules cd80 and cd86 .", "however , spaggiari et al . showed that mscs do not affect direct lps - induced maturation of dcs in cocultures , because there was no change in cd80 , cd83 , and cd86 expression .", "these results suggest that mscs exert a strong inhibitory effect on the differentiation process from monocytes to idcs but not on the lps - induced maturation of idcs to mature dcs ( mdcs ) .", "in addition , mdcs cocultured with mscs show a diminished expression of hla - dr , cd1a , cd80 , and cd86 , thus suggesting that mscs may push mdcs toward an immature state with a reduced stimulatory capacity ( figure 2 )", ". additionally , mscs affect the secretion of several cytokines that are key to dcs maturation .", "aggarwal and pittenger observed that mscs inhibit the secretion of tnf by dcs activated by lps .", "the inhibition of tnf secretion by dcs inhibits their maturation , migration to the lymph nodes , and their capacity to stimulate alloreactive t lymphocytes , because of the alteration in the expression of several receptors that are necessary to capture and process antigens .", "mscs also inhibit the dcs secretion of il-12 [ 15 , 47 , 48 ] .", "the insufficient production of il-12 is associated with the induction of t cell anergy and tolerance [ 15 , 26 , 47 ] .", "human bm - mscs that act through notch can induce the differentiation of cd34 hematopoietic progenitors into a population of regulatory dcs with specific properties : ( 1 ) the expression of high levels of il-10 mrna and low expression of il-2 mrna ; ( 2 ) the capacity to inhibit alloreactive t - cell proliferation and function ; and ( 3 ) the capacity to induce tregs differentiation characterized by expression of foxp3 and tgf1 mrna ( figure 2 ) .", "nk cells are important in innate immunity and participate in the body 's defenses against infections and cancer .", "nk cells perform their effector function through the secretion of cytokines , such as ifn , tnf , and gm - csf , and possess cytotoxic activity both spontaneous and antibody - dependent .", "nk function is regulated by the equilibrium of signals transmitted by activator and inhibitor receptors that interact with specific hla molecules on target cells .", "thus , hla - class i negative or hla - class i - mismatched cells represent potential targets of nk cells [ 27 , 50 ] .", "mscs affect the phenotype , proliferation , cytotoxic potential , and cytokine secretion of nk cells ( figure 2 ) .", "when activated by il-2 , nk cells secrete ifn , but when activated in the presence of mscs , ifn secretion significantly decreases . furthermore ,", "nk cells activated by il-2 and alloantigens in the presence of mscs show diminished proliferation and lytic activity [ 16 , 51 ] .", "il-15 is another cytokine that promotes the proliferation , survival , and effector function of nk cells , but through factor secretion , mscs can inhibit il-15 induced proliferation .", "however , mscs and nk cell contact are necessary to inhibit nk cytotoxicity in tumor cell lineages .", "few studies have analyzed the effects of mscs on b lymphocytes ; however , mscs diminish b - cell proliferation by cell cycle arrest in the g0/g1 phase and not by inducing apoptosis .", "a recent study demonstrated that effect of msc on b lymphocytes proliferation with cpg is not direct and requires presence of cd3 t cells .", "mscs can also affect b - cell differentiation because igm , igg , and iga production are diminished [ 5356 ] .", "furthermore , mscs modify the chemotactic properties of b lymphocytes , because expression changes in their chemokine receptors including cxcr4 , cxcr5 , and ccr7 were induced by mscs .", "the first molecules described in the msc - mediated immunoregulation of alloantigen - activated t lymphocytes were tgf1 and hgf .", "both cytokines can independently diminish alloantigen - activated t lymphocyte proliferation , although proliferation can be partially reestablished through blocking with antibodies [ 20 , 31 ] .", "tgf1 is involved in the msc - mediated generation of cd4cd25foxp3 tregs ( figure 1 )   and in the decreased proliferation of nk cells .", "these results suggest that tgf1 and hgf , in addition to other mechanisms , participate in the suppression of msc - mediated proliferation in mixed lymphocyte culture ( mlc ) .", "ido is an enzyme that catalyzes the conversion of the amino acid tryptophan to kynurenine .", "the inhibition of t lymphocyte proliferation is because of the exhaustion of tryptophan or the build - up of kynurenine .", "thus , the addition of exogenous tryptophan has been shown to reestablish alloantigen - activated t - cell proliferation in the presence of mscs .", "similarly , the addition of kynurenine to mlc inhibits proliferation without mscs , however such inhibition is lower when compared with that observed in cultures in the presence of mscs .", "the use of competitive inhibitors of ido reduces msc immunosuppressive effects on alloantigen - activated cd4 t lymphocytes .", "notably , the reestablished proliferation does not reach the levels observed in mlc without mscs , thus suggesting the presence and participation of additional mechanisms . the exhaustion of tryptophan by ido participates in the inhibition of th17 differentiation ;", "however , this mechanism and the build - up of kynurenine are involved in the ido generation of foxp3 tregs .", "furthermore , ido is involved in the decrease of proliferation and cytotoxic activity of nk cells activated by il-2 in the presence of mscs   and also in the inhibition of maturation and functional activity of dcs ( figure 2 ) .", "pge2 is a lipid mediator derived from the conversion of arachidonic acid to prostaglandin through cox1 and cox2 enzyme action .", "these enzymes , with pge2 , are constitutively expressed by mscs , although their expression increases in an inflammatory environment [ 14 , 17 ] .", "pge2 has been shown to diminish proliferation , stimulate the secretion of il-4 and il-10 , and promote cd4cd25foxp3 and il-10ifncd4 tregs differentiation ( figure 1 ) [ 25 , 59 ] .", "several studies have shown that pge2 is a msc effector molecule ; synthesis can be blocked with indomethacin or ns-398 , and activated t - cell proliferation increases , but not similar to levels observed of t - cell proliferation in absence of mscs [ 14 , 17 , 32 , 60 ] .", "pge2 is involved in the decrease of differentiation of monocytes into dcs   and the decrease of proliferation and cytotoxic activity of nk cells activated by il-2 in the presence of mscs ( figure 2 ) .", "it has been reported that il-10 is expressed by human   and murine   mscs and that tlr3 ligand increases the il-10 secretion by human mscs .", "however , other authors have showed that murine [ 16 , 63 ] and human mscs do not express il-10 . despite these conflicting results ,", "some studies have reported a high concentration of il-10 in the supernatant from cocultures of fetal or adult mscs and immune cells and their participation in the immunosuppression by mscs in such cocultures have been well demonstrated . in that regard", ", some authors have showed that cell - to - cell contact between bm - mscs and t cells appears vital in the concentration increase of il-10 in the supernatant [ 24 , 38 , 64 ] .", "participation of il-10 in bm - msc - mediated immunoregulation and in tregs generation has been demonstrated through the use of antibodies [ 17 , 20 ] .", "il-10 downregulates th1 cytokine expression and can stimulate the expression and secretion of hla - g5 , which is another important molecule in mcs - mediated immunoregulation .", "a recent study reported an increase in immunosuppressive capacity of cd4cd25 tregs cocultured with bm - mscs , which is due to a high expression of pd-1 on tregs stimulated by il-10 present in the coculture supernatant ( figure 1 ) .", "furthermore , il-10 is also involved in the decrease of maturation and function of dcs , inhibiting the ability of dcs to produce il-12 ( figure 2 ) .", "hla - g molecules are nonclassic hla molecules characterized by a limited allelic polymorphism and a tissue - specific expression pattern", ". there are membrane - bound isoforms ( hla - g1 , g2 , g3 , and g4 ) and soluble isoforms ( hla - g5 , g6 , and g7 ) .", "bm - mscs express the membrane - bound isoform hla - g1 and the soluble isoform hla - g5 [ 28 , 30 ] ; expression of both molecules is promoted by il-10 .", "similarly , hla - g5 stimulates the secretion of il-10 in a positive feedback loop . the simultaneous use of antibodies against both molecules nearly reestablishes proliferation of alloantigen - activated pbmc in the presence of mscs [ 28 , 67 ] . direct contact between mscs and t cells", "is required to establish the positive feedback loop and subsequent generation of an immunosuppressive environment , which is further exacerbated by the generation of the cd4cd25foxp3 tregs induced by both molecules ( figure 1 ) .", "galectins participate in the regulation of cellular homeostasis in both adaptive and innate immunity as immunostimulators or immunosuppressors .", "the silencing of galectin-1 expression using sirna   or antibodies   reestablishes proliferation of pbmc activated by mitogens or alloantigen , thus indicating that galectin-1 participates in msc immunoregulation .", "similarly , mscs express galectin-3 , a molecule known to regulate t - cell proliferation , adhesion , and migration .", "the inhibition of galectin-3 expression in mscs with sirna reduces immunosuppressive capacity on alloantigen - activated t lymphocytes .", "the authors observed that mscs express higher levels of galectin-9 in an inflammatory environment , and through the use of antibodies , the authors described the participation of galectin-9 in the msc - mediated proliferation reduction of stimulated t and b lymphocytes [ 73 , 74 ] .", "the following membrane molecules participate in msc - mediated immunoregulation : the pd-1/pd - l1 pathway , hla - g1 , jagged-1 , and adhesion molecules , such as intercellular adhesion molecule 1 ( icma - i ) and vascular cell adhesion molecule 1 ( vcam - i ) ( figure 1 ) .", "pd - l1 ( b7-h1/cd274 ) and its receptor ( pd-1/cd279 ) are components of a t lymphocyte costimulatory pathway that releases inhibitory and regulatory signals upon activation .", "the pd-1/pd - l1 pathway is activated in the event of a persistent antigenic stimulus , as occurs with self - antigens , chronic viral infection , or tumors .", "the activation of this pathway prevents autoimmunity and directly contributes to the immunosuppressive microenvironment observed in tumors through t - lymphocyte regulation . in murine models   and human msc from pl , uc , and", "bm [ 34 , 65 , 77 ] , it has been observed that ifn induce an increase of pd - l1 expression and has been demonstrated the participation of pd - l1 in msc - mediated immunosuppression of t cell proliferation through the use of monoclonal antibodies [ 29 , 75 , 76 ] .", "future studies will be necessary to determine the participation of pd - l1 expressed on mscs in the expression of foxp-3 in t cells .", "interestingly , an increase in the expression of pd - l1 receptor ( pd-1 ) has been observed in activated cd4cd25 tregs cocultured with bm - mscs , which is associated with a high immunosuppressive capacity . the same increase in pd-1", "was observed in cd4cd25 t cells , but in contrast with tregs , it is associated with apoptosis [ 65 , 77 ] .", "demonstrated that the reduced proliferation in anti - cd3/cd28-activated t lymphocytes in the presence of mscs derived from bm or fetal liver was primarily driven by cell - to - cell contact and that mscs expressed higher levels of hla - g1 in coculture .", "furthermore , the use of antibodies specific to this molecule nearly reestablished t lymphocyte proliferation and reduced concentration of il-10 in cocultures , which shows the relevance of cell - to - cell contact .", "similar studies have reported the importance of cell - to - cell contact between mscs and t cells in such mechanisms [ 24 , 38 , 64 ] .", "it appears that cell - to - cell contact in cocultures promotes expansion of cd4 t cells which produce il-10 and increase the expression of cd210 ( il-10 receptor , subunit a ) on cd4 t cells but not on cd8 t cells and mscs .", "these results suggest that through cell - to - cell contact between mscs and t cells a population of t cells whose secreted products contribute to the immunoregulatory environment generated by mscs is formed ( figure 1 ) .", "the participation of the adhesion molecules icam - i and vcam - i has been demonstrated using antibodies against both adhesion molecules , which are expressed by mscs during t - cell immunoregulation . when icam - i and vcam - i were blocked in a mouse model , anti - cd3-activated splenocyte proliferation was partially reestablished .", "additionally , the expression of icam - i and vcam - i increased when mscs were exposed to ifn .", "these results suggest that mscs increase the capacity to recruit inflammatory t - lymphocyte populations and modulate its own function toward a regulatory phenotype as was demonstrated in the th17 population .", "furthermore , adhesion molecules could be involved in t - cells immunoregulation through induction of ctla-4 expression on t cells , which has been previously demonstrated [ 79 , 80 ] .", "our laboratory recently demonstrated that cell - to - cell contact between activated cd3 lymphocytes and mscs from bm or ucb increased the expression of ctla-4 ( figure 1 ) . through different mechanisms", "it has been demonstrated that interaction between ctla-4 with cd80/b7 - 1 and cd86/b7 - 2 expressed in dc induces ido upregulation by dc .", "it is likely that a similar mechanism is present in cd4ctla-4 t cells generated in cocultures with msc [ 24 , 37 , 38 ] .", "this idea is supported by several evidences ; activated t cells express cd80 and cd86 , in particular the presence of msc induces increased expression of cd86 on cd4 t cells , and it has been shown that the interaction of these molecules with ctla-4 induced ido expression in cd4 t cells . the cell - to - cell contact between mscs and cd3 t cells is important in the immunosuppresion of b cells by mscs .", "this evidence suggest that inhibition of b - cell proliferation observed in cocultures with msc is dependent of soluble factors produced during cell - to - cell contact between t cells and mscs .", "another molecule involved in the inhibition of t lymphocyte proliferation is jagged-1 , a notch ligand .", "because jagged-1 is expressed in mscs suggests that notch signaling is involved in msc immunosuppressive functions .", "showed that when jagged-1 was blocked by antibodies , the mscs inhibitory effects were decreased on a population of alloantigen - activated cd4 lymphocytes .", "many studies suggest that mscs and immune cells have established two - way regulatory mechanisms ; thus , the activation of msc immunoregulatory properties requires the presence of derived proinflammatory cytokines from immune cells .", "similarly , as a result of this activation , factors secreted by mscs also regulate immune response .", "broad evidence demonstrates that this  activation  requires the presence of proinflammatory cytokines derived from t lymphocytes , macrophages , and nk cells , thus indicating that there are bidirectional regulatory mechanisms between mscs and immune cells .", "cytokines , such as ifn , are necessary in this process , either alone or in combination with tnf , il-1 , il-1 , or il-17 [ 14 , 17 , 85 ] ( figure 2 ) .", "the importance of an inflammatory environment for msc immunosuppressive capacity has been shown both in vitro and in vivo .", "the initial experiments indicated that when exposed to ifn , mscs induce the expression of ido [ 13 , 17 ] and pd - l1   and increase the secretion of pge2 [ 14 , 60 ] .", "similarly , the conditioned medium of msc- and il-15-activated nk cell cocultures can inhibit nk - cell proliferation , which indicates that this coculture activates msc immunoregulatory properties .", "other studies have shown that nk cells can produce ifn when they interact with mscs ( autologous or allogeneic ) , and msc exposure to ifn increases the expression of hla - i in mscs , which reduces the secretion of cytokines and cytotoxic activity of nk cells   ( figure 2 ) .", "this evidence suggests that bidirectional regulatory mechanisms drive the interaction between mscs and nk cells .", "mscs express nk receptor ligands , such as pvr , nectin-2 ( nam-1 ligands ) , and ulpbs and mica ( nkg2d ligands ) .", "because of these receptor - ligand profiles , it is likely that the interaction of nk cells and mscs ( autologous or allogeneic ) results in ifn production by nk cells , whereas this exposure of mscs to ifn increases hla-1 expression in mscs and other immunoregulatory molecules , thereby reducing the secretion of cytokines and nk cytotoxic activity . in addition to the abovementioned cytokines , a recent study reported that il-17 together with ifn and tnf increased inhibition of t - cell proliferation mediated by mscs , apparently through a synergic effect of the three cytokines , leading to a high expression of inducible nitric oxide synthase ( inos ) .", "recently suggested that mscs are not fully immunoprivileged because of their capacity to activate cells of the immune system ( nk cells , macrophages , etc . ) and may even be rejected by such immune cells .", "thus , resting mscs are immunogenic and able to promote the secretion of inflammatory cytokines , which in turn induce msc to express and secret distinct immunoregulatory molecules , allowing them to evade the immune response . in this regard , it has been demonstrated that nave mscs or primed previously with inf plus tnf are able to decrease t - cells proliferation .", "however , nave mscs induce the secretion of inf and il-2 by activated t cells at two days of coculture and this is a cellular event prior to inhibition of proliferation .", "the authors suggested that unprimed msc transiently induce proinflammatory cytokines secretion , which promote the increase of their immunoregulatory capacity . \n", "in vivo studies in mouse models suggest that mscs are effective in the treatment , but not prevention of gvhd .", "the highest survival rates were obtained when mscs were administered when serum ifn concentrations peaked .", "the injection of mscs preactivated with high concentrations of ifn was effective in gvhd prevention and resulted in 100% survival .", "however , the injection of mscs with low concentrations of ifn did not increase survival .", "the administration of mscs to patients with steroid refractory gvhd significantly improved patient outcomes [ 89 , 90 ] . however , when mscs were administered simultaneously with hematopoietic stem cells ( hsc ) , there was no change in grade ii / iv gvhd incidence and a high incidence of relapse .", "toll - like receptors ( tlrs ) are expressed by many immune cells , and their principal function is to detect pathogens .", "the activation of tlrs is essential to initiate an innate immune response and supports the adaptive immune response .", "ten tlr types have been identified in humans and each recognize specific molecular patterns associated with bacterial , viral , or fungal pathogens . the signaling pathway common to all tlrs is the activation of nf- , which controls the expression of several inflammatory cytokines and the expression of maturation markers .", "mscs express tlr-2 , 3 , 4 , 5 , 6 , 7 , and 9 , which are all functional ( except tlr-9 ) because its activation results in receptor internalization and the activation of the nf- , mapk , and akt pathways [ 62 , 84 , 94 ] .", "an in vitro population enriched with alloantigen - activated cd4 lymphocytes cultured in the presence of poly(i : c ) or lps - treated mscs , activated tlr-3 and tlr-4 in the mscs , thus inhibiting their immunosuppressive capacity .", "contradictory results by opitz et al . showed an augmentation in immunosuppressive capacity after tlr-3 and tlr-4 activation in mscs , which continued to inhibit t - cell proliferation , even in low msc  :  t cells ratio in cocultures .", "these contradictory results can be explained with additional evidence from tomchuck et al . who observed that tlr-3 activation in mscs supports the activation of the anti - inflammatory cytokines il-10 and il-12 , whereas tlr-4 activation supports the secretion of proinflammatory cytokines .", "these results were corroborated by waterman et al . who reported that tlr-3 supported msc immunosuppressive effects , whereas tlr-4 supported proinflammatory effects .", "these authors proposed that mscs could be polarized to one of two phenotypes : proinflammatory or anti - inflammatory .", "each msc population may possess unique characteristics and differ in their secretion of cytokines , differentiation capacity , extracellular matrix deposits , tgf1-signaling pathways , and expression of jagged , ido , and pge2 .", "a recent study demonstrated the secretion of proinflammatory cytokines by mscs upon activation of tlr-3 or tlr-4 .", "similarly , most literature describing the molecules that participate in immunoregulatory mechanisms are specific to bm - mscs [ 8 , 13 , 14 , 16 , 17 , 20 , 28 , 31 , 32 , 38 ] .", "few studies have used other sources such as pl [ 24 , 76 , 9799 ] , ucb [ 24 , 60 , 98 ] , uc [ 34 , 98 , 100102 ] , at [ 8 , 99 , 101103 ] , and wj [ 8 , 98 , 99 , 101 , 104 ] ( table 1 ) .", "immune cell studies that have used mscs derived from pl , at , ucb , wj , placental villi , amnion , and chorion have focused principally on t lymphocytes , one of the most studied cell types in msc immunoregulation [ 8 , 24 , 76 , 97 , 101103 , 105111 ] ( table 2 ) .", "the search for alternative sources is particularly important not only because obtaining bm is expensive and invasive , but also because of reports that msc differentiation capacity diminishes as the individual ages [ 112 , 113 ] .", "some reports have compared the immunoregulatory properties of mscs from different sources to determine which is the most viable for bm replacement . in this", "regard , our research group has demonstrated that mscs from bm and ucb , have identical immunoregulatory capacity .", "however we have shown , in the same way as other groups , that in contrast to bm - mscs , pl - derived mscs have a lower capacity [ 24 , 107 ] .", "in fact , results relating to pl are contradictory between groups [ 76 , 97 , 99 , 108 ] . in this regard ,", "results of a preclinical study for treatment of 9 patients with grades iii - iv acute gvhd with pl - mscs showed complete and partial recovery in two and four patients , respectively .", "the inconsistency of the results observed in clinical application of pl - mscs could be related to the inconsistent results obtained in vitro by various groups .", "a different study comparing uc- , ucb- , pl- , and wj - derived mscs observed differences in the capacity to express hla - g or tgf1 after activation with ifn. there were no differences observed in ido secretion .", "screening for immunosuppressive factors in wj- and bm - mscs activated with ifn or tnf has indicated differences in the postactivation secretion of ido , hgf , and pge2 , which may influence immunoregulatory properties .", "it is important to pursue comparative studies to determine whether mscs from alternative sources operate with the identical immunoregulatory mechanisms as bm - mscs , which are used in cellular therapy .", "these studies will be vital in determining alternative sources of mscs for their potential implementation at the clinical level .", "the use of stem cells to replace cells and tissues damaged by congenital or degenerative disease or trauma is called stem cell therapy . in such procedures ,", "we previously outlined that mscs have three biological properties that make them potential candidates for this use : high differentiation potential , trophic factor secretion , and immunoregulatory capacity .", "mscs are potentially applicable to many diseases , such as gvhd , autoimmune diseases and bone , cartilage , and cardiovascular diseases .", "the beneficial effects of mscs administration regarding several of the aforementioned diseases have been analyzed in animal models and phases i , ii , and iii clinical studies have been initiated [ 115 , 116 ] . in the following sections , we focus on the use of mscs in the treatment of immune - associated diseases with special emphasis on the treatment of gvhd . because of their immunoregulatory properties , bm - mscs have been applied principally in hsc transplants because mscs are capable of treating and preventing gvhd [ 89 , 117119 ] .", "after hsc transplant , gvhd presents when donor t lymphocytes recognize patient hla molecules ( alloantigen ) as nonself and mount an immune response ( allogeneic immune response ) .", "gvhd can be acute or chronic depending on the time of onset and intensity of tissue damage .", "acute gvhd ( agvhd ) appears within the first 100 days of the transplant , whereas chronic gvhd ( cgvhd ) has a later onset .", "although the exact pathophysiology is unknown , three phases are believed to describe agvhd onset : ( 1 ) the activation of host antigen presenting cells ( apcs ) by the transplant conditioning regimen ( radiotherapy and/or chemotherapy ) ; ( 2 ) the activation of t lymphocytes , which proliferate and differentiate in response to histoincompatible antigens presented by apcs ; and ( 3 ) a cellular effector and inflammatory phase .", "the final phase is a combined effect of different sectors of the immune system ( cytotoxic t lymphocytes and nk cells ) and inflammatory cytokines ( ifn , tnf , il-1 , etc . ) , which together promote inflammation and tissue damage in various organs and can cause death .", "the first clinical trial evaluating the safety and effectiveness of mscs in the treatment of gvhd was performed by frassoni et al . who observed that the coinfusion of hsc and nonirradiated mscs from the identical donor reduced the incidence and severity of gvdh in recipients of an allograft from an hla - identical sibling .", "subsequently , le blanc et al . reported the case of a 9-year - old boy with steroid refractory agvhd grade iv who was treated with haploidentical mscs from his mother .", "approximately 70 days after treatment , the child had renewed symptoms of diarrhea and high levels of bilirubin .", "the administration of a second dose of mscs significantly improved the patient 's condition , and he remained stable throughout the following 100 days .", "after this case , the identical group reported another study with the administration of mscs to 8 steroids refractory gvhd grades iii - iv patients .", "no toxic effects were observed , and survival was significantly improved compared to the control . in a multicenter phase ii study of 55 steroid", "a response was observed in 39 patients treated with mscs , of which 30 recovered completely and 9 partially recovered .", "survival was significantly improved in patients experiencing full recovery , and the mortality rate was significantly lower than that generally associated with the transplant .", "the authors suggest that the effect of the mscs was independent of the donor because mscs of hla - identical , haploidentical , and incompatible siblings had similar results .", "von bonin et al . treated 13 patients with steroid - refractory agvhd with different doses of mscs ( 15 applications ) from unrelated donors that had been expanded in medium with platelet lysate .", "only 5 patients responded to treatment and only 4 were alive after 257 days of treatment .", "similarly , mller et al . reported the treatment of 5 children with acute or cgvhd .", "mscs doses were increased in accordance with cell availability from 0.4  10 to 3.0  10/kg .", "in addition , it has been suggested that the cotransplant of hsc and bm - mscs may prevent gvhd , but there is a high rate of relapse .", "however , a clinical trial with 37 children treated with multiple doses of bm - mscs for steroid - refractory grades iii - iv agvhd reported complete response in 24 children and partial response in 8 children", ". the contradictory results may be because of differences in the method of msc expansion , the number of cells administered , number of doses given , diagnosis of the recipient ( chronic or agvhd ) , stage of gvhd development , and administration of mscs , among others . in attempts to eliminate some these variables ,", "clinical studies have been performed with a universal bm - msc preparation , such as prochymal , which has rendered positive results in the treatment of gvhd . additionally , the effects of using alternative sources of mscs , such as fetal membranes   or uc , have been studied .", "reported that , in steroid - refractory gvhd patients after msc infusion , a high frequency of cd4cd25cd127foxp3 and tr1 populations was detected .", "a decrease of th17 cells and no changes in the number of nk or b cells were observed .", "furthermore , the authors suggest an induction and maintenance of tregs , because high levels of il-2 were detected . in another similar study an increment of cd8cd28 and cd5cd19 populations and decrease of cd8cd28 and cd5cd19 b cells after bm - msc infusion in the responsive group", "the first two populations of t and b cells are related with the maintaining of peripheral tolerance or the induction of differentiation of tregs .", "similar results were obtained recently in 23 refractory cgvhd patients treated with mscs in which it was detected an increment in the population of cd19cd5il-10 b cells and a high plasma concentration of il-10 .", "it is important to mention that it has been suggested that changes in the immune microenviroment in patients may promote the risk of infections . in this regard , a retrospective cohort study showed that treatment of gvhd with msc is a risk factor for pneumonia related death .", "furthermore , remberger and ringdn reported treatment with msc increment incidence of invasive fungal infections . finally , randomized phase iii trials are necessary to determine the effectiveness of mscs in gvhd", ". msc immunosuppressive capacity may be useful in the treatment of autoimmune diseases . in these pathologies , self - antigens", "are not tolerated , and the body mounts an immune response against its own tissues and organs", ". the resulting damage can be systemic ( systemic lupus erythematosus ) or targeted to a specific organ or tissue , such as the pancreas ( type i diabetes ) , central nervous system ( multiple sclerosis ) , or joints ( rheumatoid arthritis ) [ 131133 ] .", "animal models have demonstrated that msc treatment is effective in some of these conditions , several of which have also been clinical trials , as described below [ 132 , 133 ] .", "systemic lupus erythematosus ( sle ) is a chronic autoimmune disease that affects connective tissue .", "clinical studies using allogeneic bm - mscs   and uc - mscs [ 135 , 136 ] to treat this disease have described positive outcomes and no severe side effect . in a multicenter study ,", "40 sle patients were intravenously transplanted with uc - mscs and total or partial clinical response was observed in 60% of patients .", "the beneficial effect of uc - msc administration is not permanent , because 29.2% of patients relapsed .", "although these results are interesting , it is important to mention that in the future a controlled and randomized study will be necessary to conclusively demonstrate the effectiveness of ucb - msc administration for sle treatment .", "although the mechanism through which msc improves patient condition is unknown , li et al . reported increase of cd4cd25foxp3 tregs in peripheral blood of refractory sle patients transplanted with uc - mscs , even after one month of treatment .", "additionally , a significant decrease of th17 cells since the first week and up to twelve months after transplant was observed . also , wang et al .", "detected an increase in ido activity after administration of uc - mscs in sle patients , evidenced by kynurenine concentrations .", "these results suggest participation of immunoregulatory mechanism regarding beneficial effects of msc administration in such patients . on the other hand ,", "some reports have shown that bm - msc administration in murine model of sle do not decrease the levels of autoantibodies or the mortality rates . furthermore , using the same animal model , you d et al .", "reported that administration of allogeneic bm - mscs to prevent or treat sle enhances autoantibody production and even exacerbates the disease in both experimental conditions .", "also , a study carried out in two patients showed that administration of autologous bm - mscs increased peripheral blood cd4cd25foxp3 tregs cells , but no improvement was observed in patients .", "today , there are no clinical trials in progress to assess efficacy of msc transplantation in the treatment of lupus erythematosus [ http://www.clinicaltrials.gov/ ] .", "type i diabetes is a chronic metabolic disease characterized by an autoimmune reaction against insulin - producing pancreatic  cells . in vitro studies using cocktail of growth factors or genetic manipulation have shown that bm - mscs can differentiate into insulin - producing cells . however , such transdifferentiation capacity of bm - mscs into endocrine pancreas cells in animal models has yielded contradictory results and still remains elusive . nevertheless , in animal models have been demonstrated that mscs can revert type i diabetes , enhances insulin secretion and sustain normoglycemia .", "it has been proposed that such beneficial effects are mainly due to trophic factors and immunoregulators secreted by mscs and not to their differentiation capacity . in a clinical trial ,", "the administration of insulin - producing cells derived of mscs from adipose tissue in 11 patients reduced insulin requirements over the first 2 to 4 months after intraportal infusion . in another trail ,", "wj - mscs were administrated to 15 patients and a decrease in insulin requirements and blood glucose levels was observed even after 24 months of follow - up .", "however , it is thought that msc immunoregulatory capacity prevents the destruction of the -pancreatic cells rather than their differentiation capacity to regenerate  cells . in that", "regard , intravenous administration of bm - mscs in a murine model of type 1 diabetes reversed hyperglycemia and improved pancreatic regeneration , which is associated with an increase of cd4cd25foxp3 tregs cells in spleen and pancreatic lymph nodes .", "similar results were obtained by intraperitoneal administration of mscs from at - mscs in mouse models , during early development of induced type i diabetes .", "increased secretion of insulin and decreased glucose levels in peripheral blood were observed but also inflammatory infiltrate in pancreatic islets .", "furthermore , a low frequency of cd4ifn and cd4tnf t - cells was observed in pancreatic lymph nodes ( plns ) , and in contrast , a high frequency of cd4cd25foxp3 tregs , a decrease of ifn concentration and increase in tgf1 were observed in pancreatic tissue .", "additionally , cotransplantation with bm - mscs improves engraftment of pancreatic islets in humanized diabetic mouse model .", "normoglycemia was maintained during 4 weeks after transplant which show that mscs promote functionality of transplanted islets .", "the authors observed low infiltration by cd3 t cells in the transplanted tissue and increase of cd4cd25foxp3 tregs in peripheral blood .", "future clinical trials will be required to determine if such immunological mechanisms also occur in humans .", "currently , phases i , ii , and iii clinical trials are evaluating the efficacy of autologous bm - mscs [ clinicaltrials.gov identifier : nct02057211 , nct01068951 , nct01157403 ] and uc - mscs in the treatment of type i diabetes [ clinicaltrials.gov identifier : nct01374854 ] .", "two autoimmune diseases affect the central nervous system ( cns ) : multiple sclerosis ( ms ) and amyotrophic lateral sclerosis ( als ) .", "ms is a chronic inflammatory disease characterized by the loss of myelin and axon damage .", "several studies have demonstrated in murine models that msc has positive effects for prevention and treatment of both pathologies , but it is not known what mechanisms are involved in such process . although in vitro studies show that , in the appropriate culture medium , mscs differentiate into cell types with neuronal and glial characteristics , contribution of such mechanisms for in vivo tissue regeneration is controversial . in contrast , in vitro and in vivo studies have suggested that immunoregulation and trophic factor secretion are the main mechanisms used by mscs to improve the symptoms of ms and als [ 1 , 147 ] . studies in a murine model with experimental autoimmune encephalomyelitis ( eae ) as a model for multiple sclerosis have demonstrated that the intravenous administration of bm - mscs improved the symptoms of the disease by decreasing central nervous system inflammation and demyelination .", "other studies have reported that , in eae mice that had received mscs , less infiltration by cd3 t cells and macrophages in csn was observed after pathological analysis . also , it has been detected low levels of il-17 and tnf in serum .", "observed that intravenous or intrathecal administration of bm - msc promotes generation of cd4foxp3 in cns and also an increase of il-17 mrna .", "furthermore , after administration of huc - mscs in a murine eae model , the improvement of disease activity is accompanied by an increase of cd4cd25foxp3 tregs and decrease of th17 in spleen , while in the spinal cord increased il-4 and il-10 and decreased il-1 and il-6 levels were observed . taken together , these results suggest that immunoregulation by mscs has a neuroprotector effect that reduces demyelization and axonal loss and therefore results in the improvement of eae symptoms .", "it is important to mention that in a recent study it was reported that bm - mscs transplanted in a murine eae model , cd8 t cell infiltrate was increased in cns , which exacerbated eae .", "the difference between these results could be due to disease mechanisms that underlie various models of eae .", "similarly , allogeneic mscs administration in murine models of experimental als showed an improvement in survival and motor function , in the spinal cord from msc - treated mice .", "intrathecal infusion of bm - mscs in a murine model of als delayed disease progression and prolonged survival . activated microglia secrete inflammatory molecules including tnf and nitric oxide that play an important role in als and administration of hmscs decrease microglial activation ( cd11b cells ) and concentration of tnf in the spinal cord . based on the positive results obtained in murine models ,", "clinical trials have been carried out to determine safety and efficacy of mscs for the treatment of these pathologies .", "a clinical trial in which 34 ms and als patients received intrathecal or intravenous autologous mscs reported that msc administration was safe and had an immediate immunosuppressive effect that diminished inflammation .", "a study by mazzini et al . studied the direct application of autologous mscs to the spinal cord in als patients and reported that msc administration generated no adverse side effects and was safe ; however there was no significant improvement in patients .", "currently , no study has shown an improvement in patient 's condition by msc treatment ; therefore , further clinical trials must be performed .", "several of such studies , analyzing the efficacy and safety of autologous or allogeneic mscs for the treatment of ms and als , are in progress [ http://www.clinicaltrials.gov/ ] .", "rheumatoid arthritis ( ra ) is a chronic , systemic inflammatory disorder that primarily affects joints and results in bone and cartilage destruction .", "collagen - induced arthritis ( cia ) initiated in susceptible strains of mice by immunization with native type ii collagen serves as a model of human rheumatoid arthritis .", "several authors have used this animal model to analyze msc efficacy to improve the symptoms of this disease ; however evidence remains equivocal as conflicting results have been reported .", "results in this animal model suggest that inflammatory microenvironment present in ra could reverse the immunosuppressive capacity of mscs .", "observed that msc administration does not improve the course of disease and in vitro experiments showed that tnf was responsible of reverse biological function of mscs .", "additionally , it has been reported that the intravenous administration of mscs has no effect on the progression of the disease   or make ra worse .", "similar results were reported by papadopoulou et al . who observed immunoregulatory capacity of mscs in vitro , but in vivo , they lost this capacity when they are administrated in the inflammatory ra environment . in a recent study similar immunosuppressive capacity by synovium - derived mesenchymal stem cells ( s - msc ) from ar", "both mscs are capable of decrease proliferation of pbmc activated with pha or alloantigens from healthy donors and also of autologous synovial t cells activated with pha .", "however , when cocultures are added with exogenous il-17 and/or tnf , s - mscs from ar patients or healthy donors , they lost their immunoregulatory capacity .", "in addition , it has been reported that infusion of allogeneic - related hla matched or partially matched mscs does not affect ra development , while mhc mismatched mscs exacerbate the disease activity .", "in contrast to these results , other studies have shown that administration of syngeneic , allogeneic , or xenogeneic mscs improves ra in mice models .", "thus , gonzlez et al . showed that intraperitoneal administration of human at - mscs in mice with cia reduced the incidence and severity of disease .", "improvement of ra is accompanied by a decrease both in inflammation and proinflammatory cytokine secretion ( il-1 , il-12 , il-17 , tnf , ifn , etc . ) and reduction in th1 and th17 numbers .", "in addition , in a ra murine model induced by antigens , intra - articular infusion of bm - mscs prevented cartilage damage reduced the inflammation and also decreased serum concentration of tnf .", "a few studies have been done to determine safety and efficacy of mscs administration to humans for the treatment of ra .", "a study carried out in four patients with refractory ra and treated with allogeneic mscs from bm ( 1 patient ) or uc ( 3 patient ) administered intravenously showed no adverse effects ; however clinical remissions were not detected .", "in contrast , a clinical assay with 172 patients with active ra showed that uc - msc administration is safe and that the improvement in patients is accompanied by an increase of cd4cd25foxp3 tregs in peripheral blood and a decrease of tnf secretion .", "two phases i and ii clinical trials are currently performed to evaluate the efficacy of uc - mscs [ clinicaltrials.gov identifier : nct01547091 and nct01985464 ] .", "bm - derived mscs have an immunoregulatory capacity because they can regulate the function of multiple immune system components . to fulfill this role , mscs must be activated by proinflammatory cytokines such as ifn. mscs can inhibit dcs maturation and thus prevent the activation of t lymphocytes and even more and decrease the proliferation and cytotoxic activity of nk cells . as a result of these characteristics , mscs are a promising alternative treatment for immune - related diseases .", "currently , mscs have been used in the treatment of autoimmune diseases , including gvhd , and have rendered positive results . despite this encouraging debut in clinical application ,", "it is necessary to perform more clinical trials that extend the current knowledge of the biology of msc immunoregulatory activity to optimize and control the patient 's immune response for maximum benefits .", "these studies will be relevant to clinical decisions in the treatment of gvhd , autoimmune diseases , and other illnesses with an immune component , such as cancer , in which mscs play an important role in the tumor microenvironment that favors growth as our group has previously demonstrated ."], "abstract_text": ["<S> mesenchymal stem cells ( mscs ) are multipotent cells capable of differentiation into mesenchymal lineages and that can be isolated from various tissues and easily cultivated in vitro . </S>", "<S> currently , mscs are of considerable interest because of the biological characteristics that confer high potential applicability in the clinical treatment of many diseases . specifically , because of their high immunoregulatory capacity , mscs are used as tools in cellular therapies for clinical protocols involving immune system alterations . in this review , </S>", "<S> we discuss the current knowledge about the capacity of mscs for the immunoregulation of immunocompetent cells and emphasize the effects of mscs on t cells , principal effectors of the immune response , and the immunosuppressive effects mediated by the secretion of soluble factors and membrane molecules . </S>", "<S> we also describe the mechanisms of msc immunoregulatory modulation and the participation of mscs as immune response regulators in several autoimmune diseases , and we emphasize the clinical application in graft versus host disease ( gvhd ) . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Definition and Characteristics of MSCs", "3. Immunoregulatory Properties of MSCs", "4. The Modulation of MSC Immunoregulatory Properties", "5. Immunoregulation in Alternative Sources of BM-MSCs", "6. MSCs and Clinical Applications", "7. Conclusion"], "sections": [["mesenchymal stem cells ( mscs ) , also referred to as multipotent mesenchymal stromal cells , have been a focus of recent research , partially because they are an extraordinary model for investigating the biological mechanisms that allow a cellular population to generate diverse cell types and because they are a potential tool in cellular therapies for several clinical applications .", "mscs can differentiate into mesenchymal lineages and secrete cytokines and growth factors with paracrine effects that favor the regeneration of damaged tissues [ 1 , 2 ] .", "several studies have demonstrated that mscs possess an immunoregulatory function in vitro and in vivo and that this property suggests clinical applications in the regulation of immunocompetent cell responses [ 2 , 3 ] .", "this review addresses current knowledge of the biological aspects involved in msc immunoregulatory capacity and the clinical focus of these characteristics that allows these cells to be used in the treatment of several diseases with an immune component involved .", "this review culminates with a clinical description of the diseases treated with mscs as a component of cell therapy procedures ."], ["mscs are adult stem cells that are initially isolated from bone marrow ( bm )   and can generate stromal bm components , such as adipocytes , reticular cells , and osteoblasts , whereas in conjunction with additional cellular components , mscs maintain hematopoiesis .", "mscs proliferate in vitro as adherent , colony - forming cells with a high capacity for self - renewal and proliferation [ 4 , 5 ] . because there is no definite marker of mscs ,", "the international society for cellular therapy has established minimum criteria that these in vitro cell populations must fulfill and certain characteristics to be considered mscs .", "the cells must be positive for cd105 , cd73 , and cd90 , express low levels of mhc - i , and be negative for mhc - ii , cd11b , cd14 , cd34 , cd45 , and cd31 .", "additionally , these cells must be capable of differentiation into osteoblasts , adipocytes , and chondroblasts in vitro [ 5 , 6 ] .", "mscs have been isolated from multiple tissues : skeletal muscle , adipose tissue ( at ) , synovial membranes , dental pulp , periodontal ligaments , cervical tissue , menstrual blood , wharton 's jelly ( wj ) , umbilical cord ( uc ) , umbilical cord blood ( ucb ) , amniotic fluid , placenta ( pl ) , and fetal tissues such as blood , liver , and bm [ 710 ] . in most cases ,", "isolated mscs are heterogeneous in proliferation and differentiation , although all express the characteristic msc marker profile .", "mscs cultivated in vitro possess three biological properties that qualify them for use in cellular therapy : ( a ) broad potential of differentiation , ( b ) secretion of trophic factors that favor tissue remodeling , and ( c ) immunoregulatory properties .", "furthermore , mscs differentiate into different mesodermal lineages ( adipocytes , chondrocytes , osteocytes , fibroblasts , and myocytes ) .", "because of this potential for differentiation , mscs were initially used in the treatment of imperfect osteogenesis   and myocardial damage .", "the benefits observed in these initial cell therapy protocols were thought to be the result of osteogenic and myogenic differentiation .", "the current understanding is that , in addition to diverse mesodermal differentiation capacity , msc benefits arise primarily from the secretion of trophic factors and immunoregulatory capacity [ 13 ] ."], ["mscs profoundly affect immune response through their interactions with the cellular components of the innate ( natural killer cells ( nk ) ) and adaptive ( dendritic cells ( dcs ) , b lymphocytes , and t lymphocytes ) immune system .", "msc immunoregulation can occur through cellular contact and/or the secretion of diverse factors [ 1317 ] .", "because of these properties , mscs can prevent the inappropriate activation of t lymphocytes and generate a tolerogenic environment during wound repair or stop an immune response during healing , thus contributing to the maintenance of immune homeostasis [ 2 , 3 ] .", "below , we describe the immunoregulatory effects of mscs on specific immune cells with special emphasis on the effect of mscs on t lymphocytes because of their role as effector cells in many diseases with an immune component .", "the phases in which t cells are vulnerable to msc immunoregulation , recognizing from a biological perspective that there are no obvious limits between phases , are described below .  \n", "t lymphocytes express and secrete molecules characteristic of this phase , such as cd25 , cd69 , cd38 , cytotoxic t lymphocyte antigen-4 ( ctla-4 ) , and human leukocyte antigen - dr ( hla - dr ) and in addition the cytokines interferon- ( ifn ) , tumor necrosis factor ( tnf ) , and il-2 , among others . currently , there are contradictory results regarding the effect of mscs on t lymphocyte activation .", "some studies have observed that bm - mscs prevent the expression of the early activation markers cd25 and cd69 in t cells stimulated with phytohemagglutinin ( pha ) [ 19 , 20 ] , whereas other studies describe no effect by bm - mscs on the expression of these molecules [ 16 , 21 ] .", "such contradictory results may be because of differences in the population of t lymphocytes studied . with this understanding , the activation of peripheral blood mononuclear cells ( pbmc ) with pha in the presence of bm - mscs results in lower numbers of cd4 and cd8 that express cd25 , cd38 , and cd69 . employing the identical model ,", "contradictory results report that the activation of pbmc with antibodies in the presence of bm - mscs does not modify the expression of cd25 or cd69 in cd4 and cd8 populations .", "similarly , no change was reported in the expression of cd25 , cd69 , or ctla-4 in studies of populations enriched with cd4 and cd8 lymphocytes activated by alloantigens . however , a recent study using populations enriched with cd3 t lymphocytes activated with anti - cd2/cd3/cd28 in the presence of bm - mscs showed an increase in the expression of cd69 in cd4 and cd8 t lymphocyte populations .", "the effects of mscs on the secretion of cytokines by activated t lymphocytes are also described by contradictory results . some studies", "have demonstrated that the presence of mscs diminishes [ 14 , 21 ] or increases [ 23 , 24 ] significantly the secretion of ifn by activated t lymphocytes .", "nonetheless , it has been described that the effects of mscs on ifn secretion depend on the source of the lymphocyte population studied . in this study", ", the authors demonstrated that the activation of cd3 t lymphocytes with anti - cd3/cd28 in the presence of mscs from adipose tissue resulted in an increase in ifn , which was an effect that was not observed when pbmc were activated with the identical stimulus .", "our laboratory recently demonstrated that the activation of cd3 t cells with antibodies in the presence of bm - mscs , ucb - mscs , and pl - mscs also resulted in an increase in ifn in cocultures .", "this observed effect might be related to the generation of ifn-producing t regulatory cells ( tregs ) .  \n", "the effect of mscs on the proliferation of t lymphocytes is independent of the activation method .", "the first studies to analyze the effects of bm - mscs on the proliferation of t lymphocytes used irradiated mscs that were cocultivated with alloantigen - stimulated pbmc .", "these studies reported that mscs inhibited t - cell proliferation in a dose - dependent fashion [ 13 , 16 , 17 , 19 ] .", "however , in addition to inhibiting alloantigen - induced proliferation , mscs can inhibit proliferation induced by polyclonal activators such as pha [ 14 , 26 ] or anti - cd3/cd28 [ 21 , 23 , 24 ] .", "the immunosuppressive effects of mscs have been analyzed using total populations of pbmc and populations enriched in cd3 , cd4 , or cd8 lymphocytes .", "every case has demonstrated the capacity of mscs to diminish proliferation [ 13 , 16 , 17 , 19 , 21 , 23 , 24 ] .", "immunoregulation has been shown to be independent of the induction of apoptosis [ 16 , 27 ] and is performed through mechanisms dependent and independent of cellular contact . among", "the secreted factors identified are transforming growth factor beta 1 ( tgf1 ) , hepatocyte growth factor ( hgf ) , indoleamine-2,3-dioxygenase ( ido ) , prostaglandin e2 ( pge2 ) , il-10 , and hla - g5 [ 13 , 17 , 28 ] , whereas programmed death - ligand 1 ( pd - l1 ) and hla - g1 are involved in contact - dependent mechanisms ( figure 1 ) [ 2830 ] . whether direct contact between mscs and t lymphocytes is necessary for the inhibition of t - cell proliferation remains controversial .", "some authors have suggested that mscs act via an immunosuppressive mechanism independent of cell - to - cell contact [ 31 , 32 ] , whereas others have indicated that contact is required for efficient immunoregulation [ 24 , 2830 , 33 , 34 ] .", "however , the mechanism of msc immunoregulation appears to depend on cellular populations , mode of activation , and the presence or absence of cell - to - cell contact [ 23 , 32 ] .  \n", "( 3 ) differentiation and effector function . upon activation by the presence of pathogens or signs of damage ,", "helper t cells cd4 ( th0 ) differentiate into one of the following subtypes , depending on the t - cell microenvironment : th1 , th2 , th17 , or tregs .", "each population is characterized by the secretion of a set of cytokines whose function is essential to eliminate pathogens within the organism , resolve inflammation , and maintain immune homeostasis .", "several studies have suggested that mscs modulate the differentiation , function , and balance of these subpopulations and foster the development of an anti - inflammatory immune response ( figure 1 ) [ 14 , 28 , 35 , 36 ] .", "the activation of nave t cells ( cd45ra ) in favorable conditions for the induction of th1 or th2 and in the presence of mscs results in the inhibition of ifn secretion by th1 cells and the increase of il-4 secretion by th2 cells .", "furthermore , mscs inhibit the production of proinflammatory cytokines il-17 , il-22 , ifn , and tnf and the differentiation of nave cd4 lymphocytes to th17 .", "additionally , mscs promote the secretion of il-10 and the expression of the foxp3 transcription factor , thus suggesting differentiation toward tregs ( figure 1 ) .", "similarly , in a murine model , the presence of mscs in the th1 and th17 differentiation processes favors differentiation to cd4cd25foxp3 tregs .", "this effect was not observed when mscs were added to cultures of mature th1 or th17 populations .", "these results indicate that mscs affect the differentiation and function of inflammatory t lymphocyte populations in their capacity to produce proinflammatory cytokines and also in the induction of a tregs phenotype .", "several studies have described the role of mscs in the induction of distinct tregs populations [ 14 , 28 , 3740 ] . in an initial study analyzing the participation of bm - mscs in the differentiation of tregs populations , maccario et al . observed that the presence of allogeneic and autologous mscs , with respect to the responder t lymphocyte population in mixed lymphocyte culture ( cml ) , induced a significant increase in the cd4cd25 t lymphocyte population .", "however , only allogeneic mscs favored an increase in cd4cd25ctla-4 populations . in the same year , a different study observed that pbmc activation by il-2 in the presence of mscs increased the proportion of cd4cd25 t lymphocytes .", "subsequently , prevosto et al . showed that pbmc in coculture with bm - mscs generated a population of cells that could inhibit t lymphocyte proliferation induced by alloantigens or polyclonal activators ( anti - cd3 or pha ) , and this effect required cell - to - cell contact .", "the authors observed that the increase in foxp3 mrna expression occurred only in the tregs populations derived from cd4 lymphocytes .", "mscs can induce and maintain the function and phenotype of tregs derived from cd3 , cd3cd45ro , or cd3cd45ra t lymphocyte populations . beginning with a cd3 population ,", "the authors also demonstrated that the presence of bm - mscs maintained foxp3 expression in tregs .", "furthermore , mscs can induce regulatory t type 1- ( tr1- ) like cells characterized by ifn and il-10 secretion . through an in vivo transplant - induced arteriosclerosis model ( obstructed arteries ) , jui et al .", "demonstrated that the local administration of bm - mscs could prevent this pathology through a local increase in ifn and il-10 . in a subsequent in vitro study , the identical laboratory demonstrated that bm - mscs favored the generation of tr1 lymphocytes with an il-10ifncd4 phenotype mediated by pge2 and ido ( figure 1 ) .", "msc participation in t lymphocyte subpopulation equilibrium has also been observed in human in vivo studies .", "patients who have received mscs for the treatment of gvhd show subsequent increases of cd4cd25foxp3 and tr1 populations and decreases of th17 .", "similarly , the administration of mscs to patients with systemic lupus erythematous induced an increase in cd4cd25foxp3 tregs in the peripheral blood .", "increases in this tregs population have also been observed in kidney transplant patients , which were transplanted with autologous mscs .", "these cells are derived from bm - cd34 cells in vivo and from monocytes stimulated with il-4 and granulocyte macrophage colony - stimulating factor ( gm - csf ) in vitro .", "the primary function of dcs is to process and present antigens to virgin and memory t cells , although they also interact with other immune components such as b lymphocytes and nk cells .", "the individual dcs must mature to initiate an appropriate immune response , and during the maturation process , dcs increase the membrane expression of mhc - ii and t - cell costimulatory molecules cd80 and cd86 .", "mscs can significantly reduce monocyte differentiation into dcs , affecting the upregulation of cd1a , cd40 , cd80 , cd86 , and hla - dr ( figure 2 ) [ 15 , 37 , 4648 ] .", "this reduction is performed through the secretion of factors [ 15 , 46 ] and is a reversible process because these monocytes then differentiate normally at the removal of mscs . when immature dcs ( idcs ) derived from monocytes - msc cocultures were activated with lipopolysaccharide ( lps ) to induce their final differentiation , they expressed lower levels of the maturation marker cd83 and costimulatory molecules cd80 and cd86 .", "however , spaggiari et al . showed that mscs do not affect direct lps - induced maturation of dcs in cocultures , because there was no change in cd80 , cd83 , and cd86 expression .", "these results suggest that mscs exert a strong inhibitory effect on the differentiation process from monocytes to idcs but not on the lps - induced maturation of idcs to mature dcs ( mdcs ) .", "in addition , mdcs cocultured with mscs show a diminished expression of hla - dr , cd1a , cd80 , and cd86 , thus suggesting that mscs may push mdcs toward an immature state with a reduced stimulatory capacity ( figure 2 )", ". additionally , mscs affect the secretion of several cytokines that are key to dcs maturation .", "aggarwal and pittenger observed that mscs inhibit the secretion of tnf by dcs activated by lps .", "the inhibition of tnf secretion by dcs inhibits their maturation , migration to the lymph nodes , and their capacity to stimulate alloreactive t lymphocytes , because of the alteration in the expression of several receptors that are necessary to capture and process antigens .", "mscs also inhibit the dcs secretion of il-12 [ 15 , 47 , 48 ] .", "the insufficient production of il-12 is associated with the induction of t cell anergy and tolerance [ 15 , 26 , 47 ] .", "human bm - mscs that act through notch can induce the differentiation of cd34 hematopoietic progenitors into a population of regulatory dcs with specific properties : ( 1 ) the expression of high levels of il-10 mrna and low expression of il-2 mrna ; ( 2 ) the capacity to inhibit alloreactive t - cell proliferation and function ; and ( 3 ) the capacity to induce tregs differentiation characterized by expression of foxp3 and tgf1 mrna ( figure 2 ) .", "nk cells are important in innate immunity and participate in the body 's defenses against infections and cancer .", "nk cells perform their effector function through the secretion of cytokines , such as ifn , tnf , and gm - csf , and possess cytotoxic activity both spontaneous and antibody - dependent .", "nk function is regulated by the equilibrium of signals transmitted by activator and inhibitor receptors that interact with specific hla molecules on target cells .", "thus , hla - class i negative or hla - class i - mismatched cells represent potential targets of nk cells [ 27 , 50 ] .", "mscs affect the phenotype , proliferation , cytotoxic potential , and cytokine secretion of nk cells ( figure 2 ) .", "when activated by il-2 , nk cells secrete ifn , but when activated in the presence of mscs , ifn secretion significantly decreases . furthermore ,", "nk cells activated by il-2 and alloantigens in the presence of mscs show diminished proliferation and lytic activity [ 16 , 51 ] .", "il-15 is another cytokine that promotes the proliferation , survival , and effector function of nk cells , but through factor secretion , mscs can inhibit il-15 induced proliferation .", "however , mscs and nk cell contact are necessary to inhibit nk cytotoxicity in tumor cell lineages .", "few studies have analyzed the effects of mscs on b lymphocytes ; however , mscs diminish b - cell proliferation by cell cycle arrest in the g0/g1 phase and not by inducing apoptosis .", "a recent study demonstrated that effect of msc on b lymphocytes proliferation with cpg is not direct and requires presence of cd3 t cells .", "mscs can also affect b - cell differentiation because igm , igg , and iga production are diminished [ 5356 ] .", "furthermore , mscs modify the chemotactic properties of b lymphocytes , because expression changes in their chemokine receptors including cxcr4 , cxcr5 , and ccr7 were induced by mscs .", "the first molecules described in the msc - mediated immunoregulation of alloantigen - activated t lymphocytes were tgf1 and hgf .", "both cytokines can independently diminish alloantigen - activated t lymphocyte proliferation , although proliferation can be partially reestablished through blocking with antibodies [ 20 , 31 ] .", "tgf1 is involved in the msc - mediated generation of cd4cd25foxp3 tregs ( figure 1 )   and in the decreased proliferation of nk cells .", "these results suggest that tgf1 and hgf , in addition to other mechanisms , participate in the suppression of msc - mediated proliferation in mixed lymphocyte culture ( mlc ) .", "ido is an enzyme that catalyzes the conversion of the amino acid tryptophan to kynurenine .", "the inhibition of t lymphocyte proliferation is because of the exhaustion of tryptophan or the build - up of kynurenine .", "thus , the addition of exogenous tryptophan has been shown to reestablish alloantigen - activated t - cell proliferation in the presence of mscs .", "similarly , the addition of kynurenine to mlc inhibits proliferation without mscs , however such inhibition is lower when compared with that observed in cultures in the presence of mscs .", "the use of competitive inhibitors of ido reduces msc immunosuppressive effects on alloantigen - activated cd4 t lymphocytes .", "notably , the reestablished proliferation does not reach the levels observed in mlc without mscs , thus suggesting the presence and participation of additional mechanisms . the exhaustion of tryptophan by ido participates in the inhibition of th17 differentiation ;", "however , this mechanism and the build - up of kynurenine are involved in the ido generation of foxp3 tregs .", "furthermore , ido is involved in the decrease of proliferation and cytotoxic activity of nk cells activated by il-2 in the presence of mscs   and also in the inhibition of maturation and functional activity of dcs ( figure 2 ) .", "pge2 is a lipid mediator derived from the conversion of arachidonic acid to prostaglandin through cox1 and cox2 enzyme action .", "these enzymes , with pge2 , are constitutively expressed by mscs , although their expression increases in an inflammatory environment [ 14 , 17 ] .", "pge2 has been shown to diminish proliferation , stimulate the secretion of il-4 and il-10 , and promote cd4cd25foxp3 and il-10ifncd4 tregs differentiation ( figure 1 ) [ 25 , 59 ] .", "several studies have shown that pge2 is a msc effector molecule ; synthesis can be blocked with indomethacin or ns-398 , and activated t - cell proliferation increases , but not similar to levels observed of t - cell proliferation in absence of mscs [ 14 , 17 , 32 , 60 ] .", "pge2 is involved in the decrease of differentiation of monocytes into dcs   and the decrease of proliferation and cytotoxic activity of nk cells activated by il-2 in the presence of mscs ( figure 2 ) .", "it has been reported that il-10 is expressed by human   and murine   mscs and that tlr3 ligand increases the il-10 secretion by human mscs .", "however , other authors have showed that murine [ 16 , 63 ] and human mscs do not express il-10 . despite these conflicting results ,", "some studies have reported a high concentration of il-10 in the supernatant from cocultures of fetal or adult mscs and immune cells and their participation in the immunosuppression by mscs in such cocultures have been well demonstrated . in that regard", ", some authors have showed that cell - to - cell contact between bm - mscs and t cells appears vital in the concentration increase of il-10 in the supernatant [ 24 , 38 , 64 ] .", "participation of il-10 in bm - msc - mediated immunoregulation and in tregs generation has been demonstrated through the use of antibodies [ 17 , 20 ] .", "il-10 downregulates th1 cytokine expression and can stimulate the expression and secretion of hla - g5 , which is another important molecule in mcs - mediated immunoregulation .", "a recent study reported an increase in immunosuppressive capacity of cd4cd25 tregs cocultured with bm - mscs , which is due to a high expression of pd-1 on tregs stimulated by il-10 present in the coculture supernatant ( figure 1 ) .", "furthermore , il-10 is also involved in the decrease of maturation and function of dcs , inhibiting the ability of dcs to produce il-12 ( figure 2 ) .", "hla - g molecules are nonclassic hla molecules characterized by a limited allelic polymorphism and a tissue - specific expression pattern", ". there are membrane - bound isoforms ( hla - g1 , g2 , g3 , and g4 ) and soluble isoforms ( hla - g5 , g6 , and g7 ) .", "bm - mscs express the membrane - bound isoform hla - g1 and the soluble isoform hla - g5 [ 28 , 30 ] ; expression of both molecules is promoted by il-10 .", "similarly , hla - g5 stimulates the secretion of il-10 in a positive feedback loop . the simultaneous use of antibodies against both molecules nearly reestablishes proliferation of alloantigen - activated pbmc in the presence of mscs [ 28 , 67 ] . direct contact between mscs and t cells", "is required to establish the positive feedback loop and subsequent generation of an immunosuppressive environment , which is further exacerbated by the generation of the cd4cd25foxp3 tregs induced by both molecules ( figure 1 ) .", "galectins participate in the regulation of cellular homeostasis in both adaptive and innate immunity as immunostimulators or immunosuppressors .", "the silencing of galectin-1 expression using sirna   or antibodies   reestablishes proliferation of pbmc activated by mitogens or alloantigen , thus indicating that galectin-1 participates in msc immunoregulation .", "similarly , mscs express galectin-3 , a molecule known to regulate t - cell proliferation , adhesion , and migration .", "the inhibition of galectin-3 expression in mscs with sirna reduces immunosuppressive capacity on alloantigen - activated t lymphocytes .", "the authors observed that mscs express higher levels of galectin-9 in an inflammatory environment , and through the use of antibodies , the authors described the participation of galectin-9 in the msc - mediated proliferation reduction of stimulated t and b lymphocytes [ 73 , 74 ] .", "the following membrane molecules participate in msc - mediated immunoregulation : the pd-1/pd - l1 pathway , hla - g1 , jagged-1 , and adhesion molecules , such as intercellular adhesion molecule 1 ( icma - i ) and vascular cell adhesion molecule 1 ( vcam - i ) ( figure 1 ) .", "pd - l1 ( b7-h1/cd274 ) and its receptor ( pd-1/cd279 ) are components of a t lymphocyte costimulatory pathway that releases inhibitory and regulatory signals upon activation .", "the pd-1/pd - l1 pathway is activated in the event of a persistent antigenic stimulus , as occurs with self - antigens , chronic viral infection , or tumors .", "the activation of this pathway prevents autoimmunity and directly contributes to the immunosuppressive microenvironment observed in tumors through t - lymphocyte regulation . in murine models   and human msc from pl , uc , and", "bm [ 34 , 65 , 77 ] , it has been observed that ifn induce an increase of pd - l1 expression and has been demonstrated the participation of pd - l1 in msc - mediated immunosuppression of t cell proliferation through the use of monoclonal antibodies [ 29 , 75 , 76 ] .", "future studies will be necessary to determine the participation of pd - l1 expressed on mscs in the expression of foxp-3 in t cells .", "interestingly , an increase in the expression of pd - l1 receptor ( pd-1 ) has been observed in activated cd4cd25 tregs cocultured with bm - mscs , which is associated with a high immunosuppressive capacity . the same increase in pd-1", "was observed in cd4cd25 t cells , but in contrast with tregs , it is associated with apoptosis [ 65 , 77 ] .", "demonstrated that the reduced proliferation in anti - cd3/cd28-activated t lymphocytes in the presence of mscs derived from bm or fetal liver was primarily driven by cell - to - cell contact and that mscs expressed higher levels of hla - g1 in coculture .", "furthermore , the use of antibodies specific to this molecule nearly reestablished t lymphocyte proliferation and reduced concentration of il-10 in cocultures , which shows the relevance of cell - to - cell contact .", "similar studies have reported the importance of cell - to - cell contact between mscs and t cells in such mechanisms [ 24 , 38 , 64 ] .", "it appears that cell - to - cell contact in cocultures promotes expansion of cd4 t cells which produce il-10 and increase the expression of cd210 ( il-10 receptor , subunit a ) on cd4 t cells but not on cd8 t cells and mscs .", "these results suggest that through cell - to - cell contact between mscs and t cells a population of t cells whose secreted products contribute to the immunoregulatory environment generated by mscs is formed ( figure 1 ) .", "the participation of the adhesion molecules icam - i and vcam - i has been demonstrated using antibodies against both adhesion molecules , which are expressed by mscs during t - cell immunoregulation . when icam - i and vcam - i were blocked in a mouse model , anti - cd3-activated splenocyte proliferation was partially reestablished .", "additionally , the expression of icam - i and vcam - i increased when mscs were exposed to ifn .", "these results suggest that mscs increase the capacity to recruit inflammatory t - lymphocyte populations and modulate its own function toward a regulatory phenotype as was demonstrated in the th17 population .", "furthermore , adhesion molecules could be involved in t - cells immunoregulation through induction of ctla-4 expression on t cells , which has been previously demonstrated [ 79 , 80 ] .", "our laboratory recently demonstrated that cell - to - cell contact between activated cd3 lymphocytes and mscs from bm or ucb increased the expression of ctla-4 ( figure 1 ) . through different mechanisms", "it has been demonstrated that interaction between ctla-4 with cd80/b7 - 1 and cd86/b7 - 2 expressed in dc induces ido upregulation by dc .", "it is likely that a similar mechanism is present in cd4ctla-4 t cells generated in cocultures with msc [ 24 , 37 , 38 ] .", "this idea is supported by several evidences ; activated t cells express cd80 and cd86 , in particular the presence of msc induces increased expression of cd86 on cd4 t cells , and it has been shown that the interaction of these molecules with ctla-4 induced ido expression in cd4 t cells . the cell - to - cell contact between mscs and cd3 t cells is important in the immunosuppresion of b cells by mscs .", "this evidence suggest that inhibition of b - cell proliferation observed in cocultures with msc is dependent of soluble factors produced during cell - to - cell contact between t cells and mscs .", "another molecule involved in the inhibition of t lymphocyte proliferation is jagged-1 , a notch ligand .", "because jagged-1 is expressed in mscs suggests that notch signaling is involved in msc immunosuppressive functions .", "showed that when jagged-1 was blocked by antibodies , the mscs inhibitory effects were decreased on a population of alloantigen - activated cd4 lymphocytes ."], ["many studies suggest that mscs and immune cells have established two - way regulatory mechanisms ; thus , the activation of msc immunoregulatory properties requires the presence of derived proinflammatory cytokines from immune cells .", "similarly , as a result of this activation , factors secreted by mscs also regulate immune response .", "broad evidence demonstrates that this  activation  requires the presence of proinflammatory cytokines derived from t lymphocytes , macrophages , and nk cells , thus indicating that there are bidirectional regulatory mechanisms between mscs and immune cells .", "cytokines , such as ifn , are necessary in this process , either alone or in combination with tnf , il-1 , il-1 , or il-17 [ 14 , 17 , 85 ] ( figure 2 ) .", "the importance of an inflammatory environment for msc immunosuppressive capacity has been shown both in vitro and in vivo .", "the initial experiments indicated that when exposed to ifn , mscs induce the expression of ido [ 13 , 17 ] and pd - l1   and increase the secretion of pge2 [ 14 , 60 ] .", "similarly , the conditioned medium of msc- and il-15-activated nk cell cocultures can inhibit nk - cell proliferation , which indicates that this coculture activates msc immunoregulatory properties .", "other studies have shown that nk cells can produce ifn when they interact with mscs ( autologous or allogeneic ) , and msc exposure to ifn increases the expression of hla - i in mscs , which reduces the secretion of cytokines and cytotoxic activity of nk cells   ( figure 2 ) .", "this evidence suggests that bidirectional regulatory mechanisms drive the interaction between mscs and nk cells .", "mscs express nk receptor ligands , such as pvr , nectin-2 ( nam-1 ligands ) , and ulpbs and mica ( nkg2d ligands ) .", "because of these receptor - ligand profiles , it is likely that the interaction of nk cells and mscs ( autologous or allogeneic ) results in ifn production by nk cells , whereas this exposure of mscs to ifn increases hla-1 expression in mscs and other immunoregulatory molecules , thereby reducing the secretion of cytokines and nk cytotoxic activity . in addition to the abovementioned cytokines , a recent study reported that il-17 together with ifn and tnf increased inhibition of t - cell proliferation mediated by mscs , apparently through a synergic effect of the three cytokines , leading to a high expression of inducible nitric oxide synthase ( inos ) .", "recently suggested that mscs are not fully immunoprivileged because of their capacity to activate cells of the immune system ( nk cells , macrophages , etc . ) and may even be rejected by such immune cells .", "thus , resting mscs are immunogenic and able to promote the secretion of inflammatory cytokines , which in turn induce msc to express and secret distinct immunoregulatory molecules , allowing them to evade the immune response . in this regard , it has been demonstrated that nave mscs or primed previously with inf plus tnf are able to decrease t - cells proliferation .", "however , nave mscs induce the secretion of inf and il-2 by activated t cells at two days of coculture and this is a cellular event prior to inhibition of proliferation .", "the authors suggested that unprimed msc transiently induce proinflammatory cytokines secretion , which promote the increase of their immunoregulatory capacity . \n", "in vivo studies in mouse models suggest that mscs are effective in the treatment , but not prevention of gvhd .", "the highest survival rates were obtained when mscs were administered when serum ifn concentrations peaked .", "the injection of mscs preactivated with high concentrations of ifn was effective in gvhd prevention and resulted in 100% survival .", "however , the injection of mscs with low concentrations of ifn did not increase survival .", "the administration of mscs to patients with steroid refractory gvhd significantly improved patient outcomes [ 89 , 90 ] . however , when mscs were administered simultaneously with hematopoietic stem cells ( hsc ) , there was no change in grade ii / iv gvhd incidence and a high incidence of relapse .", "toll - like receptors ( tlrs ) are expressed by many immune cells , and their principal function is to detect pathogens .", "the activation of tlrs is essential to initiate an innate immune response and supports the adaptive immune response .", "ten tlr types have been identified in humans and each recognize specific molecular patterns associated with bacterial , viral , or fungal pathogens . the signaling pathway common to all tlrs is the activation of nf- , which controls the expression of several inflammatory cytokines and the expression of maturation markers .", "mscs express tlr-2 , 3 , 4 , 5 , 6 , 7 , and 9 , which are all functional ( except tlr-9 ) because its activation results in receptor internalization and the activation of the nf- , mapk , and akt pathways [ 62 , 84 , 94 ] .", "an in vitro population enriched with alloantigen - activated cd4 lymphocytes cultured in the presence of poly(i : c ) or lps - treated mscs , activated tlr-3 and tlr-4 in the mscs , thus inhibiting their immunosuppressive capacity .", "contradictory results by opitz et al . showed an augmentation in immunosuppressive capacity after tlr-3 and tlr-4 activation in mscs , which continued to inhibit t - cell proliferation , even in low msc  :  t cells ratio in cocultures .", "these contradictory results can be explained with additional evidence from tomchuck et al . who observed that tlr-3 activation in mscs supports the activation of the anti - inflammatory cytokines il-10 and il-12 , whereas tlr-4 activation supports the secretion of proinflammatory cytokines .", "these results were corroborated by waterman et al . who reported that tlr-3 supported msc immunosuppressive effects , whereas tlr-4 supported proinflammatory effects .", "these authors proposed that mscs could be polarized to one of two phenotypes : proinflammatory or anti - inflammatory .", "each msc population may possess unique characteristics and differ in their secretion of cytokines , differentiation capacity , extracellular matrix deposits , tgf1-signaling pathways , and expression of jagged , ido , and pge2 .", "a recent study demonstrated the secretion of proinflammatory cytokines by mscs upon activation of tlr-3 or tlr-4 ."], ["similarly , most literature describing the molecules that participate in immunoregulatory mechanisms are specific to bm - mscs [ 8 , 13 , 14 , 16 , 17 , 20 , 28 , 31 , 32 , 38 ] .", "few studies have used other sources such as pl [ 24 , 76 , 9799 ] , ucb [ 24 , 60 , 98 ] , uc [ 34 , 98 , 100102 ] , at [ 8 , 99 , 101103 ] , and wj [ 8 , 98 , 99 , 101 , 104 ] ( table 1 ) .", "immune cell studies that have used mscs derived from pl , at , ucb , wj , placental villi , amnion , and chorion have focused principally on t lymphocytes , one of the most studied cell types in msc immunoregulation [ 8 , 24 , 76 , 97 , 101103 , 105111 ] ( table 2 ) .", "the search for alternative sources is particularly important not only because obtaining bm is expensive and invasive , but also because of reports that msc differentiation capacity diminishes as the individual ages [ 112 , 113 ] .", "some reports have compared the immunoregulatory properties of mscs from different sources to determine which is the most viable for bm replacement . in this", "regard , our research group has demonstrated that mscs from bm and ucb , have identical immunoregulatory capacity .", "however we have shown , in the same way as other groups , that in contrast to bm - mscs , pl - derived mscs have a lower capacity [ 24 , 107 ] .", "in fact , results relating to pl are contradictory between groups [ 76 , 97 , 99 , 108 ] . in this regard ,", "results of a preclinical study for treatment of 9 patients with grades iii - iv acute gvhd with pl - mscs showed complete and partial recovery in two and four patients , respectively .", "the inconsistency of the results observed in clinical application of pl - mscs could be related to the inconsistent results obtained in vitro by various groups .", "a different study comparing uc- , ucb- , pl- , and wj - derived mscs observed differences in the capacity to express hla - g or tgf1 after activation with ifn. there were no differences observed in ido secretion .", "screening for immunosuppressive factors in wj- and bm - mscs activated with ifn or tnf has indicated differences in the postactivation secretion of ido , hgf , and pge2 , which may influence immunoregulatory properties .", "it is important to pursue comparative studies to determine whether mscs from alternative sources operate with the identical immunoregulatory mechanisms as bm - mscs , which are used in cellular therapy .", "these studies will be vital in determining alternative sources of mscs for their potential implementation at the clinical level ."], ["the use of stem cells to replace cells and tissues damaged by congenital or degenerative disease or trauma is called stem cell therapy . in such procedures ,", "we previously outlined that mscs have three biological properties that make them potential candidates for this use : high differentiation potential , trophic factor secretion , and immunoregulatory capacity .", "mscs are potentially applicable to many diseases , such as gvhd , autoimmune diseases and bone , cartilage , and cardiovascular diseases .", "the beneficial effects of mscs administration regarding several of the aforementioned diseases have been analyzed in animal models and phases i , ii , and iii clinical studies have been initiated [ 115 , 116 ] . in the following sections , we focus on the use of mscs in the treatment of immune - associated diseases with special emphasis on the treatment of gvhd . because of their immunoregulatory properties , bm - mscs have been applied principally in hsc transplants because mscs are capable of treating and preventing gvhd [ 89 , 117119 ] .", "after hsc transplant , gvhd presents when donor t lymphocytes recognize patient hla molecules ( alloantigen ) as nonself and mount an immune response ( allogeneic immune response ) .", "gvhd can be acute or chronic depending on the time of onset and intensity of tissue damage .", "acute gvhd ( agvhd ) appears within the first 100 days of the transplant , whereas chronic gvhd ( cgvhd ) has a later onset .", "although the exact pathophysiology is unknown , three phases are believed to describe agvhd onset : ( 1 ) the activation of host antigen presenting cells ( apcs ) by the transplant conditioning regimen ( radiotherapy and/or chemotherapy ) ; ( 2 ) the activation of t lymphocytes , which proliferate and differentiate in response to histoincompatible antigens presented by apcs ; and ( 3 ) a cellular effector and inflammatory phase .", "the final phase is a combined effect of different sectors of the immune system ( cytotoxic t lymphocytes and nk cells ) and inflammatory cytokines ( ifn , tnf , il-1 , etc . ) , which together promote inflammation and tissue damage in various organs and can cause death .", "the first clinical trial evaluating the safety and effectiveness of mscs in the treatment of gvhd was performed by frassoni et al . who observed that the coinfusion of hsc and nonirradiated mscs from the identical donor reduced the incidence and severity of gvdh in recipients of an allograft from an hla - identical sibling .", "subsequently , le blanc et al . reported the case of a 9-year - old boy with steroid refractory agvhd grade iv who was treated with haploidentical mscs from his mother .", "approximately 70 days after treatment , the child had renewed symptoms of diarrhea and high levels of bilirubin .", "the administration of a second dose of mscs significantly improved the patient 's condition , and he remained stable throughout the following 100 days .", "after this case , the identical group reported another study with the administration of mscs to 8 steroids refractory gvhd grades iii - iv patients .", "no toxic effects were observed , and survival was significantly improved compared to the control . in a multicenter phase ii study of 55 steroid", "a response was observed in 39 patients treated with mscs , of which 30 recovered completely and 9 partially recovered .", "survival was significantly improved in patients experiencing full recovery , and the mortality rate was significantly lower than that generally associated with the transplant .", "the authors suggest that the effect of the mscs was independent of the donor because mscs of hla - identical , haploidentical , and incompatible siblings had similar results .", "von bonin et al . treated 13 patients with steroid - refractory agvhd with different doses of mscs ( 15 applications ) from unrelated donors that had been expanded in medium with platelet lysate .", "only 5 patients responded to treatment and only 4 were alive after 257 days of treatment .", "similarly , mller et al . reported the treatment of 5 children with acute or cgvhd .", "mscs doses were increased in accordance with cell availability from 0.4  10 to 3.0  10/kg .", "in addition , it has been suggested that the cotransplant of hsc and bm - mscs may prevent gvhd , but there is a high rate of relapse .", "however , a clinical trial with 37 children treated with multiple doses of bm - mscs for steroid - refractory grades iii - iv agvhd reported complete response in 24 children and partial response in 8 children", ". the contradictory results may be because of differences in the method of msc expansion , the number of cells administered , number of doses given , diagnosis of the recipient ( chronic or agvhd ) , stage of gvhd development , and administration of mscs , among others . in attempts to eliminate some these variables ,", "clinical studies have been performed with a universal bm - msc preparation , such as prochymal , which has rendered positive results in the treatment of gvhd . additionally , the effects of using alternative sources of mscs , such as fetal membranes   or uc , have been studied .", "reported that , in steroid - refractory gvhd patients after msc infusion , a high frequency of cd4cd25cd127foxp3 and tr1 populations was detected .", "a decrease of th17 cells and no changes in the number of nk or b cells were observed .", "furthermore , the authors suggest an induction and maintenance of tregs , because high levels of il-2 were detected . in another similar study an increment of cd8cd28 and cd5cd19 populations and decrease of cd8cd28 and cd5cd19 b cells after bm - msc infusion in the responsive group", "the first two populations of t and b cells are related with the maintaining of peripheral tolerance or the induction of differentiation of tregs .", "similar results were obtained recently in 23 refractory cgvhd patients treated with mscs in which it was detected an increment in the population of cd19cd5il-10 b cells and a high plasma concentration of il-10 .", "it is important to mention that it has been suggested that changes in the immune microenviroment in patients may promote the risk of infections . in this regard , a retrospective cohort study showed that treatment of gvhd with msc is a risk factor for pneumonia related death .", "furthermore , remberger and ringdn reported treatment with msc increment incidence of invasive fungal infections . finally , randomized phase iii trials are necessary to determine the effectiveness of mscs in gvhd", ". msc immunosuppressive capacity may be useful in the treatment of autoimmune diseases . in these pathologies , self - antigens", "are not tolerated , and the body mounts an immune response against its own tissues and organs", ". the resulting damage can be systemic ( systemic lupus erythematosus ) or targeted to a specific organ or tissue , such as the pancreas ( type i diabetes ) , central nervous system ( multiple sclerosis ) , or joints ( rheumatoid arthritis ) [ 131133 ] .", "animal models have demonstrated that msc treatment is effective in some of these conditions , several of which have also been clinical trials , as described below [ 132 , 133 ] .", "systemic lupus erythematosus ( sle ) is a chronic autoimmune disease that affects connective tissue .", "clinical studies using allogeneic bm - mscs   and uc - mscs [ 135 , 136 ] to treat this disease have described positive outcomes and no severe side effect . in a multicenter study ,", "40 sle patients were intravenously transplanted with uc - mscs and total or partial clinical response was observed in 60% of patients .", "the beneficial effect of uc - msc administration is not permanent , because 29.2% of patients relapsed .", "although these results are interesting , it is important to mention that in the future a controlled and randomized study will be necessary to conclusively demonstrate the effectiveness of ucb - msc administration for sle treatment .", "although the mechanism through which msc improves patient condition is unknown , li et al . reported increase of cd4cd25foxp3 tregs in peripheral blood of refractory sle patients transplanted with uc - mscs , even after one month of treatment .", "additionally , a significant decrease of th17 cells since the first week and up to twelve months after transplant was observed . also , wang et al .", "detected an increase in ido activity after administration of uc - mscs in sle patients , evidenced by kynurenine concentrations .", "these results suggest participation of immunoregulatory mechanism regarding beneficial effects of msc administration in such patients . on the other hand ,", "some reports have shown that bm - msc administration in murine model of sle do not decrease the levels of autoantibodies or the mortality rates . furthermore , using the same animal model , you d et al .", "reported that administration of allogeneic bm - mscs to prevent or treat sle enhances autoantibody production and even exacerbates the disease in both experimental conditions .", "also , a study carried out in two patients showed that administration of autologous bm - mscs increased peripheral blood cd4cd25foxp3 tregs cells , but no improvement was observed in patients .", "today , there are no clinical trials in progress to assess efficacy of msc transplantation in the treatment of lupus erythematosus [ http://www.clinicaltrials.gov/ ] .", "type i diabetes is a chronic metabolic disease characterized by an autoimmune reaction against insulin - producing pancreatic  cells . in vitro studies using cocktail of growth factors or genetic manipulation have shown that bm - mscs can differentiate into insulin - producing cells . however , such transdifferentiation capacity of bm - mscs into endocrine pancreas cells in animal models has yielded contradictory results and still remains elusive . nevertheless , in animal models have been demonstrated that mscs can revert type i diabetes , enhances insulin secretion and sustain normoglycemia .", "it has been proposed that such beneficial effects are mainly due to trophic factors and immunoregulators secreted by mscs and not to their differentiation capacity . in a clinical trial ,", "the administration of insulin - producing cells derived of mscs from adipose tissue in 11 patients reduced insulin requirements over the first 2 to 4 months after intraportal infusion . in another trail ,", "wj - mscs were administrated to 15 patients and a decrease in insulin requirements and blood glucose levels was observed even after 24 months of follow - up .", "however , it is thought that msc immunoregulatory capacity prevents the destruction of the -pancreatic cells rather than their differentiation capacity to regenerate  cells . in that", "regard , intravenous administration of bm - mscs in a murine model of type 1 diabetes reversed hyperglycemia and improved pancreatic regeneration , which is associated with an increase of cd4cd25foxp3 tregs cells in spleen and pancreatic lymph nodes .", "similar results were obtained by intraperitoneal administration of mscs from at - mscs in mouse models , during early development of induced type i diabetes .", "increased secretion of insulin and decreased glucose levels in peripheral blood were observed but also inflammatory infiltrate in pancreatic islets .", "furthermore , a low frequency of cd4ifn and cd4tnf t - cells was observed in pancreatic lymph nodes ( plns ) , and in contrast , a high frequency of cd4cd25foxp3 tregs , a decrease of ifn concentration and increase in tgf1 were observed in pancreatic tissue .", "additionally , cotransplantation with bm - mscs improves engraftment of pancreatic islets in humanized diabetic mouse model .", "normoglycemia was maintained during 4 weeks after transplant which show that mscs promote functionality of transplanted islets .", "the authors observed low infiltration by cd3 t cells in the transplanted tissue and increase of cd4cd25foxp3 tregs in peripheral blood .", "future clinical trials will be required to determine if such immunological mechanisms also occur in humans .", "currently , phases i , ii , and iii clinical trials are evaluating the efficacy of autologous bm - mscs [ clinicaltrials.gov identifier : nct02057211 , nct01068951 , nct01157403 ] and uc - mscs in the treatment of type i diabetes [ clinicaltrials.gov identifier : nct01374854 ] .", "two autoimmune diseases affect the central nervous system ( cns ) : multiple sclerosis ( ms ) and amyotrophic lateral sclerosis ( als ) .", "ms is a chronic inflammatory disease characterized by the loss of myelin and axon damage .", "several studies have demonstrated in murine models that msc has positive effects for prevention and treatment of both pathologies , but it is not known what mechanisms are involved in such process . although in vitro studies show that , in the appropriate culture medium , mscs differentiate into cell types with neuronal and glial characteristics , contribution of such mechanisms for in vivo tissue regeneration is controversial . in contrast , in vitro and in vivo studies have suggested that immunoregulation and trophic factor secretion are the main mechanisms used by mscs to improve the symptoms of ms and als [ 1 , 147 ] . studies in a murine model with experimental autoimmune encephalomyelitis ( eae ) as a model for multiple sclerosis have demonstrated that the intravenous administration of bm - mscs improved the symptoms of the disease by decreasing central nervous system inflammation and demyelination .", "other studies have reported that , in eae mice that had received mscs , less infiltration by cd3 t cells and macrophages in csn was observed after pathological analysis . also , it has been detected low levels of il-17 and tnf in serum .", "observed that intravenous or intrathecal administration of bm - msc promotes generation of cd4foxp3 in cns and also an increase of il-17 mrna .", "furthermore , after administration of huc - mscs in a murine eae model , the improvement of disease activity is accompanied by an increase of cd4cd25foxp3 tregs and decrease of th17 in spleen , while in the spinal cord increased il-4 and il-10 and decreased il-1 and il-6 levels were observed . taken together , these results suggest that immunoregulation by mscs has a neuroprotector effect that reduces demyelization and axonal loss and therefore results in the improvement of eae symptoms .", "it is important to mention that in a recent study it was reported that bm - mscs transplanted in a murine eae model , cd8 t cell infiltrate was increased in cns , which exacerbated eae .", "the difference between these results could be due to disease mechanisms that underlie various models of eae .", "similarly , allogeneic mscs administration in murine models of experimental als showed an improvement in survival and motor function , in the spinal cord from msc - treated mice .", "intrathecal infusion of bm - mscs in a murine model of als delayed disease progression and prolonged survival . activated microglia secrete inflammatory molecules including tnf and nitric oxide that play an important role in als and administration of hmscs decrease microglial activation ( cd11b cells ) and concentration of tnf in the spinal cord . based on the positive results obtained in murine models ,", "clinical trials have been carried out to determine safety and efficacy of mscs for the treatment of these pathologies .", "a clinical trial in which 34 ms and als patients received intrathecal or intravenous autologous mscs reported that msc administration was safe and had an immediate immunosuppressive effect that diminished inflammation .", "a study by mazzini et al . studied the direct application of autologous mscs to the spinal cord in als patients and reported that msc administration generated no adverse side effects and was safe ; however there was no significant improvement in patients .", "currently , no study has shown an improvement in patient 's condition by msc treatment ; therefore , further clinical trials must be performed .", "several of such studies , analyzing the efficacy and safety of autologous or allogeneic mscs for the treatment of ms and als , are in progress [ http://www.clinicaltrials.gov/ ] .", "rheumatoid arthritis ( ra ) is a chronic , systemic inflammatory disorder that primarily affects joints and results in bone and cartilage destruction .", "collagen - induced arthritis ( cia ) initiated in susceptible strains of mice by immunization with native type ii collagen serves as a model of human rheumatoid arthritis .", "several authors have used this animal model to analyze msc efficacy to improve the symptoms of this disease ; however evidence remains equivocal as conflicting results have been reported .", "results in this animal model suggest that inflammatory microenvironment present in ra could reverse the immunosuppressive capacity of mscs .", "observed that msc administration does not improve the course of disease and in vitro experiments showed that tnf was responsible of reverse biological function of mscs .", "additionally , it has been reported that the intravenous administration of mscs has no effect on the progression of the disease   or make ra worse .", "similar results were reported by papadopoulou et al . who observed immunoregulatory capacity of mscs in vitro , but in vivo , they lost this capacity when they are administrated in the inflammatory ra environment . in a recent study similar immunosuppressive capacity by synovium - derived mesenchymal stem cells ( s - msc ) from ar", "both mscs are capable of decrease proliferation of pbmc activated with pha or alloantigens from healthy donors and also of autologous synovial t cells activated with pha .", "however , when cocultures are added with exogenous il-17 and/or tnf , s - mscs from ar patients or healthy donors , they lost their immunoregulatory capacity .", "in addition , it has been reported that infusion of allogeneic - related hla matched or partially matched mscs does not affect ra development , while mhc mismatched mscs exacerbate the disease activity .", "in contrast to these results , other studies have shown that administration of syngeneic , allogeneic , or xenogeneic mscs improves ra in mice models .", "thus , gonzlez et al . showed that intraperitoneal administration of human at - mscs in mice with cia reduced the incidence and severity of disease .", "improvement of ra is accompanied by a decrease both in inflammation and proinflammatory cytokine secretion ( il-1 , il-12 , il-17 , tnf , ifn , etc . ) and reduction in th1 and th17 numbers .", "in addition , in a ra murine model induced by antigens , intra - articular infusion of bm - mscs prevented cartilage damage reduced the inflammation and also decreased serum concentration of tnf .", "a few studies have been done to determine safety and efficacy of mscs administration to humans for the treatment of ra .", "a study carried out in four patients with refractory ra and treated with allogeneic mscs from bm ( 1 patient ) or uc ( 3 patient ) administered intravenously showed no adverse effects ; however clinical remissions were not detected .", "in contrast , a clinical assay with 172 patients with active ra showed that uc - msc administration is safe and that the improvement in patients is accompanied by an increase of cd4cd25foxp3 tregs in peripheral blood and a decrease of tnf secretion .", "two phases i and ii clinical trials are currently performed to evaluate the efficacy of uc - mscs [ clinicaltrials.gov identifier : nct01547091 and nct01985464 ] ."], ["bm - derived mscs have an immunoregulatory capacity because they can regulate the function of multiple immune system components . to fulfill this role , mscs must be activated by proinflammatory cytokines such as ifn. mscs can inhibit dcs maturation and thus prevent the activation of t lymphocytes and even more and decrease the proliferation and cytotoxic activity of nk cells . as a result of these characteristics , mscs are a promising alternative treatment for immune - related diseases .", "currently , mscs have been used in the treatment of autoimmune diseases , including gvhd , and have rendered positive results . despite this encouraging debut in clinical application ,", "it is necessary to perform more clinical trials that extend the current knowledge of the biology of msc immunoregulatory activity to optimize and control the patient 's immune response for maximum benefits .", "these studies will be relevant to clinical decisions in the treatment of gvhd , autoimmune diseases , and other illnesses with an immune component , such as cancer , in which mscs play an important role in the tumor microenvironment that favors growth as our group has previously demonstrated ."]]}
{"article_id": "PMC5404165", "article_text": ["carpal instability is broad category consisting of various patterns of injury , with dissociative type more common .", "a 13 -year -old boy presented at 6 months following a fall with restriction of wrist movements .", "patient had sustained a closed distal one -third both bones fracture forearm fixed with k -wire , and volar lunate instability was found during sequential follow -up .", "posttraumatic carpal instability should be identified at the earliest to avoid poor hand function and morbidity associated with it .", "carpal instability and its biomechanics have always been evolving due to the advancement in the field of radiology and arthroscopy .", "lunate plays a pivotal role and acts like an intercalated segment in carpal stability , loss of anchorage of this key structure cause intercalated segment instability .", "volar intercalated segment instability ( visi ) is a less common manifestation of dissociative carpal instability .", "a 13-year - boy had sustained distal one - third radius and ulna fracture after fall on an outstretched hand .", "open reduction and k - wire fixation was performed for both bone fracture forearm ( fig .", "2 ) . he noticed restriction of dorsiflexion of wrist , while other movements normal .", "serial x - rays showed a loss of normal alignment of radio - lunate in comparison with the opposite ( fig . 3 , 4 ) .", "serial radiographs during follow - up showed a volar facing lunate with alteration of scapholunate and lunatocapitate angle and was diagnosed with a volar intercalated lunate instability ( fig .", "the patient was put on strict rehabilitation protocol and the dorsiflexion improved from 0 - 10 to 0 - 40. at 1 year follow - up patient is still having restriction of dorsiflexion ( fig .", "( a ) post - operative radiograph of right wrist posterior - anterior and ( b ) lateral view showing reduced distal one - third radius and ulna fracture with implants in situ .", "( a ) radiograph of wrist posterior - anterior and ( b ) lateral view at 2 months with uniting distal one - third radius and ulna fracture .", "( a ) radiograph of both wrist posterior - anterior view left and ( b ) right showing union of distal one - third of radius and ulna .", "loss of gilula arc with flexed scaphoid suggesting carpal instability at 6-month follow - up .", "( a ) radiograph of both wrists lateral view left and ( b ) right at 6 month follow - up showing volar facing lunate with loss of carpal alignment .", "( a ) radiograh of right wrist at 1 year follow - up , post - anterior view and ( b and c ) lateral views , ( a ) showing healed distal one - third radius and ulna fracture , signet ring sign of scaphoid and moon like appearance of lunate .", "( b ) volar facing lunate with loss of alignment between radius , lunate and capitate .", "( c ) reduction in the scapholunate angle ( 16 ) and increased capitolunate angle ( 40 ) .", "( a ) clinical photograph at 1 year follow - up showing reduction of dorsiflexion ( 0 - 40 ) and ( b ) normal palmar flexion ( 0 - 80 ) of wrist .", "carpal instability should be considered as a differential in patients presenting with chronic wrist pain and restricted movements following trauma .", "classified carpal instability into four categories and lunato - triquetral ligament is involved in stage three which leads to volar lunate instability .", "visi is a condition in which there is pathologic volar flexion of the lunate , with or without a similar posture of the other proximal row carpal bones .", "posttraumatic volar lunate instability is an entity that can be missed during the initial traumatic event because of its subtle nature .", "posttraumatic carpal stability diagnosed at the earliest avoids advanced surgical procedures such as reconstruction and fusion of joint .", "lunate instability in plain radiograph can be diagnosed with the alteration of various angles namely the loss of scapholunate angle , loss of alignment between the lunate , capitate and radius and increase in the capitolunate angle .", "treatment options include from closed reduction and k - wire fixation , dorsal capsulodesis , tenodesis , arthroscopic or open repair with reconstruction and finally arthrodesis .", "this injury could be subtle in nature before reduction and fixation that led to delay in diagnosis or iatrogenic in nature after reduction of the fracture .", "wrist function is a complex interaction of various biomechanics involving the carpal bones , ligaments , and the distal radio - ulnar complex .", "awareness of carpal instability associated with fractures or injuries around the wrist is needed by the treating surgeon to avoid delay in diagnosis and treatment . to suspect visi following a wrist injury and also following reduction with k - wire fixation .", "follow - up of patient at regular intervals to prevent morbidity due to this deformity ."], "abstract_text": ["<S> introduction : carpal instability is broad category consisting of various patterns of injury , with dissociative type more common.case report : a 13 -year -old boy presented at 6 months following a fall with restriction of wrist movements . patient had sustained a closed distal one -third both bones fracture forearm fixed with k -wire , and volar lunate instability was found during sequential follow -up.conclusion : posttraumatic carpal instability should be identified at the earliest to avoid poor hand function and morbidity associated with it . </S>"], "labels": null, "section_names": ["Introduction:", "Case Report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["carpal instability is broad category consisting of various patterns of injury , with dissociative type more common ."], ["a 13 -year -old boy presented at 6 months following a fall with restriction of wrist movements .", "patient had sustained a closed distal one -third both bones fracture forearm fixed with k -wire , and volar lunate instability was found during sequential follow -up ."], ["posttraumatic carpal instability should be identified at the earliest to avoid poor hand function and morbidity associated with it ."], ["carpal instability and its biomechanics have always been evolving due to the advancement in the field of radiology and arthroscopy .", "lunate plays a pivotal role and acts like an intercalated segment in carpal stability , loss of anchorage of this key structure cause intercalated segment instability .", "volar intercalated segment instability ( visi ) is a less common manifestation of dissociative carpal instability ."], ["a 13-year - boy had sustained distal one - third radius and ulna fracture after fall on an outstretched hand .", "open reduction and k - wire fixation was performed for both bone fracture forearm ( fig .", "2 ) . he noticed restriction of dorsiflexion of wrist , while other movements normal .", "serial x - rays showed a loss of normal alignment of radio - lunate in comparison with the opposite ( fig . 3 , 4 ) .", "serial radiographs during follow - up showed a volar facing lunate with alteration of scapholunate and lunatocapitate angle and was diagnosed with a volar intercalated lunate instability ( fig .", "the patient was put on strict rehabilitation protocol and the dorsiflexion improved from 0 - 10 to 0 - 40. at 1 year follow - up patient is still having restriction of dorsiflexion ( fig .", "( a ) post - operative radiograph of right wrist posterior - anterior and ( b ) lateral view showing reduced distal one - third radius and ulna fracture with implants in situ .", "( a ) radiograph of wrist posterior - anterior and ( b ) lateral view at 2 months with uniting distal one - third radius and ulna fracture .", "( a ) radiograph of both wrist posterior - anterior view left and ( b ) right showing union of distal one - third of radius and ulna .", "loss of gilula arc with flexed scaphoid suggesting carpal instability at 6-month follow - up .", "( a ) radiograph of both wrists lateral view left and ( b ) right at 6 month follow - up showing volar facing lunate with loss of carpal alignment .", "( a ) radiograh of right wrist at 1 year follow - up , post - anterior view and ( b and c ) lateral views , ( a ) showing healed distal one - third radius and ulna fracture , signet ring sign of scaphoid and moon like appearance of lunate .", "( b ) volar facing lunate with loss of alignment between radius , lunate and capitate .", "( c ) reduction in the scapholunate angle ( 16 ) and increased capitolunate angle ( 40 ) .", "( a ) clinical photograph at 1 year follow - up showing reduction of dorsiflexion ( 0 - 40 ) and ( b ) normal palmar flexion ( 0 - 80 ) of wrist ."], ["carpal instability should be considered as a differential in patients presenting with chronic wrist pain and restricted movements following trauma .", "classified carpal instability into four categories and lunato - triquetral ligament is involved in stage three which leads to volar lunate instability .", "visi is a condition in which there is pathologic volar flexion of the lunate , with or without a similar posture of the other proximal row carpal bones .", "posttraumatic volar lunate instability is an entity that can be missed during the initial traumatic event because of its subtle nature .", "posttraumatic carpal stability diagnosed at the earliest avoids advanced surgical procedures such as reconstruction and fusion of joint .", "lunate instability in plain radiograph can be diagnosed with the alteration of various angles namely the loss of scapholunate angle , loss of alignment between the lunate , capitate and radius and increase in the capitolunate angle .", "treatment options include from closed reduction and k - wire fixation , dorsal capsulodesis , tenodesis , arthroscopic or open repair with reconstruction and finally arthrodesis .", "this injury could be subtle in nature before reduction and fixation that led to delay in diagnosis or iatrogenic in nature after reduction of the fracture ."], ["wrist function is a complex interaction of various biomechanics involving the carpal bones , ligaments , and the distal radio - ulnar complex .", "awareness of carpal instability associated with fractures or injuries around the wrist is needed by the treating surgeon to avoid delay in diagnosis and treatment . to suspect visi following a wrist injury and also following reduction with k - wire fixation .", "follow - up of patient at regular intervals to prevent morbidity due to this deformity ."]]}
{"article_id": "PMC3432873", "article_text": ["\n preeclampsia is a disorder affecting 5 to 10% of pregnancies and is clinically characterized by new - onset hypertension and proteinuria . despite significant progress in our understanding of preeclampsia , there is a need for a reliable diagnostic biomarker for use in clinical practice . in the search for a biologically plausible biomarker , there have been attempts to reconcile clinical findings with pathologic changes in renal biopsies of preeclampsia . for example , renal pathology of preeclampsia in the kidney is classically described as  endotheliosis  , or swelling of endothelial cells in the glomerulus .", "the podocyte , a specialized visceral epithelial cell that lines and forms the slit diaphragm of the glomerular basement membrane , has traditionally been thought to be unaffected .", "however , more recent microscopic studies demonstrate that podocytes are structurally changed and harbor protein resorption droplets .", "furthermore , there is evidence that selected podocyte - specific proteins such as nephrin , synaptopodin , and glepp-1 are downregulated in preeclampsia , while vegf and flt-1 are increased [ 2 , 3 ] .", "studied the use of podocytes in the urine ( podocyturia ) as diagnostic markers and found podocyturia to be highly sensitive and specific for preeclampsia .", "we sought to study the use of podocyturia to diagnose preeclampsia and differentiate it from other conditions that may have a similar presentation in a high risk pregnancy population .", "furthermore , podocyturia was found frequently in other high risk pregnancy states such as chronic hypertension and gestational diabetes .", "\n we recruited two groups of patients all 18 years of age : uncomplicated pregnant subjects ( control group ) and women at risk for pregnancy complications ( high - risk group ) as described below from the obstetric inpatient service at jacobi medical center , bronx , ny , usa . random urine samples", "inclusion criteria for high - risk group were diagnosis of preeclampsia , chronic hypertension ( htn ) , gestational htn , type i and type ii diabetes mellitus ( dm ) , gestational dm , mixed connective tissue disease , and pregnancies with fetal chromosomal abnormalities .", "exclusion criteria were patients under the age of 18 and absence of the above - mentioned diagnosis .", "inclusion criteria for the control group were uncomplicated pregnancies and deliveries and absence of the above - mentioned high risk pregnancy states .", "exclusion criteria for the control group were < 18 years of age , preexisting high risk pregnancy states or complicated deliveries .", "diagnosis of preeclampsia fulfilled the criteria of new onset of htn with blood pressure of 140/90  mmhg after 20 weeks of gestation and proteinuria of > 300  mg of protein in a 24-hour urine specimen or 1 + protein on a urinalysis sample without evidence of another cause , such as urinary tract infection or inflammation .", "chronic htn was defined as preexisting htn or blood pressure of 140/90  mmhg before 20 weeks of gestation .", "gestational dm was defined as any degree of glucose intolerance with onset of first recognition during pregnancy .", "this study was approved by the internal review board of albert einstein college of medicine .", "urinary protein was quantified either from a 24-hour urine sample collection or extrapolated from a random urine dipstick .", "for example , when only a dipstick urine protein was available , the value was extrapolated to a 24-hour value based on the following : negative protein corresponds to less than 150  mg/24 hours , trace protein corresponds to 150  mg/24 hours , 1 + corresponds to about 200500  mg/24 hours , 2 + to 0.51.5  g/24 hours , and 3 + to 25  g/24 hours .", "twenty ml of freshly voided urine was centrifuged at 700  g for 5  min .", "the sediment pellet was carefully recovered by aspirating the supernatant , washed twice with pbs and resuspended in 1  ml of pbs .", "aliquots of 100  l of the resuspended sediment were centrifuged onto slides using the shandon cytospin 4 cytocentrifuge ( thermo electron corporation , asheville , nc ) , air - dried and fixed with 1  :  1 acetone / methanol for 10 minutes .", "the slides were immersed with pbs/1% h2o2 for 15 minutes and washed with deionized water .", "subsequently , antigen retrieval was achieved by steam - heating in a solution of citrate buffer , ph 6.0 , for 15 minutes and blocked with 10% horse serum in pbs and 2% bsa .", "slides were incubated overnight with monoclonal mouse antihuman synaptopodin antibody at 1  :  1 dilution ( gift of dr .", "peter mundel , massachusetts general hospital , boston , ma ) followed by horse anti - mouse igg at 1  :  1000 dilution ( dako inc .", "sections were then incubated in avidin - biotin complex at 1  :  25 dilution ( vector labs , burlingame , ca ) and developed using diaminobenzidine ( dab ) as chromogen .", "difference in clinical variables between more than two groups was determined by kruskal - wallis method , differences between two groups were determined by mann whitney method .", "analyses were performed with stata version 8.2 and graphpad prism version 5.02 for windows software , and results were considered statistically significant if p < 0.05 . for test characteristics of podocyturia ,", "sensitivity , specificity , positive predictive value , and negative predictive value were calculated for each high risk diagnosis .", "the diagnoses at time of urine collection were as follows : preeclampsia ( n = 28 ) , eclampsia ( n = 1 ) , chronic hypertension ( n = 3 ) , gestational hypertension ( n = 6 ) , type i dm ( n = 1 ) , type ii dm ( n = 1 ) , gestational dm ( n = 4 ) , connective tissue disorder ( n = 1 ) , marginal previa ( n = 1 ) , chromosomal anomaly ( n = 1 ) , and uncomplicated pregnancy ( n = 9 ) . the clinical characteristics of all subjects are described in table 1 .", "podocyturia ( figures 1(a ) and 1(b ) ) was present in 11 out of 29 ( 38% ) patients with preeclampsia / eclampsia , 3 out of 9 ( 33% ) with chronic and gestational htn , and 3 out of 6 ( 50% ) with gestational dm and type i / ii dm ( table 2 ) . among patients categorized as ", ", 2 ( marginal previa and chromosomal anomaly ) out of 3 patients exhibited podocyturia ( 66% ) . in contrast , 0 out of 9 patients ( 0% ) with uncomplicated pregnancies demonstrated podocyturia . based on these findings", ", we calculated the sensitivity and specificity of podocyturia for preeclampsia to be 38% and 70% , as compared to women of htn of any type , in whom it was 33% and 66% , respectively ( table 3 ) .", "the sensitivity and specificity for dm of any type were 50% and 68% , respectively .", "the positive predictive value was 57% for preeclampsia , as compared with 15% for both htn of any type and dm of any type .", "the negative predictive value , however , was poor for preeclampsia at 51% , as compared with 83% and 91% for htn of any type and dm of any type , respectively .", "podocyturia is well described in many glomerular diseases such as type i dm , iga nephropathy , lupus nephritis , and membranous nephropathy [ 5 , 6 ] .", "though endothelial injury is thought to be the main lesion in preeclampsia , more recently , derangements of the podocyte with downregulation of selected podocyte - specific proteins in renal biopsies and presence of nephrin and podocalyxin ( podocyte specific proteins ) in the urine have been described [ 2 , 7 ] . in this study", ", we sought to describe the presence of podocyturia in preeclampsia and other high risk pregnancy states using the podocyte marker synaptopodin for identification .", "though the exact reason for this discrepancy is unclear , immunofluorescent staining of podocyte - specific proteins , in general , does not appear to be an accurate tool to identify podocytes , as these podocytes may be parietal in origin   and may also be apoptotic .", "thus , the utility of urinary podocytes to detect ongoing glomerular damage in women with preeclampsia as previously suggested   is unclear .", "furthermore , our study showed presence of podocyturia in other high - risk pregnancy states such as dm ( gestational or type", "this finding is not unexpected since podocyturia and urinary podocyte mrna have been described in nonpregnant diabetic patients   and in nonpregnant hypertensive patients   respectively .", "interestingly , the number of urinary podocytes has also shown a statistically significant correlation with blood pressure but not proteinuria in preeclampsia .", "though podocyturia might help shed light on the pathophysiology of preeclampsia , we feel that its clinical utility is limited .", "both cytospin methods ( as was performed in this study ) and cultivation of podocytes in culture are fraught with difficulties and are not cost - effective .", "it may not be easy to eliminate podocyte cell debris when counting podocytes from cytospin specimen of fresh sediment .", "growing urinary podocytes in cell culture , on the other hand , are frequently limited by bacterial or fungal contamination as the urine may not have been collected under sterile conditions .", "furthermore , these cells may proliferate , undergo apoptosis , or not attach to the culture dish , thereby falsely representing the true podocyte count .", "interobserver bias poses yet another obstacle as it requires a highly trained cytologist to correctly identify these cells .", "since clinical guidelines are available to diagnose preeclampsia , some have questioned whether the addition of a urinary marker is necessary .", "we feel that the utility of this marker becomes important when the diagnosis of preeclampsia is in question and when the clinical scenario is complicated by the presence of preexisting htn , dm , or other glomerular diseases such as lupus nephritis . in those cases ,", "thus , a specific marker that is discovered through our understanding of the pathophysiology of preeclampsia remains crucial .", "our goal was to confirm the previously shown high sensitivity and specificity of podocyturia in preeclampsia .", "though a larger sample size would be ideal , we feel that of our total sample size of 56 with 29 patients diagnosed with preeclampsia / eclampsia , and 18 patients with other high - risk diagnoses , is an adequate sample size to demonstrate that podocyturia lacks sensitivity and specificity to be a diagnostic marker of preeclampsia .", "we discovered that podocyte loss is present not only in preeclampsia but in other high risk pregnancy states .", "in addition , podocyturia was not found in a majority of patients diagnosed with preeclampsia .", "however , our findings raise an important note of caution of relying on the limited findings in the literature regarding the predictive value of podocyturia in preeclampsia and encourage larger studies .", "given our current knowledge of the complex pathophysiology of the disease such as the significant role of vascular endothelial growth factor ( vegf ) signaling in maintaining a healthy endothelium and cross talk between the podocyte and vascular endothelial cell , it is unlikely that any single test or cell type will be able to predict preeclampsia ."], "abstract_text": ["<S> \n urinary podocyte ( podocyturia ) has been studied as a diagnostic marker for preeclampsia . </S>", "<S> we sought to validate its use in preeclampsia and in differentiating it from other high risk pregnancy states . </S>", "<S> we studied an obstetric population at high risk to develop preeclampsia ( study group ) and uncomplicated pregnancies ( control group ) by analyzing their urine sediment for podocytes within 24 hours of delivery . </S>", "<S> podocytes were identified by immunohistochemistry using the podocyte - specific protein synaptopodin . </S>", "<S> of the 56 patients who were enrolled , 29 patients were diagnosed with preeclampsia , 9 patients had hypertensive conditions such as chronic and gestational hypertension , 6 patients had type i / ii and gestational diabetes mellitus , 3 patients were classified as others , and 9 patients exhibited uncomplicated pregnancies . </S>", "<S> podocyturia was identified in 11 out of 29 ( 38% ) of patients with preeclampsia / eclampsia , 3 out of 9 ( 33% ) with gestational and chronic hypertension , and 3 out of 6 ( 50% ) with type i / ii and gestational diabetes mellitus . </S>", "<S> none of the 9 patients ( 0% ) with uncomplicated pregnancies demonstrated podocyturia . </S>", "<S> the sensitivity and specificity of podocyturia for preeclampsia were found to be 38% and 70% . </S>", "<S> our study showed that podocyturia does not appear to be a sensitive nor a specific marker to diagnose preeclampsia . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Limitations of the Study", "6. Conclusions"], "sections": [["\n preeclampsia is a disorder affecting 5 to 10% of pregnancies and is clinically characterized by new - onset hypertension and proteinuria . despite significant progress in our understanding of preeclampsia , there is a need for a reliable diagnostic biomarker for use in clinical practice . in the search for a biologically plausible biomarker , there have been attempts to reconcile clinical findings with pathologic changes in renal biopsies of preeclampsia . for example , renal pathology of preeclampsia in the kidney is classically described as  endotheliosis  , or swelling of endothelial cells in the glomerulus .", "the podocyte , a specialized visceral epithelial cell that lines and forms the slit diaphragm of the glomerular basement membrane , has traditionally been thought to be unaffected .", "however , more recent microscopic studies demonstrate that podocytes are structurally changed and harbor protein resorption droplets .", "furthermore , there is evidence that selected podocyte - specific proteins such as nephrin , synaptopodin , and glepp-1 are downregulated in preeclampsia , while vegf and flt-1 are increased [ 2 , 3 ] .", "studied the use of podocytes in the urine ( podocyturia ) as diagnostic markers and found podocyturia to be highly sensitive and specific for preeclampsia .", "we sought to study the use of podocyturia to diagnose preeclampsia and differentiate it from other conditions that may have a similar presentation in a high risk pregnancy population .", "furthermore , podocyturia was found frequently in other high risk pregnancy states such as chronic hypertension and gestational diabetes ."], ["\n we recruited two groups of patients all 18 years of age : uncomplicated pregnant subjects ( control group ) and women at risk for pregnancy complications ( high - risk group ) as described below from the obstetric inpatient service at jacobi medical center , bronx , ny , usa . random urine samples", "inclusion criteria for high - risk group were diagnosis of preeclampsia , chronic hypertension ( htn ) , gestational htn , type i and type ii diabetes mellitus ( dm ) , gestational dm , mixed connective tissue disease , and pregnancies with fetal chromosomal abnormalities .", "exclusion criteria were patients under the age of 18 and absence of the above - mentioned diagnosis .", "inclusion criteria for the control group were uncomplicated pregnancies and deliveries and absence of the above - mentioned high risk pregnancy states .", "exclusion criteria for the control group were < 18 years of age , preexisting high risk pregnancy states or complicated deliveries .", "diagnosis of preeclampsia fulfilled the criteria of new onset of htn with blood pressure of 140/90  mmhg after 20 weeks of gestation and proteinuria of > 300  mg of protein in a 24-hour urine specimen or 1 + protein on a urinalysis sample without evidence of another cause , such as urinary tract infection or inflammation .", "chronic htn was defined as preexisting htn or blood pressure of 140/90  mmhg before 20 weeks of gestation .", "gestational dm was defined as any degree of glucose intolerance with onset of first recognition during pregnancy .", "this study was approved by the internal review board of albert einstein college of medicine .", "urinary protein was quantified either from a 24-hour urine sample collection or extrapolated from a random urine dipstick .", "for example , when only a dipstick urine protein was available , the value was extrapolated to a 24-hour value based on the following : negative protein corresponds to less than 150  mg/24 hours , trace protein corresponds to 150  mg/24 hours , 1 + corresponds to about 200500  mg/24 hours , 2 + to 0.51.5  g/24 hours , and 3 + to 25  g/24 hours .", "twenty ml of freshly voided urine was centrifuged at 700  g for 5  min .", "the sediment pellet was carefully recovered by aspirating the supernatant , washed twice with pbs and resuspended in 1  ml of pbs .", "aliquots of 100  l of the resuspended sediment were centrifuged onto slides using the shandon cytospin 4 cytocentrifuge ( thermo electron corporation , asheville , nc ) , air - dried and fixed with 1  :  1 acetone / methanol for 10 minutes .", "the slides were immersed with pbs/1% h2o2 for 15 minutes and washed with deionized water .", "subsequently , antigen retrieval was achieved by steam - heating in a solution of citrate buffer , ph 6.0 , for 15 minutes and blocked with 10% horse serum in pbs and 2% bsa .", "slides were incubated overnight with monoclonal mouse antihuman synaptopodin antibody at 1  :  1 dilution ( gift of dr .", "peter mundel , massachusetts general hospital , boston , ma ) followed by horse anti - mouse igg at 1  :  1000 dilution ( dako inc .", "sections were then incubated in avidin - biotin complex at 1  :  25 dilution ( vector labs , burlingame , ca ) and developed using diaminobenzidine ( dab ) as chromogen .", "difference in clinical variables between more than two groups was determined by kruskal - wallis method , differences between two groups were determined by mann whitney method .", "analyses were performed with stata version 8.2 and graphpad prism version 5.02 for windows software , and results were considered statistically significant if p < 0.05 . for test characteristics of podocyturia ,", "sensitivity , specificity , positive predictive value , and negative predictive value were calculated for each high risk diagnosis ."], ["the diagnoses at time of urine collection were as follows : preeclampsia ( n = 28 ) , eclampsia ( n = 1 ) , chronic hypertension ( n = 3 ) , gestational hypertension ( n = 6 ) , type i dm ( n = 1 ) , type ii dm ( n = 1 ) , gestational dm ( n = 4 ) , connective tissue disorder ( n = 1 ) , marginal previa ( n = 1 ) , chromosomal anomaly ( n = 1 ) , and uncomplicated pregnancy ( n = 9 ) . the clinical characteristics of all subjects are described in table 1 .", "podocyturia ( figures 1(a ) and 1(b ) ) was present in 11 out of 29 ( 38% ) patients with preeclampsia / eclampsia , 3 out of 9 ( 33% ) with chronic and gestational htn , and 3 out of 6 ( 50% ) with gestational dm and type i / ii dm ( table 2 ) . among patients categorized as ", ", 2 ( marginal previa and chromosomal anomaly ) out of 3 patients exhibited podocyturia ( 66% ) . in contrast , 0 out of 9 patients ( 0% ) with uncomplicated pregnancies demonstrated podocyturia . based on these findings", ", we calculated the sensitivity and specificity of podocyturia for preeclampsia to be 38% and 70% , as compared to women of htn of any type , in whom it was 33% and 66% , respectively ( table 3 ) .", "the sensitivity and specificity for dm of any type were 50% and 68% , respectively .", "the positive predictive value was 57% for preeclampsia , as compared with 15% for both htn of any type and dm of any type .", "the negative predictive value , however , was poor for preeclampsia at 51% , as compared with 83% and 91% for htn of any type and dm of any type , respectively ."], ["podocyturia is well described in many glomerular diseases such as type i dm , iga nephropathy , lupus nephritis , and membranous nephropathy [ 5 , 6 ] .", "though endothelial injury is thought to be the main lesion in preeclampsia , more recently , derangements of the podocyte with downregulation of selected podocyte - specific proteins in renal biopsies and presence of nephrin and podocalyxin ( podocyte specific proteins ) in the urine have been described [ 2 , 7 ] . in this study", ", we sought to describe the presence of podocyturia in preeclampsia and other high risk pregnancy states using the podocyte marker synaptopodin for identification .", "though the exact reason for this discrepancy is unclear , immunofluorescent staining of podocyte - specific proteins , in general , does not appear to be an accurate tool to identify podocytes , as these podocytes may be parietal in origin   and may also be apoptotic .", "thus , the utility of urinary podocytes to detect ongoing glomerular damage in women with preeclampsia as previously suggested   is unclear .", "furthermore , our study showed presence of podocyturia in other high - risk pregnancy states such as dm ( gestational or type", "this finding is not unexpected since podocyturia and urinary podocyte mrna have been described in nonpregnant diabetic patients   and in nonpregnant hypertensive patients   respectively .", "interestingly , the number of urinary podocytes has also shown a statistically significant correlation with blood pressure but not proteinuria in preeclampsia .", "though podocyturia might help shed light on the pathophysiology of preeclampsia , we feel that its clinical utility is limited .", "both cytospin methods ( as was performed in this study ) and cultivation of podocytes in culture are fraught with difficulties and are not cost - effective .", "it may not be easy to eliminate podocyte cell debris when counting podocytes from cytospin specimen of fresh sediment .", "growing urinary podocytes in cell culture , on the other hand , are frequently limited by bacterial or fungal contamination as the urine may not have been collected under sterile conditions .", "furthermore , these cells may proliferate , undergo apoptosis , or not attach to the culture dish , thereby falsely representing the true podocyte count .", "interobserver bias poses yet another obstacle as it requires a highly trained cytologist to correctly identify these cells .", "since clinical guidelines are available to diagnose preeclampsia , some have questioned whether the addition of a urinary marker is necessary .", "we feel that the utility of this marker becomes important when the diagnosis of preeclampsia is in question and when the clinical scenario is complicated by the presence of preexisting htn , dm , or other glomerular diseases such as lupus nephritis . in those cases ,", "thus , a specific marker that is discovered through our understanding of the pathophysiology of preeclampsia remains crucial ."], ["our goal was to confirm the previously shown high sensitivity and specificity of podocyturia in preeclampsia .", "though a larger sample size would be ideal , we feel that of our total sample size of 56 with 29 patients diagnosed with preeclampsia / eclampsia , and 18 patients with other high - risk diagnoses , is an adequate sample size to demonstrate that podocyturia lacks sensitivity and specificity to be a diagnostic marker of preeclampsia ."], ["we discovered that podocyte loss is present not only in preeclampsia but in other high risk pregnancy states .", "in addition , podocyturia was not found in a majority of patients diagnosed with preeclampsia .", "however , our findings raise an important note of caution of relying on the limited findings in the literature regarding the predictive value of podocyturia in preeclampsia and encourage larger studies .", "given our current knowledge of the complex pathophysiology of the disease such as the significant role of vascular endothelial growth factor ( vegf ) signaling in maintaining a healthy endothelium and cross talk between the podocyte and vascular endothelial cell , it is unlikely that any single test or cell type will be able to predict preeclampsia ."]]}
{"article_id": "PMC3405411", "article_text": ["illness is a challenge to our physical , psychological and spiritual wellbeing that has repercussions on our identity and our social context . in a pathogenetic approach ,", "diagnostic tests are used to look for the underlying disease and treatments are aimed at removing it .", "a person is considered cured when the disease is no longer detectable and agreed parameters have normalised", ". however daily clinical practice is characterised by people who suffer from chronic illness where there is often a discrepancy between their biochemical results and the way they feel .", "good or even normal parameters do not necessarily correlate with a good perceived state of health , and vice versa .", "health is therefore a concept that goes beyond the absence of disease to include physical , mental and social wellbeing .", "salutogenesis , developed by antonovsky in the 1970s , looks at what generates health by exploring the reasons why some people stay healthy in the face of hazardous influences whilst others , faced with the same hardship fall ill .", "antonovsky s research shows how adverse events and stress can become the opportunity to generate health if certain personal characteristics are present .", "resilience to difficult situations depends on a person s sense of coherence ( soc ) , a global orientation towards life that is based on self - reliance in the face of challenges , self - confidence in one s ability to deal with demanding events and the trust that difficult events hold meaning for one s life [ 2 , 3 ] . there is a growing body of research on all age groups [ 46 ] , different socioeconomic backgrounds and across cultures   that shows how a strong soc is related to better health , healthier ageing [ 812 ] and is a protective factor against alcohol addiction despite similar rates of recreational consumption in teenagers .", "conversely , a weak soc is related to poorer health and lower mood [ 4 , 5 ] .", "although soc develops naturally in the first 30  years of life it is not a static orientation .", "it can be strengthened through personal activity and care [ 4 , 14 ] . a dynamic , personally driven element that can change through activity and care", "it is not the absence of hardship or disease that determines health but our ability to deal with them positively and assign meaning to them .", "health is not a steady state , effortlessly maintained once it has been achieved , but an active process of continuous adjustment , a subtle equilibrium between our physiological , psychological and spiritual integrity and the outer or inner influences that can strengthen or undermine this .", "the concept of health as a state of  complete well - being  advocated by the declaration of alma ata , becomes health as  the ability to adjust and self - manage  in the face of physical , emotional or personal challenges .", "a salutogenetic healthcare system needs to be oriented in such a way that illness is considered an adverse event that can become the foundation for better future health .", "methods need to be adopted that allow the ill person to be treated in all their characteristics and in context .", "each medical intervention should aim to strengthen soc and a person s physiological , psychological and spiritual resilience .", "the increasing prevalence of chronic diseases , the very different but constant environmental challenges faced by people in the developing and developed world alike , provide us with constant salutogenetic opportunities .", "person - centred medicine ( pcm ) takes into account the physical , psychological and spiritual aspects of a person in health and illness in order to individualise health promotion practices , diagnosis and treatment .", "it broadens the technological advances of biomedicine with the epistemological approach , the relationship - based care and the salutogenetic treatments of non - conventional medicine .", "complementary and alternative medicine ( cam ) and traditional medicine ( tm ) are grouped together as cam / tm or non - conventional medicine ( ncm ) .", "the term includes a variety of different medical systems and healthcare methods whose roots lie in different philosophical backgrounds and cultural origins .", "although very different from one another , they all share a holistic view of the human being as a physical , psychological and spiritual entity .", "they value the complexity of natural phenomena and study the relationship between the human being and nature in health and illness .", "their treatment systems are based on knowledge , skills and practices that restore health and encourage the self - healing abilities of the human being .", "they advocate respect for the dignity of every person and promote responsibility for keeping healthy at individual and community levels .", "this holistic , person - centred , salutogenetic approach is the reason for the growing interest in all forms of ncm by patients and healthcare workers alike .", "salutogenetic healthcare practices are personalised using the principles and methods of person - centred medicine . in salutogenetic healthcare the caregiver - person relationship", "each professional role needs to be developed to provide salutogenetic care from as early as undergraduate training . in order to have a person - centred approach , technological proficiency , solid grounding in evidence - based medicine ( ebm ) and knowledge of international protocols need to be complemented by knowledge of the epistemological systems and practical tools of ncm .", "doctors and other healthcare workers are then encouraged to the use their clinical judgment as the final decision - making tool .", "clinical judgment is central to the role of the doctor and their professionalism and can be strengthened and developed to become an accurate decision - making tool .", "this complements the application of ebm and protocols , which are based on large numbers and therefore not always applicable to particular cases .", "following protocols ensures a basic standard of care that has lead to improvements in safety on a large scale . however following them", "automatically can lead to repetitive , monotonous practice which in turn can lead to errors as well as frustration and dissatisfaction both on the part of the healthcare worker , who feels like a generic cog in a smooth machine , and the patient who does not feel personally addressed . on the contrary ,", "assessing and treating each person in their physical , psychological and spiritual uniqueness , encourages active personal involvement on the part of the both healthcare worker and the patient .", "the use of sound clinical judgment allows personalisation of guidelines and protocols to each case .", "moreover , learning and teaching about salutogenetic practices may mean that healthcare workers adopt healthier lifestyles for themselves . greater work satisfaction and active involvement", "strengthen a healthcare worker s resilience and soc , ultimately improving their health as well as that of their patients . with this person - centred method ,", "any treatment is chosen as the result of an informed choice that takes all aspects of the person into consideration .", "for any therapeutic process to become a salutogenetic experience , physical as well as psychological , social and personal factors need to be addressed at the appropriate time with attentive care to the physical emotional and spiritual needs of the person .", "the patient needs to be able to rely on the doctor or the team who take responsibility for their treatment and recovery . during rehabilitation or at other times during the process there may be a chance to bring the experience to greater consciousness , to explore the reasons that led to the event and the things that could be changed in order to prevent similar episodes from happening again .", "all this works towards developing an awareness that overcoming this difficult experience was meaningful because it allowed the development of new life skills , new coping strategies , and new confidence in one s own healing abilities .", "this change needs to be sustained over time with appropriate follow - up as habits are difficult to change and may slip if they are not followed closely and encouraged over time . in order to evaluate the success of medical interventions broadened with a salutogenetic approach , parameters need to be developed and integrated into the current evaluation systems .", "they need to have a suitable epistemological basis , a methodological approach that considers the value of small numbers , individual cases and clinical judgement .", "salutogenetic parameters such as the quality of life of patients and caregivers , sustainable change , treatment satisfaction , job satisfaction and prevention of burn - out need to be assessed as well as cost - effectiveness and technological excellence .", "health education is both a therapeutic intervention and the key form of disease prevention in salutogenetic healthcare .", "it should take place at population levels , in primary care , in hospitals , as part of any rehabilitation process , in schools and families .", "environmental obstacles that prevent people from adopting healthy lifestyles should be removed as far as possible .", "the ottawa charter advocates the implementation of eight conditions that should underlie any change in health .", "these are peace , shelter , education , food , income , a stable eco - system , sustainable resources , social justice , and equity . at the same time , individuals need to be encouraged to actively increase control over their health in order to improve it .  to reach a state of complete physical , mental and social well - being , an individual or group must be able to identify and to realize aspirations , to satisfy needs , and to change or cope with the environment  .", "encouraging locally sourced , high quality foods , encouraging biodiversity and fair - trade , has positive effects on the sustainability of primary resources and the stability of the eco - system as well as people s health . in order to be adopted by individuals in their lifestyles ,", "there should be variations between the diet of a child and that of an elderly person , of an office worker compared to that of a builder , the diet of someone who suffers from inflammatory illnesses compared to that of someone who suffers from cancer , the diet of the same person when they are well or ill .", "if diet were a better used resource in healthcare and people were taught its principles and practical applications , this may decrease the need to use medication in some cases .", "altered sleeping patterns , eating rhythms and patterns of physical activity are both a measure and a cause of pathology and health .", "variable shift workers have a higher degree of stress , of alcohol misuse and increased use of neuroleptic drugs compared to their colleagues with regular working patterns .", "many others have altered eating rhythms as they skip meals , or constantly pick at food during the day .", "it is important for people to become aware of their own biorhythms , which alter with age , gender , state of health , personal and constitutional characteristics . with the help of education ,", "improved environmental conditions and appropriate care during illness , people can adjust or at least compensate for altered biorhythms and therefore actively improve their health .", "chronic stress can strengthen or weaken a person depending on how strong or weak their resilience and soc are .", "chronic stress has been shown to be greater risk factor for developing ischaemic heart disease than hypertension or hypercholesterolaemia .", "emotions can have adverse effects on the incidence of complication and prognosis during an acute event .", "apart from reducing known risk factors and encouraging positive emotions , we can strengthen emotional resilience and soc through artistic activity in the form of painting , music , dance , drama , creative writing . a study carried out in sweden showed how music therapy improved quality of life and soc in a group of elderly people . through artistic activities", "people learn new skills and their achievements have a positive effect on their self - esteem .", "this can be applied to overcome future challenges or unknown life - situations with strengthened soc .", "the initial steps towards a salutogenetic healthcare reform , are to broaden undergraduate training and to develop pilot projects in the fields of health promotion and salutogenetic rehabilitation . the person and the caregiver - person relationship needs to be placed at the centre of the therapeutic process through the principles of person - centred medicine both in education and in practice .", "caregivers need to be given the tools to consider the person in all their aspects and characteristics through the adoption of the epistemological basis of ncm that broadens the technological advances of biomedicine .", "medical interventions should not only be aimed at removing the disease but also at improving health by strengthening a person s soc and their resilience at physical , psychological and spiritual levels . reasons for illness can be explored during rehabilitation . through health promotion programmes , during rehabilitation , in education , people are taught how to take better care of themselves by adopting individually tailored , healthier lifestyles better suited to their own physiological , emotional and personal needs .", "active involvement needs to be encouraged both on the part of the to patient and healthcare giver , the use of clinical judgment needs to be taught .", "methods for the scientific investigation of effects on small numbers or single cases need to be included in the evaluation of a person - centred , salutogenetic healthcare system alongside ebm ."], "abstract_text": ["<S> the purpose of this review is to discuss how a salutogenetic approach that takes into consideration the human being as physical , psychological and spiritual entity may provide some answers to the difficulties faced by healthcare systems . </S>", "<S> the choice of medical intervention needs to take into account the technological advances of biomedicine but tailor them to the physical , psychological and spiritual needs of the patient in the context of their biography . </S>", "<S> such person - centred medicine aims to strengthen antonovsky s concepts of resilience and sense of coherence with each therapeutic intervention so that overcoming illness becomes the foundation for better future health . </S>", "<S> appropriate evaluation parameters need to be developed and included in order to evaluate the success of interventions in a person - centred , salutogenetic system . </S>"], "labels": null, "section_names": ["Salutogenetic healthcare", "Person-centred medicine and salutogenetic healthcare", "Personalised health promotion as disease prevention in salutogenetic healthcare", "Outlook"], "sections": [["illness is a challenge to our physical , psychological and spiritual wellbeing that has repercussions on our identity and our social context . in a pathogenetic approach ,", "diagnostic tests are used to look for the underlying disease and treatments are aimed at removing it .", "a person is considered cured when the disease is no longer detectable and agreed parameters have normalised", ". however daily clinical practice is characterised by people who suffer from chronic illness where there is often a discrepancy between their biochemical results and the way they feel .", "good or even normal parameters do not necessarily correlate with a good perceived state of health , and vice versa .", "health is therefore a concept that goes beyond the absence of disease to include physical , mental and social wellbeing .", "salutogenesis , developed by antonovsky in the 1970s , looks at what generates health by exploring the reasons why some people stay healthy in the face of hazardous influences whilst others , faced with the same hardship fall ill .", "antonovsky s research shows how adverse events and stress can become the opportunity to generate health if certain personal characteristics are present .", "resilience to difficult situations depends on a person s sense of coherence ( soc ) , a global orientation towards life that is based on self - reliance in the face of challenges , self - confidence in one s ability to deal with demanding events and the trust that difficult events hold meaning for one s life [ 2 , 3 ] . there is a growing body of research on all age groups [ 46 ] , different socioeconomic backgrounds and across cultures   that shows how a strong soc is related to better health , healthier ageing [ 812 ] and is a protective factor against alcohol addiction despite similar rates of recreational consumption in teenagers .", "conversely , a weak soc is related to poorer health and lower mood [ 4 , 5 ] .", "although soc develops naturally in the first 30  years of life it is not a static orientation .", "it can be strengthened through personal activity and care [ 4 , 14 ] . a dynamic , personally driven element that can change through activity and care", "it is not the absence of hardship or disease that determines health but our ability to deal with them positively and assign meaning to them .", "health is not a steady state , effortlessly maintained once it has been achieved , but an active process of continuous adjustment , a subtle equilibrium between our physiological , psychological and spiritual integrity and the outer or inner influences that can strengthen or undermine this .", "the concept of health as a state of  complete well - being  advocated by the declaration of alma ata , becomes health as  the ability to adjust and self - manage  in the face of physical , emotional or personal challenges .", "a salutogenetic healthcare system needs to be oriented in such a way that illness is considered an adverse event that can become the foundation for better future health .", "methods need to be adopted that allow the ill person to be treated in all their characteristics and in context .", "each medical intervention should aim to strengthen soc and a person s physiological , psychological and spiritual resilience .", "the increasing prevalence of chronic diseases , the very different but constant environmental challenges faced by people in the developing and developed world alike , provide us with constant salutogenetic opportunities ."], ["person - centred medicine ( pcm ) takes into account the physical , psychological and spiritual aspects of a person in health and illness in order to individualise health promotion practices , diagnosis and treatment .", "it broadens the technological advances of biomedicine with the epistemological approach , the relationship - based care and the salutogenetic treatments of non - conventional medicine .", "complementary and alternative medicine ( cam ) and traditional medicine ( tm ) are grouped together as cam / tm or non - conventional medicine ( ncm ) .", "the term includes a variety of different medical systems and healthcare methods whose roots lie in different philosophical backgrounds and cultural origins .", "although very different from one another , they all share a holistic view of the human being as a physical , psychological and spiritual entity .", "they value the complexity of natural phenomena and study the relationship between the human being and nature in health and illness .", "their treatment systems are based on knowledge , skills and practices that restore health and encourage the self - healing abilities of the human being .", "they advocate respect for the dignity of every person and promote responsibility for keeping healthy at individual and community levels .", "this holistic , person - centred , salutogenetic approach is the reason for the growing interest in all forms of ncm by patients and healthcare workers alike .", "salutogenetic healthcare practices are personalised using the principles and methods of person - centred medicine . in salutogenetic healthcare the caregiver - person relationship", "each professional role needs to be developed to provide salutogenetic care from as early as undergraduate training . in order to have a person - centred approach , technological proficiency , solid grounding in evidence - based medicine ( ebm ) and knowledge of international protocols need to be complemented by knowledge of the epistemological systems and practical tools of ncm .", "doctors and other healthcare workers are then encouraged to the use their clinical judgment as the final decision - making tool .", "clinical judgment is central to the role of the doctor and their professionalism and can be strengthened and developed to become an accurate decision - making tool .", "this complements the application of ebm and protocols , which are based on large numbers and therefore not always applicable to particular cases .", "following protocols ensures a basic standard of care that has lead to improvements in safety on a large scale . however following them", "automatically can lead to repetitive , monotonous practice which in turn can lead to errors as well as frustration and dissatisfaction both on the part of the healthcare worker , who feels like a generic cog in a smooth machine , and the patient who does not feel personally addressed . on the contrary ,", "assessing and treating each person in their physical , psychological and spiritual uniqueness , encourages active personal involvement on the part of the both healthcare worker and the patient .", "the use of sound clinical judgment allows personalisation of guidelines and protocols to each case .", "moreover , learning and teaching about salutogenetic practices may mean that healthcare workers adopt healthier lifestyles for themselves . greater work satisfaction and active involvement", "strengthen a healthcare worker s resilience and soc , ultimately improving their health as well as that of their patients . with this person - centred method ,", "any treatment is chosen as the result of an informed choice that takes all aspects of the person into consideration .", "for any therapeutic process to become a salutogenetic experience , physical as well as psychological , social and personal factors need to be addressed at the appropriate time with attentive care to the physical emotional and spiritual needs of the person .", "the patient needs to be able to rely on the doctor or the team who take responsibility for their treatment and recovery . during rehabilitation or at other times during the process there may be a chance to bring the experience to greater consciousness , to explore the reasons that led to the event and the things that could be changed in order to prevent similar episodes from happening again .", "all this works towards developing an awareness that overcoming this difficult experience was meaningful because it allowed the development of new life skills , new coping strategies , and new confidence in one s own healing abilities .", "this change needs to be sustained over time with appropriate follow - up as habits are difficult to change and may slip if they are not followed closely and encouraged over time . in order to evaluate the success of medical interventions broadened with a salutogenetic approach , parameters need to be developed and integrated into the current evaluation systems .", "they need to have a suitable epistemological basis , a methodological approach that considers the value of small numbers , individual cases and clinical judgement .", "salutogenetic parameters such as the quality of life of patients and caregivers , sustainable change , treatment satisfaction , job satisfaction and prevention of burn - out need to be assessed as well as cost - effectiveness and technological excellence ."], ["health education is both a therapeutic intervention and the key form of disease prevention in salutogenetic healthcare .", "it should take place at population levels , in primary care , in hospitals , as part of any rehabilitation process , in schools and families .", "environmental obstacles that prevent people from adopting healthy lifestyles should be removed as far as possible .", "the ottawa charter advocates the implementation of eight conditions that should underlie any change in health .", "these are peace , shelter , education , food , income , a stable eco - system , sustainable resources , social justice , and equity . at the same time , individuals need to be encouraged to actively increase control over their health in order to improve it .  to reach a state of complete physical , mental and social well - being , an individual or group must be able to identify and to realize aspirations , to satisfy needs , and to change or cope with the environment  .", "encouraging locally sourced , high quality foods , encouraging biodiversity and fair - trade , has positive effects on the sustainability of primary resources and the stability of the eco - system as well as people s health . in order to be adopted by individuals in their lifestyles ,", "there should be variations between the diet of a child and that of an elderly person , of an office worker compared to that of a builder , the diet of someone who suffers from inflammatory illnesses compared to that of someone who suffers from cancer , the diet of the same person when they are well or ill .", "if diet were a better used resource in healthcare and people were taught its principles and practical applications , this may decrease the need to use medication in some cases .", "altered sleeping patterns , eating rhythms and patterns of physical activity are both a measure and a cause of pathology and health .", "variable shift workers have a higher degree of stress , of alcohol misuse and increased use of neuroleptic drugs compared to their colleagues with regular working patterns .", "many others have altered eating rhythms as they skip meals , or constantly pick at food during the day .", "it is important for people to become aware of their own biorhythms , which alter with age , gender , state of health , personal and constitutional characteristics . with the help of education ,", "improved environmental conditions and appropriate care during illness , people can adjust or at least compensate for altered biorhythms and therefore actively improve their health .", "chronic stress can strengthen or weaken a person depending on how strong or weak their resilience and soc are .", "chronic stress has been shown to be greater risk factor for developing ischaemic heart disease than hypertension or hypercholesterolaemia .", "emotions can have adverse effects on the incidence of complication and prognosis during an acute event .", "apart from reducing known risk factors and encouraging positive emotions , we can strengthen emotional resilience and soc through artistic activity in the form of painting , music , dance , drama , creative writing . a study carried out in sweden showed how music therapy improved quality of life and soc in a group of elderly people . through artistic activities", "people learn new skills and their achievements have a positive effect on their self - esteem .", "this can be applied to overcome future challenges or unknown life - situations with strengthened soc ."], ["the initial steps towards a salutogenetic healthcare reform , are to broaden undergraduate training and to develop pilot projects in the fields of health promotion and salutogenetic rehabilitation . the person and the caregiver - person relationship needs to be placed at the centre of the therapeutic process through the principles of person - centred medicine both in education and in practice .", "caregivers need to be given the tools to consider the person in all their aspects and characteristics through the adoption of the epistemological basis of ncm that broadens the technological advances of biomedicine .", "medical interventions should not only be aimed at removing the disease but also at improving health by strengthening a person s soc and their resilience at physical , psychological and spiritual levels . reasons for illness can be explored during rehabilitation . through health promotion programmes , during rehabilitation , in education , people are taught how to take better care of themselves by adopting individually tailored , healthier lifestyles better suited to their own physiological , emotional and personal needs .", "active involvement needs to be encouraged both on the part of the to patient and healthcare giver , the use of clinical judgment needs to be taught .", "methods for the scientific investigation of effects on small numbers or single cases need to be included in the evaluation of a person - centred , salutogenetic healthcare system alongside ebm ."]]}
{"article_id": "PMC3932252", "article_text": ["macroautophagy ( abbreviated as  autophagy  ) is a genetically regulated and evolutionarily conserved pathway for the degradation of subcellular components [ 15 ] .", "this process involves the de novo formation of cytoplasmic double membrane - bound vacuoles termed autophagosomes , which sequester cytosolic cargo for delivery to the lysosomes [ 5 , 6 ] .", "autophagic cargoes may include various subcellular targets typified by ubiquitin - modified or long - lived proteins and major cytosolic organelles ( e.g. , mitochondria and peroxisomes ) [ 79 ] .", "however , a number of other potential substrates have been identified , including lipids , nucleic acids , reticulocytes , and invading pathogens ( e.g. , intracellular bacteria , viruses , etc . ) [ 7 , 10 ] .", "the autophagic pathway proceeds through several defined steps : ( i ) the initiation phase involving the formation of an isolation membrane or phagophore , ( ii ) the elongation of the phagophore , ( iii ) the maturation of an autophagosome with assimilation of a cytosolic cargo , ( iv ) the fusion of the mature autophagosome to the lysosome , and finally ( v ) the degradation phase where the contents are digested by lysosomal proteases ( e.g. , cathepsins ) and other hydrolytic enzymes [ 15 ] ( figure 1 ) .", "autophagy has been recognized as an essential function for cell homeostasis and adaptation to environmental stress conditions including nutritional starvation , energy depletion , endoplasmic reticulum stress , oxidative stress , and hypoxia [ 1114 ] .", "furthermore , autophagy plays a vital role in innate and adaptive immune mechanisms , including resistance to pathogen infections [ 10 , 15 , 16 ] .", "the role of autophagy in diseases is an emerging area of investigation , with recent studies indicating that autophagy may exert multifunctional roles in specific diseases , with the potential for both adaptive and maladaptive outcomes .", "furthermore , deficiency or absence in autophagic function may also contribute to the pathogenesis of human diseases [ 2 , 12 , 1719 ] .", "the occurrence of autophagy in response to environmental stress , most notably starvation , is generally regarded as a cell survival mechanism [ 2022 ] . due to the often coincident appearance of morphological and biochemical markers of autophagy in cells that are dying ,", "the relationship between autophagy and cell death has been both extensively studied and speculated upon [ 2326 ] .", "autophagic cell death  to describe a form of caspase - independent necrosis - like cell death associated with accumulation of autophagosomes in cells .", "this classification is now controversial , and the casual relationship between autophagy and cell death remains unproven [ 25 , 26 ] .", "nevertheless , many studies have pointed to intimate relationships between autophagy and cellular death programs , which are not yet fully understood .", "recent studies have also examined potential cross - talk between the signaling pathways that regulate autophagy and those that regulate distinct forms of regulated cell death such as apoptosis .", "the major types of cell death which have been studied most extensively in the context of autophagy research include apoptosis , necrosis , necroptosis , and pyroptosis , as briefly summarized here .", "apoptosis denotes a regulated form of cell death that requires the coordinated action of proteases and nucleases within an intact plasma membrane .", "morphological characteristics of apoptosis include dna fragmentation , plasma membrane blebbing , cell shrinkage , and cellular decomposition into membrane - bound apoptotic bodies which are removed by phagocytosis [ 3033 ] .", "the cardinal biochemical features of apoptosis include mitochondrial dysfunction , respiratory chain inhibition , loss of inner mitochondrial membrane potential ( m ) , increased mitochondrial membrane permeability , and externalization of phosphatidylserine [ 3033 ] .", "apoptosis has a crucial function in the maintenance of tissue homeostasis under physiological conditions and also serves as a component of developmental programs and furthermore may also contribute to disease pathogenesis .", "( mitochondria - dependent ) apoptotic pathway represents a major mechanism by which exposure to harmful extracellular stimuli triggers apoptosis .", "this pathway is dependent on a proteolytic activation cascade for both regulation and execution ( i.e. , caspases ) and subject to regulation by bcl-2 family proteins . the extrinsic apoptotic pathway , which shares common downstream features with the intrinsic pathway , is defined by its dependence on receptor - ligand ( e.g. , fas - fas ligand ) interactions for initiation .", "necrosis is a type of cell death that results from acute , accidental , or nonphysiological injury [ 3033 ] .", "this type of cell death is associated with cell lysis as the consequence of membrane damage and subsequent leakage of cell constituents into the extracellular space , which may lead to local inflammation and damage to the surrounding tissue . in certain cases ,", "cell swelling or oncosis may precede necrosis . necrosis and apoptosis differ in morphological features , though the two processes are not necessarily mutually exclusive .", "both apoptosis and necrosis can occur in response to treatment with many injurious stimuli , usually in a dose - dependent fashion .", "many agents that cause apoptosis at low to moderate doses may ultimately cause necrosis at relatively higher doses .", "a number of endogenous events can determine the balance between apoptotic and necrotic death . cellular energy charge ( i.e. , atp levels )", "whereas atp is required for certain steps of caspase activation , rapid decline of cellular atp levels typically leads to necrotic cell death .", "the existence of necrotic cell death pathways regulated by an intrinsic death program distinct from that of apoptosis has also been proposed .", "necroptosis ,  that resembles necrosis has been described [ 34 , 35 ] . in this form of regulated necrosis ,", "the ligand binding of death receptors such as the tumor necrosis factor receptor 1 ( tnfr1 ) can promote the formation of a macromolecular complex ( necrosome ) , involving the receptor - interacting protein ( rip ) kinase-1 and kinase-3 that initiate necrosis [ 36 , 37 ] . increasing evidence affirms the relevance of this mode of cell death in the pathogenesis of various diseases [ 3842 ] .", "pyroptosis represents a form of cell death that is triggered by proinflammatory signals and which is associated with inflammation [ 32 , 43 , 44 ] .", "this type of cell death occurs primarily in inflammatory cells such as macrophages and may be triggered by bacterial or pathogen infections .", "caspase-1 is responsible for the maturation of proinflammatory cytokines such as il-1 and il-18 through inflammasome - dependent pathways", "cell death occurs as a result of membranous pore formation and cytoplasmic swelling and leakage of cytosolic contents .", "similar to apoptotic cells , pyroptotic cells may also display dna fragmentation and nuclear condensation .", "the molecular machinery of autophagic regulation is the subject of recent reviews [ 45 , 46 ] .", "subsequent to their identification in yeast , a number of critical autophagy - related genes ( atg ) have been identified whose gene products regulate distinct steps in the induction or progression of autophagy [ 45 , 46 ] . in brief , the autophagy pathway responds to regulation by nutrient status , including nutrient deficiency ( starvation ) and loss of energy charge .", "starvation induces autophagy through the inhibition of mammalian target of rapamycin ( mtor ) , which resides in a multiprotein complex , mtorc1 . in response to stimulation by nutrients or growth factors", ", mtorc1 negatively regulates a macromolecular substrate complex that includes ulk1 , atg13 , atg101 , and fip200 ( rb1cc1 ) , which results in autophagy suppression [ 4854 ] .", "energy depletion , which stimulates autophagy , inhibits mtorc1 , in part through activation of the amp - dependent protein kinase ( ampk ) , leading to the activation of ulk1 , an important initiating step in autophagy [ 47 , 55 , 56 ] .", "autophagy is also coregulated by a multiprotein complex consisting of beclin 1 ( homologue of yeast atg6 ) , which associates with class iii phosphatidylinositol-3-kinase ( vps34 ) and a number of additional stimulatory or inhibitory coregulatory proteins ( e.g. , atg14l , uvrag , ambra1 , and rubicon ) . in response to proautophagic stimuli ,", "the increased production of phosphatidylinositol-3-phosphate ( pi3p ) by this complex regulates autophagosome formation [ 57 , 58 ] .", "the beclin 1 complex is subject to negative regulation by the pi3  k / akt pathway   as well by binding interactions with antiapoptotic bcl-2 family proteins .", "following phagophore formation , the elongation of the autophagosome membrane requires the action of two ubiquitin - like conjugation systems : the atg5-atg12 conjugation system and the microtubule - associated protein-1 light chain 3 ( lc3 , atg8 ) conjugation system [ 61 , 62 ] .", "atg4b converts the proform of lc3b to its cytosolic free form ( lc3-i ) . in mammals ,", "the conversion of lc3-i ( and other atg8 homologues ) to its phosphatidylethanolamine - conjugated and autophagosome - membrane associated form ( i.e. , lc3-ii ) is an initiating step in autophagy [ 6366 ] .", "once thought to be relatively nonspecific , it is now believed that autophagy is a highly selective process in which distinct cellular mechanisms are employed to identify and target cargo to autophagosomes .", "such selective autophagy pathways have been identified for the turnover of mitochondria ( mitophagy ) and other organelles and the turnover of denatured protein ( aggrephagy ) . during starvation ( e.g. ,", "deprivation of glucose or growth factors or depletion of cellular energy charge ) autophagy prolongs cell survival through the degradation and recycling of cellular macromolecules .", "mice deficient in the autophagy protein atg5 are susceptible to the lethal effects of starvation .", "inhibition of autophagy by beclin 1 or atg5 knockdown , or by chemical inhibitors such as 3-methyladenine , can promote apoptosis and caspase-3 activation in starved hela cells .", "these studies have suggested a role for autophagy as a means for prolonging cell survival during starvation .", "autophagy performs a cardinal homeostatic function in the removal of damaged or dysfunctional mitochondria , in a selective process referred to as mitophagy .", "the increased turnover of mitochondria by mitophagy may occur as a result of chemical or physical stress ( e.g. , hypoxia ) .", "nix localizes in the outer mitochondrial membrane and directly interacts with mammalian atg8 homologs through its lir motif . damaged or dysfunctional", "mitochondria are recruited to the autophagosome for removal by mitophagy through a process regulated by the phosphatase and tensin homolog deleted in chromosome 10 ( pten)-induced putative kinase 1 ( pink1 ) and parkinson protein-2 ( parkin ) [ 9 , 69 , 70 ] .", "mutations in the corresponding pink1 and park2 genes are associated with recessive familial forms of parkinson 's disease . in mice , pink1 and park2 deletions", "loss of mitochondrial membrane potential and the increased production of mitochondrial reactive oxygen species ( ros ) may provide initiating signals for mitophagy .", "pink1 , a transmembrane protein , is stabilized on damaged or depolarized mitochondria . following the decline of mitochondrial membrane potential , which can be caused by chemical stress , pink1 recruits cytosolic parkin , an e3 ubiquitin protein ligase , to the mitochondria [ 69 , 70 , 73 ] .", "parkin ubiquitinates mitochondrial outer membrane proteins including porin , mitofusin , and miro [ 74 , 75 ] .", "ubiquitinated mitochondria are subsequently recognized and targeted to autophagosomes by the autophagic cargo adaptor protein p62 [ 9 , 69 , 70 ] .", "autophagy can maintain cellular protein homeostasis ( proteostasis ) by providing a mechanism for the removal of ubiquitinated protein aggregates , in a selective process termed aggrephagy .", "recent studies suggest that autophagy may provide an alternative pathway to proteolysis in addition to the ubiquitin proteasome system [ 7678 ] .", "aggrephagy requires the selective autophagy cargo adaptor p62/sqstm1 ( p62 ) which can interact with ubiquitinated proteins through a ubiquitin - associated ( uba ) domain .", "furthermore , p62 can interact with lc3 through its lir ( lc3-interacting region ) motif and thereby facilitate the targeting of ubiquitinated proteins to autophagosomes .", "the selective autophagy adaptor , nbr1 ( neighbor of brca1 gene 1 ) , promotes the formation of ubiquitin - positive protein aggregates , facilitating their sequestration and removal by aggrephagy .", "this process involves the 400  kda , pi3p - binding autophagy - linked fyve domain protein ( alfy ) , a p62-interacting protein .", "in addition to mitophagy , other forms of organelle - specific or substrate - specific autophagy have been identified and collectively may contribute to the maintenance of cellular integrity under stress .", "these include the selective autophagic degradation of peroxisomes , ribosomes , and endoplasmic reticulum fragments .", "in addition to protein , autophagic processes have been implicated in the degradation of diverse cellular biomolecules , including lipids   and rna .", "furthermore , autophagy can degrade exogenously derived substrates , most notably bacteria , virus particles , and other parasites , in a selective process termed  xenophagy  [ 10 , 15 , 16 ] . although recent studies begin to unravel the role of mitochondrial selective autophagy in cell death pathways , the role of diverse selective autophagy pathways in the modulation of cell death programs remains largely uncharted territory .", "despite a widely accepted role for autophagy in cellular survival , autophagy has also been associated with the regulation of various cell death pathways , most notably apoptosis .", "thus , autophagy may represent a failed adaptive mechanism that may have prevented death under milder conditions .", "hypothetically , excess activation of autophagy may contribute to apoptotic cell death through unchecked degradative processes .", "cells undergoing autophagy display an increase in autophagic vesicles ( i.e. , autophagosomes and autophagolysosomes ) .", "the distinctions between autophagy and apoptosis remain incompletely delineated , as the two processes are not always mutually exclusive and may occur simultaneously in the same cell type .", "recent studies suggest that factors well known to regulate apoptosis pathways also have the potential to exert regulatory activity on factors that regulate autophagy and vice - versa ( figure 2 ) .", "how these regulatory events , termed  cross - talk  , are integrated into a mechanism for the determination of cell fate yet remains incompletely understood .", "antiapoptotic bcl-2 family proteins , which downregulate apoptosis ( i.e. , bcl-2 ) by antagonizing the activity of proapoptotic proteins , can downregulate autophagy .", "binding of these bcl-2 family proteins to beclin 1 inhibits autophagy by preventing the association of beclin 1 with the class iii pi3k complex [ 57 , 60 ] .", "bnip3 is a bh3-only protein that can trigger apoptosis by sequestering antiapoptotic bcl-2 family proteins and promoting bax / bad dependent mitochondrial release of proapoptotic mediators .", "these interactions suggest that autophagy and apoptosis may be coordinately regulated by bcl-2 family proteins .", "experimental evidence also suggests that , once activated , apoptosis effector molecules may suppress autophagy ; for example , beclin 1 may be cleaved and inactivated by caspases during activation of apoptosis .", "further studies suggest that certain atg proteins may play dual roles in autophagy / apoptosis regulation ; for example , the autophagic protein atg5 may affect extrinsic apoptosis pathways through interactions with the fas - associated death domain ( fadd ) protein .", "atg5 which regulates autophagy can be subject to calpain - dependent cleavage to generate a proapoptotic truncation product ( tatg5 ) .", "this cleavage product promotes apoptosis by binding to and inhibiting antiapoptotic proteins such as bcl - xl .", "recent studies also implicate atg12 , the binding partner for atg5 required for autophagosomal elongation , as an effector of the intrinsic apoptosis pathway .", "atg12 may bind to and inactivate antiapoptotic bcl2 family proteins ( e.g. , bcl-2 and mcl-1 ) , through an interaction involving a bh2 motif , and thereby act as a proapoptotic regulator .", "recent advances have identified other regulatory targets for caspase regulation among autophagy related molecules ; for example , atg4d , a member of the atg4 family of atg8 processing enzymes , has been identified as a substrate for proapoptotic caspase-3 .", "the autophagic protein atg3 has recently been identified as a caspase-8 substrate , which is cleaved during tnf-induced apoptosis .", "the antiapoptotic protein cellular flice - like inhibitory protein ( c - flip ) can act as a negative regulator of autophagy .", "the c - flip , an endogenous inhibitor of caspase-8 processing and the extrinsic apoptotic pathway , acts to prevent the binding of atg3 to lc3 , which impairs lc3 processing .", "on the basis of what is known about the molecular cross - talk between autophagy and apoptosis it currently remains unclear whether autophagy and apoptosis are coregulated or mutually exclusive processes .", "antiapoptotic ( e.g. , bcl-2 ) as well as proapoptotic ( e.g. , caspase-3 ) molecules can downregulate autophagy by interacting with beclin 1 .", "furthermore , other caspase - dependent events have been implicated in anti - autophagy or proautophagy events .", "for example caspase processing of atg4d is proautophagy , whereas caspase processing of beclin 1 is antiautophagy .", "the definitive cellular mechanisms that control the decision to embark on each one or both of these pathways in response to specific stimuli remain unclear .", "analysis of any single isolated regulatory component ( using sirna knockdown for example ) for its potential to cross - regulate autophagy and/or apoptosis will be unlikely to answer these questions .", "thus , an integrative approach is needed to understand how the entire molecular machinery of apoptosis and autophagy are coordinated to influence cell fate decisions .", "the p53 tumor suppressor protein is a well studied regulator of cell cycle progression and apoptosis .", "p53 modulates the expression of bcl-2 family proteins ( e.g. , bax , bid ) and other apoptosis - related gene targets ( e.g. , apaf1 ) . the nuclear form of p53 targets the expression of dram ( damage regulated autophagic modulator ) , which can stimulate both autophagy and apoptosis . alternatively", ", p53 can induce autophagy through the upregulation of ampk , which downregulates the mtor pathway .", "recent studies have shown that genetic or pharmacological inhibition of p53 can also activate autophagy and have led to the identification of the cytoplasmic form of p53 as an inhibitor of autophagy .", "chemical stimuli known to induce autophagy can promote the proteasomal degradation of p53 . cellular stimulation with interferon- ( ifn- )", "induces the deacetylation of p53 , leading to suppressed bmf expression , reduced complex formation between beclin 1 and bcl2 , and enhanced autophagy .", "taken together these studies suggest a complex role of p53 in the regulation of autophagy , with opposing roles for the cytosolic and nuclear forms of p53 [ 98 , 99 ] .", "several recent studies , supported by genetic manipulation of the autophagy program , have revealed that in select toxicological models , autophagy may be associated with the promotion of apoptosis . in our recent studies", "we have found that epithelial cells subjected to cigarette smoke extract ( cse ) exposure die by activation of the extrinsic apoptosis pathway [ 100 , 101 ] .", "cse - induced cell death involved activation of the fas - dependent death - inducing signaling complex ( disc ) and downstream activation of caspases ( -8,-9,-3 ) .", "epithelial cells subjected to cse exposure concurrently responded with increased autophagosome formation and increased processing of lc3b - i to lc3b - ii in epithelial cells [ 100 , 101 ] .", "knockdown of autophagy proteins beclin 1 or lc3b inhibited apoptosis in response to cse exposure in vitro , suggesting that increased autophagy occurred in association with epithelial cell death [ 100 , 101 ] .", "further studies revealed that lc3b may act as a regulatory factor in extrinsic apoptosis in this model .", "lc3b was found to engage a complex with fas , the key component of the disc , in a fashion dependent on the lipid raft protein caveolin-1 .", "cse exposure caused the rapid dissociation of lc3b from fas , in association with the activation of apoptosis signaling . in conclusion , these results using genetic knockdown experiments have implicated a proapoptotic role for lc3b , in a specialized model of cse - induced toxicity , though the relative role of autophagic activity in promoting cell death in this model remains unclear [ 100 , 101 ] . it should be noted that cse - induced autophagy may differ from starvation - induced autophagy in that it occurs in the presence of a complex mixture of foreign matter , which may potentially alter the functionality of the autophagy response .", "thus , the concept of  toxic autophagy  may involve altered function , which may be dependent not only on whether its activation is physiological or excessive , but also on the nature of foreign substrates ( e.g. , complex xenobiotics such as tar or virus particles ) and their interactions with autophagosomes .", "further examples of coincident autophagy and apoptosis include p53-dependent autophagy through upregulation of dram , which is coincidental with upregulation of apoptosis .", "deletion of atg5 was also shown to protect cells from prodeath environmental stimuli ; however , the authors attributed this resistance to compensatory activation of chaperone - dependent autophagy , rather than inhibition of macroautophagy per se .", "these studies raise an important issue in that genetic knockdown of one specific autophagy - related factor can not establish whether autophagy was protective or not in any context , as downregulation of the target may potentially affect signaling pathways that are independent of autophagy , or alternatively , promote compensatory mechanisms , such as alternate forms of autophagy .", "the terms  autophagic cell death  or type ii programmed cell death have been previously used to refer to cell death distinct from apoptosis that occurs in association with increases in autophagosome formation and independently of caspases .", "many studies that have implicated autophagy as a cell death effector have been performed on apoptosis - compromised or caspase - deficient cells ; for example , cells treated with z - vad - fmk , a general inhibitor of caspases , or with caspase-8 and calpain inhibitors , die essentially by a nonapoptotic pathway characterized by dramatic accumulations of autophagic vacuoles [ 104107 ] .", "genetic knockdown experiments ( e.g. , beclin 1 ) suggest that autophagy contributes to cytotoxicity in these models ; however , contrasting studies , also using knockdown of autophagy proteins , have also suggested that autophagy can also protect in the context of nonapoptotic cell death induced by caspase inhibition . in baxbak mouse", "embryonic fibroblasts ( mefs ) , which can not activate intrinsic apoptosis , treatment with chemotherapeutic agents results in nonapoptotic necrosis - like cell death accompanied by excessive autophagosome formation .", "currently it remains unclear whether the process of autophagy acts as an effector or bystander of caspase - independent necrosis - like cell death , though autophagic proteins likely play an accessory role [ 25 , 26 ] .", "experiments in tumor cells have suggested the possibility of cross - talk between autophagy and necrosis in cells .", "autophagy provides a protective function to limit tumor necrosis and inflammation in response to metabolic stress .", "while autophagy acts to buffer metabolic stress , the combined impairment of apoptosis and autophagy promotes necrotic cell death in vitro and in vivo . although it remains to be determined what triggers necrosis in tumor cells , it is likely that insufficient atp production to maintain plasma - membrane integrity results in metabolic catastrophe and cell lysis [ 110 , 112 ] .", "autophagy integrates a metabolic feedback system to allow sufficient atp generation to maintain cell viability .", "enhanced autophagy by spermidine , a natural polyamine , inhibits loss of membrane integrity and release of chromatin protein high mobility group b1 ( hmgb1 ) , a biomarker of necrosis .", "however , recent consensus agrees that specific genes can regulate necrosis , which is termed necroptosis .", "the kinases receptor - interacting protein 1 ( rip1 ) and rip3 are key signaling molecules in necroptosis", ". published studies have suggested that the treatment with zvad , a caspase inhibitor with broad specificity , induced autophagy and the death of l929 cells ; and this death process required rip1 , suggesting that autophagy is involved in necroptosis . in several models , autophagy has been shown to regulate necroptosis [ 116 , 117 ] . in endothelial cells , inhibition of autophagy rescues palmitic acid - induced necroptosis . on the other hand , a recent study has demonstrated that necrostatin-1 ( nec-1 ) , a specific necroptosis inhibitor , suppressed not only necrosis but also autophagy .", "these observations suggest that autophagy may be induced by necroptosis , raising the possibility that cellular stress during cell death may lead to the induction of autophagy .", "it is tempting to speculate that so - called autophagic cell death may involve elements of necroptosis , though further research will be needed to clarify this relationship , as well as the signaling pathways linking autophagy to necroptosis .", "recent observations have revealed a relationship between autophagic proteins and inflammasome - associated proinflammatory cytokine maturation in macrophages [ 122124 ] .", "inflammasomes are cytosolic multiprotein complexes that constitute a novel inflammatory signaling mechanism and which govern the maturation and secretion of distinct proinflammatory cytokines , such as il-1 , il-18 , and il-33 .", "cytosolic receptors of the nod - like receptor ( nlr ) family ( i.e. , nlrp3 , nlrp1 ) interact with accessory proteins to form inflammasome complexes . nlrp3", "interacts with an adaptor protein [ apoptosis - associated speck like protein containing card ( asc ) ] , which recruits and activates the procaspase-1 by proteolytic cleavage .", "proinflammatory cytokine secretion ( il-1 and il-18 ) was enhanced in atg16l1 or atg7 deleted macrophages in response to lps .", "in contrast , atg16l1 or atg7 deficiency did not affect tnf and ifn- production or nf-b pathway activation in macrophages stimulated with lps .", "furthermore , atg16l1 deleted mice displayed increased susceptibility to a murine model of colitis , which could be ameliorated by anti - il-18 therapy . increased activation of il-1 and il-18", "has also been observed in macrophages and monocytes isolated from mice genetically deficient in beclin 1 and lc3b .", "cytokine activation in response to lps and atp in wild - type macrophages , as well as the amplification observed in lc3b or beclin 1 deficient macrophages , required the nlrp3 inflammasome pathway [ 123 , 124 ] .", "the mechanism by which autophagy deficiency enhanced nlrp3 inflammasome pathway activation involved mitochondrial dysfunction , including the enhanced production of mitochondrial ros and increased mitochondrial membrane permeability transition [ 122 , 123 ] .", "the pathway to caspase-1 dependent il-18 secretion in macrophages was inhibited by mitochondrial targeting antioxidants .", "these experiments suggest that autophagic proteins dampen inflammasome pathway activation by stabilizing mitochondria and/or maintaining mitochondrial quality control through autophagy .", "in contrast to negative regulation of autophagy by the inflammasome , a recent study demonstrates that autophagy induction by starvation enhances caspase-1 activation and secretion of il-1 and il-18 .", "it is possible that a distinct type of autophagy induction might differentially regulate the inflammasome pathway . taken together these studies suggest an important role for autophagic proteins in the dampening of proinflammatory responses , which warrants further investigation in models of inflammatory disease .", "in addition to confirmed negative regulatory roles of autophagy in inflammasome activation , it has been shown that stimulation of inflammasome pathways can promote autophagosome formation through activation of the gtpase ralb .", "furthermore , p62-dependent selective autophagy processes may regulate the turnover and degradation of ubiquitinated inflammasome complexes .", "further studies suggest that stimulation of plasminogen activator inhibitor-2 by toll like receptor activation suppresses nlrp3-dependent cytokines activation by promoting the autophagic degradation of nlrp3 .", "an important and unanswered question is related to whether inflammasome activation and the generation of inflammasome - associated cytokines exert downstream consequences on autophagic processing .", "pyroptosis is triggered in inflammatory cells in response to excessive inflammation by caspase-1-dependent processes , leading to release of proinflammatory cytokines ( e.g. , il-1 , il-18 , and il-33 ) from dying cells .", "recent studies suggest that macrophages activate autophagy in parallel with inflammasome activation , as a means to delay the onset of pyroptosis .", "chemical inhibition of autophagy using 3-methyladenine or inhibition of the atg4 protease resulted in increased incidence of pyroptotic cell death in activated macrophages . the impact of autophagy modulation on the regulation of pyroptosis , and the relevance of these interactions in in vivo models of inflammatory disease and sepsis , warrants further exploration .", "autophagy is generally defined as a cellular program that ensures survival under conditions of stress . the ability of autophagy to clear damaged or denatured subcellular constituents such as aggregated protein ( i.e. , aggrephagy ) as well as to maintain mitochondrial homeostasis ( i.e. , mitophagy ) appears to play important roles in the cytoprotective and homeostatic functions of autophagy . despite the homeostatic roles , autophagy is now recognized to play complex and incompletely understood roles in cell death programs ( figure 3 ) .", "furthermore , there is considerable cross - talk between the molecular regulation of autophagy and other regulated forms of cell death [ 2326 ] .", "the role of autophagy in diseases is an emerging area of investigation , with recent studies indicating that autophagy may exert multifunctional roles in specific diseases , with the potential for both adaptive and harmful outcomes .", "furthermore , deficiency or absence in autophagic function may play a pathogenic role in select human diseases [ 2 , 1719 ] .", "additional studies are needed to define the dynamic equilibrium between autophagy , apoptosis , regulated necrosis , and other modes of cell death in the context of human disease pathogenesis .", "furthermore , additional studies are needed to determine the relevance of autophagic regulation of pyroptosis in inflammatory diseases", ". an increased understanding of these relationships would be essential in the development of therapeutics targeting the autophagy pathway for the treatment of disease ."], "abstract_text": ["<S> autophagy represents a homeostatic cellular mechanism for the turnover of organelles and proteins , through a lysosome - dependent degradation pathway . during starvation </S>", "<S> , autophagy facilitates cell survival through the recycling of metabolic precursors . </S>", "<S> additionally , autophagy can modulate other vital processes such as programmed cell death ( e.g. , apoptosis ) , inflammation , and adaptive immune mechanisms and thereby influence disease pathogenesis . </S>", "<S> selective pathways can target distinct cargoes ( e.g. , mitochondria and proteins ) for autophagic degradation . at present , the causal relationship between autophagy and various forms of regulated or nonregulated cell death remains unclear . </S>", "<S> autophagy can occur in association with necrosis - like cell death triggered by caspase inhibition . </S>", "<S> autophagy and apoptosis have been shown to be coincident or antagonistic , depending on experimental context , and share cross - talk between signal transduction elements . </S>", "<S> autophagy may modulate the outcome of other regulated forms of cell death such as necroptosis . </S>", "<S> recent advances suggest that autophagy can dampen inflammatory responses , including inflammasome - dependent caspase-1 activation and maturation of proinflammatory cytokines . </S>", "<S> autophagy may also act as regulator of caspase-1 dependent cell death ( pyroptosis ) . </S>", "<S> strategies aimed at modulating autophagy may lead to therapeutic interventions for diseases in which apoptosis or other forms of regulated cell death may play a cardinal role . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Autophagy in Cellular Homeostasis", "3. Autophagy and Apoptosis", "4. Autophagy, Necrosis, and Necroptosis", "5. Autophagy, Inflammasome Activation, and Cross-Talk to Pyroptosis ", "6. Conclusions and Therapeutic Implications"], "sections": [["macroautophagy ( abbreviated as  autophagy  ) is a genetically regulated and evolutionarily conserved pathway for the degradation of subcellular components [ 15 ] .", "this process involves the de novo formation of cytoplasmic double membrane - bound vacuoles termed autophagosomes , which sequester cytosolic cargo for delivery to the lysosomes [ 5 , 6 ] .", "autophagic cargoes may include various subcellular targets typified by ubiquitin - modified or long - lived proteins and major cytosolic organelles ( e.g. , mitochondria and peroxisomes ) [ 79 ] .", "however , a number of other potential substrates have been identified , including lipids , nucleic acids , reticulocytes , and invading pathogens ( e.g. , intracellular bacteria , viruses , etc . ) [ 7 , 10 ] .", "the autophagic pathway proceeds through several defined steps : ( i ) the initiation phase involving the formation of an isolation membrane or phagophore , ( ii ) the elongation of the phagophore , ( iii ) the maturation of an autophagosome with assimilation of a cytosolic cargo , ( iv ) the fusion of the mature autophagosome to the lysosome , and finally ( v ) the degradation phase where the contents are digested by lysosomal proteases ( e.g. , cathepsins ) and other hydrolytic enzymes [ 15 ] ( figure 1 ) .", "autophagy has been recognized as an essential function for cell homeostasis and adaptation to environmental stress conditions including nutritional starvation , energy depletion , endoplasmic reticulum stress , oxidative stress , and hypoxia [ 1114 ] .", "furthermore , autophagy plays a vital role in innate and adaptive immune mechanisms , including resistance to pathogen infections [ 10 , 15 , 16 ] .", "the role of autophagy in diseases is an emerging area of investigation , with recent studies indicating that autophagy may exert multifunctional roles in specific diseases , with the potential for both adaptive and maladaptive outcomes .", "furthermore , deficiency or absence in autophagic function may also contribute to the pathogenesis of human diseases [ 2 , 12 , 1719 ] .", "the occurrence of autophagy in response to environmental stress , most notably starvation , is generally regarded as a cell survival mechanism [ 2022 ] . due to the often coincident appearance of morphological and biochemical markers of autophagy in cells that are dying ,", "the relationship between autophagy and cell death has been both extensively studied and speculated upon [ 2326 ] .", "autophagic cell death  to describe a form of caspase - independent necrosis - like cell death associated with accumulation of autophagosomes in cells .", "this classification is now controversial , and the casual relationship between autophagy and cell death remains unproven [ 25 , 26 ] .", "nevertheless , many studies have pointed to intimate relationships between autophagy and cellular death programs , which are not yet fully understood .", "recent studies have also examined potential cross - talk between the signaling pathways that regulate autophagy and those that regulate distinct forms of regulated cell death such as apoptosis .", "the major types of cell death which have been studied most extensively in the context of autophagy research include apoptosis , necrosis , necroptosis , and pyroptosis , as briefly summarized here .", "apoptosis denotes a regulated form of cell death that requires the coordinated action of proteases and nucleases within an intact plasma membrane .", "morphological characteristics of apoptosis include dna fragmentation , plasma membrane blebbing , cell shrinkage , and cellular decomposition into membrane - bound apoptotic bodies which are removed by phagocytosis [ 3033 ] .", "the cardinal biochemical features of apoptosis include mitochondrial dysfunction , respiratory chain inhibition , loss of inner mitochondrial membrane potential ( m ) , increased mitochondrial membrane permeability , and externalization of phosphatidylserine [ 3033 ] .", "apoptosis has a crucial function in the maintenance of tissue homeostasis under physiological conditions and also serves as a component of developmental programs and furthermore may also contribute to disease pathogenesis .", "( mitochondria - dependent ) apoptotic pathway represents a major mechanism by which exposure to harmful extracellular stimuli triggers apoptosis .", "this pathway is dependent on a proteolytic activation cascade for both regulation and execution ( i.e. , caspases ) and subject to regulation by bcl-2 family proteins . the extrinsic apoptotic pathway , which shares common downstream features with the intrinsic pathway , is defined by its dependence on receptor - ligand ( e.g. , fas - fas ligand ) interactions for initiation .", "necrosis is a type of cell death that results from acute , accidental , or nonphysiological injury [ 3033 ] .", "this type of cell death is associated with cell lysis as the consequence of membrane damage and subsequent leakage of cell constituents into the extracellular space , which may lead to local inflammation and damage to the surrounding tissue . in certain cases ,", "cell swelling or oncosis may precede necrosis . necrosis and apoptosis differ in morphological features , though the two processes are not necessarily mutually exclusive .", "both apoptosis and necrosis can occur in response to treatment with many injurious stimuli , usually in a dose - dependent fashion .", "many agents that cause apoptosis at low to moderate doses may ultimately cause necrosis at relatively higher doses .", "a number of endogenous events can determine the balance between apoptotic and necrotic death . cellular energy charge ( i.e. , atp levels )", "whereas atp is required for certain steps of caspase activation , rapid decline of cellular atp levels typically leads to necrotic cell death .", "the existence of necrotic cell death pathways regulated by an intrinsic death program distinct from that of apoptosis has also been proposed .", "necroptosis ,  that resembles necrosis has been described [ 34 , 35 ] . in this form of regulated necrosis ,", "the ligand binding of death receptors such as the tumor necrosis factor receptor 1 ( tnfr1 ) can promote the formation of a macromolecular complex ( necrosome ) , involving the receptor - interacting protein ( rip ) kinase-1 and kinase-3 that initiate necrosis [ 36 , 37 ] . increasing evidence affirms the relevance of this mode of cell death in the pathogenesis of various diseases [ 3842 ] .", "pyroptosis represents a form of cell death that is triggered by proinflammatory signals and which is associated with inflammation [ 32 , 43 , 44 ] .", "this type of cell death occurs primarily in inflammatory cells such as macrophages and may be triggered by bacterial or pathogen infections .", "caspase-1 is responsible for the maturation of proinflammatory cytokines such as il-1 and il-18 through inflammasome - dependent pathways", "cell death occurs as a result of membranous pore formation and cytoplasmic swelling and leakage of cytosolic contents .", "similar to apoptotic cells , pyroptotic cells may also display dna fragmentation and nuclear condensation .", "the molecular machinery of autophagic regulation is the subject of recent reviews [ 45 , 46 ] .", "subsequent to their identification in yeast , a number of critical autophagy - related genes ( atg ) have been identified whose gene products regulate distinct steps in the induction or progression of autophagy [ 45 , 46 ] . in brief , the autophagy pathway responds to regulation by nutrient status , including nutrient deficiency ( starvation ) and loss of energy charge .", "starvation induces autophagy through the inhibition of mammalian target of rapamycin ( mtor ) , which resides in a multiprotein complex , mtorc1 . in response to stimulation by nutrients or growth factors", ", mtorc1 negatively regulates a macromolecular substrate complex that includes ulk1 , atg13 , atg101 , and fip200 ( rb1cc1 ) , which results in autophagy suppression [ 4854 ] .", "energy depletion , which stimulates autophagy , inhibits mtorc1 , in part through activation of the amp - dependent protein kinase ( ampk ) , leading to the activation of ulk1 , an important initiating step in autophagy [ 47 , 55 , 56 ] .", "autophagy is also coregulated by a multiprotein complex consisting of beclin 1 ( homologue of yeast atg6 ) , which associates with class iii phosphatidylinositol-3-kinase ( vps34 ) and a number of additional stimulatory or inhibitory coregulatory proteins ( e.g. , atg14l , uvrag , ambra1 , and rubicon ) . in response to proautophagic stimuli ,", "the increased production of phosphatidylinositol-3-phosphate ( pi3p ) by this complex regulates autophagosome formation [ 57 , 58 ] .", "the beclin 1 complex is subject to negative regulation by the pi3  k / akt pathway   as well by binding interactions with antiapoptotic bcl-2 family proteins .", "following phagophore formation , the elongation of the autophagosome membrane requires the action of two ubiquitin - like conjugation systems : the atg5-atg12 conjugation system and the microtubule - associated protein-1 light chain 3 ( lc3 , atg8 ) conjugation system [ 61 , 62 ] .", "atg4b converts the proform of lc3b to its cytosolic free form ( lc3-i ) . in mammals ,", "the conversion of lc3-i ( and other atg8 homologues ) to its phosphatidylethanolamine - conjugated and autophagosome - membrane associated form ( i.e. , lc3-ii ) is an initiating step in autophagy [ 6366 ] ."], ["once thought to be relatively nonspecific , it is now believed that autophagy is a highly selective process in which distinct cellular mechanisms are employed to identify and target cargo to autophagosomes .", "such selective autophagy pathways have been identified for the turnover of mitochondria ( mitophagy ) and other organelles and the turnover of denatured protein ( aggrephagy ) . during starvation ( e.g. ,", "deprivation of glucose or growth factors or depletion of cellular energy charge ) autophagy prolongs cell survival through the degradation and recycling of cellular macromolecules .", "mice deficient in the autophagy protein atg5 are susceptible to the lethal effects of starvation .", "inhibition of autophagy by beclin 1 or atg5 knockdown , or by chemical inhibitors such as 3-methyladenine , can promote apoptosis and caspase-3 activation in starved hela cells .", "these studies have suggested a role for autophagy as a means for prolonging cell survival during starvation .", "autophagy performs a cardinal homeostatic function in the removal of damaged or dysfunctional mitochondria , in a selective process referred to as mitophagy .", "the increased turnover of mitochondria by mitophagy may occur as a result of chemical or physical stress ( e.g. , hypoxia ) .", "nix localizes in the outer mitochondrial membrane and directly interacts with mammalian atg8 homologs through its lir motif . damaged or dysfunctional", "mitochondria are recruited to the autophagosome for removal by mitophagy through a process regulated by the phosphatase and tensin homolog deleted in chromosome 10 ( pten)-induced putative kinase 1 ( pink1 ) and parkinson protein-2 ( parkin ) [ 9 , 69 , 70 ] .", "mutations in the corresponding pink1 and park2 genes are associated with recessive familial forms of parkinson 's disease . in mice , pink1 and park2 deletions", "loss of mitochondrial membrane potential and the increased production of mitochondrial reactive oxygen species ( ros ) may provide initiating signals for mitophagy .", "pink1 , a transmembrane protein , is stabilized on damaged or depolarized mitochondria . following the decline of mitochondrial membrane potential , which can be caused by chemical stress , pink1 recruits cytosolic parkin , an e3 ubiquitin protein ligase , to the mitochondria [ 69 , 70 , 73 ] .", "parkin ubiquitinates mitochondrial outer membrane proteins including porin , mitofusin , and miro [ 74 , 75 ] .", "ubiquitinated mitochondria are subsequently recognized and targeted to autophagosomes by the autophagic cargo adaptor protein p62 [ 9 , 69 , 70 ] .", "autophagy can maintain cellular protein homeostasis ( proteostasis ) by providing a mechanism for the removal of ubiquitinated protein aggregates , in a selective process termed aggrephagy .", "recent studies suggest that autophagy may provide an alternative pathway to proteolysis in addition to the ubiquitin proteasome system [ 7678 ] .", "aggrephagy requires the selective autophagy cargo adaptor p62/sqstm1 ( p62 ) which can interact with ubiquitinated proteins through a ubiquitin - associated ( uba ) domain .", "furthermore , p62 can interact with lc3 through its lir ( lc3-interacting region ) motif and thereby facilitate the targeting of ubiquitinated proteins to autophagosomes .", "the selective autophagy adaptor , nbr1 ( neighbor of brca1 gene 1 ) , promotes the formation of ubiquitin - positive protein aggregates , facilitating their sequestration and removal by aggrephagy .", "this process involves the 400  kda , pi3p - binding autophagy - linked fyve domain protein ( alfy ) , a p62-interacting protein .", "in addition to mitophagy , other forms of organelle - specific or substrate - specific autophagy have been identified and collectively may contribute to the maintenance of cellular integrity under stress .", "these include the selective autophagic degradation of peroxisomes , ribosomes , and endoplasmic reticulum fragments .", "in addition to protein , autophagic processes have been implicated in the degradation of diverse cellular biomolecules , including lipids   and rna .", "furthermore , autophagy can degrade exogenously derived substrates , most notably bacteria , virus particles , and other parasites , in a selective process termed  xenophagy  [ 10 , 15 , 16 ] . although recent studies begin to unravel the role of mitochondrial selective autophagy in cell death pathways , the role of diverse selective autophagy pathways in the modulation of cell death programs remains largely uncharted territory ."], ["despite a widely accepted role for autophagy in cellular survival , autophagy has also been associated with the regulation of various cell death pathways , most notably apoptosis .", "thus , autophagy may represent a failed adaptive mechanism that may have prevented death under milder conditions .", "hypothetically , excess activation of autophagy may contribute to apoptotic cell death through unchecked degradative processes .", "cells undergoing autophagy display an increase in autophagic vesicles ( i.e. , autophagosomes and autophagolysosomes ) .", "the distinctions between autophagy and apoptosis remain incompletely delineated , as the two processes are not always mutually exclusive and may occur simultaneously in the same cell type .", "recent studies suggest that factors well known to regulate apoptosis pathways also have the potential to exert regulatory activity on factors that regulate autophagy and vice - versa ( figure 2 ) .", "how these regulatory events , termed  cross - talk  , are integrated into a mechanism for the determination of cell fate yet remains incompletely understood .", "antiapoptotic bcl-2 family proteins , which downregulate apoptosis ( i.e. , bcl-2 ) by antagonizing the activity of proapoptotic proteins , can downregulate autophagy .", "binding of these bcl-2 family proteins to beclin 1 inhibits autophagy by preventing the association of beclin 1 with the class iii pi3k complex [ 57 , 60 ] .", "bnip3 is a bh3-only protein that can trigger apoptosis by sequestering antiapoptotic bcl-2 family proteins and promoting bax / bad dependent mitochondrial release of proapoptotic mediators .", "these interactions suggest that autophagy and apoptosis may be coordinately regulated by bcl-2 family proteins .", "experimental evidence also suggests that , once activated , apoptosis effector molecules may suppress autophagy ; for example , beclin 1 may be cleaved and inactivated by caspases during activation of apoptosis .", "further studies suggest that certain atg proteins may play dual roles in autophagy / apoptosis regulation ; for example , the autophagic protein atg5 may affect extrinsic apoptosis pathways through interactions with the fas - associated death domain ( fadd ) protein .", "atg5 which regulates autophagy can be subject to calpain - dependent cleavage to generate a proapoptotic truncation product ( tatg5 ) .", "this cleavage product promotes apoptosis by binding to and inhibiting antiapoptotic proteins such as bcl - xl .", "recent studies also implicate atg12 , the binding partner for atg5 required for autophagosomal elongation , as an effector of the intrinsic apoptosis pathway .", "atg12 may bind to and inactivate antiapoptotic bcl2 family proteins ( e.g. , bcl-2 and mcl-1 ) , through an interaction involving a bh2 motif , and thereby act as a proapoptotic regulator .", "recent advances have identified other regulatory targets for caspase regulation among autophagy related molecules ; for example , atg4d , a member of the atg4 family of atg8 processing enzymes , has been identified as a substrate for proapoptotic caspase-3 .", "the autophagic protein atg3 has recently been identified as a caspase-8 substrate , which is cleaved during tnf-induced apoptosis .", "the antiapoptotic protein cellular flice - like inhibitory protein ( c - flip ) can act as a negative regulator of autophagy .", "the c - flip , an endogenous inhibitor of caspase-8 processing and the extrinsic apoptotic pathway , acts to prevent the binding of atg3 to lc3 , which impairs lc3 processing .", "on the basis of what is known about the molecular cross - talk between autophagy and apoptosis it currently remains unclear whether autophagy and apoptosis are coregulated or mutually exclusive processes .", "antiapoptotic ( e.g. , bcl-2 ) as well as proapoptotic ( e.g. , caspase-3 ) molecules can downregulate autophagy by interacting with beclin 1 .", "furthermore , other caspase - dependent events have been implicated in anti - autophagy or proautophagy events .", "for example caspase processing of atg4d is proautophagy , whereas caspase processing of beclin 1 is antiautophagy .", "the definitive cellular mechanisms that control the decision to embark on each one or both of these pathways in response to specific stimuli remain unclear .", "analysis of any single isolated regulatory component ( using sirna knockdown for example ) for its potential to cross - regulate autophagy and/or apoptosis will be unlikely to answer these questions .", "thus , an integrative approach is needed to understand how the entire molecular machinery of apoptosis and autophagy are coordinated to influence cell fate decisions .", "the p53 tumor suppressor protein is a well studied regulator of cell cycle progression and apoptosis .", "p53 modulates the expression of bcl-2 family proteins ( e.g. , bax , bid ) and other apoptosis - related gene targets ( e.g. , apaf1 ) . the nuclear form of p53 targets the expression of dram ( damage regulated autophagic modulator ) , which can stimulate both autophagy and apoptosis . alternatively", ", p53 can induce autophagy through the upregulation of ampk , which downregulates the mtor pathway .", "recent studies have shown that genetic or pharmacological inhibition of p53 can also activate autophagy and have led to the identification of the cytoplasmic form of p53 as an inhibitor of autophagy .", "chemical stimuli known to induce autophagy can promote the proteasomal degradation of p53 . cellular stimulation with interferon- ( ifn- )", "induces the deacetylation of p53 , leading to suppressed bmf expression , reduced complex formation between beclin 1 and bcl2 , and enhanced autophagy .", "taken together these studies suggest a complex role of p53 in the regulation of autophagy , with opposing roles for the cytosolic and nuclear forms of p53 [ 98 , 99 ] .", "several recent studies , supported by genetic manipulation of the autophagy program , have revealed that in select toxicological models , autophagy may be associated with the promotion of apoptosis . in our recent studies", "we have found that epithelial cells subjected to cigarette smoke extract ( cse ) exposure die by activation of the extrinsic apoptosis pathway [ 100 , 101 ] .", "cse - induced cell death involved activation of the fas - dependent death - inducing signaling complex ( disc ) and downstream activation of caspases ( -8,-9,-3 ) .", "epithelial cells subjected to cse exposure concurrently responded with increased autophagosome formation and increased processing of lc3b - i to lc3b - ii in epithelial cells [ 100 , 101 ] .", "knockdown of autophagy proteins beclin 1 or lc3b inhibited apoptosis in response to cse exposure in vitro , suggesting that increased autophagy occurred in association with epithelial cell death [ 100 , 101 ] .", "further studies revealed that lc3b may act as a regulatory factor in extrinsic apoptosis in this model .", "lc3b was found to engage a complex with fas , the key component of the disc , in a fashion dependent on the lipid raft protein caveolin-1 .", "cse exposure caused the rapid dissociation of lc3b from fas , in association with the activation of apoptosis signaling . in conclusion , these results using genetic knockdown experiments have implicated a proapoptotic role for lc3b , in a specialized model of cse - induced toxicity , though the relative role of autophagic activity in promoting cell death in this model remains unclear [ 100 , 101 ] . it should be noted that cse - induced autophagy may differ from starvation - induced autophagy in that it occurs in the presence of a complex mixture of foreign matter , which may potentially alter the functionality of the autophagy response .", "thus , the concept of  toxic autophagy  may involve altered function , which may be dependent not only on whether its activation is physiological or excessive , but also on the nature of foreign substrates ( e.g. , complex xenobiotics such as tar or virus particles ) and their interactions with autophagosomes .", "further examples of coincident autophagy and apoptosis include p53-dependent autophagy through upregulation of dram , which is coincidental with upregulation of apoptosis .", "deletion of atg5 was also shown to protect cells from prodeath environmental stimuli ; however , the authors attributed this resistance to compensatory activation of chaperone - dependent autophagy , rather than inhibition of macroautophagy per se .", "these studies raise an important issue in that genetic knockdown of one specific autophagy - related factor can not establish whether autophagy was protective or not in any context , as downregulation of the target may potentially affect signaling pathways that are independent of autophagy , or alternatively , promote compensatory mechanisms , such as alternate forms of autophagy ."], ["the terms  autophagic cell death  or type ii programmed cell death have been previously used to refer to cell death distinct from apoptosis that occurs in association with increases in autophagosome formation and independently of caspases .", "many studies that have implicated autophagy as a cell death effector have been performed on apoptosis - compromised or caspase - deficient cells ; for example , cells treated with z - vad - fmk , a general inhibitor of caspases , or with caspase-8 and calpain inhibitors , die essentially by a nonapoptotic pathway characterized by dramatic accumulations of autophagic vacuoles [ 104107 ] .", "genetic knockdown experiments ( e.g. , beclin 1 ) suggest that autophagy contributes to cytotoxicity in these models ; however , contrasting studies , also using knockdown of autophagy proteins , have also suggested that autophagy can also protect in the context of nonapoptotic cell death induced by caspase inhibition . in baxbak mouse", "embryonic fibroblasts ( mefs ) , which can not activate intrinsic apoptosis , treatment with chemotherapeutic agents results in nonapoptotic necrosis - like cell death accompanied by excessive autophagosome formation .", "currently it remains unclear whether the process of autophagy acts as an effector or bystander of caspase - independent necrosis - like cell death , though autophagic proteins likely play an accessory role [ 25 , 26 ] .", "experiments in tumor cells have suggested the possibility of cross - talk between autophagy and necrosis in cells .", "autophagy provides a protective function to limit tumor necrosis and inflammation in response to metabolic stress .", "while autophagy acts to buffer metabolic stress , the combined impairment of apoptosis and autophagy promotes necrotic cell death in vitro and in vivo . although it remains to be determined what triggers necrosis in tumor cells , it is likely that insufficient atp production to maintain plasma - membrane integrity results in metabolic catastrophe and cell lysis [ 110 , 112 ] .", "autophagy integrates a metabolic feedback system to allow sufficient atp generation to maintain cell viability .", "enhanced autophagy by spermidine , a natural polyamine , inhibits loss of membrane integrity and release of chromatin protein high mobility group b1 ( hmgb1 ) , a biomarker of necrosis .", "however , recent consensus agrees that specific genes can regulate necrosis , which is termed necroptosis .", "the kinases receptor - interacting protein 1 ( rip1 ) and rip3 are key signaling molecules in necroptosis", ". published studies have suggested that the treatment with zvad , a caspase inhibitor with broad specificity , induced autophagy and the death of l929 cells ; and this death process required rip1 , suggesting that autophagy is involved in necroptosis . in several models , autophagy has been shown to regulate necroptosis [ 116 , 117 ] . in endothelial cells , inhibition of autophagy rescues palmitic acid - induced necroptosis . on the other hand , a recent study has demonstrated that necrostatin-1 ( nec-1 ) , a specific necroptosis inhibitor , suppressed not only necrosis but also autophagy .", "these observations suggest that autophagy may be induced by necroptosis , raising the possibility that cellular stress during cell death may lead to the induction of autophagy .", "it is tempting to speculate that so - called autophagic cell death may involve elements of necroptosis , though further research will be needed to clarify this relationship , as well as the signaling pathways linking autophagy to necroptosis ."], ["recent observations have revealed a relationship between autophagic proteins and inflammasome - associated proinflammatory cytokine maturation in macrophages [ 122124 ] .", "inflammasomes are cytosolic multiprotein complexes that constitute a novel inflammatory signaling mechanism and which govern the maturation and secretion of distinct proinflammatory cytokines , such as il-1 , il-18 , and il-33 .", "cytosolic receptors of the nod - like receptor ( nlr ) family ( i.e. , nlrp3 , nlrp1 ) interact with accessory proteins to form inflammasome complexes . nlrp3", "interacts with an adaptor protein [ apoptosis - associated speck like protein containing card ( asc ) ] , which recruits and activates the procaspase-1 by proteolytic cleavage .", "proinflammatory cytokine secretion ( il-1 and il-18 ) was enhanced in atg16l1 or atg7 deleted macrophages in response to lps .", "in contrast , atg16l1 or atg7 deficiency did not affect tnf and ifn- production or nf-b pathway activation in macrophages stimulated with lps .", "furthermore , atg16l1 deleted mice displayed increased susceptibility to a murine model of colitis , which could be ameliorated by anti - il-18 therapy . increased activation of il-1 and il-18", "has also been observed in macrophages and monocytes isolated from mice genetically deficient in beclin 1 and lc3b .", "cytokine activation in response to lps and atp in wild - type macrophages , as well as the amplification observed in lc3b or beclin 1 deficient macrophages , required the nlrp3 inflammasome pathway [ 123 , 124 ] .", "the mechanism by which autophagy deficiency enhanced nlrp3 inflammasome pathway activation involved mitochondrial dysfunction , including the enhanced production of mitochondrial ros and increased mitochondrial membrane permeability transition [ 122 , 123 ] .", "the pathway to caspase-1 dependent il-18 secretion in macrophages was inhibited by mitochondrial targeting antioxidants .", "these experiments suggest that autophagic proteins dampen inflammasome pathway activation by stabilizing mitochondria and/or maintaining mitochondrial quality control through autophagy .", "in contrast to negative regulation of autophagy by the inflammasome , a recent study demonstrates that autophagy induction by starvation enhances caspase-1 activation and secretion of il-1 and il-18 .", "it is possible that a distinct type of autophagy induction might differentially regulate the inflammasome pathway . taken together these studies suggest an important role for autophagic proteins in the dampening of proinflammatory responses , which warrants further investigation in models of inflammatory disease .", "in addition to confirmed negative regulatory roles of autophagy in inflammasome activation , it has been shown that stimulation of inflammasome pathways can promote autophagosome formation through activation of the gtpase ralb .", "furthermore , p62-dependent selective autophagy processes may regulate the turnover and degradation of ubiquitinated inflammasome complexes .", "further studies suggest that stimulation of plasminogen activator inhibitor-2 by toll like receptor activation suppresses nlrp3-dependent cytokines activation by promoting the autophagic degradation of nlrp3 .", "an important and unanswered question is related to whether inflammasome activation and the generation of inflammasome - associated cytokines exert downstream consequences on autophagic processing .", "pyroptosis is triggered in inflammatory cells in response to excessive inflammation by caspase-1-dependent processes , leading to release of proinflammatory cytokines ( e.g. , il-1 , il-18 , and il-33 ) from dying cells .", "recent studies suggest that macrophages activate autophagy in parallel with inflammasome activation , as a means to delay the onset of pyroptosis .", "chemical inhibition of autophagy using 3-methyladenine or inhibition of the atg4 protease resulted in increased incidence of pyroptotic cell death in activated macrophages . the impact of autophagy modulation on the regulation of pyroptosis , and the relevance of these interactions in in vivo models of inflammatory disease and sepsis , warrants further exploration ."], ["autophagy is generally defined as a cellular program that ensures survival under conditions of stress . the ability of autophagy to clear damaged or denatured subcellular constituents such as aggregated protein ( i.e. , aggrephagy ) as well as to maintain mitochondrial homeostasis ( i.e. , mitophagy ) appears to play important roles in the cytoprotective and homeostatic functions of autophagy . despite the homeostatic roles , autophagy is now recognized to play complex and incompletely understood roles in cell death programs ( figure 3 ) .", "furthermore , there is considerable cross - talk between the molecular regulation of autophagy and other regulated forms of cell death [ 2326 ] .", "the role of autophagy in diseases is an emerging area of investigation , with recent studies indicating that autophagy may exert multifunctional roles in specific diseases , with the potential for both adaptive and harmful outcomes .", "furthermore , deficiency or absence in autophagic function may play a pathogenic role in select human diseases [ 2 , 1719 ] .", "additional studies are needed to define the dynamic equilibrium between autophagy , apoptosis , regulated necrosis , and other modes of cell death in the context of human disease pathogenesis .", "furthermore , additional studies are needed to determine the relevance of autophagic regulation of pyroptosis in inflammatory diseases", ". an increased understanding of these relationships would be essential in the development of therapeutics targeting the autophagy pathway for the treatment of disease ."]]}
{"article_id": "PMC4194386", "article_text": ["koreich in 1981 first reported the case of a 22 year old man diagnosed as hodgkin lymphoma ( hl ) who developed hypothermia and hypotension during the course of his admission , which resolved only after initiation of chemotherapy .", "systemic symptoms are present in a third of patients at presentation and among them fever is the most common followed by night sweats and weight loss . on review of literature , we found 18 case reports with similar finding of hypotension and hypothermia associated with hl .", "most of the patients in these case reports developed hypotension and hypothermia after initiation of chemotherapy .", "only 3 out of these 18 patients were found to have hypotension at the time of presentation prior to diagnosis .", "we report the case of a patient who presented with hypotension and hypothermia and was diagnosed initially with septic shock .", "he was found to have ill - defined liver lesions on the computed tomography ( ct ) scan which were new on comparison with a recent ct abdomen done for evaluation of nephrolithiasis .", "the pathophysiology of development of hypotension and hypothermia in patients during chemotherapy or prior to diagnosis remains unexplained .", "a complex interaction between cytokines , tumor and blood vessels has been proposed as the genesis of such a presentation .", "a 61 year old man presented to emergency room with history of flank pain on right side , dysuria , urgency and frequency with occasional hematuria for 3 days associated with fever , chills and rigors .", "3 weeks before this presentation he was admitted for renal colic and was found to have a new staghorn calculus in the kidney which was managed conservatively with 7 days of oral antibiotics .", "review of systems noted a history of 40 pounds weight loss over 3 months , drenching night sweats and occasional low grade fevers for last 3 months .", "past medical history was significant for multiple episodes of renal colic secondary to nephrolithiasis treated with lithotripsy several years ago .", "social history was significant for 30 pack year history of smoking , occasional alcohol consumption and no substance abuse or high risk behavior .", "was noted to be hypotensive with a blood pressure ( bp ) of 78/49 mmhg and mean arterial pressure ( map ) of 59 mmhg .", "the hypotension was new compared with recent admission 3 weeks prior , where the bp readings were consistently above a map of 80 .", "the hypotension did not correct with bolus of 3 liters of 0.9% normal saline ( ns ) and in view of his history of dysuria and intermittent hematuria and recent diagnosis of staghorn calculus he was diagnosed with urinary tract infection ( uti ) leading to urosepsis and septic shock .", "he was admitted to medical intensive care unit ( micu ) where he was started on pressor support with norepinephrine and broad spectrum antibiotic coverage with vancomycin and piperacillin - tazobactam .", "vitals recorded at presentation were temperature of 97.3f , bp of 78/49 mmhg , map of 59 mmhg , heart rate 80/min , respiratory rate 18/min , spo2 of 98 - 99% on room air .", "general exam was significant for an averaged sized man in mild distress with mild pallor , no icterus , cyanosis or edema .", "labs were significant for hemoglobin ( hb ) of 9.2 g / dl , mean corpuscular volume ( mcv ) of 77.6 fl and leukocyte count ( wbc ) of 5.1 k/l with differential of 77% neutrophils .", "liver enzymes showed alkaline phosphatase of 212 u / l , alanine transaminase ( alt ) 80 u / l , aspartate tranaminase ( ast ) 96 u / l , total protein 3.6 g / dl and albumin 1.5 g / dl .", "urine dipstick was positive for blood ( 1 + ) , proteins ( 30 ) and glucose ( 50 ) and urine microscopy showed 109 rbc and 12 wbc .", "two sets of blood culture and urine culture were done prior to starting antibiotics which showed no growth after 5 days of incubation .", "ct scan abdomen done at presentation showed numerous ill defined lesions in the liver which were new from the ct scan done 3 weeks before for evaluation of renal colic ( figure 1 ) .", "the study redemonstrated the staghorn calculus with no evidence of obstruction , no radiographic evidence of pyelonephritis or renal abscess .", "patient received 2 days of pressor support with norepinephrine drip , following which his blood pressure improved to a map over 70 mmhg , however he continued to have intermittent episodes of hypotension which were managed with frequent boluses of 1000 to 500 ml of 0.9% ns .", "interestingly , on day 5 of admission , patient developed increased shortness of breath and became hypoxic .", "trans - thoracic echocardiogram done at bedside showed normal ejection fraction and normal inferior vena cava .", "patient was diagnosed with fluid overload secondary to frequent fluid boluses and was given one dose of 20 mg i.v .", "lasix which led to resolution of shortness of breath . during this entire stay , he continued to have intermittent episodes of hypotension with mean arterial pressure dropping to low 60 s .", "colonoscopy and esophago - gastro - duo - denoscopy ( egd ) done as part of malignancy workup , showed 2 polyps which were diagnosed as tubular adenoma and thick gastric folds with chronic gastritis on histopathology respectively .", "a liver biopsy was planned after improvement in his overall condition however on day 9 of the hospitalization patient declined the procedure and requested a break from the hospital .", "liver biopsy was deferred for a later date and patient was discharged in a stable condition . during this", "admission 4 blood cultures and 3 urine cultures did not show any growth after 5 days of incubation .", "he was discharged with oral levofloxacin to complete a course of 14 days of antibiotics for complicated uti .", "three days after being discharged from hospital , patient returned to emergency room with similar complaints of acute onset weakness and fatigue and a single episode of fever for which he received a single dose of ibuprofen at home .", "vitals at presentation showed rectal temperature of 94.1f , bp of 84/49 mmhg , mean arterial pressure of 60 mmhg , breathing at rate of 18/min , heart rate 59/min and saturating 97% on room air .", "patient was readmitted to micu with a provisional diagnosis of urosepsis and was given vancomycin and piperacillin - tazobactam . blood culture and urine culture", "done at this time again showed no growth which could support the diagnosis of sepsis .", "biopsy of the liver lesions showed extensive lymphocytic and histiocytic infiltrates with abnormally large cells and positive stains for cd15 and cd30 .", "bone marrow biopsy also showed areas of residual trilineage hematopoesis with 40% cellularity alongwith several para - trabecular infiltrates composed of large atypical cells including reed - sternberg ( rs ) cells , in a mixed inflammatory background consisting of small lymphocytes , histiocytes , eosinophils and plasma cells ( figure 2 ) .", "the immuno - histochemical stains were positive for cd15 and cd30 and negative for cd45 , cd3 and cd20 .", "a 100 cell count showed granulocytes ( 56% ) , monocytes ( 1% ) , eosinophils ( 6% ) , erythroid precursors ( 34% ) , lymphocytes ( 2% ) and plasma cells ( 1% ) .", "the presence of the characteristic rs cell in a mixed inflammatory background pointed towards a diagnosis of hl .", "the diagnosis was further confirmed by positive immune - histo - chemical ( ihc ) stain for cd15 and cd30 along - with negative ihc stain for cd45 , cd3 and cd20 .", "since no pan - t antigens were missing , the possibility of a t - cell lymphoma was very low . a ct chest for staging did not show any hilar lymphadenopathy .", "soon after the diagnosis patient was started on chemotherapy with doxorubicin , dacarbazine , vinblastine .", "bleomycin was initially withheld due to unknown pulmonary function in view of patient been active smoker and was added later after pulmonary function test turned out to be normal .", "after completion of the 1 cycle of chemotherapy the blood pressure started improving to a map of more than 80 mmhg ( figure 3 ) . at 6 month", "follow up the patient continues to be free of any episode of hypotension or hypothermia .", "reported the first patient with hl who developed hypothermia and hypotension during the course of admission , prior to initiation of chemotherapy . since then 18 more cases have been reported with hypothermia as presenting feature and in 8 out of 18 patients , hypotension has also been recorded ( table 2 ) .", "fifteen out of 18 patients reported in literature had developed these symptoms after initiation of chemotherapy with only 3 patients presenting with hypotension prior to diagnosis .", "interestingly , most of the patients described with hypothermia and hypotension have been reported to have liver metastases . to date", ", the mechanism behind hypotension and hypothermia as isolated manifestations of hl remains unexplained . in our patient", "though the presentation of the patient was highly suggestive of urosepsis , numerous blood and urine cultures did not grow any microbe which could have explained the cause of sepsis .", "moreover treatment with vancomycin and piperacillintazobactam did not lead to improvement in the condition of the patient , essentially ruling out infectious cause and sepsis as the primary cause of hypotension .", "an increased random and morning cortisol level and tsh / free t4 level ruled out adrenal insufficiency and thyroid involvement respectively .", "a normal transthoracic echocardiogram demonstrated a normal cardiac anatomy and clinically there were no signs of heart failure .", "though orthostatic maneuver was not done in our patient because of his general condition , the absence of correction of hypotension with volume repletion and fluid boluses makes hypovolemia less likely .", "blood pressure control is governed by a complex interaction between cardiac , renal , endocrine and nervous systems .", "hypotension in cancer patients has long being believed to be secondary to cytokine release from increased macrophage function .", "the cytokines implicated in causing hypotension are interleukin ( il)-1 and il-2 , tumor necrosis factor- ( tnf- ) and interferon- ( if- ) .", "although the exact mechanism of tnf- causing hypotension is not clear , it is well known that administration of tnf- leads to production of nitric oxide ( no ) which is a potent vasodilator and could lead to hypotension .", "in addition to this , cytokines also cause endothelial damage , a mechanism which is very well defined in patients with sepsis and septic shock . in patients with sepsis infection", "another important association implicated in this patient is the effect of psycho - neuroendocrine hormones in mediation of no , like melatonin , which has been shown to have counter - regulatory effect on action of free radicals , especially no .", "we postulate from the above , that the probable cause of hypotension in this patient could be release of cytokines from the tumor especially from liver metastasis , which is an extremely vascular organ .", "autonomic dysfunction is a know paraneoplastic syndrome associated with hl which raises the possibility of direct infiltration of hypothalamus or pineal gland by the tumor leading to alteration in neuroendocrine hormones .", "the loss of inhibition of counter - regulatory effects of these hormones on cytokine regulation could result in macrophage activation and no production resulting in hypotension .", "our patient never took any salicylates for his fever which rules this out as a possible cause .", "moreover , not all patients reported were taking nsaids before presentation , making this explanation less likely . in conclusion", "although , further studies are needed in support of this association , we can conclude from this case that the condition responds dramatically to aggressive chemotherapy ."], "abstract_text": ["<S> hypotension is an extremely rare manifestation of hodgkin lymphoma . </S>", "<S> we report the case of a patient who presented with new onset hypotension and was diagnosed with urosepsis and septic shock requiring pressor support for maintaining his blood pressure . </S>", "<S> computed tomography ( ct ) scan of abdomen showed liver lesions , which were new on comparison with a ct abdomen done 3 weeks back . </S>", "<S> biopsy of the liver lesions and subsequently a bone marrow biopsy showed large atypical reed - sternberg cells , positive for cd15 and cd 30 and negative for cd45 , cd3 and cd20 on immuno - histochemical staining , hence establishing the diagnosis of hodgkin lymphoma . </S>", "<S> the mechanism involved in hodgkin lymphoma causing hypotension remains anecdotal , but since it is mostly seen in patients with advanced hodgkin lymphoma , it is hypothetically related to a complex interaction between cytokines and mediators of vasodilatation . here </S>", "<S> we review relevant literature pertaining to presentation and pathogenesis of this elusive and rare association . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion and Conclusions"], "sections": [["koreich in 1981 first reported the case of a 22 year old man diagnosed as hodgkin lymphoma ( hl ) who developed hypothermia and hypotension during the course of his admission , which resolved only after initiation of chemotherapy .", "systemic symptoms are present in a third of patients at presentation and among them fever is the most common followed by night sweats and weight loss . on review of literature , we found 18 case reports with similar finding of hypotension and hypothermia associated with hl .", "most of the patients in these case reports developed hypotension and hypothermia after initiation of chemotherapy .", "only 3 out of these 18 patients were found to have hypotension at the time of presentation prior to diagnosis .", "we report the case of a patient who presented with hypotension and hypothermia and was diagnosed initially with septic shock .", "he was found to have ill - defined liver lesions on the computed tomography ( ct ) scan which were new on comparison with a recent ct abdomen done for evaluation of nephrolithiasis .", "the pathophysiology of development of hypotension and hypothermia in patients during chemotherapy or prior to diagnosis remains unexplained .", "a complex interaction between cytokines , tumor and blood vessels has been proposed as the genesis of such a presentation ."], ["a 61 year old man presented to emergency room with history of flank pain on right side , dysuria , urgency and frequency with occasional hematuria for 3 days associated with fever , chills and rigors .", "3 weeks before this presentation he was admitted for renal colic and was found to have a new staghorn calculus in the kidney which was managed conservatively with 7 days of oral antibiotics .", "review of systems noted a history of 40 pounds weight loss over 3 months , drenching night sweats and occasional low grade fevers for last 3 months .", "past medical history was significant for multiple episodes of renal colic secondary to nephrolithiasis treated with lithotripsy several years ago .", "social history was significant for 30 pack year history of smoking , occasional alcohol consumption and no substance abuse or high risk behavior .", "was noted to be hypotensive with a blood pressure ( bp ) of 78/49 mmhg and mean arterial pressure ( map ) of 59 mmhg .", "the hypotension was new compared with recent admission 3 weeks prior , where the bp readings were consistently above a map of 80 .", "the hypotension did not correct with bolus of 3 liters of 0.9% normal saline ( ns ) and in view of his history of dysuria and intermittent hematuria and recent diagnosis of staghorn calculus he was diagnosed with urinary tract infection ( uti ) leading to urosepsis and septic shock .", "he was admitted to medical intensive care unit ( micu ) where he was started on pressor support with norepinephrine and broad spectrum antibiotic coverage with vancomycin and piperacillin - tazobactam .", "vitals recorded at presentation were temperature of 97.3f , bp of 78/49 mmhg , map of 59 mmhg , heart rate 80/min , respiratory rate 18/min , spo2 of 98 - 99% on room air .", "general exam was significant for an averaged sized man in mild distress with mild pallor , no icterus , cyanosis or edema .", "labs were significant for hemoglobin ( hb ) of 9.2 g / dl , mean corpuscular volume ( mcv ) of 77.6 fl and leukocyte count ( wbc ) of 5.1 k/l with differential of 77% neutrophils .", "liver enzymes showed alkaline phosphatase of 212 u / l , alanine transaminase ( alt ) 80 u / l , aspartate tranaminase ( ast ) 96 u / l , total protein 3.6 g / dl and albumin 1.5 g / dl .", "urine dipstick was positive for blood ( 1 + ) , proteins ( 30 ) and glucose ( 50 ) and urine microscopy showed 109 rbc and 12 wbc .", "two sets of blood culture and urine culture were done prior to starting antibiotics which showed no growth after 5 days of incubation .", "ct scan abdomen done at presentation showed numerous ill defined lesions in the liver which were new from the ct scan done 3 weeks before for evaluation of renal colic ( figure 1 ) .", "the study redemonstrated the staghorn calculus with no evidence of obstruction , no radiographic evidence of pyelonephritis or renal abscess .", "patient received 2 days of pressor support with norepinephrine drip , following which his blood pressure improved to a map over 70 mmhg , however he continued to have intermittent episodes of hypotension which were managed with frequent boluses of 1000 to 500 ml of 0.9% ns .", "interestingly , on day 5 of admission , patient developed increased shortness of breath and became hypoxic .", "trans - thoracic echocardiogram done at bedside showed normal ejection fraction and normal inferior vena cava .", "patient was diagnosed with fluid overload secondary to frequent fluid boluses and was given one dose of 20 mg i.v .", "lasix which led to resolution of shortness of breath . during this entire stay , he continued to have intermittent episodes of hypotension with mean arterial pressure dropping to low 60 s .", "colonoscopy and esophago - gastro - duo - denoscopy ( egd ) done as part of malignancy workup , showed 2 polyps which were diagnosed as tubular adenoma and thick gastric folds with chronic gastritis on histopathology respectively .", "a liver biopsy was planned after improvement in his overall condition however on day 9 of the hospitalization patient declined the procedure and requested a break from the hospital .", "liver biopsy was deferred for a later date and patient was discharged in a stable condition . during this", "admission 4 blood cultures and 3 urine cultures did not show any growth after 5 days of incubation .", "he was discharged with oral levofloxacin to complete a course of 14 days of antibiotics for complicated uti .", "three days after being discharged from hospital , patient returned to emergency room with similar complaints of acute onset weakness and fatigue and a single episode of fever for which he received a single dose of ibuprofen at home .", "vitals at presentation showed rectal temperature of 94.1f , bp of 84/49 mmhg , mean arterial pressure of 60 mmhg , breathing at rate of 18/min , heart rate 59/min and saturating 97% on room air .", "patient was readmitted to micu with a provisional diagnosis of urosepsis and was given vancomycin and piperacillin - tazobactam . blood culture and urine culture", "done at this time again showed no growth which could support the diagnosis of sepsis .", "biopsy of the liver lesions showed extensive lymphocytic and histiocytic infiltrates with abnormally large cells and positive stains for cd15 and cd30 .", "bone marrow biopsy also showed areas of residual trilineage hematopoesis with 40% cellularity alongwith several para - trabecular infiltrates composed of large atypical cells including reed - sternberg ( rs ) cells , in a mixed inflammatory background consisting of small lymphocytes , histiocytes , eosinophils and plasma cells ( figure 2 ) .", "the immuno - histochemical stains were positive for cd15 and cd30 and negative for cd45 , cd3 and cd20 .", "a 100 cell count showed granulocytes ( 56% ) , monocytes ( 1% ) , eosinophils ( 6% ) , erythroid precursors ( 34% ) , lymphocytes ( 2% ) and plasma cells ( 1% ) .", "the presence of the characteristic rs cell in a mixed inflammatory background pointed towards a diagnosis of hl .", "the diagnosis was further confirmed by positive immune - histo - chemical ( ihc ) stain for cd15 and cd30 along - with negative ihc stain for cd45 , cd3 and cd20 .", "since no pan - t antigens were missing , the possibility of a t - cell lymphoma was very low . a ct chest for staging did not show any hilar lymphadenopathy .", "soon after the diagnosis patient was started on chemotherapy with doxorubicin , dacarbazine , vinblastine .", "bleomycin was initially withheld due to unknown pulmonary function in view of patient been active smoker and was added later after pulmonary function test turned out to be normal .", "after completion of the 1 cycle of chemotherapy the blood pressure started improving to a map of more than 80 mmhg ( figure 3 ) . at 6 month", "follow up the patient continues to be free of any episode of hypotension or hypothermia ."], ["reported the first patient with hl who developed hypothermia and hypotension during the course of admission , prior to initiation of chemotherapy . since then 18 more cases have been reported with hypothermia as presenting feature and in 8 out of 18 patients , hypotension has also been recorded ( table 2 ) .", "fifteen out of 18 patients reported in literature had developed these symptoms after initiation of chemotherapy with only 3 patients presenting with hypotension prior to diagnosis .", "interestingly , most of the patients described with hypothermia and hypotension have been reported to have liver metastases . to date", ", the mechanism behind hypotension and hypothermia as isolated manifestations of hl remains unexplained . in our patient", "though the presentation of the patient was highly suggestive of urosepsis , numerous blood and urine cultures did not grow any microbe which could have explained the cause of sepsis .", "moreover treatment with vancomycin and piperacillintazobactam did not lead to improvement in the condition of the patient , essentially ruling out infectious cause and sepsis as the primary cause of hypotension .", "an increased random and morning cortisol level and tsh / free t4 level ruled out adrenal insufficiency and thyroid involvement respectively .", "a normal transthoracic echocardiogram demonstrated a normal cardiac anatomy and clinically there were no signs of heart failure .", "though orthostatic maneuver was not done in our patient because of his general condition , the absence of correction of hypotension with volume repletion and fluid boluses makes hypovolemia less likely .", "blood pressure control is governed by a complex interaction between cardiac , renal , endocrine and nervous systems .", "hypotension in cancer patients has long being believed to be secondary to cytokine release from increased macrophage function .", "the cytokines implicated in causing hypotension are interleukin ( il)-1 and il-2 , tumor necrosis factor- ( tnf- ) and interferon- ( if- ) .", "although the exact mechanism of tnf- causing hypotension is not clear , it is well known that administration of tnf- leads to production of nitric oxide ( no ) which is a potent vasodilator and could lead to hypotension .", "in addition to this , cytokines also cause endothelial damage , a mechanism which is very well defined in patients with sepsis and septic shock . in patients with sepsis infection", "another important association implicated in this patient is the effect of psycho - neuroendocrine hormones in mediation of no , like melatonin , which has been shown to have counter - regulatory effect on action of free radicals , especially no .", "we postulate from the above , that the probable cause of hypotension in this patient could be release of cytokines from the tumor especially from liver metastasis , which is an extremely vascular organ .", "autonomic dysfunction is a know paraneoplastic syndrome associated with hl which raises the possibility of direct infiltration of hypothalamus or pineal gland by the tumor leading to alteration in neuroendocrine hormones .", "the loss of inhibition of counter - regulatory effects of these hormones on cytokine regulation could result in macrophage activation and no production resulting in hypotension .", "our patient never took any salicylates for his fever which rules this out as a possible cause .", "moreover , not all patients reported were taking nsaids before presentation , making this explanation less likely . in conclusion", "although , further studies are needed in support of this association , we can conclude from this case that the condition responds dramatically to aggressive chemotherapy ."]]}
{"article_id": "PMC4439505", "article_text": ["most conclusive evidences indicate that periodontal disease ( pd ) is not a conventional bacterial infection but is an inflammatory disease initiated by immune response against a group of microorganisms in susceptible hosts .", "the consequent uncontrolled inflammation causes destruction of attachment structures , being the most significant reason of tooth loss in adults from different populations . in affected tissues", "cytokines and chemokines lead to the migration of leukocytes to the periodontal tissues where these cells play an important role in pathogen destruction by releasing mediators in a local inflammatory response [ 4 , 5 ] .", "therefore , these and other mediators have been detected at elevated levels in the gingival crevicular fluid , saliva , and blood , thus being considered acceptable biomarkers for some aspects of pd [ 3 , 6 , 7 ] .", "particularly ,  proinflammatory cytokines such as interleukin-1 beta ( il-1 ) and tumor necrosis factor - alpha ( tnf- ) have been associated with pd progression   and decline after periodontal treatment .", "these cytokines are released by macrophages after bacterial infection or tissue injury and , in high concentration , stimulate the production and release of other inflammatory mediators .", "similarly , il-17 is a proinflammatory cytokine produced by activated th17 cells   that induce a distinct profile of effector cytokines which may exacerbate inflammation and tissue damage . in fact , th17 cells were found in human pd as well as il-17 expression in the alveolar bone of these subjects , suggesting a causal link between inflammation and bone destruction .", "thus , while proinflammatory cytokines are potent inducers of bone resorption and inhibitors of bone formation , immunoregulatory mediators inhibit such proinflammatory signals and suppress the activation of metalloproteinases ( mmps ) that mediate tissue destruction in pd .", "these enzymes are classified according to their catalytic domains or substrate specificity   and several studies have associated mmps regulation with pd progression , once the control of synthesis and degradation of extracellular matrix defines levels of periodontal attachment .", "moreover , since mmps production is potentiated in inflammatory conditions and these enzymes are related to pd susceptibility , the determination of the levels of inflammatory mediators in gingiva or biologic fluids became good indicators of the patient 's periodontal disease status and activity [ 18 , 19 ] .", "however , although how the inflammation develops in the gingival tissue of pd patients is largely described , little is known about the progression of this response to the alveolar bone underneath gingiva and how pd affects cytokine and mmps expression in this tissue around the extensive periodontal pocket . accordingly", ", the present study focused on the detection of the ongoing inflammatory changes in the bone and neighboring tissues directly affected by the destructive disease in pd patients .", "seventeen ( 17 ) subjects with no periodontal or systemic diseases ( controls ) and 18 patients with advanced periodontal disease ( pd ) from the periodontal clinic of uberaba university at uberaba , minas gerais , brazil , were selected for periodontal evaluation and sample collection .", "the subjects were submitted to anamnesis , clinical and periodontal exams , as proposed by the periodontal american association .", "all the clinical profile such as probing deep and clinical attachment was recorded for subjects classification into pd patients or controls ( table 1 ) .", "informed consents approved by the local ethics committee on human research ( protocol number 1230 ) were obtained from all the volunteers and the study was performed according to the declaration of helsinki , 1964 .", "bone biopsies were extracted from patients with advanced pd in which alveoloplasty was necessary before gingival suture , in cases of extraction of teeth with previous extensive periodontal damage .", "therefore , the samples were obtained after maxillary or mandibular alveolar ridge regularization , with removal of minimal bone spicule from areas adjacent to the large bone resorption regions of the previously extracted teeth .", "biopsies from control group were obtained during extraction of third molars that required osteotomy ( n = 17 ) .", "all samples used for gene expression analysis were first collected in rpmi medium supplemented with 10% of bovine fetal serum for transportation followed by washing in cold saline solution and storage into liquid nitrogen .", "samples destined for histopathology study were washed in cold saline solution and fixed in pbs - formaldehyde 10% .", "tartrate - resistant acid phosphatase ( trap ) activity was detected using a leukocyte - specific acid phosphatase kit ( sigma aldrich , st .", "briefly , samples were decalcified and embedded in paraffin and serial sections of 5  m were placed in glass slides .", "the sections were stained with trap solution for 1 hour at 37c and counterstained with mayer 's hematoxylin .", "cells stained dark red to purple with more than three nuclei on the bone surfaces were considered trap and were counted per mm of tissue . \n", "quantitative polymerase chain reaction analysis ( rt - pcr ) of tnf- , ifn- , il-10 , il-4 , and the metalloproteinases mmps 1 , 2 , 3 , and 9 mrna expression was performed in the bone tissue samples .", "primer sequences were designed using the software oligoperfect designer ( invitrogen , carlsbad , ca , usa ) .", "rna was extracted according to the manufacturer 's guidelines of rna sv total rna isolation system kit ( promega , madison , wi , usa ) and the cdna confection was performed with oligodt ( promega ) , dntps , reverse transcriptase enzyme m - mlv rt , and m - mlv buffer ( promega ) in a pcr thermocycler ptc-100 ( mj research , waltham , ma , usa ) .", "the sequences of cdna products were amplified in real - time pcr machine geneamp 7000 ( applied biosystems , foster city , ca , usa ) using the reagent faststart universal sybr green master ( rox ) ( roche , madison , wi , usa ) and specific forward / reverse primers at 0.1  g/l .", "the default pcr condition was 2 minutes at 50c , 10 minutes at 95c and fifty cycles of 15 seconds at 95c , 30 seconds at 58c , and 30 seconds at 72c .", "sequence detection software version 1.3 ( applied biosystems ) was used to analyze data after amplification .", "results were obtained as cycle threshold ( ct ) values , which were normalized using the expression of the constitutive gene -actin , following the equation 2 , according to the applied user 's bulletin # 2 , p / n 4303859 ( applied biosystems ) .", "biopsies were fixed with pbs - formaldehyde of 10% for 48  h and decalcified in pbs - edta of 0.5  m , during 30 days .", "sections ( 5  m ) were stained with hematoxylin and eosin ( he ) and analyzed by light microscopy .", "all images were captured with the digital video camera evolution mp 5 ( media cybernetics , rockville , md , usa ) mounted on a light microscopy eclipse 50i ( nikon , melville , ny , usa ) using the software image - pro plus ( media cybernetics ) .", "morphometric analysis of fibrosis was performed using the imagej software ( national institutes of health , bethesda , md ) .", "fibrous connective tissues were stained with picrosirius red and the images were captured at 400 ( 24 fields / sample ) that measured 3705.82  m .", "a grid with 100 points , each one representing 37.06  m , divided each field . for each sample ,", "2400 points were analyzed totalizing 90192  m . to calculate the percentage of collagen area , points coincident with stained area were counted .", "points were checked by the formula of hally , as described before . for immunohistochemistry ,", "deparaffinized sections were treated with 3% hydrogen peroxide in methanol for 10  min and incubated for 30  min at 90c for antigen recovery .", "the blocking of the sections was performed using 2% bovine serum albumin incubated for 30  min at room temperature .", "afterwards , they were individually incubated with anti - cytokine monoclonal antibodies specific for tgf- ( 1  :  100 ) ( r&d , hopewell , nj , usa ) and il-17 ( 1  :  20 ) ( r&d ) .", "all antibodies were diluted in 2% bsa prior to use and incubated with the samples for 2 hours at 37c .", "a secondary step was performed using biotinylated anti - mouse ig , anti - rabbit ig , and anti - goat ig from link system 002488 ( dako , carpinteria , ca , usa ) for 30  min at 37c . after being washed", ", the sections were incubated with streptavidin - peroxidase conjugated ( dako ) for 30  min .", "the number of positive cells for each cytokine was counted in 20 fields at a magnification of 400 , in a predetermined area of 0.091575  mm .", "graph pad instat and prism statistical programs were used for analysis ( graphpad , san diego , ca , usa ) . in case of normal distribution , the independent t - test was used while the nonparametric mann - whitney test was used in non - gaussian sample distribution .", "first , we performed a clinical periodontal examination and an analysis of the status of the volunteers enrolled in the study to better classify the individuals into pd or control group and to verify if these findings could be related to the next evaluations .", "data showed that both groups were balanced for gender and were different for age distribution , since chronic periodontitis was more prevalent in older individuals . regarding tobacco use ,", "4 patients from pd group were smokers , a habit known to be directly related to pd progression .", "the probing depth was not different between control and pd group ( p > 0.05 ) since there was extensive clinical attachment loss with gingival recession in pd patients , thus accounting to their classification into the advanced pd group ( table 1 ) .", "next , to verify if the periodontal and especially the bone tissue adjacent to the areas of extensive bone resorption in advanced pd presented any sign of inflammation , we performed histopathological evaluation of the he stained biopsies .", "results showed that control subjects presented a normal aspect of bone tissue with osteocytes and osteoblasts at the edge of bone matrix with no signs of alteration ( figure 1(a ) ) .", "in contrast , samples from pd group presented a focally distributed and mild inflammatory process composed predominantly by mononuclear cells with the presence of lymphocytes and plasma cells , besides a few neutrophils .", "the bone tissue did not present any significant alteration ( figure 1(b ) ) . then , since osteoclast - mediated bone resorption may be driven by local inflammation , we next aimed to verify if it could be an indicative of ongoing bone damage .", "in fact , we found increased numbers of trap - positive cells in samples from pd group when compared to controls ( figure 1(c ) ) , indicating that the inflammatory process was able to trigger osteoclast differentiation in periodontitis patients .", "previous studies from our group showed that advanced pd is characterized by elevated levels of tgf- and il-17 mrna , which may be related to disease progression [ 13 , 21 ] .", "then , in the present study , immunohistochemical analysis revealed that both groups produced tgf- and il-17 cytokines ( at protein level ) in the periodontium biopsies .", "pd subjects presented a slight augment in the density of tgf- positive cells ( figure 1(d ) ) and a significant increase was observed in the number of il-17 positive cells in comparison to the control group ( figure 1(e ) ) .", "furthermore , since the collagen matrix in the bone and connective tissue of the periodontium are one of main targets in periodontitis destruction , we determined the collagen percentage in the biopsies stained by picrosirius red . surprisingly , although pd subjects presented microscopic inflammation and osteoclast accumulation suggestive of tissue damage , a higher collagen deposition was found in this group , as observed in figure 1(f ) .", "once bone resorption is a result of the balance between anti- and proinflammatory mediators that may activate tissue destructive enzymes , we assessed the expression of cytokines and metalloproteinases ( mmps ) in the bone tissue of pd and control samples .", "the real - time pcr assays revealed that the levels of ifn- , il-10 , and il-4 mrna were not altered in the samples of subjects relative to controls ( figures 2(b)2(d ) ) .", "however , there was a strikingly elevated expression of tnf- mrna in subjects with pd compared to control group ( figure 2(a ) ) .", "importantly , this increase in the inflammatory cytokine tnf- was accompanied by augmented expression of mmp-2 mrna ( figure 3(b ) ) and there was a tendency towards elevated expression of mmp-1 and mmp-9 in pd patients ( figures 3(a ) and 3(d ) ) .", "mmp-3 was not different between the studied groups ( figure 3(c ) , p > 0.05 ) .", "furthermore , a positive and significant correlation was observed between tnf- and mmp-2 mrna expression when all the subjects of the study were evaluated together ( figure 4 , p = 0.0036 ; rs = 0.6190 ) .", "altogether , these data suggested that the local immune alterations that culminate in tooth loss are not restricted to gingiva .", "the periodontal or bone tissue next to the extensive areas of resorption presents relevant inflammatory findings indicative of periodontitis destruction and progression in susceptible individuals .", "the data presented in this study obtained from examination of sites close to areas with extensive periodontal damage showed that , as expected , inflammation in advanced pd is not restricted to gingiva . otherwise , tissues like alveolar bone that is clinically undergoing a resorption process present relevant findings that could be considered inflammatory biomarkers for disease development in susceptible patients .", "although predictable , this is the first study that showed the immune response related to periodontitis evolution directly in the damaged bone and neighboring area . interestingly , our patients with advanced pd presented a number of features characteristics of local inflammation and some of them were smokers , a habit known to be classically involved in pd progression . in spite of this , it did not seem to interfere in the disease spreading to bone tissue , since the inflammation markers analyzed were not differentially expressed by these individuals . as a next approach", ", we examined whether pd could result in histological changes in the bone resorbing areas close to the large periodontal pocket .", "also , this mild inflammation associated with trap cells could be an indicative of microbial presence and an active process of tissue damage , though not at the same extent yet as that observed in the large periodontal pocket , that led to the tooth loss .", "corroborating our data , previous studies showed that pathogenic microorganisms might colonize different sites and then spread local inflammation in case of uncontrolled pathogen burden [ 22 , 23 ] .", "the integrity of bone tissue depends on the balance between bone formation by osteoblasts and bone resorption by osteoclasts [ 24 , 25 ] . in this scenario ,", "several proinflammatory cytokines were identified as key molecules contributing to the destruction of periodontal tissue , including il-1 , tnf- , ifn- , and il-6 besides rankl [ 26 , 27 ] .", "thus , in view of our results , it is possible that the higher tnf- mrna found in our patients may have acted as an important osteoclastogenic factor by inducing the local osteoclast differentiation that could culminate in bone resorption .", "we also revealed that the number of il-17 cells was higher in pd regions than in control healthy tissues .", "such cells could be cd4 , cd8 ,  , neutrophils , natural killer cells , or others [ 2832 ] .", "a previous study demonstrated that patients with chronic periodontitis have th17 cells as well as il-17 expression in inflamed periodontal tissue , indicating the possible involvement of this cytokine in periodontal disease .", "indeed , th17 cells could exacerbate inflammatory periodontal disease by inducing metalloproteases or inflammatory mediators by gingival fibroblasts . in this context ,", "our results suggested the involvement of th17 cells in the inflammatory events triggered in the periodontal disease affected areas .", "besides , il-17 could be capable of inducing rankl , the main stimulatory factor for the differentiation and activation of osteoclasts in bone inflammatory diseases like rheumatoid arthritis and pd [ 34 , 35 ] .", "then , the higher number of osteoclasts in the areas evaluated could also be related to the presence of il-17 cells .", "the inflammatory process in pd leads to the degradation of extracellular matrix , mainly collagen , by mmps overproduction that may be induced by cytokines released from resident and inflammatory cells [ 36 , 37 ] .", "however , during the healing process that follows inflammation , collagen is also newly produced for extracellular matrix remodeling .", "therefore , the increased collagen deposition observed in our patients could result from a repair response to the initial inflammation triggered nearby the extensive bone resorption areas .", "in fact , the number of tgf- cells was slightly higher in pd group compared to controls .", "nevertheless , this cytokine could play a dual role in pd , by controlling inflammation or inducing the differentiation of th17 response [ 13 , 21 ] .", "most strikingly , as discussed before , levels of tnf- were significantly augmented in bone tissue from pd group .", "as tnf- is important for osteoclastogenesis , it is possible that this factor initiates osteoclast differentiation in subclinical inflammatory process .", "also , tnf- is produced in response to periodontopathogens , inducing the microbicidal activity of phagocytic cells , and , in the absence of the subunit p55 of tnf- receptor , mice infected with aggregatibacter actinomycetemcomitans presented less bone resorption and inflammatory response , besides increased number of bacteria .", "therefore , the higher levels of tnf- in our pd group could indicate the presence of pathogens in periodontal environment , besides accounting for the higher number of osteoclasts in these patients [ 40 , 41 ] . during active periodontitis , degradation of gingival connective tissues ,", "mainly collagen , is due to the expression of mmps in situ by inflammatory and resident cells such as fibroblasts and epithelial or endothelial cells [ 33 , 37 ] .", "however , the evaluation of mmps directly in the bone resorbing areas adjacent to the large periodontal pocket , as well as their role in the disease outcome , is unclear .", "our subjects with pd presented a mild inflammatory infiltration of cells and the expression of mmp-2 mrna was almost five times higher in samples from this group .", "this local infiltrate could be able to produce mmp-2 , based on previous results indicating that tnf- stimulates the secretion of active mmp-2 [ 42 , 43 ] , thus resulting in degradation of collagen types i , ii , and iii .", "mmp-2 expression in biopsies of subjects with pd could suggest a possible biological alteration in the turnover of connective tissue and a consequent active ongoing process of bone resorption .", "also , the degradation of collagen by mmp-2 could produce peptides important for recruitment of mononuclear and polymorphonuclear cells that could later amplify the inflammatory process . in our study ,", "although we did not evaluate mmp-2 activity in situ , we might hypothesize that the production of tnf- could upregulate membrane type metalloproteinase-1 ( mt1-mmp ) , which is able to activate the latent form of mmp-2 , thus breaking collagen in pd sites .", "such phenomenon could be confirmed by higher percentage of collagen areas , indicating a remodeling process that follows collagen degradation .", "indeed , repeated exposure to low or moderate lps concentration induced augmented cardiac fibrosis along with elevated mmps expression ( including mmp-2 ) in mice heart , thus indicating that a subclinical inflammatory response may be able to trigger tissue collagen response and alter mmps balance .", "tissues neighboring large periodontal pockets present inflammatory markers such as il-17 , tnf- , mmp-2 , and osteoclasts accumulation , which could be predictors of local bone resorption and disease spreading .", "these results support the importance of a more detailed evaluation of these regions other than gingiva , since the local immune response may represent sentinel factors relevant for disease outcome ."], "abstract_text": ["<S> chronic periodontitis is a multifactorial inflammatory disease that affects supporting structures of the teeth . </S>", "<S> although the gingival response is largely described , little is known about the immune changes in the alveolar bone and neighboring tissues that could indicate periodontal disease ( pd ) activity . </S>", "<S> then , in this study we identified the ongoing inflammatory changes and novel biomarkers for periodontitis in the tissues directly affected by the destructive disease in pd patients . </S>", "<S> samples were collected by osteotomy in 17 control subjects during extraction of third molars and 18 patients with advanced pd , in which alveoloplasty was necessary after extraction of teeth with previous extensive periodontal damage . </S>", "<S> patients presented mononuclear cells infiltration in the connective tissue next to the bone and higher fibrosis area , along with increased accumulation of il-17 + and trap+ cells . </S>", "<S> the levels of tnf- and mmp-2 mrna were also elevated compared to controls and a positive and significant correlation was observed between tnf- and mmp-2 mrna expression , considering all samples evaluated . in conclusion , nongingival tissues neighboring large periodontal pockets present inflammatory markers that could predict ongoing bone resorption and disease spreading . </S>", "<S> therefore , we suggested that the detailed evaluation of these regions could be of great importance to the assessment of disease progression . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["most conclusive evidences indicate that periodontal disease ( pd ) is not a conventional bacterial infection but is an inflammatory disease initiated by immune response against a group of microorganisms in susceptible hosts .", "the consequent uncontrolled inflammation causes destruction of attachment structures , being the most significant reason of tooth loss in adults from different populations . in affected tissues", "cytokines and chemokines lead to the migration of leukocytes to the periodontal tissues where these cells play an important role in pathogen destruction by releasing mediators in a local inflammatory response [ 4 , 5 ] .", "therefore , these and other mediators have been detected at elevated levels in the gingival crevicular fluid , saliva , and blood , thus being considered acceptable biomarkers for some aspects of pd [ 3 , 6 , 7 ] .", "particularly ,  proinflammatory cytokines such as interleukin-1 beta ( il-1 ) and tumor necrosis factor - alpha ( tnf- ) have been associated with pd progression   and decline after periodontal treatment .", "these cytokines are released by macrophages after bacterial infection or tissue injury and , in high concentration , stimulate the production and release of other inflammatory mediators .", "similarly , il-17 is a proinflammatory cytokine produced by activated th17 cells   that induce a distinct profile of effector cytokines which may exacerbate inflammation and tissue damage . in fact , th17 cells were found in human pd as well as il-17 expression in the alveolar bone of these subjects , suggesting a causal link between inflammation and bone destruction .", "thus , while proinflammatory cytokines are potent inducers of bone resorption and inhibitors of bone formation , immunoregulatory mediators inhibit such proinflammatory signals and suppress the activation of metalloproteinases ( mmps ) that mediate tissue destruction in pd .", "these enzymes are classified according to their catalytic domains or substrate specificity   and several studies have associated mmps regulation with pd progression , once the control of synthesis and degradation of extracellular matrix defines levels of periodontal attachment .", "moreover , since mmps production is potentiated in inflammatory conditions and these enzymes are related to pd susceptibility , the determination of the levels of inflammatory mediators in gingiva or biologic fluids became good indicators of the patient 's periodontal disease status and activity [ 18 , 19 ] .", "however , although how the inflammation develops in the gingival tissue of pd patients is largely described , little is known about the progression of this response to the alveolar bone underneath gingiva and how pd affects cytokine and mmps expression in this tissue around the extensive periodontal pocket . accordingly", ", the present study focused on the detection of the ongoing inflammatory changes in the bone and neighboring tissues directly affected by the destructive disease in pd patients ."], ["seventeen ( 17 ) subjects with no periodontal or systemic diseases ( controls ) and 18 patients with advanced periodontal disease ( pd ) from the periodontal clinic of uberaba university at uberaba , minas gerais , brazil , were selected for periodontal evaluation and sample collection .", "the subjects were submitted to anamnesis , clinical and periodontal exams , as proposed by the periodontal american association .", "all the clinical profile such as probing deep and clinical attachment was recorded for subjects classification into pd patients or controls ( table 1 ) .", "informed consents approved by the local ethics committee on human research ( protocol number 1230 ) were obtained from all the volunteers and the study was performed according to the declaration of helsinki , 1964 .", "bone biopsies were extracted from patients with advanced pd in which alveoloplasty was necessary before gingival suture , in cases of extraction of teeth with previous extensive periodontal damage .", "therefore , the samples were obtained after maxillary or mandibular alveolar ridge regularization , with removal of minimal bone spicule from areas adjacent to the large bone resorption regions of the previously extracted teeth .", "biopsies from control group were obtained during extraction of third molars that required osteotomy ( n = 17 ) .", "all samples used for gene expression analysis were first collected in rpmi medium supplemented with 10% of bovine fetal serum for transportation followed by washing in cold saline solution and storage into liquid nitrogen .", "samples destined for histopathology study were washed in cold saline solution and fixed in pbs - formaldehyde 10% .", "tartrate - resistant acid phosphatase ( trap ) activity was detected using a leukocyte - specific acid phosphatase kit ( sigma aldrich , st .", "briefly , samples were decalcified and embedded in paraffin and serial sections of 5  m were placed in glass slides .", "the sections were stained with trap solution for 1 hour at 37c and counterstained with mayer 's hematoxylin .", "cells stained dark red to purple with more than three nuclei on the bone surfaces were considered trap and were counted per mm of tissue . \n", "quantitative polymerase chain reaction analysis ( rt - pcr ) of tnf- , ifn- , il-10 , il-4 , and the metalloproteinases mmps 1 , 2 , 3 , and 9 mrna expression was performed in the bone tissue samples .", "primer sequences were designed using the software oligoperfect designer ( invitrogen , carlsbad , ca , usa ) .", "rna was extracted according to the manufacturer 's guidelines of rna sv total rna isolation system kit ( promega , madison , wi , usa ) and the cdna confection was performed with oligodt ( promega ) , dntps , reverse transcriptase enzyme m - mlv rt , and m - mlv buffer ( promega ) in a pcr thermocycler ptc-100 ( mj research , waltham , ma , usa ) .", "the sequences of cdna products were amplified in real - time pcr machine geneamp 7000 ( applied biosystems , foster city , ca , usa ) using the reagent faststart universal sybr green master ( rox ) ( roche , madison , wi , usa ) and specific forward / reverse primers at 0.1  g/l .", "the default pcr condition was 2 minutes at 50c , 10 minutes at 95c and fifty cycles of 15 seconds at 95c , 30 seconds at 58c , and 30 seconds at 72c .", "sequence detection software version 1.3 ( applied biosystems ) was used to analyze data after amplification .", "results were obtained as cycle threshold ( ct ) values , which were normalized using the expression of the constitutive gene -actin , following the equation 2 , according to the applied user 's bulletin # 2 , p / n 4303859 ( applied biosystems ) .", "biopsies were fixed with pbs - formaldehyde of 10% for 48  h and decalcified in pbs - edta of 0.5  m , during 30 days .", "sections ( 5  m ) were stained with hematoxylin and eosin ( he ) and analyzed by light microscopy .", "all images were captured with the digital video camera evolution mp 5 ( media cybernetics , rockville , md , usa ) mounted on a light microscopy eclipse 50i ( nikon , melville , ny , usa ) using the software image - pro plus ( media cybernetics ) .", "morphometric analysis of fibrosis was performed using the imagej software ( national institutes of health , bethesda , md ) .", "fibrous connective tissues were stained with picrosirius red and the images were captured at 400 ( 24 fields / sample ) that measured 3705.82  m .", "a grid with 100 points , each one representing 37.06  m , divided each field . for each sample ,", "2400 points were analyzed totalizing 90192  m . to calculate the percentage of collagen area , points coincident with stained area were counted .", "points were checked by the formula of hally , as described before . for immunohistochemistry ,", "deparaffinized sections were treated with 3% hydrogen peroxide in methanol for 10  min and incubated for 30  min at 90c for antigen recovery .", "the blocking of the sections was performed using 2% bovine serum albumin incubated for 30  min at room temperature .", "afterwards , they were individually incubated with anti - cytokine monoclonal antibodies specific for tgf- ( 1  :  100 ) ( r&d , hopewell , nj , usa ) and il-17 ( 1  :  20 ) ( r&d ) .", "all antibodies were diluted in 2% bsa prior to use and incubated with the samples for 2 hours at 37c .", "a secondary step was performed using biotinylated anti - mouse ig , anti - rabbit ig , and anti - goat ig from link system 002488 ( dako , carpinteria , ca , usa ) for 30  min at 37c . after being washed", ", the sections were incubated with streptavidin - peroxidase conjugated ( dako ) for 30  min .", "the number of positive cells for each cytokine was counted in 20 fields at a magnification of 400 , in a predetermined area of 0.091575  mm .", "graph pad instat and prism statistical programs were used for analysis ( graphpad , san diego , ca , usa ) . in case of normal distribution , the independent t - test was used while the nonparametric mann - whitney test was used in non - gaussian sample distribution ."], ["first , we performed a clinical periodontal examination and an analysis of the status of the volunteers enrolled in the study to better classify the individuals into pd or control group and to verify if these findings could be related to the next evaluations .", "data showed that both groups were balanced for gender and were different for age distribution , since chronic periodontitis was more prevalent in older individuals . regarding tobacco use ,", "4 patients from pd group were smokers , a habit known to be directly related to pd progression .", "the probing depth was not different between control and pd group ( p > 0.05 ) since there was extensive clinical attachment loss with gingival recession in pd patients , thus accounting to their classification into the advanced pd group ( table 1 ) .", "next , to verify if the periodontal and especially the bone tissue adjacent to the areas of extensive bone resorption in advanced pd presented any sign of inflammation , we performed histopathological evaluation of the he stained biopsies .", "results showed that control subjects presented a normal aspect of bone tissue with osteocytes and osteoblasts at the edge of bone matrix with no signs of alteration ( figure 1(a ) ) .", "in contrast , samples from pd group presented a focally distributed and mild inflammatory process composed predominantly by mononuclear cells with the presence of lymphocytes and plasma cells , besides a few neutrophils .", "the bone tissue did not present any significant alteration ( figure 1(b ) ) . then , since osteoclast - mediated bone resorption may be driven by local inflammation , we next aimed to verify if it could be an indicative of ongoing bone damage .", "in fact , we found increased numbers of trap - positive cells in samples from pd group when compared to controls ( figure 1(c ) ) , indicating that the inflammatory process was able to trigger osteoclast differentiation in periodontitis patients .", "previous studies from our group showed that advanced pd is characterized by elevated levels of tgf- and il-17 mrna , which may be related to disease progression [ 13 , 21 ] .", "then , in the present study , immunohistochemical analysis revealed that both groups produced tgf- and il-17 cytokines ( at protein level ) in the periodontium biopsies .", "pd subjects presented a slight augment in the density of tgf- positive cells ( figure 1(d ) ) and a significant increase was observed in the number of il-17 positive cells in comparison to the control group ( figure 1(e ) ) .", "furthermore , since the collagen matrix in the bone and connective tissue of the periodontium are one of main targets in periodontitis destruction , we determined the collagen percentage in the biopsies stained by picrosirius red . surprisingly , although pd subjects presented microscopic inflammation and osteoclast accumulation suggestive of tissue damage , a higher collagen deposition was found in this group , as observed in figure 1(f ) .", "once bone resorption is a result of the balance between anti- and proinflammatory mediators that may activate tissue destructive enzymes , we assessed the expression of cytokines and metalloproteinases ( mmps ) in the bone tissue of pd and control samples .", "the real - time pcr assays revealed that the levels of ifn- , il-10 , and il-4 mrna were not altered in the samples of subjects relative to controls ( figures 2(b)2(d ) ) .", "however , there was a strikingly elevated expression of tnf- mrna in subjects with pd compared to control group ( figure 2(a ) ) .", "importantly , this increase in the inflammatory cytokine tnf- was accompanied by augmented expression of mmp-2 mrna ( figure 3(b ) ) and there was a tendency towards elevated expression of mmp-1 and mmp-9 in pd patients ( figures 3(a ) and 3(d ) ) .", "mmp-3 was not different between the studied groups ( figure 3(c ) , p > 0.05 ) .", "furthermore , a positive and significant correlation was observed between tnf- and mmp-2 mrna expression when all the subjects of the study were evaluated together ( figure 4 , p = 0.0036 ; rs = 0.6190 ) .", "altogether , these data suggested that the local immune alterations that culminate in tooth loss are not restricted to gingiva .", "the periodontal or bone tissue next to the extensive areas of resorption presents relevant inflammatory findings indicative of periodontitis destruction and progression in susceptible individuals ."], ["the data presented in this study obtained from examination of sites close to areas with extensive periodontal damage showed that , as expected , inflammation in advanced pd is not restricted to gingiva . otherwise , tissues like alveolar bone that is clinically undergoing a resorption process present relevant findings that could be considered inflammatory biomarkers for disease development in susceptible patients .", "although predictable , this is the first study that showed the immune response related to periodontitis evolution directly in the damaged bone and neighboring area . interestingly , our patients with advanced pd presented a number of features characteristics of local inflammation and some of them were smokers , a habit known to be classically involved in pd progression . in spite of this , it did not seem to interfere in the disease spreading to bone tissue , since the inflammation markers analyzed were not differentially expressed by these individuals . as a next approach", ", we examined whether pd could result in histological changes in the bone resorbing areas close to the large periodontal pocket .", "also , this mild inflammation associated with trap cells could be an indicative of microbial presence and an active process of tissue damage , though not at the same extent yet as that observed in the large periodontal pocket , that led to the tooth loss .", "corroborating our data , previous studies showed that pathogenic microorganisms might colonize different sites and then spread local inflammation in case of uncontrolled pathogen burden [ 22 , 23 ] .", "the integrity of bone tissue depends on the balance between bone formation by osteoblasts and bone resorption by osteoclasts [ 24 , 25 ] . in this scenario ,", "several proinflammatory cytokines were identified as key molecules contributing to the destruction of periodontal tissue , including il-1 , tnf- , ifn- , and il-6 besides rankl [ 26 , 27 ] .", "thus , in view of our results , it is possible that the higher tnf- mrna found in our patients may have acted as an important osteoclastogenic factor by inducing the local osteoclast differentiation that could culminate in bone resorption .", "we also revealed that the number of il-17 cells was higher in pd regions than in control healthy tissues .", "such cells could be cd4 , cd8 ,  , neutrophils , natural killer cells , or others [ 2832 ] .", "a previous study demonstrated that patients with chronic periodontitis have th17 cells as well as il-17 expression in inflamed periodontal tissue , indicating the possible involvement of this cytokine in periodontal disease .", "indeed , th17 cells could exacerbate inflammatory periodontal disease by inducing metalloproteases or inflammatory mediators by gingival fibroblasts . in this context ,", "our results suggested the involvement of th17 cells in the inflammatory events triggered in the periodontal disease affected areas .", "besides , il-17 could be capable of inducing rankl , the main stimulatory factor for the differentiation and activation of osteoclasts in bone inflammatory diseases like rheumatoid arthritis and pd [ 34 , 35 ] .", "then , the higher number of osteoclasts in the areas evaluated could also be related to the presence of il-17 cells .", "the inflammatory process in pd leads to the degradation of extracellular matrix , mainly collagen , by mmps overproduction that may be induced by cytokines released from resident and inflammatory cells [ 36 , 37 ] .", "however , during the healing process that follows inflammation , collagen is also newly produced for extracellular matrix remodeling .", "therefore , the increased collagen deposition observed in our patients could result from a repair response to the initial inflammation triggered nearby the extensive bone resorption areas .", "in fact , the number of tgf- cells was slightly higher in pd group compared to controls .", "nevertheless , this cytokine could play a dual role in pd , by controlling inflammation or inducing the differentiation of th17 response [ 13 , 21 ] .", "most strikingly , as discussed before , levels of tnf- were significantly augmented in bone tissue from pd group .", "as tnf- is important for osteoclastogenesis , it is possible that this factor initiates osteoclast differentiation in subclinical inflammatory process .", "also , tnf- is produced in response to periodontopathogens , inducing the microbicidal activity of phagocytic cells , and , in the absence of the subunit p55 of tnf- receptor , mice infected with aggregatibacter actinomycetemcomitans presented less bone resorption and inflammatory response , besides increased number of bacteria .", "therefore , the higher levels of tnf- in our pd group could indicate the presence of pathogens in periodontal environment , besides accounting for the higher number of osteoclasts in these patients [ 40 , 41 ] . during active periodontitis , degradation of gingival connective tissues ,", "mainly collagen , is due to the expression of mmps in situ by inflammatory and resident cells such as fibroblasts and epithelial or endothelial cells [ 33 , 37 ] .", "however , the evaluation of mmps directly in the bone resorbing areas adjacent to the large periodontal pocket , as well as their role in the disease outcome , is unclear .", "our subjects with pd presented a mild inflammatory infiltration of cells and the expression of mmp-2 mrna was almost five times higher in samples from this group .", "this local infiltrate could be able to produce mmp-2 , based on previous results indicating that tnf- stimulates the secretion of active mmp-2 [ 42 , 43 ] , thus resulting in degradation of collagen types i , ii , and iii .", "mmp-2 expression in biopsies of subjects with pd could suggest a possible biological alteration in the turnover of connective tissue and a consequent active ongoing process of bone resorption .", "also , the degradation of collagen by mmp-2 could produce peptides important for recruitment of mononuclear and polymorphonuclear cells that could later amplify the inflammatory process . in our study ,", "although we did not evaluate mmp-2 activity in situ , we might hypothesize that the production of tnf- could upregulate membrane type metalloproteinase-1 ( mt1-mmp ) , which is able to activate the latent form of mmp-2 , thus breaking collagen in pd sites .", "such phenomenon could be confirmed by higher percentage of collagen areas , indicating a remodeling process that follows collagen degradation .", "indeed , repeated exposure to low or moderate lps concentration induced augmented cardiac fibrosis along with elevated mmps expression ( including mmp-2 ) in mice heart , thus indicating that a subclinical inflammatory response may be able to trigger tissue collagen response and alter mmps balance ."], ["tissues neighboring large periodontal pockets present inflammatory markers such as il-17 , tnf- , mmp-2 , and osteoclasts accumulation , which could be predictors of local bone resorption and disease spreading .", "these results support the importance of a more detailed evaluation of these regions other than gingiva , since the local immune response may represent sentinel factors relevant for disease outcome ."]]}
{"article_id": "PMC5083941", "article_text": ["modafinil , a novel wake - promoting drug has gained immense popularity among psychiatrists in recent times .", "now a days , its use is not limited to treat narcolepsy and other sleep wake disorders , but also in chronic fatigue syndrome , treatment resistant depression , attention deficit hyperactivity disorder and cocaine dependency.1 ) reportedly it has a much lower abuse potential but some have also suggested it to be having a definite potential for dependence.2 ) here we report a rare case of modafinil dependence who presented with hypersexuality behavior . till date , only one such similar case have been reported.3 )", "a 35 years male patient was diagnosed with bipolar affective disorder ( international classification of diseases [ icd]-10 criteria ) , since last 7 years with four episodes of depression ( characterized by low mood , decreased energy , anxiety throughout the day , depressive cognitions , decreased sleep and appetite along with occasional wish to die ) and two episodes of mania ( characterized by cheerful mood , increased activity levels , decreased sleep to only 3 hours / day , increased appetite , grandiose ideas , over - talkativeness and increased expenditure ) along with benzodiazepine dependence ( currently abstinent ) .", "he presented to our centre seeking treatment for excessive sexual desire and to limit excessive use of modafinil .", "he was on lithium ( 600 mg / day ) , carbamazepine ( 800 mg / day ) , venlafaxine ( 150 mg / day ) , and lamotrigine ( 200 mg / day ) during this period .", "exploration of the history revealed that , he was started on modafinil 200 mg / day ( 100 mg at morning and afternoon ) by a private practitioner four years earlier to improve his fluctuating sadness of mood and early morning lethargy .", "he would feel active , fresh , and would be able to work better after taking modafinil . over the next 3 years ( 2010 to 2012 ) he started to use modafinil whenever he used to feel low and escalated the dose to 400600 mg / day .", "whenever he would not take , he had strong craving for the same , felt low , lethargic , tired , aches and pains in body , decreased confidence and concentration at work .", "in mid of 2014 , inspite of good compliance to above medications , without any apparent stressors , he again started to have persistent sadness , lack of energy and easy fatigability .", "because of this , he increased dose of modafinil to 400 mg / day on his own in early morning . after taking modafinil", "so , over the next few weeks , he increased the dose to 8001,000 mg / day in divided doses .", "though there was no improvement in his mood , there was change in his sexual behavior .", "on seeing any women nearby , he started to have spontaneous erection and feel excited .", "his frequency of masturbation increased to 1012 times / day , but he would not feel satisfied unlike his premorbid self .", "in addition to these symptoms , he started interacting with commercial sex workers ( csws ) on phone , would get aroused by mere talking with them .", "he would be preoccupied with sexual fantasies and would not be able to concentrate at his work .", "he tried meditation , increased his frequency of religious activities to get rid of his sexual urges but in vain . despite being happily married for last 16 years with adequate sexual adjustment", "he would have increased desire for intercourse with females other than his wife which would further instigate guilt in his mind .", "his sleep was disturbed with restlessness and anxiety at night and he would feel fatigue throughout the day despite taking high doses of modafinil .", "his mood remained sad for most part of the day , would not enjoy the day to day activities like before and continued to harbor depressive cognitions .", "there was no associated history of cheerfulness , grandiosity , hyperactivity , overplanning , increased appetite and decreased need for sleep during this time . throughout this period", "there was neither any change in his compliance with medications nor any change in dosage of other medications .", "and he had been on venlafaxine 150 mg / day since last 2 years under the cover of three mood stabilizers , i.e. , lithium , carbamazepine , and lamotrigine with adequate dosages and normal therapeutic lithium level of 0.76 mmol / l .", "none of previous mood episodes ( both depression and mania ) had any symptoms suggestive of hypersexual behavior .", "his sexual behavior during the intake of high doses of modafinil was also not in keeping with his premorbid sexual desire and sexual activity .", "detailed evaluation of history did not suggest presence of any head injury in the recent past and presence of any associated impulsivity , obsessive compulsive behavior , substance abuse , hyperorality , cognitive decline , sleep attacks , use of aphrodisiacs and use of any other medications which could increase the sexual desire . with the above available information , a diagnosis of bipolar affective disorder with current episode mild depression ( as per icd-10 criteria ) along with an additional diagnosis of modafinil dependence and modafinil induced hypersexuality .", "physical examination , routine hematological and biochemistry investigations and electrocardiogram were within normal limits . at 8001,000 mg /", "day of modafinil , he had side effects as assessed by uku scale in areas of psychic ( score 9 ) , autonomic ( score 3 ) , other ( score 9 ) and global assessment of interference ( score 3 ) .", "he was also rated on hypersexual behavior inventory-19 ( hbi-19)4 ) where he scored 48 .", "management included a gradual and slow reduction in dosage of modafinil at the rate of 100 mg every 2 days under the cover of benzodiazepines ( clonazepam 2 mg / day ) to manage withdrawal symptoms .", "mood charting was done to assess development of any mood symptoms . over 3 weeks period", ", all the above mentioned symptoms decreased significantly ( uku total score 0 ; hbi-19 total score 19 ) and he was discharged after being counseled with structured relapse prevention programme on lithium ( 600 mg / day ) , lamotrigine ( 200 mg / day ) and venlafaxine ( 150 mg / day ) .", "in literature , we could find only two case reports on modafinil dependence at higher doses.2,5 ) as seen in the index case , modafinil seems to have some abuse potential .", "our case escalated the dose of modafinil so as to overcome fatigue , increase energy and improve concentration . later on , to overcome the fear of relapsing into depression he increased to a supratherapeutic dosage of 1,000 mg / day and started having symptoms which fulfilled the criteria of hypersexuality disorder ( as per kafka s criteria).6 ) these symptoms were temporally correlated with increment in dose of modafinil and subsided on decreasing the dose of the same in a controlled environment . hence , as neither any other drug used in the index case could be linked with such kind of behavior nor was there any switch to mania / hypomania / mixed episode which could explain such change in behavior , we concluded that the symptoms of hypersexual behavior was induced by modafinil .", "hypersexual behavior during a hypomanic / manic or mixed episode has characteristics of sexual disinhibition , severely compromised self - control , lack of guilt and shame feelings and is generally without any insight .", "in contrast , index case had guilt associated with his increased sexual desire which is very well reported in individuals with hypersexual behaviour.7,8 ) modafinil has been found to act by binding on dopamine receptors and affects dopamine uptake by dopamine transporters in the neurons .", "modafinil dependence can be explained by its dopamine uptake blockade leading to increase in dopamine concentration in the brain areas linked with reward pathways.9 ) on the other hand , a hyperdopaminergic state have been implicated in hypersexuality .", "upsurge of dopamine in the mesolimbic pathway due to use of supratherapeutic dosage of modafinil might explain for the development of hypersexuality in the index case .", "overdose of modafinil can cause insomnia , agitation , tachycardia , rise in blood pressure etc . , but in case of dependence it is more likely to cause physical withdrawal symptoms like lethargy , tremors of hands , anxiety and erratic sleep hours5 ) or only severe psychological withdrawal symptoms.10 ) modafinil has been listed under schedule iv drug in united states for regulation of its sale .", "however , in india , it is available over the counter and being regularly prescribed by many psychiatrists .", "inspite of reportedly low abuse potential , we report a case of modafinil dependence and modafinil induced hypersexuality so as to increase awareness regarding this rare side effect of modafinil and for the need of regulation on its sale ."], "abstract_text": ["<S> apart from sleep wake disorders , nowadays , modafinil is being prescribed for several psychiatric disorders including depression . despite being reported as to be having very low abuse potential , </S>", "<S> cases of modafinil dependence had come to the limelight . in this case report , we describe a 35 year old man with bipolar affective disorder while in remission who developed modafinil dependence and later on , had hypersexuality when he increased the dose of modafinil from 400 to 1,000 mg / day . </S>", "<S> existing literature suggests that modafinil when taken above prescribed doses can cause many side effects ranging from nausea , vomiting to psychotic exacerbation and mania . </S>", "<S> however , hypersexuality as a side effect of modafinil overuse is not commonly seen . </S>", "<S> the exact pathophysiological mechanism of modafinil induced hypersexuality is not clear . </S>", "<S> clinicians should be aware of possibility of modafinil leading to dependence and this rare significant side effect of modafinil . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE", "DISCUSSION"], "sections": [["modafinil , a novel wake - promoting drug has gained immense popularity among psychiatrists in recent times .", "now a days , its use is not limited to treat narcolepsy and other sleep wake disorders , but also in chronic fatigue syndrome , treatment resistant depression , attention deficit hyperactivity disorder and cocaine dependency.1 ) reportedly it has a much lower abuse potential but some have also suggested it to be having a definite potential for dependence.2 ) here we report a rare case of modafinil dependence who presented with hypersexuality behavior . till date , only one such similar case have been reported.3 )"], ["a 35 years male patient was diagnosed with bipolar affective disorder ( international classification of diseases [ icd]-10 criteria ) , since last 7 years with four episodes of depression ( characterized by low mood , decreased energy , anxiety throughout the day , depressive cognitions , decreased sleep and appetite along with occasional wish to die ) and two episodes of mania ( characterized by cheerful mood , increased activity levels , decreased sleep to only 3 hours / day , increased appetite , grandiose ideas , over - talkativeness and increased expenditure ) along with benzodiazepine dependence ( currently abstinent ) .", "he presented to our centre seeking treatment for excessive sexual desire and to limit excessive use of modafinil .", "he was on lithium ( 600 mg / day ) , carbamazepine ( 800 mg / day ) , venlafaxine ( 150 mg / day ) , and lamotrigine ( 200 mg / day ) during this period .", "exploration of the history revealed that , he was started on modafinil 200 mg / day ( 100 mg at morning and afternoon ) by a private practitioner four years earlier to improve his fluctuating sadness of mood and early morning lethargy .", "he would feel active , fresh , and would be able to work better after taking modafinil . over the next 3 years ( 2010 to 2012 ) he started to use modafinil whenever he used to feel low and escalated the dose to 400600 mg / day .", "whenever he would not take , he had strong craving for the same , felt low , lethargic , tired , aches and pains in body , decreased confidence and concentration at work .", "in mid of 2014 , inspite of good compliance to above medications , without any apparent stressors , he again started to have persistent sadness , lack of energy and easy fatigability .", "because of this , he increased dose of modafinil to 400 mg / day on his own in early morning . after taking modafinil", "so , over the next few weeks , he increased the dose to 8001,000 mg / day in divided doses .", "though there was no improvement in his mood , there was change in his sexual behavior .", "on seeing any women nearby , he started to have spontaneous erection and feel excited .", "his frequency of masturbation increased to 1012 times / day , but he would not feel satisfied unlike his premorbid self .", "in addition to these symptoms , he started interacting with commercial sex workers ( csws ) on phone , would get aroused by mere talking with them .", "he would be preoccupied with sexual fantasies and would not be able to concentrate at his work .", "he tried meditation , increased his frequency of religious activities to get rid of his sexual urges but in vain . despite being happily married for last 16 years with adequate sexual adjustment", "he would have increased desire for intercourse with females other than his wife which would further instigate guilt in his mind .", "his sleep was disturbed with restlessness and anxiety at night and he would feel fatigue throughout the day despite taking high doses of modafinil .", "his mood remained sad for most part of the day , would not enjoy the day to day activities like before and continued to harbor depressive cognitions .", "there was no associated history of cheerfulness , grandiosity , hyperactivity , overplanning , increased appetite and decreased need for sleep during this time . throughout this period", "there was neither any change in his compliance with medications nor any change in dosage of other medications .", "and he had been on venlafaxine 150 mg / day since last 2 years under the cover of three mood stabilizers , i.e. , lithium , carbamazepine , and lamotrigine with adequate dosages and normal therapeutic lithium level of 0.76 mmol / l .", "none of previous mood episodes ( both depression and mania ) had any symptoms suggestive of hypersexual behavior .", "his sexual behavior during the intake of high doses of modafinil was also not in keeping with his premorbid sexual desire and sexual activity .", "detailed evaluation of history did not suggest presence of any head injury in the recent past and presence of any associated impulsivity , obsessive compulsive behavior , substance abuse , hyperorality , cognitive decline , sleep attacks , use of aphrodisiacs and use of any other medications which could increase the sexual desire . with the above available information , a diagnosis of bipolar affective disorder with current episode mild depression ( as per icd-10 criteria ) along with an additional diagnosis of modafinil dependence and modafinil induced hypersexuality .", "physical examination , routine hematological and biochemistry investigations and electrocardiogram were within normal limits . at 8001,000 mg /", "day of modafinil , he had side effects as assessed by uku scale in areas of psychic ( score 9 ) , autonomic ( score 3 ) , other ( score 9 ) and global assessment of interference ( score 3 ) .", "he was also rated on hypersexual behavior inventory-19 ( hbi-19)4 ) where he scored 48 .", "management included a gradual and slow reduction in dosage of modafinil at the rate of 100 mg every 2 days under the cover of benzodiazepines ( clonazepam 2 mg / day ) to manage withdrawal symptoms .", "mood charting was done to assess development of any mood symptoms . over 3 weeks period", ", all the above mentioned symptoms decreased significantly ( uku total score 0 ; hbi-19 total score 19 ) and he was discharged after being counseled with structured relapse prevention programme on lithium ( 600 mg / day ) , lamotrigine ( 200 mg / day ) and venlafaxine ( 150 mg / day ) ."], ["in literature , we could find only two case reports on modafinil dependence at higher doses.2,5 ) as seen in the index case , modafinil seems to have some abuse potential .", "our case escalated the dose of modafinil so as to overcome fatigue , increase energy and improve concentration . later on , to overcome the fear of relapsing into depression he increased to a supratherapeutic dosage of 1,000 mg / day and started having symptoms which fulfilled the criteria of hypersexuality disorder ( as per kafka s criteria).6 ) these symptoms were temporally correlated with increment in dose of modafinil and subsided on decreasing the dose of the same in a controlled environment . hence , as neither any other drug used in the index case could be linked with such kind of behavior nor was there any switch to mania / hypomania / mixed episode which could explain such change in behavior , we concluded that the symptoms of hypersexual behavior was induced by modafinil .", "hypersexual behavior during a hypomanic / manic or mixed episode has characteristics of sexual disinhibition , severely compromised self - control , lack of guilt and shame feelings and is generally without any insight .", "in contrast , index case had guilt associated with his increased sexual desire which is very well reported in individuals with hypersexual behaviour.7,8 ) modafinil has been found to act by binding on dopamine receptors and affects dopamine uptake by dopamine transporters in the neurons .", "modafinil dependence can be explained by its dopamine uptake blockade leading to increase in dopamine concentration in the brain areas linked with reward pathways.9 ) on the other hand , a hyperdopaminergic state have been implicated in hypersexuality .", "upsurge of dopamine in the mesolimbic pathway due to use of supratherapeutic dosage of modafinil might explain for the development of hypersexuality in the index case .", "overdose of modafinil can cause insomnia , agitation , tachycardia , rise in blood pressure etc . , but in case of dependence it is more likely to cause physical withdrawal symptoms like lethargy , tremors of hands , anxiety and erratic sleep hours5 ) or only severe psychological withdrawal symptoms.10 ) modafinil has been listed under schedule iv drug in united states for regulation of its sale .", "however , in india , it is available over the counter and being regularly prescribed by many psychiatrists .", "inspite of reportedly low abuse potential , we report a case of modafinil dependence and modafinil induced hypersexuality so as to increase awareness regarding this rare side effect of modafinil and for the need of regulation on its sale ."]]}
{"article_id": "PMC3446081", "article_text": ["childhood constipation is a common complaint accounting for 3% of visits to pediatricians and 10 - 25% of visits to pediatric gastroenterologist[13 ] .", "commonly there is no underlying organic cause and the constipation is indentified as functional or idiopathic[4 , 5 ] .", "functional constipation is identified by rome iii criteria in children over 4 years old and adolescents having two months of at least 2 or more of the following conditions : 2 defecations in the toilet per week , 1 episode of fecal incontinence per week , history of retentive posturing or excessive volitional stool retention , history of painful or hard bowel movements , presence of a large fecal mass in the rectum , history of large diameter stools that may obstruct the toilet .", "children usually are treated with a combination of toilet training , running a bowel diary , and oral laxatives such as paraffin oil . in many patients", "there are many articles on the efficacy of probiotics in organic and functional gastrointestinal disorders .", "probiotics are live microbial food ingredients which are reported to be effective in the treatment of many forms of gastrointestinal disorders[812 ] .", "prebiotic is a selectively fermented ingredient that changes the composition and/or activity of the intestinal microflora and has shown beneficial effects in host 's health .", ", we aimed to determine the therapeutic effect of synbiotics ( combination of probiotics containing strains of l. casei , l. rhamnosus , s. thermophilus , b. breve , l. acidophilus , b. infantis , and fructooligo - saccharide as prebiotic ) on childhood constipation .", "the study was performed at children 's medical center in tehran , iran , from january to december 2009 .", "children with constipation were referred to the pediatric gastroenterology clinic for evaluation and treatment of constipation .", "one - hundred two children aged 412 years with chronic functional constipation enrolled in this study , were divided into 3 groups by simple randomization method .", "exclusion criteria were organic causes for constipation such as hirschsprung 's disease , spina bifida occulta , hypothyroidism , cystic fibrosis , neurologic abnormalities and intestinal pseudo - obstruction .", "these patients were excluded from the study by history and physical examination and rome iii criteria of childhood constipation .", "the research protocol was approved by the medical ethics committee of tehran university of medical sciences .", "label of drugs was replaced by a new label indicating drug a or b. contents of sachets or bottles were not known to the physicians or nurses involved in the study .", "group a received 1.5 ml / kg / day oral liquid paraffin plus placebo , group b received 1 sachet synbiotic per day ( restore 1x10 cfu/1 sachet , protexin co , uk ) .", "synbiotic combination consisted of probiotic strains containing l. casei , l. rhamnosus , s. thermophilus , b. breve , l. acidophilus , b. infantis , fructooligosaccharide as prebiotic , and placebo .", "group c received 1.5 ml / kg / day oral liquid paraffin and 1 sachet synbiotic per day .", "all patients in the 3 groups received drugs in bottles and sachets with similar shape , taste and color .", "frequency of bms , stool consistency , number of fecal incontinence , episodes of abdominal pain , painful defecation per week and side effects were determined in each patient before and after treatment .", "success treatment was defined as 3 bms per week , 2 incontinence per month and no abdominal pain .", "study design : from seven days prior to study and during the treatment period all children were requested to record frequency of bms , number of fecal incontinence episodes , stool consistency , abdominal pain , painful defecation and effects such as vomiting , diarrhea and oil seepage in a bowel diary . before starting treatment ,", "all children were examined and patients with fecal impaction received rectal enema ( paraffin oil 1530ml / year ) once daily for three days in order to accomplish rectal disimpaction .", "toilet training consisted of sitting on the toilet 3 times per day for 5 minutes after each meal .", "patients were not allowed to use any other kind of medication for constipation during the study period .", "clinical efficacy was recorded , patients were seen at the end of 4 weeks , and their charts were reviewed .", "outcome measures : primary outcome measures were frequency of bms per week , stool consistency , fecal incontinence episodes per week , presence of abdominal pain , and painful defecation ; secondary outcomes were success treatment and incidence of adverse effects such as vomiting , diarrhea and seepage .", "stool consistency was rated by the patients as hard , normal or watery . statistical analysis : data of baseline information were analyzed with the spss ( ver .", "continuous variables were expressed as mean ( sd ) , and categorical data were shown as frequency and percent .", "number of bms and fecal incontinence episodes in baseline information were analyzed by kruskal - wallis test .", "change of frequency of bms and fecal incontinence episodes were analyzed by non - parametric paired wilcoxon test . for assessment changes of stool consistency and abdominal pain between baseline and after treatment , the mcnemar test was used .", "totally , 102 children ( aged 412 years ) with chronic functional constipation enrolled in this study and were divided into 3 groups .", "five patients were lost to follow - up and remaining 97 patients consisted of 47.4% males and 52.6% females with a mean age of 6.32.1 years .", "as shown in table 1 , no significant differences were found with respect to demographic data and recorded baseline characteristics between the three treatment groups .", "characteristics of the patients and the main outcomes are summarized in table 1 and 2 . as shown in table 2 ,", "the frequency of bms per week increased in all groups , the highest rise occurring in group c ( p=0.03 ) .", "improvement in stool consistency and decrease in number of fecal incontinence episodes happened in all groups ( p=0.2 , p=0.3 respectively ) without any statistically significant difference .", "also , abdominal pain and painful defecation per week decreased ( p=0.6 , p=0.9 respectively ) similarly and there was no statistically significant difference between groups .", "no side effects were reported in group b but in group a 18 patients and in group c 21 patients had seepage ( p<0.001 ) .", "treatment success in group a was 24/29 ( 82.8% ) , in group b 22/31 ( 71.0% ) and in group c 28/37 ( 75.7% ) without statistically significant difference between them ( p=0.6 ) . \n ", "new investigations have shown that gut bacterial colonization has an important role in health and disease .", "there are several hypotheses about the effectiveness of this microbial flora in treatment of constipation . according to some", "investigations gut microbial flora in chronic constipation is different from that in healthy persons[17 , 18 ] .", "short chain fatty acids produced by probiotics reduce colon ph and by this mechanism enhance colon motility and finally transit time will decrease .", "there are a few rcts about probiotics in the treatment of constipation in children . in rcts study by banaszkiewicz and szajewska", "it was concluded that l. rhamnosus \n gg was not an effective adjunct to lactulose in treating constipation in children .", "bu et al evaluated the effect of l. casei rhamnosus \n lcr35 compared to magnesium oxide(mg0).comparisons of the frequency of defecation , consistency of stool and the use of lactulose or enema during the period of treatment were made among the groups .", "the patients who received mgo or probiotics had a higher defecation frequency , percentage of treatment success ( defined as 3 spontaneous defecations per week with no episodes of fecal soiling ) and less hard stool .", "there is no statistically significant difference in efficacy between mgo and lcr35 , but less abdominal pain occurred when using lcr35 . in a study by coccorullo et al", "the beneficial effects of l. reuteri ( dsm 17938 ) in infants with functional chronic constipation was evaluated .", "administration of l. reuteri had a positive effect on bowel frequency , even when there was no improvement in stool consistency and episodes of inconsolable crying episodes .", "for example , in children with constipation defined according to the rome iii criteria , administration of bifidobacteria ( b. bifidum , b. infantis , b. longum ) and lactobacilli ( l. casei , l. plantarum , l. rhamnosus ) to 20 children aged 416 years resulted in an increased frequency of bowel movements , a decreased number of fecal incontinence episodes , and reduced abdominal pain , although there was no change in stool consistency .", "it seems that our study is the first rct study which investigates the synbiotics effects in childhood constipation . according to our study , mixtures of different strains of probiotics and their specialized prebiotics are more effective than each of them alone and this combination augments their efficacy in all parameters of study , but in previous researches improvement had been seen only in some of symptoms and signs .", "also adjunctive therapy of synbiotic and liquid paraffin could be more effective to improve bms than any of them alone .", "according to this rct , synbiotic have got beneficial effects on symptoms of childhood constipation similar to liquid paraffin without any side effects and synbiotic is an effective adjunct to liquid paraffin to improve bms ."], "abstract_text": ["<S> objectiveconstipation is a common problem in children . </S>", "<S> there is some clinical evidence for the role of probiotics and prebiotics in the treatment of constipated children . </S>", "<S> this is the first study on the therapeutic effect of synbiotics ( combination of probiotics and prebiotic ) in treatment of childhood constipation.methodsin a double - blind randomized placebo controlled study 102 children aged 412 years with functional constipation were assessed according to rome iii criteria for 4 weeks . </S>", "<S> they were divided into 3 groups : group a , received 1.5 ml / kg / day oral liquid paraffin plus placebo , group b , 1 sachet synbiotic per day plus placebo and group c , 1.5 ml / kg / day oral liquid paraffin plus 1 sachet synbiotic per day . </S>", "<S> frequency of bowel movements ( bms ) , stool consistency , number of fecal incontinence episodes , abdominal pain , painful defecation per week , success of treatment and side effects were determined in each group before and after treatment.findingsthe frequency of bms per week increased in all groups ( p<0.001 ) , but it differed between groups and was higher in group c ( p=0.03 ) . </S>", "<S> stool consistency increased and number of fecal incontinence episodes , abdominal pain and painful defecation per week decreased in all groups similarly and there was statistically no difference between them . </S>", "<S> no side effects were reported in group b ; the main side effect in group a and c was seepage of oil ( p<0.001 ) . </S>", "<S> treatment success was similar in all groups without any significant difference between them ( p=0.6).conclusionthis study showed that synbiotics have positive effects on symptoms of childhood constipation without any side effects . </S>"], "labels": null, "section_names": ["Introduction", "Subjects and Methods", "Findings", "Discussion", "Conclusion", "Conflict of Interest"], "sections": [["childhood constipation is a common complaint accounting for 3% of visits to pediatricians and 10 - 25% of visits to pediatric gastroenterologist[13 ] .", "commonly there is no underlying organic cause and the constipation is indentified as functional or idiopathic[4 , 5 ] .", "functional constipation is identified by rome iii criteria in children over 4 years old and adolescents having two months of at least 2 or more of the following conditions : 2 defecations in the toilet per week , 1 episode of fecal incontinence per week , history of retentive posturing or excessive volitional stool retention , history of painful or hard bowel movements , presence of a large fecal mass in the rectum , history of large diameter stools that may obstruct the toilet .", "children usually are treated with a combination of toilet training , running a bowel diary , and oral laxatives such as paraffin oil . in many patients", "there are many articles on the efficacy of probiotics in organic and functional gastrointestinal disorders .", "probiotics are live microbial food ingredients which are reported to be effective in the treatment of many forms of gastrointestinal disorders[812 ] .", "prebiotic is a selectively fermented ingredient that changes the composition and/or activity of the intestinal microflora and has shown beneficial effects in host 's health .", ", we aimed to determine the therapeutic effect of synbiotics ( combination of probiotics containing strains of l. casei , l. rhamnosus , s. thermophilus , b. breve , l. acidophilus , b. infantis , and fructooligo - saccharide as prebiotic ) on childhood constipation ."], ["the study was performed at children 's medical center in tehran , iran , from january to december 2009 .", "children with constipation were referred to the pediatric gastroenterology clinic for evaluation and treatment of constipation .", "one - hundred two children aged 412 years with chronic functional constipation enrolled in this study , were divided into 3 groups by simple randomization method .", "exclusion criteria were organic causes for constipation such as hirschsprung 's disease , spina bifida occulta , hypothyroidism , cystic fibrosis , neurologic abnormalities and intestinal pseudo - obstruction .", "these patients were excluded from the study by history and physical examination and rome iii criteria of childhood constipation .", "the research protocol was approved by the medical ethics committee of tehran university of medical sciences .", "label of drugs was replaced by a new label indicating drug a or b. contents of sachets or bottles were not known to the physicians or nurses involved in the study .", "group a received 1.5 ml / kg / day oral liquid paraffin plus placebo , group b received 1 sachet synbiotic per day ( restore 1x10 cfu/1 sachet , protexin co , uk ) .", "synbiotic combination consisted of probiotic strains containing l. casei , l. rhamnosus , s. thermophilus , b. breve , l. acidophilus , b. infantis , fructooligosaccharide as prebiotic , and placebo .", "group c received 1.5 ml / kg / day oral liquid paraffin and 1 sachet synbiotic per day .", "all patients in the 3 groups received drugs in bottles and sachets with similar shape , taste and color .", "frequency of bms , stool consistency , number of fecal incontinence , episodes of abdominal pain , painful defecation per week and side effects were determined in each patient before and after treatment .", "success treatment was defined as 3 bms per week , 2 incontinence per month and no abdominal pain .", "study design : from seven days prior to study and during the treatment period all children were requested to record frequency of bms , number of fecal incontinence episodes , stool consistency , abdominal pain , painful defecation and effects such as vomiting , diarrhea and oil seepage in a bowel diary . before starting treatment ,", "all children were examined and patients with fecal impaction received rectal enema ( paraffin oil 1530ml / year ) once daily for three days in order to accomplish rectal disimpaction .", "toilet training consisted of sitting on the toilet 3 times per day for 5 minutes after each meal .", "patients were not allowed to use any other kind of medication for constipation during the study period .", "clinical efficacy was recorded , patients were seen at the end of 4 weeks , and their charts were reviewed .", "outcome measures : primary outcome measures were frequency of bms per week , stool consistency , fecal incontinence episodes per week , presence of abdominal pain , and painful defecation ; secondary outcomes were success treatment and incidence of adverse effects such as vomiting , diarrhea and seepage .", "stool consistency was rated by the patients as hard , normal or watery . statistical analysis : data of baseline information were analyzed with the spss ( ver .", "continuous variables were expressed as mean ( sd ) , and categorical data were shown as frequency and percent .", "number of bms and fecal incontinence episodes in baseline information were analyzed by kruskal - wallis test .", "change of frequency of bms and fecal incontinence episodes were analyzed by non - parametric paired wilcoxon test . for assessment changes of stool consistency and abdominal pain between baseline and after treatment , the mcnemar test was used ."], ["totally , 102 children ( aged 412 years ) with chronic functional constipation enrolled in this study and were divided into 3 groups .", "five patients were lost to follow - up and remaining 97 patients consisted of 47.4% males and 52.6% females with a mean age of 6.32.1 years .", "as shown in table 1 , no significant differences were found with respect to demographic data and recorded baseline characteristics between the three treatment groups .", "characteristics of the patients and the main outcomes are summarized in table 1 and 2 . as shown in table 2 ,", "the frequency of bms per week increased in all groups , the highest rise occurring in group c ( p=0.03 ) .", "improvement in stool consistency and decrease in number of fecal incontinence episodes happened in all groups ( p=0.2 , p=0.3 respectively ) without any statistically significant difference .", "also , abdominal pain and painful defecation per week decreased ( p=0.6 , p=0.9 respectively ) similarly and there was no statistically significant difference between groups .", "no side effects were reported in group b but in group a 18 patients and in group c 21 patients had seepage ( p<0.001 ) .", "treatment success in group a was 24/29 ( 82.8% ) , in group b 22/31 ( 71.0% ) and in group c 28/37 ( 75.7% ) without statistically significant difference between them ( p=0.6 ) . \n "], ["new investigations have shown that gut bacterial colonization has an important role in health and disease .", "there are several hypotheses about the effectiveness of this microbial flora in treatment of constipation . according to some", "investigations gut microbial flora in chronic constipation is different from that in healthy persons[17 , 18 ] .", "short chain fatty acids produced by probiotics reduce colon ph and by this mechanism enhance colon motility and finally transit time will decrease .", "there are a few rcts about probiotics in the treatment of constipation in children . in rcts study by banaszkiewicz and szajewska", "it was concluded that l. rhamnosus \n gg was not an effective adjunct to lactulose in treating constipation in children .", "bu et al evaluated the effect of l. casei rhamnosus \n lcr35 compared to magnesium oxide(mg0).comparisons of the frequency of defecation , consistency of stool and the use of lactulose or enema during the period of treatment were made among the groups .", "the patients who received mgo or probiotics had a higher defecation frequency , percentage of treatment success ( defined as 3 spontaneous defecations per week with no episodes of fecal soiling ) and less hard stool .", "there is no statistically significant difference in efficacy between mgo and lcr35 , but less abdominal pain occurred when using lcr35 . in a study by coccorullo et al", "the beneficial effects of l. reuteri ( dsm 17938 ) in infants with functional chronic constipation was evaluated .", "administration of l. reuteri had a positive effect on bowel frequency , even when there was no improvement in stool consistency and episodes of inconsolable crying episodes .", "for example , in children with constipation defined according to the rome iii criteria , administration of bifidobacteria ( b. bifidum , b. infantis , b. longum ) and lactobacilli ( l. casei , l. plantarum , l. rhamnosus ) to 20 children aged 416 years resulted in an increased frequency of bowel movements , a decreased number of fecal incontinence episodes , and reduced abdominal pain , although there was no change in stool consistency .", "it seems that our study is the first rct study which investigates the synbiotics effects in childhood constipation . according to our study , mixtures of different strains of probiotics and their specialized prebiotics are more effective than each of them alone and this combination augments their efficacy in all parameters of study , but in previous researches improvement had been seen only in some of symptoms and signs .", "also adjunctive therapy of synbiotic and liquid paraffin could be more effective to improve bms than any of them alone ."], ["according to this rct , synbiotic have got beneficial effects on symptoms of childhood constipation similar to liquid paraffin without any side effects and synbiotic is an effective adjunct to liquid paraffin to improve bms ."], [""]]}
{"article_id": "PMC4912650", "article_text": ["recently , the use of magnetic nanoparticles ( mnps ) in medical applications ( e.g. magnetic fluid hyperthermia ( mfh ) , magnetic drug delivery , magneto - mechanical actuation of cell surface receptors , magnetic gene transfection , and magnetic resonance imaging ( mri ) contrast improvement ) has had an upward trend .", "the principle of this technique is to destroy cancer cells by heating them up without lethal effects on normal tissues . in comparison with other treatment techniques , this technique is a physical therapy with few side effects and it can be accompanied by other therapies like radiotherapy and chemotherapy to sensitize resistant malignant cells to these therapies .", "effectiveness of the treatment is dependent on the tumor size and its location in the body .", "mnps , used as heating mediators , are magnetic materials consisting of one or more inorganic crystals . when they undergo external magnetic field , thermal energy", "the heating power of mnps depends on the particle size , size distribution , domain state , magnetic anisotropy , the viscosity of the dispersion medium and parameters of the magnetic field , including field strength ( h ) and frequency ( f ) .", "survival of the malignant cells declines when their temperature reaches 41 to 46 c . in this technique , it is therefore necessary to reach temperatures above 41 c by mnps as the heat sources .", "tumor cells are more sensitive to heat in comparison to normal cells and consequently these temperatures do not disturb the viability of normal cells . dispersing mnps in a liquid , as a carrier , leads to the production of magnetic fluids .", ". when fe is substituted by mn , co and zn in m1-xznxfe2o4 ferrites with superior magnetic properties can be produced .", "several methods are available for synthesizing mnps , but the most popular methods are coprecipitation , thermal decomposition , hydrothermal synthesis , and laser pyrolysis . in this study , we synthesized fe0.5zn0.5fe2o4 ferrite with dextrin coating by the aqueous precipitation method .", "the samples were characterized by transmission electron microscopy ( tem ) to investigate the size distribution and morphology of the nanostructure .", "magnetic properties and hysteresis loop of the mnps were studied by vibrating sample magnetometer ( vsm ) .", "fourier transform infrared ( ft - ir ) was also applied to characterize the structure and purity of the compound .", "afterwards , we injected these particles intratumorally in mice bearing implanted melanoma cancer to investigate their hyperthermia effect under amf .", "ferrite nanoparticles ( nps ) were synthesized by the aqueous precipitation route . briefly , individual metal chloride or sulfate , in an appropriate stoichiometric proportions , was dissolved in a dilute nitric acid ( 0.1 mol / l hcl ) solution and heated to 80 c .", "sodium hydroxide solution ( 4 mol / l ) was prepared separately and heated to 80 c .", "the hot nitrate precursor solution was then rapidly mixed with magnetic stirring ( final ph of 12.0 ) .", "then , the solution was cooled and the precipitate was collected by a permanent magnet and washed several times with distilled water to neutralize the supernatant .", "tem ( zeiss , em10c , 80 kv , germany ) was performed to obtain the morphology and size of the nanostructure .", "magnetic properties of ferrofluid were determined by drying the fluid and using a vsm ( meghnatis kavir co. , kashan , iran ) at room temperature .", "surface structure of the sample was identified by using a ft - ir spectroscope ( burker tensor 27 ) .", "the ft - ir spectrum was acquired in the wavenumber range of 500 - 4000 cm .", "b16/f10 melanoma cells were purchased from the pasteur institute ( iran ) and cultured in dmem ( dulbecco s modified eagle medium ) medium with 10% fbs ( fetal bovine serum ) and 1% penicillin in a humidified 37c incubator .", "four- to six - week - old inbred balb / c mice were purchased from the center of comparative and experimental medicine , shiraz university of medical sciences , shiraz , iran .", "all animals were housed in the laboratory of the center of comparative and experimental medicine under identical conditions .", "the animals were housed in special cages at a controlled temperature ( 242 c ) with weekly floor exchange .", "a 12:12 hour light - dark cycle was followed in the above - mentioned animal house center .", "all animals received care in compliance with the standard animal ethics of iran . to implant solid tumor , 1510 b16/f10 melanoma cells in pbs", "after approximately one week , the tumors appeared and the mice were followed until their tumors reached an appropriate size . to measure the tumor volume ,", "a caliper was used and the volume was computed using the following formula . where  a  and  b  are the largest and smallest diameter of tumor , respectively . after reaching an appropriate tumor size ( about 1 cm ) ,", "hypethermia study was started . in order to generate alternating magnetic field ( amf ) ,", "a horizontal solenoid coil vibrating 100 g at 515 khz ( sine wave pattern ) was used ( shiraz university , school of engineering ) .", "the solenoid had an inner diameter of 5 cm , a length of 15 cm and 14 turns .", "it consisted of a water - cooling system to avoid heat induction because of the impedance .", "a resonant h - bridge zcs inverter with a capacitor bank of 75 nf was employed to derive the coil and inject ac current .", "capacitor bank was in series with the solenoid coil , which constitutes the resonant tank .", "the inverter changed dc input voltage to a square wave alternating voltage and was applied to the resonant tank .", "sinusoidal current flowed through the coil while the frequency of the square wave voltage was adjusted to the resonant frequency of the resonant tank .", "the injected ac current generated amf whose amplitude was calculated according to the coil characteristics and the magnitude of ac current .", "where , n : number of turns , l : coil length , 0 : equal to 4*10 and is the magnetic permeability in vacuum , bm : peak value of amf , and i m : peak value of ac current . the peak value of the current was adjusted to 85 a , which was measured using gds-1072 oscilloscope .", "the maximum input power of the system was 650 w. mice bearing implanted solid tumor in proper conditions were selected and divided into four groups consisting of two mice per group .", "it included a control group ( group a ) without any treatment administration and a sham treatment group ( group b ) to investigate the effect of magnetic field on the malignant cells . the mice in group b were located in the solenoid coil for 30 min without mnps injection . for evaluating the lethal effects of the fnps on cancerous cells , the mice in group c received only intratumoral injection of fnps .", "finally , group d was chosen for treatment administration by intratumoral injection of mnps ( 200 l magnetic fluid containing 2 mg fe ) undergoing amf for 30 min .", "before administrating the experimental treatment , each mouse was anesthetized with intramuscular injection of ketamin / xylazine mix . for histological findings ,", "all treated and non - treated tumors and tissues of the spleen , kidney , and liver were removed 24 hours after the treatment . for this purpose , after dissection , the tissues were removed and each specimen was collected and fixed by immersion in 10% buffered formalin and routinely processed into paraffin .", "afterwards , sections were cut at 5 m and put in an oven for 45 minutes to remove the extra paraffin from the tissue .", "after staining with hematoxylin and eosin ( h&e ) , perl s iron staining was carried out to obtain the iron content in groups c and d. finally , each prepared slide was observed under a microscope to distinguish necrosis and inflammation areas and the distribution of mnps through the track of iron . for this purpose , the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides .", "ferrite nanoparticles ( nps ) were synthesized by the aqueous precipitation route . briefly , individual metal chloride or sulfate , in an appropriate stoichiometric proportions , was dissolved in a dilute nitric acid ( 0.1 mol / l hcl ) solution and heated to 80 c .", "sodium hydroxide solution ( 4 mol / l ) was prepared separately and heated to 80 c .", "the hot nitrate precursor solution was then rapidly mixed with magnetic stirring ( final ph of 12.0 ) .", "then , the solution was cooled and the precipitate was collected by a permanent magnet and washed several times with distilled water to neutralize the supernatant .", "tem ( zeiss , em10c , 80 kv , germany ) was performed to obtain the morphology and size of the nanostructure .", "magnetic properties of ferrofluid were determined by drying the fluid and using a vsm ( meghnatis kavir co. , kashan , iran ) at room temperature .", "surface structure of the sample was identified by using a ft - ir spectroscope ( burker tensor 27 ) .", "the ft - ir spectrum was acquired in the wavenumber range of 500 - 4000 cm .", "b16/f10 melanoma cells were purchased from the pasteur institute ( iran ) and cultured in dmem ( dulbecco s modified eagle medium ) medium with 10% fbs ( fetal bovine serum ) and 1% penicillin in a humidified 37c incubator .", "four- to six - week - old inbred balb / c mice were purchased from the center of comparative and experimental medicine , shiraz university of medical sciences , shiraz , iran .", "all animals were housed in the laboratory of the center of comparative and experimental medicine under identical conditions .", "the animals were housed in special cages at a controlled temperature ( 242 c ) with weekly floor exchange .", "a 12:12 hour light - dark cycle was followed in the above - mentioned animal house center .", "all animals received care in compliance with the standard animal ethics of iran . to implant solid tumor , 1510 b16/f10 melanoma cells in pbs", "were injected subcutaneously into the back of the neck of each mouse . after approximately one week , the tumors appeared and the mice were followed until their tumors reached an appropriate size . to measure the tumor volume ,", "a caliper was used and the volume was computed using the following formula . where  a  and  b  are the largest and smallest diameter of tumor , respectively .", "after reaching an appropriate tumor size ( about 1 cm ) , hypethermia study was started .", "in order to generate alternating magnetic field ( amf ) , a horizontal solenoid coil vibrating 100 g at 515 khz ( sine wave pattern ) was used ( shiraz university , school of engineering ) .", "the solenoid had an inner diameter of 5 cm , a length of 15 cm and 14 turns .", "it consisted of a water - cooling system to avoid heat induction because of the impedance .", "a resonant h - bridge zcs inverter with a capacitor bank of 75 nf was employed to derive the coil and inject ac current .", "capacitor bank was in series with the solenoid coil , which constitutes the resonant tank .", "the inverter changed dc input voltage to a square wave alternating voltage and was applied to the resonant tank .", "sinusoidal current flowed through the coil while the frequency of the square wave voltage was adjusted to the resonant frequency of the resonant tank . the injected ac current generated amf", "whose amplitude was calculated according to the coil characteristics and the magnitude of ac current .", "where , n : number of turns , l : coil length , 0 : equal to 4*10 and is the magnetic permeability in vacuum , bm : peak value of amf , and i m : peak value of ac current .", "the peak value of the current was adjusted to 85 a , which was measured using gds-1072 oscilloscope .", "mice bearing implanted solid tumor in proper conditions were selected and divided into four groups consisting of two mice per group .", "it included a control group ( group a ) without any treatment administration and a sham treatment group ( group b ) to investigate the effect of magnetic field on the malignant cells . the mice in group b were located in the solenoid coil for 30 min without mnps injection . for evaluating the lethal effects of the fnps on cancerous cells , the mice in group c received only intratumoral injection of fnps .", "finally , group d was chosen for treatment administration by intratumoral injection of mnps ( 200 l magnetic fluid containing 2 mg fe ) undergoing amf for 30 min .", "before administrating the experimental treatment , each mouse was anesthetized with intramuscular injection of ketamin / xylazine mix .", "for histological findings , all treated and non - treated tumors and tissues of the spleen , kidney , and liver were removed 24 hours after the treatment . for this purpose , after dissection , the tissues were removed and each specimen was collected and fixed by immersion in 10% buffered formalin and routinely processed into paraffin .", "afterwards , sections were cut at 5 m and put in an oven for 45 minutes to remove the extra paraffin from the tissue . after staining with hematoxylin and eosin ( h&e ) , perl s iron staining", "was carried out to obtain the iron content in groups c and d. finally , each prepared slide was observed under a microscope to distinguish necrosis and inflammation areas and the distribution of mnps through the track of iron . for this purpose ,", "the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides .", "magnetic properties of nps were determined by vibrating sample magnetometer ( vsm ) at room temperature .", "figure 1 depicts the m versus h curve of the dry fe0.5zn0.5fe2o4 ferrite nps with applied field between -8500 oe and 8500 oe at room temperature .", "as shown in this figure , the particle size , morphology , and agglomeration of mnps can be determined by tem images .", "m versus h curve of fe0.5zn0.5fe2o4 ferrite ( at room temperature ) indicates the single domain superparamagnetic properties of mnps .", "tem image of fe0.5zn0.5fe2o4 ferrite shows that the diameter of nanoparticles is around 9 nm . in order to confirm that dextrin was successfully coated on the surface of nps", "ft - ir spectra of fe0.5zn0.5fe2o4 ferrite shows nps were successfully covered with dextrin . for the in vivo study", ", the tumor was induced in balb / c mice via the injection of b16/f10 melanoma cells in pbs into the back of the neck of the mice . after approximately one week", ", the tumors appeared in the animals and the size of tumors was measured with a caliper ( figure 4 ) .", "figure 5 shows that the mice in groups b and d were located in the center of the solenoid and subjected to amf with 50 a / div current ( figure 6 ) for 30 minutes .", "mouse bearing implanted tumor was placed in a solenoid ( f=515 khz , h=100 g ) .", "sinusoidal current flow through the coil could be seen by oscilloscope ( 50 a / div ) .", "after 24 hours , all mice were sacrificed and each specimen of the tumors was collected and prepared for histological analyses .", "figure 7 shows stained slides of tumors in four groups . in order to document the presence of fnps between the cells and to investigate their effects on the surrounding medium , perl s iron staining was done.12,13 as shown in figure 7c , iron particles were present in malignant cells .", "a : group a ( blue arrows show malignant cells ) ; b : group b ; c : iron stained slide of mouse tumor in group c ( yellow arrow shows the presence of fe nanoparticles in malignant cells ) ; d : h&e stained slide of mouse tumor in group d. no significant difference in the necrosis extent was observed ( h&e 400 ) .", "no trace of fnps was detected in the spleen , kidney , and liver tissues and they had normal tissues , figure 8 .", "no trace of mnps was observed in the tissues of the liver ( a ) , spleen ( b ) , and kidney ( c ) ( h&e 400 ) .", "magnetic properties of nps were determined by vibrating sample magnetometer ( vsm ) at room temperature .", "figure 1 depicts the m versus h curve of the dry fe0.5zn0.5fe2o4 ferrite nps with applied field between -8500 oe and 8500 oe at room temperature .", "as shown in this figure , the particle size , morphology , and agglomeration of mnps can be determined by tem images .", "m versus h curve of fe0.5zn0.5fe2o4 ferrite ( at room temperature ) indicates the single domain superparamagnetic properties of mnps .", "tem image of fe0.5zn0.5fe2o4 ferrite shows that the diameter of nanoparticles is around 9 nm . in order to confirm that dextrin was successfully coated on the surface of nps", "for the in vivo study , the tumor was induced in balb / c mice via the injection of b16/f10 melanoma cells in pbs into the back of the neck of the mice . after approximately one week", ", the tumors appeared in the animals and the size of tumors was measured with a caliper ( figure 4 ) .", "figure 5 shows that the mice in groups b and d were located in the center of the solenoid and subjected to amf with 50 a / div current ( figure 6 ) for 30 minutes .", "mouse bearing implanted tumor was placed in a solenoid ( f=515 khz , h=100 g ) .", "sinusoidal current flow through the coil could be seen by oscilloscope ( 50 a / div ) .", "after 24 hours , all mice were sacrificed and each specimen of the tumors was collected and prepared for histological analyses .", "figure 7 shows stained slides of tumors in four groups . in order to document the presence of fnps between the cells and to investigate their effects on the surrounding medium , perl s iron staining was done.12,13 as shown in figure 7c , iron particles were present in malignant cells .", "a : group a ( blue arrows show malignant cells ) ; b : group b ; c : iron stained slide of mouse tumor in group c ( yellow arrow shows the presence of fe nanoparticles in malignant cells ) ; d : h&e stained slide of mouse tumor in group d. no significant difference in the necrosis extent was observed ( h&e 400 ) .", "no trace of fnps was detected in the spleen , kidney , and liver tissues and they had normal tissues , figure 8 .", "no trace of mnps was observed in the tissues of the liver ( a ) , spleen ( b ) , and kidney ( c ) ( h&e 400 ) .", "due to many side effects of common strategies for cancer therapy , these strategies are not considered useful for all kinds and stages of tumors . consequently , researchers are looking for a comprehensive treatment method .", "parameters that contribute to the induction of heat in this technique are the diameter of nps , anisotropy constant , fluid viscosity , and frequency and amplitude of the magnetic field . in this study , for the characterization of fnps , ft - ir , vsm , tem were done .", "the absence of hysteresis loop in this curve and non - saturated magnetization at the applied magnetic field , indicated the single domain superparamagnetic properties of mnps .", "where , m is the molecular weight of the composition ( in gram ) , ms is saturation magnetization , and 5585 is the magnetic factor . by estimating the saturation magnetization through extrapolation ( 30.16 emu / gr ) , nb was calculated to be 1.27 . according to tem image", "for in vivo study , it is necessary to coat the nps with a polymer . in this study , we used dextrin as a coating for biocompatibility of nps .", "ft - ir analysis was used to confirm that dextrin was successfully coated on the surface of mnps . in ft - ir spectrum", ", the peak at 570 cm refers to the characteristic absorption of fe - o bond of magnetic particles .", "the peaks at 1634.6 cm and 2920.5 cm corresponded to c = o bond and vibration of -ch2- groups , respectively .", "thus , according to previous studies , fnps were successfully covered with dextrin . in this study ,", "microscopic changes in tumoral cells were investigated 24 hours after treatment among the four mice groups . for this purpose , the degree of necrosis and inflammation ( 400 ) was verified on tumor slides . according to the pathology results , in the tumoral tissue , necrotic cells were easily seen in the central part of all tumors . on tumor slides belonging group c , there was no necrosis cells around nps .", "thus , it is expected that if fnps are transferred to other organs in intravenous administration , they do not make lethal effects on the normal cells . however , a previous study showed iron / iron oxide core / shell nps with porphyrin coating caused cell damage due to the generation of free radicals and magnetic hyperthermia under the influence of magnetic field .", "different degrees of necrotic cells could be seen in all specimens , but no significant difference in necrosis extent was observed among the four groups ; thus , this treatment was not powerful enough to remove the cancer cells , which may contribute to particle size .", "three mechanisms contribute to heat loss in mnps , namely , hysteresis losses , neel and brownian relaxations .", "bakoglidis et al . investigated the magnetic hyperthermia response of iron oxide nps in the size range of 5 - 18 nm .", "they divided these nps into three categories ; the superparamagnetic ( spm ) region ( as large as 10 nm ) where heating is mainly because of neel relaxation , the spm - ferromagnetic region ( 10 - 13 nm ) , and the ferromagnetic region ( > 13 nm ) where hysteresis loss mechanism is dominate in heat dissipation .", "they reported that ferromagnetic materials release heat that is more significant and hysteresis mechanism is more efficient for hyperthermia applications .", "in addition , jeun et al . reported that the heat efficiency of fe3o4 particles below 9.8 nm under 110 khz magnetic field frequency was insufficient for hyperthermia applications , and the value of specific loss power ( slp ) had an upward trend with an increase in the particle size from 11.5 to 22.5 nm under the same magnetic field .", "therefore , heat efficiency of fnps with an average diameter of 9 nm under the influence of amf ( 515 khz ) is insufficient for hyperthermia applications .", "although , ultra - small nps have a lot of potential for use in biomedical applications , it was confirmed that this np could not induce efficient heat for hyperthermia .", "however , studies show the mnp aggregate to larger cluster after systematic administration and therefore the effective hydrodynamic size of the mnp in the body is different from the initial size of nps . additionally", ", theoretical study on proton relaxation indicates that the magnetic properties of nps increase significantly upon the enhancement of the particle and the crystallite size .", "ultra - small nps can be functionalized and guided by an external magnetic field for in vivo studies and after removing the emf , dipoles return to random orientations because of the spm behavior of these nps . unlike ferromagnetic nps", "however , for the hyperthermia study , spm nps show lower heating efficiency than single domain ferromagnetic nps due to the absence or negligible amounts of hysteresis loss .", "we also surveyed the presence of nps in other organs such as the spleen , kidney , and liver .", "no trace of fnps was detected in the spleen , kidney , and liver tissues .", "thus , mnps remained in the tumor and did not transfer to other organs after intratumoral injection . in systemic administration , large particles ( > 50 nm )", "are taken up by the liver cells , but smaller ones generally circulate for a longer period .", "longer residence time in the blood stream may enhance ( enhanced permeation and retention ) epr effect .", "therefore , it is expected that in systemic administration , fnps with a diameter of 9 nm are not captured by the liver and accumulated by tumor .", "a limitation of this investigation was the use of a solenoid coil with one frequency and field strength .", "the major findings reported here illustrate that fnp ferrofluid coated with dextrin is a good candidate for in vivo approaches without any side effects on body organs .", "while fe particles were seen in malignant tumor cells , there was no trace of them in other organs .", "different amounts of necrosis were seen in the tumor tissues of all groups , but no significant difference in the necrosis extent was observed among the groups .", "therefore , although this np is a good agent for other biomedical applications , like contrast enhancement in mri , it did not induce efficient heat under external magnetic field ."], "abstract_text": ["<S> background : nowadays , magnetic nanoparticles ( mnps ) have received much attention because of their enormous potentials in many fields such as magnetic fluid hyperthermia ( mfh ) . </S>", "<S> the goal of hyperthermia is to increase the temperature of malignant cells to destroy them without any lethal effect on normal tissues . to investigate the effectiveness of cancer therapy by magnetic fluid hyperthermia , fe0.5zn0.5fe2o4 nanoparticles ( fnps ) </S>", "<S> were used to undergo external magnetic field ( f=515 khz , h=100 g ) in mice bearing implanted tumor.methods:fnps were synthesized via precipitation and characterized using transmission electron microscopy ( tem ) , vibrating sample magnetometer , and fourier transform infrared . for in vivo study , </S>", "<S> the mice bearing implanted tumor were divided into four groups ( two mice per group ) , namely , control group , amf group , mnps group , and mnps&amf group . </S>", "<S> after 24 hours , the mice were sacrificed and each tumor specimen was prepared for histological analyses . </S>", "<S> the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides.results:the mean diameter of fnps was estimated around 9 nm by tem image and m versus h curve indicates that this particle is among superparamagnetic materials . according to histological analyses , no significant difference in necrosis extent was observed among the four groups.conclusion:fnps are biocompatible and have a good size for biomedical applications . however , for mfh approach , larger diameters especially in the range of ferromagnetic particles due to hysteresis loss can induce efficient heat in the target region . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "None", "None", "None", "None", "None", "None", "Results", "None", "None", "None", "Discussion", "Conclusion"], "sections": [["recently , the use of magnetic nanoparticles ( mnps ) in medical applications ( e.g. magnetic fluid hyperthermia ( mfh ) , magnetic drug delivery , magneto - mechanical actuation of cell surface receptors , magnetic gene transfection , and magnetic resonance imaging ( mri ) contrast improvement ) has had an upward trend .", "the principle of this technique is to destroy cancer cells by heating them up without lethal effects on normal tissues . in comparison with other treatment techniques , this technique is a physical therapy with few side effects and it can be accompanied by other therapies like radiotherapy and chemotherapy to sensitize resistant malignant cells to these therapies .", "effectiveness of the treatment is dependent on the tumor size and its location in the body .", "mnps , used as heating mediators , are magnetic materials consisting of one or more inorganic crystals . when they undergo external magnetic field , thermal energy", "the heating power of mnps depends on the particle size , size distribution , domain state , magnetic anisotropy , the viscosity of the dispersion medium and parameters of the magnetic field , including field strength ( h ) and frequency ( f ) .", "survival of the malignant cells declines when their temperature reaches 41 to 46 c . in this technique , it is therefore necessary to reach temperatures above 41 c by mnps as the heat sources .", "tumor cells are more sensitive to heat in comparison to normal cells and consequently these temperatures do not disturb the viability of normal cells . dispersing mnps in a liquid , as a carrier , leads to the production of magnetic fluids .", ". when fe is substituted by mn , co and zn in m1-xznxfe2o4 ferrites with superior magnetic properties can be produced .", "several methods are available for synthesizing mnps , but the most popular methods are coprecipitation , thermal decomposition , hydrothermal synthesis , and laser pyrolysis . in this study , we synthesized fe0.5zn0.5fe2o4 ferrite with dextrin coating by the aqueous precipitation method .", "the samples were characterized by transmission electron microscopy ( tem ) to investigate the size distribution and morphology of the nanostructure .", "magnetic properties and hysteresis loop of the mnps were studied by vibrating sample magnetometer ( vsm ) .", "fourier transform infrared ( ft - ir ) was also applied to characterize the structure and purity of the compound .", "afterwards , we injected these particles intratumorally in mice bearing implanted melanoma cancer to investigate their hyperthermia effect under amf ."], ["ferrite nanoparticles ( nps ) were synthesized by the aqueous precipitation route . briefly , individual metal chloride or sulfate , in an appropriate stoichiometric proportions , was dissolved in a dilute nitric acid ( 0.1 mol / l hcl ) solution and heated to 80 c .", "sodium hydroxide solution ( 4 mol / l ) was prepared separately and heated to 80 c .", "the hot nitrate precursor solution was then rapidly mixed with magnetic stirring ( final ph of 12.0 ) .", "then , the solution was cooled and the precipitate was collected by a permanent magnet and washed several times with distilled water to neutralize the supernatant .", "tem ( zeiss , em10c , 80 kv , germany ) was performed to obtain the morphology and size of the nanostructure .", "magnetic properties of ferrofluid were determined by drying the fluid and using a vsm ( meghnatis kavir co. , kashan , iran ) at room temperature .", "surface structure of the sample was identified by using a ft - ir spectroscope ( burker tensor 27 ) .", "the ft - ir spectrum was acquired in the wavenumber range of 500 - 4000 cm .", "b16/f10 melanoma cells were purchased from the pasteur institute ( iran ) and cultured in dmem ( dulbecco s modified eagle medium ) medium with 10% fbs ( fetal bovine serum ) and 1% penicillin in a humidified 37c incubator .", "four- to six - week - old inbred balb / c mice were purchased from the center of comparative and experimental medicine , shiraz university of medical sciences , shiraz , iran .", "all animals were housed in the laboratory of the center of comparative and experimental medicine under identical conditions .", "the animals were housed in special cages at a controlled temperature ( 242 c ) with weekly floor exchange .", "a 12:12 hour light - dark cycle was followed in the above - mentioned animal house center .", "all animals received care in compliance with the standard animal ethics of iran . to implant solid tumor , 1510 b16/f10 melanoma cells in pbs", "after approximately one week , the tumors appeared and the mice were followed until their tumors reached an appropriate size . to measure the tumor volume ,", "a caliper was used and the volume was computed using the following formula . where  a  and  b  are the largest and smallest diameter of tumor , respectively . after reaching an appropriate tumor size ( about 1 cm ) ,", "hypethermia study was started . in order to generate alternating magnetic field ( amf ) ,", "a horizontal solenoid coil vibrating 100 g at 515 khz ( sine wave pattern ) was used ( shiraz university , school of engineering ) .", "the solenoid had an inner diameter of 5 cm , a length of 15 cm and 14 turns .", "it consisted of a water - cooling system to avoid heat induction because of the impedance .", "a resonant h - bridge zcs inverter with a capacitor bank of 75 nf was employed to derive the coil and inject ac current .", "capacitor bank was in series with the solenoid coil , which constitutes the resonant tank .", "the inverter changed dc input voltage to a square wave alternating voltage and was applied to the resonant tank .", "sinusoidal current flowed through the coil while the frequency of the square wave voltage was adjusted to the resonant frequency of the resonant tank .", "the injected ac current generated amf whose amplitude was calculated according to the coil characteristics and the magnitude of ac current .", "where , n : number of turns , l : coil length , 0 : equal to 4*10 and is the magnetic permeability in vacuum , bm : peak value of amf , and i m : peak value of ac current . the peak value of the current was adjusted to 85 a , which was measured using gds-1072 oscilloscope .", "the maximum input power of the system was 650 w. mice bearing implanted solid tumor in proper conditions were selected and divided into four groups consisting of two mice per group .", "it included a control group ( group a ) without any treatment administration and a sham treatment group ( group b ) to investigate the effect of magnetic field on the malignant cells . the mice in group b were located in the solenoid coil for 30 min without mnps injection . for evaluating the lethal effects of the fnps on cancerous cells , the mice in group c received only intratumoral injection of fnps .", "finally , group d was chosen for treatment administration by intratumoral injection of mnps ( 200 l magnetic fluid containing 2 mg fe ) undergoing amf for 30 min .", "before administrating the experimental treatment , each mouse was anesthetized with intramuscular injection of ketamin / xylazine mix . for histological findings ,", "all treated and non - treated tumors and tissues of the spleen , kidney , and liver were removed 24 hours after the treatment . for this purpose , after dissection , the tissues were removed and each specimen was collected and fixed by immersion in 10% buffered formalin and routinely processed into paraffin .", "afterwards , sections were cut at 5 m and put in an oven for 45 minutes to remove the extra paraffin from the tissue .", "after staining with hematoxylin and eosin ( h&e ) , perl s iron staining was carried out to obtain the iron content in groups c and d. finally , each prepared slide was observed under a microscope to distinguish necrosis and inflammation areas and the distribution of mnps through the track of iron . for this purpose , the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides ."], ["ferrite nanoparticles ( nps ) were synthesized by the aqueous precipitation route . briefly , individual metal chloride or sulfate , in an appropriate stoichiometric proportions , was dissolved in a dilute nitric acid ( 0.1 mol / l hcl ) solution and heated to 80 c .", "sodium hydroxide solution ( 4 mol / l ) was prepared separately and heated to 80 c .", "the hot nitrate precursor solution was then rapidly mixed with magnetic stirring ( final ph of 12.0 ) .", "then , the solution was cooled and the precipitate was collected by a permanent magnet and washed several times with distilled water to neutralize the supernatant ."], ["tem ( zeiss , em10c , 80 kv , germany ) was performed to obtain the morphology and size of the nanostructure .", "magnetic properties of ferrofluid were determined by drying the fluid and using a vsm ( meghnatis kavir co. , kashan , iran ) at room temperature .", "surface structure of the sample was identified by using a ft - ir spectroscope ( burker tensor 27 ) .", "the ft - ir spectrum was acquired in the wavenumber range of 500 - 4000 cm ."], ["b16/f10 melanoma cells were purchased from the pasteur institute ( iran ) and cultured in dmem ( dulbecco s modified eagle medium ) medium with 10% fbs ( fetal bovine serum ) and 1% penicillin in a humidified 37c incubator .", "four- to six - week - old inbred balb / c mice were purchased from the center of comparative and experimental medicine , shiraz university of medical sciences , shiraz , iran .", "all animals were housed in the laboratory of the center of comparative and experimental medicine under identical conditions .", "the animals were housed in special cages at a controlled temperature ( 242 c ) with weekly floor exchange .", "a 12:12 hour light - dark cycle was followed in the above - mentioned animal house center .", "all animals received care in compliance with the standard animal ethics of iran . to implant solid tumor , 1510 b16/f10 melanoma cells in pbs", "were injected subcutaneously into the back of the neck of each mouse . after approximately one week , the tumors appeared and the mice were followed until their tumors reached an appropriate size . to measure the tumor volume ,", "a caliper was used and the volume was computed using the following formula . where  a  and  b  are the largest and smallest diameter of tumor , respectively .", "after reaching an appropriate tumor size ( about 1 cm ) , hypethermia study was started ."], ["in order to generate alternating magnetic field ( amf ) , a horizontal solenoid coil vibrating 100 g at 515 khz ( sine wave pattern ) was used ( shiraz university , school of engineering ) .", "the solenoid had an inner diameter of 5 cm , a length of 15 cm and 14 turns .", "it consisted of a water - cooling system to avoid heat induction because of the impedance .", "a resonant h - bridge zcs inverter with a capacitor bank of 75 nf was employed to derive the coil and inject ac current .", "capacitor bank was in series with the solenoid coil , which constitutes the resonant tank .", "the inverter changed dc input voltage to a square wave alternating voltage and was applied to the resonant tank .", "sinusoidal current flowed through the coil while the frequency of the square wave voltage was adjusted to the resonant frequency of the resonant tank . the injected ac current generated amf", "whose amplitude was calculated according to the coil characteristics and the magnitude of ac current .", "where , n : number of turns , l : coil length , 0 : equal to 4*10 and is the magnetic permeability in vacuum , bm : peak value of amf , and i m : peak value of ac current .", "the peak value of the current was adjusted to 85 a , which was measured using gds-1072 oscilloscope ."], ["mice bearing implanted solid tumor in proper conditions were selected and divided into four groups consisting of two mice per group .", "it included a control group ( group a ) without any treatment administration and a sham treatment group ( group b ) to investigate the effect of magnetic field on the malignant cells . the mice in group b were located in the solenoid coil for 30 min without mnps injection . for evaluating the lethal effects of the fnps on cancerous cells , the mice in group c received only intratumoral injection of fnps .", "finally , group d was chosen for treatment administration by intratumoral injection of mnps ( 200 l magnetic fluid containing 2 mg fe ) undergoing amf for 30 min .", "before administrating the experimental treatment , each mouse was anesthetized with intramuscular injection of ketamin / xylazine mix ."], ["for histological findings , all treated and non - treated tumors and tissues of the spleen , kidney , and liver were removed 24 hours after the treatment . for this purpose , after dissection , the tissues were removed and each specimen was collected and fixed by immersion in 10% buffered formalin and routinely processed into paraffin .", "afterwards , sections were cut at 5 m and put in an oven for 45 minutes to remove the extra paraffin from the tissue . after staining with hematoxylin and eosin ( h&e ) , perl s iron staining", "was carried out to obtain the iron content in groups c and d. finally , each prepared slide was observed under a microscope to distinguish necrosis and inflammation areas and the distribution of mnps through the track of iron . for this purpose ,", "the necrotic surface area was estimated by using graticule ( olympus , japan ) on tumor slides ."], ["magnetic properties of nps were determined by vibrating sample magnetometer ( vsm ) at room temperature .", "figure 1 depicts the m versus h curve of the dry fe0.5zn0.5fe2o4 ferrite nps with applied field between -8500 oe and 8500 oe at room temperature .", "as shown in this figure , the particle size , morphology , and agglomeration of mnps can be determined by tem images .", "m versus h curve of fe0.5zn0.5fe2o4 ferrite ( at room temperature ) indicates the single domain superparamagnetic properties of mnps .", "tem image of fe0.5zn0.5fe2o4 ferrite shows that the diameter of nanoparticles is around 9 nm . in order to confirm that dextrin was successfully coated on the surface of nps", "ft - ir spectra of fe0.5zn0.5fe2o4 ferrite shows nps were successfully covered with dextrin . for the in vivo study", ", the tumor was induced in balb / c mice via the injection of b16/f10 melanoma cells in pbs into the back of the neck of the mice . after approximately one week", ", the tumors appeared in the animals and the size of tumors was measured with a caliper ( figure 4 ) .", "figure 5 shows that the mice in groups b and d were located in the center of the solenoid and subjected to amf with 50 a / div current ( figure 6 ) for 30 minutes .", "mouse bearing implanted tumor was placed in a solenoid ( f=515 khz , h=100 g ) .", "sinusoidal current flow through the coil could be seen by oscilloscope ( 50 a / div ) .", "after 24 hours , all mice were sacrificed and each specimen of the tumors was collected and prepared for histological analyses .", "figure 7 shows stained slides of tumors in four groups . in order to document the presence of fnps between the cells and to investigate their effects on the surrounding medium , perl s iron staining was done.12,13 as shown in figure 7c , iron particles were present in malignant cells .", "a : group a ( blue arrows show malignant cells ) ; b : group b ; c : iron stained slide of mouse tumor in group c ( yellow arrow shows the presence of fe nanoparticles in malignant cells ) ; d : h&e stained slide of mouse tumor in group d. no significant difference in the necrosis extent was observed ( h&e 400 ) .", "no trace of fnps was detected in the spleen , kidney , and liver tissues and they had normal tissues , figure 8 .", "no trace of mnps was observed in the tissues of the liver ( a ) , spleen ( b ) , and kidney ( c ) ( h&e 400 ) ."], ["magnetic properties of nps were determined by vibrating sample magnetometer ( vsm ) at room temperature .", "figure 1 depicts the m versus h curve of the dry fe0.5zn0.5fe2o4 ferrite nps with applied field between -8500 oe and 8500 oe at room temperature .", "as shown in this figure , the particle size , morphology , and agglomeration of mnps can be determined by tem images .", "m versus h curve of fe0.5zn0.5fe2o4 ferrite ( at room temperature ) indicates the single domain superparamagnetic properties of mnps .", "tem image of fe0.5zn0.5fe2o4 ferrite shows that the diameter of nanoparticles is around 9 nm . in order to confirm that dextrin was successfully coated on the surface of nps"], ["for the in vivo study , the tumor was induced in balb / c mice via the injection of b16/f10 melanoma cells in pbs into the back of the neck of the mice . after approximately one week", ", the tumors appeared in the animals and the size of tumors was measured with a caliper ( figure 4 ) .", "figure 5 shows that the mice in groups b and d were located in the center of the solenoid and subjected to amf with 50 a / div current ( figure 6 ) for 30 minutes .", "mouse bearing implanted tumor was placed in a solenoid ( f=515 khz , h=100 g ) .", "sinusoidal current flow through the coil could be seen by oscilloscope ( 50 a / div ) .", "after 24 hours , all mice were sacrificed and each specimen of the tumors was collected and prepared for histological analyses ."], ["figure 7 shows stained slides of tumors in four groups . in order to document the presence of fnps between the cells and to investigate their effects on the surrounding medium , perl s iron staining was done.12,13 as shown in figure 7c , iron particles were present in malignant cells .", "a : group a ( blue arrows show malignant cells ) ; b : group b ; c : iron stained slide of mouse tumor in group c ( yellow arrow shows the presence of fe nanoparticles in malignant cells ) ; d : h&e stained slide of mouse tumor in group d. no significant difference in the necrosis extent was observed ( h&e 400 ) .", "no trace of fnps was detected in the spleen , kidney , and liver tissues and they had normal tissues , figure 8 .", "no trace of mnps was observed in the tissues of the liver ( a ) , spleen ( b ) , and kidney ( c ) ( h&e 400 ) ."], ["due to many side effects of common strategies for cancer therapy , these strategies are not considered useful for all kinds and stages of tumors . consequently , researchers are looking for a comprehensive treatment method .", "parameters that contribute to the induction of heat in this technique are the diameter of nps , anisotropy constant , fluid viscosity , and frequency and amplitude of the magnetic field . in this study , for the characterization of fnps , ft - ir , vsm , tem were done .", "the absence of hysteresis loop in this curve and non - saturated magnetization at the applied magnetic field , indicated the single domain superparamagnetic properties of mnps .", "where , m is the molecular weight of the composition ( in gram ) , ms is saturation magnetization , and 5585 is the magnetic factor . by estimating the saturation magnetization through extrapolation ( 30.16 emu / gr ) , nb was calculated to be 1.27 . according to tem image", "for in vivo study , it is necessary to coat the nps with a polymer . in this study , we used dextrin as a coating for biocompatibility of nps .", "ft - ir analysis was used to confirm that dextrin was successfully coated on the surface of mnps . in ft - ir spectrum", ", the peak at 570 cm refers to the characteristic absorption of fe - o bond of magnetic particles .", "the peaks at 1634.6 cm and 2920.5 cm corresponded to c = o bond and vibration of -ch2- groups , respectively .", "thus , according to previous studies , fnps were successfully covered with dextrin . in this study ,", "microscopic changes in tumoral cells were investigated 24 hours after treatment among the four mice groups . for this purpose , the degree of necrosis and inflammation ( 400 ) was verified on tumor slides . according to the pathology results , in the tumoral tissue , necrotic cells were easily seen in the central part of all tumors . on tumor slides belonging group c , there was no necrosis cells around nps .", "thus , it is expected that if fnps are transferred to other organs in intravenous administration , they do not make lethal effects on the normal cells . however , a previous study showed iron / iron oxide core / shell nps with porphyrin coating caused cell damage due to the generation of free radicals and magnetic hyperthermia under the influence of magnetic field .", "different degrees of necrotic cells could be seen in all specimens , but no significant difference in necrosis extent was observed among the four groups ; thus , this treatment was not powerful enough to remove the cancer cells , which may contribute to particle size .", "three mechanisms contribute to heat loss in mnps , namely , hysteresis losses , neel and brownian relaxations .", "bakoglidis et al . investigated the magnetic hyperthermia response of iron oxide nps in the size range of 5 - 18 nm .", "they divided these nps into three categories ; the superparamagnetic ( spm ) region ( as large as 10 nm ) where heating is mainly because of neel relaxation , the spm - ferromagnetic region ( 10 - 13 nm ) , and the ferromagnetic region ( > 13 nm ) where hysteresis loss mechanism is dominate in heat dissipation .", "they reported that ferromagnetic materials release heat that is more significant and hysteresis mechanism is more efficient for hyperthermia applications .", "in addition , jeun et al . reported that the heat efficiency of fe3o4 particles below 9.8 nm under 110 khz magnetic field frequency was insufficient for hyperthermia applications , and the value of specific loss power ( slp ) had an upward trend with an increase in the particle size from 11.5 to 22.5 nm under the same magnetic field .", "therefore , heat efficiency of fnps with an average diameter of 9 nm under the influence of amf ( 515 khz ) is insufficient for hyperthermia applications .", "although , ultra - small nps have a lot of potential for use in biomedical applications , it was confirmed that this np could not induce efficient heat for hyperthermia .", "however , studies show the mnp aggregate to larger cluster after systematic administration and therefore the effective hydrodynamic size of the mnp in the body is different from the initial size of nps . additionally", ", theoretical study on proton relaxation indicates that the magnetic properties of nps increase significantly upon the enhancement of the particle and the crystallite size .", "ultra - small nps can be functionalized and guided by an external magnetic field for in vivo studies and after removing the emf , dipoles return to random orientations because of the spm behavior of these nps . unlike ferromagnetic nps", "however , for the hyperthermia study , spm nps show lower heating efficiency than single domain ferromagnetic nps due to the absence or negligible amounts of hysteresis loss .", "we also surveyed the presence of nps in other organs such as the spleen , kidney , and liver .", "no trace of fnps was detected in the spleen , kidney , and liver tissues .", "thus , mnps remained in the tumor and did not transfer to other organs after intratumoral injection . in systemic administration , large particles ( > 50 nm )", "are taken up by the liver cells , but smaller ones generally circulate for a longer period .", "longer residence time in the blood stream may enhance ( enhanced permeation and retention ) epr effect .", "therefore , it is expected that in systemic administration , fnps with a diameter of 9 nm are not captured by the liver and accumulated by tumor .", "a limitation of this investigation was the use of a solenoid coil with one frequency and field strength ."], ["the major findings reported here illustrate that fnp ferrofluid coated with dextrin is a good candidate for in vivo approaches without any side effects on body organs .", "while fe particles were seen in malignant tumor cells , there was no trace of them in other organs .", "different amounts of necrosis were seen in the tumor tissues of all groups , but no significant difference in the necrosis extent was observed among the groups .", "therefore , although this np is a good agent for other biomedical applications , like contrast enhancement in mri , it did not induce efficient heat under external magnetic field ."]]}
{"article_id": "PMC3271839", "article_text": ["it behaves highly aggressive and spreads widely throughout the skin , recurs locally , and metastasizes early .", "standard treatment with resection and adjuvant radiotherapy results in local control in approximately 50% of patients at 1 year and median survival is approximately 8 months .", "considerable evidence has implicated matrix metalloproteinases ( mmp ) in the degradation of the extracellular matrix ( ecm ) during the metastatic process .", "murakami et al .   reported the expression of mmp-2 on subcutaneous angiosarcoma in canine . in human ,", "however , there is no english report suggesting the expression of mmp-9 on human angiosarcoma of the skin . in this report", ", we describe a case of angiosarcoma in which complete remission was archived with low dose docetaxel and bisphosphonate after the standard treatment of angiosarcoma .", "a 78-year - old woman consulted us with a 6-month history of an asymptomatic nodule on her scalp .", "her nodule has been surgically resected at a private clinic 3 months before her visit .", "two weeks after the resection , skin nodules had suddenly enlarged . on her first visit ,", "physical examination revealed a skin - colored nodule with a surgery scar , teleangiectasia and purpura on her scalp ( fig .", "histologically , irregular anastomosing vascular channels lined by single layers of enlarged endothelial cells existed between collagen bundles with dense infiltration of lymphocytes ( fig .", "immunohistochemical staining revealed that these enlarged endothelial - like cells were strongly positive for cd31 and vimentin , and positive for factor viii and thrombomodulin , and negative for cd34 , s-100 , ck , sma , and desmin .", "moreover , these enlarged endothelial - like cells and tumor stromas were positive for mmp-9 ( fig .", "we used rabbit polyclonal anti - human mmp-9 antibody ( abcam , tokyo , japan ) at a dilution of 1:100 .", "in addition , we employed immunohistochemical staining for 9 cases of angiosarcomas that were histologically diagnosed in our institution , and about 78% ( 7/9 ) cases of angiosarcomas were positive for mmp9 . from these results , we diagnosed this patient as mmp-9-expressing angiosarcoma . positron emission tomography ( pet ) scans showed no evidence of metastases .", "then , we administered docetaxel monthly at 40 mg / m body surface area intravenously and weekly with oral administration of 17.5 mg sodium risedronate hydrate .", "one and a half years after the surgical treatment , there was no evidence of local recurrence or metastasis .", "in our present case , we describe a case of mmp-9-expressing angiosarcoma treated with low - dose docetaxel and bisphosphonate . like paclitaxel", ", docetaxel promotes microtubule assembly and inhibits the depolymerization of tubulin , thus stabilizing microtubules , but docetaxel has a higher potency [ 4 , 7 , 8 ] .", "however , in studies conducted in europe , docetaxel monotherapy was rather ineffective for soft - tissue sarcoma , including angiosarcoma , and could not be recommended for further use .", "mmp-9 is a stromal factor that regulates the mobilization of hematopoietic stem cells from the bone marrow niche by solubilizing the membrane - bound form of c - kitl . because it remodels the extracellular matrix and promotes the sprouting and growth of new blood vessels by making vegf available to the vegfr-2/flk receptor on endothelial cells , mmp-9 is a linchpin in tumor progression .", "moreover , recently , several reports revealed the expression of mmp-9 on tumor correlated with the progression or prognosis of several skin tumors such as malignant melanoma , squamous cell carcinoma , basal cell carcinoma , and mycosis fungoides .", "in addition , koontz et al .   reported two cases of successful treatment of angiosarcoma with the administration of the anti - vegf antibody bevacizumab along with radiotherapy prior to surgery .", "more recently , it was reported that pharmacological inhibition of mmp-9 by amino - bisphosphonate decreased pro - mmp-9 and vegf in the serum and abrogated the induction of immunosuppressive macrophages , myeloid - derived suppressor cells ( mdscs ) , in the tumor microenvironment [ 15 , 16 ] . in aggregate ,", "inhibition of mmp-9 by bisphosphonate could be one of the optimal supportive therapies for the treatment of angiosarcoma by inhibiting tumor - induced angiogenesis and by the induction of anti - tumor immunity . in conclusion ,", "the combination of docetaxel with bisphosphonate was effective for mmp-9-expressing angiosarcoma and resulted in complete remission after the standard therapy for angiosarcoma ."], "abstract_text": ["<S> we describe a 78-year - old japanese patient with angiosarcoma on the scalp . </S>", "<S> interestingly , immunohistochemical staining revealed this tumor as positive for matrix metalloproteinase 9 ( mmp-9 ) . </S>", "<S> after conventional therapy for angiosarcoma with surgical treatment and radiation therapy , we intravenously administered docetaxel at 40 mg / m2 body surface area together with oral administration of 17.5 mg sodium risedronate hydrate . </S>", "<S> one and a half years after the standard treatment , there was no evidence of local recurrence or metastasis . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["it behaves highly aggressive and spreads widely throughout the skin , recurs locally , and metastasizes early .", "standard treatment with resection and adjuvant radiotherapy results in local control in approximately 50% of patients at 1 year and median survival is approximately 8 months .", "considerable evidence has implicated matrix metalloproteinases ( mmp ) in the degradation of the extracellular matrix ( ecm ) during the metastatic process .", "murakami et al .   reported the expression of mmp-2 on subcutaneous angiosarcoma in canine . in human ,", "however , there is no english report suggesting the expression of mmp-9 on human angiosarcoma of the skin . in this report", ", we describe a case of angiosarcoma in which complete remission was archived with low dose docetaxel and bisphosphonate after the standard treatment of angiosarcoma ."], ["a 78-year - old woman consulted us with a 6-month history of an asymptomatic nodule on her scalp .", "her nodule has been surgically resected at a private clinic 3 months before her visit .", "two weeks after the resection , skin nodules had suddenly enlarged . on her first visit ,", "physical examination revealed a skin - colored nodule with a surgery scar , teleangiectasia and purpura on her scalp ( fig .", "histologically , irregular anastomosing vascular channels lined by single layers of enlarged endothelial cells existed between collagen bundles with dense infiltration of lymphocytes ( fig .", "immunohistochemical staining revealed that these enlarged endothelial - like cells were strongly positive for cd31 and vimentin , and positive for factor viii and thrombomodulin , and negative for cd34 , s-100 , ck , sma , and desmin .", "moreover , these enlarged endothelial - like cells and tumor stromas were positive for mmp-9 ( fig .", "we used rabbit polyclonal anti - human mmp-9 antibody ( abcam , tokyo , japan ) at a dilution of 1:100 .", "in addition , we employed immunohistochemical staining for 9 cases of angiosarcomas that were histologically diagnosed in our institution , and about 78% ( 7/9 ) cases of angiosarcomas were positive for mmp9 . from these results , we diagnosed this patient as mmp-9-expressing angiosarcoma . positron emission tomography ( pet ) scans showed no evidence of metastases .", "then , we administered docetaxel monthly at 40 mg / m body surface area intravenously and weekly with oral administration of 17.5 mg sodium risedronate hydrate .", "one and a half years after the surgical treatment , there was no evidence of local recurrence or metastasis ."], ["in our present case , we describe a case of mmp-9-expressing angiosarcoma treated with low - dose docetaxel and bisphosphonate . like paclitaxel", ", docetaxel promotes microtubule assembly and inhibits the depolymerization of tubulin , thus stabilizing microtubules , but docetaxel has a higher potency [ 4 , 7 , 8 ] .", "however , in studies conducted in europe , docetaxel monotherapy was rather ineffective for soft - tissue sarcoma , including angiosarcoma , and could not be recommended for further use .", "mmp-9 is a stromal factor that regulates the mobilization of hematopoietic stem cells from the bone marrow niche by solubilizing the membrane - bound form of c - kitl . because it remodels the extracellular matrix and promotes the sprouting and growth of new blood vessels by making vegf available to the vegfr-2/flk receptor on endothelial cells , mmp-9 is a linchpin in tumor progression .", "moreover , recently , several reports revealed the expression of mmp-9 on tumor correlated with the progression or prognosis of several skin tumors such as malignant melanoma , squamous cell carcinoma , basal cell carcinoma , and mycosis fungoides .", "in addition , koontz et al .   reported two cases of successful treatment of angiosarcoma with the administration of the anti - vegf antibody bevacizumab along with radiotherapy prior to surgery .", "more recently , it was reported that pharmacological inhibition of mmp-9 by amino - bisphosphonate decreased pro - mmp-9 and vegf in the serum and abrogated the induction of immunosuppressive macrophages , myeloid - derived suppressor cells ( mdscs ) , in the tumor microenvironment [ 15 , 16 ] . in aggregate ,", "inhibition of mmp-9 by bisphosphonate could be one of the optimal supportive therapies for the treatment of angiosarcoma by inhibiting tumor - induced angiogenesis and by the induction of anti - tumor immunity . in conclusion ,", "the combination of docetaxel with bisphosphonate was effective for mmp-9-expressing angiosarcoma and resulted in complete remission after the standard therapy for angiosarcoma ."]]}
{"article_id": "PMC4776578", "article_text": ["their contribution has gone beyond academic research  the team of researchers in the national institute of mental health and neurosciences ( nimhans ) , bengaluru , karnataka , india , has developed indigenous ect devices that have stood the test of time in terms of their use in clinical practice as well as in their contribution to research .", "it delivered bidirectional stimuli , and facilities for measuring various parameters of electrical dose had been incorporated in the device . with the demonstration of cognitive superiority and comparable efficacy of brief - pulse ect ,", "the national workshop on ect ( 1988 ) recommended the development of state - of - the - art ect devices with brief - pulse waveform to be available indigenously .", "a committee including the member of the bengaluru chapter of the national institution for quality and reliability , a psychiatrist ( bng ) , and a biomedical engineer ( vsc ) took up the task of developing a brief - pulse ect device indigenously .", "after several laboratory tests , the machine was finally found suitable for safe use in 1992 .", "the focus on the simplest of the models was the ease of use by psychiatrists .", "this was possible by a simple microcontroller unit that transmits a brief - pulse ( 1.5 ms as width ) of bipolar nature ( at a frequency of 125 pulses / s ) and constant current ( 800 ma ) .", "ten steps of stimulus dose was made available by adjusting the total duration of stimulus from 0.2 s to 3.6 s and keeping current , pulse width , and pulse frequency constant .", "the first two stimulus steps were 30 mc and 60 mc ; the next eight steps were in multiples of 60 mc up to 540 mc .", "the stimulus was delivered by pressing the switch provided on the hand - held electrode .", "the duration of stimulus was preprogrammed based on the total charge chosen and would be shorter if the switch was released earlier .", "the same switch also started a seconds counter to help time the observed motor seizures .", "the treating physician should keep the switch pressed to let that seconds counter on ; release of switch at the termination of seizure stops the counter and indicates the duration ( in seconds ) of the motor seizure .", "release of the switch earlier than 20 s ( failing to see the convulsion of duration longer than 20 s ) automatically would increase the stimulus dose setting to next level for re - stimulation if the doctor chooses to do so .", "features such as internal trigger to disconnect the electroencephalograph ( eeg ) electrodes from external amplifiers during stimulus helped using standby eeg / electrocardiograph ( ecg ) monitoring free of artifacts .", "this allowed changing the settings of other electrical parameters such as pulse width and frequency if required while keeping the default setting same as the earlier microcontroller - programmed steps .", "however , it gave liberty to set either 10 steps of increment as in the basic model or 20 steps increase of 15 mc as 15 , 30 , 45 , 60 ,  up to 540 mc .", "automatic upgradation of the stimulus dose , if the observed seizure was < 20 s , was preserved . by 1994 ,", "the computer monitor would display the eeg in two , four , or eight channels along with ecg .", "after eeg recording is terminated , a 10-s calibration signal of 100 v was recorded .", "there was also option of choosing different eeg settings , including number of channels , and display size ( zoom ) .", "this refined eeg and computerized model is in use in nimhans since 1993 . in 19981999", ", the option of automatic setting of threshold stimulus dose in mc , by feeding data on age , sex , and inion - nasion distance ( ind ) , was developed .", "the research related to measurement of impedance and stimulus threshold of ect in nimhans contributed to it . around the same time , feature to store ect seizure and ecg for offline analysis was made available . with popularity of bifrontal ect ( bfect ) , special concave stimulus electrodes were added .", "most recent development includes facility of administering ultra - brief - pulse ( < 0.5 ms ) ect that is expected to cause lesser cognitive dysfunction .", "the latest integrated ect machine , with four - channel eeg / ecg , has intelligent features to independently operate without interfacing with a computer system .", "eeg / ecg is displayed on 5.7 \" color graphic lcd screen , and it also has sd card memory and printer interface .", "a separate portable eeg amplifier independent of ect can remain attached to the patient and the signals can be seen on any hand - held device that has bluetooth .", "all ect machines at nimhans are periodically calibrated for pulse width , frequency , amplitude , and stimulus duration stages . the frequency and duration", ", this device has been used to administer ect to about 700800 patients annually . with each patient receiving about 67 ects on an average through his / her course ,", "the precision and flexibility of electrical parameters provided by this ect machine have fostered quality research on ect at nimhans .", "we have selected for review those studies from nimhans which have utilized specific features of this device .", "stimulus relative to seizure threshold is more critical than the absolute stimulus . a few techniques of threshold estimation included the half - age method and dose titration method but have limitations .", "the former was dependent on the model of the device while the latter might expose the patient to inadequate stimuli .", "the study at nimhans in this regard on 100 consecutive patients receiving bilateral ect ( blect ) revealed that age was the most important predictor of threshold stimulus accounting for 23% variance in multivariate analysis .", "the other significant but less robust factors were ind and severity of illness that accounted for 5% and 4% of variance in threshold , respectively . the psychotropic medications and head circumference were not noted to influence the threshold .", "a formula as mentioned below for determining stimulus threshold was devised using forward stepwise linear regression model based on age alone and was prospectively tested at the 1 and 6 ect sessions .", "t1 = 1.67 ( age ) + 48.7 t6 = 1.8 ( age ) + 71.9 t1 = threshold at 1 session , t6 = threshold at 6 session .", "the computerized version of stimulus setting allows use of this regression equation with ease . with the computerized version , ect sessions of each patient", "a similar effort was made to find the seizure threshold for unilateral ect ( ulect ) . here , age was the only significant predictor of seizure threshold ( 15% of variance ) .", "a formula based on regression analysis was developed which is as follows : threshold ( mc ) = exp ( age [ 0.015412 ] + 3.667387 ) , where unit of age is years .", "then , in an independent sample ( n = 30 ) of patients receiving ulect , the threshold was estimated ( but not administered ) using the above formula .", "the value obtained was rounded off to the nearest higher level on the ect device .", "this estimated threshold was then compared with the actual threshold stimulus which was determined by using the titration method .", "the results showed that 22 ( 73% ) patients would have developed adequate seizure with the first stimulus estimated by the formula method , but it overestimated threshold stimulus by 3060 mc in majority of them .", "moreover , it underestimated the threshold in 8 ( 27% ) patients . hence , the formula method may not be suitable for threshold estimation in ulect .", "however , if the ect administrator wishes to use it , he or she should do so with the stimulus set at one or two levels lower than the formula - estimated threshold estimation , and further titration may be done as needed .", "although stimulus at 26 times the threshold intensity is associated with greater clinical efficacy in ulect , the guidelines were less clear about this in blect .", "from 2006 , there was change in practice in nimahns and unless otherwise requested from the referring psychiatrist , all blects are administered at 1.5 times the threshold stimulus charge . before 2006 , the threshold stimulus was administered for blect .", "hence , a retrospective data analysis was done to see whether new practice has better outcome .", "the data of 100 consecutive inpatients who received blect at threshold level before the change in practice implemented were compared with that of 101 who received blect at 1.5 times threshold level after the change implemented .", "the outcome measures were the number of ects administered and the number of inpatient days after the start of ect .", "however , the change to suprathreshold blect conferred advantage in the treatment of mania : those receiving suprathreshold ect achieved clinical improvement with , on an average , 2 ects less and their inpatient days reduced by 10 days .", "ect guidelines mandate the need for seizure monitoring during ect . initially , feasibility , reliability , and validity of eeg seizure measurement were studied at nimhans using a separate paper eeg machine .", "subsequently , a paperless eeg amplifier , which was attached to a computer monitor displaying the eeg , was designed at nimhans .", "display of both eeg and the digital counter showing time duration was triggered by the ect stimulus switch ( currently , eeg monitoring is available in a display in the ect machine obviating the need for a computer attached to the ect machine ) .", "this paperless eeg was validated against a paper eeg measurement which showed high correlation in terms of seizure duration ( intraclass correlation , r = 0.99 , p < 0.001 ) . in later studies using this paperless eeg machine , unequivocal absence of epileptiform transients for five or more seconds on both channels", "the use of this paperless eeg simplified eeg monitoring by reducing mechanical problems associated with paper eeg and simplified the storing process .", "( 1998 ) monitored 158 consecutive patients referred for ect on their first ect for both motor and eeg seizures .", "around 40% of patients with prolonged eeg seizures had a seizure duration > 180 s , necessitating termination .", "motor seizure of 90 s failed to predict prolonged eeg seizures ( 120 s or more ) in 60% of occasions . a sizable minority ( 8% ) failed to obtain adequate motor seizure on the cuffed forearm although their eeg seizures were considered adequate by the study criteria ( 25 s ) .", "a similar study was conducted by mayur et al . with 232 consecutive patients posted for either blect ( 54% ) or ulect ( 46% ) . in this study ,", "16% had prolonged eeg seizures , 39% of which was missed by motor monitoring , and 7% of adequate eeg seizures were classified as inadequate by motor seizure monitoring . around 1% of sessions with ulect and 11% with blect were considered inadequate by motor seizure monitoring despite adequate eeg seizures .", "motor seizure correlated well with eeg seizure when the latter was adequate but not when prolonged .", "these findings were again replicated in a further larger study with 485 consecutive patients receiving either blect or ulect . in the later study ,", "two patients classified as having inadequate motor seizures ( < 15 s ) had prolonged eeg seizures ( > 120 s ) .", "patients with prolonged seizures were younger , had lower seizure threshold , were more likely to receive a diagnosis of mania , and were treated with lithium .", "the above studies emphasize the need for eeg monitoring during ect to prevent unnecessary re - stimulation when motor seizures monitoring incorrectly label the treatment as inadequate and also to detect and abort the prolonged seizures , which appears to be common in indian setting .", "a study was conducted on 287 consecutive consenting patients receiving either blect or ulect to explore the factors influencing ratio of motor and eeg seizure duration ( me ratio ) .", "patients were classified into low ( 0.8 ; n = 146 ) and high ( > 0.8 , n = 141 ) me - ratio groups .", "significantly , more patients in the low me - ratio group received blect , had prolonged eeg seizure , and were on concurrent lithium compared with the high me - ratio group .", "this study underscores the importance for eeg monitoring at least inpatients receiving lithium and blect .", "recent international guidelines have recognized the limitations of seizure duration as a marker of adequacy of treatment and emphasize the pattern of eeg seizure .", "a study was conducted to compare the old definition of seizure adequacy ( based on seizure duration ) with the new definition ( a seizure with polyspikes and a 3-hz activity ) in 102 computerized eeg recordings .", "this suggested that seizures meeting newer criteria almost invariably last for at least 25 s ; in the indian context , the change in the criteria would make little change in the day - to - day practice of ect .", "numerous ictal eeg markers have been elucidated to predict treatment response of ect including postictal suppression , postictal coherence and amplitude , amount of slowing and time to onset of slowing , global eeg power , largest lyapunov exponent , and strength symmetry index ( ssi ) .", "ssi was computed from the early- and mid - seizure eeg epochs using the fractal dimension ( fd ) .", "the seizures of ulect were characterized by significantly smaller ssi in both phases , despite having seizure duration comparable to blect . it was suggested that ssi may be a potential measure of seizure adequacy . in another study ,", "ssi was computed in 40 patients receiving either high - dose or low - dose bitemporal ect ( btect ) .", "ssi of the early seizure was higher in the high dose than in the low dose ect group .", "another method of analyzing eeg is to measure the components of the eeg such as its frequencies across several nonoverlapping frequency bands : delta , theta , alpha , and beta through fast fourier transform .", "the delta band of eeg is said to be relevant to the therapeutic properties of ect .", "eeg of 25 patients receiving either blect or ulect was subjected fast fourier transform , and the spectral power of the delta ( 14 hz ) band was computed .", "blect resulted in symmetrical seizure spectral power ; ulect seizures had significantly lower spectral power in the delta band on the unstimulated side .", "fd was computed for early- , mid- , and post - seizure phases of the first ect .", "fd is a geometrical method of analysis of eeg , which was calculated based on the principles described by katz .", "it is a measure of complexity of the eeg wave in the terms of amplitude and frequency .", "smaller postseizure fd after the 1 ect was associated with remission status at 2 weeks .", "smaller postictal fd was considered a marker for postictal suppression suggesting that postictal suppression may be a marker for early antidepressant response for ect .", "a similar study was conducted in 51 right - handed , drug - free patients with major depressive disorder receiving right ulects at 2.5 times their seizure threshold .", "spectral power ( db ) for 26 hz band and fd of postseizure eeg were estimated .", "smaller fd and spectral power ( i.e. greater postseizure eeg suppression ) were seen in early responders compared to the late responders .", "ictal eeg fd was calculated from 26 eeg records of patients receiving either bfect or btect for schizophrenia .", "maximum fd at the 2 or 3 ect had an inverse correlation with psychosis severity after 6 ects as well as with the number of ects provided .", "hence , maximum fd during the early part of the ect course in schizophrenia patients is predictive of greater benefit in short - term psychopathology . in another analysis of data from the sample", ", it was seen that maximum fd does not change over six ects despite decline in seizure duration over ect .", "thus , eeg morphology of blect induced seizures show little variation over the course of treatment which is in keeping with other studies .", "bispectral index ( bis ) measures the level of hypnosis ( sedation ) during anesthesia .", "bis values of awaked individuals with schizophrenia were studied through a course of bitemporal brief - pulse ect .", "it was recorded using a bis monitor ( a-2000 , aspect medical systems inc . , newton , massachusetts , usa ) , with sensor applied to the forehead on the right side .", "it was found that after six ects , 60% of patients had their resting bis values in the anesthetized / sedated range even when they are awake .", "thus , bis may not provide accurate estimation of the depth of anesthesia during ect after the initial ect sessions .", "bfect is found to be as efficacious as btect for depression but associated with fewer cognitive adverse effects .", "however , its efficacy in other indications had not been well studied and hence , it was planned in nimhans . assessing", "bfect in acute mania in double - blind randomized controlled trial showed faster decline in ymrs scores and earlier attainment of response compared to btect without differences in adverse effects .", "a double - blind , randomized controlled trial demonstrated the superior clinical as well as cognitive effects of bfect over btect in - patients with schizophrenia .", "seizure duration significantly reduced only with bfect and not with btect over the course of ect .", "this provided the indirect support to possible better anticonvulsant mechanism of action of bfect in schizophrenia .", "these studies used electrodes with inner concave surface specifically for bfect and later , we have been using these electrodes in clinical practice for bfect in nimhans .", "the effect of electrical stimulus parameters of ect on prolactin response was studied . stimulus dose , frequency of ect , and seizure variables such as seizure duration , seizure strength , pattern , and symmetry", "did not predict chance in prolactin level . similarly , none of the clinical variables including baseline severity of illness , presence of psychotic symptoms , drug - naive status , and degree of clinical improvement predicted the prolactin response .", "the prolactin response however , was significantly more in female participants which may be due to their lower seizure threshold and inherent biological predisposition .", "a study comparing low- and high - stimulus doses in a double - blind , randomized controlled design revealed that eeg seizure was of comparable duration in the two stimulus groups .", "another study with a cross - over design looking into the effect of different stimulus frequency found that lower pulse frequency ( 50 pps ) stimulus had lower seizure threshold and produced fewer subconvulsive stimulations compared with high - frequency stimulus ( 200 pps ) without altering seizure durations or the cardiovascular responses .", "the low - frequency stimulus produced similar clinical benefits as that of high frequency in subsequent study .", "thus , lowering down the frequency of pulses may be a viable option when seizure threshold is high due to various reasons .", "the lower stimulus frequency leads to longer stimulus train duration and hence , had lower rate ( mc / s ) of stimulus dissipation .", "the cardiovascular system ( cvs ) effects of ect were also studied with the features of automated cardiac monitor and ambulatory ecg system in eeg machine . to study the effect of atropine on cvs response to ect , 30 patients were randomly allocated before the third ect to receive atropine or no premedication and found that atropine contributed to 32% of the variance in rate pressure product ( rpp ) with lower values in those who did not receive atropine . however", "this study hence indicated that avoiding atropine as premedication can control rpp and thus can improve cardiac stability during ect without affecting other aspects . in a large sample of 124 patients", ", it was found that postictal rpp was significantly higher after blect than ulect even after eliminating the effect of possible confounding variables such as subconvulsions and concurrent medications .", "the difference of 10% postictal rpp increments between the right ulect and blect groups found in this study is of clinical relevance in elderly or patients at risk of cardiac complications .", "the postictal rpp may be considered as a peripheral marker of the degree of seizure generalization .", "in addition , if the hypothesis of seizure generalization to hypothalamus as a prerequisite for clinical efficacy is given consideration , then lower rpp of threshold ulect compared to blect can explain lower clinical efficacy of ulect and may reflect the less than optimal hypothalamic stimulation .", "the correlation of rpp with clinical efficacy was further supported by the lower postictal rpp with unilateral threshold ect compared to that with unilateral suprathreshold ect .", "however , atropine could mask the differential cardiovascular responses of the effective and ineffective seizures . in another study ,", "the effect of anesthetic modification of motor seizure on rpp was also explored . 1 mg / kg dose of succinylcholine improved the number of patients with successful modification compared to 0.5 mg / kg but did not influence ictal or postictal rpp .", "it thus ruled out the significant cvs alteration due to depolarizing effect of succinylcholine and 1 mg / kg dose may be used routinely without fear of exaggerated cvs response .", "it confirmed the above findings that rpp response correlates with the cerebral seizure instead of motor component of seizure .", "ictal rpp did not differ between groups with or without adequate motor seizure if eeg seizure was adequate . however , the group with inadequate eeg seizure had the least ictal rpp .", "hemodynamic response with btect and bfect were examined at five - time points starting from before anesthesia induction to 2 min after the seizure .", "the maximum values of systolic blood pressure , heart rate , and rpp were similar in both groups , suggesting that cardiovascular load is similar in bfect and btect .", "it also indicates that differential frontal lobe stimulation rather than seizure generalization may be playing a role in better efficacy of bfect over btect in schizophrenia .", "lithium given concurrently with ect can have serious interactions with various aspects of the procedure . at the same time , not combining ect and lithium in certain patients may have adverse clinical consequences . in the absence of systematically collected data on adverse effects of lithium co - prescribed with ect ,", "patients on lithium ( n=27 ) and those not on lithium ( n=28 ) were compared while they received ect .", "the sympathetic cardiovascular response was lower in those who were on lithium and the time to post - ect recovery directly correlated with serum lithium level .", "safety of lithium during ect was satisfactory particularly when serum lithium was at a lower therapeutic range in young patients without significant cardiovascular risk factors . to conclude , the indigenously developed brief - pulse ect - eeg device has been used to treat thousands of patients in the past two decades .", "summary of the findings of research conducted utilizing the specific features of the indigenously developed brief - pulse ect device * many more issues related to ect need exploration in future ; these include altering amplitude of electrical stimulus , ultra - brief ect in indications other than depression , novel techniques of different electrode 's sizes , and placements to reduce the cognitive adverse effects without compromising the clinical efficacy . the last author , mr .", "ltd . , bengaluru , karnataka , india , which manufactures the brief - pulse ect device mentioned in this review article .", "candade is the director of the company , niviqure meditech pvt . ltd . , bengaluru , karnataka , india , which manufactures the brief - pulse ect device mentioned in this review article ."], "abstract_text": ["<S> in 1993 , a device to administer brief - pulse electroconvulsive therapy was indigenously developed through collaboration between the national institution for quality and reliability and the national institute of mental health and neurosciences ( nimhans ) , bengaluru , karnataka , india . </S>", "<S> the additional feature of computerized recording of the electroencephalograph and electrocardiograph for both online and offline use had substantial clinical and research implications . over the past two decades </S>", "<S> , this device has been used extensively in different academic and nonacademic settings . </S>", "<S> a considerable body of research with clinical and heuristic interest has also emanated using this device . in this paper , we present the development of this device and follow it up with a review of research conducted at nimhans that validate the features and potentials of this device . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SEIZURE THRESHOLD", "ELECTROENCEPHALOGRAM MONITORING", "ELECTRODE PLACEMENT", "ELECTRICAL STIMULUS PARAMETERS", "CARDIOVASCULAR SYSTEM MONITORING", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["their contribution has gone beyond academic research  the team of researchers in the national institute of mental health and neurosciences ( nimhans ) , bengaluru , karnataka , india , has developed indigenous ect devices that have stood the test of time in terms of their use in clinical practice as well as in their contribution to research .", "it delivered bidirectional stimuli , and facilities for measuring various parameters of electrical dose had been incorporated in the device . with the demonstration of cognitive superiority and comparable efficacy of brief - pulse ect ,", "the national workshop on ect ( 1988 ) recommended the development of state - of - the - art ect devices with brief - pulse waveform to be available indigenously .", "a committee including the member of the bengaluru chapter of the national institution for quality and reliability , a psychiatrist ( bng ) , and a biomedical engineer ( vsc ) took up the task of developing a brief - pulse ect device indigenously .", "after several laboratory tests , the machine was finally found suitable for safe use in 1992 .", "the focus on the simplest of the models was the ease of use by psychiatrists .", "this was possible by a simple microcontroller unit that transmits a brief - pulse ( 1.5 ms as width ) of bipolar nature ( at a frequency of 125 pulses / s ) and constant current ( 800 ma ) .", "ten steps of stimulus dose was made available by adjusting the total duration of stimulus from 0.2 s to 3.6 s and keeping current , pulse width , and pulse frequency constant .", "the first two stimulus steps were 30 mc and 60 mc ; the next eight steps were in multiples of 60 mc up to 540 mc .", "the stimulus was delivered by pressing the switch provided on the hand - held electrode .", "the duration of stimulus was preprogrammed based on the total charge chosen and would be shorter if the switch was released earlier .", "the same switch also started a seconds counter to help time the observed motor seizures .", "the treating physician should keep the switch pressed to let that seconds counter on ; release of switch at the termination of seizure stops the counter and indicates the duration ( in seconds ) of the motor seizure .", "release of the switch earlier than 20 s ( failing to see the convulsion of duration longer than 20 s ) automatically would increase the stimulus dose setting to next level for re - stimulation if the doctor chooses to do so .", "features such as internal trigger to disconnect the electroencephalograph ( eeg ) electrodes from external amplifiers during stimulus helped using standby eeg / electrocardiograph ( ecg ) monitoring free of artifacts .", "this allowed changing the settings of other electrical parameters such as pulse width and frequency if required while keeping the default setting same as the earlier microcontroller - programmed steps .", "however , it gave liberty to set either 10 steps of increment as in the basic model or 20 steps increase of 15 mc as 15 , 30 , 45 , 60 ,  up to 540 mc .", "automatic upgradation of the stimulus dose , if the observed seizure was < 20 s , was preserved . by 1994 ,", "the computer monitor would display the eeg in two , four , or eight channels along with ecg .", "after eeg recording is terminated , a 10-s calibration signal of 100 v was recorded .", "there was also option of choosing different eeg settings , including number of channels , and display size ( zoom ) .", "this refined eeg and computerized model is in use in nimhans since 1993 . in 19981999", ", the option of automatic setting of threshold stimulus dose in mc , by feeding data on age , sex , and inion - nasion distance ( ind ) , was developed .", "the research related to measurement of impedance and stimulus threshold of ect in nimhans contributed to it . around the same time , feature to store ect seizure and ecg for offline analysis was made available . with popularity of bifrontal ect ( bfect ) , special concave stimulus electrodes were added .", "most recent development includes facility of administering ultra - brief - pulse ( < 0.5 ms ) ect that is expected to cause lesser cognitive dysfunction .", "the latest integrated ect machine , with four - channel eeg / ecg , has intelligent features to independently operate without interfacing with a computer system .", "eeg / ecg is displayed on 5.7 \" color graphic lcd screen , and it also has sd card memory and printer interface .", "a separate portable eeg amplifier independent of ect can remain attached to the patient and the signals can be seen on any hand - held device that has bluetooth .", "all ect machines at nimhans are periodically calibrated for pulse width , frequency , amplitude , and stimulus duration stages . the frequency and duration", ", this device has been used to administer ect to about 700800 patients annually . with each patient receiving about 67 ects on an average through his / her course ,", "the precision and flexibility of electrical parameters provided by this ect machine have fostered quality research on ect at nimhans .", "we have selected for review those studies from nimhans which have utilized specific features of this device ."], ["stimulus relative to seizure threshold is more critical than the absolute stimulus . a few techniques of threshold estimation included the half - age method and dose titration method but have limitations .", "the former was dependent on the model of the device while the latter might expose the patient to inadequate stimuli .", "the study at nimhans in this regard on 100 consecutive patients receiving bilateral ect ( blect ) revealed that age was the most important predictor of threshold stimulus accounting for 23% variance in multivariate analysis .", "the other significant but less robust factors were ind and severity of illness that accounted for 5% and 4% of variance in threshold , respectively . the psychotropic medications and head circumference were not noted to influence the threshold .", "a formula as mentioned below for determining stimulus threshold was devised using forward stepwise linear regression model based on age alone and was prospectively tested at the 1 and 6 ect sessions .", "t1 = 1.67 ( age ) + 48.7 t6 = 1.8 ( age ) + 71.9 t1 = threshold at 1 session , t6 = threshold at 6 session .", "the computerized version of stimulus setting allows use of this regression equation with ease . with the computerized version , ect sessions of each patient", "a similar effort was made to find the seizure threshold for unilateral ect ( ulect ) . here , age was the only significant predictor of seizure threshold ( 15% of variance ) .", "a formula based on regression analysis was developed which is as follows : threshold ( mc ) = exp ( age [ 0.015412 ] + 3.667387 ) , where unit of age is years .", "then , in an independent sample ( n = 30 ) of patients receiving ulect , the threshold was estimated ( but not administered ) using the above formula .", "the value obtained was rounded off to the nearest higher level on the ect device .", "this estimated threshold was then compared with the actual threshold stimulus which was determined by using the titration method .", "the results showed that 22 ( 73% ) patients would have developed adequate seizure with the first stimulus estimated by the formula method , but it overestimated threshold stimulus by 3060 mc in majority of them .", "moreover , it underestimated the threshold in 8 ( 27% ) patients . hence , the formula method may not be suitable for threshold estimation in ulect .", "however , if the ect administrator wishes to use it , he or she should do so with the stimulus set at one or two levels lower than the formula - estimated threshold estimation , and further titration may be done as needed .", "although stimulus at 26 times the threshold intensity is associated with greater clinical efficacy in ulect , the guidelines were less clear about this in blect .", "from 2006 , there was change in practice in nimahns and unless otherwise requested from the referring psychiatrist , all blects are administered at 1.5 times the threshold stimulus charge . before 2006 , the threshold stimulus was administered for blect .", "hence , a retrospective data analysis was done to see whether new practice has better outcome .", "the data of 100 consecutive inpatients who received blect at threshold level before the change in practice implemented were compared with that of 101 who received blect at 1.5 times threshold level after the change implemented .", "the outcome measures were the number of ects administered and the number of inpatient days after the start of ect .", "however , the change to suprathreshold blect conferred advantage in the treatment of mania : those receiving suprathreshold ect achieved clinical improvement with , on an average , 2 ects less and their inpatient days reduced by 10 days ."], ["ect guidelines mandate the need for seizure monitoring during ect . initially , feasibility , reliability , and validity of eeg seizure measurement were studied at nimhans using a separate paper eeg machine .", "subsequently , a paperless eeg amplifier , which was attached to a computer monitor displaying the eeg , was designed at nimhans .", "display of both eeg and the digital counter showing time duration was triggered by the ect stimulus switch ( currently , eeg monitoring is available in a display in the ect machine obviating the need for a computer attached to the ect machine ) .", "this paperless eeg was validated against a paper eeg measurement which showed high correlation in terms of seizure duration ( intraclass correlation , r = 0.99 , p < 0.001 ) . in later studies using this paperless eeg machine , unequivocal absence of epileptiform transients for five or more seconds on both channels", "the use of this paperless eeg simplified eeg monitoring by reducing mechanical problems associated with paper eeg and simplified the storing process .", "( 1998 ) monitored 158 consecutive patients referred for ect on their first ect for both motor and eeg seizures .", "around 40% of patients with prolonged eeg seizures had a seizure duration > 180 s , necessitating termination .", "motor seizure of 90 s failed to predict prolonged eeg seizures ( 120 s or more ) in 60% of occasions . a sizable minority ( 8% ) failed to obtain adequate motor seizure on the cuffed forearm although their eeg seizures were considered adequate by the study criteria ( 25 s ) .", "a similar study was conducted by mayur et al . with 232 consecutive patients posted for either blect ( 54% ) or ulect ( 46% ) . in this study ,", "16% had prolonged eeg seizures , 39% of which was missed by motor monitoring , and 7% of adequate eeg seizures were classified as inadequate by motor seizure monitoring . around 1% of sessions with ulect and 11% with blect were considered inadequate by motor seizure monitoring despite adequate eeg seizures .", "motor seizure correlated well with eeg seizure when the latter was adequate but not when prolonged .", "these findings were again replicated in a further larger study with 485 consecutive patients receiving either blect or ulect . in the later study ,", "two patients classified as having inadequate motor seizures ( < 15 s ) had prolonged eeg seizures ( > 120 s ) .", "patients with prolonged seizures were younger , had lower seizure threshold , were more likely to receive a diagnosis of mania , and were treated with lithium .", "the above studies emphasize the need for eeg monitoring during ect to prevent unnecessary re - stimulation when motor seizures monitoring incorrectly label the treatment as inadequate and also to detect and abort the prolonged seizures , which appears to be common in indian setting .", "a study was conducted on 287 consecutive consenting patients receiving either blect or ulect to explore the factors influencing ratio of motor and eeg seizure duration ( me ratio ) .", "patients were classified into low ( 0.8 ; n = 146 ) and high ( > 0.8 , n = 141 ) me - ratio groups .", "significantly , more patients in the low me - ratio group received blect , had prolonged eeg seizure , and were on concurrent lithium compared with the high me - ratio group .", "this study underscores the importance for eeg monitoring at least inpatients receiving lithium and blect .", "recent international guidelines have recognized the limitations of seizure duration as a marker of adequacy of treatment and emphasize the pattern of eeg seizure .", "a study was conducted to compare the old definition of seizure adequacy ( based on seizure duration ) with the new definition ( a seizure with polyspikes and a 3-hz activity ) in 102 computerized eeg recordings .", "this suggested that seizures meeting newer criteria almost invariably last for at least 25 s ; in the indian context , the change in the criteria would make little change in the day - to - day practice of ect .", "numerous ictal eeg markers have been elucidated to predict treatment response of ect including postictal suppression , postictal coherence and amplitude , amount of slowing and time to onset of slowing , global eeg power , largest lyapunov exponent , and strength symmetry index ( ssi ) .", "ssi was computed from the early- and mid - seizure eeg epochs using the fractal dimension ( fd ) .", "the seizures of ulect were characterized by significantly smaller ssi in both phases , despite having seizure duration comparable to blect . it was suggested that ssi may be a potential measure of seizure adequacy . in another study ,", "ssi was computed in 40 patients receiving either high - dose or low - dose bitemporal ect ( btect ) .", "ssi of the early seizure was higher in the high dose than in the low dose ect group .", "another method of analyzing eeg is to measure the components of the eeg such as its frequencies across several nonoverlapping frequency bands : delta , theta , alpha , and beta through fast fourier transform .", "the delta band of eeg is said to be relevant to the therapeutic properties of ect .", "eeg of 25 patients receiving either blect or ulect was subjected fast fourier transform , and the spectral power of the delta ( 14 hz ) band was computed .", "blect resulted in symmetrical seizure spectral power ; ulect seizures had significantly lower spectral power in the delta band on the unstimulated side .", "fd was computed for early- , mid- , and post - seizure phases of the first ect .", "fd is a geometrical method of analysis of eeg , which was calculated based on the principles described by katz .", "it is a measure of complexity of the eeg wave in the terms of amplitude and frequency .", "smaller postseizure fd after the 1 ect was associated with remission status at 2 weeks .", "smaller postictal fd was considered a marker for postictal suppression suggesting that postictal suppression may be a marker for early antidepressant response for ect .", "a similar study was conducted in 51 right - handed , drug - free patients with major depressive disorder receiving right ulects at 2.5 times their seizure threshold .", "spectral power ( db ) for 26 hz band and fd of postseizure eeg were estimated .", "smaller fd and spectral power ( i.e. greater postseizure eeg suppression ) were seen in early responders compared to the late responders .", "ictal eeg fd was calculated from 26 eeg records of patients receiving either bfect or btect for schizophrenia .", "maximum fd at the 2 or 3 ect had an inverse correlation with psychosis severity after 6 ects as well as with the number of ects provided .", "hence , maximum fd during the early part of the ect course in schizophrenia patients is predictive of greater benefit in short - term psychopathology . in another analysis of data from the sample", ", it was seen that maximum fd does not change over six ects despite decline in seizure duration over ect .", "thus , eeg morphology of blect induced seizures show little variation over the course of treatment which is in keeping with other studies .", "bispectral index ( bis ) measures the level of hypnosis ( sedation ) during anesthesia .", "bis values of awaked individuals with schizophrenia were studied through a course of bitemporal brief - pulse ect .", "it was recorded using a bis monitor ( a-2000 , aspect medical systems inc . , newton , massachusetts , usa ) , with sensor applied to the forehead on the right side .", "it was found that after six ects , 60% of patients had their resting bis values in the anesthetized / sedated range even when they are awake .", "thus , bis may not provide accurate estimation of the depth of anesthesia during ect after the initial ect sessions ."], ["bfect is found to be as efficacious as btect for depression but associated with fewer cognitive adverse effects .", "however , its efficacy in other indications had not been well studied and hence , it was planned in nimhans . assessing", "bfect in acute mania in double - blind randomized controlled trial showed faster decline in ymrs scores and earlier attainment of response compared to btect without differences in adverse effects .", "a double - blind , randomized controlled trial demonstrated the superior clinical as well as cognitive effects of bfect over btect in - patients with schizophrenia .", "seizure duration significantly reduced only with bfect and not with btect over the course of ect .", "this provided the indirect support to possible better anticonvulsant mechanism of action of bfect in schizophrenia .", "these studies used electrodes with inner concave surface specifically for bfect and later , we have been using these electrodes in clinical practice for bfect in nimhans ."], ["the effect of electrical stimulus parameters of ect on prolactin response was studied . stimulus dose , frequency of ect , and seizure variables such as seizure duration , seizure strength , pattern , and symmetry", "did not predict chance in prolactin level . similarly , none of the clinical variables including baseline severity of illness , presence of psychotic symptoms , drug - naive status , and degree of clinical improvement predicted the prolactin response .", "the prolactin response however , was significantly more in female participants which may be due to their lower seizure threshold and inherent biological predisposition .", "a study comparing low- and high - stimulus doses in a double - blind , randomized controlled design revealed that eeg seizure was of comparable duration in the two stimulus groups .", "another study with a cross - over design looking into the effect of different stimulus frequency found that lower pulse frequency ( 50 pps ) stimulus had lower seizure threshold and produced fewer subconvulsive stimulations compared with high - frequency stimulus ( 200 pps ) without altering seizure durations or the cardiovascular responses .", "the low - frequency stimulus produced similar clinical benefits as that of high frequency in subsequent study .", "thus , lowering down the frequency of pulses may be a viable option when seizure threshold is high due to various reasons .", "the lower stimulus frequency leads to longer stimulus train duration and hence , had lower rate ( mc / s ) of stimulus dissipation ."], ["the cardiovascular system ( cvs ) effects of ect were also studied with the features of automated cardiac monitor and ambulatory ecg system in eeg machine . to study the effect of atropine on cvs response to ect , 30 patients were randomly allocated before the third ect to receive atropine or no premedication and found that atropine contributed to 32% of the variance in rate pressure product ( rpp ) with lower values in those who did not receive atropine . however", "this study hence indicated that avoiding atropine as premedication can control rpp and thus can improve cardiac stability during ect without affecting other aspects . in a large sample of 124 patients", ", it was found that postictal rpp was significantly higher after blect than ulect even after eliminating the effect of possible confounding variables such as subconvulsions and concurrent medications .", "the difference of 10% postictal rpp increments between the right ulect and blect groups found in this study is of clinical relevance in elderly or patients at risk of cardiac complications .", "the postictal rpp may be considered as a peripheral marker of the degree of seizure generalization .", "in addition , if the hypothesis of seizure generalization to hypothalamus as a prerequisite for clinical efficacy is given consideration , then lower rpp of threshold ulect compared to blect can explain lower clinical efficacy of ulect and may reflect the less than optimal hypothalamic stimulation .", "the correlation of rpp with clinical efficacy was further supported by the lower postictal rpp with unilateral threshold ect compared to that with unilateral suprathreshold ect .", "however , atropine could mask the differential cardiovascular responses of the effective and ineffective seizures . in another study ,", "the effect of anesthetic modification of motor seizure on rpp was also explored . 1 mg / kg dose of succinylcholine improved the number of patients with successful modification compared to 0.5 mg / kg but did not influence ictal or postictal rpp .", "it thus ruled out the significant cvs alteration due to depolarizing effect of succinylcholine and 1 mg / kg dose may be used routinely without fear of exaggerated cvs response .", "it confirmed the above findings that rpp response correlates with the cerebral seizure instead of motor component of seizure .", "ictal rpp did not differ between groups with or without adequate motor seizure if eeg seizure was adequate . however , the group with inadequate eeg seizure had the least ictal rpp .", "hemodynamic response with btect and bfect were examined at five - time points starting from before anesthesia induction to 2 min after the seizure .", "the maximum values of systolic blood pressure , heart rate , and rpp were similar in both groups , suggesting that cardiovascular load is similar in bfect and btect .", "it also indicates that differential frontal lobe stimulation rather than seizure generalization may be playing a role in better efficacy of bfect over btect in schizophrenia .", "lithium given concurrently with ect can have serious interactions with various aspects of the procedure . at the same time , not combining ect and lithium in certain patients may have adverse clinical consequences . in the absence of systematically collected data on adverse effects of lithium co - prescribed with ect ,", "patients on lithium ( n=27 ) and those not on lithium ( n=28 ) were compared while they received ect .", "the sympathetic cardiovascular response was lower in those who were on lithium and the time to post - ect recovery directly correlated with serum lithium level .", "safety of lithium during ect was satisfactory particularly when serum lithium was at a lower therapeutic range in young patients without significant cardiovascular risk factors . to conclude , the indigenously developed brief - pulse ect - eeg device has been used to treat thousands of patients in the past two decades .", "summary of the findings of research conducted utilizing the specific features of the indigenously developed brief - pulse ect device * many more issues related to ect need exploration in future ; these include altering amplitude of electrical stimulus , ultra - brief ect in indications other than depression , novel techniques of different electrode 's sizes , and placements to reduce the cognitive adverse effects without compromising the clinical efficacy . the last author , mr .", "ltd . , bengaluru , karnataka , india , which manufactures the brief - pulse ect device mentioned in this review article ."], [""], ["candade is the director of the company , niviqure meditech pvt . ltd . , bengaluru , karnataka , india , which manufactures the brief - pulse ect device mentioned in this review article ."]]}
{"article_id": "PMC4647058", "article_text": ["the many genetic studies performed in recent years showed that genes such as interleukin 23 receptor ( il23r ) and il12b and tumor necrosis factor alpha ( tnf ) are closely associated with psoriasis and related diseases such as rheumatoid arthritis , psoriatic arthritis , and crohn 's disease .", "human leukocyte antigen c ( hla - c)0602 is the allele most closely associated with this disease .", "the age at onset of psoriasis follows a bimodal distribution : type i psoriasis appears before the age of 40 years ( early - onset ) , with a peak at 1622 years ; type ii psoriasis appears after the age of 40 years ( late - onset ) , with a peak at 5760 years .", "type i psoriasis has been associated with several single - nucleotide polymorphisms ( snps ) in genes associated with the immune response ( table 1 ) .", "for example , hla - c0602 is more strongly associated with type i psoriasis than with type ii psoriasis .", "although several association studies have already been performed in psoriasis in both populations ( type i or type ii psoriasis patients ) , knowledge of age at onset remains limited and controversial ( table 1 ) .", "therefore , we performed a candidate gene study , where we evaluated genetic susceptibility to type i or type ii psoriasis in patients with moderate - to - severe chronic plaque psoriasis . this approach may help us to identify snps previously associated with psoriasis or other autoimmune diseases   that are specific to type i or type ii psoriasis .", "furthermore , our genetic study could improve our understanding of psoriasis and of its etiology and pathogenesis .", "we recruited 198 caucasian patients with moderate - to - severe plaque type psoriasis ( psoriasis area and severity index > 10 ) who attended the department of dermatology in four university hospitals in madrid between 16/10/2007 and 17/12/2012 .", "five samples did not fulfill the quality criteria of the human genotyping unit - cegen ( cegen , spanish national cancer research centre , madrid , spain ) , and 2 samples had insufficient volume .", "we also included 197 healthy volunteers ( controls ) recruited between 10/01/2011 and 14/12/2012 from the clinical pharmacology service ( hospital universitario de la princesa , madrid , spain ) .", "all the volunteers were caucasian and had no personal or family history of psoriasis ( at least 2 generations ) .", "the protocol fulfilled spanish law on biomedical research and was approved by the ethics committee for clinical investigation of hospital universitario de la princesa .", "all controls and patients gave their written informed consent to donate a sample for investigation .", "we preselected 320 snps based on an extensive review of 449 articles describing the association between polymorphisms and psoriasis and response to biological drugs and psoriasis and related inflammatory diseases ( rheumatoid arthritis , psoriatic arthritis , and crohn 's disease ) .", "we finally selected 192 snps based on minor allele frequency ( 0.05 ) and on the results of studies performed in caucasians and psoriatic patients .", "information on the 173 snps analyzed can be found in supplementary table s1 , which is published in .", "dna was obtained from samples using an automatic dna extractor ( magna pure system , roche applied science , usa ) and its concentration was quantified in nanodrop nd-1000 spectrophotometer ( wilmington , usa ) . the extracted dna was stored at 80c in the clinical pharmacology service until use .", "a total of 196 samples from patients ( 2 samples of 198 cases had insufficient volume ) and 197 samples from controls were sent to the human genotyping unit - cegen to genotype 192 snps .", "the analysis was performed using the illumina veracode genotyping platform . if fluorescence was low or the genotype clusters were undifferentiated , the snps were removed .", "in addition , if the call rate was less than 95% of the average of the 192 snps analyzed , the samples were removed .", "since cegen quality criteria were not met in 19 snps and 5 patients , we finally analyzed 173 snps in 191 patients and 197 controls .", "the statistical analysis was performed to compare the following stratified populations : patients with type i psoriasis ( n = 155 ) or type ii psoriasis ( n = 36 ) versus controls ( n = 197 ) and patients with type i psoriasis versus cases with type ii psoriasis .", "hardy - weinberg equilibrium was tested for all the snps analyzed using the snpstats program .", "snps that were not in hardy - weinberg equilibrium in controls were removed from the subsequent analysis .", "we constructed various logistic regression models depending on the main types of inheritance ( codominant , dominant , recessive , and additive ) . in the additive model", ", the presence of 2 mutant alleles confers double the risk of 1 mutant allele .", "the results were adjusted for rs12191877 ( snp that is strongly associated with the hla - c0602 allele and is highly prevalent in our population ) [ 3 , 35 ] .", "subsequently , snps with p < 0.1 in the univariate analysis ( adjusted for rs12191877 ) were included in a multivariate logistic regression model to adjust for relevant confounding factors ( spss 15.0 ) .", "the results of the univariate analysis were adjusted for rs12191877 , except when we compared patients with type i psoriasis and patients with type ii psoriasis ( the influence of rs12191877 was not very relevant ) .", "we expressed the results as the odds ratio ( or ) , 95% confidence interval , and p value .", "the study population included 155 patients with moderate - to - severe chronic plaque type i psoriasis ( 92 men and 63 women ) , 36 patients with type ii psoriasis ( 19 men and 17 women ) , and 197 controls ( 98 men and 99 women ) .", "the mean age was 46.01  13.11 years in patients with type i psoriasis ( 45.72  11.69 in men and 46.43  15.04 in women ) , 67.72  11.85 years in patients with type ii psoriasis ( 65.95  11.18 in men and 69.71  12.59 in women ) , and 24.51  4.29 years in the controls ( 25.07  4.94 in men and 23.95  3.46 in women ) .", "the mean age at onset of psoriasis was 23.31  8.52 in patients with type i psoriasis and 52.58  10.45 in patients with type ii psoriasis .", "a total of 192 snps were analyzed ( see supplementary table s1 published in ) .", "one snp was monomorphic ( rs165161 in the junb gene ) and was excluded from the statistical analysis .", "all the minor allele frequencies were in hardy - weinberg equilibrium except 9 snps in the controls and 12 snps in the patients ( see supplementary table s1 published in ) .", "the 9 snps which were not in hardy - weinberg equilibrium in controls were removed from the statistical analysis .", "our findings showed an association between type i psoriasis and 10 snps ( n = 155 versus n = 197 controls ) : rs1634517 ( ccl4l ) , rs1975974 ( c17orf51 ) , rs12720356 ( tyk2 ) , rs1800925 ( il13 ) , and rs6908425 ( cdkal1 ) decreased the risk of psoriasis 2.94-fold , 2.08-fold , 10-fold , 100-fold , and 2.44-fold , respectively ; and rs2282276 ( clmn ) , rs10782001 ( fbxl19 ) , rs3792876 ( slc22a4 ) , rs12191877 ( hla - c ) , and rs13437088 ( hla - b / mica ) increased the risk of psoriasis 3.90-fold , 2.10-fold , 3.75-fold , 30.54-fold , and 2.52-fold , respectively ( table 2 ) .", "however , comparison of 36 patients with type ii psoriasis and 197 controls revealed no significant association ( results not shown ) .", "four snps were associated with significant decreases in the risk of type i psoriasis ( n = 155 ) compared with type ii psoriasis ( n = 36 ) , namely , rs191190 ( tnfr1 ; 126.08-fold ) , rs361525 ( tnf- ; 190.76-fold ) , and rs10499194 and rs6920220 ( tnfaip3 ; 155.02-fold and 19.14-fold , resp . ) .", "we also found 5 snps that were associated with a significant increase in the risk of type i psoriasis , namely , rs1801274", "( fcgr2a ; 5.26-fold ) , rs763361 ( cd226 ; 33.3-fold ) , rs12459358 ( psors6 ; 11.11-fold ) , rs12191877 ( hla - c ; 12.5-fold ) , and rs1576 ( cchcr1 ; 166.66-fold ) ( table 3 ) .", "about 75% of patients with chronic plaque psoriasis have type i psoriasis before age 40 , whereas a lower number of patients develop psoriasis at around 5060 years .", "our results are consistent with these findings , since 79.06% of our patients developed psoriasis before the age of 40 .", "when we compared patients with type i psoriasis and controls , we found 10 significant snps in clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , hla - c , and hla - b / mica . the hla - c0602 allele is", "a risk factor for psoriasis   and has been associated with both type i [ 69 ] and type ii psoriasis . in one study ,", "85.3% of patients with type i psoriasis had this allele , whereas only 14.7% of patients with type ii psoriasis were carriers .", "other authors found an association between rs10484554 ( hla - c ) and type i psoriasis compared with type ii psoriasis ( or = 3.24 in type i ) .", "the hla - c gene was associated with type i psoriasis ( p = 2.97e  18 for rs1265181 , p = 2.58e  15 for rs12191877 , p = 1.84e  15 for rs4406273 , and p = 1.10e  07 for rs2395029 ) , but not with type ii psoriasis after application of the bonferroni correction .", "in addition , our results showed significant differences in rs12191877 ( hla - c ) in patients with type i psoriasis ( p = 2.50e  19 ) .", "however , we did not find this association in patients with type ii psoriasis , probably owing to the small sample size in this group ( n = 36 ) .", "found an association between rs1295685 in the il13 gene and type i psoriasis ( p = 2.47e  03 ) .", "our results showed an association between another snp in il13 ( rs1800925 ) and type i psoriasis ( p = 0.034 ) .", "in addition , munir et al . did not obtain significant results when they compared controls with type ii psoriasis or type i psoriasis with type ii psoriasis .", "both snps in il13 have been associated with predisposition to psoriasis [ 36 , 37 ] .", "our comparison of patients with type i psoriasis and controls is the first to obtain significant results for a series of snps in type i psoriasis , although the snps have already been associated with the risk of psoriasis .", "rs10782001 in fbxl19 , rs1975974 in c17orf51 , rs12720356 in tyk2 [ 3 , 39 ] , rs3792876 in slc22a4 , rs6908425 in cdkal1 , and rs13437088 in hla - b / mica   have previously been associated with psoriasis , but not with type i psoriasis .", "furthermore , snps in clmn ( rs2282276 ) and ccl4l ( rs1634517 ) have not been associated with psoriasis or age at onset .", "we found no significant differences between patients with type ii psoriasis and controls owing to the small sample size ( n = 36 ) .", "comparison between patients with type i psoriasis and patients with type ii psoriasis revealed significant associations for the following genes : fcgr2a , tnfr1 , cd226 , psors6 , tnfaip3 , hla - c , tnf- , and cchcr1 .", "polymorphisms in cchcr1 ( 386 and 404 , cchcr1ww allele ) have been associated with type i psoriasis [ 9 , 31 ] .", "we found significant differences between rs1576 in cchcr1 and age at onset . in a study comparing controls ( 54.8% ) and patients with psoriasis type ii ( 66.0% ) , allen et al", "analyzed rs763361 in cd226 in patients with early - onset psoriasis and patients with late - onset psoriasis , although they found no associations .", "rs12459358 in psors6 has been associated with type i psoriasis ( g risk allele , or = 1.47 and p = 0.005 ) .", "in contrast , our data showed an association between the t allele and type i psoriasis ( or = 11.11 ; p = 0.026 ) .", "rs361525 ( 238 ) in the tnf gene has been associated with susceptibility to psoriasis , and the a allele was more frequent in male patients with type i psoriasis ( p = 2e  07 ) [ 15 , 22 ] .", "we found significant results in rs361525 ( tnf- ) when we compared patients with type i psoriasis and patients with type ii psoriasis , although we found no gender differences .", "other authors confirmed our association with type i psoriasis in caucasian [ 20 , 23 ] and mongolian patients .", "baran et al . found no significant differences between rs1800629 in the 308 promoter ( tnf ) and type i or type ii psoriasis .", "likewise , rs12191877 in hla - c has been associated with increased risk of psoriasis .", "compared patients with type i psoriasis and patients with type ii psoriasis and obtained significant results for rs1265181 , rs4406273 , and rs12191877 in hla - c .", "we replicated these results in rs12191877 ( t allele risk ; p = 0.045 ) .", "rs191190 in tnfr1   and rs10499194 in tnfaip3   have been associated with psoriasis , but not with age of onset .", "moreover , rs1801274 in fcgr2a and rs6920220 in tnfaip3 have not been studied in patients with psoriasis according to age of onset .", "given the small sample size in the group with type ii psoriasis in our study , our results should be interpreted with caution .", "our results highlight the role of the immune system in psoriasis and enhance our understanding of pathogenic mechanisms .", "second , the sample size was limited by the number of study patients treated in the dermatology department , thus making it difficult to detect snps with a low probability of causing psoriasis .", "third , since the snps were selected based on a literature review , several major snps may not yet have been investigated . in conclusion ,", "our study confirmed an association between rs12191877 ( hla - c ) and type i psoriasis and between type i and type ii psoriasis patients .", "ours is the first study to show an association between clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , and hla - b / mica and type i psoriasis .", "in addition , psors6 and tnf have been described as more prevalent genes in type i psoriasis and we showed a significant association when we compared type i psoriasis and type ii psoriasis .", "ours is the first study to identify an association between fcgr2a , tnfr1 , cd226 , tnfaip3 , and cchcr1 and age at onset of psoriasis ."], "abstract_text": ["<S> psoriasis is a chronic skin disease in which genetics play a major role . </S>", "<S> although many genome - wide association studies have been performed in psoriasis , knowledge of the age at onset remains limited . </S>", "<S> therefore , we analyzed 173 single - nucleotide polymorphisms in genes associated with psoriasis and other autoimmune diseases in patients with moderate - to - severe plaque psoriasis type i ( early - onset , < 40 years ) or type ii ( late - onset , 40 years ) and healthy controls . </S>", "<S> moreover , we performed a comparison between patients with type i psoriasis and patients with type ii psoriasis . </S>", "<S> our comparison of a stratified population with type i psoriasis ( n = 155 ) and healthy controls ( n = 197 ) is the first to reveal a relationship between the clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , and hla - b / mica genes . when we compared type i psoriasis with type ii psoriasis ( n = 36 ) , we found a significant association between age at onset and the genes psors6 , tnf- , fcgr2a , tnfr1 , cd226 , hla - c , tnfaip3 , and cchcr1 . </S>", "<S> moreover , we replicated the association between rs12191877 ( hla - c ) and type i psoriasis and between type i and type ii psoriasis . </S>", "<S> our findings highlight the role of genetics in age of onset of psoriasis . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion"], "sections": [["the many genetic studies performed in recent years showed that genes such as interleukin 23 receptor ( il23r ) and il12b and tumor necrosis factor alpha ( tnf ) are closely associated with psoriasis and related diseases such as rheumatoid arthritis , psoriatic arthritis , and crohn 's disease .", "human leukocyte antigen c ( hla - c)0602 is the allele most closely associated with this disease .", "the age at onset of psoriasis follows a bimodal distribution : type i psoriasis appears before the age of 40 years ( early - onset ) , with a peak at 1622 years ; type ii psoriasis appears after the age of 40 years ( late - onset ) , with a peak at 5760 years .", "type i psoriasis has been associated with several single - nucleotide polymorphisms ( snps ) in genes associated with the immune response ( table 1 ) .", "for example , hla - c0602 is more strongly associated with type i psoriasis than with type ii psoriasis .", "although several association studies have already been performed in psoriasis in both populations ( type i or type ii psoriasis patients ) , knowledge of age at onset remains limited and controversial ( table 1 ) .", "therefore , we performed a candidate gene study , where we evaluated genetic susceptibility to type i or type ii psoriasis in patients with moderate - to - severe chronic plaque psoriasis . this approach may help us to identify snps previously associated with psoriasis or other autoimmune diseases   that are specific to type i or type ii psoriasis .", "furthermore , our genetic study could improve our understanding of psoriasis and of its etiology and pathogenesis ."], ["we recruited 198 caucasian patients with moderate - to - severe plaque type psoriasis ( psoriasis area and severity index > 10 ) who attended the department of dermatology in four university hospitals in madrid between 16/10/2007 and 17/12/2012 .", "five samples did not fulfill the quality criteria of the human genotyping unit - cegen ( cegen , spanish national cancer research centre , madrid , spain ) , and 2 samples had insufficient volume .", "we also included 197 healthy volunteers ( controls ) recruited between 10/01/2011 and 14/12/2012 from the clinical pharmacology service ( hospital universitario de la princesa , madrid , spain ) .", "all the volunteers were caucasian and had no personal or family history of psoriasis ( at least 2 generations ) .", "the protocol fulfilled spanish law on biomedical research and was approved by the ethics committee for clinical investigation of hospital universitario de la princesa .", "all controls and patients gave their written informed consent to donate a sample for investigation .", "we preselected 320 snps based on an extensive review of 449 articles describing the association between polymorphisms and psoriasis and response to biological drugs and psoriasis and related inflammatory diseases ( rheumatoid arthritis , psoriatic arthritis , and crohn 's disease ) .", "we finally selected 192 snps based on minor allele frequency ( 0.05 ) and on the results of studies performed in caucasians and psoriatic patients .", "information on the 173 snps analyzed can be found in supplementary table s1 , which is published in .", "dna was obtained from samples using an automatic dna extractor ( magna pure system , roche applied science , usa ) and its concentration was quantified in nanodrop nd-1000 spectrophotometer ( wilmington , usa ) . the extracted dna was stored at 80c in the clinical pharmacology service until use .", "a total of 196 samples from patients ( 2 samples of 198 cases had insufficient volume ) and 197 samples from controls were sent to the human genotyping unit - cegen to genotype 192 snps .", "the analysis was performed using the illumina veracode genotyping platform . if fluorescence was low or the genotype clusters were undifferentiated , the snps were removed .", "in addition , if the call rate was less than 95% of the average of the 192 snps analyzed , the samples were removed .", "since cegen quality criteria were not met in 19 snps and 5 patients , we finally analyzed 173 snps in 191 patients and 197 controls .", "the statistical analysis was performed to compare the following stratified populations : patients with type i psoriasis ( n = 155 ) or type ii psoriasis ( n = 36 ) versus controls ( n = 197 ) and patients with type i psoriasis versus cases with type ii psoriasis .", "hardy - weinberg equilibrium was tested for all the snps analyzed using the snpstats program .", "snps that were not in hardy - weinberg equilibrium in controls were removed from the subsequent analysis .", "we constructed various logistic regression models depending on the main types of inheritance ( codominant , dominant , recessive , and additive ) . in the additive model", ", the presence of 2 mutant alleles confers double the risk of 1 mutant allele .", "the results were adjusted for rs12191877 ( snp that is strongly associated with the hla - c0602 allele and is highly prevalent in our population ) [ 3 , 35 ] .", "subsequently , snps with p < 0.1 in the univariate analysis ( adjusted for rs12191877 ) were included in a multivariate logistic regression model to adjust for relevant confounding factors ( spss 15.0 ) .", "the results of the univariate analysis were adjusted for rs12191877 , except when we compared patients with type i psoriasis and patients with type ii psoriasis ( the influence of rs12191877 was not very relevant ) .", "we expressed the results as the odds ratio ( or ) , 95% confidence interval , and p value ."], ["the study population included 155 patients with moderate - to - severe chronic plaque type i psoriasis ( 92 men and 63 women ) , 36 patients with type ii psoriasis ( 19 men and 17 women ) , and 197 controls ( 98 men and 99 women ) .", "the mean age was 46.01  13.11 years in patients with type i psoriasis ( 45.72  11.69 in men and 46.43  15.04 in women ) , 67.72  11.85 years in patients with type ii psoriasis ( 65.95  11.18 in men and 69.71  12.59 in women ) , and 24.51  4.29 years in the controls ( 25.07  4.94 in men and 23.95  3.46 in women ) .", "the mean age at onset of psoriasis was 23.31  8.52 in patients with type i psoriasis and 52.58  10.45 in patients with type ii psoriasis .", "a total of 192 snps were analyzed ( see supplementary table s1 published in ) .", "one snp was monomorphic ( rs165161 in the junb gene ) and was excluded from the statistical analysis .", "all the minor allele frequencies were in hardy - weinberg equilibrium except 9 snps in the controls and 12 snps in the patients ( see supplementary table s1 published in ) .", "the 9 snps which were not in hardy - weinberg equilibrium in controls were removed from the statistical analysis .", "our findings showed an association between type i psoriasis and 10 snps ( n = 155 versus n = 197 controls ) : rs1634517 ( ccl4l ) , rs1975974 ( c17orf51 ) , rs12720356 ( tyk2 ) , rs1800925 ( il13 ) , and rs6908425 ( cdkal1 ) decreased the risk of psoriasis 2.94-fold , 2.08-fold , 10-fold , 100-fold , and 2.44-fold , respectively ; and rs2282276 ( clmn ) , rs10782001 ( fbxl19 ) , rs3792876 ( slc22a4 ) , rs12191877 ( hla - c ) , and rs13437088 ( hla - b / mica ) increased the risk of psoriasis 3.90-fold , 2.10-fold , 3.75-fold , 30.54-fold , and 2.52-fold , respectively ( table 2 ) .", "however , comparison of 36 patients with type ii psoriasis and 197 controls revealed no significant association ( results not shown ) .", "four snps were associated with significant decreases in the risk of type i psoriasis ( n = 155 ) compared with type ii psoriasis ( n = 36 ) , namely , rs191190 ( tnfr1 ; 126.08-fold ) , rs361525 ( tnf- ; 190.76-fold ) , and rs10499194 and rs6920220 ( tnfaip3 ; 155.02-fold and 19.14-fold , resp . ) .", "we also found 5 snps that were associated with a significant increase in the risk of type i psoriasis , namely , rs1801274", "( fcgr2a ; 5.26-fold ) , rs763361 ( cd226 ; 33.3-fold ) , rs12459358 ( psors6 ; 11.11-fold ) , rs12191877 ( hla - c ; 12.5-fold ) , and rs1576 ( cchcr1 ; 166.66-fold ) ( table 3 ) ."], ["about 75% of patients with chronic plaque psoriasis have type i psoriasis before age 40 , whereas a lower number of patients develop psoriasis at around 5060 years .", "our results are consistent with these findings , since 79.06% of our patients developed psoriasis before the age of 40 .", "when we compared patients with type i psoriasis and controls , we found 10 significant snps in clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , hla - c , and hla - b / mica . the hla - c0602 allele is", "a risk factor for psoriasis   and has been associated with both type i [ 69 ] and type ii psoriasis . in one study ,", "85.3% of patients with type i psoriasis had this allele , whereas only 14.7% of patients with type ii psoriasis were carriers .", "other authors found an association between rs10484554 ( hla - c ) and type i psoriasis compared with type ii psoriasis ( or = 3.24 in type i ) .", "the hla - c gene was associated with type i psoriasis ( p = 2.97e  18 for rs1265181 , p = 2.58e  15 for rs12191877 , p = 1.84e  15 for rs4406273 , and p = 1.10e  07 for rs2395029 ) , but not with type ii psoriasis after application of the bonferroni correction .", "in addition , our results showed significant differences in rs12191877 ( hla - c ) in patients with type i psoriasis ( p = 2.50e  19 ) .", "however , we did not find this association in patients with type ii psoriasis , probably owing to the small sample size in this group ( n = 36 ) .", "found an association between rs1295685 in the il13 gene and type i psoriasis ( p = 2.47e  03 ) .", "our results showed an association between another snp in il13 ( rs1800925 ) and type i psoriasis ( p = 0.034 ) .", "in addition , munir et al . did not obtain significant results when they compared controls with type ii psoriasis or type i psoriasis with type ii psoriasis .", "both snps in il13 have been associated with predisposition to psoriasis [ 36 , 37 ] .", "our comparison of patients with type i psoriasis and controls is the first to obtain significant results for a series of snps in type i psoriasis , although the snps have already been associated with the risk of psoriasis .", "rs10782001 in fbxl19 , rs1975974 in c17orf51 , rs12720356 in tyk2 [ 3 , 39 ] , rs3792876 in slc22a4 , rs6908425 in cdkal1 , and rs13437088 in hla - b / mica   have previously been associated with psoriasis , but not with type i psoriasis .", "furthermore , snps in clmn ( rs2282276 ) and ccl4l ( rs1634517 ) have not been associated with psoriasis or age at onset .", "we found no significant differences between patients with type ii psoriasis and controls owing to the small sample size ( n = 36 ) .", "comparison between patients with type i psoriasis and patients with type ii psoriasis revealed significant associations for the following genes : fcgr2a , tnfr1 , cd226 , psors6 , tnfaip3 , hla - c , tnf- , and cchcr1 .", "polymorphisms in cchcr1 ( 386 and 404 , cchcr1ww allele ) have been associated with type i psoriasis [ 9 , 31 ] .", "we found significant differences between rs1576 in cchcr1 and age at onset . in a study comparing controls ( 54.8% ) and patients with psoriasis type ii ( 66.0% ) , allen et al", "analyzed rs763361 in cd226 in patients with early - onset psoriasis and patients with late - onset psoriasis , although they found no associations .", "rs12459358 in psors6 has been associated with type i psoriasis ( g risk allele , or = 1.47 and p = 0.005 ) .", "in contrast , our data showed an association between the t allele and type i psoriasis ( or = 11.11 ; p = 0.026 ) .", "rs361525 ( 238 ) in the tnf gene has been associated with susceptibility to psoriasis , and the a allele was more frequent in male patients with type i psoriasis ( p = 2e  07 ) [ 15 , 22 ] .", "we found significant results in rs361525 ( tnf- ) when we compared patients with type i psoriasis and patients with type ii psoriasis , although we found no gender differences .", "other authors confirmed our association with type i psoriasis in caucasian [ 20 , 23 ] and mongolian patients .", "baran et al . found no significant differences between rs1800629 in the 308 promoter ( tnf ) and type i or type ii psoriasis .", "likewise , rs12191877 in hla - c has been associated with increased risk of psoriasis .", "compared patients with type i psoriasis and patients with type ii psoriasis and obtained significant results for rs1265181 , rs4406273 , and rs12191877 in hla - c .", "we replicated these results in rs12191877 ( t allele risk ; p = 0.045 ) .", "rs191190 in tnfr1   and rs10499194 in tnfaip3   have been associated with psoriasis , but not with age of onset .", "moreover , rs1801274 in fcgr2a and rs6920220 in tnfaip3 have not been studied in patients with psoriasis according to age of onset .", "given the small sample size in the group with type ii psoriasis in our study , our results should be interpreted with caution .", "our results highlight the role of the immune system in psoriasis and enhance our understanding of pathogenic mechanisms .", "second , the sample size was limited by the number of study patients treated in the dermatology department , thus making it difficult to detect snps with a low probability of causing psoriasis .", "third , since the snps were selected based on a literature review , several major snps may not yet have been investigated . in conclusion ,", "our study confirmed an association between rs12191877 ( hla - c ) and type i psoriasis and between type i and type ii psoriasis patients .", "ours is the first study to show an association between clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , and hla - b / mica and type i psoriasis .", "in addition , psors6 and tnf have been described as more prevalent genes in type i psoriasis and we showed a significant association when we compared type i psoriasis and type ii psoriasis .", "ours is the first study to identify an association between fcgr2a , tnfr1 , cd226 , tnfaip3 , and cchcr1 and age at onset of psoriasis ."]]}
{"article_id": "PMC5260609", "article_text": ["a case showing sustained structural and functional responses 2 years after a single treatment with iluvien ( 0.2 g / day fluocinolone acetonide , fac ) despite suboptimal responses to ranibizumab .", "a 68-year - old female patient with diabetic macular oedema ( dme ) from type 2 diabetes mellitus was first diagnosed in october 2010 and had a baseline visual acuity ( va ) of 46 early treatment diabetic retinopathy study ( etdrs ) letters in the left eye .", "the patient was treated with 11 intravitreal injections of ranibizumab ( 5 in combination with a small - interfering rna agent ) , and by march 2014 , va and cft were largely unchanged ( 55 etdrs letters and 774 microns ) .", "the patient was treated with iluvien as she had a pseudophakic lens and a clearly suboptimal response to the prior therapy with ranibizumab .", "an implant releasing fac at a dosage of 0.2 g / day was administered in march 2014 , and the optical coherence tomography indicated that the macula was dry after 7 days ( cft was below 300 microns ) .", "va improved by 5 letters within 7 days and by 15 letters within 14 days , and this was maintained after 24 months . throughout the duration of this study , the intraocular pressure was 22 mm hg , and no glaucoma medication was administered .", "in real - life uk practice , this dme patient showed a suboptimal response to multiple intravitreal injections of ranibizumab . when subsequently treated with a single injection of iluvien", ", there were large and rapid improvements in va and cft that were maintained for the following 2 years .", "worldwide diabetic macular edema ( dme ) is reported to have a prevalence of 6.8% and results in vision loss .", "dme management can be difficult , with only around 15% of the patients achieving significant vision recovery with laser , the current standard of care .", "a number of pharmacotherapies are now licensed for the treatment of dme in europe , including anti - vascular endothelial growth factor agents and corticosteroid therapies , which are used to target vascular endothelial growth pathways and inflammatory pathways , respectively . in diabetes ,", "leukocytes release free radicals and enzymes that cause direct damage to endothelial cells , increasing blood retinal barrier leakage .", "they also release cytokines , including vascular endothelial growth factor , tumor necrosis factor- , and interleukin-6 , which act through various signaling pathways to increase vascular permeability .", "the release of cytokines with a corticosteroid  such as dexamethasone , triamcinolone or fluocinolone  has been shown to reduce dme and rehabilitate vision in several pivotal clinical studies .", "iluvien contains the corticosteroid fluocinolone acetonide ( fac ) and uses microdosingtm technology to release fac at a daily rate of 0.2 g for up to 3 years .", "this differs significantly from both the dexamethasone delivery system and that of triamcinolone , which are reported to have a duration of action of only up to 3 and 4 months , respectively .", "the effectiveness of iluvien was demonstrated in the fame trials ( nct00344968 ) in which patients with dme who had previously shown an insufficient response to laser were treated with either a 0.2 g / day ( iluvien ) or 0.5 g / day fac implant and were compared against a sham - control treated population .", "both fame trials independently met their primary endpoint and showed that significantly more patients with chronic dme treated with the 0.2 g / day fac implant experienced a 15-letter improvement in visual acuity ( va ) at month 24 than sham - control patients ( pooled data ; 34.4 vs. 13.4% ; p < 0.001 ) and that this benefit was sustained to month 36 .", "functional improvements were accompanied by rapid and sustained improvements in central foveal thickness ( cft ) . in europe ,", "iluvien is indicated for the treatment of vision impairment associated with chronic diabetic macular oedema considered insufficiently responsive to available therapies . since then", "miss clare bailey presented the real world outcomes from a uk audit of patients treated with iluvien at the annual meeting of the royal college of ophthalmologists in birmingham , uk , between june 24 and 26 , 2016 .", "this unpublished interim analysis showed that the majority of treated eyes ( 69.4% of 290 eyes ) maintained or improved va .", "to date , however , relatively limited data with a longer - term follow - up of the functional and structural responses following treatment with iluvien have been reported .", "the case described herein provides data for both va and cft over a 24-month follow - up period .", "a 68-year - old caucasian female with type 2 diabetes mellitus had diffuse dme diagnosed in her left eye in october 2010 .", "the treatment history is summarised in table 1 and was previously reported when only 6 months of follow - up were available . at diagnosis , va was 46 early treatment diabetic retinopathy study ( etdrs ) letters , converted from snellen va using the equation 85 + 50 x log ( snellen fraction ) , cft was 712 microns , and intraocular pressure ( iop ) was 22 mm hg .", "the patient had phacoemulsification plus intraocular lens insertion in the left eye in february 2012 .", "intravitreal triamcinolone acetonide was also administered to treat the persistent dme postoperatively . at the first postoperative visit ,", "va was 70 etdrs letters , cft was 278 microns , and iop was 20 mm hg . over the next 5 months , va ( 50 etdrs letters ) and cft ( 805 microns ) progressively worsened , and the patient was admitted into the matisse trial in september 2012 to test the effects of ranibizumab plus a small - interfering rna agent .", "between october 2012 and march 2014 , the patient received 5 injections of ranibizumab plus the small - interfering rna agent and then subsequently listed for 2 further courses of 3 intravitreal injections of ranibizumab . by the end of this stage of treatment , by 1 month after the final injection of ranibizumab , the dme had recurred , va was 55 etdrs letters , cft was 774 microns , and iop was 17 mm hg .", "the patient 's dme was now chronic ( over 18 months of duration ) , relapsing , and her crt had fluctuated wildly through the different treatment phases , which is believed to have a deleterious effect on the function of the retina and , more specifically , on the visual function due to the presence of submacular fluid .", "it was at this stage that the patient was treated with iluvien according to nice ta301 ( please see http://www.nice.org.uk/guidance/ta301/chapter/1-guidance ) .", "figure 1 shows the response of va and cft after a single iluvien implant without any further treatment to this eye over the next 2 years . within 7 days", ", va had increased to 61 etdrs letters and cft had decreased to 267 microns . within 2 weeks , va had improved further to 70 etdrs letters , which is the legal requirement to hold a driving license in the uk .", "these early improvements were then largely maintained throughout the next 24 months without further intervention , as shown in figure 1 .", "figure 2 shows optical coherence tomography scans before and after iluvien implantation , illustrating the structural improvement that occurred alongside the functional improvement in vision .", "over the course of the 2-year period , iop remained below 21 mm hg , and no iop - lowering medication was required .", "this is the first case that the authors are aware of , which shows that a single injection of the 0.2 g / day fac implant leads to rapid improvements ( within 7 days ) in va and cft that are maintained over 2 years of follow - up .", "this was despite 11 previous injections of ranibizumab , which failed to control the dme prior to treatment with iluvien .", "the functional and structural responses presented herein are consistent with those reported in the fame trials , with rapid and marked responses observed early and being sustained in the long term", ". this patient will continue to be monitored to see if the responses reported in controlled clinical trials are replicated with a single injection in real - life uk practice over a 36-month period .", "this article does not contain any new studies with human or animal subjects performed by any of the authors .", "f. quhill has attended advisory boards and speaker engagements and has been remunerated for these by alimera sciences ."], "abstract_text": ["<S> importancea case showing sustained structural and functional responses 2 years after a single treatment with iluvien ( 0.2 g / day fluocinolone acetonide , fac ) despite suboptimal responses to ranibizumab.observationsa 68-year - old female patient with diabetic macular oedema ( dme ) from type 2 diabetes mellitus was first diagnosed in october 2010 and had a baseline visual acuity ( va ) of 46 early treatment diabetic retinopathy study ( etdrs ) letters in the left eye . </S>", "<S> central foveal thickness ( cft ) was 712 microns . </S>", "<S> the patient was treated with 11 intravitreal injections of ranibizumab ( 5 in combination with a small - interfering rna agent ) , and by march 2014 , va and cft were largely unchanged ( 55 etdrs letters and 774 microns ) . </S>", "<S> the patient was treated with iluvien as she had a pseudophakic lens and a clearly suboptimal response to the prior therapy with ranibizumab . </S>", "<S> an implant releasing fac at a dosage of 0.2 g / day was administered in march 2014 , and the optical coherence tomography indicated that the macula was dry after 7 days ( cft was below 300 microns ) . </S>", "<S> this was sustained at 6 , 12 , and 24 months after the treatment . </S>", "<S> va improved by 5 letters within 7 days and by 15 letters within 14 days , and this was maintained after 24 months . throughout the duration of this study , the intraocular pressure was 22 mm hg , and no glaucoma medication was administered.conclusions and relevancein real - life uk practice , this dme patient showed a suboptimal response to multiple intravitreal injections of ranibizumab . when subsequently treated with a single injection of iluvien , there were large and rapid improvements in va and cft that were maintained for the following 2 years . </S>"], "labels": null, "section_names": ["Importance", "Observations", "Conclusions and Relevance", "Background", "Case Presentation", "Conclusion", "Statement of Ethics", "Disclosure Statement"], "sections": [["a case showing sustained structural and functional responses 2 years after a single treatment with iluvien ( 0.2 g / day fluocinolone acetonide , fac ) despite suboptimal responses to ranibizumab ."], ["a 68-year - old female patient with diabetic macular oedema ( dme ) from type 2 diabetes mellitus was first diagnosed in october 2010 and had a baseline visual acuity ( va ) of 46 early treatment diabetic retinopathy study ( etdrs ) letters in the left eye .", "the patient was treated with 11 intravitreal injections of ranibizumab ( 5 in combination with a small - interfering rna agent ) , and by march 2014 , va and cft were largely unchanged ( 55 etdrs letters and 774 microns ) .", "the patient was treated with iluvien as she had a pseudophakic lens and a clearly suboptimal response to the prior therapy with ranibizumab .", "an implant releasing fac at a dosage of 0.2 g / day was administered in march 2014 , and the optical coherence tomography indicated that the macula was dry after 7 days ( cft was below 300 microns ) .", "va improved by 5 letters within 7 days and by 15 letters within 14 days , and this was maintained after 24 months . throughout the duration of this study , the intraocular pressure was 22 mm hg , and no glaucoma medication was administered ."], ["in real - life uk practice , this dme patient showed a suboptimal response to multiple intravitreal injections of ranibizumab . when subsequently treated with a single injection of iluvien", ", there were large and rapid improvements in va and cft that were maintained for the following 2 years ."], ["worldwide diabetic macular edema ( dme ) is reported to have a prevalence of 6.8% and results in vision loss .", "dme management can be difficult , with only around 15% of the patients achieving significant vision recovery with laser , the current standard of care .", "a number of pharmacotherapies are now licensed for the treatment of dme in europe , including anti - vascular endothelial growth factor agents and corticosteroid therapies , which are used to target vascular endothelial growth pathways and inflammatory pathways , respectively . in diabetes ,", "leukocytes release free radicals and enzymes that cause direct damage to endothelial cells , increasing blood retinal barrier leakage .", "they also release cytokines , including vascular endothelial growth factor , tumor necrosis factor- , and interleukin-6 , which act through various signaling pathways to increase vascular permeability .", "the release of cytokines with a corticosteroid  such as dexamethasone , triamcinolone or fluocinolone  has been shown to reduce dme and rehabilitate vision in several pivotal clinical studies .", "iluvien contains the corticosteroid fluocinolone acetonide ( fac ) and uses microdosingtm technology to release fac at a daily rate of 0.2 g for up to 3 years .", "this differs significantly from both the dexamethasone delivery system and that of triamcinolone , which are reported to have a duration of action of only up to 3 and 4 months , respectively .", "the effectiveness of iluvien was demonstrated in the fame trials ( nct00344968 ) in which patients with dme who had previously shown an insufficient response to laser were treated with either a 0.2 g / day ( iluvien ) or 0.5 g / day fac implant and were compared against a sham - control treated population .", "both fame trials independently met their primary endpoint and showed that significantly more patients with chronic dme treated with the 0.2 g / day fac implant experienced a 15-letter improvement in visual acuity ( va ) at month 24 than sham - control patients ( pooled data ; 34.4 vs. 13.4% ; p < 0.001 ) and that this benefit was sustained to month 36 .", "functional improvements were accompanied by rapid and sustained improvements in central foveal thickness ( cft ) . in europe ,", "iluvien is indicated for the treatment of vision impairment associated with chronic diabetic macular oedema considered insufficiently responsive to available therapies . since then", "miss clare bailey presented the real world outcomes from a uk audit of patients treated with iluvien at the annual meeting of the royal college of ophthalmologists in birmingham , uk , between june 24 and 26 , 2016 .", "this unpublished interim analysis showed that the majority of treated eyes ( 69.4% of 290 eyes ) maintained or improved va .", "to date , however , relatively limited data with a longer - term follow - up of the functional and structural responses following treatment with iluvien have been reported .", "the case described herein provides data for both va and cft over a 24-month follow - up period ."], ["a 68-year - old caucasian female with type 2 diabetes mellitus had diffuse dme diagnosed in her left eye in october 2010 .", "the treatment history is summarised in table 1 and was previously reported when only 6 months of follow - up were available . at diagnosis , va was 46 early treatment diabetic retinopathy study ( etdrs ) letters , converted from snellen va using the equation 85 + 50 x log ( snellen fraction ) , cft was 712 microns , and intraocular pressure ( iop ) was 22 mm hg .", "the patient had phacoemulsification plus intraocular lens insertion in the left eye in february 2012 .", "intravitreal triamcinolone acetonide was also administered to treat the persistent dme postoperatively . at the first postoperative visit ,", "va was 70 etdrs letters , cft was 278 microns , and iop was 20 mm hg . over the next 5 months , va ( 50 etdrs letters ) and cft ( 805 microns ) progressively worsened , and the patient was admitted into the matisse trial in september 2012 to test the effects of ranibizumab plus a small - interfering rna agent .", "between october 2012 and march 2014 , the patient received 5 injections of ranibizumab plus the small - interfering rna agent and then subsequently listed for 2 further courses of 3 intravitreal injections of ranibizumab . by the end of this stage of treatment , by 1 month after the final injection of ranibizumab , the dme had recurred , va was 55 etdrs letters , cft was 774 microns , and iop was 17 mm hg .", "the patient 's dme was now chronic ( over 18 months of duration ) , relapsing , and her crt had fluctuated wildly through the different treatment phases , which is believed to have a deleterious effect on the function of the retina and , more specifically , on the visual function due to the presence of submacular fluid .", "it was at this stage that the patient was treated with iluvien according to nice ta301 ( please see http://www.nice.org.uk/guidance/ta301/chapter/1-guidance ) .", "figure 1 shows the response of va and cft after a single iluvien implant without any further treatment to this eye over the next 2 years . within 7 days", ", va had increased to 61 etdrs letters and cft had decreased to 267 microns . within 2 weeks , va had improved further to 70 etdrs letters , which is the legal requirement to hold a driving license in the uk .", "these early improvements were then largely maintained throughout the next 24 months without further intervention , as shown in figure 1 .", "figure 2 shows optical coherence tomography scans before and after iluvien implantation , illustrating the structural improvement that occurred alongside the functional improvement in vision .", "over the course of the 2-year period , iop remained below 21 mm hg , and no iop - lowering medication was required ."], ["this is the first case that the authors are aware of , which shows that a single injection of the 0.2 g / day fac implant leads to rapid improvements ( within 7 days ) in va and cft that are maintained over 2 years of follow - up .", "this was despite 11 previous injections of ranibizumab , which failed to control the dme prior to treatment with iluvien .", "the functional and structural responses presented herein are consistent with those reported in the fame trials , with rapid and marked responses observed early and being sustained in the long term", ". this patient will continue to be monitored to see if the responses reported in controlled clinical trials are replicated with a single injection in real - life uk practice over a 36-month period ."], ["this article does not contain any new studies with human or animal subjects performed by any of the authors ."], ["f. quhill has attended advisory boards and speaker engagements and has been remunerated for these by alimera sciences ."]]}
{"article_id": "PMC4807908", "article_text": ["patients were recruited from the epilepsy clinic at austin health , from the practices of investigators , and by referral for epilepsy genetics research from around australia and internationally .", "informed consent was obtained from the patient or his or her parent or legal guardian .", "the study was approved by the institutional review boards of austin health and the university of washington .", "we used molecular inversion probes to capture all exons and 5 base pairs of flanking intronic depdc5 sequence ; next - generation sequencing and data analysis were performed as described previously in 130 patients with epileptic spasms of unknown etiology .", "known epileptic encephalopathy genes had been excluded in many cases ( unpublished data , carvill et al . , january 2015 ) .", "we considered only nonsynonymous , splice site , or frameshift variants that were present at an allele frequency < 1% in 61,000 exomes of the exome aggregation consortium ( exac ) data set ( to exclude single nucleotide polymorphisms ) ( http://exac.broadinstitute.org/ ) for further analysis and performed segregation analysis for these rare variants in available family members .", "we considered truncating variants to be pathogenic and missense variants that were either inherited from an affected parent or arose de novo to be possibly pathogenic .", "maternity / paternity was confirmed using the powerplex s5 system ( promega , madison , wi ) .", "we included an additional novel depdc5 case , identified through commercial genetic testing ( d : ii:1 ) , and additional phenotypic data on cousins from our earlier report ( family e ) .", "patients were recruited from the epilepsy clinic at austin health , from the practices of investigators , and by referral for epilepsy genetics research from around australia and internationally .", "informed consent was obtained from the patient or his or her parent or legal guardian .", "the study was approved by the institutional review boards of austin health and the university of washington .", "we used molecular inversion probes to capture all exons and 5 base pairs of flanking intronic depdc5 sequence ; next - generation sequencing and data analysis were performed as described previously in 130 patients with epileptic spasms of unknown etiology .", "known epileptic encephalopathy genes had been excluded in many cases ( unpublished data , carvill et al . , january 2015 ) .", "we considered only nonsynonymous , splice site , or frameshift variants that were present at an allele frequency < 1% in 61,000 exomes of the exome aggregation consortium ( exac ) data set ( to exclude single nucleotide polymorphisms ) ( http://exac.broadinstitute.org/ ) for further analysis and performed segregation analysis for these rare variants in available family members .", "we considered truncating variants to be pathogenic and missense variants that were either inherited from an affected parent or arose de novo to be possibly pathogenic .", "maternity / paternity was confirmed using the powerplex s5 system ( promega , madison , wi ) .", "we included an additional novel depdc5 case , identified through commercial genetic testing ( d : ii:1 ) , and additional phenotypic data on cousins from our earlier report ( family e ) .", "we sequenced all depdc5 target base pairs to a depth of 50 at a median of 90% across all samples .", "we identified likely pathogenic variants in 3 of 130 ( 2.3% ) patients with epileptic spasms of unknown etiology ( table 1 , figures 1 and 2 ) .", "a total of 92 of 130 patients had west syndrome , while 38 had epileptic spasms in association with other epileptic encephalopathies . in 2 cases ( families a and c ) , the depdc5 variants occurred de novo .", "patient b : iii:2 inherited a truncating mutation not seen in controls from a father with frontal lobe epilepsy .", "patient c : iii:3 , who was of chinese descent , had a de novo missense variant that disrupted a highly conserved nucleotide , which was predicted to be damaging by 2 of 3 in silico tools ( table 1 ) .", "this variant occurred in 71 individuals in exac , including 66 of 8,732 individuals of east asian descent ( minor allele frequency [ maf ] 0.7% ) .", "clinical and molecular features in patients with depdc5 variants and epileptic spasms we describe 6 patients in 5 families with depdc5 variants and spasms .", "variants arose de novo in 2 cases ( families a and c ) and were inherited in 3 ( families b , d , and e ) .", "upper panel of the schematic shows all previously reported truncating mutations ( black ) and those described in this study ( red ) .", "lower panel shows all missense mutations in previous studies ( black ) and those described in this study ( red ) , numbers in parentheses show the highest population maf from the exac data set , and black lines show the missense variants present in exac and variable frequencies .", "many of the missense mutations described in patients are present at appreciable frequencies in controls , and there are many missense variants across the gene", ". it will be vital to perform functional experiments to test the functional effect of these variants , to understand whether and how they cause disease , and to understand the incomplete penetrance that is a common feature of this disorder .", "we describe 2 additional families with inherited depdc5 variants in whom 1 or more affected family members presented with spasms ( table 1 , figure 1 ) .", "family d , ascertained through commercial testing , had a tyr281phe variant , which is highly conserved and predicted to be damaging by 2 of 3 tools ( table 1 ) .", "this variant was present in 5 of 67,552 ( maf 0.0007% ) individuals of non - finnish european descent .", "family e carries a splice site mutation , and we describe 2 patients with infantile spasms from this previously reported family .", "spasms were the presenting seizure type in each case , with onset at 612 weeks in 5 cases and late onset at 2 years , 8 months in 1 .", "two had easily controlled spasms , both with offset at 6 months . in 4 cases , spasms had focal electroclinical features .", "eeg features included multifocal epileptiform abnormalities , generalized paroxysmal fast activity , and slow spike waves ( figure e-1 at neurology.org/ng ) .", "three cases had later focal seizures with impaired awareness and onset from 9 months to 13 years ; all were refractory .", "one patient , e : iv:2 , had normal intellect and no developmental regression with spasm onset ; at 34 years , she was a professional .", "brain mri revealed temporal focal cortical dysplasia ( fcd ) in a : iii:2 and frontal fcd in d : ii:1 ( figure e-2 ) .", "d : ii:1 had a histologically confirmed left frontal fcd type iia ( figure e-3 ) .", "after anatomic left frontal lobectomy , the patient was seizure - free for 6 months and then had return of head drops and tonic seizures .", "we sequenced all depdc5 target base pairs to a depth of 50 at a median of 90% across all samples .", "we identified likely pathogenic variants in 3 of 130 ( 2.3% ) patients with epileptic spasms of unknown etiology ( table 1 , figures 1 and 2 ) .", "a total of 92 of 130 patients had west syndrome , while 38 had epileptic spasms in association with other epileptic encephalopathies . in 2 cases ( families a and c ) , the depdc5 variants occurred de novo .", "patient b : iii:2 inherited a truncating mutation not seen in controls from a father with frontal lobe epilepsy .", "patient c : iii:3 , who was of chinese descent , had a de novo missense variant that disrupted a highly conserved nucleotide , which was predicted to be damaging by 2 of 3 in silico tools ( table 1 ) .", "this variant occurred in 71 individuals in exac , including 66 of 8,732 individuals of east asian descent ( minor allele frequency [ maf ] 0.7% ) .", "clinical and molecular features in patients with depdc5 variants and epileptic spasms we describe 6 patients in 5 families with depdc5 variants and spasms .", "variants arose de novo in 2 cases ( families a and c ) and were inherited in 3 ( families b , d , and e ) .", "upper panel of the schematic shows all previously reported truncating mutations ( black ) and those described in this study ( red ) .", "lower panel shows all missense mutations in previous studies ( black ) and those described in this study ( red ) , numbers in parentheses show the highest population maf from the exac data set , and black lines show the missense variants present in exac and variable frequencies .", "many of the missense mutations described in patients are present at appreciable frequencies in controls , and there are many missense variants across the gene", ". it will be vital to perform functional experiments to test the functional effect of these variants , to understand whether and how they cause disease , and to understand the incomplete penetrance that is a common feature of this disorder .", "we describe 2 additional families with inherited depdc5 variants in whom 1 or more affected family members presented with spasms ( table 1 , figure 1 ) .", "family d , ascertained through commercial testing , had a tyr281phe variant , which is highly conserved and predicted to be damaging by 2 of 3 tools ( table 1 ) .", "this variant was present in 5 of 67,552 ( maf 0.0007% ) individuals of non - finnish european descent .", "family e carries a splice site mutation , and we describe 2 patients with infantile spasms from this previously reported family .", "spasms were the presenting seizure type in each case , with onset at 612 weeks in 5 cases and late onset at 2 years , 8 months in 1 .", "two had easily controlled spasms , both with offset at 6 months . in 4 cases , spasms had focal electroclinical features .", "eeg features included multifocal epileptiform abnormalities , generalized paroxysmal fast activity , and slow spike waves ( figure e-1 at neurology.org/ng ) .", "three cases had later focal seizures with impaired awareness and onset from 9 months to 13 years ; all were refractory .", "one patient , e : iv:2 , had normal intellect and no developmental regression with spasm onset ; at 34 years , she was a professional .", "brain mri revealed temporal focal cortical dysplasia ( fcd ) in a : iii:2 and frontal fcd in d : ii:1 ( figure e-2 ) .", "d : ii:1 had a histologically confirmed left frontal fcd type iia ( figure e-3 ) .", "after anatomic left frontal lobectomy , the patient was seizure - free for 6 months and then had return of head drops and tonic seizures .", "we first identified depdc5 in familial focal epilepsy , and here we show its relevance to epileptic spasms , illustrating the convergence of phenotypes in genetic mutations of the mtor pathway .", "our findings suggest that greater significance should be attributed to a family history of focal seizures in patients with epileptic spasms .", "affected family members had focal epilepsies emanating from different cortical regions , consistent with the pattern of ffevf .", "infantile spasms have an identifiable etiology in 60% cases and may include hypoxic - ischemic or metabolic encephalopathies , malformations , infection , and chromosomal anomalies .", "a family history of spasms is rare but has been described in conditions such as tsc and specific genetic diseases such as arx .", "the importance of de novo mutations has recently been emphasized , with a pathogenic mutation attributed to a single gene identified in 5%16% of cases ( n = 268 from 3 studies using next - generation sequencing technologies ) ( table e-1 ) .", "the most frequently mutated genes were stxbp1 ( n = 6 ) , cdkl5 ( n = 2 ) , kcnq2 ( n = 2 ) , and alg13 ( n = 2 ) .", "our finding of a depdc5 variant in up to 2.3% of patients in our cohort suggests that this gene may be one of the more frequent genes associated with epileptic spasms , taking into account that this cohort had been previously screened for many of the known genes .", "as only 1 patient showed classic hypsarrhythmia , the cohort may have some fundamental differences from other studies of infantile spasms in which hypsarrhythmia is essential for inclusion .", "we identified 3 truncating mutations : 1 occurred de novo and the remaining 2 were inherited .", "this is in keeping with previous reports in which the overwhelming majority of pathogenic depdc5 mutations resulted in premature truncation of the protein ( figure 2 ) , suggesting that depdc5 mutations cause disease by haploinsufficiency of the protein .", "this is further supported by the presence of only 15 truncating variants in the 61,000 exomes in exac .", "however , we report 2 missense variants , pro1031his ( maf 0.7% ) and tyr281phe ( maf 0.007% ) , and there are more than 400 missense variants in exac .", "tyr281phe is exceedingly rare , and incomplete penetrance of depdc5 mutations may explain the presence of these variants in the population .", "however , the 0.7% maf of pro1031his ( arose de novo in family c ) seems too high in this population to be explained solely by incomplete penetrance , and this result needs to be interpreted with caution .", "this may also be true for several reported missense mutations also present in controls at low frequencies ( figure 2 )", ". it will be important to develop robust functional experiments to assess the pathogenicity of these missense variants in the future .", "we have combined a targeted resequencing approach in 130 patients and results from clinical diagnostics and extended phenotyping in a known mutation - positive family to determine several notable features that highlight the variability of onset and outcome of depdc5-associated spasms .", "our findings expand the depdc5 phenotypic spectrum to include more severe epilepsies presenting with spasms .", "first , the outcome may be excellent with normal intellect , although most of our patients had intellectual disability with or without autistic features .", "third , 1 patient had later onset of spasms in the third year with further cognitive decline in adolescence .", "fourth , 2 patients had malformations with fcd ; in 1 , surgery resulted in seizure improvement .", "surprisingly , only 1 patient showed hypsarrhythmia on eeg ; however , all had highly abnormal eegs with abundant epileptiform activity , which can be associated with epileptic spasms .", "the absence of classic hypsarrhythmia means that these patients differ from those with west syndrome , which has been the focus of many recent genomic studies . of note", ", spasms were present in patients with fcd ( 2 patients ) and those without fcd ( 3 patients ) after careful scrutiny of the mri .", "because many patients with focal epilepsies and depdc5 mutations have normal mri , the presence of detectable lesions is not necessary for the development of seizures .", "rather , loss - of - function mutations in depdc5 , an inhibitor of the mtor pathway , presumably lead to excessive signaling of this pathway , which has many functions that could conceivably contribute to hyperexcitability .", "this scenario would be analogous to tsc , in which a second mutation in the mtor regulators tsc1 and tsc2 is reported in some tumors of patients with tsc . in patients with depdc5 mutations ,", "second hit  could occur either on the other allele or on another gene involved in the mtor pathway .", "alternatively , an acquired cause , such as a human papillomavirus , has been conjectured to be a ", "deep targeted or even whole - exome sequencing should be performed on resected tissue to explore this hypothesis .", "given the incomplete penetrance of depdc5 mutations and the discovery of both inherited and de novo mutations , molecular approaches for epileptic spasms should interrogate both inheritance patterns .", "the detection of inherited mutations has important reproductive counseling implications for families of children with spasms , which needs to incorporate increased risk for comorbidities such as intellectual disability and autism spectrum disorders .", "the recognition of spasms in depdc5 epilepsies parallels other mtoropathies such as tsc and ste20-related kinase adaptor alpha syndrome .", "although many depdc5 epilepsies are milder , the convergence of the phenotypic spectrum and molecular pathways suggests that targeted mtor therapies may benefit patients with the more severe depdc5 disorders .", "g.l.c . : drafted / revised the manuscript , study design , acquisition of data , and data analysis . d.e.c . : drafted / revised the manuscript , acquisition of data , and data analysis . b.m.r . :", ", s.f.b . , and j. sullivan : acquisition of data and data analysis . i.e.s . and h.c.m .", ": drafted / revised the manuscript , study design , acquisition of data , and data analysis .", "this work was supported by funding from the nih ( ninds 5r01ns069605 ) to h.c.m .", "is supported by a postdoctoral fellowship from the epilepsy foundation and the lennox and lombroso fund and the nih ( ninds 1k99ns089858 ) .", "i.e.s . , s.f.b . , and l.d . are supported by a national health and medical research council of australia ( nhmrc ) program grant .", "is supported by an nhmrc practitioner fellowship and l.d . is supported by an nhmrc career development fellowship ( 1032603 ) .", "the work was also supported by the university of washington intellectual and developmental disabilities research center genetics core ( nih u54hd083091 ) .", "dr . carvill is a member of the scientific advisory board of ambry genetics and has received grants from the nih / national institute of neurological disorders and stroke , the epilepsy foundation and lennox and lombroso trust , and citizens united for research in epilepsy .", "crompton has received research support from the national health and medical research council of australia .", "mcmahon has received research support from the national health and medical research council of australia .", "howell has received research support from the national health and medical research council gustav nossal postgraduate scholarship and her husband holds stock options for general electric ( medical imaging equipment ) .", "mandelstam has been an employee of the royal children 's hospital , the melbourne brain centre , and the university of melbourne .", "dr . leventer has received honoraria for travel from the asia oceania congress of child neurology and has received research support from the national health and medical research council project grant 1068278 , the national health and medical research council project grant 1059666 , and campbell edwards trust research support .", "mullen has been an employee of the florey institute of neuroscience and mental health , the university of melbourne , australia austin health , and australia eastern health ; has received travel funding from the american epilepsy society young investigator award ; and has received research support from the nhmrc early career fellowship and nhmrc project grant .", "berkovic has served on the scientific advisory board of ucb pharma ; has served on the editorial boards of brain , epileptic disorders , and lancet neurology ; has received research support from the national health and medical research council of australia ; has received honoraria from ucb ; has a patent for pcdh19 testing planned and is one of the inventors listed on a patent held by bionomics inc . on diagnostic testing of using the scn1a gene", "; has received payment for development of educational presentations from ucb pharma , novartis pharmaceuticals , sanofi - aventis , and janssen - cilag ; and has been a consultant to bionomics and athena diagnostics .", "dr . sullivan has been the chair of sab for nonprofit entity pcdh19 alliance ; has received honoraria from zogenix ; and has received research support from marinus pharmaceuticals .", "scheffer has served on scientific advisory boards for ucb and janssen - cilag emea ; has served on the editorial boards of the annals of neurology , neurology , epilepsy currents , progress in epileptic disorders series , virtual neuro centre , and epileptic disorders ; holds patents for methods of treatment and diagnosis of epilepsy by detecting mutations in the scn1a gene , a diagnostic method for epilepsy , mutations in ion channels , diagnostic and treatment methods relating to autosomal dominant nocturnal frontal lobe epilepsy ( pending ) , gene and mutations thereof associated with seizure disorders , a gene and mutations thereof associated with seizure and movement disorders , and diagnostic and therapeutic methods for efmr ; may accrue future revenue on pending patent wo61/010176 ( filed : 2008 ) : therapeutic compound ; has received speaker honoraria from glaxosmithkline , athena diagnostics , ucb , biocodex , and janssen - cilag emea ; has received funding for travel from athena diagnostics , ucb , biocodex , glaxosmithkline , janssen - cilag emea , aoccn taiwan , weizmann institute , the american academy of neurology , irccs oasi maria ss , sanofi china , qbi university of queensland , international league against epilepsy , australian academy of science , commonwealth department of industry , westmead hospital , perpetual , university of california , matthew 's friends , sbs , pts for nfle conference , turkish child neurology association , european congress on epileptology , international child neurology association , ucb , movement disorder society , international epilepsy congress , university of auckland , world congress of neurology , epileptic disorders , eisai , aoccn , sanofi , and transgenomic ; and has received research support from the national health and medical research council of australia , nih , australian research council , health research council of new zealand , cure , american epilepsy society , us department of defense autism spectrum disorder research program , the jack brockhoff foundation , the shepherd foundation , perpetual charitable trustees , the university of melbourne , the epilepsy association of tasmania , melbourne neurosciences institute , weizmann institute , cure sudep award , and perpetual philanthropic services .", "dr . mefford has received research support from the nih / national institute of neurological disorders and stroke , the burroughs wellcome fund , the american thoracic society foundation partner award , and simon 's foundation for autism research ; has served on the editorial board of science translational medicine , is a consultant for sera prognostics and the simons foundation ( sfari gene advisory board ) ; and has served on the scientific advisory boards of the lennox gastaut foundation and supporting families with koolen - de vries syndrome ."], "abstract_text": ["<S> objective : to assess the presence of depdc5 mutations in a cohort of patients with epileptic spasms.methods:we performed depdc5 resequencing in 130 patients with spasms , segregation analysis of variants of interest , and detailed clinical assessment of patients with possibly and likely pathogenic variants.results:we identified 3 patients with variants in depdc5 in the cohort of 130 patients with spasms . </S>", "<S> we also describe 3 additional patients with depdc5 alterations and epileptic spasms : 2 from a previously described family and a third ascertained by clinical testing . </S>", "<S> overall , we describe 6 patients from 5 families with spasms and depdc5 variants ; 2 arose de novo and 3 were familial . </S>", "<S> two individuals had focal cortical dysplasia . </S>", "<S> clinical outcome was highly variable.conclusions:while recent molecular findings in epileptic spasms emphasize the contribution of de novo mutations , we highlight the relevance of inherited mutations in the setting of a family history of focal epilepsies . </S>", "<S> we also illustrate the utility of clinical diagnostic testing and detailed phenotypic evaluation in characterizing the constellation of phenotypes associated with depdc5 alterations . </S>", "<S> we expand this phenotypic spectrum to include epileptic spasms , aligning depdc5 epilepsies more with the recognized features of other mtoropathies . </S>"], "labels": null, "section_names": ["METHODS", "Standard protocol approvals, registrations, and patient consents.", "Targeted resequencing of ", "Accession numbers.", "RESULTS", "Molecular analysis.", "Clinical characterization.", "DISCUSSION", "Supplementary Material", "AUTHOR CONTRIBUTIONS", "STUDY FUNDING", "DISCLOSURE", "ONLINE RESOURCES"], "sections": [["patients were recruited from the epilepsy clinic at austin health , from the practices of investigators , and by referral for epilepsy genetics research from around australia and internationally .", "informed consent was obtained from the patient or his or her parent or legal guardian .", "the study was approved by the institutional review boards of austin health and the university of washington .", "we used molecular inversion probes to capture all exons and 5 base pairs of flanking intronic depdc5 sequence ; next - generation sequencing and data analysis were performed as described previously in 130 patients with epileptic spasms of unknown etiology .", "known epileptic encephalopathy genes had been excluded in many cases ( unpublished data , carvill et al . , january 2015 ) .", "we considered only nonsynonymous , splice site , or frameshift variants that were present at an allele frequency < 1% in 61,000 exomes of the exome aggregation consortium ( exac ) data set ( to exclude single nucleotide polymorphisms ) ( http://exac.broadinstitute.org/ ) for further analysis and performed segregation analysis for these rare variants in available family members .", "we considered truncating variants to be pathogenic and missense variants that were either inherited from an affected parent or arose de novo to be possibly pathogenic .", "maternity / paternity was confirmed using the powerplex s5 system ( promega , madison , wi ) .", "we included an additional novel depdc5 case , identified through commercial genetic testing ( d : ii:1 ) , and additional phenotypic data on cousins from our earlier report ( family e ) ."], ["patients were recruited from the epilepsy clinic at austin health , from the practices of investigators , and by referral for epilepsy genetics research from around australia and internationally .", "informed consent was obtained from the patient or his or her parent or legal guardian .", "the study was approved by the institutional review boards of austin health and the university of washington ."], ["we used molecular inversion probes to capture all exons and 5 base pairs of flanking intronic depdc5 sequence ; next - generation sequencing and data analysis were performed as described previously in 130 patients with epileptic spasms of unknown etiology .", "known epileptic encephalopathy genes had been excluded in many cases ( unpublished data , carvill et al . , january 2015 ) .", "we considered only nonsynonymous , splice site , or frameshift variants that were present at an allele frequency < 1% in 61,000 exomes of the exome aggregation consortium ( exac ) data set ( to exclude single nucleotide polymorphisms ) ( http://exac.broadinstitute.org/ ) for further analysis and performed segregation analysis for these rare variants in available family members .", "we considered truncating variants to be pathogenic and missense variants that were either inherited from an affected parent or arose de novo to be possibly pathogenic .", "maternity / paternity was confirmed using the powerplex s5 system ( promega , madison , wi ) .", "we included an additional novel depdc5 case , identified through commercial genetic testing ( d : ii:1 ) , and additional phenotypic data on cousins from our earlier report ( family e ) ."], [""], ["we sequenced all depdc5 target base pairs to a depth of 50 at a median of 90% across all samples .", "we identified likely pathogenic variants in 3 of 130 ( 2.3% ) patients with epileptic spasms of unknown etiology ( table 1 , figures 1 and 2 ) .", "a total of 92 of 130 patients had west syndrome , while 38 had epileptic spasms in association with other epileptic encephalopathies . in 2 cases ( families a and c ) , the depdc5 variants occurred de novo .", "patient b : iii:2 inherited a truncating mutation not seen in controls from a father with frontal lobe epilepsy .", "patient c : iii:3 , who was of chinese descent , had a de novo missense variant that disrupted a highly conserved nucleotide , which was predicted to be damaging by 2 of 3 in silico tools ( table 1 ) .", "this variant occurred in 71 individuals in exac , including 66 of 8,732 individuals of east asian descent ( minor allele frequency [ maf ] 0.7% ) .", "clinical and molecular features in patients with depdc5 variants and epileptic spasms we describe 6 patients in 5 families with depdc5 variants and spasms .", "variants arose de novo in 2 cases ( families a and c ) and were inherited in 3 ( families b , d , and e ) .", "upper panel of the schematic shows all previously reported truncating mutations ( black ) and those described in this study ( red ) .", "lower panel shows all missense mutations in previous studies ( black ) and those described in this study ( red ) , numbers in parentheses show the highest population maf from the exac data set , and black lines show the missense variants present in exac and variable frequencies .", "many of the missense mutations described in patients are present at appreciable frequencies in controls , and there are many missense variants across the gene", ". it will be vital to perform functional experiments to test the functional effect of these variants , to understand whether and how they cause disease , and to understand the incomplete penetrance that is a common feature of this disorder .", "we describe 2 additional families with inherited depdc5 variants in whom 1 or more affected family members presented with spasms ( table 1 , figure 1 ) .", "family d , ascertained through commercial testing , had a tyr281phe variant , which is highly conserved and predicted to be damaging by 2 of 3 tools ( table 1 ) .", "this variant was present in 5 of 67,552 ( maf 0.0007% ) individuals of non - finnish european descent .", "family e carries a splice site mutation , and we describe 2 patients with infantile spasms from this previously reported family .", "spasms were the presenting seizure type in each case , with onset at 612 weeks in 5 cases and late onset at 2 years , 8 months in 1 .", "two had easily controlled spasms , both with offset at 6 months . in 4 cases , spasms had focal electroclinical features .", "eeg features included multifocal epileptiform abnormalities , generalized paroxysmal fast activity , and slow spike waves ( figure e-1 at neurology.org/ng ) .", "three cases had later focal seizures with impaired awareness and onset from 9 months to 13 years ; all were refractory .", "one patient , e : iv:2 , had normal intellect and no developmental regression with spasm onset ; at 34 years , she was a professional .", "brain mri revealed temporal focal cortical dysplasia ( fcd ) in a : iii:2 and frontal fcd in d : ii:1 ( figure e-2 ) .", "d : ii:1 had a histologically confirmed left frontal fcd type iia ( figure e-3 ) .", "after anatomic left frontal lobectomy , the patient was seizure - free for 6 months and then had return of head drops and tonic seizures ."], ["we sequenced all depdc5 target base pairs to a depth of 50 at a median of 90% across all samples .", "we identified likely pathogenic variants in 3 of 130 ( 2.3% ) patients with epileptic spasms of unknown etiology ( table 1 , figures 1 and 2 ) .", "a total of 92 of 130 patients had west syndrome , while 38 had epileptic spasms in association with other epileptic encephalopathies . in 2 cases ( families a and c ) , the depdc5 variants occurred de novo .", "patient b : iii:2 inherited a truncating mutation not seen in controls from a father with frontal lobe epilepsy .", "patient c : iii:3 , who was of chinese descent , had a de novo missense variant that disrupted a highly conserved nucleotide , which was predicted to be damaging by 2 of 3 in silico tools ( table 1 ) .", "this variant occurred in 71 individuals in exac , including 66 of 8,732 individuals of east asian descent ( minor allele frequency [ maf ] 0.7% ) .", "clinical and molecular features in patients with depdc5 variants and epileptic spasms we describe 6 patients in 5 families with depdc5 variants and spasms .", "variants arose de novo in 2 cases ( families a and c ) and were inherited in 3 ( families b , d , and e ) .", "upper panel of the schematic shows all previously reported truncating mutations ( black ) and those described in this study ( red ) .", "lower panel shows all missense mutations in previous studies ( black ) and those described in this study ( red ) , numbers in parentheses show the highest population maf from the exac data set , and black lines show the missense variants present in exac and variable frequencies .", "many of the missense mutations described in patients are present at appreciable frequencies in controls , and there are many missense variants across the gene", ". it will be vital to perform functional experiments to test the functional effect of these variants , to understand whether and how they cause disease , and to understand the incomplete penetrance that is a common feature of this disorder .", "we describe 2 additional families with inherited depdc5 variants in whom 1 or more affected family members presented with spasms ( table 1 , figure 1 ) .", "family d , ascertained through commercial testing , had a tyr281phe variant , which is highly conserved and predicted to be damaging by 2 of 3 tools ( table 1 ) .", "this variant was present in 5 of 67,552 ( maf 0.0007% ) individuals of non - finnish european descent .", "family e carries a splice site mutation , and we describe 2 patients with infantile spasms from this previously reported family ."], ["spasms were the presenting seizure type in each case , with onset at 612 weeks in 5 cases and late onset at 2 years , 8 months in 1 .", "two had easily controlled spasms , both with offset at 6 months . in 4 cases , spasms had focal electroclinical features .", "eeg features included multifocal epileptiform abnormalities , generalized paroxysmal fast activity , and slow spike waves ( figure e-1 at neurology.org/ng ) .", "three cases had later focal seizures with impaired awareness and onset from 9 months to 13 years ; all were refractory .", "one patient , e : iv:2 , had normal intellect and no developmental regression with spasm onset ; at 34 years , she was a professional .", "brain mri revealed temporal focal cortical dysplasia ( fcd ) in a : iii:2 and frontal fcd in d : ii:1 ( figure e-2 ) .", "d : ii:1 had a histologically confirmed left frontal fcd type iia ( figure e-3 ) .", "after anatomic left frontal lobectomy , the patient was seizure - free for 6 months and then had return of head drops and tonic seizures ."], ["we first identified depdc5 in familial focal epilepsy , and here we show its relevance to epileptic spasms , illustrating the convergence of phenotypes in genetic mutations of the mtor pathway .", "our findings suggest that greater significance should be attributed to a family history of focal seizures in patients with epileptic spasms .", "affected family members had focal epilepsies emanating from different cortical regions , consistent with the pattern of ffevf .", "infantile spasms have an identifiable etiology in 60% cases and may include hypoxic - ischemic or metabolic encephalopathies , malformations , infection , and chromosomal anomalies .", "a family history of spasms is rare but has been described in conditions such as tsc and specific genetic diseases such as arx .", "the importance of de novo mutations has recently been emphasized , with a pathogenic mutation attributed to a single gene identified in 5%16% of cases ( n = 268 from 3 studies using next - generation sequencing technologies ) ( table e-1 ) .", "the most frequently mutated genes were stxbp1 ( n = 6 ) , cdkl5 ( n = 2 ) , kcnq2 ( n = 2 ) , and alg13 ( n = 2 ) .", "our finding of a depdc5 variant in up to 2.3% of patients in our cohort suggests that this gene may be one of the more frequent genes associated with epileptic spasms , taking into account that this cohort had been previously screened for many of the known genes .", "as only 1 patient showed classic hypsarrhythmia , the cohort may have some fundamental differences from other studies of infantile spasms in which hypsarrhythmia is essential for inclusion .", "we identified 3 truncating mutations : 1 occurred de novo and the remaining 2 were inherited .", "this is in keeping with previous reports in which the overwhelming majority of pathogenic depdc5 mutations resulted in premature truncation of the protein ( figure 2 ) , suggesting that depdc5 mutations cause disease by haploinsufficiency of the protein .", "this is further supported by the presence of only 15 truncating variants in the 61,000 exomes in exac .", "however , we report 2 missense variants , pro1031his ( maf 0.7% ) and tyr281phe ( maf 0.007% ) , and there are more than 400 missense variants in exac .", "tyr281phe is exceedingly rare , and incomplete penetrance of depdc5 mutations may explain the presence of these variants in the population .", "however , the 0.7% maf of pro1031his ( arose de novo in family c ) seems too high in this population to be explained solely by incomplete penetrance , and this result needs to be interpreted with caution .", "this may also be true for several reported missense mutations also present in controls at low frequencies ( figure 2 )", ". it will be important to develop robust functional experiments to assess the pathogenicity of these missense variants in the future .", "we have combined a targeted resequencing approach in 130 patients and results from clinical diagnostics and extended phenotyping in a known mutation - positive family to determine several notable features that highlight the variability of onset and outcome of depdc5-associated spasms .", "our findings expand the depdc5 phenotypic spectrum to include more severe epilepsies presenting with spasms .", "first , the outcome may be excellent with normal intellect , although most of our patients had intellectual disability with or without autistic features .", "third , 1 patient had later onset of spasms in the third year with further cognitive decline in adolescence .", "fourth , 2 patients had malformations with fcd ; in 1 , surgery resulted in seizure improvement .", "surprisingly , only 1 patient showed hypsarrhythmia on eeg ; however , all had highly abnormal eegs with abundant epileptiform activity , which can be associated with epileptic spasms .", "the absence of classic hypsarrhythmia means that these patients differ from those with west syndrome , which has been the focus of many recent genomic studies . of note", ", spasms were present in patients with fcd ( 2 patients ) and those without fcd ( 3 patients ) after careful scrutiny of the mri .", "because many patients with focal epilepsies and depdc5 mutations have normal mri , the presence of detectable lesions is not necessary for the development of seizures .", "rather , loss - of - function mutations in depdc5 , an inhibitor of the mtor pathway , presumably lead to excessive signaling of this pathway , which has many functions that could conceivably contribute to hyperexcitability .", "this scenario would be analogous to tsc , in which a second mutation in the mtor regulators tsc1 and tsc2 is reported in some tumors of patients with tsc . in patients with depdc5 mutations ,", "second hit  could occur either on the other allele or on another gene involved in the mtor pathway .", "alternatively , an acquired cause , such as a human papillomavirus , has been conjectured to be a ", "deep targeted or even whole - exome sequencing should be performed on resected tissue to explore this hypothesis .", "given the incomplete penetrance of depdc5 mutations and the discovery of both inherited and de novo mutations , molecular approaches for epileptic spasms should interrogate both inheritance patterns .", "the detection of inherited mutations has important reproductive counseling implications for families of children with spasms , which needs to incorporate increased risk for comorbidities such as intellectual disability and autism spectrum disorders .", "the recognition of spasms in depdc5 epilepsies parallels other mtoropathies such as tsc and ste20-related kinase adaptor alpha syndrome .", "although many depdc5 epilepsies are milder , the convergence of the phenotypic spectrum and molecular pathways suggests that targeted mtor therapies may benefit patients with the more severe depdc5 disorders ."], [""], ["g.l.c . : drafted / revised the manuscript , study design , acquisition of data , and data analysis . d.e.c . : drafted / revised the manuscript , acquisition of data , and data analysis . b.m.r . :", ", s.f.b . , and j. sullivan : acquisition of data and data analysis . i.e.s . and h.c.m .", ": drafted / revised the manuscript , study design , acquisition of data , and data analysis ."], ["this work was supported by funding from the nih ( ninds 5r01ns069605 ) to h.c.m .", "is supported by a postdoctoral fellowship from the epilepsy foundation and the lennox and lombroso fund and the nih ( ninds 1k99ns089858 ) .", "i.e.s . , s.f.b . , and l.d . are supported by a national health and medical research council of australia ( nhmrc ) program grant .", "is supported by an nhmrc practitioner fellowship and l.d . is supported by an nhmrc career development fellowship ( 1032603 ) .", "the work was also supported by the university of washington intellectual and developmental disabilities research center genetics core ( nih u54hd083091 ) ."], ["dr . carvill is a member of the scientific advisory board of ambry genetics and has received grants from the nih / national institute of neurological disorders and stroke , the epilepsy foundation and lennox and lombroso trust , and citizens united for research in epilepsy .", "crompton has received research support from the national health and medical research council of australia .", "mcmahon has received research support from the national health and medical research council of australia .", "howell has received research support from the national health and medical research council gustav nossal postgraduate scholarship and her husband holds stock options for general electric ( medical imaging equipment ) .", "mandelstam has been an employee of the royal children 's hospital , the melbourne brain centre , and the university of melbourne .", "dr . leventer has received honoraria for travel from the asia oceania congress of child neurology and has received research support from the national health and medical research council project grant 1068278 , the national health and medical research council project grant 1059666 , and campbell edwards trust research support .", "mullen has been an employee of the florey institute of neuroscience and mental health , the university of melbourne , australia austin health , and australia eastern health ; has received travel funding from the american epilepsy society young investigator award ; and has received research support from the nhmrc early career fellowship and nhmrc project grant .", "berkovic has served on the scientific advisory board of ucb pharma ; has served on the editorial boards of brain , epileptic disorders , and lancet neurology ; has received research support from the national health and medical research council of australia ; has received honoraria from ucb ; has a patent for pcdh19 testing planned and is one of the inventors listed on a patent held by bionomics inc . on diagnostic testing of using the scn1a gene", "; has received payment for development of educational presentations from ucb pharma , novartis pharmaceuticals , sanofi - aventis , and janssen - cilag ; and has been a consultant to bionomics and athena diagnostics .", "dr . sullivan has been the chair of sab for nonprofit entity pcdh19 alliance ; has received honoraria from zogenix ; and has received research support from marinus pharmaceuticals .", "scheffer has served on scientific advisory boards for ucb and janssen - cilag emea ; has served on the editorial boards of the annals of neurology , neurology , epilepsy currents , progress in epileptic disorders series , virtual neuro centre , and epileptic disorders ; holds patents for methods of treatment and diagnosis of epilepsy by detecting mutations in the scn1a gene , a diagnostic method for epilepsy , mutations in ion channels , diagnostic and treatment methods relating to autosomal dominant nocturnal frontal lobe epilepsy ( pending ) , gene and mutations thereof associated with seizure disorders , a gene and mutations thereof associated with seizure and movement disorders , and diagnostic and therapeutic methods for efmr ; may accrue future revenue on pending patent wo61/010176 ( filed : 2008 ) : therapeutic compound ; has received speaker honoraria from glaxosmithkline , athena diagnostics , ucb , biocodex , and janssen - cilag emea ; has received funding for travel from athena diagnostics , ucb , biocodex , glaxosmithkline , janssen - cilag emea , aoccn taiwan , weizmann institute , the american academy of neurology , irccs oasi maria ss , sanofi china , qbi university of queensland , international league against epilepsy , australian academy of science , commonwealth department of industry , westmead hospital , perpetual , university of california , matthew 's friends , sbs , pts for nfle conference , turkish child neurology association , european congress on epileptology , international child neurology association , ucb , movement disorder society , international epilepsy congress , university of auckland , world congress of neurology , epileptic disorders , eisai , aoccn , sanofi , and transgenomic ; and has received research support from the national health and medical research council of australia , nih , australian research council , health research council of new zealand , cure , american epilepsy society , us department of defense autism spectrum disorder research program , the jack brockhoff foundation , the shepherd foundation , perpetual charitable trustees , the university of melbourne , the epilepsy association of tasmania , melbourne neurosciences institute , weizmann institute , cure sudep award , and perpetual philanthropic services .", "dr . mefford has received research support from the nih / national institute of neurological disorders and stroke , the burroughs wellcome fund , the american thoracic society foundation partner award , and simon 's foundation for autism research ; has served on the editorial board of science translational medicine , is a consultant for sera prognostics and the simons foundation ( sfari gene advisory board ) ; and has served on the scientific advisory boards of the lennox gastaut foundation and supporting families with koolen - de vries syndrome ."], [""]]}
{"article_id": "PMC5139928", "article_text": ["previous studies have shown a close relationship between bone density and success of implant treatment [ 27 ] .", "bone quality is determined by cortical bone thickness , the amount of trabeculae and mineralization   and can be evaluated by bone density assessment .", "several methods have been proposed for bone quality assessment ; among which , volumetric imaging techniques are the most feasible .", "these techniques enable the clinicians to assess bone density without any superimposition , which is a major limitation of plain radiography .", "computed tomography ( ct ) is a useful method for bone density assessment prior to dental implant surgery   and has been accepted as the gold standard for such evaluations .", "cone - beam computed tomography ( cbct ) has become a widely used imaging modality for pre - implant assessment in the recent years . compared with ct scan", ", cbct images provide superior resolution and lower patient radiation dose . despite the wide application of cbct in implant dentistry ,", "some studies advocate it as a reliable substitute for ct in this respect and confirm its validity [ 1,2,6,7,1316 ] while others have brought it into question .", "hounsfield unit ( hu ) is the standard scheme for scaling the reconstructed attenuation coefficient in medical ct systems . in cbct systems ,", "the gray values are used to represent the reconstructed values , although it has not yet been proposed as a standard system . in the present study ,", "the correlation between the gray values obtained from cbct and the hus in ct images was evaluated and the effect of the type of tissue , tissue thickness , acquisition parameters and location was assessed in this regard .", "in this in vitro diagnostic study , we compared the tissue density values on cbct and ct images in four different tissue phantoms with two different thicknesses , two different acquisition settings and in three locations in the phantoms .", "the phantoms used in this study simulated different densities namely hard tissue , soft tissue , water and air .", "hard tissue equivalent phantom was made of bovine bone powder mixed with epoxy resin with the proportion of 0.55:1 in the laboratory of nuclear engineering department of shahid beheshti university .", "soft tissue equivalent phantom consisted of a 1 cm - thick plexiglas slab , suggested by the medical engineering and medical physics department of shahid beheshti university .", "they were fixed over each other and placed in a clear plastic cylindrical container ( biokips , komax industrial co. ltd . ,", "the phantoms were positioned at the center of the field of view ( fov ) . for easy repeatability ,", "the phantoms were scanned three times to increase precision using ge bright speed 16 slice multi detector ct scan system ( general electric , schenectady , new york , usa ) with two different exposure settings .", "it was also scanned three times with two different acquisition settings using three different cbct units namely scanora 3d ( soredex , helsinki , finland ) , newtom vg ( afp , verona , italy ) and pro - max 3d ( planmeca , helsinki , finland ) .", "exposure parameters for different units in the first phase , six different series of scans were obtained by each unit , three with high and three with low acquisition settings , all in full thickness . in the second phase ,", "the new series of scans were obtained by the same units and the same acquisition parameters but with half thickness . in order to overcome the effect of various computer programs , all ct and cbct images were imported into a single computer program ( workstation 2.1 merge emed ,", "the ct number of ct images and the voxel value in cbct images were evaluated .", "three definite locations were selected on each image : at the middle ( m ; 270 mm from the periphery ) , on the periphery ( p ) , and an intermediate point between these two ( int ; 140 mm from the periphery ) on the radius of phantom with a constant region of interest ( roi ; fig .", "these points were selected on a slice and distributed to all using a software option to evaluate the same location in all slices and scans .", "the same was done for the half thickness images . an oral and maxillofacial radiologist observed and recorded the values in all images .", "evaluation of hus in hard tissue phantom with merge e - film software in three times of scanning in full thickness phantom .", "the relationship between ct numbers on ct scans and the gray values of cbct images was studied by anova ( p<0.05 ) .", "the effects of the type of material , acquisition parameters , phantoms thickness and location of the assessed point on the phantoms were also studied .", "the relationship between ct numbers on ct scans and the gray values of cbct images was studied by anova ( p<0.05 ) .", "the effects of the type of material , acquisition parameters , phantoms thickness and location of the assessed point on the phantoms were also studied .", "in the first evaluation , the interaction effect of the variables was found to be significant using results were not the same in different conditions and each variable had to be examined separately .", "the results of anova used to compare the cbct and ct data are shown in tables 2 and 3 .", "comparison of voxel values in cbct systems with hounsfield units in ct in full thickness phantoms ns : non - significant s : significant comparison of voxel values in cbct systems with hounsfield units in ct in half thickness phantoms ns : non - significant s : significant", "bone quality and quantity assessment is critical for pre - implant surgeries ; thus , reliable and precise radiographic evaluations are essential . however , despite wide use of cbct in different fields of dentistry , it is still not a valid method for bone quality assessment [ 4,1416,1921 ] .", "object location in cbct systems , object volume , acquisition parameters and some other factors have been evaluated in previous studies and controversial results have been reported [ 7,8,10,11,1316 ] . in this study ,", "the diagnostic accuracy of three cbct systems was compared with that of ct as the gold standard , and the effect of four factors including different tissue phantoms , object volume size , acquisition parameters and location of the objects was assessed . as the results showed , none of the cbct systems revealed the precise bone density as the gold standard . in this study ,", "different tissue phantoms were used to evaluate diagnostic accuracy of cbct systems in different density ranges .", "review of the literature revealed that only mah et al ,   used standard phantoms .", "heterogeneity of the hard tissue and air phantoms was a limitation of our study and was due to the production process .", "they were handmade in a laboratory and even in the best conditions , porosities were seen .", "however , in the clinical setting , the tissues are not homogenous ; thus , this factor may not be a real limitation for generalization of results to the clinical setting .", "however , in this study , the results showed no significant effect of this restriction by comparing the data of the four phantoms .", "the effect of tissue thickness is one of the main subjects for researchers to asses in density studies .", "in the first phase , the complete thickness and in the second phase , half - thickness samples were evaluated , which showed no considerable effect on assessment of the tissue density .", "the results were different in the two phases and there was a significant difference in results compared with ct system .", "katsumata et al ,   showed that the thicker the tissues , the more precise the values of tissue density .", "they discussed that beam characteristics may be a more important factor than the tissue thickness , which is because of the 360-degree rotation of the system and changes in the intensity of the x - ray beam while passing through different parts of an object , which causes maximum and minimum x - ray intensities .", "considering the importance of this factor , we used different acquisition parameters to evaluate the importance of the effect of intensity of x - ray beam on tissue density . in our study , despite the changes in the values , no significant difference was shown between the various image acquisition parameters of cbct systems and all values were different from the ct numbers .", "parsa et al ,   found a significant difference between ct and cbct values and showed changes in cbct values with changes in acquisition parameters .", "gray values increased with increase in size of fov in accuitomo system but this increase was obtained with an increase in volume in newtom system .", "this discrepancy between the behaviors of the two systems could be attributed to the variability in reconstruction and post - processing methods applied by the two manufacturers .", "however , one theory suggests that the tissue density of an object is more effective than the beam intensity and characteristics in showing the actual tissue density . to evaluate and interpret the cbct and ct images", ", we used only one software which can show the same level of tissue density in both systems , the point which was confirmed by ghasemi et al . by doing so", "mah et al ,   who used 11 types of cbct systems also used only one software program ( on demand 3d ) to match the observing conditions and roi in all sections .", "this fact is important in density assessment studies and can affect the results . because of the variable geometric characteristics of different systems , the density values and the selected roi are variable in different software programs provided by the systems .", "katsumata et al ,   emphasized that the selected roi should be the same in all sections with at least 3% error . in their study , ct images were also examined by the same software program to achieve the same situation in all conditions .", "nevertheless , mah et al ,   believed that human error is inevitable in selecting the roi .", "however , no specific method for precise selection of an area in different conditions has been suggested in studies except in a study by naitoh et al ,   who emphasized on selection of equal points via observation . by choosing three different points in an object", ", we wanted to evaluate the effect of object location in the fov on tissue density .", "legravere et al ,   did not report any significant difference in different locations in the tube field in their study , which was in contrast to the results of oliveira et al ,   who noticed that the correlation between the object density and ct number in cbct systems was not uniform through the dental arch .", "we found no significant difference between different locations in the diversity between the voxel values and hus .", "hua et al ,   found that some artifacts and scatter radiation are responsible for inefficacy of cbct for density assessment , which are inevitable because of the design of cbct systems and their detectors .", "considering the inefficacy of cbct for density evaluation , the x - ray beam heterogeneity in cbct can affect hu values , and lead to absence of a clear relationship between voxel value in cbct and bone mineral density provided by dual x - ray absorptiometry .", "the artifacts such as beam hardening or heel effect can decrease the validity of these values .", "several studies mentioned the difference between the cbct values and the ct values and in most cases , the reported cbct values were higher than the ct values .", "parsa et al ,   mentioned higher gray value in their study and believed that it may be due to the increased noise level , scattering and artefacts specific to the scanning technology .", "scarf and farman   proposed that although a sort of association was seen between the provided hus by ct and voxel value in cbct , the variability in measurements by cbct was higher than that by ct .", "however , legraver et al ,   found a linear relationship in r ( coefficient of relationship between two variables ) between the values in cbct and ct , and indicated that the cbct values were generally higher . in our study , the provided values by cbct were higher than the ct values in one of the systems ( newtom vg 3d ) , which is similar to the results of legrave et al ,   who used the same system for the same purpose .", "according to this point of view , the difference between the values in our study can be the result of different levels of energy in the three systems ; because the systems with a lower power result in lower intensity of x - ray beam and subsequently less tissue penetration depth . in order to create parallel conditions between the experimental phases and the clinical setting , the suitable acquisition parameters for each system were determined by an expert operator .", "thus , the levels of kvp and mas were specific and different in the three systems .", "haristory et al ,   studied the effect of different exposure parameters on the voxel value of cbct and found a strong relationship between cbct and ct using r value .", "however , because of different results in cbct , they suggested that the use of calibration phantom is necessary before imaging to ensure accurate bone density values . despite the afore - mentioned results , some studies showed inefficacy of this system in providing accurate values of bone density and found a linear relationship", "; thus , a high correlation between the results of cbct and ct was achieved . a study confirming the optimal efficacy of cbct systems to estimate bone density introduced a conversion coefficient .", "nomura et al ,   reported a relationship between two systems in evaluating bone mineral density and voxel value , but because of the nonlinear regression obtained in their study , further studies were suggested .", "however , a linear relationship and high correlation coefficient between these values have been shown in several studies . by applying the attenuation coefficient in an equation , mah et al ,  ", "obtained the hu values of tested materials from the voxel values with only a small difference from the actual hu values .", "considering all the above , we can say that our methodology , which is one reason for the uniqueness of this study , affected the results , and our results do not completely reflect the efficacy of the systems tested . as recently stated by pauwels et al ,   it is logical to postulate that although attempts have been made to correct the gray level variability , quantitative use of values provided by cbct should be generally avoided at this time .", "according to the results , the cbct systems were not able to show the accurate value of tissue density and the factors such as type of tissue ( hard , soft , water , air ) , thickness ( full against half ) , image acquisition conditions ( high settings against low settings ) and object location ( middle , peripheral and intermediate ) did not affect density evaluation by cbct systems ."], "abstract_text": ["<S> objectives : bone quality and quantity assessment is one of the most important steps in implant treatment planning . </S>", "<S> different methods such as computed tomography ( ct ) and recently suggested cone beam computed tomography ( cbct ) with lower radiation dose and less time and cost are used for bone density assessment . </S>", "<S> this in vitro study aimed to compare the tissue density values in hounsfield units ( hus ) in cbct and ct scans of different tissue phantoms with two different thicknesses , two different image acquisition settings and in three locations in the phantoms.materials and methods : four different tissue phantoms namely hard tissue , soft tissue , air and water were scanned by three different cbct and a ct system in two thicknesses ( full and half ) and two image acquisition settings ( high and low kvp and ma ) . </S>", "<S> the images were analyzed at three sites ( middle , periphery and intermediate ) using efilm software . </S>", "<S> the difference in density values was analyzed by anova and correction coefficient test ( p<0.05).results : there was a significant difference between density values in cbct and ct scans in most situations , and cbct values were not similar to ct values in any of the phantoms in different thicknesses and acquisition parameters or the three different sites . </S>", "<S> the correction coefficients confirmed the results.conclusions:cbct is not reliable for tissue density assessment . </S>", "<S> the results were not affected by changes in thickness , acquisition parameters or locations . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Statistical analysis:", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["in dental implant treatments , the outcome is influenced by both bone quality and quantity .", "previous studies have shown a close relationship between bone density and success of implant treatment [ 27 ] .", "bone quality is determined by cortical bone thickness , the amount of trabeculae and mineralization   and can be evaluated by bone density assessment .", "several methods have been proposed for bone quality assessment ; among which , volumetric imaging techniques are the most feasible .", "these techniques enable the clinicians to assess bone density without any superimposition , which is a major limitation of plain radiography .", "computed tomography ( ct ) is a useful method for bone density assessment prior to dental implant surgery   and has been accepted as the gold standard for such evaluations .", "cone - beam computed tomography ( cbct ) has become a widely used imaging modality for pre - implant assessment in the recent years . compared with ct scan", ", cbct images provide superior resolution and lower patient radiation dose . despite the wide application of cbct in implant dentistry ,", "some studies advocate it as a reliable substitute for ct in this respect and confirm its validity [ 1,2,6,7,1316 ] while others have brought it into question .", "hounsfield unit ( hu ) is the standard scheme for scaling the reconstructed attenuation coefficient in medical ct systems . in cbct systems ,", "the gray values are used to represent the reconstructed values , although it has not yet been proposed as a standard system . in the present study ,", "the correlation between the gray values obtained from cbct and the hus in ct images was evaluated and the effect of the type of tissue , tissue thickness , acquisition parameters and location was assessed in this regard ."], ["in this in vitro diagnostic study , we compared the tissue density values on cbct and ct images in four different tissue phantoms with two different thicknesses , two different acquisition settings and in three locations in the phantoms .", "the phantoms used in this study simulated different densities namely hard tissue , soft tissue , water and air .", "hard tissue equivalent phantom was made of bovine bone powder mixed with epoxy resin with the proportion of 0.55:1 in the laboratory of nuclear engineering department of shahid beheshti university .", "soft tissue equivalent phantom consisted of a 1 cm - thick plexiglas slab , suggested by the medical engineering and medical physics department of shahid beheshti university .", "they were fixed over each other and placed in a clear plastic cylindrical container ( biokips , komax industrial co. ltd . ,", "the phantoms were positioned at the center of the field of view ( fov ) . for easy repeatability ,", "the phantoms were scanned three times to increase precision using ge bright speed 16 slice multi detector ct scan system ( general electric , schenectady , new york , usa ) with two different exposure settings .", "it was also scanned three times with two different acquisition settings using three different cbct units namely scanora 3d ( soredex , helsinki , finland ) , newtom vg ( afp , verona , italy ) and pro - max 3d ( planmeca , helsinki , finland ) .", "exposure parameters for different units in the first phase , six different series of scans were obtained by each unit , three with high and three with low acquisition settings , all in full thickness . in the second phase ,", "the new series of scans were obtained by the same units and the same acquisition parameters but with half thickness . in order to overcome the effect of various computer programs , all ct and cbct images were imported into a single computer program ( workstation 2.1 merge emed ,", "the ct number of ct images and the voxel value in cbct images were evaluated .", "three definite locations were selected on each image : at the middle ( m ; 270 mm from the periphery ) , on the periphery ( p ) , and an intermediate point between these two ( int ; 140 mm from the periphery ) on the radius of phantom with a constant region of interest ( roi ; fig .", "these points were selected on a slice and distributed to all using a software option to evaluate the same location in all slices and scans .", "the same was done for the half thickness images . an oral and maxillofacial radiologist observed and recorded the values in all images .", "evaluation of hus in hard tissue phantom with merge e - film software in three times of scanning in full thickness phantom .", "the relationship between ct numbers on ct scans and the gray values of cbct images was studied by anova ( p<0.05 ) .", "the effects of the type of material , acquisition parameters , phantoms thickness and location of the assessed point on the phantoms were also studied ."], ["the relationship between ct numbers on ct scans and the gray values of cbct images was studied by anova ( p<0.05 ) .", "the effects of the type of material , acquisition parameters , phantoms thickness and location of the assessed point on the phantoms were also studied ."], ["in the first evaluation , the interaction effect of the variables was found to be significant using results were not the same in different conditions and each variable had to be examined separately .", "the results of anova used to compare the cbct and ct data are shown in tables 2 and 3 .", "comparison of voxel values in cbct systems with hounsfield units in ct in full thickness phantoms ns : non - significant s : significant comparison of voxel values in cbct systems with hounsfield units in ct in half thickness phantoms ns : non - significant s : significant"], ["bone quality and quantity assessment is critical for pre - implant surgeries ; thus , reliable and precise radiographic evaluations are essential . however , despite wide use of cbct in different fields of dentistry , it is still not a valid method for bone quality assessment [ 4,1416,1921 ] .", "object location in cbct systems , object volume , acquisition parameters and some other factors have been evaluated in previous studies and controversial results have been reported [ 7,8,10,11,1316 ] . in this study ,", "the diagnostic accuracy of three cbct systems was compared with that of ct as the gold standard , and the effect of four factors including different tissue phantoms , object volume size , acquisition parameters and location of the objects was assessed . as the results showed , none of the cbct systems revealed the precise bone density as the gold standard . in this study ,", "different tissue phantoms were used to evaluate diagnostic accuracy of cbct systems in different density ranges .", "review of the literature revealed that only mah et al ,   used standard phantoms .", "heterogeneity of the hard tissue and air phantoms was a limitation of our study and was due to the production process .", "they were handmade in a laboratory and even in the best conditions , porosities were seen .", "however , in the clinical setting , the tissues are not homogenous ; thus , this factor may not be a real limitation for generalization of results to the clinical setting .", "however , in this study , the results showed no significant effect of this restriction by comparing the data of the four phantoms .", "the effect of tissue thickness is one of the main subjects for researchers to asses in density studies .", "in the first phase , the complete thickness and in the second phase , half - thickness samples were evaluated , which showed no considerable effect on assessment of the tissue density .", "the results were different in the two phases and there was a significant difference in results compared with ct system .", "katsumata et al ,   showed that the thicker the tissues , the more precise the values of tissue density .", "they discussed that beam characteristics may be a more important factor than the tissue thickness , which is because of the 360-degree rotation of the system and changes in the intensity of the x - ray beam while passing through different parts of an object , which causes maximum and minimum x - ray intensities .", "considering the importance of this factor , we used different acquisition parameters to evaluate the importance of the effect of intensity of x - ray beam on tissue density . in our study , despite the changes in the values , no significant difference was shown between the various image acquisition parameters of cbct systems and all values were different from the ct numbers .", "parsa et al ,   found a significant difference between ct and cbct values and showed changes in cbct values with changes in acquisition parameters .", "gray values increased with increase in size of fov in accuitomo system but this increase was obtained with an increase in volume in newtom system .", "this discrepancy between the behaviors of the two systems could be attributed to the variability in reconstruction and post - processing methods applied by the two manufacturers .", "however , one theory suggests that the tissue density of an object is more effective than the beam intensity and characteristics in showing the actual tissue density . to evaluate and interpret the cbct and ct images", ", we used only one software which can show the same level of tissue density in both systems , the point which was confirmed by ghasemi et al . by doing so", "mah et al ,   who used 11 types of cbct systems also used only one software program ( on demand 3d ) to match the observing conditions and roi in all sections .", "this fact is important in density assessment studies and can affect the results . because of the variable geometric characteristics of different systems , the density values and the selected roi are variable in different software programs provided by the systems .", "katsumata et al ,   emphasized that the selected roi should be the same in all sections with at least 3% error . in their study , ct images were also examined by the same software program to achieve the same situation in all conditions .", "nevertheless , mah et al ,   believed that human error is inevitable in selecting the roi .", "however , no specific method for precise selection of an area in different conditions has been suggested in studies except in a study by naitoh et al ,   who emphasized on selection of equal points via observation . by choosing three different points in an object", ", we wanted to evaluate the effect of object location in the fov on tissue density .", "legravere et al ,   did not report any significant difference in different locations in the tube field in their study , which was in contrast to the results of oliveira et al ,   who noticed that the correlation between the object density and ct number in cbct systems was not uniform through the dental arch .", "we found no significant difference between different locations in the diversity between the voxel values and hus .", "hua et al ,   found that some artifacts and scatter radiation are responsible for inefficacy of cbct for density assessment , which are inevitable because of the design of cbct systems and their detectors .", "considering the inefficacy of cbct for density evaluation , the x - ray beam heterogeneity in cbct can affect hu values , and lead to absence of a clear relationship between voxel value in cbct and bone mineral density provided by dual x - ray absorptiometry .", "the artifacts such as beam hardening or heel effect can decrease the validity of these values .", "several studies mentioned the difference between the cbct values and the ct values and in most cases , the reported cbct values were higher than the ct values .", "parsa et al ,   mentioned higher gray value in their study and believed that it may be due to the increased noise level , scattering and artefacts specific to the scanning technology .", "scarf and farman   proposed that although a sort of association was seen between the provided hus by ct and voxel value in cbct , the variability in measurements by cbct was higher than that by ct .", "however , legraver et al ,   found a linear relationship in r ( coefficient of relationship between two variables ) between the values in cbct and ct , and indicated that the cbct values were generally higher . in our study , the provided values by cbct were higher than the ct values in one of the systems ( newtom vg 3d ) , which is similar to the results of legrave et al ,   who used the same system for the same purpose .", "according to this point of view , the difference between the values in our study can be the result of different levels of energy in the three systems ; because the systems with a lower power result in lower intensity of x - ray beam and subsequently less tissue penetration depth . in order to create parallel conditions between the experimental phases and the clinical setting , the suitable acquisition parameters for each system were determined by an expert operator .", "thus , the levels of kvp and mas were specific and different in the three systems .", "haristory et al ,   studied the effect of different exposure parameters on the voxel value of cbct and found a strong relationship between cbct and ct using r value .", "however , because of different results in cbct , they suggested that the use of calibration phantom is necessary before imaging to ensure accurate bone density values . despite the afore - mentioned results , some studies showed inefficacy of this system in providing accurate values of bone density and found a linear relationship", "; thus , a high correlation between the results of cbct and ct was achieved . a study confirming the optimal efficacy of cbct systems to estimate bone density introduced a conversion coefficient .", "nomura et al ,   reported a relationship between two systems in evaluating bone mineral density and voxel value , but because of the nonlinear regression obtained in their study , further studies were suggested .", "however , a linear relationship and high correlation coefficient between these values have been shown in several studies . by applying the attenuation coefficient in an equation , mah et al ,  ", "obtained the hu values of tested materials from the voxel values with only a small difference from the actual hu values .", "considering all the above , we can say that our methodology , which is one reason for the uniqueness of this study , affected the results , and our results do not completely reflect the efficacy of the systems tested . as recently stated by pauwels et al ,   it is logical to postulate that although attempts have been made to correct the gray level variability , quantitative use of values provided by cbct should be generally avoided at this time ."], ["according to the results , the cbct systems were not able to show the accurate value of tissue density and the factors such as type of tissue ( hard , soft , water , air ) , thickness ( full against half ) , image acquisition conditions ( high settings against low settings ) and object location ( middle , peripheral and intermediate ) did not affect density evaluation by cbct systems ."]]}
{"article_id": "PMC2561156", "article_text": ["damage of the cell membrane system , especially the plasma membrane , is one of the primary events in heavy metal toxic action in plants .", "disruption of membrane integrity is thought to be an effect of a complex interaction between heavy metals and functional groups of membranes .", "it is well known that metal ions are easily bound to both the sulphhydryl groups of proteins and the hydroxyl part of phospholipids ( devi and prasad , 1999 ) .", "they can also replace the calcium ions at essential sites on the cell membranes ( breckle and kahle , 1991 ) .", "all those events result in an increase of a non - specific membrane permeability and the parallel decrease of specific transporting activities which disrupt the ionic homeostasis and , subsequently , the activities of many enzymes crucial for basic cell metabolism .", "the atp - dependent proton pump of the plasma membrane has a central function in the regulation of ion homeostasis in the cytosol .", "this enzyme is encoded by a multigene family ( portillo , 2000 ; arango et al . , 2003 ) and", "the expression of at least some isogenes is differentially regulated according to tissue type and developmental stage ( oufattole et al . , 2000 ) .", "many studies have also shown changes in the gene expression of the plasma membrane h - atpase in response to a variety of environmental factors , including salt stress ( binzel , 1995 ) , dehydration ( surowy and boyer , 1991 ) , light conditions ( harms et al . , 1994 ) , mechanical stress ( oufattole et al . , 2000 ) , and externally applied hormones ( fras et al . , 1996 ) .", "increasing evidence indicates that , besides the genetic regulation of the proton pump , its activity might be fast modulated post - translationally at the protein level , mainly through reversible phosphorylation ( schaller and sussman , 1988 ; portillo , 2000 ) .", "this important role in the regulation of the plasma membrane h - atpase has its autoinhibitory domain in the c - terminal region of the enzyme ( jahn et al . ,", "1997 ; oecking et al . , 1997 ; baunsgaard et al . , 1998 ;", "the c - terminal tail interacts with the catalytic region of the enzyme , limiting its activity ( portillo , 2000 ) .", "many data have suggested that the activation of the plant plasma membrane h - atpase is mediated through phosphorylation of the thr-948 in the enzyme , which allows the binding of 14 - 3 - 3 protein ( svennelid et al . , 1999 ) .", "this causes displacement of the c - terminal tail and activates the pm h - atpase ( palmgren , 2001 ; arango et al . , 2003 ) .", "dephosphorylation of the enzyme protein , mediated by the specific phosphatases belonging to the pp2 and followed by the release of 14 - 3 - 3 protein from the complex , inactivates h - atpase . to date , data concerning heavy metal action on the plasma membrane h - atpase are very limited .", "only a few observations have indicated that enzyme activity was decreased under heavy metal stresses ( kennedy and gonsalves , 1989 ; fodor et al . , 1995 ) .", "however , no attempts were undertaken to elucidate the mechanisms involved in this inhibition . in this work ,", "to explain the mechanism of metal action on the plasma membrane proton pump , the hydrolytic and transporting activities of h - atpase were measured simultaneously with the expression of genes encoding the enzyme .", "moreover , the effect of cantharidin , a specific inhibitor of phosphatases , on the inhibitory action of metals on the h - atpase was determined .", "since the activity of the plasma membrane proton pump depends on atp , the changes in its level were measured in plants treated with metals .", "finally , the accumulation of cadmium , copper , and nickel in cucumber roots was asseyed to estimate to what degree the efficiency of metal absorption may affect the h - atpase activity .", "krak ) , germinated in darkness at 25 c for 48 h , were transferred to a nutrient medium for 6 d. the nutrient solution contained 1 mm k2so4 , 0.2 mm ca(h2po4)2.h2o ; 1.5 mm caso4.0.5h2o , 0.3 mm mgso4.7h2o , and microelements 75 m ferric citrate , 10 m mnso4.5h2o , 5 m h3bo4 , 1 m cuso4.5 h2o , 0.01 m znso4.7h2o , 0.05 m na2moo4.2h2o .", "after this the plants were exposed for 2 h to the 0.33 mm mes - naoh ( ph 5.5 ) solution without ( control ) or with 10 m cd , cu or ni and 100 m cd , cu or ni .", "the plants were grown hydroponically with a 16 h photoperiod ( 180 mol m s ) at 25 c during the day and 22 c during the night .", "plasma membrane ( pm ) vesicles were isolated from cucumber root microsomes by phase partitioning according to the procedure by larsson ( 1985 ) and modified by kobus ( 1995 ) . a 16 g phase system containing 6.2% ( w / w ) dextran t500 , 6.2% ( w / w ) polyethylene glycol 3350 ,", "330 mm sorbitol , 5 mm kcl , 5 mm btp - mes ( ph 7.5 ) was used .", "the plasma membranes obtained by this procedure were mainly composed of right - side - out vesicles and were used to determine the hydrolytic atpase activity .", "part of the vesicles was turned to the inside - out oriented form by the method of johansson et al .", "( 1995 ) and used for the measurements of atp - dependent h transport in plasma membranes .", "the hydrolytic activity of the vanadate - sensitive atpase ( pm - h - atpase ) was determined according to the procedure of gallagher and leonard ( 1982 ) , as modified by sze ( 1985 ) .", "the reaction mixture contained 50 g of protein ( plasma membrane ) , 33 mm tris - mes ( ph 7.5 ) , 3 mm atp , 2.5 mm mgso4 , 50 mm kcl , 1 mm nan3 , 0.1 mm na2moo4 , 50 mm nano3 , 200 m na3vo4 , and 0.02% triton x-100 .", "pm h - atpase activity was expressed as the difference between the activity measured in the absence and in the presence of na3vo4 .", "the pi released during the reaction was determined according to ames ( 1966 ) with 0.2% ( w / v ) sodium dodecyl sulphate included to prevent precipitation ( dulley , 1975 ) .", "h transport activity was measured spectrophotometrically as the change in acridine orange absorbance at 495 nm ( a495 ) according to kobus and buczek ( 1995 ) .", "the assay medium contained : pm vesicles ( about 50 g of protein ) , 25 mm btp - mes ( ph 7.5 ) , 330 mm sorbitol , 50 mm kcl , 0.1% bsa , 10 m acridine orange , and 0.05% brij 58 .", "proton transport was initiated by the addition of 3 mm mg - atp . for every combination , passive proton movement through the membrane", "protein was measured according to bradford ( 1976 ) in the presence of 0.02% triton x-100 with bovine serum albumin as the standard .", "determination of adenosine triphosphate ( atp ) was made according to glaab and kaiser ( 1999 ) . the fresh root material ( 1 g ) was ground in liquid nitrogen and 5 cm of 4.5% perchloric acid was added and mixed until it thawed . the mixture was then supplemented with 0.125 cm 2 mm tris and centrifuged for 5 min at 5000 g. the ph of the supernatant was adjusted to 7.4 with 5 mol dm k2co3 , and recentrifuged .", "firefly luciferin - luciferase assay using a td-20/20 luminometer ( turner designs , usa ) .", "total and symplastic metal levels were determined spectrophotometrically ( perkin - elmer 3300 ) in fresh root tissues digested with concentrated hno3 in a microwave .", "prior to the determination of the symplastic metal pool after 2 h exposure to cu , cd or ni , the roots were washed with 5 mm pbcl2 at 0 c for 30 min , in order to remove the extracellular bound ions ( harrison et al . , 1979 ) .", "the apoplastic pool was calculated from the differences between the total and the symplastic metal level .", "total rna was isolated from 50 mg of roots using tri reagent ( sigma ) . to evaluate the expression of the pm - h - atpase gene ( csha3 ) , semi - quantitative rt - pcr analysis with specific primers for each gene", "was performed ( titan one tube rt - pcr system , roche , germany ) . as an internal standard", "the following specific primers were designed for the pm - h - atpase gene ( cucumber root h - atpase csha3 , accession number ef375892 ) 5-aagtttgtggggttcatgtggaat-3 ( forward primer ) ; 5-gtaagaggaagtgactctccagtc-3 ( reverse primer ) , and for actin the primers were 5-ccgttctgtccctctacgctagtg-3 ( forward primer ) ; 5-ggaactgctctttgcagtctcgag-3 ( reverse primer ) .", "for the cdna reaction 18 cycles were run ( uno ii thermoblock , biometra , germany ) , which corresponded to the exponential phase for the pm - h - atpase and actin genes .", "each cycle was composed of a 30 s denaturation at 94 c , 30 s of annealing at 59 c , a 2 min 40 s extension at 68 c followed by 7 min of prolonged extension at 68 c .", "the gel images were digitally captured with a sony xc - st50ce camera and analysed using the biocapt version 99 program . for western blot analysis", ", 20 g of plasma membrane proteins were incubated in sds buffer containing 2% ( w / v ) sds , 80 mm dtt , 40% ( w / v ) glycerol , 5 mm pmsf , 10 mm tris , 1 mm edta , and 0.05% ( w / v ) bromophenol blue for 30 min at room temperature and separated on 7.5% sds - polyacrylamide gels ( laemmli , 1970 ) . after 1 h of electrophoresis at room temperature ( 25 ma ) proteins", "were electrotransferred at room temperature ( 60 v , 200 ma ) for 1.5 h to nitrocellulose using a sigma - aldrich sv10-eb10 blotting apparatus .", "transfer buffer contained 25 mm tris , 150 mm gly , and 10% ( v / v ) methanol .", "to identify the plasma membrane h - atpase , the blots were incubated overnight at 8 c with monoclonal antibody against plasma membrane h - atpase , ( 46e5b11d ) kindly provided by w michalke ( universitt freiburg , germany ) .", "after repeated washing the nitrocellulose membrane was incubated at room temperature for 1 h with 1:4000 diluted secondary antibody ( anti - mouse , conjugated to horseradish peroxidase , sigma - aldrich ) and visualized by staining with dab .", "phosphorylation of plasma membrane h - atpase was detected with the antibody against phosphothreonine ( rabbit polyclonal to phosphothreonine , abcam ) used at a concentration of 2 g ml after overnight incubation at 8 c .", "the membranes were rinsed and incubated for 1 h at room temperature with 5000-fold secondary antibody conjugated to horseradish peroxidase ( goat polyclonal to rabbit igg , abcam ) .", "data reported in the figures are the results from at least three experiments performed on independent preparation .", "data reported in the figures are the results from at least three experiments performed on independent preparation .", "treatment of the cucumber seedlings with heavy metals ( cd , cu and ni ) changed the hydrolytic and transporting activities of the plasma membrane h - atpase ( figs 1 , 2 ) .", "the effect of 10 m cd and cu was similar , and both metals decreased hydrolytic activity by about 30% ( fig .", "1 , white bars ) , whereas 10 m ni increased the hydrolysis of atp in plasma membrane vesicles by about 30% ( fig .", "higher concentrations of every metal ( 100 m ) in nutrient solution caused the distinct inhibition of h - atpase activity ( fig .", "the greatest inhibition of atp hydrolysis was observed in plasma membranes isolated from cucumber roots treated with cd ( about 60% ) , while in the case of cu and ni it was 45% and 20% , respectively .", "all metals at both concentrations affected the transporting activity of the plasma membrane proton pump in a similar manner ( fig .", "effect of cd , cu and ni on the hydrolytic activity of h - atpase in plasma membrane vesicles .", "the plasma membrane was isolated from the control roots ( control ) , and roots treated for 2 h with 10 m metals ( white bars ) or with 100 m metals ( grey bars ) .", "hydrolytic activity of h - atpase was measured as described in the materials and methods .", "effect of cd , cu and ni on the proton transport activities measured in the plasma membrane vesicles .", "the plasma membranes ( 50 g of protein ) were isolated from control roots ( control ) , roots treated for 2 h with 10 m ( a , open symbols ) or with 100 m ( b , closed symbols ) of cd , cu or ni . after equilibration of membranes with the reaction medium ( at least for 5 min ) , vesicle acidification was initiated by the addition of mg - atp to give a final concentration of 3 mm .", "the formation of ph gradient in the vesicles was monitored as the changes in acridine orange absorbance ( a495 ) .", "the values presented are representative for the results obtained in three independent experiments with each experiment done in triplicate .", "the results in the inner diagrams ( the steady - state of h - transport ) are means sd from those three independent experiments . the effect of metals on the plasma membrane proton pump could comprise the expression levels of a gene ( mrna ) as well as the enzyme protein level . to verify whether an alteration of enzyme activity under heavy metals was caused by changes in gene expression", ", the level of specific transcript of cucumis sativus plasma membrane atpase ( genbank accession number ef375892 ) was determined using rt - pcr approaches .", "the transcript level of the constitutively expressed actin gene was used as the internal standard .", "3a , c ) and as a ratio of the pm - h - atpase gene signal in the selected line compared with the actin gene signal ( fig .", "transcript levels of the csha3 gene in roots treated with 10 m cd , cu and ni were similar to the control , indicating that the metal effect on plasma membrane h - atpase in our short - term experiments did not involve the gene level ( fig .", "also , higher concentrations ( 100 m ) of cu and ni did not affect the expression of the csha3 gene , whereas in roots treated with 100 m cd the level of the transcript markedly declined ( fig .", "expression of the pm - h - atpase gene in cucumber roots treated with 10 m cd , cu or ni ( a , b ) and 100 m cd , cu or ni ( c , d ) .", "total rna was isolated from cucumber roots untreated ( control , white bar ) or treated for 2 h with 10 or 100 m heavy metals ( grey bars ) . to evaluate the expression of pm - h - atpase and actin ( internal standard ) genes , semi - quantitative rt - pcr ( titan one tube rt - pcr system , roche ) with specific primers for each gene was performed .", "ethidium bromide - stained bands for the atpase transcripts were quantified with respect to the band of actin .", "rt - pcr results are presented as the gel image ( a , c ) and as a ratio of pm - h - atpase gene signal in the selected line to the actin gene signal ( b , d ) .", "all experiments were repeated three times independently with comparable results ( sd ) . since the activity of h - atpase", "can also be rapidly changed through reversible protein phosphorylation , it was tested if cantharidin , the specific inhibitor of protein phospatases ( pp2a , pp1 ) , could affect the inhibitory effect of metals on the enzyme activity .", "when cucumber plants were exposed to 100 m cd , cu and ni in the presence of cantharidin the metal - dependent inhibition was partially diminished ( fig .", "4 ) suggesting that the effect of metals could involve dephosphorylation of h - atpase protein .", "effect of metals and cantharidin on the hydrolytic activity of h - atpase in plasma membranes of cucumber roots .", "the plasma membranes were isolated from the control roots ( control ) , from roots treated for 2 h with 100 m cd , cu and ni , ( white bars ) or from roots treated for 2 h with metals and 50 m cantharidin ( grey bars ) . hydrolytic activity of h - atpase was measured as described in the materials and methods .", "data are expressed as a percentage of the enzyme activity determined in plasma membranes of control roots .", "the differences in phosphorylation of plasma membrane h - atpase after metal treatment of plants ( 2 h with 100 m cd , cu and ni ) were also confirmed with the antibody against phosphothreonine ( fig .", "5b ) . to verify the specificity of the antibody against phosphoamino acids , a control with antibody against plasma membrane h - atpase , 46e5b11d , was used ( fig .", "figure 5b shows that the phosphorylation of plasma membrane atpase decreased in plants treated with heavy metals compared with the control .", "this result strongly indicates that , under heavy metal stress , dephosphorylation of plasma membrane h - atpase occurs .", "western blot of plasma membrane protein ( obtained from control plants or plants treated with 100 m cd , cu , ni ) with antibodies raised against plasma membrane h - atpase -46e5b11d ( a ) , and phosphothreonine ( b ) .", "therefore the amount of atp in tissues could be one of the factors limiting an active phospho - enzyme level .", "for that reason , atp levels in cucumber roots treated for 2 h with heavy metals were also measured .", "in general , atp contents in roots of plants stressed with every metal were lower than in the control ( fig . 6 ) .", "plants were exposed for 2 h to the mes - naoh solution without ( control , white bar ) or with 10 m cd , cu or ni ( grey bars ) and 100 m cd , cu or ni ( black bars ) .", "the data represent an average ( sd ) of three experiments ; each assay was performed three times .", "plasma membrane functions are rapidly altered by heavy metals present in the environment at high concentrations .", "the first diagnostic symptom of membrane damage by heavy metals is an increase in its permeability ( fodor et al . , 1995 ) with a subsequent disturbance in the ionic balance of the cell .", "it is mainly caused by metal - induced changes in the composition of the membrane lipids and the saturation of fatty acids ( de vos et al . , 1993 ) .", "however , the action of metals is much more complex and comprise various events including the oxidation and cross - linking of protein thiols resulting in an inhibition of the key membrane proteins ( harms et al . , 1994 ) .", "one of the membrane enzymes which is altered in plants stressed with metals seems to be the h - atpase , the only proton pump operating in plasma membranes and playing a central role in the regulation of ion homeostasis .", "it was shown that the hydrolytic activity of h - atpase in roots of different plants was inhibited by cd ( kennedy and gonsalves , 1989 ; fodor et al . , 1995 ) as well as cu ( burzyski and kolano , 2003 ) .", "the short - term treatment of cucumber with cd or cu reduced the hydrolysis of atp in the plasma membranes of root cells ( fig .", "moreover , the atp - dependent proton transport in membranes isolated from roots stressed with metals was also inhibited in a similar manner ( fig .", "the effect of metals on both hydrolytic and transporting activities was dependent on their concentrations in the nutrient solution and correlated well with an accumulation in the symplast of the cells ( table 1 ) .", "a similar effect was observed in roots treated with higher concentration of ni ( 100 m ) , whereas lower concentrations of the metal slightly elevated both activities of the plasma membrane proton pump .", "comparable observations were made earlier by burzyski and buczek ( 1994 ) and by ros et al .", "( 1992 ) for the plasma membrane atpase in cucumber and rice grown with nickel .", "interestingly , the action of nickel on the proton pumps was parallel to its effect on plant growth : low concentration of ni stimulated the growth of various crop species , while higher concentrations of the metal inhibited the growth of plants ( brown et al .", "since one of the main function of plasma membrane h - atpase is the regulation of growth ( rayle and cleland , 1992 ) , it could be suggested that the enhanced growth of plants subjected to low concentrations of ni and the lowered growth in the presence of higher concentrations of ni is , at least partially , a result of the modification of plasma membrane h - atpase activity by nickel .", "( 2005 ) also observed the increase of plasma membrane h - atpase activity when plants were subjected to another metal  aluminium .", "results from real - time rt - pcr and immunodetection analysis indicated that the stimulation of the plasma membrane proton pump activity by al , in soybean roots , is caused by transcriptional and translational regulation .", "total , symplastic and apoplastic metal levels in root tissues after 2 h of cucumber exposition to cd , cu or ni 10 seedlings were incubated in 50 cm of 10 or 100 mol dm of metal chloride solutions ( ph 5.5 ) .", "cd , cu or ni levels were determined as described in the materials and methods .", "as mentioned above , the effect of metals on the plasma membrane proton pump was correlated with their concentration in the nutrient solution . however ,", "possible reasons for the differential action of the metals on enzyme activity , in spite of similarities in their uptake , is not clear . the strongest inhibition of h - atpase was obtained after treatment of plants with cadmium .", "there is no correlation between enzyme protein inhibition and lewis acidity of metal ions or when the inhibition by metals is compared with the various ligands ( hydroxyl , carboxyl , phosphoryl , amino , sulphydryl ) present in cells ( filippis , 1979 ) .", "heavy metal toxicity can be due to membrane distortion as shown earlier by the altered lipid content ( burzyski and kolano , 2003 ) .", "1995 ) have indicated that saturation of fatty acid in the membrane of plants growing with metals increased , whereas the level of sterols significantly decreased . since lipid composition and membrane fluidity", "are considered as significant factors regulating the plasma membrane h - atpase ( hernandez et al . , 2002 ) , an inactivation of the proton pump could be the result of the metal - induced changes in the plasma membrane .", "inhibition of the plasma membrane proton pump in root cells under heavy metal stress could also result from alteration at the transcriptional as well as the post - transcriptional level .", "the plasma membrane h - atpase is encoded by a multigene family and it is generally accepted that the expression of at least some of them is differentially regulated in response to a variety of environmental stresses .", "thus it could be assumed that heavy metals also affect the activity of the proton pump through an alteration of specific gene expression .", "however , using genetic approaches , it has been proved that , among the metals used in these experiments , only cadmium markedly decreased the level of csha3 mrna .", "those observations are in agreement with other findings that show that cd is a very effective modulator ( up- and down - regulation ) of plant gene expression ( fusco et al . , 2005 ; kovalchuk et al . ,", "( 2005 ) analysed the global genome expression in plants exposed to cd and have shown that there are at least 65 up - regulated and 338 down - regulated genes .", "also in brassica juncea l. , known as a hyperaccumulator plant , 73 transcript - derived fragments were identified as cd - responsive ( fusco et al . , 2005 ) .", "among them 52 genes have been identified as encoding proteins with varied physiological functions ( transcriptional factors , expression regulators , stress - responsive proteins , and proteins involved in general metabolism ) .", "such results indicate that the response of plants to cd stress at the genetic level is very broad .", "the other metals used in our experiments ( cu and ni ) did not change the level of plasma membrane h - atpase transcripts in cucumber roots ( fig .", "thus the alteration of the plasma membrane proton pump activity in cucumber roots stressed with those metals seem not to involve the gene expression level .", "however , h - atpase is encoded by a gene family and only one isoform was analysed at the transcript level .", "evidence has been presented that the plasma membrane h - atpase activity is rapidly modulated through the phosphorylation / dephosphorylation mechanism ( schaller and sussman , 1988 ; kobus and janicka - russak , 2004 , janicka - russak and kobus , 2006 ) .", "the activation of the enzyme is due to its phosphorylation followed by binding of 14 - 3 - 3 protein , whereas the dephosphorylation of h - atpase by specific phosphatases belonging to the pp2 , and the release of the 14 - 3 - 3 protein from the complex , inactivates the enzyme . to determine if the metal - induced inhibition of the plasma membrane proton pump in cucumber roots was attributed to its post - translational modification , the effect on the membrane proton pump of cantharidin , a specific inhibitor of protein phosphatases ,", "as shown , cantharidin applied together with 100 m cd , cu and ni distinctly diminished the inhibitory effect of the metals ( fig .", "4 ) , suggesting that the metals altered the proton pump activity mainly via dephosphorylation of its protein .", "the level of h - atpase phosporylation was distinctly lower in plasma membranes from cucumber roots treated with cd , cu or ni ( fig .", "5 ) demonstrating that alteration of the plasma membrane proton pump under heavy metals is due to dephosphorylation of the enzyme protein . considering that our plants ( control plants and those treated with heavy metals ) were exposed for 2 h to mes - naoh without ca in solution , a set of supplementary experiments with plants exposed to metals in mes - naoh with or without 200 m ca ( caso4 )", "nevertheless no differences were found in both the hydrolytic and the transporting activity of the h - atpase .", "also western blot analysis of plasma membranes with the antibody against phosphothreonine showed that the presence of calcium in the solution did not affect the metal action on h - atpase phosphorylation in the short - term experiments presented in this paper ( data not shown ) .", "as protein phosphorylation depends on the energetic status of the cell , the level of endogenous atp was measured .", "each metal treatment decreased the amount of the adenosine triphosphate in root tissues , suggesting that , besides the dephosphorylation intensity of the proton pump , the atp level is also an important factor in enzyme deactivation under heavy metals stresses .", "it is known that the main source of atp in root tissues is mitochondrial respiration . until now", ", the effect of heavy metals on respiration and atp content in plant cells has not been sufficiently studied .", "however , it was revealed that cd treatment of plants significantly reduced the oxygen consumption by roots ( reese and roberts , 1985 ) as well as disordering the electron transport chain ( kessler and brand , 1994 ) .", "thus it is suspected that altered respiration could enhance the inhibition of plasma membrane proton pump activity in plants treated with metals .", "there is the possible action of heavy metals on atpase activity through decreasing the energy charge of the tissue .", "such a correlation is indicated since the endogenous amount of atp , the substrate for plasma membrane h - atpase , decreased under heavy metal stress .", "in addition , it is well known that endogenous atp influences atpase activity , not only as the substrate but also through its function as an activator .", "since protein phosphorylation depends on the endogenous atp level , its reduction upon the addition of heavy metals is in line with our hypothesis .", "data of experiments with cantharidin and western blot with antibodies against phosphothreonine support this supposition .", "we think that some changes in plant cells initiated under heavy metals lead to an increase in the activity of specific phosphatases , responsible for the dephosphorylation of h - atpase protein . taken together", ", the data presented suggest that alterations of the plasma membrane proton pump under heavy metal stresses are mainly due to the post - translational modification of its protein . only in the case of cadmium action ,"], "abstract_text": ["<S> the effect of heavy metals on the modification of plasma membrane h+-atpase ( ec 3.6.3.14 ) activity in cucumber roots was studied . in plants stressed for 2 h with 10 m or 100 m cd , cu or ni the hydrolytic as well as </S>", "<S> the transporting activity of h+-atpase in the plasma membranes of root cells was decreased . </S>", "<S> transcript levels of cucumis sativus plasma membrane h+-atpase in roots treated with 10 m cd , cu , or ni as well as with 100 m cu or ni were similar to the control , indicating that the action of metals did not involve the gene expression level . only in roots exposed to 100 m cd was the level of plasma membrane h+-atpase mrna markedly decreased . </S>", "<S> the inhibition of the plasma membrane proton pump caused by 100 m cd , cu and ni was partially diminished in the presence of cantharidin , a specific inhibitor of protein phosphatases . </S>", "<S> western blot analysis with the antibody against phosphothreonine confirmed that decreased activity of plasma membrane h+-atpase under heavy metals resulted from dephosphorylation of the enzyme protein . </S>", "<S> taken together , these data strongly indicated that alteration of the enzyme under heavy metal stresses was mainly due to the post - translational modification of its proteins in short - term experiments . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Statistics", "Results", "Discussion"], "sections": [["damage of the cell membrane system , especially the plasma membrane , is one of the primary events in heavy metal toxic action in plants .", "disruption of membrane integrity is thought to be an effect of a complex interaction between heavy metals and functional groups of membranes .", "it is well known that metal ions are easily bound to both the sulphhydryl groups of proteins and the hydroxyl part of phospholipids ( devi and prasad , 1999 ) .", "they can also replace the calcium ions at essential sites on the cell membranes ( breckle and kahle , 1991 ) .", "all those events result in an increase of a non - specific membrane permeability and the parallel decrease of specific transporting activities which disrupt the ionic homeostasis and , subsequently , the activities of many enzymes crucial for basic cell metabolism .", "the atp - dependent proton pump of the plasma membrane has a central function in the regulation of ion homeostasis in the cytosol .", "this enzyme is encoded by a multigene family ( portillo , 2000 ; arango et al . , 2003 ) and", "the expression of at least some isogenes is differentially regulated according to tissue type and developmental stage ( oufattole et al . , 2000 ) .", "many studies have also shown changes in the gene expression of the plasma membrane h - atpase in response to a variety of environmental factors , including salt stress ( binzel , 1995 ) , dehydration ( surowy and boyer , 1991 ) , light conditions ( harms et al . , 1994 ) , mechanical stress ( oufattole et al . , 2000 ) , and externally applied hormones ( fras et al . , 1996 ) .", "increasing evidence indicates that , besides the genetic regulation of the proton pump , its activity might be fast modulated post - translationally at the protein level , mainly through reversible phosphorylation ( schaller and sussman , 1988 ; portillo , 2000 ) .", "this important role in the regulation of the plasma membrane h - atpase has its autoinhibitory domain in the c - terminal region of the enzyme ( jahn et al . ,", "1997 ; oecking et al . , 1997 ; baunsgaard et al . , 1998 ;", "the c - terminal tail interacts with the catalytic region of the enzyme , limiting its activity ( portillo , 2000 ) .", "many data have suggested that the activation of the plant plasma membrane h - atpase is mediated through phosphorylation of the thr-948 in the enzyme , which allows the binding of 14 - 3 - 3 protein ( svennelid et al . , 1999 ) .", "this causes displacement of the c - terminal tail and activates the pm h - atpase ( palmgren , 2001 ; arango et al . , 2003 ) .", "dephosphorylation of the enzyme protein , mediated by the specific phosphatases belonging to the pp2 and followed by the release of 14 - 3 - 3 protein from the complex , inactivates h - atpase . to date , data concerning heavy metal action on the plasma membrane h - atpase are very limited .", "only a few observations have indicated that enzyme activity was decreased under heavy metal stresses ( kennedy and gonsalves , 1989 ; fodor et al . , 1995 ) .", "however , no attempts were undertaken to elucidate the mechanisms involved in this inhibition . in this work ,", "to explain the mechanism of metal action on the plasma membrane proton pump , the hydrolytic and transporting activities of h - atpase were measured simultaneously with the expression of genes encoding the enzyme .", "moreover , the effect of cantharidin , a specific inhibitor of phosphatases , on the inhibitory action of metals on the h - atpase was determined .", "since the activity of the plasma membrane proton pump depends on atp , the changes in its level were measured in plants treated with metals .", "finally , the accumulation of cadmium , copper , and nickel in cucumber roots was asseyed to estimate to what degree the efficiency of metal absorption may affect the h - atpase activity ."], ["krak ) , germinated in darkness at 25 c for 48 h , were transferred to a nutrient medium for 6 d. the nutrient solution contained 1 mm k2so4 , 0.2 mm ca(h2po4)2.h2o ; 1.5 mm caso4.0.5h2o , 0.3 mm mgso4.7h2o , and microelements 75 m ferric citrate , 10 m mnso4.5h2o , 5 m h3bo4 , 1 m cuso4.5 h2o , 0.01 m znso4.7h2o , 0.05 m na2moo4.2h2o .", "after this the plants were exposed for 2 h to the 0.33 mm mes - naoh ( ph 5.5 ) solution without ( control ) or with 10 m cd , cu or ni and 100 m cd , cu or ni .", "the plants were grown hydroponically with a 16 h photoperiod ( 180 mol m s ) at 25 c during the day and 22 c during the night .", "plasma membrane ( pm ) vesicles were isolated from cucumber root microsomes by phase partitioning according to the procedure by larsson ( 1985 ) and modified by kobus ( 1995 ) . a 16 g phase system containing 6.2% ( w / w ) dextran t500 , 6.2% ( w / w ) polyethylene glycol 3350 ,", "330 mm sorbitol , 5 mm kcl , 5 mm btp - mes ( ph 7.5 ) was used .", "the plasma membranes obtained by this procedure were mainly composed of right - side - out vesicles and were used to determine the hydrolytic atpase activity .", "part of the vesicles was turned to the inside - out oriented form by the method of johansson et al .", "( 1995 ) and used for the measurements of atp - dependent h transport in plasma membranes .", "the hydrolytic activity of the vanadate - sensitive atpase ( pm - h - atpase ) was determined according to the procedure of gallagher and leonard ( 1982 ) , as modified by sze ( 1985 ) .", "the reaction mixture contained 50 g of protein ( plasma membrane ) , 33 mm tris - mes ( ph 7.5 ) , 3 mm atp , 2.5 mm mgso4 , 50 mm kcl , 1 mm nan3 , 0.1 mm na2moo4 , 50 mm nano3 , 200 m na3vo4 , and 0.02% triton x-100 .", "pm h - atpase activity was expressed as the difference between the activity measured in the absence and in the presence of na3vo4 .", "the pi released during the reaction was determined according to ames ( 1966 ) with 0.2% ( w / v ) sodium dodecyl sulphate included to prevent precipitation ( dulley , 1975 ) .", "h transport activity was measured spectrophotometrically as the change in acridine orange absorbance at 495 nm ( a495 ) according to kobus and buczek ( 1995 ) .", "the assay medium contained : pm vesicles ( about 50 g of protein ) , 25 mm btp - mes ( ph 7.5 ) , 330 mm sorbitol , 50 mm kcl , 0.1% bsa , 10 m acridine orange , and 0.05% brij 58 .", "proton transport was initiated by the addition of 3 mm mg - atp . for every combination , passive proton movement through the membrane", "protein was measured according to bradford ( 1976 ) in the presence of 0.02% triton x-100 with bovine serum albumin as the standard .", "determination of adenosine triphosphate ( atp ) was made according to glaab and kaiser ( 1999 ) . the fresh root material ( 1 g ) was ground in liquid nitrogen and 5 cm of 4.5% perchloric acid was added and mixed until it thawed . the mixture was then supplemented with 0.125 cm 2 mm tris and centrifuged for 5 min at 5000 g. the ph of the supernatant was adjusted to 7.4 with 5 mol dm k2co3 , and recentrifuged .", "firefly luciferin - luciferase assay using a td-20/20 luminometer ( turner designs , usa ) .", "total and symplastic metal levels were determined spectrophotometrically ( perkin - elmer 3300 ) in fresh root tissues digested with concentrated hno3 in a microwave .", "prior to the determination of the symplastic metal pool after 2 h exposure to cu , cd or ni , the roots were washed with 5 mm pbcl2 at 0 c for 30 min , in order to remove the extracellular bound ions ( harrison et al . , 1979 ) .", "the apoplastic pool was calculated from the differences between the total and the symplastic metal level .", "total rna was isolated from 50 mg of roots using tri reagent ( sigma ) . to evaluate the expression of the pm - h - atpase gene ( csha3 ) , semi - quantitative rt - pcr analysis with specific primers for each gene", "was performed ( titan one tube rt - pcr system , roche , germany ) . as an internal standard", "the following specific primers were designed for the pm - h - atpase gene ( cucumber root h - atpase csha3 , accession number ef375892 ) 5-aagtttgtggggttcatgtggaat-3 ( forward primer ) ; 5-gtaagaggaagtgactctccagtc-3 ( reverse primer ) , and for actin the primers were 5-ccgttctgtccctctacgctagtg-3 ( forward primer ) ; 5-ggaactgctctttgcagtctcgag-3 ( reverse primer ) .", "for the cdna reaction 18 cycles were run ( uno ii thermoblock , biometra , germany ) , which corresponded to the exponential phase for the pm - h - atpase and actin genes .", "each cycle was composed of a 30 s denaturation at 94 c , 30 s of annealing at 59 c , a 2 min 40 s extension at 68 c followed by 7 min of prolonged extension at 68 c .", "the gel images were digitally captured with a sony xc - st50ce camera and analysed using the biocapt version 99 program . for western blot analysis", ", 20 g of plasma membrane proteins were incubated in sds buffer containing 2% ( w / v ) sds , 80 mm dtt , 40% ( w / v ) glycerol , 5 mm pmsf , 10 mm tris , 1 mm edta , and 0.05% ( w / v ) bromophenol blue for 30 min at room temperature and separated on 7.5% sds - polyacrylamide gels ( laemmli , 1970 ) . after 1 h of electrophoresis at room temperature ( 25 ma ) proteins", "were electrotransferred at room temperature ( 60 v , 200 ma ) for 1.5 h to nitrocellulose using a sigma - aldrich sv10-eb10 blotting apparatus .", "transfer buffer contained 25 mm tris , 150 mm gly , and 10% ( v / v ) methanol .", "to identify the plasma membrane h - atpase , the blots were incubated overnight at 8 c with monoclonal antibody against plasma membrane h - atpase , ( 46e5b11d ) kindly provided by w michalke ( universitt freiburg , germany ) .", "after repeated washing the nitrocellulose membrane was incubated at room temperature for 1 h with 1:4000 diluted secondary antibody ( anti - mouse , conjugated to horseradish peroxidase , sigma - aldrich ) and visualized by staining with dab .", "phosphorylation of plasma membrane h - atpase was detected with the antibody against phosphothreonine ( rabbit polyclonal to phosphothreonine , abcam ) used at a concentration of 2 g ml after overnight incubation at 8 c .", "the membranes were rinsed and incubated for 1 h at room temperature with 5000-fold secondary antibody conjugated to horseradish peroxidase ( goat polyclonal to rabbit igg , abcam ) .", "data reported in the figures are the results from at least three experiments performed on independent preparation ."], ["data reported in the figures are the results from at least three experiments performed on independent preparation ."], ["treatment of the cucumber seedlings with heavy metals ( cd , cu and ni ) changed the hydrolytic and transporting activities of the plasma membrane h - atpase ( figs 1 , 2 ) .", "the effect of 10 m cd and cu was similar , and both metals decreased hydrolytic activity by about 30% ( fig .", "1 , white bars ) , whereas 10 m ni increased the hydrolysis of atp in plasma membrane vesicles by about 30% ( fig .", "higher concentrations of every metal ( 100 m ) in nutrient solution caused the distinct inhibition of h - atpase activity ( fig .", "the greatest inhibition of atp hydrolysis was observed in plasma membranes isolated from cucumber roots treated with cd ( about 60% ) , while in the case of cu and ni it was 45% and 20% , respectively .", "all metals at both concentrations affected the transporting activity of the plasma membrane proton pump in a similar manner ( fig .", "effect of cd , cu and ni on the hydrolytic activity of h - atpase in plasma membrane vesicles .", "the plasma membrane was isolated from the control roots ( control ) , and roots treated for 2 h with 10 m metals ( white bars ) or with 100 m metals ( grey bars ) .", "hydrolytic activity of h - atpase was measured as described in the materials and methods .", "effect of cd , cu and ni on the proton transport activities measured in the plasma membrane vesicles .", "the plasma membranes ( 50 g of protein ) were isolated from control roots ( control ) , roots treated for 2 h with 10 m ( a , open symbols ) or with 100 m ( b , closed symbols ) of cd , cu or ni . after equilibration of membranes with the reaction medium ( at least for 5 min ) , vesicle acidification was initiated by the addition of mg - atp to give a final concentration of 3 mm .", "the formation of ph gradient in the vesicles was monitored as the changes in acridine orange absorbance ( a495 ) .", "the values presented are representative for the results obtained in three independent experiments with each experiment done in triplicate .", "the results in the inner diagrams ( the steady - state of h - transport ) are means sd from those three independent experiments . the effect of metals on the plasma membrane proton pump could comprise the expression levels of a gene ( mrna ) as well as the enzyme protein level . to verify whether an alteration of enzyme activity under heavy metals was caused by changes in gene expression", ", the level of specific transcript of cucumis sativus plasma membrane atpase ( genbank accession number ef375892 ) was determined using rt - pcr approaches .", "the transcript level of the constitutively expressed actin gene was used as the internal standard .", "3a , c ) and as a ratio of the pm - h - atpase gene signal in the selected line compared with the actin gene signal ( fig .", "transcript levels of the csha3 gene in roots treated with 10 m cd , cu and ni were similar to the control , indicating that the metal effect on plasma membrane h - atpase in our short - term experiments did not involve the gene level ( fig .", "also , higher concentrations ( 100 m ) of cu and ni did not affect the expression of the csha3 gene , whereas in roots treated with 100 m cd the level of the transcript markedly declined ( fig .", "expression of the pm - h - atpase gene in cucumber roots treated with 10 m cd , cu or ni ( a , b ) and 100 m cd , cu or ni ( c , d ) .", "total rna was isolated from cucumber roots untreated ( control , white bar ) or treated for 2 h with 10 or 100 m heavy metals ( grey bars ) . to evaluate the expression of pm - h - atpase and actin ( internal standard ) genes , semi - quantitative rt - pcr ( titan one tube rt - pcr system , roche ) with specific primers for each gene was performed .", "ethidium bromide - stained bands for the atpase transcripts were quantified with respect to the band of actin .", "rt - pcr results are presented as the gel image ( a , c ) and as a ratio of pm - h - atpase gene signal in the selected line to the actin gene signal ( b , d ) .", "all experiments were repeated three times independently with comparable results ( sd ) . since the activity of h - atpase", "can also be rapidly changed through reversible protein phosphorylation , it was tested if cantharidin , the specific inhibitor of protein phospatases ( pp2a , pp1 ) , could affect the inhibitory effect of metals on the enzyme activity .", "when cucumber plants were exposed to 100 m cd , cu and ni in the presence of cantharidin the metal - dependent inhibition was partially diminished ( fig .", "4 ) suggesting that the effect of metals could involve dephosphorylation of h - atpase protein .", "effect of metals and cantharidin on the hydrolytic activity of h - atpase in plasma membranes of cucumber roots .", "the plasma membranes were isolated from the control roots ( control ) , from roots treated for 2 h with 100 m cd , cu and ni , ( white bars ) or from roots treated for 2 h with metals and 50 m cantharidin ( grey bars ) . hydrolytic activity of h - atpase was measured as described in the materials and methods .", "data are expressed as a percentage of the enzyme activity determined in plasma membranes of control roots .", "the differences in phosphorylation of plasma membrane h - atpase after metal treatment of plants ( 2 h with 100 m cd , cu and ni ) were also confirmed with the antibody against phosphothreonine ( fig .", "5b ) . to verify the specificity of the antibody against phosphoamino acids , a control with antibody against plasma membrane h - atpase , 46e5b11d , was used ( fig .", "figure 5b shows that the phosphorylation of plasma membrane atpase decreased in plants treated with heavy metals compared with the control .", "this result strongly indicates that , under heavy metal stress , dephosphorylation of plasma membrane h - atpase occurs .", "western blot of plasma membrane protein ( obtained from control plants or plants treated with 100 m cd , cu , ni ) with antibodies raised against plasma membrane h - atpase -46e5b11d ( a ) , and phosphothreonine ( b ) .", "therefore the amount of atp in tissues could be one of the factors limiting an active phospho - enzyme level .", "for that reason , atp levels in cucumber roots treated for 2 h with heavy metals were also measured .", "in general , atp contents in roots of plants stressed with every metal were lower than in the control ( fig . 6 ) .", "plants were exposed for 2 h to the mes - naoh solution without ( control , white bar ) or with 10 m cd , cu or ni ( grey bars ) and 100 m cd , cu or ni ( black bars ) .", "the data represent an average ( sd ) of three experiments ; each assay was performed three times ."], ["plasma membrane functions are rapidly altered by heavy metals present in the environment at high concentrations .", "the first diagnostic symptom of membrane damage by heavy metals is an increase in its permeability ( fodor et al . , 1995 ) with a subsequent disturbance in the ionic balance of the cell .", "it is mainly caused by metal - induced changes in the composition of the membrane lipids and the saturation of fatty acids ( de vos et al . , 1993 ) .", "however , the action of metals is much more complex and comprise various events including the oxidation and cross - linking of protein thiols resulting in an inhibition of the key membrane proteins ( harms et al . , 1994 ) .", "one of the membrane enzymes which is altered in plants stressed with metals seems to be the h - atpase , the only proton pump operating in plasma membranes and playing a central role in the regulation of ion homeostasis .", "it was shown that the hydrolytic activity of h - atpase in roots of different plants was inhibited by cd ( kennedy and gonsalves , 1989 ; fodor et al . , 1995 ) as well as cu ( burzyski and kolano , 2003 ) .", "the short - term treatment of cucumber with cd or cu reduced the hydrolysis of atp in the plasma membranes of root cells ( fig .", "moreover , the atp - dependent proton transport in membranes isolated from roots stressed with metals was also inhibited in a similar manner ( fig .", "the effect of metals on both hydrolytic and transporting activities was dependent on their concentrations in the nutrient solution and correlated well with an accumulation in the symplast of the cells ( table 1 ) .", "a similar effect was observed in roots treated with higher concentration of ni ( 100 m ) , whereas lower concentrations of the metal slightly elevated both activities of the plasma membrane proton pump .", "comparable observations were made earlier by burzyski and buczek ( 1994 ) and by ros et al .", "( 1992 ) for the plasma membrane atpase in cucumber and rice grown with nickel .", "interestingly , the action of nickel on the proton pumps was parallel to its effect on plant growth : low concentration of ni stimulated the growth of various crop species , while higher concentrations of the metal inhibited the growth of plants ( brown et al .", "since one of the main function of plasma membrane h - atpase is the regulation of growth ( rayle and cleland , 1992 ) , it could be suggested that the enhanced growth of plants subjected to low concentrations of ni and the lowered growth in the presence of higher concentrations of ni is , at least partially , a result of the modification of plasma membrane h - atpase activity by nickel .", "( 2005 ) also observed the increase of plasma membrane h - atpase activity when plants were subjected to another metal  aluminium .", "results from real - time rt - pcr and immunodetection analysis indicated that the stimulation of the plasma membrane proton pump activity by al , in soybean roots , is caused by transcriptional and translational regulation .", "total , symplastic and apoplastic metal levels in root tissues after 2 h of cucumber exposition to cd , cu or ni 10 seedlings were incubated in 50 cm of 10 or 100 mol dm of metal chloride solutions ( ph 5.5 ) .", "cd , cu or ni levels were determined as described in the materials and methods .", "as mentioned above , the effect of metals on the plasma membrane proton pump was correlated with their concentration in the nutrient solution . however ,", "possible reasons for the differential action of the metals on enzyme activity , in spite of similarities in their uptake , is not clear . the strongest inhibition of h - atpase was obtained after treatment of plants with cadmium .", "there is no correlation between enzyme protein inhibition and lewis acidity of metal ions or when the inhibition by metals is compared with the various ligands ( hydroxyl , carboxyl , phosphoryl , amino , sulphydryl ) present in cells ( filippis , 1979 ) .", "heavy metal toxicity can be due to membrane distortion as shown earlier by the altered lipid content ( burzyski and kolano , 2003 ) .", "1995 ) have indicated that saturation of fatty acid in the membrane of plants growing with metals increased , whereas the level of sterols significantly decreased . since lipid composition and membrane fluidity", "are considered as significant factors regulating the plasma membrane h - atpase ( hernandez et al . , 2002 ) , an inactivation of the proton pump could be the result of the metal - induced changes in the plasma membrane .", "inhibition of the plasma membrane proton pump in root cells under heavy metal stress could also result from alteration at the transcriptional as well as the post - transcriptional level .", "the plasma membrane h - atpase is encoded by a multigene family and it is generally accepted that the expression of at least some of them is differentially regulated in response to a variety of environmental stresses .", "thus it could be assumed that heavy metals also affect the activity of the proton pump through an alteration of specific gene expression .", "however , using genetic approaches , it has been proved that , among the metals used in these experiments , only cadmium markedly decreased the level of csha3 mrna .", "those observations are in agreement with other findings that show that cd is a very effective modulator ( up- and down - regulation ) of plant gene expression ( fusco et al . , 2005 ; kovalchuk et al . ,", "( 2005 ) analysed the global genome expression in plants exposed to cd and have shown that there are at least 65 up - regulated and 338 down - regulated genes .", "also in brassica juncea l. , known as a hyperaccumulator plant , 73 transcript - derived fragments were identified as cd - responsive ( fusco et al . , 2005 ) .", "among them 52 genes have been identified as encoding proteins with varied physiological functions ( transcriptional factors , expression regulators , stress - responsive proteins , and proteins involved in general metabolism ) .", "such results indicate that the response of plants to cd stress at the genetic level is very broad .", "the other metals used in our experiments ( cu and ni ) did not change the level of plasma membrane h - atpase transcripts in cucumber roots ( fig .", "thus the alteration of the plasma membrane proton pump activity in cucumber roots stressed with those metals seem not to involve the gene expression level .", "however , h - atpase is encoded by a gene family and only one isoform was analysed at the transcript level .", "evidence has been presented that the plasma membrane h - atpase activity is rapidly modulated through the phosphorylation / dephosphorylation mechanism ( schaller and sussman , 1988 ; kobus and janicka - russak , 2004 , janicka - russak and kobus , 2006 ) .", "the activation of the enzyme is due to its phosphorylation followed by binding of 14 - 3 - 3 protein , whereas the dephosphorylation of h - atpase by specific phosphatases belonging to the pp2 , and the release of the 14 - 3 - 3 protein from the complex , inactivates the enzyme . to determine if the metal - induced inhibition of the plasma membrane proton pump in cucumber roots was attributed to its post - translational modification , the effect on the membrane proton pump of cantharidin , a specific inhibitor of protein phosphatases ,", "as shown , cantharidin applied together with 100 m cd , cu and ni distinctly diminished the inhibitory effect of the metals ( fig .", "4 ) , suggesting that the metals altered the proton pump activity mainly via dephosphorylation of its protein .", "the level of h - atpase phosporylation was distinctly lower in plasma membranes from cucumber roots treated with cd , cu or ni ( fig .", "5 ) demonstrating that alteration of the plasma membrane proton pump under heavy metals is due to dephosphorylation of the enzyme protein . considering that our plants ( control plants and those treated with heavy metals ) were exposed for 2 h to mes - naoh without ca in solution , a set of supplementary experiments with plants exposed to metals in mes - naoh with or without 200 m ca ( caso4 )", "nevertheless no differences were found in both the hydrolytic and the transporting activity of the h - atpase .", "also western blot analysis of plasma membranes with the antibody against phosphothreonine showed that the presence of calcium in the solution did not affect the metal action on h - atpase phosphorylation in the short - term experiments presented in this paper ( data not shown ) .", "as protein phosphorylation depends on the energetic status of the cell , the level of endogenous atp was measured .", "each metal treatment decreased the amount of the adenosine triphosphate in root tissues , suggesting that , besides the dephosphorylation intensity of the proton pump , the atp level is also an important factor in enzyme deactivation under heavy metals stresses .", "it is known that the main source of atp in root tissues is mitochondrial respiration . until now", ", the effect of heavy metals on respiration and atp content in plant cells has not been sufficiently studied .", "however , it was revealed that cd treatment of plants significantly reduced the oxygen consumption by roots ( reese and roberts , 1985 ) as well as disordering the electron transport chain ( kessler and brand , 1994 ) .", "thus it is suspected that altered respiration could enhance the inhibition of plasma membrane proton pump activity in plants treated with metals .", "there is the possible action of heavy metals on atpase activity through decreasing the energy charge of the tissue .", "such a correlation is indicated since the endogenous amount of atp , the substrate for plasma membrane h - atpase , decreased under heavy metal stress .", "in addition , it is well known that endogenous atp influences atpase activity , not only as the substrate but also through its function as an activator .", "since protein phosphorylation depends on the endogenous atp level , its reduction upon the addition of heavy metals is in line with our hypothesis .", "data of experiments with cantharidin and western blot with antibodies against phosphothreonine support this supposition .", "we think that some changes in plant cells initiated under heavy metals lead to an increase in the activity of specific phosphatases , responsible for the dephosphorylation of h - atpase protein . taken together", ", the data presented suggest that alterations of the plasma membrane proton pump under heavy metal stresses are mainly due to the post - translational modification of its protein . only in the case of cadmium action ,"]]}
{"article_id": "PMC5081467", "article_text": ["it is well known that they provide structural and metabolic support for neuronal networks , but a growing body of evidence indicates that they also play an active role in modulating neuronal activity .", "astrocytes make close contact with perisynaptic regions , forming a functional structure called the \" tripartite synapse , \" together with presynaptic and postsynaptic nerve terminals .", "indeed , one astrocyte in the hippocampus makes contact with tens of thousands of synapses .", "it is well established that astrocytes clear away excessive neurotransmitters and ions released from synaptic clefts through uptake via specific transporters and channels .", "for example , astrocytes remove excess extracellular glutamate using sodium - dependent glutamate transporters , such as the glutamate aspartate transporter and glutamate type 1 transporter .", "excessive glutamate is cytotoxic to neurons , causing an influx of calcium that far exceeds physiological levels and triggering the activation of enzymes and signaling proteins that are detrimental to neurons .", "evidence from a number of studies indicates that astrocytes release signaling molecules , the so - called \" gliotransmitters , \" such as glutamate , gaba , d - serine , and atp , into the extracellular milieu in response to extracellular stimuli ( fig .", "gliotransmitter release often results from the activation of g protein - coupled receptors ( gpcrs ) that trigger downstream signaling cascades in astrocytes , including phospholipase c , adenylate cyclase , inositol 1,4,5-trisphosphate ( ip3 ) , and cause an intracellular calcium increase .", "gliotransmitters facilitate or inhibit the excitability and synaptic transmission of neighboring neurons , and the outcome of their release is dependent on the site of action and types of activated receptors . through the use of volume - sensitive organic anion channels , gap junction hemichannels , p2x7 , bestrophin-1 , and reverse - orientation plasma membrane glutamate transporters , diverse mechanisms for gliotransmitter release", "have been identified in astrocytes , including calcium - dependent exocytotic vesicular release as well as non - exocytotic mechanisms .", "although accumulating evidence suggests a coupling between various intracellular changes in astrocytes , such as intracellular calcium increase and gliotransmitter release , there is also evidence against this view ; thus , the mechanisms underlying astrocyte - neuronal communication are still debated . in this review ,", "we present recent studies that have using optogenetic and chemogenetic approaches to explore the function of astrocytes and gliotransmitters .", "optogenetics is a novel biological technique based on a variety of light - sensitive proteins called opsins , which include microbial ion channels and ion pumps as well as engineered gpcrs ( fig .", "1 ) . following absorption of a specific wavelength of light , an opsin undergoes a conformational change that triggers diverse cellular changes in opsin - expressing cells .", "some of the opsins induce the translocation of ions , and others activate intracellular signaling cascades , such as g protein - mediated signaling .", "since most of these opsins do not exist in experimental - model organisms , and photostimulation itself has a negligible effect on cells and tissues , optogenetics has instead been used as a powerful experimental tool to manipulate specific populations of cells both in vitro and in vivo by means of a combinatorial approach of cell type - specific promoters and additional genetic tricks .", "this technique has also enabled the manipulation of cellular activity with millisecond - scale temporal precision .", "the time - resolved stimulation has made possible the revelation of causal relationships between manipulated cellular activity and functional outcomes , particularly in the study of neuronal circuits mediating specific behaviors .", "opsins have been modified to generate mutants and chimeric proteins with diverse features , including their intracellular effects , optimal wavelengths of light for activation , and temporal dynamics in activation and inactivation ; thus , they offer great flexibility in designing experiments and conducting more refined manipulations .", "channelrhodopsin-2 ( chr2 ) , originally identified in green algae , is a cation channel that becomes permeable to positively charged ions such as proton and sodium when it is stimulated with blue light .", "when it is expressed in neurons , photostimulation elicits an influx of cations , which causes depolarization and the firing of action potentials in the stimulated cells .", "an influx of protons though chr2 can also acidify the cytosolic compartment of photostimulated cells . in the study of neurons ,", "the frequency and duration of neuronal spiking can be easily controlled using variants of chr2 , such as chr2(h134r ) , chr2(c128s ) , cheta , and step function opsin ( sfo ) .", "for example , cheta can drive ultrafast spiking up to 200 hz , and sfo can elicit prolonged , bi - stable , sub - threshold depolarization of membranes . some light - gated cation channels , such as calcium - translocating channelrhodopsin ( catch ) and liglur ,", "are more permeable to calcium than chr2 , and therefore they have been preferentially used in studies exploring the role of intracellular calcium .", "liglur is a mutated ionotropic glutamate receptor 6 containing its ligand attached to an optically switchable tether called maleimide - azobenzene - glutamate .", "halorhodopsin is an opsin identified from archaea which , when stimulated with yellow light , pumps chloride ions into cells .", "when halorhodopsin is expressed in neurons , photostimulation promotes an influx of chloride ions that results in hyperpolarization and the inhibition of the firing of action potentials in the stimulated cells .", "archaerhodopsins , such as arch and archt , are light - driven outward proton pumps that inhibit the firing of action potentials during photostimulation when they are expressed in neurons ; the efflux of protons can also cause alkalization of the cytosol . finally , optoxrs , such as opto1ar and opto2ar , are chimeric gpcrs in which the intracellular loops of rhodopsin are replaced with those of other gpcrs , such as adrenergic receptors and dopamine receptors .", "photostimulation can initiate diverse intracellular signaling cascades in target cells , depending on the type of g protein replacing the intracellular loops of rhodopsin .", "thus , these opsins enable the acute activation of different gpcr signaling pathways in cultured cells and animals .", "chemogenetics is based on engineered proteins , such as gpcrs and ligand - gated ion channels , that are no longer responsive or only very weakly responsive to their endogenous ligands but strongly respond to synthetic chemical ligands that are otherwise biologically inert .", "for example , hm3dq , one of the designer receptors exclusively activated by designer drugs ( dreadds ) , is generated by multiple cycles of randomized mutagenesis of the human m3 muscarinic receptor , which is linked to the gq protein .", "it is neither sensitive to the endogenous muscarinic acetylcholine receptor ligand acetylcholine nor is it constitutively active , but it is strongly activated in response to a synthetic ligand , clozapine - n - oxide ( cno ) , with nanomolar potency . in response to cno , hm3dq can induce an enhancement of neuronal excitability that can lead to burst - like firing .", "thus , it is one of the most frequently used chemogenetic tools to activate neurons .", "another dreadd molecule , hm4di , is a mutant of the gi - coupled human m4 muscarinic receptor that responds to cno . upon an application of the chemical agonist", ", hm4di activates the g subunit of the gi protein , which then stimulates g protein inwardly rectifying potassium channels ( girk ) , causing an efflux of potassium and a resulting robust hyperpolarization when it is expressed in neurons .", "thus , hm4di has been used to silence spontaneous and depolarization - evoked neuronal firing .", "this goal is often attained by injecting a virus ( e.g. , adeno - associated virus ( aav ) or lentivirus ) that encodes an effector into a target region in the brain or other tissue .", "alternatively , the effector can be expressed as a transgene in a genetically engineered mouse line . by using cell type - specific promoters , such as the astrocyte - specific glial fibrillary acid protein ( gfap ) promoter ,", "the effector 's expression can be restricted to a specific population ( or more than one population ) of cells .", "an intersectional strategy based on a combination of specific promoters and genetic tools , such as cre- and flippase - mediated recombination , can further restrict the effector expression to specific subpopulations .", "in addition , other genetic tricks , such as the use of tetracycline - dependent transcriptional regulation , have been used to achieve temporal control as well as amplification of effector expression .", "optogenetics can deliver photostimulation directly to target cells and manipulate cellular activity acutely and reversibly .", "in contrast , chemogenetics is ideal for a prolonged manipulation of cellular activity in the range of minutes to days , depending on the route of ligand delivery and the pharmacokinetic properties of the synthetic ligand(s ) used .", "optogenetics is excellent in generating spiking patterns that mimic the endogenous firing responses of neurons by using a pulse generator that produces lights with different frequencies and pulse durations .", "in addition , photostimulation can be delivered to different subcellular locations such as the soma and nerve terminals , a useful feature for studying neuronal circuits in the brain .", "on the other hand , chemogenetics is less invasive in experimental animals and hampers animal behaviors only marginally , if at all , because it requires neither the installation of a fiber - optic cable within the brain nor a connection of the cable to a light source , such as a laser or a light - emitting diode ( led ) .", "furthermore , some synthetic ligands for chemogenetics , such as cno , can be delivered via the animal 's water and/or food as well as by systemic injection , permitting the delivery of the ligand with minimal disturbance of the animals , particularly in the case of chronic manipulation .", "optogenetic and chemogenetic techniques have been most frequently used to investigate neuronal circuits , but they also have been used to study non - neuronal cells in the brain and peripheral tissues . in the following section , we will summarize the approaches and findings of recent studies that have employed these techniques to reveal the function of astrocytes ( table 1 and 2 ) .", "studies using primary astrocytes and immortal astrocyte cell lines have shown that optogenetic stimulation can elicit an elevation of intracellular calcium and subsequent release of gliotransmitters that can activate adjacent astrocytes as well as neurons .", "have reported that photostimulation of chr2-expressing primary astrocytes using a led can elicit an intracellular calcium increase and electrophysiological responses not only in the stimulated cells but also in co - cultured astrocytes and neurons that do not express chr2 . the calcium response in chr2-negative cells", "is suppressed by the application of antagonists of n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptors in the bath solution , suggesting that the response in chr2-negative cells is mediated by glutamate released from chr2-expressing astrocytes .", "similar results have been obtained in another study , in which the coupling has been demonstrated between an intracellular calcium increase in astrocytes and glutamate release .", "have performed fluorescent calcium imaging in primary cultures of mouse cortical astrocytes and shown that photostimulation of astrocytes expressing liglur can elicit calcium transients not only in stimulated liglur - expressing cells but also in neighboring astrocytes that do not express the opsin . the calcium response in liglur - negative astrocytes", "is affected by antagonists of glutamate receptors , but not by a gap junction blocker or an antagonist of extracellular atp signaling , suggesting the involvement of glutamate in the communication between astrocytes .", "further experiments have shown that the calcium transients in liglur - negative astrocytes are inhibited by an anion channel blocker but are unaffected by an inhibitor of v - atpase , which blocks exocytosis , suggesting that liglur - evoked glutamate release is mediated by anion channels . in an attempt to investigate whether intracellular ionic alteration in astrocytes triggers gliotransmitter release , ono and coworkers have co - cultured an astrocytic cell line and a neuronal cell line ; in response to photostimulation , the chr2-expressing astrocytes exhibited diverse cellular changes , including an increase in intracellular sodium and calcium , intracellular acidification , glutamate release , and inhibition of proliferation .", "a short period of photostimulation ( for minutes ) elicited calcium transients in the co - cultured chr2-negative neurons , whereas a long period of stimulation for several days resulted in apoptotic responses in the neurons .", "thus , this study has demonstrated that activation of astrocytes releases glutamate which , in turn , provokes an intracellular calcium increase and cytotoxic cell death . to mimic", "gpcr - mediated signaling events occurring in astrocytes in response to extracellular neurotransmitters and neuromodulators , figueiredo et al .", "have expressed gpcr - based opsins , such as opto1ar and opto2ar , in astrocytes to activate gq- and gs - mediated signaling cascades , respectively .", "photostimulation elicited an intracellular calcium increase in astrocytes expressing either opsin , which was blocked by apyrase , an enzyme hydrolyzing extracellular atp , as well as by pharmacological blockers for the corresponding intracellular signaling cascade , such as inhibitors of phospholipase c and adenylate cyclase ; these data indicate that a large portion of the calcium rise that was evoked by the activation of either opsin was a result of the autocrine action of extracellular atp .", "this study has demonstrated that gpcr - based opsins can be effectively used in the study of astrocytic gpcr - mediated signaling .", "tetracycline - dependent expression , using the so - called tet - off system , has been employed in several studies to generate transgenic mouse models expressing an opsin . in the tet - off system", ", the tetracycline - controlled transcriptional activator ( tta ) binds to a tta - responsive promoter sequence ( teto ) to induce the expression of a downstream gene . when bound to the tetracycline derivative doxycycline , tta undergoes a conformational change that prevents tta from binding to teto , inhibiting the transcription of a target gene .", "thus , this system enables a reversible control of gene expression produced by treatment with doxycycline .", "tanaka and coworkers have generated a mouse line by knocking in a transgene cassette encoding teto - driven chr2(c128s ) downstream of a housekeeping gene , -actin , to obtain a high level expression .", "the knockin mice have been crossed to tta driver lines in which tta is driven by cell type - specific promoters , the mlc1 , plp , and iba-1 promoters , to induce chr2(c128s ) expression in astrocytes , oligodendrocytes , and microglia , respectively .", "the double - transgenic mice containing mlc1-driven tta and teto - driven chr2(c128s ) have been used to reveal the role of bergman glia ( bg ) , a specialized subtype of astrocytes in the cerebellum , in modulating the activity of purkinje neurons .", "first , photostimulation of acute brain slices prepared from the transgenic mice was found to elicit current responses from chr2-expressing bg , suggesting that chr2 is expressed in glial cells to a level sufficient for electrophysiological responses .", "second , photostimulation of the cerebellum using a fiber - optic cable installed above the skull , to avoid the generation of injury - induced reactive gliosis , was found to be sufficient to evoke an induction of a surrogate marker for cellular activation , c - fos , in chr2-expressing bg .", "third , photostimulation of chr2-expressing bg in acute cerebellar slices was shown to be sufficient to trigger glutamate release and firing of nearby purkinje cells ( pcs ) , resulting in long - term plasticity between parallel fibers and pcs .", "finally , in vivo photostimulation of glia cells using fiber - optic cable inserted into the cerebellar flocculus was found to cause pupil dilation as well as perturbation of smooth eye pursuit of visual stimuli in head - fixed mice .", "have recently demonstrated using the same mouse line that neuronal damage in the mouse model of ischemia can be exacerbated by optogenetically induced acidosis and attenuated by alkalization of the cytosolic compartment of astrocytes . under ischemic conditions , such as deprivation of oxygen and glucose ,", "cerebellar bg exhibited intracellular acidosis and glutamate release , followed by an inward excitatory current in the surrounding pcs .", "the response in pcs was inhibited by a cocktail of glutamate receptor and transporter blockers , suggesting the involvement of glutamate in the interaction between bg and pcs .", "acidosis induced in bg by optogenetic stimulation of chr2(c128s ) was sufficient to evoke an inward excitatory current in the adjacent pcs .", "the response in pcs was inhibited by a non - competitive ampa and kainate receptor antagonist , confirming the involvement of glutamate in the signaling between bg and pc . in contrast , an efflux of proton from bg produced by optogenetic stimulation of a light - gated outward proton pump , archt , led to a reduction in the inward currents in the pcs elicited by the deprivation of oxygen and glucose .", "furthermore , in vivo photostimulation of archt - expressing bg caused a substantial reduction in cerebellar infarction following a local thrombosis - caused ischemic stroke , whereas control mice without archt activation exhibited severe neuronal death under the same conditions .", "taken together , the results of this study have demonstrated that ischemic injury causes glial acidosis , which , in turn , releases glutamate into the extracellular space and causes ischemic neuronal death .", "have demonstrated that in vivo optogenetic stimulation of cortical astrocytes elicits a rapid , robust , and widespread increase in cerebral blood flow ( cbf ) .", "the increased cbf was abolished by an application of the inward rectifier potassium channel blocker , bacl2 , on the exposed cortex , indicating the importance of potassium signaling in astrocytic modulation of cbf .", "in contrast , the study found that neither astrocytic intracellular calcium signaling nor glutamate release was involved in the increase in cbf .", "a number of studies have employed virally mediated expression of opsins to manipulate astrocytes , despite the possibility of inducing reactive gliosis as a result of viral infection .", "for example , an aav encoding gfap promoter - driven chr2 has been used to reveal a causal relationship between the activity of astrocytes and visual processing in the primary visual cortex ( v1 ) . although a previous study had shown that astrocytes in the visual cortex respond to visual sensory stimuli , their roles had not been clearly determined because of the difficulty in selectively manipulating astrocytes among the heterogeneous populations of cells in the region .", "perea et al . reported that in vivo optogenetic stimulation of astrocytes in the v1 enhanced the spontaneous firing of a population of inhibitory neurons expressing parvalbumin , and this firing was suppressed by treatment with an antagonist of type 1a metabotropic glutamate receptors , suggesting the involvement of glutamate in astrocyte - mediated visual processing . in contrast , optical stimulation of astrocytes had mixed effects in terms of activation and inhibition on excitatory neurons and another population of inhibitory neurons expressing somatostatin .", "finally , in vivo optogenetic stimulation of astrocytes in the v1 strongly affected the responses of neuronal populations to visual stimuli .", "optogenetic manipulations have revealed the involvement of other signaling molecules released by astrocytes , such as extracellular atp and l - lactate , in modulating the activity of neurons in the brainstem .", "have reported that astrocytes in the ventral surface of the medulla oblongata ( vs ) are exquisitely ph - sensitive . in response to a decrease in ph in anesthetized rats , astrocytes residing near the vs exhibited an intracellular calcium increase .", "furthermore , a decrease in ph in brainstem slices elicited a sustained atp release in the vs region , as well as extracellular atp - dependent calcium responses in vs astrocytes . to mimic ph - elicited calcium excitation in astrocytes ,", "an aav encoding enhanced gfap promoter - driven chr2(h134r ) was injected into the brainstem . in organotypic brainstem slices ,", "photostimulation elicited not only calcium transients in chr2-expressing astrocytes but also long - lasting depolarization in adjacent chemo - sensitive neurons in the retrotrapezoid nucleus ( rtn ) .", "rtn neurons have been found to play an important role in monitoring glucose concentrations , ph , and partial pressure of co2 .", "either apyrase or an antagonist of extracellular atp receptor blocked the response of the rtn neurons , suggesting that extracellular atp mediates the interaction between astrocytes and adjacent neurons .", "finally , in vivo unilateral optogenetic stimulation of astrocytes in anesthetized , vagotomized , and artificially ventilated rats elicited a robust respiratory activity from hypocapnic apnea and an increase in phrenic nerve amplitude , which was suppressed by an antagonist of the extracellular atp receptor ; these results indicate that astrocytes are critical components of the central respiratory and chemosensory functions , and extracellular atp is a key molecule in the signaling between astrocytes and neighboring neurons in the rtn .", "the same group of researchers has investigated the astrocytic modulation of norepinephrine ( ne ) release in the locus coeruleus ( lc ) using an aav encoding gfap promoter - driven chr2(h134r ) .", "evidence existed to suggest that l - lactate is involved in the process , but the exact mechanism was unclear .", "photostimulation of chr2-expressing astrocytes in organotypic cultured brain slices elicited delayed depolarization and increased firing rates in norepinephrine ( ne)-ergic neurons .", "pharmacological interventions that reduce the level of l - lactate suppressed light - induced depolarization of neergic neurons , suggesting that astrocytes activate neergic neurons via l - lactate .", "indeed , the application of l - lactate to brain slices provoked similar electrophysiological responses in neergic neurons and caused ne release from the activated neurons . finally , optogenetic activation of astrocytes using either opto2ar or chr2(h134r ) was sufficient to trigger ne release .", "thus , this study clearly demonstrated that activated astrocytes in the lc release l - lactate , which then triggers ne release from neergic neurons .", "a similar viral approach was used by gradinaru et al . to examine whether local astrocytes contribute to the therapeutic effect of deep - brain stimulation ( dbs ) delivered to the subthalamic nucleus ( stn ) to relieve tremor in parkinson 's disease . to deliver photostimulation and measure neuronal activity from a parkinsonian rodent model ,", "optrode recordings were performed in anesthetized rats , in which 6-hydroxydopamine ( 6-ohda ) had been unilaterally injected into the right medial forebrain bundle to cause a loss of nigral dopaminergic cells .", "6-ohda - treated animals displayed rotations ipsilateral to the lesion as a result of specific deficits in contralateral limb function , which became more obvious when amphetamine was administered to the subjects to increase locomotion .", "this study revealed that photostimulation of chr2-expressing astrocytes in the stn can reversibly inhibit firing of stn neurons in 6-ohda - treated animals ; this treatment , however , failed to cause any changes in pathological motor behavior in parkinsonian rats , suggesting that astrocytes are unlikely to be critical players in the dbs - elicited effects .", "optogenetic manipulation has been used in two recent studies to examine the role of astrocytes in sleep .", "have reported that optogenetic activation of the posterior hypothalamus using chr2(h134r ) promotes both rapid and non - rapid eye movement sleep .", "on the other hand , yamashita et al . have reported that in vivo optogenetic stimulation of chr2-expressing astrocytes in the anterior cingulate cortex results in a significant increase in wakefulness as well as disturbance of non - rapid eye movement sleep . in a very recent study reported by poskanzer and yuste , the role of neocortical astrocytes in the control of cortical circuit functions", "was examined using in vivo two - photon calcium imaging based on the genetic calcium indicator gcamp6s , together with electrophysiological recording from cortical neurons . to examine the causal relationship between the calcium signaling in astrocytes and neuronal activity in the v1 ,", "an aav encoding cre - dependent arch was injected into the v1 of transgenic mice expressing gfap promoter - driven cre , which resulted in astrocyte - specific expression of the opsin . when expressed in neurons", ", arch hyperpolarizes membrane potentials and inhibits neuronal firing by pumping protons out of neurons in response to yellow - light photostimulation .", "surprisingly , photostimulation of arch in the astrocytes triggered calcium transients that were specifically localized to the processes of astrocytes and largely undetected in the soma .", "in contrast , neighboring arch - negative cells failed to exhibit a calcium response during photostimulation .", "a previous study has reported that arch - mediated stimulation of cerebellar bg increases the intracellular ph as the result of an efflux of protons out of cells under oxygen - glucose - deprived conditions .", "in contrast , poskanzer and yuste found no significant changes in ph in stimulated astrocytes as well as in surrounding cells in the v1 .", "it is not clear whether this discrepancy is a byproduct of cell - type specificity .", "finally , local field potential recordings have revealed that in vivo optogenetic stimulation of astrocytes in the v1 results in calcium transients , followed by a brief increase in extracellular glutamate and a shift in neuronal firing patterns in v1 from a desynchronized state to the synchronized slow oscillation - dominated state .", "among diverse chemogenetic effectors , dreadds , such as hm3dq , have been used most frequently in studies focusing on astrocytes . as in optogenetic approaches , astrocyte - specific expression of chemogenetic proteins has been achieved by using viral and transgenic delivery in combination with astrocyte - specific promoters such as the gfap and mlc1 promoters . to manipulate gq - coupled receptor signaling in astrocytes , fiacco et al .", "generated a bi - transgenic mouse line encoding gfap promoter - driven tta and teto promoter - driven mas - related g protein - coupled receptor a1 ( mrgpra1 ) to express gpcr selectively in astrocytes .", "mrgpra1 can be activated by rf amides , such as a peptide phe - leu - arg - phe amide ( flrf ) . since endogenous mrgpra1", "is specifically expressed in dorsal root ganglion neurons but not in the brain , this protein is a useful molecular tool for manipulating neurons and glia in the brain when it is exogenously expressed in these cells .", "an infusion of flrf into acute hippocampal slices prepared from the transgenic mice elicited a robust calcium increase in widespread astrocytes , suggesting that mrgpra1 is able to activate the gq - coupled intracellular signaling pathway .", "it is particularly interesting that such a widespread calcium rise in astrocytes failed to affect neuronal activity in the same slices .", "this finding is at odds with other studies , questioning the hypothesis that an astrocytic calcium increase causes the release of gliotransmitters which , in turn , activate nearby neurons . in a follow - up study using margpra1 mice together with mice lacking inositol 1,4,5-trisphosphate receptor 2 ( ip3 r2 ) ,", "the astrocyte - specific ip3 receptor isoform , the same group of researchers further confirmed that activation of gq protein - coupled signaling affects neither spontaneous excitatory postsynaptic currents nor the induction and maintenance of long - term potentiation in ca1 hippocampal neurons .", "in another study performed by the same group of researchers , bonder and mccarthy reported that hm3dq can be selectively expressed in astrocytes by injecting aav incorporating cre - dependent hm3dq into the visual cortex of transgenic mice encoding gfap promoter - driven cre , and they have used this system to investigate whether the astrocytic calcium elevation triggers vasodilation and a change in local blood flow in the cortex .", "activation of hm3dq with cno was sufficient to increase the intracellular calcium level in astrocytes but not to alter the basal blood flow in the visual cortex .", "the study also reported the absence of a temporal correlation between the astrocytic calcium increase and the change in cortical blood flow following either visual stimulation or a startle - evoking air puff .", "furthermore , genetic deletion of ip3 r2 did not affect neurovascular coupling , suggesting that gq signaling and ip3-dependent calcium elevation in astrocytes do not mediate vasodilation in the visual cortex .", "the gfap - driven mrgpra1 mouse line was used in a more recent study of cao and coworkers to investigate the role of astrocytic atp release in depression - like behaviors .", "application of the mrgpra1 agonist flrf elicited not only a robust increase in intracellular calcium in mrgpra1-expressing primary astrocytes but also a 2.5-fold increase in the atp concentration in the culture medium .", "furthermore , mrgpra1 mice infused with flrf into the cerebral ventricle exhibited a substantial reduction in depression - like behavior elicited in the murine paradigm of chronic social - defeat stress .", "together with other results reported in the study , these findings suggest that endogenous atp released from astrocytes can induce antidepressant - like behavior . whether intracellular calcium increases in astrocytes can affect the activity of neighboring neurons was untested in the study .", "for instance , sweger et al . developed a mouse line expressing an engineered k - opioid receptor ( ro1 ) in gfap - expressing astrocytes by crossing transgenic mice encoding gfap promoter - driven tta mice with another line encoding teto promoter - driven ro1 on the background of a genetic deletion of the endogenous k - opioid receptor ( kor ) .", "ro1 is a gi - coupled gpcr that is insensitive to endogenous ligands of kor , such as dynorphin , but highly sensitive to a synthetic ligand of the k - opioid receptor , spiradoline .", "ro1-expressing transgenic mice developed hydrocephalus and accumulation of cerebrospinal fluid in the ventricular system , even in the absence of a synthetic ligand , suggesting that ro1 is constitutively active in this mouse model ; unfortunately , this constitutive activity negates one of main features of chemogenetics , its temporal controllability , and limits the use of this model .", "the same group of researchers has described another transgenic mouse line expressing gfap promoter - driven hm3dq .", "systemic treatment of these mice with cno elicited a number of physiological changes that are controlled by the autonomic nervous system , including cardiovascular function , saliva formation , and homeostasis of body temperature .", "furthermore , hm3dq - expressing mice receiving cno exhibited substantial changes in activity - related behaviors and motor coordination .", "thus , these findings indicate the critical role of astrocytes in a broad range of basic physiological functions .", "interestingly , the physiological and behavioral changes were unaffected by genetic deletion of ip3 r2 , suggesting that astrocytic ip3-mediated calcium increase is dispensable for hm3dq - elicited responses .", "the gfap promoter - driven hm3dq mice have been used to study glial cells outside of the brain .", "examined the potential role of the enteric glia , which are astrocyte - like peripheral glial cells surrounding enteric neurons in the gut .", "an application of cno to the ileal and colonic myenteric plexus prepared from transgenic mice not only elicited and intracellular calcium increase in astrocytes but also triggered contraction of the ileum and colon to a degree similar to that elicited by direct stimulation of smooth muscle or electrical stimulation of enteric neurons .", "the contraction was abolished by the application of tetradotoxin , indicating the involvement of neuronal activation in the process .", "these findings have demonstrated that astrocytes in the gut play a critical role in the contractions of intestinal smooth muscle .", "the mechanism by which activation of gq - coupled receptor in astrocytes leads to activation of enteric neurons remains unknown .", "have reported two new transgenic mouse lines expressing hm3dq , depending on cre and flippase - mediated recombination . when crossed to cre or flp driver lines ,", "the new mouse lines permit the selective expression of hm3dq in a population of cells that express either cre or flippase .", "in addition , the intersectional strategy involving both cre- and flippas - edependent recombination further restricts hm3dq expression in a specific subpopulation . this group reported that a systemic application of cno to mice expressing hm3dq in gfap - expressing cells elicits hypothermia , confirming the efficacy of the new mouse line by reproducing the previous finding .", "finally , the chemogenetic approach has also been applied to reveal the function of gs - coupled signaling in longterm memory in normal animals and the alzheimer animal model .", "for example , double - transgenic mice encoding gfap promoter - driven tta and teto - driven rs1 have been generated to acutely modulate gs - coupled receptor activity .", "rs1 is the human gs - coupled 5-ht4b serotonin receptor with a point mutation that renders this receptor insensitive to serotonin but highly sensitive to a synthetic ligand , gr-125487 .", "activation of gs signaling by systemic delivery of gr-125487 impairs the performance of transgenic mice in the morris water maze as well as a novel object - recognition task .", "orr et al . found that rs1 is constitutively active in this mouse model , driving the gs signaling pathway even in the absence of the synthetic ligand .", "thus far , only a small number of studies focusing on glia have used a virally mediated method to achieve the expression of chemogenetic proteins . for example , an aav encoding gfap promoter - driven hm3dq or hm4di has been stereotactically injected into the arcuate nucleus of the mouse brain to investigate the potential role of medial basal hypothalamic astrocytes in regulating food intake . in the feeding assay , hm3dq - expressing mice receiving cno exhibited a significant reduction in both baseline feeding and ghrelin - elicited hyperphagia , whereas hm4di - expressing mice receiving cno showed substantially enhanced and prolonged ghrelin - evoked feeding . in contrast , following the cno treatment , leptin - induced anorexia was facilitated in hm3dq - expressing mice but suppressed in hm4di - expressing mice .", "thus , this study employing two different chemogenetic actuators that recruit different downstream signaling molecules clearly demonstrated that astrocytes in the arcuate nucleus exert bi - directional regulation of food consumption .", "an aav virus expressing gfap promoter - driven hm3dq was injected into the rat nucleus accumbens core ( nacore ) in two recent studies in order to investigate the contribution of glial cells and extracellular glutamate to substance abuse and motivation .", "reported that an application of cno elicited an elevation of the intracellular calcium level in hm3dq - expressing primary astrocytes and a decrease in motivation for self - administration of ethanol after 3 weeks of abstinence .", "showed that intracranial or systemic administration of cno triggered an increase in extracellular glutamate in the nacore .", "furthermore , hm3dq - expressing rats receiving intraperitoneal cno exhibited a significant reduction in the cueinduced reinstatement of cocaine seeking .", "neurons have always been a main focus of brain research , and non - neuronal cells that make up the majority of brain cells , such as glial cells , have not received much attention until recently .", "studies of glia have revealed that they do not merely provide food and support to neurons ; rather , they play an important role in brain function . in order to understand astrocytic function ,", "it is critical to be able to control their intracellular activity in a native context .", "since glia are intermingled with neurons in the brain and they express receptors and ion channels that are also expressed in neurons , it is difficult to perform such manipulation selectively in glial cells , while leaving neighboring neurons unaffected .", "optogenetics and chemogenetics are novel manipulation techniques based on genetically encoded effector molecules , such as specific ion channels and gpcrs , that respond to exogenously delivered light stimuli or synthetic ligands , but are unresponsive to endogenous molecules . in combination with cell type - specific promoters and other genetic tools , expression of effector molecules can be restricted to specific cell types .", "thus , the features of spatial and temporal control make it possible to perform a time - resolved functional manipulation in a specific population of cells .", "optogenetics and chemogenetics have been used most extensively in the study of neuronal circuits and behavior , but they have also been employed in a number of studies focusing on glial cells , mainly astrocytes .", "such studies have demonstrated that astrocytes play a critical role not only in a variety of basic physiological responses , including visual processing , norepinephrine release , breathing , cerebral blood flow , feeding , memory , and sleep , but also in pathological conditions , including drug addition , depression , and ischemia .", "those studies have further revealed the importance of gliotransmitters , such as extracellular glutamate , atp , and l - lactate , that modulate excitability and synaptic transmission in neighboring neurons .", "however , it is still debatable whether astrocytic release of gliotransmitters is a calcium - dependent process .", "in addition , the exact molecular mechanisms governing gliotransmitter release from astrocytes remains to be revealed . a combinatorial approach of advanced functional manipulation techniques such as optogenetics and chemogenetics , together with pharmacological and molecular genetic methods , can further our understanding of glial function in health and disease , including neurodevelopment , neurodegenerative disorders , and neuroinflammatory conditions ."], "abstract_text": ["<S> the brain consists of heterogeneous populations of neuronal and non - neuronal cells . </S>", "<S> the revelation of their connections and interactions is fundamental to understanding normal brain functions as well as abnormal changes in pathological conditions . </S>", "<S> optogenetics and chemogenetics have been developed to allow functional manipulations both in vitro and in vivo to examine causal relationships between cellular changes and functional outcomes . </S>", "<S> these techniques are based on genetically encoded effector molecules that respond exclusively to exogenous stimuli , such as a certain wavelength of light or a synthetic ligand . </S>", "<S> activation of effector molecules provokes diverse intracellular changes , such as an influx or efflux of ions , depolarization or hyperpolarization of membranes , and activation of intracellular signaling cascades . </S>", "<S> optogenetics and chemogenetics have been applied mainly to the study of neuronal circuits , but their use in studying non - neuronal cells has been gradually increasing . here </S>", "<S> we introduce recent studies that have employed optogenetics and chemogenetics to reveal the function of astrocytes and gliotransmitters . </S>"], "labels": null, "section_names": ["INTRODUCTION", "OPTOGENETICS AND CHEMOGENETICS", "OPTOGENETIC STIMULATION OF CULTURED ASTROCYTES", "OPTOGENETIC STIMULATION OF ASTROCYTES USING TRANSGENIC MICE", "OPTOGENETIC STIMULATION OF ASTROCYTES EXPRESSING VIRALLY DELIVERED OPSINS", "CHEMOGENETIC STIMULATION OF ASTROCYTES IN TRANSGENIC MICE", "CHEMOGENETIC STIMULATION OF ASTROCYTES EXPRESSING VIRALLY DELIVERED EFFECTORS", "CONCLUDING REMARKS"], "sections": [["it is well known that they provide structural and metabolic support for neuronal networks , but a growing body of evidence indicates that they also play an active role in modulating neuronal activity .", "astrocytes make close contact with perisynaptic regions , forming a functional structure called the \" tripartite synapse , \" together with presynaptic and postsynaptic nerve terminals .", "indeed , one astrocyte in the hippocampus makes contact with tens of thousands of synapses .", "it is well established that astrocytes clear away excessive neurotransmitters and ions released from synaptic clefts through uptake via specific transporters and channels .", "for example , astrocytes remove excess extracellular glutamate using sodium - dependent glutamate transporters , such as the glutamate aspartate transporter and glutamate type 1 transporter .", "excessive glutamate is cytotoxic to neurons , causing an influx of calcium that far exceeds physiological levels and triggering the activation of enzymes and signaling proteins that are detrimental to neurons .", "evidence from a number of studies indicates that astrocytes release signaling molecules , the so - called \" gliotransmitters , \" such as glutamate , gaba , d - serine , and atp , into the extracellular milieu in response to extracellular stimuli ( fig .", "gliotransmitter release often results from the activation of g protein - coupled receptors ( gpcrs ) that trigger downstream signaling cascades in astrocytes , including phospholipase c , adenylate cyclase , inositol 1,4,5-trisphosphate ( ip3 ) , and cause an intracellular calcium increase .", "gliotransmitters facilitate or inhibit the excitability and synaptic transmission of neighboring neurons , and the outcome of their release is dependent on the site of action and types of activated receptors . through the use of volume - sensitive organic anion channels , gap junction hemichannels , p2x7 , bestrophin-1 , and reverse - orientation plasma membrane glutamate transporters , diverse mechanisms for gliotransmitter release", "have been identified in astrocytes , including calcium - dependent exocytotic vesicular release as well as non - exocytotic mechanisms .", "although accumulating evidence suggests a coupling between various intracellular changes in astrocytes , such as intracellular calcium increase and gliotransmitter release , there is also evidence against this view ; thus , the mechanisms underlying astrocyte - neuronal communication are still debated . in this review ,", "we present recent studies that have using optogenetic and chemogenetic approaches to explore the function of astrocytes and gliotransmitters ."], ["optogenetics is a novel biological technique based on a variety of light - sensitive proteins called opsins , which include microbial ion channels and ion pumps as well as engineered gpcrs ( fig .", "1 ) . following absorption of a specific wavelength of light , an opsin undergoes a conformational change that triggers diverse cellular changes in opsin - expressing cells .", "some of the opsins induce the translocation of ions , and others activate intracellular signaling cascades , such as g protein - mediated signaling .", "since most of these opsins do not exist in experimental - model organisms , and photostimulation itself has a negligible effect on cells and tissues , optogenetics has instead been used as a powerful experimental tool to manipulate specific populations of cells both in vitro and in vivo by means of a combinatorial approach of cell type - specific promoters and additional genetic tricks .", "this technique has also enabled the manipulation of cellular activity with millisecond - scale temporal precision .", "the time - resolved stimulation has made possible the revelation of causal relationships between manipulated cellular activity and functional outcomes , particularly in the study of neuronal circuits mediating specific behaviors .", "opsins have been modified to generate mutants and chimeric proteins with diverse features , including their intracellular effects , optimal wavelengths of light for activation , and temporal dynamics in activation and inactivation ; thus , they offer great flexibility in designing experiments and conducting more refined manipulations .", "channelrhodopsin-2 ( chr2 ) , originally identified in green algae , is a cation channel that becomes permeable to positively charged ions such as proton and sodium when it is stimulated with blue light .", "when it is expressed in neurons , photostimulation elicits an influx of cations , which causes depolarization and the firing of action potentials in the stimulated cells .", "an influx of protons though chr2 can also acidify the cytosolic compartment of photostimulated cells . in the study of neurons ,", "the frequency and duration of neuronal spiking can be easily controlled using variants of chr2 , such as chr2(h134r ) , chr2(c128s ) , cheta , and step function opsin ( sfo ) .", "for example , cheta can drive ultrafast spiking up to 200 hz , and sfo can elicit prolonged , bi - stable , sub - threshold depolarization of membranes . some light - gated cation channels , such as calcium - translocating channelrhodopsin ( catch ) and liglur ,", "are more permeable to calcium than chr2 , and therefore they have been preferentially used in studies exploring the role of intracellular calcium .", "liglur is a mutated ionotropic glutamate receptor 6 containing its ligand attached to an optically switchable tether called maleimide - azobenzene - glutamate .", "halorhodopsin is an opsin identified from archaea which , when stimulated with yellow light , pumps chloride ions into cells .", "when halorhodopsin is expressed in neurons , photostimulation promotes an influx of chloride ions that results in hyperpolarization and the inhibition of the firing of action potentials in the stimulated cells .", "archaerhodopsins , such as arch and archt , are light - driven outward proton pumps that inhibit the firing of action potentials during photostimulation when they are expressed in neurons ; the efflux of protons can also cause alkalization of the cytosol . finally , optoxrs , such as opto1ar and opto2ar , are chimeric gpcrs in which the intracellular loops of rhodopsin are replaced with those of other gpcrs , such as adrenergic receptors and dopamine receptors .", "photostimulation can initiate diverse intracellular signaling cascades in target cells , depending on the type of g protein replacing the intracellular loops of rhodopsin .", "thus , these opsins enable the acute activation of different gpcr signaling pathways in cultured cells and animals .", "chemogenetics is based on engineered proteins , such as gpcrs and ligand - gated ion channels , that are no longer responsive or only very weakly responsive to their endogenous ligands but strongly respond to synthetic chemical ligands that are otherwise biologically inert .", "for example , hm3dq , one of the designer receptors exclusively activated by designer drugs ( dreadds ) , is generated by multiple cycles of randomized mutagenesis of the human m3 muscarinic receptor , which is linked to the gq protein .", "it is neither sensitive to the endogenous muscarinic acetylcholine receptor ligand acetylcholine nor is it constitutively active , but it is strongly activated in response to a synthetic ligand , clozapine - n - oxide ( cno ) , with nanomolar potency . in response to cno , hm3dq can induce an enhancement of neuronal excitability that can lead to burst - like firing .", "thus , it is one of the most frequently used chemogenetic tools to activate neurons .", "another dreadd molecule , hm4di , is a mutant of the gi - coupled human m4 muscarinic receptor that responds to cno . upon an application of the chemical agonist", ", hm4di activates the g subunit of the gi protein , which then stimulates g protein inwardly rectifying potassium channels ( girk ) , causing an efflux of potassium and a resulting robust hyperpolarization when it is expressed in neurons .", "thus , hm4di has been used to silence spontaneous and depolarization - evoked neuronal firing .", "this goal is often attained by injecting a virus ( e.g. , adeno - associated virus ( aav ) or lentivirus ) that encodes an effector into a target region in the brain or other tissue .", "alternatively , the effector can be expressed as a transgene in a genetically engineered mouse line . by using cell type - specific promoters , such as the astrocyte - specific glial fibrillary acid protein ( gfap ) promoter ,", "the effector 's expression can be restricted to a specific population ( or more than one population ) of cells .", "an intersectional strategy based on a combination of specific promoters and genetic tools , such as cre- and flippase - mediated recombination , can further restrict the effector expression to specific subpopulations .", "in addition , other genetic tricks , such as the use of tetracycline - dependent transcriptional regulation , have been used to achieve temporal control as well as amplification of effector expression .", "optogenetics can deliver photostimulation directly to target cells and manipulate cellular activity acutely and reversibly .", "in contrast , chemogenetics is ideal for a prolonged manipulation of cellular activity in the range of minutes to days , depending on the route of ligand delivery and the pharmacokinetic properties of the synthetic ligand(s ) used .", "optogenetics is excellent in generating spiking patterns that mimic the endogenous firing responses of neurons by using a pulse generator that produces lights with different frequencies and pulse durations .", "in addition , photostimulation can be delivered to different subcellular locations such as the soma and nerve terminals , a useful feature for studying neuronal circuits in the brain .", "on the other hand , chemogenetics is less invasive in experimental animals and hampers animal behaviors only marginally , if at all , because it requires neither the installation of a fiber - optic cable within the brain nor a connection of the cable to a light source , such as a laser or a light - emitting diode ( led ) .", "furthermore , some synthetic ligands for chemogenetics , such as cno , can be delivered via the animal 's water and/or food as well as by systemic injection , permitting the delivery of the ligand with minimal disturbance of the animals , particularly in the case of chronic manipulation .", "optogenetic and chemogenetic techniques have been most frequently used to investigate neuronal circuits , but they also have been used to study non - neuronal cells in the brain and peripheral tissues . in the following section , we will summarize the approaches and findings of recent studies that have employed these techniques to reveal the function of astrocytes ( table 1 and 2 ) ."], ["studies using primary astrocytes and immortal astrocyte cell lines have shown that optogenetic stimulation can elicit an elevation of intracellular calcium and subsequent release of gliotransmitters that can activate adjacent astrocytes as well as neurons .", "have reported that photostimulation of chr2-expressing primary astrocytes using a led can elicit an intracellular calcium increase and electrophysiological responses not only in the stimulated cells but also in co - cultured astrocytes and neurons that do not express chr2 . the calcium response in chr2-negative cells", "is suppressed by the application of antagonists of n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) receptors in the bath solution , suggesting that the response in chr2-negative cells is mediated by glutamate released from chr2-expressing astrocytes .", "similar results have been obtained in another study , in which the coupling has been demonstrated between an intracellular calcium increase in astrocytes and glutamate release .", "have performed fluorescent calcium imaging in primary cultures of mouse cortical astrocytes and shown that photostimulation of astrocytes expressing liglur can elicit calcium transients not only in stimulated liglur - expressing cells but also in neighboring astrocytes that do not express the opsin . the calcium response in liglur - negative astrocytes", "is affected by antagonists of glutamate receptors , but not by a gap junction blocker or an antagonist of extracellular atp signaling , suggesting the involvement of glutamate in the communication between astrocytes .", "further experiments have shown that the calcium transients in liglur - negative astrocytes are inhibited by an anion channel blocker but are unaffected by an inhibitor of v - atpase , which blocks exocytosis , suggesting that liglur - evoked glutamate release is mediated by anion channels . in an attempt to investigate whether intracellular ionic alteration in astrocytes triggers gliotransmitter release , ono and coworkers have co - cultured an astrocytic cell line and a neuronal cell line ; in response to photostimulation , the chr2-expressing astrocytes exhibited diverse cellular changes , including an increase in intracellular sodium and calcium , intracellular acidification , glutamate release , and inhibition of proliferation .", "a short period of photostimulation ( for minutes ) elicited calcium transients in the co - cultured chr2-negative neurons , whereas a long period of stimulation for several days resulted in apoptotic responses in the neurons .", "thus , this study has demonstrated that activation of astrocytes releases glutamate which , in turn , provokes an intracellular calcium increase and cytotoxic cell death . to mimic", "gpcr - mediated signaling events occurring in astrocytes in response to extracellular neurotransmitters and neuromodulators , figueiredo et al .", "have expressed gpcr - based opsins , such as opto1ar and opto2ar , in astrocytes to activate gq- and gs - mediated signaling cascades , respectively .", "photostimulation elicited an intracellular calcium increase in astrocytes expressing either opsin , which was blocked by apyrase , an enzyme hydrolyzing extracellular atp , as well as by pharmacological blockers for the corresponding intracellular signaling cascade , such as inhibitors of phospholipase c and adenylate cyclase ; these data indicate that a large portion of the calcium rise that was evoked by the activation of either opsin was a result of the autocrine action of extracellular atp .", "this study has demonstrated that gpcr - based opsins can be effectively used in the study of astrocytic gpcr - mediated signaling ."], ["tetracycline - dependent expression , using the so - called tet - off system , has been employed in several studies to generate transgenic mouse models expressing an opsin . in the tet - off system", ", the tetracycline - controlled transcriptional activator ( tta ) binds to a tta - responsive promoter sequence ( teto ) to induce the expression of a downstream gene . when bound to the tetracycline derivative doxycycline , tta undergoes a conformational change that prevents tta from binding to teto , inhibiting the transcription of a target gene .", "thus , this system enables a reversible control of gene expression produced by treatment with doxycycline .", "tanaka and coworkers have generated a mouse line by knocking in a transgene cassette encoding teto - driven chr2(c128s ) downstream of a housekeeping gene , -actin , to obtain a high level expression .", "the knockin mice have been crossed to tta driver lines in which tta is driven by cell type - specific promoters , the mlc1 , plp , and iba-1 promoters , to induce chr2(c128s ) expression in astrocytes , oligodendrocytes , and microglia , respectively .", "the double - transgenic mice containing mlc1-driven tta and teto - driven chr2(c128s ) have been used to reveal the role of bergman glia ( bg ) , a specialized subtype of astrocytes in the cerebellum , in modulating the activity of purkinje neurons .", "first , photostimulation of acute brain slices prepared from the transgenic mice was found to elicit current responses from chr2-expressing bg , suggesting that chr2 is expressed in glial cells to a level sufficient for electrophysiological responses .", "second , photostimulation of the cerebellum using a fiber - optic cable installed above the skull , to avoid the generation of injury - induced reactive gliosis , was found to be sufficient to evoke an induction of a surrogate marker for cellular activation , c - fos , in chr2-expressing bg .", "third , photostimulation of chr2-expressing bg in acute cerebellar slices was shown to be sufficient to trigger glutamate release and firing of nearby purkinje cells ( pcs ) , resulting in long - term plasticity between parallel fibers and pcs .", "finally , in vivo photostimulation of glia cells using fiber - optic cable inserted into the cerebellar flocculus was found to cause pupil dilation as well as perturbation of smooth eye pursuit of visual stimuli in head - fixed mice .", "have recently demonstrated using the same mouse line that neuronal damage in the mouse model of ischemia can be exacerbated by optogenetically induced acidosis and attenuated by alkalization of the cytosolic compartment of astrocytes . under ischemic conditions , such as deprivation of oxygen and glucose ,", "cerebellar bg exhibited intracellular acidosis and glutamate release , followed by an inward excitatory current in the surrounding pcs .", "the response in pcs was inhibited by a cocktail of glutamate receptor and transporter blockers , suggesting the involvement of glutamate in the interaction between bg and pcs .", "acidosis induced in bg by optogenetic stimulation of chr2(c128s ) was sufficient to evoke an inward excitatory current in the adjacent pcs .", "the response in pcs was inhibited by a non - competitive ampa and kainate receptor antagonist , confirming the involvement of glutamate in the signaling between bg and pc . in contrast , an efflux of proton from bg produced by optogenetic stimulation of a light - gated outward proton pump , archt , led to a reduction in the inward currents in the pcs elicited by the deprivation of oxygen and glucose .", "furthermore , in vivo photostimulation of archt - expressing bg caused a substantial reduction in cerebellar infarction following a local thrombosis - caused ischemic stroke , whereas control mice without archt activation exhibited severe neuronal death under the same conditions .", "taken together , the results of this study have demonstrated that ischemic injury causes glial acidosis , which , in turn , releases glutamate into the extracellular space and causes ischemic neuronal death .", "have demonstrated that in vivo optogenetic stimulation of cortical astrocytes elicits a rapid , robust , and widespread increase in cerebral blood flow ( cbf ) .", "the increased cbf was abolished by an application of the inward rectifier potassium channel blocker , bacl2 , on the exposed cortex , indicating the importance of potassium signaling in astrocytic modulation of cbf .", "in contrast , the study found that neither astrocytic intracellular calcium signaling nor glutamate release was involved in the increase in cbf ."], ["a number of studies have employed virally mediated expression of opsins to manipulate astrocytes , despite the possibility of inducing reactive gliosis as a result of viral infection .", "for example , an aav encoding gfap promoter - driven chr2 has been used to reveal a causal relationship between the activity of astrocytes and visual processing in the primary visual cortex ( v1 ) . although a previous study had shown that astrocytes in the visual cortex respond to visual sensory stimuli , their roles had not been clearly determined because of the difficulty in selectively manipulating astrocytes among the heterogeneous populations of cells in the region .", "perea et al . reported that in vivo optogenetic stimulation of astrocytes in the v1 enhanced the spontaneous firing of a population of inhibitory neurons expressing parvalbumin , and this firing was suppressed by treatment with an antagonist of type 1a metabotropic glutamate receptors , suggesting the involvement of glutamate in astrocyte - mediated visual processing . in contrast , optical stimulation of astrocytes had mixed effects in terms of activation and inhibition on excitatory neurons and another population of inhibitory neurons expressing somatostatin .", "finally , in vivo optogenetic stimulation of astrocytes in the v1 strongly affected the responses of neuronal populations to visual stimuli .", "optogenetic manipulations have revealed the involvement of other signaling molecules released by astrocytes , such as extracellular atp and l - lactate , in modulating the activity of neurons in the brainstem .", "have reported that astrocytes in the ventral surface of the medulla oblongata ( vs ) are exquisitely ph - sensitive . in response to a decrease in ph in anesthetized rats , astrocytes residing near the vs exhibited an intracellular calcium increase .", "furthermore , a decrease in ph in brainstem slices elicited a sustained atp release in the vs region , as well as extracellular atp - dependent calcium responses in vs astrocytes . to mimic ph - elicited calcium excitation in astrocytes ,", "an aav encoding enhanced gfap promoter - driven chr2(h134r ) was injected into the brainstem . in organotypic brainstem slices ,", "photostimulation elicited not only calcium transients in chr2-expressing astrocytes but also long - lasting depolarization in adjacent chemo - sensitive neurons in the retrotrapezoid nucleus ( rtn ) .", "rtn neurons have been found to play an important role in monitoring glucose concentrations , ph , and partial pressure of co2 .", "either apyrase or an antagonist of extracellular atp receptor blocked the response of the rtn neurons , suggesting that extracellular atp mediates the interaction between astrocytes and adjacent neurons .", "finally , in vivo unilateral optogenetic stimulation of astrocytes in anesthetized , vagotomized , and artificially ventilated rats elicited a robust respiratory activity from hypocapnic apnea and an increase in phrenic nerve amplitude , which was suppressed by an antagonist of the extracellular atp receptor ; these results indicate that astrocytes are critical components of the central respiratory and chemosensory functions , and extracellular atp is a key molecule in the signaling between astrocytes and neighboring neurons in the rtn .", "the same group of researchers has investigated the astrocytic modulation of norepinephrine ( ne ) release in the locus coeruleus ( lc ) using an aav encoding gfap promoter - driven chr2(h134r ) .", "evidence existed to suggest that l - lactate is involved in the process , but the exact mechanism was unclear .", "photostimulation of chr2-expressing astrocytes in organotypic cultured brain slices elicited delayed depolarization and increased firing rates in norepinephrine ( ne)-ergic neurons .", "pharmacological interventions that reduce the level of l - lactate suppressed light - induced depolarization of neergic neurons , suggesting that astrocytes activate neergic neurons via l - lactate .", "indeed , the application of l - lactate to brain slices provoked similar electrophysiological responses in neergic neurons and caused ne release from the activated neurons . finally , optogenetic activation of astrocytes using either opto2ar or chr2(h134r ) was sufficient to trigger ne release .", "thus , this study clearly demonstrated that activated astrocytes in the lc release l - lactate , which then triggers ne release from neergic neurons .", "a similar viral approach was used by gradinaru et al . to examine whether local astrocytes contribute to the therapeutic effect of deep - brain stimulation ( dbs ) delivered to the subthalamic nucleus ( stn ) to relieve tremor in parkinson 's disease . to deliver photostimulation and measure neuronal activity from a parkinsonian rodent model ,", "optrode recordings were performed in anesthetized rats , in which 6-hydroxydopamine ( 6-ohda ) had been unilaterally injected into the right medial forebrain bundle to cause a loss of nigral dopaminergic cells .", "6-ohda - treated animals displayed rotations ipsilateral to the lesion as a result of specific deficits in contralateral limb function , which became more obvious when amphetamine was administered to the subjects to increase locomotion .", "this study revealed that photostimulation of chr2-expressing astrocytes in the stn can reversibly inhibit firing of stn neurons in 6-ohda - treated animals ; this treatment , however , failed to cause any changes in pathological motor behavior in parkinsonian rats , suggesting that astrocytes are unlikely to be critical players in the dbs - elicited effects .", "optogenetic manipulation has been used in two recent studies to examine the role of astrocytes in sleep .", "have reported that optogenetic activation of the posterior hypothalamus using chr2(h134r ) promotes both rapid and non - rapid eye movement sleep .", "on the other hand , yamashita et al . have reported that in vivo optogenetic stimulation of chr2-expressing astrocytes in the anterior cingulate cortex results in a significant increase in wakefulness as well as disturbance of non - rapid eye movement sleep . in a very recent study reported by poskanzer and yuste , the role of neocortical astrocytes in the control of cortical circuit functions", "was examined using in vivo two - photon calcium imaging based on the genetic calcium indicator gcamp6s , together with electrophysiological recording from cortical neurons . to examine the causal relationship between the calcium signaling in astrocytes and neuronal activity in the v1 ,", "an aav encoding cre - dependent arch was injected into the v1 of transgenic mice expressing gfap promoter - driven cre , which resulted in astrocyte - specific expression of the opsin . when expressed in neurons", ", arch hyperpolarizes membrane potentials and inhibits neuronal firing by pumping protons out of neurons in response to yellow - light photostimulation .", "surprisingly , photostimulation of arch in the astrocytes triggered calcium transients that were specifically localized to the processes of astrocytes and largely undetected in the soma .", "in contrast , neighboring arch - negative cells failed to exhibit a calcium response during photostimulation .", "a previous study has reported that arch - mediated stimulation of cerebellar bg increases the intracellular ph as the result of an efflux of protons out of cells under oxygen - glucose - deprived conditions .", "in contrast , poskanzer and yuste found no significant changes in ph in stimulated astrocytes as well as in surrounding cells in the v1 .", "it is not clear whether this discrepancy is a byproduct of cell - type specificity .", "finally , local field potential recordings have revealed that in vivo optogenetic stimulation of astrocytes in the v1 results in calcium transients , followed by a brief increase in extracellular glutamate and a shift in neuronal firing patterns in v1 from a desynchronized state to the synchronized slow oscillation - dominated state ."], ["among diverse chemogenetic effectors , dreadds , such as hm3dq , have been used most frequently in studies focusing on astrocytes . as in optogenetic approaches , astrocyte - specific expression of chemogenetic proteins has been achieved by using viral and transgenic delivery in combination with astrocyte - specific promoters such as the gfap and mlc1 promoters . to manipulate gq - coupled receptor signaling in astrocytes , fiacco et al .", "generated a bi - transgenic mouse line encoding gfap promoter - driven tta and teto promoter - driven mas - related g protein - coupled receptor a1 ( mrgpra1 ) to express gpcr selectively in astrocytes .", "mrgpra1 can be activated by rf amides , such as a peptide phe - leu - arg - phe amide ( flrf ) . since endogenous mrgpra1", "is specifically expressed in dorsal root ganglion neurons but not in the brain , this protein is a useful molecular tool for manipulating neurons and glia in the brain when it is exogenously expressed in these cells .", "an infusion of flrf into acute hippocampal slices prepared from the transgenic mice elicited a robust calcium increase in widespread astrocytes , suggesting that mrgpra1 is able to activate the gq - coupled intracellular signaling pathway .", "it is particularly interesting that such a widespread calcium rise in astrocytes failed to affect neuronal activity in the same slices .", "this finding is at odds with other studies , questioning the hypothesis that an astrocytic calcium increase causes the release of gliotransmitters which , in turn , activate nearby neurons . in a follow - up study using margpra1 mice together with mice lacking inositol 1,4,5-trisphosphate receptor 2 ( ip3 r2 ) ,", "the astrocyte - specific ip3 receptor isoform , the same group of researchers further confirmed that activation of gq protein - coupled signaling affects neither spontaneous excitatory postsynaptic currents nor the induction and maintenance of long - term potentiation in ca1 hippocampal neurons .", "in another study performed by the same group of researchers , bonder and mccarthy reported that hm3dq can be selectively expressed in astrocytes by injecting aav incorporating cre - dependent hm3dq into the visual cortex of transgenic mice encoding gfap promoter - driven cre , and they have used this system to investigate whether the astrocytic calcium elevation triggers vasodilation and a change in local blood flow in the cortex .", "activation of hm3dq with cno was sufficient to increase the intracellular calcium level in astrocytes but not to alter the basal blood flow in the visual cortex .", "the study also reported the absence of a temporal correlation between the astrocytic calcium increase and the change in cortical blood flow following either visual stimulation or a startle - evoking air puff .", "furthermore , genetic deletion of ip3 r2 did not affect neurovascular coupling , suggesting that gq signaling and ip3-dependent calcium elevation in astrocytes do not mediate vasodilation in the visual cortex .", "the gfap - driven mrgpra1 mouse line was used in a more recent study of cao and coworkers to investigate the role of astrocytic atp release in depression - like behaviors .", "application of the mrgpra1 agonist flrf elicited not only a robust increase in intracellular calcium in mrgpra1-expressing primary astrocytes but also a 2.5-fold increase in the atp concentration in the culture medium .", "furthermore , mrgpra1 mice infused with flrf into the cerebral ventricle exhibited a substantial reduction in depression - like behavior elicited in the murine paradigm of chronic social - defeat stress .", "together with other results reported in the study , these findings suggest that endogenous atp released from astrocytes can induce antidepressant - like behavior . whether intracellular calcium increases in astrocytes can affect the activity of neighboring neurons was untested in the study .", "for instance , sweger et al . developed a mouse line expressing an engineered k - opioid receptor ( ro1 ) in gfap - expressing astrocytes by crossing transgenic mice encoding gfap promoter - driven tta mice with another line encoding teto promoter - driven ro1 on the background of a genetic deletion of the endogenous k - opioid receptor ( kor ) .", "ro1 is a gi - coupled gpcr that is insensitive to endogenous ligands of kor , such as dynorphin , but highly sensitive to a synthetic ligand of the k - opioid receptor , spiradoline .", "ro1-expressing transgenic mice developed hydrocephalus and accumulation of cerebrospinal fluid in the ventricular system , even in the absence of a synthetic ligand , suggesting that ro1 is constitutively active in this mouse model ; unfortunately , this constitutive activity negates one of main features of chemogenetics , its temporal controllability , and limits the use of this model .", "the same group of researchers has described another transgenic mouse line expressing gfap promoter - driven hm3dq .", "systemic treatment of these mice with cno elicited a number of physiological changes that are controlled by the autonomic nervous system , including cardiovascular function , saliva formation , and homeostasis of body temperature .", "furthermore , hm3dq - expressing mice receiving cno exhibited substantial changes in activity - related behaviors and motor coordination .", "thus , these findings indicate the critical role of astrocytes in a broad range of basic physiological functions .", "interestingly , the physiological and behavioral changes were unaffected by genetic deletion of ip3 r2 , suggesting that astrocytic ip3-mediated calcium increase is dispensable for hm3dq - elicited responses .", "the gfap promoter - driven hm3dq mice have been used to study glial cells outside of the brain .", "examined the potential role of the enteric glia , which are astrocyte - like peripheral glial cells surrounding enteric neurons in the gut .", "an application of cno to the ileal and colonic myenteric plexus prepared from transgenic mice not only elicited and intracellular calcium increase in astrocytes but also triggered contraction of the ileum and colon to a degree similar to that elicited by direct stimulation of smooth muscle or electrical stimulation of enteric neurons .", "the contraction was abolished by the application of tetradotoxin , indicating the involvement of neuronal activation in the process .", "these findings have demonstrated that astrocytes in the gut play a critical role in the contractions of intestinal smooth muscle .", "the mechanism by which activation of gq - coupled receptor in astrocytes leads to activation of enteric neurons remains unknown .", "have reported two new transgenic mouse lines expressing hm3dq , depending on cre and flippase - mediated recombination . when crossed to cre or flp driver lines ,", "the new mouse lines permit the selective expression of hm3dq in a population of cells that express either cre or flippase .", "in addition , the intersectional strategy involving both cre- and flippas - edependent recombination further restricts hm3dq expression in a specific subpopulation . this group reported that a systemic application of cno to mice expressing hm3dq in gfap - expressing cells elicits hypothermia , confirming the efficacy of the new mouse line by reproducing the previous finding .", "finally , the chemogenetic approach has also been applied to reveal the function of gs - coupled signaling in longterm memory in normal animals and the alzheimer animal model .", "for example , double - transgenic mice encoding gfap promoter - driven tta and teto - driven rs1 have been generated to acutely modulate gs - coupled receptor activity .", "rs1 is the human gs - coupled 5-ht4b serotonin receptor with a point mutation that renders this receptor insensitive to serotonin but highly sensitive to a synthetic ligand , gr-125487 .", "activation of gs signaling by systemic delivery of gr-125487 impairs the performance of transgenic mice in the morris water maze as well as a novel object - recognition task .", "orr et al . found that rs1 is constitutively active in this mouse model , driving the gs signaling pathway even in the absence of the synthetic ligand ."], ["thus far , only a small number of studies focusing on glia have used a virally mediated method to achieve the expression of chemogenetic proteins . for example , an aav encoding gfap promoter - driven hm3dq or hm4di has been stereotactically injected into the arcuate nucleus of the mouse brain to investigate the potential role of medial basal hypothalamic astrocytes in regulating food intake . in the feeding assay , hm3dq - expressing mice receiving cno exhibited a significant reduction in both baseline feeding and ghrelin - elicited hyperphagia , whereas hm4di - expressing mice receiving cno showed substantially enhanced and prolonged ghrelin - evoked feeding . in contrast , following the cno treatment , leptin - induced anorexia was facilitated in hm3dq - expressing mice but suppressed in hm4di - expressing mice .", "thus , this study employing two different chemogenetic actuators that recruit different downstream signaling molecules clearly demonstrated that astrocytes in the arcuate nucleus exert bi - directional regulation of food consumption .", "an aav virus expressing gfap promoter - driven hm3dq was injected into the rat nucleus accumbens core ( nacore ) in two recent studies in order to investigate the contribution of glial cells and extracellular glutamate to substance abuse and motivation .", "reported that an application of cno elicited an elevation of the intracellular calcium level in hm3dq - expressing primary astrocytes and a decrease in motivation for self - administration of ethanol after 3 weeks of abstinence .", "showed that intracranial or systemic administration of cno triggered an increase in extracellular glutamate in the nacore .", "furthermore , hm3dq - expressing rats receiving intraperitoneal cno exhibited a significant reduction in the cueinduced reinstatement of cocaine seeking ."], ["neurons have always been a main focus of brain research , and non - neuronal cells that make up the majority of brain cells , such as glial cells , have not received much attention until recently .", "studies of glia have revealed that they do not merely provide food and support to neurons ; rather , they play an important role in brain function . in order to understand astrocytic function ,", "it is critical to be able to control their intracellular activity in a native context .", "since glia are intermingled with neurons in the brain and they express receptors and ion channels that are also expressed in neurons , it is difficult to perform such manipulation selectively in glial cells , while leaving neighboring neurons unaffected .", "optogenetics and chemogenetics are novel manipulation techniques based on genetically encoded effector molecules , such as specific ion channels and gpcrs , that respond to exogenously delivered light stimuli or synthetic ligands , but are unresponsive to endogenous molecules . in combination with cell type - specific promoters and other genetic tools , expression of effector molecules can be restricted to specific cell types .", "thus , the features of spatial and temporal control make it possible to perform a time - resolved functional manipulation in a specific population of cells .", "optogenetics and chemogenetics have been used most extensively in the study of neuronal circuits and behavior , but they have also been employed in a number of studies focusing on glial cells , mainly astrocytes .", "such studies have demonstrated that astrocytes play a critical role not only in a variety of basic physiological responses , including visual processing , norepinephrine release , breathing , cerebral blood flow , feeding , memory , and sleep , but also in pathological conditions , including drug addition , depression , and ischemia .", "those studies have further revealed the importance of gliotransmitters , such as extracellular glutamate , atp , and l - lactate , that modulate excitability and synaptic transmission in neighboring neurons .", "however , it is still debatable whether astrocytic release of gliotransmitters is a calcium - dependent process .", "in addition , the exact molecular mechanisms governing gliotransmitter release from astrocytes remains to be revealed . a combinatorial approach of advanced functional manipulation techniques such as optogenetics and chemogenetics , together with pharmacological and molecular genetic methods , can further our understanding of glial function in health and disease , including neurodevelopment , neurodegenerative disorders , and neuroinflammatory conditions ."]]}
{"article_id": "PMC4412552", "article_text": ["the online version of this article ( doi:10.1007/s10953 - 014 - 0276-y ) contains supplementary material , which is available to authorized users .", "in recent years , the deep desulfurization of diesel fuel has become the most studied process with different techniques ( extraction , liquid  liquid separation , oxidative desulfurization , adsorption ) .", "the emission of sulfur from petrol and diesel oils , which is linked to acid rain , plays a crucial role in pollution problems of large conglomerates .", "thus , the usa and european countries have issued regulations regarding sulfur content in fuels [ 1 , 2 ] . due to this situation ,", "the european union approved a new directive stating that the content of total sulfur in european gasoline and diesel fuels from 2010 onwards must be at a maximum concentration level of 10  ppm .", "ionic liquids ( ils ) have the ability to extract aromatic sulfur - containing compounds at ambient conditions .", "additionally , ils are immiscible with the fuel , are non - volatile and can be regenerated and recycled by solvent washing .", "oxidative desulfurization in future years probably will bring better results than simple liquid  liquid separation , however , first the best ils must be chosen . at present , the hydrodesulfurization ( hds ) processes is the established method used in some industrial technologies to remove organic sulfur from fuels .", "however , to achieve low sulfur targets with current hds technology , higher temperatures , higher pressures , larger reactor volumes , and more active catalysts are needed .", "the hds process does not purify fuels of polycyclic organic sulfides such as thiophene , benzothiophene , methyldibenzothiophenes , 4,6-dibenzothiophenethiols , thioethers , and disulfides .", "extraction desulfurization , which has begun to be popular , especially with ils , has the potential for being an alternative and future complementary technology for deep desulfurization [ 410 ] . in order to solve this problem , extractive liquid", " liquid equilibrium ( lle ) desulfurization with ils has been proposed [ 7 , 1018 ] .", "the 1-alkylpiperidinium - based , or pyrrolidinium - based ils with different anions , or 1-alkylcyanopyridinium - based ils , have been recently studied in our laboratory in ternary lle { il  +  thiophene , or benzothiophene  +  heptane ) with high selectivities .", "attractive extraction parameters were presented as well for 1-ethyl-3-methylimidazolium bis{(trifluoromethyl)sulfonyl}imide , [ emim][ntf2 ] ( , and references cited therein ) , 1-ethyl-3-methylimidazolium acetate , [ emim][oac ] , 1-ethyl-3-methylimidazolium thiocyanate , [ emim][scn ] , and 1,3-dimethylimidazolium methylphosphonate [ dmim][mp ] .", "this work is a continuation of our systematic studies on the physicochemical properties and the extraction abilities of piperidinium - based ils ( and references cited therein ) .", "proposed by us are new interaction parameters for the group contribution method modified unifac for the piperidnium - based ils , predicted attractive infinite dilution selectivity , and capacity of piperidinium - based ils ( alkane chain , n  =  36 ) in the thiophene / heptane separation problem at t  =  328.15  k. to our best knowledge , the phosphonium - based il ( tributyl - methylphosphonium methylsulfate , [ p1,4,4,4][ch3so4 ] ) was measured in ternary lle for the separation of thiophene from cyclohexane at t  =  298.15  k with very low selectivities in a range of 1.5 to 5.4 .", "better results were obtained with deep eutectic solvents , des , containing phosphonium - based ils with ethylene glycol .", "des is composed of methyltripentylphosphonium bromide , [ p1,5,5,5][br ] , and ethylene glycol ( as a hydrogen bond donor ) showed selectivities of about s  =  60100 for ternary lle at t  =  318  k for benzene / hexane separation .", "poorer results were estimated with des composed of tetrabutylphosphonium bromide , [ p4,4,4,4][br ] , and ethylene glycol for the separation of toluene / heptane .", "usually , the results of separation processes for aliphatic / aromatic hydrocarbons provide good information for the separation of aliphatic / aromatic sulfur compounds .", "we can expect similar or even better results for the chosen il . on the other side ,", "there are very good results obtained with methylphosphonate   and diethylphosphate   anions of ils in ternary lle ( il  +  thiophene  +  heptane ) mixtures . in this work", "we report experimental ternary lle data for one additional piperidinium - based il , { 1-pentyl-1-methylpiperidinium bis{(trifluoromethyl)sylfonyl}imide [ c5mpip][ntf2 ] , for comparison with measured earlier 1-propyl , or 1-butyl- , or 1-hexyl-1-methylpiperidinium bis{(trifluoromethyl)sylfonyl}imide .", "moreover , tributylethylphosphonium diethylphosphate , [ p2,4,4,4][dep ] was chosen to check the influence of the anion .", "the [ dep ] anion in [ emim][dep ] shows interesting results for thiophene extraction from hexane at t  =  298.15  k .", "the solvents heptane , thiophene , and benzothiophene used in this work are model compounds for fuel and sulfur organic hydrocarbons , respectively .", "the ternary systems { il ( 1 )  +  thiophene , or benzothiophene ( 2 )  +  heptane ( 3 ) } were investigated at t  =  308.15  k and p  =  101.33  kpa . the experimental tie - lines for four ternary mixtures", "the solute distribution ratio and the extractive selectivity were determined from the experimental data , and are compared to the literature data .", "the ils studied , [ c1c5pip][ntf2 ] and [ p2,4,4,4][dep ] , were purchased from iolitec .", "the names , abbreviations , structures , measured densities and mass fraction of ils are listed in table  1 .", "the names , cas numbers , sources , mass fraction purities , purification method , water content , and measured and literature densities of all chemicals used are shown in table  1s in the supplementary material information .", "the samples of ils were dried for 24  h at 300  k under reduced pressure to remove volatile impurities and trace amounts of water .", "thiophene and benzothiophene were stored over freshly activated molecular sieves of type 4   ( union carbide ) .", "the densities for all substances were measured at t  =  298.15 and 101.33  kpa . the method and uncertainties", "have been described previously .table  1list of investigated ionic liquids : structure , name , abbreviation of name , molar mass ( m ) and densitycationanionname , abbreviationm/(gmol)exp .", "density /gcm ( 298.15  k ; 101.33  kpa ) \n\n\n\n 1-pentyl-1-methylpiperidinium bis{(trifluoromethyl ) sulfonyl}imide [ c1c5pip][ntf2]450.461.35016 \n\n\n\n tributylethylphosphonium diethylphosphate , [ p2,4,4,4][dep]384.471.0089 list of investigated ionic liquids : structure , name , abbreviation of name , molar mass ( m ) and density the water content was analyzed by the karl - fischer titration ( method titroline kf ) .", "the sample of il , or solvent , was dissolved in methanol and titrated in steps of 0.0025  cm .", "the error in the water content is 10    10 in mass fraction for the 3  cm of injected il .", "the water content in solvents used was less than 350    10 in mass fraction . to obtain the experimental lle tie - lines , mixtures with compositions inside the immiscible region of the systems", "the vessel was tightly closed to avoid losses by evaporation or pickup of moisture from the atmosphere .", "the jackets were connected to a thermostatic water bath ( lauda alpha ) to maintain a constant temperature of t  =  308.15  k ( 0.05 ) .", "the mixtures were stirred for 6  h to reach thermodynamic equilibrium and after a minimum of 12  h were analyzed .", "after the phase separation , samples of about ( 0.10.3 )    10 cm were taken from both phases using glass syringes with coupled stainless steel needles .", "a sample of the phase was placed in an ampoule with a capacity of 2    10 cm .", "next , acetone ( 1.0  cm ) was added to the samples to avoid phase splitting and to maintain a homogeneous mixture .", "because of the low vapor pressure , the ils used in this work can not be analyzed by gc .", "thus , only thiophene or benzothiophene and heptane were analyzed ; the mass fraction of the third component , the il , was determined by subtracting the mole fractions of the two other components from unity .", "the compositions were analyzed by gas chromatography ( perkinelmer clarus 580 gc equipped with auto sampler and fid and tcd detectors ) .", "the capillary column of the chromatograph was protected with a pre - column to avoid the non - volatile ionic liquid reaching the column in the case of a leak from the glass wool in the liner .", "the totalchrom workstation software was used to obtain the chromatographic areas for the thiophene , or benzothiophene , heptane and the internal standard propan-1-ol .", "details of the operational conditions of the apparatus are reported in table  2s in the supplementary material .", "the estimated uncertainty in the determination of mole fraction compositions is    0.003 for compositions of the hydrocarbon - rich phase and 0.005 for compositions of the il - rich phase .", "the equilibrium compositions of the experimental tie - line ends in ternary systems of four mixtures { il ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k and p  =  101.33  kpa are reported in table  2 .", "experimental solubilities for [ c1c5pip][ntf2 ] and [ p2,4,4,4][dep ] in heptane at t  =  308.15  k are totally different from each other . in the binary { il ( 1 )  +  heptane ( 3 )", "} system complete liquid miscibility ( solubility of heptane in the il ) is up to mole fraction of heptane \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{il } } $ $ \\end{document}x3il  =  0.089 and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{il } } $ $ \\end{document}x3il  =  0.456 for [ c1c5pip][ntf2 ] and [ p2,4,4,4][dep ] , respectively .", "the solubility of heptane is much larger in [ p2,4,4,4][dep ] than that in [ c1c5pip][ntf2 ] .", "the piperidinium - based il shows much lower solubility of heptane in the il . in comparison with piperidinium - based il measured by us earlier", ", heptane shows higher solubility in [ c1c5pip][ntf2 ] than in [ c1c3pip][ntf2 ] ( \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{il } } $ $ \\end{document}x3il  =  0.051 , at t  =  308.15  k ) .", "this effect is due to an increase in the van der waals interactions between the hydrocarbon chain of the cation and heptane.table  2compositions of experimental tie lines , solute distribution ratios ,  , and selectivity , s , for ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpahydrocarbon - rich phaseil - rich phase \n  \n\n s \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{1}^{\\text{i } } $ $ \\end{document}x1i \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{i } } $ $ \\end{document}x2i \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{1}^{\\text{ii } } $ $ \\end{document}x1ii \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{ii } } $ $ \\end{document}x2ii \n [ c1c5pip][ntf2 ]  +  thiophene  +  heptane  0.0000.0000.9110.000  0.0000.0570.7700.1422.4926.7  0.0000.1110.6630.2502.2523.0  0.0000.1910.5640.3521.8417.7  0.0000.2300.5230.3921.7015.4  0.0000.3530.4240.4901.3910.4  0.0000.4470.3760.5421.218.2  0.0000.5300.3370.5821.106.4  0.0000.6960.2810.6500.934.1 ", "0.0000.8000.2410.7020.883.1  0.0000.8910.2130.7500.842.5  0.0001.0000.1860.8140.81[c1c5pip][ntf2 ]  +  benzothiophene  +  heptane  0.0000.0000.9110.000  0.0000.0250.8030.1094.3648.3  0.0000.0520.7050.2104.0445.0  0.0000.0920.5890.3263.5437.9  0.0000.1610.4440.4722.9329.3  0.0000.2430.3830.5312.1919.2  0.0000.3460.2990.6111.7712.8  0.0000.4600.2360.6741.478.8  0.0000.5530.2080.6961.265.9 ", "0.0000.7660.1440.7580.992.4  0.0000.8530.0960.8190.961.7  0.0000.9150.0600.8730.951.2[p2,4,4,4][dep ]  +  thiophene  +  heptane  0.0020.0000.5440.000  0.0020.0160.5150.0432.696.0  0.0040.0430.4430.1042.425.1  0.0050.0790.3760.1702.154.3  0.0080.1120.3230.2141.913.6  0.0090.1480.2740.2571.743.1  0.0080.1720.2440.2801.632.8  0.0100.1990.2010.3011.512.4  0.0130.2120.1870.3091.462.2  0.0130.2390.1600.3261.362.0[p2,4,4,4][dep ]  +  benzothiophene  +  heptane  0.0020.0000.5480.000  0.0060.0150.4950.0624.139.1  0.0070.0440.4180.1633.708.4  0.0090.0680.3690.2243.297.5  0.0060.0920.3270.2773.016.9  0.0110.1200.2870.3232.696.0  0.0060.1670.2180.3802.284.7  0.0140.2410.1710.4221.753.2  0.0140.2710.1470.4351.612.7 ", "0.0150.3110.1150.4531.462.3standard uncertainties are : u(x )  <  0.003 , u(t )  =  0.05  k compositions of experimental tie lines , solute distribution ratios ,  , and selectivity , s , for ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpa standard uncertainties are : u(x )  <  0.003 , u(t )  =  0.05  k the solubility of thiophene at t  =  308.15  k is equal to \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{il } } $ $ \\end{document}x2il  =  0.814 for [ c1c5pip][ntf2 ] ( x2il  =  0.797 for [ c1c3pip][ntf2 ] at t  =  298.15  k , the influence of temperature is minimal ; the largest solubility of thiophene in the piperidinium - based il was observed for c1c6pip][ntf2 ] ) .", "complete miscibility with thiophene was observed for [ p2,4,4,4][dep ] . in the binary system with benzothiophene ,", "the solubility of benzothiophene in [ c1c5pip][ntf2 ] at t  =  308.15  k is equal to \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{il } } $ $ \\end{document}x2il  =  0.873 ( \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{il } } $ $ \\end{document}x2il  =  0.945 for [ c1c3pip][ntf2 ] at t  =  308.15  k .", "immiscibility is observed in the { thiophene , or benzothiophene ( 2 )  +  heptane ( 3 ) } binary mixture , as was reported previously .", "the determined experimental tie - lines for the ternary lle systems are plotted in figs .  1 , 2 , 3 , 4 for thiophene and benzothiophene , respectively", ". figures  1 , 2 , 3 , 4 show that the two - phase region is much larger for [ c1c5pip][ntf2 ] than that for [ p2,4,4,4][dep].fig . ", "1plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  kfig . ", "2plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  kfig . ", "3plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  kfig . ", "4plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k the results obtained in this work show that the more suitable il for the separation of thiophene , or benzothiophene from heptane , is [ c1c5pip][ntf2 ] because of its much larger selectivity ( s ) and the comparable solute distribution ratio (  ) .", "these parameters are defined as follows:1\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\beta = \\frac{{x _ { 2}^{\\text{ii } } } } { { x _ { 2}^{\\text{i } } } } $ $ \\end{document}=x2iix2i2\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ s = \\frac{{x _ { 2}^{\\text{ii } } \\cdot x _ { 3}^{\\text{i } } } } { { x _ { 2}^{\\text{i } } \\cdot x _ { 3}^{\\text{ii } } } } $ $ \\end{document}s = x2iix3ix2ix3iiwhere x is the mole fraction ; superscripts i and ii refer to the heptane - rich phase and the il - rich phase , respectively .", "the values of  and s are listed in table  2 for thiophene and benzothiophene .", "figures  5 and 6 present measured values of  and s for ils for thiophene and benzothiophene.fig . ", "5plot of the selectivity ( s ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  kfig . ", "6plot of the solute distribution ratio (  ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k plot of the selectivity ( s ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k plot of the solute distribution ratio (  ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k the values presented in table  2 show that the distribution ratio coefficient are in the range of 0.812.41 , 0.954.36 , 1.362.69 and 1.464.13 for [ c1c5pip][ntf2]/thiophene , [ c1c5pip][ntf2]/benzothiophene , [ p2,4,4,4][dep]/thiophene and [ p2,4,4,4][dep]/benzothiophene , respectively .", "the selectivities of the separation in the system thiophene or benzothiophene / heptane is quite high for [ c1c5pip][ntf2 ] and very low for [ p2,4,4,4][dep ] .", "the values listed in table  2 for the best tie - lines are : 26.7 , 48.3 , 6.0 and 9.1 for [ c1c5pip][ntf2]/thiophene , [ c1c5pip][ntf2]/benzothiophene , [ p2,4,4,4][dep]/thiophene and [ p2,4,4,4][dep]/benzothiophene , respectively . in this work", "the effect of the alkane chain length on the cation of the piperidinium - based il was examined for comparison with previously measured data for [ c1c3pip][ntf2]/thiophene , [ c1c4pip][ntf2]/thiophene , and [ c1c6pip][ntf2]/thiophene at t  =  298.15  k , and of [ c1c3pip][ntf2]/benzothiophene at t  =  308.15  k   ( the influence of temperature is not large ) .", "the characteristic extraction parameters obtained in this work are compared to the few previously described in the open literature in table  3 .", "unfortunately , the selectivity for [ c1c5pip][ntf2 ] obtained in this work is slightly worse than that for [ c1c3pip][ntf2 ] measured by us earlier .", "the values of selectivity presented for 1-alkylcyanopyridinium - based ils at t  =  308.15  k measured in our earlier work   are also larger than those for piperidinium - based ils   ( see table  3).table  3comparison of solute distribution ratio (  ) and selectivity ( s ) for sulfur compounds extractionillle system \n", "t / k \n  \n max \n\n s \n max \n ref.[c1c5pip][ntf2]il  +  thiophene  +  heptane3082.4926.7this work[c1c5pip][ntf2]il  +  benzothiophene  +  heptane3084.3648.3this work[c1c3pip][ntf2]il  +  thiophene  +  heptane2982.5060.3[c1c3pip][ntf2]il  +  benzothiophene  +  heptane3085.3693.0[coc2mpip][ntf2 ] \n il  +  thiophene  +  heptane2982.6462.9[coc2mpip][fap ] \n il  +  thiophene  +  heptane2984.0056.8[bcnpy][ntf2 ] \n il  +  thiophene  +  heptane3081.9362.2[bcnpy][ntf2 ] \n il  +  benzothiophene  +  heptane3083.50117.1[p2,4,4,4][dep]il  +  thiophene  +  heptane3082.696.0this work[p2,4,4,4][dep]il  +  benzothiophene  +  heptane3084.139.1this work[emim][dep ] \n il  +  thiophene  +  hexane2982.8848.8[p1,4,4,4][ch3so4]il  +  thiophene  +  cyclohexane2981.385.38[dmim][mp ] \n il  +  thiophene  +  heptane2980.421,756 \n 1-(2-methoxyethyl)-1-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide \n 1-(2-methoxyethyl)-1-methylpiperidinium trifluorotris(perfluoroethyl)phosphate \n 1-butyl-4-cyanopyridinium bis{(trifluoromethyl)sulfonyl}imide \n 1-ethyl-3-methylimidazolium diethylphosphate \n 1,3-dimethylimidazolium methylphosphonate comparison of solute distribution ratio (  ) and selectivity ( s ) for sulfur compounds extraction \n 1-(2-methoxyethyl)-1-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide \n 1-(2-methoxyethyl)-1-methylpiperidinium trifluorotris(perfluoroethyl)phosphate \n 1-butyl-4-cyanopyridinium bis{(trifluoromethyl)sulfonyl}imide \n 1-ethyl-3-methylimidazolium diethylphosphate \n 1,3-dimethylimidazolium methylphosphonate the selectivities for [ c1c3pip][ntf2 ] are comparable to those for 4-(2-methoxyethyl)-4-methylpiperidinium trifluorotris(perfluoroethyl)phosphate [ coc2mpip][fap ] ils at t  =  298.15  k , or to 4-(2-methoxyethyl)-4-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide [ coc2mpip][ntf2 ] .", "the extraction results for [ p2,4,4,4][dep ] are very low and similar to [ p1,4,4,4][ch3so4 ] .", "it can be definitely concluded that phosphonium - based cations are not suitable for these separation processes .", "however , for the diethylphosphate anion [ dep ] and imidazolium - based cation [ emim ] , the results are comparable to those obtained in this work with [ c1c5pip][ntf2 ] but with a lower  value   ( see table  3 ) .", "it can be also seen from figs .  5 and 6 that  and s decrease as the solute mole fraction ( thiophene , or benzothiophene ) in the heptane phase increases , for all systems , when going through the tie - line end compositions .", "the ternary lle data measured in this study were correlated ( the tie - line correlation ) using the well known non - random liquid equation , nrtl .", "the equations and algorithms used for the calculation of the compositions in both phases follow the method described by walas .", "the objective function f(p ) was used to minimize the difference between the experimental and calculated compositions:3\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ f(p ) = \\sum\\limits_{i = 1}^{n } { \\left [ { x_{2}^{{{\\text{i,}}exp } } - x _ { 2}^{\\text{i , calc } } \\left ( { pt } \\right ) } \\right]^{2 } + \\left [ { x_{3}^{{{\\text{i,}}exp } } - x_{3}^{\\text{i , calc } } \\left ( { pt } \\right ) } \\right]}^{2 } + \\left [ { x_{2}^{{{\\text{ii,}}exp } } - x _ { 2}^{\\text{ii , calc } } \\left ( { pt } \\right ) } \\right]^{2 } + \\left [ { x_{3}^{{{\\text{ii,}}exp } } - x _ { 3}^{\\text{ii , calc } } \\left ( { pt } \\right ) } \\right]^{2 } $ $ \\end{document}f(p)=i=1nx2i , exp - x2i , calcpt2+x3i , exp - x3i , calcpt2+x2ii , exp - x2ii , calcpt2+x3ii , exp - x3ii , calcpt2where p is the set of parameters vector , n is the number of experimental points , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{{{\\text{i}},exp } } $ $ \\end{document}x2i , exp , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{{{\\text{i}},exp } } $ $ \\end{document}x3i , exp and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x _ { 2i}^{{{\\text{i}},{\\text{calc } } } } \\left ( { pt } \\right ) $ $ \\end{document}x2ii , calcpt , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{i , calc } } \\left ( { pt } \\right ) $ $ \\end{document}x3i , calcpt are the experimental and calculated mole fractions of one phase , and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{{{\\text{ii}},exp } } $ $ \\end{document}x2ii , exp , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{{{\\text{ii,}}exp } } $ $ \\end{document}x3ii , exp\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x _ { 2}^{\\text{ii , calc } } \\left ( { pt } \\right ) $ $ \\end{document}x2ii , calcpt , and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x _ { 3}^{\\text{ii , calc } } \\left ( { pt } \\right ) $ $ \\end{document}x3ii , calcpt are the experimental and calculated mole fractions of the second phase .", "the binary parameters of each constituent were regressed by minimizing the sum of the squares of the differences between the experimental and calculated mole fractions of each component of both liquid phases for each ternary system .", "the value of the non - randomness parameter , ij , was optimized in order to obtain the best model fit .", "the correlated parameters are given in table  4 along with the root mean square deviations ( rmsd ) .", "the rmsd values , which are a measure of the precision of the correlation , were calculated according the equation:4\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ { \\text{rmsd } } = \\left ( { \\sum\\limits_{i } { \\sum\\limits_{l } { \\sum\\limits_{m } { \\left [ { x_{ilm}^{exp } - x_{ilm}^{\\text{calc } } } \\right]^{2 } /6k } } } } \\right)^{1/2 } $ $ \\end{document}rmsd=ilmxilmexp - xilmcalc2/6k1/2where x is the mole fraction and the subscripts i , l , and m designate the component , phase , and tie - line , respectively .", "the experimental and calculated lle data agreed relatively well.table  4binary interaction parameters , parameter  \n ij and root mean square deviation (  \n x ) for the nrtl equation for the ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpa \n ij \n g \n 12/(jmol)g \n 21/(jmol ) \n  \n ij \n rmsd  \n x \n [ c1c5pip][ntf2 ]  +  thiophene  +  heptane  128261.1123225.940.20.007  132536.4315361.68  23460.97475.45[c1c5pip][ntf2 ]  +  benzothiophene  +  heptane  125698.7312608.560.20.007  135142.9534672.57  232498.842232.66[p2,4,4,4][dep ]  +  thiophene  +  heptane  125566.067459.650.20.003  132785.5519670.14  233351.226008.81[p2,4,4,4][dep ]  +  benzothiophene  +  heptane  129265.5911427.530.20.004  132823.0616531.68  231048.012808.72 binary interaction parameters , parameter  \n ij and root mean square deviation (  \n x ) for the nrtl equation for the ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpa", "liquid phase equilibrium data were measured in this study for the extraction of thiophene or benzothiophene from heptane using two ils . four ternary systems { il  +  thiophene or benzothiophene  +  heptane } were analytically determined using gc for the composition analysis at temperature t  =  308.15  k at ambient pressure .", "it has been demonstrated that the 1-pentyl-1-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide il is much more effective than the phosphonium - based il for extraction of thiophene or benzothiophene from alkanes .", "sulfur compounds can be extracted easily by piperidinium - based ils , leading to low sulfur content in fuels .", "our earlier experimental results revealed that the solubility of sulfur compounds in the il increases as the alkyl chain length increases .", "the capacity of extraction , described in terms of the selectivity and the solute distribution ratio coefficients , was calculated for all ternary systems and compared to the published data used in similar extraction problems .", "based on the values obtained , [ c1c5pip][ntf2 ] was found to be useful for the extraction of sulfur compounds from alkanes ; however , it is not as good as [ c1c3pip][ntf2 ] measured previously . the selectivity and the solute distribution ratio decrease as the mole fraction of thiophene or benzothiophene in the heptane - rich phase increases . the best selectivity ( s )", "is observed for very low mole fractions of s - compounds in the hydrocarbon - rich phase \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ ( x_{2}^{\\text{hc } } \\ , = \\,0.0 5 ) $ $ \\end{document}(x2hc=0.05 ) ( see fig . ", "5 ) , which may be compared with the results of the hds method for the removal of the s - compounds . the experimental data in this work", "the non - randomness parameter was also determined through the reduction of the experimental data .", "the model exhibited an excellent fit to the data with the average rmsd values between 0.003 and 0.007 . \n"], "abstract_text": ["<S> in this work , the desulfurization ability of alkyl - piperidinium - based and phosphonium - based ionic liquids ( ils ) for ( thiophene or benzothiophene  +  heptane ) mixtures are studied . with this aim , ternary liquid </S>", "<S>  liquid phase equilibrium data ( lle ) have been obtained for mixtures of { il ( 1 )  +  thiophene , or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k and p  =  101.33  kpa . for this study 1-pentyl-1-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide , [ c1c5pip][ntf2 ] , and tributylethylphosphonium diethylphosphate , [ p2,4,4,4][dep ] , </S>", "<S> were used . </S>", "<S> the suitability of these ils as solvents for extractive desulfurization has been evaluated in terms of the solute distribution ratio and selectivity . </S>", "<S> immiscibility was observed in the binary liquid systems of ( thiophene , or benzothiophene  +  heptane ) with both ils . </S>", "<S> one of the studied ils , [ c1c5pip][ntf2 ] , shows high distribution ratios and high selectivities for extraction of sulfur compounds . </S>", "<S> the data obtained have been correlated with the non - random two liquid nrtl model . </S>", "<S> the experimental tie - lines and the phase compositions in mole fractions in the ternary systems were calculated with an average root mean square deviation of 0.0052.electronic supplementary materialthe online version of this article ( doi:10.1007/s10953 - 014 - 0276-y ) contains supplementary material , which is available to authorized users . </S>"], "labels": null, "section_names": ["Electronic supplementary material", "Introduction", "Experimental", "Results and Discussion", "Data Correlation", "Conclusions", "Electronic Supplementary Material"], "sections": [["the online version of this article ( doi:10.1007/s10953 - 014 - 0276-y ) contains supplementary material , which is available to authorized users ."], ["in recent years , the deep desulfurization of diesel fuel has become the most studied process with different techniques ( extraction , liquid  liquid separation , oxidative desulfurization , adsorption ) .", "the emission of sulfur from petrol and diesel oils , which is linked to acid rain , plays a crucial role in pollution problems of large conglomerates .", "thus , the usa and european countries have issued regulations regarding sulfur content in fuels [ 1 , 2 ] . due to this situation ,", "the european union approved a new directive stating that the content of total sulfur in european gasoline and diesel fuels from 2010 onwards must be at a maximum concentration level of 10  ppm .", "ionic liquids ( ils ) have the ability to extract aromatic sulfur - containing compounds at ambient conditions .", "additionally , ils are immiscible with the fuel , are non - volatile and can be regenerated and recycled by solvent washing .", "oxidative desulfurization in future years probably will bring better results than simple liquid  liquid separation , however , first the best ils must be chosen . at present , the hydrodesulfurization ( hds ) processes is the established method used in some industrial technologies to remove organic sulfur from fuels .", "however , to achieve low sulfur targets with current hds technology , higher temperatures , higher pressures , larger reactor volumes , and more active catalysts are needed .", "the hds process does not purify fuels of polycyclic organic sulfides such as thiophene , benzothiophene , methyldibenzothiophenes , 4,6-dibenzothiophenethiols , thioethers , and disulfides .", "extraction desulfurization , which has begun to be popular , especially with ils , has the potential for being an alternative and future complementary technology for deep desulfurization [ 410 ] . in order to solve this problem , extractive liquid", " liquid equilibrium ( lle ) desulfurization with ils has been proposed [ 7 , 1018 ] .", "the 1-alkylpiperidinium - based , or pyrrolidinium - based ils with different anions , or 1-alkylcyanopyridinium - based ils , have been recently studied in our laboratory in ternary lle { il  +  thiophene , or benzothiophene  +  heptane ) with high selectivities .", "attractive extraction parameters were presented as well for 1-ethyl-3-methylimidazolium bis{(trifluoromethyl)sulfonyl}imide , [ emim][ntf2 ] ( , and references cited therein ) , 1-ethyl-3-methylimidazolium acetate , [ emim][oac ] , 1-ethyl-3-methylimidazolium thiocyanate , [ emim][scn ] , and 1,3-dimethylimidazolium methylphosphonate [ dmim][mp ] .", "this work is a continuation of our systematic studies on the physicochemical properties and the extraction abilities of piperidinium - based ils ( and references cited therein ) .", "proposed by us are new interaction parameters for the group contribution method modified unifac for the piperidnium - based ils , predicted attractive infinite dilution selectivity , and capacity of piperidinium - based ils ( alkane chain , n  =  36 ) in the thiophene / heptane separation problem at t  =  328.15  k. to our best knowledge , the phosphonium - based il ( tributyl - methylphosphonium methylsulfate , [ p1,4,4,4][ch3so4 ] ) was measured in ternary lle for the separation of thiophene from cyclohexane at t  =  298.15  k with very low selectivities in a range of 1.5 to 5.4 .", "better results were obtained with deep eutectic solvents , des , containing phosphonium - based ils with ethylene glycol .", "des is composed of methyltripentylphosphonium bromide , [ p1,5,5,5][br ] , and ethylene glycol ( as a hydrogen bond donor ) showed selectivities of about s  =  60100 for ternary lle at t  =  318  k for benzene / hexane separation .", "poorer results were estimated with des composed of tetrabutylphosphonium bromide , [ p4,4,4,4][br ] , and ethylene glycol for the separation of toluene / heptane .", "usually , the results of separation processes for aliphatic / aromatic hydrocarbons provide good information for the separation of aliphatic / aromatic sulfur compounds .", "we can expect similar or even better results for the chosen il . on the other side ,", "there are very good results obtained with methylphosphonate   and diethylphosphate   anions of ils in ternary lle ( il  +  thiophene  +  heptane ) mixtures . in this work", "we report experimental ternary lle data for one additional piperidinium - based il , { 1-pentyl-1-methylpiperidinium bis{(trifluoromethyl)sylfonyl}imide [ c5mpip][ntf2 ] , for comparison with measured earlier 1-propyl , or 1-butyl- , or 1-hexyl-1-methylpiperidinium bis{(trifluoromethyl)sylfonyl}imide .", "moreover , tributylethylphosphonium diethylphosphate , [ p2,4,4,4][dep ] was chosen to check the influence of the anion .", "the [ dep ] anion in [ emim][dep ] shows interesting results for thiophene extraction from hexane at t  =  298.15  k .", "the solvents heptane , thiophene , and benzothiophene used in this work are model compounds for fuel and sulfur organic hydrocarbons , respectively .", "the ternary systems { il ( 1 )  +  thiophene , or benzothiophene ( 2 )  +  heptane ( 3 ) } were investigated at t  =  308.15  k and p  =  101.33  kpa . the experimental tie - lines for four ternary mixtures", "the solute distribution ratio and the extractive selectivity were determined from the experimental data , and are compared to the literature data ."], ["the ils studied , [ c1c5pip][ntf2 ] and [ p2,4,4,4][dep ] , were purchased from iolitec .", "the names , abbreviations , structures , measured densities and mass fraction of ils are listed in table  1 .", "the names , cas numbers , sources , mass fraction purities , purification method , water content , and measured and literature densities of all chemicals used are shown in table  1s in the supplementary material information .", "the samples of ils were dried for 24  h at 300  k under reduced pressure to remove volatile impurities and trace amounts of water .", "thiophene and benzothiophene were stored over freshly activated molecular sieves of type 4   ( union carbide ) .", "the densities for all substances were measured at t  =  298.15 and 101.33  kpa . the method and uncertainties", "have been described previously .table  1list of investigated ionic liquids : structure , name , abbreviation of name , molar mass ( m ) and densitycationanionname , abbreviationm/(gmol)exp .", "density /gcm ( 298.15  k ; 101.33  kpa ) \n\n\n\n 1-pentyl-1-methylpiperidinium bis{(trifluoromethyl ) sulfonyl}imide [ c1c5pip][ntf2]450.461.35016 \n\n\n\n tributylethylphosphonium diethylphosphate , [ p2,4,4,4][dep]384.471.0089 list of investigated ionic liquids : structure , name , abbreviation of name , molar mass ( m ) and density the water content was analyzed by the karl - fischer titration ( method titroline kf ) .", "the sample of il , or solvent , was dissolved in methanol and titrated in steps of 0.0025  cm .", "the error in the water content is 10    10 in mass fraction for the 3  cm of injected il .", "the water content in solvents used was less than 350    10 in mass fraction . to obtain the experimental lle tie - lines , mixtures with compositions inside the immiscible region of the systems", "the vessel was tightly closed to avoid losses by evaporation or pickup of moisture from the atmosphere .", "the jackets were connected to a thermostatic water bath ( lauda alpha ) to maintain a constant temperature of t  =  308.15  k ( 0.05 ) .", "the mixtures were stirred for 6  h to reach thermodynamic equilibrium and after a minimum of 12  h were analyzed .", "after the phase separation , samples of about ( 0.10.3 )    10 cm were taken from both phases using glass syringes with coupled stainless steel needles .", "a sample of the phase was placed in an ampoule with a capacity of 2    10 cm .", "next , acetone ( 1.0  cm ) was added to the samples to avoid phase splitting and to maintain a homogeneous mixture .", "because of the low vapor pressure , the ils used in this work can not be analyzed by gc .", "thus , only thiophene or benzothiophene and heptane were analyzed ; the mass fraction of the third component , the il , was determined by subtracting the mole fractions of the two other components from unity .", "the compositions were analyzed by gas chromatography ( perkinelmer clarus 580 gc equipped with auto sampler and fid and tcd detectors ) .", "the capillary column of the chromatograph was protected with a pre - column to avoid the non - volatile ionic liquid reaching the column in the case of a leak from the glass wool in the liner .", "the totalchrom workstation software was used to obtain the chromatographic areas for the thiophene , or benzothiophene , heptane and the internal standard propan-1-ol .", "details of the operational conditions of the apparatus are reported in table  2s in the supplementary material .", "the estimated uncertainty in the determination of mole fraction compositions is    0.003 for compositions of the hydrocarbon - rich phase and 0.005 for compositions of the il - rich phase ."], ["the equilibrium compositions of the experimental tie - line ends in ternary systems of four mixtures { il ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k and p  =  101.33  kpa are reported in table  2 .", "experimental solubilities for [ c1c5pip][ntf2 ] and [ p2,4,4,4][dep ] in heptane at t  =  308.15  k are totally different from each other . in the binary { il ( 1 )  +  heptane ( 3 )", "} system complete liquid miscibility ( solubility of heptane in the il ) is up to mole fraction of heptane \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{il } } $ $ \\end{document}x3il  =  0.089 and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{il } } $ $ \\end{document}x3il  =  0.456 for [ c1c5pip][ntf2 ] and [ p2,4,4,4][dep ] , respectively .", "the solubility of heptane is much larger in [ p2,4,4,4][dep ] than that in [ c1c5pip][ntf2 ] .", "the piperidinium - based il shows much lower solubility of heptane in the il . in comparison with piperidinium - based il measured by us earlier", ", heptane shows higher solubility in [ c1c5pip][ntf2 ] than in [ c1c3pip][ntf2 ] ( \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{il } } $ $ \\end{document}x3il  =  0.051 , at t  =  308.15  k ) .", "this effect is due to an increase in the van der waals interactions between the hydrocarbon chain of the cation and heptane.table  2compositions of experimental tie lines , solute distribution ratios ,  , and selectivity , s , for ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpahydrocarbon - rich phaseil - rich phase \n  \n\n s \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{1}^{\\text{i } } $ $ \\end{document}x1i \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{i } } $ $ \\end{document}x2i \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{1}^{\\text{ii } } $ $ \\end{document}x1ii \n\n \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{ii } } $ $ \\end{document}x2ii \n [ c1c5pip][ntf2 ]  +  thiophene  +  heptane  0.0000.0000.9110.000  0.0000.0570.7700.1422.4926.7  0.0000.1110.6630.2502.2523.0  0.0000.1910.5640.3521.8417.7  0.0000.2300.5230.3921.7015.4  0.0000.3530.4240.4901.3910.4  0.0000.4470.3760.5421.218.2  0.0000.5300.3370.5821.106.4  0.0000.6960.2810.6500.934.1 ", "0.0000.8000.2410.7020.883.1  0.0000.8910.2130.7500.842.5  0.0001.0000.1860.8140.81[c1c5pip][ntf2 ]  +  benzothiophene  +  heptane  0.0000.0000.9110.000  0.0000.0250.8030.1094.3648.3  0.0000.0520.7050.2104.0445.0  0.0000.0920.5890.3263.5437.9  0.0000.1610.4440.4722.9329.3  0.0000.2430.3830.5312.1919.2  0.0000.3460.2990.6111.7712.8  0.0000.4600.2360.6741.478.8  0.0000.5530.2080.6961.265.9 ", "0.0000.7660.1440.7580.992.4  0.0000.8530.0960.8190.961.7  0.0000.9150.0600.8730.951.2[p2,4,4,4][dep ]  +  thiophene  +  heptane  0.0020.0000.5440.000  0.0020.0160.5150.0432.696.0  0.0040.0430.4430.1042.425.1  0.0050.0790.3760.1702.154.3  0.0080.1120.3230.2141.913.6  0.0090.1480.2740.2571.743.1  0.0080.1720.2440.2801.632.8  0.0100.1990.2010.3011.512.4  0.0130.2120.1870.3091.462.2  0.0130.2390.1600.3261.362.0[p2,4,4,4][dep ]  +  benzothiophene  +  heptane  0.0020.0000.5480.000  0.0060.0150.4950.0624.139.1  0.0070.0440.4180.1633.708.4  0.0090.0680.3690.2243.297.5  0.0060.0920.3270.2773.016.9  0.0110.1200.2870.3232.696.0  0.0060.1670.2180.3802.284.7  0.0140.2410.1710.4221.753.2  0.0140.2710.1470.4351.612.7 ", "0.0150.3110.1150.4531.462.3standard uncertainties are : u(x )  <  0.003 , u(t )  =  0.05  k compositions of experimental tie lines , solute distribution ratios ,  , and selectivity , s , for ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpa standard uncertainties are : u(x )  <  0.003 , u(t )  =  0.05  k the solubility of thiophene at t  =  308.15  k is equal to \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{il } } $ $ \\end{document}x2il  =  0.814 for [ c1c5pip][ntf2 ] ( x2il  =  0.797 for [ c1c3pip][ntf2 ] at t  =  298.15  k , the influence of temperature is minimal ; the largest solubility of thiophene in the piperidinium - based il was observed for c1c6pip][ntf2 ] ) .", "complete miscibility with thiophene was observed for [ p2,4,4,4][dep ] . in the binary system with benzothiophene ,", "the solubility of benzothiophene in [ c1c5pip][ntf2 ] at t  =  308.15  k is equal to \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{il } } $ $ \\end{document}x2il  =  0.873 ( \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{\\text{il } } $ $ \\end{document}x2il  =  0.945 for [ c1c3pip][ntf2 ] at t  =  308.15  k .", "immiscibility is observed in the { thiophene , or benzothiophene ( 2 )  +  heptane ( 3 ) } binary mixture , as was reported previously .", "the determined experimental tie - lines for the ternary lle systems are plotted in figs .  1 , 2 , 3 , 4 for thiophene and benzothiophene , respectively", ". figures  1 , 2 , 3 , 4 show that the two - phase region is much larger for [ c1c5pip][ntf2 ] than that for [ p2,4,4,4][dep].fig . ", "1plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  kfig . ", "2plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  kfig . ", "3plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  kfig . ", "4plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k plot of the experimental ( filled circle , gray solid lines ) results versus values calculated with the nrtl equation ( square , black dotted lines ) for the composition tie lines of the ternary system { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k the results obtained in this work show that the more suitable il for the separation of thiophene , or benzothiophene from heptane , is [ c1c5pip][ntf2 ] because of its much larger selectivity ( s ) and the comparable solute distribution ratio (  ) .", "these parameters are defined as follows:1\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\beta = \\frac{{x _ { 2}^{\\text{ii } } } } { { x _ { 2}^{\\text{i } } } } $ $ \\end{document}=x2iix2i2\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ s = \\frac{{x _ { 2}^{\\text{ii } } \\cdot x _ { 3}^{\\text{i } } } } { { x _ { 2}^{\\text{i } } \\cdot x _ { 3}^{\\text{ii } } } } $ $ \\end{document}s = x2iix3ix2ix3iiwhere x is the mole fraction ; superscripts i and ii refer to the heptane - rich phase and the il - rich phase , respectively .", "the values of  and s are listed in table  2 for thiophene and benzothiophene .", "figures  5 and 6 present measured values of  and s for ils for thiophene and benzothiophene.fig . ", "5plot of the selectivity ( s ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  kfig . ", "6plot of the solute distribution ratio (  ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k plot of the selectivity ( s ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k plot of the solute distribution ratio (  ) as a function of the mole fraction of solute in the hydrocarbon - rich phase for the ternary systems : closed circle { [ c1c5pip][ntf2 ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } filled square { [ c1c5pip][ntf2 ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } open circle { [ p2,4,4,4][dep ] ( 1 )  +  thiophene ( 2 )  +  heptane ( 3 ) } , and open square { [ p2,4,4,4][dep ] ( 1 )  +  benzothiophene ( 2 )  +  heptane ( 3 ) } , at t  =  308.15  k the values presented in table  2 show that the distribution ratio coefficient are in the range of 0.812.41 , 0.954.36 , 1.362.69 and 1.464.13 for [ c1c5pip][ntf2]/thiophene , [ c1c5pip][ntf2]/benzothiophene , [ p2,4,4,4][dep]/thiophene and [ p2,4,4,4][dep]/benzothiophene , respectively .", "the selectivities of the separation in the system thiophene or benzothiophene / heptane is quite high for [ c1c5pip][ntf2 ] and very low for [ p2,4,4,4][dep ] .", "the values listed in table  2 for the best tie - lines are : 26.7 , 48.3 , 6.0 and 9.1 for [ c1c5pip][ntf2]/thiophene , [ c1c5pip][ntf2]/benzothiophene , [ p2,4,4,4][dep]/thiophene and [ p2,4,4,4][dep]/benzothiophene , respectively . in this work", "the effect of the alkane chain length on the cation of the piperidinium - based il was examined for comparison with previously measured data for [ c1c3pip][ntf2]/thiophene , [ c1c4pip][ntf2]/thiophene , and [ c1c6pip][ntf2]/thiophene at t  =  298.15  k , and of [ c1c3pip][ntf2]/benzothiophene at t  =  308.15  k   ( the influence of temperature is not large ) .", "the characteristic extraction parameters obtained in this work are compared to the few previously described in the open literature in table  3 .", "unfortunately , the selectivity for [ c1c5pip][ntf2 ] obtained in this work is slightly worse than that for [ c1c3pip][ntf2 ] measured by us earlier .", "the values of selectivity presented for 1-alkylcyanopyridinium - based ils at t  =  308.15  k measured in our earlier work   are also larger than those for piperidinium - based ils   ( see table  3).table  3comparison of solute distribution ratio (  ) and selectivity ( s ) for sulfur compounds extractionillle system \n", "t / k \n  \n max \n\n s \n max \n ref.[c1c5pip][ntf2]il  +  thiophene  +  heptane3082.4926.7this work[c1c5pip][ntf2]il  +  benzothiophene  +  heptane3084.3648.3this work[c1c3pip][ntf2]il  +  thiophene  +  heptane2982.5060.3[c1c3pip][ntf2]il  +  benzothiophene  +  heptane3085.3693.0[coc2mpip][ntf2 ] \n il  +  thiophene  +  heptane2982.6462.9[coc2mpip][fap ] \n il  +  thiophene  +  heptane2984.0056.8[bcnpy][ntf2 ] \n il  +  thiophene  +  heptane3081.9362.2[bcnpy][ntf2 ] \n il  +  benzothiophene  +  heptane3083.50117.1[p2,4,4,4][dep]il  +  thiophene  +  heptane3082.696.0this work[p2,4,4,4][dep]il  +  benzothiophene  +  heptane3084.139.1this work[emim][dep ] \n il  +  thiophene  +  hexane2982.8848.8[p1,4,4,4][ch3so4]il  +  thiophene  +  cyclohexane2981.385.38[dmim][mp ] \n il  +  thiophene  +  heptane2980.421,756 \n 1-(2-methoxyethyl)-1-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide \n 1-(2-methoxyethyl)-1-methylpiperidinium trifluorotris(perfluoroethyl)phosphate \n 1-butyl-4-cyanopyridinium bis{(trifluoromethyl)sulfonyl}imide \n 1-ethyl-3-methylimidazolium diethylphosphate \n 1,3-dimethylimidazolium methylphosphonate comparison of solute distribution ratio (  ) and selectivity ( s ) for sulfur compounds extraction \n 1-(2-methoxyethyl)-1-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide \n 1-(2-methoxyethyl)-1-methylpiperidinium trifluorotris(perfluoroethyl)phosphate \n 1-butyl-4-cyanopyridinium bis{(trifluoromethyl)sulfonyl}imide \n 1-ethyl-3-methylimidazolium diethylphosphate \n 1,3-dimethylimidazolium methylphosphonate the selectivities for [ c1c3pip][ntf2 ] are comparable to those for 4-(2-methoxyethyl)-4-methylpiperidinium trifluorotris(perfluoroethyl)phosphate [ coc2mpip][fap ] ils at t  =  298.15  k , or to 4-(2-methoxyethyl)-4-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide [ coc2mpip][ntf2 ] .", "the extraction results for [ p2,4,4,4][dep ] are very low and similar to [ p1,4,4,4][ch3so4 ] .", "it can be definitely concluded that phosphonium - based cations are not suitable for these separation processes .", "however , for the diethylphosphate anion [ dep ] and imidazolium - based cation [ emim ] , the results are comparable to those obtained in this work with [ c1c5pip][ntf2 ] but with a lower  value   ( see table  3 ) .", "it can be also seen from figs .  5 and 6 that  and s decrease as the solute mole fraction ( thiophene , or benzothiophene ) in the heptane phase increases , for all systems , when going through the tie - line end compositions ."], ["the ternary lle data measured in this study were correlated ( the tie - line correlation ) using the well known non - random liquid equation , nrtl .", "the equations and algorithms used for the calculation of the compositions in both phases follow the method described by walas .", "the objective function f(p ) was used to minimize the difference between the experimental and calculated compositions:3\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ f(p ) = \\sum\\limits_{i = 1}^{n } { \\left [ { x_{2}^{{{\\text{i,}}exp } } - x _ { 2}^{\\text{i , calc } } \\left ( { pt } \\right ) } \\right]^{2 } + \\left [ { x_{3}^{{{\\text{i,}}exp } } - x_{3}^{\\text{i , calc } } \\left ( { pt } \\right ) } \\right]}^{2 } + \\left [ { x_{2}^{{{\\text{ii,}}exp } } - x _ { 2}^{\\text{ii , calc } } \\left ( { pt } \\right ) } \\right]^{2 } + \\left [ { x_{3}^{{{\\text{ii,}}exp } } - x _ { 3}^{\\text{ii , calc } } \\left ( { pt } \\right ) } \\right]^{2 } $ $ \\end{document}f(p)=i=1nx2i , exp - x2i , calcpt2+x3i , exp - x3i , calcpt2+x2ii , exp - x2ii , calcpt2+x3ii , exp - x3ii , calcpt2where p is the set of parameters vector , n is the number of experimental points , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{{{\\text{i}},exp } } $ $ \\end{document}x2i , exp , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{{{\\text{i}},exp } } $ $ \\end{document}x3i , exp and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x _ { 2i}^{{{\\text{i}},{\\text{calc } } } } \\left ( { pt } \\right ) $ $ \\end{document}x2ii , calcpt , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{\\text{i , calc } } \\left ( { pt } \\right ) $ $ \\end{document}x3i , calcpt are the experimental and calculated mole fractions of one phase , and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{2}^{{{\\text{ii}},exp } } $ $ \\end{document}x2ii , exp , \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x_{3}^{{{\\text{ii,}}exp } } $ $ \\end{document}x3ii , exp\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x _ { 2}^{\\text{ii , calc } } \\left ( { pt } \\right ) $ $ \\end{document}x2ii , calcpt , and \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ x _ { 3}^{\\text{ii , calc } } \\left ( { pt } \\right ) $ $ \\end{document}x3ii , calcpt are the experimental and calculated mole fractions of the second phase .", "the binary parameters of each constituent were regressed by minimizing the sum of the squares of the differences between the experimental and calculated mole fractions of each component of both liquid phases for each ternary system .", "the value of the non - randomness parameter , ij , was optimized in order to obtain the best model fit .", "the correlated parameters are given in table  4 along with the root mean square deviations ( rmsd ) .", "the rmsd values , which are a measure of the precision of the correlation , were calculated according the equation:4\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ { \\text{rmsd } } = \\left ( { \\sum\\limits_{i } { \\sum\\limits_{l } { \\sum\\limits_{m } { \\left [ { x_{ilm}^{exp } - x_{ilm}^{\\text{calc } } } \\right]^{2 } /6k } } } } \\right)^{1/2 } $ $ \\end{document}rmsd=ilmxilmexp - xilmcalc2/6k1/2where x is the mole fraction and the subscripts i , l , and m designate the component , phase , and tie - line , respectively .", "the experimental and calculated lle data agreed relatively well.table  4binary interaction parameters , parameter  \n ij and root mean square deviation (  \n x ) for the nrtl equation for the ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpa \n ij \n g \n 12/(jmol)g \n 21/(jmol ) \n  \n ij \n rmsd  \n x \n [ c1c5pip][ntf2 ]  +  thiophene  +  heptane  128261.1123225.940.20.007  132536.4315361.68  23460.97475.45[c1c5pip][ntf2 ]  +  benzothiophene  +  heptane  125698.7312608.560.20.007  135142.9534672.57  232498.842232.66[p2,4,4,4][dep ]  +  thiophene  +  heptane  125566.067459.650.20.003  132785.5519670.14  233351.226008.81[p2,4,4,4][dep ]  +  benzothiophene  +  heptane  129265.5911427.530.20.004  132823.0616531.68  231048.012808.72 binary interaction parameters , parameter  \n ij and root mean square deviation (  \n x ) for the nrtl equation for the ternary systems { [ c1c5pip][ntf2 ] or [ p2,4,4,4][dep ] ( 1 )  +  thiophene or benzothiophene ( 2 )  +  heptane ( 3 ) } at t  =  308.15  k , p  =  101.33  kpa"], ["liquid phase equilibrium data were measured in this study for the extraction of thiophene or benzothiophene from heptane using two ils . four ternary systems { il  +  thiophene or benzothiophene  +  heptane } were analytically determined using gc for the composition analysis at temperature t  =  308.15  k at ambient pressure .", "it has been demonstrated that the 1-pentyl-1-methylpiperidinium bis{(trifluoromethyl)sulfonyl}imide il is much more effective than the phosphonium - based il for extraction of thiophene or benzothiophene from alkanes .", "sulfur compounds can be extracted easily by piperidinium - based ils , leading to low sulfur content in fuels .", "our earlier experimental results revealed that the solubility of sulfur compounds in the il increases as the alkyl chain length increases .", "the capacity of extraction , described in terms of the selectivity and the solute distribution ratio coefficients , was calculated for all ternary systems and compared to the published data used in similar extraction problems .", "based on the values obtained , [ c1c5pip][ntf2 ] was found to be useful for the extraction of sulfur compounds from alkanes ; however , it is not as good as [ c1c3pip][ntf2 ] measured previously . the selectivity and the solute distribution ratio decrease as the mole fraction of thiophene or benzothiophene in the heptane - rich phase increases . the best selectivity ( s )", "is observed for very low mole fractions of s - compounds in the hydrocarbon - rich phase \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ ( x_{2}^{\\text{hc } } \\ , = \\,0.0 5 ) $ $ \\end{document}(x2hc=0.05 ) ( see fig . ", "5 ) , which may be compared with the results of the hds method for the removal of the s - compounds . the experimental data in this work", "the non - randomness parameter was also determined through the reduction of the experimental data .", "the model exhibited an excellent fit to the data with the average rmsd values between 0.003 and 0.007 . \n"], [""]]}
{"article_id": "PMC4508536", "article_text": ["higher carriage rates are seen in diabetics , intravenous drug users ( ivdu ) , hiv and dialysis patients .", "all patients with s. aureus bacteremia should undergo transthoracic echocardiography ( tte ) , since s. aureus bacteremia is associated with heart valve involvement in 25% of the cases .", "nevertheless , tee has been shown to be superior to tte for the diagnosis of infective endocarditis ( ie ) , identifying small vegetations and abscesses .", "all infective foci must be identified and removed as soon as possible ; however , foci are not always obvious and long - term antimicrobial therapy might be necessary .", "nafcillin is a well - established agent for serious systemic non methicillin - resistant s. aureus ( mrsa ) infections and has been reported as superior over vancomycin .", "we present a very interesting case of a rapidly progressive methicillin - sensitive staphylococcus aureus infection , for which empirical treatment with vancomycin and initial treatment with nafcillin did not stop further dissemination despite adequate mic , taking longer than usual to respond to adequate treatment .", "a 64-year - old male patient with a past medical history of hypertension , hiatal hernia and osteoarthritis presented to the emergency department with a chief complaint of acute worsening of his chronic lower back pain for two weeks and progressive weakness in lower extremities .", "he used to ambulate with a cane and later used a walker for several days , but recently he felt non - ambulant .", "cardiac examination showed a regular rate and rhythm with normal s1 and s2 and no murmurs .", "on further review of systems he reported having chronic bilateral knee pain related with osteoarthritis and a congenital deformation of his right knee .", "he was a smoker of 40 pack - years , occasional user of alcohol and marijuana , but denied ever using intravenous drugs and toxicology was positive only for oxycodone , which he used for chronic lumbar pain for several years .", "laboratory exams displayed a leukocytosis of 25,500 with 89% neutrophils , no bands , sedimentation rate of 44 , lactic acid 1.6 , anion gap 18 .", "thoracic and lumbar spine computed tomography ( ct ) scan showed multilevel central canal and bilateral neural foraminal compromise , but did not show evidence of abscess .", "partially visualized lungs showed a cavitary lesion in the superior segment of the left lower lobe , measuring 1.4 cm with circumferential thick wall , suggestive of septic emboli versus tuberculosis , and left inferior renal pole abnormalities suggestive of multiple infarctions .", "the patient was started empirically on vancomycin 15 mg / kg iv q 12h , while waiting for sensitivities .", "the tte performed after bacteremia diagnosis showed an ejection fraction of 65% with normal valves and no vegetations . on day two of hospitalization the clinical picture worsened as the patient suddenly developed an altered mental status and nuchal rigidity .", "lumbar puncture confirmed meningitis with a cerebrospinal fluid leukocytosis of 1157 ( neutrophil 95% ) and culture positive for s. aureus .", "testing for hiv , herpes simplex virus ( hsv ) and tuberculin skin test ( ppd ) were all negative .", "spine magnetic resonance imaging ( mri ) showed osteomyelitis at t12-l1 and previously seen ( in ct scan ) renal infarcts .", "the patient continued to be febrile despite pathogen susceptible to vancomycin with mic < 2 mg / ml , trough previous to 4 dose 11 , repeated trough 18.4 , repeat blood cultures at 48 and 96 hours remained negative .", "six days later he had clinical deterioration with tachypnea , hypoxia , new systolic 2/6 murmur , louder over cardiac apex area , and bilateral respiratory crackles .", "at this point , the patient was switched to nafcillin 2 g iv q 4h when blood culture results confirmed methicillin susceptibility on day 3 of admission .", "head mri showed multiple infarcts in a non - vascular pattern secondary to septic embolisms ( figure 1 ) .", "the tee showed severe mitral and tricuspid regurgitations , with 1.5 cm mobile vegetation on the posterior leaflet of the mitral valve .", "the patient was transferred to the intensive care unit due to the complicated picture of mssa bacteremia , ie , osteomyelitis , meningitis , ischemic stroke , renal and pulmonary infarcts secondary to septic emboli .", "there were indications for emergent mitral valve replacement , however given his recent finding of embolic stroke ; this was not feasible due to high mortality risk .", "follow up tte showed worsening mitral and tricuspid valve involvement , therefore mitral and tricuspid valve replacements were performed , four weeks from ie diagnosis .", "he completed 8 weeks of nafcillin ( given his vertebral involvement and unknown source ) .", "after 2 months of hospitalization , patient was discharged home with a dual - chamber pacemaker due to persistent 3rd degree atrio - ventricular block , post surgery .", "staphylococcus aureus is one of the leading agents of infection among adult patients . when s. aureus invades deep structures it often metastasize hematogenously to other areas and organs with significant morbidity .", "infection caused by community acquired mssa strains are characterized by severe clinical course with increased incidence of endocarditis and organ failure .", "two important questions should be asked to the patient with the intention to identify focal source and consider additional necessary diagnostic test .", "ivdu related infection can manifest as an initially insidious presentation that later complicates to be an aggressive metastatic disease .", "we presented a unique case since our patient was not an ivdu nor an initial focus of infection was identified .", "additionally , the progression from negative to positive echocardiogram findings of valvular vegetations highlights the high virulence of this community - acquired pathogen , since severe valvular regurgitation or insufficiency should be equally observed on both types of echocardiograms . altered mental status in such patients", "a mri is crucial to confirm the diagnosis with the visualization of a non - vascular pattern .", "mssa meningitis is a serious infection , which can occurs in patients without risk factors or immunosuppression . like meningitis ,", "stroke secondary ca - mssa is rarely seen , regardless of the severity of infection .", "high level of clinical suspicion is needed in such patients , as back pain could be the only reliable predictor of an added spine infection .", "when treating for gram - positive cocci bacteremia , empirical antibiotic therapy should provide coverage against staphylococci , usually with vancomycin to cover mrsa .", "nafcillin / oxacillin remains the antibiotic of choice for treating infections caused by mssa once culture and sensitivity results confirm it , because nafcillin is superior to vancomycin in preventing persistent or relapsing mssa bacteremia . in the case of endocarditis", ", studies have demonstrated that vancomycin ( versus nafcillin ) is significantly associated with relapse given its slow bactericidal activity .", "for our case , the reasons why empirical treatment with vancomycin and initial treatment with nafcillin , did not stop further dissemination remained unknown . nevertheless , continued treatment with nafcillin eventually did resolve the infection .", "we have to consider that , even though the mic remains the only satisfactory in vitro measurement of the intrinsic activity of antimicrobials , the test has always been open for criticism since it is performed with the use of artificial media and fixed concentrations , under conditions that may be very different from those in the actual site of infection .", "there is evidence that support the concept of the principal determinant of efficacy of beta lactams to be the time for which the drug levels exceed the mic at the site of infection , not just and mic < 2 mg / ml by itself .", "also , bacterial strains have been detected to have a mbc ( minimum bactericidal concentration ) many times higher than the mic , ( known as phenotypic tolerance ) , isolated in vitro gram - positive bacteria causing slower clinical response .", "the inoculum effect may be clinically relevant since the number of bacteria at the site of infection may much higher that the traditionally used for susceptibility testing .", "although the benefit of such concentration is questionable since our patient s infection was multifocal .", "our laboratory was not able to analyze serum nafcillin concentrations ; hence , samples would have to send to a referral laboratory .", "a community acquired pathogen , even when part of the normal flora could be virulent enough to cause end organ damage from head to toe .", "the use of head mri as a pre evaluation tool for ie - related urgent valve surgery is imperative , to investigate whether such preoperative findings affect postoperative outcomes .", "the presence of characteristic cranial mri lesions may prompt early diagnosis of infective source and lead to the adequate management . beyond the mic , the time the drug level exceeds the mic , the phenotypic tolerance and the inoculum effects may be reasons why empirical treatment with vancomycin and initial treatment with nafcillin did not stop further dissemination , but , eventually , clearing the infection ."], "abstract_text": ["<S> methicillin - sensitive staphylococcus aureus ( mssa ) meningitis is a rare disease when not related to neurosurgery : there are only few reported cases in the literature to date . </S>", "<S> we describe a case that highlights not only meningeal but also diffuse and rapidly progressive systemic involvement with multi - organ failure . </S>", "<S> a 64-year - old male presented to our hospital with a chief complaint of acute worsening of his usual chronic lower back pain , progressive weakness in lower extremities and subjective fevers at home . </S>", "<S> hospital course demonstrated mssa bacteremia , of questionable source , that resulted in endocarditis affecting right and left heart in a patient with no history of intravenous drug use . </S>", "<S> the case was complicated by septic emboli to systemic circulation involving the kidneys , vertebral spine , lungs and brain with consequent meningitis and stroke , even when treated empirically with vancomycin and then switched to nafcillin as indicated . even though mssa infections are well known , there are very few case reports describing such an acute - simultaneous - manifestation of multi - end - organ failure , including meningitis and stroke . </S>", "<S> our case , also presented with an uncommon manifestation of persistent infection dissemination despite adequate antibiotic treatment . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusions"], "sections": [["higher carriage rates are seen in diabetics , intravenous drug users ( ivdu ) , hiv and dialysis patients .", "all patients with s. aureus bacteremia should undergo transthoracic echocardiography ( tte ) , since s. aureus bacteremia is associated with heart valve involvement in 25% of the cases .", "nevertheless , tee has been shown to be superior to tte for the diagnosis of infective endocarditis ( ie ) , identifying small vegetations and abscesses .", "all infective foci must be identified and removed as soon as possible ; however , foci are not always obvious and long - term antimicrobial therapy might be necessary .", "nafcillin is a well - established agent for serious systemic non methicillin - resistant s. aureus ( mrsa ) infections and has been reported as superior over vancomycin .", "we present a very interesting case of a rapidly progressive methicillin - sensitive staphylococcus aureus infection , for which empirical treatment with vancomycin and initial treatment with nafcillin did not stop further dissemination despite adequate mic , taking longer than usual to respond to adequate treatment ."], ["a 64-year - old male patient with a past medical history of hypertension , hiatal hernia and osteoarthritis presented to the emergency department with a chief complaint of acute worsening of his chronic lower back pain for two weeks and progressive weakness in lower extremities .", "he used to ambulate with a cane and later used a walker for several days , but recently he felt non - ambulant .", "cardiac examination showed a regular rate and rhythm with normal s1 and s2 and no murmurs .", "on further review of systems he reported having chronic bilateral knee pain related with osteoarthritis and a congenital deformation of his right knee .", "he was a smoker of 40 pack - years , occasional user of alcohol and marijuana , but denied ever using intravenous drugs and toxicology was positive only for oxycodone , which he used for chronic lumbar pain for several years .", "laboratory exams displayed a leukocytosis of 25,500 with 89% neutrophils , no bands , sedimentation rate of 44 , lactic acid 1.6 , anion gap 18 .", "thoracic and lumbar spine computed tomography ( ct ) scan showed multilevel central canal and bilateral neural foraminal compromise , but did not show evidence of abscess .", "partially visualized lungs showed a cavitary lesion in the superior segment of the left lower lobe , measuring 1.4 cm with circumferential thick wall , suggestive of septic emboli versus tuberculosis , and left inferior renal pole abnormalities suggestive of multiple infarctions .", "the patient was started empirically on vancomycin 15 mg / kg iv q 12h , while waiting for sensitivities .", "the tte performed after bacteremia diagnosis showed an ejection fraction of 65% with normal valves and no vegetations . on day two of hospitalization the clinical picture worsened as the patient suddenly developed an altered mental status and nuchal rigidity .", "lumbar puncture confirmed meningitis with a cerebrospinal fluid leukocytosis of 1157 ( neutrophil 95% ) and culture positive for s. aureus .", "testing for hiv , herpes simplex virus ( hsv ) and tuberculin skin test ( ppd ) were all negative .", "spine magnetic resonance imaging ( mri ) showed osteomyelitis at t12-l1 and previously seen ( in ct scan ) renal infarcts .", "the patient continued to be febrile despite pathogen susceptible to vancomycin with mic < 2 mg / ml , trough previous to 4 dose 11 , repeated trough 18.4 , repeat blood cultures at 48 and 96 hours remained negative .", "six days later he had clinical deterioration with tachypnea , hypoxia , new systolic 2/6 murmur , louder over cardiac apex area , and bilateral respiratory crackles .", "at this point , the patient was switched to nafcillin 2 g iv q 4h when blood culture results confirmed methicillin susceptibility on day 3 of admission .", "head mri showed multiple infarcts in a non - vascular pattern secondary to septic embolisms ( figure 1 ) .", "the tee showed severe mitral and tricuspid regurgitations , with 1.5 cm mobile vegetation on the posterior leaflet of the mitral valve .", "the patient was transferred to the intensive care unit due to the complicated picture of mssa bacteremia , ie , osteomyelitis , meningitis , ischemic stroke , renal and pulmonary infarcts secondary to septic emboli .", "there were indications for emergent mitral valve replacement , however given his recent finding of embolic stroke ; this was not feasible due to high mortality risk .", "follow up tte showed worsening mitral and tricuspid valve involvement , therefore mitral and tricuspid valve replacements were performed , four weeks from ie diagnosis .", "he completed 8 weeks of nafcillin ( given his vertebral involvement and unknown source ) .", "after 2 months of hospitalization , patient was discharged home with a dual - chamber pacemaker due to persistent 3rd degree atrio - ventricular block , post surgery ."], ["staphylococcus aureus is one of the leading agents of infection among adult patients . when s. aureus invades deep structures it often metastasize hematogenously to other areas and organs with significant morbidity .", "infection caused by community acquired mssa strains are characterized by severe clinical course with increased incidence of endocarditis and organ failure .", "two important questions should be asked to the patient with the intention to identify focal source and consider additional necessary diagnostic test .", "ivdu related infection can manifest as an initially insidious presentation that later complicates to be an aggressive metastatic disease .", "we presented a unique case since our patient was not an ivdu nor an initial focus of infection was identified .", "additionally , the progression from negative to positive echocardiogram findings of valvular vegetations highlights the high virulence of this community - acquired pathogen , since severe valvular regurgitation or insufficiency should be equally observed on both types of echocardiograms . altered mental status in such patients", "a mri is crucial to confirm the diagnosis with the visualization of a non - vascular pattern .", "mssa meningitis is a serious infection , which can occurs in patients without risk factors or immunosuppression . like meningitis ,", "stroke secondary ca - mssa is rarely seen , regardless of the severity of infection .", "high level of clinical suspicion is needed in such patients , as back pain could be the only reliable predictor of an added spine infection .", "when treating for gram - positive cocci bacteremia , empirical antibiotic therapy should provide coverage against staphylococci , usually with vancomycin to cover mrsa .", "nafcillin / oxacillin remains the antibiotic of choice for treating infections caused by mssa once culture and sensitivity results confirm it , because nafcillin is superior to vancomycin in preventing persistent or relapsing mssa bacteremia . in the case of endocarditis", ", studies have demonstrated that vancomycin ( versus nafcillin ) is significantly associated with relapse given its slow bactericidal activity .", "for our case , the reasons why empirical treatment with vancomycin and initial treatment with nafcillin , did not stop further dissemination remained unknown . nevertheless , continued treatment with nafcillin eventually did resolve the infection .", "we have to consider that , even though the mic remains the only satisfactory in vitro measurement of the intrinsic activity of antimicrobials , the test has always been open for criticism since it is performed with the use of artificial media and fixed concentrations , under conditions that may be very different from those in the actual site of infection .", "there is evidence that support the concept of the principal determinant of efficacy of beta lactams to be the time for which the drug levels exceed the mic at the site of infection , not just and mic < 2 mg / ml by itself .", "also , bacterial strains have been detected to have a mbc ( minimum bactericidal concentration ) many times higher than the mic , ( known as phenotypic tolerance ) , isolated in vitro gram - positive bacteria causing slower clinical response .", "the inoculum effect may be clinically relevant since the number of bacteria at the site of infection may much higher that the traditionally used for susceptibility testing .", "although the benefit of such concentration is questionable since our patient s infection was multifocal .", "our laboratory was not able to analyze serum nafcillin concentrations ; hence , samples would have to send to a referral laboratory ."], ["a community acquired pathogen , even when part of the normal flora could be virulent enough to cause end organ damage from head to toe .", "the use of head mri as a pre evaluation tool for ie - related urgent valve surgery is imperative , to investigate whether such preoperative findings affect postoperative outcomes .", "the presence of characteristic cranial mri lesions may prompt early diagnosis of infective source and lead to the adequate management . beyond the mic , the time the drug level exceeds the mic , the phenotypic tolerance and the inoculum effects may be reasons why empirical treatment with vancomycin and initial treatment with nafcillin did not stop further dissemination , but , eventually , clearing the infection ."]]}
{"article_id": "PMC3590747", "article_text": ["the use of nanomaterials in medicine involves the applications of nanoparticles and manufactured nanosystems to provide regeneration at the cellular and tissue levels .", "they encapsulate therapeutic agents and typically carry multiple targeting motifs such as hemostasis [ 1 , 2 ] . for instance", ", ellis - behnke and coworkers   introduced a unique nanomedicinal method to stop bleeding using a self - assembling peptide that establishes a nanofiber barrier and incorporates it into the surrounding tissue to form an extracellular matrix [ 1 , 2 ] .  ", "biomaterials used as tissue engineering scaffolds have specific physical properties and might form fibrous networks similar to collagenous extracellular matrix .", "they also can be programmed to carry chemical and physical cues to provide bioactivity for cell - materials interactions . in the search for more improved bioactive materials for tissue engineering purposes , peptide amphiphile ( pa ) molecules are good candidates to bring scaffold properties and bioactivity together [ 3 , 4 ] .", "hydrophobic region of the pa is composed of fatty acids , which are packed against aqueous medium inducing self - assembly of the molecule to form cylindrical nanofibers . charged amino acids on hydrophilic peptide region of the pa molecules are responsible for triggering self - assembly upon isolation of charges .", "neutralization of charge density on amino acids causes aggregation of pa molecules and formation of nanofibrous networks at microscale .", "encapsulation of water by these nanofiber networks results in constitution of self - supporting gel at macroscale .", "peptide part of the pa molecules can be synthesized in a way to include bioactive epitopes or biofunctional chemical groups , which are eventually presented to cells on nanofibers .", "pa gels carrying these types of functionalities have been studied thoroughly and proven to be active in terms of inducing angiogenesis , neuronal regeneration , and cartilage and bone tissue formation [ 710 ] .", "charge neutralization mechanism also allows us to use bioactive molecules with high negative or positive charge density , such as heparin , dna , or oligonucleotides for inducing gel formation , while exploiting their bioactivity [ 6 , 11 , 12 ] .", "the use of medicinal plants as remedies for numerous disorders has formed the basis of current medicinal approach .", "various plants are used ethnomedicinally for prevention of excessive bleeding and as wound dressing to staunch blood flow .", "ankaferd hemostat , a topical hemostatic agent of plant origin , has recently been registered for the management of clinical hemorrhages when the conventional methods to control bleeding by ligature and/or hemostatic measures are ineffective [ 1416 ] .", "ankaferd blood stopper ( abs ) includes standardized preparation of the plants thymus vulgaris , glycyrrhiza glabra , vitis vinifera , alpinia officinarum , and urtica dioica [ 17 , 18 ] .", "abs provides vital erythroid aggregation covering the entire physiological hemostatic process via a unique protein network depending primarily on the interactions between abs and blood proteins , particularly with fibrinogen gamma and prothrombin [ 16 , 1922 ] .", "vital erythroid aggregation takes place with the spectrin and ankyrin receptors on the surface of red blood cells ( rbcs ) .", "those rbc proteins and the required atp bioenergy are included in the abs protein library [ 23 , 24 ] .", "on the other hand , controlled clinical trials indicated the safety and efficacy of topical ankaferd hemostat in distinct clinical backgrounds [ 19 , 2531 ] .   in this work", ", we present a chimeric hemostatic agent , abs nanohemostat , via combining a self - assembling pa molecule with the traditional ankaferd hemostat .", "the first step of our research was the synthesis of the specific self - assembling peptide molecules capable of being a part of the combined abs nanohemostat compound .", "the second step was the assembly of the peptide nanofibers and abs to generate the abs nanohemostat .", "the third step of our study was testing the in vivo hemostatic effects of abs nanohemostat in comparison with the traditional ankaferd in a previously established and published controlled surgical experimental trial in kidney tissue .", "the ultimate aim of the present study is , therefore , to test the efficacy of abs nanohemostat formed by combination of a self - assembling pa molecule and ankaferd hemostat for controlling surgical bleeding due to renal injury during partial nephrectomy ( pn ) and to compare its hemostatic effects to the traditional surgical and ankaferd hemostat groups .", "9-fluorenylmethoxycarbonyl ( fmoc ) , ter . butoxycarbonyl ( boc ) protected amino acids , rink amide mbha resin , and 2-(1h - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) were purchased from novabiochem or abcr .", "the other chemicals were purchased from fisher , merck , alfa aesar , or aldrich and used as provided .", "amino acid couplings were done with 2 equivalents of fmoc protected amino acid , 1.95 equivalents hbtu , and 3 equivalents of n , n - diisopropylethylamine ( diea ) for 2 hours .", "fmoc removal was performed with 20% piperidine / dimethylformamide ( dmf ) solution for 20  min .", "cleavage of the peptides from the resin was carried out with a mixture of tfa  :  tis  :  h2o in ratio of 95  :  2.5  :  2.5 for 2  h. excess tfa was removed by rotary evaporation .", "the remaining viscous peptide solution was triturated with ice - cold ether , and the resulting white product was dried under vacuum .", "pa molecules were characterized by liquid chromatography - mass spectrometry ( lc - ms ) ( figure 1 ) .", "mass spectrum was obtained with agilent lc - ms equipped with agilent zorbax extend - c18 2.1  50  mm column .", "a gradient of ( a ) water ( 0.1% formic acid ) and ( b ) acetonitrile ( 0.1% formic acid ) was used .  ", "it is composed of a lauryl ( c12 ) group , hydrophobic region of the pa , and a peptide sequence .", "vvag peptide sequence is used as -sheet inducer that causes nanofiber formation , while the lysine ( k ) residue is protonated at physiological ph and increases solvation of pa molecule in aqueous solution at ph 7 .  ", "figure 2 shows both the chemical structure of the pa ( lauryl - vvagk - am ) and the schematic representation and abs nanohemostat formation .", "1  10  m peptide solutions were measured from 300  nm to 190  nm , data interval and data pitch being 0.1  nm and scanning speed being 100  nm / min , all measurements with three accumulations . digital integration time ( dit )", "was selected as 1  sec , band width as 1  nm , and the sensitivity was standard .", "oscillatory rheology measurements were performed with anton paar physica rm301 rheometer operating with a 25  mm parallel plate configuration at 25c .", "each sample of 100  l total volume with a final peptide concentration of 1  wt% was carefully loaded on the center of the lower plate and incubated for 15  min before measuring .", "after equilibration , the upper plate was lowered to a gap distance of 0.5 mm .", "storage moduli ( g ) and loss moduli ( g ) values were scanned from 100  rad / s to 0.1  rad / s of angular frequency , with a 0.5% shear strain .", "sem experiments were performed with fei nova nanosem 230 , using the etd detector at low vacuum mode with 30  kev beam energy .", "small amounts of gels with a final peptide concentration of 1% were put on a metal mesh , dried at critical point ( 1072  psi , 31c ) with tousimis autosamdri-815 b series c critical point dryer and coated with 6  nm au - pd .  ", "figure 3 shows sem images of pa gel with and without ankaferd at ph 10 .", "an amount of 250  l of pa was mixed with 250  l abs solution on glass slide and incubated at room temperature for 30  min for self - supporting gel formation . due to anionic molecules in ankaferd solution ,", "all animal experimentations described in this paper were carried out in accordance with national guidelines for the use and care of  laboratory animals and were approved by the local animal review and ethics committee .", "all procedures were in full compliance with turkish law 6343/2 , veterinary medicine deontology regulation 6.7.26 , and with the helsinki declaration of world medical association recommendations on animal studies .", "the animals were obtained from the center of medical experimental research of ankara training and research hospital .", "the rats were housed in stainless steel cages in an animal room maintained at a temperature of 2224c with 12-hour light / dark periods .", "all were fed with the same amount of laboratory pellet diet and with water supplied ad libitum for a minimum of 5 days before procedure .", "a total of 24 wistar rats weighing 200 to 300  g were divided into 4 groups of 6 each and underwent pn .", "all operations were performed under total anesthesia with injection of 50  mg / kg intramuscular ketamine hydrochloride .", "the lower third of the left kidney was resected in guillotine fashion with a single stroke of an amputating knife .", "( i ) group 1 ( g1 ) is the left pn with hilar vascular control including intracorporeal suturing of the renal parenchyma and collecting duct ( control group ) . ", "( ii ) group 2 ( g2 ) is the conventional pn with only 0.5  ml traditional ankaferd hemostat ( abs ) application without suturing . ", "( iii ) group 3 ( g3 ) is the conventional pn with abs ( 0.25  ml ) + peptide ( 0.25  ml ) gel ( abs nanohemostat ) mixture application with no suturing . ", "( iv ) group 4 ( g4 ) is the conventional pn with only 0.5  ml peptide solution application .", "( i ) group 1 ( g1 ) is the left pn with hilar vascular control including intracorporeal suturing of the renal parenchyma and collecting duct ( control group ) .  ", "( ii ) group 2 ( g2 ) is the conventional pn with only 0.5  ml traditional ankaferd hemostat ( abs ) application without suturing .", "( iii ) group 3 ( g3 ) is the conventional pn with abs ( 0.25  ml ) + peptide ( 0.25  ml ) gel ( abs nanohemostat ) mixture application with no suturing .", "( iv ) group 4 ( g4 ) is the conventional pn with only 0.5  ml peptide solution application .", "two objective parameters were recorded during the surgical procedure : warm ischemia time ( wit ) and amount of bleeding ( aob ) .", "second ,  while the aob was measured with the bleeding area ( cm ) onto the sponges .", "all the rats were allowed to feed and drink water for the following 4 weeks .", "after that , each rat was sacrificed , and total nephrectomy was performed for histopathological examination .", "wi started with clamping the renal artery and vein and finished with taking the clamp out . in g1 ,", "traditional hemostasis method was used as compression onto the renal excised area and suturing the renal vessels and collecting duct with absorbable sutures ( figures 4(a ) and 4(b ) ) . in g2 , 2  ml of abs was dropped to the amputated renal margin steadily until bleeding stopped ( figures 4(c ) and 4(d ) ) . in g3 , abs ( 2  ml ) + nanopeptide gel mixture ( abs nanohemostat )", "other hemostatic methods including sponges , surgicel , electrocautery , and any other sources were not used to control bleeding in the present study .  ", "coronal 1 to 2  mm thick slices of the kidney were fixed in 4% formaldehyde and embedded in paraffin , and 3 to 4  m thick tissue sections were stained with hematoxylin and eosin .", "sections were examined for several parameters including erythroid aggregation , fibrosis , calcification , inflammatory cells , and siderophages . for each case , a global estimation of parenchymal damage was done and semiquantitatively graded on a scale in which 0 was no abnormality , and 1 + , 2 + , and 3 + represented mild , moderate , and severe abnormalities , respectively .", "data analysis was performed using ibm statistical package for the social sciences ( spss ) statistics software .", "comparisons of numerical variables among four groups were initially performed using the kruskal - wallis test and mann - whitney u - test with the bonferroni correction used for post hoc analysis to compare the groups separately . a p value of < 0.0083 ( 0.05/6 ) was deemed statistically significant in multiple comparisons .", "the percentages of histopathological features between study groups were compared using the chi - squared test . a p value of < 0.05 was considered as statistically significant .", "  in this work , we synthesized a positively charged pa molecule as shown in figure 2 .", "storage and loss moduli measurement by oscillatory rheometry was performed in order to characterize the mechanical properties of the gel .", "storage moduli , showing stiffness of gel , were found to be several folds higher than loss moduli , indicating a self - supporting gel formation ( figure 5 ) .", "moreover , storage modulus value was comparable to control gel formed by elevating ph of pa solution .", "circular dichroism spectroscopy was used to investigate the secondary structure of the pa and ankaferd mixture .", "characteristic -sheet formation was observed in pa - ankaferd solution , which was similar to pa solution at high ph .", "morphology of the gel was examined by using sem which showed nanofibrous network structure similar to pa gels reported before and pa samples at high ph .", "the ph 10 solutions for pa molecule were studied to analyze the peptide gels alone .", "the pa molecules were dissolved at ph 7 , and they self - assembled into nanofibers forming gels upon addition of ankaferd solution or by increasing the ph to 10 . left lower pole pn was surgically performed successfully to each rat in all groups . the kidney size and", "the same surgical equipments were used , and rommel clamps succeeded the warm ischemia during all surgical procedures .   ", "mean  sd wits were 232.8  56.3 , 65.6  11.4 , 75.5  17.2 , and 58.1  17.6 seconds in g1 to g4 , respectively .", "a significant difference was detected between g1 and g2 , 3 , and 4 , while no difference was observed among the three groups ; g1 , g2 , and g3 ( table 1 ) . in g1 ,", "renal parenchyma injury that caused the longer wit and repaired with primary suture was present in one rat . in g4 , more amount of nanopeptide agent was used to stop the bleeding in one rat following the warm ischemia . apart from these two cases , no complication was observed during the operations .", "fibrosis was not different among the groups ( p > 0.05 ) , while inflammation was detected to be significantly different in g3 and g4 ( p = 0.04 ) .", "erythrocyte aggregation , especially in glomerular field ( figure 7 ) , was confirmed to be significantly higher in g3 ( abs nanohemostat ) compared with the other groups ( p = 0.03 ) .", "hemosiderin was also detected in g3 ; however , no significant difference was detected among the groups . in g4 ,", "bleeding and congestion without erythrocyte aggregation were confirmed with significant calcification ( p < 0.001 ) ( figure 8) .  ", "we confirmed the significant demonstrative aggregation of red blood cells in abs nanohemostat group as shown in abs group .", "giant cell reaction , thyroidization , fibroblast activation , and microvascular proliferation were not detected .", "in this study , we revealed that pa - ankaferd gel mixture ( abs nanohemostat ) is effective as traditional ankaferd hemostat .", "moreover , this unique nanomedicine proves itself as an effective and easily applicable alternative hemostatic method which can successfully be used even in complicated hemorrhagic situations such as aggressive surgical tissue bleeding due to pn .  ", "several hemostatic agents are preferred to control external and internal bleedings ; yet , commercially available products are not sufficiently effective or fast acting to achieve hemostasis in extreme occasions .", "ankaferd hemostat is a topical hemostatic agent of plant origin , including molecules with high density of negative and positive charges , and is proven to work as an efficient hemostatic agent [ 1416 ] .", "controlled clinical trials indicated the safety and efficacy of topical ankaferd hemostat in distinct clinical backgrounds [ 19 , 2531 ] . in this work , we aimed to assemble a nanostructured scaffold material with hemostatic activity in the pa and ankaferd gel formed upon mixing soluble pa molecule with ankaferd solution ( abs nanohemostat ) .", "thus , we hypothesized that , while reducing blood loss , nanofiber network will serve as a scaffold for infiltrating cells to wound area , and eventually tissue regeneration rate will be enhanced . sustaining hemostasis in clinical hemorrhages is a challenging task and requires extensive effort to stabilize traumatic and surgical injuries .", "our experimental animal model is therefore clinically relevant since bleeding is one of the most important major complications leading to the morbidity and even mortality during pn for renal masses . providing hemostasis following", "the renal mass excision is the most important step of pn , and various hemostatic agents were used to stop bleeding during this operation [ 3335 ] .", "although the complication rates including bleeding are slightly higher than those of open radical nephrectomy , the advantage of renal preservation is more evident . likewise , laparoscopic pn , including hilar control , suture repair of the collecting system , suture ligation of blood vessels , and capsular closure , is also a valuable urological intervention .", "bleeding and ischemic renal damage due to warm ischemia period are the most important complications following surgery . in order to decrease wit and pnt", "several agents have been investigated for their hemostatic potential in managing vascular injury and many have also been evaluated for their efficacy on repairing the collecting system injury . in our results , abs and abs nanohemostat applications onto the transected kidney area provided active hemostasis in pn with significant decrease in wits and pnts , comparable with suture group . with more application number of abs products ,", "however , the absence of urinoma in the groups applied with abs may reveal the effect of abs on collecting system repair following one month .", "these favorable findings lead to the active hemostasis and regular healing of transected kidney without adherence and urinoma around the kidney . in our studies , we demonstrated foreign material reaction in conventional pn model but not observed in the abs groups .", "however , it could be speculated that the topical abs administration onto the sutured line might prevent foreign biological reactions ( fbrs ) against the suture by the way of affecting biological pathways .", "the anti - infective effects [ 3638 ] of abs represent a putative biological mode of action on the hypothetical fbr .  ", "the hemostatic effect of local abs application in a patient who underwent an open pn has already been shown .", "abs was successfully applied to stop bleeding without suturing the renal parenchyma , which provided active hemostasis in pn on the transected kidney area .", "the surgical and histopathological findings about pn in the present trial and our previous research   suggested similar results regarding the renal tissue effects of abs .", "moreover , nanotechnologically generated chimeric abs nanohemostat was observed to increase the duration of the contact of ankaferd hemostatic agent with the bleeding area during pn via the controlled targeted release of the topical hemostatic agent to the tissue . controlled clinical studiesconducted to evaluate the effectiveness of abs in distinct states of bleeding disorders documented the safety and efficacy of traditional ankaferd hemostat in comparison to conventional antihemorrhagic medications .", "the first randomized controlled clinical study was reported by teker et al .   in 49 patients with anterior epistaxis . in this study , patients were randomly grouped to receive the hemostasis technique by means of either abs wet tampon or phenylephrine impregnated gauze tampon for anterior epistaxis control .", "abs successfully treated all bleeding intensity with one application ( for 5  m ) .", "abs patients experienced fewer rebleeding rates within 7 days compared to phenylephrine patients ( 8.3 versus 20% , p < 0.05 ) . in patients to whom abs was applied , significant differences in effective control of anterior epistaxis", "similarly , another clinical trial has also demonstrated the effectiveness of abs in children undergoing tonsillectomy . in this prospective controlled study ,", "the success of abs and the traditional knot - tie approach to reach hemostasis for patients undergoing tonsillectomy was evaluated .", "forty - seven consecutive patients undergoing cold knife dissection tonsillectomy were studied , in all of whom abs wet tampon was used for right side tonsil hemorrhage and knot - tie technique for left side tonsil hemorrhage .", "the abs side was reported to have shorter hemostasis time after tonsil removal than the knot - tie side ( 3.19  0.74  min versus 7.29  2.33  min , p < 0.01 ) and less blood loss ( 1.57  2.26  ml versus 14.04  7.23  ml , p < 0.01 ) .", "the author concluded that abs is not only safe and efficient , but also it decreases intraoperative bleeding and reduces operating time compared to the traditional hemostasis methods after cold knife dissection tonsillectomy .", "the effectiveness of abs in cancer patients was also reported in a study by al et al . .", "sixty - nine cancer patients that were admitted for port insertion to a university hospital were randomized either to take a wet compress form of abs or regular dry sterile sponges to stop the bleeding that occurs during the clinically indicated vascular port insertion .", "the average time needed to stop the bleeding was 32.97  29.9  s for abs group and 123.75  47.5  s for dry sponge group .", "abs was proven to stop local bleeding in a shorter time , with a lower recurrence rate in comparison with the sterile sponge .", "additional clinical studies about antihemorrhagic efficacy of abs on the bleeding due to adenoidectomy , [ 28 , 40 ] the surgical bleeding of thyroidectomy ,   gastrointestinal bleedings [ 14 , 29 ] , and dental bleedings   were performed with similar beneficial outcomes .", "our promising experimental results about abs nanohemostat in this study cast novel studies to search clinically measurable end - points regarding the hemostatic efficiency of this unique chimeric nanomedicine .", "the findings of our present study represent a starting point to investigate clinical antihemorrhagic effects of abs nanohemostat . the ability of abs to induce formation of a protein network not only makes it an effective hemostatic agent , but also confers anti - infective , antineoplastic , and healing modulator properties to the extract [ 1416 ] .", "those pleiotropic effects were ascribed to the abs - induced alterations in apoptosis , angiogenesis , cellular proliferation , and cellular dynamics [ 46 ] . in previous investigations ,", "histopathological examination of the damaged vascular structures revealed abs - induced red blood cell aggregates supporting the hypothesis that abs - induced formation of the protein network with vital erythroid aggregation covers the entire physiological hemostatic process [ 42 , 43 ] .", "we have observed the same structures in the kidney tissue in the present study . in the current study ,", "abs nanohemostat has led to a more pronounced erythroid aggregation at the tissue level in comparison to the traditional ankaferd hemostat .", "however , clinical antihemorrhagic efficiency of ankaferd solution seems to be superior to the chimeric ankaferd nanohemostat .", "in summary , abs nanohemostat has comparable hemostatic efficacy to the traditional ankaferd hemostat in the pn experimental model .", "these observations prompt the design of future experimental and clinical studies focusing on the antibleeding and vascular repair effects of this novel hemostatic nanomedicine .", "the production of abs nanohemostat in gel formation is also important for using the hemostatic material during the laparoscopic urologic surgery .", "the steady scaffold onto the resected area with abs nanohemostat predicts the intracorporeal use of abs hemostat as a promising agent .", "likewise , future controlled studies are needed to shed further light on the expanding spectrum of the effects of abs compounds in hemostasis and related areas ."], "abstract_text": ["<S> \n purpose . using the classical ankaferd blood stopper ( abs ) solution to create active hemostasis during partial nephrectomy ( pn ) may not be so effective due to insufficient contact surface between the abs hemostatic liquid agent and the bleeding area . in order to broaden the contact surface , we generated a chimeric hemostatic agent , abs nanohemostat , via combining a self - assembling peptide amphiphile molecule with the traditional ankaferd hemostat . </S>", "<S> materials and methods . in order to generate abs nanohemostat , a positively charged peptide amphiphile ( pa ) molecule was synthesized by using solid phase peptide synthesis . for animal experiments </S>", "<S> , 24 wistar rats were divided into the following 4 groups : group 1 : control ; group 2 : conventional pn with only 0.5  ml ankaferd hemostat ; group 3 : conventional pn with abs + peptide gel ; group 4 : conventional pn with only 0.5  ml peptide solution . </S>", "<S> results . </S>", "<S> mean warm ischemia times ( wits ) were 232.8     56.3 , 65.6  11.4 , 75.5  17.2 , and 58.1  17.6 seconds in group 1 to group 4 , respectively . </S>", "<S> fibrosis was not different among the groups , while inflammation was detected to be significantly different in g3 and g4 . </S>", "<S> conclusions . </S>", "<S> abs nanohemostat has comparable hemostatic efficacy to the traditional ankaferd hemostat in the partial nephrectomy experimental model . </S>", "<S> elucidation of the cellular and tissue effects of this chimeric compound may establish a catalytic spark and open new avenues for novel experimental and clinical studies in the battlefield of hemostasis . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["the use of nanomaterials in medicine involves the applications of nanoparticles and manufactured nanosystems to provide regeneration at the cellular and tissue levels .", "they encapsulate therapeutic agents and typically carry multiple targeting motifs such as hemostasis [ 1 , 2 ] . for instance", ", ellis - behnke and coworkers   introduced a unique nanomedicinal method to stop bleeding using a self - assembling peptide that establishes a nanofiber barrier and incorporates it into the surrounding tissue to form an extracellular matrix [ 1 , 2 ] .  ", "biomaterials used as tissue engineering scaffolds have specific physical properties and might form fibrous networks similar to collagenous extracellular matrix .", "they also can be programmed to carry chemical and physical cues to provide bioactivity for cell - materials interactions . in the search for more improved bioactive materials for tissue engineering purposes , peptide amphiphile ( pa ) molecules are good candidates to bring scaffold properties and bioactivity together [ 3 , 4 ] .", "hydrophobic region of the pa is composed of fatty acids , which are packed against aqueous medium inducing self - assembly of the molecule to form cylindrical nanofibers . charged amino acids on hydrophilic peptide region of the pa molecules are responsible for triggering self - assembly upon isolation of charges .", "neutralization of charge density on amino acids causes aggregation of pa molecules and formation of nanofibrous networks at microscale .", "encapsulation of water by these nanofiber networks results in constitution of self - supporting gel at macroscale .", "peptide part of the pa molecules can be synthesized in a way to include bioactive epitopes or biofunctional chemical groups , which are eventually presented to cells on nanofibers .", "pa gels carrying these types of functionalities have been studied thoroughly and proven to be active in terms of inducing angiogenesis , neuronal regeneration , and cartilage and bone tissue formation [ 710 ] .", "charge neutralization mechanism also allows us to use bioactive molecules with high negative or positive charge density , such as heparin , dna , or oligonucleotides for inducing gel formation , while exploiting their bioactivity [ 6 , 11 , 12 ] .", "the use of medicinal plants as remedies for numerous disorders has formed the basis of current medicinal approach .", "various plants are used ethnomedicinally for prevention of excessive bleeding and as wound dressing to staunch blood flow .", "ankaferd hemostat , a topical hemostatic agent of plant origin , has recently been registered for the management of clinical hemorrhages when the conventional methods to control bleeding by ligature and/or hemostatic measures are ineffective [ 1416 ] .", "ankaferd blood stopper ( abs ) includes standardized preparation of the plants thymus vulgaris , glycyrrhiza glabra , vitis vinifera , alpinia officinarum , and urtica dioica [ 17 , 18 ] .", "abs provides vital erythroid aggregation covering the entire physiological hemostatic process via a unique protein network depending primarily on the interactions between abs and blood proteins , particularly with fibrinogen gamma and prothrombin [ 16 , 1922 ] .", "vital erythroid aggregation takes place with the spectrin and ankyrin receptors on the surface of red blood cells ( rbcs ) .", "those rbc proteins and the required atp bioenergy are included in the abs protein library [ 23 , 24 ] .", "on the other hand , controlled clinical trials indicated the safety and efficacy of topical ankaferd hemostat in distinct clinical backgrounds [ 19 , 2531 ] .   in this work", ", we present a chimeric hemostatic agent , abs nanohemostat , via combining a self - assembling pa molecule with the traditional ankaferd hemostat .", "the first step of our research was the synthesis of the specific self - assembling peptide molecules capable of being a part of the combined abs nanohemostat compound .", "the second step was the assembly of the peptide nanofibers and abs to generate the abs nanohemostat .", "the third step of our study was testing the in vivo hemostatic effects of abs nanohemostat in comparison with the traditional ankaferd in a previously established and published controlled surgical experimental trial in kidney tissue .", "the ultimate aim of the present study is , therefore , to test the efficacy of abs nanohemostat formed by combination of a self - assembling pa molecule and ankaferd hemostat for controlling surgical bleeding due to renal injury during partial nephrectomy ( pn ) and to compare its hemostatic effects to the traditional surgical and ankaferd hemostat groups ."], ["9-fluorenylmethoxycarbonyl ( fmoc ) , ter . butoxycarbonyl ( boc ) protected amino acids , rink amide mbha resin , and 2-(1h - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) were purchased from novabiochem or abcr .", "the other chemicals were purchased from fisher , merck , alfa aesar , or aldrich and used as provided .", "amino acid couplings were done with 2 equivalents of fmoc protected amino acid , 1.95 equivalents hbtu , and 3 equivalents of n , n - diisopropylethylamine ( diea ) for 2 hours .", "fmoc removal was performed with 20% piperidine / dimethylformamide ( dmf ) solution for 20  min .", "cleavage of the peptides from the resin was carried out with a mixture of tfa  :  tis  :  h2o in ratio of 95  :  2.5  :  2.5 for 2  h. excess tfa was removed by rotary evaporation .", "the remaining viscous peptide solution was triturated with ice - cold ether , and the resulting white product was dried under vacuum .", "pa molecules were characterized by liquid chromatography - mass spectrometry ( lc - ms ) ( figure 1 ) .", "mass spectrum was obtained with agilent lc - ms equipped with agilent zorbax extend - c18 2.1  50  mm column .", "a gradient of ( a ) water ( 0.1% formic acid ) and ( b ) acetonitrile ( 0.1% formic acid ) was used .  ", "it is composed of a lauryl ( c12 ) group , hydrophobic region of the pa , and a peptide sequence .", "vvag peptide sequence is used as -sheet inducer that causes nanofiber formation , while the lysine ( k ) residue is protonated at physiological ph and increases solvation of pa molecule in aqueous solution at ph 7 .  ", "figure 2 shows both the chemical structure of the pa ( lauryl - vvagk - am ) and the schematic representation and abs nanohemostat formation .", "1  10  m peptide solutions were measured from 300  nm to 190  nm , data interval and data pitch being 0.1  nm and scanning speed being 100  nm / min , all measurements with three accumulations . digital integration time ( dit )", "was selected as 1  sec , band width as 1  nm , and the sensitivity was standard .", "oscillatory rheology measurements were performed with anton paar physica rm301 rheometer operating with a 25  mm parallel plate configuration at 25c .", "each sample of 100  l total volume with a final peptide concentration of 1  wt% was carefully loaded on the center of the lower plate and incubated for 15  min before measuring .", "after equilibration , the upper plate was lowered to a gap distance of 0.5 mm .", "storage moduli ( g ) and loss moduli ( g ) values were scanned from 100  rad / s to 0.1  rad / s of angular frequency , with a 0.5% shear strain .", "sem experiments were performed with fei nova nanosem 230 , using the etd detector at low vacuum mode with 30  kev beam energy .", "small amounts of gels with a final peptide concentration of 1% were put on a metal mesh , dried at critical point ( 1072  psi , 31c ) with tousimis autosamdri-815 b series c critical point dryer and coated with 6  nm au - pd .  ", "figure 3 shows sem images of pa gel with and without ankaferd at ph 10 .", "an amount of 250  l of pa was mixed with 250  l abs solution on glass slide and incubated at room temperature for 30  min for self - supporting gel formation . due to anionic molecules in ankaferd solution ,", "all animal experimentations described in this paper were carried out in accordance with national guidelines for the use and care of  laboratory animals and were approved by the local animal review and ethics committee .", "all procedures were in full compliance with turkish law 6343/2 , veterinary medicine deontology regulation 6.7.26 , and with the helsinki declaration of world medical association recommendations on animal studies .", "the animals were obtained from the center of medical experimental research of ankara training and research hospital .", "the rats were housed in stainless steel cages in an animal room maintained at a temperature of 2224c with 12-hour light / dark periods .", "all were fed with the same amount of laboratory pellet diet and with water supplied ad libitum for a minimum of 5 days before procedure .", "a total of 24 wistar rats weighing 200 to 300  g were divided into 4 groups of 6 each and underwent pn .", "all operations were performed under total anesthesia with injection of 50  mg / kg intramuscular ketamine hydrochloride .", "the lower third of the left kidney was resected in guillotine fashion with a single stroke of an amputating knife .", "( i ) group 1 ( g1 ) is the left pn with hilar vascular control including intracorporeal suturing of the renal parenchyma and collecting duct ( control group ) . ", "( ii ) group 2 ( g2 ) is the conventional pn with only 0.5  ml traditional ankaferd hemostat ( abs ) application without suturing . ", "( iii ) group 3 ( g3 ) is the conventional pn with abs ( 0.25  ml ) + peptide ( 0.25  ml ) gel ( abs nanohemostat ) mixture application with no suturing . ", "( iv ) group 4 ( g4 ) is the conventional pn with only 0.5  ml peptide solution application .", "( i ) group 1 ( g1 ) is the left pn with hilar vascular control including intracorporeal suturing of the renal parenchyma and collecting duct ( control group ) .  ", "( ii ) group 2 ( g2 ) is the conventional pn with only 0.5  ml traditional ankaferd hemostat ( abs ) application without suturing .", "( iii ) group 3 ( g3 ) is the conventional pn with abs ( 0.25  ml ) + peptide ( 0.25  ml ) gel ( abs nanohemostat ) mixture application with no suturing .", "( iv ) group 4 ( g4 ) is the conventional pn with only 0.5  ml peptide solution application .", "two objective parameters were recorded during the surgical procedure : warm ischemia time ( wit ) and amount of bleeding ( aob ) .", "second ,  while the aob was measured with the bleeding area ( cm ) onto the sponges .", "all the rats were allowed to feed and drink water for the following 4 weeks .", "after that , each rat was sacrificed , and total nephrectomy was performed for histopathological examination .", "wi started with clamping the renal artery and vein and finished with taking the clamp out . in g1 ,", "traditional hemostasis method was used as compression onto the renal excised area and suturing the renal vessels and collecting duct with absorbable sutures ( figures 4(a ) and 4(b ) ) . in g2 , 2  ml of abs was dropped to the amputated renal margin steadily until bleeding stopped ( figures 4(c ) and 4(d ) ) . in g3 , abs ( 2  ml ) + nanopeptide gel mixture ( abs nanohemostat )", "other hemostatic methods including sponges , surgicel , electrocautery , and any other sources were not used to control bleeding in the present study .  ", "coronal 1 to 2  mm thick slices of the kidney were fixed in 4% formaldehyde and embedded in paraffin , and 3 to 4  m thick tissue sections were stained with hematoxylin and eosin .", "sections were examined for several parameters including erythroid aggregation , fibrosis , calcification , inflammatory cells , and siderophages . for each case , a global estimation of parenchymal damage was done and semiquantitatively graded on a scale in which 0 was no abnormality , and 1 + , 2 + , and 3 + represented mild , moderate , and severe abnormalities , respectively .", "data analysis was performed using ibm statistical package for the social sciences ( spss ) statistics software .", "comparisons of numerical variables among four groups were initially performed using the kruskal - wallis test and mann - whitney u - test with the bonferroni correction used for post hoc analysis to compare the groups separately . a p value of < 0.0083 ( 0.05/6 ) was deemed statistically significant in multiple comparisons .", "the percentages of histopathological features between study groups were compared using the chi - squared test . a p value of < 0.05 was considered as statistically significant ."], ["  in this work , we synthesized a positively charged pa molecule as shown in figure 2 .", "storage and loss moduli measurement by oscillatory rheometry was performed in order to characterize the mechanical properties of the gel .", "storage moduli , showing stiffness of gel , were found to be several folds higher than loss moduli , indicating a self - supporting gel formation ( figure 5 ) .", "moreover , storage modulus value was comparable to control gel formed by elevating ph of pa solution .", "circular dichroism spectroscopy was used to investigate the secondary structure of the pa and ankaferd mixture .", "characteristic -sheet formation was observed in pa - ankaferd solution , which was similar to pa solution at high ph .", "morphology of the gel was examined by using sem which showed nanofibrous network structure similar to pa gels reported before and pa samples at high ph .", "the ph 10 solutions for pa molecule were studied to analyze the peptide gels alone .", "the pa molecules were dissolved at ph 7 , and they self - assembled into nanofibers forming gels upon addition of ankaferd solution or by increasing the ph to 10 . left lower pole pn was surgically performed successfully to each rat in all groups . the kidney size and", "the same surgical equipments were used , and rommel clamps succeeded the warm ischemia during all surgical procedures .   ", "mean  sd wits were 232.8  56.3 , 65.6  11.4 , 75.5  17.2 , and 58.1  17.6 seconds in g1 to g4 , respectively .", "a significant difference was detected between g1 and g2 , 3 , and 4 , while no difference was observed among the three groups ; g1 , g2 , and g3 ( table 1 ) . in g1 ,", "renal parenchyma injury that caused the longer wit and repaired with primary suture was present in one rat . in g4 , more amount of nanopeptide agent was used to stop the bleeding in one rat following the warm ischemia . apart from these two cases , no complication was observed during the operations .", "fibrosis was not different among the groups ( p > 0.05 ) , while inflammation was detected to be significantly different in g3 and g4 ( p = 0.04 ) .", "erythrocyte aggregation , especially in glomerular field ( figure 7 ) , was confirmed to be significantly higher in g3 ( abs nanohemostat ) compared with the other groups ( p = 0.03 ) .", "hemosiderin was also detected in g3 ; however , no significant difference was detected among the groups . in g4 ,", "bleeding and congestion without erythrocyte aggregation were confirmed with significant calcification ( p < 0.001 ) ( figure 8) .  ", "we confirmed the significant demonstrative aggregation of red blood cells in abs nanohemostat group as shown in abs group .", "giant cell reaction , thyroidization , fibroblast activation , and microvascular proliferation were not detected ."], ["in this study , we revealed that pa - ankaferd gel mixture ( abs nanohemostat ) is effective as traditional ankaferd hemostat .", "moreover , this unique nanomedicine proves itself as an effective and easily applicable alternative hemostatic method which can successfully be used even in complicated hemorrhagic situations such as aggressive surgical tissue bleeding due to pn .  ", "several hemostatic agents are preferred to control external and internal bleedings ; yet , commercially available products are not sufficiently effective or fast acting to achieve hemostasis in extreme occasions .", "ankaferd hemostat is a topical hemostatic agent of plant origin , including molecules with high density of negative and positive charges , and is proven to work as an efficient hemostatic agent [ 1416 ] .", "controlled clinical trials indicated the safety and efficacy of topical ankaferd hemostat in distinct clinical backgrounds [ 19 , 2531 ] . in this work , we aimed to assemble a nanostructured scaffold material with hemostatic activity in the pa and ankaferd gel formed upon mixing soluble pa molecule with ankaferd solution ( abs nanohemostat ) .", "thus , we hypothesized that , while reducing blood loss , nanofiber network will serve as a scaffold for infiltrating cells to wound area , and eventually tissue regeneration rate will be enhanced . sustaining hemostasis in clinical hemorrhages is a challenging task and requires extensive effort to stabilize traumatic and surgical injuries .", "our experimental animal model is therefore clinically relevant since bleeding is one of the most important major complications leading to the morbidity and even mortality during pn for renal masses . providing hemostasis following", "the renal mass excision is the most important step of pn , and various hemostatic agents were used to stop bleeding during this operation [ 3335 ] .", "although the complication rates including bleeding are slightly higher than those of open radical nephrectomy , the advantage of renal preservation is more evident . likewise , laparoscopic pn , including hilar control , suture repair of the collecting system , suture ligation of blood vessels , and capsular closure , is also a valuable urological intervention .", "bleeding and ischemic renal damage due to warm ischemia period are the most important complications following surgery . in order to decrease wit and pnt", "several agents have been investigated for their hemostatic potential in managing vascular injury and many have also been evaluated for their efficacy on repairing the collecting system injury . in our results , abs and abs nanohemostat applications onto the transected kidney area provided active hemostasis in pn with significant decrease in wits and pnts , comparable with suture group . with more application number of abs products ,", "however , the absence of urinoma in the groups applied with abs may reveal the effect of abs on collecting system repair following one month .", "these favorable findings lead to the active hemostasis and regular healing of transected kidney without adherence and urinoma around the kidney . in our studies , we demonstrated foreign material reaction in conventional pn model but not observed in the abs groups .", "however , it could be speculated that the topical abs administration onto the sutured line might prevent foreign biological reactions ( fbrs ) against the suture by the way of affecting biological pathways .", "the anti - infective effects [ 3638 ] of abs represent a putative biological mode of action on the hypothetical fbr .  ", "the hemostatic effect of local abs application in a patient who underwent an open pn has already been shown .", "abs was successfully applied to stop bleeding without suturing the renal parenchyma , which provided active hemostasis in pn on the transected kidney area .", "the surgical and histopathological findings about pn in the present trial and our previous research   suggested similar results regarding the renal tissue effects of abs .", "moreover , nanotechnologically generated chimeric abs nanohemostat was observed to increase the duration of the contact of ankaferd hemostatic agent with the bleeding area during pn via the controlled targeted release of the topical hemostatic agent to the tissue . controlled clinical studiesconducted to evaluate the effectiveness of abs in distinct states of bleeding disorders documented the safety and efficacy of traditional ankaferd hemostat in comparison to conventional antihemorrhagic medications .", "the first randomized controlled clinical study was reported by teker et al .   in 49 patients with anterior epistaxis . in this study , patients were randomly grouped to receive the hemostasis technique by means of either abs wet tampon or phenylephrine impregnated gauze tampon for anterior epistaxis control .", "abs successfully treated all bleeding intensity with one application ( for 5  m ) .", "abs patients experienced fewer rebleeding rates within 7 days compared to phenylephrine patients ( 8.3 versus 20% , p < 0.05 ) . in patients to whom abs was applied , significant differences in effective control of anterior epistaxis", "similarly , another clinical trial has also demonstrated the effectiveness of abs in children undergoing tonsillectomy . in this prospective controlled study ,", "the success of abs and the traditional knot - tie approach to reach hemostasis for patients undergoing tonsillectomy was evaluated .", "forty - seven consecutive patients undergoing cold knife dissection tonsillectomy were studied , in all of whom abs wet tampon was used for right side tonsil hemorrhage and knot - tie technique for left side tonsil hemorrhage .", "the abs side was reported to have shorter hemostasis time after tonsil removal than the knot - tie side ( 3.19  0.74  min versus 7.29  2.33  min , p < 0.01 ) and less blood loss ( 1.57  2.26  ml versus 14.04  7.23  ml , p < 0.01 ) .", "the author concluded that abs is not only safe and efficient , but also it decreases intraoperative bleeding and reduces operating time compared to the traditional hemostasis methods after cold knife dissection tonsillectomy .", "the effectiveness of abs in cancer patients was also reported in a study by al et al . .", "sixty - nine cancer patients that were admitted for port insertion to a university hospital were randomized either to take a wet compress form of abs or regular dry sterile sponges to stop the bleeding that occurs during the clinically indicated vascular port insertion .", "the average time needed to stop the bleeding was 32.97  29.9  s for abs group and 123.75  47.5  s for dry sponge group .", "abs was proven to stop local bleeding in a shorter time , with a lower recurrence rate in comparison with the sterile sponge .", "additional clinical studies about antihemorrhagic efficacy of abs on the bleeding due to adenoidectomy , [ 28 , 40 ] the surgical bleeding of thyroidectomy ,   gastrointestinal bleedings [ 14 , 29 ] , and dental bleedings   were performed with similar beneficial outcomes .", "our promising experimental results about abs nanohemostat in this study cast novel studies to search clinically measurable end - points regarding the hemostatic efficiency of this unique chimeric nanomedicine .", "the findings of our present study represent a starting point to investigate clinical antihemorrhagic effects of abs nanohemostat . the ability of abs to induce formation of a protein network not only makes it an effective hemostatic agent , but also confers anti - infective , antineoplastic , and healing modulator properties to the extract [ 1416 ] .", "those pleiotropic effects were ascribed to the abs - induced alterations in apoptosis , angiogenesis , cellular proliferation , and cellular dynamics [ 46 ] . in previous investigations ,", "histopathological examination of the damaged vascular structures revealed abs - induced red blood cell aggregates supporting the hypothesis that abs - induced formation of the protein network with vital erythroid aggregation covers the entire physiological hemostatic process [ 42 , 43 ] .", "we have observed the same structures in the kidney tissue in the present study . in the current study ,", "abs nanohemostat has led to a more pronounced erythroid aggregation at the tissue level in comparison to the traditional ankaferd hemostat .", "however , clinical antihemorrhagic efficiency of ankaferd solution seems to be superior to the chimeric ankaferd nanohemostat ."], ["in summary , abs nanohemostat has comparable hemostatic efficacy to the traditional ankaferd hemostat in the pn experimental model .", "these observations prompt the design of future experimental and clinical studies focusing on the antibleeding and vascular repair effects of this novel hemostatic nanomedicine .", "the production of abs nanohemostat in gel formation is also important for using the hemostatic material during the laparoscopic urologic surgery .", "the steady scaffold onto the resected area with abs nanohemostat predicts the intracorporeal use of abs hemostat as a promising agent .", "likewise , future controlled studies are needed to shed further light on the expanding spectrum of the effects of abs compounds in hemostasis and related areas ."]]}
{"article_id": "PMC4242935", "article_text": ["falls are one of the leading injury - related accidents involving older adults whose muscular \n strength , proprioceptive sense , and bodily coordination are compromised with aging1 .", "the elderly lack obstacle negotiation \n capacity and are prone to trip over commonly encountered environmental barriers such as door \n sills , pavement blocks , and safety bumps2 . \n in a local community , 33% of elderly people were reported to experience falls , of which \n 42.4% suffered falls3 .", "individuals with a history of falls have fears of repeated falls , depression , and anxiety , \n all of which lead to decreased physical activities4 .", "these negative experiences result in reduced physical capacity in \n terms of muscular strength , bodily flexibility , and coordination , rendering the affected \n elderly even more susceptible to falls in a vicious cycle5 .", "notably , elderly women are more at risk of secondary injury such as \n severe fractures after falling than elderly men because of their low bone density after \n menopause6 .", "interventions for fall prevention include exercise , education , environmental improvements , \n and medication7,8,9 , of which exercise has been \n used in fall prevention programs in many ways , as it takes less time and money to implement \n and programs are easy to set up .", "carter et al.10 reported in a \n bibliographical study on exercise intervention that exercise improved leg strength 44.4% and \n balance ability 37.5% in older adults and that as weakness of muscle strength in the trunk \n was more important than that in the lower extremities , strengthening of lumbar function can \n improve functional stability , leading to increased balance ability , gait ability , and \n prevention of falling11 , 12 .", "core stabilization exercise ( cse ) can improve balance control ability by reinforcing \n intersegmental muscles in the multifidus , transversus abdominis , and rotators13 and physicopsychological functions in a \n harmonious way by stimulating proprioception powerfully when it is accompanied by swiss ball \n exercises , improving balance sense , and maintenance ability14 .", "previous studies on falling in elderly adults involving cse have \n been limited to balance ability or gait ability15 , \n 16 , and there have been only a few \n studies on obstacles as an environmental factor or psychological factors .", "therefore , this study evaluated changes in the physicopsychological functions of elderly \n women with a fear of falling in negotiating obstacles such as a doorstep ( 5.2  cm ) of a \n bathroom , where falling occurs frequently , through a performance - oriented mobility \n assessment ( poma ) in order to understand whether cse is valid as an efficient exercise to \n prevent and control falling in consideration of the physicopsychological functions of \n elderly women .", "the subjects of the study were 20 elderly women above the age of 65 who were able to walk \n independently , did not have experience participating in regular balance training more than \n twice a week within the past 6 months , had a score higher than 24 on the mini - mental status \n examination - korea ( mmse - k ) , had no limits on exercise performance due to visual or \n musculoskeletal disorders and no previous experience of falling , and scored lower than 19 on \n the tinetti poma17 .", "the purpose and \n methods of this study were explained to all the participants , who read and signed an \n informed consent from that revealed all the details of the study protocol , which were \n approved by the ethics committee of kangwon national university ( no .", "the subjects selected were divided into an experimental group and a control group by \n drawing lots , and the average age , height , and weight in the experimental group were \n 73.203.46  years , 152.154.29  cm , and 57.787.95  kg , while those in the control group were \n 71.003.50  years , 149.836.45  cm , and 53.8011.04  kg .", "there were no significant differences \n among the groups with regard to age , height , or weight ( p>0.05 ) .", "the core stabilization exercise program proposed by jeffrey18 and hesari et al.19 was applied to the experimental group and cse was composed of three \n steps .", "level 1 consisted of abdominal hollowing in a supine position , abdominal hollowing in \n a prone position , abdominal hollowing in a quadruped position , abdominal hollowing in a \n supine position while curling the feet , abdominal hollowing in a prone position while \n curling the feet , and modified side bridging .", "level 2 consisted of bridging with abdominal \n hollowing , pelvic bridging , the dying bug with abdominal hollowing , and abdominal hollowing \n while seated on a swiss ball .", "level 3 consisted of pelvic bridging with a swiss ball , bird \n dog exercise , twists on a swiss ball , and bird dog exercises on a swiss ball .", "the \n experimental group performed a core stabilization program consisting of three levels for \n 30  min , three times per week on alternate days , for 6 weeks .", "this program consisted of three \n levels , and the subjects began at exercise level 1 and proceeded to the next level according \n to the protocol for the day .", "level 1 included static holds in a stable environment , level 2 \n included dynamic movements in a stable environment , and level 3 included dynamic movements \n in an unstable environment , such as on a swiss ball , and resisted dynamic movements in an \n unstable environment . before the program and 6 weeks after the program , the tinetti poma , crossing velocity ( cv ) , \n maximum vertical heel clearance ( mvhc ) and knee flexion angle , which are related to physical \n functions , were measured , and with respect to psychological functions , depression and fear \n of falling were measured .", "the tinetti poma is an instrument used to decide the degree of \n fear of falling and balance and mobility in elderly adults consisting of items scored on a \n 3-point scale .", "the maximum score is 28 , with 16 points for balance and 12 points for gait . \n", "if a subject scores below 19 , fear of falling is high , and scores of 19 to 24 indicate an \n intermediate degree for fear of falling ; scores of 25 to 28 indicate no fear of falling . for \n the crossing velocity ( cv ) , the horizontal distance from the point at which the leading limb \n leaves the ground to the point at which the heel returns to the ground", "again is divided by \n the time taken20 . for the maximum \n vertical heel clearance ( mvhc ) ,", "the vertical distance from the height of the obstacle before \n obstacle negotiation to the ball of the leading limb was measured21 .", "the obstacle was 60  cm long , \n 10  cm wide , and 5.2  cm high .", "the subjects began their gait 5  m from the obstacle and \n continued on 3  m after obstacle negotiation .", "cv and mvhc were measured with the use of dartfish \n software ( dartfish , fribourg , switzerland)23 , and the results were output in a data table . to analyze \n depression , the ces - d ( center for epidemiological studies - depression scale ) was used .", "it is \n scored on a 4-point scale ( 03 ) , and includes a total of 20 questions .", "the higher the score , \n the higher the depression24 . for fear of \n falling , the fofq ( fear of falling questionnaire ) was used .", "it is scored on a 4-point scale \n ( 14 ) and includes a total of 11 questions .", "the higher the score , the higher the fear of \n falling25 . for statistical analysis of the results , spss 18.0 was used .", "to explain the differences in measurement variables \n according to the measurement period between exercise groups , a 2-way anova with repeated \n measure was used , and the level of statistical significance was =0.05 .", "repeated measure anova to analyze changes in the poma , cv , mvhc , kf , depression , and fof \n according to the measurement periods showed that there was a statistically significant \n difference in the interaction between time and the groups and that the changes in poma , cv , \n mvhc , kf , depression , and fof according to time differed ( p<0.001 ) ( table 1table 1.changes in physical and psychological function variablesgroupbeforeafterpoma ( score)eg ( n=10)17.601.3419.301.57**cg ( n=10)17.801.4017.601.43cv ( m / sec)eg ( n=10)1.560.211.600.21*cg ( n=10)1.560.101.550.09mvhc ( cm)eg ( n=10)7.421.387.021.33*cg ( n=10)7.361.627.311.74kf ( angle)eg ( n=10)111.003.16108.604.06*cg ( n=10)110.803.77110.303.16depression ( score)eg ( n=10)27.103.6724.503.34**cg ( n=10)26.002.5825.203.00fear of falling ( score)eg ( n=10)22.501.4319.801.69*cg ( n=10)22.603.2721.903.54meansd .", "eg , experimental group ; cg , control group ; poma , performance - oriented \n mobility assessment ; cv , crossing velocity ; mvhc , maximum vertical heel clearance ; kf , \n knee flexion .", "eg , experimental group ; cg , control group ; poma , performance - oriented \n mobility assessment ; cv , crossing velocity ; mvhc , maximum vertical heel clearance ; kf , \n knee flexion . * p<0.05 ; * * p<0.01", "older adults have low balance and stability due to physiological and functional decreases \n that occur with ageing26 , and to \n compensate for balance and stability in gait , cadence and stride length decrease27 . in the case of obstacle negotiation ,", "when \n the leading foot encounters obstacles , the center of the body moves forward and fear of \n falling increases28", ". in this case , the \n mvhc , joint angle , and cv are important measures to assess the ability of elderly adults to \n safely cross obstacles during gait5 , 29 , 30 .", "although falling is not always accompanied by physical injury , fear \n of falling again can be used to predict falling in elderly adults5 .", "fear of falling leads to a decrease in activity and low \n self - esteem for independent behavior4 and \n has direct negative effects that result in decreased balance and gait disorder31 , 32 .", "cse is training to improve the stability of the trunk by inducing a correcting reaction and \n an equilibrium reaction through balance training of the flexors and extensors , and as it has \n been judged to have an effect on physicopsychological functions in obstacle negotiation , a \n clinical study was conducted .", "the results of the present study showed that the tinetti poma , cv , mvhc , and knee flexion \n angle were significantly improved .", "esculier et al.33 provided lumbar stability training through a wii fit program for \n parkinson s patients for 6 weeks and administered the tinetti poma .", "chou et al.20 reported that the cv was slow for elderly adults with \n lower balance ability .", "weerdesteyn et al.21 provided balance , gait , and coordination training for elderly \n adults and reported that foot clearance decreased , which matched with the results of the \n present study .", "park and lee22 reported \n that the maximum knee flexion angle of normal adults with lumbar stability was significantly \n lower in comparison with that of elderly adults with lumbar instability , which partially \n matched with the results of the present study .", "the study by lee et al.34 , who reported that a falling prevention program decreased \n the gds ( geriatric depression scale ) significantly , was in agreement with the study by duque \n et al.35 , who reported that fear of \n falling by elderly adults who experienced falling significantly decreased as a result of \n balance training using a virtual reality system .", "such results indicate that stability of the trunk was secured , as cse decreased the sway \n area of the center of mass . through harmonious exercise of the limbs based on physical \n stability", ", the subjects could negotiate obstacles precisely and easily , had higher \n self - esteem and had less fear of falling and depression .", "it is suggested that the 6-week cse \n decreased excessive obstacle gait with physical instability in elderly women and improved \n their physicopsychological functions involving obstacle gait ."], "abstract_text": ["<S> [ purpose ] the aim of the present study was to investigate the effects of core stability \n exercise ( cse ) on the physical and psychological functions of elderly women while \n negotiating general obstacles . [ subjects and methods ] after allocating 10 elderly women \n each to the core stability training group and the control group , we carried out \n performance - oriented mobility assessment ( poma ) and measured crossing velocity ( cv ) , \n maximum vertical heel clearance ( mvhc ) , and knee flexion angle for assessing physical \n performances . </S>", "<S> we evaluated depression and fear of falling for assessing psychological \n functions . </S>", "<S> [ results ] relative to the control group , the core stability training group \n showed statistically significant overall changes after the training session : an increase \n in poma scores , faster cv , lower mvhc , and a decrease in knee flexion angle . </S>", "<S> furthermore , \n depression and fear of falling decreased significantly . </S>", "<S> [ conclusion ] cse can have a \n positive effect on the improvement of physical and psychological performances of older \n women who are vulnerable to falls as they negotiate everyday obstacles . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["falls are one of the leading injury - related accidents involving older adults whose muscular \n strength , proprioceptive sense , and bodily coordination are compromised with aging1 .", "the elderly lack obstacle negotiation \n capacity and are prone to trip over commonly encountered environmental barriers such as door \n sills , pavement blocks , and safety bumps2 . \n in a local community , 33% of elderly people were reported to experience falls , of which \n 42.4% suffered falls3 .", "individuals with a history of falls have fears of repeated falls , depression , and anxiety , \n all of which lead to decreased physical activities4 .", "these negative experiences result in reduced physical capacity in \n terms of muscular strength , bodily flexibility , and coordination , rendering the affected \n elderly even more susceptible to falls in a vicious cycle5 .", "notably , elderly women are more at risk of secondary injury such as \n severe fractures after falling than elderly men because of their low bone density after \n menopause6 .", "interventions for fall prevention include exercise , education , environmental improvements , \n and medication7,8,9 , of which exercise has been \n used in fall prevention programs in many ways , as it takes less time and money to implement \n and programs are easy to set up .", "carter et al.10 reported in a \n bibliographical study on exercise intervention that exercise improved leg strength 44.4% and \n balance ability 37.5% in older adults and that as weakness of muscle strength in the trunk \n was more important than that in the lower extremities , strengthening of lumbar function can \n improve functional stability , leading to increased balance ability , gait ability , and \n prevention of falling11 , 12 .", "core stabilization exercise ( cse ) can improve balance control ability by reinforcing \n intersegmental muscles in the multifidus , transversus abdominis , and rotators13 and physicopsychological functions in a \n harmonious way by stimulating proprioception powerfully when it is accompanied by swiss ball \n exercises , improving balance sense , and maintenance ability14 .", "previous studies on falling in elderly adults involving cse have \n been limited to balance ability or gait ability15 , \n 16 , and there have been only a few \n studies on obstacles as an environmental factor or psychological factors .", "therefore , this study evaluated changes in the physicopsychological functions of elderly \n women with a fear of falling in negotiating obstacles such as a doorstep ( 5.2  cm ) of a \n bathroom , where falling occurs frequently , through a performance - oriented mobility \n assessment ( poma ) in order to understand whether cse is valid as an efficient exercise to \n prevent and control falling in consideration of the physicopsychological functions of \n elderly women ."], ["the subjects of the study were 20 elderly women above the age of 65 who were able to walk \n independently , did not have experience participating in regular balance training more than \n twice a week within the past 6 months , had a score higher than 24 on the mini - mental status \n examination - korea ( mmse - k ) , had no limits on exercise performance due to visual or \n musculoskeletal disorders and no previous experience of falling , and scored lower than 19 on \n the tinetti poma17 .", "the purpose and \n methods of this study were explained to all the participants , who read and signed an \n informed consent from that revealed all the details of the study protocol , which were \n approved by the ethics committee of kangwon national university ( no .", "the subjects selected were divided into an experimental group and a control group by \n drawing lots , and the average age , height , and weight in the experimental group were \n 73.203.46  years , 152.154.29  cm , and 57.787.95  kg , while those in the control group were \n 71.003.50  years , 149.836.45  cm , and 53.8011.04  kg .", "there were no significant differences \n among the groups with regard to age , height , or weight ( p>0.05 ) .", "the core stabilization exercise program proposed by jeffrey18 and hesari et al.19 was applied to the experimental group and cse was composed of three \n steps .", "level 1 consisted of abdominal hollowing in a supine position , abdominal hollowing in \n a prone position , abdominal hollowing in a quadruped position , abdominal hollowing in a \n supine position while curling the feet , abdominal hollowing in a prone position while \n curling the feet , and modified side bridging .", "level 2 consisted of bridging with abdominal \n hollowing , pelvic bridging , the dying bug with abdominal hollowing , and abdominal hollowing \n while seated on a swiss ball .", "level 3 consisted of pelvic bridging with a swiss ball , bird \n dog exercise , twists on a swiss ball , and bird dog exercises on a swiss ball .", "the \n experimental group performed a core stabilization program consisting of three levels for \n 30  min , three times per week on alternate days , for 6 weeks .", "this program consisted of three \n levels , and the subjects began at exercise level 1 and proceeded to the next level according \n to the protocol for the day .", "level 1 included static holds in a stable environment , level 2 \n included dynamic movements in a stable environment , and level 3 included dynamic movements \n in an unstable environment , such as on a swiss ball , and resisted dynamic movements in an \n unstable environment . before the program and 6 weeks after the program , the tinetti poma , crossing velocity ( cv ) , \n maximum vertical heel clearance ( mvhc ) and knee flexion angle , which are related to physical \n functions , were measured , and with respect to psychological functions , depression and fear \n of falling were measured .", "the tinetti poma is an instrument used to decide the degree of \n fear of falling and balance and mobility in elderly adults consisting of items scored on a \n 3-point scale .", "the maximum score is 28 , with 16 points for balance and 12 points for gait . \n", "if a subject scores below 19 , fear of falling is high , and scores of 19 to 24 indicate an \n intermediate degree for fear of falling ; scores of 25 to 28 indicate no fear of falling . for \n the crossing velocity ( cv ) , the horizontal distance from the point at which the leading limb \n leaves the ground to the point at which the heel returns to the ground", "again is divided by \n the time taken20 . for the maximum \n vertical heel clearance ( mvhc ) ,", "the vertical distance from the height of the obstacle before \n obstacle negotiation to the ball of the leading limb was measured21 .", "the obstacle was 60  cm long , \n 10  cm wide , and 5.2  cm high .", "the subjects began their gait 5  m from the obstacle and \n continued on 3  m after obstacle negotiation .", "cv and mvhc were measured with the use of dartfish \n software ( dartfish , fribourg , switzerland)23 , and the results were output in a data table . to analyze \n depression , the ces - d ( center for epidemiological studies - depression scale ) was used .", "it is \n scored on a 4-point scale ( 03 ) , and includes a total of 20 questions .", "the higher the score , \n the higher the depression24 . for fear of \n falling , the fofq ( fear of falling questionnaire ) was used .", "it is scored on a 4-point scale \n ( 14 ) and includes a total of 11 questions .", "the higher the score , the higher the fear of \n falling25 . for statistical analysis of the results , spss 18.0 was used .", "to explain the differences in measurement variables \n according to the measurement period between exercise groups , a 2-way anova with repeated \n measure was used , and the level of statistical significance was =0.05 ."], ["repeated measure anova to analyze changes in the poma , cv , mvhc , kf , depression , and fof \n according to the measurement periods showed that there was a statistically significant \n difference in the interaction between time and the groups and that the changes in poma , cv , \n mvhc , kf , depression , and fof according to time differed ( p<0.001 ) ( table 1table 1.changes in physical and psychological function variablesgroupbeforeafterpoma ( score)eg ( n=10)17.601.3419.301.57**cg ( n=10)17.801.4017.601.43cv ( m / sec)eg ( n=10)1.560.211.600.21*cg ( n=10)1.560.101.550.09mvhc ( cm)eg ( n=10)7.421.387.021.33*cg ( n=10)7.361.627.311.74kf ( angle)eg ( n=10)111.003.16108.604.06*cg ( n=10)110.803.77110.303.16depression ( score)eg ( n=10)27.103.6724.503.34**cg ( n=10)26.002.5825.203.00fear of falling ( score)eg ( n=10)22.501.4319.801.69*cg ( n=10)22.603.2721.903.54meansd .", "eg , experimental group ; cg , control group ; poma , performance - oriented \n mobility assessment ; cv , crossing velocity ; mvhc , maximum vertical heel clearance ; kf , \n knee flexion .", "eg , experimental group ; cg , control group ; poma , performance - oriented \n mobility assessment ; cv , crossing velocity ; mvhc , maximum vertical heel clearance ; kf , \n knee flexion . * p<0.05 ; * * p<0.01"], ["older adults have low balance and stability due to physiological and functional decreases \n that occur with ageing26 , and to \n compensate for balance and stability in gait , cadence and stride length decrease27 . in the case of obstacle negotiation ,", "when \n the leading foot encounters obstacles , the center of the body moves forward and fear of \n falling increases28", ". in this case , the \n mvhc , joint angle , and cv are important measures to assess the ability of elderly adults to \n safely cross obstacles during gait5 , 29 , 30 .", "although falling is not always accompanied by physical injury , fear \n of falling again can be used to predict falling in elderly adults5 .", "fear of falling leads to a decrease in activity and low \n self - esteem for independent behavior4 and \n has direct negative effects that result in decreased balance and gait disorder31 , 32 .", "cse is training to improve the stability of the trunk by inducing a correcting reaction and \n an equilibrium reaction through balance training of the flexors and extensors , and as it has \n been judged to have an effect on physicopsychological functions in obstacle negotiation , a \n clinical study was conducted .", "the results of the present study showed that the tinetti poma , cv , mvhc , and knee flexion \n angle were significantly improved .", "esculier et al.33 provided lumbar stability training through a wii fit program for \n parkinson s patients for 6 weeks and administered the tinetti poma .", "chou et al.20 reported that the cv was slow for elderly adults with \n lower balance ability .", "weerdesteyn et al.21 provided balance , gait , and coordination training for elderly \n adults and reported that foot clearance decreased , which matched with the results of the \n present study .", "park and lee22 reported \n that the maximum knee flexion angle of normal adults with lumbar stability was significantly \n lower in comparison with that of elderly adults with lumbar instability , which partially \n matched with the results of the present study .", "the study by lee et al.34 , who reported that a falling prevention program decreased \n the gds ( geriatric depression scale ) significantly , was in agreement with the study by duque \n et al.35 , who reported that fear of \n falling by elderly adults who experienced falling significantly decreased as a result of \n balance training using a virtual reality system .", "such results indicate that stability of the trunk was secured , as cse decreased the sway \n area of the center of mass . through harmonious exercise of the limbs based on physical \n stability", ", the subjects could negotiate obstacles precisely and easily , had higher \n self - esteem and had less fear of falling and depression .", "it is suggested that the 6-week cse \n decreased excessive obstacle gait with physical instability in elderly women and improved \n their physicopsychological functions involving obstacle gait ."]]}
{"article_id": "PMC4143597", "article_text": ["a stroke is defined by the who as  rapidly developing clinical signs of a focal ( or global ) disturbance of cerebral function , with symptoms lasting 24  h or longer or leading to death , with no apparent cause other than of vascular origin  . taking into consideration the cause , the mechanism and the character of morphological lesions ,", "there are two types of stroke : ischemic and haemorrhagic [ 2 , 3 ] .", "eighty percent of strokes are of ischemic origin [ 2 , 4 ] , and their most common cause is thrombosis of the atherosclerotic internal carotid artery ( ica ) and/or cerebral arteries ( ca)20  % .", "a stroke , including an ischemic one , is of particular interest to the forensic pathologist aiming on establishing the cause of death and its mechanism , rather than for giving an opinion in cases of suspected medical malpractice .", "neck and/or head trauma may very rarely constitute a cause of thrombosis of the ica and ca without any pre - existing pathology [ 58 ] .", "it most commonly results from direct , penetrating or non - penetrating ( blunt ) neck trauma in the region of large cervical vessels ; less frequently it is attributed to indirect head trauma . in such cases", "the autopsy result and available clinical information has to allow the forensic pathologist to prove the existence of neck and/or head trauma and establish its causal connection with ica thrombosis and exclude other , known , non - traumatic causes of the latter .", "we present two cases of ica thrombosis with concomitant middle cerebral artery ( mca ) thrombosis secondary to the thrombus present in the ica lumen , in which the postmortem examination , the medical records regarding hospitalization and records of investigation indicated trauma as the cause of thrombosis .", "a 57-year old male carpenter sustained an injury in an occupational accident in the carpenter s warehouse .", "he was struck very hard in the face , in the region of his left cheek by an irregular - shaped wood fragment which looked like a big splinter ( measuring about 26  cm in length , and with the greatest cross - sectional dimensions of 3.5    1.5  cm ) that broke off from the board being processed on the carpenter s machine ( fig .  1 ) .", "first aid was provided in the emergency department in the local hospital , where the presence of a large , wooden fragment that thrust deep into the left cheek and penetrated through the parapharyngeal space into the neck was diagnosed . on admission", "he was then transferred to the otolaryngological ward , where a physical examination , including an evaluation of his neurological status revealed : maintained consciousness , narrow and symmetric pupils , circulatory and respiratory sufficiency and bp of 180/80  mmhg .", "the ct examination revealed the presence of a foreign body penetrating the left maxillary sinus with a comminuted fracture of the anterior and inferior wall below the external surface of the base of the skull , and a left occipital condyle fracture with concomitant breaking and impression into the posterior cranial fossa and soft tissues of the neck .", "no cerebral lesions , including traumatic , or features of raised intracranial pressure were observed ( fig .  2 ) .", "the ct angiography allowed the diagnosis of an occlusion of the ica by a thrombus localised at the level of the bifurcation of the common carotid artery ( cca ) and at the level of c1 , adjacent to the foreign body .", "the laboratory findings were as follows : d - dimer 20921.25  g / l ( n  <  500 ) ; pt and aptt within normal limits .", "once the diagnostic procedures were finished , a surgical intervention was performed on the day of admission .", "the preparation of the internal jugular vein revealed the medial translocation and compression of the carotid vessels . the foreign body was removed .", "post - interventional ct revealed the thrombotic occlusion of the left ica and mca and a hypodense area supplied by the mca and the concomitant left hemisphere oedema of the brain .", "the patient died on the 6th post - operative day exhibiting symptoms of intracranial hypertension and a brain oedema.fig . ", "2case 1 : localisation of the foreign body ( ct ) case 1 :  wooden splinter  removed during the surgical procedure case 1 : localisation of the foreign body ( ct )", "the findings of the medico - legal autopsy were as follows : a thrombus occluding the left ica and the proximal segment of the left mca , a large infarction area in the temporal lobe and adjacent parts of the frontal and occipital lobes of the left hemisphere of the brain , a large brain oedema with features of subfalcine herniation and transtentorian herniation , a comminuted fracture of the left maxilla and an occipital bone fracture in the region of the occipital condyle with the fragment impression .", "the histopathological examination revealed the occlusion of the left ica by the thrombus ( h  +  e ( fig . ", "3 ) , masson , gomori , verhoeff and ptah ) , focal intimal haemorrhages and features of a mechanical injury  the rupture of the artery wall in the form of an irregular fissure in the tunica media ( fig .  4 ) .", "the samples of the macroscopically changed left cerebral hemisphere revealed ischemic necrosis with polymorphonuclear leukocytes reaction , oedema and hyperaemia , while secondary , focal , non - reactive perivascular haemorrhages were found in the brainstem .", "other organs showed no significant histopathological lesions , apart from morphological indices of circulatory disturbances.fig . ", "3case 1 : the internal carotid artery wall with the adjacent thrombus ( h  +  e)fig . ", "4case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h  +  e and b verhoeff ) case 1 : the internal carotid artery wall with the adjacent thrombus ( h  +  e ) case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h  +  e and b verhoeff )", "a 32-year old female patient was brought to the hospital after being battered what resulted in police intervention . on admission", "the patient was conscious , complaining about a left upper extremity contracture that started 2  weeks prior to admission .", "she was generally healthy , with no chronic diseases , taking no medication , apart from being a smoker .", "for the previous month she had been repeatedly battered by her partner (  about twice a week  ) with a fist and open hand to the face , neck and back , as well as pulled by the hair .", "the last battery that took place 1  day prior to admission resulted in a short - term loss of consciousness . on admission", "the following body injuries were described in her medical record : resorbing bruises on the face , including a wound of the lower lip mucosa , and the upper extremities , . on admission", "the patient was sleepy with left - sided hemiparesis with symptoms concerning mainly the upper extremity , blood pressure was 110/80  mmhg , a fundus examination revealed no pathological lesions .", "d - dimer 1 200  ng / ml ( n  <  500 ) , aptt slightly shortened 25.69  s ( n : 2836 ) , aptt ratio and pt within normal limits .", "ct revealed the presence of a hypodense area in the right hemisphere region supplied by the mca , whereas mri scan confirmed the presence of an ischemic brain damage .", "thrombotic occlusion of the ica and mca was visible in mri ( fig .  5 ) and angioct ( fig . ", ", the patient died on the 12th day after admission presenting symptoms of raised intracranial pressure with a concomitant brainstem injury secondary to a brain oedema.fig . ", "5case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri)fig . ", "6case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri ) case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) the findings of the medico - legal autopsy were as follows : a thrombus occluding the right ica and the mca , thrombosis of the internal jugular veins , as well as the superior sagittal sinus and transverse sinus , an extensive infarction area in the right hemisphere region supplied by the mca ; a brain oedema with features of right - sided subfalcine herniation and transtentorian herniation , carotid and basal ca with no atherosclerotic lesions .", "a histopathological examination revealed an occlusion of the right ica by the thrombus ( h  +  e , masson , gomori , verhoeff and ptah)a muscular type artery with normal wall structure , the lumen filled with thrombus fragments , mechanical injury of the arterial wall ", "rupture of the intima and tunica media ( fig .  7 ) . internal jugular veins  normal wall structure , occluded by thrombi .", "there were dominating circulatory disturbances in the brain in the form of very significant capillary - venous hyperaemia resulting from an impaired cerebral venous return secondary to jugular vein and dural sinuses thrombosis.fig . ", "7case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h  +  e and b verhoeff ) case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h  +  e and b verhoeff )", "observed in both cases , focal ischemic brain injury was secondary to the thrombosis of the ica and mca .", "cerebral vascular lesions may be causally associated with the sustained head and/or neck trauma with concomitant ica thrombosis and ischemic brain damage [ 5 , 713 ] .", "they can , however , accompany head and/or neck trauma , yet be independent of trauma and have other morbid origins [ 2 , 4 ] . in both cases we considered the most common , known , possible non - traumatic causes for ica thrombosis , including atherosclerosis , which is the most common cause for ica and ca thrombosis .", "the endothelium and the connective tissue cap injury expose the blood coagulation activating factors , which induce thrombus formation .", "the endothelium injury itself with exposure of the basal membrane may predispose to thrombus formation as well .", "the presence of stable atherosclerotic plaque may be complicated by arterial thrombosis , and this refers to cerebral vessels as well .", "however , vessel stenosis has to be significant , i.e. the blood flow transforms from being laminar into post - stenotic turbulent [ 1417 ] both the in vivo neuroradiologic diagnostic imaging modalities , including ct angiography , and the postmortem examination with a histopathological assessment did not reveal the atherosclerosis of ica or mca in any of the discussed cases . other factors favouring carotid arteries thrombosis include : hypertension , diabetes , hypercholesterolaemia , obesity and smoking [ 2 , 4 , 18 ] .", "such factors accelerate the course of atherosclerosis , of which a common complication is thrombosis .", "the analysis of both patients medical records , including the laboratory findings obtained during hospitalisation allowed diabetes and lipid metabolism disorders to be ruled out .", "the woman did not manifest features of hypertension , whereas unstable aterial pressure values , i.e. labile hypertension , was observed in the male patient .", "no lesions in the small - sized cerebral vessels that could have been responsible for the ischemic stroke were found during the postmortem examination , including the neuropathologic examination , within the ischemic , as well as in the non - ischemic areas .", "no arteritis , amyloid angiopathy , vascular malformations nor lesions observed in the course of hypertensive encephalopathy were found .", "the presence of a thrombus inside the ica and/or ca , and secondary focal ischemic brain damage may result from embolism [ 2 , 4 , 18 , 19 ] .", "the most common source of such embolic material are thrombi localised in the left side of the heart ( auricle of the left atrium in patients with atrial fibrillation ) , parietal thrombi in the left ventricle whose formation is associated with myocardial infarction or more frequently aneurysm of the heart in the area of an extensive post - infarction scar or left ventricle dilatation , e.g. in the course of dilated cardiomyopathy or endocarditis , especially of bacterial origin", "the embolism in the ica and ca may then develop in patients with atrial septal defect and deep vein thrombosis .", "the postmortem examination did not , however , reveal any of these conditions in the discussed patients .", "a significantly less common cause of stroke are haematological disorders , which are responsible for about 1  % of all strokes and occur slightly more frequently in young subjects .", "the laboratory findings performed during hospitalisation and autopsies allowed haematological disorders to be ruled out as factors predisposing to thrombosis .", "the elevation of d - dimer level was the sole abnormal parameter observed on the coagulation profile and was associated with present ica and mca thrombosis in both cases .", "oral contraceptives ( oc ) should be considered as a possible cause for thrombosis in the female patient .", "oc are described in neurology as one of the ethological factors of ica and ca thrombosis . in the presented case the anamnesis collected on the patient s admission to", "the hospital indicated that she did not take any medication , most probably including oc .", "however , should she have taken oc , they did not significantly influence the development of ica thrombosis .", "arterial thrombosis in such cases is very rare in women below 35  years of age [ 21 , 22 ] .", "the annual incidence of ischemic stroke in women in this age group is 13 cases/100 000 women .", "the following risk factors are considered to increase the risk of developing thrombosis in subjects using oc : hypertension , diabetes , hyperlipidemia and smoking [ 2025 ] .", "oc predispose more strongly to the development of venous , rather then arterial thrombosis , which results from the haemodynamic properties of blood flow in these two types of vessels .", "other risk factors for venous thrombosis are slowed blood flow and thrombophilia , whereas vessel wall injury ( endothelium ) , including the atherosclerotic plaque are characteristic risk factors for arterial thrombosis . despite doubts concerning her taking", "oc , the patient s age ( 32  years old ) and lack of other , above - mentioned risk factors and also atherosclerosis allow oc to be ruled out as a possible cause of ica and mca thrombosis .", "the above - mentioned exclusions and the circumstances preceding the disease established in the course of the investigation indicate the traumatic background of the ica thrombosis in both cases .", "the ct and ct angiography performed in the first patient revealed the presence of a foreign body adjacent to the left ica on the level of c1 , whereas medial translocation and compression of carotid vessels caused by the foreign body were found during the surgical procedure .", "the histopathological examination revealed features of mechanical arterial injury , namely the rupture of the tunica media with focal haemorrhages into the intima and tunica media .", "the above - mentioned conditions indicate direct trauma as the cause of thrombosis . apart from the arterial occlusion by the thrombus found in the histopathological sections obtained from the second patient , the mechanical injury to the arterial wall in the form of the intima and tunica media rupture were also noted .", "such findings and the information obtained during investigation indicate indirect trauma as the cause of arterial thrombosis .", "post - traumatic ica thrombosis may result from a penetrating ( e.g. knife , bullet or other foreign body ", "splinter , as in case 1 ) or non - penetrating ( blunt ) trauma .", "there are two types of the latter , namely direct ones concerning the neck in the region of the carotid artery , and indirect concerning distant regions , e.g. head .", "penetrating neck trauma caused by the above - mentioned tools , leads to arterial wall injury in the form of contusion or vessel wall dissection with the formation of intramural haematoma , outer arterial layer damage ( adventitia ) and the formation of pseudoaneurysm or segmental vessel spasm .", "suppurative inflammation may also develop in the periarterial space , should the patient survive long enough after the trauma .", "the above - mentioned primary vessel wall injuries , associated with an injury limited to at least endothelium and intima , predispose to the development of thrombosis with subsequent vessel occlusion and ischemic stroke .", "the ica segment localised a few centimetres over the cca bifurcation is the vessel segment most commonly injured in the course of non - penetrating ( blunt ) ica traumas , both direct and indirect .", "they result in the rupture of the intima and/or tunica media , with the possible formation of intramural haematoma , i.e. a condition called dissecting aneurysm .", "the thrombotic occlusion of the vessel lumen occurs a few hours or even days or weeks after the trauma [ 5 , 27 , 28 ] .", "six to ten percent of patients manifest such symptoms within the first hour after trauma , another 50  % after 10  h and in 5773  % of cases the asymptomatic period lasts more than 24  h. only 1735  % of such patients develop ischemic symptoms after such a period .", "the above - mentioned facts indicate that in some cases of traumatic ica thrombosis there is a variably long asymptomatic period between the trauma and the manifestation of neurological symptoms of ischemic brain damage .", "some authors compare it to  intervallum lucidum  occurring in patients with epi- and/or subdural haematoma .", "it results from thrombus propagation within the damaged vessel and may hamper the process of establishing the causal connection between the trauma and the ica thrombosis , especially , as according to some authors , the process itself may last up to several years [ 8 , 9 , 27 ] , which in our opinion is hardly probable . according to unterharnscheidt   traumatic ica thrombosis may result from : ( 1 ) direct neck trauma ( e.g. boxers ) ; ( 2 ) direct head trauma ( including insignificant ) with force transfer to the neck region ; ( 3 ) oral cavity trauma ; ( 4 ) fractures of the neuro- and splanchnocranium bones ; ( 5 ) whiplash injuries ; ( 6 ) strangling or other manual procedures in the carotid artery region ( e.g. massage ) and ( 7 ) compression by seatbelt in the course of road traffic accidents .", "non - penetrating ica injuries result from blunt head and/or neck trauma . a head trauma with a violent and powerful head rotation and hyperextension of the cervical spine results in some topographic alterations , including the ica course .", "it may lead to ica compression by the transverse processes of c3 or c1c2 vertebral bodies and predisposes to thrombosis due to the secondary rupture of intima and tunica media by an intact adventitia .", "ica thrombosis may result from neck trauma , as well . by certain head positions , in which the artery is covered only with skin and fascia ,", "it would be pressed against the c1 and c2 or transverse processes of c3c5 with secondary damage to intima , and even tunica media . in both of the discussed cases the thrombosis could have been caused by a segmental artery spasm on the injury site , which is impossible to establish during a postmortem examination . in 1974 crissey and bernstein   identified four basic mechanisms of ica injuries , and thus four types of damage .", "type i damage results from a direct blow to the neck and most commonly concerns older patients , frequently having advanced ica atherosclerosis .", "a similar mechanism occurs by a sudden powerful hyperflexion of the cervical spine with a compression of the ica between the mandible and spine .", "type ii ica damage is caused by the hyperextension of the cervical spine with a head and neck rotation in the opposite direction .", "such a mechanism results in injury to the ica segment that is overstretched over the lateral masses of the first two cervical vertebrae .", "the discussed mechanisms of ica injuries , apart from a direct blow to its region , are most commonly observed in motorcyclists participating in road traffic accidents .", "type iii ica damage is most commonly observed in children and accompanies the intraoral trauma caused by a foreign body e.g. by falling on a hand - held or more frequently mouth - held object , e.g. pencil .", "the establishment of the causal connection between neck and/or head trauma and ica thrombosis and its cerebral consequences requires the exclusion of non - traumatic factors in the first line  disease - associated , being the most common cause of thrombosis .", "this is very important as such trauma is most frequently a result of a criminal offence and brings the perpetrator to criminal and civil liability .", "the establishment of a causal connection may be problematic or even impossible in victims who apart from traumatic injuries suffer from diseases or factors predisposing to thrombosis ."], "abstract_text": ["<S> the following manuscript presents two cases of ischemic stroke secondary to traumatic internal carotid artery thrombosis with concomitant middle cerebral artery thrombosis occurring very rarely in the medico - legal practice . penetrating neck trauma due to an occupational accident and multiple head and neck trauma secondary to battery </S>", "<S> were described . </S>", "<S> the autopsy and histopathological examination as well as the analysis of available medical records , including radiological examinations , and records of investigation indicated the sustained trauma to be the cause of the thrombosis . </S>"], "labels": null, "section_names": ["Introduction", "Case 1", "Case 2", "Discussion", "Conclusions"], "sections": [["a stroke is defined by the who as  rapidly developing clinical signs of a focal ( or global ) disturbance of cerebral function , with symptoms lasting 24  h or longer or leading to death , with no apparent cause other than of vascular origin  . taking into consideration the cause , the mechanism and the character of morphological lesions ,", "there are two types of stroke : ischemic and haemorrhagic [ 2 , 3 ] .", "eighty percent of strokes are of ischemic origin [ 2 , 4 ] , and their most common cause is thrombosis of the atherosclerotic internal carotid artery ( ica ) and/or cerebral arteries ( ca)20  % .", "a stroke , including an ischemic one , is of particular interest to the forensic pathologist aiming on establishing the cause of death and its mechanism , rather than for giving an opinion in cases of suspected medical malpractice .", "neck and/or head trauma may very rarely constitute a cause of thrombosis of the ica and ca without any pre - existing pathology [ 58 ] .", "it most commonly results from direct , penetrating or non - penetrating ( blunt ) neck trauma in the region of large cervical vessels ; less frequently it is attributed to indirect head trauma . in such cases", "the autopsy result and available clinical information has to allow the forensic pathologist to prove the existence of neck and/or head trauma and establish its causal connection with ica thrombosis and exclude other , known , non - traumatic causes of the latter .", "we present two cases of ica thrombosis with concomitant middle cerebral artery ( mca ) thrombosis secondary to the thrombus present in the ica lumen , in which the postmortem examination , the medical records regarding hospitalization and records of investigation indicated trauma as the cause of thrombosis ."], ["a 57-year old male carpenter sustained an injury in an occupational accident in the carpenter s warehouse .", "he was struck very hard in the face , in the region of his left cheek by an irregular - shaped wood fragment which looked like a big splinter ( measuring about 26  cm in length , and with the greatest cross - sectional dimensions of 3.5    1.5  cm ) that broke off from the board being processed on the carpenter s machine ( fig .  1 ) .", "first aid was provided in the emergency department in the local hospital , where the presence of a large , wooden fragment that thrust deep into the left cheek and penetrated through the parapharyngeal space into the neck was diagnosed . on admission", "he was then transferred to the otolaryngological ward , where a physical examination , including an evaluation of his neurological status revealed : maintained consciousness , narrow and symmetric pupils , circulatory and respiratory sufficiency and bp of 180/80  mmhg .", "the ct examination revealed the presence of a foreign body penetrating the left maxillary sinus with a comminuted fracture of the anterior and inferior wall below the external surface of the base of the skull , and a left occipital condyle fracture with concomitant breaking and impression into the posterior cranial fossa and soft tissues of the neck .", "no cerebral lesions , including traumatic , or features of raised intracranial pressure were observed ( fig .  2 ) .", "the ct angiography allowed the diagnosis of an occlusion of the ica by a thrombus localised at the level of the bifurcation of the common carotid artery ( cca ) and at the level of c1 , adjacent to the foreign body .", "the laboratory findings were as follows : d - dimer 20921.25  g / l ( n  <  500 ) ; pt and aptt within normal limits .", "once the diagnostic procedures were finished , a surgical intervention was performed on the day of admission .", "the preparation of the internal jugular vein revealed the medial translocation and compression of the carotid vessels . the foreign body was removed .", "post - interventional ct revealed the thrombotic occlusion of the left ica and mca and a hypodense area supplied by the mca and the concomitant left hemisphere oedema of the brain .", "the patient died on the 6th post - operative day exhibiting symptoms of intracranial hypertension and a brain oedema.fig . ", "2case 1 : localisation of the foreign body ( ct ) case 1 :  wooden splinter  removed during the surgical procedure case 1 : localisation of the foreign body ( ct )", "the findings of the medico - legal autopsy were as follows : a thrombus occluding the left ica and the proximal segment of the left mca , a large infarction area in the temporal lobe and adjacent parts of the frontal and occipital lobes of the left hemisphere of the brain , a large brain oedema with features of subfalcine herniation and transtentorian herniation , a comminuted fracture of the left maxilla and an occipital bone fracture in the region of the occipital condyle with the fragment impression .", "the histopathological examination revealed the occlusion of the left ica by the thrombus ( h  +  e ( fig . ", "3 ) , masson , gomori , verhoeff and ptah ) , focal intimal haemorrhages and features of a mechanical injury  the rupture of the artery wall in the form of an irregular fissure in the tunica media ( fig .  4 ) .", "the samples of the macroscopically changed left cerebral hemisphere revealed ischemic necrosis with polymorphonuclear leukocytes reaction , oedema and hyperaemia , while secondary , focal , non - reactive perivascular haemorrhages were found in the brainstem .", "other organs showed no significant histopathological lesions , apart from morphological indices of circulatory disturbances.fig . ", "3case 1 : the internal carotid artery wall with the adjacent thrombus ( h  +  e)fig . ", "4case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h  +  e and b verhoeff ) case 1 : the internal carotid artery wall with the adjacent thrombus ( h  +  e ) case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h  +  e and b verhoeff )"], ["a 32-year old female patient was brought to the hospital after being battered what resulted in police intervention . on admission", "the patient was conscious , complaining about a left upper extremity contracture that started 2  weeks prior to admission .", "she was generally healthy , with no chronic diseases , taking no medication , apart from being a smoker .", "for the previous month she had been repeatedly battered by her partner (  about twice a week  ) with a fist and open hand to the face , neck and back , as well as pulled by the hair .", "the last battery that took place 1  day prior to admission resulted in a short - term loss of consciousness . on admission", "the following body injuries were described in her medical record : resorbing bruises on the face , including a wound of the lower lip mucosa , and the upper extremities , . on admission", "the patient was sleepy with left - sided hemiparesis with symptoms concerning mainly the upper extremity , blood pressure was 110/80  mmhg , a fundus examination revealed no pathological lesions .", "d - dimer 1 200  ng / ml ( n  <  500 ) , aptt slightly shortened 25.69  s ( n : 2836 ) , aptt ratio and pt within normal limits .", "ct revealed the presence of a hypodense area in the right hemisphere region supplied by the mca , whereas mri scan confirmed the presence of an ischemic brain damage .", "thrombotic occlusion of the ica and mca was visible in mri ( fig .  5 ) and angioct ( fig . ", ", the patient died on the 12th day after admission presenting symptoms of raised intracranial pressure with a concomitant brainstem injury secondary to a brain oedema.fig . ", "5case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri)fig . ", "6case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri ) case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) the findings of the medico - legal autopsy were as follows : a thrombus occluding the right ica and the mca , thrombosis of the internal jugular veins , as well as the superior sagittal sinus and transverse sinus , an extensive infarction area in the right hemisphere region supplied by the mca ; a brain oedema with features of right - sided subfalcine herniation and transtentorian herniation , carotid and basal ca with no atherosclerotic lesions .", "a histopathological examination revealed an occlusion of the right ica by the thrombus ( h  +  e , masson , gomori , verhoeff and ptah)a muscular type artery with normal wall structure , the lumen filled with thrombus fragments , mechanical injury of the arterial wall ", "rupture of the intima and tunica media ( fig .  7 ) . internal jugular veins  normal wall structure , occluded by thrombi .", "there were dominating circulatory disturbances in the brain in the form of very significant capillary - venous hyperaemia resulting from an impaired cerebral venous return secondary to jugular vein and dural sinuses thrombosis.fig . ", "7case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h  +  e and b verhoeff ) case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h  +  e and b verhoeff )"], ["observed in both cases , focal ischemic brain injury was secondary to the thrombosis of the ica and mca .", "cerebral vascular lesions may be causally associated with the sustained head and/or neck trauma with concomitant ica thrombosis and ischemic brain damage [ 5 , 713 ] .", "they can , however , accompany head and/or neck trauma , yet be independent of trauma and have other morbid origins [ 2 , 4 ] . in both cases we considered the most common , known , possible non - traumatic causes for ica thrombosis , including atherosclerosis , which is the most common cause for ica and ca thrombosis .", "the endothelium and the connective tissue cap injury expose the blood coagulation activating factors , which induce thrombus formation .", "the endothelium injury itself with exposure of the basal membrane may predispose to thrombus formation as well .", "the presence of stable atherosclerotic plaque may be complicated by arterial thrombosis , and this refers to cerebral vessels as well .", "however , vessel stenosis has to be significant , i.e. the blood flow transforms from being laminar into post - stenotic turbulent [ 1417 ] both the in vivo neuroradiologic diagnostic imaging modalities , including ct angiography , and the postmortem examination with a histopathological assessment did not reveal the atherosclerosis of ica or mca in any of the discussed cases . other factors favouring carotid arteries thrombosis include : hypertension , diabetes , hypercholesterolaemia , obesity and smoking [ 2 , 4 , 18 ] .", "such factors accelerate the course of atherosclerosis , of which a common complication is thrombosis .", "the analysis of both patients medical records , including the laboratory findings obtained during hospitalisation allowed diabetes and lipid metabolism disorders to be ruled out .", "the woman did not manifest features of hypertension , whereas unstable aterial pressure values , i.e. labile hypertension , was observed in the male patient .", "no lesions in the small - sized cerebral vessels that could have been responsible for the ischemic stroke were found during the postmortem examination , including the neuropathologic examination , within the ischemic , as well as in the non - ischemic areas .", "no arteritis , amyloid angiopathy , vascular malformations nor lesions observed in the course of hypertensive encephalopathy were found .", "the presence of a thrombus inside the ica and/or ca , and secondary focal ischemic brain damage may result from embolism [ 2 , 4 , 18 , 19 ] .", "the most common source of such embolic material are thrombi localised in the left side of the heart ( auricle of the left atrium in patients with atrial fibrillation ) , parietal thrombi in the left ventricle whose formation is associated with myocardial infarction or more frequently aneurysm of the heart in the area of an extensive post - infarction scar or left ventricle dilatation , e.g. in the course of dilated cardiomyopathy or endocarditis , especially of bacterial origin", "the embolism in the ica and ca may then develop in patients with atrial septal defect and deep vein thrombosis .", "the postmortem examination did not , however , reveal any of these conditions in the discussed patients .", "a significantly less common cause of stroke are haematological disorders , which are responsible for about 1  % of all strokes and occur slightly more frequently in young subjects .", "the laboratory findings performed during hospitalisation and autopsies allowed haematological disorders to be ruled out as factors predisposing to thrombosis .", "the elevation of d - dimer level was the sole abnormal parameter observed on the coagulation profile and was associated with present ica and mca thrombosis in both cases .", "oral contraceptives ( oc ) should be considered as a possible cause for thrombosis in the female patient .", "oc are described in neurology as one of the ethological factors of ica and ca thrombosis . in the presented case the anamnesis collected on the patient s admission to", "the hospital indicated that she did not take any medication , most probably including oc .", "however , should she have taken oc , they did not significantly influence the development of ica thrombosis .", "arterial thrombosis in such cases is very rare in women below 35  years of age [ 21 , 22 ] .", "the annual incidence of ischemic stroke in women in this age group is 13 cases/100 000 women .", "the following risk factors are considered to increase the risk of developing thrombosis in subjects using oc : hypertension , diabetes , hyperlipidemia and smoking [ 2025 ] .", "oc predispose more strongly to the development of venous , rather then arterial thrombosis , which results from the haemodynamic properties of blood flow in these two types of vessels .", "other risk factors for venous thrombosis are slowed blood flow and thrombophilia , whereas vessel wall injury ( endothelium ) , including the atherosclerotic plaque are characteristic risk factors for arterial thrombosis . despite doubts concerning her taking", "oc , the patient s age ( 32  years old ) and lack of other , above - mentioned risk factors and also atherosclerosis allow oc to be ruled out as a possible cause of ica and mca thrombosis .", "the above - mentioned exclusions and the circumstances preceding the disease established in the course of the investigation indicate the traumatic background of the ica thrombosis in both cases .", "the ct and ct angiography performed in the first patient revealed the presence of a foreign body adjacent to the left ica on the level of c1 , whereas medial translocation and compression of carotid vessels caused by the foreign body were found during the surgical procedure .", "the histopathological examination revealed features of mechanical arterial injury , namely the rupture of the tunica media with focal haemorrhages into the intima and tunica media .", "the above - mentioned conditions indicate direct trauma as the cause of thrombosis . apart from the arterial occlusion by the thrombus found in the histopathological sections obtained from the second patient , the mechanical injury to the arterial wall in the form of the intima and tunica media rupture were also noted .", "such findings and the information obtained during investigation indicate indirect trauma as the cause of arterial thrombosis .", "post - traumatic ica thrombosis may result from a penetrating ( e.g. knife , bullet or other foreign body ", "splinter , as in case 1 ) or non - penetrating ( blunt ) trauma .", "there are two types of the latter , namely direct ones concerning the neck in the region of the carotid artery , and indirect concerning distant regions , e.g. head .", "penetrating neck trauma caused by the above - mentioned tools , leads to arterial wall injury in the form of contusion or vessel wall dissection with the formation of intramural haematoma , outer arterial layer damage ( adventitia ) and the formation of pseudoaneurysm or segmental vessel spasm .", "suppurative inflammation may also develop in the periarterial space , should the patient survive long enough after the trauma .", "the above - mentioned primary vessel wall injuries , associated with an injury limited to at least endothelium and intima , predispose to the development of thrombosis with subsequent vessel occlusion and ischemic stroke .", "the ica segment localised a few centimetres over the cca bifurcation is the vessel segment most commonly injured in the course of non - penetrating ( blunt ) ica traumas , both direct and indirect .", "they result in the rupture of the intima and/or tunica media , with the possible formation of intramural haematoma , i.e. a condition called dissecting aneurysm .", "the thrombotic occlusion of the vessel lumen occurs a few hours or even days or weeks after the trauma [ 5 , 27 , 28 ] .", "six to ten percent of patients manifest such symptoms within the first hour after trauma , another 50  % after 10  h and in 5773  % of cases the asymptomatic period lasts more than 24  h. only 1735  % of such patients develop ischemic symptoms after such a period .", "the above - mentioned facts indicate that in some cases of traumatic ica thrombosis there is a variably long asymptomatic period between the trauma and the manifestation of neurological symptoms of ischemic brain damage .", "some authors compare it to  intervallum lucidum  occurring in patients with epi- and/or subdural haematoma .", "it results from thrombus propagation within the damaged vessel and may hamper the process of establishing the causal connection between the trauma and the ica thrombosis , especially , as according to some authors , the process itself may last up to several years [ 8 , 9 , 27 ] , which in our opinion is hardly probable . according to unterharnscheidt   traumatic ica thrombosis may result from : ( 1 ) direct neck trauma ( e.g. boxers ) ; ( 2 ) direct head trauma ( including insignificant ) with force transfer to the neck region ; ( 3 ) oral cavity trauma ; ( 4 ) fractures of the neuro- and splanchnocranium bones ; ( 5 ) whiplash injuries ; ( 6 ) strangling or other manual procedures in the carotid artery region ( e.g. massage ) and ( 7 ) compression by seatbelt in the course of road traffic accidents .", "non - penetrating ica injuries result from blunt head and/or neck trauma . a head trauma with a violent and powerful head rotation and hyperextension of the cervical spine results in some topographic alterations , including the ica course .", "it may lead to ica compression by the transverse processes of c3 or c1c2 vertebral bodies and predisposes to thrombosis due to the secondary rupture of intima and tunica media by an intact adventitia .", "ica thrombosis may result from neck trauma , as well . by certain head positions , in which the artery is covered only with skin and fascia ,", "it would be pressed against the c1 and c2 or transverse processes of c3c5 with secondary damage to intima , and even tunica media . in both of the discussed cases the thrombosis could have been caused by a segmental artery spasm on the injury site , which is impossible to establish during a postmortem examination . in 1974 crissey and bernstein   identified four basic mechanisms of ica injuries , and thus four types of damage .", "type i damage results from a direct blow to the neck and most commonly concerns older patients , frequently having advanced ica atherosclerosis .", "a similar mechanism occurs by a sudden powerful hyperflexion of the cervical spine with a compression of the ica between the mandible and spine .", "type ii ica damage is caused by the hyperextension of the cervical spine with a head and neck rotation in the opposite direction .", "such a mechanism results in injury to the ica segment that is overstretched over the lateral masses of the first two cervical vertebrae .", "the discussed mechanisms of ica injuries , apart from a direct blow to its region , are most commonly observed in motorcyclists participating in road traffic accidents .", "type iii ica damage is most commonly observed in children and accompanies the intraoral trauma caused by a foreign body e.g. by falling on a hand - held or more frequently mouth - held object , e.g. pencil ."], ["the establishment of the causal connection between neck and/or head trauma and ica thrombosis and its cerebral consequences requires the exclusion of non - traumatic factors in the first line  disease - associated , being the most common cause of thrombosis .", "this is very important as such trauma is most frequently a result of a criminal offence and brings the perpetrator to criminal and civil liability .", "the establishment of a causal connection may be problematic or even impossible in victims who apart from traumatic injuries suffer from diseases or factors predisposing to thrombosis ."]]}
{"article_id": "PMC3531944", "article_text": ["erythromelalgia ( em ) is a rare disease characterized by painful swelling and erythema on the extremities [ 1 , 2 , 3 ] .", "the symptoms are often triggered by minor trauma , exercise , increased skin temperature , or heat exposure and often alleviated by cooling and elevation of the involved extremity .", "there are three distinct types : primary em , secondary em and em with thrombocythemia .", "primary em is observed most commonly in patients aged less than 30 years without underlying disorders .", "secondary em is associated with several autoimmune disorders [ e.g. systemic lupus erythematosus ( sle ) , insulin - dependent diabetes mellitus ] .", "in contrast , em with thrombocythemia is usually treated with aspirin , since the burning pain is relatively easy to control . in this report", ", we describe a case of adult - onset em successfully controlled with steroid pulse and pregabalin .", "a 68-year - old japanese woman presented at our outpatient clinic with a 10-year history of painful , recurrent erythema on her bilateral hands . there was no erythema on her feet .", "she had been diagnosed with idiopathic em and treated with oral intake of vitamin e 150 mg / day , transamin capsules 750 mg / day , prostaglandin e1 15 g / day and aspirin 100 mg / day , without any improvement . on her initial visit , physical examination revealed dark erythematous swelling on her bilateral hands ( fig .", "the biochemical profile revealed normal levels of antinuclear antibodies ( 40 ) and a slightly decreased igg level ( 702 mg / dl ) .", "no specific autoantibodies ( e.g. dsdna , ssdna , ss - a , ss - b ) were detected .", "prothrombin time , activated partial thromboplastin time , serum fibrinogen , and platelet count were within normal ranges .", "the patient was found to be euthyroid by a measurement of the basal tsh and free t4 levels as well as by trh provocative test .", "internal malignancy was excluded at the internal department . from the above findings , we diagnosed the patient as having adult - onset em of unknown origin .", "we administered methylprednisolone sodium succinate 1,000 mg / day intravenously for 3 days , and after that we treated her with oral prednisolone 60 , 40 and 20 mg / day for 2 days each .", "two days after the administration of methylprednisolone sodium succinate , eruption and pain had improved ( fig . 1b ) .", "the visual analog scale ( vas ) score improved from 10 to 2 ( fig .", "however , 6 days after therapy start , her pain gradually developed again ( vas score 2 to 5 ) .", "the vas score improved again , and the patient has been able to control her pain for half a year , although slight erythema could still be observed .", "em is a rare condition characterized by episodic bouts of burning pain , erythema , and edema , most commonly involving the extremities .", "symptoms are often triggered by minor trauma , exercise , or heat exposure and often alleviated by cooling and elevation of the involved extremity .", "several reports have suggested successful treatments for em [ 2 , 3 , 4 , 5 ] , but the optimal therapy is still under discussion . in this report", ", we describe a case of adult - onset em successfully controlled with steroid pulse and pregabalin as evaluated by vas .", "previous reports suggested an association between secondary forms of em and autoimmune disorders [ 3 , 6 , 7 ] .", "indeed , there is evidence that secondary em might be associated with an autoimmune condition in which there are autoantibodies directed against the sodium channel encoded for by the scn9a gene .", "in addition , jackson and oates   reported a case of autoimmune - related em ; however , these autoantibodies were not suggested to be directly associated with em like sle . on the contrary , the primary form of em", "is associated with a mutation in the sodium channel nav1.7 , which is selectively expressed within the dorsal root ganglion and sympathetic ganglia and encoded by the scn9a gene [ 3 , 8 ] .", "once diagnosis is established , potential secondary causes must be excluded [ 6 , 9 ] .", "other causes associated with em include sle , raynaud 's disease , pernicious anemia , thrombotic thrombocytopenic purpura , infectious mononucleosis , metabolic , endocrine , and vascular origin and diabetic neuropathy [ 6 , 7 , 9 ] .", "although there was no evidence for autoimmune - associated em in our present case , late onset of em might indicate the possible involvement of autoantibodies .", "in addition , suh et al .   reported that adult - onset idiopathic em could be successfully treated with corticosteroids and pentazocine . furthermore , pfund et al .  ", "for the above reasons , we decided to administer steroid pulse and pregabalin , and monitored the patient 's pain with vas .", "vas is a simple assessment tool consisting of a 10-cm line with 0 on one end , representing no pain , and 10 on the other end , representing the worst pain ever experienced , which a patient marks to indicate the severity of his or her pain ( fig .", ", the vas score and erythema on the hands immediately improved after administration of high - dose methylprednisolone sodium succinate and were maintained by oral pregabalin .", "although we describe a single case and the observation is limited , our case might suggest a possible , optimal therapy for adult - onset em ."], "abstract_text": ["<S> adult - onset erythromelalgia ( em ) is a rare disease characterized by episodic bouts of burning pain and erythema for which the optimal therapy is unclear . in this report </S>", "<S> , we describe a 68-year - old japanese woman with adult - onset em . </S>", "<S> intravenous administration of methylprednisolone sodium succinate 1,000 mg / day dramatically improved her pain as evaluated by the visual analog scale . </S>", "<S> although the patient 's pain gradually developed again , it could be controlled with pregabalin . </S>", "<S> our present case might suggest a possible , optimal therapy for adult - onset em . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["erythromelalgia ( em ) is a rare disease characterized by painful swelling and erythema on the extremities [ 1 , 2 , 3 ] .", "the symptoms are often triggered by minor trauma , exercise , increased skin temperature , or heat exposure and often alleviated by cooling and elevation of the involved extremity .", "there are three distinct types : primary em , secondary em and em with thrombocythemia .", "primary em is observed most commonly in patients aged less than 30 years without underlying disorders .", "secondary em is associated with several autoimmune disorders [ e.g. systemic lupus erythematosus ( sle ) , insulin - dependent diabetes mellitus ] .", "in contrast , em with thrombocythemia is usually treated with aspirin , since the burning pain is relatively easy to control . in this report", ", we describe a case of adult - onset em successfully controlled with steroid pulse and pregabalin ."], ["a 68-year - old japanese woman presented at our outpatient clinic with a 10-year history of painful , recurrent erythema on her bilateral hands . there was no erythema on her feet .", "she had been diagnosed with idiopathic em and treated with oral intake of vitamin e 150 mg / day , transamin capsules 750 mg / day , prostaglandin e1 15 g / day and aspirin 100 mg / day , without any improvement . on her initial visit , physical examination revealed dark erythematous swelling on her bilateral hands ( fig .", "the biochemical profile revealed normal levels of antinuclear antibodies ( 40 ) and a slightly decreased igg level ( 702 mg / dl ) .", "no specific autoantibodies ( e.g. dsdna , ssdna , ss - a , ss - b ) were detected .", "prothrombin time , activated partial thromboplastin time , serum fibrinogen , and platelet count were within normal ranges .", "the patient was found to be euthyroid by a measurement of the basal tsh and free t4 levels as well as by trh provocative test .", "internal malignancy was excluded at the internal department . from the above findings , we diagnosed the patient as having adult - onset em of unknown origin .", "we administered methylprednisolone sodium succinate 1,000 mg / day intravenously for 3 days , and after that we treated her with oral prednisolone 60 , 40 and 20 mg / day for 2 days each .", "two days after the administration of methylprednisolone sodium succinate , eruption and pain had improved ( fig . 1b ) .", "the visual analog scale ( vas ) score improved from 10 to 2 ( fig .", "however , 6 days after therapy start , her pain gradually developed again ( vas score 2 to 5 ) .", "the vas score improved again , and the patient has been able to control her pain for half a year , although slight erythema could still be observed ."], ["em is a rare condition characterized by episodic bouts of burning pain , erythema , and edema , most commonly involving the extremities .", "symptoms are often triggered by minor trauma , exercise , or heat exposure and often alleviated by cooling and elevation of the involved extremity .", "several reports have suggested successful treatments for em [ 2 , 3 , 4 , 5 ] , but the optimal therapy is still under discussion . in this report", ", we describe a case of adult - onset em successfully controlled with steroid pulse and pregabalin as evaluated by vas .", "previous reports suggested an association between secondary forms of em and autoimmune disorders [ 3 , 6 , 7 ] .", "indeed , there is evidence that secondary em might be associated with an autoimmune condition in which there are autoantibodies directed against the sodium channel encoded for by the scn9a gene .", "in addition , jackson and oates   reported a case of autoimmune - related em ; however , these autoantibodies were not suggested to be directly associated with em like sle . on the contrary , the primary form of em", "is associated with a mutation in the sodium channel nav1.7 , which is selectively expressed within the dorsal root ganglion and sympathetic ganglia and encoded by the scn9a gene [ 3 , 8 ] .", "once diagnosis is established , potential secondary causes must be excluded [ 6 , 9 ] .", "other causes associated with em include sle , raynaud 's disease , pernicious anemia , thrombotic thrombocytopenic purpura , infectious mononucleosis , metabolic , endocrine , and vascular origin and diabetic neuropathy [ 6 , 7 , 9 ] .", "although there was no evidence for autoimmune - associated em in our present case , late onset of em might indicate the possible involvement of autoantibodies .", "in addition , suh et al .   reported that adult - onset idiopathic em could be successfully treated with corticosteroids and pentazocine . furthermore , pfund et al .  ", "for the above reasons , we decided to administer steroid pulse and pregabalin , and monitored the patient 's pain with vas .", "vas is a simple assessment tool consisting of a 10-cm line with 0 on one end , representing no pain , and 10 on the other end , representing the worst pain ever experienced , which a patient marks to indicate the severity of his or her pain ( fig .", ", the vas score and erythema on the hands immediately improved after administration of high - dose methylprednisolone sodium succinate and were maintained by oral pregabalin .", "although we describe a single case and the observation is limited , our case might suggest a possible , optimal therapy for adult - onset em ."]]}
{"article_id": "PMC5045955", "article_text": ["  discharging patient from intensive care unit ( icu ) is a difficult and complex process \n because icu nurses need to provide a lot of critical information to patients and delegate \n many heavy responsibilities to medical - surgical nurses .", "moreover , more than one third of patients who are discharged form icu , \n develop serious complications and are readmitted to icu .", "studies have shown that approximately 10% of patients who are discharged \n from icu are readmitted to this unit before discharging from the hospital .   moreover ,", "despite great technological advances in healthcare provision , icu readmission \n rate has increased during the past two decades .", "the netherlands intensive care \n evaluation committee 2011 reported that a total of 75000 patients have been admitted to icus \n so far . considering a mean hospital stay of patients in one day , about", "1.6 million euros can \n be saved yearly by decreasing the icu readmission rate by only one percent .  ", "moreover ; icu readmissions are associated with increased length of hospital stay ( 3547 \n vs. 1621 days ) , elevated mortality rate , and increased risk for developing nosocomial \n infections . on the other hand , given the growing demand for advanced medical \n technologies , the increasing number of elderly people with complicated health problems , and \n the limited number of icu beds , prioritizing icu hospitalizations and using icu equipments \n in an optimal way are crucial .   accordingly , special attention has been paid in the last decade to developing strategies \n for improving patient outcomes once getting discharged from icu .", "an innovative strategy for these purposes was the introduction \n of a new nursing role entitled  liaison nurse  .  ", "liaison nurse strategy was originally designed in 2001 in several hospitals located in \n australia .", "the primary goal of this \n strategy is the facilitating of post - icu care provision , minimizing post - icu complications , \n identifying severely - ill patients who need intensive care , and reducing icu readmission and \n hospital mortality rates .", "liaison nurses are \n expected to manage post - icu care , provide scientific and educational support to other \n nurses , establish relationship between icu and ward nurses , and provide intensive nursing \n care to patients who are in medical - surgical wards .", "accordingly , they can \n maintain the continuity and the quality of care which finally improve patient \n outcomes .", "given the novelty of the \n nurse liaison strategy , few studies have investigated its effects on patient \n outcomes .", "moreover , there are still \n controversies about the effectiveness of this strategy for improving patient outcomes .", "the \n aim of the study was to investigate the effect of liaison nurse on patient outcomes after \n icu discharge .", "  this was a single blinded randomized controlled trial that the patients did nt know to \n selecting for which groups of control and intervention .", "the study population consisted of all patients who had been recently \n transferred from icus to medical - surgical wards of two teaching hospitals located in tehran , \n iran .", "sample size was calculated using the altman s nomogram and the results of a study \n conducted by chaboyer et al .  ", "accordingly , with considering  of 0.05 ,  of 0.1 , and a power of 0.9 , the sample size was \n determined to be 40 patients in each group .  ", "participants were recruited by the convenience sampling and were randomly allocated to \n either the control or the experimental groups regardless of the hospitals ( figure 1 ) .", "flow diagram of subject progress through the phases of randomized trial   the inclusion criteria were having an age of 1885 years , having no cardiac dysrhythmia , \n and having no immune disorders , leucopenia , or cancer .", "if patients developed conditions that \n the liaison nurse could not or was not privileged to manage , they were excluded from the \n study .", "accordingly , the exclusion criteria were experiencing cardiac arrest and need for \n resuscitation , developing hemodynamic instability , being transferred to other clinical \n settings , and discharging from hospital with personal consent .  ", "patients in the experimental group received the study intervention by researcher for three \n consecutive days : when patient was transferred from icu to medical - surgical wards , the \n liaison nurse of the study introduced herself to patient and his or her family members . \n", "then , she started collecting data about patients condition through reading their medical \n records .  ", "moreover , the liaison nurse as researcher monitored patients baseline vital signs and \n informed them about the characteristics of medical - surgical wards and nurses , how to access \n nurses in these wards , differences between these wards and icus , and the reason for \n transferring patients from icu to these wards .", "in addition , the liaison nurse taught them \n about the dietary regimen , the importance of daily self - care activities , and how to cope \n with new conditions .", "the liaison nurse also answered patients questions and helped them to \n perform their self - care activities .", "patients vital signs and clinical conditions were \n monitored and documented twice a day for three days by a checklist .", "medical - surgical nurses \n were also informed about patients conditions , their care plan , and their educational needs . \n", "after the intervention , patients vital signs , level of consciousness , length of hospital \n stay , need for re - hospitalization in icu , and satisfaction with care were measured and \n documented by the checklist .", "the liaison nurse was collected study data by using three tools ; a ) a checklist , b ) the \n early warning score , and c ) the patient satisfaction instrument .", "the patient assessment tool consisted of \n items on the functions of : the cardiovascular system : heart rate , blood pressure , edema , and capillary filling \n time ; the nervous system : the history of cerebrovascular accident and the level of \n consciousness was determined by glasgow coma scale ;   the respiratory system : percutaneous oxygen saturation , body temperature , cough , the \n color and the volume of sputum ;   the digestive system : route of nutrition , appetite , pattern of elimination , and weight ; \n  the urinary system : the color and the volume of urine and the serum levels of \n electrolytes , urea , creatinine , albumin , and also fasting and postprandial glucose ;   the musculoskeletal system : mobility , joint mobility and range of motion , independence \n in doing daily activities , and muscular strength .   ", ", for the early \n diagnosis of life - threatening conditions and significant alterations in patients state of \n health .", "ews assesses patient s \n clinical condition based on items such as respiratory rate , heart rate , level of \n consciousness , body temperature , and systolic blood pressure .", "each item is scored on a \n four - point scale from zero to three ( figure 2 ) .", "the \n total score of the instrument is interpreted as follows : scores 02 : the nurse continues providing routine care and monitors vital signs every \n twelve hours ;   score 3 : the nurse immediately modifies the vital sign monitoring schedule from every \n twelve hours to every two hours .", "if the problem persists after three rounds of vital \n signs monitoring , the nurse needs to monitor patient and provide intensive care ;    score 4 : the nurse reports patient s condition to physician performs the continues \n monitoring of vital signs , and measures intake and output ( i / o ) every two hours ;    score 5 : the nurse monitors patient s vital signs and blood oxygen saturation hourly \n and reports patient s condition to the resuscitation team .", "if the patient acquires a \n score of 5 at three subsequent rounds of monitoring , the nurse reports the condition to \n physician to decide on transferring patient to icu ;    score 6 : the nurse reports patient s condition to both physician and resuscitation \n team when the patient is transferred to icu .  ", "this figure shows early warning score   the researcher used the 25-item patient satisfaction instrument ( psi ) for measuring \n patient satisfaction with care .", ", this \n instrument consists of three domains including nurses professional practice ( seven items ) , \n patients confidence in nurses ( eleven items ) , and nurses patient education services ( seven \n items ) .", "items are scored on a five - point likert scale ranging from 1 ( completely disagree ) \n to 5 ( completely agree ) .", "we invited ten icu nurses who had the work experience of more than fifteen years to \n evaluate the content validity of the checklist .", "the reliability of the ews had been \n previously determined using the guttman split - half method which yielded a correlation \n coefficient of 0.868 .", "hajinezhad et al . , evaluated the reliability of the psi and reported a \n coronbach s alpha of 0.90 for the instrument .  ", "the normality of the variables were assessed by using the \n kolmogrov - smirnov test .", "the chi - square , the independent - samples t - test , and the repeated \n measures analysis of variance ( anova ) tests were used for comparing the study groups in \n terms of the demographic characteristics , the length of hospital stay , ews , and patient \n satisfaction .", "this study was approved by the ethics committee of baqiyatallah university of medical \n sciences , tehran , iran .", "  the age of participants ranged from 22 to 82 years with a mean of 59.5 ( 14.24 ) years .", "the results of the chi - square test \n revealed that the intervention and control groups did not differ significantly in terms of \n demographic characteristics such as gender , education , employment , reason for \n hospitalization , and history of previous hospitalization in icu ( p > 0.05 ) ( table 1 ) .   the results of the independent - samples t - test showed that there were no significant \n differences between the two groups regarding heart rate , respiratory rate , systolic blood \n pressure , post - icu level of consciousness , satisfaction with care , and the length of \n hospitalization in medical - surgical wards ( p>0.05 ) . results of variables associated with \n hospitalization of patients are shows in table2 .  ", "moreover , the results of the independent - samples t and the repeated measures anova tests \n indicated that the study groups did not differ significantly regarding the post - icu ews \n scores ( p>0.05)(table 3 ) .", "however , the results of \n the independent - samples t - test showed that the difference between control and intervention \n groups in terms of body temperature was statistically significant ( p<0.05 ) .", "  this study was conducted to investigate the effect of liaison nurse on post - icu patient \n outcomes .", "the findings revealed that liaison nurse had no significant effect on icu \n re - hospitalization rate and the length of post - icu hospital stay . in line with this study , \n doric et al .", ", reported that liaison nurse s services did not significantly affect the rate \n of icu re - hospitalization and the length of hospitalization stay", "mean ( sd ) , f = repeated measures anova   however , green and edmonds found that a five - year care plan provided by liaison nurses \n significantly reduced the rate of icu re - hospitalization  from 2.3% to 0.5% .", ", also reported that liaison \n nurse strategy reduced the rate of pediatric icu re - hospitalization from 5.4% to \n 4.8% . according to chaboyer et al .", ", \n contradictions in terms of the effect of liaison nurse strategy on patient outcomes can be \n attributed to factors such as differences in patients clinical conditions , complexity and \n diversity of post - icu care , differences in the length and the types of liaison nurses \n interventions , and differences in the types , scale , and the management systems of the \n settings of the studies", ", noted that liaison nurses can produce more significant effects on patient outcomes \n in large - scale hospitals that have several icus and higher icu admission and discharge \n rates .  ", "the findings of our study revealed that liaison nurse strategy had no significant effect \n on patient satisfaction .", "chaboyer et al . , also reported the similar findings . however , coffin et al .", ", found that 99% of their \n participants were satisfied with liaison nurses care services .", "this contradiction is probably related to the differences in \n liaison nurses clinical competence and communication skills .", "we also found that the care \n services provided by liaison nurse only decreased patients body temperature and had no \n significant effects on heart rate , respiratory rate , blood pressure , and level of \n consciousness .", "doric et al . , noted that liaison nurses can positively affect patients \n survival and empowerment .", "to the best of \n our knowledge , none of the previous studies investigated the effects of liaison nurse \n strategy on patients vital signs and level of consciousness .  ", "this \n study had also limited budget and also was required to be completed in a short period of \n time .", "accordingly , designing and conducting long - term and large - scale studies may produce \n different results and more persuasive evidence .", "  study findings suggest that care services provided by an icu liaison nurse has limited \n effects on patient outcomes .  ", "however , according the contradiction among the studies , further studies are needed for \n exploring factors that improve care quality and patient outcomes as well as for providing \n clear evidence about the effectiveness of liaison nurses ."], "abstract_text": ["<S> \n introduction : recent studies suggest that liaison nurse \n intervention might be effective to solve the gap between intensive care unit and wards , \n but little studies are known about the effect of this intervention . </S>", "<S> the aim of this study \n was to investigate the effect of liaison nurse service on patient outcomes after \n discharging from intensive care unit . </S>", "<S> methods : in this single blinded randomized controlled trial , a \n total of 80 patients were selected by convenience sampling method from two teaching \n hospitals located in tehran , iran . </S>", "<S> patients were randomly allocated to either the \n experimental or the control groups . </S>", "<S> patients in the experimental group received post - icu \n care from a liaison nurse and patients in the control group received the routine care . \n </S>", "<S> after the intervention , patients vital signs , level of consciousness , length of hospital \n stay , need for re - hospitalization in icu , and satisfaction with care were measure . </S>", "<S> data \n were analyzed by spss ver.13 software . \n </S>", "<S> results : none of the participants experienced icu \n re - hospitalization . according to the result and </S>", "<S> there were no significant differences \n between the study groups regarding heart rate , respiratory rate , systolic blood pressure , \n post - icu level of consciousness , satisfaction with care , and length of hospitalization in \n medical - surgical wards . </S>", "<S> however , the study groups differed significantly in terms of body \n temperature . \n </S>", "<S> conclusion : care services provided by an icu liaison nurse has \n limited effects on patient outcomes . </S>", "<S> however , considering the contradictions among the \n studies , further studies are needed for providing clear evidence about the effectiveness \n of the liaison nurse strategy . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Results", "Discussion", "Conclusion", "Acknowledgments", "Ethical issues", "Conflict of interest"], "sections": [["  discharging patient from intensive care unit ( icu ) is a difficult and complex process \n because icu nurses need to provide a lot of critical information to patients and delegate \n many heavy responsibilities to medical - surgical nurses .", "moreover , more than one third of patients who are discharged form icu , \n develop serious complications and are readmitted to icu .", "studies have shown that approximately 10% of patients who are discharged \n from icu are readmitted to this unit before discharging from the hospital .   moreover ,", "despite great technological advances in healthcare provision , icu readmission \n rate has increased during the past two decades .", "the netherlands intensive care \n evaluation committee 2011 reported that a total of 75000 patients have been admitted to icus \n so far . considering a mean hospital stay of patients in one day , about", "1.6 million euros can \n be saved yearly by decreasing the icu readmission rate by only one percent .  ", "moreover ; icu readmissions are associated with increased length of hospital stay ( 3547 \n vs. 1621 days ) , elevated mortality rate , and increased risk for developing nosocomial \n infections . on the other hand , given the growing demand for advanced medical \n technologies , the increasing number of elderly people with complicated health problems , and \n the limited number of icu beds , prioritizing icu hospitalizations and using icu equipments \n in an optimal way are crucial .   accordingly , special attention has been paid in the last decade to developing strategies \n for improving patient outcomes once getting discharged from icu .", "an innovative strategy for these purposes was the introduction \n of a new nursing role entitled  liaison nurse  .  ", "liaison nurse strategy was originally designed in 2001 in several hospitals located in \n australia .", "the primary goal of this \n strategy is the facilitating of post - icu care provision , minimizing post - icu complications , \n identifying severely - ill patients who need intensive care , and reducing icu readmission and \n hospital mortality rates .", "liaison nurses are \n expected to manage post - icu care , provide scientific and educational support to other \n nurses , establish relationship between icu and ward nurses , and provide intensive nursing \n care to patients who are in medical - surgical wards .", "accordingly , they can \n maintain the continuity and the quality of care which finally improve patient \n outcomes .", "given the novelty of the \n nurse liaison strategy , few studies have investigated its effects on patient \n outcomes .", "moreover , there are still \n controversies about the effectiveness of this strategy for improving patient outcomes .", "the \n aim of the study was to investigate the effect of liaison nurse on patient outcomes after \n icu discharge ."], ["  this was a single blinded randomized controlled trial that the patients did nt know to \n selecting for which groups of control and intervention .", "the study population consisted of all patients who had been recently \n transferred from icus to medical - surgical wards of two teaching hospitals located in tehran , \n iran .", "sample size was calculated using the altman s nomogram and the results of a study \n conducted by chaboyer et al .  ", "accordingly , with considering  of 0.05 ,  of 0.1 , and a power of 0.9 , the sample size was \n determined to be 40 patients in each group .  ", "participants were recruited by the convenience sampling and were randomly allocated to \n either the control or the experimental groups regardless of the hospitals ( figure 1 ) .", "flow diagram of subject progress through the phases of randomized trial   the inclusion criteria were having an age of 1885 years , having no cardiac dysrhythmia , \n and having no immune disorders , leucopenia , or cancer .", "if patients developed conditions that \n the liaison nurse could not or was not privileged to manage , they were excluded from the \n study .", "accordingly , the exclusion criteria were experiencing cardiac arrest and need for \n resuscitation , developing hemodynamic instability , being transferred to other clinical \n settings , and discharging from hospital with personal consent .  ", "patients in the experimental group received the study intervention by researcher for three \n consecutive days : when patient was transferred from icu to medical - surgical wards , the \n liaison nurse of the study introduced herself to patient and his or her family members . \n", "then , she started collecting data about patients condition through reading their medical \n records .  ", "moreover , the liaison nurse as researcher monitored patients baseline vital signs and \n informed them about the characteristics of medical - surgical wards and nurses , how to access \n nurses in these wards , differences between these wards and icus , and the reason for \n transferring patients from icu to these wards .", "in addition , the liaison nurse taught them \n about the dietary regimen , the importance of daily self - care activities , and how to cope \n with new conditions .", "the liaison nurse also answered patients questions and helped them to \n perform their self - care activities .", "patients vital signs and clinical conditions were \n monitored and documented twice a day for three days by a checklist .", "medical - surgical nurses \n were also informed about patients conditions , their care plan , and their educational needs . \n", "after the intervention , patients vital signs , level of consciousness , length of hospital \n stay , need for re - hospitalization in icu , and satisfaction with care were measured and \n documented by the checklist .", "the liaison nurse was collected study data by using three tools ; a ) a checklist , b ) the \n early warning score , and c ) the patient satisfaction instrument .", "the patient assessment tool consisted of \n items on the functions of : the cardiovascular system : heart rate , blood pressure , edema , and capillary filling \n time ; the nervous system : the history of cerebrovascular accident and the level of \n consciousness was determined by glasgow coma scale ;   the respiratory system : percutaneous oxygen saturation , body temperature , cough , the \n color and the volume of sputum ;   the digestive system : route of nutrition , appetite , pattern of elimination , and weight ; \n  the urinary system : the color and the volume of urine and the serum levels of \n electrolytes , urea , creatinine , albumin , and also fasting and postprandial glucose ;   the musculoskeletal system : mobility , joint mobility and range of motion , independence \n in doing daily activities , and muscular strength .   ", ", for the early \n diagnosis of life - threatening conditions and significant alterations in patients state of \n health .", "ews assesses patient s \n clinical condition based on items such as respiratory rate , heart rate , level of \n consciousness , body temperature , and systolic blood pressure .", "each item is scored on a \n four - point scale from zero to three ( figure 2 ) .", "the \n total score of the instrument is interpreted as follows : scores 02 : the nurse continues providing routine care and monitors vital signs every \n twelve hours ;   score 3 : the nurse immediately modifies the vital sign monitoring schedule from every \n twelve hours to every two hours .", "if the problem persists after three rounds of vital \n signs monitoring , the nurse needs to monitor patient and provide intensive care ;    score 4 : the nurse reports patient s condition to physician performs the continues \n monitoring of vital signs , and measures intake and output ( i / o ) every two hours ;    score 5 : the nurse monitors patient s vital signs and blood oxygen saturation hourly \n and reports patient s condition to the resuscitation team .", "if the patient acquires a \n score of 5 at three subsequent rounds of monitoring , the nurse reports the condition to \n physician to decide on transferring patient to icu ;    score 6 : the nurse reports patient s condition to both physician and resuscitation \n team when the patient is transferred to icu .  ", "this figure shows early warning score   the researcher used the 25-item patient satisfaction instrument ( psi ) for measuring \n patient satisfaction with care .", ", this \n instrument consists of three domains including nurses professional practice ( seven items ) , \n patients confidence in nurses ( eleven items ) , and nurses patient education services ( seven \n items ) .", "items are scored on a five - point likert scale ranging from 1 ( completely disagree ) \n to 5 ( completely agree ) .", "we invited ten icu nurses who had the work experience of more than fifteen years to \n evaluate the content validity of the checklist .", "the reliability of the ews had been \n previously determined using the guttman split - half method which yielded a correlation \n coefficient of 0.868 .", "hajinezhad et al . , evaluated the reliability of the psi and reported a \n coronbach s alpha of 0.90 for the instrument .  ", "the normality of the variables were assessed by using the \n kolmogrov - smirnov test .", "the chi - square , the independent - samples t - test , and the repeated \n measures analysis of variance ( anova ) tests were used for comparing the study groups in \n terms of the demographic characteristics , the length of hospital stay , ews , and patient \n satisfaction .", "this study was approved by the ethics committee of baqiyatallah university of medical \n sciences , tehran , iran ."], ["  the age of participants ranged from 22 to 82 years with a mean of 59.5 ( 14.24 ) years .", "the results of the chi - square test \n revealed that the intervention and control groups did not differ significantly in terms of \n demographic characteristics such as gender , education , employment , reason for \n hospitalization , and history of previous hospitalization in icu ( p > 0.05 ) ( table 1 ) .   the results of the independent - samples t - test showed that there were no significant \n differences between the two groups regarding heart rate , respiratory rate , systolic blood \n pressure , post - icu level of consciousness , satisfaction with care , and the length of \n hospitalization in medical - surgical wards ( p>0.05 ) . results of variables associated with \n hospitalization of patients are shows in table2 .  ", "moreover , the results of the independent - samples t and the repeated measures anova tests \n indicated that the study groups did not differ significantly regarding the post - icu ews \n scores ( p>0.05)(table 3 ) .", "however , the results of \n the independent - samples t - test showed that the difference between control and intervention \n groups in terms of body temperature was statistically significant ( p<0.05 ) ."], ["  this study was conducted to investigate the effect of liaison nurse on post - icu patient \n outcomes .", "the findings revealed that liaison nurse had no significant effect on icu \n re - hospitalization rate and the length of post - icu hospital stay . in line with this study , \n doric et al .", ", reported that liaison nurse s services did not significantly affect the rate \n of icu re - hospitalization and the length of hospitalization stay", "mean ( sd ) , f = repeated measures anova   however , green and edmonds found that a five - year care plan provided by liaison nurses \n significantly reduced the rate of icu re - hospitalization  from 2.3% to 0.5% .", ", also reported that liaison \n nurse strategy reduced the rate of pediatric icu re - hospitalization from 5.4% to \n 4.8% . according to chaboyer et al .", ", \n contradictions in terms of the effect of liaison nurse strategy on patient outcomes can be \n attributed to factors such as differences in patients clinical conditions , complexity and \n diversity of post - icu care , differences in the length and the types of liaison nurses \n interventions , and differences in the types , scale , and the management systems of the \n settings of the studies", ", noted that liaison nurses can produce more significant effects on patient outcomes \n in large - scale hospitals that have several icus and higher icu admission and discharge \n rates .  ", "the findings of our study revealed that liaison nurse strategy had no significant effect \n on patient satisfaction .", "chaboyer et al . , also reported the similar findings . however , coffin et al .", ", found that 99% of their \n participants were satisfied with liaison nurses care services .", "this contradiction is probably related to the differences in \n liaison nurses clinical competence and communication skills .", "we also found that the care \n services provided by liaison nurse only decreased patients body temperature and had no \n significant effects on heart rate , respiratory rate , blood pressure , and level of \n consciousness .", "doric et al . , noted that liaison nurses can positively affect patients \n survival and empowerment .", "to the best of \n our knowledge , none of the previous studies investigated the effects of liaison nurse \n strategy on patients vital signs and level of consciousness .  ", "this \n study had also limited budget and also was required to be completed in a short period of \n time .", "accordingly , designing and conducting long - term and large - scale studies may produce \n different results and more persuasive evidence ."], ["  study findings suggest that care services provided by an icu liaison nurse has limited \n effects on patient outcomes .  ", "however , according the contradiction among the studies , further studies are needed for \n exploring factors that improve care quality and patient outcomes as well as for providing \n clear evidence about the effectiveness of liaison nurses ."], [""], [""], [""]]}
{"article_id": "PMC3499899", "article_text": ["by the turn of the century , it was clear that the vertical transmission of hiv to children could effectively be prevented with the appropriate use of short courses of antiretroviral therapy ( art ) . in their landmark paper on the issue , de cock et al .", "noted that few other aspects of hiv had , by that time , demonstrated results as dramatic as perinatal prevention .", "they argued that prevention of infection among children  requires hiv and aids to be addressed as a disease of the family and the community and leads to consideration of other interventions , such as reproductive health care for women and support for children orphaned by the epidemic  [ 1 , p. 1181", ", the critical policy and programme issues needing to be addressed were highlighted as : increased hiv counselling and testing , expanded use of the most effective drug regimens and the prevention of transmission through breastfeeding . since then , all three issues have been resolved to the point of creating conditions feasible for the virtual prevention of hiv transmission from parent to child through pregnancy , delivery and infant feeding .", "more effective drugs are available , there is more integration among services and the drug regimens recommended are more comprehensive and better targeted .", "hiv counseling and testing ( hct ) no longer depends only on individuals stepping out of the line to be tested and risking gossip and stigmatization for doing so . in 2007 , the world health organization ( who ) recommended provider - initiated testing and counselling ( pitc )   to streamline and normalize the process .", "pitc also helps to increase the uptake of prevention of mother - to - child transmission services ( pmtct ) , more appropriately called prevention of vertical transmission ( pvt ) , in order not to place blame unintentionally on women .", "home - based testing similarly expands services and provides for families to be tested together , as does couples testing , guidelines for which have recently been published by who . up until recently , pmtct programmes predominantly focussed on the prevention of hiv transmission to infants .", "however , successfully enrolling eligible pregnant women into treatment and retaining them on treatment is proving to be the most effective approach to protecting mothers , their children  as well as their partners .", "the 2012 who pmtct technical update   recommends a single universal regimen both to treat pregnant women living with hiv and to prevent transmission to her baby ( so - called option b ) .", "this approach simplifies service delivery , aligns and links with art programmes enabling women to more easily transition between services and specifically targets the prevention of maternal mortality and vertical transmission  both of which are disproportionately attributable to the poor health of pregnant women in need of treatment .", "extended prophylaxis given to infants or the continued treatment of their mothers also renders breastfeeding safer .", "given the dramatically increased vulnerability of young children whose mothers die or who suffer serious ill - health , treatment for women not only protects women 's wellbeing but also safeguards their children .", "moreover , providing early treatment reduces hiv transmission between serodifferent couples . scientific and technical knowledge and programme evaluations of pvt", "have increased dramatically , as indicated by an overview of published literature on the topic between 1990 and 2010 ( figure 1 ) .", "average number of clinical , public health and programmatic papers on pvt published every five years from 1990 to 2009 . despite pitc , some 10% to 20% of women refuse testing and another 20% or so either directly or indirectly avoid pvt prescriptions , procedures and follow - up . when women are not fully informed of the benefits , they may perceive pitc to be coercive . the need or perceived requirement to get their partners consent   and the cost of transport and out - of - pocket expenses are some of the reasons women give for declining pvt prescriptions and procedures .", "a recent review highlights the loss of women between vertically provided pregnancy- and hiv - related services and stresses the need for both integration and  family - focussed care  .", "together with health service characteristics such as the availability of test kits and drugs and the negative attitudes of some healthcare providers towards women living with hiv , progress to prevent hiv infections among young children and to ensure the wellbeing of their mothers is slower than was hoped .", "we believe that what is needed , in addition to developments already highlighted , is to implement recommendation by de cock et al . that hiv and aids be addressed as a disease of the family and the community and that hiv interventions be integrated across the lifecycle of women , families and children .", "assessments of progress towards the millennium development goals show that the effects of hiv and aids are especially severe for the survival and wellbeing of mothers and young children .", "in addition , pvt is central to global hiv prevention efforts and remains the one area of prevention in which resounding success can be achieved .", "however , given current knowledge and technology to prevent almost all parent to child transmission of hiv , scale up of pvt and universal access to prevention and treatment has been slow , with especial concern for women and children 's health in the worst affected countries .", "support for a concerted global effort to prevent hiv transmission from parent to child has been growing [ 2023 ] . at the 2011 united nations", "general assembly high level meeting on aids , world leaders committed to work together to achieve this goal .", "the aims of  countdown to zero : global plan towards the elimination of new hiv infections among children by 2015 and keeping their mothers alive  are to achieve a 90% reduction in the number of new hiv infections among children and a 50% reduction in aids - related deaths among pregnant women .", "this is the boldest plan to date to protect children from hiv and to safeguard their families , and it offers unprecedented opportunities to change the way the health sector , donors , governments and others work with affected people and communities to change the course of the epidemic .", "however , a modelling exercise , published before the launch of the plan , laid out how high a mountain had to be climbed to reach the targets . based on data from 25 countries with the highest numbers of pregnant women living with hiv , mahy et al .", "estimated that even if : ( 1 ) more effective drug regimens were implemented , ( 2 ) the current unmet need for family planning among women living with hiv was wholly met and ( 3 ) breastfeeding was limited to 12 months to curb hiv transmission , the number of new child infections averted ( reckoned to be 79% ) would still fall short of the target .", "what is critical , they argued , was for high coverage to be reached on all aspects of the pvt programme ( with far fewer women lost to follow - up at each stage of what is called the ", "pmtct cascade  ) , safer feeding practices adopted and implementation of a comprehensive approach . such a comprehensive approach must include meeting the family planning needs of couples affected by hiv and reducing new hiv infections among women of reproductive age . that is , giving effect to all four prongs of pvt . these are ( 1 ) primary prevention of hiv among women of reproductive age ; ( 2 ) reducing the unmet need for family planning among women living with hiv ; ( 3 ) scaling up more efficacious arv regimens for women living with hiv and hiv - exposed infants and ( 4 ) expanding treatment and care for women , children and their families . the challenges to expanding comprehensive approaches , even in the highest burden regions , differ by country and by area within countries .", "ninety - one percent of pregnant women living with hiv in 2009 are in 22 countries , all but one in africa . however , huge differences exist between these 22 countries , with some classified as middle - income ( such as india and south africa ) and others low - income ( malawi and mozambique ) ; some stable states ( for example , cameroon and ethiopia ) and others with fragile political regimes ( democratic republic of congo and zimbabwe ) .", "there are also differences in health service access ( at least one antenatal visit : 97% in botswana , 39% in chad ) , hiv incidence ( 1.68 in south africa , 0.02 in india ) , unmet need for family planning ( 41% in uganda , 7% in namibia ) , coverage with any arv regimen ( 95% in botswana , 6% in the democratic republic of congo ) and median duration of breastfeeding in the general population ( 34 months in rwanda , 16 in south africa ) .", "unicef has developed fact sheets on the status of national pvt responses in these 22 countries as at 2010 . from the assembled data ,", "it is clear that some countries , such as angola , must increase pvt services in antenatal care , especially in rural areas .", "others , such as nigeria , must expand the low reach of and access to antenatal services  most of which are relatively well provisioned for pvt programmes  especially amongst poor and rural women .", "south africa must bolster the prevention of new hiv infections among young women and improve the quality of pvt services , including the retention of women across the range of vertical prevention services . in all countries , big gaps , inefficiencies and poor quality services must be addressed .", "as has been recognized for some time , though , to reach the targets requires actions , not only within and by health services but also by affected women , their partners , within families and communities and in the wider society . in turn , this engagement is only possible under enabling political , legal , material and social conditions .", "the choices of women are affected , and their options limited , by their husbands and partners , their families and what they perceive other people think about them and how their neighbours and friends treat them . in turn ,", "social norms are influenced by laws and policies , cultural beliefs , economic conditions and the range , quality and helpfulness of the services they have at their disposal .", "the same concentric circles of influence have been noted in other areas of health intervention , with the following quotation taken from cardiovascular disease :  just as we have learned that it is difficult to change the behavior of individuals without changing the communities in which they live , we may be learning that it is difficult to change the behavior of whole communities without changing their broader social environment as well  [ 27 , p. 1391", "scale - up emphasizes the importance of supporting community - based programmes and achieving integration with family planning , as well as other aspects of sexual and reproductive health and tuberculosis services , in order to create or enable facilitative conditions for individual and social actions ( demand ) to complement health service provision ( supply ) .", "these programmes include the following : developing policy and legal frameworks , guidelines , tools and competencies to link services to community - based providers;defining standard packages of services to improve maternal , neonatal and child survival and health , at both health facility and at the community level;building capacity with technical and financial support to community - based organizations to deliver pvt and hiv services at facilities and in communities;promoting the active engagement of people living with hiv in advocacy and planning and delivering services;promoting male - friendly models of delivering hiv services and the participation of men in pvt and in hiv care for children ; anddeveloping and implementing policies and programmes to reduce hiv - related violence , stigma and discrimination in the context of pvt and hiv care for children , including supporting women to disclose their hiv status to partners and family members [ 19 , p. 2223 ] . \n  developing policy and legal frameworks , guidelines , tools and competencies to link services to community - based providers ; defining standard packages of services to improve maternal , neonatal and child survival and health , at both health facility and at the community level ; building capacity with technical and financial support to community - based organizations to deliver pvt and hiv services at facilities and in communities ; promoting the active engagement of people living with hiv in advocacy and planning and delivering services ; promoting male - friendly models of delivering hiv services and the participation of men in pvt and in hiv care for children ; and developing and implementing policies and programmes to reduce hiv - related violence , stigma and discrimination in the context of pvt and hiv care for children , including supporting women to disclose their hiv status to partners and family members [ 19 , p. 2223 ] .", "there has been no assessment of country responses to these guidance points or progress in achieving greater women , family and community involvement in pvt .", "it is troubling that recommendations for community action are considerably diluted in  countdown to zero  .", "they include less specific strategies , such as developing community charters , ensuring participation of all stakeholders , maximizing community assets and identifying solutions to stigma .", "there is an emerging consensus that many hiv prevention programmes do not succeed specifically because they do not engage communities .", "instead , they  are conceived by external experts and imposed on communities in  top - down ways . as a result", ", they fail to resonate with the worldviews and perceived needs and interests of their target groupings , or to take adequate account of the complex social relations into which programmes are inserted  [ 28 , p. 1570", ". too often , suggest campbell and cornish , target communities are seen as passive recipients , as  objects  of intervention .", "but community engagement and participation is essential for several reasons : ( 1 ) because it is the most effective way to deliver acceptable messages and services to  hard - to - reach  groups ; ( 2 ) because it is one of the main ways of engendering a sense of agency with which to build individual and collective health promotion ; and ( 3 ) because the overall shortage of health workers means that community volunteers and workers are needed to help deliver hiv prevention , treatment and care to those who need it .", "the avahan experience in india , the largest prevention programme ever undertaken , demonstrates the value of involving communities , including the private sector , an oft - forgotten constituency in the public domain . at a broader societal level ,", "laws and policies within which health services are funded and delivered emanate from social values and are subject to public opinion  which , in turn influences and is influenced by all forms of media .", "given the importance of engaging women , families and communities to achieve an end to hiv transmission from parent to child , this special issue takes stock of current knowledge and good practice in community action to create an enabling environment , expand access and improve care of women and children in pvt programmes , as well as to reach men .", "the papers review and organize what is known to date , document examples of community mobilization , and the reach and effectiveness of community workers linked to health facilities .", "they draw attention to gender inequalities , the ways in which affected groups and their networks can expand services and the importance of their experiences and voices to unsettle complacency and compel changes in attitudes and behaviour .", "several things stand out from these papers : firstly , how determined community groups are to be part of the response to hiv and how energetic and innovative are their practices .", "secondly , how large - scale some community activities are : for example , in uganda , 750 groups of people living with hiv , working in larger coalitions , assisted 1.3 million people to access hiv - related health services ( gitau - mburu , this issue ) ; mothers2mothers reports having more than 250,000 patient encounters per month , seeing nearly 24,000 new hiv - positive women per month in 714 sites .", "thirdly , how comprehensive community - based services try to be as they integrate their activities to meet individual needs and family concerns ( see kim et al . and patel et al .", "this issue ) , and fourth , the urgent need that community actors have for financial , policy , programme and personal support for their work ( see dhlamini et al .", "however , what is also evident , especially from the review papers , is how little published research there is and , as a result , how limited our knowledge and use of community processes in increasing demand and supply of services and their role in the creation of an enabling environment .", "in addition to the papers , including the reviews and helpful conceptual frameworks offered by , amongst others , buzsa et al .", "firstly , the joint initiators of the global plan , dr michel sidibe , executive director or unaids , and dr eric goosby , global aids coordinator responsible for the implementation of pepfar , have provided a foreword . by doing so", ", they emphasize the importance of community engagement and action , and their commitment , within the global plan , to achieve it .", "it is a massive expression of support for community action , and it manifests the seriousness of their determination .", "the second unique feature is that , in addition to academic peer - reviewers , nine women living with hiv , recommended for their professional expertise and personal experiences by the international community of women with hiv / aids ( icw ) and the global network of people living with aids ( gnp+ ) were invited to review terminology .", "their perspectives demonstrated the ways in which language can distort understanding , fuel discrimination and deeply hurt those who are directly affected by hiv .", "although the comments of this group of reviewers were discretionary unless independently endorsed by the journal 's standard editorial process , all authors responded seriously to the issues raised .", "this is the first time , of which we are aware , that a scientific journal has engaged the people at the receiving end of science , policy and service in a mutual effort to find common ground and move forward together . in this sense", ", the special issue embraces the commitment to include community even though the common ground between science and experience is uneven , a consequence of both low use and potential misunderstanding .", "the special issue closes with an account of the experiences of people living with hiv , male and female recipients of our services , and an annotation by women living with hiv and their networks on  language , identity and hiv .", " almost all the papers take as their starting point the role of community engagement in the following areas : expanded reach and supply of services , increased uptake , enhanced adherence to treatment and care regimes , improved retention in programmes and better psychosocial wellbeing of women , children and their families within an enabling environment .", "reviews of published research and of best practice experience endorse the need for community action in pvt .", "community action strategies can expand the reach of services to women and their families , increase access , boost adherence and retention , and support affected children and ensure they receive treatment . by and large ,", "the mechanisms for community action involve the engagement , enrolment and collaboration of a diverse range of community - based volunteers , workers , counselors and social actors through independent and integrated community- and facility - based programmes .", "stand - out examples from the papers themselves and the work they cite are couples and home - based testing and prevention programmes [ 3133 ] , mentor and peer counselors ; support groups and community forums   and community - based financing mechanisms that address barriers due to transport and other costs .", "more and more effort is being made , with success , to involve men , a strategy strongly endorsed by affected women ( anderson et al .", "less apparent , although model studies and programmes can be found , are efforts to substantially change the social and policy environment through legal reform , mass media , community activism and demands for and implementation of mechanisms to increase accountability at the local and international levels . in a recent review of community accountability mechanisms , molyneux et al .", "identify three basic approaches : committees and groups , report cards and patient charters . while some impressive findings are reported for committees and groups , patient charters are less promising , offering only guidelines rather than consequences for abuses of patient rights and poor quality of health services .", "one of the most impressive effects of the use of report cards to improve healthcare is given by bjrkman and svensson from a randomized field experiment in fifty communities in nine districts in uganda .", "local organizations facilitated agreements between community members and private and government health providers on which submitted reports were based .", "a pre- and post - survey of 5000 households after one year suggested that the accountability mechanism was highly effective .", "health workers were rated as making more effort , there were large increases in utilization and health outcomes , including in child growth and a reduction of under - five mortality by 33% .", "it is clear that research in this area needs to be done and project and programme evaluations must be improved to pass the scrutiny of peers and be published . while there are many promising practices of the effectiveness of community action , the evidence base is still very limited .", "in this special issue , several different conceptual frameworks are used to review and or describe community approaches .", "( this issue ) employ a social ecological framework that links the individual with peers and family , community and the broader social , cultural and economic environment in overlapping concentric circles .", "as they indicate , such conceptual frameworks are useful for illustrating the relationships between determinants that are proximal ( downstream ) or distal ( upstream ) to the person as previously described by latkin and knowlton .", "gulaid and kiragu ( this issue ) use the distinction made by rosato et al . between participation , mobilization and empowerment in an analysis of their roles in maternal , newborn and child health .", "see community empowerment being built up through layers of personal and collective action , starting from the individual , to small mutual groups , community organizations , partnerships and coalitions and social and political action .", "however , they conclude with several fundamental questions , unanswered also with respect to pvt .", "is community participation an essential prerequisite for better health outcomes or simply a useful but non - essential companion to the delivery of treatments and preventive health education ? if essential , then is it only a transitional strategy for the poorest and most deprived populations but largely irrelevant once healthcare systems are established ; or is it the critical missing component and the reason we are failing to achieve ( in this case ) the millennium development goals 4 and 5 regarding maternal and child mortality ?", "community means many different things and we speak simultaneously of the international or donor community and a particular local community ; we refer to community - based , community - oriented and services delivered in , for and by community ( as opposed to health facility ) .", "refer to four categories of implicit constructions of community in health projects : community as setting ( usually geographic ) , community as target ( groups as compared to individuals ) , community as resource ( frequently in terms of participation and support ) and community as agent ( meeting day - to - day needs ) . writing in 1968 of his attempts to develop a unitary approach to community intervention , jack rothman notes ", "it was as if i had packed a large and assorted pile of conceptual clothing into a cognitive suitcase and found there was a sock or the end of a tie sticking out after i had pressed it closed  [ 45 , p. 27 ] . criticized for ignoring culture as a factor in determining the shape of community responses , rothman 's conception remains a cornerstone for understanding community action . in short", "the first aims to improve service delivery through community development , bringing people together to solve local problems on a cooperative self - help basis . in this approach , participation , inclusion and consensus are critical .", "the second is a rationalistic approach used in policy and planning as evidenced , for example , in task shifting arguments for the role of community action .", "the third approach , social action , aims to  aid the oppressed , promote social justice , and change society  adopted , for instance , as the foundation of many aids activist groups [ 45 , p. 27 ] . in practice , the three approaches may be interwoven and differentially phased , with one approach giving way to another .", "the treatment action campaign , gnp+ and physicians for social justice , as examples , combine assumptions , goals and processes of all three approaches , in different ways and at different times .", "these approaches , argues rothman ,  can all be applied in a way to pursue social change and human betterment ", "in fact , they have to draw from one another because each has inherent limitations for which the others can compensate .", "campbell and cornish also argue for alignment of different approaches to community action but draw attention to the contexts in which such action can flourish .", "funding , though needed , may impose expectations and constraints that undermine the participatory ethos that is necessary for community action .", "similarly , mass media approaches frequently represent the views of western agencies , for example , of gender or sex work , and may miss opportunities for affected groups to challenge stigma and establish their symbolic legitimacy .", "lastly , community action depends also on a relational context , in which marginalized groups must have opportunities to interact with powerful local actors in their efforts to protect their health and receive services .", "campbell and cornish advocate what they call  fourth generation  social mobilization , mobilization for supportive conditions for community action at local , national and international levels .", "here there is a role for donors , development agencies and governments to consider how their policies and actions support or sabotage community action . in addition", ", they suggest , this will require that researchers also shift their attention  upwards  to examine , explicate and hold accountable the international community , as well as focussing ", "downwards on the successes and failures of marginalized local communities  [ 28 , p. 1578", "what is clear is that community engagement and action is necessary for the global plan to succeed .", "communities , holding hands with health services , are needed to contribute to creating demand for services , to help supply services and to assist in establishing an enabling environment for both to occur with maximal effectiveness ."], "abstract_text": ["<S> virtual prevention of hiv transmission from parents to children is possible . </S>", "<S> this is cause for hope and renewed energy for prevention in general . </S>", "<S> the global plan is the most concerted and ambitious plan to date to protect children and to promote their care . </S>", "<S> but the inspiring and much appreciated global targets can not be achieved , nor will they be realized in spirit in addition to form , without joint action between health services , affected women , their partners , families and communities and the wider society . in turn , this engagement is only possible under enabling political , legal , material and social conditions . much has already been achieved , and community engagement can everywhere be seen in efforts to increase demand , to supply services and to create and improve enabling environments . </S>", "<S> some of these initiatives are highly organized and expansive , with demonstrated success . </S>", "<S> others are local but essential adjuncts to health services . </S>", "<S> the nature of this engagement varies because the challenges are different across countries and parts of countries . to be sustained and effective </S>", "<S> , community action must simultaneously be inclusive and supportive for those people who are affected , it must be appreciated and assigned a place within the broad systemic response , and it must promote and defend social justice . </S>"], "labels": null, "section_names": ["Introduction: virtual prevention of HIV transmission from parent to child", "Countdown to Zero: the Global Plan", "Community engagement and action", "Quo vadis?", "Competing interests"], "sections": [["by the turn of the century , it was clear that the vertical transmission of hiv to children could effectively be prevented with the appropriate use of short courses of antiretroviral therapy ( art ) . in their landmark paper on the issue , de cock et al .", "noted that few other aspects of hiv had , by that time , demonstrated results as dramatic as perinatal prevention .", "they argued that prevention of infection among children  requires hiv and aids to be addressed as a disease of the family and the community and leads to consideration of other interventions , such as reproductive health care for women and support for children orphaned by the epidemic  [ 1 , p. 1181", ", the critical policy and programme issues needing to be addressed were highlighted as : increased hiv counselling and testing , expanded use of the most effective drug regimens and the prevention of transmission through breastfeeding . since then , all three issues have been resolved to the point of creating conditions feasible for the virtual prevention of hiv transmission from parent to child through pregnancy , delivery and infant feeding .", "more effective drugs are available , there is more integration among services and the drug regimens recommended are more comprehensive and better targeted .", "hiv counseling and testing ( hct ) no longer depends only on individuals stepping out of the line to be tested and risking gossip and stigmatization for doing so . in 2007 , the world health organization ( who ) recommended provider - initiated testing and counselling ( pitc )   to streamline and normalize the process .", "pitc also helps to increase the uptake of prevention of mother - to - child transmission services ( pmtct ) , more appropriately called prevention of vertical transmission ( pvt ) , in order not to place blame unintentionally on women .", "home - based testing similarly expands services and provides for families to be tested together , as does couples testing , guidelines for which have recently been published by who . up until recently , pmtct programmes predominantly focussed on the prevention of hiv transmission to infants .", "however , successfully enrolling eligible pregnant women into treatment and retaining them on treatment is proving to be the most effective approach to protecting mothers , their children  as well as their partners .", "the 2012 who pmtct technical update   recommends a single universal regimen both to treat pregnant women living with hiv and to prevent transmission to her baby ( so - called option b ) .", "this approach simplifies service delivery , aligns and links with art programmes enabling women to more easily transition between services and specifically targets the prevention of maternal mortality and vertical transmission  both of which are disproportionately attributable to the poor health of pregnant women in need of treatment .", "extended prophylaxis given to infants or the continued treatment of their mothers also renders breastfeeding safer .", "given the dramatically increased vulnerability of young children whose mothers die or who suffer serious ill - health , treatment for women not only protects women 's wellbeing but also safeguards their children .", "moreover , providing early treatment reduces hiv transmission between serodifferent couples . scientific and technical knowledge and programme evaluations of pvt", "have increased dramatically , as indicated by an overview of published literature on the topic between 1990 and 2010 ( figure 1 ) .", "average number of clinical , public health and programmatic papers on pvt published every five years from 1990 to 2009 . despite pitc , some 10% to 20% of women refuse testing and another 20% or so either directly or indirectly avoid pvt prescriptions , procedures and follow - up . when women are not fully informed of the benefits , they may perceive pitc to be coercive . the need or perceived requirement to get their partners consent   and the cost of transport and out - of - pocket expenses are some of the reasons women give for declining pvt prescriptions and procedures .", "a recent review highlights the loss of women between vertically provided pregnancy- and hiv - related services and stresses the need for both integration and  family - focussed care  .", "together with health service characteristics such as the availability of test kits and drugs and the negative attitudes of some healthcare providers towards women living with hiv , progress to prevent hiv infections among young children and to ensure the wellbeing of their mothers is slower than was hoped .", "we believe that what is needed , in addition to developments already highlighted , is to implement recommendation by de cock et al . that hiv and aids be addressed as a disease of the family and the community and that hiv interventions be integrated across the lifecycle of women , families and children ."], ["assessments of progress towards the millennium development goals show that the effects of hiv and aids are especially severe for the survival and wellbeing of mothers and young children .", "in addition , pvt is central to global hiv prevention efforts and remains the one area of prevention in which resounding success can be achieved .", "however , given current knowledge and technology to prevent almost all parent to child transmission of hiv , scale up of pvt and universal access to prevention and treatment has been slow , with especial concern for women and children 's health in the worst affected countries .", "support for a concerted global effort to prevent hiv transmission from parent to child has been growing [ 2023 ] . at the 2011 united nations", "general assembly high level meeting on aids , world leaders committed to work together to achieve this goal .", "the aims of  countdown to zero : global plan towards the elimination of new hiv infections among children by 2015 and keeping their mothers alive  are to achieve a 90% reduction in the number of new hiv infections among children and a 50% reduction in aids - related deaths among pregnant women .", "this is the boldest plan to date to protect children from hiv and to safeguard their families , and it offers unprecedented opportunities to change the way the health sector , donors , governments and others work with affected people and communities to change the course of the epidemic .", "however , a modelling exercise , published before the launch of the plan , laid out how high a mountain had to be climbed to reach the targets . based on data from 25 countries with the highest numbers of pregnant women living with hiv , mahy et al .", "estimated that even if : ( 1 ) more effective drug regimens were implemented , ( 2 ) the current unmet need for family planning among women living with hiv was wholly met and ( 3 ) breastfeeding was limited to 12 months to curb hiv transmission , the number of new child infections averted ( reckoned to be 79% ) would still fall short of the target .", "what is critical , they argued , was for high coverage to be reached on all aspects of the pvt programme ( with far fewer women lost to follow - up at each stage of what is called the ", "pmtct cascade  ) , safer feeding practices adopted and implementation of a comprehensive approach . such a comprehensive approach must include meeting the family planning needs of couples affected by hiv and reducing new hiv infections among women of reproductive age . that is , giving effect to all four prongs of pvt . these are ( 1 ) primary prevention of hiv among women of reproductive age ; ( 2 ) reducing the unmet need for family planning among women living with hiv ; ( 3 ) scaling up more efficacious arv regimens for women living with hiv and hiv - exposed infants and ( 4 ) expanding treatment and care for women , children and their families . the challenges to expanding comprehensive approaches , even in the highest burden regions , differ by country and by area within countries .", "ninety - one percent of pregnant women living with hiv in 2009 are in 22 countries , all but one in africa . however , huge differences exist between these 22 countries , with some classified as middle - income ( such as india and south africa ) and others low - income ( malawi and mozambique ) ; some stable states ( for example , cameroon and ethiopia ) and others with fragile political regimes ( democratic republic of congo and zimbabwe ) .", "there are also differences in health service access ( at least one antenatal visit : 97% in botswana , 39% in chad ) , hiv incidence ( 1.68 in south africa , 0.02 in india ) , unmet need for family planning ( 41% in uganda , 7% in namibia ) , coverage with any arv regimen ( 95% in botswana , 6% in the democratic republic of congo ) and median duration of breastfeeding in the general population ( 34 months in rwanda , 16 in south africa ) .", "unicef has developed fact sheets on the status of national pvt responses in these 22 countries as at 2010 . from the assembled data ,", "it is clear that some countries , such as angola , must increase pvt services in antenatal care , especially in rural areas .", "others , such as nigeria , must expand the low reach of and access to antenatal services  most of which are relatively well provisioned for pvt programmes  especially amongst poor and rural women .", "south africa must bolster the prevention of new hiv infections among young women and improve the quality of pvt services , including the retention of women across the range of vertical prevention services . in all countries , big gaps , inefficiencies and poor quality services must be addressed .", "as has been recognized for some time , though , to reach the targets requires actions , not only within and by health services but also by affected women , their partners , within families and communities and in the wider society . in turn , this engagement is only possible under enabling political , legal , material and social conditions .", "the choices of women are affected , and their options limited , by their husbands and partners , their families and what they perceive other people think about them and how their neighbours and friends treat them . in turn ,", "social norms are influenced by laws and policies , cultural beliefs , economic conditions and the range , quality and helpfulness of the services they have at their disposal .", "the same concentric circles of influence have been noted in other areas of health intervention , with the following quotation taken from cardiovascular disease :  just as we have learned that it is difficult to change the behavior of individuals without changing the communities in which they live , we may be learning that it is difficult to change the behavior of whole communities without changing their broader social environment as well  [ 27 , p. 1391", "scale - up emphasizes the importance of supporting community - based programmes and achieving integration with family planning , as well as other aspects of sexual and reproductive health and tuberculosis services , in order to create or enable facilitative conditions for individual and social actions ( demand ) to complement health service provision ( supply ) .", "these programmes include the following : developing policy and legal frameworks , guidelines , tools and competencies to link services to community - based providers;defining standard packages of services to improve maternal , neonatal and child survival and health , at both health facility and at the community level;building capacity with technical and financial support to community - based organizations to deliver pvt and hiv services at facilities and in communities;promoting the active engagement of people living with hiv in advocacy and planning and delivering services;promoting male - friendly models of delivering hiv services and the participation of men in pvt and in hiv care for children ; anddeveloping and implementing policies and programmes to reduce hiv - related violence , stigma and discrimination in the context of pvt and hiv care for children , including supporting women to disclose their hiv status to partners and family members [ 19 , p. 2223 ] . \n  developing policy and legal frameworks , guidelines , tools and competencies to link services to community - based providers ; defining standard packages of services to improve maternal , neonatal and child survival and health , at both health facility and at the community level ; building capacity with technical and financial support to community - based organizations to deliver pvt and hiv services at facilities and in communities ; promoting the active engagement of people living with hiv in advocacy and planning and delivering services ; promoting male - friendly models of delivering hiv services and the participation of men in pvt and in hiv care for children ; and developing and implementing policies and programmes to reduce hiv - related violence , stigma and discrimination in the context of pvt and hiv care for children , including supporting women to disclose their hiv status to partners and family members [ 19 , p. 2223 ] .", "there has been no assessment of country responses to these guidance points or progress in achieving greater women , family and community involvement in pvt .", "it is troubling that recommendations for community action are considerably diluted in  countdown to zero  .", "they include less specific strategies , such as developing community charters , ensuring participation of all stakeholders , maximizing community assets and identifying solutions to stigma .", "there is an emerging consensus that many hiv prevention programmes do not succeed specifically because they do not engage communities .", "instead , they  are conceived by external experts and imposed on communities in  top - down ways . as a result", ", they fail to resonate with the worldviews and perceived needs and interests of their target groupings , or to take adequate account of the complex social relations into which programmes are inserted  [ 28 , p. 1570", ". too often , suggest campbell and cornish , target communities are seen as passive recipients , as  objects  of intervention .", "but community engagement and participation is essential for several reasons : ( 1 ) because it is the most effective way to deliver acceptable messages and services to  hard - to - reach  groups ; ( 2 ) because it is one of the main ways of engendering a sense of agency with which to build individual and collective health promotion ; and ( 3 ) because the overall shortage of health workers means that community volunteers and workers are needed to help deliver hiv prevention , treatment and care to those who need it .", "the avahan experience in india , the largest prevention programme ever undertaken , demonstrates the value of involving communities , including the private sector , an oft - forgotten constituency in the public domain . at a broader societal level ,", "laws and policies within which health services are funded and delivered emanate from social values and are subject to public opinion  which , in turn influences and is influenced by all forms of media ."], ["given the importance of engaging women , families and communities to achieve an end to hiv transmission from parent to child , this special issue takes stock of current knowledge and good practice in community action to create an enabling environment , expand access and improve care of women and children in pvt programmes , as well as to reach men .", "the papers review and organize what is known to date , document examples of community mobilization , and the reach and effectiveness of community workers linked to health facilities .", "they draw attention to gender inequalities , the ways in which affected groups and their networks can expand services and the importance of their experiences and voices to unsettle complacency and compel changes in attitudes and behaviour .", "several things stand out from these papers : firstly , how determined community groups are to be part of the response to hiv and how energetic and innovative are their practices .", "secondly , how large - scale some community activities are : for example , in uganda , 750 groups of people living with hiv , working in larger coalitions , assisted 1.3 million people to access hiv - related health services ( gitau - mburu , this issue ) ; mothers2mothers reports having more than 250,000 patient encounters per month , seeing nearly 24,000 new hiv - positive women per month in 714 sites .", "thirdly , how comprehensive community - based services try to be as they integrate their activities to meet individual needs and family concerns ( see kim et al . and patel et al .", "this issue ) , and fourth , the urgent need that community actors have for financial , policy , programme and personal support for their work ( see dhlamini et al .", "however , what is also evident , especially from the review papers , is how little published research there is and , as a result , how limited our knowledge and use of community processes in increasing demand and supply of services and their role in the creation of an enabling environment .", "in addition to the papers , including the reviews and helpful conceptual frameworks offered by , amongst others , buzsa et al .", "firstly , the joint initiators of the global plan , dr michel sidibe , executive director or unaids , and dr eric goosby , global aids coordinator responsible for the implementation of pepfar , have provided a foreword . by doing so", ", they emphasize the importance of community engagement and action , and their commitment , within the global plan , to achieve it .", "it is a massive expression of support for community action , and it manifests the seriousness of their determination .", "the second unique feature is that , in addition to academic peer - reviewers , nine women living with hiv , recommended for their professional expertise and personal experiences by the international community of women with hiv / aids ( icw ) and the global network of people living with aids ( gnp+ ) were invited to review terminology .", "their perspectives demonstrated the ways in which language can distort understanding , fuel discrimination and deeply hurt those who are directly affected by hiv .", "although the comments of this group of reviewers were discretionary unless independently endorsed by the journal 's standard editorial process , all authors responded seriously to the issues raised .", "this is the first time , of which we are aware , that a scientific journal has engaged the people at the receiving end of science , policy and service in a mutual effort to find common ground and move forward together . in this sense", ", the special issue embraces the commitment to include community even though the common ground between science and experience is uneven , a consequence of both low use and potential misunderstanding .", "the special issue closes with an account of the experiences of people living with hiv , male and female recipients of our services , and an annotation by women living with hiv and their networks on  language , identity and hiv .", " almost all the papers take as their starting point the role of community engagement in the following areas : expanded reach and supply of services , increased uptake , enhanced adherence to treatment and care regimes , improved retention in programmes and better psychosocial wellbeing of women , children and their families within an enabling environment .", "reviews of published research and of best practice experience endorse the need for community action in pvt .", "community action strategies can expand the reach of services to women and their families , increase access , boost adherence and retention , and support affected children and ensure they receive treatment . by and large ,", "the mechanisms for community action involve the engagement , enrolment and collaboration of a diverse range of community - based volunteers , workers , counselors and social actors through independent and integrated community- and facility - based programmes .", "stand - out examples from the papers themselves and the work they cite are couples and home - based testing and prevention programmes [ 3133 ] , mentor and peer counselors ; support groups and community forums   and community - based financing mechanisms that address barriers due to transport and other costs .", "more and more effort is being made , with success , to involve men , a strategy strongly endorsed by affected women ( anderson et al .", "less apparent , although model studies and programmes can be found , are efforts to substantially change the social and policy environment through legal reform , mass media , community activism and demands for and implementation of mechanisms to increase accountability at the local and international levels . in a recent review of community accountability mechanisms , molyneux et al .", "identify three basic approaches : committees and groups , report cards and patient charters . while some impressive findings are reported for committees and groups , patient charters are less promising , offering only guidelines rather than consequences for abuses of patient rights and poor quality of health services .", "one of the most impressive effects of the use of report cards to improve healthcare is given by bjrkman and svensson from a randomized field experiment in fifty communities in nine districts in uganda .", "local organizations facilitated agreements between community members and private and government health providers on which submitted reports were based .", "a pre- and post - survey of 5000 households after one year suggested that the accountability mechanism was highly effective .", "health workers were rated as making more effort , there were large increases in utilization and health outcomes , including in child growth and a reduction of under - five mortality by 33% .", "it is clear that research in this area needs to be done and project and programme evaluations must be improved to pass the scrutiny of peers and be published . while there are many promising practices of the effectiveness of community action , the evidence base is still very limited ."], ["in this special issue , several different conceptual frameworks are used to review and or describe community approaches .", "( this issue ) employ a social ecological framework that links the individual with peers and family , community and the broader social , cultural and economic environment in overlapping concentric circles .", "as they indicate , such conceptual frameworks are useful for illustrating the relationships between determinants that are proximal ( downstream ) or distal ( upstream ) to the person as previously described by latkin and knowlton .", "gulaid and kiragu ( this issue ) use the distinction made by rosato et al . between participation , mobilization and empowerment in an analysis of their roles in maternal , newborn and child health .", "see community empowerment being built up through layers of personal and collective action , starting from the individual , to small mutual groups , community organizations , partnerships and coalitions and social and political action .", "however , they conclude with several fundamental questions , unanswered also with respect to pvt .", "is community participation an essential prerequisite for better health outcomes or simply a useful but non - essential companion to the delivery of treatments and preventive health education ? if essential , then is it only a transitional strategy for the poorest and most deprived populations but largely irrelevant once healthcare systems are established ; or is it the critical missing component and the reason we are failing to achieve ( in this case ) the millennium development goals 4 and 5 regarding maternal and child mortality ?", "community means many different things and we speak simultaneously of the international or donor community and a particular local community ; we refer to community - based , community - oriented and services delivered in , for and by community ( as opposed to health facility ) .", "refer to four categories of implicit constructions of community in health projects : community as setting ( usually geographic ) , community as target ( groups as compared to individuals ) , community as resource ( frequently in terms of participation and support ) and community as agent ( meeting day - to - day needs ) . writing in 1968 of his attempts to develop a unitary approach to community intervention , jack rothman notes ", "it was as if i had packed a large and assorted pile of conceptual clothing into a cognitive suitcase and found there was a sock or the end of a tie sticking out after i had pressed it closed  [ 45 , p. 27 ] . criticized for ignoring culture as a factor in determining the shape of community responses , rothman 's conception remains a cornerstone for understanding community action . in short", "the first aims to improve service delivery through community development , bringing people together to solve local problems on a cooperative self - help basis . in this approach , participation , inclusion and consensus are critical .", "the second is a rationalistic approach used in policy and planning as evidenced , for example , in task shifting arguments for the role of community action .", "the third approach , social action , aims to  aid the oppressed , promote social justice , and change society  adopted , for instance , as the foundation of many aids activist groups [ 45 , p. 27 ] . in practice , the three approaches may be interwoven and differentially phased , with one approach giving way to another .", "the treatment action campaign , gnp+ and physicians for social justice , as examples , combine assumptions , goals and processes of all three approaches , in different ways and at different times .", "these approaches , argues rothman ,  can all be applied in a way to pursue social change and human betterment ", "in fact , they have to draw from one another because each has inherent limitations for which the others can compensate .", "campbell and cornish also argue for alignment of different approaches to community action but draw attention to the contexts in which such action can flourish .", "funding , though needed , may impose expectations and constraints that undermine the participatory ethos that is necessary for community action .", "similarly , mass media approaches frequently represent the views of western agencies , for example , of gender or sex work , and may miss opportunities for affected groups to challenge stigma and establish their symbolic legitimacy .", "lastly , community action depends also on a relational context , in which marginalized groups must have opportunities to interact with powerful local actors in their efforts to protect their health and receive services .", "campbell and cornish advocate what they call  fourth generation  social mobilization , mobilization for supportive conditions for community action at local , national and international levels .", "here there is a role for donors , development agencies and governments to consider how their policies and actions support or sabotage community action . in addition", ", they suggest , this will require that researchers also shift their attention  upwards  to examine , explicate and hold accountable the international community , as well as focussing ", "downwards on the successes and failures of marginalized local communities  [ 28 , p. 1578", "what is clear is that community engagement and action is necessary for the global plan to succeed .", "communities , holding hands with health services , are needed to contribute to creating demand for services , to help supply services and to assist in establishing an enabling environment for both to occur with maximal effectiveness ."], [""]]}
{"article_id": "PMC5054260", "article_text": ["the regulation of gene expression in the brain plays an important role in behavioral plasticity and decision making in response to external stimuli .", "social insects such as honey bees represent good models to study the relationship between brain gene expression ( i.e. neurogenomics ) and behavioral modulations , , .", "insect societies are composed of reproductive females ( queens ) , males and non - reproductive workers that each display a remarkably distinct behavioral repertoire .", "workers in particular exhibit striking patterns of division of labor and behavioral maturation that are crucial for colony survival and growth , which generally consist of a sequence of behaviors known as temporal polyethism , from nursing and nest construction to nest guarding and food foraging .", "several external factors may modify this behavioral sequence , such as food availability and colony demography .", "pathogen infections also accelerate maturation towards early foraging , a change in behavior that is considered to function as a form of social immunity in insect societies .", "pathogens such as microsporidian and viruses have been shown to accelerate temporal polyethism in honey bee colonies , ,   as well as dramatically altering brain gene expression , including genes involved in neural function and foraging behavior . among the multiple pathogens infecting honey bees , the gut parasite nosema ceranae ,", "a microsporidia which recently switched from its original host , the eastern honey bee apis cerana , to the western honey bee a. mellifera , is one of the most prevalent parasite of honey bees in europe .", "honey bees are also infected by several rna viruses , and one of the most prevalent is the positive single strand rna virus , black queen cell virus ( bqcv ) . in a previous study , we showed that bqcv and n. ceranae interact synergistically to increase worker bee mortality . using samples from the same experiment , we sequenced the brain transcriptome of worker bees infected by the two pathogens , alone or in combination , and compared it to control bees . here", ", we explore the genome - wide response of worker bee brains to experimental infection .", "workers honey bees apis mellifera carnica originated from colonies located in halle ( saale ) , germany .", "colonies had been treated to control varroa mites with varidol ( amitraz ; tolnagro , hungary ) the previous fall , six months before the experiment .", "two day - old worker honey bees were experimentally infected individually with 10 \n n. ceranae spores and 1.4    10 genome equivalents of bqcv per os , alone or in combination ( see details in ) .", "bees were kept 13  days post - infection in metal cages ( 10    10    6  cm ) comprising 30 individuals from the same colonies and treatment , with an 8  cm piece of organic beeswax .", "cages were held in incubators at 30  c    1 and 50% rh and bees were fed 50% sucrose solution ad libitum following standardized guidelines .", "three replicates using three different honey bee colonies were used within a treatment , and the same colonies were used across treatments . at the end of the experiment ,", "rna was extracted from a pool of four brains ( from all four treatments and three replicates except co - infection , for which only two replicates could be analyzed ) using trizol and the rneasy mini kit ( qiagen ) .", "sample preparation was performed using the dge dpnii sample preparation kit ( illumina , inc .", ", san diego , ca , usa ) with 2  g rna . library preparation of mrna was perform following the truseq stranded mrna sample preparation kit from illumina . image analysis and base calling were performed using the hiseq control software and real - time analysis component .", "the quality of the data was assessed using fastqc from the babraham institute and the illumina software sav ( sequence analysis viewer ) .", "reads were mapped to the apis mellifera genome ( amel_4.5 ) using the eland_rna module of casava 1.8.2 .", "ncbi annotation file ( seq_gene.md.gz ; 2012 - 12 - 17 ) was used to generate splice junctions automatically .", "reads were also aligned to a set of contaminants , including the ribosomal rnas , the phix genome ( illumina control ) and the illumina adapters .", "gene counting was performed with htseq count ( union mode ) . as the sequencing was strand - specific ,", "the reads were mapped to the opposite strand of the gene . before any statistical analysis , genes with <  15 reads summed across all the analyzed samples were filtered out . differentially expressed genes were identified using the bioconductor r package edger 2.6.2 and the upper quartile normalization method . genes with adjusted p - value <  5% ( according to the benjamini - hochberg fdr method ) were declared differentially expressed . functional analysis based on", "overlap tests with previously published brain transcriptomes upon parasitism with varroa mites and n. ceranae \n  were performed using a hypergeometric test .", "workers honey bees apis mellifera carnica originated from colonies located in halle ( saale ) , germany .", "colonies had been treated to control varroa mites with varidol ( amitraz ; tolnagro , hungary ) the previous fall , six months before the experiment .", "two day - old worker honey bees were experimentally infected individually with 10 \n n. ceranae spores and 1.4    10 genome equivalents of bqcv per os , alone or in combination ( see details in ) .", "bees were kept 13  days post - infection in metal cages ( 10    10    6  cm ) comprising 30 individuals from the same colonies and treatment , with an 8  cm piece of organic beeswax .", "cages were held in incubators at 30  c    1 and 50% rh and bees were fed 50% sucrose solution ad libitum following standardized guidelines .", "three replicates using three different honey bee colonies were used within a treatment , and the same colonies were used across treatments .", "at the end of the experiment , bees were flash frozen in liquid nitrogen and brains dissected on dry ice .", "rna was extracted from a pool of four brains ( from all four treatments and three replicates except co - infection , for which only two replicates could be analyzed ) using trizol and the rneasy mini kit ( qiagen ) .", "sample preparation was performed using the dge dpnii sample preparation kit ( illumina , inc . , san diego , ca , usa ) with 2  g rna . library preparation of mrna was perform following the truseq stranded mrna sample preparation kit from illumina .", "image analysis and base calling were performed using the hiseq control software and real - time analysis component .", "the quality of the data was assessed using fastqc from the babraham institute and the illumina software sav ( sequence analysis viewer ) .", "reads were mapped to the apis mellifera genome ( amel_4.5 ) using the eland_rna module of casava 1.8.2 .", "ncbi annotation file ( seq_gene.md.gz ; 2012 - 12 - 17 ) was used to generate splice junctions automatically .", "reads were also aligned to a set of contaminants , including the ribosomal rnas , the phix genome ( illumina control ) and the illumina adapters .", "gene counting was performed with htseq count ( union mode ) . as the sequencing was strand - specific ,", "the reads were mapped to the opposite strand of the gene . before any statistical analysis ,", "genes with <  15 reads summed across all the analyzed samples were filtered out . differentially expressed genes were identified using the bioconductor r package edger 2.6.2 and the upper quartile normalization method . genes with adjusted p - value <  5% ( according to the benjamini - hochberg fdr method ) were declared differentially expressed . functional analysis based on", "overlap tests with previously published brain transcriptomes upon parasitism with varroa mites and n. ceranae \n  were performed using a hypergeometric test .", "a total of 875,319,378 reads were generated , with an average of 79,574,489 per replicate (   5,023,017 sem ) .", "after quality control and alignment to the a. mellifera genome , 397,450,508 reads ( average per replicate : 36,131,864    3,629,065 reads ) were uniquely assigned to exons and used for statistical analysis of the host response to the experimental inoculation treatments ( details in supplementary table 1 ) .", "the number of genes showing significant changes in expression level upon infection was markedly different between treatments . while 144 genes where differentially expressed in brains of workers infected by bqcv , only 13 genes had a different level of expression in brains of bees infected by n. ceranae ( fig .", "co - infection with the two pathogens induced the differential expression of 67 genes , including 29 genes that were also differentially expressed in brain of worker bees infected by bqcv , and 6 genes also differentially expressed in brain of bees infected by n. ceranae ; 31 genes were differentially expressed in co - infected bees only . among the latter , three cytochrome oxidase p450 genes ( loc408453 , cyp4g11 and loc412209 ) and two genes coding for odorant binding proteins ( obp4 and obp18 )", "conversely , the gene coding for the protein yellow - x1 ( loc724293 ) was significantly up - regulated in co - infected bees . finally , one gene coding for a heat shock protein ( loc724488 ) was consistently down - regulated after all pathogen treatments , including co - infection", ". the functional analysis of genes differentially regulated showed no significantly overrepresented go terms in workers bees infected with n. ceranae or co - infected with both pathogens .", "however , we found a significant overrepresentation of genes involved in immune functions that were differentially expressed in brains of bees infected with bqcv ( benjamini - hochberg corrected p  =  0.039 ) .", "several genes from the toll and imd pathways were up - regulated , such as the antimicrobial peptides abaecin ( loc406144 ) , apidaecin ( apid1 and apid73 ) and hymenoptaecin ( loc406142 ) , but also rel , lys-2 , the drosophila homolog of pirk ( loc100578156 ) , and the gene coding for the pathogen recognition protein pgrp - s2 .", "more importantly , two genes from the rnai antiviral pathway , ago2 and dicer ( loc726766 ) , were found up - regulated in both treatments including bqcv .", "although no functional group associated to brain and neuronal activities were overrepresented in the list of differentially expressed genes , we found the chemosensory protein csp6 , a light sensitive protein ( lop2 ) , the heat shock cognate protein hsc70 - 4 and a neuropeptide cchamide-1 receptor - like ( loc411632 ) to decrease in expression upon infection by bqcv .", "infection by n. ceranae induced lower expression of the chemosensory protein csp1 but increased expression of the neurotransmitter nt-4 .", "a complete list of genes exhibiting significant differential expression between treatments and control is available in supplementary table 2 .", "comparison with a previous study investigating effect of the varroa mite and n. ceranae on honey bee worker brain gene expression   revealed significant overlaps , with 27 and 2 genes found in response to varroa / bqcv and both n. ceranae studies , respectively , in independent experiments ( supplementary table 3 ) .", "this study reports the transcriptome responses of the honey bee brain to two different pathogens , an rna virus and a gut microsporidia .", "we found that bqcv induces a more dramatic change in gene expression than the microsporidia n. ceranae in brains .", "the main reason is likely the capacity of the virus to reach and infect the central nervous system of its host , while n. ceranae is strictly restricted to the gut of honey bees .", "such a difference of impact was also observed in a previous study comparing the effect of the varroa mite when transmitting another rna virus , deformed wing virus ( dwv ) , and the same microsporidia n. ceranae \n .", "interestingly , we found that genes from the antibacterial / antifungal pathways toll and imd , including several amps , where not triggered upon infection with n. ceranae , but rather involved in the response to bqcv .", "although observed in other model insect species , the involvement of these pathways in the antiviral response remains to be elucidated .", "more importantly , we confirmed experimentally the role of the rnai genes dicer and ago2 in the antiviral response of honey bees , as previously observed in response to another virus .", "an important aspect of our transcriptome study is that the response of the host to co - infection with two pathogens was also analyzed .", "bqcv and n. \n ceranae have been shown to interact synergistically in honey bee workers , with co - infection significantly decreasing host survival . with these transcriptome sequences we identify candidate genes involved in pathogen interactions .", "for instance , genes involved in the behavior and responses to external stimuli such as yellow - x1 \n  and odorant binding proteins   were found significantly differentially expressed in co - infected bees only .", "we believe that such data will provide important resource for research on honey bee diseases , and more generally on insect host - pathogen and pathogen - pathogen interactions .", "the following are the supplementary data related to this article.supplementary table 1summary of our illumina sequencing results of brain transcripts of honey bees experimentally inoculated with bqcv , nosema ceranae , both or neither.supplementary table 1supplementary table 2list of genes differentially expressed in worker honey bee brains upon experimental infection by bqcv , n. ceranae or both in comparison to controls .", "fdr indicate the corrected p - value associated with the differential expression level in each pathogen treatment against controls .", "bold values show significant differential expression ( fdr  <  0.05).supplementary table 2supplementary table 3overlap of differentially regulated genes with a similar study .", "our list of genes differentially expressed in brains after bqcv infection was compared with the list of genes differentially expressed in brains after varroa mite parasitism in mcdonnell et al . , while our list of genes differentially expressed in brains after n. ceranae infection was compared with the list of genes differentially expressed in brains also after n. ceranae infection in mcdonnell et al . .", "* indicate significantly more overlap than expected by chance ( hypergeometric test ; p  <  0.002 at most ) .", "reference of the study : mcdonnell cm , alaux c , parrinello h , desvignes j - p , crauser d , durbesson e , beslay d and le conte y   ecto- and endoparasite induce similar chemical and brain neurogenomic responses in the honey bee ( apis mellifera ) . bmc ecology , 13 : 25 .", "doi : 10.1186/1472 - 6785 - 13 - 25.supplementary table 3 summary of our illumina sequencing results of brain transcripts of honey bees experimentally inoculated with bqcv , nosema ceranae , both or neither .", "list of genes differentially expressed in worker honey bee brains upon experimental infection by bqcv , n. ceranae or both in comparison to controls .", "fdr indicate the corrected p - value associated with the differential expression level in each pathogen treatment against controls .", "our list of genes differentially expressed in brains after bqcv infection was compared with the list of genes differentially expressed in brains after varroa mite parasitism in mcdonnell et al . , while our list of genes differentially expressed in brains after n. ceranae infection was compared with the list of genes differentially expressed in brains also after n. ceranae infection in mcdonnell et al . .", "* indicate significantly more overlap than expected by chance ( hypergeometric test ; p  <  0.002 at most ) .", "reference of the study : mcdonnell cm , alaux c , parrinello h , desvignes j - p , crauser d , durbesson e , beslay d and le conte y   ecto- and endoparasite induce similar chemical and brain neurogenomic responses in the honey bee ( apis mellifera ) . bmc ecology , 13 : 25 ."], "abstract_text": ["<S> regulation of gene expression in the brain plays an important role in behavioral plasticity and decision making in response to external stimuli . </S>", "<S> however , both can be severely affected by environmental factors , such as parasites and pathogens . in honey bees , the emergence and re - emergence of pathogens and potential for pathogen co - infection and interaction </S>", "<S> have been suggested as major components that significantly impaired social behavior and survival . to understand how the honey bee is affected and responds to interacting pathogens , we co - infected workers with two prevalent pathogens of different nature , the positive single strand rna virus black queen cell virus ( bqcv ) , and the microsporidia nosema ceranae , and explored gene expression changes in brains upon single infections and co - infections . </S>", "<S> our data provide an important resource for research on honey bee diseases , and more generally on insect host - pathogen and pathogen - pathogen interactions . </S>", "<S> raw and processed data are publicly available in the ncbi / geo database : ( http://www.ncbi.nlm.nih.gov/geo/ ) under accession number gse81664 . </S>"], "labels": null, "section_names": ["Direct link to deposited data", "Introduction", "Materials and methods", "Experimental infections", "RNA-sequencing", "Data analysis", "Results", "Discussion"], "sections": [[""], ["the regulation of gene expression in the brain plays an important role in behavioral plasticity and decision making in response to external stimuli .", "social insects such as honey bees represent good models to study the relationship between brain gene expression ( i.e. neurogenomics ) and behavioral modulations , , .", "insect societies are composed of reproductive females ( queens ) , males and non - reproductive workers that each display a remarkably distinct behavioral repertoire .", "workers in particular exhibit striking patterns of division of labor and behavioral maturation that are crucial for colony survival and growth , which generally consist of a sequence of behaviors known as temporal polyethism , from nursing and nest construction to nest guarding and food foraging .", "several external factors may modify this behavioral sequence , such as food availability and colony demography .", "pathogen infections also accelerate maturation towards early foraging , a change in behavior that is considered to function as a form of social immunity in insect societies .", "pathogens such as microsporidian and viruses have been shown to accelerate temporal polyethism in honey bee colonies , ,   as well as dramatically altering brain gene expression , including genes involved in neural function and foraging behavior . among the multiple pathogens infecting honey bees , the gut parasite nosema ceranae ,", "a microsporidia which recently switched from its original host , the eastern honey bee apis cerana , to the western honey bee a. mellifera , is one of the most prevalent parasite of honey bees in europe .", "honey bees are also infected by several rna viruses , and one of the most prevalent is the positive single strand rna virus , black queen cell virus ( bqcv ) . in a previous study , we showed that bqcv and n. ceranae interact synergistically to increase worker bee mortality . using samples from the same experiment , we sequenced the brain transcriptome of worker bees infected by the two pathogens , alone or in combination , and compared it to control bees . here", ", we explore the genome - wide response of worker bee brains to experimental infection ."], ["workers honey bees apis mellifera carnica originated from colonies located in halle ( saale ) , germany .", "colonies had been treated to control varroa mites with varidol ( amitraz ; tolnagro , hungary ) the previous fall , six months before the experiment .", "two day - old worker honey bees were experimentally infected individually with 10 \n n. ceranae spores and 1.4    10 genome equivalents of bqcv per os , alone or in combination ( see details in ) .", "bees were kept 13  days post - infection in metal cages ( 10    10    6  cm ) comprising 30 individuals from the same colonies and treatment , with an 8  cm piece of organic beeswax .", "cages were held in incubators at 30  c    1 and 50% rh and bees were fed 50% sucrose solution ad libitum following standardized guidelines .", "three replicates using three different honey bee colonies were used within a treatment , and the same colonies were used across treatments . at the end of the experiment ,", "rna was extracted from a pool of four brains ( from all four treatments and three replicates except co - infection , for which only two replicates could be analyzed ) using trizol and the rneasy mini kit ( qiagen ) .", "sample preparation was performed using the dge dpnii sample preparation kit ( illumina , inc .", ", san diego , ca , usa ) with 2  g rna . library preparation of mrna was perform following the truseq stranded mrna sample preparation kit from illumina . image analysis and base calling were performed using the hiseq control software and real - time analysis component .", "the quality of the data was assessed using fastqc from the babraham institute and the illumina software sav ( sequence analysis viewer ) .", "reads were mapped to the apis mellifera genome ( amel_4.5 ) using the eland_rna module of casava 1.8.2 .", "ncbi annotation file ( seq_gene.md.gz ; 2012 - 12 - 17 ) was used to generate splice junctions automatically .", "reads were also aligned to a set of contaminants , including the ribosomal rnas , the phix genome ( illumina control ) and the illumina adapters .", "gene counting was performed with htseq count ( union mode ) . as the sequencing was strand - specific ,", "the reads were mapped to the opposite strand of the gene . before any statistical analysis , genes with <  15 reads summed across all the analyzed samples were filtered out . differentially expressed genes were identified using the bioconductor r package edger 2.6.2 and the upper quartile normalization method . genes with adjusted p - value <  5% ( according to the benjamini - hochberg fdr method ) were declared differentially expressed . functional analysis based on", "overlap tests with previously published brain transcriptomes upon parasitism with varroa mites and n. ceranae \n  were performed using a hypergeometric test ."], ["workers honey bees apis mellifera carnica originated from colonies located in halle ( saale ) , germany .", "colonies had been treated to control varroa mites with varidol ( amitraz ; tolnagro , hungary ) the previous fall , six months before the experiment .", "two day - old worker honey bees were experimentally infected individually with 10 \n n. ceranae spores and 1.4    10 genome equivalents of bqcv per os , alone or in combination ( see details in ) .", "bees were kept 13  days post - infection in metal cages ( 10    10    6  cm ) comprising 30 individuals from the same colonies and treatment , with an 8  cm piece of organic beeswax .", "cages were held in incubators at 30  c    1 and 50% rh and bees were fed 50% sucrose solution ad libitum following standardized guidelines .", "three replicates using three different honey bee colonies were used within a treatment , and the same colonies were used across treatments ."], ["at the end of the experiment , bees were flash frozen in liquid nitrogen and brains dissected on dry ice .", "rna was extracted from a pool of four brains ( from all four treatments and three replicates except co - infection , for which only two replicates could be analyzed ) using trizol and the rneasy mini kit ( qiagen ) .", "sample preparation was performed using the dge dpnii sample preparation kit ( illumina , inc . , san diego , ca , usa ) with 2  g rna . library preparation of mrna was perform following the truseq stranded mrna sample preparation kit from illumina ."], ["image analysis and base calling were performed using the hiseq control software and real - time analysis component .", "the quality of the data was assessed using fastqc from the babraham institute and the illumina software sav ( sequence analysis viewer ) .", "reads were mapped to the apis mellifera genome ( amel_4.5 ) using the eland_rna module of casava 1.8.2 .", "ncbi annotation file ( seq_gene.md.gz ; 2012 - 12 - 17 ) was used to generate splice junctions automatically .", "reads were also aligned to a set of contaminants , including the ribosomal rnas , the phix genome ( illumina control ) and the illumina adapters .", "gene counting was performed with htseq count ( union mode ) . as the sequencing was strand - specific ,", "the reads were mapped to the opposite strand of the gene . before any statistical analysis ,", "genes with <  15 reads summed across all the analyzed samples were filtered out . differentially expressed genes were identified using the bioconductor r package edger 2.6.2 and the upper quartile normalization method . genes with adjusted p - value <  5% ( according to the benjamini - hochberg fdr method ) were declared differentially expressed . functional analysis based on", "overlap tests with previously published brain transcriptomes upon parasitism with varroa mites and n. ceranae \n  were performed using a hypergeometric test ."], ["a total of 875,319,378 reads were generated , with an average of 79,574,489 per replicate (   5,023,017 sem ) .", "after quality control and alignment to the a. mellifera genome , 397,450,508 reads ( average per replicate : 36,131,864    3,629,065 reads ) were uniquely assigned to exons and used for statistical analysis of the host response to the experimental inoculation treatments ( details in supplementary table 1 ) .", "the number of genes showing significant changes in expression level upon infection was markedly different between treatments . while 144 genes where differentially expressed in brains of workers infected by bqcv , only 13 genes had a different level of expression in brains of bees infected by n. ceranae ( fig .", "co - infection with the two pathogens induced the differential expression of 67 genes , including 29 genes that were also differentially expressed in brain of worker bees infected by bqcv , and 6 genes also differentially expressed in brain of bees infected by n. ceranae ; 31 genes were differentially expressed in co - infected bees only . among the latter , three cytochrome oxidase p450 genes ( loc408453 , cyp4g11 and loc412209 ) and two genes coding for odorant binding proteins ( obp4 and obp18 )", "conversely , the gene coding for the protein yellow - x1 ( loc724293 ) was significantly up - regulated in co - infected bees . finally , one gene coding for a heat shock protein ( loc724488 ) was consistently down - regulated after all pathogen treatments , including co - infection", ". the functional analysis of genes differentially regulated showed no significantly overrepresented go terms in workers bees infected with n. ceranae or co - infected with both pathogens .", "however , we found a significant overrepresentation of genes involved in immune functions that were differentially expressed in brains of bees infected with bqcv ( benjamini - hochberg corrected p  =  0.039 ) .", "several genes from the toll and imd pathways were up - regulated , such as the antimicrobial peptides abaecin ( loc406144 ) , apidaecin ( apid1 and apid73 ) and hymenoptaecin ( loc406142 ) , but also rel , lys-2 , the drosophila homolog of pirk ( loc100578156 ) , and the gene coding for the pathogen recognition protein pgrp - s2 .", "more importantly , two genes from the rnai antiviral pathway , ago2 and dicer ( loc726766 ) , were found up - regulated in both treatments including bqcv .", "although no functional group associated to brain and neuronal activities were overrepresented in the list of differentially expressed genes , we found the chemosensory protein csp6 , a light sensitive protein ( lop2 ) , the heat shock cognate protein hsc70 - 4 and a neuropeptide cchamide-1 receptor - like ( loc411632 ) to decrease in expression upon infection by bqcv .", "infection by n. ceranae induced lower expression of the chemosensory protein csp1 but increased expression of the neurotransmitter nt-4 .", "a complete list of genes exhibiting significant differential expression between treatments and control is available in supplementary table 2 .", "comparison with a previous study investigating effect of the varroa mite and n. ceranae on honey bee worker brain gene expression   revealed significant overlaps , with 27 and 2 genes found in response to varroa / bqcv and both n. ceranae studies , respectively , in independent experiments ( supplementary table 3 ) ."], ["this study reports the transcriptome responses of the honey bee brain to two different pathogens , an rna virus and a gut microsporidia .", "we found that bqcv induces a more dramatic change in gene expression than the microsporidia n. ceranae in brains .", "the main reason is likely the capacity of the virus to reach and infect the central nervous system of its host , while n. ceranae is strictly restricted to the gut of honey bees .", "such a difference of impact was also observed in a previous study comparing the effect of the varroa mite when transmitting another rna virus , deformed wing virus ( dwv ) , and the same microsporidia n. ceranae \n .", "interestingly , we found that genes from the antibacterial / antifungal pathways toll and imd , including several amps , where not triggered upon infection with n. ceranae , but rather involved in the response to bqcv .", "although observed in other model insect species , the involvement of these pathways in the antiviral response remains to be elucidated .", "more importantly , we confirmed experimentally the role of the rnai genes dicer and ago2 in the antiviral response of honey bees , as previously observed in response to another virus .", "an important aspect of our transcriptome study is that the response of the host to co - infection with two pathogens was also analyzed .", "bqcv and n. \n ceranae have been shown to interact synergistically in honey bee workers , with co - infection significantly decreasing host survival . with these transcriptome sequences we identify candidate genes involved in pathogen interactions .", "for instance , genes involved in the behavior and responses to external stimuli such as yellow - x1 \n  and odorant binding proteins   were found significantly differentially expressed in co - infected bees only .", "we believe that such data will provide important resource for research on honey bee diseases , and more generally on insect host - pathogen and pathogen - pathogen interactions .", "the following are the supplementary data related to this article.supplementary table 1summary of our illumina sequencing results of brain transcripts of honey bees experimentally inoculated with bqcv , nosema ceranae , both or neither.supplementary table 1supplementary table 2list of genes differentially expressed in worker honey bee brains upon experimental infection by bqcv , n. ceranae or both in comparison to controls .", "fdr indicate the corrected p - value associated with the differential expression level in each pathogen treatment against controls .", "bold values show significant differential expression ( fdr  <  0.05).supplementary table 2supplementary table 3overlap of differentially regulated genes with a similar study .", "our list of genes differentially expressed in brains after bqcv infection was compared with the list of genes differentially expressed in brains after varroa mite parasitism in mcdonnell et al . , while our list of genes differentially expressed in brains after n. ceranae infection was compared with the list of genes differentially expressed in brains also after n. ceranae infection in mcdonnell et al . .", "* indicate significantly more overlap than expected by chance ( hypergeometric test ; p  <  0.002 at most ) .", "reference of the study : mcdonnell cm , alaux c , parrinello h , desvignes j - p , crauser d , durbesson e , beslay d and le conte y   ecto- and endoparasite induce similar chemical and brain neurogenomic responses in the honey bee ( apis mellifera ) . bmc ecology , 13 : 25 .", "doi : 10.1186/1472 - 6785 - 13 - 25.supplementary table 3 summary of our illumina sequencing results of brain transcripts of honey bees experimentally inoculated with bqcv , nosema ceranae , both or neither .", "list of genes differentially expressed in worker honey bee brains upon experimental infection by bqcv , n. ceranae or both in comparison to controls .", "fdr indicate the corrected p - value associated with the differential expression level in each pathogen treatment against controls .", "our list of genes differentially expressed in brains after bqcv infection was compared with the list of genes differentially expressed in brains after varroa mite parasitism in mcdonnell et al . , while our list of genes differentially expressed in brains after n. ceranae infection was compared with the list of genes differentially expressed in brains also after n. ceranae infection in mcdonnell et al . .", "* indicate significantly more overlap than expected by chance ( hypergeometric test ; p  <  0.002 at most ) .", "reference of the study : mcdonnell cm , alaux c , parrinello h , desvignes j - p , crauser d , durbesson e , beslay d and le conte y   ecto- and endoparasite induce similar chemical and brain neurogenomic responses in the honey bee ( apis mellifera ) . bmc ecology , 13 : 25 ."]]}
{"article_id": "PMC2447759", "article_text": ["it is well known that genes can be silenced by antisense rna oligonucleotides called small interfering rna ( sirna ) ( 1,2 ) . in order to design an efficient sirna sequence , empirical rules based on the features of the sirna sequence", "have been discovered , including , for example , low g / c content , lack of self - structure , preference of a at position 3 , absence of g or c at position 19 and asymmetry in the stability of the terminal base pairs ( 310 ) .", "the self - structure of the target and oligonucleotide is also an important consideration for the effective binding ( 1115 ) .", "it is desirable to select an oligonucleotide having high accessibility to the target - binding site and low duplex stability . here", ", the oligowalk server , which predicts efficient sirna sequences using an accessibility calculation with a convenient web interface , is described .", "overall , the positive predictive value of the server is 0.786 , meaning that 78.6% of the sirnas selected by the server will be efficient at silencing ( 16 ) .", "the positive predictive value was determined by testing against a database of sirna experiments conducted under diverse experimental conditions ( 17 ) . in the calculation of the oligowalk server ,", "unimolecular and bimolecular self - structures for the sirna are considered along with unimolecular self - structure in the target at the oligonucleotide binding region ( 16 ) .", "oligowalk predicts the free energy changes ( g ) involved in these equilibrium states ( 18 ) .", "the predicted thermodynamics ( g ) , plus the oligonucleotide sequence features ( 19 ) , are then utilized to predict sirna efficacy for candidate sirna sequences ( 16 ) , which are generally 19 nucleotide duplexes with 3 dinucleotide dangling ends ( 7 ) .", "a support vector machine ( svm ) program ( 20 ) is embedded in the server to take the thermodynamic and sequence features as input .", "the svm classification model used in the server has been proven to be able to predict efficient sirna ( greater than 70% inhibition of the target mrna expression ) with high accuracy ( 16 ) .", "the svm was trained on a sirna database that contains 2431 experimental results conducted in human cells at 37c ( 10 ) .", "the output of the oligowalk server is a table of sirna candidates , showing the sirna sequences and the probabilities of being efficient ( having silencing efficacy larger than 70% ) .", "each of the free energy change terms for each candidate is also listed in a separate table .", "the oligowalk server uses the cgi ( common gateway interface ) module of perl for taking user input and submitting calculations from the homepage .", "the input of oligowalk server is the rna sequence of the target gene . only a , u , t , g and c are the acceptable types of nucleotides in the sequence ( the server will replace the nucleotide t with u for calculations ) , and the maximum sequence length is 10 000 nucleotides .", "an email address is required because the server sends an email to the user when the calculation is completed .", "submit query , the server generates a list of efficient sirna candidates for the target gene .", "jobs are submitted by the server to a cluster of seven nodes with 3.2 or 3.4 ghz pentium 4 processors running fedora linux ( http://fedoraproject.org/ ) , managed by sun grid engine ( http://gridengine.sunsource.net/ ) .", "when the calculation is complete , an html ( hypertext markup language ) page is generated with links to tables containing predicted sirna efficacy data and thermodynamic binding data . in the sirna efficacy table ( figure 1 ) , the sequences of sirna candidates are ranked in the output list by their probabilities of being efficient sirna .", "the probabilities are predicted by a svm embedded in the web server for selecting efficient sirna .", "the classification model ( 16 ) used in the svm was trained with a publically available database ( 10 ) , using thermodynamic and sequence features of sirna candidates . the position number of each sirna candidate is also listed in the table as the index of the 5 most base in the target - binding region . \n", "the sequences of sirna are ranked from top to end by their probabilities of being efficient ( antisense efficacy larger than 70% ) . a table of sirna candidates generated by oligowalk server .", "the sequences of sirna are ranked from top to end by their probabilities of being efficient ( antisense efficacy larger than 70% ) .", "in addition , the predicted equilibrium thermodynamics table is generated as a reference for advanced users . in the table", " overall ( in kcal / mol ) is the overall free energy change of oligonucleotide - target binding ,   when all contributions are considered , including breaking target and oligonucleotide self - structures ( 18 ) .", " duplex ( in kcal / mol ) is the free energy change of hybridized duplex between oligonucleotide and target ( antisense  sense duplex ) , .", "the value is independent of oligonucleotide concentration because it is a standard free energy change . ", "tm - dup ( in c ) is the melting temperature in degrees for the duplex formation of oligonucleotide and target . ", "break - targ. ( in kcal / mol ) is the free energy cost to open the intramolecular target base pairs for oligonucleotide binding , .", "a more negative number indicates higher free energy cost , which is unfavorable for oligonucleotide - target binding . ", "intraoligo ( in kcal / mol ) is the free energy change of intramolecular oligonucleotide structure , .", "it usually has a negative value or , if there is no favor - able intramolecular structure , it is zero . ", "interoligo ( in kcal / mol ) is the free energy change of intermolecular oligonucleotide structure , .", "antisense bimolecular structure , which decreases the oligonucleotide - target ( antisense  sense ) binding affinity .  end_diff. ( in kcal / mol ) is the free energy difference between the 5 and 3 end of the antisense strand of sirna , with windows of two base pairs .", "functional sirna prefer to have an unstable 5 end ( 3 ) , which means a positive end_diff .", " prefilter_score is the score calculated with a method based on the empirical rules by reynolds et al . ( 7 ) .", "( 7 ) , except for the melting temperature of intramolecular oligonucleotide self - structure because the free energy ( 21 ) and enthalpy parameters ( 22 ) used by oligowalk are more recent .", "when calculating the prefilter score , 57c is used as the cutoff of the intramolecular oligonucleotide melting temperature , as suggested in another study ( 23 ) . as an example", ", the prediction of the webserver is compared with experimental results in figure 2 . in the experiment ( 3 ) , sirna were tested for efficacy against the target mrna , human cyclophilin ( genbank i d : m60857 ) , at 37c .", "the inhibition efficacy of each sirna is defined as 100% minus the percentage of mrna level after sirna application as compared to matched control .", "the prediction result is the probability of being efficient ( having inhibition efficacy larger than 70% ) , which is calculated with the server . in figure 2 , most of the sirna with high inhibition efficacy are predicted to have high probability of being efficient . \n", "figure 2.a comparison between the prediction and experimental result . in blue is the experimental result ( inhibition efficacy ) for an mrna target , human cyclophilin ( genbank i d : m60857 ) , at 37c ( 3 ) .", "the inhibition efficacy is defined as 100% minus the percent level of mrna expression after sirna application as compared to a control . in red", "is the probability ( ranging from 01 ) of being efficient ( having inhibition efficacy larger than 70% ) , as predicted by oligowalk .", "a comparison between the prediction and experimental result . in blue is the experimental result ( inhibition efficacy ) for an mrna target , human cyclophilin ( genbank i d : m60857 ) , at 37c ( 3 ) .", "the inhibition efficacy is defined as 100% minus the percent level of mrna expression after sirna application as compared to a control . in red", "is the probability ( ranging from 01 ) of being efficient ( having inhibition efficacy larger than 70% ) , as predicted by oligowalk .", "advanced options ( figure 3 ) are available for users who understand the underlying calculations and would like to test novel hypotheses . the option form is written in html , embedded with javascript controlling the options so that they are context - aware .", "the oligonucleotide concentration does not affect the result of sirna sequence design , because the inputs to the svm are standard free energy changes , g . concentration changes do , however , alter the overall free energy change ( ) , which is provided to the user with the thermodynamic details table .", "the sense  antisense duplex free energy changes are calculated without considering the self - structures of the target . this mode is not recommended for an accurate sirna prediction .", "it is more rigorous to consider the accessibility of the target and oligonucleotide self - structures because binding affinity is lost to open existing base pairs in the target and oligonucleotide .", "the second mode is to break local structure . in this case , the structure of target is fixed and only the base pairs on the binding site will be broken without refolding the global structure of target . the final and most rigorous mode is to refold the target rna . in this mode ,", "the rna target is folded before oligonucleotide binding and refolded afterwards for each possible oligonucleotide to consider complete equilibration .", "the default setting of sirna design is shown in the figure . if the target rna secondary structure is considered , three different prediction methods are available to calculate the free energy change of target self - structure .", "the first method is optimal structure prediction , where only the optimal structure ( lowest free energy structure ) of the target is considered to calculate the free energy cost of opening the base pairs of binding region .", "the second method considers a set of suboptimal structures to determine the free energy cost .", "each structure 's free energy cost is weighted according to the free energy change of the structure to arrive at the ensemble cost .", "for this option , at most 1000 suboptimal structures ( within 10% free energy difference from the optimal structure ) are generated with a heuristic method ( 24 ) .", "the number of suboptimal structures will be listed in the output table if the target is folded with suboptimal structure prediction method .", "constrained target structure is the refolded structure where the binding region is forced to be single - stranded , so that the oligonucleotide can bind to it .", "this is the most rigorous method because it considers every possible secondary structure in the folding ensemble , with boltzmann weight .", "the structure prediction only folds a certain total number ( folding size ) of nucleotides centered at the binding region .", "the user can define this number , but the largest folding size is 1000 nucleotides for the webserver in order to save compute time .", "users can define longer folding sizes by downloading and installing the oligowalk program to a local machine .", "( 7 ) can be used to rule out nonefficient sirna candidates before folding the target sequence , i.e. the sirna sequences having score less than six points will not be considered for the folding step .", "it is suggested to turn on the prefilter option to save considerable computation time ( table 1 ) .", "furthermore , the scan region can be redefined if the user is interested only in a specific region of the target . \n", "table 1.calculation size and time of oligowalk server for different target sequencestarget mrna ( genbank id)sequence length ( nucleotide)time ( h : min : sec)memory ( mb)nm_0205487300:57:1793m608578513:55:53110nm_00287012116:09:25112nm_00246721896:36:42113aj27221234606:53:05117the benchmarks were performed with the default options : the oligonucleotide was a 19 base rna ; the folding size of the target was 800 nucleotides centering on the binding site ( full length if the whole target has less than 800 nt ) ; the partition function calculation was conducted ; the entire mrna was scanned ; and the prefilter was on . the time cost changes little for long sequence because the prefilter ( 7 ) is on and number of candidates being folded is limited to about the same number for each sequence.the calculations were submitted and benchmarked on the oligowalk web server ( http://rna.urmc.rochester.edu/servers/oligowalk ) .", "each node has a 3.2 or 3.4 ghz pentium 4 processor running fedora linux.the calculation for sequences less than 800 nucleotides is relatively fast because the dynamic programming arrays are reused for calculations of short sequences ( 16 ) .", "calculation size and time of oligowalk server for different target sequences the benchmarks were performed with the default options : the oligonucleotide was a 19 base rna ; the folding size of the target was 800 nucleotides centering on the binding site ( full length if the whole target has less than 800 nt ) ; the partition function calculation was conducted ; the entire mrna was scanned ; and the prefilter was on .", "the time cost changes little for long sequence because the prefilter ( 7 ) is on and number of candidates being folded is limited to about the same number for each sequence .", "the calculation for sequences less than 800 nucleotides is relatively fast because the dynamic programming arrays are reused for calculations of short sequences ( 16 ) .", "the oligowalk web server predicts the hybridization thermodynamics of an oligonucleotide binding to a complementary target rna using the most recent rna folding parameters ( 21 , 22 ) .", "it predicts efficient sirna with high accuracy using a transparent implementation of an svm ( 16 ) , which considers both sequence and thermodynamic features .", "the calculation time and memory size of oligowalk are shown in table 1 for a sample of mrna sequences .", "the prefilter ( 7 ) that uses local sequence information to narrow down the list of sirna candidates before calculating the equilibrium affinity is used by default .", "for example , the server takes 3 h and 43 min for a complete scan of all possible sirnas on an mrna having 730 nucleotides using the partition function calculation . for the same sequence ,", "the algorithm time scales o(mn ) and the memory use scales o(n ) ( table 1 ) , m is the number of candidates and n is the value of folding size .", "the time and memory costs change little with sequence because the same folding size ( e.g. 800 nucleotides ) is used and the prefilter ( 7 ) is turned on , which limits the number of candidates to be folded in a way that is apparently independent of target length .", "there is currently significant interest in using sirna for both basic science and medical research .", "the fact that not all sirna duplexes will function in silencing means that there is a significant cost in trial and error for sirna design ."], "abstract_text": ["<S> given an mrna sequence as input , the oligowalk web server generates a list of small interfering rna ( sirna ) candidate sequences , ranked by the probability of being efficient sirna ( silencing efficacy greater than 70% ) . </S>", "<S> to accomplish this , the server predicts the free energy changes of the hybridization of an sirna to a target mrna , considering both sirna and mrna self - structure . </S>", "<S> the free energy changes of the structures are rigorously calculated using a partition function calculation . by changing advanced options , the free energy changes </S>", "<S> can also be calculated using less rigorous lowest free energy structure or suboptimal structure prediction methods for the purpose of comparison . </S>", "<S> considering the predicted free energy changes and local sirna sequence features , the server selects efficient sirna with high accuracy using a support vector machine . </S>", "<S> on average , the fraction of efficient sirnas selected by the server that will be efficient at silencing is 78.6% . </S>", "<S> the oligowalk web server is freely accessible through internet at http://rna.urmc.rochester.edu/servers/oligowalk . </S>"], "labels": null, "section_names": ["INTRODUCTION", "OLIGOWALK SERVER INPUT", "OLIGOWALK SERVER OUTPUT", "ADVANCED OPTIONS", "CONCLUSIONS"], "sections": [["it is well known that genes can be silenced by antisense rna oligonucleotides called small interfering rna ( sirna ) ( 1,2 ) . in order to design an efficient sirna sequence , empirical rules based on the features of the sirna sequence", "have been discovered , including , for example , low g / c content , lack of self - structure , preference of a at position 3 , absence of g or c at position 19 and asymmetry in the stability of the terminal base pairs ( 310 ) .", "the self - structure of the target and oligonucleotide is also an important consideration for the effective binding ( 1115 ) .", "it is desirable to select an oligonucleotide having high accessibility to the target - binding site and low duplex stability . here", ", the oligowalk server , which predicts efficient sirna sequences using an accessibility calculation with a convenient web interface , is described .", "overall , the positive predictive value of the server is 0.786 , meaning that 78.6% of the sirnas selected by the server will be efficient at silencing ( 16 ) .", "the positive predictive value was determined by testing against a database of sirna experiments conducted under diverse experimental conditions ( 17 ) . in the calculation of the oligowalk server ,", "unimolecular and bimolecular self - structures for the sirna are considered along with unimolecular self - structure in the target at the oligonucleotide binding region ( 16 ) .", "oligowalk predicts the free energy changes ( g ) involved in these equilibrium states ( 18 ) .", "the predicted thermodynamics ( g ) , plus the oligonucleotide sequence features ( 19 ) , are then utilized to predict sirna efficacy for candidate sirna sequences ( 16 ) , which are generally 19 nucleotide duplexes with 3 dinucleotide dangling ends ( 7 ) .", "a support vector machine ( svm ) program ( 20 ) is embedded in the server to take the thermodynamic and sequence features as input .", "the svm classification model used in the server has been proven to be able to predict efficient sirna ( greater than 70% inhibition of the target mrna expression ) with high accuracy ( 16 ) .", "the svm was trained on a sirna database that contains 2431 experimental results conducted in human cells at 37c ( 10 ) .", "the output of the oligowalk server is a table of sirna candidates , showing the sirna sequences and the probabilities of being efficient ( having silencing efficacy larger than 70% ) .", "each of the free energy change terms for each candidate is also listed in a separate table ."], ["the oligowalk server uses the cgi ( common gateway interface ) module of perl for taking user input and submitting calculations from the homepage .", "the input of oligowalk server is the rna sequence of the target gene . only a , u , t , g and c are the acceptable types of nucleotides in the sequence ( the server will replace the nucleotide t with u for calculations ) , and the maximum sequence length is 10 000 nucleotides .", "an email address is required because the server sends an email to the user when the calculation is completed .", "submit query , the server generates a list of efficient sirna candidates for the target gene .", "jobs are submitted by the server to a cluster of seven nodes with 3.2 or 3.4 ghz pentium 4 processors running fedora linux ( http://fedoraproject.org/ ) , managed by sun grid engine ( http://gridengine.sunsource.net/ ) ."], ["when the calculation is complete , an html ( hypertext markup language ) page is generated with links to tables containing predicted sirna efficacy data and thermodynamic binding data . in the sirna efficacy table ( figure 1 ) , the sequences of sirna candidates are ranked in the output list by their probabilities of being efficient sirna .", "the probabilities are predicted by a svm embedded in the web server for selecting efficient sirna .", "the classification model ( 16 ) used in the svm was trained with a publically available database ( 10 ) , using thermodynamic and sequence features of sirna candidates . the position number of each sirna candidate is also listed in the table as the index of the 5 most base in the target - binding region . \n", "the sequences of sirna are ranked from top to end by their probabilities of being efficient ( antisense efficacy larger than 70% ) . a table of sirna candidates generated by oligowalk server .", "the sequences of sirna are ranked from top to end by their probabilities of being efficient ( antisense efficacy larger than 70% ) .", "in addition , the predicted equilibrium thermodynamics table is generated as a reference for advanced users . in the table", " overall ( in kcal / mol ) is the overall free energy change of oligonucleotide - target binding ,   when all contributions are considered , including breaking target and oligonucleotide self - structures ( 18 ) .", " duplex ( in kcal / mol ) is the free energy change of hybridized duplex between oligonucleotide and target ( antisense  sense duplex ) , .", "the value is independent of oligonucleotide concentration because it is a standard free energy change . ", "tm - dup ( in c ) is the melting temperature in degrees for the duplex formation of oligonucleotide and target . ", "break - targ. ( in kcal / mol ) is the free energy cost to open the intramolecular target base pairs for oligonucleotide binding , .", "a more negative number indicates higher free energy cost , which is unfavorable for oligonucleotide - target binding . ", "intraoligo ( in kcal / mol ) is the free energy change of intramolecular oligonucleotide structure , .", "it usually has a negative value or , if there is no favor - able intramolecular structure , it is zero . ", "interoligo ( in kcal / mol ) is the free energy change of intermolecular oligonucleotide structure , .", "antisense bimolecular structure , which decreases the oligonucleotide - target ( antisense  sense ) binding affinity .  end_diff. ( in kcal / mol ) is the free energy difference between the 5 and 3 end of the antisense strand of sirna , with windows of two base pairs .", "functional sirna prefer to have an unstable 5 end ( 3 ) , which means a positive end_diff .", " prefilter_score is the score calculated with a method based on the empirical rules by reynolds et al . ( 7 ) .", "( 7 ) , except for the melting temperature of intramolecular oligonucleotide self - structure because the free energy ( 21 ) and enthalpy parameters ( 22 ) used by oligowalk are more recent .", "when calculating the prefilter score , 57c is used as the cutoff of the intramolecular oligonucleotide melting temperature , as suggested in another study ( 23 ) . as an example", ", the prediction of the webserver is compared with experimental results in figure 2 . in the experiment ( 3 ) , sirna were tested for efficacy against the target mrna , human cyclophilin ( genbank i d : m60857 ) , at 37c .", "the inhibition efficacy of each sirna is defined as 100% minus the percentage of mrna level after sirna application as compared to matched control .", "the prediction result is the probability of being efficient ( having inhibition efficacy larger than 70% ) , which is calculated with the server . in figure 2 , most of the sirna with high inhibition efficacy are predicted to have high probability of being efficient . \n", "figure 2.a comparison between the prediction and experimental result . in blue is the experimental result ( inhibition efficacy ) for an mrna target , human cyclophilin ( genbank i d : m60857 ) , at 37c ( 3 ) .", "the inhibition efficacy is defined as 100% minus the percent level of mrna expression after sirna application as compared to a control . in red", "is the probability ( ranging from 01 ) of being efficient ( having inhibition efficacy larger than 70% ) , as predicted by oligowalk .", "a comparison between the prediction and experimental result . in blue is the experimental result ( inhibition efficacy ) for an mrna target , human cyclophilin ( genbank i d : m60857 ) , at 37c ( 3 ) .", "the inhibition efficacy is defined as 100% minus the percent level of mrna expression after sirna application as compared to a control . in red", "is the probability ( ranging from 01 ) of being efficient ( having inhibition efficacy larger than 70% ) , as predicted by oligowalk ."], ["advanced options ( figure 3 ) are available for users who understand the underlying calculations and would like to test novel hypotheses . the option form is written in html , embedded with javascript controlling the options so that they are context - aware .", "the oligonucleotide concentration does not affect the result of sirna sequence design , because the inputs to the svm are standard free energy changes , g . concentration changes do , however , alter the overall free energy change ( ) , which is provided to the user with the thermodynamic details table .", "the sense  antisense duplex free energy changes are calculated without considering the self - structures of the target . this mode is not recommended for an accurate sirna prediction .", "it is more rigorous to consider the accessibility of the target and oligonucleotide self - structures because binding affinity is lost to open existing base pairs in the target and oligonucleotide .", "the second mode is to break local structure . in this case , the structure of target is fixed and only the base pairs on the binding site will be broken without refolding the global structure of target . the final and most rigorous mode is to refold the target rna . in this mode ,", "the rna target is folded before oligonucleotide binding and refolded afterwards for each possible oligonucleotide to consider complete equilibration .", "the default setting of sirna design is shown in the figure . if the target rna secondary structure is considered , three different prediction methods are available to calculate the free energy change of target self - structure .", "the first method is optimal structure prediction , where only the optimal structure ( lowest free energy structure ) of the target is considered to calculate the free energy cost of opening the base pairs of binding region .", "the second method considers a set of suboptimal structures to determine the free energy cost .", "each structure 's free energy cost is weighted according to the free energy change of the structure to arrive at the ensemble cost .", "for this option , at most 1000 suboptimal structures ( within 10% free energy difference from the optimal structure ) are generated with a heuristic method ( 24 ) .", "the number of suboptimal structures will be listed in the output table if the target is folded with suboptimal structure prediction method .", "constrained target structure is the refolded structure where the binding region is forced to be single - stranded , so that the oligonucleotide can bind to it .", "this is the most rigorous method because it considers every possible secondary structure in the folding ensemble , with boltzmann weight .", "the structure prediction only folds a certain total number ( folding size ) of nucleotides centered at the binding region .", "the user can define this number , but the largest folding size is 1000 nucleotides for the webserver in order to save compute time .", "users can define longer folding sizes by downloading and installing the oligowalk program to a local machine .", "( 7 ) can be used to rule out nonefficient sirna candidates before folding the target sequence , i.e. the sirna sequences having score less than six points will not be considered for the folding step .", "it is suggested to turn on the prefilter option to save considerable computation time ( table 1 ) .", "furthermore , the scan region can be redefined if the user is interested only in a specific region of the target . \n", "table 1.calculation size and time of oligowalk server for different target sequencestarget mrna ( genbank id)sequence length ( nucleotide)time ( h : min : sec)memory ( mb)nm_0205487300:57:1793m608578513:55:53110nm_00287012116:09:25112nm_00246721896:36:42113aj27221234606:53:05117the benchmarks were performed with the default options : the oligonucleotide was a 19 base rna ; the folding size of the target was 800 nucleotides centering on the binding site ( full length if the whole target has less than 800 nt ) ; the partition function calculation was conducted ; the entire mrna was scanned ; and the prefilter was on . the time cost changes little for long sequence because the prefilter ( 7 ) is on and number of candidates being folded is limited to about the same number for each sequence.the calculations were submitted and benchmarked on the oligowalk web server ( http://rna.urmc.rochester.edu/servers/oligowalk ) .", "each node has a 3.2 or 3.4 ghz pentium 4 processor running fedora linux.the calculation for sequences less than 800 nucleotides is relatively fast because the dynamic programming arrays are reused for calculations of short sequences ( 16 ) .", "calculation size and time of oligowalk server for different target sequences the benchmarks were performed with the default options : the oligonucleotide was a 19 base rna ; the folding size of the target was 800 nucleotides centering on the binding site ( full length if the whole target has less than 800 nt ) ; the partition function calculation was conducted ; the entire mrna was scanned ; and the prefilter was on .", "the time cost changes little for long sequence because the prefilter ( 7 ) is on and number of candidates being folded is limited to about the same number for each sequence .", "the calculation for sequences less than 800 nucleotides is relatively fast because the dynamic programming arrays are reused for calculations of short sequences ( 16 ) ."], ["the oligowalk web server predicts the hybridization thermodynamics of an oligonucleotide binding to a complementary target rna using the most recent rna folding parameters ( 21 , 22 ) .", "it predicts efficient sirna with high accuracy using a transparent implementation of an svm ( 16 ) , which considers both sequence and thermodynamic features .", "the calculation time and memory size of oligowalk are shown in table 1 for a sample of mrna sequences .", "the prefilter ( 7 ) that uses local sequence information to narrow down the list of sirna candidates before calculating the equilibrium affinity is used by default .", "for example , the server takes 3 h and 43 min for a complete scan of all possible sirnas on an mrna having 730 nucleotides using the partition function calculation . for the same sequence ,", "the algorithm time scales o(mn ) and the memory use scales o(n ) ( table 1 ) , m is the number of candidates and n is the value of folding size .", "the time and memory costs change little with sequence because the same folding size ( e.g. 800 nucleotides ) is used and the prefilter ( 7 ) is turned on , which limits the number of candidates to be folded in a way that is apparently independent of target length .", "there is currently significant interest in using sirna for both basic science and medical research .", "the fact that not all sirna duplexes will function in silencing means that there is a significant cost in trial and error for sirna design ."]]}
{"article_id": "PMC4155239", "article_text": ["stroke is a disease that causes sudden local neurological deficit symptoms due to a \n cerebrovascular accident1 .", "stroke may \n decrease postural control and balance ability due to sensory disability , motor disturbance , \n muscular weakness , and asymmetric postures resulting from central nervous system damage2", ". reduced postural control and balance \n ability decreases overall physical function , making independent activities of daily living \n difficult for stroke patients , and increases the risk of falls .", "therefore , improving stroke \n patients balance ability is an important therapeutic objective3 . with regard to this issue", ", a recent cross - sectional study \n revealed that stroke patients trunk control ability was correlated with balance , gait , and \n functional abilities4 .", "exercises intended to improve stroke patients trunk control have been extensively studied \n in recent years .", "verheyden et al.2 \n reported that a group of stroke patients that performed trunk exercises on plinths showed \n better improvement in trunk control than a group that received only conventional physical \n therapy .", "pereira et al.5 conducted trunk \n flexion and extension , and compared trunk muscle activities measured by semg on the affected \n and unaffected sides of stroke patients .", "karthikbabu et al.6 studied the effect of exercise using swiss balls on the improvement \n of trunk muscles .", "although many exercise programs for trunk control have been implemented , \n there are few programs that can improve subjects balance while maintaining their interest . \n", "after the first horse riding studies were performed with patients with neurological damage , \n hippotherapy was used only to treat children with neurological disorders , mainly cerebral \n palsy , to improve their postural control and promote their normal development and functional \n recovery7 , 8 .", "the horse s gait is repetitive and rhythmic , and \n causes the center of gravity to move in anterior - posterior , lateral , and upward - downward \n directions .", "since the patterns of these movements are similar to those of the movements of \n the trunk and pelvis during human gait , such exercise is expected to make the subjects feel \n as if they were walking9 .", "hippotherapy is \n effective in terms of psychological motivation because it arouses subjects interest in \n participating in the therapy9 , 10 , and helps in the recovery of many physical functions . \n", "hippotherapy increases the range of joint motion , strengthens muscles , and improves muscle \n tone , postural control , and balance and equilibrium ability .", "improvements in postural \n control and balance ability are achieved through stimulation of the upper motor neurons \n through proprioceptive sensory stimulation inputs11 , \n 12 . despite these therapeutic effects , \n", "no studies have been conducted on the changes in muscles related to stroke patients \n stability and balance after hippotherapy . in this study , the improvement in stroke patients \n trunk control and changes in abdominal muscle thicknesses after horse riding simulation \n training were examined .", "the purpose of this study was explained to patients diagnosed with stroke at d general \n hospital in busan , south korea .", "patients willing to participate in the study were recruited , \n and those who satisfied the selection criteria were randomly assigned to a control or \n experimental group ( 15 patients per group ) .", "subject selection criteria were as follows : patients with hemiplegia and no previous \n experience with hippotherapy who could follow verbal instructions , sit and walk \n independently ( regardless of the use of walking aids ) , and had no restriction in the range \n of joint motion .", "then , the \n experimental group performed horse riding simulation training , while the control group \n performed trunk exercises using swiss balls .", "training was performed for 30  min per session , \n three sessions per week for a total of eight weeks10 .", "subjects general characteristic ( n = 30)groupcontrol group(n = 15)experimental group(n = 15)age ( years)56.57.555.16.1height ( cm)163.38.2165.18.5weight ( kg)63.58.565.49.8sex ( male / female)8 / 77 / 8cause of brain damage(cerebral hemorrhage /cerebral \n", "infarction)8 / 76 / 9affected side ( right / left)9 / 610 / 5data are mean  sd . all subjects agreed in writing to participate in this study after receiving a detailed \n explanation of the experimental procedure , and possible side effects of the intervention . \n the experiment was conducted after receiving approval from the human subjects research \n ethics committee of dong - eui medical center ( demcirb-2013 - 1001 ) .", "the horse riding simulator ( eu6441 core muscle trainer ; panasonic , japan ) used is designed \n to simulate the effects of horse riding exercises in an indoor environment using \n three - directional movements ( anterior - posterior , lateral , upward - downward ) , and has three \n different programs and nine levels of exercise intensity .", "a therapist and an assistant \n monitored each subject during horse riding training . a balance ability measuring and training system ( an analysis system with biofeedback , \n ap1153 ; biorescue , france ) was used to evaluate subjects balance ability .", "static balance \n ability ( center of pressure [ cop ] path length and cop travel speed ) was measured while \n subjects stood for 60  s with their eyes open .", "the subjects were prevented from seeing the \n monitor to avoid visual feedback , and the position of the feet was maintained during \n repeated measurements to eliminate errors resulting from changes in foot positions .", "all \n tests were performed three times , and average values were calculated . ultrasonic imaging equipment ( sonoace x4 ; medison , korea )", "subjects were instructed to bend their knees to 90 and to maintain a \n comfortable posture in a supine position .", "linear probes were positioned on both sides , \n midway between the bottom of the rib cage and the top of the iliac crest .", "the \n thicknesses of the external oblique , internal oblique , and transversus abdominis muscles \n were measured three times and their average values were calculated .", "the paired t - test was conducted to \n compare the results before and after the intervention within each group and the independent \n t - test was used to compare the exercise and control groups before and after the experiment . \n", "the experimental but not the control group showed statistically significant differences \n ( p<0.01 ) between the cop path lengths of before and after the intervention .", "the \n differences between the two groups were not significant before the experiment , but were \n statistically significant ( p<0.05 ) after the intervention ( table 2table 2 .", "changes in static balance following horse riding simulation traininggrouppre - testpost- testcenter of pressure path length ( cm)control14.55.914.45.8experimental15.95.112.24.3 center of pressure travel speed ( mm / s)control0.70.30.70.2experimental0.70.30.60.2   different superscripts within the same columns indicate significant \n differences ( p<0.05 ) ) .   different superscripts within the same columns indicate significant \n differences ( p<0.05 ) the experimental , but not the control group , showed statistically significant differences \n ( p<0.05 ) between the speeds of the cop travel of before and after the intervention .", "there \n was no significant difference between the control and the experimental group before the \n experiment , but the speed of cop travel decreased more in the experimental group than in the \n control group after the experiment , and this difference was statistically significant \n ( p<0.05 ; table 2 ) .", "changes in abdominal muscle thicknesses after training were observed in the experimental \n group . before training , tra and io did not show any significant differences between the \n affected and unaffected sides .", "in contrast , eo showed significant differences : 5.71.6  mm on \n the unaffected side and 4.71.4  mm on the affected side ( p<0.05 ) .", "after eight weeks of \n training , tra , io , and eo on the unaffected side did not show any changes compared to before \n the training .", "tra and io on the affected side did not show any changes after training , but the eo \n thickness significantly increased from 4.71.4  mm to 5.21.6  mm .", "total abdominal muscle \n thickness differed significantly before training between the affected and unaffected sides \n ( 13.52.7  mm and 14.33.0  mm , respectively ) .", "after eight weeks of training , both sides \n showed slight increases , with final values of 14.12.9  mm on the affected side and \n 14.35.8  mm on the unaffected side , but these differences were not statistically significant \n ( p>0.05 ; table 3table 3 .", "changes in abdominal muscle thickness following horse riding simulation training \n ( experimental group ; n = 15)sidepre - testpost - testtraa2.50.82.70.9na2.50.62.60.6ioa6.30.156.31.6na6.00.166.11.8eoa4.71.4 5.21.6na5.71.65.71.4totala13.52.7 14.12.9na14.33.014.35.8a , affected side ; na , non - affected side ; abdominal muscle thicknesses unit :  mm . \n", "different superscripts within the same columns indicate significant differences \n ( p<0.05 ) ) . a , affected side ; na , non - affected side ; abdominal muscle thicknesses unit :  mm . \n", "this study examined the effects of horse riding simulation training on the improvement of \n balance ability ( which is an important issue for stroke patients ) , and the changes in \n abdominal muscle thicknesses of stroke patients . the distance and speed of center of gravity \n movements", "were measured using a biorescue system , and changes in abdominal muscle \n thicknesses were observed by ultrasonography .", "the cop path lengths of before and after the intervention were similar in the control group \n ( p>0.05 ) , but significantly decreased in the experimental group after horse riding \n training .", "the changes in the speed of cop travel showed that body sway remarkably decreased \n in the experimental group after training in comparison with the control group .", "these changes \n presumably occurred because the trunks of the subjects became more stable as a result of \n horse riding simulation training , reducing body sway and shaking .", "regarding abdominal muscle thicknesses , eo was thinner before training on the affected side \n than on the unaffected side , resulting in a difference in total abdominal muscle thickness \n on the affected and unaffected sides .", "this is consistent with the results of the study by \n english et al.14 , which measured the \n abdominal muscles of stroke patients ( 65  years old or older ) at rest .", "although the mean age \n of the subjects in our present study was somewhat lower ( 55.16.1  years ) , similar results \n were obtained .", "even \n though eo is mainly mobilized during trunk rotation movements , pereria et al.5 reported that eo was activated when stroke \n patients performed exercises that involved lifting of both legs , and this reportedly \n occurred to compensate for the inactivity of the rectus abdominis .", "similarly , in our present \n study the eo muscle may have activated more to compensate for the weakness of the rectus \n abdominal muscles on the affected side of the stroke patients .", "another reason for higher eo \n activation on the affected side could be that it is located closer to the surface layer than \n tra and io , which would affect thickness changes because horse riding simulation exercises \n induce multi - directional movements .", "although diverse balance training programs for stroke patients have been reported , \n therapeutic approaches using horse riding have rarely been studied with stroke patients as \n subjects .", "some studies have been conducted with persons with neurological disorders , in \n particular children with cerebral palsy7 , 8 . therefore , comparison of our \n present results with those of previous studies was difficult .", "future studies of horse riding \n simulation may lead to an improvement in stroke patients balance ability .", "horse riding \n simulation training can be fun and interesting for patients , which may increase their \n motivation to actively participate in exercise program and thus enhance the improvement of \n neurologic functions9 , 10 .", "second , ultrasonic measurement was conducted in a steady state in a supine \n position .", "third , since the damaged sites and symptoms differed among subjects , standard \n subjects could not be selected .", "therefore , in future studies , the effects of hippotherapy \n on stroke patients should be examined with a more efficient control and the selection of a \n wide range of subjects .", "additional information should be provided by comparison ( during \n ultrasonic measurement ) of the degrees of contraction on the affected and unaffected \n sides . in conclusion ,", "horse riding simulation training may reduce the distance and speed of the \n center of gravity movements of stroke patients and reduce the asymmetry of the abdominal \n muscles . to improve stroke patients balance ability , diverse uses of horse riding \n simulation training should be considered ."], "abstract_text": ["<S>   [ purpose ] the purpose of this study was to assess the effects of horse riding simulation \n training on changes in balance ability and abdominal muscle thicknesses of stroke \n patients . </S>", "<S> [ subjects ] thirty stroke patients with hemiplegia were recruited , and they were \n randomly assigned to a control or experimental group . [ methods ] the experimental group \n performed horse riding simulation training , whereas the control group performed trunk \n exercises for 8 weeks . </S>", "<S> balance ability was measured using a biorescue system . </S>", "<S> the \n thicknesses of subjects external oblique , internal oblique , and transversus abdominis \n muscles were measured by ultrasonic imaging . [ results ] in the experimental group , balance \n ability </S>", "<S> was significantly improved after training . </S>", "<S> similarly , the thickness of the \n abdominal muscles on the affected side changed after training in the experimental group , \n whereas the control group showed no statistically significant changes . [ conclusion ] </S>", "<S> we \n suggest that horse riding simulation training is more effective than trunk exercises at \n reducing the center of pressure path length and travel speed and improving the asymmetry \n of the abdominal muscles of stroke patients . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["stroke is a disease that causes sudden local neurological deficit symptoms due to a \n cerebrovascular accident1 .", "stroke may \n decrease postural control and balance ability due to sensory disability , motor disturbance , \n muscular weakness , and asymmetric postures resulting from central nervous system damage2", ". reduced postural control and balance \n ability decreases overall physical function , making independent activities of daily living \n difficult for stroke patients , and increases the risk of falls .", "therefore , improving stroke \n patients balance ability is an important therapeutic objective3 . with regard to this issue", ", a recent cross - sectional study \n revealed that stroke patients trunk control ability was correlated with balance , gait , and \n functional abilities4 .", "exercises intended to improve stroke patients trunk control have been extensively studied \n in recent years .", "verheyden et al.2 \n reported that a group of stroke patients that performed trunk exercises on plinths showed \n better improvement in trunk control than a group that received only conventional physical \n therapy .", "pereira et al.5 conducted trunk \n flexion and extension , and compared trunk muscle activities measured by semg on the affected \n and unaffected sides of stroke patients .", "karthikbabu et al.6 studied the effect of exercise using swiss balls on the improvement \n of trunk muscles .", "although many exercise programs for trunk control have been implemented , \n there are few programs that can improve subjects balance while maintaining their interest . \n", "after the first horse riding studies were performed with patients with neurological damage , \n hippotherapy was used only to treat children with neurological disorders , mainly cerebral \n palsy , to improve their postural control and promote their normal development and functional \n recovery7 , 8 .", "the horse s gait is repetitive and rhythmic , and \n causes the center of gravity to move in anterior - posterior , lateral , and upward - downward \n directions .", "since the patterns of these movements are similar to those of the movements of \n the trunk and pelvis during human gait , such exercise is expected to make the subjects feel \n as if they were walking9 .", "hippotherapy is \n effective in terms of psychological motivation because it arouses subjects interest in \n participating in the therapy9 , 10 , and helps in the recovery of many physical functions . \n", "hippotherapy increases the range of joint motion , strengthens muscles , and improves muscle \n tone , postural control , and balance and equilibrium ability .", "improvements in postural \n control and balance ability are achieved through stimulation of the upper motor neurons \n through proprioceptive sensory stimulation inputs11 , \n 12 . despite these therapeutic effects , \n", "no studies have been conducted on the changes in muscles related to stroke patients \n stability and balance after hippotherapy . in this study , the improvement in stroke patients \n trunk control and changes in abdominal muscle thicknesses after horse riding simulation \n training were examined ."], ["the purpose of this study was explained to patients diagnosed with stroke at d general \n hospital in busan , south korea .", "patients willing to participate in the study were recruited , \n and those who satisfied the selection criteria were randomly assigned to a control or \n experimental group ( 15 patients per group ) .", "subject selection criteria were as follows : patients with hemiplegia and no previous \n experience with hippotherapy who could follow verbal instructions , sit and walk \n independently ( regardless of the use of walking aids ) , and had no restriction in the range \n of joint motion .", "then , the \n experimental group performed horse riding simulation training , while the control group \n performed trunk exercises using swiss balls .", "training was performed for 30  min per session , \n three sessions per week for a total of eight weeks10 .", "subjects general characteristic ( n = 30)groupcontrol group(n = 15)experimental group(n = 15)age ( years)56.57.555.16.1height ( cm)163.38.2165.18.5weight ( kg)63.58.565.49.8sex ( male / female)8 / 77 / 8cause of brain damage(cerebral hemorrhage /cerebral \n", "infarction)8 / 76 / 9affected side ( right / left)9 / 610 / 5data are mean  sd . all subjects agreed in writing to participate in this study after receiving a detailed \n explanation of the experimental procedure , and possible side effects of the intervention . \n the experiment was conducted after receiving approval from the human subjects research \n ethics committee of dong - eui medical center ( demcirb-2013 - 1001 ) .", "the horse riding simulator ( eu6441 core muscle trainer ; panasonic , japan ) used is designed \n to simulate the effects of horse riding exercises in an indoor environment using \n three - directional movements ( anterior - posterior , lateral , upward - downward ) , and has three \n different programs and nine levels of exercise intensity .", "a therapist and an assistant \n monitored each subject during horse riding training . a balance ability measuring and training system ( an analysis system with biofeedback , \n ap1153 ; biorescue , france ) was used to evaluate subjects balance ability .", "static balance \n ability ( center of pressure [ cop ] path length and cop travel speed ) was measured while \n subjects stood for 60  s with their eyes open .", "the subjects were prevented from seeing the \n monitor to avoid visual feedback , and the position of the feet was maintained during \n repeated measurements to eliminate errors resulting from changes in foot positions .", "all \n tests were performed three times , and average values were calculated . ultrasonic imaging equipment ( sonoace x4 ; medison , korea )", "subjects were instructed to bend their knees to 90 and to maintain a \n comfortable posture in a supine position .", "linear probes were positioned on both sides , \n midway between the bottom of the rib cage and the top of the iliac crest .", "the \n thicknesses of the external oblique , internal oblique , and transversus abdominis muscles \n were measured three times and their average values were calculated .", "the paired t - test was conducted to \n compare the results before and after the intervention within each group and the independent \n t - test was used to compare the exercise and control groups before and after the experiment . \n"], ["the experimental but not the control group showed statistically significant differences \n ( p<0.01 ) between the cop path lengths of before and after the intervention .", "the \n differences between the two groups were not significant before the experiment , but were \n statistically significant ( p<0.05 ) after the intervention ( table 2table 2 .", "changes in static balance following horse riding simulation traininggrouppre - testpost- testcenter of pressure path length ( cm)control14.55.914.45.8experimental15.95.112.24.3 center of pressure travel speed ( mm / s)control0.70.30.70.2experimental0.70.30.60.2   different superscripts within the same columns indicate significant \n differences ( p<0.05 ) ) .   different superscripts within the same columns indicate significant \n differences ( p<0.05 ) the experimental , but not the control group , showed statistically significant differences \n ( p<0.05 ) between the speeds of the cop travel of before and after the intervention .", "there \n was no significant difference between the control and the experimental group before the \n experiment , but the speed of cop travel decreased more in the experimental group than in the \n control group after the experiment , and this difference was statistically significant \n ( p<0.05 ; table 2 ) .", "changes in abdominal muscle thicknesses after training were observed in the experimental \n group . before training , tra and io did not show any significant differences between the \n affected and unaffected sides .", "in contrast , eo showed significant differences : 5.71.6  mm on \n the unaffected side and 4.71.4  mm on the affected side ( p<0.05 ) .", "after eight weeks of \n training , tra , io , and eo on the unaffected side did not show any changes compared to before \n the training .", "tra and io on the affected side did not show any changes after training , but the eo \n thickness significantly increased from 4.71.4  mm to 5.21.6  mm .", "total abdominal muscle \n thickness differed significantly before training between the affected and unaffected sides \n ( 13.52.7  mm and 14.33.0  mm , respectively ) .", "after eight weeks of training , both sides \n showed slight increases , with final values of 14.12.9  mm on the affected side and \n 14.35.8  mm on the unaffected side , but these differences were not statistically significant \n ( p>0.05 ; table 3table 3 .", "changes in abdominal muscle thickness following horse riding simulation training \n ( experimental group ; n = 15)sidepre - testpost - testtraa2.50.82.70.9na2.50.62.60.6ioa6.30.156.31.6na6.00.166.11.8eoa4.71.4 5.21.6na5.71.65.71.4totala13.52.7 14.12.9na14.33.014.35.8a , affected side ; na , non - affected side ; abdominal muscle thicknesses unit :  mm . \n", "different superscripts within the same columns indicate significant differences \n ( p<0.05 ) ) . a , affected side ; na , non - affected side ; abdominal muscle thicknesses unit :  mm . \n"], ["this study examined the effects of horse riding simulation training on the improvement of \n balance ability ( which is an important issue for stroke patients ) , and the changes in \n abdominal muscle thicknesses of stroke patients . the distance and speed of center of gravity \n movements", "were measured using a biorescue system , and changes in abdominal muscle \n thicknesses were observed by ultrasonography .", "the cop path lengths of before and after the intervention were similar in the control group \n ( p>0.05 ) , but significantly decreased in the experimental group after horse riding \n training .", "the changes in the speed of cop travel showed that body sway remarkably decreased \n in the experimental group after training in comparison with the control group .", "these changes \n presumably occurred because the trunks of the subjects became more stable as a result of \n horse riding simulation training , reducing body sway and shaking .", "regarding abdominal muscle thicknesses , eo was thinner before training on the affected side \n than on the unaffected side , resulting in a difference in total abdominal muscle thickness \n on the affected and unaffected sides .", "this is consistent with the results of the study by \n english et al.14 , which measured the \n abdominal muscles of stroke patients ( 65  years old or older ) at rest .", "although the mean age \n of the subjects in our present study was somewhat lower ( 55.16.1  years ) , similar results \n were obtained .", "even \n though eo is mainly mobilized during trunk rotation movements , pereria et al.5 reported that eo was activated when stroke \n patients performed exercises that involved lifting of both legs , and this reportedly \n occurred to compensate for the inactivity of the rectus abdominis .", "similarly , in our present \n study the eo muscle may have activated more to compensate for the weakness of the rectus \n abdominal muscles on the affected side of the stroke patients .", "another reason for higher eo \n activation on the affected side could be that it is located closer to the surface layer than \n tra and io , which would affect thickness changes because horse riding simulation exercises \n induce multi - directional movements .", "although diverse balance training programs for stroke patients have been reported , \n therapeutic approaches using horse riding have rarely been studied with stroke patients as \n subjects .", "some studies have been conducted with persons with neurological disorders , in \n particular children with cerebral palsy7 , 8 . therefore , comparison of our \n present results with those of previous studies was difficult .", "future studies of horse riding \n simulation may lead to an improvement in stroke patients balance ability .", "horse riding \n simulation training can be fun and interesting for patients , which may increase their \n motivation to actively participate in exercise program and thus enhance the improvement of \n neurologic functions9 , 10 .", "second , ultrasonic measurement was conducted in a steady state in a supine \n position .", "third , since the damaged sites and symptoms differed among subjects , standard \n subjects could not be selected .", "therefore , in future studies , the effects of hippotherapy \n on stroke patients should be examined with a more efficient control and the selection of a \n wide range of subjects .", "additional information should be provided by comparison ( during \n ultrasonic measurement ) of the degrees of contraction on the affected and unaffected \n sides . in conclusion ,", "horse riding simulation training may reduce the distance and speed of the \n center of gravity movements of stroke patients and reduce the asymmetry of the abdominal \n muscles . to improve stroke patients balance ability , diverse uses of horse riding \n simulation training should be considered ."]]}
{"article_id": "PMC4450547", "article_text": ["a number of gases are produced endogenously in humans and have roles in the pathology of various diseases . among these gases , nitric oxide ( no ) and carbon monoxide ( co ) have vital roles in the physiology of the body . since the last decade", ", hydrogen sulfide ( h2s ) has captured the interest of scientists because of its significant role in different systems of the body .", "h2s has been known as a toxic gas with rotten egg smell for more than 300 years . as a toxicant ,", "some studies have also reported the toxic effects of h2s on the central nervous system ( cns ) and respiratory system .", "h2s is endogenously produced from two sulfur - containing amino acids l - cysteine and l - methionine by the two enzymes cystathionine  lyse ( cse ) and cystathionine  synthase ( cbs ) as shown in figure 1a and b. recently , a third enzyme , namely , 3-mercaptopyruvate sulfur transferase ( 3mst ) , along with cysteine aminotransferase ( cat ) , which is similar to aspartate amino transferase , has been shown to produce h2s in brain .", "3mst is produced by schematic chain reaction from 3-mercaptopyruvate which is also produced by cat from cysteine and -ketoglutarate[figure 1 ] . \n ", "( a - c ) the schematic presentation of h2s production in the mammalians body by involving three different pathways both the enzymes cse and cbs are present in mammalian cells and tissues .", "previously , cse has been reported to be responsible for the production of h2s in the cardiovascular system ( cvs ) and kidneys , whereas cbs performs the same function in cns .", "few studies have supported that cse is dominant in cvs , whereas cbs is dominant in cns .", "both cbs and cse have been reported to be present in the kidneys , predominantly in predominantly cortical thymoma .", "however , the mechanism of its production still remains unclear ; whether or not the production of h2s is induced by cse or cbs .", "expression of both enzymes in the kidneys can lead us to conclude about the production of h2s by both enzymes .", "the third enzyme , 3mst , is responsible for h2s production and has also been reported to be present in endothelial cells in the thoracic aorta .", "recent studies have shown that greater than 90% of the total h2s is produced in the brain by 3mst .", "based on the literature , different enzymes are involved in the production of h2s in different parts of body ; for example , h2s is predominantly produced in the heart by cse , both cbs and cse in the kidneys , and 3mst in the brain by .", "dl - propargylglycine ( dl - pag ) is an inhibitor of cse , whereas aminooxyacetic acid is an inhibitor of cbs .", "both inhibitors are nonselective and may be responsible for the inhibition of other enzymes . some selective inhibitors can elaborate the mechanisms of h2s . some mechanisms for long - term control production of h2s are coming to light .", "long - term regulation of h2s seems to be dependent on s - adenosyl - l  methionine , which activates cbs and ultimately leads to the production of h2s[figure 1c ] .", "the endogenous concentration of circulating h2s is 50160 m in rats , bovines , and humans .", "h2s hyperpolarizes the membranes of localized cells , modulates the neuronal excitability , relaxes the smooth muscles , and controls cell apoptosis or proliferation . in one study , normal concentration of h2s in wistar kyoto ( wky ) rats was measured at 10 m ; however , other studies have demonstrated that the plasma level of h2s is 50 m", ". the tissue level of h2s has been thought to be higher than its plasma level .", "for example , the physiological concentration of h2s in the brain has been documented at 50160 m .", "the h2s level decreased below the normal level in the body if coronary heart disease spontaneously hypertensive rats ( shrs ) ; however , the level increased in diabetes and circulatory shock . in carrageenan - induced inflammation model , concentration is increased .", "increase in the concentration of h2s has also been observed in acute pancreatitis , hemorrhagic shock , and in endotoxin shock .", "the vasorelaxant effect of h2s has been proven , which shows that h2s relaxes the isolated aorta at a concentration as low as 18 m and 60 m if pretreated with 20 mm kcl or phe .", "a number of gases are produced endogenously in humans and have roles in the pathology of various diseases . among these gases , nitric oxide ( no ) and carbon monoxide ( co ) have vital roles in the physiology of the body . since the last decade", ", hydrogen sulfide ( h2s ) has captured the interest of scientists because of its significant role in different systems of the body .", "h2s has been known as a toxic gas with rotten egg smell for more than 300 years . as a toxicant ,", "some studies have also reported the toxic effects of h2s on the central nervous system ( cns ) and respiratory system .", "h2s is endogenously produced from two sulfur - containing amino acids l - cysteine and l - methionine by the two enzymes cystathionine  lyse ( cse ) and cystathionine  synthase ( cbs ) as shown in figure 1a and b. recently , a third enzyme , namely , 3-mercaptopyruvate sulfur transferase ( 3mst ) , along with cysteine aminotransferase ( cat ) , which is similar to aspartate amino transferase , has been shown to produce h2s in brain .", "3mst is produced by schematic chain reaction from 3-mercaptopyruvate which is also produced by cat from cysteine and -ketoglutarate[figure 1 ] . \n ", "( a - c ) the schematic presentation of h2s production in the mammalians body by involving three different pathways both the enzymes cse and cbs are present in mammalian cells and tissues .", "previously , cse has been reported to be responsible for the production of h2s in the cardiovascular system ( cvs ) and kidneys , whereas cbs performs the same function in cns .", "few studies have supported that cse is dominant in cvs , whereas cbs is dominant in cns .", "both cbs and cse have been reported to be present in the kidneys , predominantly in predominantly cortical thymoma .", "however , the mechanism of its production still remains unclear ; whether or not the production of h2s is induced by cse or cbs .", "expression of both enzymes in the kidneys can lead us to conclude about the production of h2s by both enzymes .", "the third enzyme , 3mst , is responsible for h2s production and has also been reported to be present in endothelial cells in the thoracic aorta .", "recent studies have shown that greater than 90% of the total h2s is produced in the brain by 3mst .", "based on the literature , different enzymes are involved in the production of h2s in different parts of body ; for example , h2s is predominantly produced in the heart by cse , both cbs and cse in the kidneys , and 3mst in the brain by .", "dl - propargylglycine ( dl - pag ) is an inhibitor of cse , whereas aminooxyacetic acid is an inhibitor of cbs .", "both inhibitors are nonselective and may be responsible for the inhibition of other enzymes . some selective inhibitors can elaborate the mechanisms of h2s . some mechanisms for long - term control production of h2s are coming to light .", "long - term regulation of h2s seems to be dependent on s - adenosyl - l  methionine , which activates cbs and ultimately leads to the production of h2s[figure 1c ] .", "the endogenous concentration of circulating h2s is 50160 m in rats , bovines , and humans .", "h2s hyperpolarizes the membranes of localized cells , modulates the neuronal excitability , relaxes the smooth muscles , and controls cell apoptosis or proliferation . in one study , normal concentration of h2s in wistar kyoto ( wky ) rats was measured at 10 m ; however , other studies have demonstrated that the plasma level of h2s is 50 m", ". the tissue level of h2s has been thought to be higher than its plasma level .", "for example , the physiological concentration of h2s in the brain has been documented at 50160 m .", "the h2s level decreased below the normal level in the body if coronary heart disease spontaneously hypertensive rats ( shrs ) ; however , the level increased in diabetes and circulatory shock . in carrageenan - induced inflammation model , concentration is increased .", "increase in the concentration of h2s has also been observed in acute pancreatitis , hemorrhagic shock , and in endotoxin shock .", "the vasorelaxant effect of h2s has been proven , which shows that h2s relaxes the isolated aorta at a concentration as low as 18 m and 60 m if pretreated with 20 mm kcl or phe .", "the role of h2s seems to be partially solved by evaluating different mechanisms ; however , researchers have shifted their interest to other gaseous transmitters , such as no and carbon monoxide .", "studying the role of h2s in hypertension chronologically , we were first informed that h2s produces vasorelaxation on rat aortic tissue in vitro . in succeeding research , the vasorelaxant effect of h2s", "has been attributed to the opening of the atp - sensitive potassium ( katp ) channel ; this effect was mimicked by pinacidil ( a katp channel agonist ) and antagonized by glibenclamide ( a katp channel antagonist ) .", "blood pressure reduction by intravenous ( iv ) bolus injection of h2s was 1230 mm of hg .", "the study under discussion revealed that aortic rings were relaxed by 63%  2.2% at a dose of 180 m , if pretreated with phe .", "the vasorelaxation of h2s was partially dependent on endothelium and mostly through direct effects on the smooth muscle cells . to further exclude the involvement of no ,", "the vasorelaxant effects of h2s are not blocked by 1h-(1,2,4)oxadiazolo[4,3-a ] quinoxalin-1-one ( odq ) ( an inhibitor of guanylyl cyclase ) .", "the katp channel mechanism was confirmed by inducing contractions in the aortic tissue through treating the tissue with low k ( 20 mm ) and high k ( 100 mm ) ; vasorelaxation produced by h2s on aortic tissues was observed when pretreated with low k ( 20 mm ) and high k ( 100 mm ) .", "the maximum vasorelaxation produced by h2s was 90%  8.2% and 19%  3.9% when pretreated with low k ( 20 mm ) and high k ( 100 mm ) , respectively .", "hydrogen sulfide produces more relaxation with low k ( 20 mm ) because of k conductance .", "the k channel opener effect was further verified using 10 mm tetraethylammonium and 100 nm of charybdotoxin or 100 nm iberiotoxin ( specific inhibitors of kca channel ) , completely inhibiting h2s - induced relaxation . in upcoming years", ", the vasorelaxation produced by h2s was proven to be through a different mechanism than that of no and co. furthermore , the vasorelaxation effect on the vascular tissue by no is reduced when pretreated with h2s .", "this study also suggests that an additive response can be achieved using no and h2s .", "a study elaborated that the cardioprotective action of h2s mediated by katp channel opening is caused by the physiological production of h2s in the heart .", "this study has proven the negative inotropic effects of h2s both in vivo and in vitro , and concluded that h2s can effectively prevent hypertension in rats when induced by l - n - nitro arginine methyl ester . nitric oxide and", "deficiency in no and co has been proven to contribute to the pathogenesis of hypertension .", "h2s has also been reported to play the same role in hypertension because of similar biological activities . in subsequent studies , the level of h2s in the plasma and its production rate in seriously hypertensive rats ( shr ) was lower than that in wky rats . therefore , cse is a specific enzyme for h2s production in the thoracic aorta and its decreased activity in hypertension may lead to less production of h2s , resulting in decreased circulating level of h2s .", "the above - mentioned theory proposes that less activity and decreased transcription of cse results in decreased circulating aortic h2s level and that vasoconstriction is a dominant phenomenon over vasorelaxant one .", "sodium hydrogen sulfide ( nahs ) has been selected as an exogenous source ofh2s because of to four reasons .", "( 1 ) na dissociates from hs in a nahs solution , then hs associates with h and produces h2s , regardless if the h2s solution was prepared by bubbling h2s gas or dissolving nahs . in physiological saline ,", "about one - third of h2s exists in the undissociated form ( h2s ) , and the remaining two - thirds is hs - at equilibrium with h2s .", "( 2 ) nahs enables a more accurate and reproducible measurement of h2s concentrations in a solution than by bubbling h2s gas . ( 3 ) the influence of 1 mm or less sodium ion on the physiological experiments is negligible .", "( 4 ) nahs at concentrations used in the current study does not change the ph of the medium .", "the results of one study suggest that upregulation of h2s results in the reduction of sbp in the shr + nahs group ( 158.13  12.52", "mm vs. 183.57  11.8 mm of hg ) . a study demonstrated that an iv bolus injection of h2s at 2.8 mol / kg and 14 mol / kg body weight results in a decrease in the mean arterial blood pressure of rats by 12.5  2.1 mmhg and 29.8  7.6 mmhg , respectively .", "therefore , the physiological concentration of h2s is responsible for maintaining the mean arterial blood pressure .", "however , the expression of cse activity decreases with the onset of disease . based on the findings , the finding that the physiological concentration of h2s is responsible for the normal function of cvs is a matter of great interest .", "diseases like hypertension occur when the concentration of h2s decreases ; however , the opposite occurs in hemorrhagic shock . in a study , induction of hemorrhagic shock results in a prolonged decrease in the mean arterial pressure ( map ) and heart rate ( hr ) .", "however , data suggest that vasoconstriction is responsible for a hemorrhagic shock because of vasopressin , noradrenaline , and angiotensin ii .", "a reasonable number of previous studies have shown that excessive formation of inducible nitric oxide synthase ( inos ) in hypotension is responsible for hemorrhagic shock ; thus , the concentration of h2s increases in the induction of hemorrhagic shock .", "treatment with inhibitors of h2s - producing enzymes cse and dl - pag , a suicidal inhibitor , resulted in the rapid and partial restoration of map and hr .", "previously , glibenclamide ( a k channel blocker ) has been proven to perform the same partial but equally effective function after hemorrhagic shock in a rat .", "a comparative study was conducted to compare the effect of h2s with pag and -cyano - l - alanine , a reversible inhibitor of cse , on the blood pressure of rats subjected to hemorrhagic shock .", "after many years of earning good repute among researchers , h2s research started to intermingle with each other .", "two different schools of thought exist between interchangeable production of h2s and no . in 1997", ", a study has proven that a low concentration of h2s increases the relaxation of smooth muscles by 13-folds .", "this study also explained that low concentration ( 30 m ) of h2s enhances the vasodilator effect of no .", "therefore , a synergistic response between h2s and no can be the therapeutic outcome in hypertension .", "this study further elaborated that no - induced vasorelaxation is specifically for h2s , but no can not potentiate the vasorelaxant effect of h2s .", "another study has proven that no is responsible for the upregulated production of h2s in rats in a dose - dependent manner ( 1100 m ) , as shown in the following figure .", "this study claims that production of h2s by no is narrated by two mechanisms : ( 1 ) no increases cse activity , which ultimately leads to the production of h2s , and ( 2 ) no increases the activity of protein kinase , which is dependent on cyclic guanosine monophosphate , thereby increasing cse protein .", "a growing body of evidence has proven that h2s as a cofactor is responsible for the generation of no from nitrite . in his previous studies in 2002 , documented the interaction between hydrogen sulfide and no resulting in the formation of the intermediate compound nitrosothiol , which releases no through hemolysis . in his next experimentation , proposed the mechanism of no production from sodium nitroprusside . in 2006 , has proven that the interaction between two gasotransmitters results in the formation of nitrosothiol and the release of only a small portion of no unless an antioxidant is involved .", "these findings contradict those of other studies , which demonstrate that the interaction between no and h2s enhances vasodilatation . in the battle of the interchangeable production of h2s and no ,", "another finding cropped up , supporting the claim that h2s is responsible for the direct inhibition of endothelial nitric oxide synthase ( enos ) .", "this theory is born out of the result of the first school of thought , which proposed that no can potentiate the response of h2s , but no can do this .", "has elaborated that h2s is responsible for the inhibition of enos , as well as inos and nnos .", "they further strengthened the results of their study by explaining that h2s and tetrahydrobiopterin ( bh4 ) reverse the h2s inhibition of enos and nnos , but not inos .", "a study in 2006 elaborated that the intermediate complex formed from the interaction between h2s and no has no vasorelaxant effect ; h2s was responsible for the regulation of no .", "this study concluded that h2s inhibits the vasorelaxant effect of no . a decrease in h2s concentration results in over stimulation of  adrenoreceptor .", "the h2s has also been reported to inhibit the l - type ca channels in cardiomyocyte , which leads to a decrease in contractility , thereby revealing the ca channel - blocking mechanism of h2s . in the same year", ", h2s has been found to have the ability to block the angiotensin - converting enzyme and responsible for additive vasorelaxant response leading to the inhibition of angiotensin ii production , ultimately resulting in reduced degradation of bradykinin . in a related study in 2010 , reported that enos and neuronal nitric oxide synthase ( nos ) activation is important to produce a cardioprotective response .", "the cardioprotective response of the nos family mentioned in is contrary to the results by .", "another area of interest is the formation of the intermediate complex nitrosothiol , which is responsible for the vasorelaxant effect in the blood vessels through interaction between two gasotransmitters , h2s and no .", "the presence of this intermediate molecule , nitrosothiol , has not been fully confirmed and has been considered as a possible molecule for the said action .", "a previously mentioned study has stated that the intermediate molecule is nitrosothiol but has no vasorelaxant effect in vivo and in vitro .", "another school of thought has reported that the interaction between no and h2s leads to the formation of the nitroxyl group ; nitroxyl has been reported to be involved in positive inotropic and vasodilation activities .", "these results support the evidence that hno / no produce positive inotropic and lusitropic effects ( independent of -adrenergic stimulation ) in a failing heart .", "most recently , a group of researchers studied the role of h2s in hypertension along with diabetes and emphasized that h2s improves the renal blood flow by reducing renal vasculature resistance through vasodilation .", "the same group of researchers extended their study to investigate the role of h2s in cardiac protection in dihydroxycortisone acetate - induced hypertension and shr combined with diabetes .", "in view of the above - mentioned literature review , many things remain to be explored and many questions still to be answered .", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect?concentration of h2s decreased in hypertension .", "is it a cause or consequence of the disease?h2s acts by vasodilatation , so  blocking effect can be investigatedbh4 and h2s effects in hypertension can be studiedthe molecular mechanism of h2s can be studied using reverse transcription polymerase chain reactionsome selective inhibitors can be introduced to avoid inhibition of unwanted enzymesreleasers of h2s introduced as present releasers are inconsistent and have sustained - release property . \n ", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect ?", "h2s acts by vasodilatation , so  blocking effect can be investigated bh4 and h2s effects in hypertension can be studied the molecular mechanism of h2s can be studied using reverse transcription polymerase chain reaction some selective inhibitors can be introduced to avoid inhibition of unwanted enzymes releasers of h2s introduced as present releasers are inconsistent and have sustained - release property .", "in view of the above - mentioned literature review , many things remain to be explored and many questions still to be answered .", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect?concentration of h2s decreased in hypertension .", "is it a cause or consequence of the disease?h2s acts by vasodilatation , so  blocking effect can be investigatedbh4 and h2s effects in hypertension can be studiedthe molecular mechanism of h2s can be studied using reverse transcription polymerase chain reactionsome selective inhibitors can be introduced to avoid inhibition of unwanted enzymesreleasers of h2s introduced as present releasers are inconsistent and have sustained - release property . \n ", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect ?", "h2s acts by vasodilatation , so  blocking effect can be investigated bh4 and h2s effects in hypertension can be studied the molecular mechanism of h2s can be studied using reverse transcription polymerase chain reaction some selective inhibitors can be introduced to avoid inhibition of unwanted enzymes releasers of h2s introduced as present releasers are inconsistent and have sustained - release property .", "in view of the above - mentioned literature review , many things remain to be explored and many questions still to be answered .", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect?concentration of h2s decreased in hypertension .", "is it a cause or consequence of the disease?h2s acts by vasodilatation , so  blocking effect can be investigatedbh4 and h2s effects in hypertension can be studiedthe molecular mechanism of h2s can be studied using reverse transcription polymerase chain reactionsome selective inhibitors can be introduced to avoid inhibition of unwanted enzymesreleasers of h2s introduced as present releasers are inconsistent and have sustained - release property . \n ", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect ?", "h2s acts by vasodilatation , so  blocking effect can be investigated bh4 and h2s effects in hypertension can be studied the molecular mechanism of h2s can be studied using reverse transcription polymerase chain reaction some selective inhibitors can be introduced to avoid inhibition of unwanted enzymes releasers of h2s introduced as present releasers are inconsistent and have sustained - release property ."], "abstract_text": ["<S> in the family of gas transmitters , hydrogen sulfide ( h2s ) is yet not adequately researched . </S>", "<S> known for its rotten egg smell and adverse effects on the brain , lungs , and kidneys for more than 300 years , the vasorelaxant effects of h2s on blood vessel was first observed in 1997 . since then , research continued to explore the possible therapeutic effects of h2s in hypertension , inflammation , pancreatitis , different types of shock , diabetes , and heart failure . </S>", "<S> however , a considerable amount of efforts are yet needed to elucidate the mechanisms involved in the therapeutic effects of h2s , such as nitric oxide - dependent or independent vasodilation in hypertension and regression of left ventricular hypertrophy . </S>", "<S> more than a decade of good repute among researchers , h2s research has certain results that need to be clarified or reevaluated . </S>", "<S> h2s produces its response by multiple modes of action , such as opening the atp - sensitive potassium channel , angiotensin - converting enzyme inhibition , and calcium channel blockade . </S>", "<S> h2s is endogenously produced from two sulfur - containing amino acids l - cysteine and l - methionine by the two enzymes cystathionine  lyase and cystathionine  synthase . </S>", "<S> recently , the third enzyme , 3-mercaptopyruvate sulfur transferase , along with cysteine aminotransferase , which is similar to aspartate aminotransferase , has been found to produce h2s in the brain . </S>", "<S> the h2s has interested researchers , and a great deal of information is being generated every year . </S>", "<S> this review aims to provide an update on the developments in the research of h2s in hypertension amid the ambiguity in defining the exact role of h2s in hypertension because of insufficient number of research results on this area . </S>", "<S> this critical review on the role of h2s in hypertension will clarify the gray areas and highlight its future prospects . </S>"], "labels": null, "section_names": ["Introduction", "History and Background of Hydrogen Sulfide", "Role of Hydrogen Sulfide in Hypertension", "None", "Future Prospects"], "sections": [["a number of gases are produced endogenously in humans and have roles in the pathology of various diseases . among these gases , nitric oxide ( no ) and carbon monoxide ( co ) have vital roles in the physiology of the body . since the last decade", ", hydrogen sulfide ( h2s ) has captured the interest of scientists because of its significant role in different systems of the body .", "h2s has been known as a toxic gas with rotten egg smell for more than 300 years . as a toxicant ,", "some studies have also reported the toxic effects of h2s on the central nervous system ( cns ) and respiratory system .", "h2s is endogenously produced from two sulfur - containing amino acids l - cysteine and l - methionine by the two enzymes cystathionine  lyse ( cse ) and cystathionine  synthase ( cbs ) as shown in figure 1a and b. recently , a third enzyme , namely , 3-mercaptopyruvate sulfur transferase ( 3mst ) , along with cysteine aminotransferase ( cat ) , which is similar to aspartate amino transferase , has been shown to produce h2s in brain .", "3mst is produced by schematic chain reaction from 3-mercaptopyruvate which is also produced by cat from cysteine and -ketoglutarate[figure 1 ] . \n ", "( a - c ) the schematic presentation of h2s production in the mammalians body by involving three different pathways both the enzymes cse and cbs are present in mammalian cells and tissues .", "previously , cse has been reported to be responsible for the production of h2s in the cardiovascular system ( cvs ) and kidneys , whereas cbs performs the same function in cns .", "few studies have supported that cse is dominant in cvs , whereas cbs is dominant in cns .", "both cbs and cse have been reported to be present in the kidneys , predominantly in predominantly cortical thymoma .", "however , the mechanism of its production still remains unclear ; whether or not the production of h2s is induced by cse or cbs .", "expression of both enzymes in the kidneys can lead us to conclude about the production of h2s by both enzymes .", "the third enzyme , 3mst , is responsible for h2s production and has also been reported to be present in endothelial cells in the thoracic aorta .", "recent studies have shown that greater than 90% of the total h2s is produced in the brain by 3mst .", "based on the literature , different enzymes are involved in the production of h2s in different parts of body ; for example , h2s is predominantly produced in the heart by cse , both cbs and cse in the kidneys , and 3mst in the brain by .", "dl - propargylglycine ( dl - pag ) is an inhibitor of cse , whereas aminooxyacetic acid is an inhibitor of cbs .", "both inhibitors are nonselective and may be responsible for the inhibition of other enzymes . some selective inhibitors can elaborate the mechanisms of h2s . some mechanisms for long - term control production of h2s are coming to light .", "long - term regulation of h2s seems to be dependent on s - adenosyl - l  methionine , which activates cbs and ultimately leads to the production of h2s[figure 1c ] .", "the endogenous concentration of circulating h2s is 50160 m in rats , bovines , and humans .", "h2s hyperpolarizes the membranes of localized cells , modulates the neuronal excitability , relaxes the smooth muscles , and controls cell apoptosis or proliferation . in one study , normal concentration of h2s in wistar kyoto ( wky ) rats was measured at 10 m ; however , other studies have demonstrated that the plasma level of h2s is 50 m", ". the tissue level of h2s has been thought to be higher than its plasma level .", "for example , the physiological concentration of h2s in the brain has been documented at 50160 m .", "the h2s level decreased below the normal level in the body if coronary heart disease spontaneously hypertensive rats ( shrs ) ; however , the level increased in diabetes and circulatory shock . in carrageenan - induced inflammation model , concentration is increased .", "increase in the concentration of h2s has also been observed in acute pancreatitis , hemorrhagic shock , and in endotoxin shock .", "the vasorelaxant effect of h2s has been proven , which shows that h2s relaxes the isolated aorta at a concentration as low as 18 m and 60 m if pretreated with 20 mm kcl or phe ."], ["a number of gases are produced endogenously in humans and have roles in the pathology of various diseases . among these gases , nitric oxide ( no ) and carbon monoxide ( co ) have vital roles in the physiology of the body . since the last decade", ", hydrogen sulfide ( h2s ) has captured the interest of scientists because of its significant role in different systems of the body .", "h2s has been known as a toxic gas with rotten egg smell for more than 300 years . as a toxicant ,", "some studies have also reported the toxic effects of h2s on the central nervous system ( cns ) and respiratory system .", "h2s is endogenously produced from two sulfur - containing amino acids l - cysteine and l - methionine by the two enzymes cystathionine  lyse ( cse ) and cystathionine  synthase ( cbs ) as shown in figure 1a and b. recently , a third enzyme , namely , 3-mercaptopyruvate sulfur transferase ( 3mst ) , along with cysteine aminotransferase ( cat ) , which is similar to aspartate amino transferase , has been shown to produce h2s in brain .", "3mst is produced by schematic chain reaction from 3-mercaptopyruvate which is also produced by cat from cysteine and -ketoglutarate[figure 1 ] . \n ", "( a - c ) the schematic presentation of h2s production in the mammalians body by involving three different pathways both the enzymes cse and cbs are present in mammalian cells and tissues .", "previously , cse has been reported to be responsible for the production of h2s in the cardiovascular system ( cvs ) and kidneys , whereas cbs performs the same function in cns .", "few studies have supported that cse is dominant in cvs , whereas cbs is dominant in cns .", "both cbs and cse have been reported to be present in the kidneys , predominantly in predominantly cortical thymoma .", "however , the mechanism of its production still remains unclear ; whether or not the production of h2s is induced by cse or cbs .", "expression of both enzymes in the kidneys can lead us to conclude about the production of h2s by both enzymes .", "the third enzyme , 3mst , is responsible for h2s production and has also been reported to be present in endothelial cells in the thoracic aorta .", "recent studies have shown that greater than 90% of the total h2s is produced in the brain by 3mst .", "based on the literature , different enzymes are involved in the production of h2s in different parts of body ; for example , h2s is predominantly produced in the heart by cse , both cbs and cse in the kidneys , and 3mst in the brain by .", "dl - propargylglycine ( dl - pag ) is an inhibitor of cse , whereas aminooxyacetic acid is an inhibitor of cbs .", "both inhibitors are nonselective and may be responsible for the inhibition of other enzymes . some selective inhibitors can elaborate the mechanisms of h2s . some mechanisms for long - term control production of h2s are coming to light .", "long - term regulation of h2s seems to be dependent on s - adenosyl - l  methionine , which activates cbs and ultimately leads to the production of h2s[figure 1c ] .", "the endogenous concentration of circulating h2s is 50160 m in rats , bovines , and humans .", "h2s hyperpolarizes the membranes of localized cells , modulates the neuronal excitability , relaxes the smooth muscles , and controls cell apoptosis or proliferation . in one study , normal concentration of h2s in wistar kyoto ( wky ) rats was measured at 10 m ; however , other studies have demonstrated that the plasma level of h2s is 50 m", ". the tissue level of h2s has been thought to be higher than its plasma level .", "for example , the physiological concentration of h2s in the brain has been documented at 50160 m .", "the h2s level decreased below the normal level in the body if coronary heart disease spontaneously hypertensive rats ( shrs ) ; however , the level increased in diabetes and circulatory shock . in carrageenan - induced inflammation model , concentration is increased .", "increase in the concentration of h2s has also been observed in acute pancreatitis , hemorrhagic shock , and in endotoxin shock .", "the vasorelaxant effect of h2s has been proven , which shows that h2s relaxes the isolated aorta at a concentration as low as 18 m and 60 m if pretreated with 20 mm kcl or phe ."], ["the role of h2s seems to be partially solved by evaluating different mechanisms ; however , researchers have shifted their interest to other gaseous transmitters , such as no and carbon monoxide .", "studying the role of h2s in hypertension chronologically , we were first informed that h2s produces vasorelaxation on rat aortic tissue in vitro . in succeeding research , the vasorelaxant effect of h2s", "has been attributed to the opening of the atp - sensitive potassium ( katp ) channel ; this effect was mimicked by pinacidil ( a katp channel agonist ) and antagonized by glibenclamide ( a katp channel antagonist ) .", "blood pressure reduction by intravenous ( iv ) bolus injection of h2s was 1230 mm of hg .", "the study under discussion revealed that aortic rings were relaxed by 63%  2.2% at a dose of 180 m , if pretreated with phe .", "the vasorelaxation of h2s was partially dependent on endothelium and mostly through direct effects on the smooth muscle cells . to further exclude the involvement of no ,", "the vasorelaxant effects of h2s are not blocked by 1h-(1,2,4)oxadiazolo[4,3-a ] quinoxalin-1-one ( odq ) ( an inhibitor of guanylyl cyclase ) .", "the katp channel mechanism was confirmed by inducing contractions in the aortic tissue through treating the tissue with low k ( 20 mm ) and high k ( 100 mm ) ; vasorelaxation produced by h2s on aortic tissues was observed when pretreated with low k ( 20 mm ) and high k ( 100 mm ) .", "the maximum vasorelaxation produced by h2s was 90%  8.2% and 19%  3.9% when pretreated with low k ( 20 mm ) and high k ( 100 mm ) , respectively .", "hydrogen sulfide produces more relaxation with low k ( 20 mm ) because of k conductance .", "the k channel opener effect was further verified using 10 mm tetraethylammonium and 100 nm of charybdotoxin or 100 nm iberiotoxin ( specific inhibitors of kca channel ) , completely inhibiting h2s - induced relaxation . in upcoming years", ", the vasorelaxation produced by h2s was proven to be through a different mechanism than that of no and co. furthermore , the vasorelaxation effect on the vascular tissue by no is reduced when pretreated with h2s .", "this study also suggests that an additive response can be achieved using no and h2s .", "a study elaborated that the cardioprotective action of h2s mediated by katp channel opening is caused by the physiological production of h2s in the heart .", "this study has proven the negative inotropic effects of h2s both in vivo and in vitro , and concluded that h2s can effectively prevent hypertension in rats when induced by l - n - nitro arginine methyl ester . nitric oxide and", "deficiency in no and co has been proven to contribute to the pathogenesis of hypertension .", "h2s has also been reported to play the same role in hypertension because of similar biological activities . in subsequent studies , the level of h2s in the plasma and its production rate in seriously hypertensive rats ( shr ) was lower than that in wky rats . therefore , cse is a specific enzyme for h2s production in the thoracic aorta and its decreased activity in hypertension may lead to less production of h2s , resulting in decreased circulating level of h2s .", "the above - mentioned theory proposes that less activity and decreased transcription of cse results in decreased circulating aortic h2s level and that vasoconstriction is a dominant phenomenon over vasorelaxant one .", "sodium hydrogen sulfide ( nahs ) has been selected as an exogenous source ofh2s because of to four reasons .", "( 1 ) na dissociates from hs in a nahs solution , then hs associates with h and produces h2s , regardless if the h2s solution was prepared by bubbling h2s gas or dissolving nahs . in physiological saline ,", "about one - third of h2s exists in the undissociated form ( h2s ) , and the remaining two - thirds is hs - at equilibrium with h2s .", "( 2 ) nahs enables a more accurate and reproducible measurement of h2s concentrations in a solution than by bubbling h2s gas . ( 3 ) the influence of 1 mm or less sodium ion on the physiological experiments is negligible .", "( 4 ) nahs at concentrations used in the current study does not change the ph of the medium .", "the results of one study suggest that upregulation of h2s results in the reduction of sbp in the shr + nahs group ( 158.13  12.52", "mm vs. 183.57  11.8 mm of hg ) . a study demonstrated that an iv bolus injection of h2s at 2.8 mol / kg and 14 mol / kg body weight results in a decrease in the mean arterial blood pressure of rats by 12.5  2.1 mmhg and 29.8  7.6 mmhg , respectively .", "therefore , the physiological concentration of h2s is responsible for maintaining the mean arterial blood pressure .", "however , the expression of cse activity decreases with the onset of disease . based on the findings , the finding that the physiological concentration of h2s is responsible for the normal function of cvs is a matter of great interest .", "diseases like hypertension occur when the concentration of h2s decreases ; however , the opposite occurs in hemorrhagic shock . in a study , induction of hemorrhagic shock results in a prolonged decrease in the mean arterial pressure ( map ) and heart rate ( hr ) .", "however , data suggest that vasoconstriction is responsible for a hemorrhagic shock because of vasopressin , noradrenaline , and angiotensin ii .", "a reasonable number of previous studies have shown that excessive formation of inducible nitric oxide synthase ( inos ) in hypotension is responsible for hemorrhagic shock ; thus , the concentration of h2s increases in the induction of hemorrhagic shock .", "treatment with inhibitors of h2s - producing enzymes cse and dl - pag , a suicidal inhibitor , resulted in the rapid and partial restoration of map and hr .", "previously , glibenclamide ( a k channel blocker ) has been proven to perform the same partial but equally effective function after hemorrhagic shock in a rat .", "a comparative study was conducted to compare the effect of h2s with pag and -cyano - l - alanine , a reversible inhibitor of cse , on the blood pressure of rats subjected to hemorrhagic shock .", "after many years of earning good repute among researchers , h2s research started to intermingle with each other .", "two different schools of thought exist between interchangeable production of h2s and no . in 1997", ", a study has proven that a low concentration of h2s increases the relaxation of smooth muscles by 13-folds .", "this study also explained that low concentration ( 30 m ) of h2s enhances the vasodilator effect of no .", "therefore , a synergistic response between h2s and no can be the therapeutic outcome in hypertension .", "this study further elaborated that no - induced vasorelaxation is specifically for h2s , but no can not potentiate the vasorelaxant effect of h2s .", "another study has proven that no is responsible for the upregulated production of h2s in rats in a dose - dependent manner ( 1100 m ) , as shown in the following figure .", "this study claims that production of h2s by no is narrated by two mechanisms : ( 1 ) no increases cse activity , which ultimately leads to the production of h2s , and ( 2 ) no increases the activity of protein kinase , which is dependent on cyclic guanosine monophosphate , thereby increasing cse protein .", "a growing body of evidence has proven that h2s as a cofactor is responsible for the generation of no from nitrite . in his previous studies in 2002 , documented the interaction between hydrogen sulfide and no resulting in the formation of the intermediate compound nitrosothiol , which releases no through hemolysis . in his next experimentation , proposed the mechanism of no production from sodium nitroprusside . in 2006 , has proven that the interaction between two gasotransmitters results in the formation of nitrosothiol and the release of only a small portion of no unless an antioxidant is involved .", "these findings contradict those of other studies , which demonstrate that the interaction between no and h2s enhances vasodilatation . in the battle of the interchangeable production of h2s and no ,", "another finding cropped up , supporting the claim that h2s is responsible for the direct inhibition of endothelial nitric oxide synthase ( enos ) .", "this theory is born out of the result of the first school of thought , which proposed that no can potentiate the response of h2s , but no can do this .", "has elaborated that h2s is responsible for the inhibition of enos , as well as inos and nnos .", "they further strengthened the results of their study by explaining that h2s and tetrahydrobiopterin ( bh4 ) reverse the h2s inhibition of enos and nnos , but not inos .", "a study in 2006 elaborated that the intermediate complex formed from the interaction between h2s and no has no vasorelaxant effect ; h2s was responsible for the regulation of no .", "this study concluded that h2s inhibits the vasorelaxant effect of no . a decrease in h2s concentration results in over stimulation of  adrenoreceptor .", "the h2s has also been reported to inhibit the l - type ca channels in cardiomyocyte , which leads to a decrease in contractility , thereby revealing the ca channel - blocking mechanism of h2s . in the same year", ", h2s has been found to have the ability to block the angiotensin - converting enzyme and responsible for additive vasorelaxant response leading to the inhibition of angiotensin ii production , ultimately resulting in reduced degradation of bradykinin . in a related study in 2010 , reported that enos and neuronal nitric oxide synthase ( nos ) activation is important to produce a cardioprotective response .", "the cardioprotective response of the nos family mentioned in is contrary to the results by .", "another area of interest is the formation of the intermediate complex nitrosothiol , which is responsible for the vasorelaxant effect in the blood vessels through interaction between two gasotransmitters , h2s and no .", "the presence of this intermediate molecule , nitrosothiol , has not been fully confirmed and has been considered as a possible molecule for the said action .", "a previously mentioned study has stated that the intermediate molecule is nitrosothiol but has no vasorelaxant effect in vivo and in vitro .", "another school of thought has reported that the interaction between no and h2s leads to the formation of the nitroxyl group ; nitroxyl has been reported to be involved in positive inotropic and vasodilation activities .", "these results support the evidence that hno / no produce positive inotropic and lusitropic effects ( independent of -adrenergic stimulation ) in a failing heart .", "most recently , a group of researchers studied the role of h2s in hypertension along with diabetes and emphasized that h2s improves the renal blood flow by reducing renal vasculature resistance through vasodilation .", "the same group of researchers extended their study to investigate the role of h2s in cardiac protection in dihydroxycortisone acetate - induced hypertension and shr combined with diabetes .", "in view of the above - mentioned literature review , many things remain to be explored and many questions still to be answered .", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect?concentration of h2s decreased in hypertension .", "is it a cause or consequence of the disease?h2s acts by vasodilatation , so  blocking effect can be investigatedbh4 and h2s effects in hypertension can be studiedthe molecular mechanism of h2s can be studied using reverse transcription polymerase chain reactionsome selective inhibitors can be introduced to avoid inhibition of unwanted enzymesreleasers of h2s introduced as present releasers are inconsistent and have sustained - release property . \n ", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect ?", "h2s acts by vasodilatation , so  blocking effect can be investigated bh4 and h2s effects in hypertension can be studied the molecular mechanism of h2s can be studied using reverse transcription polymerase chain reaction some selective inhibitors can be introduced to avoid inhibition of unwanted enzymes releasers of h2s introduced as present releasers are inconsistent and have sustained - release property ."], ["in view of the above - mentioned literature review , many things remain to be explored and many questions still to be answered .", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect?concentration of h2s decreased in hypertension .", "is it a cause or consequence of the disease?h2s acts by vasodilatation , so  blocking effect can be investigatedbh4 and h2s effects in hypertension can be studiedthe molecular mechanism of h2s can be studied using reverse transcription polymerase chain reactionsome selective inhibitors can be introduced to avoid inhibition of unwanted enzymesreleasers of h2s introduced as present releasers are inconsistent and have sustained - release property . \n ", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect ?", "h2s acts by vasodilatation , so  blocking effect can be investigated bh4 and h2s effects in hypertension can be studied the molecular mechanism of h2s can be studied using reverse transcription polymerase chain reaction some selective inhibitors can be introduced to avoid inhibition of unwanted enzymes releasers of h2s introduced as present releasers are inconsistent and have sustained - release property ."], ["in view of the above - mentioned literature review , many things remain to be explored and many questions still to be answered .", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect?concentration of h2s decreased in hypertension .", "is it a cause or consequence of the disease?h2s acts by vasodilatation , so  blocking effect can be investigatedbh4 and h2s effects in hypertension can be studiedthe molecular mechanism of h2s can be studied using reverse transcription polymerase chain reactionsome selective inhibitors can be introduced to avoid inhibition of unwanted enzymesreleasers of h2s introduced as present releasers are inconsistent and have sustained - release property . \n ", "future studies must verify whether the intermediate molecule formed as a result of interaction between no and h2s is nitroxyl or nitrosthiol or both .", "if both molecules are present ; then , which one is responsible for the vasorelaxant effect ?", "h2s acts by vasodilatation , so  blocking effect can be investigated bh4 and h2s effects in hypertension can be studied the molecular mechanism of h2s can be studied using reverse transcription polymerase chain reaction some selective inhibitors can be introduced to avoid inhibition of unwanted enzymes releasers of h2s introduced as present releasers are inconsistent and have sustained - release property ."]]}
{"article_id": "PMC3466900", "article_text": ["ischemic heart disease is rare during pregnancy , occurring in approximately one in 10,000 live births .", "the risk of myocardial infarction ( mi ) in pregnancy is reported from one per 37,500 to 6.2 per 100,000 deliveries.13 the diagnosis is by clinical findings , electrocardiogram ( ecg ) , and measurement of the serum level of the cardiac specific contractile protein , troponin 1.4 , 5 the most common forms of angina are stable and unstable angina , which are usually due to atherosclerosis . but coronary spasm , coronary dissection , and thrombus have been reported as other causes.6 , 7 prinzmetal s angina ( variant angina ) is rare and it accounts for only 2 out of every 100 cases of angina .", "we report a pregnant woman who presented with signs and symptoms of acute mi and whose ecg findings and troponin 1 level were compatible with mi , leading to the final diagnosis of prinzmetal s angina .", "a 35-year - old woman , gravid 3 ( with a history of two cesarean section operations ) , developed acute mi at the 30 week of gestation .", "she was a non - smoker and had no history of drug abuse or systemic disease .", "she referred to the emergency ward due to a sudden , severe pain in the neck and between the two scapulas that had started during sleep at 5 am , with cold sweat and nausea .", "her blood pressure was 200/150 mm hg ; she had a history of increased blood pressure of two weeks duration .", "the patient also had tachycardia , rales in the bases of the lungs , signs of anteroseptal mi in the ecg ( figure 1 ) , and increased troponin 1 blood level .", "we performed a cesarean section operation because of severe preeclampsia , repeat cesarean section , and transverse lie .", "four days after delivery , the patient s blood pressure was 140/80 mmhg and coronary angiography showed total occlusion of the second obtuse marginal artery ( om2 ) in addition to diffuse spasm of the left circumflex coronary artery ( lcx ) ( figure 2 ) .", "at the seventh day after delivery in the ward , the patient had chest pain and st - segment elevation in the inferior leads ( figure 3 ) as well as signs of pulmonary edema .", "the ecg changes reversed after intravenous trinitroglycerin ( figure 4 ) . based on the ecg , the diagnosis was inferior wall ischemia .", "we performed angiography for a second time and surprisingly the lcx and om2 coronary arteries were normal and there was narrowing in the rca ( figure 5 ) .", "we conclude that this discrepancy between the first and second ecgs and angiographic findings were due to prinzmetal s angina .", "a few days later , the patient was discharged ( blood pressure = 130/80 mmhg ) on diltiazem , atorvastatin , captopril , nitrocontin , furosemide , and clopidogrel while the ecg showed previous anteroseptal mi ( figure 6 ) .", "an ecg three months after discharge showed the same findings too ( figure 7 ) .", "mi in pregnancy is rare but can produce significant maternal and neonatal morbidity . prompt diagnosis and immediate therapy are necessary to lower the high likelihood of the mortality of mother and fetus .", "the highest incidence of mi seems to occur in the third trimester and in multigravidas older than 33 years old .", "acute mi in pregnancy is commonly located in the anterior wall , and acute maternal death rate is reported to be 1930%.13 , 8 complications of pregnancy that are significantly associated with acute mi are preeclampsia , postpartum hemorrhage , postpartum infection , and fluid and electrolyte imbalance.1 , 9 shock from postpartum hemorrhage,10 ergonovine,11 and prostaglandin e1 ( pge1)12 is reported as a risk factors for mi in pregnancy . our patient was a 35-year - old , obese , multiparous , pregnant woman with preeclampsia , and her symptoms of mi appeared at rest in the early hours of morning .", "prinzmetal s angina usually occurs at rest and happens between the midnight and early morning .", "people with prinzmetal s angina are often younger than those with the other forms of angina .", "the gold standard for diagnosis is coronary angiography with injection of provocative agents ( like ergonovine ) into the coronary arteries .", ". also administration of intracoronary nitroglycerin in cases of vasospasm can cause marked diffuse vasodilatation.13 treatment with calcium - channel blockers or nitrates eliminates spasm in most of these patients .", "in our patient , the discrepancy between the first and second angiographic findings was the confirmatory evidence that she had prinzmetal s angina .", "this conclusion was derived from the fact that the first angiographic examination showed that the lad coronary artery was normal despite the definite occurrence of an mi in its territory , thus suggesting transient spasm .", "furthermore , a subsequent bout of inferior wall ischemia with confirmation of transient spasm by angiography was another piece of confirmatory evidence .", "last but not least , asymptomatic rca spasm , detected in the last angiography , was another clue to the propensity of the coronary arteries to vasospasm , which is the sine qua non of prinzmetal s angina ."], "abstract_text": ["<S> acute myocardial infarction ( mi ) during pregnancy is rare and mi due to prinzmetal s angina is much rarer . </S>", "<S> we present a 35-year - old , obese , multigravida , and pre - eclamptic woman , who developed acute anterior wall mi at the 30th week of gestation . on coronary angiography , </S>", "<S> the second obtuse marginal branch was totally occluded and the right coronary artery ( rca ) was normal . </S>", "<S> three days later , she had chest pain and st elevation in the inferior leads . on second angiography , there was narrowing in the rca , while the obtuse marginal branch was patent . </S>", "<S> we presume that this discrepancy between the first and second electrocardiograms and angiographic findings was due to prinzmetal s angina . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["ischemic heart disease is rare during pregnancy , occurring in approximately one in 10,000 live births .", "the risk of myocardial infarction ( mi ) in pregnancy is reported from one per 37,500 to 6.2 per 100,000 deliveries.13 the diagnosis is by clinical findings , electrocardiogram ( ecg ) , and measurement of the serum level of the cardiac specific contractile protein , troponin 1.4 , 5 the most common forms of angina are stable and unstable angina , which are usually due to atherosclerosis . but coronary spasm , coronary dissection , and thrombus have been reported as other causes.6 , 7 prinzmetal s angina ( variant angina ) is rare and it accounts for only 2 out of every 100 cases of angina .", "we report a pregnant woman who presented with signs and symptoms of acute mi and whose ecg findings and troponin 1 level were compatible with mi , leading to the final diagnosis of prinzmetal s angina ."], ["a 35-year - old woman , gravid 3 ( with a history of two cesarean section operations ) , developed acute mi at the 30 week of gestation .", "she was a non - smoker and had no history of drug abuse or systemic disease .", "she referred to the emergency ward due to a sudden , severe pain in the neck and between the two scapulas that had started during sleep at 5 am , with cold sweat and nausea .", "her blood pressure was 200/150 mm hg ; she had a history of increased blood pressure of two weeks duration .", "the patient also had tachycardia , rales in the bases of the lungs , signs of anteroseptal mi in the ecg ( figure 1 ) , and increased troponin 1 blood level .", "we performed a cesarean section operation because of severe preeclampsia , repeat cesarean section , and transverse lie .", "four days after delivery , the patient s blood pressure was 140/80 mmhg and coronary angiography showed total occlusion of the second obtuse marginal artery ( om2 ) in addition to diffuse spasm of the left circumflex coronary artery ( lcx ) ( figure 2 ) .", "at the seventh day after delivery in the ward , the patient had chest pain and st - segment elevation in the inferior leads ( figure 3 ) as well as signs of pulmonary edema .", "the ecg changes reversed after intravenous trinitroglycerin ( figure 4 ) . based on the ecg , the diagnosis was inferior wall ischemia .", "we performed angiography for a second time and surprisingly the lcx and om2 coronary arteries were normal and there was narrowing in the rca ( figure 5 ) .", "we conclude that this discrepancy between the first and second ecgs and angiographic findings were due to prinzmetal s angina .", "a few days later , the patient was discharged ( blood pressure = 130/80 mmhg ) on diltiazem , atorvastatin , captopril , nitrocontin , furosemide , and clopidogrel while the ecg showed previous anteroseptal mi ( figure 6 ) .", "an ecg three months after discharge showed the same findings too ( figure 7 ) ."], ["mi in pregnancy is rare but can produce significant maternal and neonatal morbidity . prompt diagnosis and immediate therapy are necessary to lower the high likelihood of the mortality of mother and fetus .", "the highest incidence of mi seems to occur in the third trimester and in multigravidas older than 33 years old .", "acute mi in pregnancy is commonly located in the anterior wall , and acute maternal death rate is reported to be 1930%.13 , 8 complications of pregnancy that are significantly associated with acute mi are preeclampsia , postpartum hemorrhage , postpartum infection , and fluid and electrolyte imbalance.1 , 9 shock from postpartum hemorrhage,10 ergonovine,11 and prostaglandin e1 ( pge1)12 is reported as a risk factors for mi in pregnancy . our patient was a 35-year - old , obese , multiparous , pregnant woman with preeclampsia , and her symptoms of mi appeared at rest in the early hours of morning .", "prinzmetal s angina usually occurs at rest and happens between the midnight and early morning .", "people with prinzmetal s angina are often younger than those with the other forms of angina .", "the gold standard for diagnosis is coronary angiography with injection of provocative agents ( like ergonovine ) into the coronary arteries .", ". also administration of intracoronary nitroglycerin in cases of vasospasm can cause marked diffuse vasodilatation.13 treatment with calcium - channel blockers or nitrates eliminates spasm in most of these patients ."], ["in our patient , the discrepancy between the first and second angiographic findings was the confirmatory evidence that she had prinzmetal s angina .", "this conclusion was derived from the fact that the first angiographic examination showed that the lad coronary artery was normal despite the definite occurrence of an mi in its territory , thus suggesting transient spasm .", "furthermore , a subsequent bout of inferior wall ischemia with confirmation of transient spasm by angiography was another piece of confirmatory evidence .", "last but not least , asymptomatic rca spasm , detected in the last angiography , was another clue to the propensity of the coronary arteries to vasospasm , which is the sine qua non of prinzmetal s angina ."]]}
{"article_id": "PMC5353406", "article_text": ["moderate or severe mitral regurgitation ( mr ) is the most frequent valve disease in the usa and the second most common form of valvular heart disease needing surgery in europe", ". degenerative mitral valve disease ( mvd ) is the leading cause of organic mr in western countries .", "its mechanism is primarily characterized by mitral valve prolapse ( mvp ) due to excessive leaflet movement ( 2 mm abnormal systolic movement beyond the saddle - shaped annular level ) , as defined by carpentier ( type ii ) .", "the anatomic lesions associated with the degenerative mr etiology encompass a wide pathological spectrum , from fibroelastic deficiency ( fed ) to extensive myxomatous disease .", "carpentier described fed as a condition associated with a fibrillin deficiency that often leads to the rupture of one or more thinned and elongated chordae .", "this process usually , but not exclusively , involves the middle scallop of the posterior leaflet with the myxomatous degeneration limited to the prolapsing segment .", "in contrast to fed , myxomatous disease , often called barlow disease ( bd ) , is characterized by diffuse excess tissue , with large valves comprising multiple degenerated segments presenting as thick , mostly with elongated chordae ; severe mitral annulus ( ma ) dilatation associated with different degrees of annular calcification is a frequent finding in patients with bd , and subvalvular fibrosis and calcification of the papillary muscles , usually the anterior , may also occur .", "myxomatous degeneration is defined by the accumulation of mucopolysaccharides responsible for the thickening and  proliferative aspect of valvular tissue .", "even if molecular disorders and pathophysiologic pathways may not be uniform , there is a continuum between the different anatomic aspects of degenerative mr .", "surgery is the treatment of choice in degenerative mr , improving symptoms and preventing heart failure . since the 1970s", ", mitral valve ( mv ) repair has emerged as the optimal treatment , preferred to traditional valve replacement , as it improves outcome and reduces mortality of patients with severe organic mr by about 70% .", "recent studies and guidelines have underlined the importance of early surgical intervention to preserve long - term left ventricular ( lv ) function in severe mr .", "indeed , the best short- and long - term results are obtained in asymptomatic patients operated in advanced repair centers with low operative mortality ( < 1% ) and high repair rates ( 80 - 90% ) .", "these results emphasize the importance of early detection and detailed assessment of valve morphology and mr .", "moreover , in the recent years there has been an increasing interest towards non - surgical solutions to severe mr , with the percutaneous mitraclip system , mimicking the surgical alfieri stitch by placing a clip on the beating heart , being the most well established , with results not inferior to surgical correction in selected patients .", "patient selection is a crucial step of the mitraclip procedure and several anatomic criteria should be fulfilled to ensure the suitability of the device to mv anatomy . in this complex scenario , echocardiography is the method of choice to evaluate patients with degenerative mr ; it allows the accurate assessment of mr severity , etiology , mechanisms and anatomic lesions , lv and left atrial remodeling , and consequently defines the indication and the probability of successful mv repair .", "three - dimensional ( 3d ) echocardiographic imaging has represented a major innovation in cardiovascular ultrasound .", "recent guidelines stated that real - time three - dimensional ( rt3d ) imaging modalities are useful in the presentation of realistic views of heart valves and are ideal for interrogating the anatomy and function of each of the individual components of the mv apparatus , including annulus , leaflets , chordae and papillary muscles . both qualitative and quantitative evaluations of degenerative mvd", "all publications considered for this review were identified accessing the medline database via pubmed . the search strategy employed a number of free - text keywords and controlled vocabulary terms including , but not limited to", ", the following concepts : 3d echocardiography ; mitral valve ; degenerative mitral valve disease ; mitral valve repair ; percutaneous mitral valve repair . fields of search were limited to the title and abstract and restricted to the english language .", "all the retrieved studies were screened for inclusion using a hierarchical approach assessing title , abstract and manuscript .", "additional searches were conducted after screening the references of all selected studies . as a direct consequence of a series of technological breakthrough , such as the introduction of the 3d matrix technology with its improved spatial resolution compared to previously available sparse array transducers , rt3d echocardiography demonstrated feasibility and accuracy in mv assessment .", "the enhanced visualization of morphologic and pathologic aspects of the mv apparatus in degenerative mr has contributed to our knowledge of mv pathology , with unquestionable benefits compared to traditional two - dimensional ( 2d ) echocardiography .", "qualitative morphological evaluation of the mvp may be accurately assessed both by 3d transthoracic echocardiography ( tte ) and transesophageal echocardiography ( tee ) and the recent data showed that rt3d , either tte or tee , is superior to the corresponding 2d techniques in the description of mv pathology [ figure 1 ] .", "example of head - to - head comparison between 3d tte ( left ) and 3d tee ( right ) datasets in two cases of a2 prolapse ( above ) and p2 prolapse ( below ) , imaged from the surgical view ( left atrium ) 3d evaluation may be easily integrated into a standard 2d examination in the assessment of qualitative morphology of the mv .", "indeed , the possibility of visualizing mv leaflets , commissures , annulus calcifications and subvalvular structures in different and unique planes , both from the atrium or ventricle , with access to  en face  views , enhances the understanding of this complex apparatus .", "diagnostic accuracy of rt3d tte vs surgical inspection in the identification of prolapsing segments has been investigated by tamborini et al .", "the overall accuracy of the method was high ( sensitivity 92.5% , specificity 96% , accuracy 95% ) ; sensitivity was slightly lower for antero - lateral commissure , p1 and a1 , while specificity was very high in all the segments .", "even focusing on the differences in rt3d tte accuracy in the setting of simple ( isolated p2 prolapse or p2 associated with p1 or p3 ) and complex cases ( lesions involving the entire posterior leaflet , lesions of the anterior leaflet , bileaflet prolapse , and commissural involvement ) , the accuracy was very high , 98% and 93% respectively , even if slightly lower in complex lesions .", "all these data were in accordance with previous findings . as it concerns rt3d tee , there is a substantial body of literature demonstrating the additional value of 3d over 2d in the evaluation of patients with mvp .", "the majority of these studies showed that 3d echocardiographic findings correlate closely to surgical findings , achieving an exact anatomic description in approximately 90% to 95% of segments , whereas 2d echocardiography is less accurate .", ", underlined that in agreement with previous works , the incremental value of 3d modality over conventional 2d can be better appreciated in complex disease involving both leaflets or the anterior leaflet alone .", "la canna et al . , demonstrated that rt3d tee provided more accurate mapping of mv prolapse than 2d tee and rt3d tte imaging , adding quantitative recognition of dominant and secondary lesions and mv anatomy details in 222 patients undergoing surgical repair for mr secondary to prolapse .", "recently , hien et al . , investigated whether the known benefit of rt3d tte over 2d tee imaging for the evaluation of mvp would be valuable among novice echocardiographers .", "they demonstrated that both expert and novice echocardiographers were able to provide more accurate descriptions of the mv prolapse with rt3d than with 2d tee images , and this benefit was three times more important for inexperienced operators . with the advances of rt3d echocardiography together with the development of dedicated rt3d software capable of providing several parameters ( annular diameters and area , annular height and planarity , saddle - shaped morphology , leaflet size , coaptation geometry ) , high - resolution , full - volume imaging and quantification of morphology of the entire mitral apparatus have become feasible [ figure 2 ] .", "several studies demonstrated that the use of rt3d allows improving the evaluation of the mv apparatus both qualitatively , as previously illustrated , but also quantitatively .", "in fact , the mv has a complex 3d morphology and attempts to use 2d images to comprehend a complex 3d structure have provided an incomplete picture .", "in particular , the normal ma is characterized by a nonplanar saddle shape that has been suggested to be important for alleviating the mechanical stress on mitral leaflets and chordae tendinae imposed by lv pressure . moreover ,", "the geometry of leaflet surface and coaptation , magnitude of leaflet billowing , and deformation of subvalvular structures are important morphological features in mvp and they can be only partially assessed with 2d imaging .", "chandra et al . , hypothesized that rt3de - derived measurements of valvular anatomy could be used to characterize mvd in an objective way .", "morphological analysis in the mvd group revealed a progressive increase in multiple parameters from normal subjects to fed to bd , allowing an accurate diagnosis of these entities .", "three - dimensional billowing height with a cutoff value of 1.0 mm differentiated mvd from normal subjects , and billowing volume with a cutoff value of 1.15 ml differentiated between fed and bd .", "these findings could support the adoption of preoperative automated clinical decision - making strategies based on reliable quantitative parameters , with important surgical implications .", "example of 3d tee and corresponding reconstruction of mitral annulus and leaflets in a patient with fibroelastic deficiency ( left ) and in two patients with barlow disease ( right ) . in the first case ,", "the two barlow cases with the same method are characterized by multiple scallop involvement of both leaflets identified by the red surfaces moreover , grewal et al .", ", investigated mitral annular size , shape , and motion 6 times over the cardiac cycle using rt3de in patients with degenerative mvd compared with normal subjects and patients with ischemic mr and demonstrated that degenerative mvd annulus is still a dynamic structure but behaves considerably different from normal patients with loss of early - systolic area contraction and saddle - shape deepening .", "maffessanti et al . , characterized the mv 3d morphology using rt3d tee in the presence of severe mr associated with fed or bd , immediately before and after mv repair , in comparison with a control group .", "they found that the annular enlargement after mr is not isotropic , being more pronounced in the anteroposterior and intercommissural directions rather than along the mitral height , resulting in flattening of the mv in both fed and bd groups with higher values in the bd group .", "postoperatively , annular diameters and mv area were significantly smaller compared with preoperative values ; similar changes were found in the exposed areas of both leaflets .", "more recently , lee et al . , demonstrated that patients with mvp - related mr had more dilated mitral annulus , a flattening of annular saddle shape , redundant leaflet surfaces , greater leaflet billow volume and billow height , longer lengths from papillary muscles to coaptation and more frequent chordal rupture compared to normal subjects and non - prolapse mr patients .", "interestingly , the association between annular flattening and the increased frequency of chordal rupture suggests that the loss of the annular saddle shape may predispose to chordal elongation and rupture as a result of excessive chordal tension .", "as a direct consequence of a series of technological breakthrough , such as the introduction of the 3d matrix technology with its improved spatial resolution compared to previously available sparse array transducers , rt3d echocardiography demonstrated feasibility and accuracy in mv assessment .", "the enhanced visualization of morphologic and pathologic aspects of the mv apparatus in degenerative mr has contributed to our knowledge of mv pathology , with unquestionable benefits compared to traditional two - dimensional ( 2d ) echocardiography .", "qualitative morphological evaluation of the mvp may be accurately assessed both by 3d transthoracic echocardiography ( tte ) and transesophageal echocardiography ( tee ) and the recent data showed that rt3d , either tte or tee , is superior to the corresponding 2d techniques in the description of mv pathology [ figure 1 ] .", "example of head - to - head comparison between 3d tte ( left ) and 3d tee ( right ) datasets in two cases of a2 prolapse ( above ) and p2 prolapse ( below ) , imaged from the surgical view ( left atrium ) 3d evaluation may be easily integrated into a standard 2d examination in the assessment of qualitative morphology of the mv .", "indeed , the possibility of visualizing mv leaflets , commissures , annulus calcifications and subvalvular structures in different and unique planes , both from the atrium or ventricle , with access to  en face  views , enhances the understanding of this complex apparatus .", "diagnostic accuracy of rt3d tte vs surgical inspection in the identification of prolapsing segments has been investigated by tamborini et al .", "the overall accuracy of the method was high ( sensitivity 92.5% , specificity 96% , accuracy 95% ) ; sensitivity was slightly lower for antero - lateral commissure , p1 and a1 , while specificity was very high in all the segments . even focusing on the differences in rt3d tte accuracy in the setting of simple ( isolated p2 prolapse or p2 associated with p1 or p3 ) and complex cases ( lesions involving the entire posterior leaflet , lesions of the anterior leaflet , bileaflet prolapse , and commissural involvement ) , the accuracy was very high , 98% and 93% respectively , even if slightly lower in complex lesions .", "all these data were in accordance with previous findings . as it concerns rt3d tee , there is a substantial body of literature demonstrating the additional value of 3d over 2d in the evaluation of patients with mvp .", "the majority of these studies showed that 3d echocardiographic findings correlate closely to surgical findings , achieving an exact anatomic description in approximately 90% to 95% of segments , whereas 2d echocardiography is less accurate .", ", underlined that in agreement with previous works , the incremental value of 3d modality over conventional 2d can be better appreciated in complex disease involving both leaflets or the anterior leaflet alone .", "la canna et al . , demonstrated that rt3d tee provided more accurate mapping of mv prolapse than 2d tee and rt3d tte imaging , adding quantitative recognition of dominant and secondary lesions and mv anatomy details in 222 patients undergoing surgical repair for mr secondary to prolapse .", "recently , hien et al . , investigated whether the known benefit of rt3d tte over 2d tee imaging for the evaluation of mvp would be valuable among novice echocardiographers .", "they demonstrated that both expert and novice echocardiographers were able to provide more accurate descriptions of the mv prolapse with rt3d than with 2d tee images , and this benefit was three times more important for inexperienced operators . with the advances of rt3d echocardiography together with the development of dedicated rt3d software capable of providing several parameters ( annular diameters and area , annular height and planarity , saddle - shaped morphology , leaflet size , coaptation geometry ) , high - resolution , full - volume imaging and quantification of morphology of the entire mitral apparatus have become feasible [ figure 2 ] .", "several studies demonstrated that the use of rt3d allows improving the evaluation of the mv apparatus both qualitatively , as previously illustrated , but also quantitatively .", "in fact , the mv has a complex 3d morphology and attempts to use 2d images to comprehend a complex 3d structure have provided an incomplete picture .", "in particular , the normal ma is characterized by a nonplanar saddle shape that has been suggested to be important for alleviating the mechanical stress on mitral leaflets and chordae tendinae imposed by lv pressure . moreover ,", "the geometry of leaflet surface and coaptation , magnitude of leaflet billowing , and deformation of subvalvular structures are important morphological features in mvp and they can be only partially assessed with 2d imaging .", "chandra et al . , hypothesized that rt3de - derived measurements of valvular anatomy could be used to characterize mvd in an objective way .", "morphological analysis in the mvd group revealed a progressive increase in multiple parameters from normal subjects to fed to bd , allowing an accurate diagnosis of these entities .", "three - dimensional billowing height with a cutoff value of 1.0 mm differentiated mvd from normal subjects , and billowing volume with a cutoff value of 1.15 ml differentiated between fed and bd .", "these findings could support the adoption of preoperative automated clinical decision - making strategies based on reliable quantitative parameters , with important surgical implications .", "example of 3d tee and corresponding reconstruction of mitral annulus and leaflets in a patient with fibroelastic deficiency ( left ) and in two patients with barlow disease ( right ) . in the first case ,", "the two barlow cases with the same method are characterized by multiple scallop involvement of both leaflets identified by the red surfaces moreover , grewal et al . ,", "investigated mitral annular size , shape , and motion 6 times over the cardiac cycle using rt3de in patients with degenerative mvd compared with normal subjects and patients with ischemic mr and demonstrated that degenerative mvd annulus is still a dynamic structure but behaves considerably different from normal patients with loss of early - systolic area contraction and saddle - shape deepening .", "maffessanti et al . , characterized the mv 3d morphology using rt3d tee in the presence of severe mr associated with fed or bd , immediately before and after mv repair , in comparison with a control group .", "they found that the annular enlargement after mr is not isotropic , being more pronounced in the anteroposterior and intercommissural directions rather than along the mitral height , resulting in flattening of the mv in both fed and bd groups with higher values in the bd group .", "postoperatively , annular diameters and mv area were significantly smaller compared with preoperative values ; similar changes were found in the exposed areas of both leaflets .", "more recently , lee et al . , demonstrated that patients with mvp - related mr had more dilated mitral annulus , a flattening of annular saddle shape , redundant leaflet surfaces , greater leaflet billow volume and billow height , longer lengths from papillary muscles to coaptation and more frequent chordal rupture compared to normal subjects and non - prolapse mr patients .", "interestingly , the association between annular flattening and the increased frequency of chordal rupture suggests that the loss of the annular saddle shape may predispose to chordal elongation and rupture as a result of excessive chordal tension .", "the prognosis and the best timing for surgical treatment in degenerative mvd strongly depend on the accurate quantification of mr severity , which claims for accurate , feasible and reproducible techniques . despite these premises ,", "recent technological development has made rt3d echocardiography the ideal technique for the evaluation of mr severity , in particular , in all those cases where the hypotheses at the basis of 2d estimate are not met .", "mr grading can be obtained , in accordance with standard 2d echocardiography , by means of vena contracta area ( vca ) , effective regurgitant orifice area ( eroa ) using 3d proximal isovelocity surface area ( pisa ) or mr volume measurement [ figure 3 ] . detailed and comprehensive discussions on mr quantitative evaluation", "two examples of the complementary use of 3d color doppler in two different patients with a more common and typical patterns ( left case p2 prolapse ) and an uncommon case with multiple scallop involvement .", "3d tte color doppler dataset ( left ) clearly facilitates the identification of the origin of the jet ( p2 ) and its measurement while in the complex case ( right ) tee identifies multiple regurgitant jets 3d vca can be easily obtained , without any a priori shape assumption , acquiring a 3d color doppler dataset , and then cutting the volumetric dataset till the best en face view of the regurgitant jet is obtained ; vca is measured as the mr jet planimetry .", "several studies have shown vca estimated by rt3d echocardiography to be more accurate and reproducible than 2d echocardiography - derived vca , when compared with other techniques such as cardiovascular magnetic resonance .", "moreover , 3d - vca measurements appear accurate even in asymmetric regurgitant orifices and unaffected by the etiology or eccentricity of mr .", "a cutoff of 0.41 cm using 3d - vca showed 82% sensitivity and 97% specificity in differentiating moderate from severe mr .", "further , using the planimetric 3d - vca and sampling the regurgitant jet with the continuous wave doppler , mr volume can be obtained multiplying the anatomical regurgitant orifice area and the velocity time integral , similarly to what is done routinely using 2d echocardiography .", "the major limitation affecting the 3d - vca technique is related to the limited temporal and spatial resolutions , which may prevent the accurate measurement of vca .", "the anatomic severity of mr can be assessed by 3d color doppler using the pisa technique .", "the theoretical benefit of the 3d pisa technique is the ability to measure the 3d surface of the proximal flow convergence region ( pfcr ) or to obtain the largest radius of the pfcr using 3d navigation , possibly increasing the accuracy of the eroa calculation .", "although the use of 3d data may improve the accuracy of the eroa calculation , it still requires assumptions about the shape of the pfcr .", "two studies attempted to obtain a 3d surface area , one using measurement of multiple radial planes of the pfcr in an in vitro model to reconstruct subsequently the total surface area , and the second using multiple linear measurements to reconstruct the 3d surface area .", "recently , a completely 3d - based method for mr quantification has been proposed and validated against cardiovascular magnetic resonance .", "the mr volume is obtained subtracting the left ventricular ( lv ) outflow from the mv inflow ; both flows are calculated based on 3d color - doppler acquisitions , using advanced algorithm for the estimate of the flow rate in planes close to the mv or lv outflow tract", ". this can be more accurate and reproducible than 2d pulsed - wave doppler - based methods . despite several studies", "have clearly shown the benefit of rt3d echocardiography , both tee and tte , for the mr quantification when compared to standard 2d echocardiographic methods , the lack of extensive , independent and ideally multicentric validation studies , limits the widespread use of the above mentioned techniques .", "guidelines on management of valvular heart diseases underlined that mv repair should be considered even in asymptomatic patients when the likelihood of a successful repair is high and in presence of a low operative risk .", "it is clear that the likelihood of a durable and safe mv repair strongly depends on the anatomy and morphology of the mv , and similarly the complexity of the surgical procedure is related to the complexity of the mv lesions . in a paper by tamborini et al .", ", the relationship between the extent of mv lesions and the complexity of the surgical procedures has been investigated .", "the results clearly demonstrated that simple lesions , as assessed by rt3d echo , are very likely to be treated with simple techniques ; while in almost half of the patients with complex lesion , complex surgical repair was performed by the surgeon .", "moreover , biaggi et al . , showed that quantitative mv characteristics , as assessed by rt3d tee rather than 2d tee , determined the complexity of mv repair , being involvement of the anterior leaflet , bileaflet prolapse , severe annular calcification , increased annular dimensions and leaflet height anatomic predictors of a lower likelihood of successful repair .", "thus , an accurate pre - operative rt3d tte evaluation , eventually using ad hoc post - processing software , may facilitate surgical planning allowing a tailored approach to each case .", "several publications have underlined the importance of mv annuloplasty during repair of degenerative mvd to optimize repair durability .", "using rt3d tee , demonstrated the presence of an enlarged posterior ma in patients with significant mr due to degenerative mvd , while there was no evidence that the intertrigonal distance is abnormal in these patients leading to the conclusion that posterior annular reduction with a flexible device at the time of mv repair is important , and that altering the anterior intertrigonal portion of the ma is unnecessary .", "caiani et al . , investigated , by rt3d tte , the changes in ma dynamics and the long - term effects induced by annuloplasty in mv organic prolapse undergoing repair using either an incomplete flexible band or a complete semi - rigid ring .", "both rings showed similar dynamic changes and , at 6 months , the ma area change during the cardiac cycle was depressed compared to control groups , independently of the implanted prosthetic ring .", "they concluded that the main factor affecting ma function is the undersizing due to the ring , which restricts ma geometry and limits the natural ma motion .", "most of the investigation focused on the mv and based rt3d echocardiography aimed at evaluating mv morphology as an isolated structure .", "however , veronesi et al . , investigated , using custom software , the relationship between mv and the aortic annulus .", "they demonstrated that in patients with mr , the dynamic mitral - aortic coupling was preserved despite the altered morphology and function of the mv . in these patients , mv repair with", "annuloplasty led to smaller and less pulsatile ma , with altered spatial displacement of the mv - aortic annulus complex .", "despite surgical correction of mr has demonstrated optimal outcomes in terms of efficiency , safety and residual mr , it is often not feasible in high - risk patients . according to the results of the euroheart survey , approximately half of the symptomatic patients with severe mr of functional or degenerative origin are denied surgery , and the likelihood of surgery denial increases with lv dysfunction , age and presence of comorbidities .", "chiam et al . , classified the proposed technologies into those targeting the chordae , the leaflets or the ma , both indirectly via the coronary sinus or directly true an edge - to - edge repair . among the latter ,", "the mitraclip system is the more diffused and studied , and is currently under ce mark in europe and investigational use in the usa and parts of asia .", "the mitraclip system mimics the surgical mv repair procedure introduced by alfieri , who treated a patient with an anterior prolapse by placing a pledgeted stitch to approximate the middle portions of the mv leaflets , creating a double orifice mv and successfully reducing the mr .", "the everest trials ( i and ii ) and the pivotal studies established the feasibility , safety and hemodynamic improvements in the large majority of patients , despite less effective at reducing mr than conventional surgery and thus with a higher rate of reintervention and/or surgical operation . currently , catheter - based mv clip repair is guided by standard , 2d tee .", "however , 2d tee is limited in the direct visualization of major mv pathology , the assessment of the 3d location of the catheter - clip system during the procedure , and the effect of the procedure on 3d mv morphology .", "rt3d tee was found helpful in several catheter - based procedure such as closure of atrial septal defects and transcatheter aortic valve implantation and , providing a unique  en face  visualization of mv anatomy and mr orifice , that is demonstrated to facilitate the transcatheter closure of mitral prosthesis paravalvular leaks .", "biner et al . , hypothesized that the combined use of 2d and 3d tee would also have additional value over 2d imaging in evaluating mr for catheter - based mv clip repair and guidance of the procedure .", "they demonstrated that combined 2d and 3d tee imaging was associated with a shorter time to first clip deployment and with a general reduction in the total procedure time when compared to traditional 2d tee guidance .", "they also stressed that rt3d tee is valuable in determining the precise orientation of the clip arms guiding the interventional cardiologist till the clip is positioned perpendicularly between the two central scallops , without the need for multiple verifications and cross - checks of 2d planes .", "moreover , the use of 3d color doppler could allow a better identification of the site of origin and the degree of any residual mr .", ", identified thickened anterior mitral leaflet and more severe mr to be the echocardiographic predictors associated with a higher likelihood of a dual clips procedure . recently , faletra et al .", ", showed side - by - side images obtained by fluoroscopy , 2d tee , and rt 3d tee . while essential data during the procedure were provided by the use of the 2d standard imaging techniques and the capture of mitral leaflets", "was always guided by 2d tee , they demonstrated that in almost every step of the mitralclip procedure , rt 3d tee may provide new additional useful data : the more precise anatomic information , the fine details of the devices and the precise relationship of catheters / clip with surrounding anatomic structures , may enhance the confidence of imaging interpretation and eventually improve the efficiency of the procedure .", "a crucial step during the percutaneous edge - to - edge repair of the mv is the assessment of clip attachment to the mv leaflets , which may be challenging using the 2d tee . in the everest", "i trial , single leaflet clip detachment occurred in 9% of the patients , demonstrating the need for new techniques that allow a more reliable assessment of clip attachment .", "braun et al . , developed a novel method for the assessment of clip attachment using intraprocedural 3d tee .", "they tried to quantify exactly the portion of mv leaflets fixed into the clip by measuring the distance between the lowermost part of the leaflet and the top edge of the clip .", "they found that the frequency of clip complications ( partial clip detachment or displacement ) was higher in patients imaged by 2d tee compared to patients imaged by 3d tee .", "hence , 3d imaging using volumetric analysis in addition to standard techniques may help to confirm a secure clip attachment during the mv clipping procedure .", "rt3d tee data of 55 patients ( 14 patients with degenerative mvd ) acquired during the mitraclip procedure immediately before and after clip placement have been recently published with the aim of assessing changes in annulus diameter and area .", "the results demonstrated that mitraclip can produce immediate reductions in ma size and tenting in functional mr .", "in contrast , similar changes were not observed in organic mr , and this different remodeling between the two etiologies of mr may be important to improve procedural strategies .", "scandura et al . , demonstrated that the mitraclip system led to left cardiac chamber reverse remodeling , with significant improvement in lv size and function , similarly to what observed in surgical restoration , both in patients with prolapsed - related mr and functional mr .", "all these results support the importance of 3d imaging for pre - procedural patient selection and prediction of procedural effectiveness .", "three - dimensional echocardiography represents a major technological breakthrough in the field of cardiovascular imaging , as it allows visualizing cardiac structures in their morphological complexity without the need for multiple acquisitions and mental reconstruction . during the past ten years", "a substantial body of literature has proven the potentials of 3d tee and tee in the evaluation of mv prolapse . moreover ,", "this imaging modality has not been limited to investigational studies , but several evidences have enlightened the benefits that 3d echocardiography can add to the daily clinical practice . furthermore , 3d echocardiography plays a pivotal role in understanding the pathophysiology of mv disease , better defining surgical strategy , guiding percutaneous procedure and improving the communication inside the heart team ."], "abstract_text": ["<S> degenerative mitral valve disease ( mvd ) is the leading cause of organic mitral regurgitation ( mr ) , one of the most common valvular heart disease in western countries . </S>", "<S> substantial progresses in the surgical treatment of degenerative mvd have improved life expectancy of patients with significant mr . however , prognosis , surgical decision and timing of surgery strongly depend on the accurate characterization of mitral valve ( mv ) anatomy and pathology and on the precise quantification of mr . three - dimensional ( 3d ) echocardiography , a major technological breakthrough in the field of cardiovascular imaging , provides several advantages over two - dimensional ( 2d ) imaging in the qualitative and quantitative evaluations of mv apparatus . </S>", "<S> in this review , we focus on the contribution of this new modality to the diagnosis of degenerative mvd , the quantitative assessment of mr severity , the selection and monitoring of surgical and percutaneous procedures , the evaluation of procedural outcomes . the results of a systematic and exhaustive search of the existing literature , restricted to real - time 3d echocardiography in adults , are here reported . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "3D qualitative and quantitative evaluations of degenerative mitral valve disease", "QUANTIFICATION OF MR", "IMPLICATIONS FOR MV SURGERY", "IMPLICATIONS FOR MV PERCUTANEOUS REPAIR", "CONCLUSIONS"], "sections": [["moderate or severe mitral regurgitation ( mr ) is the most frequent valve disease in the usa and the second most common form of valvular heart disease needing surgery in europe", ". degenerative mitral valve disease ( mvd ) is the leading cause of organic mr in western countries .", "its mechanism is primarily characterized by mitral valve prolapse ( mvp ) due to excessive leaflet movement ( 2 mm abnormal systolic movement beyond the saddle - shaped annular level ) , as defined by carpentier ( type ii ) .", "the anatomic lesions associated with the degenerative mr etiology encompass a wide pathological spectrum , from fibroelastic deficiency ( fed ) to extensive myxomatous disease .", "carpentier described fed as a condition associated with a fibrillin deficiency that often leads to the rupture of one or more thinned and elongated chordae .", "this process usually , but not exclusively , involves the middle scallop of the posterior leaflet with the myxomatous degeneration limited to the prolapsing segment .", "in contrast to fed , myxomatous disease , often called barlow disease ( bd ) , is characterized by diffuse excess tissue , with large valves comprising multiple degenerated segments presenting as thick , mostly with elongated chordae ; severe mitral annulus ( ma ) dilatation associated with different degrees of annular calcification is a frequent finding in patients with bd , and subvalvular fibrosis and calcification of the papillary muscles , usually the anterior , may also occur .", "myxomatous degeneration is defined by the accumulation of mucopolysaccharides responsible for the thickening and  proliferative aspect of valvular tissue .", "even if molecular disorders and pathophysiologic pathways may not be uniform , there is a continuum between the different anatomic aspects of degenerative mr .", "surgery is the treatment of choice in degenerative mr , improving symptoms and preventing heart failure . since the 1970s", ", mitral valve ( mv ) repair has emerged as the optimal treatment , preferred to traditional valve replacement , as it improves outcome and reduces mortality of patients with severe organic mr by about 70% .", "recent studies and guidelines have underlined the importance of early surgical intervention to preserve long - term left ventricular ( lv ) function in severe mr .", "indeed , the best short- and long - term results are obtained in asymptomatic patients operated in advanced repair centers with low operative mortality ( < 1% ) and high repair rates ( 80 - 90% ) .", "these results emphasize the importance of early detection and detailed assessment of valve morphology and mr .", "moreover , in the recent years there has been an increasing interest towards non - surgical solutions to severe mr , with the percutaneous mitraclip system , mimicking the surgical alfieri stitch by placing a clip on the beating heart , being the most well established , with results not inferior to surgical correction in selected patients .", "patient selection is a crucial step of the mitraclip procedure and several anatomic criteria should be fulfilled to ensure the suitability of the device to mv anatomy . in this complex scenario , echocardiography is the method of choice to evaluate patients with degenerative mr ; it allows the accurate assessment of mr severity , etiology , mechanisms and anatomic lesions , lv and left atrial remodeling , and consequently defines the indication and the probability of successful mv repair .", "three - dimensional ( 3d ) echocardiographic imaging has represented a major innovation in cardiovascular ultrasound .", "recent guidelines stated that real - time three - dimensional ( rt3d ) imaging modalities are useful in the presentation of realistic views of heart valves and are ideal for interrogating the anatomy and function of each of the individual components of the mv apparatus , including annulus , leaflets , chordae and papillary muscles . both qualitative and quantitative evaluations of degenerative mvd"], ["all publications considered for this review were identified accessing the medline database via pubmed . the search strategy employed a number of free - text keywords and controlled vocabulary terms including , but not limited to", ", the following concepts : 3d echocardiography ; mitral valve ; degenerative mitral valve disease ; mitral valve repair ; percutaneous mitral valve repair . fields of search were limited to the title and abstract and restricted to the english language .", "all the retrieved studies were screened for inclusion using a hierarchical approach assessing title , abstract and manuscript .", "additional searches were conducted after screening the references of all selected studies . as a direct consequence of a series of technological breakthrough , such as the introduction of the 3d matrix technology with its improved spatial resolution compared to previously available sparse array transducers , rt3d echocardiography demonstrated feasibility and accuracy in mv assessment .", "the enhanced visualization of morphologic and pathologic aspects of the mv apparatus in degenerative mr has contributed to our knowledge of mv pathology , with unquestionable benefits compared to traditional two - dimensional ( 2d ) echocardiography .", "qualitative morphological evaluation of the mvp may be accurately assessed both by 3d transthoracic echocardiography ( tte ) and transesophageal echocardiography ( tee ) and the recent data showed that rt3d , either tte or tee , is superior to the corresponding 2d techniques in the description of mv pathology [ figure 1 ] .", "example of head - to - head comparison between 3d tte ( left ) and 3d tee ( right ) datasets in two cases of a2 prolapse ( above ) and p2 prolapse ( below ) , imaged from the surgical view ( left atrium ) 3d evaluation may be easily integrated into a standard 2d examination in the assessment of qualitative morphology of the mv .", "indeed , the possibility of visualizing mv leaflets , commissures , annulus calcifications and subvalvular structures in different and unique planes , both from the atrium or ventricle , with access to  en face  views , enhances the understanding of this complex apparatus .", "diagnostic accuracy of rt3d tte vs surgical inspection in the identification of prolapsing segments has been investigated by tamborini et al .", "the overall accuracy of the method was high ( sensitivity 92.5% , specificity 96% , accuracy 95% ) ; sensitivity was slightly lower for antero - lateral commissure , p1 and a1 , while specificity was very high in all the segments .", "even focusing on the differences in rt3d tte accuracy in the setting of simple ( isolated p2 prolapse or p2 associated with p1 or p3 ) and complex cases ( lesions involving the entire posterior leaflet , lesions of the anterior leaflet , bileaflet prolapse , and commissural involvement ) , the accuracy was very high , 98% and 93% respectively , even if slightly lower in complex lesions .", "all these data were in accordance with previous findings . as it concerns rt3d tee , there is a substantial body of literature demonstrating the additional value of 3d over 2d in the evaluation of patients with mvp .", "the majority of these studies showed that 3d echocardiographic findings correlate closely to surgical findings , achieving an exact anatomic description in approximately 90% to 95% of segments , whereas 2d echocardiography is less accurate .", ", underlined that in agreement with previous works , the incremental value of 3d modality over conventional 2d can be better appreciated in complex disease involving both leaflets or the anterior leaflet alone .", "la canna et al . , demonstrated that rt3d tee provided more accurate mapping of mv prolapse than 2d tee and rt3d tte imaging , adding quantitative recognition of dominant and secondary lesions and mv anatomy details in 222 patients undergoing surgical repair for mr secondary to prolapse .", "recently , hien et al . , investigated whether the known benefit of rt3d tte over 2d tee imaging for the evaluation of mvp would be valuable among novice echocardiographers .", "they demonstrated that both expert and novice echocardiographers were able to provide more accurate descriptions of the mv prolapse with rt3d than with 2d tee images , and this benefit was three times more important for inexperienced operators . with the advances of rt3d echocardiography together with the development of dedicated rt3d software capable of providing several parameters ( annular diameters and area , annular height and planarity , saddle - shaped morphology , leaflet size , coaptation geometry ) , high - resolution , full - volume imaging and quantification of morphology of the entire mitral apparatus have become feasible [ figure 2 ] .", "several studies demonstrated that the use of rt3d allows improving the evaluation of the mv apparatus both qualitatively , as previously illustrated , but also quantitatively .", "in fact , the mv has a complex 3d morphology and attempts to use 2d images to comprehend a complex 3d structure have provided an incomplete picture .", "in particular , the normal ma is characterized by a nonplanar saddle shape that has been suggested to be important for alleviating the mechanical stress on mitral leaflets and chordae tendinae imposed by lv pressure . moreover ,", "the geometry of leaflet surface and coaptation , magnitude of leaflet billowing , and deformation of subvalvular structures are important morphological features in mvp and they can be only partially assessed with 2d imaging .", "chandra et al . , hypothesized that rt3de - derived measurements of valvular anatomy could be used to characterize mvd in an objective way .", "morphological analysis in the mvd group revealed a progressive increase in multiple parameters from normal subjects to fed to bd , allowing an accurate diagnosis of these entities .", "three - dimensional billowing height with a cutoff value of 1.0 mm differentiated mvd from normal subjects , and billowing volume with a cutoff value of 1.15 ml differentiated between fed and bd .", "these findings could support the adoption of preoperative automated clinical decision - making strategies based on reliable quantitative parameters , with important surgical implications .", "example of 3d tee and corresponding reconstruction of mitral annulus and leaflets in a patient with fibroelastic deficiency ( left ) and in two patients with barlow disease ( right ) . in the first case ,", "the two barlow cases with the same method are characterized by multiple scallop involvement of both leaflets identified by the red surfaces moreover , grewal et al .", ", investigated mitral annular size , shape , and motion 6 times over the cardiac cycle using rt3de in patients with degenerative mvd compared with normal subjects and patients with ischemic mr and demonstrated that degenerative mvd annulus is still a dynamic structure but behaves considerably different from normal patients with loss of early - systolic area contraction and saddle - shape deepening .", "maffessanti et al . , characterized the mv 3d morphology using rt3d tee in the presence of severe mr associated with fed or bd , immediately before and after mv repair , in comparison with a control group .", "they found that the annular enlargement after mr is not isotropic , being more pronounced in the anteroposterior and intercommissural directions rather than along the mitral height , resulting in flattening of the mv in both fed and bd groups with higher values in the bd group .", "postoperatively , annular diameters and mv area were significantly smaller compared with preoperative values ; similar changes were found in the exposed areas of both leaflets .", "more recently , lee et al . , demonstrated that patients with mvp - related mr had more dilated mitral annulus , a flattening of annular saddle shape , redundant leaflet surfaces , greater leaflet billow volume and billow height , longer lengths from papillary muscles to coaptation and more frequent chordal rupture compared to normal subjects and non - prolapse mr patients .", "interestingly , the association between annular flattening and the increased frequency of chordal rupture suggests that the loss of the annular saddle shape may predispose to chordal elongation and rupture as a result of excessive chordal tension ."], ["as a direct consequence of a series of technological breakthrough , such as the introduction of the 3d matrix technology with its improved spatial resolution compared to previously available sparse array transducers , rt3d echocardiography demonstrated feasibility and accuracy in mv assessment .", "the enhanced visualization of morphologic and pathologic aspects of the mv apparatus in degenerative mr has contributed to our knowledge of mv pathology , with unquestionable benefits compared to traditional two - dimensional ( 2d ) echocardiography .", "qualitative morphological evaluation of the mvp may be accurately assessed both by 3d transthoracic echocardiography ( tte ) and transesophageal echocardiography ( tee ) and the recent data showed that rt3d , either tte or tee , is superior to the corresponding 2d techniques in the description of mv pathology [ figure 1 ] .", "example of head - to - head comparison between 3d tte ( left ) and 3d tee ( right ) datasets in two cases of a2 prolapse ( above ) and p2 prolapse ( below ) , imaged from the surgical view ( left atrium ) 3d evaluation may be easily integrated into a standard 2d examination in the assessment of qualitative morphology of the mv .", "indeed , the possibility of visualizing mv leaflets , commissures , annulus calcifications and subvalvular structures in different and unique planes , both from the atrium or ventricle , with access to  en face  views , enhances the understanding of this complex apparatus .", "diagnostic accuracy of rt3d tte vs surgical inspection in the identification of prolapsing segments has been investigated by tamborini et al .", "the overall accuracy of the method was high ( sensitivity 92.5% , specificity 96% , accuracy 95% ) ; sensitivity was slightly lower for antero - lateral commissure , p1 and a1 , while specificity was very high in all the segments . even focusing on the differences in rt3d tte accuracy in the setting of simple ( isolated p2 prolapse or p2 associated with p1 or p3 ) and complex cases ( lesions involving the entire posterior leaflet , lesions of the anterior leaflet , bileaflet prolapse , and commissural involvement ) , the accuracy was very high , 98% and 93% respectively , even if slightly lower in complex lesions .", "all these data were in accordance with previous findings . as it concerns rt3d tee , there is a substantial body of literature demonstrating the additional value of 3d over 2d in the evaluation of patients with mvp .", "the majority of these studies showed that 3d echocardiographic findings correlate closely to surgical findings , achieving an exact anatomic description in approximately 90% to 95% of segments , whereas 2d echocardiography is less accurate .", ", underlined that in agreement with previous works , the incremental value of 3d modality over conventional 2d can be better appreciated in complex disease involving both leaflets or the anterior leaflet alone .", "la canna et al . , demonstrated that rt3d tee provided more accurate mapping of mv prolapse than 2d tee and rt3d tte imaging , adding quantitative recognition of dominant and secondary lesions and mv anatomy details in 222 patients undergoing surgical repair for mr secondary to prolapse .", "recently , hien et al . , investigated whether the known benefit of rt3d tte over 2d tee imaging for the evaluation of mvp would be valuable among novice echocardiographers .", "they demonstrated that both expert and novice echocardiographers were able to provide more accurate descriptions of the mv prolapse with rt3d than with 2d tee images , and this benefit was three times more important for inexperienced operators . with the advances of rt3d echocardiography together with the development of dedicated rt3d software capable of providing several parameters ( annular diameters and area , annular height and planarity , saddle - shaped morphology , leaflet size , coaptation geometry ) , high - resolution , full - volume imaging and quantification of morphology of the entire mitral apparatus have become feasible [ figure 2 ] .", "several studies demonstrated that the use of rt3d allows improving the evaluation of the mv apparatus both qualitatively , as previously illustrated , but also quantitatively .", "in fact , the mv has a complex 3d morphology and attempts to use 2d images to comprehend a complex 3d structure have provided an incomplete picture .", "in particular , the normal ma is characterized by a nonplanar saddle shape that has been suggested to be important for alleviating the mechanical stress on mitral leaflets and chordae tendinae imposed by lv pressure . moreover ,", "the geometry of leaflet surface and coaptation , magnitude of leaflet billowing , and deformation of subvalvular structures are important morphological features in mvp and they can be only partially assessed with 2d imaging .", "chandra et al . , hypothesized that rt3de - derived measurements of valvular anatomy could be used to characterize mvd in an objective way .", "morphological analysis in the mvd group revealed a progressive increase in multiple parameters from normal subjects to fed to bd , allowing an accurate diagnosis of these entities .", "three - dimensional billowing height with a cutoff value of 1.0 mm differentiated mvd from normal subjects , and billowing volume with a cutoff value of 1.15 ml differentiated between fed and bd .", "these findings could support the adoption of preoperative automated clinical decision - making strategies based on reliable quantitative parameters , with important surgical implications .", "example of 3d tee and corresponding reconstruction of mitral annulus and leaflets in a patient with fibroelastic deficiency ( left ) and in two patients with barlow disease ( right ) . in the first case ,", "the two barlow cases with the same method are characterized by multiple scallop involvement of both leaflets identified by the red surfaces moreover , grewal et al . ,", "investigated mitral annular size , shape , and motion 6 times over the cardiac cycle using rt3de in patients with degenerative mvd compared with normal subjects and patients with ischemic mr and demonstrated that degenerative mvd annulus is still a dynamic structure but behaves considerably different from normal patients with loss of early - systolic area contraction and saddle - shape deepening .", "maffessanti et al . , characterized the mv 3d morphology using rt3d tee in the presence of severe mr associated with fed or bd , immediately before and after mv repair , in comparison with a control group .", "they found that the annular enlargement after mr is not isotropic , being more pronounced in the anteroposterior and intercommissural directions rather than along the mitral height , resulting in flattening of the mv in both fed and bd groups with higher values in the bd group .", "postoperatively , annular diameters and mv area were significantly smaller compared with preoperative values ; similar changes were found in the exposed areas of both leaflets .", "more recently , lee et al . , demonstrated that patients with mvp - related mr had more dilated mitral annulus , a flattening of annular saddle shape , redundant leaflet surfaces , greater leaflet billow volume and billow height , longer lengths from papillary muscles to coaptation and more frequent chordal rupture compared to normal subjects and non - prolapse mr patients .", "interestingly , the association between annular flattening and the increased frequency of chordal rupture suggests that the loss of the annular saddle shape may predispose to chordal elongation and rupture as a result of excessive chordal tension ."], ["the prognosis and the best timing for surgical treatment in degenerative mvd strongly depend on the accurate quantification of mr severity , which claims for accurate , feasible and reproducible techniques . despite these premises ,", "recent technological development has made rt3d echocardiography the ideal technique for the evaluation of mr severity , in particular , in all those cases where the hypotheses at the basis of 2d estimate are not met .", "mr grading can be obtained , in accordance with standard 2d echocardiography , by means of vena contracta area ( vca ) , effective regurgitant orifice area ( eroa ) using 3d proximal isovelocity surface area ( pisa ) or mr volume measurement [ figure 3 ] . detailed and comprehensive discussions on mr quantitative evaluation", "two examples of the complementary use of 3d color doppler in two different patients with a more common and typical patterns ( left case p2 prolapse ) and an uncommon case with multiple scallop involvement .", "3d tte color doppler dataset ( left ) clearly facilitates the identification of the origin of the jet ( p2 ) and its measurement while in the complex case ( right ) tee identifies multiple regurgitant jets 3d vca can be easily obtained , without any a priori shape assumption , acquiring a 3d color doppler dataset , and then cutting the volumetric dataset till the best en face view of the regurgitant jet is obtained ; vca is measured as the mr jet planimetry .", "several studies have shown vca estimated by rt3d echocardiography to be more accurate and reproducible than 2d echocardiography - derived vca , when compared with other techniques such as cardiovascular magnetic resonance .", "moreover , 3d - vca measurements appear accurate even in asymmetric regurgitant orifices and unaffected by the etiology or eccentricity of mr .", "a cutoff of 0.41 cm using 3d - vca showed 82% sensitivity and 97% specificity in differentiating moderate from severe mr .", "further , using the planimetric 3d - vca and sampling the regurgitant jet with the continuous wave doppler , mr volume can be obtained multiplying the anatomical regurgitant orifice area and the velocity time integral , similarly to what is done routinely using 2d echocardiography .", "the major limitation affecting the 3d - vca technique is related to the limited temporal and spatial resolutions , which may prevent the accurate measurement of vca .", "the anatomic severity of mr can be assessed by 3d color doppler using the pisa technique .", "the theoretical benefit of the 3d pisa technique is the ability to measure the 3d surface of the proximal flow convergence region ( pfcr ) or to obtain the largest radius of the pfcr using 3d navigation , possibly increasing the accuracy of the eroa calculation .", "although the use of 3d data may improve the accuracy of the eroa calculation , it still requires assumptions about the shape of the pfcr .", "two studies attempted to obtain a 3d surface area , one using measurement of multiple radial planes of the pfcr in an in vitro model to reconstruct subsequently the total surface area , and the second using multiple linear measurements to reconstruct the 3d surface area .", "recently , a completely 3d - based method for mr quantification has been proposed and validated against cardiovascular magnetic resonance .", "the mr volume is obtained subtracting the left ventricular ( lv ) outflow from the mv inflow ; both flows are calculated based on 3d color - doppler acquisitions , using advanced algorithm for the estimate of the flow rate in planes close to the mv or lv outflow tract", ". this can be more accurate and reproducible than 2d pulsed - wave doppler - based methods . despite several studies", "have clearly shown the benefit of rt3d echocardiography , both tee and tte , for the mr quantification when compared to standard 2d echocardiographic methods , the lack of extensive , independent and ideally multicentric validation studies , limits the widespread use of the above mentioned techniques ."], ["guidelines on management of valvular heart diseases underlined that mv repair should be considered even in asymptomatic patients when the likelihood of a successful repair is high and in presence of a low operative risk .", "it is clear that the likelihood of a durable and safe mv repair strongly depends on the anatomy and morphology of the mv , and similarly the complexity of the surgical procedure is related to the complexity of the mv lesions . in a paper by tamborini et al .", ", the relationship between the extent of mv lesions and the complexity of the surgical procedures has been investigated .", "the results clearly demonstrated that simple lesions , as assessed by rt3d echo , are very likely to be treated with simple techniques ; while in almost half of the patients with complex lesion , complex surgical repair was performed by the surgeon .", "moreover , biaggi et al . , showed that quantitative mv characteristics , as assessed by rt3d tee rather than 2d tee , determined the complexity of mv repair , being involvement of the anterior leaflet , bileaflet prolapse , severe annular calcification , increased annular dimensions and leaflet height anatomic predictors of a lower likelihood of successful repair .", "thus , an accurate pre - operative rt3d tte evaluation , eventually using ad hoc post - processing software , may facilitate surgical planning allowing a tailored approach to each case .", "several publications have underlined the importance of mv annuloplasty during repair of degenerative mvd to optimize repair durability .", "using rt3d tee , demonstrated the presence of an enlarged posterior ma in patients with significant mr due to degenerative mvd , while there was no evidence that the intertrigonal distance is abnormal in these patients leading to the conclusion that posterior annular reduction with a flexible device at the time of mv repair is important , and that altering the anterior intertrigonal portion of the ma is unnecessary .", "caiani et al . , investigated , by rt3d tte , the changes in ma dynamics and the long - term effects induced by annuloplasty in mv organic prolapse undergoing repair using either an incomplete flexible band or a complete semi - rigid ring .", "both rings showed similar dynamic changes and , at 6 months , the ma area change during the cardiac cycle was depressed compared to control groups , independently of the implanted prosthetic ring .", "they concluded that the main factor affecting ma function is the undersizing due to the ring , which restricts ma geometry and limits the natural ma motion .", "most of the investigation focused on the mv and based rt3d echocardiography aimed at evaluating mv morphology as an isolated structure .", "however , veronesi et al . , investigated , using custom software , the relationship between mv and the aortic annulus .", "they demonstrated that in patients with mr , the dynamic mitral - aortic coupling was preserved despite the altered morphology and function of the mv . in these patients , mv repair with", "annuloplasty led to smaller and less pulsatile ma , with altered spatial displacement of the mv - aortic annulus complex ."], ["despite surgical correction of mr has demonstrated optimal outcomes in terms of efficiency , safety and residual mr , it is often not feasible in high - risk patients . according to the results of the euroheart survey , approximately half of the symptomatic patients with severe mr of functional or degenerative origin are denied surgery , and the likelihood of surgery denial increases with lv dysfunction , age and presence of comorbidities .", "chiam et al . , classified the proposed technologies into those targeting the chordae , the leaflets or the ma , both indirectly via the coronary sinus or directly true an edge - to - edge repair . among the latter ,", "the mitraclip system is the more diffused and studied , and is currently under ce mark in europe and investigational use in the usa and parts of asia .", "the mitraclip system mimics the surgical mv repair procedure introduced by alfieri , who treated a patient with an anterior prolapse by placing a pledgeted stitch to approximate the middle portions of the mv leaflets , creating a double orifice mv and successfully reducing the mr .", "the everest trials ( i and ii ) and the pivotal studies established the feasibility , safety and hemodynamic improvements in the large majority of patients , despite less effective at reducing mr than conventional surgery and thus with a higher rate of reintervention and/or surgical operation . currently , catheter - based mv clip repair is guided by standard , 2d tee .", "however , 2d tee is limited in the direct visualization of major mv pathology , the assessment of the 3d location of the catheter - clip system during the procedure , and the effect of the procedure on 3d mv morphology .", "rt3d tee was found helpful in several catheter - based procedure such as closure of atrial septal defects and transcatheter aortic valve implantation and , providing a unique  en face  visualization of mv anatomy and mr orifice , that is demonstrated to facilitate the transcatheter closure of mitral prosthesis paravalvular leaks .", "biner et al . , hypothesized that the combined use of 2d and 3d tee would also have additional value over 2d imaging in evaluating mr for catheter - based mv clip repair and guidance of the procedure .", "they demonstrated that combined 2d and 3d tee imaging was associated with a shorter time to first clip deployment and with a general reduction in the total procedure time when compared to traditional 2d tee guidance .", "they also stressed that rt3d tee is valuable in determining the precise orientation of the clip arms guiding the interventional cardiologist till the clip is positioned perpendicularly between the two central scallops , without the need for multiple verifications and cross - checks of 2d planes .", "moreover , the use of 3d color doppler could allow a better identification of the site of origin and the degree of any residual mr .", ", identified thickened anterior mitral leaflet and more severe mr to be the echocardiographic predictors associated with a higher likelihood of a dual clips procedure . recently , faletra et al .", ", showed side - by - side images obtained by fluoroscopy , 2d tee , and rt 3d tee . while essential data during the procedure were provided by the use of the 2d standard imaging techniques and the capture of mitral leaflets", "was always guided by 2d tee , they demonstrated that in almost every step of the mitralclip procedure , rt 3d tee may provide new additional useful data : the more precise anatomic information , the fine details of the devices and the precise relationship of catheters / clip with surrounding anatomic structures , may enhance the confidence of imaging interpretation and eventually improve the efficiency of the procedure .", "a crucial step during the percutaneous edge - to - edge repair of the mv is the assessment of clip attachment to the mv leaflets , which may be challenging using the 2d tee . in the everest", "i trial , single leaflet clip detachment occurred in 9% of the patients , demonstrating the need for new techniques that allow a more reliable assessment of clip attachment .", "braun et al . , developed a novel method for the assessment of clip attachment using intraprocedural 3d tee .", "they tried to quantify exactly the portion of mv leaflets fixed into the clip by measuring the distance between the lowermost part of the leaflet and the top edge of the clip .", "they found that the frequency of clip complications ( partial clip detachment or displacement ) was higher in patients imaged by 2d tee compared to patients imaged by 3d tee .", "hence , 3d imaging using volumetric analysis in addition to standard techniques may help to confirm a secure clip attachment during the mv clipping procedure .", "rt3d tee data of 55 patients ( 14 patients with degenerative mvd ) acquired during the mitraclip procedure immediately before and after clip placement have been recently published with the aim of assessing changes in annulus diameter and area .", "the results demonstrated that mitraclip can produce immediate reductions in ma size and tenting in functional mr .", "in contrast , similar changes were not observed in organic mr , and this different remodeling between the two etiologies of mr may be important to improve procedural strategies .", "scandura et al . , demonstrated that the mitraclip system led to left cardiac chamber reverse remodeling , with significant improvement in lv size and function , similarly to what observed in surgical restoration , both in patients with prolapsed - related mr and functional mr .", "all these results support the importance of 3d imaging for pre - procedural patient selection and prediction of procedural effectiveness ."], ["three - dimensional echocardiography represents a major technological breakthrough in the field of cardiovascular imaging , as it allows visualizing cardiac structures in their morphological complexity without the need for multiple acquisitions and mental reconstruction . during the past ten years", "a substantial body of literature has proven the potentials of 3d tee and tee in the evaluation of mv prolapse . moreover ,", "this imaging modality has not been limited to investigational studies , but several evidences have enlightened the benefits that 3d echocardiography can add to the daily clinical practice . furthermore , 3d echocardiography plays a pivotal role in understanding the pathophysiology of mv disease , better defining surgical strategy , guiding percutaneous procedure and improving the communication inside the heart team ."]]}
{"article_id": "PMC4249997", "article_text": ["surgical resections such as unilateral temporal lobectomy and amygdalo - hippo - campectomy have an established place in the management of carefully selected patients with refractory localisation - related epilepsy .", "adverse cognitive sequelae of epilepsy surgery have been well - recognised since the seminal report of scoville and milner in 1957 documenting the dense anterograde amnesia in patient h.m .", "such amnesia has been observed on occasion following unilateral surgery , reflecting preoperative damage in the unoperated , contralateral , temporal lobe , a finding which mandates careful preoperative assessment of the non - operated hemisphere , for example using sodium amytal ( wada ) testing and/or functional neuroimaging , to try to ensure cognitive function is preserved post - operatively .", "we present a patient with refractory epilepsy who , following initially apparently successful unilateral temporal lobectomy , developed recurrent seizures and profound amnesia , and in whom subsequent investigations unexpectedly suggested an autoimmune aetiology .", "a 36-year - old right - handed female was referred to our centre for assessment of epileptic seizures and cognitive impairment . at the age of 33 ,", "she had undergone a right ( non - dominant ) temporal lobectomy for refractory complex partial seizures , performed at another neuroscience centre with an established epilepsy surgery programme .", "the patient 's seizures began at the age of 15 years ; there was no history of childhood febrile convulsions .", "seizures were characterised by dj vu , absence , and automatisms , and were thought to arise in the right temporal lobe .", "mr imaging appearances were equivocal , with right temporal lobe changes thought to represent either sclerosis or a possible dysplastic lesion . because of the refractory nature of the seizures , pre - operative workup was undertaken , including fdg - pet which showed reduced uptake of tracer in the right temporal lobe .", "intracranial eeg ( subtemporal strips ) confirmed complex partial seizures arising from the lateral right temporal cortex , but there also seemed to be subclinical events arising from the left side .", "a sodium amytal test performed prior to surgery confirmed that the patient was left - hemisphere dominant for language and that both hemispheres supported memory function .", "however , 34 months post - operatively the patient deteriorated with further frequent complex partial seizures . additionally , she was noted to have symptoms suggestive of both anterograde and retrograde amnesia . by this time", "cognitive assessment included administration of cognitive screening instruments which showed impaired performance : on the mini - mental state examination ( mmse ) , she scored 23/30 ; on the addenbrooke 's cognitive examination - revised ( ace - r ) , she scored 74/100 , with 12/26 on the memory components ; and on the montreal cognitive assessment , she scored 23/30 ( normal 26/30 ) . on the repeatable battery for the assessment of neuropsychological status ( rbans ) , her delayed memory scores fell within the extremely low range .", "this impairment was for both verbal and visual material , with a subtle indication of slightly higher levels of delayed recall with visual information ( list recall total score = 0 ; story recall total score = 1 ; figure recall total score = 2 ) .", "other domains assessed by the rbans showed the immediate memory to be in the borderline range , attention was low average , whilst language and visuospatial / constructional abilities were relatively preserved ( table 1 ; left - hand column ) .", "in addition to the evidence of right temporal lobectomy , this also showed a high signal change in the left temporal lobe involving the hippocampus ( fig .", "serological testing revealed a very high titre of antibodies directed against glutamic acid decarboxylase ( gad ) . in sum , these investigations suggested a diagnosis of anti - gad limbic encephalitis ( le ) . over the next 2 years , the patient was empirically treated with various immunomodulatory interventions including intravenous methylprednisolone , plasma exchange , and two infusions of rituximab , all without obvious clinical improvement in either seizures or cognitive function .", "three years after surgery , prior to embarking on a treatment trial of intravenous immunoglobulin at our centre , the patient was still receiving polytherapy for epileptic seizures ( levetiracetam , pregabalin , clonazepam , and lacosamide ) . on cognitive testing", ", she now scored 17/30 on the mmse and 63/100 on the ace - r with 8/26 on the memory components . repeating the rbans ,", "the cognitive profile was little changed , with the delayed memory score still being extremely low ( table 1 ; right - hand column ) , again affecting both verbal and visual material , with the latter still at slightly higher levels ( list recall total score = 2 ; story recall total score = 2 ; figure recall total score = 8) .", "behaviourally , she used external memory aids to record daily events since she had no recall of these after only a brief period of time .", "antibodies to gad have been associated with various neurological syndromes , including stiff person syndrome , cerebellar ataxia , epilepsy , paraneoplastic syndromes ( encephalomyelitis , cerebellar ataxia , and le ) , idiopathic le , and myasthenia gravis . from the epilepsy perspective , in a cohort of 253 epilepsy patients , liimatainen et al .   detected anti - gad antibodies in 15 patients ( 5.9 vs. 1.5% in 200 controls ) , most of them ( 90% ) with temporal lobe epilepsy . in a study of patients with adult - onset ( > 30 years ) temporal lobe epilepsy , anti - gad antibodies", "were found in 5 out of 42 , with evidence for pharmacoresistant epilepsy with associated memory impairment and other autoimmune diseases .", "temporal lobe epilepsy with anti - gad antibodies may not therefore be a rare condition , especially amongst the treatment - refractory patients referred for surgical evaluation .", "the precise pathogenic sequence of events in our patient remains uncertain ; a number of potential explanations exist .", "it is possible that she had two separate problems , namely mesial temporal sclerosis followed by adult - onset anti - gad antibody le .", "if this is so , it might be speculated whether her temporal lobectomy surgery might have unmasked epitopes which initiated an autoimmune response that produced anti - gad antibodies and hence le .", "another possibility is that the entire syndrome was due to anti - gad le , albeit very long - lived .", "defined non - paraneoplastic anti - gad le as a chronic non - remitting disorder , with antibody titres remaining high after intravenous methylprednisolone . moreover , none of their patients became seizure free despite intense anti - epileptic drug therapy , unlike the situation in le associated with voltage - gated potassium channel ( vgkc / lgi1 ) antibodies [ 10 , 11 ] .", "furthermore , cognitive impairments did not improve after treatment in anti - gad le . in this context , it is of note that the neuropathological examination of our patient 's temporal lobectomy specimen found evidence of chronic inflammatory change consisting almost exclusively of t cells with associated microglial activation , suggesting a chronic encephalitic process in addition to hippocampal sclerosis .", "to date there is little information on the efficacy of intravenous immunoglobulin in anti - gad le .", "response has been reported in new - onset focal epilepsy , but the chances of success must be doubtful in chronic epilepsy , as in our patient . this case may illustrate that autoimmune processes , rather than surgery , may cause bilateral hippocampal pathology resulting in profound amnesia , behaviourally akin to that seen in the classic amnesic patient h.m .", "[ 14 , 15 ] . following bilateral anterior temporal lobectomy for intractable seizures with partial hippocampal removal , h.m . developed a profound anterograde amnesia for episodic autobiographical material but with preserved general intelligence , attention , working memory , language and perceptual skills .", "in contrast , the neuropsychological outcome following unilateral non - dominant hemisphere temporal lobectomy , as undergone by our patient , is usually confined to material - specific ( i.e. visual rather than verbal ) memory impairments .", "we recommend that the possibility of anti - gad le needs to be considered in all patients with refractory epilepsy of presumed temporal lobe origin , including those being considered for epilepsy surgery , especially those whose clinical course is not typical for mesial temporal sclerosis ."], "abstract_text": ["<S> we describe a patient who developed significant cognitive decline with profound amnesia following non - dominant temporal lobectomy for refractory seizures , in whom the original suspicion of structural pathology was revised following the discovery of clinical and neuropathological markers of inflammation , neuropsychological evidence of bilateral involvement , and high titres of antibodies directed against glutamic acid decarboxylase ( gad ) . </S>", "<S> this case adds to the evidence that the diagnosis of non - paraneoplastic anti - gad limbic encephalitis merits consideration in any patient with a refractory seizure disorder and cognitive decline . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion"], "sections": [["surgical resections such as unilateral temporal lobectomy and amygdalo - hippo - campectomy have an established place in the management of carefully selected patients with refractory localisation - related epilepsy .", "adverse cognitive sequelae of epilepsy surgery have been well - recognised since the seminal report of scoville and milner in 1957 documenting the dense anterograde amnesia in patient h.m .", "such amnesia has been observed on occasion following unilateral surgery , reflecting preoperative damage in the unoperated , contralateral , temporal lobe , a finding which mandates careful preoperative assessment of the non - operated hemisphere , for example using sodium amytal ( wada ) testing and/or functional neuroimaging , to try to ensure cognitive function is preserved post - operatively .", "we present a patient with refractory epilepsy who , following initially apparently successful unilateral temporal lobectomy , developed recurrent seizures and profound amnesia , and in whom subsequent investigations unexpectedly suggested an autoimmune aetiology ."], ["a 36-year - old right - handed female was referred to our centre for assessment of epileptic seizures and cognitive impairment . at the age of 33 ,", "she had undergone a right ( non - dominant ) temporal lobectomy for refractory complex partial seizures , performed at another neuroscience centre with an established epilepsy surgery programme .", "the patient 's seizures began at the age of 15 years ; there was no history of childhood febrile convulsions .", "seizures were characterised by dj vu , absence , and automatisms , and were thought to arise in the right temporal lobe .", "mr imaging appearances were equivocal , with right temporal lobe changes thought to represent either sclerosis or a possible dysplastic lesion . because of the refractory nature of the seizures , pre - operative workup was undertaken , including fdg - pet which showed reduced uptake of tracer in the right temporal lobe .", "intracranial eeg ( subtemporal strips ) confirmed complex partial seizures arising from the lateral right temporal cortex , but there also seemed to be subclinical events arising from the left side .", "a sodium amytal test performed prior to surgery confirmed that the patient was left - hemisphere dominant for language and that both hemispheres supported memory function .", "however , 34 months post - operatively the patient deteriorated with further frequent complex partial seizures . additionally , she was noted to have symptoms suggestive of both anterograde and retrograde amnesia . by this time", "cognitive assessment included administration of cognitive screening instruments which showed impaired performance : on the mini - mental state examination ( mmse ) , she scored 23/30 ; on the addenbrooke 's cognitive examination - revised ( ace - r ) , she scored 74/100 , with 12/26 on the memory components ; and on the montreal cognitive assessment , she scored 23/30 ( normal 26/30 ) . on the repeatable battery for the assessment of neuropsychological status ( rbans ) , her delayed memory scores fell within the extremely low range .", "this impairment was for both verbal and visual material , with a subtle indication of slightly higher levels of delayed recall with visual information ( list recall total score = 0 ; story recall total score = 1 ; figure recall total score = 2 ) .", "other domains assessed by the rbans showed the immediate memory to be in the borderline range , attention was low average , whilst language and visuospatial / constructional abilities were relatively preserved ( table 1 ; left - hand column ) .", "in addition to the evidence of right temporal lobectomy , this also showed a high signal change in the left temporal lobe involving the hippocampus ( fig .", "serological testing revealed a very high titre of antibodies directed against glutamic acid decarboxylase ( gad ) . in sum , these investigations suggested a diagnosis of anti - gad limbic encephalitis ( le ) . over the next 2 years , the patient was empirically treated with various immunomodulatory interventions including intravenous methylprednisolone , plasma exchange , and two infusions of rituximab , all without obvious clinical improvement in either seizures or cognitive function .", "three years after surgery , prior to embarking on a treatment trial of intravenous immunoglobulin at our centre , the patient was still receiving polytherapy for epileptic seizures ( levetiracetam , pregabalin , clonazepam , and lacosamide ) . on cognitive testing", ", she now scored 17/30 on the mmse and 63/100 on the ace - r with 8/26 on the memory components . repeating the rbans ,", "the cognitive profile was little changed , with the delayed memory score still being extremely low ( table 1 ; right - hand column ) , again affecting both verbal and visual material , with the latter still at slightly higher levels ( list recall total score = 2 ; story recall total score = 2 ; figure recall total score = 8) .", "behaviourally , she used external memory aids to record daily events since she had no recall of these after only a brief period of time ."], ["antibodies to gad have been associated with various neurological syndromes , including stiff person syndrome , cerebellar ataxia , epilepsy , paraneoplastic syndromes ( encephalomyelitis , cerebellar ataxia , and le ) , idiopathic le , and myasthenia gravis . from the epilepsy perspective , in a cohort of 253 epilepsy patients , liimatainen et al .   detected anti - gad antibodies in 15 patients ( 5.9 vs. 1.5% in 200 controls ) , most of them ( 90% ) with temporal lobe epilepsy . in a study of patients with adult - onset ( > 30 years ) temporal lobe epilepsy , anti - gad antibodies", "were found in 5 out of 42 , with evidence for pharmacoresistant epilepsy with associated memory impairment and other autoimmune diseases .", "temporal lobe epilepsy with anti - gad antibodies may not therefore be a rare condition , especially amongst the treatment - refractory patients referred for surgical evaluation .", "the precise pathogenic sequence of events in our patient remains uncertain ; a number of potential explanations exist .", "it is possible that she had two separate problems , namely mesial temporal sclerosis followed by adult - onset anti - gad antibody le .", "if this is so , it might be speculated whether her temporal lobectomy surgery might have unmasked epitopes which initiated an autoimmune response that produced anti - gad antibodies and hence le .", "another possibility is that the entire syndrome was due to anti - gad le , albeit very long - lived .", "defined non - paraneoplastic anti - gad le as a chronic non - remitting disorder , with antibody titres remaining high after intravenous methylprednisolone . moreover , none of their patients became seizure free despite intense anti - epileptic drug therapy , unlike the situation in le associated with voltage - gated potassium channel ( vgkc / lgi1 ) antibodies [ 10 , 11 ] .", "furthermore , cognitive impairments did not improve after treatment in anti - gad le . in this context , it is of note that the neuropathological examination of our patient 's temporal lobectomy specimen found evidence of chronic inflammatory change consisting almost exclusively of t cells with associated microglial activation , suggesting a chronic encephalitic process in addition to hippocampal sclerosis .", "to date there is little information on the efficacy of intravenous immunoglobulin in anti - gad le .", "response has been reported in new - onset focal epilepsy , but the chances of success must be doubtful in chronic epilepsy , as in our patient . this case may illustrate that autoimmune processes , rather than surgery , may cause bilateral hippocampal pathology resulting in profound amnesia , behaviourally akin to that seen in the classic amnesic patient h.m .", "[ 14 , 15 ] . following bilateral anterior temporal lobectomy for intractable seizures with partial hippocampal removal , h.m . developed a profound anterograde amnesia for episodic autobiographical material but with preserved general intelligence , attention , working memory , language and perceptual skills .", "in contrast , the neuropsychological outcome following unilateral non - dominant hemisphere temporal lobectomy , as undergone by our patient , is usually confined to material - specific ( i.e. visual rather than verbal ) memory impairments .", "we recommend that the possibility of anti - gad le needs to be considered in all patients with refractory epilepsy of presumed temporal lobe origin , including those being considered for epilepsy surgery , especially those whose clinical course is not typical for mesial temporal sclerosis ."]]}
{"article_id": "PMC3341426", "article_text": ["however , cardiac metastasis is diagnosed in less than 1% of patients with malignant melanoma because less than 10% of these patients present with cardiac symptoms.1 - 3 ) identification of cardiac metastasis from melanoma usually means that the patient is suffering systemic metastasis . unlike typical cardiac metastasized patients , we report a first case of a patient with a metastatic malignant melanoma in the heart without an identifiable primary source or additional metastasis in korea .", "a 59-year - old woman was admitted for cough and pleuritic chest pain with no history of malignancy or heart disease . her initial blood pressure was 110/70 mm hg , pulse rate 70 beats / min , respiratory rate 20/min , and body temperature was 36.1. jugular veins were not distended .", "laboratory studies , including a complete blood count , liver , and chemical profiles were in normal ranges .", "chest computed tomography showed a large amount of pericardial effusion and a mass in the right atrium ( ra ) ( fig .", "transthoracic echocardiography showed a large mass measuring 4231 mm in the ra , which did not obstruct tricuspid valve flow .", "effusion analysis showed a red blood cell count of 1.910/mm and a white blood cell count of 3300/mm ( lymphocytes , 55% ; neutrophils , 14% ) .", "cardiac mri also revealed a large mass surrounding ascending aorta spread into transverse sinus and around pulmonary trunk ( fig .", "considering the risk of open heart surgery and poor prognosis of extensive cardiac metastasis , pericardial window operation and epicardial mass biopsy were performed to relieve symptoms and confirm the pathological diagnosis . pathological analysis including immunohistochemistry revealed the final diagnosis to be malignant melanoma ( fig .", "positron emission tomography revealed no other distant organ metastasis except for heart and mediastinal lymph nodes .", "malignant melanoma has aggressive biological behavior and the greatest tendency for metastasis to the heart.1 ) although autopsy studies reported an incidence of 50% to 71% , cardiac metastasis is diagnosed in less than 1% of patients with malignant melanoma because less than 10% of these patients present with cardiac symptoms.2 ) such metastases most frequently occur after multifocal hematological dissemination and may develop anywhere in the heart.3 ) melanotic metastases can invade the wall of any of the 4 cardiac chambers , and the ra is involved most frequently.4 ) the clinical signs and symptoms of cardiac metastasis are unclear and non - specific , although when present , the clinical signs and symptoms include fatigue , weakness , pericardial effusion , congestive heart failure , cardiac arrhythmia , superior vena cava syndrome , right ventricular outflow and inflow obstruction , and transient ischemic attack.5 ) however , patients with malignant melanoma who have cardiac metastases may present symptoms only caused by tumors in other organ systems . although cardiac involvement occurs during the course of the disease , it is rare that the initial manifestation is cardiac metastasis .", "a tumor 's anatomic location and extent of invasion determine the feasibility of surgical intervention , which should optimally be performed during the early stages of the disease.6 ) a complete resection of an intracardiac melanoma prevents potential morbidities that are associated with progressive intracardiac growth , such as superior vena cava syndrome , right ventricular outflow and inflow obstruction , dysrhythmia , cardiac tamponade , and heart failure.6 ) even when total resection is not possible , conservative surgery can relieve symptoms and prevent imminent cardiac failure .", "conservative surgery improves the quality of a patient 's life , as in our patient 's case .", "although more than 90% of melanomas have a cutaneous origin,7 ) melanomas may sometimes present metastatically in the absence of a primary lesion , termed melanomas of unknown primary origin .", "most authors estimate that 2 - 6% of patients are diagnosed with metastatic melanoma of unknown primary site .", "in particular , such a metastatic melanoma in the heart such as in this patient without a known primary cutaneous origin is a rare presentation and chiefly an anecdotal finding of metastatic melanoma .", "several reported cases can be found in the literature about cardiac involvement with cutaneous primary malignant melanoma and multiple metastasis in korea , as well as worldwide.8)9 ) as far as we know , malignant cardiac melanoma without a primary origin has not yet been reported in korea .", "the survival of patients with unknown primary melanoma was demonstrated to be similar to that of patients with known primary tumors when corresponding stages were compared.10 - 12 ) those patients with metastases to any other visceral sites are described to have a 1 year survival rate of 41% and a median survival of approximately 6 months.11)13)14 ) accordingly , we expected that the outcome of this patient might be very unfavorable .", "although total resection was not possible because of extensive cardiac metastasis , a pericardial window operation could resolve her symptoms .", "after surgery , chemotherapy was planned , including cisplatin , dacarbazine , casmustine , and tamoxifen . however , the patient refused further chemotherapy after completion of two sessions .", "as only few reports about cardiac metastasis of malignant melanoma without cutaneous origin have been published , we are uncertain if conservative surgery is associated with prolonged survival and if the role of surgery for survival is worth further investigation ."], "abstract_text": ["<S> malignant melanoma has a very high propensity to metastasize to the heart . however , melanoma may sometimes present as a metastatic lesion in the absence of a primary lesion , which are called melanomas of unknown primary origin . </S>", "<S> we report a case in which a patient presented with a metastatic maligant melanoma in the right atrium with pericardial effusion and without a primary origin . </S>"], "labels": null, "section_names": ["Introduction", "Case", "Discussion"], "sections": [["however , cardiac metastasis is diagnosed in less than 1% of patients with malignant melanoma because less than 10% of these patients present with cardiac symptoms.1 - 3 ) identification of cardiac metastasis from melanoma usually means that the patient is suffering systemic metastasis . unlike typical cardiac metastasized patients , we report a first case of a patient with a metastatic malignant melanoma in the heart without an identifiable primary source or additional metastasis in korea ."], ["a 59-year - old woman was admitted for cough and pleuritic chest pain with no history of malignancy or heart disease . her initial blood pressure was 110/70 mm hg , pulse rate 70 beats / min , respiratory rate 20/min , and body temperature was 36.1. jugular veins were not distended .", "laboratory studies , including a complete blood count , liver , and chemical profiles were in normal ranges .", "chest computed tomography showed a large amount of pericardial effusion and a mass in the right atrium ( ra ) ( fig .", "transthoracic echocardiography showed a large mass measuring 4231 mm in the ra , which did not obstruct tricuspid valve flow .", "effusion analysis showed a red blood cell count of 1.910/mm and a white blood cell count of 3300/mm ( lymphocytes , 55% ; neutrophils , 14% ) .", "cardiac mri also revealed a large mass surrounding ascending aorta spread into transverse sinus and around pulmonary trunk ( fig .", "considering the risk of open heart surgery and poor prognosis of extensive cardiac metastasis , pericardial window operation and epicardial mass biopsy were performed to relieve symptoms and confirm the pathological diagnosis . pathological analysis including immunohistochemistry revealed the final diagnosis to be malignant melanoma ( fig .", "positron emission tomography revealed no other distant organ metastasis except for heart and mediastinal lymph nodes ."], ["malignant melanoma has aggressive biological behavior and the greatest tendency for metastasis to the heart.1 ) although autopsy studies reported an incidence of 50% to 71% , cardiac metastasis is diagnosed in less than 1% of patients with malignant melanoma because less than 10% of these patients present with cardiac symptoms.2 ) such metastases most frequently occur after multifocal hematological dissemination and may develop anywhere in the heart.3 ) melanotic metastases can invade the wall of any of the 4 cardiac chambers , and the ra is involved most frequently.4 ) the clinical signs and symptoms of cardiac metastasis are unclear and non - specific , although when present , the clinical signs and symptoms include fatigue , weakness , pericardial effusion , congestive heart failure , cardiac arrhythmia , superior vena cava syndrome , right ventricular outflow and inflow obstruction , and transient ischemic attack.5 ) however , patients with malignant melanoma who have cardiac metastases may present symptoms only caused by tumors in other organ systems . although cardiac involvement occurs during the course of the disease , it is rare that the initial manifestation is cardiac metastasis .", "a tumor 's anatomic location and extent of invasion determine the feasibility of surgical intervention , which should optimally be performed during the early stages of the disease.6 ) a complete resection of an intracardiac melanoma prevents potential morbidities that are associated with progressive intracardiac growth , such as superior vena cava syndrome , right ventricular outflow and inflow obstruction , dysrhythmia , cardiac tamponade , and heart failure.6 ) even when total resection is not possible , conservative surgery can relieve symptoms and prevent imminent cardiac failure .", "conservative surgery improves the quality of a patient 's life , as in our patient 's case .", "although more than 90% of melanomas have a cutaneous origin,7 ) melanomas may sometimes present metastatically in the absence of a primary lesion , termed melanomas of unknown primary origin .", "most authors estimate that 2 - 6% of patients are diagnosed with metastatic melanoma of unknown primary site .", "in particular , such a metastatic melanoma in the heart such as in this patient without a known primary cutaneous origin is a rare presentation and chiefly an anecdotal finding of metastatic melanoma .", "several reported cases can be found in the literature about cardiac involvement with cutaneous primary malignant melanoma and multiple metastasis in korea , as well as worldwide.8)9 ) as far as we know , malignant cardiac melanoma without a primary origin has not yet been reported in korea .", "the survival of patients with unknown primary melanoma was demonstrated to be similar to that of patients with known primary tumors when corresponding stages were compared.10 - 12 ) those patients with metastases to any other visceral sites are described to have a 1 year survival rate of 41% and a median survival of approximately 6 months.11)13)14 ) accordingly , we expected that the outcome of this patient might be very unfavorable .", "although total resection was not possible because of extensive cardiac metastasis , a pericardial window operation could resolve her symptoms .", "after surgery , chemotherapy was planned , including cisplatin , dacarbazine , casmustine , and tamoxifen . however , the patient refused further chemotherapy after completion of two sessions .", "as only few reports about cardiac metastasis of malignant melanoma without cutaneous origin have been published , we are uncertain if conservative surgery is associated with prolonged survival and if the role of surgery for survival is worth further investigation ."]]}
{"article_id": "PMC3830322", "article_text": ["vitamin d deficiency ( vdd ) is defined as serum 25-hydroxy vitamin d ( 25ohd ) levels < 20 ng / ml .", "vdd has been documented in more than 90% across all age groups and both sexes from india .", "classical manifestations of vdd is described as rickets / osteomalacia , which manifest as bony deformity / pain , decreased bone mineral density ( bmd ) , increased risk of fracture and is associated with raised alkaline phosphatase and parathormone ( pth ) . however , secondary hyperparathyroidism ( shpt ) is observed in < 50% of subjects in indian and us population .", "subjects with same levels of serum 25ohd have varied clinical and biochemical abnormalities including some showing no abnormalities .", "this raises logical question  do all subjects with vdd have clinical disease according to this definition ?  the main physiological function of vitamin d is maintenance of calcium homeostasis by its effect on calcium absorption and bone health in association with parathyroid gland .", "calcium is absorbed actively in the duodenum through transcellular ( active transport-80% ) process , which is vitamin d dependent , whereas passive absorption is a paracellular ( passive diffusion-20% ) process , which occurs throughout intestine independent of vitamin d and is dependent on concentration of calcium in the intestinal tract .", "total fraction of calcium absorbed from total intake can vary from 20 to 80% . in the event of decreased calcium availability from intake ,", "calcium is released from bone under the effect of vitamin d - pth system to maintain its homeostasis .", "as per the recent institute of medicine ( iom ) statement  the data currently suggest that fractional calcium absorption ( fca ) reaches a maximum between 12 and 20 ng / ml in both children and adults .", " in most of the studies reviewed by iom , the baseline serum 25ohd was > 10 ng / ml and there was no correlation of serum 25ohd levels with calcium absorption .", "when we have plotted mean basal serum 25ohd levels in various studies and mean fca , there was a significant inverse correlation ( r = 0.75 , p = 0.001 ) .", "there is only one study among elderly that has assessed the relation of calcium absorption and base line serum 25ohd levels ranging from 4 to 20 ng / ml .", "this study has clearly shown that calcium absorption decreases in the ranges from 4 to 8 ng / ml and not > 8 ng / ml .", "similarly , in the most studies related to vitamin d supplementation , basal serum 25ohd levels were > 8 ng / ml .", "only one study carried out in subjects with 25ohd level of 4 ng / ml showed an increase of 21% with change in 25ohd", "level to 24 ng / ml , whereas in those with the increase in 25ohd level from 8 to 28 ng / ml only 3% increase of calcium absorption occurred", ". there is no correlation of mean change in fca ( increase or decrease ) with either mean basal serum 25ohd levels ( r = 0.122 , p = 0.754 ) or increment in serum 25ohd levels .", "these data clearly shows that the maximum calcium absorption capacity is reached when serum 25ohd levels are > 8 ng / ml . logically to maintain calcium homeostasis in the face of vdd , the first body will try to absorb maximum available calcium , rather than affecting bone .", "hence , calcium absorption is the first most important adaptive mechanism in patients with vdd .", "high fca ( 54 - 63% ) has been reported from the region of china with low calcium intake ( < 500 mg ) compared with 25 - 34% in us children with high intake of calcium ( > 900 mg ) .", "this suggests that the body tries to adapt to the calcium availability to bodies requirement by adjusting fca .", "the conventional explanation of homeostasis is by systemic adaptation , in which decreased calcium intake results in decreased calcium absorption , which leads to increase in pth levels .", "the pth up regulates the 1- hydroxylase enzyme , leading to increase generation of 1,25-dihydroxyvitamin d ( 1,25(oh ) 2d ) levels and increased calcium absorption and bone resorption .", "however , as deduced from the above discussion , the body has a tremendous reserve to increase the fca in the face of decrease in calcium intake .", "this suggests that calcium absorption can be kept static over a wide range of calcium intake and serum 25ohd levels by local intestinal adaptation .", "we hypothesize that the first adaptive mechanism in calcium homeostasis is local rather than systemic .", "it consists of calcium sensing receptor ( casr ) on intestinal brush border , which senses calcium in intestinal cells and negatively affect vitamin d system in intestinal cells to decrease active transcellular calcium transport .", "it also facilitates passive paracellular diffusion of calcium in the intestine , which is less efficient process . on the contrary , when there is decreased calcium intake , this feedback inhibition is removed and vitamin d dependent active calcium absorption will increase , maintaining calcium homeostasis .", "furthermore , there may be some genetic or epigenetic alteration in genes of 1- hydroxylase enzyme , which decreases efficiency of active vitamin d generation or vitamin d receptor ( vdr ) genotype affecting calcium absorption . in subjects with efficient vdr genotype for calcium absorption , local adaptation", "will be maintained at lower levels of serum 25ohd and vice versa will also be true .", "the interaction between casr and vitamin d system in intestinal cells (  intestinal calcistat  ) will decide the level of serum 25ohd at which calcium absorption can be maintained according to the need of the body or becomes suboptimal in a given individual indicating failure of local adaptation .", "firstly , decrease in calcium intake < 250 mg , which can not be overcome by increasing fca .", "this will manifest as calcium deficiency rickets on the face of normal vitamin d levels .", "secondly , mutation in casr , if  activating ,  may lead to decreased calcium absorption and if  inactivating ,  increased calcium absorption .", "thirdly , decreased supply of substrate below critical levels ( serum 25ohd < 8 ng / ml ) will lead to vdd rickets .", "however , this level can vary according to interaction between casr and vitamin d system in an individual-intestinal calcistat .  finally , genetic mutation in 1- hydroxylase [ vitamin d resistant rickets - i ( vdrr - i ) ] or vdr ( vdrr - ii ) will also lead to failure of local adaptive response .", "this will also explain the observation in vdrr - i and ii , where high intake of calcium can overcome most of the clinical manifestation of the disease .", "with very high intake , casrs will get saturated and will enhance passive ( paracellular ) calcium absorption , which will be able to fulfill the requirement of body . with increasing severity of vdd , there will be decrease in calcium absorption .", "the calcium levels will now be maintained by bone resorption , rather than increasing calcium absorption , which is currently believed .", "hence , generally held belief that increase in pth will increase calcium absorption through generation of active vitamin d metabolites , is wrong .", "this puts pth as a marker for systemic vdd or failure of local adaptation by the  intestinal calcistat . ", "the above hypothesis of the  intestinal calcistat  explains the vide variation observed in literature about relation between serum 25ohd , pth , calcium absorption and bmd . according to this hypothesis , subjects with low serum 25ohd who have normal  intestinal calcistat ", "will absorb required amount of calcium and will not mount systemic adaptive response in the form of increase in pth and 1,25(oh ) 2d levels , hence will have lower 1,25(oh ) 2d levels than those with failure of adaptation and bmd will not be affected .", "subjects with adaptive failure will have higher 1,25(oh ) 2d levels and will have lower bmd .", "this is further supported by observation that patients with similar low serum 25ohd levels ( < 10 ng / ml ) , bmd was lower in subjects with shpt .", "this will also explain the observation that about 50% of subjects with vdd do nt mount pth response because they have adequate local adaptation in  intestinal calcistat .", " this will also explains that why there is no substantial increase in calcium absorption in with vitamin d supplementation because basal level of serum 25ohd is sufficient to supple enough substrate for generation of active vitamin d metabolites .", "this brings us to question that should we define vdd with a value of serum 25ohd in isolation ? it is obvious from the above discussion that there are adaptive mechanisms to overcome low vitamin d levels , which can be operative over a wide range of serum 25ohd levels .", "failure of adaptive mechanism will lead to clinical and biochemical evidence of vdd . among them", "hence , the subjects with vdd defined by low vitamin d levels ( < 20 ng / ml or < 30 ng / ml ) according to the current definition with normal pth and bmd will not have any clinical and biochemical consequence of low vitamin d levels and vice versa subjects with similar levels of serum 25ohd with raised pth or low bmd are likely to be vdd . what should we call subjects with low serum 25ohd levels without evidence of shpt or low bmd ?", "should we call them subclinical vdd , compensated vdd , asymptomatic vdd or not call them vdd at all .", "further studies are required to define adverse biological consequences of vdd in this group and effects of vitamin d supplementation and comparing them with the population who already had adverse biological effects of vdd ."], "abstract_text": ["<S> the main physiological function of vitamin d is maintenance of calcium homeostasis by its effect on calcium absorption , and bone health in association with parathyroid gland . </S>", "<S> vitamin d deficiency ( vdd ) is defined as serum 25-hydroxy vitamin d ( 25ohd ) levels < 20 ng / ml . </S>", "<S> do all subjects with vdd have clinical disease according to this definition ? </S>", "<S> we hypothesize that there exist an intestinal calcistat , which controls the calcium absorption independent of pth levels . </S>", "<S> it consists of calcium sensing receptor ( casr ) on intestinal brush border , which senses calcium in intestinal cells and vitamin d system in intestinal cells . </S>", "<S> casr dampens the generation of active vitamin d metabolite in intestinal cells and decrease active transcellular calcium transport . </S>", "<S> it also facilitates passive paracellular diffusion of calcium in intestine . </S>", "<S> this local adaptation adjusts the fractional calcium absorption according the body requirement . </S>", "<S> failure of local adaptation due to decreased calcium intake , decreased supply of 25ohd , mutation in casr or vitamin d system decreases systemic calcium levels and systemic adaptations comes into the play . </S>", "<S> systemic adaptations consist of rise in pth and increase in active vitamin d metabolites . </S>", "<S> these adaptations lead to bone resorption and maintenance of calcium homeostasis . </S>", "<S> not all subjects with varying levels of vdd manifest with secondary hyperparathyroidism and decreased in bone mineral density . </S>", "<S> we suggest that rise in pth is first indicator of vdd along with decrease in bmd depending on duration of vdd . </S>", "<S> hence , subjects with any degree of vdd with normal pth and bmd should not be labeled as vitamin d deficient . </S>", "<S> these subjects can be called subclinical vdd , and further studies are required to assess beneficial effect of vitamin d supplementation in this subset of population . </S>"], "labels": null, "section_names": ["I", "V", "I", "E", "C"], "sections": [["vitamin d deficiency ( vdd ) is defined as serum 25-hydroxy vitamin d ( 25ohd ) levels < 20 ng / ml .", "vdd has been documented in more than 90% across all age groups and both sexes from india .", "classical manifestations of vdd is described as rickets / osteomalacia , which manifest as bony deformity / pain , decreased bone mineral density ( bmd ) , increased risk of fracture and is associated with raised alkaline phosphatase and parathormone ( pth ) . however , secondary hyperparathyroidism ( shpt ) is observed in < 50% of subjects in indian and us population .", "subjects with same levels of serum 25ohd have varied clinical and biochemical abnormalities including some showing no abnormalities .", "this raises logical question  do all subjects with vdd have clinical disease according to this definition ?  the main physiological function of vitamin d is maintenance of calcium homeostasis by its effect on calcium absorption and bone health in association with parathyroid gland .", "calcium is absorbed actively in the duodenum through transcellular ( active transport-80% ) process , which is vitamin d dependent , whereas passive absorption is a paracellular ( passive diffusion-20% ) process , which occurs throughout intestine independent of vitamin d and is dependent on concentration of calcium in the intestinal tract .", "total fraction of calcium absorbed from total intake can vary from 20 to 80% . in the event of decreased calcium availability from intake ,", "calcium is released from bone under the effect of vitamin d - pth system to maintain its homeostasis ."], ["as per the recent institute of medicine ( iom ) statement  the data currently suggest that fractional calcium absorption ( fca ) reaches a maximum between 12 and 20 ng / ml in both children and adults .", " in most of the studies reviewed by iom , the baseline serum 25ohd was > 10 ng / ml and there was no correlation of serum 25ohd levels with calcium absorption .", "when we have plotted mean basal serum 25ohd levels in various studies and mean fca , there was a significant inverse correlation ( r = 0.75 , p = 0.001 ) .", "there is only one study among elderly that has assessed the relation of calcium absorption and base line serum 25ohd levels ranging from 4 to 20 ng / ml .", "this study has clearly shown that calcium absorption decreases in the ranges from 4 to 8 ng / ml and not > 8 ng / ml .", "similarly , in the most studies related to vitamin d supplementation , basal serum 25ohd levels were > 8 ng / ml .", "only one study carried out in subjects with 25ohd level of 4 ng / ml showed an increase of 21% with change in 25ohd", "level to 24 ng / ml , whereas in those with the increase in 25ohd level from 8 to 28 ng / ml only 3% increase of calcium absorption occurred", ". there is no correlation of mean change in fca ( increase or decrease ) with either mean basal serum 25ohd levels ( r = 0.122 , p = 0.754 ) or increment in serum 25ohd levels .", "these data clearly shows that the maximum calcium absorption capacity is reached when serum 25ohd levels are > 8 ng / ml . logically to maintain calcium homeostasis in the face of vdd , the first body will try to absorb maximum available calcium , rather than affecting bone .", "hence , calcium absorption is the first most important adaptive mechanism in patients with vdd .", "high fca ( 54 - 63% ) has been reported from the region of china with low calcium intake ( < 500 mg ) compared with 25 - 34% in us children with high intake of calcium ( > 900 mg ) .", "this suggests that the body tries to adapt to the calcium availability to bodies requirement by adjusting fca ."], ["the conventional explanation of homeostasis is by systemic adaptation , in which decreased calcium intake results in decreased calcium absorption , which leads to increase in pth levels .", "the pth up regulates the 1- hydroxylase enzyme , leading to increase generation of 1,25-dihydroxyvitamin d ( 1,25(oh ) 2d ) levels and increased calcium absorption and bone resorption .", "however , as deduced from the above discussion , the body has a tremendous reserve to increase the fca in the face of decrease in calcium intake .", "this suggests that calcium absorption can be kept static over a wide range of calcium intake and serum 25ohd levels by local intestinal adaptation .", "we hypothesize that the first adaptive mechanism in calcium homeostasis is local rather than systemic .", "it consists of calcium sensing receptor ( casr ) on intestinal brush border , which senses calcium in intestinal cells and negatively affect vitamin d system in intestinal cells to decrease active transcellular calcium transport .", "it also facilitates passive paracellular diffusion of calcium in the intestine , which is less efficient process . on the contrary , when there is decreased calcium intake , this feedback inhibition is removed and vitamin d dependent active calcium absorption will increase , maintaining calcium homeostasis .", "furthermore , there may be some genetic or epigenetic alteration in genes of 1- hydroxylase enzyme , which decreases efficiency of active vitamin d generation or vitamin d receptor ( vdr ) genotype affecting calcium absorption . in subjects with efficient vdr genotype for calcium absorption , local adaptation", "will be maintained at lower levels of serum 25ohd and vice versa will also be true .", "the interaction between casr and vitamin d system in intestinal cells (  intestinal calcistat  ) will decide the level of serum 25ohd at which calcium absorption can be maintained according to the need of the body or becomes suboptimal in a given individual indicating failure of local adaptation .", "firstly , decrease in calcium intake < 250 mg , which can not be overcome by increasing fca .", "this will manifest as calcium deficiency rickets on the face of normal vitamin d levels .", "secondly , mutation in casr , if  activating ,  may lead to decreased calcium absorption and if  inactivating ,  increased calcium absorption .", "thirdly , decreased supply of substrate below critical levels ( serum 25ohd < 8 ng / ml ) will lead to vdd rickets .", "however , this level can vary according to interaction between casr and vitamin d system in an individual-intestinal calcistat .  finally , genetic mutation in 1- hydroxylase [ vitamin d resistant rickets - i ( vdrr - i ) ] or vdr ( vdrr - ii ) will also lead to failure of local adaptive response .", "this will also explain the observation in vdrr - i and ii , where high intake of calcium can overcome most of the clinical manifestation of the disease .", "with very high intake , casrs will get saturated and will enhance passive ( paracellular ) calcium absorption , which will be able to fulfill the requirement of body . with increasing severity of vdd , there will be decrease in calcium absorption .", "the calcium levels will now be maintained by bone resorption , rather than increasing calcium absorption , which is currently believed .", "hence , generally held belief that increase in pth will increase calcium absorption through generation of active vitamin d metabolites , is wrong .", "this puts pth as a marker for systemic vdd or failure of local adaptation by the  intestinal calcistat . "], ["the above hypothesis of the  intestinal calcistat  explains the vide variation observed in literature about relation between serum 25ohd , pth , calcium absorption and bmd . according to this hypothesis , subjects with low serum 25ohd who have normal  intestinal calcistat ", "will absorb required amount of calcium and will not mount systemic adaptive response in the form of increase in pth and 1,25(oh ) 2d levels , hence will have lower 1,25(oh ) 2d levels than those with failure of adaptation and bmd will not be affected .", "subjects with adaptive failure will have higher 1,25(oh ) 2d levels and will have lower bmd .", "this is further supported by observation that patients with similar low serum 25ohd levels ( < 10 ng / ml ) , bmd was lower in subjects with shpt .", "this will also explain the observation that about 50% of subjects with vdd do nt mount pth response because they have adequate local adaptation in  intestinal calcistat .", " this will also explains that why there is no substantial increase in calcium absorption in with vitamin d supplementation because basal level of serum 25ohd is sufficient to supple enough substrate for generation of active vitamin d metabolites ."], ["this brings us to question that should we define vdd with a value of serum 25ohd in isolation ? it is obvious from the above discussion that there are adaptive mechanisms to overcome low vitamin d levels , which can be operative over a wide range of serum 25ohd levels .", "failure of adaptive mechanism will lead to clinical and biochemical evidence of vdd . among them", "hence , the subjects with vdd defined by low vitamin d levels ( < 20 ng / ml or < 30 ng / ml ) according to the current definition with normal pth and bmd will not have any clinical and biochemical consequence of low vitamin d levels and vice versa subjects with similar levels of serum 25ohd with raised pth or low bmd are likely to be vdd . what should we call subjects with low serum 25ohd levels without evidence of shpt or low bmd ?", "should we call them subclinical vdd , compensated vdd , asymptomatic vdd or not call them vdd at all .", "further studies are required to define adverse biological consequences of vdd in this group and effects of vitamin d supplementation and comparing them with the population who already had adverse biological effects of vdd ."]]}
{"article_id": "PMC2809293", "article_text": ["the penn diabetes heart study ( pdhs ) is an ongoing , cross - sectional study of individuals with type 2 diabetes ( aged 3575 years ) without clinical evidence of cvd ( myocardial infarction , coronary revascularization or angiographic disease , positive stress test , clinical peripheral arterial disease or peripheral arterial revascularization , stroke , or transient ischemic attack ) .", "the university of pennsylvania institutional review board approved the study protocol , and all subjects gave written informed consent ( 4 ) . the ankle - brachial index ( abi )", "was calculated by dividing the average of the ankle pressures by the highest brachial pressure .", "participants with an abi > 1.4 ( n = 13 ) reflecting noncompressible arteries were excluded ( 5 ) .", "each subject had the estimated glomerular filtration rate ( egfr ) calculated based on the abbreviated modification of diet in renal disease study equation ( 6 ) .", "plasma levels of fetuin - a ( 4.3 and 10.2% , respectively , intra - assay and inter - assay coefficient of variation ) were measured by enzyme - linked immunosorbent assay ( biovendor laboratory medicine , modrice , czech republic ) .", "high - sensitivity c - reactive protein ( hscrp ) was measured by immunoturbidimetric assay ( wako , osaka , japan ) .", "student 's t test was used for normal continuous variables and the kruskal - wallis for nonnormal distributed variables .", "the association between a 1-sd change in fetuin - a and pad was examined using unadjusted and multivariable adjusted logistic regression models as described in table 1 .", "analyses were performed on stata 10.0 software ( stata , college station , tx ) .", "association between fetuin - a and pad data are odds ratios ( 95% ci ) and were obtained by logistic regression model .", "model 2 : model 1 + smoking , hypertension , hypercholesterolemia , metabolic syndrome , a1c , framingham risk score ( % ) , and medications ( ace inhibitors , aspirin , statins , insulin , metformin , sulfonylureas , and thiazolidinediones ) .", "the ankle - brachial index ( abi ) was calculated by dividing the average of the ankle pressures by the highest brachial pressure .", "participants with an abi > 1.4 ( n = 13 ) reflecting noncompressible arteries were excluded ( 5 ) .", "each subject had the estimated glomerular filtration rate ( egfr ) calculated based on the abbreviated modification of diet in renal disease study equation ( 6 ) .", "plasma levels of fetuin - a ( 4.3 and 10.2% , respectively , intra - assay and inter - assay coefficient of variation ) were measured by enzyme - linked immunosorbent assay ( biovendor laboratory medicine , modrice , czech republic ) . high - sensitivity c - reactive protein ( hscrp )", "student 's t test was used for normal continuous variables and the kruskal - wallis for nonnormal distributed variables .", "the association between a 1-sd change in fetuin - a and pad was examined using unadjusted and multivariable adjusted logistic regression models as described in table 1 .", "analyses were performed on stata 10.0 software ( stata , college station , tx ) .", "association between fetuin - a and pad data are odds ratios ( 95% ci ) and were obtained by logistic regression model .", "model 2 : model 1 + smoking , hypertension , hypercholesterolemia , metabolic syndrome , a1c , framingham risk score ( % ) , and medications ( ace inhibitors , aspirin , statins , insulin , metformin , sulfonylureas , and thiazolidinediones ) .", "among 738 subjects in the study sample , pad prevalence was 5.1% , the mean age was 58.7  9.3 years , 37.1% were female , 63.2% were caucasians , and 32.1% were african americans .", "the median fetuin - a level was 292.4 ng / ml ( interquartile range 115.5 ) . individuals with pad had a significantly lower fetuin - a level compared with individuals without pad ( 269.3 vs. 293.4 , p < 0.001 ) .", "no significant differences were noted in other cardiovascular risk factors , metabolic syndrome , kidney function , inflammation , bmi , serum albumin ( the only surrogate marker of liver function collected on the pdhs ) , or medication use .", "fetuin - a levels decreased consistently ( p for trend < 0.02 ) across abi clinically relevant cut points ( < 0.7 , 0.70.9 , 0.91.1 , and 1.11.4 ) ( 7 ) ( see supplemental fig . 1 in the online appendix [ available at http://care.diabetesjournals.org/cgi/content/full/dc09-1541/dc1 ] ) . the odds of pad were significantly increased for each sd decrease in fetuin - a ( odds ratio 1.6 [ 95% ci 1.12.3 ] , p = 0.001 ) and the association persisted in incremental models that adjusted fully for age , sex , race , kidney function , cardiovascular risk factors , medication use , and hscrp ( 1.6 [ 1.0582.5 ] , p = 0.03 ) ( table 1 ) .", "a similar trend was noted among subjects with egfr > 80 ml  min  1.73 m ( 1.9 [ 1.03.4 ] , p = 0.05 ) , suggesting that our findings were not confounded by the presence of moderate kidney disease .", "finally , in a fully adjusted subgroup analysis , participants with hscrp levels <3 mg / dl ( n = 460 ) had higher odds of pad ( 2.6 [ 1.45.0 ] , p = 0.002 ) , whereas subjects with high hscrp levels ( 3 mg / dl ; n = 234 ) did not ( 0.82 [ 0.41.6 ] , p = 0.59 ) .", "low levels of fetuin - a have been linked to medial arterial calcification and flow limiting aortic stenosis in humans ( 8,9 ) . in this study , we demonstrate that lower levels of fetuin - a are associated with pad in subjects with type 2 diabetes . to our knowledge , this is the first study to report such a relationship in the absence of advanced kidney disease or prevalent cvd .", "notably , in analysis stratified by the centers for disease control and prevention / american heart association  defined hscrp risk strata ( 10 ) , fetuin - a conferred increased odds of pad in subjects with hscrp <3 mg / dl and also in participants with interleukin ( il)-6 levels below the median ( data not shown ) .", "this is consistent with studies reporting an increased cvd risk mortality with fetuin - a deficiency independent of hscrp and il-6 ( 11 ) and points to a unique role for this negative acute - phase protein as a biomarker of pad beyond traditional and novel cardiovascular risk factors . remarkably , animal models appear to track with our clinical observations . in mice lacking the fetuin - a gene ,", "the aorta was found to be spared of calcification and fibrosis , whereas peripheral vessels in the skin and kidney showed evidence of extensive calcification ( 2 ) , and the small artery involvement preceded the renal impairment ( 3 ) . however , in the absence of direct imaging data , we are unable to define the type of vascular phenotype ( intimal calcification or medial calcification ) that account for the observed association .", "an abi of < 0.9 is 95% sensitive and 99% specific for a stenotic lesion ( > 50% ) ( 12 ) .", "therefore , we assume that some degree of eccentric atherosclerotic calcification contributes to the association observed while acknowledging that multiple types of vascular calcification may coexist in type 2 diabetes ( 13 ) . nonalcoholic liver disease and other phenotypes related to insulin resistance , including type 2 diabetes ( 14 ) , are associated with higher levels of fetuin - a ( 15 ) .", "we controlled for most potential confounding factors and found no attenuation of the inverse association of fetuin - a with pad .", "in particular , because of this inverse relationship , a significant confounding effect was not expected by any condition associated with high fetuin - a levels .", "finally , our study is limited by cross - sectional design , which limits causal inferences . in summary ,", "low plasma fetuin - a levels are associated with pad in type 2 diabetes independent of traditional and contemporary risk factors .", "our findings suggest a unique role for fetuin - a deficiency as a biomarker of pad in the setting of type 2 diabetes ."], "abstract_text": ["<S> objectivefetuin - a is an inhibitor of vascular calcification and a mediator of insulin resistance . </S>", "<S> this study evaluated the association of plasma fetuin - a and peripheral arterial disease ( pad).research design and methodsa total of 738 individuals with type 2 diabetes ( mean age 58.7 years , 37.1% female ) without known cardiovascular or kidney disease were included in this cross - sectional analysis.resultssubjects with pad had a significantly lower fetuin - a ( 264.3 vs. 293.4 ng / dl , p < 0.001 ) . in multivariable analysis , a 1-sd decrease in fetuin - a increased the odds of pad ( odds ratio 1.6 , p = 0.02 ) . </S>", "<S> subgroup analysis revealed an increased odds even in subjects with glomerular filtration rate > 80 ( odds ratio 1.9 , p = 0.05 ) or high - sensitivity c - reactive protein <3 mg / dl ( odds ratio 2.7 , p = 0.002).conclusionslower circulating fetuin - a is associated with pad in type 2 diabetes beyond traditional and novel cardiovascular risk factors . </S>", "<S> our findings suggest a potentially unique role for fetuin - a deficiency as a biomarker of pad in patients with type 2 diabetes . </S>"], "labels": null, "section_names": ["RESEARCH DESIGN AND METHODS", "Evaluated parameters", "Statistical analysis", "RESULTS", "CONCLUSIONS", "Supplementary Material"], "sections": [["the penn diabetes heart study ( pdhs ) is an ongoing , cross - sectional study of individuals with type 2 diabetes ( aged 3575 years ) without clinical evidence of cvd ( myocardial infarction , coronary revascularization or angiographic disease , positive stress test , clinical peripheral arterial disease or peripheral arterial revascularization , stroke , or transient ischemic attack ) .", "the university of pennsylvania institutional review board approved the study protocol , and all subjects gave written informed consent ( 4 ) . the ankle - brachial index ( abi )", "was calculated by dividing the average of the ankle pressures by the highest brachial pressure .", "participants with an abi > 1.4 ( n = 13 ) reflecting noncompressible arteries were excluded ( 5 ) .", "each subject had the estimated glomerular filtration rate ( egfr ) calculated based on the abbreviated modification of diet in renal disease study equation ( 6 ) .", "plasma levels of fetuin - a ( 4.3 and 10.2% , respectively , intra - assay and inter - assay coefficient of variation ) were measured by enzyme - linked immunosorbent assay ( biovendor laboratory medicine , modrice , czech republic ) .", "high - sensitivity c - reactive protein ( hscrp ) was measured by immunoturbidimetric assay ( wako , osaka , japan ) .", "student 's t test was used for normal continuous variables and the kruskal - wallis for nonnormal distributed variables .", "the association between a 1-sd change in fetuin - a and pad was examined using unadjusted and multivariable adjusted logistic regression models as described in table 1 .", "analyses were performed on stata 10.0 software ( stata , college station , tx ) .", "association between fetuin - a and pad data are odds ratios ( 95% ci ) and were obtained by logistic regression model .", "model 2 : model 1 + smoking , hypertension , hypercholesterolemia , metabolic syndrome , a1c , framingham risk score ( % ) , and medications ( ace inhibitors , aspirin , statins , insulin , metformin , sulfonylureas , and thiazolidinediones ) ."], ["the ankle - brachial index ( abi ) was calculated by dividing the average of the ankle pressures by the highest brachial pressure .", "participants with an abi > 1.4 ( n = 13 ) reflecting noncompressible arteries were excluded ( 5 ) .", "each subject had the estimated glomerular filtration rate ( egfr ) calculated based on the abbreviated modification of diet in renal disease study equation ( 6 ) .", "plasma levels of fetuin - a ( 4.3 and 10.2% , respectively , intra - assay and inter - assay coefficient of variation ) were measured by enzyme - linked immunosorbent assay ( biovendor laboratory medicine , modrice , czech republic ) . high - sensitivity c - reactive protein ( hscrp )"], ["student 's t test was used for normal continuous variables and the kruskal - wallis for nonnormal distributed variables .", "the association between a 1-sd change in fetuin - a and pad was examined using unadjusted and multivariable adjusted logistic regression models as described in table 1 .", "analyses were performed on stata 10.0 software ( stata , college station , tx ) .", "association between fetuin - a and pad data are odds ratios ( 95% ci ) and were obtained by logistic regression model .", "model 2 : model 1 + smoking , hypertension , hypercholesterolemia , metabolic syndrome , a1c , framingham risk score ( % ) , and medications ( ace inhibitors , aspirin , statins , insulin , metformin , sulfonylureas , and thiazolidinediones ) ."], ["among 738 subjects in the study sample , pad prevalence was 5.1% , the mean age was 58.7  9.3 years , 37.1% were female , 63.2% were caucasians , and 32.1% were african americans .", "the median fetuin - a level was 292.4 ng / ml ( interquartile range 115.5 ) . individuals with pad had a significantly lower fetuin - a level compared with individuals without pad ( 269.3 vs. 293.4 , p < 0.001 ) .", "no significant differences were noted in other cardiovascular risk factors , metabolic syndrome , kidney function , inflammation , bmi , serum albumin ( the only surrogate marker of liver function collected on the pdhs ) , or medication use .", "fetuin - a levels decreased consistently ( p for trend < 0.02 ) across abi clinically relevant cut points ( < 0.7 , 0.70.9 , 0.91.1 , and 1.11.4 ) ( 7 ) ( see supplemental fig . 1 in the online appendix [ available at http://care.diabetesjournals.org/cgi/content/full/dc09-1541/dc1 ] ) . the odds of pad were significantly increased for each sd decrease in fetuin - a ( odds ratio 1.6 [ 95% ci 1.12.3 ] , p = 0.001 ) and the association persisted in incremental models that adjusted fully for age , sex , race , kidney function , cardiovascular risk factors , medication use , and hscrp ( 1.6 [ 1.0582.5 ] , p = 0.03 ) ( table 1 ) .", "a similar trend was noted among subjects with egfr > 80 ml  min  1.73 m ( 1.9 [ 1.03.4 ] , p = 0.05 ) , suggesting that our findings were not confounded by the presence of moderate kidney disease .", "finally , in a fully adjusted subgroup analysis , participants with hscrp levels <3 mg / dl ( n = 460 ) had higher odds of pad ( 2.6 [ 1.45.0 ] , p = 0.002 ) , whereas subjects with high hscrp levels ( 3 mg / dl ; n = 234 ) did not ( 0.82 [ 0.41.6 ] , p = 0.59 ) ."], ["low levels of fetuin - a have been linked to medial arterial calcification and flow limiting aortic stenosis in humans ( 8,9 ) . in this study , we demonstrate that lower levels of fetuin - a are associated with pad in subjects with type 2 diabetes . to our knowledge , this is the first study to report such a relationship in the absence of advanced kidney disease or prevalent cvd .", "notably , in analysis stratified by the centers for disease control and prevention / american heart association  defined hscrp risk strata ( 10 ) , fetuin - a conferred increased odds of pad in subjects with hscrp <3 mg / dl and also in participants with interleukin ( il)-6 levels below the median ( data not shown ) .", "this is consistent with studies reporting an increased cvd risk mortality with fetuin - a deficiency independent of hscrp and il-6 ( 11 ) and points to a unique role for this negative acute - phase protein as a biomarker of pad beyond traditional and novel cardiovascular risk factors . remarkably , animal models appear to track with our clinical observations . in mice lacking the fetuin - a gene ,", "the aorta was found to be spared of calcification and fibrosis , whereas peripheral vessels in the skin and kidney showed evidence of extensive calcification ( 2 ) , and the small artery involvement preceded the renal impairment ( 3 ) . however , in the absence of direct imaging data , we are unable to define the type of vascular phenotype ( intimal calcification or medial calcification ) that account for the observed association .", "an abi of < 0.9 is 95% sensitive and 99% specific for a stenotic lesion ( > 50% ) ( 12 ) .", "therefore , we assume that some degree of eccentric atherosclerotic calcification contributes to the association observed while acknowledging that multiple types of vascular calcification may coexist in type 2 diabetes ( 13 ) . nonalcoholic liver disease and other phenotypes related to insulin resistance , including type 2 diabetes ( 14 ) , are associated with higher levels of fetuin - a ( 15 ) .", "we controlled for most potential confounding factors and found no attenuation of the inverse association of fetuin - a with pad .", "in particular , because of this inverse relationship , a significant confounding effect was not expected by any condition associated with high fetuin - a levels .", "finally , our study is limited by cross - sectional design , which limits causal inferences . in summary ,", "low plasma fetuin - a levels are associated with pad in type 2 diabetes independent of traditional and contemporary risk factors .", "our findings suggest a unique role for fetuin - a deficiency as a biomarker of pad in the setting of type 2 diabetes ."], [""]]}
{"article_id": "PMC4022822", "article_text": ["collecting duct carcinoma ( cdc ) of the kidney is an unusual variant of renal cell carcinoma ( rcc ) , accountings for less than 1% of all renal cancers .", "cdc arises precisely from the principal cells lining distal collecting ducts of epithelium and distal renal tubules that originates from mesonephros . considering that urothelial carcinoma originating from the ureter , pelvis , or calices also arises from the mesonephros", ", cdc might be similar to urothelial carcinoma and its radiologic and pathologic findings differ from those of other rccs .", "recent publications have pointed out the histological heterogeneity of this neoplasm and its extensive histological overlapping with high grade papillary tumors and urothelial carcinoma .", "accurate diagnosis is important for proper management . in diagnosis of cdc , it is important to distinguish between invasive papillary rcc and urothelial carcinoma .", "positive immunohistochemical staining for distal tubules and collecting duct markers is helpful indiscrimination of cdc from the more commonly diagnosed clear cell rcc of proximal nephron origin .", "cdc generally expresses broad spectrum keratins and high molecular weight ( hmw ) cytokeratin , which is expressed in the lower nephron and the urothelium .", "it also shows positive staining with e - cadherin , epithelial membrane antigen , cke12 , and ck19 .", "however , cd10 , c - kit , and a - methylacylcoa racemase ( amacr ) show no staining .", "in contrast , papillary rcc showed positive results for cd10 and amacr , and it appears to be different from cdc .", "however , this immunohistochemistry is not specific and may be seen in medullary carcinomas and in urothelial carcinoma , including those arising in the renal pelvis .", "although the gross and microscopic features of the tumor are well established , diagnostic confusion can still occur .", "40% of patients have already developed metastatic lesions , including lymphnodes , lungs , or adrenal glands .", "clinical outcome is poor , with 66% of patients dying of the disease within two years after diagnosis .", "various treatments have been proposed , including radiation therapy , immunotherapy , and some combinations of chemotherapy , however , results have been unsatisfactory . to date , no standard therapy for cdc has been established .", "the aim of this study is to conduct an investigation of the clinicopathologic findings of cdc and to determine their correlation with the disease status and prognosis .", "we retrospectively reviewed 35 patients diagnosed with cdc at eight korean medical centers from 1996 to 2009 .", "data on gender , age , initial symptoms , and laboratory findings , including complete blood count profile , calcium , and urine analysis , pathological features , treatment , and patient outcome were obtained from patient medical records .", "diagnosis of cdc was made by examination of a nephrectomy specimen in 27 cases and by renal biopsy in eight .", "tumors were staged according to the 2002 american joint committee on cancer ( ajcc ) tnm stage classification .", "this study was approved by the institutional review board ( irb ) from each participating institution .", "tumor response after treatment was re - evaluated using the response evaluation criteria in solid tumors ( recist ver .", "progression free survival ( pfs ) was estimated from the date treatment began to the date when disease progression was recognized , or the date of the last follow - up visit , or the date of death . overall survival ( os )", "was estimated from the date of diagnosis to the date of death from any cause or the last follow - up visit .", "the cox regression model was used for multivariate analysis with factors that had been used in univariate ( log rank ) analysis of os and pfs .", "p - values less than 0.05 were considered statistically significant and all p - values correspond to two - sided significance tests .", "the median age of patients was 56 years ( range , 29 to 82 years ) and 74% of the patients were male .", "of 32 symptomatic patients , 16 and 11 experienced pain and gross hematuria . other presenting symptoms included weight loss , microscopic hematuria , and a palpable mass .", "seventeen patients had a tumor size of 7 cm or less , and 10 patients had a tumor size of 7 cm or greater .", "the median level of hemoglobin and calcium was 12.5 g / dl ( range , 8.9 to 18.3 g / dl ) and 9.30 g / dl ( range , 7.9 to 11.1 g / dl ) , respectively . according to the immunohistochemistry finding ,", "cdc expressed cytokeratin in nine patients ( 26% ) , hmw - cytokeratin in 14 ( 40% ) , low molecular weight - cytokeratin in three ( 8.6% ) , and cke12f in one ( 2.9% ) .", "it also expressed cd10 in five ( 14.3% ) and vimentin in 11 ( 31.4% ) . at diagnosis , nine , two , four , and 19 patients had tnm stage i , ii , iii , and iv , respectively .", "eight patients had two or more metastatic sites of the bone ( 44% ) , lungs ( 39% ) , liver ( 16% ) , and lymph nodes ( 11% ) as the most common sites . with a median follow - up period of 15.8 months ( range , 0.6 to 88.4 months ) , 14 ( 40% )", "deaths were reported . during the median follow - up period of 15.8 months , 14 patients died , while nine patients ( 25.7% ) were lost in the follow - up .", "twenty seven of the 35 patients underwent nephrectomy for initial treatment ( curative surgery in 17 , and palliative in 10 ) , three patients received chemotherapy , and four patients did not receive any treatment ( fig .", "palliative chemotherapy was administered for 22 persons , who were composed of eight of 14 relapsed patients , eight of 10 patients who were in stage iv and underwent palliative surgery , and four patients who did not undergo an operation ( fig .", "median pfs and os for all patients were 5.8 months ( 95% confidence interval [ ci ] , 3.5 to 9.2 months ) and 54.4 months ( 95% ci , 0 to 109.2 months ) , respectively ( figs . 2 and 3a ) .", "the os of the patients with stages i - iii was 69.9 months ( 95% ci , 54.0 to 85.8 months ) , while that of patients with stage iv was 8.6 months , which showed a statistical significant difference ( p=0.01 ) ( fig .", "3b ) . in addition , among patients with stage iv , the os of patients who received a palliative treatment ( immunotherapy , chemotherapy , or targeted therapy ) was 18.4 months , which was higher than the os of patients without treatment of 4.5 months .", "the pfs of patients with stages i - iii was 6.9 months ( 95% ci , 1.3 to 12.4 months ) .", "recurrence occurred in 14 patients , 82% of the 17 patients who underwent a curative surgery , and their average recurrence period was 5.9 months , with a short pfs and a high relapse rate . using the log - rank method , no relationship was demonstrated between survival end points ( pfs and os ) and explanatory covariates , including patients ' age , gender , and initial calcium level , except for hemoglobin ( p=0.005 and p=0.193 , respectively ) and initial tnm stage ( p=0.022 and p=0.002 , respectively ) .", "results of multivariate regression analysis using a cox 's proportional hazards model showed that tnm stage ( i - iii vs. iv ; hazard ratio , 4.58 ; 95% ci , 1.301 to 16.135 ; p=0.018 ) was an independent prognostic factor for survival of cdc ( table 3 ) .", "the frequency of cdc is within 1% of the entire rcc and its radiologic and pathologic findings differ from those of other rccs . in 1976 ,", "mancilla - jimenez et al .   reported on 34 cases of papillary rcc and postulated a collecting duct origin for three of these tumors based on the findings of atypical hyperplastic changes in adjacent collecting tubules .", "this is the first report on cdc based on medical records from eight institutions in korea . in japan ,", "a retrospective survey was conducted in order to analyze the nature of cdc . in the study ,", "the central pathologists confirmed cdc in 81 of 120 cases diagnosed as cdc at 66 institutions .", "it was a large - scale nationwide survey with an advantage of a multi - institutional central review .", "on the other hand , in this study , 35 patients were selected from eight different organizations nationwide in korea .", "although a pathological central review was not performed , there was significant detailed information on each case with the pattern of cases and treatment outcomes .", "thus , based on such information , the results were evaluated with regard to the types of post operational treatment and the drugs used as palliative treatment and the responses .", "our results are in agreement with those of previous reports showing that the median age was 56 years ( range , 29 to 82 years ) and that males comprised 74% of the patient population . in our study , cdc expressed cytokeratin , hmw - cytokeratin , and cke12 in many cases , however , it also expressed cd10 and vimentin , which is generally expressed in the upper nephron , and not in the lower nephron .", "ninety - one percent of patients had symptoms and the most common presenting symptoms were pain , hematuria , and weight loss . at diagnosis , 19 ( 54% )", "patients were tnm stage iv , and the median os period of patients with stage iv was 9.29 months ( 95% ci , 0.0 to 26.78 months ) .", "a summary of the clinical data on cdc gathered from published series and case reports is shown in table 4 . due to the rarity of its occurrence , optimal treatment for cdc has not been established . despite past reports on striking responses to cytokines , currently , immunotherapy only has an historical role .", "cdc might be distinct from conventional rcc and share embryological origins and biological features with urothelial carcinoma .", "therefore , even if trials comparing immunotherapy with chemotherapy have not been conducted , chemotherapy currently represents the most used therapeutic approach .", "however , it remains unclear whether this carcinoma should be managed with a treatment similar to that for urothelial cell carcinoma or rcc .", "multiple chemotherapeutic and/or immunotherapeutic regimens have been tried for treatment of cdc ( table 4 ) .", "these data appear to suggest that chemotherapy and immunotherapy may offer only limited benefits to a selected group of patients . in our study ,", "surgical treatment was performed as the initial treatment in 77% of patients . however , recurrence occurred in most patients who underwent surgery and a palliative treatment was administered in 75% of patients .", "most patients with advanced or recurrent disease were treated with immunotherapy , chemotherapy , radiation therapy , or targeted therapy .", "the most commonly used agents included interferon , gemcitabine , cisplatin / carboplatin , and sunitinib .", "it seems that patients with stages i - iii had a high relapse rate with a short pfs of 6.9 months , while seven patients ( 58% ) with stages i - iii survived for a long time with patients in the no evidence of disease state , contributing to the increase of the os , so that there was a discrepancy between the pfs and the os .", "most of the long - term survivors were in stages i - iii and those who received palliative treatment after a relapse , and the treatments administered to these patients included target therapy as well as immunotherapy and chemotherapy . due to the small number of patients , the correlation between the prognosis and the treatment could not be known .", "however , it can be assumed that palliative treatment takes the role of extending survival . in paticular ,", "the current standard therapy against rcc is the targeted therapy , and though it is recognized as a different disease from rcc , there were some cdc patients who were treated with sunitinib , temsirolimus , or other targeted agents , different from the past . according to an analysis of clinical aspects , treatment and prognosis in the records of seven cdc patients diagnosed with rcc in procopio 's study and included in patients treated with the target therapy , five persons showed survival of four months while two patients showed long - term survival of 49 months and 19 months , respectively . in this study of the patients who were recently diagnosed and received a target therapy ,", "one patient for whom sunitinib was used finally died , but showed a partial response during treatment .", "cdc is an aggressive disease with poor prognosis , however , like some patients in this study who survived for a long period of time , a study on predictive markers by which the outcomes of prognosis and therapy , especially target therapy as well as their clinical features can be predicted is needed .", "pfs and os were short , however , there were some long - term survivors , therefore , additional research on the predictive markers of several clinical , pathological differences and their treatments will be needed ."], "abstract_text": ["<S> purposecollecting duct carcinoma ( cdc ) of the kidney is an aggressive disease with a poor prognosis , accountings for less than 1% of all renal cancers . to date , no standard therapy for cdc has been established . </S>", "<S> the aim of this study is an investigation of clinicopathologic findings of cdc and correlation of the disease status with a prognosis.materials and methodsfrom 1996 to 2009 , 35 patients with cdc were treated at eight medical centers . </S>", "<S> the diagnosis of cdc was made based on nephrectomy in 27 cases and renal biopsy in eight cases.resultsmedian pfs and os for all patients were 5.8 months ( 95% ci 3.5 to 9.2 ) and 54.4 months ( 95% ci 0 to 109.2 ) , respectively . </S>", "<S> the os of patients with stages i - iii was 69.9 months ( 95% ci 54.0 to 85.8 ) , while that of patients with stage iv was 8.6 months ( 95% ci 0 to 23.3 ) , which showed a statistically significant difference ( p=0.01 ) . in addition , among patients with stage iv , the os of patients who received a palliative treatment ( immunotherapy , chemotherapy , or targeted therapy ) was 18.4 months , which was higher than the os of patients without treatment of 4.5 months.conclusioncdc is a highly aggressive form of renal cell carcinoma . despite most of the treatments , pfs and os were short , however , there were some long - term survivors , therefore , conduct of additional research on the predictive markers of the several clinical , pathological differences and their treatments will be necessary . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion", "Conclusion"], "sections": [["collecting duct carcinoma ( cdc ) of the kidney is an unusual variant of renal cell carcinoma ( rcc ) , accountings for less than 1% of all renal cancers .", "cdc arises precisely from the principal cells lining distal collecting ducts of epithelium and distal renal tubules that originates from mesonephros . considering that urothelial carcinoma originating from the ureter , pelvis , or calices also arises from the mesonephros", ", cdc might be similar to urothelial carcinoma and its radiologic and pathologic findings differ from those of other rccs .", "recent publications have pointed out the histological heterogeneity of this neoplasm and its extensive histological overlapping with high grade papillary tumors and urothelial carcinoma .", "accurate diagnosis is important for proper management . in diagnosis of cdc , it is important to distinguish between invasive papillary rcc and urothelial carcinoma .", "positive immunohistochemical staining for distal tubules and collecting duct markers is helpful indiscrimination of cdc from the more commonly diagnosed clear cell rcc of proximal nephron origin .", "cdc generally expresses broad spectrum keratins and high molecular weight ( hmw ) cytokeratin , which is expressed in the lower nephron and the urothelium .", "it also shows positive staining with e - cadherin , epithelial membrane antigen , cke12 , and ck19 .", "however , cd10 , c - kit , and a - methylacylcoa racemase ( amacr ) show no staining .", "in contrast , papillary rcc showed positive results for cd10 and amacr , and it appears to be different from cdc .", "however , this immunohistochemistry is not specific and may be seen in medullary carcinomas and in urothelial carcinoma , including those arising in the renal pelvis .", "although the gross and microscopic features of the tumor are well established , diagnostic confusion can still occur .", "40% of patients have already developed metastatic lesions , including lymphnodes , lungs , or adrenal glands .", "clinical outcome is poor , with 66% of patients dying of the disease within two years after diagnosis .", "various treatments have been proposed , including radiation therapy , immunotherapy , and some combinations of chemotherapy , however , results have been unsatisfactory . to date , no standard therapy for cdc has been established .", "the aim of this study is to conduct an investigation of the clinicopathologic findings of cdc and to determine their correlation with the disease status and prognosis ."], ["we retrospectively reviewed 35 patients diagnosed with cdc at eight korean medical centers from 1996 to 2009 .", "data on gender , age , initial symptoms , and laboratory findings , including complete blood count profile , calcium , and urine analysis , pathological features , treatment , and patient outcome were obtained from patient medical records .", "diagnosis of cdc was made by examination of a nephrectomy specimen in 27 cases and by renal biopsy in eight .", "tumors were staged according to the 2002 american joint committee on cancer ( ajcc ) tnm stage classification .", "this study was approved by the institutional review board ( irb ) from each participating institution .", "tumor response after treatment was re - evaluated using the response evaluation criteria in solid tumors ( recist ver .", "progression free survival ( pfs ) was estimated from the date treatment began to the date when disease progression was recognized , or the date of the last follow - up visit , or the date of death . overall survival ( os )", "was estimated from the date of diagnosis to the date of death from any cause or the last follow - up visit .", "the cox regression model was used for multivariate analysis with factors that had been used in univariate ( log rank ) analysis of os and pfs .", "p - values less than 0.05 were considered statistically significant and all p - values correspond to two - sided significance tests ."], ["the median age of patients was 56 years ( range , 29 to 82 years ) and 74% of the patients were male .", "of 32 symptomatic patients , 16 and 11 experienced pain and gross hematuria . other presenting symptoms included weight loss , microscopic hematuria , and a palpable mass .", "seventeen patients had a tumor size of 7 cm or less , and 10 patients had a tumor size of 7 cm or greater .", "the median level of hemoglobin and calcium was 12.5 g / dl ( range , 8.9 to 18.3 g / dl ) and 9.30 g / dl ( range , 7.9 to 11.1 g / dl ) , respectively . according to the immunohistochemistry finding ,", "cdc expressed cytokeratin in nine patients ( 26% ) , hmw - cytokeratin in 14 ( 40% ) , low molecular weight - cytokeratin in three ( 8.6% ) , and cke12f in one ( 2.9% ) .", "it also expressed cd10 in five ( 14.3% ) and vimentin in 11 ( 31.4% ) . at diagnosis , nine , two , four , and 19 patients had tnm stage i , ii , iii , and iv , respectively .", "eight patients had two or more metastatic sites of the bone ( 44% ) , lungs ( 39% ) , liver ( 16% ) , and lymph nodes ( 11% ) as the most common sites . with a median follow - up period of 15.8 months ( range , 0.6 to 88.4 months ) , 14 ( 40% )", "deaths were reported . during the median follow - up period of 15.8 months , 14 patients died , while nine patients ( 25.7% ) were lost in the follow - up .", "twenty seven of the 35 patients underwent nephrectomy for initial treatment ( curative surgery in 17 , and palliative in 10 ) , three patients received chemotherapy , and four patients did not receive any treatment ( fig .", "palliative chemotherapy was administered for 22 persons , who were composed of eight of 14 relapsed patients , eight of 10 patients who were in stage iv and underwent palliative surgery , and four patients who did not undergo an operation ( fig .", "median pfs and os for all patients were 5.8 months ( 95% confidence interval [ ci ] , 3.5 to 9.2 months ) and 54.4 months ( 95% ci , 0 to 109.2 months ) , respectively ( figs . 2 and 3a ) .", "the os of the patients with stages i - iii was 69.9 months ( 95% ci , 54.0 to 85.8 months ) , while that of patients with stage iv was 8.6 months , which showed a statistical significant difference ( p=0.01 ) ( fig .", "3b ) . in addition , among patients with stage iv , the os of patients who received a palliative treatment ( immunotherapy , chemotherapy , or targeted therapy ) was 18.4 months , which was higher than the os of patients without treatment of 4.5 months .", "the pfs of patients with stages i - iii was 6.9 months ( 95% ci , 1.3 to 12.4 months ) .", "recurrence occurred in 14 patients , 82% of the 17 patients who underwent a curative surgery , and their average recurrence period was 5.9 months , with a short pfs and a high relapse rate . using the log - rank method , no relationship was demonstrated between survival end points ( pfs and os ) and explanatory covariates , including patients ' age , gender , and initial calcium level , except for hemoglobin ( p=0.005 and p=0.193 , respectively ) and initial tnm stage ( p=0.022 and p=0.002 , respectively ) .", "results of multivariate regression analysis using a cox 's proportional hazards model showed that tnm stage ( i - iii vs. iv ; hazard ratio , 4.58 ; 95% ci , 1.301 to 16.135 ; p=0.018 ) was an independent prognostic factor for survival of cdc ( table 3 ) ."], ["the frequency of cdc is within 1% of the entire rcc and its radiologic and pathologic findings differ from those of other rccs . in 1976 ,", "mancilla - jimenez et al .   reported on 34 cases of papillary rcc and postulated a collecting duct origin for three of these tumors based on the findings of atypical hyperplastic changes in adjacent collecting tubules .", "this is the first report on cdc based on medical records from eight institutions in korea . in japan ,", "a retrospective survey was conducted in order to analyze the nature of cdc . in the study ,", "the central pathologists confirmed cdc in 81 of 120 cases diagnosed as cdc at 66 institutions .", "it was a large - scale nationwide survey with an advantage of a multi - institutional central review .", "on the other hand , in this study , 35 patients were selected from eight different organizations nationwide in korea .", "although a pathological central review was not performed , there was significant detailed information on each case with the pattern of cases and treatment outcomes .", "thus , based on such information , the results were evaluated with regard to the types of post operational treatment and the drugs used as palliative treatment and the responses .", "our results are in agreement with those of previous reports showing that the median age was 56 years ( range , 29 to 82 years ) and that males comprised 74% of the patient population . in our study , cdc expressed cytokeratin , hmw - cytokeratin , and cke12 in many cases , however , it also expressed cd10 and vimentin , which is generally expressed in the upper nephron , and not in the lower nephron .", "ninety - one percent of patients had symptoms and the most common presenting symptoms were pain , hematuria , and weight loss . at diagnosis , 19 ( 54% )", "patients were tnm stage iv , and the median os period of patients with stage iv was 9.29 months ( 95% ci , 0.0 to 26.78 months ) .", "a summary of the clinical data on cdc gathered from published series and case reports is shown in table 4 . due to the rarity of its occurrence , optimal treatment for cdc has not been established . despite past reports on striking responses to cytokines , currently , immunotherapy only has an historical role .", "cdc might be distinct from conventional rcc and share embryological origins and biological features with urothelial carcinoma .", "therefore , even if trials comparing immunotherapy with chemotherapy have not been conducted , chemotherapy currently represents the most used therapeutic approach .", "however , it remains unclear whether this carcinoma should be managed with a treatment similar to that for urothelial cell carcinoma or rcc .", "multiple chemotherapeutic and/or immunotherapeutic regimens have been tried for treatment of cdc ( table 4 ) .", "these data appear to suggest that chemotherapy and immunotherapy may offer only limited benefits to a selected group of patients . in our study ,", "surgical treatment was performed as the initial treatment in 77% of patients . however , recurrence occurred in most patients who underwent surgery and a palliative treatment was administered in 75% of patients .", "most patients with advanced or recurrent disease were treated with immunotherapy , chemotherapy , radiation therapy , or targeted therapy .", "the most commonly used agents included interferon , gemcitabine , cisplatin / carboplatin , and sunitinib .", "it seems that patients with stages i - iii had a high relapse rate with a short pfs of 6.9 months , while seven patients ( 58% ) with stages i - iii survived for a long time with patients in the no evidence of disease state , contributing to the increase of the os , so that there was a discrepancy between the pfs and the os .", "most of the long - term survivors were in stages i - iii and those who received palliative treatment after a relapse , and the treatments administered to these patients included target therapy as well as immunotherapy and chemotherapy . due to the small number of patients , the correlation between the prognosis and the treatment could not be known .", "however , it can be assumed that palliative treatment takes the role of extending survival . in paticular ,", "the current standard therapy against rcc is the targeted therapy , and though it is recognized as a different disease from rcc , there were some cdc patients who were treated with sunitinib , temsirolimus , or other targeted agents , different from the past . according to an analysis of clinical aspects , treatment and prognosis in the records of seven cdc patients diagnosed with rcc in procopio 's study and included in patients treated with the target therapy , five persons showed survival of four months while two patients showed long - term survival of 49 months and 19 months , respectively . in this study of the patients who were recently diagnosed and received a target therapy ,", "one patient for whom sunitinib was used finally died , but showed a partial response during treatment .", "cdc is an aggressive disease with poor prognosis , however , like some patients in this study who survived for a long period of time , a study on predictive markers by which the outcomes of prognosis and therapy , especially target therapy as well as their clinical features can be predicted is needed ."], ["pfs and os were short , however , there were some long - term survivors , therefore , additional research on the predictive markers of several clinical , pathological differences and their treatments will be needed ."]]}
{"article_id": "PMC3284312", "article_text": ["after approval by the institutional review board of the federal university of so paulo , brazil , 500 patients with dm from the diabetic foot outpatient clinic of orthopaedic department of the federal university of so paulo were enrolled in a prospective study to evaluate the results of minimally invasive surgery to treat forefoot ulcerations .", "patients were included for surgery in the study if they had a history of dm type 2 for at least 5 years duration , decreased plantar sensation ( detected with 10 g semmes  weinstein monofilament ) , dorsiflexion range of motion of the ankle equal to or less than 10 measured via goniometer ( fig .", "1 ) , palpable distal pulse , and history of forefoot ulceration healed with proper treatment ( 10 , 11 ) .", "patients were excluded from surgery if they had an infected ulceration , dorsiflexion of the ankle greater than 10 , and absent distal pulse on clinical examination .", "fifty - two patients met the study criteria and the average age was 66.4 years .", "a modification of white 's surgical technique was performed to address the equinus contracture with three hemisections of the achilles tendon , with specific configuration according to the clinical presentation of the hindfoot and ankle ( 10 ) .", "the proximal and distal hemisections were made at the same side of the achilles tendon , and the intermediate cut was performed at the opposite side .", "a valgus hindfoot received proximal and distal hemisections at the lateral side of the tendon , whereas a varus hindfoot was addressed with medial side hemisections ( fig .", "2 ) . the postoperative care included plaster short - leg cast immobilization to maintain maximal dorsiflexion . after 1 week", ", a total contact cast with full weight bearing as tolerated was applied for 6 weeks .", "physical therapy was then initiated to maintain ankle position , calf strengthening , proprioceptive improvement , and gait training in an accommodative walking boot for an additional 6 weeks ( 8 , 12 ) . \n fig , 2 .", "a modification of white 's surgical technique was performed to address the equinus contracture with three hemisections of the achilles tendon , with specific configuration according to the clinical presentation of the hindfoot and ankle ( 10 ) .", "the proximal and distal hemisections were made at the same side of the achilles tendon , and the intermediate cut was performed at the opposite side .", "a valgus hindfoot received proximal and distal hemisections at the lateral side of the tendon , whereas a varus hindfoot was addressed with medial side hemisections ( fig .", "2 ) . the postoperative care included plaster short - leg cast immobilization to maintain maximal dorsiflexion . after 1 week", ", a total contact cast with full weight bearing as tolerated was applied for 6 weeks .", "physical therapy was then initiated to maintain ankle position , calf strengthening , proprioceptive improvement , and gait training in an accommodative walking boot for an additional 6 weeks ( 8 , 12 ) . \n", "fifty - two patients in the study underwent surgical treatment consisting of percutaneous achilles tendon lengthening through a modified white 's technique to treat forefoot ulcerations .", "forty - eight patients ( 92% ) had no recurrence of ulcerations during the follow - up and demonstrated improved foot function .", "follow - up was performed through a multidisciplinary team to maintain metabolic and glycemic control , provide education strategies to prevent recurrence of lesions , and also physical therapy for adequate function and protection .", "patients with recurrent forefoot ulcerations ( n=4 feet ) , which occurred in the same location as the original ulcer , also underwent a percutaneous distal metatarsal osteotomy ( fig .", "3 ) of the associated metatarsal and were permitted full weight bearing in a total contact cast for 4 weeks .", "thereafter , walking boots and custom shoes were prescribed . during the study , none of the patients presented with infection , pain , or necrosis .", "diabetes mellitus is a chronic disease with potential complications that are responsible for high rates of morbidity and mortality .", "the presence of plantar foot ulcerations in these patients increases the risk of infection , sepsis , and amputation .", "early detection , multidisciplinary treatment , and education are critical for preventing any related lower extremity amputations .", "our study demonstrated success in 92% of patients for prevention of recurrent forefoot ulceration in patients with ankle equinus , suggesting the usefulness of a minimally invasive approach for percutaneous lengthening of the achilles tendon in these patients .", "dm can affect all components of the nervous system , including light touch sensation , motor control , pain recognition , proprioception , and autonomic function .", "absence of protective sensation and the presence of foot deformities are the clinical factors with the highest correlation for the development of foot ulcerations and eventual lower extremity amputation ( 8 , 1315 ) .", "in fact , 85% of lower limb amputations are preceded by plantar ulcerations ( 16 ) .", "prevention and early treatment of these lesions can maintain satisfactory biomechanical and functional longevity of the lower extremity .", "longer duration of dm is associated with the development of calf shortening due to structural changes , glycated collagen of tendon fibers , and the presence of peripheral sensory neuropathy , contributing strong risk factors for forefoot ulceration .", "clinical and imaging assays demonstrate that these patients often present with heterogeneous patterns of tendon fibers and sometimes contain internal calcifications .", "anatomic physiologic patterns show significant modification within the calcaneal tendon that becomes a solid tendon ( 12 , 1719 ) . as a consequence , patients present with limited range of motion of the ankle , physiologic loss of collagen elasticity , gait alterations , and inability of the foot to act as an energy absorber leading to elevated forefoot pressures ( 9 , 18 , 19 ) .", "diabetic prophylactic foot surgery plays an important role in establishing optimal biomechanical function as well as preventing and treating foot ulcerations ( 8 , 11 , 12 , 17 ) .", "our study illustrates that percutaneous achilles tendon lengthening with a modification of white 's original technique constitutes an important mechanism to restore appropriate range of motion to the ankle , improving gait quality , and preventing recurrent forefoot ulcerations .", "when necessary in cases of ulcer recurrence after achilles tendon lengthening , the percutaneous distal metatarsal osteotomy has been recommended ( 9 ) .", "we believed that the ulcer recurrences were associated with anatomic disturbance of the involved metatarsal such as elongation and plantar flexion ( 9 ) .", "this study also highlights successful treatment of forefoot ulcerations and prevention of recurrence during a follow - up period in a large cohort of subjects via a multidisciplinary team approach .", "prophylactic surgery of the diabetic foot is a viable option in advanced treatment clinics to avoid foot complications and treat forefoot ulceration in diabetic patients . in an effort to reduce forefoot pressures and", "subsequently prevent ulcer recurrence , our study demonstrates an effective , reproducible surgical technique for percutaneous achilles tendon lengthening with good tolerance by patients , and minimal complications .", "the authors have not received any funding or benefits from industry to conduct this study ."], "abstract_text": ["<S> complications of diabetes mellitus constitute the most common indications for hospitalization and non - traumatic amputations in the usa . the most important risk factors for the development of diabetic foot ulcerations include the presence of peripheral neuropathy , vasculopathy , limited joint mobility , and pre - existing foot deformities . in our study , 500 diabetic patients treated for plantar forefoot ulcerations were enrolled in a prospective study from 2000 to 2008 at the federal university of so paulo , brazil . </S>", "<S> fifty - two patients in the study met the criteria and underwent surgical treatment consisting of percutaneous achilles tendon lengthening to treat plantar forefoot ulcerations . </S>", "<S> the postoperative follow - up demonstrated prevention of recurrent foot ulcerations in 92% of these diabetic patients that maintained an improved foot function . in conclusion , </S>", "<S> our study supports that identification and treatment of ankle equinus in the diabetic population may potentially lead to decreased patient morbidity , including reduced risk for both reulceration , and potential lower extremity amputation . </S>"], "labels": null, "section_names": ["Materials and methods", "Operative technique and postoperative care", "Results", "Discussion", "Conclusion", "Conflict of interest and funding"], "sections": [["after approval by the institutional review board of the federal university of so paulo , brazil , 500 patients with dm from the diabetic foot outpatient clinic of orthopaedic department of the federal university of so paulo were enrolled in a prospective study to evaluate the results of minimally invasive surgery to treat forefoot ulcerations .", "patients were included for surgery in the study if they had a history of dm type 2 for at least 5 years duration , decreased plantar sensation ( detected with 10 g semmes  weinstein monofilament ) , dorsiflexion range of motion of the ankle equal to or less than 10 measured via goniometer ( fig .", "1 ) , palpable distal pulse , and history of forefoot ulceration healed with proper treatment ( 10 , 11 ) .", "patients were excluded from surgery if they had an infected ulceration , dorsiflexion of the ankle greater than 10 , and absent distal pulse on clinical examination .", "fifty - two patients met the study criteria and the average age was 66.4 years .", "a modification of white 's surgical technique was performed to address the equinus contracture with three hemisections of the achilles tendon , with specific configuration according to the clinical presentation of the hindfoot and ankle ( 10 ) .", "the proximal and distal hemisections were made at the same side of the achilles tendon , and the intermediate cut was performed at the opposite side .", "a valgus hindfoot received proximal and distal hemisections at the lateral side of the tendon , whereas a varus hindfoot was addressed with medial side hemisections ( fig .", "2 ) . the postoperative care included plaster short - leg cast immobilization to maintain maximal dorsiflexion . after 1 week", ", a total contact cast with full weight bearing as tolerated was applied for 6 weeks .", "physical therapy was then initiated to maintain ankle position , calf strengthening , proprioceptive improvement , and gait training in an accommodative walking boot for an additional 6 weeks ( 8 , 12 ) . \n fig , 2 ."], ["a modification of white 's surgical technique was performed to address the equinus contracture with three hemisections of the achilles tendon , with specific configuration according to the clinical presentation of the hindfoot and ankle ( 10 ) .", "the proximal and distal hemisections were made at the same side of the achilles tendon , and the intermediate cut was performed at the opposite side .", "a valgus hindfoot received proximal and distal hemisections at the lateral side of the tendon , whereas a varus hindfoot was addressed with medial side hemisections ( fig .", "2 ) . the postoperative care included plaster short - leg cast immobilization to maintain maximal dorsiflexion . after 1 week", ", a total contact cast with full weight bearing as tolerated was applied for 6 weeks .", "physical therapy was then initiated to maintain ankle position , calf strengthening , proprioceptive improvement , and gait training in an accommodative walking boot for an additional 6 weeks ( 8 , 12 ) . \n"], ["fifty - two patients in the study underwent surgical treatment consisting of percutaneous achilles tendon lengthening through a modified white 's technique to treat forefoot ulcerations .", "forty - eight patients ( 92% ) had no recurrence of ulcerations during the follow - up and demonstrated improved foot function .", "follow - up was performed through a multidisciplinary team to maintain metabolic and glycemic control , provide education strategies to prevent recurrence of lesions , and also physical therapy for adequate function and protection .", "patients with recurrent forefoot ulcerations ( n=4 feet ) , which occurred in the same location as the original ulcer , also underwent a percutaneous distal metatarsal osteotomy ( fig .", "3 ) of the associated metatarsal and were permitted full weight bearing in a total contact cast for 4 weeks .", "thereafter , walking boots and custom shoes were prescribed . during the study , none of the patients presented with infection , pain , or necrosis ."], ["diabetes mellitus is a chronic disease with potential complications that are responsible for high rates of morbidity and mortality .", "the presence of plantar foot ulcerations in these patients increases the risk of infection , sepsis , and amputation .", "early detection , multidisciplinary treatment , and education are critical for preventing any related lower extremity amputations .", "our study demonstrated success in 92% of patients for prevention of recurrent forefoot ulceration in patients with ankle equinus , suggesting the usefulness of a minimally invasive approach for percutaneous lengthening of the achilles tendon in these patients .", "dm can affect all components of the nervous system , including light touch sensation , motor control , pain recognition , proprioception , and autonomic function .", "absence of protective sensation and the presence of foot deformities are the clinical factors with the highest correlation for the development of foot ulcerations and eventual lower extremity amputation ( 8 , 1315 ) .", "in fact , 85% of lower limb amputations are preceded by plantar ulcerations ( 16 ) .", "prevention and early treatment of these lesions can maintain satisfactory biomechanical and functional longevity of the lower extremity .", "longer duration of dm is associated with the development of calf shortening due to structural changes , glycated collagen of tendon fibers , and the presence of peripheral sensory neuropathy , contributing strong risk factors for forefoot ulceration .", "clinical and imaging assays demonstrate that these patients often present with heterogeneous patterns of tendon fibers and sometimes contain internal calcifications .", "anatomic physiologic patterns show significant modification within the calcaneal tendon that becomes a solid tendon ( 12 , 1719 ) . as a consequence , patients present with limited range of motion of the ankle , physiologic loss of collagen elasticity , gait alterations , and inability of the foot to act as an energy absorber leading to elevated forefoot pressures ( 9 , 18 , 19 ) .", "diabetic prophylactic foot surgery plays an important role in establishing optimal biomechanical function as well as preventing and treating foot ulcerations ( 8 , 11 , 12 , 17 ) .", "our study illustrates that percutaneous achilles tendon lengthening with a modification of white 's original technique constitutes an important mechanism to restore appropriate range of motion to the ankle , improving gait quality , and preventing recurrent forefoot ulcerations .", "when necessary in cases of ulcer recurrence after achilles tendon lengthening , the percutaneous distal metatarsal osteotomy has been recommended ( 9 ) .", "we believed that the ulcer recurrences were associated with anatomic disturbance of the involved metatarsal such as elongation and plantar flexion ( 9 ) .", "this study also highlights successful treatment of forefoot ulcerations and prevention of recurrence during a follow - up period in a large cohort of subjects via a multidisciplinary team approach ."], ["prophylactic surgery of the diabetic foot is a viable option in advanced treatment clinics to avoid foot complications and treat forefoot ulceration in diabetic patients . in an effort to reduce forefoot pressures and", "subsequently prevent ulcer recurrence , our study demonstrates an effective , reproducible surgical technique for percutaneous achilles tendon lengthening with good tolerance by patients , and minimal complications ."], ["the authors have not received any funding or benefits from industry to conduct this study ."]]}
{"article_id": "PMC3888855", "article_text": ["a 40-year - old woman , 169 cm , 57 kg , underwent laparoscopic right adrenalectomy due to an adrenal pheochromocytoma .", "a 7.5 fr 3-lumen catheter was inserted under ultrasonographic guidance ( linear probe : ust-5546 , 5 - 10 mhz , machine : prosound ssd-4400 , aloka medical , co. , ltd . ,", "tokyo , japan ) into the right internal jugular vein , and lung sliding was confirmed in both thoraces .", "next , the patient was placed into the left lateral decubitus position . before incision , etco2 , pmax , and peripheral oxygen saturation ( spo2 )", "pneumoperitoneum was established using co2 , intraabdominal pressure was maintained < 15 mmhg , and pmax was 23 cmh2o at the time .", "thirty minutes after inducing pneumoperitoneum , the etco2 and pmax levels gradually increased to 43 mmhg and 30 cmh2o , respectively .", "however , the right side of the chest demonstrated no sounds . immediately , the fraction of inspired oxygen ( fio2 ) increased from 0.5 to 0.8 , and the endotracheal tube was withdrawn back to 2 cm in order to rule out endobronchial intubation . because the depth of the endotracheal tube was 18 cm at the incisor teeth and confirmed the depth of endotracheal tube by cuff palpation at the sternal notch , we supposed that it was enough to rule out one - lung intubation without using bronchoscope .", "spo2 was maintained at between 98 - 100% , though the right thorax was still silent .", "1a ) , however there was no sliding with barcode sign in the right lung ( fig . 1b ) .", "although the surgeon simultaneously inspected the diaphragm in detail , no apparent injuries or defects were found . because the vital signs were stable and spo2 and etco2 were normal , as indicated by an increased fio2 and respiratory rate , the operation was laparoscopically completed .", "when the operation was concluded , a positive end - expiratory pressure ( peep ) of 5 cmh2o was applied to inflate the collapsed lung .", "the patient was examined using ultrasonography whilst in the supine position , and at the same time , portable chest radiography was called to assess a pneumothroax .", "the ultrasonographic probe was placed on the right lower lateral chest wall between anterior and mid axillary line ( fig .", "2 ) , and we identified the \" lung point \" sign , which can be used to diagnose pneumothorax . also , chest radiography indicated a < 30% collapse of the right lung ( fig .", "auscultation of the right lung improved , as well as the appearance of lung sliding sign and the disappearance of the lung point sign in whole right hemithorax within about 30 minutes after the end of the operation .", "no hemodynamic instability was noted , and spo2 was maintained at 100% using only medical air .", "hence , pneumthorax was considered to be capnothorax , and the patient was extubated without the insertion of a chest tube .", "the patient was transferred to the postanesthetic care unit ( pacu ) and did not complain about any respiratory disturbance .", "a 39-year - old man , 165 cm , 63 kg , was scheduled for living right donor nephrectomy via hand - assisted laparoscopic surgery ( hals ) .", "the induction of anesthesia with thiopental sodium , fentanyl , vecuronium and desflurane was uneventful .", "after co2-pneumoperitoneum was induced , the intraabdominal pressure was maintained < 14 mmhg , and etco2 , pmax , and spo2 were 33 mmhg , 23 cmh2o and 100% , respectively .", "when the upper pole of the kidney was approached , the diaphragm was injured by the dissector , which was immediately repaired .", "the surgeon inquired about the status of the right lung because the right hemidiaphragm was bulging .", "the lung sounds in the right chest were decreased on auscultation , and etco2 and pmax were increased to 42 mmhg and 30 cmh2o , respectively .", ", we evaluated the right thorax using ultrasonography ( linear probe : l25x , 6 - 12 mhz , machine : m - turbo system , sonosite inc . ,", "bothell , wa , usa ) in the operative field , and the \" lung point \" sign was found at the right lower anterior axillary line ( fig .", ", a small amount of pneumothorax was expected . however , because the hemodynamic parameters were maintained in a normal range , the operation was continued via hals and a peep of 5 cmh2o was applied . at the end of the surgery ,", "the surgeon requested a portable chest radiography and we concurrently examined the patient using ultrasonography in the supine position .", "3b ) , and the range of the pneumothorax which was indicated by the lung point sign was gradually diminished . because signs of cardiopulmonary instability were not found and spo2 was maintained without an increase in fio2 , the insertion of a chest tube was not necessary and we safely extubated the endotracheal tube .", "the patient was transferred to the pacu and did not complain of any respiratory discomfort .", "a 40-year - old woman , 169 cm , 57 kg , underwent laparoscopic right adrenalectomy due to an adrenal pheochromocytoma .", "a 7.5 fr 3-lumen catheter was inserted under ultrasonographic guidance ( linear probe : ust-5546 , 5 - 10 mhz , machine : prosound ssd-4400 , aloka medical , co. , ltd . ,", "tokyo , japan ) into the right internal jugular vein , and lung sliding was confirmed in both thoraces .", "next , the patient was placed into the left lateral decubitus position . before incision , etco2 , pmax , and peripheral oxygen saturation ( spo2 )", "pneumoperitoneum was established using co2 , intraabdominal pressure was maintained < 15 mmhg , and pmax was 23 cmh2o at the time .", "thirty minutes after inducing pneumoperitoneum , the etco2 and pmax levels gradually increased to 43 mmhg and 30 cmh2o , respectively .", "however , the right side of the chest demonstrated no sounds . immediately , the fraction of inspired oxygen ( fio2 ) increased from 0.5 to 0.8 , and the endotracheal tube was withdrawn back to 2 cm in order to rule out endobronchial intubation . because the depth of the endotracheal tube was 18 cm at the incisor teeth and confirmed the depth of endotracheal tube by cuff palpation at the sternal notch , we supposed that it was enough to rule out one - lung intubation without using bronchoscope .", "spo2 was maintained at between 98 - 100% , though the right thorax was still silent .", "1a ) , however there was no sliding with barcode sign in the right lung ( fig . 1b ) .", "although the surgeon simultaneously inspected the diaphragm in detail , no apparent injuries or defects were found . because the vital signs were stable and spo2 and etco2 were normal , as indicated by an increased fio2 and respiratory rate , the operation was laparoscopically completed .", "when the operation was concluded , a positive end - expiratory pressure ( peep ) of 5 cmh2o was applied to inflate the collapsed lung .", "the patient was examined using ultrasonography whilst in the supine position , and at the same time , portable chest radiography was called to assess a pneumothroax .", "the ultrasonographic probe was placed on the right lower lateral chest wall between anterior and mid axillary line ( fig .", "2 ) , and we identified the \" lung point \" sign , which can be used to diagnose pneumothorax . also , chest radiography indicated a < 30% collapse of the right lung ( fig .", "auscultation of the right lung improved , as well as the appearance of lung sliding sign and the disappearance of the lung point sign in whole right hemithorax within about 30 minutes after the end of the operation .", "no hemodynamic instability was noted , and spo2 was maintained at 100% using only medical air .", "hence , pneumthorax was considered to be capnothorax , and the patient was extubated without the insertion of a chest tube .", "the patient was transferred to the postanesthetic care unit ( pacu ) and did not complain about any respiratory disturbance .", "a 39-year - old man , 165 cm , 63 kg , was scheduled for living right donor nephrectomy via hand - assisted laparoscopic surgery ( hals ) .", "the induction of anesthesia with thiopental sodium , fentanyl , vecuronium and desflurane was uneventful .", "after co2-pneumoperitoneum was induced , the intraabdominal pressure was maintained < 14 mmhg , and etco2 , pmax , and spo2 were 33 mmhg , 23 cmh2o and 100% , respectively .", "when the upper pole of the kidney was approached , the diaphragm was injured by the dissector , which was immediately repaired .", "the surgeon inquired about the status of the right lung because the right hemidiaphragm was bulging .", "the lung sounds in the right chest were decreased on auscultation , and etco2 and pmax were increased to 42 mmhg and 30 cmh2o , respectively .", ", we evaluated the right thorax using ultrasonography ( linear probe : l25x , 6 - 12 mhz , machine : m - turbo system , sonosite inc . ,", "bothell , wa , usa ) in the operative field , and the \" lung point \" sign was found at the right lower anterior axillary line ( fig .", "however , because the hemodynamic parameters were maintained in a normal range , the operation was continued via hals and a peep of 5 cmh2o was applied . at the end of the surgery ,", "the surgeon requested a portable chest radiography and we concurrently examined the patient using ultrasonography in the supine position .", "3b ) , and the range of the pneumothorax which was indicated by the lung point sign was gradually diminished . because signs of cardiopulmonary instability were not found and spo2 was maintained without an increase in fio2 , the insertion of a chest tube was not necessary and we safely extubated the endotracheal tube .", "the patient was transferred to the pacu and did not complain of any respiratory discomfort .", "patients under general anesthesia may develop pneumothorax due to a variety of reasons , such as barotraumas , rupture of the lung bullae or needle insertion into the pleural cavity .", "capnothorax due to co2 during laparoscopic surgery is rare , but , it has been described as a complication of almost every type of laparoscopic surgery [ 1 - 3,6 - 8 ] .", "it has been speculated that an abnormal diaphragm caused by congenital failure of fusion ( e.g. , sternal , costal , and lumbar portions ) , weak point ( e.g. , around the aorta , vena cava , and esophagus ) , congenital defects or iatrogenic injuries by the instrument can lead to capnothorax [ 1 - 3,6 ] . because the creation of pneumoperitoneum and positional changes can demonstrate several potential physiological alterations , the accurate diagnosis of pneumothorax is more difficult during laparoscopic surgery than other surgical procedure .", "when pneumothorax is suspected , it is traditionally diagnosed by portable chest radiography in the operating room .", "however , chest radiography may take a considerable amount of time and demonstrates a sensitivity of 31.8 - 75.5% for the diagnosis of pneumothorax [ 10 - 12 ] .", "in many different fields , lung ultrasonography is often used to diagnose pathological conditions of the lung . in particular , ultrasonography has received attention as a very effective method for confirming pneumothorax at the bedside in the operating room . in numerous studies ,", "ultrasonography has been reported as superior to conventional portable chest radiography for the detection of pneumothorax [ 4,10 - 13 ] .", "the presence or absence of characteristic ultrasonographic signs are key to a correct diagnosis , i.e. , lung sliding , lung pulse , comet - tail artifact , barcode sign ( stratosphere sign ) , seashore sign and lung point sign . among these signs ,", "however , the absence of lung sliding does not always indicate pneumothorax due to several mechanical factors ( e.g. , poor technique , inappropriate ultrasound probe frequency , the presence of dynamic noise filters ) and the patient 's affliction ( e.g. , main bronchial intubation , emphysema , atelectasis , pleural calcification , pleural adhesion , lung fibrosis ) . for these reasons ,", "more specific ultrasonographic methods are needed to confirm pneumothorax . in a particular area of the thorax", ", the ultrasonographic patterns of pneumothorax and the normal lung alternatively appear depending on the respiratory cycle .", "this can be explained by a slight increase in pulmonary volume during inspiration and can be observed in two ultrasonographic modes : real - time ( b - mode ) and time - motion ( m - mode ) . in b - mode , the lung point sign can be found by observing lung - sliding and absent of lung - sliding alternately at a same pleural line .", "analogous patterns also can be found in m - mode . seashore sign and barcode sign", "can be observed in turn , and these signs demonstrate the specificity of 100% for the diagnosis of pneumothorax .", "furthermore , by serially observing the location of the sign , the extension of pneumothorax may be identified . despite this wide spread practice , there are few studies about the relationship between the lung point sign and the extension of pneumothorax", ". when it moves to the lower lateral chest wall , the volume of pneumothorax expands and the patient may need to have a chest tube immediately inserted . on the other hand , movement of the sign from the lower lateral chest to the anterior chest indicates an improvement in pneumothorax ( fig .", "3 ) . if capnothorax is highly suspected and cardiopulmonary instability is not severe , alteration of the \" lung point \" sign can be used to identify improvements in pneumothorax .", "in addition , for faster recovery of capnothorax , application of peep is supportive method to absorb co2 more readily from the pleural cavity .", "therefore , a chest tube insertion , which can cause injury to other structures in the thorax , may not be necessary in these cases . in conclusion", ", the lung point sign may predict the range and amount of pneumothorax during laparoscopic surgery .", "ultrasonography can not only help to diagnose pneumothorax , but can also be used to decide treatment of pneumothorax .", "most notably , in the case of capnothorax which resolves spontaneously as time goes on , the unnecessary insertion of a chest tube may be avoided by observing alterations in the lung point sign ."], "abstract_text": ["<S> pneumothorax during general anesthesia is more difficult to diagnose compared with that of non - anesthetized patient . </S>", "<S> furthermore , the early diagnosis of pneumothorax is to some extent difficult due to co2-pneumoperitoneum during laparoscopic surgery . </S>", "<S> the use of ultrasonography to diagnose pneumothorax has increased in a variety of situations , demonstrating a better diagnostic rate than conventional chest radiography . here , </S>", "<S> we report two cases of intraoperative capnothorax that were confirmed using the m - mode \" lung point \" sign . </S>", "<S> however , the insertion of a chest tube could have been avoided because the spontaneous resolution of capnothorax was quickly identified using bedside lung ultrasonography . </S>"], "labels": null, "section_names": ["Case Reports", "Case 1", "Case 2", "Discussion"], "sections": [["a 40-year - old woman , 169 cm , 57 kg , underwent laparoscopic right adrenalectomy due to an adrenal pheochromocytoma .", "a 7.5 fr 3-lumen catheter was inserted under ultrasonographic guidance ( linear probe : ust-5546 , 5 - 10 mhz , machine : prosound ssd-4400 , aloka medical , co. , ltd . ,", "tokyo , japan ) into the right internal jugular vein , and lung sliding was confirmed in both thoraces .", "next , the patient was placed into the left lateral decubitus position . before incision , etco2 , pmax , and peripheral oxygen saturation ( spo2 )", "pneumoperitoneum was established using co2 , intraabdominal pressure was maintained < 15 mmhg , and pmax was 23 cmh2o at the time .", "thirty minutes after inducing pneumoperitoneum , the etco2 and pmax levels gradually increased to 43 mmhg and 30 cmh2o , respectively .", "however , the right side of the chest demonstrated no sounds . immediately , the fraction of inspired oxygen ( fio2 ) increased from 0.5 to 0.8 , and the endotracheal tube was withdrawn back to 2 cm in order to rule out endobronchial intubation . because the depth of the endotracheal tube was 18 cm at the incisor teeth and confirmed the depth of endotracheal tube by cuff palpation at the sternal notch , we supposed that it was enough to rule out one - lung intubation without using bronchoscope .", "spo2 was maintained at between 98 - 100% , though the right thorax was still silent .", "1a ) , however there was no sliding with barcode sign in the right lung ( fig . 1b ) .", "although the surgeon simultaneously inspected the diaphragm in detail , no apparent injuries or defects were found . because the vital signs were stable and spo2 and etco2 were normal , as indicated by an increased fio2 and respiratory rate , the operation was laparoscopically completed .", "when the operation was concluded , a positive end - expiratory pressure ( peep ) of 5 cmh2o was applied to inflate the collapsed lung .", "the patient was examined using ultrasonography whilst in the supine position , and at the same time , portable chest radiography was called to assess a pneumothroax .", "the ultrasonographic probe was placed on the right lower lateral chest wall between anterior and mid axillary line ( fig .", "2 ) , and we identified the \" lung point \" sign , which can be used to diagnose pneumothorax . also , chest radiography indicated a < 30% collapse of the right lung ( fig .", "auscultation of the right lung improved , as well as the appearance of lung sliding sign and the disappearance of the lung point sign in whole right hemithorax within about 30 minutes after the end of the operation .", "no hemodynamic instability was noted , and spo2 was maintained at 100% using only medical air .", "hence , pneumthorax was considered to be capnothorax , and the patient was extubated without the insertion of a chest tube .", "the patient was transferred to the postanesthetic care unit ( pacu ) and did not complain about any respiratory disturbance .", "a 39-year - old man , 165 cm , 63 kg , was scheduled for living right donor nephrectomy via hand - assisted laparoscopic surgery ( hals ) .", "the induction of anesthesia with thiopental sodium , fentanyl , vecuronium and desflurane was uneventful .", "after co2-pneumoperitoneum was induced , the intraabdominal pressure was maintained < 14 mmhg , and etco2 , pmax , and spo2 were 33 mmhg , 23 cmh2o and 100% , respectively .", "when the upper pole of the kidney was approached , the diaphragm was injured by the dissector , which was immediately repaired .", "the surgeon inquired about the status of the right lung because the right hemidiaphragm was bulging .", "the lung sounds in the right chest were decreased on auscultation , and etco2 and pmax were increased to 42 mmhg and 30 cmh2o , respectively .", ", we evaluated the right thorax using ultrasonography ( linear probe : l25x , 6 - 12 mhz , machine : m - turbo system , sonosite inc . ,", "bothell , wa , usa ) in the operative field , and the \" lung point \" sign was found at the right lower anterior axillary line ( fig .", ", a small amount of pneumothorax was expected . however , because the hemodynamic parameters were maintained in a normal range , the operation was continued via hals and a peep of 5 cmh2o was applied . at the end of the surgery ,", "the surgeon requested a portable chest radiography and we concurrently examined the patient using ultrasonography in the supine position .", "3b ) , and the range of the pneumothorax which was indicated by the lung point sign was gradually diminished . because signs of cardiopulmonary instability were not found and spo2 was maintained without an increase in fio2 , the insertion of a chest tube was not necessary and we safely extubated the endotracheal tube .", "the patient was transferred to the pacu and did not complain of any respiratory discomfort ."], ["a 40-year - old woman , 169 cm , 57 kg , underwent laparoscopic right adrenalectomy due to an adrenal pheochromocytoma .", "a 7.5 fr 3-lumen catheter was inserted under ultrasonographic guidance ( linear probe : ust-5546 , 5 - 10 mhz , machine : prosound ssd-4400 , aloka medical , co. , ltd . ,", "tokyo , japan ) into the right internal jugular vein , and lung sliding was confirmed in both thoraces .", "next , the patient was placed into the left lateral decubitus position . before incision , etco2 , pmax , and peripheral oxygen saturation ( spo2 )", "pneumoperitoneum was established using co2 , intraabdominal pressure was maintained < 15 mmhg , and pmax was 23 cmh2o at the time .", "thirty minutes after inducing pneumoperitoneum , the etco2 and pmax levels gradually increased to 43 mmhg and 30 cmh2o , respectively .", "however , the right side of the chest demonstrated no sounds . immediately , the fraction of inspired oxygen ( fio2 ) increased from 0.5 to 0.8 , and the endotracheal tube was withdrawn back to 2 cm in order to rule out endobronchial intubation . because the depth of the endotracheal tube was 18 cm at the incisor teeth and confirmed the depth of endotracheal tube by cuff palpation at the sternal notch , we supposed that it was enough to rule out one - lung intubation without using bronchoscope .", "spo2 was maintained at between 98 - 100% , though the right thorax was still silent .", "1a ) , however there was no sliding with barcode sign in the right lung ( fig . 1b ) .", "although the surgeon simultaneously inspected the diaphragm in detail , no apparent injuries or defects were found . because the vital signs were stable and spo2 and etco2 were normal , as indicated by an increased fio2 and respiratory rate , the operation was laparoscopically completed .", "when the operation was concluded , a positive end - expiratory pressure ( peep ) of 5 cmh2o was applied to inflate the collapsed lung .", "the patient was examined using ultrasonography whilst in the supine position , and at the same time , portable chest radiography was called to assess a pneumothroax .", "the ultrasonographic probe was placed on the right lower lateral chest wall between anterior and mid axillary line ( fig .", "2 ) , and we identified the \" lung point \" sign , which can be used to diagnose pneumothorax . also , chest radiography indicated a < 30% collapse of the right lung ( fig .", "auscultation of the right lung improved , as well as the appearance of lung sliding sign and the disappearance of the lung point sign in whole right hemithorax within about 30 minutes after the end of the operation .", "no hemodynamic instability was noted , and spo2 was maintained at 100% using only medical air .", "hence , pneumthorax was considered to be capnothorax , and the patient was extubated without the insertion of a chest tube .", "the patient was transferred to the postanesthetic care unit ( pacu ) and did not complain about any respiratory disturbance ."], ["a 39-year - old man , 165 cm , 63 kg , was scheduled for living right donor nephrectomy via hand - assisted laparoscopic surgery ( hals ) .", "the induction of anesthesia with thiopental sodium , fentanyl , vecuronium and desflurane was uneventful .", "after co2-pneumoperitoneum was induced , the intraabdominal pressure was maintained < 14 mmhg , and etco2 , pmax , and spo2 were 33 mmhg , 23 cmh2o and 100% , respectively .", "when the upper pole of the kidney was approached , the diaphragm was injured by the dissector , which was immediately repaired .", "the surgeon inquired about the status of the right lung because the right hemidiaphragm was bulging .", "the lung sounds in the right chest were decreased on auscultation , and etco2 and pmax were increased to 42 mmhg and 30 cmh2o , respectively .", ", we evaluated the right thorax using ultrasonography ( linear probe : l25x , 6 - 12 mhz , machine : m - turbo system , sonosite inc . ,", "bothell , wa , usa ) in the operative field , and the \" lung point \" sign was found at the right lower anterior axillary line ( fig .", "however , because the hemodynamic parameters were maintained in a normal range , the operation was continued via hals and a peep of 5 cmh2o was applied . at the end of the surgery ,", "the surgeon requested a portable chest radiography and we concurrently examined the patient using ultrasonography in the supine position .", "3b ) , and the range of the pneumothorax which was indicated by the lung point sign was gradually diminished . because signs of cardiopulmonary instability were not found and spo2 was maintained without an increase in fio2 , the insertion of a chest tube was not necessary and we safely extubated the endotracheal tube .", "the patient was transferred to the pacu and did not complain of any respiratory discomfort ."], ["patients under general anesthesia may develop pneumothorax due to a variety of reasons , such as barotraumas , rupture of the lung bullae or needle insertion into the pleural cavity .", "capnothorax due to co2 during laparoscopic surgery is rare , but , it has been described as a complication of almost every type of laparoscopic surgery [ 1 - 3,6 - 8 ] .", "it has been speculated that an abnormal diaphragm caused by congenital failure of fusion ( e.g. , sternal , costal , and lumbar portions ) , weak point ( e.g. , around the aorta , vena cava , and esophagus ) , congenital defects or iatrogenic injuries by the instrument can lead to capnothorax [ 1 - 3,6 ] . because the creation of pneumoperitoneum and positional changes can demonstrate several potential physiological alterations , the accurate diagnosis of pneumothorax is more difficult during laparoscopic surgery than other surgical procedure .", "when pneumothorax is suspected , it is traditionally diagnosed by portable chest radiography in the operating room .", "however , chest radiography may take a considerable amount of time and demonstrates a sensitivity of 31.8 - 75.5% for the diagnosis of pneumothorax [ 10 - 12 ] .", "in many different fields , lung ultrasonography is often used to diagnose pathological conditions of the lung . in particular , ultrasonography has received attention as a very effective method for confirming pneumothorax at the bedside in the operating room . in numerous studies ,", "ultrasonography has been reported as superior to conventional portable chest radiography for the detection of pneumothorax [ 4,10 - 13 ] .", "the presence or absence of characteristic ultrasonographic signs are key to a correct diagnosis , i.e. , lung sliding , lung pulse , comet - tail artifact , barcode sign ( stratosphere sign ) , seashore sign and lung point sign . among these signs ,", "however , the absence of lung sliding does not always indicate pneumothorax due to several mechanical factors ( e.g. , poor technique , inappropriate ultrasound probe frequency , the presence of dynamic noise filters ) and the patient 's affliction ( e.g. , main bronchial intubation , emphysema , atelectasis , pleural calcification , pleural adhesion , lung fibrosis ) . for these reasons ,", "more specific ultrasonographic methods are needed to confirm pneumothorax . in a particular area of the thorax", ", the ultrasonographic patterns of pneumothorax and the normal lung alternatively appear depending on the respiratory cycle .", "this can be explained by a slight increase in pulmonary volume during inspiration and can be observed in two ultrasonographic modes : real - time ( b - mode ) and time - motion ( m - mode ) . in b - mode , the lung point sign can be found by observing lung - sliding and absent of lung - sliding alternately at a same pleural line .", "analogous patterns also can be found in m - mode . seashore sign and barcode sign", "can be observed in turn , and these signs demonstrate the specificity of 100% for the diagnosis of pneumothorax .", "furthermore , by serially observing the location of the sign , the extension of pneumothorax may be identified . despite this wide spread practice , there are few studies about the relationship between the lung point sign and the extension of pneumothorax", ". when it moves to the lower lateral chest wall , the volume of pneumothorax expands and the patient may need to have a chest tube immediately inserted . on the other hand , movement of the sign from the lower lateral chest to the anterior chest indicates an improvement in pneumothorax ( fig .", "3 ) . if capnothorax is highly suspected and cardiopulmonary instability is not severe , alteration of the \" lung point \" sign can be used to identify improvements in pneumothorax .", "in addition , for faster recovery of capnothorax , application of peep is supportive method to absorb co2 more readily from the pleural cavity .", "therefore , a chest tube insertion , which can cause injury to other structures in the thorax , may not be necessary in these cases . in conclusion", ", the lung point sign may predict the range and amount of pneumothorax during laparoscopic surgery .", "ultrasonography can not only help to diagnose pneumothorax , but can also be used to decide treatment of pneumothorax .", "most notably , in the case of capnothorax which resolves spontaneously as time goes on , the unnecessary insertion of a chest tube may be avoided by observing alterations in the lung point sign ."]]}
{"article_id": "PMC5080203", "article_text": ["imbalance of muscles around the knee can cause changes in the alignment of the knee \n joint1 .", "therefore , to prevent knee \n injuries in females , improved muscle balance around the knee during dynamic motions such as \n landing and balanced strengthening of lower limb muscles are necessary .", "kim et al.1 found kinematic differences in landing \n motions between male and female college students and reported that the valgus angles of \n females were more increased than those of males during a vertical landing motion .", "the \n decline squat exercise moves the line of gravity backwards and increases external momentum \n during extension in knee joints2 ; thus , \n the squat exercise on a decline board has had great effectiveness in reducing knee pain due \n to the selective strengthening of lower extremity muscles3 .", "this study investigated the effect of the single - leg , lateral \n oblique , decline squat exercise on sacroiliac ( si ) joint pain with knee pain .", "a 39-year - old female had severe pain in the right medial buttock and right anterior knee . \n", "she complained of continuous low back pain for 8 months , and examination revealed that her \n pelvis was tilted posteriorly .", "the patient was unable to sleep in the side - lying position \n due to the pain .", "the purpose and methods of the study were explained to the participant \n before her inclusion in the study , and she provided informed consent according to the \n principles of the declaration of helsinki .", "the pelvic inclination was measured with a \n palpation meter ( palm ; performance attainment associates , st .", "paul , mn , usa ) by one \n examiner . at the initial assessment ,", "the anterior pelvic tilt angles were 3.5 and 6.5 on \n the right and left sides ( normal range , 11  4 ) , respectively .", "on palpation of the right \n medial buttock and right anterior patella , the visual analog scale ( vas ) scores were 7/10 \n and 6/10 , respectively .", "the si joint pain provocation tests used in this study were the \n gaenslen test and patrick test .", "the single - leg , lateral oblique , decline squat exercise is \n performed with descent to 90 knee flexion , followed by ascent to the initial position at \n the individual s natural speed .", "this study assessed the anterior pelvic tilt angle and the response to \n pain provocation tests before and after 4 weeks of performing the exercise .", "following the course of exercise , the anterior pelvic tilt angles were 8 and 10 on the \n right and left sides , respectively , and were higher than the initial values ( 3.5 and 6.5 ) . \n", "on palpation of the right medial buttock and right knee , the initial vas scores of 7/10 and \n 6/10 decreased to 2/10 for both regions .", "the decline squat exercise induces anterior pelvic tilt as the trunk is pushed backward to \n compensate for the feeling of the trunk tilting forward2 , 3 .", "the subject in the current study also experienced \n considerable reduction in lateral knee pain , due to decreased tension in the tensor fasciae \n latae or iliotibial band of the lateral knee .", "in particular , the pain in the si joint also \n subsided after the course of exercise .", "the decline board used for the single - leg , lateral \n oblique , decline squat exercise induced foot supination , which promoted ideal alignment of \n the hip and pelvis , preventing knee valgus .", "in addition , the single - leg position can \n activate muscles that provide pelvic stability more effectively than the two - leg \n position4 .", "manual pelvic compression was \n demonstrated to be a compensatory strategy to enhance the force closure mechanism and thus \n normalize the altered motor responses5 . \n", "the stability of the si joint through form and/or force closure mechanisms was proposed to \n facilitate load transfer to the pelvis5 . \n", "therefore , the single - leg , lateral oblique , decline squat exercise can be effective for \n treating si joint pain with knee pain caused by an abnormal quadriceps angle in females ."], "abstract_text": ["<S> [ purpose ] this study investigated the effect of the single - leg , lateral oblique , decline \n squat exercise on sacroiliac joint pain with knee pain . [ subjects and methods ] a \n 39-year - old female had severe pain in the right medial buttock and right anterior knee . \n </S>", "<S> this study assessed the anterior pelvic tilt angle and pain provocation tests before and \n after single - leg , lateral oblique , decline squat exercise for 4 weeks . </S>", "<S> [ results ] following \n the course of exercise , the anterior pelvic tilt angles were increased , and the visual \n analog scale pain scores for both the right buttock and right knee were 2/10 . [ conclusion ] \n single - leg </S>", "<S> , lateral oblique , decline squat exercise may be effective for treating si joint \n pain with knee pain in females . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["imbalance of muscles around the knee can cause changes in the alignment of the knee \n joint1 .", "therefore , to prevent knee \n injuries in females , improved muscle balance around the knee during dynamic motions such as \n landing and balanced strengthening of lower limb muscles are necessary .", "kim et al.1 found kinematic differences in landing \n motions between male and female college students and reported that the valgus angles of \n females were more increased than those of males during a vertical landing motion .", "the \n decline squat exercise moves the line of gravity backwards and increases external momentum \n during extension in knee joints2 ; thus , \n the squat exercise on a decline board has had great effectiveness in reducing knee pain due \n to the selective strengthening of lower extremity muscles3 .", "this study investigated the effect of the single - leg , lateral \n oblique , decline squat exercise on sacroiliac ( si ) joint pain with knee pain ."], ["a 39-year - old female had severe pain in the right medial buttock and right anterior knee . \n", "she complained of continuous low back pain for 8 months , and examination revealed that her \n pelvis was tilted posteriorly .", "the patient was unable to sleep in the side - lying position \n due to the pain .", "the purpose and methods of the study were explained to the participant \n before her inclusion in the study , and she provided informed consent according to the \n principles of the declaration of helsinki .", "the pelvic inclination was measured with a \n palpation meter ( palm ; performance attainment associates , st .", "paul , mn , usa ) by one \n examiner . at the initial assessment ,", "the anterior pelvic tilt angles were 3.5 and 6.5 on \n the right and left sides ( normal range , 11  4 ) , respectively .", "on palpation of the right \n medial buttock and right anterior patella , the visual analog scale ( vas ) scores were 7/10 \n and 6/10 , respectively .", "the si joint pain provocation tests used in this study were the \n gaenslen test and patrick test .", "the single - leg , lateral oblique , decline squat exercise is \n performed with descent to 90 knee flexion , followed by ascent to the initial position at \n the individual s natural speed .", "this study assessed the anterior pelvic tilt angle and the response to \n pain provocation tests before and after 4 weeks of performing the exercise ."], ["following the course of exercise , the anterior pelvic tilt angles were 8 and 10 on the \n right and left sides , respectively , and were higher than the initial values ( 3.5 and 6.5 ) . \n", "on palpation of the right medial buttock and right knee , the initial vas scores of 7/10 and \n 6/10 decreased to 2/10 for both regions ."], ["the decline squat exercise induces anterior pelvic tilt as the trunk is pushed backward to \n compensate for the feeling of the trunk tilting forward2 , 3 .", "the subject in the current study also experienced \n considerable reduction in lateral knee pain , due to decreased tension in the tensor fasciae \n latae or iliotibial band of the lateral knee .", "in particular , the pain in the si joint also \n subsided after the course of exercise .", "the decline board used for the single - leg , lateral \n oblique , decline squat exercise induced foot supination , which promoted ideal alignment of \n the hip and pelvis , preventing knee valgus .", "in addition , the single - leg position can \n activate muscles that provide pelvic stability more effectively than the two - leg \n position4 .", "manual pelvic compression was \n demonstrated to be a compensatory strategy to enhance the force closure mechanism and thus \n normalize the altered motor responses5 . \n", "the stability of the si joint through form and/or force closure mechanisms was proposed to \n facilitate load transfer to the pelvis5 . \n", "therefore , the single - leg , lateral oblique , decline squat exercise can be effective for \n treating si joint pain with knee pain caused by an abnormal quadriceps angle in females ."]]}
{"article_id": "PMC373458", "article_text": ["stemming from the seminal work by cannon and selye , stress is defined as \" an alteration in the body 's hormonal and neuronal secretions caused by the central nervous system in response to a perceived threat \" and a stressor is defined as \" a change in an organism 's internal or external environment which is perceived by the organism as threatening \" [ 1 - 3 ] . within this context , psychosocial stressors like social conflict , social isolation", "the most profound change that occurs with individual housing is an increase in aggression of males seen in both mice and rats following even relatively brief periods of individual housing .", "for example , in one study it was found that group size per se had limited long - term effects on pathophysiological measures of social stress , although it had a significant influence on many aspects of behavior when rats were first introduced into their groups . over weeks", "18 , single housed rats continued to spend much more time apparently attempting to escape ( sniffing and chewing at the bars and suddenly dashing around their cage ) while those housed in groups spent more time sleeping and feeding .", "the objective of the present study was to examine whether social isolation in growing male rats affected 24-h variations of activity of the hypophysial gonadal axis .", "indeed , stressors have been shown to modify gonadotropin and testosterone secretion , acute stressors activating and chronic stressors suppressing the activity of the hypophysial gonadal axis .", "it must be noted that , except for some exceptions , studies on the subject were performed at single time - points , generally in the morning , a serious drawback in view of the circadian nature of hormone release for most of the hormones considered . in this study", "we measured the daily pattern of plasma prolactin , luteinizing hormone ( lh ) , follicle - stimulating hormone ( fsh ) and testosterone levels at 6 different time points within a 24-h cycle in growing male rats kept in isolation or group - caged for 30 days .", "thirty five day - old male wistar rats were kept under standard conditions of controlled light ( 12:12 h light / dark schedule ; lights on at 0800 h ) and temperature ( 22  2c ) .", "rats were either put in individual cages ( isolated group ) or left in cages of 45 animals each .", "all animals had free access to food and water for the 30 days of the study .", "the experiments were conducted in accordance with the guidelines of the international council for laboratory animal science ( iclas ) .", "groups of 68 rats were killed by decapitation under conditions of minimal stress , at six different time intervals every 4 h throughout a 24-h cycle starting at 0900 h. blood was collected from the trunk wound in heparinized tubes and was centrifuged at 1500  g for 15 min . the plasma was collected and stored at -20c until further analysis .", "plasma prolactin , fsh and lh levels were measured by a homologous specific double antibody ria , using materials kindly supplied by the niddk 's national hormone and pituitary program .", "sensitivities of the rias were 45 , 9 and 45 pg / ml for prolactin , fsh and lh using the niddk rat prolactin rp-3 , rat fsh - rp-2 and rat lh - rp-3 , respectively .", "results were expressed as ng / ml for prolactin and as pg / ml for fsh and lh .", "plasma testosterone levels were measured by using a commercial kit ( icn pharmaceuticals , inc .", "sensitivity of the assay was 0.2 ng / ml and the intraassay coefficient of variation was 5% , as previously described ; results were expressed as ng testosterone / ml .", "statistical analysis of results was performed by a two - way factorial analysis of variance ( anova ) . generally , the analysis included assessment of the group effect ( i.e. the occurrence of differences in mean values between isolated and control rats ) , of time - of - day effects ( the occurrence of daily changes ) and of the interaction between the two factors ( manipulation and time , from which inference about differences in timing and amplitude could be obtained ) .", "post - hoc tukey - kramer 's multiple comparisons tests were then employed to show which time points were significantly different within each experimental group to define existence of peaks .", "figure 1 shows the levels of prolactin throughout the day in isolated and control rats .", "a factorial anova for main effects indicated a significant 74% decrease of circulating prolactin in isolated rats ( f1,75= 75.9 , p < 0.00001 ) and the occurrence of significant time of day changes ( f5,75= 18.8 , p < 0.00001 ) .", "the maximum seen in control animals at the beginning of the activity span was no longer detected in isolated rats ( fig .", "1 ) , as indicated both by a significant interaction between time of day and the experimental procedure in the factorial anova ( f5,75= 9.23 , p < 0.00001 ) and by post - hoc tukey - kramer 's tests ( fig .", "1 ) . effect of isolation on 24-h changes of plasma prolactin concentration in young male rats .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "letters indicate the existence of significant differences between time points within each group after a tukey - kramer 's multiple comparisons test , as follows : p < 0.01 vs. 0900 , 1300 , 0100 and 0500 h. for further statistical analysis , see text .", "globally , this manoeuvre augmented fsh levels by 64% ( f1,79= 8.31 , p < 0.005 , factorial anova ) with absence of significant changes as a function of time of day ( f= 1.95 , p > 0.9 ) . as shown in fig .", "3 , this pattern differed in the case of plasma lh levels , i.e. isolation decreased circulating lh to 51% of controls ( f1,78= 4.99 , p < 0.03 , factorial anova ) without any significant effect of time of day ( f= 1.41 , p > 0.2 ) ( fig .", "no significant correlation between fsh and concentration values was found , high fsh and lh levels being distributed randomly among animals ( results not shown ) .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "isolation brought about a 34% decrease of plasma testosterone ( factorial anova , f1,77= 58.8 , p < 0.00001 ) .", "significant effects of time of day ( f5,77= 8.71 , p < 0.00001 ) and a significant interaction \" time of day  treatment \" occurred ( f5,77= 21.9 , p < 0.00001 ) , i.e. , the maximum in circulating testosterone took place at 1700 h in controls and at 2100 h in isolated rats and the decrease of plasma testosterone in isolated rats was seen only during the light phase of daily photoperiod ( fig .", "4 ) . effect of isolation on 24-h changes of plasma testosterone concentration in young male rats .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "letters indicate the existence of significant differences between time points within each group after a tukey - kramer 's multiple comparisons test , as follows : p < 0.01 vs. all time points . for further statistical analysis ,", "our results indicate that social isolation of young male rats for 30 days brings about changes in the 24-h variation of pituitary - testicular function .", "overall , the secretion of prolactin , lh and testosterone decreased whereas that of fsh augmented in isolated rats .", "the maximum in prolactin seen in group - caged rats at the beginning of the activity span was not observed in isolated rats . in addition , the maximum in circulating testosterone taking place at the second part of rest span in controls was phase - delayed to light - dark transition in isolated rats .", "a decrease of plasma testosterone in isolated rats was seen only during the light phase of daily photoperiod .", "solitary housing of usually social animals such as rats and mice causes complex neurobiological changes .", "socially isolated animals exhibit a decrease in the electrical activity of neurons within the hypothalamus and have lower basal plasma corticosterone levels than do animals raised in social conditions .", "although this could be interpreted as indicating less psychosocial stress in isolation , individual housing of animals is associated with an increase in aggression of males , hyperresponsiveness to several stressors   and an increase in time spent attempting to escape and decrease in time spent sleeping and feeding .", "decreases in plasma levels of prolactin were found in subordinate hamsters after exposure to social conflict   and in isolated male hamsters as compared to hamsters with a family .", "therefore , the decrease levels of plasma prolactin herein described after a 1-month isolation of growing male rats agrees with the reported modifications of prolactin seen in isolated hamsters .", "prolactin is a versatile compound that has a dual function  as a circulating hormone and as a cytokine .", "the prolactin receptor is a member of the cytokine receptor superfamily , linked to activation of signaling pathways that promote cell growth and survival . through these mechanisms prolactin regulates diverse physiological functions via its effects on cellular processes such as proliferation , differentiation , and cell survival .", "in addition , prolactin may represent a peripheral regulatory factor for reproductive function in males , and/or a feedback mechanism that signals cns centers controlling sexual arousal and behavior .", "for example , studies on sexual hormonal response in males demonstrated that plasma prolactin concentrations are substantially increased for over 1 h following orgasm in men but unchanged following sexual arousal without orgasm .", "evidence exists for a brain prolactin receptor - mediated anxiolytic action and for inhibitory actions on the reactivity of the hypothalamic - pituitary - adrenal axis and the neurohypophysial oxytocin system .", "hyperprolactinemia in males induces hypogonadism by inhibiting gonadotropin - releasing hormone pulsatile secretion and , consequently , fsh , lh and testosterone release .", "this leads to spermatogenic arrest , impaired motility , and sperm quality and results in morphologic alterations of the testes similar to those observed in prepubertal testes .", "the present results on decreased prolactin levels in isolated male rats disagree with the previously reported increase in prolactin levels in a similar psychosocial stress paradigm .", "some conditions of the experiments , like the age of rats , i.e. , growing rats in this study vs. adult rats in the study by gambardella and colleagues , could explain this discrepancy .", "the changes in gonadotropin secretion found in isolated rats include a decrease of plasma lh and an increase of plasma fsh levels .", "the reduction in circulating lh was accompanied by a concomitant reduction of testosterone . since fsh levels augmented in isolated rats , the possible decrease of testicular inhibin should be considered .", "temporal organization is an important feature of biological systems and its main function is to facilitate adaptation of the organism to the environment .", "the daily variation of biological variables arises from an internal time - keeping system , and the major action of the environment is to synchronize this internal clock to a period of exactly 24 h. the light - dark cycle , food , ambient temperature , scents and social cues have been identified as environmental synchronizers or \" zeitgebers \" in rats .", "stress is also capable of perturbing temporal organization by affecting the shape and amplitude of a rhythm or by modifying the intrinsic oscillatory mechanism itself .", "in particular , social stress in rodents has been found to cause disruptions of the body temperature , heart rate and locomotor activity rhythms [ 31 - 33 ] .", "further experiments are needed to assess whether the changes in amplitude as well in timing of 24-h rhythms of prolactin and testosterone secretion seen in socially isolated rats can be attributed to an effect on the endogenous clock that modulates the circadian variation of pituitary testicular hormones or to a masking effect on some output(s ) of the clock .", "likewise , to what extent the differences between group- and single - housed rats in the plasma levels of the various hormones can affect reproduction or the immune response should be further explored .", "our results concerning fsh plasma levels indicate that the increase in secretion induced by isolation is several fold larger than the daily variation in group - housed animals ( fig .", "2 ) , which suggests that measurements of fsh secretion in isolated animals may misrepresent natural pituitary function in this species .", "secretion of prolactin , lh and testosterone decreases , and secretion of fsh increases , in isolated rats .", "the maximum in prolactin seen in group - caged rats at the beginning of the activity span is not observed in isolated rats .", "the maximum in circulating testosterone taking place at the second part of the rest span in controls is phase - delayed to the light - dark transition in isolated rats .", "this work was supported by grants from dges , spain , fundacin rodrguez - pascual , spain , agencia nacional de promocin cientfica y tecnolgica , argentina ( pict 6153 ) , fundacin bunge y born , argentina and fundacin antorchas , argentina .", "the gift of the reagents to measure prolactin , lh and fsh levels by the niddk 's national hormone and pituitary program and dr ."], "abstract_text": ["<S> backgroundthis work analyzes the effect of social isolation ( a mild stressor ) on the 24-h variation of pituitary - testicular function in young wistar rats , assessed by measuring circulating levels of prolactin , fsh , lh and testosterone.methodsanimals were either individually caged or kept in groups ( 45 animals per cage ) under a 12:12 h light - dark cycle ( lights on at 0800 h ) for 30 days starting on day 35 of life . </S>", "<S> rats were killed at 4-h intervals during a 24-h cycle , beginning at 0900 h.resultsisolation brought about a decrease in prolactin , lh and testosterone secretion and an increase of fsh secretion . in isolated rats </S>", "<S> the 24-h secretory pattern of prolactin and testosterone became modified , i.e. , the maximum in prolactin seen in control animals at the beginning of the activity span was no longer detected , whereas the maximum in circulating testosterone taking place at 1700 h in controls was phase - delayed to 2100 h in isolated rats.conclusionsocial isolation affects the 24-h variation of pituitary - testicular function in young rats . </S>", "<S> secretion of prolactin , lh and testosterone decreases , and secretion of fsh increases , in isolated rats . </S>", "<S> the maximum in prolactin seen in group - caged rats at the beginning of the activity span is not observed in isolated rats . </S>", "<S> the maximum in circulating testosterone taking place at the second part of the rest span in controls is phase - delayed to the light - dark transition in isolated rats . </S>"], "labels": null, "section_names": ["Background", "Methods", "Results", "Discussion", "Conclusions", "Competing interests", "Authors' contributions", "Acknowledgements"], "sections": [["stemming from the seminal work by cannon and selye , stress is defined as \" an alteration in the body 's hormonal and neuronal secretions caused by the central nervous system in response to a perceived threat \" and a stressor is defined as \" a change in an organism 's internal or external environment which is perceived by the organism as threatening \" [ 1 - 3 ] . within this context , psychosocial stressors like social conflict , social isolation", "the most profound change that occurs with individual housing is an increase in aggression of males seen in both mice and rats following even relatively brief periods of individual housing .", "for example , in one study it was found that group size per se had limited long - term effects on pathophysiological measures of social stress , although it had a significant influence on many aspects of behavior when rats were first introduced into their groups . over weeks", "18 , single housed rats continued to spend much more time apparently attempting to escape ( sniffing and chewing at the bars and suddenly dashing around their cage ) while those housed in groups spent more time sleeping and feeding .", "the objective of the present study was to examine whether social isolation in growing male rats affected 24-h variations of activity of the hypophysial gonadal axis .", "indeed , stressors have been shown to modify gonadotropin and testosterone secretion , acute stressors activating and chronic stressors suppressing the activity of the hypophysial gonadal axis .", "it must be noted that , except for some exceptions , studies on the subject were performed at single time - points , generally in the morning , a serious drawback in view of the circadian nature of hormone release for most of the hormones considered . in this study", "we measured the daily pattern of plasma prolactin , luteinizing hormone ( lh ) , follicle - stimulating hormone ( fsh ) and testosterone levels at 6 different time points within a 24-h cycle in growing male rats kept in isolation or group - caged for 30 days ."], ["thirty five day - old male wistar rats were kept under standard conditions of controlled light ( 12:12 h light / dark schedule ; lights on at 0800 h ) and temperature ( 22  2c ) .", "rats were either put in individual cages ( isolated group ) or left in cages of 45 animals each .", "all animals had free access to food and water for the 30 days of the study .", "the experiments were conducted in accordance with the guidelines of the international council for laboratory animal science ( iclas ) .", "groups of 68 rats were killed by decapitation under conditions of minimal stress , at six different time intervals every 4 h throughout a 24-h cycle starting at 0900 h. blood was collected from the trunk wound in heparinized tubes and was centrifuged at 1500  g for 15 min . the plasma was collected and stored at -20c until further analysis .", "plasma prolactin , fsh and lh levels were measured by a homologous specific double antibody ria , using materials kindly supplied by the niddk 's national hormone and pituitary program .", "sensitivities of the rias were 45 , 9 and 45 pg / ml for prolactin , fsh and lh using the niddk rat prolactin rp-3 , rat fsh - rp-2 and rat lh - rp-3 , respectively .", "results were expressed as ng / ml for prolactin and as pg / ml for fsh and lh .", "plasma testosterone levels were measured by using a commercial kit ( icn pharmaceuticals , inc .", "sensitivity of the assay was 0.2 ng / ml and the intraassay coefficient of variation was 5% , as previously described ; results were expressed as ng testosterone / ml .", "statistical analysis of results was performed by a two - way factorial analysis of variance ( anova ) . generally , the analysis included assessment of the group effect ( i.e. the occurrence of differences in mean values between isolated and control rats ) , of time - of - day effects ( the occurrence of daily changes ) and of the interaction between the two factors ( manipulation and time , from which inference about differences in timing and amplitude could be obtained ) .", "post - hoc tukey - kramer 's multiple comparisons tests were then employed to show which time points were significantly different within each experimental group to define existence of peaks ."], ["figure 1 shows the levels of prolactin throughout the day in isolated and control rats .", "a factorial anova for main effects indicated a significant 74% decrease of circulating prolactin in isolated rats ( f1,75= 75.9 , p < 0.00001 ) and the occurrence of significant time of day changes ( f5,75= 18.8 , p < 0.00001 ) .", "the maximum seen in control animals at the beginning of the activity span was no longer detected in isolated rats ( fig .", "1 ) , as indicated both by a significant interaction between time of day and the experimental procedure in the factorial anova ( f5,75= 9.23 , p < 0.00001 ) and by post - hoc tukey - kramer 's tests ( fig .", "1 ) . effect of isolation on 24-h changes of plasma prolactin concentration in young male rats .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "letters indicate the existence of significant differences between time points within each group after a tukey - kramer 's multiple comparisons test , as follows : p < 0.01 vs. 0900 , 1300 , 0100 and 0500 h. for further statistical analysis , see text .", "globally , this manoeuvre augmented fsh levels by 64% ( f1,79= 8.31 , p < 0.005 , factorial anova ) with absence of significant changes as a function of time of day ( f= 1.95 , p > 0.9 ) . as shown in fig .", "3 , this pattern differed in the case of plasma lh levels , i.e. isolation decreased circulating lh to 51% of controls ( f1,78= 4.99 , p < 0.03 , factorial anova ) without any significant effect of time of day ( f= 1.41 , p > 0.2 ) ( fig .", "no significant correlation between fsh and concentration values was found , high fsh and lh levels being distributed randomly among animals ( results not shown ) .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "isolation brought about a 34% decrease of plasma testosterone ( factorial anova , f1,77= 58.8 , p < 0.00001 ) .", "significant effects of time of day ( f5,77= 8.71 , p < 0.00001 ) and a significant interaction \" time of day  treatment \" occurred ( f5,77= 21.9 , p < 0.00001 ) , i.e. , the maximum in circulating testosterone took place at 1700 h in controls and at 2100 h in isolated rats and the decrease of plasma testosterone in isolated rats was seen only during the light phase of daily photoperiod ( fig .", "4 ) . effect of isolation on 24-h changes of plasma testosterone concentration in young male rats .", "groups of 68 rats were killed by decapitation at 6 different time intervals throughout a 24 h cycle .", "letters indicate the existence of significant differences between time points within each group after a tukey - kramer 's multiple comparisons test , as follows : p < 0.01 vs. all time points . for further statistical analysis ,"], ["our results indicate that social isolation of young male rats for 30 days brings about changes in the 24-h variation of pituitary - testicular function .", "overall , the secretion of prolactin , lh and testosterone decreased whereas that of fsh augmented in isolated rats .", "the maximum in prolactin seen in group - caged rats at the beginning of the activity span was not observed in isolated rats . in addition , the maximum in circulating testosterone taking place at the second part of rest span in controls was phase - delayed to light - dark transition in isolated rats .", "a decrease of plasma testosterone in isolated rats was seen only during the light phase of daily photoperiod .", "solitary housing of usually social animals such as rats and mice causes complex neurobiological changes .", "socially isolated animals exhibit a decrease in the electrical activity of neurons within the hypothalamus and have lower basal plasma corticosterone levels than do animals raised in social conditions .", "although this could be interpreted as indicating less psychosocial stress in isolation , individual housing of animals is associated with an increase in aggression of males , hyperresponsiveness to several stressors   and an increase in time spent attempting to escape and decrease in time spent sleeping and feeding .", "decreases in plasma levels of prolactin were found in subordinate hamsters after exposure to social conflict   and in isolated male hamsters as compared to hamsters with a family .", "therefore , the decrease levels of plasma prolactin herein described after a 1-month isolation of growing male rats agrees with the reported modifications of prolactin seen in isolated hamsters .", "prolactin is a versatile compound that has a dual function  as a circulating hormone and as a cytokine .", "the prolactin receptor is a member of the cytokine receptor superfamily , linked to activation of signaling pathways that promote cell growth and survival . through these mechanisms prolactin regulates diverse physiological functions via its effects on cellular processes such as proliferation , differentiation , and cell survival .", "in addition , prolactin may represent a peripheral regulatory factor for reproductive function in males , and/or a feedback mechanism that signals cns centers controlling sexual arousal and behavior .", "for example , studies on sexual hormonal response in males demonstrated that plasma prolactin concentrations are substantially increased for over 1 h following orgasm in men but unchanged following sexual arousal without orgasm .", "evidence exists for a brain prolactin receptor - mediated anxiolytic action and for inhibitory actions on the reactivity of the hypothalamic - pituitary - adrenal axis and the neurohypophysial oxytocin system .", "hyperprolactinemia in males induces hypogonadism by inhibiting gonadotropin - releasing hormone pulsatile secretion and , consequently , fsh , lh and testosterone release .", "this leads to spermatogenic arrest , impaired motility , and sperm quality and results in morphologic alterations of the testes similar to those observed in prepubertal testes .", "the present results on decreased prolactin levels in isolated male rats disagree with the previously reported increase in prolactin levels in a similar psychosocial stress paradigm .", "some conditions of the experiments , like the age of rats , i.e. , growing rats in this study vs. adult rats in the study by gambardella and colleagues , could explain this discrepancy .", "the changes in gonadotropin secretion found in isolated rats include a decrease of plasma lh and an increase of plasma fsh levels .", "the reduction in circulating lh was accompanied by a concomitant reduction of testosterone . since fsh levels augmented in isolated rats , the possible decrease of testicular inhibin should be considered .", "temporal organization is an important feature of biological systems and its main function is to facilitate adaptation of the organism to the environment .", "the daily variation of biological variables arises from an internal time - keeping system , and the major action of the environment is to synchronize this internal clock to a period of exactly 24 h. the light - dark cycle , food , ambient temperature , scents and social cues have been identified as environmental synchronizers or \" zeitgebers \" in rats .", "stress is also capable of perturbing temporal organization by affecting the shape and amplitude of a rhythm or by modifying the intrinsic oscillatory mechanism itself .", "in particular , social stress in rodents has been found to cause disruptions of the body temperature , heart rate and locomotor activity rhythms [ 31 - 33 ] .", "further experiments are needed to assess whether the changes in amplitude as well in timing of 24-h rhythms of prolactin and testosterone secretion seen in socially isolated rats can be attributed to an effect on the endogenous clock that modulates the circadian variation of pituitary testicular hormones or to a masking effect on some output(s ) of the clock .", "likewise , to what extent the differences between group- and single - housed rats in the plasma levels of the various hormones can affect reproduction or the immune response should be further explored .", "our results concerning fsh plasma levels indicate that the increase in secretion induced by isolation is several fold larger than the daily variation in group - housed animals ( fig .", "2 ) , which suggests that measurements of fsh secretion in isolated animals may misrepresent natural pituitary function in this species ."], ["secretion of prolactin , lh and testosterone decreases , and secretion of fsh increases , in isolated rats .", "the maximum in prolactin seen in group - caged rats at the beginning of the activity span is not observed in isolated rats .", "the maximum in circulating testosterone taking place at the second part of the rest span in controls is phase - delayed to the light - dark transition in isolated rats ."], [""], [""], ["this work was supported by grants from dges , spain , fundacin rodrguez - pascual , spain , agencia nacional de promocin cientfica y tecnolgica , argentina ( pict 6153 ) , fundacin bunge y born , argentina and fundacin antorchas , argentina .", "the gift of the reagents to measure prolactin , lh and fsh levels by the niddk 's national hormone and pituitary program and dr ."]]}
{"article_id": "PMC3302998", "article_text": ["punctate inner choroidopathy ( pic ) is an idiopathic disorder representing one point on a spectrum of the multifocal choroiditides .", "the visual outcome is good unless complicated by submacular choroidal neovascularization ( cnv ) , which may occur bilaterally .", "treatment options of cnv in pic include argon laser photocoagulation , local or systemic corticosteroids or other immunosuppressant agents , intravitreal anti - vegf agents , photodynamic therapy ( pdt ) , and submacular surgery .", "in cnv cases with subfoveal involvement , argon laser photocoagulation has been associated with poor visual outcome . before the introduction of anti - vascular endothelial growth factor ( vegf ) agents ,", "surgical excision was the therapeutic option of last resort and only employed after conventional management with corticosteroids or pdt had failed .", "various angiogenic factors have been implicated in the pathogenesis of cnv , with vegf putatively the key player .", "the advent of targeted pharmacologic inhibition of vegf has been a major advance in the management of cnv secondary to age - related macular degeneration ( amd ) .", "there are no randomized clinical trials providing evidence for the efficacy of intravitreal anti - vegf agents in the treatment of cnv in conditions other than amd .", "nevertheless , given their excellent safety profile , these agents are widely used in all subfoveal cnv cases regardless of etiology , including pic [ 2 , 3 ] . it remains unclear why cnv responds in different ways to anti - vegf treatment .", "a multifactorial etiology of ocular angiogenesis , including inflammation and mediators other than vegf , as well as the different levels of vascular maturation of the neovascular complex , has been proposed . to date , there is no report of the surgical management and ultrastructural study of cnv secondary to choroiditis following intravitreal anti - vegf treatment .", "we report herein on the clinical outcome of bilateral surgical excisions of cnv in a pic case which failed anti - vegf treatment .", "electron microscopic evaluation was performed on the excised specimens in an attempt to elucidate the pathogenesis of the condition and the possible mechanisms of treatment failure .", "the cnv regressed following one intravitreal bevacizumab injection with an improvement in vision to 20/25 .", "it recurred 17  months later with a decrease in vision to 20/40 , and a second injection was administered with no response .", "dilated fundus examination revealed small punctate yellowish chorioretinal lesions bilaterally consistent with choroiditis ( fig . ", "1b , c ) . optical coherence tomography ( oct ) revealed that the lesion was associated with adjacent intraretinal cystic spaces ( fig .  2a , top).fig . ", "a preoperative color photograph demonstrates multiple , small , yellowish punctate lesions at the level of the choroid .", "c late phase of the angiogram shows leakage from the cnv along with multiple punctate hyperfluorescent lesions in the peripapillary area and the periphery .", "d fundus photograph 1  month after surgical excision of cnv showing rpe atrophyfig . ", "a ( top ) oct before surgical excision of cnv showing hyperreflective area at the level of the rpe corresponding to the cnv .", "c ( bottom ) oct 15  months after surgical excision of cnv right eye .", "a preoperative color photograph demonstrates multiple , small , yellowish punctate lesions at the level of the choroid .", "c late phase of the angiogram shows leakage from the cnv along with multiple punctate hyperfluorescent lesions in the peripapillary area and the periphery .", "d fundus photograph 1  month after surgical excision of cnv showing rpe atrophy right eye .", "a ( top ) oct before surgical excision of cnv showing hyperreflective area at the level of the rpe corresponding to the cnv .", "c ( bottom ) oct 15  months after surgical excision of cnv surgical removal of the cnv 3  months after the second injection led to improvement in vision to 20/20 - 1 with resolution of metamorphopsia ( figs . ", "there has been no recurrence at 23  months of follow - up after surgery .", "cnv developed in the left eye 2  years after failed pharmacotherapy in the right eye .", "3d ) led to resolution of metamorphopsia and improvement of vision to 20/20 at a 3-month follow - up .", "six months following surgery , visual acuity decreased to 20/63 - 2 secondary to recurrence .", "informed consent was obtained from the patient regarding evaluating the surgical specimens obtained by light and electron microscopy.fig . ", "a color fundus photograph 3  months after second injection of bevacizumab shows a grayish subfoveal cnv membrane .", "b early phase of the fluorescein angiogram reveals hyperfluorescence corresponding to the cnv lesion with hypofluorescent borders corresponding to the pigmented borders of the lesion .", "a color fundus photograph 3  months after second injection of bevacizumab shows a grayish subfoveal cnv membrane .", "b early phase of the fluorescein angiogram reveals hyperfluorescence corresponding to the cnv lesion with hypofluorescent borders corresponding to the pigmented borders of the lesion .", "the specimens obtained were evaluated by light microscopy in 1-m plastic sections stained with toluidine blue and by transmission electron microscopic examination of ultrathin sections prepared according to standard techniques ( a mixture of 2.5% glutaraldehyde and 2% formaldehyde fixation buffered in 0.1  m cacodylate followed by 2% osmium fixation in an aqueous solution ) .", "the first specimen was obtained from the right eye 20  months after the introduction of anti - vegf therapy , 3  months after the most recent intravitreal bevacizumab injection .", "the light microscopic evaluation of epon sections revealed a hypocellular membrane featuring multiple widely separated capillaries in the midst of a fibrillar matrix without apparent inflammatory cells ( fig . ", "some of the vessels contained erythrocytes , which were not detected in the extracellular space . at one edge of the membrane ,", "4a right submacular surgical specimen consists of collagen with many capillary channels ( arrows ) , some of which have erythrocytes in their lumens ( crossed arrows ) .", "scattered pigment granules are observed , with a concentration of pigment epithelial cells ( pe ) toward the bottom left ( toluidine blue , 200 ) .", "b a nonfunctioning capillary exhibits attenuation and fragmentation of the endothelial ( en ) cell layer in the absence of pericytes . note the persistence of erythrocytes ( er ) within the lumen , which is filled with microfilaments ( f ) .", "h histiocyte , pe retinal pigment epithelial cell process , ma extracellular fibrocollagenous matrix ( em 2,600 ) . c a viable neovascular unit displays endothelium ( e ) with focal fenestrations ( f ) and elongated lumen ( l ) .", "both the endothelial cells and surrounding pericytes ( p ) are clothed by a basement membrane .", "the matrix ( ma ) is looser in this region and contains a macrophage ( m ) with complex phagolysosomes ( v ) and surface villi ( vi ) .", "( em 5,800 ) . d multilaminar membrane ( m ) from the left eye has formed above bruch s membrane ( bm ) and contains small neovascular units ( arrows ) .", "( toluidine blue , 200 ) a right submacular surgical specimen consists of collagen with many capillary channels ( arrows ) , some of which have erythrocytes in their lumens ( crossed arrows ) .", "scattered pigment granules are observed , with a concentration of pigment epithelial cells ( pe ) toward the bottom left ( toluidine blue , 200 ) .", "b a nonfunctioning capillary exhibits attenuation and fragmentation of the endothelial ( en ) cell layer in the absence of pericytes .", "note the persistence of erythrocytes ( er ) within the lumen , which is filled with microfilaments ( f ) .", "h histiocyte , pe retinal pigment epithelial cell process , ma extracellular fibrocollagenous matrix ( em 2,600 ) . c a viable neovascular unit displays endothelium ( e ) with focal fenestrations ( f ) and elongated lumen ( l ) .", "both the endothelial cells and surrounding pericytes ( p ) are clothed by a basement membrane .", "the matrix ( ma ) is looser in this region and contains a macrophage ( m ) with complex phagolysosomes ( v ) and surface villi ( vi ) .", "d multilaminar membrane ( m ) from the left eye has formed above bruch s membrane ( bm ) and contains small neovascular units ( arrows ) .", "( toluidine blue , 200 ) at the ultrastructural level , no photoreceptors or other retinal elements were detected .", "the predominant one contained occasional intraluminal erythrocytes but was for the most part occluded with microfibrils ( fig . ", "the second less common type of capillary had a well - defined pericytic layer ; only one such capillary was identified in the specimen ( fig . ", "the pigment epithelial cell cluster exhibited a peculiar fine clumping of the nuclear chromatin and disruption of the plasmalemmas , presumably degenerative phenomena , causing the extracellular release of melanin granules .", "the membranous tissue excised from the left eye 5  months after initiation of therapy and 3  months after most recent intravitreal injection comprised the inner choroid with its choriocapillaris , bruch s membrane , and a vascularized subretinal multilaminar cellular membrane with many capillary - type units ( fig . ", "epon - embedded , light microscopic sections , no break was detected in bruch s membrane that demonstrated a vascular channel connecting the choriocapillaris with the subretinal neovascular units .", "the inner choroid harbored a distinct focus of chronic inflammatory cells without follicular organization . by transmission electron microscopy , the pigment epithelium consisted of one or two layers at the center of the membrane with neovascular channels distributed among them in the absence of inflammation ( fig . ", "the rpe cells and neovascular channels in the middle of the lesion also interfaced with broad processes of mueller cells without the interposition of photoreceptors or their degenerative remains ( fig . ", "the mueller cells were characterized by multiform mitochondria , abundant profiles of smooth surfaced endoplasmic reticulum , and the absence of pigment granules .", "poorly pigmented displaced rpe cells contained rare pigment granules , profiles of rough surfaced endoplasmic reticulum and manifested segments of basement membrane material and disorganized surface villi .", "extravasated erythrocytes , macrophages , lymphocytes , and conspicuous extracellular matrix material were not found in the membrane , except for the last which was limited to the pericapillary zone .", "the viable neoformed intramembranous capillaries displayed patent lumens with endothelial cell fenestrae and uniformly enveloping pericytes , both ensheathed by basement membranes ( fig . ", "5a in the left eye , above bruch s membrane ( bm ) with its elastic layer and subjacent choriocapillaris ( ch ) , are two to three layers of pigment epithelial cells ( pe ) with loss of polarity .", "the pe cells interface with broad processes of mueller cells ( mc ) in the upper half of the field with myriad mitochondria .", "inset : fenestrations ( arrows ) of the endothelial cells of the neoformed capillary and a thick surrounding basement membrane ( bm ) ( em 1,950 ) .", "c at the edge of the membrane , pigment epithelial cells ( pe ) with basement membrane ( bm ) are positioned on bruch s membrane ( bm ) . within the cytoplasm of the central pe cell", "is a degenerated compact whorled membranous body ( arrow ) , another of which is also shown toward the right in the cytoplasm of an indeterminate cell ( crossed arrow ) .", "the underlying choriocapillaris ( cc ) with its endothelial cells ( e ) is patent .", "a large melanophage ( m ) contains complex phagolysosomes ( crossed arrows ) of engulfed uveal melanin .", "e extravasated erythrocyte ( em 1,450 ) a in the left eye , above bruch s membrane ( bm ) with its elastic layer and subjacent choriocapillaris ( ch ) , are two to three layers of pigment epithelial cells ( pe ) with loss of polarity .", "the pe cells interface with broad processes of mueller cells ( mc ) in the upper half of the field with myriad mitochondria .", "inset : fenestrations ( arrows ) of the endothelial cells of the neoformed capillary and a thick surrounding basement membrane ( bm ) ( em 1,950 ) .", "c at the edge of the membrane , pigment epithelial cells ( pe ) with basement membrane ( bm ) are positioned on bruch s membrane ( bm ) . within the cytoplasm of the central pe cell is a degenerated compact whorled membranous body ( arrow ) , another of which is also shown toward the right in the cytoplasm of an indeterminate cell ( crossed arrow ) .", "the underlying choriocapillaris ( cc ) with its endothelial cells ( e ) is patent .", "a large melanophage ( m ) contains complex phagolysosomes ( crossed arrows ) of engulfed uveal melanin .", "e extravasated erythrocyte ( em 1,450 ) at the edge of the membrane , a single layer of dyspolar pigment epithelial cells without apical villi was surrounded by whorled membranous bodies of various sizes , some of which were engulfed by the rpe cells and others by macrophages ( fig . ", "the focus of inner choroidal inflammatory cells was composed of mature small lymphocytes with clumped chromatin and scant cytoplasm without intermixed histiocytes and plasma cells ( fig . ", "the choroidal melanocytes were focally damaged and had released their small pigment granules extracellularly , which had attracted melanophages ( fig . ", "this report documents for the first time the healing response following anti - vegf treatment of cnvs and highlights possible causes of treatment failure .", "we also analyzed the clinical and anatomical implications of surgical removal of cnvs following treatment failure of anti - vegf agents .", "there is one other report in the literature of excision of a cnv membrane following intravitreal injection of the anti - vegf agent bevacizumab , in which only avascular fibrous tissue was described pathologically .", "the submacular membranes removed from both eyes in our case had significantly different light and electron microscopic features . that from the right eye , which had been followed and treated over a span of 20  months prior to surgical excision ( in comparison with 5  months for the left eye ) , was remarkably hypocellular and mainly composed of abundant fibrillocollagenous material .", "apart from the presence of rare elongated processes of degenerating pigment epithelial cells or an occasional macrophage , lymphocytes and plasma cells were absent .", "a cluster of polygonal pigment epithelial cells at the membrane s base near bruch s membrane displayed disrupted plasmalemmas and a disturbed nuclear chromatin pattern of fine clumping compatible with incipient dissolution . the ability of the rpe cells to undergo fibrous and even osseous metaplasia is well recognized .", "the vascular endothelial cells of the membrane s capillaries were attenuated and fragmented , and their lumens were filled with microfibrils .", "only one capillary appeared functional with healthier looking constituent cells that included fenestrated endothelial cells and well - preserved pericytes . in this zone , the surrounding extracellular matrix was looser , suggesting that such vessels were of more recent origin than the others and probably related to the recurrence .", "in contrast , the submacular membrane that was removed from the left eye 5  months after initiation of treatment displayed virtually no extracellular fibrillar matrix material .", "the obviously viable and intact vascular endothelial cells displayed fenestrae and were consistently surrounded by pericytes .", "the fenestrae support an origin of the neovascular units from the choriocapillaris , which normally possesses this feature .", "there was no evidence of a lymphohistiocytic inflammatory component accompanying the subretinal neovascularization throughout most of the membrane , as also described in a previous immunohistochemical study of cnv in pic .", ", however , reported moderate to prominent lymphocytic infiltration in four out of the six cnv specimens studied . the pigment epithelial cells in the left membrane remained for the most part confined to one or two layers at the base of the membrane and were in direct apposition to broad processes of mueller cells without the interposition of photoreceptors or their degenerative remnants .", "the latter were discovered at the borders of the membrane in the form of whorled lamellated membranous bodies probably representing degenerative products of outer segments .", "this finding suggests a primary degenerative mechanism rather than an infarction , which would have produced a more complete and broader effacement of cellular detail with a prominent inflammatory cell infiltrate . finally , the fortuitous discovery of lymphocytes at the level of the inner choroid with sparing of the choriocapillaris lends , for the fist time , ultrastructural electron microscopic support to the hypothesis that pic is an inflammatory disease , with the inflammation originating in the choroid .", "there are no other em studies of tissue excised from a pic case that include choroid manifesting inflammatory infiltration .", "thus , there was a striking difference in the response of the two submacular membranes to bevacizumab treatment . while different durations of the two membranes before their removal created concomitant differences in the amount of collagen deposition , the chief ultrastructural differential feature was the pronounced regression of the neoformed vessels in the right eye that lacked pericytes ( in comparison with those in the left eye that possessed them ) . this phenomenon could be a primary feature of the neovascular units themselves or reflect a preferential secondary loss during the membrane s evolution or treatment .", "the well - preserved architecture of the endothelial - lined vascular channels with a normal accompaniment of pericytes in the left eye intimates that anti - vegf treatment had no effect on them .", "the finding of one capillary in the right specimen with fenestrated endothelial cells and well - preserved pericytes , while all other capillaries which were occluded manifested fragmented endothelial cells and lacked pericytes , may signify that it represented cnv nonresponsiveness to the most recent anti - vegf treatment 3  months prior to surgery .", "this is further supported by the fact that in this zone the surrounding extracellular matrix was looser , suggesting more recent origin and less deposition of collagen as explained above .", "the capillaries with fragmented endothelial cells most likely represent the regressed cnv treated with anti - vegf agents 20  months prior to surgery .", "pericyte - poor neovascular units have been shown to be more susceptible to anti - vegf agents than pericyte - rich ones .", "we therefore hypothesize that failure to respond to anti - vegf treatment in the left eye of our patient may be associated with the uniform presence of a pericytic component in the neovascular channels .", "these observations indicate that the presence of pericytes may act as a survival factor for endothelial cells and therefore increase resistance to anti - vegf therapy .", "context , dual vegf and platelet - derived growth factor - targeted therapy has been shown to be more effective in inhibiting in vivo tumor growth than either agent alone .", "choroidal lymphocytic infiltration was found in the left subretinal excision which included a segment of choriocapillaris and surrounding inner choroidal tissue .", "this finding should not be totally unexpected despite the absence of overt clinical intraocular inflammation .", "none of the eyes had undergone prior surgery or photodynamic therapy that could have triggered inflammation .", "the presence of a focus of choroidal inflammation is also suggestive of the possibility that anti - vegf treatment of cnvs may fail because of underlying persistent inflammation .", "this provides another basis for considering a combination of therapeutic modalities in addition to anti - vegf factors to improve the efficacy of treatment in cnvs secondary to pic .", "this is the first report showing persistent choroidal inflammation after anti - vegf treatment that could possibly provoke recurrent cnvs , in contradistinction to inflammation in the membrane itself .", "available data on treatment of cnv associated to pic are derived from small nonrandomized case series .", "local and systemic treatment with steroids has not been proven effective in the treatment of cnv associated with pic , even when administered at high doses for many months [ 6 , 10 ] .", "more recent small prospective studies of pdt in combination with either systemic steroids or 4  mg intravitreal triamcinolone of five and four eyes , respectively , showed decreased activity of cnv lesions [ 11 , 12 ] .", "three out of the four patients who received intravitreal triamcinolone experienced improvement of two or more lines of vision and one patient lost two or more lines at 1-year follow - up .", "we should , however , take into account the high rate of side effects of systemic or local corticosteroid treatment .", "regarding the surgical management outcome of this case , prior anti - vegf treatment did not appear to complicate surgical excision . in our series of three submacular surgeries in two eyes ,", "there were no adverse events peri- or postoperatively , but the number is too small for any definite conclusions .", "this high rate of recurrence in our series is in accord with the results of the submacular surgery trial ( sst ) trial in which recurrent cnv developed in 58% of surgically treated eyes by the 24-month examination .", "recurrences occurred in four of six eyes with cnv associated with pic following surgical excision in the series reported by olsen et al . .", "following reports from the sst trial for cnv that showed no benefit in preventing visual loss and a high rate of adverse events , submacular surgery has been virtually abandoned .", "sst reported on both type i membranes , which grow below the rpe and are typically secondary to amd , and type ii membranes , which grow subretinally .", "pic is one of the conditions , along with an idiopathic category , myopia , and presumed ocular histoplasmosis syndrome , that are complicated by type ii cnvs .", "the sst trial showed that surgical excision of cnv in pohs and idiopathic cases probably has a more favorable visual outcome in the subgroup of eyes with initial visual acuity 20/100 or worse .", "a successful outcome was defined as a visual acuity better , or no more than one line worse , than at baseline . as a result , a smaller benefit of surgery , such as reduction of risk of loss of three lines of vision , which many trials have been designed to detect , could not be confirmed .", "also , 25% of patients were 60  years or older ; results may be more favorable in a younger cohort of patients , as large case series have suggested .", "still , submacular surgery carries significant risks and should be considered only in selected cases nonresponsive or nontolerant to other treatment modalities . in conclusion , this report provides evidence that inhibition of vegf alone can not be relied upon to induce stable and lasting regression of subretinal neovascularization in pic .", "this is the first pathological study employing human tissue that points to pericytes as a potential critical target with the aggravating influence of inner choroidal chronic inflammation .", "this article is distributed under the terms of the creative commons attribution license which permits any use , distribution and reproduction in any medium , provided the original author(s ) and source are credited ."], "abstract_text": ["<S> purposethis study aims to describe surgical management results and the pathologic features of choroidal neovascularization ( cnv ) secondary to punctate inner choroidopathy ( pic ) following anti - vascular endothelial growth factor treatment.designthis study is a case report on the surgical management and ultrastructural study of choroidal neovascularization.methodsclinicopathologic and ultrastructural report of cnv membranes excised from both eyes of one patient was presented.resultsthe right eye responded to bevacizumab , and recurrence 17  months later did not ; the left eye never responded . </S>", "<S> excision of the active cnvs was performed 3  months after the last injection . in the right eye </S>", "<S> , there was no recurrence 23  months after surgery . in the left eye </S>", "<S> , cnv recurred after 6  months , with no response to bevacizumab . </S>", "<S> electron microscopy revealed subretinal neovascular tissue and , additionally , bruch 's membrane and inner choroid in the left . in the right eye , lumens of many neovascular channels were occluded by microfibrils and pericytes were infrequent . in the left eye , </S>", "<S> patent cnv units with pericytes were present . </S>", "<S> there were scattered macrophages but no lymphocytes in either membrane . </S>", "<S> an inner focal choroidal lymphocytic infiltrate was discovered.conclusionssubmacular surgery did not cause complications following treatment with bevacizumab . </S>", "<S> mostly nonfunctional capillaries in the right membrane failed to display pericytes . </S>", "<S> the left membrane , which was unresponsive to bevacizumab , displayed well - formed neovascular units consistently exhibiting pericytes . </S>", "<S> a focus of inner choroidal lymphocytic infiltration was found in the left eye despite the absence of overt clinical intraocular inflammation . </S>", "<S> this is the first pathological study employing human tissue that points to pericytes as a potential critical therapeutic target with the aggravating influence of inner choroidal chronic inflammation in pic . </S>"], "labels": null, "section_names": ["Introduction", "Clinical findings", "Light and electron microscopic findings", "Discussion", "Open Access"], "sections": [["punctate inner choroidopathy ( pic ) is an idiopathic disorder representing one point on a spectrum of the multifocal choroiditides .", "the visual outcome is good unless complicated by submacular choroidal neovascularization ( cnv ) , which may occur bilaterally .", "treatment options of cnv in pic include argon laser photocoagulation , local or systemic corticosteroids or other immunosuppressant agents , intravitreal anti - vegf agents , photodynamic therapy ( pdt ) , and submacular surgery .", "in cnv cases with subfoveal involvement , argon laser photocoagulation has been associated with poor visual outcome . before the introduction of anti - vascular endothelial growth factor ( vegf ) agents ,", "surgical excision was the therapeutic option of last resort and only employed after conventional management with corticosteroids or pdt had failed .", "various angiogenic factors have been implicated in the pathogenesis of cnv , with vegf putatively the key player .", "the advent of targeted pharmacologic inhibition of vegf has been a major advance in the management of cnv secondary to age - related macular degeneration ( amd ) .", "there are no randomized clinical trials providing evidence for the efficacy of intravitreal anti - vegf agents in the treatment of cnv in conditions other than amd .", "nevertheless , given their excellent safety profile , these agents are widely used in all subfoveal cnv cases regardless of etiology , including pic [ 2 , 3 ] . it remains unclear why cnv responds in different ways to anti - vegf treatment .", "a multifactorial etiology of ocular angiogenesis , including inflammation and mediators other than vegf , as well as the different levels of vascular maturation of the neovascular complex , has been proposed . to date , there is no report of the surgical management and ultrastructural study of cnv secondary to choroiditis following intravitreal anti - vegf treatment .", "we report herein on the clinical outcome of bilateral surgical excisions of cnv in a pic case which failed anti - vegf treatment .", "electron microscopic evaluation was performed on the excised specimens in an attempt to elucidate the pathogenesis of the condition and the possible mechanisms of treatment failure ."], ["the cnv regressed following one intravitreal bevacizumab injection with an improvement in vision to 20/25 .", "it recurred 17  months later with a decrease in vision to 20/40 , and a second injection was administered with no response .", "dilated fundus examination revealed small punctate yellowish chorioretinal lesions bilaterally consistent with choroiditis ( fig . ", "1b , c ) . optical coherence tomography ( oct ) revealed that the lesion was associated with adjacent intraretinal cystic spaces ( fig .  2a , top).fig . ", "a preoperative color photograph demonstrates multiple , small , yellowish punctate lesions at the level of the choroid .", "c late phase of the angiogram shows leakage from the cnv along with multiple punctate hyperfluorescent lesions in the peripapillary area and the periphery .", "d fundus photograph 1  month after surgical excision of cnv showing rpe atrophyfig . ", "a ( top ) oct before surgical excision of cnv showing hyperreflective area at the level of the rpe corresponding to the cnv .", "c ( bottom ) oct 15  months after surgical excision of cnv right eye .", "a preoperative color photograph demonstrates multiple , small , yellowish punctate lesions at the level of the choroid .", "c late phase of the angiogram shows leakage from the cnv along with multiple punctate hyperfluorescent lesions in the peripapillary area and the periphery .", "d fundus photograph 1  month after surgical excision of cnv showing rpe atrophy right eye .", "a ( top ) oct before surgical excision of cnv showing hyperreflective area at the level of the rpe corresponding to the cnv .", "c ( bottom ) oct 15  months after surgical excision of cnv surgical removal of the cnv 3  months after the second injection led to improvement in vision to 20/20 - 1 with resolution of metamorphopsia ( figs . ", "there has been no recurrence at 23  months of follow - up after surgery .", "cnv developed in the left eye 2  years after failed pharmacotherapy in the right eye .", "3d ) led to resolution of metamorphopsia and improvement of vision to 20/20 at a 3-month follow - up .", "six months following surgery , visual acuity decreased to 20/63 - 2 secondary to recurrence .", "informed consent was obtained from the patient regarding evaluating the surgical specimens obtained by light and electron microscopy.fig . ", "a color fundus photograph 3  months after second injection of bevacizumab shows a grayish subfoveal cnv membrane .", "b early phase of the fluorescein angiogram reveals hyperfluorescence corresponding to the cnv lesion with hypofluorescent borders corresponding to the pigmented borders of the lesion .", "a color fundus photograph 3  months after second injection of bevacizumab shows a grayish subfoveal cnv membrane .", "b early phase of the fluorescein angiogram reveals hyperfluorescence corresponding to the cnv lesion with hypofluorescent borders corresponding to the pigmented borders of the lesion ."], ["the specimens obtained were evaluated by light microscopy in 1-m plastic sections stained with toluidine blue and by transmission electron microscopic examination of ultrathin sections prepared according to standard techniques ( a mixture of 2.5% glutaraldehyde and 2% formaldehyde fixation buffered in 0.1  m cacodylate followed by 2% osmium fixation in an aqueous solution ) .", "the first specimen was obtained from the right eye 20  months after the introduction of anti - vegf therapy , 3  months after the most recent intravitreal bevacizumab injection .", "the light microscopic evaluation of epon sections revealed a hypocellular membrane featuring multiple widely separated capillaries in the midst of a fibrillar matrix without apparent inflammatory cells ( fig . ", "some of the vessels contained erythrocytes , which were not detected in the extracellular space . at one edge of the membrane ,", "4a right submacular surgical specimen consists of collagen with many capillary channels ( arrows ) , some of which have erythrocytes in their lumens ( crossed arrows ) .", "scattered pigment granules are observed , with a concentration of pigment epithelial cells ( pe ) toward the bottom left ( toluidine blue , 200 ) .", "b a nonfunctioning capillary exhibits attenuation and fragmentation of the endothelial ( en ) cell layer in the absence of pericytes . note the persistence of erythrocytes ( er ) within the lumen , which is filled with microfilaments ( f ) .", "h histiocyte , pe retinal pigment epithelial cell process , ma extracellular fibrocollagenous matrix ( em 2,600 ) . c a viable neovascular unit displays endothelium ( e ) with focal fenestrations ( f ) and elongated lumen ( l ) .", "both the endothelial cells and surrounding pericytes ( p ) are clothed by a basement membrane .", "the matrix ( ma ) is looser in this region and contains a macrophage ( m ) with complex phagolysosomes ( v ) and surface villi ( vi ) .", "( em 5,800 ) . d multilaminar membrane ( m ) from the left eye has formed above bruch s membrane ( bm ) and contains small neovascular units ( arrows ) .", "( toluidine blue , 200 ) a right submacular surgical specimen consists of collagen with many capillary channels ( arrows ) , some of which have erythrocytes in their lumens ( crossed arrows ) .", "scattered pigment granules are observed , with a concentration of pigment epithelial cells ( pe ) toward the bottom left ( toluidine blue , 200 ) .", "b a nonfunctioning capillary exhibits attenuation and fragmentation of the endothelial ( en ) cell layer in the absence of pericytes .", "note the persistence of erythrocytes ( er ) within the lumen , which is filled with microfilaments ( f ) .", "h histiocyte , pe retinal pigment epithelial cell process , ma extracellular fibrocollagenous matrix ( em 2,600 ) . c a viable neovascular unit displays endothelium ( e ) with focal fenestrations ( f ) and elongated lumen ( l ) .", "both the endothelial cells and surrounding pericytes ( p ) are clothed by a basement membrane .", "the matrix ( ma ) is looser in this region and contains a macrophage ( m ) with complex phagolysosomes ( v ) and surface villi ( vi ) .", "d multilaminar membrane ( m ) from the left eye has formed above bruch s membrane ( bm ) and contains small neovascular units ( arrows ) .", "( toluidine blue , 200 ) at the ultrastructural level , no photoreceptors or other retinal elements were detected .", "the predominant one contained occasional intraluminal erythrocytes but was for the most part occluded with microfibrils ( fig . ", "the second less common type of capillary had a well - defined pericytic layer ; only one such capillary was identified in the specimen ( fig . ", "the pigment epithelial cell cluster exhibited a peculiar fine clumping of the nuclear chromatin and disruption of the plasmalemmas , presumably degenerative phenomena , causing the extracellular release of melanin granules .", "the membranous tissue excised from the left eye 5  months after initiation of therapy and 3  months after most recent intravitreal injection comprised the inner choroid with its choriocapillaris , bruch s membrane , and a vascularized subretinal multilaminar cellular membrane with many capillary - type units ( fig . ", "epon - embedded , light microscopic sections , no break was detected in bruch s membrane that demonstrated a vascular channel connecting the choriocapillaris with the subretinal neovascular units .", "the inner choroid harbored a distinct focus of chronic inflammatory cells without follicular organization . by transmission electron microscopy , the pigment epithelium consisted of one or two layers at the center of the membrane with neovascular channels distributed among them in the absence of inflammation ( fig . ", "the rpe cells and neovascular channels in the middle of the lesion also interfaced with broad processes of mueller cells without the interposition of photoreceptors or their degenerative remains ( fig . ", "the mueller cells were characterized by multiform mitochondria , abundant profiles of smooth surfaced endoplasmic reticulum , and the absence of pigment granules .", "poorly pigmented displaced rpe cells contained rare pigment granules , profiles of rough surfaced endoplasmic reticulum and manifested segments of basement membrane material and disorganized surface villi .", "extravasated erythrocytes , macrophages , lymphocytes , and conspicuous extracellular matrix material were not found in the membrane , except for the last which was limited to the pericapillary zone .", "the viable neoformed intramembranous capillaries displayed patent lumens with endothelial cell fenestrae and uniformly enveloping pericytes , both ensheathed by basement membranes ( fig . ", "5a in the left eye , above bruch s membrane ( bm ) with its elastic layer and subjacent choriocapillaris ( ch ) , are two to three layers of pigment epithelial cells ( pe ) with loss of polarity .", "the pe cells interface with broad processes of mueller cells ( mc ) in the upper half of the field with myriad mitochondria .", "inset : fenestrations ( arrows ) of the endothelial cells of the neoformed capillary and a thick surrounding basement membrane ( bm ) ( em 1,950 ) .", "c at the edge of the membrane , pigment epithelial cells ( pe ) with basement membrane ( bm ) are positioned on bruch s membrane ( bm ) . within the cytoplasm of the central pe cell", "is a degenerated compact whorled membranous body ( arrow ) , another of which is also shown toward the right in the cytoplasm of an indeterminate cell ( crossed arrow ) .", "the underlying choriocapillaris ( cc ) with its endothelial cells ( e ) is patent .", "a large melanophage ( m ) contains complex phagolysosomes ( crossed arrows ) of engulfed uveal melanin .", "e extravasated erythrocyte ( em 1,450 ) a in the left eye , above bruch s membrane ( bm ) with its elastic layer and subjacent choriocapillaris ( ch ) , are two to three layers of pigment epithelial cells ( pe ) with loss of polarity .", "the pe cells interface with broad processes of mueller cells ( mc ) in the upper half of the field with myriad mitochondria .", "inset : fenestrations ( arrows ) of the endothelial cells of the neoformed capillary and a thick surrounding basement membrane ( bm ) ( em 1,950 ) .", "c at the edge of the membrane , pigment epithelial cells ( pe ) with basement membrane ( bm ) are positioned on bruch s membrane ( bm ) . within the cytoplasm of the central pe cell is a degenerated compact whorled membranous body ( arrow ) , another of which is also shown toward the right in the cytoplasm of an indeterminate cell ( crossed arrow ) .", "the underlying choriocapillaris ( cc ) with its endothelial cells ( e ) is patent .", "a large melanophage ( m ) contains complex phagolysosomes ( crossed arrows ) of engulfed uveal melanin .", "e extravasated erythrocyte ( em 1,450 ) at the edge of the membrane , a single layer of dyspolar pigment epithelial cells without apical villi was surrounded by whorled membranous bodies of various sizes , some of which were engulfed by the rpe cells and others by macrophages ( fig . ", "the focus of inner choroidal inflammatory cells was composed of mature small lymphocytes with clumped chromatin and scant cytoplasm without intermixed histiocytes and plasma cells ( fig . ", "the choroidal melanocytes were focally damaged and had released their small pigment granules extracellularly , which had attracted melanophages ( fig . "], ["this report documents for the first time the healing response following anti - vegf treatment of cnvs and highlights possible causes of treatment failure .", "we also analyzed the clinical and anatomical implications of surgical removal of cnvs following treatment failure of anti - vegf agents .", "there is one other report in the literature of excision of a cnv membrane following intravitreal injection of the anti - vegf agent bevacizumab , in which only avascular fibrous tissue was described pathologically .", "the submacular membranes removed from both eyes in our case had significantly different light and electron microscopic features . that from the right eye , which had been followed and treated over a span of 20  months prior to surgical excision ( in comparison with 5  months for the left eye ) , was remarkably hypocellular and mainly composed of abundant fibrillocollagenous material .", "apart from the presence of rare elongated processes of degenerating pigment epithelial cells or an occasional macrophage , lymphocytes and plasma cells were absent .", "a cluster of polygonal pigment epithelial cells at the membrane s base near bruch s membrane displayed disrupted plasmalemmas and a disturbed nuclear chromatin pattern of fine clumping compatible with incipient dissolution . the ability of the rpe cells to undergo fibrous and even osseous metaplasia is well recognized .", "the vascular endothelial cells of the membrane s capillaries were attenuated and fragmented , and their lumens were filled with microfibrils .", "only one capillary appeared functional with healthier looking constituent cells that included fenestrated endothelial cells and well - preserved pericytes . in this zone , the surrounding extracellular matrix was looser , suggesting that such vessels were of more recent origin than the others and probably related to the recurrence .", "in contrast , the submacular membrane that was removed from the left eye 5  months after initiation of treatment displayed virtually no extracellular fibrillar matrix material .", "the obviously viable and intact vascular endothelial cells displayed fenestrae and were consistently surrounded by pericytes .", "the fenestrae support an origin of the neovascular units from the choriocapillaris , which normally possesses this feature .", "there was no evidence of a lymphohistiocytic inflammatory component accompanying the subretinal neovascularization throughout most of the membrane , as also described in a previous immunohistochemical study of cnv in pic .", ", however , reported moderate to prominent lymphocytic infiltration in four out of the six cnv specimens studied . the pigment epithelial cells in the left membrane remained for the most part confined to one or two layers at the base of the membrane and were in direct apposition to broad processes of mueller cells without the interposition of photoreceptors or their degenerative remnants .", "the latter were discovered at the borders of the membrane in the form of whorled lamellated membranous bodies probably representing degenerative products of outer segments .", "this finding suggests a primary degenerative mechanism rather than an infarction , which would have produced a more complete and broader effacement of cellular detail with a prominent inflammatory cell infiltrate . finally , the fortuitous discovery of lymphocytes at the level of the inner choroid with sparing of the choriocapillaris lends , for the fist time , ultrastructural electron microscopic support to the hypothesis that pic is an inflammatory disease , with the inflammation originating in the choroid .", "there are no other em studies of tissue excised from a pic case that include choroid manifesting inflammatory infiltration .", "thus , there was a striking difference in the response of the two submacular membranes to bevacizumab treatment . while different durations of the two membranes before their removal created concomitant differences in the amount of collagen deposition , the chief ultrastructural differential feature was the pronounced regression of the neoformed vessels in the right eye that lacked pericytes ( in comparison with those in the left eye that possessed them ) . this phenomenon could be a primary feature of the neovascular units themselves or reflect a preferential secondary loss during the membrane s evolution or treatment .", "the well - preserved architecture of the endothelial - lined vascular channels with a normal accompaniment of pericytes in the left eye intimates that anti - vegf treatment had no effect on them .", "the finding of one capillary in the right specimen with fenestrated endothelial cells and well - preserved pericytes , while all other capillaries which were occluded manifested fragmented endothelial cells and lacked pericytes , may signify that it represented cnv nonresponsiveness to the most recent anti - vegf treatment 3  months prior to surgery .", "this is further supported by the fact that in this zone the surrounding extracellular matrix was looser , suggesting more recent origin and less deposition of collagen as explained above .", "the capillaries with fragmented endothelial cells most likely represent the regressed cnv treated with anti - vegf agents 20  months prior to surgery .", "pericyte - poor neovascular units have been shown to be more susceptible to anti - vegf agents than pericyte - rich ones .", "we therefore hypothesize that failure to respond to anti - vegf treatment in the left eye of our patient may be associated with the uniform presence of a pericytic component in the neovascular channels .", "these observations indicate that the presence of pericytes may act as a survival factor for endothelial cells and therefore increase resistance to anti - vegf therapy .", "context , dual vegf and platelet - derived growth factor - targeted therapy has been shown to be more effective in inhibiting in vivo tumor growth than either agent alone .", "choroidal lymphocytic infiltration was found in the left subretinal excision which included a segment of choriocapillaris and surrounding inner choroidal tissue .", "this finding should not be totally unexpected despite the absence of overt clinical intraocular inflammation .", "none of the eyes had undergone prior surgery or photodynamic therapy that could have triggered inflammation .", "the presence of a focus of choroidal inflammation is also suggestive of the possibility that anti - vegf treatment of cnvs may fail because of underlying persistent inflammation .", "this provides another basis for considering a combination of therapeutic modalities in addition to anti - vegf factors to improve the efficacy of treatment in cnvs secondary to pic .", "this is the first report showing persistent choroidal inflammation after anti - vegf treatment that could possibly provoke recurrent cnvs , in contradistinction to inflammation in the membrane itself .", "available data on treatment of cnv associated to pic are derived from small nonrandomized case series .", "local and systemic treatment with steroids has not been proven effective in the treatment of cnv associated with pic , even when administered at high doses for many months [ 6 , 10 ] .", "more recent small prospective studies of pdt in combination with either systemic steroids or 4  mg intravitreal triamcinolone of five and four eyes , respectively , showed decreased activity of cnv lesions [ 11 , 12 ] .", "three out of the four patients who received intravitreal triamcinolone experienced improvement of two or more lines of vision and one patient lost two or more lines at 1-year follow - up .", "we should , however , take into account the high rate of side effects of systemic or local corticosteroid treatment .", "regarding the surgical management outcome of this case , prior anti - vegf treatment did not appear to complicate surgical excision . in our series of three submacular surgeries in two eyes ,", "there were no adverse events peri- or postoperatively , but the number is too small for any definite conclusions .", "this high rate of recurrence in our series is in accord with the results of the submacular surgery trial ( sst ) trial in which recurrent cnv developed in 58% of surgically treated eyes by the 24-month examination .", "recurrences occurred in four of six eyes with cnv associated with pic following surgical excision in the series reported by olsen et al . .", "following reports from the sst trial for cnv that showed no benefit in preventing visual loss and a high rate of adverse events , submacular surgery has been virtually abandoned .", "sst reported on both type i membranes , which grow below the rpe and are typically secondary to amd , and type ii membranes , which grow subretinally .", "pic is one of the conditions , along with an idiopathic category , myopia , and presumed ocular histoplasmosis syndrome , that are complicated by type ii cnvs .", "the sst trial showed that surgical excision of cnv in pohs and idiopathic cases probably has a more favorable visual outcome in the subgroup of eyes with initial visual acuity 20/100 or worse .", "a successful outcome was defined as a visual acuity better , or no more than one line worse , than at baseline . as a result , a smaller benefit of surgery , such as reduction of risk of loss of three lines of vision , which many trials have been designed to detect , could not be confirmed .", "also , 25% of patients were 60  years or older ; results may be more favorable in a younger cohort of patients , as large case series have suggested .", "still , submacular surgery carries significant risks and should be considered only in selected cases nonresponsive or nontolerant to other treatment modalities . in conclusion , this report provides evidence that inhibition of vegf alone can not be relied upon to induce stable and lasting regression of subretinal neovascularization in pic .", "this is the first pathological study employing human tissue that points to pericytes as a potential critical target with the aggravating influence of inner choroidal chronic inflammation ."], ["this article is distributed under the terms of the creative commons attribution license which permits any use , distribution and reproduction in any medium , provided the original author(s ) and source are credited ."]]}
{"article_id": "PMC4672720", "article_text": ["hemolysis , elevated liver enzymes , and low platelet count ( hellp ) syndrome is a severe manifestation of a hypertensive disorder of pregnancy called pre - eclampsia .", "it affects about 10% to 20% of patients with severe pre - eclampsia ( 0.5% to 0.9% of all pregnancies ) and causes significant mortality and morbidity , which increases in accordance with the severity of this syndrome .", "this syndrome is associated with increased maternal risk of developing morbidities , including cerebrovascular complications , hemorrhage , pulmonary edema , retinal detachment , hematoma / hepatic rupture , acute renal failure , liver failure , intravascular coagulopathy , placental abruption , and sepsis [ 35 ] .", "perinatal / infant morbidity and mortality rates are higher in pregnant women with hellp syndrome .", "the preterm delivery rate is about 70% in hellp syndrome patients with about 15% of cases requiring parturition before the 27 week of gestation .", "sufferers may refrain from further pregnancies and need psychological support , and those who attempt further pregnancies have higher risk of gestational hypertension .", "previously , immediate delivery was indicated for patients diagnosed with hellp syndrome , which often resulted in significant maternal and neonatal morbidity and/or mortality .", "later it was recognized that antepartum administration of high - dose corticosteroids can stabilize the disease indicators and prolong the gestation .", "although many studies have demonstrated that cort use helps raise the platelet count and reduce elevated liver enzymes , results are not consistent across all studies .", "moreover , evidence regarding the role of corticosteroids in improving maternal morbidity and mortality is not clear .", "the present study , therefore , carried out a systematic review of the relevant studies and performed a meta - analysis of all related parameters for the sake of evaluating the efficacy of cort therapy observed in studies with controlled designs .", "important features of the method used for the present study are summarized in table 1 .", "several electronic databases were searched for the acquisition of required study reports by using the most relevant mesh and keywords in different logical combinations and phrases .", "the inclusion criterion was  the studies examining the efficacy of cort therapy to treat hellp patients either in a prospective or retrospective controlled design. some studies were , however , excluded by the following exclusion criteria ( table 1 ) .", "important information including outcome measures and outcomes , dosage and mode of administration of cort , and obstetric and demographic characteristics were obtained from identified papers and organized on datasheets .", "meta - analyses of mean difference and odds ratio were carried out under the random - effects model .", "fifteen studies [ 1226 ] fulfilled were eligible and were included in the meta - analysis .", "of the included studies , 8 were randomized controlled trials and 7 were retrospective analyses .", "the overall population of this meta - analysis is 675 cort treated and 787 control hellp patients .", "age of the cort treated and control patients as mean sd ( range ) was 26.945.8 ( 23.2633.54 ) years and 26.355.7 ( 23.1630.93.3 ) years , respectively .", "gestation duration was 32.273.7 ( 29.13.535.12.9 ) weeks in cort treated and 32.423.8 ( 27.63.335.52.6 ) weeks in control hellp patients . in the cort treated group ,", "least asymmetry was visible from the visual inspection of the funnel plots , indicative of almost no publication bias in this area of research ( figure 2 ) .", "the mean difference [ 95% confidence interval ] in the change from baseline between cort treated patients and controls was 38.08 [ 15.71 , 60.45]10/l ; p=0.0009 ( figure 3 ) . on the other hand ,", "the mean differences in the changes from baseline between cort treated and controls were 0.44 [ 0.76 , 0.12 ] iu / ml ; p=0.007 for ldh ( figure 4 ) and 143.34 [ 278.69 , 7.99 ] iu / l ; p=0.04 for alt . however , the decrease in ast levels was not statistically significant in cort - treated patients in comparison with controls ( 48.50 [ 114.32 , 17.32 ] iu / l ; p=0.15 ; table 2 ) .", "blood transfusion rate was significantly lower in cort - treated patients ( odds ratio [ 95% ci ] : 0.42 [ 0.24 , 0.76 ] ; p=0.004 .", "hospital / icu stay was also significantly lower in cort - treated patients ( mean difference : 1.79 [ 3.54 , 0.05 ] ; p=0.04 ) .", "there was no significant difference between cort - treated and control patients in the incidence of cesarean deliveries ( odds ratio [ 95% ci ] : 1.25 [ 0.95 , 1.63 ] ; p=0.11 ) , prevalence of infections ( 0.78 [ 0.19 , 3.15 ] ; p=0.73 ; table 2 ) , birth weight ( mean difference : 0.09 [ 0.11 , 0.28 ] ; p=0.38 ) , infant respiratory distress incidence ( odds ratio : 1.13 [ 0.50 , 2.53 ] ; p=0.78 ) and maternal mortality ( odds ratio : 1.27 [ 0.45 , 3.60 ] ; p=0.65 ) ( table 2 ) . among the included studies , infant mortality was 23% in cort - treated patients and 8.3% in controls   and 4% in cort - treated patients and 0% in controls .", "perinatal death was 0% in cort - treated patients and 3% in controls . despite lower frequency of morbid conditions in hellp patients treated with cort ( 318 vs. 418 ) , there was no significant difference in the incidence of overall morbidity between the groups ( odds ratio : 0.79 [ 0.58 , 1.08 ] ; p=0.14 ) .", "morbid complications observed in 1 or more studies included pulmonary edema ( 3.6% ) , intraventricular hemorrhage ( 18% , disseminated intravascular coagulation ( 15% ) , endomyometritis ( 9% ) , ascites ( 13.3% ) , hematoma ( 3.3% ) , acute renal failure and other renal pathologies ( 14% , necrotizing colitis ( 12% , bronchopulmonary dysplasia ( 80% ) , intraventricular hematoma ( 20% ) , infant thrombocytopenia ( 13% ) , apgar score less than 7 ( 18% ) , and other hematological ( 36% ) , neurological ( 12% ) , and cardiopulmonary complications ( 33% ) .", "this meta - analysis of the studies with variable research designs revealed that in comparison with controls , cort therapy significantly improved the platelet count , ldh , and alt , as well as reducing ast levels non - significantly in patients with hellp syndrome .", "moreover , blood transfusion rate and hospital / icu stay were significantly lower in cort - treated patients .", "however , there was no significant difference in the maternal mortality , overall morbidity , birth weight , or infant respiratory distress between cort - treated and control patients .", "platelet count and serum ldh levels are reliable indicators of hellp severity , and recovery and longer recovery time is required for more severe cases .", "corticosteroids are thought to prevent platelet consumption and erythrocyte destruction by stabilizing the vascular endothelium and effectually reducing blood product administration requirements .", "the recovery of platelets is reported to start as earlier as 12 hours after cort administration .", "the hellp syndrome , especially in the postpartum period , is associated with high maternal morbidity .", "class 1 hellp syndrome patients are at higher risk of maternal mortality , and delay in the diagnosis worsens prognosis . despite improvements in biological parameters of hellp syndrome , most of the studies reported that cort treatment does not reduce maternal morbidity .", "the present study also found no significant difference between cort - treated and control hellp patients in the incidence of overall morbidity in a meta - analysis of 8 studies presenting 15 morbid conditions .", "the morbid conditions observed in the present study were also reported by many studies not included in this meta - analysis .", "the morbidities not reported herein include abruptio placentae , retinal detachment , adult respiratory distress syndrome , and hypoxic ischemic encephalopathy .", "it is believed that an imbalance between proangiogenic and antiangiogenic factors and increased proinflammatory cytokines play an important role in women with preeclampsia and hellp syndrome .", "higher circulating levels of anti - angiogenic proteins secreted by the placenta , such as soluble fms - like tyrosine kinase 1 ( sflt1 ) and soluble endoglin , are found in preeclampsia patients .", "dexamethasone has been demonstrated to significantly decrease sflt-1 , soluble endoglin , il-6 , and tnf- after 24 hours of treatment in hellp patients .", "these soluble factors are known to stimulate angiotensin ii receptor ( at1-aa ) production and increase endothelin 1 , which are known to play a pathophysiological role in gestational hypertension .", "these findings suggest that targeting immunomodulators of hellp syndrome pathology may be a novel therapeutic research strategy .", "management of hellp syndrome requires earlier diagnosis , mother - fetus status examination , stabilization of the indicators and symptoms , delivery at optimal time , and postpartum care in order to reduce maternal morbidity and mortality .", "a rather longer postpartum recovery period may be required for patients with progressively worsening hellp syndrome .", "corticosteroid therapy is a cost - effective medication that can be administered via different routes and reduces the length of hospitalization as compared to other treatments , such as platelet transfusion . in the present study , on average , cort therapy reduced hospital / icu", "stay by about 3 days in comparison with controls and this difference was statistically significant in the meta - analysis of 7 studies .", "thus , corticosteroids can be beneficial in carefully selected hellp patients without apparent adverse effects to mother or fetus / neonate .", "firstly , studies with varying designs were included because none of a particular design could make sufficient data available .", "secondly , clinical and methodological heterogeneity of the sample population in the form of factors such as the severity of hellp syndrome , time of cort administration , and dosage and duration of cort administration in recruited patients may have affected overall outcomes .", "although , the random - effects model was used to interpret the results , but multi - center randomized controlled trials will be required for clarification of these results .", "thirdly , the effect of some statistical procedures used to impute missing data may also have had a slight impact , as not all studies provided measures of dispersal values of the effect size of change in indicators following cort / placebo treatments .", "corticosteroid administration to hellp patients improves platelet count and the serum levels of ldh , besides reducing hospital / icu stay and blood transfusion rate .", "however , these indices are not significantly associated with maternal mortality and overall morbidity prevalence ."], "abstract_text": ["<S> backgroundhemolysis , elevated liver enzymes , and low platelet count ( hellp ) syndrome is a severe condition of pregnancy that is associated with significant morbidity and mortality . </S>", "<S> corticoteroid ( cort ) therapy is common in the management of hellp syndrome . </S>", "<S> this study evaluates the efficacy of cort therapy to patients with hellp syndrome.material/methodsa literature search was carried out in multiple electronic databases . </S>", "<S> meta - analyses of means difference and odds ratio were carried under the random - effects model.resultsfifteen studies ( 675 cort treated and 787 control hellp patients ) were included . </S>", "<S> cort treatment significantly improved platelet count ( mean difference between cort treated and controls in changes from baseline , md : 38.08 [ 15.71 , 60.45]109 ; p=0.0009 ) , lactic dehydrogenase ( ldh ) levels ( md : 440 [ 760 , 120 ] iu / l ; p=0.007 ) , and alanine aminotransferase ( alt ) levels ( md : 143.34 [ 278.69 , 7.99 ] iu / l ; p=0.04 ) but the decrease in aspartate aminotransferase ( ast ) levels was not statistically significant ( md : 48.50 [ 114.32 , 17.32 ] iu / l ; p=0.15 ) . </S>", "<S> corticosteroid treatment was also associated with significantly less blood transfusion rate ( odds ratio , or : 0.42 [ 0.24 , 0.76 ] ; p=0.004 ) and hospital / icu stay ( md : 1.79 [ 3.54 , 0.05 ] days ; p=0.04 ) . maternal mortality ( or : 1.27 [ 0.45 , 3.60 ] ; p=0.65 ) , birth weight ( md : 0.09 [ 0.11 , 0.28 ] ; p=0.38 ) and the prevalence of morbid conditions ( or : 0.79 [ 0.58 , 1.08 ] ; p=0.14 ) did not differ significantly between both groups.conclusionscorticosteroid administration to hellp patients improves platelet count , and the serum levels of ldh and alt , and reduces hospital / icu stay and blood transfusion rate , but is not significantly associated with better maternal mortality and overall morbidity . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Results", "Discussion", "Conclusions", "Supplementary materials"], "sections": [["hemolysis , elevated liver enzymes , and low platelet count ( hellp ) syndrome is a severe manifestation of a hypertensive disorder of pregnancy called pre - eclampsia .", "it affects about 10% to 20% of patients with severe pre - eclampsia ( 0.5% to 0.9% of all pregnancies ) and causes significant mortality and morbidity , which increases in accordance with the severity of this syndrome .", "this syndrome is associated with increased maternal risk of developing morbidities , including cerebrovascular complications , hemorrhage , pulmonary edema , retinal detachment , hematoma / hepatic rupture , acute renal failure , liver failure , intravascular coagulopathy , placental abruption , and sepsis [ 35 ] .", "perinatal / infant morbidity and mortality rates are higher in pregnant women with hellp syndrome .", "the preterm delivery rate is about 70% in hellp syndrome patients with about 15% of cases requiring parturition before the 27 week of gestation .", "sufferers may refrain from further pregnancies and need psychological support , and those who attempt further pregnancies have higher risk of gestational hypertension .", "previously , immediate delivery was indicated for patients diagnosed with hellp syndrome , which often resulted in significant maternal and neonatal morbidity and/or mortality .", "later it was recognized that antepartum administration of high - dose corticosteroids can stabilize the disease indicators and prolong the gestation .", "although many studies have demonstrated that cort use helps raise the platelet count and reduce elevated liver enzymes , results are not consistent across all studies .", "moreover , evidence regarding the role of corticosteroids in improving maternal morbidity and mortality is not clear .", "the present study , therefore , carried out a systematic review of the relevant studies and performed a meta - analysis of all related parameters for the sake of evaluating the efficacy of cort therapy observed in studies with controlled designs ."], ["important features of the method used for the present study are summarized in table 1 .", "several electronic databases were searched for the acquisition of required study reports by using the most relevant mesh and keywords in different logical combinations and phrases .", "the inclusion criterion was  the studies examining the efficacy of cort therapy to treat hellp patients either in a prospective or retrospective controlled design. some studies were , however , excluded by the following exclusion criteria ( table 1 ) .", "important information including outcome measures and outcomes , dosage and mode of administration of cort , and obstetric and demographic characteristics were obtained from identified papers and organized on datasheets .", "meta - analyses of mean difference and odds ratio were carried out under the random - effects model ."], ["fifteen studies [ 1226 ] fulfilled were eligible and were included in the meta - analysis .", "of the included studies , 8 were randomized controlled trials and 7 were retrospective analyses .", "the overall population of this meta - analysis is 675 cort treated and 787 control hellp patients .", "age of the cort treated and control patients as mean sd ( range ) was 26.945.8 ( 23.2633.54 ) years and 26.355.7 ( 23.1630.93.3 ) years , respectively .", "gestation duration was 32.273.7 ( 29.13.535.12.9 ) weeks in cort treated and 32.423.8 ( 27.63.335.52.6 ) weeks in control hellp patients . in the cort treated group ,", "least asymmetry was visible from the visual inspection of the funnel plots , indicative of almost no publication bias in this area of research ( figure 2 ) .", "the mean difference [ 95% confidence interval ] in the change from baseline between cort treated patients and controls was 38.08 [ 15.71 , 60.45]10/l ; p=0.0009 ( figure 3 ) . on the other hand ,", "the mean differences in the changes from baseline between cort treated and controls were 0.44 [ 0.76 , 0.12 ] iu / ml ; p=0.007 for ldh ( figure 4 ) and 143.34 [ 278.69 , 7.99 ] iu / l ; p=0.04 for alt . however , the decrease in ast levels was not statistically significant in cort - treated patients in comparison with controls ( 48.50 [ 114.32 , 17.32 ] iu / l ; p=0.15 ; table 2 ) .", "blood transfusion rate was significantly lower in cort - treated patients ( odds ratio [ 95% ci ] : 0.42 [ 0.24 , 0.76 ] ; p=0.004 .", "hospital / icu stay was also significantly lower in cort - treated patients ( mean difference : 1.79 [ 3.54 , 0.05 ] ; p=0.04 ) .", "there was no significant difference between cort - treated and control patients in the incidence of cesarean deliveries ( odds ratio [ 95% ci ] : 1.25 [ 0.95 , 1.63 ] ; p=0.11 ) , prevalence of infections ( 0.78 [ 0.19 , 3.15 ] ; p=0.73 ; table 2 ) , birth weight ( mean difference : 0.09 [ 0.11 , 0.28 ] ; p=0.38 ) , infant respiratory distress incidence ( odds ratio : 1.13 [ 0.50 , 2.53 ] ; p=0.78 ) and maternal mortality ( odds ratio : 1.27 [ 0.45 , 3.60 ] ; p=0.65 ) ( table 2 ) . among the included studies , infant mortality was 23% in cort - treated patients and 8.3% in controls   and 4% in cort - treated patients and 0% in controls .", "perinatal death was 0% in cort - treated patients and 3% in controls . despite lower frequency of morbid conditions in hellp patients treated with cort ( 318 vs. 418 ) , there was no significant difference in the incidence of overall morbidity between the groups ( odds ratio : 0.79 [ 0.58 , 1.08 ] ; p=0.14 ) .", "morbid complications observed in 1 or more studies included pulmonary edema ( 3.6% ) , intraventricular hemorrhage ( 18% , disseminated intravascular coagulation ( 15% ) , endomyometritis ( 9% ) , ascites ( 13.3% ) , hematoma ( 3.3% ) , acute renal failure and other renal pathologies ( 14% , necrotizing colitis ( 12% , bronchopulmonary dysplasia ( 80% ) , intraventricular hematoma ( 20% ) , infant thrombocytopenia ( 13% ) , apgar score less than 7 ( 18% ) , and other hematological ( 36% ) , neurological ( 12% ) , and cardiopulmonary complications ( 33% ) ."], ["this meta - analysis of the studies with variable research designs revealed that in comparison with controls , cort therapy significantly improved the platelet count , ldh , and alt , as well as reducing ast levels non - significantly in patients with hellp syndrome .", "moreover , blood transfusion rate and hospital / icu stay were significantly lower in cort - treated patients .", "however , there was no significant difference in the maternal mortality , overall morbidity , birth weight , or infant respiratory distress between cort - treated and control patients .", "platelet count and serum ldh levels are reliable indicators of hellp severity , and recovery and longer recovery time is required for more severe cases .", "corticosteroids are thought to prevent platelet consumption and erythrocyte destruction by stabilizing the vascular endothelium and effectually reducing blood product administration requirements .", "the recovery of platelets is reported to start as earlier as 12 hours after cort administration .", "the hellp syndrome , especially in the postpartum period , is associated with high maternal morbidity .", "class 1 hellp syndrome patients are at higher risk of maternal mortality , and delay in the diagnosis worsens prognosis . despite improvements in biological parameters of hellp syndrome , most of the studies reported that cort treatment does not reduce maternal morbidity .", "the present study also found no significant difference between cort - treated and control hellp patients in the incidence of overall morbidity in a meta - analysis of 8 studies presenting 15 morbid conditions .", "the morbid conditions observed in the present study were also reported by many studies not included in this meta - analysis .", "the morbidities not reported herein include abruptio placentae , retinal detachment , adult respiratory distress syndrome , and hypoxic ischemic encephalopathy .", "it is believed that an imbalance between proangiogenic and antiangiogenic factors and increased proinflammatory cytokines play an important role in women with preeclampsia and hellp syndrome .", "higher circulating levels of anti - angiogenic proteins secreted by the placenta , such as soluble fms - like tyrosine kinase 1 ( sflt1 ) and soluble endoglin , are found in preeclampsia patients .", "dexamethasone has been demonstrated to significantly decrease sflt-1 , soluble endoglin , il-6 , and tnf- after 24 hours of treatment in hellp patients .", "these soluble factors are known to stimulate angiotensin ii receptor ( at1-aa ) production and increase endothelin 1 , which are known to play a pathophysiological role in gestational hypertension .", "these findings suggest that targeting immunomodulators of hellp syndrome pathology may be a novel therapeutic research strategy .", "management of hellp syndrome requires earlier diagnosis , mother - fetus status examination , stabilization of the indicators and symptoms , delivery at optimal time , and postpartum care in order to reduce maternal morbidity and mortality .", "a rather longer postpartum recovery period may be required for patients with progressively worsening hellp syndrome .", "corticosteroid therapy is a cost - effective medication that can be administered via different routes and reduces the length of hospitalization as compared to other treatments , such as platelet transfusion . in the present study , on average , cort therapy reduced hospital / icu", "stay by about 3 days in comparison with controls and this difference was statistically significant in the meta - analysis of 7 studies .", "thus , corticosteroids can be beneficial in carefully selected hellp patients without apparent adverse effects to mother or fetus / neonate .", "firstly , studies with varying designs were included because none of a particular design could make sufficient data available .", "secondly , clinical and methodological heterogeneity of the sample population in the form of factors such as the severity of hellp syndrome , time of cort administration , and dosage and duration of cort administration in recruited patients may have affected overall outcomes .", "although , the random - effects model was used to interpret the results , but multi - center randomized controlled trials will be required for clarification of these results .", "thirdly , the effect of some statistical procedures used to impute missing data may also have had a slight impact , as not all studies provided measures of dispersal values of the effect size of change in indicators following cort / placebo treatments ."], ["corticosteroid administration to hellp patients improves platelet count and the serum levels of ldh , besides reducing hospital / icu stay and blood transfusion rate .", "however , these indices are not significantly associated with maternal mortality and overall morbidity prevalence ."], [""]]}
{"article_id": "PMC4322381", "article_text": ["during the last few years , dual - source high - pitch imaging has become increasingly important in the daily routine of radiology departments . in dual - source computed tomography ( ct ) imaging two ", "sources means that two x - ray tubes are in use at an angle of approximately 90 degrees to each other .", "after introduction of the second generation dual - source ct , about four years ago , increasing pitch ( pitch = table mm / n * t mm , where n = no", ". of slices and t = slice thickness mm ) values above the former traditional technical limit of 1.5 in a single - source ct , became possible .", "when high - pitch imaging is performed , one of the major advantages , compared to conventional single source normal pitch imaging , is the image acquisition time . in high - pitch imaging", "this makes high - pitch imaging the technique of choice , especially when regions of the body that are normally subject to motion artifacts have to be examined .", "therefore , in clinical practice there are some major regions of interest where high - pitch imaging can demonstrate its advantages , such as , in imaging of the heart , the great thoracic vessels , and the lungs , in patients who have problems holding their breath . as our clinical experience in high - pitched imaging shows", " especially in non - electrocardiogram ( ekg)-gated high - pitch imaging  the amount of energy that can be applied may be limited by patient habitus . in high - pitch imaging of the lung ,", "these energy factors are not relevant , but in imaging of the upper abdominal tract the maximum applicable energy may cause problems , leading to diagnostic issues . therefore , the aim of this study was to examine the maximum current levels that can be used in dual - source high - pitch ct during imaging of the upper abdominal tract .", "an upper - abdominal phantom was placed on the ct table and no alterations were made to this experimental setup during all the ct examinations [ figure 1 ] .", "this was followed by a series of high - pitch examinations , beginning with a pitch of 3.2 [ table 1 ] .", "this value was reduced in steps of 0.2 , with each successive test , ending at a pitch of 1.6 in the final examination [ tables 2 and 3 ] .", "all investigations were carried out in the same scan range , once with a tube voltage of 120 kv , and once with 100 kv ( modes 1 and 2 , table 1 ) .", "the tube charge in mas (= ma multiplied by the rotation time ) was always adjusted to the maximum that the ct machine could perform at , without using the dose modulation software ( care dose 4d ) , so that the exact tube maximum could be reached . to achieve the maximum current , the mas value", "one step downward from the level producing an error - message was chosen for the examinations , and this was taken as the maximum current that the ct machine was able to apply .", "examination protocols high - pitch examinations at 100 kv high - pitch examinations at 120 kv the images obtained were reconstructed in a conventional filtered back - projection recon kernel b30 , as frequently used for imaging soft tissue .", "the images were assessed in slices / increments of 5 mm/5 mm as transversal cross - sectional images . for further evaluation coronal and sagittal images in slices / increments of 2 mm/2 mm were reconstructed in the same manner . as objective measures of image quality of the examination series , several region - of - interest ( roi ) measurements , using the circle tool and a picture archiving and communication system ( pacs ) workstation ( centricity 4.2 , general electric healthcare , dornstadt , germany ) , were performed by a radiologist , who had four years of experience in ct .", "the mean attenuation values and standard deviation were recorded and displayed in hounsfield units ( hu ) .", "the background noise ( bn ) was determined as the standard deviation of air measured in front of the phantom .", "the attenuation at different locations in the phantom were measured ( liver parenchyma , liver metastases ( hyper- and hypo - dense examples ) , muscle tissue , cutaneous fat tissue , cutaneous metastases ( hyper- and hypodense examples ) , as also the air in front of the phantom ) .", "the roi measurements were drawn as large as possible , to include as much of the corresponding area of interest as feasible . to minimize bias from single measurements", ", we calculated the average of four measurements for each roi . on the basis of these measurements , the signal - to - noise ratio ( snr )", "was determined according to the following equation : snr = attenuation / bn . to calculate the contrast - to - noise ratio ( cnr)- value , we measured the attenuation and the hu and sd of attenuation of the surrounding muscle tissue ( roi muscle ) compared with the attenuation of the hyperdense subcutaneous lesions [ figures 2 and 3 ] .", "cnr was calculated as cnr = ( ( roi subclesion - roi muscle)/image noise ) .", "images at different pitch values ( 100 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ; b30 kernel ) . images at different pitch values ( 120 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ) .", "subjective image quality rating was conducted in a blind fashion by two independent radiologists , with three and four years of experience , respectively , in general ct imaging .", "the rating was done according to a five - point scale as follows : 1 = excellent / no artifacts , 2 = good / hardly any artifacts , 3 = moderate / few artifacts , 4 = fair / many artifacts , 5 = unacceptable .", "exemplary : attenuation values , snr , cnr , subjective image quality score , dlp ( dose length product ) and ctdivol ( computed tomography dose index ) .", "analyses were performed using a dedicated software ( bias 9.14 , epsilon verlag , germany ) .", "a cohen 's kappa analysis was performed to determine the interobserver agreement for subjective image quality scoring .", "as objective measures of image quality of the examination series , several region - of - interest ( roi ) measurements , using the circle tool and a picture archiving and communication system ( pacs ) workstation ( centricity 4.2 , general electric healthcare , dornstadt , germany ) , were performed by a radiologist , who had four years of experience in ct .", "the mean attenuation values and standard deviation were recorded and displayed in hounsfield units ( hu ) .", "the background noise ( bn ) was determined as the standard deviation of air measured in front of the phantom .", "the attenuation at different locations in the phantom were measured ( liver parenchyma , liver metastases ( hyper- and hypo - dense examples ) , muscle tissue , cutaneous fat tissue , cutaneous metastases ( hyper- and hypodense examples ) , as also the air in front of the phantom ) .", "the roi measurements were drawn as large as possible , to include as much of the corresponding area of interest as feasible . to minimize bias from single measurements", "on the basis of these measurements , the signal - to - noise ratio ( snr ) was determined according to the following equation : snr = attenuation / bn . to calculate the contrast - to - noise ratio ( cnr)- value , we measured the attenuation and the hu and sd of attenuation of the surrounding muscle tissue ( roi muscle ) compared with the attenuation of the hyperdense subcutaneous lesions [ figures 2 and 3 ] .", ". images at different pitch values ( 100 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ; b30 kernel ) .", "images at different pitch values ( 120 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ) .", "subjective image quality rating was conducted in a blind fashion by two independent radiologists , with three and four years of experience , respectively , in general ct imaging .", "the rating was done according to a five - point scale as follows : 1 = excellent / no artifacts , 2 = good / hardly any artifacts , 3 = moderate / few artifacts , 4 = fair / many artifacts , 5 = unacceptable .", "exemplary : attenuation values , snr , cnr , subjective image quality score , dlp ( dose length product ) and ctdivol ( computed tomography dose index ) .", "analyses were performed using a dedicated software ( bias 9.14 , epsilon verlag , germany ) . for statistical comparison", "a cohen 's kappa analysis was performed to determine the interobserver agreement for subjective image quality scoring .", "it became apparent that the maximum applicable energy , especially at increasing pitch levels , was limited . at a pitch factor of 3.2 it was possible to apply a maximum of 142 mas at 120 kv and 114 mas at 100 kv .", "all measurements were performed without the use of a dose modulating software ( care dose 4d , siemens , forchheim , germany ) to obtain a value for the maximum energy that the ct machine was able to deliver . as the applicable energy decreased , while increasing the pitch factor , the image noise increased .", "the snr and cnr values decreased , while the pitch factors increased [ figures 4 and 5 ] .", "when discussing the increasing imaging noise , we should consider the reduction in image quality .", "as expected , in the subjective image analysis , we saw a near linear correlation between the increase in pitch factor and a corresponding decrease in the applicable energy , to decreasing image quality ) .", "the linear correlation did display a dip in the curve , and this dip occurred at a pitch factor of 2.8 [ figure 6 ] .", "therefore , performing examinations at a pitch factor of 2.8 resulted in a better image quality compared to the pitch values nearby .", "thus , this remained an interesting point for future investigations , although we could not immediately envisage why at that particular pitch value the image quality was rated to be better compared to the other values .", "therefore , there might be an advantage in conducting a study exploiting a more overlapping dataset . to reduce the image noise seen in high - pitch imaging", ", it had already been proved that iterative reconstruction algorithms had positive effects on image quality . in our study", ", we reconstructed the images in the b30 kernel ( medium - smooth kernel ) , which were often used for abdominal imaging .", "with regard to the image noise , we calculated the snr , cnr , and additionally evaluated the image quality in grades from one to five , as explained above [ figures 46 ] .", "the interobserver agreement was good , with a value of cohen 's kappa of 0.8 .", "subjective image quality at different pitch factors ( quality rating scale from 1 to 5 = y - axis : 1 = excellent image quality , 5 = worst image quality ) .", "the red line represents the 100 kv group , the blue line represents the 120 kv group .", "the main result of our study was that the energy in dual source high - pitch mode was definitely limited .", "as shown in tables 2 and 3 , the maximum applicable current that could be supplied by the machine , especially beyond a pitch of 3.0 , had to be kept in mind when imaging at those pitch values .", "the scanner studied here was often working at its absolute limits ; these limits were a result of the accelerated rotation time combined with fast table movement .", "together , the time during which radiation was applied to the patient , and consequently the image acquisition time during which the detector was absorbing the x - rays , remained so brief that the tubes were often working at a current above 1000 ma .", "therefore , when we tried to go beyond these limits , the scanner produced a malfunction message", ". thus , this represented one major cause for the dose savings described in the recent literature . in a conventional single - source mode ,", "a ct machine was able to deliver as much energy as was needed , and so gained a constant noise - level , and this could be adjusted using the dose - modulation software ( such as care dose 4d ) . in high - pitch mode ,", "the machine , at some point during examination of the patient , was not able to deliver the energy that was necessary and so a malfunction message was displayed , and as a result the examination had to be performed with  under radiation in some areas of the body , resulting in less total radiation exposure . in our examination , we wanted to rule out the limits of the scanner , so we performed all our scans without the use of the dose - modulating software .", "the use of this software would have meant that we would have been unable to produce valid results and it would not have been possible to draw conclusions that would have been relevant to clinical practice from our results .", "this was on account of various patient features , especially the body mass index , as this was the major influential factor of the dose - modulation software , which adjusted the energy levels to keep the noise levels stable .", "when the dose - modulating software is used , the tube or the scanner can be adjusted to higher mas values , as we observed , and this is in agreement with the recent publications in the field of high - pitch imaging . however , even in the field of high - pitch imaging , when using the dose - modulating software , the energy is still limited .", "naturally , determining these values by using the dose - modulation software is more complicated , because other influential factors have to be kept in mind ( e.g. , patient size , weight , and the region that will be examined ) .", "overall , for the evaluation of the maximum applicable energy when using dose modulating software , further studies have to be performed , but in our view the study here may represent a first step . with respect to the image quality rating , looking at figure 6 , there is a dip in the curve at pitch values between 2.6 and 2.8 .", "we can not explain why at exactly these pitch values the image quality is rated better compared to the other values .", "thus , it may be advantageous to conduct a study utilizing a more overlapping dataset . performing examinations at a pitch factor between 2.6 and 2.8", "resulted in an image quality nearly equal to a pitch factor of 2.2 . in recent times , many studies concerning high - pitched imaging have been published .", "most of them focus on imaging of the heart and the vessels of the thorax . in some studies", ", the limitation of energy is described and the scanner is adjusted to values that the machine is able to perform , however , a controlled study for the maximum applicable energy has not yet been published . in our study", "we wanted to determine the limits of high - pitch imaging and the consequences for image quality .", "as the ongoing progress in a multi - slice ct enables us to use a more sophisticated ct scanner technology , one of the recent technologies uses wide detector arrays , up to 16 cm , to cover a wide area of the body with one rotation .", "however , this is a different approach when compared to a dual - source ct , because this remains a one - tube detector system , whereas , in a dual - source ct there are two tube - detector systems in use , at about 90-degree angles to each other .", "one limitation of our study was that we did not compare the use of the dose - modulating software with the dose modulating software mode when it was not used . however , our main goal was to show how much energy could be applied in a stable setting , and therefore , we performed our examinations without the dose modulation software .", "of course , the energy levels when using the dose modulating software would be limited too .", "another limitation of the ct scanner is the width of the second detector , which only covers 33 cm .", "another limitation we show in our study is the maximum energy that can be applied to the patient . in a conventional single source mode , the maximum applicable energy is not generally a concern , because the scanner , depending on the rotation time , is normally able to deliver as much energy as is needed to examine the patient , irrespective of their height and weight . in high - pitch imaging", "one limitation of our study was that we did not compare the use of the dose - modulating software with the dose modulating software mode when it was not used . however , our main goal was to show how much energy could be applied in a stable setting , and therefore , we performed our examinations without the dose modulation software .", "of course , the energy levels when using the dose modulating software would be limited too .", "another limitation of the ct scanner is the width of the second detector , which only covers 33 cm .", "another limitation we show in our study is the maximum energy that can be applied to the patient . in a conventional single source mode , the maximum applicable energy is not generally a concern , because the scanner , depending on the rotation time , is normally able to deliver as much energy as is needed to examine the patient , irrespective of their height and weight . in high - pitch imaging", "high - pitch imaging , in departments where a ct machine that is able to work in this mode is available , is already a standard examination tool for selected clinical indications .", "as the high - pitch mode is used more routinely , it becomes increasingly important to examine the limitations of high - pitch imaging .", "therefore , in some cases , high - pitch imaging is a good solution for imaging patients , but in other cases , especially in abdominal imaging , it may be desirable to apply more energy than the ct is able to deliver . altogether , not every clinical question can be answered in a way that it could be in conventional single source mode ct .", "the limited energy , as well as the detector width , have to be kept in mind when examining at high - pitch ."], "abstract_text": ["<S> objectives : the aim of our study was to find out how much energy is applicable in second - generation dual source high - pitch computed tomography ( ct ) in imaging of the abdomen.materials and methods : we examined an upper abdominal phantom using a somatom definition flash ct - scanner ( siemens , forchheim , germany ) . the study protocol consisted of a scan - series at 100 kv and 120 kv . in each scan series </S>", "<S> we started with a pitch of 3.2 and reduced it in steps of 0.2 , until a pitch of 1.6 was reached . </S>", "<S> the current was adjusted to the maximum the scanner could achieve . </S>", "<S> energy values , image noise , image quality , and radiation exposure were evaluated.results:for a pitch of 3.2 the maximum applicable current was 142 mas at 120 kv and in 100 kv the maximum applicable current was 114 mas . for conventional abdominal imaging , current levels of 200 to 260 mas are generally used . to achieve similar current levels , we had to decrease the pitch to 1.8 at 100 kv  at this pitch we could perform our imaging at 204 mas . at a pitch of 2.2 in 120 kv </S>", "<S> we could apply a current of 206 mas.conclusion:we conclude our study by stating that if there is a need for a higher current , we have to reduce the pitch . in a high - pitch dual source ct </S>", "<S> , we always have to remember where our main focus is , so we can adjust the pitch to the energy we need in the area of the body that has to be imaged , to find answers to the clinical question being raised . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Measurements", "Statistical analysis", "RESULTS", "DISCUSSION", "Limitations", "CONCLUSION"], "sections": [["during the last few years , dual - source high - pitch imaging has become increasingly important in the daily routine of radiology departments . in dual - source computed tomography ( ct ) imaging two ", "sources means that two x - ray tubes are in use at an angle of approximately 90 degrees to each other .", "after introduction of the second generation dual - source ct , about four years ago , increasing pitch ( pitch = table mm / n * t mm , where n = no", ". of slices and t = slice thickness mm ) values above the former traditional technical limit of 1.5 in a single - source ct , became possible .", "when high - pitch imaging is performed , one of the major advantages , compared to conventional single source normal pitch imaging , is the image acquisition time . in high - pitch imaging", "this makes high - pitch imaging the technique of choice , especially when regions of the body that are normally subject to motion artifacts have to be examined .", "therefore , in clinical practice there are some major regions of interest where high - pitch imaging can demonstrate its advantages , such as , in imaging of the heart , the great thoracic vessels , and the lungs , in patients who have problems holding their breath . as our clinical experience in high - pitched imaging shows", " especially in non - electrocardiogram ( ekg)-gated high - pitch imaging  the amount of energy that can be applied may be limited by patient habitus . in high - pitch imaging of the lung ,", "these energy factors are not relevant , but in imaging of the upper abdominal tract the maximum applicable energy may cause problems , leading to diagnostic issues . therefore , the aim of this study was to examine the maximum current levels that can be used in dual - source high - pitch ct during imaging of the upper abdominal tract ."], ["an upper - abdominal phantom was placed on the ct table and no alterations were made to this experimental setup during all the ct examinations [ figure 1 ] .", "this was followed by a series of high - pitch examinations , beginning with a pitch of 3.2 [ table 1 ] .", "this value was reduced in steps of 0.2 , with each successive test , ending at a pitch of 1.6 in the final examination [ tables 2 and 3 ] .", "all investigations were carried out in the same scan range , once with a tube voltage of 120 kv , and once with 100 kv ( modes 1 and 2 , table 1 ) .", "the tube charge in mas (= ma multiplied by the rotation time ) was always adjusted to the maximum that the ct machine could perform at , without using the dose modulation software ( care dose 4d ) , so that the exact tube maximum could be reached . to achieve the maximum current , the mas value", "one step downward from the level producing an error - message was chosen for the examinations , and this was taken as the maximum current that the ct machine was able to apply .", "examination protocols high - pitch examinations at 100 kv high - pitch examinations at 120 kv the images obtained were reconstructed in a conventional filtered back - projection recon kernel b30 , as frequently used for imaging soft tissue .", "the images were assessed in slices / increments of 5 mm/5 mm as transversal cross - sectional images . for further evaluation coronal and sagittal images in slices / increments of 2 mm/2 mm were reconstructed in the same manner . as objective measures of image quality of the examination series , several region - of - interest ( roi ) measurements , using the circle tool and a picture archiving and communication system ( pacs ) workstation ( centricity 4.2 , general electric healthcare , dornstadt , germany ) , were performed by a radiologist , who had four years of experience in ct .", "the mean attenuation values and standard deviation were recorded and displayed in hounsfield units ( hu ) .", "the background noise ( bn ) was determined as the standard deviation of air measured in front of the phantom .", "the attenuation at different locations in the phantom were measured ( liver parenchyma , liver metastases ( hyper- and hypo - dense examples ) , muscle tissue , cutaneous fat tissue , cutaneous metastases ( hyper- and hypodense examples ) , as also the air in front of the phantom ) .", "the roi measurements were drawn as large as possible , to include as much of the corresponding area of interest as feasible . to minimize bias from single measurements", ", we calculated the average of four measurements for each roi . on the basis of these measurements , the signal - to - noise ratio ( snr )", "was determined according to the following equation : snr = attenuation / bn . to calculate the contrast - to - noise ratio ( cnr)- value , we measured the attenuation and the hu and sd of attenuation of the surrounding muscle tissue ( roi muscle ) compared with the attenuation of the hyperdense subcutaneous lesions [ figures 2 and 3 ] .", "cnr was calculated as cnr = ( ( roi subclesion - roi muscle)/image noise ) .", "images at different pitch values ( 100 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ; b30 kernel ) . images at different pitch values ( 120 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ) .", "subjective image quality rating was conducted in a blind fashion by two independent radiologists , with three and four years of experience , respectively , in general ct imaging .", "the rating was done according to a five - point scale as follows : 1 = excellent / no artifacts , 2 = good / hardly any artifacts , 3 = moderate / few artifacts , 4 = fair / many artifacts , 5 = unacceptable .", "exemplary : attenuation values , snr , cnr , subjective image quality score , dlp ( dose length product ) and ctdivol ( computed tomography dose index ) .", "analyses were performed using a dedicated software ( bias 9.14 , epsilon verlag , germany ) .", "a cohen 's kappa analysis was performed to determine the interobserver agreement for subjective image quality scoring ."], ["as objective measures of image quality of the examination series , several region - of - interest ( roi ) measurements , using the circle tool and a picture archiving and communication system ( pacs ) workstation ( centricity 4.2 , general electric healthcare , dornstadt , germany ) , were performed by a radiologist , who had four years of experience in ct .", "the mean attenuation values and standard deviation were recorded and displayed in hounsfield units ( hu ) .", "the background noise ( bn ) was determined as the standard deviation of air measured in front of the phantom .", "the attenuation at different locations in the phantom were measured ( liver parenchyma , liver metastases ( hyper- and hypo - dense examples ) , muscle tissue , cutaneous fat tissue , cutaneous metastases ( hyper- and hypodense examples ) , as also the air in front of the phantom ) .", "the roi measurements were drawn as large as possible , to include as much of the corresponding area of interest as feasible . to minimize bias from single measurements", "on the basis of these measurements , the signal - to - noise ratio ( snr ) was determined according to the following equation : snr = attenuation / bn . to calculate the contrast - to - noise ratio ( cnr)- value , we measured the attenuation and the hu and sd of attenuation of the surrounding muscle tissue ( roi muscle ) compared with the attenuation of the hyperdense subcutaneous lesions [ figures 2 and 3 ] .", ". images at different pitch values ( 100 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ; b30 kernel ) .", "images at different pitch values ( 120 kv , a = pitch 3.2 ; b = pitch 2.8 , c = pitch 2.2 ) .", "subjective image quality rating was conducted in a blind fashion by two independent radiologists , with three and four years of experience , respectively , in general ct imaging .", "the rating was done according to a five - point scale as follows : 1 = excellent / no artifacts , 2 = good / hardly any artifacts , 3 = moderate / few artifacts , 4 = fair / many artifacts , 5 = unacceptable ."], ["exemplary : attenuation values , snr , cnr , subjective image quality score , dlp ( dose length product ) and ctdivol ( computed tomography dose index ) .", "analyses were performed using a dedicated software ( bias 9.14 , epsilon verlag , germany ) . for statistical comparison", "a cohen 's kappa analysis was performed to determine the interobserver agreement for subjective image quality scoring ."], ["it became apparent that the maximum applicable energy , especially at increasing pitch levels , was limited . at a pitch factor of 3.2 it was possible to apply a maximum of 142 mas at 120 kv and 114 mas at 100 kv .", "all measurements were performed without the use of a dose modulating software ( care dose 4d , siemens , forchheim , germany ) to obtain a value for the maximum energy that the ct machine was able to deliver . as the applicable energy decreased , while increasing the pitch factor , the image noise increased .", "the snr and cnr values decreased , while the pitch factors increased [ figures 4 and 5 ] .", "when discussing the increasing imaging noise , we should consider the reduction in image quality .", "as expected , in the subjective image analysis , we saw a near linear correlation between the increase in pitch factor and a corresponding decrease in the applicable energy , to decreasing image quality ) .", "the linear correlation did display a dip in the curve , and this dip occurred at a pitch factor of 2.8 [ figure 6 ] .", "therefore , performing examinations at a pitch factor of 2.8 resulted in a better image quality compared to the pitch values nearby .", "thus , this remained an interesting point for future investigations , although we could not immediately envisage why at that particular pitch value the image quality was rated to be better compared to the other values .", "therefore , there might be an advantage in conducting a study exploiting a more overlapping dataset . to reduce the image noise seen in high - pitch imaging", ", it had already been proved that iterative reconstruction algorithms had positive effects on image quality . in our study", ", we reconstructed the images in the b30 kernel ( medium - smooth kernel ) , which were often used for abdominal imaging .", "with regard to the image noise , we calculated the snr , cnr , and additionally evaluated the image quality in grades from one to five , as explained above [ figures 46 ] .", "the interobserver agreement was good , with a value of cohen 's kappa of 0.8 .", "subjective image quality at different pitch factors ( quality rating scale from 1 to 5 = y - axis : 1 = excellent image quality , 5 = worst image quality ) .", "the red line represents the 100 kv group , the blue line represents the 120 kv group ."], ["the main result of our study was that the energy in dual source high - pitch mode was definitely limited .", "as shown in tables 2 and 3 , the maximum applicable current that could be supplied by the machine , especially beyond a pitch of 3.0 , had to be kept in mind when imaging at those pitch values .", "the scanner studied here was often working at its absolute limits ; these limits were a result of the accelerated rotation time combined with fast table movement .", "together , the time during which radiation was applied to the patient , and consequently the image acquisition time during which the detector was absorbing the x - rays , remained so brief that the tubes were often working at a current above 1000 ma .", "therefore , when we tried to go beyond these limits , the scanner produced a malfunction message", ". thus , this represented one major cause for the dose savings described in the recent literature . in a conventional single - source mode ,", "a ct machine was able to deliver as much energy as was needed , and so gained a constant noise - level , and this could be adjusted using the dose - modulation software ( such as care dose 4d ) . in high - pitch mode ,", "the machine , at some point during examination of the patient , was not able to deliver the energy that was necessary and so a malfunction message was displayed , and as a result the examination had to be performed with  under radiation in some areas of the body , resulting in less total radiation exposure . in our examination , we wanted to rule out the limits of the scanner , so we performed all our scans without the use of the dose - modulating software .", "the use of this software would have meant that we would have been unable to produce valid results and it would not have been possible to draw conclusions that would have been relevant to clinical practice from our results .", "this was on account of various patient features , especially the body mass index , as this was the major influential factor of the dose - modulation software , which adjusted the energy levels to keep the noise levels stable .", "when the dose - modulating software is used , the tube or the scanner can be adjusted to higher mas values , as we observed , and this is in agreement with the recent publications in the field of high - pitch imaging . however , even in the field of high - pitch imaging , when using the dose - modulating software , the energy is still limited .", "naturally , determining these values by using the dose - modulation software is more complicated , because other influential factors have to be kept in mind ( e.g. , patient size , weight , and the region that will be examined ) .", "overall , for the evaluation of the maximum applicable energy when using dose modulating software , further studies have to be performed , but in our view the study here may represent a first step . with respect to the image quality rating , looking at figure 6 , there is a dip in the curve at pitch values between 2.6 and 2.8 .", "we can not explain why at exactly these pitch values the image quality is rated better compared to the other values .", "thus , it may be advantageous to conduct a study utilizing a more overlapping dataset . performing examinations at a pitch factor between 2.6 and 2.8", "resulted in an image quality nearly equal to a pitch factor of 2.2 . in recent times , many studies concerning high - pitched imaging have been published .", "most of them focus on imaging of the heart and the vessels of the thorax . in some studies", ", the limitation of energy is described and the scanner is adjusted to values that the machine is able to perform , however , a controlled study for the maximum applicable energy has not yet been published . in our study", "we wanted to determine the limits of high - pitch imaging and the consequences for image quality .", "as the ongoing progress in a multi - slice ct enables us to use a more sophisticated ct scanner technology , one of the recent technologies uses wide detector arrays , up to 16 cm , to cover a wide area of the body with one rotation .", "however , this is a different approach when compared to a dual - source ct , because this remains a one - tube detector system , whereas , in a dual - source ct there are two tube - detector systems in use , at about 90-degree angles to each other .", "one limitation of our study was that we did not compare the use of the dose - modulating software with the dose modulating software mode when it was not used . however , our main goal was to show how much energy could be applied in a stable setting , and therefore , we performed our examinations without the dose modulation software .", "of course , the energy levels when using the dose modulating software would be limited too .", "another limitation of the ct scanner is the width of the second detector , which only covers 33 cm .", "another limitation we show in our study is the maximum energy that can be applied to the patient . in a conventional single source mode , the maximum applicable energy is not generally a concern , because the scanner , depending on the rotation time , is normally able to deliver as much energy as is needed to examine the patient , irrespective of their height and weight . in high - pitch imaging"], ["one limitation of our study was that we did not compare the use of the dose - modulating software with the dose modulating software mode when it was not used . however , our main goal was to show how much energy could be applied in a stable setting , and therefore , we performed our examinations without the dose modulation software .", "of course , the energy levels when using the dose modulating software would be limited too .", "another limitation of the ct scanner is the width of the second detector , which only covers 33 cm .", "another limitation we show in our study is the maximum energy that can be applied to the patient . in a conventional single source mode , the maximum applicable energy is not generally a concern , because the scanner , depending on the rotation time , is normally able to deliver as much energy as is needed to examine the patient , irrespective of their height and weight . in high - pitch imaging"], ["high - pitch imaging , in departments where a ct machine that is able to work in this mode is available , is already a standard examination tool for selected clinical indications .", "as the high - pitch mode is used more routinely , it becomes increasingly important to examine the limitations of high - pitch imaging .", "therefore , in some cases , high - pitch imaging is a good solution for imaging patients , but in other cases , especially in abdominal imaging , it may be desirable to apply more energy than the ct is able to deliver . altogether , not every clinical question can be answered in a way that it could be in conventional single source mode ct .", "the limited energy , as well as the detector width , have to be kept in mind when examining at high - pitch ."]]}
{"article_id": "PMC2839988", "article_text": ["inherited breast cancers are ( pleiotropic ) expressions of mutations in a number of distinct genes causing other cancers as well . to some extent", ", the biological function of the different genes is known , and to some extent the ways breast cancers are produced when this function is lacking , have been described .", "the cancers associated with different genetic syndromes occur at different ages , they may differ with respect to tumour characteristics , they have different prognosis , and they respond differently to prophylactic and treatment modalities . following the clinical genetic work - up to provide health care to a woman possibly at risk for inherited breast cancer , there is a multistep approach .", "if she is demonstrated to be at risk , the preventive / treatment modalities she needs are a consequence of which subgroup of the inherited breast cancers she is at risk with . in most european countries , it is the understanding to discuss health care as what is to be offered to any woman who needs it .", "thus , the question is not what might be done or what money possibly can buy - it is about what is reasonable and affordable .", "it is also about tradition and culture - what is possible in the current socio - ethical context .", "the first detailed scientific description of inherited breast - ovarian cancer was given by paul broca in 1866 .", "he demonstrated the transmission of the assumed underlying genetic defect , its expressions , the age - related and sex - limited penetrance , and the possibilities of modifying environmental and genetic factors . not until 10 years ago , we learned that the syndrome is produced by brca1 mutations .", "brca2 mutations produce breast cancers in a similar prevalence and at a similar age , but with completely different tumour characteristics and with a different set of associated cancers .", "a number of additional genes cause multiorgan cancer syndromes when mutated , including mutations in tp53 , pten , atm and chek2 .", "heterozygous state for atm mutation as a predisposition to breast cancer is debated , and the chek2 syndrome needs further evaluation .", "some   assume that there have to be more dominantly inherited breast cancer genes , others disagree and conclude that they may be recessive , low penetrant or multifactorially interacting , but not dominantly inherited with high penetrance .", "all cancer genetic clinics are aware that \" inherited breast cancer \" outside demonstrated brca mutation carrying syndromes may be a fiction , nevertheless all such clinics have defined a large volume of \" inherited breast cancer \" cases defined by family history but lacking demonstrated dna mutations and referred them to follow - up examinations . we have demonstrated a brca mutation in but a small fraction of our at - risk families , and we have excluded brca mutations in a number of large dominantly inherited breast cancer pedigrees .", "none of the models for probability calculations for recurrence risk of breast cancer in breast cancer kindreds are valid after a brca mutation have been excluded - they all assume that the patient is selected from an untested population", ". however , many cancer genetic centres continue to estimate the probability that a given woman is a mutation carrier , after she has been tested and found not to be so .", "this makes sense if you - as we do - believe there are more genes .", "whatever we may believe , the calculations are wrong , because they can not be interpreted without correcting the probability estimates for the testing performed prior to the calculations .", "moreover , our activity of nesting up all the large mutation - carrying families will remove a substantial part of the mutation carriers from the population before the remaining familial clusters are referred to genetic counselling : for each passing day , the next familial cluster of breast cancer referred is less likely to harbour one of our founder brca1 mutations .", ". this may in principle be due to four factors : ( 1 ) selection biases , ( 2 ) improper algorithms to calculate penetrances , ( 3 ) true differences between the mutations examined and/or ( 4 ) environmental or genetic modifiers of penetrance .", "because the first series obviously had selection biases and because many of the families were not tested ( carrier status for relatives was assumed , calculated upon and thereafter presented as results ) , the methodological problems may have been major .", "studies based upon testing rather than assuming carrier status , and studies employing sophisticated statistics to eliminate methodological problems , now agree that penetrance for breast cancer is high for all brca1 or brca2 truncating mutations .", "breast and ovarian cancers are competitive causes of death in a brca1/2 mutation carrier , and both are caused by the same mutation .", "estimating the penetrance of breast cancer ( or ovarian cancer ) implies the methodological problem of informed censoring : whatever you do is methodologically wrong , because you censor the data with an argument dependent on what you are examining .", "because there is no universally \" correct \" method , the solution is to formulate explicit questions specifying the assumptions to the answers .", "if a figure for probability for a healthy mutation carrier to contract ovarian cancer is looked for , one must censor out all mutation carriers when they contract another ( breast ) cancer .", "doing so , lifetime penetrance for ovarian cancer is high in brca1 mutation carriers , possibly as high as for breast cancer .", "two of the reasons for the low penetrance for ovarian cancer in some studies may be the combined effect that the families were selected for by the presence of breast cancer , and the fact that you may not contract ovarian cancer after having died of breast cancer .", "in addition , there may be biological differences between different mutations in the same genes .", "the exercise of pooling a number of small families without excluding the index cases used to ascertain the families , with different mutations , without actually testing the relatives but assuming their carrier status , without specifying the questions addressed and how the data are censored to answer that question , may give results of low practical value .", "a statistical flaw has made some mistakenly conclude that brca1-associated cancers have prognosis similar to other breast cancers .", "the flaw is that according to oncological standards for randomized trials to evaluate effects of treatment modalities , the brca1 cancers have been compared to controls selected for similar prognostic tumour characteristics ( oestrogen receptor , histopathological grade , etc . ) . in this way", ", brca1 cancers have been matched with a subset of patients demonstrated to have bad prognostic signs ( over - parameterization ) .", "the question of whether brca1 mutation carriers have even worse prognosis is debated , but all studies agree that brca1 cancers have worse prognosis than age - matched controls . because a germline mutation is always prior to the tumour it causes", "possibly , the mathematical models are pertinent , but the way the results are presented may seduce the readers to conceptional misunderstandings . brca1-associated breast cancers are , as a group , different from all other defined groups of breast cancers [ 6 - 8 ] .", "the picture is so clear that all exceptions may be sporadic cancers caused by different mechanisms in brca1 mutations carriers ( age - related sporadic breast cancer may occur in brca1 mutation carriers as well ) .", "the brca1-associated breast cancers are hormone receptor negative , of histopathological high grade , and they are close to never appear as precancers ( dcis ) when diagnosed clinically or by mammography .", "attempts of early diagnosis to achieve early treatment was initially considered successful : the tumours diagnosed were small and often without spread .", "it turned out , however , that the prognosis was not as good as hoped for according to the stage at diagnosis : retrospective series before any attempt on early diagnosis and treatment demonstrated 5-year survival of 63% for invasive brca1 cancers .", "the results of the biomed2 prospective series included a 5-year survival of 63% for invasive brca1 cancers , point estimate was no effect at all .", "prophylactic mastectomy is an alternative , but with severe implications both on personal and professional ethical levels , and it is resource demanding . a new attempt on secondary prophylaxis has been implemented in most centres : mri obviously has the capability of demonstrating tumours invisible in mammography .", "we do not , however , at present know whether or not mri may diagnose the tumours before they have biologically achieved their bad prognostic propensities .", "we ask for time - out to retrieve this figure before we consider the alternatives .", "early diagnosis and treatment to improve prognosis for inherited ovarian cancer was undertaken by ultrasound and cea125 in many centres .", "it did not work - no report claims substantial improvement in survival . actually , there are few reports on survival , most reports mention cancer with spread at diagnosis but give no survival data . however , it became clear that brca1-associated ovarian cancer seldom occurs before the age of 40 .", "combined with the finding that oophorectomy at that age reduced not only ovarian cancer risk by more than 90% , but also reduced breast cancer risk in brca1 mutation carriers - even in those using hormone replacement therapy   - most centres advocate prophylactic oophorectomy past childbearing ages .", "in contrast to prophylactic mastectomy where uptake is low , the majority of postmenopausal brca1 carriers seem to choose oophorectomy .", "if a mean to early diagnosis and cure for ovarian cancer appears , we may soon have no brca1 carriers left to evaluate the effects , because there may not be many ovaries left in the mutation carriers aged over 40 years .", "moreover , the disease is so lethal and prophylactic oophorectomy past childbearing ages seems to be so well tolerated , that it would be hard to suggest a trial .", "in contrast to the differences in breast cancer phenotype , ovarian cancer caused by brca2 seems similar to that caused by brca1 , besides that the penetrance may be lower and disease onset later in brca2 carriers .", "one report concluded that oophorectomy in brca1 carriers contracting cancer improved the prognosis of the breast cancer .", "this observation needs to be supported by an independent series , but it is in keeping with the beneficial effect of prophylactic oophorectomy to breast cancer risk in the same group .", "breast cancers in inherited / familial non - brca1/2 carriers have good prognosis ( about 90% 5-year survival ) inside early detection programmes applying annual clinical mammography .", "moreover , a number of cases are demonstrated as precancers ( dcis ) , and they have reportedly 100% event - free 5-year survival .", "few studies have been presented on prospective survival in brca2-associated breast cancers , but these cancers seem to be comparable to sporadic breast cancers beside the young age of onset .", "there is no indication that the prognosis should be worse than that of non - brca1/2-associated breast cancers . because all non - brca1 carriers ( including the brca2 carriers ) predominantly contract hormone receptor - positive breast cancers , they should theoretically benefit from receptor blocking agents ( like tamoxifen ) and oestrogen production blocking agents ( aromatase inhibitors ) .", "most agree that by now it would be reasonable to suggest such chemoprevention to these groups , but there is no agreement on which compound and exactly which group to address .", "it is advocated to give various regimens of such treatment under strict control to evaluate the effects .", "that is where we are today : trials and discussions , but no consensus on applying chemoprevention as standard health care .", "it may be expected that because as a group they have receptor - negative cancers , they should not respond either to oestrogen blockers or to oestrogen .", ", oophorectomy prevents breast cancer , tamoxifen prevents contralateral breast cancer , and oral contraceptives induce breast cancer .", "it seems , however , irrational to suggest oestrogen blockers to prevent oestrogen receptor - negative tumours . in oncology ,", "the scientific standard is a randomized trial . as is evident from the reports ( beside chemoprevention ) mentioned above , we have no randomized trials .", "you can not randomize a woman to mastectomy , and you can not deprive a mutation - carrying woman from any potential life - saving health care available .", "we are faced with the challenge of doing science without randomized trials , and we can not ( as the mammographic screening of older women ) go and get the families we want for research .", "this leaves us with series subjected to a number of ascertainment biases , and we should interpret the results with caution . because we are outside the framework of randomized trials , we may be better off not discussing exact figures in single reports , but rather focus on the main results , methods employed and whether or not the empirical facts are in keeping with the current paradigms for understanding . in this perspective , it may seem that we need to reconsider our paradigms for brca1-associated breast cancer .", "for the other groups , early diagnosis and treatment works as expected , improving early diagnosis may hopefully further improve the results obtained so far , and the principles of chemoprevention may be projected from sporadic cancers . in conclusion to the facts discussed above ,", "most clinical genetic centres relating to inherited breast cancer today advocate annual mammography from the age of 30 onwards to women at risk for inherited non - brca1 breast cancer .", "it is agreed that besides brca1 carriers , oestrogen blockers / aromatase inhibitors may be beneficial - but there is no agreement on exactly how to implement such chemoprevention .", "we are now hoping mri to be better to avoid large numbers of prophylactic mastectomies .", "we are confused by the data on the effect of hormones and hormone blockers in brca1 carriers , and we all hope for chemoprevention to make the unpleasant discussion of prophylactic surgery superfluous if early detection and treatment does not work .", "early diagnosis does not work for ovarian cancers , but oophorectomy is beneficial and advocated at the end of childbearing ages . as is the case with most scientific reports , this contribution has focused on unsolved problems .", "we may remember , however , that we actually have about 90% 5-year survival of non - brca1 breast cancer with today 's means , and the great majority of inherited breast cancers belong to this group .", "prophylactic oophorectomy at the end of childbearing ages in brca1 carriers reduces morbidity and mortality by more than 50% .", "the systematic attempts to prevent and cure inherited breast cancer have been undertaken for but about 10 years .", "the results are actually good , which is reflected in the high compliance from the affected kindreds .", "the attempts to prevent and cure inherited breast cancer is an example of a consumer - driven activity based on knowledge and collaboration from the patients needing our care . actually , based on knowledge two brca1 carriers with small invasive tumours without spread this year have asked me for immediate chemotherapy .", "trials we can not impose upon them , may soon be initiated by patients who want to know .", "the physicians and the researchers have the role of producing , filing , retrieving and communicating knowledge as appropriate .", "the choice what to do , however , should be the patient 's choice , there are no scientific arguments as to whether or not to undergo prophylactic oophorectomy . as we all know , there are more arguments about what to do with your life than the doctors ' suggestions .", "the high compliance to our advice during the last decade most probably reflects that the families have identified their problems long ago and were waiting for our care .", "the high compliance to genetic testing obviously reflects the opinion that our activity may prevent and cure . to maintain the high compliance", "it is our obligation , however , to ensure that the advocated options are actually available to each single patient .", "in addition , we may produce arguments to advocate some options because they have consequences to our liking .", "the suggestions in table 1 may be agreed by most , and some would go further and actively advocate prophylactic mastectomy in brca1 carriers and chemoprevention for the rest .", "genetic counselling is to present information and options so that any given patient may be supported in exploring her values to make her choices .", "the challenge is to support the patients who make choices not corresponding with your own priorities .", "primary and secondary prevention for women at risk for inherited breast or breast - ovarian cancer"], "abstract_text": ["<S> inherited breast - ovarian cancer was described in 1866 . </S>", "<S> the underlying genetic defects in brca1/2 were demonstrated 128 years later . </S>", "<S> we now have 10 years of experience with genetic testing in brca kindreds . </S>", "<S> the majority of breast cancer kindreds ( familial breast cancer ) do not demonstrate ovarian cancer and are not associated with brca mutations . </S>", "<S> the effect of early diagnosis and treatment is monitored through international collaborations.brca1-associated breast cancer is biologically different from other breast cancers , including a worse prognosis . </S>", "<S> brca2-associated breast cancer is , beside early onset , in many ways similar to sporadic breast cancer . </S>", "<S> mammography screening of the high risk groups aiming at early diagnosis and treatment , seems promising for familial breast cancer and for brca2-associated breast cancer , but numbers included for brca2 carriers are limited . </S>", "<S> brca1-carriers have worse prognosis , and the potential benefit of mri for early diagnosis is now being explored . </S>", "<S> early diagnosis and treatment of ovarian cancer does not substantially improve survival , and prophylactic oophorectomy at the end of childbearing ages is advocated . </S>", "<S> prophylactic mastectomy is debated , and we may await the results of mri trials before recommending this option . </S>", "<S> familial breast cancer and brca2-associated breast cancers are often oestrogen receptor positive , and may be prevented by oestrogen blockers / inhibitors . </S>", "<S> oophorectomy prevents ovarian cancer , and may possibly prevent both receptor positive and receptor negative breast cancer as well , also while using hrt . </S>", "<S> oral contraceptives may reduce ovarian cancer risk and increase breast cancer risk , irrespective of initial risk and genetic subgroup . </S>"], "labels": null, "section_names": ["None"], "sections": [["inherited breast cancers are ( pleiotropic ) expressions of mutations in a number of distinct genes causing other cancers as well . to some extent", ", the biological function of the different genes is known , and to some extent the ways breast cancers are produced when this function is lacking , have been described .", "the cancers associated with different genetic syndromes occur at different ages , they may differ with respect to tumour characteristics , they have different prognosis , and they respond differently to prophylactic and treatment modalities . following the clinical genetic work - up to provide health care to a woman possibly at risk for inherited breast cancer , there is a multistep approach .", "if she is demonstrated to be at risk , the preventive / treatment modalities she needs are a consequence of which subgroup of the inherited breast cancers she is at risk with . in most european countries , it is the understanding to discuss health care as what is to be offered to any woman who needs it .", "thus , the question is not what might be done or what money possibly can buy - it is about what is reasonable and affordable .", "it is also about tradition and culture - what is possible in the current socio - ethical context .", "the first detailed scientific description of inherited breast - ovarian cancer was given by paul broca in 1866 .", "he demonstrated the transmission of the assumed underlying genetic defect , its expressions , the age - related and sex - limited penetrance , and the possibilities of modifying environmental and genetic factors . not until 10 years ago , we learned that the syndrome is produced by brca1 mutations .", "brca2 mutations produce breast cancers in a similar prevalence and at a similar age , but with completely different tumour characteristics and with a different set of associated cancers .", "a number of additional genes cause multiorgan cancer syndromes when mutated , including mutations in tp53 , pten , atm and chek2 .", "heterozygous state for atm mutation as a predisposition to breast cancer is debated , and the chek2 syndrome needs further evaluation .", "some   assume that there have to be more dominantly inherited breast cancer genes , others disagree and conclude that they may be recessive , low penetrant or multifactorially interacting , but not dominantly inherited with high penetrance .", "all cancer genetic clinics are aware that \" inherited breast cancer \" outside demonstrated brca mutation carrying syndromes may be a fiction , nevertheless all such clinics have defined a large volume of \" inherited breast cancer \" cases defined by family history but lacking demonstrated dna mutations and referred them to follow - up examinations . we have demonstrated a brca mutation in but a small fraction of our at - risk families , and we have excluded brca mutations in a number of large dominantly inherited breast cancer pedigrees .", "none of the models for probability calculations for recurrence risk of breast cancer in breast cancer kindreds are valid after a brca mutation have been excluded - they all assume that the patient is selected from an untested population", ". however , many cancer genetic centres continue to estimate the probability that a given woman is a mutation carrier , after she has been tested and found not to be so .", "this makes sense if you - as we do - believe there are more genes .", "whatever we may believe , the calculations are wrong , because they can not be interpreted without correcting the probability estimates for the testing performed prior to the calculations .", "moreover , our activity of nesting up all the large mutation - carrying families will remove a substantial part of the mutation carriers from the population before the remaining familial clusters are referred to genetic counselling : for each passing day , the next familial cluster of breast cancer referred is less likely to harbour one of our founder brca1 mutations .", ". this may in principle be due to four factors : ( 1 ) selection biases , ( 2 ) improper algorithms to calculate penetrances , ( 3 ) true differences between the mutations examined and/or ( 4 ) environmental or genetic modifiers of penetrance .", "because the first series obviously had selection biases and because many of the families were not tested ( carrier status for relatives was assumed , calculated upon and thereafter presented as results ) , the methodological problems may have been major .", "studies based upon testing rather than assuming carrier status , and studies employing sophisticated statistics to eliminate methodological problems , now agree that penetrance for breast cancer is high for all brca1 or brca2 truncating mutations .", "breast and ovarian cancers are competitive causes of death in a brca1/2 mutation carrier , and both are caused by the same mutation .", "estimating the penetrance of breast cancer ( or ovarian cancer ) implies the methodological problem of informed censoring : whatever you do is methodologically wrong , because you censor the data with an argument dependent on what you are examining .", "because there is no universally \" correct \" method , the solution is to formulate explicit questions specifying the assumptions to the answers .", "if a figure for probability for a healthy mutation carrier to contract ovarian cancer is looked for , one must censor out all mutation carriers when they contract another ( breast ) cancer .", "doing so , lifetime penetrance for ovarian cancer is high in brca1 mutation carriers , possibly as high as for breast cancer .", "two of the reasons for the low penetrance for ovarian cancer in some studies may be the combined effect that the families were selected for by the presence of breast cancer , and the fact that you may not contract ovarian cancer after having died of breast cancer .", "in addition , there may be biological differences between different mutations in the same genes .", "the exercise of pooling a number of small families without excluding the index cases used to ascertain the families , with different mutations , without actually testing the relatives but assuming their carrier status , without specifying the questions addressed and how the data are censored to answer that question , may give results of low practical value .", "a statistical flaw has made some mistakenly conclude that brca1-associated cancers have prognosis similar to other breast cancers .", "the flaw is that according to oncological standards for randomized trials to evaluate effects of treatment modalities , the brca1 cancers have been compared to controls selected for similar prognostic tumour characteristics ( oestrogen receptor , histopathological grade , etc . ) . in this way", ", brca1 cancers have been matched with a subset of patients demonstrated to have bad prognostic signs ( over - parameterization ) .", "the question of whether brca1 mutation carriers have even worse prognosis is debated , but all studies agree that brca1 cancers have worse prognosis than age - matched controls . because a germline mutation is always prior to the tumour it causes", "possibly , the mathematical models are pertinent , but the way the results are presented may seduce the readers to conceptional misunderstandings . brca1-associated breast cancers are , as a group , different from all other defined groups of breast cancers [ 6 - 8 ] .", "the picture is so clear that all exceptions may be sporadic cancers caused by different mechanisms in brca1 mutations carriers ( age - related sporadic breast cancer may occur in brca1 mutation carriers as well ) .", "the brca1-associated breast cancers are hormone receptor negative , of histopathological high grade , and they are close to never appear as precancers ( dcis ) when diagnosed clinically or by mammography .", "attempts of early diagnosis to achieve early treatment was initially considered successful : the tumours diagnosed were small and often without spread .", "it turned out , however , that the prognosis was not as good as hoped for according to the stage at diagnosis : retrospective series before any attempt on early diagnosis and treatment demonstrated 5-year survival of 63% for invasive brca1 cancers .", "the results of the biomed2 prospective series included a 5-year survival of 63% for invasive brca1 cancers , point estimate was no effect at all .", "prophylactic mastectomy is an alternative , but with severe implications both on personal and professional ethical levels , and it is resource demanding . a new attempt on secondary prophylaxis has been implemented in most centres : mri obviously has the capability of demonstrating tumours invisible in mammography .", "we do not , however , at present know whether or not mri may diagnose the tumours before they have biologically achieved their bad prognostic propensities .", "we ask for time - out to retrieve this figure before we consider the alternatives .", "early diagnosis and treatment to improve prognosis for inherited ovarian cancer was undertaken by ultrasound and cea125 in many centres .", "it did not work - no report claims substantial improvement in survival . actually , there are few reports on survival , most reports mention cancer with spread at diagnosis but give no survival data . however , it became clear that brca1-associated ovarian cancer seldom occurs before the age of 40 .", "combined with the finding that oophorectomy at that age reduced not only ovarian cancer risk by more than 90% , but also reduced breast cancer risk in brca1 mutation carriers - even in those using hormone replacement therapy   - most centres advocate prophylactic oophorectomy past childbearing ages .", "in contrast to prophylactic mastectomy where uptake is low , the majority of postmenopausal brca1 carriers seem to choose oophorectomy .", "if a mean to early diagnosis and cure for ovarian cancer appears , we may soon have no brca1 carriers left to evaluate the effects , because there may not be many ovaries left in the mutation carriers aged over 40 years .", "moreover , the disease is so lethal and prophylactic oophorectomy past childbearing ages seems to be so well tolerated , that it would be hard to suggest a trial .", "in contrast to the differences in breast cancer phenotype , ovarian cancer caused by brca2 seems similar to that caused by brca1 , besides that the penetrance may be lower and disease onset later in brca2 carriers .", "one report concluded that oophorectomy in brca1 carriers contracting cancer improved the prognosis of the breast cancer .", "this observation needs to be supported by an independent series , but it is in keeping with the beneficial effect of prophylactic oophorectomy to breast cancer risk in the same group .", "breast cancers in inherited / familial non - brca1/2 carriers have good prognosis ( about 90% 5-year survival ) inside early detection programmes applying annual clinical mammography .", "moreover , a number of cases are demonstrated as precancers ( dcis ) , and they have reportedly 100% event - free 5-year survival .", "few studies have been presented on prospective survival in brca2-associated breast cancers , but these cancers seem to be comparable to sporadic breast cancers beside the young age of onset .", "there is no indication that the prognosis should be worse than that of non - brca1/2-associated breast cancers . because all non - brca1 carriers ( including the brca2 carriers ) predominantly contract hormone receptor - positive breast cancers , they should theoretically benefit from receptor blocking agents ( like tamoxifen ) and oestrogen production blocking agents ( aromatase inhibitors ) .", "most agree that by now it would be reasonable to suggest such chemoprevention to these groups , but there is no agreement on which compound and exactly which group to address .", "it is advocated to give various regimens of such treatment under strict control to evaluate the effects .", "that is where we are today : trials and discussions , but no consensus on applying chemoprevention as standard health care .", "it may be expected that because as a group they have receptor - negative cancers , they should not respond either to oestrogen blockers or to oestrogen .", ", oophorectomy prevents breast cancer , tamoxifen prevents contralateral breast cancer , and oral contraceptives induce breast cancer .", "it seems , however , irrational to suggest oestrogen blockers to prevent oestrogen receptor - negative tumours . in oncology ,", "the scientific standard is a randomized trial . as is evident from the reports ( beside chemoprevention ) mentioned above , we have no randomized trials .", "you can not randomize a woman to mastectomy , and you can not deprive a mutation - carrying woman from any potential life - saving health care available .", "we are faced with the challenge of doing science without randomized trials , and we can not ( as the mammographic screening of older women ) go and get the families we want for research .", "this leaves us with series subjected to a number of ascertainment biases , and we should interpret the results with caution . because we are outside the framework of randomized trials , we may be better off not discussing exact figures in single reports , but rather focus on the main results , methods employed and whether or not the empirical facts are in keeping with the current paradigms for understanding . in this perspective , it may seem that we need to reconsider our paradigms for brca1-associated breast cancer .", "for the other groups , early diagnosis and treatment works as expected , improving early diagnosis may hopefully further improve the results obtained so far , and the principles of chemoprevention may be projected from sporadic cancers . in conclusion to the facts discussed above ,", "most clinical genetic centres relating to inherited breast cancer today advocate annual mammography from the age of 30 onwards to women at risk for inherited non - brca1 breast cancer .", "it is agreed that besides brca1 carriers , oestrogen blockers / aromatase inhibitors may be beneficial - but there is no agreement on exactly how to implement such chemoprevention .", "we are now hoping mri to be better to avoid large numbers of prophylactic mastectomies .", "we are confused by the data on the effect of hormones and hormone blockers in brca1 carriers , and we all hope for chemoprevention to make the unpleasant discussion of prophylactic surgery superfluous if early detection and treatment does not work .", "early diagnosis does not work for ovarian cancers , but oophorectomy is beneficial and advocated at the end of childbearing ages . as is the case with most scientific reports , this contribution has focused on unsolved problems .", "we may remember , however , that we actually have about 90% 5-year survival of non - brca1 breast cancer with today 's means , and the great majority of inherited breast cancers belong to this group .", "prophylactic oophorectomy at the end of childbearing ages in brca1 carriers reduces morbidity and mortality by more than 50% .", "the systematic attempts to prevent and cure inherited breast cancer have been undertaken for but about 10 years .", "the results are actually good , which is reflected in the high compliance from the affected kindreds .", "the attempts to prevent and cure inherited breast cancer is an example of a consumer - driven activity based on knowledge and collaboration from the patients needing our care . actually , based on knowledge two brca1 carriers with small invasive tumours without spread this year have asked me for immediate chemotherapy .", "trials we can not impose upon them , may soon be initiated by patients who want to know .", "the physicians and the researchers have the role of producing , filing , retrieving and communicating knowledge as appropriate .", "the choice what to do , however , should be the patient 's choice , there are no scientific arguments as to whether or not to undergo prophylactic oophorectomy . as we all know , there are more arguments about what to do with your life than the doctors ' suggestions .", "the high compliance to our advice during the last decade most probably reflects that the families have identified their problems long ago and were waiting for our care .", "the high compliance to genetic testing obviously reflects the opinion that our activity may prevent and cure . to maintain the high compliance", "it is our obligation , however , to ensure that the advocated options are actually available to each single patient .", "in addition , we may produce arguments to advocate some options because they have consequences to our liking .", "the suggestions in table 1 may be agreed by most , and some would go further and actively advocate prophylactic mastectomy in brca1 carriers and chemoprevention for the rest .", "genetic counselling is to present information and options so that any given patient may be supported in exploring her values to make her choices .", "the challenge is to support the patients who make choices not corresponding with your own priorities .", "primary and secondary prevention for women at risk for inherited breast or breast - ovarian cancer"]]}
{"article_id": "PMC2717437", "article_text": ["the past 15 years have seen major advances in our understanding of severity assessment in community - acquired pneumonia ( cap ) .", "prognostic tools have been promoted to guide all major management decisions in cap , including admission to the critical care unit .", "several recent studies , including the study by renaud and colleagues , have challenged us to re - evaluate how we consider severe cap , a concept for which there is still no universally accepted definition . since the development of the pneumonia severity index in 1997 , severe cap has been considered in terms of a patient 's risk of 30-day mortality  determined by a combination of age , co - morbidities and physiological parameters measured on admission .", "the two most widely used scores , the pneumonia severity index and the curb65 score , were developed to predict 30-day mortality .", "it is recognised that the majority of pneumonia mortality occurs in older people , however , and that many patients who die are treated palliatively .", "nearly 50% of all deaths in patients with pneumonia and more than one - quarter of deaths within 30 days are related to co - morbidities rather than being directly pneumonia related .", "these scores therefore have important limitations arising from the use of 30-day mortality as an outcome .", "the scores may underestimate severity in young people   and they perform less well when considering outcomes such as intensive care unit ( icu ) admission or requirement for mechanical ventilation or vasopressor support [ 7 - 9 ] .", "as few as 20% of patients in the highest pneumonia severity index class ( class v ) require icu admission , illustrating the system 's limited value for the critical care community .", "there is a growing consensus that icu admission and , more specifically , mechanical ventilation or vasopressor support are more useful outcomes than 30-day mortality to define severe cap and to identify the most acute ill patients [ 7,10 - 12 ] .", "the requirement for mechanical ventilation or vaso - pressor support is preferred to simply using icu admission , as evidence suggests that icu admission rates and criteria vary widely across different healthcare systems .", "this helps to explain why we see icu admission rates of 17% in spain   compared with 8.7% in the uk or 4% in hong kong .", "the revised british thoracic society cap guidelines are due to be published in 2009 and will recommend using the curb65 criteria to determine icu admission . the infectious disease society of america  american thoracic society guidelines recommend the revised american thoracic society criteria , which comprise two major criteria ( the requirement for mechanical ventilation and vasopressor support ) or three minor criteria ( comprising respiratory rate , pao2/fio2 ratio , multilobar infiltrates , confusion , uraemia , leucopenia , thrombocytopenia , hypothermia and hypotension requiring aggressive fluid resuscitation ) . alongside these criteria , renaud and colleagues , charles and colleagues   and espana and colleagues  ", "it is reassuring that the high - risk features identified in each of these studies are similar , with acidosis , systolic blood pressure , respiratory rate , uraemia , confusion , hypoxaemia and multilobar infiltrates featuring in each of the derived scores .", "the abundance of severity criteria , however , reveals the lack of consensus over which patients should be initially managed in the icu .", "delayed transfer to the icu is associated with increased mortality , and therefore early recognition of these patients is important .", "the risk of early admission to intensive care unit score has been shown to predict patients with delayed admission to the icu .", "this group probably consists of patients in whom severity was underestimated on admission , of patients with treatment failure and of patients with unstable co - morbidities and nosocomial superinfection .", "all of the new scores are complex , making them difficult to implement in clinical practice .", "evidence suggests that current severity criteria , such as the curb65 score , are under - utilised .", "it may therefore be impractical to expect staff to use the curb65 score to decide on the site of care , then use smart - cop or the american thoracic society criteria to decide whether a patient requires icu care , and then use the risk of early admission to intensive care unit score to assess their risk of requiring icu subsequently .", "a perfect scoring system may not exist , but it should ideally predict both 30-day mortality and the requirement for mechanical ventilation or vasopressor support . the scoring system should be simple , composed of the fewest possible factors , and easy to remember in a busy emergency department", ". the system should function equally well in older patients and young patients , and should be based on physiological derangement and organ dysfunction rather than on age or co - morbidities .", "the hope is that future studies can identify physiological scoring systems or biomarkers that can achieve these goals and provide an effective adjunct to clinical judgement in the early management of cap .", "cap : community - acquired pneumonia ; curb65 : confusion , urea > 7 mmol / l , respiratory rate  30/min , blood pressure < 90 mmhg systolic and/or diastolic blood pressure  60 mmhg , and age  65 years ; icu : intensive care unit ; smart - cop : systolic blood pressure , multilobar chest radiograph involvement , albumin , respiratory rate , tachycardia , confusion , oxygenation and arterial ph ."], "abstract_text": ["<S> the past 15 years have seen major advances in our understanding of severity assessment in community - acquired pneumonia ( cap ) . </S>", "<S> prognostic tools have been promoted to guide all major management decisions in cap , including admission to the critical care unit . </S>", "<S> several recent studies , including the study by renaud and colleagues , have challenged us to re - evaluate how we consider severe cap , a concept for which there is still no universally accepted definition . </S>", "<S> existing severity scores such as the pneumonia severity index and the curb65 score are designed to predict 30-day mortality . as a result , they are heavily weighted by age and co - morbidity . </S>", "<S> they perform less well when predicting other outcomes such as requirement for icu admission and are of limited use in the critical care environment . </S>", "<S> this commentary discusses recent attempts to develop useful severity criteria to guide the use of icu resources in patients with severe cap . </S>"], "labels": null, "section_names": ["None", "Abbreviations", "Competing interests"], "sections": [["the past 15 years have seen major advances in our understanding of severity assessment in community - acquired pneumonia ( cap ) .", "prognostic tools have been promoted to guide all major management decisions in cap , including admission to the critical care unit .", "several recent studies , including the study by renaud and colleagues , have challenged us to re - evaluate how we consider severe cap , a concept for which there is still no universally accepted definition . since the development of the pneumonia severity index in 1997 , severe cap has been considered in terms of a patient 's risk of 30-day mortality  determined by a combination of age , co - morbidities and physiological parameters measured on admission .", "the two most widely used scores , the pneumonia severity index and the curb65 score , were developed to predict 30-day mortality .", "it is recognised that the majority of pneumonia mortality occurs in older people , however , and that many patients who die are treated palliatively .", "nearly 50% of all deaths in patients with pneumonia and more than one - quarter of deaths within 30 days are related to co - morbidities rather than being directly pneumonia related .", "these scores therefore have important limitations arising from the use of 30-day mortality as an outcome .", "the scores may underestimate severity in young people   and they perform less well when considering outcomes such as intensive care unit ( icu ) admission or requirement for mechanical ventilation or vasopressor support [ 7 - 9 ] .", "as few as 20% of patients in the highest pneumonia severity index class ( class v ) require icu admission , illustrating the system 's limited value for the critical care community .", "there is a growing consensus that icu admission and , more specifically , mechanical ventilation or vasopressor support are more useful outcomes than 30-day mortality to define severe cap and to identify the most acute ill patients [ 7,10 - 12 ] .", "the requirement for mechanical ventilation or vaso - pressor support is preferred to simply using icu admission , as evidence suggests that icu admission rates and criteria vary widely across different healthcare systems .", "this helps to explain why we see icu admission rates of 17% in spain   compared with 8.7% in the uk or 4% in hong kong .", "the revised british thoracic society cap guidelines are due to be published in 2009 and will recommend using the curb65 criteria to determine icu admission . the infectious disease society of america  american thoracic society guidelines recommend the revised american thoracic society criteria , which comprise two major criteria ( the requirement for mechanical ventilation and vasopressor support ) or three minor criteria ( comprising respiratory rate , pao2/fio2 ratio , multilobar infiltrates , confusion , uraemia , leucopenia , thrombocytopenia , hypothermia and hypotension requiring aggressive fluid resuscitation ) . alongside these criteria , renaud and colleagues , charles and colleagues   and espana and colleagues  ", "it is reassuring that the high - risk features identified in each of these studies are similar , with acidosis , systolic blood pressure , respiratory rate , uraemia , confusion , hypoxaemia and multilobar infiltrates featuring in each of the derived scores .", "the abundance of severity criteria , however , reveals the lack of consensus over which patients should be initially managed in the icu .", "delayed transfer to the icu is associated with increased mortality , and therefore early recognition of these patients is important .", "the risk of early admission to intensive care unit score has been shown to predict patients with delayed admission to the icu .", "this group probably consists of patients in whom severity was underestimated on admission , of patients with treatment failure and of patients with unstable co - morbidities and nosocomial superinfection .", "all of the new scores are complex , making them difficult to implement in clinical practice .", "evidence suggests that current severity criteria , such as the curb65 score , are under - utilised .", "it may therefore be impractical to expect staff to use the curb65 score to decide on the site of care , then use smart - cop or the american thoracic society criteria to decide whether a patient requires icu care , and then use the risk of early admission to intensive care unit score to assess their risk of requiring icu subsequently .", "a perfect scoring system may not exist , but it should ideally predict both 30-day mortality and the requirement for mechanical ventilation or vasopressor support . the scoring system should be simple , composed of the fewest possible factors , and easy to remember in a busy emergency department", ". the system should function equally well in older patients and young patients , and should be based on physiological derangement and organ dysfunction rather than on age or co - morbidities .", "the hope is that future studies can identify physiological scoring systems or biomarkers that can achieve these goals and provide an effective adjunct to clinical judgement in the early management of cap ."], ["cap : community - acquired pneumonia ; curb65 : confusion , urea > 7 mmol / l , respiratory rate  30/min , blood pressure < 90 mmhg systolic and/or diastolic blood pressure  60 mmhg , and age  65 years ; icu : intensive care unit ; smart - cop : systolic blood pressure , multilobar chest radiograph involvement , albumin , respiratory rate , tachycardia , confusion , oxygenation and arterial ph ."], [""]]}
{"article_id": "PMC3844257", "article_text": ["stroke is still a major cause of death and long - term disability worldwide and it is associated with significant clinical and socioeconomical problems . despite the continuous efforts to develop the new pharmacological strategies , there is no effective neuroprotective therapy so far for ischemic stroke .", "novel approaches are needed to improve the recovery and quality of life of stroke patients .", "development of tissue damage after ischemic insult is dependent not only on duration and intensity of the blood flow reduction , but also on flow independent mechanisms , especially in the peri - infarct brain area . the blood flow dependent mechanisms of tissue damage develop in brain ischemic focus in the short time after onset of blood flow reduction . at that time cell death", "is a consequence of the acute energy failure and permanent anoxic cells depolarization is induced by loss of ionic gradients .", "a few hours later , the infarct expands into the adjacent penumbra , and cellular damage is mainly triggered by excitotoxicity , mitochondrial disturbances , reactive oxygen species production , and programmed cell death [ 1 , 2 ] .  ", "excitotoxicity is a pathologic process based on massive activation of ampa and nmda receptors in the brain .", "inappropriate activation of ampa and nmda receptors is a trigger for subsequent dysregulation of calcium ions homeostasis in the neurons and finally results in neuronal loss .", "massive activation of those receptors is observed in many cns disorders including stroke , epilepsy , multiple sclerosis , amyotrophic lateral sclerosis , parkinson , alzheimer , and huntington diseases .", "the most important factor leading to ampa and nmda upregulation is glutamate . glutamate is an important neurotransmitter in physiological concentration , but in pathologically high concentration it is neurotoxic [ 35 ] .   lately", ", it is suggested that stroke triggers immune responses leading to inflammatory cell activation and infiltration of cerebral parenchyma . in the stroke brain upregulation of a variety of cytotoxic agents like cytokines , matrix metalloproteinases ( mmps ) , nitric oxide ( no ) , and more ros can be detected [ 68 ] .", "there is also upregulation of expression of some chemokines like ccl2 in the csf [ 9 , 10 ] and serum of patients with stroke .", "studies in experimental stroke ( middle cerebral artery occlusion model ( mcao ) ) confirmed involvement of chemokine ccl2 and its receptors ccr2 in stroke development .", "reported no difference in the level of ccl5   but montecucco and colleagues detected increased expression of plasma ccl5 in symptomatic as compared with asymptomatic patients .", "moreover , canou - poitrine confirmed that , higher systemic levels of ccl5 and cxcl10 in asymptomatic men are independent predictors of ischemic stroke .", "there is also a recent report from tokami et al . supporting the concept that ccl5 may be neuroprotective during stroke development .", "they showed upregulation of ccl5 but not ccl2 , ccl3 , and ccl4 on day 0 in stroke patients .", "this upregulation correlated with plasma concentrations of neuroprotective factors bdnf , egf , and vegf .", "other data from mcao model also showed upregulation of several chemokines and their receptors including ccl7 , cxcl10 , ccl20 , and chemokine receptors cxcr4   and ccr6 .  ", "et-1 induced model of stroke has been previously described by anthony et al . who induced the acute rat cerebral blood volume changes after intravenous and intracranial injections of this vasoconstrictor [ 21 , 22 ] .", "after microinjection of et-1 into selected brain regions they observed using magnetic resonance imaging ( mri ) an acute reduction of local perfusion in the injected hemisphere , loss of neurons in the grey matter and a macrophage / microglia and astrocyte response .", "after injection of et-1 into the cortical white matter , those authors observed amyloid precursor protein - positive immunostaining ( indicative of axonal disruption ) and an increase in tau-1 immunostaining in oligodendrocytes .", "similar to the grey matter lesions , no neutrophils were present and macrophage / microglia response did not occur .", "additionally , no breakdown in the blood - brain barrier was detected in the white and grey matter .   in this study expression of several chemokines including : ccl2 , ccl3 , ccl5 , and cxcl2 as well as expression of markers of neuroinflammation like cd3 , f4/80 , and il-1 beta was studied .", "correlation of this expression with intensity of early neurodegeneration detected in the brain during the et-1 induced model of stroke was also analyzed .", "in all experiments , 8- to 12-week - old female sjl / j mice ( n = five for each time point ) were used .", "all animals were housed at the animal facility of the medical university of lodz , lodz , poland , under standard conditions .", "all experiments in this study have been approved by the local ethics committee for affairs experiments on animals .", "animal stroke model was induced by stereotactic , intracerebral injection of endothelin-1 ( et-1 , sigma - aldrich , poznan , poland , ) ( 20  pmol in 1  l of pbs per mouse ) into the left hemisphere of the brain .", "prior injection mice were anaesthetized with mixture of ketamine ( 1,15  mg , biowet , pulawy , poland ) and ksylazine ( 0,1  mg , biowet , pulawy , poland ) per mouse .", "after complete anaesthetization mice were placed in stereotactic frame ( david kopf instruments , ca , usa ) , skin on the head was cut , and a small hole in the skull was made using surgical drill .", "et-1 was administered with a hamilton syringe ( 32 g needle ) ( hamilton company , bonaduz , gr , switzerland ) .", "site of injection ( a-2  mm , l-1 , 2  mm , d-2 , 5  mm ) was selected using the stereotactic atlas ", "tissue samples were collected 24 and 72 hours after the model induction . as a controls , brains from uninjected mice and from mice injected in the same way with pbs were used .   to obtain rna animals , were perfused with a saline solution .", "tissues were weighed and then homogenized using a mechanical homogenizer ultra turrax ( ika , staufen , germany ) .", "tissues were homogenized in a volume of 1ml of trizol ls reagent ( gibco brl , invitrogen , carlsbad , ca , usa ) .", "rna was isolated from the homogenates with trizol ls reagent using phenol - chloroform method described by chomczynski and sacchi ( chomczynski and sacchi , 1987 ) . after rna isolation", ", its concentration was estimated using the photometric method ( biophotometr plus , eppendorf company , wien , austria ) . to obtain the proteins for the elisa assay", "harvested organs were weighed using a laboratory balance ( radwag radom , poland ) and homogenized using a mechanical homogenizer ultra turrax ( ika ) .", "homogenisation was performed in a volume of 1  ml hepes buffer ph 7.4 containing : hepes 20  mm ; edta 1.5  mm ; benzamidyn 0.5  mm ; chicken egg owoinhibitor 10  ug / ml pmsf ( phenylmetylsulfonyl fluoride ) 0.1  mm ( sigma - aldrich , poznan , poland ) .", "supernatants were obtained after centrifugation ( 20 000  g , time 30 minutes at 4c mpw , warsaw , poland ) .", "analysis of the rna expression was performed using the corbett real - time pcr machine rotor gene 3000 apparatus ( corbett research , sydney , australia ) .", "the key enzyme used in this reaction was taq polymerase with activity of 5  u / ml .", "additional reaction components were buffer for polymerase , 25  mm mgcl2 , 10  mm dntps , fluorescent dye evagreen ( biomibo , warsaw , poland ) , 10  m primers specific to the duplicated sequences , and rnase / dnase free water . for each reaction 2  l of cdna derived from the reverse transcription reaction", "was used and the total volume was 20  l . as a control histone h3 gene and reference rna ( qpcr mouse reference total cellular rna , stratagene , la jolla , ca , usa ) were used", ". quantitative analysis of gene expression at the protein level was performed using elisa method with commercially available immunoenzymatic quantikine kits ( r & d systems , mn , usa ) .", "each set consisted of 96 well plates coated by manufacturer , standard proteins used to prepare the calibration , secondary and tertiary antibodies combined with the horseradish peroxidase enzyme , and washing buffer and color substrate for peroxidase .", "the assay procedure was performed according to the protocol provided by the manufacturer . after stopping the color reaction protein concentration", "was evaluated using a photometric reader victor2 wallac 1420 ( perkinelmer , waltham , ma , usa ) with for 450  nm filters , corrected at 595  nm . all samples were analyzed in duplicates .", "quantitative assessment of the intensity of neurodegeneration was performed using elisa method with primary antibodies directed against phosphorylated neurofilaments .", "the first step was the coating of 96 well maxisorb microtitre plate ( nunc , roskilde , denmark ) with monoclonal anti - nfh antibodies ( smi35r , sternberger monoclonals , convance princeton , nj , usa ) and overnight incubation at 4c .", "the next day tested samples and standard curve samples ( neurofilament 200  kd , progen , heidelberg , germany ) were added . as a secondary antibody rabbit polyclonal antineurofilament 200 antibody ( sigma - aldrich , poznan , poland ) was used . as a tertiary antibody swine antibodies against rabbit immunoglobulin conjugated with horseradish peroxidase ( dako , glostrup , denmark ) were used .", "the final step was the addition of color substrate for horseradish peroxidase , which was 3,35,5-tetramethylbenzidine ( sigma - aldrich , poznan , poland ) .", "inhibition of the reaction was performed with 1  m hcl and finally color photometric assessment was done using victor2 reader wallac 1420 ( perkinelmer , waltham , ma , usa ) .", "the analysis was performed using 450  nm filter with correction at 595  nm . all samples were analyzed in duplicates and the concentration of nfh was determined by referring to the standard curve .", "assessment of the localization and severity of neurodegeneration at the level of protein was performed using the fluorescent fluoro - jade c dye ( chemicon , millipore , warsaw , poland ) .", "animals were perfused with 4% buffered formalin solution and tissues samples were embedded in paraffin blocks .", "10  m thick sections were applied to a polished super frost slides ( menzel - glaser braunschweig , germany ) . before final staining paraffin", "fluoro - jade c staining was performed according to the protocol provided by the manufacturer ( chemicon ) . for staining of nuclei sections", "were counterstained using the blue fluorescent dye dapi ( sigma - aldrich , poznan , poland ) . then the tissue was mounted and coverslipped using dpx ( sigma - aldrich , poznan , poland ) .   for the analysis and acquisition of images an inverted microscope axioobserver a1 ( carl zeiss inc . ,", "the following lenses made by carl zeiss inc . were used : plan - achromat : 4x/0.10 , a- plan 10x/0.25 ph1 ; ld a - plan 20x/0.3 ; ld plan - neofluar 40x/0.6 , ph2 korr . the images were obtained with a digital camera , axiocam mrc5 ( carl zeiss group , goettingen , germany ) attached to the microscope . for image acquisition we used axio - vision rel .", "nonparametric kruskal - wallis and mann - whitney tests were used . for correlation analyses kendal", "significant upregulation of expression of t cell line marker - cd3 was observed in the et-1-injected hemisphere at 72  h after injection ( p = 0.03 , mann - whitney test ) ( figure 1(a ) ) . at that time a significant difference in expression of cd3 was detected between ipsilateral and contralateral hemispheres ( p = 0.019 , mann - whitney test ) ( figure 1(a ) ) .", "expression of cd3 in ipsilateral hemisphere was also increased when compared to pbs injected hemisphere ( p = 0.28 , mann - whitney test ) .   the expression of monocyte / macrophage lineage marker f4/80 was significantly elevated only in et-1 injected hemisphere at 72 hours after injection . at that time", "significant difference was observed in expression of f4/80 between et-1 injected hemisphere and untreated control group ( p = 0.022 ; mann - whitney test ) ( figure 1(b ) ) .", "we detected also a significant difference in expression of f4/80 between ipsilateral and contralateral hemispheres of et-1 injected mice at 72 hours after injection ( p = 0.035 , mann - whitney test ) ( figure 1(b ) ) .  ", "upregulation of cytokine il-1 expression was observed in et-1 injected hemispheres only at 24  h after injection .", "significant difference in expression of il-1 was observed between contralateral hemispheres and normal control group at 72 hours after injection ( p = 0.03 and 0.019 , resp . ;", "we detected also a significant difference in expression of il-1 between ipsilateral and contralateral hemispheres of et-1 injected mice 72 hours after injection ( p = 0.019 , p = 0.019 , resp . ;", "upregulation of chemokine ccl2 expression was observed in ipsilateral hemispheres at 24 and 72  h after injection of et-1 ( p = 0.005 , p = 0.005 , resp . ;", "mann - whitney test ) ( figure 2(a ) ) . at 24  h ccl2 expression in ipsilateral hemisphere was significantly higher than at 72  h ( p = 0.019 ; mann - whitney test ) .", "significant difference in ccl2 expression after et-1 injection was also observed between ipsilateral and contralateral hemispheres at 24  h and 72  h ( p = 0.012 and 0.036 , resp . ;", "we also showed that during early stage of et-1-injection stroke model expression of ccl3 is significantly upregulated at 24 and 72  h after model induction ( figure 2(b ) ) .", "there was significant upregulation of ccl3 expression in ipsilateral hemispheres of et-1 injected mice in comparison to normal controls and contralateral hemispheres at 24  h after injection ( p = 0.008 and 0.012 , resp . ;", "mann - whitney test ) . at 72  h after model induction we observed significant upregulation of ccl3 expression in et-1-injected hemispheres in comparison to normal brains and contralateral hemispheres ( p = 0.014 and 0.019 , resp . ; mann - whitney test ) ( figure 2(b ) ) .  ", "ccl5 was observed in et-1-injected hemispheres in comparison to uninjected animals at 24 and 72  h ( p = 0.008 , and 0.008 resp . ;", "significant difference was also observed in ccl5 expression between ipsilateral and contralateral hemispheres at 24  h and 72  h after injection of et-1 ( p = 0.008 and 0.012 , resp . ;", "mann - whitney test ) ( figure 2(c ) ) .   initially increasing expression of cxcl2 in et-1 injected hemispheres", "was detected with the peak at 24  h and subsequent decrease at 72  h but still significantly higher than in normal controls ( p = 0.036 , and 0.036 , resp . ;", "mann - whitney test ) ( figure 2(d ) ) . at 24  h cxcl2 expression in ipsilateral hemisphere was significantly higher than at 72  h ( p = 0.012 ; mann - whitney test ) and then at 24  h after et-1 injection in contralateral hemispheres ( p = 0.012 ; mann - whitney test ) ( figure 2(d ) ) .  ", "the expression of cxcl12 in the et-1 and pbs - injected brains and normal controls did not show a significant difference between analysed groups ( data not shown ) .", "the most severe neurodegeneration was observed in et-1-injected hemispheres at 24 and 72  h after model induction ( p = 0.019 and 0.029 , resp . ;", "there was also increased neurodegeneration in contralateral hemispheres of et-1 injected mice at 24 and 72  h , but it was significantly lower than in ipsilateral hemispheres ( p = 0.03 , and 0.03 , resp . ;", "the localization of ischemic lesion was detected in et-1 injected ipsilateral hemispheres using cresyl violet staining ( figure 3(b ) , large box ) . inside the ischemic focus injured neurons", "were abundant ( detected by fluoro - jade and marked by arrows ) cells nuclei counterstained with dapi are marked on the picture by arrowheads ( figure 3(c ) ) .", "we observed the positive correlation between expression of lymphocyte lineage marker cd3 ( kendall tau = 0,62 ; p = 0.0004 ) ( figure 4(a ) ) as well as monocyte / macrophage lineage marker f4/80 ( kendall tau = 0,56 ; p = 0.0007 ) ( figure 4(b ) ) and the severity of neurodegeneration in et-1 injected brain hemispheres .  ", "although the expression of several studied chemotactic inflammatory mediators ( chemokines ccl2 , ccl3 , ccl5 , and cxcl2 ) was significantly increased in the early stage of this stroke model , there was no clear correlation between this expression and intensity of neurodegeneration ( data not shown ) .", "in this study we analyzed potential relationship between neuroinflammation and neurodegeneration in experimental model of ischemic stroke induced by intracerebral et-1 injection .", "we focused on a group of proinflammatory chemokines , especially the classical representatives of ccl subfamily .", "the reason for selecting these chemokines was their confirmed participation in pathogenesis of many central nervous system diseases . during the first few days of the experimental brain ischemia we observed increasing neurodegeneration in et-1 injected hemisphere .", "there are several studies showing that neurodegeneration occurs early during brain ischaemia [ 23 , 24 ] . at that time also", "the relationship between those two processes is complex and still requires further studies . to measure the intensity of neuroinflammation the expression of inflammatory cells markers (", "cd3 for t cells and f4/80 for monocytes / macrophages ) has been measured at the same time .", "this analysis showed increased lymphocyte migration to ischemic brain hemisphere at 72  h after model induction .", "similarly , infiltration of ipsilateral hemisphere by monocytes / macrophages was significantly increased at 72  h after initiation of brain ischemia .", "comparable observation was reported by others who showed the presence of macrophages / activated microglia at 72  h after intracerebral injection of et-1 to rat brain . in another study using mcao stroke model , the influx of mononuclear cells to the site of brain ischemia was recorded between 2 and 15 days after model induction .  ", "the presence of neuroinflammation during early brain ischemia was also confirmed in our study by elevated expression of inflammatory mediator - cytokine il-1. its presence was observed as early as 24  h after model induction .", "in several cell types including astrocytes , microglia , neurons , and endothelium . in another study increased production of il-1 was reported even at 36 hours after induction of brain ischemia .", "the peak of this expression was observed at 12  h , and it returned to baseline level after 5 days .", "other studies confirmed also that inflammatory mediators , such as il-1 and tnf , are important contributors to cns neural tissue damage induced by ischemia [ 28 , 29 ] . in our stroke model analysis of the relationship between the infiltration of ischemic hemisphere by mononuclear inflammatory cells and", "this may suggest that there is close connection between neuroinflammation and neurodegeneration in ischemic stroke .", "migration of inflammatory cells from the blood to the ischemic brain may be at least partially induced by chemotactic cytokines - chemokines . to study this concept", "the highest expression of ccl2 was observed in our model at 24  h after initiation of brain ischemia .", "increased expression of ccl2 was still observed at 72  h but was at that time significantly lower . increased expression of ccl2 in mcao model in the ipsilateral hemisphere was observed on neurons at 12  h and on astrocytes at 24  h after cardiac arrest , suggesting that these cells are the potential source of ccl2 during ischemic stroke .", "minami and satoh using double in situ hybridization method pointed to microglia as the cellular source of ccl2 during mcao .", "it was shown in another mcao study that ccl2 leads to infiltration of the cns by monocytes and thus enhances brain damage induced by ischemia . also in human stroke patients", "elevated level of ccl2 was detected in cerebrospinal fluid and serum [ 9 , 11 ] .  ", "the highest ccl3 expression was detected at 24  h after et-1 injection . at 72  h", "this expression was still increased but it was much lower than at 24  h. also at the protein level we observed significant increase in ccl3 production in the ischemic hemisphere .", "our results are in line with the report by gourmala et al . who observed an increase in ccl3 expression at mrna level already at 1  h after mcao in rats , with peak expression at 816  h .", "in addition , they observed higher expression of ccl3 during temporary mcao than in permanent mcao , suggesting the impact of reperfusion on the neuroinflammation in the damaged tissue .", "gourmala et al . using in situ hybridization localized the expression of ccl3 on microglial cells / macrophages during brain ischemia . in addition , another studies have concluded that ccl3 application to the brain ventricles after complete mcao enhances mcao harmful effects .  ", "we observed almost 46-fold and 30-fold increase in ccl5 expression at 24  h and 72  h , respectively , after induction of et-1 induced stroke model .", "there are only a few reports concerning the role of ccl5 in the development of ischemic stroke .", "it was suggested that ccl5 mediates blood - brain - barrier ( bbb ) disruption and cns tissue damage as well as inflammation after reperfusion during mcao model .", "these data were not confirmed by tokami and colleagues who observed neuroprotective effect of ccl5 in ischemic stroke suggesting that ccl5 is expressed during stroke mostly in neurons .   in et-1 induced experimental stroke a significant increase in cxcl2 expression at 24  h after brain ischemia induction was also observed .", "observed increased expression of cxcl2 in the brain of rats with the permanent mcao   as well as in the brain and spleen during temporary mcao in mice .", "in other study increased cxcl2 expression was observed at 6  h of reperfusion and decreased by almost half at 22  h after reperfusion .", "vikman et al . showed increased cxcl2 expression in the brain vessels in the model of subarachnoid haemorrhage and in organotypic cultures .", "cxcl2 involvement in the inflammatory process in the cns during mcao was also confirmed in a scid mice .", "mcao induced in scid mice led to development of significantly reduced area of brain damage and lower inflammatory infiltration in ipsilateral hemispheres .", "reduced expression of many inflammatory mediators including cxcl2 was also observed in t- and b - cell - deficient mice mcao study .", "unfortunately , therapy of ischemic stroke with cxcl2 receptor - cxcr2 antagonists sb225002 was not successful . in report presented by copin et al .", "the cxcl1/cxcl2 chemokine - binding protein evasin-3 treatment was associated with reduction in neutrophilic inflammation in mice mcao model .", "although in our study the expression of several studied chemokines was significantly increased at the early phase of et-1 induced stroke model , no clear correlation of this expression with neurodegeneration was observed .", "that they suggest that inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration .", "upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke ."], "abstract_text": ["<S> neurodegeneration is a hallmark of most of the central nervous system ( cns ) disorders including stroke . </S>", "<S> recently inflammation has been implicated in pathogenesis of neurodegeneration and neurodegenerative diseases . </S>", "<S> the aim of this study was analysis of expression of several inflammatory markers and its correlation with development of neurodegeneration during the early stage of experimental stroke . </S>", "<S> ischemic stroke model was induced by stereotaxic intracerebral injection of vasoconstricting agent endothelin-1 ( et-1 ) . </S>", "<S> it was observed that neurodegeneration appears very early in that model and correlates well with migration of inflammatory lymphocytes and macrophages to the brain . </S>", "<S> although the expression of several studied chemotactic cytokines ( chemokines ) was significantly increased at the early phase of et-1 induced stroke model , no clear correlation of this expression with neurodegeneration was observed . </S>", "<S> these data may indicate that chemokines do not induce neurodegeneration directly . </S>", "<S> upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke . </S>", "<S> inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Material and Methods", "3. Results", "4. Discussion"], "sections": [["stroke is still a major cause of death and long - term disability worldwide and it is associated with significant clinical and socioeconomical problems . despite the continuous efforts to develop the new pharmacological strategies , there is no effective neuroprotective therapy so far for ischemic stroke .", "novel approaches are needed to improve the recovery and quality of life of stroke patients .", "development of tissue damage after ischemic insult is dependent not only on duration and intensity of the blood flow reduction , but also on flow independent mechanisms , especially in the peri - infarct brain area . the blood flow dependent mechanisms of tissue damage develop in brain ischemic focus in the short time after onset of blood flow reduction . at that time cell death", "is a consequence of the acute energy failure and permanent anoxic cells depolarization is induced by loss of ionic gradients .", "a few hours later , the infarct expands into the adjacent penumbra , and cellular damage is mainly triggered by excitotoxicity , mitochondrial disturbances , reactive oxygen species production , and programmed cell death [ 1 , 2 ] .  ", "excitotoxicity is a pathologic process based on massive activation of ampa and nmda receptors in the brain .", "inappropriate activation of ampa and nmda receptors is a trigger for subsequent dysregulation of calcium ions homeostasis in the neurons and finally results in neuronal loss .", "massive activation of those receptors is observed in many cns disorders including stroke , epilepsy , multiple sclerosis , amyotrophic lateral sclerosis , parkinson , alzheimer , and huntington diseases .", "the most important factor leading to ampa and nmda upregulation is glutamate . glutamate is an important neurotransmitter in physiological concentration , but in pathologically high concentration it is neurotoxic [ 35 ] .   lately", ", it is suggested that stroke triggers immune responses leading to inflammatory cell activation and infiltration of cerebral parenchyma . in the stroke brain upregulation of a variety of cytotoxic agents like cytokines , matrix metalloproteinases ( mmps ) , nitric oxide ( no ) , and more ros can be detected [ 68 ] .", "there is also upregulation of expression of some chemokines like ccl2 in the csf [ 9 , 10 ] and serum of patients with stroke .", "studies in experimental stroke ( middle cerebral artery occlusion model ( mcao ) ) confirmed involvement of chemokine ccl2 and its receptors ccr2 in stroke development .", "reported no difference in the level of ccl5   but montecucco and colleagues detected increased expression of plasma ccl5 in symptomatic as compared with asymptomatic patients .", "moreover , canou - poitrine confirmed that , higher systemic levels of ccl5 and cxcl10 in asymptomatic men are independent predictors of ischemic stroke .", "there is also a recent report from tokami et al . supporting the concept that ccl5 may be neuroprotective during stroke development .", "they showed upregulation of ccl5 but not ccl2 , ccl3 , and ccl4 on day 0 in stroke patients .", "this upregulation correlated with plasma concentrations of neuroprotective factors bdnf , egf , and vegf .", "other data from mcao model also showed upregulation of several chemokines and their receptors including ccl7 , cxcl10 , ccl20 , and chemokine receptors cxcr4   and ccr6 .  ", "et-1 induced model of stroke has been previously described by anthony et al . who induced the acute rat cerebral blood volume changes after intravenous and intracranial injections of this vasoconstrictor [ 21 , 22 ] .", "after microinjection of et-1 into selected brain regions they observed using magnetic resonance imaging ( mri ) an acute reduction of local perfusion in the injected hemisphere , loss of neurons in the grey matter and a macrophage / microglia and astrocyte response .", "after injection of et-1 into the cortical white matter , those authors observed amyloid precursor protein - positive immunostaining ( indicative of axonal disruption ) and an increase in tau-1 immunostaining in oligodendrocytes .", "similar to the grey matter lesions , no neutrophils were present and macrophage / microglia response did not occur .", "additionally , no breakdown in the blood - brain barrier was detected in the white and grey matter .   in this study expression of several chemokines including : ccl2 , ccl3 , ccl5 , and cxcl2 as well as expression of markers of neuroinflammation like cd3 , f4/80 , and il-1 beta was studied .", "correlation of this expression with intensity of early neurodegeneration detected in the brain during the et-1 induced model of stroke was also analyzed ."], ["in all experiments , 8- to 12-week - old female sjl / j mice ( n = five for each time point ) were used .", "all animals were housed at the animal facility of the medical university of lodz , lodz , poland , under standard conditions .", "all experiments in this study have been approved by the local ethics committee for affairs experiments on animals .", "animal stroke model was induced by stereotactic , intracerebral injection of endothelin-1 ( et-1 , sigma - aldrich , poznan , poland , ) ( 20  pmol in 1  l of pbs per mouse ) into the left hemisphere of the brain .", "prior injection mice were anaesthetized with mixture of ketamine ( 1,15  mg , biowet , pulawy , poland ) and ksylazine ( 0,1  mg , biowet , pulawy , poland ) per mouse .", "after complete anaesthetization mice were placed in stereotactic frame ( david kopf instruments , ca , usa ) , skin on the head was cut , and a small hole in the skull was made using surgical drill .", "et-1 was administered with a hamilton syringe ( 32 g needle ) ( hamilton company , bonaduz , gr , switzerland ) .", "site of injection ( a-2  mm , l-1 , 2  mm , d-2 , 5  mm ) was selected using the stereotactic atlas ", "tissue samples were collected 24 and 72 hours after the model induction . as a controls , brains from uninjected mice and from mice injected in the same way with pbs were used .   to obtain rna animals , were perfused with a saline solution .", "tissues were weighed and then homogenized using a mechanical homogenizer ultra turrax ( ika , staufen , germany ) .", "tissues were homogenized in a volume of 1ml of trizol ls reagent ( gibco brl , invitrogen , carlsbad , ca , usa ) .", "rna was isolated from the homogenates with trizol ls reagent using phenol - chloroform method described by chomczynski and sacchi ( chomczynski and sacchi , 1987 ) . after rna isolation", ", its concentration was estimated using the photometric method ( biophotometr plus , eppendorf company , wien , austria ) . to obtain the proteins for the elisa assay", "harvested organs were weighed using a laboratory balance ( radwag radom , poland ) and homogenized using a mechanical homogenizer ultra turrax ( ika ) .", "homogenisation was performed in a volume of 1  ml hepes buffer ph 7.4 containing : hepes 20  mm ; edta 1.5  mm ; benzamidyn 0.5  mm ; chicken egg owoinhibitor 10  ug / ml pmsf ( phenylmetylsulfonyl fluoride ) 0.1  mm ( sigma - aldrich , poznan , poland ) .", "supernatants were obtained after centrifugation ( 20 000  g , time 30 minutes at 4c mpw , warsaw , poland ) .", "analysis of the rna expression was performed using the corbett real - time pcr machine rotor gene 3000 apparatus ( corbett research , sydney , australia ) .", "the key enzyme used in this reaction was taq polymerase with activity of 5  u / ml .", "additional reaction components were buffer for polymerase , 25  mm mgcl2 , 10  mm dntps , fluorescent dye evagreen ( biomibo , warsaw , poland ) , 10  m primers specific to the duplicated sequences , and rnase / dnase free water . for each reaction 2  l of cdna derived from the reverse transcription reaction", "was used and the total volume was 20  l . as a control histone h3 gene and reference rna ( qpcr mouse reference total cellular rna , stratagene , la jolla , ca , usa ) were used", ". quantitative analysis of gene expression at the protein level was performed using elisa method with commercially available immunoenzymatic quantikine kits ( r & d systems , mn , usa ) .", "each set consisted of 96 well plates coated by manufacturer , standard proteins used to prepare the calibration , secondary and tertiary antibodies combined with the horseradish peroxidase enzyme , and washing buffer and color substrate for peroxidase .", "the assay procedure was performed according to the protocol provided by the manufacturer . after stopping the color reaction protein concentration", "was evaluated using a photometric reader victor2 wallac 1420 ( perkinelmer , waltham , ma , usa ) with for 450  nm filters , corrected at 595  nm . all samples were analyzed in duplicates .", "quantitative assessment of the intensity of neurodegeneration was performed using elisa method with primary antibodies directed against phosphorylated neurofilaments .", "the first step was the coating of 96 well maxisorb microtitre plate ( nunc , roskilde , denmark ) with monoclonal anti - nfh antibodies ( smi35r , sternberger monoclonals , convance princeton , nj , usa ) and overnight incubation at 4c .", "the next day tested samples and standard curve samples ( neurofilament 200  kd , progen , heidelberg , germany ) were added . as a secondary antibody rabbit polyclonal antineurofilament 200 antibody ( sigma - aldrich , poznan , poland ) was used . as a tertiary antibody swine antibodies against rabbit immunoglobulin conjugated with horseradish peroxidase ( dako , glostrup , denmark ) were used .", "the final step was the addition of color substrate for horseradish peroxidase , which was 3,35,5-tetramethylbenzidine ( sigma - aldrich , poznan , poland ) .", "inhibition of the reaction was performed with 1  m hcl and finally color photometric assessment was done using victor2 reader wallac 1420 ( perkinelmer , waltham , ma , usa ) .", "the analysis was performed using 450  nm filter with correction at 595  nm . all samples were analyzed in duplicates and the concentration of nfh was determined by referring to the standard curve .", "assessment of the localization and severity of neurodegeneration at the level of protein was performed using the fluorescent fluoro - jade c dye ( chemicon , millipore , warsaw , poland ) .", "animals were perfused with 4% buffered formalin solution and tissues samples were embedded in paraffin blocks .", "10  m thick sections were applied to a polished super frost slides ( menzel - glaser braunschweig , germany ) . before final staining paraffin", "fluoro - jade c staining was performed according to the protocol provided by the manufacturer ( chemicon ) . for staining of nuclei sections", "were counterstained using the blue fluorescent dye dapi ( sigma - aldrich , poznan , poland ) . then the tissue was mounted and coverslipped using dpx ( sigma - aldrich , poznan , poland ) .   for the analysis and acquisition of images an inverted microscope axioobserver a1 ( carl zeiss inc . ,", "the following lenses made by carl zeiss inc . were used : plan - achromat : 4x/0.10 , a- plan 10x/0.25 ph1 ; ld a - plan 20x/0.3 ; ld plan - neofluar 40x/0.6 , ph2 korr . the images were obtained with a digital camera , axiocam mrc5 ( carl zeiss group , goettingen , germany ) attached to the microscope . for image acquisition we used axio - vision rel .", "nonparametric kruskal - wallis and mann - whitney tests were used . for correlation analyses kendal"], ["significant upregulation of expression of t cell line marker - cd3 was observed in the et-1-injected hemisphere at 72  h after injection ( p = 0.03 , mann - whitney test ) ( figure 1(a ) ) . at that time a significant difference in expression of cd3 was detected between ipsilateral and contralateral hemispheres ( p = 0.019 , mann - whitney test ) ( figure 1(a ) ) .", "expression of cd3 in ipsilateral hemisphere was also increased when compared to pbs injected hemisphere ( p = 0.28 , mann - whitney test ) .   the expression of monocyte / macrophage lineage marker f4/80 was significantly elevated only in et-1 injected hemisphere at 72 hours after injection . at that time", "significant difference was observed in expression of f4/80 between et-1 injected hemisphere and untreated control group ( p = 0.022 ; mann - whitney test ) ( figure 1(b ) ) .", "we detected also a significant difference in expression of f4/80 between ipsilateral and contralateral hemispheres of et-1 injected mice at 72 hours after injection ( p = 0.035 , mann - whitney test ) ( figure 1(b ) ) .  ", "upregulation of cytokine il-1 expression was observed in et-1 injected hemispheres only at 24  h after injection .", "significant difference in expression of il-1 was observed between contralateral hemispheres and normal control group at 72 hours after injection ( p = 0.03 and 0.019 , resp . ;", "we detected also a significant difference in expression of il-1 between ipsilateral and contralateral hemispheres of et-1 injected mice 72 hours after injection ( p = 0.019 , p = 0.019 , resp . ;", "upregulation of chemokine ccl2 expression was observed in ipsilateral hemispheres at 24 and 72  h after injection of et-1 ( p = 0.005 , p = 0.005 , resp . ;", "mann - whitney test ) ( figure 2(a ) ) . at 24  h ccl2 expression in ipsilateral hemisphere was significantly higher than at 72  h ( p = 0.019 ; mann - whitney test ) .", "significant difference in ccl2 expression after et-1 injection was also observed between ipsilateral and contralateral hemispheres at 24  h and 72  h ( p = 0.012 and 0.036 , resp . ;", "we also showed that during early stage of et-1-injection stroke model expression of ccl3 is significantly upregulated at 24 and 72  h after model induction ( figure 2(b ) ) .", "there was significant upregulation of ccl3 expression in ipsilateral hemispheres of et-1 injected mice in comparison to normal controls and contralateral hemispheres at 24  h after injection ( p = 0.008 and 0.012 , resp . ;", "mann - whitney test ) . at 72  h after model induction we observed significant upregulation of ccl3 expression in et-1-injected hemispheres in comparison to normal brains and contralateral hemispheres ( p = 0.014 and 0.019 , resp . ; mann - whitney test ) ( figure 2(b ) ) .  ", "ccl5 was observed in et-1-injected hemispheres in comparison to uninjected animals at 24 and 72  h ( p = 0.008 , and 0.008 resp . ;", "significant difference was also observed in ccl5 expression between ipsilateral and contralateral hemispheres at 24  h and 72  h after injection of et-1 ( p = 0.008 and 0.012 , resp . ;", "mann - whitney test ) ( figure 2(c ) ) .   initially increasing expression of cxcl2 in et-1 injected hemispheres", "was detected with the peak at 24  h and subsequent decrease at 72  h but still significantly higher than in normal controls ( p = 0.036 , and 0.036 , resp . ;", "mann - whitney test ) ( figure 2(d ) ) . at 24  h cxcl2 expression in ipsilateral hemisphere was significantly higher than at 72  h ( p = 0.012 ; mann - whitney test ) and then at 24  h after et-1 injection in contralateral hemispheres ( p = 0.012 ; mann - whitney test ) ( figure 2(d ) ) .  ", "the expression of cxcl12 in the et-1 and pbs - injected brains and normal controls did not show a significant difference between analysed groups ( data not shown ) .", "the most severe neurodegeneration was observed in et-1-injected hemispheres at 24 and 72  h after model induction ( p = 0.019 and 0.029 , resp . ;", "there was also increased neurodegeneration in contralateral hemispheres of et-1 injected mice at 24 and 72  h , but it was significantly lower than in ipsilateral hemispheres ( p = 0.03 , and 0.03 , resp . ;", "the localization of ischemic lesion was detected in et-1 injected ipsilateral hemispheres using cresyl violet staining ( figure 3(b ) , large box ) . inside the ischemic focus injured neurons", "were abundant ( detected by fluoro - jade and marked by arrows ) cells nuclei counterstained with dapi are marked on the picture by arrowheads ( figure 3(c ) ) .", "we observed the positive correlation between expression of lymphocyte lineage marker cd3 ( kendall tau = 0,62 ; p = 0.0004 ) ( figure 4(a ) ) as well as monocyte / macrophage lineage marker f4/80 ( kendall tau = 0,56 ; p = 0.0007 ) ( figure 4(b ) ) and the severity of neurodegeneration in et-1 injected brain hemispheres .  ", "although the expression of several studied chemotactic inflammatory mediators ( chemokines ccl2 , ccl3 , ccl5 , and cxcl2 ) was significantly increased in the early stage of this stroke model , there was no clear correlation between this expression and intensity of neurodegeneration ( data not shown ) ."], ["in this study we analyzed potential relationship between neuroinflammation and neurodegeneration in experimental model of ischemic stroke induced by intracerebral et-1 injection .", "we focused on a group of proinflammatory chemokines , especially the classical representatives of ccl subfamily .", "the reason for selecting these chemokines was their confirmed participation in pathogenesis of many central nervous system diseases . during the first few days of the experimental brain ischemia we observed increasing neurodegeneration in et-1 injected hemisphere .", "there are several studies showing that neurodegeneration occurs early during brain ischaemia [ 23 , 24 ] . at that time also", "the relationship between those two processes is complex and still requires further studies . to measure the intensity of neuroinflammation the expression of inflammatory cells markers (", "cd3 for t cells and f4/80 for monocytes / macrophages ) has been measured at the same time .", "this analysis showed increased lymphocyte migration to ischemic brain hemisphere at 72  h after model induction .", "similarly , infiltration of ipsilateral hemisphere by monocytes / macrophages was significantly increased at 72  h after initiation of brain ischemia .", "comparable observation was reported by others who showed the presence of macrophages / activated microglia at 72  h after intracerebral injection of et-1 to rat brain . in another study using mcao stroke model , the influx of mononuclear cells to the site of brain ischemia was recorded between 2 and 15 days after model induction .  ", "the presence of neuroinflammation during early brain ischemia was also confirmed in our study by elevated expression of inflammatory mediator - cytokine il-1. its presence was observed as early as 24  h after model induction .", "in several cell types including astrocytes , microglia , neurons , and endothelium . in another study increased production of il-1 was reported even at 36 hours after induction of brain ischemia .", "the peak of this expression was observed at 12  h , and it returned to baseline level after 5 days .", "other studies confirmed also that inflammatory mediators , such as il-1 and tnf , are important contributors to cns neural tissue damage induced by ischemia [ 28 , 29 ] . in our stroke model analysis of the relationship between the infiltration of ischemic hemisphere by mononuclear inflammatory cells and", "this may suggest that there is close connection between neuroinflammation and neurodegeneration in ischemic stroke .", "migration of inflammatory cells from the blood to the ischemic brain may be at least partially induced by chemotactic cytokines - chemokines . to study this concept", "the highest expression of ccl2 was observed in our model at 24  h after initiation of brain ischemia .", "increased expression of ccl2 was still observed at 72  h but was at that time significantly lower . increased expression of ccl2 in mcao model in the ipsilateral hemisphere was observed on neurons at 12  h and on astrocytes at 24  h after cardiac arrest , suggesting that these cells are the potential source of ccl2 during ischemic stroke .", "minami and satoh using double in situ hybridization method pointed to microglia as the cellular source of ccl2 during mcao .", "it was shown in another mcao study that ccl2 leads to infiltration of the cns by monocytes and thus enhances brain damage induced by ischemia . also in human stroke patients", "elevated level of ccl2 was detected in cerebrospinal fluid and serum [ 9 , 11 ] .  ", "the highest ccl3 expression was detected at 24  h after et-1 injection . at 72  h", "this expression was still increased but it was much lower than at 24  h. also at the protein level we observed significant increase in ccl3 production in the ischemic hemisphere .", "our results are in line with the report by gourmala et al . who observed an increase in ccl3 expression at mrna level already at 1  h after mcao in rats , with peak expression at 816  h .", "in addition , they observed higher expression of ccl3 during temporary mcao than in permanent mcao , suggesting the impact of reperfusion on the neuroinflammation in the damaged tissue .", "gourmala et al . using in situ hybridization localized the expression of ccl3 on microglial cells / macrophages during brain ischemia . in addition , another studies have concluded that ccl3 application to the brain ventricles after complete mcao enhances mcao harmful effects .  ", "we observed almost 46-fold and 30-fold increase in ccl5 expression at 24  h and 72  h , respectively , after induction of et-1 induced stroke model .", "there are only a few reports concerning the role of ccl5 in the development of ischemic stroke .", "it was suggested that ccl5 mediates blood - brain - barrier ( bbb ) disruption and cns tissue damage as well as inflammation after reperfusion during mcao model .", "these data were not confirmed by tokami and colleagues who observed neuroprotective effect of ccl5 in ischemic stroke suggesting that ccl5 is expressed during stroke mostly in neurons .   in et-1 induced experimental stroke a significant increase in cxcl2 expression at 24  h after brain ischemia induction was also observed .", "observed increased expression of cxcl2 in the brain of rats with the permanent mcao   as well as in the brain and spleen during temporary mcao in mice .", "in other study increased cxcl2 expression was observed at 6  h of reperfusion and decreased by almost half at 22  h after reperfusion .", "vikman et al . showed increased cxcl2 expression in the brain vessels in the model of subarachnoid haemorrhage and in organotypic cultures .", "cxcl2 involvement in the inflammatory process in the cns during mcao was also confirmed in a scid mice .", "mcao induced in scid mice led to development of significantly reduced area of brain damage and lower inflammatory infiltration in ipsilateral hemispheres .", "reduced expression of many inflammatory mediators including cxcl2 was also observed in t- and b - cell - deficient mice mcao study .", "unfortunately , therapy of ischemic stroke with cxcl2 receptor - cxcr2 antagonists sb225002 was not successful . in report presented by copin et al .", "the cxcl1/cxcl2 chemokine - binding protein evasin-3 treatment was associated with reduction in neutrophilic inflammation in mice mcao model .", "although in our study the expression of several studied chemokines was significantly increased at the early phase of et-1 induced stroke model , no clear correlation of this expression with neurodegeneration was observed .", "that they suggest that inhibition of inflammatory cell accumulation in the brain at the early stage of stroke may lead to amelioration of ischemic neurodegeneration .", "upregulated in the ischemic brain chemokines may be a potential target for future therapies reducing inflammatory cell migration to the brain in early stroke ."]]}
{"article_id": "PMC3114585", "article_text": ["stress urinary incontinence ( sui ) is defined as the complaint of involuntary leakage on effort or exertion or on sneezing or coughing . although sui is not a life - threatening condition", "surgical therapy is employed in patients who have severe degrees of sui or those patients in whom conservative or pharmacological treatments have failed .", "sling procedures for genuine sui are today the mainstay of treatment and have been used for over a century with first procedure reported by schultze in 1888 .", "the integral theory of female urinary continence described by petros and ulmsten redefined the modern approach to anti - incontinence surgery and ushered the era of the midurethral sling .", "the concept was applied clinically by placing the sling to a more distal location beneath the urethra then as compared to the previous techniques .", "subsequently excellent results were presented using suprapubic arc system ( sparc , american medical systems inc .", "some authors have even termed midurethral sling surgery as the new gold - standard for sui . with the available midurethral slings , the trocar has to be passed blindly either from above ( suprapubically ) or below ( transvaginally ) , which increases the chances of injury to the pelvic organs and blood vessels .", "shlomo raz modified the technique of sling placement with opening the endopelvic fascia and passing the needle under controlled digital palpation , thereby decreasing the chances of injury to surrounding pelvic organs as well as significantly decreasing cost using tailor - made mesh .", "their finger - guided passing of needle was similar to the raz procedure of bladder neck suspension .", "we present our experience with the use of large pore polypropylene mesh / polypropylene - polyglactin mesh as a pubovaginal sling ( midurethral ) in the treatment of sui with the modified raz technique .", "a retrospective analysis was performed of consecutive 53 patients of pure sui who underwent midurethral slings procedure with the modified technique from june 2003 to december 2008 at our institute .", "preoperative evaluation included a history , physical examination , urine microscopic examination , and culture .", "severity of sui was defined by the number of pads used by the patients per day as mild ( < 2 ) , moderate ( 24 ) , and severe ( > 4 ) .", "patients were examined in lithotomy and standing positions to demonstrate sui on cough test and valsalva maneuver .", "four patients of our series underwent multichannel urodynamic examination for history suggestive of mixed incontinence in accordance with nice guidelines .", "all patients were counseled regarding the need of postoperative clean intermittent catheterization ( cic ) and transient voiding dysfunction .", "vaginal tissue was atrophic in four patients and they were preoperatively treated with local estrogen cream .", "twenty patients gave previous history of hysterectomy ; others had history of undergoing gynecological procedures , details of which were not available .", "none of the patients had previous history of surgery for incontinence or pelvic organ prolapsed .", "eight patients had grade 1 cystocele and two patients had grade 1 rectocele , which did not require treatment .", "five patients underwent simultaneous vaginal hysterectomy for gynecological indications . a polypropylene mesh ( prolene )", "/polypropylene - polyglactin mesh ( vipro ) measuring 1  10 cm ( ethicon , johnson and johnson , usa ) was fashioned from commercially available 15  7.5 cm mesh .", "this strip was soaked in antibiotic saline and stitched at all four corners to 1 - 0 polyglactin suture .", "16 fr foley catheter was placed in urinary bladder and balloon was palpated at bladder neck to estimate urethral length .", "two percent of xylocaine with adrenaline was infiltrated in the vaginal mucosa overlying the urethra .", "a 1.52 cm mid - line incision was made over the midurethra ( 1.5 cm proximal to external urethral meatus ) .", "vaginal mucosal flaps were dissected on either side extending into avascular plane , until endopelvic fascia was reached .", "blunt dissection was carried out in retropubic space by inserting a finger and the bladder was swept medially .", "two small punctures were made suprapubically and a double - pronged needle ( cook urological inc . ,", "indiana , usa ) is passed under finger control through the fascia and retropubic space .", "a marking catgut suture is placed in the center of the mesh along its length to ensure that mesh placement is equidistant .", "the polyglactin suture was pulled and tension adjusted by placing the tip of an artery forceps while positioning sling against midurethra .", "vaginal pack was removed after 24 hours of surgery and providine - iodine vaginal pressary was advised for 5 days .", "if residual urine was more than 50 ml , patient was advised cic . in sexually active patients ,", "the patients were followed up with history and clinical examination by the operating surgeon in the opd .", "social dryness was defined as patient requiring 1 pad per day and acceptable leak while carrying out routine tasks .", "five of the patients also underwent simultaneous vaginal hysterectomy for gynecological indications with no increase in morbidity .", "mean duration of follow - up was 46.1 months ( 1278 months ) [ table 1 ] .", "forty - five ( 85% ) patients were completely dry and eight ( 15% ) were socially dry at the end of the follow - up .", "four patients failed voiding trial and were advised cic , which was later discontinued after 38 weeks , when their pvr fell to < 50 ml .", "two patients complained of dull aching lower abdominal pain , which was relieved by administration of oral analgesic agents .", "five of our patients complained of mild dyspareunia , which was transient and did not require treatment [ table 2 ] .", "none of the patients reported significant voiding dysfunction , infection , nonhealing , or erosion of the sling till their last follow - up [ table 2 ] .", "over last few years , many procedures using autologous material ( rectus sheath , fascia lata ) or synthetic material ( polypropylene , mersilene ) have been reported in literature", ". continued refinements in materials were sought to identify an ideal compound for use in transvaginal slings that would be inert , sterile , noncarcinogenic , and mechanically durable .", "synthetic materials have the advantage of being readily available and do not require harvesting from another site .", "this decreases the operative time , discomfort , and potential donor site complications after the surgery .", "histological and clinical studies have shown that polypropylene is a synthetic material that is well - tolerated by the body , with little exposure of the patient to infection and vaginal or urethral erosion .", "cure rates using synthetic slings have been shown to be around 7395% . in a review of contemporary literature ,", "daneshgari et al . have found complication rates ranging from 4.3% to 75.1% for retropubic midurethral slings .", "they have quoted postoperative obstruction ranging from 1.9% to 19.7% from various series . in these patients ,", "resolution is commonly spontaneous ; the intervening period can be managed with cic or indwelling catheter .", "a recent meta - analysis showed that tvt outperformed burch colposuspension both in terms of postoperative continence rates , whereas success rate efficacies were similar after tvt and pubovaginal slings . comparing tvt to the other retropubic tension - free midurethral vaginal slings , tvt was more efficacious than both intravaginal slingplasty ( ivs ) and sparc .", "some authors have even described midurethral slings as the new gold - standard for the treatment of female sui .", "the reasons for popularity of these procedures are effectiveness , ease , and low rate of serious complications .", "a recent meta - analysis of complications of these procedures have highlighted significantly high rates of bladder perforation after tvt .", "have questioned the authenticity of reported complication rates and have described major complications and even 10 deaths as retrieved after systemic search of food and drug administration ( fda ) manufacturer and user facility device experience ( maude ) database .", "rodriguez and raz have described a mid - distal urethral sling procedure in which distal urethra is defined as anything distal to the pubourethral ligament .", "they have explained the mechanism of action of tvt procedure by providing support as well as contributing to normal function of distal urethral complex ( composed of the pubourethral ligaments , intrinsic sphincteric mechanism , extrinsic sphincter , and levator muscles located immediately distal to the pubourethral ligaments ) .", "furthermore , they have enumerated the drawbacks of current midurethral sling systems as being blind procedures with consequently higher incidences of major complications . in their modification", ", a sling is refashioned from commercially available mesh , which is cheap , does not require any special instrumentation , and is placed only within the retropubic space .", "the optimal surgical approach should minimize the risk of damage to the bladder neck , vagina , and urethra .", "this is achieved by developing retropubic space with blunt dissection and passing double - pronged needle under finger guidance .", "the procedure should augment the urethral resistance during sudden increase in the intra - abdominal pressure without preventing normal decreases in urethral pressure during voiding . placing a sling beneath", "the urethra increases the urethral compression and provides a plate for receiving the transmitted intra - abdominal pressure to the bladder neck and proximal urethra .", "the safety of the procedure has also been demonstrated in their series with no incidence of major complications .", "conventional guidelines recommended multichannel urodynamic studies in sui patients planned for surgery . with the advent of midurethral slings , which have shown to be effective in all types of urinary incontinence , studies have questioned the routine use of urodynamic parameters like valsalva leak point pressure in predicting outcome of sling surgery .", "have proposed that standard use of urodynamic investigation in the preoperative workup of midurethral slings needs to be revisited . in our series", "we had cure rate in all the 53 patients till their last follow - up .", "sling procedures that are successful at 6 months are likely to remain successful for many years . in our series , all the patients had a follow - up of more than 12 months and all these patients were doing well till their last follow - up . in a comprehensive meta - analysis of complications of midurethral slings , novara et al .", "have found the incidences of various complications  bladder / vaginal perforation 2.919.31% ; hematoma 1.453.9% , uti 3.77.5% , and cic in 77.5% of cases in various rcts and non - randomized studies .", "rodriguez et al . have described pelvic hematoma ( not requiring treatment ) in 0.33% and suprapubic pain in 0.66% of cases .", "three of their patients required cic for maximum of 3 months after which they were all spontaneously voiding . in our series , four ( 7.5% )", "patients required cic after failed voiding trial , which was discontinued after a period of 38 weeks .", "we attribute this to be the procedure - related local tissue edema / pain , which gradually subsided .", "other minor complications in our series were hematoma ( not requiring treatment ) in 1.8% , uti in 3.7% , low back pain in 3.7% , and dyspareunia in 9.4% of cases .", "major concerns about the erosion of the sling into the urinary tract have been diminished as a result of meticulous detail in placing the mesh through a small incision and tying the mesh loosely so as to avoid excessive compression and ischemia .", "we did not encounter any incidence of mesh erosion into the urethra in our series of patients .", "we faced none of the major injuries like vascular injury , bowel injury , necrotizing fasciitis , sepsis , or death , a fact emphasized by the shlomo raz group .", "one standard - sized polypropylene mesh costs around inr 1800 and polypropylene - polyglactin ( vipro ) mesh costs around inr 2300 .", "this is economically friendlier as compared to custom - made mesh systems commercially available , ranging from inr 18,000 to 25,000 , especially in developing country like india . the limitations of this study are lack of objective analysis and quantification of sui .", "outcomes were based on patients interview on opd basis by the operating surgeon and not by patient - driven questionnaires , possibly influencing the overall results . despite the limitations of the study", ", we believe this procedure is a cost - effective alternative to other minimally invasive procedures using commercially available kits and with comparable outcomes .", "polypropylene mesh as midurethral slings by modified raz technique is cost - effective , safe , and has acceptable complication rates .", "although our series is a not big enough to draw any formal conclusions , but we can safely infer that the results of this procedure are comparable to other techniques used in patients with pure sui ."], "abstract_text": ["<S> objectives : we report our experience of pure stress urinary incontinence ( sui ) treated by midurethral synthetic sling placement by modified raz technique.materials and methods : fifty - three patients with pure sui operated at our institute between june 2003 and december 2008 were included in this study . </S>", "<S> midurethral sling tape , fashioned from commercially available large pore synthetic mesh , was placed using the modified raz technique . </S>", "<S> the technique consisted of placing the tape within retropubic space using double - pronged needle , which is passed under finger control through the fascia and retropubic space . </S>", "<S> outcomes were assessed on the basis of patient 's interview in follow - up opd.results:mean age was 57.68 ( 2869 ) years . </S>", "<S> forty - five ( 85% ) patients were totally dry and eight ( 15% ) socially dry at the end of the follow - up . </S>", "<S> mean operative time was 46.5 + 11.3 minutes ( 3580 minutes ) . </S>", "<S> none of the patients required blood transfusion or had bladder / bowel injury . </S>", "<S> mean duration of hospital stay was 2.17 days ( 24 days ) . </S>", "<S> mean duration of follow - up was 46.1 months ( 1278 months).conclusions : modified raz technique is safe and cost - effective for placing midurethral sling for genuine stress incontinence . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["stress urinary incontinence ( sui ) is defined as the complaint of involuntary leakage on effort or exertion or on sneezing or coughing . although sui is not a life - threatening condition", "surgical therapy is employed in patients who have severe degrees of sui or those patients in whom conservative or pharmacological treatments have failed .", "sling procedures for genuine sui are today the mainstay of treatment and have been used for over a century with first procedure reported by schultze in 1888 .", "the integral theory of female urinary continence described by petros and ulmsten redefined the modern approach to anti - incontinence surgery and ushered the era of the midurethral sling .", "the concept was applied clinically by placing the sling to a more distal location beneath the urethra then as compared to the previous techniques .", "subsequently excellent results were presented using suprapubic arc system ( sparc , american medical systems inc .", "some authors have even termed midurethral sling surgery as the new gold - standard for sui . with the available midurethral slings , the trocar has to be passed blindly either from above ( suprapubically ) or below ( transvaginally ) , which increases the chances of injury to the pelvic organs and blood vessels .", "shlomo raz modified the technique of sling placement with opening the endopelvic fascia and passing the needle under controlled digital palpation , thereby decreasing the chances of injury to surrounding pelvic organs as well as significantly decreasing cost using tailor - made mesh .", "their finger - guided passing of needle was similar to the raz procedure of bladder neck suspension .", "we present our experience with the use of large pore polypropylene mesh / polypropylene - polyglactin mesh as a pubovaginal sling ( midurethral ) in the treatment of sui with the modified raz technique ."], ["a retrospective analysis was performed of consecutive 53 patients of pure sui who underwent midurethral slings procedure with the modified technique from june 2003 to december 2008 at our institute .", "preoperative evaluation included a history , physical examination , urine microscopic examination , and culture .", "severity of sui was defined by the number of pads used by the patients per day as mild ( < 2 ) , moderate ( 24 ) , and severe ( > 4 ) .", "patients were examined in lithotomy and standing positions to demonstrate sui on cough test and valsalva maneuver .", "four patients of our series underwent multichannel urodynamic examination for history suggestive of mixed incontinence in accordance with nice guidelines .", "all patients were counseled regarding the need of postoperative clean intermittent catheterization ( cic ) and transient voiding dysfunction .", "vaginal tissue was atrophic in four patients and they were preoperatively treated with local estrogen cream .", "twenty patients gave previous history of hysterectomy ; others had history of undergoing gynecological procedures , details of which were not available .", "none of the patients had previous history of surgery for incontinence or pelvic organ prolapsed .", "eight patients had grade 1 cystocele and two patients had grade 1 rectocele , which did not require treatment .", "five patients underwent simultaneous vaginal hysterectomy for gynecological indications . a polypropylene mesh ( prolene )", "/polypropylene - polyglactin mesh ( vipro ) measuring 1  10 cm ( ethicon , johnson and johnson , usa ) was fashioned from commercially available 15  7.5 cm mesh .", "this strip was soaked in antibiotic saline and stitched at all four corners to 1 - 0 polyglactin suture .", "16 fr foley catheter was placed in urinary bladder and balloon was palpated at bladder neck to estimate urethral length .", "two percent of xylocaine with adrenaline was infiltrated in the vaginal mucosa overlying the urethra .", "a 1.52 cm mid - line incision was made over the midurethra ( 1.5 cm proximal to external urethral meatus ) .", "vaginal mucosal flaps were dissected on either side extending into avascular plane , until endopelvic fascia was reached .", "blunt dissection was carried out in retropubic space by inserting a finger and the bladder was swept medially .", "two small punctures were made suprapubically and a double - pronged needle ( cook urological inc . ,", "indiana , usa ) is passed under finger control through the fascia and retropubic space .", "a marking catgut suture is placed in the center of the mesh along its length to ensure that mesh placement is equidistant .", "the polyglactin suture was pulled and tension adjusted by placing the tip of an artery forceps while positioning sling against midurethra .", "vaginal pack was removed after 24 hours of surgery and providine - iodine vaginal pressary was advised for 5 days .", "if residual urine was more than 50 ml , patient was advised cic . in sexually active patients ,", "the patients were followed up with history and clinical examination by the operating surgeon in the opd .", "social dryness was defined as patient requiring 1 pad per day and acceptable leak while carrying out routine tasks ."], ["five of the patients also underwent simultaneous vaginal hysterectomy for gynecological indications with no increase in morbidity .", "mean duration of follow - up was 46.1 months ( 1278 months ) [ table 1 ] .", "forty - five ( 85% ) patients were completely dry and eight ( 15% ) were socially dry at the end of the follow - up .", "four patients failed voiding trial and were advised cic , which was later discontinued after 38 weeks , when their pvr fell to < 50 ml .", "two patients complained of dull aching lower abdominal pain , which was relieved by administration of oral analgesic agents .", "five of our patients complained of mild dyspareunia , which was transient and did not require treatment [ table 2 ] .", "none of the patients reported significant voiding dysfunction , infection , nonhealing , or erosion of the sling till their last follow - up [ table 2 ] ."], ["over last few years , many procedures using autologous material ( rectus sheath , fascia lata ) or synthetic material ( polypropylene , mersilene ) have been reported in literature", ". continued refinements in materials were sought to identify an ideal compound for use in transvaginal slings that would be inert , sterile , noncarcinogenic , and mechanically durable .", "synthetic materials have the advantage of being readily available and do not require harvesting from another site .", "this decreases the operative time , discomfort , and potential donor site complications after the surgery .", "histological and clinical studies have shown that polypropylene is a synthetic material that is well - tolerated by the body , with little exposure of the patient to infection and vaginal or urethral erosion .", "cure rates using synthetic slings have been shown to be around 7395% . in a review of contemporary literature ,", "daneshgari et al . have found complication rates ranging from 4.3% to 75.1% for retropubic midurethral slings .", "they have quoted postoperative obstruction ranging from 1.9% to 19.7% from various series . in these patients ,", "resolution is commonly spontaneous ; the intervening period can be managed with cic or indwelling catheter .", "a recent meta - analysis showed that tvt outperformed burch colposuspension both in terms of postoperative continence rates , whereas success rate efficacies were similar after tvt and pubovaginal slings . comparing tvt to the other retropubic tension - free midurethral vaginal slings , tvt was more efficacious than both intravaginal slingplasty ( ivs ) and sparc .", "some authors have even described midurethral slings as the new gold - standard for the treatment of female sui .", "the reasons for popularity of these procedures are effectiveness , ease , and low rate of serious complications .", "a recent meta - analysis of complications of these procedures have highlighted significantly high rates of bladder perforation after tvt .", "have questioned the authenticity of reported complication rates and have described major complications and even 10 deaths as retrieved after systemic search of food and drug administration ( fda ) manufacturer and user facility device experience ( maude ) database .", "rodriguez and raz have described a mid - distal urethral sling procedure in which distal urethra is defined as anything distal to the pubourethral ligament .", "they have explained the mechanism of action of tvt procedure by providing support as well as contributing to normal function of distal urethral complex ( composed of the pubourethral ligaments , intrinsic sphincteric mechanism , extrinsic sphincter , and levator muscles located immediately distal to the pubourethral ligaments ) .", "furthermore , they have enumerated the drawbacks of current midurethral sling systems as being blind procedures with consequently higher incidences of major complications . in their modification", ", a sling is refashioned from commercially available mesh , which is cheap , does not require any special instrumentation , and is placed only within the retropubic space .", "the optimal surgical approach should minimize the risk of damage to the bladder neck , vagina , and urethra .", "this is achieved by developing retropubic space with blunt dissection and passing double - pronged needle under finger guidance .", "the procedure should augment the urethral resistance during sudden increase in the intra - abdominal pressure without preventing normal decreases in urethral pressure during voiding . placing a sling beneath", "the urethra increases the urethral compression and provides a plate for receiving the transmitted intra - abdominal pressure to the bladder neck and proximal urethra .", "the safety of the procedure has also been demonstrated in their series with no incidence of major complications .", "conventional guidelines recommended multichannel urodynamic studies in sui patients planned for surgery . with the advent of midurethral slings , which have shown to be effective in all types of urinary incontinence , studies have questioned the routine use of urodynamic parameters like valsalva leak point pressure in predicting outcome of sling surgery .", "have proposed that standard use of urodynamic investigation in the preoperative workup of midurethral slings needs to be revisited . in our series", "we had cure rate in all the 53 patients till their last follow - up .", "sling procedures that are successful at 6 months are likely to remain successful for many years . in our series , all the patients had a follow - up of more than 12 months and all these patients were doing well till their last follow - up . in a comprehensive meta - analysis of complications of midurethral slings , novara et al .", "have found the incidences of various complications  bladder / vaginal perforation 2.919.31% ; hematoma 1.453.9% , uti 3.77.5% , and cic in 77.5% of cases in various rcts and non - randomized studies .", "rodriguez et al . have described pelvic hematoma ( not requiring treatment ) in 0.33% and suprapubic pain in 0.66% of cases .", "three of their patients required cic for maximum of 3 months after which they were all spontaneously voiding . in our series , four ( 7.5% )", "patients required cic after failed voiding trial , which was discontinued after a period of 38 weeks .", "we attribute this to be the procedure - related local tissue edema / pain , which gradually subsided .", "other minor complications in our series were hematoma ( not requiring treatment ) in 1.8% , uti in 3.7% , low back pain in 3.7% , and dyspareunia in 9.4% of cases .", "major concerns about the erosion of the sling into the urinary tract have been diminished as a result of meticulous detail in placing the mesh through a small incision and tying the mesh loosely so as to avoid excessive compression and ischemia .", "we did not encounter any incidence of mesh erosion into the urethra in our series of patients .", "we faced none of the major injuries like vascular injury , bowel injury , necrotizing fasciitis , sepsis , or death , a fact emphasized by the shlomo raz group .", "one standard - sized polypropylene mesh costs around inr 1800 and polypropylene - polyglactin ( vipro ) mesh costs around inr 2300 .", "this is economically friendlier as compared to custom - made mesh systems commercially available , ranging from inr 18,000 to 25,000 , especially in developing country like india . the limitations of this study are lack of objective analysis and quantification of sui .", "outcomes were based on patients interview on opd basis by the operating surgeon and not by patient - driven questionnaires , possibly influencing the overall results . despite the limitations of the study", ", we believe this procedure is a cost - effective alternative to other minimally invasive procedures using commercially available kits and with comparable outcomes ."], ["polypropylene mesh as midurethral slings by modified raz technique is cost - effective , safe , and has acceptable complication rates .", "although our series is a not big enough to draw any formal conclusions , but we can safely infer that the results of this procedure are comparable to other techniques used in patients with pure sui ."]]}
{"article_id": "PMC3135062", "article_text": ["this causes misalignment between the sleep and wake propensities that are controlled by hypothalamic circadian pacemaker   and results into shift work sleep disorder ( swsd ) .", "the reported incidence of swsd in india is about 44.8% of night - shift workers and 35.8% of rotating workers .", "swsd is characterized by persistent excessive sleepiness during night work and insomnia when attempting sleep in the daytime .", "individuals with swsd have significantly higher incidence of sleepiness - related accidents , absenteeism , depression , and missed family and social activities as compared with other night - shift workers .", "it is also associated with higher incidence of ulcers , cardiovascular disease , and deficit in cognition and psychomotor performance [ 4 , 5 ] .  ", "the pharmacological management of swsd involves treatment with modafinil that has been shown to improve wakefulness and ability to sustain attention in these patients .", "however , despite the half - life of 15 hours , the wakefulness promoting effect of modafinil is found to be ill - sustained in the last one third of night shift hours .", "the lack of efficacy in the early morning hours and undue patient confidence in the drug can result into excessive sleepiness while commuting home .", "armodafinil , the chirally pure r - enantiomer of modafinil , approved by us fda in 2007 has half - life ( t1/2 = 15 hours ) three times longer than its s - enantiomer ( t1/2 = 3 hours ) .", "despite the same half lives , comparison of the equivalent ( 200  mg ) doses of modafinil and armodafinil , in humans has revealed that armodafinil sustains higher plasma concentrations 614  hrs postadministration than that of racemic modafinil with longer maintenance of wakefulness [ 810 ] .  ", "this was a randomized , comparative , double - blind , and multicentric study comparing the effects of modafinil 200  mg with armodafinil 150  mg in indian patients of swsd .", "prior approval was obtained from drug controller general of india ( dcgi ) and appropriate ethics committees .", "the study was conducted in accordance of good clinical practice guidelines ( issued by central drugs standard control organization , government of india ) and according to the declaration of helsinki .", "the trial was registered at the clinical trials registry , india ( http://www.ctri.in/ ) . after obtaining written informed consent , patients of either sex , aged between 18 and 60 years , attending outpatient clinics of the authors , and suffering from excessive sleepiness associated with swsd ( assessed basis patient ' primary complaint and using the diagnostic criteria adopted from international classification for sleep disorders   ( table 1 ) ) were enrolled .  ", "patients were working at least five night shifts every month for 12 hours or less , with 6 hours or more working between 10  p.m. and 8  a.m. and at least three shifts occurring consecutively .  ", "the major exclusion criteria were patients with significant liver or kidney or heart diseases , patients with clinically significant , uncontrolled psychiatric or medical condition , patients with known history of hypersensitivity to formulation , patients operating an automobile or hazardous machinery , caffeine consumption averaging more than 600  mg / day within 1 week of baseline , use of other concomitant medications which inhibit , induce , or are metabolized by cyp450 , patients using sedative or cns acting drugs or medication liable to affect outcome of the study ( e.g. , antihistamines , selective serotonin reuptake inhibitors , tricyclic antidepressants , lithium , anti - psychotics , anticonvulsants , monoamine oxidase inhibitor , benzodiazepines , psychostimulants , and anticoagulants ) , pregnant and lactating mothers , females of reproductive age and expecting   pregnancy or using steroidal contraception , and patients with alcohol or drug abuse .", "this was a multi - centric , randomized , comparative , and double - blind parallel group , clinical trial conducted over 18 sites across india .", "1 ratio for test and reference products online at http://www.randomization.com/. patients received orally either armodafinil 150  mg tablet ( emcure pharmaceuticals ltd . , india ) or modafinil 200  mg tablet ( from commercial source ) one hour prior to start of every night shift for 12 weeks .", "the test formulation was earlier found to be bioequivalent to the us fda - approved formulation of armodafinil , in 26 healthy indian volunteers .", "the tablets of armodafinil and modafinil were identical in shape , size , and color and were dispensed in coded , identical , and opaque packs to conceal identity and maintain blinding .", "patients were evaluated for sleepiness score based on stanford sleepiness scale ( sss ) at baseline , 4 weeks , 8 weeks , and 12 weeks .", "all the assessments were done in the morning hours at the end of three consecutive night shifts .", "the primary efficacy endpoint was proportion of patients showing at least 2 grades of improvement ( responder ) based on sss in both groups .", "the other efficacy variables included improvement in mean sss grades compared to baseline , compliance to therapy , and patients ' as well as physicians ' global assessment for efficacy .", "global assessment of efficacy was performed using the following grades : ( i ) excellent = reduction of > 75% of symptoms , ( ii ) good = reduction of 5175% of symptoms , ( iii ) fair = reduction of 2650% of symptoms , and ( iv ) poor = no improvement or reduction in < 25% of symptoms .  ", "patients ' compliance to the therapy was calculated in percentage by using following formula : ( number of tablets actually taken 100)/number of tablets supposed to be taken .", "a general and detailed systemic examination was performed for all patients during each study visit .", "blood samples were collected at baseline and at the end of the study for complete hemograms , liver function tests , renal function tests , lipid profile , and fasting blood glucose levels .", "electrocardiograms were performed for all patients at baseline and at the end of the study .", "tolerability was assessed by recording patients ' global assessment about the tolerability of the drug and percent of the patients experiencing any drug - related adverse events .", "the global assessment of tolerability was performed using following grades : ( i ) excellent = no adverse drug reaction , ( ii ) good = mild adverse drug reaction but no interference with normal lifestyle , ( iii ) fair = mild adverse drug reaction which interference with normal lifestyle .", "however , benefits of drug therapy outweigh the inconvenience , ( iv ) poor = drug withdrawn .", "prestudy calculations showed that a sample size of 100 in each group would have 80% power to detect a difference of at least 19% in responder rate with a significance level ( alpha ) of.05 ( two tailed ) .  ", "was done by comparing the proportion of patients showing excellent and good response against proportion of patients showing fair and poor response .", "for all statistical tests , a p value of less than or equal to 0.05 was considered as significant , after correction for any multiple comparisons .", "two hundred and eleven patients of swsd were recruited with 105 subjects in armodafinil group and 106 subjects in the modafinil group ( figure 1 ) .", "both modafinil and armodafinil significantly improved sleepiness grades as compared to baseline ( p < .0001 ) ( figure 2 ) .", "responder rates with armodafinil ( 72.12% ) and modafinil ( 74.29% ) were comparable ( p = .76 ) . at the end of therapy ,", "compliance in both modafinil group ( 99.31%  3.06% ) and armodafinil group ( 99.13%  2.35% ) was found to be good and comparable ( p = .63 ) indicating adequate patient adherence to therapy .", "both physicians ' and patients ' assessment of efficacy was found to be comparable between armodafinil and modafinil group ( figure 3 ) .", "the intention - to - treat analysis showed that the adverse event incidences in modafinil ( 40.57% ) and armodafinil ( 42.87% ) groups were similar ( p = .78 ) .  ", "the adverse effect profile of both drugs was found to be similar with headache , nausea , and dry mouth being the common adverse effects ( table 3 ) .  ", "no incidences of accidents or absenteeism from work were noted during the study period as assessed from patient history .  ", "physicians ' and patients ' assessment of tolerability was found to be comparable between armodafinil and modafinil group ( figure 4 ) . the baseline and after - therapy biochemical values were within normal range and similar between two groups , except that there was slight increase in mean sgpt in both armodafinil and modafinil groups as compared to baseline ( p = .008 and  .0007 ) without inter - group significance and mean blood urea value in armodafinil group increased ( p = .002 ) compared to baseline . however , the increased values were within normal limits . in both groups ,", "electrocardiograms were within normal at baseline and after completion of therapy in all patients .  ", "the adverse events that led to discontinuation were palpitation , anxiety , hypertension , depression , nervousness , and depressed mood in a patient receiving armodafinil and vomiting along with dizziness in another patient receiving modafinil .", "the present study confirms the efficacy of armodafinil 150  mg in patients of swsd .", "the efficacy of armodafinil was found to be comparable to 200  mg of modafinil in maintaining wakefulness .", "both modafinil and armodafinil caused a slight increase in liver enzymes , and armodafinil caused a slight increase in blood urea nitrogen .", "this was not of clinical significance as the increased values were within normal laboratory limits .", "armodafinil 150  mg was comparable to modafinil 200  mg , which indicates that armodafinil is 1.33 time more potent than racemic modafinil .  ", "the use of r - enantiomer of modafinil avoids unnecessary use of s - isomer and exerts less metabolic load on the body .   in previous studies , 200  mg of armodafinil", "was shown to provide more sustained plasma concentrations late in the day as compared to 200  mg of modafinil and monophasic plasma elimination kinetics as compared biphasic for modafinil .", "we chose the 150  mg dose of armodafinil , as this was the approved dosage for the present indication .", "our study demonstrated no difference in the efficacy of 150  mg of armodafinil over 200  mg of modafinil .  ", "the comparative efficacy of 200  mg of armodafinil with modafinil in swsd has not yet been assessed .  ", "a limitation of the present study is that the assessment of sleep latency and polysomnography throughout the nightshift could not be done due to unavailability of patients and investigators .", "the study did not demonstrate any difference in efficacy and safety between armodafinil 150  mg   and   modafinil 200  mg , and both drugs were comparable ."], "abstract_text": ["<S> \n aim . to compare the efficacy and safety of armodafinil , the r - enantiomer of modafinil , with modafinil in patients of shift work sleep disorder ( swsd ) . </S>", "<S> material and methods . </S>", "<S> this was a 12-week , randomized , comparative , double - blind , multicentric , parallel - group study in 211 patients of swsd , receiving armodafinil ( 150  mg ) or modafinil ( 200  mg ) one hour prior to the night shift . </S>", "<S> outcome measures . </S>", "<S> efficacy was assessed by change in stanford sleepiness score ( sss ) by at least 2 grades ( responder ) and global assessment for efficacy . </S>", "<S> safety was assessed by incidence of adverse events , change in laboratory parameters , ecg , and global assessment of tolerability . </S>", "<S> results . </S>", "<S> both modafinil and armodafinil significantly improved sleepiness mean grades as compared to baseline ( p < .0001 ) . </S>", "<S> responder rates with armodafinil ( 72.12% ) and modafinil ( 74.29% ) were comparable ( p = .76 ) . </S>", "<S> adverse event incidences were comparable . conclusion . </S>", "<S> armodafinil was found to be safe and effective in the treatment of swsd in indian patients . </S>", "<S> the study did not demonstrate any difference in efficacy and safety of armodafinil 150 mg and modafinil 200  mg . </S>"], "labels": null, "section_names": ["1. Introduction ", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion", " Disclosure"], "sections": [["this causes misalignment between the sleep and wake propensities that are controlled by hypothalamic circadian pacemaker   and results into shift work sleep disorder ( swsd ) .", "the reported incidence of swsd in india is about 44.8% of night - shift workers and 35.8% of rotating workers .", "swsd is characterized by persistent excessive sleepiness during night work and insomnia when attempting sleep in the daytime .", "individuals with swsd have significantly higher incidence of sleepiness - related accidents , absenteeism , depression , and missed family and social activities as compared with other night - shift workers .", "it is also associated with higher incidence of ulcers , cardiovascular disease , and deficit in cognition and psychomotor performance [ 4 , 5 ] .  ", "the pharmacological management of swsd involves treatment with modafinil that has been shown to improve wakefulness and ability to sustain attention in these patients .", "however , despite the half - life of 15 hours , the wakefulness promoting effect of modafinil is found to be ill - sustained in the last one third of night shift hours .", "the lack of efficacy in the early morning hours and undue patient confidence in the drug can result into excessive sleepiness while commuting home .", "armodafinil , the chirally pure r - enantiomer of modafinil , approved by us fda in 2007 has half - life ( t1/2 = 15 hours ) three times longer than its s - enantiomer ( t1/2 = 3 hours ) .", "despite the same half lives , comparison of the equivalent ( 200  mg ) doses of modafinil and armodafinil , in humans has revealed that armodafinil sustains higher plasma concentrations 614  hrs postadministration than that of racemic modafinil with longer maintenance of wakefulness [ 810 ] .  ", "this was a randomized , comparative , double - blind , and multicentric study comparing the effects of modafinil 200  mg with armodafinil 150  mg in indian patients of swsd ."], ["prior approval was obtained from drug controller general of india ( dcgi ) and appropriate ethics committees .", "the study was conducted in accordance of good clinical practice guidelines ( issued by central drugs standard control organization , government of india ) and according to the declaration of helsinki .", "the trial was registered at the clinical trials registry , india ( http://www.ctri.in/ ) . after obtaining written informed consent , patients of either sex , aged between 18 and 60 years , attending outpatient clinics of the authors , and suffering from excessive sleepiness associated with swsd ( assessed basis patient ' primary complaint and using the diagnostic criteria adopted from international classification for sleep disorders   ( table 1 ) ) were enrolled .  ", "patients were working at least five night shifts every month for 12 hours or less , with 6 hours or more working between 10  p.m. and 8  a.m. and at least three shifts occurring consecutively .  ", "the major exclusion criteria were patients with significant liver or kidney or heart diseases , patients with clinically significant , uncontrolled psychiatric or medical condition , patients with known history of hypersensitivity to formulation , patients operating an automobile or hazardous machinery , caffeine consumption averaging more than 600  mg / day within 1 week of baseline , use of other concomitant medications which inhibit , induce , or are metabolized by cyp450 , patients using sedative or cns acting drugs or medication liable to affect outcome of the study ( e.g. , antihistamines , selective serotonin reuptake inhibitors , tricyclic antidepressants , lithium , anti - psychotics , anticonvulsants , monoamine oxidase inhibitor , benzodiazepines , psychostimulants , and anticoagulants ) , pregnant and lactating mothers , females of reproductive age and expecting   pregnancy or using steroidal contraception , and patients with alcohol or drug abuse .", "this was a multi - centric , randomized , comparative , and double - blind parallel group , clinical trial conducted over 18 sites across india .", "1 ratio for test and reference products online at http://www.randomization.com/. patients received orally either armodafinil 150  mg tablet ( emcure pharmaceuticals ltd . , india ) or modafinil 200  mg tablet ( from commercial source ) one hour prior to start of every night shift for 12 weeks .", "the test formulation was earlier found to be bioequivalent to the us fda - approved formulation of armodafinil , in 26 healthy indian volunteers .", "the tablets of armodafinil and modafinil were identical in shape , size , and color and were dispensed in coded , identical , and opaque packs to conceal identity and maintain blinding .", "patients were evaluated for sleepiness score based on stanford sleepiness scale ( sss ) at baseline , 4 weeks , 8 weeks , and 12 weeks .", "all the assessments were done in the morning hours at the end of three consecutive night shifts .", "the primary efficacy endpoint was proportion of patients showing at least 2 grades of improvement ( responder ) based on sss in both groups .", "the other efficacy variables included improvement in mean sss grades compared to baseline , compliance to therapy , and patients ' as well as physicians ' global assessment for efficacy .", "global assessment of efficacy was performed using the following grades : ( i ) excellent = reduction of > 75% of symptoms , ( ii ) good = reduction of 5175% of symptoms , ( iii ) fair = reduction of 2650% of symptoms , and ( iv ) poor = no improvement or reduction in < 25% of symptoms .  ", "patients ' compliance to the therapy was calculated in percentage by using following formula : ( number of tablets actually taken 100)/number of tablets supposed to be taken .", "a general and detailed systemic examination was performed for all patients during each study visit .", "blood samples were collected at baseline and at the end of the study for complete hemograms , liver function tests , renal function tests , lipid profile , and fasting blood glucose levels .", "electrocardiograms were performed for all patients at baseline and at the end of the study .", "tolerability was assessed by recording patients ' global assessment about the tolerability of the drug and percent of the patients experiencing any drug - related adverse events .", "the global assessment of tolerability was performed using following grades : ( i ) excellent = no adverse drug reaction , ( ii ) good = mild adverse drug reaction but no interference with normal lifestyle , ( iii ) fair = mild adverse drug reaction which interference with normal lifestyle .", "however , benefits of drug therapy outweigh the inconvenience , ( iv ) poor = drug withdrawn .", "prestudy calculations showed that a sample size of 100 in each group would have 80% power to detect a difference of at least 19% in responder rate with a significance level ( alpha ) of.05 ( two tailed ) .  ", "was done by comparing the proportion of patients showing excellent and good response against proportion of patients showing fair and poor response .", "for all statistical tests , a p value of less than or equal to 0.05 was considered as significant , after correction for any multiple comparisons ."], ["two hundred and eleven patients of swsd were recruited with 105 subjects in armodafinil group and 106 subjects in the modafinil group ( figure 1 ) .", "both modafinil and armodafinil significantly improved sleepiness grades as compared to baseline ( p < .0001 ) ( figure 2 ) .", "responder rates with armodafinil ( 72.12% ) and modafinil ( 74.29% ) were comparable ( p = .76 ) . at the end of therapy ,", "compliance in both modafinil group ( 99.31%  3.06% ) and armodafinil group ( 99.13%  2.35% ) was found to be good and comparable ( p = .63 ) indicating adequate patient adherence to therapy .", "both physicians ' and patients ' assessment of efficacy was found to be comparable between armodafinil and modafinil group ( figure 3 ) .", "the intention - to - treat analysis showed that the adverse event incidences in modafinil ( 40.57% ) and armodafinil ( 42.87% ) groups were similar ( p = .78 ) .  ", "the adverse effect profile of both drugs was found to be similar with headache , nausea , and dry mouth being the common adverse effects ( table 3 ) .  ", "no incidences of accidents or absenteeism from work were noted during the study period as assessed from patient history .  ", "physicians ' and patients ' assessment of tolerability was found to be comparable between armodafinil and modafinil group ( figure 4 ) . the baseline and after - therapy biochemical values were within normal range and similar between two groups , except that there was slight increase in mean sgpt in both armodafinil and modafinil groups as compared to baseline ( p = .008 and  .0007 ) without inter - group significance and mean blood urea value in armodafinil group increased ( p = .002 ) compared to baseline . however , the increased values were within normal limits . in both groups ,", "electrocardiograms were within normal at baseline and after completion of therapy in all patients .  ", "the adverse events that led to discontinuation were palpitation , anxiety , hypertension , depression , nervousness , and depressed mood in a patient receiving armodafinil and vomiting along with dizziness in another patient receiving modafinil ."], ["the present study confirms the efficacy of armodafinil 150  mg in patients of swsd .", "the efficacy of armodafinil was found to be comparable to 200  mg of modafinil in maintaining wakefulness .", "both modafinil and armodafinil caused a slight increase in liver enzymes , and armodafinil caused a slight increase in blood urea nitrogen .", "this was not of clinical significance as the increased values were within normal laboratory limits .", "armodafinil 150  mg was comparable to modafinil 200  mg , which indicates that armodafinil is 1.33 time more potent than racemic modafinil .  ", "the use of r - enantiomer of modafinil avoids unnecessary use of s - isomer and exerts less metabolic load on the body .   in previous studies , 200  mg of armodafinil", "was shown to provide more sustained plasma concentrations late in the day as compared to 200  mg of modafinil and monophasic plasma elimination kinetics as compared biphasic for modafinil .", "we chose the 150  mg dose of armodafinil , as this was the approved dosage for the present indication .", "our study demonstrated no difference in the efficacy of 150  mg of armodafinil over 200  mg of modafinil .  ", "the comparative efficacy of 200  mg of armodafinil with modafinil in swsd has not yet been assessed .  ", "a limitation of the present study is that the assessment of sleep latency and polysomnography throughout the nightshift could not be done due to unavailability of patients and investigators ."], ["the study did not demonstrate any difference in efficacy and safety between armodafinil 150  mg   and   modafinil 200  mg , and both drugs were comparable ."], [""]]}
{"article_id": "PMC4439680", "article_text": ["lignocaine ( n - diethylaminoacetyl-2,6-xylidide ) was first synthesized by lofgen a swedish chemist in 1943 and was first introduced into clinical use in 1948 .", "lignocaine hydrochloride ( c14h22n2o.hcl ) is most soluble in water and so this is most commonly used injectable solution as local anesthetic agents .", "the efficacy profile of lidocaine as a local anesthetic is characterized by a rapid onset of action and intermediate duration of efficacy .", "lidocaine or lignocaine along with adrenaline has the advantage of a rapid onset of action .", "epinephrine ( adrenaline ) vasoconstricts arteries , reducing bleeding and also delays the resorption of lidocaine , almost doubling the duration of anesthesia . for surface anesthesia ,", "several available formulations can be used , e.g. for endoscopies , before intubations , etc .", "topical lidocaine has been shown in some patients to relieve the pain of postherpetic neuralgia ( a complication of shingles ) , though not enough study evidence exists to recommend it as a first - line treatment .", "although not completely curing the disorder , it has been shown to reduce the effects by around two - thirds .", "lidocaine is also the most important class-1b antiarrhythmic drug ; it is used intravenously for the treatment of ventricular arrhythmias ( for acute myocardial infarction , digoxin poisoning , cardioversion , or cardiac catheterization ) if amiodarone is not available or contraindicated .", "lidocaine should be given for this indication after defibrillation , cardiopulmonary resuscitation , and vasopressors have been initiated .", "a routine prophylactic administration is no longer recommended for acute cardiac infarction ; the overall benefit of this measure is not convincing .", "the onset of action of lidocaine is about 4590 s and its duration is 1020 min .", "it is about 95% metabolized ( dealkylated ) in the liver mainly by cyp3a4 to the pharmacologically active metabolites monoethylglycinexylidide ( megx ) and then subsequently to the inactive glycine xylidide .", "megx has a longer half - life than lidocaine but also is a less potent sodium channel blocker .", "the elimination half - life of lidocaine is biphasic and around 90120 min in most patients .", "this may be prolonged in patients with hepatic impairment ( average 343 min ) or congestive heart failure ( average 136 min ) .", "lidocaine is excreted in the urine ( 90% as metabolites and 10% as unchanged drug ) .", "bupivacaine ( 1-butyl-2 , 6- pipecoloxylidide ) was first synthesized in 1957 by ekenstam , a scandinavian chemist , and was first introduced into clinical use in 1963 .", "it is the longer side chain with four methylene groups on the piperidine ring that is responsible for the different properties of bupivacaine when compared to lignocaine .", "it has a molecular weight of 288.4 and an empirical formula of c18h28n2o and exists as white crystalline base with a melting point of 251258c .", "the base is not soluble in water , but the acid salt , bupivacaine hydrochloride ( c18h28n2o.hcl ) is slightly soluble .", "the rate of systemic absorption of bupivacaine and other local anesthetics is dependent upon the dose and concentration of drug administered , the route of administration , the vascularity of the administration site , and the presence or absence of epinephrine in the preparation .", "duration of action ( route and dose - dependent ) : 29 h. half - life in neonates is 8.1 h and in adults is 2.7 h. time to peak plasma concentration ( for peripheral , epidural or caudal block ) is 3045 min .", "the rate of systemic absorption of local anesthetics is dependent upon the total dose and concentration of drug administered , the route of administration , the vascularity of the administration site , and the presence or absence of epinephrine in the anesthetic solution .", "a dilute concentration of epinephrine ( 1:200,000 or 5 mcg / ml ) usually reduces the rate of absorption and peak plasma concentration of bupivacaine , permitting the use of moderately larger total doses and sometimes prolonging the duration of action .", "the onset of action with bupivacaine is rapid and anesthesia is long lasting . the duration of anesthesia is significantly longer with bupivacaine than with any other commonly used local anesthetic .", "it has also been noted that there is a period of analgesia that persists after the return of sensation , during which time the need for strong analgesics is reduced .", "local anesthetics are bound to plasma proteins in varying degrees . in general , the lower the plasma concentration of drug the higher the percentage of drug bound to plasma proteins .", "the rate and degree of diffusion are governed by ( 1 ) the degree of plasma protein binding , ( 2 ) the degree of ionization , and ( 3 ) the degree of lipid solubility", ". fetal / maternal ratios of local anesthetics appear to be inversely related to the degree of plasma protein binding because only the free , unbound drug is available for placental transfer .", "bupivacaine with a high protein binding capacity ( 95% ) has a low fetal / maternal ratio ( 0.20.4 ) . depending upon the route of administration ,", "local anesthetics are distributed to some extent to all body tissues , with high concentrations found in highly perfused organs such as the liver , lungs , heart , and brain .", "pharmacokinetic studies on the plasma profile of bupivacaine after direct intravenous injection suggest a three - compartment open model .", "the second compartment represents the equilibration of the drug throughout the highly perfused organs such as the brain , myocardium , lungs , kidneys , and liver .", "the third compartment represents an equilibration of the drug with poorly perfused tissues , such as muscle and fat .", "the elimination of drug from tissue distribution depends largely upon the ability of binding sites in the circulation to carry it to the liver where it is metabolized .", "after injection of bupivacaine hydrochloride for caudal , epidural , or peripheral nerve block in man , peak levels of bupivacaine in the blood are reached in 3045 min , followed by a decline to insignificant levels during the next 36 h. various pharmacokinetic parameters of the local anesthetics can be significantly altered by the presence of hepatic or renal disease , addition of epinephrine , factors affecting urinary ph , renal blood flow , the route of drug administration , and the age of the patient .", "the half - life of bupivacaine in adults is 2.7 h and in neonates 8.1 h. in clinical studies , elderly patients reached the maximal spread of analgesia and maximal motor blockade more rapidly than younger patients .", "amide - type local anesthetics such as bupivacaine are metabolized primarily in the liver via conjugation with glucuronic acid .", "patients with hepatic disease , especially those with severe hepatic disease , may be more susceptible to the potential toxicities of the amide - type local anesthetics .", "when administered in recommended doses and concentrations , bupivacaine hydrochloride does not ordinarily produce irritation or tissue damage and does not cause methemoglobinemia .", "local anesthetics block the generation and the conduction of nerve impulses , presumably by increasing the threshold for electrical excitation in the nerve , by slowing the propagation of the nerve impulse , and by reducing the rate of rise of the action potential . in general ,", "the progression of anesthesia is related to the diameter , myelination , and conduction velocity of affected nerve fibers .", "clinically , the order of loss of nerve function is as follows : ( 1 ) pain , ( 2 ) temperature , ( 3 ) touch , ( 4 ) proprioception , and ( 5 ) skeletal muscle tone .", "systemic absorption of local anesthetics produces effects on the cardiovascular and central nervous systems ( cns ) . at blood concentrations achieved with normal therapeutic doses , changes in cardiac conduction ,", "however , toxic blood concentrations depress cardiac conduction and excitability , which may lead to atrioventricular block , ventricular arrhythmias , and cardiac arrest , sometimes resulting in fatalities . in addition", ", myocardial contractility is depressed , and peripheral vasodilation occurs , leading to decreased cardiac output and arterial blood pressure .", "recent clinical reports and animal research suggest that these cardiovascular changes are more likely to occur after unintended intravascular injection of bupivacaine", "apparent central stimulation is manifested as restlessness , tremors and shivering progressing to convulsions , followed by depression and coma progressing ultimately to respiratory arrest . however , the local anesthetics have a primary depressant effect on the medulla and on higher centers", "in most of the studies 0.5% bupivacaine with adrenaline 1:200,000 and 2% lignocaine with adrenaline 1:200,000 were used as local anesthetic agents for third molar surgery as they are roughly equipotent .", "studies have proved that bupivacaine is superior to lidocaine plus diflunisal in controlling postoperative pain after lower third molar surgery .", "surprisingly there have been records of patients with no or mild pain at 8 h after the bupivacaine procedure was almost twice as many as after the lidocaine procedure , but is explained by the reason for preference being shorter duration of the anesthetic agent . in an australian study , most of the patients preferred the long - acting anesthetic agent bupivacaine .", "many authors suggest that it seems reasonable that the combination of a long - acting local anesthetic with a weak analgesic is more efficient in relieving pain than the combination of a rather short - acting local anesthetic and a weak analgesic .", "bupivacaine is considered to have a therapeutic ratio of 2:0 while lignocaine in combination with adrenaline has a therapeutic ratio of 2:3 .", "however , it has shown that the injection route alters the relative toxicity of local anesthetics . in a study by de jong and bonin in 1980 they found out that the bupivacaine has a greater therapeutic ratio than lignocaine when used for surgical removal of impacted third molars .", "studies have proved that long - acting bupivacaine can be safely administered for surgical removal of lower third molar and it does have a long period of nalgesia postoperatively compared to lidocaine , but the cardio depressant property of bupivacaine should be kept in mind and should be administered judicially .", "even studies have also shown that bupivacaine combined with methylprednisolone reduced the postoperative pain and swelling compared with the use of lidocaine and placebo , lidocaine and methylprednisolone , or bupivacaine and placebo .", "right now clinical trials are going on bupivacaine versus lidocaine anesthesia under university of british columbia 2014 with the idea that the longer duration of anesthesia offered by bupivacaine when administered preoperatively in elective outpatient hand surgeries , will offer more effective postoperative pain control compared to using lidocaine only .", "it has been found that both bupivacaine and lignocaine have their merits and demerits but beyond any doubt it has been proven by the clinical trials that bupivacaine provides better and prolonged analgesia and anesthesia post operatively during minor surgical procedures done at chair side along with surgical removal of impacted third molars .", "hence , bupivacaine can be regularly used as the anaesthetic solution along with adrenaline 1:200,000 for surgical removal of impacted third molars provided care being taken regarding the dosage and the cardiodepressant property of bupivacaine . right now , further studies are going on ."], "abstract_text": ["<S> one of the most important goal in minor surgical procedures is to achieve proper and sufficient anesthesia and analgesia preoperatively , intraoperatively and in the immediate postoperative period . </S>", "<S> several local anesthetic agents have been cited in the literature and studied . </S>", "<S> bupivacaine is one of the most common long - acting anesthetic agents being used for surgical removal of impacted third molars . </S>", "<S> lignocaine is one of the commonest short - acting anesthetic agents being used for the same procedure . in this review article , </S>", "<S> the analgesic and anesthetic abilities of the bupivacaine versus lignocaine have been reviewed while surgical removal of impacted third molars . </S>"], "labels": null, "section_names": ["Literature Review", "Discussion", "Conclusion"], "sections": [["lignocaine ( n - diethylaminoacetyl-2,6-xylidide ) was first synthesized by lofgen a swedish chemist in 1943 and was first introduced into clinical use in 1948 .", "lignocaine hydrochloride ( c14h22n2o.hcl ) is most soluble in water and so this is most commonly used injectable solution as local anesthetic agents .", "the efficacy profile of lidocaine as a local anesthetic is characterized by a rapid onset of action and intermediate duration of efficacy .", "lidocaine or lignocaine along with adrenaline has the advantage of a rapid onset of action .", "epinephrine ( adrenaline ) vasoconstricts arteries , reducing bleeding and also delays the resorption of lidocaine , almost doubling the duration of anesthesia . for surface anesthesia ,", "several available formulations can be used , e.g. for endoscopies , before intubations , etc .", "topical lidocaine has been shown in some patients to relieve the pain of postherpetic neuralgia ( a complication of shingles ) , though not enough study evidence exists to recommend it as a first - line treatment .", "although not completely curing the disorder , it has been shown to reduce the effects by around two - thirds .", "lidocaine is also the most important class-1b antiarrhythmic drug ; it is used intravenously for the treatment of ventricular arrhythmias ( for acute myocardial infarction , digoxin poisoning , cardioversion , or cardiac catheterization ) if amiodarone is not available or contraindicated .", "lidocaine should be given for this indication after defibrillation , cardiopulmonary resuscitation , and vasopressors have been initiated .", "a routine prophylactic administration is no longer recommended for acute cardiac infarction ; the overall benefit of this measure is not convincing .", "the onset of action of lidocaine is about 4590 s and its duration is 1020 min .", "it is about 95% metabolized ( dealkylated ) in the liver mainly by cyp3a4 to the pharmacologically active metabolites monoethylglycinexylidide ( megx ) and then subsequently to the inactive glycine xylidide .", "megx has a longer half - life than lidocaine but also is a less potent sodium channel blocker .", "the elimination half - life of lidocaine is biphasic and around 90120 min in most patients .", "this may be prolonged in patients with hepatic impairment ( average 343 min ) or congestive heart failure ( average 136 min ) .", "lidocaine is excreted in the urine ( 90% as metabolites and 10% as unchanged drug ) .", "bupivacaine ( 1-butyl-2 , 6- pipecoloxylidide ) was first synthesized in 1957 by ekenstam , a scandinavian chemist , and was first introduced into clinical use in 1963 .", "it is the longer side chain with four methylene groups on the piperidine ring that is responsible for the different properties of bupivacaine when compared to lignocaine .", "it has a molecular weight of 288.4 and an empirical formula of c18h28n2o and exists as white crystalline base with a melting point of 251258c .", "the base is not soluble in water , but the acid salt , bupivacaine hydrochloride ( c18h28n2o.hcl ) is slightly soluble .", "the rate of systemic absorption of bupivacaine and other local anesthetics is dependent upon the dose and concentration of drug administered , the route of administration , the vascularity of the administration site , and the presence or absence of epinephrine in the preparation .", "duration of action ( route and dose - dependent ) : 29 h. half - life in neonates is 8.1 h and in adults is 2.7 h. time to peak plasma concentration ( for peripheral , epidural or caudal block ) is 3045 min .", "the rate of systemic absorption of local anesthetics is dependent upon the total dose and concentration of drug administered , the route of administration , the vascularity of the administration site , and the presence or absence of epinephrine in the anesthetic solution .", "a dilute concentration of epinephrine ( 1:200,000 or 5 mcg / ml ) usually reduces the rate of absorption and peak plasma concentration of bupivacaine , permitting the use of moderately larger total doses and sometimes prolonging the duration of action .", "the onset of action with bupivacaine is rapid and anesthesia is long lasting . the duration of anesthesia is significantly longer with bupivacaine than with any other commonly used local anesthetic .", "it has also been noted that there is a period of analgesia that persists after the return of sensation , during which time the need for strong analgesics is reduced .", "local anesthetics are bound to plasma proteins in varying degrees . in general , the lower the plasma concentration of drug the higher the percentage of drug bound to plasma proteins .", "the rate and degree of diffusion are governed by ( 1 ) the degree of plasma protein binding , ( 2 ) the degree of ionization , and ( 3 ) the degree of lipid solubility", ". fetal / maternal ratios of local anesthetics appear to be inversely related to the degree of plasma protein binding because only the free , unbound drug is available for placental transfer .", "bupivacaine with a high protein binding capacity ( 95% ) has a low fetal / maternal ratio ( 0.20.4 ) . depending upon the route of administration ,", "local anesthetics are distributed to some extent to all body tissues , with high concentrations found in highly perfused organs such as the liver , lungs , heart , and brain .", "pharmacokinetic studies on the plasma profile of bupivacaine after direct intravenous injection suggest a three - compartment open model .", "the second compartment represents the equilibration of the drug throughout the highly perfused organs such as the brain , myocardium , lungs , kidneys , and liver .", "the third compartment represents an equilibration of the drug with poorly perfused tissues , such as muscle and fat .", "the elimination of drug from tissue distribution depends largely upon the ability of binding sites in the circulation to carry it to the liver where it is metabolized .", "after injection of bupivacaine hydrochloride for caudal , epidural , or peripheral nerve block in man , peak levels of bupivacaine in the blood are reached in 3045 min , followed by a decline to insignificant levels during the next 36 h. various pharmacokinetic parameters of the local anesthetics can be significantly altered by the presence of hepatic or renal disease , addition of epinephrine , factors affecting urinary ph , renal blood flow , the route of drug administration , and the age of the patient .", "the half - life of bupivacaine in adults is 2.7 h and in neonates 8.1 h. in clinical studies , elderly patients reached the maximal spread of analgesia and maximal motor blockade more rapidly than younger patients .", "amide - type local anesthetics such as bupivacaine are metabolized primarily in the liver via conjugation with glucuronic acid .", "patients with hepatic disease , especially those with severe hepatic disease , may be more susceptible to the potential toxicities of the amide - type local anesthetics .", "when administered in recommended doses and concentrations , bupivacaine hydrochloride does not ordinarily produce irritation or tissue damage and does not cause methemoglobinemia ."], ["local anesthetics block the generation and the conduction of nerve impulses , presumably by increasing the threshold for electrical excitation in the nerve , by slowing the propagation of the nerve impulse , and by reducing the rate of rise of the action potential . in general ,", "the progression of anesthesia is related to the diameter , myelination , and conduction velocity of affected nerve fibers .", "clinically , the order of loss of nerve function is as follows : ( 1 ) pain , ( 2 ) temperature , ( 3 ) touch , ( 4 ) proprioception , and ( 5 ) skeletal muscle tone .", "systemic absorption of local anesthetics produces effects on the cardiovascular and central nervous systems ( cns ) . at blood concentrations achieved with normal therapeutic doses , changes in cardiac conduction ,", "however , toxic blood concentrations depress cardiac conduction and excitability , which may lead to atrioventricular block , ventricular arrhythmias , and cardiac arrest , sometimes resulting in fatalities . in addition", ", myocardial contractility is depressed , and peripheral vasodilation occurs , leading to decreased cardiac output and arterial blood pressure .", "recent clinical reports and animal research suggest that these cardiovascular changes are more likely to occur after unintended intravascular injection of bupivacaine", "apparent central stimulation is manifested as restlessness , tremors and shivering progressing to convulsions , followed by depression and coma progressing ultimately to respiratory arrest . however , the local anesthetics have a primary depressant effect on the medulla and on higher centers", "in most of the studies 0.5% bupivacaine with adrenaline 1:200,000 and 2% lignocaine with adrenaline 1:200,000 were used as local anesthetic agents for third molar surgery as they are roughly equipotent .", "studies have proved that bupivacaine is superior to lidocaine plus diflunisal in controlling postoperative pain after lower third molar surgery .", "surprisingly there have been records of patients with no or mild pain at 8 h after the bupivacaine procedure was almost twice as many as after the lidocaine procedure , but is explained by the reason for preference being shorter duration of the anesthetic agent . in an australian study , most of the patients preferred the long - acting anesthetic agent bupivacaine .", "many authors suggest that it seems reasonable that the combination of a long - acting local anesthetic with a weak analgesic is more efficient in relieving pain than the combination of a rather short - acting local anesthetic and a weak analgesic .", "bupivacaine is considered to have a therapeutic ratio of 2:0 while lignocaine in combination with adrenaline has a therapeutic ratio of 2:3 .", "however , it has shown that the injection route alters the relative toxicity of local anesthetics . in a study by de jong and bonin in 1980 they found out that the bupivacaine has a greater therapeutic ratio than lignocaine when used for surgical removal of impacted third molars .", "studies have proved that long - acting bupivacaine can be safely administered for surgical removal of lower third molar and it does have a long period of nalgesia postoperatively compared to lidocaine , but the cardio depressant property of bupivacaine should be kept in mind and should be administered judicially .", "even studies have also shown that bupivacaine combined with methylprednisolone reduced the postoperative pain and swelling compared with the use of lidocaine and placebo , lidocaine and methylprednisolone , or bupivacaine and placebo .", "right now clinical trials are going on bupivacaine versus lidocaine anesthesia under university of british columbia 2014 with the idea that the longer duration of anesthesia offered by bupivacaine when administered preoperatively in elective outpatient hand surgeries , will offer more effective postoperative pain control compared to using lidocaine only ."], ["it has been found that both bupivacaine and lignocaine have their merits and demerits but beyond any doubt it has been proven by the clinical trials that bupivacaine provides better and prolonged analgesia and anesthesia post operatively during minor surgical procedures done at chair side along with surgical removal of impacted third molars .", "hence , bupivacaine can be regularly used as the anaesthetic solution along with adrenaline 1:200,000 for surgical removal of impacted third molars provided care being taken regarding the dosage and the cardiodepressant property of bupivacaine . right now , further studies are going on ."]]}
{"article_id": "PMC4840268", "article_text": ["coccidia are a diverse group of protozoan parasites within the phylum apicomplexa and include pathogenic species of significance to animal and human health .", "atoxoplasma ) , primarily parasitize a single host throughout their life cycle and include important avian pathogens ( mcdougald , 1998 , barta et  al . , 2005 , berto et al . , 2011 ) .", "heteroxenous coccidian protozoa including toxoplasma gondii , neospora caninum and sarcocystis neurona infect multiple hosts throughout their life cycle and their transmission is facilitated by predator ", "prey relationships ( sibley , 2003 ) . in the intermediate host ( often herbivorous or omnivorous ) ,", "infectious stages are formed within tissue cysts that can be ingested by the definitive host ( often a carnivorous predator ) .", "the definitive host is so called because the parasite life cycle is perpetuated through sexual multiplication in these animals .", "definitive hosts shed environmentally - resistant oocysts , ( or sporocysts from sporulated oocysts in the case of s.  neurona ) , which are infective after sporulation for intermediate hosts that ingest fecally - contaminated food or water ( sibley , 2003 ) . although the only known definitive hosts of t.  gondii , n.  caninum and s.  neurona are terrestrial animals , specifically felids , canids and opossums ( didelphis spp . ) , respectively , there is evidence that marine mammals can also become infected with these and other related coccidia ( dubey et  al . , 2000 ,", ", 2001a , dubey et  al . , 2001b , tenter et  al . , 2000 , dubey et  al . , 2003 ,", ", 2011 , gibson et  al . , 2011 , carlson - bremer et  al . ,", "the most likely modes of transmission of these pathogens to aquatic animals are via ingestion of water - borne oocysts or sporocysts originating in land - based surface runoff , or infected prey ( miller et  al . , 2002 , conrad et  al . , 2005 , massie et  al . , 2010 , gibson et  al . , 2011 ,", "one of the most serious consequences of s.  neurona and t.  gondii infection in marine mammals is fatal protozoal encephalitis which has been described most commonly in cetaceans , including southern sea otters ( enhydra lutris nereis ) , california sea lions ( zalophus californianus ) and pacific harbor seals ( phoca vitulana richardsi ) ( thomas and cole , 1996 , lapointe et  al . , 1998 , cole et  al . , 2000 , miller et  al", ", 2001a , dubey et  al . , 2003 , kreuder et  al . , 2003 , miller et  al . , 2004 , miller et  al . , 2009 ,", "miller et  al . , 2010 , gibson et  al . , 2011 ) .", "more recently , severe myositis in a hospitalized california sea lion was recognized as a new clinical syndrome associated with s.  neurona infection ( carlson - bremer et  al . , 2012b ) .", "pathogenic infections with other , less well characterized , tissue - encysting coccidia have also been documented in marine mammals in association with protozoal lymphadenitis , hepatitis , myocarditis , encephalitis and non - suppurative necrotizing meningoencephalitis ( dubey et  al . , 2003 , lapointe et  al . , 2003 ,", "california sea lions inhabit waters of the pacific coast of north america between southwestern canada and baja california ( lowry et  al . , 1992 ) . as long - lived coastal residents with large fat stores and piscivorous prey preferences that are shared with humans , sea lions have the potential to act as sentinel species , or indicators of aquatic ecosystem and human health ( bossart , 2011 ) . during postmortem examination of rescued california sea lions that died at the marine mammal center ( tmmc , sausalito , california ) , sexual and asexual stages of three novel coccidia with genetic similarity to neospora spp .", "were identified in sea lion enterocytes , and these organisms were putatively named coccidia a , b and c ( colegrove et  al . , 2011 , carlson - bremer et  al . , 2012a ) .", "subsequent fecal analysis of stranded and rehabilitated sea lions sampled between 2007 and 2009 confirmed that sea lions shed coccidia a and b , particularly as yearlings , and that shedding of coccidia a could be detected in feces for up to 22 days ( carlson - bremer et  al . , 2012a ) .", "evidence to date suggests that california sea lions act as both definitive and intermediate hosts of novel protozoa in the absence of clinical signs or pathologic evidence of disseminated infection . yet , the identification of severe protozoal hepatitis , myocarditis and encephalitis in a neonatal harbor seal infected with coccidia c is of particular concern because it indicates that sea lions may be capable of shedding coccidian protozoa that are pathogenic to harbor seals and perhaps other aquatic wildlife ( colegrove et  al . , 2011 ) . a better understanding of the biology , epidemiology , and pathogenesis of tissue - encysting coccidian organisms that parasitize marine mammals is needed to properly assess the risks and burden of protozoal disease in aquatic ecosystems such as the pacific coast of north america . as part of ongoing health surveillance in aquatic species of the pacific ocean , and accompanying studies of pathogen pollution in near - coastal california waters", ", we examined the diversity of coccidian parasites shed by hospitalized and free - ranging sea lions at coastal haul - out locations in central california .", "in addition , we characterized the phenotypes of sea lion - derived coccidian protozoa in both a mouse model of pathogenesis and in mammalian cell culture .", "staff at tmmc collected fecal samples from individual california sea lions ( csl ) stranded along the central and northern california coast between september 2010 and may 2012 as previously described ( carlson - bremer et  al . , 2012a ) .", "samples were typically collected within the first week of hospital admission , although some animals were sampled up to 3 weeks later . animals and corresponding fecal samples", "when coccidial oocysts were identified at tmmc , one or more fecal samples from an individual animal were shipped on cold packs to the university of california davis for oocyst harvesting and genetic characterization .", "age classifications for sea lions ( adult , subadult , juvenile , yearling , and pup ) were estimated as previously described ( carlson - bremer et  al . , 2012a ) . between may 2011 and october 2012 , sea lion fecal samples from free - ranging animals", "were also collected from coastal haul out sites at ao nuevo state park ( 37.108 , 122.337 ) , white rock ( 35.532 , 121.088 ) , and seal rock at point lobos ( 36.516 , 122.336 ) , placed into individual 50  ml conical vials and kept at 4  c during transport to uc davis for oocyst detection , harvesting and genetic characterization .", "coccidial oocysts were harvested from fecal samples by a double centrifugation flotation method as described by dabritz and co - authors ( dabritz et  al . , 2007 ) with some modifications .", "a detailed protocol for oocyst harvesting from sea lions is available in the supplemental information associated with this article ( see supplementary data ) .", "when quantities were sufficient , a portion of freshly - harvested oocysts were not frozen , but were reserved and sporulated for bioassay in mice and inoculation into cell cultures .", "newly - harvested oocysts were washed two times in sterile pbs , and the final pellet resuspended in 5  ml pbs , to which was added clorox bleach at a final concentration of 10% . following 20  min incubation with occasional gentle mixing , the sample was washed 3 times in 50  ml molecular biology grade water , with each centrifugation at 1000  g for 10  min .", "after final centrifugation , supernatant was carefully removed and the pellet mixed with 35  ml pbs ( higher volume for samples with higher oocyst concentration )", ". approximately 1  ml of oocyst suspension in pbs was added to 5  ml filtered seawater ( 0.22  m , acrodisc syringe filters , pall corporation ) with 250  l amphotericin b ( 250  g / ml ) in a t-25 tissue culture flask with ventilated lid .", "each flask was set on a gentle rocker at rt and visually inspected every 2 days by light microscopy for evidence of sporulation .", "sporulated oocysts were washed two times in 40  ml sterile pbs , counted , and prepared for oral inoculation into mice . for excystation and preparation for cell culture ,", "sporulated oocysts were suspended in 0.5  ml warm ( 37  c ) dulbecco 's minimum essential media ( dmem , gibco ) , supplemented with 10% ( v / v ) heat - inactivated fetal bovine serum , 2  mm l - glutamine , 100  u / ml penicillin , 100  g / ml streptomycin , 10  mm hepes and 55  m 2-mercaptethanol and transferred to 2  ml screw cap tubes containing 350  mg of 200400  mm sterile acid - washed glass beads ( thermofisher scientific ) .", "oocyst disruption and sporozoite release was accomplished through vortexing in the presence of glass beads for 2030  s. to confirm the presence of free sporozoites , a 5  l aliquot of the suspension was examined following each 10  s of vortexing .", "when sporozoites were visible , the sample was immediately transferred to monolayer cultures of embryonic monkey kidney ( ma104 ) cells ( biowhittaker , walkersville , md ) and incubated for 2  h at 37  c in 5% co2 , after which the inoculum was removed and replaced with fresh 37  c prepared dmem .", "cells were checked 3 times weekly for parasite growth alongside uninoculated ma104  cells . when zoites became visible in cell culture ,", "supernatant was collected and pelleted for dna extraction , cryopreservation , and antigen slide preparation for serological testing . during microscopic analysis of oocysts isolated from sea lion feces ,", "multiple photomicrographs were taken at 4001000 magnification using a camera ( spot rt3 scientific digital ccd camera , spot imaging solutions  , sterling heights , mi ) mounted on a zeiss axioskop epifluorescent microscope equipped with a uv emission filter set ( emitter 460/50  nm band pass filter ; chroma # 11000 v3 ) .", "micrographs were used to measure the long axis and short axis lengths of oocysts ( in unsporulated and sporulated forms ) and sporocysts ( in sporulated forms ) using spot advanced software ( spot imaging solutions , sterling heights , mi , usa ) .", "the mean and standard deviation of 1020 sporulated and unsporulated oocysts were calculated separately for each coccidian species .", "in oocyst - inoculated cell cultures , approximately 10 zoites per culture were measured at 200 magnification from photomicrographs using spot software .", "all animal experiments were conducted with the approval and oversight of the institutional animal care and use committee at the university of california , davis , which is accredited by the association for assessment and accreditation of laboratory animal care , international ( iacuc # 17501 ) .", "mouse strains were chosen based on known susceptibility to t.  gondii ( rytel and jones , 1966 , villegas et  al . , 2002 , gavrilescu and denkers , 2003 ) .", "prior to inoculation , 68 week old female mice were screened by the indirect fluorescent antibody test ( ifat , see methods below ) using a 1:40 serum dilution to ensure seronegativity to t.  gondii , n.  caninum and s.  neurona .", "approximately 1000 sporulated oocysts were inoculated into two mice of each genetic background : b6.129s7-ifng / j ( c57bl/6 background ) , cba / caj ( the jackson laboratory , bar harbor , me ) and swiss webster ( cfw ) ( charles river laboratories , wilmington , ma ) .", "of the two mice in each genetic background group , one was orally inoculated by gastric lavage and the other was subcutaneously inoculated in the interscapular region as previously described ( fritz et  al . , 2012 ) .", "control mice of each genetic background were given inocula free of oocysts consisting of clinicare canine / feline liquid diet ( abbott health care ) for gastric lavage controls or pbs for subcutaneous controls .", "mice were checked once per week for clinical signs of disease and were bled at 3 and 6 weeks post - infection for ifat serology .", "mice were humanely euthanized by co2 asphyxiation for necropsy at 5070 days post - inoculation , at which time additional blood was collected from the heart cavity for ifat serology . during necropsy ,", "portions of the tongue , heart , and brain were collected , divided into three pieces , and frozen at  80  c until dna extraction .", "a portion of the mouse brains was homogenized , trypsinized and placed over monolayer cultures of ma104  cells for coccidial parasite detection as previously described ( miller et  al . , 2001b ) .", "remaining mouse tissues were placed in 10% formalin and submitted to the california animal health and food safety laboratory ( cahfs , davis , ca ) for histological and immunohistochemical analysis .", "the following mouse tissues were fixed in 10% neutral buffered formalin and examined histologically : liver , kidney , heart , lung , spleen , subcutaneous fat , skeletal muscle , pancreas , salivary gland , uterus , lymph node , thymus , multiple sections of gastrointestinal tract and brain , gonad , trachea , adrenal gland .", "formalin - fixed tissues were trimmed , routinely processed for paraffin embedding , sectioned at 5  m , and stained with hematoxylin and eosin .", "immunohistochemical staining for the presence of t.  gondii , s. neurona and n. caninum antigen was performed as previously described ( miller et  al . , 2001b ) on selected sections of formalin - fixed , paraffin - embedded tissues .", "antigen slides for ifat analysis were prepared with parasites harvested from the supernatants of ma104 cultures containing visible zoites using previously described methods ( miller et  al . , 2001b ) .", "mouse blood was collected by venipuncture , cardiac puncture or retro - orbital bleed , allowed to clot , and centrifuged at 1000  g for 10  min .", "sea lion serum was similarly separated after collection via venipuncture of the caudal gluteal vein during hospitalization at tmmc and was shipped to uc davis for ifa testing .", "serum was serially diluted in pbs from 1:40 to 1:81,920 and screened for immunoreactivity to t.  gondii , s.  neurona and n.  caninum antigen using a fluorescein isothiocyanate ( fitc)-conjugated rabbit anti - canine igg ( jackson immunoresearch laboratories , west grove , pa ) .", "mouse sera were screened at a 1:40 dilution for immunoreactivity to antigens of the same three parasites using fitc - conjugated goat anti - mouse igg ( jackson immunoresearch laboratories ) .", "test controls consisted of previously analyzed animal sera with titers ranging from 1:12801:10,240 ( positive controls ) or titers < 1:40 ( negative controls ) .", "sea lion and mouse serum specimens that showed no reactivity at 1:40 dilution were considered test - negative .", "for the purpose of this study , sea lion sera that were reactive to t.  gondii antigen at 1:640 dilution were considered test - positive to identify sea lions likely infected with t.  gondii .", "sea lion sera that were reactive to s.  neurona antigen at 1:320 were considered test - positive as has been used previously to identify s.  neurona infection in sympatric species ( kreuder et  al . , 2005 ,", "prior to dna extraction , frozen , pelleted oocysts that had been harvested from sea lion feces were subjected to a single freeze ", "dna was extracted from oocysts and mouse tissues using the qiagen dneasy blood and tissue kit using manufacturer 's recommendation for animal tissues .", "two out of three mouse tissue sections were analyzed per tissue type per mouse , with preference for mice that demonstrated histological lesions , leading to a total of 164 individual samples , including controls , which were extracted and analyzed by pcr .", "negative controls ( water ) were included in the extraction process to monitor for environmental contamination . for molecular characterization of coccidian dna in oocysts , mouse tissues and zoite cultures , nested pcr", "was performed using pan - coccidian primers that span the internal transcribed spacer 1 ( its-1 ) region , and s.  neurona / s . ", "falcatula - specific primers ( its1500 ) that amplify 500  bp of the its1 region in those organisms ( miller et  al . , 2009 , gibson et  al . , 2011 ) .", "for characterization of s.  neurona - like dna , pcr protocols were employed to amplify microsatellites sn7 ( nested pcr ) and sn9 ( hemi - nested pcr ) , ( asmundsson and rosenthal , 2006 , rejmanek et  al . , 2010 ) and surface antigens snsag3 , snsag4 ( nested pcr ) ( rejmanek et  al . , 2010 ) and either snsag1 , snsag5 , or snsag6 which amplify using snsag1 - 5 - 6 primers ( wendte et  al . , 2010 ) .", "additional primers were used to amplify a secondary region of s.  neurona its-1 , approximately 150  bp upstream of the area amplified by its1500 primers : snits1-f , ccgggatgatgtcgtcaag and snits1-r , acagatgatgtgccccgc ( carlson - bremer , 2011 ) .", "all pcr reactions were performed using qiagen hotstar taq plus ( qiagen , valencia , ca ) according to manufacturer 's recommendations , utilizing 5  l of template dna .", "the following reaction conditions were used for all primer sets : initial denaturation at 95  c for 5  min , followed by 35 cycles at 94  c for 40  s , 58  c for 40  s ( external primers or non - nested pcr ) or 59  c for 40  s ( internal primers for nested / hemi - nested pcr ) , and 72  c for 90  s , followed by a 10  min extension at 72  c . in the case of nested or hemi - nested pcr protocols , 1  l of first round pcr product was added to the second round master mix .", "positive controls for pcr included either hammondia heydorni dna isolated from a domestic dog , neospora hughesi isolated from a horse , and/or s.  neurona strain snucd-1 ( rejmanek et  al .", "pcr products were purified using the qiaquick pcr purification kit ( qiagen ) and were sequenced by the ucdna sequencing facility ( davis , ca ) . forward and reverse sequences were assembled into contigs using geneious pro v. 5.3.4 ( drummond et  al . , 2009 ) .", "consensus sequences were aligned to reference sequences obtained from ncbi ( http://www.ncbi.nlm.nih.gov/nuccore ) using muscle or mafft ( katoh et  al . , 2002 , edgar , 2004 ) with default parameters , followed by manual editing .", "posterior sets of phylogenetic trees were generated using mrbayes ( huelsenbeck and ronquist , 2001 ) implemented in geneious pro v. 5.3.4 and run for 1,000,000 generations using the gtr  +  g nucleotide substitution model .", "trees were sampled every 1000 generations and 25% of trees were discarded from the initial burn - in period .", "pairwise genetic distance values were determined through uncorrected - p distance matrices generated in paup * ( sinaeur associates inc . ) using prepared alignments of 420  bp for its-1 .", "individual sequences were analyzed using blast ( basic local alignment search tool ) available on the ncbi website http://blast.ncbi.nlm.nih.gov/blast.cgi .", "kp999999 and kp990539 for t.  gondii m4 and ao11 , respectively ) , and snsag3 ( kr011915 for sarcocystis sp .", "groups were compared for significant differences using the two - tailed two - sample test of proportions ( stata / ic v. 11.0 , statacorp lp , college station , tx ) .", "fecal samples collected from 139 sea lions stranded in nine counties located on the california coast between 2010 and 2012 were screened upon hospitalization for the presence of coccidian oocysts . the majority of animals stranded in monterey ( n  =  48 ) , san luis obispo ( n  =  36 ) and santa cruz ( n  =  32 ) counties , followed by san mateo ( n  =  7 ) , san francisco ( n  =  5 ) , sonoma ( n  =  4 ) , marin ( n  =  4 ) , mendocino ( n  =  2 ) and humboldt ( n  =  1 ) .", "coccidian oocysts were identified in the feces of 16 of the 139 ( 11.5% ) sea lions sampled the majority of which were from monterey ( 8.3% , 4/48 ) and san luis obispo ( 13.9% , 5/36 ) counties .", "only one of the 32 stranded animals ( 3.1% ) from santa cruz county was shedding oocysts .", "table  1 describes the clinical status and results of serological testing and coccidian oocyst dna analysis for the 16 sea lions that had oocysts detected in their feces upon presentation at tmmc .", "oocysts observed in the feces of 16 stranded sea lions ( table  1 ) were 810  m in diameter .", "sporocysts were not observed by light microscopy following fecal flotation in any of the sea lions sampled .", "a similar proportion of males ( 9/89 ) and females ( 7/51 ) shed oocysts ( p  =  0.518 ) .", "yearlings most commonly shed oocysts ( 8/33 , 24.2% ) followed by subadults ( 2/16 , 12.5% ) , pups ( 2/19 , 10.5% ) , juveniles ( 3/50 , 6.0% ) and adults ( 1/21 , 4.8% ) .", "the proportion of yearlings shedding oocysts ( 8/33 ) was significantly greater than the proportion of all other age classes shedding oocysts ( 8/106 ; p  =  0.009 ) .", "1 shows the close relationship of coccidian dna sequences amplified from fecal samples of the stranded hospitalized sea lions in this study ( e.g. csl ncmmc1047 humboldt and csl9878 monterey ) to n.  caninum and dna sequences amplified from marine mammal fecal samples and tissues in previous studies ( colegrove et  al .", ", 2011 , gibson et  al . , 2011 , carlson - bremer et  al . ,", "2011 ) , coccidia a ( csl ncmmc1047 ) and b ( csl9878 ) its-1 sequences shared 79.6% and 78.8% pairwise", "nucleotide similarity , respectively , to n.  caninum and 74.1% and 71.8% similarity , respectively , to t.  gondii . based on sequence analysis of the its-1 locus from the 16 hospitalized sea lions that were shedding coccidia during this study ,", "6 ( 37.5% ) animals were confirmed to be shedding coccidia a , 6 ( 37.5% ) were shedding coccidia b , and 2 ( 12.5% ) were shedding both a and b simultaneously , indicating dual infections ( table  1 ) .", "sera from 5 ( 31.3% ) of these sea lions had antibodies to t.  gondii antigens detectable by ifat , but only one of these animals ( csl9830 ) had an antibody titer above 1:640 ( table  1 ) .", "one animal from monterey ( csl9878 ) was seroreactive to s.  neurona antigen at a 1:2560 dilution .", "no animals tested during this time period were seroreactive to n.  caninum despite the apparent genetic relatedness of coccidia a and b to neospora spp . at the its-1 locus ( fig", ".  1 ) ( colegrove et  al . , 2011 , carlson - bremer et  al . ,", "unsporulated oocysts had a round to ovoid shape and autofluoresced under uv illumination ( fig . ", "2 ) as observed for t.  gondii and other coccidian oocysts ( lindquist et  al . , 2003 ) . unsporulated coccidia a oocysts averaged 9.9    0.59  m in length and 9.5    0.69  m in width (", "n  =  20 oocysts from csl ncmmc 1047 , measured at 200 magnification ) .", "unsporulated coccidia b oocysts averaged 8.9    0.31  m in length and 8.5    0.51  m in width (", "n  =  20 oocysts from csl9878 , measured at 1000 magnification ) ( fig .  2 ) .", "coccidian oocysts a and b were previously identified in the feces of stranded california sea lions surveyed at tmmc between 2007 and 2009 ( carlson - bremer et  al . , 2012a ) . here , we summarized shedding prevalence for all sea lions sampled between 2007 and 2012 ( table  2 ) . over the six year period", ", 558 sea lions were sampled , 31 ( 5.6% ) of which were shedding coccidia a or b , in similar frequency .", "hospitalized stranded sea lions were typically diagnosed with malnutrition and trauma with no apparent clinical effects attributed to protozoal infection ( table  1 ) . between august 2011 and september 2012 ,", "california sea lion fecal samples were collected from coastal haul - out sites located at ao nuevo state reserve", "( n  =  48 ) , white rock ( n  =  94 ) and point lobos ( n  =  70 ) and the samples were screened for oocysts .", "the prevalence of oocysts identified by microscopy during isolation was 8.3% ( 4/48 ) at the ao nuevo haul - out site , 3.2% ( 3/94 ) at white rock , and 8.6% ( 6/70 ) at point lobos ( table  3 ) .", "sporocysts were not observed by light microscopy following fecal flotation in any of the free - ranging sea lion samples . dna amplification and", "sequence alignment revealed that both coccidia a and b were present in fecal samples collected at ao nuevo and point lobos , while only coccidia a was identified in samples collected at white rock ( table  3 ) .", "fecal sample 11 collected at ao nuevo state park ( ao11 ) represented a novel sequence type with 9798% pairwise similarity at the its-1 locus to previously published coccidian dna isolates in guadalupe fur seal tissues ( see neospora sp .", "the csl ao11 isolate had 92% pairwise similarity to coccidia a and b and shared even less similarity ( 87.7% ) to coccidia c at the its-1 locus .", "this sequence as well as representative sequences of coccidia a and b dna isolated from field - collected specimens ( csl ao53 and csl ao50 , respectively ) are shown in fig .  1 , and the geographic distribution of coccidian oocyst species shed by free - ranging animals is shown in fig . ", "when sufficient quantities were available , oocysts harvested from fecal samples were sporulated and inoculated into mice to investigate coccidian parasite infectivity and pathogenesis .", "sporulated coccidia a oocysts were , on average , 9.8    0.83  m in length and 8.6    0.68  m in width , with sporocysts measuring 5.3    0.69  m long and 4.0    0.53  m wide ( n  =  20 oocysts from csl ncmmc1047 , measured at 4001000 magnification ) ( fig . ", "sporulated coccidia b oocysts were 9.9    0.88  m in length and 9.0    1.2  m in width , with sporocysts measuring 6.0    0.4  m long and 4.6    0.5  m wide (", "n  =  10 oocysts from csl9878 , measured at 4001000 magnification ) ( fig . ", "sufficient quantities of oocysts from animals that were shedding either coccidia a or b ( or both ) including csl9830 , csl9878 , csl ncmmc1047 , csl10089 , csl10092 , and csl10100 , were available for mouse bioassay .", "all mice were seronegative for t.  gondii , s.  neurona and n.  caninum prior to inoculation and at 3 weeks and 6 weeks post - inoculation , as well as at the time of necropsy ( 5070 days post - inoculation ) .", "no clinical signs were observed in coccidia a- and b- inoculated or mock - inoculated control mice throughout the observation period .", "mouse tissues were generally unremarkable histologically , although one or more of the following histological changes were observed in approximately 40% of mice : slight nephritis , slight pneumonia , inflammation in the lung , spleen or lymph node , and steatitis .", "mouse brain , heart and tongue tissues tested negative for the presence of coccidia a or b dna using pan - coccidian primers that span the its-1 region .", "in an effort to propagate and isolate coccidian protozoa identified by light microscopy in sea lion feces , sporulated oocysts from csl10089 ( coccidia a ) , csl10092 ( coccidia a & b ) , csl10100 ( coccidia a & b ) , and csl10266 ( coccidia a ) were prepared and inoculated into flasks containing ma104  cells .", "all cultures were fed and examined for the presence of live coccidian zoites three times per week .", "after 14 days , 21 days and 35 days of observation , cell cultures inoculated with sporulated oocysts from csl10089 , csl10100 , and csl10092 , respectively , showed propagating organisms with morphologic similarity to sarcocystis spp .", "extracellular zoites were 811  m in length and 12  m in width when measured with spot advanced software at 200 magnification .", "schizonts were seen periodically as the culture matured and the number of free zoites increased ; suggesting that these were merozoites produced by schizogony .", "free zoites had active , circumaxial motility characteristic of s.  neurona merozoites observed in  vitro ( miller et  al . , 2001a ) .", "similar organisms did not grow in cultures inoculated with sporulated oocysts from a fourth sea lion , csl10266 or in un - inoculated cultures of ma104  cells ( table  4 ) . by ifat , antigen slides prepared from zoite cultures of all three sea lions , csl10089 , csl10092 and csl10100 , tested positive using serum from a horse with equine protozoal myeloencephalitis ( fig . ", "the horse 's s.  neurona infection was confirmed by ifat and western blot on serum ( 1:320 titer to laboratory strain snucd-1 ) and cerebral spinal fluid , and by immunohistochemistry used to detect s.  neurona merozoites in the lumbar spine .", "in addition , all three zoite isolates from sea lion oocyst cultures reacted to sera from hospitalized sea lion csl9878 ( table  4 ) which was itself seroreactive to merozoites of snucd-1 ( 1:2560 ) ( table  1 and fig . ", "sea lion culture antigen was ifat - negative ( < 1:40 ) to goat serum containing t.  gondii - reactive antibodies , bovine fetal serum containing n.  caninum - reactive antibodies , and , as shown in fig . ", "5e , to horse serum that was seronegative to s.  neurona and n.  hughesi .", "as soon as zoites were observed , supernatant from the ma104  cell cultures inoculated with sporulated , excysted oocysts from the fecal samples of csl10089 , csl10100 and csl10092 were collected for molecular characterization approximately once per week for 1114 weeks .", "pan - coccidian primers that span the its-1 region produced a 1000  bp band when dna was amplified from zoites taken from cultures from the 3 sea lions throughout their collection period .", "this is the expected band size for amplification of s.  neurona dna using pan - coccidian its-1 primers ( gibson et  al . , 2011 ) .", "in addition , a second band 400  bp in size was amplified simultaneously in culture supernatant of csl10100 and csl10092 using the pan - coccidian primers , but only in the first week of sample collection following zoite observation ( table  4 ) .", "four hundred basepairs is the expected band size for amplification of coccidia a , b or c using pan - coccidian its-1 primers ( colegrove et  al . , 2011 , gibson et  al . , 2011 ) .", "in culture csl10266 , in which no zoites ( only oocysts ) were observed , only the 400  bp band was amplified by pcr during the first two weeks of incubation .", "subsequent supernatant samples of culture csl10266 and all uninoculated ma104 control cultures were pcr - negative ( table  4 ) .", "sequence analysis confirmed that the 400  bp amplicon dna generated in early sampling of cultures csl10100 , csl10092 and csl10266 was identical to dna amplified from frozen , pelleted oocysts originally harvested from the same animal ; namely , coccidia a alone ( in the case of csl10092 and csl10266 ) or a dual infection with coccidia a and b ( in the case of csl10100 ) ( table  4 ) . by blast analysis , good quality partial sequences ( 700  bp ; abbreviated due to the presence of a homopolymer region ) generated from the 1000  bp amplicon in csl10089 , csl10092 and csl10100 zoite cultures were 99% similar to s. neurona dna isolated from horses ( af204230 & af081944 ) and from a skunk ( ay082648 ) ( see supplementary data ) .", "dinucleotide mixtures in the its-1 sequences indicated the presence of at least two s.  neurona - like genotypes in the sea lion cultures .", "these polymorphic regions shared nucleotide similarity to horse strains snucd-1 ( af081944 ) and sn - mu1 ( af204230 ) , sea otter strains so3639 ( dq084486 ) and so5259 , and a skunk isolate ( ay082648 ) ( see supplementary data ) . to further characterize the s.  neurona - like organisms", "cultured from sea lion feces , we amplified seven additional genetic loci in dna extracted from zoites in the sea lion oocyst culture supernatant .", "table  4 displays the pcr and sequencing results for these additional loci . using its1500 primers , with known specificity for s.  neurona or sarcocystis falcatula", "( gibson et  al . , 2011 ) , a 500  bp product was consistently amplified in all culture supernatant samples from csl10089 , csl10092 , csl10100 in which zoites had been observed . by blast analysis ,", "good quality partial its1500 amplicon sequences ( 300  bp , abbreviated due to the presence of a homopolymer region ) were 98% identical to s.  neurona isolated in southern sea otters including isolates so3528 , so3539 , so3485 , and so3501 ( genbank dq084485  dq084488 ) .", "microsatellite primers amplified 150  bp products containing di - nucleotide repeats ca17 ( sn7 ) and gt18 ( sn9 ) found in s.  neurona strain sn - mu1 and other s.  neurona strains isolated from opossums , sea otters , and cats ( rejmanek et  al . , 2010 ) .", "merozoite surface antigen sag1 gene sequences of csl10089 , csl10092 and csl10100 , amplified by snsag 1 - 5 - 6 primers , were 100% identical across 959  bp to s.  neurona strain snucd-1 ( af401682 ) ( ellison et  al . , 2002 ) and 99% similar across 968  bp to the representative sequence of the snsag1 allele amplified in samples from terrestrial and marine mammals ( gq851951 ) ( wendte et  al . , 2010 ) .", "sea lion cultures csl10089 , csl10092 and csl10100 were identical to each other across a 948  bp sequence of the merozoite surface antigen gene snsag4 and 100% identical to sequences of s.  neurona isolated from a sea otter ( so3106 , gq851957 ) and an opossum ( op134 , gq386979 ) . a unique sequence in sea lion s.  neurona - like isolates was identified in the merozoite surface antigen 3 ( snsag3 ) locus in which csl10089 was confirmed by bi - directional sequencing to contain an at insertion at positions 519520 ( table  5 ) .", "the same insert was observed in single direction sequencing for csl10092 but could not be confirmed in csl10100 due to poor sequence quality .", "the same insert was not identified in snsag3 sequences available in genbank originating in a variety of host species including sea otters , opossums and horses ( table  5 ) .", "lack of the position 519520 insert in laboratory strain snucd-1 , derived from a horse in california ( table  5 ) , ruled out the possibility that s.  neurona growth in sea lion oocyst cultures was due to laboratory contamination . at other polymorphic snsag3 nucleotide positions , including 239 and 1059 , sea lion s.  neurona - like isolates were similar to published opossum and sea otter isolates ( table  5 ) . in light of the unexpected findings revealed during cell culture analysis of fecal samples from stranded hospitalized sea lions , we screened all dna extracted from frozen , pelleted oocysts of both hospitalized and field - collected fecal samples using s.  neurona / s .  falcatula - specific its1500 primers . while none of the additional frozen , pelleted oocyst samples collected from hospitalized animals were pcr - positive for s.  neurona - like dna ( including those remaining for csl10089 , csl10092 and csl10100 ) , two fecal samples collected at point lobos ( fig .  3 , samples pl11 and pl27 ) produced 500  bp products in the its1500 pcr reaction ( table  3 ) .", "additional primers ( sn - its ) targeting the its-1 region of s.  neurona produced amplicons with sequences identical to hospitalized sea lion zoites collected in the present study as well as s.  neurona isolated in other animals ( see supplementary data ) .", "all other attempts to amplify s.  neurona in samples pl11 and pl27 using alternative primers failed with the exception of microsatellite sn7 which , in sample pl27 , produced a sequence containing the di - nucleotide repeat ca17 found in s.  neurona and zoites isolated in csl10089 , csl10092 and csl10100 ( table  4 ) .", "the investigation of infectious diseases in coastal aquatic wildlife allows us to probe the relationships between terrestrial - origin pathogen distribution , patterns of overland water runoff , and the risk of pathogen pollution in the coastal and ocean environment .", "the present research was undertaken to investigate the role of california sea lions in the dispersal and life cycle of terrestrial and aquatic coccidian parasites that pose a risk to marine mammal health , and to genetically and phenotypically characterize protozoa shed by sea lions .", "building on data acquired from previous protozoal pathogen surveillance efforts in california sea lions from the coastal waters of california ( carlson - bremer et  al . , 2012a ) , we confirmed that oocysts ( 810  m in size ) that are morphologically similar to , but genetically distinct from , t.  gondii and n.  caninum are frequently shed by stranded sea lions that are hospitalized and by free - ranging populations .", "genetic variability in the partial its-1 region of these coccidia a and b isolates was not observed within this study or in previous reports ( colegrove et  al . , 2011 , carlson - bremer et  al . ,", "consistent with other studies of stranded sea lions , shedding of coccidia a and b occurred primarily in yearlings , and those shedding oocysts had stranded due to causes other than protozoal infection .", "while our data further establish that sea lions are naturally asymptomatic definitive hosts of coccidia a and b , the pathogenic potential of these protozoa in other aquatic species requires further investigation , particularly considering the known virulence of coccidia c and other novel tissue - cyst forming coccidia in harbor seals and guadalupe fur seals ( colegrove et  al .", "phylogenetic analyses of the its-1 locus performed here and elsewhere ( gibson et  al . , 2011 , carlson - bremer et  al . , 2012a ) demonstrate that coccidia a , b , c , csl ao11 , and isolates from a harbor seal and guadalupe fur seals share a common ancestor with n.  caninum .", "morphologically , however , coccidia a and b measured in this study were smaller than n.  caninum oocysts and had < 80% pairwise similarity to n.  caninum at the its-1 locus . while unsporulated coccidia a and b oocysts shed by sea lions are 810  m in diameter , unsporulated n.  caninum oocysts shed by dogs are typically 1011  m in diameter ( mcallister et  al . , 1998 ) .", "sporulated n.  caninum oocysts are reportedly an average of 11.7    11.3  m ( lindsay et  al . , 1999 ) while sporulated forms of coccidia a and b are an average of 9.8    8.6  m , and 9.9    9.0  m , respectively . by comparison ,", "unsporulated t.  gondii oocysts are 10    12  m in diameter and sporulated forms are 11    13  m in diameter ( dubey et  al . , 1998 ) .", "more detailed genetic and morphologic studies ( e.g. whole genome sequencing , electron microscopy , and histological examination of life stages in situ ) are necessary to clarify the taxonomy of novel coccidia identified in aquatic animals , and will likely reveal that these organisms belong to either new neospora species or a new genus within the family sarcocystidae . at the phenotypic level , we observed that the sporulated oocysts of coccidia a and b were not cultivable in the african green monkey kidney cell line , ma104 , commonly used to propagate other tissue - cyst - forming coccidia including t.  gondii , n. caninum and s.  neurona ( miller et  al .", ", 2001a , miller et  al . , 2004 , holmberg et  al . , 2006 ) . while coccidia a dna was detectable by pcr in a culture prepared from oocyst", "isolate csl10266 for up to two weeks after inoculation , no protozoal organisms were ever observed . in ma104 cultures of oocyst", "isolates csl10092 and csl10100 , coccidia a and b dna as well as s.  neurona - like dna were amplified for up to 56 weeks post - inoculation ; after which time only s.  neurona - like dna and live s.  neurona - like zoites were detected for the duration of observation .", "while it is possible in the latter cases that coccidia a and b did not replicate in  vitro due to competition from the sarcosystsis sp . during co - culture , we hypothesize , rather , that the ma104  cell line may not be susceptible to infection with coccidia a and b , or the culture conditions were not optimal for these parasites .", "other cell culture systems should be investigated for their propagation , particularly in the interest of developing a serological assay for the purpose of screening marine mammals for infections with these and other potentially pathogenic protozoa .", "coccidia a and b did not cause disease in mice with known susceptibility to t.  gondii including b6.129s7-ifng / j , cba / caj and swiss webster ( cfw ) ( rytel and jones , 1966 , villegas et  al . , 2002 , gavrilescu and denkers , 2003 ) .", "surprisingly , coccidia a and b were not pathogenic to cba / caj mice despite previous studies showing that cba / ca mice can be used as a model of n.  caninum pathogenesis ( mcguire et  al .", "these findings , taken together with the lack of growth in ma104  cells or reactivity of serum from coccidia a- and b - infected sea lions with n.  caninum , t.  gondii or s.  neurona antigen by ifat , is further evidence of the taxonomic divergence of the sea lion parasites from known tissue - cyst forming coccidia with terrestrial life cycles . a significant outcome of this study was the discovery of s.  neurona or s.  neurona - like zoites growing in cell cultures inoculated with fecal material ( including sporulated , excysted oocysts of coccidia a and b ) prepared from three stranded hospitalized sea lions sampled between 2010 and 2012 .", "the cultivation of live protozoal zoites with strong genetic similarity to s.  neurona across multiple genes during parasite isolation from sea lion feces , particularly in the absence of microscopic evidence of sarcocystis sp .", "a possible explanation for these findings is that samples taken for microscopic observation during fecal flotation only represent a small portion of the overall feces sampled and the overall pool of oocysts being shed . if the original pool of isolated protozoa from feces consisted of a mixture of variable numbers of coccidian oocysts ( coccidia a and/or b ) and sporocysts ( s.  neurona - like ) , it is possible that the subdivision of the oocyst pool for flotation and microscopy , pelleting / freezing , sporulation for mouse bioassay , and sporulation / excystation for cell culture , could explain discrepancies in the ability to detect different parasites , depending on the sensitivity of each assay .", "we hypothesize that a small number of s.  neurona - like sporocysts existed in the pool of protozoa prepared for cell culture , but were not present in the subsamples designated for microscopy , pelleting and freezing or mouse bioassay .", "in addition , in  vitro cultivation may have facilitated the expansion of small numbers of s.  neurona sporozoites that were able to develop into schizonts that produced merozoites .", "the isolation of s.  neurona - like dna from feces of two free - ranging sea lions at point lobos indicates that the organism may occur naturally in sea lions , as opposed to occurring via nosocomial exposure in the hospitalized setting .", "exposure of sea lions to s.  neurona , or an s.  neurona - like organism , most likely occurs in coastal waters contaminated with opossum feces transported from land - to - sea via freshwater run - off ( carlson - bremer et  al .", ", 2012b , shapiro et  al . , 2012 ) or may be concentrated in sea lion prey , as has been shown with t.  gondii shed by terrestrial felids ( miller et  al . , 2002 , johnson et  al . , 2009 ) .", "although the extent of environmental contamination with s.  neurona sporocyts has not been documented , infections in southern sea otters and a number of other marine mammals of the pacific northwest have been reported ( miller et  al .", ", 2001b , miller et  al . , 2009 , miller et  al . , 2010 ,", "all mouse tissues were pcr - negative for s.  neurona using its1500 primers , indicating that the sea lion s.  neurona - like sporocysts were either not present in the inoculum , were present but below the threshold of infection , or the mice utilized were not susceptible . because of the similarity of coccidia a and b to t.  gondii , mice in this study were selected for their susceptibility to t.  gondii , not s.  neurona .", "shed by sea lions would benefit from the utilization of interferon gamma gene knock - out mice ( e.g. c57bl/6-black ) with well - characterized susceptibility to s.  neurona ( dubey et  al .", "sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis  a serious and progressively debilitating neurological disease in horses and is one of several coccidian parasites known to cause protozoal encephalitis in pacific harbor seals , southern sea otters , and other marine mammals ( lapointe et  al .", ", 1998 , rosonke et  al . , 1999 , lindsay et  al . , 2000 , lindsay et  al . , 2001 ,", "miller et  al . , 2001a , lapointe et  al . , 2003 , thomas et  al . , 2007 , gibson et  al . , 2011 , dubey et  al . , 2015", "although not frequently reported in the literature , when s.  neurona infections do occur in california sea lions , they are either clinically irrelevant ( gibson et  al . , 2011 ) or associated with myositis ( carlson - bremer et  al . , 2012b ) .", "tissue cysts caused by other sarcocystis spp . , including sarcocystis canis - like organisms , have been detected in sea lion muscle ( mense et  al .", ", 1992 , miller , 2008 ) and liver tissue ( mense et  al . , 1992 ,", "the three animals that shed s.  neurona - like protozoa were admitted to tmmc for malnutrition or trauma , and had no overt signs of protozoal infection .", "lack of serum titers to s.  neurona in these animals indicated that they had not yet mounted a detectable antibody response , or that they never had systemic infections .", "an elevated s.  neurona titer ( 1:2560 ) was detected in another hospitalized sea lion , csl9878 , but this animal also had no signs of disease and was not shedding detectable levels of s.  neurona - like sporocysts or dna at the time of sampling for this study .", "overall , findings from this study suggest that california sea lions are likely acting as mechanical vectors of s.  neurona or an s.  neurona - like organism , similar to the way canines can mechanically transmit t.  gondii ( lindsay et  al . , 1997 ) .", "using a mouse bioassay to detect the presence of infectious t.  gondii oocysts in canine tissues and feces , lindsay et  al .", "sporulated t.  gondii oocysts can traverse the canine intestinal tract and be excreted in an infectious state  .", "several experimental outcomes indicate that an s. neurona - like organism is excreted by sea lions in low concentrations , which is expected during mechanical transmission : ( 1 ) s.  neurona - like sporocysts were not observed by light microscopy following fecal flotation ( 2 ) s.  neurona - like dna was not amplified from concentrated oocysts ( pellets ) isolated from hospitalized sea lion fecal samples , ( 3 ) s.  neurona - like dna was only amplified by pcr on high copy number genes in concentrated oocysts isolated from feces of free - ranging animals , and ( 4 ) s.  neurona - like merozoites were only observed after long - term cultivation in ma104  cells .", "the genetic similarity of sea lion s.  neurona - like isolates to previously characterized protozoa in aquatic and terrestrial animals across much of the genome is not surprising considering the extensive overlap in genetic identity in s.  neurona isolates from the tissues of opossums , sea otters and horses that was previously demonstrated ( rejmanek et  al . , 2010 ,", "the key snsag3 polymorphism that we identified in the zoites from sea lion fecal samples may represent a novel s.  neurona genotype which , in the future , may facilitate identification of the origin of infection .", "this is particularly important as we seek to understand the flow of terrestrial pathogens into oceans and risk factors for s.  neurona infections in other aquatic species .", "continued investigations into the pathogenesis of s.  neurona in sea lions through necropsy and systematic surveillance for s.  neurona exposure , shedding , and disease will help clarify the importance of this host species in parasite transmission within the pacific coastal ecosystem . in conclusion , continued surveillance for tissue - cyst forming coccidia in marine mammals should remain a priority for programs that monitor the population health of aquatic wildlife .", "efforts to explore the distribution , pathogenesis and genetic diversity of sarcocystidae in land and sea isolates promise to improve our understanding of the interconnectedness between terrestrial and aquatic microbial diseases , and to direct interventions that will benefit our oceans and their inhabitants ."], "abstract_text": ["<S> tissue - cyst forming coccidia in the family sarcocystidae are etiologic agents of protozoal encephalitis in marine mammals including the federally listed southern sea otter ( enhydra lutris ) . </S>", "<S> california sea lions ( zalophus californianus ) , whose coastal habitat overlaps with sea otters , are definitive hosts for coccidian protozoa provisionally named coccidia a , b and c. while coccidia a and b have unknown clinical effects on aquatic wildlife hosts , coccidia c is associated with severe protozoal disease in harbor seals ( phoca vitulina ) . in this study , we conducted surveillance for protozoal infection and fecal shedding in hospitalized and free - ranging california sea lions on the pacific coast and examined oocyst morphology and phenotypic characteristics of isolates via mouse bioassay and cell culture . </S>", "<S> coccidia a and b were shed in similar frequency , particularly by yearlings . </S>", "<S> oocysts shed by one free - ranging sea lion sampled at ao nuevo state park in california were previously unidentified in sea lions and were most similar to coccidia infecting guadalupe fur seals ( arctocephalus townsendi ) diagnosed with protozoal disease in oregon ( usa ) . </S>", "<S> sporulated coccidia a and b oocysts did not replicate in three strains of mice or in african green monkey kidney cells . </S>", "<S> however , cultivation experiments revealed that the inoculum of fecally - derived coccidia a and b oocysts additionally contained organisms with genetic and antigenic similarity to sarcocystis neurona ; despite the absence of detectable free sporocysts in fecal samples by microscopic examination . </S>", "<S> in addition to the further characterization of coccidia a and b in free - ranging and hospitalized sea lions , these results provide evidence of a new role for sea lions as putative mechanical vectors of s.  neurona , or s.  neurona - like species . </S>", "<S> future work is needed to clarify the distribution , taxonomical status , and pathogenesis of these parasites in sea lions and other marine mammals that share their the near - shore marine environment . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Results", "Discussion"], "sections": [["coccidia are a diverse group of protozoan parasites within the phylum apicomplexa and include pathogenic species of significance to animal and human health .", "atoxoplasma ) , primarily parasitize a single host throughout their life cycle and include important avian pathogens ( mcdougald , 1998 , barta et  al . , 2005 , berto et al . , 2011 ) .", "heteroxenous coccidian protozoa including toxoplasma gondii , neospora caninum and sarcocystis neurona infect multiple hosts throughout their life cycle and their transmission is facilitated by predator ", "prey relationships ( sibley , 2003 ) . in the intermediate host ( often herbivorous or omnivorous ) ,", "infectious stages are formed within tissue cysts that can be ingested by the definitive host ( often a carnivorous predator ) .", "the definitive host is so called because the parasite life cycle is perpetuated through sexual multiplication in these animals .", "definitive hosts shed environmentally - resistant oocysts , ( or sporocysts from sporulated oocysts in the case of s.  neurona ) , which are infective after sporulation for intermediate hosts that ingest fecally - contaminated food or water ( sibley , 2003 ) . although the only known definitive hosts of t.  gondii , n.  caninum and s.  neurona are terrestrial animals , specifically felids , canids and opossums ( didelphis spp . ) , respectively , there is evidence that marine mammals can also become infected with these and other related coccidia ( dubey et  al . , 2000 ,", ", 2001a , dubey et  al . , 2001b , tenter et  al . , 2000 , dubey et  al . , 2003 ,", ", 2011 , gibson et  al . , 2011 , carlson - bremer et  al . ,", "the most likely modes of transmission of these pathogens to aquatic animals are via ingestion of water - borne oocysts or sporocysts originating in land - based surface runoff , or infected prey ( miller et  al . , 2002 , conrad et  al . , 2005 , massie et  al . , 2010 , gibson et  al . , 2011 ,", "one of the most serious consequences of s.  neurona and t.  gondii infection in marine mammals is fatal protozoal encephalitis which has been described most commonly in cetaceans , including southern sea otters ( enhydra lutris nereis ) , california sea lions ( zalophus californianus ) and pacific harbor seals ( phoca vitulana richardsi ) ( thomas and cole , 1996 , lapointe et  al . , 1998 , cole et  al . , 2000 , miller et  al", ", 2001a , dubey et  al . , 2003 , kreuder et  al . , 2003 , miller et  al . , 2004 , miller et  al . , 2009 ,", "miller et  al . , 2010 , gibson et  al . , 2011 ) .", "more recently , severe myositis in a hospitalized california sea lion was recognized as a new clinical syndrome associated with s.  neurona infection ( carlson - bremer et  al . , 2012b ) .", "pathogenic infections with other , less well characterized , tissue - encysting coccidia have also been documented in marine mammals in association with protozoal lymphadenitis , hepatitis , myocarditis , encephalitis and non - suppurative necrotizing meningoencephalitis ( dubey et  al . , 2003 , lapointe et  al . , 2003 ,", "california sea lions inhabit waters of the pacific coast of north america between southwestern canada and baja california ( lowry et  al . , 1992 ) . as long - lived coastal residents with large fat stores and piscivorous prey preferences that are shared with humans , sea lions have the potential to act as sentinel species , or indicators of aquatic ecosystem and human health ( bossart , 2011 ) . during postmortem examination of rescued california sea lions that died at the marine mammal center ( tmmc , sausalito , california ) , sexual and asexual stages of three novel coccidia with genetic similarity to neospora spp .", "were identified in sea lion enterocytes , and these organisms were putatively named coccidia a , b and c ( colegrove et  al . , 2011 , carlson - bremer et  al . , 2012a ) .", "subsequent fecal analysis of stranded and rehabilitated sea lions sampled between 2007 and 2009 confirmed that sea lions shed coccidia a and b , particularly as yearlings , and that shedding of coccidia a could be detected in feces for up to 22 days ( carlson - bremer et  al . , 2012a ) .", "evidence to date suggests that california sea lions act as both definitive and intermediate hosts of novel protozoa in the absence of clinical signs or pathologic evidence of disseminated infection . yet , the identification of severe protozoal hepatitis , myocarditis and encephalitis in a neonatal harbor seal infected with coccidia c is of particular concern because it indicates that sea lions may be capable of shedding coccidian protozoa that are pathogenic to harbor seals and perhaps other aquatic wildlife ( colegrove et  al . , 2011 ) . a better understanding of the biology , epidemiology , and pathogenesis of tissue - encysting coccidian organisms that parasitize marine mammals is needed to properly assess the risks and burden of protozoal disease in aquatic ecosystems such as the pacific coast of north america . as part of ongoing health surveillance in aquatic species of the pacific ocean , and accompanying studies of pathogen pollution in near - coastal california waters", ", we examined the diversity of coccidian parasites shed by hospitalized and free - ranging sea lions at coastal haul - out locations in central california .", "in addition , we characterized the phenotypes of sea lion - derived coccidian protozoa in both a mouse model of pathogenesis and in mammalian cell culture ."], ["staff at tmmc collected fecal samples from individual california sea lions ( csl ) stranded along the central and northern california coast between september 2010 and may 2012 as previously described ( carlson - bremer et  al . , 2012a ) .", "samples were typically collected within the first week of hospital admission , although some animals were sampled up to 3 weeks later . animals and corresponding fecal samples", "when coccidial oocysts were identified at tmmc , one or more fecal samples from an individual animal were shipped on cold packs to the university of california davis for oocyst harvesting and genetic characterization .", "age classifications for sea lions ( adult , subadult , juvenile , yearling , and pup ) were estimated as previously described ( carlson - bremer et  al . , 2012a ) . between may 2011 and october 2012 , sea lion fecal samples from free - ranging animals", "were also collected from coastal haul out sites at ao nuevo state park ( 37.108 , 122.337 ) , white rock ( 35.532 , 121.088 ) , and seal rock at point lobos ( 36.516 , 122.336 ) , placed into individual 50  ml conical vials and kept at 4  c during transport to uc davis for oocyst detection , harvesting and genetic characterization .", "coccidial oocysts were harvested from fecal samples by a double centrifugation flotation method as described by dabritz and co - authors ( dabritz et  al . , 2007 ) with some modifications .", "a detailed protocol for oocyst harvesting from sea lions is available in the supplemental information associated with this article ( see supplementary data ) .", "when quantities were sufficient , a portion of freshly - harvested oocysts were not frozen , but were reserved and sporulated for bioassay in mice and inoculation into cell cultures .", "newly - harvested oocysts were washed two times in sterile pbs , and the final pellet resuspended in 5  ml pbs , to which was added clorox bleach at a final concentration of 10% . following 20  min incubation with occasional gentle mixing , the sample was washed 3 times in 50  ml molecular biology grade water , with each centrifugation at 1000  g for 10  min .", "after final centrifugation , supernatant was carefully removed and the pellet mixed with 35  ml pbs ( higher volume for samples with higher oocyst concentration )", ". approximately 1  ml of oocyst suspension in pbs was added to 5  ml filtered seawater ( 0.22  m , acrodisc syringe filters , pall corporation ) with 250  l amphotericin b ( 250  g / ml ) in a t-25 tissue culture flask with ventilated lid .", "each flask was set on a gentle rocker at rt and visually inspected every 2 days by light microscopy for evidence of sporulation .", "sporulated oocysts were washed two times in 40  ml sterile pbs , counted , and prepared for oral inoculation into mice . for excystation and preparation for cell culture ,", "sporulated oocysts were suspended in 0.5  ml warm ( 37  c ) dulbecco 's minimum essential media ( dmem , gibco ) , supplemented with 10% ( v / v ) heat - inactivated fetal bovine serum , 2  mm l - glutamine , 100  u / ml penicillin , 100  g / ml streptomycin , 10  mm hepes and 55  m 2-mercaptethanol and transferred to 2  ml screw cap tubes containing 350  mg of 200400  mm sterile acid - washed glass beads ( thermofisher scientific ) .", "oocyst disruption and sporozoite release was accomplished through vortexing in the presence of glass beads for 2030  s. to confirm the presence of free sporozoites , a 5  l aliquot of the suspension was examined following each 10  s of vortexing .", "when sporozoites were visible , the sample was immediately transferred to monolayer cultures of embryonic monkey kidney ( ma104 ) cells ( biowhittaker , walkersville , md ) and incubated for 2  h at 37  c in 5% co2 , after which the inoculum was removed and replaced with fresh 37  c prepared dmem .", "cells were checked 3 times weekly for parasite growth alongside uninoculated ma104  cells . when zoites became visible in cell culture ,", "supernatant was collected and pelleted for dna extraction , cryopreservation , and antigen slide preparation for serological testing . during microscopic analysis of oocysts isolated from sea lion feces ,", "multiple photomicrographs were taken at 4001000 magnification using a camera ( spot rt3 scientific digital ccd camera , spot imaging solutions  , sterling heights , mi ) mounted on a zeiss axioskop epifluorescent microscope equipped with a uv emission filter set ( emitter 460/50  nm band pass filter ; chroma # 11000 v3 ) .", "micrographs were used to measure the long axis and short axis lengths of oocysts ( in unsporulated and sporulated forms ) and sporocysts ( in sporulated forms ) using spot advanced software ( spot imaging solutions , sterling heights , mi , usa ) .", "the mean and standard deviation of 1020 sporulated and unsporulated oocysts were calculated separately for each coccidian species .", "in oocyst - inoculated cell cultures , approximately 10 zoites per culture were measured at 200 magnification from photomicrographs using spot software .", "all animal experiments were conducted with the approval and oversight of the institutional animal care and use committee at the university of california , davis , which is accredited by the association for assessment and accreditation of laboratory animal care , international ( iacuc # 17501 ) .", "mouse strains were chosen based on known susceptibility to t.  gondii ( rytel and jones , 1966 , villegas et  al . , 2002 , gavrilescu and denkers , 2003 ) .", "prior to inoculation , 68 week old female mice were screened by the indirect fluorescent antibody test ( ifat , see methods below ) using a 1:40 serum dilution to ensure seronegativity to t.  gondii , n.  caninum and s.  neurona .", "approximately 1000 sporulated oocysts were inoculated into two mice of each genetic background : b6.129s7-ifng / j ( c57bl/6 background ) , cba / caj ( the jackson laboratory , bar harbor , me ) and swiss webster ( cfw ) ( charles river laboratories , wilmington , ma ) .", "of the two mice in each genetic background group , one was orally inoculated by gastric lavage and the other was subcutaneously inoculated in the interscapular region as previously described ( fritz et  al . , 2012 ) .", "control mice of each genetic background were given inocula free of oocysts consisting of clinicare canine / feline liquid diet ( abbott health care ) for gastric lavage controls or pbs for subcutaneous controls .", "mice were checked once per week for clinical signs of disease and were bled at 3 and 6 weeks post - infection for ifat serology .", "mice were humanely euthanized by co2 asphyxiation for necropsy at 5070 days post - inoculation , at which time additional blood was collected from the heart cavity for ifat serology . during necropsy ,", "portions of the tongue , heart , and brain were collected , divided into three pieces , and frozen at  80  c until dna extraction .", "a portion of the mouse brains was homogenized , trypsinized and placed over monolayer cultures of ma104  cells for coccidial parasite detection as previously described ( miller et  al . , 2001b ) .", "remaining mouse tissues were placed in 10% formalin and submitted to the california animal health and food safety laboratory ( cahfs , davis , ca ) for histological and immunohistochemical analysis .", "the following mouse tissues were fixed in 10% neutral buffered formalin and examined histologically : liver , kidney , heart , lung , spleen , subcutaneous fat , skeletal muscle , pancreas , salivary gland , uterus , lymph node , thymus , multiple sections of gastrointestinal tract and brain , gonad , trachea , adrenal gland .", "formalin - fixed tissues were trimmed , routinely processed for paraffin embedding , sectioned at 5  m , and stained with hematoxylin and eosin .", "immunohistochemical staining for the presence of t.  gondii , s. neurona and n. caninum antigen was performed as previously described ( miller et  al . , 2001b ) on selected sections of formalin - fixed , paraffin - embedded tissues .", "antigen slides for ifat analysis were prepared with parasites harvested from the supernatants of ma104 cultures containing visible zoites using previously described methods ( miller et  al . , 2001b ) .", "mouse blood was collected by venipuncture , cardiac puncture or retro - orbital bleed , allowed to clot , and centrifuged at 1000  g for 10  min .", "sea lion serum was similarly separated after collection via venipuncture of the caudal gluteal vein during hospitalization at tmmc and was shipped to uc davis for ifa testing .", "serum was serially diluted in pbs from 1:40 to 1:81,920 and screened for immunoreactivity to t.  gondii , s.  neurona and n.  caninum antigen using a fluorescein isothiocyanate ( fitc)-conjugated rabbit anti - canine igg ( jackson immunoresearch laboratories , west grove , pa ) .", "mouse sera were screened at a 1:40 dilution for immunoreactivity to antigens of the same three parasites using fitc - conjugated goat anti - mouse igg ( jackson immunoresearch laboratories ) .", "test controls consisted of previously analyzed animal sera with titers ranging from 1:12801:10,240 ( positive controls ) or titers < 1:40 ( negative controls ) .", "sea lion and mouse serum specimens that showed no reactivity at 1:40 dilution were considered test - negative .", "for the purpose of this study , sea lion sera that were reactive to t.  gondii antigen at 1:640 dilution were considered test - positive to identify sea lions likely infected with t.  gondii .", "sea lion sera that were reactive to s.  neurona antigen at 1:320 were considered test - positive as has been used previously to identify s.  neurona infection in sympatric species ( kreuder et  al . , 2005 ,", "prior to dna extraction , frozen , pelleted oocysts that had been harvested from sea lion feces were subjected to a single freeze ", "dna was extracted from oocysts and mouse tissues using the qiagen dneasy blood and tissue kit using manufacturer 's recommendation for animal tissues .", "two out of three mouse tissue sections were analyzed per tissue type per mouse , with preference for mice that demonstrated histological lesions , leading to a total of 164 individual samples , including controls , which were extracted and analyzed by pcr .", "negative controls ( water ) were included in the extraction process to monitor for environmental contamination . for molecular characterization of coccidian dna in oocysts , mouse tissues and zoite cultures , nested pcr", "was performed using pan - coccidian primers that span the internal transcribed spacer 1 ( its-1 ) region , and s.  neurona / s . ", "falcatula - specific primers ( its1500 ) that amplify 500  bp of the its1 region in those organisms ( miller et  al . , 2009 , gibson et  al . , 2011 ) .", "for characterization of s.  neurona - like dna , pcr protocols were employed to amplify microsatellites sn7 ( nested pcr ) and sn9 ( hemi - nested pcr ) , ( asmundsson and rosenthal , 2006 , rejmanek et  al . , 2010 ) and surface antigens snsag3 , snsag4 ( nested pcr ) ( rejmanek et  al . , 2010 ) and either snsag1 , snsag5 , or snsag6 which amplify using snsag1 - 5 - 6 primers ( wendte et  al . , 2010 ) .", "additional primers were used to amplify a secondary region of s.  neurona its-1 , approximately 150  bp upstream of the area amplified by its1500 primers : snits1-f , ccgggatgatgtcgtcaag and snits1-r , acagatgatgtgccccgc ( carlson - bremer , 2011 ) .", "all pcr reactions were performed using qiagen hotstar taq plus ( qiagen , valencia , ca ) according to manufacturer 's recommendations , utilizing 5  l of template dna .", "the following reaction conditions were used for all primer sets : initial denaturation at 95  c for 5  min , followed by 35 cycles at 94  c for 40  s , 58  c for 40  s ( external primers or non - nested pcr ) or 59  c for 40  s ( internal primers for nested / hemi - nested pcr ) , and 72  c for 90  s , followed by a 10  min extension at 72  c . in the case of nested or hemi - nested pcr protocols , 1  l of first round pcr product was added to the second round master mix .", "positive controls for pcr included either hammondia heydorni dna isolated from a domestic dog , neospora hughesi isolated from a horse , and/or s.  neurona strain snucd-1 ( rejmanek et  al .", "pcr products were purified using the qiaquick pcr purification kit ( qiagen ) and were sequenced by the ucdna sequencing facility ( davis , ca ) . forward and reverse sequences were assembled into contigs using geneious pro v. 5.3.4 ( drummond et  al . , 2009 ) .", "consensus sequences were aligned to reference sequences obtained from ncbi ( http://www.ncbi.nlm.nih.gov/nuccore ) using muscle or mafft ( katoh et  al . , 2002 , edgar , 2004 ) with default parameters , followed by manual editing .", "posterior sets of phylogenetic trees were generated using mrbayes ( huelsenbeck and ronquist , 2001 ) implemented in geneious pro v. 5.3.4 and run for 1,000,000 generations using the gtr  +  g nucleotide substitution model .", "trees were sampled every 1000 generations and 25% of trees were discarded from the initial burn - in period .", "pairwise genetic distance values were determined through uncorrected - p distance matrices generated in paup * ( sinaeur associates inc . ) using prepared alignments of 420  bp for its-1 .", "individual sequences were analyzed using blast ( basic local alignment search tool ) available on the ncbi website http://blast.ncbi.nlm.nih.gov/blast.cgi .", "kp999999 and kp990539 for t.  gondii m4 and ao11 , respectively ) , and snsag3 ( kr011915 for sarcocystis sp .", "groups were compared for significant differences using the two - tailed two - sample test of proportions ( stata / ic v. 11.0 , statacorp lp , college station , tx ) ."], ["fecal samples collected from 139 sea lions stranded in nine counties located on the california coast between 2010 and 2012 were screened upon hospitalization for the presence of coccidian oocysts . the majority of animals stranded in monterey ( n  =  48 ) , san luis obispo ( n  =  36 ) and santa cruz ( n  =  32 ) counties , followed by san mateo ( n  =  7 ) , san francisco ( n  =  5 ) , sonoma ( n  =  4 ) , marin ( n  =  4 ) , mendocino ( n  =  2 ) and humboldt ( n  =  1 ) .", "coccidian oocysts were identified in the feces of 16 of the 139 ( 11.5% ) sea lions sampled the majority of which were from monterey ( 8.3% , 4/48 ) and san luis obispo ( 13.9% , 5/36 ) counties .", "only one of the 32 stranded animals ( 3.1% ) from santa cruz county was shedding oocysts .", "table  1 describes the clinical status and results of serological testing and coccidian oocyst dna analysis for the 16 sea lions that had oocysts detected in their feces upon presentation at tmmc .", "oocysts observed in the feces of 16 stranded sea lions ( table  1 ) were 810  m in diameter .", "sporocysts were not observed by light microscopy following fecal flotation in any of the sea lions sampled .", "a similar proportion of males ( 9/89 ) and females ( 7/51 ) shed oocysts ( p  =  0.518 ) .", "yearlings most commonly shed oocysts ( 8/33 , 24.2% ) followed by subadults ( 2/16 , 12.5% ) , pups ( 2/19 , 10.5% ) , juveniles ( 3/50 , 6.0% ) and adults ( 1/21 , 4.8% ) .", "the proportion of yearlings shedding oocysts ( 8/33 ) was significantly greater than the proportion of all other age classes shedding oocysts ( 8/106 ; p  =  0.009 ) .", "1 shows the close relationship of coccidian dna sequences amplified from fecal samples of the stranded hospitalized sea lions in this study ( e.g. csl ncmmc1047 humboldt and csl9878 monterey ) to n.  caninum and dna sequences amplified from marine mammal fecal samples and tissues in previous studies ( colegrove et  al .", ", 2011 , gibson et  al . , 2011 , carlson - bremer et  al . ,", "2011 ) , coccidia a ( csl ncmmc1047 ) and b ( csl9878 ) its-1 sequences shared 79.6% and 78.8% pairwise", "nucleotide similarity , respectively , to n.  caninum and 74.1% and 71.8% similarity , respectively , to t.  gondii . based on sequence analysis of the its-1 locus from the 16 hospitalized sea lions that were shedding coccidia during this study ,", "6 ( 37.5% ) animals were confirmed to be shedding coccidia a , 6 ( 37.5% ) were shedding coccidia b , and 2 ( 12.5% ) were shedding both a and b simultaneously , indicating dual infections ( table  1 ) .", "sera from 5 ( 31.3% ) of these sea lions had antibodies to t.  gondii antigens detectable by ifat , but only one of these animals ( csl9830 ) had an antibody titer above 1:640 ( table  1 ) .", "one animal from monterey ( csl9878 ) was seroreactive to s.  neurona antigen at a 1:2560 dilution .", "no animals tested during this time period were seroreactive to n.  caninum despite the apparent genetic relatedness of coccidia a and b to neospora spp . at the its-1 locus ( fig", ".  1 ) ( colegrove et  al . , 2011 , carlson - bremer et  al . ,", "unsporulated oocysts had a round to ovoid shape and autofluoresced under uv illumination ( fig . ", "2 ) as observed for t.  gondii and other coccidian oocysts ( lindquist et  al . , 2003 ) . unsporulated coccidia a oocysts averaged 9.9    0.59  m in length and 9.5    0.69  m in width (", "n  =  20 oocysts from csl ncmmc 1047 , measured at 200 magnification ) .", "unsporulated coccidia b oocysts averaged 8.9    0.31  m in length and 8.5    0.51  m in width (", "n  =  20 oocysts from csl9878 , measured at 1000 magnification ) ( fig .  2 ) .", "coccidian oocysts a and b were previously identified in the feces of stranded california sea lions surveyed at tmmc between 2007 and 2009 ( carlson - bremer et  al . , 2012a ) . here , we summarized shedding prevalence for all sea lions sampled between 2007 and 2012 ( table  2 ) . over the six year period", ", 558 sea lions were sampled , 31 ( 5.6% ) of which were shedding coccidia a or b , in similar frequency .", "hospitalized stranded sea lions were typically diagnosed with malnutrition and trauma with no apparent clinical effects attributed to protozoal infection ( table  1 ) . between august 2011 and september 2012 ,", "california sea lion fecal samples were collected from coastal haul - out sites located at ao nuevo state reserve", "( n  =  48 ) , white rock ( n  =  94 ) and point lobos ( n  =  70 ) and the samples were screened for oocysts .", "the prevalence of oocysts identified by microscopy during isolation was 8.3% ( 4/48 ) at the ao nuevo haul - out site , 3.2% ( 3/94 ) at white rock , and 8.6% ( 6/70 ) at point lobos ( table  3 ) .", "sporocysts were not observed by light microscopy following fecal flotation in any of the free - ranging sea lion samples . dna amplification and", "sequence alignment revealed that both coccidia a and b were present in fecal samples collected at ao nuevo and point lobos , while only coccidia a was identified in samples collected at white rock ( table  3 ) .", "fecal sample 11 collected at ao nuevo state park ( ao11 ) represented a novel sequence type with 9798% pairwise similarity at the its-1 locus to previously published coccidian dna isolates in guadalupe fur seal tissues ( see neospora sp .", "the csl ao11 isolate had 92% pairwise similarity to coccidia a and b and shared even less similarity ( 87.7% ) to coccidia c at the its-1 locus .", "this sequence as well as representative sequences of coccidia a and b dna isolated from field - collected specimens ( csl ao53 and csl ao50 , respectively ) are shown in fig .  1 , and the geographic distribution of coccidian oocyst species shed by free - ranging animals is shown in fig . ", "when sufficient quantities were available , oocysts harvested from fecal samples were sporulated and inoculated into mice to investigate coccidian parasite infectivity and pathogenesis .", "sporulated coccidia a oocysts were , on average , 9.8    0.83  m in length and 8.6    0.68  m in width , with sporocysts measuring 5.3    0.69  m long and 4.0    0.53  m wide ( n  =  20 oocysts from csl ncmmc1047 , measured at 4001000 magnification ) ( fig . ", "sporulated coccidia b oocysts were 9.9    0.88  m in length and 9.0    1.2  m in width , with sporocysts measuring 6.0    0.4  m long and 4.6    0.5  m wide (", "n  =  10 oocysts from csl9878 , measured at 4001000 magnification ) ( fig . ", "sufficient quantities of oocysts from animals that were shedding either coccidia a or b ( or both ) including csl9830 , csl9878 , csl ncmmc1047 , csl10089 , csl10092 , and csl10100 , were available for mouse bioassay .", "all mice were seronegative for t.  gondii , s.  neurona and n.  caninum prior to inoculation and at 3 weeks and 6 weeks post - inoculation , as well as at the time of necropsy ( 5070 days post - inoculation ) .", "no clinical signs were observed in coccidia a- and b- inoculated or mock - inoculated control mice throughout the observation period .", "mouse tissues were generally unremarkable histologically , although one or more of the following histological changes were observed in approximately 40% of mice : slight nephritis , slight pneumonia , inflammation in the lung , spleen or lymph node , and steatitis .", "mouse brain , heart and tongue tissues tested negative for the presence of coccidia a or b dna using pan - coccidian primers that span the its-1 region .", "in an effort to propagate and isolate coccidian protozoa identified by light microscopy in sea lion feces , sporulated oocysts from csl10089 ( coccidia a ) , csl10092 ( coccidia a & b ) , csl10100 ( coccidia a & b ) , and csl10266 ( coccidia a ) were prepared and inoculated into flasks containing ma104  cells .", "all cultures were fed and examined for the presence of live coccidian zoites three times per week .", "after 14 days , 21 days and 35 days of observation , cell cultures inoculated with sporulated oocysts from csl10089 , csl10100 , and csl10092 , respectively , showed propagating organisms with morphologic similarity to sarcocystis spp .", "extracellular zoites were 811  m in length and 12  m in width when measured with spot advanced software at 200 magnification .", "schizonts were seen periodically as the culture matured and the number of free zoites increased ; suggesting that these were merozoites produced by schizogony .", "free zoites had active , circumaxial motility characteristic of s.  neurona merozoites observed in  vitro ( miller et  al . , 2001a ) .", "similar organisms did not grow in cultures inoculated with sporulated oocysts from a fourth sea lion , csl10266 or in un - inoculated cultures of ma104  cells ( table  4 ) . by ifat , antigen slides prepared from zoite cultures of all three sea lions , csl10089 , csl10092 and csl10100 , tested positive using serum from a horse with equine protozoal myeloencephalitis ( fig . ", "the horse 's s.  neurona infection was confirmed by ifat and western blot on serum ( 1:320 titer to laboratory strain snucd-1 ) and cerebral spinal fluid , and by immunohistochemistry used to detect s.  neurona merozoites in the lumbar spine .", "in addition , all three zoite isolates from sea lion oocyst cultures reacted to sera from hospitalized sea lion csl9878 ( table  4 ) which was itself seroreactive to merozoites of snucd-1 ( 1:2560 ) ( table  1 and fig . ", "sea lion culture antigen was ifat - negative ( < 1:40 ) to goat serum containing t.  gondii - reactive antibodies , bovine fetal serum containing n.  caninum - reactive antibodies , and , as shown in fig . ", "5e , to horse serum that was seronegative to s.  neurona and n.  hughesi .", "as soon as zoites were observed , supernatant from the ma104  cell cultures inoculated with sporulated , excysted oocysts from the fecal samples of csl10089 , csl10100 and csl10092 were collected for molecular characterization approximately once per week for 1114 weeks .", "pan - coccidian primers that span the its-1 region produced a 1000  bp band when dna was amplified from zoites taken from cultures from the 3 sea lions throughout their collection period .", "this is the expected band size for amplification of s.  neurona dna using pan - coccidian its-1 primers ( gibson et  al . , 2011 ) .", "in addition , a second band 400  bp in size was amplified simultaneously in culture supernatant of csl10100 and csl10092 using the pan - coccidian primers , but only in the first week of sample collection following zoite observation ( table  4 ) .", "four hundred basepairs is the expected band size for amplification of coccidia a , b or c using pan - coccidian its-1 primers ( colegrove et  al . , 2011 , gibson et  al . , 2011 ) .", "in culture csl10266 , in which no zoites ( only oocysts ) were observed , only the 400  bp band was amplified by pcr during the first two weeks of incubation .", "subsequent supernatant samples of culture csl10266 and all uninoculated ma104 control cultures were pcr - negative ( table  4 ) .", "sequence analysis confirmed that the 400  bp amplicon dna generated in early sampling of cultures csl10100 , csl10092 and csl10266 was identical to dna amplified from frozen , pelleted oocysts originally harvested from the same animal ; namely , coccidia a alone ( in the case of csl10092 and csl10266 ) or a dual infection with coccidia a and b ( in the case of csl10100 ) ( table  4 ) . by blast analysis , good quality partial sequences ( 700  bp ; abbreviated due to the presence of a homopolymer region ) generated from the 1000  bp amplicon in csl10089 , csl10092 and csl10100 zoite cultures were 99% similar to s. neurona dna isolated from horses ( af204230 & af081944 ) and from a skunk ( ay082648 ) ( see supplementary data ) .", "dinucleotide mixtures in the its-1 sequences indicated the presence of at least two s.  neurona - like genotypes in the sea lion cultures .", "these polymorphic regions shared nucleotide similarity to horse strains snucd-1 ( af081944 ) and sn - mu1 ( af204230 ) , sea otter strains so3639 ( dq084486 ) and so5259 , and a skunk isolate ( ay082648 ) ( see supplementary data ) . to further characterize the s.  neurona - like organisms", "cultured from sea lion feces , we amplified seven additional genetic loci in dna extracted from zoites in the sea lion oocyst culture supernatant .", "table  4 displays the pcr and sequencing results for these additional loci . using its1500 primers , with known specificity for s.  neurona or sarcocystis falcatula", "( gibson et  al . , 2011 ) , a 500  bp product was consistently amplified in all culture supernatant samples from csl10089 , csl10092 , csl10100 in which zoites had been observed . by blast analysis ,", "good quality partial its1500 amplicon sequences ( 300  bp , abbreviated due to the presence of a homopolymer region ) were 98% identical to s.  neurona isolated in southern sea otters including isolates so3528 , so3539 , so3485 , and so3501 ( genbank dq084485  dq084488 ) .", "microsatellite primers amplified 150  bp products containing di - nucleotide repeats ca17 ( sn7 ) and gt18 ( sn9 ) found in s.  neurona strain sn - mu1 and other s.  neurona strains isolated from opossums , sea otters , and cats ( rejmanek et  al . , 2010 ) .", "merozoite surface antigen sag1 gene sequences of csl10089 , csl10092 and csl10100 , amplified by snsag 1 - 5 - 6 primers , were 100% identical across 959  bp to s.  neurona strain snucd-1 ( af401682 ) ( ellison et  al . , 2002 ) and 99% similar across 968  bp to the representative sequence of the snsag1 allele amplified in samples from terrestrial and marine mammals ( gq851951 ) ( wendte et  al . , 2010 ) .", "sea lion cultures csl10089 , csl10092 and csl10100 were identical to each other across a 948  bp sequence of the merozoite surface antigen gene snsag4 and 100% identical to sequences of s.  neurona isolated from a sea otter ( so3106 , gq851957 ) and an opossum ( op134 , gq386979 ) . a unique sequence in sea lion s.  neurona - like isolates was identified in the merozoite surface antigen 3 ( snsag3 ) locus in which csl10089 was confirmed by bi - directional sequencing to contain an at insertion at positions 519520 ( table  5 ) .", "the same insert was observed in single direction sequencing for csl10092 but could not be confirmed in csl10100 due to poor sequence quality .", "the same insert was not identified in snsag3 sequences available in genbank originating in a variety of host species including sea otters , opossums and horses ( table  5 ) .", "lack of the position 519520 insert in laboratory strain snucd-1 , derived from a horse in california ( table  5 ) , ruled out the possibility that s.  neurona growth in sea lion oocyst cultures was due to laboratory contamination . at other polymorphic snsag3 nucleotide positions , including 239 and 1059 , sea lion s.  neurona - like isolates were similar to published opossum and sea otter isolates ( table  5 ) . in light of the unexpected findings revealed during cell culture analysis of fecal samples from stranded hospitalized sea lions , we screened all dna extracted from frozen , pelleted oocysts of both hospitalized and field - collected fecal samples using s.  neurona / s .  falcatula - specific its1500 primers . while none of the additional frozen , pelleted oocyst samples collected from hospitalized animals were pcr - positive for s.  neurona - like dna ( including those remaining for csl10089 , csl10092 and csl10100 ) , two fecal samples collected at point lobos ( fig .  3 , samples pl11 and pl27 ) produced 500  bp products in the its1500 pcr reaction ( table  3 ) .", "additional primers ( sn - its ) targeting the its-1 region of s.  neurona produced amplicons with sequences identical to hospitalized sea lion zoites collected in the present study as well as s.  neurona isolated in other animals ( see supplementary data ) .", "all other attempts to amplify s.  neurona in samples pl11 and pl27 using alternative primers failed with the exception of microsatellite sn7 which , in sample pl27 , produced a sequence containing the di - nucleotide repeat ca17 found in s.  neurona and zoites isolated in csl10089 , csl10092 and csl10100 ( table  4 ) ."], ["the investigation of infectious diseases in coastal aquatic wildlife allows us to probe the relationships between terrestrial - origin pathogen distribution , patterns of overland water runoff , and the risk of pathogen pollution in the coastal and ocean environment .", "the present research was undertaken to investigate the role of california sea lions in the dispersal and life cycle of terrestrial and aquatic coccidian parasites that pose a risk to marine mammal health , and to genetically and phenotypically characterize protozoa shed by sea lions .", "building on data acquired from previous protozoal pathogen surveillance efforts in california sea lions from the coastal waters of california ( carlson - bremer et  al . , 2012a ) , we confirmed that oocysts ( 810  m in size ) that are morphologically similar to , but genetically distinct from , t.  gondii and n.  caninum are frequently shed by stranded sea lions that are hospitalized and by free - ranging populations .", "genetic variability in the partial its-1 region of these coccidia a and b isolates was not observed within this study or in previous reports ( colegrove et  al . , 2011 , carlson - bremer et  al . ,", "consistent with other studies of stranded sea lions , shedding of coccidia a and b occurred primarily in yearlings , and those shedding oocysts had stranded due to causes other than protozoal infection .", "while our data further establish that sea lions are naturally asymptomatic definitive hosts of coccidia a and b , the pathogenic potential of these protozoa in other aquatic species requires further investigation , particularly considering the known virulence of coccidia c and other novel tissue - cyst forming coccidia in harbor seals and guadalupe fur seals ( colegrove et  al .", "phylogenetic analyses of the its-1 locus performed here and elsewhere ( gibson et  al . , 2011 , carlson - bremer et  al . , 2012a ) demonstrate that coccidia a , b , c , csl ao11 , and isolates from a harbor seal and guadalupe fur seals share a common ancestor with n.  caninum .", "morphologically , however , coccidia a and b measured in this study were smaller than n.  caninum oocysts and had < 80% pairwise similarity to n.  caninum at the its-1 locus . while unsporulated coccidia a and b oocysts shed by sea lions are 810  m in diameter , unsporulated n.  caninum oocysts shed by dogs are typically 1011  m in diameter ( mcallister et  al . , 1998 ) .", "sporulated n.  caninum oocysts are reportedly an average of 11.7    11.3  m ( lindsay et  al . , 1999 ) while sporulated forms of coccidia a and b are an average of 9.8    8.6  m , and 9.9    9.0  m , respectively . by comparison ,", "unsporulated t.  gondii oocysts are 10    12  m in diameter and sporulated forms are 11    13  m in diameter ( dubey et  al . , 1998 ) .", "more detailed genetic and morphologic studies ( e.g. whole genome sequencing , electron microscopy , and histological examination of life stages in situ ) are necessary to clarify the taxonomy of novel coccidia identified in aquatic animals , and will likely reveal that these organisms belong to either new neospora species or a new genus within the family sarcocystidae . at the phenotypic level , we observed that the sporulated oocysts of coccidia a and b were not cultivable in the african green monkey kidney cell line , ma104 , commonly used to propagate other tissue - cyst - forming coccidia including t.  gondii , n. caninum and s.  neurona ( miller et  al .", ", 2001a , miller et  al . , 2004 , holmberg et  al . , 2006 ) . while coccidia a dna was detectable by pcr in a culture prepared from oocyst", "isolate csl10266 for up to two weeks after inoculation , no protozoal organisms were ever observed . in ma104 cultures of oocyst", "isolates csl10092 and csl10100 , coccidia a and b dna as well as s.  neurona - like dna were amplified for up to 56 weeks post - inoculation ; after which time only s.  neurona - like dna and live s.  neurona - like zoites were detected for the duration of observation .", "while it is possible in the latter cases that coccidia a and b did not replicate in  vitro due to competition from the sarcosystsis sp . during co - culture , we hypothesize , rather , that the ma104  cell line may not be susceptible to infection with coccidia a and b , or the culture conditions were not optimal for these parasites .", "other cell culture systems should be investigated for their propagation , particularly in the interest of developing a serological assay for the purpose of screening marine mammals for infections with these and other potentially pathogenic protozoa .", "coccidia a and b did not cause disease in mice with known susceptibility to t.  gondii including b6.129s7-ifng / j , cba / caj and swiss webster ( cfw ) ( rytel and jones , 1966 , villegas et  al . , 2002 , gavrilescu and denkers , 2003 ) .", "surprisingly , coccidia a and b were not pathogenic to cba / caj mice despite previous studies showing that cba / ca mice can be used as a model of n.  caninum pathogenesis ( mcguire et  al .", "these findings , taken together with the lack of growth in ma104  cells or reactivity of serum from coccidia a- and b - infected sea lions with n.  caninum , t.  gondii or s.  neurona antigen by ifat , is further evidence of the taxonomic divergence of the sea lion parasites from known tissue - cyst forming coccidia with terrestrial life cycles . a significant outcome of this study was the discovery of s.  neurona or s.  neurona - like zoites growing in cell cultures inoculated with fecal material ( including sporulated , excysted oocysts of coccidia a and b ) prepared from three stranded hospitalized sea lions sampled between 2010 and 2012 .", "the cultivation of live protozoal zoites with strong genetic similarity to s.  neurona across multiple genes during parasite isolation from sea lion feces , particularly in the absence of microscopic evidence of sarcocystis sp .", "a possible explanation for these findings is that samples taken for microscopic observation during fecal flotation only represent a small portion of the overall feces sampled and the overall pool of oocysts being shed . if the original pool of isolated protozoa from feces consisted of a mixture of variable numbers of coccidian oocysts ( coccidia a and/or b ) and sporocysts ( s.  neurona - like ) , it is possible that the subdivision of the oocyst pool for flotation and microscopy , pelleting / freezing , sporulation for mouse bioassay , and sporulation / excystation for cell culture , could explain discrepancies in the ability to detect different parasites , depending on the sensitivity of each assay .", "we hypothesize that a small number of s.  neurona - like sporocysts existed in the pool of protozoa prepared for cell culture , but were not present in the subsamples designated for microscopy , pelleting and freezing or mouse bioassay .", "in addition , in  vitro cultivation may have facilitated the expansion of small numbers of s.  neurona sporozoites that were able to develop into schizonts that produced merozoites .", "the isolation of s.  neurona - like dna from feces of two free - ranging sea lions at point lobos indicates that the organism may occur naturally in sea lions , as opposed to occurring via nosocomial exposure in the hospitalized setting .", "exposure of sea lions to s.  neurona , or an s.  neurona - like organism , most likely occurs in coastal waters contaminated with opossum feces transported from land - to - sea via freshwater run - off ( carlson - bremer et  al .", ", 2012b , shapiro et  al . , 2012 ) or may be concentrated in sea lion prey , as has been shown with t.  gondii shed by terrestrial felids ( miller et  al . , 2002 , johnson et  al . , 2009 ) .", "although the extent of environmental contamination with s.  neurona sporocyts has not been documented , infections in southern sea otters and a number of other marine mammals of the pacific northwest have been reported ( miller et  al .", ", 2001b , miller et  al . , 2009 , miller et  al . , 2010 ,", "all mouse tissues were pcr - negative for s.  neurona using its1500 primers , indicating that the sea lion s.  neurona - like sporocysts were either not present in the inoculum , were present but below the threshold of infection , or the mice utilized were not susceptible . because of the similarity of coccidia a and b to t.  gondii , mice in this study were selected for their susceptibility to t.  gondii , not s.  neurona .", "shed by sea lions would benefit from the utilization of interferon gamma gene knock - out mice ( e.g. c57bl/6-black ) with well - characterized susceptibility to s.  neurona ( dubey et  al .", "sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis  a serious and progressively debilitating neurological disease in horses and is one of several coccidian parasites known to cause protozoal encephalitis in pacific harbor seals , southern sea otters , and other marine mammals ( lapointe et  al .", ", 1998 , rosonke et  al . , 1999 , lindsay et  al . , 2000 , lindsay et  al . , 2001 ,", "miller et  al . , 2001a , lapointe et  al . , 2003 , thomas et  al . , 2007 , gibson et  al . , 2011 , dubey et  al . , 2015", "although not frequently reported in the literature , when s.  neurona infections do occur in california sea lions , they are either clinically irrelevant ( gibson et  al . , 2011 ) or associated with myositis ( carlson - bremer et  al . , 2012b ) .", "tissue cysts caused by other sarcocystis spp . , including sarcocystis canis - like organisms , have been detected in sea lion muscle ( mense et  al .", ", 1992 , miller , 2008 ) and liver tissue ( mense et  al . , 1992 ,", "the three animals that shed s.  neurona - like protozoa were admitted to tmmc for malnutrition or trauma , and had no overt signs of protozoal infection .", "lack of serum titers to s.  neurona in these animals indicated that they had not yet mounted a detectable antibody response , or that they never had systemic infections .", "an elevated s.  neurona titer ( 1:2560 ) was detected in another hospitalized sea lion , csl9878 , but this animal also had no signs of disease and was not shedding detectable levels of s.  neurona - like sporocysts or dna at the time of sampling for this study .", "overall , findings from this study suggest that california sea lions are likely acting as mechanical vectors of s.  neurona or an s.  neurona - like organism , similar to the way canines can mechanically transmit t.  gondii ( lindsay et  al . , 1997 ) .", "using a mouse bioassay to detect the presence of infectious t.  gondii oocysts in canine tissues and feces , lindsay et  al .", "sporulated t.  gondii oocysts can traverse the canine intestinal tract and be excreted in an infectious state  .", "several experimental outcomes indicate that an s. neurona - like organism is excreted by sea lions in low concentrations , which is expected during mechanical transmission : ( 1 ) s.  neurona - like sporocysts were not observed by light microscopy following fecal flotation ( 2 ) s.  neurona - like dna was not amplified from concentrated oocysts ( pellets ) isolated from hospitalized sea lion fecal samples , ( 3 ) s.  neurona - like dna was only amplified by pcr on high copy number genes in concentrated oocysts isolated from feces of free - ranging animals , and ( 4 ) s.  neurona - like merozoites were only observed after long - term cultivation in ma104  cells .", "the genetic similarity of sea lion s.  neurona - like isolates to previously characterized protozoa in aquatic and terrestrial animals across much of the genome is not surprising considering the extensive overlap in genetic identity in s.  neurona isolates from the tissues of opossums , sea otters and horses that was previously demonstrated ( rejmanek et  al . , 2010 ,", "the key snsag3 polymorphism that we identified in the zoites from sea lion fecal samples may represent a novel s.  neurona genotype which , in the future , may facilitate identification of the origin of infection .", "this is particularly important as we seek to understand the flow of terrestrial pathogens into oceans and risk factors for s.  neurona infections in other aquatic species .", "continued investigations into the pathogenesis of s.  neurona in sea lions through necropsy and systematic surveillance for s.  neurona exposure , shedding , and disease will help clarify the importance of this host species in parasite transmission within the pacific coastal ecosystem . in conclusion , continued surveillance for tissue - cyst forming coccidia in marine mammals should remain a priority for programs that monitor the population health of aquatic wildlife .", "efforts to explore the distribution , pathogenesis and genetic diversity of sarcocystidae in land and sea isolates promise to improve our understanding of the interconnectedness between terrestrial and aquatic microbial diseases , and to direct interventions that will benefit our oceans and their inhabitants ."]]}
{"article_id": "PMC4353011", "article_text": ["bacterial infections \n as well as the emergence and spread of antibiotic \n resistance in human pathogens are serious public health problems in \n hospital and community settings across the globe .", "new strategies to prevent and treat bacterial infections \n are needed , including methods to overcome antibacterial resistance \n that results from the outer membrane permeability barrier in gram - negative \n organisms and targeting approaches that afford species- or pathogen - specific \n therapeutics .", "almost \n all \n bacterial species have a metabolic iron requirement and therefore \n employ various strategies to acquire this metal ion when colonizing .", "siderophores are high - affinity fe(iii ) chelators that are \n produced by bacteria under conditions of iron limitation , such as \n those encountered in the vertebrate host , to scavenge this metal ion \n from the environment .", "siderophore producers also \n express dedicated ferric siderophore import machinery and employ \n various mechanisms to release siderophore - bound iron following \n cellular uptake ( e.g. , reductive and/or hydrolytic release mediated \n by reductases and/or esterases , respectively ) .", "numerous studies support the importance of siderophore - based \n iron acquisition during bacterial infections .", "thus , the potential of using siderophores , or targeting siderophore biosynthetic and transport machineries , in therapeutic development continues to attract significant interest . \n of particular relevance to the advances described herein are prior \n investigations pertaining to the development of siderophore ", "this strategy has received particular attention for the delivery \n of antibiotics into gram - negative bacteria because these organisms \n are inherently less sensitive to many antibiotics used in the clinic \n as a result of the outer membrane permeability barrier .", "both native siderophores and synthetic siderophore mimics \n have been evaluated as platforms for therapeutic development . in the clinic , the native siderophore desferrioxamine \n b is used for iron - chelation therapy in patients with iron overload . \n", "several antibiotic small molecules found in nature called  sideromycins  \n provide inspiration for synthetic siderophore ", "the sideromycins are secondary \n metabolites comprised of a siderophore moiety and a toxic cargo ; \n the siderophore portion targets sideromycins to bacterial strains \n expressing the appropriate siderophore receptor .", "antibiotic conjugate produced by a clinical \n isolate of klebsiella pneumoniae , is an 84-residue \n antibacterial peptide with a glucosylated enterobactin ( ent , figure 1 ) derivative attached to its c - terminus that exhibits \n enhanced antibacterial activity against strains expressing the enterobactin \n receptor fepa . from the standpoints of \n antibacterial activity and therapeutic potential , studies of synthetic \n siderophore ", "antibiotic conjugates have provided the \n community with mixed results , causing some skepticism about the potential \n of siderophore - based approaches despite the successful utilization \n of such molecules by nature .", "structures of enterobactin ( 1 , \n ent ) and a generalized \n enterobactin  cargo conjugate .", "many of the failures encountered with early and recent studies \n of siderophore - based antibiotic delivery may be attributed , \n at least in part , to ( i ) use of non - native siderophores with \n relatively low fe(iii ) affinities and/or compromised receptor recognition ; ( ii ) modification of antibiotics such that the \n antibacterial activity is attenuated or lost completely ; ( iii ) bridging the siderophores and antibiotics with problematic \n linkers , including linkers designed for drug release that are either \n too stable or too labile , the latter of which promotes premature release ; and ( iv ) antibiotic resistance .", "nevertheless , \n the lessons of many unsuccessful studies highlight the complexity \n of siderophore - based therapeutic development and provide a foundation \n for inventing improved next - generation approaches .", "many of the issues \n described above may be overcome by careful molecular design and biological \n evaluation .", "in particular , the selection of appropriate native siderophore \n platforms and modification of these platforms in ways that do not \n compromise iron binding or receptor recognition , installation of an \n antibacterial cargo in such a manner that antibacterial activity is \n retained , and the development and application of assays that afford \n insight into the fate of siderophore  antibiotic conjugates \n are critical to the overall success of this approach .", "along such lines , \n a recent and insightful study by pfizer addressed complications associated \n with using relatively low - molecular - weight siderophore mimics in vivo .", "their results indicate \n that competition between the siderophore - conjugated monobactam \n mb-1 and native siderophores resulted in poor in vivo efficacy against pseudomonas aeruginosa and provide \n support for designing and evaluating siderophore ", "one recent \n and successful example based on a native siderophore platform \n is a mycobactin ", "artemisinin conjugate that exhibits enhanced \n antibacterial activity against mycobacterium tuberculosis compared to unmodified artemisinin .", "enterobactin ( ent , figure 1 ) is a triscatecholate \n siderophore biosynthesized by enteric bacteria and used for \n iron acquisition in the vertebrate host . motivated by the importance of ent in the host / microbe interaction \n as well as the decades of investigations pertaining to its ( bio)synthesis , \n coordination chemistry , and biology , in prior work we reported a synthetic \n route to monofunctionalized ent platforms .", "moreover , we established that the native ent platform , when monofunctionalized \n at the c5 position of one catecholate ring ( figure 1 ) , affords delivery of nontoxic small - molecule cargo across \n the outer membrane of gram - negative organisms that express ent uptake \n machinery ( e.g. , fepabcdg of escherichia coli ) .", "as described herein , this proof - of - concept study \n motivated us to demonstrate that ent effectively delivers antibacterial \n cargo to organisms that utilize ent for iron acquisition , thereby \n providing antibiotic targeting to specific sub - populations and a means \n to address antibiotic resistance that results from the gram - negative \n outer membrane permeability barrier . in this work", ", we present \n the syntheses and characterization of \n siderophore  antibiotic conjugates based on the native \n ent platform that harbor the clinically relevant -lactam antibiotics \n ampicillin ( amp ) and amoxicillin ( amx ) .", "these antibiotics block cell \n wall biosynthesis by inhibiting transpeptidases , also named \n penicillin binding proteins ( pbps ) , located in the periplasm of e. coli .", "we report that the ent--lactam conjugates \n exhibit significantly enhanced antibacterial activity ( up to 1000-fold ) \n against pathogenic e. coli and provide more rapid \n cell - killing than the parent -lactams as a result of ent - mediated \n delivery to the periplasm . moreover ,", "in proof - of - concept studies for \n species - specific killing , these conjugates selectively kill e. coli in the presence of staphylococcus aureus , a gram - positive organism that is more susceptible to the parent \n -lactams .", "these studies support the notion that native siderophore \n platforms provide an effective means to target molecular cargo to \n siderophore - utilizing organisms and to hijack siderophore \n uptake machinery to deliver cargos , including antibiotics , across \n the outer membrane permeability barrier of gram - negative microbes .", "dimethylformamide ( dmf ) and dichloromethane \n ( ch2cl2 ) were obtained from a vac solvent purification \n system ( vacuum atmospheres ) .", "anhydrous dimethyl sulfoxide ( dmso ) was \n purchased from sigma - aldrich and used as received .", "l - ent 1 , the d - enantiomer of benzyl - protected \n ent - co2h 2 , and the benzyl - protected ent - peg3-n33 were synthesized according to \n previously reported procedures .", "11-azido-3,6,9-trioxaundecan-1-amine \n was purchased from fluka . all other chemicals and solvents were purchased \n from sigma - aldrich or alfa aesar in the highest available purity and \n used as received .", "benzyl - protected ent - azide 3 ( 80 mg , 55 mol ) and pentamethylbenzene ( pmb , 147 mg , \n 990 mol ) were dissolved in 5 ml of anhydrous ch2cl2 to give a light yellow solution .", "this solution was \n cooled to 78 c in an acetone / dry ice bath under n2 , and bcl3 ( 660 l of 1 m solution in ch2cl2 , 660 mol ) was added slowly along the \n flask wall .", "after the solution was stirred for 1.5 h , dipea ( 300 l , \n 1.73 mmol ) was added to the flask , followed by meoh ( 2 ml ) to quench \n the reaction . the reaction was then warmed to room temperature , and \n the solvents were removed under reduced pressure . the resulting white \n solid was dissolved in 5:3 meoh/1,4-dioxane and purified by preparative \n hplc ( 33% b for 5 min and 3360% b over 20 min , 10 ml / min ) . \n", "the product eluted at 17 min and was lyophilized to yield compound 4 as white solid ( 13.9 mg , 28% ) .", "h nmr ( dmso - d6 , 500 mhz ) :  3.353.57 ( 16h , \n m ) , 4.384.41 ( 3h , m ) , 4.634.69 ( 3h , m ) , 4.894.96 \n ( 3h , m ) , 6.74 ( 2h , dd , j = 7.5 , 8.0 hz ) , 6.97 ( 2h , \n d , j = 7.5 hz ) , 7.35 ( 2h , d , j = \n 8.0 hz ) , 7.46 ( 1h , s ) , 7.94 ( 1h , s ) , 8.338.35 ( 1h , m ) , 9.12 \n ( 2h , d , j = 6.0 hz ) , 9.29 ( 1h , d , j = 6.0 hz ) , 9.44 ( 2h , bs ) , 9.76 ( 1h , bs ) , 11.6 ( 2h , bs ) , 11.9 ( 1h , \n bs ) .", "c nmr ( cdcl3 , 125 mhz ) :  50.1 , \n 51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 , 115.3 , 115.4 , 115.4 , \n 117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 , 148.7 , 148.7 , 150.8 , \n 166.0 , 168.4 , 169.1 , 169.6 , 169.7 .", "ir ( kbr disk , cm ) : 3389 , 2954 , 2928 , 2868 , 2111 , 1754 , 1645 , 1589 , 1535 , 1460 , 1384 , \n 1329 , 1266 , 1176 , 1132 , 1074 , 992 , 846 .", "11-azido-3,6,9-trioxaundecan-1-amine \n ( 36 l , 181 mol ) and d - bn6ent - cooh \n ( 2 , 177 mg , 142 mol ) were dissolved in 5 ml of \n dry ch2cl2 .", "pyaop ( 147 mg , 283 mol ) and \n dipea ( 98.5 l , 568 mol ) were added to give a light yellow \n solution .", "the reaction was stirred for 4 h at room temperature and \n concentrated , and the crude product was purified by preparative tlc \n ( 50% etoac / ch2cl2 ) to afford 5 as \n white foam ( 159 mg , 77% ) .", "h nmr ( dmso - d6 , 500 mhz ) :  3.33 \n ( 2h , j = 5.2 hz ) , 3.623.69 ( 14h , m ) , 4.024.06 \n ( 3h , m ) , 4.154.18 ( 3h , m ) , 4.914.94 ( 3h , m ) , 5.045.21 \n ( 12h , m ) , 6.96 ( 1h , s ) , 7.117.45 ( 36h , m ) , 7.657.67 \n ( 2h , m ) , 7.857.85 ( 1h ,", "m ) , 7.977.97 ( 1h , m ) , 8.508.54 \n ( 3h , m ) .", "c nmr ( cdcl3 , 125 mhz ) :  25.6 , \n 29.5 , 38.8 , 40.0 , 45.3 , 51.3 , 51.4 , 63.9 , 64.1 , 69.8 , 39.8 , 70.0 , \n 70.3 , 70.4 , 70.4 , 71.2 , 71.2 , 76.3 , 76.3 , 116.8 , 117.5 , 120.4 , 123.0 , \n 124.3 , 125.4 , 126.1 , 126.2 , 127.6 , 127.6 , 127.9 , 128.2 , 128.3 , 128.4 , \n 128.4 , 128.5 , 128.5 , 128.6 , 128.6 , 128.8 , 128.8 , 128.9 , 129.0 , 130.1 , \n 135.4 , 135.7 , 135.9 , 136.0 , 136.1 , 146.8 , 146.9 , 149.1 , 151.6 , 151.8 , \n 164.3 , 164.9 , 164.9 , 165.8 , 168.9 , 169.0 , 169.1 , 176.2 . ir ( kbr disk , \n cm ) : 3357 , 3062 , 3032 , 2958 , 2923 , 2859 , 2104 , \n 1751 , 1551 , 1576 , 1515 , 1455 , 1375 , 1345 , 1299 , 1264 , 1204 , 1126 , \n 1082 , 1040 , 1018 , 957 , 915 , 854 , 811 . hrms ( esi ) :", "compound 6 was synthesized \n from 5 ( 153 mg , 105 mol ) following the same procedure \n as for compound 4 .", "the crude reaction was purified by \n preparative hplc ( 33% b for 5 min and 3360% b over 20 min , \n 10 ml / min ) .", "the product eluted at 17.0 min and was lyophilized to \n yield compound 6 as white solid ( 31 mg , 33% ) .", "h nmr ( cdcl3 , 500 mhz ) :  3.343.56 ( 16h , \n m ) , 4.394.41 ( 3h , m ) , 4.614.66 ( 3h , m ) , 4.884.94 \n ( 3h , m ) , 6.74 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h , \n d , j = 7.8 hz ) , 7.33 ( 2h , d , j = \n 7.8 hz ) , 7.44 ( 1h , s ) , 7.91 ( 1h , s ) , 8.318.33 ( 1h , m ) , 9.129.13 \n ( 2h , m ) , 9.279.28 ( 1h , m ) , 9.50 ( 2h , bs ) , 9.84 ( 1h , bs ) , 11.6 \n ( 2h , bs ) , 11.9 ( 1h , bs ) .", "c nmr ( cdcl3 , 125 \n mhz ) :  50.1 , 51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 , \n 115.3 , 115.4 , 115.4 , 117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 , \n 148.7 , 148.7 , 150.8 , 166.0 , 168.4 , 169.1 , 169.6 , 169.7 .", "ir ( kbr disk , \n cm ) : 3390 , 2958 , 2925 , 2863 , 2110 , 1754 , 1645 , \n 1589 , 1535 , 1460 , 1384 , 1342 , 1262 , 1176 , 1117 , 1074 , 841 , 800 .", "hrms \n ( esi ) : [ m+na]m / z calcd \n 936.2506 , found 936.2512 .", "5-hexynoic acid ( 113 l , 1.00 \n mmol ) and thionyl chloride ( 1.00 ml , 13.8 mmol ) were combined and \n refluxed for 1 h. the reaction was cooled to room temperature and \n concentrated under reduced pressure , and the resulting crude acyl \n chloride was dissolved in acetone ( 0.5 ml ) and carried on to the next \n step without purification .", "ampicillin sodium salt ( 186 mg , 0.500 mmol ) \n was dissolved in a solution of nahco3 ( 210 mg , 2.5 mmol ) \n in 4:1 water / acetone ( 2.5 ml ) and cooled on ice , to which the acyl \n chloride was added slowly with stirring .", "the reaction was subsequently \n warmed to room temperature and stirred for 1 h. water ( 3 ml ) was added \n to the reaction , and the aqueous phase was washed with etoac ( 2  \n 10 ml ) , acidified to ph 2 by addition of hcl , and extracted with etoac \n ( 20 ml ) .", "the resulting organic phase was washed with cold water ( 2 \n  5 ml ) , dried over na2so4 , and concentrated \n under reduced pressure . the crude reaction was triturated with hexanes , \n which afforded a yellow solid ( 180 mg , 77% ) .", "h nmr ( dmso - d6 , \n 500 mhz ) :  1.41 ( 3h , s ) , 1.55 ( 3h , s ) , 1.641.69 ( 2h , \n m ) , 2.132.16 ( 2h , m ) , 2.292.32 ( 2h , m ) , 2.772.78 \n ( 1h , m ) , 4.20 ( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) , \n 5.52 ( 1h , dd , j = 4.0 , 8.0 hz ) , 5.70 ( 1h , d , j = 8.0 hz ) , 7.257.43 ( 5h , m ) , 8.57 ( 1h , d , j = 8.0 hz ) , 9.11 ( 1h , d , j = 8.0 hz ) .", "c nmr ( dmso - d6 , 125 mhz ) :  \n 17.4 , 24.4 , 26.6 , 30.4 , 33.8 , 55.5 , 58.1 , 63.7 , 67.3 , 70.3 , 71.5 , \n 84.2 , 127.2 , 127.6 , 128.2 , 138.2 , 169.0 , 170.2 , 171.5 , 173.5 .", "ir ( kbr \n disk , cm ) : 3297 , 3058 , 3023 , 2970 , 2937 , 2863 , \n 2626 , 2526 , 2120 , 1780 , 1688 , 1518,1455 , 1437 , 1390 , 1373 , 1324 , 1295 , \n 1208 , 1139 , 1027 , 1001 , 843 .", "compound 8 was synthesized \n as described for compound 7 except that amoxicillin ( 760 \n mg , 2.1 mmol ) was used instead of ampicillin sodium salt .", "compound 8 was obtained as light yellow solid ( 533 mg , 56% ) after trituration \n and employed without further purification .", "h nmr ( dmso - d6 , 500 mhz ) : \n  1.42 ( 3h , s ) , 1.56 ( 3h , s ) , 1.631.68 ( 2h , m ) , 2.122.15 \n ( 2h , m ) , 2.252.29 ( 2h , m ) , 2.762.78 ( 1h , m ) , 4.19 \n ( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) , 5.515.54 \n ( 2h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) , 7.19 ( 2h , d , j = 8.5 hz ) , 8.41 ( 1h , d , j = 8.0 hz ) , \n 8.93 ( 1h , d , j = 8.0 hz ) , 9.40 ( 1h , bs ) .", "c nmr ( dmso - d6 , 125 mhz ) :  17.6 , \n 24.5 , 26.8 , 30.4 , 34.0 , 55.4 , 58.2 , 63.9 , 71.6 , 71.7 , 84.4 , 115.1 , \n 128.4 , 128.7 , 128.8 , 157.0 , 169.2 , 170.9 , 171.7 , 173.8 . ir ( kbr disk , \n cm ) : 3356 , 3294 , 3045 , 2970 , 2928 , 1770 , 1738 , \n 1650 , 1615 , 1515 , 1457 , 1373 , 1208 , 1009 , 945 , 839 , 815 . hrms ( esi ) : \n [ m+na]m / z calcd 460.1537 , \n found 460.1534 .", "compound 7 ( 60 mg , 0.13 \n mmol ) was dissolved in 1:1 h2o / mecn ( 5 ml ) , and tfa was \n added to a final concentration of 1% .", "the solution was incubated at \n 37 c for 24 h and purified by preparative hplc ( 2050% \n b over 25 min , 10 ml / min ) , which afforded a white powder ( 12 mg , 25% ) . \n", "the white powder is a diastereomeric mixture of products , and no further \n separation was performed .", "h nmr ( dmso - d6 , 500 mhz ) :  ( mixture of two diastereomers ) 1.221.23 \n ( 3h , pair of s ) , 1.541.58 ( 3h , pair of s ) , 1.621.68 \n ( 2h , m ) , 2.122.14 ( 2h , m ) , 2.282.30 ( 2h , m ) , 2.77 \n ( 1h , s ) , 3.183.24 ( 0.5h , m ) , 3.303.36 ( 0.5h , m ) , 3.443.49 \n ( 0.5h , m ) , 3.553.60 ( 0.5h , m ) , 3.92 ( 0.5h , s ) , 4.01 ( 0.5h , \n s ) , 4.67 ( 0.5h , dd , j = 6.7 , 6.5 hz ) , 4.78 ( 0.5h , \n dd , j = 5.2 , 5.2 hz ) , 5.455.48 ( 1h , m ) , 7.277.40 \n ( 5h , m ) , 8.508.61 ( 2h , m ) .", "c nmr ( dmso - d6 , 125 mhz ) :  ( mixture of two diastereomers ) \n 18.1 , 25.0 , 27.6 , 28.1 , 28.4 , 29.5 , 34.4 , 42.2 , 56.8 , 56.8 , 72.1 , \n 72.2 , 84.8 , 127.9 , 128.2 , 128.9 , 139.2 , 139.4 , 171.2 , 171.4 , 172.0 . \n", "ir ( kbr disk , cm ) : 3297 , 3071 , 3041 , 2967 , 2938 , \n 2535 , 2124 , 1734 , 1653 , 1527 , 1456 , 1427 , 1375 , 1299 , 1199 , 1137 , \n 1070 , 1027 , 836 . hrms ( esi ) :", "compound 10 was synthesized \n as described for compound 9 except that compound 8 was used instead of 7 ( 60 mg , 0.13 mmol ) .", "the \n product was purified by preparative hplc ( 2050% b over 25 \n min , 10 ml / min ) , and obtained as white powder ( 14.5 mg , 24% ) .", "h nmr ( dmso - d6 , 500 mhz ) :  \n ( mixture of two diastereomers ) 1.26 ( 3h , s ) , 1.551.59 ( 3h , \n pair of s ) , 1.611.67 ( 2h , m ) , 2.122.14 ( 2h , m ) , 2.242.27 \n ( 2h , m ) , 2.77 ( 1h , s ) , 3.203.26 ( 0.5h , m ) , 3.343.39 \n ( 0.5h , m ) , 3.423.47 ( 0.5h , m ) , 3.563.60 ( 0.5h , m ) , \n 4.03 ( 0.5h , s ) , 4.12 ( 0.5h , s ) , 4.68 ( 0.5h , dd , j = 6.5 , 6.5 hz ) , 4.79 ( 0.5h , dd , j = 5.5 , 5.5 hz ) , \n 5.285.31 ( 1h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) , \n 7.17 ( 2h , d , j = 8.5 hz ) , 8.368.50 ( 3h , m ) .", "c nmr ( dmso - d6 , 125 mhz ) :  \n ( mixture of two diastereomers ) 17.5 , 24.3 , 26.9 , 27.5 , 27.7 , 28.8 , \n 33.8 , 41.4 , 55.7 , 55.8 , 71.4 , 71.6 , 84.2 , 114.8 , 115.0 , 128.5 , 128.7 , \n 128.8 , 156.9 , 158.3 , 158.6 , 171.2 , 171.3 .", "ir ( kbr disk , cm ) : 3301 , 3071 , 3028 , 2973 , 2928 , 2548 , 2111 , 1737 , 1662 , 1606 , 1593 , \n 1515 , 1435 , 1377 , 1197 , 1139 , 837 . hrms ( esi ) :", "ampicillin - alkyne 7 ( 120 l \n of an 80 mm solution in dmso , 9.6 mol ) and \n ent - peg3-n34 ( 250 l of \n a 13 mm solution in 1,4-dioxane , 3.3 mol ) were combined , and \n 400 l of dmso was added .", "cuso4 ( 100 l of \n a 90 mm solution in water , 9.0 mol ) and tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta , 200 l of \n a 50 mm solution in dmso , 10 mol ) were combined , and 100 l \n of dmso was added to give a blue - green solution , to which naasc ( 400 \n l of a 90 mm solution in water , 36.0 mol ) was added . \n this solution became light yellow and was immediately added to the \n alkyne / azide solution .", "the reaction was shaken on a benchtop rotator \n for 2 h at room temperature , diluted by 3- to 4-fold with 1:1 mecn / water , \n centrifuged ( 13,000 rpm  10 min , 4 c ) , and purified by \n semi - preparative hplc ( 20% b for 5 min and 20%50% b over 11 \n min , 4 ml / min ; 0.005% tfa was used in the solvent system to prevent \n decomposition of the -lactam ) .", "the hplc fractions containing 11 were collected manually and flash frozen in liquid n2 immediately after collection to prevent -lactam decomposition . \n", "h nmr ( dmso - d6 , 500 mhz ) :  1.40 \n ( 3h , s ) , 1.54 ( 3h , s ) , 1.781.81 ( 2h , m ) , 2.27 ( 2h , t , j = 6.8 hz ) , 2.58 ( 2h , t , j = 6.5 hz ) , \n 3.48 ( 12h , m ) , 3.76 ( 2h , s ) , 4.19 ( 1h , s ) , 4.384.44 ( 5h , m ) , \n 4.644.66 ( 3h , m ) , 4.914.92 ( 3h , m ) , 5.39 ( 1h , d , j = 3.5 hz ) , 5.515.52 ( 1h , m ) , 5.72 ( 1h , d , j = 7.5 hz ) , 6.74 ( 2h , dd , j = 7.8 , 7.8 \n hz ) , 6.96 ( 2h , d , j = 7.5 hz ) , 7.267.35 ( 5h , \n m ) , 7.427.45 ( 3h , m ) , 7.81 ( 1h , s ) , 7.92 ( 1h , s ) , 8.33 ( 1h , \n s ) , 8.55 ( 1h , d , j = 7.5 hz ) , 9.12 ( 3h , d , j = 7.0 hz ) , 9.29 ( 1h , d , j = 6.5 hz ) , \n 9.42 ( 2h , bs ) , 9.74 ( 1h , s ) , 11.6 ( 2h , s ) , 11.9 ( 1h , bs ) , 13.35 ( 1h , \n bs )", ". hrms ( esi ) : [ m+na]m / z calcd 1379.4021 , found 1379.4046 .", "compound 12 was \n synthesized as described for 11 except that compound 8 was used instead of compound 7 .", "h nmr ( dmso - d6 , 500 mhz ) :  1.40 ( 3h , s ) , 1.54 ( 3h , \n s ) , 1.781.80 ( 2h , m ) , 2.23 ( 2h , t , j = 6.5 \n hz ) , 2.57 ( 2h , t , j = 6.5 hz ) , 3.47 ( 12h , m ) , 3.76 \n ( 2h , bs ) , 4.18 ( 1h , s ) , 4.394.43 ( 5h , m ) , 4.634.65 \n ( 3h , m ) , 4.90 ( 3h , bs ) , 5.38 ( 1h , s ) , 5.525.56 ( 2h , m ) , 6.68 \n ( 2h , d , j = 8.5 hz ) , 6.73 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h , d , j = 7.5 ) , 7.19 ( 2h , \n d , j = 8.5 hz ) , 7.33 ( 2h , d , j = \n 7.5 hz ) , 7.44 ( 1h , s ) , 7.80 ( 1h , s ) , 7.92 ( 1h , s ) , 8.338.39 \n ( 2h , m ) , 8.94 ( 1h , d , j = 8.0 hz ) , 9.119.12 \n ( 2h , m ) , 9.29 ( 1h , bs ) , 9.389.43 ( 3h , m ) , 9.75 ( 1h , s ) , 11.6 \n ( 2h , bs ) , 11.9 ( 1h , bs ) .", "compound 13 was \n synthesized as described for 11 except \n that compound 6 was used instead of compound 4 .", "hrms ( esi ) : \n [ m+na]m / z calcd 1379.4021 , \n found 1379.4022 .", "compound 14 was synthesized as described for 12 except \n that compound 6 was used instead of compound 4 .", "hrms ( esi ) : \n [ m+na]m / z calcd 1395.3970 , \n found 1395.3995 .", "compound 15 was synthesized as described for 11 except that compound 9 was used instead of compound 7 .", "hrms ( esi ) : [ m+h]m / z calcd 1331.4409 , found \n 1331.4389 .", "compound 16 was synthesized as described for 12 except that compound 10 was used instead of compound 8 .", "hrms ( esi ) : [ m+na]m / z calcd 1369.4177 , found \n 1369.4191 .", "general microbiology materials and methods , including \n details of \n ent - amp / amx stock solution preparation and storage , are provided as supporting information .", "overnight cultures of \n the bacterial strains ( table s1 ) were prepared \n in 15-ml polypropylene tubes by inoculating 5 ml of lb media with \n the appropriate freezer stock .", "the overnight cultures were incubated \n at 37 c for 1618 h in a tabletop incubator shaker set \n at 150 rpm and housing a beaker of water .", "each overnight culture was \n diluted 1:100 into 5 ml of fresh lb media containing 200 m \n 2,2-dipyridyl ( dp ) and incubated at 37 c with shaking \n at 150 rpm until od600 reached 0.6 .", "g / l ) with or without 200 m \n dp to achieve an od600 value of 0.001 .", "a 90-l aliquot \n of the diluted culture was combined with a 10-l aliquot of \n a 10 solution of the antibiotic or ent - antibiotic conjugate \n in a 96-well plate , and the covered plate was wrapped in parafilm \n and incubated at 30 c with shaking at 150 rpm for 19 h in a \n tabletop incubator housing a beaker of water .", "bacterial growth was \n determined by measuring od600 ( end point analysis ) using \n a biotek synergy ht plate reader .", "each well condition was prepared \n in duplicate and at least three independent replicates using two different \n synthetic batches of each conjugate were conducted on different days . \n", "the resulting mean od600 values are reported , and the error \n bars are the standard error of the mean ( sem ) obtained from the independent \n replicates .", "these assays were performed with e. coli atcc \n 35218 and k. pneumoniae atcc 13883 following the \n general procedure except that sulbactam ( sb ) or potassium clavulanate \n ( pc ) were mixed with ampicillin or amoxicillin and the ent - amp or \n ent - amx conjugates , respectively .", "the molar ratios of the inhibitor/-lactam \n mixtures were sulbactam / amp or ent - amp , 1.5:1 , and potassium clavulanate / amx \n or ent - amx , 0.9:1 .", "these assays were \n performed with e. coli k-12 and \n cft073 following the general procedure except that varying concentrations \n ( 1100 m ) of synthetic l - ent were mixed with \n ent - amp / amx .", "this assay is based on a published protocol and was conducted with e. coli cft073 . \n", "each overnight culture was serially diluted into \n m9 minimal medium to provide 1010 cfu / ml . \n", "lipocalin 2 was diluted in pbs to a concentration of 10 m upon \n arrival , aliquoted , and stored at 20 c until use .", "a \n 90-l aliquot of the diluted culture was added to each well \n of a 96-well plate that contained varying concentrations of lipocalin \n 2 , ent - amp , and ent , and the final volume was adjusted to 100 l \n with sterile pbs .", "the 96-well plate was incubated at 37 c for \n 24 h in a tabletop incubator set at 150 rpm , and bacterial growth \n was determined by measuring od600 using a plate reader . \n", "each well condition was repeated at least three times independently \n on different days and with different batches of lipocalin 2 .", "the resulting \n mean od600 is reported , and the error bars are the sem .", "a 5-ml overnight culture of e. coli k-12 or cft073 was grown in lb ( vide supra ) and diluted 1:100 into 5 ml of fresh lb media containing 200 m \n dp , and this culture was incubated at 37 c with shaking at 150 \n rpm in a tabletop incubator housing a beaker of water until od600 reached 0.3 .", "the culture was centrifuged ( 3000 \n rpm  10 min , rt ) , and the resulting pellet was washed twice \n by resuspension in 50% mhb and centrifugation ( 3000 rpm  10 \n min , rt ) .", "the resulting pellet was resuspended in 50% mhb with or \n without dp , and the od600 was adjusted to 0.3 .", "a 90-l \n aliquot of the resulting culture was mixed with a 10-l aliquot \n of a 10 solution of amp / amx or the ent - amp / amx in a 96-well \n plate , which was covered , wrapped in parafilm , and incubated at 37 \n c with shaking at 150 rpm .", "the od600 values were \n recorded at t = 0 , 1 , 2 , and 3 h by using a plate \n reader . in a parallel experiment ,", "a 10-l aliquot of the culture \n was taken at t = 0 , 1 , 2 , and 3 h , serially diluted \n by using sterile phosphate - buffered saline ( pbs ) , and plated on lb - agar \n plates for colony counting ( cfu / ml ) .", "the resulting \n mean od600 or cfu / ml is reported , and the error bars are \n the sem .", "a 5 ml overnight culture of each \n bacterial strain was grown in lb , diluted 1:100 into 5 ml of fresh \n lb media containing 200 m dp , and incubated at 37 c with \n shaking at 150 rpm in a tabletop incubator housing a beaker of water \n until od600 reached 0.6 .", "each mid - log - phase culture was \n diluted to 10 cfu / ml in 50% mhb with or without 200 m \n dp . for experiments requiring a mixture of two species ,", "a 1:1 mixture \n was prepared ( 10 cfu / ml for each strain ) in 50% mhb with \n or without 200 m dp from the mid - log - phase cultures . to confirm \n cfu / ml of each culture ,", "the single- and double - species cultures were \n serially diluted by using sterile pbs , and aliquots were plated on \n a chrom - uti plate (  starter - culture plate  ) .", "for each \n cell - killing experiment , a 90-l aliquot of each culture was \n combined with a 10-l aliquot of a 10 m solution of the \n antibiotic or ent - antibiotic conjugate in a 96-well plate , which was \n covered , wrapped in parafilm , and incubated at 30 c with shaking \n at 150 rpm for 19 h. bacterial growth was assayed both by measuring \n od600 using the plate reader and by plating on chrom - uti \n plates after serial dilution (  assay plate  ) .", "each well \n condition was repeated at least three times independently on different \n days .", "the resulting mean od600 is reported , and the error \n bars are the sem .", "we aimed to harness our enterobactin - mediated \n cargo delivery strategy to enable the transport of toxic cargo across \n the outer membrane of e. coli . to address this goal , \n we linked the -lactam antibiotics ampicillin ( amp ) and amoxicillin \n ( amx ) to a monofunctionalized ent scaffold where ent is derivatized \n at the c5 position of one catechol ring via a flexible and stable \n peg3 linker .", "we selected amp and amx as antibacterial cargo \n for several reasons : these molecules are commercially available and \n amenable to synthetic modification , retain antibacterial activity \n when appropriately modified , possess periplasmic targets in gram - negative \n bacteria and must cross the outer membrane to be active against these \n species , and have relatively low molecular weights .", "we selected low - molecular - weight \n antibiotics because our prior studies of ent - mediated cargo transport \n indicated that the ent transport machinery of e. coli k-12 imports ent - cargo conjugates harboring relatively small cargos \n ( e.g. , cyclohexane , naphthalene , phenylmethylbenzene ) \n to the cytosol readily , whereas large cargos ( e.g. , vancomycin ) are \n not transported to the cytosol .", "moreover , \n in prior studies , various -lactams including amp and amx have \n been linked to simple catechols and more complex catechol - containing siderophores \n or mimics thereof , which provides the \n opportunity to compare the outcomes obtained for different siderophore - inspired \n design strategies . in scheme 1", ", we present the syntheses of \n ent - amp 11 and ent - amx 12 , which feature \n installation of alkyne - modified -lactam warheads onto ent - azide 4 via copper - catalyzed azide - alkyne cycloaddition ( hereafter \n click reaction ) in the final step .", "the catechol moieties of benzyl - protected \n ent - azide 3 were deprotected by using bcl3 at 78 c to achieve ent - azide 4 as a white \n powder in 28% yield following purification by reverse - phase preparative \n hplc . catalytic hydrogenation using hydrogen gas and a pd / c catalyst \n is typically employed to deprotect ent catechols ; however , we observed that amp / amx decompose under these conditions \n and poison the pd / c catalyst .", "moreover , deprotection of the enterobactin \n catechols prior to installing the -lactams requires preservation \n of the azide moiety , and we therefore employed bcl3 for \n this reaction .", "initial attempts at assembling ent - amp / amx using standard \n conditions for the copper - catalyzed click reactions with ent - azide 4 and the alkyne - modified -lactams 7/8 , prepared by thionyl chloride coupling of 5-hexynoic acid \n to the amino group of amp / amx , failed because of copper - mediated -lactam \n decomposition .", "this problem was overcome \n by including the metal - ion chelator tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta ) in the click reactions , and ent - amp and ent - amx were obtained as \n white powders in high purity and yields of 66% and 76% , respectively , \n following semi - preparative hplc purification .", "it was necessary to \n perform hplc purification with eluents containing only 0.005% tfa \n to prevent decomposition of the acid - sensitive -lactam moieties . \n", "this synthetic route was likewise employed to prepare the d - enantiomers of ent - amp / amx 13 and 14 ( scheme 1 ) .", "deprotected ent - azide 4 enables alkyne - functionalized \n molecules to be covalently linked to ent via a click reaction , including \n molecules that are incompatible with reaction conditions required \n to deprotect the ent catechols .", "moreover , the deprotected ent - azide \n may be employed to append ent to surfaces , other materials , or biomolecules \n harboring alkyne groups .", "indeed , very \n few examples employing the copper(i)-catalyzed click reaction with \n fused -lactams are reported in the literature , and it is likely that this paucity stems from the fact that -lactams \n are incompatible with standard conditions for copper - catalyzed azide - alkyne \n cycloaddition .", "the conditions defined \n in this work employing tbta allow for copper - catalyzed triazole formation \n and preserve the -lactam warhead . in the absence of fe(iii ) , \n ent - amp / amx exhibit an absorption band centered at 316 nm resulting \n from catecholate absorption and the solutions are colorless ( meoh , \n rt ) .", "addition of 1.0 equiv of fe(iii ) to methanolic solutions of ent - amp \n and ent - amx causes the solution to immediately change from colorless \n to purple - red , and a broad absorption feature in the 400700 \n nm range appears ( figure s1 ) , confirming \n that both ent - amp / amx readily chelate fe(iii ) . to ascertain whether ent - amp / amx provide antibacterial activity \n against e. coli , including pathogenic strains , we preformed antimicrobial activity assays using \n six strains ( table s1 , figures 2 , s2s7 ) .", "e. coli atcc 25922 is a laboratory susceptibility test strain \n originally obtained as a clinical isolate .", "coli uti89 and \n cft073 are both pathogens of the human \n urinary tract ( upec).e .", "coli atcc 35401 ( serotype o78:h11 ) is an enterotoxigenic ( etec ) strain \n that was isolated from human feces .", "e. coli atcc \n 43895 ( serotype o157:h7 ) is an enterohemorrhagic ( ehec ) strain that \n was isolated from raw hamburger meat implicated in a hemorrhagic colitis \n outbreak .", "both etec and ehec strains produce virulence factors and \n toxins and cause diarrhea in humans .", "all e. coli strains biosynthesize ent for iron acquisition and \n express the ent receptor fepa .", "some e. coli strains \n employed in this work also have the capacity to produce and utilize \n salmochelins , c - glucoyslated ent derivatives .", "these molecules are produced by salmonella spp . \n and pathogenic e. coli strains for iron acquisition . \n", "the iroa gene cluster ( irobcden ) \n encodes proteins required for the biosynthesis and transport of salmochelins , \n and iron is the outer membrane receptor for salmochelins encoded by \n the iroa cluster .", "studies with salmonella indicate that iron has the ability to transport \n ent as well as its glucosylated forms . of the strains considered in this work , e. coli cft073 and uti89 harbor the iroa gene cluster .", "e. coli h9049 does not produce salmochelins , and a blast search using available e. \n coli genomes reveals that the e. coli 43985 \n genome does not contain the iroa cluster .", "the genome \n for e. coli atcc 25922 is unpublished ; however , this \n strain is reported to be sensitive to lipocalin-2 ( vide infra ) , which suggests that its genome does \n not encode the iroa cluster .", "lastly , it should be noted that e. coli cft073 is \n celebrated for having redundant iron import machineries , and this \n strain also harbors the iha gene , which encodes the \n outer membrane ent receptor iha that is distinct from fepa .", "antibacterial activity of ent - amp / amx against various e. \n coli strains that include human pathogens .", "all assays \n were performed in 50% mhb medium supplemented with 200 m dp \n to provide iron - limiting conditions ( mean  sem , n  3 ) .", "the data for assays performed in the absence of dp \n are presented in figures s2s7 .", "we performed antibacterial activity \n assays using a 10-fold dilution \n series to compare the abilities of ent - amp / amx and unmodified amp / amx \n to kill e. coli ( figures 2 , s2s7 ) .", "these assays were conducted \n in 50% mhb and in the absence or presence of 200 m dp .", "the \n latter growth conditions provide iron limitation and result in expression \n of the ent uptake machinery fepabcdg .", "amp / amx exhibit minimum inhibitory \n concentration ( mic ) values of 10 m against these e. \n coli strains regardless of the presence of dp in the growth \n medium ( figures 2 , s2s7 ) .", "all six e. coli strains are more susceptible \n to ent - amp / amx than amp / amx under conditions of iron limitation ( figure 2 ) .", "based on the 10-fold dilution series , ent - amp / amx \n are 100-fold more potent against e. coli 25922 , uti89 , \n and h9049 under iron - limiting conditions .", "although the mic values \n for 35401 and 43895 are only ca . 10-fold higher than for amp / amx in \n this assay , a significant reduction in growth is observed at 100 nm \n ent - amp / amx , whereas this concentration of amp / amx affords no growth \n inhibition .", "the enhanced sensitivity of e. coli cft073 \n to ent - amp / amx is remarkable .", "this strain exhibits the greatest sensitivity \n to ent - amp / amx , providing a 1000-fold decreased mic value ( 10 nm ) , \n and growth inhibition in the presence of 1 nm of the conjugate . \n", "moreover , in the absence of dp , e. coli cft073 exhibits \n the greatest susceptibility to ent - amp / amx ( figures \n s2s7 ) .", "a noteworthy characteristic of cft073 is its \n multiple mechanisms for iron acquisition , and we hypothesize that \n the presence of multiple receptors that recognize and transport ent \n ( fepa , iron , iha ) contributes to this enhanced sensitivity .", "cft073 \n and uti89 both express iron , which may indicate that iha is responsible \n for the enhanced susceptibility of cft073 ; however , we can not rule \n out the possibility that the conserved receptors function differently \n or exhibit different expression levels depending on the strain . moreover", ", \n other unappreciated mechanisms may contribute to the potent bactericidal \n action exhibited by ent - amp / amx against cft073 and other strains . to gain further insight into the mechanism \n of ent - amp / amx antibacterial action", ", we performed a series of experiments \n with the standard laboratory strain e. coli k-12 . \n", "we employed the same 10-fold dilution series to compare the activities \n of ent - amp / amx and amp / amx against k-12 . in the absence of dp ,", "ent - amp / amx \n and amp / amx exhibit comparable mic values , with complete killing observed \n at 10 m . at lower concentrations , ent - amp / amx exhibit \n slightly greater antibacterial activity than unmodified amp / amx ( figure 3a ) .", "this phenomenon is most evident at a conjugate / drug \n concentration of 1 m , where ent - amp / amx inhibit e. \n coli k-12 growth to varying degrees and amp / amx do not affect \n bacterial growth . under conditions of iron limitation ,", "a 100-fold \n reduction in mic value ( 10 m to 100 nm ) for ent - amp / amx is \n observed , and ca .", "50% growth inhibition occurs at 10 nm of each conjugate \n ( figure 3b ) .", "these trends are comparable to \n those observed for e. coli atcc 25922 , h9049 , and \n uti89 ( figures 2 , s2 , s3 , \n and s5 ) .", "when the antibacterial activity assay was performed \n using a 1:1 ratio of native l - ent and amp / amx , no reduction \n of amp / amx mic values was observed ( figure 3c ) , which suggests the conjugation between ent and the -lactams \n is required for the enhanced bactericidal action", ". moreover , treatment \n of e. coli k-12 with the iron - bound forms of ent - amp / amx , \n obtained by pre - incubating each conjugate with 1 equiv of ferric chloride , \n afforded the same mic values as observed for apo ent - amp / amx .", "these \n results indicate that the enhanced antibacterial activity does not \n result from iron chelation in the growth media ( figure 3d ) . in total", ", the data obtained for e. coli k-12 as well as the six other e. coli strains demonstrate \n that the antibacterial activity of ent - amp / amx against e. \n coli k-12 is enhanced under conditions of iron limitation \n and support a model of ent - mediated delivery of antibacterial cargo \n to the e. coli periplasm . antibacterial activity \n of ent - amp / amx against wild - type and mutant e. coli k-12 .", "( a , b ) growth inhibition of e. coli k-12 by \n amp / amx and ent - amp / amx in the absence ( a ) and presence \n ( b ) of dp .", "( c ) growth inhibition of e. coli k-12 \n treated with a 1:1 molar ratio of ent / amp and ent / amx .", "( d ) growth \n inhibition of e. coli k-12 treated with ferric ent - amp / amx . \n", "( e  g ) growth inhibition of fepa- ( e ) , fepc- ( f ) , and fes- ( g ) by amp / amx and \n ent - amp / amx .", "( h , i ) growth of e. coli k-12 in the \n presence of 1 m ent - amp / amx (  conjugate  ) and \n mixtures of ent - amp / amx ( 1 m ) and 1 , 5 , or 20 equiv of exogenous \n ent in the absence ( h ) and presence ( i ) of dp . the * * indicates od600 < 0.01 .", "all assays were performed in 50% mhb medium \n with or without 200 m dp ( see panels ) ( mean  sem ,", "the data for additional assays performed \n in the absence of dp are presented in figure s8 . to evaluate transport of ent - amp / amx \n into e. coli , we investigated the antimicrobial activity \n of ent - amp / amx and amp / amx against three single - gene knockout e. coli k-12 strains obtained from the keio collection , fepa- , fepc- , and fes- ( figures 3e ", "we selected these mutants to ascertain \n how components of the enterobactin transport and processing machinery \n contribute to ent - amp / amx antibacterial activity .", "e. colifepa- lacks the outer membrane ent receptor fepa \n that allows periplasmic delivery , fepc- lacks the \n atpase component of the inner membrane ent permease that transports \n ent into the cytosol , and e. colifes- lacks the cytoplasmic esterase fes responsible for hydrolysis of \n the ent macrolactone for iron release . on the basis of our studies \n with wild - type k-12 and other e. coli strains , we \n hypothesized that the activity of ent - amp / amx would be attenuated \n for the fepa- mutant .", "moreover , we questioned whether \n loss of fepc or fes would modulate the antimicrobial activity .", "overnight cultures of fepa- and fepc- reached od600 values ( 0.15 ) similar to that observed \n for wild - type k-12 when grown in 50% mhb supplemented with 200 m \n dp .", "in contrast , the fes- strain exhibited a severe \n growth defect under these conditions ( od600  0.04 ) . \n", "treatment of fepa- with ent - amp / amx afforded the \n same mic values as for amp / amx ( figure 3e ) \n and hence a 100-fold reduction in activity as compared to wild - type \n k-12 .", "iron deprivation is deleterious to e. coli , \n and we contend that the growth inhibition observed at 10 m \n ent - amp / amx results from iron starvation rather than an antibacterial \n activity of the amp / amx cargo .", "analysis of 50% mhb by inductively \n coupled plasma optical emission spectroscopy ( icp - oes ) revealed a \n total iron concentration of ca .", "4 m ( table \n s2 ) , which can be compared to 10 m of a high - affinity \n extracellular iron chelator .", "indeed , we previously observed similar \n growth inhibition of fepa- with 10 m of an \n ent - vancomycin conjugate ( extracellular iron chelation ) and also 10 \n m d - ent ( iron chelator that can not be used for iron \n acquisition ) under these growth conditions .", "these data confirm that fepa is essential for the potent antibacterial \n activity of ent - amp / amx against e. coli k-12 .", "in contrast to fepa- , the growth of fepc- and fes- was completely inhibited with 100 nm ent - amp / amx \n under iron - limiting conditions ( figure 3f , g ) , \n comparable to what was observed for the wild - type strain . the targets \n of -lactam antibiotics are penicillin binding proteins ( pbps ) , \n which are located in the periplasm of gram - negative bacteria .", "after \n crossing the outer membrane through fepa , ent - amp / amx enter the periplasm \n where covalent capture by the pbps presumably occurs .", "thus , it is \n reasonable that the downstream ent transport and processing steps \n involving fepcdg and fes do not affect the antimicrobial activity \n of the conjugates if they are trapped in the periplasm as a result \n of pbp binding .", "although it is possible that the ent uptake machinery \n ( e.g. , periplasmic binding protein fepb ) competes with the pbps for \n ent - amp / amx , no improved antibacterial activities were observed for \n the fepc- and fes- mutants compared \n to wild - type k-12 .", "this observation indicates that ent - amp / amx bind \n to pbps and are trapped in the periplasm .", "it should be noted that \n the fepb- mutant , which lacks the periplasmic binding \n protein , was also considered in this work ; however , this strain exhibited \n a severe growth defect and afforded inconsistent results . to probe \n interaction between fepa and ent - amp / amx , we performed \n growth inhibition assays employing mixtures of ent - amp / amx and varying \n concentrations of unmodified ent ( figure 3h ", "when these assays were performed under conditions of iron limitation , \n the presence of exogenous ent attenuated the antibacterial activity \n of ent - amp / amx .", "a 1:1 molar ratio of ent - amx / ent afforded an od600 value comparable to that of the untreated control , whereas \n higher equivalents of ent were required to block the antibacterial \n action of ent - amp .", "the origins of this difference are unclear and \n may indicate that the hydroxyl group of amx has a negative effect \n on the transport efficiency of the conjugate . in total , these ent \n addition assays suggest that competition for ent - amp / amx and ent occurs \n at the receptor(s ) and that the conjugates are delivered into the \n bacteria via the same uptake machinery as ent .", "l - serine is a biosynthetic \n building block for ent , and a role \n for chiral recognition in ent transport has been probed in prior studies . in one series of investigations ,", "e. coli fepa was \n found to bind ferric l - ent and ferric d - ent with \n similar affinities ( kd = 21 and 17 nm , \n respectively ; ascertained by measuring the binding of fe - loaded siderophores to e. coli bn1071 cells ) .", "a lack of transport of ferric d - ent \n into e. coli bn1071 was also reported in this work . \n", "a later study probed ent uptake in bacillus subtilis , and transport of both l- and d - ent analogues \n was observed to occur with similar efficiency .", "2457 t to hydrolyze l - ent and d - ent was \n evaluated , and fes did not accept d - ent as a substrate . as \n a result of the transport studies in b. subtilis and \n enzymatic activity assays with s. flexneri fes , a \n model in which both ent enantiomers are transported and chiral", "taken together , these studies suggest that the ability \n to transport d - ent may vary between species and even between \n strains of a given species , and more studies are required to address \n such possibilities .", "based on the observation that e. coli fepa binds d - ent and that b. subtilis transports d - ent analogues , we synthesized the d - enantiomers of \n the ent--lactam conjugates ( 13 and 14 , scheme 1 ) and evaluated the antibacterial \n activity of these conjugates against e. coli k-12 , \n 25922 , cft073 , 35401 , and 43895 ( figures s9s13 ) .", "d - ent - amp / amx exhibited reduced antibacterial activity \n relative to ent - amp / amx for k-12 , 25922 , cft073 , and 43895 . under \n conditions of iron limitation ,", "complete growth inhibition was observed \n with 1 m d - ent - amp / amx for e. coli k-12 , 25922 , and 43895 , compared to 100 nm for ent - amp / amx .", "likewise , \n a 10-fold reduction in antibacterial activity was observed for e. coli cft073 , where 100 nm d - ent was required \n to inhibit growth completely .", "in contrast , a negligible difference \n in antibacterial activity of the l- and d - isomers \n was observed for e. coli 35401 .", "regardless of enantiomer , \n all four ent - amp / amx conjugates provide enhanced antibacterial activity \n against these e. coli strains relative to amp / amx . \n", "nevertheless , these data suggest that d - ent - amp / amx are less \n readily transported into various e. coli strains \n than the l - isomers .", "although this modification provides no \n appreciable benefit for this -lactam delivery system with periplasmic \n targets , it is possible that ent - antibiotic conjugates based on d - ent may be desirable for delivering cargos to the cytosol , \n precluding concomitant delivery of nutrient fe(iii ) . with support for ent - mediated delivery of ent - amp /", "amx \n to the e. coli periplasm , we sought to confirm the \n essentiality of the -lactam warheads in antibacterial action . \n", "we therefore designed and prepared hydrolyzed ent - amp / amx analogues 15 and 16 ( scheme 2 ) where \n the -lactam structure is destroyed .", "hydrolysis of the amp / amx - alkynes 7 and 8 was achieved in the presence of 1% tfa \n with heating at 37 c , and the decomposition products 9 and 10 were obtained as diastereomeric mixtures ( scheme 2 ) .", "the formation of these species followed the reported \n degradation pathways for ampicillin , where hydrolysis and subsequent \n decarboxylation occur .", "the diastereomeric \n mixtures were employed to prepare the hydrolyzed conjugates ent - hydro - amp 15 and ent - hydro - amx 16 via a copper - catalyzed \n click reaction .", "( a , b ) antibacterial activity assays against e. coli k-12 ( a ) and cft073 ( b ) using ent - amp / amx and ent - hydro - amp / amx . \n", "( c ) antibacterial activity assays against e. coli atcc 35218 , which expresses a class a serine -lactamase , \n using ent - amp / amx in the absence and presence of the -lactamase \n inhibitors potassium clavulanate ( pc ) and sulbactam ( sb ) .", "all assays \n were performed in 50% mhb supplemented with 200 m dp ( mean \n  sem , n  3 ) .", "we employed ent - hydro - amp / amx in antibacterial activity assays \n against e. coli k-12 and observed negligible growth \n inhibition ( figure 4a ) . when e. coli cft073 was treated with ent - hydro - amp / amx in the presence of dp", "this result indicates that ent - hydro - amp / amx \n are transported into the cytoplasm of e. coli cft073 , \n where nutrient iron is released .", "we also performed a series \n of antibacterial activity assays with e. coli atcc \n 35218 , a strain that expresses a class a serine \n -lactamase .", "similar to unmodified amp / amx , ent - amp / amx were \n inactive against e. coli atcc 35218 ( mic > 10 \n m ) \n in the absence and presence of dp ( figures 4c and s14 ) .", "slight growth inhibition was \n observed at 10 m under conditions of iron limitation , which \n may be attributed to iron chelation .", "the addition of -lactamase \n inhibitors restored the activities of ent - amp / amx and amp / amx , and \n the conjugates exhibited greater antibacterial activity than the parent \n antibiotics ( figures 4c and s14 ) . in total , the assays with ent - hydro - amp / amx and strains \n expressing -lactamase demonstrate that an intact -lactam \n is required for the antibacterial activity of ent - amp / amx .", "moreover , \n these studies indicate that the -lactams retain their original \n function and inhibit pbps when conjugated to ent .", "the remarkable sensitivity of e. coli cft073 to \n ent - amp / amx ( figure 2c ) motivated us to investigate \n the relative cell - killing kinetics \n of ent - amp / amx and amp / amx to determine whether these conjugates kill e. coli cft073 more rapidly than the unmodified drugs . for \n comparison between e. coli strains ,", "ent - amp / amx \n provide more rapid cell death than unmodified amp / amx ( figure 5 ) , and this behavior is most apparent for e. coli cft073 , where the od600 value was almost \n reduced to the baseline value after 1 h incubation with 5 m \n ent - amp / amx , corresponding to a 2-fold log reduction in cfu / ml . in \n contrast , the change in od600 and cfu / ml for e. \n coli cft073 treated with 50 m unmodified amp / amx is \n negligible over this time period .", "the time - kill kinetics for e. coli k-12 , conducted with 50 m of both unmodified \n and modified -lactams , indicate a slight increase in kill kinetics \n for ent - amp / amx relative to amp / amx , and that the kinetics of cell - killing \n are slower for k-12 than cft073 ( figure 5 ) . \n", "these results support a model whereby ent modification facilitates \n uptake of amp / amx relative to the unmodified drugs .", "this effect is \n more dramatic for e. coli cft073 than k-12 , which \n is in accordance with the enhanced antibacterial activity observed \n for cft073 relative to the other e. coli strains \n considered in this work .", "time - kill kinetic assays for treatment of e. coli k-12 ( top panel ) and cft073 ( bottom panel ) with \n amp / amx and ent - amp / amx .", "e. coli k-12 ( 10 cfu / ml ) was treated \n with 50 m of amp / amx or 50 m ent - amp / amx .", "e. \n coli cft073 ( 10 cfu / ml ) was treated with \n 50 m of amp / amx or 5 m ent - amp / amx .", "the assays were \n conducted in 50% mhb medium containing 200 m dp at 37 c \n ( mean  sem , n = 3 ) . to determine whether ent - amp / amx exhibit broad - spectrum or species - selective \n activity , we performed antibacterial activity assays with two additional \n gram - negative and two gram - positive species in both the absence and \n presence of dp .", "these species include klebsiella pneumoniae atcc 13883 , pseudomonas aeruginosa pao1 , s. aureus atcc 25923 , and bacillus cereus atcc 14579 .", "k. pneumoniae is a gram - negative species \n that biosynthesizes and utilizes ent for iron acquisition .", "p. aeruginosa is a gram - negative bacterium that captures \n ent as a xenosiderophore and expresses two ent receptors \n pfea and pira.s .", "aureus and b. cereus are both gram - positive bacterial species , and the ability to utilize \n ferric ent as an iron source is reported for both species .", "in contrast to gram - negative bacteria , where the pbps are located \n in the periplasm , the targets of -lactam antibiotics are in \n the extracellular peptidoglycan of gram - positive organisms .", "k. pneumoniae atcc 13883 has a chromosomally encoded \n class a -lactamase ( shv-1 ) and lacks sensitivity to amp / amx . \n", "we observed no effect of 100 m amp / amx on k. pneumoniae growth under our assay conditions ( figure s15 ) .", "only 50% growth inhibition was observed when k. \n pneumoniae was treated with high concentrations ( 10 m ) \n of ent - amp / amx in the absence of dp , and the ent - amp conjugate provided \n the greatest activity under conditions of iron limitation with 90% \n growth inhibition at 10 m .", "when -lactamase inhibitors \n were included in the assays , k. pneumoniae exhibited \n greater sensitivity to amp / amx ( mic = 100 m ) and ent - amp / amx \n ( mic = 10 m ) ; however , we observed some growth inhibitory activity \n of the -lactamase inhibitor sulbactam alone under these assay \n conditions ( 100 m sb , figure s15 ) .", "thus , the possibility of a synergistic effect from the inhibitors \n and conjugates can not be ruled out completely .", "the lack of activity \n of ent - amp / amx against k. pneumoniae atcc 13883 is \n reminiscent of results obtained during investigations of amp / amx - functionalized \n tripodal triscatecholate ligands .", "these \n compounds were inactive against k. pneumoniae , and \n the behavior was attributed to either an inability of the k. pneumoniae iron transport machinery to import the conjugates \n or the development of resistance over the course of the assay .", "an \n alternative explanation is that -lactamase expression by k. pneumoniae resulted in inactivation of the -lactams .", "the p. aeruginosa pao1 strain employed in this \n work exhibited little sensitivity to both amp / amx and the conjugates \n under the antibacterial assay conditions ( figure \n s16 ) .", "amp / amx exhibited no activity up to 100 m , whereas \n ent - amp / amx provided growth inhibition at 10 m in both the \n absence and presence of dp . whether these results indicate that ent - amp / amx \n will be ineffective against multiple p. aeruginosa strains is unclear .", "p. aeruginosa strains exhibit \n different phenotypes , and highly variable and strain - dependent mic \n values have been reported for triscatecholate-lactam \n conjugates against p. aeruginosa .", "we previously reported that p. aeruginosa pao1 imports ent - cargo conjugates , and \n we speculate that the lack of activity observed for this strain stems \n from its inherent insensitivity to amp / amx .", "b. cereus atcc 14579 was also insensitive to amp / amx , \n which only afforded growth inhibition at 100 m .", "some growth \n inhibition was observed for b. cereus treated with \n 10 m ent - amp / amx , which may result from iron sequestration \n ( figure s17 ) .", "s. aureus atcc 25923 is susceptible to amp / amx , with complete growth inhibition \n observed at 1 m . in this case , a 10-fold reduction in antibacterial \n activity was observed for ent - amp / amx relative to unmodified amp / amx \n ( figure 6 ) . although the origins of this attenuation \n are unclear , we speculate that ent - amp / amx may have trouble penetrating \n the thick peptidoglycan of s. aureus .", "an alternative \n possibility is that recognition of ent - amp / amx by the s. aureus ent receptor diverts the -lactams from the pbps . in \n total , the results from these assays indicate that ent - amp / amx \n exhibit antibacterial activity enhancements that are species - selective , \n providing increased potency against e. coli strains \n and not for the other strains evaluated in this work . we therefore \n reasoned that ent - amp / amx , at low concentrations , should selectively \n kill e. coli in the presence of other less sensitive \n species .", "we treated co - cultures of e. coli cft073 \n and s. aureus with ent - amp / amx or amp / amx and analyzed \n the species composition following a 19-h incubation using harty - uti \n plates .", "these agar plates are employed in medical microbiology laboratories \n for the diagnosis of urinary tract infections and provide species \n identification by the colony color . when grown on harty - uti plates ,", "s. aureus are off - white and e. coli are \n purple - pink . in figure 6 , we present representative \n images of the colonies that resulted from treating co - cultures of e. coli and s. aureus with amp / amx or ent - amp / amx . \n in the absence of antibiotic ,", "the cultures provide a mixture of off - white \n and purple - pink colonies , indicating that both e. coli and s. aureus grow when cultured together .", "when \n the co - cultures are treated with 1 m amp / amx , only purple - pink \n colonies are present , which reveals that only e. coli survives .", "in contrast , treatment of the co - cultures with 1 m \n ent - amp / amx results in only off - white colonies from s. aureus .", "these comparisons demonstrate that ent - amp / amx selectively kill e. coli in the presence of s. aureus and \n that the siderophore modification reverses the inherent species \n selectivity of the parent antibiotics .", "achieving such species - selective \n and single - pathogen antibiotic targeting is an important goal and \n unmet need for pharmaceutical development that will allow for treating \n disease with minimal perturbation to the commensal microbiota .", "ent - amp / amx \n selectively kill e. coli cft073 in \n the presence of s. aureus atcc 25923 .", "( a , b ) antimicrobial \n activity assays against s. aureus atcc 25923 in the \n absence ( a ) and presence ( b ) of 200 m dp .", "( c , d ) bacterial growth \n monitored by od600 for cultures of e. coli only , s. aureus only , and 1:1 e. coli / s .", "aureus mixtures treated with amp / amx or ent - amp / amx \n in the absence ( c ) and presence ( d ) of 200 m dp .", "( e ) representative photographs of colonies \n from mixed cultures of e. coli cft073 and s. aureus atcc 29523 treated with ent - amp / amx ( 1 m ) \n or amp / amx ( 1 m ) in the presence of 200 m dp .", "all assays \n were conducted in 50% mhb medium ( t = 19 h , 30 c ) \n ( mean  sem , n  3 for a  d ) .", "we evaluated the cytotoxicity of ent - amp \n against the human t84 colon \n epithelial cell line .", "cell survival was evaluated by mtt assay after \n a 24 h treatment with apo or iron - bound ent - amp , amp , or ent .", "the \n iron - bound forms were assayed to determine whether iron chelation \n in the growth medium is a factor .", "no cytoxicity was observed for amp \n or ent - amp , whereas apo ent itself decreased the survival of t84 cells \n by approximately 30% at the highest concentration evaluated . when \n pre - loaded with fe(iii ) ,", "percent cell survival quantified by mtt assay after \n a 24 h treatment with apo or iron - bound ent , ent - amp , and the parent \n antibiotic amp in the absence and presence of 1 equiv of fe(iii ) ( mean \n  sem , n = 3 ) .", "lipocalin-2 \n ( lcn2 , also known as siderocalin or ngal ) is a 22-kda protein produced \n and released by neutrophils and epithelial cells .", "it has a hydrophobic \n binding pocket and coordinates ferric ent with sub - nanomolar affinity . by sequestering ferric ent", ", this host - defense protein contributes \n to the metal - withholding response and prevents bacterial acquisition \n of this essential nutrient . to determine whether lcn2 also binds ent - amp / amx \n and thereby blocks antibacterial activity ,", "we performed antibacterial \n activity assays with e. coli cft073 in m9 minimal \n medium supplemented with lcn2 or bovine serum albumin ( bsa ) . under \n these conditions , up to 1 m of lcn2 had no effect on the growth \n of e. coli cft073 . addition of 1 m lcn2 to \n the medium rescued the growth of e. coli cft073 treated \n with 100 nm of ent - amp ( figure 8) whereas addition \n of 1 m bsa had no effect on ent - amp cell killing .", "these results \n suggest that lcn2 binds ent - amp and blocks its recognition and uptake . \n", "to ascertain whether lcn2 binds ent - amp in the presence of exogenous \n ent , we performed a series of experiments where e. coli cft073 were treated with fixed concentrations of ent - amp ( 100 nm ) \n and lcn2 ( 1 m ) and the concentration of ent was varied ( 0 , \n 0.5 , and 1 m ) .", "moreover , when \n ent - amp was combined with a 1:1 molar ratio of ent and lcn2 at 10-fold \n excess over the conjugate , no e. coli growth was \n observed , which suggests that lcn2 preferentially binds ent .", "prior \n work demonstrated that lcn2 can not bind glucosylated ent , which was \n attributed to a steric clash between the glucose moieties and the \n ent binding site of the protein , and \n decreased hydrophobicity of the siderophore may also be a factor . \n", "thus , our data suggest that the nature of linker attachment at c5 \n and the peg3 moiety of ent - amp / amx do no abrogate lcn2 \n binding as effectively as the glucose moieties exhibited by the salmochelins", ". antibacterial \n activity of ent - amp against e. coli cft073 in the \n presence of lcn2 or bsa .", "( a ) e. coli cft073 treated \n with 100 nm ent - amp and varying concentrations of \n lcn2 or bsa control .", "( b ) e. coli cft073 treated with \n ent - amp , varying concentration of ent , and varying concentrations \n of lcn2 or bsa control .", "the assays were performed in m9 minimal medium \n ( 24 h , 37 c ) ( mean  sem , n  3 ) . \n", "ent - amp / amx are two siderophore-lactam \n conjugates \n based on the native enterobactin scaffold .", "these molecules hijack \n siderophore - based iron uptake pathways and provide potent antibacterial \n activity against various e. coli strains , including \n human pathogens .", "our investigations of ent - amp / amx establish the following : \n ( i ) ent - amp / amx provide up to 1000-fold enhanced antibacterial activity \n against e. coli strains ; ( ii ) ent - amp / amx are transported \n into e. coli by fepa and potentially other catecholate \n siderophore receptors ( iron , iha ) employed by pathogenic ctf073 \n and uti98 ; ( iii ) ent - amp / amx are captured by pbps in the periplasm , \n which results in pbp inhibition and cell death ; ( iv ) selective killing \n of e. coli in the presence of less susceptible organisms \n such as s. aureus is achieved because of the enterobactin \n scaffold ; ( v ) ent - amp / amx exhibit negligible cytotoxicity to human \n t84 intestinal epithelial cells ; and ( vi ) although lcn2 has the ability \n to bind ent - amp / amx , this siderophore - scavenging protein prefers \n to capture native ent . in total", ", these studies demonstrate that modification \n of antibiotic cargo with the native enterobactin platform provides \n many desirable features for antibiotic delivery and efficacy .", "the \n large molecular weight of the conjugates resulting from the native \n ent scaffold ( as opposed to a smaller mimic ) enhances rather than \n diminishes uptake for gram - negative e. coli .", "moreover , \n we observed no evidence for the development of resistance to ent - amp / amx \n over the course of the antibacterial activity assays performed during \n these investigations .", "our studies confirm that the enhanced \n antibacterial activity observed \n for ent - amp / amx requires both enterobactin recognition by outer membrane \n receptors and an intact -lactam moiety .", "the results are reminiscent of the dramatic \n antibacterial activity enhancements observed for albomycin , a secondary \n metabolite produced by actinomyces subtropicus .", "albomycin \n is comprised of the siderophore ferrichrome and a trna synthetase \n inhibitor , and it exhibits antimicrobial activities that are 30,000-fold \n greater than those of the unmodified trna synthetase inhibitor against e. coli and s. aureus . nonetheless , the ent - amp / amx cell - killing mechanism may \n be more complex than only more efficient -lactam delivery across \n the gram - negative outer membrane .", "binding of ent - amp / amx to the pbps \n presumably results in accumulation of ferric enterobactin in the e. coli periplasm for some period of time , which may have \n deleterious consequences .", "a recent study of an e. colitolc- mutant revealed that enterobactin accumulation \n in the periplasm affords growth defects and abnormal cellular morphologies .", "a fascinating observation that stems \n from our current work is the \n variable susceptibilities and responses of different e. coli strains to ent - amp / amx , which contrast the effects of unmodified \n amp / amx .", "such differences are manifest in the mic values to some degree \n and time - kill kinetics ; however , the results presented in figure 2 indicate that mic values alone do not provide a \n full description of how ent - amp / amx susceptibility differs between e. coli strains .", "these results suggest underlying complexity \n in microbial physiology related to iron - uptake pathways that can not \n be fully explained by the presence or absence of a gene for a particular \n receptor ( i.e. , fepa , iron ) .", "the heightened sensitivity of uropathogenic e. coli cft073 is particularly noteworthy , and it will be \n interesting to decipher the physiological origins of this effect as \n well as the differential behavior of various e. coli pathogens toward ent - antibiotic conjugates . there is a clear \n and unmet need for new antibacterial agents to \n treat bacterial infections in humans , including antibiotics that target \n specific bacterial sub - populations . preventing undesirable consequences of antibiotic treatment on the \n commensal microbiota , which contributes to human health in beneficial \n ways , is a challenge that needs to be addressed .", "such targeted therapeutics will be valuable not only for treating \n bacterial infections when the causative agent is known ( e.g. , urinary \n tract infection and e. coli , cystic fibrosis lung \n infection and p. aeruginosa ) but also for other pathologies \n that involve microbial dysbiosis , such as irritable bowel disease .", "our studies of ent - amp / amx provide one step \n toward addressing species - specific antibiotic targeting as well as \n overcoming gram - negative outer membrane permeability . from the standpoint \n of the host environment , commensal e. coli employ \n ent for acquiring iron in the host , and thus further elaboration of \n this strategy to specifically target pathogenicity and evade host \n responses ( e.g. , lcn2 ) is desirable ."], "abstract_text": ["<S> the design , synthesis , and characterization \n of enterobactin  </S>", "<S> antibiotic \n conjugates , hereafter ent - amp / amx , where the -lactam antibiotics \n ampicillin ( amp ) and amoxicillin ( amx ) are linked to a monofunctionalized \n enterobactin scaffold via a stable poly(ethylene glycol ) linker are \n reported . under conditions of iron limitation , </S>", "<S> these siderophore - modified \n antibiotics provide enhanced antibacterial activity against escherichia coli strains , including uropathogenic e. coli cft073 and uti89 , enterohemorrhagic e. coli o157:h7 , and enterotoxigenic e. coli o78:h11 , compared to the parent -lactams . </S>", "<S> studies with e. coli k-12 derivatives defective in ferric enterobactin \n transport reveal that the enhanced antibacterial activity observed \n for this strain requires the outer membrane ferric enterobactin transporter \n fepa . a remarkable 1000-fold decrease in minimum inhibitory concentration \n ( mic ) value is observed for uropathogenic e. coli cft073 relative to amp / amx , and time - kill kinetic studies demonstrate \n that ent - amp / amx kill this strain more rapidly at 10-fold lower concentrations \n than the parent antibiotics . </S>", "<S> moreover , ent - amp and ent - amx selectively \n kill e. coli cft073 co - cultured with other bacterial \n species such as staphylococcus aureus , and ent - amp \n exhibits low cytotoxicity against human t84 intestinal cells in both \n the apo and iron - bound forms . </S>", "<S> these studies demonstrate that the native \n enterobactin platform provides a means to effectively deliver antibacterial \n cargo across the outer membrane permeability barrier of gram - negative \n pathogens utilizing enterobactin for iron acquisition . </S>"], "labels": null, "section_names": ["Introduction", "Experimental Section", "Results and Discussion", "Summary and Perspectives"], "sections": [["bacterial infections \n as well as the emergence and spread of antibiotic \n resistance in human pathogens are serious public health problems in \n hospital and community settings across the globe .", "new strategies to prevent and treat bacterial infections \n are needed , including methods to overcome antibacterial resistance \n that results from the outer membrane permeability barrier in gram - negative \n organisms and targeting approaches that afford species- or pathogen - specific \n therapeutics .", "almost \n all \n bacterial species have a metabolic iron requirement and therefore \n employ various strategies to acquire this metal ion when colonizing .", "siderophores are high - affinity fe(iii ) chelators that are \n produced by bacteria under conditions of iron limitation , such as \n those encountered in the vertebrate host , to scavenge this metal ion \n from the environment .", "siderophore producers also \n express dedicated ferric siderophore import machinery and employ \n various mechanisms to release siderophore - bound iron following \n cellular uptake ( e.g. , reductive and/or hydrolytic release mediated \n by reductases and/or esterases , respectively ) .", "numerous studies support the importance of siderophore - based \n iron acquisition during bacterial infections .", "thus , the potential of using siderophores , or targeting siderophore biosynthetic and transport machineries , in therapeutic development continues to attract significant interest . \n of particular relevance to the advances described herein are prior \n investigations pertaining to the development of siderophore ", "this strategy has received particular attention for the delivery \n of antibiotics into gram - negative bacteria because these organisms \n are inherently less sensitive to many antibiotics used in the clinic \n as a result of the outer membrane permeability barrier .", "both native siderophores and synthetic siderophore mimics \n have been evaluated as platforms for therapeutic development . in the clinic , the native siderophore desferrioxamine \n b is used for iron - chelation therapy in patients with iron overload . \n", "several antibiotic small molecules found in nature called  sideromycins  \n provide inspiration for synthetic siderophore ", "the sideromycins are secondary \n metabolites comprised of a siderophore moiety and a toxic cargo ; \n the siderophore portion targets sideromycins to bacterial strains \n expressing the appropriate siderophore receptor .", "antibiotic conjugate produced by a clinical \n isolate of klebsiella pneumoniae , is an 84-residue \n antibacterial peptide with a glucosylated enterobactin ( ent , figure 1 ) derivative attached to its c - terminus that exhibits \n enhanced antibacterial activity against strains expressing the enterobactin \n receptor fepa . from the standpoints of \n antibacterial activity and therapeutic potential , studies of synthetic \n siderophore ", "antibiotic conjugates have provided the \n community with mixed results , causing some skepticism about the potential \n of siderophore - based approaches despite the successful utilization \n of such molecules by nature .", "structures of enterobactin ( 1 , \n ent ) and a generalized \n enterobactin  cargo conjugate .", "many of the failures encountered with early and recent studies \n of siderophore - based antibiotic delivery may be attributed , \n at least in part , to ( i ) use of non - native siderophores with \n relatively low fe(iii ) affinities and/or compromised receptor recognition ; ( ii ) modification of antibiotics such that the \n antibacterial activity is attenuated or lost completely ; ( iii ) bridging the siderophores and antibiotics with problematic \n linkers , including linkers designed for drug release that are either \n too stable or too labile , the latter of which promotes premature release ; and ( iv ) antibiotic resistance .", "nevertheless , \n the lessons of many unsuccessful studies highlight the complexity \n of siderophore - based therapeutic development and provide a foundation \n for inventing improved next - generation approaches .", "many of the issues \n described above may be overcome by careful molecular design and biological \n evaluation .", "in particular , the selection of appropriate native siderophore \n platforms and modification of these platforms in ways that do not \n compromise iron binding or receptor recognition , installation of an \n antibacterial cargo in such a manner that antibacterial activity is \n retained , and the development and application of assays that afford \n insight into the fate of siderophore  antibiotic conjugates \n are critical to the overall success of this approach .", "along such lines , \n a recent and insightful study by pfizer addressed complications associated \n with using relatively low - molecular - weight siderophore mimics in vivo .", "their results indicate \n that competition between the siderophore - conjugated monobactam \n mb-1 and native siderophores resulted in poor in vivo efficacy against pseudomonas aeruginosa and provide \n support for designing and evaluating siderophore ", "one recent \n and successful example based on a native siderophore platform \n is a mycobactin ", "artemisinin conjugate that exhibits enhanced \n antibacterial activity against mycobacterium tuberculosis compared to unmodified artemisinin .", "enterobactin ( ent , figure 1 ) is a triscatecholate \n siderophore biosynthesized by enteric bacteria and used for \n iron acquisition in the vertebrate host . motivated by the importance of ent in the host / microbe interaction \n as well as the decades of investigations pertaining to its ( bio)synthesis , \n coordination chemistry , and biology , in prior work we reported a synthetic \n route to monofunctionalized ent platforms .", "moreover , we established that the native ent platform , when monofunctionalized \n at the c5 position of one catecholate ring ( figure 1 ) , affords delivery of nontoxic small - molecule cargo across \n the outer membrane of gram - negative organisms that express ent uptake \n machinery ( e.g. , fepabcdg of escherichia coli ) .", "as described herein , this proof - of - concept study \n motivated us to demonstrate that ent effectively delivers antibacterial \n cargo to organisms that utilize ent for iron acquisition , thereby \n providing antibiotic targeting to specific sub - populations and a means \n to address antibiotic resistance that results from the gram - negative \n outer membrane permeability barrier . in this work", ", we present \n the syntheses and characterization of \n siderophore  antibiotic conjugates based on the native \n ent platform that harbor the clinically relevant -lactam antibiotics \n ampicillin ( amp ) and amoxicillin ( amx ) .", "these antibiotics block cell \n wall biosynthesis by inhibiting transpeptidases , also named \n penicillin binding proteins ( pbps ) , located in the periplasm of e. coli .", "we report that the ent--lactam conjugates \n exhibit significantly enhanced antibacterial activity ( up to 1000-fold ) \n against pathogenic e. coli and provide more rapid \n cell - killing than the parent -lactams as a result of ent - mediated \n delivery to the periplasm . moreover ,", "in proof - of - concept studies for \n species - specific killing , these conjugates selectively kill e. coli in the presence of staphylococcus aureus , a gram - positive organism that is more susceptible to the parent \n -lactams .", "these studies support the notion that native siderophore \n platforms provide an effective means to target molecular cargo to \n siderophore - utilizing organisms and to hijack siderophore \n uptake machinery to deliver cargos , including antibiotics , across \n the outer membrane permeability barrier of gram - negative microbes ."], ["dimethylformamide ( dmf ) and dichloromethane \n ( ch2cl2 ) were obtained from a vac solvent purification \n system ( vacuum atmospheres ) .", "anhydrous dimethyl sulfoxide ( dmso ) was \n purchased from sigma - aldrich and used as received .", "l - ent 1 , the d - enantiomer of benzyl - protected \n ent - co2h 2 , and the benzyl - protected ent - peg3-n33 were synthesized according to \n previously reported procedures .", "11-azido-3,6,9-trioxaundecan-1-amine \n was purchased from fluka . all other chemicals and solvents were purchased \n from sigma - aldrich or alfa aesar in the highest available purity and \n used as received .", "benzyl - protected ent - azide 3 ( 80 mg , 55 mol ) and pentamethylbenzene ( pmb , 147 mg , \n 990 mol ) were dissolved in 5 ml of anhydrous ch2cl2 to give a light yellow solution .", "this solution was \n cooled to 78 c in an acetone / dry ice bath under n2 , and bcl3 ( 660 l of 1 m solution in ch2cl2 , 660 mol ) was added slowly along the \n flask wall .", "after the solution was stirred for 1.5 h , dipea ( 300 l , \n 1.73 mmol ) was added to the flask , followed by meoh ( 2 ml ) to quench \n the reaction . the reaction was then warmed to room temperature , and \n the solvents were removed under reduced pressure . the resulting white \n solid was dissolved in 5:3 meoh/1,4-dioxane and purified by preparative \n hplc ( 33% b for 5 min and 3360% b over 20 min , 10 ml / min ) . \n", "the product eluted at 17 min and was lyophilized to yield compound 4 as white solid ( 13.9 mg , 28% ) .", "h nmr ( dmso - d6 , 500 mhz ) :  3.353.57 ( 16h , \n m ) , 4.384.41 ( 3h , m ) , 4.634.69 ( 3h , m ) , 4.894.96 \n ( 3h , m ) , 6.74 ( 2h , dd , j = 7.5 , 8.0 hz ) , 6.97 ( 2h , \n d , j = 7.5 hz ) , 7.35 ( 2h , d , j = \n 8.0 hz ) , 7.46 ( 1h , s ) , 7.94 ( 1h , s ) , 8.338.35 ( 1h , m ) , 9.12 \n ( 2h , d , j = 6.0 hz ) , 9.29 ( 1h , d , j = 6.0 hz ) , 9.44 ( 2h , bs ) , 9.76 ( 1h , bs ) , 11.6 ( 2h , bs ) , 11.9 ( 1h , \n bs ) .", "c nmr ( cdcl3 , 125 mhz ) :  50.1 , \n 51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 , 115.3 , 115.4 , 115.4 , \n 117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 , 148.7 , 148.7 , 150.8 , \n 166.0 , 168.4 , 169.1 , 169.6 , 169.7 .", "ir ( kbr disk , cm ) : 3389 , 2954 , 2928 , 2868 , 2111 , 1754 , 1645 , 1589 , 1535 , 1460 , 1384 , \n 1329 , 1266 , 1176 , 1132 , 1074 , 992 , 846 .", "11-azido-3,6,9-trioxaundecan-1-amine \n ( 36 l , 181 mol ) and d - bn6ent - cooh \n ( 2 , 177 mg , 142 mol ) were dissolved in 5 ml of \n dry ch2cl2 .", "pyaop ( 147 mg , 283 mol ) and \n dipea ( 98.5 l , 568 mol ) were added to give a light yellow \n solution .", "the reaction was stirred for 4 h at room temperature and \n concentrated , and the crude product was purified by preparative tlc \n ( 50% etoac / ch2cl2 ) to afford 5 as \n white foam ( 159 mg , 77% ) .", "h nmr ( dmso - d6 , 500 mhz ) :  3.33 \n ( 2h , j = 5.2 hz ) , 3.623.69 ( 14h , m ) , 4.024.06 \n ( 3h , m ) , 4.154.18 ( 3h , m ) , 4.914.94 ( 3h , m ) , 5.045.21 \n ( 12h , m ) , 6.96 ( 1h , s ) , 7.117.45 ( 36h , m ) , 7.657.67 \n ( 2h , m ) , 7.857.85 ( 1h ,", "m ) , 7.977.97 ( 1h , m ) , 8.508.54 \n ( 3h , m ) .", "c nmr ( cdcl3 , 125 mhz ) :  25.6 , \n 29.5 , 38.8 , 40.0 , 45.3 , 51.3 , 51.4 , 63.9 , 64.1 , 69.8 , 39.8 , 70.0 , \n 70.3 , 70.4 , 70.4 , 71.2 , 71.2 , 76.3 , 76.3 , 116.8 , 117.5 , 120.4 , 123.0 , \n 124.3 , 125.4 , 126.1 , 126.2 , 127.6 , 127.6 , 127.9 , 128.2 , 128.3 , 128.4 , \n 128.4 , 128.5 , 128.5 , 128.6 , 128.6 , 128.8 , 128.8 , 128.9 , 129.0 , 130.1 , \n 135.4 , 135.7 , 135.9 , 136.0 , 136.1 , 146.8 , 146.9 , 149.1 , 151.6 , 151.8 , \n 164.3 , 164.9 , 164.9 , 165.8 , 168.9 , 169.0 , 169.1 , 176.2 . ir ( kbr disk , \n cm ) : 3357 , 3062 , 3032 , 2958 , 2923 , 2859 , 2104 , \n 1751 , 1551 , 1576 , 1515 , 1455 , 1375 , 1345 , 1299 , 1264 , 1204 , 1126 , \n 1082 , 1040 , 1018 , 957 , 915 , 854 , 811 . hrms ( esi ) :", "compound 6 was synthesized \n from 5 ( 153 mg , 105 mol ) following the same procedure \n as for compound 4 .", "the crude reaction was purified by \n preparative hplc ( 33% b for 5 min and 3360% b over 20 min , \n 10 ml / min ) .", "the product eluted at 17.0 min and was lyophilized to \n yield compound 6 as white solid ( 31 mg , 33% ) .", "h nmr ( cdcl3 , 500 mhz ) :  3.343.56 ( 16h , \n m ) , 4.394.41 ( 3h , m ) , 4.614.66 ( 3h , m ) , 4.884.94 \n ( 3h , m ) , 6.74 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h , \n d , j = 7.8 hz ) , 7.33 ( 2h , d , j = \n 7.8 hz ) , 7.44 ( 1h , s ) , 7.91 ( 1h , s ) , 8.318.33 ( 1h , m ) , 9.129.13 \n ( 2h , m ) , 9.279.28 ( 1h , m ) , 9.50 ( 2h , bs ) , 9.84 ( 1h , bs ) , 11.6 \n ( 2h , bs ) , 11.9 ( 1h , bs ) .", "c nmr ( cdcl3 , 125 \n mhz ) :  50.1 , 51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 , \n 115.3 , 115.4 , 115.4 , 117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 , \n 148.7 , 148.7 , 150.8 , 166.0 , 168.4 , 169.1 , 169.6 , 169.7 .", "ir ( kbr disk , \n cm ) : 3390 , 2958 , 2925 , 2863 , 2110 , 1754 , 1645 , \n 1589 , 1535 , 1460 , 1384 , 1342 , 1262 , 1176 , 1117 , 1074 , 841 , 800 .", "hrms \n ( esi ) : [ m+na]m / z calcd \n 936.2506 , found 936.2512 .", "5-hexynoic acid ( 113 l , 1.00 \n mmol ) and thionyl chloride ( 1.00 ml , 13.8 mmol ) were combined and \n refluxed for 1 h. the reaction was cooled to room temperature and \n concentrated under reduced pressure , and the resulting crude acyl \n chloride was dissolved in acetone ( 0.5 ml ) and carried on to the next \n step without purification .", "ampicillin sodium salt ( 186 mg , 0.500 mmol ) \n was dissolved in a solution of nahco3 ( 210 mg , 2.5 mmol ) \n in 4:1 water / acetone ( 2.5 ml ) and cooled on ice , to which the acyl \n chloride was added slowly with stirring .", "the reaction was subsequently \n warmed to room temperature and stirred for 1 h. water ( 3 ml ) was added \n to the reaction , and the aqueous phase was washed with etoac ( 2  \n 10 ml ) , acidified to ph 2 by addition of hcl , and extracted with etoac \n ( 20 ml ) .", "the resulting organic phase was washed with cold water ( 2 \n  5 ml ) , dried over na2so4 , and concentrated \n under reduced pressure . the crude reaction was triturated with hexanes , \n which afforded a yellow solid ( 180 mg , 77% ) .", "h nmr ( dmso - d6 , \n 500 mhz ) :  1.41 ( 3h , s ) , 1.55 ( 3h , s ) , 1.641.69 ( 2h , \n m ) , 2.132.16 ( 2h , m ) , 2.292.32 ( 2h , m ) , 2.772.78 \n ( 1h , m ) , 4.20 ( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) , \n 5.52 ( 1h , dd , j = 4.0 , 8.0 hz ) , 5.70 ( 1h , d , j = 8.0 hz ) , 7.257.43 ( 5h , m ) , 8.57 ( 1h , d , j = 8.0 hz ) , 9.11 ( 1h , d , j = 8.0 hz ) .", "c nmr ( dmso - d6 , 125 mhz ) :  \n 17.4 , 24.4 , 26.6 , 30.4 , 33.8 , 55.5 , 58.1 , 63.7 , 67.3 , 70.3 , 71.5 , \n 84.2 , 127.2 , 127.6 , 128.2 , 138.2 , 169.0 , 170.2 , 171.5 , 173.5 .", "ir ( kbr \n disk , cm ) : 3297 , 3058 , 3023 , 2970 , 2937 , 2863 , \n 2626 , 2526 , 2120 , 1780 , 1688 , 1518,1455 , 1437 , 1390 , 1373 , 1324 , 1295 , \n 1208 , 1139 , 1027 , 1001 , 843 .", "compound 8 was synthesized \n as described for compound 7 except that amoxicillin ( 760 \n mg , 2.1 mmol ) was used instead of ampicillin sodium salt .", "compound 8 was obtained as light yellow solid ( 533 mg , 56% ) after trituration \n and employed without further purification .", "h nmr ( dmso - d6 , 500 mhz ) : \n  1.42 ( 3h , s ) , 1.56 ( 3h , s ) , 1.631.68 ( 2h , m ) , 2.122.15 \n ( 2h , m ) , 2.252.29 ( 2h , m ) , 2.762.78 ( 1h , m ) , 4.19 \n ( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) , 5.515.54 \n ( 2h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) , 7.19 ( 2h , d , j = 8.5 hz ) , 8.41 ( 1h , d , j = 8.0 hz ) , \n 8.93 ( 1h , d , j = 8.0 hz ) , 9.40 ( 1h , bs ) .", "c nmr ( dmso - d6 , 125 mhz ) :  17.6 , \n 24.5 , 26.8 , 30.4 , 34.0 , 55.4 , 58.2 , 63.9 , 71.6 , 71.7 , 84.4 , 115.1 , \n 128.4 , 128.7 , 128.8 , 157.0 , 169.2 , 170.9 , 171.7 , 173.8 . ir ( kbr disk , \n cm ) : 3356 , 3294 , 3045 , 2970 , 2928 , 1770 , 1738 , \n 1650 , 1615 , 1515 , 1457 , 1373 , 1208 , 1009 , 945 , 839 , 815 . hrms ( esi ) : \n [ m+na]m / z calcd 460.1537 , \n found 460.1534 .", "compound 7 ( 60 mg , 0.13 \n mmol ) was dissolved in 1:1 h2o / mecn ( 5 ml ) , and tfa was \n added to a final concentration of 1% .", "the solution was incubated at \n 37 c for 24 h and purified by preparative hplc ( 2050% \n b over 25 min , 10 ml / min ) , which afforded a white powder ( 12 mg , 25% ) . \n", "the white powder is a diastereomeric mixture of products , and no further \n separation was performed .", "h nmr ( dmso - d6 , 500 mhz ) :  ( mixture of two diastereomers ) 1.221.23 \n ( 3h , pair of s ) , 1.541.58 ( 3h , pair of s ) , 1.621.68 \n ( 2h , m ) , 2.122.14 ( 2h , m ) , 2.282.30 ( 2h , m ) , 2.77 \n ( 1h , s ) , 3.183.24 ( 0.5h , m ) , 3.303.36 ( 0.5h , m ) , 3.443.49 \n ( 0.5h , m ) , 3.553.60 ( 0.5h , m ) , 3.92 ( 0.5h , s ) , 4.01 ( 0.5h , \n s ) , 4.67 ( 0.5h , dd , j = 6.7 , 6.5 hz ) , 4.78 ( 0.5h , \n dd , j = 5.2 , 5.2 hz ) , 5.455.48 ( 1h , m ) , 7.277.40 \n ( 5h , m ) , 8.508.61 ( 2h , m ) .", "c nmr ( dmso - d6 , 125 mhz ) :  ( mixture of two diastereomers ) \n 18.1 , 25.0 , 27.6 , 28.1 , 28.4 , 29.5 , 34.4 , 42.2 , 56.8 , 56.8 , 72.1 , \n 72.2 , 84.8 , 127.9 , 128.2 , 128.9 , 139.2 , 139.4 , 171.2 , 171.4 , 172.0 . \n", "ir ( kbr disk , cm ) : 3297 , 3071 , 3041 , 2967 , 2938 , \n 2535 , 2124 , 1734 , 1653 , 1527 , 1456 , 1427 , 1375 , 1299 , 1199 , 1137 , \n 1070 , 1027 , 836 . hrms ( esi ) :", "compound 10 was synthesized \n as described for compound 9 except that compound 8 was used instead of 7 ( 60 mg , 0.13 mmol ) .", "the \n product was purified by preparative hplc ( 2050% b over 25 \n min , 10 ml / min ) , and obtained as white powder ( 14.5 mg , 24% ) .", "h nmr ( dmso - d6 , 500 mhz ) :  \n ( mixture of two diastereomers ) 1.26 ( 3h , s ) , 1.551.59 ( 3h , \n pair of s ) , 1.611.67 ( 2h , m ) , 2.122.14 ( 2h , m ) , 2.242.27 \n ( 2h , m ) , 2.77 ( 1h , s ) , 3.203.26 ( 0.5h , m ) , 3.343.39 \n ( 0.5h , m ) , 3.423.47 ( 0.5h , m ) , 3.563.60 ( 0.5h , m ) , \n 4.03 ( 0.5h , s ) , 4.12 ( 0.5h , s ) , 4.68 ( 0.5h , dd , j = 6.5 , 6.5 hz ) , 4.79 ( 0.5h , dd , j = 5.5 , 5.5 hz ) , \n 5.285.31 ( 1h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) , \n 7.17 ( 2h , d , j = 8.5 hz ) , 8.368.50 ( 3h , m ) .", "c nmr ( dmso - d6 , 125 mhz ) :  \n ( mixture of two diastereomers ) 17.5 , 24.3 , 26.9 , 27.5 , 27.7 , 28.8 , \n 33.8 , 41.4 , 55.7 , 55.8 , 71.4 , 71.6 , 84.2 , 114.8 , 115.0 , 128.5 , 128.7 , \n 128.8 , 156.9 , 158.3 , 158.6 , 171.2 , 171.3 .", "ir ( kbr disk , cm ) : 3301 , 3071 , 3028 , 2973 , 2928 , 2548 , 2111 , 1737 , 1662 , 1606 , 1593 , \n 1515 , 1435 , 1377 , 1197 , 1139 , 837 . hrms ( esi ) :", "ampicillin - alkyne 7 ( 120 l \n of an 80 mm solution in dmso , 9.6 mol ) and \n ent - peg3-n34 ( 250 l of \n a 13 mm solution in 1,4-dioxane , 3.3 mol ) were combined , and \n 400 l of dmso was added .", "cuso4 ( 100 l of \n a 90 mm solution in water , 9.0 mol ) and tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta , 200 l of \n a 50 mm solution in dmso , 10 mol ) were combined , and 100 l \n of dmso was added to give a blue - green solution , to which naasc ( 400 \n l of a 90 mm solution in water , 36.0 mol ) was added . \n this solution became light yellow and was immediately added to the \n alkyne / azide solution .", "the reaction was shaken on a benchtop rotator \n for 2 h at room temperature , diluted by 3- to 4-fold with 1:1 mecn / water , \n centrifuged ( 13,000 rpm  10 min , 4 c ) , and purified by \n semi - preparative hplc ( 20% b for 5 min and 20%50% b over 11 \n min , 4 ml / min ; 0.005% tfa was used in the solvent system to prevent \n decomposition of the -lactam ) .", "the hplc fractions containing 11 were collected manually and flash frozen in liquid n2 immediately after collection to prevent -lactam decomposition . \n", "h nmr ( dmso - d6 , 500 mhz ) :  1.40 \n ( 3h , s ) , 1.54 ( 3h , s ) , 1.781.81 ( 2h , m ) , 2.27 ( 2h , t , j = 6.8 hz ) , 2.58 ( 2h , t , j = 6.5 hz ) , \n 3.48 ( 12h , m ) , 3.76 ( 2h , s ) , 4.19 ( 1h , s ) , 4.384.44 ( 5h , m ) , \n 4.644.66 ( 3h , m ) , 4.914.92 ( 3h , m ) , 5.39 ( 1h , d , j = 3.5 hz ) , 5.515.52 ( 1h , m ) , 5.72 ( 1h , d , j = 7.5 hz ) , 6.74 ( 2h , dd , j = 7.8 , 7.8 \n hz ) , 6.96 ( 2h , d , j = 7.5 hz ) , 7.267.35 ( 5h , \n m ) , 7.427.45 ( 3h , m ) , 7.81 ( 1h , s ) , 7.92 ( 1h , s ) , 8.33 ( 1h , \n s ) , 8.55 ( 1h , d , j = 7.5 hz ) , 9.12 ( 3h , d , j = 7.0 hz ) , 9.29 ( 1h , d , j = 6.5 hz ) , \n 9.42 ( 2h , bs ) , 9.74 ( 1h , s ) , 11.6 ( 2h , s ) , 11.9 ( 1h , bs ) , 13.35 ( 1h , \n bs )", ". hrms ( esi ) : [ m+na]m / z calcd 1379.4021 , found 1379.4046 .", "compound 12 was \n synthesized as described for 11 except that compound 8 was used instead of compound 7 .", "h nmr ( dmso - d6 , 500 mhz ) :  1.40 ( 3h , s ) , 1.54 ( 3h , \n s ) , 1.781.80 ( 2h , m ) , 2.23 ( 2h , t , j = 6.5 \n hz ) , 2.57 ( 2h , t , j = 6.5 hz ) , 3.47 ( 12h , m ) , 3.76 \n ( 2h , bs ) , 4.18 ( 1h , s ) , 4.394.43 ( 5h , m ) , 4.634.65 \n ( 3h , m ) , 4.90 ( 3h , bs ) , 5.38 ( 1h , s ) , 5.525.56 ( 2h , m ) , 6.68 \n ( 2h , d , j = 8.5 hz ) , 6.73 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h , d , j = 7.5 ) , 7.19 ( 2h , \n d , j = 8.5 hz ) , 7.33 ( 2h , d , j = \n 7.5 hz ) , 7.44 ( 1h , s ) , 7.80 ( 1h , s ) , 7.92 ( 1h , s ) , 8.338.39 \n ( 2h , m ) , 8.94 ( 1h , d , j = 8.0 hz ) , 9.119.12 \n ( 2h , m ) , 9.29 ( 1h , bs ) , 9.389.43 ( 3h , m ) , 9.75 ( 1h , s ) , 11.6 \n ( 2h , bs ) , 11.9 ( 1h , bs ) .", "compound 13 was \n synthesized as described for 11 except \n that compound 6 was used instead of compound 4 .", "hrms ( esi ) : \n [ m+na]m / z calcd 1379.4021 , \n found 1379.4022 .", "compound 14 was synthesized as described for 12 except \n that compound 6 was used instead of compound 4 .", "hrms ( esi ) : \n [ m+na]m / z calcd 1395.3970 , \n found 1395.3995 .", "compound 15 was synthesized as described for 11 except that compound 9 was used instead of compound 7 .", "hrms ( esi ) : [ m+h]m / z calcd 1331.4409 , found \n 1331.4389 .", "compound 16 was synthesized as described for 12 except that compound 10 was used instead of compound 8 .", "hrms ( esi ) : [ m+na]m / z calcd 1369.4177 , found \n 1369.4191 .", "general microbiology materials and methods , including \n details of \n ent - amp / amx stock solution preparation and storage , are provided as supporting information .", "overnight cultures of \n the bacterial strains ( table s1 ) were prepared \n in 15-ml polypropylene tubes by inoculating 5 ml of lb media with \n the appropriate freezer stock .", "the overnight cultures were incubated \n at 37 c for 1618 h in a tabletop incubator shaker set \n at 150 rpm and housing a beaker of water .", "each overnight culture was \n diluted 1:100 into 5 ml of fresh lb media containing 200 m \n 2,2-dipyridyl ( dp ) and incubated at 37 c with shaking \n at 150 rpm until od600 reached 0.6 .", "g / l ) with or without 200 m \n dp to achieve an od600 value of 0.001 .", "a 90-l aliquot \n of the diluted culture was combined with a 10-l aliquot of \n a 10 solution of the antibiotic or ent - antibiotic conjugate \n in a 96-well plate , and the covered plate was wrapped in parafilm \n and incubated at 30 c with shaking at 150 rpm for 19 h in a \n tabletop incubator housing a beaker of water .", "bacterial growth was \n determined by measuring od600 ( end point analysis ) using \n a biotek synergy ht plate reader .", "each well condition was prepared \n in duplicate and at least three independent replicates using two different \n synthetic batches of each conjugate were conducted on different days . \n", "the resulting mean od600 values are reported , and the error \n bars are the standard error of the mean ( sem ) obtained from the independent \n replicates .", "these assays were performed with e. coli atcc \n 35218 and k. pneumoniae atcc 13883 following the \n general procedure except that sulbactam ( sb ) or potassium clavulanate \n ( pc ) were mixed with ampicillin or amoxicillin and the ent - amp or \n ent - amx conjugates , respectively .", "the molar ratios of the inhibitor/-lactam \n mixtures were sulbactam / amp or ent - amp , 1.5:1 , and potassium clavulanate / amx \n or ent - amx , 0.9:1 .", "these assays were \n performed with e. coli k-12 and \n cft073 following the general procedure except that varying concentrations \n ( 1100 m ) of synthetic l - ent were mixed with \n ent - amp / amx .", "this assay is based on a published protocol and was conducted with e. coli cft073 . \n", "each overnight culture was serially diluted into \n m9 minimal medium to provide 1010 cfu / ml . \n", "lipocalin 2 was diluted in pbs to a concentration of 10 m upon \n arrival , aliquoted , and stored at 20 c until use .", "a \n 90-l aliquot of the diluted culture was added to each well \n of a 96-well plate that contained varying concentrations of lipocalin \n 2 , ent - amp , and ent , and the final volume was adjusted to 100 l \n with sterile pbs .", "the 96-well plate was incubated at 37 c for \n 24 h in a tabletop incubator set at 150 rpm , and bacterial growth \n was determined by measuring od600 using a plate reader . \n", "each well condition was repeated at least three times independently \n on different days and with different batches of lipocalin 2 .", "the resulting \n mean od600 is reported , and the error bars are the sem .", "a 5-ml overnight culture of e. coli k-12 or cft073 was grown in lb ( vide supra ) and diluted 1:100 into 5 ml of fresh lb media containing 200 m \n dp , and this culture was incubated at 37 c with shaking at 150 \n rpm in a tabletop incubator housing a beaker of water until od600 reached 0.3 .", "the culture was centrifuged ( 3000 \n rpm  10 min , rt ) , and the resulting pellet was washed twice \n by resuspension in 50% mhb and centrifugation ( 3000 rpm  10 \n min , rt ) .", "the resulting pellet was resuspended in 50% mhb with or \n without dp , and the od600 was adjusted to 0.3 .", "a 90-l \n aliquot of the resulting culture was mixed with a 10-l aliquot \n of a 10 solution of amp / amx or the ent - amp / amx in a 96-well \n plate , which was covered , wrapped in parafilm , and incubated at 37 \n c with shaking at 150 rpm .", "the od600 values were \n recorded at t = 0 , 1 , 2 , and 3 h by using a plate \n reader . in a parallel experiment ,", "a 10-l aliquot of the culture \n was taken at t = 0 , 1 , 2 , and 3 h , serially diluted \n by using sterile phosphate - buffered saline ( pbs ) , and plated on lb - agar \n plates for colony counting ( cfu / ml ) .", "the resulting \n mean od600 or cfu / ml is reported , and the error bars are \n the sem .", "a 5 ml overnight culture of each \n bacterial strain was grown in lb , diluted 1:100 into 5 ml of fresh \n lb media containing 200 m dp , and incubated at 37 c with \n shaking at 150 rpm in a tabletop incubator housing a beaker of water \n until od600 reached 0.6 .", "each mid - log - phase culture was \n diluted to 10 cfu / ml in 50% mhb with or without 200 m \n dp . for experiments requiring a mixture of two species ,", "a 1:1 mixture \n was prepared ( 10 cfu / ml for each strain ) in 50% mhb with \n or without 200 m dp from the mid - log - phase cultures . to confirm \n cfu / ml of each culture ,", "the single- and double - species cultures were \n serially diluted by using sterile pbs , and aliquots were plated on \n a chrom - uti plate (  starter - culture plate  ) .", "for each \n cell - killing experiment , a 90-l aliquot of each culture was \n combined with a 10-l aliquot of a 10 m solution of the \n antibiotic or ent - antibiotic conjugate in a 96-well plate , which was \n covered , wrapped in parafilm , and incubated at 30 c with shaking \n at 150 rpm for 19 h. bacterial growth was assayed both by measuring \n od600 using the plate reader and by plating on chrom - uti \n plates after serial dilution (  assay plate  ) .", "each well \n condition was repeated at least three times independently on different \n days .", "the resulting mean od600 is reported , and the error \n bars are the sem ."], ["we aimed to harness our enterobactin - mediated \n cargo delivery strategy to enable the transport of toxic cargo across \n the outer membrane of e. coli . to address this goal , \n we linked the -lactam antibiotics ampicillin ( amp ) and amoxicillin \n ( amx ) to a monofunctionalized ent scaffold where ent is derivatized \n at the c5 position of one catechol ring via a flexible and stable \n peg3 linker .", "we selected amp and amx as antibacterial cargo \n for several reasons : these molecules are commercially available and \n amenable to synthetic modification , retain antibacterial activity \n when appropriately modified , possess periplasmic targets in gram - negative \n bacteria and must cross the outer membrane to be active against these \n species , and have relatively low molecular weights .", "we selected low - molecular - weight \n antibiotics because our prior studies of ent - mediated cargo transport \n indicated that the ent transport machinery of e. coli k-12 imports ent - cargo conjugates harboring relatively small cargos \n ( e.g. , cyclohexane , naphthalene , phenylmethylbenzene ) \n to the cytosol readily , whereas large cargos ( e.g. , vancomycin ) are \n not transported to the cytosol .", "moreover , \n in prior studies , various -lactams including amp and amx have \n been linked to simple catechols and more complex catechol - containing siderophores \n or mimics thereof , which provides the \n opportunity to compare the outcomes obtained for different siderophore - inspired \n design strategies . in scheme 1", ", we present the syntheses of \n ent - amp 11 and ent - amx 12 , which feature \n installation of alkyne - modified -lactam warheads onto ent - azide 4 via copper - catalyzed azide - alkyne cycloaddition ( hereafter \n click reaction ) in the final step .", "the catechol moieties of benzyl - protected \n ent - azide 3 were deprotected by using bcl3 at 78 c to achieve ent - azide 4 as a white \n powder in 28% yield following purification by reverse - phase preparative \n hplc . catalytic hydrogenation using hydrogen gas and a pd / c catalyst \n is typically employed to deprotect ent catechols ; however , we observed that amp / amx decompose under these conditions \n and poison the pd / c catalyst .", "moreover , deprotection of the enterobactin \n catechols prior to installing the -lactams requires preservation \n of the azide moiety , and we therefore employed bcl3 for \n this reaction .", "initial attempts at assembling ent - amp / amx using standard \n conditions for the copper - catalyzed click reactions with ent - azide 4 and the alkyne - modified -lactams 7/8 , prepared by thionyl chloride coupling of 5-hexynoic acid \n to the amino group of amp / amx , failed because of copper - mediated -lactam \n decomposition .", "this problem was overcome \n by including the metal - ion chelator tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta ) in the click reactions , and ent - amp and ent - amx were obtained as \n white powders in high purity and yields of 66% and 76% , respectively , \n following semi - preparative hplc purification .", "it was necessary to \n perform hplc purification with eluents containing only 0.005% tfa \n to prevent decomposition of the acid - sensitive -lactam moieties . \n", "this synthetic route was likewise employed to prepare the d - enantiomers of ent - amp / amx 13 and 14 ( scheme 1 ) .", "deprotected ent - azide 4 enables alkyne - functionalized \n molecules to be covalently linked to ent via a click reaction , including \n molecules that are incompatible with reaction conditions required \n to deprotect the ent catechols .", "moreover , the deprotected ent - azide \n may be employed to append ent to surfaces , other materials , or biomolecules \n harboring alkyne groups .", "indeed , very \n few examples employing the copper(i)-catalyzed click reaction with \n fused -lactams are reported in the literature , and it is likely that this paucity stems from the fact that -lactams \n are incompatible with standard conditions for copper - catalyzed azide - alkyne \n cycloaddition .", "the conditions defined \n in this work employing tbta allow for copper - catalyzed triazole formation \n and preserve the -lactam warhead . in the absence of fe(iii ) , \n ent - amp / amx exhibit an absorption band centered at 316 nm resulting \n from catecholate absorption and the solutions are colorless ( meoh , \n rt ) .", "addition of 1.0 equiv of fe(iii ) to methanolic solutions of ent - amp \n and ent - amx causes the solution to immediately change from colorless \n to purple - red , and a broad absorption feature in the 400700 \n nm range appears ( figure s1 ) , confirming \n that both ent - amp / amx readily chelate fe(iii ) . to ascertain whether ent - amp / amx provide antibacterial activity \n against e. coli , including pathogenic strains , we preformed antimicrobial activity assays using \n six strains ( table s1 , figures 2 , s2s7 ) .", "e. coli atcc 25922 is a laboratory susceptibility test strain \n originally obtained as a clinical isolate .", "coli uti89 and \n cft073 are both pathogens of the human \n urinary tract ( upec).e .", "coli atcc 35401 ( serotype o78:h11 ) is an enterotoxigenic ( etec ) strain \n that was isolated from human feces .", "e. coli atcc \n 43895 ( serotype o157:h7 ) is an enterohemorrhagic ( ehec ) strain that \n was isolated from raw hamburger meat implicated in a hemorrhagic colitis \n outbreak .", "both etec and ehec strains produce virulence factors and \n toxins and cause diarrhea in humans .", "all e. coli strains biosynthesize ent for iron acquisition and \n express the ent receptor fepa .", "some e. coli strains \n employed in this work also have the capacity to produce and utilize \n salmochelins , c - glucoyslated ent derivatives .", "these molecules are produced by salmonella spp . \n and pathogenic e. coli strains for iron acquisition . \n", "the iroa gene cluster ( irobcden ) \n encodes proteins required for the biosynthesis and transport of salmochelins , \n and iron is the outer membrane receptor for salmochelins encoded by \n the iroa cluster .", "studies with salmonella indicate that iron has the ability to transport \n ent as well as its glucosylated forms . of the strains considered in this work , e. coli cft073 and uti89 harbor the iroa gene cluster .", "e. coli h9049 does not produce salmochelins , and a blast search using available e. \n coli genomes reveals that the e. coli 43985 \n genome does not contain the iroa cluster .", "the genome \n for e. coli atcc 25922 is unpublished ; however , this \n strain is reported to be sensitive to lipocalin-2 ( vide infra ) , which suggests that its genome does \n not encode the iroa cluster .", "lastly , it should be noted that e. coli cft073 is \n celebrated for having redundant iron import machineries , and this \n strain also harbors the iha gene , which encodes the \n outer membrane ent receptor iha that is distinct from fepa .", "antibacterial activity of ent - amp / amx against various e. \n coli strains that include human pathogens .", "all assays \n were performed in 50% mhb medium supplemented with 200 m dp \n to provide iron - limiting conditions ( mean  sem , n  3 ) .", "the data for assays performed in the absence of dp \n are presented in figures s2s7 .", "we performed antibacterial activity \n assays using a 10-fold dilution \n series to compare the abilities of ent - amp / amx and unmodified amp / amx \n to kill e. coli ( figures 2 , s2s7 ) .", "these assays were conducted \n in 50% mhb and in the absence or presence of 200 m dp .", "the \n latter growth conditions provide iron limitation and result in expression \n of the ent uptake machinery fepabcdg .", "amp / amx exhibit minimum inhibitory \n concentration ( mic ) values of 10 m against these e. \n coli strains regardless of the presence of dp in the growth \n medium ( figures 2 , s2s7 ) .", "all six e. coli strains are more susceptible \n to ent - amp / amx than amp / amx under conditions of iron limitation ( figure 2 ) .", "based on the 10-fold dilution series , ent - amp / amx \n are 100-fold more potent against e. coli 25922 , uti89 , \n and h9049 under iron - limiting conditions .", "although the mic values \n for 35401 and 43895 are only ca . 10-fold higher than for amp / amx in \n this assay , a significant reduction in growth is observed at 100 nm \n ent - amp / amx , whereas this concentration of amp / amx affords no growth \n inhibition .", "the enhanced sensitivity of e. coli cft073 \n to ent - amp / amx is remarkable .", "this strain exhibits the greatest sensitivity \n to ent - amp / amx , providing a 1000-fold decreased mic value ( 10 nm ) , \n and growth inhibition in the presence of 1 nm of the conjugate . \n", "moreover , in the absence of dp , e. coli cft073 exhibits \n the greatest susceptibility to ent - amp / amx ( figures \n s2s7 ) .", "a noteworthy characteristic of cft073 is its \n multiple mechanisms for iron acquisition , and we hypothesize that \n the presence of multiple receptors that recognize and transport ent \n ( fepa , iron , iha ) contributes to this enhanced sensitivity .", "cft073 \n and uti89 both express iron , which may indicate that iha is responsible \n for the enhanced susceptibility of cft073 ; however , we can not rule \n out the possibility that the conserved receptors function differently \n or exhibit different expression levels depending on the strain . moreover", ", \n other unappreciated mechanisms may contribute to the potent bactericidal \n action exhibited by ent - amp / amx against cft073 and other strains . to gain further insight into the mechanism \n of ent - amp / amx antibacterial action", ", we performed a series of experiments \n with the standard laboratory strain e. coli k-12 . \n", "we employed the same 10-fold dilution series to compare the activities \n of ent - amp / amx and amp / amx against k-12 . in the absence of dp ,", "ent - amp / amx \n and amp / amx exhibit comparable mic values , with complete killing observed \n at 10 m . at lower concentrations , ent - amp / amx exhibit \n slightly greater antibacterial activity than unmodified amp / amx ( figure 3a ) .", "this phenomenon is most evident at a conjugate / drug \n concentration of 1 m , where ent - amp / amx inhibit e. \n coli k-12 growth to varying degrees and amp / amx do not affect \n bacterial growth . under conditions of iron limitation ,", "a 100-fold \n reduction in mic value ( 10 m to 100 nm ) for ent - amp / amx is \n observed , and ca .", "50% growth inhibition occurs at 10 nm of each conjugate \n ( figure 3b ) .", "these trends are comparable to \n those observed for e. coli atcc 25922 , h9049 , and \n uti89 ( figures 2 , s2 , s3 , \n and s5 ) .", "when the antibacterial activity assay was performed \n using a 1:1 ratio of native l - ent and amp / amx , no reduction \n of amp / amx mic values was observed ( figure 3c ) , which suggests the conjugation between ent and the -lactams \n is required for the enhanced bactericidal action", ". moreover , treatment \n of e. coli k-12 with the iron - bound forms of ent - amp / amx , \n obtained by pre - incubating each conjugate with 1 equiv of ferric chloride , \n afforded the same mic values as observed for apo ent - amp / amx .", "these \n results indicate that the enhanced antibacterial activity does not \n result from iron chelation in the growth media ( figure 3d ) . in total", ", the data obtained for e. coli k-12 as well as the six other e. coli strains demonstrate \n that the antibacterial activity of ent - amp / amx against e. \n coli k-12 is enhanced under conditions of iron limitation \n and support a model of ent - mediated delivery of antibacterial cargo \n to the e. coli periplasm . antibacterial activity \n of ent - amp / amx against wild - type and mutant e. coli k-12 .", "( a , b ) growth inhibition of e. coli k-12 by \n amp / amx and ent - amp / amx in the absence ( a ) and presence \n ( b ) of dp .", "( c ) growth inhibition of e. coli k-12 \n treated with a 1:1 molar ratio of ent / amp and ent / amx .", "( d ) growth \n inhibition of e. coli k-12 treated with ferric ent - amp / amx . \n", "( e  g ) growth inhibition of fepa- ( e ) , fepc- ( f ) , and fes- ( g ) by amp / amx and \n ent - amp / amx .", "( h , i ) growth of e. coli k-12 in the \n presence of 1 m ent - amp / amx (  conjugate  ) and \n mixtures of ent - amp / amx ( 1 m ) and 1 , 5 , or 20 equiv of exogenous \n ent in the absence ( h ) and presence ( i ) of dp . the * * indicates od600 < 0.01 .", "all assays were performed in 50% mhb medium \n with or without 200 m dp ( see panels ) ( mean  sem ,", "the data for additional assays performed \n in the absence of dp are presented in figure s8 . to evaluate transport of ent - amp / amx \n into e. coli , we investigated the antimicrobial activity \n of ent - amp / amx and amp / amx against three single - gene knockout e. coli k-12 strains obtained from the keio collection , fepa- , fepc- , and fes- ( figures 3e ", "we selected these mutants to ascertain \n how components of the enterobactin transport and processing machinery \n contribute to ent - amp / amx antibacterial activity .", "e. colifepa- lacks the outer membrane ent receptor fepa \n that allows periplasmic delivery , fepc- lacks the \n atpase component of the inner membrane ent permease that transports \n ent into the cytosol , and e. colifes- lacks the cytoplasmic esterase fes responsible for hydrolysis of \n the ent macrolactone for iron release . on the basis of our studies \n with wild - type k-12 and other e. coli strains , we \n hypothesized that the activity of ent - amp / amx would be attenuated \n for the fepa- mutant .", "moreover , we questioned whether \n loss of fepc or fes would modulate the antimicrobial activity .", "overnight cultures of fepa- and fepc- reached od600 values ( 0.15 ) similar to that observed \n for wild - type k-12 when grown in 50% mhb supplemented with 200 m \n dp .", "in contrast , the fes- strain exhibited a severe \n growth defect under these conditions ( od600  0.04 ) . \n", "treatment of fepa- with ent - amp / amx afforded the \n same mic values as for amp / amx ( figure 3e ) \n and hence a 100-fold reduction in activity as compared to wild - type \n k-12 .", "iron deprivation is deleterious to e. coli , \n and we contend that the growth inhibition observed at 10 m \n ent - amp / amx results from iron starvation rather than an antibacterial \n activity of the amp / amx cargo .", "analysis of 50% mhb by inductively \n coupled plasma optical emission spectroscopy ( icp - oes ) revealed a \n total iron concentration of ca .", "4 m ( table \n s2 ) , which can be compared to 10 m of a high - affinity \n extracellular iron chelator .", "indeed , we previously observed similar \n growth inhibition of fepa- with 10 m of an \n ent - vancomycin conjugate ( extracellular iron chelation ) and also 10 \n m d - ent ( iron chelator that can not be used for iron \n acquisition ) under these growth conditions .", "these data confirm that fepa is essential for the potent antibacterial \n activity of ent - amp / amx against e. coli k-12 .", "in contrast to fepa- , the growth of fepc- and fes- was completely inhibited with 100 nm ent - amp / amx \n under iron - limiting conditions ( figure 3f , g ) , \n comparable to what was observed for the wild - type strain . the targets \n of -lactam antibiotics are penicillin binding proteins ( pbps ) , \n which are located in the periplasm of gram - negative bacteria .", "after \n crossing the outer membrane through fepa , ent - amp / amx enter the periplasm \n where covalent capture by the pbps presumably occurs .", "thus , it is \n reasonable that the downstream ent transport and processing steps \n involving fepcdg and fes do not affect the antimicrobial activity \n of the conjugates if they are trapped in the periplasm as a result \n of pbp binding .", "although it is possible that the ent uptake machinery \n ( e.g. , periplasmic binding protein fepb ) competes with the pbps for \n ent - amp / amx , no improved antibacterial activities were observed for \n the fepc- and fes- mutants compared \n to wild - type k-12 .", "this observation indicates that ent - amp / amx bind \n to pbps and are trapped in the periplasm .", "it should be noted that \n the fepb- mutant , which lacks the periplasmic binding \n protein , was also considered in this work ; however , this strain exhibited \n a severe growth defect and afforded inconsistent results . to probe \n interaction between fepa and ent - amp / amx , we performed \n growth inhibition assays employing mixtures of ent - amp / amx and varying \n concentrations of unmodified ent ( figure 3h ", "when these assays were performed under conditions of iron limitation , \n the presence of exogenous ent attenuated the antibacterial activity \n of ent - amp / amx .", "a 1:1 molar ratio of ent - amx / ent afforded an od600 value comparable to that of the untreated control , whereas \n higher equivalents of ent were required to block the antibacterial \n action of ent - amp .", "the origins of this difference are unclear and \n may indicate that the hydroxyl group of amx has a negative effect \n on the transport efficiency of the conjugate . in total , these ent \n addition assays suggest that competition for ent - amp / amx and ent occurs \n at the receptor(s ) and that the conjugates are delivered into the \n bacteria via the same uptake machinery as ent .", "l - serine is a biosynthetic \n building block for ent , and a role \n for chiral recognition in ent transport has been probed in prior studies . in one series of investigations ,", "e. coli fepa was \n found to bind ferric l - ent and ferric d - ent with \n similar affinities ( kd = 21 and 17 nm , \n respectively ; ascertained by measuring the binding of fe - loaded siderophores to e. coli bn1071 cells ) .", "a lack of transport of ferric d - ent \n into e. coli bn1071 was also reported in this work . \n", "a later study probed ent uptake in bacillus subtilis , and transport of both l- and d - ent analogues \n was observed to occur with similar efficiency .", "2457 t to hydrolyze l - ent and d - ent was \n evaluated , and fes did not accept d - ent as a substrate . as \n a result of the transport studies in b. subtilis and \n enzymatic activity assays with s. flexneri fes , a \n model in which both ent enantiomers are transported and chiral", "taken together , these studies suggest that the ability \n to transport d - ent may vary between species and even between \n strains of a given species , and more studies are required to address \n such possibilities .", "based on the observation that e. coli fepa binds d - ent and that b. subtilis transports d - ent analogues , we synthesized the d - enantiomers of \n the ent--lactam conjugates ( 13 and 14 , scheme 1 ) and evaluated the antibacterial \n activity of these conjugates against e. coli k-12 , \n 25922 , cft073 , 35401 , and 43895 ( figures s9s13 ) .", "d - ent - amp / amx exhibited reduced antibacterial activity \n relative to ent - amp / amx for k-12 , 25922 , cft073 , and 43895 . under \n conditions of iron limitation ,", "complete growth inhibition was observed \n with 1 m d - ent - amp / amx for e. coli k-12 , 25922 , and 43895 , compared to 100 nm for ent - amp / amx .", "likewise , \n a 10-fold reduction in antibacterial activity was observed for e. coli cft073 , where 100 nm d - ent was required \n to inhibit growth completely .", "in contrast , a negligible difference \n in antibacterial activity of the l- and d - isomers \n was observed for e. coli 35401 .", "regardless of enantiomer , \n all four ent - amp / amx conjugates provide enhanced antibacterial activity \n against these e. coli strains relative to amp / amx . \n", "nevertheless , these data suggest that d - ent - amp / amx are less \n readily transported into various e. coli strains \n than the l - isomers .", "although this modification provides no \n appreciable benefit for this -lactam delivery system with periplasmic \n targets , it is possible that ent - antibiotic conjugates based on d - ent may be desirable for delivering cargos to the cytosol , \n precluding concomitant delivery of nutrient fe(iii ) . with support for ent - mediated delivery of ent - amp /", "amx \n to the e. coli periplasm , we sought to confirm the \n essentiality of the -lactam warheads in antibacterial action . \n", "we therefore designed and prepared hydrolyzed ent - amp / amx analogues 15 and 16 ( scheme 2 ) where \n the -lactam structure is destroyed .", "hydrolysis of the amp / amx - alkynes 7 and 8 was achieved in the presence of 1% tfa \n with heating at 37 c , and the decomposition products 9 and 10 were obtained as diastereomeric mixtures ( scheme 2 ) .", "the formation of these species followed the reported \n degradation pathways for ampicillin , where hydrolysis and subsequent \n decarboxylation occur .", "the diastereomeric \n mixtures were employed to prepare the hydrolyzed conjugates ent - hydro - amp 15 and ent - hydro - amx 16 via a copper - catalyzed \n click reaction .", "( a , b ) antibacterial activity assays against e. coli k-12 ( a ) and cft073 ( b ) using ent - amp / amx and ent - hydro - amp / amx . \n", "( c ) antibacterial activity assays against e. coli atcc 35218 , which expresses a class a serine -lactamase , \n using ent - amp / amx in the absence and presence of the -lactamase \n inhibitors potassium clavulanate ( pc ) and sulbactam ( sb ) .", "all assays \n were performed in 50% mhb supplemented with 200 m dp ( mean \n  sem , n  3 ) .", "we employed ent - hydro - amp / amx in antibacterial activity assays \n against e. coli k-12 and observed negligible growth \n inhibition ( figure 4a ) . when e. coli cft073 was treated with ent - hydro - amp / amx in the presence of dp", "this result indicates that ent - hydro - amp / amx \n are transported into the cytoplasm of e. coli cft073 , \n where nutrient iron is released .", "we also performed a series \n of antibacterial activity assays with e. coli atcc \n 35218 , a strain that expresses a class a serine \n -lactamase .", "similar to unmodified amp / amx , ent - amp / amx were \n inactive against e. coli atcc 35218 ( mic > 10 \n m ) \n in the absence and presence of dp ( figures 4c and s14 ) .", "slight growth inhibition was \n observed at 10 m under conditions of iron limitation , which \n may be attributed to iron chelation .", "the addition of -lactamase \n inhibitors restored the activities of ent - amp / amx and amp / amx , and \n the conjugates exhibited greater antibacterial activity than the parent \n antibiotics ( figures 4c and s14 ) . in total , the assays with ent - hydro - amp / amx and strains \n expressing -lactamase demonstrate that an intact -lactam \n is required for the antibacterial activity of ent - amp / amx .", "moreover , \n these studies indicate that the -lactams retain their original \n function and inhibit pbps when conjugated to ent .", "the remarkable sensitivity of e. coli cft073 to \n ent - amp / amx ( figure 2c ) motivated us to investigate \n the relative cell - killing kinetics \n of ent - amp / amx and amp / amx to determine whether these conjugates kill e. coli cft073 more rapidly than the unmodified drugs . for \n comparison between e. coli strains ,", "ent - amp / amx \n provide more rapid cell death than unmodified amp / amx ( figure 5 ) , and this behavior is most apparent for e. coli cft073 , where the od600 value was almost \n reduced to the baseline value after 1 h incubation with 5 m \n ent - amp / amx , corresponding to a 2-fold log reduction in cfu / ml . in \n contrast , the change in od600 and cfu / ml for e. \n coli cft073 treated with 50 m unmodified amp / amx is \n negligible over this time period .", "the time - kill kinetics for e. coli k-12 , conducted with 50 m of both unmodified \n and modified -lactams , indicate a slight increase in kill kinetics \n for ent - amp / amx relative to amp / amx , and that the kinetics of cell - killing \n are slower for k-12 than cft073 ( figure 5 ) . \n", "these results support a model whereby ent modification facilitates \n uptake of amp / amx relative to the unmodified drugs .", "this effect is \n more dramatic for e. coli cft073 than k-12 , which \n is in accordance with the enhanced antibacterial activity observed \n for cft073 relative to the other e. coli strains \n considered in this work .", "time - kill kinetic assays for treatment of e. coli k-12 ( top panel ) and cft073 ( bottom panel ) with \n amp / amx and ent - amp / amx .", "e. coli k-12 ( 10 cfu / ml ) was treated \n with 50 m of amp / amx or 50 m ent - amp / amx .", "e. \n coli cft073 ( 10 cfu / ml ) was treated with \n 50 m of amp / amx or 5 m ent - amp / amx .", "the assays were \n conducted in 50% mhb medium containing 200 m dp at 37 c \n ( mean  sem , n = 3 ) . to determine whether ent - amp / amx exhibit broad - spectrum or species - selective \n activity , we performed antibacterial activity assays with two additional \n gram - negative and two gram - positive species in both the absence and \n presence of dp .", "these species include klebsiella pneumoniae atcc 13883 , pseudomonas aeruginosa pao1 , s. aureus atcc 25923 , and bacillus cereus atcc 14579 .", "k. pneumoniae is a gram - negative species \n that biosynthesizes and utilizes ent for iron acquisition .", "p. aeruginosa is a gram - negative bacterium that captures \n ent as a xenosiderophore and expresses two ent receptors \n pfea and pira.s .", "aureus and b. cereus are both gram - positive bacterial species , and the ability to utilize \n ferric ent as an iron source is reported for both species .", "in contrast to gram - negative bacteria , where the pbps are located \n in the periplasm , the targets of -lactam antibiotics are in \n the extracellular peptidoglycan of gram - positive organisms .", "k. pneumoniae atcc 13883 has a chromosomally encoded \n class a -lactamase ( shv-1 ) and lacks sensitivity to amp / amx . \n", "we observed no effect of 100 m amp / amx on k. pneumoniae growth under our assay conditions ( figure s15 ) .", "only 50% growth inhibition was observed when k. \n pneumoniae was treated with high concentrations ( 10 m ) \n of ent - amp / amx in the absence of dp , and the ent - amp conjugate provided \n the greatest activity under conditions of iron limitation with 90% \n growth inhibition at 10 m .", "when -lactamase inhibitors \n were included in the assays , k. pneumoniae exhibited \n greater sensitivity to amp / amx ( mic = 100 m ) and ent - amp / amx \n ( mic = 10 m ) ; however , we observed some growth inhibitory activity \n of the -lactamase inhibitor sulbactam alone under these assay \n conditions ( 100 m sb , figure s15 ) .", "thus , the possibility of a synergistic effect from the inhibitors \n and conjugates can not be ruled out completely .", "the lack of activity \n of ent - amp / amx against k. pneumoniae atcc 13883 is \n reminiscent of results obtained during investigations of amp / amx - functionalized \n tripodal triscatecholate ligands .", "these \n compounds were inactive against k. pneumoniae , and \n the behavior was attributed to either an inability of the k. pneumoniae iron transport machinery to import the conjugates \n or the development of resistance over the course of the assay .", "an \n alternative explanation is that -lactamase expression by k. pneumoniae resulted in inactivation of the -lactams .", "the p. aeruginosa pao1 strain employed in this \n work exhibited little sensitivity to both amp / amx and the conjugates \n under the antibacterial assay conditions ( figure \n s16 ) .", "amp / amx exhibited no activity up to 100 m , whereas \n ent - amp / amx provided growth inhibition at 10 m in both the \n absence and presence of dp . whether these results indicate that ent - amp / amx \n will be ineffective against multiple p. aeruginosa strains is unclear .", "p. aeruginosa strains exhibit \n different phenotypes , and highly variable and strain - dependent mic \n values have been reported for triscatecholate-lactam \n conjugates against p. aeruginosa .", "we previously reported that p. aeruginosa pao1 imports ent - cargo conjugates , and \n we speculate that the lack of activity observed for this strain stems \n from its inherent insensitivity to amp / amx .", "b. cereus atcc 14579 was also insensitive to amp / amx , \n which only afforded growth inhibition at 100 m .", "some growth \n inhibition was observed for b. cereus treated with \n 10 m ent - amp / amx , which may result from iron sequestration \n ( figure s17 ) .", "s. aureus atcc 25923 is susceptible to amp / amx , with complete growth inhibition \n observed at 1 m . in this case , a 10-fold reduction in antibacterial \n activity was observed for ent - amp / amx relative to unmodified amp / amx \n ( figure 6 ) . although the origins of this attenuation \n are unclear , we speculate that ent - amp / amx may have trouble penetrating \n the thick peptidoglycan of s. aureus .", "an alternative \n possibility is that recognition of ent - amp / amx by the s. aureus ent receptor diverts the -lactams from the pbps . in \n total , the results from these assays indicate that ent - amp / amx \n exhibit antibacterial activity enhancements that are species - selective , \n providing increased potency against e. coli strains \n and not for the other strains evaluated in this work . we therefore \n reasoned that ent - amp / amx , at low concentrations , should selectively \n kill e. coli in the presence of other less sensitive \n species .", "we treated co - cultures of e. coli cft073 \n and s. aureus with ent - amp / amx or amp / amx and analyzed \n the species composition following a 19-h incubation using harty - uti \n plates .", "these agar plates are employed in medical microbiology laboratories \n for the diagnosis of urinary tract infections and provide species \n identification by the colony color . when grown on harty - uti plates ,", "s. aureus are off - white and e. coli are \n purple - pink . in figure 6 , we present representative \n images of the colonies that resulted from treating co - cultures of e. coli and s. aureus with amp / amx or ent - amp / amx . \n in the absence of antibiotic ,", "the cultures provide a mixture of off - white \n and purple - pink colonies , indicating that both e. coli and s. aureus grow when cultured together .", "when \n the co - cultures are treated with 1 m amp / amx , only purple - pink \n colonies are present , which reveals that only e. coli survives .", "in contrast , treatment of the co - cultures with 1 m \n ent - amp / amx results in only off - white colonies from s. aureus .", "these comparisons demonstrate that ent - amp / amx selectively kill e. coli in the presence of s. aureus and \n that the siderophore modification reverses the inherent species \n selectivity of the parent antibiotics .", "achieving such species - selective \n and single - pathogen antibiotic targeting is an important goal and \n unmet need for pharmaceutical development that will allow for treating \n disease with minimal perturbation to the commensal microbiota .", "ent - amp / amx \n selectively kill e. coli cft073 in \n the presence of s. aureus atcc 25923 .", "( a , b ) antimicrobial \n activity assays against s. aureus atcc 25923 in the \n absence ( a ) and presence ( b ) of 200 m dp .", "( c , d ) bacterial growth \n monitored by od600 for cultures of e. coli only , s. aureus only , and 1:1 e. coli / s .", "aureus mixtures treated with amp / amx or ent - amp / amx \n in the absence ( c ) and presence ( d ) of 200 m dp .", "( e ) representative photographs of colonies \n from mixed cultures of e. coli cft073 and s. aureus atcc 29523 treated with ent - amp / amx ( 1 m ) \n or amp / amx ( 1 m ) in the presence of 200 m dp .", "all assays \n were conducted in 50% mhb medium ( t = 19 h , 30 c ) \n ( mean  sem , n  3 for a  d ) .", "we evaluated the cytotoxicity of ent - amp \n against the human t84 colon \n epithelial cell line .", "cell survival was evaluated by mtt assay after \n a 24 h treatment with apo or iron - bound ent - amp , amp , or ent .", "the \n iron - bound forms were assayed to determine whether iron chelation \n in the growth medium is a factor .", "no cytoxicity was observed for amp \n or ent - amp , whereas apo ent itself decreased the survival of t84 cells \n by approximately 30% at the highest concentration evaluated . when \n pre - loaded with fe(iii ) ,", "percent cell survival quantified by mtt assay after \n a 24 h treatment with apo or iron - bound ent , ent - amp , and the parent \n antibiotic amp in the absence and presence of 1 equiv of fe(iii ) ( mean \n  sem , n = 3 ) .", "lipocalin-2 \n ( lcn2 , also known as siderocalin or ngal ) is a 22-kda protein produced \n and released by neutrophils and epithelial cells .", "it has a hydrophobic \n binding pocket and coordinates ferric ent with sub - nanomolar affinity . by sequestering ferric ent", ", this host - defense protein contributes \n to the metal - withholding response and prevents bacterial acquisition \n of this essential nutrient . to determine whether lcn2 also binds ent - amp / amx \n and thereby blocks antibacterial activity ,", "we performed antibacterial \n activity assays with e. coli cft073 in m9 minimal \n medium supplemented with lcn2 or bovine serum albumin ( bsa ) . under \n these conditions , up to 1 m of lcn2 had no effect on the growth \n of e. coli cft073 . addition of 1 m lcn2 to \n the medium rescued the growth of e. coli cft073 treated \n with 100 nm of ent - amp ( figure 8) whereas addition \n of 1 m bsa had no effect on ent - amp cell killing .", "these results \n suggest that lcn2 binds ent - amp and blocks its recognition and uptake . \n", "to ascertain whether lcn2 binds ent - amp in the presence of exogenous \n ent , we performed a series of experiments where e. coli cft073 were treated with fixed concentrations of ent - amp ( 100 nm ) \n and lcn2 ( 1 m ) and the concentration of ent was varied ( 0 , \n 0.5 , and 1 m ) .", "moreover , when \n ent - amp was combined with a 1:1 molar ratio of ent and lcn2 at 10-fold \n excess over the conjugate , no e. coli growth was \n observed , which suggests that lcn2 preferentially binds ent .", "prior \n work demonstrated that lcn2 can not bind glucosylated ent , which was \n attributed to a steric clash between the glucose moieties and the \n ent binding site of the protein , and \n decreased hydrophobicity of the siderophore may also be a factor . \n", "thus , our data suggest that the nature of linker attachment at c5 \n and the peg3 moiety of ent - amp / amx do no abrogate lcn2 \n binding as effectively as the glucose moieties exhibited by the salmochelins", ". antibacterial \n activity of ent - amp against e. coli cft073 in the \n presence of lcn2 or bsa .", "( a ) e. coli cft073 treated \n with 100 nm ent - amp and varying concentrations of \n lcn2 or bsa control .", "( b ) e. coli cft073 treated with \n ent - amp , varying concentration of ent , and varying concentrations \n of lcn2 or bsa control .", "the assays were performed in m9 minimal medium \n ( 24 h , 37 c ) ( mean  sem , n  3 ) . \n"], ["ent - amp / amx are two siderophore-lactam \n conjugates \n based on the native enterobactin scaffold .", "these molecules hijack \n siderophore - based iron uptake pathways and provide potent antibacterial \n activity against various e. coli strains , including \n human pathogens .", "our investigations of ent - amp / amx establish the following : \n ( i ) ent - amp / amx provide up to 1000-fold enhanced antibacterial activity \n against e. coli strains ; ( ii ) ent - amp / amx are transported \n into e. coli by fepa and potentially other catecholate \n siderophore receptors ( iron , iha ) employed by pathogenic ctf073 \n and uti98 ; ( iii ) ent - amp / amx are captured by pbps in the periplasm , \n which results in pbp inhibition and cell death ; ( iv ) selective killing \n of e. coli in the presence of less susceptible organisms \n such as s. aureus is achieved because of the enterobactin \n scaffold ; ( v ) ent - amp / amx exhibit negligible cytotoxicity to human \n t84 intestinal epithelial cells ; and ( vi ) although lcn2 has the ability \n to bind ent - amp / amx , this siderophore - scavenging protein prefers \n to capture native ent . in total", ", these studies demonstrate that modification \n of antibiotic cargo with the native enterobactin platform provides \n many desirable features for antibiotic delivery and efficacy .", "the \n large molecular weight of the conjugates resulting from the native \n ent scaffold ( as opposed to a smaller mimic ) enhances rather than \n diminishes uptake for gram - negative e. coli .", "moreover , \n we observed no evidence for the development of resistance to ent - amp / amx \n over the course of the antibacterial activity assays performed during \n these investigations .", "our studies confirm that the enhanced \n antibacterial activity observed \n for ent - amp / amx requires both enterobactin recognition by outer membrane \n receptors and an intact -lactam moiety .", "the results are reminiscent of the dramatic \n antibacterial activity enhancements observed for albomycin , a secondary \n metabolite produced by actinomyces subtropicus .", "albomycin \n is comprised of the siderophore ferrichrome and a trna synthetase \n inhibitor , and it exhibits antimicrobial activities that are 30,000-fold \n greater than those of the unmodified trna synthetase inhibitor against e. coli and s. aureus . nonetheless , the ent - amp / amx cell - killing mechanism may \n be more complex than only more efficient -lactam delivery across \n the gram - negative outer membrane .", "binding of ent - amp / amx to the pbps \n presumably results in accumulation of ferric enterobactin in the e. coli periplasm for some period of time , which may have \n deleterious consequences .", "a recent study of an e. colitolc- mutant revealed that enterobactin accumulation \n in the periplasm affords growth defects and abnormal cellular morphologies .", "a fascinating observation that stems \n from our current work is the \n variable susceptibilities and responses of different e. coli strains to ent - amp / amx , which contrast the effects of unmodified \n amp / amx .", "such differences are manifest in the mic values to some degree \n and time - kill kinetics ; however , the results presented in figure 2 indicate that mic values alone do not provide a \n full description of how ent - amp / amx susceptibility differs between e. coli strains .", "these results suggest underlying complexity \n in microbial physiology related to iron - uptake pathways that can not \n be fully explained by the presence or absence of a gene for a particular \n receptor ( i.e. , fepa , iron ) .", "the heightened sensitivity of uropathogenic e. coli cft073 is particularly noteworthy , and it will be \n interesting to decipher the physiological origins of this effect as \n well as the differential behavior of various e. coli pathogens toward ent - antibiotic conjugates . there is a clear \n and unmet need for new antibacterial agents to \n treat bacterial infections in humans , including antibiotics that target \n specific bacterial sub - populations . preventing undesirable consequences of antibiotic treatment on the \n commensal microbiota , which contributes to human health in beneficial \n ways , is a challenge that needs to be addressed .", "such targeted therapeutics will be valuable not only for treating \n bacterial infections when the causative agent is known ( e.g. , urinary \n tract infection and e. coli , cystic fibrosis lung \n infection and p. aeruginosa ) but also for other pathologies \n that involve microbial dysbiosis , such as irritable bowel disease .", "our studies of ent - amp / amx provide one step \n toward addressing species - specific antibiotic targeting as well as \n overcoming gram - negative outer membrane permeability . from the standpoint \n of the host environment , commensal e. coli employ \n ent for acquiring iron in the host , and thus further elaboration of \n this strategy to specifically target pathogenicity and evade host \n responses ( e.g. , lcn2 ) is desirable ."]]}
{"article_id": "PMC3604872", "article_text": ["huntington 's disease ( hd ) is a dominantly inherited neurodegenerative disease caused by mutations in the it-15 gene on chromosome 4 , which was subsequently renamed htt .", "more specifically , the disease is linked to expansion of the cag repeat tract of this gene , which codes for polyglutamine repeats in the huntingtin protein .", "george huntington provided the classic description of the disorder in 1872 from cases in the east hampton area of long island , n.y .", "the best known manifestation is chorea , which consists of random , fluid involuntary hyperkinetic movements .", "the term is derived from the greek word o , meaning a traditional circle dance .", "it is important to note that , while movements in chorea may resemble a dance , they are not rhythmic or repetitive .", "psychiatric manifestations may include irritability , anxiety , obsessive compulsive disorder , paranoid ideation , and psychosis .", "depression is the most common psychiatric feature of hd and suicidal ideation occurs in 510% of hd patients .", "other neurologic manifestations include eye movement abnormalities , cognitive disturbance , pendular knee reflexes , motor impersistence , and postural instability .", "we describe a mild case of adult onset hd early in the disease course along with mri and diffusion tensor imaging ( dti ) features . increased fractional anisotropy ( fa ) of striatum", "the patient is a 50-year - old right - handed caucasian male who was diagnosed with hd at age 47 by an outside movement disorders specialist .", "the patient received a clinical diagnosis of hd given mild neurological findings in the context of genetically confirmed family history .", "there is a history of depression since his diagnosis , which has become worse over the past week .", "he was admitted for psychiatric evaluation due to suicidal ideation with plans to overdose on sleeping pills .", "in addition , he complains of mild cognitive disturbance for at least one year , with difficulty processing information in distracting environments .", "he has a history of alcohol abuse and quit alcoholics anonymous about six months ago .", "his medications include paroxetine 30 mg q.h.s . , aripiprazole 5 mg q.a.m . , naltrexone 50 mg q.a.m . ,", "memantine 10 mg b.i.d . , and trazodone 200 mg q.h.s . upon examination , he was alert and oriented to person , place , and time . speech was fluent .", "cranial nerve examination was significant for saccadic pursuit , slow saccade initiation and velocity in the horizontal and vertical directions , and motor impersistence exhibited by inability to fully protrude the tongue for five seconds .", "there was only mild intermittent chorea in the fingertips bilaterally and rare lower facial chorea .", "he improved after one week of inpatient psychotherapy and was released for outpatient psychiatric and neurologic follow - up .", "images were obtained on a 3.0-tesla phillips achieva mri system . bicaudate ratio ( bcr ) , an imaging measure that correlates with", "caudate atrophy in hd , was obtained by measuring the shortest inter - caudate distance ( seen as indentation of the frontal horns ) on axial slice and dividing it by the distance between the outer tables of the skull along the same line , multiplied by 100 .", "phillips fibertrak software was used to analyze dti values on regions of interest which were defined as caudate head bilaterally and putamen bilaterally on axial slices .", "1 ) . axial bcr in the patient was increased compared to control at 16 versus 12 , respectively .", "2 ) was increased in the bilateral caudate and putamen versus control ( table 1 ) .", "depression and suicidality were managed with inpatient psychiatry admission and psychotherapy , followed by outpatient therapy .", "subtle neurologic manifestations included slowing of saccade imitation and velocity , saccadic pursuit , difficulty with tongue protrusion due to motor impersistence , pendular knee reflexes , and postural instability demonstrated by the inability to maintain tandem stance .", "tetrabenazine is fda approved for the treatment of hd chorea and its efficacy is supported by clinical evidence .", "caudate atrophy and ex vacuo dilatation of the frontal horns , which is typical for hd , was visualized .", "increase in fa in white matter is linked to more coherent fiber orientation and normal structure , but in striatal grey matter increased fa is due to neurodegeneration .", "propose that increased fa in striatal grey matter structures is due to the loss of efferent fibers which in turn leads to increased coherence .", "for example , in hd there is preferential loss of putamino - pallidal connections that are oriented mediolaterally as relative preservation of anterior - posterior and dorsal - ventral cortical - putaminal fibers .", "hence , loss of these mediolateral fibers may lead to more uniform fiber orientation and higher fa values . in conclusion ,", "we have presented a patient who is early in the course of huntington 's disease and who already exhibits basal ganglia neurodegeneration with atrophy and changes in fa .", "dr . fekete received honoraria from medlink , inc . , and serves as a consultant for teva neuroscience , inc . , and lundbeck , llc ."], "abstract_text": ["<S> huntington 's disease ( hd ) is a dominantly inherited neurodegenerative disease best known for chorea . </S>", "<S> the disorder includes numerous other clinical features including mood disorder , eye movement abnormalities , cognitive disturbance , pendular knee reflexes , motor impersistence , and postural instability . </S>", "<S> we describe a mild case of hd early in the disease course with depression and subtle neurological manifestations . </S>", "<S> in addition , we review mri and diffusion tensor imaging features in this patient . the bicaudate ratio , a measure of caudate atrophy , was increased . </S>", "<S> fractional anisotropy values of the bilateral caudate and putamen were increased , signifying neurodegeneration of these structures in hd . </S>"], "labels": null, "section_names": ["Introduction", "Case Presentation", "Materials and Methods", "Results", "Discussion", "Disclosure Statement"], "sections": [["huntington 's disease ( hd ) is a dominantly inherited neurodegenerative disease caused by mutations in the it-15 gene on chromosome 4 , which was subsequently renamed htt .", "more specifically , the disease is linked to expansion of the cag repeat tract of this gene , which codes for polyglutamine repeats in the huntingtin protein .", "george huntington provided the classic description of the disorder in 1872 from cases in the east hampton area of long island , n.y .", "the best known manifestation is chorea , which consists of random , fluid involuntary hyperkinetic movements .", "the term is derived from the greek word o , meaning a traditional circle dance .", "it is important to note that , while movements in chorea may resemble a dance , they are not rhythmic or repetitive .", "psychiatric manifestations may include irritability , anxiety , obsessive compulsive disorder , paranoid ideation , and psychosis .", "depression is the most common psychiatric feature of hd and suicidal ideation occurs in 510% of hd patients .", "other neurologic manifestations include eye movement abnormalities , cognitive disturbance , pendular knee reflexes , motor impersistence , and postural instability .", "we describe a mild case of adult onset hd early in the disease course along with mri and diffusion tensor imaging ( dti ) features . increased fractional anisotropy ( fa ) of striatum"], ["the patient is a 50-year - old right - handed caucasian male who was diagnosed with hd at age 47 by an outside movement disorders specialist .", "the patient received a clinical diagnosis of hd given mild neurological findings in the context of genetically confirmed family history .", "there is a history of depression since his diagnosis , which has become worse over the past week .", "he was admitted for psychiatric evaluation due to suicidal ideation with plans to overdose on sleeping pills .", "in addition , he complains of mild cognitive disturbance for at least one year , with difficulty processing information in distracting environments .", "he has a history of alcohol abuse and quit alcoholics anonymous about six months ago .", "his medications include paroxetine 30 mg q.h.s . , aripiprazole 5 mg q.a.m . , naltrexone 50 mg q.a.m . ,", "memantine 10 mg b.i.d . , and trazodone 200 mg q.h.s . upon examination , he was alert and oriented to person , place , and time . speech was fluent .", "cranial nerve examination was significant for saccadic pursuit , slow saccade initiation and velocity in the horizontal and vertical directions , and motor impersistence exhibited by inability to fully protrude the tongue for five seconds .", "there was only mild intermittent chorea in the fingertips bilaterally and rare lower facial chorea .", "he improved after one week of inpatient psychotherapy and was released for outpatient psychiatric and neurologic follow - up ."], ["images were obtained on a 3.0-tesla phillips achieva mri system . bicaudate ratio ( bcr ) , an imaging measure that correlates with", "caudate atrophy in hd , was obtained by measuring the shortest inter - caudate distance ( seen as indentation of the frontal horns ) on axial slice and dividing it by the distance between the outer tables of the skull along the same line , multiplied by 100 .", "phillips fibertrak software was used to analyze dti values on regions of interest which were defined as caudate head bilaterally and putamen bilaterally on axial slices ."], ["1 ) . axial bcr in the patient was increased compared to control at 16 versus 12 , respectively .", "2 ) was increased in the bilateral caudate and putamen versus control ( table 1 ) ."], ["depression and suicidality were managed with inpatient psychiatry admission and psychotherapy , followed by outpatient therapy .", "subtle neurologic manifestations included slowing of saccade imitation and velocity , saccadic pursuit , difficulty with tongue protrusion due to motor impersistence , pendular knee reflexes , and postural instability demonstrated by the inability to maintain tandem stance .", "tetrabenazine is fda approved for the treatment of hd chorea and its efficacy is supported by clinical evidence .", "caudate atrophy and ex vacuo dilatation of the frontal horns , which is typical for hd , was visualized .", "increase in fa in white matter is linked to more coherent fiber orientation and normal structure , but in striatal grey matter increased fa is due to neurodegeneration .", "propose that increased fa in striatal grey matter structures is due to the loss of efferent fibers which in turn leads to increased coherence .", "for example , in hd there is preferential loss of putamino - pallidal connections that are oriented mediolaterally as relative preservation of anterior - posterior and dorsal - ventral cortical - putaminal fibers .", "hence , loss of these mediolateral fibers may lead to more uniform fiber orientation and higher fa values . in conclusion ,", "we have presented a patient who is early in the course of huntington 's disease and who already exhibits basal ganglia neurodegeneration with atrophy and changes in fa ."], ["dr . fekete received honoraria from medlink , inc . , and serves as a consultant for teva neuroscience , inc . , and lundbeck , llc ."]]}
{"article_id": "PMC4341291", "article_text": ["because motor weakness is one of the most serious disabling sequelae of stroke , so it is important to elucidate the motor recovery mechanism for successful rehabilitation .", "many studies have attempted to elucidate the motor recovery mechanism of stroke and several motor recovery mechanisms have been suggested .", "however , the majority of these studies focus on cerebral infarct and relatively little is known about the motor recovery mechanism of intracerebral hemorrhage ( ich ) .", "diffusion tensor imaging ( dti ) can help to investigate the motor recovery mechanism of ich by enabling the direct visualization and estimation of the corticospinal tract ( cst ) .", ", we report on a patient with ich who showed a change in the origin of an injured cst from the premotor cortex ( pmc ) to the primary motor cortex ( m1 ) .", "an 86-year - old woman presented with complete paralysis of the right upper and lower extremities , which occurred at the onset of spontaneous ich ( motricity index ( mi ) : 0 ( full mark : 100 ) ) ( table 1 ) .", "longitudinal changes in motor function t2-weighted mr images showed a hematoma in the left fronto - parietal lobe , including m1 , at 1 month after onset , that resolved at 5 months after onset ( figure 1a ) . from 1 month to 5 months after onset , she received comprehensive rehabilitative management , which included the administration of neurotrophic drugs ( ropinirole , bromocriptine , levodopa , and amantadine ) , movement therapy , and neuromuscular electrical stimulation of the affected finger extensors and ankle dorsiflexors .", "movement therapy focused on improving the motor functions of the right upper and lower extremities and included physical and occupational therapy sessions five times per week .", "weakness of her left extremities improved from an mi score of 0 point at 1 month to 27 points at 2 months and to 52 points ( 5 months after ich onset ) after 4 months of rehabilitation . as a result", ", she was able to extend the affected fingers against gravity and to walk independently on even ground .", "brain magnetic resonance images , diffusion tensor imaging ( dti ) and transcranial magnetic stimulation results of an 86-year - old female patient with a hematoma in the left fronto - parietal lobe .", "( a ) t2-weighted magnetic resonance images showed a hematoma in the left fronto - parietal lobe including the primary motor cortex at 1 month after onset , which was resolved at 5 months after onset .", "dti results showed that at 1month and 5 months after intracerebral hemorrhage , the right csts originated from the cerebral cortex , including the primary motor cortex , and then passed through the known cst pathway . in the left ( affected ) hemisphere", ", the csts originated from the left premotor cortex at 1 month after onset , and from the left primary motor cortex and premotor cortex at 5 months after onset .", "a motor evoked potential of low amplitude ( 100 v ) was obtained from right abductor pollicis brevis muscle by the affected ( left ) hemisphere stimulation at 1 month after onset . however , motor evoked potential amplitude was increased to the normal range ( 3.8 mv ) at 5 months after onset .", "ich volume was measured on ct image and t2-weighted mri images through picture archived communication system ( pacs , marotech , korea ) .", "the maximum width ( x ) , length ( y ) and height ( z ) of lesion at the level where a hematoma was clearly seen were measured .", "the ich volume was calculated according to the formula : the volume of ich was 37.19 mv on the ct images at the ich onset and 26.71 mv on the mr images at 1 month after onset .", "diffusion tensor images were acquired using a synergy - l sensitivity encoding ( sense ) head coil on a 1.5-t philips gyroscan intera system ( hoffman - laroche , mijdrecht , the netherlands ) using a single - shot echo planar imaging with a navigator echo .", "for each of the 32 noncollinear diffusion - sensitizing gradients , 60 contiguous slices ( matrix = 192  192 , field of vision = 240 mm , repetition time / echo time = 10 726/76 ms , b = 600 mm / s , number of excitations = 1 , thickness = 2.5 mm ) were acquired .", "three - dimensional reconstructions of fiber tracts were obtained using the dti task card software ( philips extended mr work space 2.6.3 ) ( threshold fractional anisotropy ( fa ) = 0.15 , angle = 45 ) .", "fiber tracts passing through both region of interests ( cst portion of the anterior mid - pons and low - pons on the color map ) were designated final tracts of interest . at 1 month and 5 months after ich onset , dti results showed that in the right ( non - affected ) hemisphere , the right cst originated from the cerebral cortex ( including m1 ) and passed through the known cst pathway .", "however , in the left ( affected ) hemisphere , the cst originated from the left pmc at 1 month and from the left m1 and pmc at 5 months ( figure 1b ) .", "tms was performed using a magstim novametrix 200 magnetic stimulator equipped with a 9-cm mean diameter circular coil ( novametrix medical systems inc , wallingford , ct , usa ) .", "the left hemisphere was stimulated by a counterclockwise current and the right hemisphere was stimulated by a clockwise current .", "motor - evoked potentials ( meps ) were obtained from both abductor pollicis brevis muscles in a relaxed state . excitatory threshold ( et )", "was defined as the minimum stimulus required to elicit an mep with a peak - to - peak amplitude of 50 v or greater during two of four attempts .", "each site was stimulated three times with inter - stimulus intervals of  10 seconds , and shortest latency and average peak to peak amplitude were estimated .", "mep ( latency : 23.5 ms ; amplitude : 100 v ; et : 100% ) was evoked from the affected ( left ) hemisphere during the tms study conducted at 1 month after ich onset , but the amplitude increased to the normal range at 5 months after ich onset ( latency : 19.7 ms ; amplitude : 3.8 mv ; et : 70% ) ( figure 1c ) .", "ich volume was measured on ct image and t2-weighted mri images through picture archived communication system ( pacs , marotech , korea ) . the maximum width ( x ) ,", "length ( y ) and height ( z ) of lesion at the level where a hematoma was clearly seen were measured .", "the ich volume was calculated according to the formula : the volume of ich was 37.19 mv on the ct images at the ich onset and 26.71 mv on the mr images at 1 month after onset .", "diffusion tensor images were acquired using a synergy - l sensitivity encoding ( sense ) head coil on a 1.5-t philips gyroscan intera system ( hoffman - laroche , mijdrecht , the netherlands ) using a single - shot echo planar imaging with a navigator echo .", "for each of the 32 noncollinear diffusion - sensitizing gradients , 60 contiguous slices ( matrix = 192  192 , field of vision = 240 mm , repetition time / echo time = 10 726/76 ms , b = 600 mm / s , number of excitations = 1 , thickness = 2.5 mm ) were acquired .", "three - dimensional reconstructions of fiber tracts were obtained using the dti task card software ( philips extended mr work space 2.6.3 ) ( threshold fractional anisotropy ( fa ) = 0.15 , angle = 45 ) .", "fiber tracts passing through both region of interests ( cst portion of the anterior mid - pons and low - pons on the color map ) were designated final tracts of interest . at 1 month and 5 months after ich onset , dti results showed that in the right ( non - affected ) hemisphere , the right cst originated from the cerebral cortex ( including m1 ) and passed through the known cst pathway .", "however , in the left ( affected ) hemisphere , the cst originated from the left pmc at 1 month and from the left m1 and pmc at 5 months ( figure 1b ) .", "tms was performed using a magstim novametrix 200 magnetic stimulator equipped with a 9-cm mean diameter circular coil ( novametrix medical systems inc , wallingford , ct , usa ) .", "the left hemisphere was stimulated by a counterclockwise current and the right hemisphere was stimulated by a clockwise current .", "motor - evoked potentials ( meps ) were obtained from both abductor pollicis brevis muscles in a relaxed state . excitatory threshold ( et )", "was defined as the minimum stimulus required to elicit an mep with a peak - to - peak amplitude of 50 v or greater during two of four attempts .", "each site was stimulated three times with inter - stimulus intervals of  10 seconds , and shortest latency and average peak to peak amplitude were estimated .", "mep ( latency : 23.5 ms ; amplitude : 100 v ; et : 100% ) was evoked from the affected ( left ) hemisphere during the tms study conducted at 1 month after ich onset , but the amplitude increased to the normal range at 5 months after ich onset ( latency : 19.7 ms ; amplitude : 3.8 mv ; et : 70% ) ( figure 1c ) .", "in this patient , we investigated changes of the injured left cst and found that the injured cst seemed to recover as detected by dti , tms and clinical observation . at 1 month after ich onset , the left cst originated from the pmc by dti , and the mep obtained at the right hand showed low amplitude and a latency compatible with that of the cst .", "by contrast , 5-month results revealed that the left cst originated from the left m1 by dti and that mep amplitude had improved to the normal range by tms .", "the m1 is the major origin of the cst , although the cst is known to originate from the extensive cerebral cortex , including the pmc , the primary somatosensory cortex , and the prefrontal cortex . on the other hand ,", "consequently , the dti and tms results of this patient suggest that the severely injured left cst had recovered over 4 months in terms of its origin and fiber number .", "furthermore , clinically , the finding that the patient was able to flex and extend against gravity at 5 months after ich onset provides additional evidence of left cst recovery . as a result", ", right extremity motor functions seemed to have been recovered due to reorganization of the injured left cst originating from the left pmc to left m1 .", "it is also possible that compression of the cst into the left pmc recovered in concert with hematoma resolution . for the motor recovery mechanism in ich ,", "several dti based studies have described recovery of an injured cst or the contribution made to recovery by the contralateral unaffected cst . during the last 5 years", ", a number of serial dti studies have described the recovery of injured cst after ich .", "jang et al demonstrated the recovery process of an injured cst in a patient with a subcortical ich by serial dti .", "the patient presented with complete paralysis of right extremities at onset , but over 16 months , motor functions of affected extremities slowly recovered to nearly normal .", "furthermore , dtt showed that the origin of the cst had changed from the posterior parietal cortex at 1 month to the primary somatosensory cortex at 4 months and m1 at 16 months .", "the authors suggested that recovery of the origin of the damaged cst was due to a process of normalization from the parietal cortex to m1 .", "by contrast , our patient showed a change in the origin of the injured cst from the pmc to m1 .", "this study described changes of an injured cst that occurred in concert with motor recovery in a patient with ich . to the best of our knowledge ,", "this is the first longitudinal study to demonstrate a change in the origin of an injured cst from the pmc to m1 in ich .", "results from this study suggest a motor recovery mechanism of ich and the important implications regarding brain plasticity and brain rehabilitation after ich .", "however , this study is obviously limited by case numbers , and further complementary studies involving larger case numbers are warranted ."], "abstract_text": ["<S> many studies have attempted to elucidate the motor recovery mechanism of stroke , but the majority of these studies focus on cerebral infarct and relatively little is known about the motor recovery mechanism of intracerebral hemorrhage . in this study , we report on a patient with intracerebral hemorrhage who displayed a change in injured corticospinal tract originating from the premotor cortex to the primary motor cortex on diffusion tensor imaging . an 86-year - old woman presented with complete paralysis of the right extremities following spontaneous intracerebral hemorrhage in the left frontoparietal cortex . </S>", "<S> the patient showed motor recovery , to the extent of being able to extend affected fingers against gravity and to walk independently on even ground at 5 months after onset . </S>", "<S> diffusion tensor imaging showed that the left corticospinal tract originated from the premotor cortex at 1 month after intracerebral hemorrhage and from the left primary motor cortex and premotor cortex at 5 months after intracerebral hemorrhage . </S>", "<S> the change of injured corticospinal tract originating from the premotor cortex to the primary motor cortex suggests motor recovery of intracerebral hemorrhage . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "Clinical evaluation", "DTI examination", "Transcranial magnetic stimulation (TMS)", "DISCUSSION"], "sections": [["because motor weakness is one of the most serious disabling sequelae of stroke , so it is important to elucidate the motor recovery mechanism for successful rehabilitation .", "many studies have attempted to elucidate the motor recovery mechanism of stroke and several motor recovery mechanisms have been suggested .", "however , the majority of these studies focus on cerebral infarct and relatively little is known about the motor recovery mechanism of intracerebral hemorrhage ( ich ) .", "diffusion tensor imaging ( dti ) can help to investigate the motor recovery mechanism of ich by enabling the direct visualization and estimation of the corticospinal tract ( cst ) .", ", we report on a patient with ich who showed a change in the origin of an injured cst from the premotor cortex ( pmc ) to the primary motor cortex ( m1 ) ."], ["an 86-year - old woman presented with complete paralysis of the right upper and lower extremities , which occurred at the onset of spontaneous ich ( motricity index ( mi ) : 0 ( full mark : 100 ) ) ( table 1 ) .", "longitudinal changes in motor function t2-weighted mr images showed a hematoma in the left fronto - parietal lobe , including m1 , at 1 month after onset , that resolved at 5 months after onset ( figure 1a ) . from 1 month to 5 months after onset , she received comprehensive rehabilitative management , which included the administration of neurotrophic drugs ( ropinirole , bromocriptine , levodopa , and amantadine ) , movement therapy , and neuromuscular electrical stimulation of the affected finger extensors and ankle dorsiflexors .", "movement therapy focused on improving the motor functions of the right upper and lower extremities and included physical and occupational therapy sessions five times per week .", "weakness of her left extremities improved from an mi score of 0 point at 1 month to 27 points at 2 months and to 52 points ( 5 months after ich onset ) after 4 months of rehabilitation . as a result", ", she was able to extend the affected fingers against gravity and to walk independently on even ground .", "brain magnetic resonance images , diffusion tensor imaging ( dti ) and transcranial magnetic stimulation results of an 86-year - old female patient with a hematoma in the left fronto - parietal lobe .", "( a ) t2-weighted magnetic resonance images showed a hematoma in the left fronto - parietal lobe including the primary motor cortex at 1 month after onset , which was resolved at 5 months after onset .", "dti results showed that at 1month and 5 months after intracerebral hemorrhage , the right csts originated from the cerebral cortex , including the primary motor cortex , and then passed through the known cst pathway . in the left ( affected ) hemisphere", ", the csts originated from the left premotor cortex at 1 month after onset , and from the left primary motor cortex and premotor cortex at 5 months after onset .", "a motor evoked potential of low amplitude ( 100 v ) was obtained from right abductor pollicis brevis muscle by the affected ( left ) hemisphere stimulation at 1 month after onset . however , motor evoked potential amplitude was increased to the normal range ( 3.8 mv ) at 5 months after onset .", "ich volume was measured on ct image and t2-weighted mri images through picture archived communication system ( pacs , marotech , korea ) .", "the maximum width ( x ) , length ( y ) and height ( z ) of lesion at the level where a hematoma was clearly seen were measured .", "the ich volume was calculated according to the formula : the volume of ich was 37.19 mv on the ct images at the ich onset and 26.71 mv on the mr images at 1 month after onset .", "diffusion tensor images were acquired using a synergy - l sensitivity encoding ( sense ) head coil on a 1.5-t philips gyroscan intera system ( hoffman - laroche , mijdrecht , the netherlands ) using a single - shot echo planar imaging with a navigator echo .", "for each of the 32 noncollinear diffusion - sensitizing gradients , 60 contiguous slices ( matrix = 192  192 , field of vision = 240 mm , repetition time / echo time = 10 726/76 ms , b = 600 mm / s , number of excitations = 1 , thickness = 2.5 mm ) were acquired .", "three - dimensional reconstructions of fiber tracts were obtained using the dti task card software ( philips extended mr work space 2.6.3 ) ( threshold fractional anisotropy ( fa ) = 0.15 , angle = 45 ) .", "fiber tracts passing through both region of interests ( cst portion of the anterior mid - pons and low - pons on the color map ) were designated final tracts of interest . at 1 month and 5 months after ich onset , dti results showed that in the right ( non - affected ) hemisphere , the right cst originated from the cerebral cortex ( including m1 ) and passed through the known cst pathway .", "however , in the left ( affected ) hemisphere , the cst originated from the left pmc at 1 month and from the left m1 and pmc at 5 months ( figure 1b ) .", "tms was performed using a magstim novametrix 200 magnetic stimulator equipped with a 9-cm mean diameter circular coil ( novametrix medical systems inc , wallingford , ct , usa ) .", "the left hemisphere was stimulated by a counterclockwise current and the right hemisphere was stimulated by a clockwise current .", "motor - evoked potentials ( meps ) were obtained from both abductor pollicis brevis muscles in a relaxed state . excitatory threshold ( et )", "was defined as the minimum stimulus required to elicit an mep with a peak - to - peak amplitude of 50 v or greater during two of four attempts .", "each site was stimulated three times with inter - stimulus intervals of  10 seconds , and shortest latency and average peak to peak amplitude were estimated .", "mep ( latency : 23.5 ms ; amplitude : 100 v ; et : 100% ) was evoked from the affected ( left ) hemisphere during the tms study conducted at 1 month after ich onset , but the amplitude increased to the normal range at 5 months after ich onset ( latency : 19.7 ms ; amplitude : 3.8 mv ; et : 70% ) ( figure 1c ) ."], ["ich volume was measured on ct image and t2-weighted mri images through picture archived communication system ( pacs , marotech , korea ) . the maximum width ( x ) ,", "length ( y ) and height ( z ) of lesion at the level where a hematoma was clearly seen were measured .", "the ich volume was calculated according to the formula : the volume of ich was 37.19 mv on the ct images at the ich onset and 26.71 mv on the mr images at 1 month after onset ."], ["diffusion tensor images were acquired using a synergy - l sensitivity encoding ( sense ) head coil on a 1.5-t philips gyroscan intera system ( hoffman - laroche , mijdrecht , the netherlands ) using a single - shot echo planar imaging with a navigator echo .", "for each of the 32 noncollinear diffusion - sensitizing gradients , 60 contiguous slices ( matrix = 192  192 , field of vision = 240 mm , repetition time / echo time = 10 726/76 ms , b = 600 mm / s , number of excitations = 1 , thickness = 2.5 mm ) were acquired .", "three - dimensional reconstructions of fiber tracts were obtained using the dti task card software ( philips extended mr work space 2.6.3 ) ( threshold fractional anisotropy ( fa ) = 0.15 , angle = 45 ) .", "fiber tracts passing through both region of interests ( cst portion of the anterior mid - pons and low - pons on the color map ) were designated final tracts of interest . at 1 month and 5 months after ich onset , dti results showed that in the right ( non - affected ) hemisphere , the right cst originated from the cerebral cortex ( including m1 ) and passed through the known cst pathway .", "however , in the left ( affected ) hemisphere , the cst originated from the left pmc at 1 month and from the left m1 and pmc at 5 months ( figure 1b ) ."], ["tms was performed using a magstim novametrix 200 magnetic stimulator equipped with a 9-cm mean diameter circular coil ( novametrix medical systems inc , wallingford , ct , usa ) .", "the left hemisphere was stimulated by a counterclockwise current and the right hemisphere was stimulated by a clockwise current .", "motor - evoked potentials ( meps ) were obtained from both abductor pollicis brevis muscles in a relaxed state . excitatory threshold ( et )", "was defined as the minimum stimulus required to elicit an mep with a peak - to - peak amplitude of 50 v or greater during two of four attempts .", "each site was stimulated three times with inter - stimulus intervals of  10 seconds , and shortest latency and average peak to peak amplitude were estimated .", "mep ( latency : 23.5 ms ; amplitude : 100 v ; et : 100% ) was evoked from the affected ( left ) hemisphere during the tms study conducted at 1 month after ich onset , but the amplitude increased to the normal range at 5 months after ich onset ( latency : 19.7 ms ; amplitude : 3.8 mv ; et : 70% ) ( figure 1c ) ."], ["in this patient , we investigated changes of the injured left cst and found that the injured cst seemed to recover as detected by dti , tms and clinical observation . at 1 month after ich onset , the left cst originated from the pmc by dti , and the mep obtained at the right hand showed low amplitude and a latency compatible with that of the cst .", "by contrast , 5-month results revealed that the left cst originated from the left m1 by dti and that mep amplitude had improved to the normal range by tms .", "the m1 is the major origin of the cst , although the cst is known to originate from the extensive cerebral cortex , including the pmc , the primary somatosensory cortex , and the prefrontal cortex . on the other hand ,", "consequently , the dti and tms results of this patient suggest that the severely injured left cst had recovered over 4 months in terms of its origin and fiber number .", "furthermore , clinically , the finding that the patient was able to flex and extend against gravity at 5 months after ich onset provides additional evidence of left cst recovery . as a result", ", right extremity motor functions seemed to have been recovered due to reorganization of the injured left cst originating from the left pmc to left m1 .", "it is also possible that compression of the cst into the left pmc recovered in concert with hematoma resolution . for the motor recovery mechanism in ich ,", "several dti based studies have described recovery of an injured cst or the contribution made to recovery by the contralateral unaffected cst . during the last 5 years", ", a number of serial dti studies have described the recovery of injured cst after ich .", "jang et al demonstrated the recovery process of an injured cst in a patient with a subcortical ich by serial dti .", "the patient presented with complete paralysis of right extremities at onset , but over 16 months , motor functions of affected extremities slowly recovered to nearly normal .", "furthermore , dtt showed that the origin of the cst had changed from the posterior parietal cortex at 1 month to the primary somatosensory cortex at 4 months and m1 at 16 months .", "the authors suggested that recovery of the origin of the damaged cst was due to a process of normalization from the parietal cortex to m1 .", "by contrast , our patient showed a change in the origin of the injured cst from the pmc to m1 .", "this study described changes of an injured cst that occurred in concert with motor recovery in a patient with ich . to the best of our knowledge ,", "this is the first longitudinal study to demonstrate a change in the origin of an injured cst from the pmc to m1 in ich .", "results from this study suggest a motor recovery mechanism of ich and the important implications regarding brain plasticity and brain rehabilitation after ich .", "however , this study is obviously limited by case numbers , and further complementary studies involving larger case numbers are warranted ."]]}
{"article_id": "PMC4657800", "article_text": ["colorectal cancer ( crc ) is one of most common cancers and leading causes of mortality in the usa , accounting for approximately 136,000 new cases and 50,000 deaths per year.1 for metastatic or unresectable crc , standard first- and second - line treatments typically involve a combination of cytotoxic chemotherapies ( eg , folfiri [ 5- fluorouracil + oxaliplatin + irinotecan])2,3 and molecular targeted agents ( eg , bevacizumab , cetuximab , panitumumab),46 which can help to improve progression - free survival and overall survival .", "however , due to the nature of the disease , many patients will progress through guideline - recommended standard regimens while maintaining a good performance status .", "regorafenib , an oral multikinase inhibitor , which targets angiogenic , stromal , and oncogenic receptor tyrosine kinases , was approved by the us food and drug administration in 2012 for the treatment of patients with metastatic crc who have been previously treated with fluoropyrimidine- , oxaliplatin- , and irinotecan - based chemotherapy , an anti  vascular endothelial growth factor ( vegf ) therapy and , if kras wild - type , an anti  epidermal growth factor receptor ( egfr ) therapy.7 this approval was based on the correct study , which demonstrated a median overall survival of 6.4 months with regorafenib vs 5.0 months with placebo ( hazard ratio , 0.77 ; 95% confidence interval , 0.640.94 ; p=0.0052).8 among the most common adverse effects were fatigue , hand ", "a case of an elderly patient on regorafenib who achieved stable disease for over 11 months with strategic dose modifications was presented .", "no ethics approval was needed for the care of this patient as all treatments were in accordance with institutional best practices . verbal consent to appear in this case study was given by the patient to dr seery . a 73-year - old indian female with a past medical history of hypertension and hyper cholesterolemia", "the patient had no history of smoking , alcohol consumption , or any family history of cancer .", "the patient initially had symptoms of hemoptysis for approximately 2 weeks and then subsequently developed hematochezia for 3 days .", "due to these symptoms , the patient underwent a colonoscopy , which revealed left - sided colon cancer .", "a left hemicolectomy was performed ; pathology revealed a stage iiib well - differentiated , kras wild - type adenocarcinoma , with one out of 15 lymph nodes testing positive .", "the patient moved back to india and did not receive adjuvant chemotherapy due to patient preference . in march 2011", ", the patient returned to the usa with complaints of mild abdominal distention , and a subsequent positron emission tomography / computed tomography ( pet / ct ) scan showed evidence of recurrence , with ovarian and peritoneal metastases .", "the patient began treatment with capecitabine , oxaliplatin , and bevacizumab , and developed an anaphylactic reaction in the sixth cycle .", "she continued with single - agent capecitabine for an additional two cycles and completed therapy in june 2011 as there was no further evidence of disease on the pet / ct scans .", "the patient did not receive any additional therapy until early 2012 when she was restaged and was again noted to have diffuse metastatic disease in the peritoneum , with imaging consistent with moderate to extensive carcinomatosis .", "the patient received cetuximab and folfiri from february 2012 through august 2012 and achieved an excellent response , with the pet / ct scan showing no evidence of measurable disease , with some minimal hypodensities in the liver that were not pet avid .", "the patient did well until april 2013 when she experienced increased abdominal distention , bloating , and difficulty with defecation .", "the patient received a repeat ct scan that showed progression of the disease , with a large pleural - based mass in the right lower lobe with bilateral pleural effusions , ascites , and multiple peritoneal implants , as well as omental infiltration along with a possible l3 lytic lesion .", "also , the patient s carcinoembryonic antigen ( cea ) values had increased to 23 . as a result", ", the patient was restarted on cetuximab and folfiri in may 2013 ; however , a pet / ct scan 2 months later revealed a mixed response , with persistent fluorodeoxyglucose - avid right pleural disease , but also some areas of decreased activity with stable nodal disease in the left axilla , retroperitoneum , and pelvis , and increased activity in the left inguinal and right retrocrural nodes .", "the patient continued with cetuximab and folfiri until august 2013 , and a repeat pet / ct scan in september 2013 revealed progressive disease in the right pleural mass along with increase in size of the retrocrural and left inguinal lymph nodes and liver ( figure 1a ) .", "the patient was started on regorafenib 160 mg daily on september 12 , 2013 , but required a treatment interruption due to hypertension with consistent systolic blood pressure readings in the range of 170 mm hg and diastolic blood pressure in the range of 100 mm hg , as well as grade 3 fatigue during cycle 1 .", "the patient was started on antihypertensive medications , and regorafenib was held for 2 weeks to resolve these adverse events .", "the dose was then reduced again due to grade 3 fatigue , and the patient s dose was stabilized at 80 mg daily .", "a repeat pet / ct scan in february 2014 showed a mixed response , with increased activity in non - target lesions noted in multiple omental , peritoneal , and pelvic lesions , but decreased activity in target lesions , specifically the thoracic and liver lesions , with new activity in the right tenth rib ( figure 1b ) .", "specifically , a right pleural - based mass had decreased from 27.6 mm to 26.7 mm , and standardized uptake value decreased from 14.4 to 3.4 in conjunction with a decrease in disease measuring from 58.0 mm to 43.6 mm in the central pelvic mesentery superior extending to the uterine fundus .", "the patient s cea decreased to 3.7 , while eastern cooperative oncology group ( ecog ) performance status improved from 2 to 1 , and treatment was therefore continued at 80 mg daily .", "the patient s most recent pet / ct scan in august 2014 showed frank progression of disease , with an increase in number and activity of multiple metastatic pulmonary and hepatic lesions along with increased activity in omental and mesenteric implants .", "as of august 2015 , the patient has been on regorafenib for 11 months with progression ; however , her ecog performance status remains at 1 , and she would like to continue taking the medication as she does not have any side effects and feels her quality of life has improved .", "verbal consent was obtained from the patient prior to her departing our facility for hospice care .", "the patient s identification has been protected and any information which could potentially be used to reveal the identity of the patient has been removed .", "we reported on a case of an elderly woman with kras wild - type , refractory metastatic colon cancer who has received multiple chemotherapy regimens , most recently regorafenib for 11 months with minimal side effects and palliation of symptoms .", "the report shows that regorafenib can provide a decrease in tumor burden and improve quality of life with prolonged survival if an appropriate dose is used in terms of balancing side effects with benefits of symptom palliation .", "moreover , the report shows that continuing therapy beyond disease progression is no longer an absolute contraindication .", "this case report additionally demonstrates that close and frequent follow - ups by the primary oncologist is needed in order to monitor side effects and to find the effective and tolerable dose . in the correct study ,", "the frequency of adverse events was shown to peak during the first cycle and gradually taper off with subsequent cycles.8 this report suggests that regorafenib can be a viable option for patients who still have a good performance status and have progressed through other regimens .", "importantly , patient and financial support services are in place through the reach program to help patients manage the cost of this therapy ."], "abstract_text": ["<S> regorafenib , an oral multikinase inhibitor , was approved in september 2012 by the us food and drug administration for the treatment of patients with metastatic colorectal cancer . since this time , </S>", "<S> however , few case reports outlining real - world usage have been published in the literature . here , we detail the clinical history of an elderly woman with kras wild - type colon cancer who received regorafenib after prior treatment with other agents . </S>", "<S> we show that by employing dose modification strategies to address adverse events , this patient was able to remain on therapy for 11 months and achieve stable disease . </S>"], "labels": null, "section_names": ["Introduction", "Case report", "Ethics", "Conclusion"], "sections": [["colorectal cancer ( crc ) is one of most common cancers and leading causes of mortality in the usa , accounting for approximately 136,000 new cases and 50,000 deaths per year.1 for metastatic or unresectable crc , standard first- and second - line treatments typically involve a combination of cytotoxic chemotherapies ( eg , folfiri [ 5- fluorouracil + oxaliplatin + irinotecan])2,3 and molecular targeted agents ( eg , bevacizumab , cetuximab , panitumumab),46 which can help to improve progression - free survival and overall survival .", "however , due to the nature of the disease , many patients will progress through guideline - recommended standard regimens while maintaining a good performance status .", "regorafenib , an oral multikinase inhibitor , which targets angiogenic , stromal , and oncogenic receptor tyrosine kinases , was approved by the us food and drug administration in 2012 for the treatment of patients with metastatic crc who have been previously treated with fluoropyrimidine- , oxaliplatin- , and irinotecan - based chemotherapy , an anti  vascular endothelial growth factor ( vegf ) therapy and , if kras wild - type , an anti  epidermal growth factor receptor ( egfr ) therapy.7 this approval was based on the correct study , which demonstrated a median overall survival of 6.4 months with regorafenib vs 5.0 months with placebo ( hazard ratio , 0.77 ; 95% confidence interval , 0.640.94 ; p=0.0052).8 among the most common adverse effects were fatigue , hand ", "a case of an elderly patient on regorafenib who achieved stable disease for over 11 months with strategic dose modifications was presented ."], ["no ethics approval was needed for the care of this patient as all treatments were in accordance with institutional best practices . verbal consent to appear in this case study was given by the patient to dr seery . a 73-year - old indian female with a past medical history of hypertension and hyper cholesterolemia", "the patient had no history of smoking , alcohol consumption , or any family history of cancer .", "the patient initially had symptoms of hemoptysis for approximately 2 weeks and then subsequently developed hematochezia for 3 days .", "due to these symptoms , the patient underwent a colonoscopy , which revealed left - sided colon cancer .", "a left hemicolectomy was performed ; pathology revealed a stage iiib well - differentiated , kras wild - type adenocarcinoma , with one out of 15 lymph nodes testing positive .", "the patient moved back to india and did not receive adjuvant chemotherapy due to patient preference . in march 2011", ", the patient returned to the usa with complaints of mild abdominal distention , and a subsequent positron emission tomography / computed tomography ( pet / ct ) scan showed evidence of recurrence , with ovarian and peritoneal metastases .", "the patient began treatment with capecitabine , oxaliplatin , and bevacizumab , and developed an anaphylactic reaction in the sixth cycle .", "she continued with single - agent capecitabine for an additional two cycles and completed therapy in june 2011 as there was no further evidence of disease on the pet / ct scans .", "the patient did not receive any additional therapy until early 2012 when she was restaged and was again noted to have diffuse metastatic disease in the peritoneum , with imaging consistent with moderate to extensive carcinomatosis .", "the patient received cetuximab and folfiri from february 2012 through august 2012 and achieved an excellent response , with the pet / ct scan showing no evidence of measurable disease , with some minimal hypodensities in the liver that were not pet avid .", "the patient did well until april 2013 when she experienced increased abdominal distention , bloating , and difficulty with defecation .", "the patient received a repeat ct scan that showed progression of the disease , with a large pleural - based mass in the right lower lobe with bilateral pleural effusions , ascites , and multiple peritoneal implants , as well as omental infiltration along with a possible l3 lytic lesion .", "also , the patient s carcinoembryonic antigen ( cea ) values had increased to 23 . as a result", ", the patient was restarted on cetuximab and folfiri in may 2013 ; however , a pet / ct scan 2 months later revealed a mixed response , with persistent fluorodeoxyglucose - avid right pleural disease , but also some areas of decreased activity with stable nodal disease in the left axilla , retroperitoneum , and pelvis , and increased activity in the left inguinal and right retrocrural nodes .", "the patient continued with cetuximab and folfiri until august 2013 , and a repeat pet / ct scan in september 2013 revealed progressive disease in the right pleural mass along with increase in size of the retrocrural and left inguinal lymph nodes and liver ( figure 1a ) .", "the patient was started on regorafenib 160 mg daily on september 12 , 2013 , but required a treatment interruption due to hypertension with consistent systolic blood pressure readings in the range of 170 mm hg and diastolic blood pressure in the range of 100 mm hg , as well as grade 3 fatigue during cycle 1 .", "the patient was started on antihypertensive medications , and regorafenib was held for 2 weeks to resolve these adverse events .", "the dose was then reduced again due to grade 3 fatigue , and the patient s dose was stabilized at 80 mg daily .", "a repeat pet / ct scan in february 2014 showed a mixed response , with increased activity in non - target lesions noted in multiple omental , peritoneal , and pelvic lesions , but decreased activity in target lesions , specifically the thoracic and liver lesions , with new activity in the right tenth rib ( figure 1b ) .", "specifically , a right pleural - based mass had decreased from 27.6 mm to 26.7 mm , and standardized uptake value decreased from 14.4 to 3.4 in conjunction with a decrease in disease measuring from 58.0 mm to 43.6 mm in the central pelvic mesentery superior extending to the uterine fundus .", "the patient s cea decreased to 3.7 , while eastern cooperative oncology group ( ecog ) performance status improved from 2 to 1 , and treatment was therefore continued at 80 mg daily .", "the patient s most recent pet / ct scan in august 2014 showed frank progression of disease , with an increase in number and activity of multiple metastatic pulmonary and hepatic lesions along with increased activity in omental and mesenteric implants .", "as of august 2015 , the patient has been on regorafenib for 11 months with progression ; however , her ecog performance status remains at 1 , and she would like to continue taking the medication as she does not have any side effects and feels her quality of life has improved ."], ["verbal consent was obtained from the patient prior to her departing our facility for hospice care .", "the patient s identification has been protected and any information which could potentially be used to reveal the identity of the patient has been removed ."], ["we reported on a case of an elderly woman with kras wild - type , refractory metastatic colon cancer who has received multiple chemotherapy regimens , most recently regorafenib for 11 months with minimal side effects and palliation of symptoms .", "the report shows that regorafenib can provide a decrease in tumor burden and improve quality of life with prolonged survival if an appropriate dose is used in terms of balancing side effects with benefits of symptom palliation .", "moreover , the report shows that continuing therapy beyond disease progression is no longer an absolute contraindication .", "this case report additionally demonstrates that close and frequent follow - ups by the primary oncologist is needed in order to monitor side effects and to find the effective and tolerable dose . in the correct study ,", "the frequency of adverse events was shown to peak during the first cycle and gradually taper off with subsequent cycles.8 this report suggests that regorafenib can be a viable option for patients who still have a good performance status and have progressed through other regimens .", "importantly , patient and financial support services are in place through the reach program to help patients manage the cost of this therapy ."]]}
{"article_id": "PMC4075217", "article_text": ["the treatment and surgical background of gynaecomastia has history of evolution over and above 60 years . in 1928 , dufourmental described a semi circular intra - areolar incision which was later documented by webster in 1946 , who recognised the fact that the conspicuous scarring left by older techniques is more embarrassing than the original condition .", "the categories were classified by simon in 1973 and later on , liposuction was introduced in 1983 .", "ultrasound - assisted liposuction remains the accepted standard that gained much popularity for addressing dense adipose tissue for the management of relatively glandular or fibrous gynaecomastia .", "it is significant to mention the wide range of alternatives and advancements with minimally invasive approaches developed to eliminate the condition of gynaecomastia in a more refined way .", "it has been observed that liposuction technique alone has its limitation with removal of adipose tissue only and unable to address the problem of tough glandular component ; therefore , combining liposuction with different kinds of direct incisions was introduced to deliver the fibrous architecture of the breast by many authors .", "a minimally invasive approach was advocated by some authors that consist of minimal access incisions in either peri - areolar , circum - areolar or trans - areolar region , while some proposed arthroscopic shavers to break down the fibrous capsule of the glandular tissues and deliver it with liposuction .", "but they have few drawbacks such as less access to the fibro - glandular tissue due to the minimal incision , visible unattractive scarring at nipple areola complex left after the peri - areolar incision as shown in figure 1 , dermal necrosis and subsequent scarring due to arthroscopic shavers and poor results . scarring left after peri - areolar incision", "the gynaecomastia surgery is basically sought for aesthetic purpose and such operation should not leave any tell - tale signs of the procedure .", "there had to be a solution for the smooth delivery of fibro glandular tissues without added scarring .", "this article presents an innovative approach that consists of a criss - cross trans - nipple incision to retrieve the fibro - glandular tissues following liposuction method for excellent outcomes .", "no additional scarring is visible , even on the operation table and even in a close up view right after the procedure with this trans - nipple approach . in all the cases ,", "the trans - nipple incision is proved to give  no signs of surgery  over the nipple areola complex after a recovery period of three to six months following the surgical procedure .", "between the duration of january 2012 to october 2013 , 28 candidates suffering from various degrees of gynaecomastia ( except grade-4 , which definitively required skin excision ) were surgically treated by this method .", "after proper consultation and examination , it was concluded that a surgical approach would be needed and all of them underwent the process of routine medical investigations for the anaesthesia fitness .", "right before the procedure , markings were made to limit the boundaries of treatment [ figure 2 ] .", "the procedure started with liposuction to retrieve adipose tissues through a tiny hole ( approx 6 - 7 mm ) at both sides of the chest using number 4 liposuction canullae .", "after performing thorough liposuction of the marked area , a small criss - cross incision was made right on the top of the nipple with the help of no-11 blade [ figure 3 ] .", "pre - operative marking of the treatment boundaries for liposuction the criss - cross incision at the centre of the nipple through a fine curved hemostat , the tough fibro - glandular tissues were delivered while creating slight pressure with thumb and forefinger ( broad pinching ) over the base of nipple areola area [ figure 4a and 4b ]", ". only tough tethering bands , which obstructed the path , were cut with a fine curved scissors [ figure 4c ] .", "care was taken to keep the point of scissors away from the dermis of areola .", "an amount of 3 - 5 mm tissue beneath the areolar skin was preserved to prevent any vascular complications [ video 1 ] .", "removal fibro - glandular tissues through the incision cutting the tethering band through scissors keeping tip away from areolar skin to preserve at least 3 - 5 mm tissue beneath areolar skin the incision was then closed with 4 - 0 monocryl in a single purse - string suture manner [ figure 5 ] .", "the liposuction incisions were left open to drain any collection of fluid without any freshening of margins or stitches .", "the compression garment was applied on the operation table right after the procedure and was replaced with new compression garment on seventh day of the surgery when the patient was called for first follow up .", "all the patients were advised to do proper massage of the chest area after 7 days of procedure with any moisturiser of their choice and to wear the compression garment for the next three months following the surgery .", "serial medical photographs - front , lateral and semi - lateral views were taken prior to the procedure , after three , six and twelve months from the surgery to assess the final outcomes .", "it was observed in a close view that there was no visible scar on the nipple areola complex even on the operation table after the surgery [ figure 6 ] and on long - term follow - up of six months and one year [ figures 7 and 8 ] .", "none of the 28 patients reported any alteration of sensation in the nipple areola region after 3 to 4 months .", "trans - nipple removal of fibro - glandular tissue is an easy , innovative approach in gynaecomastia surgery that removes all the tough fibro - glandular tissue without leaving any visible scars over the nipple areola complex .", "it produces the most rewarding results and maximum satisfaction to the patient right from their procedure .", "close - up view right after the surgery showing practically no disturbance of nipple - areola complex architecture example cases showing follow - up between six months to one year after surgery . see the close up view of practically scar less nipple - areola complex after recovery", "gynaecosmastia usually requires no attention , unless patient feels uncomfortable or embarrassed due to aesthetic reasons .", "chances of having this condition can be same in a slender patient as well as in a healthy or over - weight patient .", "the difference is only the degree or severity that can be noticed differently in obese patients or who are relatively fat can develop significant visibility or enlargement .", "though the root cause of this problem is unknown in most of the cases and commonly idiopathic , heredity , consumption of certain drugs , obesity or hormonal changes can be one of the various other reasons .", "many techniques and refined approaches have been advocated through the years to address the issue that leave visible scars . through the recent years", ", the surgical techniques that have been supported by many authors advocate liposuction or ultrasound - assisted liposuction alone or in combination with direct incision of the breast tissue through a peri - areolar incision or a pull - through technique , and various other methods have been advocated to eliminate remnant tough fibro - glandular tissue of gynaecomastia and achieve more aesthetic results .", "it is observed that gynaecomastia causes a great mental discomfort and impairs the self - confidence of the patient .", "they tend to over react and sometimes become extra - conscious about their condition and start to notice even a mild elevation which is left in most cases of liposuction alone methods", ". this slight elevation can be acceptable for normal people but becomes great matter of mental stress for the patients suffering from this problem .", "it has been noticed that slight bulges are left as remnant after doing liposuction and it is evident on operation table during the surgery that significant elevation is left after doing liposuction only [ figure 9a ] .", "this elevation is formed by the leftover fibro - glandular tissues that causes visible bulges and can not be retrieved through only liposuction .", "that is why liposuction alone can not serve the purpose as it has its limitations and is only ideal for removal of adipose tissue . in most of the cases where patients are more conscious even about a mild elevation", ", there remains a need of removal of this fibro - glandular tissue to achieve flat chest appearance [ figure 9b ] .", "the invasive approaches along with liposuction are able to address this issue but they leave some conspicuous scarring ( peri - areolar or trans - areolar incisions ) or loss of sensation of the nipple areola complex , compromised vascualrity of overlying skin and subsequent complication ( arthroscopic shavers or modified liposuction shaver canullae ) , leading to inadequate outcomes . per - operative", "view showing visible bulge below nipple - areola complex just after completing liposuction due to remaining tough fibro - glandular tissue per - operative view of same case showing flat chest just after removal of tough fibro - glandular tissues through trans - nipple approach we consider a trans - areolar incision to remove these tough glandular tissues to achieve flat chest and eliminate any elevation caused by fibro glandular component over the nipple areola complex [ figure 9c ] .", "our technique is relatively simple and retrieves any volume of the tough fibro - glandular breast tissue without leaving any visible scars on the nipple areola complex , unlike peri - areolar incision or other invasive techniques .", "the tough fibro - glandular tissues are removed through a criss - cross incision over the centre of nipple right after performing liposuction of the marked area .", "the key to prevent any complication is to cut only the tethering band coming in way of delivery of fibro - glandular tissue and preservation of 3 - 5 mm tissue beneath the areolar skin .", "the technique offers the perfect control over nipple position as there are absolutely no chances to re - positioning or traction on the nipple due to subsequent scar that sometimes causes nipple malpositioning or inversion in peri - areolar incision technique . on table final result right after the procedure in our study , the presented patients had different categories of gynaecomastia and patient - a [ figure 10a ] delivered little amount of tough fibro - glandular component ( approx 50 g ) and while patient - b [ figure 10b ] delivered huge amount ( approx 250 g ) .", "both patients were operated with the same modality of treatment , i.e. , liposuction for adiposal component and trans - nipple approach for removal of tough fibro - glandular tissue to treat their condition and discharged on the same day of their operation . both were advised to do regular massage and wear compression garment for three to four months .", "they both enjoyed flat chest , very faint and thin scar of liposuction incision site and practically invisible scars over the nipple areola complex after 6 months of follow up .", "example patients - a ( before and after ) with fewer amounts of fibro - glandular tissues example patient - b ( before and after ) with large amount of fibro - glandular tissues", "trans - nipple approach is an easy , innovative and practically excellent technique to remove any amount of the residual glandular fibrous tissue after liposuction without leaving any additional visible scars on the nipple - areola complex in gynaecomastia surgery and results in best aesthetic outcomes .", "all the 28 patients who were operated by this approach reported very satisfactory aesthetic outcomes after their recovery .", "the procedure offers a promising approach for all the patients of gynaecomastia , especially for the patients who want to keep secret of having any surgical procedure done to their chest ."], "abstract_text": ["<S> context : the established techniques that have been used to treat gynaecomastia are said to have relatively less patient satisfaction rate as they leave some visible scars or mild elevation over the nipple areola complex , resulting in aesthetically unsatisfactory results . </S>", "<S> even the slightest elevation or smallest scar over nipple areola complex leave patients extremely self conscious and in a dilemma of having a second intervention to get rid of that blemish.aims:the aim of the study is to achieve - a flat chest without adding a scar and with no chances of re - occurrence of the condition . </S>", "<S> this article suggests an innovative approach to address the problem.materials and methods : the author presents trans - nipple incision approach for the delivery of fibro - glandular tissue component following liposuction for maximum patient satisfaction . </S>", "<S> this method consists of a unique small criss - cross incision right on the nipple itself for retrieving any volume of tough fibro - glandular tissues . between the duration of january 2012 to october 2013 </S>", "<S> , 28 male patients of different ages were operated with this technique.results:the surgery resulted in well - shaped , symmetric chest contour without any visible elevation or additional scars on nipple areola complex . </S>", "<S> no complications were noticed in any of the patients.conclusions:the presented technique is proved to have a high patient satisfaction rate and to be promising method to achieve good aesthetic results in gynaecomastia surgery . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION", "See Video on: "], "sections": [["the treatment and surgical background of gynaecomastia has history of evolution over and above 60 years . in 1928 , dufourmental described a semi circular intra - areolar incision which was later documented by webster in 1946 , who recognised the fact that the conspicuous scarring left by older techniques is more embarrassing than the original condition .", "the categories were classified by simon in 1973 and later on , liposuction was introduced in 1983 .", "ultrasound - assisted liposuction remains the accepted standard that gained much popularity for addressing dense adipose tissue for the management of relatively glandular or fibrous gynaecomastia .", "it is significant to mention the wide range of alternatives and advancements with minimally invasive approaches developed to eliminate the condition of gynaecomastia in a more refined way .", "it has been observed that liposuction technique alone has its limitation with removal of adipose tissue only and unable to address the problem of tough glandular component ; therefore , combining liposuction with different kinds of direct incisions was introduced to deliver the fibrous architecture of the breast by many authors .", "a minimally invasive approach was advocated by some authors that consist of minimal access incisions in either peri - areolar , circum - areolar or trans - areolar region , while some proposed arthroscopic shavers to break down the fibrous capsule of the glandular tissues and deliver it with liposuction .", "but they have few drawbacks such as less access to the fibro - glandular tissue due to the minimal incision , visible unattractive scarring at nipple areola complex left after the peri - areolar incision as shown in figure 1 , dermal necrosis and subsequent scarring due to arthroscopic shavers and poor results . scarring left after peri - areolar incision", "the gynaecomastia surgery is basically sought for aesthetic purpose and such operation should not leave any tell - tale signs of the procedure .", "there had to be a solution for the smooth delivery of fibro glandular tissues without added scarring .", "this article presents an innovative approach that consists of a criss - cross trans - nipple incision to retrieve the fibro - glandular tissues following liposuction method for excellent outcomes .", "no additional scarring is visible , even on the operation table and even in a close up view right after the procedure with this trans - nipple approach . in all the cases ,", "the trans - nipple incision is proved to give  no signs of surgery  over the nipple areola complex after a recovery period of three to six months following the surgical procedure ."], ["between the duration of january 2012 to october 2013 , 28 candidates suffering from various degrees of gynaecomastia ( except grade-4 , which definitively required skin excision ) were surgically treated by this method .", "after proper consultation and examination , it was concluded that a surgical approach would be needed and all of them underwent the process of routine medical investigations for the anaesthesia fitness .", "right before the procedure , markings were made to limit the boundaries of treatment [ figure 2 ] .", "the procedure started with liposuction to retrieve adipose tissues through a tiny hole ( approx 6 - 7 mm ) at both sides of the chest using number 4 liposuction canullae .", "after performing thorough liposuction of the marked area , a small criss - cross incision was made right on the top of the nipple with the help of no-11 blade [ figure 3 ] .", "pre - operative marking of the treatment boundaries for liposuction the criss - cross incision at the centre of the nipple through a fine curved hemostat , the tough fibro - glandular tissues were delivered while creating slight pressure with thumb and forefinger ( broad pinching ) over the base of nipple areola area [ figure 4a and 4b ]", ". only tough tethering bands , which obstructed the path , were cut with a fine curved scissors [ figure 4c ] .", "care was taken to keep the point of scissors away from the dermis of areola .", "an amount of 3 - 5 mm tissue beneath the areolar skin was preserved to prevent any vascular complications [ video 1 ] .", "removal fibro - glandular tissues through the incision cutting the tethering band through scissors keeping tip away from areolar skin to preserve at least 3 - 5 mm tissue beneath areolar skin the incision was then closed with 4 - 0 monocryl in a single purse - string suture manner [ figure 5 ] .", "the liposuction incisions were left open to drain any collection of fluid without any freshening of margins or stitches .", "the compression garment was applied on the operation table right after the procedure and was replaced with new compression garment on seventh day of the surgery when the patient was called for first follow up .", "all the patients were advised to do proper massage of the chest area after 7 days of procedure with any moisturiser of their choice and to wear the compression garment for the next three months following the surgery .", "serial medical photographs - front , lateral and semi - lateral views were taken prior to the procedure , after three , six and twelve months from the surgery to assess the final outcomes ."], ["it was observed in a close view that there was no visible scar on the nipple areola complex even on the operation table after the surgery [ figure 6 ] and on long - term follow - up of six months and one year [ figures 7 and 8 ] .", "none of the 28 patients reported any alteration of sensation in the nipple areola region after 3 to 4 months .", "trans - nipple removal of fibro - glandular tissue is an easy , innovative approach in gynaecomastia surgery that removes all the tough fibro - glandular tissue without leaving any visible scars over the nipple areola complex .", "it produces the most rewarding results and maximum satisfaction to the patient right from their procedure .", "close - up view right after the surgery showing practically no disturbance of nipple - areola complex architecture example cases showing follow - up between six months to one year after surgery . see the close up view of practically scar less nipple - areola complex after recovery"], ["gynaecosmastia usually requires no attention , unless patient feels uncomfortable or embarrassed due to aesthetic reasons .", "chances of having this condition can be same in a slender patient as well as in a healthy or over - weight patient .", "the difference is only the degree or severity that can be noticed differently in obese patients or who are relatively fat can develop significant visibility or enlargement .", "though the root cause of this problem is unknown in most of the cases and commonly idiopathic , heredity , consumption of certain drugs , obesity or hormonal changes can be one of the various other reasons .", "many techniques and refined approaches have been advocated through the years to address the issue that leave visible scars . through the recent years", ", the surgical techniques that have been supported by many authors advocate liposuction or ultrasound - assisted liposuction alone or in combination with direct incision of the breast tissue through a peri - areolar incision or a pull - through technique , and various other methods have been advocated to eliminate remnant tough fibro - glandular tissue of gynaecomastia and achieve more aesthetic results .", "it is observed that gynaecomastia causes a great mental discomfort and impairs the self - confidence of the patient .", "they tend to over react and sometimes become extra - conscious about their condition and start to notice even a mild elevation which is left in most cases of liposuction alone methods", ". this slight elevation can be acceptable for normal people but becomes great matter of mental stress for the patients suffering from this problem .", "it has been noticed that slight bulges are left as remnant after doing liposuction and it is evident on operation table during the surgery that significant elevation is left after doing liposuction only [ figure 9a ] .", "this elevation is formed by the leftover fibro - glandular tissues that causes visible bulges and can not be retrieved through only liposuction .", "that is why liposuction alone can not serve the purpose as it has its limitations and is only ideal for removal of adipose tissue . in most of the cases where patients are more conscious even about a mild elevation", ", there remains a need of removal of this fibro - glandular tissue to achieve flat chest appearance [ figure 9b ] .", "the invasive approaches along with liposuction are able to address this issue but they leave some conspicuous scarring ( peri - areolar or trans - areolar incisions ) or loss of sensation of the nipple areola complex , compromised vascualrity of overlying skin and subsequent complication ( arthroscopic shavers or modified liposuction shaver canullae ) , leading to inadequate outcomes . per - operative", "view showing visible bulge below nipple - areola complex just after completing liposuction due to remaining tough fibro - glandular tissue per - operative view of same case showing flat chest just after removal of tough fibro - glandular tissues through trans - nipple approach we consider a trans - areolar incision to remove these tough glandular tissues to achieve flat chest and eliminate any elevation caused by fibro glandular component over the nipple areola complex [ figure 9c ] .", "our technique is relatively simple and retrieves any volume of the tough fibro - glandular breast tissue without leaving any visible scars on the nipple areola complex , unlike peri - areolar incision or other invasive techniques .", "the tough fibro - glandular tissues are removed through a criss - cross incision over the centre of nipple right after performing liposuction of the marked area .", "the key to prevent any complication is to cut only the tethering band coming in way of delivery of fibro - glandular tissue and preservation of 3 - 5 mm tissue beneath the areolar skin .", "the technique offers the perfect control over nipple position as there are absolutely no chances to re - positioning or traction on the nipple due to subsequent scar that sometimes causes nipple malpositioning or inversion in peri - areolar incision technique . on table final result right after the procedure in our study , the presented patients had different categories of gynaecomastia and patient - a [ figure 10a ] delivered little amount of tough fibro - glandular component ( approx 50 g ) and while patient - b [ figure 10b ] delivered huge amount ( approx 250 g ) .", "both patients were operated with the same modality of treatment , i.e. , liposuction for adiposal component and trans - nipple approach for removal of tough fibro - glandular tissue to treat their condition and discharged on the same day of their operation . both were advised to do regular massage and wear compression garment for three to four months .", "they both enjoyed flat chest , very faint and thin scar of liposuction incision site and practically invisible scars over the nipple areola complex after 6 months of follow up .", "example patients - a ( before and after ) with fewer amounts of fibro - glandular tissues example patient - b ( before and after ) with large amount of fibro - glandular tissues"], ["trans - nipple approach is an easy , innovative and practically excellent technique to remove any amount of the residual glandular fibrous tissue after liposuction without leaving any additional visible scars on the nipple - areola complex in gynaecomastia surgery and results in best aesthetic outcomes .", "all the 28 patients who were operated by this approach reported very satisfactory aesthetic outcomes after their recovery .", "the procedure offers a promising approach for all the patients of gynaecomastia , especially for the patients who want to keep secret of having any surgical procedure done to their chest ."], [""]]}
{"article_id": "PMC4641483", "article_text": ["the natural history of acute myeloid leukamia ( aml ) is typically unfavorable with rapid progression leading to death in the absence of specific therapy .", "spontaneous remission ( sr ) is a rare but well - documented event with hundreds of cases published to date . despite reported cases and progress of conventional cytogenetic and molecular biology ,", "ever since the generalization and rapid start of induction chemotherapy after acute myeloid leukemia diagnosis , sr has been very rarely observed . in particular , aml with monocytic differentiation ( aml m5 ) represents the largest subgroup of aml with spontaneous remission reported in the literature , with seven cases published since 1980 17 but the existence of a link between aml m5 and sr remains elusive .", "for the first time , we report the cases of three patients with similar genetic characteristics diagnosed with aml m5 according to the fab classification , who underwent rapid and transient sr .", "aml diagnoses were established by conventional morphologic , histologic , cytochemical , immunophenotypic , and genetic criteria .", "metaphases were obtained and analyzed using standard techniques of colchicine arrest , hypotonic treatment , and 3:1 v / v methanol / acetic acid fixation .", "karyotypes were reported using the international system for human cytogenetic nomenclature , based on the analysis of at least 20 metaphases when possible .", "dna was extracted from bone marrow using proteinase k , followed by salt and ethanol precipitation and stored at 20c in 10 mmol / l tris  cl 1 mmol / l edta ( ph 8) buffer .", "pcr reactions were performed on 100 ng of dna using npm1-f ( 5- tgg ttc ctt aac cac att tct tt-3 ) and npm1-r ( 5- ttc cat aca tac tta aaa cca agc a-3 ) to exon 12 of npm1 .", ", two fragments were amplified using pp1-f : 5-tcgccatgccgggagaactctaac-3 ; pp1-r : 5-ctggtaagggaagaggccggccag-3 and pp2-f : 5-ccgctggtgatcaagcagga-3 ; pp2-r : 5-gggcaagcctcgagatcc-3 primers .", "only the hotspot region was sequenced for flt3 ( flt3-f : 5-ccaggaacgtgcttgtcacccac-3 and flt3-r : 5-tcaaaaatgcaccacagtgag-3 ) .", "all the pcr products were visualized on 1.5% agarose gel electrophoresis after gel red staining .", "all pcr - amplified samples were purified by standard methods and sequenced on abi prism 3130 genetic analyzer ( life technologies , carlsbad , ca ) .", "in january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue .", "physical examination showed reduced performance status ( ecog = ; 2 ) and high grade fever ( 40c ) but no sign of organ infiltration and no lymphadenopathy .", "a full - body ct scan revealed no abnormalities . upon admission , blood cell count showed moderate pancytopenia ( hb 10 g / dl , platelets 45 g / l and wbc 2", "bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei .", "the bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase ( mpo ) .", "an empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence , normalization of hemogram values and a complete sr documented by bone marrow examination ( blasts < 5% ) .", "no colony - stimulating factors were administered and the patient was able to return home .", "second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80% , mpo negative , esterase - positive monoblastic cells .", "the patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen .", "npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient ( c.861_862instgtc ; identified in cosmic database as cosm20861 ) .", "no mutations were found in either cebpa or flt3 ( neither internal duplication ( it d ) nor mutations in tyrosine kinase domain ( tkd ) ) .", "a 33-year - old male patient with no medical history was admitted in january 2013 .", "he had presented fever , fatigue , moderate weight loss , and anorexia for 1 month before hand .", "g / l with 5% of blast cells ) , and normal platelet count ( 175", "bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis .", "a bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 + , cd117 + , cd15 + , cd34 , cd163 , and mpo blast cells .", "the karyotype was normal , fish analysis showed no mll rearrangements , and molecular biology identified a type j npm1 mutation ( an insertion of tatg , fig .", "1 ) without flt3-itd , flt3-tkd , or cebpa mutation . due to the patient 's septic condition ,", "antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started .", "sr was observed in the following days with normalization of complete blood count ( cbc ) and clearance of blasts cells in the blood and bone marrow .", "blast cell morphology , cytogenetics , and molecular biology were identical to that of first diagnosis .", "the pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine .", "a 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia .", "this patient had a history of high blood pressure , diabetes , and high cholesterol .", "cbc revealed a hemoglobin level of 8.8 g / dl , a white blood cell count of 21 g / l with 8% of neutrophils and 15", "bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts , and 41% of promonocytes .", "the karyotype was normal , fish analysis identified no mll rearrangements , and molecular biology revealed a type a npm1 mutation ( duplication of tctg , fig .", "2 ) with neither flt3-itd nor flt3-tkd , nor cebpa mutation . antibiotic therapy with amoxicillin / clavulanic acid", "was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts ( despite persistence of approximately 15% of dystrophic monocytes ) . given the sr , the patient was allowed to return home .", "flow cytometry performed on the bone marrow identified a cd13 + , cd33 + , cd117 + , cd64 + , and cd14 + blast population , in favor of a monocytic differentiation .", "cytogenetics and molecular biology were identical to that of the initial diagnosis . following aml type chemotherapy ,", "electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow .", "in january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue .", "physical examination showed reduced performance status ( ecog = ; 2 ) and high grade fever ( 40c ) but no sign of organ infiltration and no lymphadenopathy .", "a full - body ct scan revealed no abnormalities . upon admission , blood cell count showed moderate pancytopenia ( hb 10 g / dl , platelets 45 g / l and wbc 2", "bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei .", "the bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase ( mpo ) .", "an empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence , normalization of hemogram values and a complete sr documented by bone marrow examination ( blasts < 5% ) .", "no colony - stimulating factors were administered and the patient was able to return home .", "second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80% , mpo negative , esterase - positive monoblastic cells .", "the patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen .", "npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient ( c.861_862instgtc ; identified in cosmic database as cosm20861 ) .", "no mutations were found in either cebpa or flt3 ( neither internal duplication ( it d ) nor mutations in tyrosine kinase domain ( tkd ) ) .", "a 33-year - old male patient with no medical history was admitted in january 2013 .", "he had presented fever , fatigue , moderate weight loss , and anorexia for 1 month before hand .", "g / l with 5% of blast cells ) , and normal platelet count ( 175", "bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis .", "a bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 + , cd117 + , cd15 + , cd34 , cd163 , and mpo blast cells .", "the karyotype was normal , fish analysis showed no mll rearrangements , and molecular biology identified a type j npm1 mutation ( an insertion of tatg , fig .", "1 ) without flt3-itd , flt3-tkd , or cebpa mutation . due to the patient 's septic condition ,", "antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started .", "sr was observed in the following days with normalization of complete blood count ( cbc ) and clearance of blasts cells in the blood and bone marrow .", "blast cell morphology , cytogenetics , and molecular biology were identical to that of first diagnosis .", "the pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine .", "a 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia .", "this patient had a history of high blood pressure , diabetes , and high cholesterol .", "cbc revealed a hemoglobin level of 8.8 g / dl , a white blood cell count of 21 g / l with 8% of neutrophils and 15", "bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts , and 41% of promonocytes .", "the karyotype was normal , fish analysis identified no mll rearrangements , and molecular biology revealed a type a npm1 mutation ( duplication of tctg , fig .", "antibiotic therapy with amoxicillin / clavulanic acid was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts ( despite persistence of approximately 15% of dystrophic monocytes ) . given the sr , the patient was allowed to return home .", "flow cytometry performed on the bone marrow identified a cd13 + , cd33 + , cd117 + , cd64 + , and cd14 + blast population , in favor of a monocytic differentiation .", "cytogenetics and molecular biology were identical to that of the initial diagnosis . following aml type chemotherapy , the patient is alive , in good health and in cr .", "electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow .", "spontaneous remission of acute myeloid leukemia is an extremely rare and almost always transient event , with a mean duration in the literature of 7.7 months ( range 136 ) 8 . since 1979 , approximately thirty cases of aml sr have been reported 8,9 , although the underlying mechanisms involved remain unclear . a potential role of bacterial and/or fungal infections and blood transfusions was suggested in sr occurrence 10 by triggering an immune and antileukemic response .", "activation of the immune system and cytokines such as tumor necrosis factor ( tnf ) , interferon gamma ( ifn- ) , and interleukin-2 ( il-2 ) released during infections , in conjunction with an increase in the antibody - mediated cytotoxicity of natural killer ( nk ) cells and cytotoxic activity of t lymphocytes and macrophages , may play a role in the occurrence of sr 1,2,11,12 .", "indeed , immune response might be a potential mechanism of prolonged disease control : in 2012 , the case of a german patient who presented aml with a 10-year - long sr was reported . in this patient , it was demonstrated in vitro that nk cells displayed cytotoxic activity against the k562 myeloid leukemia cell line with upregulation of cd107a in flow cytometry 13 . in addition , infection is often accompanied by a hypergammaglobulinemia , which has been associated with aml sr in some cases 13 .", "the potentially beneficial effect of systemic infections in aml led to preliminary trials using bacterial extract inoculations or vaccination programs , which did not show a significant effect on survival or occurrence of remission 14,15 .", "our patients did not receive any blood transfusions but all presented with infections that responded to antibiotic treatment with normalization of cbc and occurrence of sr .", "unfortunately , no blood cytokine monitoring was performed in our three patients . to date , most cases of aml sr have been described in patients with normal karyotypes 3,11 . to our knowledge , this is the first report of three cases of sr in aml m5 according to the fab classification with normal karyotype and npm1 mutations .", "npm1-mutated aml represents a separate entity included in the aml who classification since 2008 , with a favorable prognosis in patients with normal karyotype aml ( aml - nk ) in the absence of the flt3-itd mutation 16 .", "the npm1 mutation is the most common genetic alteration found in aml - nk and is present in approximately one - third of adult aml 17 .", "npm1 mutations are located in the last exon ( exon 12 ) and consist in duplication or insertion of small nucleotide sequences . more than 50 mutations have been reported to date , leading to a npm1 protein variant with modification of the last seven residues , as well as four additional residues 13,17,18 .", "physiologically , npm1 encodes a nucleocytoplasmic shuttling protein ( npm1wt ) involved in the regulation of ribosome genesis and centrosome duplication 19 .", "npm1 mutation is associated with the aberrant cytoplasmic localization of npm1 , thereby disrupting its function and perturbing multiple cellular pathways 18,20 .", "the potential role of npm1 mutations in the occurrence of sr is not known but it has been previously reported that patients with aml - nk and mutated npm1 had a better response rate to induction chemotherapy 17,18 which could advocate for increased chemosensitivity of blast cells with npm1 mutation .", "recent findings suggest that npm1 mutations are associated with increased sensitivity to oxidative stress and that mutant npm1 is a target of arsenic trioxide - mediated oxidative stress 21,22 .", "systemic infections , usually described in cases of sr , are associated with exuberant activation of the immune system and high production of reactive oxygen species ( ros ) .", "thus , we hypothesize that the three cases of sr presented herein might be linked by the presence of mutant npm1 , and therefore be more susceptible to oxidative stress than cases with wild - type npm1 .", "moreover , we can speculate that there is a link between sr and ros that still need to be demonstrated .", "sr is an exceptional phenomenon , often transient and relatively well documented but the underlying molecular mechanisms are still unknown . npm1-mutated aml m5 patients who developed spontaneous remission after antibiotic treatment of febrile neutropenia present at diagnosis seem to form a subtype with favorable prognosis and chemo sensitive disease .", "a better understanding of this rare process could uncover new potential therapeutic targets for acute leukemia ."], "abstract_text": ["<S> key clinical messagepatients with npm1-mutated aml m5 who develop spontaneous remission ( sr ) after antibiotic therapy at diagnosis seem to form a favorable prognosis and chemo sensitive subtype . </S>", "<S> we report three cases of aml m5 patients with the same genotype that experienced transient sr and are now leukemia free after standard treatment . </S>"], "labels": null, "section_names": ["Introduction", "Patients and Methods", "Case Reports", "Patient 1", "Patient 2", "Patient 3", "Discussion", "Conflict of interest"], "sections": [["the natural history of acute myeloid leukamia ( aml ) is typically unfavorable with rapid progression leading to death in the absence of specific therapy .", "spontaneous remission ( sr ) is a rare but well - documented event with hundreds of cases published to date . despite reported cases and progress of conventional cytogenetic and molecular biology ,", "ever since the generalization and rapid start of induction chemotherapy after acute myeloid leukemia diagnosis , sr has been very rarely observed . in particular , aml with monocytic differentiation ( aml m5 ) represents the largest subgroup of aml with spontaneous remission reported in the literature , with seven cases published since 1980 17 but the existence of a link between aml m5 and sr remains elusive .", "for the first time , we report the cases of three patients with similar genetic characteristics diagnosed with aml m5 according to the fab classification , who underwent rapid and transient sr ."], ["aml diagnoses were established by conventional morphologic , histologic , cytochemical , immunophenotypic , and genetic criteria .", "metaphases were obtained and analyzed using standard techniques of colchicine arrest , hypotonic treatment , and 3:1 v / v methanol / acetic acid fixation .", "karyotypes were reported using the international system for human cytogenetic nomenclature , based on the analysis of at least 20 metaphases when possible .", "dna was extracted from bone marrow using proteinase k , followed by salt and ethanol precipitation and stored at 20c in 10 mmol / l tris  cl 1 mmol / l edta ( ph 8) buffer .", "pcr reactions were performed on 100 ng of dna using npm1-f ( 5- tgg ttc ctt aac cac att tct tt-3 ) and npm1-r ( 5- ttc cat aca tac tta aaa cca agc a-3 ) to exon 12 of npm1 .", ", two fragments were amplified using pp1-f : 5-tcgccatgccgggagaactctaac-3 ; pp1-r : 5-ctggtaagggaagaggccggccag-3 and pp2-f : 5-ccgctggtgatcaagcagga-3 ; pp2-r : 5-gggcaagcctcgagatcc-3 primers .", "only the hotspot region was sequenced for flt3 ( flt3-f : 5-ccaggaacgtgcttgtcacccac-3 and flt3-r : 5-tcaaaaatgcaccacagtgag-3 ) .", "all the pcr products were visualized on 1.5% agarose gel electrophoresis after gel red staining .", "all pcr - amplified samples were purified by standard methods and sequenced on abi prism 3130 genetic analyzer ( life technologies , carlsbad , ca ) ."], ["in january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue .", "physical examination showed reduced performance status ( ecog = ; 2 ) and high grade fever ( 40c ) but no sign of organ infiltration and no lymphadenopathy .", "a full - body ct scan revealed no abnormalities . upon admission , blood cell count showed moderate pancytopenia ( hb 10 g / dl , platelets 45 g / l and wbc 2", "bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei .", "the bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase ( mpo ) .", "an empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence , normalization of hemogram values and a complete sr documented by bone marrow examination ( blasts < 5% ) .", "no colony - stimulating factors were administered and the patient was able to return home .", "second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80% , mpo negative , esterase - positive monoblastic cells .", "the patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen .", "npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient ( c.861_862instgtc ; identified in cosmic database as cosm20861 ) .", "no mutations were found in either cebpa or flt3 ( neither internal duplication ( it d ) nor mutations in tyrosine kinase domain ( tkd ) ) .", "a 33-year - old male patient with no medical history was admitted in january 2013 .", "he had presented fever , fatigue , moderate weight loss , and anorexia for 1 month before hand .", "g / l with 5% of blast cells ) , and normal platelet count ( 175", "bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis .", "a bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 + , cd117 + , cd15 + , cd34 , cd163 , and mpo blast cells .", "the karyotype was normal , fish analysis showed no mll rearrangements , and molecular biology identified a type j npm1 mutation ( an insertion of tatg , fig .", "1 ) without flt3-itd , flt3-tkd , or cebpa mutation . due to the patient 's septic condition ,", "antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started .", "sr was observed in the following days with normalization of complete blood count ( cbc ) and clearance of blasts cells in the blood and bone marrow .", "blast cell morphology , cytogenetics , and molecular biology were identical to that of first diagnosis .", "the pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine .", "a 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia .", "this patient had a history of high blood pressure , diabetes , and high cholesterol .", "cbc revealed a hemoglobin level of 8.8 g / dl , a white blood cell count of 21 g / l with 8% of neutrophils and 15", "bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts , and 41% of promonocytes .", "the karyotype was normal , fish analysis identified no mll rearrangements , and molecular biology revealed a type a npm1 mutation ( duplication of tctg , fig .", "2 ) with neither flt3-itd nor flt3-tkd , nor cebpa mutation . antibiotic therapy with amoxicillin / clavulanic acid", "was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts ( despite persistence of approximately 15% of dystrophic monocytes ) . given the sr , the patient was allowed to return home .", "flow cytometry performed on the bone marrow identified a cd13 + , cd33 + , cd117 + , cd64 + , and cd14 + blast population , in favor of a monocytic differentiation .", "cytogenetics and molecular biology were identical to that of the initial diagnosis . following aml type chemotherapy ,", "electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow ."], ["in january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue .", "physical examination showed reduced performance status ( ecog = ; 2 ) and high grade fever ( 40c ) but no sign of organ infiltration and no lymphadenopathy .", "a full - body ct scan revealed no abnormalities . upon admission , blood cell count showed moderate pancytopenia ( hb 10 g / dl , platelets 45 g / l and wbc 2", "bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei .", "the bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase ( mpo ) .", "an empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence , normalization of hemogram values and a complete sr documented by bone marrow examination ( blasts < 5% ) .", "no colony - stimulating factors were administered and the patient was able to return home .", "second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80% , mpo negative , esterase - positive monoblastic cells .", "the patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen .", "npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient ( c.861_862instgtc ; identified in cosmic database as cosm20861 ) .", "no mutations were found in either cebpa or flt3 ( neither internal duplication ( it d ) nor mutations in tyrosine kinase domain ( tkd ) ) ."], ["a 33-year - old male patient with no medical history was admitted in january 2013 .", "he had presented fever , fatigue , moderate weight loss , and anorexia for 1 month before hand .", "g / l with 5% of blast cells ) , and normal platelet count ( 175", "bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis .", "a bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 + , cd117 + , cd15 + , cd34 , cd163 , and mpo blast cells .", "the karyotype was normal , fish analysis showed no mll rearrangements , and molecular biology identified a type j npm1 mutation ( an insertion of tatg , fig .", "1 ) without flt3-itd , flt3-tkd , or cebpa mutation . due to the patient 's septic condition ,", "antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started .", "sr was observed in the following days with normalization of complete blood count ( cbc ) and clearance of blasts cells in the blood and bone marrow .", "blast cell morphology , cytogenetics , and molecular biology were identical to that of first diagnosis .", "the pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine ."], ["a 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia .", "this patient had a history of high blood pressure , diabetes , and high cholesterol .", "cbc revealed a hemoglobin level of 8.8 g / dl , a white blood cell count of 21 g / l with 8% of neutrophils and 15", "bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts , and 41% of promonocytes .", "the karyotype was normal , fish analysis identified no mll rearrangements , and molecular biology revealed a type a npm1 mutation ( duplication of tctg , fig .", "antibiotic therapy with amoxicillin / clavulanic acid was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts ( despite persistence of approximately 15% of dystrophic monocytes ) . given the sr , the patient was allowed to return home .", "flow cytometry performed on the bone marrow identified a cd13 + , cd33 + , cd117 + , cd64 + , and cd14 + blast population , in favor of a monocytic differentiation .", "cytogenetics and molecular biology were identical to that of the initial diagnosis . following aml type chemotherapy , the patient is alive , in good health and in cr .", "electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow ."], ["spontaneous remission of acute myeloid leukemia is an extremely rare and almost always transient event , with a mean duration in the literature of 7.7 months ( range 136 ) 8 . since 1979 , approximately thirty cases of aml sr have been reported 8,9 , although the underlying mechanisms involved remain unclear . a potential role of bacterial and/or fungal infections and blood transfusions was suggested in sr occurrence 10 by triggering an immune and antileukemic response .", "activation of the immune system and cytokines such as tumor necrosis factor ( tnf ) , interferon gamma ( ifn- ) , and interleukin-2 ( il-2 ) released during infections , in conjunction with an increase in the antibody - mediated cytotoxicity of natural killer ( nk ) cells and cytotoxic activity of t lymphocytes and macrophages , may play a role in the occurrence of sr 1,2,11,12 .", "indeed , immune response might be a potential mechanism of prolonged disease control : in 2012 , the case of a german patient who presented aml with a 10-year - long sr was reported . in this patient , it was demonstrated in vitro that nk cells displayed cytotoxic activity against the k562 myeloid leukemia cell line with upregulation of cd107a in flow cytometry 13 . in addition , infection is often accompanied by a hypergammaglobulinemia , which has been associated with aml sr in some cases 13 .", "the potentially beneficial effect of systemic infections in aml led to preliminary trials using bacterial extract inoculations or vaccination programs , which did not show a significant effect on survival or occurrence of remission 14,15 .", "our patients did not receive any blood transfusions but all presented with infections that responded to antibiotic treatment with normalization of cbc and occurrence of sr .", "unfortunately , no blood cytokine monitoring was performed in our three patients . to date , most cases of aml sr have been described in patients with normal karyotypes 3,11 . to our knowledge , this is the first report of three cases of sr in aml m5 according to the fab classification with normal karyotype and npm1 mutations .", "npm1-mutated aml represents a separate entity included in the aml who classification since 2008 , with a favorable prognosis in patients with normal karyotype aml ( aml - nk ) in the absence of the flt3-itd mutation 16 .", "the npm1 mutation is the most common genetic alteration found in aml - nk and is present in approximately one - third of adult aml 17 .", "npm1 mutations are located in the last exon ( exon 12 ) and consist in duplication or insertion of small nucleotide sequences . more than 50 mutations have been reported to date , leading to a npm1 protein variant with modification of the last seven residues , as well as four additional residues 13,17,18 .", "physiologically , npm1 encodes a nucleocytoplasmic shuttling protein ( npm1wt ) involved in the regulation of ribosome genesis and centrosome duplication 19 .", "npm1 mutation is associated with the aberrant cytoplasmic localization of npm1 , thereby disrupting its function and perturbing multiple cellular pathways 18,20 .", "the potential role of npm1 mutations in the occurrence of sr is not known but it has been previously reported that patients with aml - nk and mutated npm1 had a better response rate to induction chemotherapy 17,18 which could advocate for increased chemosensitivity of blast cells with npm1 mutation .", "recent findings suggest that npm1 mutations are associated with increased sensitivity to oxidative stress and that mutant npm1 is a target of arsenic trioxide - mediated oxidative stress 21,22 .", "systemic infections , usually described in cases of sr , are associated with exuberant activation of the immune system and high production of reactive oxygen species ( ros ) .", "thus , we hypothesize that the three cases of sr presented herein might be linked by the presence of mutant npm1 , and therefore be more susceptible to oxidative stress than cases with wild - type npm1 .", "moreover , we can speculate that there is a link between sr and ros that still need to be demonstrated .", "sr is an exceptional phenomenon , often transient and relatively well documented but the underlying molecular mechanisms are still unknown . npm1-mutated aml m5 patients who developed spontaneous remission after antibiotic treatment of febrile neutropenia present at diagnosis seem to form a subtype with favorable prognosis and chemo sensitive disease .", "a better understanding of this rare process could uncover new potential therapeutic targets for acute leukemia ."], [""]]}
{"article_id": "PMC4614206", "article_text": ["rare cancers are not rare at all . despite the relatively low incidence of each type of cancer , rare cancers combined account for 20%-30% of cancer cases and deaths .", "every year , more than 500,000 people are diagnosed with a rare form of cancer in the european union , and more than 4 million are living with the diagnosis of rare cancer .", "survival rates for rare cancers are lower than those for common cancers ( 5 years ; 47% vs. 65% ) .", "therefore , rare cancers pose a significant burden and should be recognized as a public health priority .", "patients and their families , as well as clinicians , researchers , and policy makers involved in cancer care are faced with the profound challenges and difficulties imposed by these forms of cancer .", "in fact , it is estimated that thousands of patients with these  forgotten cancers  are paying a high price as a result of repeated misdiagnosis and receiving inappropriate treatment .", "some may need to travel long distances in order to obtain the necessary treatment , while many struggle to find information about their disease and find their consultation time insufficient .", "clinicians also face unique challenges resulting from the lack of sufficient evidence , and researchers face challenges from insufficient funding or low number of research subjects . from the perspective of policy makers ,", "the disproportionate occurrence of such forms of cancer in younger and minority populations , unequal access to quality care , and geographic variation in survival rates create additional challenges in terms of health disparities .", "several workshops held in the united states and the eu promoting epidemiological research on rare and understudied cancer are notable examples of recent progress in this area .", "consequently , a multi - stakeholder initiative dedicated to improving rare cancer care was launched , and several recommendations were made by professional societies .", "these initiatives and recommendations reflect the joint effort of research communities , clinicians , policy makers , patients , and industry partners , and workshop attendees are often advocates and supporters of proposed recommendations and thus may not represent the views of the  average  clinician or researcher .", "in addition , several recommendations lack firm scientific evidence or support , and may be up for debate .", "for example , it remains unclear as to whether patients with rare cancers treated in high volume centers show better survival .", "in addition , there is some evidence that the public do not have a societal preference for treating rare diseases over common ones , implying that the degree of rarity of a disease does not justify prioritization ( e.g. , the special market access status of orphan drugs ) .", "moreover , proponents assert that incentives promoting use of orphan drugs have resulted in market failures", ". experts also disagree on whether current incentives for research focusing on rare diseases are adequate . to the best of our knowledge", ", there are no currently available quantitative data on the experiences and opinions of physicians involved in cancer care .", "therefore , in this study involving nationally representative samples of oncologists , we sought to investigate perceived difficulties with regard to rare cancer care and potential solutions endorsed by oncologists .", "this study was part of a nationwide survey conducted in order to explore views regarding medical care and treatments among physicians involved in cancer care .", "physicians in the national cancer center and 12 government - designated regional cancer centers across korea participated in the survey .", "the study was approved by the institutional review board of the national cancer center , korea .", "of the 901 cancer care physicians invited to participate in the study , 680 agreed ( 75.5% participation rate ) to do so and completed the study survey . among them", ", those who directly see cancer patients for diagnosis and treatment were asked to answer questions regarding rare cancer issues , and 175 physicians who provided clinical support to oncologists ( e.g. , radiologists , pathologists , cardiologists , rehabilitation specialists , pain specialists , and psychiatrists ) were excluded from the study .", "in addition , 30 oncologists who did not answer the questionnaire regarding rare cancer issues and 52 physicians who reported that they do not see rare cancer patients at all were also excluded from the study .", "three additional oncologists were excluded from the analyses due to their high rates of missing responses (  50% of items on rare cancer questions ) , leaving 420 oncologists comprising the study s eventual sample .", "we developed a questionnaire based on themes identified from various scientific and lay literature regarding rare cancer care [ 4,5,7,13 - 19 ] .", "survey questions covered : ( 1 ) the proportion of rare cancer patients in participants practices , ( 2 ) personal experiences of difficulties in treatment of rare cancers , and ( 3 ) participants endorsements of potential solutions for improving rare cancer care ( appendix 1 ) . in our instructions to study participants , we addressed the current lack of a unified definition of rare cancer to ensure that they understood the survey s content :  although there is presently no clear definition of  rare cancer, it is commonly defined as a cancer with an incidence of 5 per 100,000 .", "please answer the following questions , supposing that they do not apply to common cancers ( e.g. , cancers of the stomach , lung , liver , colon , breast , cervix , thyroid , prostate , gallbladder , and pancreas ) but may apply to cancers with uncommon histologies in common cancer sites . ", "other issues related to rare cancer care that we explored through the survey included lack of clinical experience and need for referral , availability of approved drugs and need to promote off - label treatments , reimbursement issues , lack of research evidence and guidelines , research funding and clinical trials of pharmaceutical companies , support and incentives from the government , and the need for an ( inter)national registry [ 4,16 - 18 ] . when answering the questions regarding difficulties faced by oncologists and solutions for improvement of care survey physicians were allowed to choose multiple answers that apply without limitation .", "in addition , a blank space was provided for them to share any personal experiences or opinions regarding rare cancer treatment .", "data on participants age , sex , specialty , years from board certification , and patient volume ( average number of outpatients per week ) were also collected .", "chi - square tests were performed for comparison of responses according to participants specialties and the proportions of rare cancer patients in their practices .", "correlations between responses to the items were further explored to determine associations between perceived barriers and recommendations .", "12.0 ( statacorp , college station , tx ) , and a p < 0.05 was considered statistically significant .", "this study was part of a nationwide survey conducted in order to explore views regarding medical care and treatments among physicians involved in cancer care .", "physicians in the national cancer center and 12 government - designated regional cancer centers across korea participated in the survey .", "the study was approved by the institutional review board of the national cancer center , korea .", "of the 901 cancer care physicians invited to participate in the study , 680 agreed ( 75.5% participation rate ) to do so and completed the study survey . among them", ", those who directly see cancer patients for diagnosis and treatment were asked to answer questions regarding rare cancer issues , and 175 physicians who provided clinical support to oncologists ( e.g. , radiologists , pathologists , cardiologists , rehabilitation specialists , pain specialists , and psychiatrists ) were excluded from the study .", "in addition , 30 oncologists who did not answer the questionnaire regarding rare cancer issues and 52 physicians who reported that they do not see rare cancer patients at all were also excluded from the study .", "three additional oncologists were excluded from the analyses due to their high rates of missing responses (  50% of items on rare cancer questions ) , leaving 420 oncologists comprising the study s eventual sample .", "we developed a questionnaire based on themes identified from various scientific and lay literature regarding rare cancer care [ 4,5,7,13 - 19 ] .", "survey questions covered : ( 1 ) the proportion of rare cancer patients in participants practices , ( 2 ) personal experiences of difficulties in treatment of rare cancers , and ( 3 ) participants endorsements of potential solutions for improving rare cancer care ( appendix 1 ) . in our instructions to study participants , we addressed the current lack of a unified definition of rare cancer to ensure that they understood the survey s content :  although there is presently no clear definition of  rare cancer, it is commonly defined as a cancer with an incidence of 5 per 100,000 .", "please answer the following questions , supposing that they do not apply to common cancers ( e.g. , cancers of the stomach , lung , liver , colon , breast , cervix , thyroid , prostate , gallbladder , and pancreas ) but may apply to cancers with uncommon histologies in common cancer sites . ", "other issues related to rare cancer care that we explored through the survey included lack of clinical experience and need for referral , availability of approved drugs and need to promote off - label treatments , reimbursement issues , lack of research evidence and guidelines , research funding and clinical trials of pharmaceutical companies , support and incentives from the government , and the need for an ( inter)national registry [ 4,16 - 18 ] .", "when answering the questions regarding difficulties faced by oncologists and solutions for improvement of care survey physicians were allowed to choose multiple answers that apply without limitation .", "in addition , a blank space was provided for them to share any personal experiences or opinions regarding rare cancer treatment .", "data on participants age , sex , specialty , years from board certification , and patient volume ( average number of outpatients per week ) were also collected .", "chi - square tests were performed for comparison of responses according to participants specialties and the proportions of rare cancer patients in their practices .", "correlations between responses to the items were further explored to determine associations between perceived barriers and recommendations .", "12.0 ( statacorp , college station , tx ) , and a p < 0.05 was considered statistically significant .", "the mean age of the oncologists was 43.0 years , and the average number of years since board certification was 12.0 years .", "the sample comprised surgical oncologists ( 57.1% ) , medical oncologists ( 36.2% ) , and radiation oncologists ( 6.7% ) ( table 1 ) .", "overall , the proportions of rare cancer patients seen by the oncologists were < 1% for 38.3% and 1%-5% for 39.1% .", "less than a quarter of oncologists answered that rare cancer patients constituted > 5% of the patients in their practice , except for pediatric medical oncologists , 60% of whom noted that rare cancer patients comprised the majority of their patient population ( table 2 ) .", "difficulties reported by more than half of the respondents included lack of standard treatment guidelines ( 65.7% ) , insufficient personal experience with rare cancer treatments ( 65.2% ) , and lack of evidence regarding rare cancer treatments ( 54.1% ) .", "relatively little clinical experience was higher for those who rarely see rare cancer patients ( p < 0.001 ) .", "reimbursements for drug treatments ( 44.5% ) , and unavailability of sufficient approved treatment options ( 39.8% ) were also commonly reported , particularly by medical oncologists ( p < 0.001 ) .", "less than 20% of participants felt that investments from pharmaceutical companies and clinical trials were lacking ( 18.2% ) , or that more research funding from the government was needed ( 17.1% ) , although oncologists who frequently saw rare cancer patients were more likely to endorse these measures as solutions for better rare cancer care ( p < 0.05 ) ( table 3 ) .", "more than half of our study participants noted the need for development of clinical practice guidelines ( 66.0% ) , and more flexible reimbursement guidelines for treatment of rare cancers ( 52.9% ) .", "more than 30% of respondents felt that the establishment of a national registry ( 47.4% ) , a referral system to high volume centers for accumulating treatment experience ( 35.5% ) , research incentives ( 33.8% ) , and government initiative and support for research in rare cancer ( 30.2% ) would improve patient care . only a small proportion of respondents endorsed solutions involving off - label treatments for rare cancers ( 21.0% ) and legislation mandating budget allocations for development of drugs for treatment of rare cancers ( 13.1% ) ( table 4 ) .", "the mean age of the oncologists was 43.0 years , and the average number of years since board certification was 12.0 years .", "the sample comprised surgical oncologists ( 57.1% ) , medical oncologists ( 36.2% ) , and radiation oncologists ( 6.7% ) ( table 1 ) .", "overall , the proportions of rare cancer patients seen by the oncologists were < 1% for 38.3% and 1%-5% for 39.1% .", "less than a quarter of oncologists answered that rare cancer patients constituted > 5% of the patients in their practice , except for pediatric medical oncologists , 60% of whom noted that rare cancer patients comprised the majority of their patient population ( table 2 ) .", "difficulties reported by more than half of the respondents included lack of standard treatment guidelines ( 65.7% ) , insufficient personal experience with rare cancer treatments ( 65.2% ) , and lack of evidence regarding rare cancer treatments ( 54.1% ) .", "relatively little clinical experience was higher for those who rarely see rare cancer patients ( p < 0.001 ) .", "reimbursements for drug treatments ( 44.5% ) , and unavailability of sufficient approved treatment options ( 39.8% ) were also commonly reported , particularly by medical oncologists ( p < 0.001 ) .", "less than 20% of participants felt that investments from pharmaceutical companies and clinical trials were lacking ( 18.2% ) , or that more research funding from the government was needed ( 17.1% ) , although oncologists who frequently saw rare cancer patients were more likely to endorse these measures as solutions for better rare cancer care ( p < 0.05 ) ( table 3 ) .", "more than half of our study participants noted the need for development of clinical practice guidelines ( 66.0% ) , and more flexible reimbursement guidelines for treatment of rare cancers ( 52.9% ) .", "more than 30% of respondents felt that the establishment of a national registry ( 47.4% ) , a referral system to high volume centers for accumulating treatment experience ( 35.5% ) , research incentives ( 33.8% ) , and government initiative and support for research in rare cancer ( 30.2% ) would improve patient care . only a small proportion of respondents endorsed solutions involving off - label treatments for rare cancers ( 21.0% ) and legislation mandating budget allocations for development of drugs for treatment of rare cancers ( 13.1% ) ( table 4 ) .", "to the best of our knowledge , our study was the first to investigate the consensus among oncologists regarding rare cancer patient care .", "the strengths of our study include its nationally representative samples of oncologists with various specialties and experiences in treatment of rare cancers , which enabled a comparison of responses across subgroups .", "the lack of standard treatment guidelines was the most common difficulty in treating rare cancers noted by oncologists , regardless of their specialty .", "in addition , development of clinical practice guidelines for rare cancers was the most frequently endorsed item for improvement of rare cancer care .", "agencies including the national comprehensive cancer network ( nccn ) and national institute for health and care excellence ( nice ) are publishing more clinical guidelines on cancers , which currently do not yet cover most rare cancers .", "the lack of established guidelines for rare cancer treatment often leaves oncologists with no clear direction in treating patients with rare cancers and in making treatment decisions based on empirical evidence .", "indeed , the world health organization ( who )   and european society for medical oncology ( esmo )   have suggested implementing guidelines for medical and psychosocial care .", "insufficient personal experience in rare cancer treatment was the second most common difficulty faced by oncologists , and was related to endorsement of referrals to high volume centers ( =0.23 , p < 0.001 ) .", "centralization of diagnosis and multidisciplinary treatment at specialized centers have been advocated by professional societies , government bodies , and even insurers , who assume that such measures will improve outcomes . however ,", "among our study participants , the endorsement of referrals to high volume centers was relatively low ( 35.5% ) , particularly among those who saw a higher proportion of rare cancer patients ( p=0.02 ) . in the blank space in which they could express their views and offer suggestions , several oncologists proposed that rare cancer treatments be administered in regional cancer centers rather than in centralized venues , and suggested the need for information sharing , registry establishment , and collaborative research .", "further studies are warranted regarding the optimal degree of centralization in treatment of rare cancers , and the full potential of information technology / telemedicine   as a viable alternative to centralization .", "insufficient approved treatment options and issues pertaining to reimbursement were also frequently reported , both of which were found to be inter - related ( =0.51 , data not shown ) .", "these two barriers were also strongly related to their endorsement for encouragement of off - label treatment for rare cancer treatment and relief of reimbursement guidelines for rare cancer treatment ( =0.20 - 0.45 , p < 0.001 ) .", "medical oncologists , whose main treatment modalities were anti - cancer drugs , were more likely to perceive these as being obstacles to quality rare cancer patient care and called for necessary improvements .", "clinical trials on rare disease treatment are rarely conducted due to the lack of commercial incentives .", "when conducted , trials are likely to be underpowered due to a small number of available patients .", "therefore , off - label anti - cancer drugs are often requested by patients or family caregivers , are justified or regarded as essential in certain conditions when there is compelling biological plausibility as to their efficacy , and were supported by professional guidelines and us reimbursement policies . in our study , while the need for flexible reimbursement guidelines was acknowledged by over half of the oncologists , only about 20% agreed with the idea of promoting off - label medication use .", "such seemingly discrepant attitudes were also reported in a us survey ; while 61% of oncologists expressed their belief that off - protocol treatment use should be discouraged among patients , only 31% agreed that such treatments should not be available .", "this implies that while oncologists prefer greater flexibility in cancer drug use / treatments , they also feel uncomfortable trying unproven therapies .", "in fact , professional oncology societies have called for timely production of  standard medical compendia  enlisting off - label uses judged to be legitimate .", "insufficient research evidence was also among the most commonly perceived difficulties in rare cancer care .", "rare cancers typically receive little scientific attention   and have suffered from difficulties in patient accrual as well as underfunding due to being low in priority .", "therefore , platforms for collaborative research and sustained funding mechanisms have been advocated , including registries or research networks at the national or multinational level , federal or government funding for rare disease research projects , and financial support from industry partners for clinical research or regulatory registration activities for specific rare diseases . among such potential solutions ,", "the development of a national rare cancer registry was supported by nearly half of the oncologists , implying that they agree with the idea that registries constitute key instruments increasing empirical evidence on rare diseases .", "in contrast , only a small proportion of oncologists mentioned insufficient research funding or clinical trials as difficulties .", "in addition , endorsement for incentives for rare cancer research and government funding was only moderate , and oncologists generally opposed the enactment of legislation mandating pharmaceutical companies allocate funds for rare cancer treatment .", "however , we found no negative free statements on the idea and found many others supporting such ideas .", "in addition , these solutions were often endorsed by oncologists who frequently saw rare cancer patients .", "therefore , we supposed that the low endorsement rates reflected low personal needs for such measures rather than objections to these ideas .", "first , we did not provide specific lists of rare cancers , as definitions vary from one set of guidelines to another and can differ according to the determining criterion [ 1 - 3,9 ] .", "therefore , the proportion of patients with rare cancers in their practice estimated by the oncologists may not be accurate .", "second , the majority of survey respondents did not frequently see rare cancer patients , and thus might not have clear opinions regarding many issues in rare cancer care .", "however , in the real world they would be the first physicians whom patients with rare cancers see and therefore their opinions were critical in development of national policies and strategies .", "third , the experiences of oncologists in treatment of rare cancers as well as their attitudes towards treatments would be specific to each country as these are largely determined by health care delivery and reimbursement systems .", "in summary , oncologists faced various difficulties in treatment of patients with rare cancers , including the lack of clinical practice guidelines and personal experience , lack of approved treatment options , and reimbursement issues .", "they were generally supportive of recent recommendations by multi - stakeholder initiatives as well as professional societies for development of clear clinical practice guidelines , flexible reimbursement guidelines , and a national rare cancer registry . however , there was only moderate endorsement for referrals to high - volume centers or encouragement of off - label treatments .", "insights into the general attitudes of oncologists gained through our nationwide survey of representative samples would be helpful in development of clinical practices and public health policies in rare cancer treatment and research ."], "abstract_text": ["<S> purposein response to the challenges and difficulties imposed by rare cancers , multi - stakeholder initiatives dedicated to improving rare cancer care was launched , and several recommendations were made by professional societies . </S>", "<S> however , these primarily reflect the view of the advocates and supporters , and may not represent the views of the  average  clinician or researcher . in this study </S>", "<S> , we sought to investigate perceived difficulties with regard to rare cancer care and potential solutions endorsed by oncologists.materials and methodsa representative sample of 420 oncologists recruited in 13 cancer centers participated in a nationwide survey.resultsoncologists faced various difficulties in treatment of patients with rare cancers , including the lack of clinical practice guidelines ( 65.7% ) and personal experience ( 65.2% ) , lack of approved treatment options ( 39.8% ) , and reimbursement issues ( 44.5% ) . </S>", "<S> they were generally supportive of recent recommendations by multi - stakeholder initiatives as well as professional societies for development of clear clinical practice guidelines ( 66.0% ) , flexible reimbursement guidelines ( 52.9% ) , and a national rare cancer registry ( 47.4% ) . </S>", "<S> however , there was only moderate endorsement for referrals to high - volume centers ( 35.5% ) and encouragement of off - label treatments ( 21.0%).conclusioninsights into the general attitudes of oncologists gained through our nationwide survey of representative samples would be helpful in development of clinical practices and public health policies in rare cancer treatment and research . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "1. Study design and subjects", "2. Measures", "3. Statistical analysis", "Results", "1. Characteristics of participants", "2. Difficulties faced by oncologists in rare cancer care", "3. Potential solutions for improvement of rare cancer care", "Discussion", "Conclusion"], "sections": [["rare cancers are not rare at all . despite the relatively low incidence of each type of cancer , rare cancers combined account for 20%-30% of cancer cases and deaths .", "every year , more than 500,000 people are diagnosed with a rare form of cancer in the european union , and more than 4 million are living with the diagnosis of rare cancer .", "survival rates for rare cancers are lower than those for common cancers ( 5 years ; 47% vs. 65% ) .", "therefore , rare cancers pose a significant burden and should be recognized as a public health priority .", "patients and their families , as well as clinicians , researchers , and policy makers involved in cancer care are faced with the profound challenges and difficulties imposed by these forms of cancer .", "in fact , it is estimated that thousands of patients with these  forgotten cancers  are paying a high price as a result of repeated misdiagnosis and receiving inappropriate treatment .", "some may need to travel long distances in order to obtain the necessary treatment , while many struggle to find information about their disease and find their consultation time insufficient .", "clinicians also face unique challenges resulting from the lack of sufficient evidence , and researchers face challenges from insufficient funding or low number of research subjects . from the perspective of policy makers ,", "the disproportionate occurrence of such forms of cancer in younger and minority populations , unequal access to quality care , and geographic variation in survival rates create additional challenges in terms of health disparities .", "several workshops held in the united states and the eu promoting epidemiological research on rare and understudied cancer are notable examples of recent progress in this area .", "consequently , a multi - stakeholder initiative dedicated to improving rare cancer care was launched , and several recommendations were made by professional societies .", "these initiatives and recommendations reflect the joint effort of research communities , clinicians , policy makers , patients , and industry partners , and workshop attendees are often advocates and supporters of proposed recommendations and thus may not represent the views of the  average  clinician or researcher .", "in addition , several recommendations lack firm scientific evidence or support , and may be up for debate .", "for example , it remains unclear as to whether patients with rare cancers treated in high volume centers show better survival .", "in addition , there is some evidence that the public do not have a societal preference for treating rare diseases over common ones , implying that the degree of rarity of a disease does not justify prioritization ( e.g. , the special market access status of orphan drugs ) .", "moreover , proponents assert that incentives promoting use of orphan drugs have resulted in market failures", ". experts also disagree on whether current incentives for research focusing on rare diseases are adequate . to the best of our knowledge", ", there are no currently available quantitative data on the experiences and opinions of physicians involved in cancer care .", "therefore , in this study involving nationally representative samples of oncologists , we sought to investigate perceived difficulties with regard to rare cancer care and potential solutions endorsed by oncologists ."], ["this study was part of a nationwide survey conducted in order to explore views regarding medical care and treatments among physicians involved in cancer care .", "physicians in the national cancer center and 12 government - designated regional cancer centers across korea participated in the survey .", "the study was approved by the institutional review board of the national cancer center , korea .", "of the 901 cancer care physicians invited to participate in the study , 680 agreed ( 75.5% participation rate ) to do so and completed the study survey . among them", ", those who directly see cancer patients for diagnosis and treatment were asked to answer questions regarding rare cancer issues , and 175 physicians who provided clinical support to oncologists ( e.g. , radiologists , pathologists , cardiologists , rehabilitation specialists , pain specialists , and psychiatrists ) were excluded from the study .", "in addition , 30 oncologists who did not answer the questionnaire regarding rare cancer issues and 52 physicians who reported that they do not see rare cancer patients at all were also excluded from the study .", "three additional oncologists were excluded from the analyses due to their high rates of missing responses (  50% of items on rare cancer questions ) , leaving 420 oncologists comprising the study s eventual sample .", "we developed a questionnaire based on themes identified from various scientific and lay literature regarding rare cancer care [ 4,5,7,13 - 19 ] .", "survey questions covered : ( 1 ) the proportion of rare cancer patients in participants practices , ( 2 ) personal experiences of difficulties in treatment of rare cancers , and ( 3 ) participants endorsements of potential solutions for improving rare cancer care ( appendix 1 ) . in our instructions to study participants , we addressed the current lack of a unified definition of rare cancer to ensure that they understood the survey s content :  although there is presently no clear definition of  rare cancer, it is commonly defined as a cancer with an incidence of 5 per 100,000 .", "please answer the following questions , supposing that they do not apply to common cancers ( e.g. , cancers of the stomach , lung , liver , colon , breast , cervix , thyroid , prostate , gallbladder , and pancreas ) but may apply to cancers with uncommon histologies in common cancer sites . ", "other issues related to rare cancer care that we explored through the survey included lack of clinical experience and need for referral , availability of approved drugs and need to promote off - label treatments , reimbursement issues , lack of research evidence and guidelines , research funding and clinical trials of pharmaceutical companies , support and incentives from the government , and the need for an ( inter)national registry [ 4,16 - 18 ] . when answering the questions regarding difficulties faced by oncologists and solutions for improvement of care survey physicians were allowed to choose multiple answers that apply without limitation .", "in addition , a blank space was provided for them to share any personal experiences or opinions regarding rare cancer treatment .", "data on participants age , sex , specialty , years from board certification , and patient volume ( average number of outpatients per week ) were also collected .", "chi - square tests were performed for comparison of responses according to participants specialties and the proportions of rare cancer patients in their practices .", "correlations between responses to the items were further explored to determine associations between perceived barriers and recommendations .", "12.0 ( statacorp , college station , tx ) , and a p < 0.05 was considered statistically significant ."], ["this study was part of a nationwide survey conducted in order to explore views regarding medical care and treatments among physicians involved in cancer care .", "physicians in the national cancer center and 12 government - designated regional cancer centers across korea participated in the survey .", "the study was approved by the institutional review board of the national cancer center , korea .", "of the 901 cancer care physicians invited to participate in the study , 680 agreed ( 75.5% participation rate ) to do so and completed the study survey . among them", ", those who directly see cancer patients for diagnosis and treatment were asked to answer questions regarding rare cancer issues , and 175 physicians who provided clinical support to oncologists ( e.g. , radiologists , pathologists , cardiologists , rehabilitation specialists , pain specialists , and psychiatrists ) were excluded from the study .", "in addition , 30 oncologists who did not answer the questionnaire regarding rare cancer issues and 52 physicians who reported that they do not see rare cancer patients at all were also excluded from the study .", "three additional oncologists were excluded from the analyses due to their high rates of missing responses (  50% of items on rare cancer questions ) , leaving 420 oncologists comprising the study s eventual sample ."], ["we developed a questionnaire based on themes identified from various scientific and lay literature regarding rare cancer care [ 4,5,7,13 - 19 ] .", "survey questions covered : ( 1 ) the proportion of rare cancer patients in participants practices , ( 2 ) personal experiences of difficulties in treatment of rare cancers , and ( 3 ) participants endorsements of potential solutions for improving rare cancer care ( appendix 1 ) . in our instructions to study participants , we addressed the current lack of a unified definition of rare cancer to ensure that they understood the survey s content :  although there is presently no clear definition of  rare cancer, it is commonly defined as a cancer with an incidence of 5 per 100,000 .", "please answer the following questions , supposing that they do not apply to common cancers ( e.g. , cancers of the stomach , lung , liver , colon , breast , cervix , thyroid , prostate , gallbladder , and pancreas ) but may apply to cancers with uncommon histologies in common cancer sites . ", "other issues related to rare cancer care that we explored through the survey included lack of clinical experience and need for referral , availability of approved drugs and need to promote off - label treatments , reimbursement issues , lack of research evidence and guidelines , research funding and clinical trials of pharmaceutical companies , support and incentives from the government , and the need for an ( inter)national registry [ 4,16 - 18 ] .", "when answering the questions regarding difficulties faced by oncologists and solutions for improvement of care survey physicians were allowed to choose multiple answers that apply without limitation .", "in addition , a blank space was provided for them to share any personal experiences or opinions regarding rare cancer treatment .", "data on participants age , sex , specialty , years from board certification , and patient volume ( average number of outpatients per week ) were also collected ."], ["chi - square tests were performed for comparison of responses according to participants specialties and the proportions of rare cancer patients in their practices .", "correlations between responses to the items were further explored to determine associations between perceived barriers and recommendations .", "12.0 ( statacorp , college station , tx ) , and a p < 0.05 was considered statistically significant ."], ["the mean age of the oncologists was 43.0 years , and the average number of years since board certification was 12.0 years .", "the sample comprised surgical oncologists ( 57.1% ) , medical oncologists ( 36.2% ) , and radiation oncologists ( 6.7% ) ( table 1 ) .", "overall , the proportions of rare cancer patients seen by the oncologists were < 1% for 38.3% and 1%-5% for 39.1% .", "less than a quarter of oncologists answered that rare cancer patients constituted > 5% of the patients in their practice , except for pediatric medical oncologists , 60% of whom noted that rare cancer patients comprised the majority of their patient population ( table 2 ) .", "difficulties reported by more than half of the respondents included lack of standard treatment guidelines ( 65.7% ) , insufficient personal experience with rare cancer treatments ( 65.2% ) , and lack of evidence regarding rare cancer treatments ( 54.1% ) .", "relatively little clinical experience was higher for those who rarely see rare cancer patients ( p < 0.001 ) .", "reimbursements for drug treatments ( 44.5% ) , and unavailability of sufficient approved treatment options ( 39.8% ) were also commonly reported , particularly by medical oncologists ( p < 0.001 ) .", "less than 20% of participants felt that investments from pharmaceutical companies and clinical trials were lacking ( 18.2% ) , or that more research funding from the government was needed ( 17.1% ) , although oncologists who frequently saw rare cancer patients were more likely to endorse these measures as solutions for better rare cancer care ( p < 0.05 ) ( table 3 ) .", "more than half of our study participants noted the need for development of clinical practice guidelines ( 66.0% ) , and more flexible reimbursement guidelines for treatment of rare cancers ( 52.9% ) .", "more than 30% of respondents felt that the establishment of a national registry ( 47.4% ) , a referral system to high volume centers for accumulating treatment experience ( 35.5% ) , research incentives ( 33.8% ) , and government initiative and support for research in rare cancer ( 30.2% ) would improve patient care . only a small proportion of respondents endorsed solutions involving off - label treatments for rare cancers ( 21.0% ) and legislation mandating budget allocations for development of drugs for treatment of rare cancers ( 13.1% ) ( table 4 ) ."], ["the mean age of the oncologists was 43.0 years , and the average number of years since board certification was 12.0 years .", "the sample comprised surgical oncologists ( 57.1% ) , medical oncologists ( 36.2% ) , and radiation oncologists ( 6.7% ) ( table 1 ) .", "overall , the proportions of rare cancer patients seen by the oncologists were < 1% for 38.3% and 1%-5% for 39.1% .", "less than a quarter of oncologists answered that rare cancer patients constituted > 5% of the patients in their practice , except for pediatric medical oncologists , 60% of whom noted that rare cancer patients comprised the majority of their patient population ( table 2 ) ."], ["difficulties reported by more than half of the respondents included lack of standard treatment guidelines ( 65.7% ) , insufficient personal experience with rare cancer treatments ( 65.2% ) , and lack of evidence regarding rare cancer treatments ( 54.1% ) .", "relatively little clinical experience was higher for those who rarely see rare cancer patients ( p < 0.001 ) .", "reimbursements for drug treatments ( 44.5% ) , and unavailability of sufficient approved treatment options ( 39.8% ) were also commonly reported , particularly by medical oncologists ( p < 0.001 ) .", "less than 20% of participants felt that investments from pharmaceutical companies and clinical trials were lacking ( 18.2% ) , or that more research funding from the government was needed ( 17.1% ) , although oncologists who frequently saw rare cancer patients were more likely to endorse these measures as solutions for better rare cancer care ( p < 0.05 ) ( table 3 ) ."], ["more than half of our study participants noted the need for development of clinical practice guidelines ( 66.0% ) , and more flexible reimbursement guidelines for treatment of rare cancers ( 52.9% ) .", "more than 30% of respondents felt that the establishment of a national registry ( 47.4% ) , a referral system to high volume centers for accumulating treatment experience ( 35.5% ) , research incentives ( 33.8% ) , and government initiative and support for research in rare cancer ( 30.2% ) would improve patient care . only a small proportion of respondents endorsed solutions involving off - label treatments for rare cancers ( 21.0% ) and legislation mandating budget allocations for development of drugs for treatment of rare cancers ( 13.1% ) ( table 4 ) ."], ["to the best of our knowledge , our study was the first to investigate the consensus among oncologists regarding rare cancer patient care .", "the strengths of our study include its nationally representative samples of oncologists with various specialties and experiences in treatment of rare cancers , which enabled a comparison of responses across subgroups .", "the lack of standard treatment guidelines was the most common difficulty in treating rare cancers noted by oncologists , regardless of their specialty .", "in addition , development of clinical practice guidelines for rare cancers was the most frequently endorsed item for improvement of rare cancer care .", "agencies including the national comprehensive cancer network ( nccn ) and national institute for health and care excellence ( nice ) are publishing more clinical guidelines on cancers , which currently do not yet cover most rare cancers .", "the lack of established guidelines for rare cancer treatment often leaves oncologists with no clear direction in treating patients with rare cancers and in making treatment decisions based on empirical evidence .", "indeed , the world health organization ( who )   and european society for medical oncology ( esmo )   have suggested implementing guidelines for medical and psychosocial care .", "insufficient personal experience in rare cancer treatment was the second most common difficulty faced by oncologists , and was related to endorsement of referrals to high volume centers ( =0.23 , p < 0.001 ) .", "centralization of diagnosis and multidisciplinary treatment at specialized centers have been advocated by professional societies , government bodies , and even insurers , who assume that such measures will improve outcomes . however ,", "among our study participants , the endorsement of referrals to high volume centers was relatively low ( 35.5% ) , particularly among those who saw a higher proportion of rare cancer patients ( p=0.02 ) . in the blank space in which they could express their views and offer suggestions , several oncologists proposed that rare cancer treatments be administered in regional cancer centers rather than in centralized venues , and suggested the need for information sharing , registry establishment , and collaborative research .", "further studies are warranted regarding the optimal degree of centralization in treatment of rare cancers , and the full potential of information technology / telemedicine   as a viable alternative to centralization .", "insufficient approved treatment options and issues pertaining to reimbursement were also frequently reported , both of which were found to be inter - related ( =0.51 , data not shown ) .", "these two barriers were also strongly related to their endorsement for encouragement of off - label treatment for rare cancer treatment and relief of reimbursement guidelines for rare cancer treatment ( =0.20 - 0.45 , p < 0.001 ) .", "medical oncologists , whose main treatment modalities were anti - cancer drugs , were more likely to perceive these as being obstacles to quality rare cancer patient care and called for necessary improvements .", "clinical trials on rare disease treatment are rarely conducted due to the lack of commercial incentives .", "when conducted , trials are likely to be underpowered due to a small number of available patients .", "therefore , off - label anti - cancer drugs are often requested by patients or family caregivers , are justified or regarded as essential in certain conditions when there is compelling biological plausibility as to their efficacy , and were supported by professional guidelines and us reimbursement policies . in our study , while the need for flexible reimbursement guidelines was acknowledged by over half of the oncologists , only about 20% agreed with the idea of promoting off - label medication use .", "such seemingly discrepant attitudes were also reported in a us survey ; while 61% of oncologists expressed their belief that off - protocol treatment use should be discouraged among patients , only 31% agreed that such treatments should not be available .", "this implies that while oncologists prefer greater flexibility in cancer drug use / treatments , they also feel uncomfortable trying unproven therapies .", "in fact , professional oncology societies have called for timely production of  standard medical compendia  enlisting off - label uses judged to be legitimate .", "insufficient research evidence was also among the most commonly perceived difficulties in rare cancer care .", "rare cancers typically receive little scientific attention   and have suffered from difficulties in patient accrual as well as underfunding due to being low in priority .", "therefore , platforms for collaborative research and sustained funding mechanisms have been advocated , including registries or research networks at the national or multinational level , federal or government funding for rare disease research projects , and financial support from industry partners for clinical research or regulatory registration activities for specific rare diseases . among such potential solutions ,", "the development of a national rare cancer registry was supported by nearly half of the oncologists , implying that they agree with the idea that registries constitute key instruments increasing empirical evidence on rare diseases .", "in contrast , only a small proportion of oncologists mentioned insufficient research funding or clinical trials as difficulties .", "in addition , endorsement for incentives for rare cancer research and government funding was only moderate , and oncologists generally opposed the enactment of legislation mandating pharmaceutical companies allocate funds for rare cancer treatment .", "however , we found no negative free statements on the idea and found many others supporting such ideas .", "in addition , these solutions were often endorsed by oncologists who frequently saw rare cancer patients .", "therefore , we supposed that the low endorsement rates reflected low personal needs for such measures rather than objections to these ideas .", "first , we did not provide specific lists of rare cancers , as definitions vary from one set of guidelines to another and can differ according to the determining criterion [ 1 - 3,9 ] .", "therefore , the proportion of patients with rare cancers in their practice estimated by the oncologists may not be accurate .", "second , the majority of survey respondents did not frequently see rare cancer patients , and thus might not have clear opinions regarding many issues in rare cancer care .", "however , in the real world they would be the first physicians whom patients with rare cancers see and therefore their opinions were critical in development of national policies and strategies .", "third , the experiences of oncologists in treatment of rare cancers as well as their attitudes towards treatments would be specific to each country as these are largely determined by health care delivery and reimbursement systems ."], ["in summary , oncologists faced various difficulties in treatment of patients with rare cancers , including the lack of clinical practice guidelines and personal experience , lack of approved treatment options , and reimbursement issues .", "they were generally supportive of recent recommendations by multi - stakeholder initiatives as well as professional societies for development of clear clinical practice guidelines , flexible reimbursement guidelines , and a national rare cancer registry . however , there was only moderate endorsement for referrals to high - volume centers or encouragement of off - label treatments .", "insights into the general attitudes of oncologists gained through our nationwide survey of representative samples would be helpful in development of clinical practices and public health policies in rare cancer treatment and research ."]]}
{"article_id": "PMC4616070", "article_text": ["many problems occur while we are trying to improve the quality of life and material wealth \n by the development of modern society . among them", ", air pollution is a serious problem in \n western and developing countries . long - term exposure to air pollution has a high correlation \n with the incidence of cardiovascular disease , respiratory diseases , and diabetes1,2,3 , threatening health . in particular , air \n pollution is a major cause of respiratory diseases such as asthma and allergic diseases4 , 5 and \n it has a negative effect on the healthy growth and development of growing children and \n adolescents .", "another problem caused by the development of modern society is the reduction in physical \n activity across all generations throughout the world .", "the time spent in physical activity by \n children has decreased significantly compared to before6 , 7 .", "the time spent using \n visual media and watching tv has increased8 , 9 , and physical inactivity due to excessive \n academic pressure and the influence of parents10 , has increased obesity , incorrect posture , and muscle weakness . in \n addition , energy consumption has increased due to environmental , metabolic , and genetic \n factors11 .", "thus , children living in \n modern society are showing gradually decreasing physical fitness . the change in the environment due to", "the development of modern society is a threat to the \n health of growing children . according to the korea education development institute , \n", "respiratory system abnormalities have been much higher than those other systems over the \n last three years . reduced basic physical fitness and reduced pulmonary functions are \n seriously threating the physical development of korean elementary , middle , and high school \n students .", "although the physical fitness and lung health of growing children and adolescents is \n threatened , some studies have reported a relationship between air pollution and pulmonary \n function12 , 13 .", "however , there has been insufficient research on the relationship \n between exercise and lung function .", "most studies on exercise have only suggested lung \n function is improved by regular exercise14 , 15 .", "the present study is to our knowledge the \n first to investigate the relationship between basic physical fitness and lung function .", "the purpose of the present study was to determine the relationship between basic physical \n fitness and pulmonary function in healthy korean school students to enable us to present an \n alternative method for improving their pulmonary function .", "a total 240 healthy children and adolescents aged 617  years who lived in busan in korea \n were recruited for the present study .", "the participants had a body mass index ( bmi ) < \n 25  kg / m , and no respiratory system abnormalities .", "the sample comprised 20 \n healthy students ( 10 boys and 10 girls ) of each age from 6 to 17 .", "the participants who did \n not meet the bmi criteria or who showed significantly low pulmonary test values were \n excluded , and replacements were recruited in order to conduct the analysis with the same \n numbers of subjects .", "the participants were divided into the early period of elementary \n school ( 68 y ) , the late period of elementary school ( 911 y ) , the period of middle school \n ( 1214 y ) , and the period of high school ( 1517 y ) in order to investigate the rapid changes \n in body composition and basic physical fitness with growth .", "all participants gave their \n informed consent and the experimental protocol was approved by the ethical committee of \n dong - a university .", "body composition including height , weight , bmi , and body fat ( % ) was measured using a body \n composition analyzer venus 5.5 ( jawon medical , korea ) while subjects were fully relaxed . \n", "muscle strength , muscle power , flexibility , and balance were measured to evaluate basic \n physical fitness .", "all participants were given a full explanation about the correct posture \n and procedure of each measurement .", "hand - grip strength was evaluated using a grip - d grip strength dynamometer \n ( takei , japan ) with 0.1  kg accuracy of both the right and left hands .", "flexibility was \n evaluated by measuring sit and reach using a helmas iii trunk forward flexion instrument \n ( o2run , korea ) with 0.1  cm accuracy .", "muscle power was evaluated by measuring the sargent \n jump height using a helmas iii sargent jump instrument ( o2run , korea ) with 0.1  cm accuracy . \n", "balance was evaluated by measuring the eyes - closed single - leg standing time using a helmas \n iii blind single - leg stand instrument ( o2run , korea ) with 0.1 sec accuracy , and the \n participants performed the test using their preferred leg .", "pulmonary function tests were conducted under standard laboratory conditions ( temperature : \n 2225  c , relative humidity : 5560% ) .", "all spirometric tests were conducted by the same \n technician to reduce inter - observer variability and to prevent the failure of the \n measurement due to the young age of the subjects .", "the participants were given sufficient \n explanation about the method and instrument use , and the tests were performed in a sitting \n position while wearing a nose clip . the forced vital capacity ( fvc ) and the forced \n expiratory volume in one second ( fev1 ) were measured using a quark pft ( cosmed , italy ) .", "all \n of the pulmonary tests were conducted following the standards presented by the american \n thoracic society / european respiratory society16 .", "the data were analyzed using the statistical package for the social sciences ( spss version \n 22.00 ) and the results are presented as the mean  standard deviation .", "the significance of \n differences between boys and girls were examined using the independent t - test .", "the relationships between body composition and pulmonary \n function , and basic physical fitness and pulmonary function were analyzed using simple \n linear regression analysis .", "table 1table 1.the differences in body composition between groups ( gender and age ) and within \n group ( n=240)groupsage ( yrs)height ( cm)weight ( kg)bmi ( kg / m)body fat ( % ) malefemalemalefemalemalefemalemalefemaleperiod of early elementary school6123.7 4.43121.6 4.9624.8 4.5622.9 3.4816.1 2.0415.4 1.748.9 3.0113.0 5.067127.7 4.66126.2 3.9728.8 4.3726.2 5.0717.6 2.2016.4 2.619.6 4.4216.5 3.618133.0 4.57133.9 5.4631.7 6.6730.8 5.4117.8 2.9117.2 2.4310.5 6.1717.9 3.461,2<3**1<2<3***1<3 * 1,2<3**period of late elementary school9137.3 4.62139.6 5.7135.9 7.7634.8 6.7718.9 3.1817.6 2.2313.1 5.9617.8 4.5110143.9 5.80147.1 9.4245.2 11.9236.8 10.1121.6 4.1216.7 2.8818.2 7.8017.0 4.7611155.5 6.65154.5 2.8648.2 11.5946.8 6.1419.9 3.9919.6 2.2014.7 3.8221.4 5.171<2<3***1<2<3***1<3 * 1,2<3**period of middle school12164.9 6.43158.3 4.9149.3 10.8347.4 6.9519.5 2.9718.9 2.5913.1 8.4720.4 4.0813165.9 3.41159.1 3.3055.1 11.1250.8 6.4520.0 3.9320.3 2.1612.8 7.6021.0 5.8114169.6 5.17158.0 3.0864.9 10.9153.2 5.4421.7 1.7921.3 2.0716.8 3.9721.1 6.401,2<3*period of high school15176.5 7.30164.1 3.8160.3 7.9258.6 2.2419.3 1.4921.8 1.5311.5 4.8126.1 2.9416174.3 5.63162.5 5.8057.6 3.5453.1 5.8720.6 0.5720.0 1.4514.7 3.0024.2 2.3617172.8 4.01163.1 5.0162.4 8.1857.0 5.3220.9 2.3821.4 1.5715.5 5.8326.1 2.06values represent means  sd .", "bmi : body mass index . * : significant difference within \n group ( * p<0.05 , * * p<0.01 , * * * p<0.001 ) shows the differences in body composition between groups ( gender and age ) and \n within groups .", "height was significantly different in the early period of elementary school \n ( boys : p<0.01 , girls : p<0.001 ) and the late period of elementary school ( p<0.001 \n for both boys and girls ) .", "weight was significantly different in the periods of early and \n late elementary and middle school for boys ( p<0.05 ) , and in the periods of early and late \n elementary school for girls ( p<0.01 ) .", "* : significant difference within \n group ( * p<0.05 , * * p<0.01 , * * * p<0.001 ) table 2table 2.the differences in basic physical fitness between groups ( gender and age ) and \n within group ( n=240)groupsage ( yrs)basic physical fitnesspulmonary functionright hand grip ( kg)left hand grip ( kg)sit and reach ( cm)sargent jump ( cm ) single - leg stance ( sec)fvc ( l)fev1 ( l)boysperiod of early elementary school68.42.508.81.954.75.2823.23.808.46.071.360.321.300.20710.62.3610.52.678.05.8326.64.5615.911.371.730.451.540.4089.32.6010.92.594.44.8226.02.5910.97.181.840.351.720.44period of late elementary school912.51.4714.13.325.66.3528.13.6925.819.002.000.381.850.411017.82.7517.02.765.66.7230.15.6544.224.672.550.442.220.321119.14.1018.73.897.99.1738.56.5933.118.672.590.532.390.531<3***1<2,3 * 1,2<3**1<2,3 * 1<3*period of middle school1220.97.5221.89.112.29.6238.55.5226.317.333.430.703.190.621326.64.3029.94.385.27.6141.26.5626.222.533.540.563.530.291436.69.3937.97.137.89.1145.35.7530.813.514.270.493.840.511<2,3**1,2<3**1,2<3*period of high school1531.96.1232.67.117.05.6343.64.4131.425.804.180.654.010.571636.30.3538.50.8512.12.2543.50.5052.56.504.600.724.390.661736.83.0438.32.8611.59.5147.69.8236.422.784.120.703.850.65girlsperiod of early elementary school67.71.878.52.059.43.3122.14.1411.17.901.460.261.230.2178.81.589.21.9510.83.7825.96.0916.315.781.430.271.310.2789.31.829.33.456.13.7525.03.134.32.091.660.281.570.283<1*period of late elementary school910.23.0310.93.226.46.2327.94.4223.917.791.830.411.580.311012.72.6813.84.009.88.5430.04.8329.621.722.040.591.930.531116.83.5317.43.9910.87.4030.48.6735.422.812.540.392.330.341<3***1,2<3**1<3 * 1<3*period of middle school1218.92.5520.01.9313.48.5333.65.8512.65.732.630.442.490.371321.74.8322.64.8310.38.7329.82.5727.820.372.610.322.580.241420.12.7021.73.6311.78.3428.44.0337.422.332.510.572.300.50period of high school1523.62.4225.53.9910.911.3129.87.8023.319.162.780.372.680.371622.23.9324.14.0415.310.6629.64.1330.215.853.010.472.850.371720.23.2323.03.4110.311.7028.95.0524.76.582.920.212.810.16values represent means  sd .", "fvc : forced vital capacity , fev1 : forced expiratory \n volume in 1 second . * : significant difference within group ( * p<0.05 , * * p<0.01 , \n * * * p<0.001 ) shows the differences in basic physical fitness and pulmonary function between \n and within groups . for boys , right and left hand - grip strength were significantly different \n in the late period of elementary school ( right : p<0.001 , left : < 0.05 ) and middle \n school ( p<0.01 ) .", "the sargent jump height was only significantly different in the late \n period of elementary school ( p<0.01 ) for boys .", "in the case of girls , right and left \n hand - grip strength significantly different in the late period of elementary school \n ( p<0.01 ) , and the sit and reach distance was significantly different in the early period \n of elementary school ( p<0.01 ) .", "for boys , fvc was significantly different in the late \n period of elementary school and middle school ( p<0.05 ) , and fev1 was significantly \n different only in the late period of elementary school ( p<0.05 ) , and for girls , fvc and \n fev1 were significantly different in the late period of elementary school ( p<0.05 ) .", "fvc : forced vital capacity , fev1 : forced expiratory \n volume in 1 second . * : significant difference within group ( * p<0.05 , * * p<0.01 , \n * * * p<0.001 ) table 3table 3.the results of simple linear regression analysis between pulmonary function and \n body compositionfvcfev1rrboysheight0.827***0.758***weight0.677***0.658***bmi0.168***0.144***body fat ( % ) 0.048 * 0.037girlsheight0.756***0.764***weight0.728***0.733***bmi0.455***0.462***body fat ( % ) 0.392***0.400 * * * * p<0.05 , * * p<0.01 , * * * p<0.001 shows the results of the simple linear regression analysis that was performed \n for fvc and fev1 against height , weight , bmi , and body fat ( % ) . for boys ,", "fvc significantly \n correlated with height ( r=0.827 , p<0.001 ) , weight ( r=0.677 , \n p<0.001 ) , bmi ( r=0.168 , p<0.001 ) , and percent body fat \n ( r=0.048 , p<0.05 ) in descending order .", "fev1 significantly correlated with \n height ( r=0.758 , p<0.001 ) , weight ( r=0.658 , p<0.001 ) , and bmi \n ( r=0.144 , p<0.001 ) ; however , there was no significant correlation with \n percent body fat ( r=0.037 , p=0.077 ) . in the case of girls ,", "fvc significantly \n correlated with height ( r=0.756 , p<0.001 ) , weight ( r=0.728 , \n p<0.001 ) , bmi ( r=0.455 , p<0.001 ) , and percent body fat \n ( r=0.392 , p<0.001 ) in descending order .", "fev1 significantly correlated with \n height ( r=0.764 , p<0.001 ) , weight ( r=0.733 , p<0.001 ) , bmi \n ( r=0.462 , p<0.001 ) , and percent body fat ( r=0.400 , p<0.001 ) \n in descending order . * p<0.05 , * * p<0.01 , * * * p<0.001 the results of the comparison between boys and girls revealed that height and weight were \n similarly correlated .", "however , bmi ( fvc : 0.455 vs. 0.168 , fev1 : 0.462 vs. 0.144 ) and percent \n body fat ( fvc : 0.392 vs. 0.048 , fev1 : 0.400 vs. 0.037 ) showed higher correlations with \n pulmonary function in girls than in boys .", "table 4table 4.the results of simple linear regression analysis between pulmonary function and \n basic physical fitnessvariablefvcfev1rrboysright hand grip strength 0.774***0.794***left hand grip strength 0.747***0.762***sit and reach distance 0.0420.039sargent jump height0.573***0.584***single - leg standing time 0.058 * 0.048*girlsright hand grip strength 0.619***0.652***left hand grip strength 0.607***0.641***sit and reach distance 0.0200.015sargent jump height0.129**0.121**single - leg standing time 0.0180.020 * p<0.05 , * * p<0.01 , * * * p<0.001 shows the results of the simple linear regression analysis that was performed \n for fvc and fev1 against right hand - grip strength , left hand - grip strength , sit and reach \n distance , sargent jump height , and single - leg standing time .", "for boys , both fvc and fev1 \n showed high correlations with right hand - grip strength ( r=0.774 , \n r=0.794 , p<0.001 ) , left hand - grip strength ( r=0.747 , \n r=0.762 , p<0.001 ) , sargent jump height ( r=0.573 , \n r=0.584 , p<0.001 ) , and single - leg standing time ( r=0.058 , \n r=0.048 , p<0.05 ) in descending order . in the case of girls , both fvc and \n fev1 showed high correlations with right hand - grip strength ( r=0.619 , \n r=0.652 , p<0.001 ) , left hand - grip strength ( r=0.607 , \n r=0.641 , p<0.001 ) , and sargent jump height ( r=0.129 , \n r=0.121 , p<0.01 ) in descending order , whereas the sit and reach distance \n and single - leg standing time showed no significant correlations .", "* p<0.05 , * * p<0.01 , * * * p<0.001 the results of boys and girls were similarly in descending order .", "however , basic physical \n fitness parameters were more highly correlated with pulmonary function for boys than for \n girls , especially the sargent jump height ( fvc : 0.573 vs. 0.129 , fev1 : 0.584 vs. 0.121 ) .", "the purpose of this study was to determine the relationship between basic physical fitness \n and pulmonary function in healthy korean school students , in order to present an alternative \n method for improving their pulmonary function .", "an investigation of the development of korean students was conducted by the ministry of \n education in 2014 .", "the results show that boys grow 56  cm per year from an average height of 121.5  cm \n in the first grade of elementary school .", "the biggest growth was found among 6th grade \n elementary school students and 1st grade middle school students .", "girls grow around 6  cm per year from an average height of 120.3  cm \n in the first grade of elementary school .", "their degree of growth decreases rapidly after 6th \n grade elementary school compared to boys .", "body weight of boys showed the biggest differences \n between the first and second grades of middle school ( 5.9  kg ) , and between the 5th and 6th \n grades of elementary school among girls ( 5.5  kg ) .", "the results of the present study show that \n the height of both boys and girls rapidly increased in the elementary school period . \n", "however , weight dramatically increased in the period of elementary school and middle school \n among boys , and in the period of elementary school among girls .", "the growth of children and \n adolescents who participated in this study seems to be representative of korean children and \n adolescents since the results appear to be similar to the findings of the ministry of \n education .", "however , bmi was somewhat different since we selected healthy students , excluding \n obese children as subjects . basic physical fitness is closely related to health .", "especially , it has a negative \n correlation with the prevalence of obesity17 , hypertension18 , \n and cardiovascular disease19 . in this \n respect ,", "the korean government has implemented the physical activity promotion system ( paps ) \n in order to systematically measure the physique and fitness , as well as to prescribe \n physical activity for individuals , highlighting the need of regular exercise . however , \n accurate measurement , evaluation , and prescription of exercise seem insufficient . in the \n present study ,", "basic physical fitness was measured using the correct postures and methods by \n a fully trained technician .", "muscle strength increased sharply in the late period of \n elementary and middle school in boys , and in the late period of elementary school in girls . \n", "in addition , muscle power of boys increased sharply only in the late period of elementary \n school . pulmonary function", "was reported as being correlated with age20 , 21 , in addition , height , \n weight , area of body surface , percent body fat , smoking status , and residential environment \n also have effects on spirometry22,23,24,25 .", "height is a factor positively influencing \n lung function at all ages26 , whereas age \n and body fat mass sometimes have an inverse correlation in accordance with the age of \n subjects .", "the present study revealed that pulmonary function increased with age for subjects \n during the growth period .", "in addition , pulmonary function was influenced by height , weight , \n bmi , and percent body fat , in descending order .", "height , area of \n body surface , and weight correlate with pulmonary function in descending order27 , a result which is consistent with the \n results of our present study .", "girls showed higher correlation than boys between percent body \n fat and pulmonary function in the present study .", "however , the percent body fat showed slight \n or no correlation with pulmonary function for boys , a result which differs from those of \n previous studies .", "rossi et al.28 found \n that body fat and lung function showed an inverse correlation in obese female adults . \n", "gonzalez - barcala et al.29 conducted a \n study of children aged 6 to 18  years whose bmi was under 30  kg / m .", "they suggested \n that lung function can be difference depending on individual differences . in the present \n study , subjects had normal weight and bmi < 25  kg / m .", "park et al.30 reported that it was difficult to explain \n the effects of bmi , percent body fat , and muscle mass on pulmonary function .", "therefore , \n different results of pulmonary function could be occurred depending on the body composition \n of the individual subjects .", "thus , aerobic exercise and resistance \n exercise are effective ways of improving lung function . besides", "the present study \n revealed that pulmonary function is highly correlated with right hand - grip strength , left \n hand - grip strength , and sargent jump height in descending order .", "therefore , exercises for \n muscle strength and power could be effective at improving school students pulmonary \n function . in summary , in order to improve the pulmonary function of growing children and adolescents , \n", "aerobic exercise and exercise programs to increase muscle strength and power are needed , and \n they would be especially effective if they were to begin in the late period of elementary \n school when the muscle strength and power of students are rapidly increasing ."], "abstract_text": ["<S> [ purpose ] the purpose of the present study was to determine whether there was a \n correlation between basic physical fitness and pulmonary function in korean school \n students , to present an alternative method for improving their pulmonary function . \n </S>", "<S> [ subjects and methods ] two hundred forty healthy students aged 617  years performed \n physical fitness tests of hand - grip strength , sit and reach , sargent jump , single leg \n stance , and pulmonary function tests of forced vital capacity ( fvc ) and forced expiratory \n volume in one second ( fev1 ) using a quark pft . </S>", "<S> [ results ] muscle strength and power of boys \n improved in the late period of elementary school and middle school </S>", "<S> . muscle strength of \n girls improved in the late period of elementary school . </S>", "<S> analysis of factors affecting \n pulmonary function revealed that height , weight , bmi , and body fat significantly \n correlated with spirometric parameters . </S>", "<S> right hand - grip strength , left hand - grip strength , \n and sargent jump also significantly correlated with fvc and fev1 . </S>", "<S> [ conclusion ] in order to \n improve the pulmonary function of children and adolescents , aerobic exercise and an \n exercise program to increase muscle strength and power is needed , and it should start in \n the late period of elementary school when muscle strength and power are rapidly \n increasing . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["many problems occur while we are trying to improve the quality of life and material wealth \n by the development of modern society . among them", ", air pollution is a serious problem in \n western and developing countries . long - term exposure to air pollution has a high correlation \n with the incidence of cardiovascular disease , respiratory diseases , and diabetes1,2,3 , threatening health . in particular , air \n pollution is a major cause of respiratory diseases such as asthma and allergic diseases4 , 5 and \n it has a negative effect on the healthy growth and development of growing children and \n adolescents .", "another problem caused by the development of modern society is the reduction in physical \n activity across all generations throughout the world .", "the time spent in physical activity by \n children has decreased significantly compared to before6 , 7 .", "the time spent using \n visual media and watching tv has increased8 , 9 , and physical inactivity due to excessive \n academic pressure and the influence of parents10 , has increased obesity , incorrect posture , and muscle weakness . in \n addition , energy consumption has increased due to environmental , metabolic , and genetic \n factors11 .", "thus , children living in \n modern society are showing gradually decreasing physical fitness . the change in the environment due to", "the development of modern society is a threat to the \n health of growing children . according to the korea education development institute , \n", "respiratory system abnormalities have been much higher than those other systems over the \n last three years . reduced basic physical fitness and reduced pulmonary functions are \n seriously threating the physical development of korean elementary , middle , and high school \n students .", "although the physical fitness and lung health of growing children and adolescents is \n threatened , some studies have reported a relationship between air pollution and pulmonary \n function12 , 13 .", "however , there has been insufficient research on the relationship \n between exercise and lung function .", "most studies on exercise have only suggested lung \n function is improved by regular exercise14 , 15 .", "the present study is to our knowledge the \n first to investigate the relationship between basic physical fitness and lung function .", "the purpose of the present study was to determine the relationship between basic physical \n fitness and pulmonary function in healthy korean school students to enable us to present an \n alternative method for improving their pulmonary function ."], ["a total 240 healthy children and adolescents aged 617  years who lived in busan in korea \n were recruited for the present study .", "the participants had a body mass index ( bmi ) < \n 25  kg / m , and no respiratory system abnormalities .", "the sample comprised 20 \n healthy students ( 10 boys and 10 girls ) of each age from 6 to 17 .", "the participants who did \n not meet the bmi criteria or who showed significantly low pulmonary test values were \n excluded , and replacements were recruited in order to conduct the analysis with the same \n numbers of subjects .", "the participants were divided into the early period of elementary \n school ( 68 y ) , the late period of elementary school ( 911 y ) , the period of middle school \n ( 1214 y ) , and the period of high school ( 1517 y ) in order to investigate the rapid changes \n in body composition and basic physical fitness with growth .", "all participants gave their \n informed consent and the experimental protocol was approved by the ethical committee of \n dong - a university .", "body composition including height , weight , bmi , and body fat ( % ) was measured using a body \n composition analyzer venus 5.5 ( jawon medical , korea ) while subjects were fully relaxed . \n", "muscle strength , muscle power , flexibility , and balance were measured to evaluate basic \n physical fitness .", "all participants were given a full explanation about the correct posture \n and procedure of each measurement .", "hand - grip strength was evaluated using a grip - d grip strength dynamometer \n ( takei , japan ) with 0.1  kg accuracy of both the right and left hands .", "flexibility was \n evaluated by measuring sit and reach using a helmas iii trunk forward flexion instrument \n ( o2run , korea ) with 0.1  cm accuracy .", "muscle power was evaluated by measuring the sargent \n jump height using a helmas iii sargent jump instrument ( o2run , korea ) with 0.1  cm accuracy . \n", "balance was evaluated by measuring the eyes - closed single - leg standing time using a helmas \n iii blind single - leg stand instrument ( o2run , korea ) with 0.1 sec accuracy , and the \n participants performed the test using their preferred leg .", "pulmonary function tests were conducted under standard laboratory conditions ( temperature : \n 2225  c , relative humidity : 5560% ) .", "all spirometric tests were conducted by the same \n technician to reduce inter - observer variability and to prevent the failure of the \n measurement due to the young age of the subjects .", "the participants were given sufficient \n explanation about the method and instrument use , and the tests were performed in a sitting \n position while wearing a nose clip . the forced vital capacity ( fvc ) and the forced \n expiratory volume in one second ( fev1 ) were measured using a quark pft ( cosmed , italy ) .", "all \n of the pulmonary tests were conducted following the standards presented by the american \n thoracic society / european respiratory society16 .", "the data were analyzed using the statistical package for the social sciences ( spss version \n 22.00 ) and the results are presented as the mean  standard deviation .", "the significance of \n differences between boys and girls were examined using the independent t - test .", "the relationships between body composition and pulmonary \n function , and basic physical fitness and pulmonary function were analyzed using simple \n linear regression analysis ."], ["table 1table 1.the differences in body composition between groups ( gender and age ) and within \n group ( n=240)groupsage ( yrs)height ( cm)weight ( kg)bmi ( kg / m)body fat ( % ) malefemalemalefemalemalefemalemalefemaleperiod of early elementary school6123.7 4.43121.6 4.9624.8 4.5622.9 3.4816.1 2.0415.4 1.748.9 3.0113.0 5.067127.7 4.66126.2 3.9728.8 4.3726.2 5.0717.6 2.2016.4 2.619.6 4.4216.5 3.618133.0 4.57133.9 5.4631.7 6.6730.8 5.4117.8 2.9117.2 2.4310.5 6.1717.9 3.461,2<3**1<2<3***1<3 * 1,2<3**period of late elementary school9137.3 4.62139.6 5.7135.9 7.7634.8 6.7718.9 3.1817.6 2.2313.1 5.9617.8 4.5110143.9 5.80147.1 9.4245.2 11.9236.8 10.1121.6 4.1216.7 2.8818.2 7.8017.0 4.7611155.5 6.65154.5 2.8648.2 11.5946.8 6.1419.9 3.9919.6 2.2014.7 3.8221.4 5.171<2<3***1<2<3***1<3 * 1,2<3**period of middle school12164.9 6.43158.3 4.9149.3 10.8347.4 6.9519.5 2.9718.9 2.5913.1 8.4720.4 4.0813165.9 3.41159.1 3.3055.1 11.1250.8 6.4520.0 3.9320.3 2.1612.8 7.6021.0 5.8114169.6 5.17158.0 3.0864.9 10.9153.2 5.4421.7 1.7921.3 2.0716.8 3.9721.1 6.401,2<3*period of high school15176.5 7.30164.1 3.8160.3 7.9258.6 2.2419.3 1.4921.8 1.5311.5 4.8126.1 2.9416174.3 5.63162.5 5.8057.6 3.5453.1 5.8720.6 0.5720.0 1.4514.7 3.0024.2 2.3617172.8 4.01163.1 5.0162.4 8.1857.0 5.3220.9 2.3821.4 1.5715.5 5.8326.1 2.06values represent means  sd .", "bmi : body mass index . * : significant difference within \n group ( * p<0.05 , * * p<0.01 , * * * p<0.001 ) shows the differences in body composition between groups ( gender and age ) and \n within groups .", "height was significantly different in the early period of elementary school \n ( boys : p<0.01 , girls : p<0.001 ) and the late period of elementary school ( p<0.001 \n for both boys and girls ) .", "weight was significantly different in the periods of early and \n late elementary and middle school for boys ( p<0.05 ) , and in the periods of early and late \n elementary school for girls ( p<0.01 ) .", "* : significant difference within \n group ( * p<0.05 , * * p<0.01 , * * * p<0.001 ) table 2table 2.the differences in basic physical fitness between groups ( gender and age ) and \n within group ( n=240)groupsage ( yrs)basic physical fitnesspulmonary functionright hand grip ( kg)left hand grip ( kg)sit and reach ( cm)sargent jump ( cm ) single - leg stance ( sec)fvc ( l)fev1 ( l)boysperiod of early elementary school68.42.508.81.954.75.2823.23.808.46.071.360.321.300.20710.62.3610.52.678.05.8326.64.5615.911.371.730.451.540.4089.32.6010.92.594.44.8226.02.5910.97.181.840.351.720.44period of late elementary school912.51.4714.13.325.66.3528.13.6925.819.002.000.381.850.411017.82.7517.02.765.66.7230.15.6544.224.672.550.442.220.321119.14.1018.73.897.99.1738.56.5933.118.672.590.532.390.531<3***1<2,3 * 1,2<3**1<2,3 * 1<3*period of middle school1220.97.5221.89.112.29.6238.55.5226.317.333.430.703.190.621326.64.3029.94.385.27.6141.26.5626.222.533.540.563.530.291436.69.3937.97.137.89.1145.35.7530.813.514.270.493.840.511<2,3**1,2<3**1,2<3*period of high school1531.96.1232.67.117.05.6343.64.4131.425.804.180.654.010.571636.30.3538.50.8512.12.2543.50.5052.56.504.600.724.390.661736.83.0438.32.8611.59.5147.69.8236.422.784.120.703.850.65girlsperiod of early elementary school67.71.878.52.059.43.3122.14.1411.17.901.460.261.230.2178.81.589.21.9510.83.7825.96.0916.315.781.430.271.310.2789.31.829.33.456.13.7525.03.134.32.091.660.281.570.283<1*period of late elementary school910.23.0310.93.226.46.2327.94.4223.917.791.830.411.580.311012.72.6813.84.009.88.5430.04.8329.621.722.040.591.930.531116.83.5317.43.9910.87.4030.48.6735.422.812.540.392.330.341<3***1,2<3**1<3 * 1<3*period of middle school1218.92.5520.01.9313.48.5333.65.8512.65.732.630.442.490.371321.74.8322.64.8310.38.7329.82.5727.820.372.610.322.580.241420.12.7021.73.6311.78.3428.44.0337.422.332.510.572.300.50period of high school1523.62.4225.53.9910.911.3129.87.8023.319.162.780.372.680.371622.23.9324.14.0415.310.6629.64.1330.215.853.010.472.850.371720.23.2323.03.4110.311.7028.95.0524.76.582.920.212.810.16values represent means  sd .", "fvc : forced vital capacity , fev1 : forced expiratory \n volume in 1 second . * : significant difference within group ( * p<0.05 , * * p<0.01 , \n * * * p<0.001 ) shows the differences in basic physical fitness and pulmonary function between \n and within groups . for boys , right and left hand - grip strength were significantly different \n in the late period of elementary school ( right : p<0.001 , left : < 0.05 ) and middle \n school ( p<0.01 ) .", "the sargent jump height was only significantly different in the late \n period of elementary school ( p<0.01 ) for boys .", "in the case of girls , right and left \n hand - grip strength significantly different in the late period of elementary school \n ( p<0.01 ) , and the sit and reach distance was significantly different in the early period \n of elementary school ( p<0.01 ) .", "for boys , fvc was significantly different in the late \n period of elementary school and middle school ( p<0.05 ) , and fev1 was significantly \n different only in the late period of elementary school ( p<0.05 ) , and for girls , fvc and \n fev1 were significantly different in the late period of elementary school ( p<0.05 ) .", "fvc : forced vital capacity , fev1 : forced expiratory \n volume in 1 second . * : significant difference within group ( * p<0.05 , * * p<0.01 , \n * * * p<0.001 ) table 3table 3.the results of simple linear regression analysis between pulmonary function and \n body compositionfvcfev1rrboysheight0.827***0.758***weight0.677***0.658***bmi0.168***0.144***body fat ( % ) 0.048 * 0.037girlsheight0.756***0.764***weight0.728***0.733***bmi0.455***0.462***body fat ( % ) 0.392***0.400 * * * * p<0.05 , * * p<0.01 , * * * p<0.001 shows the results of the simple linear regression analysis that was performed \n for fvc and fev1 against height , weight , bmi , and body fat ( % ) . for boys ,", "fvc significantly \n correlated with height ( r=0.827 , p<0.001 ) , weight ( r=0.677 , \n p<0.001 ) , bmi ( r=0.168 , p<0.001 ) , and percent body fat \n ( r=0.048 , p<0.05 ) in descending order .", "fev1 significantly correlated with \n height ( r=0.758 , p<0.001 ) , weight ( r=0.658 , p<0.001 ) , and bmi \n ( r=0.144 , p<0.001 ) ; however , there was no significant correlation with \n percent body fat ( r=0.037 , p=0.077 ) . in the case of girls ,", "fvc significantly \n correlated with height ( r=0.756 , p<0.001 ) , weight ( r=0.728 , \n p<0.001 ) , bmi ( r=0.455 , p<0.001 ) , and percent body fat \n ( r=0.392 , p<0.001 ) in descending order .", "fev1 significantly correlated with \n height ( r=0.764 , p<0.001 ) , weight ( r=0.733 , p<0.001 ) , bmi \n ( r=0.462 , p<0.001 ) , and percent body fat ( r=0.400 , p<0.001 ) \n in descending order . * p<0.05 , * * p<0.01 , * * * p<0.001 the results of the comparison between boys and girls revealed that height and weight were \n similarly correlated .", "however , bmi ( fvc : 0.455 vs. 0.168 , fev1 : 0.462 vs. 0.144 ) and percent \n body fat ( fvc : 0.392 vs. 0.048 , fev1 : 0.400 vs. 0.037 ) showed higher correlations with \n pulmonary function in girls than in boys .", "table 4table 4.the results of simple linear regression analysis between pulmonary function and \n basic physical fitnessvariablefvcfev1rrboysright hand grip strength 0.774***0.794***left hand grip strength 0.747***0.762***sit and reach distance 0.0420.039sargent jump height0.573***0.584***single - leg standing time 0.058 * 0.048*girlsright hand grip strength 0.619***0.652***left hand grip strength 0.607***0.641***sit and reach distance 0.0200.015sargent jump height0.129**0.121**single - leg standing time 0.0180.020 * p<0.05 , * * p<0.01 , * * * p<0.001 shows the results of the simple linear regression analysis that was performed \n for fvc and fev1 against right hand - grip strength , left hand - grip strength , sit and reach \n distance , sargent jump height , and single - leg standing time .", "for boys , both fvc and fev1 \n showed high correlations with right hand - grip strength ( r=0.774 , \n r=0.794 , p<0.001 ) , left hand - grip strength ( r=0.747 , \n r=0.762 , p<0.001 ) , sargent jump height ( r=0.573 , \n r=0.584 , p<0.001 ) , and single - leg standing time ( r=0.058 , \n r=0.048 , p<0.05 ) in descending order . in the case of girls , both fvc and \n fev1 showed high correlations with right hand - grip strength ( r=0.619 , \n r=0.652 , p<0.001 ) , left hand - grip strength ( r=0.607 , \n r=0.641 , p<0.001 ) , and sargent jump height ( r=0.129 , \n r=0.121 , p<0.01 ) in descending order , whereas the sit and reach distance \n and single - leg standing time showed no significant correlations .", "* p<0.05 , * * p<0.01 , * * * p<0.001 the results of boys and girls were similarly in descending order .", "however , basic physical \n fitness parameters were more highly correlated with pulmonary function for boys than for \n girls , especially the sargent jump height ( fvc : 0.573 vs. 0.129 , fev1 : 0.584 vs. 0.121 ) ."], ["the purpose of this study was to determine the relationship between basic physical fitness \n and pulmonary function in healthy korean school students , in order to present an alternative \n method for improving their pulmonary function .", "an investigation of the development of korean students was conducted by the ministry of \n education in 2014 .", "the results show that boys grow 56  cm per year from an average height of 121.5  cm \n in the first grade of elementary school .", "the biggest growth was found among 6th grade \n elementary school students and 1st grade middle school students .", "girls grow around 6  cm per year from an average height of 120.3  cm \n in the first grade of elementary school .", "their degree of growth decreases rapidly after 6th \n grade elementary school compared to boys .", "body weight of boys showed the biggest differences \n between the first and second grades of middle school ( 5.9  kg ) , and between the 5th and 6th \n grades of elementary school among girls ( 5.5  kg ) .", "the results of the present study show that \n the height of both boys and girls rapidly increased in the elementary school period . \n", "however , weight dramatically increased in the period of elementary school and middle school \n among boys , and in the period of elementary school among girls .", "the growth of children and \n adolescents who participated in this study seems to be representative of korean children and \n adolescents since the results appear to be similar to the findings of the ministry of \n education .", "however , bmi was somewhat different since we selected healthy students , excluding \n obese children as subjects . basic physical fitness is closely related to health .", "especially , it has a negative \n correlation with the prevalence of obesity17 , hypertension18 , \n and cardiovascular disease19 . in this \n respect ,", "the korean government has implemented the physical activity promotion system ( paps ) \n in order to systematically measure the physique and fitness , as well as to prescribe \n physical activity for individuals , highlighting the need of regular exercise . however , \n accurate measurement , evaluation , and prescription of exercise seem insufficient . in the \n present study ,", "basic physical fitness was measured using the correct postures and methods by \n a fully trained technician .", "muscle strength increased sharply in the late period of \n elementary and middle school in boys , and in the late period of elementary school in girls . \n", "in addition , muscle power of boys increased sharply only in the late period of elementary \n school . pulmonary function", "was reported as being correlated with age20 , 21 , in addition , height , \n weight , area of body surface , percent body fat , smoking status , and residential environment \n also have effects on spirometry22,23,24,25 .", "height is a factor positively influencing \n lung function at all ages26 , whereas age \n and body fat mass sometimes have an inverse correlation in accordance with the age of \n subjects .", "the present study revealed that pulmonary function increased with age for subjects \n during the growth period .", "in addition , pulmonary function was influenced by height , weight , \n bmi , and percent body fat , in descending order .", "height , area of \n body surface , and weight correlate with pulmonary function in descending order27 , a result which is consistent with the \n results of our present study .", "girls showed higher correlation than boys between percent body \n fat and pulmonary function in the present study .", "however , the percent body fat showed slight \n or no correlation with pulmonary function for boys , a result which differs from those of \n previous studies .", "rossi et al.28 found \n that body fat and lung function showed an inverse correlation in obese female adults . \n", "gonzalez - barcala et al.29 conducted a \n study of children aged 6 to 18  years whose bmi was under 30  kg / m .", "they suggested \n that lung function can be difference depending on individual differences . in the present \n study , subjects had normal weight and bmi < 25  kg / m .", "park et al.30 reported that it was difficult to explain \n the effects of bmi , percent body fat , and muscle mass on pulmonary function .", "therefore , \n different results of pulmonary function could be occurred depending on the body composition \n of the individual subjects .", "thus , aerobic exercise and resistance \n exercise are effective ways of improving lung function . besides", "the present study \n revealed that pulmonary function is highly correlated with right hand - grip strength , left \n hand - grip strength , and sargent jump height in descending order .", "therefore , exercises for \n muscle strength and power could be effective at improving school students pulmonary \n function . in summary , in order to improve the pulmonary function of growing children and adolescents , \n", "aerobic exercise and exercise programs to increase muscle strength and power are needed , and \n they would be especially effective if they were to begin in the late period of elementary \n school when the muscle strength and power of students are rapidly increasing ."]]}
{"article_id": "PMC55320", "article_text": ["the idea of a constitutively expressed death machinery in each cell has given way to the notion that survival factors repress this machinery and , if such factors are unavailable , cells default into death [ 1 , 2 , 3 ] . this theory is supported by findings showing that many forms of programmed cell death do not require mrna or protein synthesis .", "in fact , mrna and protein synthesis inhibitors can induce apoptosis , suggesting that in some cases transcriptional activity might actually impede cell death [ 4 , 5 ] . to identify genes that are transcriptionally regulated in cells undergoing apoptosis by survival factor deprivation", "gene trapping involves introduction of a reporter gene into a random collection of chromosomal sites , including transcriptionally active regions . by selecting for gene expression ,", "recombinants are obtained in which the reporter gene is fused to the regulatory elements of an endogenous gene .", "transcripts generated by these fusions faithfully reflect the activity of a disrupted cellular gene and serve as a molecular tag to clone any gene linked to a specific function [ 6 , 7 ] . to identify genes that are transiently expressed during a biological process", ", we developed a strategy , which makes use of the site - specific recombination system cre / loxp . by combining gene trap mutagenesis with site - specific recombination", ", it is possible to uncouple a trapped cellular promoter from a transduced reporter gene .", "this enables the recovery of recombinants even in the absence of an active cellular promoter and thus allows selection for integrations into transiently expressed genes [ 8 , 9 ] .", "we employed this strategy to isolate genes induced in hematopoietic cells ( fdcp1 ) undergoing apoptosis by growth factor withdrawal .", "briefly , the interleukin-3 ( il-3)-dependent hematopoietic cells ( floxil3 ) expressing a reporter plasmid encoding hsv - thymidine - kinase , neomycin - phosphotransferase and murine il-3 , were transduced with a retroviral gene trap vector carrying coding sequences for cre recombinase ( cre ) in the u3 region .", "activation of cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the floxil3 cells to factor independence .", "selection for autonomous growth yielded recombinants in which cre sequences in the u3 region were expressed from upstream cellular promoters . as", "the expression of the marker genes is independent of the trapped cellular promoter , genes could be identified that were transiently induced by il-3 withdrawal ( figure 1 ) .", "( a ) u3cre gene trap activation from integrations in genes induced by il-3 withdrawal excises the tkneo fusion gene , which is flanked by loxp sites from the reporter plasmid pgklxtkneoil3 .", "this places the il-3 cdna immediately downstream of the pgk promoter and enables its expression .", "cre , cre recombinase ; pol ii , rna polymerase ii ; pgk , phosphoglycerate kinase promoter ; lx , loxp target sequence for cre recombinase ; tkneo , hsv - thymidine - kinase / neomycin - phosphotransferase fusion gene ; pa , bovine growth hormone polyadenylation sequence ; il-3 , murine interleukin-3 cdna .", "u3cre - infected floxil3 cells were first selected in g418 to eliminate integrations into constitutively expressed genes .", "the g418-resistant cells were then plated into semisolid ( agar ) cultures in the absence of il-3 .", "analysis of several recombinants obtained by this method indicated that cells upregulate survival genes before committing to cell death . as a result , cells receiving a transient apoptotic stimulus exhibit improved survival in a growth factor deficient environment .", "from a floxil3 gene trap integration library consisting of approximately 2  10 unique proviral integrations , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal .", "cellular sequences adjacent to u3cre integration sites ( gene trap sequence tags , gtsts ) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers .", "ninety clones yielded informative gtsts ( > 30 nucleotides ) , of which 15 ( 17% ) belonged to known genes , 10 ( 11% ) matched single ests or full cdnas with unknown function and 65 ( 72% ) had no match within the public databases ( figure 2 ) .", "interestingly , the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector .", "for example , only 782 ( 46% ) of 1,687 gtsts recovered from embryonic stem ( es ) cells expressing a u3 geo gene trap virus were novel .", "this difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases .", "figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat ( ltr ) , most integrations occurred in or near 5 ' exons , although integrations further downstream were observed occasionally . to confirm transcriptional induction by il-3 withdrawal", ", polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes .", "( a ) gene trap position in previously characterized genes . gtsts were either obtained by 5'race or inverse genomic pcr .", "exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers .", "polyadenylated rnas ( 5 g per lane ) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels , blotted onto nylon filters and hybridized to p -labeled gene- or exon ( net1)-specific probes .", "as summarized in table 1 , a significant number of the trapped known genes encode proteins involved in cell growth and survival .", "for example , the guanine nucleotide exchange factors ( gefs ) for the rho - family gtpases net1 and h - pem2 ( kiaa0424 ) [ 10 , 11 ] belong to a large family of proteins that regulate cell growth and can transform cells in culture .", "transformation is dependent on the highly conserved dbl homology ( dh ) domain and in most cases requires amino - terminal truncation of the protein [ 13 , 14 ] .", "interestingly , a novel splice variant of net1 , net1a , which was isolated in these experiments , encodes an amino - terminally truncated protein that is transforming in its native form ( table 1 ) .", "thus , when overexpressed in nih3t3 fibroblasts , net1a but not net1 produces transformed foci with typical rho morphology ( f.w . and h.v.m .", "in addition to the dh domain , rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase ( pi3 kinase ) signal transduction pathway .", "the latter is responsible for mediating survival signals , particularly in hematopoietic cells , and is directly activated by il-3 .", "surprisingly , two members of this pathway , the phosphatidylinositol 4/5 phosphate kinases ( pip5 kinases ) type i ( which corresponds to human pip5 kinase type i ) and type i , were induced by il-3 withdrawal ( table 1 ) .", "pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate ( pip2 ) , which enhances cell survival by at least three mechanisms .", "moreover , overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- ( tnf- ) .", "second , pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate ( pip3 ) , which activates the survival kinase akt .", "akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad , caspase 9 and the forkhead family transcription factor fkhrl1 , which induces the expression of fas ligand [ 17 , 18 , 19 , 20 ] .", "third , pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane .", "as shown previously , apoptotic signals can cause membrane depolarization , which leads to cell death by directly inhibiting potassium channels .", "summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 ( ebp1 ) .", "although less well characterized than the above proteins , p38-g4 is absent from go cells and may stimulate cell - cycle progression [ 23 , 24 ] .", "it includes the il-12 receptor , which inhibits fas - mediated apoptosis in certain types of t lymphocytes   and the transcription factor yb-1 , which represses fas gene expression .", "interestingly , yb-1 also activates the expression of the multidrug resistance gene ( mdr1 ) whose product , the p glycoprotein , is an efflux pump that eliminates a variety of toxins from the cell , including proapoptotic anticancer drugs .", "similarly cytoprotective is vmat2 , the protein product of the vesicular monoamine transporter gene , which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [ 28 , 29 ] . finally , il-3 withdrawal induced transcription of several dna repair genes .", "these were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes .", "while alkb protects dna from damage by alkylating agents , rec8 and rad50 are dna double - strand break repair proteins [ 31 , 32 , 33 ] .", "only a few genes upregulated by il-3 withdrawal ( 4 out of 15 ) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis ( table 1 ) . however , the low frequency with which such genes were recovered underscores the specificity of the gene trap approach . to further study transcriptional regulation during growth factor starvation , we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays ( clontech ) .", "atlas arrays contain cdna probes for 588 genes involved in development , oncogenesis , dna repair , tumor suppression and apoptosis .", "figure 4a shows that of the 588 genes displayed on the arrays , only 12 were differentially expressed after factor deprivation .", "differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting ( figure 4c ) .", "( a ) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h ( left ) and 2 h ( right ) of il-3 deprivation .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimage 1.5 software .", "( b ) relative changes in gene expression on a log scale . 1 , no regulation ; >", "1 , upregulation ; < 1 , downregulation . note that the apparent downregulation of bcl-2 could not be confirmed on northern blots .", "induction of c - kit and chop10 ( asterisked ) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation .", "( c ) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software .", "poly(a ) rna ( 2.5 g ) prepared after 0 , 2 , 4 , 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels , blotted onto nylon filters and hybridized with p - labeled gene - specific probes .", "similar to the trapping results , several genes with survival functions were upregulated in the absence of il-3 . among these", "were the survival kinase akt , the c - kit receptor tyrosine kinase , the potent inhibitor of apoptosis flipl , and the il-3-receptor itself [ 34 , 35 , 36 ] .", "moreover , survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [ 37 , 38 ] . in confirmation of previous results , however , the expression of several survival genes appeared il-3 dependent .", "thus , transcripts for antiapoptotic pim-1 , c - myc and bcl - xl proteins were repressed in the absence of il-3 .", "finally , only two genes with arguably proapoptotic functions ( that is , jund and chop10 ) were upregulated in these experiments ( figure 4c ) .", "taken together , the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death .", "the activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival .", "accordingly , a transient factor deprivation should , by upregulating anti - apoptotic gene expression , reduce the fdcp1 cell 's need for il-3 . to test this", ", we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 .", "figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts , indicating that survival mechanisms were induced by factor deprivation . whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation , it gradually subsided with prolonged il-3 deprivation .", "after incubation for 0 , 1 , 2 and 4 h , il-3 was added at the indicated concentrations .", "1  10 cells were deprived of il-3 for 1 and 2 h , respectively .", "after re - adding il-3 for 4 h , cells were washed and incubated without il-3 .", "after 6 , 9 and 12 h factor deprivation , the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry .", ", we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . to this end , il-3 was initially withdrawn from fdcp1 cells for 1 or 2 hours .", "subsequently , il-3 was re - added for 4 hours only to be removed again for 6 , 9 and 12 hours . at these time points cells", "figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts , indicating that apoptotic prestimulation increases resistance to factor deprivation . like the clonal survival assays , the cell protective effect was highest after a prestimulation of 2 hours . on the basis of these results ,", "we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death .", "from a floxil3 gene trap integration library consisting of approximately 2  10 unique proviral integrations , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal .", "cellular sequences adjacent to u3cre integration sites ( gene trap sequence tags , gtsts ) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers .", "ninety clones yielded informative gtsts ( > 30 nucleotides ) , of which 15 ( 17% ) belonged to known genes , 10 ( 11% ) matched single ests or full cdnas with unknown function and 65 ( 72% ) had no match within the public databases ( figure 2 ) .", "interestingly , the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector .", "for example , only 782 ( 46% ) of 1,687 gtsts recovered from embryonic stem ( es ) cells expressing a u3 geo gene trap virus were novel .", "this difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases .", "figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat ( ltr )", ", most integrations occurred in or near 5 ' exons , although integrations further downstream were observed occasionally . to confirm transcriptional induction by il-3 withdrawal", ", polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes .", "( a ) gene trap position in previously characterized genes . gtsts were either obtained by 5'race or inverse genomic pcr .", "exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers .", "polyadenylated rnas ( 5 g per lane ) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels , blotted onto nylon filters and hybridized to p -labeled gene- or exon ( net1)-specific probes .", "as summarized in table 1 , a significant number of the trapped known genes encode proteins involved in cell growth and survival .", "for example , the guanine nucleotide exchange factors ( gefs ) for the rho - family gtpases net1 and h - pem2 ( kiaa0424 ) [ 10 , 11 ] belong to a large family of proteins that regulate cell growth and can transform cells in culture .", "transformation is dependent on the highly conserved dbl homology ( dh ) domain and in most cases requires amino - terminal truncation of the protein [ 13 , 14 ] .", "interestingly , a novel splice variant of net1 , net1a , which was isolated in these experiments , encodes an amino - terminally truncated protein that is transforming in its native form ( table 1 ) .", "thus , when overexpressed in nih3t3 fibroblasts , net1a but not net1 produces transformed foci with typical rho morphology ( f.w . and h.v.m . , unpublished data ) .", "in addition to the dh domain , rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase ( pi3 kinase ) signal transduction pathway .", "the latter is responsible for mediating survival signals , particularly in hematopoietic cells , and is directly activated by il-3 .", "surprisingly , two members of this pathway , the phosphatidylinositol 4/5 phosphate kinases ( pip5 kinases ) type i ( which corresponds to human pip5 kinase type i ) and type i , were induced by il-3 withdrawal ( table 1 ) .", "pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate ( pip2 ) , which enhances cell survival by at least three mechanisms .", "moreover , overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- ( tnf- ) .", "second , pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate ( pip3 ) , which activates the survival kinase akt .", "akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad , caspase 9 and the forkhead family transcription factor fkhrl1 , which induces the expression of fas ligand [ 17 , 18 , 19 , 20 ] .", "third , pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane .", "as shown previously , apoptotic signals can cause membrane depolarization , which leads to cell death by directly inhibiting potassium channels .", "summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 ( ebp1 ) .", "although less well characterized than the above proteins , p38-g4 is absent from go cells and may stimulate cell - cycle progression [ 23 , 24 ] .", "it includes the il-12 receptor , which inhibits fas - mediated apoptosis in certain types of t lymphocytes   and the transcription factor yb-1 , which represses fas gene expression .", "interestingly , yb-1 also activates the expression of the multidrug resistance gene ( mdr1 ) whose product , the p glycoprotein , is an efflux pump that eliminates a variety of toxins from the cell , including proapoptotic anticancer drugs .", "similarly cytoprotective is vmat2 , the protein product of the vesicular monoamine transporter gene , which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [ 28 , 29 ] . finally , il-3 withdrawal induced transcription of several dna repair genes .", "these were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes .", "while alkb protects dna from damage by alkylating agents , rec8 and rad50 are dna double - strand break repair proteins [ 31 , 32 , 33 ] .", "only a few genes upregulated by il-3 withdrawal ( 4 out of 15 ) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis ( table 1 ) . however , the low frequency with which such genes were recovered underscores the specificity of the gene trap approach .", "to further study transcriptional regulation during growth factor starvation , we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays ( clontech ) .", "atlas arrays contain cdna probes for 588 genes involved in development , oncogenesis , dna repair , tumor suppression and apoptosis .", "figure 4a shows that of the 588 genes displayed on the arrays , only 12 were differentially expressed after factor deprivation .", "differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting ( figure 4c ) .", "( a ) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h ( left ) and 2 h ( right ) of il-3 deprivation .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimage 1.5 software .", "( b ) relative changes in gene expression on a log scale . 1 , no regulation ; > 1 , upregulation ; < 1 , downregulation . note that the apparent downregulation of bcl-2 could not be confirmed on northern blots .", "induction of c - kit and chop10 ( asterisked ) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation .", "( c ) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software .", "poly(a ) rna ( 2.5 g ) prepared after 0 , 2 , 4 , 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels , blotted onto nylon filters and hybridized with p - labeled gene - specific probes .", "similar to the trapping results , several genes with survival functions were upregulated in the absence of il-3 . among these", "were the survival kinase akt , the c - kit receptor tyrosine kinase , the potent inhibitor of apoptosis flipl , and the il-3-receptor itself [ 34 , 35 , 36 ] .", "moreover , survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [ 37 , 38 ] . in confirmation of previous results , however , the expression of several survival genes appeared il-3 dependent .", "thus , transcripts for antiapoptotic pim-1 , c - myc and bcl - xl proteins were repressed in the absence of il-3 .", "finally , only two genes with arguably proapoptotic functions ( that is , jund and chop10 ) were upregulated in these experiments ( figure 4c ) . taken together", ", the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death .", "the activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival .", "accordingly , a transient factor deprivation should , by upregulating anti - apoptotic gene expression , reduce the fdcp1 cell 's need for il-3 . to test this", ", we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 .", "figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts , indicating that survival mechanisms were induced by factor deprivation . whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation , it gradually subsided with prolonged il-3 deprivation .", "after incubation for 0 , 1 , 2 and 4 h , il-3 was added at the indicated concentrations .", "1  10 cells were deprived of il-3 for 1 and 2 h , respectively .", "after re - adding il-3 for 4 h , cells were washed and incubated without il-3 .", "after 6 , 9 and 12 h factor deprivation , the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry .", ", we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . to this end", "subsequently , il-3 was re - added for 4 hours only to be removed again for 6 , 9 and 12 hours . at these time points cells were stained with annexin and apoptosis was quantified by flow cytometry .", "figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts , indicating that apoptotic prestimulation increases resistance to factor deprivation . like the clonal survival assays , the cell protective effect was highest after a prestimulation of 2 hours . on the basis of these results ,", "we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death .", "a gene trap strategy was used to identify genes induced in hematopoietic cells undergoing apoptosis by growth factor withdrawal .", "this approach enables identification of transiently expressed genes that are difficult to isolate by standard methodology .", "a comparatively large proportion of unknown genes were recovered , which underscores the strategy 's potential for isolating novel genes .", "in contrast to conventional gene trapping , which tags and disrupts random genes , the cre / loxp approach allows enrichment for genes induced by specific biological stimuli .", "as most regulatory genes are expressed in a temporally and spatially restricted manner , we believe that the combination of gene trap mutagenesis and site - specific recombination provides a sound alternative for functional gene analysis in the post - genomic era . as judged by the small fraction of recombinants recovered after growth factor deprivation ( that is , 124 of 2  10 or 0.006% )   and by the identity of the trapped", "known genes , the strategy seems highly specific for regulatory genes induced during programmed cell death .", "accordingly , the majority of genes upregulated by il-3 withdrawal were associated with cell death and survival .", "although this was similar on the cdna arrays , the differentially expressed genes recovered with the two methods were quite different .", "thus , with the exception of yb1 and rad50 , none of the genes displayed on the arrays were tagged by gene trap mutagenesis .", "moreover , yb1 and rad50 , despite being recovered in the gene trap approach as induced by il-3 withdrawal , appeared constitutively expressed or even downregulated on both arrays and northern blots , respectively ( figures 3,4 ) .", "first , the integration library used here covered only 25% of the genome . as only seven genes were induced on the arrays ( those encoding akt , il-3 receptor , glutathione - s - transferase , flipl , c - kit , jund and chop10 ) their recovery from an unsaturated library was unlikely .", "second , transient gene trapping as opposed to cdna hybridization has no bias towards highly expressed genes .", "third , the gene trap strategy selects for real gene inductions and , unlike the arrays and northern blots , is independent of pre - existing steady - state mrna levels subjected to post - transcriptional regulation . taken together", ", these considerations suggest that for the functional analysis of the mammalian genome , gene trapping effectively complements cdna - based strategies , including cdna arrays , which are unable to distinguish between transcriptional and/or post - transcriptional changes in gene expression . in terms of apoptosis", "whereas in most cells conflicts between prosurvival and apoptotic signals are carried out post - translationally by well characterized proteins ( for example , bcl-2 family members , caspases ) , there is increasing evidence for transcriptional regulators of apoptosis capable of tilting the balance between the constitutively expressed pro- and anti - apoptotic proteins [ 20 , 41 ] .", "these transcriptional regulators of apoptosis - still largely unknown - are likely to confer tissue specificity on the apoptotic process .", "the identity of such regulators is of considerable interest as they could provide valuable targets for prospective anti - neoplastic and/or anti - degenerative drugs .", "most genes identified in this study encode cell protective and/or pro - survival functions . in line with this ,", "the biological experiments described here have shown such functions to materialize in cells exposed to apoptotic prestimulation .", "accordingly , cells receiving stimulation exhibited improved survival and reduced apoptosis in a growth factor deprived environment .", "interestingly , a similar cytoprotective phenomenon known as ischemic preconditioning has been observed in animal models . in this , a short , sublethal period of ischemia induces profound resistance to subsequent ischemic events .", "thus , induction of anti - apoptotic gene expression prior to a lethal stimulus seems to raise the threshold required for that stimulus to be effective .", "this provides an additional safety mechanism that can prevent unwanted loss of cells exposed only accidentally to apoptotic stimulation . in summary", ", the present experiments have shown that hematopoietic cells undergoing apoptosis by il-3 withdrawal activate survival genes that do impede cell death .", "this suggests that apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals , rather than a simple death by default .", "inverse pcr from genomic dnas was performed using the cre - specific primers described previously   and a combination of the blunt end restriction enzymes sspi and hincii .", "5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer 's instructions .", "amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector ( promega ) as described previously .", "fdcp1 cells were propagated at concentrations of 2  10 cells / ml in dulbecco 's modified eagle 's medium ( dmem ; gibco ) , supplemented with 10% ( v / v ) fetal bovine serum ( boehringer - mannheim ) and 5 ng / ml recombinant mouse il-3 ( peprotech ) unless indicated otherwise .", "agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% ( v / v ) fetal bovine serum and 0.6% ( w / v ) bacto - agar ( difco ) in double distilled water as previously described .", "apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit ( roche ) and the manufacturer 's instructions .", "poly(a ) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array ( clontech ) according to the manufacturer 's instructions .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimagetm 1.5 software ( clontech ) . for northern blots , 2.5 g of poly(a )", "was fractionated in 1% formaldehyde - agarose gels , transferred onto hybond n membranes ( amersham ) and hybridized to specific p - dctp - labeled probes produced by random priming ( rediprime , amersham ) .", "cellular sequences adjacent to proviral integrations ( gene trap sequence tags ; gtsts ) were searched in the ncbi / nih genomic databases   using the blastn algorithm .", "inverse pcr from genomic dnas was performed using the cre - specific primers described previously   and a combination of the blunt end restriction enzymes sspi and hincii .", "5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer 's instructions .", "amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector ( promega ) as described previously .", "fdcp1 cells were propagated at concentrations of 2  10 cells / ml in dulbecco 's modified eagle 's medium ( dmem ; gibco ) , supplemented with 10% ( v / v ) fetal bovine serum ( boehringer - mannheim ) and 5 ng / ml recombinant mouse il-3 ( peprotech ) unless indicated otherwise .", "agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% ( v / v ) fetal bovine serum and 0.6% ( w / v ) bacto - agar ( difco ) in double distilled water as previously described .", "apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit ( roche ) and the manufacturer 's instructions .", "poly(a ) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array ( clontech ) according to the manufacturer 's instructions .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimagetm 1.5 software ( clontech ) . for northern blots , 2.5 g of poly(a )", "was fractionated in 1% formaldehyde - agarose gels , transferred onto hybond n membranes ( amersham ) and hybridized to specific p - dctp - labeled probes produced by random priming ( rediprime , amersham ) .", "cellular sequences adjacent to proviral integrations ( gene trap sequence tags ; gtsts ) were searched in the ncbi / nih genomic databases   using the blastn algorithm .", "this work was supported in part by grants from deutsche krebshilfe and the deutsche forschungsgemeinschaft , bonn to h.v.m and by schering ag , berlin ."], "abstract_text": ["<S> backgroundthe existence of a constitutively expressed machinery for death in individual cells has led to the notion that survival factors repress this machinery and , if such factors are unavailable , cells die by default . in many cells , however , mrna and protein synthesis inhibitors induce apoptosis , suggesting that in some cases transcriptional activity might actually impede cell death . </S>", "<S> to identify transcriptional mechanisms that interfere with cell death and survival , we combined gene trap mutagenesis with site - specific recombination ( cre / loxp system ) to isolate genes from cells undergoing apoptosis by growth factor deprivation.resultsfrom an integration library consisting of approximately 2  106 unique proviral integrations obtained by infecting the interleukin-3 ( il-3)-dependent hematopoietic cell line - floxil3 - with u3cre gene trap virus , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal . </S>", "<S> of 102 cellular sequences adjacent to u3cre integration sites , 17% belonged to known genes , 11% matched single expressed sequence tags ( ests ) or full cdnas with unknown function and 72% had no match within the public databases . </S>", "<S> most of the known genes recovered in this analysis encoded proteins with survival functions.conclusionswe have shown that hematopoietic cells undergoing apoptosis after withdrawal of il-3 activate survival genes that impede cell death . </S>", "<S> this results in reduced apoptosis and improved survival of cells treated with a transient apoptotic stimulus . </S>", "<S> thus , apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals , rather than a simple death by default . </S>"], "labels": null, "section_names": ["Background", "Results", "A majority of gene trap sequence tags recovered following IL-3 withdrawal correspond to unknown genes", "Most genes induced by IL-3 withdrawal exhibit cell-protective functions", "Mouse cDNA expression arrays reveal differential expression of cell death and survival genes following IL-3 withdrawal", "Apoptotic prestimulation improves survival in the absence of IL-3", "Discussion", "Materials and methods", "Amplification and cloning of upstream sequences", "Cell cultures and apoptosis assays", "Mouse cDNA expression arrays and northern blot hybridizations", "Database searches", "Acknowledgements"], "sections": [["the idea of a constitutively expressed death machinery in each cell has given way to the notion that survival factors repress this machinery and , if such factors are unavailable , cells default into death [ 1 , 2 , 3 ] . this theory is supported by findings showing that many forms of programmed cell death do not require mrna or protein synthesis .", "in fact , mrna and protein synthesis inhibitors can induce apoptosis , suggesting that in some cases transcriptional activity might actually impede cell death [ 4 , 5 ] . to identify genes that are transcriptionally regulated in cells undergoing apoptosis by survival factor deprivation", "gene trapping involves introduction of a reporter gene into a random collection of chromosomal sites , including transcriptionally active regions . by selecting for gene expression ,", "recombinants are obtained in which the reporter gene is fused to the regulatory elements of an endogenous gene .", "transcripts generated by these fusions faithfully reflect the activity of a disrupted cellular gene and serve as a molecular tag to clone any gene linked to a specific function [ 6 , 7 ] . to identify genes that are transiently expressed during a biological process", ", we developed a strategy , which makes use of the site - specific recombination system cre / loxp . by combining gene trap mutagenesis with site - specific recombination", ", it is possible to uncouple a trapped cellular promoter from a transduced reporter gene .", "this enables the recovery of recombinants even in the absence of an active cellular promoter and thus allows selection for integrations into transiently expressed genes [ 8 , 9 ] .", "we employed this strategy to isolate genes induced in hematopoietic cells ( fdcp1 ) undergoing apoptosis by growth factor withdrawal .", "briefly , the interleukin-3 ( il-3)-dependent hematopoietic cells ( floxil3 ) expressing a reporter plasmid encoding hsv - thymidine - kinase , neomycin - phosphotransferase and murine il-3 , were transduced with a retroviral gene trap vector carrying coding sequences for cre recombinase ( cre ) in the u3 region .", "activation of cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the floxil3 cells to factor independence .", "selection for autonomous growth yielded recombinants in which cre sequences in the u3 region were expressed from upstream cellular promoters . as", "the expression of the marker genes is independent of the trapped cellular promoter , genes could be identified that were transiently induced by il-3 withdrawal ( figure 1 ) .", "( a ) u3cre gene trap activation from integrations in genes induced by il-3 withdrawal excises the tkneo fusion gene , which is flanked by loxp sites from the reporter plasmid pgklxtkneoil3 .", "this places the il-3 cdna immediately downstream of the pgk promoter and enables its expression .", "cre , cre recombinase ; pol ii , rna polymerase ii ; pgk , phosphoglycerate kinase promoter ; lx , loxp target sequence for cre recombinase ; tkneo , hsv - thymidine - kinase / neomycin - phosphotransferase fusion gene ; pa , bovine growth hormone polyadenylation sequence ; il-3 , murine interleukin-3 cdna .", "u3cre - infected floxil3 cells were first selected in g418 to eliminate integrations into constitutively expressed genes .", "the g418-resistant cells were then plated into semisolid ( agar ) cultures in the absence of il-3 .", "analysis of several recombinants obtained by this method indicated that cells upregulate survival genes before committing to cell death . as a result , cells receiving a transient apoptotic stimulus exhibit improved survival in a growth factor deficient environment ."], ["from a floxil3 gene trap integration library consisting of approximately 2  10 unique proviral integrations , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal .", "cellular sequences adjacent to u3cre integration sites ( gene trap sequence tags , gtsts ) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers .", "ninety clones yielded informative gtsts ( > 30 nucleotides ) , of which 15 ( 17% ) belonged to known genes , 10 ( 11% ) matched single ests or full cdnas with unknown function and 65 ( 72% ) had no match within the public databases ( figure 2 ) .", "interestingly , the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector .", "for example , only 782 ( 46% ) of 1,687 gtsts recovered from embryonic stem ( es ) cells expressing a u3 geo gene trap virus were novel .", "this difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases .", "figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat ( ltr ) , most integrations occurred in or near 5 ' exons , although integrations further downstream were observed occasionally . to confirm transcriptional induction by il-3 withdrawal", ", polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes .", "( a ) gene trap position in previously characterized genes . gtsts were either obtained by 5'race or inverse genomic pcr .", "exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers .", "polyadenylated rnas ( 5 g per lane ) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels , blotted onto nylon filters and hybridized to p -labeled gene- or exon ( net1)-specific probes .", "as summarized in table 1 , a significant number of the trapped known genes encode proteins involved in cell growth and survival .", "for example , the guanine nucleotide exchange factors ( gefs ) for the rho - family gtpases net1 and h - pem2 ( kiaa0424 ) [ 10 , 11 ] belong to a large family of proteins that regulate cell growth and can transform cells in culture .", "transformation is dependent on the highly conserved dbl homology ( dh ) domain and in most cases requires amino - terminal truncation of the protein [ 13 , 14 ] .", "interestingly , a novel splice variant of net1 , net1a , which was isolated in these experiments , encodes an amino - terminally truncated protein that is transforming in its native form ( table 1 ) .", "thus , when overexpressed in nih3t3 fibroblasts , net1a but not net1 produces transformed foci with typical rho morphology ( f.w . and h.v.m .", "in addition to the dh domain , rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase ( pi3 kinase ) signal transduction pathway .", "the latter is responsible for mediating survival signals , particularly in hematopoietic cells , and is directly activated by il-3 .", "surprisingly , two members of this pathway , the phosphatidylinositol 4/5 phosphate kinases ( pip5 kinases ) type i ( which corresponds to human pip5 kinase type i ) and type i , were induced by il-3 withdrawal ( table 1 ) .", "pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate ( pip2 ) , which enhances cell survival by at least three mechanisms .", "moreover , overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- ( tnf- ) .", "second , pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate ( pip3 ) , which activates the survival kinase akt .", "akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad , caspase 9 and the forkhead family transcription factor fkhrl1 , which induces the expression of fas ligand [ 17 , 18 , 19 , 20 ] .", "third , pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane .", "as shown previously , apoptotic signals can cause membrane depolarization , which leads to cell death by directly inhibiting potassium channels .", "summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 ( ebp1 ) .", "although less well characterized than the above proteins , p38-g4 is absent from go cells and may stimulate cell - cycle progression [ 23 , 24 ] .", "it includes the il-12 receptor , which inhibits fas - mediated apoptosis in certain types of t lymphocytes   and the transcription factor yb-1 , which represses fas gene expression .", "interestingly , yb-1 also activates the expression of the multidrug resistance gene ( mdr1 ) whose product , the p glycoprotein , is an efflux pump that eliminates a variety of toxins from the cell , including proapoptotic anticancer drugs .", "similarly cytoprotective is vmat2 , the protein product of the vesicular monoamine transporter gene , which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [ 28 , 29 ] . finally , il-3 withdrawal induced transcription of several dna repair genes .", "these were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes .", "while alkb protects dna from damage by alkylating agents , rec8 and rad50 are dna double - strand break repair proteins [ 31 , 32 , 33 ] .", "only a few genes upregulated by il-3 withdrawal ( 4 out of 15 ) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis ( table 1 ) . however , the low frequency with which such genes were recovered underscores the specificity of the gene trap approach . to further study transcriptional regulation during growth factor starvation , we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays ( clontech ) .", "atlas arrays contain cdna probes for 588 genes involved in development , oncogenesis , dna repair , tumor suppression and apoptosis .", "figure 4a shows that of the 588 genes displayed on the arrays , only 12 were differentially expressed after factor deprivation .", "differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting ( figure 4c ) .", "( a ) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h ( left ) and 2 h ( right ) of il-3 deprivation .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimage 1.5 software .", "( b ) relative changes in gene expression on a log scale . 1 , no regulation ; >", "1 , upregulation ; < 1 , downregulation . note that the apparent downregulation of bcl-2 could not be confirmed on northern blots .", "induction of c - kit and chop10 ( asterisked ) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation .", "( c ) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software .", "poly(a ) rna ( 2.5 g ) prepared after 0 , 2 , 4 , 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels , blotted onto nylon filters and hybridized with p - labeled gene - specific probes .", "similar to the trapping results , several genes with survival functions were upregulated in the absence of il-3 . among these", "were the survival kinase akt , the c - kit receptor tyrosine kinase , the potent inhibitor of apoptosis flipl , and the il-3-receptor itself [ 34 , 35 , 36 ] .", "moreover , survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [ 37 , 38 ] . in confirmation of previous results , however , the expression of several survival genes appeared il-3 dependent .", "thus , transcripts for antiapoptotic pim-1 , c - myc and bcl - xl proteins were repressed in the absence of il-3 .", "finally , only two genes with arguably proapoptotic functions ( that is , jund and chop10 ) were upregulated in these experiments ( figure 4c ) .", "taken together , the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death .", "the activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival .", "accordingly , a transient factor deprivation should , by upregulating anti - apoptotic gene expression , reduce the fdcp1 cell 's need for il-3 . to test this", ", we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 .", "figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts , indicating that survival mechanisms were induced by factor deprivation . whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation , it gradually subsided with prolonged il-3 deprivation .", "after incubation for 0 , 1 , 2 and 4 h , il-3 was added at the indicated concentrations .", "1  10 cells were deprived of il-3 for 1 and 2 h , respectively .", "after re - adding il-3 for 4 h , cells were washed and incubated without il-3 .", "after 6 , 9 and 12 h factor deprivation , the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry .", ", we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . to this end , il-3 was initially withdrawn from fdcp1 cells for 1 or 2 hours .", "subsequently , il-3 was re - added for 4 hours only to be removed again for 6 , 9 and 12 hours . at these time points cells", "figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts , indicating that apoptotic prestimulation increases resistance to factor deprivation . like the clonal survival assays , the cell protective effect was highest after a prestimulation of 2 hours . on the basis of these results ,", "we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death ."], ["from a floxil3 gene trap integration library consisting of approximately 2  10 unique proviral integrations , we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal .", "cellular sequences adjacent to u3cre integration sites ( gene trap sequence tags , gtsts ) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers .", "ninety clones yielded informative gtsts ( > 30 nucleotides ) , of which 15 ( 17% ) belonged to known genes , 10 ( 11% ) matched single ests or full cdnas with unknown function and 65 ( 72% ) had no match within the public databases ( figure 2 ) .", "interestingly , the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector .", "for example , only 782 ( 46% ) of 1,687 gtsts recovered from embryonic stem ( es ) cells expressing a u3 geo gene trap virus were novel .", "this difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases ."], ["figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat ( ltr )", ", most integrations occurred in or near 5 ' exons , although integrations further downstream were observed occasionally . to confirm transcriptional induction by il-3 withdrawal", ", polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes .", "( a ) gene trap position in previously characterized genes . gtsts were either obtained by 5'race or inverse genomic pcr .", "exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers .", "polyadenylated rnas ( 5 g per lane ) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels , blotted onto nylon filters and hybridized to p -labeled gene- or exon ( net1)-specific probes .", "as summarized in table 1 , a significant number of the trapped known genes encode proteins involved in cell growth and survival .", "for example , the guanine nucleotide exchange factors ( gefs ) for the rho - family gtpases net1 and h - pem2 ( kiaa0424 ) [ 10 , 11 ] belong to a large family of proteins that regulate cell growth and can transform cells in culture .", "transformation is dependent on the highly conserved dbl homology ( dh ) domain and in most cases requires amino - terminal truncation of the protein [ 13 , 14 ] .", "interestingly , a novel splice variant of net1 , net1a , which was isolated in these experiments , encodes an amino - terminally truncated protein that is transforming in its native form ( table 1 ) .", "thus , when overexpressed in nih3t3 fibroblasts , net1a but not net1 produces transformed foci with typical rho morphology ( f.w . and h.v.m . , unpublished data ) .", "in addition to the dh domain , rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase ( pi3 kinase ) signal transduction pathway .", "the latter is responsible for mediating survival signals , particularly in hematopoietic cells , and is directly activated by il-3 .", "surprisingly , two members of this pathway , the phosphatidylinositol 4/5 phosphate kinases ( pip5 kinases ) type i ( which corresponds to human pip5 kinase type i ) and type i , were induced by il-3 withdrawal ( table 1 ) .", "pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate ( pip2 ) , which enhances cell survival by at least three mechanisms .", "moreover , overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- ( tnf- ) .", "second , pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate ( pip3 ) , which activates the survival kinase akt .", "akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad , caspase 9 and the forkhead family transcription factor fkhrl1 , which induces the expression of fas ligand [ 17 , 18 , 19 , 20 ] .", "third , pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane .", "as shown previously , apoptotic signals can cause membrane depolarization , which leads to cell death by directly inhibiting potassium channels .", "summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 ( ebp1 ) .", "although less well characterized than the above proteins , p38-g4 is absent from go cells and may stimulate cell - cycle progression [ 23 , 24 ] .", "it includes the il-12 receptor , which inhibits fas - mediated apoptosis in certain types of t lymphocytes   and the transcription factor yb-1 , which represses fas gene expression .", "interestingly , yb-1 also activates the expression of the multidrug resistance gene ( mdr1 ) whose product , the p glycoprotein , is an efflux pump that eliminates a variety of toxins from the cell , including proapoptotic anticancer drugs .", "similarly cytoprotective is vmat2 , the protein product of the vesicular monoamine transporter gene , which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [ 28 , 29 ] . finally , il-3 withdrawal induced transcription of several dna repair genes .", "these were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes .", "while alkb protects dna from damage by alkylating agents , rec8 and rad50 are dna double - strand break repair proteins [ 31 , 32 , 33 ] .", "only a few genes upregulated by il-3 withdrawal ( 4 out of 15 ) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis ( table 1 ) . however , the low frequency with which such genes were recovered underscores the specificity of the gene trap approach ."], ["to further study transcriptional regulation during growth factor starvation , we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays ( clontech ) .", "atlas arrays contain cdna probes for 588 genes involved in development , oncogenesis , dna repair , tumor suppression and apoptosis .", "figure 4a shows that of the 588 genes displayed on the arrays , only 12 were differentially expressed after factor deprivation .", "differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting ( figure 4c ) .", "( a ) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h ( left ) and 2 h ( right ) of il-3 deprivation .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimage 1.5 software .", "( b ) relative changes in gene expression on a log scale . 1 , no regulation ; > 1 , upregulation ; < 1 , downregulation . note that the apparent downregulation of bcl-2 could not be confirmed on northern blots .", "induction of c - kit and chop10 ( asterisked ) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation .", "( c ) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software .", "poly(a ) rna ( 2.5 g ) prepared after 0 , 2 , 4 , 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels , blotted onto nylon filters and hybridized with p - labeled gene - specific probes .", "similar to the trapping results , several genes with survival functions were upregulated in the absence of il-3 . among these", "were the survival kinase akt , the c - kit receptor tyrosine kinase , the potent inhibitor of apoptosis flipl , and the il-3-receptor itself [ 34 , 35 , 36 ] .", "moreover , survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [ 37 , 38 ] . in confirmation of previous results , however , the expression of several survival genes appeared il-3 dependent .", "thus , transcripts for antiapoptotic pim-1 , c - myc and bcl - xl proteins were repressed in the absence of il-3 .", "finally , only two genes with arguably proapoptotic functions ( that is , jund and chop10 ) were upregulated in these experiments ( figure 4c ) . taken together", ", the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death ."], ["the activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival .", "accordingly , a transient factor deprivation should , by upregulating anti - apoptotic gene expression , reduce the fdcp1 cell 's need for il-3 . to test this", ", we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 .", "figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts , indicating that survival mechanisms were induced by factor deprivation . whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation , it gradually subsided with prolonged il-3 deprivation .", "after incubation for 0 , 1 , 2 and 4 h , il-3 was added at the indicated concentrations .", "1  10 cells were deprived of il-3 for 1 and 2 h , respectively .", "after re - adding il-3 for 4 h , cells were washed and incubated without il-3 .", "after 6 , 9 and 12 h factor deprivation , the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry .", ", we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . to this end", "subsequently , il-3 was re - added for 4 hours only to be removed again for 6 , 9 and 12 hours . at these time points cells were stained with annexin and apoptosis was quantified by flow cytometry .", "figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts , indicating that apoptotic prestimulation increases resistance to factor deprivation . like the clonal survival assays , the cell protective effect was highest after a prestimulation of 2 hours . on the basis of these results ,", "we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death ."], ["a gene trap strategy was used to identify genes induced in hematopoietic cells undergoing apoptosis by growth factor withdrawal .", "this approach enables identification of transiently expressed genes that are difficult to isolate by standard methodology .", "a comparatively large proportion of unknown genes were recovered , which underscores the strategy 's potential for isolating novel genes .", "in contrast to conventional gene trapping , which tags and disrupts random genes , the cre / loxp approach allows enrichment for genes induced by specific biological stimuli .", "as most regulatory genes are expressed in a temporally and spatially restricted manner , we believe that the combination of gene trap mutagenesis and site - specific recombination provides a sound alternative for functional gene analysis in the post - genomic era . as judged by the small fraction of recombinants recovered after growth factor deprivation ( that is , 124 of 2  10 or 0.006% )   and by the identity of the trapped", "known genes , the strategy seems highly specific for regulatory genes induced during programmed cell death .", "accordingly , the majority of genes upregulated by il-3 withdrawal were associated with cell death and survival .", "although this was similar on the cdna arrays , the differentially expressed genes recovered with the two methods were quite different .", "thus , with the exception of yb1 and rad50 , none of the genes displayed on the arrays were tagged by gene trap mutagenesis .", "moreover , yb1 and rad50 , despite being recovered in the gene trap approach as induced by il-3 withdrawal , appeared constitutively expressed or even downregulated on both arrays and northern blots , respectively ( figures 3,4 ) .", "first , the integration library used here covered only 25% of the genome . as only seven genes were induced on the arrays ( those encoding akt , il-3 receptor , glutathione - s - transferase , flipl , c - kit , jund and chop10 ) their recovery from an unsaturated library was unlikely .", "second , transient gene trapping as opposed to cdna hybridization has no bias towards highly expressed genes .", "third , the gene trap strategy selects for real gene inductions and , unlike the arrays and northern blots , is independent of pre - existing steady - state mrna levels subjected to post - transcriptional regulation . taken together", ", these considerations suggest that for the functional analysis of the mammalian genome , gene trapping effectively complements cdna - based strategies , including cdna arrays , which are unable to distinguish between transcriptional and/or post - transcriptional changes in gene expression . in terms of apoptosis", "whereas in most cells conflicts between prosurvival and apoptotic signals are carried out post - translationally by well characterized proteins ( for example , bcl-2 family members , caspases ) , there is increasing evidence for transcriptional regulators of apoptosis capable of tilting the balance between the constitutively expressed pro- and anti - apoptotic proteins [ 20 , 41 ] .", "these transcriptional regulators of apoptosis - still largely unknown - are likely to confer tissue specificity on the apoptotic process .", "the identity of such regulators is of considerable interest as they could provide valuable targets for prospective anti - neoplastic and/or anti - degenerative drugs .", "most genes identified in this study encode cell protective and/or pro - survival functions . in line with this ,", "the biological experiments described here have shown such functions to materialize in cells exposed to apoptotic prestimulation .", "accordingly , cells receiving stimulation exhibited improved survival and reduced apoptosis in a growth factor deprived environment .", "interestingly , a similar cytoprotective phenomenon known as ischemic preconditioning has been observed in animal models . in this , a short , sublethal period of ischemia induces profound resistance to subsequent ischemic events .", "thus , induction of anti - apoptotic gene expression prior to a lethal stimulus seems to raise the threshold required for that stimulus to be effective .", "this provides an additional safety mechanism that can prevent unwanted loss of cells exposed only accidentally to apoptotic stimulation . in summary", ", the present experiments have shown that hematopoietic cells undergoing apoptosis by il-3 withdrawal activate survival genes that do impede cell death .", "this suggests that apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals , rather than a simple death by default ."], ["inverse pcr from genomic dnas was performed using the cre - specific primers described previously   and a combination of the blunt end restriction enzymes sspi and hincii .", "5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer 's instructions .", "amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector ( promega ) as described previously .", "fdcp1 cells were propagated at concentrations of 2  10 cells / ml in dulbecco 's modified eagle 's medium ( dmem ; gibco ) , supplemented with 10% ( v / v ) fetal bovine serum ( boehringer - mannheim ) and 5 ng / ml recombinant mouse il-3 ( peprotech ) unless indicated otherwise .", "agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% ( v / v ) fetal bovine serum and 0.6% ( w / v ) bacto - agar ( difco ) in double distilled water as previously described .", "apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit ( roche ) and the manufacturer 's instructions .", "poly(a ) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array ( clontech ) according to the manufacturer 's instructions .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimagetm 1.5 software ( clontech ) . for northern blots , 2.5 g of poly(a )", "was fractionated in 1% formaldehyde - agarose gels , transferred onto hybond n membranes ( amersham ) and hybridized to specific p - dctp - labeled probes produced by random priming ( rediprime , amersham ) .", "cellular sequences adjacent to proviral integrations ( gene trap sequence tags ; gtsts ) were searched in the ncbi / nih genomic databases   using the blastn algorithm ."], ["inverse pcr from genomic dnas was performed using the cre - specific primers described previously   and a combination of the blunt end restriction enzymes sspi and hincii .", "5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer 's instructions .", "amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector ( promega ) as described previously ."], ["fdcp1 cells were propagated at concentrations of 2  10 cells / ml in dulbecco 's modified eagle 's medium ( dmem ; gibco ) , supplemented with 10% ( v / v ) fetal bovine serum ( boehringer - mannheim ) and 5 ng / ml recombinant mouse il-3 ( peprotech ) unless indicated otherwise .", "agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% ( v / v ) fetal bovine serum and 0.6% ( w / v ) bacto - agar ( difco ) in double distilled water as previously described .", "apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit ( roche ) and the manufacturer 's instructions ."], ["poly(a ) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array ( clontech ) according to the manufacturer 's instructions .", "filters were scanned with a phosphoimager ( molecular dynamics ) and analyzed with the atlasimagetm 1.5 software ( clontech ) . for northern blots , 2.5 g of poly(a )", "was fractionated in 1% formaldehyde - agarose gels , transferred onto hybond n membranes ( amersham ) and hybridized to specific p - dctp - labeled probes produced by random priming ( rediprime , amersham ) ."], ["cellular sequences adjacent to proviral integrations ( gene trap sequence tags ; gtsts ) were searched in the ncbi / nih genomic databases   using the blastn algorithm ."], ["this work was supported in part by grants from deutsche krebshilfe and the deutsche forschungsgemeinschaft , bonn to h.v.m and by schering ag , berlin ."]]}
{"article_id": "PMC5050381", "article_text": ["noise - induced hidden hearing loss ( nihhl ) refers to any functional impairment seen in subjects with noise exposing history but no permanent threshold shift ( pts ) .", "this is different from the conventional definition of noise - induced hearing loss ( nihl ) , which is based on changes in auditory sensitivity or threshold shift .", "therefore , noise exposure recommendations are based on the likelihood that a particular dose of exposure will result in a pts .", "physiologically , variations in auditory sensitivity following exposure to noise are largely due to the functional status of outer hair cells ( ohcs ) in the cochlea , which provide mechanical amplification of soft sounds [ 2 , 3 ] .", "noise exposures that result in only a temporary threshold shift ( tts ) have a reversible impact on ohc function , which is manifested by the recovery of otoacoustic emissions ( oae ) [ 46 ] and cochlear microphonics ( cm ) [ 711 ] .", "the functional changes in these measures parallel the recovery of hearing thresholds , as well as the repair of structures such as stereocilia and the tectorial membrane [ 7 , 12 ] .", "by contrast , noise exposure at higher levels and/or for longer durations can cause permanent damage to , or even the death of , ohcs and , hence , lead to pts .", "therefore , the ohcs and the structures surrounding them , including the tectorial membrane and the supporting cells , are considered to be the major loci of cochlear damage that result in noise - induced threshold shifts [ 13 , 14 ] . although some early reports claimed that reversible noise - induced ihc pathologies were responsible for tts [ 15 , 16 ] , ihcs are relatively insensitive to noise - induced cell death .", "however , it has long been recognized that the synapse between ihcs and primary spiral ganglion neurons ( sgns ) can be damaged by noise [ 1719 ] .", "these early studies showed that this manifests mainly as damage to the postsynaptic terminals ; however , there is clear evidence from more recent studies that noise induces damage to both pre- and postsynaptic structures .", "more importantly , disruption of the synapses can be permanent , resulting in degenerative death of sgns .", "the finding that damage to ribbon synapses can occur without pts is significant because of the potential impact of such damage on hearing function .", "because the physiological damage is not accompanied by a permanent shift in hearing threshold , it would likely be missed by a standard ( i.e. , threshold - based ) hearing assessment and has thus been referred to as nihhl .", "nihhl first manifests as reduced output of the auditory nerve at high sound levels , without affecting the hearing threshold .", "this reduction has been found in both animals [ 6 , 2023 ] and human subjects with a history of noise exposure but with normal audiograms . since the thresholds of the auditory nerve remain unchanged , the function relating compound action potentials ( cap ) amplitude with sound levels in nihhl animal is different from that in animals with threshold changes .", "schematic curves of cap input / output functions are presented in figure 1 for a comparison across normal control and those with different pathologies .", "theoretically , if the damage is restricted to ohcs , the major change in cap input / output ( i / o ) curve is restricted around threshold and the amplitude reaches the control value at high sound levels . in the case of nihhl , cap reduction is mainly at high sound level , with no difference at low sound level , suggesting a suprathreshold deficit .", "when the damage occurs at both ohcs and the ihc - sgn synapses , the reduction of cap amplitude is seen across all sound levels . as nihhl is initiated at the synapse between the ihcs and sgns , which silences the auditory nerve fibers ( anfs ) that extend from them , the corresponding disorder is categorized as a cochlear neuropathy ( i.e. , cochlear synaptopathy ) [ 25 , 26 ] .", "presumably , the reduction in the amplitude of the auditory nerve response without threshold elevation is due to selective loss of anfs that have high thresholds , which is supported by single - unit recording studies [ 20 , 27 ] . given the important features of those low - spontaneous - rate anfs in auditory coding , the neuropathy or synaptopathy in hidden hearing loss is not simply a reduction in the number of functional anfs .", "furthermore , the synaptopathy in nihhl is likely to be related to the synaptic repair after initial damage by noise , rather than a simple initial loss . in addition , the functional deficits seen in nihhl may also involve the contribution from central auditory plasticity [ 26 , 2832 ] . in this review", ", we summarize the available data for noise - induced damage and repair around ihc  sgn synapses and discuss the evidence for the contributions of cochlear malfunction and central plasticity to nihhl .", "accumulated evidence has shown that the synapses between ihcs and type - i sgns are sensitive to noise and the damage to this synapse is likely to be the bases for nihhl .", "the synapse is characterized by presynaptic dense bodies termed ribbons [ 3335 ] , which are spherical or ellipsoidal in shape , 100200  nm in diameter , and surrounded by synaptic vesicles .", "the ribbons are built up from ribeye protein subunits [ 37 , 38 ] and anchored to the active zone of the presynaptic membrane via bassoon proteins [ 3941 ] .", "the functional role of ribbons has been recognized as tethering and conveying synaptic vesicles to the active zones [ 42 , 43 ] , where the release of neurotransmitters at these synapses is modulated by a specific l - type calcium ion channel ( i.e. , cav1.3 ) [ 44 , 45 ] .", "noise exposure causes damage to both the presynaptic ribbons and postsynaptic nerve terminals of the ribbon synapses [ 6 , 22 , 23 , 4648 ] .", "the damaged synapses exhibit various degrees of swelling of the terminals , resulting in disruption of the synaptic connections between ihcs and sgns [ 20 , 46 , 48 ] .", "immunohistological staining has revealed similar losses for ribbons and terminals [ 6 , 22 , 23 , 49 ] .", "the mechanism for the damage to the postsynaptic terminal is glutamate - mediated excitotoxicity ( reviewed in ) .", "one possible mechanism of ribbon loss is the loss of cell - cell contact that is required for the maintenance of the pre- and postsynaptic complexes [ 5053 ] .", "our electron microscopy evaluations did not reveal any residual presynaptic complexes without ribbon and postsynaptic terminals .", "therefore , it is likely that the entire presynaptic structure breaks down when the postsynaptic terminal is damaged .", "a brick assembly model , in which a ribbon is built up from multiple ribeye subunits , has been proposed for ribbon construction in retina photoreceptor cells . moreover ,", "the ribbons in retina sensorial cells can be partially broken down by light , but they rapidly reassemble in the dark , probably serving as a mechanism of adaption to bright light [ 5458 ] . in the retina , the ribbon size appears to be a determining factor for the quantity of neurotransmitter released .", "however , the dynamic disassembling / reassembling process has not been identified in the cochlea , and changes in the ribbon size and the relationship with the release of neurotransmitters have not been investigated in the cochlea .", "additionally , disassembly and reassembly , as well as ribbon size , are modulated by ca signaling involving cav - channels , presynaptic ca levels and storage , and guanylate cyclase - activating protein-2 ( gcap2 ; see the review by schmitz ) .", "interestingly , optical stimulation of photoreceptors causes hyperpolarization of the presynaptic membrane and a decrease in [ ca]i , as opposed to depolarization and the large increase in [ ca]i in ihcs in response to sound .", "the decrease in [ ca]i in photoreceptor cells is followed by a conformational change of gcap2 , which results in the disassembly of the ribbons . in the cochlea", ", it is not known whether there is a gcap - mediated pathway that controls ribbon size . as the membrane potential of ihcs", "is depolarized with increasing sound levels , resulting in an influx of ca , the role of ca in ribbon assembly is unlikely to be the same as it is in the retina .", "the first quantitative study of noise - induced ribbon synapse damage in cba mice   reported that the number of ribbon synapses was reduced to 40% compared with the control 1 day after brief noise exposure that did not lead to pts .", "the synapse count recovered to 50% within 1 week , but no further recovery was observed , and this 50% loss of synapses was considered permanent .", "sgn death observed 2 years after the noise was found to match the 50% permanent loss of synapses .", "however , a study on guinea pigs carried out by the same research group found a similar loss of ribbon synapses 2 weeks after exposure to noise that did not cause pts , but this study found a much smaller final loss of sgns .", "this suggests that some sgns , which had originally lost their synapses with ihcs , survived and reestablished synapses with ihcs .", "our studies on guinea pigs have revealed a clear recovery in the synapse count following a massive initial loss induced by noise exposure that did not lead to pts [ 22 , 23 ] .", "although this recovery was not complete , approximately 50% of the initial loss of paired ribbon and postsynaptic density ( psd ) puncta in the basal half of the cochleae was seen 1 day after noise , and the loss was recovered to < 20% within 1 month . comparing the aforementioned data from mice and guinea pigs", ", it appears that there may be some species difference in the ability to regenerate synapses following noise - induced hearing damage . however , a recent study of c57 mice reported that the loss of ribbon synapses induced by non - pts - inducing noise was largely reversible .", "this discrepancy in synapse regeneration following noise exposure requires further investigation . in a recent review", ", it was argued that the recovery of ctbp2/psd counts in guinea pig cochleae following noise exposure reported in our studies may be attributable to up / downregulation of the synaptic protein rather than regeneration of synaptic connections .", "however , there are several lines of evidence for the possibility of synapse repair following noise - induced damage .", "first , it has been reported that plastic changes occur in the presynaptic component , including the existence of multiple presynaptic ribbons around an active zone   and the changes in the size and location of ribbons following noise exposure .", "second , the change in the amplitude of the compound action potential ( cap ) corresponded to the changes in ribbon / psd counts : a large initial reduction in cap amplitude and synapse counts were followed by a significant recovery after the noise exposure .", "third , changes in many single - anf coding activities were not seen at the time that the synapses were damaged but rather manifested later ( see section 3 ) with the recovery in both the cap and synapse number , suggesting that those changes occurred in the anfs that connect ihcs via repaired / reestablished synapses .", "further work is required to determine the mechanisms and factors that influence the repair of both pre- and postsynaptic components .", "ribbon synapses exhibit spatial differences around ihcs ; that is , the synapses at the modiolar side of an ihc have relatively small ribbons but larger postsynaptic terminals , whereas those at the pillar side have relatively large ribbons but smaller terminals .", "liberman et al . reported that anfs are functionally categorized by their spontaneous rate ( sr ) , which is inversely related to the fiber 's threshold and dynamic range [ 6365 ] .", "it is widely accepted that low - sr anfs exhibit synapses with ihcs on their modiolar side , whereas high - sr units exhibit synapses on the pillar side ( this is based on data obtained using intracellular tracer injections ) .", "the low - sr units are considered critical for hearing in noisy environments due to their larger dynamic range , higher thresholds , and the ability to follow the quick change of the amplitude of acoustic signals . by contrast , high - sr units are responsible for the sensitivity to quiet sounds and are saturated by high - level background noise [ 26 , 63 , 64 , 67 , 68 ] . in nihhl ,", "low - sr anfs appear to be more vulnerable to noise than high - sr units .", "selective loss of low - sr anfs has been found following exposure to noise that did not lead to pts .", "presumably , this selective loss of low - sr units should produce coding deficits , which can be predicted based on the unique features of those units .", "however , no coding deficits were examined and reported in this study . on the other hand ,", "we reported a time delay in the development of coding deficits by single anfs in guinea pigs following a similar noise exposure that did not cause pts ; these deficits were attributed to intensity coding and temporal coding as summarized in sections 3.1 and 3.2 .", "intensity coding in the cochlea is defined as the ability of anfs to encode the sound intensity or the change of sound intensity .", "this ability is determined primarily by the spike rate ( or the change of spike rate ) of individual anf in response to sound intensity change and the number of functional anfs .", "therefore , the intensity coding deficits can be evaluated in both evoked field potential and single - unit recordings .", "deficits in intensity coding were first suggested by a reduction in wave i of the auditory brainstem response ( abr ) [ 6 , 49 ] , as well as a reduction in the amplitude of the cap , as this is likely due to the loss of functional anfs following synapse disruption .", "the fact that the reduction is more significant at higher sound levels has been considered evidence for selective damage to low - sr fibers , which have higher thresholds [ 25 , 26 , 69 ] .", "the deterioration in intensity coding following no - pts noise exposure was manifested as a reduction in the driven spike rates ( peak , sustained , and total rates ) of anf units that were tested only at one sound level .", "such changes are significant only in low - sr anf units and are seen at a later time rather than immediately following exposure .", "this time delay in the development of coding deficits suggests that ( 1 ) the reduction in driven spike rates occurs in the anfs to which the synaptic connections to the ihcs are reestablished following the initial disruption and ( 2 ) the repaired synapses are functionally abnormal , with less efficient neurotransmitter release .", "temporal processing ability in the cochlea as well as in the whole auditory pathway is defined as the ability to follow the quick change of acoustic signals . in human subjects ,", "the process involves both bottom - up and top - down mechanisms ; but in animal models , only bottom - up mechanisms are tested ( see reviewed by ) .", "many different tests have been used to detect the bottom - up mechanisms of temporal coding , some of them based on the peristimulatory changes of firing rate showing latency and adaptation . as reviewed above , the major function of presynaptic ribbons in ihcs is to facilitate the synaptic transmission .", "indeed , such deficits were manifested as an increase in response latency of anfs in animals with nihhl .", "this was first demonstrated as a significant delay in cap peak latency   and then further supported by the delayed latency of peak in psth ( or peak latency ) of anfs in our single - unit study . in another very recent report , such delay was reported in abr as the marker of cochlear synaptopathy .", "we also found a reduction in the ratio of peak to sustained rates in animals that were exposed to noise .", "this ratio is considered an index of the ability of a neuron to encode dynamic signal changes ( see review by ) . using a paired - click paradigm", ", we found that the anf response of noise - exposed animals to the second click recovered more slowly from the masking effect of the first click .", "these results reveal poorer coding to the transient features of acoustic signals by anfs , which were examined in previous studies to show the deterioration in of temporal coding in animals with bassoon mutation [ 39 , 41 ] .", "whereas an increase in peak latency was seen shortly after exposure to noise , changes in the peak rate and the peak / sustained spike ratio , as well as a slower recovery of the spike rate to the second click , were not seen until later , suggesting an association between the deficits and the synapse repair .", "a temporal deficit in phase - locking responses has been proposed based on selective loss of low - sr units and the functional features of this group of anfs [ 25 , 26 , 69 ] , but it has not been tested at the single - unit level .", "so far , there appear to be two models for the development of coding deficits in nihhl .", "one model suggests that the coding deficit or synaptopathy is simply due to the loss of low - sr anfs .", "since those units have unique functions in signal coding , the loss of those functions is predicted as the consequences .", "that is , the coding deficits are developed as the result of unhealthy synaptic repair after initial disruption .", "we found that the noise - induced synaptic damage in guinea pigs under nihhl is largely repairable , leaving only a small amount of synapses not being reestablished .", "therefore , the coding deficits or synaptopathy can not be simply attributed to the loss of sr units . since the coding deficits are seen at the time when the synapse counts are largely recovered , we believe that the coding deficits likely occur in the repaired synapses ( most of them innervating low - sr anfs ) .", "studies are needed to verify which model is more likely the case in human subjects .", "it has long been recognized that subjects with normal audiograms may have perceptual difficulties , and this is especially true in the elderly . age - related hearing loss with threshold elevation", "is termed peripheral presbycusis , whereas the perceptual difficulties seen in the elderly without threshold shift are usually termed central presbycusis . for example , temporal processing deficits and difficulties of hearing in noisy environments are two major problems experienced by the elderly .", "these problems were recognized long before the discovery of cochlear damage associated with nihhl and were considered to be the result of  central auditory processing disorders  [ 7175 ] .", "it was generally accepted that any perceptual deficits observed without changes to hearing thresholds and cognitive functioning can be attributed to central dysfunctions .", "based on recent progress in functional deficits in cochlear coding , such separation between peripheral and central presbycusis is likely to be incorrect .", "the so - called central presbycusis may , at least in part , result from disorders in the auditory periphery .", "the coding deficits related to the loss of low - sr anfs had been described as a type of auditory neuropathy and/or synaptopathy even before any of the predicted deficits were identified .", "data on changes in the sr distributions of anfs suggest the reestablishment of synapses following an initial disruption that was selective to low - sr units . although our data revealed abnormalities in some aspects of coding in the auditory nerve , further work is required to investigate coding deficits in nihhl .", "such studies can not be replaced by speculation based on the selective loss of low - sr fibers ; for example , one can not be certain how the auditory nerve changes its response to amplitude modulation until it is measured at the single - unit level .", "two possibilities must be considered : ( 1 ) the surviving anfs may change their function and ( 2 ) the initially lost low - sr fibers may be repaired but with changed function .", "it should be noted that there is now a tendency in the literature to consider nihhl to be a purely peripheral issue , a result of the overcorrection of the  central presbycusis .", " however , despite the strong evidence for a peripheral contribution , the central contribution to the problems seen in nihhl should not be neglected . in other words", ", it may be more constructive to assume that there are both peripheral and central contributions to nihhl .", "it is well known that hearing loss ( with elevated threshold ) can induce central changes , which can result in deteriorations in signal processing .", "studies aiming to distinguish the role of central plasticity from that of ribbon synapse damage are rare .", "one such report found that an increase in central gain was responsible for tinnitus in human subjects with typical damage seen in nihhl ( i.e. , reduced auditory nerve input to the brain ( measured as a smaller abr wave i ) ) but normal hearing threshold . in an earlier study in rats ,", "tinnitus was found 6 months after exposure to noise that caused minimal loss of hair cells and pts but significant loss of anfs .", "one of the central impacts of hearing loss due to damage to peripheral auditory organ is imbalance between excitation and inhibition , resulting in hyperactivity and/or hyperresponsiveness in the central auditory system ( see reviews in [ 7780 ] ) .", "the types of hearing loss producing such central enhancement include cochlear ablation , drug- and noise - induced damage . while direct effect of drugs and noise on central neurons needs to be differentiated , a similarity across those hearing loss models is the reduction of cochlea output to the auditory brain , which may be the main initial factor causing the imbalance between excitation and inhibition . in this sense ,", "while most of studies in central plasticity using nihl model correlated the central enhancement with the amount of threshold shifts [ 29 , 8185 ] , at least one study has reported central enhancement in mice exposed briefly to noise at a moderate level that did not cause pts , presumably producing only nihhl .", "unfortunately , the reduction in auditory input from the cochlea was not quantified in this study . taken together ,", "available data suggest that cochlear damage , with or without threshold elevation , can lead to central plasticity by reducing input from the auditory nerve .", "further work is required to establish the central contribution to coding / perception difficulties in nihhl , and previous studies on central processing disorders in subjects with nihl should be reevaluated to differentiate the central contributions from the peripheral ones . in a brief summary", ", we use figure 2 to summarize the available data for the mechanisms of perception difficulty experienced by subjects with history of noise exposure but normal or near normal thresholds . in this schematic diagram , we include the two potential models of noise - induced synaptopathy in cochleae . in model 1 , the coding deficits are speculated based on the role of low - sr anfs in signal coding .", "both models result in a reduction in the cochlear output to the auditory brain , which in turn will result in plastic reorganization of the brain .", "auditory signal processing disorders experienced by subjects with long - term nihhl should include what are inherited from the coding deficits developed in the auditory peripheral and those associated with the plastic changes of auditory brain .", "although more studies on the impact of noise on human hearing showing no changes in auditory sensitivity are required , evidence suggesting the occurrence of nihhl in human subjects is being accumulated .", "this is supported by thorough research on the signal perception deficits experienced by subjects with a history of noise exposure but normal thresholds .", "since the deficits are demonstrated at suprathreshold levels , it is clear that normal hearing thresholds do not guarantee normal hearing functions , especially in subjects with history of noise exposure [ 24 , 86 ] .", "the second line of evidence is the reduction in the output of the auditory nerve in subjects with a history of exposure to noise .", "interestingly , the combination of a reduction in wave i and an increase in wave v / i ratio may be considered evidence of increased central gain and is likely responsible for the generation of tinnitus in hidden hearing loss [ 32 , 87 , 88 ] .", "the third line of evidence comes from the age - related sgn degeneration seen in the examination of human temporal bones .", "unfortunately , there is , as yet , no clear human evidence that degeneration of sgns is expedited by exposure to noise that does not cause threshold elevation .", "the clinical implications of nihhl are manifested by the fact that noise exposure causing nihhl occurs frequently in daily life and impacts much more general population .", "such noise exposure has been generally considered to be safe according to current safety standards for exposure to noise .", "the evidence from the studies reviewed here indicates that the resulting damage to the ribbon synapses from noise that did not induce pts can be repaired even though the repair is incomplete .", "more importantly , the signal coding deficits are developed in association with the synapse repair . since the damage and repair occur repeatedly , the damage on signal coding can be accumulated during aging and likely contributes to the perceptual difficulties experienced by the elderly .", "this impact of noise exposure on signal coding is obviously different from the contribution made by the hearing loss defined by threshold shifts . in future", ", the coding deficits and related synaptic repair in nihhl should be further investigated in a laboratory setting .", "since the ribbon synapse is the first gating point for temporal processing in auditory pathway , the observed coding deficits suggest a clear peripheral origin for the decline in temporal processing and perceptual difficulties during aging . whether and how the synaptic damage will impact the central auditory processing need to be investigated in a manner that is clearly differentiated from the impact of hearing threshold shift .", "moreover , the coding function of anfs should be observed over a long period of time following exposure to noise to determine whether the coding deficits are temporary or persistent .", "we are currently collecting data using electron microscopy , as well as conducting an analysis on the potential changes of the molecular structures of ribbons and psds , in an attempt to elucidate the morphological / molecular mechanisms responsible for functional changes of repaired ribbon synapses .", "it would also be interesting to understand the reasons for the extreme sensitivity of low - sr synapses to noise , as well as elucidate possible methods to prevent damage .", "laboratory studies should also aim to explore the mechanisms of synaptic repair in the cochlea , as well as reveal the factors that influence repair in order to promote it . to translate the knowledge to clinic", ", investigation is needed to establish good measures for detecting nihhl in human subjects . although abr wave i is useful for evaluating synaptopathy caused by noise that does not induce pts , its reliability and sensitivity are questionable in human subjects where the abr amplitude is small , and other methods should be explored .", "a very recent report suggests the use of abr latency as the marker of nihhl .", "the study tested human subjects with normal hearing thresholds and reported a big variation in the threshold of envelope interaural timing difference , which was negatively correlated with the shift of abr wave v latency by background noise : the higher the threshold ( poorer sensitivity ) , the smaller the shift .", "the observation of the latency shift with masking is supported by the fact that the low - sr anfs have longer latency than high - sr fibers and are resistant to background noise [ 91 , 92 ] .", "it is not clear why the study did not report the change in wave v amplitude by masking .", "theoretically , the masking should produce greater reduction in wave v amplitude in subjects with selective loss of low - sr units .", "moreover , no information about the history of noise exposure was reported and it is not clear whether the poorer performance in temporal cue detection was due to noise - induced synaptopathy or other reasons . to date , the most promising methods for diagnosing cochlear synaptopathy are related to selective loss of low - sr anfs , the subcortical steady state responses ( sssr ) [ 93 , 94 ] .", "based on the animal studies , this test should be carried out using amplitude - modulated signals at relatively high intensity and a shallow modulation depth .", "the input intensity of the driving signal should fall within the saturation range of the high - sr fibers .", "high frequency carrier waves with a high intensity and with shallow amplitude modulation are especially useful for evaluating the function of low - sr fibers .", "this is supported by modeling the loss of low - sr fibers . to differentiate the sssr contribution from the auditory nerve from that of central neurons", "a recent mouse study found that the modulation frequency close to 1  khz was optimum with a high frequency carrier without concern of modulation depth .", "however , a recent human study reported a successful detection of the low - sr unit loss using off - frequency maskers and a shallow modulation depth ."], "abstract_text": ["<S> recent studies on animal models have shown that noise exposure that does not lead to permanent threshold shift ( pts ) can cause considerable damage around the synapses between inner hair cells ( ihcs ) and type - i afferent auditory nerve fibers ( anfs ) . </S>", "<S> disruption of these synapses not only disables the innervated anfs but also results in the slow degeneration of spiral ganglion neurons if the synapses are not reestablished . </S>", "<S> such a loss of anfs should result in signal coding deficits , which are exacerbated by the bias of the damage toward synapses connecting low - spontaneous - rate ( sr ) anfs , which are known to be vital for signal coding in noisy background . as there is no pts , these functional deficits </S>", "<S> can not be detected using routine audiological evaluations and may be unknown to subjects who have them . </S>", "<S> such functional deficits in hearing without changes in sensitivity are generally called  noise - induced hidden hearing loss ( nihhl ) .  here , we provide a brief review to address several critical issues related to nihhl : ( 1 ) the mechanism of noise induced synaptic damage , ( 2 ) reversibility of the synaptic damage , ( 3 ) the functional deficits as the nature of nihhl in animal studies , ( 4 ) evidence of nihhl in human subjects , and ( 5 ) peripheral and central contribution of nihhl . </S>"], "labels": null, "section_names": ["1. Noise-Induced Hidden Hearing Loss (NIHHL)", "2. Noise-Induced Damage and Repair around Cochlear Ribbon Synapses", "3. Cochlear Coding Deficits in Hidden Hearing Loss", "4. Association of Coding Deficits with Unhealthy Synaptic Repair ", "5. Central and Peripheral Contributions to NIHHL", "6. Clinical Implications and Future Directions"], "sections": [["noise - induced hidden hearing loss ( nihhl ) refers to any functional impairment seen in subjects with noise exposing history but no permanent threshold shift ( pts ) .", "this is different from the conventional definition of noise - induced hearing loss ( nihl ) , which is based on changes in auditory sensitivity or threshold shift .", "therefore , noise exposure recommendations are based on the likelihood that a particular dose of exposure will result in a pts .", "physiologically , variations in auditory sensitivity following exposure to noise are largely due to the functional status of outer hair cells ( ohcs ) in the cochlea , which provide mechanical amplification of soft sounds [ 2 , 3 ] .", "noise exposures that result in only a temporary threshold shift ( tts ) have a reversible impact on ohc function , which is manifested by the recovery of otoacoustic emissions ( oae ) [ 46 ] and cochlear microphonics ( cm ) [ 711 ] .", "the functional changes in these measures parallel the recovery of hearing thresholds , as well as the repair of structures such as stereocilia and the tectorial membrane [ 7 , 12 ] .", "by contrast , noise exposure at higher levels and/or for longer durations can cause permanent damage to , or even the death of , ohcs and , hence , lead to pts .", "therefore , the ohcs and the structures surrounding them , including the tectorial membrane and the supporting cells , are considered to be the major loci of cochlear damage that result in noise - induced threshold shifts [ 13 , 14 ] . although some early reports claimed that reversible noise - induced ihc pathologies were responsible for tts [ 15 , 16 ] , ihcs are relatively insensitive to noise - induced cell death .", "however , it has long been recognized that the synapse between ihcs and primary spiral ganglion neurons ( sgns ) can be damaged by noise [ 1719 ] .", "these early studies showed that this manifests mainly as damage to the postsynaptic terminals ; however , there is clear evidence from more recent studies that noise induces damage to both pre- and postsynaptic structures .", "more importantly , disruption of the synapses can be permanent , resulting in degenerative death of sgns .", "the finding that damage to ribbon synapses can occur without pts is significant because of the potential impact of such damage on hearing function .", "because the physiological damage is not accompanied by a permanent shift in hearing threshold , it would likely be missed by a standard ( i.e. , threshold - based ) hearing assessment and has thus been referred to as nihhl .", "nihhl first manifests as reduced output of the auditory nerve at high sound levels , without affecting the hearing threshold .", "this reduction has been found in both animals [ 6 , 2023 ] and human subjects with a history of noise exposure but with normal audiograms . since the thresholds of the auditory nerve remain unchanged , the function relating compound action potentials ( cap ) amplitude with sound levels in nihhl animal is different from that in animals with threshold changes .", "schematic curves of cap input / output functions are presented in figure 1 for a comparison across normal control and those with different pathologies .", "theoretically , if the damage is restricted to ohcs , the major change in cap input / output ( i / o ) curve is restricted around threshold and the amplitude reaches the control value at high sound levels . in the case of nihhl , cap reduction is mainly at high sound level , with no difference at low sound level , suggesting a suprathreshold deficit .", "when the damage occurs at both ohcs and the ihc - sgn synapses , the reduction of cap amplitude is seen across all sound levels . as nihhl is initiated at the synapse between the ihcs and sgns , which silences the auditory nerve fibers ( anfs ) that extend from them , the corresponding disorder is categorized as a cochlear neuropathy ( i.e. , cochlear synaptopathy ) [ 25 , 26 ] .", "presumably , the reduction in the amplitude of the auditory nerve response without threshold elevation is due to selective loss of anfs that have high thresholds , which is supported by single - unit recording studies [ 20 , 27 ] . given the important features of those low - spontaneous - rate anfs in auditory coding , the neuropathy or synaptopathy in hidden hearing loss is not simply a reduction in the number of functional anfs .", "furthermore , the synaptopathy in nihhl is likely to be related to the synaptic repair after initial damage by noise , rather than a simple initial loss . in addition , the functional deficits seen in nihhl may also involve the contribution from central auditory plasticity [ 26 , 2832 ] . in this review", ", we summarize the available data for noise - induced damage and repair around ihc  sgn synapses and discuss the evidence for the contributions of cochlear malfunction and central plasticity to nihhl ."], ["accumulated evidence has shown that the synapses between ihcs and type - i sgns are sensitive to noise and the damage to this synapse is likely to be the bases for nihhl .", "the synapse is characterized by presynaptic dense bodies termed ribbons [ 3335 ] , which are spherical or ellipsoidal in shape , 100200  nm in diameter , and surrounded by synaptic vesicles .", "the ribbons are built up from ribeye protein subunits [ 37 , 38 ] and anchored to the active zone of the presynaptic membrane via bassoon proteins [ 3941 ] .", "the functional role of ribbons has been recognized as tethering and conveying synaptic vesicles to the active zones [ 42 , 43 ] , where the release of neurotransmitters at these synapses is modulated by a specific l - type calcium ion channel ( i.e. , cav1.3 ) [ 44 , 45 ] .", "noise exposure causes damage to both the presynaptic ribbons and postsynaptic nerve terminals of the ribbon synapses [ 6 , 22 , 23 , 4648 ] .", "the damaged synapses exhibit various degrees of swelling of the terminals , resulting in disruption of the synaptic connections between ihcs and sgns [ 20 , 46 , 48 ] .", "immunohistological staining has revealed similar losses for ribbons and terminals [ 6 , 22 , 23 , 49 ] .", "the mechanism for the damage to the postsynaptic terminal is glutamate - mediated excitotoxicity ( reviewed in ) .", "one possible mechanism of ribbon loss is the loss of cell - cell contact that is required for the maintenance of the pre- and postsynaptic complexes [ 5053 ] .", "our electron microscopy evaluations did not reveal any residual presynaptic complexes without ribbon and postsynaptic terminals .", "therefore , it is likely that the entire presynaptic structure breaks down when the postsynaptic terminal is damaged .", "a brick assembly model , in which a ribbon is built up from multiple ribeye subunits , has been proposed for ribbon construction in retina photoreceptor cells . moreover ,", "the ribbons in retina sensorial cells can be partially broken down by light , but they rapidly reassemble in the dark , probably serving as a mechanism of adaption to bright light [ 5458 ] . in the retina , the ribbon size appears to be a determining factor for the quantity of neurotransmitter released .", "however , the dynamic disassembling / reassembling process has not been identified in the cochlea , and changes in the ribbon size and the relationship with the release of neurotransmitters have not been investigated in the cochlea .", "additionally , disassembly and reassembly , as well as ribbon size , are modulated by ca signaling involving cav - channels , presynaptic ca levels and storage , and guanylate cyclase - activating protein-2 ( gcap2 ; see the review by schmitz ) .", "interestingly , optical stimulation of photoreceptors causes hyperpolarization of the presynaptic membrane and a decrease in [ ca]i , as opposed to depolarization and the large increase in [ ca]i in ihcs in response to sound .", "the decrease in [ ca]i in photoreceptor cells is followed by a conformational change of gcap2 , which results in the disassembly of the ribbons . in the cochlea", ", it is not known whether there is a gcap - mediated pathway that controls ribbon size . as the membrane potential of ihcs", "is depolarized with increasing sound levels , resulting in an influx of ca , the role of ca in ribbon assembly is unlikely to be the same as it is in the retina .", "the first quantitative study of noise - induced ribbon synapse damage in cba mice   reported that the number of ribbon synapses was reduced to 40% compared with the control 1 day after brief noise exposure that did not lead to pts .", "the synapse count recovered to 50% within 1 week , but no further recovery was observed , and this 50% loss of synapses was considered permanent .", "sgn death observed 2 years after the noise was found to match the 50% permanent loss of synapses .", "however , a study on guinea pigs carried out by the same research group found a similar loss of ribbon synapses 2 weeks after exposure to noise that did not cause pts , but this study found a much smaller final loss of sgns .", "this suggests that some sgns , which had originally lost their synapses with ihcs , survived and reestablished synapses with ihcs .", "our studies on guinea pigs have revealed a clear recovery in the synapse count following a massive initial loss induced by noise exposure that did not lead to pts [ 22 , 23 ] .", "although this recovery was not complete , approximately 50% of the initial loss of paired ribbon and postsynaptic density ( psd ) puncta in the basal half of the cochleae was seen 1 day after noise , and the loss was recovered to < 20% within 1 month . comparing the aforementioned data from mice and guinea pigs", ", it appears that there may be some species difference in the ability to regenerate synapses following noise - induced hearing damage . however , a recent study of c57 mice reported that the loss of ribbon synapses induced by non - pts - inducing noise was largely reversible .", "this discrepancy in synapse regeneration following noise exposure requires further investigation . in a recent review", ", it was argued that the recovery of ctbp2/psd counts in guinea pig cochleae following noise exposure reported in our studies may be attributable to up / downregulation of the synaptic protein rather than regeneration of synaptic connections .", "however , there are several lines of evidence for the possibility of synapse repair following noise - induced damage .", "first , it has been reported that plastic changes occur in the presynaptic component , including the existence of multiple presynaptic ribbons around an active zone   and the changes in the size and location of ribbons following noise exposure .", "second , the change in the amplitude of the compound action potential ( cap ) corresponded to the changes in ribbon / psd counts : a large initial reduction in cap amplitude and synapse counts were followed by a significant recovery after the noise exposure .", "third , changes in many single - anf coding activities were not seen at the time that the synapses were damaged but rather manifested later ( see section 3 ) with the recovery in both the cap and synapse number , suggesting that those changes occurred in the anfs that connect ihcs via repaired / reestablished synapses .", "further work is required to determine the mechanisms and factors that influence the repair of both pre- and postsynaptic components ."], ["ribbon synapses exhibit spatial differences around ihcs ; that is , the synapses at the modiolar side of an ihc have relatively small ribbons but larger postsynaptic terminals , whereas those at the pillar side have relatively large ribbons but smaller terminals .", "liberman et al . reported that anfs are functionally categorized by their spontaneous rate ( sr ) , which is inversely related to the fiber 's threshold and dynamic range [ 6365 ] .", "it is widely accepted that low - sr anfs exhibit synapses with ihcs on their modiolar side , whereas high - sr units exhibit synapses on the pillar side ( this is based on data obtained using intracellular tracer injections ) .", "the low - sr units are considered critical for hearing in noisy environments due to their larger dynamic range , higher thresholds , and the ability to follow the quick change of the amplitude of acoustic signals . by contrast , high - sr units are responsible for the sensitivity to quiet sounds and are saturated by high - level background noise [ 26 , 63 , 64 , 67 , 68 ] . in nihhl ,", "low - sr anfs appear to be more vulnerable to noise than high - sr units .", "selective loss of low - sr anfs has been found following exposure to noise that did not lead to pts .", "presumably , this selective loss of low - sr units should produce coding deficits , which can be predicted based on the unique features of those units .", "however , no coding deficits were examined and reported in this study . on the other hand ,", "we reported a time delay in the development of coding deficits by single anfs in guinea pigs following a similar noise exposure that did not cause pts ; these deficits were attributed to intensity coding and temporal coding as summarized in sections 3.1 and 3.2 .", "intensity coding in the cochlea is defined as the ability of anfs to encode the sound intensity or the change of sound intensity .", "this ability is determined primarily by the spike rate ( or the change of spike rate ) of individual anf in response to sound intensity change and the number of functional anfs .", "therefore , the intensity coding deficits can be evaluated in both evoked field potential and single - unit recordings .", "deficits in intensity coding were first suggested by a reduction in wave i of the auditory brainstem response ( abr ) [ 6 , 49 ] , as well as a reduction in the amplitude of the cap , as this is likely due to the loss of functional anfs following synapse disruption .", "the fact that the reduction is more significant at higher sound levels has been considered evidence for selective damage to low - sr fibers , which have higher thresholds [ 25 , 26 , 69 ] .", "the deterioration in intensity coding following no - pts noise exposure was manifested as a reduction in the driven spike rates ( peak , sustained , and total rates ) of anf units that were tested only at one sound level .", "such changes are significant only in low - sr anf units and are seen at a later time rather than immediately following exposure .", "this time delay in the development of coding deficits suggests that ( 1 ) the reduction in driven spike rates occurs in the anfs to which the synaptic connections to the ihcs are reestablished following the initial disruption and ( 2 ) the repaired synapses are functionally abnormal , with less efficient neurotransmitter release .", "temporal processing ability in the cochlea as well as in the whole auditory pathway is defined as the ability to follow the quick change of acoustic signals . in human subjects ,", "the process involves both bottom - up and top - down mechanisms ; but in animal models , only bottom - up mechanisms are tested ( see reviewed by ) .", "many different tests have been used to detect the bottom - up mechanisms of temporal coding , some of them based on the peristimulatory changes of firing rate showing latency and adaptation . as reviewed above , the major function of presynaptic ribbons in ihcs is to facilitate the synaptic transmission .", "indeed , such deficits were manifested as an increase in response latency of anfs in animals with nihhl .", "this was first demonstrated as a significant delay in cap peak latency   and then further supported by the delayed latency of peak in psth ( or peak latency ) of anfs in our single - unit study . in another very recent report , such delay was reported in abr as the marker of cochlear synaptopathy .", "we also found a reduction in the ratio of peak to sustained rates in animals that were exposed to noise .", "this ratio is considered an index of the ability of a neuron to encode dynamic signal changes ( see review by ) . using a paired - click paradigm", ", we found that the anf response of noise - exposed animals to the second click recovered more slowly from the masking effect of the first click .", "these results reveal poorer coding to the transient features of acoustic signals by anfs , which were examined in previous studies to show the deterioration in of temporal coding in animals with bassoon mutation [ 39 , 41 ] .", "whereas an increase in peak latency was seen shortly after exposure to noise , changes in the peak rate and the peak / sustained spike ratio , as well as a slower recovery of the spike rate to the second click , were not seen until later , suggesting an association between the deficits and the synapse repair .", "a temporal deficit in phase - locking responses has been proposed based on selective loss of low - sr units and the functional features of this group of anfs [ 25 , 26 , 69 ] , but it has not been tested at the single - unit level ."], ["so far , there appear to be two models for the development of coding deficits in nihhl .", "one model suggests that the coding deficit or synaptopathy is simply due to the loss of low - sr anfs .", "since those units have unique functions in signal coding , the loss of those functions is predicted as the consequences .", "that is , the coding deficits are developed as the result of unhealthy synaptic repair after initial disruption .", "we found that the noise - induced synaptic damage in guinea pigs under nihhl is largely repairable , leaving only a small amount of synapses not being reestablished .", "therefore , the coding deficits or synaptopathy can not be simply attributed to the loss of sr units . since the coding deficits are seen at the time when the synapse counts are largely recovered , we believe that the coding deficits likely occur in the repaired synapses ( most of them innervating low - sr anfs ) .", "studies are needed to verify which model is more likely the case in human subjects ."], ["it has long been recognized that subjects with normal audiograms may have perceptual difficulties , and this is especially true in the elderly . age - related hearing loss with threshold elevation", "is termed peripheral presbycusis , whereas the perceptual difficulties seen in the elderly without threshold shift are usually termed central presbycusis . for example , temporal processing deficits and difficulties of hearing in noisy environments are two major problems experienced by the elderly .", "these problems were recognized long before the discovery of cochlear damage associated with nihhl and were considered to be the result of  central auditory processing disorders  [ 7175 ] .", "it was generally accepted that any perceptual deficits observed without changes to hearing thresholds and cognitive functioning can be attributed to central dysfunctions .", "based on recent progress in functional deficits in cochlear coding , such separation between peripheral and central presbycusis is likely to be incorrect .", "the so - called central presbycusis may , at least in part , result from disorders in the auditory periphery .", "the coding deficits related to the loss of low - sr anfs had been described as a type of auditory neuropathy and/or synaptopathy even before any of the predicted deficits were identified .", "data on changes in the sr distributions of anfs suggest the reestablishment of synapses following an initial disruption that was selective to low - sr units . although our data revealed abnormalities in some aspects of coding in the auditory nerve , further work is required to investigate coding deficits in nihhl .", "such studies can not be replaced by speculation based on the selective loss of low - sr fibers ; for example , one can not be certain how the auditory nerve changes its response to amplitude modulation until it is measured at the single - unit level .", "two possibilities must be considered : ( 1 ) the surviving anfs may change their function and ( 2 ) the initially lost low - sr fibers may be repaired but with changed function .", "it should be noted that there is now a tendency in the literature to consider nihhl to be a purely peripheral issue , a result of the overcorrection of the  central presbycusis .", " however , despite the strong evidence for a peripheral contribution , the central contribution to the problems seen in nihhl should not be neglected . in other words", ", it may be more constructive to assume that there are both peripheral and central contributions to nihhl .", "it is well known that hearing loss ( with elevated threshold ) can induce central changes , which can result in deteriorations in signal processing .", "studies aiming to distinguish the role of central plasticity from that of ribbon synapse damage are rare .", "one such report found that an increase in central gain was responsible for tinnitus in human subjects with typical damage seen in nihhl ( i.e. , reduced auditory nerve input to the brain ( measured as a smaller abr wave i ) ) but normal hearing threshold . in an earlier study in rats ,", "tinnitus was found 6 months after exposure to noise that caused minimal loss of hair cells and pts but significant loss of anfs .", "one of the central impacts of hearing loss due to damage to peripheral auditory organ is imbalance between excitation and inhibition , resulting in hyperactivity and/or hyperresponsiveness in the central auditory system ( see reviews in [ 7780 ] ) .", "the types of hearing loss producing such central enhancement include cochlear ablation , drug- and noise - induced damage . while direct effect of drugs and noise on central neurons needs to be differentiated , a similarity across those hearing loss models is the reduction of cochlea output to the auditory brain , which may be the main initial factor causing the imbalance between excitation and inhibition . in this sense ,", "while most of studies in central plasticity using nihl model correlated the central enhancement with the amount of threshold shifts [ 29 , 8185 ] , at least one study has reported central enhancement in mice exposed briefly to noise at a moderate level that did not cause pts , presumably producing only nihhl .", "unfortunately , the reduction in auditory input from the cochlea was not quantified in this study . taken together ,", "available data suggest that cochlear damage , with or without threshold elevation , can lead to central plasticity by reducing input from the auditory nerve .", "further work is required to establish the central contribution to coding / perception difficulties in nihhl , and previous studies on central processing disorders in subjects with nihl should be reevaluated to differentiate the central contributions from the peripheral ones . in a brief summary", ", we use figure 2 to summarize the available data for the mechanisms of perception difficulty experienced by subjects with history of noise exposure but normal or near normal thresholds . in this schematic diagram , we include the two potential models of noise - induced synaptopathy in cochleae . in model 1 , the coding deficits are speculated based on the role of low - sr anfs in signal coding .", "both models result in a reduction in the cochlear output to the auditory brain , which in turn will result in plastic reorganization of the brain .", "auditory signal processing disorders experienced by subjects with long - term nihhl should include what are inherited from the coding deficits developed in the auditory peripheral and those associated with the plastic changes of auditory brain ."], ["although more studies on the impact of noise on human hearing showing no changes in auditory sensitivity are required , evidence suggesting the occurrence of nihhl in human subjects is being accumulated .", "this is supported by thorough research on the signal perception deficits experienced by subjects with a history of noise exposure but normal thresholds .", "since the deficits are demonstrated at suprathreshold levels , it is clear that normal hearing thresholds do not guarantee normal hearing functions , especially in subjects with history of noise exposure [ 24 , 86 ] .", "the second line of evidence is the reduction in the output of the auditory nerve in subjects with a history of exposure to noise .", "interestingly , the combination of a reduction in wave i and an increase in wave v / i ratio may be considered evidence of increased central gain and is likely responsible for the generation of tinnitus in hidden hearing loss [ 32 , 87 , 88 ] .", "the third line of evidence comes from the age - related sgn degeneration seen in the examination of human temporal bones .", "unfortunately , there is , as yet , no clear human evidence that degeneration of sgns is expedited by exposure to noise that does not cause threshold elevation .", "the clinical implications of nihhl are manifested by the fact that noise exposure causing nihhl occurs frequently in daily life and impacts much more general population .", "such noise exposure has been generally considered to be safe according to current safety standards for exposure to noise .", "the evidence from the studies reviewed here indicates that the resulting damage to the ribbon synapses from noise that did not induce pts can be repaired even though the repair is incomplete .", "more importantly , the signal coding deficits are developed in association with the synapse repair . since the damage and repair occur repeatedly , the damage on signal coding can be accumulated during aging and likely contributes to the perceptual difficulties experienced by the elderly .", "this impact of noise exposure on signal coding is obviously different from the contribution made by the hearing loss defined by threshold shifts . in future", ", the coding deficits and related synaptic repair in nihhl should be further investigated in a laboratory setting .", "since the ribbon synapse is the first gating point for temporal processing in auditory pathway , the observed coding deficits suggest a clear peripheral origin for the decline in temporal processing and perceptual difficulties during aging . whether and how the synaptic damage will impact the central auditory processing need to be investigated in a manner that is clearly differentiated from the impact of hearing threshold shift .", "moreover , the coding function of anfs should be observed over a long period of time following exposure to noise to determine whether the coding deficits are temporary or persistent .", "we are currently collecting data using electron microscopy , as well as conducting an analysis on the potential changes of the molecular structures of ribbons and psds , in an attempt to elucidate the morphological / molecular mechanisms responsible for functional changes of repaired ribbon synapses .", "it would also be interesting to understand the reasons for the extreme sensitivity of low - sr synapses to noise , as well as elucidate possible methods to prevent damage .", "laboratory studies should also aim to explore the mechanisms of synaptic repair in the cochlea , as well as reveal the factors that influence repair in order to promote it . to translate the knowledge to clinic", ", investigation is needed to establish good measures for detecting nihhl in human subjects . although abr wave i is useful for evaluating synaptopathy caused by noise that does not induce pts , its reliability and sensitivity are questionable in human subjects where the abr amplitude is small , and other methods should be explored .", "a very recent report suggests the use of abr latency as the marker of nihhl .", "the study tested human subjects with normal hearing thresholds and reported a big variation in the threshold of envelope interaural timing difference , which was negatively correlated with the shift of abr wave v latency by background noise : the higher the threshold ( poorer sensitivity ) , the smaller the shift .", "the observation of the latency shift with masking is supported by the fact that the low - sr anfs have longer latency than high - sr fibers and are resistant to background noise [ 91 , 92 ] .", "it is not clear why the study did not report the change in wave v amplitude by masking .", "theoretically , the masking should produce greater reduction in wave v amplitude in subjects with selective loss of low - sr units .", "moreover , no information about the history of noise exposure was reported and it is not clear whether the poorer performance in temporal cue detection was due to noise - induced synaptopathy or other reasons . to date , the most promising methods for diagnosing cochlear synaptopathy are related to selective loss of low - sr anfs , the subcortical steady state responses ( sssr ) [ 93 , 94 ] .", "based on the animal studies , this test should be carried out using amplitude - modulated signals at relatively high intensity and a shallow modulation depth .", "the input intensity of the driving signal should fall within the saturation range of the high - sr fibers .", "high frequency carrier waves with a high intensity and with shallow amplitude modulation are especially useful for evaluating the function of low - sr fibers .", "this is supported by modeling the loss of low - sr fibers . to differentiate the sssr contribution from the auditory nerve from that of central neurons", "a recent mouse study found that the modulation frequency close to 1  khz was optimum with a high frequency carrier without concern of modulation depth .", "however , a recent human study reported a successful detection of the low - sr unit loss using off - frequency maskers and a shallow modulation depth ."]]}
{"article_id": "PMC4719364", "article_text": ["total talar dislocations are uncommon injuries and usually seen following high velocity injuries . total talar dislocations", "we hereby report a closed total talar dislocation in a 25 year old male without an associated fracture around ankle .", "a 25 year old male presented to orthopaedic causality with injury to right ankle following a road traffic accident .", "patient complained of severe pain and deformity of ankle following injury . on examination ankle was deformed and swollen .", "plain radiographs of right ankle joint revealed total anterolateral dislocation of talus without any accompanying ankle fracture .", "total talar dislocations are very rare injuries and should be treated as impending open fractures .", "there is no consensus on treatment of such complex injuries as very few cases have been reported in literature .", "talar fracture dislocations are very rare injuries accounting for 0.06% of all dislocation and 2% of all talar injuries and are usually associated with malleolar fracture or a talar fracture itself[1 - 2 ] .", "total dislocation of the talus bone from all of its three joints ( tibiotalar , subtalar , and talonavicular ) without a fracture is a extremely rare injury[3 - 5 ] .", "these type of injuries are usually caused by high velocity injury with disruption of almost all ligaments and capsular attachments of the talus .", "it usually leads to degenerative changes in neighbouring joints and frequently avascular necrosis is a predictable outcome .", "we present a case of total talus dislocation without any fracture which was treated by closed reduction and immobilization for six weeks .", "a 25 years old male presented to orthopaedic causality with injury to right ankle following a road traffic accident .", "patient complained of severe pain and deformity of ankle following injury . on examination ankle was deformed and swollen with very tense skin .", "plain radiographs revealed total anterolateral i dislocation of talus without accompanying ankle fracture [ fig.3 , 4 ] , ct scan with 3d reconstruction also confirmed our radiographic findings [ fig.5 ] . under spinal anaesthesia and fluoroscopic guidance ,", "closed reduction was performed by longitudinal traction and pushing the talus in the posteromedial direction . following", "a 3 mm k - wire was passed from calcaneum to tibia through talus [ fig.6 , 7 ] .", "plain radiographs at one year follow up showed no signs of avascular necrosis of talus or arthritis of subtalar joint .", "pre- operative clinical photograph showing talar dislocation pre operative clinical photograph showing abrasion over lateral malleolus .", "proposed treatments for total talus dislocation varied from primary talectomy or arthodesis to closed reduction and below knee cast .", "the few case reports found in the literature and the non - existent guidelines add to the confusion regarding the best method of treatment .", "depending on whether the foot comes to supination or pronation the dislocation can be either anterolateral ( the most usual ) or posteromedial .", "this type of injury is supposed to be in continuation of subtalar dislocation when the force magnifies and continues .", "dislocation of subtalar joint is the first stage of the injury . when the force progress , talonavicular joint dislocates and finally tibiotalar joint dislocation occurs . because of all capsular and ligamentous attachments of the talus are ruptured in this injury and dependence of talus bone vascularization on these ligaments , avascular necrosis of the talus is mostly predictable .", "there is a deference of opinion regarding best method of treatment for closed talar dislocations .", "most of the authors suggest open reduction for open talar dislocations [ table 1 ] .", "ritsema et al suggested open reduction as the best choice of treatment for closed injuries .", "anterolateral incision is preferred approach for open reduction as it gives an excellent approach to ankle joint .", "taymaz and gunal reported a case of closed total talus dislocation treated by closed means with excellent result .", "hadji et al . also reported good result after closed reduction of a complete talus dislocation on a three year follow - up .", "total talar dislocations are very rare injuries seen following a high velocity injuries and are associated with ankle fractures .", "closed reduction at the earliest is the best method of treatment for closed talar dislocations ."], "abstract_text": ["<S> introduction : total talar dislocations are uncommon injuries and usually seen following high velocity injuries . </S>", "<S> total talar dislocations ( missing talus ) without fractures around the ankle are extremely rare . </S>", "<S> there are no consensuses on the best treatment of such injuries . </S>", "<S> to best of our knowledge very few cases have been reported in literature . </S>", "<S> we hereby report a closed total talar dislocation in a 25 year old male without an associated fracture around ankle . </S>", "<S> he was managed with emergency closed reduction and below knee splint . at one year </S>", "<S> follow up there were no complications.case report : a 25 year old male presented to orthopaedic causality with injury to right ankle following a road traffic accident . </S>", "<S> patient complained of severe pain and deformity of ankle following injury . on examination ankle was deformed and swollen . </S>", "<S> plain radiographs of right ankle joint revealed total anterolateral dislocation of talus without any accompanying ankle fracture . </S>", "<S> ct scan with 3d reconstruction also confirmed our radiographic findings . under spinal anaesthesia and fluoroscopic guidance closed reduction </S>", "<S> was performed . to maintain reduction </S>", "<S> a 3 mm k - wire was passed from calcaneum to tibia through talus . </S>", "<S> a posterior below knee splint was applied . </S>", "<S> patient was followed every three months till one year . at one year follow up </S>", "<S> patient had good range of motion at ankle and subtalar joint.conclusion:total talar dislocations are very rare injuries and should be treated as impending open fractures . </S>", "<S> there is no consensus on treatment of such complex injuries as very few cases have been reported in literature . </S>"], "labels": null, "section_names": ["Introduction:", "Case Report:", "Conclusion:", "Introduction", "Case Report", "Discussion", "Conclusion"], "sections": [["total talar dislocations are uncommon injuries and usually seen following high velocity injuries . total talar dislocations", "we hereby report a closed total talar dislocation in a 25 year old male without an associated fracture around ankle ."], ["a 25 year old male presented to orthopaedic causality with injury to right ankle following a road traffic accident .", "patient complained of severe pain and deformity of ankle following injury . on examination ankle was deformed and swollen .", "plain radiographs of right ankle joint revealed total anterolateral dislocation of talus without any accompanying ankle fracture ."], ["total talar dislocations are very rare injuries and should be treated as impending open fractures .", "there is no consensus on treatment of such complex injuries as very few cases have been reported in literature ."], ["talar fracture dislocations are very rare injuries accounting for 0.06% of all dislocation and 2% of all talar injuries and are usually associated with malleolar fracture or a talar fracture itself[1 - 2 ] .", "total dislocation of the talus bone from all of its three joints ( tibiotalar , subtalar , and talonavicular ) without a fracture is a extremely rare injury[3 - 5 ] .", "these type of injuries are usually caused by high velocity injury with disruption of almost all ligaments and capsular attachments of the talus .", "it usually leads to degenerative changes in neighbouring joints and frequently avascular necrosis is a predictable outcome .", "we present a case of total talus dislocation without any fracture which was treated by closed reduction and immobilization for six weeks ."], ["a 25 years old male presented to orthopaedic causality with injury to right ankle following a road traffic accident .", "patient complained of severe pain and deformity of ankle following injury . on examination ankle was deformed and swollen with very tense skin .", "plain radiographs revealed total anterolateral i dislocation of talus without accompanying ankle fracture [ fig.3 , 4 ] , ct scan with 3d reconstruction also confirmed our radiographic findings [ fig.5 ] . under spinal anaesthesia and fluoroscopic guidance ,", "closed reduction was performed by longitudinal traction and pushing the talus in the posteromedial direction . following", "a 3 mm k - wire was passed from calcaneum to tibia through talus [ fig.6 , 7 ] .", "plain radiographs at one year follow up showed no signs of avascular necrosis of talus or arthritis of subtalar joint .", "pre- operative clinical photograph showing talar dislocation pre operative clinical photograph showing abrasion over lateral malleolus ."], ["proposed treatments for total talus dislocation varied from primary talectomy or arthodesis to closed reduction and below knee cast .", "the few case reports found in the literature and the non - existent guidelines add to the confusion regarding the best method of treatment .", "depending on whether the foot comes to supination or pronation the dislocation can be either anterolateral ( the most usual ) or posteromedial .", "this type of injury is supposed to be in continuation of subtalar dislocation when the force magnifies and continues .", "dislocation of subtalar joint is the first stage of the injury . when the force progress , talonavicular joint dislocates and finally tibiotalar joint dislocation occurs . because of all capsular and ligamentous attachments of the talus are ruptured in this injury and dependence of talus bone vascularization on these ligaments , avascular necrosis of the talus is mostly predictable .", "there is a deference of opinion regarding best method of treatment for closed talar dislocations .", "most of the authors suggest open reduction for open talar dislocations [ table 1 ] .", "ritsema et al suggested open reduction as the best choice of treatment for closed injuries .", "anterolateral incision is preferred approach for open reduction as it gives an excellent approach to ankle joint .", "taymaz and gunal reported a case of closed total talus dislocation treated by closed means with excellent result .", "hadji et al . also reported good result after closed reduction of a complete talus dislocation on a three year follow - up ."], ["total talar dislocations are very rare injuries seen following a high velocity injuries and are associated with ankle fractures .", "closed reduction at the earliest is the best method of treatment for closed talar dislocations ."]]}
{"article_id": "PMC4540885", "article_text": ["they have been suggested as the \n main cause of time off from work , reduced school performance , and low quality of life1,2,3 .", "furthermore , they have led to personal , \n familial and societal burdens , and significant healthcare problems globally4 , 5 . \n", "the prevalence of headaches is estimated at 13% of the united states population6 , 20% of the australians1 , and migraines are estimated at 11% , with tension - type \n headaches at 78% of the population world - wide5 , 7 . according to the international headache society , headaches can generally be divided into \n two categories which are primary and secondary headaches on the basis of the underlying \n pathology8 .", "primary headaches are not \n associated with pre - existing medical conditions and there are three types : migraines , \n tension - headaches and cluster - headaches6 . \n", "the remaining headache - sufferers \n discontinue medications due to adverse side - effects or excessive use of abortive \n medications .", "these can lead to a refractory condition of medication overuse headache , which \n means a consequent worsening of the headaches10 . as a result of these shortcomings ,", "complementary and alternative \n medicine has recently become common practice in current headache management6 , 10,11,12,13 .", "yoga exercises are considered to be complementary and alternative medicine and are \n practiced by approximately 5% of the adult population in the united states and 12% of \n australians for alleviating headaches14 . \n", "yoga has been reported \n as a safe and cost - effective intervention for managing pain1 , 14 .", "evidence for the \n efficacy of yoga exercise for a number of conditions is emerging .", "a growing body of evidence \n also supports the belief that yoga benefits physical and psychosocial health through the \n mechanisms of down - regulation of the hypothalamic - pituitary - adrenal axis and the sympathetic \n nervous system15,16,17 . as a result ,", "yoga plays \n an important role in reducing sympathetic activity , increasing parasympathetic activity , \n improving quality of life , and decreasing pain levels18 , 19 .", "as stated , there is \n evidence of the benefit of yoga in reducing pain20 , \n 21 .", "however , rigorous methodology and \n quality of the evidence needs to be examined to establish whether or not we can assert yoga \n can be used as a complementary and alternative therapy for sufferers of headaches22 , 23 .", "therefore , the aim of this review was to assess the evidence for the \n effectiveness of yoga exercises in the management of primary headaches .", "the review was planned and conducted in accordance with the preferred reporting items for \n systematic reviews and meta - analyses ( prisma ) guidelines24 , and the consolidated standards of reporting trials ( consort ) \n guidelines for reporting parallel group randomized trials25 .", "the cochrane library , cinahl , embase , psycinfo , pubmed , and \n koreamed electronic databases were searched to identify rcts published between 1966 and \n january 2015 .", "all \n potentially eligible studies were retrieved and the full texts of the articles were reviewed \n to determine whether they met the following selection criteria . to be eligible , studies had to meet the following conditions .", "1 ) population : participants \n in the trials had to meet diagnostic criteria according to the international classification \n of headache disorders , 3rd edition ( beta version ) published by the international headache \n society 20138 ; primary headaches .", "2 ) \n intervention : randomized controlled trials were included that used yoga as an intervention \n to review or reduce symptoms associated with headaches or migraines compared with no yoga . \n", "3 ) outcomes : primary outcomes were headache intensity , frequency , and duration ; secondary \n outcomes were anxiety and depression scores , and symptomatic medication use .", "quality \n assessment of the articles was conducted using the critical appraisal , cochrane risk of bias \n tool for rcts , which was recommended by the cochrane handbook for systematic reviews of \n interventions26 .", "the cochrane risk of \n bias tool is a six - item list designed to assess sequence generation , allocation concealment , \n blinding , incomplete outcome data , selective outcome reporting , and other potential sources \n of bias .", "each item is rated as  yes  ,  no  , or  unclear  .", "according to the cochrane \n handbook , the quality of clinical trials can be divided into three levels27 . when the study design fully meets the \n preceding six criteria , it is considered a level , which means a low risk of bias .", "b level is \n assigned when one or more criteria are partly met , and when one or more criteria are not \n met , the study is defined as c level , implying high risk of bias .", "a total of 179 titles related to the search terms were screened . among these , there were 32 \n potential trials identified from chinal , 52 from koreamed , 43 from psycinfo and 52 from the \n pubmed databases .", "after the titles had been retrieved a total of 121 studies were excluded \n either because they were duplicates or they were case studies , commentaries , review \n articles , or had no target concepts , which means no headaches or migraines .", "thirty - four potential trials were identified in the search \n conducted in january 2015 .", "thirty - four potentially relevant papers were retrieved for \n evaluation of the full text .", "after evaluation of the 34 full texts , 33 studies were \n excluded , because 30 studies had no randomized trials and 3 studies had no full text of rct . \n", ". the characteristics of the included study are also presented ( table 1table 1.characteristics of included randomized controlled trialsauthor , year , location participantsinterventionsoutcome measuresmain resultsadverse events limitationsexperimental groupcontrol grouppopulation  sample sizen  ( n ; eg / cg)n  mean age ( years ; \n eg / cg)n  drop out n ( % ) interventions  delivery method  duration \n interventionistjohn et al.,2013 , india/ rajasthangeneral person72 ( 36/36)34.2 ( 34.3/34.2)7 \n ( 9.7)yoga postures , pranayama , kriya 60 min per day , 5 days \n per week 3 months yoga therapistself - care education once a month 3 months handouts headache intensityheadache frequencyheadache \n durationanxiety - depression scoressymptomatic medication use(p<0.001)(p<0.001)(p<0.001)(p<0.001)noneabsence of a placebo groups.no blinding.all \n subjective outcome measures.no long - term follow - up dataeg : experimental group ; cg : control group ; n : number ) . flowchart of included studies through the literature searches eg : experimental group ; cg : control group ;", "participants were recruited \n from a headache clinic of the nmp medical research institute by advertising in local \n newspapers .", "the participants mean age \n was 34.2  years , and they had primary headaches with migraines . the yoga program comprised yoga postures , breathing and pranayama , and kriya etc .", "yoga \n postures included physical exercises such the stretching of the neck , shoulder and back \n muscles , followed by relaxation , toning , strengthening , and flexibility .", "breathing and \n pranayama means conscious breathing , kriya was practiced as a jalaneti ( nasal water \n cleansing ) and kapalbhanti ( forced exhalations ) .", "program length , frequency , and duration of \n one trial was 60 minutes a day , 5 days per week for 3 months .", "one trial was identified that compared a control group with a yoga intervention group and \n evaluated the effect on headaches .", "headache intensity ( p<0.001 ) , headache frequency ( p<0.001 ) , anxiety and \n depression scores ( p<0.001 ) , and symptomatic medication use ( p<0.001 ) were \n significantly lower in the intervention group then in the control group ( table 1 ) .", "neither included trial reported data on adverse effects \n of treatment ( table 1 ) .", "assessments of each methodological quality item of the one included trial are described \n ( table 2table 2.methodological quality summary of included trialsstudy , yearrandom allocationallocation concealmentblindingincomplete outcomeselective reportingother biasquality leveljohn et al .", "the quality of the one trial was level b. + : criteria met ; - : criteria not met ; ? : unclear whether criteria were met", "the purpose of this review was to assess the evidence for the effectiveness of yoga \n interventions for primary headaches when compared to no yoga .", "a meta - analysis combining \n results from all the trials was not possible because only one study was identified .", "its interventions included yoga poses , \n pranayama , and kriya to manage headaches or migraines .", "one trial reported a significant \n decrease in headache intensity , headache frequency , anxiety and depression scores , and \n symptomatic medication use in the trained group .", "if required , participants were allowed to \n take acute medication prescribed by neurologists during the trial .", "the effects of the \n medication could have diminished the efficacy of yoga exercises for alleviating headaches . \n in spite of", "both groups having received medication , reduction in the outcome of the yoga \n group was significantly higher than that of the control group .", "as stated in previous \n studies , these results support yoga practice as a means of evidence - based positive \n management of headaches or migraines20 , 22 , 28 , 29 .", "the quality rating of the trial included \n in this review had a moderate methodological quality , and the trial did not mention \n blinding . however , no strong conclusion can be made due to the number of small trials and \n other methodological considerations .", "major strengths of this group of studies include the study , the use of randomization , and \n the quality of measurement tools .", "second , the trial did not mention blinding , lack of \n which may have threatened the internal validity of the trial .", "third , all the outcome \n measurements were questioner - based and subjective ; objective parameters were lacking . \n therefore , evidence - based research employing objective outcome parameters is needed to \n identify the efficacy of integrated yoga therapy for headaches .", "fourth , the trial had no \n long - term follow - up data concerning the durability of the treatment effect . finally , the \n generalizability of the findings was limited due to the number of small trials and their \n partially limited quality . however ,", "this one trial does provide a strong basis for future \n studies and suggests that yoga exercises could provide a safe , cost - effective therapy for \n the growing public health issue of headaches .", "furthermore , this review contributes to the \n development of knowledge in physical therapy about how sufferers with primary headaches can \n manage themselves . in conclusion , although this review retrieved only a limited number of small trials , of \n partially limited quality , its findings suggest that yoga practice can effectively alleviate \n symptoms associated with primary headaches .", "however further rigorous methodological and \n high - quality rcts are needed to confirm and further comprehend the effects of standardized \n yoga programs aiming to control pain intensity and frequency , symptoms , and medication use \n etc ."], "abstract_text": ["<S> [ purpose ] to assess the evidence for the effectiveness of yoga exercises in the \n management of headaches . </S>", "<S> [ subjects and methods ] a search was conducted of six electronic \n databases to identify randomized controlled trials ( rcts ) reporting the effects of yogic \n intervention on headaches published in any language before january 2015 . </S>", "<S> quality \n assessment was conducted using the cochrane risk of bias tool . </S>", "<S> [ results ] one potential \n trial was identified and included in this review . </S>", "<S> the quality critical appraisal indicated \n a moderate risk of bias . </S>", "<S> the available data could only be included as a narrative \n description . </S>", "<S> headache intensity and frequency , anxiety and depression scores , and \n symptomatic medication use were significantly lower in the yoga group compared to the \n control group . </S>", "<S> [ conclusion ] there is evidence from one rct that yoga exercises may be \n beneficial for headaches . </S>", "<S> however , the findings should be interpreted with caution due to \n the small number of rcts . </S>", "<S> therefore , further rigorous methodological and high quality rcts \n are required to investigate the hypothesis that yoga exercises alleviate headaches , and to \n confirm and further comprehend the effects of standardized yoga programs on headaches . </S>"], "labels": null, "section_names": ["INTRODUCTION", "SUBJECTS AND METHODS", "RESULTS", "DISCUSSION"], "sections": [["they have been suggested as the \n main cause of time off from work , reduced school performance , and low quality of life1,2,3 .", "furthermore , they have led to personal , \n familial and societal burdens , and significant healthcare problems globally4 , 5 . \n", "the prevalence of headaches is estimated at 13% of the united states population6 , 20% of the australians1 , and migraines are estimated at 11% , with tension - type \n headaches at 78% of the population world - wide5 , 7 . according to the international headache society , headaches can generally be divided into \n two categories which are primary and secondary headaches on the basis of the underlying \n pathology8 .", "primary headaches are not \n associated with pre - existing medical conditions and there are three types : migraines , \n tension - headaches and cluster - headaches6 . \n", "the remaining headache - sufferers \n discontinue medications due to adverse side - effects or excessive use of abortive \n medications .", "these can lead to a refractory condition of medication overuse headache , which \n means a consequent worsening of the headaches10 . as a result of these shortcomings ,", "complementary and alternative \n medicine has recently become common practice in current headache management6 , 10,11,12,13 .", "yoga exercises are considered to be complementary and alternative medicine and are \n practiced by approximately 5% of the adult population in the united states and 12% of \n australians for alleviating headaches14 . \n", "yoga has been reported \n as a safe and cost - effective intervention for managing pain1 , 14 .", "evidence for the \n efficacy of yoga exercise for a number of conditions is emerging .", "a growing body of evidence \n also supports the belief that yoga benefits physical and psychosocial health through the \n mechanisms of down - regulation of the hypothalamic - pituitary - adrenal axis and the sympathetic \n nervous system15,16,17 . as a result ,", "yoga plays \n an important role in reducing sympathetic activity , increasing parasympathetic activity , \n improving quality of life , and decreasing pain levels18 , 19 .", "as stated , there is \n evidence of the benefit of yoga in reducing pain20 , \n 21 .", "however , rigorous methodology and \n quality of the evidence needs to be examined to establish whether or not we can assert yoga \n can be used as a complementary and alternative therapy for sufferers of headaches22 , 23 .", "therefore , the aim of this review was to assess the evidence for the \n effectiveness of yoga exercises in the management of primary headaches ."], ["the review was planned and conducted in accordance with the preferred reporting items for \n systematic reviews and meta - analyses ( prisma ) guidelines24 , and the consolidated standards of reporting trials ( consort ) \n guidelines for reporting parallel group randomized trials25 .", "the cochrane library , cinahl , embase , psycinfo , pubmed , and \n koreamed electronic databases were searched to identify rcts published between 1966 and \n january 2015 .", "all \n potentially eligible studies were retrieved and the full texts of the articles were reviewed \n to determine whether they met the following selection criteria . to be eligible , studies had to meet the following conditions .", "1 ) population : participants \n in the trials had to meet diagnostic criteria according to the international classification \n of headache disorders , 3rd edition ( beta version ) published by the international headache \n society 20138 ; primary headaches .", "2 ) \n intervention : randomized controlled trials were included that used yoga as an intervention \n to review or reduce symptoms associated with headaches or migraines compared with no yoga . \n", "3 ) outcomes : primary outcomes were headache intensity , frequency , and duration ; secondary \n outcomes were anxiety and depression scores , and symptomatic medication use .", "quality \n assessment of the articles was conducted using the critical appraisal , cochrane risk of bias \n tool for rcts , which was recommended by the cochrane handbook for systematic reviews of \n interventions26 .", "the cochrane risk of \n bias tool is a six - item list designed to assess sequence generation , allocation concealment , \n blinding , incomplete outcome data , selective outcome reporting , and other potential sources \n of bias .", "each item is rated as  yes  ,  no  , or  unclear  .", "according to the cochrane \n handbook , the quality of clinical trials can be divided into three levels27 . when the study design fully meets the \n preceding six criteria , it is considered a level , which means a low risk of bias .", "b level is \n assigned when one or more criteria are partly met , and when one or more criteria are not \n met , the study is defined as c level , implying high risk of bias ."], ["a total of 179 titles related to the search terms were screened . among these , there were 32 \n potential trials identified from chinal , 52 from koreamed , 43 from psycinfo and 52 from the \n pubmed databases .", "after the titles had been retrieved a total of 121 studies were excluded \n either because they were duplicates or they were case studies , commentaries , review \n articles , or had no target concepts , which means no headaches or migraines .", "thirty - four potential trials were identified in the search \n conducted in january 2015 .", "thirty - four potentially relevant papers were retrieved for \n evaluation of the full text .", "after evaluation of the 34 full texts , 33 studies were \n excluded , because 30 studies had no randomized trials and 3 studies had no full text of rct . \n", ". the characteristics of the included study are also presented ( table 1table 1.characteristics of included randomized controlled trialsauthor , year , location participantsinterventionsoutcome measuresmain resultsadverse events limitationsexperimental groupcontrol grouppopulation  sample sizen  ( n ; eg / cg)n  mean age ( years ; \n eg / cg)n  drop out n ( % ) interventions  delivery method  duration \n interventionistjohn et al.,2013 , india/ rajasthangeneral person72 ( 36/36)34.2 ( 34.3/34.2)7 \n ( 9.7)yoga postures , pranayama , kriya 60 min per day , 5 days \n per week 3 months yoga therapistself - care education once a month 3 months handouts headache intensityheadache frequencyheadache \n durationanxiety - depression scoressymptomatic medication use(p<0.001)(p<0.001)(p<0.001)(p<0.001)noneabsence of a placebo groups.no blinding.all \n subjective outcome measures.no long - term follow - up dataeg : experimental group ; cg : control group ; n : number ) . flowchart of included studies through the literature searches eg : experimental group ; cg : control group ;", "participants were recruited \n from a headache clinic of the nmp medical research institute by advertising in local \n newspapers .", "the participants mean age \n was 34.2  years , and they had primary headaches with migraines . the yoga program comprised yoga postures , breathing and pranayama , and kriya etc .", "yoga \n postures included physical exercises such the stretching of the neck , shoulder and back \n muscles , followed by relaxation , toning , strengthening , and flexibility .", "breathing and \n pranayama means conscious breathing , kriya was practiced as a jalaneti ( nasal water \n cleansing ) and kapalbhanti ( forced exhalations ) .", "program length , frequency , and duration of \n one trial was 60 minutes a day , 5 days per week for 3 months .", "one trial was identified that compared a control group with a yoga intervention group and \n evaluated the effect on headaches .", "headache intensity ( p<0.001 ) , headache frequency ( p<0.001 ) , anxiety and \n depression scores ( p<0.001 ) , and symptomatic medication use ( p<0.001 ) were \n significantly lower in the intervention group then in the control group ( table 1 ) .", "neither included trial reported data on adverse effects \n of treatment ( table 1 ) .", "assessments of each methodological quality item of the one included trial are described \n ( table 2table 2.methodological quality summary of included trialsstudy , yearrandom allocationallocation concealmentblindingincomplete outcomeselective reportingother biasquality leveljohn et al .", "the quality of the one trial was level b. + : criteria met ; - : criteria not met ; ? : unclear whether criteria were met"], ["the purpose of this review was to assess the evidence for the effectiveness of yoga \n interventions for primary headaches when compared to no yoga .", "a meta - analysis combining \n results from all the trials was not possible because only one study was identified .", "its interventions included yoga poses , \n pranayama , and kriya to manage headaches or migraines .", "one trial reported a significant \n decrease in headache intensity , headache frequency , anxiety and depression scores , and \n symptomatic medication use in the trained group .", "if required , participants were allowed to \n take acute medication prescribed by neurologists during the trial .", "the effects of the \n medication could have diminished the efficacy of yoga exercises for alleviating headaches . \n in spite of", "both groups having received medication , reduction in the outcome of the yoga \n group was significantly higher than that of the control group .", "as stated in previous \n studies , these results support yoga practice as a means of evidence - based positive \n management of headaches or migraines20 , 22 , 28 , 29 .", "the quality rating of the trial included \n in this review had a moderate methodological quality , and the trial did not mention \n blinding . however , no strong conclusion can be made due to the number of small trials and \n other methodological considerations .", "major strengths of this group of studies include the study , the use of randomization , and \n the quality of measurement tools .", "second , the trial did not mention blinding , lack of \n which may have threatened the internal validity of the trial .", "third , all the outcome \n measurements were questioner - based and subjective ; objective parameters were lacking . \n therefore , evidence - based research employing objective outcome parameters is needed to \n identify the efficacy of integrated yoga therapy for headaches .", "fourth , the trial had no \n long - term follow - up data concerning the durability of the treatment effect . finally , the \n generalizability of the findings was limited due to the number of small trials and their \n partially limited quality . however ,", "this one trial does provide a strong basis for future \n studies and suggests that yoga exercises could provide a safe , cost - effective therapy for \n the growing public health issue of headaches .", "furthermore , this review contributes to the \n development of knowledge in physical therapy about how sufferers with primary headaches can \n manage themselves . in conclusion , although this review retrieved only a limited number of small trials , of \n partially limited quality , its findings suggest that yoga practice can effectively alleviate \n symptoms associated with primary headaches .", "however further rigorous methodological and \n high - quality rcts are needed to confirm and further comprehend the effects of standardized \n yoga programs aiming to control pain intensity and frequency , symptoms , and medication use \n etc ."]]}
{"article_id": "PMC3165150", "article_text": ["staphylococcus ( s. ) intermedius and s. pseudintermedius are both staphylococcus intermedius group ( sig ) members .", "these microorganisms are opportunistic pathogens that cause skin and nosocomial infections in dogs and cats .", "s. pseudintermedius is the predominant sig species in korea and until recently was misclassified as s. intermedius .", "increased identification of methicillin - resistant sig ( mrsig ) isolates has emphasized the importance of public health precautions for veterinary staff and pet owners .", "many current studies have evaluated methicillin - resistant characteristics and the staphylococcal cassette chromosome mec ( sccmec ) of sig isolates , particularly s. pseudintermedius .", "although type i to type viii sccmec types have been identified , there is no significant studies have been conducted to examine sccmec types isolated from veterinary hospitals located nationwide in korea .", "sccmec types may produce staphylococcal exotoxins including staphylococcal enterotoxins ( ses ) , exfoliative toxins ( ets ) including s. intermedius exfoliative toxin ( siet ) , and toxic shock syndrome toxin 1 ( tsst 1 ) ; these toxins are associated with atopic dermatitis , mastitis , food poisoning , canine pyoderma , and chronic otits .", "siet ( encoded by the siet gene ) , first described by terauchi et al . , plays a potential role in the pathogenesis of canine pyoderma and chronic otitis .", "there may also be a greater chance of sig isolates to colonization and/or expand in veterinary staff , companion animals , and hospital environments .", "therefore , this study is focused on the genetic identification of staphylococcal exotoxins , sccmec types , and genetic relatedness by pulsed - field gel electrophoresis ( pfge ) of the sig isolates in korea .", "samples of s. pseudintermedius ( n = 167 ) and s. intermedius ( n = 11 ) were isolated ; their identity was confirmed by gram - staining and biochemical testing such as coagulase , dnase production and hydrolysis", ". polymerase chain reaction ( pcr ) was carried out with primers targeting s. intermedius nuclease and 16s rrna genes . to differentiate s. pseudintermedius from s. intermedius and s. delphini , pcr - restriction fragment length polymorphism analysis was performed using pta gene - specific primers ( pta_f1:5'-aaa gac aaa ctt tca ggt aa-3 ' , and pta_r1 : 5'-gca taa aca agc att gta ccg-3 ' ) , and the restriction enzyme mboi ( new england biolabs , usa ) .", "samples were collected from eight veterinary hospitals from four different regions in korea between november 2006 and january 2010 as previously described . pcr analysis to detect the methicillin - resistance gene ( meca )", "pcr was also performed to amplify genes encoding sea ( sea ) , seb ( seb ) , sec ( sec ) , sed ( sed ) , see ( see ) , seg ( seg ) , seh ( seh ) , sei ( sei ) , tsst 1(tsst-1 ) and ets ( eta , etb , etd , and siet ) using primers and pcr conditions used in other previous studies .", "fri913 , fri 361 , fri 472 , fri 569 , mnhoch , and rn4220 strains were used as either positive or negative controls for superantigen genes . a superantigen gene obtained in this study", "was further analyzed by dna sequencing using the vector nti align x program ( invitrogen , usa ) .", "all mrsig isolates including 49 methicillin - resistant s. pseudintermedius ( mrsp ) and three methicillin - resistant s. intermedius ( mrsi ) were subsequently classified as sccmec type i to viii by either single or multiplex pcr assays using protocols in other previous studies including primers and pcr conditions .", "genomic dna plug kits ( bio - rad , usa ) with smai ( new england biolabs , usa ) digestion were used for the pfge analysis of 39 s. pseudintermedius isolates from a private referral veterinary hospital collected at different times ( eight isolates from november 2006 , 24 isolates from april 2008 , four isolates from june 2009 , and three isolates from october 2009 ) and six s. intermedius isolates ( all collected during april 2008 ) .", "pfge was performed according to protocol used in previous study   and the manufacture 's instruction ( bio - rad , usa ) except for an initial pulse time of 5 sec and final time of 40 sec for 21 h .", "analysis of the sig isolates ( n = 178 ) for ses and tsst 1 genes showed that only a single s. pseudintermedius isolate from a veterinary staff member ( h1 - 23 ) harbored the enterotoxin c ( sec ) gene ( fig .", "2 ) revealed that the amplified sec gene the canine type c se gene ( seccaine ) .", "a total of 166 out of 178 sig isolates ( 155 s. pseudintermedius and 11 s. intermedius ) isolated from veterinary staff members ( n = 38 , 95% ) , companion animals ( n = 107 , 93% ) , and veterinary hospitals ( n = 21 , 91.3% ) harbored siet originally detected in s. intermedius .", "however , other et genes such as eta , etb , and etd , known to originate from s. aureus , were not identified .", "sccmec typing of the 49 mrsp isolates identified 38 type v isolates , one type iv isolate , and 10 non - identifiable isolates .", "pfge analysis showed that sig isolates from a private referral animal hospital ( collected between 2006 and 2009 ) recovered from veterinary staff , companion animals , and the environment had the same band patterns ( fig .", "3 ) . six s. pseudintermedius isolates ( two from companion animals collected in september 2006 , three from veterinary staff members , and one from companion animals collected in april 2008 ) and two s. pseudintermedius isolates ( one from a veterinary hospital environment isolated in april 2008 , and one from a companion animal isolated in june 2009 ) showed the same band patterns ( fig .", "since sig is closely related to s. aureus , studies have been performed to determine whether sig has adapted the eta , etb and etd toxins from s. aureus by pcr test with specific primers .", "however , 166 out of 178 ( 93.3% ) sig isolates harbored the siet toxin originating from s. intermedius .", "this result and those of other studies   imply that the majority of sig isolates harbor the siet gene .", "although the siet gene was present in 93.3% of the sig isolates ( from 108 dogs and two cats ) in this study , only 14 dogs had a history of various skin disease including allergy and prolonged inflammation lesion in skin ( n = 13 ) or otitis ( n = 1 ) ( data not shown ) .", "therefore , other factors such as the general health of an animal and existence of other sig virulence factors may play an important role in outbreaks of various kinds of skin disease or otitis .", "the sec gene was detected in a single isolate in the present study , which was identified as seccanine by dna sequencing .", "however , this isolate was isolated from a veterinary staff member , and no additional seccanine isolates were identified in the veterinary hospital where this individual worked .", "this suggested that the isolate might be transmitted from an in- or outgoing companion animal with which the veterinary staff was in contact .", "the low incidence of toxins in this study could be secondary to the small number of isolates collected from companion animals that a history of skin disease or otitis .", "only 11.3% of s. pseudintermedius in a previous study had exotoxins although all samples were taken from patients diagnosed with pyoderma or chronic otitis and referred to a veterinary teaching hospital .", "although the majority of sccmec types were type v ( 78.9% ) , one isolate was type iv . in a previous study ,", "23 isolates ( 85.2% ) , three isolates ( 11.1% ) and one isolate ( 3.7% ) from veterinarians , staff , students , companion animals and environment in the veterinary hospitals were determined to be an mrsp hybrid sccmec type i~ii , type v , or non - identifiable , respectively .", "a previous european and north american study   identified 75 hybrid sccmec type ii~iii isolates ( 72.8% ) , two type iii isolates ( 1.9% ) , six type iv isolates ( 5.8% ) , 14 type v isolates ( 13.6% ) , four type vii isolates ( 3.9% ) , and two non - identifiable isolates ( 1.9% ) from diseased and healthy dogs in veterinary diagnostic laboratories of different countries .", "this demonstrated that the majority of mrsig isolates in korea harbor the sccmec type v whereas the hybrid type ii~iii is the main sccmec type found in veterinary hospitals in japan , europe , and north america . in the present study , pfge analysis of the 39 s. pseudintermedius and six s. intermedius isolates from a private referral veterinary hospital ( collected during november 2006 , april 2008 , june 2009 , and october 2009 ) showed that 20 isolates ( lines 1~6 , lines 7~9 , lines 10~15 , lines 16~18 , and lines 19~20 ) had the same band patterns .", "moreover , some isolates obtained on different sampling dates showed the same band patterns ( six isolates from lines 12~17 , and two isolates from lines 21~22 ) .", "these results suggest potential contamination or expansion of s. pseudintermedius and s. intermedus isolates among veterinary staff , companion animals , and veterinary hospital environments , and colonization by these specific strains for more than 13 or 18 months in the same hospital . in conclusion ,", "eta , etb , etd genes were not detected but siet toxin was found in 166 isolates in the current study .", "pfge analysis results of isolated from h animal hospital showed that s. pseudintermedius isolates collected in over a period of 13 and 18 months from veterinary staff , companion animals , and the hospital environment had the same band patterns .", "s. pseudintermedius infections in humans , spread of mrsp populations , and association of sig with canine pyoderma and chronic otitis   has been previously reported .", "therefore , sig , especially mrsig , may have significant clinical implications for companion animals with skin infections or chronic otitis that is of concern for veterinary staff , companion animal owners , and healthy companion animals ."], "abstract_text": ["<S> the staphylococcus ( s. ) intermedius group ( sig ) has been a main research subject in recent years . </S>", "<S> s. pseudintermedius causes pyoderma and otitis in companion animals as well as foodborne diseases . to prevent sig - associated infection and disease outbreaks , identification of both staphylococcal exotoxins and staphylococcal cassette chromosome mec ( sccmec ) types among sig isolates may be helpful . in this study </S>", "<S> , it was found that a single isolate ( one out of 178 sig isolates examined ) harbored the canine enterotoxin sec gene . </S>", "<S> however , the s. intermedius exfoliative toxin gene was found in 166 sig isolates although the s. aureus - derived exfoliative toxin genes , such as eta , etb and etd , were not detected . </S>", "<S> sccmec typing resulted in classifying one isolate as sccmec type iv , 41 isolates as type v ( including three s. intermedius isolates ) , and 10 isolates as non - classifiable . </S>", "<S> genetic relatedness of all s. pseudintermedius isolates recovered from veterinary staff , companion animals , and hospital environments was determined by pulsed - field gel electrophoresis . </S>", "<S> strains having the same band patterns were detected in s. pseudintermedius isolates collected at 13 and 18 months , suggesting possible colonization and/or expansion of a specific s. pseudintermedius strain in a veterinary hospital . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Discussion"], "sections": [["staphylococcus ( s. ) intermedius and s. pseudintermedius are both staphylococcus intermedius group ( sig ) members .", "these microorganisms are opportunistic pathogens that cause skin and nosocomial infections in dogs and cats .", "s. pseudintermedius is the predominant sig species in korea and until recently was misclassified as s. intermedius .", "increased identification of methicillin - resistant sig ( mrsig ) isolates has emphasized the importance of public health precautions for veterinary staff and pet owners .", "many current studies have evaluated methicillin - resistant characteristics and the staphylococcal cassette chromosome mec ( sccmec ) of sig isolates , particularly s. pseudintermedius .", "although type i to type viii sccmec types have been identified , there is no significant studies have been conducted to examine sccmec types isolated from veterinary hospitals located nationwide in korea .", "sccmec types may produce staphylococcal exotoxins including staphylococcal enterotoxins ( ses ) , exfoliative toxins ( ets ) including s. intermedius exfoliative toxin ( siet ) , and toxic shock syndrome toxin 1 ( tsst 1 ) ; these toxins are associated with atopic dermatitis , mastitis , food poisoning , canine pyoderma , and chronic otits .", "siet ( encoded by the siet gene ) , first described by terauchi et al . , plays a potential role in the pathogenesis of canine pyoderma and chronic otitis .", "there may also be a greater chance of sig isolates to colonization and/or expand in veterinary staff , companion animals , and hospital environments .", "therefore , this study is focused on the genetic identification of staphylococcal exotoxins , sccmec types , and genetic relatedness by pulsed - field gel electrophoresis ( pfge ) of the sig isolates in korea ."], ["samples of s. pseudintermedius ( n = 167 ) and s. intermedius ( n = 11 ) were isolated ; their identity was confirmed by gram - staining and biochemical testing such as coagulase , dnase production and hydrolysis", ". polymerase chain reaction ( pcr ) was carried out with primers targeting s. intermedius nuclease and 16s rrna genes . to differentiate s. pseudintermedius from s. intermedius and s. delphini , pcr - restriction fragment length polymorphism analysis was performed using pta gene - specific primers ( pta_f1:5'-aaa gac aaa ctt tca ggt aa-3 ' , and pta_r1 : 5'-gca taa aca agc att gta ccg-3 ' ) , and the restriction enzyme mboi ( new england biolabs , usa ) .", "samples were collected from eight veterinary hospitals from four different regions in korea between november 2006 and january 2010 as previously described . pcr analysis to detect the methicillin - resistance gene ( meca )", "pcr was also performed to amplify genes encoding sea ( sea ) , seb ( seb ) , sec ( sec ) , sed ( sed ) , see ( see ) , seg ( seg ) , seh ( seh ) , sei ( sei ) , tsst 1(tsst-1 ) and ets ( eta , etb , etd , and siet ) using primers and pcr conditions used in other previous studies .", "fri913 , fri 361 , fri 472 , fri 569 , mnhoch , and rn4220 strains were used as either positive or negative controls for superantigen genes . a superantigen gene obtained in this study", "was further analyzed by dna sequencing using the vector nti align x program ( invitrogen , usa ) .", "all mrsig isolates including 49 methicillin - resistant s. pseudintermedius ( mrsp ) and three methicillin - resistant s. intermedius ( mrsi ) were subsequently classified as sccmec type i to viii by either single or multiplex pcr assays using protocols in other previous studies including primers and pcr conditions .", "genomic dna plug kits ( bio - rad , usa ) with smai ( new england biolabs , usa ) digestion were used for the pfge analysis of 39 s. pseudintermedius isolates from a private referral veterinary hospital collected at different times ( eight isolates from november 2006 , 24 isolates from april 2008 , four isolates from june 2009 , and three isolates from october 2009 ) and six s. intermedius isolates ( all collected during april 2008 ) .", "pfge was performed according to protocol used in previous study   and the manufacture 's instruction ( bio - rad , usa ) except for an initial pulse time of 5 sec and final time of 40 sec for 21 h ."], ["analysis of the sig isolates ( n = 178 ) for ses and tsst 1 genes showed that only a single s. pseudintermedius isolate from a veterinary staff member ( h1 - 23 ) harbored the enterotoxin c ( sec ) gene ( fig .", "2 ) revealed that the amplified sec gene the canine type c se gene ( seccaine ) .", "a total of 166 out of 178 sig isolates ( 155 s. pseudintermedius and 11 s. intermedius ) isolated from veterinary staff members ( n = 38 , 95% ) , companion animals ( n = 107 , 93% ) , and veterinary hospitals ( n = 21 , 91.3% ) harbored siet originally detected in s. intermedius .", "however , other et genes such as eta , etb , and etd , known to originate from s. aureus , were not identified .", "sccmec typing of the 49 mrsp isolates identified 38 type v isolates , one type iv isolate , and 10 non - identifiable isolates .", "pfge analysis showed that sig isolates from a private referral animal hospital ( collected between 2006 and 2009 ) recovered from veterinary staff , companion animals , and the environment had the same band patterns ( fig .", "3 ) . six s. pseudintermedius isolates ( two from companion animals collected in september 2006 , three from veterinary staff members , and one from companion animals collected in april 2008 ) and two s. pseudintermedius isolates ( one from a veterinary hospital environment isolated in april 2008 , and one from a companion animal isolated in june 2009 ) showed the same band patterns ( fig ."], ["since sig is closely related to s. aureus , studies have been performed to determine whether sig has adapted the eta , etb and etd toxins from s. aureus by pcr test with specific primers .", "however , 166 out of 178 ( 93.3% ) sig isolates harbored the siet toxin originating from s. intermedius .", "this result and those of other studies   imply that the majority of sig isolates harbor the siet gene .", "although the siet gene was present in 93.3% of the sig isolates ( from 108 dogs and two cats ) in this study , only 14 dogs had a history of various skin disease including allergy and prolonged inflammation lesion in skin ( n = 13 ) or otitis ( n = 1 ) ( data not shown ) .", "therefore , other factors such as the general health of an animal and existence of other sig virulence factors may play an important role in outbreaks of various kinds of skin disease or otitis .", "the sec gene was detected in a single isolate in the present study , which was identified as seccanine by dna sequencing .", "however , this isolate was isolated from a veterinary staff member , and no additional seccanine isolates were identified in the veterinary hospital where this individual worked .", "this suggested that the isolate might be transmitted from an in- or outgoing companion animal with which the veterinary staff was in contact .", "the low incidence of toxins in this study could be secondary to the small number of isolates collected from companion animals that a history of skin disease or otitis .", "only 11.3% of s. pseudintermedius in a previous study had exotoxins although all samples were taken from patients diagnosed with pyoderma or chronic otitis and referred to a veterinary teaching hospital .", "although the majority of sccmec types were type v ( 78.9% ) , one isolate was type iv . in a previous study ,", "23 isolates ( 85.2% ) , three isolates ( 11.1% ) and one isolate ( 3.7% ) from veterinarians , staff , students , companion animals and environment in the veterinary hospitals were determined to be an mrsp hybrid sccmec type i~ii , type v , or non - identifiable , respectively .", "a previous european and north american study   identified 75 hybrid sccmec type ii~iii isolates ( 72.8% ) , two type iii isolates ( 1.9% ) , six type iv isolates ( 5.8% ) , 14 type v isolates ( 13.6% ) , four type vii isolates ( 3.9% ) , and two non - identifiable isolates ( 1.9% ) from diseased and healthy dogs in veterinary diagnostic laboratories of different countries .", "this demonstrated that the majority of mrsig isolates in korea harbor the sccmec type v whereas the hybrid type ii~iii is the main sccmec type found in veterinary hospitals in japan , europe , and north america . in the present study , pfge analysis of the 39 s. pseudintermedius and six s. intermedius isolates from a private referral veterinary hospital ( collected during november 2006 , april 2008 , june 2009 , and october 2009 ) showed that 20 isolates ( lines 1~6 , lines 7~9 , lines 10~15 , lines 16~18 , and lines 19~20 ) had the same band patterns .", "moreover , some isolates obtained on different sampling dates showed the same band patterns ( six isolates from lines 12~17 , and two isolates from lines 21~22 ) .", "these results suggest potential contamination or expansion of s. pseudintermedius and s. intermedus isolates among veterinary staff , companion animals , and veterinary hospital environments , and colonization by these specific strains for more than 13 or 18 months in the same hospital . in conclusion ,", "eta , etb , etd genes were not detected but siet toxin was found in 166 isolates in the current study .", "pfge analysis results of isolated from h animal hospital showed that s. pseudintermedius isolates collected in over a period of 13 and 18 months from veterinary staff , companion animals , and the hospital environment had the same band patterns .", "s. pseudintermedius infections in humans , spread of mrsp populations , and association of sig with canine pyoderma and chronic otitis   has been previously reported .", "therefore , sig , especially mrsig , may have significant clinical implications for companion animals with skin infections or chronic otitis that is of concern for veterinary staff , companion animal owners , and healthy companion animals ."]]}
{"article_id": "PMC3296390", "article_text": ["increasing life expectancy has been accompanied by improvements in the health and quality of life of the middle - aged and elderly and knowing that women live one third of their lives during menopause . apart from age , decreased well being , increased symptomatology , and natural menopause contribute to significant reduction in sexual activity .", "physical changes , such as urogenital ageing and musculoskeletal changes which occur just before or after menopause , affect sexual functioning .", "on the other hand , some studies have shown that having sexual activity may have positive effect on health improvement and protection of the natural physique of postmenopausal women . in a global study ,", "nicolosi et al . , found that 39% of women were affected by at least one sexual dysfunction , while in a survey , only 14% of americans aged 4080 said that they d been asked by their physicians about sexual difficulties during the past 3 year .", "beliefs and practices that influence a woman 's perception of menopause can also influence her sexual function in many societies .", "there is a belief that older women become  sexually retired  after menopause and are , therefore , expected to be less sexually active . on the other hand", ", some women may feel freedom because of the end of the reproductive age and also less responsibility about children . in our society ,", "lots of women are embarrassed to ask questions about their sexual function because of some cultural taboos .", "no information exists on sexual functioning of postmenopausal women who are living in amol , a city in iran .", "therefore , we have conducted a research to determine the frequency of sexual activity , satisfaction and sexual dysfunction among postmenopausal women .", "this is a cross - sectional study that has been done on 280 postmenopausal women .", "exclusion criteria include known disease that have effect on sexual function , such as thyroid disease , heart - pulmonary diseases , supra renal disorders , diabetes , infectious diseases , genitalia injury , use of some medications such as antidepressants , antiblood pressure , opioids , hrt ( hormonal replacement therapy ) , surgery resulting in menopause , stress experience or living without spouse , sexual dysfunction in spouse , etc .", "a semi structured questionnaire in the persian language was designed to obtain information on the sexual functioning of postmenopausal women .", "the development and design of the questionnaire was done following extensive literature review of related studies and fsfi ( female sexual function index ) to ascertain face validity of the questionnaire .", "the content of the questionnaire was reviewed by 5 academic faculty members ( psychologist , nursing , and midwifery department faculty members ) .", "reliability co - efficient ( cronbach 's alpha ) was 0.82 , which indicated that the instrument has a good reliability .", "the questionnaire had two parts , demographic characters and sexual function . to assess the desire , arousal , orgasm , sexual pain , lubrication of vagina , their satisfaction , and spouse 's satisfaction about these women 's sexual function ,", "two questions were considered and each question was scored from 0 ( no problem ) to 4 ( worst problem ) .", "total score was from 048 and based on total score , subjects were classified into three groups : 024 normal , 2536 borderline , and 3748 severe .", "the mean age of the subjects and their spouses in this study was ( 55.9  6.02 , 61.1  7.83 ) years and average time from their last date of menstruation was 7.5  5.43 years .", "90% of the subjects had primary school education , and majority of them mentioned their economic status as good [ table 1 ] .", "mean of some socio demographic factors among selected subjects \n in our study 56.4% of subjects had sexual dysfunction in different phases .", "more than half of them had severe dysfunction based on our scoring system , one third were facing borderline dysfunction .", "it can be seen from the figure that desire and arousal dysfunction are predominant in females .", "based on these observations it is obvious that that menopause women are high risk for sexual dysfunction .", "sexual dysfunction is a broad collection of concerns that occur in menopausal women and can be effective in their quality of life .", "70% reported decrease in frequency of intercourse after menopause.41.8% of women revealed that their spouses sexual interest decreased , while frequency of sexual desire decrease among women was twice more than their spouse ; more than half of the subjects reported that spouses sexual interest was more than their interest.52.6% of them had noticed changes in vaginal stretching for full penetration and half of them reported some problems with their sexual function .", "menopausal changes suggest that most mid - life women report a range of somatic , emotional , and vasomotor conditions including irritability and vaginal dryness . the research on whether these conditions are caused by hormonal fluctuations due to menopause , aging , or life stressors of midlife is inconclusive.31.9% of respondents revealed that their spouses were unsatisfactory with their sexual function , and 52.5% mentioned that sexual problems had an effect on their relationship to their husbands.55.6% of subjects had painful intercourse after menopause .", "more than half of them had severe dysfunction based on our scoring system , one third were facing borderline dysfunction .", "it can be seen from the figure that desire and arousal dysfunction are predominant in females .", "based on these observations it is obvious that that menopause women are high risk for sexual dysfunction .", "sexual dysfunction is a broad collection of concerns that occur in menopausal women and can be effective in their quality of life .", "41.8% of women revealed that their spouses sexual interest decreased , while frequency of sexual desire decrease among women was twice more than their spouse ; more than half of the subjects reported that spouses sexual interest was more than their interest .", "52.6% of them had noticed changes in vaginal stretching for full penetration and half of them reported some problems with their sexual function .", "menopausal changes suggest that most mid - life women report a range of somatic , emotional , and vasomotor conditions including irritability and vaginal dryness . the research on whether these conditions are caused by hormonal fluctuations due to menopause , aging , or life stressors of midlife is inconclusive .", "31.9% of respondents revealed that their spouses were unsatisfactory with their sexual function , and 52.5% mentioned that sexual problems had an effect on their relationship to their husbands .", "in this study , 70.3% of subjects reported a decrease in sexual activity after menopause .", "this incidence is somewhat similar to park 's study in korea and dhillon 's study in malaysia .", "it is , however , different from dennerstein 's study in australia where 31% of respondents reported a decrease in sexual activity with nearly two - third reporting no change .", "the reason for the differences between the two studies may be related to the age range of the study population and it seems that in our study subject 's mean age are higher than dennerstein 's study . in this study ,", "56.4% of subjects had sexual dysfunction but according to arman , et al . , the frequency of sexual dysfunction in isfahan was 72.4% , while the frequency of sexual dysfunction among middle - aged and elder women in addis 's study was 45% and in bonnie 's study was 20% .", "possible explanations for the higher frequencies in our study include sociocultural differences , genetic ( racial ) differences and religions differences .", "a study in australia revealed all types of sexual dysfunction increased significantly with age and apart from age , sexual dysfunction significantly increased with advancing menopausal years . also other studies showed that natural sexual activity will be reduced on becoming older . on the other hand", "women reported either an absent or decreased sexual desire that is the same as kaboody 's study which was done in 2002 in kermanshah ( 70% ) .", "hayes et al . , found the proportion of european women with low desire increased from 11% amongst women aged 2029 years to 53% amongst women aged 6070 years .", ", found women aged 6170 years had a 2.8-fold higher risk for sexual desire disorder as compared to those aged 1230 years .", "although we did not find any association between dyspareunia and number of sexual intercourses , frequencies showed women with dyspareunia had a lower intercourse frequency .", "in a study in malaysia relationship between number of intercourse and dyspareunia was significant . in our study ,", "frequency of dyspareunia was 55.6% and it is higher than studies that have been done in other parts of the world , such as australia 12% , taiwan32% , pakistan 16.9% , and turkey 16%.[21719 ] reason for this high difference is unknown .", "decrease in vaginal lubrication during sexual activity or menopausal atrophic changes can be reasons for pain during sexual intercourse .", "prevalence of vaginal dryness in singapore was 20.7% , thailand 20.7% , and 23.6% among taiwanese women .", "one of the reasons for high incidence in our study could be due to the age range of our study population .", "recent evidence suggests that the declining vaginal secretion might also be due to the effect of age and hypoandrogenism . a negative correlation between age and the density of androgen receptor expression", "has been reported , suggesting that androgen might also be involved in the regulation of vaginal blood flow and secretions . in this study", ", we found significant relationship between sexual desire loss and number of sexual intercourses during the last year ( p=0.000 ) .", "we found association between the number of sexual intercourse with some variables , such as subject 's education level and their spouse 's education level ( p=0.000 , p=0.006 ) and these findings were similar to addis et al . , findings .", "a negative correlation was noticed between the age of spouse and the number of sexual intercourse ( p=0.000 ) and also between age of the spouse and the sexual desire of the women ( p<0.01 ) similar to the kelantanese women in malaysia .", "there were no association between sexual dysfunction and occupation , income , number of children .", "arousal problems were 80% and it was similar to another study in iran that was done by arman et al . , in their study arousal problems was 75.3% .", "52.5% of the subjects mentioned that sexual problems had an effect on their relationship with their husbands , also nicolosi et al . , study found that 76% of the women agreed that satisfactory sex is essential to maintain relationships .", "iranian traditional sexual beliefs persist and moreover the meaning of sexuality in iranian society is based on religious minds , which makes them feel themselves passive in sexual activities .", "these women avoid showing their sexual emotion because of lack of knowledge and traditional beliefs . nowadays", "promotion of quality of life in elder women is one of the most important issues .", "so it seems to need more investigations because sexual activity and satisfaction is one of the important aspect of human life which is largely ignored , especially in developing countries ."], "abstract_text": ["<S> background : sexual activity is an important part of the human being 's life but this instinct could be influenced by some factors such as diseases , drug using , aging , and menopause . but </S>", "<S> information about that is limited.aim:the aim of this study is to determine the status of sexual activity among married menopausal women in amol , iran.materials and methods : this descriptive analytical study was conducted to describe the sexual activity and sexual dysfunction of women after menopause . </S>", "<S> data were collected from health centers in amol from 280 married women using a questionnaire ( self - completed or by interview).finding : mean age of subjects were 55.9  6.02 years . </S>", "<S> 23.4% of subjects reported that their sexual intercourse had been low . </S>", "<S> 70% of subjects reported a decrease in their sexual activities after menopause . </S>", "<S> sexual dysfunctions includes sexual desire disorder 80% arousal dysfunction 80% , orgasmic dysfunction 25% , dyspareunia 55.6% , and lack of sexual satisfaction 43.2%.conclusion : findings revealed high percentage of sexual desire disorder and sexual arousal disorder in menopausal women . </S>", "<S> therefore , we should have emphasis on counseling and education about sexual activities during the menopause period . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "FINDINGS", "DISCUSSION"], "sections": [["increasing life expectancy has been accompanied by improvements in the health and quality of life of the middle - aged and elderly and knowing that women live one third of their lives during menopause . apart from age , decreased well being , increased symptomatology , and natural menopause contribute to significant reduction in sexual activity .", "physical changes , such as urogenital ageing and musculoskeletal changes which occur just before or after menopause , affect sexual functioning .", "on the other hand , some studies have shown that having sexual activity may have positive effect on health improvement and protection of the natural physique of postmenopausal women . in a global study ,", "nicolosi et al . , found that 39% of women were affected by at least one sexual dysfunction , while in a survey , only 14% of americans aged 4080 said that they d been asked by their physicians about sexual difficulties during the past 3 year .", "beliefs and practices that influence a woman 's perception of menopause can also influence her sexual function in many societies .", "there is a belief that older women become  sexually retired  after menopause and are , therefore , expected to be less sexually active . on the other hand", ", some women may feel freedom because of the end of the reproductive age and also less responsibility about children . in our society ,", "lots of women are embarrassed to ask questions about their sexual function because of some cultural taboos .", "no information exists on sexual functioning of postmenopausal women who are living in amol , a city in iran .", "therefore , we have conducted a research to determine the frequency of sexual activity , satisfaction and sexual dysfunction among postmenopausal women ."], ["this is a cross - sectional study that has been done on 280 postmenopausal women .", "exclusion criteria include known disease that have effect on sexual function , such as thyroid disease , heart - pulmonary diseases , supra renal disorders , diabetes , infectious diseases , genitalia injury , use of some medications such as antidepressants , antiblood pressure , opioids , hrt ( hormonal replacement therapy ) , surgery resulting in menopause , stress experience or living without spouse , sexual dysfunction in spouse , etc .", "a semi structured questionnaire in the persian language was designed to obtain information on the sexual functioning of postmenopausal women .", "the development and design of the questionnaire was done following extensive literature review of related studies and fsfi ( female sexual function index ) to ascertain face validity of the questionnaire .", "the content of the questionnaire was reviewed by 5 academic faculty members ( psychologist , nursing , and midwifery department faculty members ) .", "reliability co - efficient ( cronbach 's alpha ) was 0.82 , which indicated that the instrument has a good reliability .", "the questionnaire had two parts , demographic characters and sexual function . to assess the desire , arousal , orgasm , sexual pain , lubrication of vagina , their satisfaction , and spouse 's satisfaction about these women 's sexual function ,", "two questions were considered and each question was scored from 0 ( no problem ) to 4 ( worst problem ) .", "total score was from 048 and based on total score , subjects were classified into three groups : 024 normal , 2536 borderline , and 3748 severe ."], ["the mean age of the subjects and their spouses in this study was ( 55.9  6.02 , 61.1  7.83 ) years and average time from their last date of menstruation was 7.5  5.43 years .", "90% of the subjects had primary school education , and majority of them mentioned their economic status as good [ table 1 ] .", "mean of some socio demographic factors among selected subjects \n in our study 56.4% of subjects had sexual dysfunction in different phases .", "more than half of them had severe dysfunction based on our scoring system , one third were facing borderline dysfunction .", "it can be seen from the figure that desire and arousal dysfunction are predominant in females .", "based on these observations it is obvious that that menopause women are high risk for sexual dysfunction .", "sexual dysfunction is a broad collection of concerns that occur in menopausal women and can be effective in their quality of life .", "70% reported decrease in frequency of intercourse after menopause.41.8% of women revealed that their spouses sexual interest decreased , while frequency of sexual desire decrease among women was twice more than their spouse ; more than half of the subjects reported that spouses sexual interest was more than their interest.52.6% of them had noticed changes in vaginal stretching for full penetration and half of them reported some problems with their sexual function .", "menopausal changes suggest that most mid - life women report a range of somatic , emotional , and vasomotor conditions including irritability and vaginal dryness . the research on whether these conditions are caused by hormonal fluctuations due to menopause , aging , or life stressors of midlife is inconclusive.31.9% of respondents revealed that their spouses were unsatisfactory with their sexual function , and 52.5% mentioned that sexual problems had an effect on their relationship to their husbands.55.6% of subjects had painful intercourse after menopause .", "more than half of them had severe dysfunction based on our scoring system , one third were facing borderline dysfunction .", "it can be seen from the figure that desire and arousal dysfunction are predominant in females .", "based on these observations it is obvious that that menopause women are high risk for sexual dysfunction .", "sexual dysfunction is a broad collection of concerns that occur in menopausal women and can be effective in their quality of life .", "41.8% of women revealed that their spouses sexual interest decreased , while frequency of sexual desire decrease among women was twice more than their spouse ; more than half of the subjects reported that spouses sexual interest was more than their interest .", "52.6% of them had noticed changes in vaginal stretching for full penetration and half of them reported some problems with their sexual function .", "menopausal changes suggest that most mid - life women report a range of somatic , emotional , and vasomotor conditions including irritability and vaginal dryness . the research on whether these conditions are caused by hormonal fluctuations due to menopause , aging , or life stressors of midlife is inconclusive .", "31.9% of respondents revealed that their spouses were unsatisfactory with their sexual function , and 52.5% mentioned that sexual problems had an effect on their relationship to their husbands ."], ["in this study , 70.3% of subjects reported a decrease in sexual activity after menopause .", "this incidence is somewhat similar to park 's study in korea and dhillon 's study in malaysia .", "it is , however , different from dennerstein 's study in australia where 31% of respondents reported a decrease in sexual activity with nearly two - third reporting no change .", "the reason for the differences between the two studies may be related to the age range of the study population and it seems that in our study subject 's mean age are higher than dennerstein 's study . in this study ,", "56.4% of subjects had sexual dysfunction but according to arman , et al . , the frequency of sexual dysfunction in isfahan was 72.4% , while the frequency of sexual dysfunction among middle - aged and elder women in addis 's study was 45% and in bonnie 's study was 20% .", "possible explanations for the higher frequencies in our study include sociocultural differences , genetic ( racial ) differences and religions differences .", "a study in australia revealed all types of sexual dysfunction increased significantly with age and apart from age , sexual dysfunction significantly increased with advancing menopausal years . also other studies showed that natural sexual activity will be reduced on becoming older . on the other hand", "women reported either an absent or decreased sexual desire that is the same as kaboody 's study which was done in 2002 in kermanshah ( 70% ) .", "hayes et al . , found the proportion of european women with low desire increased from 11% amongst women aged 2029 years to 53% amongst women aged 6070 years .", ", found women aged 6170 years had a 2.8-fold higher risk for sexual desire disorder as compared to those aged 1230 years .", "although we did not find any association between dyspareunia and number of sexual intercourses , frequencies showed women with dyspareunia had a lower intercourse frequency .", "in a study in malaysia relationship between number of intercourse and dyspareunia was significant . in our study ,", "frequency of dyspareunia was 55.6% and it is higher than studies that have been done in other parts of the world , such as australia 12% , taiwan32% , pakistan 16.9% , and turkey 16%.[21719 ] reason for this high difference is unknown .", "decrease in vaginal lubrication during sexual activity or menopausal atrophic changes can be reasons for pain during sexual intercourse .", "prevalence of vaginal dryness in singapore was 20.7% , thailand 20.7% , and 23.6% among taiwanese women .", "one of the reasons for high incidence in our study could be due to the age range of our study population .", "recent evidence suggests that the declining vaginal secretion might also be due to the effect of age and hypoandrogenism . a negative correlation between age and the density of androgen receptor expression", "has been reported , suggesting that androgen might also be involved in the regulation of vaginal blood flow and secretions . in this study", ", we found significant relationship between sexual desire loss and number of sexual intercourses during the last year ( p=0.000 ) .", "we found association between the number of sexual intercourse with some variables , such as subject 's education level and their spouse 's education level ( p=0.000 , p=0.006 ) and these findings were similar to addis et al . , findings .", "a negative correlation was noticed between the age of spouse and the number of sexual intercourse ( p=0.000 ) and also between age of the spouse and the sexual desire of the women ( p<0.01 ) similar to the kelantanese women in malaysia .", "there were no association between sexual dysfunction and occupation , income , number of children .", "arousal problems were 80% and it was similar to another study in iran that was done by arman et al . , in their study arousal problems was 75.3% .", "52.5% of the subjects mentioned that sexual problems had an effect on their relationship with their husbands , also nicolosi et al . , study found that 76% of the women agreed that satisfactory sex is essential to maintain relationships .", "iranian traditional sexual beliefs persist and moreover the meaning of sexuality in iranian society is based on religious minds , which makes them feel themselves passive in sexual activities .", "these women avoid showing their sexual emotion because of lack of knowledge and traditional beliefs . nowadays", "promotion of quality of life in elder women is one of the most important issues .", "so it seems to need more investigations because sexual activity and satisfaction is one of the important aspect of human life which is largely ignored , especially in developing countries ."]]}
{"article_id": "PMC4220378", "article_text": ["the tubularized incised plate urethroplasty ( tipu ) repair is the procedure of choice for distal and mid - shaft hypospadias and is increasingly being used for proximal hypospadias and redo repairs .", "post - operatively , urethrocutaneous fistula is the most common complication . to decrease fistula rate , an additional tissue cover after", "neourethra reconstruction has been described using transverse island dorsal subcutaneous flap , de - epithelialized skin flaps , lateral dartos flap , double dartos flaps , double breasted de - epithelialized penile skin flap , tunica vaginalis , and corpus spongiosum .", "the use of corpus spongiosum as an intervening layer over the neourethra is less frequent .", "we evaluated the efficacy of spongioplasty alone after mobilization of corpus spongiosum in prevention of urethral fistula in patients undergoing tip repair for hypospadias .", "a prospective clinical study was carried out at our institution between june 2010 and march 2012 .", "the parents ( for minors ) and patients were informed about the merits and complications of the procedure and a written informed consent was obtained .", "intra - operatively , we divided the corpus spongiosum into three categories depending on the appearance and vascularity .", "poorly developed - thin spongiosal tissue with decreased vascularity ; the diameter of the neourethra covered by spongiosum after spongioplasty was less than the proximal healthy urethra [ figure 1 ] .", "poorly developed corpus spongiosum ( a ) mobilized spongiosum on left side with minimal spongiosum tissue ( b ) mobilized spongiosum on right with minimal spongiosum tissue ( c ) hypoplastic urethra with mobilized spongiosum on both sides with minimal spongiosum tissue ( d ) after completion of spongioplasty , proximal normal urethra bulkier than distal neourethra with spongioplasty moderately developed - average thickness and vascularity of spongiosal tissue ; the diameter of the neourethra covered by spongiosum after spongioplasty was almost equal to that of the proximal healthy urethra [ figure 2 ] .", "moderately developed corpus spongiosum ( a and b ) both side mobilized spongiosum with moderate spongiosum tissue ( c and d ) spongioplasty of mobilized spongiosum well - developed - robust , thick spongiosum with good vascularity ; the diameter of the neourethra covered by spongiosum was greater than that of the proximal healthy urethra [ figure 3 ] .", "well - developed corpus spongiosum ( a - c ) both side mobilized spongiosum with robust healthy spongiosum tissue ( d ) spongioplasty of mobilized spongiosum an u - shaped incision is made encircling the meatus up to the corona , preserving the urethral plate and then extended circumferentially around the corona .", "penile de - gloving is done when required , up to the root of the penis by creating a plane at the level of buck 's fascia .", "a gittes test is carried out to evaluate the chordee after penile de - gloving .", "starting well proximal to the hypospadiac meatus , a plane is created between the buck 's fascia and tunica albuginea [ figure 4 ] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . from lateral to medial , the spongiosum is dissected off the underlying corpora cavernosa .", "mobilization is done well beneath the true urethral plate on both sides , lifting off the midline spongiosum and urethral plate [ figure 5 ] from the corpora cavernosa when required , to correct chordee .", "dissection is performed with care , avoiding damage to the corpus spongiosum or corpus cavernosum .", "the proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee .", "the spongiosal pillars spread beneath the glans wings on each side , care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees , which leaves healthy , thick glans wings yet intact spongiosum when done correctly [ figure 6 ] .", "the urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone ( pds ) suture after a dorsal incision to create a new urethra .", "the mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [ figure 2d ] . a 6-to 10 fr urethral catheter , depending on patient age", "no additional cover of dartos , tunica vaginalis or de - epithelialized skin was used .", "skin is sutured with 7 - 0 pds interrupted suture . showing creation of plane proximal to meatus between tunica and buck 's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ .", "patients were evaluated at follow - up visits after 1 , 3 , 6 , 12 months and then yearly .", "results were evaluated in terms of patients , parents and surgeon 's satisfaction , keeping in mind the stream , cosmesis and lack of complications .", "if at 3 months follow - up the stream was narrow , we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube .", "an u - shaped incision is made encircling the meatus up to the corona , preserving the urethral plate and then extended circumferentially around the corona .", "penile de - gloving is done when required , up to the root of the penis by creating a plane at the level of buck 's fascia .", "a gittes test is carried out to evaluate the chordee after penile de - gloving .", "starting well proximal to the hypospadiac meatus , a plane is created between the buck 's fascia and tunica albuginea [ figure 4 ] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . from lateral to medial , the spongiosum is dissected off the underlying corpora cavernosa .", "mobilization is done well beneath the true urethral plate on both sides , lifting off the midline spongiosum and urethral plate [ figure 5 ] from the corpora cavernosa when required , to correct chordee .", "dissection is performed with care , avoiding damage to the corpus spongiosum or corpus cavernosum .", "the proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee .", "adequacy of curvature correction is confirmed by gittes test . since the spongiosal pillars spread beneath the glans wings on each side , care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees , which leaves healthy , thick glans wings yet intact spongiosum when done correctly [ figure 6 ] .", "the urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone ( pds ) suture after a dorsal incision to create a new urethra .", "the mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [ figure 2d ] . a 6-to 10 fr urethral catheter , depending on patient age", "no additional cover of dartos , tunica vaginalis or de - epithelialized skin was used .", "showing creation of plane proximal to meatus between tunica and buck 's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ .", "patients were evaluated at follow - up visits after 1 , 3 , 6 , 12 months and then yearly .", "results were evaluated in terms of patients , parents and surgeon 's satisfaction , keeping in mind the stream , cosmesis and lack of complications .", "if at 3 months follow - up the stream was narrow , we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube .", "an u - shaped incision is made encircling the meatus up to the corona , preserving the urethral plate and then extended circumferentially around the corona .", "penile de - gloving is done when required , up to the root of the penis by creating a plane at the level of buck 's fascia .", "a gittes test is carried out to evaluate the chordee after penile de - gloving .", "starting well proximal to the hypospadiac meatus , a plane is created between the buck 's fascia and tunica albuginea [ figure 4 ] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . from lateral to medial , the spongiosum is dissected off the underlying corpora cavernosa .", "mobilization is done well beneath the true urethral plate on both sides , lifting off the midline spongiosum and urethral plate [ figure 5 ] from the corpora cavernosa when required , to correct chordee .", "dissection is performed with care , avoiding damage to the corpus spongiosum or corpus cavernosum .", "the proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee .", "adequacy of curvature correction is confirmed by gittes test . since the spongiosal pillars spread beneath the glans wings on each side , care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees , which leaves healthy , thick glans wings yet intact spongiosum when done correctly [ figure 6 ] .", "the urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone ( pds ) suture after a dorsal incision to create a new urethra .", "the mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [ figure 2d ] . a 6-to 10 fr urethral catheter , depending on patient age", "no additional cover of dartos , tunica vaginalis or de - epithelialized skin was used .", "showing creation of plane proximal to meatus between tunica and buck 's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ .", "patients were evaluated at follow - up visits after 1 , 3 , 6 , 12 months and then yearly .", "results were evaluated in terms of patients , parents and surgeon 's satisfaction , keeping in mind the stream , cosmesis and lack of complications .", "if at 3 months follow - up the stream was narrow , we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube .", "the age range of the patients was 4 months to 38 years ( mean 11.53 years ) .", "the hospital stay ranged from 8 - 10 days and period of follow - up was 6 months to 2 years .", "the hypospadias was distal , mid and proximal in 81 , 12 , and 20 cases respectively .", "correction of chordee was carried out by penile de - gloving alone in 5 , mobilization of urethral plate with spongiosum in 22 and combination of both in 45 cases .", "on correlating the type of hypospadias with development of spongiosum ; more proximal hypospadias was associated with poorer development of the spongiosum ( p = 0.05 ) [ table 1 ] .", "two patients had meatal stenosis with fistula and seven patients had urethral fistula ( clavien - dindo grade 3b ) alone with an overall complication rate of 7.96% .", "fistulas occurred in 2 cases each of distal and mid hypospadias and 5 cases of proximal hypospadias .", "complications were higher in proximal hypospadias as compared to distal ( p = 0.0019 ) [ table 1 ] .", "correlation of type of hypospadias , development of spongiosum and complications three ( one each in distal , mid and proximal ) out of thirteen ( 23.03% ) cases with poorly developed spongiosum and six ( distal 1 , mid 1 and proximal 4 ) out of 53 ( 11.32% ) cases with moderately developed spongiosum developed a fistula while none of the cases with well - developed spongiosum developed a fistula .", "one out of 53 ( 1.88% ) cases with moderately developed spongiosum and 1/13 ( 7.69% ) cases with poorly developed spongiosum developed meatal stenosis .", "thus , poorer spongiosum was associated with greater number of complications ( p = 0.011 ) [ table 1 ] .", "none of the patients developed a stricture urethra and all patients / parents were satisfied with cosmesis .", "all nine patients with urethral fistula were operated upon 6 - 12 months later and the fistulae repaired with no further sequelae .", "the male urethra develops from the urethral folds as they fuse in the midline after the 6 week of fetal life .", "the endodermally derived distal solid urethral plate must canalize to form the distal most glanular part of the urethra .", "the urethra forms sequentially after the proximal urethral folds fuse and the mesenchyme within the urethral folds forms the corpus spongiosum . according to the classic theory , embryologically , the last step in urethra formation is the joining of the main endodermal urethral channel with the ectoderm as it invades the glans .", "the normal and hypospadiac penises differ in the development of glans and urethra , especially their vascularity . in hypospadias ,", "there is failed urethral fold fusion and ectodermal intrusion , histologically characterized by an extremely vascular area with large endothelial lined sinuses around the abortive urethral spongiosum and abnormal glans .", "the degree of hypoplasia of corpus spongiosum depends upon the severity of hypospadias and proliferation of mesodermal tissue .", "we classified the spongiosum according to its development and vascularity as poorly developed , moderately developed and well - developed .", "further , it may act as a guide to new surgeons attempting hypospadias about the feasibility of spongioplasty in a particular case and whether an additional layer may be required .", "none of the patients with well - developed spongiosum in distal hypospadias had any complication .", "complications in proximal hypospadias with well - developed spongiosum were also very low as compared to poorly developed spongiosum cases .", "most importantly , poorer was the spongiosum , greater was the number of complications ( p = 0.011 ) . on analysis of our results , we found that more proximal meatus was associated with poorer development of the spongiosum ( p = 0.05 ) and proximal hypospadias was associated with greater incidence of complications ( p = 0.0019 ) .", "however , not all hypospadias with poorly developed spongiosum had complications , suggesting that other factors like development and width of urethral plate also play a role in the outcome of the repair .", "since none of the patients of distal hypospadias with well - developed spongiosum had fistula , dorsal dartos cover in such patients may be omitted and the prepuce can be utilized for preputioplasty .", "however in patients with poorly developed spongiosa , an additional interposing layer like dartos or tunica should be used .", "since its first description by snodgrass for repair of hypospadias in 1994 , tip has gained widespread acceptance due to its versatility , low complication rate and reliable creation of vertically oriented meatus , with excellent cosmetic outcome .", "some authors recommend tipu as the technique to be considered for primary and re - operative repair of distal and some midshaft hypospadias cases with selected proximal ones .", "the common complications reported after tipu repair include fistulae , urethral stricture , meatal stenosis , persistent chordee , infections and wound dehiscence with development of urethrocutaneous fistula being the most common .", "the causes of fistulae are local infection , ischemia , an inadequate procedure , poor tissue healing and distal obstruction due to meatal stenosis / encrustation which promotes fistulae formation . to decrease the incidence of post - operative fistulae , many modifications in the tip repair have been tried .", "however , no single surgical technique can be considered to be a clear winner to prevent the formation of fistulae .", "one of the most important factors in reducing fistula formation with the tip technique is the introduction of a protective intermediate layer between the neourethra and the skin .", "dorsal , lateral , single or double dartos flaps , ventral based dartos flap , scrotal dartos , de - epithelized local penile skin , preputial flap , paraurethral tissue , spongioplasty , or tunica vaginalis flaps have all been used for the same . out of all these", ", the dorsal dartos flap is used by most surgeons , but there is still no consensus over the ideal interposing tissue in tipu .", "because of edema , necrosis of skin , hematoma and torque with mobilization of the dorsal dartos flap , its role as an interposing tissue has been questioned .", "the corpus spongiosum provides a well - vascularized , spongy protective covering to the normal urethra .", "it may aid in the natural propulsion of urine and semen . using this natural , locally available tissue to cover", "spongioplasty also decreases the tension on the suture line of urethroplasty in the midline , especially during erections .", "the use of spongiosal tissue as an intermediate layer between the urethra and skin was described in 2000 by yerkes and beaudoin in separate studies .", "yerkes reported no fistulae in any of her patients for whom spongioplasty was implemented in a number of urethral repair techniques , whereas beaudoin described the anatomical characteristics of the spongiosal layer in the hypospadic penis and implemented spongioplasty in patients who underwent urethral tubularization . in 2003 , dodat et al", ". showed no fistula formation in any of the 51 patients who underwent tipu and spongioplasty .", "el - sherbiny et al . reported that covering the neourethra with a flap or spongioplasty minimized the risk of fistula formation . in a series of 500 cases , sarhan et al .", "used a dartos flap , spongioplasty , or a combination of these techniques in addition to tip .", "they observed a statistically significant decrease in fistula formation in cases in which spongioplasty was implemented compared with cases in which it was not . in the present series we had an acceptable overall fistula rate of 7.96% with no complications in distal hypospadias with well - developed spongiosum where we did not use dorsal / ventral dartos or any other tissue in addition with spongioplasty .", "these results are comparable with other studies where dorsal dartos has been used with or without spongioplasty .", "since the results of well - developed spongioplasty alone in distal hypospadias were very good , additional cover with dorsal dartos can be omitted .", "report that various studies using dartos based flaps , tunica vaginalis and subcutaneous tissue flaps have a fistula rate of 2 - 33% .", "when spongioplasty alone was used as an interposing layer , in various series complications ranged from 4% to 40% with 14.2% complication rate in 197 cases whereas in the present series they were seen in 7.96 % cases [ table 2 ] .", "series using spongioplasty alone for hypospadias repair our technique of spongioplasty is similar to those mentioned earlier in that we mobilize the urethral plate with the spongiosum completely off the corpora cavernosa starting laterally and moving medially .", "furthermore we mobilized the spongiosal pillars into the glans obliquely at an angle of 45 almost up to the tip of the glans .", "the mobilization of urethral plate with spongiosum have added advantages of correction of torsion and ventral curvature , urethral plate can be tubularized without tension on suture line and many a times without incising the urethral plate .", "complete spongioplasty if done properly as outlined above restores a near normal urethra and provides good support .", "it is a healthy , vascular cover which probably decreases tension on the midline sutures especially during erections .", "better is the spongiosum , lesser are the chances of post - operative fistulae formation .", "the strengths of our study are that it is prospective , cases belonged to similar socio - economic status , procedures were performed by a single surgeon in similar circumstances and with adequate follow - up .", "the weaknesses are that the criteria for development of spongiosum are subjective so there are chances of error in judgment , especially in well and moderately developed spongiosum . to the best of our knowledge , this is the only series in current literature with such a large number of patients undergoing spongioplasty alone with tip repair .", "a more proximal hypospadias is associated with poorer spongiosum and this is likely to increase the chances of complications . better developed and thicker spongiosum results in lower incidence of fistula formation after tipu .", "spongioplasty reconstructs a near normal urethra as per anatomical principles of surgery with least complications .", "finally , it adds an extra layer of locally available healthy vascular tissue avoiding dissection for local flaps .", "we recommend spongioplasty be incorporated as an essential step in all patients undergoing tip repair for hypospadias ."], "abstract_text": ["<S> introduction : additional cover after neourethra formation to decrease the fistula rate , has been described using the dartos , tunica , denuded skin and corpus spongiosum . </S>", "<S> the use of corpus spongiosum alone to cover the neourethra is infrequent . </S>", "<S> the objective of this study was to evaluate the efficacy of spongioplasty alone as an intervening layer in the prevention of urethral fistula following tubularized incised plate urethroplasty ( tipu).materials and methods : a prospective study was performed including 113 primary hypospadias cases undergoing tipu with spongioplasty from june 2010 to march 2012 . </S>", "<S> correction of chordee was carried out by penile degloving alone in 5 , mobilization of urethral plate with spongiosum in 22 and combination of both in 45 cases . </S>", "<S> intra - operatively , spongiosum was taken to be poorly developed if it was thin and fibrous , moderate if good spongiosal tissue with good vascularization and well - developed if healthy robust spongiosum , which became bulkier than native spongiosum after tubularisation . </S>", "<S> spongioplasty was done in a single layer after mobilization of spongiosum , starting just proximal to the native meatus and into the glans distally.results:the mean age of the patients was 11.53 years . </S>", "<S> the type of hypospadias was distal , mid and proximal in 81 , 12 and 20 cases respectively . </S>", "<S> spongiosum was poorly developed in 13 , moderate in 53 and well - developed in 47 cases . </S>", "<S> the mean hospital stay was 8 - 10 days and follow - up ranged from 6 months to 2 years . </S>", "<S> urethral fistula was seen in six patients ( 11.3% ) with moderate spongiosum ( distal 1 , mid 1 and proximal 4 ) , and three ( 23.03% ) with poorly developed spongiosum ( one each in distal , mid and proximal ) with an overall 7.96% fistula rate . </S>", "<S> none of the patients with well - developed spongiosum developed a fistula . </S>", "<S> poorer spongiosum correlated with a greater number of complications ( p = 0.011 ) . </S>", "<S> five out of thirteen cases with poor spongiosum ( 38.46% ) had proximal hypospadias , i.e. more proximal was the hypospadias , poorer was the development of the spongiosum ( p = 0.05 ) . </S>", "<S> meatal stenosis was seen in two patients ( 1.76% ) with proximal hypospadias , one with moderate and the other with poorly developed spongiosum . </S>", "<S> more proximal was the hypospadias , greater were the number of complications ( p = 0.0019).conclusion : tipu with spongioplasty reconstructs a near normal urethra with low complications . </S>", "<S> better developed and thicker spongiosum results in lower incidence of fistula and meatal stenosis . </S>", "<S> more proximal hypospadias is associated with poorer spongiosum . </S>", "<S> we recommend spongioplasty to be incorporated as an essential step in all patients undergoing tubularized incised - plate repair for hypospadias . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "None", "Surgical technique", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["the tubularized incised plate urethroplasty ( tipu ) repair is the procedure of choice for distal and mid - shaft hypospadias and is increasingly being used for proximal hypospadias and redo repairs .", "post - operatively , urethrocutaneous fistula is the most common complication . to decrease fistula rate , an additional tissue cover after", "neourethra reconstruction has been described using transverse island dorsal subcutaneous flap , de - epithelialized skin flaps , lateral dartos flap , double dartos flaps , double breasted de - epithelialized penile skin flap , tunica vaginalis , and corpus spongiosum .", "the use of corpus spongiosum as an intervening layer over the neourethra is less frequent .", "we evaluated the efficacy of spongioplasty alone after mobilization of corpus spongiosum in prevention of urethral fistula in patients undergoing tip repair for hypospadias ."], ["a prospective clinical study was carried out at our institution between june 2010 and march 2012 .", "the parents ( for minors ) and patients were informed about the merits and complications of the procedure and a written informed consent was obtained .", "intra - operatively , we divided the corpus spongiosum into three categories depending on the appearance and vascularity .", "poorly developed - thin spongiosal tissue with decreased vascularity ; the diameter of the neourethra covered by spongiosum after spongioplasty was less than the proximal healthy urethra [ figure 1 ] .", "poorly developed corpus spongiosum ( a ) mobilized spongiosum on left side with minimal spongiosum tissue ( b ) mobilized spongiosum on right with minimal spongiosum tissue ( c ) hypoplastic urethra with mobilized spongiosum on both sides with minimal spongiosum tissue ( d ) after completion of spongioplasty , proximal normal urethra bulkier than distal neourethra with spongioplasty moderately developed - average thickness and vascularity of spongiosal tissue ; the diameter of the neourethra covered by spongiosum after spongioplasty was almost equal to that of the proximal healthy urethra [ figure 2 ] .", "moderately developed corpus spongiosum ( a and b ) both side mobilized spongiosum with moderate spongiosum tissue ( c and d ) spongioplasty of mobilized spongiosum well - developed - robust , thick spongiosum with good vascularity ; the diameter of the neourethra covered by spongiosum was greater than that of the proximal healthy urethra [ figure 3 ] .", "well - developed corpus spongiosum ( a - c ) both side mobilized spongiosum with robust healthy spongiosum tissue ( d ) spongioplasty of mobilized spongiosum an u - shaped incision is made encircling the meatus up to the corona , preserving the urethral plate and then extended circumferentially around the corona .", "penile de - gloving is done when required , up to the root of the penis by creating a plane at the level of buck 's fascia .", "a gittes test is carried out to evaluate the chordee after penile de - gloving .", "starting well proximal to the hypospadiac meatus , a plane is created between the buck 's fascia and tunica albuginea [ figure 4 ] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . from lateral to medial , the spongiosum is dissected off the underlying corpora cavernosa .", "mobilization is done well beneath the true urethral plate on both sides , lifting off the midline spongiosum and urethral plate [ figure 5 ] from the corpora cavernosa when required , to correct chordee .", "dissection is performed with care , avoiding damage to the corpus spongiosum or corpus cavernosum .", "the proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee .", "the spongiosal pillars spread beneath the glans wings on each side , care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees , which leaves healthy , thick glans wings yet intact spongiosum when done correctly [ figure 6 ] .", "the urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone ( pds ) suture after a dorsal incision to create a new urethra .", "the mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [ figure 2d ] . a 6-to 10 fr urethral catheter , depending on patient age", "no additional cover of dartos , tunica vaginalis or de - epithelialized skin was used .", "skin is sutured with 7 - 0 pds interrupted suture . showing creation of plane proximal to meatus between tunica and buck 's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ .", "patients were evaluated at follow - up visits after 1 , 3 , 6 , 12 months and then yearly .", "results were evaluated in terms of patients , parents and surgeon 's satisfaction , keeping in mind the stream , cosmesis and lack of complications .", "if at 3 months follow - up the stream was narrow , we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube ."], ["an u - shaped incision is made encircling the meatus up to the corona , preserving the urethral plate and then extended circumferentially around the corona .", "penile de - gloving is done when required , up to the root of the penis by creating a plane at the level of buck 's fascia .", "a gittes test is carried out to evaluate the chordee after penile de - gloving .", "starting well proximal to the hypospadiac meatus , a plane is created between the buck 's fascia and tunica albuginea [ figure 4 ] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . from lateral to medial , the spongiosum is dissected off the underlying corpora cavernosa .", "mobilization is done well beneath the true urethral plate on both sides , lifting off the midline spongiosum and urethral plate [ figure 5 ] from the corpora cavernosa when required , to correct chordee .", "dissection is performed with care , avoiding damage to the corpus spongiosum or corpus cavernosum .", "the proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee .", "adequacy of curvature correction is confirmed by gittes test . since the spongiosal pillars spread beneath the glans wings on each side , care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees , which leaves healthy , thick glans wings yet intact spongiosum when done correctly [ figure 6 ] .", "the urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone ( pds ) suture after a dorsal incision to create a new urethra .", "the mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [ figure 2d ] . a 6-to 10 fr urethral catheter , depending on patient age", "no additional cover of dartos , tunica vaginalis or de - epithelialized skin was used .", "showing creation of plane proximal to meatus between tunica and buck 's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ .", "patients were evaluated at follow - up visits after 1 , 3 , 6 , 12 months and then yearly .", "results were evaluated in terms of patients , parents and surgeon 's satisfaction , keeping in mind the stream , cosmesis and lack of complications .", "if at 3 months follow - up the stream was narrow , we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube ."], ["an u - shaped incision is made encircling the meatus up to the corona , preserving the urethral plate and then extended circumferentially around the corona .", "penile de - gloving is done when required , up to the root of the penis by creating a plane at the level of buck 's fascia .", "a gittes test is carried out to evaluate the chordee after penile de - gloving .", "starting well proximal to the hypospadiac meatus , a plane is created between the buck 's fascia and tunica albuginea [ figure 4 ] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . from lateral to medial , the spongiosum is dissected off the underlying corpora cavernosa .", "mobilization is done well beneath the true urethral plate on both sides , lifting off the midline spongiosum and urethral plate [ figure 5 ] from the corpora cavernosa when required , to correct chordee .", "dissection is performed with care , avoiding damage to the corpus spongiosum or corpus cavernosum .", "the proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee .", "adequacy of curvature correction is confirmed by gittes test . since the spongiosal pillars spread beneath the glans wings on each side , care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees , which leaves healthy , thick glans wings yet intact spongiosum when done correctly [ figure 6 ] .", "the urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone ( pds ) suture after a dorsal incision to create a new urethra .", "the mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [ figure 2d ] . a 6-to 10 fr urethral catheter , depending on patient age", "no additional cover of dartos , tunica vaginalis or de - epithelialized skin was used .", "showing creation of plane proximal to meatus between tunica and buck 's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ .", "patients were evaluated at follow - up visits after 1 , 3 , 6 , 12 months and then yearly .", "results were evaluated in terms of patients , parents and surgeon 's satisfaction , keeping in mind the stream , cosmesis and lack of complications .", "if at 3 months follow - up the stream was narrow , we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube ."], ["the age range of the patients was 4 months to 38 years ( mean 11.53 years ) .", "the hospital stay ranged from 8 - 10 days and period of follow - up was 6 months to 2 years .", "the hypospadias was distal , mid and proximal in 81 , 12 , and 20 cases respectively .", "correction of chordee was carried out by penile de - gloving alone in 5 , mobilization of urethral plate with spongiosum in 22 and combination of both in 45 cases .", "on correlating the type of hypospadias with development of spongiosum ; more proximal hypospadias was associated with poorer development of the spongiosum ( p = 0.05 ) [ table 1 ] .", "two patients had meatal stenosis with fistula and seven patients had urethral fistula ( clavien - dindo grade 3b ) alone with an overall complication rate of 7.96% .", "fistulas occurred in 2 cases each of distal and mid hypospadias and 5 cases of proximal hypospadias .", "complications were higher in proximal hypospadias as compared to distal ( p = 0.0019 ) [ table 1 ] .", "correlation of type of hypospadias , development of spongiosum and complications three ( one each in distal , mid and proximal ) out of thirteen ( 23.03% ) cases with poorly developed spongiosum and six ( distal 1 , mid 1 and proximal 4 ) out of 53 ( 11.32% ) cases with moderately developed spongiosum developed a fistula while none of the cases with well - developed spongiosum developed a fistula .", "one out of 53 ( 1.88% ) cases with moderately developed spongiosum and 1/13 ( 7.69% ) cases with poorly developed spongiosum developed meatal stenosis .", "thus , poorer spongiosum was associated with greater number of complications ( p = 0.011 ) [ table 1 ] .", "none of the patients developed a stricture urethra and all patients / parents were satisfied with cosmesis .", "all nine patients with urethral fistula were operated upon 6 - 12 months later and the fistulae repaired with no further sequelae ."], ["the male urethra develops from the urethral folds as they fuse in the midline after the 6 week of fetal life .", "the endodermally derived distal solid urethral plate must canalize to form the distal most glanular part of the urethra .", "the urethra forms sequentially after the proximal urethral folds fuse and the mesenchyme within the urethral folds forms the corpus spongiosum . according to the classic theory , embryologically , the last step in urethra formation is the joining of the main endodermal urethral channel with the ectoderm as it invades the glans .", "the normal and hypospadiac penises differ in the development of glans and urethra , especially their vascularity . in hypospadias ,", "there is failed urethral fold fusion and ectodermal intrusion , histologically characterized by an extremely vascular area with large endothelial lined sinuses around the abortive urethral spongiosum and abnormal glans .", "the degree of hypoplasia of corpus spongiosum depends upon the severity of hypospadias and proliferation of mesodermal tissue .", "we classified the spongiosum according to its development and vascularity as poorly developed , moderately developed and well - developed .", "further , it may act as a guide to new surgeons attempting hypospadias about the feasibility of spongioplasty in a particular case and whether an additional layer may be required .", "none of the patients with well - developed spongiosum in distal hypospadias had any complication .", "complications in proximal hypospadias with well - developed spongiosum were also very low as compared to poorly developed spongiosum cases .", "most importantly , poorer was the spongiosum , greater was the number of complications ( p = 0.011 ) . on analysis of our results , we found that more proximal meatus was associated with poorer development of the spongiosum ( p = 0.05 ) and proximal hypospadias was associated with greater incidence of complications ( p = 0.0019 ) .", "however , not all hypospadias with poorly developed spongiosum had complications , suggesting that other factors like development and width of urethral plate also play a role in the outcome of the repair .", "since none of the patients of distal hypospadias with well - developed spongiosum had fistula , dorsal dartos cover in such patients may be omitted and the prepuce can be utilized for preputioplasty .", "however in patients with poorly developed spongiosa , an additional interposing layer like dartos or tunica should be used .", "since its first description by snodgrass for repair of hypospadias in 1994 , tip has gained widespread acceptance due to its versatility , low complication rate and reliable creation of vertically oriented meatus , with excellent cosmetic outcome .", "some authors recommend tipu as the technique to be considered for primary and re - operative repair of distal and some midshaft hypospadias cases with selected proximal ones .", "the common complications reported after tipu repair include fistulae , urethral stricture , meatal stenosis , persistent chordee , infections and wound dehiscence with development of urethrocutaneous fistula being the most common .", "the causes of fistulae are local infection , ischemia , an inadequate procedure , poor tissue healing and distal obstruction due to meatal stenosis / encrustation which promotes fistulae formation . to decrease the incidence of post - operative fistulae , many modifications in the tip repair have been tried .", "however , no single surgical technique can be considered to be a clear winner to prevent the formation of fistulae .", "one of the most important factors in reducing fistula formation with the tip technique is the introduction of a protective intermediate layer between the neourethra and the skin .", "dorsal , lateral , single or double dartos flaps , ventral based dartos flap , scrotal dartos , de - epithelized local penile skin , preputial flap , paraurethral tissue , spongioplasty , or tunica vaginalis flaps have all been used for the same . out of all these", ", the dorsal dartos flap is used by most surgeons , but there is still no consensus over the ideal interposing tissue in tipu .", "because of edema , necrosis of skin , hematoma and torque with mobilization of the dorsal dartos flap , its role as an interposing tissue has been questioned .", "the corpus spongiosum provides a well - vascularized , spongy protective covering to the normal urethra .", "it may aid in the natural propulsion of urine and semen . using this natural , locally available tissue to cover", "spongioplasty also decreases the tension on the suture line of urethroplasty in the midline , especially during erections .", "the use of spongiosal tissue as an intermediate layer between the urethra and skin was described in 2000 by yerkes and beaudoin in separate studies .", "yerkes reported no fistulae in any of her patients for whom spongioplasty was implemented in a number of urethral repair techniques , whereas beaudoin described the anatomical characteristics of the spongiosal layer in the hypospadic penis and implemented spongioplasty in patients who underwent urethral tubularization . in 2003 , dodat et al", ". showed no fistula formation in any of the 51 patients who underwent tipu and spongioplasty .", "el - sherbiny et al . reported that covering the neourethra with a flap or spongioplasty minimized the risk of fistula formation . in a series of 500 cases , sarhan et al .", "used a dartos flap , spongioplasty , or a combination of these techniques in addition to tip .", "they observed a statistically significant decrease in fistula formation in cases in which spongioplasty was implemented compared with cases in which it was not . in the present series we had an acceptable overall fistula rate of 7.96% with no complications in distal hypospadias with well - developed spongiosum where we did not use dorsal / ventral dartos or any other tissue in addition with spongioplasty .", "these results are comparable with other studies where dorsal dartos has been used with or without spongioplasty .", "since the results of well - developed spongioplasty alone in distal hypospadias were very good , additional cover with dorsal dartos can be omitted .", "report that various studies using dartos based flaps , tunica vaginalis and subcutaneous tissue flaps have a fistula rate of 2 - 33% .", "when spongioplasty alone was used as an interposing layer , in various series complications ranged from 4% to 40% with 14.2% complication rate in 197 cases whereas in the present series they were seen in 7.96 % cases [ table 2 ] .", "series using spongioplasty alone for hypospadias repair our technique of spongioplasty is similar to those mentioned earlier in that we mobilize the urethral plate with the spongiosum completely off the corpora cavernosa starting laterally and moving medially .", "furthermore we mobilized the spongiosal pillars into the glans obliquely at an angle of 45 almost up to the tip of the glans .", "the mobilization of urethral plate with spongiosum have added advantages of correction of torsion and ventral curvature , urethral plate can be tubularized without tension on suture line and many a times without incising the urethral plate .", "complete spongioplasty if done properly as outlined above restores a near normal urethra and provides good support .", "it is a healthy , vascular cover which probably decreases tension on the midline sutures especially during erections .", "better is the spongiosum , lesser are the chances of post - operative fistulae formation .", "the strengths of our study are that it is prospective , cases belonged to similar socio - economic status , procedures were performed by a single surgeon in similar circumstances and with adequate follow - up .", "the weaknesses are that the criteria for development of spongiosum are subjective so there are chances of error in judgment , especially in well and moderately developed spongiosum . to the best of our knowledge , this is the only series in current literature with such a large number of patients undergoing spongioplasty alone with tip repair ."], ["a more proximal hypospadias is associated with poorer spongiosum and this is likely to increase the chances of complications . better developed and thicker spongiosum results in lower incidence of fistula formation after tipu .", "spongioplasty reconstructs a near normal urethra as per anatomical principles of surgery with least complications .", "finally , it adds an extra layer of locally available healthy vascular tissue avoiding dissection for local flaps .", "we recommend spongioplasty be incorporated as an essential step in all patients undergoing tip repair for hypospadias ."]]}
{"article_id": "PMC4418247", "article_text": ["premenstrual syndrome ( pms ) is a common disorder of young and middle - aged women , characterized by emotional and physical symptoms that consistently recur in a cyclic manner during the luteal phase of the ms and typically abate by menopause .", "the symptoms which exacerbate during the premenstrual period may be present in about 20 - 40% of women , though some studies quote higher figures .", "premenstrual dysphoric disorder ( pmdd ) is characterized by a constellation of affective and somatic symptoms manifested during late luteal phase and resolve shortly after the onset of menses . unlike other mood disorders , the mood disturbances associated with pmdd are cyclical and tightly linked to the menstrual cycle ( ms ) ; hence , the occurrence of symptoms ceases during pregnancy and after menopause . up to 85% of menstruating women", "more than 200 symptoms have been associated with pms , but irritability , tension , and dysphoria are the most prominent and consistently described .", "symptoms relieve within 4 days of the onset of menses and do not recur until at least day 13 of the ms . during premenstrual period , onset of a depressive episode may be observed .", "significant behavioral symptoms ( depression , aggression , agitated depression , irritability and so on ) occur during pms and do interfere with personal , social , and occupational functioning .", "overall , little data are available on the presence of pms and pmdd in indian population which houses about a sixth of the world female population and the largest adolescent female population .", "moreover , studies have not dealt with rural setting , although 70% of women in india reside in rural areas .", "there is a suggestion that differences exist in the premenstrual symptoms and severity thereof in the urban and rural areas .", "therefore , this study was undertaken in order to evaluate the presence of pms and pmdd and find their correlation with depression in a female population primarily comprising nursing students and staff in a rural setting .", "the current prospective observational study was carried out at a rural tertiary care medical college and hospital located in wardha , vidharbha region of maharashtra , by the department of psychiatry .", "female nursing students or staff aged between 18 and 40 years , who were likely to stay within the institution for the study period was included . those subjects with diagnosed chronic illnesses like epilepsy , migraine , anemia , thyroid disease , polycystic ovarian disease ; or those using oral contraceptive pills , any hormonal drugs , propranolol or antihypertensives", "the students and the staff members ( total : 118 ) were divided into batches of 25 and were given a short sensitizing lecture focusing on the symptomatology and identification of pms , pmdd , and depressive disorder .", "the participants rated 17 symptoms of penn daily symptom report scoring sheets on a daily basis .", "the menstrual period ( day 1 to end of menstrual flow ) and premenstrual period ( 5 days prior to 1 day prior to start of menstrual flow ) were marked on the scoring sheet .", "weekly mobile short message service and workplace / classroom reminders were sent to the participants .", "subjects with features suggestive of pmdd were further investigated for the presence of pmdd using diagnostic and statistical manual of mental disorders , 4 edition , text revision ( dsm - iv tr ) diagnostic research criteria .", "information was gathered through a structured clinical interview by one of the authors ( rr ) . at the end of the study period ,", "the participants were asked to rate their depressive symptoms if any , on the primary care evaluation of mental disorders ( prime md ) depression screen .", "the dsm - iv tr criteria for depression were applied to those scoring above the cutoff on prime md , and hamilton depression rating scale was applied to assess the severity of depression .", "penn daily symptom report : is a self - administered checklist enquiring 17 symptoms that are rated on a likert scale of 0 ( not at all ) to 4 ( very severe ) .", "the scale is short and reliable , valid with good internal consistency and used in a primary care setting .", "primary care evaluation of mental disorders is a self - reported screening instrument for depression designed to facilitate the recognition and diagnosis of the most common mental disorders in primary care patients .", "the depression screen has 9 questions with 4 possible responses ( not at all to every day ) .", "if the response to five or more questions is yes , with one of the first two questions being answered as affirmative , a diagnosis of depression can be considered .", "hamilton rating scale for depression ( ham - d ) : has 17 items with a total score ranging from 0 to 50 , is widely used to assess the symptoms of depression .", "ratings are made on the basis of the clinical interview , plus any additional available information .", "diagnostic and statistical manual of mental disorders , 4 edition , text revision research criteria for pmdd require the presence of symptoms during the last week of most menstrual cycles of the preceding year which begin to remit after the onset of the follicular phase . for a diagnosis", ", 5 out of 11 symptoms should be present and with at least one of them being markedly depressed mood , hopelessness or self - deprecating thoughts ; marked anxiety , tension or feelings of being ", "keyed up  or  on the edge ,  marked affective lability ; or persistent and marked anger , irritability or increased interpersonal conflicts .", "symptoms should be associated with socio - occupational impairment , not merely be an exacerbation of another disorder , and the criteria should be confirmed by prospective ratings of at least 2 months .", "using spss 19.0 , descriptive statistics were applied for analysis of demographic and clinical variables ; inferential statistics to access relationships between the demographic and clinical variables and pmdd and pms symptoms .", "the current prospective observational study was carried out at a rural tertiary care medical college and hospital located in wardha , vidharbha region of maharashtra , by the department of psychiatry .", "female nursing students or staff aged between 18 and 40 years , who were likely to stay within the institution for the study period was included . those subjects with diagnosed chronic illnesses like epilepsy , migraine , anemia , thyroid disease , polycystic ovarian disease ; or those using oral contraceptive pills , any hormonal drugs , propranolol or antihypertensives", "the students and the staff members ( total : 118 ) were divided into batches of 25 and were given a short sensitizing lecture focusing on the symptomatology and identification of pms , pmdd , and depressive disorder .", "the participants rated 17 symptoms of penn daily symptom report scoring sheets on a daily basis .", "the menstrual period ( day 1 to end of menstrual flow ) and premenstrual period ( 5 days prior to 1 day prior to start of menstrual flow ) were marked on the scoring sheet .", "the days not ascribed to menstrual or premenstrual period was considered as  other  period .", "weekly mobile short message service and workplace / classroom reminders were sent to the participants .", "subjects with features suggestive of pmdd were further investigated for the presence of pmdd using diagnostic and statistical manual of mental disorders , 4 edition , text revision ( dsm - iv tr ) diagnostic research criteria .", "information was gathered through a structured clinical interview by one of the authors ( rr ) . at the end of the study period ,", "the participants were asked to rate their depressive symptoms if any , on the primary care evaluation of mental disorders ( prime md ) depression screen .", "the dsm - iv tr criteria for depression were applied to those scoring above the cutoff on prime md , and hamilton depression rating scale was applied to assess the severity of depression .", "penn daily symptom report : is a self - administered checklist enquiring 17 symptoms that are rated on a likert scale of 0 ( not at all ) to 4 ( very severe ) .", "the scale is short and reliable , valid with good internal consistency and used in a primary care setting .", "primary care evaluation of mental disorders is a self - reported screening instrument for depression designed to facilitate the recognition and diagnosis of the most common mental disorders in primary care patients .", "the depression screen has 9 questions with 4 possible responses ( not at all to every day ) .", "if the response to five or more questions is yes , with one of the first two questions being answered as affirmative , a diagnosis of depression can be considered .", "hamilton rating scale for depression ( ham - d ) : has 17 items with a total score ranging from 0 to 50 , is widely used to assess the symptoms of depression .", "ratings are made on the basis of the clinical interview , plus any additional available information .", "diagnostic and statistical manual of mental disorders , 4 edition , text revision research criteria for pmdd require the presence of symptoms during the last week of most menstrual cycles of the preceding year which begin to remit after the onset of the follicular phase . for a diagnosis", ", 5 out of 11 symptoms should be present and with at least one of them being markedly depressed mood , hopelessness or self - deprecating thoughts ; marked anxiety , tension or feelings of being ", "keyed up  or  on the edge ,  marked affective lability ; or persistent and marked anger , irritability or increased interpersonal conflicts .", "symptoms should be associated with socio - occupational impairment , not merely be an exacerbation of another disorder , and the criteria should be confirmed by prospective ratings of at least 2 months .", "using spss 19.0 , descriptive statistics were applied for analysis of demographic and clinical variables ; inferential statistics to access relationships between the demographic and clinical variables and pmdd and pms symptoms .", "a total of 118 subjects was approached , of which 100 consented to the study and 18 refused consent .", "the mean age of the sample was 21.83 years ( 4.85 ) with a range of 18 - 37 years .", "the mss were regular in most of the participants , and their duration ranged from 26 to 30 days .", "demographic and clinical details the phenomenology in relation to the ms is shown in table 2 .", "overall , fatigue , mood swings , and irritability were the most common symptoms in the premenstrual phase ; fatigue , cramps , and body aches were the most common symptoms in the menstrual phase and concentration difficulties , headache , and fatigue were the most common symptoms in the other periods .", "when compared to premenstrual and menstrual phase , the lesser number of symptoms were present in the other time periods .", "phenomenology of symptoms during premenstrual and menstrual phase and other times according to penn daily symptom report as per the criteria of penn daily symptom report , the majority of females had some premenstrual symptoms ( 67 participants ) .", "of these , 26 participants had some premenstrual distress , 20 had mild pms , 10 had moderate pms , 8 had severe pms , and 3 had very severe pms .", "the diagnosis of pmdd according to dsm - iv criteria was present in 10 participants .", "the diagnosis of pmdd according to dsm - iv tr and premenstrual symptom severity ( measured by penn daily symptom report ) concurred with each other ( mann - whitney u = 12.5 , p < 0.001 )", ". the severity of premenstrual symptoms did not have significant correlation with age , socioeconomic status or cycle length ( kendall tau b of 0.005 , 0.039 , and 0.089 with p = 0.950 , 0.669 and 0.264 , respectively ) .", "however , premenstrual symptom severity and the presence of pmdd diagnosis were associated with lesser age of menarche ( kendall tau b = 0.170 , p = 0.038 and mann - whitney u = 139.5 , p < 0.001 , respectively ) .", "major depressive disorder according to dsm - iv tr criteria was present in 13 of them ( i.e. , 46.4% of those with depressive symptoms ) , and the remaining 15 had subsyndromal depression . among the 13 with major depressive disorder , 4 had mild depression (", "ham d scores 7 - 17 ) , 5 had moderate depression ( ham score 18 - 24 ) , and 4 had severe depression ( ham d score of > 24 ) .", "severity of premenstrual symptoms and depressive symptoms the relationship of depressive symptoms to that of premenstrual symptoms is shown in table 4 .", "significant relationship was found between the diagnosis of pmdd and the presence of depression according to prime md as well as the presence of dsm - iv tr diagnosis of depression (  = 5.644 , p = 0.027 and  = 7.162 , p = 0.024 , respectively ) .", "furthermore , there was a significant relationship between the severity of premenstrual symptoms and the presence of a diagnosis of depression according to dsm - iv tr ( mann - whitney u = 315 , p = 0.008 ) .", "some pms symptoms were observed in 67% ; diagnosis of pmdd in 10% ; depressive symptoms in 28% of the sample .", "the diagnosis of major depression was significantly associated with the severity of pms symptoms as well as the presence of pmdd .", "study takes a systematic look at the occurrence of premenstrual symptoms and pmdd in a rural area ( usually neglected population in any country ) .", "some of the key demographic variables have been looked into , and the association with a diagnosis of major depression has been explored .", "the sensitization lecture imparted as a part of the methodology could itself be indirect means of creating awareness of the same in the community .", "highly selective sample comprising rural nursing students and staff from one academic institute that limits the generalizability of the findings ; small sample size ; longitudinal relations between potential confounders such as examination stress and outcome were not assessed ; and the presence of medical and gynecological disorders were not systematically evaluated or ruled out which may have influenced the symptom profile encountered with pms .", "the diagnosis of depression was based upon clinical interview , and structured instruments like mini international neuropsychiatric interview were not used .", "the recruitment was through purposive sampling and only those women who consented to participate were enrolled .", "this study in a relatively young female population finds that distressing premenstrual symptoms are fairly common and present in about two - thirds of the studied population .", "a cross - cultural study comprising 14 culturally different areas in 10 countries reported prevalence of 23 - 34% in non - western cultures ; 71 - 73% in western countries .", "however , recent studies from asian countries including egypt , saudi arabia , and japan show a high proportion of women do suffer from premenstrual symptoms .", "the differences in reported rates of premenstrual symptoms depend to some extent on the definitions used methods of data collection , sampling technique , and the type of study population .", "the occurrence of pmdd in our study ( 10% ) is in line with published literature .", "pmdd , as per the dsm - iv criteria , is found to occur in 3 - 8% of women and these figures have been closely replicated in several epidemiological studies and surveys .", "gehlert and hartlage found that prevalence rates for pmdd , even when dsm - iv criteria are used , significantly vary depending upon the method of measuring symptom change", ". the restrictive nature of the dsm - iv pmdd criteria , particularly the requirement of an arbitrary cutoff point of at least 5 severe symptoms , remains controversial .", "a question arises as to whether there are a substantial proportion of symptomatic women in the general population who have premenstrual impairment and distress , and may need treatment but do not meet dsm - iv criteria for pmdd due to lesser number of symptoms reported . in this", "wittchen et al . found 35.3% of women had four or more premenstrual symptoms , and prevalence of sub - threshold pmdd was 18.6% . looking at studies from india , pmdd has been reported in about 10% of the population .", "mchichi alami et al . remarked on the high rates of depressive symptoms such as low mood , fatigue , and sleep abnormalities in those women with pmdd .", "similar results of increased rates of depression in those suffering from pms have been found by other authors . typically , irritable and depressed moods increased in the late luteal phase .", "our results concur with previous findings as we observed presence of depressive symptoms to be associated with premenstrual symptoms , as well as pmdd .", "though a wide range of symptoms have been associated with premenstrual complaints , we evaluated a specific set of a - priori 17 symptoms .", "we found that increased appetite , sleep disorders ( insomnia and hypersomnia ) , fatigue , feeling of lack of energy , and decrease of interest for everyday activity were quite common .", "some other studies have reported different frequencies and rank order of symptoms with breast tenderness and muscle pain as predominant complaints .", "the reason why different symptoms are reported even when using scales of similar items may be partially attributable to cultural differences and prior bias toward a set of symptomatology due to preconceived notions about premenstrual problems and symptoms .", "some pms symptoms were observed in 67% ; diagnosis of pmdd in 10% ; depressive symptoms in 28% of the sample .", "the diagnosis of major depression was significantly associated with the severity of pms symptoms as well as the presence of pmdd .", "study takes a systematic look at the occurrence of premenstrual symptoms and pmdd in a rural area ( usually neglected population in any country ) .", "some of the key demographic variables have been looked into , and the association with a diagnosis of major depression has been explored .", "the sensitization lecture imparted as a part of the methodology could itself be indirect means of creating awareness of the same in the community .", "highly selective sample comprising rural nursing students and staff from one academic institute that limits the generalizability of the findings ; small sample size ; longitudinal relations between potential confounders such as examination stress and outcome were not assessed ; and the presence of medical and gynecological disorders were not systematically evaluated or ruled out which may have influenced the symptom profile encountered with pms .", "the diagnosis of depression was based upon clinical interview , and structured instruments like mini international neuropsychiatric interview were not used .", "the recruitment was through purposive sampling and only those women who consented to participate were enrolled .", "this study in a relatively young female population finds that distressing premenstrual symptoms are fairly common and present in about two - thirds of the studied population .", "a cross - cultural study comprising 14 culturally different areas in 10 countries reported prevalence of 23 - 34% in non - western cultures ; 71 - 73% in western countries .", "however , recent studies from asian countries including egypt , saudi arabia , and japan show a high proportion of women do suffer from premenstrual symptoms .", "the differences in reported rates of premenstrual symptoms depend to some extent on the definitions used methods of data collection , sampling technique , and the type of study population .", "the occurrence of pmdd in our study ( 10% ) is in line with published literature .", "pmdd , as per the dsm - iv criteria , is found to occur in 3 - 8% of women and these figures have been closely replicated in several epidemiological studies and surveys .", "gehlert and hartlage found that prevalence rates for pmdd , even when dsm - iv criteria are used , significantly vary depending upon the method of measuring symptom change", ". the restrictive nature of the dsm - iv pmdd criteria , particularly the requirement of an arbitrary cutoff point of at least 5 severe symptoms , remains controversial .", "a question arises as to whether there are a substantial proportion of symptomatic women in the general population who have premenstrual impairment and distress , and may need treatment but do not meet dsm - iv criteria for pmdd due to lesser number of symptoms reported . in this", "regard , chawla et al . reported that 12.6% of women met full pmdd criteria for one cycle .", "found 35.3% of women had four or more premenstrual symptoms , and prevalence of sub - threshold pmdd was 18.6% .", "looking at studies from india , pmdd has been reported in about 10% of the population .", "mchichi alami et al . remarked on the high rates of depressive symptoms such as low mood , fatigue , and sleep abnormalities in those women with pmdd .", "similar results of increased rates of depression in those suffering from pms have been found by other authors . typically , irritable and depressed moods increased in the late luteal phase .", "our results concur with previous findings as we observed presence of depressive symptoms to be associated with premenstrual symptoms , as well as pmdd .", "though a wide range of symptoms have been associated with premenstrual complaints , we evaluated a specific set of a - priori 17 symptoms .", "we found that increased appetite , sleep disorders ( insomnia and hypersomnia ) , fatigue , feeling of lack of energy , and decrease of interest for everyday activity were quite common .", "some other studies have reported different frequencies and rank order of symptoms with breast tenderness and muscle pain as predominant complaints .", "the reason why different symptoms are reported even when using scales of similar items may be partially attributable to cultural differences and prior bias toward a set of symptomatology due to preconceived notions about premenstrual problems and symptoms .", "concurrent depression is increased by the severity of pms symptoms and the presence of pmdd . gynecologist needs to screen such subjects for depression and refer to mental - health professional early , in routine clinical practice .", "there is a need for conducting larger prospective studies focusing on pmdd and its relation with depressive disorder ."], "abstract_text": ["<S> background : premenstrual syndrome ( pms ) , premenstrual dysphoric disorder ( pmdd ) and depressive disorder are fairly common ; symptoms do overlap , often under - identified and under - emphasized , particularly in rural india.objective:the objective was to assess the occurrence of pms and pmdd in a sample of students and staff of a nursing college and to find their correlation with depression.materials and methods : a prospective cohort study ; tertiary care hospital in rural india ( wardha , maharashtra ) ; 118 female nursing students or staff aged between 18 and 40 years , who were likely to stay within the institution for the study period . </S>", "<S> the participants were rated on penn daily symptom report prospectively for a period of 3-month . those who scored positive were applied diagnostic and statistical manual of mental disorders , 4th edition , text revision ( dsm - iv tr ) criteria for pmdd ; and were applied primary care evaluation of mental disorders depression screening followed by dsm - iv tr criteria for depression . </S>", "<S> severity of depression was measured using hamilton depression rating scale.results:main outcome measures were frequency and severity of depression in individuals with pms and pmdd and their clinical and sociodemographic correlation . </S>", "<S> the age range of the sample was 18 - 37 years . </S>", "<S> some pms symptoms were observed in 67% ; diagnosis of pmdd in 10% ; depressive symptoms in 28% of the sample . </S>", "<S> 46.4% of those with depressive symptoms had major depression . </S>", "<S> the diagnosis of major depression was significantly associated with the severity of pms symptoms as well as the presence of pmdd.conclusion:premenstrual syndrome is present in a substantial proportion of young females . </S>", "<S> concurrent depression is increased by the severity of pms symptoms and the presence of pmdd . </S>", "<S> gynecologist needs to screen such subjects for depression and refer to mental - health professional early , in routine clinical practice . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Setting and participants", "Procedure", "Instruments", "RESULTS", "DISCUSSION", "Main findings", "Strengths and limitations of the study", "The limitations of the study are", "Interpretation", "CONCLUSION"], "sections": [["premenstrual syndrome ( pms ) is a common disorder of young and middle - aged women , characterized by emotional and physical symptoms that consistently recur in a cyclic manner during the luteal phase of the ms and typically abate by menopause .", "the symptoms which exacerbate during the premenstrual period may be present in about 20 - 40% of women , though some studies quote higher figures .", "premenstrual dysphoric disorder ( pmdd ) is characterized by a constellation of affective and somatic symptoms manifested during late luteal phase and resolve shortly after the onset of menses . unlike other mood disorders , the mood disturbances associated with pmdd are cyclical and tightly linked to the menstrual cycle ( ms ) ; hence , the occurrence of symptoms ceases during pregnancy and after menopause . up to 85% of menstruating women", "more than 200 symptoms have been associated with pms , but irritability , tension , and dysphoria are the most prominent and consistently described .", "symptoms relieve within 4 days of the onset of menses and do not recur until at least day 13 of the ms . during premenstrual period , onset of a depressive episode may be observed .", "significant behavioral symptoms ( depression , aggression , agitated depression , irritability and so on ) occur during pms and do interfere with personal , social , and occupational functioning .", "overall , little data are available on the presence of pms and pmdd in indian population which houses about a sixth of the world female population and the largest adolescent female population .", "moreover , studies have not dealt with rural setting , although 70% of women in india reside in rural areas .", "there is a suggestion that differences exist in the premenstrual symptoms and severity thereof in the urban and rural areas .", "therefore , this study was undertaken in order to evaluate the presence of pms and pmdd and find their correlation with depression in a female population primarily comprising nursing students and staff in a rural setting ."], ["the current prospective observational study was carried out at a rural tertiary care medical college and hospital located in wardha , vidharbha region of maharashtra , by the department of psychiatry .", "female nursing students or staff aged between 18 and 40 years , who were likely to stay within the institution for the study period was included . those subjects with diagnosed chronic illnesses like epilepsy , migraine , anemia , thyroid disease , polycystic ovarian disease ; or those using oral contraceptive pills , any hormonal drugs , propranolol or antihypertensives", "the students and the staff members ( total : 118 ) were divided into batches of 25 and were given a short sensitizing lecture focusing on the symptomatology and identification of pms , pmdd , and depressive disorder .", "the participants rated 17 symptoms of penn daily symptom report scoring sheets on a daily basis .", "the menstrual period ( day 1 to end of menstrual flow ) and premenstrual period ( 5 days prior to 1 day prior to start of menstrual flow ) were marked on the scoring sheet .", "weekly mobile short message service and workplace / classroom reminders were sent to the participants .", "subjects with features suggestive of pmdd were further investigated for the presence of pmdd using diagnostic and statistical manual of mental disorders , 4 edition , text revision ( dsm - iv tr ) diagnostic research criteria .", "information was gathered through a structured clinical interview by one of the authors ( rr ) . at the end of the study period ,", "the participants were asked to rate their depressive symptoms if any , on the primary care evaluation of mental disorders ( prime md ) depression screen .", "the dsm - iv tr criteria for depression were applied to those scoring above the cutoff on prime md , and hamilton depression rating scale was applied to assess the severity of depression .", "penn daily symptom report : is a self - administered checklist enquiring 17 symptoms that are rated on a likert scale of 0 ( not at all ) to 4 ( very severe ) .", "the scale is short and reliable , valid with good internal consistency and used in a primary care setting .", "primary care evaluation of mental disorders is a self - reported screening instrument for depression designed to facilitate the recognition and diagnosis of the most common mental disorders in primary care patients .", "the depression screen has 9 questions with 4 possible responses ( not at all to every day ) .", "if the response to five or more questions is yes , with one of the first two questions being answered as affirmative , a diagnosis of depression can be considered .", "hamilton rating scale for depression ( ham - d ) : has 17 items with a total score ranging from 0 to 50 , is widely used to assess the symptoms of depression .", "ratings are made on the basis of the clinical interview , plus any additional available information .", "diagnostic and statistical manual of mental disorders , 4 edition , text revision research criteria for pmdd require the presence of symptoms during the last week of most menstrual cycles of the preceding year which begin to remit after the onset of the follicular phase . for a diagnosis", ", 5 out of 11 symptoms should be present and with at least one of them being markedly depressed mood , hopelessness or self - deprecating thoughts ; marked anxiety , tension or feelings of being ", "keyed up  or  on the edge ,  marked affective lability ; or persistent and marked anger , irritability or increased interpersonal conflicts .", "symptoms should be associated with socio - occupational impairment , not merely be an exacerbation of another disorder , and the criteria should be confirmed by prospective ratings of at least 2 months .", "using spss 19.0 , descriptive statistics were applied for analysis of demographic and clinical variables ; inferential statistics to access relationships between the demographic and clinical variables and pmdd and pms symptoms ."], ["the current prospective observational study was carried out at a rural tertiary care medical college and hospital located in wardha , vidharbha region of maharashtra , by the department of psychiatry .", "female nursing students or staff aged between 18 and 40 years , who were likely to stay within the institution for the study period was included . those subjects with diagnosed chronic illnesses like epilepsy , migraine , anemia , thyroid disease , polycystic ovarian disease ; or those using oral contraceptive pills , any hormonal drugs , propranolol or antihypertensives"], ["the students and the staff members ( total : 118 ) were divided into batches of 25 and were given a short sensitizing lecture focusing on the symptomatology and identification of pms , pmdd , and depressive disorder .", "the participants rated 17 symptoms of penn daily symptom report scoring sheets on a daily basis .", "the menstrual period ( day 1 to end of menstrual flow ) and premenstrual period ( 5 days prior to 1 day prior to start of menstrual flow ) were marked on the scoring sheet .", "the days not ascribed to menstrual or premenstrual period was considered as  other  period .", "weekly mobile short message service and workplace / classroom reminders were sent to the participants .", "subjects with features suggestive of pmdd were further investigated for the presence of pmdd using diagnostic and statistical manual of mental disorders , 4 edition , text revision ( dsm - iv tr ) diagnostic research criteria .", "information was gathered through a structured clinical interview by one of the authors ( rr ) . at the end of the study period ,", "the participants were asked to rate their depressive symptoms if any , on the primary care evaluation of mental disorders ( prime md ) depression screen .", "the dsm - iv tr criteria for depression were applied to those scoring above the cutoff on prime md , and hamilton depression rating scale was applied to assess the severity of depression ."], ["penn daily symptom report : is a self - administered checklist enquiring 17 symptoms that are rated on a likert scale of 0 ( not at all ) to 4 ( very severe ) .", "the scale is short and reliable , valid with good internal consistency and used in a primary care setting .", "primary care evaluation of mental disorders is a self - reported screening instrument for depression designed to facilitate the recognition and diagnosis of the most common mental disorders in primary care patients .", "the depression screen has 9 questions with 4 possible responses ( not at all to every day ) .", "if the response to five or more questions is yes , with one of the first two questions being answered as affirmative , a diagnosis of depression can be considered .", "hamilton rating scale for depression ( ham - d ) : has 17 items with a total score ranging from 0 to 50 , is widely used to assess the symptoms of depression .", "ratings are made on the basis of the clinical interview , plus any additional available information .", "diagnostic and statistical manual of mental disorders , 4 edition , text revision research criteria for pmdd require the presence of symptoms during the last week of most menstrual cycles of the preceding year which begin to remit after the onset of the follicular phase . for a diagnosis", ", 5 out of 11 symptoms should be present and with at least one of them being markedly depressed mood , hopelessness or self - deprecating thoughts ; marked anxiety , tension or feelings of being ", "keyed up  or  on the edge ,  marked affective lability ; or persistent and marked anger , irritability or increased interpersonal conflicts .", "symptoms should be associated with socio - occupational impairment , not merely be an exacerbation of another disorder , and the criteria should be confirmed by prospective ratings of at least 2 months .", "using spss 19.0 , descriptive statistics were applied for analysis of demographic and clinical variables ; inferential statistics to access relationships between the demographic and clinical variables and pmdd and pms symptoms ."], ["a total of 118 subjects was approached , of which 100 consented to the study and 18 refused consent .", "the mean age of the sample was 21.83 years ( 4.85 ) with a range of 18 - 37 years .", "the mss were regular in most of the participants , and their duration ranged from 26 to 30 days .", "demographic and clinical details the phenomenology in relation to the ms is shown in table 2 .", "overall , fatigue , mood swings , and irritability were the most common symptoms in the premenstrual phase ; fatigue , cramps , and body aches were the most common symptoms in the menstrual phase and concentration difficulties , headache , and fatigue were the most common symptoms in the other periods .", "when compared to premenstrual and menstrual phase , the lesser number of symptoms were present in the other time periods .", "phenomenology of symptoms during premenstrual and menstrual phase and other times according to penn daily symptom report as per the criteria of penn daily symptom report , the majority of females had some premenstrual symptoms ( 67 participants ) .", "of these , 26 participants had some premenstrual distress , 20 had mild pms , 10 had moderate pms , 8 had severe pms , and 3 had very severe pms .", "the diagnosis of pmdd according to dsm - iv criteria was present in 10 participants .", "the diagnosis of pmdd according to dsm - iv tr and premenstrual symptom severity ( measured by penn daily symptom report ) concurred with each other ( mann - whitney u = 12.5 , p < 0.001 )", ". the severity of premenstrual symptoms did not have significant correlation with age , socioeconomic status or cycle length ( kendall tau b of 0.005 , 0.039 , and 0.089 with p = 0.950 , 0.669 and 0.264 , respectively ) .", "however , premenstrual symptom severity and the presence of pmdd diagnosis were associated with lesser age of menarche ( kendall tau b = 0.170 , p = 0.038 and mann - whitney u = 139.5 , p < 0.001 , respectively ) .", "major depressive disorder according to dsm - iv tr criteria was present in 13 of them ( i.e. , 46.4% of those with depressive symptoms ) , and the remaining 15 had subsyndromal depression . among the 13 with major depressive disorder , 4 had mild depression (", "ham d scores 7 - 17 ) , 5 had moderate depression ( ham score 18 - 24 ) , and 4 had severe depression ( ham d score of > 24 ) .", "severity of premenstrual symptoms and depressive symptoms the relationship of depressive symptoms to that of premenstrual symptoms is shown in table 4 .", "significant relationship was found between the diagnosis of pmdd and the presence of depression according to prime md as well as the presence of dsm - iv tr diagnosis of depression (  = 5.644 , p = 0.027 and  = 7.162 , p = 0.024 , respectively ) .", "furthermore , there was a significant relationship between the severity of premenstrual symptoms and the presence of a diagnosis of depression according to dsm - iv tr ( mann - whitney u = 315 , p = 0.008 ) ."], ["some pms symptoms were observed in 67% ; diagnosis of pmdd in 10% ; depressive symptoms in 28% of the sample .", "the diagnosis of major depression was significantly associated with the severity of pms symptoms as well as the presence of pmdd .", "study takes a systematic look at the occurrence of premenstrual symptoms and pmdd in a rural area ( usually neglected population in any country ) .", "some of the key demographic variables have been looked into , and the association with a diagnosis of major depression has been explored .", "the sensitization lecture imparted as a part of the methodology could itself be indirect means of creating awareness of the same in the community .", "highly selective sample comprising rural nursing students and staff from one academic institute that limits the generalizability of the findings ; small sample size ; longitudinal relations between potential confounders such as examination stress and outcome were not assessed ; and the presence of medical and gynecological disorders were not systematically evaluated or ruled out which may have influenced the symptom profile encountered with pms .", "the diagnosis of depression was based upon clinical interview , and structured instruments like mini international neuropsychiatric interview were not used .", "the recruitment was through purposive sampling and only those women who consented to participate were enrolled .", "this study in a relatively young female population finds that distressing premenstrual symptoms are fairly common and present in about two - thirds of the studied population .", "a cross - cultural study comprising 14 culturally different areas in 10 countries reported prevalence of 23 - 34% in non - western cultures ; 71 - 73% in western countries .", "however , recent studies from asian countries including egypt , saudi arabia , and japan show a high proportion of women do suffer from premenstrual symptoms .", "the differences in reported rates of premenstrual symptoms depend to some extent on the definitions used methods of data collection , sampling technique , and the type of study population .", "the occurrence of pmdd in our study ( 10% ) is in line with published literature .", "pmdd , as per the dsm - iv criteria , is found to occur in 3 - 8% of women and these figures have been closely replicated in several epidemiological studies and surveys .", "gehlert and hartlage found that prevalence rates for pmdd , even when dsm - iv criteria are used , significantly vary depending upon the method of measuring symptom change", ". the restrictive nature of the dsm - iv pmdd criteria , particularly the requirement of an arbitrary cutoff point of at least 5 severe symptoms , remains controversial .", "a question arises as to whether there are a substantial proportion of symptomatic women in the general population who have premenstrual impairment and distress , and may need treatment but do not meet dsm - iv criteria for pmdd due to lesser number of symptoms reported . in this", "wittchen et al . found 35.3% of women had four or more premenstrual symptoms , and prevalence of sub - threshold pmdd was 18.6% . looking at studies from india , pmdd has been reported in about 10% of the population .", "mchichi alami et al . remarked on the high rates of depressive symptoms such as low mood , fatigue , and sleep abnormalities in those women with pmdd .", "similar results of increased rates of depression in those suffering from pms have been found by other authors . typically , irritable and depressed moods increased in the late luteal phase .", "our results concur with previous findings as we observed presence of depressive symptoms to be associated with premenstrual symptoms , as well as pmdd .", "though a wide range of symptoms have been associated with premenstrual complaints , we evaluated a specific set of a - priori 17 symptoms .", "we found that increased appetite , sleep disorders ( insomnia and hypersomnia ) , fatigue , feeling of lack of energy , and decrease of interest for everyday activity were quite common .", "some other studies have reported different frequencies and rank order of symptoms with breast tenderness and muscle pain as predominant complaints .", "the reason why different symptoms are reported even when using scales of similar items may be partially attributable to cultural differences and prior bias toward a set of symptomatology due to preconceived notions about premenstrual problems and symptoms ."], ["some pms symptoms were observed in 67% ; diagnosis of pmdd in 10% ; depressive symptoms in 28% of the sample .", "the diagnosis of major depression was significantly associated with the severity of pms symptoms as well as the presence of pmdd ."], ["study takes a systematic look at the occurrence of premenstrual symptoms and pmdd in a rural area ( usually neglected population in any country ) .", "some of the key demographic variables have been looked into , and the association with a diagnosis of major depression has been explored .", "the sensitization lecture imparted as a part of the methodology could itself be indirect means of creating awareness of the same in the community ."], ["highly selective sample comprising rural nursing students and staff from one academic institute that limits the generalizability of the findings ; small sample size ; longitudinal relations between potential confounders such as examination stress and outcome were not assessed ; and the presence of medical and gynecological disorders were not systematically evaluated or ruled out which may have influenced the symptom profile encountered with pms .", "the diagnosis of depression was based upon clinical interview , and structured instruments like mini international neuropsychiatric interview were not used .", "the recruitment was through purposive sampling and only those women who consented to participate were enrolled ."], ["this study in a relatively young female population finds that distressing premenstrual symptoms are fairly common and present in about two - thirds of the studied population .", "a cross - cultural study comprising 14 culturally different areas in 10 countries reported prevalence of 23 - 34% in non - western cultures ; 71 - 73% in western countries .", "however , recent studies from asian countries including egypt , saudi arabia , and japan show a high proportion of women do suffer from premenstrual symptoms .", "the differences in reported rates of premenstrual symptoms depend to some extent on the definitions used methods of data collection , sampling technique , and the type of study population .", "the occurrence of pmdd in our study ( 10% ) is in line with published literature .", "pmdd , as per the dsm - iv criteria , is found to occur in 3 - 8% of women and these figures have been closely replicated in several epidemiological studies and surveys .", "gehlert and hartlage found that prevalence rates for pmdd , even when dsm - iv criteria are used , significantly vary depending upon the method of measuring symptom change", ". the restrictive nature of the dsm - iv pmdd criteria , particularly the requirement of an arbitrary cutoff point of at least 5 severe symptoms , remains controversial .", "a question arises as to whether there are a substantial proportion of symptomatic women in the general population who have premenstrual impairment and distress , and may need treatment but do not meet dsm - iv criteria for pmdd due to lesser number of symptoms reported . in this", "regard , chawla et al . reported that 12.6% of women met full pmdd criteria for one cycle .", "found 35.3% of women had four or more premenstrual symptoms , and prevalence of sub - threshold pmdd was 18.6% .", "looking at studies from india , pmdd has been reported in about 10% of the population .", "mchichi alami et al . remarked on the high rates of depressive symptoms such as low mood , fatigue , and sleep abnormalities in those women with pmdd .", "similar results of increased rates of depression in those suffering from pms have been found by other authors . typically , irritable and depressed moods increased in the late luteal phase .", "our results concur with previous findings as we observed presence of depressive symptoms to be associated with premenstrual symptoms , as well as pmdd .", "though a wide range of symptoms have been associated with premenstrual complaints , we evaluated a specific set of a - priori 17 symptoms .", "we found that increased appetite , sleep disorders ( insomnia and hypersomnia ) , fatigue , feeling of lack of energy , and decrease of interest for everyday activity were quite common .", "some other studies have reported different frequencies and rank order of symptoms with breast tenderness and muscle pain as predominant complaints .", "the reason why different symptoms are reported even when using scales of similar items may be partially attributable to cultural differences and prior bias toward a set of symptomatology due to preconceived notions about premenstrual problems and symptoms ."], ["concurrent depression is increased by the severity of pms symptoms and the presence of pmdd . gynecologist needs to screen such subjects for depression and refer to mental - health professional early , in routine clinical practice .", "there is a need for conducting larger prospective studies focusing on pmdd and its relation with depressive disorder ."]]}
{"article_id": "PMC3783811", "article_text": ["benign neoplasms of the salivary glands are frequently encountered in dental practice . these account for 3% of the tumors involving the head and neck .", "the majority of them occur in the parotid gland , and 80% of them are benign . of these benign neoplasms ,", "50 - 80% are pleomorphic adenomas and 5 - 20% are warthin 's tumors ( wt ) .", "however , warthin 's tumor is the most frequent monomorphic adenoma of the major salivary glands .", "this is a curious benign neoplasm with its intimidating histological name , papillary cyst adenoma lymphomatosum .", "however , the eponym wt has been extensively used ever since aldred warthin reported two cases of this tumor in 1929 .", "earlier in the literature this was also referred to as adeno - lymphoma , papillary cyst adenoma , cystadeno - lymphoma , and epitheliolymphoid cyst .", "wt is generally a disease of elderly men , with the highest incidence in the sixth and seventh decades and the male : female ratio is 4.6:1 .", "the typical features on cytology of wt include oncocytic cells in cohesive , monolayered sheets ; background lymphocytes ; and amorphous , cystic debris .", "histopathologically , it has a cystic appearance with a double layer of oncocytes surrounding a lymphoid stroma .", "the following case presentation deals with wt of the left parotid gland and highlights its clinicopathologic concepts along with its therapeutic management .", "a 65-year - old male patient visited the department of oral medicine , with the chief complaint of swelling below the left ear lobe since six years .", "patient was a known smoker since the past 25 years and there was no history of alcohol consumption . on examination , the lesion extended from the left ear lobule to the lower border of the ramus of the mandible superoinferiorly and also extended behind the left ear [ figure 1 ] .", "it was approximately 5 cm in greatest dimensions ; smooth contoured , was firm in consistency and had well - defined borders .", "stimulation of the parotid glands yielded normal salivary flow with normal consistency , quantity and color .", "based on the history and clinical examination , a provisional diagnosis of warthin 's tumor was given .", "a differential diagnosis of pleomorphic adenoma , a low - grade parotid malignancy , lipoma and neurofibroma arising in the salivary gland were included .", "the investigatory workup included complete hemogram , extra - oral radiograph , ultrasonography , computed tomography and excisional biopsy of the lesion .", "ultrasonographic finding showed a well - defined hypoechoic mass in the lower pole of the left parotid gland .", "the rest of the parotid gland parenchyma was normal and there was no evidence of ductal dilatation . computed tomography examination revealed a rounded and well - defined cystic lesion involving the superficial lobe of the left parotid gland [ figure 3 ] .", "ultrasonograph showing well - defined hypoechoic mass computed tomography examination showing the lesion later , excisional biopsy of the lesion was planned using partial parotidectomy as the technique of choice [ figure 4 ] .", "the tissue obtained was fixed in 10% of neutral buffered formalin , and processed routinely .", "the sections stained with hematoxylin and eosin revealed cystic spaces lined by a papillary epithelial proliferation which was bilayered .", "the cells of the epithelial lining appeared intensely eosinophilic . at the core of papillary projections", "a variable amount of lymphoid tissue with mature lymphocytes was observed [ figure 5 ] .", "tumor after superficial parotidectomy microscopic picture ( 10 ) the patient did not present with any post - surgical complications .", "the most accepted hypothesis about the origin of wt is that it develops from salivary duct inclusions in the lymph nodes , after the embryonic development of the parotid gland .", "this hypothesis is further supported by the frequent detection of salivary gland tissue in the peri- and intraparotidal lymph nodes . in the parotid region , lymph nodes", "the tumors presenting epithelial differentiations similar to those observed in wt develop outside lymph nodes and have no lymphoid stromal component .", "benign tumors have only rarely been associated with cigarette smoking , which focuses attention on the nature of the underlying neoplastic process and how it may differ from other benign tumors .", "although generally believed to be an adenoma , wt , as suggested by allegra , may be a delayed hypersensitivity reaction .", "an interesting fact that caught the attention of the pathologists is that a decline in the incidence in men and a concurrent increased incidence in women has been observed in recent years .", "the change is probably due to decline in the smoking habit in men and a reverse trend in women .", "the increased frequency of adenolymphoma has been ascribed to the association of adenolymphoma with smoking and the proportional increase in female smokers .", "studies conducted among atomic bomb survivors suggest that radiation may also be implicated in the tumorigenesis", "barr virus ( ebv ) , because of the ebv  dna found in tumor cells in some studies has not been substantiated .", "clinically , wt occurs almost exclusively in the parotid glands , in its superficial lobe and rarely in the deeper lobe ( 10% ) .", "the other preferred locations include the buccal mucosa , submaxillary gland , lip and palate .", "the patients can be asymptomatic or can have facial pain , rarely , facial nerve palsy may be seen in tumors associated with inflammation and fibrosis , which can be mistaken for malignant tumor .", "ipsilateral earache , tinnitus and deafness are uncommon ear symptoms that might be seen in some patients .", "the size is variable , from a few millimeters to centimeters , averaging 2 to 4 cm in diameter , with a preferred location in the lower pole of the gland ( in the jaw angle ) .", "it has been reported predominantly in whites , less frequently in orientals , and rarely in blacks .", "the incidence rate is higher than that of salivary gland cancer but is lower than that of benign mixed tumors ( pleomorphic adenoma ) .", "macroscopically wt presents as a spherical or ovoid mass , with a dense fibrous capsule and displaying multiple cystic compartments filled with a viscous yellow or dull brown material . however , eveson and cawson found 77% cases with an incomplete capsule , a full capsule in 8% and 16% tumors in which there was no evidence of capsule .", "the cytological smears in our case showed variable amounts of cellularity , ranging from barely optimum cellularity to occasional hypercellularity .", "there was an admixture of epithelial fragments , occasional single epithelial cells , and abundant lymphocytes noted in a granular cystic background .", "the epithelial cells were oncocytic in appearance with large nuclei , prominent nucleoli , and moderately abundant granular cytoplasm .", "since wt can be multifocal , a preoperative diagnosis by means of fine needle aspiration biopsy is mandatory and complete bilateral screening of the gland by mri is needed to program surgery .", ". an experienced cytopathologist can reliably distinguish malignant salivary pathologies from benign , but a histological classification based on only aspiration is an unrealistic goal .", "computerized tomography and magnetic resonance imaging enable accurate assessment of tumor extension , compression or infiltration of adjacent structures , presence of nodal metastases and better planning of the therapeutic approach .", "dynamic dual - phase scinti - scanning with technetium-99 , a recognized method of identifying adenolymphoma , could be used more frequently in these selected patient groups .", "lesion vascularity on initial power doppler examination is often relatively sparse , but wt that did contain areas of vascularity on initial examination showed a reduction in this vascularity as the tumor size reduced . with regard to luminal cells of the tumor lining the lymphoid stroma the cells reveal a similar aspect to the striated ducts of the normal salivary glands and have numerous mitochondria . these cells , called oxifile or oncocytic cells are swollen epithelial cells , with abundant eosinophilic granular cytoplasm , rich in mitochondria and enzymes .", "an increased number of oncocytic cells are also observed in the normal salivary glands once the person gets older .", "the diffuse proliferation of the oncocytes without other changes has no pathologic significance and is called oncocytosis or oncocytic metaplasia .", "the epithelial cells , the oncocytes , are disposed on two layers , a luminal layer of oncocytic columnar cells , supported by a discontinuous layer of oncocytic basal cells .", "the nuclei of the luminal cells appear uniform and display palisading towards the free surface .", "the basal cells possess round to oval nuclei , centrally located , small , with conspicuous nucleoli .", "the lumen of the cysts contains thick proteinaceous secretions , cellular debris , cholesterol crystals , and sometimes , laminated bodies that resemble corpora amylacea .", "seifert recognizes four subtypes : subtype 1 ( classic wt ) is 50% epithelial ( 77% of all wt ) ; subtype 2 ( stroma - poor ) is 70 - 80% epithelial ( 14% cases ) ; subtype 3 ( stroma - rich ) is only 20 - 30% epithelial ( 2% ) ; and subtype 4 is characterized by extensive squamous metaplasia .", "however , presence of cellular atypia and a pseudoinfiltrative appearance of the metaplastic squamous epithelium in the residual tumor often can be mistaken for squamous cell or mucoepidermoid carcinoma .", "squamous metaplasia of wt usually lacks keratinization , which is seen in most squamous cell carcinoma .", "in contrast to low - grade mucoepidermoid carcinoma , there is no definite infiltrative growth and the tumor cells appear more frankly squamous .", "a differential diagnosis must be made also with a variant of papillary thyroid carcinoma recently reported as  warthin - like  .", "the microscopic characteristic is a prominent lymphoid stroma and oncocytic metaplasia of the epithelium , but the nuclei have chromatin clearing , inclusion and groove - formation and the epithelial cells show immunohistochemical expression of thyroglobulin .", "the differential diagnosis of this malignancy should be performed preferably with pleomorphic adenoma and cystoadenoma .", "the anatomico - pathological diagnosis is generally easy , but it also should be distinguished from canalicular adenoma , sialadenoma as well as from branchial cyst when involving the parotid gland .", "sunardhi - widyaputra and van darmne in 1993 immunohistochemically studied the presence of tenacin , a molecule in the mesenchyme of salivary glands believed to play a role in the embryogenesis and development of tumors , in papillary cystadenoma lymphomatosum and in oncocytoma .", "they found the protein to be abundant in papillary cystadenoma lymphomatosum , prominent in the proximity of the basement membrane , beneath the oncocytic epithelial components .", "tenacin staining in oncocytoma was focal although oncocytes are the actively proliferating cells in this tumor .", "the presence of oncocytic myoepithelial cells both in papillary cystadenoma lymphomatosum and in oncocytoma surrounded by tenacin suggested that both tumors may arise from stem cells that are capable of differentiating into aberrant epithelial cells ( oncocytes ) , myoepithelial cells in variable proportion or both .", "recent molecular studies have shown that the epithelial component is polyclonal and does not exhibit clonal allelic losses , suggesting that this tumor is not a true neoplasm .", "recent studies have also reported the presence of b - cells ( cd20 ) , nk ( cd56 ) and t ( cd3 ) , including helper subtypes ( cd4 ) and suppressor ( cd8 ) in the tumor 's stroma , something similar to that of normal or reactive lymph nodes . also , it was found that cd20-positive b - lymphocytes were located in the germ centers and peripheral b - area while cd3-positive t - lymphocytes are located interfollicularly .", "surgeons are traditionalists , and the early experience of our peers has colored current surgical opinion and slowed the introduction of conservative surgery for the benign parotid lump .", "this situation is now changing , and centers with experience of treating parotid tumors increasingly recognize that benign tumors can be removed safely by techniques much less invasive than a formal parotidectomy .", "this surgical modality is based on meticulous dissection immediately outside the tumor capsule with preservation of the facial nerves .", "in view of the possible association of wt with extra - salivary neoplasms , extensive workup of the patients harboring multiple wt is , therefore , indicated and long - term follow - up is mandatory , due to the possible occurrence of metachronous salivary and extra - salivary tumors even after prolonged time intervals .", "rarely , either the epithelial or lymphoid component of wt can undergo malignant transformation with an estimated incidence of less than 0.1% . in order of frequency ,", "the commonest carcinomas are squamous cell carcinoma , oncocytic carcinoma , adenocarcinoma , undifferentiated carcinoma , mucoepidermoid carcinoma and merkel cell carcinoma .", "complications must be unusual and of low frequency for the surgical resection of a wt , including some complications considered of minor importance , such as paresis of the ear lobe resulting from manipulation and/or section of the auricularis magnus branch of the superficial cervical plexus .", "the auricularis magnus nerve , in its path toward the ear lobe , may pass through the tumor , hampering the dissection .", "another complication of lesser importance is the change of facial contour due to resection of a large portion of the parotid gland ."], "abstract_text": ["<S> warthin 's tumor undoubtedly is the most frequent monomorphic adenoma of the major salivary glands . </S>", "<S> clinically , it appears as a slow - growing tumor often fluctuant on palpation due to its cystic nature . </S>", "<S> the treatment of choice is complete excision with wide tumor - free margins . </S>", "<S> this article highlights a case of warthin 's tumor of the parotid gland in an elderly male patient along with a review of the literature on the aforementioned pathology . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION"], "sections": [["benign neoplasms of the salivary glands are frequently encountered in dental practice . these account for 3% of the tumors involving the head and neck .", "the majority of them occur in the parotid gland , and 80% of them are benign . of these benign neoplasms ,", "50 - 80% are pleomorphic adenomas and 5 - 20% are warthin 's tumors ( wt ) .", "however , warthin 's tumor is the most frequent monomorphic adenoma of the major salivary glands .", "this is a curious benign neoplasm with its intimidating histological name , papillary cyst adenoma lymphomatosum .", "however , the eponym wt has been extensively used ever since aldred warthin reported two cases of this tumor in 1929 .", "earlier in the literature this was also referred to as adeno - lymphoma , papillary cyst adenoma , cystadeno - lymphoma , and epitheliolymphoid cyst .", "wt is generally a disease of elderly men , with the highest incidence in the sixth and seventh decades and the male : female ratio is 4.6:1 .", "the typical features on cytology of wt include oncocytic cells in cohesive , monolayered sheets ; background lymphocytes ; and amorphous , cystic debris .", "histopathologically , it has a cystic appearance with a double layer of oncocytes surrounding a lymphoid stroma .", "the following case presentation deals with wt of the left parotid gland and highlights its clinicopathologic concepts along with its therapeutic management ."], ["a 65-year - old male patient visited the department of oral medicine , with the chief complaint of swelling below the left ear lobe since six years .", "patient was a known smoker since the past 25 years and there was no history of alcohol consumption . on examination , the lesion extended from the left ear lobule to the lower border of the ramus of the mandible superoinferiorly and also extended behind the left ear [ figure 1 ] .", "it was approximately 5 cm in greatest dimensions ; smooth contoured , was firm in consistency and had well - defined borders .", "stimulation of the parotid glands yielded normal salivary flow with normal consistency , quantity and color .", "based on the history and clinical examination , a provisional diagnosis of warthin 's tumor was given .", "a differential diagnosis of pleomorphic adenoma , a low - grade parotid malignancy , lipoma and neurofibroma arising in the salivary gland were included .", "the investigatory workup included complete hemogram , extra - oral radiograph , ultrasonography , computed tomography and excisional biopsy of the lesion .", "ultrasonographic finding showed a well - defined hypoechoic mass in the lower pole of the left parotid gland .", "the rest of the parotid gland parenchyma was normal and there was no evidence of ductal dilatation . computed tomography examination revealed a rounded and well - defined cystic lesion involving the superficial lobe of the left parotid gland [ figure 3 ] .", "ultrasonograph showing well - defined hypoechoic mass computed tomography examination showing the lesion later , excisional biopsy of the lesion was planned using partial parotidectomy as the technique of choice [ figure 4 ] .", "the tissue obtained was fixed in 10% of neutral buffered formalin , and processed routinely .", "the sections stained with hematoxylin and eosin revealed cystic spaces lined by a papillary epithelial proliferation which was bilayered .", "the cells of the epithelial lining appeared intensely eosinophilic . at the core of papillary projections", "a variable amount of lymphoid tissue with mature lymphocytes was observed [ figure 5 ] .", "tumor after superficial parotidectomy microscopic picture ( 10 ) the patient did not present with any post - surgical complications ."], ["the most accepted hypothesis about the origin of wt is that it develops from salivary duct inclusions in the lymph nodes , after the embryonic development of the parotid gland .", "this hypothesis is further supported by the frequent detection of salivary gland tissue in the peri- and intraparotidal lymph nodes . in the parotid region , lymph nodes", "the tumors presenting epithelial differentiations similar to those observed in wt develop outside lymph nodes and have no lymphoid stromal component .", "benign tumors have only rarely been associated with cigarette smoking , which focuses attention on the nature of the underlying neoplastic process and how it may differ from other benign tumors .", "although generally believed to be an adenoma , wt , as suggested by allegra , may be a delayed hypersensitivity reaction .", "an interesting fact that caught the attention of the pathologists is that a decline in the incidence in men and a concurrent increased incidence in women has been observed in recent years .", "the change is probably due to decline in the smoking habit in men and a reverse trend in women .", "the increased frequency of adenolymphoma has been ascribed to the association of adenolymphoma with smoking and the proportional increase in female smokers .", "studies conducted among atomic bomb survivors suggest that radiation may also be implicated in the tumorigenesis", "barr virus ( ebv ) , because of the ebv  dna found in tumor cells in some studies has not been substantiated .", "clinically , wt occurs almost exclusively in the parotid glands , in its superficial lobe and rarely in the deeper lobe ( 10% ) .", "the other preferred locations include the buccal mucosa , submaxillary gland , lip and palate .", "the patients can be asymptomatic or can have facial pain , rarely , facial nerve palsy may be seen in tumors associated with inflammation and fibrosis , which can be mistaken for malignant tumor .", "ipsilateral earache , tinnitus and deafness are uncommon ear symptoms that might be seen in some patients .", "the size is variable , from a few millimeters to centimeters , averaging 2 to 4 cm in diameter , with a preferred location in the lower pole of the gland ( in the jaw angle ) .", "it has been reported predominantly in whites , less frequently in orientals , and rarely in blacks .", "the incidence rate is higher than that of salivary gland cancer but is lower than that of benign mixed tumors ( pleomorphic adenoma ) .", "macroscopically wt presents as a spherical or ovoid mass , with a dense fibrous capsule and displaying multiple cystic compartments filled with a viscous yellow or dull brown material . however , eveson and cawson found 77% cases with an incomplete capsule , a full capsule in 8% and 16% tumors in which there was no evidence of capsule .", "the cytological smears in our case showed variable amounts of cellularity , ranging from barely optimum cellularity to occasional hypercellularity .", "there was an admixture of epithelial fragments , occasional single epithelial cells , and abundant lymphocytes noted in a granular cystic background .", "the epithelial cells were oncocytic in appearance with large nuclei , prominent nucleoli , and moderately abundant granular cytoplasm .", "since wt can be multifocal , a preoperative diagnosis by means of fine needle aspiration biopsy is mandatory and complete bilateral screening of the gland by mri is needed to program surgery .", ". an experienced cytopathologist can reliably distinguish malignant salivary pathologies from benign , but a histological classification based on only aspiration is an unrealistic goal .", "computerized tomography and magnetic resonance imaging enable accurate assessment of tumor extension , compression or infiltration of adjacent structures , presence of nodal metastases and better planning of the therapeutic approach .", "dynamic dual - phase scinti - scanning with technetium-99 , a recognized method of identifying adenolymphoma , could be used more frequently in these selected patient groups .", "lesion vascularity on initial power doppler examination is often relatively sparse , but wt that did contain areas of vascularity on initial examination showed a reduction in this vascularity as the tumor size reduced . with regard to luminal cells of the tumor lining the lymphoid stroma the cells reveal a similar aspect to the striated ducts of the normal salivary glands and have numerous mitochondria . these cells , called oxifile or oncocytic cells are swollen epithelial cells , with abundant eosinophilic granular cytoplasm , rich in mitochondria and enzymes .", "an increased number of oncocytic cells are also observed in the normal salivary glands once the person gets older .", "the diffuse proliferation of the oncocytes without other changes has no pathologic significance and is called oncocytosis or oncocytic metaplasia .", "the epithelial cells , the oncocytes , are disposed on two layers , a luminal layer of oncocytic columnar cells , supported by a discontinuous layer of oncocytic basal cells .", "the nuclei of the luminal cells appear uniform and display palisading towards the free surface .", "the basal cells possess round to oval nuclei , centrally located , small , with conspicuous nucleoli .", "the lumen of the cysts contains thick proteinaceous secretions , cellular debris , cholesterol crystals , and sometimes , laminated bodies that resemble corpora amylacea .", "seifert recognizes four subtypes : subtype 1 ( classic wt ) is 50% epithelial ( 77% of all wt ) ; subtype 2 ( stroma - poor ) is 70 - 80% epithelial ( 14% cases ) ; subtype 3 ( stroma - rich ) is only 20 - 30% epithelial ( 2% ) ; and subtype 4 is characterized by extensive squamous metaplasia .", "however , presence of cellular atypia and a pseudoinfiltrative appearance of the metaplastic squamous epithelium in the residual tumor often can be mistaken for squamous cell or mucoepidermoid carcinoma .", "squamous metaplasia of wt usually lacks keratinization , which is seen in most squamous cell carcinoma .", "in contrast to low - grade mucoepidermoid carcinoma , there is no definite infiltrative growth and the tumor cells appear more frankly squamous .", "a differential diagnosis must be made also with a variant of papillary thyroid carcinoma recently reported as  warthin - like  .", "the microscopic characteristic is a prominent lymphoid stroma and oncocytic metaplasia of the epithelium , but the nuclei have chromatin clearing , inclusion and groove - formation and the epithelial cells show immunohistochemical expression of thyroglobulin .", "the differential diagnosis of this malignancy should be performed preferably with pleomorphic adenoma and cystoadenoma .", "the anatomico - pathological diagnosis is generally easy , but it also should be distinguished from canalicular adenoma , sialadenoma as well as from branchial cyst when involving the parotid gland .", "sunardhi - widyaputra and van darmne in 1993 immunohistochemically studied the presence of tenacin , a molecule in the mesenchyme of salivary glands believed to play a role in the embryogenesis and development of tumors , in papillary cystadenoma lymphomatosum and in oncocytoma .", "they found the protein to be abundant in papillary cystadenoma lymphomatosum , prominent in the proximity of the basement membrane , beneath the oncocytic epithelial components .", "tenacin staining in oncocytoma was focal although oncocytes are the actively proliferating cells in this tumor .", "the presence of oncocytic myoepithelial cells both in papillary cystadenoma lymphomatosum and in oncocytoma surrounded by tenacin suggested that both tumors may arise from stem cells that are capable of differentiating into aberrant epithelial cells ( oncocytes ) , myoepithelial cells in variable proportion or both .", "recent molecular studies have shown that the epithelial component is polyclonal and does not exhibit clonal allelic losses , suggesting that this tumor is not a true neoplasm .", "recent studies have also reported the presence of b - cells ( cd20 ) , nk ( cd56 ) and t ( cd3 ) , including helper subtypes ( cd4 ) and suppressor ( cd8 ) in the tumor 's stroma , something similar to that of normal or reactive lymph nodes . also , it was found that cd20-positive b - lymphocytes were located in the germ centers and peripheral b - area while cd3-positive t - lymphocytes are located interfollicularly .", "surgeons are traditionalists , and the early experience of our peers has colored current surgical opinion and slowed the introduction of conservative surgery for the benign parotid lump .", "this situation is now changing , and centers with experience of treating parotid tumors increasingly recognize that benign tumors can be removed safely by techniques much less invasive than a formal parotidectomy .", "this surgical modality is based on meticulous dissection immediately outside the tumor capsule with preservation of the facial nerves .", "in view of the possible association of wt with extra - salivary neoplasms , extensive workup of the patients harboring multiple wt is , therefore , indicated and long - term follow - up is mandatory , due to the possible occurrence of metachronous salivary and extra - salivary tumors even after prolonged time intervals .", "rarely , either the epithelial or lymphoid component of wt can undergo malignant transformation with an estimated incidence of less than 0.1% . in order of frequency ,", "the commonest carcinomas are squamous cell carcinoma , oncocytic carcinoma , adenocarcinoma , undifferentiated carcinoma , mucoepidermoid carcinoma and merkel cell carcinoma .", "complications must be unusual and of low frequency for the surgical resection of a wt , including some complications considered of minor importance , such as paresis of the ear lobe resulting from manipulation and/or section of the auricularis magnus branch of the superficial cervical plexus .", "the auricularis magnus nerve , in its path toward the ear lobe , may pass through the tumor , hampering the dissection .", "another complication of lesser importance is the change of facial contour due to resection of a large portion of the parotid gland ."]]}
{"article_id": "PMC3546754", "article_text": ["patients with diabetes are at increased risk of morbidity and mortality from cardiovascular disease ( cvd ) compared to normoglycemic controls.1 several additional factors may be associated with this increased risk , including persistent hyperglycemia , untreated hypertension , and dyslipidemia characterized by raised serum triglycerides ( tg ) and low concentrations of high - density lipoprotein cholesterol ( hdl - c).2 the reduction in cardiovascular events produced by statins in patients with diabetes is broadly consistent with that in normoglycemic , dyslipidemic controls . in the collaborative", "atorvastatin diabetes study ( cards ) , a primary prevention study in type 2 diabetes mellitus ( t2 dm ) , atorvastatin reduced coronary heart disease events by 36% , stroke by 48% , and mortality by 27%.3 in a meta - analysis of secondary prevention studies in t2 dm , statins reduced cardiovascular events by 28% and strokes by 32%.4 despite this efficacy , a considerable residual cardiovascular risk remains among patients receiving statin therapy.2,5 hypertriglyceridemia ( > 1.7 mmol / l ) alters the composition of atherogenic particles and may make an important pathophysiological contribution to the residual cardiovascular risk.6 indeed , the current european atherosclerosis society consensus statement recommends treating hypertriglyceridemia of 1.7 mmol / l.6 statins , as well as lowering ldl - c , reduce blood triglyceride concentrations by up to 40%.7,8 only a few previous studies have evaluated hypertriglyceridemia in statin - treated patients with t2dm.9,10 however , there is little data evaluating these patients in primary care in the uk .", "therefore , this audit assessed the prevalence of residual hyperlipidemia and the potential need for additional lipid - lowering therapy in individuals with t2 dm treated with statins in primary care in the uk .", "this cross - sectional , observational study audited all patients with diabetes from the first 40 primary care practices that agreed to participate from an existing panel established by an independent clinical research organization ( quintiles commercial uk limited , bracknell , uk ) .", "prescriptions issued were assessed during the preceding 4 months and repeat prescriptions were included . after excluding patients with forms of diabetes other than t2 dm , the patients were stratified according to the european society of cardiology ( esc ) ", "feasible  treatment targets in patients with diabetes : total cholesterol ( tc ) of < 4 mmol / l , ldl - c of < 2.0", "mmol / l , hdl - c of > 1.0 mmol / l ( male ) or hdl - c > 1.2 mmol / l ( female ) , and fasting tg of < 1.7 mmol / l.11 due to the relatively small numbers of patients receiving combination therapy or monotherapy with lipid - lowering drugs other than statins , this analysis focused on target attainment among patients taking statin monotherapy .", "descriptive statistics were performed using microsoft excel 2010 ( microsoft , redmond , wa ) .", "due to limitations in the demographic data ( see  discussion  ) , inferential and comparative statistical analyses were not performed .", "the audit captured data on 14,652 patients with diagnosed diabetes from 40 practices . on average ,", "89.5% ( n = 13,108 ) had t2 dm ( median age : 66 years old , range : 17106 ; 44.7% female ) .", "approximately one - fifth ( 22.2% ; 2916 ) of the total t2 dm cohort had not received a prescription for lipid - lowering therapy during the 4 months before the audit .", "for example , only around one - fifth were within the target values for total cholesterol and ldl - c . of patients with t2 dm who received lipid - lowering therapy , 94.7% ( 9647 )", "approximately half of the patients who received statin monotherapy attained the esc treatment targets for total cholesterol , ldl - c , hdl - c , or tg ( table 3 ) .", "indeed , irrespective of the individual statin used , around half of patients receiving statin monotherapy attained the esc treatment targets ( table 2 ) .", "table 4 summarizes the dose distribution of each statin in t2 dm patients taking monotherapy .", "the audit captured data on 14,652 patients with diagnosed diabetes from 40 practices . on average ,", "89.5% ( n = 13,108 ) had t2 dm ( median age : 66 years old , range : 17106 ; 44.7% female ) .", "approximately one - fifth ( 22.2% ; 2916 ) of the total t2 dm cohort had not received a prescription for lipid - lowering therapy during the 4 months before the audit .", "for example , only around one - fifth were within the target values for total cholesterol and ldl - c .", "of patients with t2 dm who received lipid - lowering therapy , 94.7% ( 9647 ) received statin monotherapy , which was usually simvastatin or atorvastatin ( table 2 ) .", "approximately half of the patients who received statin monotherapy attained the esc treatment targets for total cholesterol , ldl - c , hdl - c , or tg ( table 3 ) .", "indeed , irrespective of the individual statin used , around half of patients receiving statin monotherapy attained the esc treatment targets ( table 2 ) .", "table 4 summarizes the dose distribution of each statin in t2 dm patients taking monotherapy .", "this large , cross - sectional , observational , systematic audit showed that , first , dyslipidemia is common among people with t2 dm in the uk and , secondly , that lipid concentrations generally and tg in particular remain elevated despite statin monotherapy .", "indeed , just over a fifth of t2 dm patients were not receiving lipid - lowering therapy , despite up to approximately 80% of these subjects showing evidence of dyslipidemia ( table 1 ) .", "persistent hypertriglyceridemia was common , occurring in half of those on statin monotherapy ( table 2 ) .", "this audit is , as far as the authors are aware , the first large study performed solely from the perspective of primary care in the uk that assesses triglyceride concentrations in patients with t2 dm receiving statin monotherapy .", "the results confirm the findings of the dyslipidemia international study ( dysis ) , which stratified patients according to the presence or absence of cvd . in patients with diagnosed cvd", ", 37.5% did not reach the ldl - c target , 38.3% were not at the hdl - c target , and 49.1% did not achieve the triglyceride target . among patients without cvd ,", "the proportions were 45.0% , 30.4% , and 44.8% respectively . only around a quarter ( 25.2% and 25.8% with and without cvd , respectively ) achieved all three targets.9 in another study , 20.9% of 182 patients with t2 dm taking statins showed hypertriglyceridemia ( 2.3", "< 2.5 mmol / l ( 13.7% ) , ldl - c < 2.0 mmol / l ( 8.8% ) , and hdl - c  0.9 mmol / l ( 6.0%).10 in the present study , simvastatin 40 mg was the most frequently prescribed statin dose .", "however , 35.2% of simvastatin users took 20 mg or less , 24.7% of atorvastatin users took 10 mg , 28.2% of pravastatin users took 20 mg , and 72.2% of rosuvastatin users took 10 mg or less ( table 4 ) .", "for example , simvastatin was the most widely prescribed statin in the dysis , in which the most common regimen was 2040 mg / day simvastatin equivalent.9 in a swedish study , 76% of simvastatin users took 20 mg or less , 48% of atorvastatin users took 10 mg , 55% of pravastatin users took 20 mg , and 76% of rosuvastatin users took 10 mg or less.12 the cross - sectional nature of the present audit and lack of clinical detail about each patient precluded an examination of any dose  response relationship with statins or ascertaining of whether the low dose was clinically justified ( eg , due to concurrent conditions or adverse events ) .", "nevertheless , the data and previous studies suggest that a considerable proportion of patients receives low doses of statin and , as a result , may be undertreated .", "as such , there may be scope to intensify statin regimens in many t2 dm patients with dyslipidemia who are managed in primary care .", "the major limitation of this study is the absence of any information regarding the possible contribution made by potential confounders ( such as coexisting obesity , dietary issues , secondary hypertriglyceridemia , impaired renal and hepatic function , and suboptimal glycemic control ) to the suboptimal management of triglyceride levels .", "for example , more potent statins ( atorvastatin , rosuvastatin , and pitavastatin ) produce a  robust lowering  of tg , especially at high doses and in patients with hypertriglyceridemia.13 however , in this audit , a numerically smaller proportion of patients receiving more potent statins ( atorvastatin and rosuvastatin ) attained the treatment targets than those taking simvastatin .", "similarly , the nature of the audit meant that we could not ascertain whether the subjects had relatively treatment - resistant dyslipidemia or were in the titration phase , or whether there were any differences between patients receiving statins and the group that did not receive any lipid - lowering medication .", "moreover , we could not correlate hba1c levels or compliance with hypoglycemic medication with use of statins or triglyceride levels .", "however , the present study is based on data from a large number of patients and , therefore , probably accurately represents the proportions of people with t2 dm who attain lipid targets currently in uk primary care . as such , the audit underscores the need to ascertain the reasons why patients failed to attain the treatment targets and to consider intensified intervention .", "the esc guidelines specify treatment targets based on fasting triglyceride levels . however , the audit was unable to ascertain whether triglyceride levels were measured in the fed or fasting state .", "however , postprandial lipids , partially hydrolyzed chylomicron , and very - ldl remnants apparently accelerate atherogenesis by exacerbating endothelial dysfunction , augmenting numbers of atherogenic small ldl particles , and promoting thrombosis and inflammation .", "arguably , therefore , postprandial tg more accurately predict vascular risk than fasting levels.14 as a result , both fed and fasting hypertriglyceridemia are associated with increased cvd risk .", "the high prevalence of hypertriglyceridemia identified in this audit suggests that tg are often undertreated .", "numerous secondary causes  including hypothyroidism , renal impairment , hepatic inflammation , and poor glycemic control  may increase tg and should be diagnosed and managed.15 however , in this study , the number of patients who had secondary hypertriglyceridemia and the extent to which concurrent conditions influenced the likelihood of attaining the treatment targets could not be assessed . despite these caveats , the results of the present study have implications for management .", "for example , clinicians should continue to emphasize that all patients should follow dietary advice and exercise regularly to improve glycemic control and lipid profiles , as well as implementing interventions to optimize compliance with lifestyle changes , and lipid - lowering and other medications .", "the audit showed that relatively few patients with t2 dm received statins in combination with ezetimibe , fibrate , omega 3 polyunsaturated fatty acids , or nicotinic acid .", "as many patients show dyslipidemic profiles that are associated with an increased risk of cvd despite statin treatment , clinicians need to consider intensifying statin regimens , offering additional lipid - modifying therapies generally , or regimens specifically aimed at lowering tg levels ( such as pharmaceutical grade , highly purified omega 3-acid ethyl esters ) , particularly to improve the management of diabetic dyslipidemia .", "t2 dm patients managed in uk primary care commonly show persistent lipid abnormalities . in this study , over a fifth of t2 dm patients were found to be not receiving lipid - lowering therapy .", "clinicians need to implement interventions to optimize compliance with lipid - lowering and other medications .", "clinicians also need to consider intensifying statin regimens , prescribing additional lipid - modifying therapies , and specific treatments aimed at triglyceride lowering to improve dyslipidemia control in statin - treated patients with t2 dm ."], "abstract_text": ["<S> purposethis paper reports the results of an audit that assessed the prevalence of residual hypertriglyceridemia and the potential need for intensified management among patients with statin - treated type 2 diabetes mellitus ( t2 dm ) in primary care in the uk.patients and methodsa cross - sectional , observational , systematic audit of patients with diagnosed diabetes from 40 primary care practices was undertaken . </S>", "<S> the audit collected basic demographic information and data on prescriptions issued during the preceding 4 months . </S>", "<S> t2 dm patients were stratified according to the proportion that attained european society of cardiology treatment targets.resultsthe audit collected data from 14,652 patients with diagnosed diabetes : 89.5% ( n = 13,108 ) of the total cohort had t2 dm . </S>", "<S> of the people with t2 dm , 22.2% ( 2916 ) were not currently receiving lipid - lowering therapy . </S>", "<S> up to approximately 80% of these people showed evidence of dyslipidemia . among the group that received lipid - lowering therapy , 94.7% ( 9647 ) were on statin monotherapy , which was usually simvastatin ( 69.5% of patients receiving statin monotherapy ; 6707 ) . </S>", "<S> the currently available statins were prescribed , with the most common dose being 40 mg simvastatin ( 44.2% ; 4267 ) . </S>", "<S> irrespective of the statin used , around half of the patients receiving statin monotherapy did not attain the european society of cardiology treatment targets for triglycerides , low - density lipoprotein , high - density lipoprotein , and total cholesterol.conclusiont2dm patients managed in uk primary care commonly show persistent lipid abnormalities . </S>", "<S> clinicians need to optimize compliance with lipid - lowering and other medications . </S>", "<S> clinicians also need to consider intensifying statin regimens , prescribing additional lipid- modifying therapies , and specific treatments aimed at triglyceride lowering to improve dyslipidemia control in statin - treated patients with t2 dm . </S>"], "labels": null, "section_names": ["Introduction", "Patients and methods", "Results", "Study population", "Patients with T2DM who were not currently receiving lipid-lowering therapy", "Patients with T2DM who received lipid-lowering therapy", "Discussion", "Conclusion"], "sections": [["patients with diabetes are at increased risk of morbidity and mortality from cardiovascular disease ( cvd ) compared to normoglycemic controls.1 several additional factors may be associated with this increased risk , including persistent hyperglycemia , untreated hypertension , and dyslipidemia characterized by raised serum triglycerides ( tg ) and low concentrations of high - density lipoprotein cholesterol ( hdl - c).2 the reduction in cardiovascular events produced by statins in patients with diabetes is broadly consistent with that in normoglycemic , dyslipidemic controls . in the collaborative", "atorvastatin diabetes study ( cards ) , a primary prevention study in type 2 diabetes mellitus ( t2 dm ) , atorvastatin reduced coronary heart disease events by 36% , stroke by 48% , and mortality by 27%.3 in a meta - analysis of secondary prevention studies in t2 dm , statins reduced cardiovascular events by 28% and strokes by 32%.4 despite this efficacy , a considerable residual cardiovascular risk remains among patients receiving statin therapy.2,5 hypertriglyceridemia ( > 1.7 mmol / l ) alters the composition of atherogenic particles and may make an important pathophysiological contribution to the residual cardiovascular risk.6 indeed , the current european atherosclerosis society consensus statement recommends treating hypertriglyceridemia of 1.7 mmol / l.6 statins , as well as lowering ldl - c , reduce blood triglyceride concentrations by up to 40%.7,8 only a few previous studies have evaluated hypertriglyceridemia in statin - treated patients with t2dm.9,10 however , there is little data evaluating these patients in primary care in the uk .", "therefore , this audit assessed the prevalence of residual hyperlipidemia and the potential need for additional lipid - lowering therapy in individuals with t2 dm treated with statins in primary care in the uk ."], ["this cross - sectional , observational study audited all patients with diabetes from the first 40 primary care practices that agreed to participate from an existing panel established by an independent clinical research organization ( quintiles commercial uk limited , bracknell , uk ) .", "prescriptions issued were assessed during the preceding 4 months and repeat prescriptions were included . after excluding patients with forms of diabetes other than t2 dm , the patients were stratified according to the european society of cardiology ( esc ) ", "feasible  treatment targets in patients with diabetes : total cholesterol ( tc ) of < 4 mmol / l , ldl - c of < 2.0", "mmol / l , hdl - c of > 1.0 mmol / l ( male ) or hdl - c > 1.2 mmol / l ( female ) , and fasting tg of < 1.7 mmol / l.11 due to the relatively small numbers of patients receiving combination therapy or monotherapy with lipid - lowering drugs other than statins , this analysis focused on target attainment among patients taking statin monotherapy .", "descriptive statistics were performed using microsoft excel 2010 ( microsoft , redmond , wa ) .", "due to limitations in the demographic data ( see  discussion  ) , inferential and comparative statistical analyses were not performed ."], ["the audit captured data on 14,652 patients with diagnosed diabetes from 40 practices . on average ,", "89.5% ( n = 13,108 ) had t2 dm ( median age : 66 years old , range : 17106 ; 44.7% female ) .", "approximately one - fifth ( 22.2% ; 2916 ) of the total t2 dm cohort had not received a prescription for lipid - lowering therapy during the 4 months before the audit .", "for example , only around one - fifth were within the target values for total cholesterol and ldl - c . of patients with t2 dm who received lipid - lowering therapy , 94.7% ( 9647 )", "approximately half of the patients who received statin monotherapy attained the esc treatment targets for total cholesterol , ldl - c , hdl - c , or tg ( table 3 ) .", "indeed , irrespective of the individual statin used , around half of patients receiving statin monotherapy attained the esc treatment targets ( table 2 ) .", "table 4 summarizes the dose distribution of each statin in t2 dm patients taking monotherapy ."], ["the audit captured data on 14,652 patients with diagnosed diabetes from 40 practices . on average ,", "89.5% ( n = 13,108 ) had t2 dm ( median age : 66 years old , range : 17106 ; 44.7% female ) ."], ["approximately one - fifth ( 22.2% ; 2916 ) of the total t2 dm cohort had not received a prescription for lipid - lowering therapy during the 4 months before the audit .", "for example , only around one - fifth were within the target values for total cholesterol and ldl - c ."], ["of patients with t2 dm who received lipid - lowering therapy , 94.7% ( 9647 ) received statin monotherapy , which was usually simvastatin or atorvastatin ( table 2 ) .", "approximately half of the patients who received statin monotherapy attained the esc treatment targets for total cholesterol , ldl - c , hdl - c , or tg ( table 3 ) .", "indeed , irrespective of the individual statin used , around half of patients receiving statin monotherapy attained the esc treatment targets ( table 2 ) .", "table 4 summarizes the dose distribution of each statin in t2 dm patients taking monotherapy ."], ["this large , cross - sectional , observational , systematic audit showed that , first , dyslipidemia is common among people with t2 dm in the uk and , secondly , that lipid concentrations generally and tg in particular remain elevated despite statin monotherapy .", "indeed , just over a fifth of t2 dm patients were not receiving lipid - lowering therapy , despite up to approximately 80% of these subjects showing evidence of dyslipidemia ( table 1 ) .", "persistent hypertriglyceridemia was common , occurring in half of those on statin monotherapy ( table 2 ) .", "this audit is , as far as the authors are aware , the first large study performed solely from the perspective of primary care in the uk that assesses triglyceride concentrations in patients with t2 dm receiving statin monotherapy .", "the results confirm the findings of the dyslipidemia international study ( dysis ) , which stratified patients according to the presence or absence of cvd . in patients with diagnosed cvd", ", 37.5% did not reach the ldl - c target , 38.3% were not at the hdl - c target , and 49.1% did not achieve the triglyceride target . among patients without cvd ,", "the proportions were 45.0% , 30.4% , and 44.8% respectively . only around a quarter ( 25.2% and 25.8% with and without cvd , respectively ) achieved all three targets.9 in another study , 20.9% of 182 patients with t2 dm taking statins showed hypertriglyceridemia ( 2.3", "< 2.5 mmol / l ( 13.7% ) , ldl - c < 2.0 mmol / l ( 8.8% ) , and hdl - c  0.9 mmol / l ( 6.0%).10 in the present study , simvastatin 40 mg was the most frequently prescribed statin dose .", "however , 35.2% of simvastatin users took 20 mg or less , 24.7% of atorvastatin users took 10 mg , 28.2% of pravastatin users took 20 mg , and 72.2% of rosuvastatin users took 10 mg or less ( table 4 ) .", "for example , simvastatin was the most widely prescribed statin in the dysis , in which the most common regimen was 2040 mg / day simvastatin equivalent.9 in a swedish study , 76% of simvastatin users took 20 mg or less , 48% of atorvastatin users took 10 mg , 55% of pravastatin users took 20 mg , and 76% of rosuvastatin users took 10 mg or less.12 the cross - sectional nature of the present audit and lack of clinical detail about each patient precluded an examination of any dose  response relationship with statins or ascertaining of whether the low dose was clinically justified ( eg , due to concurrent conditions or adverse events ) .", "nevertheless , the data and previous studies suggest that a considerable proportion of patients receives low doses of statin and , as a result , may be undertreated .", "as such , there may be scope to intensify statin regimens in many t2 dm patients with dyslipidemia who are managed in primary care .", "the major limitation of this study is the absence of any information regarding the possible contribution made by potential confounders ( such as coexisting obesity , dietary issues , secondary hypertriglyceridemia , impaired renal and hepatic function , and suboptimal glycemic control ) to the suboptimal management of triglyceride levels .", "for example , more potent statins ( atorvastatin , rosuvastatin , and pitavastatin ) produce a  robust lowering  of tg , especially at high doses and in patients with hypertriglyceridemia.13 however , in this audit , a numerically smaller proportion of patients receiving more potent statins ( atorvastatin and rosuvastatin ) attained the treatment targets than those taking simvastatin .", "similarly , the nature of the audit meant that we could not ascertain whether the subjects had relatively treatment - resistant dyslipidemia or were in the titration phase , or whether there were any differences between patients receiving statins and the group that did not receive any lipid - lowering medication .", "moreover , we could not correlate hba1c levels or compliance with hypoglycemic medication with use of statins or triglyceride levels .", "however , the present study is based on data from a large number of patients and , therefore , probably accurately represents the proportions of people with t2 dm who attain lipid targets currently in uk primary care . as such , the audit underscores the need to ascertain the reasons why patients failed to attain the treatment targets and to consider intensified intervention .", "the esc guidelines specify treatment targets based on fasting triglyceride levels . however , the audit was unable to ascertain whether triglyceride levels were measured in the fed or fasting state .", "however , postprandial lipids , partially hydrolyzed chylomicron , and very - ldl remnants apparently accelerate atherogenesis by exacerbating endothelial dysfunction , augmenting numbers of atherogenic small ldl particles , and promoting thrombosis and inflammation .", "arguably , therefore , postprandial tg more accurately predict vascular risk than fasting levels.14 as a result , both fed and fasting hypertriglyceridemia are associated with increased cvd risk .", "the high prevalence of hypertriglyceridemia identified in this audit suggests that tg are often undertreated .", "numerous secondary causes  including hypothyroidism , renal impairment , hepatic inflammation , and poor glycemic control  may increase tg and should be diagnosed and managed.15 however , in this study , the number of patients who had secondary hypertriglyceridemia and the extent to which concurrent conditions influenced the likelihood of attaining the treatment targets could not be assessed . despite these caveats , the results of the present study have implications for management .", "for example , clinicians should continue to emphasize that all patients should follow dietary advice and exercise regularly to improve glycemic control and lipid profiles , as well as implementing interventions to optimize compliance with lifestyle changes , and lipid - lowering and other medications .", "the audit showed that relatively few patients with t2 dm received statins in combination with ezetimibe , fibrate , omega 3 polyunsaturated fatty acids , or nicotinic acid .", "as many patients show dyslipidemic profiles that are associated with an increased risk of cvd despite statin treatment , clinicians need to consider intensifying statin regimens , offering additional lipid - modifying therapies generally , or regimens specifically aimed at lowering tg levels ( such as pharmaceutical grade , highly purified omega 3-acid ethyl esters ) , particularly to improve the management of diabetic dyslipidemia ."], ["t2 dm patients managed in uk primary care commonly show persistent lipid abnormalities . in this study , over a fifth of t2 dm patients were found to be not receiving lipid - lowering therapy .", "clinicians need to implement interventions to optimize compliance with lipid - lowering and other medications .", "clinicians also need to consider intensifying statin regimens , prescribing additional lipid - modifying therapies , and specific treatments aimed at triglyceride lowering to improve dyslipidemia control in statin - treated patients with t2 dm ."]]}
{"article_id": "PMC4629304", "article_text": ["approximately 95% occur in the neck and axilla , while the remaining 5% are found in the chest and abdomen .", "lymphangiomas of the abdomen are rare ( accounting for 1 per 100,000 hospital admissions ) , but have been reported in the mesentery , retroperitoneum , gastrointestinal tract and intra - abdominal solid viscera .", "the clinical presentation of lymphangiomas varies from incidental discovery on imaging to presenting with an acute abdomen .", "mesenteric lymphangiomas , in particular , can result in complications such as intestinal obstruction or volvulus leading to infarction .", "this case report documents a lymphangioma of the small bowel mesentery , incidentally discovered on pelvic ultrasound and initially thought to represent a complex cystic adnexal mass .", "a 42-year - old female presented with a history of chronic iron deficiency anemia and menorrhagia for several years .", "ultrasound was technically difficult , but demonstrated multiple fibroids [ figure 1 ] and a simple cyst within the left ovary .", "the right ovary was not visualized , but ultrasound demonstrated a complex cystic right  adnexal mass  measuring 9.7  5.2  6.3 cm that was presumed to originate from the right ovary [ figure 2 ] .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "transabdominal ultrasound ( a ) sagittal and ( b ) transverse scans demonstrate a bulky uterus with a subserosal fibroid ( white arrows ) seen in both sagittal and transverse planes .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery underwent pelvic ultrasound for investigation of menorrhagia and was found to have a complex cystic right adnexal mass .", "transabdominal ultrasound demonstrates a cystic lesion with numerous thin septae ( curved arrows ) and good through transmission ( straight arrows ) .", "this confirmed multiple uterine fibroids , t1 bright foci within the left ovary suggestive of an ovarian endometrioma , as well as a dilated left fallopian tube with t1 high signal consistent with a hematosalpinx .", "was only partially visualized on the pelvic mri , but was deemed to originate from the abdomen rather than the ovary , which was tethered to the uterus [ figure 3 ] .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "axial t2 images ( a and b ) demonstrate tethering of right ovary ( arrows ) to the uterus posteriorly .", "this study demonstrated a cystic mesenteric lesion with high t2 signal and low t1 signal and measuring 17.6  6.8  8.7 cm .", "the lesion insinuated around adjacent vessels within the small bowel mesentery and demonstrated multiple thin internal septations [ figure 4a ] .", "mass effect was notably absent , and the cisterna chyli and thoracic duct were not enlarged . the lesion was considered to represent a mesenteric lymphangioma .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "( a ) coronal fast imaging with steady - state precession ( fisp ) demonstrates a multiloculated cystic lesion with numerous septae ( straight arrows ) , lacking mass effect .", "( b ) sagittal t2 ( 13 months later ) of lesion , shows increase in size of lesion with thin - walled septae ( straight arrows ) and insinuating around the mesenteric vessels ( curved arrow ) .", "a 13-month follow - up mri examination showed that the lymphangioma had increased in size compared to the initial examination , measuring 17.6  8.4  11.4 cm and extending from the uncinate process of the pancreas to the uterine fundus [ figure 4b ] . the increase in size prompted a surgical referral .", "furthermore , the cystic fluid of the lymphangioma demonstrated signal loss on the t1 out - of - phase sequence compared to the t1 in - phase sequence , consistent with microscopic lipid content . the signal intensity on t1 in - phase was 78.4 and on t1 out - of - phase was 56.2 , thus denoting a 28.3% signal intensity loss between phases .", "this finding is compatible with the presence of chylous fluid and is a very suggestive feature of a lymphangioma [ figure 5 ] .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "corresponding axial t1 ( a ) in - phase and ( b ) out - of - phase mr images demonstrate the cystic lymphangioma ( straight arrows ) with signal intensity loss on the out - of - phase image ( b ) compared to the in - phase image , indicative of microscopic lipid content of chylous fluid .", "lymphangiomas are low - flow vascular malformations that develop as a result of failure of communication of lymph sacs with the venous drainage system .", "it is postulated that these lesions arise secondary to inflammatory processes , surgery , or radiotherapy that lead to lymphatic obstruction .", "lymphangiomas are histologically divided into several types : simple capillary , cavernous , and cystic lymphangiomas .", "cysts may contain serous , chylous , bloody fluid or even purulent fluid in the case of secondary infection .", "the presence of emulsified fats within chylous fluid most likely explains the signal loss on the t1 out - of - phase mr sequence compared to the t1 in - phase mr sequence in our study .", "intra - abdominal lymphangiomas mostly occur in the mesentery followed by the omentum , mesocolon , and retroperitoneum .", "involvement of solid viscera has been described in the literature in the liver , spleen , pancreas , adrenals , kidneys , bladder , and the ovaries .", "the clinical symptoms of a mesenteric lymphangioma are non - specific and include abdominal pain , vomiting , and constipation , making a specific diagnosis practically impossible on clinical grounds .", "the majority of lymphangiomas are discovered incidentally on imaging for the investigation of unrelated clinical indications .", "the differential diagnosis includes a wide range of cystic intra - abdominal lesions , ranging from pancreatic pseudocysts to abdominal tuberculosis , hydatid disease , or malignancies such as mucinous carcinomatosis .", "the patient did not have a history of previous pancreatitis to suggest pseudocysts or risk factors for pancreatitis such as gallstones or alcohol use .", "abdominal tuberculosis and malignancy were excluded largely on clinical grounds , as the patient was relatively healthy with no clinical complaints other than menorrhagia .", "hydatid disease was unlikely , given the absence of daughter cysts and calcifications , and the insinuating nature of the lesion lacking mass effect despite its size .", "the absence of aggressive features on imaging , such as invasion or solid component , made malignancy unlikely .", "a combination of imaging modalities may be helpful in confirming the diagnosis , with mri being the preferred examination of choice .", "typical imaging findings are those of a thin - walled , multiloculated cystic lesion lacking solid components or mural nodularity .", "lymphangiomas typically show t2 high signal fluid , while signal loss may be visualized on chemical shift t1-weighted imaging due to microscopic fat content from chylous fluid  this is a very suggestive finding for a lymphangioma occurring in 2030% of cases , although rarely it may be found in other limited differentials such as lymphoceles , lymphoepithelial cysts , and pancreatic pseudocysts .", "they typically show little in the way of mass effect and generally insinuate around adjacent structures .", "the imaging algorithm for the work - up of lymphangiomas would depend on the local resources and availability of imaging modalities such as us , ct , and mri . in our institution , cystic lesions that are either indeterminate or suspicious for lymphangiomas on us or ct are referred to mri for further characterization . on mri ,", "the presence of a thin - walled , multiloculated cystic lesion in the abdomen , without mass effect on adjacent structures , is suggestive of the diagnosis after relevant differential diagnoses have been excluded on clinical , laboratory , and imaging grounds .", "the presence of signal loss on chemical shift imaging is very suggestive of the diagnosis .", "if definitive confirmation is required , imaging - guided diagnostic aspiration of the cyst fluid could be performed .", "secondary infection , bowel obstruction , volvulus , and rupture with hemorrhage are a few recognized complications .", "so , to date , radical excision remains the definitive treatment , in particular , to prevent recurrence .", "in conclusion , intra - abdominal lymphangiomas are rare entities , which although are typically asymptomatic , can result in life - threatening complications . in our case , the lesion was discovered incidentally on imaging for an unrelated clinical indication and demonstrated interval growth at 1 year warranting a surgical referral . as of the present time , the surgical consultation has not yet been conducted and our diagnosis is based on imaging . our findings were those of a thin - walled multiloculated cystic lesion , lacking both mass effect and enhancement , and which insinuated around vessels within the small bowel mesentery .", "the cyst fluid showed signal loss on chemical shift t1-weighted mr imaging due to the microscopic fat content of chylous fluid  a very suggestive finding for a lymphangioma that helps to differentiate it from other etiologies of cystic lesions in the abdomen ."], "abstract_text": ["<S> lymphangiomas are benign lesions of mesenchymal origin . </S>", "<S> although more commonly encountered in the head and neck , intra - abdominal lymphangiomas are a rare entity that typically present as multiloculated intra - abdominal cystic lesions that are often incidentally discovered on imaging . </S>", "<S> this case report discusses such a case . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["approximately 95% occur in the neck and axilla , while the remaining 5% are found in the chest and abdomen .", "lymphangiomas of the abdomen are rare ( accounting for 1 per 100,000 hospital admissions ) , but have been reported in the mesentery , retroperitoneum , gastrointestinal tract and intra - abdominal solid viscera .", "the clinical presentation of lymphangiomas varies from incidental discovery on imaging to presenting with an acute abdomen .", "mesenteric lymphangiomas , in particular , can result in complications such as intestinal obstruction or volvulus leading to infarction .", "this case report documents a lymphangioma of the small bowel mesentery , incidentally discovered on pelvic ultrasound and initially thought to represent a complex cystic adnexal mass ."], ["a 42-year - old female presented with a history of chronic iron deficiency anemia and menorrhagia for several years .", "ultrasound was technically difficult , but demonstrated multiple fibroids [ figure 1 ] and a simple cyst within the left ovary .", "the right ovary was not visualized , but ultrasound demonstrated a complex cystic right  adnexal mass  measuring 9.7  5.2  6.3 cm that was presumed to originate from the right ovary [ figure 2 ] .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "transabdominal ultrasound ( a ) sagittal and ( b ) transverse scans demonstrate a bulky uterus with a subserosal fibroid ( white arrows ) seen in both sagittal and transverse planes .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery underwent pelvic ultrasound for investigation of menorrhagia and was found to have a complex cystic right adnexal mass .", "transabdominal ultrasound demonstrates a cystic lesion with numerous thin septae ( curved arrows ) and good through transmission ( straight arrows ) .", "this confirmed multiple uterine fibroids , t1 bright foci within the left ovary suggestive of an ovarian endometrioma , as well as a dilated left fallopian tube with t1 high signal consistent with a hematosalpinx .", "was only partially visualized on the pelvic mri , but was deemed to originate from the abdomen rather than the ovary , which was tethered to the uterus [ figure 3 ] .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "axial t2 images ( a and b ) demonstrate tethering of right ovary ( arrows ) to the uterus posteriorly .", "this study demonstrated a cystic mesenteric lesion with high t2 signal and low t1 signal and measuring 17.6  6.8  8.7 cm .", "the lesion insinuated around adjacent vessels within the small bowel mesentery and demonstrated multiple thin internal septations [ figure 4a ] .", "mass effect was notably absent , and the cisterna chyli and thoracic duct were not enlarged . the lesion was considered to represent a mesenteric lymphangioma .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "( a ) coronal fast imaging with steady - state precession ( fisp ) demonstrates a multiloculated cystic lesion with numerous septae ( straight arrows ) , lacking mass effect .", "( b ) sagittal t2 ( 13 months later ) of lesion , shows increase in size of lesion with thin - walled septae ( straight arrows ) and insinuating around the mesenteric vessels ( curved arrow ) .", "a 13-month follow - up mri examination showed that the lymphangioma had increased in size compared to the initial examination , measuring 17.6  8.4  11.4 cm and extending from the uncinate process of the pancreas to the uterine fundus [ figure 4b ] . the increase in size prompted a surgical referral .", "furthermore , the cystic fluid of the lymphangioma demonstrated signal loss on the t1 out - of - phase sequence compared to the t1 in - phase sequence , consistent with microscopic lipid content . the signal intensity on t1 in - phase was 78.4 and on t1 out - of - phase was 56.2 , thus denoting a 28.3% signal intensity loss between phases .", "this finding is compatible with the presence of chylous fluid and is a very suggestive feature of a lymphangioma [ figure 5 ] .", "42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery .", "corresponding axial t1 ( a ) in - phase and ( b ) out - of - phase mr images demonstrate the cystic lymphangioma ( straight arrows ) with signal intensity loss on the out - of - phase image ( b ) compared to the in - phase image , indicative of microscopic lipid content of chylous fluid ."], ["lymphangiomas are low - flow vascular malformations that develop as a result of failure of communication of lymph sacs with the venous drainage system .", "it is postulated that these lesions arise secondary to inflammatory processes , surgery , or radiotherapy that lead to lymphatic obstruction .", "lymphangiomas are histologically divided into several types : simple capillary , cavernous , and cystic lymphangiomas .", "cysts may contain serous , chylous , bloody fluid or even purulent fluid in the case of secondary infection .", "the presence of emulsified fats within chylous fluid most likely explains the signal loss on the t1 out - of - phase mr sequence compared to the t1 in - phase mr sequence in our study .", "intra - abdominal lymphangiomas mostly occur in the mesentery followed by the omentum , mesocolon , and retroperitoneum .", "involvement of solid viscera has been described in the literature in the liver , spleen , pancreas , adrenals , kidneys , bladder , and the ovaries .", "the clinical symptoms of a mesenteric lymphangioma are non - specific and include abdominal pain , vomiting , and constipation , making a specific diagnosis practically impossible on clinical grounds .", "the majority of lymphangiomas are discovered incidentally on imaging for the investigation of unrelated clinical indications .", "the differential diagnosis includes a wide range of cystic intra - abdominal lesions , ranging from pancreatic pseudocysts to abdominal tuberculosis , hydatid disease , or malignancies such as mucinous carcinomatosis .", "the patient did not have a history of previous pancreatitis to suggest pseudocysts or risk factors for pancreatitis such as gallstones or alcohol use .", "abdominal tuberculosis and malignancy were excluded largely on clinical grounds , as the patient was relatively healthy with no clinical complaints other than menorrhagia .", "hydatid disease was unlikely , given the absence of daughter cysts and calcifications , and the insinuating nature of the lesion lacking mass effect despite its size .", "the absence of aggressive features on imaging , such as invasion or solid component , made malignancy unlikely .", "a combination of imaging modalities may be helpful in confirming the diagnosis , with mri being the preferred examination of choice .", "typical imaging findings are those of a thin - walled , multiloculated cystic lesion lacking solid components or mural nodularity .", "lymphangiomas typically show t2 high signal fluid , while signal loss may be visualized on chemical shift t1-weighted imaging due to microscopic fat content from chylous fluid  this is a very suggestive finding for a lymphangioma occurring in 2030% of cases , although rarely it may be found in other limited differentials such as lymphoceles , lymphoepithelial cysts , and pancreatic pseudocysts .", "they typically show little in the way of mass effect and generally insinuate around adjacent structures .", "the imaging algorithm for the work - up of lymphangiomas would depend on the local resources and availability of imaging modalities such as us , ct , and mri . in our institution , cystic lesions that are either indeterminate or suspicious for lymphangiomas on us or ct are referred to mri for further characterization . on mri ,", "the presence of a thin - walled , multiloculated cystic lesion in the abdomen , without mass effect on adjacent structures , is suggestive of the diagnosis after relevant differential diagnoses have been excluded on clinical , laboratory , and imaging grounds .", "the presence of signal loss on chemical shift imaging is very suggestive of the diagnosis .", "if definitive confirmation is required , imaging - guided diagnostic aspiration of the cyst fluid could be performed .", "secondary infection , bowel obstruction , volvulus , and rupture with hemorrhage are a few recognized complications .", "so , to date , radical excision remains the definitive treatment , in particular , to prevent recurrence ."], ["in conclusion , intra - abdominal lymphangiomas are rare entities , which although are typically asymptomatic , can result in life - threatening complications . in our case , the lesion was discovered incidentally on imaging for an unrelated clinical indication and demonstrated interval growth at 1 year warranting a surgical referral . as of the present time , the surgical consultation has not yet been conducted and our diagnosis is based on imaging . our findings were those of a thin - walled multiloculated cystic lesion , lacking both mass effect and enhancement , and which insinuated around vessels within the small bowel mesentery .", "the cyst fluid showed signal loss on chemical shift t1-weighted mr imaging due to the microscopic fat content of chylous fluid  a very suggestive finding for a lymphangioma that helps to differentiate it from other etiologies of cystic lesions in the abdomen ."], [""], [""]]}
{"article_id": "PMC3026981", "article_text": ["broomrapes ( orobanche spp . ) are fully parasitic flowering plants that lack chlorophyll ; hence they penetrate roots of susceptible hosts , removing water , minerals and sugars .", "( egyptian broomrape ) attacks dicotyledonous crops cultivated around the mediterranean , causing massive yield losses .", "broomrape attached to the host by means of tubercle , a swollen organ which may be simple or composite . with the exception of the case of transgenic target - site , herbicide - resistant host plants , meant to be a temporary measure until other effective control means are found .", "orobanche generally maintains a close relationship with the host and so it is unreasonable to attack it using herbicides because the latter may adversely affect the nontarget host . despite research on orobanche spp .", "for over three decades , yield losses still abound because there is no sustainable method for controlling the parasite .  \n fusarium compactum ( wollenw . )", "the inundative biocontrol approach , with repeated applications of the biocontrol agent , generates a state of equilibrium with a very low level of weed density as a result of the artificial inoculation of the biocontrol agent but the fungi are not sufficiently virulent for field release , regardless of the amount used .", "a series of experiments were conducted using f.   compactum , a biological control agent that infects orobanche without affecting the roots of tomato .", "pectins are complex polysaccharides and are one of the major components of the plant cell wall of dicotyledonous plants , where they control the ionic status , cell expansion , and separation .", "some cellulase producing fungi includes acremonium sp , aspergillus spp and fusarium sp , , trichoderma spp .", "[ 6 , 7 ] , zymomonas , and mutant penicillium . in this paper ,", "a hydrolytic enzyme plays an important role in the pathogenicity of plants by facilitating fungal penetration through the host cell wall [ 10 , 11 ] .", "experiments using mycoherbicidal organisms plus pectinase ( ec 3.2.1.15 )   or cellulase ( ec 3.2.1.4 )   indicate that enzyme enhances the weed control of pathogenic fungi . here", ", pectinolytic and cellulolytic enzymes have been used to enhance the virulence of f.   compactum on tomato plants infested with broomrape .", "a semiaxenic polyethylene bag system was used that allowed easy visual observation of the fungal infection of the tubercles .", "this study reports that the addition of pectinase and cellulase alone or in mixtures enhanced the virulence of f. compactum on broomrape .", "\n f. compactum was cultured on potato glucose agar ( pda , pronadisa ) in petri dishes incubated at 25c .", "subcultures were grown in 100  ml potato glucose broth ( pdb , pronadisa ) in 250  ml erlenmeyer flasks .  ", "the cultures were left on a rotary shaker ( brunswick scientific ) at 150  rpm for 48  h. f.   compactum mycelia were harvested on miracloth ( calbiochem , la jolla , ca ) , rinsed with distilled water to remove remaining spores and excess medium , and harvested by vacuum filtration .", "the washed hyphae were chopped at 6,000  rpm for 2  min with a homogenizer ( ika t18 basic ultra - turrax usa ) , resuspended in sterile water , and the propagule concentrations of chopped mycelia were estimated after serial dilution and plating .", "surface sterilizing seeds ensure that the fungal infection on the seeds is from deliberate infection .", "thus , about 13  mg seeds in small bags formed of miracloth were wetted and surface sterilized in 80% ethanol for 1  min and in a mixture of 1% sodium hypochlorite in 0.01% aqueous tween 20 for 10  min .  ", "tomato transplant plugs at the two to three leaf stages in speedling insert trays were purchased from hishtil inc , ashkelon , israel .  ", "the pathogenicity of f.   compactum was tested in the semiaxenic polyethylene bag system ; briefly about 13  mg of dry surface - disinfected seeds ( up to 1,500 ) were sprinkled on wet whatman gf / a glass - fiber sheets ( whatman int .  ", "the broomrape seeds were conditioned for a 7  d period on the wet glass - fiber sheets .", "a tomato seedling with three or four expanded leaves and washed roots was fixed inside each polyethylene bag containing conditioned broomrape seeds .", "the plant roots in each bag were moistened by capillary action with forty  ml of modified hoagland 's solution in the base of each bag . modified hoagland 's solution", "fourteen - hour photoperiods were provided by a photosynthetically active light intensity of 65  e / m / s ( li - cor , inc . , photometer , model li-188b ) produced by six 40  w cool white fluorescent tubes suspended 35 cm above the benches . two  ml of 5-g  ml gr-24 ( synthetic germination stimulant ) were added to each bag with a pipette to augment the tomato root exudates .", "the broomrape seeds germinated , attached to tomato roots , and formed small tubercles during the following 2 weeks .         ", "allocation of treatment to orobanche - infested tomato plants were in such a way that the tubercle numbers and sizes were almost the same .", "the virulence of the fungus was determined with and without various concentrations of either pectinase ( ex fungal origin , 1.1  u  mg , sigma ) and/or cellulase ( cellulysin , ex trichoderma viride , 10  u  mg calbiochem - behring corp . , la jolla , ca 92037 ) .", "the effect of cellulase concentration ( 10 to 20  u  ml ) on tubercle death was similarly determined at a constant inoculum level . thereafter , the virulence of the fungus with the two enzymes was determined in combination at varying ratios .  ", "control plants were mock - inoculated with either sterile distilled water containing 0.01% tween 20 or 4 to 20  u  ml of single or combined enzyme preparations but without fungal mycelia .", "tubercles on the tomato plants infested with broomrape were counted and the diameters were measured with a ruler , with the assumption that the tubercles are perfectly spherical . the treatments consisted of f. compactum or f. compactum plus cellulase ( 4 to 20  u  ml ) .", "one  ml of aqueous fungal cellulase samples ( 10  units  mg ) , freshly made for each experiment as a solution containing 10  u  ml , and was checked for pectinase activity .", "pectinase activity was measured in a reaction mixture consisting of 533  l of 1% polygalacturonic acid ( pectin ) , 400  l of 50  mm sodium acetate buffer at ph 5.0 and 67  l of the cellulase .", "the mixture was incubated at 37c for 10  min as outlined by tonukari et al . .", "a 100-l aliquot of the reaction mixture was mixed with 1.5  ml of 1% 4-hydroxybenzhydrazide ( fluka , fluorescence grade ) in 0.5  m  naoh .", "the mixture was heated at 100c for 10  min , and cooled on ice water .", "absorbance was measured at 410  nm against the zero time blank as outlined by lever , using a spectrophotometer with a versamax tunable microplate reader .", "pectinase activity of the cellulase was calculated from a standard curve prepared with a d - galacturonic acid ( sigma ) .", "one unit of enzyme forms 1  mol of galacturonic acid from polygalacturonic acid in 1  min under the conditions of the assay .", "broomrape tubercle deaths were recorded at 24  h intervals after fungal inoculation , for 8 to 11  d.   tubercles were visually scored as healthy ( translucent , dense , and intact ) , infected ( diseased ) , or dead ( black and soft ) .", "the levels of infection caused by f.   compactum plus pectinase in both experiments were always better than those achieved by the fungus without pectinase ( figures 1(a ) and 1(b ) ) .", "the numbers of broomrape tubercles infected continuously increased over time throughout the period of observation .", "this was not only due to inoculum buildup in the tomato root system but also to the enzyme action .", "this study wanted to ascertain whether the added pectinase would have an effect on suboptimal f.   compactum inoculum . therefore", ", subthreshold levels of inoculum ( 10  propagules  ml ) were investigated in the presence of pectinase .", "f.   compactum alone killed 30% of broomrape tubercles at the suboptimal inoculum levels ( figure 1(a ) ) . at the lowest inoculation density ,", "f.   compactum ( 3.84  10  propagules  ml ) combined with 11  u  ml pectinase killed more broomrape tubercles than the f.   compactum alone ( figures 1(a ) and 1(b ) ) .", "the killing of broomrape tubercles indicated that pectinase enhanced the virulence of f.   compactum ( figure 2 ) .", "since both enzymes separately enhanced the severity of tubercle infection , it was ascertained whether the pathogenicity of f.   compactum ( 1.4  10  propagules  ml ) on broomrape tubercles could be synergistic through the joint action of the enzymes ( 10  u  ml of each enzyme ) ( figure 3 ) .", "the broomrape tubercles on the tomato roots were large and healthy in the absence of fungal treatment but , at times , a few naturally brown tubercles could be seen ( figure 3(a ) ) .", "f.   compactum alone infected all the inoculated tubercles but did not kill any significant number ( figure 3(b ) ) .", "infested tomato roots that were inoculated with mycelia plus pectinase ( 20  u  ml ) ( figure 3(c ) ) had over 50% tubercles dead one week after treatment .", "those inoculated with mycelia plus cellulase ( 20  u  ml ) ( figure 3(d ) ) had above 60% mortality .", "a mixture of both enzymes with f.   compactum increased fungal infection of broomrape by f.   compactum ( figure 3(e ) ) .", "the response of the infected tubercles varied from 100% kill to mild infection , depending on tubercle size .  ", "mycelia plus cellulase ( 20  u  ml ) mix provoked about the same level of infection as mycelia in solution with cellulase and pectinase ( 10  u  ml of each enzyme ) ( figure 3(f ) ) .", "it is logical to think collective mixing of pectinase and cellulase to f. compactum will effectively enhance the fungal biocontrol potential : thus , it was tested . at various enzyme compositions ,", "the f. compactum treatment ( 1.05  10  propagules  ml ) that caused only a hypersensitive reaction ( 9% death ) on the tubercles caused about 35 to 85% tubercle death when the tubercles were treated with mixtures that contained mycelia , pectinase , and cellulase in various ratios ( figure 4 ) .", "higher tubercle infection by f. compactum was observed with a high ratio of cellulase to pectinase .  ", "beginning from day 4 after f. compactum inoculation on broomrape infested tomato plants ; statistically significant differences among treatments were observed ( figure 4 ) .", "broomrape infested roots coinoculated with chopped mycelia plus pectinase and cellulase had substantial tubercle infection and subsequent large numbers of dead tubercles ( 3585% ) ( figure 4 ) .", "this system ensures easy examination of seedling attachment to the tomato roots and easy observation of tubercle infection in the course of the experiment .", "the activity catalyzed by an enzyme is a measure of the amount of enzyme present .", "values pooled from three sets of experiment showed that in a 1  mg ( 10  u  mg ) cellulase preparation , 0.055  mg pectinase ( 1.1  u  mg ) is present .", "this means the 10  u cellulase has a 0.06  u of detectable pectinase .", "the basis for this study stems from previous studies   which examines single enzyme - assisted effect on fungi pathogenicity .", "the orobanche were at three main phases of their life cycle ( seed , germination , and parasitic phases ) at fungal inoculation .", "tubercles are usually present at the germination phase and later become parasitic . in this study ,", "in apparently healthy tubercles , it may be possible to find diseased tubercles ( as depicted in figure 2 ) ; such tubercles deaths were induced by accidental infestation . in total , these results demonstrate that exogenous pectinase as well as cellulase can contribute to pathogenicity .", "the mycelia plus enzyme mix killed some broomrape tubercles , thus they could not form new seeds to replenish the orobanche seed bank .", "the rationale of approach is also applicable to soil grown tubercles where the presence of enzyme may be exogenous .", "this is well taken care of in nature because the tubercles are borne on the roots and roots are mostly below the soil surface .", "the temperature in the growth chamber was kept at 25c to induce infection by f. compactum .   as demonstrated in this study ,", "descriptions of actions of cell wall degrading enzymes are given by wanjiru et al . .", "f. compactum can infect the tubercle by degrading the cell wall components and invading the tissues and cells .", "pectinolytic and cellulolytic activities of the enzymes ( in this case of fungal origin ) result in a loss of structural integrity in the tubercle and characteristic damage .", "the enzyme substrate range and mechanism of action could partly explain the interaction between f. compactum and the enzymes on broomrape tubercles .", "the pathogenicity of f. compactum plus the enzyme mix observed could rarely be observed without pectinase and cellulase at the inoculum level used .", "besides , neither pectinase nor cellulase was ordinarily able to injure and kill broomrape at the concentration used ( max . 20  u  ml ) , demonstrating that the broomrape tubercle infection and death observed are due to the addition of a mycoherbicidal organism .", "the role of pectinase and cellulase in the degradation of cell wall material as observed in this study corresponds with the reports of many authors [ 10 , 11 , 18 , 19 ] . in the previous paper ,", "the results reported by sasaki and nagayama   showed that the fungi pathogenicity was not always proportional to the enzyme activity .", "it is known that the level of -glucosidase in an enzyme preparation may affect the result of cellulase assays , for example , for the estimation of activities of extracellular cellulase enzymes produced by trichoderma . as pointed out by kumpoun and motomura", ", the pectinase used in their study is mainly pectinase , but some glycosidases were also present .", "the presence of -glucosidase or other enzymes , such as cellobiose phosphorylase , which are required for cellobiose metabolism and to enhance cellulose hydrolysis but which are not , strictly speaking , cellulases or rhamnosidase and -glucosidase activities in pectinase   could further complicates research findings .", "care must be taken in interpreting the results of pathogenicity tests as the enzyme preparation may contain a battery of enzymes .", "this result may suggest the need to study the presence of other enzymes in an enzyme of interest even if the enzyme of interest is from a commercial source .     ", "contrary to some of the findings associated with enhanced f.   compactum infection on broomrape , f. oxysporum pathogenicity on broomrape was not enhanced by pectinase and/or cellulase .", "thus , although f. oxysporum and f.   compactum belong to the same genus , they may have different mycoherbicidal mechanisms .", "mycelia of f.   compactum did not penetrate nor show apparent damage to the tomato roots   as also observed in this study . in conclusion , pectinolytic and cellulolytic activities are widely exhibited by bacteria and fungi .", "the enzymatic activities can predispose broomrape tubercles to infection by fungi , in this case , f.   compactum .", "the latter fact adds to the commercial value of f.   compactum as a potential mycoherbicide when sufficiently virulent ."], "abstract_text": ["<S> the use of enzyme could facilitate pathogen penetration into plant host . here </S>", "<S> the combination of cellulase and pectinase was ascertained on the pathogenicity of f. compactum ( 1.4  106  propagules ml1 ) on broomrape tubercles . </S>", "<S> f.   compactum alone infected all the inoculated tubercles but did not kill any significant number . </S>", "<S> infested tomato roots that were inoculated with mycelia plus pectinase ( 20  u  ml1 ) had over 50% tubercles dead one week after treatment . those inoculated with mycelia plus cellulase ( 20  u  ml1 ) had above 60% mortality . </S>", "<S> mixtures of mycelial plus the two enzymes ( 10  u  ml1 of each enzyme ) showed synergy . </S>", "<S> the activity catalyzed by an enzyme is a measure of the amount of enzyme present . </S>", "<S> it was shown that , in a 1  mg ( 10  u  mg1 ) cellulase used , 0.055  mg pectinase ( 1.1  u  mg1 ) is present . </S>", "<S> this explains why mycelial plus cellulase mix contends with mycelial plus the two enzymes . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion"], "sections": [["broomrapes ( orobanche spp . ) are fully parasitic flowering plants that lack chlorophyll ; hence they penetrate roots of susceptible hosts , removing water , minerals and sugars .", "( egyptian broomrape ) attacks dicotyledonous crops cultivated around the mediterranean , causing massive yield losses .", "broomrape attached to the host by means of tubercle , a swollen organ which may be simple or composite . with the exception of the case of transgenic target - site , herbicide - resistant host plants , meant to be a temporary measure until other effective control means are found .", "orobanche generally maintains a close relationship with the host and so it is unreasonable to attack it using herbicides because the latter may adversely affect the nontarget host . despite research on orobanche spp .", "for over three decades , yield losses still abound because there is no sustainable method for controlling the parasite .  \n fusarium compactum ( wollenw . )", "the inundative biocontrol approach , with repeated applications of the biocontrol agent , generates a state of equilibrium with a very low level of weed density as a result of the artificial inoculation of the biocontrol agent but the fungi are not sufficiently virulent for field release , regardless of the amount used .", "a series of experiments were conducted using f.   compactum , a biological control agent that infects orobanche without affecting the roots of tomato .", "pectins are complex polysaccharides and are one of the major components of the plant cell wall of dicotyledonous plants , where they control the ionic status , cell expansion , and separation .", "some cellulase producing fungi includes acremonium sp , aspergillus spp and fusarium sp , , trichoderma spp .", "[ 6 , 7 ] , zymomonas , and mutant penicillium . in this paper ,", "a hydrolytic enzyme plays an important role in the pathogenicity of plants by facilitating fungal penetration through the host cell wall [ 10 , 11 ] .", "experiments using mycoherbicidal organisms plus pectinase ( ec 3.2.1.15 )   or cellulase ( ec 3.2.1.4 )   indicate that enzyme enhances the weed control of pathogenic fungi . here", ", pectinolytic and cellulolytic enzymes have been used to enhance the virulence of f.   compactum on tomato plants infested with broomrape .", "a semiaxenic polyethylene bag system was used that allowed easy visual observation of the fungal infection of the tubercles .", "this study reports that the addition of pectinase and cellulase alone or in mixtures enhanced the virulence of f. compactum on broomrape ."], ["\n f. compactum was cultured on potato glucose agar ( pda , pronadisa ) in petri dishes incubated at 25c .", "subcultures were grown in 100  ml potato glucose broth ( pdb , pronadisa ) in 250  ml erlenmeyer flasks .  ", "the cultures were left on a rotary shaker ( brunswick scientific ) at 150  rpm for 48  h. f.   compactum mycelia were harvested on miracloth ( calbiochem , la jolla , ca ) , rinsed with distilled water to remove remaining spores and excess medium , and harvested by vacuum filtration .", "the washed hyphae were chopped at 6,000  rpm for 2  min with a homogenizer ( ika t18 basic ultra - turrax usa ) , resuspended in sterile water , and the propagule concentrations of chopped mycelia were estimated after serial dilution and plating .", "surface sterilizing seeds ensure that the fungal infection on the seeds is from deliberate infection .", "thus , about 13  mg seeds in small bags formed of miracloth were wetted and surface sterilized in 80% ethanol for 1  min and in a mixture of 1% sodium hypochlorite in 0.01% aqueous tween 20 for 10  min .  ", "tomato transplant plugs at the two to three leaf stages in speedling insert trays were purchased from hishtil inc , ashkelon , israel .  ", "the pathogenicity of f.   compactum was tested in the semiaxenic polyethylene bag system ; briefly about 13  mg of dry surface - disinfected seeds ( up to 1,500 ) were sprinkled on wet whatman gf / a glass - fiber sheets ( whatman int .  ", "the broomrape seeds were conditioned for a 7  d period on the wet glass - fiber sheets .", "a tomato seedling with three or four expanded leaves and washed roots was fixed inside each polyethylene bag containing conditioned broomrape seeds .", "the plant roots in each bag were moistened by capillary action with forty  ml of modified hoagland 's solution in the base of each bag . modified hoagland 's solution", "fourteen - hour photoperiods were provided by a photosynthetically active light intensity of 65  e / m / s ( li - cor , inc . , photometer , model li-188b ) produced by six 40  w cool white fluorescent tubes suspended 35 cm above the benches . two  ml of 5-g  ml gr-24 ( synthetic germination stimulant ) were added to each bag with a pipette to augment the tomato root exudates .", "the broomrape seeds germinated , attached to tomato roots , and formed small tubercles during the following 2 weeks .         ", "allocation of treatment to orobanche - infested tomato plants were in such a way that the tubercle numbers and sizes were almost the same .", "the virulence of the fungus was determined with and without various concentrations of either pectinase ( ex fungal origin , 1.1  u  mg , sigma ) and/or cellulase ( cellulysin , ex trichoderma viride , 10  u  mg calbiochem - behring corp . , la jolla , ca 92037 ) .", "the effect of cellulase concentration ( 10 to 20  u  ml ) on tubercle death was similarly determined at a constant inoculum level . thereafter , the virulence of the fungus with the two enzymes was determined in combination at varying ratios .  ", "control plants were mock - inoculated with either sterile distilled water containing 0.01% tween 20 or 4 to 20  u  ml of single or combined enzyme preparations but without fungal mycelia .", "tubercles on the tomato plants infested with broomrape were counted and the diameters were measured with a ruler , with the assumption that the tubercles are perfectly spherical . the treatments consisted of f. compactum or f. compactum plus cellulase ( 4 to 20  u  ml ) .", "one  ml of aqueous fungal cellulase samples ( 10  units  mg ) , freshly made for each experiment as a solution containing 10  u  ml , and was checked for pectinase activity .", "pectinase activity was measured in a reaction mixture consisting of 533  l of 1% polygalacturonic acid ( pectin ) , 400  l of 50  mm sodium acetate buffer at ph 5.0 and 67  l of the cellulase .", "the mixture was incubated at 37c for 10  min as outlined by tonukari et al . .", "a 100-l aliquot of the reaction mixture was mixed with 1.5  ml of 1% 4-hydroxybenzhydrazide ( fluka , fluorescence grade ) in 0.5  m  naoh .", "the mixture was heated at 100c for 10  min , and cooled on ice water .", "absorbance was measured at 410  nm against the zero time blank as outlined by lever , using a spectrophotometer with a versamax tunable microplate reader .", "pectinase activity of the cellulase was calculated from a standard curve prepared with a d - galacturonic acid ( sigma ) .", "one unit of enzyme forms 1  mol of galacturonic acid from polygalacturonic acid in 1  min under the conditions of the assay .", "broomrape tubercle deaths were recorded at 24  h intervals after fungal inoculation , for 8 to 11  d.   tubercles were visually scored as healthy ( translucent , dense , and intact ) , infected ( diseased ) , or dead ( black and soft ) ."], ["the levels of infection caused by f.   compactum plus pectinase in both experiments were always better than those achieved by the fungus without pectinase ( figures 1(a ) and 1(b ) ) .", "the numbers of broomrape tubercles infected continuously increased over time throughout the period of observation .", "this was not only due to inoculum buildup in the tomato root system but also to the enzyme action .", "this study wanted to ascertain whether the added pectinase would have an effect on suboptimal f.   compactum inoculum . therefore", ", subthreshold levels of inoculum ( 10  propagules  ml ) were investigated in the presence of pectinase .", "f.   compactum alone killed 30% of broomrape tubercles at the suboptimal inoculum levels ( figure 1(a ) ) . at the lowest inoculation density ,", "f.   compactum ( 3.84  10  propagules  ml ) combined with 11  u  ml pectinase killed more broomrape tubercles than the f.   compactum alone ( figures 1(a ) and 1(b ) ) .", "the killing of broomrape tubercles indicated that pectinase enhanced the virulence of f.   compactum ( figure 2 ) .", "since both enzymes separately enhanced the severity of tubercle infection , it was ascertained whether the pathogenicity of f.   compactum ( 1.4  10  propagules  ml ) on broomrape tubercles could be synergistic through the joint action of the enzymes ( 10  u  ml of each enzyme ) ( figure 3 ) .", "the broomrape tubercles on the tomato roots were large and healthy in the absence of fungal treatment but , at times , a few naturally brown tubercles could be seen ( figure 3(a ) ) .", "f.   compactum alone infected all the inoculated tubercles but did not kill any significant number ( figure 3(b ) ) .", "infested tomato roots that were inoculated with mycelia plus pectinase ( 20  u  ml ) ( figure 3(c ) ) had over 50% tubercles dead one week after treatment .", "those inoculated with mycelia plus cellulase ( 20  u  ml ) ( figure 3(d ) ) had above 60% mortality .", "a mixture of both enzymes with f.   compactum increased fungal infection of broomrape by f.   compactum ( figure 3(e ) ) .", "the response of the infected tubercles varied from 100% kill to mild infection , depending on tubercle size .  ", "mycelia plus cellulase ( 20  u  ml ) mix provoked about the same level of infection as mycelia in solution with cellulase and pectinase ( 10  u  ml of each enzyme ) ( figure 3(f ) ) .", "it is logical to think collective mixing of pectinase and cellulase to f. compactum will effectively enhance the fungal biocontrol potential : thus , it was tested . at various enzyme compositions ,", "the f. compactum treatment ( 1.05  10  propagules  ml ) that caused only a hypersensitive reaction ( 9% death ) on the tubercles caused about 35 to 85% tubercle death when the tubercles were treated with mixtures that contained mycelia , pectinase , and cellulase in various ratios ( figure 4 ) .", "higher tubercle infection by f. compactum was observed with a high ratio of cellulase to pectinase .  ", "beginning from day 4 after f. compactum inoculation on broomrape infested tomato plants ; statistically significant differences among treatments were observed ( figure 4 ) .", "broomrape infested roots coinoculated with chopped mycelia plus pectinase and cellulase had substantial tubercle infection and subsequent large numbers of dead tubercles ( 3585% ) ( figure 4 ) .", "this system ensures easy examination of seedling attachment to the tomato roots and easy observation of tubercle infection in the course of the experiment .", "the activity catalyzed by an enzyme is a measure of the amount of enzyme present .", "values pooled from three sets of experiment showed that in a 1  mg ( 10  u  mg ) cellulase preparation , 0.055  mg pectinase ( 1.1  u  mg ) is present .", "this means the 10  u cellulase has a 0.06  u of detectable pectinase .", "the basis for this study stems from previous studies   which examines single enzyme - assisted effect on fungi pathogenicity .", "the orobanche were at three main phases of their life cycle ( seed , germination , and parasitic phases ) at fungal inoculation .", "tubercles are usually present at the germination phase and later become parasitic . in this study ,", "in apparently healthy tubercles , it may be possible to find diseased tubercles ( as depicted in figure 2 ) ; such tubercles deaths were induced by accidental infestation . in total , these results demonstrate that exogenous pectinase as well as cellulase can contribute to pathogenicity .", "the mycelia plus enzyme mix killed some broomrape tubercles , thus they could not form new seeds to replenish the orobanche seed bank .", "the rationale of approach is also applicable to soil grown tubercles where the presence of enzyme may be exogenous .", "this is well taken care of in nature because the tubercles are borne on the roots and roots are mostly below the soil surface .", "the temperature in the growth chamber was kept at 25c to induce infection by f. compactum .   as demonstrated in this study ,", "descriptions of actions of cell wall degrading enzymes are given by wanjiru et al . .", "f. compactum can infect the tubercle by degrading the cell wall components and invading the tissues and cells .", "pectinolytic and cellulolytic activities of the enzymes ( in this case of fungal origin ) result in a loss of structural integrity in the tubercle and characteristic damage .", "the enzyme substrate range and mechanism of action could partly explain the interaction between f. compactum and the enzymes on broomrape tubercles .", "the pathogenicity of f. compactum plus the enzyme mix observed could rarely be observed without pectinase and cellulase at the inoculum level used .", "besides , neither pectinase nor cellulase was ordinarily able to injure and kill broomrape at the concentration used ( max . 20  u  ml ) , demonstrating that the broomrape tubercle infection and death observed are due to the addition of a mycoherbicidal organism .", "the role of pectinase and cellulase in the degradation of cell wall material as observed in this study corresponds with the reports of many authors [ 10 , 11 , 18 , 19 ] . in the previous paper ,", "the results reported by sasaki and nagayama   showed that the fungi pathogenicity was not always proportional to the enzyme activity .", "it is known that the level of -glucosidase in an enzyme preparation may affect the result of cellulase assays , for example , for the estimation of activities of extracellular cellulase enzymes produced by trichoderma . as pointed out by kumpoun and motomura", ", the pectinase used in their study is mainly pectinase , but some glycosidases were also present .", "the presence of -glucosidase or other enzymes , such as cellobiose phosphorylase , which are required for cellobiose metabolism and to enhance cellulose hydrolysis but which are not , strictly speaking , cellulases or rhamnosidase and -glucosidase activities in pectinase   could further complicates research findings .", "care must be taken in interpreting the results of pathogenicity tests as the enzyme preparation may contain a battery of enzymes .", "this result may suggest the need to study the presence of other enzymes in an enzyme of interest even if the enzyme of interest is from a commercial source .     ", "contrary to some of the findings associated with enhanced f.   compactum infection on broomrape , f. oxysporum pathogenicity on broomrape was not enhanced by pectinase and/or cellulase .", "thus , although f. oxysporum and f.   compactum belong to the same genus , they may have different mycoherbicidal mechanisms .", "mycelia of f.   compactum did not penetrate nor show apparent damage to the tomato roots   as also observed in this study . in conclusion , pectinolytic and cellulolytic activities are widely exhibited by bacteria and fungi .", "the enzymatic activities can predispose broomrape tubercles to infection by fungi , in this case , f.   compactum .", "the latter fact adds to the commercial value of f.   compactum as a potential mycoherbicide when sufficiently virulent ."]]}
{"article_id": "PMC3617651", "article_text": ["isotopic signatures of environmental plutonium are generally used to assess the origin of the material . while decay counting is restricted to pu , pu , and pu", ", all mass spectrometric methods can in principle detect the isotopes pu ( t1/2  =  24.1  ka ) , pu ( 6.65  ka ) , pu ( 14.4 a ) , pu ( 373  ka ) , and pu ( 80.8  ma ) .", "however , there are several publications available concerning isotopic concentrations of the minor plutonium isotopes pu , pu , and pu in environmental samples .  ", "has assessed the global distribution of pu and pu , while   uses pu to distinguish chernobyl plutonium from global fallout . in   we", "have shown how the isotopic vector of pu ( pu , pu , pu , pu ) can be measured by combining the respective best suited method for each isotope . due to the strong interference of u in mass spectrometers , pu can generally be measured with alpha - spectrometry only . also ,", "while the combined activity of pu and pu can be determined efficiently by decay counting , their similar -energies are not readily separated , thus a mass spectrometer is usually required to determine the isotopic ratio pu / pu . the short half - life of pu renders -decay counting by lsc ( liquid scintillation counting ) the most efficient method .", "for the long lived isotopes pu and pu only mass spectrometric methods are suitable . among the mass spectrometric methods , ams provides the most sensitive measurements , probably since in most cases the limit is imposed by background from u rather than by detection efficiency . the destruction of molecules by stripping and the combination of several spectrometers , permits ams to suppress background better than other methods .", "if only ams is used , pu can not be measured , and a lower efficiency is achieved for pu . however , since no additional sample processing is required , the measurement of pu can be done at very little additional effort if the concentration of pu in the sample is high enough . during the last few years", ", we have carried out several studies on environmental pu [ 57 ] . in most cases , the measurement of the pu / pu ratio was the main interest . however ,", "if we encountered samples with sufficiently high count rates for these isotopes , we additionally performed measurements of the minor pu isotopes pu , pu , and in some cases even pu . dating with plutonium can be done very precisely by using the ratio am / pu ( e.g. ) .", "the parent nuclide pu ( t1/2  =  14.4  yr ) and the daughter am are different chemical elements and thus this ratio yields the date of either the irradiation or that of the last chemical separation .", "while this is a useful method for applications in the scope of nuclear safeguards , chemical fractionation occurring while the sample resides in the environment compromises its use for environmental studies . without the presence of the daughter isotope", "am in the sample , the age since the irradiation can be assessed only if the initial isotopic abundance of pu is known .", "this abundance , however , depends strongly on the production process , and thus is generally ambiguous .", "we propose to use the isotopic ratios of the other plutonium isotopes to estimate the initial pu content . to check the accuracy of this estimate ,", "we have applied the method to measured literature values for a thermonuclear weapons test , and for the chernobyl accident where we adopt the numbers obtained by   given by .", "additionally , we have simulated thermal neutron irradiation of u. to demonstrate the practical applicability , we have measured environmental samples of different origin .", "the short lived nuclide pu ( t1/2  =  5  h ) decays in a reactor before pu is produced , which results in cm being the isotope of mass 244 produced in a reactor . in a nuclear explosion , however , in which all nuclear reactions happen on a time scale of microseconds , pu can be produced directly via neutron captures on plutonium isotopes , and also by captures on uranium and subsequent -decays after the explosion .", "results have been published for the thermonuclear weapons test  ivy mike  ( pu / pu  =  ( 11.8    0.7 )    10 )   and for the low - yield test ", "( pu / pu  =  ( 2.3    0.4 )    10 ) .", "local weapons test fallout in bikini atoll sediments and soils has been measured with ams recently by , yielding pu / pu ratios between 2.8    10 and 5.7    10 . in the outer layer of a deep - sea manganese nodule a pu / pu ratio of approximately 1 ", "  10 has been observed ( taken from fig . 3 of ) , which the authors attribute to a global fallout .", "samples were obtained from three different projects . in   depth profiles of pu and pu ( among others ) were obtained from the region of nassfeld ( salzburg , austria ) . while cs and sr in this region stem from the reactor accident in chernobyl , pu / pu and am / pu activity ratios as well as the pu / pu isotope ratio determined by ams identified global fallout as the source of plutonium .", "the samples used for this work ( t2f and t2 g in ) were collected on the mountain pasture  nafeld - alm  at an altitude of 2530  m asl , at 47.018  n , 13.012  e on august 25th , 1999 .", "the chemical separation of the samples ( separating am from pu ) was carried out in summer 2006 , the ams measurement in august 2006 .", "the discharge history of pu from sellafield is well documented . though releases extended from 19521992 , the peak discharge occurred between 1970 and 1980 ( fig .", "1 ) . a marine sediment core was collected in 1993 at 54.416  n , 3.563  w by the research vessel gauss , federal maritime and hydrographic agency , germany .", "this core was investigated for a number of isotopes in   and recently for pu isotopes and u with ams at vera . generally , the chronology of the 48  cm long core is unclear , since mixing of the sediment is suggested by the oceanographic situation of the sampling site and hence   expects an  average integral ratio  of the discharges in the core ; despite this , clearly discriminated peaks in the pu , pu , and pu are observed in . for the present work ,", "the ams sputter targets prepared for   were re - measured for pu , pu , pu , pu and pu . the chemical sample preparation was performed in november 2009 , while the ams re - measurement was done in december 2011 . for some of the samples , pu / pu and pu / pu data have already been given in , however the data in the present manuscript stems from the new measurement .", "the garigliano nuclear power plant was situated at sessa aurunca near the garigliano river in campania , italy .", "it consisted of a boiling water reactor with a thermal power of 506  mw , and was in operation from 1964 to 1978 , and is presently in the decommissioning phase .", "though mainly operated with 2.3% enriched uo2 , mixed oxide fuel , consisting of uo2 and puo2 , was also used . during operation and decommissioning ,", "a core from the sediment inside the ( now dry ) drain channel ( 41.257  n ;", "13.832  e ) and another one from the entry point of the channel into the river was collected in may 2006 ; pu was separated in autumn 2006 , and the ams measurement was performed in august 2007 . based on the pu depth profiles ,", "in studies on the yield of heavy elements in thermonuclear explosions   a general exponential trend with increasing mass was observed .", "this trend can be understood under the simplifying assumption of an identical cross section for all involved ( n, ) reactions ; the chance to produce a mass n masses above the initial nucleus is then the probability of absorbing exactly n neutrons , which is described by a poison distribution . as long as the neutron absorption probability is small ,", "furthermore , the higher binding energy of even - even nuclei leads to a higher abundance of isotopes with even masses ; a factor of 1.58 was observed for the  ivy mike  device .", "thus the initial ratio between mass 241 and 239 will be the same as that between mass 242 and 240:(1)241pu239pu0=242pu240puand thus we obtain the time t since irradiation(2)t = t1/2ln2ln(241pu239pu)(242pu240pu)with t1/2  =  14.4  yr the half - life of pu . despite the present - day availability of improved nuclear data for a more detailed simulation , calculation of the actual isotope yields", "is complicated by the fact that a constant neutron flux throughout the uranium or plutonium used in the devices can not be assumed .", "the resultant distribution will generally exhibit a higher yield of the heavier masses ( more frequently produced in the high flux spots ) than indicated by the lighter masses .", "simulations are possible for simplified cases , especially for thermal neutrons , which corresponds roughly to the case of reactor production .", "additionally , we assume that the duration of the irradiation is much shorter than the half life of pu .", "both neutron capture and fission are taken into account , the corresponding cross sections were taken from the endf / b - vii.0 database .", "the system of differential equations was solved numerically with mathematica ( wolfram research , champaign , il , usa ) for different assumed burn - up levels .", "the methods to extract the plutonium from the environmental samples varied between the different projects ; the ( dry ) sample mass was 1  g for sellafield , 212  g for garigliano , and 20  g for salzburg samples , respectively . generally , the samples were leached , and plutonium was extracted with ion exchange .", "after co - precipitation with 1 to 3  mg iron , the sample was calcined to produce plutonium oxide embedded in an iron oxide matrix . the ams facility vera is especially equipped for the measurement of heavy ions .", "uranium pilot beams are used for tuning the spectrometer , which is then scaled to the various pu isotopes for the measurement .", "the switching times between the different isotopes is typically 15  s , therefore fast variations of the ion source output are not completely averaged out and contribute to the uncertainty of the measured isotopic ratios .", "the high - energy beam analysers are designed to allow the transport of 5  +  actinide ions at a terminal voltage of 3  mv .", "oxygen is used as a stripper gas which results in a stripping yield of 5% for this charge state . for", "most previously published data a time - of - flight ( tof ) detector with 25% efficiency was used to further suppress potential background from u ; but we have never observed such background , so measurements can also be carried out without tof , resulting in a theoretical detection efficiency of 4    10 . however , under typical sputter source conditions , for 10 pu atoms in the sputter target we obtain a count rate of 30  s ( for puo2 only 1/10 of this count rate is observed ) . since the sputter targets last for many hours , the available accelerator beam time usually limits the detection efficiency .", "we hope that we can improve on this by reducing the amount of iron matrix used , which requires new handling procedures .", "counting statistics were no limitation for the sellafield marine core samples , since their concentration of radionuclides is exceptionally high ; as already reported in , to avoid saturation of the detectors by the very high count rates we had to reduce the source output strongly , by running the cesium reservoir essentially at room temperature instead of the usual 150  c . therefore , these sputter targets appeared almost untouched after the measurement .", "all data was normalized to a pulser running at a fixed frequency to correct for any detector dead time that arose as a result of high counting rates .", "since all pu isotopes are measured in the same ionization chamber , we assume the same ion optical transmission .", "the ams device can in principle introduce large fractionation , since each isotope is essentially measured with a different tuning of the ( relatively complex ) mass spectrometer , and the tunings may differ in quality . since we obtain our plutonium tunings by scaling from the u pilot beam the heavier plutonium masses are more likely to suffer from ion optical losses .", "the resultant uncertainty is difficult to assess , since no plutonium standard with isotopic ratios suitable for ams is available so far , which would allow to monitor and correct machine fractionation for every measurement .", "however , reference materials measured in the past suggest a typical precision better than 10% . to monitor whether the beam tuning degrades as a result of drift in the components , the u / u ratio of our in - house uranium standard vienna - kku is measured using three sputter targets per turn of the sample wheel .", "it should be noted that systematic trends in the ion optical transmission of the different pu isotopes will partly cancel out in the double - ratio used in eq .", "we expect little fractionation during chemical sample preparation , and blank material allows any laboratory contamination to be traced .", "the beam analysers will not distinguish between the in - growing am and the remaining pu .", "however , the time span between the separation of am and the ams measurement is still relevant .", "am ( most likely ) has a different negative ion yield to pu , which introduces an uncertainty into the mass 241 determination .", "we are not aware of any published ionization yield for am , but   has investigated other monoxide ions of actinides : the relative negative - ion formation probability of tho , uo , npo , and puo is approximately 1:3:5:7 . as an example , we assume that am does form negative monoxide ions even two times better than pu , and that the measurement is carried out one year after chemical separation . since 5% of pu will have decayed into am , the observed count rate for mass 241 will appear 5% higher , which corresponds to a 1  yr lower age . as another extreme example , if we assume that am does not form any negative monoxide ions at all ( which is definitively not the case , see ) , the sample will appear one year older - as it actually is .", "since no information is available on the actual ion yield of am , which is moreover expected to vary with the matrix of the sputter target and with ion source conditions , we assume an additional uncertainty of 1  year for the age determination per year between separation and measurement .", "fig . 2 and table 1 shows the results measured in this work , in comparison to the numbers given for the  ivy mike", "43 counts of pu were successfully measured on salzburg soil within 1.5  h. the resultant isotopic ratio for pu / pu is ( 5.7    1.0 )    10 .", "a contribution of reactor material in our samples , in particular from the chernobyl accident , could cause a lower ratio .", "however , a significant contribution from chernobyl was excluded in . additionally , while a pu / pu ratio of 2.7    10 is expected for chernobyl debris in 2011 ,", "our measured ratio of ( 1.43    0.06 )    10 is more than one order of magnitude lower , and lies perfectly on the eye guide for global fallout ( through masses 239 and 242 of salzburg soil ) .", "we think that our value better represents the northern hemisphere global fallout ratio than that taken from . a possible explanation for the higher ratio observed in the deep sea crust ( va132 , 918 n , 14603 w , 4830  m depth ) would be influence from the various nuclear test sites in the south pacific .", "we think this assumption is supported by the pu / pu of 0.25 observed in the nodule sample ( also taken from fig .", "3 in ) , while the accepted northern hemisphere global fallout average is 0.185 ; this value is also observed in our salzburg soil samples .", "( 2 ) for the measurements from literature and the simulations for thermal neutrons is shown in table 2 and fig .", "since all ratios were calculated for the time of production , or corrected for pu decay , an age of 0 is expected in all cases .", "while the mike test appears 6  years older than expected , the simulated thermal neutron results appear too young , and the deviation grows with the assumed burn - up .", "the chernobyl data appears 5  years too young ; a more accurate estimate is obtained if we assume production by thermal neutrons . in this case", "( 2 ) , but fit the simulation to the measured pu / pu ratio , which results in a total thermal neutron fluence of 0.0025 b , and in an estimated age deviating by less than 2  years from the known calendar date of the chernobyl explosion .", "this agreement is surprising , considering that the processes in the actual reactor deviated significantly from a simple thermal neutron irradiation .", "for the salzburg soil , an age of 52.5    1.5  yr is obtained , corresponding to the year 1954 .", "this age is 8  yr before the maximum of the nuclear weapons tests in 1963 , and the deviation is similar to that for the ivy mike test . if this trend holds for all material produced in nuclear explosions , a correction could be applied reducing the deviation .", "the garigliano results suffer from a larger statistical uncertainty , due to lower pu concentration in the samples .", "the age is 42    8  yr , which corresponds to the year 1965 , in agreement with expectations . our new pu / pu data for the sellafield core agrees with previous measurements on the same sputter targets presented in , while the three pu / pu ratios given there have significantly lower precision .", "however , one sample ( st60  k ) shows a deviation of about 25% from the previously published data , which is much more than allowed by counting statistics ( see table 1 ) .", "we attribute this outlier to variations in the transmission of pu through the spectrometer , limiting the reproducibility of the present method .", "since a deviation of 25% , corresponding to a dating error of about 5  years , is relevant for the present work , this emphasizes the need for an ams standard which would allow to trace and correct for such variations .", "our sellafield dataset is well suited to illustrate the advantage of using the pu information for dating .", "( 2 ) compared to an estimate of the initial pu / pu which is based only on pu / pu:(3)241pu239pu0=240pu239pu2 while the age shift due to the neglected even ", "odd offset of this simpler method could probably be handled , the pu information establishes a monotonous age - depth curve for the sediment core , however the data covers only a short time span of seven years .", "the time range 1977 to 1984 falls well into the later part of the sellafield discharges ; however , if the age is calibrated against a simulated thermal neutron irradiation , a fluence of 0.0015 b fits best , and leads on an estimated age correction of 5  years .", "the conventional approach towards source assessment of an unknown material is to measure elemental concentrations and isotopic ratios , and to compare them with a database of all viable sources .", "this works also for environmental plutonium isotopes , and probably would lead to the same source assignments as the method proposed in this work .", "however , we think that the calculation of  predictive  values like burn - up , irradiation date , or production mode ( i.e. reactor vs. explosion ) makes the assessment more straightforward . the measurement of pu , pu , pu , pu and pu allows for more precise assessment of the history and origin of environmental plutonium .", "additionally we have shown that the use of pu and pu allows dating of the time of irradiation accurate to within 6  years , while our data on the irish sea sediment core suggest that relative dating of material from the same source is possible with a precision of less than 2  years .", "the information contained in the other plutonium isotopes may allow further improvement in the precision .", "when a reactor origin can be assumed , possibly based on the absence of pu , and by calibrating the data against simulated thermal neutron data with the same pu / pu , an accuracy of 2  years is suggested by our data ."], "abstract_text": ["<S> vera , the vienna environmental research accelerator , is especially equipped for the measurement of actinides , and performs a growing number of measurements on environmental samples . </S>", "<S> while ams is not the optimum method for each particular plutonium isotope , the possibility to measure 239pu , 240pu , 241pu , 242pu and 244pu on the same ams sputter target is a great simplification . </S>", "<S> we have obtained a first result on the global fallout value of 244pu/239pu  =  ( 5.7    </S>", "<S> 1.0 )    105 based on soil samples from salzburg prefecture , austria . </S>", "<S> furthermore , we suggest using the 242pu/240pu ratio as an estimate of the initial 241pu/239pu ratio , which allows dating of the time of irradiation based solely on pu isotopes . we have checked the validity of this estimate using literature data , simulations , and environmental samples from soil from the salzburg prefecture ( austria ) , from the shut down garigliano nuclear power plant ( sessa aurunca , italy ) and from the irish sea near the sellafield nuclear facility . </S>", "<S> the maximum deviation of the estimated dates from the expected ages is 6  years , while relative dating of material from the same source seems to be possible with a precision of less than 2  years . </S>", "<S> additional information carried by the minor plutonium isotopes may allow further improvements of the precision of the method . </S>"], "labels": null, "section_names": ["Introduction", "Samples", "Methods", "Results and discussion", "Conclusions"], "sections": [["isotopic signatures of environmental plutonium are generally used to assess the origin of the material . while decay counting is restricted to pu , pu , and pu", ", all mass spectrometric methods can in principle detect the isotopes pu ( t1/2  =  24.1  ka ) , pu ( 6.65  ka ) , pu ( 14.4 a ) , pu ( 373  ka ) , and pu ( 80.8  ma ) .", "however , there are several publications available concerning isotopic concentrations of the minor plutonium isotopes pu , pu , and pu in environmental samples .  ", "has assessed the global distribution of pu and pu , while   uses pu to distinguish chernobyl plutonium from global fallout . in   we", "have shown how the isotopic vector of pu ( pu , pu , pu , pu ) can be measured by combining the respective best suited method for each isotope . due to the strong interference of u in mass spectrometers , pu can generally be measured with alpha - spectrometry only . also ,", "while the combined activity of pu and pu can be determined efficiently by decay counting , their similar -energies are not readily separated , thus a mass spectrometer is usually required to determine the isotopic ratio pu / pu . the short half - life of pu renders -decay counting by lsc ( liquid scintillation counting ) the most efficient method .", "for the long lived isotopes pu and pu only mass spectrometric methods are suitable . among the mass spectrometric methods , ams provides the most sensitive measurements , probably since in most cases the limit is imposed by background from u rather than by detection efficiency . the destruction of molecules by stripping and the combination of several spectrometers , permits ams to suppress background better than other methods .", "if only ams is used , pu can not be measured , and a lower efficiency is achieved for pu . however , since no additional sample processing is required , the measurement of pu can be done at very little additional effort if the concentration of pu in the sample is high enough . during the last few years", ", we have carried out several studies on environmental pu [ 57 ] . in most cases , the measurement of the pu / pu ratio was the main interest . however ,", "if we encountered samples with sufficiently high count rates for these isotopes , we additionally performed measurements of the minor pu isotopes pu , pu , and in some cases even pu . dating with plutonium can be done very precisely by using the ratio am / pu ( e.g. ) .", "the parent nuclide pu ( t1/2  =  14.4  yr ) and the daughter am are different chemical elements and thus this ratio yields the date of either the irradiation or that of the last chemical separation .", "while this is a useful method for applications in the scope of nuclear safeguards , chemical fractionation occurring while the sample resides in the environment compromises its use for environmental studies . without the presence of the daughter isotope", "am in the sample , the age since the irradiation can be assessed only if the initial isotopic abundance of pu is known .", "this abundance , however , depends strongly on the production process , and thus is generally ambiguous .", "we propose to use the isotopic ratios of the other plutonium isotopes to estimate the initial pu content . to check the accuracy of this estimate ,", "we have applied the method to measured literature values for a thermonuclear weapons test , and for the chernobyl accident where we adopt the numbers obtained by   given by .", "additionally , we have simulated thermal neutron irradiation of u. to demonstrate the practical applicability , we have measured environmental samples of different origin .", "the short lived nuclide pu ( t1/2  =  5  h ) decays in a reactor before pu is produced , which results in cm being the isotope of mass 244 produced in a reactor . in a nuclear explosion , however , in which all nuclear reactions happen on a time scale of microseconds , pu can be produced directly via neutron captures on plutonium isotopes , and also by captures on uranium and subsequent -decays after the explosion .", "results have been published for the thermonuclear weapons test  ivy mike  ( pu / pu  =  ( 11.8    0.7 )    10 )   and for the low - yield test ", "( pu / pu  =  ( 2.3    0.4 )    10 ) .", "local weapons test fallout in bikini atoll sediments and soils has been measured with ams recently by , yielding pu / pu ratios between 2.8    10 and 5.7    10 . in the outer layer of a deep - sea manganese nodule a pu / pu ratio of approximately 1 ", "  10 has been observed ( taken from fig . 3 of ) , which the authors attribute to a global fallout ."], ["samples were obtained from three different projects . in   depth profiles of pu and pu ( among others ) were obtained from the region of nassfeld ( salzburg , austria ) . while cs and sr in this region stem from the reactor accident in chernobyl , pu / pu and am / pu activity ratios as well as the pu / pu isotope ratio determined by ams identified global fallout as the source of plutonium .", "the samples used for this work ( t2f and t2 g in ) were collected on the mountain pasture  nafeld - alm  at an altitude of 2530  m asl , at 47.018  n , 13.012  e on august 25th , 1999 .", "the chemical separation of the samples ( separating am from pu ) was carried out in summer 2006 , the ams measurement in august 2006 .", "the discharge history of pu from sellafield is well documented . though releases extended from 19521992 , the peak discharge occurred between 1970 and 1980 ( fig .", "1 ) . a marine sediment core was collected in 1993 at 54.416  n , 3.563  w by the research vessel gauss , federal maritime and hydrographic agency , germany .", "this core was investigated for a number of isotopes in   and recently for pu isotopes and u with ams at vera . generally , the chronology of the 48  cm long core is unclear , since mixing of the sediment is suggested by the oceanographic situation of the sampling site and hence   expects an  average integral ratio  of the discharges in the core ; despite this , clearly discriminated peaks in the pu , pu , and pu are observed in . for the present work ,", "the ams sputter targets prepared for   were re - measured for pu , pu , pu , pu and pu . the chemical sample preparation was performed in november 2009 , while the ams re - measurement was done in december 2011 . for some of the samples , pu / pu and pu / pu data have already been given in , however the data in the present manuscript stems from the new measurement .", "the garigliano nuclear power plant was situated at sessa aurunca near the garigliano river in campania , italy .", "it consisted of a boiling water reactor with a thermal power of 506  mw , and was in operation from 1964 to 1978 , and is presently in the decommissioning phase .", "though mainly operated with 2.3% enriched uo2 , mixed oxide fuel , consisting of uo2 and puo2 , was also used . during operation and decommissioning ,", "a core from the sediment inside the ( now dry ) drain channel ( 41.257  n ;", "13.832  e ) and another one from the entry point of the channel into the river was collected in may 2006 ; pu was separated in autumn 2006 , and the ams measurement was performed in august 2007 . based on the pu depth profiles ,"], ["in studies on the yield of heavy elements in thermonuclear explosions   a general exponential trend with increasing mass was observed .", "this trend can be understood under the simplifying assumption of an identical cross section for all involved ( n, ) reactions ; the chance to produce a mass n masses above the initial nucleus is then the probability of absorbing exactly n neutrons , which is described by a poison distribution . as long as the neutron absorption probability is small ,", "furthermore , the higher binding energy of even - even nuclei leads to a higher abundance of isotopes with even masses ; a factor of 1.58 was observed for the  ivy mike  device .", "thus the initial ratio between mass 241 and 239 will be the same as that between mass 242 and 240:(1)241pu239pu0=242pu240puand thus we obtain the time t since irradiation(2)t = t1/2ln2ln(241pu239pu)(242pu240pu)with t1/2  =  14.4  yr the half - life of pu . despite the present - day availability of improved nuclear data for a more detailed simulation , calculation of the actual isotope yields", "is complicated by the fact that a constant neutron flux throughout the uranium or plutonium used in the devices can not be assumed .", "the resultant distribution will generally exhibit a higher yield of the heavier masses ( more frequently produced in the high flux spots ) than indicated by the lighter masses .", "simulations are possible for simplified cases , especially for thermal neutrons , which corresponds roughly to the case of reactor production .", "additionally , we assume that the duration of the irradiation is much shorter than the half life of pu .", "both neutron capture and fission are taken into account , the corresponding cross sections were taken from the endf / b - vii.0 database .", "the system of differential equations was solved numerically with mathematica ( wolfram research , champaign , il , usa ) for different assumed burn - up levels .", "the methods to extract the plutonium from the environmental samples varied between the different projects ; the ( dry ) sample mass was 1  g for sellafield , 212  g for garigliano , and 20  g for salzburg samples , respectively . generally , the samples were leached , and plutonium was extracted with ion exchange .", "after co - precipitation with 1 to 3  mg iron , the sample was calcined to produce plutonium oxide embedded in an iron oxide matrix . the ams facility vera is especially equipped for the measurement of heavy ions .", "uranium pilot beams are used for tuning the spectrometer , which is then scaled to the various pu isotopes for the measurement .", "the switching times between the different isotopes is typically 15  s , therefore fast variations of the ion source output are not completely averaged out and contribute to the uncertainty of the measured isotopic ratios .", "the high - energy beam analysers are designed to allow the transport of 5  +  actinide ions at a terminal voltage of 3  mv .", "oxygen is used as a stripper gas which results in a stripping yield of 5% for this charge state . for", "most previously published data a time - of - flight ( tof ) detector with 25% efficiency was used to further suppress potential background from u ; but we have never observed such background , so measurements can also be carried out without tof , resulting in a theoretical detection efficiency of 4    10 . however , under typical sputter source conditions , for 10 pu atoms in the sputter target we obtain a count rate of 30  s ( for puo2 only 1/10 of this count rate is observed ) . since the sputter targets last for many hours , the available accelerator beam time usually limits the detection efficiency .", "we hope that we can improve on this by reducing the amount of iron matrix used , which requires new handling procedures .", "counting statistics were no limitation for the sellafield marine core samples , since their concentration of radionuclides is exceptionally high ; as already reported in , to avoid saturation of the detectors by the very high count rates we had to reduce the source output strongly , by running the cesium reservoir essentially at room temperature instead of the usual 150  c . therefore , these sputter targets appeared almost untouched after the measurement .", "all data was normalized to a pulser running at a fixed frequency to correct for any detector dead time that arose as a result of high counting rates .", "since all pu isotopes are measured in the same ionization chamber , we assume the same ion optical transmission .", "the ams device can in principle introduce large fractionation , since each isotope is essentially measured with a different tuning of the ( relatively complex ) mass spectrometer , and the tunings may differ in quality . since we obtain our plutonium tunings by scaling from the u pilot beam the heavier plutonium masses are more likely to suffer from ion optical losses .", "the resultant uncertainty is difficult to assess , since no plutonium standard with isotopic ratios suitable for ams is available so far , which would allow to monitor and correct machine fractionation for every measurement .", "however , reference materials measured in the past suggest a typical precision better than 10% . to monitor whether the beam tuning degrades as a result of drift in the components , the u / u ratio of our in - house uranium standard vienna - kku is measured using three sputter targets per turn of the sample wheel .", "it should be noted that systematic trends in the ion optical transmission of the different pu isotopes will partly cancel out in the double - ratio used in eq .", "we expect little fractionation during chemical sample preparation , and blank material allows any laboratory contamination to be traced .", "the beam analysers will not distinguish between the in - growing am and the remaining pu .", "however , the time span between the separation of am and the ams measurement is still relevant .", "am ( most likely ) has a different negative ion yield to pu , which introduces an uncertainty into the mass 241 determination .", "we are not aware of any published ionization yield for am , but   has investigated other monoxide ions of actinides : the relative negative - ion formation probability of tho , uo , npo , and puo is approximately 1:3:5:7 . as an example , we assume that am does form negative monoxide ions even two times better than pu , and that the measurement is carried out one year after chemical separation . since 5% of pu will have decayed into am , the observed count rate for mass 241 will appear 5% higher , which corresponds to a 1  yr lower age . as another extreme example , if we assume that am does not form any negative monoxide ions at all ( which is definitively not the case , see ) , the sample will appear one year older - as it actually is .", "since no information is available on the actual ion yield of am , which is moreover expected to vary with the matrix of the sputter target and with ion source conditions , we assume an additional uncertainty of 1  year for the age determination per year between separation and measurement ."], ["fig . 2 and table 1 shows the results measured in this work , in comparison to the numbers given for the  ivy mike", "43 counts of pu were successfully measured on salzburg soil within 1.5  h. the resultant isotopic ratio for pu / pu is ( 5.7    1.0 )    10 .", "a contribution of reactor material in our samples , in particular from the chernobyl accident , could cause a lower ratio .", "however , a significant contribution from chernobyl was excluded in . additionally , while a pu / pu ratio of 2.7    10 is expected for chernobyl debris in 2011 ,", "our measured ratio of ( 1.43    0.06 )    10 is more than one order of magnitude lower , and lies perfectly on the eye guide for global fallout ( through masses 239 and 242 of salzburg soil ) .", "we think that our value better represents the northern hemisphere global fallout ratio than that taken from . a possible explanation for the higher ratio observed in the deep sea crust ( va132 , 918 n , 14603 w , 4830  m depth ) would be influence from the various nuclear test sites in the south pacific .", "we think this assumption is supported by the pu / pu of 0.25 observed in the nodule sample ( also taken from fig .", "3 in ) , while the accepted northern hemisphere global fallout average is 0.185 ; this value is also observed in our salzburg soil samples .", "( 2 ) for the measurements from literature and the simulations for thermal neutrons is shown in table 2 and fig .", "since all ratios were calculated for the time of production , or corrected for pu decay , an age of 0 is expected in all cases .", "while the mike test appears 6  years older than expected , the simulated thermal neutron results appear too young , and the deviation grows with the assumed burn - up .", "the chernobyl data appears 5  years too young ; a more accurate estimate is obtained if we assume production by thermal neutrons . in this case", "( 2 ) , but fit the simulation to the measured pu / pu ratio , which results in a total thermal neutron fluence of 0.0025 b , and in an estimated age deviating by less than 2  years from the known calendar date of the chernobyl explosion .", "this agreement is surprising , considering that the processes in the actual reactor deviated significantly from a simple thermal neutron irradiation .", "for the salzburg soil , an age of 52.5    1.5  yr is obtained , corresponding to the year 1954 .", "this age is 8  yr before the maximum of the nuclear weapons tests in 1963 , and the deviation is similar to that for the ivy mike test . if this trend holds for all material produced in nuclear explosions , a correction could be applied reducing the deviation .", "the garigliano results suffer from a larger statistical uncertainty , due to lower pu concentration in the samples .", "the age is 42    8  yr , which corresponds to the year 1965 , in agreement with expectations . our new pu / pu data for the sellafield core agrees with previous measurements on the same sputter targets presented in , while the three pu / pu ratios given there have significantly lower precision .", "however , one sample ( st60  k ) shows a deviation of about 25% from the previously published data , which is much more than allowed by counting statistics ( see table 1 ) .", "we attribute this outlier to variations in the transmission of pu through the spectrometer , limiting the reproducibility of the present method .", "since a deviation of 25% , corresponding to a dating error of about 5  years , is relevant for the present work , this emphasizes the need for an ams standard which would allow to trace and correct for such variations .", "our sellafield dataset is well suited to illustrate the advantage of using the pu information for dating .", "( 2 ) compared to an estimate of the initial pu / pu which is based only on pu / pu:(3)241pu239pu0=240pu239pu2 while the age shift due to the neglected even ", "odd offset of this simpler method could probably be handled , the pu information establishes a monotonous age - depth curve for the sediment core , however the data covers only a short time span of seven years .", "the time range 1977 to 1984 falls well into the later part of the sellafield discharges ; however , if the age is calibrated against a simulated thermal neutron irradiation , a fluence of 0.0015 b fits best , and leads on an estimated age correction of 5  years ."], ["the conventional approach towards source assessment of an unknown material is to measure elemental concentrations and isotopic ratios , and to compare them with a database of all viable sources .", "this works also for environmental plutonium isotopes , and probably would lead to the same source assignments as the method proposed in this work .", "however , we think that the calculation of  predictive  values like burn - up , irradiation date , or production mode ( i.e. reactor vs. explosion ) makes the assessment more straightforward . the measurement of pu , pu , pu , pu and pu allows for more precise assessment of the history and origin of environmental plutonium .", "additionally we have shown that the use of pu and pu allows dating of the time of irradiation accurate to within 6  years , while our data on the irish sea sediment core suggest that relative dating of material from the same source is possible with a precision of less than 2  years .", "the information contained in the other plutonium isotopes may allow further improvement in the precision .", "when a reactor origin can be assumed , possibly based on the absence of pu , and by calibrating the data against simulated thermal neutron data with the same pu / pu , an accuracy of 2  years is suggested by our data ."]]}
{"article_id": "PMC3740650", "article_text": ["since the introduction of tooth whitening by haywood and heymann in 1989 , this trend is getting more popular .", "there are a few types of bleaching methods , but all of them share a common principle of the decomposition of peroxides from hydrogen peroxide or its compounds such as carbamide peroxide ( cp ) into unstable free radicals . these radicals further breakdown into large pigmented molecules either through oxidation or reduction reaction .", "the oxidation or reduction process changes the chemical structure of the interacting organic substances of the tooth , which results in the change in color .", "furthermore , at 45% concentration of cp in different intracoronal tooth - bleaching procedures , maleknejad ( 2012 ) reported an increase in the diameter of dentinal tubules , and it also promoted alterations in the mineral content of the dentine .", "types of bleaching methods include nonvital bleaching , in - office professional bleaching , and home bleaching .", "nightguard home bleaching uses a relatively low level of whitening agent and is applied to the teeth via a custom - fabricated mouthguard and is worn at night for a duration of at least two weeks .", "a lot of research has been carried out to identify the effects of bleaching on the tooth surface and dental restorative materials .", "jacob ( 2007 ) reported that bleaching with cp may alter the marginal leakage of resin composite restoration ; however , amalgam restorations were not adversely affected in vitro .", "it was reported that the bleaching agent , regardless of the whitening products used , will reduce the microhardness of the enamel and promote an increase in surface roughness .", "hence , it is widely used for anterior and also posterior restorations . due to this", "some studies concluded that application of home bleaching agent gels may cause surface softening of composite resins , whereas others stated that there are no significant changes in surface microhardness found after the application of 15% cp on composite resin .", "furthermore , a study was done to evaluate the effects of bleaching on toothbrush abrasion in three types of composites .", "it was concluded that nano - filled resin composites are to be used for restoration when bleaching treatment is required .", "there is a significant and positive correlation found between surface roughness and vital bacterial adhesion .", "it appears that bleaching agents may affect the adherence of certain cariogenic micro - organisms to the outer surfaces of composite resin restorations .", "roughness has a major impact on aesthetic appearance , discoloration of restorations , plaque accumulation , secondary caries , and gingival irritation .", "the experimental composite tested in this study was a locally produced dental restoration nanocomposite ( kelfil ) .", "this nanocomposite was produced by the school of dental sciences ( ppsg ) , universiti sains malaysia ( usm ) , kelantan , malaysia .", "the nanosilica fillers , synthesized locally by a sol - gel process , were in the size range of 1020 nm .", "the experimental nanocomposite ( enc2 ) contained 35 wt% of nanosilica fillers . this material ( enc2 )", "the aim of this study was to study the effects of home bleaching on the hardness and surface morphology of a universal nanocomposite , an anterior nanocomposite , and a nanohybrid composite .", "two commercially available resin - based composites , based on bisphenol a - glycidyl methacrylate / triethyleneglycol dimethacrylate / ethoxylated bisphenol - a - dimethacrylate ( bis - gma)/ethoxylated bisphenol - a dimethacrylate ( bis - ema)/triethylene glycol dimethacrylate ( tegdma ) ] resin chemistry ( filtek z350 ; 3 m espe , st .", "paul , mn , usa and tph 3 ; dentsply caulk , milford , de , usa ) and one experimental resin - based composite based on bis - gma / urethane dimethacrylate ( udma)/tegdma resin chemistry ( kelfil ) were used in the present study [ table 1 ] .", "all composites were packed into acrylic molds ( 4 mm diameter  2 mm thick ) which were placed on a mylar strip .", "the excess composites were removed using another mylar strip on top of the mold and pressed flat with a glass slab .", "eighteen samples of each composite material were prepared , with a total at 54 samples .", "all samples were light - cured for 20 seconds from each top and bottom surface using elipar freelight 2 ( 3 m espe st .", "all samples were then polished using sof - lex ( 3 m espe , st .", "four different roughness of disks were used , which were coarse ( 55 m ) , medium ( 40 m ) , fine ( 24 m ) , and ultrafine ( 8 m ) , using a mandrel on a slow - speed handpiece , 10 seconds per disk without water .", "conscious efforts were made to standardize strokes , downward forces , and number of strokes for each disk .", "all samples were stored in a distilled water bath at 37 c for 24 hours prior to the treatment procedure .", "composites resin and bleaching agents tested the samples from each composite resin material were further divided into three groups ; groups 1 , 2 and 3 with n = 6 .", "samples in group 1 as the control group were not bleached but stored in shaking water bath sw23 ( julabo , seelbach , germany ) for 14 days at 37 c of distilled water .", "samples in group 2 were subjected to opalescence pf ( ultradent product , south jordan , ut , usa ) 10% cp for eight hours per day , for 14 days according to the suggested wear time of the manufacturer .", "samples in group 3 were bleached with opalescence pf ( ultradent product , south jordan , ut , usa ) 20% cp for two hours per day for a total of 14 days . in groups 2 and 3 ,", "prior to each bleaching procedure , the samples were taken out from the distilled water bath , air - dried with an air jet spray for 60 seconds .", "the bleaching agent was applied on one surface of the sample using a microbrush ( kerr corporation , california , usa ) and left for the duration suggested by the manufacturer . after each bleaching procedure", ", the samples were washed with a water jet spray for 60 seconds before they were stored back in distilled water bath and ready for the next bleaching procedure the next day .", "all samples were subjected to surface roughness testing using atomic force microscopy ( afm ) ( ambios technology , california , usa ) .", "five different areas were selected randomly with a scan area of 40  40 m and resolution 512  512 pixels to obtain surface roughness values ( ra ) .", "then , three - dimensional ( 3d ) images with 10  10 m sizes were acquired for each group of materials .", "after surface roughness testing , all samples were subjected to hardness testing using a vickers hardness tester , model vm50 ( fie , maharashtra state , india ) .", "the samples were placed underneath the indenter and 1 kg load was applied for a dwell time of 15 seconds .", "every sample was indented three times at three different points , and the mean readings were recorded .", "all statistical analysis was conducted at a significance level of p < 0.05 using mann - whitney test and kruskal - wallis test .", "all statistical analysis was conducted at a significance level of p < 0.05 using mann - whitney test and kruskal - wallis test .", "the results of the statistical analysis using kruskal - wallis test are presented in tables 2 and 3 .", "table 4 reveals the result of mann - whitney test analysis of kelfil and table 5 is the result of mann - whitney test of tph 3 .", "median roughness number ( ra , nm ) and interquartile range of the three tested composite resins after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range of the three tested composite resins after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range for kelfil after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range for tph 3 after 14 days of bleaching with 10 and 20% cp table 2 shows the roughness number ( ra ) of the three types of composites that were bleached with 10 and 20% cp home bleaching agents .", "there was no significant change in roughness in all three composites after 14 days of bleaching with 10 and 20% cp compared to the control group .", "table 3 illustrates the results of all the samples that were subjected to the vickers hardness test .", "kelfil showed significant change in hardness with a p value of 0.001 and there was also a significant change in tph 3 with a p value of 0.021 .", "table 4 shows that there was a significant change in kelfil after 14 days of bleaching .", "there was a significant increase in the surface hardness after bleaching with 10% cp home bleaching agent .", "however , there was a significant decrease after bleaching with 20% cp home bleaching agent .", "there was also a significant decrease in the surface hardness of kelfil after bleaching with 20% cp when compared to the group of samples bleached with 10% cp .", "table 5 reveals a significant change in the surface hardness of tph 3 after being subjected to 14 days of bleaching .", "there was a significant decrease in the surface hardness in the groups bleached with 10 and 20% cp compared to the control group .", "however , there was no significant change when the group was bleached with 10% cp compared to the group bleached with 20% cp .", "in this study , filtek z350 , a commercialized and widely used nanocomposite did not show any significant changes in surface hardness when compared to its control group , and also to groups of samples bleached with either 10 or 20% cp .", "however , there were significant changes in the vickers hardness number for both kelfil and tph 3 that had been subjected to 14 days of bleaching with either 10 or 20% cp .", "for kelfil , there was a significant increase in hardness after bleaching for 14 days with 10% cp but a significant decrease in hardness with 20% cp .", "after the curing process of a composite resin , a postpolymerization process continues to occur up to a certain period of time , which increases the hardness of the composite . in this study , the bleaching process was possibly being carried out at the same time as the postpolymerization process .", "the postpolymerization process in kelfil was observed with the 10% cp , and it was more prominent than the bleaching process ; hence the increase in its surface hardness .", ". nevertheless , with 20% cp , the bleaching process was more dominant than the postpolymerization process in all the three composites tested .", "the increase in hardness with 10% cp was observed in both filtek z350 and kelfil , and it maybe due to the similarities in their filler structure . in tph 3 , there was a significant decrease when comparison was made between the control group and 10% cp group , and also between the control group and 20% cp .", "this significant decrease is consistent with the conclusion drawn from other studies . as a general rule , the higher the filler loading , the greater the physical properties of the composite resins .", "it has a lower filler loading of 29% by volume compared to filtek z350 with the filler loading of 78.5% by weight or 59.5% by volume which is suitable for anterior and also posterior composite resin restorations .", "the lower filler loading of kelfil may lead to loosely packed inorganic fillers , which in turn caused the significant decrease in the vickers hardness number .", "for tph 3 , there was a significant decrease in vickers hardness number in samples that were subjected to 14 days of bleaching with both 10 and 20% cp .", "it was concluded that nanohybrid resins generally presented inferior properties compared to the nanofilled composite .", "tph 3 has 58% volume filler loading , which is similar to filtek z350 . the possible explanation for the significant reduction in the surface hardness might be due to the presence of bis - gma monomer .", "resin composites are reported to be highly susceptible to chemical softening due to the presence of bis - gma monomer if the chemicals have a solubility parameter ranging from 1.82  10 to 2.97  10 ( j / m ) . in the case of tph 3", ", the content of bis - gma in its matrix might be higher than in filtek z350 .", "another possible explanation is the degree to which the filler is bonded to the resin matrix .", "although there was a significant decrease in the vickers hardness numbers of kelfil and tph 3 , there were no significant changes in the roughness number in all specimens after 14 days of bleaching with either 10 or 20% cp opalescence pf home bleaching agents .", "this is consistent with the conclusion that no statistically significant differences were detected for any material .", "the cantilever sensor of afm senses any irregularities at the surface of the specimen ; however , as all the composites tested have similar nano - sized fillers , approximately 20 nm , there was no significant difference between them .", "furthermore , all samples were polished with the same polishing system , sof - lex disks which may contribute to a similar surface finish in the composite resins even after bleaching treatment .", "the 3d images of afm [ figures 13 ] show that there was no plucking out of filler particles after the bleaching treatment .", "afm topography 40  40 m of z350 after bleaching with 20% cp afm topography 40  40 m of kelfil after bleaching with 20% cp afm topography 40  40 m of tph 3 after bleaching with 20% cp generally , the surface roughness of all composites tested has a reading below 0.2 m or 200 nm .", "ra above 0.2 m results in an increase in the accumulation of plaque and a higher risk for caries and periodontal inflammation . according to chung in 1994 , when ra was lower than 1m ,", "therefore , all composites surfaces evaluated after bleaching treatment have demonstrated a smooth surface , which from the clinical point of view , present no risk of accumulation of plaque . from this study", ", it can be inferred that following bleaching , the experimental anterior composite and nanohybrid composite may need to be replaced , due to the reduction in their surface hardness .", "however , this has to be further investigated with in vitro studies evaluating the effects of saliva , and controlled clinical trials are necessary to determine any clinical implication .", "based on the result of this study , it can be concluded that 14 days of bleaching with 10 or 20% cp home bleaching agents did not cause any changes in surface roughness of the three tested composites .", "the afm evaluation of surface roughness showed that both bleaching agents yielded ra values below 200 nm for all materials tested , which poses no risk of the accumulation of plaque .", "the performance of kelfil after bleaching treatment is similar to that of the commercial nanocomposites .", "the afm evaluation of surface roughness observed in the 3d images promises to be an effective technique .", "nanocomposite and nanohybrid composite resins bleached with 10 or 20% cp bleaching agents result in the same outcome . on the other hand ,"], "abstract_text": ["<S> objective : home bleaching agents may not be safe for composite resins . the purpose of this study was to evaluate the effects of 10 and 20% opalescence pf home bleaching agents on the surface roughness and hardness of universal nanocomposite ( filtek z350 ) , anterior nanocomposite ( kelfil ) , and nanohybrid composite </S>", "<S> ( tph 3).materials and methods : fifty - four composite resin samples with 18 samples for each type of composite resin were prepared using acrylic molds ( 4  2 mm ) . </S>", "<S> each type of composite resin was further divided into three groups [ n = 6 controls were placed in distilled water for 14 days and the other two groups of n = 6 were bleached with 10 and 20% carbamide peroxide ( cp ) , respectively for 14 days ] . </S>", "<S> surface hardness of the composite resin was tested with a vickers hardness tester , whereas surface roughness was tested with atomic force microscopy ( afm).results : there were significant changes in the surface hardness of kelfil and tph 3 </S>", "<S> . however , all the tested materials showed no significant changes in the surface roughness.conclusion:after 14 days of home bleaching treatment , there was no adverse effect on the surface roughness of all three composite resins , although the surface hardness for kelfil and tph 3 were significantly reduced . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "Statistical analysis", "RESULTS", "DISCUSSION", "CONCLUSIONS"], "sections": [["since the introduction of tooth whitening by haywood and heymann in 1989 , this trend is getting more popular .", "there are a few types of bleaching methods , but all of them share a common principle of the decomposition of peroxides from hydrogen peroxide or its compounds such as carbamide peroxide ( cp ) into unstable free radicals . these radicals further breakdown into large pigmented molecules either through oxidation or reduction reaction .", "the oxidation or reduction process changes the chemical structure of the interacting organic substances of the tooth , which results in the change in color .", "furthermore , at 45% concentration of cp in different intracoronal tooth - bleaching procedures , maleknejad ( 2012 ) reported an increase in the diameter of dentinal tubules , and it also promoted alterations in the mineral content of the dentine .", "types of bleaching methods include nonvital bleaching , in - office professional bleaching , and home bleaching .", "nightguard home bleaching uses a relatively low level of whitening agent and is applied to the teeth via a custom - fabricated mouthguard and is worn at night for a duration of at least two weeks .", "a lot of research has been carried out to identify the effects of bleaching on the tooth surface and dental restorative materials .", "jacob ( 2007 ) reported that bleaching with cp may alter the marginal leakage of resin composite restoration ; however , amalgam restorations were not adversely affected in vitro .", "it was reported that the bleaching agent , regardless of the whitening products used , will reduce the microhardness of the enamel and promote an increase in surface roughness .", "hence , it is widely used for anterior and also posterior restorations . due to this", "some studies concluded that application of home bleaching agent gels may cause surface softening of composite resins , whereas others stated that there are no significant changes in surface microhardness found after the application of 15% cp on composite resin .", "furthermore , a study was done to evaluate the effects of bleaching on toothbrush abrasion in three types of composites .", "it was concluded that nano - filled resin composites are to be used for restoration when bleaching treatment is required .", "there is a significant and positive correlation found between surface roughness and vital bacterial adhesion .", "it appears that bleaching agents may affect the adherence of certain cariogenic micro - organisms to the outer surfaces of composite resin restorations .", "roughness has a major impact on aesthetic appearance , discoloration of restorations , plaque accumulation , secondary caries , and gingival irritation .", "the experimental composite tested in this study was a locally produced dental restoration nanocomposite ( kelfil ) .", "this nanocomposite was produced by the school of dental sciences ( ppsg ) , universiti sains malaysia ( usm ) , kelantan , malaysia .", "the nanosilica fillers , synthesized locally by a sol - gel process , were in the size range of 1020 nm .", "the experimental nanocomposite ( enc2 ) contained 35 wt% of nanosilica fillers . this material ( enc2 )", "the aim of this study was to study the effects of home bleaching on the hardness and surface morphology of a universal nanocomposite , an anterior nanocomposite , and a nanohybrid composite ."], ["two commercially available resin - based composites , based on bisphenol a - glycidyl methacrylate / triethyleneglycol dimethacrylate / ethoxylated bisphenol - a - dimethacrylate ( bis - gma)/ethoxylated bisphenol - a dimethacrylate ( bis - ema)/triethylene glycol dimethacrylate ( tegdma ) ] resin chemistry ( filtek z350 ; 3 m espe , st .", "paul , mn , usa and tph 3 ; dentsply caulk , milford , de , usa ) and one experimental resin - based composite based on bis - gma / urethane dimethacrylate ( udma)/tegdma resin chemistry ( kelfil ) were used in the present study [ table 1 ] .", "all composites were packed into acrylic molds ( 4 mm diameter  2 mm thick ) which were placed on a mylar strip .", "the excess composites were removed using another mylar strip on top of the mold and pressed flat with a glass slab .", "eighteen samples of each composite material were prepared , with a total at 54 samples .", "all samples were light - cured for 20 seconds from each top and bottom surface using elipar freelight 2 ( 3 m espe st .", "all samples were then polished using sof - lex ( 3 m espe , st .", "four different roughness of disks were used , which were coarse ( 55 m ) , medium ( 40 m ) , fine ( 24 m ) , and ultrafine ( 8 m ) , using a mandrel on a slow - speed handpiece , 10 seconds per disk without water .", "conscious efforts were made to standardize strokes , downward forces , and number of strokes for each disk .", "all samples were stored in a distilled water bath at 37 c for 24 hours prior to the treatment procedure .", "composites resin and bleaching agents tested the samples from each composite resin material were further divided into three groups ; groups 1 , 2 and 3 with n = 6 .", "samples in group 1 as the control group were not bleached but stored in shaking water bath sw23 ( julabo , seelbach , germany ) for 14 days at 37 c of distilled water .", "samples in group 2 were subjected to opalescence pf ( ultradent product , south jordan , ut , usa ) 10% cp for eight hours per day , for 14 days according to the suggested wear time of the manufacturer .", "samples in group 3 were bleached with opalescence pf ( ultradent product , south jordan , ut , usa ) 20% cp for two hours per day for a total of 14 days . in groups 2 and 3 ,", "prior to each bleaching procedure , the samples were taken out from the distilled water bath , air - dried with an air jet spray for 60 seconds .", "the bleaching agent was applied on one surface of the sample using a microbrush ( kerr corporation , california , usa ) and left for the duration suggested by the manufacturer . after each bleaching procedure", ", the samples were washed with a water jet spray for 60 seconds before they were stored back in distilled water bath and ready for the next bleaching procedure the next day .", "all samples were subjected to surface roughness testing using atomic force microscopy ( afm ) ( ambios technology , california , usa ) .", "five different areas were selected randomly with a scan area of 40  40 m and resolution 512  512 pixels to obtain surface roughness values ( ra ) .", "then , three - dimensional ( 3d ) images with 10  10 m sizes were acquired for each group of materials .", "after surface roughness testing , all samples were subjected to hardness testing using a vickers hardness tester , model vm50 ( fie , maharashtra state , india ) .", "the samples were placed underneath the indenter and 1 kg load was applied for a dwell time of 15 seconds .", "every sample was indented three times at three different points , and the mean readings were recorded .", "all statistical analysis was conducted at a significance level of p < 0.05 using mann - whitney test and kruskal - wallis test ."], ["all statistical analysis was conducted at a significance level of p < 0.05 using mann - whitney test and kruskal - wallis test ."], ["the results of the statistical analysis using kruskal - wallis test are presented in tables 2 and 3 .", "table 4 reveals the result of mann - whitney test analysis of kelfil and table 5 is the result of mann - whitney test of tph 3 .", "median roughness number ( ra , nm ) and interquartile range of the three tested composite resins after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range of the three tested composite resins after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range for kelfil after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range for tph 3 after 14 days of bleaching with 10 and 20% cp table 2 shows the roughness number ( ra ) of the three types of composites that were bleached with 10 and 20% cp home bleaching agents .", "there was no significant change in roughness in all three composites after 14 days of bleaching with 10 and 20% cp compared to the control group .", "table 3 illustrates the results of all the samples that were subjected to the vickers hardness test .", "kelfil showed significant change in hardness with a p value of 0.001 and there was also a significant change in tph 3 with a p value of 0.021 .", "table 4 shows that there was a significant change in kelfil after 14 days of bleaching .", "there was a significant increase in the surface hardness after bleaching with 10% cp home bleaching agent .", "however , there was a significant decrease after bleaching with 20% cp home bleaching agent .", "there was also a significant decrease in the surface hardness of kelfil after bleaching with 20% cp when compared to the group of samples bleached with 10% cp .", "table 5 reveals a significant change in the surface hardness of tph 3 after being subjected to 14 days of bleaching .", "there was a significant decrease in the surface hardness in the groups bleached with 10 and 20% cp compared to the control group .", "however , there was no significant change when the group was bleached with 10% cp compared to the group bleached with 20% cp ."], ["in this study , filtek z350 , a commercialized and widely used nanocomposite did not show any significant changes in surface hardness when compared to its control group , and also to groups of samples bleached with either 10 or 20% cp .", "however , there were significant changes in the vickers hardness number for both kelfil and tph 3 that had been subjected to 14 days of bleaching with either 10 or 20% cp .", "for kelfil , there was a significant increase in hardness after bleaching for 14 days with 10% cp but a significant decrease in hardness with 20% cp .", "after the curing process of a composite resin , a postpolymerization process continues to occur up to a certain period of time , which increases the hardness of the composite . in this study , the bleaching process was possibly being carried out at the same time as the postpolymerization process .", "the postpolymerization process in kelfil was observed with the 10% cp , and it was more prominent than the bleaching process ; hence the increase in its surface hardness .", ". nevertheless , with 20% cp , the bleaching process was more dominant than the postpolymerization process in all the three composites tested .", "the increase in hardness with 10% cp was observed in both filtek z350 and kelfil , and it maybe due to the similarities in their filler structure . in tph 3 , there was a significant decrease when comparison was made between the control group and 10% cp group , and also between the control group and 20% cp .", "this significant decrease is consistent with the conclusion drawn from other studies . as a general rule , the higher the filler loading , the greater the physical properties of the composite resins .", "it has a lower filler loading of 29% by volume compared to filtek z350 with the filler loading of 78.5% by weight or 59.5% by volume which is suitable for anterior and also posterior composite resin restorations .", "the lower filler loading of kelfil may lead to loosely packed inorganic fillers , which in turn caused the significant decrease in the vickers hardness number .", "for tph 3 , there was a significant decrease in vickers hardness number in samples that were subjected to 14 days of bleaching with both 10 and 20% cp .", "it was concluded that nanohybrid resins generally presented inferior properties compared to the nanofilled composite .", "tph 3 has 58% volume filler loading , which is similar to filtek z350 . the possible explanation for the significant reduction in the surface hardness might be due to the presence of bis - gma monomer .", "resin composites are reported to be highly susceptible to chemical softening due to the presence of bis - gma monomer if the chemicals have a solubility parameter ranging from 1.82  10 to 2.97  10 ( j / m ) . in the case of tph 3", ", the content of bis - gma in its matrix might be higher than in filtek z350 .", "another possible explanation is the degree to which the filler is bonded to the resin matrix .", "although there was a significant decrease in the vickers hardness numbers of kelfil and tph 3 , there were no significant changes in the roughness number in all specimens after 14 days of bleaching with either 10 or 20% cp opalescence pf home bleaching agents .", "this is consistent with the conclusion that no statistically significant differences were detected for any material .", "the cantilever sensor of afm senses any irregularities at the surface of the specimen ; however , as all the composites tested have similar nano - sized fillers , approximately 20 nm , there was no significant difference between them .", "furthermore , all samples were polished with the same polishing system , sof - lex disks which may contribute to a similar surface finish in the composite resins even after bleaching treatment .", "the 3d images of afm [ figures 13 ] show that there was no plucking out of filler particles after the bleaching treatment .", "afm topography 40  40 m of z350 after bleaching with 20% cp afm topography 40  40 m of kelfil after bleaching with 20% cp afm topography 40  40 m of tph 3 after bleaching with 20% cp generally , the surface roughness of all composites tested has a reading below 0.2 m or 200 nm .", "ra above 0.2 m results in an increase in the accumulation of plaque and a higher risk for caries and periodontal inflammation . according to chung in 1994 , when ra was lower than 1m ,", "therefore , all composites surfaces evaluated after bleaching treatment have demonstrated a smooth surface , which from the clinical point of view , present no risk of accumulation of plaque . from this study", ", it can be inferred that following bleaching , the experimental anterior composite and nanohybrid composite may need to be replaced , due to the reduction in their surface hardness .", "however , this has to be further investigated with in vitro studies evaluating the effects of saliva , and controlled clinical trials are necessary to determine any clinical implication ."], ["based on the result of this study , it can be concluded that 14 days of bleaching with 10 or 20% cp home bleaching agents did not cause any changes in surface roughness of the three tested composites .", "the afm evaluation of surface roughness showed that both bleaching agents yielded ra values below 200 nm for all materials tested , which poses no risk of the accumulation of plaque .", "the performance of kelfil after bleaching treatment is similar to that of the commercial nanocomposites .", "the afm evaluation of surface roughness observed in the 3d images promises to be an effective technique .", "nanocomposite and nanohybrid composite resins bleached with 10 or 20% cp bleaching agents result in the same outcome . on the other hand ,"]]}
{"article_id": "PMC4164061", "article_text": ["penile squamous cell carcinoma ( pscc ) , the predominant histological type ( > 95% ) of penile cancer , is a relatively rare malignant tumor in western countries and japan .", "pscc is a rare disease , making it difficult to establish a standard of care in any of the clinical stages , particularly in advanced disease .", "multimodal treatments , including surgery , chemotherapy , and radiation therapy , should be considered for patients with advanced pscc . however , the optimal chemotherapeutic regimen is unknown , although cisplatin - containing chemotherapy is the mainstay of combination chemotherapy .", "recent studies have suggested that taxanes in combination with cisplatin and fluorouracil ( 5-fu ) have a significant effect on unresectable and recurrent penile cancer .", "malignant wound , results in a decline in the quality of life because of the presence of bleeding , exudates , and/or strong odor .", "mohs chemosurgery is a technique of chemical fixation of a cutaneous tumor and subsequent excision .", "several studies have shown the efficacy of mohs paste for maintaining malignant wounds in advanced squamous cell carcinoma of the breast , skin , head , and neck [ 4 , 5 ] .", "we report a case of a pscc with advanced lymph node metastasis treated with combination therapy consisting of taxane - based chemotherapy , irradiation , and mohs paste .", "an 80-year - old male presented to a community hospital with pain and redness in his left inguinal region .", "he had undergone penectomy and bilateral inguinal lymphadenectomy 1 year ago because of primary pscc ( pt1pn0 ) .", "a large solid mass with bleeding and a smelly exudate was observed in his left inguinal region .", "macroscopically , the tumor mass of the left inguinal region was 7.0 cm in diameter with an ulcer , redness , and exudate ( fig .", "a ct scan revealed a huge mass ( 5.9  5.8 cm ) in the left inguinal region along with his left femoral vessels ( fig .", "he was administered combination therapy consisting of a taxane - based chemotherapy , irradiation , and mohs chemosurgery .", "the chemotherapy regimen consisted of 60 mg / m docetaxel administered over 3 h on day 1 ; 750 mg / m 5-fu on days 15 ; and 70 mg / m cisplatin on day 4 .", "the patient also received 50-gy external - beam radiation therapy to the left inguinal region , initiated on the same day of chemotherapy .", "mohs paste was applied every morning with a surrounding gauze to avoid attaching with normal skin ( as previously reported ) .", "mohs paste included 50 ml of zinc chloride - saturated aqueous solution , 10 g of zinc powder , and 15 ml of glycerin .", "radiation therapy was temporarily deferred for 12 days beginning on day 9 because of neutropenia and general fatigue .", "the necrotic tissue fell off and the wound flattened on day 20 after starting chemotherapy .", "2b ) , and a ct scan showed that the tumor had decreased by 70% in diameter at 1 month after the first course of chemotherapy ( fig .", "3b ) . because of socio - economic reasons , the patient declined additional treatment .", "there was no progression or metastasis on the chest and abdominal ct for 8 months .", "however , the wound grew and became erosive again 10 months after discharge , and the patient died because of the progression of local recurrence 1 year after chemotherapy treatment .", "in our patient with inguinal recurrence of pscc , multimodal treatment , consisting of combination chemotherapy , irradiation , and mohs chemosurgery , was responsible for the tumor shrinkage and the successful local control of a malignant wound .", "although this field is limited by a paucity of clinical trials or prospective data , the available single institutional retrospective reviews indicate that multiagent cisplatin - based chemotherapy regimens fight significantly against pscc . in 1991 , dexeus et al .  ", "reported a triple - drug chemotherapy regimen , which soon became a standard regimen for pscc with tolerable adverse effects .", "the regimen consisted of 20 mg / m cisplatin on days 26 , 200 mg / m methotrexate on days 1 and 15 , and 10 mg / m bleomycin on days 26 .", "they reported a response in 10 of 14 ( 71% ) patients ( with moderate side effects ) . in their follow - up studies ,", "a lower rate of complete responses with severe toxicities was reported [ 8 , 9 ] . with respect to scc of the head and neck , in which pscc is historically similar", ", a randomized trial revealed that the addition of docetaxel significantly improved progression - free , and overall survival in patients with unresectable scc compared to the standard regimen of cisplatin and 5-fu ; therefore , docetaxel , cisplatin and 5-fu have become the current standard induction regimen for advanced scc of the head and neck . in pscc , pizzocaro et al .  ", "they reported a high activity of this regimen , with 5 of 6 treated patients showing a response .", "based on these findings , we chose docetaxel for taxane - based combination therapy for the patient , although there are few reports demonstrating its efficacy against pscc .", "further studies are needed to determine which taxane has a better response and less toxicity in patients with pscc .", "radiation therapy has been used for many years in the treatment of pscc for the primary tumor , inguinal metastases and distal metastases .", "it may also play a role in the treatment of locally advanced penile cancer , particularly when inguinal adenopathy is initially unresectable .", "there are no randomized trials that have evaluated the impact of radiation on the prognosis and the local control of patients with inguinal metastasis originating from pscc .", "however , ravi et al .   revealed that palliative radiation therapy showed an amelioration of symptoms in 56% of patients with inguinal metastasis in growing pscc .", "in addition , the radiosensitizing effect of cisplatin , 5-fu , and docetaxel has been demonstrated in several types of cancers [ 13 , 14 , 15 ] .", "therefore , it is plausible that radiation in combination with cisplatin , 5-fu , and the docetaxel regimen is a promising option for multimodal treatment of pscc .", "malignant wounds from primary or metastatic carcinoma are generally incurable , and palliative methods to manage the wounds are needed .", "frederic e. mohs developed and published a technique for the chemical fixation of a cutaneous tumor in 1941 .", "mohs paste is effective for the hemostasis of bleeding , odors , and exudates , thereby contributing to the patient 's quality of life . in japan , kakimoto et al", ".   reported on 5 patients with breast cancer who were successfully treated with mohs paste .", "showed the efficacy of radical surgery followed by systematic therapy and mohs paste in patients with breast cancer . in our case ,", "the tumor started to decrease in size after treatment and the necrotic tumor fell off by day 20 , without any resection . to the best of our knowledge ,", "this is the first report demonstrating the advantage of mohs paste for inguinal recurrence of pscc .", "our result shows that mohs paste may be an effective and reliable option for multimodal treatment of inguinal recurrence .", "it is difficult to define which treatment modality was the most effective for the patient , because we used a combination of 3 different modalities .", "although our result suggests that each treatment was synergistically effective , it is also important to delineate which modality is the most effective and then choose a suitable modality for each patient with pscc .", "secondly , the socio - economic background of the patient did not allow him to continue systemic treatment and/or maintenance therapy .", "the study showed that paclitaxel was well tolerated , with a moderate activity against pscc .", "therefore , the patient may have been able to avoid cancer progression for a much longer time if he had received additional maintenance chemotherapy . in conclusion , we report a case of successful local control of a recurrent inguinal mass of pscc treated with multimodal therapy . to the best of our knowledge ,", "combination treatment with taxane - based chemotherapy , external beam radiation therapy , and mohs paste is a reliable option for the induction therapy for recurrent pscc .", "further studies are needed to investigate maintenance treatment after effective induction therapy for recurrent pscc ."], "abstract_text": ["<S> abstractpenile squamous cell carcinoma ( pscc ) is a rare disease , making it difficult to establish a standard of care , particularly in the advanced stage . </S>", "<S> we report a case of pscc with advanced lymph node metastasis treated with multimodal therapy consisting of combination chemotherapy , irradiation , and chemosurgery using mohs zinc chloride - containing paste . </S>", "<S> an 80-year - old male with a past history of local treatment for penile cancer presented with a large painful inguinal mass with an ulcer and exudates . </S>", "<S> the patient underwent multimodal treatment with combination chemotherapy , irradiation , and mohs paste . the combination chemotherapy consisted of cisplatin , 5-fluorouracil , and docetaxel . </S>", "<S> the patient received 50-gy external - beam radiation therapy to the left inguinal region along with daily local treatment with mohs paste . </S>", "<S> after the initiation of treatment , the pain and bleeding in the inguinal region considerably ameliorated . the wound became dry and flattened 20 days after the initiation of chemotherapy . </S>", "<S> a ct scan showed that the tumor had decreased 70% in diameter 1 month after the initiation of chemotherapy . </S>", "<S> after the first course of chemotherapy , the patient and his family decided not to continue treatment because of socio - economic reasons . </S>", "<S> the patient underwent no additional treatments ; nevertheless , he had no local progression of the inguinal tumors for 8 months . </S>", "<S> we report a case of successful local control of recurrent inguinal pscc treated with multimodal therapy . </S>", "<S> combination treatment with taxane - based chemotherapy , external - beam radiation therapy , and mohs paste is an option for the management of recurrent pscc . </S>"], "labels": null, "section_names": ["Introduction", "Case Report", "Discussion", "Disclosure Statement"], "sections": [["penile squamous cell carcinoma ( pscc ) , the predominant histological type ( > 95% ) of penile cancer , is a relatively rare malignant tumor in western countries and japan .", "pscc is a rare disease , making it difficult to establish a standard of care in any of the clinical stages , particularly in advanced disease .", "multimodal treatments , including surgery , chemotherapy , and radiation therapy , should be considered for patients with advanced pscc . however , the optimal chemotherapeutic regimen is unknown , although cisplatin - containing chemotherapy is the mainstay of combination chemotherapy .", "recent studies have suggested that taxanes in combination with cisplatin and fluorouracil ( 5-fu ) have a significant effect on unresectable and recurrent penile cancer .", "malignant wound , results in a decline in the quality of life because of the presence of bleeding , exudates , and/or strong odor .", "mohs chemosurgery is a technique of chemical fixation of a cutaneous tumor and subsequent excision .", "several studies have shown the efficacy of mohs paste for maintaining malignant wounds in advanced squamous cell carcinoma of the breast , skin , head , and neck [ 4 , 5 ] .", "we report a case of a pscc with advanced lymph node metastasis treated with combination therapy consisting of taxane - based chemotherapy , irradiation , and mohs paste ."], ["an 80-year - old male presented to a community hospital with pain and redness in his left inguinal region .", "he had undergone penectomy and bilateral inguinal lymphadenectomy 1 year ago because of primary pscc ( pt1pn0 ) .", "a large solid mass with bleeding and a smelly exudate was observed in his left inguinal region .", "macroscopically , the tumor mass of the left inguinal region was 7.0 cm in diameter with an ulcer , redness , and exudate ( fig .", "a ct scan revealed a huge mass ( 5.9  5.8 cm ) in the left inguinal region along with his left femoral vessels ( fig .", "he was administered combination therapy consisting of a taxane - based chemotherapy , irradiation , and mohs chemosurgery .", "the chemotherapy regimen consisted of 60 mg / m docetaxel administered over 3 h on day 1 ; 750 mg / m 5-fu on days 15 ; and 70 mg / m cisplatin on day 4 .", "the patient also received 50-gy external - beam radiation therapy to the left inguinal region , initiated on the same day of chemotherapy .", "mohs paste was applied every morning with a surrounding gauze to avoid attaching with normal skin ( as previously reported ) .", "mohs paste included 50 ml of zinc chloride - saturated aqueous solution , 10 g of zinc powder , and 15 ml of glycerin .", "radiation therapy was temporarily deferred for 12 days beginning on day 9 because of neutropenia and general fatigue .", "the necrotic tissue fell off and the wound flattened on day 20 after starting chemotherapy .", "2b ) , and a ct scan showed that the tumor had decreased by 70% in diameter at 1 month after the first course of chemotherapy ( fig .", "3b ) . because of socio - economic reasons , the patient declined additional treatment .", "there was no progression or metastasis on the chest and abdominal ct for 8 months .", "however , the wound grew and became erosive again 10 months after discharge , and the patient died because of the progression of local recurrence 1 year after chemotherapy treatment ."], ["in our patient with inguinal recurrence of pscc , multimodal treatment , consisting of combination chemotherapy , irradiation , and mohs chemosurgery , was responsible for the tumor shrinkage and the successful local control of a malignant wound .", "although this field is limited by a paucity of clinical trials or prospective data , the available single institutional retrospective reviews indicate that multiagent cisplatin - based chemotherapy regimens fight significantly against pscc . in 1991 , dexeus et al .  ", "reported a triple - drug chemotherapy regimen , which soon became a standard regimen for pscc with tolerable adverse effects .", "the regimen consisted of 20 mg / m cisplatin on days 26 , 200 mg / m methotrexate on days 1 and 15 , and 10 mg / m bleomycin on days 26 .", "they reported a response in 10 of 14 ( 71% ) patients ( with moderate side effects ) . in their follow - up studies ,", "a lower rate of complete responses with severe toxicities was reported [ 8 , 9 ] . with respect to scc of the head and neck , in which pscc is historically similar", ", a randomized trial revealed that the addition of docetaxel significantly improved progression - free , and overall survival in patients with unresectable scc compared to the standard regimen of cisplatin and 5-fu ; therefore , docetaxel , cisplatin and 5-fu have become the current standard induction regimen for advanced scc of the head and neck . in pscc , pizzocaro et al .  ", "they reported a high activity of this regimen , with 5 of 6 treated patients showing a response .", "based on these findings , we chose docetaxel for taxane - based combination therapy for the patient , although there are few reports demonstrating its efficacy against pscc .", "further studies are needed to determine which taxane has a better response and less toxicity in patients with pscc .", "radiation therapy has been used for many years in the treatment of pscc for the primary tumor , inguinal metastases and distal metastases .", "it may also play a role in the treatment of locally advanced penile cancer , particularly when inguinal adenopathy is initially unresectable .", "there are no randomized trials that have evaluated the impact of radiation on the prognosis and the local control of patients with inguinal metastasis originating from pscc .", "however , ravi et al .   revealed that palliative radiation therapy showed an amelioration of symptoms in 56% of patients with inguinal metastasis in growing pscc .", "in addition , the radiosensitizing effect of cisplatin , 5-fu , and docetaxel has been demonstrated in several types of cancers [ 13 , 14 , 15 ] .", "therefore , it is plausible that radiation in combination with cisplatin , 5-fu , and the docetaxel regimen is a promising option for multimodal treatment of pscc .", "malignant wounds from primary or metastatic carcinoma are generally incurable , and palliative methods to manage the wounds are needed .", "frederic e. mohs developed and published a technique for the chemical fixation of a cutaneous tumor in 1941 .", "mohs paste is effective for the hemostasis of bleeding , odors , and exudates , thereby contributing to the patient 's quality of life . in japan , kakimoto et al", ".   reported on 5 patients with breast cancer who were successfully treated with mohs paste .", "showed the efficacy of radical surgery followed by systematic therapy and mohs paste in patients with breast cancer . in our case ,", "the tumor started to decrease in size after treatment and the necrotic tumor fell off by day 20 , without any resection . to the best of our knowledge ,", "this is the first report demonstrating the advantage of mohs paste for inguinal recurrence of pscc .", "our result shows that mohs paste may be an effective and reliable option for multimodal treatment of inguinal recurrence .", "it is difficult to define which treatment modality was the most effective for the patient , because we used a combination of 3 different modalities .", "although our result suggests that each treatment was synergistically effective , it is also important to delineate which modality is the most effective and then choose a suitable modality for each patient with pscc .", "secondly , the socio - economic background of the patient did not allow him to continue systemic treatment and/or maintenance therapy .", "the study showed that paclitaxel was well tolerated , with a moderate activity against pscc .", "therefore , the patient may have been able to avoid cancer progression for a much longer time if he had received additional maintenance chemotherapy . in conclusion , we report a case of successful local control of a recurrent inguinal mass of pscc treated with multimodal therapy . to the best of our knowledge ,", "combination treatment with taxane - based chemotherapy , external beam radiation therapy , and mohs paste is a reliable option for the induction therapy for recurrent pscc .", "further studies are needed to investigate maintenance treatment after effective induction therapy for recurrent pscc ."], [""]]}
{"article_id": "PMC3803124", "article_text": ["the majority of women living with hiv are in their reproductive years ( ages 1549 ) [ 1 , 2 ] .", "the dramatic decrease in the risk of mother - to - child hiv transmission ( mtct ) is leading to normality in the lives of couples affected by hiv , who want own children . in europe", ", the reduction in mtct to less than 1% is mainly due to highly active antiretroviral therapy ( haart ) .", "effective haart is resulting in suppressed viral load ( vl ) ; thus , a vaginal birth can be as safe as a planned caesarean section [ 3 , 4 ] .", "avoidance of breastfeeding and postnatal neonatal postexposure prophylaxis ( pep ) further supports the effective reduction in mtct [ 35 ] .", "the literature suggests that there is no increased rate of fetal malformations due to the hiv infection or haart [ 6 , 7 ] . a pregnant woman with hiv infection", "large studies of noninvasive prenatal screening have already indicated that it will lead to a decrease of invasive prenatal screening procedures such as amniocentesis ( ac ) or chorionic villi biopsy ( cvs ) .", "if invasive prenatal testing is necessary , it can be done , but in these circumstances , haart should be started prior to the procedure to suppress the vl below the limit of detection . in these cases", "current , evidence suggests that mtct is very unlikely ; however , studies reporting on the risk of mtct in invasive prenatal testing are limited due to small study size .", "haart is given during pregnancy for two reasons , first to women with an own indication for haart ( they require treatment for their own health ) and secondly to pregnant women starting therapy purely as a prophylactic treatment to reduce mtct .  ", "the aim of our study was to investigate if pregnant hiv - positive women get referred for special prenatal ultrasound screening services in our tertiary referral center , but also if and at what point the prenatal ultrasonography is performed .", "pregnant hiv - positive women usually have a combined antenatal care in a tertiary referral center and with their own gynaecologists .   as well as the prevalence of prenatal ultrasound screening , prenatal , and postnatal finding was recorded .", "we hypothesized that the fetal anomaly rate in women with hiv - infection is as low as in all other pregnancies ( 35% ) [ 12 , 13 ] .", "hiv - positive pregnant women who presented in our tertiary referral center between january 1 , 2002 and december 31 , 2012 were included in this retrospective cohort study .  ", "three categories were used : very preterm delivery ( 24 + 0 to 33 + 6 weeks of gestation ) , preterm delivery ( 34 + 0 to 36 + 6 weeks of gestation ) , and term delivery ( 37 weeks of gestation ) .  ", "all data regarding early prenatal screening ( as , e.g. , nuchal translucency measurements ) and fetal anomaly scan at 20 weeks of gestation or at first presentation in our center were recorded . only scans which were performed in our center", "were included , reflecting the fact that hiv - positive pregnant women are high risk pregnancies , and high - risk pregnancies are referred to a tertiary center or an equivalent specialized center for prenatal screening [ 1417 ] .", "an early anomaly scan was defined as a first trimester scan ; in the study period , the fetal nuchal thickness was assessed ; a formal nuchal translucency measurement was included if measured by appropriately qualified sonographers .", "a fetal anomaly scan was defined as a detailed scan in the second trimester ( usually between 20 to 22 weeks of gestation ) .", "all the scans performed at a later gestation in our department prior to birth are recorded separately as late scans in the third trimester .", "malformations were any fetal / neonatal disease , which required either surgery or special pediatric care including chromosomal anomalies .", "all cases with an ac , mtct , and any intrauterine or postnatal death were evaluated .", "maternal information included age , ethnicity , gestational age at delivery , gravidity and parity , haart already before the pregnancy , vl ( copies / ml ) , cd4 count ( cells/l ) prior to birth , and other risk factors such as coinfection with hcv .", "the last recorded vl prior to the delivery was used and classified in three risk groups . in the study , a vl below 50 is considered as negative / undetectable .", "the last cd4 count prior to birth was noted , and again three categories were used .", "the mode of delivery was classified as ( 1 ) planned caesarean section ; ( 2 ) in cases of rupture of membranes and/or contraction it was recorded as elective caesarean section in labour ; ( 3 ) emergency caesarean section ; ( 4 ) caesarean section after planned vaginal birth ; ( 5 ) vaginal birth ; ( 6 ) unplanned vaginal birth and ( 7 ) instrumental vaginal delivery . in the unit the first planned vaginal birth was recorded in 2009 . before that time , women were offered elective caesarean section at around 37 + 0 weeks of gestation [ 3 , 8 , 19 ] . with evidence for the safety of the vaginal birth with undetectable vl ,", "the policy in the unit shifted towards planned vaginal birth , and if caesarean section was offered in these cases , the delivery was delayed until > 37 weeks of gestation according to the german - austrian guidelines [ 3 , 4 , 8 ] .", "the following neonatal data were included : apgar score , arterial cord ph ( aph ) , cord base excess ( be ) and neonatal weight ( stratified according to 10th , 1090th and > 90th percentile ) .", "a weight below the 10th percentile was considered to be intrauterine growth retardation ( iugr ) .  ", "information about scan findings was obtained from the record of the ultrasound department , and further information was collected from maternal case notes , pediatric notes , and discharge letters .  ", "ethics approval for the retrospective study was obtained from the ethics committee at the j. w. goethe university , frankfurt ( number 30/13 ) . for categorical variables and nominal variables ,", "frequency tables were used for descriptive statistical analysis . for ordinal and quantitative data , mean and", "these data were further analyzed using the wilcoxon - mann - whitney test , kruskal - wallis test , spearman - correlation , chi - test , and fisher 's exact test as appropriate .", "in addition , multivariate logistic regression analysis was performed to identify factors associated with a woman having an early anomaly scan .  ", "overall 330 pregnancies were recorded , with 322 singleton pregnancies ( 97.6% ) and in eight twin gestations ( 2.4% ) .", "one twin pregnancy was conceived due to ivf with first diagnosis of the hiv - infection in the early second trimester .", "there were 122 preterm deliveries ( 36.5% ) and 90 ( 26.9% ) of these were between 34 and 36 + 6 weeks of gestation .", "maternal and neonatal characteristics are presented in table 1 , stratified by pregnancy duration in table 2 .  ", "two thirds of women ( 66.4% ) were of african ethnicity . in one quarter of women ,", "more than three quarters 257 ( 77.4% ) of the births were elective caesarean section . in 29 cases ( 8.7% ) , women delivered vaginally .", "the cd4 count ( cells/l ) prior to birth was in the majority of 175 ( 62.5% ) 350 , in 76 ( 27% ) between 200 and 349 , and in 30 ( 10.7% ) < 200 . the vl ( copies / ml ) in most women 168 ( 55.8% ) was suppressed below 50 copies in 88 ( 29.2% ) 50399 and in 45 ( 15% ) 400 .", "one hundred and eight women ( 37.4% ) were on no haart treatment at the beginning of the pregnancy . in 25 ( 8.9% ) , a positive anti - hcv test was recorded .", "thirty newborns ( 9% ) were classified as below the 10th percentile . in 100 of the 330 pregnancies ( 30.5% )", "the nuchal translucency was measured in 67 ( 20.3% ) of the 330 cases ( nt median 1.22  mm ( range 0.63  mm ) ) . a multivariate analysis for factors influencing a woman having an early anomaly scan ( table 3 ) showed that african ethnicity and first diagnosis of hiv during the ongoing pregnancy were factors which significantly could be related to not having early prenatal ultrasound screening ( figure 1 ) .  ", "invasive testing ( ac ) was done in three ( 0.9% ) of 330 cases .", "only one case was done at 25 weeks in our department , and we started haart and performed the ac after vl was fully suppressed .", "two cases were done for advanced maternal age without control of vl and without specific precautions for example , haart , and both revealed a normal karyotype . in all of three cases , no mtct occurred . in the second trimester in 252 ( 74.5% ) of 330 , a detailed anomaly scan at 2022 weeks was done . in 18 ( 5.5% ) patients ,", "the scan was performed in the third trimester due to late presentation in our unit . in table 4 ,", "fetal and neonatal malformations as well as chromosomal anomalies are presented . in seven cases of 330 cases ( 2.1% ) , we diagnosed a fetal malformation .", "postnatally , all of the seven cases were confirmed , and eight further malformations and two cases with trisomy 21 were detected .", "the chromosomal anomalies were not suspected . both women , 33 and 39 years of age , had no early scan or biochemical screening but a scan in our unit ( late in the second trimester with no anomalies seen ) .", "there were three cases with a skin tag , one nevus sebaceous of the occiput , and one case with a socalled sucking blister on the hand , all considered to be minor .", "each of these cases had at least one scan in our department prior to the birth .", "however , the sucking blister and the nevus were leading to an upgrade in neonatal pep due to breaking down of protective skin barrier , and one newborn presented with a small omphalocele which was not seen prior to birth .", ". the overall fetal malformation rate ( including the minor anomalies ) was 4.5% .   in table 5 ,", "the fetal and postnatal mortalities are recorded . in our cohort , we had six cases of intrauterine or postnatal loss and all were born by caesarean section .", "all of the three newborns were delivered by caesarean section , and all were preterm ( 33 + 6 , 36 + 3 and 36 + 4 weeks of gestation ) . in all cases ,", "the vl was detectable , all women were on haart , and one woman was coinfected with hcv .", "one woman had already a vertically infected child , and she had a poor compliance .", "there are conflicting results regarding the risks for hiv - positive mothers for possible adverse effects in their pregnancies [ 5 , 6 ] . in our study", ", we confirm the low fetal malformation rate of 4.5% in women living with hiv .", "there are different national registers collecting data on haart and pregnancy outcome ( e.g. , apr : antiviral pregnancy registry ; nshpc : national study of hiv in pregnancy and childhood ( uk ) ; ecs : european collaborative cohort ; epf  french perinatal cohort ) [ 4 , 2124 ] .", "these registers confirm the same malformation rate in women taking haart as in the general population ( 35% ) [ 12 , 13 ] .", "the postnatal anomalies were minor ones ( skin tag , sucking blister ) or missed due to minimal extend ( omphalocele ) .", "the two cases with postnatal trisomy 21 were missed prenatally but were not seen in typical screening periods . there were no anomalies in the unscreened population .", "a change in treatment policies is evident over the 11 years of the study , reflected in the changes in delivery mode over time and the gestational age at delivery .", "a high preterm delivery rate is confirmed by other groups . in our population , 26.9% are late preterm deliveries ( 3436 + 6 weeks of gestation ) and are mostly iatrogenic due to early caesarean section as in other studies and in the past .", "the updated national german - austrian guidelines now delay caesarean section to term in women with suppressed vl .", "the numbers of women with fully suppressed vl ( vl < 50 copies / ml ) ( p < 0.001 ) and cd4 cells 350 ( p > 0.20 ) prior to birth increased over the last years .  ", "the first is early screening which should take place between 11 + 0 and 14 + 0 weeks of gestation .", "this early screening was introduced by nicolaidis in 1992 as a combined method of screening ( including ultrasound screening and two maternal biochemical markers : free human chorionic gonadotropin ( free hcg ) and pregnancy - associated plasma protein a ( papp - a ) [ 2628 ] . in germany", "this test is not covered by the national health system and there for is paid by the woman herself . usually at that time", "an early anomaly ultrasound scan can be performed , which is covered by the health system .", "the second screening interval is the anomaly scan at 2022 weeks of gestation [ 17 , 29 ]", ".   the prevalence of first trimester screening of 97.5% in a low risk general population has been demonstrated .  ", "we demonstrate that prenatal screening is offered and available , but that the early screening interval is missed , as only 30.5% women get referred for early anomaly scan .", "even so some women may have chosen not to undergo testing for ethical and cultural reasons . as a limitation of our data", "it could be that the screening which is done at the community - based care is missed , but as indicated usually , it warrants a referral to a highly qualified and specially trained team [ 16 , 29 ] . in our study ,", "population the majority of 188 ( 66.4% ) women were of african origin , and in 79 ( 24.2% ) , the diagnosis hiv - infection occurred in the pregnancy , both factors were significantly related to having no early prenatal screening .", "tariq et al . are reporting on late booking for antenatal care in non - caucasian women compared with caucasian women regardless of time of diagnosis of hiv - infection .", "three cases of mtct are low ( 0.9% ) and confirmed by other groups ( reporting mtct rates of 0.1%1.3% ) [ 3 , 4 ] .", "however , looking back in our data , the viral control has improved dramatically over the last 11 years ; in 55.8% of all pregnancies , the vl is < 50", ".   national health systems vary , and a complete first trimester screening ( with inclusion of biochemical serum markers ) has not been established on a national basis for high risk pregnancies in some european countries . in our cohort , two cases of trisomy 21 occurred , and the question remains open if these two cases could have been traced in a complete first trimester screening . in one pregnancy , an ac was performed due to suspected chromosomal anomaly , which revealed a normal karyotype .", "due to the time required to initiate haart and to have a suppressed vl in hiv - positive pregnancies , invasive testing will be very likely to happen in the second trimester which will then raise the difficult ethical questions about late termination of pregnancy when an abnormal result is obtained [ 32 , 33 ] .", "first trimester screening ( including maternal markers as free hcg and papp - a ) has been investigated in pregnant women living with hiv .", "some groups feel that maternal markers could be less reliable than those in hiv - negative women [ 34 , 35 ] . in our study data from 2002 to 2012 , in the first years , the nuchal translucency was assessed but not formally measured .", "this could be due to the delay in having certified specially trained sonographers involved .   in the future ,", "the new methods of chromosome - selective sequencing of maternal plasma cell - free fetal dna ( cfdna ) in noninvasive prenatal testing ( nipt ) are valuable especially for our study group due to no risk of mtct . at present", "this interesting method is not widely available , and more data of this new method are needed ."], "abstract_text": ["<S> \n objective . to assess the prevalence of prenatal screening and of adverse outcome in high - risk pregnancies due to maternal hiv infection </S>", "<S> . study design . </S>", "<S> the prevalence of prenatal screening in 330 pregnancies of hiv - positive women attending the department for prenatal screening and/or during labour between january 1 , 2002 and december 31 , 2012 , was recorded . </S>", "<S> screening results were compared with the postnatal outcome and maternal morbidity , and mother - to - child transmission ( mtct ) was evaluated . results . </S>", "<S> one hundred of 330 women ( 30.5% ) had an early anomaly scan , 252 ( 74.5% ) had a detailed scan at 2022 weeks , 18 ( 5.5% ) had a detailed scan prior to birth , and three ( 0.9% ) had an amniocentesis . in seven cases ( 2.12% ) , </S>", "<S> a fetal anomaly was detected prenatally and confirmed postnatally , while in eight ( 2.42% ) an anomaly was only detected postnatally , even though a prenatal scan was performed . </S>", "<S> there were no anomalies in the unscreened group . </S>", "<S> mtct occurred in three cases ( 0.9% ) and seven fetal and neonatal deaths ( 2.1% ) were reported . </S>", "<S> conclusion . </S>", "<S> the overall prevalence of prenatal ultrasound screening in our cohort is 74.5% , but often the opportunity for prenatal ultrasonography in the first trimester is missed . in general , the aim should be to offer prenatal ultrasonography in the first trimester in all pregnancies . </S>", "<S> this allows early reassurance or if fetal disease is suspected , further steps can be taken . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Method", "3. Results", "4. Conclusion "], "sections": [["the majority of women living with hiv are in their reproductive years ( ages 1549 ) [ 1 , 2 ] .", "the dramatic decrease in the risk of mother - to - child hiv transmission ( mtct ) is leading to normality in the lives of couples affected by hiv , who want own children . in europe", ", the reduction in mtct to less than 1% is mainly due to highly active antiretroviral therapy ( haart ) .", "effective haart is resulting in suppressed viral load ( vl ) ; thus , a vaginal birth can be as safe as a planned caesarean section [ 3 , 4 ] .", "avoidance of breastfeeding and postnatal neonatal postexposure prophylaxis ( pep ) further supports the effective reduction in mtct [ 35 ] .", "the literature suggests that there is no increased rate of fetal malformations due to the hiv infection or haart [ 6 , 7 ] . a pregnant woman with hiv infection", "large studies of noninvasive prenatal screening have already indicated that it will lead to a decrease of invasive prenatal screening procedures such as amniocentesis ( ac ) or chorionic villi biopsy ( cvs ) .", "if invasive prenatal testing is necessary , it can be done , but in these circumstances , haart should be started prior to the procedure to suppress the vl below the limit of detection . in these cases", "current , evidence suggests that mtct is very unlikely ; however , studies reporting on the risk of mtct in invasive prenatal testing are limited due to small study size .", "haart is given during pregnancy for two reasons , first to women with an own indication for haart ( they require treatment for their own health ) and secondly to pregnant women starting therapy purely as a prophylactic treatment to reduce mtct .  ", "the aim of our study was to investigate if pregnant hiv - positive women get referred for special prenatal ultrasound screening services in our tertiary referral center , but also if and at what point the prenatal ultrasonography is performed .", "pregnant hiv - positive women usually have a combined antenatal care in a tertiary referral center and with their own gynaecologists .   as well as the prevalence of prenatal ultrasound screening , prenatal , and postnatal finding was recorded .", "we hypothesized that the fetal anomaly rate in women with hiv - infection is as low as in all other pregnancies ( 35% ) [ 12 , 13 ] ."], ["hiv - positive pregnant women who presented in our tertiary referral center between january 1 , 2002 and december 31 , 2012 were included in this retrospective cohort study .  ", "three categories were used : very preterm delivery ( 24 + 0 to 33 + 6 weeks of gestation ) , preterm delivery ( 34 + 0 to 36 + 6 weeks of gestation ) , and term delivery ( 37 weeks of gestation ) .  ", "all data regarding early prenatal screening ( as , e.g. , nuchal translucency measurements ) and fetal anomaly scan at 20 weeks of gestation or at first presentation in our center were recorded . only scans which were performed in our center", "were included , reflecting the fact that hiv - positive pregnant women are high risk pregnancies , and high - risk pregnancies are referred to a tertiary center or an equivalent specialized center for prenatal screening [ 1417 ] .", "an early anomaly scan was defined as a first trimester scan ; in the study period , the fetal nuchal thickness was assessed ; a formal nuchal translucency measurement was included if measured by appropriately qualified sonographers .", "a fetal anomaly scan was defined as a detailed scan in the second trimester ( usually between 20 to 22 weeks of gestation ) .", "all the scans performed at a later gestation in our department prior to birth are recorded separately as late scans in the third trimester .", "malformations were any fetal / neonatal disease , which required either surgery or special pediatric care including chromosomal anomalies .", "all cases with an ac , mtct , and any intrauterine or postnatal death were evaluated .", "maternal information included age , ethnicity , gestational age at delivery , gravidity and parity , haart already before the pregnancy , vl ( copies / ml ) , cd4 count ( cells/l ) prior to birth , and other risk factors such as coinfection with hcv .", "the last recorded vl prior to the delivery was used and classified in three risk groups . in the study , a vl below 50 is considered as negative / undetectable .", "the last cd4 count prior to birth was noted , and again three categories were used .", "the mode of delivery was classified as ( 1 ) planned caesarean section ; ( 2 ) in cases of rupture of membranes and/or contraction it was recorded as elective caesarean section in labour ; ( 3 ) emergency caesarean section ; ( 4 ) caesarean section after planned vaginal birth ; ( 5 ) vaginal birth ; ( 6 ) unplanned vaginal birth and ( 7 ) instrumental vaginal delivery . in the unit the first planned vaginal birth was recorded in 2009 . before that time , women were offered elective caesarean section at around 37 + 0 weeks of gestation [ 3 , 8 , 19 ] . with evidence for the safety of the vaginal birth with undetectable vl ,", "the policy in the unit shifted towards planned vaginal birth , and if caesarean section was offered in these cases , the delivery was delayed until > 37 weeks of gestation according to the german - austrian guidelines [ 3 , 4 , 8 ] .", "the following neonatal data were included : apgar score , arterial cord ph ( aph ) , cord base excess ( be ) and neonatal weight ( stratified according to 10th , 1090th and > 90th percentile ) .", "a weight below the 10th percentile was considered to be intrauterine growth retardation ( iugr ) .  ", "information about scan findings was obtained from the record of the ultrasound department , and further information was collected from maternal case notes , pediatric notes , and discharge letters .  ", "ethics approval for the retrospective study was obtained from the ethics committee at the j. w. goethe university , frankfurt ( number 30/13 ) . for categorical variables and nominal variables ,", "frequency tables were used for descriptive statistical analysis . for ordinal and quantitative data , mean and", "these data were further analyzed using the wilcoxon - mann - whitney test , kruskal - wallis test , spearman - correlation , chi - test , and fisher 's exact test as appropriate .", "in addition , multivariate logistic regression analysis was performed to identify factors associated with a woman having an early anomaly scan .  "], ["overall 330 pregnancies were recorded , with 322 singleton pregnancies ( 97.6% ) and in eight twin gestations ( 2.4% ) .", "one twin pregnancy was conceived due to ivf with first diagnosis of the hiv - infection in the early second trimester .", "there were 122 preterm deliveries ( 36.5% ) and 90 ( 26.9% ) of these were between 34 and 36 + 6 weeks of gestation .", "maternal and neonatal characteristics are presented in table 1 , stratified by pregnancy duration in table 2 .  ", "two thirds of women ( 66.4% ) were of african ethnicity . in one quarter of women ,", "more than three quarters 257 ( 77.4% ) of the births were elective caesarean section . in 29 cases ( 8.7% ) , women delivered vaginally .", "the cd4 count ( cells/l ) prior to birth was in the majority of 175 ( 62.5% ) 350 , in 76 ( 27% ) between 200 and 349 , and in 30 ( 10.7% ) < 200 . the vl ( copies / ml ) in most women 168 ( 55.8% ) was suppressed below 50 copies in 88 ( 29.2% ) 50399 and in 45 ( 15% ) 400 .", "one hundred and eight women ( 37.4% ) were on no haart treatment at the beginning of the pregnancy . in 25 ( 8.9% ) , a positive anti - hcv test was recorded .", "thirty newborns ( 9% ) were classified as below the 10th percentile . in 100 of the 330 pregnancies ( 30.5% )", "the nuchal translucency was measured in 67 ( 20.3% ) of the 330 cases ( nt median 1.22  mm ( range 0.63  mm ) ) . a multivariate analysis for factors influencing a woman having an early anomaly scan ( table 3 ) showed that african ethnicity and first diagnosis of hiv during the ongoing pregnancy were factors which significantly could be related to not having early prenatal ultrasound screening ( figure 1 ) .  ", "invasive testing ( ac ) was done in three ( 0.9% ) of 330 cases .", "only one case was done at 25 weeks in our department , and we started haart and performed the ac after vl was fully suppressed .", "two cases were done for advanced maternal age without control of vl and without specific precautions for example , haart , and both revealed a normal karyotype . in all of three cases , no mtct occurred . in the second trimester in 252 ( 74.5% ) of 330 , a detailed anomaly scan at 2022 weeks was done . in 18 ( 5.5% ) patients ,", "the scan was performed in the third trimester due to late presentation in our unit . in table 4 ,", "fetal and neonatal malformations as well as chromosomal anomalies are presented . in seven cases of 330 cases ( 2.1% ) , we diagnosed a fetal malformation .", "postnatally , all of the seven cases were confirmed , and eight further malformations and two cases with trisomy 21 were detected .", "the chromosomal anomalies were not suspected . both women , 33 and 39 years of age , had no early scan or biochemical screening but a scan in our unit ( late in the second trimester with no anomalies seen ) .", "there were three cases with a skin tag , one nevus sebaceous of the occiput , and one case with a socalled sucking blister on the hand , all considered to be minor .", "each of these cases had at least one scan in our department prior to the birth .", "however , the sucking blister and the nevus were leading to an upgrade in neonatal pep due to breaking down of protective skin barrier , and one newborn presented with a small omphalocele which was not seen prior to birth .", ". the overall fetal malformation rate ( including the minor anomalies ) was 4.5% .   in table 5 ,", "the fetal and postnatal mortalities are recorded . in our cohort , we had six cases of intrauterine or postnatal loss and all were born by caesarean section .", "all of the three newborns were delivered by caesarean section , and all were preterm ( 33 + 6 , 36 + 3 and 36 + 4 weeks of gestation ) . in all cases ,", "the vl was detectable , all women were on haart , and one woman was coinfected with hcv .", "one woman had already a vertically infected child , and she had a poor compliance ."], ["there are conflicting results regarding the risks for hiv - positive mothers for possible adverse effects in their pregnancies [ 5 , 6 ] . in our study", ", we confirm the low fetal malformation rate of 4.5% in women living with hiv .", "there are different national registers collecting data on haart and pregnancy outcome ( e.g. , apr : antiviral pregnancy registry ; nshpc : national study of hiv in pregnancy and childhood ( uk ) ; ecs : european collaborative cohort ; epf  french perinatal cohort ) [ 4 , 2124 ] .", "these registers confirm the same malformation rate in women taking haart as in the general population ( 35% ) [ 12 , 13 ] .", "the postnatal anomalies were minor ones ( skin tag , sucking blister ) or missed due to minimal extend ( omphalocele ) .", "the two cases with postnatal trisomy 21 were missed prenatally but were not seen in typical screening periods . there were no anomalies in the unscreened population .", "a change in treatment policies is evident over the 11 years of the study , reflected in the changes in delivery mode over time and the gestational age at delivery .", "a high preterm delivery rate is confirmed by other groups . in our population , 26.9% are late preterm deliveries ( 3436 + 6 weeks of gestation ) and are mostly iatrogenic due to early caesarean section as in other studies and in the past .", "the updated national german - austrian guidelines now delay caesarean section to term in women with suppressed vl .", "the numbers of women with fully suppressed vl ( vl < 50 copies / ml ) ( p < 0.001 ) and cd4 cells 350 ( p > 0.20 ) prior to birth increased over the last years .  ", "the first is early screening which should take place between 11 + 0 and 14 + 0 weeks of gestation .", "this early screening was introduced by nicolaidis in 1992 as a combined method of screening ( including ultrasound screening and two maternal biochemical markers : free human chorionic gonadotropin ( free hcg ) and pregnancy - associated plasma protein a ( papp - a ) [ 2628 ] . in germany", "this test is not covered by the national health system and there for is paid by the woman herself . usually at that time", "an early anomaly ultrasound scan can be performed , which is covered by the health system .", "the second screening interval is the anomaly scan at 2022 weeks of gestation [ 17 , 29 ]", ".   the prevalence of first trimester screening of 97.5% in a low risk general population has been demonstrated .  ", "we demonstrate that prenatal screening is offered and available , but that the early screening interval is missed , as only 30.5% women get referred for early anomaly scan .", "even so some women may have chosen not to undergo testing for ethical and cultural reasons . as a limitation of our data", "it could be that the screening which is done at the community - based care is missed , but as indicated usually , it warrants a referral to a highly qualified and specially trained team [ 16 , 29 ] . in our study ,", "population the majority of 188 ( 66.4% ) women were of african origin , and in 79 ( 24.2% ) , the diagnosis hiv - infection occurred in the pregnancy , both factors were significantly related to having no early prenatal screening .", "tariq et al . are reporting on late booking for antenatal care in non - caucasian women compared with caucasian women regardless of time of diagnosis of hiv - infection .", "three cases of mtct are low ( 0.9% ) and confirmed by other groups ( reporting mtct rates of 0.1%1.3% ) [ 3 , 4 ] .", "however , looking back in our data , the viral control has improved dramatically over the last 11 years ; in 55.8% of all pregnancies , the vl is < 50", ".   national health systems vary , and a complete first trimester screening ( with inclusion of biochemical serum markers ) has not been established on a national basis for high risk pregnancies in some european countries . in our cohort , two cases of trisomy 21 occurred , and the question remains open if these two cases could have been traced in a complete first trimester screening . in one pregnancy , an ac was performed due to suspected chromosomal anomaly , which revealed a normal karyotype .", "due to the time required to initiate haart and to have a suppressed vl in hiv - positive pregnancies , invasive testing will be very likely to happen in the second trimester which will then raise the difficult ethical questions about late termination of pregnancy when an abnormal result is obtained [ 32 , 33 ] .", "first trimester screening ( including maternal markers as free hcg and papp - a ) has been investigated in pregnant women living with hiv .", "some groups feel that maternal markers could be less reliable than those in hiv - negative women [ 34 , 35 ] . in our study data from 2002 to 2012 , in the first years , the nuchal translucency was assessed but not formally measured .", "this could be due to the delay in having certified specially trained sonographers involved .   in the future ,", "the new methods of chromosome - selective sequencing of maternal plasma cell - free fetal dna ( cfdna ) in noninvasive prenatal testing ( nipt ) are valuable especially for our study group due to no risk of mtct . at present", "this interesting method is not widely available , and more data of this new method are needed ."]]}
{"article_id": "PMC4451559", "article_text": ["diabetes mellitus is a global health problem and an important cause of mortality and morbidity in many countries .", "the trend of increasing diabetes prevalence seems to prevail among developing countries . in brazil , diabetes affected 11.3 million people in 2011 , and this number is likely to triple by 2030 .", "estimates suggest that the diabetes rate in less developed countries will increase by 69% between 2010 and 2030 .", "diabetes imposes a burden for society such as high socioeconomic costs that have an impact on productivity as well as life and health quality .", "this situation seems to be worse in developing countries , where the healthcare system often fails to meet demand .", "studies have concluded that a western dietary pattern , sedentary lifestyle , and genetic factors play a central role in diabetes development .", "the brazilian ministry of health has followed the world health organization 's recommendations and has taken some actions to monitor diabetes such as an annual telephone - based survey .", "socioeconomic disparities might contribute to some degree of heterogeneity in measures of prevalence between regions .", "a study demonstrated that diabetes prevalence across the brazilian states ranged from 11% to 25% , with an overall rate of 16% in 2001 .", "brasilia , the capital of brazil , is located in the central - west region of the country .", "the city has the highest human development index in brazil , but it has one of the highest levels of social inequality compared with other brazilian regions [ 11 , 12 ] . these characteristics of brasilia warrant further investigation in many aspects , including the health status of its population .", "thus , the goal of this study was to estimate the prevalence of diabetes and its associated risk factors in adults of brasilia , brazil .", "the present cross - sectional population - based study was conducted in brasilia , brazil , from february to may 2012 .", "the sample size was calculated based on an estimation of 16% of self - reported diabetes cases . considering a 95% confidence interval ( ci ) , precision of 2.25% , and a design effect of 1.8 ,", "we added 10% of the sample size to compensate for any eventual attrition , which resulted in a final sample of 2,019 individuals .", "participants were selected by a two - stage probability sampling process by cluster and were stratified by sex and age .", "a total of 220 census tracts were randomly selected from 3,886 urban tracts with more than 200 inhabitants .", "up to 10 households were selected from each census tract . in total , one adult per household was selected following the predefined quotas of sex and age to answer the interview . trained professionals surveyed all of the participants in their homes using a semistructured questionnaire . to ensure reliability , 20% of the interviews were audited by telephone . to test the understanding and acceptability of the questionnaire ,", "150 pilot interviews were held prior to data collection . the dependent variable was self - reported diabetes .", "independent variables included demographic characteristics ( age group , sex , marital status , living arrangements , and household location ) , socioeconomic characteristics ( level of education , occupation , and social class ) , chronic health conditions ( self - reported hypertension , depression , respiratory diseases , cardiovascular diseases , and other chronic diseases ) , access to healthcare ( health insurance , medical consultation , and hospitalization ) , and perceived health status ( mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) .", "the stratification was based on the brazilian criterion of economic classification , which defines five classes , with  a  being the wealthiest group and  e  being the poorest . in all of the analyses ,", "self - reported diabetes prevalence in the population was then calculated at a 95% ci . to identify factors related to diabetes prevalence , we calculated prevalence ratios ( pr ) using bivariate analysis and calculated the adjusted pr by a poisson regression model with robust variance . in this model ,", "we preferred to use this more conservative model that included all of the variables to allow for better confounding adjustment .", "other models that included only the most significant variables were tested and did not change the significance of the variables .", "associations were considered to be statistically significant when p < 0.05 . the stata software version 10.1 was used for all of the calculations .", "approximately 60% of the participants were women , and 57% were aged between 35 and 60 years .", "most of the participants belonged to economic class  c ,  had completed high school , were married or cohabitating , lived with at least one more person in the household , and dwelled in a satellite town .", "diabetes was self - reported by 10.1% ( 95% ci : 8.5%11.6% ) of the adult population in brasilia .", "table 1 depicts diabetes prevalence and prevalence ratios ( pr ) before and after adjustment by poisson regression .", "the age group of 3565 years , hypertension , respiratory disease , cardiovascular disease , and pain / discomfort were significantly associated with diabetes .", "sex , marital status , living arrangements , social class , education level , employability , living location , health insurance , medical consultation , hospitalization , physical mobility , self - care , usual activities , and anxiety / depression revealed no significant association . \n", "figure 2 illustrates differences in diabetes prevalence between all persons and the population with comorbidities .", "diabetes prevalence in the age range 3065 years is higher among individuals with cardiovascular disease , followed by those with hypertension and those with respiratory diseases .", "this result suggests that the likelihood of diabetes increases with age and is greater in persons with comorbidities .", "an age of 35 years and over , presence of pain or discomfort , cardiovascular disease , hypertension , and respiratory disease were positively associated with diabetes in the adult population of brasilia", ". the main limitations of our study were the self - reported assessments of the primary outcome and independent variables .", "self - reported diabetes might be a source of bias because individuals need to be aware of the diagnosis prior to answering , which could result in disease underestimation .", "however , performing a clinical test for diagnosing diabetes is not always possible in population - based studies .", "thus , self - reported answers regarding diabetes have been a common practice according to the literature [ 17 , 18 ] .", "another shortcoming was the cross - sectional design of the study , which hampers a causal relationship between diabetes and the significantly associated factors identified herein .", "a previous population - based study developed in brazil in 2008 used telephone interviews to investigate self - reported diabetes prevalence and found low prevalence rates in brasilia .", "another study found that brasilia was the region with the highest diabetes prevalence compared with other brazilian regions from 2002 to 2007 .", "research identified a significant increase in self - reported diabetes in the brazilian population because it ascended from 3.3% in 1998 to 5.3% in 2008 . in south and central america ,", "the estimated diabetes prevalence in 2013 was 8.0% ; brazil demonstrated the highest prevalence , followed by colombia and argentina .", "the variability of diabetes prevalence may be due to a poorer diet and a lack of physical activity , or it could be related to better access to diagnostic testing . as expected , our results demonstrated that the likelihood of having diabetes increases with age . from a healthcare policy perspective , diabetes prevention and management programs should target young people and not only the elderly population .", "diabetes prevalence was higher among individuals with cardiovascular disease , hypertension , and respiratory disease compared with the general population .", "there is convincing evidence of the association between diabetes and hypertension , which increases the risk of a cardiovascular event .", "a 2003 study conducted in so luis , a city located in one of the poorest areas of brazil , observed a positive association between diabetes and hypertension . a cross - sectional study conducted between 2004 and 2005 in so jose do rio preto , a city in the brazilian southeast region , revealed that the diabetes prevalence was almost threefold higher in a population of hypertensive individuals compared with the general population . a cohort study performed in women between 1988 and 1996 throughout 11 states in the united states found that chronic obstructive pulmonary disease was a diabetes risk factor . a retrospective cohort study conducted in northern", "california reported that individuals with diabetes are at a greater risk of developing asthma , chronic obstructive pulmonary disease , fibrosis , and pneumonia .", "in contrast , a systematic review of 10 studies suggests that growing up in a socioeconomically disadvantaged environment may contribute to diabetes in later life .", "an australian study also described a positive association between socioeconomic variables and diabetes in adults aged 45 years and over .", "the perceived health dimensions physical mobility , self - care , usual activities , and anxiety / depression were not associated with diabetes in our sample . in 2012 , a literature review found that diabetes was considered a potential risk factor for the poor performance of daily life activities among individuals aged 50 years and over . a study conducted with older adult", "other than depression , this finding might depict an association between diabetes and activities of daily living , which may be developed at older ages .", "diabetes is a common health condition in adults living in brasilia and is positively associated with older age , cardiovascular disease , hypertension , respiratory disease , and presence of pain or discomfort ."], "abstract_text": ["<S> \n aim . </S>", "<S> the aim of this study was to estimate the prevalence of diabetes and its associated risk factors in adults from brasilia , brazil . methods . </S>", "<S> the present cross - sectional population - based study consisted of interviews with individuals aged 1865 years . </S>", "<S> participants were selected through two - stage probability sampling by clusters and stratified by sex and age . </S>", "<S> demographic and clinical data were collected directly with participants from february to may 2012 . </S>", "<S> self - reported diabetes prevalence was calculated at a 95% confidence interval ( ci ) . </S>", "<S> prevalence ratios ( pr ) were adjusted by poisson regression with robust variance . </S>", "<S> results . in all , 1,820 individuals were interviewed . </S>", "<S> diabetes prevalence in the adult population of brasilia was 10.1% ( 95% ci , 8.5%11.6% ) . </S>", "<S> variables associated with diabetes were an age between 35 and 49 years ( pr = 1.83 ; 95% ci , 1.192.82 ) or 50 and 65 years ( pr = 1.95 ; 95% ci , 1.173.23 ) , hypertension ( pr = 4.04 ; 95% ci , 2.666.13 ) , respiratory disease ( pr = 1.67 ; 95% ci , 1.112.50 ) , cardiovascular disease ( pr = 1.74 ; 95% ci , 1.152.63 ) , and pain / discomfort ( pr = 1.71 ; 95% ci , 1.212.41 ) . conclusion . </S>", "<S> diabetes is a prevalent condition in adults living in brasilia , and disease risk increases with age and comorbidities . </S>", "<S> future health policies should focus on screening programs and prevention for the more vulnerable groups . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["diabetes mellitus is a global health problem and an important cause of mortality and morbidity in many countries .", "the trend of increasing diabetes prevalence seems to prevail among developing countries . in brazil , diabetes affected 11.3 million people in 2011 , and this number is likely to triple by 2030 .", "estimates suggest that the diabetes rate in less developed countries will increase by 69% between 2010 and 2030 .", "diabetes imposes a burden for society such as high socioeconomic costs that have an impact on productivity as well as life and health quality .", "this situation seems to be worse in developing countries , where the healthcare system often fails to meet demand .", "studies have concluded that a western dietary pattern , sedentary lifestyle , and genetic factors play a central role in diabetes development .", "the brazilian ministry of health has followed the world health organization 's recommendations and has taken some actions to monitor diabetes such as an annual telephone - based survey .", "socioeconomic disparities might contribute to some degree of heterogeneity in measures of prevalence between regions .", "a study demonstrated that diabetes prevalence across the brazilian states ranged from 11% to 25% , with an overall rate of 16% in 2001 .", "brasilia , the capital of brazil , is located in the central - west region of the country .", "the city has the highest human development index in brazil , but it has one of the highest levels of social inequality compared with other brazilian regions [ 11 , 12 ] . these characteristics of brasilia warrant further investigation in many aspects , including the health status of its population .", "thus , the goal of this study was to estimate the prevalence of diabetes and its associated risk factors in adults of brasilia , brazil ."], ["the present cross - sectional population - based study was conducted in brasilia , brazil , from february to may 2012 .", "the sample size was calculated based on an estimation of 16% of self - reported diabetes cases . considering a 95% confidence interval ( ci ) , precision of 2.25% , and a design effect of 1.8 ,", "we added 10% of the sample size to compensate for any eventual attrition , which resulted in a final sample of 2,019 individuals .", "participants were selected by a two - stage probability sampling process by cluster and were stratified by sex and age .", "a total of 220 census tracts were randomly selected from 3,886 urban tracts with more than 200 inhabitants .", "up to 10 households were selected from each census tract . in total , one adult per household was selected following the predefined quotas of sex and age to answer the interview . trained professionals surveyed all of the participants in their homes using a semistructured questionnaire . to ensure reliability , 20% of the interviews were audited by telephone . to test the understanding and acceptability of the questionnaire ,", "150 pilot interviews were held prior to data collection . the dependent variable was self - reported diabetes .", "independent variables included demographic characteristics ( age group , sex , marital status , living arrangements , and household location ) , socioeconomic characteristics ( level of education , occupation , and social class ) , chronic health conditions ( self - reported hypertension , depression , respiratory diseases , cardiovascular diseases , and other chronic diseases ) , access to healthcare ( health insurance , medical consultation , and hospitalization ) , and perceived health status ( mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) .", "the stratification was based on the brazilian criterion of economic classification , which defines five classes , with  a  being the wealthiest group and  e  being the poorest . in all of the analyses ,", "self - reported diabetes prevalence in the population was then calculated at a 95% ci . to identify factors related to diabetes prevalence , we calculated prevalence ratios ( pr ) using bivariate analysis and calculated the adjusted pr by a poisson regression model with robust variance . in this model ,", "we preferred to use this more conservative model that included all of the variables to allow for better confounding adjustment .", "other models that included only the most significant variables were tested and did not change the significance of the variables .", "associations were considered to be statistically significant when p < 0.05 . the stata software version 10.1 was used for all of the calculations ."], ["approximately 60% of the participants were women , and 57% were aged between 35 and 60 years .", "most of the participants belonged to economic class  c ,  had completed high school , were married or cohabitating , lived with at least one more person in the household , and dwelled in a satellite town .", "diabetes was self - reported by 10.1% ( 95% ci : 8.5%11.6% ) of the adult population in brasilia .", "table 1 depicts diabetes prevalence and prevalence ratios ( pr ) before and after adjustment by poisson regression .", "the age group of 3565 years , hypertension , respiratory disease , cardiovascular disease , and pain / discomfort were significantly associated with diabetes .", "sex , marital status , living arrangements , social class , education level , employability , living location , health insurance , medical consultation , hospitalization , physical mobility , self - care , usual activities , and anxiety / depression revealed no significant association . \n", "figure 2 illustrates differences in diabetes prevalence between all persons and the population with comorbidities .", "diabetes prevalence in the age range 3065 years is higher among individuals with cardiovascular disease , followed by those with hypertension and those with respiratory diseases .", "this result suggests that the likelihood of diabetes increases with age and is greater in persons with comorbidities ."], ["an age of 35 years and over , presence of pain or discomfort , cardiovascular disease , hypertension , and respiratory disease were positively associated with diabetes in the adult population of brasilia", ". the main limitations of our study were the self - reported assessments of the primary outcome and independent variables .", "self - reported diabetes might be a source of bias because individuals need to be aware of the diagnosis prior to answering , which could result in disease underestimation .", "however , performing a clinical test for diagnosing diabetes is not always possible in population - based studies .", "thus , self - reported answers regarding diabetes have been a common practice according to the literature [ 17 , 18 ] .", "another shortcoming was the cross - sectional design of the study , which hampers a causal relationship between diabetes and the significantly associated factors identified herein .", "a previous population - based study developed in brazil in 2008 used telephone interviews to investigate self - reported diabetes prevalence and found low prevalence rates in brasilia .", "another study found that brasilia was the region with the highest diabetes prevalence compared with other brazilian regions from 2002 to 2007 .", "research identified a significant increase in self - reported diabetes in the brazilian population because it ascended from 3.3% in 1998 to 5.3% in 2008 . in south and central america ,", "the estimated diabetes prevalence in 2013 was 8.0% ; brazil demonstrated the highest prevalence , followed by colombia and argentina .", "the variability of diabetes prevalence may be due to a poorer diet and a lack of physical activity , or it could be related to better access to diagnostic testing . as expected , our results demonstrated that the likelihood of having diabetes increases with age . from a healthcare policy perspective , diabetes prevention and management programs should target young people and not only the elderly population .", "diabetes prevalence was higher among individuals with cardiovascular disease , hypertension , and respiratory disease compared with the general population .", "there is convincing evidence of the association between diabetes and hypertension , which increases the risk of a cardiovascular event .", "a 2003 study conducted in so luis , a city located in one of the poorest areas of brazil , observed a positive association between diabetes and hypertension . a cross - sectional study conducted between 2004 and 2005 in so jose do rio preto , a city in the brazilian southeast region , revealed that the diabetes prevalence was almost threefold higher in a population of hypertensive individuals compared with the general population . a cohort study performed in women between 1988 and 1996 throughout 11 states in the united states found that chronic obstructive pulmonary disease was a diabetes risk factor . a retrospective cohort study conducted in northern", "california reported that individuals with diabetes are at a greater risk of developing asthma , chronic obstructive pulmonary disease , fibrosis , and pneumonia .", "in contrast , a systematic review of 10 studies suggests that growing up in a socioeconomically disadvantaged environment may contribute to diabetes in later life .", "an australian study also described a positive association between socioeconomic variables and diabetes in adults aged 45 years and over .", "the perceived health dimensions physical mobility , self - care , usual activities , and anxiety / depression were not associated with diabetes in our sample . in 2012 , a literature review found that diabetes was considered a potential risk factor for the poor performance of daily life activities among individuals aged 50 years and over . a study conducted with older adult", "other than depression , this finding might depict an association between diabetes and activities of daily living , which may be developed at older ages ."], ["diabetes is a common health condition in adults living in brasilia and is positively associated with older age , cardiovascular disease , hypertension , respiratory disease , and presence of pain or discomfort ."]]}
{"article_id": "PMC4620262", "article_text": ["natural killer t ( nkt ) cells are innate - like lymphocytes typified by coexpression of receptors characteristic of natural killer and conventional t cells . as such , murine nkt cells generally bear ly49 receptors , nkg2 family of receptors , cd94 , and nk1.1 ( the latter only being expressed in specific strains , including the commonly used c57bl/6 ) .", "human nkt cells often express similar surface molecules including cd56 , cd161 , cd94 , nkg2d , and nkg2a .", "both human and mouse nkt cells display a variety of stimulatory and inhibitory t cell - associated receptors and ligands ( e.g. , cd28 and cd154 ) , whose expression depends on the activation status of the cell .", "finally , both human and murine nkt populations include cd4 and cd4cd8 ( double negative ; dn ) subpopulations ; while cd8 nkt cells are found in humans , they are rare in mice . the t cell receptors ( tcrs ) expressed by nkt cells recognize the conserved and nonpolymorphic mhc class i - like molecule , cd1d . unlike classical mhc class i - like molecules , the expression of cd1d", "is largely restricted to cells of bone marrow origin including antigen presenting cells ( apcs ) such as dendritic cells ( dcs ) , macrophages , and b cells .", "furthermore , the cd1d molecule ( via heterodimerization with 2-microglobulin ) specializes in displaying lipid moieties rather than protein polypeptides .", "importantly , intact expression of cd1d is critical for the development of nkt cell populations , as cd1d/ mice are devoid of these cells .", "nkt cells are further subclassified into type i or ii lineages , depending on the composition of their tcr and the cd1d - presented glycolipid antigens to which they respond .", "type i or invariant nkt ( inkt ) cells express canonical tcr chains comprised of specific gene segments ( v14-j18 in mice and v24-j18 in humans ) that preferentially pair with specific tcr chains ( v8 , v7 , or v2 in mice and v11 in humans ) .", "these invariant tcr pairings confer reactivity to cd1d and a restricted array of presented glycolipid antigens .", "the dependence of inkt cells on the v14-j18-comprised tcr is demonstrated by v14 tcr transgenic mice , in which a higher frequency and number of inkt cells are observed , and also j18/ mice , in which no mature inkt cells develop . despite the conserved use of the invariant tcr , inkt cell populations are phenotypically ( e.g. , presence or absence of cd4 expression ) and functionally ( e.g. , preferential production of certain cytokines , such as il-17 ) diverse .", "the prototypical ( and first discovered ) inkt cell stimulatory glycolipid , alpha - galactosylceramide ( -galcer ) , was identified during a screening for compounds from marine sponges ( agelas species ) with antitumor activity . since this initial discovery ,", "a number of naturally occurring and synthetic lipid antigens have been described to bind cd1d and activate inkt cells .", "these cells are now typically identified using cd1d tetramers loaded with -galcer or its synthetic analogs ( e.g. , pbs-57 ; ) .", "in contrast , type ii or variant nkt ( vnkt ) cells bear a more diverse array of tcr and  chains and have been shown to recognize sulfatide moieties presented by cd1d .", "more recently , type ii nkt cells have also begun to be better characterized through development of cd1d tetramers loaded with sulfatide [ 9 , 10 ] , but these cells are still less well characterized than their invariant brethren . given", "that far more is known regarding the antitumor activity of inkt cells , we will predominantly focus our attention on these cells .", "inkt cells develop in the thymus , by originating from cd4cd8 double positive ( dp ) thymocytes .", "positive selection of inkt cells is mediated by homotypic interactions of dp cells and recognition of glycolipid antigen - cd1d complexes [ 1114 ] ; however , the nature of the self - antigens involved in this process remains somewhat elusive", ". like conventional t cells , maturation of inkt cells at the dp stage and beyond depends on the ability to construct a functional tcr and intact signaling . as such ,", "inkt cells are profoundly diminished or absent in mice lacking expression of rag , cd3 , lck , zap-70 , slp-76 , itk , lat , or vav [ 1521 ] .", "transcriptionally , development of inkt cells at the dp stage is regulated by the transcription factor rort , which prolongs the survival of dp thymocytes by upregulating bcl - xl , to allow sufficient time for distal tcr gene segment rearrangements to occur [ 22 , 23 ] .", "more recent studies have shown that heb , the e protein family of basic helix - loop - helix transcription factors , regulates inkt cell development by regulating rort and bcl - xl mrna .", "finally , the absence of the transcription factor runx1 also blocks inkt cell development at the earliest detectable inkt cell - committed subset .", "inkt cell development at the dp stage also critically depends on the signals generated by engagement of the signaling lymphocyte activation molecule ( slam ) family of surface receptors , which are expressed on developing inkt cells , as well as conventional dp thymocytes .", "slam family receptor signaling is transduced by the adaptor molecule sap ( slam - associated protein ) , which in turn binds to the tyrosine kinase fyn , and results in propagation of a phosphorylation signal [ 25 , 26 ] . accordingly , inkt cells fail to develop in mice and humans bearing mutations in the gene that encodes for sap [ 2729 ] , in mice lacking fyn or expressing a mutant version of sap that can not bind fyn [ 23 , 30 ] , in mice in which both ly108 and slam signaling are simultaneously disrupted , and in those lacking the transcription factor cmyb ( which is necessary for appropriate expression of sap and certain slam family members ) . taken together", ", these studies establish the importance of the slam - sap - fyn signaling axis in inkt cell development .", "following positive selection , inkt cells undergo distinct stages of maturation that are characterized by the sequential acquisition of cd24 , cd44 , and nk1.1 : cd24cd44nk1.1 ( stage 0 ) , cd24cd44nk1.1 ( stage 1 ) , cd24cd44nk1.1 ( stage 2 ) , and finally cd24cd44nk1.1 ( stage 3 ) . as these cells progress through these developmental stages", ", they begin to upregulate nk cell markers ( e.g. , nkg2d and ly49 receptors ) , cd69 , and cd122 and acquire distinct effector functions ( e.g. , production of il-4 , ifn- , perforin , and granzymes ) .", "one of the key regulators of inkt cell development and acquisition of an effector / memory phenotype and functions is the broad complex tramtrack bric - a - brac - zinc finger transcription factor plzf , whose expression is highest in stage 0 and 1 populations [ 36 , 37 ] .", "plzf - deficient animals exhibit a severe reduction in inkt cell number and plzf - deficient inkt cells fail to cosecrete th1 and th2 cytokines upon stimulation [ 36 , 37 ] .", "recently , it was demonstrated that the lethal-7 microrna posttranscriptionally regulate plzf expression and inkt cell effector functions .", "the transcription factor t - bet is indispensable for the final maturation stages of inkt cells [ 39 , 40 ] and absence of this transcription factor results in reduced inkt cell numbers due to developmental blockade at stage 2 .", "t - bet - deficient inkt cells fail to proliferate in response to il-15 as they lack surface expression of cd122 , a component of the il-15 receptor .", "in addition , t - bet - deficient inkt cells fail to produce ifn- in response to tcr stimulation and exhibit defective cytolytic activity [ 39 , 40 ] as t - bet directly regulates the activation of genes associated with mature inkt cell functions , such as perforin , cd178 , and ifn- . as inkt cells progress to stage 1 , a proportion of cells downregulate cd4 , giving rise to dn inkt cells .", "generation of the cd4 inkt cell lineage and production of th2-type cytokines is critically regulated by the transcription factor gata-3 .", "similar to plzf - deficient inkt cells , gata-3 deficient inkt cells fail to produce th1 or th2 cytokines in response to -galcer .", "recent studies have identified a unique subpopulation of nk1.1cd4 inkt cells that are transcriptionally regulated by rort and capable of producing large quantities of il-17 upon stimulation . as such ,", "inkt cells are also sometimes classified into nkt1 , nkt2 , and nkt17 based on their cytokine production profiles and respective expression of t - bet , gata-3 , and rort [ 43 , 44 ] .", "finally , mechanistic target of rapamycin ( mtor ) signaling has also been shown to be important for inkt cell lineage diversification and acquisition of effector functions [ 4548 ] , and loss of mtor2 may result in loss of nkt17 cells . taken together , these recent studies provide new insights into the transcriptional regulation of inkt cell maturation and functional differentiation .", "the importance of inkt cells in mediating protection against tumors is highlighted by several findings .", "first , a number of independent studies have shown a decrease in the number of inkt cells in the peripheral blood of patients with a variety of cancers and even precancerous myelodysplastic syndromes [ 4951 ] . moreover ,", "the inkt cells that persist appear to have decreased proliferative and functional responses [ 5254 ] .", "interestingly , an increased frequency of peripheral blood inkt cells in cancer patients portends a more favorable response to therapy [ 55 , 56 ] . while these observations identify an association between inkt cell numbers and/or function and development of malignancy , they do not provide a direct causal link", "this link has been established in a number of mouse studies in which the biology of the host and initiation of tumors can be more systematically manipulated via gene knockouts , antibody depletion strategies , and adoptive transfer of various lymphocyte populations into cancer - predisposed or tumor - challenged hosts . in mice that are prone to development of tumors due to loss of one allele of a tumor suppressor ( p53+/ ) , absence of inkt cells ( by virtue of genetic knockout of the j18 gene segment or cd1d ) results in earlier and more frequent development of tumors and thus shorter survival , when compared to inkt - sufficient littermates .", "similarly , treatment of cd1d/ and j18/ mice with a carcinogen resulted in increased incidence and earlier onset of tumors in comparison to treated wild type mice .", "conversely , administration of -galcer to mice controlled the growth and metastasis of adoptively transferred [ 59 , 60 ] or carcinogen - induced [ 61 , 62 ] or spontaneous   tumors .", "moreover , adoptive transfer of inkt cells into j18/ inkt cell - deficient mice prevented the growth of subcutaneous sarcomas .", "finally , adoptive transfer of small numbers of purified inkt cells into lymphocyte - deficient nod - scid - il2r/ ( nsg ) mice was sufficient to protect mice from challenge with a cd1d tumor .", "these findings collectively argue that inkt cells play a central and nonredundant role in the response to tumors .", "further studies would shed light on the mechanisms by which inkt cells exert these antitumor effects .", "engagement of the invariant tcr by cd1d / glycolipid antigen complexes results in inkt cell activation , an event that is typified by rapid and robust production of a variety of cytokines and chemokines , including  but not limited to  il-2 , il-4 , il-10 , il-13 il-17 , ifn- , tnf , tgf , gm - csf , rantes , eotaxin , mip-1 , and mip-1 [ 65 , 66 ] . the nature and magnitude of the inkt cell cytokine response", "depend on the glycolipid antigen ; for example , -galcer - mediated inkt cell activation elicits a strong ifn--dominated cytokine response , while och ( a synthetic analog of -galcer with a truncated lipid chain ) elicits a response with significantly higher level of il-4 production .", "the rapidity of this cytokine response is attributed to the semiactivated state of inkt cells and the presence of preformed cytosolic mrna for a variety of cytokines .", "indeed , administration of -galcer to inkt cell - sufficient , but not inkt cell - deficient , mice results in polyclonal activation of conventional t , b , and nk cells within 3 - 4 hours   and also eventually leads to the mobilization of macrophages and neutrophils .", "intriguingly , it was previously believed that mammalian species are incapable of producing glycolipids ( such as -galcer ) , in which the sugar moiety is attached via an o - linkage to the ceramide backbone in an alpha - anomeric configuration . despite the absence of -glucosyl or -galactosyl transferases in mammals , recent findings indicate that a small percentage of the glycolipids that are constitutively presented by mammalian cd1d are indeed -anomeric . whether the percentage of cd1d - presented -anomeric glycolipids is altered in tumor tissues", "nonetheless , following encounter with cd1d / antigen complexes displayed by apcs , inkt cells not only produce cytokines but also upregulate surface expression of cd154 ( see figure 1(a ) ) .", "ligation of apc - expressed cd40 is especially important for mediating subsequent maturation and functional activation of dcs , subsequent upregulation of cd80 and cd86 , and amplified production of ifn- [ 72 , 73 ] .", "in addition , the ligation of the chemokine receptor cxcr6 on inkt cells by cxcl16 expressed on apcs also provides costimulatory signals resulting in robust -galcer - induced inkt cell activation .", "importantly , matured dcs are potent producers of il-12 , which induces sustained ifn- production by inkt cells [ 7577 ] .", "the importance of inkt cells in il-12-mediated tumor rejection was effectively demonstrated by the defective clearance of a variety of tumors in j18/ mice .", "mature dcs also support the priming and activation of cd8 t cells , culminating in optimal effector and memory cell formation [ 72 , 78 ] .", "finally , the sustained release of ifn- by inkt cells leads to activation and proliferation of nk cells and nk cell secretion of ifn-. the combination of cytokines ( e.g. , il-2 , il-12 , and ifn- ) as a result of inkt cell activation also leads to upregulation of death - inducing ligands ( e.g. , cd178 or cd253 ) on nk cells and cd8 t cells [ 79 , 80 ] .", "these sequential activation events are believed to be critical for the -galcer - induced inkt cell - mediated antitumor effects [ 76 , 81 , 82 ] . as such , inkt cells not only bridge the activation of innate and adaptive immunity , but also indirectly potentiate the antitumor activity of other cytotoxic effector lymphocytes .", "tumor establishment and growth are believed to be intricately modulated by a myriad of soluble and contact - derived signals obtained from the tumor microenvironment ( tme ) , which consists of the tumor cells themselves , tumor - infiltrating lymphocytes ( tils ) , and stromal cells that communicate in a dynamic and bidirectional manner .", "in addition to their indirect modulation of other effector lymphocyte populations , inkt cells may also regulate tumor growth via their effects on the tme ( see figure 1(b ) ) . indeed following intravenous administration ,", "inkt cells were shown to represent a significant percentage of the tils in patients with head and neck carcinomas [ 83 , 84 ] .", "importantly , higher frequency of tumor - infiltrating inkt cells correlated with overall and disease - free survival as an independent prognostic factor in primary colorectal cancer patients   and with tumor regression in head and neck carcinomas .", "conversely , in patients with primary hepatocellular or metastatic cancer , cd4 inkt cells that produced high levels of th2-type cytokines and had low cytolytic activity were enriched within the tumor and appeared to inhibit the expansion of antigen - specific cd8 t cells , suggesting that these particular inkt cells may contribute to generate an immunosuppressive microenvironment . in experimental studies ,", "cotransfer of human monocytes and inkt cells to tumor - bearing nod - scid mice suppressed tumor growth when compared with mice that received monocytes alone .", "importantly , inkt cells can target tumor supportive cells such as tumor - associated macrophages ( tams ) , a highly plastic monocyte - derived subset of inflammatory cells that can exert immunosuppressive functions , and promote tumor proliferation and matrix turnover [ 88 , 89 ] .", "indeed tams are known to produce il-6 , a cytokine that appears to promote the proliferation of many solid tumors , including neuroblastomas and breast and prostate carcinomas .", "found that macrophage density correlated positively with microvessel counts and negatively with patient relapse - free survival .", "since tams cross - present neuroblastomaderived endogenous cd1d ligand(s ) , they can be specifically recognized and killed by inkt cells in an il-15-dependent process .", "other potential inkt cell tme targets include myeloid - derived suppressor cells ( mdscs ) .", "mdscs have been found to accumulate in the blood , lymph nodes , and bone marrow and at tumor sites in most patients and experimental animals with cancer and inhibit both adaptive and innate immunity .", "the absence of inkt cells in mice during influenza virus infection resulted in the expansion of mdscs , high viral titer , and increased mortality .", "the adoptive transfer of inkt cells abolished the suppressive activity of mdscs and restored virus - specific immune responses , resulting in reduced viral titers and increased rates of host survival .", "thus , certain populations of inkt cells may help alter the tme via their effects on tams and mdscs , to help create a tumor - suppressive or immune - permissive milieu .", "in addition to their indirect control of tumor growth , inkt cells can mediate direct killing of tumor targets ( see figure 1(c ) ) .", "inkt cells alone , or in combination with nk cells , have been shown to kill a variety of tumor targets in vitro [ 6 , 93 , 94 ] .", "while this mechanism of killing appears to be dependent on the presence of stimulatory glycolipids and cd1d [ 95 , 96 ] , inkt cell cytotoxicity also appears to be triggered via ligation of nkg2d by target - expressed stress ligands .", "it remains to be seen whether mult1 , the newly identified shed form of high affinity nkg2d ligand that triggers nk - mediated tumor rejection in mice , also activates inkt cells .", "consistent with their direct cytotoxic capacity , inkt cells express perforin and granzymes , as well as cd178 [ 34 , 96 , 99 , 100 ] . in our hands ,", "blockade of cd1d - mediated lipid antigen presentation , disruption of t cell receptor ( tcr ) signaling , or loss of perforin expression was found to significantly reduce inkt cell killing in vitro .", "moreover , we demonstrated that inkt cells alone were sufficient for control of the growth of a t cell lymphoma in vivo that preferentially relies on perforin and the adaptor protein sap [ 64 , 69 ] .", "mechanistically , inkt cells rely on sap for formation of stable conjugates with the tumor targets as well as proper orientation of the lytic machinery at the immunological synapse . despite the majority of studies implicating inkt cells as having an antitumor role , a limited number of studies also implicate inkt cells as suppressing antitumor responses , but", "these paradoxic responses may be related to the level of tumor cd1d expression [ 102 , 103 ] .", "alternatively , these differences may stem from the fact that  contrary to the use of c57bl/6 mice in the previously discussed studies ", "these last two studies were performed in balb / c mice , in which there is a predominance of il-4-producing th2 phenotype inkt cells .", "interestingly , the antitumor responses of inkt cells may be regulated by the activity of type ii nkt cells .", "demonstrated that type ii variant nkt ( vnkt ) cells were sufficient for the downregulation of tumor immunosurveillance and relapse growth of a model fibrosarcoma in an antigen - dependent manner , while a second study found that activation of vnkt cells with sulfatide antigen could suppress the activation of inkt cells .", "conversely , type ii vnkt cells were , in at least one study , suggested to promote the antitumor activity of cpg oligodeoxynucleotides .", "this suppression appears to be mediated through a contact- and il-10-dependent mechanism [ 109 , 110 ] .", "indeed , induction of treg cells suppressed the protective effect of adoptive transfer of inkt cells into j18/ mice .", "consistent with these findings , depletion of treg cells or short - term elimination of their suppressive activity results in enhanced inkt cell - mediated antitumor responses and increased nk and cd8 t cell activation and ifn- production .", "interestingly , the ability of treg cells to suppress inkt cell proliferation depends on the degree of invariant tcr agonism , such that responses to weak ( e.g. , och ) , but not strong ( e.g. , -galcer ) , agonists were effectively suppressed . when viewed collectively , these findings suggest that inkt cells possess inherent capacity for direct cytotoxicity but their antigenic exposure may modulate whether their antitumor effects can be suppressed by treg and vnkt cells .", "given the preponderance of evidence suggesting that the activation of inkt cells provides protection against the growth and metastasis of a variety of tumors , safety of -galcer administration was examined in a phase i trial .", "while administration of -galcer was well tolerated at a range of doses , no clinical responses were observed in patients with advanced solid tumors . on the heels of this study ,", "nieda et al . showed that treatment of metastatic cancer patients with -galcer - pulsed immature monocyte - derived dcs resulted in dramatic increases in serum ifn- and il-12 and activation of nk and t cells in the majority of subjects .", "importantly , this phase i trial also documented reduction in tumor biomarkers and tumor necrosis in several patients .", "these findings were extended in a study of a small number of patients , in which the -galcer - pulsed dcs were matured prior to adoptive transfer .", "this study demonstrated a > 100-fold increase in blood inkt cell numbers in all patients , and this increase was long - lived ( > 6 months ) .", "a number of subsequent clinical trials , all with limited number of patients with advanced head and neck or non - small cell lung cancers , have since employed similar strategies of adoptive transfer of -galcer - pulsed apcs [ 115118 ] .", "collectively , these studies demonstrate increases in blood ifn- levels and inkt cells in some but not all patients , stabilization of disease in a few of the subjects , and absence of severe treatment - related toxicities . in a different approach ,", "chemotherapy - refractory 5 lymphoma patients were treated with autologous peripheral blood mononuclear cells ( pbmcs ) stimulated with anti - cd3 , il-2 , and ifn-. this ex vivo stimulation resulted in enrichment of nkt cells ( to ~20% on average ) , and this cell fraction was shown to possess the highest cytotoxic capacity in vitro .", "of the nine patients who received adoptive transfer of these cells , two showed partial responses and two others had stabilization of disease .", "two subsequent studies by motohashi et al . evaluated the adoptive transfer of ex vivo expanded inkt cell - enriched cells to patients with advanced cancer . in the first ,", "6 patients with advanced non - small cell lung cancer were treated with either a low or a high dose of ex vivo expanded inkt cells .", "of the 3 patients treated with the high dose , all had an increase in the frequency of ifn--producing pbmcs and 2 showed expansion of inkt cells . although no clinical responses were observed in this study , a follow - up trial of 17 patients with advanced head and neck cancers treated with a high dose of inkt cell - enriched autologous pbmcs showed a significant increase in ifn--producing pbmcs in 10 of 17 patients .", "importantly , while none of these patients displayed tumor regression , 5 had disease stabilization and the mean survival time for the subjects with higher frequencies of ifn--producing pbmcs was tripled above those with low percentages of ifn--producing pbmcs ( 29.3 versus 9.7 months ) .", "administered both in vitro expanded inkt cells and -galcer - pulsed apcs to patients with advanced head and neck squamous cell carcinomas .", "treatment increased the frequencies of inkt cells and ifn--producing pbmcs , and a partial clinical response or disease stabilization was observed in 7 of 8 patients . although the responses in these studies have not been profound , it must be noted that these inkt cell - based immunotherapies have all been conducted on patients with advanced malignancies  often those in whom standard chemotherapy , irradiation , and/or surgical excision treatments had failed .", "future studies of inkt cell - based immunotherapy may be able to take advantage of two recent technologies .", "as mentioned previously , many malignancies are associated with a decrease in the numbers and proliferative capacity of peripheral blood inkt cells . in order to circumvent the difficulty of being able to expand these infrequent and potentially defective cells from patients , watarai et al .", "generated induced pluripotent stem ( ips ) cells from mature inkt cells and then expanded large numbers of inkt cells from these established ips cells .", "ips - nkt cells generated in this fashion were demonstrated to be able to activate and expand antigen - specific cd8 t cell responses to limit the growth of leukemia in mice   without inducing graft versus host disease ( gvhd ) .", "described inkt cells engineered to express cars bearing specificity for gd2 , a highly expressed moiety on neuroblastoma cells . in their studies , they showed that inkt cells expanded from the pbmcs of healthy human donors and transduced with retroviral car constructs could protect humanized nsg mice against metastatic neuroblastoma without inducing gvhd .", "whether these two technologies could be combined to generate functional car - bearing ips - nkt cells remains to be seen .", "inkt cells are innate - like effector lymphocytes that not only are directly cytotoxic , but also possess the unique ability to nucleate the antitumor responses of other effector lymphocytes and alter the cellular and angiogenic makeup of the tumor microenvironment . as such , the promise of an effective inkt cell - based immunotherapy can only be realized by devising and evaluating strategies that simultaneously maximize each of these antitumor effector mechanisms . the challenge for the future will thus be to identify these strategies and apply them to tumors against which inkt cells wield the most optimal responses ."], "abstract_text": ["<S> natural killer t ( nkt ) cells are innate - like lymphocytes that were first described in the late 1980s . since their initial description , </S>", "<S> numerous studies have collectively shed light on their development and effector function . </S>", "<S> these studies have highlighted the unique requirements for the activation of these lymphocytes and the functional responses that distinguish these cells from other effector lymphocyte populations such as conventional t cells and nk cells . </S>", "<S> this body of literature suggests that nkt cells play diverse nonredundant roles in a number of disease processes , including the initiation and propagation of airway hyperreactivity , protection against a variety of pathogens , development of autoimmunity , and mediation of allograft responses . in this review , however , we focus on the role of a specific lineage of nkt cells in antitumor immunity . </S>", "<S> specifically , we describe the development of invariant nkt ( inkt ) cells and the factors that are critical for their acquisition of effector function . </S>", "<S> next , we delineate the mechanisms by which inkt cells influence and modulate the activity of other immune cells to directly or indirectly affect tumor growth </S>", "<S> . finally , we review the successes and failures of clinical trials employing inkt cell - based immunotherapies and explore the future prospects for the use of such strategies . </S>"], "labels": null, "section_names": ["1. Introduction", "2. iNKT Cell Development and Acquisition of Effector Function", "3. iNKT Cells and Antitumor Immunity", "4. iNKT Cell-Based Immunotherapy", "5. Concluding Remarks"], "sections": [["natural killer t ( nkt ) cells are innate - like lymphocytes typified by coexpression of receptors characteristic of natural killer and conventional t cells . as such , murine nkt cells generally bear ly49 receptors , nkg2 family of receptors , cd94 , and nk1.1 ( the latter only being expressed in specific strains , including the commonly used c57bl/6 ) .", "human nkt cells often express similar surface molecules including cd56 , cd161 , cd94 , nkg2d , and nkg2a .", "both human and mouse nkt cells display a variety of stimulatory and inhibitory t cell - associated receptors and ligands ( e.g. , cd28 and cd154 ) , whose expression depends on the activation status of the cell .", "finally , both human and murine nkt populations include cd4 and cd4cd8 ( double negative ; dn ) subpopulations ; while cd8 nkt cells are found in humans , they are rare in mice . the t cell receptors ( tcrs ) expressed by nkt cells recognize the conserved and nonpolymorphic mhc class i - like molecule , cd1d . unlike classical mhc class i - like molecules , the expression of cd1d", "is largely restricted to cells of bone marrow origin including antigen presenting cells ( apcs ) such as dendritic cells ( dcs ) , macrophages , and b cells .", "furthermore , the cd1d molecule ( via heterodimerization with 2-microglobulin ) specializes in displaying lipid moieties rather than protein polypeptides .", "importantly , intact expression of cd1d is critical for the development of nkt cell populations , as cd1d/ mice are devoid of these cells .", "nkt cells are further subclassified into type i or ii lineages , depending on the composition of their tcr and the cd1d - presented glycolipid antigens to which they respond .", "type i or invariant nkt ( inkt ) cells express canonical tcr chains comprised of specific gene segments ( v14-j18 in mice and v24-j18 in humans ) that preferentially pair with specific tcr chains ( v8 , v7 , or v2 in mice and v11 in humans ) .", "these invariant tcr pairings confer reactivity to cd1d and a restricted array of presented glycolipid antigens .", "the dependence of inkt cells on the v14-j18-comprised tcr is demonstrated by v14 tcr transgenic mice , in which a higher frequency and number of inkt cells are observed , and also j18/ mice , in which no mature inkt cells develop . despite the conserved use of the invariant tcr , inkt cell populations are phenotypically ( e.g. , presence or absence of cd4 expression ) and functionally ( e.g. , preferential production of certain cytokines , such as il-17 ) diverse .", "the prototypical ( and first discovered ) inkt cell stimulatory glycolipid , alpha - galactosylceramide ( -galcer ) , was identified during a screening for compounds from marine sponges ( agelas species ) with antitumor activity . since this initial discovery ,", "a number of naturally occurring and synthetic lipid antigens have been described to bind cd1d and activate inkt cells .", "these cells are now typically identified using cd1d tetramers loaded with -galcer or its synthetic analogs ( e.g. , pbs-57 ; ) .", "in contrast , type ii or variant nkt ( vnkt ) cells bear a more diverse array of tcr and  chains and have been shown to recognize sulfatide moieties presented by cd1d .", "more recently , type ii nkt cells have also begun to be better characterized through development of cd1d tetramers loaded with sulfatide [ 9 , 10 ] , but these cells are still less well characterized than their invariant brethren . given", "that far more is known regarding the antitumor activity of inkt cells , we will predominantly focus our attention on these cells ."], ["inkt cells develop in the thymus , by originating from cd4cd8 double positive ( dp ) thymocytes .", "positive selection of inkt cells is mediated by homotypic interactions of dp cells and recognition of glycolipid antigen - cd1d complexes [ 1114 ] ; however , the nature of the self - antigens involved in this process remains somewhat elusive", ". like conventional t cells , maturation of inkt cells at the dp stage and beyond depends on the ability to construct a functional tcr and intact signaling . as such ,", "inkt cells are profoundly diminished or absent in mice lacking expression of rag , cd3 , lck , zap-70 , slp-76 , itk , lat , or vav [ 1521 ] .", "transcriptionally , development of inkt cells at the dp stage is regulated by the transcription factor rort , which prolongs the survival of dp thymocytes by upregulating bcl - xl , to allow sufficient time for distal tcr gene segment rearrangements to occur [ 22 , 23 ] .", "more recent studies have shown that heb , the e protein family of basic helix - loop - helix transcription factors , regulates inkt cell development by regulating rort and bcl - xl mrna .", "finally , the absence of the transcription factor runx1 also blocks inkt cell development at the earliest detectable inkt cell - committed subset .", "inkt cell development at the dp stage also critically depends on the signals generated by engagement of the signaling lymphocyte activation molecule ( slam ) family of surface receptors , which are expressed on developing inkt cells , as well as conventional dp thymocytes .", "slam family receptor signaling is transduced by the adaptor molecule sap ( slam - associated protein ) , which in turn binds to the tyrosine kinase fyn , and results in propagation of a phosphorylation signal [ 25 , 26 ] . accordingly , inkt cells fail to develop in mice and humans bearing mutations in the gene that encodes for sap [ 2729 ] , in mice lacking fyn or expressing a mutant version of sap that can not bind fyn [ 23 , 30 ] , in mice in which both ly108 and slam signaling are simultaneously disrupted , and in those lacking the transcription factor cmyb ( which is necessary for appropriate expression of sap and certain slam family members ) . taken together", ", these studies establish the importance of the slam - sap - fyn signaling axis in inkt cell development .", "following positive selection , inkt cells undergo distinct stages of maturation that are characterized by the sequential acquisition of cd24 , cd44 , and nk1.1 : cd24cd44nk1.1 ( stage 0 ) , cd24cd44nk1.1 ( stage 1 ) , cd24cd44nk1.1 ( stage 2 ) , and finally cd24cd44nk1.1 ( stage 3 ) . as these cells progress through these developmental stages", ", they begin to upregulate nk cell markers ( e.g. , nkg2d and ly49 receptors ) , cd69 , and cd122 and acquire distinct effector functions ( e.g. , production of il-4 , ifn- , perforin , and granzymes ) .", "one of the key regulators of inkt cell development and acquisition of an effector / memory phenotype and functions is the broad complex tramtrack bric - a - brac - zinc finger transcription factor plzf , whose expression is highest in stage 0 and 1 populations [ 36 , 37 ] .", "plzf - deficient animals exhibit a severe reduction in inkt cell number and plzf - deficient inkt cells fail to cosecrete th1 and th2 cytokines upon stimulation [ 36 , 37 ] .", "recently , it was demonstrated that the lethal-7 microrna posttranscriptionally regulate plzf expression and inkt cell effector functions .", "the transcription factor t - bet is indispensable for the final maturation stages of inkt cells [ 39 , 40 ] and absence of this transcription factor results in reduced inkt cell numbers due to developmental blockade at stage 2 .", "t - bet - deficient inkt cells fail to proliferate in response to il-15 as they lack surface expression of cd122 , a component of the il-15 receptor .", "in addition , t - bet - deficient inkt cells fail to produce ifn- in response to tcr stimulation and exhibit defective cytolytic activity [ 39 , 40 ] as t - bet directly regulates the activation of genes associated with mature inkt cell functions , such as perforin , cd178 , and ifn- . as inkt cells progress to stage 1 , a proportion of cells downregulate cd4 , giving rise to dn inkt cells .", "generation of the cd4 inkt cell lineage and production of th2-type cytokines is critically regulated by the transcription factor gata-3 .", "similar to plzf - deficient inkt cells , gata-3 deficient inkt cells fail to produce th1 or th2 cytokines in response to -galcer .", "recent studies have identified a unique subpopulation of nk1.1cd4 inkt cells that are transcriptionally regulated by rort and capable of producing large quantities of il-17 upon stimulation . as such ,", "inkt cells are also sometimes classified into nkt1 , nkt2 , and nkt17 based on their cytokine production profiles and respective expression of t - bet , gata-3 , and rort [ 43 , 44 ] .", "finally , mechanistic target of rapamycin ( mtor ) signaling has also been shown to be important for inkt cell lineage diversification and acquisition of effector functions [ 4548 ] , and loss of mtor2 may result in loss of nkt17 cells . taken together , these recent studies provide new insights into the transcriptional regulation of inkt cell maturation and functional differentiation ."], ["the importance of inkt cells in mediating protection against tumors is highlighted by several findings .", "first , a number of independent studies have shown a decrease in the number of inkt cells in the peripheral blood of patients with a variety of cancers and even precancerous myelodysplastic syndromes [ 4951 ] . moreover ,", "the inkt cells that persist appear to have decreased proliferative and functional responses [ 5254 ] .", "interestingly , an increased frequency of peripheral blood inkt cells in cancer patients portends a more favorable response to therapy [ 55 , 56 ] . while these observations identify an association between inkt cell numbers and/or function and development of malignancy , they do not provide a direct causal link", "this link has been established in a number of mouse studies in which the biology of the host and initiation of tumors can be more systematically manipulated via gene knockouts , antibody depletion strategies , and adoptive transfer of various lymphocyte populations into cancer - predisposed or tumor - challenged hosts . in mice that are prone to development of tumors due to loss of one allele of a tumor suppressor ( p53+/ ) , absence of inkt cells ( by virtue of genetic knockout of the j18 gene segment or cd1d ) results in earlier and more frequent development of tumors and thus shorter survival , when compared to inkt - sufficient littermates .", "similarly , treatment of cd1d/ and j18/ mice with a carcinogen resulted in increased incidence and earlier onset of tumors in comparison to treated wild type mice .", "conversely , administration of -galcer to mice controlled the growth and metastasis of adoptively transferred [ 59 , 60 ] or carcinogen - induced [ 61 , 62 ] or spontaneous   tumors .", "moreover , adoptive transfer of inkt cells into j18/ inkt cell - deficient mice prevented the growth of subcutaneous sarcomas .", "finally , adoptive transfer of small numbers of purified inkt cells into lymphocyte - deficient nod - scid - il2r/ ( nsg ) mice was sufficient to protect mice from challenge with a cd1d tumor .", "these findings collectively argue that inkt cells play a central and nonredundant role in the response to tumors .", "further studies would shed light on the mechanisms by which inkt cells exert these antitumor effects .", "engagement of the invariant tcr by cd1d / glycolipid antigen complexes results in inkt cell activation , an event that is typified by rapid and robust production of a variety of cytokines and chemokines , including  but not limited to  il-2 , il-4 , il-10 , il-13 il-17 , ifn- , tnf , tgf , gm - csf , rantes , eotaxin , mip-1 , and mip-1 [ 65 , 66 ] . the nature and magnitude of the inkt cell cytokine response", "depend on the glycolipid antigen ; for example , -galcer - mediated inkt cell activation elicits a strong ifn--dominated cytokine response , while och ( a synthetic analog of -galcer with a truncated lipid chain ) elicits a response with significantly higher level of il-4 production .", "the rapidity of this cytokine response is attributed to the semiactivated state of inkt cells and the presence of preformed cytosolic mrna for a variety of cytokines .", "indeed , administration of -galcer to inkt cell - sufficient , but not inkt cell - deficient , mice results in polyclonal activation of conventional t , b , and nk cells within 3 - 4 hours   and also eventually leads to the mobilization of macrophages and neutrophils .", "intriguingly , it was previously believed that mammalian species are incapable of producing glycolipids ( such as -galcer ) , in which the sugar moiety is attached via an o - linkage to the ceramide backbone in an alpha - anomeric configuration . despite the absence of -glucosyl or -galactosyl transferases in mammals , recent findings indicate that a small percentage of the glycolipids that are constitutively presented by mammalian cd1d are indeed -anomeric . whether the percentage of cd1d - presented -anomeric glycolipids is altered in tumor tissues", "nonetheless , following encounter with cd1d / antigen complexes displayed by apcs , inkt cells not only produce cytokines but also upregulate surface expression of cd154 ( see figure 1(a ) ) .", "ligation of apc - expressed cd40 is especially important for mediating subsequent maturation and functional activation of dcs , subsequent upregulation of cd80 and cd86 , and amplified production of ifn- [ 72 , 73 ] .", "in addition , the ligation of the chemokine receptor cxcr6 on inkt cells by cxcl16 expressed on apcs also provides costimulatory signals resulting in robust -galcer - induced inkt cell activation .", "importantly , matured dcs are potent producers of il-12 , which induces sustained ifn- production by inkt cells [ 7577 ] .", "the importance of inkt cells in il-12-mediated tumor rejection was effectively demonstrated by the defective clearance of a variety of tumors in j18/ mice .", "mature dcs also support the priming and activation of cd8 t cells , culminating in optimal effector and memory cell formation [ 72 , 78 ] .", "finally , the sustained release of ifn- by inkt cells leads to activation and proliferation of nk cells and nk cell secretion of ifn-. the combination of cytokines ( e.g. , il-2 , il-12 , and ifn- ) as a result of inkt cell activation also leads to upregulation of death - inducing ligands ( e.g. , cd178 or cd253 ) on nk cells and cd8 t cells [ 79 , 80 ] .", "these sequential activation events are believed to be critical for the -galcer - induced inkt cell - mediated antitumor effects [ 76 , 81 , 82 ] . as such , inkt cells not only bridge the activation of innate and adaptive immunity , but also indirectly potentiate the antitumor activity of other cytotoxic effector lymphocytes .", "tumor establishment and growth are believed to be intricately modulated by a myriad of soluble and contact - derived signals obtained from the tumor microenvironment ( tme ) , which consists of the tumor cells themselves , tumor - infiltrating lymphocytes ( tils ) , and stromal cells that communicate in a dynamic and bidirectional manner .", "in addition to their indirect modulation of other effector lymphocyte populations , inkt cells may also regulate tumor growth via their effects on the tme ( see figure 1(b ) ) . indeed following intravenous administration ,", "inkt cells were shown to represent a significant percentage of the tils in patients with head and neck carcinomas [ 83 , 84 ] .", "importantly , higher frequency of tumor - infiltrating inkt cells correlated with overall and disease - free survival as an independent prognostic factor in primary colorectal cancer patients   and with tumor regression in head and neck carcinomas .", "conversely , in patients with primary hepatocellular or metastatic cancer , cd4 inkt cells that produced high levels of th2-type cytokines and had low cytolytic activity were enriched within the tumor and appeared to inhibit the expansion of antigen - specific cd8 t cells , suggesting that these particular inkt cells may contribute to generate an immunosuppressive microenvironment . in experimental studies ,", "cotransfer of human monocytes and inkt cells to tumor - bearing nod - scid mice suppressed tumor growth when compared with mice that received monocytes alone .", "importantly , inkt cells can target tumor supportive cells such as tumor - associated macrophages ( tams ) , a highly plastic monocyte - derived subset of inflammatory cells that can exert immunosuppressive functions , and promote tumor proliferation and matrix turnover [ 88 , 89 ] .", "indeed tams are known to produce il-6 , a cytokine that appears to promote the proliferation of many solid tumors , including neuroblastomas and breast and prostate carcinomas .", "found that macrophage density correlated positively with microvessel counts and negatively with patient relapse - free survival .", "since tams cross - present neuroblastomaderived endogenous cd1d ligand(s ) , they can be specifically recognized and killed by inkt cells in an il-15-dependent process .", "other potential inkt cell tme targets include myeloid - derived suppressor cells ( mdscs ) .", "mdscs have been found to accumulate in the blood , lymph nodes , and bone marrow and at tumor sites in most patients and experimental animals with cancer and inhibit both adaptive and innate immunity .", "the absence of inkt cells in mice during influenza virus infection resulted in the expansion of mdscs , high viral titer , and increased mortality .", "the adoptive transfer of inkt cells abolished the suppressive activity of mdscs and restored virus - specific immune responses , resulting in reduced viral titers and increased rates of host survival .", "thus , certain populations of inkt cells may help alter the tme via their effects on tams and mdscs , to help create a tumor - suppressive or immune - permissive milieu .", "in addition to their indirect control of tumor growth , inkt cells can mediate direct killing of tumor targets ( see figure 1(c ) ) .", "inkt cells alone , or in combination with nk cells , have been shown to kill a variety of tumor targets in vitro [ 6 , 93 , 94 ] .", "while this mechanism of killing appears to be dependent on the presence of stimulatory glycolipids and cd1d [ 95 , 96 ] , inkt cell cytotoxicity also appears to be triggered via ligation of nkg2d by target - expressed stress ligands .", "it remains to be seen whether mult1 , the newly identified shed form of high affinity nkg2d ligand that triggers nk - mediated tumor rejection in mice , also activates inkt cells .", "consistent with their direct cytotoxic capacity , inkt cells express perforin and granzymes , as well as cd178 [ 34 , 96 , 99 , 100 ] . in our hands ,", "blockade of cd1d - mediated lipid antigen presentation , disruption of t cell receptor ( tcr ) signaling , or loss of perforin expression was found to significantly reduce inkt cell killing in vitro .", "moreover , we demonstrated that inkt cells alone were sufficient for control of the growth of a t cell lymphoma in vivo that preferentially relies on perforin and the adaptor protein sap [ 64 , 69 ] .", "mechanistically , inkt cells rely on sap for formation of stable conjugates with the tumor targets as well as proper orientation of the lytic machinery at the immunological synapse . despite the majority of studies implicating inkt cells as having an antitumor role , a limited number of studies also implicate inkt cells as suppressing antitumor responses , but", "these paradoxic responses may be related to the level of tumor cd1d expression [ 102 , 103 ] .", "alternatively , these differences may stem from the fact that  contrary to the use of c57bl/6 mice in the previously discussed studies ", "these last two studies were performed in balb / c mice , in which there is a predominance of il-4-producing th2 phenotype inkt cells .", "interestingly , the antitumor responses of inkt cells may be regulated by the activity of type ii nkt cells .", "demonstrated that type ii variant nkt ( vnkt ) cells were sufficient for the downregulation of tumor immunosurveillance and relapse growth of a model fibrosarcoma in an antigen - dependent manner , while a second study found that activation of vnkt cells with sulfatide antigen could suppress the activation of inkt cells .", "conversely , type ii vnkt cells were , in at least one study , suggested to promote the antitumor activity of cpg oligodeoxynucleotides .", "this suppression appears to be mediated through a contact- and il-10-dependent mechanism [ 109 , 110 ] .", "indeed , induction of treg cells suppressed the protective effect of adoptive transfer of inkt cells into j18/ mice .", "consistent with these findings , depletion of treg cells or short - term elimination of their suppressive activity results in enhanced inkt cell - mediated antitumor responses and increased nk and cd8 t cell activation and ifn- production .", "interestingly , the ability of treg cells to suppress inkt cell proliferation depends on the degree of invariant tcr agonism , such that responses to weak ( e.g. , och ) , but not strong ( e.g. , -galcer ) , agonists were effectively suppressed . when viewed collectively , these findings suggest that inkt cells possess inherent capacity for direct cytotoxicity but their antigenic exposure may modulate whether their antitumor effects can be suppressed by treg and vnkt cells ."], ["given the preponderance of evidence suggesting that the activation of inkt cells provides protection against the growth and metastasis of a variety of tumors , safety of -galcer administration was examined in a phase i trial .", "while administration of -galcer was well tolerated at a range of doses , no clinical responses were observed in patients with advanced solid tumors . on the heels of this study ,", "nieda et al . showed that treatment of metastatic cancer patients with -galcer - pulsed immature monocyte - derived dcs resulted in dramatic increases in serum ifn- and il-12 and activation of nk and t cells in the majority of subjects .", "importantly , this phase i trial also documented reduction in tumor biomarkers and tumor necrosis in several patients .", "these findings were extended in a study of a small number of patients , in which the -galcer - pulsed dcs were matured prior to adoptive transfer .", "this study demonstrated a > 100-fold increase in blood inkt cell numbers in all patients , and this increase was long - lived ( > 6 months ) .", "a number of subsequent clinical trials , all with limited number of patients with advanced head and neck or non - small cell lung cancers , have since employed similar strategies of adoptive transfer of -galcer - pulsed apcs [ 115118 ] .", "collectively , these studies demonstrate increases in blood ifn- levels and inkt cells in some but not all patients , stabilization of disease in a few of the subjects , and absence of severe treatment - related toxicities . in a different approach ,", "chemotherapy - refractory 5 lymphoma patients were treated with autologous peripheral blood mononuclear cells ( pbmcs ) stimulated with anti - cd3 , il-2 , and ifn-. this ex vivo stimulation resulted in enrichment of nkt cells ( to ~20% on average ) , and this cell fraction was shown to possess the highest cytotoxic capacity in vitro .", "of the nine patients who received adoptive transfer of these cells , two showed partial responses and two others had stabilization of disease .", "two subsequent studies by motohashi et al . evaluated the adoptive transfer of ex vivo expanded inkt cell - enriched cells to patients with advanced cancer . in the first ,", "6 patients with advanced non - small cell lung cancer were treated with either a low or a high dose of ex vivo expanded inkt cells .", "of the 3 patients treated with the high dose , all had an increase in the frequency of ifn--producing pbmcs and 2 showed expansion of inkt cells . although no clinical responses were observed in this study , a follow - up trial of 17 patients with advanced head and neck cancers treated with a high dose of inkt cell - enriched autologous pbmcs showed a significant increase in ifn--producing pbmcs in 10 of 17 patients .", "importantly , while none of these patients displayed tumor regression , 5 had disease stabilization and the mean survival time for the subjects with higher frequencies of ifn--producing pbmcs was tripled above those with low percentages of ifn--producing pbmcs ( 29.3 versus 9.7 months ) .", "administered both in vitro expanded inkt cells and -galcer - pulsed apcs to patients with advanced head and neck squamous cell carcinomas .", "treatment increased the frequencies of inkt cells and ifn--producing pbmcs , and a partial clinical response or disease stabilization was observed in 7 of 8 patients . although the responses in these studies have not been profound , it must be noted that these inkt cell - based immunotherapies have all been conducted on patients with advanced malignancies  often those in whom standard chemotherapy , irradiation , and/or surgical excision treatments had failed .", "future studies of inkt cell - based immunotherapy may be able to take advantage of two recent technologies .", "as mentioned previously , many malignancies are associated with a decrease in the numbers and proliferative capacity of peripheral blood inkt cells . in order to circumvent the difficulty of being able to expand these infrequent and potentially defective cells from patients , watarai et al .", "generated induced pluripotent stem ( ips ) cells from mature inkt cells and then expanded large numbers of inkt cells from these established ips cells .", "ips - nkt cells generated in this fashion were demonstrated to be able to activate and expand antigen - specific cd8 t cell responses to limit the growth of leukemia in mice   without inducing graft versus host disease ( gvhd ) .", "described inkt cells engineered to express cars bearing specificity for gd2 , a highly expressed moiety on neuroblastoma cells . in their studies , they showed that inkt cells expanded from the pbmcs of healthy human donors and transduced with retroviral car constructs could protect humanized nsg mice against metastatic neuroblastoma without inducing gvhd .", "whether these two technologies could be combined to generate functional car - bearing ips - nkt cells remains to be seen ."], ["inkt cells are innate - like effector lymphocytes that not only are directly cytotoxic , but also possess the unique ability to nucleate the antitumor responses of other effector lymphocytes and alter the cellular and angiogenic makeup of the tumor microenvironment . as such , the promise of an effective inkt cell - based immunotherapy can only be realized by devising and evaluating strategies that simultaneously maximize each of these antitumor effector mechanisms . the challenge for the future will thus be to identify these strategies and apply them to tumors against which inkt cells wield the most optimal responses ."]]}
{"article_id": "PMC5167333", "article_text": ["as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset .", "technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors ."], "abstract_text": ["<S> abstractthe kinetics of formation of the valence tautomers ( tpfc)mniv(ola)]n+ [ where la = znii , caii , sciii , ybiii , b(c6f5)3 , and trifluoroacetic acid ( tfa ) ; tpfc=5,10,15tris(pentafluorophenyl ) corrole ] from ( tpfc)mnv(o ) were followed by uv / vis spectroscopy , giving secondorder rate constants ranging over five orders of magnitude from 102 for ca to 103 \n m \n 1  s1 for sc . </S>", "<S> hydrogen atom transfer ( hat ) rates from 2,4ditertbutyl phenol ( 2,4dtbp ) to the various lewis acid valence tautomers of manganese oxo corrole complexes were evaluated and compared . for la = tfa , </S>", "<S> sciii , or ybiii , the rate constants of hat were comparable to unactivated ( tpfc)mnv(o ) . </S>", "<S> however , with la = b(c6f5)3 , znii , and caii , 6 , 21 , and 31fold rate enhancements were observed , respectively . remarkably , [ ( tpfc)mniv(oca)]2 + gave the most enhancement despite its rate of formation being the slowest . </S>", "<S> comparisons of hat rate constants among the various lewis acid tautomers revealed that both size and charge are important . </S>", "<S> this study underscores how valence may affect the reactivity of highvalent manganeseoxo compounds and sheds light on nature 's choice of ca in the activation of mnoxo in the oxygenevolving complex . </S>"], "labels": null, "section_names": ["Supporting information"], "sections": [["as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset .", "technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors ."]]}
{"article_id": "PMC4909374", "article_text": ["clinicians rely on patients recall of their symptoms to adjust therapies and monitor responses , but the accuracy of patients recall , particularly between clinic visits , is poor.1 patients recall recent symptoms more clearly than earlier , more remote symptoms , and more severe symptoms more clearly than mild symptoms.2 recognition of day - to - day variability in symptoms is also important for revealing important trends ( improvement or worsening ) , but measuring such variability is difficult without close tracking of symptoms .", "daily symptom variation is important because it may obscure the ability to detect signal ( of improvement ) from the noise ( of random daily variation ) . taken together , these challenges highlight the problems of symptom recall as the basis for making treatment recommendations , and either altering or escalating therapeutic interventions.1,3 inflammatory bowel disease ( ibd ) causes chronic inflammation of the gastrointestinal tract , often resulting in severe symptoms . despite optimal treatment , over 40 percent of patients", "continue to have symptoms that reduce their quality of life ( qol ) such as persistent fatigue,4,5 abdominal bloating , or fecal urgency.6 careful attention to a patient s symptomatic response to therapies using paper symptom logs is often inadequate or too cumbersome to provide sufficient evidence to guide treatment.7 improved methods are needed to ease symptom data collection and to facilitate conducting individual ( n - of-1 ) therapeutic trials to improve symptoms and qol .", "currently , 78 percent of teenage patients carry mobile devices capable of easily collecting and transmitting symptom data on a daily basis.8 there is growing interest among patients ( e.g. , the  quantified self  community)5,9,10 and physicians1115 in leveraging mobile technologies for improving patient outcomes . according to a recent study by the pew research center ,", "over 40 percent of americans who have a chronic health condition track their symptoms or health indicators such as headaches , blood sugar , blood pressure , etc.5 but there is little evidence of the impact that frequent monitoring of self - reported data has on patient qol or other health outcomes.16 while some limited progress has been made in integrating formalized patient - reported outcome ( pro ) measures into clinical practice , these early efforts provide little evidence to guide the effective integration of pros into real - world patient care setting.16,17 efforts at implementation are made more difficult by technical limitations ( e.g. , many care centers lack electronic tools to collect pros , and completing repeated online surveys are not sustainable for most patients over time ) . most published studies utilizing pros in real - world patient care settings have demonstrated that the pros have little or no impact on patient outcomes.16 this may be due to a lack of validated tools to monitor patient symptoms or qol reliably and prospectively in real time.16 standardized qol indicators for general assessment have not been studied widely for assessing patients in clinical practice .", "the patient - reported outcomes measurement information system ( promis ) survey instruments may be appropriate for this purpose , but promis surveys have been validated only in population studies.18,19 promis tools have not been studied in individual patients or over short periods , and have not been broadly incorporated into routine patient care . as part of an ongoing study exploring innovations in health care , we evaluated a prototype tool to determine if continuous symptom tracking aids in improving patients and physicians understanding of symptom patterns and if tracking can help patients and physicians work together to better target treatments.20,21 we also attempted to determine if promis measures can be used in individual patients to track qol over short periods , as opposed to populations of patients for which they were developed .", "we report , here , important learning from a small number of patients who completed prototype testing of this tool .", "this study was conducted as part of the ongoing work of the collaborative chronic care network ( c3n ) project that is developing innovations to facilitate more continuous , collaborative relationships between patients and families with chronic illness and their physicians .", "the tools described here emerged from a structured design process involving patients , families , clinicians , and innovators that entails testing novel concepts in small - scale prototypes and larger pilot studies prior to deployment.21 this manuscript describes the prototype testing of the novel concept .", "we took advantage of the collaborative network of patients , physicians , and researchers who participate in the improvecarenow network to engage participants.21 we recruited adolescent patients with ibd who were engaged in their care and were interested in further improving their symptoms and qol . each patient", "( along with a parent if under 18 years old ) met with his or her pediatric gastroenterologist and a researcher at the time of study enrollment to discuss the patient s medical history and to plan the details of the patient s individual study .", "the researcher and patient agreed upon a means of assessing symptoms by selecting from a list of preassembled measures .", "they were allowed to create their own symptom measure if none was available to fit their needs .", "we used a daily , automated text - message service to prompt patients to submit symptom scores via text message .", "we did not limit the number of questions that could be asked per day , but suggested each patient select no more than three to four . at study enrollment ,", "the questions were delivered each day at this prespecified time , and did not vary from day to day or with responses to prior questions .", "we also used the promis survey instruments to measure fatigue and pain interference across all patients.22,23 these short surveys have been extensively validated in a wide range of chronic medical conditions and children.18,19,24 the promis surveys query information on specific outcomes for the preceding seven days .", "the results were converted to t - scores , where a score of 50 represents the mean score of the general pediatric calibration sample ( standard deviation 10).25 t - scores greater than 50 represent worse fatigue or pain interference .", "we deployed the appropriate promis survey weekly via a web - based interface to each patient .", "we compared individual patients promis survey results with their average symptom scores from the same period .", "we used individual - level , statistical process control methods for time - sequence data to monitor the patient s daily symptom data and weekly promis survey data as they were collected over time.26 we established each patient s individual baseline pattern of symptom variation , or  common cause variation .  symptom data points that fell outside three sigma control limits were noted as  special cause variation ,  or variation outside stable baseline variability.26,27 these data , along with calculated control limits , were graphed on a microsoft excel spreadsheet for visual representation of data for patient and physician review . for prototype testing , we used a heavily manual process  including creating microsoft excel graphs by hand and sharing data with patients and providers via email  that was possible for only a small number of patients .", "the purpose of this type of manual prototype was to inform more robust pilot testing facilitated by automated tools being developed by the research team .", "patients were given access to their own data and were provided with graphical displays of their data at each meeting .", "physicians and patients jointly reviewed the data on a regular basis in prearranged consultations ( in person or by phone , according to each family and physician s preferences ) .", "these meetings were opportunities for patients and physicians to learn from patterns of data and to make treatment decisions if they felt this was appropriate .", "this study and the consent procedures were approved by the institutional review boards of both the university of michigan health system ( hum00051651 ) and cincinnati children s hospital medical center ( 20103380 ) .", "all adult participants provided written informed consent to participate in this study . written informed consent was obtained from parents of minors and written assent from minors themselves for participation in this study .", "patients tracked symptoms in order to monitor for changes in disease status , to generate hypotheses as to what makes their symptoms better or worse , or as part of a specific n - of-1 study .", "we present three patients who tracked their symptoms , prior to implementation of n - of-1 testing , and for whom symptom patterns led to important revelations .", "a 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years .", "she had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol .", "she had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . in collaboration with her physician , she decided to track her total number of daily stools and the number of stools waking her from sleep each night .", "she historically awoke to have a bowel movement from three to six times per night on average . during her baseline observation period", ", there was a span of nine consecutive nights with fewer episodes of nocturnal stools ( fig .", "1 ) . however , she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab , which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern .", "however , this postinfusion improvement was not reproduced after her subsequent four infliximab infusions , during which time her daily symptoms were continuing to be recorded .", "she did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician ( previously illustrated in kaplan et al.20 ) . during those times when she experienced decreased nighttime bowel movements , she also felt less fatigued ( fig .", "2 ) . upon reviewing these data she confirmed that she felt less fatigued and more energetic .", "the patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects .", "both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency .", "over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved ( fig .", "3 , green dashed ovals ) , though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score ( fig .", "after three months of monitoring , the patient developed an exacerbation of the disease ( fig .", "after he was diagnosed with crohn s disease , his running pace decreased and had not returned to his pre - illness baseline , and he was interested in assessing his running potential after starting treatment with biological therapy . at his enrollment in this study , his baseline promis fatigue t - score was 35.7 , indicating that he was less fatigued than the average healthy individual .", "he received his first infliximab infusion on may 25 , 2011 , with induction regimen completed by july 6 , 2011 , ( usually consisting of three infusions spread over six weeks ) .", "his number of daily bowel movements decreased within four days of completing the induction regimen , and attained a steady state along with a special cause ( fig .", "his running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile , indicated by a special cause shift as well .", "a 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years .", "she had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol .", "she had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . in collaboration with her physician", ", she decided to track her total number of daily stools and the number of stools waking her from sleep each night .", "she historically awoke to have a bowel movement from three to six times per night on average . during her baseline observation period", ", there was a span of nine consecutive nights with fewer episodes of nocturnal stools ( fig .", "1 ) . however , she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab , which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern .", "however , this postinfusion improvement was not reproduced after her subsequent four infliximab infusions , during which time her daily symptoms were continuing to be recorded .", "she did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician ( previously illustrated in kaplan et al.20 ) . during those times when she experienced decreased nighttime bowel movements , she also felt less fatigued ( fig .", "2 ) . upon reviewing these data she confirmed that she felt less fatigued and more energetic .", "the patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects .", "both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency .", "over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved ( fig .", "3 , green dashed ovals ) , though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score ( fig .", "after three months of monitoring , the patient developed an exacerbation of the disease ( fig .", "after he was diagnosed with crohn s disease , his running pace decreased and had not returned to his pre - illness baseline , and he was interested in assessing his running potential after starting treatment with biological therapy . at his enrollment in this study , his baseline promis fatigue t - score was 35.7 , indicating that he was less fatigued than the average healthy individual .", "he received his first infliximab infusion on may 25 , 2011 , with induction regimen completed by july 6 , 2011 , ( usually consisting of three infusions spread over six weeks ) .", "his number of daily bowel movements decreased within four days of completing the induction regimen , and attained a steady state along with a special cause ( fig .", "his running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile , indicated by a special cause shift as well .", "we found that monitoring daily symptom data detects the daily variation in patients symptoms . establishing a patient s background pattern of symptom variation can serve as a baseline from which to make subsequent comparisons when making changes in diet or medications .", "significant ( special cause ) deviations in symptoms from the baseline pattern are also associated with variation in promis instrument assessments .", "our data from these patients suggest that the promis measures of fatigue and pain , in particular , are responsive to changes in patient symptoms over short periods and on the individual level .", "these findings lend face validity to daily remote monitoring of patient symptoms , and pave the way for broader use of the promis instruments for use in patient - centered improvement research . using standard promis instruments , in addition to more customized patient symptoms , may enable greater learning across patients because measures are well validated and standardized in pediatric populations .", "we also found that knowledge about daily symptom variation and promis assessments of key outcomes may be clinically relevant .", "improving patients and physicians understanding of symptom patterns for the purpose of tailoring treatments to the patients needs is essential for understanding response to attempted therapies .", "it is also essential to measure symptoms that are important to patients in order to help patients achieve good qol even if other traditional measures show minimal symptoms .", "this work is important and expands upon the very few previous studies of the impact of daily monitoring on outcomes for chronic diseases.16 the most widely documented evidence is for asthma in which daily monitoring is associated with improved asthma control.28 there is also evidence that daily monitoring via mobile phone of adults with heart failure results in improved qol , improved self - care,12 and  in one study  decreased mortality.29 similarly , in insulin - dependent diabetes mellitus , daily monitoring leads to improved glucose control , more symptom - free days , and decreased perceived disease - related anxiety.13 despite limited evidence of benefit from mobile symptom tracking in some studies , the majority of studies do not demonstrate a measurable improvement in patient outcomes.16 one significant issue that has not previously been addressed is the day - to - day variability of patient symptoms . as we ve seen in our study ,", "there is substantial daily variability in symptoms , which may obscure the detection of improvements , when they occur .", "the work reported here builds on previous studies by developing tools that enable daily symptom tracking to better understand and detect changes in patient symptoms .", "understanding the nuances of symptom variation  at baseline , and in response to therapeutic challenges  is critical for targeting therapies for improvement and for determining the individual efficacy of therapeutic changes .", "comparing daily symptom data with weekly promis surveys provides internal consistency of the pro measures collected for each patient .", "together , the collection of these pro data from patients facilitates real - time feedback to patients and their physicians about the effectiveness of therapies .", "these methods offer substantial improvements over routine clinical practice and over prior mobile health ( mhealth ) studies lacking such feedback .", "these were highly engaged patients who volunteered for this study ( as is the case in many studies ) .", "it is possible that daily or weekly symptom measurement may not be feasible in less - engaged patients .", "however since 75 percent of young people have mobile phones and 88 percent of them send and respond to numerous text messages daily ( typical 14 to 17 year olds send and receive 100 text messages per day),30 it remains to be determined how important engagement with care is to these assessments .", "there is also some concern that daily monitoring may lead to increased attention to symptoms and worsening of perceived physical health.31 however others have demonstrated that patients with chronic constipation who monitored daily symptoms had no worse outcomes than those monitored weekly in a prospective randomized clinical trial.32 others found improved outcomes with daily monitoring for irritable bowel syndrome , asthma , and heart failure.12,13,33 it will be important to determine if certain patients may benefit more from these methods and if others should avoid these methods to minimize attention to symptoms .", "note that a patient with longstanding daily symptoms may have a significant improvement in symptoms without being aware of such changes ( as was seen with patient 1 ) .", "conversely , perceived improvement may not actually represent real improvement , but may be misattributed background variation  which , when graphically evaluated , represents common cause variation ( as seen with patient 2 ) .", "this was seen with the initial improvement in nocturnal stools following infliximab infusion ( patient 1 ) , which was not reproduced after subsequent infusions .", "it is tempting to infer a causal relationship between two events because of their close proximity in timing , even if the association is coincidental .", "the accuracy of the interpretation of associations has direct implications for how most physicians and patients routinely test therapies by ad hoc trial and error .", "it is only with careful monitoring of outcomes , and with replication ( through experimentation ) , that we can distinguish between causal relationships or associations.34,35 this study also highlights the importance of tracking the symptoms that patients feel are most important to them .", "the example of patient 3 , whose gastrointestinal symptoms and fatigue were not severe , illustrates this additional benefit of our individualized , patient - centered approach . by closely monitoring the symptom that was most important to the patient ( his running pace ) ,", "he and his physician were able to determine when a significant improvement occurred in the outcome that was of greatest importance to the patient .", "our findings in this early study highlight the difficulty in assessing treatment efficacy based on casual assessment of symptom relief for an individual patient .", "these findings are of great importance to improving the physician s ability to adequately understand their patient s disease activity and the symptoms contributing to qol for the purpose of tailoring treatments to the needs of the patient .", "this applies when new therapies are initiated and also during changing , modifying , weaning off , or withdrawing therapies ( as could be the case with steroid weaning for ibd treatment ) .", "these new methods offer the potential to support more collaborative decision - making and to improve clinicians and patients abilities to identify the right treatments both through informal learning and planned experimentation with individual patient therapeutic ( n - of-1 ) trials .", "we must have a clear understanding of the effects of an intervention ( medical , dietary , behavioral , etc . ) on the patient s symptoms . only with a clear understanding of symptom variation , and an ability to discern a meaningful change in symptom pattern from the background variation , can we fully understand the effects of therapies on symptoms and know with certainty whether or not a therapy improves symptoms and qol .", "the new methods described here are the building blocks for transforming the doctor - patient relationship to become more collaborative and to provide improved evidence for understanding individual patient treatment efficacy for better treatment individualization .", "based on the success of daily tracking using customized and standardized measures ( promis measures ) , and the utility of graphical display of symptoms using statistical process control , we are developing more advanced technology ( web platform and mobile app ) to enable tracking and real - time visualization of this data for the patient and provider .", "we are also examining ways to better support patients and providers in using pro data to enable formal n - of-1 studies of pediatric patients with ibd .", "n - of-1 studies are well established as a method to improve patient symptoms . however , until now these methods have been limited in their scope of use .", "we are currently expanding these methods that we have described here to develop the means of conducting n - of-1 studies concurrently with larger numbers of patients who may have different complaints and different treatments .", "these methods have the potential to benefit patients broadly and to improve patient - physician engagement and communication across a wide range of clinical settings .", "clinicians and patients should consider the use of mobile daily symptom data collection to improve care and outcomes .", "daily symptom tracking can facilitate improved informal learning as well as rigorous n - of-1 experimental studies to objectively assess individual treatment efficacy and individualize patient care.34,35 prototype studies have provided proof of concept so that these methods can be further expanded to enable more robust learning designed to improve symptoms and qol for pediatric patients with ibd and others with differing chronic health conditions ."], "abstract_text": ["<S> introduction : improving symptoms for patients with chronic illness is difficult due to poor recall and imprecise assessments of therapeutic response to inform treatment decisions . </S>", "<S> daily variation in symptoms may obscure subtle improvement or lead to erroneous associations between symptom changes and alteration in medication or dietary regimens . </S>", "<S> this may lead to mistaken impressions of treatment efficacy ( or inefficacy ) . mobile health technologies that collect daily patient reported outcome ( pro ) data have the potential to improve care by providing more detailed information for clinical decision - making in practice and may facilitate conducting single subject ( n - of-1 ) trials.methods:interrupted time series to prototype mobile health enabled data collection for three patients . </S>", "<S> we recruited pediatric patients with established inflammatory bowel disease who had persistent symptoms . based on their self - identified most troubling symptoms , </S>", "<S> patients were sent customized , daily - automated text messages to assess the extent of their symptoms . </S>", "<S> standardized , pro measurement information system ( promis ) surveys were deployed weekly . </S>", "<S> individual statistical process control charts were used to assess variation . </S>", "<S> patients met with physicians regularly to interpret their data jointly.results:we report the experience of 3 patients with inflammatory bowel disease , each with different symptoms . </S>", "<S> daily symptom monitoring uncovered important patterns , some of which even patients were unaware before reviewing their symptom data . </S>", "<S> important associations were found between symptom variation and changes in medications and diet . </S>", "<S> promis survey results assessed longitudinally accurately reflected changes in patient symptoms.conclusions:we demonstrated how pros can be implemented in practice . monitoring and analyzing daily symptom data , using both customized and standard pros , has the potential to detect meaningful variation in symptom patterns , which can inform clinical decision - making or can facilitate conducting formal n - of-1 trials to further improve outcomes . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results", "Patient 1", "Patient 2", "Patient 3", "Discussion", "Conclusion"], "sections": [["clinicians rely on patients recall of their symptoms to adjust therapies and monitor responses , but the accuracy of patients recall , particularly between clinic visits , is poor.1 patients recall recent symptoms more clearly than earlier , more remote symptoms , and more severe symptoms more clearly than mild symptoms.2 recognition of day - to - day variability in symptoms is also important for revealing important trends ( improvement or worsening ) , but measuring such variability is difficult without close tracking of symptoms .", "daily symptom variation is important because it may obscure the ability to detect signal ( of improvement ) from the noise ( of random daily variation ) . taken together , these challenges highlight the problems of symptom recall as the basis for making treatment recommendations , and either altering or escalating therapeutic interventions.1,3 inflammatory bowel disease ( ibd ) causes chronic inflammation of the gastrointestinal tract , often resulting in severe symptoms . despite optimal treatment , over 40 percent of patients", "continue to have symptoms that reduce their quality of life ( qol ) such as persistent fatigue,4,5 abdominal bloating , or fecal urgency.6 careful attention to a patient s symptomatic response to therapies using paper symptom logs is often inadequate or too cumbersome to provide sufficient evidence to guide treatment.7 improved methods are needed to ease symptom data collection and to facilitate conducting individual ( n - of-1 ) therapeutic trials to improve symptoms and qol .", "currently , 78 percent of teenage patients carry mobile devices capable of easily collecting and transmitting symptom data on a daily basis.8 there is growing interest among patients ( e.g. , the  quantified self  community)5,9,10 and physicians1115 in leveraging mobile technologies for improving patient outcomes . according to a recent study by the pew research center ,", "over 40 percent of americans who have a chronic health condition track their symptoms or health indicators such as headaches , blood sugar , blood pressure , etc.5 but there is little evidence of the impact that frequent monitoring of self - reported data has on patient qol or other health outcomes.16 while some limited progress has been made in integrating formalized patient - reported outcome ( pro ) measures into clinical practice , these early efforts provide little evidence to guide the effective integration of pros into real - world patient care setting.16,17 efforts at implementation are made more difficult by technical limitations ( e.g. , many care centers lack electronic tools to collect pros , and completing repeated online surveys are not sustainable for most patients over time ) . most published studies utilizing pros in real - world patient care settings have demonstrated that the pros have little or no impact on patient outcomes.16 this may be due to a lack of validated tools to monitor patient symptoms or qol reliably and prospectively in real time.16 standardized qol indicators for general assessment have not been studied widely for assessing patients in clinical practice .", "the patient - reported outcomes measurement information system ( promis ) survey instruments may be appropriate for this purpose , but promis surveys have been validated only in population studies.18,19 promis tools have not been studied in individual patients or over short periods , and have not been broadly incorporated into routine patient care . as part of an ongoing study exploring innovations in health care , we evaluated a prototype tool to determine if continuous symptom tracking aids in improving patients and physicians understanding of symptom patterns and if tracking can help patients and physicians work together to better target treatments.20,21 we also attempted to determine if promis measures can be used in individual patients to track qol over short periods , as opposed to populations of patients for which they were developed .", "we report , here , important learning from a small number of patients who completed prototype testing of this tool ."], ["this study was conducted as part of the ongoing work of the collaborative chronic care network ( c3n ) project that is developing innovations to facilitate more continuous , collaborative relationships between patients and families with chronic illness and their physicians .", "the tools described here emerged from a structured design process involving patients , families , clinicians , and innovators that entails testing novel concepts in small - scale prototypes and larger pilot studies prior to deployment.21 this manuscript describes the prototype testing of the novel concept .", "we took advantage of the collaborative network of patients , physicians , and researchers who participate in the improvecarenow network to engage participants.21 we recruited adolescent patients with ibd who were engaged in their care and were interested in further improving their symptoms and qol . each patient", "( along with a parent if under 18 years old ) met with his or her pediatric gastroenterologist and a researcher at the time of study enrollment to discuss the patient s medical history and to plan the details of the patient s individual study .", "the researcher and patient agreed upon a means of assessing symptoms by selecting from a list of preassembled measures .", "they were allowed to create their own symptom measure if none was available to fit their needs .", "we used a daily , automated text - message service to prompt patients to submit symptom scores via text message .", "we did not limit the number of questions that could be asked per day , but suggested each patient select no more than three to four . at study enrollment ,", "the questions were delivered each day at this prespecified time , and did not vary from day to day or with responses to prior questions .", "we also used the promis survey instruments to measure fatigue and pain interference across all patients.22,23 these short surveys have been extensively validated in a wide range of chronic medical conditions and children.18,19,24 the promis surveys query information on specific outcomes for the preceding seven days .", "the results were converted to t - scores , where a score of 50 represents the mean score of the general pediatric calibration sample ( standard deviation 10).25 t - scores greater than 50 represent worse fatigue or pain interference .", "we deployed the appropriate promis survey weekly via a web - based interface to each patient .", "we compared individual patients promis survey results with their average symptom scores from the same period .", "we used individual - level , statistical process control methods for time - sequence data to monitor the patient s daily symptom data and weekly promis survey data as they were collected over time.26 we established each patient s individual baseline pattern of symptom variation , or  common cause variation .  symptom data points that fell outside three sigma control limits were noted as  special cause variation ,  or variation outside stable baseline variability.26,27 these data , along with calculated control limits , were graphed on a microsoft excel spreadsheet for visual representation of data for patient and physician review . for prototype testing , we used a heavily manual process  including creating microsoft excel graphs by hand and sharing data with patients and providers via email  that was possible for only a small number of patients .", "the purpose of this type of manual prototype was to inform more robust pilot testing facilitated by automated tools being developed by the research team .", "patients were given access to their own data and were provided with graphical displays of their data at each meeting .", "physicians and patients jointly reviewed the data on a regular basis in prearranged consultations ( in person or by phone , according to each family and physician s preferences ) .", "these meetings were opportunities for patients and physicians to learn from patterns of data and to make treatment decisions if they felt this was appropriate .", "this study and the consent procedures were approved by the institutional review boards of both the university of michigan health system ( hum00051651 ) and cincinnati children s hospital medical center ( 20103380 ) .", "all adult participants provided written informed consent to participate in this study . written informed consent was obtained from parents of minors and written assent from minors themselves for participation in this study ."], ["patients tracked symptoms in order to monitor for changes in disease status , to generate hypotheses as to what makes their symptoms better or worse , or as part of a specific n - of-1 study .", "we present three patients who tracked their symptoms , prior to implementation of n - of-1 testing , and for whom symptom patterns led to important revelations .", "a 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years .", "she had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol .", "she had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . in collaboration with her physician , she decided to track her total number of daily stools and the number of stools waking her from sleep each night .", "she historically awoke to have a bowel movement from three to six times per night on average . during her baseline observation period", ", there was a span of nine consecutive nights with fewer episodes of nocturnal stools ( fig .", "1 ) . however , she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab , which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern .", "however , this postinfusion improvement was not reproduced after her subsequent four infliximab infusions , during which time her daily symptoms were continuing to be recorded .", "she did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician ( previously illustrated in kaplan et al.20 ) . during those times when she experienced decreased nighttime bowel movements , she also felt less fatigued ( fig .", "2 ) . upon reviewing these data she confirmed that she felt less fatigued and more energetic .", "the patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects .", "both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency .", "over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved ( fig .", "3 , green dashed ovals ) , though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score ( fig .", "after three months of monitoring , the patient developed an exacerbation of the disease ( fig .", "after he was diagnosed with crohn s disease , his running pace decreased and had not returned to his pre - illness baseline , and he was interested in assessing his running potential after starting treatment with biological therapy . at his enrollment in this study , his baseline promis fatigue t - score was 35.7 , indicating that he was less fatigued than the average healthy individual .", "he received his first infliximab infusion on may 25 , 2011 , with induction regimen completed by july 6 , 2011 , ( usually consisting of three infusions spread over six weeks ) .", "his number of daily bowel movements decreased within four days of completing the induction regimen , and attained a steady state along with a special cause ( fig .", "his running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile , indicated by a special cause shift as well ."], ["a 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years .", "she had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol .", "she had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . in collaboration with her physician", ", she decided to track her total number of daily stools and the number of stools waking her from sleep each night .", "she historically awoke to have a bowel movement from three to six times per night on average . during her baseline observation period", ", there was a span of nine consecutive nights with fewer episodes of nocturnal stools ( fig .", "1 ) . however , she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab , which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern .", "however , this postinfusion improvement was not reproduced after her subsequent four infliximab infusions , during which time her daily symptoms were continuing to be recorded .", "she did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician ( previously illustrated in kaplan et al.20 ) . during those times when she experienced decreased nighttime bowel movements , she also felt less fatigued ( fig .", "2 ) . upon reviewing these data she confirmed that she felt less fatigued and more energetic .", "the patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects ."], ["both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency .", "over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved ( fig .", "3 , green dashed ovals ) , though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score ( fig .", "after three months of monitoring , the patient developed an exacerbation of the disease ( fig ."], ["after he was diagnosed with crohn s disease , his running pace decreased and had not returned to his pre - illness baseline , and he was interested in assessing his running potential after starting treatment with biological therapy . at his enrollment in this study , his baseline promis fatigue t - score was 35.7 , indicating that he was less fatigued than the average healthy individual .", "he received his first infliximab infusion on may 25 , 2011 , with induction regimen completed by july 6 , 2011 , ( usually consisting of three infusions spread over six weeks ) .", "his number of daily bowel movements decreased within four days of completing the induction regimen , and attained a steady state along with a special cause ( fig .", "his running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile , indicated by a special cause shift as well ."], ["we found that monitoring daily symptom data detects the daily variation in patients symptoms . establishing a patient s background pattern of symptom variation can serve as a baseline from which to make subsequent comparisons when making changes in diet or medications .", "significant ( special cause ) deviations in symptoms from the baseline pattern are also associated with variation in promis instrument assessments .", "our data from these patients suggest that the promis measures of fatigue and pain , in particular , are responsive to changes in patient symptoms over short periods and on the individual level .", "these findings lend face validity to daily remote monitoring of patient symptoms , and pave the way for broader use of the promis instruments for use in patient - centered improvement research . using standard promis instruments , in addition to more customized patient symptoms , may enable greater learning across patients because measures are well validated and standardized in pediatric populations .", "we also found that knowledge about daily symptom variation and promis assessments of key outcomes may be clinically relevant .", "improving patients and physicians understanding of symptom patterns for the purpose of tailoring treatments to the patients needs is essential for understanding response to attempted therapies .", "it is also essential to measure symptoms that are important to patients in order to help patients achieve good qol even if other traditional measures show minimal symptoms .", "this work is important and expands upon the very few previous studies of the impact of daily monitoring on outcomes for chronic diseases.16 the most widely documented evidence is for asthma in which daily monitoring is associated with improved asthma control.28 there is also evidence that daily monitoring via mobile phone of adults with heart failure results in improved qol , improved self - care,12 and  in one study  decreased mortality.29 similarly , in insulin - dependent diabetes mellitus , daily monitoring leads to improved glucose control , more symptom - free days , and decreased perceived disease - related anxiety.13 despite limited evidence of benefit from mobile symptom tracking in some studies , the majority of studies do not demonstrate a measurable improvement in patient outcomes.16 one significant issue that has not previously been addressed is the day - to - day variability of patient symptoms . as we ve seen in our study ,", "there is substantial daily variability in symptoms , which may obscure the detection of improvements , when they occur .", "the work reported here builds on previous studies by developing tools that enable daily symptom tracking to better understand and detect changes in patient symptoms .", "understanding the nuances of symptom variation  at baseline , and in response to therapeutic challenges  is critical for targeting therapies for improvement and for determining the individual efficacy of therapeutic changes .", "comparing daily symptom data with weekly promis surveys provides internal consistency of the pro measures collected for each patient .", "together , the collection of these pro data from patients facilitates real - time feedback to patients and their physicians about the effectiveness of therapies .", "these methods offer substantial improvements over routine clinical practice and over prior mobile health ( mhealth ) studies lacking such feedback .", "these were highly engaged patients who volunteered for this study ( as is the case in many studies ) .", "it is possible that daily or weekly symptom measurement may not be feasible in less - engaged patients .", "however since 75 percent of young people have mobile phones and 88 percent of them send and respond to numerous text messages daily ( typical 14 to 17 year olds send and receive 100 text messages per day),30 it remains to be determined how important engagement with care is to these assessments .", "there is also some concern that daily monitoring may lead to increased attention to symptoms and worsening of perceived physical health.31 however others have demonstrated that patients with chronic constipation who monitored daily symptoms had no worse outcomes than those monitored weekly in a prospective randomized clinical trial.32 others found improved outcomes with daily monitoring for irritable bowel syndrome , asthma , and heart failure.12,13,33 it will be important to determine if certain patients may benefit more from these methods and if others should avoid these methods to minimize attention to symptoms .", "note that a patient with longstanding daily symptoms may have a significant improvement in symptoms without being aware of such changes ( as was seen with patient 1 ) .", "conversely , perceived improvement may not actually represent real improvement , but may be misattributed background variation  which , when graphically evaluated , represents common cause variation ( as seen with patient 2 ) .", "this was seen with the initial improvement in nocturnal stools following infliximab infusion ( patient 1 ) , which was not reproduced after subsequent infusions .", "it is tempting to infer a causal relationship between two events because of their close proximity in timing , even if the association is coincidental .", "the accuracy of the interpretation of associations has direct implications for how most physicians and patients routinely test therapies by ad hoc trial and error .", "it is only with careful monitoring of outcomes , and with replication ( through experimentation ) , that we can distinguish between causal relationships or associations.34,35 this study also highlights the importance of tracking the symptoms that patients feel are most important to them .", "the example of patient 3 , whose gastrointestinal symptoms and fatigue were not severe , illustrates this additional benefit of our individualized , patient - centered approach . by closely monitoring the symptom that was most important to the patient ( his running pace ) ,", "he and his physician were able to determine when a significant improvement occurred in the outcome that was of greatest importance to the patient .", "our findings in this early study highlight the difficulty in assessing treatment efficacy based on casual assessment of symptom relief for an individual patient .", "these findings are of great importance to improving the physician s ability to adequately understand their patient s disease activity and the symptoms contributing to qol for the purpose of tailoring treatments to the needs of the patient .", "this applies when new therapies are initiated and also during changing , modifying , weaning off , or withdrawing therapies ( as could be the case with steroid weaning for ibd treatment ) .", "these new methods offer the potential to support more collaborative decision - making and to improve clinicians and patients abilities to identify the right treatments both through informal learning and planned experimentation with individual patient therapeutic ( n - of-1 ) trials .", "we must have a clear understanding of the effects of an intervention ( medical , dietary , behavioral , etc . ) on the patient s symptoms . only with a clear understanding of symptom variation , and an ability to discern a meaningful change in symptom pattern from the background variation , can we fully understand the effects of therapies on symptoms and know with certainty whether or not a therapy improves symptoms and qol .", "the new methods described here are the building blocks for transforming the doctor - patient relationship to become more collaborative and to provide improved evidence for understanding individual patient treatment efficacy for better treatment individualization .", "based on the success of daily tracking using customized and standardized measures ( promis measures ) , and the utility of graphical display of symptoms using statistical process control , we are developing more advanced technology ( web platform and mobile app ) to enable tracking and real - time visualization of this data for the patient and provider .", "we are also examining ways to better support patients and providers in using pro data to enable formal n - of-1 studies of pediatric patients with ibd .", "n - of-1 studies are well established as a method to improve patient symptoms . however , until now these methods have been limited in their scope of use .", "we are currently expanding these methods that we have described here to develop the means of conducting n - of-1 studies concurrently with larger numbers of patients who may have different complaints and different treatments .", "these methods have the potential to benefit patients broadly and to improve patient - physician engagement and communication across a wide range of clinical settings ."], ["clinicians and patients should consider the use of mobile daily symptom data collection to improve care and outcomes .", "daily symptom tracking can facilitate improved informal learning as well as rigorous n - of-1 experimental studies to objectively assess individual treatment efficacy and individualize patient care.34,35 prototype studies have provided proof of concept so that these methods can be further expanded to enable more robust learning designed to improve symptoms and qol for pediatric patients with ibd and others with differing chronic health conditions ."]]}
{"article_id": "PMC3010668", "article_text": ["the vast majority of cancers in the oral cavity and in the head and neck are squamous cell carcinomas ( sccs ) .", "it is the sixth most common cancer worldwide , and its incidence is rising in industrialized nations [ 1 , 2 ] .", "similarly , other benign lesions of the oral cavity such as lichen planus may have a prevalence of 0.52% in the general population and may have a risk of malignant transformation of 1% .", "many benign oral mucosal lesions are not cancerous which presents a clinical dilemma to the physician .", "furthermore , precancerous lesions such as leukoplakia may exhibit mild structural alterations in the mucosa that can be difficult to distinguish from normal healthy tissue .", "currently , obtaining histopathology via biopsy is the gold standard of diagnosis ; however , this procedure can pose significant morbidity to the patient such as the risk of bleeding , wound infection , and potentially impairment of speech and swallowing if multiple biopsies are performed .", "moreover , it becomes a clinical challenge to monitor patients for progression of diffuse dysplasia or leukoplakia , and many of them may require multiple biopsies over many years .", "the discomfort of biopsy and compromisation of tissue integrity can lead to problems with future biopsy interpretation or in the case of laryngeal biopsy , considerable problems in individuals with high vocal demands .", "subsequently , any technique that can yield histopathological information without injuring tissue has obvious advantages over biopsy .", "detailed examinations of the texture , color , contour , and extent of mucosal lesions have been performed utilizing many instruments such as the hopkins ' rod - lens scopes , flexible endoscopes , direct laryngoscopes , and advances in microlaryngoscopic visualization techniques . however , these methods are limited by their inability to provide histopathological data during the clinical examination . as a result , over the last decade", ", technological advances in optical imaging detection techniques have emerged with a variety of methods employed to facilitate detailed examination and provision of histopathological information of mucosal lesions .", "examples of such novel optical techniques include : aminolevulinic acid - induced fluorecence , autofluorescence , confocal endomicroscopy , and contact endoscopy .", "aminolevulinic acid - induced fluorescence is a technique whereby neoplastic cells undergo preferential fluorescence after aminolevulinic acid ( ala ) has been applied to the mucosa surface . in the presence of ala ,", "once this  dye - like  substance has been applied , mucosa containing neoplastic cells will fluoresce orange red and normal mucosa will retain the normal green fluorescence .", "coupled with autofluorescence , several authors have noted that these techniques can diagnose laryngeal carcinoma and dysplasia with good accuracy .", "autofluorescence was first described in identification of neoplastic cells of the larynx by harris et al . .", "certain molecules then transform into photonic energy , which is emitted as long - wave scattered light which can be detected .", "the autofluorescence imaging method detects the fluorecence given off by the different concentrations of fluorophores seen in normal and neoplastic mucosa .", "thus , autofluorescence videoendoscopy for photodiagnosis of head and neck squamous cell carcinomas has been described as being quite accurate with good sensitivity and specificity in several studies [ 616 ] . unlike ala and autofluorescence where histological detail is not appreciated , other optical techniques such as narrow - band imaging endoscopy ( nbie )", "nbie uses filtered light with wavelengths preferentially corresponding to peaks of absorption of hemoglobin to enhance superficial neoplasms based on their neoangiogenic pattern .", "these light wavelengths penetrate superficial mucosal and deep submucosal layers to enhance capillary and submucosal vasculature .", "the obtained image is further enhanced by using high - definition television ( hdtv ) .", "carcinomas can then be identified based on the changes in the microvascular pattern of the mucosal lesion .", "several studies have shown good sensitivity , specificity , negative and positive predictive value , and accuracy in detection of squamous cell carcinoma of the oral cavity , oropharynx , larynx , and esophagus [ 1720 ] .", "however , instead of solely relying on neoangiogenic patterns for diagnosis of carcinoma , further histological detail can be obtained with the use of confocal endoscopy which is an in vivo optical imaging method whereby mucosal lesions can undergo significant magnification to allow examination of cellular histology .", "this technique also allows reconstruction of three - dimensional structures based on the acquired images .", "utilization of various stains to help highlight cellular structures has been tried by some authors to distinguish normal from invasive carcinoma cells .", "the utility of this new technology is highlighted in its capability to distinguish between benign or low - grade mucosal dysplasia thereby potentially reducing unnecessary biopsies .  ", "contact endoscopy is another novel noninvasive optical diagnostic imaging method that allows in vivo and in situ examination of the cellular architecture of the superficial layers of the mucosal epithelium .  ", "magnified images are obtained using hopkins ' rod - lens endoscope placed on the surface of the dye stained mucosal tissue .", "this technique allows assessment of precancerous and cancerous lesions in vivo and has significant potential in the histopathologic diagnosis of many suspicious head and neck mucosal lesions without tissue biopsy .   ", "ce was originally described and used by hamou in 1979 as a technique for visualization of cervical and uterine epithelial cells for screening and diagnosis of cervical and uterine pathology .", "the first reported use of ce in otolaryngology head and neck surgery was by andrea et al .", "as a diagnostic tool in the evaluation of various pathologies in the larynx in the 1990s [ 4 , 2330 ] . they were able to visualize and diagnose laryngeal mucosal pathology from the magnification of vocal fold epithelium and microvasculature during microlaryngoscopy after staining the vocal cords with methylene blue dye .  ", "diameters , of these scopes come as either 4  mm or 5.5  mm and lengths of 23  cm and 18  cm . straight forward", "( o ) and forward - oblique telescopes ( 30 ) are also available , and all are capable of 1x , 60x , and 150x magnification .", "these endoscopes require a high intensity xenon light source , and images can be digitally captured for real - time photographic and video documentation , figures 1 and 2 .", "the most basic technique of ce involves staining of the superficial cells of the mucosa with a contrast dye , 1% methylene blue ( mb ) after which the magnifying endoscope ( karl storz 8715  aa , tuttlingen , germany ) 0 is then placed in contact against the mucosal surface , and the documented magnified cytological images ( at 60x or 150x ) are then recorded , figure 3 .", "both a cytopathologist and an otolaryngologist can then assess these images , comparable to histology , figure 4 .", "contact endoscopy and its efficacy in head and neck oncology , advantages , limitations , and future potential diagnostic utility will be briefly reviewed in this article .", "the literature search was conducted using the following key terms :  contact endoscopy  ,  contact microlaryngoscopy  ,  aminolevulinic acid induced fluorecence  ,  autofluorescence  ,  confocal endomicroscopy  ,  oral mucosa  ,  oral cavity  , ", "larynx  ,  oropharynx  ,  hypopharynx  ,  head and neck carcinoma  ,  leukoplakia  , and  lichen planus .", "relevant search terms and combinations using boolean operators were performed , and relevant article selection was limited to the prospective , human and english studies without restriction to year of publication .", "examined 42 consecutive patients at a tertiary care center with suspicious lesions of larynx , pharynx , and esophagus under general anesthesia .", "the results obtained by the cytopathologist and otolaryngologist were based on images generated from the ce .  ", "they found that the more experienced the examiner , the higher the sensitivity of ce was in the diagnostic differentiation of benign versus malignant mucosal lesions .", "they included 142 patients undergoing microlaryngoscopy at their institution with various laryngeal diseases all underwent ce and subsequent biopsy for histopathological diagnosis .", "all malignant lesions identified by ce was confirmed by histopathology , but ce did not identify malignancy in 10 patients diagnosed histopathologically thus giving ce a sensitivity of 79.6% , specificity of 100% , and accuracy of 93% .", "their results were based on computer - assisted analysis of all ce images based certain nuclear morphometric parameters to determine benign from malignant lesions .", "thus , based on their computer - assisted analysis of ce images , their sensitivity was 91% and specificity 81% .", "all patients were examined with contact rhinoscopes under local anesthesia and biopsy of the area under examination was done . in all 5 cases of malignancy , ce and histological diagnosis directly correlated with each other .", "most significantly , for the prediction of persistent and recurrent disease , sensitivity and specificity for ce was 100% with an accuracy of 92.1% .  ", "pilot study examined 83 patients using both autofluorescence and contact endoscopy during microlaryngoscopy . for contact endoscopy ,", "the calculated sensitivity was 94.7 , specificity of 95.5 and an accuracy of 94% . in summary ,", "authors of the above prospective trials have obtained the following results : a sensitivity of 79100% , a specificity of 81100% , and an accuracy of 8894% .", "overall , it appears that sensitivity , specificity , and accuracy of ce are similar across the trials .", "since the development of contact endoscopy , this technology has been used successfully by several authors in analyzing and diagnosing various pathologies of the larynx , oral cavity , oropharynx , and nasopharynx via  real - time ", "examination of mucosal cytological detail [ 4 , 2631 , 3336 ] . despite its introduction into otolaryngology", ", ce has yet to find a place in routine clinical practice despite its potential advantages . from the above clinical trials", ", ce appears to have good sensitivity , specificity , and accuracy as a noninvasive method for distinguishing between benign and malignant mucosal lesions in the head and neck .", "however , some authors state that it may be difficult for ce to detect mild ( grade i ) mucosal dysplasia because most of the cellular anomalies occur in at the level of the basal epithelium , and this technique can only examine cellular architecture found at the superficial epithelial layers [ 4 , 21 ] .   despite this limitation ,", "most significantly , ce accurate ability to diagnose and tease out the histological differences between squamous metaplasia , atypia , and carcinoma even in the presence of irradiated mucosa was highlighted by the study performed by pak et al . .", "at present , most authors seem to agree that it has significant potential as a noninvasive detection method that could play a role as a future substitute for histological examination .", "most significantly , it offers a noninvasive , rapid , and repeatable in vivo assessment of the cytological architecture while avoiding the need for an invasive biopsy and its associated risks .", "ce provides immediate results , with the possibility of examining multiple mucosal areas in a short time .", "ce can also assess a wider surface mucosal area , providing more information than a selected histological section taken by biopsy .", "it also avoids tissue damage and alteration of cellular architecture which may occur in the biopsy and histological preparation .", "this noninvasive technique also helps to direct the site of biopsy by identifying areas with cellular atypia and thus avoiding the need for multiple biopsies .", "subsequently , this results in a dramatic improvement of the diagnostic yield of the biopsy .", "other potential roles of ce include the rapid diagnosis of benign and malignant mucosal lesions in an outpatient or operating room setting , surveillance , guided biopsies , and intraoperative evaluation of tumor resection margins .   despite its advantages", "most notably , ce can only evaluate the most superficial cell layer of the mucosal epithelium .", "this is most likely due to a number of factors including ( i ) poor penetration of methylene blue which only stains a few superficial layers , ( ii ) short focal distance of the scope ( i.e. , ce can only assess to a depth of 80  um at 60x magnification and 30  um at 150x magnification ) , and ( iii ) optical artifact at high magnification due to glare from light reflected from cells not in focus .", "subsequently , assessment of submucosal lesions or lesions occupying deeper cell layers becomes more difficult [ 4 , 27 , 28 , 30 , 32 , 33 ] .", "the lack of depth of penetration prevents the evaluation of important histological information especially when vertical extent of dysplasia is crucial in distinguishing the different grades of dysplasia from carcinoma in situ and invasive carcinoma . as a result", ", these factors could affect the sensitivity of ce , thus accounting for some of the false negative diagnostic results noted by authors .", "the potential impact of ce missing a malignant lesion needs to be taken into consideration if this technology is to one day substitute histopathology .", "future investigation into better penetrating dyes , advances in digital optics , and image enhancements will eventually allow better vertical staining and increased resolution of the deeper cell layers which would translate ce in becoming a much more sensitive and accurate diagnostic tool .   a pilot study conducted at our institution also investigated some of the limitations and potential advantages of ce in the evaluation of head and neck mucosal lesions . from our preliminary experience , technical difficulties with line of sight , access difficulties to mucosal surfaces ,", "scope positioning , and problems with consistent image quality due to artifact were consistent with those found by previous authors [ 30 , 33 ] .  ", "our pilot study also demonstrated that although ce is a simple , rapid , repeatable , noninvasive examination performed with standard equipment , there is a learning curve associated with its use .", "however , once one is accustomed with this detection system , ce can be performed almost as quickly as an outpatient flexible fiberoptic nasopharyngoscopic examination .   in conclusion , in vivo assessment of head and neck mucosal pathology may be applied to ( i ) early detection of premalignant and malignant lesions , ( ii ) serial follow - up examinations of suspicious lesions such as leukoplakia and lichen planus , and ( iii ) assessment of resection margins . despite its limitations", ", ce represents a promising optical technology that may afford reliable , accurate , and noninvasive in vivo assessment of cytological pathology . prospective investigation with ce", "we hypothesize that future study will demonstrate improved sensitivity , specificity , and accuracy of contact endoscopy in the diagnosis of head and neck tumors ."], "abstract_text": ["<S> \n background . </S>", "<S> there are a variety of described noninvasive optical detection techniques for evaluation of head and neck mucosal lesions . </S>", "<S> contact endoscopy is a promising method of in vivo microscopic examination whereby a rigid telescope is placed on a previously dye - stained mucosa allowing evaluation of the superficial cell layers of the epithelium . </S>", "<S> this technique produces real - time , magnified images of cellular architecture of surface mucosa comparable to histology without the need for biopsy . in this review </S>", "<S> , we will briefly summarize the efficacy of ce in the detection of precancerous and cancerous mucosal lesions and its potential as a novel technique in early diagnosis , monitoring , and preoperative assessment of mucosal lesions of the head and neck . </S>", "<S> methods . </S>", "<S> pubmed , medline , and cochrane search revealed five prospective articles on contact endoscopy for the diagnosis of mucosal lesions in the head and neck . </S>", "<S> results . </S>", "<S> the literature search yielded five prospective studies examining contact endoscopy for the diagnosis of benign versus malignant head and neck mucosal lesions . </S>", "<S> these reported a sensitivity and specificity of 77100% , specificity of 66100% and an accuracy of 7292% . </S>", "<S> conclusion . </S>", "<S> contact endoscopy is a promising optical technology that may be a useful adjunct in the evaluation and diagnosis of benign and malignant head and neck mucosal lesions . </S>", "<S> future prospective randomized double - blind studies of this detection method are required . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion"], "sections": [["the vast majority of cancers in the oral cavity and in the head and neck are squamous cell carcinomas ( sccs ) .", "it is the sixth most common cancer worldwide , and its incidence is rising in industrialized nations [ 1 , 2 ] .", "similarly , other benign lesions of the oral cavity such as lichen planus may have a prevalence of 0.52% in the general population and may have a risk of malignant transformation of 1% .", "many benign oral mucosal lesions are not cancerous which presents a clinical dilemma to the physician .", "furthermore , precancerous lesions such as leukoplakia may exhibit mild structural alterations in the mucosa that can be difficult to distinguish from normal healthy tissue .", "currently , obtaining histopathology via biopsy is the gold standard of diagnosis ; however , this procedure can pose significant morbidity to the patient such as the risk of bleeding , wound infection , and potentially impairment of speech and swallowing if multiple biopsies are performed .", "moreover , it becomes a clinical challenge to monitor patients for progression of diffuse dysplasia or leukoplakia , and many of them may require multiple biopsies over many years .", "the discomfort of biopsy and compromisation of tissue integrity can lead to problems with future biopsy interpretation or in the case of laryngeal biopsy , considerable problems in individuals with high vocal demands .", "subsequently , any technique that can yield histopathological information without injuring tissue has obvious advantages over biopsy .", "detailed examinations of the texture , color , contour , and extent of mucosal lesions have been performed utilizing many instruments such as the hopkins ' rod - lens scopes , flexible endoscopes , direct laryngoscopes , and advances in microlaryngoscopic visualization techniques . however , these methods are limited by their inability to provide histopathological data during the clinical examination . as a result , over the last decade", ", technological advances in optical imaging detection techniques have emerged with a variety of methods employed to facilitate detailed examination and provision of histopathological information of mucosal lesions .", "examples of such novel optical techniques include : aminolevulinic acid - induced fluorecence , autofluorescence , confocal endomicroscopy , and contact endoscopy .", "aminolevulinic acid - induced fluorescence is a technique whereby neoplastic cells undergo preferential fluorescence after aminolevulinic acid ( ala ) has been applied to the mucosa surface . in the presence of ala ,", "once this  dye - like  substance has been applied , mucosa containing neoplastic cells will fluoresce orange red and normal mucosa will retain the normal green fluorescence .", "coupled with autofluorescence , several authors have noted that these techniques can diagnose laryngeal carcinoma and dysplasia with good accuracy .", "autofluorescence was first described in identification of neoplastic cells of the larynx by harris et al . .", "certain molecules then transform into photonic energy , which is emitted as long - wave scattered light which can be detected .", "the autofluorescence imaging method detects the fluorecence given off by the different concentrations of fluorophores seen in normal and neoplastic mucosa .", "thus , autofluorescence videoendoscopy for photodiagnosis of head and neck squamous cell carcinomas has been described as being quite accurate with good sensitivity and specificity in several studies [ 616 ] . unlike ala and autofluorescence where histological detail is not appreciated , other optical techniques such as narrow - band imaging endoscopy ( nbie )", "nbie uses filtered light with wavelengths preferentially corresponding to peaks of absorption of hemoglobin to enhance superficial neoplasms based on their neoangiogenic pattern .", "these light wavelengths penetrate superficial mucosal and deep submucosal layers to enhance capillary and submucosal vasculature .", "the obtained image is further enhanced by using high - definition television ( hdtv ) .", "carcinomas can then be identified based on the changes in the microvascular pattern of the mucosal lesion .", "several studies have shown good sensitivity , specificity , negative and positive predictive value , and accuracy in detection of squamous cell carcinoma of the oral cavity , oropharynx , larynx , and esophagus [ 1720 ] .", "however , instead of solely relying on neoangiogenic patterns for diagnosis of carcinoma , further histological detail can be obtained with the use of confocal endoscopy which is an in vivo optical imaging method whereby mucosal lesions can undergo significant magnification to allow examination of cellular histology .", "this technique also allows reconstruction of three - dimensional structures based on the acquired images .", "utilization of various stains to help highlight cellular structures has been tried by some authors to distinguish normal from invasive carcinoma cells .", "the utility of this new technology is highlighted in its capability to distinguish between benign or low - grade mucosal dysplasia thereby potentially reducing unnecessary biopsies .  ", "contact endoscopy is another novel noninvasive optical diagnostic imaging method that allows in vivo and in situ examination of the cellular architecture of the superficial layers of the mucosal epithelium .  ", "magnified images are obtained using hopkins ' rod - lens endoscope placed on the surface of the dye stained mucosal tissue .", "this technique allows assessment of precancerous and cancerous lesions in vivo and has significant potential in the histopathologic diagnosis of many suspicious head and neck mucosal lesions without tissue biopsy .   ", "ce was originally described and used by hamou in 1979 as a technique for visualization of cervical and uterine epithelial cells for screening and diagnosis of cervical and uterine pathology .", "the first reported use of ce in otolaryngology head and neck surgery was by andrea et al .", "as a diagnostic tool in the evaluation of various pathologies in the larynx in the 1990s [ 4 , 2330 ] . they were able to visualize and diagnose laryngeal mucosal pathology from the magnification of vocal fold epithelium and microvasculature during microlaryngoscopy after staining the vocal cords with methylene blue dye .  ", "diameters , of these scopes come as either 4  mm or 5.5  mm and lengths of 23  cm and 18  cm . straight forward", "( o ) and forward - oblique telescopes ( 30 ) are also available , and all are capable of 1x , 60x , and 150x magnification .", "these endoscopes require a high intensity xenon light source , and images can be digitally captured for real - time photographic and video documentation , figures 1 and 2 .", "the most basic technique of ce involves staining of the superficial cells of the mucosa with a contrast dye , 1% methylene blue ( mb ) after which the magnifying endoscope ( karl storz 8715  aa , tuttlingen , germany ) 0 is then placed in contact against the mucosal surface , and the documented magnified cytological images ( at 60x or 150x ) are then recorded , figure 3 .", "both a cytopathologist and an otolaryngologist can then assess these images , comparable to histology , figure 4 .", "contact endoscopy and its efficacy in head and neck oncology , advantages , limitations , and future potential diagnostic utility will be briefly reviewed in this article ."], ["the literature search was conducted using the following key terms :  contact endoscopy  ,  contact microlaryngoscopy  ,  aminolevulinic acid induced fluorecence  ,  autofluorescence  ,  confocal endomicroscopy  ,  oral mucosa  ,  oral cavity  , ", "larynx  ,  oropharynx  ,  hypopharynx  ,  head and neck carcinoma  ,  leukoplakia  , and  lichen planus .", "relevant search terms and combinations using boolean operators were performed , and relevant article selection was limited to the prospective , human and english studies without restriction to year of publication ."], ["examined 42 consecutive patients at a tertiary care center with suspicious lesions of larynx , pharynx , and esophagus under general anesthesia .", "the results obtained by the cytopathologist and otolaryngologist were based on images generated from the ce .  ", "they found that the more experienced the examiner , the higher the sensitivity of ce was in the diagnostic differentiation of benign versus malignant mucosal lesions .", "they included 142 patients undergoing microlaryngoscopy at their institution with various laryngeal diseases all underwent ce and subsequent biopsy for histopathological diagnosis .", "all malignant lesions identified by ce was confirmed by histopathology , but ce did not identify malignancy in 10 patients diagnosed histopathologically thus giving ce a sensitivity of 79.6% , specificity of 100% , and accuracy of 93% .", "their results were based on computer - assisted analysis of all ce images based certain nuclear morphometric parameters to determine benign from malignant lesions .", "thus , based on their computer - assisted analysis of ce images , their sensitivity was 91% and specificity 81% .", "all patients were examined with contact rhinoscopes under local anesthesia and biopsy of the area under examination was done . in all 5 cases of malignancy , ce and histological diagnosis directly correlated with each other .", "most significantly , for the prediction of persistent and recurrent disease , sensitivity and specificity for ce was 100% with an accuracy of 92.1% .  ", "pilot study examined 83 patients using both autofluorescence and contact endoscopy during microlaryngoscopy . for contact endoscopy ,", "the calculated sensitivity was 94.7 , specificity of 95.5 and an accuracy of 94% . in summary ,", "authors of the above prospective trials have obtained the following results : a sensitivity of 79100% , a specificity of 81100% , and an accuracy of 8894% .", "overall , it appears that sensitivity , specificity , and accuracy of ce are similar across the trials ."], ["since the development of contact endoscopy , this technology has been used successfully by several authors in analyzing and diagnosing various pathologies of the larynx , oral cavity , oropharynx , and nasopharynx via  real - time ", "examination of mucosal cytological detail [ 4 , 2631 , 3336 ] . despite its introduction into otolaryngology", ", ce has yet to find a place in routine clinical practice despite its potential advantages . from the above clinical trials", ", ce appears to have good sensitivity , specificity , and accuracy as a noninvasive method for distinguishing between benign and malignant mucosal lesions in the head and neck .", "however , some authors state that it may be difficult for ce to detect mild ( grade i ) mucosal dysplasia because most of the cellular anomalies occur in at the level of the basal epithelium , and this technique can only examine cellular architecture found at the superficial epithelial layers [ 4 , 21 ] .   despite this limitation ,", "most significantly , ce accurate ability to diagnose and tease out the histological differences between squamous metaplasia , atypia , and carcinoma even in the presence of irradiated mucosa was highlighted by the study performed by pak et al . .", "at present , most authors seem to agree that it has significant potential as a noninvasive detection method that could play a role as a future substitute for histological examination .", "most significantly , it offers a noninvasive , rapid , and repeatable in vivo assessment of the cytological architecture while avoiding the need for an invasive biopsy and its associated risks .", "ce provides immediate results , with the possibility of examining multiple mucosal areas in a short time .", "ce can also assess a wider surface mucosal area , providing more information than a selected histological section taken by biopsy .", "it also avoids tissue damage and alteration of cellular architecture which may occur in the biopsy and histological preparation .", "this noninvasive technique also helps to direct the site of biopsy by identifying areas with cellular atypia and thus avoiding the need for multiple biopsies .", "subsequently , this results in a dramatic improvement of the diagnostic yield of the biopsy .", "other potential roles of ce include the rapid diagnosis of benign and malignant mucosal lesions in an outpatient or operating room setting , surveillance , guided biopsies , and intraoperative evaluation of tumor resection margins .   despite its advantages", "most notably , ce can only evaluate the most superficial cell layer of the mucosal epithelium .", "this is most likely due to a number of factors including ( i ) poor penetration of methylene blue which only stains a few superficial layers , ( ii ) short focal distance of the scope ( i.e. , ce can only assess to a depth of 80  um at 60x magnification and 30  um at 150x magnification ) , and ( iii ) optical artifact at high magnification due to glare from light reflected from cells not in focus .", "subsequently , assessment of submucosal lesions or lesions occupying deeper cell layers becomes more difficult [ 4 , 27 , 28 , 30 , 32 , 33 ] .", "the lack of depth of penetration prevents the evaluation of important histological information especially when vertical extent of dysplasia is crucial in distinguishing the different grades of dysplasia from carcinoma in situ and invasive carcinoma . as a result", ", these factors could affect the sensitivity of ce , thus accounting for some of the false negative diagnostic results noted by authors .", "the potential impact of ce missing a malignant lesion needs to be taken into consideration if this technology is to one day substitute histopathology .", "future investigation into better penetrating dyes , advances in digital optics , and image enhancements will eventually allow better vertical staining and increased resolution of the deeper cell layers which would translate ce in becoming a much more sensitive and accurate diagnostic tool .   a pilot study conducted at our institution also investigated some of the limitations and potential advantages of ce in the evaluation of head and neck mucosal lesions . from our preliminary experience , technical difficulties with line of sight , access difficulties to mucosal surfaces ,", "scope positioning , and problems with consistent image quality due to artifact were consistent with those found by previous authors [ 30 , 33 ] .  ", "our pilot study also demonstrated that although ce is a simple , rapid , repeatable , noninvasive examination performed with standard equipment , there is a learning curve associated with its use .", "however , once one is accustomed with this detection system , ce can be performed almost as quickly as an outpatient flexible fiberoptic nasopharyngoscopic examination .   in conclusion , in vivo assessment of head and neck mucosal pathology may be applied to ( i ) early detection of premalignant and malignant lesions , ( ii ) serial follow - up examinations of suspicious lesions such as leukoplakia and lichen planus , and ( iii ) assessment of resection margins . despite its limitations", ", ce represents a promising optical technology that may afford reliable , accurate , and noninvasive in vivo assessment of cytological pathology . prospective investigation with ce", "we hypothesize that future study will demonstrate improved sensitivity , specificity , and accuracy of contact endoscopy in the diagnosis of head and neck tumors ."]]}
{"article_id": "PMC4207111", "article_text": ["the first historical study of quality assessment in the medical field was reported by ernest amory codman , md , of massachusetts general hospital in 1920 . to support his  end results", "theory, he made public the results of the review of his own hospital in a privately published book ,  a study in hospital efficiency ,  in which he emphasized the importance of patient follow - up and quality assessment .", "he helped to found the hospital standardization program , which eventually became the joint commission on accreditation of healthcare organizations in 1987 , and the joint commission in 2007 , with the motto  helping health care organizations help patients. from the 1970s through the 1980s , the rapid increase in medical lawsuits and the medical malpractice insurance crisis promoted risk management of medical practices in the united states . in 1989 , the society for thoracic surgeons ( sts ) started to establish national databases   as an initiative to improve quality and patient safety among cardiothoracic surgeons and to respond to strong public opinion about the importance of accountability . in 1997 , an initiative was begun to improve data quality and auditing , and staff were hired to support these efforts . in the sts congenital heart surgery", "database data specification , ( http://www.sts.org/sites/default/files/documents/congenitaldataspecsv3_22.pdf ) , the patient national identification ( social security number ) is listed , but this field should be collected in compliance with state / local privacy laws .", "the sts national database complies with the health insurance portability and accountability act , and the federal government protects the sts national database . in 1998 , the sts contracted with the duke clinical research institute ( dcri ) for data warehousing and data analysis . in 1999 , the institute of medicine published a report titled  to err is human : building a safe health system ,  which stated that 44,000 to 98,000 persons die in hospitals as a result of medical errors that could have been prevented .", "today , the management of the national database is one of the most important tasks of the sts .", "the database contains three components : adult cardiac surgery , general thoracic surgery and congenital heart surgery ( fig .", "the sts was the first professional organization to seek approval for its measures from the national quality forum ( nqf ) , a multi - stakeholder health policy organization head - quartered in washington , dc . in this manner , the sts has gained a positive reputation with the government and with health policy organizations .", "in addition , in 2010 , the sts started to publicly report isolated coronary artery bypass grafting ( cabg ) composite star ratings not only on its own website but also on a consumer report website ( www.consumerreportshealth.org ) . later , public reporting of aortic valve surgery ( avr ) and cabg+avr began , and this year , will be extended to congenital heart surgery .", "the nqf has been releasing quality indicators in medical fields under the rubric of  nqf - endorsed standards ( http://www.quality-forum.org/home.aspx ) .", "participation in the sts national database,  operative mortality stratified by the five sts - eacts ( european association for cardiothoracic surgery ) mortality categories, and  risk adjustment in congenital heart surgery ( rachs-1 ) pediatric heart surgery mortality . the sts states on its website that  sts believes the public has a right to know the quality of the surgical outcomes , and considers public reporting an ethical responsibility of the specialty .", "in turn , in 1998 , at the 7th annual meeting of the asian society for cardiovascular and thoracic surgery in singapore , the need for an asian cardiovascular surgery database was discussed .", "first , a database ad hoc committee was formed by the japanese society for cardiovascular surgery ( jscvs ) and the japanese association for thoracic surgery ( jats ) ( table 1 ) .", "moreover , quality improvement of cardiovascular surgery has been discussed by the members of the board of jscvs and jats since early 2000 . in pursuit of this goal ,", "three committees were organized by the jscvs and jats among its academic groups : 1 ) a board certification committee , 2 ) a center aggregation committee , and 3 ) a nurse practitioner and physician assistant committee . in 2000 , before this movement , the japan cardiovascular surgery database ( jcvsd ) was established with close ties to the jscvs and jats .", "the jcvsd and jscvs invited the founder of the sts national database to discuss starting the construction of the database", ". the jcvsd established input items comparable to those of the sts national database . in the congenital heart surgery database , the common terminologies and the definitions of congenital heart diseases published in the  annals of thoracic surgery  were adopted , and 193 input items were established in the japan congenital cardiovascular surgery database ( jccvsd ) .", "thus , the congenital heart surgery databases in the united states , europe , and japan were integrated by using common language in these databases . as a result ,", "although the results were not reported , for example , the discharge mortality in the jccvsd was 0.2% , 0.7% , 3.6% , 7% , and 17.6% for rachs-1 categories 1 , 2 , 3 , 4 , and 5/6 , respectively , during 2008 to 2010 ( fig .", "this result is comparable to that reported from the sts congenital heart surgery database . unlike the sts national database , the jcvsd employed web - based data collection .", "data on adult cardiac surgery ( japan adult cardiovascular surgery database [ jacvsd ] ) were collected beginning in 2001 by five participating units and data on congenital heart surgery ( jccvsd ) were collected beginning in 2008 by seven units .", "jcvsd required informed consent from each patient according to the  opt - in rule to comply with the private information protection law . for web - data transmission ,", "high level secure socket layer was adopted for coding of the individual patient s information .", "the jacvsd and jccvsd grew to become national databases by the end of 2013 ( fig .", "the most recent annual number of submitted procedures are 49,507 in jacvsd and 10,835 in jccvsd .", "twenty frequently cited papers dealing with topics such as risk models of isolated coronary bypass surgery , thoracic aortic surgery , and valve surgery   have been published in indexed international journals .", "the performance of the congenital heart surgery risk model as measured by the c - index is over 0.8 . on the basis of these risk models ,", ", adult cardiac surgeons can estimate the 30-day mortality rate , in - hospital mortality rate , and major complication rate after inputting the patient s covariates before the surgical procedure .", "japanscore contributes to obtaining adequate informed consent from the patients and the families , leading to increased satisfaction . in addition , benchmark reports have been released as support tools for quality improvement of participating institutions . in japan , many adult cardiac surgeons learn about the risks faced by their patients , as well as their own performance as a surgeon , through the risk - adjusted mortality and benchmark report . to ensure fairness and transparency in evidence - based medicine ( ebm ) , the jcvsd organized a data access and usage working group .", "this working group meets twice a year , and requests 100% of their data during at least for the immediate 2-years . after the working group accepts an application , the department of health quality assessment ( hqa ) of the university of tokyo   analyzes the newly submitted data .", "the role of the hqa is similar to that of the dcri for the sts national database .", "the members of the working group include two to three adult cardiac surgeons ; visits to 70 sites have been carried out so far .", "recently the hqa reported the details and outcomes of the site visits to the jccvsd . in 2011 ,", "the jcvsd started to collect a participation fee of 10,000 yen per year for each section in the jacvsd and the jccvsd .", "the total number of sections was 658 ( 541 in the jacvsd , 117 in the jccvsd ) by the end of april 2014 .", "this participation fee is much lower than that required by the sts national database ; however , it is an important financial resource , especially for site visits . in 2011 , the japanese board of cardiovascular surgery ( jbcvs ) decided to adopt the data of the jacvsd and the jccvsd for board certification . in 2013 , there were 162 new applicants and 1,003 renewals .", "the jbcvs held its first web - based and paperless review in september 2013 . compared with the previously employed review method that relied on the submission of operation records , the web - based and paperless review method had higher quality , lower cost , and required less time", "in 2010 , the jcvsd served as the basis for the establishment of the national clinical database ( ncd ) in japan , which includes clinician - initiated databases reflecting all surgical fields .", "data collection with the same security level of jcvsd as mentioned above to protect the individual patient s information . through the central institutional review board in the university of tokyo , an  opt - out rule was adopted , and informed consent became unnecessary .", "the ncd is governed by a committee whose members are representatives of medical associations related to surgery , such as the japan surgical society ( jss ) , jscvs , jats , the japanese association for chest surgery , the japanese society of gastroenterological surgery , the japanese society of pediatric surgeons , the japanese society of vascular surgery , the japanese society of endocrine surgery , the japanese society for mammary cancer , and the japanese thyroid association .", "the ncd establishes the surgical board certification system for the jss , which requires 13 input items at the first level in the hierarchy of specialties .", "six board certification systems , including the jbcvs and the databases of nine academic associations , are set at the second level as subspecialties .", "the main server was transferred from the hqa to the university hospital medical information network ( umin ) with a mirror - image backup .", "the hqa focused on data analysis and site visits , whereas the umin is responsible for data warehousing .", "the ncd uses cutting - edge statistical techniques to detect any trace of data inconsistency .", "the participating associations have supported the ncd financially and the database has grown rapidly ; the total number of participating hospitals is 4105 , and the number of cumulative procedures was 4,138,000 at the end of april 2014 .", "the participating associations will release , or have released , their own risk models [ 1618 ] , and papers have been published based on data from the ncd .", "the administrative database , diagnosis procedure combination ( dpc)/par - diem payment system ( pdps ) was introduced in japan by the japanese ministry of health , labour and welfare ( mhlw , a government agency ) in april 2003 to comprehensively assess fixed daily payments and to control medical expenditure in the acute setting based on the quality assessment .", "the number of participating hospitals by the end of april 2014 was 1,585 , including all advanced - treatment hospitals , that is , university hospitals .", "in japan , total health care expenditures have been increasing by 1 trillion yen annually , and health care expenditures make up 9.5% of the gross domestic product , which puts japan in the 16th position of the 34 member countries of the organization for economic cooperation and development . on the other hand ,", "the population aging rate in japan is over 24% , which is the highest rate in the world .", "changes in population makeup and the growing proportion of elderly persons are the underlying issues relating to rising health care expenditures , and successive cabinet office members and the mhlw have set policy directions to address this national issue .", "quality improvement , quality assessment , and the pay - for - performance system provide methods to control medical expenditures .", "the quality and outcomes framework ( qof ) , a system for the performance management and payment of genaral practitioners ( gps ) in the national health service in england , wales , scotland and northern ireland was introduced as part of the new general medical services contract in april 2004 .", "in contrast , in the united states , the agency of healthcare research and quality ( ahrq ) has defined ", "never events or errors of medical care for which medicare , the government healthcare insurance for aged and disabled persons , does not pay . in the c.", "walton lillehei lecture of the 49th sts annual meeting , the director of the ahrq emphasized that the federal government will pay for the quality , not for the volume . in japan ,", "the ncd and dpc / pdps could play complementary functions for quality assessment through adequate risk adjustment and the complete enumeration of procedures in various surgical fields . in the future ,", "balancing professional autonomy and administrative leadership might be a recurrent issue for quality assessment and quality improvement in japan .", "recently , the japanese association of cardiovascular intervention and therapeutics proposed to the ncd a comparative study between percutaneous coronary intervention and cabg that would use well - tested statistical methods such as propensity score matching .", "thus , the participation of units from nonsurgical fields , such as medical therapy , intervention , radiation therapy , and chemotherapy , will facilitate risk stratification of each treatment modality , and will contribute to the search for the best management of diseases and patients .", "a longitudinal follow - up database is needed for the design of such studies , and it is under construction .", "recently , the pharmaceutical and medical device agency ( pmda ) , a consultative organization of the mhlw , suggested to enroll in the jacvsd and perform follow - ups on the use of artificial valves for trans - aortic valve implantation .", "the pmda recognized the completeness and reliability of the data of the jacvsd , and from a cost - performance point of view , the pmda decided to outsource the post - market surveillance of newly covered medical devices in the cardiovascular surgical field . this demonstrates how the national database could contribute to the post - marketing surveillance of drugs and medical devices , and could help control randomized trials and multicenter studies .", "the ncd will start to collect fees from participating hospitals according to the total number of enrolled surgical procedures .", "clerical assistants have been widely employed throughout the country , which has gradually lightened the data input workload of young surgeons . governmental support and some government funds", "clinicians are responsible for patient safety and quality improvement , and the database will aid in achieving these goals . as reinertsen   stated , to truly improve quality , the system must , 1 ) eliminate unnecessary variation ( standardize processes ) , and 2 ) achieve and document continuous improvement ( in care processes and outcomes ) . in recent years , the importance of  certainty , not excellence of operations , and that of the concept of structure , process and outcome   have been emphasized , and multiple approaches , for instance , postgraduate education systems , reporting systems of malpractice to prevent recurrence , introduction of information technology , introduction of simulators , ebm , and other techniques , have been used for patient safety .", "since it is methodologically based on the jcvsd , the ncd represents an interface between medical databases and board certification systems , which is its point of difference from the sts national database . in 2014 , a new organization for medical board certification was established in japan that , beginning in 2017 , will certify all medical boards in close collaboration with medical associations .", "this new organization will adopt the standards of the jcvsd and the ncd for evaluating the clinical practices of applicants .", "for the assessment of medical outcomes and quality , the jcvsd and the ncd will continue to be the sole reliable data source for surgical fields in japan , where medical system reform will be implemented quickly and based on professional autonomy .", "the national database is fundamental for quality improvement , patient safety , and the adequate control of medical expenditures in the country ."], "abstract_text": ["<S> the jcvsd ( japan cardiovascular surgery database ) was organized in 2000 to improve the quality of cardiovascular surgery in japan . </S>", "<S> web - based data harvesting on adult cardiac surgery was started ( japan adult cardiovascular surgery database , jacvsd ) in 2001 , and on congenital heart surgery ( japan congenital cardiovascular surgery database , jccvsd ) in 2008 . </S>", "<S> both databases grew to become national databases by the end of 2013 . </S>", "<S> this was influenced by the success of the society for thoracic surgeons national database , which contains comparable input items . in 2011 </S>", "<S> , the japanese board of cardiovascular surgery announced that the jacvsd and jccvsd data are to be used for board certification , which improved the quality of the first paperless and web - based board certification review undertaken in 2013 . </S>", "<S> these changes led to a further step . in 2011 , the national clinical database ( ncd ) was organized to investigate the feasibility of clinical databases in other medical fields , especially surgery . in the ncd , </S>", "<S> the board certification system of the japan surgical society , the basic association of surgery was set as the first level in the hierarchy of specialties , and nine associations and six board certification systems were set at the second level as subspecialties . </S>", "<S> the ncd grew rapidly , and now covers 95% of total surgical procedures . </S>", "<S> the participating associations will release or have released risk models , and studies that use  </S>", "<S> big data from these databases have been published . </S>", "<S> the national databases have contributed to evidence - based medicine , to the accountability of medical professionals , and to quality assessment and quality improvement of surgery in japan . </S>"], "labels": null, "section_names": ["INTRODUCTION: THE SOCIETY FOR THORACIC SURGEONS NATIONAL DATABASE", "THE JAPAN CARDIOVASCULAR SURGERY DATABASE", "THE NATIONAL CLINICAL DATABASE", "NATIONAL CLINICAL DATABASES AND HEALTH SERVICES", "PERSPECTIVES", "CONCLUSION"], "sections": [["the first historical study of quality assessment in the medical field was reported by ernest amory codman , md , of massachusetts general hospital in 1920 . to support his  end results", "theory, he made public the results of the review of his own hospital in a privately published book ,  a study in hospital efficiency ,  in which he emphasized the importance of patient follow - up and quality assessment .", "he helped to found the hospital standardization program , which eventually became the joint commission on accreditation of healthcare organizations in 1987 , and the joint commission in 2007 , with the motto  helping health care organizations help patients. from the 1970s through the 1980s , the rapid increase in medical lawsuits and the medical malpractice insurance crisis promoted risk management of medical practices in the united states . in 1989 , the society for thoracic surgeons ( sts ) started to establish national databases   as an initiative to improve quality and patient safety among cardiothoracic surgeons and to respond to strong public opinion about the importance of accountability . in 1997 , an initiative was begun to improve data quality and auditing , and staff were hired to support these efforts . in the sts congenital heart surgery", "database data specification , ( http://www.sts.org/sites/default/files/documents/congenitaldataspecsv3_22.pdf ) , the patient national identification ( social security number ) is listed , but this field should be collected in compliance with state / local privacy laws .", "the sts national database complies with the health insurance portability and accountability act , and the federal government protects the sts national database . in 1998 , the sts contracted with the duke clinical research institute ( dcri ) for data warehousing and data analysis . in 1999 , the institute of medicine published a report titled  to err is human : building a safe health system ,  which stated that 44,000 to 98,000 persons die in hospitals as a result of medical errors that could have been prevented .", "today , the management of the national database is one of the most important tasks of the sts .", "the database contains three components : adult cardiac surgery , general thoracic surgery and congenital heart surgery ( fig .", "the sts was the first professional organization to seek approval for its measures from the national quality forum ( nqf ) , a multi - stakeholder health policy organization head - quartered in washington , dc . in this manner , the sts has gained a positive reputation with the government and with health policy organizations .", "in addition , in 2010 , the sts started to publicly report isolated coronary artery bypass grafting ( cabg ) composite star ratings not only on its own website but also on a consumer report website ( www.consumerreportshealth.org ) . later , public reporting of aortic valve surgery ( avr ) and cabg+avr began , and this year , will be extended to congenital heart surgery .", "the nqf has been releasing quality indicators in medical fields under the rubric of  nqf - endorsed standards ( http://www.quality-forum.org/home.aspx ) .", "participation in the sts national database,  operative mortality stratified by the five sts - eacts ( european association for cardiothoracic surgery ) mortality categories, and  risk adjustment in congenital heart surgery ( rachs-1 ) pediatric heart surgery mortality . the sts states on its website that  sts believes the public has a right to know the quality of the surgical outcomes , and considers public reporting an ethical responsibility of the specialty ."], ["in turn , in 1998 , at the 7th annual meeting of the asian society for cardiovascular and thoracic surgery in singapore , the need for an asian cardiovascular surgery database was discussed .", "first , a database ad hoc committee was formed by the japanese society for cardiovascular surgery ( jscvs ) and the japanese association for thoracic surgery ( jats ) ( table 1 ) .", "moreover , quality improvement of cardiovascular surgery has been discussed by the members of the board of jscvs and jats since early 2000 . in pursuit of this goal ,", "three committees were organized by the jscvs and jats among its academic groups : 1 ) a board certification committee , 2 ) a center aggregation committee , and 3 ) a nurse practitioner and physician assistant committee . in 2000 , before this movement , the japan cardiovascular surgery database ( jcvsd ) was established with close ties to the jscvs and jats .", "the jcvsd and jscvs invited the founder of the sts national database to discuss starting the construction of the database", ". the jcvsd established input items comparable to those of the sts national database . in the congenital heart surgery database , the common terminologies and the definitions of congenital heart diseases published in the  annals of thoracic surgery  were adopted , and 193 input items were established in the japan congenital cardiovascular surgery database ( jccvsd ) .", "thus , the congenital heart surgery databases in the united states , europe , and japan were integrated by using common language in these databases . as a result ,", "although the results were not reported , for example , the discharge mortality in the jccvsd was 0.2% , 0.7% , 3.6% , 7% , and 17.6% for rachs-1 categories 1 , 2 , 3 , 4 , and 5/6 , respectively , during 2008 to 2010 ( fig .", "this result is comparable to that reported from the sts congenital heart surgery database . unlike the sts national database , the jcvsd employed web - based data collection .", "data on adult cardiac surgery ( japan adult cardiovascular surgery database [ jacvsd ] ) were collected beginning in 2001 by five participating units and data on congenital heart surgery ( jccvsd ) were collected beginning in 2008 by seven units .", "jcvsd required informed consent from each patient according to the  opt - in rule to comply with the private information protection law . for web - data transmission ,", "high level secure socket layer was adopted for coding of the individual patient s information .", "the jacvsd and jccvsd grew to become national databases by the end of 2013 ( fig .", "the most recent annual number of submitted procedures are 49,507 in jacvsd and 10,835 in jccvsd .", "twenty frequently cited papers dealing with topics such as risk models of isolated coronary bypass surgery , thoracic aortic surgery , and valve surgery   have been published in indexed international journals .", "the performance of the congenital heart surgery risk model as measured by the c - index is over 0.8 . on the basis of these risk models ,", ", adult cardiac surgeons can estimate the 30-day mortality rate , in - hospital mortality rate , and major complication rate after inputting the patient s covariates before the surgical procedure .", "japanscore contributes to obtaining adequate informed consent from the patients and the families , leading to increased satisfaction . in addition , benchmark reports have been released as support tools for quality improvement of participating institutions . in japan , many adult cardiac surgeons learn about the risks faced by their patients , as well as their own performance as a surgeon , through the risk - adjusted mortality and benchmark report . to ensure fairness and transparency in evidence - based medicine ( ebm ) , the jcvsd organized a data access and usage working group .", "this working group meets twice a year , and requests 100% of their data during at least for the immediate 2-years . after the working group accepts an application , the department of health quality assessment ( hqa ) of the university of tokyo   analyzes the newly submitted data .", "the role of the hqa is similar to that of the dcri for the sts national database .", "the members of the working group include two to three adult cardiac surgeons ; visits to 70 sites have been carried out so far .", "recently the hqa reported the details and outcomes of the site visits to the jccvsd . in 2011 ,", "the jcvsd started to collect a participation fee of 10,000 yen per year for each section in the jacvsd and the jccvsd .", "the total number of sections was 658 ( 541 in the jacvsd , 117 in the jccvsd ) by the end of april 2014 .", "this participation fee is much lower than that required by the sts national database ; however , it is an important financial resource , especially for site visits . in 2011 , the japanese board of cardiovascular surgery ( jbcvs ) decided to adopt the data of the jacvsd and the jccvsd for board certification . in 2013 , there were 162 new applicants and 1,003 renewals .", "the jbcvs held its first web - based and paperless review in september 2013 . compared with the previously employed review method that relied on the submission of operation records , the web - based and paperless review method had higher quality , lower cost , and required less time"], ["in 2010 , the jcvsd served as the basis for the establishment of the national clinical database ( ncd ) in japan , which includes clinician - initiated databases reflecting all surgical fields .", "data collection with the same security level of jcvsd as mentioned above to protect the individual patient s information . through the central institutional review board in the university of tokyo , an  opt - out rule was adopted , and informed consent became unnecessary .", "the ncd is governed by a committee whose members are representatives of medical associations related to surgery , such as the japan surgical society ( jss ) , jscvs , jats , the japanese association for chest surgery , the japanese society of gastroenterological surgery , the japanese society of pediatric surgeons , the japanese society of vascular surgery , the japanese society of endocrine surgery , the japanese society for mammary cancer , and the japanese thyroid association .", "the ncd establishes the surgical board certification system for the jss , which requires 13 input items at the first level in the hierarchy of specialties .", "six board certification systems , including the jbcvs and the databases of nine academic associations , are set at the second level as subspecialties .", "the main server was transferred from the hqa to the university hospital medical information network ( umin ) with a mirror - image backup .", "the hqa focused on data analysis and site visits , whereas the umin is responsible for data warehousing .", "the ncd uses cutting - edge statistical techniques to detect any trace of data inconsistency .", "the participating associations have supported the ncd financially and the database has grown rapidly ; the total number of participating hospitals is 4105 , and the number of cumulative procedures was 4,138,000 at the end of april 2014 .", "the participating associations will release , or have released , their own risk models [ 1618 ] , and papers have been published based on data from the ncd ."], ["the administrative database , diagnosis procedure combination ( dpc)/par - diem payment system ( pdps ) was introduced in japan by the japanese ministry of health , labour and welfare ( mhlw , a government agency ) in april 2003 to comprehensively assess fixed daily payments and to control medical expenditure in the acute setting based on the quality assessment .", "the number of participating hospitals by the end of april 2014 was 1,585 , including all advanced - treatment hospitals , that is , university hospitals .", "in japan , total health care expenditures have been increasing by 1 trillion yen annually , and health care expenditures make up 9.5% of the gross domestic product , which puts japan in the 16th position of the 34 member countries of the organization for economic cooperation and development . on the other hand ,", "the population aging rate in japan is over 24% , which is the highest rate in the world .", "changes in population makeup and the growing proportion of elderly persons are the underlying issues relating to rising health care expenditures , and successive cabinet office members and the mhlw have set policy directions to address this national issue .", "quality improvement , quality assessment , and the pay - for - performance system provide methods to control medical expenditures .", "the quality and outcomes framework ( qof ) , a system for the performance management and payment of genaral practitioners ( gps ) in the national health service in england , wales , scotland and northern ireland was introduced as part of the new general medical services contract in april 2004 .", "in contrast , in the united states , the agency of healthcare research and quality ( ahrq ) has defined ", "never events or errors of medical care for which medicare , the government healthcare insurance for aged and disabled persons , does not pay . in the c.", "walton lillehei lecture of the 49th sts annual meeting , the director of the ahrq emphasized that the federal government will pay for the quality , not for the volume . in japan ,", "the ncd and dpc / pdps could play complementary functions for quality assessment through adequate risk adjustment and the complete enumeration of procedures in various surgical fields . in the future ,", "balancing professional autonomy and administrative leadership might be a recurrent issue for quality assessment and quality improvement in japan ."], ["recently , the japanese association of cardiovascular intervention and therapeutics proposed to the ncd a comparative study between percutaneous coronary intervention and cabg that would use well - tested statistical methods such as propensity score matching .", "thus , the participation of units from nonsurgical fields , such as medical therapy , intervention , radiation therapy , and chemotherapy , will facilitate risk stratification of each treatment modality , and will contribute to the search for the best management of diseases and patients .", "a longitudinal follow - up database is needed for the design of such studies , and it is under construction .", "recently , the pharmaceutical and medical device agency ( pmda ) , a consultative organization of the mhlw , suggested to enroll in the jacvsd and perform follow - ups on the use of artificial valves for trans - aortic valve implantation .", "the pmda recognized the completeness and reliability of the data of the jacvsd , and from a cost - performance point of view , the pmda decided to outsource the post - market surveillance of newly covered medical devices in the cardiovascular surgical field . this demonstrates how the national database could contribute to the post - marketing surveillance of drugs and medical devices , and could help control randomized trials and multicenter studies .", "the ncd will start to collect fees from participating hospitals according to the total number of enrolled surgical procedures .", "clerical assistants have been widely employed throughout the country , which has gradually lightened the data input workload of young surgeons . governmental support and some government funds"], ["clinicians are responsible for patient safety and quality improvement , and the database will aid in achieving these goals . as reinertsen   stated , to truly improve quality , the system must , 1 ) eliminate unnecessary variation ( standardize processes ) , and 2 ) achieve and document continuous improvement ( in care processes and outcomes ) . in recent years , the importance of  certainty , not excellence of operations , and that of the concept of structure , process and outcome   have been emphasized , and multiple approaches , for instance , postgraduate education systems , reporting systems of malpractice to prevent recurrence , introduction of information technology , introduction of simulators , ebm , and other techniques , have been used for patient safety .", "since it is methodologically based on the jcvsd , the ncd represents an interface between medical databases and board certification systems , which is its point of difference from the sts national database . in 2014 , a new organization for medical board certification was established in japan that , beginning in 2017 , will certify all medical boards in close collaboration with medical associations .", "this new organization will adopt the standards of the jcvsd and the ncd for evaluating the clinical practices of applicants .", "for the assessment of medical outcomes and quality , the jcvsd and the ncd will continue to be the sole reliable data source for surgical fields in japan , where medical system reform will be implemented quickly and based on professional autonomy .", "the national database is fundamental for quality improvement , patient safety , and the adequate control of medical expenditures in the country ."]]}
{"article_id": "PMC3362706", "article_text": ["migraine is a disabling neurological disorder considered by the world health organization as the 19th leading cause of all years lived with disability among both males and females of all ages , and as the 12th leading cause of years lived with disability among females of all ages .", "apart from pain , the disability caused by migraine is aggravated by accompanying symptomatology , such as gastro - intestinal symptoms , the most common being nausea and vomiting , to such an extent that their presence is one of the diagnostic criteria for migraine .", "a telephone interview survey of 500 self - reported migraine sufferers found that nausea occurred in more than 90  % of all migraineurs ; nearly one - third of these had nausea during every attack .", "vomiting occurred in almost 70  % of all migraineurs ; nearly one - third of these vomited in the majority of attacks .", "indeed , 30.5  % of those who had nausea , reported that it interfered with their ability to take their oral migraine medication . the american study ii stated that 73  % of the migraineurs studied reported to have suffered from nausea during attacks and that 29  % had vomited .", "one of the most interesting approaches to nausea adopted by traditional chinese medicine and , in particular , by acupuncture is the stimulation of the acupoint pc6 neiguan .", "indeed , there is documented evidence as to the efficacy of stimulating this point to alleviate chemotherapy - induced nausea and vomiting ( cinv ) , postoperative nausea and vomiting ( ponv ) and motion sickness , both with acupuncture and acupressure . however , to the best of our knowledge , there are no studies in indexed medical literature as to the efficacy of treating pc6 acupoint for gastrointestinal symptoms in migraine attacks and particularly for nausea . therefore , our preliminary study aimed at verifying if pressure applied to the point pc6 was effective on the presence of nausea during migraine attacks .", "a total of 40 female patients were enrolled into this study , after having given their informed consent , and all were suffering from migraine without aura , diagnosed according to the criteria established by the international classification of headache disorders ( ichd - ii ) .", "the patients were examined at the women s headache center , department of gynaecology and obstetrics of turin university .", "inclusion criteria were : at least two migraine attacks per month for a 1-year period before enrollment ; no more than 15  days of pain per month .", "none of the patients were on prophylactic therapy , but were allowed to continue taking their usual symptomatic treatment .", "the patients medical history had to include the presence of nausea as accompanying symptomatology of their migraine , documented by a diary noting at least 1  month of attacks with nausea , prior to the inclusion in the study .", "subjects taking antiemetics to control their nausea , whether as a single product or present as a compound in a combination product for the control of migraine , were excluded from the study .", "the patients enrolled were asked to fill in a dedicated diary recording the details of the length and intensity of the migraine attacks along with the accompanying symptomatology , paying particular attention to the presence of nausea . a device known as the sea - band", "the sea - bands are elastic wristbands with a 1  cm protruding round plastic button ; these devices apply continual pressure to the pc6 acupuncture point with the aim of decreasing , or completely eliminating nausea ( fig .  1 ) .", "the pc6 point , also called neiguan , is located on the anterior surface of the forearm , 3 fingers widths up from the first wrist crease and between the tendons of the flexor carpi radialis and palmaris longus .", "the sea - bands were applied bilaterally on both wrists on the neiguan point , starting from the onset of the migraine attack and left in place for no less than 4  h , or for the whole attack period.fig . ", "1the localization of the point pc6 neiguan and the correct positioning of the sea - bands the localization of the point pc6 neiguan and the correct positioning of the sea - bands the patients were asked to document a total of six migraine attacks : three without the use of the sea - band wristbands ( phase c , control ) and three with the application of the sea - bands ( phase sb ) .", "the sequence of the treatment given for the attacks ( with , or without sea - bands ) was chosen at random according to a scheme provided by the computer and was applied to each single patient .", "the section of the diary provided that covered the symptom of nausea was detailed to include information as to the time of symptom onset and symptom resolution , the intensity of nausea at the onset ( t0 ) , at 30 ( t1 ) , 60 ( t2 ) , 120 ( t3 ) and 240 ( t4 ) minutes evaluated on a scale from 0 to 10 , where 0 indicated no nausea and 10 the maximum sensation of nausea .", "diary analysis was carried out by an impartial operator who did not know in which attacks the sea - bands were used or not . in this preliminary study ,", "the average values of nausea in phases c and sb were calculated at different times throughout the study and a statistical evaluation of the differences between the values obtained in t0 , t1 , t2 , t3 and t4 in the two groups studied was performed using a non - parametric friedman test for repeated measures .", "moreover , a non - parametric wilcoxon test for paired data was always performed for each level of the variable   time to evaluate the difference between phase c and phase sb .", "this test also took into consideration the average intensity of the three attacks in each of the two phases .", "all values given in the following text are reported as arithmetic means ( sem ) .", "all analyses were performed using the statistical package for the social sciences ( spss ) software program .", "only 32 patients ( mean age 39.65  years , range 1961 ) completed the study .", "four patients were lost to follow - up , three handed over a diary with incomplete , unreliable data and one patient did not suffer from any migraine attacks in the 3-month observation period .", "the friedman test for repeated measures showed a highly statistically significant reduction in the intensity of nausea in the sb group ( p  <  0.001 ) during treatment ( at t1 , t2 , t3 and t4 ) .", "the wilcoxon test for paired data showed that the nausea intensities were significantly higher in phase c than in phase sb ( fig . ", "2 ) : after 30  min ( t1 c 5.55    0.36 vs. t1 sb 4.6    0.40 , p  =  0.006 ) , 60  min ( t2 c 4.93    0.33 vs. t2 sb 3.11    0.40 p ", "<  0.001 ) , 120  min ( t3 c 3.48    0.35 vs. t3 sb 1.89    0.31 p ", "<  0.001 ) and 240  min ( t4 c 2.05    0.28 vs. t4 sb 0.93    0.23 p  <  0.001 ) .", "there was no difference between groups at t0 ( t0 c 5.96    0.38 vs. t0 sb 6.36    0.35 ; p  =  0.276).fig . ", "2average values of nausea score before treatment ( t0 ) , after 30  min ( t1 ) , after 60  min ( t2 ) , after 120  min ( t3 ) , after 240  min ( t4 ) , in phase sb ( black columns ) and in phase c ( white columns ) .", "non - parametric wilcoxon test for paired data at t0 , t1 , t2 , t3 and t4 : at t0 p  =  0.276 , n.s . ; at t1 * p  =  0.006 ; at t2 , t3 and t4 * * p  <  0.001 average values of nausea score before treatment ( t0 ) , after 30  min ( t1 ) , after 60  min ( t2 ) , after 120  min ( t3 ) , after 240  min ( t4 ) , in phase sb ( black columns ) and in phase c ( white columns ) .", "non - parametric wilcoxon test for paired data at t0 , t1 , t2 , t3 and t4 : at t0 p  =  0.276 , n.s . ; at t1 * p  =  0.006 ; at t2 , t3 and t4 * *", "p  <  0.001 the number of patients who reported having had at least a 50  % reduction in the nausea score was : 0/32 at 30  min in phase c and 7/32 in phase sb ( chi square test : p  =  0.16 rr 0.43 ; ci 95  % 0.320.58 ) ; 1/32 at 60  min in phase c and 15/32 in phase sb ( chi square test : p  <  0.001 rr 0.37 , ci 95  % 0.250.56 ) ; 11/32 at 120  min in phase c and 23/32 in phase sb ( chi square test : p  =  0.003 rr 0.44 , ci 95  % 0.240.80 ) ; 21/32 at 240  min in phase c and 27/32 in phase sb ( chi square test : p  =  0.083 rr 0.55 , ci 95  % 0.251.1 ) .", "moreover , when the consistency of the treatment ( response in at least two out of three treated attacks ) is taken into consideration , it was reached : in 9 patients ( 28  % ) at 60  min ; in 13 ( 40  % ) at 120  min and in 19 ( 59  % ) at 240  min .", "noteworthy , the nausea was significantly reduced by acupressure in 3/3 attacks : in 5/32 patients ( 15  % ) at 60  min ; in 10/32 ( 31  % ) at 120  min and in 17/32 ( 53  % ) at 240  min .", "some studies have reported that nausea was present in 73 to more than 90  % of the subjects studied and that almost one - third of these experienced nausea during every attack . moreover", ", 30.5  % of the subjects who reported nausea indicated that its severity even interfered with their ability to take their oral migraine medication [ 3 , 4 ] .", "traditional chinese medicine and especially acupuncture , stimulates some points that can be considered extremely valid from the point of view of nausea and/or vomiting control .", "in particular , the treatment of the acupoint pc6 neiguan may be applied to this aim , even with the application of acupressure alone , as has been validated by various studies .", "international literature reports numerous studies on the efficacy of stimulating the acupuncture point pc6 and its capacity to reduce nausea under various clinical conditions . a cochrane review on ponv concluded by stating that , compared with sham treatment , acupoint stimulation significantly reduces nausea ( rr 0.71 , 95  % ci 0.610.83 ) and the need for rescue antiemetics ( rr 0.69 , 95  % ci 0.570.83 ) . from a cochrane review on cinv", ", it emerged that acupressure is effective for both mean and worst acute nausea severity , and , therefore , acupressure is able to offer a no - cost , convenient , self - administered intervention for chemotherapy patients to reduce acute nausea .", "on the basis of the data obtained in this study , the application of acupressure for the control of nausea during a migraine attack seems to be justified .", "indeed , the application of the sea - bands on the acupoint pc6 neiguan was observed to be effective in the control of nausea .", "the average nausea scores drop in the sb phase from 6.36    0.35 in t0 , to 4.60    0.39 in t1 , to 3.11    0.40 in t2 , to 1.88    0.31 in t3 and to 0.92    0.22 in t4 . at each time step taken into consideration after the application of the sea - bands , there was a statistically significant improvement over the non - treated phases .", "moreover , there was a high percentage of responders to the treatment : i.e. 46.8  % at 60  min ; 71.8  % at 120  min ; 84.3  % at 240  min with a consistent response over time .", "even when the fact that our study is both preliminary and open is taken into consideration , the results obtained seem to be encouraging and advocate the continuous application of pc6 acupressure in all migraine attacks with the accompanying symptom of nausea .", "further controlled studies are , of course , required to validate the findings of this study ."], "abstract_text": ["<S> migraine is a disabling neurological disorder , aggravated by accompanying symptomatology , such as nausea . </S>", "<S> one of the most interesting approaches to nausea adopted by traditional chinese medicine is the stimulation of the acupoint pc6 neiguan . </S>", "<S> actually there are no studies in medical literature as to the efficacy of treating pc6 acupoint for gastrointestinal symptoms in migraine attacks . </S>", "<S> our study aimed at verifying if pressure applied to the acupoint pc6 was effective on nausea during migraine . </S>", "<S> forty female patients suffering from migraine without aura were enrolled , if nausea was always present as accompanying symptomatology of their migraine . </S>", "<S> the patients were treated randomly for a total of six migraine attacks : three with the application of a device , the sea - band wristband , which applies continual pressure to the pc6 acupoint ( phase sb ) , and three without it ( phase c ) . </S>", "<S> the intensities of nausea at the onset , at 30 , 60 , 120 and 240  min were evaluated on a scale from 0 to 10 . </S>", "<S> the values were always significantly lower in phase sb than in phase c. also the number of patients who reported at least a 50  % reduction in the nausea score was significantly higher in phase sb than in phase c at 30 , 60 and 120  min . moreover , the consistency of the treatment ( response in at least two out of three treated attacks ) was reached in 28  % patients at 60  min ; in 40  % at 120  min and 59  % at 240  min . </S>", "<S> our results encourage the application of pc6 acupressure for the treatment of migraine - associated nausea . </S>"], "labels": null, "section_names": ["Introduction", "Patients and methods", "Results", "Discussion"], "sections": [["migraine is a disabling neurological disorder considered by the world health organization as the 19th leading cause of all years lived with disability among both males and females of all ages , and as the 12th leading cause of years lived with disability among females of all ages .", "apart from pain , the disability caused by migraine is aggravated by accompanying symptomatology , such as gastro - intestinal symptoms , the most common being nausea and vomiting , to such an extent that their presence is one of the diagnostic criteria for migraine .", "a telephone interview survey of 500 self - reported migraine sufferers found that nausea occurred in more than 90  % of all migraineurs ; nearly one - third of these had nausea during every attack .", "vomiting occurred in almost 70  % of all migraineurs ; nearly one - third of these vomited in the majority of attacks .", "indeed , 30.5  % of those who had nausea , reported that it interfered with their ability to take their oral migraine medication . the american study ii stated that 73  % of the migraineurs studied reported to have suffered from nausea during attacks and that 29  % had vomited .", "one of the most interesting approaches to nausea adopted by traditional chinese medicine and , in particular , by acupuncture is the stimulation of the acupoint pc6 neiguan .", "indeed , there is documented evidence as to the efficacy of stimulating this point to alleviate chemotherapy - induced nausea and vomiting ( cinv ) , postoperative nausea and vomiting ( ponv ) and motion sickness , both with acupuncture and acupressure . however , to the best of our knowledge , there are no studies in indexed medical literature as to the efficacy of treating pc6 acupoint for gastrointestinal symptoms in migraine attacks and particularly for nausea . therefore , our preliminary study aimed at verifying if pressure applied to the point pc6 was effective on the presence of nausea during migraine attacks ."], ["a total of 40 female patients were enrolled into this study , after having given their informed consent , and all were suffering from migraine without aura , diagnosed according to the criteria established by the international classification of headache disorders ( ichd - ii ) .", "the patients were examined at the women s headache center , department of gynaecology and obstetrics of turin university .", "inclusion criteria were : at least two migraine attacks per month for a 1-year period before enrollment ; no more than 15  days of pain per month .", "none of the patients were on prophylactic therapy , but were allowed to continue taking their usual symptomatic treatment .", "the patients medical history had to include the presence of nausea as accompanying symptomatology of their migraine , documented by a diary noting at least 1  month of attacks with nausea , prior to the inclusion in the study .", "subjects taking antiemetics to control their nausea , whether as a single product or present as a compound in a combination product for the control of migraine , were excluded from the study .", "the patients enrolled were asked to fill in a dedicated diary recording the details of the length and intensity of the migraine attacks along with the accompanying symptomatology , paying particular attention to the presence of nausea . a device known as the sea - band", "the sea - bands are elastic wristbands with a 1  cm protruding round plastic button ; these devices apply continual pressure to the pc6 acupuncture point with the aim of decreasing , or completely eliminating nausea ( fig .  1 ) .", "the pc6 point , also called neiguan , is located on the anterior surface of the forearm , 3 fingers widths up from the first wrist crease and between the tendons of the flexor carpi radialis and palmaris longus .", "the sea - bands were applied bilaterally on both wrists on the neiguan point , starting from the onset of the migraine attack and left in place for no less than 4  h , or for the whole attack period.fig . ", "1the localization of the point pc6 neiguan and the correct positioning of the sea - bands the localization of the point pc6 neiguan and the correct positioning of the sea - bands the patients were asked to document a total of six migraine attacks : three without the use of the sea - band wristbands ( phase c , control ) and three with the application of the sea - bands ( phase sb ) .", "the sequence of the treatment given for the attacks ( with , or without sea - bands ) was chosen at random according to a scheme provided by the computer and was applied to each single patient .", "the section of the diary provided that covered the symptom of nausea was detailed to include information as to the time of symptom onset and symptom resolution , the intensity of nausea at the onset ( t0 ) , at 30 ( t1 ) , 60 ( t2 ) , 120 ( t3 ) and 240 ( t4 ) minutes evaluated on a scale from 0 to 10 , where 0 indicated no nausea and 10 the maximum sensation of nausea .", "diary analysis was carried out by an impartial operator who did not know in which attacks the sea - bands were used or not . in this preliminary study ,", "the average values of nausea in phases c and sb were calculated at different times throughout the study and a statistical evaluation of the differences between the values obtained in t0 , t1 , t2 , t3 and t4 in the two groups studied was performed using a non - parametric friedman test for repeated measures .", "moreover , a non - parametric wilcoxon test for paired data was always performed for each level of the variable   time to evaluate the difference between phase c and phase sb .", "this test also took into consideration the average intensity of the three attacks in each of the two phases .", "all values given in the following text are reported as arithmetic means ( sem ) .", "all analyses were performed using the statistical package for the social sciences ( spss ) software program ."], ["only 32 patients ( mean age 39.65  years , range 1961 ) completed the study .", "four patients were lost to follow - up , three handed over a diary with incomplete , unreliable data and one patient did not suffer from any migraine attacks in the 3-month observation period .", "the friedman test for repeated measures showed a highly statistically significant reduction in the intensity of nausea in the sb group ( p  <  0.001 ) during treatment ( at t1 , t2 , t3 and t4 ) .", "the wilcoxon test for paired data showed that the nausea intensities were significantly higher in phase c than in phase sb ( fig . ", "2 ) : after 30  min ( t1 c 5.55    0.36 vs. t1 sb 4.6    0.40 , p  =  0.006 ) , 60  min ( t2 c 4.93    0.33 vs. t2 sb 3.11    0.40 p ", "<  0.001 ) , 120  min ( t3 c 3.48    0.35 vs. t3 sb 1.89    0.31 p ", "<  0.001 ) and 240  min ( t4 c 2.05    0.28 vs. t4 sb 0.93    0.23 p  <  0.001 ) .", "there was no difference between groups at t0 ( t0 c 5.96    0.38 vs. t0 sb 6.36    0.35 ; p  =  0.276).fig . ", "2average values of nausea score before treatment ( t0 ) , after 30  min ( t1 ) , after 60  min ( t2 ) , after 120  min ( t3 ) , after 240  min ( t4 ) , in phase sb ( black columns ) and in phase c ( white columns ) .", "non - parametric wilcoxon test for paired data at t0 , t1 , t2 , t3 and t4 : at t0 p  =  0.276 , n.s . ; at t1 * p  =  0.006 ; at t2 , t3 and t4 * * p  <  0.001 average values of nausea score before treatment ( t0 ) , after 30  min ( t1 ) , after 60  min ( t2 ) , after 120  min ( t3 ) , after 240  min ( t4 ) , in phase sb ( black columns ) and in phase c ( white columns ) .", "non - parametric wilcoxon test for paired data at t0 , t1 , t2 , t3 and t4 : at t0 p  =  0.276 , n.s . ; at t1 * p  =  0.006 ; at t2 , t3 and t4 * *", "p  <  0.001 the number of patients who reported having had at least a 50  % reduction in the nausea score was : 0/32 at 30  min in phase c and 7/32 in phase sb ( chi square test : p  =  0.16 rr 0.43 ; ci 95  % 0.320.58 ) ; 1/32 at 60  min in phase c and 15/32 in phase sb ( chi square test : p  <  0.001 rr 0.37 , ci 95  % 0.250.56 ) ; 11/32 at 120  min in phase c and 23/32 in phase sb ( chi square test : p  =  0.003 rr 0.44 , ci 95  % 0.240.80 ) ; 21/32 at 240  min in phase c and 27/32 in phase sb ( chi square test : p  =  0.083 rr 0.55 , ci 95  % 0.251.1 ) .", "moreover , when the consistency of the treatment ( response in at least two out of three treated attacks ) is taken into consideration , it was reached : in 9 patients ( 28  % ) at 60  min ; in 13 ( 40  % ) at 120  min and in 19 ( 59  % ) at 240  min .", "noteworthy , the nausea was significantly reduced by acupressure in 3/3 attacks : in 5/32 patients ( 15  % ) at 60  min ; in 10/32 ( 31  % ) at 120  min and in 17/32 ( 53  % ) at 240  min ."], ["some studies have reported that nausea was present in 73 to more than 90  % of the subjects studied and that almost one - third of these experienced nausea during every attack . moreover", ", 30.5  % of the subjects who reported nausea indicated that its severity even interfered with their ability to take their oral migraine medication [ 3 , 4 ] .", "traditional chinese medicine and especially acupuncture , stimulates some points that can be considered extremely valid from the point of view of nausea and/or vomiting control .", "in particular , the treatment of the acupoint pc6 neiguan may be applied to this aim , even with the application of acupressure alone , as has been validated by various studies .", "international literature reports numerous studies on the efficacy of stimulating the acupuncture point pc6 and its capacity to reduce nausea under various clinical conditions . a cochrane review on ponv concluded by stating that , compared with sham treatment , acupoint stimulation significantly reduces nausea ( rr 0.71 , 95  % ci 0.610.83 ) and the need for rescue antiemetics ( rr 0.69 , 95  % ci 0.570.83 ) . from a cochrane review on cinv", ", it emerged that acupressure is effective for both mean and worst acute nausea severity , and , therefore , acupressure is able to offer a no - cost , convenient , self - administered intervention for chemotherapy patients to reduce acute nausea .", "on the basis of the data obtained in this study , the application of acupressure for the control of nausea during a migraine attack seems to be justified .", "indeed , the application of the sea - bands on the acupoint pc6 neiguan was observed to be effective in the control of nausea .", "the average nausea scores drop in the sb phase from 6.36    0.35 in t0 , to 4.60    0.39 in t1 , to 3.11    0.40 in t2 , to 1.88    0.31 in t3 and to 0.92    0.22 in t4 . at each time step taken into consideration after the application of the sea - bands , there was a statistically significant improvement over the non - treated phases .", "moreover , there was a high percentage of responders to the treatment : i.e. 46.8  % at 60  min ; 71.8  % at 120  min ; 84.3  % at 240  min with a consistent response over time .", "even when the fact that our study is both preliminary and open is taken into consideration , the results obtained seem to be encouraging and advocate the continuous application of pc6 acupressure in all migraine attacks with the accompanying symptom of nausea .", "further controlled studies are , of course , required to validate the findings of this study ."]]}
{"article_id": "PMC2901954", "article_text": ["my experience in state government has been that after natural or manmade point - source disasters , a governor reflexively turns to the office of emergency management , the department of public safety ( or homeland security ) , or the national guard for advice and counsel .", "the staffs of these agencies , however , have neither the expertise necessary to guide the response to an epidemic nor an established , ongoing communications and surveillance system with hospitals , laboratories , and medical providers .", "it is essential , therefore , to establish a formal process that allows public health and medical experts to assist elected officials in analyzing and interpreting information about the outbreak and in coordinating the public health response to the outbreak .", "guidance for fiscal year 2002 supplemental funds for public health preparedness and response for bioterrorism issued by the centers for disease control and prevention ( cdc ) requires that a state establish an advisory committee that includes representatives from health departments , first responders , hospitals , and voluntary organizations such as the red cross ( 11 ) . in colorado", "this advisory committee includes not only the nine groups listed in the cdc announcement but also the presidents of the state board of health , state medical society , and state hospital association ; the state veterinarian ; a wildlife disease specialist ; a medical examiner ; a specialist in posttraumatic stress management ; a pharmacist member of the board of pharmacy ; the attorney general ; the chief public information officer for the state health department ; and , as an ex - officio member , the chief of the colorado national guard ( 3 ) .", "these persons were named to the committee because they possess useful expertise or connections to the community .", "the statute authorizing the formation of the committee provided legal immunity to members for their advice ( 4 ) , and the members pledged that they would attend the committee meetings during a bioterrorist attack rather than report to their regular jobs . by meeting regularly", "the committee members learn about each other s skills , experience , and roles and develop a working relationship that , by itself , can be extremely valuable during a crisis .", "one notable absence in the composition of the advisory committee is representation from federal agencies , such as cdc , the federal emergency management agency , the environmental protection agency , and the federal bureau of investigation . although these agencies can not , as a practical matter , attend meetings in every state and large municipality , during a crisis they will have an integral role , and disputes are more likely if the leaders are meeting for the first time in a highly stressful situation .", "for example , local - state - federal disagreements occurred in the management of the pneumonic plague epidemic in los angeles in 1924 , the last instance of person - to - person transmission of plague in the united states , as well as during the anthrax outbreak in 2001 ( 12,13 ) .", "some existing state regulations , which in normal times are intended to ensure quality medical care , could hinder community efforts during a bioterrorist attack .", "for example , consideration should be given to modifying , for a limited period through executive orders , the regulations that control the prescription and dispensing of medicine , licensing of physicians and nurses , and transfer of patients between hospitals .", "providing antibiotics or vaccinations in mass clinics and obtaining the services of retired or out - of - state physicians and nurses may be necessary .", "in colorado , executive orders that address these concerns have been drafted by the governor s technical advisory committee .", "the orders would permit a ) health - care providers other than pharmacists and physicians , such as nurses and emergency management technicians , to dispense medications , b ) medicines to be distributed without an identified patient s name on the packet or bottle , c ) practice of medicine and nursing by professionals who are not currently licensed in colorado , provided the practice is restricted to caring for epidemic - associated illnesses and the persons are working under the supervision of a licensed practitioner ( who is given legal immunity for the supervisee s work ) , and d ) persons seeking medical care at one facility to be redirected to another facility without initial assessment or stabilization attempt if the initial hospital is unable to care for any more persons or if a specific facility ( established or temporary ) has been directed to receive epidemic patients , e.g. , those with smallpox .", "these draft orders must still be tailored to the actual emergency and signed by the governor , but the background legal work can be completed ahead of time . two additional features of the colorado bioterrorism statute exist ; these features were designed to encourage volunteers and remove legal barriers to cooperation among institutions and agencies .", "first , the statutory definition of  civil defense worker  was modified to include a  physician , health care provider , public health worker , or emergency medical service provider who is ordered by the governor  to provide specific medical or public health services during and related to an emergency epidemic and who complies with this order without pay or other consideration  ( 7 ) . with this amendment", ", civil defense workers may receive compensation for injury , including illness caused by bioterrorism , which is suffered as a result of civil defense service .", "second , the statute provides that  persons and entities [ including hospitals ] that in good faith comply completely with board of health rules regarding the emergency epidemic and executive orders  shall be immune from civil or criminal liability for any action taken to comply with the executive order or rule  and that the state shall provide  compensation for property  if the property was commandeered or otherwise used in coping with an emergency epidemic   ( 4 ) .", "to ensure that a sufficient number of health - care providers , laboratory technicians , public health epidemiologists , and administrative support workers show up for work during a bioterrorist attack , appropriate personal protection ( e.g. , respiratory protection , vaccination , or chemoprophylaxis ) for the worker and , probably , for household members of the worker are essential . when performing nonstandard work , the worker may also need legal protection , as discussed above .", "plans for a bioterrorist attack should include these factors and be written by the employer who knows how the agency operates and is staffed because people work for an agency , hospital , or institution , not a region .", "nonetheless , coordination of resources should develop mutual aid agreements with neighboring jurisdictions and integrate single institution or agency plans into community , regional , or statewide plans .", "in the 2000 colorado bioterrorism statute , the state board of health was given the new authority to promulgate rules requiring each state and local health department , general or critical access hospital , and managed - care organization to write a plan for responding to bioterrorism ( 7 ) .", "while hospitals and health departments may have previously written plans for managing mass casualties resulting from aircraft , bus , or train crashes or natural disasters , such plans need to be modified to include consideration of the special circumstances of bioterrorism ( e.g. , chemoprophylaxis and personal protective equipment for workers , infection control , and handling of laboratory specimens ) .", "because pandemic influenza may pose challenges to the medical and public health systems similar to those of bioterrorism , a single plan for both types of epidemics should be drafted .", "during  typical  outbreaks of communicable diseases , clear and timely communication by the state health department with multiple local health departments and hospitals can be a challenge . in a bioterrorist attack", ", the communications challenge will likely be greater because many more persons and agencies will be involved .", "the telephone system may not have sufficient capacity for the increased demand or it may be damaged and disorganized , as happened during the response to the attacks on the world trade centers in new york city in september 2001 ( 14 ) .", "furthermore , a large , sometimes overwhelming , number of inquiries made by members of the public to the public health agency usually occur during public health crises , and therefore , administrative plans for a bioterrorist event should include consideration of this workload .", "rather than relying on hospital personnel , public health agencies may find it advantageous to station their own personnel with mobile telephone or radio communications equipment in individual hospitals to assure that public health agencies get the information they need as rapidly as possible .", "accomplishing this may require an executive order of the governor that commandeers two - way radios .", "in colorado , board of health regulations require the state and local health departments to include assignment of employees to hospitals in the agency s emergency plan ( 8) .", "disease reporting requires specification of what to report in what manner and timeframe to which parties .", "a first legal step in this process is to require immediate reporting of any suspected or confirmed illness , syndrome , or outbreak caused by any potential bioterrorist agent .", "for example , colorado regulations were modified in 1999 so that cases of plague , which had been required to be reported within 24 hours of diagnosis by telephone , fax , or through a web - based system , were to be reported immediately only by telephone to an on - call person if the physician or hospital suspected the case was related to a bioterrorist event ( 9 ) .", "disease surveillance systems are critical not only for the initial detection of an outbreak but also for monitoring the extent and spread of the outbreak and for determining when it is over .", "managing a large outbreak would require gathering information from contact tracing and source - of - exposure investigations as well as information about the availability of critical medicine , medical equipment , and the handling of corpses .", "these information needs are much different than those needed for early detection of an attack .", "therefore , legal authority for surveillance should be modified as necessary to ensure collection of all information that could be needed by the public health agency to fulfill its duties throughout the epidemic .", "this legal authority may include requirements for groups that do not commonly report information , such as pharmacists , to provide it .", "administrative public health orders restricting personal behavior of persons with certain diseases , such as tuberculosis , are relatively common in this country ( 15 ) . such orders are usually hand - delivered to a specific person(s ) , and the restrictions are removed after a specified period , such as after one incubation period or when an ill person is no longer infectious .", "another type of public health order might involve work restriction , e.g. , health - care providers who can not demonstrate evidence of immunity to a vaccine - preventable disease are not permitted to work during an outbreak of such disease .", "few , if any states , however , have experience issuing and enforcing large - scale quarantine orders that last more than 12 days . orders restricting large numbers of contacts of cases of plague to home were issued in florence , italy , in 1630 and described in the 1999 book , galileo s daughter ( 16 ) .", "the enforcement of orders restricting the movement of residents of an entire town in which there was an outbreak of viral hemorrhagic fever was depicted in the 1996 movie , outbreak .", "the images of severe disease and enforced quarantine are similar in the book and movie and are plausible and disturbing to lay audiences .", "a more recent , well - documented example of a large - scale movement restriction was the british epidemic of foot - and - mouth disease of 2001 , which affected many farms and businesses and led to the quarantine and slaughter of 4 million sheep , cattle , and pigs for disease control purposes ( 17 ) .", "in all three examples , a decentralized quarantine was imposed . in general , the advantage of a decentralized strategy ( e.g. , persons are restricted to home ) is that it may reduce the risk for transmission of disease because fewer persons congregate .", "alternatively , the centralized strategy ( e.g. , restricted persons are taken to a sports arena , auditorium , theater , school , or hospital ) is seemingly easier for the government to care for restricted persons and to enforce the order but could allow contagious and noncontagious persons to come into contact with each other .", "another example of large - scale quarantine occurred in los angeles in 1924 during the last epidemic of pneumonic plague in this country ( 12 ) .", "three days after the first 15 cases in this outbreak became known to public health officials , eight city blocks that housed approximately 2,500 mexicans were placed in quarantine .", "public health nurses were sent to the area to make house - to - house inspections to identify new cases , and all patients with suspected cases in the area were examined by physicians at the patient s home and then sent to the county hospital .", "all persons who lived at addresses where cases had occurred were quarantined in the county general hospital , and a spanish - speaking priest and social workers were placed in the area to reassure and calm the residents .", "the quarantine actions taken in this outbreak were a combination of centralized and decentralized strategies .", "as has been discussed by barbera et al . ( 18 ) , numerous concerns regarding large - scale quarantine exist .", "all states currently have in place varying degrees of legal authority enabling isolation , quarantine , or travel restrictions if needed to maintain the welfare and safety of the public .", "drafting restrictive orders in advance is less helpful than with the other types of orders discussed above because restrictive orders require more tailoring to the specific circumstances and parameters of an outbreak .", "factors such as duration and location of restriction are dependent on what the bioterrorist agent is , how it is transmitted , how widely the agent has been disseminated , whether exposed persons can be personally identified , and what resources are available to care for restricted persons .", "not drafting such orders in advance , however , means that they may be written during the turmoil of multiple agencies trying to control an outbreak .", "authorities should never hesitate to revise the orders on the basis of on updated information . at the end of the operation topoff exercise , for instance , when the governor had issued a travel restriction order for all of metropolitan denver and cdc", "had quarantined the entire state of colorado , such orders created many unforeseen problems , including how to enforce the orders , maintain essential community services , and distribute foods and prescription medicines .", "accurate and substantive information given to the public by credible public health and medical experts can do much to allay the fears of the public and encourage their cooperation and participation in constructive , organized community response efforts ( 19,20 ) .", "i have discussed a number of ideas about legal and administrative preparations for a bioterrorist attack , but more work can be done , including development of strategies addressing issues related to mental health , disposal of corpses , performing forensic autopsies , signing death certificates , and managing potential animal vectors of disease .", "i have not discussed the sharing of medical and epidemiologic information between public health agencies and law enforcement agencies , such as the federal bureau of investigation . under normal circumstances , public health officials typically argue that release of disease surveillance information to the criminal justice system will discourage persons with reportable conditions from disclosing to public health officials where they have been and with whom they have had contact . however , a bioterrorist attack is not a routine event , and i recommend that state and local public health agencies review the laws and regulations governing the confidentiality of disease surveillance records and develop a legal and administrative protocol for sharing pertinent and relevant information with law enforcement agencies during a bioterrorist attack ( 21 ) . finally , i have not discussed the protection of civil liberties and due process for persons affected by executive orders of the governor and public health officials .", "this is an important and difficult issue , especially when well persons are quarantined solely on the basis of their having visited , worked , or resided in a particular location at a particular time , as opposed to having had face - to - face contact with a known contagious person .", "public health officials and attorneys general should review existing safeguards for the protection of civil liberties and determine whether modifications need to be made for the special circumstances created by a bioterrorist attack ."], "abstract_text": ["<S> this article proposes and discusses legal and administrative preparations for a bioterrorist attack . to perform the duties expected of public health agencies during a disease outbreak caused by bioterrorism </S>", "<S> , an agency must have a sufficient number of employees and providers at work and a good communications system between staff in the central offices of the public health agency and those in outlying or neighboring agencies and hospitals . </S>", "<S> the article proposes strategies for achieving these objectives as well as for removing legal barriers that discourage agencies , institutions , and persons from working together for the overall good of the community . </S>", "<S> issues related to disease surveillance and special considerations regarding public health restrictive orders are discussed . </S>"], "labels": null, "section_names": ["Obtaining Expert Advice", "Removing Legal Barriers", "Requiring Plans for Bioterrorist Events", "Ensuring Good Communications", "Disease Reporting and Surveillance", "Restrictive Measures, Isolation, and Quarantine", "Conclusions"], "sections": [["my experience in state government has been that after natural or manmade point - source disasters , a governor reflexively turns to the office of emergency management , the department of public safety ( or homeland security ) , or the national guard for advice and counsel .", "the staffs of these agencies , however , have neither the expertise necessary to guide the response to an epidemic nor an established , ongoing communications and surveillance system with hospitals , laboratories , and medical providers .", "it is essential , therefore , to establish a formal process that allows public health and medical experts to assist elected officials in analyzing and interpreting information about the outbreak and in coordinating the public health response to the outbreak .", "guidance for fiscal year 2002 supplemental funds for public health preparedness and response for bioterrorism issued by the centers for disease control and prevention ( cdc ) requires that a state establish an advisory committee that includes representatives from health departments , first responders , hospitals , and voluntary organizations such as the red cross ( 11 ) . in colorado", "this advisory committee includes not only the nine groups listed in the cdc announcement but also the presidents of the state board of health , state medical society , and state hospital association ; the state veterinarian ; a wildlife disease specialist ; a medical examiner ; a specialist in posttraumatic stress management ; a pharmacist member of the board of pharmacy ; the attorney general ; the chief public information officer for the state health department ; and , as an ex - officio member , the chief of the colorado national guard ( 3 ) .", "these persons were named to the committee because they possess useful expertise or connections to the community .", "the statute authorizing the formation of the committee provided legal immunity to members for their advice ( 4 ) , and the members pledged that they would attend the committee meetings during a bioterrorist attack rather than report to their regular jobs . by meeting regularly", "the committee members learn about each other s skills , experience , and roles and develop a working relationship that , by itself , can be extremely valuable during a crisis .", "one notable absence in the composition of the advisory committee is representation from federal agencies , such as cdc , the federal emergency management agency , the environmental protection agency , and the federal bureau of investigation . although these agencies can not , as a practical matter , attend meetings in every state and large municipality , during a crisis they will have an integral role , and disputes are more likely if the leaders are meeting for the first time in a highly stressful situation .", "for example , local - state - federal disagreements occurred in the management of the pneumonic plague epidemic in los angeles in 1924 , the last instance of person - to - person transmission of plague in the united states , as well as during the anthrax outbreak in 2001 ( 12,13 ) ."], ["some existing state regulations , which in normal times are intended to ensure quality medical care , could hinder community efforts during a bioterrorist attack .", "for example , consideration should be given to modifying , for a limited period through executive orders , the regulations that control the prescription and dispensing of medicine , licensing of physicians and nurses , and transfer of patients between hospitals .", "providing antibiotics or vaccinations in mass clinics and obtaining the services of retired or out - of - state physicians and nurses may be necessary .", "in colorado , executive orders that address these concerns have been drafted by the governor s technical advisory committee .", "the orders would permit a ) health - care providers other than pharmacists and physicians , such as nurses and emergency management technicians , to dispense medications , b ) medicines to be distributed without an identified patient s name on the packet or bottle , c ) practice of medicine and nursing by professionals who are not currently licensed in colorado , provided the practice is restricted to caring for epidemic - associated illnesses and the persons are working under the supervision of a licensed practitioner ( who is given legal immunity for the supervisee s work ) , and d ) persons seeking medical care at one facility to be redirected to another facility without initial assessment or stabilization attempt if the initial hospital is unable to care for any more persons or if a specific facility ( established or temporary ) has been directed to receive epidemic patients , e.g. , those with smallpox .", "these draft orders must still be tailored to the actual emergency and signed by the governor , but the background legal work can be completed ahead of time . two additional features of the colorado bioterrorism statute exist ; these features were designed to encourage volunteers and remove legal barriers to cooperation among institutions and agencies .", "first , the statutory definition of  civil defense worker  was modified to include a  physician , health care provider , public health worker , or emergency medical service provider who is ordered by the governor  to provide specific medical or public health services during and related to an emergency epidemic and who complies with this order without pay or other consideration  ( 7 ) . with this amendment", ", civil defense workers may receive compensation for injury , including illness caused by bioterrorism , which is suffered as a result of civil defense service .", "second , the statute provides that  persons and entities [ including hospitals ] that in good faith comply completely with board of health rules regarding the emergency epidemic and executive orders  shall be immune from civil or criminal liability for any action taken to comply with the executive order or rule  and that the state shall provide  compensation for property  if the property was commandeered or otherwise used in coping with an emergency epidemic   ( 4 ) ."], ["to ensure that a sufficient number of health - care providers , laboratory technicians , public health epidemiologists , and administrative support workers show up for work during a bioterrorist attack , appropriate personal protection ( e.g. , respiratory protection , vaccination , or chemoprophylaxis ) for the worker and , probably , for household members of the worker are essential . when performing nonstandard work , the worker may also need legal protection , as discussed above .", "plans for a bioterrorist attack should include these factors and be written by the employer who knows how the agency operates and is staffed because people work for an agency , hospital , or institution , not a region .", "nonetheless , coordination of resources should develop mutual aid agreements with neighboring jurisdictions and integrate single institution or agency plans into community , regional , or statewide plans .", "in the 2000 colorado bioterrorism statute , the state board of health was given the new authority to promulgate rules requiring each state and local health department , general or critical access hospital , and managed - care organization to write a plan for responding to bioterrorism ( 7 ) .", "while hospitals and health departments may have previously written plans for managing mass casualties resulting from aircraft , bus , or train crashes or natural disasters , such plans need to be modified to include consideration of the special circumstances of bioterrorism ( e.g. , chemoprophylaxis and personal protective equipment for workers , infection control , and handling of laboratory specimens ) .", "because pandemic influenza may pose challenges to the medical and public health systems similar to those of bioterrorism , a single plan for both types of epidemics should be drafted ."], ["during  typical  outbreaks of communicable diseases , clear and timely communication by the state health department with multiple local health departments and hospitals can be a challenge . in a bioterrorist attack", ", the communications challenge will likely be greater because many more persons and agencies will be involved .", "the telephone system may not have sufficient capacity for the increased demand or it may be damaged and disorganized , as happened during the response to the attacks on the world trade centers in new york city in september 2001 ( 14 ) .", "furthermore , a large , sometimes overwhelming , number of inquiries made by members of the public to the public health agency usually occur during public health crises , and therefore , administrative plans for a bioterrorist event should include consideration of this workload .", "rather than relying on hospital personnel , public health agencies may find it advantageous to station their own personnel with mobile telephone or radio communications equipment in individual hospitals to assure that public health agencies get the information they need as rapidly as possible .", "accomplishing this may require an executive order of the governor that commandeers two - way radios .", "in colorado , board of health regulations require the state and local health departments to include assignment of employees to hospitals in the agency s emergency plan ( 8) ."], ["disease reporting requires specification of what to report in what manner and timeframe to which parties .", "a first legal step in this process is to require immediate reporting of any suspected or confirmed illness , syndrome , or outbreak caused by any potential bioterrorist agent .", "for example , colorado regulations were modified in 1999 so that cases of plague , which had been required to be reported within 24 hours of diagnosis by telephone , fax , or through a web - based system , were to be reported immediately only by telephone to an on - call person if the physician or hospital suspected the case was related to a bioterrorist event ( 9 ) .", "disease surveillance systems are critical not only for the initial detection of an outbreak but also for monitoring the extent and spread of the outbreak and for determining when it is over .", "managing a large outbreak would require gathering information from contact tracing and source - of - exposure investigations as well as information about the availability of critical medicine , medical equipment , and the handling of corpses .", "these information needs are much different than those needed for early detection of an attack .", "therefore , legal authority for surveillance should be modified as necessary to ensure collection of all information that could be needed by the public health agency to fulfill its duties throughout the epidemic .", "this legal authority may include requirements for groups that do not commonly report information , such as pharmacists , to provide it ."], ["administrative public health orders restricting personal behavior of persons with certain diseases , such as tuberculosis , are relatively common in this country ( 15 ) . such orders are usually hand - delivered to a specific person(s ) , and the restrictions are removed after a specified period , such as after one incubation period or when an ill person is no longer infectious .", "another type of public health order might involve work restriction , e.g. , health - care providers who can not demonstrate evidence of immunity to a vaccine - preventable disease are not permitted to work during an outbreak of such disease .", "few , if any states , however , have experience issuing and enforcing large - scale quarantine orders that last more than 12 days . orders restricting large numbers of contacts of cases of plague to home were issued in florence , italy , in 1630 and described in the 1999 book , galileo s daughter ( 16 ) .", "the enforcement of orders restricting the movement of residents of an entire town in which there was an outbreak of viral hemorrhagic fever was depicted in the 1996 movie , outbreak .", "the images of severe disease and enforced quarantine are similar in the book and movie and are plausible and disturbing to lay audiences .", "a more recent , well - documented example of a large - scale movement restriction was the british epidemic of foot - and - mouth disease of 2001 , which affected many farms and businesses and led to the quarantine and slaughter of 4 million sheep , cattle , and pigs for disease control purposes ( 17 ) .", "in all three examples , a decentralized quarantine was imposed . in general , the advantage of a decentralized strategy ( e.g. , persons are restricted to home ) is that it may reduce the risk for transmission of disease because fewer persons congregate .", "alternatively , the centralized strategy ( e.g. , restricted persons are taken to a sports arena , auditorium , theater , school , or hospital ) is seemingly easier for the government to care for restricted persons and to enforce the order but could allow contagious and noncontagious persons to come into contact with each other .", "another example of large - scale quarantine occurred in los angeles in 1924 during the last epidemic of pneumonic plague in this country ( 12 ) .", "three days after the first 15 cases in this outbreak became known to public health officials , eight city blocks that housed approximately 2,500 mexicans were placed in quarantine .", "public health nurses were sent to the area to make house - to - house inspections to identify new cases , and all patients with suspected cases in the area were examined by physicians at the patient s home and then sent to the county hospital .", "all persons who lived at addresses where cases had occurred were quarantined in the county general hospital , and a spanish - speaking priest and social workers were placed in the area to reassure and calm the residents .", "the quarantine actions taken in this outbreak were a combination of centralized and decentralized strategies .", "as has been discussed by barbera et al . ( 18 ) , numerous concerns regarding large - scale quarantine exist .", "all states currently have in place varying degrees of legal authority enabling isolation , quarantine , or travel restrictions if needed to maintain the welfare and safety of the public .", "drafting restrictive orders in advance is less helpful than with the other types of orders discussed above because restrictive orders require more tailoring to the specific circumstances and parameters of an outbreak .", "factors such as duration and location of restriction are dependent on what the bioterrorist agent is , how it is transmitted , how widely the agent has been disseminated , whether exposed persons can be personally identified , and what resources are available to care for restricted persons .", "not drafting such orders in advance , however , means that they may be written during the turmoil of multiple agencies trying to control an outbreak .", "authorities should never hesitate to revise the orders on the basis of on updated information . at the end of the operation topoff exercise , for instance , when the governor had issued a travel restriction order for all of metropolitan denver and cdc", "had quarantined the entire state of colorado , such orders created many unforeseen problems , including how to enforce the orders , maintain essential community services , and distribute foods and prescription medicines ."], ["accurate and substantive information given to the public by credible public health and medical experts can do much to allay the fears of the public and encourage their cooperation and participation in constructive , organized community response efforts ( 19,20 ) .", "i have discussed a number of ideas about legal and administrative preparations for a bioterrorist attack , but more work can be done , including development of strategies addressing issues related to mental health , disposal of corpses , performing forensic autopsies , signing death certificates , and managing potential animal vectors of disease .", "i have not discussed the sharing of medical and epidemiologic information between public health agencies and law enforcement agencies , such as the federal bureau of investigation . under normal circumstances , public health officials typically argue that release of disease surveillance information to the criminal justice system will discourage persons with reportable conditions from disclosing to public health officials where they have been and with whom they have had contact . however , a bioterrorist attack is not a routine event , and i recommend that state and local public health agencies review the laws and regulations governing the confidentiality of disease surveillance records and develop a legal and administrative protocol for sharing pertinent and relevant information with law enforcement agencies during a bioterrorist attack ( 21 ) . finally , i have not discussed the protection of civil liberties and due process for persons affected by executive orders of the governor and public health officials .", "this is an important and difficult issue , especially when well persons are quarantined solely on the basis of their having visited , worked , or resided in a particular location at a particular time , as opposed to having had face - to - face contact with a known contagious person .", "public health officials and attorneys general should review existing safeguards for the protection of civil liberties and determine whether modifications need to be made for the special circumstances created by a bioterrorist attack ."]]}
{"article_id": "PMC3625579", "article_text": ["the name of mucosa - associated lymphoid tissue ( malt ) lymphoma was first established in 1983 by isaacson and du . from the beginning , it was adopted well and is still used in an unchanged form .", "marginal zone lymphoma of malt is , apart from diffuse large b - cell lymphoma , the most frequent type of lymphoma that occurs in the stomach .", "what is important is that it can develop in almost every organ and tissue , for instance lungs , breast , thyroid gland , bladder , skin , or orbital adnexa .", "it is an indolent type , but clinical outcomes and response to treatment vary among patients .", "malt lymphoma arises from the extranodal sites reach in b - lymphocytes , which appears in response to chronic antigenic stimulation caused by infection ( helicobacter pylori ) or autoimmune process ( hashimoto disease ) .", "this disorder is the best example of how infectious pathogens and genetic abnormalities lead to malignant transformation .", "gastric malt lymphoma pathogenesis is a complex process including many gene alternations that result in cancer appearance .", "better understanding of the background of the disease is crucial for discovering new prognostic factors , helpful in deciding when more aggressive treatment should be employed .", "the incidence of malignant lymphomas is at the rate of 3%-4% of all malignancy worldwide and has been increasing during the last 50 years . lately", ", some stabilization in the number of diagnosis was observed , but only in developed countries .", "malignant lymphomas are observed to be more frequent in north america , australia , and europe than in asia and africa .", "malt lymphomas determine almost 7% of all non - hodgkin 's lymphoma , and at least 40% is primarily located in stomach .", "it is confirmed that gastric malt lymphoma occurs in younger patients than the rest of malignant lymphomas .", "the malt lymphoma is mainly a disease of older adults , with a median age of 60 years .", ", there is much higher proportions of malt lymphomas , which can be caused by more frequent prevalence of helicobacter pylori in this region of the world .", "although 90% of population worldwide have confirmed bacteria colonization , only 2% will develop malignant lymphoma .", "it was confirmed by weber et al .   that almost 90% of patients with gastric malt lymphoma are infected with helicobacter pylori .", "this curved bacillus , previously called campylobacter pyloridis , is a gram negative pathogen found in the stomach .", "although over 80% of people are asymptomatic , chronic infection can lead to gastritis , gastric and duodenal ulcer , gastric adenocarcinoma , and malt lymphoma [ 5 , 6 ] .", "nowadays , it is widely accepted that helicobacter pylori gastritis is crucial in an evolution of malt lymphoma localized in stomach .", "it was confirmed by several studies that chronic gastric inflammation causes constant antigenic stimulation , which leads to clonal expansion of b - cell lymphocytes [ 7 , 8 ] . in the gastric mucosal cells ,", "there are elevated levels of some cytokines , including proliferation - inducing ligand ( april ) , which belongs to the tumour necrosis factor ( tnf ) family .", "april is produced by macrophages present in the gastric malt infiltrate , located close to the neoplastic cells .", "april may also induce b - cells transformation and the progression to the diffuse large b - cell lymphoma ( figure 1 ) .", "they come either from t cells or directly by the antigenic autostimulation of lymphoma cells .", "gastric inflammation causes the appearance of a large number of macrophages , which , under a helicobacter pylori infection , release large amounts of april .", "importantly , a number of april - producing macrophages significantly decrease in complete remission after eradication therapy .", "other pathogens , are also suspected to play an important role in malt lymphoma pathogenesis .", "there are bacteria such as campylobacter jejuni , borrelia burgdorferi , and chlamydia psittaci and viruses like hepatitis c virus ( hcv ) that are potentially responsible for oncogenesis .", "these pathogens were found in histological material , but so far no strong evidences were established . patients with autoimmune disease have for sure higher risk of developing malt lymphoma .", "autoreactive b cells infiltrate the healthy organs and create lymphoid infiltrate similar to normal malt tissue with huge amount of reactive clonal b lymphocytes .", "this situation is observed in salivary gland in patients with diagnosis of sjgren syndrome and in the thyroid gland in hashimoto disease .", "sjgren syndrome is associated with 44 times increased risk of lymphoma , whereas hashimoto 's thyroiditis causes 70 times increased risk of thyroid lymphoma .  ", "some of them are proven to be strongly associated with the disease , but some are still not confirmed .", "it is believed , that on the background of chronic inflammation not only reactive b - cells are stimulated but also activated neutrophils which can lead to production of oxygen species . as a result , this genotoxins provoke dna damages , which are responsible for mutations and transformations of genetic material .  ", "it originates from a fusion of two proteins : apoptosis inhibitor 2 ( api2 ) and paracaspase malt lymphoma - translocation gene 1 ( malt1 ) .", "what is more important is that while t(11;18)(q21;q21 ) is detected , no other chromosome abnormality can be found .", "unfortunately , positive cases do not response to helicobacter pylori eradication , but , in contrast , they do not transform to more aggressive diffusive large b - cell lymphoma .", "it is known that complete remission can be seen in at least 20% of patients with t(11;18)(q21;q21 ) .", "the incidence of positivity for this translocation malt lymphoma is at approximately 20% in europe [ 16 , 17 ] but is not as common in the united states where only 5% are positive .", "moreover , it is usually connected with an advanced stage of disease and poor outcomes .", "bcl-10 gene is relocated from chromosome 1 to 14 , which in consequence triggers overexpression of bcl-10 protein also known as ciper , carmen , or me10 . in healthy organisms , higher expression is observed in lymph nodes , spleen , and testis .", "so far , it is believed that bcl-10 protein expression is responsible for proliferative effects [ 19 , 20 ] .  ", "the t(14;18)(q32;q21)(igh - bcl-2 ) is commonly present in follicular lymphoma , in about 20% of diffuse large b - cell lymphoma and sometimes in chronic lymphocytic leukemia .", "although this aberration is extremely rare in other types of lymphomas , it can be found in some cases of gastric malt lymphoma .", "bcl-2 is an antiapoptotic protein , which helps in survival and expansion of clonal b cells .", "so far , the role of t(14;18 ) in gastric malt lymphoma is not fully understood .", "overexpression of bcl-2 is found not only in translocation positive patients but also in the negative ones .", "it is believed that similar to other types of lymphomas , t(14;18)(igh - bcl-2 )  must coexist with other genetic abnormalities in order to develop neoplasm .", "t(3;14)(p14;q32)(igh - foxp1 ) is a newly described abnormality present in patients with malt lymphoma .", "the first study showed that positivity for this translocation is approximately 10% of all malt lymphoma patients .", "the most recent studies described the presence of t(3;14)(p14;q32 ) in diffuse large b - cell lymphoma , outside the lymph nodes especially [ 23 , 24 ] .", "only one study , so far , confirmed the existence of this translocation in gastric malt lymphoma   which involved bad clinical outcomes .", "in pathogenesis of malt lymphoma , the above described translocation promotes oncogenesis by similar well - known mechanism .", "the majority of them involve the same pathway , which leads to antigen receptor - mediated activation of nfb .", "this is a crucial transcript factor which plays a key role in malt lymphogenesis [ 26 , 27 ] .", "it regulates processes connected with b - cell development , growth , and survival by production of cytokines and growth factors , for example , tnf- family ( baff ) .", "latest studies have shown that b - cell activation in malt lymphoma can be strictly connected with tnf family .", "it can be also responsible for activation of cell apoptosis [ 28 , 29 ] .", "it is observed that in patients with higher baff levels in serum , the prognosis and survival are much worse .", "based on recent knowledge about genetic abnormalities in gastric malt lymphoma , there is a model of multistep pathogenesis . on", "the background of chronic inflammation and antigenic stimulation occurs genetic instability . as a result , many possible translocation and unbalanced aberrations are observed .", "gastric malt lymphoma can be long - time asymptomatic or associated with dyspepsia , abdominal pain , vomiting , diarrhea , obstruction , and nausea . sometimes bleeding from gastrointestinal tract or even perforation", "may occur while extensive lesions are present . as a result , symptoms of anemia like paleness , weakness , or easy fatigue can be observed .", "b symptoms ( weight loss , unexplained fever , and night sweats ) in gastric malt lymphoma are very rare , but the most common of the above is weight loss . a prompt diagnosis is crucial , but , unfortunately , it is usually made by incidence .", "patients with early stage of disease have usually low tumor growth and minimal possibility to spread .", "in contrast , patients with advanced stage of disease can undergo transformation to more aggressive lymphoma and may become resistant to treatment .   not only symptoms but also endoscopic picture can be inconclusive . difficulties often arise to differentiate between chronic gastritis or ulcer from an early - stage lymphoma . in order to confirm the diagnosis ,", "there always must be made pcr or fish analysis for t(11;18 ) , which is important to separate groups that will not respond to standard treatment .", "characteristic for gastric malt lymphoma are lymphoepithelial lesions ( lel ) with the presence of mainly two types of cells : neoplastic centrocyte - like or small lymphoid .", "there is no specific immunohistochemical profile typical for gastric malt lymphoma diagnosis . in 50% of patients , there is coexpression of cd43/bcl2 .", "neoplastic cells are positive for cd-20 and negative for cd-10 , cd-23 , and cyclin d1 .", "if it is negative in histochemistry , rapid urea breath test or fecal antigen test have to be made . another analysis", "to prove absence of helicobacter pylori infection is serological test for caga antibodies and helicobacter pylori - igg antibodies .", "sometimes , there is possibility to detect other helicobacter species , for example , heilmannii or felis .", "before taking any decision on how aggressive the treatment should be , it is extremely important to perform a complete staging of the disease .", "what is more important is that risk factors and individual parameters , which can affect later therapy , are crucial .", "medical history must include information about the age , time of the first symptoms , the family history , and medical condition .", "the most important factor that we rely on during choosing method of treatment is clinical stage of the patient . during physical examination ,", "it is important to remember about waldeyer 's ring , which is mandatory in every gastric lymphoma patient . staging in gastric malt lymphoma is similar to that in other types of lymphomas . according to recent european society for medical oncology ( esmo ) recommendations , it should include morphology with basic biochemical studies .", "if the blood cell count is lower , it can be caused by infiltration of bone marrow .", "biochemical tests can detect liver or kidney problems , which can be important before the beginning of a chemotherapy .", "lactate dehydrogenase ( ldh ) and 2-mikroglobulin are prognostic factors and will be abnormally high in patients with fast - growing tumor .", "every newly diagnosed patient should be examined in case of certain viral infections that can affect treatment , such as hepatitis b and c or human immunodeficiency virus ( hiv ) .   in every case ,", "computed tomography ( ct ) scans of neck , chest , abdomen , and pelvis , which are crucial to evaluate enlarged lymph nodes , should be performed .", "core needle biopsy of bone marrow is made to diagnose possible infiltration of neoplastic cells .", "it was confirmed that 15% of gastric malt lymphoma patients have lymphoma cells in bone marrow .", "positron emission tomography ( pet ) has still not confirmed clinical necessity , but it can be extremely helpful in controversial cases . moreover , during staging procedures of gastric malt lymphoma , gastroduodenal endoscopy must be made .", "biopsies are taken from different sites of gastrointestinal tract ( e.g. , stomach , duodenum , and gastroesophageal junction ) and every location that looks suspicious .  ", "most often , ann arbour staging is employed , which describe the extend of all types of non - hodgkin lymphoma in adults .", "thus , staging of gastric lymphoma based upon the ann arbor system includes stage i e , which is disease limited to the stomach without nodal spread .", "stage ii e1 is tumor in the stomach with spread to adjacent contiguous lymph nodes .", "stage ii e2 is tumor in the stomach with spread to lymph nodes that are noncontiguous with the primary tumor .", "moreover , if the spleen is affected , we add s. if the person has any of the b symptoms , we add letter b , and if is asymptomatic , we assign a ( table 1 ) .", "prognostic factors in gastric malt lymphoma are similar to the value for non - hodgkin b - cells lymphoma .", "factors that determine poor outcome are age , high level of ldh in serum , higher ecog performance status , stages iii and iv in ann - arbour scale , white blood count , and more than one extranodal site .", "it was observed that patients with nodal invasion has difficulty with complete remission after eradication treatment .", "for instance patients , with t(11;18)(q21;q21 ) especially , are resistant to the first line therapy , and remission rate was lower than that in patients of api2-malt1 negative ( 78% versus 22.2% ; p = 0.0001 ) .", "only one study so far proved that the presence of t(3;14)(p14;q32 ) is connected with poor clinical outcomes of patients with gastric malt lymphoma .", "while helicobacter pylori plays a main role in the pathogenesis of malt lymphoma , it is also crucial in approach to the treatment . according to current international guidelines , first line treatment for localized helicobacter", "the treatment may be used with every highly effective antibiotics against helicobacter pylori , taking into consideration the locally expected antibiotic resistance . if there is no response to the therapy above , second line triple or quadruple therapy is used .", "it was reported that after two lines of treatment , 99.8% of patients were cured from gastritis . in a large study of 1408 patients , remission after eradication treatment in early stage", "unfortunately , in 5%10% of gastric malt lymphoma patients , we can not confirm helicobacter pylori infection .", "moreover , more than 30% patients are resistant to first line treatment , and 30% of them have t(11;18)(q21;q21 ) .", "treatment for this patients should be chosen individually depending on the clinical stage of disease . for those who have stable disease without any symptoms ,", "radiotherapy , chemotherapy , and/or surgery can be considered after unsuccessful eradication treatment . further recommended", "surgery is considered to be a standard therapy in therapy of patients with gastric malt lymphomas , but , recently , the value of this therapy has been not confirmed .", "even if the lymphoma is localized at early stage , the gastrectomy should be rather extensive due to the nature of the disease .", "moreover , it is a major surgery and can be associated with serious complications and worsen a quality of life .", "german multicenter study group ( gmsg ) presented no difference between survival in patients treated with gastrectomy compared to eradication ( overall survival rate 82% to 84% ) .", "what is more important is that there were observed 50% long - term complications were observed after surgery . in few studies", "the use of a modest dose of involved fields was performed on resistant - to - eradication therapy patients with early - stage disease .", "the dose was 2535  gy to the stomach and perigastric nodes for the period of 4 weeks [ 42 , 43 ] . compared to surgery , no serious long - term complications and toxicity were observed . only nausea and anorexia were present during the time of radiotherapy . for a long time", "it was believed that gastric malt lymphoma is just a localized disease and that surgery and radiotherapy are the best treatment strategy .", "now , when it is well known that it is disseminated disorder chemotherapy , it became more important .", "still , there are no standard recommendations for relapse or progressive patients after therapy and for those with late stage of the disease from the beginning .", "it was observed that chemotherapy alone is more effective than surgery apart from some cases with gastric obstruction .", "complete remission ( cr ) after oral monochemotherapy with cyclophosphamide was 83% in a study by nakamura and coworkers .", "unfortunately , patients with positive translocation t(11 ; 18 ) are resistant to second line therapy with oral monochemotherapy with alkylating agents .", "nucleoside analogs are confirmed to be effective in treatment of different kinds of indolent lymphomas .", "a polychemotherapy with fludarabine and mitoxantrone ( fm ) has a very good effect on patients with gastric malt lymphoma in both first and second line treatment .", "the complete remission after 4 cycles achieved 84% of investigated and all of them reacted to the treatment .", "after 2-cda , there were observed complications such as toxicities of 3 and 4 grade of who , mainly leukopenia , infections , and secondary neoplastic disease .", "nowadays , immunotherapy became an extremely important part of treatment of non - hodgkin lymphomas .", "it is a chimeric mouse / human monoclonal antibody specified to cd20 antigen expressed on the surface of b lymphocytes .", "now it is widely used alone or in combination with chemotherapeutic drugs in many types of b - cell non - hodgkin lymphomas .", "rituximab binds to cd20 antigen and activates the lysis of b cells by mediating cytotoxicity of complement dependent ( cdc ) and cell - mediated cytotoxicity antibody dependent ( adcc ) .", "the role of this drug is still not clear in gastric malt lymphoma . in 2003 , there was a first - phase study by conconi et al .  ", "the cr was observed in 29% and overall response rate ( orr ) was 64% .", "the toxicity of this treatment was moderate or even mild , but the relapse rate was 36% .", "an important fact is that patients with translocation t(11;18 ) are responsive to rituximab treatment [ 58 , 59 ] .", "what is more important is that in a study by the international extranodal lymphoma study group ( ielsg ) , it was confirmed that chlorambucil in combination with rituximab was more effective than chlorambucil alone .", "the conclusion is that rituximab may a benefit in individual patients , but for the majority it is not sufficient when used alone .", "the efficacy of the combination of rituximab with chlorambucil was evaluated in a randomized study ( comparator was chlorambucil alone ) by the international extranodal lymphoma study group ( ielsg ) in gastric malt lymphomas that had failed antibiotics and in nongastric malt lymphomas .", "the preliminary report   showed that the 5-year event - free survival was significantly better for patients treated with chlorambucil plus rituximab .", "there were also studies by raderer et al . with cycles generally used in more aggressive lymphomas .", "twenty - six patients were administrated rituximab plus cyclophosphamide , doxorubicin or mitoxantrone , vincristine , and prednisone .", "lately , bortezomib , the first therapeutic proteasome inhibitor , was examined by kiesewetter et al . in 2012 with cr in 33% and pr in 27.8% .", "the results on phase ii studies with chemotherapy and immunotherapy are shown in table 2 .", "outcomes in gastric malt lymphoma patients with progressive , disseminated disease are very comparable with outcomes in follicular lymphoma .", "although gastric malt lymphoma has a very favorable outcome , it is still important to have a proper followup", ". it is possible that the disease will return even after 5 years of complete remission .", "the relapse can be due to reinfection of helicobcater pylori . in a study by zullo et al .", "the followup is obligatory in patients with gastric malt lymphoma to identify early phase of the recurrence of the disease . to confirm a complete remission ,", "although , there are no specified recommendations for a followup , the biopsy of gastric sites should be made every 6 months in first two years , and later once a year for the next five years .", "systemic followup consist of blood tests and minimal adequate radiological and ultrasound and should be made at least once a year in the first 5 years .", "the transformation in more aggressive lymphoma is low at the level of 0.05% , but there is a higher risk of occurrence of secondary neoplasm   and gastric cancer .", "these studies confirm that patients with gastric malt lymphoma need a long - term followup not only to detect early recurrence but also to find secondary disease .", "recently , enormous progress has been made in better understanding of pathogenesis of gastric malt lymphoma .", "it has a great influence on the development of new and more effective treatment strategy .", "still , not enough clinical trials are performed due to rare expression and high effectiveness of first line treatment of gastric malt lymphoma .", "what is more important is that early diagnosis of gastric malt lymphoma is extremely important . while the symptoms are unspecific or not , always during the endoscopic exam the complete histological biopsies must be taken to make diagnosis correctly . the less advanced the stage of the disease , the bigger the chances to achieve complete remission"], "abstract_text": ["<S> nowadays , it is believed that the main role in the development of gastric mucosa - associated lymphoid tissue ( malt ) lymphoma plays helicobacter pylori infection . </S>", "<S> this world - wide distributed bacteria is in charge of most cases of not only upper gastrointestinal tract disorders but also some of extragastric problems . </S>", "<S> constant stimulation of the immune system causes a b - lymphocytes proliferation , which is considered to be responsible for the neoplastic transformation . on the other hand , </S>", "<S> there are 10%20% of patients who do not respond to helicobacter pylori eradication treatment . </S>", "<S> this group has often a chromosome translocation , which suggests that there is another unknown , so far , pathogenetic mechanism of malt lymphoma . </S>", "<S> majority of genetic abnormalities are connected with nuclear factor-b ( nf-b ) pathway , which activates the uncontrolled proliferation of neoplastic cells . </S>", "<S> translocations already described in studies are t(11;18)(q21;q21 ) , which is the most common , t(14;18)(q32;q21 ) , t(14;18)(q32;q21 ) , and t(3;14)(p14.1;q32 ) . </S>", "<S> this non - hodgkin 's lymphoma is an indolent type originated outside lymph nodes . in more than 50% of cases </S>", "<S> , it occurs in the stomach . </S>", "<S> occasionally , it can be found in salivary and thyroid gland , lung , breast , bladder , skin , or any other place in the human body . this paper is a review of the current knowledge on etiology , pathogenesis , treatment , and follow - up of gastric malt lymphoma . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Epidemiology", "3. Pathogenesis", "4. Symptoms and Diagnosis", "5. Staging and Risk Factors", "6. Treatment", "7. Followup", "8. Conclusions"], "sections": [["the name of mucosa - associated lymphoid tissue ( malt ) lymphoma was first established in 1983 by isaacson and du . from the beginning , it was adopted well and is still used in an unchanged form .", "marginal zone lymphoma of malt is , apart from diffuse large b - cell lymphoma , the most frequent type of lymphoma that occurs in the stomach .", "what is important is that it can develop in almost every organ and tissue , for instance lungs , breast , thyroid gland , bladder , skin , or orbital adnexa .", "it is an indolent type , but clinical outcomes and response to treatment vary among patients .", "malt lymphoma arises from the extranodal sites reach in b - lymphocytes , which appears in response to chronic antigenic stimulation caused by infection ( helicobacter pylori ) or autoimmune process ( hashimoto disease ) .", "this disorder is the best example of how infectious pathogens and genetic abnormalities lead to malignant transformation .", "gastric malt lymphoma pathogenesis is a complex process including many gene alternations that result in cancer appearance .", "better understanding of the background of the disease is crucial for discovering new prognostic factors , helpful in deciding when more aggressive treatment should be employed ."], ["the incidence of malignant lymphomas is at the rate of 3%-4% of all malignancy worldwide and has been increasing during the last 50 years . lately", ", some stabilization in the number of diagnosis was observed , but only in developed countries .", "malignant lymphomas are observed to be more frequent in north america , australia , and europe than in asia and africa .", "malt lymphomas determine almost 7% of all non - hodgkin 's lymphoma , and at least 40% is primarily located in stomach .", "it is confirmed that gastric malt lymphoma occurs in younger patients than the rest of malignant lymphomas .", "the malt lymphoma is mainly a disease of older adults , with a median age of 60 years .", ", there is much higher proportions of malt lymphomas , which can be caused by more frequent prevalence of helicobacter pylori in this region of the world ."], ["although 90% of population worldwide have confirmed bacteria colonization , only 2% will develop malignant lymphoma .", "it was confirmed by weber et al .   that almost 90% of patients with gastric malt lymphoma are infected with helicobacter pylori .", "this curved bacillus , previously called campylobacter pyloridis , is a gram negative pathogen found in the stomach .", "although over 80% of people are asymptomatic , chronic infection can lead to gastritis , gastric and duodenal ulcer , gastric adenocarcinoma , and malt lymphoma [ 5 , 6 ] .", "nowadays , it is widely accepted that helicobacter pylori gastritis is crucial in an evolution of malt lymphoma localized in stomach .", "it was confirmed by several studies that chronic gastric inflammation causes constant antigenic stimulation , which leads to clonal expansion of b - cell lymphocytes [ 7 , 8 ] . in the gastric mucosal cells ,", "there are elevated levels of some cytokines , including proliferation - inducing ligand ( april ) , which belongs to the tumour necrosis factor ( tnf ) family .", "april is produced by macrophages present in the gastric malt infiltrate , located close to the neoplastic cells .", "april may also induce b - cells transformation and the progression to the diffuse large b - cell lymphoma ( figure 1 ) .", "they come either from t cells or directly by the antigenic autostimulation of lymphoma cells .", "gastric inflammation causes the appearance of a large number of macrophages , which , under a helicobacter pylori infection , release large amounts of april .", "importantly , a number of april - producing macrophages significantly decrease in complete remission after eradication therapy .", "other pathogens , are also suspected to play an important role in malt lymphoma pathogenesis .", "there are bacteria such as campylobacter jejuni , borrelia burgdorferi , and chlamydia psittaci and viruses like hepatitis c virus ( hcv ) that are potentially responsible for oncogenesis .", "these pathogens were found in histological material , but so far no strong evidences were established . patients with autoimmune disease have for sure higher risk of developing malt lymphoma .", "autoreactive b cells infiltrate the healthy organs and create lymphoid infiltrate similar to normal malt tissue with huge amount of reactive clonal b lymphocytes .", "this situation is observed in salivary gland in patients with diagnosis of sjgren syndrome and in the thyroid gland in hashimoto disease .", "sjgren syndrome is associated with 44 times increased risk of lymphoma , whereas hashimoto 's thyroiditis causes 70 times increased risk of thyroid lymphoma .  ", "some of them are proven to be strongly associated with the disease , but some are still not confirmed .", "it is believed , that on the background of chronic inflammation not only reactive b - cells are stimulated but also activated neutrophils which can lead to production of oxygen species . as a result , this genotoxins provoke dna damages , which are responsible for mutations and transformations of genetic material .  ", "it originates from a fusion of two proteins : apoptosis inhibitor 2 ( api2 ) and paracaspase malt lymphoma - translocation gene 1 ( malt1 ) .", "what is more important is that while t(11;18)(q21;q21 ) is detected , no other chromosome abnormality can be found .", "unfortunately , positive cases do not response to helicobacter pylori eradication , but , in contrast , they do not transform to more aggressive diffusive large b - cell lymphoma .", "it is known that complete remission can be seen in at least 20% of patients with t(11;18)(q21;q21 ) .", "the incidence of positivity for this translocation malt lymphoma is at approximately 20% in europe [ 16 , 17 ] but is not as common in the united states where only 5% are positive .", "moreover , it is usually connected with an advanced stage of disease and poor outcomes .", "bcl-10 gene is relocated from chromosome 1 to 14 , which in consequence triggers overexpression of bcl-10 protein also known as ciper , carmen , or me10 . in healthy organisms , higher expression is observed in lymph nodes , spleen , and testis .", "so far , it is believed that bcl-10 protein expression is responsible for proliferative effects [ 19 , 20 ] .  ", "the t(14;18)(q32;q21)(igh - bcl-2 ) is commonly present in follicular lymphoma , in about 20% of diffuse large b - cell lymphoma and sometimes in chronic lymphocytic leukemia .", "although this aberration is extremely rare in other types of lymphomas , it can be found in some cases of gastric malt lymphoma .", "bcl-2 is an antiapoptotic protein , which helps in survival and expansion of clonal b cells .", "so far , the role of t(14;18 ) in gastric malt lymphoma is not fully understood .", "overexpression of bcl-2 is found not only in translocation positive patients but also in the negative ones .", "it is believed that similar to other types of lymphomas , t(14;18)(igh - bcl-2 )  must coexist with other genetic abnormalities in order to develop neoplasm .", "t(3;14)(p14;q32)(igh - foxp1 ) is a newly described abnormality present in patients with malt lymphoma .", "the first study showed that positivity for this translocation is approximately 10% of all malt lymphoma patients .", "the most recent studies described the presence of t(3;14)(p14;q32 ) in diffuse large b - cell lymphoma , outside the lymph nodes especially [ 23 , 24 ] .", "only one study , so far , confirmed the existence of this translocation in gastric malt lymphoma   which involved bad clinical outcomes .", "in pathogenesis of malt lymphoma , the above described translocation promotes oncogenesis by similar well - known mechanism .", "the majority of them involve the same pathway , which leads to antigen receptor - mediated activation of nfb .", "this is a crucial transcript factor which plays a key role in malt lymphogenesis [ 26 , 27 ] .", "it regulates processes connected with b - cell development , growth , and survival by production of cytokines and growth factors , for example , tnf- family ( baff ) .", "latest studies have shown that b - cell activation in malt lymphoma can be strictly connected with tnf family .", "it can be also responsible for activation of cell apoptosis [ 28 , 29 ] .", "it is observed that in patients with higher baff levels in serum , the prognosis and survival are much worse .", "based on recent knowledge about genetic abnormalities in gastric malt lymphoma , there is a model of multistep pathogenesis . on", "the background of chronic inflammation and antigenic stimulation occurs genetic instability . as a result , many possible translocation and unbalanced aberrations are observed ."], ["gastric malt lymphoma can be long - time asymptomatic or associated with dyspepsia , abdominal pain , vomiting , diarrhea , obstruction , and nausea . sometimes bleeding from gastrointestinal tract or even perforation", "may occur while extensive lesions are present . as a result , symptoms of anemia like paleness , weakness , or easy fatigue can be observed .", "b symptoms ( weight loss , unexplained fever , and night sweats ) in gastric malt lymphoma are very rare , but the most common of the above is weight loss . a prompt diagnosis is crucial , but , unfortunately , it is usually made by incidence .", "patients with early stage of disease have usually low tumor growth and minimal possibility to spread .", "in contrast , patients with advanced stage of disease can undergo transformation to more aggressive lymphoma and may become resistant to treatment .   not only symptoms but also endoscopic picture can be inconclusive . difficulties often arise to differentiate between chronic gastritis or ulcer from an early - stage lymphoma . in order to confirm the diagnosis ,", "there always must be made pcr or fish analysis for t(11;18 ) , which is important to separate groups that will not respond to standard treatment .", "characteristic for gastric malt lymphoma are lymphoepithelial lesions ( lel ) with the presence of mainly two types of cells : neoplastic centrocyte - like or small lymphoid .", "there is no specific immunohistochemical profile typical for gastric malt lymphoma diagnosis . in 50% of patients , there is coexpression of cd43/bcl2 .", "neoplastic cells are positive for cd-20 and negative for cd-10 , cd-23 , and cyclin d1 .", "if it is negative in histochemistry , rapid urea breath test or fecal antigen test have to be made . another analysis", "to prove absence of helicobacter pylori infection is serological test for caga antibodies and helicobacter pylori - igg antibodies .", "sometimes , there is possibility to detect other helicobacter species , for example , heilmannii or felis ."], ["before taking any decision on how aggressive the treatment should be , it is extremely important to perform a complete staging of the disease .", "what is more important is that risk factors and individual parameters , which can affect later therapy , are crucial .", "medical history must include information about the age , time of the first symptoms , the family history , and medical condition .", "the most important factor that we rely on during choosing method of treatment is clinical stage of the patient . during physical examination ,", "it is important to remember about waldeyer 's ring , which is mandatory in every gastric lymphoma patient . staging in gastric malt lymphoma is similar to that in other types of lymphomas . according to recent european society for medical oncology ( esmo ) recommendations , it should include morphology with basic biochemical studies .", "if the blood cell count is lower , it can be caused by infiltration of bone marrow .", "biochemical tests can detect liver or kidney problems , which can be important before the beginning of a chemotherapy .", "lactate dehydrogenase ( ldh ) and 2-mikroglobulin are prognostic factors and will be abnormally high in patients with fast - growing tumor .", "every newly diagnosed patient should be examined in case of certain viral infections that can affect treatment , such as hepatitis b and c or human immunodeficiency virus ( hiv ) .   in every case ,", "computed tomography ( ct ) scans of neck , chest , abdomen , and pelvis , which are crucial to evaluate enlarged lymph nodes , should be performed .", "core needle biopsy of bone marrow is made to diagnose possible infiltration of neoplastic cells .", "it was confirmed that 15% of gastric malt lymphoma patients have lymphoma cells in bone marrow .", "positron emission tomography ( pet ) has still not confirmed clinical necessity , but it can be extremely helpful in controversial cases . moreover , during staging procedures of gastric malt lymphoma , gastroduodenal endoscopy must be made .", "biopsies are taken from different sites of gastrointestinal tract ( e.g. , stomach , duodenum , and gastroesophageal junction ) and every location that looks suspicious .  ", "most often , ann arbour staging is employed , which describe the extend of all types of non - hodgkin lymphoma in adults .", "thus , staging of gastric lymphoma based upon the ann arbor system includes stage i e , which is disease limited to the stomach without nodal spread .", "stage ii e1 is tumor in the stomach with spread to adjacent contiguous lymph nodes .", "stage ii e2 is tumor in the stomach with spread to lymph nodes that are noncontiguous with the primary tumor .", "moreover , if the spleen is affected , we add s. if the person has any of the b symptoms , we add letter b , and if is asymptomatic , we assign a ( table 1 ) .", "prognostic factors in gastric malt lymphoma are similar to the value for non - hodgkin b - cells lymphoma .", "factors that determine poor outcome are age , high level of ldh in serum , higher ecog performance status , stages iii and iv in ann - arbour scale , white blood count , and more than one extranodal site .", "it was observed that patients with nodal invasion has difficulty with complete remission after eradication treatment .", "for instance patients , with t(11;18)(q21;q21 ) especially , are resistant to the first line therapy , and remission rate was lower than that in patients of api2-malt1 negative ( 78% versus 22.2% ; p = 0.0001 ) .", "only one study so far proved that the presence of t(3;14)(p14;q32 ) is connected with poor clinical outcomes of patients with gastric malt lymphoma ."], ["while helicobacter pylori plays a main role in the pathogenesis of malt lymphoma , it is also crucial in approach to the treatment . according to current international guidelines , first line treatment for localized helicobacter", "the treatment may be used with every highly effective antibiotics against helicobacter pylori , taking into consideration the locally expected antibiotic resistance . if there is no response to the therapy above , second line triple or quadruple therapy is used .", "it was reported that after two lines of treatment , 99.8% of patients were cured from gastritis . in a large study of 1408 patients , remission after eradication treatment in early stage", "unfortunately , in 5%10% of gastric malt lymphoma patients , we can not confirm helicobacter pylori infection .", "moreover , more than 30% patients are resistant to first line treatment , and 30% of them have t(11;18)(q21;q21 ) .", "treatment for this patients should be chosen individually depending on the clinical stage of disease . for those who have stable disease without any symptoms ,", "radiotherapy , chemotherapy , and/or surgery can be considered after unsuccessful eradication treatment . further recommended", "surgery is considered to be a standard therapy in therapy of patients with gastric malt lymphomas , but , recently , the value of this therapy has been not confirmed .", "even if the lymphoma is localized at early stage , the gastrectomy should be rather extensive due to the nature of the disease .", "moreover , it is a major surgery and can be associated with serious complications and worsen a quality of life .", "german multicenter study group ( gmsg ) presented no difference between survival in patients treated with gastrectomy compared to eradication ( overall survival rate 82% to 84% ) .", "what is more important is that there were observed 50% long - term complications were observed after surgery . in few studies", "the use of a modest dose of involved fields was performed on resistant - to - eradication therapy patients with early - stage disease .", "the dose was 2535  gy to the stomach and perigastric nodes for the period of 4 weeks [ 42 , 43 ] . compared to surgery , no serious long - term complications and toxicity were observed . only nausea and anorexia were present during the time of radiotherapy . for a long time", "it was believed that gastric malt lymphoma is just a localized disease and that surgery and radiotherapy are the best treatment strategy .", "now , when it is well known that it is disseminated disorder chemotherapy , it became more important .", "still , there are no standard recommendations for relapse or progressive patients after therapy and for those with late stage of the disease from the beginning .", "it was observed that chemotherapy alone is more effective than surgery apart from some cases with gastric obstruction .", "complete remission ( cr ) after oral monochemotherapy with cyclophosphamide was 83% in a study by nakamura and coworkers .", "unfortunately , patients with positive translocation t(11 ; 18 ) are resistant to second line therapy with oral monochemotherapy with alkylating agents .", "nucleoside analogs are confirmed to be effective in treatment of different kinds of indolent lymphomas .", "a polychemotherapy with fludarabine and mitoxantrone ( fm ) has a very good effect on patients with gastric malt lymphoma in both first and second line treatment .", "the complete remission after 4 cycles achieved 84% of investigated and all of them reacted to the treatment .", "after 2-cda , there were observed complications such as toxicities of 3 and 4 grade of who , mainly leukopenia , infections , and secondary neoplastic disease .", "nowadays , immunotherapy became an extremely important part of treatment of non - hodgkin lymphomas .", "it is a chimeric mouse / human monoclonal antibody specified to cd20 antigen expressed on the surface of b lymphocytes .", "now it is widely used alone or in combination with chemotherapeutic drugs in many types of b - cell non - hodgkin lymphomas .", "rituximab binds to cd20 antigen and activates the lysis of b cells by mediating cytotoxicity of complement dependent ( cdc ) and cell - mediated cytotoxicity antibody dependent ( adcc ) .", "the role of this drug is still not clear in gastric malt lymphoma . in 2003 , there was a first - phase study by conconi et al .  ", "the cr was observed in 29% and overall response rate ( orr ) was 64% .", "the toxicity of this treatment was moderate or even mild , but the relapse rate was 36% .", "an important fact is that patients with translocation t(11;18 ) are responsive to rituximab treatment [ 58 , 59 ] .", "what is more important is that in a study by the international extranodal lymphoma study group ( ielsg ) , it was confirmed that chlorambucil in combination with rituximab was more effective than chlorambucil alone .", "the conclusion is that rituximab may a benefit in individual patients , but for the majority it is not sufficient when used alone .", "the efficacy of the combination of rituximab with chlorambucil was evaluated in a randomized study ( comparator was chlorambucil alone ) by the international extranodal lymphoma study group ( ielsg ) in gastric malt lymphomas that had failed antibiotics and in nongastric malt lymphomas .", "the preliminary report   showed that the 5-year event - free survival was significantly better for patients treated with chlorambucil plus rituximab .", "there were also studies by raderer et al . with cycles generally used in more aggressive lymphomas .", "twenty - six patients were administrated rituximab plus cyclophosphamide , doxorubicin or mitoxantrone , vincristine , and prednisone .", "lately , bortezomib , the first therapeutic proteasome inhibitor , was examined by kiesewetter et al . in 2012 with cr in 33% and pr in 27.8% .", "the results on phase ii studies with chemotherapy and immunotherapy are shown in table 2 .", "outcomes in gastric malt lymphoma patients with progressive , disseminated disease are very comparable with outcomes in follicular lymphoma ."], ["although gastric malt lymphoma has a very favorable outcome , it is still important to have a proper followup", ". it is possible that the disease will return even after 5 years of complete remission .", "the relapse can be due to reinfection of helicobcater pylori . in a study by zullo et al .", "the followup is obligatory in patients with gastric malt lymphoma to identify early phase of the recurrence of the disease . to confirm a complete remission ,", "although , there are no specified recommendations for a followup , the biopsy of gastric sites should be made every 6 months in first two years , and later once a year for the next five years .", "systemic followup consist of blood tests and minimal adequate radiological and ultrasound and should be made at least once a year in the first 5 years .", "the transformation in more aggressive lymphoma is low at the level of 0.05% , but there is a higher risk of occurrence of secondary neoplasm   and gastric cancer .", "these studies confirm that patients with gastric malt lymphoma need a long - term followup not only to detect early recurrence but also to find secondary disease ."], ["recently , enormous progress has been made in better understanding of pathogenesis of gastric malt lymphoma .", "it has a great influence on the development of new and more effective treatment strategy .", "still , not enough clinical trials are performed due to rare expression and high effectiveness of first line treatment of gastric malt lymphoma .", "what is more important is that early diagnosis of gastric malt lymphoma is extremely important . while the symptoms are unspecific or not , always during the endoscopic exam the complete histological biopsies must be taken to make diagnosis correctly . the less advanced the stage of the disease , the bigger the chances to achieve complete remission"]]}
{"article_id": "PMC3221551", "article_text": ["airprom : airway disease , predicting outcomes through patient specific computational modeling ( fp7 ) ; bioshare - eu : biobank standardization and harmonization for research excellence in the european union ( fp7 ) ; ict : information communication technology ; medall : mechanisms of the development of allergy ( fp7 ) ; naepp - epr3 : national asthma education and prevention program , expert report 3 ; ncd : non - communicable disease ; p4 , predictive , preventive , personalized and participatory ; u - biopred : unbiased biomarkers in prediction of respiratory disease outcomes ( fp7 ) ; un : united nations ; who : world health organization .", "the authors declare that they have no competing interests in relation to the content of this article .", "may 2008 : 61st world health assembly . who recommended a worldwide priority policy on ncd prevention and control ( 2008 to 2013 ) , including cardiovascular disease , cancer , chronic respiratory diseases   and diabetes , not least because they often have common environmental risk factors .", "may 2010 : united nations ( un ) general assembly unanimously adopted resolution a / res/64/265 : ' tackling ncds constitutes one of the major challenges for sustainable development in the 21st century ' .", "december 2010 : the council of the european union adopted conclusions based on innovative and global approaches for ncds in public health and healthcare systems to further develop population - based and patient - centered policies .", "2010 : us center for disease control and prevention ( cdc )   says that ' an essential strategy for keeping older adults healthy is preventing ncds and reducing associated complications ' .", "the classical definition of prevention   includes : primary prevention : to avoid the development of disease .", "secondary prevention : recognize a disease before it results in morbidity ( or co - morbidity ) . tertiary prevention : to reduce the negative impact of established disease by restoring function and reducing disease - related complications . expanding on the traditional model of prevention ,", "gordon   proposed a three - tiered preventative intervention classification system on the basis of the population for whom the measure is advisable based on a cost - benefit analysis : universal prevention addresses the entire population ( for example , national , local community , school , and district ) and aims to prevent or delay risk factor exposure .", "all individuals , without screening , are provided with information and skills necessary to prevent the problem .", "the subgroups may be distinguished by characteristics such as age , gender , family history , or economic status . indicated prevention involves a screening process . according to these definitions", ", health promotion   should be used for primary universal and selective prevention strategies , whereas p4 medicine ( predictive , preventive , personalized and participatory )   should be used for primary , secondary and tertiary indicated prevention strategies . to prevent the occurrence of ncds by implementing effective action at societal and individual levels : to detect and diagnose disease at an early stage , when it can be controlled effectively . to stratify patients into groups , enabling the selection of optimal therapy . to reduce adverse drug reactions through the predictive or early assessment of individual drug responses and assessing genes leading to ineffective drug metabolism . to improve the selection of new biochemical targets for drug discovery . to reduce the time , cost , and failure rate of clinical trials for new therapies . to shift the emphasis in medicine from reaction to prevention and from disease to wellness .", "part of the conceptual work presented has received support from the european commission fp7 projects airprom ( grant agreement fp7 270194 ) , bioshare - eu ( grant agreement fp7 261433 ) , medall ( grant agreement fp7 264357 ) , synergy - copd ( grant agreement ) and u - biopred ( grant agreement imi 115010 ) .", "jb , jma , ac - t , fk , mlk , sp , cp and ca were supported by medall ; pjs , i m a and kfc were supported by u - biopred ; jr and aa were supported by synergy - copd ; cb was supported by airprom ; ac - t was supported by bioshare - eu .", "the positions , proposals and ideas expressed in this paper have been discussed by several authors ( ca , zc , lh , ab ,", "jb , ac , sa , dc , dn ) during the inaugural event of the european institute for systems biology and medicine of the systemoscope international consortium at the biovision world life sciences forum in lyon on 28 march 2011 ."], "abstract_text": ["<S> we propose an innovative , integrated , cost - effective health system to combat major non - communicable diseases ( ncds ) , including cardiovascular , chronic respiratory , metabolic , rheumatologic and neurologic disorders and cancers , which together are the predominant health problem of the 21st century . </S>", "<S> this proposed holistic strategy involves comprehensive patient - centered integrated care and multi - scale , multi - modal and multi - level systems approaches to tackle ncds as a common group of diseases . rather than studying each disease individually </S>", "<S> , it will take into account their intertwined gene - environment , socio - economic interactions and co - morbidities that lead to individual - specific complex phenotypes . </S>", "<S> it will implement a road map for predictive , preventive , personalized and participatory ( p4 ) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information . </S>", "<S> it will be supported by strategic partnerships involving all stakeholders , including general practitioners associated with patient - centered care . </S>", "<S> this systems medicine strategy , which will take a holistic approach to disease , is designed to allow the results to be used globally , taking into account the needs and specificities of local economies and health systems . </S>"], "labels": null, "section_names": ["Abbreviations", "Competing interests", "None", "Acknowledgements"], "sections": [["airprom : airway disease , predicting outcomes through patient specific computational modeling ( fp7 ) ; bioshare - eu : biobank standardization and harmonization for research excellence in the european union ( fp7 ) ; ict : information communication technology ; medall : mechanisms of the development of allergy ( fp7 ) ; naepp - epr3 : national asthma education and prevention program , expert report 3 ; ncd : non - communicable disease ; p4 , predictive , preventive , personalized and participatory ; u - biopred : unbiased biomarkers in prediction of respiratory disease outcomes ( fp7 ) ; un : united nations ; who : world health organization ."], ["the authors declare that they have no competing interests in relation to the content of this article ."], ["may 2008 : 61st world health assembly . who recommended a worldwide priority policy on ncd prevention and control ( 2008 to 2013 ) , including cardiovascular disease , cancer , chronic respiratory diseases   and diabetes , not least because they often have common environmental risk factors .", "may 2010 : united nations ( un ) general assembly unanimously adopted resolution a / res/64/265 : ' tackling ncds constitutes one of the major challenges for sustainable development in the 21st century ' .", "december 2010 : the council of the european union adopted conclusions based on innovative and global approaches for ncds in public health and healthcare systems to further develop population - based and patient - centered policies .", "2010 : us center for disease control and prevention ( cdc )   says that ' an essential strategy for keeping older adults healthy is preventing ncds and reducing associated complications ' .", "the classical definition of prevention   includes : primary prevention : to avoid the development of disease .", "secondary prevention : recognize a disease before it results in morbidity ( or co - morbidity ) . tertiary prevention : to reduce the negative impact of established disease by restoring function and reducing disease - related complications . expanding on the traditional model of prevention ,", "gordon   proposed a three - tiered preventative intervention classification system on the basis of the population for whom the measure is advisable based on a cost - benefit analysis : universal prevention addresses the entire population ( for example , national , local community , school , and district ) and aims to prevent or delay risk factor exposure .", "all individuals , without screening , are provided with information and skills necessary to prevent the problem .", "the subgroups may be distinguished by characteristics such as age , gender , family history , or economic status . indicated prevention involves a screening process . according to these definitions", ", health promotion   should be used for primary universal and selective prevention strategies , whereas p4 medicine ( predictive , preventive , personalized and participatory )   should be used for primary , secondary and tertiary indicated prevention strategies . to prevent the occurrence of ncds by implementing effective action at societal and individual levels : to detect and diagnose disease at an early stage , when it can be controlled effectively . to stratify patients into groups , enabling the selection of optimal therapy . to reduce adverse drug reactions through the predictive or early assessment of individual drug responses and assessing genes leading to ineffective drug metabolism . to improve the selection of new biochemical targets for drug discovery . to reduce the time , cost , and failure rate of clinical trials for new therapies . to shift the emphasis in medicine from reaction to prevention and from disease to wellness ."], ["part of the conceptual work presented has received support from the european commission fp7 projects airprom ( grant agreement fp7 270194 ) , bioshare - eu ( grant agreement fp7 261433 ) , medall ( grant agreement fp7 264357 ) , synergy - copd ( grant agreement ) and u - biopred ( grant agreement imi 115010 ) .", "jb , jma , ac - t , fk , mlk , sp , cp and ca were supported by medall ; pjs , i m a and kfc were supported by u - biopred ; jr and aa were supported by synergy - copd ; cb was supported by airprom ; ac - t was supported by bioshare - eu .", "the positions , proposals and ideas expressed in this paper have been discussed by several authors ( ca , zc , lh , ab ,", "jb , ac , sa , dc , dn ) during the inaugural event of the european institute for systems biology and medicine of the systemoscope international consortium at the biovision world life sciences forum in lyon on 28 march 2011 ."]]}
{"article_id": "PMC3432379", "article_text": ["the adventitia is more than just the outermost layer of the artery ; it is now known to play a critical role in vascular remodeling and other important processes of the artery [ 13 ] .", "recent attention to the role of the adventitia in vascular remodeling has increased reporting of common carotid artery interadventitial diameter ( iad ) , a noninvasive measure of vascular geometry and health .", "the results from one of these studies , an ancillary of the study of women 's health across the nation ( swan ) called swan heart , suggest that declining endogenous estrogen that accompanies the menopausal transition has a direct effect on the peripheral vasculature .", "the study found lower levels of estradiol were significantly associated with larger common carotid artery iad even after adjustment for cardiovascular risk factors .", "other studies have shown that larger iad is associated with increasing age [ 57 ] , cardiovascular risk factors [ 46 , 813 ] , prevalent cardiovascular disease [ 12 , 14 , 15 ] , and incident cardiovascular events .", "thus , the increase in iad observed with declining endogenous estrogen   suggests that lower levels of endogenous estrogens are associated with a less healthy vasculature .  ", "the strong association between iad and endogenous estrogen suggests that a similar association may exist with the use of exogenous estrogen .", "the purpose of this study was to determine whether postmenopausal current ht users had significantly different iad than those who were former users of ht in the women on the move through activity and nutrition ( woman ) randomized trial .", "we also wanted to determine if there were differences between other measures of vascular health .", "this study evaluates cross - sectional associations using measurements from the baseline visit of the clinical trial ( clinical trials registry number : nct 00023543 ) .", "the woman trial tested the ability of nonpharmacological lifestyle intervention to modify cardiovascular risk factors in postmenopausal women .", "the study recruited 508 eligible african american and caucasian women from allegheny county , pa , between april 2002 and october 2003 through direct mailings .", "eligible women were postmenopausal , between 52 and 62 years of age , able to walk , currently using ht , and willing to participate in either intervention group regardless of assignment and had a waist circumference 80  cm , a body mass index ( bmi ) between 25.0 and 39.9  kg / m , blood pressure < 160/95  mmhg , and low density lipoprotein ( ldl ) cholesterol between 100 and 160  mg / dl .", "women were ineligible if they were taking medication for cholesterol , diagnosed with or on medication for diabetes , diagnosed with a psychotic disorder , or suffering from depression .", "the results of the women 's health initiative estrogen / progestin arm were published in the middle of recruitment ; as a result the eligibility criterion of current use of ht was modified to current or recent history of hormone use .", "recent history of hormone use was defined as prior use of at least 2 years within 6 months of randomization .", "the decision to remain on ht was determined by the participant and her physician . at baseline ,", "40% of the women had discontinued use of ht ( these women will be referred to as former ht users ) and 60% remained on ht ( these women will be referred to as current ht users ) . for those who discontinued use of ht the median time off therapy was 7 months prior to study randomization .", "common carotid artery intima media thickness ( imt ) , iad , lumen diameter ( ld ) , and plaque were assessed by b - mode ultrasound using a toshiba ssa-270a duplex scanner ( toshiba american medical systems , tustin , ca , usa ) with a 5  mhz - linear array transducer .", "right and left carotid images were taken of the near and far walls of the distal common carotid artery 1  cm proximal to the carotid bulb .", "imt was defined as the distance from the lumen - intimal interface to the medial - adventitial interface ( figure 1 ) .", "iad was defined as the distance from the adventitial - medial interface on the near wall to the medial - adventitial interface on the far wall ( figure 1 ) .", "ld was defined as the distance from the intima - lumen interface of the near wall to the lumen - intima interface of the far wall ( figure 1 ) . using a semiautomated edge detection software ,", "the interfaces were traced electronically over the distal cca and a computer generated measurement was obtained for each pixel in the area of interest ; these measurements were averaged to determine imt , iad , and ld used for this analysis .", "a reproducibility study , conducted in 20 women who were similar to the women in the current study , provided an intraclass correlation of 0.98 for imt and 0.99 for iad .", "the reproducibility study took place at the same lab and used the same equipment and readers as the current study .", "the presence of plaque was determined for each of the 5 segments of the left and right carotid artery ( distal and proximal cca , carotid bulb , and proximal internal and external carotid artery ) .", "plaque was defined as a distinct area protruding into the vessel lumen at least 50% thicker than the adjacent imt .", "the first of two prerandomization screening visits included a 12-hour fasting blood draw , physical measures of height , weight , waist circumference , blood pressure , the long distance corridor walk , medical , physical activity , and weight history .", "conventional enzymatic methods were used to obtain total cholesterol , high density lipoprotein ( hdl ) cholesterol , and triglyceride concentrations from the blood samples . low density lipoprotein ( ldl ) cholesterol was estimated using the friedewald equation .", "medical history included history of drug , vitamin / mineral supplement , and alcohol use .", "common carotid artery iad , ld , imt and plaque were measured at the second screening visit .  ", "seventeen women had incomplete data for the calculation of iad or imt and were excluded leaving 491 women for analysis .", "means and standard deviations are presented for normally distributed variables and medians and 25th and 75th percentiles are provided for nonparametric variables ; dichotomous variables are presented as percents .", "differences between the current ht users and the former ht users were determined using chi - square analyses for categorical variables and t - tests and wilcoxon - rank sum tests for continuous variables .  ", "simple linear regression was used to assess univariate associations between iad and ld with ht and the following cardiovascular risk factors : age , race , systolic blood pressure , diastolic blood pressure , pulse pressure , bmi , weight , height , waist circumference , total cholesterol , ldl and hdl cholesterol , triglycerides , glucose , insulin , smoking status , and antihypertensive medication use .", "when collinearity between covariates was suspected ( r > 0.4 ) , the variable most strongly correlated to iad or ld was selected for the analysis .", "the following variables were collinear : glucose and insulin ; systolic blood pressure and pulse pressure ; bmi , weight , and waist circumference ; weight and height ; total cholesterol and ldl . based on spearman", "correlation results glucose , pulse pressure , weight , and ldl were chosen for the multivariable models .", "multivariable linear regression was used to test for the following predetermined covariates : age , race , pulse pressure , and smoking status .", "in addition , any statistically significant variable in the univariate analysis and any variable that differed by ht use status were also tested .", "total cholesterol , hdl , ldl , glucose , and insulin differed by ht use status ; addition of these variables did not alter the regression model so they are not presented in the results .", "the median ( 25th , 75th percentiles ) age of the women was 57 ( 55 , 60 ) years , median bmi was 30 ( 28 , 34 ) kg / m ; 11% were african american and 6% were current smokers .", "there were 197 former ht users and 294 current ht users at the time of the baseline carotid ultrasound scan .", "former ht users were older and had a higher percent of african americans ( table 1 ) .", "overall former ht users had a significantly worse cardiovascular disease risk profile than current ht users : higher total cholesterol , higher ldl cholesterol , higher glucose and insulin , and lower hdl cholesterol ( table 1 ) .", "there were , however , no differences by ht status in blood pressure , measures of general or central obesity , and smoking status ( table 1 ) .", "the mean iad was 6.94  mm for former ht users and 6.79  mm for current ht users ( p = 0.001 , table 2 ) .", "ld was also significantly larger in the former ht users than in the current ht users ( 5.44  mm versus 5.31  mm , p = 0.002 , table 2 )", ". however , imt and presence of plaque were not different between the two groups ( table 2 ) .  ", "simple linear regression showed that in addition to former ht use , larger iad was significantly associated with greater systolic blood pressure , pulse pressure , bmi , weight , height , waist circumference ( all p < 0.0001 ) , glucose , insulin ( both p = 0.001 ) , age , caucasian race , current nonsmoking status and use of antihypertensive medications ( p < 0.05 ) ( table 3 ) .", "the most parsimonious model in the multivariate analysis revealed that higher pulse pressure , higher weight and former ht use were the key factors independently associated with larger iad .", "the model was also run forcing age , race , and smoking status ( table 4 ) . in this model ,", "hormone therapy , pulse pressure , and weight remained significantly associated with iad ( all p < 0.01 , table 4 ) .", "the model was also run controlling for antihypertensive medication use , but this variable fell out of the multivariable model when pulse pressure was added .", "current ht use was associated with a 0.14  mm smaller iad ( table 4 ) .", "african american women had a 0.05  mm smaller iad than the caucasian women , although this was not significant ( table 4 ) .", "current cigarette smokers had 0.18  mm smaller iad than current nonsmokers , with borderline significance ( table 4 ) .   ", "former ht use , greater bmi , weight , height , waist circumference , insulin ( all p < 0.01 ) , glucose , and pulse pressure ( both p < 0.05 ) were associated with larger ld in univariate linear regression ( table 3 ) . the variables that yielded the best multivariate model to explain larger ld were former ht use , higher pulse pressure , and higher weight .", "when age , race and smoking were forced in the model only ht use and weight remained significantly associated with ld ( table 4 ) ; the same results were seen when antihypertensive medication was added to the model ( data not shown ) .  ", "current ht use was associated with a 0.13  mm smaller ld ( table 4 ) .", "each kg of weight was associated with 0.009  mm larger ld ( table 4 ) .", "postmenopausal current ht users had statistically significant smaller iad than the former ht users ; this relationship remained significant after adjustment for known cardiovascular risk factors .", "the current ht users also had statistically significant smaller ld than the former ht users .", "in contrast , imt and plaque were not statistically different between current ht users and former ht users .", "it also demonstrates the value of measuring iad and ld in this type of study .", "the adventitia , the most outer layer of the artery , is composed of supportive connective tissue , fibroblasts , collagen , and elastin fibers .", "estrogen is known to preserve arterial structure by slowing elastin and connective tissue degradation , and by slowing age- and estrogen - related increases in collagen which lead to increased vascular stiffening .", "a small diameter reflects a healthy vasculature that is able to maintain an optimal balance of shear and tensile stress [ 1921 ] .", "this can make the artery vulnerable to injury and atherosclerotic development [ 1 , 11 ] .  ", "the results of the current study , specifically the association of current exogenous estrogen use with smaller iad , is in line with the swan heart study   that showed an association between higher levels of endogenous estrogen and smaller iad .", "the current study observed a 0.15  mm difference in iad between the current and former ht users .", "a longitudinal study observed 0.03  mm increase in iad each year for women ( with similar mean age and mean height as the women in the current study ) .", "so the difference in iad observed in the current study translates to the change in iad expected over 5 years ( 0.03  mm / year 5 years = 0.15  mm ) .  ", "these findings agree with the results of a cross - sectional study that found smaller ld among non - oral ( percutaneous gel or transdermal patch ) ht users compared to ht non - users .", "together , the findings from iad and ld may suggest the positive effect of estrogen on the vasculature through maintenance of vascular structure and function .   both the current study and the swan heart study found that larger diameter was associated with older age , higher systolic blood pressure , higher glucose , and higher insulin : all risk factors for cvd .", "additional supporting evidence that enlarged diameter is an indicator of poor vascular health come from several studies showing enlarged iad is associated with cardiovascular disease risk factors [ 46 , 813 ] , increased imt [ 5 , 12 ] , plaque [ 5 , 12 , 23 ] , and prevalent [ 12 , 14 , 15 ] and incident cvd .", "polak et al . recently published an article that identified a positive relationship between iad and left ventricular mass , an indicator of left ventricular hypertrophy .", "each 1 gram difference in left ventricular mass was associated with 0.006  mm larger iad in a multiethnic population of women after adjustment for height , weight , and imt .  ", "arterial diameter differences in current ht users and former ht users were observed in this study but differences in imt were not .", "consistent with our findings , a cross - sectional study of an american cohort from the atherosclerosis risk in communities ( aric ) study , found no significant difference in imt by ht use .", "the women in the aric study were of similar age to the women in this study and also had an undefined ht regimen that was predetermined by the woman and her physician prior to the study .", "selection bias may be present in both studies since women who chose to go on ht or women who chose to continue ht may have been different from the women who did not chose ht .", "a longitudinal study of oral therapy with one year of follow - up did not find a difference in imt progression in ht users and non - users .  ", "three studies evaluated differences in imt by ht and age [ 22 , 26 , 27 ] .", "the women were dichotomized into younger versus older ( using 55 or 60 as the age cut - point ) .", "significant differences in imt were observed only in the older women who had longer use of ht than the younger women [ 22 , 26 , 27 ] .", "this may suggest that the effects of estrogen on imt are evident after long - term use .", "other explanations are that the differences observed are attributed to differing vascular effects of oral ht compared to transdermal ht , and fewer years on ht in the negative studies than the positive studies .", "one study that compared oral and non - oral therapy found transdermal ht had greater statistically significant effects on imt than oral ht . the aric study and this study participants used oral ht , were relatively younger and had fewer years on ht compared to the positive studies .", "a limitation of this study is that the ht regimen was varied since the dose , hormone composition ( estrogen only or estrogen plus progestin ) , and form were chosen prior to the study by the participant and her health care provider .", "a standard dose and regimen of ht would be easier to evaluate and compare this study to previous studies", ". this would likely be especially true for the imt results that were not significant in this study .", "another limitation is that the adherence to ht and the level of estrogen or estradiol in the current users and former users was not assessed in this study .", "although the women reported ht use we do not know their adherence rates or the level of estrogen metabolites present during the ultrasound measurements . in the future ,", "assessment of estradiol levels should be included to improve our understanding of carotid diameter associations and dose - related effects .", "strengths of this study are that it fills a gap in the literature , the methods used are valid and reliable , the lab that performed the ultrasound measures has high quality control , and it is one of the first to evaluate iad and ht .", "this study demonstrates the importance of iad and ld as more sensitive indicators of vascular health than imt .", "high resolution b - mode ultrasound is a valid and reliable detector of structural atherosclerotic changes of the arterial walls .", "the ultrasound measures in this study were performed with excellent reproducibility ( class intra correlations of 0.98 for imt and 0.99 for iad ) and continuous quality control to ensure reliable and valid data .", "in conclusion , these data suggest that current ht use is associated with vascular geometry in the postmenopausal women independent of cardiovascular risk factors", ". it also demonstrates the importance of measuring iad and ld in postmenopausal women with differing ht use .", "these measures should be included in addition to imt to provide a more complete story of vascular response and health ."], "abstract_text": ["<S> arterial diameter is an underutilized indicator of vascular health . </S>", "<S> we hypothesized that interadventitial and lumen diameter of the common carotid artery would be better indicators of vascular health than carotid plaque or intima media thickness ( imt ) . </S>", "<S> participants were 491 overweight or obese , postmenopausal women who were former or current hormone therapy ( ht ) users , 5262 years , with waist circumference > 80  cm . </S>", "<S> we evaluated cross - sectional associations of cardiovascular risk factors with carotid measures , by ht status . </S>", "<S> former ht users had a worse cardiovascular profile than current ht users : larger adventitial ( 6.94  mm versus 6.79  mm ) and lumen diameter ( 5.44  mm versus 5.31  mm , both p < 0.01 ) independent of cardiovascular risk factors ; imt and plaque were similar . </S>", "<S> larger diameters were best explained by former ht use , higher pulse pressure , and greater weight . </S>", "<S> independent of potential confounders , overweight and obese postmenopausal former ht users had larger carotid diameters than current ht users . </S>", "<S> carotid diameter should be considered in studies of ht . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Methods", "3. Results", "4. Discussion", "5. Conclusions"], "sections": [["the adventitia is more than just the outermost layer of the artery ; it is now known to play a critical role in vascular remodeling and other important processes of the artery [ 13 ] .", "recent attention to the role of the adventitia in vascular remodeling has increased reporting of common carotid artery interadventitial diameter ( iad ) , a noninvasive measure of vascular geometry and health .", "the results from one of these studies , an ancillary of the study of women 's health across the nation ( swan ) called swan heart , suggest that declining endogenous estrogen that accompanies the menopausal transition has a direct effect on the peripheral vasculature .", "the study found lower levels of estradiol were significantly associated with larger common carotid artery iad even after adjustment for cardiovascular risk factors .", "other studies have shown that larger iad is associated with increasing age [ 57 ] , cardiovascular risk factors [ 46 , 813 ] , prevalent cardiovascular disease [ 12 , 14 , 15 ] , and incident cardiovascular events .", "thus , the increase in iad observed with declining endogenous estrogen   suggests that lower levels of endogenous estrogens are associated with a less healthy vasculature .  ", "the strong association between iad and endogenous estrogen suggests that a similar association may exist with the use of exogenous estrogen .", "the purpose of this study was to determine whether postmenopausal current ht users had significantly different iad than those who were former users of ht in the women on the move through activity and nutrition ( woman ) randomized trial .", "we also wanted to determine if there were differences between other measures of vascular health ."], ["this study evaluates cross - sectional associations using measurements from the baseline visit of the clinical trial ( clinical trials registry number : nct 00023543 ) .", "the woman trial tested the ability of nonpharmacological lifestyle intervention to modify cardiovascular risk factors in postmenopausal women .", "the study recruited 508 eligible african american and caucasian women from allegheny county , pa , between april 2002 and october 2003 through direct mailings .", "eligible women were postmenopausal , between 52 and 62 years of age , able to walk , currently using ht , and willing to participate in either intervention group regardless of assignment and had a waist circumference 80  cm , a body mass index ( bmi ) between 25.0 and 39.9  kg / m , blood pressure < 160/95  mmhg , and low density lipoprotein ( ldl ) cholesterol between 100 and 160  mg / dl .", "women were ineligible if they were taking medication for cholesterol , diagnosed with or on medication for diabetes , diagnosed with a psychotic disorder , or suffering from depression .", "the results of the women 's health initiative estrogen / progestin arm were published in the middle of recruitment ; as a result the eligibility criterion of current use of ht was modified to current or recent history of hormone use .", "recent history of hormone use was defined as prior use of at least 2 years within 6 months of randomization .", "the decision to remain on ht was determined by the participant and her physician . at baseline ,", "40% of the women had discontinued use of ht ( these women will be referred to as former ht users ) and 60% remained on ht ( these women will be referred to as current ht users ) . for those who discontinued use of ht the median time off therapy was 7 months prior to study randomization .", "common carotid artery intima media thickness ( imt ) , iad , lumen diameter ( ld ) , and plaque were assessed by b - mode ultrasound using a toshiba ssa-270a duplex scanner ( toshiba american medical systems , tustin , ca , usa ) with a 5  mhz - linear array transducer .", "right and left carotid images were taken of the near and far walls of the distal common carotid artery 1  cm proximal to the carotid bulb .", "imt was defined as the distance from the lumen - intimal interface to the medial - adventitial interface ( figure 1 ) .", "iad was defined as the distance from the adventitial - medial interface on the near wall to the medial - adventitial interface on the far wall ( figure 1 ) .", "ld was defined as the distance from the intima - lumen interface of the near wall to the lumen - intima interface of the far wall ( figure 1 ) . using a semiautomated edge detection software ,", "the interfaces were traced electronically over the distal cca and a computer generated measurement was obtained for each pixel in the area of interest ; these measurements were averaged to determine imt , iad , and ld used for this analysis .", "a reproducibility study , conducted in 20 women who were similar to the women in the current study , provided an intraclass correlation of 0.98 for imt and 0.99 for iad .", "the reproducibility study took place at the same lab and used the same equipment and readers as the current study .", "the presence of plaque was determined for each of the 5 segments of the left and right carotid artery ( distal and proximal cca , carotid bulb , and proximal internal and external carotid artery ) .", "plaque was defined as a distinct area protruding into the vessel lumen at least 50% thicker than the adjacent imt .", "the first of two prerandomization screening visits included a 12-hour fasting blood draw , physical measures of height , weight , waist circumference , blood pressure , the long distance corridor walk , medical , physical activity , and weight history .", "conventional enzymatic methods were used to obtain total cholesterol , high density lipoprotein ( hdl ) cholesterol , and triglyceride concentrations from the blood samples . low density lipoprotein ( ldl ) cholesterol was estimated using the friedewald equation .", "medical history included history of drug , vitamin / mineral supplement , and alcohol use .", "common carotid artery iad , ld , imt and plaque were measured at the second screening visit .  ", "seventeen women had incomplete data for the calculation of iad or imt and were excluded leaving 491 women for analysis .", "means and standard deviations are presented for normally distributed variables and medians and 25th and 75th percentiles are provided for nonparametric variables ; dichotomous variables are presented as percents .", "differences between the current ht users and the former ht users were determined using chi - square analyses for categorical variables and t - tests and wilcoxon - rank sum tests for continuous variables .  ", "simple linear regression was used to assess univariate associations between iad and ld with ht and the following cardiovascular risk factors : age , race , systolic blood pressure , diastolic blood pressure , pulse pressure , bmi , weight , height , waist circumference , total cholesterol , ldl and hdl cholesterol , triglycerides , glucose , insulin , smoking status , and antihypertensive medication use .", "when collinearity between covariates was suspected ( r > 0.4 ) , the variable most strongly correlated to iad or ld was selected for the analysis .", "the following variables were collinear : glucose and insulin ; systolic blood pressure and pulse pressure ; bmi , weight , and waist circumference ; weight and height ; total cholesterol and ldl . based on spearman", "correlation results glucose , pulse pressure , weight , and ldl were chosen for the multivariable models .", "multivariable linear regression was used to test for the following predetermined covariates : age , race , pulse pressure , and smoking status .", "in addition , any statistically significant variable in the univariate analysis and any variable that differed by ht use status were also tested .", "total cholesterol , hdl , ldl , glucose , and insulin differed by ht use status ; addition of these variables did not alter the regression model so they are not presented in the results ."], ["the median ( 25th , 75th percentiles ) age of the women was 57 ( 55 , 60 ) years , median bmi was 30 ( 28 , 34 ) kg / m ; 11% were african american and 6% were current smokers .", "there were 197 former ht users and 294 current ht users at the time of the baseline carotid ultrasound scan .", "former ht users were older and had a higher percent of african americans ( table 1 ) .", "overall former ht users had a significantly worse cardiovascular disease risk profile than current ht users : higher total cholesterol , higher ldl cholesterol , higher glucose and insulin , and lower hdl cholesterol ( table 1 ) .", "there were , however , no differences by ht status in blood pressure , measures of general or central obesity , and smoking status ( table 1 ) .", "the mean iad was 6.94  mm for former ht users and 6.79  mm for current ht users ( p = 0.001 , table 2 ) .", "ld was also significantly larger in the former ht users than in the current ht users ( 5.44  mm versus 5.31  mm , p = 0.002 , table 2 )", ". however , imt and presence of plaque were not different between the two groups ( table 2 ) .  ", "simple linear regression showed that in addition to former ht use , larger iad was significantly associated with greater systolic blood pressure , pulse pressure , bmi , weight , height , waist circumference ( all p < 0.0001 ) , glucose , insulin ( both p = 0.001 ) , age , caucasian race , current nonsmoking status and use of antihypertensive medications ( p < 0.05 ) ( table 3 ) .", "the most parsimonious model in the multivariate analysis revealed that higher pulse pressure , higher weight and former ht use were the key factors independently associated with larger iad .", "the model was also run forcing age , race , and smoking status ( table 4 ) . in this model ,", "hormone therapy , pulse pressure , and weight remained significantly associated with iad ( all p < 0.01 , table 4 ) .", "the model was also run controlling for antihypertensive medication use , but this variable fell out of the multivariable model when pulse pressure was added .", "current ht use was associated with a 0.14  mm smaller iad ( table 4 ) .", "african american women had a 0.05  mm smaller iad than the caucasian women , although this was not significant ( table 4 ) .", "current cigarette smokers had 0.18  mm smaller iad than current nonsmokers , with borderline significance ( table 4 ) .   ", "former ht use , greater bmi , weight , height , waist circumference , insulin ( all p < 0.01 ) , glucose , and pulse pressure ( both p < 0.05 ) were associated with larger ld in univariate linear regression ( table 3 ) . the variables that yielded the best multivariate model to explain larger ld were former ht use , higher pulse pressure , and higher weight .", "when age , race and smoking were forced in the model only ht use and weight remained significantly associated with ld ( table 4 ) ; the same results were seen when antihypertensive medication was added to the model ( data not shown ) .  ", "current ht use was associated with a 0.13  mm smaller ld ( table 4 ) .", "each kg of weight was associated with 0.009  mm larger ld ( table 4 ) ."], ["postmenopausal current ht users had statistically significant smaller iad than the former ht users ; this relationship remained significant after adjustment for known cardiovascular risk factors .", "the current ht users also had statistically significant smaller ld than the former ht users .", "in contrast , imt and plaque were not statistically different between current ht users and former ht users .", "it also demonstrates the value of measuring iad and ld in this type of study .", "the adventitia , the most outer layer of the artery , is composed of supportive connective tissue , fibroblasts , collagen , and elastin fibers .", "estrogen is known to preserve arterial structure by slowing elastin and connective tissue degradation , and by slowing age- and estrogen - related increases in collagen which lead to increased vascular stiffening .", "a small diameter reflects a healthy vasculature that is able to maintain an optimal balance of shear and tensile stress [ 1921 ] .", "this can make the artery vulnerable to injury and atherosclerotic development [ 1 , 11 ] .  ", "the results of the current study , specifically the association of current exogenous estrogen use with smaller iad , is in line with the swan heart study   that showed an association between higher levels of endogenous estrogen and smaller iad .", "the current study observed a 0.15  mm difference in iad between the current and former ht users .", "a longitudinal study observed 0.03  mm increase in iad each year for women ( with similar mean age and mean height as the women in the current study ) .", "so the difference in iad observed in the current study translates to the change in iad expected over 5 years ( 0.03  mm / year 5 years = 0.15  mm ) .  ", "these findings agree with the results of a cross - sectional study that found smaller ld among non - oral ( percutaneous gel or transdermal patch ) ht users compared to ht non - users .", "together , the findings from iad and ld may suggest the positive effect of estrogen on the vasculature through maintenance of vascular structure and function .   both the current study and the swan heart study found that larger diameter was associated with older age , higher systolic blood pressure , higher glucose , and higher insulin : all risk factors for cvd .", "additional supporting evidence that enlarged diameter is an indicator of poor vascular health come from several studies showing enlarged iad is associated with cardiovascular disease risk factors [ 46 , 813 ] , increased imt [ 5 , 12 ] , plaque [ 5 , 12 , 23 ] , and prevalent [ 12 , 14 , 15 ] and incident cvd .", "polak et al . recently published an article that identified a positive relationship between iad and left ventricular mass , an indicator of left ventricular hypertrophy .", "each 1 gram difference in left ventricular mass was associated with 0.006  mm larger iad in a multiethnic population of women after adjustment for height , weight , and imt .  ", "arterial diameter differences in current ht users and former ht users were observed in this study but differences in imt were not .", "consistent with our findings , a cross - sectional study of an american cohort from the atherosclerosis risk in communities ( aric ) study , found no significant difference in imt by ht use .", "the women in the aric study were of similar age to the women in this study and also had an undefined ht regimen that was predetermined by the woman and her physician prior to the study .", "selection bias may be present in both studies since women who chose to go on ht or women who chose to continue ht may have been different from the women who did not chose ht .", "a longitudinal study of oral therapy with one year of follow - up did not find a difference in imt progression in ht users and non - users .  ", "three studies evaluated differences in imt by ht and age [ 22 , 26 , 27 ] .", "the women were dichotomized into younger versus older ( using 55 or 60 as the age cut - point ) .", "significant differences in imt were observed only in the older women who had longer use of ht than the younger women [ 22 , 26 , 27 ] .", "this may suggest that the effects of estrogen on imt are evident after long - term use .", "other explanations are that the differences observed are attributed to differing vascular effects of oral ht compared to transdermal ht , and fewer years on ht in the negative studies than the positive studies .", "one study that compared oral and non - oral therapy found transdermal ht had greater statistically significant effects on imt than oral ht . the aric study and this study participants used oral ht , were relatively younger and had fewer years on ht compared to the positive studies .", "a limitation of this study is that the ht regimen was varied since the dose , hormone composition ( estrogen only or estrogen plus progestin ) , and form were chosen prior to the study by the participant and her health care provider .", "a standard dose and regimen of ht would be easier to evaluate and compare this study to previous studies", ". this would likely be especially true for the imt results that were not significant in this study .", "another limitation is that the adherence to ht and the level of estrogen or estradiol in the current users and former users was not assessed in this study .", "although the women reported ht use we do not know their adherence rates or the level of estrogen metabolites present during the ultrasound measurements . in the future ,", "assessment of estradiol levels should be included to improve our understanding of carotid diameter associations and dose - related effects .", "strengths of this study are that it fills a gap in the literature , the methods used are valid and reliable , the lab that performed the ultrasound measures has high quality control , and it is one of the first to evaluate iad and ht .", "this study demonstrates the importance of iad and ld as more sensitive indicators of vascular health than imt .", "high resolution b - mode ultrasound is a valid and reliable detector of structural atherosclerotic changes of the arterial walls .", "the ultrasound measures in this study were performed with excellent reproducibility ( class intra correlations of 0.98 for imt and 0.99 for iad ) and continuous quality control to ensure reliable and valid data ."], ["in conclusion , these data suggest that current ht use is associated with vascular geometry in the postmenopausal women independent of cardiovascular risk factors", ". it also demonstrates the importance of measuring iad and ld in postmenopausal women with differing ht use .", "these measures should be included in addition to imt to provide a more complete story of vascular response and health ."]]}
{"article_id": "PMC3329792", "article_text": ["prostate cancer ( pca ) is the most common solid organ malignancy in american men with global statistics mirroring those found in the united states . screening with prostate specific antigen ( psa )", "has resulted in a significant stage migration such that the majority of new cases of pca are now detected while the disease is still clinically localized .", "these patients can choose from several treatment options and must weigh the potential morbidity of each treatment modality on their quality of life . in the vast majority of cases , urologists are the primary physicians that diagnose these patients with pca and are typically involved in the initial work up , discussion of all treatment options , and counseling of patients .", "urologists are not only intimately involved with the treatment of the primary disease but also the consequential treatment - related complications while overseeing the long - term followup of these patients .", "several centers have been recently established where urologists partner with radiation oncologists acquire ownership interest in intensity - modulated radiation therapy ( imrt ) equipment and provide integrated prostate cancer care . although not yet validated in the literature , this may allow for improved quality of care and decreased cost .", "unfortunately , these efforts have been much maligned in both the media and radiation oncology literature as conduits to increased revenue for the urologists with only debatable patient benefit [ 3 , 4 ] .", "these reports have not yet been supported by data . after acquiring financial interest in an integrated prostate cancer center , we sought to evaluate whether our investment in imrt resulted in an increased utilization of radiation therapy in our patients with newly diagnosed prostate cancer .", "  in september of 2008 , we acquired financial interest in an integrated pca center offering imrt . following institutional board approval , we identified all patients who were diagnosed with pca in the 12 months before and after the center became operational .", "newly diagnosed cases of pca were identified by searching our electronic medical record using both prostate biopsy cpt codes ( transrectal ultrasound - guided [ trus ] needle biopsy of the prostate , 55700/76942/76872 ) and the icd-9 prostate cancer diagnostic code ( prostate cancer , 185.0 ) .", "all men were diagnosed with pca after pathologic review of biopsy needle cores obtained after trus .", "indications for biopsy included elevated psa , abnormal digital rectal exam , abnormal pca 3 test , and/or strong family history of pca .", "prostate biopsies were performed utilizing a routine sextant pattern with at least 12 cores obtained . in cases where clinically indicated ,", "the medical records of these patients were retrospectively reviewed and the data pertaining to the patients ' demographics , cancer parameters , and initial pca treatment modality were extracted .", "patients were assigned to two discrete groups based on the date of their pca diagnosis as it related to the date of the first availability of imrt at our integrated prostate center .", "all consecutive patients diagnosed with pca on a biopsy within 12 months prior to availability of imrt constituted the pre - investment group and all consecutive patients diagnosed with prostate cancer on a biopsy in the 12 months following initiation of imrt services constituted the post - investment group .", "the primary treatments received were designated as active surveillance ( as ) , brachytherapy ( bt ) , radiation therapy ( xrt ) , radical prostatectomy ( rp ) , and androgen deprivation therapy ( adt ) .", "treatment data were available for all patients and were stratified by the patient 's age and gleason score .", "the age of 70 years old served as a cut - off point as in our clinical practice most of these patients are deemed suboptimal surgical candidates because of increased risk of postoperative urinary incontinence and erectile dysfunction .", "our integrated pca center was established in collaboration with an academic radiation oncology department from a national cancer institute ( nci ) designated cancer center with a nationwide reputation for clinical and academic excellence .", "the radiation oncologists that treat our patients are not employed by the cancer center , but serve as full time academic faculty and have no financial interest in imrt .", "radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of radiation therapy .", "the center also employs a nurse - practitioner charged exclusively with the coordination and support of on - going clinical research programs .", "patients who were referred to us for second opinion with biopsy - proven pca as well as men referred to us specifically for robotic - assisted laparoscopic radical prostatectomy were excluded to eliminate the potential biases resulting from the treatment recommendations rendered by an outside urologist .", "the overwhelming majority of these patients choose surgical therapy and their inclusion would skew the results toward lower utilization rates of xrt .", "unpaired t - tests , chi - squared tests , and fisher 's exact tests were implemented as appropriate .", "  a total of 344 patients were diagnosed with pca on trus biopsy over the designated 24-month time period . of the total patient population , 198 men were diagnosed with pca in the 12 months preceding availability of imrt , while 146 men constituted the post - investment group .", "patient and cancer characteristics were similar between the two groups ( table 1 and figure 1 ) .", "the percentage of patients with gleason 7  pca was higher in the post - investment group but did not reach statistical significance , p = 0.073 ( figure 1 ) .", "overall , the use of radiation therapy for those patients with newly diagnosed pca following investment in imrt ( 20.55% versus 20.71% , p = 1.00 ) was similar between the two patient populations ( table 2 and figure 2 ) .", "the number of patients treated with rp ( 67.81% versus 71.72% , p = 0.729 ) , as ( 9.59% versus 4.55% , p = 0.177 ) , adt ( 1.37% versus 2.02% , p = 0.999 ) , and bt ( 0.68% versus 1.01% , p = 0.999 ) were not significantly different between post- and pre - investment groups . while overall treatment trends afford succinct analysis , clinical decisions regarding treatment of pca are often driven by a multitude of patient - specific factors .", "as such , the data was analyzed stratifying both gleason score and age ( 70 years old serving as a cutoff point ) .", "treatments stratified by patient age are shown in table 3 and figure 3 . despite the increased incidence of gleason score 7 disease in the post - investment group", ", there was no significant difference between the groups in all treatment patterns in men less than 70 years of age ( table 3 ) .", "an increase was found in the use of xrt in men older than 70 years of age in the pre - investment group ( 45.45% ) as compared to men following acquisition of imrt ( 55.32% ) , but this did not reach statistical significance ( p = 0.355 ) . analyzed by age and gleason score simultaneously , there was no difference between the treatment groups in patients younger than 70 regardless of the gleason score , table 4(a ) and figure 4(a ) . for men 70 years or older with gleason 6 disease , there was a trend toward increased use of as ( 34.78% versus 15.79% ) and decreased use of rp ( 21.74% versus 31.58% ) , but did not reach statistical significance ( table 4(b ) ) .", "there was no difference ( 43.48% versus 42.11% ) in the use of xrt for men over 70 with gleason 6 disease . for patients with gleason 7 disease", ", there was a statistically significant increase in the utilization of xrt ( pre - investment 41.38% versus post - investment 68.42% , p = 0.035 ) and decrease in the use of rp in the post - investment group ( 15.79% versus 55.17% , p = 0.006 ) seen in table 4(b ) and figure 4(b ) .", "there was no difference between treatment groups in men over 70 with gleason 8 disease .", "  external beam radiation therapy is widely used and is an effective treatment option for localized pca .", "there is now level i evidence that high - dose radiation therapy decreases the risk of biochemical failure in men with clinically localized prostate cancer as compared to conventional dose conformal radiation [ 57 ] .", "this improvement , however , comes at a cost of increased gastrointestinal ( gi ) and genitourinary ( gu ) toxicity .", "observed that 2% of men receiving high - dose radiation experienced acute urinary or rectal morbidity of radiation therapy oncology group ( rtog ) grade 3 or greater .", "intensity modulated radiation therapy allows for delivery of radiation with greater conformality to the target volume compared with traditional 3 d technique .", "several randomized trials have shown that imrt reduced gi and gu toxicity compared with 3 d conformal radiation [ 810 ] .", "were able to deliver 81  gy with less than 2% of grade 2 rectal morbidity and no grade 4 or greater rectal complications in patients with clinically localized pca .", "furthermore , imrt has been shown to reduce the acute and late gi toxicity of patients treated with high - dose radiation therapy and adjuvant androgen deprivation as compared to 3 d conformal radiotherapy .", "this reduction in gu and gi morbidity has made imrt extremely popular in the delivery of high - dose external beam radiation for patients with clinically localized pca in the united states . since 2004", "several large urology groups in partnership with radiation and medical oncologists have established centers of integrated prostate cancer care .", "the integrated care model is patient - centered and disease specific , where the equipment and the staff are dedicated to the treatment of pca and no other disease entity .", "although yet to be validated , this model may potentially result in better recognition and management of treatment - related complications , improved access to care , and increased experience with each treatment modality and thus better clinical outcomes .", "recently , these centers have become the targets of intense criticism [ 3 , 4 ] .", "the detractor 's claim that integrated pca care centers lead to self - referral by financially motivated urologists and radiation oncologists and result in over - utilization of imrt contributing to the increased cost of health care .", "they further claim that these centers have a negative impact on residency training in radiation oncology by shifting patients away from the academic radiation oncology training programs .", "unfortunately , these claims are not substantiated by data , but rather rely on indirect analysis of medicare claims and a 12% negative impact report from a single 3-point questionnaire survey of 81 radiation oncology training programs [ 3 , 4 ] . to our knowledge , this is a first study conducted to directly evaluate whether financial interest in imrt as part of the integrated prostate cancer care model changed treatment recommendations for newly diagnosed patients with prostate cancer .", "we compared the distribution of treatments choices of all consecutive patients diagnosed with prostate cancer on a biopsy in our practice during a 12-month period prior to acquiring financial interest in imrt to a 12-month period following that acquisition .", "our analysis revealed that overall there was a small , but statistically insignificant decrease in the use of radiation therapy and radical prostatectomy and a small increase in the use of active surveillance following investment in imrt .", "the increased use of active surveillance is likely due to the emergence of data from several large trials supporting the safety and efficacy of this approach in appropriately selected patients [ 1113 ] .", "once the data were stratified by gleason score and patient age , a statistically significant increase in the use of radiation was found in men over 70 with gleason 7 disease ( 41.3% versus 68.3% ) .", "however , because of the overall low number of patients in this subgroup , this increase was due to a single patient difference between the groups ( 12 versus 13 ) .", "these findings are not surprising as we believe that several important attributes of our integrated prostate cancer program provide for many patient benefits without the recently theorized , yet unsubstantiated risks of overutilization of imrt .", "as previously described , our center was established in collaboration with an academic radiation oncology department from an nci designated cancer center with a nationwide reputation for clinical and academic excellence .", "the radiation oncologists that treat our patients are not employed by the integrated prostate cancer center , but rather serve as full - time academic faculty and have no financial interest in imrt .", "furthermore , the final determination on whether a patient is an appropriate candidate for primary or adjuvant radiation therapy is made entirely by the treating radiation oncologist .", "radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of imrt , thus deriving an educational benefit from this partnership .", "the center employs a nurse - practitioner charged exclusively with coordination and support of the clinical research program .", "additionally , we are privileged to have cme accreditation by our state medical society and conduct regularly scheduled multidisciplinary morbidity and mortality conferences and discussions of challenging cases . finally , we maintain a very high volume surgical program that ranks second in the number of radical prostatectomies performed annually in the greater philadelphia , pa , usa .", "first , this study is underpowered due to a fairly low number of patients and therefore the results of our statistical analysis must withstand the test of a larger trial for our conclusions to be validated .", "second , even though we attempted to minimize limitations of the retrospective study design by including all consecutive patients diagnosed with prostate cancer within the 24-month period , the selection bias inherent in retrospective study design was not completely eliminated .", "third , we did not include patients undergoing adjuvant or salvage radiation therapy in the trial and therefore did not ascertain the effect of financial interest in imrt on utilization of radiation in these patients .", "finally , our findings may not be applicable to other integrated prostate cancer centers because of the unique structure of our specific program .", "financial interest in imrt does not result in an increased utilization of radiation therapy in the treatment of newly diagnosed patients with clinically localized prostate cancer in our integrated prostate cancer center ."], "abstract_text": ["<S> \n objective . as recent participants in an integrated prostate cancer ( pca ) care center , we sought to evaluate whether financial investment in an intensity - modulated radiation therapy ( imrt ) center resulted in an increased utilization of radiation therapy in our patients with newly diagnosed pca . </S>", "<S> materials & methods . following institutional review board approval </S>", "<S> , we retrospectively reviewed the records of all consecutive patients who were diagnosed with prostate cancer in the 12 months prior to and after investment in imrt . </S>", "<S> primary treatment modalities included active surveillance ( as ) , brachytherapy ( bt ) , radiation therapy ( xrt ) , radical prostatectomy ( rp ) , and androgen deprivation therapy ( adt ) . </S>", "<S> treatment data were available for all patients and were compared between the two groups . </S>", "<S> results . </S>", "<S> a total of 344 patients with newly diagnosed pca were evaluated over the designated time period . </S>", "<S> the pre - investment group totaled 198 patients , while 146 patients constituted the post - investment group . among all </S>", "<S> patients evaluated , there was a similar rate in the use of xrt ( 20.71% versus 20.55% , p = 1.000 ) pre- and post - investment in imrt . </S>", "<S> conclusions . </S>", "<S> financial interest in imrt by urologists does not impact overall utilization rates among patients with newly diagnosed pca at our center . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion", "5. Conclusion"], "sections": [["prostate cancer ( pca ) is the most common solid organ malignancy in american men with global statistics mirroring those found in the united states . screening with prostate specific antigen ( psa )", "has resulted in a significant stage migration such that the majority of new cases of pca are now detected while the disease is still clinically localized .", "these patients can choose from several treatment options and must weigh the potential morbidity of each treatment modality on their quality of life . in the vast majority of cases , urologists are the primary physicians that diagnose these patients with pca and are typically involved in the initial work up , discussion of all treatment options , and counseling of patients .", "urologists are not only intimately involved with the treatment of the primary disease but also the consequential treatment - related complications while overseeing the long - term followup of these patients .", "several centers have been recently established where urologists partner with radiation oncologists acquire ownership interest in intensity - modulated radiation therapy ( imrt ) equipment and provide integrated prostate cancer care . although not yet validated in the literature , this may allow for improved quality of care and decreased cost .", "unfortunately , these efforts have been much maligned in both the media and radiation oncology literature as conduits to increased revenue for the urologists with only debatable patient benefit [ 3 , 4 ] .", "these reports have not yet been supported by data . after acquiring financial interest in an integrated prostate cancer center , we sought to evaluate whether our investment in imrt resulted in an increased utilization of radiation therapy in our patients with newly diagnosed prostate cancer ."], ["  in september of 2008 , we acquired financial interest in an integrated pca center offering imrt . following institutional board approval , we identified all patients who were diagnosed with pca in the 12 months before and after the center became operational .", "newly diagnosed cases of pca were identified by searching our electronic medical record using both prostate biopsy cpt codes ( transrectal ultrasound - guided [ trus ] needle biopsy of the prostate , 55700/76942/76872 ) and the icd-9 prostate cancer diagnostic code ( prostate cancer , 185.0 ) .", "all men were diagnosed with pca after pathologic review of biopsy needle cores obtained after trus .", "indications for biopsy included elevated psa , abnormal digital rectal exam , abnormal pca 3 test , and/or strong family history of pca .", "prostate biopsies were performed utilizing a routine sextant pattern with at least 12 cores obtained . in cases where clinically indicated ,", "the medical records of these patients were retrospectively reviewed and the data pertaining to the patients ' demographics , cancer parameters , and initial pca treatment modality were extracted .", "patients were assigned to two discrete groups based on the date of their pca diagnosis as it related to the date of the first availability of imrt at our integrated prostate center .", "all consecutive patients diagnosed with pca on a biopsy within 12 months prior to availability of imrt constituted the pre - investment group and all consecutive patients diagnosed with prostate cancer on a biopsy in the 12 months following initiation of imrt services constituted the post - investment group .", "the primary treatments received were designated as active surveillance ( as ) , brachytherapy ( bt ) , radiation therapy ( xrt ) , radical prostatectomy ( rp ) , and androgen deprivation therapy ( adt ) .", "treatment data were available for all patients and were stratified by the patient 's age and gleason score .", "the age of 70 years old served as a cut - off point as in our clinical practice most of these patients are deemed suboptimal surgical candidates because of increased risk of postoperative urinary incontinence and erectile dysfunction .", "our integrated pca center was established in collaboration with an academic radiation oncology department from a national cancer institute ( nci ) designated cancer center with a nationwide reputation for clinical and academic excellence .", "the radiation oncologists that treat our patients are not employed by the cancer center , but serve as full time academic faculty and have no financial interest in imrt .", "radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of radiation therapy .", "the center also employs a nurse - practitioner charged exclusively with the coordination and support of on - going clinical research programs .", "patients who were referred to us for second opinion with biopsy - proven pca as well as men referred to us specifically for robotic - assisted laparoscopic radical prostatectomy were excluded to eliminate the potential biases resulting from the treatment recommendations rendered by an outside urologist .", "the overwhelming majority of these patients choose surgical therapy and their inclusion would skew the results toward lower utilization rates of xrt .", "unpaired t - tests , chi - squared tests , and fisher 's exact tests were implemented as appropriate ."], ["  a total of 344 patients were diagnosed with pca on trus biopsy over the designated 24-month time period . of the total patient population , 198 men were diagnosed with pca in the 12 months preceding availability of imrt , while 146 men constituted the post - investment group .", "patient and cancer characteristics were similar between the two groups ( table 1 and figure 1 ) .", "the percentage of patients with gleason 7  pca was higher in the post - investment group but did not reach statistical significance , p = 0.073 ( figure 1 ) .", "overall , the use of radiation therapy for those patients with newly diagnosed pca following investment in imrt ( 20.55% versus 20.71% , p = 1.00 ) was similar between the two patient populations ( table 2 and figure 2 ) .", "the number of patients treated with rp ( 67.81% versus 71.72% , p = 0.729 ) , as ( 9.59% versus 4.55% , p = 0.177 ) , adt ( 1.37% versus 2.02% , p = 0.999 ) , and bt ( 0.68% versus 1.01% , p = 0.999 ) were not significantly different between post- and pre - investment groups . while overall treatment trends afford succinct analysis , clinical decisions regarding treatment of pca are often driven by a multitude of patient - specific factors .", "as such , the data was analyzed stratifying both gleason score and age ( 70 years old serving as a cutoff point ) .", "treatments stratified by patient age are shown in table 3 and figure 3 . despite the increased incidence of gleason score 7 disease in the post - investment group", ", there was no significant difference between the groups in all treatment patterns in men less than 70 years of age ( table 3 ) .", "an increase was found in the use of xrt in men older than 70 years of age in the pre - investment group ( 45.45% ) as compared to men following acquisition of imrt ( 55.32% ) , but this did not reach statistical significance ( p = 0.355 ) . analyzed by age and gleason score simultaneously , there was no difference between the treatment groups in patients younger than 70 regardless of the gleason score , table 4(a ) and figure 4(a ) . for men 70 years or older with gleason 6 disease , there was a trend toward increased use of as ( 34.78% versus 15.79% ) and decreased use of rp ( 21.74% versus 31.58% ) , but did not reach statistical significance ( table 4(b ) ) .", "there was no difference ( 43.48% versus 42.11% ) in the use of xrt for men over 70 with gleason 6 disease . for patients with gleason 7 disease", ", there was a statistically significant increase in the utilization of xrt ( pre - investment 41.38% versus post - investment 68.42% , p = 0.035 ) and decrease in the use of rp in the post - investment group ( 15.79% versus 55.17% , p = 0.006 ) seen in table 4(b ) and figure 4(b ) .", "there was no difference between treatment groups in men over 70 with gleason 8 disease ."], ["  external beam radiation therapy is widely used and is an effective treatment option for localized pca .", "there is now level i evidence that high - dose radiation therapy decreases the risk of biochemical failure in men with clinically localized prostate cancer as compared to conventional dose conformal radiation [ 57 ] .", "this improvement , however , comes at a cost of increased gastrointestinal ( gi ) and genitourinary ( gu ) toxicity .", "observed that 2% of men receiving high - dose radiation experienced acute urinary or rectal morbidity of radiation therapy oncology group ( rtog ) grade 3 or greater .", "intensity modulated radiation therapy allows for delivery of radiation with greater conformality to the target volume compared with traditional 3 d technique .", "several randomized trials have shown that imrt reduced gi and gu toxicity compared with 3 d conformal radiation [ 810 ] .", "were able to deliver 81  gy with less than 2% of grade 2 rectal morbidity and no grade 4 or greater rectal complications in patients with clinically localized pca .", "furthermore , imrt has been shown to reduce the acute and late gi toxicity of patients treated with high - dose radiation therapy and adjuvant androgen deprivation as compared to 3 d conformal radiotherapy .", "this reduction in gu and gi morbidity has made imrt extremely popular in the delivery of high - dose external beam radiation for patients with clinically localized pca in the united states . since 2004", "several large urology groups in partnership with radiation and medical oncologists have established centers of integrated prostate cancer care .", "the integrated care model is patient - centered and disease specific , where the equipment and the staff are dedicated to the treatment of pca and no other disease entity .", "although yet to be validated , this model may potentially result in better recognition and management of treatment - related complications , improved access to care , and increased experience with each treatment modality and thus better clinical outcomes .", "recently , these centers have become the targets of intense criticism [ 3 , 4 ] .", "the detractor 's claim that integrated pca care centers lead to self - referral by financially motivated urologists and radiation oncologists and result in over - utilization of imrt contributing to the increased cost of health care .", "they further claim that these centers have a negative impact on residency training in radiation oncology by shifting patients away from the academic radiation oncology training programs .", "unfortunately , these claims are not substantiated by data , but rather rely on indirect analysis of medicare claims and a 12% negative impact report from a single 3-point questionnaire survey of 81 radiation oncology training programs [ 3 , 4 ] . to our knowledge , this is a first study conducted to directly evaluate whether financial interest in imrt as part of the integrated prostate cancer care model changed treatment recommendations for newly diagnosed patients with prostate cancer .", "we compared the distribution of treatments choices of all consecutive patients diagnosed with prostate cancer on a biopsy in our practice during a 12-month period prior to acquiring financial interest in imrt to a 12-month period following that acquisition .", "our analysis revealed that overall there was a small , but statistically insignificant decrease in the use of radiation therapy and radical prostatectomy and a small increase in the use of active surveillance following investment in imrt .", "the increased use of active surveillance is likely due to the emergence of data from several large trials supporting the safety and efficacy of this approach in appropriately selected patients [ 1113 ] .", "once the data were stratified by gleason score and patient age , a statistically significant increase in the use of radiation was found in men over 70 with gleason 7 disease ( 41.3% versus 68.3% ) .", "however , because of the overall low number of patients in this subgroup , this increase was due to a single patient difference between the groups ( 12 versus 13 ) .", "these findings are not surprising as we believe that several important attributes of our integrated prostate cancer program provide for many patient benefits without the recently theorized , yet unsubstantiated risks of overutilization of imrt .", "as previously described , our center was established in collaboration with an academic radiation oncology department from an nci designated cancer center with a nationwide reputation for clinical and academic excellence .", "the radiation oncologists that treat our patients are not employed by the integrated prostate cancer center , but rather serve as full - time academic faculty and have no financial interest in imrt .", "furthermore , the final determination on whether a patient is an appropriate candidate for primary or adjuvant radiation therapy is made entirely by the treating radiation oncologist .", "radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of imrt , thus deriving an educational benefit from this partnership .", "the center employs a nurse - practitioner charged exclusively with coordination and support of the clinical research program .", "additionally , we are privileged to have cme accreditation by our state medical society and conduct regularly scheduled multidisciplinary morbidity and mortality conferences and discussions of challenging cases . finally , we maintain a very high volume surgical program that ranks second in the number of radical prostatectomies performed annually in the greater philadelphia , pa , usa .", "first , this study is underpowered due to a fairly low number of patients and therefore the results of our statistical analysis must withstand the test of a larger trial for our conclusions to be validated .", "second , even though we attempted to minimize limitations of the retrospective study design by including all consecutive patients diagnosed with prostate cancer within the 24-month period , the selection bias inherent in retrospective study design was not completely eliminated .", "third , we did not include patients undergoing adjuvant or salvage radiation therapy in the trial and therefore did not ascertain the effect of financial interest in imrt on utilization of radiation in these patients .", "finally , our findings may not be applicable to other integrated prostate cancer centers because of the unique structure of our specific program ."], ["financial interest in imrt does not result in an increased utilization of radiation therapy in the treatment of newly diagnosed patients with clinically localized prostate cancer in our integrated prostate cancer center ."]]}
{"article_id": "PMC4759865", "article_text": ["neurofibromatosis type-1 ( nf-1 ) is a multisystem , autosomal dominant disorder of peripheral nerves affecting nearly 1/3000 individuals worldwide.1 it was first described by a german pathologist , friedrich daniel von recklinghausen .", "inherited or spontaneous mutation of the neurofibromin gene located on chromosome 17 is responsible for this diverse disorder .", "common skeletal manifestations of nf-1 include spinal deformities , congenital tibial dysplasia ( congenital bowing and pseudarthrosis ) , sphenoidal dysplasia and cystic lesions in bones . pathological fracture of the acetabulum with anterior dislocation of hip secondary to osseous involvement of the acetabulum , femoral head , and pubic rami has never been documented in a case of nf-1 .", "a 16-year - old boy presented with the complaints of pain in the left hip associated with the inability to bear weight following a trivial fall . on examination , the affected limb was 1.5 cm short , abducted and externally rotated .", "on general examination , patient had 8 caf au lait spots over the body , bilateral axillary freckles and multiple palpable neurofibromas in the subcutaneous tissues of forearm , thighs and back [ figure 1 ] .", "patient met three out of seven criteria described for the diagnosis of nf-1 [ table 1].2 plain radiograph and computed tomography scan of pelvis revealed an ill - defined lytic lesion causing pathological fracture - dislocation of the left hip [ figure 2 ] .", "magnetic resonance imaging ( mri ) showed additional soft tissue involvement and joint effusion [ figure 3 ] .", "ultrasound guided fine - needle aspiration cytology showed scanty cellularity with round to oval cells having minimal pleomorphism ; hyperchromatic nucleus and moderate cytoplasm with spindle cells and osteoblasts .", "clinical photograph showing skin lesions - caf au lait spots ( black arrows ) and axillary freckling ( white arrow ) criteria for diagnosis of nf-1 ( at least 2 or more features ) preoperative radiograph anteroposterior view ( a ) and computed tomography scan ( b ) of pelvis showing an ill - defined lytic lesion destroying anterior column of acetabulum , pubic rami and part of the femoral head coronal ( a ) and axial ( b ) sections of magnetic resonance imaging of pelvis showing expansile lytic lesion of acetabulum and pubic rami with soft tissue involvement and joint effusion a wide local excision followed by arthrodesis of the joint was planned .", "considering the extent of bony and soft tissue involvement , we used a modification of the ilioinguinal and iliofemoral approach to have a wide exposure .", "we used the conventional ilioinguinal incision and combined it with femoral part of the iliofemoral incision [ figure 4a ] .", "on exposing the pelvis , anatomy was distorted . the deformed femur head and acetabulum with deficient pubic rami", "there was extensive soft tissue involvement adjacent to the acetabulum and lower part of the ilium .", "the entire acetabulum with 2 - 3 cm clear margin of the ilium was resected along with the abnormal soft tissue .", "iliofemoral arthrodesis was done using a 14 hole stainless steel dynamic compression plate [ figures 4c  d , and 5 ] .", "intraoperative photographs showing ( a ) skin incision ( b ) deformed femoral head ( c ) 14 hole dcp ( d ) iliofemoral arthrodesis postoperative radiograph after tumor resection and iliofemoral arthrodesis histopathology revealed dense collagenous tissue cores with spindle cells having blunt nuclei with minimal atypia and no mitosis or necrosis [ figure 6a ] . on immunohistochemistry , cells were s-100 positive and of neural origin [ figure 6b ] .", "partial weight bearing was allowed at 6 weeks and full weight bearing at 10 weeks . at 1-year followup ,", "the patient was comfortable , pain free , able to ambulate unassisted , stand on one limb , sit and climb stairs without any difficulty [ figure 7 ] .", "( a ) histopathological photomicrograph showing dense collagenous tissue cores and spindle cells with blunt nuclei .", "( b ) immunohistochemistry showing neural marker s-100 positivity clinical photographs showing functional outcome at 1-year followup", "type 1 neurofibromatosis or von recklinghausen disease , is a multisystem disorder that primarily affects the cell growth of neural tissue and characterized by involvement of skin , peripheral nerves , subcutaneous tissue , eyes , and skeletal system .", "although involvement of the musculoskeletal system is common , there have been only a few cases of subluxation / dislocation of hip in patients with nf [ table 3].34567891011121314 on reviewing the literature , the etiology of hip instability leading to pathological subluxation / dislocation in patients with nf-1 can be classified as local and remote .", "most of the cases are secondary to local ( intra and peri - articular ) neurofibromas , which can result in mass effect , bony erosions ( ilium , acetabulum , and femoral neck ) , acetabular dysplasia , narrowing of the femoral neck , coxa valga , increased femoral neck offset , capsuloligamentous laxity , and synovial membrane proliferation.3458101113 remote causes of hip instability include intra spinal neurofibromas / schwannomas leading to motor deficit ( hip abductor weakness ) or sensory deficit ( charcot 's neuropathic arthropathy ) , limb length discrepancies secondary to hemi - hypertrophy of lower limb and abnormal biomechanical alteration in the spinopelvic alignment due to scoliosis.6791011 endo et al . described anterior subluxation of hip secondary to decreased femoral head coverage resulting from decreased lumbosacral lordosis and posterior pelvic inclination following scoliosis correction.11 until date , there has been no case of nf-1 reported in the literature with pathological fracture of the acetabulum with anterior dislocation of hip attributable to a neurofibroma involving the acetabulum , pubic rami and femoral head .", "orthopedic manifestations of nf-1 comprehensive literature review on published cases of hip dislocation / subluxation in nf-1 the various treatment options described for pathological hip dislocations in nf-1 include closed reduction , open reduction , shelf operation with fascia lata tenorraphy , rotational acetabular osteotomy with femoral varus osteotomy , girdle stone resection , total hip replacement with the trochanteric distalisation.791114 the rate of re dislocation is very high in most of the cases , subsequently requiring a secondary surgical procedure for stabilization . since only a handful of cases have been described in the literature , it is difficult to comment upon the best line of management . in our case ,", "arthrodesis was the best possible option since the bone stock after tumor resection was so inadequate that none of the above mentioned procedures could be tried .", "moreover , the fear of redislocation , which might necessitate repeated surgeries , was negated .", "a combination of ilioinguinal and iliofemoral approach was employed to have a better exposure of the hip joint .", "the neurofibromatous tumors associated with nf-1 are usually benign ; however , there is a 2 - 5% chance of malignant transformation , especially with plexiform neurofibromas.15 plexiform neurofibromas are diffuse , poorly defined nerve sheath tumors arising from multiple nerve fascicles and surrounding tissues .", "they are more prone for hemorrhage , dysfunction , pain , disfigurement , and malignant transformation.16 there was no clinical or radiological evidence of recurrence or malignant transformation in our patient at 1-year followup .", "subtle clinical signs such as skin patches , axillary freckling , and subcutaneous neurofibromas can be easily missed .", "orthopedic surgeons must be aware about the various management options available and tailor them as per the needs of their patient .", "iliofemoral arthrodesis offered a good functional outcome with improved quality of life in our case ."], "abstract_text": ["<S> skeletal neurofibromatosis ( nf ) commonly manifests as scoliosis and tibial dysplasias . </S>", "<S> nf affecting the pelvic girdle is extremely rare . </S>", "<S> pathological fracture of the acetabulum leading to anterior hip dislocation in a patient with nf-1 has never been reported in the literature . </S>", "<S> the paper presents the clinical symptomatology , the course of management and the successful outcome of such a rare case of nf-1 . </S>", "<S> histopathological and immunohistochemistry studies showing abundant spindle cells , which are s-100 positive and of neural origin are the classical hallmarks of neurofibromatous lesions . </S>", "<S> tumor resection and iliofemoral arthrodesis can be considered as a valid option in young patients with pathological fracture dislocation of the acetabulum . </S>"], "labels": null, "section_names": ["I", "C", "D"], "sections": [["neurofibromatosis type-1 ( nf-1 ) is a multisystem , autosomal dominant disorder of peripheral nerves affecting nearly 1/3000 individuals worldwide.1 it was first described by a german pathologist , friedrich daniel von recklinghausen .", "inherited or spontaneous mutation of the neurofibromin gene located on chromosome 17 is responsible for this diverse disorder .", "common skeletal manifestations of nf-1 include spinal deformities , congenital tibial dysplasia ( congenital bowing and pseudarthrosis ) , sphenoidal dysplasia and cystic lesions in bones . pathological fracture of the acetabulum with anterior dislocation of hip secondary to osseous involvement of the acetabulum , femoral head , and pubic rami has never been documented in a case of nf-1 ."], ["a 16-year - old boy presented with the complaints of pain in the left hip associated with the inability to bear weight following a trivial fall . on examination , the affected limb was 1.5 cm short , abducted and externally rotated .", "on general examination , patient had 8 caf au lait spots over the body , bilateral axillary freckles and multiple palpable neurofibromas in the subcutaneous tissues of forearm , thighs and back [ figure 1 ] .", "patient met three out of seven criteria described for the diagnosis of nf-1 [ table 1].2 plain radiograph and computed tomography scan of pelvis revealed an ill - defined lytic lesion causing pathological fracture - dislocation of the left hip [ figure 2 ] .", "magnetic resonance imaging ( mri ) showed additional soft tissue involvement and joint effusion [ figure 3 ] .", "ultrasound guided fine - needle aspiration cytology showed scanty cellularity with round to oval cells having minimal pleomorphism ; hyperchromatic nucleus and moderate cytoplasm with spindle cells and osteoblasts .", "clinical photograph showing skin lesions - caf au lait spots ( black arrows ) and axillary freckling ( white arrow ) criteria for diagnosis of nf-1 ( at least 2 or more features ) preoperative radiograph anteroposterior view ( a ) and computed tomography scan ( b ) of pelvis showing an ill - defined lytic lesion destroying anterior column of acetabulum , pubic rami and part of the femoral head coronal ( a ) and axial ( b ) sections of magnetic resonance imaging of pelvis showing expansile lytic lesion of acetabulum and pubic rami with soft tissue involvement and joint effusion a wide local excision followed by arthrodesis of the joint was planned .", "considering the extent of bony and soft tissue involvement , we used a modification of the ilioinguinal and iliofemoral approach to have a wide exposure .", "we used the conventional ilioinguinal incision and combined it with femoral part of the iliofemoral incision [ figure 4a ] .", "on exposing the pelvis , anatomy was distorted . the deformed femur head and acetabulum with deficient pubic rami", "there was extensive soft tissue involvement adjacent to the acetabulum and lower part of the ilium .", "the entire acetabulum with 2 - 3 cm clear margin of the ilium was resected along with the abnormal soft tissue .", "iliofemoral arthrodesis was done using a 14 hole stainless steel dynamic compression plate [ figures 4c  d , and 5 ] .", "intraoperative photographs showing ( a ) skin incision ( b ) deformed femoral head ( c ) 14 hole dcp ( d ) iliofemoral arthrodesis postoperative radiograph after tumor resection and iliofemoral arthrodesis histopathology revealed dense collagenous tissue cores with spindle cells having blunt nuclei with minimal atypia and no mitosis or necrosis [ figure 6a ] . on immunohistochemistry , cells were s-100 positive and of neural origin [ figure 6b ] .", "partial weight bearing was allowed at 6 weeks and full weight bearing at 10 weeks . at 1-year followup ,", "the patient was comfortable , pain free , able to ambulate unassisted , stand on one limb , sit and climb stairs without any difficulty [ figure 7 ] .", "( a ) histopathological photomicrograph showing dense collagenous tissue cores and spindle cells with blunt nuclei .", "( b ) immunohistochemistry showing neural marker s-100 positivity clinical photographs showing functional outcome at 1-year followup"], ["type 1 neurofibromatosis or von recklinghausen disease , is a multisystem disorder that primarily affects the cell growth of neural tissue and characterized by involvement of skin , peripheral nerves , subcutaneous tissue , eyes , and skeletal system .", "although involvement of the musculoskeletal system is common , there have been only a few cases of subluxation / dislocation of hip in patients with nf [ table 3].34567891011121314 on reviewing the literature , the etiology of hip instability leading to pathological subluxation / dislocation in patients with nf-1 can be classified as local and remote .", "most of the cases are secondary to local ( intra and peri - articular ) neurofibromas , which can result in mass effect , bony erosions ( ilium , acetabulum , and femoral neck ) , acetabular dysplasia , narrowing of the femoral neck , coxa valga , increased femoral neck offset , capsuloligamentous laxity , and synovial membrane proliferation.3458101113 remote causes of hip instability include intra spinal neurofibromas / schwannomas leading to motor deficit ( hip abductor weakness ) or sensory deficit ( charcot 's neuropathic arthropathy ) , limb length discrepancies secondary to hemi - hypertrophy of lower limb and abnormal biomechanical alteration in the spinopelvic alignment due to scoliosis.6791011 endo et al . described anterior subluxation of hip secondary to decreased femoral head coverage resulting from decreased lumbosacral lordosis and posterior pelvic inclination following scoliosis correction.11 until date , there has been no case of nf-1 reported in the literature with pathological fracture of the acetabulum with anterior dislocation of hip attributable to a neurofibroma involving the acetabulum , pubic rami and femoral head .", "orthopedic manifestations of nf-1 comprehensive literature review on published cases of hip dislocation / subluxation in nf-1 the various treatment options described for pathological hip dislocations in nf-1 include closed reduction , open reduction , shelf operation with fascia lata tenorraphy , rotational acetabular osteotomy with femoral varus osteotomy , girdle stone resection , total hip replacement with the trochanteric distalisation.791114 the rate of re dislocation is very high in most of the cases , subsequently requiring a secondary surgical procedure for stabilization . since only a handful of cases have been described in the literature , it is difficult to comment upon the best line of management . in our case ,", "arthrodesis was the best possible option since the bone stock after tumor resection was so inadequate that none of the above mentioned procedures could be tried .", "moreover , the fear of redislocation , which might necessitate repeated surgeries , was negated .", "a combination of ilioinguinal and iliofemoral approach was employed to have a better exposure of the hip joint .", "the neurofibromatous tumors associated with nf-1 are usually benign ; however , there is a 2 - 5% chance of malignant transformation , especially with plexiform neurofibromas.15 plexiform neurofibromas are diffuse , poorly defined nerve sheath tumors arising from multiple nerve fascicles and surrounding tissues .", "they are more prone for hemorrhage , dysfunction , pain , disfigurement , and malignant transformation.16 there was no clinical or radiological evidence of recurrence or malignant transformation in our patient at 1-year followup .", "subtle clinical signs such as skin patches , axillary freckling , and subcutaneous neurofibromas can be easily missed .", "orthopedic surgeons must be aware about the various management options available and tailor them as per the needs of their patient .", "iliofemoral arthrodesis offered a good functional outcome with improved quality of life in our case ."]]}
{"article_id": "PMC4970421", "article_text": ["ramus can be used to differentiate between male and female strongly expresses univariate sexual dimorphism .", "when skeletal sex determination is considered , metric analyses on the radiographs are often found to be of superior value owing to their objectivity , accuracy , and reproducibility .", "after both of these bony areas , mandible remains next in the human which will also help us in the identification of age , sex , and race .", "humphrey et al . emphasized that almost any site of mandibular bone deposition , or resorption , or remodeling for that matter , seems to have a potential for becoming sexually dimorphic .", "hence , mandibular condyle and ramus , in particular , are generally the most sexually dimorphic as they are the sites associated with the greatest morphological changes in size and remodeling during growth . among various radiographic technique the orthopantomography ( opg ) is still used as one of the measure in determination of sex wherein , the morphology of mandibular ramus is studied .", "hence , this study was taken to understand the sexual dimorphism , using digital opg .", "this study aims to determine the usefulness of mandibular ramus as an aid in sex determination .", "this study aims to determine the usefulness of mandibular ramus as an aid in sex determination .", "a short retrospective study was conducted of sixty males and sixty females , using orthopantomographs which were taken using kodak 8000c digital panoramic and cephalometric system ( 73 kvp , 12 ma , 13.9 s ) .", "patients with a history of extraction , fracture , and any other severe developmental disturbances leading to variation in the size of mandible were excluded from the study .", "all the mandibular ramus measurements will be carried out using carestream health , inc , 2010 , kodak 8000c digital panoramic and cephalometric system ( 73 kvp , 12 ma , 13.9 s ) with master view 3.0 software . the ethical clearance obtained from the institution review board . the following variables were measured using mouse - driven method by moving the mouse and drawing lines using chosen points on the digital panoramic radiograph [ figures 1 and 2 ] .", "mandibular ramus measurements with five variables mandibular ramus measurements of female patient on orthopantomograph the sex could be determined from calculations using the equations given below : ( table 1 ) 10 dmale : 185.622 + 1.361 ( max", ". ramus breadth ) + 1.087 ( min.ramus breadth ) + 2.253 ( condylar height )  0.717 ( projective height of ramus ) + 0.081 ( coronoid height ) dfemale : 161.761 + 1.276 ( max .", "ramus breadth ) + 0.948 ( min.ramus breadth ) + 2.220 ( condylar height )  0.753 ( projective height of ramus ) + 0.063 ( coronoid height ) \n maximum ramus breadth : the distance between the most anterior point on the mandibular ramus and a line connecting the most posterior point on the condyle and the angle of jawminimum ramus breadth : smallest anterior  posterior diameter of the ramuscondylar height / maximum ramus height : height of the ramus of the mandible from the most superior point on the mandibular condyle to the tubercle , or most protruding portion of the inferior border of the ramusprojective height of ramus : projective height of ramus between the highest point of the mandibular condyle and lower margin of the bonecoronoid height : projective distance between coronion and lower wall of the bone . \n ", "maximum ramus breadth : the distance between the most anterior point on the mandibular ramus and a line connecting the most posterior point on the condyle and the angle of jaw minimum ramus breadth : smallest anterior  posterior diameter of the ramus condylar height / maximum ramus height : height of the ramus of the mandible from the most superior point on the mandibular condyle to the tubercle , or most protruding portion of the inferior border of the ramus projective height of ramus : projective height of ramus between the highest point of the mandibular condyle and lower margin of the bone coronoid height : projective distance between coronion and lower wall of the bone .", "the data were analyzed by the discriminant function analysis using fischer exact test [ table 1 ] .", "mean measurements in males and females descriptive statistics shows mean values were higher in males compared to females .", "f - statistic values indicated that highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth .", "maximum ramus breadth , condylar height , and projective height of ramus were statistically significant with p < 0.05 as shown in table 2 .", "descriptive statistics sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53% , and sex was accurately determined in 36 cases out of sixty female mandibular measurements with an accuracy rate of 60% [ table 3 ] .", "the data were analyzed by the discriminant function analysis using fischer exact test [ table 1 ] .", "mean measurements in males and females descriptive statistics shows mean values were higher in males compared to females .", "f - statistic values indicated that highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth .", "maximum ramus breadth , condylar height , and projective height of ramus were statistically significant with p < 0.05 as shown in table 2 .", "descriptive statistics sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53% , and sex was accurately determined in 36 cases out of sixty female mandibular measurements with an accuracy rate of 60% [ table 3 ] .", "determination of sex by morphological assessment has remained as one of the oldest approaches in forensic anthropology and medico - legal examinations . the method may vary and depend on the available bones and their conditions .", "the identification of sex is of significance in cases of mass fatality incidents where bodies are damaged beyond recognition .", "when entire adult skeleton is available for analysis , sex can be determined up to 100% accuracy ( pelvis ) . however , in cases of mass disasters where usually fragmented bones are found , sex determination with 100% accuracy is not possible , and it depends largely on the available parts of skeleton .", "however , in cases where intact skull is not found , mandible may play a vital role in sex determination as it is the most dimorphic bone of skull .", "anthropometry of the face and intraoral regions can help in the field of forensic odontology when common forensic data are unavailable .", "the disadvantages opg technique is unequal magnification and geometric distortion , which causes many problems .", "these distortions are the result of the horizontal movement of the film and x - ray source .", "the position of the movement object is within the focal trough , the size , and shape of the resultant images keeps changing .", "panoramic radiographic technique remains as quite sensitive to positioning errors because of relatively narrow image layer .", "objects which are outside the focal trough are blurred , magnified or reduced in sizes which are sometimes distorted .", "a study conducted by kambylafkas et al . concluded that the evaluation of total ramal height is reliable , and an asymmetry of more than 6% is an indication of atrue asymmetry using panoramic radiograph . in this study , asymmetry noted was 7% using panoramic radiograph .", "found mandibular ramus height to be the best parameter in their study , with 75.8% accuracy . in our study ,", "mandibular ramus height found to be the best parameter with statistically significant with p = 0.005 . a study conducted by saini , et al .", "showed that coronoid height possessed the best potential for sex determination on indian people with the accuracy of 74.1% , and the combination of it with minimum ramus breadth , maximum ramus breadth , and/or mandibular ramus length will show significant sexual dimorphism with an overall accuracy of 80.2% . in this study , all five variables that is maximum ramus breadth , minimum ramus breadth , condylar height , projective height of ramus , coronoid height showed prediction accuracy of 53% for males and prediction accuracy of 60% for females .", "another study conducted by indira et al . on mandibular ramus measurements were subjected to discriminant function analysis .", "each of the five variables measured on mandibular ramus using orthopantomograph showed statistically significant sex differences between sexes , indicating that ramus expresses strong sexual dimorphism .", "the mandibular ramus demonstrated greatest univariate sexual dimorphism in terms of minimum ramus breadth , condylar height , followed by projective height of ramus .", "in our study , highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth .", "maximum ramus breadth , condylar height , projective height of ramus was statistically significant with the p < 0.05 .", "shivaprakash and vijaykumar conducted a study in diagnosing in the sex by observing the mandibular ramus posterior flexure .", "sex was accurately determined in 44 cases out of 55 male mandibles with an accuracy rate of 80% , and sex was accurately determined in 35 cases out of 49 female mandibles with accuracy rate of 71% . in our study ,", "sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53% and sex was accurately determined in 36 cases out of sixty female mandibular measurements with accuracy rate of 60% .", "the mandibular ramus height can be considered another valuable tool in gender determination with the help of opg . however , further studies with larger sample and less magnification errors are needed to be taken up in future ."], "abstract_text": ["<S> aims and objectives:(1 ) to determine the usefulness of mandibular ramus as an aid in sex determination . </S>", "<S> ( 2 ) to evaluate anteroposterior | superioinferior angle of mandibular condyle.materials and methods : a retrospective study was conducted using orthopantomographs of 60 males and 60 females , which were taken using kodak 8000c digital panoramic and cephalometric system ( 73 kvp , 12 ma , 13.9 s ) . </S>", "<S> the age group ranged between 18  45 years . </S>", "<S> mandibular ramus measurements were carried out using master view 3.0 software . </S>", "<S> the measurements of the mandibular ramus will be subjected to discriminant function analysis.results:maximum ramus breadth , minimum ramus breadth , condylar height , projective height of ramus coronoid height were calculated for both the sexes differently with the formula & analyzed with discriminant function analysis using fischer exact test . </S>", "<S> the p value was statistically significant with the p value < 0.05 for the following parameters max . </S>", "<S> ramus breadth , condylar height and projective height of ramus.conclusion:mandibular ramus measurements can be a useful tool for gender determination . </S>"], "labels": null, "section_names": ["Introduction", "Aims and objectives", "Materials and Methods", "Statistical analysis", "Discussion", "Conclusion", "Financial support and sponsorship", "Conflicts of interest"], "sections": [["dentofacial radiography has become a routine procedure in the dental , medical , and hospital clinics . where in radiographs are taken at different periods during the lifetime of large segments of the population . whereas the determination of sex is an important aspect of forensic anthropology and vital in medico - legal investigations . among various measures mandibular", "ramus can be used to differentiate between male and female strongly expresses univariate sexual dimorphism .", "when skeletal sex determination is considered , metric analyses on the radiographs are often found to be of superior value owing to their objectivity , accuracy , and reproducibility .", "after both of these bony areas , mandible remains next in the human which will also help us in the identification of age , sex , and race .", "humphrey et al . emphasized that almost any site of mandibular bone deposition , or resorption , or remodeling for that matter , seems to have a potential for becoming sexually dimorphic .", "hence , mandibular condyle and ramus , in particular , are generally the most sexually dimorphic as they are the sites associated with the greatest morphological changes in size and remodeling during growth . among various radiographic technique the orthopantomography ( opg ) is still used as one of the measure in determination of sex wherein , the morphology of mandibular ramus is studied .", "hence , this study was taken to understand the sexual dimorphism , using digital opg .", "this study aims to determine the usefulness of mandibular ramus as an aid in sex determination ."], ["this study aims to determine the usefulness of mandibular ramus as an aid in sex determination ."], ["a short retrospective study was conducted of sixty males and sixty females , using orthopantomographs which were taken using kodak 8000c digital panoramic and cephalometric system ( 73 kvp , 12 ma , 13.9 s ) .", "patients with a history of extraction , fracture , and any other severe developmental disturbances leading to variation in the size of mandible were excluded from the study .", "all the mandibular ramus measurements will be carried out using carestream health , inc , 2010 , kodak 8000c digital panoramic and cephalometric system ( 73 kvp , 12 ma , 13.9 s ) with master view 3.0 software . the ethical clearance obtained from the institution review board . the following variables were measured using mouse - driven method by moving the mouse and drawing lines using chosen points on the digital panoramic radiograph [ figures 1 and 2 ] .", "mandibular ramus measurements with five variables mandibular ramus measurements of female patient on orthopantomograph the sex could be determined from calculations using the equations given below : ( table 1 ) 10 dmale : 185.622 + 1.361 ( max", ". ramus breadth ) + 1.087 ( min.ramus breadth ) + 2.253 ( condylar height )  0.717 ( projective height of ramus ) + 0.081 ( coronoid height ) dfemale : 161.761 + 1.276 ( max .", "ramus breadth ) + 0.948 ( min.ramus breadth ) + 2.220 ( condylar height )  0.753 ( projective height of ramus ) + 0.063 ( coronoid height ) \n maximum ramus breadth : the distance between the most anterior point on the mandibular ramus and a line connecting the most posterior point on the condyle and the angle of jawminimum ramus breadth : smallest anterior  posterior diameter of the ramuscondylar height / maximum ramus height : height of the ramus of the mandible from the most superior point on the mandibular condyle to the tubercle , or most protruding portion of the inferior border of the ramusprojective height of ramus : projective height of ramus between the highest point of the mandibular condyle and lower margin of the bonecoronoid height : projective distance between coronion and lower wall of the bone . \n ", "maximum ramus breadth : the distance between the most anterior point on the mandibular ramus and a line connecting the most posterior point on the condyle and the angle of jaw minimum ramus breadth : smallest anterior  posterior diameter of the ramus condylar height / maximum ramus height : height of the ramus of the mandible from the most superior point on the mandibular condyle to the tubercle , or most protruding portion of the inferior border of the ramus projective height of ramus : projective height of ramus between the highest point of the mandibular condyle and lower margin of the bone coronoid height : projective distance between coronion and lower wall of the bone .", "the data were analyzed by the discriminant function analysis using fischer exact test [ table 1 ] .", "mean measurements in males and females descriptive statistics shows mean values were higher in males compared to females .", "f - statistic values indicated that highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth .", "maximum ramus breadth , condylar height , and projective height of ramus were statistically significant with p < 0.05 as shown in table 2 .", "descriptive statistics sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53% , and sex was accurately determined in 36 cases out of sixty female mandibular measurements with an accuracy rate of 60% [ table 3 ] ."], ["the data were analyzed by the discriminant function analysis using fischer exact test [ table 1 ] .", "mean measurements in males and females descriptive statistics shows mean values were higher in males compared to females .", "f - statistic values indicated that highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth .", "maximum ramus breadth , condylar height , and projective height of ramus were statistically significant with p < 0.05 as shown in table 2 .", "descriptive statistics sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53% , and sex was accurately determined in 36 cases out of sixty female mandibular measurements with an accuracy rate of 60% [ table 3 ] ."], ["determination of sex by morphological assessment has remained as one of the oldest approaches in forensic anthropology and medico - legal examinations . the method may vary and depend on the available bones and their conditions .", "the identification of sex is of significance in cases of mass fatality incidents where bodies are damaged beyond recognition .", "when entire adult skeleton is available for analysis , sex can be determined up to 100% accuracy ( pelvis ) . however , in cases of mass disasters where usually fragmented bones are found , sex determination with 100% accuracy is not possible , and it depends largely on the available parts of skeleton .", "however , in cases where intact skull is not found , mandible may play a vital role in sex determination as it is the most dimorphic bone of skull .", "anthropometry of the face and intraoral regions can help in the field of forensic odontology when common forensic data are unavailable .", "the disadvantages opg technique is unequal magnification and geometric distortion , which causes many problems .", "these distortions are the result of the horizontal movement of the film and x - ray source .", "the position of the movement object is within the focal trough , the size , and shape of the resultant images keeps changing .", "panoramic radiographic technique remains as quite sensitive to positioning errors because of relatively narrow image layer .", "objects which are outside the focal trough are blurred , magnified or reduced in sizes which are sometimes distorted .", "a study conducted by kambylafkas et al . concluded that the evaluation of total ramal height is reliable , and an asymmetry of more than 6% is an indication of atrue asymmetry using panoramic radiograph . in this study , asymmetry noted was 7% using panoramic radiograph .", "found mandibular ramus height to be the best parameter in their study , with 75.8% accuracy . in our study ,", "mandibular ramus height found to be the best parameter with statistically significant with p = 0.005 . a study conducted by saini , et al .", "showed that coronoid height possessed the best potential for sex determination on indian people with the accuracy of 74.1% , and the combination of it with minimum ramus breadth , maximum ramus breadth , and/or mandibular ramus length will show significant sexual dimorphism with an overall accuracy of 80.2% . in this study , all five variables that is maximum ramus breadth , minimum ramus breadth , condylar height , projective height of ramus , coronoid height showed prediction accuracy of 53% for males and prediction accuracy of 60% for females .", "another study conducted by indira et al . on mandibular ramus measurements were subjected to discriminant function analysis .", "each of the five variables measured on mandibular ramus using orthopantomograph showed statistically significant sex differences between sexes , indicating that ramus expresses strong sexual dimorphism .", "the mandibular ramus demonstrated greatest univariate sexual dimorphism in terms of minimum ramus breadth , condylar height , followed by projective height of ramus .", "in our study , highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth .", "maximum ramus breadth , condylar height , projective height of ramus was statistically significant with the p < 0.05 .", "shivaprakash and vijaykumar conducted a study in diagnosing in the sex by observing the mandibular ramus posterior flexure .", "sex was accurately determined in 44 cases out of 55 male mandibles with an accuracy rate of 80% , and sex was accurately determined in 35 cases out of 49 female mandibles with accuracy rate of 71% . in our study ,", "sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53% and sex was accurately determined in 36 cases out of sixty female mandibular measurements with accuracy rate of 60% ."], ["the mandibular ramus height can be considered another valuable tool in gender determination with the help of opg . however , further studies with larger sample and less magnification errors are needed to be taken up in future ."], [""], [""]]}
{"article_id": "PMC4745543", "article_text": ["they form an integral part of indian diet , especially for those who have type 2 diabetes for whom white rice is considered less desirable because of its high gi .", "chapattis and other flatbreads are popular in europe also where they form a part of daily diet among members of ethnic minority groups who follow traditional dietary patterns .", "chapattis are made from whole - wheat flour and cooked on hot flat open griddles .", "they can also be prepared by substituting wheat flour with other cereal or legume flours at different levels .", "incorporation of cereal brans at proportions up to 10% has resulted in good quality chapattis .", "the color and appearance of chapattis were found to be good with substitution of wheat flour with up to 10% cereal brans .", "generally , chapatti is prepared from whole - wheat flour obtained by grinding wheat in a disk mill ( locally known as chakki ) .", "chapatti quality can be assessed from its softness and flexibility which may be affected by flour protein quantity and quality .", "the chapatti quality is also influenced by the dough consistency , which in turn depends mainly on the quantity of water added .", "bran is the hard outer layer of cereal grains , rich in a myriad of healthy phytochemicals , namely , phenolics , flavonoids , glucans , and pigments .", "unfortunately , these nutrition - rich components are often discarded during milling out of ignorance , organoleptic reasons , and rancidity problems . knowing the phytochemical constituents and pharmacological profile of bran is expected to give insight to their potential application in promotion of health .", "cereal brans , the by - products obtained in large amounts in grain milling industry , considered as inedible material for humans , is mostly used as animal feed .", "however , brans are concentrated source of dietary fibre and other nutrients ( proteins , b - vitamins , and minerals ) .", "brans are generally composed mainly of insoluble cellulose and hemicellulose , with only about 5 percent soluble fibre , and have little hypercholesterolemia effect .", "bran contributes a pleasing , sweet , nutty flavour when added as a flavour enhancer in a variety of food products .", "commercial wheat flour and oat bran ( baggry 's india ltd . , new delhi , india ) were purchased from local market .", "chappati was prepared by the addition of cereal brans ( wheat , oat , and rice ) singly and in combination ( w  :  r  :  o :  : 2  :  1.5  :  1.5 ) to wheat flour at 5 and 10% bran supplementation . \n chapatti .", "cereal brans singly and in combination at 5 and 10% level were added to wheat flour and required quantity of water which were mixed manually to obtain dough of suitable consistency .", "the dough was divided into four equal parts and moulded into circular chapattis of 15.0  cm in diameter with rolling pin and board .", "traditional home baking procedure was followed to bake chapattis on iron plate ( tawa ) .", "chapattis were cooled and comparative evaluation was done using the following criteria which also included observations on dough handling properties .  \n\n", "characteristic \n\n score grade \n dough handling  nonsticky  sticky  slightly sticky  very sticky  puffing of chapatti  full  partial  nil . \n ", "the instrument was calibrated with the user supplied black plate calibration standard that was used for zero setting .", "the instruments were placed on the plate and three exposures at different places were conducted .", "readings were displayed as a , b , and l color parameters according to the cielab system of color measurement .", "the a value ranges from 100 ( redness ) to + 100 ( greenness ) and the b value ranges from 100 ( blueness ) to + 100 ( yellowness ) , while the l value , indicating the measure of lightness , ranges from 0 ( black ) to 100 ( white ) .", "was evaluated by using texture analyser ( stable micro systems , model ta - hdi , uk ) .", "one strip at a time was placed on the centre of the sample holder and the blade was allowed to cut the chapatti strip .", "the force ( n ) required to cut chapatti strip into two pieces was recorded .", "bran enriched products such as extruded snacks , breakfast cereal - porridge , and chapatti were evaluated for sensory attributes ( appearance , colour , texture , flavor , and overall acceptability ) through a panel of semi - trained judges using 9-point hedonic scale .", "water activity of bran enriched products was estimated using water activity meter having hygrolab 3 bench - top indicator ( rotrogenic company ) .", "standard aoac procedure   was followed for free fatty acids determination in cereal bran enriched products .", "product sample ( 5  g ) was taken in flask and 50  ml benzene was added and kept for 30  min for extraction of free fatty acids . after extraction , 5  ml extract , 5  ml benzene , 10  ml alcohol , and phenolphthalein as indicator were taken in flask and titrated against 0.02  n koh till light pink colour disappeared : \n ( 1)%ffa  ( %  oleic  acid )  = 2820.02  n  kohml  of  alkali  useddilution  factor1000wt  of  sample  taken  100 ,  moisture content by method of aacc 2000 ,  total plate count by method of maturin and peeler .", "moisture content by method of aacc 2000 , total plate count by method of maturin and peeler .", "data collected from the aforesaid experiments was subjected to statistical analysis for standard error and duncan 's multiple range test using minitab software .", "the quality evaluation of chapattis prepared by different bran enriched levels is mentioned in table 1 .", "the pooled scores obtained by the various bran enriched levels of chapattis for appearance , color , texture , and flavor were 7.92 , 7.18 , 7.68 , and 8.10 for wheat bran , rice bran , oat bran , and bran in combination enriched chapattis , respectively .", "the overall acceptability at 5 and 10 percent level of supplementation was 7.65 and 7.80 , respectively .", "reported that chapattis , prepared by the addition of 10% bran , showed better performance and were quite comparable with whole - wheat flour regarding the proximate components and sensory attributes .", "dough handling characteristics of bran enriched chapattis do not show much variation with respect to type of bran used . except for rice bran", "incorporated dough for chapatti ( slightly sticky ) , all others showed nonsticky behavior during dough development .", "all types of bran enriched chapattis showed full puffing except 10% rice bran enriched chapattis in which partial puffing during chapatti preparation was visualised .", "the data presented in table 2 depicted color and texture analysis of bran enriched chapatti . statistically significant ( p  0.05 ) difference was observed in l value of bran enriched chapatti", ". l value of various cereal bran enriched chapattis was 64.37 , 59.12 , 60.04 , and 61.92 for wheat , rice , oat , and bran in combination , respectively .", "l value showed decreasing trend with increase in level of supplementation of cereal brans in chapattis .", "the l value of 66.83 , 61.59 , and 61.14 was observed at 0 , 5 , and 10% level of supplementation , which means slightly lower brightness at higher levels of supplementation .", "a value of wheat , rice , oat , and bran in combination was 4.18 , 5.24 , 4.71 , and 4.28 , respectively .", "with increase in level of supplementation , a value ( redness ) increased from 3.14 at 0% level of supplementation to 5.11 at 10% level .", "altan et al .   stated that , among the color parameters , the l and a values showed marked changes due to addition of tomato pomace . an increase in tomato pomace level decreased the l value of the sample and increased the a value of samples . also , increasing bran level supplementation resulted in a decrease in the b value of chapattis .", "a negative correlation was found between a value and b value of the enriched chapattis . cutting force ( n ) reflects the texture of the chapattis and it stimulates the biting action of the human teeth on chapattis . cutting force ( n ) of various bran enriched chapattis varied as 6.56  n , 5.92  n , 5.30  n , and 5.99 ", "cutting force increased due to presence of more fibres at higher enrichment levels . at 0 , 5 , and 10 percent level of supplementation , the corresponding cutting force ( n ) was 5.25 , 5.91 , and 5.97  n , respectively .", "manu and prasada rao   reported that cutting force of chapattis prepared from different wheat varieties ranged from 4.22 to 6.96  n. hemalatha et al .  ", "also reported that the cutting force ( n ) for chapattis made from different wheat varieties ranged between 4.22 and 6.67 .", "the variation in cutting force might be because of variation in protein and fibre content of brans which determine the resistance offered by the samples . \n", "increase in fibre content might have increased the water holding capacity of chapattis and hence increased moisture content ( % ) with addition of bran .", "reported that moisture content ( % ) of bran enriched chapattis was 31.0 percent while control had moisture content of 30.2 percent .", "maximum water activity was observed in rice bran enriched chapattis ( 0.462 ) which was statistically at par with water activity of bran in combination enriched chapattis ( 0.455 ) .", "water activity of oat and wheat bran enriched chapattis was 0.429 and 0.406 , respectively .", "it was observed from data that water activity of samples was positively correlated with moisture content and followed the same pattern .", "manthey et al .   reported that water activity of bran / fibre enriched pasta increased with bran supplementation over control .", "the increase in water activity is correlated with increase in moisture content at higher levels of bran supplementation .", "the data pertaining to free fatty acids ( % ) is presented in table 3 .", "the free fatty acids ( % ) of bran enriched chapattis ranged from 0.057 to 0.085 .", "the highest free fatty acids were recorded in rice bran enriched chapattis ( 0.085% ) , being the lowest in wheat bran enriched chapattis ( 0.057 ) .", "it is also evident from the table that , with increase in level of bran supplementation , free fatty acids increased significantly .", "the free fatty acids ( % ) at 5 and 10 percent level of supplementation were 0.067 and 0.079 percent , respectively .", "khan et al .   reported similar results regarding free fatty acids while studying development and evaluation of long shelf life ambient stable chapattis .", "a significant variation ( p  0.05 ) was observed in total plate count of bran extruded chapattis .", "total plate count of enriched chapattis varied from 6.5 to 17  10  cfu / g .", "it is also evident from the table that with increase in bran supplementation level , a slight increase in total plate content was observed .", "the mean value of total plate content for 5 and 10% level of supplementation was 10.25  10 and 12.5  10  cfu / g , respectively .", "frazier and westhoff   reported that total plate content increased from 2.3  10 to 3.4  10  cfu / g for flour .", "quality characteristics for chapatti revealed that dough handling and puffing of bran enriched chapattis prepared by 5 and 10% level of bran supplementation did not vary significantly .", "all types of bran enriched chapattis except rice bran enriched chapattis showed nonsticky behavior during dough handling .", "rice bran enriched chapatti recorded maximum moisture ( % ) , water activity , and free fatty acids ( % ) . with increase in level of supplementation , moisture , water activity , and free fatty acids increased .", "the future emphasis can be given on development of functional flatbreads which has got increased demand due to increase in health conscious consumer base ."], "abstract_text": ["<S> cereal brans singly and in combination were blended at varying levels ( 5 and 10% ) for development of chapattis . cereal bran enriched chapattis were assessed for quality and physicochemical characteristics . on the basis of quality assessment , </S>", "<S> 10% enrichment level for chapatti was the best . </S>", "<S> moisture content , water activity , and free fatty acids remained stable during the study period . </S>", "<S> quality assessment and physicochemical characteristics of bran enriched chapattis carried out revealed that dough handling and puffing of bran enriched chapattis prepared by 5 and 10% level of bran supplementation did not vary significantly . </S>", "<S> all types of bran enriched chapattis except rice bran enriched chapattis showed nonsticky behavior during dough handling . </S>", "<S> bran enriched chapattis exhibited full puffing character during preparation . </S>", "<S> the sensory attributes showed that both 5 and 10% bran supplemented chapattis were acceptable . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "4. Conclusions"], "sections": [["they form an integral part of indian diet , especially for those who have type 2 diabetes for whom white rice is considered less desirable because of its high gi .", "chapattis and other flatbreads are popular in europe also where they form a part of daily diet among members of ethnic minority groups who follow traditional dietary patterns .", "chapattis are made from whole - wheat flour and cooked on hot flat open griddles .", "they can also be prepared by substituting wheat flour with other cereal or legume flours at different levels .", "incorporation of cereal brans at proportions up to 10% has resulted in good quality chapattis .", "the color and appearance of chapattis were found to be good with substitution of wheat flour with up to 10% cereal brans .", "generally , chapatti is prepared from whole - wheat flour obtained by grinding wheat in a disk mill ( locally known as chakki ) .", "chapatti quality can be assessed from its softness and flexibility which may be affected by flour protein quantity and quality .", "the chapatti quality is also influenced by the dough consistency , which in turn depends mainly on the quantity of water added .", "bran is the hard outer layer of cereal grains , rich in a myriad of healthy phytochemicals , namely , phenolics , flavonoids , glucans , and pigments .", "unfortunately , these nutrition - rich components are often discarded during milling out of ignorance , organoleptic reasons , and rancidity problems . knowing the phytochemical constituents and pharmacological profile of bran is expected to give insight to their potential application in promotion of health .", "cereal brans , the by - products obtained in large amounts in grain milling industry , considered as inedible material for humans , is mostly used as animal feed .", "however , brans are concentrated source of dietary fibre and other nutrients ( proteins , b - vitamins , and minerals ) .", "brans are generally composed mainly of insoluble cellulose and hemicellulose , with only about 5 percent soluble fibre , and have little hypercholesterolemia effect .", "bran contributes a pleasing , sweet , nutty flavour when added as a flavour enhancer in a variety of food products ."], ["commercial wheat flour and oat bran ( baggry 's india ltd . , new delhi , india ) were purchased from local market .", "chappati was prepared by the addition of cereal brans ( wheat , oat , and rice ) singly and in combination ( w  :  r  :  o :  : 2  :  1.5  :  1.5 ) to wheat flour at 5 and 10% bran supplementation . \n chapatti .", "cereal brans singly and in combination at 5 and 10% level were added to wheat flour and required quantity of water which were mixed manually to obtain dough of suitable consistency .", "the dough was divided into four equal parts and moulded into circular chapattis of 15.0  cm in diameter with rolling pin and board .", "traditional home baking procedure was followed to bake chapattis on iron plate ( tawa ) .", "chapattis were cooled and comparative evaluation was done using the following criteria which also included observations on dough handling properties .  \n\n", "characteristic \n\n score grade \n dough handling  nonsticky  sticky  slightly sticky  very sticky  puffing of chapatti  full  partial  nil . \n ", "the instrument was calibrated with the user supplied black plate calibration standard that was used for zero setting .", "the instruments were placed on the plate and three exposures at different places were conducted .", "readings were displayed as a , b , and l color parameters according to the cielab system of color measurement .", "the a value ranges from 100 ( redness ) to + 100 ( greenness ) and the b value ranges from 100 ( blueness ) to + 100 ( yellowness ) , while the l value , indicating the measure of lightness , ranges from 0 ( black ) to 100 ( white ) .", "was evaluated by using texture analyser ( stable micro systems , model ta - hdi , uk ) .", "one strip at a time was placed on the centre of the sample holder and the blade was allowed to cut the chapatti strip .", "the force ( n ) required to cut chapatti strip into two pieces was recorded .", "bran enriched products such as extruded snacks , breakfast cereal - porridge , and chapatti were evaluated for sensory attributes ( appearance , colour , texture , flavor , and overall acceptability ) through a panel of semi - trained judges using 9-point hedonic scale .", "water activity of bran enriched products was estimated using water activity meter having hygrolab 3 bench - top indicator ( rotrogenic company ) .", "standard aoac procedure   was followed for free fatty acids determination in cereal bran enriched products .", "product sample ( 5  g ) was taken in flask and 50  ml benzene was added and kept for 30  min for extraction of free fatty acids . after extraction , 5  ml extract , 5  ml benzene , 10  ml alcohol , and phenolphthalein as indicator were taken in flask and titrated against 0.02  n koh till light pink colour disappeared : \n ( 1)%ffa  ( %  oleic  acid )  = 2820.02  n  kohml  of  alkali  useddilution  factor1000wt  of  sample  taken  100 ,  moisture content by method of aacc 2000 ,  total plate count by method of maturin and peeler .", "moisture content by method of aacc 2000 , total plate count by method of maturin and peeler .", "data collected from the aforesaid experiments was subjected to statistical analysis for standard error and duncan 's multiple range test using minitab software ."], ["the quality evaluation of chapattis prepared by different bran enriched levels is mentioned in table 1 .", "the pooled scores obtained by the various bran enriched levels of chapattis for appearance , color , texture , and flavor were 7.92 , 7.18 , 7.68 , and 8.10 for wheat bran , rice bran , oat bran , and bran in combination enriched chapattis , respectively .", "the overall acceptability at 5 and 10 percent level of supplementation was 7.65 and 7.80 , respectively .", "reported that chapattis , prepared by the addition of 10% bran , showed better performance and were quite comparable with whole - wheat flour regarding the proximate components and sensory attributes .", "dough handling characteristics of bran enriched chapattis do not show much variation with respect to type of bran used . except for rice bran", "incorporated dough for chapatti ( slightly sticky ) , all others showed nonsticky behavior during dough development .", "all types of bran enriched chapattis showed full puffing except 10% rice bran enriched chapattis in which partial puffing during chapatti preparation was visualised .", "the data presented in table 2 depicted color and texture analysis of bran enriched chapatti . statistically significant ( p  0.05 ) difference was observed in l value of bran enriched chapatti", ". l value of various cereal bran enriched chapattis was 64.37 , 59.12 , 60.04 , and 61.92 for wheat , rice , oat , and bran in combination , respectively .", "l value showed decreasing trend with increase in level of supplementation of cereal brans in chapattis .", "the l value of 66.83 , 61.59 , and 61.14 was observed at 0 , 5 , and 10% level of supplementation , which means slightly lower brightness at higher levels of supplementation .", "a value of wheat , rice , oat , and bran in combination was 4.18 , 5.24 , 4.71 , and 4.28 , respectively .", "with increase in level of supplementation , a value ( redness ) increased from 3.14 at 0% level of supplementation to 5.11 at 10% level .", "altan et al .   stated that , among the color parameters , the l and a values showed marked changes due to addition of tomato pomace . an increase in tomato pomace level decreased the l value of the sample and increased the a value of samples . also , increasing bran level supplementation resulted in a decrease in the b value of chapattis .", "a negative correlation was found between a value and b value of the enriched chapattis . cutting force ( n ) reflects the texture of the chapattis and it stimulates the biting action of the human teeth on chapattis . cutting force ( n ) of various bran enriched chapattis varied as 6.56  n , 5.92  n , 5.30  n , and 5.99 ", "cutting force increased due to presence of more fibres at higher enrichment levels . at 0 , 5 , and 10 percent level of supplementation , the corresponding cutting force ( n ) was 5.25 , 5.91 , and 5.97  n , respectively .", "manu and prasada rao   reported that cutting force of chapattis prepared from different wheat varieties ranged from 4.22 to 6.96  n. hemalatha et al .  ", "also reported that the cutting force ( n ) for chapattis made from different wheat varieties ranged between 4.22 and 6.67 .", "the variation in cutting force might be because of variation in protein and fibre content of brans which determine the resistance offered by the samples . \n", "increase in fibre content might have increased the water holding capacity of chapattis and hence increased moisture content ( % ) with addition of bran .", "reported that moisture content ( % ) of bran enriched chapattis was 31.0 percent while control had moisture content of 30.2 percent .", "maximum water activity was observed in rice bran enriched chapattis ( 0.462 ) which was statistically at par with water activity of bran in combination enriched chapattis ( 0.455 ) .", "water activity of oat and wheat bran enriched chapattis was 0.429 and 0.406 , respectively .", "it was observed from data that water activity of samples was positively correlated with moisture content and followed the same pattern .", "manthey et al .   reported that water activity of bran / fibre enriched pasta increased with bran supplementation over control .", "the increase in water activity is correlated with increase in moisture content at higher levels of bran supplementation .", "the data pertaining to free fatty acids ( % ) is presented in table 3 .", "the free fatty acids ( % ) of bran enriched chapattis ranged from 0.057 to 0.085 .", "the highest free fatty acids were recorded in rice bran enriched chapattis ( 0.085% ) , being the lowest in wheat bran enriched chapattis ( 0.057 ) .", "it is also evident from the table that , with increase in level of bran supplementation , free fatty acids increased significantly .", "the free fatty acids ( % ) at 5 and 10 percent level of supplementation were 0.067 and 0.079 percent , respectively .", "khan et al .   reported similar results regarding free fatty acids while studying development and evaluation of long shelf life ambient stable chapattis .", "a significant variation ( p  0.05 ) was observed in total plate count of bran extruded chapattis .", "total plate count of enriched chapattis varied from 6.5 to 17  10  cfu / g .", "it is also evident from the table that with increase in bran supplementation level , a slight increase in total plate content was observed .", "the mean value of total plate content for 5 and 10% level of supplementation was 10.25  10 and 12.5  10  cfu / g , respectively .", "frazier and westhoff   reported that total plate content increased from 2.3  10 to 3.4  10  cfu / g for flour ."], ["quality characteristics for chapatti revealed that dough handling and puffing of bran enriched chapattis prepared by 5 and 10% level of bran supplementation did not vary significantly .", "all types of bran enriched chapattis except rice bran enriched chapattis showed nonsticky behavior during dough handling .", "rice bran enriched chapatti recorded maximum moisture ( % ) , water activity , and free fatty acids ( % ) . with increase in level of supplementation , moisture , water activity , and free fatty acids increased .", "the future emphasis can be given on development of functional flatbreads which has got increased demand due to increase in health conscious consumer base ."]]}
{"article_id": "PMC2708983", "article_text": ["glaucoma is a progressive optic neuropathy leading to permanent visual loss that is often associated with elevated intraocular pressure ( iop ) .", "primary open - angle glaucoma ( poag ) is the most common type of glaucoma .", "normal tension glaucoma ( ntg ) is an important subset of poag ; while many poag patients have high iop,1 patients with ntg have statistically normal iop.24 the prevalence of ntg is higher among the japanese population than among caucasians , and recent studies reported that 92% of poag patients in japan had ntg.58 the diagnosis of glaucoma is based on a combination of factors including optic nerve damage and specific field defects for which iop is the only treatable risk factor .", "ntg , however , tends to be underdiagnosed because of an accompanying high myopia in many cases and the lack of elevated iop .", "although relatively higher iop , myopia , and older age are known to be factors associated with the development of ntg,9 they are not pathognomonic and there remains a growing interest in the identification of pathogenetic factors associated with ntg .", "glaucoma is genetically heterogeneous and the detection of susceptibility genes could provide useful information for early diagnosis of glaucoma . to date , over 30 genetic loci for glaucoma have been identified by linkage analysis in multiple pedigrees ; 1012 14 loci of poag , 3 loci of primary congenital glaucoma ( pcg ) , and 1 locus of pigment dispersion syndrome have been designated glc1a - glc1n , glc3a - glc3c , and gpds1 ( with approval from the hugo genome nomenclature committee ) , respectively . among them , glc3a harbors cytochrome p450 , family 1 , subfamily b , polypeptide 1 ( cyp1b1).13 cyp1b1 sapans 8.5 kb on chromosome 2p21 with three exons , and mutation of this gene is a major cause of pcg.10,13 most cyp1b1 mutations are genetic insertions , deletions , or missense mutations , indicating that cyp1b1 is relatively susceptible to recombination events.10 cyp1b1 has also been found to be involved in the development of juvenile - onset glaucoma ( juvenile open angle glaucoma [ joag]).1416 in addition , recent studies have shown that cyp1b1 mutations are related to poag in several ethnic groups,10 and there have been reports of delayed expression of a cyp1b1 mutation and coexistence of pcg and poag in the same pedigree.17,18 these recent studies suggest that cyp1b1 mutations in the glc3a locus may contribute to a broader range of glaucoma phenotypes than the pcg phenotype alone , including ntg . in this study , with prospective snp analysis of the cyp1b1 gene in ntg patients in mind , we performed comprehensive microsatellite mapping in and around the glc3a locus and investigated the disease - susceptibility of this locus in ntg patients .", "we recruited 243 japanese subjects from the yokohama city university , yamanashi university , gifu university , kobe university , yamaguchi university , kumamoto university , hokkaido university , tokyo university , niigata university , kanazawa university , hiroshima university , tajimi municipal hospital , and tokai university in japan .", "of these subjects , 142 were diagnosed with ntg , and 101 were control subjects .", "the control subjects were of the same age and sex as the ntg patients , and they were not affected by glaucoma or any ophthalmological or systemic diseases that could result in optic nerve or visual field changes .", "furthermore , the control cases either had no myopia or had mild myopia with refractive errors of 3.00 d or less .", "all patients and control subjects were of japanese ethnicity , with similar social backgrounds and residing in the same urban area .", "informed consent was obtained from all patients , and the study was conducted in accordance with the declaration of helsinki and subsequent revisions thereof .", "ntg patients enrolled in this study were diagnosed as such if the patient had the following conditions : glaucomatous changes in the optic nerve head with or without retinal nerve fiber layer defect and corresponding glaucomatous visual field defects ; normal open angle with angle width greater than shaffer grade 2 ; absence of intraocular pressure greater than 21 mmhg on repeated examination by goldmann applanation tonometry without medication ; neurologic , rhinologic , and general medical examinations including magnetic resonance imaging that failed to disclose any pathology responsible for optic nerve change .", "glaucomatous optic nerve abnormality was diagnosed when the vertical cup / disc ratio of the optic nerve head was more than or equal to 0.7 , or the rim width at the superior portion ( 111 hour ) or inferior portion ( 57 hour ) was less than or equal to 0.1 of disc diameter , or the difference of the vertical cup / disc ratio was greater than or equal to 0.2 between both eyes , or a nerve fiber layer defect was found .", "glaucomatous visual field defect was defined on a hemifield basis using a reliable field data examined by the humphrey static visual field analyser ( carl zeiss meditec , oberkochen , germany , ) c-30 - 2 program according to the anderson and patella s criteria ; 19 the hemifield was judged abnormal when the pattern deviation probability plot showed a cluster of three or more nonedge - contiguous points having sensitivity with a probability of less than 5% in the upper or lower hemifield , and in one of these with a probability of less than 1% . the following inclusion and exclusion criteria", "we excluded individuals who were diagnosed under 20 or over 60 years of age and who had 8.0 d or higher myopic refractive error of spherical equivalence .", "the selection criteria of hfa mean deviation were stratified depending on the subjects age in order to minimize the effect of aging on retinal ganglion cell loss and subsequent visual field defect(s ) ( i ) no limitation if the patient was diagnosed under 50 years of age ( ii ) 10.00 db or worse in at least one eye if the patient was diagnosed between 50 and 55 years of age ( iii ) 15.00 db or worse in at least one eye if the patient was diagnosed above 55 years of age . in this study ,", "the cases exhibiting a comparatively early onset were selected as they suggest that genetic factors may show stronger involvement . during diagnosis ,", "patients whose refraction values had changed due to cataract surgery , refractive surgery , or other intervention were excluded from the study . in cases where a glaucomatous visual field defect was present only in one eye , the refraction value and glaucomatous visual field defect of the affected eye were adopted . in cases", "where a glaucomatous visual field defect was present in both eyes , the refraction value and glaucomatous visual field defect of the more severely affected eye were adopted .", "genomic dna was extracted using the qiaamp dna blood mini kit ( qiagen , hilden , germany ) or the guanidine method . in this association study , we selected 13 highly polymorphic microsatellite markers that are located in and around the glc3a locus as shown in figure 1 .", "the markers were determined based on the national center for biotechnology information for fine mapping .", "polymerase chain reaction ( pcr ) was performed in a reaction mixture with a total volume of 12.5 l containing pcr buffer , genomic dna , 0.2 mm dinucleotide triphosphates ( dntps ) , 0.5 m primers , and 0.35 u taq polymerase .", "the pcr conditions were as follows : 94 c for 5 min , followed by 30 cycles of denaturation at 94 c for 30 sec , annealing at 56 c for 30 sec , extension at 72 c for 1 min , and a final elongation step at 72 c for 10 min .", "the reaction was carried out in a pcr thermal cycler ( geneamp system 9700 , applied biosystems , foster city , ca , usa ) .", "the forward primer was labelled at the 5 end with 6-fam , vic , pet , or ned ( sigma - aldrich , st .", "louis , mo , usa ) ( table 1 ) . to determine the number of microsatellite repeats", ", the pcr products were denatured at 97 c for 2 min , mixed with formamide , and electrophoresed using an abi3130 genetic analyser ( applied biosystems ) .", "the number of microsatellite repeats was estimated automatically using the genescan 672 software ( applied biosystems ) by the local southern method with a size marker of gs500 tamra ( applied biosystems ) .", "the significance of allelic frequencies between the patient and control groups was evaluated by fisher s exact test .", "the probability of association was corrected by the bonferroni inequality method , ie , by multiplying the obtained p values with the number of alleles compared .", "statistical analyses were performed on a computer using the spss software ( version 10.1 ; spss inc . ,", "haplotype frequencies and linkage disequilibrium ( ld ) in the multi - locus analyses were calculated using pypop.20 haplotype frequencies were estimated using the iterative expectation - maximization algorithm .", "we recruited 243 japanese subjects from the yokohama city university , yamanashi university , gifu university , kobe university , yamaguchi university , kumamoto university , hokkaido university , tokyo university , niigata university , kanazawa university , hiroshima university , tajimi municipal hospital , and tokai university in japan .", "of these subjects , 142 were diagnosed with ntg , and 101 were control subjects .", "the control subjects were of the same age and sex as the ntg patients , and they were not affected by glaucoma or any ophthalmological or systemic diseases that could result in optic nerve or visual field changes .", "furthermore , the control cases either had no myopia or had mild myopia with refractive errors of 3.00 d or less .", "all patients and control subjects were of japanese ethnicity , with similar social backgrounds and residing in the same urban area .", "informed consent was obtained from all patients , and the study was conducted in accordance with the declaration of helsinki and subsequent revisions thereof .", "ntg patients enrolled in this study were diagnosed as such if the patient had the following conditions : glaucomatous changes in the optic nerve head with or without retinal nerve fiber layer defect and corresponding glaucomatous visual field defects ; normal open angle with angle width greater than shaffer grade 2 ; absence of intraocular pressure greater than 21 mmhg on repeated examination by goldmann applanation tonometry without medication ; neurologic , rhinologic , and general medical examinations including magnetic resonance imaging that failed to disclose any pathology responsible for optic nerve change .", "glaucomatous optic nerve abnormality was diagnosed when the vertical cup / disc ratio of the optic nerve head was more than or equal to 0.7 , or the rim width at the superior portion ( 111 hour ) or inferior portion ( 57 hour ) was less than or equal to 0.1 of disc diameter , or the difference of the vertical cup / disc ratio was greater than or equal to 0.2 between both eyes , or a nerve fiber layer defect was found .", "glaucomatous visual field defect was defined on a hemifield basis using a reliable field data examined by the humphrey static visual field analyser ( carl zeiss meditec , oberkochen , germany , ) c-30 - 2 program according to the anderson and patella s criteria ; 19 the hemifield was judged abnormal when the pattern deviation probability plot showed a cluster of three or more nonedge - contiguous points having sensitivity with a probability of less than 5% in the upper or lower hemifield , and in one of these with a probability of less than 1% . the following inclusion and exclusion criteria", "we excluded individuals who were diagnosed under 20 or over 60 years of age and who had 8.0 d or higher myopic refractive error of spherical equivalence .", "the selection criteria of hfa mean deviation were stratified depending on the subjects age in order to minimize the effect of aging on retinal ganglion cell loss and subsequent visual field defect(s ) ( i ) no limitation if the patient was diagnosed under 50 years of age ( ii ) 10.00 db or worse in at least one eye if the patient was diagnosed between 50 and 55 years of age ( iii ) 15.00 db or worse in at least one eye if the patient was diagnosed above 55 years of age . in this study ,", "the cases exhibiting a comparatively early onset were selected as they suggest that genetic factors may show stronger involvement . during diagnosis ,", "patients whose refraction values had changed due to cataract surgery , refractive surgery , or other intervention were excluded from the study . in cases where a glaucomatous visual field defect was present only in one eye , the refraction value and glaucomatous visual field defect of the affected eye were adopted . in cases", "where a glaucomatous visual field defect was present in both eyes , the refraction value and glaucomatous visual field defect of the more severely affected eye were adopted .", "genomic dna was extracted using the qiaamp dna blood mini kit ( qiagen , hilden , germany ) or the guanidine method . in this association study , we selected 13 highly polymorphic microsatellite markers that are located in and around the glc3a locus as shown in figure 1 .", "the markers were determined based on the national center for biotechnology information for fine mapping .", "polymerase chain reaction ( pcr ) was performed in a reaction mixture with a total volume of 12.5 l containing pcr buffer , genomic dna , 0.2 mm dinucleotide triphosphates ( dntps ) , 0.5 m primers , and 0.35 u taq polymerase .", "the pcr conditions were as follows : 94 c for 5 min , followed by 30 cycles of denaturation at 94 c for 30 sec , annealing at 56 c for 30 sec , extension at 72 c for 1 min , and a final elongation step at 72 c for 10 min .", "the reaction was carried out in a pcr thermal cycler ( geneamp system 9700 , applied biosystems , foster city , ca , usa ) .", "the forward primer was labelled at the 5 end with 6-fam , vic , pet , or ned ( sigma - aldrich , st .", "louis , mo , usa ) ( table 1 ) . to determine the number of microsatellite repeats , the pcr products were denatured at 97 c for 2 min , mixed with formamide , and electrophoresed using an abi3130 genetic analyser ( applied biosystems ) .", "the number of microsatellite repeats was estimated automatically using the genescan 672 software ( applied biosystems ) by the local southern method with a size marker of gs500 tamra ( applied biosystems ) .", "the significance of allelic frequencies between the patient and control groups was evaluated by fisher s exact test .", "the probability of association was corrected by the bonferroni inequality method , ie , by multiplying the obtained p values with the number of alleles compared .", "statistical analyses were performed on a computer using the spss software ( version 10.1 ; spss inc . ,", "haplotype frequencies and linkage disequilibrium ( ld ) in the multi - locus analyses were calculated using pypop.20 haplotype frequencies were estimated using the iterative expectation - maximization algorithm .", "patient age range was 2158 years ( mean 49.1  9.7 ) ; 47.2% were male and 52.8% were female .", "the mean refraction value was 3.74  3.02 diopters ( d ) , and the mean deviation observed in the humphrey static visual field determination ( carl zeiss meditec , oberkochen , germany ) was 10.14  8.04 db .", "we genotyped 13 polymorphic microsatellite markers in and around the glc3a locus in 142 patients and 101 controls ( figure 1 ) .", "the observed and expected frequencies of each genotype for the 13 markers in the case and control subjects were in hardy  weinberg equilibrium ( data not shown ) .", "only two adjacent markers , d2s0416i and d2s0425i , were significantly positive , as shown in table 2 , and the frequency of the 444 allele of d2s0416i and the 258 allele of d2s0425i were decreased in cases compared to controls ( p = 0.022 , or = 0.59 and p = 0.034 , or = 0.42 , respectively ) .", "however , this statistical significance disappeared ( pc > 0.05 ) when evaluated by bonferroni correction .", "the magnitude of ld between these two markers was low , with pair - wise d = 0.25 , and the comparison of haplotype consisting of two alleles ( d2s0416i_444 and d2s0425i_258 ) rendered no significant difference between cases and controls ( cases vs controls = 3.5% vs 7.3% , p = 0.055 ) ( data not shown ) .", "the purpose of this study was to investigate whether the glc3a locus is associated with ntg in japanese subjects , based on results from recent studies reporting that the cyp1b1 gene , located at the glc3a locus on chromosome 2p21 , could be a causative gene in poag as well as pcg . to this end , we genotyped 13 microsatellite markers in and around the glc3a locus . here", "we report a lack of association between the glc3a locus and ntg in japanese patients , suggesting that genetic variation at this locus may not play an important role in the development of ntg .", "sarfarazi and colleagues mapped glc3a as a putative pcg locus to 2p21 in 11 turkish families , and the d2s177 microsatellite , located 270 kb telomeric to the cyp1b1 gene , showed a maximal lod score of 9.40.22 bejjani and colleagues also reported that the maximal lod score was 15.76 with d2s177 in 25 saudi arabian pcg families.23 recently , it has been reported that the d2s177 allele was associated with cyp1b1 mutations related to pcg.24 therefore , d2s177 may be potentially useful as a marker for genetic events associated with glaucoma , such as cyp1b1 mutations . in the present study", ", we did not find any significant association between d2s177 alleles and ntg , suggesting that cyp1b1 mutations may not be a risk factor for ntg .", "it has been hypothesized that mutations of cyp1b1 cause developmental abnormality in the structure and function of the anterior ocular segment.25 however , ntg patients have statistically normal iop with no abnormality in the structure of anterior ocular segment , suggesting that cyp1b1 is a potential disease susceptibility gene of pcg , poag , and joag , but not ntg . on the other hand", ", we found a weak association between two adjacent markers , d2s0416i and d2s0425i , and ntg , although this link did not reach statistical significance when corrected .", "the nearest gene of d2s0416i is the sos1 ( son of sevenless homolog 1 [ drosophila ] ) gene , which is about 40 kb from the marker .", "sos1 encodes a protein that is a guanine nucleotide exchange factor for ras proteins,26 and mutations in this gene are associated with gingival fibromatosis and noonan syndrome.2729 another marker , d2s0425i , is within the cdkl4 ( cyclin - dependent kinase - like 4 ) gene , a member of the ser / thr protein kinase family . currently , there are no reports suggesting any connection between these two genes and ntg .", "previous studies reported that mutations in genes such as optic atrophy 1 ( opa1),3032 apolipoprotein e ( apoe),33,34 and optineurin ( optn)3537 are implicated in ntg .", "recently , wd repeat - domain 36 ( wdr36),38 endothelin receptor type a ( ednra)39,40 methylenetetrahydrofolate reductase ( mthfr),41 and -1-adrenergic receptor ( adrb1)42 have also been reported as risk factors for ntg .", "however , associations between these genes and ntg were not strong or not often replicated in other populations,4347 suggesting that there may be other unknown genetic factors having more powerful effects on the development of ntg than those identified so far . in conclusion , we performed an association analysis of the glc3a locus including the cyp1b1 gene using microsatellite markers in ntg patients , but the marker involved in ntg was not detected in this locus . in ntg", "patients , diagnosis is usually made after a visual field defect has occurred , due to few subjective symptoms and normal iop . therefore it is necessary to identify a disease susceptibility gene and elucidate pathogenic mechanisms of ntg for early diagnosis , prevention , and therapeutic development ."], "abstract_text": ["<S> purpose : to investigate whether the glc3a locus harboring the cyp1b1 gene is associated with normal tension glaucoma ( ntg ) in japanese patients.materials and methods : one hundred forty - two japanese patients with ntg and 101 japanese healthy controls were recruited . </S>", "<S> patients exhibiting a comparatively early onset were selected as this suggests that genetic factors may show stronger involvement . </S>", "<S> genotyping and assessment of allelic diversity was performed on 13 highly polymorphic microsatellite markers in and around the glc3a locus.results:there were decreased frequencies of the 444 allele of d2s0416i and the 258 allele of d2s0425i in cases compared to controls ( p = 0.022 and p = 0.034 , respectively ) . </S>", "<S> however , this statistical significance disappeared when corrected ( pc > 0.05 ) . </S>", "<S> we did not find any significant association between the remaining 11 microsatellite markers , including d2s177 , which may be associated with cyp1b1 , and ntg ( p > 0.05).conclusions : our study showed no association between the glca3 locus and ntg , suggesting that the cyp1b1 gene , which is reportedly involved in a range of glaucoma phenotypes , may not be an associated factor in the pathogenesis of ntg . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Subjects", "Analysis of repeat polymorphisms for 13 microsatellite markers", "Statistical analysis", "Results", "Discussion"], "sections": [["glaucoma is a progressive optic neuropathy leading to permanent visual loss that is often associated with elevated intraocular pressure ( iop ) .", "primary open - angle glaucoma ( poag ) is the most common type of glaucoma .", "normal tension glaucoma ( ntg ) is an important subset of poag ; while many poag patients have high iop,1 patients with ntg have statistically normal iop.24 the prevalence of ntg is higher among the japanese population than among caucasians , and recent studies reported that 92% of poag patients in japan had ntg.58 the diagnosis of glaucoma is based on a combination of factors including optic nerve damage and specific field defects for which iop is the only treatable risk factor .", "ntg , however , tends to be underdiagnosed because of an accompanying high myopia in many cases and the lack of elevated iop .", "although relatively higher iop , myopia , and older age are known to be factors associated with the development of ntg,9 they are not pathognomonic and there remains a growing interest in the identification of pathogenetic factors associated with ntg .", "glaucoma is genetically heterogeneous and the detection of susceptibility genes could provide useful information for early diagnosis of glaucoma . to date , over 30 genetic loci for glaucoma have been identified by linkage analysis in multiple pedigrees ; 1012 14 loci of poag , 3 loci of primary congenital glaucoma ( pcg ) , and 1 locus of pigment dispersion syndrome have been designated glc1a - glc1n , glc3a - glc3c , and gpds1 ( with approval from the hugo genome nomenclature committee ) , respectively . among them , glc3a harbors cytochrome p450 , family 1 , subfamily b , polypeptide 1 ( cyp1b1).13 cyp1b1 sapans 8.5 kb on chromosome 2p21 with three exons , and mutation of this gene is a major cause of pcg.10,13 most cyp1b1 mutations are genetic insertions , deletions , or missense mutations , indicating that cyp1b1 is relatively susceptible to recombination events.10 cyp1b1 has also been found to be involved in the development of juvenile - onset glaucoma ( juvenile open angle glaucoma [ joag]).1416 in addition , recent studies have shown that cyp1b1 mutations are related to poag in several ethnic groups,10 and there have been reports of delayed expression of a cyp1b1 mutation and coexistence of pcg and poag in the same pedigree.17,18 these recent studies suggest that cyp1b1 mutations in the glc3a locus may contribute to a broader range of glaucoma phenotypes than the pcg phenotype alone , including ntg . in this study , with prospective snp analysis of the cyp1b1 gene in ntg patients in mind , we performed comprehensive microsatellite mapping in and around the glc3a locus and investigated the disease - susceptibility of this locus in ntg patients ."], ["we recruited 243 japanese subjects from the yokohama city university , yamanashi university , gifu university , kobe university , yamaguchi university , kumamoto university , hokkaido university , tokyo university , niigata university , kanazawa university , hiroshima university , tajimi municipal hospital , and tokai university in japan .", "of these subjects , 142 were diagnosed with ntg , and 101 were control subjects .", "the control subjects were of the same age and sex as the ntg patients , and they were not affected by glaucoma or any ophthalmological or systemic diseases that could result in optic nerve or visual field changes .", "furthermore , the control cases either had no myopia or had mild myopia with refractive errors of 3.00 d or less .", "all patients and control subjects were of japanese ethnicity , with similar social backgrounds and residing in the same urban area .", "informed consent was obtained from all patients , and the study was conducted in accordance with the declaration of helsinki and subsequent revisions thereof .", "ntg patients enrolled in this study were diagnosed as such if the patient had the following conditions : glaucomatous changes in the optic nerve head with or without retinal nerve fiber layer defect and corresponding glaucomatous visual field defects ; normal open angle with angle width greater than shaffer grade 2 ; absence of intraocular pressure greater than 21 mmhg on repeated examination by goldmann applanation tonometry without medication ; neurologic , rhinologic , and general medical examinations including magnetic resonance imaging that failed to disclose any pathology responsible for optic nerve change .", "glaucomatous optic nerve abnormality was diagnosed when the vertical cup / disc ratio of the optic nerve head was more than or equal to 0.7 , or the rim width at the superior portion ( 111 hour ) or inferior portion ( 57 hour ) was less than or equal to 0.1 of disc diameter , or the difference of the vertical cup / disc ratio was greater than or equal to 0.2 between both eyes , or a nerve fiber layer defect was found .", "glaucomatous visual field defect was defined on a hemifield basis using a reliable field data examined by the humphrey static visual field analyser ( carl zeiss meditec , oberkochen , germany , ) c-30 - 2 program according to the anderson and patella s criteria ; 19 the hemifield was judged abnormal when the pattern deviation probability plot showed a cluster of three or more nonedge - contiguous points having sensitivity with a probability of less than 5% in the upper or lower hemifield , and in one of these with a probability of less than 1% . the following inclusion and exclusion criteria", "we excluded individuals who were diagnosed under 20 or over 60 years of age and who had 8.0 d or higher myopic refractive error of spherical equivalence .", "the selection criteria of hfa mean deviation were stratified depending on the subjects age in order to minimize the effect of aging on retinal ganglion cell loss and subsequent visual field defect(s ) ( i ) no limitation if the patient was diagnosed under 50 years of age ( ii ) 10.00 db or worse in at least one eye if the patient was diagnosed between 50 and 55 years of age ( iii ) 15.00 db or worse in at least one eye if the patient was diagnosed above 55 years of age . in this study ,", "the cases exhibiting a comparatively early onset were selected as they suggest that genetic factors may show stronger involvement . during diagnosis ,", "patients whose refraction values had changed due to cataract surgery , refractive surgery , or other intervention were excluded from the study . in cases where a glaucomatous visual field defect was present only in one eye , the refraction value and glaucomatous visual field defect of the affected eye were adopted . in cases", "where a glaucomatous visual field defect was present in both eyes , the refraction value and glaucomatous visual field defect of the more severely affected eye were adopted .", "genomic dna was extracted using the qiaamp dna blood mini kit ( qiagen , hilden , germany ) or the guanidine method . in this association study , we selected 13 highly polymorphic microsatellite markers that are located in and around the glc3a locus as shown in figure 1 .", "the markers were determined based on the national center for biotechnology information for fine mapping .", "polymerase chain reaction ( pcr ) was performed in a reaction mixture with a total volume of 12.5 l containing pcr buffer , genomic dna , 0.2 mm dinucleotide triphosphates ( dntps ) , 0.5 m primers , and 0.35 u taq polymerase .", "the pcr conditions were as follows : 94 c for 5 min , followed by 30 cycles of denaturation at 94 c for 30 sec , annealing at 56 c for 30 sec , extension at 72 c for 1 min , and a final elongation step at 72 c for 10 min .", "the reaction was carried out in a pcr thermal cycler ( geneamp system 9700 , applied biosystems , foster city , ca , usa ) .", "the forward primer was labelled at the 5 end with 6-fam , vic , pet , or ned ( sigma - aldrich , st .", "louis , mo , usa ) ( table 1 ) . to determine the number of microsatellite repeats", ", the pcr products were denatured at 97 c for 2 min , mixed with formamide , and electrophoresed using an abi3130 genetic analyser ( applied biosystems ) .", "the number of microsatellite repeats was estimated automatically using the genescan 672 software ( applied biosystems ) by the local southern method with a size marker of gs500 tamra ( applied biosystems ) .", "the significance of allelic frequencies between the patient and control groups was evaluated by fisher s exact test .", "the probability of association was corrected by the bonferroni inequality method , ie , by multiplying the obtained p values with the number of alleles compared .", "statistical analyses were performed on a computer using the spss software ( version 10.1 ; spss inc . ,", "haplotype frequencies and linkage disequilibrium ( ld ) in the multi - locus analyses were calculated using pypop.20 haplotype frequencies were estimated using the iterative expectation - maximization algorithm ."], ["we recruited 243 japanese subjects from the yokohama city university , yamanashi university , gifu university , kobe university , yamaguchi university , kumamoto university , hokkaido university , tokyo university , niigata university , kanazawa university , hiroshima university , tajimi municipal hospital , and tokai university in japan .", "of these subjects , 142 were diagnosed with ntg , and 101 were control subjects .", "the control subjects were of the same age and sex as the ntg patients , and they were not affected by glaucoma or any ophthalmological or systemic diseases that could result in optic nerve or visual field changes .", "furthermore , the control cases either had no myopia or had mild myopia with refractive errors of 3.00 d or less .", "all patients and control subjects were of japanese ethnicity , with similar social backgrounds and residing in the same urban area .", "informed consent was obtained from all patients , and the study was conducted in accordance with the declaration of helsinki and subsequent revisions thereof .", "ntg patients enrolled in this study were diagnosed as such if the patient had the following conditions : glaucomatous changes in the optic nerve head with or without retinal nerve fiber layer defect and corresponding glaucomatous visual field defects ; normal open angle with angle width greater than shaffer grade 2 ; absence of intraocular pressure greater than 21 mmhg on repeated examination by goldmann applanation tonometry without medication ; neurologic , rhinologic , and general medical examinations including magnetic resonance imaging that failed to disclose any pathology responsible for optic nerve change .", "glaucomatous optic nerve abnormality was diagnosed when the vertical cup / disc ratio of the optic nerve head was more than or equal to 0.7 , or the rim width at the superior portion ( 111 hour ) or inferior portion ( 57 hour ) was less than or equal to 0.1 of disc diameter , or the difference of the vertical cup / disc ratio was greater than or equal to 0.2 between both eyes , or a nerve fiber layer defect was found .", "glaucomatous visual field defect was defined on a hemifield basis using a reliable field data examined by the humphrey static visual field analyser ( carl zeiss meditec , oberkochen , germany , ) c-30 - 2 program according to the anderson and patella s criteria ; 19 the hemifield was judged abnormal when the pattern deviation probability plot showed a cluster of three or more nonedge - contiguous points having sensitivity with a probability of less than 5% in the upper or lower hemifield , and in one of these with a probability of less than 1% . the following inclusion and exclusion criteria", "we excluded individuals who were diagnosed under 20 or over 60 years of age and who had 8.0 d or higher myopic refractive error of spherical equivalence .", "the selection criteria of hfa mean deviation were stratified depending on the subjects age in order to minimize the effect of aging on retinal ganglion cell loss and subsequent visual field defect(s ) ( i ) no limitation if the patient was diagnosed under 50 years of age ( ii ) 10.00 db or worse in at least one eye if the patient was diagnosed between 50 and 55 years of age ( iii ) 15.00 db or worse in at least one eye if the patient was diagnosed above 55 years of age . in this study ,", "the cases exhibiting a comparatively early onset were selected as they suggest that genetic factors may show stronger involvement . during diagnosis ,", "patients whose refraction values had changed due to cataract surgery , refractive surgery , or other intervention were excluded from the study . in cases where a glaucomatous visual field defect was present only in one eye , the refraction value and glaucomatous visual field defect of the affected eye were adopted . in cases", "where a glaucomatous visual field defect was present in both eyes , the refraction value and glaucomatous visual field defect of the more severely affected eye were adopted ."], ["genomic dna was extracted using the qiaamp dna blood mini kit ( qiagen , hilden , germany ) or the guanidine method . in this association study , we selected 13 highly polymorphic microsatellite markers that are located in and around the glc3a locus as shown in figure 1 .", "the markers were determined based on the national center for biotechnology information for fine mapping .", "polymerase chain reaction ( pcr ) was performed in a reaction mixture with a total volume of 12.5 l containing pcr buffer , genomic dna , 0.2 mm dinucleotide triphosphates ( dntps ) , 0.5 m primers , and 0.35 u taq polymerase .", "the pcr conditions were as follows : 94 c for 5 min , followed by 30 cycles of denaturation at 94 c for 30 sec , annealing at 56 c for 30 sec , extension at 72 c for 1 min , and a final elongation step at 72 c for 10 min .", "the reaction was carried out in a pcr thermal cycler ( geneamp system 9700 , applied biosystems , foster city , ca , usa ) .", "the forward primer was labelled at the 5 end with 6-fam , vic , pet , or ned ( sigma - aldrich , st .", "louis , mo , usa ) ( table 1 ) . to determine the number of microsatellite repeats , the pcr products were denatured at 97 c for 2 min , mixed with formamide , and electrophoresed using an abi3130 genetic analyser ( applied biosystems ) .", "the number of microsatellite repeats was estimated automatically using the genescan 672 software ( applied biosystems ) by the local southern method with a size marker of gs500 tamra ( applied biosystems ) ."], ["the significance of allelic frequencies between the patient and control groups was evaluated by fisher s exact test .", "the probability of association was corrected by the bonferroni inequality method , ie , by multiplying the obtained p values with the number of alleles compared .", "statistical analyses were performed on a computer using the spss software ( version 10.1 ; spss inc . ,", "haplotype frequencies and linkage disequilibrium ( ld ) in the multi - locus analyses were calculated using pypop.20 haplotype frequencies were estimated using the iterative expectation - maximization algorithm ."], ["patient age range was 2158 years ( mean 49.1  9.7 ) ; 47.2% were male and 52.8% were female .", "the mean refraction value was 3.74  3.02 diopters ( d ) , and the mean deviation observed in the humphrey static visual field determination ( carl zeiss meditec , oberkochen , germany ) was 10.14  8.04 db .", "we genotyped 13 polymorphic microsatellite markers in and around the glc3a locus in 142 patients and 101 controls ( figure 1 ) .", "the observed and expected frequencies of each genotype for the 13 markers in the case and control subjects were in hardy  weinberg equilibrium ( data not shown ) .", "only two adjacent markers , d2s0416i and d2s0425i , were significantly positive , as shown in table 2 , and the frequency of the 444 allele of d2s0416i and the 258 allele of d2s0425i were decreased in cases compared to controls ( p = 0.022 , or = 0.59 and p = 0.034 , or = 0.42 , respectively ) .", "however , this statistical significance disappeared ( pc > 0.05 ) when evaluated by bonferroni correction .", "the magnitude of ld between these two markers was low , with pair - wise d = 0.25 , and the comparison of haplotype consisting of two alleles ( d2s0416i_444 and d2s0425i_258 ) rendered no significant difference between cases and controls ( cases vs controls = 3.5% vs 7.3% , p = 0.055 ) ( data not shown ) ."], ["the purpose of this study was to investigate whether the glc3a locus is associated with ntg in japanese subjects , based on results from recent studies reporting that the cyp1b1 gene , located at the glc3a locus on chromosome 2p21 , could be a causative gene in poag as well as pcg . to this end , we genotyped 13 microsatellite markers in and around the glc3a locus . here", "we report a lack of association between the glc3a locus and ntg in japanese patients , suggesting that genetic variation at this locus may not play an important role in the development of ntg .", "sarfarazi and colleagues mapped glc3a as a putative pcg locus to 2p21 in 11 turkish families , and the d2s177 microsatellite , located 270 kb telomeric to the cyp1b1 gene , showed a maximal lod score of 9.40.22 bejjani and colleagues also reported that the maximal lod score was 15.76 with d2s177 in 25 saudi arabian pcg families.23 recently , it has been reported that the d2s177 allele was associated with cyp1b1 mutations related to pcg.24 therefore , d2s177 may be potentially useful as a marker for genetic events associated with glaucoma , such as cyp1b1 mutations . in the present study", ", we did not find any significant association between d2s177 alleles and ntg , suggesting that cyp1b1 mutations may not be a risk factor for ntg .", "it has been hypothesized that mutations of cyp1b1 cause developmental abnormality in the structure and function of the anterior ocular segment.25 however , ntg patients have statistically normal iop with no abnormality in the structure of anterior ocular segment , suggesting that cyp1b1 is a potential disease susceptibility gene of pcg , poag , and joag , but not ntg . on the other hand", ", we found a weak association between two adjacent markers , d2s0416i and d2s0425i , and ntg , although this link did not reach statistical significance when corrected .", "the nearest gene of d2s0416i is the sos1 ( son of sevenless homolog 1 [ drosophila ] ) gene , which is about 40 kb from the marker .", "sos1 encodes a protein that is a guanine nucleotide exchange factor for ras proteins,26 and mutations in this gene are associated with gingival fibromatosis and noonan syndrome.2729 another marker , d2s0425i , is within the cdkl4 ( cyclin - dependent kinase - like 4 ) gene , a member of the ser / thr protein kinase family . currently , there are no reports suggesting any connection between these two genes and ntg .", "previous studies reported that mutations in genes such as optic atrophy 1 ( opa1),3032 apolipoprotein e ( apoe),33,34 and optineurin ( optn)3537 are implicated in ntg .", "recently , wd repeat - domain 36 ( wdr36),38 endothelin receptor type a ( ednra)39,40 methylenetetrahydrofolate reductase ( mthfr),41 and -1-adrenergic receptor ( adrb1)42 have also been reported as risk factors for ntg .", "however , associations between these genes and ntg were not strong or not often replicated in other populations,4347 suggesting that there may be other unknown genetic factors having more powerful effects on the development of ntg than those identified so far . in conclusion , we performed an association analysis of the glc3a locus including the cyp1b1 gene using microsatellite markers in ntg patients , but the marker involved in ntg was not detected in this locus . in ntg", "patients , diagnosis is usually made after a visual field defect has occurred , due to few subjective symptoms and normal iop . therefore it is necessary to identify a disease susceptibility gene and elucidate pathogenic mechanisms of ntg for early diagnosis , prevention , and therapeutic development ."]]}
{"article_id": "PMC3222027", "article_text": ["recently , a single center study conducted by oiva and coworkers and published in critical care   demonstrated that phospho - specific whole blood flow cytometry could be used to assess activated signaling path - ways in leukocytes isolated from pancreatitis patients .", "pancreatitis is an inflammatory process in which pancreatic enzymes are activated leading to autodigestion of the pancreas and adjacent tissues . during pernicious cases ,", "the initial inflammatory response is excessive and can result in tissue injury , organ failure , and death . to counter this inflammation ,", "these steps can include leukocyte apoptosis , anti - inflammatory cytokine production , and anergy .", "when patients become leukopenic and immunosuppressed , they often become susceptible to infections with organisms to which a healthy patient would normally be resistant .", "although therapy aimed at blunting the initial over - exuberant inflammation may be beneficial if applied early , it may also be detrimental by impairing the host 's ability to fight infection that is a common cause of death in these patients . to complicate the potential use of immune - modulating therapy ,", "inflammatory and anti - inflammatory processes can occur along different time - lines for the varied leukocyte subsets .", "for example , during immune response , t lymphocytes might be undergoing apoptosis while neutrophils are primed to produce reactive oxygen species .", "thus , during prolonged pancreatitis , leukocyte subsets may be concurrently exhibiting both pro- and anti - inflammatory processes .", "as noted above , a potential promising therapy of pancreatitis is modulation of the immune response .", "this is a difficult undertaking as the host response will be affected by a number of variables , including genetic background , co - morbidities , age , gender , and so on .", "in addition , the patient 's immune response will vary during the course of the disease .", "thus , what is needed for immunotherapy to be practical is a rapid , robust measure of the host immune system . in the manuscript published by oiva and co - workers", ", the authors determined the signaling profiles of circulating leukocytes isolated from pancreatitis patients using phospho - specific whole blood flow cytometry .", "this is a powerful new technology that allows for simultaneous single - cell determination of leukocyte subsets using cell surface markers as well as intracellular protein phosphorylation .", "this work represents a continuation of work published previously . in this earlier work , oiva and co - workers", "demonstrated that stimulated monocytes isolated from patients with acute pancreatitis had decreased phosphorylated erk 1/2 , nf-b , and stat1 and 3 .", "the authors concluded that these changes could lead to impaired monocyte recruitment as well as increased susceptibility to infections .", "here , changes in activated t lymphocyte p38 , nf-b , stat1 and stat6 activity was observed that could be interpreted as being pro- or anti - inflammatory .", "interestingly , they determined that patients ' lymphocytes exhibited decreased phosphorylated nf-b and stat1 and increased phosphorylation of p38 and stat6 , suggesting a transition from a th1 to th2 phenotype .", "additionally , this minimally invasive methodology could be used to generate immune status within 6 hours under ideal conditions .", "thus , this methodology represents an essential step prior to targeting the underlying immune response to pancreatitis .", "although beyond the scope of the report , a potentially necessary step after immune status determination , and prior to treatment with inflammatory altering treatments , would be to first treat the whole blood cells isolated from the patients with the potential therapeutic agent and evaluate the immune effector cell response .", "phospho - specific flow cytometry could also be used to determine if the leukocytes responded in a way that would be beneficial .", "thus , the minimally invasive , relatively quick methodology developed in this paper could be utilized to determine immune status as well as provide a method to test potential therapies .", "one unavoidable limitation to the report is that the signaling processes were determined using only peripheral leukocytes .", "a possibility exists that peripheral leukocytes may not respond similarly to ex vivo stimuli as leukocytes isolated from the inflammatory site(s ) . when feasible , future studies need to be undertaken to compare the response of peripheral leukocytes to these tissue leukocytes .", "rapid results from whole blood phospho - specific flow cytometry during pancreatitis will allow for immune status determination , likely improve early diagnosis and provide a rational basis for immune targeting therapies .", "this work was supported in part by national institutes of health grants gm44118 and gm55194 ( to rsh ) , gm72760 ( to ccc ) , and the alan a and edith l wolff foundation ."], "abstract_text": ["<S> recently , a single center study conducted by oiva and coworkers and published in critical care demonstrated that phospho - specific whole blood flow cytometry could be used to assess activated signaling pathways in leukocytes isolated from pancreatitis patients . </S>", "<S> the authors demonstrated that this methodology had the potential to determine the current status of a patient 's immune state . </S>", "<S> although the experimental cohort was clinically homogeneous , the observed data were heterogeneous . </S>", "<S> altogether , these results suggest that prior to administering immune - modulatory therapies in inflammatory diseases , it will be beneficial to first determine immune status </S>", "<S> . rapid results from whole blood phospho - specific flow cytometry may allow for determination of immune status , improve early diagnosis , and provide a rational basis for immunomodulatory therapies . </S>"], "labels": null, "section_names": ["None", "Abbreviations", "Competing interests", "Acknowledgements"], "sections": [["recently , a single center study conducted by oiva and coworkers and published in critical care   demonstrated that phospho - specific whole blood flow cytometry could be used to assess activated signaling path - ways in leukocytes isolated from pancreatitis patients .", "pancreatitis is an inflammatory process in which pancreatic enzymes are activated leading to autodigestion of the pancreas and adjacent tissues . during pernicious cases ,", "the initial inflammatory response is excessive and can result in tissue injury , organ failure , and death . to counter this inflammation ,", "these steps can include leukocyte apoptosis , anti - inflammatory cytokine production , and anergy .", "when patients become leukopenic and immunosuppressed , they often become susceptible to infections with organisms to which a healthy patient would normally be resistant .", "although therapy aimed at blunting the initial over - exuberant inflammation may be beneficial if applied early , it may also be detrimental by impairing the host 's ability to fight infection that is a common cause of death in these patients . to complicate the potential use of immune - modulating therapy ,", "inflammatory and anti - inflammatory processes can occur along different time - lines for the varied leukocyte subsets .", "for example , during immune response , t lymphocytes might be undergoing apoptosis while neutrophils are primed to produce reactive oxygen species .", "thus , during prolonged pancreatitis , leukocyte subsets may be concurrently exhibiting both pro- and anti - inflammatory processes .", "as noted above , a potential promising therapy of pancreatitis is modulation of the immune response .", "this is a difficult undertaking as the host response will be affected by a number of variables , including genetic background , co - morbidities , age , gender , and so on .", "in addition , the patient 's immune response will vary during the course of the disease .", "thus , what is needed for immunotherapy to be practical is a rapid , robust measure of the host immune system . in the manuscript published by oiva and co - workers", ", the authors determined the signaling profiles of circulating leukocytes isolated from pancreatitis patients using phospho - specific whole blood flow cytometry .", "this is a powerful new technology that allows for simultaneous single - cell determination of leukocyte subsets using cell surface markers as well as intracellular protein phosphorylation .", "this work represents a continuation of work published previously . in this earlier work , oiva and co - workers", "demonstrated that stimulated monocytes isolated from patients with acute pancreatitis had decreased phosphorylated erk 1/2 , nf-b , and stat1 and 3 .", "the authors concluded that these changes could lead to impaired monocyte recruitment as well as increased susceptibility to infections .", "here , changes in activated t lymphocyte p38 , nf-b , stat1 and stat6 activity was observed that could be interpreted as being pro- or anti - inflammatory .", "interestingly , they determined that patients ' lymphocytes exhibited decreased phosphorylated nf-b and stat1 and increased phosphorylation of p38 and stat6 , suggesting a transition from a th1 to th2 phenotype .", "additionally , this minimally invasive methodology could be used to generate immune status within 6 hours under ideal conditions .", "thus , this methodology represents an essential step prior to targeting the underlying immune response to pancreatitis .", "although beyond the scope of the report , a potentially necessary step after immune status determination , and prior to treatment with inflammatory altering treatments , would be to first treat the whole blood cells isolated from the patients with the potential therapeutic agent and evaluate the immune effector cell response .", "phospho - specific flow cytometry could also be used to determine if the leukocytes responded in a way that would be beneficial .", "thus , the minimally invasive , relatively quick methodology developed in this paper could be utilized to determine immune status as well as provide a method to test potential therapies .", "one unavoidable limitation to the report is that the signaling processes were determined using only peripheral leukocytes .", "a possibility exists that peripheral leukocytes may not respond similarly to ex vivo stimuli as leukocytes isolated from the inflammatory site(s ) . when feasible , future studies need to be undertaken to compare the response of peripheral leukocytes to these tissue leukocytes .", "rapid results from whole blood phospho - specific flow cytometry during pancreatitis will allow for immune status determination , likely improve early diagnosis and provide a rational basis for immune targeting therapies ."], [""], [""], ["this work was supported in part by national institutes of health grants gm44118 and gm55194 ( to rsh ) , gm72760 ( to ccc ) , and the alan a and edith l wolff foundation ."]]}
{"article_id": "PMC4073566", "article_text": ["these misconceptions have a significant influence on the day - to - day life including the search for treatment in times of illness .", "a number of studies have reported that misconceptions and inadequate knowledge present significant barriers to effective management of diabetes .", "it is imperative for physicians to understand myths and misconceptions in a particular community about a disease to improve patient care , especially when dealing with chronic diseases like diabetes .", "diabetes mellitus ( dm ) is one of the most common noncommunicable diseases in the world and its prevalence is increasing dramatically .", "currently , there are around 285 million diabetic patients around the world , and the numbers are predicted to rise to 439 million by 2030 , with the largest increase in the developing rather than in the developed world .", "its dramatic increase began a few decades ago with the rapid urbanization and development in the country .", "studies in the 1980s showed a trend towards an increase among adult saudis especially females . a large study of saudi patients from 1995 to 2000 revealed prevalence of 23.7% .", "however , a study in 2011 showed a significant rise in prevalence reaching 34.1% in men and 27.6% in women .", "correct knowledge about diabetes and its management has an enormous impact on attitude and practice of diabetic patients .", "a study of diabetic patients in new york , reported that patients with diabetes frequently had beliefs of the disease and medication that were false and even dangerous .", "it is imperative that awareness of diabetes and its correct management be created and various myths and misconceptions surrounding its course and management be removed .", "a few excellent studies about prevailing misconceptions about diabetes have already been carried out in the saudi population , more specifically in female diabetic teachers , in the eastern region , western region and qasim region .", "however , no studies have examined in detail the determinants of the misconceptions of diabetic patients .", "therefore , this study was undertaken to identify the determinants of prevailing misconceptions on diabetes and the management of diabetic patients registered at a diabetes clinic of a tertiary care hospital in the eastern region of saudi arabia .", "this cross - sectional study was carried out at the diabetes clinic of a tertiary care hospital , in eastern saudi arabia .", "this clinic has a registered patient population of 2000 diabetic patients . at a confidence level of 95% ,", "response distribution of 50% and accepted margin of error of 10% , we calculated a sample size of 200 .", "a table of random numbers was used to select patients from the medical record numbers of the registered patients of the diabetes clinic .", "the inclusion criteria were all saudi patients who had been registered at the diabetes clinic of the tertiary care hospital in saudi arabia for 6 months or more since diagnosis and on continuous treatment .", "no freshly diagnosed patient was included . in the same vein uncooperative or nonconsenting patients , as well as patients with any illness ( physical or psychological ) that was likely to influence reliable , valid responses to the interviewer 's questions were excluded .", "the same interviewer spoke to all the selected patients on their scheduled follow - up visits after taking their informed consent .", "when a prospective participant refused to take part , the next patient on the list of random numbers was asked until the sample size was completed .", "all attendees were interviewed in standardized conditions with prior information to ensure valid reliable responses .", "the data collection instrument was an interviewer - filled questionnaire prepared in accordance with  brief illness perception questionnaire  and  belief about medicines questionnaire . ", "the demographic and classification data included age categories ( < 20 , 21 - 40 , 41 - 60 or > 60 years ) , gender ( male and female ) , area of residence ( urban or rural ) , education ( primary , middle , high school , graduate and above ) , family history of diabetes ( present or absent ) , type of diabetes ( type 1 or type 2 ) , time since diagnosis ( < 5 , 6 - 10 , 11 - 15 or > 15 years ) , type of treatment ( oral hypoglycemic or insulin ) , self - monitoring ( yes or no ) and diet control as per doctor 's advice ( yes or no ) . in order to establish validity and reliability of the questionnaire ,", "a pilot study was conducted in a sub - sample of attendees who were not included in the study proper .", "necessary changes were made in the questionnaire as well as the interview style as necessary .", "questions on the commonest reported misconceptions relating to etiology , types , pathogenesis , day - to - day life , diet and treatment of diabetes were included after a review of studies from saudi arabia , usa , india , nepal , and pakistan .", "an answer in  yes  to a misconception question was considered a misconception and a score of one was given .", "the total number of misconception questions was 36 ; therefore , the possible maximum score for any patient was 0 - 36 .", "low ( scores 0 - 12 ) , moderate ( scores 13 - 24 ) , and high ( scores of 24 - 36 ) .", "chi - square test was used to determine the association of the socio - demographic variables with the three categories of misconception scores .", "stepwise logistic regression analysis was applied to the socio - demographic variables showing significant association with the misconception score by chi - square test and values of -coefficient , chi - square , odds ratio , and confidence interval were reported .", "a total of 200 responses for each item in the questionnaire were recorded from 200 subjects .", "the distribution of subjects in the age groups was 32 ( 16% ) in < 20 years , 62 ( 31% ) in 21 - 40 years , 81 ( 40.5% ) between 41 and 60 years and 25 ( 12.5% ) in > 60 years .", "the subjects from urban areas were 168 ( 84% ) and those from rural areas were 32 ( 16% ) .", "those with the highest level of education were 7 ( 3.5% ) who had up to postgraduation , 36 ( 18% ) were university graduates , 76 ( 38% ) high school , 43 ( 21.5% ) middle , 29 ( 14.5% ) primary and 9 ( 4.5% ) illiterate .", "there was a family history of diabetes in 147 ( 73.5% ) and no history in 53 ( 26.5% ) .", "those with type 1 diabetics were 78 ( 39% ) while 122 ( 61% ) had type 2 diabetes .", "the time since diagnosis was < 5 years in 73 ( 36.5% ) , 6 - 10 years in 72 ( 36% ) , 11 - 15 years in 21 ( 10.5% ) and > 15 years in 34 ( 17% ) subjects .", "the type of treatment was insulin in 105 ( 52.5% ) and oral hypoglycemics in 95 ( 47.5% ) indicating that many type 2 diabetic patients were also on insulin .", "self - monitoring of blood glucose was done by 124 ( 62% ) of the subjects while 76 ( 38% ) did not .", "diet control was used by 112 ( 56% ) subjects while 88 ( 44% ) did not .", "a total of 167 ( 83.5% ) had received formal  diabetes awareness education  while 33 ( 16.5% ) had not .", "the total misconception score was low ( 0 - 12 ) in 115 ( 57.5% ) , moderate ( 13 - 24 ) in 77 ( 38.5% ) and high ( 24 - 36 ) in 8 ( 4% ) respondents ( n = 200 ) .", "table 1 demonstrates the frequency distribution of respondents according to misconceptions about etiology , types and pathogenesis of diabetes .", "the most common misconceptions identified on etiology , types and pathogenesis were that  overweight causes diabetes  ( 89% ) ,  diabetes is only a hereditary disease  ( 80.5% ) and  eating too much sugar causes diabetes  ( 69% ) .", "in addition , this table shows the distribution of subjects with reference to misconceptions about daily life .", "the most frequent misconceptions about the daily life of diabetics were that  diabetics can not lead a normal social life ", "frequency of respondents with misconceptions about etiology , types , pathogenesis and day - to - day life of diabetes ( n=200 ) table 2 illustrates the distribution of respondents according to misconceptions about diet .", "the second misconception on treatment was that  there is no need to take medicines when blood glucose is normal ", "table 3 shows the association of the selected socio - demographic variables on the misconception score .", "it shows that female gender , living in a rural area , little or no education , < 5 or > 15 years since diagnosis , lack of self - monitoring , poor compliance with dietary control and no diabetes education were significantly ( p < 0.05 ) associated with moderate or high misconception score .", "however , age , family history of diabetes , type of diabetes and type of treatment were not significantly associated with the misconception score .", "frequency of respondents with misconceptions about diet and treatment ( n=200 ) association of misconception score category with sociodemographic variables table 4 gives stepwise logistic regression to determine the predictive value of independent variables [ showing significant association as per table 3 ] with the misconception score ( dependent variable ) .", "it indicates that diabetes education , gender , education and time since diagnosis are good predictors of misconception score .", "every chronic disease necessitates long - term commitment from the patient , family and health care professionals .", "health care is a full package that includes the proper education of society in general and the patient in particular , about all aspects of the disease in question .", "a label of  no definitive cure  on chronic diseases generates many myths and misconceptions .", "these misconceptions are affected by socio - demographic factors and are propagated by opportunists who take advantage of patients to market their products .", "understanding the myths and misconceptions about a disease , like dm , is important for the provision of excellent care and health education to both patients and healthy individuals .", "these myths and misconceptions are generally about forbidden foods , the use of herbs , life - style changes , side - effects of treatment and so - called  dependence on medicines .", " they usually interfere with self - management plans for diabetic patients leading to undesirable complications .", "the frequency of common misconceptions reported by other studies is more or less similar to what our report presents .", "a previous study in eastern saudi arabia that was carried out on adult male attendees of primary health centers found misconceptions about the etiology of diabetes in 21.2% of those studied , about general concepts of diabetes in 13.8% and diet in 10.7% of the patients .", "however , our study population was different , in that all of them were diabetics .", "a study in western saudi arabia reported that the top misconceptions that diabetic patients had were that  oral medications might be more effective than insulin ,   medications might cause habituation and serious complications ,   the efficacy of medications depends on their cost ,   cure is expected following a short course of treatment  and  a diabetic could eat anything as long as medications were taken . ", "the prevailing myths reported in the population of qassim region of saudi arabia were that  consuming sugar results in diabetes , ", " diabetics should avoid sweets ,   some type of dates do not increase sugar level ,   honey intake does n't increase sugar level ,  and  diabetes in its early stages can affect sexual performance .  determinants of myths and misconceptions are the factors that are directly related to generation or propagation of these wrong beliefs .", "knowledge of the determinants of any misconception , attitude or behavior is very important to the management of disease .", "our results indicate that around 42.5% of the subjects had a moderate to high misconception score , indicating that there was much room for improvement in our patients ' education .", "the mean misconception score was 10.29  4.92 , while 38.5% had moderate ( 1324 ) and 4% high ( 24 - 36 ) misconception scores .", "the factors which had a significant association with high misconception scores were female gender , rural residence , little or no education , < 5 years or > 15 years since diagnosis , no self - monitoring , poor diet control and no prior education about diabetes . the relationships of different determinants of misconceptions studied in our study are discussed below .", "female gender was found to be significantly ( p < 0.05 ) associated with moderate to high misconception score .", "this was not a totally unexpected finding due to social norms of a conservative society with significantly less exposure of women to information .", "this finding gave us a clear task to target our female patients to provide them with the correct knowledge about diabetes and its management .", "a study carried out on female school teachers of alkhobar showed that their understanding of diabetes was inadequate .", "similarly , a sudanese study also found poor compliance to therapy , poor glycemic control and poor knowledge about diabetes in women as compared to men .", "this association was also reported in misconception studies carried out in new york and in india . however , a study carried out in qasim did not find gender to be associated with myths and misconceptions .", "the proportion of patients from rural areas was very low in our study as a result of the saudi government 's policy of ensuring that patients access health facilities nearest to their homes .", "however , the proportion of patients with moderate to high misconception scores was higher in patients from rural areas .", "this shows that certain socio - demographic factors render people from those areas more vulnerable to misconceptions .", "however , there are other associated factors that can modify beliefs of urban populations as well . in a study carried out in a low income population of new york ,", "a survey carried out in the 4 largest city of india , chennai , on around 26000 subjects concluded that even though there had been diabetes education campaigns from as far back as 1948 , the level of lack of knowledge and misconceptions was unacceptable .", "as expected , the proportion of subjects with moderate to high misconception was highest in the illiterate group , and this decreased as the level of education rose .", "the study by sabra et al . in eastern saudi arabia found high misconception scores in 23.5% of illiterate or those who could only read - and - write .", "a study in new york found that participants with less than a high school education were more likely to have misconceptions .", "a study of myths and misconceptions in the qasim region also found significant differences in responses according to educational status .", "this necessitates the identification of the less educated from the beginning in order to design special educational programs that suit their respective levels of education .", "time since diagnosis was found to be significantly associated ( p < 0.05 ) with misconception scores . it also had a high predictive value for the misconception score .", "patients who had been diagnosed with diabetes > 15 years before were more prone to have a moderate to high misconception score , and the chances of having misconceptions decreased in categories as times since diagnosis lessened", ". this might be due to better current diabetes awareness programs as well as the ease of access to information for freshly diagnosed diabetics who are curious .", "the knowledge of the subjects presenting for the 1 time has generally been reported to be inadequate and other studies have indicated this relationship .", "the patients who reported that they were controlling their diabetes by self - monitoring of blood glucose had low misconception scores .", "this group comprised 62% of the total number of patients , about two - thirds of whom had low misconception scores . on the other hand , two - thirds of those who were not self - monitoring had moderate to high misconception scores .", "this shows that making the patient take charge of his or her diabetes control develops an interested attitude .", "in addition , a clear picture of alterations in blood sugar by medicines , diet control and life - style changes help in removing of unfounded baseless misconceptions . no other study has reported this association . however", ", a study from western saudi arabia has shown an association of  discontinuity of treatment  with a high level of misconception .", "noncompliance to the recommended diet control was found to be a major determinant of misconceptions .", "surprisingly , most of patients with the highest misconception scores ( 7 out of 8) reported to have little or no diet control .", "this reflects a general carefree attitude of these patients to all aspects of life - style changes associated with the management of diabetes .", "however , most of the patients using the recommended diet control were found to have less misconception scores .", "this suggests that we must identify patients with a carefree attitude from the beginning and design proper education programs , with psychotherapy sessions if possible for them .", "the majority of the patients ( 83.5% ) reported some formal instruction by health care professionals about diabetes and its management .", "as expected most of the subjects who had undergone a proper education about diabetes ( presentation , management and life - style changes ) had low misconception scores .", "not surprisingly , this was the most significant single determinant of removing misconceptions about diabetes .", "the results also indicated that there was room for improvement in the education of the large proportion of patients with moderate to high misconception scores . in our study , family history of diabetes , type of diabetes and type of treatment", "were not found to have any significant association with misconception scores . as in our study", "similarly , the age of our subjects did not show a significant association with misconception score . however , the proportion of subjects with moderate to high misconception scores was highest in the categories of the youngest and the oldest patients ( < 20 and > 60 years ) .", "though not statistically significant , it still shows that we must make an extra effort to educate these two vulnerable age groups about their disease .", "suboptimal knowledge and beliefs are potentially modifiable and are logical targets for educational interventions to improve diabetes self - management .", "knowledge of all the identified determinants of moderate to high misconception scores will help in streamlining the awareness programs for patients in accordance with these factors .", "similarly , it will act as a guideline for other units in our region to develop better patient education programs .", "if the patients are given proper guidance and education on diabetes care , there would be a significant improvement in their life - style which would in turn help in producing good glycemic control .", "we conclude that myths and misconceptions about diabetes and its management are common in our patients .", "the strongest determinants of the misconceptions in our study population are female gender , rural area of residence , illiteracy or little education , < 5 or > 15 years since diagnosis , no self - monitoring of blood glucose , poor diet control and no education about diabetes .", "therefore , diabetes educational programs should focus on individuals with one or more of these predictors ."], "abstract_text": ["<S> objective : to identify the determinants of misconceptions about diabetes in patients registered with a diabetes clinic at a tertiary care hospital in eastern saudi arabia.materials and methods : this cross - sectional survey was carried out at a diabetes clinic of a tertiary care hospital in eastern saudi arabia , from january to december 2012 . </S>", "<S> a total of 200 diabetic patients were interviewed using a questionnaire comprising 36 popular misconceptions . </S>", "<S> the total misconception score was calculated and categorized into low ( 0 - 12 ) , moderate ( 13 - 24 ) and high ( 25 - 36 ) scores . </S>", "<S> the association of misconception score with various potential determinants was calculated using chi - square test . </S>", "<S> step - wise logistic regression was applied to the variables showing significant association with the misconception score in order to identify the determinants of misconceptions.results:the mean age was 39.62  16.7 and 112 ( 56% ) subjects were females . </S>", "<S> type 1 diabetics were 78 ( 39% ) , while 122 ( 61% ) had type 2 diabetes . </S>", "<S> insulin was being used by 105 ( 52.5% ) , 124 ( 62% ) were self - monitoring blood glucose and 112 ( 56% ) were using diet control . </S>", "<S> formal education on diabetes awareness had been received by 167 ( 83.5% ) before the interview . </S>", "<S> the mean misconception score was 10.29  4.92 with 115 ( 57.5% ) subjects had low misconception scores ( < 12/36 ) . on the chi - square test , female gender , rural area of residence , little or no education , < 5 or > 15 years since diagnosis , no self - monitoring , no dietary control and no diabetes education were all significantly ( p < 0.05 ) associated with higher misconception scores . </S>", "<S> step - wise logistic regression suggested that diabetes education , gender , education and time since diagnosis were significant ( p < 0.05 ) predictors of misconception scores.conclusions:the strongest determinants of misconceptions about diabetes in our study population were female gender , rural area of residence , illiteracy or little education , < 5 or > 15 years since diagnosis , no self - monitoring , no diet control and no education about diabetes . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "DISCUSSION", "CONCLUSION"], "sections": [["these misconceptions have a significant influence on the day - to - day life including the search for treatment in times of illness .", "a number of studies have reported that misconceptions and inadequate knowledge present significant barriers to effective management of diabetes .", "it is imperative for physicians to understand myths and misconceptions in a particular community about a disease to improve patient care , especially when dealing with chronic diseases like diabetes .", "diabetes mellitus ( dm ) is one of the most common noncommunicable diseases in the world and its prevalence is increasing dramatically .", "currently , there are around 285 million diabetic patients around the world , and the numbers are predicted to rise to 439 million by 2030 , with the largest increase in the developing rather than in the developed world .", "its dramatic increase began a few decades ago with the rapid urbanization and development in the country .", "studies in the 1980s showed a trend towards an increase among adult saudis especially females . a large study of saudi patients from 1995 to 2000 revealed prevalence of 23.7% .", "however , a study in 2011 showed a significant rise in prevalence reaching 34.1% in men and 27.6% in women .", "correct knowledge about diabetes and its management has an enormous impact on attitude and practice of diabetic patients .", "a study of diabetic patients in new york , reported that patients with diabetes frequently had beliefs of the disease and medication that were false and even dangerous .", "it is imperative that awareness of diabetes and its correct management be created and various myths and misconceptions surrounding its course and management be removed .", "a few excellent studies about prevailing misconceptions about diabetes have already been carried out in the saudi population , more specifically in female diabetic teachers , in the eastern region , western region and qasim region .", "however , no studies have examined in detail the determinants of the misconceptions of diabetic patients .", "therefore , this study was undertaken to identify the determinants of prevailing misconceptions on diabetes and the management of diabetic patients registered at a diabetes clinic of a tertiary care hospital in the eastern region of saudi arabia ."], ["this cross - sectional study was carried out at the diabetes clinic of a tertiary care hospital , in eastern saudi arabia .", "this clinic has a registered patient population of 2000 diabetic patients . at a confidence level of 95% ,", "response distribution of 50% and accepted margin of error of 10% , we calculated a sample size of 200 .", "a table of random numbers was used to select patients from the medical record numbers of the registered patients of the diabetes clinic .", "the inclusion criteria were all saudi patients who had been registered at the diabetes clinic of the tertiary care hospital in saudi arabia for 6 months or more since diagnosis and on continuous treatment .", "no freshly diagnosed patient was included . in the same vein uncooperative or nonconsenting patients , as well as patients with any illness ( physical or psychological ) that was likely to influence reliable , valid responses to the interviewer 's questions were excluded .", "the same interviewer spoke to all the selected patients on their scheduled follow - up visits after taking their informed consent .", "when a prospective participant refused to take part , the next patient on the list of random numbers was asked until the sample size was completed .", "all attendees were interviewed in standardized conditions with prior information to ensure valid reliable responses .", "the data collection instrument was an interviewer - filled questionnaire prepared in accordance with  brief illness perception questionnaire  and  belief about medicines questionnaire . ", "the demographic and classification data included age categories ( < 20 , 21 - 40 , 41 - 60 or > 60 years ) , gender ( male and female ) , area of residence ( urban or rural ) , education ( primary , middle , high school , graduate and above ) , family history of diabetes ( present or absent ) , type of diabetes ( type 1 or type 2 ) , time since diagnosis ( < 5 , 6 - 10 , 11 - 15 or > 15 years ) , type of treatment ( oral hypoglycemic or insulin ) , self - monitoring ( yes or no ) and diet control as per doctor 's advice ( yes or no ) . in order to establish validity and reliability of the questionnaire ,", "a pilot study was conducted in a sub - sample of attendees who were not included in the study proper .", "necessary changes were made in the questionnaire as well as the interview style as necessary .", "questions on the commonest reported misconceptions relating to etiology , types , pathogenesis , day - to - day life , diet and treatment of diabetes were included after a review of studies from saudi arabia , usa , india , nepal , and pakistan .", "an answer in  yes  to a misconception question was considered a misconception and a score of one was given .", "the total number of misconception questions was 36 ; therefore , the possible maximum score for any patient was 0 - 36 .", "low ( scores 0 - 12 ) , moderate ( scores 13 - 24 ) , and high ( scores of 24 - 36 ) .", "chi - square test was used to determine the association of the socio - demographic variables with the three categories of misconception scores .", "stepwise logistic regression analysis was applied to the socio - demographic variables showing significant association with the misconception score by chi - square test and values of -coefficient , chi - square , odds ratio , and confidence interval were reported ."], ["a total of 200 responses for each item in the questionnaire were recorded from 200 subjects .", "the distribution of subjects in the age groups was 32 ( 16% ) in < 20 years , 62 ( 31% ) in 21 - 40 years , 81 ( 40.5% ) between 41 and 60 years and 25 ( 12.5% ) in > 60 years .", "the subjects from urban areas were 168 ( 84% ) and those from rural areas were 32 ( 16% ) .", "those with the highest level of education were 7 ( 3.5% ) who had up to postgraduation , 36 ( 18% ) were university graduates , 76 ( 38% ) high school , 43 ( 21.5% ) middle , 29 ( 14.5% ) primary and 9 ( 4.5% ) illiterate .", "there was a family history of diabetes in 147 ( 73.5% ) and no history in 53 ( 26.5% ) .", "those with type 1 diabetics were 78 ( 39% ) while 122 ( 61% ) had type 2 diabetes .", "the time since diagnosis was < 5 years in 73 ( 36.5% ) , 6 - 10 years in 72 ( 36% ) , 11 - 15 years in 21 ( 10.5% ) and > 15 years in 34 ( 17% ) subjects .", "the type of treatment was insulin in 105 ( 52.5% ) and oral hypoglycemics in 95 ( 47.5% ) indicating that many type 2 diabetic patients were also on insulin .", "self - monitoring of blood glucose was done by 124 ( 62% ) of the subjects while 76 ( 38% ) did not .", "diet control was used by 112 ( 56% ) subjects while 88 ( 44% ) did not .", "a total of 167 ( 83.5% ) had received formal  diabetes awareness education  while 33 ( 16.5% ) had not .", "the total misconception score was low ( 0 - 12 ) in 115 ( 57.5% ) , moderate ( 13 - 24 ) in 77 ( 38.5% ) and high ( 24 - 36 ) in 8 ( 4% ) respondents ( n = 200 ) .", "table 1 demonstrates the frequency distribution of respondents according to misconceptions about etiology , types and pathogenesis of diabetes .", "the most common misconceptions identified on etiology , types and pathogenesis were that  overweight causes diabetes  ( 89% ) ,  diabetes is only a hereditary disease  ( 80.5% ) and  eating too much sugar causes diabetes  ( 69% ) .", "in addition , this table shows the distribution of subjects with reference to misconceptions about daily life .", "the most frequent misconceptions about the daily life of diabetics were that  diabetics can not lead a normal social life ", "frequency of respondents with misconceptions about etiology , types , pathogenesis and day - to - day life of diabetes ( n=200 ) table 2 illustrates the distribution of respondents according to misconceptions about diet .", "the second misconception on treatment was that  there is no need to take medicines when blood glucose is normal ", "table 3 shows the association of the selected socio - demographic variables on the misconception score .", "it shows that female gender , living in a rural area , little or no education , < 5 or > 15 years since diagnosis , lack of self - monitoring , poor compliance with dietary control and no diabetes education were significantly ( p < 0.05 ) associated with moderate or high misconception score .", "however , age , family history of diabetes , type of diabetes and type of treatment were not significantly associated with the misconception score .", "frequency of respondents with misconceptions about diet and treatment ( n=200 ) association of misconception score category with sociodemographic variables table 4 gives stepwise logistic regression to determine the predictive value of independent variables [ showing significant association as per table 3 ] with the misconception score ( dependent variable ) .", "it indicates that diabetes education , gender , education and time since diagnosis are good predictors of misconception score ."], ["every chronic disease necessitates long - term commitment from the patient , family and health care professionals .", "health care is a full package that includes the proper education of society in general and the patient in particular , about all aspects of the disease in question .", "a label of  no definitive cure  on chronic diseases generates many myths and misconceptions .", "these misconceptions are affected by socio - demographic factors and are propagated by opportunists who take advantage of patients to market their products .", "understanding the myths and misconceptions about a disease , like dm , is important for the provision of excellent care and health education to both patients and healthy individuals .", "these myths and misconceptions are generally about forbidden foods , the use of herbs , life - style changes , side - effects of treatment and so - called  dependence on medicines .", " they usually interfere with self - management plans for diabetic patients leading to undesirable complications .", "the frequency of common misconceptions reported by other studies is more or less similar to what our report presents .", "a previous study in eastern saudi arabia that was carried out on adult male attendees of primary health centers found misconceptions about the etiology of diabetes in 21.2% of those studied , about general concepts of diabetes in 13.8% and diet in 10.7% of the patients .", "however , our study population was different , in that all of them were diabetics .", "a study in western saudi arabia reported that the top misconceptions that diabetic patients had were that  oral medications might be more effective than insulin ,   medications might cause habituation and serious complications ,   the efficacy of medications depends on their cost ,   cure is expected following a short course of treatment  and  a diabetic could eat anything as long as medications were taken . ", "the prevailing myths reported in the population of qassim region of saudi arabia were that  consuming sugar results in diabetes , ", " diabetics should avoid sweets ,   some type of dates do not increase sugar level ,   honey intake does n't increase sugar level ,  and  diabetes in its early stages can affect sexual performance .  determinants of myths and misconceptions are the factors that are directly related to generation or propagation of these wrong beliefs .", "knowledge of the determinants of any misconception , attitude or behavior is very important to the management of disease .", "our results indicate that around 42.5% of the subjects had a moderate to high misconception score , indicating that there was much room for improvement in our patients ' education .", "the mean misconception score was 10.29  4.92 , while 38.5% had moderate ( 1324 ) and 4% high ( 24 - 36 ) misconception scores .", "the factors which had a significant association with high misconception scores were female gender , rural residence , little or no education , < 5 years or > 15 years since diagnosis , no self - monitoring , poor diet control and no prior education about diabetes . the relationships of different determinants of misconceptions studied in our study are discussed below .", "female gender was found to be significantly ( p < 0.05 ) associated with moderate to high misconception score .", "this was not a totally unexpected finding due to social norms of a conservative society with significantly less exposure of women to information .", "this finding gave us a clear task to target our female patients to provide them with the correct knowledge about diabetes and its management .", "a study carried out on female school teachers of alkhobar showed that their understanding of diabetes was inadequate .", "similarly , a sudanese study also found poor compliance to therapy , poor glycemic control and poor knowledge about diabetes in women as compared to men .", "this association was also reported in misconception studies carried out in new york and in india . however , a study carried out in qasim did not find gender to be associated with myths and misconceptions .", "the proportion of patients from rural areas was very low in our study as a result of the saudi government 's policy of ensuring that patients access health facilities nearest to their homes .", "however , the proportion of patients with moderate to high misconception scores was higher in patients from rural areas .", "this shows that certain socio - demographic factors render people from those areas more vulnerable to misconceptions .", "however , there are other associated factors that can modify beliefs of urban populations as well . in a study carried out in a low income population of new york ,", "a survey carried out in the 4 largest city of india , chennai , on around 26000 subjects concluded that even though there had been diabetes education campaigns from as far back as 1948 , the level of lack of knowledge and misconceptions was unacceptable .", "as expected , the proportion of subjects with moderate to high misconception was highest in the illiterate group , and this decreased as the level of education rose .", "the study by sabra et al . in eastern saudi arabia found high misconception scores in 23.5% of illiterate or those who could only read - and - write .", "a study in new york found that participants with less than a high school education were more likely to have misconceptions .", "a study of myths and misconceptions in the qasim region also found significant differences in responses according to educational status .", "this necessitates the identification of the less educated from the beginning in order to design special educational programs that suit their respective levels of education .", "time since diagnosis was found to be significantly associated ( p < 0.05 ) with misconception scores . it also had a high predictive value for the misconception score .", "patients who had been diagnosed with diabetes > 15 years before were more prone to have a moderate to high misconception score , and the chances of having misconceptions decreased in categories as times since diagnosis lessened", ". this might be due to better current diabetes awareness programs as well as the ease of access to information for freshly diagnosed diabetics who are curious .", "the knowledge of the subjects presenting for the 1 time has generally been reported to be inadequate and other studies have indicated this relationship .", "the patients who reported that they were controlling their diabetes by self - monitoring of blood glucose had low misconception scores .", "this group comprised 62% of the total number of patients , about two - thirds of whom had low misconception scores . on the other hand , two - thirds of those who were not self - monitoring had moderate to high misconception scores .", "this shows that making the patient take charge of his or her diabetes control develops an interested attitude .", "in addition , a clear picture of alterations in blood sugar by medicines , diet control and life - style changes help in removing of unfounded baseless misconceptions . no other study has reported this association . however", ", a study from western saudi arabia has shown an association of  discontinuity of treatment  with a high level of misconception .", "noncompliance to the recommended diet control was found to be a major determinant of misconceptions .", "surprisingly , most of patients with the highest misconception scores ( 7 out of 8) reported to have little or no diet control .", "this reflects a general carefree attitude of these patients to all aspects of life - style changes associated with the management of diabetes .", "however , most of the patients using the recommended diet control were found to have less misconception scores .", "this suggests that we must identify patients with a carefree attitude from the beginning and design proper education programs , with psychotherapy sessions if possible for them .", "the majority of the patients ( 83.5% ) reported some formal instruction by health care professionals about diabetes and its management .", "as expected most of the subjects who had undergone a proper education about diabetes ( presentation , management and life - style changes ) had low misconception scores .", "not surprisingly , this was the most significant single determinant of removing misconceptions about diabetes .", "the results also indicated that there was room for improvement in the education of the large proportion of patients with moderate to high misconception scores . in our study , family history of diabetes , type of diabetes and type of treatment", "were not found to have any significant association with misconception scores . as in our study", "similarly , the age of our subjects did not show a significant association with misconception score . however , the proportion of subjects with moderate to high misconception scores was highest in the categories of the youngest and the oldest patients ( < 20 and > 60 years ) .", "though not statistically significant , it still shows that we must make an extra effort to educate these two vulnerable age groups about their disease .", "suboptimal knowledge and beliefs are potentially modifiable and are logical targets for educational interventions to improve diabetes self - management .", "knowledge of all the identified determinants of moderate to high misconception scores will help in streamlining the awareness programs for patients in accordance with these factors .", "similarly , it will act as a guideline for other units in our region to develop better patient education programs .", "if the patients are given proper guidance and education on diabetes care , there would be a significant improvement in their life - style which would in turn help in producing good glycemic control ."], ["we conclude that myths and misconceptions about diabetes and its management are common in our patients .", "the strongest determinants of the misconceptions in our study population are female gender , rural area of residence , illiteracy or little education , < 5 or > 15 years since diagnosis , no self - monitoring of blood glucose , poor diet control and no education about diabetes .", "therefore , diabetes educational programs should focus on individuals with one or more of these predictors ."]]}
{"article_id": "PMC3743341", "article_text": ["thoracic injury can result in a wide range of clinical manifestations depending on the structures involved like chest wall , diaphragm , mediastinum , trachea , lungs parenchyma etc .", "the non penetrating trauma to the lungs manifests as contusions , lacerations of pulmonary parenchyma , pneumatocele formation , hematomas , and fractures of trachea and bronchi .", "while pulmonary contusion is commonly associated with blunt thoracic trauma , appearance of cavitary lesions variably described as traumatic pulmonary pesudocysts , ( tpp ) , traumatic pneumatoceles , traumatic lung cysts are rare , developing in less than 3% of patients with pulmonary parenchymal injuries .", "a 26 year old unmarried male was referred to us with history of left sided chest pain for two days .", "he had consulted at local hospital immediately after trauma . a skiagram chest taken there [ figure 1 ] showed non homogenous opacity with central lucency in the region of left hilum . on examination ,", "the patient gave history of trauma - a tractor wheel hitting him on the anterior chest wall , while he was lying down ; following which he developed the pain .", "there was no history of breathlessness , fever , cough , hemoptysis , cough or expectoration .", "there was no history suggestive of bronchial asthma or exposure to pets at home or work place .", "the margins of the lesion were now thinned out and smooth [ figure 2 ] .", "chest radiograph on day 1 - pa view showing non homogenous opacity with central translucency near left hilum day 3 - chest radiograph pa view showing multiple cavities near left hilum his sputum for afb was negative .", "other lab investigation revealed haemoglobin 14.4 , leukocyte count 7900 , and differential count of 78 % polymorphs and 19% lymphocytes .", "his serum was non reactive for hiv antibodies . a ct scan of thorax done on 03 - 09 - 2011 showed a single smooth walled cavity abutting left chest wall with surrounding areas of ground glass haziness , probably suggestive of lung contusion [ figure 3 ] . computed tomogram of chest showing smooth walled cavity in left upper lobe with surrounding ground glass opacity of lung contusion fibre optic bronchoscopy", "was performed to achieve a microbiological diagnosis and to inspect the trachea - bronchial tree in view of previous reports being inconclusive .", "bronchoscopy revealed a bleeding spot in left main bronchus , but no active bleeding was seen .", "bronchial brushings taken from left upper lobe were negative for malignant cells and afb , and the lavage fluid also was sterile .", "in view of the patient 's history of ancedent recent trauma , negative past and present history for any infectious process , and negative microbiological reports , a diagnosis of post traumatic pulmonary pseudo cyst ( tpp ) was made .", "serial follow up chest skiagram showed rapid resolution of the cavities [ figure 4 ] without any antibiotics , thus confirming our diagnosis of tpp .", "among the various names given to the cavitary appearance of pulmonary lacerations , the term traumatic pulmonary pseudo cysts ( tpp ) appears to be the best nomenclature because the wall of these lesions are formed by the inter lobar interstitial connective tissue and shows no epithelial lining or bronchial wall elements .", "the rarity of the entity can be estimated by the fact that there have been only around 10 large case series with 8 or more patients published since 1967 .", "tpp following blunt or non penetrating trauma develops through a mechanism that allows transmission of high compressive forces to the lung parenchyma .", "retraction of normal lung elastic tissue from contusion induced cavities permits the escape of air and fluid into it .", "laceration of pulmonary parenchyma and appearance of cavity may also occur if there is closure of the glottis at the moment of injury which may prevent fast exit of the air from compressed lung segment .", "the parenchyma and/or interstitium get lacerated in a bursting manner resulting in a cavity formation .", "resolution of a pulmonary hematoma or drainage into a bronchus may result in development of tpp at a later stage and it is called secondary tpp .", "tpp can occur at any age , but they are more often seen in children and young adults , probably because of greater compliance of chest wall which permits a larger transmission of force of impact to the parenchyma .", "sorsdahl and powell reported that 85% of the patients with tpp were under the age of 30 years with male predominance .", "impact velocity and degree of chest wall displacement may play an important role in the development of tpp after blunt trauma to the chest .", "a high velocity impact causes peripheral tpps while a low velocity high displacement impact produces central parenchymal and major bronchial disruption .", "tpp is a clinical entity that manifests itself with minor clinical and major radiological signs .", "the usual clinical manifestations include hemoptysis , occurring in about half of the cases , cough , dyspnoea , chest pain , fever and leucocytosis .", "tpps may appear immediately or within a few hours after injury and their sizes range from 2 to 14 cm in diameter tpp can be differentiated from cavitating hematomas on the basis of radiographic appearance of air within the tpp 's within 48 hours .", "tpps may be identifiable on chest radiography but ct scans are superior for detecting them .", "unlike other cystic lesions and cavities , the size , shape , and nature of wall of tpps change relatively quickly . therefore", ", serial skiagrams of chest , done over several days can help to differentiate pseudo cysts from other lesions .", "found a greater resolution time in blood filled tpps and those with > 2 cm in size .", "they may be seen on the site of injury or on the opposite side secondary to countre coup effects .", "the majority of tpps are found in the lower lobes , the ct appearance of single or multiple thin walled cystic lesions with air space consolidation of the surrounding lung parenchyma in the backdrop of antecedent trauma is diagnostic .", "differential diagnosis includes ruptured oesophagus , or herniation of viscera , post pneumonia pneumatocele , tuberculosis cavity , cavitating bronchial carcinoma , lung abscess , bronchogenic cysts , and pulmonary sequestration .", "a history of trauma , rapid sequential changes over days on chest skiagram , and presence of contusion on the base of lesion usually delineates any confusion . if the cavitary lesion does not decrease with time other aetiology must be considered .", "conservative management is recommended as long as evidence of a decrease in size of the lesion occurs within 6 weeks after trauma in adults and 3 - 4 months in children .", "the use of antibiotics is controversial and may be used more to provide simple reassurance that the pseudo cyst wo nt get infected .", "tpp 's can be complicated and may require surgery . they may rupture and cause a secondary pneumothorax that may require tube thoracostomy .", "the indications for diagnostic and therapeutic bronchoscopy are endobronchial bleeding , thick sputum , large air leak , mediastinal emphysema and lobar collapse .", "the approach to an infected pseudo cyst is similar to that for a lung abscess .", "if an infected pseudo cyst is larger than 2 cm , or there are unremitting signs of sepsis after 72 hours of antibiotics , the pseudo cysts may be drained percutaneously .", "early lobectomy may be considered for complex tpp 's with extensive lung abscess surrounded by necrotic parenchyma , failure of bronchoscopic treatment of massive airway bleeding , infected pseudo cyst more than 6 cm in size , or no response to more conservative treatment .", "video assisted thoracoscopic surgery ( vats ) may be considered for managing a persistent air leak , hemothorax due to pseudo cyst rupture , failure of lung expansion , progressive enlargement of pseudo cyst and compression of lung parenchyma .", "late thoracotomy has been reported ( lobectomy , cystotomy , capitonnage ) up to 6 months after trauma because of pneumonic infiltration and persistent cavitary size .", "our patient presented with few abrasions on anterior chest wall and history of blunt trauma to chest wall of one day duration .", "there was no previous history of any respiratory illness or unconsciousness during or after trauma .", "the immediate chest skiagram revealed an area of consolidation with central area of lucency around left hilar region .", "there was no associated cough , fever , expectoration , hemoptysis or breathlessness which clinically ruled out an infective process .", "serial skiagrams over next 24 hours showed a rapid development of a cavity which regressed spontaneously and significantly over next 7 days without any antibiotics .", "ct thorax done was also suggestive of tpp , in view of a cavity adjacent to anterior chest wall with surrounding contusion .", "exclusion of other infectious diseases like tuberculosis , lung abscess , by absence of relevant clinical history , sputum negativity on zn staining , sterile pyogenic culture , negative mantoux test , negative microbiology of bal , and cytology of bronchial brushing led us to the diagnosis of tpp which was well supported by the available literature .", "it is generally a self limiting benign condition , diagnosed by excluding other conditions which may present with similar radiological manifestations .", "a typical history of blunt trauma to chest , rapid radiological changes of the cavity and exclusion of other cavitary pulmonary diseases by adequate laboratory work helps in diagnosing this rare condition .", "the disease at times might become complicated and becomes life threatening ; hence , should be closely monitored and followed up ."], "abstract_text": ["<S> blunt thoracic trauma manifests in various ways , depending on the structures injured and type of injury . commonly manifested as parenchymal contusion , at times , pseudacavitation may also been seen on the chest x ray . </S>", "<S> they are to be differentiated from other causes of pulmonary cavitations which are often done based on history . </S>", "<S> the so called pulmonary pseudo cysts usually have a benign course and needs only observation . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "REVIEW OF LITERATURE", "DISCUSSION", "CONCLUSION"], "sections": [["thoracic injury can result in a wide range of clinical manifestations depending on the structures involved like chest wall , diaphragm , mediastinum , trachea , lungs parenchyma etc .", "the non penetrating trauma to the lungs manifests as contusions , lacerations of pulmonary parenchyma , pneumatocele formation , hematomas , and fractures of trachea and bronchi .", "while pulmonary contusion is commonly associated with blunt thoracic trauma , appearance of cavitary lesions variably described as traumatic pulmonary pesudocysts , ( tpp ) , traumatic pneumatoceles , traumatic lung cysts are rare , developing in less than 3% of patients with pulmonary parenchymal injuries ."], ["a 26 year old unmarried male was referred to us with history of left sided chest pain for two days .", "he had consulted at local hospital immediately after trauma . a skiagram chest taken there [ figure 1 ] showed non homogenous opacity with central lucency in the region of left hilum . on examination ,", "the patient gave history of trauma - a tractor wheel hitting him on the anterior chest wall , while he was lying down ; following which he developed the pain .", "there was no history of breathlessness , fever , cough , hemoptysis , cough or expectoration .", "there was no history suggestive of bronchial asthma or exposure to pets at home or work place .", "the margins of the lesion were now thinned out and smooth [ figure 2 ] .", "chest radiograph on day 1 - pa view showing non homogenous opacity with central translucency near left hilum day 3 - chest radiograph pa view showing multiple cavities near left hilum his sputum for afb was negative .", "other lab investigation revealed haemoglobin 14.4 , leukocyte count 7900 , and differential count of 78 % polymorphs and 19% lymphocytes .", "his serum was non reactive for hiv antibodies . a ct scan of thorax done on 03 - 09 - 2011 showed a single smooth walled cavity abutting left chest wall with surrounding areas of ground glass haziness , probably suggestive of lung contusion [ figure 3 ] . computed tomogram of chest showing smooth walled cavity in left upper lobe with surrounding ground glass opacity of lung contusion fibre optic bronchoscopy", "was performed to achieve a microbiological diagnosis and to inspect the trachea - bronchial tree in view of previous reports being inconclusive .", "bronchoscopy revealed a bleeding spot in left main bronchus , but no active bleeding was seen .", "bronchial brushings taken from left upper lobe were negative for malignant cells and afb , and the lavage fluid also was sterile .", "in view of the patient 's history of ancedent recent trauma , negative past and present history for any infectious process , and negative microbiological reports , a diagnosis of post traumatic pulmonary pseudo cyst ( tpp ) was made .", "serial follow up chest skiagram showed rapid resolution of the cavities [ figure 4 ] without any antibiotics , thus confirming our diagnosis of tpp ."], ["among the various names given to the cavitary appearance of pulmonary lacerations , the term traumatic pulmonary pseudo cysts ( tpp ) appears to be the best nomenclature because the wall of these lesions are formed by the inter lobar interstitial connective tissue and shows no epithelial lining or bronchial wall elements .", "the rarity of the entity can be estimated by the fact that there have been only around 10 large case series with 8 or more patients published since 1967 .", "tpp following blunt or non penetrating trauma develops through a mechanism that allows transmission of high compressive forces to the lung parenchyma .", "retraction of normal lung elastic tissue from contusion induced cavities permits the escape of air and fluid into it .", "laceration of pulmonary parenchyma and appearance of cavity may also occur if there is closure of the glottis at the moment of injury which may prevent fast exit of the air from compressed lung segment .", "the parenchyma and/or interstitium get lacerated in a bursting manner resulting in a cavity formation .", "resolution of a pulmonary hematoma or drainage into a bronchus may result in development of tpp at a later stage and it is called secondary tpp .", "tpp can occur at any age , but they are more often seen in children and young adults , probably because of greater compliance of chest wall which permits a larger transmission of force of impact to the parenchyma .", "sorsdahl and powell reported that 85% of the patients with tpp were under the age of 30 years with male predominance .", "impact velocity and degree of chest wall displacement may play an important role in the development of tpp after blunt trauma to the chest .", "a high velocity impact causes peripheral tpps while a low velocity high displacement impact produces central parenchymal and major bronchial disruption .", "tpp is a clinical entity that manifests itself with minor clinical and major radiological signs .", "the usual clinical manifestations include hemoptysis , occurring in about half of the cases , cough , dyspnoea , chest pain , fever and leucocytosis .", "tpps may appear immediately or within a few hours after injury and their sizes range from 2 to 14 cm in diameter tpp can be differentiated from cavitating hematomas on the basis of radiographic appearance of air within the tpp 's within 48 hours .", "tpps may be identifiable on chest radiography but ct scans are superior for detecting them .", "unlike other cystic lesions and cavities , the size , shape , and nature of wall of tpps change relatively quickly . therefore", ", serial skiagrams of chest , done over several days can help to differentiate pseudo cysts from other lesions .", "found a greater resolution time in blood filled tpps and those with > 2 cm in size .", "they may be seen on the site of injury or on the opposite side secondary to countre coup effects .", "the majority of tpps are found in the lower lobes , the ct appearance of single or multiple thin walled cystic lesions with air space consolidation of the surrounding lung parenchyma in the backdrop of antecedent trauma is diagnostic .", "differential diagnosis includes ruptured oesophagus , or herniation of viscera , post pneumonia pneumatocele , tuberculosis cavity , cavitating bronchial carcinoma , lung abscess , bronchogenic cysts , and pulmonary sequestration .", "a history of trauma , rapid sequential changes over days on chest skiagram , and presence of contusion on the base of lesion usually delineates any confusion . if the cavitary lesion does not decrease with time other aetiology must be considered .", "conservative management is recommended as long as evidence of a decrease in size of the lesion occurs within 6 weeks after trauma in adults and 3 - 4 months in children .", "the use of antibiotics is controversial and may be used more to provide simple reassurance that the pseudo cyst wo nt get infected .", "tpp 's can be complicated and may require surgery . they may rupture and cause a secondary pneumothorax that may require tube thoracostomy .", "the indications for diagnostic and therapeutic bronchoscopy are endobronchial bleeding , thick sputum , large air leak , mediastinal emphysema and lobar collapse .", "the approach to an infected pseudo cyst is similar to that for a lung abscess .", "if an infected pseudo cyst is larger than 2 cm , or there are unremitting signs of sepsis after 72 hours of antibiotics , the pseudo cysts may be drained percutaneously .", "early lobectomy may be considered for complex tpp 's with extensive lung abscess surrounded by necrotic parenchyma , failure of bronchoscopic treatment of massive airway bleeding , infected pseudo cyst more than 6 cm in size , or no response to more conservative treatment .", "video assisted thoracoscopic surgery ( vats ) may be considered for managing a persistent air leak , hemothorax due to pseudo cyst rupture , failure of lung expansion , progressive enlargement of pseudo cyst and compression of lung parenchyma .", "late thoracotomy has been reported ( lobectomy , cystotomy , capitonnage ) up to 6 months after trauma because of pneumonic infiltration and persistent cavitary size ."], ["our patient presented with few abrasions on anterior chest wall and history of blunt trauma to chest wall of one day duration .", "there was no previous history of any respiratory illness or unconsciousness during or after trauma .", "the immediate chest skiagram revealed an area of consolidation with central area of lucency around left hilar region .", "there was no associated cough , fever , expectoration , hemoptysis or breathlessness which clinically ruled out an infective process .", "serial skiagrams over next 24 hours showed a rapid development of a cavity which regressed spontaneously and significantly over next 7 days without any antibiotics .", "ct thorax done was also suggestive of tpp , in view of a cavity adjacent to anterior chest wall with surrounding contusion .", "exclusion of other infectious diseases like tuberculosis , lung abscess , by absence of relevant clinical history , sputum negativity on zn staining , sterile pyogenic culture , negative mantoux test , negative microbiology of bal , and cytology of bronchial brushing led us to the diagnosis of tpp which was well supported by the available literature ."], ["it is generally a self limiting benign condition , diagnosed by excluding other conditions which may present with similar radiological manifestations .", "a typical history of blunt trauma to chest , rapid radiological changes of the cavity and exclusion of other cavitary pulmonary diseases by adequate laboratory work helps in diagnosing this rare condition .", "the disease at times might become complicated and becomes life threatening ; hence , should be closely monitored and followed up ."]]}
{"article_id": "PMC4273007", "article_text": [". however , cerebral phaeohyphomycosis ( cp ) caused by darkly pigmented fungi appears to be a common exception to this rule because about one - half of this fungal infection occurred in patients with no underlying disease or risk factors .", "cp is a very rare cause of brain abscess , but is often a fatal disease regardless of immune status14,17,23 ) .", "the authors illustrate a 75-year - old , immunocompetent male patient who had a single brain abscess from dematiaceous fungi . to the authors ' knowledge , this is the first case of cp in korea .", "a 75-year - old male , a resident of a rural area and a farmer by occupation , visited our outpatient clinic with the symptoms of poor cognition and memory decline over 2 weeks .", "he denied any history of fever , headache , blurred vision , vomiting or seizure .", "there were no laboratory abnormalities including leukocytosis or c - reactive protein rising . upon the neurologic examination", ", he was conscious and there were no neurologic deficits except intermittent expressive dysphasia and disorientation .", "brain magnetic resonance imaging ( mri ) was performed because of suspicion of some type of dementia .", "it showed a 20 mm sized nodular enhancing mass with peritumoral edema in the left frontal lobe .", "high - grade glioma or metastatic tumor was initially presumed based on his age and progressive symptoms .", "the lesion appeared to be white and took the form of a relatively hard mass with a clear boundary , permitting radical excision of the mass ( fig .", ". septated hyphae and melanin pigments were confirmed at fontana - masson stain consistent with cp ( fig .", "the patient was started on intravenous amphotericin b at a dose of 68 mg daily .", "after 10 days , he was switched to 270 mg of intravenous voriconazole twice a day because of the elevation of serum creatinine .", "he took the injection for 8 weeks , followed by oral voriconazole 200 mg twice a day for 2 months .", "a follow - up brain mri 3 weeks after surgical excision demonstrated a significant resolution of the edema", ". ongoing resolution of the lesion was found on the latest follow - up mri ( fig .", "he showed dramatic improvement in his symptoms including disorientation and memory disturbance after completion of surgery and antifungal therapy .", "cp is a rare infection caused by darkly pigmented fungi , namely dematiaceous fungi23 ) .", "dematiaceous fungi represent a group of filamentous molds that contain melanin pigment in their cell walls3,6,14,17 ) .", "rhinocladiella mackenziei ( formerly ramichloridium mackenziei ) is the second most common cause of cp , which is exclusively endemic in the middle east area17,22 ) .", "because of this , occupational predisposition has been reported in agricultural workers , especially farmers due to risk of soil exposure17,23 ) .", "cp commonly occurs in the second and third decades of life with male predominance , except rhinocladiella mackenziei which affects adults with a median age of 62 years without male predominance3,13,23 ) .", "the most unique characteristic of cp is its occurrence irrelevant to the immune status of the host15,17,19,23 ) .", "even though immunodeficiency may play a role as a risk factor , there are many reports of this infection in immunocompetent individuals similar to the patient in this report12,15,17,19,25 ) .", "the portal entry to brain is unclear , although several possible routes have been suggested , such as hematogenous dissemination of inhaled spores or accidental skin inoculation as well as direct extension from adjacent paranasal sinuses or ears2,6,10,14,15,19,22,23 ) .", "pathogenesis of cp is associated with the presence of melanin as a virulence factor that provides advantages in evading host defense and crossing the blood - brain barrier by binding to hydrolytic enzyme14,16,17 ) .", "clinical spectrum of phaeohyphomycosis was listed as a variable , ranging from solitary subcutaneous nodules to a life - threatening infection5,11,16,17,18 ) . in the central nervous system ( cns )", "about 70 - 80% of cases typically manifest as a single brain abscess particularly on the frontal lobe ( 52% ) like in our case , while multiple brain abscesses can be seen in immunocompromised patients3,6,17,19 )", ". the diagnosis of cp can be difficult because dematiaceous fungi are often considered contaminants when identified in culture .", "furthermore , the pathogen can not always be cultured and isolated from the serum or cerebrospinal fluid ( csf)12,21,24 ) .", "no molecular techniques are available to speedily identify these fungi even to the genus level17 ) .", "only the tissue examination can be useful to identify irregularly swollen hyphae with yeast - like structure and to confirm the presence of dematiaceous hyphae in melanin - specific fontana - masson stain14,17 ) .", "unfortunately in this case , fungus was not identified in the culture of surgical specimen , therefore , the species that causes cp could not be detected .", "meanwhile , the brain mri reveals a ring - enhancing lesion with a low - attenuation core , suggesting the presence of necrosis or pus10,19 ) . in cases where high - grade glioma or metastasis is mimicked by irregular and variably contrast - enhancing lesions ,", "imaging findings of this patient were more suggestive of a glioma than an abscess , because nodular heterogeneity on contrast injection mimicked the images seen in high - grade tumors .", "consequently , surgical biopsy is essential for the diagnosis of cp . because of the rarity of the cases", "complete excision of brain lesions may provide better results than simple aspiration unless the lesion is multiple or is located within the eloquent area of the brain3,17 ) .", "antifungal agents are generally used in combination of amphotericin b , 5-flucytosine and itraconazole because it is associated with improved survival rates3,14,17,20 ) .", "voriconazole can be used as alternative to itraconazole because of its good penetration into both csf and brain tissue17,22 ) .", "duration of taking the medications is still unknown because most reported patients expired during treatment except a few survivors who received voriconazole for about 12 months8,19 ) .", "in addition , posaconazole may be a potent drug when pathogen is rhinocladiella mackenziei1,3,8 ) . in this case ,", "amphotericin b has fatal side effects such as nephrotoxicity , therefore , close observation on kidney function is needed .", "mortality rate approaches 100% in untreated patients , while that of treated cases as high as 65% to 73% despite the aggressive treatment6,9,10,17,19,23 ) .", "interestingly , mortality rate did not differ significantly between immunocompromised and immunocompetent patients ( 75% vs. 71%)12,17 ) .", "fortunately , the patient reported here had a good response to surgery and chemotherapy and showed fine recovery without any sequela .", "solitary lesion and the good general condition of the patient , together with an aggressive therapeutic approach , are therefore inferred to contribute to a favorable outcome .", "further studies are necessary to find more potentially useful antifungal regimen for these refractory infections and to investigate more detailed pathophysiology and prognostic factors to increase the survival rate .", "cp is rare disease , but challenging one with high mortality rate , particularly when the cns is affected .", "as shown in this report , complete resection and adequate antifungal therapy are the most recommended modality for patients with cp - related abscess to this time ."], "abstract_text": ["<S> cerebral phaeohyphomycosis ( cp ) is a very rare but serious form of central nervous system fungal infection that is caused by dematiaceous fungi . </S>", "<S> it is commonly associated with poor prognosis irrespective of the immune status of the patient . in this study , the authors describe the first case of cp in korea that occurred in a 75-year - old man without immunodeficiency and showed favorable outcome after surgical excision and antifungal therapy . </S>", "<S> in addition , the authors herein review the literature regarding characteristics of this rare clinical entity with previously reported cases . </S>"], "labels": null, "section_names": ["INTRODUCTION", "CASE REPORT", "DISCUSSION", "CONCLUSION"], "sections": [[". however , cerebral phaeohyphomycosis ( cp ) caused by darkly pigmented fungi appears to be a common exception to this rule because about one - half of this fungal infection occurred in patients with no underlying disease or risk factors .", "cp is a very rare cause of brain abscess , but is often a fatal disease regardless of immune status14,17,23 ) .", "the authors illustrate a 75-year - old , immunocompetent male patient who had a single brain abscess from dematiaceous fungi . to the authors ' knowledge , this is the first case of cp in korea ."], ["a 75-year - old male , a resident of a rural area and a farmer by occupation , visited our outpatient clinic with the symptoms of poor cognition and memory decline over 2 weeks .", "he denied any history of fever , headache , blurred vision , vomiting or seizure .", "there were no laboratory abnormalities including leukocytosis or c - reactive protein rising . upon the neurologic examination", ", he was conscious and there were no neurologic deficits except intermittent expressive dysphasia and disorientation .", "brain magnetic resonance imaging ( mri ) was performed because of suspicion of some type of dementia .", "it showed a 20 mm sized nodular enhancing mass with peritumoral edema in the left frontal lobe .", "high - grade glioma or metastatic tumor was initially presumed based on his age and progressive symptoms .", "the lesion appeared to be white and took the form of a relatively hard mass with a clear boundary , permitting radical excision of the mass ( fig .", ". septated hyphae and melanin pigments were confirmed at fontana - masson stain consistent with cp ( fig .", "the patient was started on intravenous amphotericin b at a dose of 68 mg daily .", "after 10 days , he was switched to 270 mg of intravenous voriconazole twice a day because of the elevation of serum creatinine .", "he took the injection for 8 weeks , followed by oral voriconazole 200 mg twice a day for 2 months .", "a follow - up brain mri 3 weeks after surgical excision demonstrated a significant resolution of the edema", ". ongoing resolution of the lesion was found on the latest follow - up mri ( fig .", "he showed dramatic improvement in his symptoms including disorientation and memory disturbance after completion of surgery and antifungal therapy ."], ["cp is a rare infection caused by darkly pigmented fungi , namely dematiaceous fungi23 ) .", "dematiaceous fungi represent a group of filamentous molds that contain melanin pigment in their cell walls3,6,14,17 ) .", "rhinocladiella mackenziei ( formerly ramichloridium mackenziei ) is the second most common cause of cp , which is exclusively endemic in the middle east area17,22 ) .", "because of this , occupational predisposition has been reported in agricultural workers , especially farmers due to risk of soil exposure17,23 ) .", "cp commonly occurs in the second and third decades of life with male predominance , except rhinocladiella mackenziei which affects adults with a median age of 62 years without male predominance3,13,23 ) .", "the most unique characteristic of cp is its occurrence irrelevant to the immune status of the host15,17,19,23 ) .", "even though immunodeficiency may play a role as a risk factor , there are many reports of this infection in immunocompetent individuals similar to the patient in this report12,15,17,19,25 ) .", "the portal entry to brain is unclear , although several possible routes have been suggested , such as hematogenous dissemination of inhaled spores or accidental skin inoculation as well as direct extension from adjacent paranasal sinuses or ears2,6,10,14,15,19,22,23 ) .", "pathogenesis of cp is associated with the presence of melanin as a virulence factor that provides advantages in evading host defense and crossing the blood - brain barrier by binding to hydrolytic enzyme14,16,17 ) .", "clinical spectrum of phaeohyphomycosis was listed as a variable , ranging from solitary subcutaneous nodules to a life - threatening infection5,11,16,17,18 ) . in the central nervous system ( cns )", "about 70 - 80% of cases typically manifest as a single brain abscess particularly on the frontal lobe ( 52% ) like in our case , while multiple brain abscesses can be seen in immunocompromised patients3,6,17,19 )", ". the diagnosis of cp can be difficult because dematiaceous fungi are often considered contaminants when identified in culture .", "furthermore , the pathogen can not always be cultured and isolated from the serum or cerebrospinal fluid ( csf)12,21,24 ) .", "no molecular techniques are available to speedily identify these fungi even to the genus level17 ) .", "only the tissue examination can be useful to identify irregularly swollen hyphae with yeast - like structure and to confirm the presence of dematiaceous hyphae in melanin - specific fontana - masson stain14,17 ) .", "unfortunately in this case , fungus was not identified in the culture of surgical specimen , therefore , the species that causes cp could not be detected .", "meanwhile , the brain mri reveals a ring - enhancing lesion with a low - attenuation core , suggesting the presence of necrosis or pus10,19 ) . in cases where high - grade glioma or metastasis is mimicked by irregular and variably contrast - enhancing lesions ,", "imaging findings of this patient were more suggestive of a glioma than an abscess , because nodular heterogeneity on contrast injection mimicked the images seen in high - grade tumors .", "consequently , surgical biopsy is essential for the diagnosis of cp . because of the rarity of the cases", "complete excision of brain lesions may provide better results than simple aspiration unless the lesion is multiple or is located within the eloquent area of the brain3,17 ) .", "antifungal agents are generally used in combination of amphotericin b , 5-flucytosine and itraconazole because it is associated with improved survival rates3,14,17,20 ) .", "voriconazole can be used as alternative to itraconazole because of its good penetration into both csf and brain tissue17,22 ) .", "duration of taking the medications is still unknown because most reported patients expired during treatment except a few survivors who received voriconazole for about 12 months8,19 ) .", "in addition , posaconazole may be a potent drug when pathogen is rhinocladiella mackenziei1,3,8 ) . in this case ,", "amphotericin b has fatal side effects such as nephrotoxicity , therefore , close observation on kidney function is needed .", "mortality rate approaches 100% in untreated patients , while that of treated cases as high as 65% to 73% despite the aggressive treatment6,9,10,17,19,23 ) .", "interestingly , mortality rate did not differ significantly between immunocompromised and immunocompetent patients ( 75% vs. 71%)12,17 ) .", "fortunately , the patient reported here had a good response to surgery and chemotherapy and showed fine recovery without any sequela .", "solitary lesion and the good general condition of the patient , together with an aggressive therapeutic approach , are therefore inferred to contribute to a favorable outcome .", "further studies are necessary to find more potentially useful antifungal regimen for these refractory infections and to investigate more detailed pathophysiology and prognostic factors to increase the survival rate ."], ["cp is rare disease , but challenging one with high mortality rate , particularly when the cns is affected .", "as shown in this report , complete resection and adequate antifungal therapy are the most recommended modality for patients with cp - related abscess to this time ."]]}
{"article_id": "PMC3649162", "article_text": ["cephalopelvic disproportion ( cpd ) in labour occurs when there is a mismatch between the size of the fetus and the dimensions of the maternal pelvis .", "the factors which mainly influence the outcome of the delivery can be summarised as the three  ps  of the labour : passageway , passenger , and power of the uterus .", "the passageway component of this trinity has been investigated by pelvimetry which measures the maternal bony pelvic dimensions , with very little emphasis on its shape or pelvic floor muscles . during the last decades , the use of pelvimetry has been discouraged , but at present , no replacing methods to evaluate the maternal pelvis have been introduced .", "the benefits of vaginal deliveries are well known when no risk factors are present   even after previous cesarean section ( cs ) [ 5 , 6 ] . on the other hand , unplanned interventions during labour such as acute or emergency cesarean sections as well as operative vaginal delivery increase both maternal and fetal morbidities   as does a prolonged second stage of the delivery .", "the safety and the accuracy of the measurements obtained in pelvimetry have improved in the era of the mri technology [ 9 , 10 ] .", "it is also in the interest of the mother and her physician to minimize the number of unplanned interventions during labour .", "the purpose of this observational cohort study was to evaluate whether pelvic measurements , especially pelvic outlet , displayed any association with operative vaginal deliveries and the duration of the second stage of the delivery .", "this retrospective study was approved by the ethical committee of north - carelian central hospital .", "it investigated caucasian women , that had been examined by x - ray or mri pelvimetry during 20002008 in north - carelian central hospital .", "eligibility criteria included that pelvimetric and fetal measurements had been recorded . in the operative delivery group ,", "there were no signs of fetal distress in cardiotocography , inertia was not diagnosed , and there was no malpresentation .", "originally , 915 women were screened for possible inclusion , but 429 women were excluded because of breech presentation .", "a total of 486 patients with the fetus in the cephalic presentation were screened in the study , but those 234 women that went through elective or acute cesarean section were excluded from the analysis .", "the clinical indication for pelvimetry was breech presentation , or if the fetus was in cephalic presentation , the indication was suspected cephalopelvic disproportion in clinical examination .", "the findings that referred to cpd in clinical examination were clinically small pelvis , unengaged presentation , or suspected macrosomia .", "pelvimetric measurements were found in all patients , as required by the inclusion criteria . there were 252 participants with fetal cephalic presentation delivered vaginally , of whom 184 women delivered spontaneously and 68 women went through operative vaginal delivery with vacuum extraction . of this latter group of women , in 26 patients , the vacuum extraction was undertaken primarily because of fetal distress and inertia , and these patients were excluded from the final analysis , leaving 42 women in the operative vaginal delivery group .", "thus , the total number of participants evaluated in the final stage of this study was 226 .", "the obstetric and radiologic data were collected from patients ' medical records by the author ( uk ) and transferred into a commercially available worksheet ( excel , microsoft 2003 , ireland ) .", "the following pelvimetric parameters were recorded : in the pelvic inlet , anteroposterior ( conjugata vera ) and transverse diameters and in outlet , interspinous diameter and sagittal diameter from the surface of the pubic symphysis to the surface of the sacrum measured at the spinous level .", "pelvic inlet and outlet circumferences were calculated from the pelvic anteroposterior and transverse diameters using the formula ( ap + dt  1.57 ) .", "until the year of 2003 , all pelvimetries were performed with an x - ray technique , and from the year 2004 onwards , they were performed with magnetic resonance imaging ( mri ) . during the transition period", ", both x - ray and mri pelvimetries were performed to verify the repeatability of the measurement results . already at the beginning of 1990 , in order to minimize the variability in pelvimetric measurements , they were centralized so that instead of being conducted by several radiologists , they were conducted by trained obstetricians . when the mri pelvimetry was taken into clinical practice , there was one radiologist with previous experience of mri pelvimetry , and during a two - year period ( 20042006 ) , three radiologists and further three obstetricians were also trained to measure the images . in this evaluation of the diagnostic accuracy of the pelvimetry in vaginal deliveries ,", "patients were divided into subgroups according to the size of the fetus and also by the parity to evaluate the variability reflecting differences in patient groups . for statistical analysis , we used spss 17.0 ( spss inc . , 2009 ,", "receiver operating characteristic ( roc )   curves were established , and the area under curve ( auc ) values with significances were calculated .", "pelvimetric measurements were found in all patients , as required by the inclusion criteria . in the spontaneous vaginal delivery group ,", "most of the nulliparous patients were sent to maternity clinics consultation because of suspected disproportion . in the multiparous group ,", "24% had delivered by cs and 37% by operative vaginal delivery . in the operative vaginal delivery group ,", "of the nine multiparous patients , six had delivered by cs in their previous pregnancy and two had had previous vaginal operative delivery .", "the demographic data of these 226 patients subdivided according to the route of delivery are shown in table 1 .", "patients were further subdivided into two subgroups according to the infant 's weight and the mode of delivery .", "the maternal pelvic inlet and outlet sizes and duration of the first and second stages of the delivery by the mode of delivery in infant weight subgroups are shown in table 2 .", "the mean maternal outlet ( sd ) was 3613 ( 20 )  mm in all , 351 ( 17 )  mm in infant weight < 3700  g , and 369.5 ( 17.7 ) mm in infant weight 3700  g groups .", "no clinically or statistically significant differences in the pelvic sizes were found between the modes of delivery within the subgroups . between the subgroups ,", "the size of the maternal pelvic size was 4%-5% larger in the mothers with infant weight 3700  g. the duration of the second stage of the delivery was 54 minutes longer ( p < 0.01 ) in the operative vaginal delivery group amounting to a 45-minute longer duration ( p = 0.01 ) in infant weight < 3700  g and 62 minutes longer ( p < 0.01 ) in infant weight 3700  g group .", "the one - minute apgar scores were above 8 in all groups with the exception of those with infant weight less than 3700  g in the operative vaginal group , where the mean of apgar score at one minute was 7.8  1.8 .", "the receiver operating characteristic curve analysis for pelvic inlet and outlet as a diagnostic test for the mode of vaginal delivery is shown in figures 2(a ) and 2(b ) .", "the area under the curve ( auc ) for the pelvic inlet was 0.566 with the p value of 0.18 and 95% confidence interval ( ci ) of 0.4650.667 . for pelvic outlet ,", "the main finding of this study was that the maternal bony pelvic dimensions displayed virtually no correlation to the need for operative vaginal deliveries .", "the indications for intervention in vaginal deliveries were chosen on clinical grounds as evidenced by the fact that there was an association between the duration of the second stage of the delivery and the size of the pelvic outlet . if the delivery had reached the second stage , it was probable that the uterine  power  played a more significant role in the overall outcome than either the  passageway  or the  passenger  . on the other hand , pelvic floor muscles , the three - dimensional shape of the bony pelvis , or other soft tissues", "the pelvimetry was performed in most of the patients because of suspected disproportion , or an intervention had been required in a previous labour .", "of those patients that had previous cs and were now exposed to the trial of labour , over 80% delivered spontaneously , and less than 20% required an operative vaginal delivery .", "this is in agreement with previous studies [ 6 , 13 ] . of those women", "that had had a previous operative vaginal labour , only 5% underwent repeated vaginally assisted delivery .", "this may have been due to the fact that the patients were chosen for the trial of labour correctly irrespective of the previous operative delivery .", "there were no statistically significant differences between the size of the maternal inlet or outlet in the spontaneous and the operative vaginal delivery groups .", "when patients were divided into subgroups according to the infant weight , the maternal inlet was 4.7% and the outlet was 5.1% larger in the infant weight 3700  g subgroup among those who delivered spontaneously compared to those vaginally assisted .", "the duration of the first stage of the delivery was longer in the smaller infant group , whereas the second stage was shorter than in the larger infant group .", "in the two delivery subgroups , the duration of the second stage of the delivery was significantly longer in operative vaginal delivery group than in spontaneous vaginal delivery group .", "the apgar scores were acceptable in all delivery groups referring to the fact that both spontaneous and operative vaginal deliveries were uncomplicated and severe shoulder dystocia was not present .", "the apgar 1-minute scores were lower in the operative vaginal delivery group than in spontaneous vaginal delivery group when infant weight was < 3700  g. these results refer to the fact that operative vaginal delivery increases the time of the second stage of the delivery and decreases the apgar 1-minute scores .", "the roc curve analysis for maternal pelvic inlet and outlet revealed that both inlet and outlet had only a fair prognostic value in predicting the mode of the vaginal delivery .", "the poor predictive value of pelvimetry to predict protracted labor is a well - known fact from previous studies , whereas the evidence on the need of vaginal operative deliveries is less extensively evaluated .", "the data did not reveal in detail the fluency of the operative deliveries . as mentioned earlier ,", "therefore , it was not possible to evaluate the influence of the pelvic dimensions on the severe dystocia .", "for that kind of study , the cohort examined here study is too small due to the rare incidence of severe dystocia .", "accordingly , due to the retrospective nature of the study , no blinding was present , and the caregivers were aware of the pelvimetric measurements during the labour .", "furthermore , the study women were at high risk of operative deliveries due to the inclusion criteria , but in clinical care , this would be the group eligible for pelvic assessment before delivery . in conclusion", ", our study revealed that maternal bony pelvic dimensions , either pelvic inlet or outlet , were not associated with the need for operative vaginal deliveries .", "it was more likely that other factors related to the maternal perineal soft tissue , maternal resources , and the passenger were the reasons leading to operative vaginal deliveries .", "subsequently , we can not recommend that caregivers use pelvimetric measurements to predict the outcome of the second stage of the labour .", "observational studies with larger cohorts would be needed , if one wished to investigate whether the maternal bony pelvic size has any effect on severe dystocia .", "in addition , the three - dimensional shape of the bony pelvis and the soft tissues are worth considering in future studies ."], "abstract_text": ["<S> \n objective . to evaluate whether pelvic measurements have any association with operative vaginal deliveries and the duration of the second stage of \n the delivery </S>", "<S> . study design . a retrospective study of pregnant women at an increased risk of fetal - pelvic disproportion during 20002008 in north - carelian central hospital . </S>", "<S> the mode of the vaginal delivery was chosen to represent the reference standard . </S>", "<S> the target condition was spontaneous vaginal delivery . </S>", "<S> patients were divided into subgroups according to the size of the fetus and also by the parity to evaluate the variability reflecting differences in patient groups . </S>", "<S> receiver operating characteristic ( roc ) curves were established . </S>", "<S> results . a total of 226 participants with fetal cephalic presentation delivered vaginally ; of these </S>", "<S> , 184 women delivered spontaneously , and 42 women required operative vaginal delivery with vacuum extraction . </S>", "<S> there were no clinically or statistically significant differences between the size of the maternal pelvic outlet and the different modes of delivery types within these subgroups . </S>", "<S> with respect to the pelvic inlet and outlet , the areas under the curve in roc were 0.566 with the \n p value of 0.18 and 95% confidence interval ( ci ) of 0.4650.667 and 0.573 ( 95% ci : 0.4840.622 ; p = 0.14 ) . </S>", "<S> conclusions . </S>", "<S> the maternal bony pelvic dimensions exhibited virtually no correlation with the need for operative vaginal deliveries . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Conclusions"], "sections": [["cephalopelvic disproportion ( cpd ) in labour occurs when there is a mismatch between the size of the fetus and the dimensions of the maternal pelvis .", "the factors which mainly influence the outcome of the delivery can be summarised as the three  ps  of the labour : passageway , passenger , and power of the uterus .", "the passageway component of this trinity has been investigated by pelvimetry which measures the maternal bony pelvic dimensions , with very little emphasis on its shape or pelvic floor muscles . during the last decades , the use of pelvimetry has been discouraged , but at present , no replacing methods to evaluate the maternal pelvis have been introduced .", "the benefits of vaginal deliveries are well known when no risk factors are present   even after previous cesarean section ( cs ) [ 5 , 6 ] . on the other hand , unplanned interventions during labour such as acute or emergency cesarean sections as well as operative vaginal delivery increase both maternal and fetal morbidities   as does a prolonged second stage of the delivery .", "the safety and the accuracy of the measurements obtained in pelvimetry have improved in the era of the mri technology [ 9 , 10 ] .", "it is also in the interest of the mother and her physician to minimize the number of unplanned interventions during labour .", "the purpose of this observational cohort study was to evaluate whether pelvic measurements , especially pelvic outlet , displayed any association with operative vaginal deliveries and the duration of the second stage of the delivery ."], ["this retrospective study was approved by the ethical committee of north - carelian central hospital .", "it investigated caucasian women , that had been examined by x - ray or mri pelvimetry during 20002008 in north - carelian central hospital .", "eligibility criteria included that pelvimetric and fetal measurements had been recorded . in the operative delivery group ,", "there were no signs of fetal distress in cardiotocography , inertia was not diagnosed , and there was no malpresentation .", "originally , 915 women were screened for possible inclusion , but 429 women were excluded because of breech presentation .", "a total of 486 patients with the fetus in the cephalic presentation were screened in the study , but those 234 women that went through elective or acute cesarean section were excluded from the analysis .", "the clinical indication for pelvimetry was breech presentation , or if the fetus was in cephalic presentation , the indication was suspected cephalopelvic disproportion in clinical examination .", "the findings that referred to cpd in clinical examination were clinically small pelvis , unengaged presentation , or suspected macrosomia .", "pelvimetric measurements were found in all patients , as required by the inclusion criteria . there were 252 participants with fetal cephalic presentation delivered vaginally , of whom 184 women delivered spontaneously and 68 women went through operative vaginal delivery with vacuum extraction . of this latter group of women , in 26 patients , the vacuum extraction was undertaken primarily because of fetal distress and inertia , and these patients were excluded from the final analysis , leaving 42 women in the operative vaginal delivery group .", "thus , the total number of participants evaluated in the final stage of this study was 226 .", "the obstetric and radiologic data were collected from patients ' medical records by the author ( uk ) and transferred into a commercially available worksheet ( excel , microsoft 2003 , ireland ) .", "the following pelvimetric parameters were recorded : in the pelvic inlet , anteroposterior ( conjugata vera ) and transverse diameters and in outlet , interspinous diameter and sagittal diameter from the surface of the pubic symphysis to the surface of the sacrum measured at the spinous level .", "pelvic inlet and outlet circumferences were calculated from the pelvic anteroposterior and transverse diameters using the formula ( ap + dt  1.57 ) .", "until the year of 2003 , all pelvimetries were performed with an x - ray technique , and from the year 2004 onwards , they were performed with magnetic resonance imaging ( mri ) . during the transition period", ", both x - ray and mri pelvimetries were performed to verify the repeatability of the measurement results . already at the beginning of 1990 , in order to minimize the variability in pelvimetric measurements , they were centralized so that instead of being conducted by several radiologists , they were conducted by trained obstetricians . when the mri pelvimetry was taken into clinical practice , there was one radiologist with previous experience of mri pelvimetry , and during a two - year period ( 20042006 ) , three radiologists and further three obstetricians were also trained to measure the images . in this evaluation of the diagnostic accuracy of the pelvimetry in vaginal deliveries ,", "patients were divided into subgroups according to the size of the fetus and also by the parity to evaluate the variability reflecting differences in patient groups . for statistical analysis , we used spss 17.0 ( spss inc . , 2009 ,", "receiver operating characteristic ( roc )   curves were established , and the area under curve ( auc ) values with significances were calculated ."], ["pelvimetric measurements were found in all patients , as required by the inclusion criteria . in the spontaneous vaginal delivery group ,", "most of the nulliparous patients were sent to maternity clinics consultation because of suspected disproportion . in the multiparous group ,", "24% had delivered by cs and 37% by operative vaginal delivery . in the operative vaginal delivery group ,", "of the nine multiparous patients , six had delivered by cs in their previous pregnancy and two had had previous vaginal operative delivery .", "the demographic data of these 226 patients subdivided according to the route of delivery are shown in table 1 .", "patients were further subdivided into two subgroups according to the infant 's weight and the mode of delivery .", "the maternal pelvic inlet and outlet sizes and duration of the first and second stages of the delivery by the mode of delivery in infant weight subgroups are shown in table 2 .", "the mean maternal outlet ( sd ) was 3613 ( 20 )  mm in all , 351 ( 17 )  mm in infant weight < 3700  g , and 369.5 ( 17.7 ) mm in infant weight 3700  g groups .", "no clinically or statistically significant differences in the pelvic sizes were found between the modes of delivery within the subgroups . between the subgroups ,", "the size of the maternal pelvic size was 4%-5% larger in the mothers with infant weight 3700  g. the duration of the second stage of the delivery was 54 minutes longer ( p < 0.01 ) in the operative vaginal delivery group amounting to a 45-minute longer duration ( p = 0.01 ) in infant weight < 3700  g and 62 minutes longer ( p < 0.01 ) in infant weight 3700  g group .", "the one - minute apgar scores were above 8 in all groups with the exception of those with infant weight less than 3700  g in the operative vaginal group , where the mean of apgar score at one minute was 7.8  1.8 .", "the receiver operating characteristic curve analysis for pelvic inlet and outlet as a diagnostic test for the mode of vaginal delivery is shown in figures 2(a ) and 2(b ) .", "the area under the curve ( auc ) for the pelvic inlet was 0.566 with the p value of 0.18 and 95% confidence interval ( ci ) of 0.4650.667 . for pelvic outlet ,"], ["the main finding of this study was that the maternal bony pelvic dimensions displayed virtually no correlation to the need for operative vaginal deliveries .", "the indications for intervention in vaginal deliveries were chosen on clinical grounds as evidenced by the fact that there was an association between the duration of the second stage of the delivery and the size of the pelvic outlet . if the delivery had reached the second stage , it was probable that the uterine  power  played a more significant role in the overall outcome than either the  passageway  or the  passenger  . on the other hand , pelvic floor muscles , the three - dimensional shape of the bony pelvis , or other soft tissues", "the pelvimetry was performed in most of the patients because of suspected disproportion , or an intervention had been required in a previous labour .", "of those patients that had previous cs and were now exposed to the trial of labour , over 80% delivered spontaneously , and less than 20% required an operative vaginal delivery .", "this is in agreement with previous studies [ 6 , 13 ] . of those women", "that had had a previous operative vaginal labour , only 5% underwent repeated vaginally assisted delivery .", "this may have been due to the fact that the patients were chosen for the trial of labour correctly irrespective of the previous operative delivery .", "there were no statistically significant differences between the size of the maternal inlet or outlet in the spontaneous and the operative vaginal delivery groups .", "when patients were divided into subgroups according to the infant weight , the maternal inlet was 4.7% and the outlet was 5.1% larger in the infant weight 3700  g subgroup among those who delivered spontaneously compared to those vaginally assisted .", "the duration of the first stage of the delivery was longer in the smaller infant group , whereas the second stage was shorter than in the larger infant group .", "in the two delivery subgroups , the duration of the second stage of the delivery was significantly longer in operative vaginal delivery group than in spontaneous vaginal delivery group .", "the apgar scores were acceptable in all delivery groups referring to the fact that both spontaneous and operative vaginal deliveries were uncomplicated and severe shoulder dystocia was not present .", "the apgar 1-minute scores were lower in the operative vaginal delivery group than in spontaneous vaginal delivery group when infant weight was < 3700  g. these results refer to the fact that operative vaginal delivery increases the time of the second stage of the delivery and decreases the apgar 1-minute scores .", "the roc curve analysis for maternal pelvic inlet and outlet revealed that both inlet and outlet had only a fair prognostic value in predicting the mode of the vaginal delivery .", "the poor predictive value of pelvimetry to predict protracted labor is a well - known fact from previous studies , whereas the evidence on the need of vaginal operative deliveries is less extensively evaluated .", "the data did not reveal in detail the fluency of the operative deliveries . as mentioned earlier ,", "therefore , it was not possible to evaluate the influence of the pelvic dimensions on the severe dystocia .", "for that kind of study , the cohort examined here study is too small due to the rare incidence of severe dystocia .", "accordingly , due to the retrospective nature of the study , no blinding was present , and the caregivers were aware of the pelvimetric measurements during the labour .", "furthermore , the study women were at high risk of operative deliveries due to the inclusion criteria , but in clinical care , this would be the group eligible for pelvic assessment before delivery . in conclusion", ", our study revealed that maternal bony pelvic dimensions , either pelvic inlet or outlet , were not associated with the need for operative vaginal deliveries .", "it was more likely that other factors related to the maternal perineal soft tissue , maternal resources , and the passenger were the reasons leading to operative vaginal deliveries .", "subsequently , we can not recommend that caregivers use pelvimetric measurements to predict the outcome of the second stage of the labour .", "observational studies with larger cohorts would be needed , if one wished to investigate whether the maternal bony pelvic size has any effect on severe dystocia .", "in addition , the three - dimensional shape of the bony pelvis and the soft tissues are worth considering in future studies ."]]}
{"article_id": "PMC2761158", "article_text": ["a close relationship between tmj and structures of the middle ear110 has been explained by the presence of the anterior mallear ligament ( aml ) , the sphenomandibular ligament ( sml ) and the discomallear ligament ( dml).2,3,8,1113 in the past , aml was accepted as an initial ligament lying between the middle ear and articular disc ( ad ) of tmj.13 however , further investigations discovered that two distinct ligaments form the aml.12 the anatomic relations and functional properties of dml , aml , and sml are described comprehensively in well - documented studies.5,8,12,14 on the other hand , different opinions exist about histological characteristics of these ligaments.6,8,9,12,1417 various investigators have recognized aml to be consisted of muscular fibers , fibrotic tissue bands , fibro - elastic bands and", "lastly collagenous fibers since the last century.3,6,14,16,17 the dml is a free ligament , lying between the malleus and the tmj,5,8,12,18 which was shown to have a total structural difference from the aml.19 these ligaments were exposed to various traction and stretching tests in the cadaveric studies to discover whether they were responsible for mallear mobility by experimental simulation of discal or condylar translation.6,9,1719 a number of researches supposed that these ligaments were comprised of collagenous fibres9,12 whereas the others supported that only fibro - elastic fibres could transmit traction forces of tmj and cause mobility of the mallear ossicle.6,8 for that reason , we conducted a histological study on the ligaments between the ad and the malleus to clarify the weight of histological properties on mobility of the mallear ossicle in the middle ear .", "15 adult skulls , fixed in a formalin solution , with undamaged tmjs were used in the presented study .", "the cadavers were obtained from gulhane military medical academy , department of anatomy , ankara , turkey .", "then malleus and incus were explored by entering middle ear cavity through the arcuate eminence . with protection of malleus and incus , petrous part of the temporal bone", "the components of middle ear , the ligaments lying anterior to malleus , the ad , and the capsule were explored .", "anterior surface of the petrous part was removed subsequently to follow posteromedial section of tmj .", "little forceps were used to collect bone remnants . by this way , posteromedial and retrodiscal tissues of tmj were completely exposed .", "aml and dml were excised and the intact ones were processed for histologic examination to evaluate their histologic structure and relations with surrounding tissues .", "the ligaments , which were already fixed in 10% buffered formal - saline solution were sectioned longitudinally ( 710 m thickness ) for examination under light microscopy .", "the sections were stained with verhoff s van gieson s stain ( vvg ) ( for elastic fibres ) .", "the sections were visualised at x2.5 and x10 magnifications under a leica dmi 6000 microscope ( leica , germany ) using the software leica application suite ( leica , germany ) .", "by vvg stain , the elastic fibres are observed in blue colour and the collagen fibres in red colour .", "histologically , the sml and the aml were composed of collagen bundles and fibrocytes between them .", "the collagenous fibres were running as filaments in some part of the stained specimens , or they were dispersed in denser fashion as thick bands ( figure 1 ) .", "however , no evidence of elastic fibres was found in none of the examined sections .", "nuclei of fibrocytes and collagenous filaments straddling perpendicularly were observed clearly in the thicker collagenous bands .", "in addition , lumens of capillary vessels were observed particularly between collagenous bands ( figure 1 ) .", "the dml was constituted of rich collagenous fibres in analysed sections ( figures 3 and 4 ) .", "interestingly , one specimen , that was harvested between petrotympanic fissures and at the insertion sites of dml to articular disc - capsular ligament unit , contained elastic fibres dispersed as cotton - bowls amid the collagenous filaments ( figures 3 and 4 ) .", "however , no evidence of elastic structure was found in the part of the dml spanning between the malleus and petrotympanic fissure .", "the collagenous fibres were running as filaments in some part of the stained specimens , or they were dispersed in denser fashion as thick bands ( figure 1 ) .", "however , no evidence of elastic fibres was found in none of the examined sections .", "nuclei of fibrocytes and collagenous filaments straddling perpendicularly were observed clearly in the thicker collagenous bands .", "in addition , lumens of capillary vessels were observed particularly between collagenous bands ( figure 1 ) .", "the dml was constituted of rich collagenous fibres in analysed sections ( figures 3 and 4 ) .", "interestingly , one specimen , that was harvested between petrotympanic fissures and at the insertion sites of dml to articular disc - capsular ligament unit , contained elastic fibres dispersed as cotton - bowls amid the collagenous filaments ( figures 3 and 4 ) .", "however , no evidence of elastic structure was found in the part of the dml spanning between the malleus and petrotympanic fissure .", "mammalian meckel s cartilage , which is derived from the first brachial arch , consists of calcified and uncalcified segments.20 it is well known that the anterior portion that is anterior to the mental foramen and the auricular portion of meckel s cartilage contribute to bone formation of the mandible , the malleus and incus by endochondral ossification.20 by contrast , the midportion of meckel s cartilage , in the soft tissue , eventually forms the sml.21 aml and dml were demonstrated , in many studies , lying from malleus to petrotympanic fissure.8,12,14 such a relationship of the ligaments with adjacent anatomic structures set a hypothesis about possible damage to the middle ear ossicles and the tympanic membrane.12,19 that hypothesis was based on the fact that mallear movement would be responsible for such a dilemma that might be caused by invasive tmj surgery or anterior displacement of the ad.19,22 another hypothesis for this phenomenon was role of extreme distraction of mandibular ramus that incorporated in stretching of aml by intense traction of sml.19 the previous studies mentioned that application of strain forces on dml caused mobility of mallear ossicle.6,9,23 mallear movement by sml stretching was observed in specimens among 50 cadavers.12 kim stretched dml and found no movement ; in contrary , noteworthy mobility of the malleus was obtained by aml stretching.17 the same findings were proved in our studies in which we detected movement in one third of 15 cadavers.19 in the light of these studies , histologic properties of these ligaments were thought to have a possible role on the mallear movement . at the same time", "the stretching tests are partially doubtful , since the adherences or the bony impingements in the petrotympanic fissure are removed during cadaveric dissections to free these ligaments .", "therefore , the reported studies may be suspicious to some extent since mallear movement would not have occurred unless full unearthing or bony dissection for these ligaments was accomplished in the middle cranial fossa . that important fact made us to investigate the ligaments histologically to establish a theory for explaining mallear movement by grounds of the previous stretching tests and histological properties of the filaments .", "the most recent view about histologic properties of aml and dml was that they consisted of collagenous filaments.15 in our study , we found exactly the same findings .", "the elastic fibres were dispersed throughout the stroma of ligament as cotton - bowl appearance .", "the ligaments of any filament composition connected to the malleus can initiate mallear mobility without taking into account the adherences in the petrotympanic fissure .", "however , the abundant elastic fibres in the dml may be responsible for compensation of strain forces on this ligament and may reflect minor forces to the mallear head .", "the collagenous ligaments are lacking such a compensatory mechanism and may reproduce major movement of malleus by direct transformation of strain forces .", "this hypothesis could be accepted when histological characteristics of the ligaments are judged . accordingly , the same mechanism was observed in the first part of this study that covered anatomic and functional aspects of these ligaments .", "the aml forms  pulling  effect on the structures which it is inserting by initiation and termination of a traction force .", "theoretically , this special configuration is not expected to form the same strain forces as the aml does which means that the dml has a less disimpaction effect on the malleus ."], "abstract_text": ["<S> objectivesto examine histological aspects of the ligaments between the middle ear and temporomandibular joint and suppose a theoretical role of their structural characteristics on mobility of mallear ossicle.methodsthe ligaments were obtained by microdissection of middle cranial fossa on both sites of 15 cadavers fixed in formalin solution and were sectioned longitudinally ( 710 m thickness ) . </S>", "<S> the sections were stained with verhoff s van gieson s stain ( vvg ) for demonstration of elastic fibers and visualized at x2.5 and x10 magnifications under light microscopy.resultsanterior mallear ligament ( aml ) and sphenomandibular ligaments ( sml ) were consisted of collagen fibres in analyzed specimens . </S>", "<S> the discomallear ligament ( dml ) was constituted of rich collagenous fibres . </S>", "<S> one specimen of dml harvested between petrotympanic fissure and retrodiscal - capsular intersection site contained elastic fibers dispersed in cotton - bowl appearance between collagen fibers . in the light of functional tests performed in previous studies </S>", "<S> , it could be extrapolated that presence of elastic fibers in the dml may prevent excessive forces conducted to mallear head by elongation of elastic fibers.conclusionscollagenous fibres have no ability to stretch along their axis which may lack compensatory mechanism to prevent mallear head mobility . </S>"], "labels": null, "section_names": ["INTRODUCTION", "MATERIALS AND METHODS", "RESULTS", "AML and SML", "DML", "DISCUSSION", "CONCLUSIONS"], "sections": [["a close relationship between tmj and structures of the middle ear110 has been explained by the presence of the anterior mallear ligament ( aml ) , the sphenomandibular ligament ( sml ) and the discomallear ligament ( dml).2,3,8,1113 in the past , aml was accepted as an initial ligament lying between the middle ear and articular disc ( ad ) of tmj.13 however , further investigations discovered that two distinct ligaments form the aml.12 the anatomic relations and functional properties of dml , aml , and sml are described comprehensively in well - documented studies.5,8,12,14 on the other hand , different opinions exist about histological characteristics of these ligaments.6,8,9,12,1417 various investigators have recognized aml to be consisted of muscular fibers , fibrotic tissue bands , fibro - elastic bands and", "lastly collagenous fibers since the last century.3,6,14,16,17 the dml is a free ligament , lying between the malleus and the tmj,5,8,12,18 which was shown to have a total structural difference from the aml.19 these ligaments were exposed to various traction and stretching tests in the cadaveric studies to discover whether they were responsible for mallear mobility by experimental simulation of discal or condylar translation.6,9,1719 a number of researches supposed that these ligaments were comprised of collagenous fibres9,12 whereas the others supported that only fibro - elastic fibres could transmit traction forces of tmj and cause mobility of the mallear ossicle.6,8 for that reason , we conducted a histological study on the ligaments between the ad and the malleus to clarify the weight of histological properties on mobility of the mallear ossicle in the middle ear ."], ["15 adult skulls , fixed in a formalin solution , with undamaged tmjs were used in the presented study .", "the cadavers were obtained from gulhane military medical academy , department of anatomy , ankara , turkey .", "then malleus and incus were explored by entering middle ear cavity through the arcuate eminence . with protection of malleus and incus , petrous part of the temporal bone", "the components of middle ear , the ligaments lying anterior to malleus , the ad , and the capsule were explored .", "anterior surface of the petrous part was removed subsequently to follow posteromedial section of tmj .", "little forceps were used to collect bone remnants . by this way , posteromedial and retrodiscal tissues of tmj were completely exposed .", "aml and dml were excised and the intact ones were processed for histologic examination to evaluate their histologic structure and relations with surrounding tissues .", "the ligaments , which were already fixed in 10% buffered formal - saline solution were sectioned longitudinally ( 710 m thickness ) for examination under light microscopy .", "the sections were stained with verhoff s van gieson s stain ( vvg ) ( for elastic fibres ) .", "the sections were visualised at x2.5 and x10 magnifications under a leica dmi 6000 microscope ( leica , germany ) using the software leica application suite ( leica , germany ) ."], ["by vvg stain , the elastic fibres are observed in blue colour and the collagen fibres in red colour .", "histologically , the sml and the aml were composed of collagen bundles and fibrocytes between them .", "the collagenous fibres were running as filaments in some part of the stained specimens , or they were dispersed in denser fashion as thick bands ( figure 1 ) .", "however , no evidence of elastic fibres was found in none of the examined sections .", "nuclei of fibrocytes and collagenous filaments straddling perpendicularly were observed clearly in the thicker collagenous bands .", "in addition , lumens of capillary vessels were observed particularly between collagenous bands ( figure 1 ) .", "the dml was constituted of rich collagenous fibres in analysed sections ( figures 3 and 4 ) .", "interestingly , one specimen , that was harvested between petrotympanic fissures and at the insertion sites of dml to articular disc - capsular ligament unit , contained elastic fibres dispersed as cotton - bowls amid the collagenous filaments ( figures 3 and 4 ) .", "however , no evidence of elastic structure was found in the part of the dml spanning between the malleus and petrotympanic fissure ."], ["the collagenous fibres were running as filaments in some part of the stained specimens , or they were dispersed in denser fashion as thick bands ( figure 1 ) .", "however , no evidence of elastic fibres was found in none of the examined sections .", "nuclei of fibrocytes and collagenous filaments straddling perpendicularly were observed clearly in the thicker collagenous bands .", "in addition , lumens of capillary vessels were observed particularly between collagenous bands ( figure 1 ) ."], ["the dml was constituted of rich collagenous fibres in analysed sections ( figures 3 and 4 ) .", "interestingly , one specimen , that was harvested between petrotympanic fissures and at the insertion sites of dml to articular disc - capsular ligament unit , contained elastic fibres dispersed as cotton - bowls amid the collagenous filaments ( figures 3 and 4 ) .", "however , no evidence of elastic structure was found in the part of the dml spanning between the malleus and petrotympanic fissure ."], ["mammalian meckel s cartilage , which is derived from the first brachial arch , consists of calcified and uncalcified segments.20 it is well known that the anterior portion that is anterior to the mental foramen and the auricular portion of meckel s cartilage contribute to bone formation of the mandible , the malleus and incus by endochondral ossification.20 by contrast , the midportion of meckel s cartilage , in the soft tissue , eventually forms the sml.21 aml and dml were demonstrated , in many studies , lying from malleus to petrotympanic fissure.8,12,14 such a relationship of the ligaments with adjacent anatomic structures set a hypothesis about possible damage to the middle ear ossicles and the tympanic membrane.12,19 that hypothesis was based on the fact that mallear movement would be responsible for such a dilemma that might be caused by invasive tmj surgery or anterior displacement of the ad.19,22 another hypothesis for this phenomenon was role of extreme distraction of mandibular ramus that incorporated in stretching of aml by intense traction of sml.19 the previous studies mentioned that application of strain forces on dml caused mobility of mallear ossicle.6,9,23 mallear movement by sml stretching was observed in specimens among 50 cadavers.12 kim stretched dml and found no movement ; in contrary , noteworthy mobility of the malleus was obtained by aml stretching.17 the same findings were proved in our studies in which we detected movement in one third of 15 cadavers.19 in the light of these studies , histologic properties of these ligaments were thought to have a possible role on the mallear movement . at the same time", "the stretching tests are partially doubtful , since the adherences or the bony impingements in the petrotympanic fissure are removed during cadaveric dissections to free these ligaments .", "therefore , the reported studies may be suspicious to some extent since mallear movement would not have occurred unless full unearthing or bony dissection for these ligaments was accomplished in the middle cranial fossa . that important fact made us to investigate the ligaments histologically to establish a theory for explaining mallear movement by grounds of the previous stretching tests and histological properties of the filaments .", "the most recent view about histologic properties of aml and dml was that they consisted of collagenous filaments.15 in our study , we found exactly the same findings .", "the elastic fibres were dispersed throughout the stroma of ligament as cotton - bowl appearance .", "the ligaments of any filament composition connected to the malleus can initiate mallear mobility without taking into account the adherences in the petrotympanic fissure .", "however , the abundant elastic fibres in the dml may be responsible for compensation of strain forces on this ligament and may reflect minor forces to the mallear head .", "the collagenous ligaments are lacking such a compensatory mechanism and may reproduce major movement of malleus by direct transformation of strain forces .", "this hypothesis could be accepted when histological characteristics of the ligaments are judged . accordingly , the same mechanism was observed in the first part of this study that covered anatomic and functional aspects of these ligaments ."], ["the aml forms  pulling  effect on the structures which it is inserting by initiation and termination of a traction force .", "theoretically , this special configuration is not expected to form the same strain forces as the aml does which means that the dml has a less disimpaction effect on the malleus ."]]}
{"article_id": "PMC5288758", "article_text": ["a key issue in biomedicine and biotechnology is to control processes in cells with the highest specificity without unwanted side effects of the intervening agents .", "gene transcription is universally controlled by structural elements within the gene , cisacting regulatory elements , which specifically bind transcription factors.1 among these cisacting elements , the core promoters bind general transcription factors of the general transcription machinery , whereas enhancer elements and proximal promoter elements bind sequencespecific transcription factors , which activate transcription by stimulating transcription initiation at the core promoter .", "targeting these enhancer and proximal promoter elements with , for example , synthetic sequencespecific transcription factors , represents a way to activate the transcription of only the genes harboring these elements . yet", "however , unlike enhancer elements , which act independently of their distance to the core promoter , the effect of proximal promoter elements depends on the distance between the transcription factor binding site and the core promoter.2 , 3 transcription factors are modular proteins composed of a dna binding domain ( dbd ) , which specifically recognizes the response element , and a transactivation domain ( tad ) , which directly or indirectly interacts with the general transcription machinery at the core promoter .", "therefore , if one could engineer an artificial transcription factor with a fixed distance between the dbd and the tad that matches the distance between the proximal promoter element and the core promoter of a particular gene ( figure  1 ) , one would potentially have a much more specific tool to selectively activate the transcription of this gene . \n ", "a )  a dnabinding domain ( dbd ) and transactivation domain ( tad ) are separated by a rigid don of a length matching the distance between the transcription factor binding site ( tfbs ) and the core promoter of gene  a. upon binding of the dbd moiety to the tfbs , the tad would be ideally positioned to activate the transcription of gene  a. the length of the don does not match the distance between the tfbs and core promoter in gene  b. therefore , gene  b is predicted not to be activated by the construct .", "b )  as a test case , a rigid , tubular don ( don1 ) derivatized with the suicide ligands benzylguanine ( bg ) at one end and chlorohexane ( ch ) at the other end is functionalized with a bgreactive , snaptagged version of the dbd of the yeast gal4 transcription factor and with a chreactive , halotagged version of the vp16 tad to generate don  gv . don ", "gv is then microinjected in zebrafish embryos together with a luciferase reporter gene construct , in which five copies of the consensus binding site for gal4 ( 5uas ) are separated from the core promoter by a spacer sequence predicted to match the length of the don .", "they are readily available by folding a long singlestranded dna ( ssdna ) molecule with a designed set of short synthetic  staplestrand ", "oligonucleotides.5 , 6 , 7 dna origami nanostructures ( dons ) have typical dimensions in the 10100  nm regime , which perfectly match those of large supramolecular protein complexes , such as the molecular machinery involved in gene regulation , cell signaling , or cell division.8 more importantly , these dimensions are within the range of the typical distances between proximal promoter elements and core promoters .", "furthermore , dons have unique structuredirecting properties , because they can be used as scaffolds for the precise arrangement of nonnucleic acid components such as proteins or colloidal nanoparticles.9 , 10 it is , therefore , not surprising that there is increasing activity to explore dons as tools to manipulate and analyze cellular functions.11 along this line , efforts are underway to study the stability and function of dons under in  vitro and in  vivo conditions .", "for instance , it has been demonstrated that dons are stable reagents under cell culturing conditions.12 , 13 , 14 dons are functional as immuneactivating programmable adjuvants,15 , 16 and they can be used as drugdelivery vehicles to circumvent drug resistance.17 , 18 , 19 , 20 these studies have suggested that intracellular don uptake is usually dependent on endocytosis.18 furthermore , dons can bind to the outside of the cells to stimulate cellular transmembrane receptors.21 , 22 , 23 \n  despite these exciting reports , the exploitation of dons for the analysis and manipulation of cells is still in its infancy , and there is a strong demand for further insights into the complex interactions of these synthetic biomacromolecules with the machinery of living systems . towards this goal , we report here the microinjection of a proteinfunctionalized don construct into zebrafish embryos ( figure  1 ) .", "our working hypothesis is that using a don to control the distance between a dbd and a tad in order to match the distance between a proximal promoter element and a core promoter would enable the selective activation of a downstream gene . as a test system , we took advantage of the gal4uas system .", "this binary expression system has been widely used in many model species to drive the expression of genes .", "the first component of the binary system consists of a synthetic transcription factor composed of the dbd of the yeast gal4 transcription factor fused to the tad of the herpes simplex vp16 protein .", "the second component is comprised of a synthetic gene with a tandem array of the dna binding sites for the galvp16 chimera , called the upstream activating sequence or uas ( cgg agtac t gtcct ccg),24 upstream of a minimal core promoter .", "this uas promoter controls the expression of downstream cloned genes with dependence on the gal4vp16 chimeric transcription factor.25 it is known that gal4vp16 fusion proteins generate robust expression from the uas responder genes in transient and stable activation assays in zebrafish.26 to allow for a quantitative assessment of transcription , we chose the firefly luciferase reporter gene as a regulated gene . to test the effect of the distance between the uas and the core promoter in the reporter plasmid ,", "they were separated from each other by a variable spacer ( grey region of the reporter gene , figure  1 ) .", "the reporter plasmid was constructed by gibson cloning27 by sequentially aligning five copies of the uas , a spacer , a minimal cmv ( cytomegalie virus ) promoter and the firefly reporter gene28 ( detailed plasmid map shown in figure  s1 ) .", "we reasoned that , upon injection into zebrafish embryo , the level of expression of the firefly luciferase reporter should then depend on the distance between the dbd ( gal4 ) and the tad ( vp16 ) physically connected to each other through a don spacer in the supramolecular construct don ", "to prepare the don  gv construct , we assembled a threedimensional 811148  nm rigidrod tubular don1 ( figure  2  a ) by using the singlestranded 5438  nt template 109z5 , prepared as previously described.29 \n  design and analysis of dna origami nanostructures .", "b )  position  ( top ) and chemical structure ( bottom ) of the staple strands used for the incorporation of the suicide ligands benzylguanine  ( bg ) and chlorohexane  ( ch ) at 5  ends of the selected staple oligonucleotides .", "c )  ethidium bromidestained 0.75  % agarose gel of don samples before  ( not purified , np ) and after  ( purified , p ) peg purification .", "the supernatant ( s ) removed after precipitation contains the nonincorporated staple strands ( red arrow ) .", "e )  height profiles obtained from afm images along the crosssections are marked in blue and red in panel ( d ) .", "the boxwhisker plots show the results of the statistical analysis of 60  particles to determine their dimensions along their axes .", "the values for x and y were determined from the crosssections , whereas the z values were determined from the particle 's heights . to enable the selective attachment of the gal4dbd and vp16 protein at the two ends of the rod", ", we incorporated at the appropriate positions pairs of the smallmolecule suicide ligands benzylguanine ( bg ) and chlorohexane ( ch ) , to which snap and halotagged proteins , respectively , can be ligated.30 the bg and chmodified staple strands were prepared from nhsactivated precursors , as previously described,30 , 31 and characterized by using gel electrophoresis ( figure  s2 ) .", "the assembly of don1 was achieved by temperaturedependent annealing . in brief , the singlestranded dna plasmid and the staple strands at a 1:20 molar ratio were heated to 95  c and the annealing was performed by decreasing the temperature from 85 to 65  c at 1  c per cycle with each step held for 10  min and , subsequently , from 65 to 25  c at 1  c per cycle with each step held for 15  min .", "the assembled don1 samples were purified through polyethylene glycol precipitation32 and their integrity and correct folding was confirmed by gel electrophoresis ( figure  2  c ) , fretdependent annealing studies ( figure  s3 ) , and afm analysis ( figure  2  d ) .", "statistical analysis of the afm images ( figure  2  e ) revealed that the observed length ( 1465  nm ) , thickness ( 173  nm ) , and height ( 71  nm ) were in agreement with the theoretically calculated values of 148  nm length , 11  nm thickness , and 8  nm height for the rods , as depicted in figure  2  a. to synthesize the regulatory proteindecorated construct don ", "gv ( figure  1 ) , the required proteins were produced by recombinant expression .", "we cloned gal4dbd cterminally fused to the snaptag through a flexible ( ggggs)3 linker ( figure  s4 ) .", "the corresponding protein gal4snap was overexpressed in e.  coli and purified to homogeneity by fast protein liquid chromatography ( fplc , figure  s5 ) . as native gal4 functions as a homodimer", "furthermore , the functionality of the novel gal4snap fusion protein to bind the uas recognition sequence was demonstrated by using a microbeadbased pulldown assay ( figure  s7 ) .", "the vp16 tad was cloned with the halotag fused to its nterminus ( figure  s4 ) .", "the corresponding halovp16 protein was overexpressed in e.  coli , purified to homogeneity by fplc , and its identity was verified with western blot analysis ( figure  s8 ) . having both regulatory proteins available as pure and bioconjugatable reagents ( figure  3  a )", "to this end , a cy5labeled don1 ( don1 ) was prepared as described above and mixed with 25  molar equivalents of gal4snap and halovp16 .", "several controls , which included don2 , a construct identical to don1 but lacking the bg and ch ligands , and samples lacking one of the two proteins were prepared in a similar manner .", "the reaction products were characterized by using gel electrophoresis ( figure  3  b ) , which clearly indicated that don ", "the experiments also indicated the absence of unspecific binding between gal4snap , halovp16 , and the don . \n ", "a )  electrophoretic analysis of the conjugation of the two ch and bgmodified staple strands with the recombinant proteins halovp16 and gal4snap to be used as tad and dbd , respectively , in don  gv ( see figure  2  b ) .", "this is a 16  % sds gel , 20  v  cm , 1  h , stained with coomassie ; m :  page ruler prestained protein ladder .", "the appearance of a second band with lower electrophoretic mobility upon incubation with the ligandconjugated dna oligonucleotides ( sample  2 , 3 , 5 , and 6 ) indicates the successful conjugation of the protein with the respective staples .", "b )  halovp16 and gal4snap do not bind to don2 , a construct lacking ch and bgmodified staple strands ( lanes  15 ) .", "in contrast , don1 containing the ch and bg modifications can bind both of the proteins , as indicated from the shift of the bands in lanes  79 .", "note that the conjugation with halovp16 ( lane  8) induces only slight electrophoretic mobility shifts , presumably owing to net negative charge of vp16 ( pi=4.4 ) .", "this is a 0.75  % agarose gel , 70  v , 3  h , visualized by cy5 fluorescence imaging  ( top ) and ethidium bromide staining  ( bottom ) .", "we then started to analyze the behavior of the dons in  vivo in the zebrafish embryo . to this end , we used a zebrafish line stably expressing gfpfused histone h2a33 that labels the cells nuclei and , thereby , facilitates the determination of the dons subcellular localization .", "don1 ( approximately 500  pl , 250  nm ) was injected into the yolk of onecellstage embryos and the embryos were imaged 6  h later ( figure  4  a ) . \n ", "onecell stage embryos from a zebrafish line stably expressing gfpfused histone h2a were used,33 which allows the direct localization of the cell nuclei  ( green channel ) .", "the cy5labeled constructs  ( red channel ) were microinjected into the yolk with don1 alone  ( a ) , the proteindecorated don ( don1gv ; b ) or don1gv along with the reporter plasmid  ( c ) . then , at 6  h postfertilization , the embryos were embedded in agarose and analyzed by using confocal fluorescence microscopy .", "three channels were recorded : the brightfield image  ( i ) , the signal from the gfphistone h2 in green  ( ii ) , and the cy5 signal from the dons in red  ( iii ) .", "the highintensity signal in the red channel shows that the large dna constructs were internalized at a high rate from the yolk .", "in addition , the high magnification  ( bottom images ) clearly indicates that the internalized dons are mostly cytosolic  ( orange arrow ) , with very little to no nuclear localization  ( white arrow ) .", "note that the presence of neither the proteins nor the reporter plasmid influenced the internalization or the subcellular distribution of the dons .", "scale bars :  30  m and 10  m for low and high magnification , respectively .", "the injected embryos survived and developed normally , suggesting that the introduced nanomaterial was well tolerated .", "this result shows that don1 has no obvious deleterious effects on early embryonic development and is indeed efficiently internalized from the yolk ( figure  4  a , panel  iii )", ". however , comparison of the nuclei position ( gfp signal , panel  ii ) with the don fluorescent signal ( panel  iii ) indicates that the don localizes mainly to the cytosol .", "gv injected alone or together with the reporter plasmid ( figures  4  b and 4  c , respectively ) .", "the don  gv also had no obvious effect on early embryonic development and was internalized by the cells .", "furthermore , it was only detected in the cytosol , irrespective of the presence of the reporter plasmid ( figures  4  b and 4  c ) .", "gv had no effect on the expression of the reporter gene , as compared to the don1 ( data not shown ) .", "spherical particles with a diameter below 26  nm freely diffuse into the nucleus , whereas larger particles require a nuclear localization signal ( nls ) to actively translocate through the nuclear pore complex , with an upper size limit of about 39  nm.34 the gal4dbd used to functionalize the don  gv harbors an intrinsic nuclear localization signal.35 however , it was apparently not sufficient to drive the don  gv to the nucleus .", "recently , it was shown that functionalization with an nls increases the nuclear uptake of 510  nm100300  nm rodshaped polymeric", "gv is unlikely because of its size , but rather to a low efficiency of the gal4dbd nls in the context of the construct .", "stronger  nls as a peptide covalently attached to the don might increase its nuclear uptake .", "we report here on an ambitious concept for controlling gene transcription in a distancedependent manner in the developing zebrafish embryo , taking advantage of designed protein  dna origami nanostructures .", "although we could clearly demonstrate the successful synthesis of our target construct , applications in  vivo require further developments . nonetheless , we believe that our study represents an important contribution to the exploitation of dons for the analysis and manipulation of cells in  vivo", ". we demonstrated , for the first time , that microinjection in the yolk of the zebrafish embryo enables intracellular distribution in most of the cells of large origami constructs harboring active proteins precisely arranged in a 3d fashion .", "furthermore , we demonstrated that the don constructs are not toxic for the embryos and allowed normal development , at least in the early stages .", "we foresee that this technology could be applied to deliver complex multiprotein constructs to the cytosol and maybe also to the nuclei by means of strong nuclear localization signals .", "as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset .", "technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors ."], "abstract_text": ["<S> abstractthe unique structuredirecting properties of dna origami nanostructures ( dons ) show great potential to specifically manipulate intracellular processes . </S>", "<S> we report an innovative concept to selectively activate the transcription of a single gene in the developing zebrafish embryo . </S>", "<S> we reason that engineering a designer transcription factor in which a rigid don imposes a fixed distance between the dnabinding domain ( dbd ) and the transactivation domain ( tad ) would allow the selective activation of a gene harboring the same distance between the corresponding transcription factor binding site and the core promoter . as a test case , a rigid tubular don was designed to separate the dbd of the gal4 transcription factor and the vp16 viral protein as a tad . </S>", "<S> this construct was microinjected in the yolk of onecellstage zebrafish embryos , together with a reporter plasmid to assess its functionality . </S>", "<S> the large don was efficiently distributed to cells of the developing embryo and showed no signs of toxicity . however , because the don showed only a cytosolic localization , it did not activate transcription of the reporter gene . </S>", "<S> although this work clearly demonstrates that don microinjection enables the intracellular distribution of multiprotein architectures in most of the cells of the developing zebrafish embryo , further refinements are necessary to enable selective gene activation in  vivo . </S>"], "labels": null, "section_names": ["Introduction", "Results and Discussion", "Conclusions", "Experimental Section", "Conflict of interest", "Supporting information"], "sections": [["a key issue in biomedicine and biotechnology is to control processes in cells with the highest specificity without unwanted side effects of the intervening agents .", "gene transcription is universally controlled by structural elements within the gene , cisacting regulatory elements , which specifically bind transcription factors.1 among these cisacting elements , the core promoters bind general transcription factors of the general transcription machinery , whereas enhancer elements and proximal promoter elements bind sequencespecific transcription factors , which activate transcription by stimulating transcription initiation at the core promoter .", "targeting these enhancer and proximal promoter elements with , for example , synthetic sequencespecific transcription factors , represents a way to activate the transcription of only the genes harboring these elements . yet", "however , unlike enhancer elements , which act independently of their distance to the core promoter , the effect of proximal promoter elements depends on the distance between the transcription factor binding site and the core promoter.2 , 3 transcription factors are modular proteins composed of a dna binding domain ( dbd ) , which specifically recognizes the response element , and a transactivation domain ( tad ) , which directly or indirectly interacts with the general transcription machinery at the core promoter .", "therefore , if one could engineer an artificial transcription factor with a fixed distance between the dbd and the tad that matches the distance between the proximal promoter element and the core promoter of a particular gene ( figure  1 ) , one would potentially have a much more specific tool to selectively activate the transcription of this gene . \n ", "a )  a dnabinding domain ( dbd ) and transactivation domain ( tad ) are separated by a rigid don of a length matching the distance between the transcription factor binding site ( tfbs ) and the core promoter of gene  a. upon binding of the dbd moiety to the tfbs , the tad would be ideally positioned to activate the transcription of gene  a. the length of the don does not match the distance between the tfbs and core promoter in gene  b. therefore , gene  b is predicted not to be activated by the construct .", "b )  as a test case , a rigid , tubular don ( don1 ) derivatized with the suicide ligands benzylguanine ( bg ) at one end and chlorohexane ( ch ) at the other end is functionalized with a bgreactive , snaptagged version of the dbd of the yeast gal4 transcription factor and with a chreactive , halotagged version of the vp16 tad to generate don  gv . don ", "gv is then microinjected in zebrafish embryos together with a luciferase reporter gene construct , in which five copies of the consensus binding site for gal4 ( 5uas ) are separated from the core promoter by a spacer sequence predicted to match the length of the don .", "they are readily available by folding a long singlestranded dna ( ssdna ) molecule with a designed set of short synthetic  staplestrand ", "oligonucleotides.5 , 6 , 7 dna origami nanostructures ( dons ) have typical dimensions in the 10100  nm regime , which perfectly match those of large supramolecular protein complexes , such as the molecular machinery involved in gene regulation , cell signaling , or cell division.8 more importantly , these dimensions are within the range of the typical distances between proximal promoter elements and core promoters .", "furthermore , dons have unique structuredirecting properties , because they can be used as scaffolds for the precise arrangement of nonnucleic acid components such as proteins or colloidal nanoparticles.9 , 10 it is , therefore , not surprising that there is increasing activity to explore dons as tools to manipulate and analyze cellular functions.11 along this line , efforts are underway to study the stability and function of dons under in  vitro and in  vivo conditions .", "for instance , it has been demonstrated that dons are stable reagents under cell culturing conditions.12 , 13 , 14 dons are functional as immuneactivating programmable adjuvants,15 , 16 and they can be used as drugdelivery vehicles to circumvent drug resistance.17 , 18 , 19 , 20 these studies have suggested that intracellular don uptake is usually dependent on endocytosis.18 furthermore , dons can bind to the outside of the cells to stimulate cellular transmembrane receptors.21 , 22 , 23 \n  despite these exciting reports , the exploitation of dons for the analysis and manipulation of cells is still in its infancy , and there is a strong demand for further insights into the complex interactions of these synthetic biomacromolecules with the machinery of living systems . towards this goal , we report here the microinjection of a proteinfunctionalized don construct into zebrafish embryos ( figure  1 ) .", "our working hypothesis is that using a don to control the distance between a dbd and a tad in order to match the distance between a proximal promoter element and a core promoter would enable the selective activation of a downstream gene . as a test system , we took advantage of the gal4uas system .", "this binary expression system has been widely used in many model species to drive the expression of genes .", "the first component of the binary system consists of a synthetic transcription factor composed of the dbd of the yeast gal4 transcription factor fused to the tad of the herpes simplex vp16 protein .", "the second component is comprised of a synthetic gene with a tandem array of the dna binding sites for the galvp16 chimera , called the upstream activating sequence or uas ( cgg agtac t gtcct ccg),24 upstream of a minimal core promoter .", "this uas promoter controls the expression of downstream cloned genes with dependence on the gal4vp16 chimeric transcription factor.25 it is known that gal4vp16 fusion proteins generate robust expression from the uas responder genes in transient and stable activation assays in zebrafish.26 to allow for a quantitative assessment of transcription , we chose the firefly luciferase reporter gene as a regulated gene . to test the effect of the distance between the uas and the core promoter in the reporter plasmid ,", "they were separated from each other by a variable spacer ( grey region of the reporter gene , figure  1 ) .", "the reporter plasmid was constructed by gibson cloning27 by sequentially aligning five copies of the uas , a spacer , a minimal cmv ( cytomegalie virus ) promoter and the firefly reporter gene28 ( detailed plasmid map shown in figure  s1 ) .", "we reasoned that , upon injection into zebrafish embryo , the level of expression of the firefly luciferase reporter should then depend on the distance between the dbd ( gal4 ) and the tad ( vp16 ) physically connected to each other through a don spacer in the supramolecular construct don "], ["to prepare the don  gv construct , we assembled a threedimensional 811148  nm rigidrod tubular don1 ( figure  2  a ) by using the singlestranded 5438  nt template 109z5 , prepared as previously described.29 \n  design and analysis of dna origami nanostructures .", "b )  position  ( top ) and chemical structure ( bottom ) of the staple strands used for the incorporation of the suicide ligands benzylguanine  ( bg ) and chlorohexane  ( ch ) at 5  ends of the selected staple oligonucleotides .", "c )  ethidium bromidestained 0.75  % agarose gel of don samples before  ( not purified , np ) and after  ( purified , p ) peg purification .", "the supernatant ( s ) removed after precipitation contains the nonincorporated staple strands ( red arrow ) .", "e )  height profiles obtained from afm images along the crosssections are marked in blue and red in panel ( d ) .", "the boxwhisker plots show the results of the statistical analysis of 60  particles to determine their dimensions along their axes .", "the values for x and y were determined from the crosssections , whereas the z values were determined from the particle 's heights . to enable the selective attachment of the gal4dbd and vp16 protein at the two ends of the rod", ", we incorporated at the appropriate positions pairs of the smallmolecule suicide ligands benzylguanine ( bg ) and chlorohexane ( ch ) , to which snap and halotagged proteins , respectively , can be ligated.30 the bg and chmodified staple strands were prepared from nhsactivated precursors , as previously described,30 , 31 and characterized by using gel electrophoresis ( figure  s2 ) .", "the assembly of don1 was achieved by temperaturedependent annealing . in brief , the singlestranded dna plasmid and the staple strands at a 1:20 molar ratio were heated to 95  c and the annealing was performed by decreasing the temperature from 85 to 65  c at 1  c per cycle with each step held for 10  min and , subsequently , from 65 to 25  c at 1  c per cycle with each step held for 15  min .", "the assembled don1 samples were purified through polyethylene glycol precipitation32 and their integrity and correct folding was confirmed by gel electrophoresis ( figure  2  c ) , fretdependent annealing studies ( figure  s3 ) , and afm analysis ( figure  2  d ) .", "statistical analysis of the afm images ( figure  2  e ) revealed that the observed length ( 1465  nm ) , thickness ( 173  nm ) , and height ( 71  nm ) were in agreement with the theoretically calculated values of 148  nm length , 11  nm thickness , and 8  nm height for the rods , as depicted in figure  2  a. to synthesize the regulatory proteindecorated construct don ", "gv ( figure  1 ) , the required proteins were produced by recombinant expression .", "we cloned gal4dbd cterminally fused to the snaptag through a flexible ( ggggs)3 linker ( figure  s4 ) .", "the corresponding protein gal4snap was overexpressed in e.  coli and purified to homogeneity by fast protein liquid chromatography ( fplc , figure  s5 ) . as native gal4 functions as a homodimer", "furthermore , the functionality of the novel gal4snap fusion protein to bind the uas recognition sequence was demonstrated by using a microbeadbased pulldown assay ( figure  s7 ) .", "the vp16 tad was cloned with the halotag fused to its nterminus ( figure  s4 ) .", "the corresponding halovp16 protein was overexpressed in e.  coli , purified to homogeneity by fplc , and its identity was verified with western blot analysis ( figure  s8 ) . having both regulatory proteins available as pure and bioconjugatable reagents ( figure  3  a )", "to this end , a cy5labeled don1 ( don1 ) was prepared as described above and mixed with 25  molar equivalents of gal4snap and halovp16 .", "several controls , which included don2 , a construct identical to don1 but lacking the bg and ch ligands , and samples lacking one of the two proteins were prepared in a similar manner .", "the reaction products were characterized by using gel electrophoresis ( figure  3  b ) , which clearly indicated that don ", "the experiments also indicated the absence of unspecific binding between gal4snap , halovp16 , and the don . \n ", "a )  electrophoretic analysis of the conjugation of the two ch and bgmodified staple strands with the recombinant proteins halovp16 and gal4snap to be used as tad and dbd , respectively , in don  gv ( see figure  2  b ) .", "this is a 16  % sds gel , 20  v  cm , 1  h , stained with coomassie ; m :  page ruler prestained protein ladder .", "the appearance of a second band with lower electrophoretic mobility upon incubation with the ligandconjugated dna oligonucleotides ( sample  2 , 3 , 5 , and 6 ) indicates the successful conjugation of the protein with the respective staples .", "b )  halovp16 and gal4snap do not bind to don2 , a construct lacking ch and bgmodified staple strands ( lanes  15 ) .", "in contrast , don1 containing the ch and bg modifications can bind both of the proteins , as indicated from the shift of the bands in lanes  79 .", "note that the conjugation with halovp16 ( lane  8) induces only slight electrophoretic mobility shifts , presumably owing to net negative charge of vp16 ( pi=4.4 ) .", "this is a 0.75  % agarose gel , 70  v , 3  h , visualized by cy5 fluorescence imaging  ( top ) and ethidium bromide staining  ( bottom ) .", "we then started to analyze the behavior of the dons in  vivo in the zebrafish embryo . to this end , we used a zebrafish line stably expressing gfpfused histone h2a33 that labels the cells nuclei and , thereby , facilitates the determination of the dons subcellular localization .", "don1 ( approximately 500  pl , 250  nm ) was injected into the yolk of onecellstage embryos and the embryos were imaged 6  h later ( figure  4  a ) . \n ", "onecell stage embryos from a zebrafish line stably expressing gfpfused histone h2a were used,33 which allows the direct localization of the cell nuclei  ( green channel ) .", "the cy5labeled constructs  ( red channel ) were microinjected into the yolk with don1 alone  ( a ) , the proteindecorated don ( don1gv ; b ) or don1gv along with the reporter plasmid  ( c ) . then , at 6  h postfertilization , the embryos were embedded in agarose and analyzed by using confocal fluorescence microscopy .", "three channels were recorded : the brightfield image  ( i ) , the signal from the gfphistone h2 in green  ( ii ) , and the cy5 signal from the dons in red  ( iii ) .", "the highintensity signal in the red channel shows that the large dna constructs were internalized at a high rate from the yolk .", "in addition , the high magnification  ( bottom images ) clearly indicates that the internalized dons are mostly cytosolic  ( orange arrow ) , with very little to no nuclear localization  ( white arrow ) .", "note that the presence of neither the proteins nor the reporter plasmid influenced the internalization or the subcellular distribution of the dons .", "scale bars :  30  m and 10  m for low and high magnification , respectively .", "the injected embryos survived and developed normally , suggesting that the introduced nanomaterial was well tolerated .", "this result shows that don1 has no obvious deleterious effects on early embryonic development and is indeed efficiently internalized from the yolk ( figure  4  a , panel  iii )", ". however , comparison of the nuclei position ( gfp signal , panel  ii ) with the don fluorescent signal ( panel  iii ) indicates that the don localizes mainly to the cytosol .", "gv injected alone or together with the reporter plasmid ( figures  4  b and 4  c , respectively ) .", "the don  gv also had no obvious effect on early embryonic development and was internalized by the cells .", "furthermore , it was only detected in the cytosol , irrespective of the presence of the reporter plasmid ( figures  4  b and 4  c ) .", "gv had no effect on the expression of the reporter gene , as compared to the don1 ( data not shown ) .", "spherical particles with a diameter below 26  nm freely diffuse into the nucleus , whereas larger particles require a nuclear localization signal ( nls ) to actively translocate through the nuclear pore complex , with an upper size limit of about 39  nm.34 the gal4dbd used to functionalize the don  gv harbors an intrinsic nuclear localization signal.35 however , it was apparently not sufficient to drive the don  gv to the nucleus .", "recently , it was shown that functionalization with an nls increases the nuclear uptake of 510  nm100300  nm rodshaped polymeric", "gv is unlikely because of its size , but rather to a low efficiency of the gal4dbd nls in the context of the construct .", "stronger  nls as a peptide covalently attached to the don might increase its nuclear uptake ."], ["we report here on an ambitious concept for controlling gene transcription in a distancedependent manner in the developing zebrafish embryo , taking advantage of designed protein  dna origami nanostructures .", "although we could clearly demonstrate the successful synthesis of our target construct , applications in  vivo require further developments . nonetheless , we believe that our study represents an important contribution to the exploitation of dons for the analysis and manipulation of cells in  vivo", ". we demonstrated , for the first time , that microinjection in the yolk of the zebrafish embryo enables intracellular distribution in most of the cells of large origami constructs harboring active proteins precisely arranged in a 3d fashion .", "furthermore , we demonstrated that the don constructs are not toxic for the embryos and allowed normal development , at least in the early stages .", "we foresee that this technology could be applied to deliver complex multiprotein constructs to the cytosol and maybe also to the nuclei by means of strong nuclear localization signals ."], [""], [""], ["as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset .", "technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors ."]]}
{"article_id": "PMC4739229", "article_text": ["visfatin , also known as nicotinamide phosphoribosyltransferase ( nampt ) as well as pre - b - cell colony - enhancing factor , is a multifaceted protein with suggested enzymatic , immunological , and metabolic properties .", "visfatin has been analyzed in hypo- and hyperthyroidism in in vitro and in vivo studies , but results are inconclusive .", "in addition , nampt level was found to be elevated in many autoimmune diseases , that is , rheumatoid arthritis , systemic lupus erythematosus , inflammatory bowel diseases , and psoriasis [ 25 ] .", "visfatin also positively correlates with activity and severity of rheumatoid arthritis and psoriasis [ 2 , 5 ] .", "we have recently found an overexpression of nampt in leukocytes of patients with graves ' ophthalmopathy with corresponding increased serum concentration ( accepted manuscript ) .", "our findings suggest that visfatin might be involved in autoimmune processes in thyroid diseases . in our opinion", ", the controversial findings of visfatin in thyroid hormone deficiency may arise from the heterogeneity of thyroid dysfunction .", "we hypothesized that regulation of visfatin in hypothyroidism might be altered by coexisting chronic autoimmune thyroiditis , since high visfatin levels were observed in other autoimmune diseases . to answer the question about the influence of coexisting chronic autoimmune inflammation on visfatin level", ", we analyzed its serum concentration among hypothyroid patients with chronic autoimmune thyroiditis and in patients after thyroidectomy , who were negative for thyroid antibodies .", "this is a prospective case - control study of 118 subjects . the autoimmune study group ( ait ) consisted of 39 patients newly diagnosed with hypothyroidism in a course of chronic autoimmune thyroiditis .", "the nonautoimmune study group ( tt ) consisted of 40 patients thyroidectomized due to the differentiated thyroid cancer staged pt1 .", "tt patients were evaluated five years after radioiodine remnant ablation and were negative for thyroglobulin and radioiodine uptake in a whole body scintigraphy ( wbs ) .", "they achieved endogenous tsh stimulation and became hypothyroid after l - t4 withdrawal for at least 4 weeks .", "the control group comprised 39 healthy volunteers adjusted for age , sex , and bmi with normal thyroid function and negative thyroid antibodies .", "exclusion criteria consisted of other autoimmune diseases , active neoplastic disease , diabetes mellitus , and infection , which were reported to alter visfatin level .", "fasting blood samples were taken for visfatin , tsh , free thyroxine ( ft4 ) , free triiodothyronine ( ft3 ) , antithyroperoxidase antibodies ( tpoab ) , antithyroglobulin antibodies ( tgab ) , glucose , and insulin levels . in tt group", "tsh , ft4 , and ft3 were measured using the electrochemiluminescence technique in cobas e 601 ( norm ranges : tsh 0.274.2 ", "tpoab and tgab were measured by radioimmunoassay ( norm range : < 34  iu / ml and < 115  iu / ml , resp . ) .", "glucose level was assessed with the use of hitachi cobas e601 chemiluminescent analyzer ( roche diagnostics ) and insulin concentration was assessed using elisa kit from phoenix pharmaceuticals .", "the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) was calculated .", "the study was approved by the local ethics committee , and informed consent was signed by every subject .", "statistical analysis was performed with medcalc version 12.1.3.0 ( medcalc software , mariakerke , belgium ) .", "variables with normal distribution were compared between three groups with one - way analysis of variance .", "if data did not follow normal distribution , comparison of the analyzed parameters between three groups was performed with the kruskal - wallis test .", "simple regression analysis was used to test for the relationships between them . before inclusion to this statistical analysis ,", "furthermore , stepwise multiple regression analysis was employed to investigate the influence of various parameters on visfatin serum concentration [ age , bmi , ft3 , autoimmunity ( yes / no ) , homa - ir ] .", "variables were entered into the model if their associated p - values were less than 0.05 and then sequentially removed if their associated p - values became greater than 0.2 .", "tests were considered to be statistically significant if p - value was lower than 0.05 .", "clinical and laboratory data of the study groups and the control group are shown in table 1 .", "the highest visfatin serum concentration was in ait group , and healthy controls had visfatin level higher than tt ( p = 0.0001 ) ( figure 1 ) .", "three groups did not differ in age , sex , bmi , fasting glucose , and insulin levels , homa - ir .", "they had statistically different tsh , ft4 , ft3 , and tgab levels ( table 1 ) .", "simple linear regression analysis revealed that visfatin serum concentration was significantly associated with autoimmunity (  = 0.1014 ; p = 0.003 ) , ft4 (  = 0.05412 ; p = 0.048 ) , ft3 (  = 0.05242 ; p = 0.038 ) , and tpoab (  = 0.0002 ; p = 0.0025 ) ( figure 2 ) .", "there was no association between visfatin and age , sex , bmi , tsh , tgab , fasting insulin and glucose levels , and homa - ir ( table 2 ) .", "in the stepwise multiple regression analysis we confirmed the association between serum visfatin level and autoimmunity ( coefficient = 3.8461 ; p = 0.0001 ) , and ft3 ( coefficient = 0.4198 ; p = 0.0441 ) , whereas age , bmi , and homa - ir did not contribute significantly .", "in separate stepwise multiple regression analysis we confirmed the association of serum visfatin concentration with autoimmunity ( coefficient = 4.1105 ; p = 0.0001 ) and ft4 ( coefficient = 0.1397 ; p = 0.038 ) , whereas age , bmi , and homa - ir did not enter the model .", "similarly , association of visfatin with tpoab ( coefficient = 0.0057 ; p = 0.0163 ) was observed with adjustment for age , bmi , ft3 , and homa - ir in multivariate regression analysis .", "to date , visfatin serum concentration in hypothyroidism has been analyzed in a few studies .", "reported elevated level of this adipocytokine in hypothyroidism with further increase after restoration of thyroid function .", "ozkaya et al . observed that visfatin level decreased after recovery . in those articles the etiology of hypothyroidism varied from chronic autoimmune thyroiditis , postpartum thyroiditis to thyroid function insufficiency after radioiodine treatment or after thyroidectomy .", "hence , to date autoimmune status of studied patients has not been taken into consideration .", "we hypothesized that these controversial findings might result from heterogeneity of study groups . to answer the question ,", "whether coexisting autoimmune inflammation influences visfatin level in hypothyroid patients , we analyzed its serum concentration in chronic autoimmune thyroiditis and thyroidectomized patients negative for thyroid antibodies . since we have previously proved that visfatin mrna expression is increased in thyroid malignancies and is correlated with tumor stage , we recruited only those patients who did not have any features of active neoplastic disease .", "we also recruited the control group adjusted for age , sex , and bmi . to the best of our knowledge ,", "this is the first study addressing the changes in the release of visfatin in thyroid autoimmunity .", "we came to interesting results indicating that visfatin serum concentration in hypothyroid patients is associated with both autoimmunity and free thyroid hormones level ( ft4 , ft3 ) .", "visfatin has been recognized as a cytokine with a broad range of immune and inflammatory activities , including induction of inflammatory cytokines , and regulation of macrophage and lymphocyte proliferation .", "visfatin stimulates the production of proinflammatory cytokines ( il-6 , tnf- , and il-1 ) and potentially acts as a chemotactic factor for monocytes .", "furthermore , its expression is upregulated by il-6 , tnf- , and il-1 [ 1012 ] .", "enhanced mrna expression of visfatin was observed in inflamed mucosa of patients with inflammatory bowel disease ( ibd ) .", "further analysis identified that antigen presenting cells ( i.e. , macrophages , dendritic cells ) might be a main source of this protein in ibd .", "visfatin has also potency for activation of t cells by upregulation of costimulatory molecules ( cd40 , cd54 , and cd80 ) on monocytes .", "we observed the positive association between visfatin and tpoab , and the latter is considered the best serological marker of chronic autoimmune thyroiditis .", "furthermore , tpoab contribute to thyroid destruction through antibody- and complement - dependent cell - cytotoxicity [ 13 , 14 ] . the first mechanism is associated with mononuclear cell infiltration of thyroid stroma .", "in addition , th1-derived cytokines ( il-2 , tnf- , and inf ) were found to be elevated in patients with chronic autoimmune thyroiditis .", "also , il-1 and tnf- have been recently reported to discriminate chronic autoimmune hypothyroid children from healthy controls .", "altogether , association of visfatin with tpoab observed in our study further supports our hypothesis that visfatin might be involved in the pathogenesis of chronic autoimmune thyroiditis .", "our findings about the association of visfatin with ft3 levels are in accordance with the results of in vitro and in vivo studies .", "however , controversial results whether t3 stimulates or downregulates the production of visfatin were found .", "in contrast , caixs et al . did not find any relationship between visfatin and free thyroid hormones . in vitro experiment", "showed the nonlinear regulation of visfatin mrna expression in the 3t3-l1 cell culture model affected by t3 .", "since our study groups significantly differed with free thyroid hormones levels , we were able to analyze visfatin concentration in a broad spectrum of ft3 and ft4 .", "therefore , we might suggest that pattern of visfatin changes varies in different ft3 concentration . according to our observations , as well as other authors", ", visfatin did not reflect insulin resistance assessed by homa - ir [ 18 , 19 ] .", "our study might then prove that visfatin in hypothyroidism depends on thyroid hormones level and coexisting autoimmunity .", "we may assume that these two factors should be taken into consideration to assess visfatin level in patients with thyroid dysfunction .", "in addition , the possible involvement of visfatin in pathogenesis of chronic autoimmune thyroiditis needs further research .", "the main limitation of our study is its cross - sectional design that does not enable us to reveal the causal pathways of relationship between visfatin and autoimmune thyroiditis .", "however , we used very strict criteria of exclusion to limit the possible influence of other known factors such as diabetes mellitus , other autoimmune processes , infection , and active neoplastic disease .", "our nonautoimmune group with hypothyroidism had been thyroidectomized at least 5 years earlier and did not have any clinical nor laboratory features of active thyroid cancer ."], "abstract_text": ["<S> we hypothesized that regulation of visfatin in hypothyroidism might be altered by coexisting chronic autoimmune thyroiditis . </S>", "<S> this is a prospective case - control study of 118 subjects . the autoimmune study group ( ait ) consisted of 39 patients newly diagnosed with hypothyroidism in a course of chronic autoimmune thyroiditis . </S>", "<S> the nonautoimmune study group ( tt ) consisted of 40 patients thyroidectomized due to the differentiated thyroid cancer staged pt1 . </S>", "<S> the control group comprised 39 healthy volunteers adjusted for age , sex , and bmi with normal thyroid function and negative thyroid antibodies . </S>", "<S> exclusion criteria consisted of other autoimmune diseases , active neoplastic disease , diabetes mellitus , and infection , which were reported to alter visfatin level . </S>", "<S> fasting blood samples were taken for visfatin , tsh , free thyroxine ( ft4 ) , free triiodothyronine ( ft3 ) , antithyroperoxidase antibodies ( tpoab ) , antithyroglobulin antibodies ( tgab ) , glucose , and insulin levels . </S>", "<S> the highest visfatin serum concentration was in ait group , and healthy controls had visfatin level higher than tt ( p = 0.0001 ) . </S>", "<S> simple linear regression analysis revealed that visfatin serum concentration was significantly associated with autoimmunity (  = 0.1014 ; p = 0.003 ) , ft4 (  = 0.05412 ; p = 0.048 ) , ft3 (  = 0.05242 ; p = 0.038 ) , and tpoab (  = 0.0002 ; p = 0.0025 ) , and the relationships were further confirmed in the multivariate regression analysis . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Patients and Methods", "3. Results", "4. Discussion", "Limitation of the Study"], "sections": [["visfatin , also known as nicotinamide phosphoribosyltransferase ( nampt ) as well as pre - b - cell colony - enhancing factor , is a multifaceted protein with suggested enzymatic , immunological , and metabolic properties .", "visfatin has been analyzed in hypo- and hyperthyroidism in in vitro and in vivo studies , but results are inconclusive .", "in addition , nampt level was found to be elevated in many autoimmune diseases , that is , rheumatoid arthritis , systemic lupus erythematosus , inflammatory bowel diseases , and psoriasis [ 25 ] .", "visfatin also positively correlates with activity and severity of rheumatoid arthritis and psoriasis [ 2 , 5 ] .", "we have recently found an overexpression of nampt in leukocytes of patients with graves ' ophthalmopathy with corresponding increased serum concentration ( accepted manuscript ) .", "our findings suggest that visfatin might be involved in autoimmune processes in thyroid diseases . in our opinion", ", the controversial findings of visfatin in thyroid hormone deficiency may arise from the heterogeneity of thyroid dysfunction .", "we hypothesized that regulation of visfatin in hypothyroidism might be altered by coexisting chronic autoimmune thyroiditis , since high visfatin levels were observed in other autoimmune diseases . to answer the question about the influence of coexisting chronic autoimmune inflammation on visfatin level", ", we analyzed its serum concentration among hypothyroid patients with chronic autoimmune thyroiditis and in patients after thyroidectomy , who were negative for thyroid antibodies ."], ["this is a prospective case - control study of 118 subjects . the autoimmune study group ( ait ) consisted of 39 patients newly diagnosed with hypothyroidism in a course of chronic autoimmune thyroiditis .", "the nonautoimmune study group ( tt ) consisted of 40 patients thyroidectomized due to the differentiated thyroid cancer staged pt1 .", "tt patients were evaluated five years after radioiodine remnant ablation and were negative for thyroglobulin and radioiodine uptake in a whole body scintigraphy ( wbs ) .", "they achieved endogenous tsh stimulation and became hypothyroid after l - t4 withdrawal for at least 4 weeks .", "the control group comprised 39 healthy volunteers adjusted for age , sex , and bmi with normal thyroid function and negative thyroid antibodies .", "exclusion criteria consisted of other autoimmune diseases , active neoplastic disease , diabetes mellitus , and infection , which were reported to alter visfatin level .", "fasting blood samples were taken for visfatin , tsh , free thyroxine ( ft4 ) , free triiodothyronine ( ft3 ) , antithyroperoxidase antibodies ( tpoab ) , antithyroglobulin antibodies ( tgab ) , glucose , and insulin levels . in tt group", "tsh , ft4 , and ft3 were measured using the electrochemiluminescence technique in cobas e 601 ( norm ranges : tsh 0.274.2 ", "tpoab and tgab were measured by radioimmunoassay ( norm range : < 34  iu / ml and < 115  iu / ml , resp . ) .", "glucose level was assessed with the use of hitachi cobas e601 chemiluminescent analyzer ( roche diagnostics ) and insulin concentration was assessed using elisa kit from phoenix pharmaceuticals .", "the estimate of insulin resistance by homeostasis model assessment ( homa - ir ) was calculated .", "the study was approved by the local ethics committee , and informed consent was signed by every subject .", "statistical analysis was performed with medcalc version 12.1.3.0 ( medcalc software , mariakerke , belgium ) .", "variables with normal distribution were compared between three groups with one - way analysis of variance .", "if data did not follow normal distribution , comparison of the analyzed parameters between three groups was performed with the kruskal - wallis test .", "simple regression analysis was used to test for the relationships between them . before inclusion to this statistical analysis ,", "furthermore , stepwise multiple regression analysis was employed to investigate the influence of various parameters on visfatin serum concentration [ age , bmi , ft3 , autoimmunity ( yes / no ) , homa - ir ] .", "variables were entered into the model if their associated p - values were less than 0.05 and then sequentially removed if their associated p - values became greater than 0.2 .", "tests were considered to be statistically significant if p - value was lower than 0.05 ."], ["clinical and laboratory data of the study groups and the control group are shown in table 1 .", "the highest visfatin serum concentration was in ait group , and healthy controls had visfatin level higher than tt ( p = 0.0001 ) ( figure 1 ) .", "three groups did not differ in age , sex , bmi , fasting glucose , and insulin levels , homa - ir .", "they had statistically different tsh , ft4 , ft3 , and tgab levels ( table 1 ) .", "simple linear regression analysis revealed that visfatin serum concentration was significantly associated with autoimmunity (  = 0.1014 ; p = 0.003 ) , ft4 (  = 0.05412 ; p = 0.048 ) , ft3 (  = 0.05242 ; p = 0.038 ) , and tpoab (  = 0.0002 ; p = 0.0025 ) ( figure 2 ) .", "there was no association between visfatin and age , sex , bmi , tsh , tgab , fasting insulin and glucose levels , and homa - ir ( table 2 ) .", "in the stepwise multiple regression analysis we confirmed the association between serum visfatin level and autoimmunity ( coefficient = 3.8461 ; p = 0.0001 ) , and ft3 ( coefficient = 0.4198 ; p = 0.0441 ) , whereas age , bmi , and homa - ir did not contribute significantly .", "in separate stepwise multiple regression analysis we confirmed the association of serum visfatin concentration with autoimmunity ( coefficient = 4.1105 ; p = 0.0001 ) and ft4 ( coefficient = 0.1397 ; p = 0.038 ) , whereas age , bmi , and homa - ir did not enter the model .", "similarly , association of visfatin with tpoab ( coefficient = 0.0057 ; p = 0.0163 ) was observed with adjustment for age , bmi , ft3 , and homa - ir in multivariate regression analysis ."], ["to date , visfatin serum concentration in hypothyroidism has been analyzed in a few studies .", "reported elevated level of this adipocytokine in hypothyroidism with further increase after restoration of thyroid function .", "ozkaya et al . observed that visfatin level decreased after recovery . in those articles the etiology of hypothyroidism varied from chronic autoimmune thyroiditis , postpartum thyroiditis to thyroid function insufficiency after radioiodine treatment or after thyroidectomy .", "hence , to date autoimmune status of studied patients has not been taken into consideration .", "we hypothesized that these controversial findings might result from heterogeneity of study groups . to answer the question ,", "whether coexisting autoimmune inflammation influences visfatin level in hypothyroid patients , we analyzed its serum concentration in chronic autoimmune thyroiditis and thyroidectomized patients negative for thyroid antibodies . since we have previously proved that visfatin mrna expression is increased in thyroid malignancies and is correlated with tumor stage , we recruited only those patients who did not have any features of active neoplastic disease .", "we also recruited the control group adjusted for age , sex , and bmi . to the best of our knowledge ,", "this is the first study addressing the changes in the release of visfatin in thyroid autoimmunity .", "we came to interesting results indicating that visfatin serum concentration in hypothyroid patients is associated with both autoimmunity and free thyroid hormones level ( ft4 , ft3 ) .", "visfatin has been recognized as a cytokine with a broad range of immune and inflammatory activities , including induction of inflammatory cytokines , and regulation of macrophage and lymphocyte proliferation .", "visfatin stimulates the production of proinflammatory cytokines ( il-6 , tnf- , and il-1 ) and potentially acts as a chemotactic factor for monocytes .", "furthermore , its expression is upregulated by il-6 , tnf- , and il-1 [ 1012 ] .", "enhanced mrna expression of visfatin was observed in inflamed mucosa of patients with inflammatory bowel disease ( ibd ) .", "further analysis identified that antigen presenting cells ( i.e. , macrophages , dendritic cells ) might be a main source of this protein in ibd .", "visfatin has also potency for activation of t cells by upregulation of costimulatory molecules ( cd40 , cd54 , and cd80 ) on monocytes .", "we observed the positive association between visfatin and tpoab , and the latter is considered the best serological marker of chronic autoimmune thyroiditis .", "furthermore , tpoab contribute to thyroid destruction through antibody- and complement - dependent cell - cytotoxicity [ 13 , 14 ] . the first mechanism is associated with mononuclear cell infiltration of thyroid stroma .", "in addition , th1-derived cytokines ( il-2 , tnf- , and inf ) were found to be elevated in patients with chronic autoimmune thyroiditis .", "also , il-1 and tnf- have been recently reported to discriminate chronic autoimmune hypothyroid children from healthy controls .", "altogether , association of visfatin with tpoab observed in our study further supports our hypothesis that visfatin might be involved in the pathogenesis of chronic autoimmune thyroiditis .", "our findings about the association of visfatin with ft3 levels are in accordance with the results of in vitro and in vivo studies .", "however , controversial results whether t3 stimulates or downregulates the production of visfatin were found .", "in contrast , caixs et al . did not find any relationship between visfatin and free thyroid hormones . in vitro experiment", "showed the nonlinear regulation of visfatin mrna expression in the 3t3-l1 cell culture model affected by t3 .", "since our study groups significantly differed with free thyroid hormones levels , we were able to analyze visfatin concentration in a broad spectrum of ft3 and ft4 .", "therefore , we might suggest that pattern of visfatin changes varies in different ft3 concentration . according to our observations , as well as other authors", ", visfatin did not reflect insulin resistance assessed by homa - ir [ 18 , 19 ] .", "our study might then prove that visfatin in hypothyroidism depends on thyroid hormones level and coexisting autoimmunity .", "we may assume that these two factors should be taken into consideration to assess visfatin level in patients with thyroid dysfunction .", "in addition , the possible involvement of visfatin in pathogenesis of chronic autoimmune thyroiditis needs further research ."], ["the main limitation of our study is its cross - sectional design that does not enable us to reveal the causal pathways of relationship between visfatin and autoimmune thyroiditis .", "however , we used very strict criteria of exclusion to limit the possible influence of other known factors such as diabetes mellitus , other autoimmune processes , infection , and active neoplastic disease .", "our nonautoimmune group with hypothyroidism had been thyroidectomized at least 5 years earlier and did not have any clinical nor laboratory features of active thyroid cancer ."]]}
{"article_id": "PMC3560678", "article_text": ["diastolic heart failure ( dhf ) is a clinical syndrome characterized by the symptoms and signs of heart failure , a preserved ejection fraction and abnormal diastolic function .", "the percentage of patients with dhf in epidemiological studies ranges from 4071% ( mean 56% ) , but in hospital - based cohort studies it is slightly lower , ranging from 2455% ( mean 41% ) .", "elderliness , hypertension with left ventricular ( lv ) hypertrophy , pathologies such as diabetes , obesity , coronary artery disease , new onset atrial fibrillation , and others are commonly associated with dhf [ 24 ] .", "cardiotrophin-1 ( ct-1 ) is a member of the interleukin ( il)-6 cytokine family that shares the transmembrane signaling protein , glycoprotein ( gp ) 130 , as a receptor .", "ct-1 mrna is expressed in adult human heart , skeletal muscle , ovary , colon , prostate and testis and in fetal kidney and lung .", "ct-1 has hypertrophic and cytoprotective actions on the cardiac myocytes and may play an important role in other organ systems .", "the plasma concentration of ct-1 is increased in various cardiovascular diseases such as congestive heart failure , hypertension , valvular heart disease , acute coronary syndrome , and cardiomyopathies [ 818 ] . although , the prognostic importance of ct-1 in various cardiovascular diseases , including congestive heart failure , is well - known , there is limited data about ct-1 in patients with dhf .", "the purpose of the present study was to determine if ct-1 levels are significantly different in dhf patients compared to controls and to investigate the relationship between ct-1 and echocardiographic parameters .", "fifty - seven consecutive patients ( mean age 578 years , 24 ( 42% ) males ) diagnosed with dhf in our clinic and 33 controls ( mean age 557 years , 12 ( 36% ) males ) were included in the study .", "dhf was diagnosed when symptoms ( dyspnea not associated with any other cause ) and signs ( rale or peripheral edema ) of heart failure were observed along with a preserved lv ejection fraction ( lvef ) ( 50% ) and evidence of diastolic dysfunction .", "the control group was formed from volunteer subjects admitted to our clinic who did not have heart failure symptoms and signs and who had a preserved lvef . in order to exclude other causes of dyspnea", ", all patients underwent physical and laboratory examinations , including serum hemoglobin and thyroid hormones , chest radiogram and spirometry .", "patients with systolic heart failure , moderate or severe valvular stenosis or regurgitation , congenital heart disease , atrial fibrillation , chronic obstructive pulmonary disease , malignancy , and other extracellular fluid - increasing diseases , such as hypothyroidism and liver cirrhosis , were excluded from the study .", "all of the study participants underwent echocardiographic evaluation ( 2.5 mhz transducer , philips envisor c , bothell , washington , usa ) .", "standardized projections and measurements were performed for the evaluation of cardiac anatomy , ventricular function and valve competence .", "lv mass index was obtained by dividing the lv mass by the body surface area .", "the following conventional mitral inflow pulse wave doppler parameters were measured : peak velocity of early diastolic filling ( e ) , late filling ( a ) , and deceleration time ( dt ) of the e - wave velocity and isovolumetric relaxation time ( ivrt ) .", "these parameters were obtained from the apical four - chamber view with a 1 to 3 mm sample volume placed between the mitral leaflet tips during diastole .", "pulmonary venous flow parameters were also measured : peak systolic velocity ( ps ) , peak anterograde diastolic velocity ( pd ) and the ps / pd ratio .", "these parameters were obtained from the apical four - chamber view with a 2 to 3 mm sample volume placed 1 cm into the pulmonary vein .", "tissue doppler parameters were measured : peak systolic mitral annular velocity ( sm ) and early diastolic mitral annular velocity ( em ) and late diastolic mitral velocity ( am ) .", "these parameters were obtained from the apical four - chamber view with a 2 to 5 mm sample volume placed 1 cm within the septal and lateral insertions of the mitral leaflets .", "the mean of 3 or more measurements was used for analysis of the doppler data .", "the ratio of mitral peak velocity of early diastolic filling to early diastolic mitral annular velocity ( e / em ) was calculated for the lateral and septal annulus and the mean of the lateral and septal e / em were also determined .", "as previously described , the formula ( 1.24(e / em)+1.9 ) was used to estimate pulmonary capillary wedge pressure ( pcwp ) .", "patients with mean e / em 8 or a change in e / a ratio with the valsalva maneuver of < 0.5 were excluded from the study .", "diastolic dysfunction was defined as em < am if em was less than 10 cm / sec in lateral mitral annulus or less than 8 cm / sec in septal mitral annulus .", "blood samples were obtained during admission for routine chemistry , including ct-1 and n - terminal pro - b - type natriuretic peptide ( nt - probnp ) following an overnight fast .", "ct-1 values were measured with a sandwich enzyme immunoassay method ( organon teknika microwell system reader 230 s , germany ) in our hospital laboratory .", "nt - probnp analyses were made by the electrochemiluminescence immunoassay method ( cobas 6000 analyzer , roche diagnostics gmbh , mannheim , germany ) .", "the cockcroft - gault formula was used to calculate creatinine clearance . according to kolmogorov - smirnov normality test", ", 2 independent sample t tests were used to compare the normally distributed independent variables between the 2 groups , and mann - whitney u test was used to compare the non - normally distributed independent variables between the 2 groups . normally distributed continuous data were expressed as mean  standard deviation ( sd ) ; non - normally distributed continuous variables were presented as median and interquartile range ( iqr ) [ quartile l to quartile 3 ] .", "linear regression analyses were used to determine the effect of age , creatinine clearance , systolic blood pressure , left atrium diameter , and lv mass index on log ct-1 and log nt - probnp .", "statistical analysis was performed by using commercial software ( ibm spss statistics 19 , spss inc .", "fifty - seven consecutive patients ( mean age 578 years , 24 ( 42% ) males ) diagnosed with dhf in our clinic and 33 controls ( mean age 557 years , 12 ( 36% ) males ) were included in the study .", "dhf was diagnosed when symptoms ( dyspnea not associated with any other cause ) and signs ( rale or peripheral edema ) of heart failure were observed along with a preserved lv ejection fraction ( lvef ) ( 50% ) and evidence of diastolic dysfunction .", "the control group was formed from volunteer subjects admitted to our clinic who did not have heart failure symptoms and signs and who had a preserved lvef . in order to exclude other causes of dyspnea", ", all patients underwent physical and laboratory examinations , including serum hemoglobin and thyroid hormones , chest radiogram and spirometry .", "patients with systolic heart failure , moderate or severe valvular stenosis or regurgitation , congenital heart disease , atrial fibrillation , chronic obstructive pulmonary disease , malignancy , and other extracellular fluid - increasing diseases , such as hypothyroidism and liver cirrhosis , were excluded from the study .", "all of the study participants underwent echocardiographic evaluation ( 2.5 mhz transducer , philips envisor c , bothell , washington , usa ) .", "standardized projections and measurements were performed for the evaluation of cardiac anatomy , ventricular function and valve competence .", "lv mass index was obtained by dividing the lv mass by the body surface area .", "the following conventional mitral inflow pulse wave doppler parameters were measured : peak velocity of early diastolic filling ( e ) , late filling ( a ) , and deceleration time ( dt ) of the e - wave velocity and isovolumetric relaxation time ( ivrt ) .", "these parameters were obtained from the apical four - chamber view with a 1 to 3 mm sample volume placed between the mitral leaflet tips during diastole .", "pulmonary venous flow parameters were also measured : peak systolic velocity ( ps ) , peak anterograde diastolic velocity ( pd ) and the ps / pd ratio .", "these parameters were obtained from the apical four - chamber view with a 2 to 3 mm sample volume placed 1 cm into the pulmonary vein .", "tissue doppler parameters were measured : peak systolic mitral annular velocity ( sm ) and early diastolic mitral annular velocity ( em ) and late diastolic mitral velocity ( am ) .", "these parameters were obtained from the apical four - chamber view with a 2 to 5 mm sample volume placed 1 cm within the septal and lateral insertions of the mitral leaflets .", "the mean of 3 or more measurements was used for analysis of the doppler data .", "the ratio of mitral peak velocity of early diastolic filling to early diastolic mitral annular velocity ( e / em ) was calculated for the lateral and septal annulus and the mean of the lateral and septal e / em were also determined .", "as previously described , the formula ( 1.24(e / em)+1.9 ) was used to estimate pulmonary capillary wedge pressure ( pcwp ) .", "patients with mean e / em 8 or a change in e / a ratio with the valsalva maneuver of < 0.5 were excluded from the study .", "diastolic dysfunction was defined as em < am if em was less than 10 cm / sec in lateral mitral annulus or less than 8 cm / sec in septal mitral annulus .", "blood samples were obtained during admission for routine chemistry , including ct-1 and n - terminal pro - b - type natriuretic peptide ( nt - probnp ) following an overnight fast .", "ct-1 values were measured with a sandwich enzyme immunoassay method ( organon teknika microwell system reader 230 s , germany ) in our hospital laboratory .", "nt - probnp analyses were made by the electrochemiluminescence immunoassay method ( cobas 6000 analyzer , roche diagnostics gmbh , mannheim , germany ) .", "according to kolmogorov - smirnov normality test , 2 independent sample t tests were used to compare the normally distributed independent variables between the 2 groups , and mann - whitney u test was used to compare the non - normally distributed independent variables between the 2 groups .", "normally distributed continuous data were expressed as mean  standard deviation ( sd ) ; non - normally distributed continuous variables were presented as median and interquartile range ( iqr ) [ quartile l to quartile 3 ] .", "linear regression analyses were used to determine the effect of age , creatinine clearance , systolic blood pressure , left atrium diameter , and lv mass index on log ct-1 and log nt - probnp .", "statistical analysis was performed by using commercial software ( ibm spss statistics 19 , spss inc .", "there were no significant differences between the patient and control groups with regard to age , sex , hypertension , diabetes , cad , smoking , medications , body mass index , fasting blood glucose , thyroid status , lipid profile , creatinine clearance , serum creatinine and hemoglobin levels ( table 1 ) .", "the patient group had a higher lv posterior wall thickness and a larger left atrial size , but differences in lvef , chamber sizes , and mass index remained insignificant ( tables 1 and 2 ) .", "arterial blood pressures were also not different between the 2 groups . ct-1 and nt - probnp were significantly higher in the patient group ( ct-1 : 11.30 [ 8.0916.51 ] fmol / ml vs. 17.5 [ 8.9528.74 ] fmol / ml , p=0.017 ; nt - probnp : 64 [ 27.595 ] pg / ml vs. 82 [ 55.5241 ] pg / ml , p=0.009 ) ( table 1 ) .", "the mitral e , lateral , septal and mean e / em and the pcwp estimated from each of the e / em measurements were all significantly higher , whereas ps / pd , lateral and septal em and lateral and septal em / am were significantly lower in the patient group ( table 2 ) .", "ct-1 positively correlated with nt - probnp ( p=0.001 , r=0.349 ) , mean e / em ( p=0.003 , r=0.307 ) , and estimated mean pcwp ( p=0.001 , r=0.308 ) ( figure 1 ) . according to bivariate linear regression analyses , log ct-1 was predicted by left atrium diameter and log nt - probnp was predicted by age , creatinine clearance , and left atrium diameter ( table 3 ) . in multiple linear regression analysis ,", "log ct-1 and log nt - probnp were predicted only by left atrium diameter ( table 4 ) .", "the data of this study shows that the ct-1 is elevated in dhf patients and is associated with nt - probnp and estimated lv filling pressures in dhf patients .", "the ct-1 protein expression is constitutive not only in the heart , but also in the pulmonary , renal , gastrointestinal , cerebral , and muscular tissues .", "cardiac myocytes and cardiac fibroblasts may produce ct-1 in situations of biomechanical stress and under exposure to humoral factors such as angiotensin ii and norepineprine [ 2325 ] .", "ventricular stretch caused by pressure or volume overload is thought to be the major stimulus of myocardial ct-1 release .", "previous studies have found that ct-1 promotes myocardial structural changes , later participating in the progression of lv remodeling , which results in lv failure in various diseases such as hypertensive heart disease , coronary artery disease , aortic stenosis , and dilated cardiomyopathy .", "both bnp and ct-1 have a beneficial effect not only on the myocardium but also on hemodynamic variables .", "therefore , ongoing stimulation of bnp and ct-1 caused by ventricular stretch and circulating cytokines , promote structural remodeling and may become maladaptive with the progression of heart failure .", "ct-1 levels correlate with the severity of the heart failure and has been shown to be an independent predictor of mortality in chronic heart failure .", "plasma ct-1 has high diagnostic efficacy for heart failure ( at concentration of 68 fmol / ml , sensitivity and specificity were 95% and 82.5% , respectively ) .", "talwar et al found that plasma ct-1 levels measured shortly after an acute myocardial infarction serve as a strong independent predictor of lv systolic dysfunction . in another study , talwar et al .", "found that changes in ct-1 levels may reflect early changes in ventricular physiology that occur in the early part of the heart failure process before they can be detected echocardiographically .", "it was also found that ct-1 is significantly increased in patients with moderate to severe mitral regurgitation despite having normal lv systolic function and no hypertrophy .", "they were unable to demonstrate a similar increase in subjects with tricuspid or aortic regurgitation .", "in our study , we showed ct-1 elevation in a group of dhf patients with normal lv systolic function and no significant hypertrophy , but having more dilated left atrium than controls . considering these results", ", it may be speculated that left atrial wall stretch is a more important and earlier stimulation for ct-1 secretion rather than ventricular wall stretch .", "it has already been shown that ct-1 mrna was detected in both atria and ventricles . in a series of studies performed by lopez et al", ", they concluded that ct-1 is a more sensitive and specific biomarker than nt - pro bnp for detection of the inappropriateness of lv mass and lv dysfunction in hypertension , and nt - pro bnp remains a useful diagnostic tool for hypertensive heart disease only when lv systolic dysfunction is present .", "it was also shown that elevated ct-1 levels represent an earlier stage of the same neurohumoral cascade that results in elevated plasma bnp .", ", both ct-1 and nt - pro bnp were found to be more elevated in dhf patients than in control subjects , and there was a positive correlation between ct-1 and nt - pro bnp .", "previous studies show that elevated nt - probnp values is diagnostic for dhf and associated with elevated lv filling pressures .", "it has been found that there is a strong correlation between nt - probnp and e / em , and a threshold of 269.1 pg / ml of nt - probnp predicted an e / em > 15 with 90% sensitivity and 73% specificity in dhf .", "the positive correlation between elevated ct-1 and nt - probnp , e / em and pcwp in our study implies that pressure overload represented as increases in lv filling pressures could be the main underlying mechanism for ct-1 secretion in patients with dhf .", "a series of studies performed by lopez et al concluded that ct-1 is associated with systolic and diastolic dysfunction in hypertensive patients . in the other study they found that the ratio of peak velocity of early diastolic filling to peak velocity of late filling of mitral inflow ( e / a ) is the only parameter that differed in the hypertensive group , while dt and ivrt remained unchanged .", "significant association was found between normalization of ct-1 and regression of lv hypertrophy and increment of e / a . in their studies", "they only used conventional doppler parameters in order to evaluate diastolic function , and e / a was the only parameter found to be different from the control group . in our study", "chronic ct-1 treatment resulted in the development of insulin resistance as judged by a decrease in insulin - stimulated glucose uptake .", "we found high glucose levels in dhf patients , but this difference was insignificant , probably due to small sample size .", "second , despite the evidences that e / em is an effective noninvasive predictor of lv filling pressures , we did not measure lv pressures directly .", "it was already known that adipose tissue can be recognized as a source of ct-1 , which could account for the high circulating levels of ct-1 in patients with metabolic syndrome .", "finally , ct-1 mrna is expressed in other organs , and potential additional sources of circulating ct-1 can not be excluded in patients with dhf .", "this increase was associated with nt - probnp and estimated lv filling pressures in dhf patients .", "our results suggest that diastolic dysfunction and subsequent pressure increase at the left side of the heart may be responsible for ct-1 increase in dhf patients", ". however , further studies are required to elucidate the underlying mechanism for the ct-1 increase in these patients ."], "abstract_text": ["<S> summarybackgroundcardiotrophin-1 ( ct-1 ) is a member of the interleukin ( il-6 ) family of cytokines and is increased in various cardiovascular diseases , including chronic heart failure . </S>", "<S> the aim of the study was to determine if plasma ct-1 is associated with diastolic heart failure ( dhf ) and to investigate the relationship between ct-1 and echocardiographic parameters.material/methodsfifty-seven consecutive patients ( mean age 578 years , 24 males ) diagnosed with dhf in our clinic and 33 controls ( mean age 557 years , 12 males ) were included in the study . </S>", "<S> all study participants underwent echocardiographic evaluation and blood samples were obtained.resultsct-1 and nt - probnp values were significantly higher in dhf subjects than in controls ( 11.30 [ 8.0916.51 ] vs. 17.5 [ 8.9528.74 ] fmol / ml , p=0.017 and 64 [ 27.595 ] vs. 82 [ 55.5241 ] pg / ml , p=0.009 , respectively ) </S>", "<S> . the mitral peak velocity of early diastolic filling ( e ) , mean ratio of e to early diastolic mitral annular velocity ( e / em ) , and the pulmonary capillary wedge pressure ( pcwp ) estimated from e / em measurements were all significantly higher in the patient group ( 62.2714.69 vs. 75.6718.85 cm / sec , 6.401.48 vs. 10.303.48 , and 10 [ 911]vs . </S>", "<S> 14[1216 ] mmhg , p0.001 for all ) . </S>", "<S> lateral and septal em were significantly lower in the patient group ( 10.691.87 vs. 8.692.00 cm / sec and 8.911.22 vs. 6.651.58 cm / sec , p<0.001 for both ) . </S>", "<S> ct-1 positively correlated with nt - probnp ( p=0.001 , r=0.349 ) , mean e / em ( p=0.003 , r=0.307 ) , and estimated mean pcwp ( p=0.001 , r=0.308).conclusionsct-1 is elevated in patients with dhf and is associated with nt - probnp and estimated left ventricular filling pressures . </S>"], "labels": null, "section_names": ["Background", "Material and Methods", "Study population", "Echocardiographic measurements", "Biochemical measurements", "Statistical analysis", "Results", "Discussion", "Conclusions"], "sections": [["diastolic heart failure ( dhf ) is a clinical syndrome characterized by the symptoms and signs of heart failure , a preserved ejection fraction and abnormal diastolic function .", "the percentage of patients with dhf in epidemiological studies ranges from 4071% ( mean 56% ) , but in hospital - based cohort studies it is slightly lower , ranging from 2455% ( mean 41% ) .", "elderliness , hypertension with left ventricular ( lv ) hypertrophy , pathologies such as diabetes , obesity , coronary artery disease , new onset atrial fibrillation , and others are commonly associated with dhf [ 24 ] .", "cardiotrophin-1 ( ct-1 ) is a member of the interleukin ( il)-6 cytokine family that shares the transmembrane signaling protein , glycoprotein ( gp ) 130 , as a receptor .", "ct-1 mrna is expressed in adult human heart , skeletal muscle , ovary , colon , prostate and testis and in fetal kidney and lung .", "ct-1 has hypertrophic and cytoprotective actions on the cardiac myocytes and may play an important role in other organ systems .", "the plasma concentration of ct-1 is increased in various cardiovascular diseases such as congestive heart failure , hypertension , valvular heart disease , acute coronary syndrome , and cardiomyopathies [ 818 ] . although , the prognostic importance of ct-1 in various cardiovascular diseases , including congestive heart failure , is well - known , there is limited data about ct-1 in patients with dhf .", "the purpose of the present study was to determine if ct-1 levels are significantly different in dhf patients compared to controls and to investigate the relationship between ct-1 and echocardiographic parameters ."], ["fifty - seven consecutive patients ( mean age 578 years , 24 ( 42% ) males ) diagnosed with dhf in our clinic and 33 controls ( mean age 557 years , 12 ( 36% ) males ) were included in the study .", "dhf was diagnosed when symptoms ( dyspnea not associated with any other cause ) and signs ( rale or peripheral edema ) of heart failure were observed along with a preserved lv ejection fraction ( lvef ) ( 50% ) and evidence of diastolic dysfunction .", "the control group was formed from volunteer subjects admitted to our clinic who did not have heart failure symptoms and signs and who had a preserved lvef . in order to exclude other causes of dyspnea", ", all patients underwent physical and laboratory examinations , including serum hemoglobin and thyroid hormones , chest radiogram and spirometry .", "patients with systolic heart failure , moderate or severe valvular stenosis or regurgitation , congenital heart disease , atrial fibrillation , chronic obstructive pulmonary disease , malignancy , and other extracellular fluid - increasing diseases , such as hypothyroidism and liver cirrhosis , were excluded from the study .", "all of the study participants underwent echocardiographic evaluation ( 2.5 mhz transducer , philips envisor c , bothell , washington , usa ) .", "standardized projections and measurements were performed for the evaluation of cardiac anatomy , ventricular function and valve competence .", "lv mass index was obtained by dividing the lv mass by the body surface area .", "the following conventional mitral inflow pulse wave doppler parameters were measured : peak velocity of early diastolic filling ( e ) , late filling ( a ) , and deceleration time ( dt ) of the e - wave velocity and isovolumetric relaxation time ( ivrt ) .", "these parameters were obtained from the apical four - chamber view with a 1 to 3 mm sample volume placed between the mitral leaflet tips during diastole .", "pulmonary venous flow parameters were also measured : peak systolic velocity ( ps ) , peak anterograde diastolic velocity ( pd ) and the ps / pd ratio .", "these parameters were obtained from the apical four - chamber view with a 2 to 3 mm sample volume placed 1 cm into the pulmonary vein .", "tissue doppler parameters were measured : peak systolic mitral annular velocity ( sm ) and early diastolic mitral annular velocity ( em ) and late diastolic mitral velocity ( am ) .", "these parameters were obtained from the apical four - chamber view with a 2 to 5 mm sample volume placed 1 cm within the septal and lateral insertions of the mitral leaflets .", "the mean of 3 or more measurements was used for analysis of the doppler data .", "the ratio of mitral peak velocity of early diastolic filling to early diastolic mitral annular velocity ( e / em ) was calculated for the lateral and septal annulus and the mean of the lateral and septal e / em were also determined .", "as previously described , the formula ( 1.24(e / em)+1.9 ) was used to estimate pulmonary capillary wedge pressure ( pcwp ) .", "patients with mean e / em 8 or a change in e / a ratio with the valsalva maneuver of < 0.5 were excluded from the study .", "diastolic dysfunction was defined as em < am if em was less than 10 cm / sec in lateral mitral annulus or less than 8 cm / sec in septal mitral annulus .", "blood samples were obtained during admission for routine chemistry , including ct-1 and n - terminal pro - b - type natriuretic peptide ( nt - probnp ) following an overnight fast .", "ct-1 values were measured with a sandwich enzyme immunoassay method ( organon teknika microwell system reader 230 s , germany ) in our hospital laboratory .", "nt - probnp analyses were made by the electrochemiluminescence immunoassay method ( cobas 6000 analyzer , roche diagnostics gmbh , mannheim , germany ) .", "the cockcroft - gault formula was used to calculate creatinine clearance . according to kolmogorov - smirnov normality test", ", 2 independent sample t tests were used to compare the normally distributed independent variables between the 2 groups , and mann - whitney u test was used to compare the non - normally distributed independent variables between the 2 groups . normally distributed continuous data were expressed as mean  standard deviation ( sd ) ; non - normally distributed continuous variables were presented as median and interquartile range ( iqr ) [ quartile l to quartile 3 ] .", "linear regression analyses were used to determine the effect of age , creatinine clearance , systolic blood pressure , left atrium diameter , and lv mass index on log ct-1 and log nt - probnp .", "statistical analysis was performed by using commercial software ( ibm spss statistics 19 , spss inc ."], ["fifty - seven consecutive patients ( mean age 578 years , 24 ( 42% ) males ) diagnosed with dhf in our clinic and 33 controls ( mean age 557 years , 12 ( 36% ) males ) were included in the study .", "dhf was diagnosed when symptoms ( dyspnea not associated with any other cause ) and signs ( rale or peripheral edema ) of heart failure were observed along with a preserved lv ejection fraction ( lvef ) ( 50% ) and evidence of diastolic dysfunction .", "the control group was formed from volunteer subjects admitted to our clinic who did not have heart failure sy